Synthesis and antiviral activity of novel spirocyclic nucleosides

Alexander J. A. Cobb *a, Antonio Dell’Isola a, Ban O. Abdulsattar§ b, Matthew M. W. McLachlan c, Benjamin W. Neuman b, Christin Müller d, Kenneth Shankland a, Hawaa M. N. Al-Mulla b, Alexander W. D. Binks b and Warren Elvidge b
aSchool of Chemistry, Food and Pharmacy (SCFP), University of Reading, Whiteknights, Reading, Berks RG6 6AD, UK
bSchool of Biological Sciences, University of Reading, Whiteknights, Reading, Berks RG6 6AJ, UK
cSyngenta, Jealott's Hill International Research Centre, Bracknell, Berks RG42 6EY, UK
dInstitut für Medizinische Virologie, Justus-Liebig-Universität Giessen, Schubertstraße 81, 35392 Giessen, Germany

Received 4th June 2018 , Accepted 13th October 2018

First published on 17th October 2018


The synthesis of a number of spirocyclic ribonucleosides containing either a triazolic or azetidinic system is described, along with two analogous phosphonate derivatives of the former. These systems were constructed from the same β-D-psicofuranose starting material. The triazole spirocyclic nucleosides were constructed using the 1-azido-1-hydroxymethyl derived sugars, where the primary alcohol was alkylated with a range of propargyl bromides, whereas the azetidine systems orginated from the corresponding 1-cyano-1-hydroxymethyl sugars. Owing to their close similarity with ribavirin, the library of compounds were investigated for their antiviral properties using MHV (Murine Hepatitis Virus) as a model.


Introduction

Ribavirin 1 (also known as rebatol® and virazole®), a 1,2,4-triazole nucleoside, discovered by Witkowski and co-workers in 1972,1 exhibits a broad spectrum of antiviral activity against both DNA and RNA viruses and has been used for the treatment of a variety of viral infections, such as severe respiratory syncytial virus (RSV) infection, lassa fever, influenza A and B, measles and mumps.2 Today ribavirin is the standard care for the treatment of chronic hepatitis C in combination with PEGylated interferon-α (Fig. 1).3
image file: c8nj02777c-f1.tif
Fig. 1 Ribavirin 1, and its phosphorylated active form.

Despite its potent antiviral activity, the use of ribavirin is limited by adverse effects – mainly associated with haemolytic anaemia.4 In plasma, the nucleoside is transported into erythrocytes by suitable transporters and converted into its phosphate forms (RMP, RDP and RTP). These then accumulate owing to the lack the phosphatases needed to hydrolyse them, leading to a relative deficiency of adenosine triphosphate and subsequent extravascular haemolysis.5

Therefore, driven by the need to find safer and more specific IMP mimics, we set out to develop a range of spirocyclic nucleosides, the first class containing a [1,2,3]-triazolyl moiety (accessed via an intramolecular [1,3]-dipolar cycloaddition) and the second an azetidine functionality (accessed via intramolecular SN2 reaction) around the spirocyclic anomeric centre (Fig. 2) and which show remarkable structural similarity to ribavirin. Nucleosides where the conformational restriction involves the nucleobase are known as cyclonucleosides, and have garnered interest on a number of fronts, including agrochemical (e.g. hydantocidin),6 as well as being of synthetic interest.7,8


image file: c8nj02777c-f2.tif
Fig. 2 Spirocyclic nucleosides targeted.

Nucleosides restricted in this way can often show a greater specificity towards their enzymatic target, and this can result in enhanced biological activity.9

Results and discussion

The starting point for the synthesis of both spirocyclic systems was β-D-fructopyranose 4, which was converted to D-psicopyranose 5 in a straightforward and previously reported three step procedure.10 This intermediate was then converted to the corresponding furanose 6 using amberlyst A15 resin in acetone (Scheme 1). The subsequent protecting group used on the 6′OH-position was found to be hugely influential on the next glycosidation step. In the case of azide insertion, the benzoate ester 7 sufficed, but for the nitrile insertion, the benzylated intermediate 8 was required for efficient reaction (Scheme 1). These glycosidations were achieved through the use of trimethylsilyl triflate and either TMS azide for compound 9, or TMS nitrile for compound 11.11
image file: c8nj02777c-s1.tif
Scheme 1 Access to psicofuranosyl derivatives building blocks 10 and 11.

The former resulted in the trimethylsilyl ether which required acidic methanolysis to give the primary alcohol 10, but fortunately this was very high yielding. Also satisfyingly, only the desired anomer was produced and this is assumed to be a result of the intermediate oxonium ion being very hindered on the α-face owing to the presence of the acetonide, in addition to the product being the more stable isomer owing to a positive anomeric effect.

[1,2,3]-Triazolooxazines

With building block 10 in hand, the alkylation of the primary alcohol to form the alkynyl-azido compound 2 was undertaken. A variety of bases were used to achieve this, initially with propargyl bromide as the electrophile (Table 1). Using sodium hydride in THF (entry 1), two products were isolated.
Table 1 Alkylation study for the synthesis of azido-alkyne 2aa

image file: c8nj02777c-u1.tif

Entry Base Solvent Yield 2a (%) Yield 12 (%)
a Reactions were performed at 0 °C for 2 h.
1 NaH THF 11 6
2 NaH/TBAI CH3CN 22 6
3 LiHMDS THF
4 NaHMDS THF 11 70
5 KHMDS THF 16 73
6 BEMP CH3CN 54


The first was identified as the required product 2a, which was obtained in a disappointing 11% yield, and the second – obtained in 6% yield – was the hydrolysed diol 12. None of the other metal bases screened gave the desired compound in any useful yield, and interestingly KHMDS in THF gave predominantly the diol 12. We then tested the non-metallic phosphazene base BEMP in acetonitrile (entry 6), and pleasingly this gave exclusively the azido-alkynyl product 2a in 54% yield.

With the optimised alkylation conditions in place, a range of propargyl bromides 15a–f were used to obtain the corresponding propargylic ethers 2. These were prepared from commercially available aryl iodides and propargyl alcohol using a two-step process involving Sonogashira coupling12 followed by conversion of the resulting 3-arylprop-2-ynyl alcohols 14a–f to their corresponding bromides under Appel conditions (Table 2).

Table 2 Preparation of 3-arylprop-2-ynyl bromides 15a–f

image file: c8nj02777c-u2.tif

Entry Ar Product Yield, % Product Yield, %
1 Ph 14a nd 15a 83
2 4-Cl-C6H4 14b 68 15b 76
3 4-MeO-C6H4 14c 84 15c 56
4 4-F-C6H4 14d 86 15d 85
5 3-F-C6H4 14e 81 15e 70
6 2-F-C6H4 14f 55 15f 49


The O-alkylation reaction was then performed using the optimized conditions described (Table 3). The crude alkynyl-azido intermediates 2a–k underwent efficient intramolecular 1,3-dipolar cycloaddition upon heating in toluene for 24 h,13 resulting in the novel protected anomeric spironucleoside library 16a–k.

Table 3 Alkylation and 1,3-dipolar cycloaddition to access the spirocyclic nucleoside systema

image file: c8nj02777c-u3.tif

Entry R Product Overall yield, %
a Overall isolated yield for alkylation and cycloaddition.
1 H 16a 51
2 Me 16b 53
3 Et 16c 43
4 2-Napthyl 16d 59
5 Ph 16e 44
6 4-Cl-C6H4 16f 45
7 4-MeO-C6H4 16g 43
8 4-F-C6H4 16h 43
9 3-F-C6H4 16i 39
10 2-F-C6H4 16j 45
11 n-Pentyl 16k 36


Debenzoylation of these compounds, using a 7 M solution of ammonia in methanol gave intermediates 17a–k. X-ray diffraction on a single crystal of 17a was our first glimpse of the spirocyclic nature of these novel systems (Fig. 3). Finally, these acetonide systems were deprotected with acidic resin (Dowex® 50 W) to give straightforward access to anomeric spironucleosides 18a–k in satisfactory yield (Table 4).14


image file: c8nj02777c-f3.tif
Fig. 3 X-ray crystal structure of 17a (CCDC 1840501).
Table 4 Final deprotection steps to obtain anomeric spirocyclic nucleosides 18a–ka

image file: c8nj02777c-u4.tif

Entry R Product Overall yield, %
a Overall isolated yield for benzoate and acetonide deprotection.
1 H 18a 56
2 Me 18b 47
3 Et 18c 51
4 2-Napthyl 18d 50
5 Ph 18e 67
6 4-Cl-C6H4 18f 63
7 4-MeO-C6H4 18g 52
8 4-F-C6H4 18h 80
9 3-F-C6H4 18i 74
10 2-F-C6H4 18j 73
11 n-Pentyl 18k 69


Spiro-[1,2,3]-triazolooxazine nucleoside phosphonates

As has been mentioned, the active form of ribavirin is the 5′-phosphate, and as such nucleoside analogues containing the phosphonate moiety as a bioisostere of this have achieved reasonable success.15 As such we sought to also make the phosphonate 22 as a part of our library (Scheme 2). This was achieved through Dess–Martin oxidation of the 5′-OH in 17a to the corresponding aldehyde 19 in 80% yield, followed by Horner–Wadsworth–Emmons reaction using tetraethyl methylenediphosphonate to give the corresponding vinyl phosphonate 20 in a 5[thin space (1/6-em)]:[thin space (1/6-em)]2 E/Z ratio. High pressure (4 bar) reduction in methanol gave the corresponding alkyl phosphonate 21 in 61% yield. This underwent deprotection, first of the acetonide using Dowex resin as described previously, followed by deprotection of the phosphonate ester to the corresponding phosphonic acid 23 using trimethylsilyl bromide and 2,6-lutidine in dichloromethane in a 12% overall yield for the two final steps (Scheme 3).
image file: c8nj02777c-s2.tif
Scheme 2 Synthesis of phosphonate 23.

image file: c8nj02777c-s3.tif
Scheme 3 Synthesis of azetidinic systems.

Spirocyclic azetidines

Another class of spirocyclic nucleoside we were interested in was based on the azetidinic system as described previously by Fuentes and co-workers.16 In this work, they took the N-,O-methyl sulfonate 25a and subjected it to sodium hydride in DMF, whereupon intramolecular cyclisation of the sulfonamide anion onto the O-mesylate resulting in the azetidinic system 26a. We conducted the same reaction for tosyl and nosyl compounds 25b and 25c. Subsequently, for the mesyl, and tosyl systems (26a and 26b respectively), we achieved deprotection of the acetonide in the usual way, followed by removal of the benzyl protecting group at high pressure to give spirocylic azetidines 27a and 27b. The configuration of the tosyl derivative 27b was determined by X-ray crystallography (Fig. 4). The nosyl derivative 26c was synthesized due to the ease with which it can be removed from the nitrogen atom on which it resides compared to the mesyl and tosyl compounds to expose the secondary amine. However, we were unable to deprotect this compound fully to the corresponding triol but did managed to convert it to the secondary amine intermediate 28. This possibly represents a useful scaffold to generate a library with structurally diverse spironucleosides.
image file: c8nj02777c-f4.tif
Fig. 4 X-ray crystal structure of novel tosyl azetidinic system 27b (CCDC 1840502).

Biological evaluation

Coronaviruses are an important family of human and veterinary pathogens that can cause enteric and respiratory infections, including severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV), which are amongst the most lethal viral infections currently known. Infection with the model coronavirus Murine hepatitis virus (MHV) can also lead to gastroenteritis, nephritis, hepatitis, encephalitis, and progressive demyelinating disease, depending on the animal model, virus strain and inoculation route used.17 Coronaviruses are considered the largest and most complex RNA viruses known, encoding an unusually wide array of proteins that interact with or modify viral RNA.18 Since coronaviruses are enveloped viruses with a positive-sense RNA genome, they are predicted to be sensitive to RNA-like drugs,19 and some nucleosides, such as ribavirin 1, have anti-coronaviral activity.

As a result, MHV has been chosen as a proving ground for the novel nucleoside analogues described in this study for antiviral activity. In order to test for antiviral effects, MHV was grown on mouse 17Cl-1 cells that had been pre-treated with the experimental compounds at a concentration of 1 mM 3 h before inoculation to allow time for drug uptake and potential phosphorylation, inoculated with 10 infectious units of virus per cell for 1 h to ensure as many cells as possible were infected, rinsed with saline to remove any virus that had not entered a cell yet, and incubated again with the same amount of experimental compound for 14 h. The amount of MHV released from infected cells usually peaks at about 14 h after infection (Fig. 5A). The amount of virus released from infected cells was then measured by plaque assay. Two of the treatments, 18b and 18f reduced the amount of MHV that was released by about tenfold (Fig. 5B). Unfortunately, no significant activity was seen for compound 27b or the phosphonate analogue, compound 22.


image file: c8nj02777c-f5.tif
Fig. 5 Effects of spirocyclic nucleosides on release of infectious virus. (A) Experimental procedure used to test antiviral efficacy. Mouse 17Cl-1 cells were pre-treated with compounds 3 h before inoculation with the virus MHV. After 14 h virus growth was measured by plaque assay. (B) Effects of 1 mM treatment on virus release, normalized to virus release from infected untreated cells.

MHV infection in 17Cl-1 cells normally results in formation of large multinucleate syncytia starting about 6 h after infection, followed by detachment of cells from the culture flask and widespread cell death by 14 h after infection.

Compounds were tested for side effects on cell growth (Fig. 6A) and then for the ability to protect cells from overt signs of infection (Fig. 6B). Selected compounds were tested for side effects on cell viability and growth over three days of treatment by MTT assay. The most effective experimental compound 18f did not show any significant toxicity at concentrations of 1 mM and below (Fig. 6C). The concentration that produced a 50% reduction in cell viability in these assays was greater than 1 mM for each of the experimental compounds tested, demonstrating that the compounds are relatively non-toxic.


image file: c8nj02777c-f6.tif
Fig. 6 Effects of spirocyclic nucleoside treatment on cells. The experimental procedures used to test for effects on cell viability (A) and the appearance of virus-induced cytopathic effects in mouse 17Cl-1 cells (B) are shown. The effects of experimental compounds on cell viability were assessed by MTT assay 24 hours after treatment (C). Cytopathic effects due to MHV infection were assessed by counting visible nuclei in cells and syncytia after infection in the presence of absence of experimental compounds (D). Representative examples of cells from each treatment group are shown (E) to illustrate the appearance of typical MHV cytopathic effects including formation of multinucleate syncytia (most apparent in 2.5× magnified insets from infected 250 μM, 500 μM and 1 mM treatment groups) and loss of cells due to cell destruction (apparent in the infected 0 μM and 150 μM treatment groups).

Compound 18f was then screened for the ability to protect cells from MHV-induced cytopathology, including cell fusion and detachment from the culture flask. The 17Cl-1 cells were pre-treated with 18f 3 h before infection, rinsed after 24 h to remove any dead or detached cells, and surviving adherent cells were photographed 24 h after infection. Treatment with 1 mM compound 18f resulted in a dose-dependent reduction in both syncytium formation and detachment of infected cells (Fig. 6D and E).

From these data it was concluded that 18f exerted a limited protective effect on treated cells at concentrations of 250 μM, and appeared to completely protect treated cells from overt cytopathic effects of virus infection at 2 mM. This also demonstrated that the apparent antiviral activity of 18f was not simply an artefact of cytotoxicity. More detailed dose–response experiments were performed for four of the experimental compounds in order to better gauge their antiviral potential. The two treatments that were previously identified as most effective, compound 18b and 18f, strongly reduced virus growth at 1 mM and 2 mM concentrations, but did not significantly inhibit virus growth at 0.1 mM or lower concentrations (Fig. 7) similar to the dose-effectiveness profile of the antiviral nucleoside ribavirin (Fig. 8). Indeed, the low activity of ribavirin against MHV has been well documented.20–22 Two groups using different cell culture methods demonstrated an approximately ten-fold reduction of MHV growth in the presence of 41 micromolar ribavirin,22 and a greater than ten-fold reduction in MHV growth in the presence of a 40 micromolar conjugate of human hemoglobin and ribavirin bound to haptoglobin.20 However, ribavirin treatment has also been shown to reduce the severity of MHV-induced disease in mice and alter the cytokine profile in infected mice.20–22 In the investigation of our spirocyclic systems, pre-treatment with 2 mM of 18f produced the strongest antiviral effects, resulting in approximately one million-fold reduction of MHV growth. Unfortunately, no significant activity was seen for compounds 23 or 27b at any of the concentrations tested.


image file: c8nj02777c-f7.tif
Fig. 7 Dose-dependent inhibition of virus growth. The experimental protocol was performed as described in Fig. 6A.

image file: c8nj02777c-f8.tif
Fig. 8 Ribavirin has the similarly low mM cytotoxicity as for our compounds and reduced virus titre in a dose-dependent manner.

A further experiment was performed in order to learn more about the mechanism of 18f antiviral activity by evolving drug resistance. MHV was serially passaged eight times on 17Cl-1 cells, which had been pre-treated with 1 mM 18f, a concentration that reproducibly reduced viral growth by about 90%. Previous work on antiviral compounds suggested that these conditions were appropriate for the selection of drug-resistant coronavirus within about five passages.23 MHV growth in the presence of 18f was consistently reduced by about 90% compared to the virus produced in untreated control cells, and did not develop resistance (data not shown). These results suggest that the mechanism of action of 18f is unclear, and that effects of 18f on the cell cannot be ruled out as a potential explanation of the antiviral effects.

Conclusions

In conclusion, we have synthesized an array of spirocyclic ribonucleosides from a common precursor, the protected psicose derivative 6. Both triazolic and azetidinic systems were accessed, as well as phosphate derivatives. Some of these agents showed promising activity towards MHV (Mouse Hepatitis Virus), with the most promising being the triazolic system 18f.

Experimental

Materials and methods

All moisture-sensitive reactions were carried out under an atmosphere of nitrogen. All solvents were analytical grade purity, dried over standard drying agents and stored over 3 Å molecular sieves. All commercially available chemicals were purchased from Sigma Aldrich and used as supplied unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC), which was performed on Merck aluminium backed plates coated with 0.2 mm silica gel 60 F254. The spots were visualised using UV light (254 nm) and then by charring with 10% sulfuric acid in methanol. Column chromatography was carried out using silica gel 60 Å (35–70 μm). 1H-NMR spectra were recorded in suitable deuterated solvents (CDCl3, CD3OD) using a Bruker DPX 400 (400 MHz) or a Bruker Avance III 400 (400 MHz) or a Bruker Avance II+ 500 (500 MHz) spectrometer. In all cases, tetramethylsilane (TMS) was used as internal standard for calibrating chemical shifts (δ), which were quoted in parts per million. Abbreviations were used for the following multiplicities: s, singlet; d, doublet; dd, doublet of doublets; ddd, double double doublet; dt, doublet of triplets; t, triplet; td, triplet of doublets, tt, triplet of triplets; q, quartet; m, multiplet and the coupling constants J were quoted in Hz. 13C-NMR spectra were recorded at 100 MHz on a DPX 400 or Avance III 400 or 125 MHz on a Avance II+ 500 in suitable deuterated solvents (CDCl3, CD3OD). Assignments were confirmed by homonuclear 2D COSY and heteronuclear 2D correlated experiments (1H,13CHSQC, 1H,13C-HMBC). Infrared spectra were recorded on a Perkin-Elmer FT-IR spectrometer as a thin film. The absorptions are quoted in wavenumbers (cm−1). Mass spectrometry data was recorded on a Thermo Scientific LTQ Orbitrap XL using electrospray ionisation (ESI) conditions. Optical rotations were recorded with a Perkin-Elmer 341 polarimeter using a sodium lamp (D line, 589 nm) and mercury lamp (yellow line, 578 nm) as the source of polarized light. They were measured at 20 ± 2 °C in the stated solvent and are quoted in units of 10−1 deg cm2 g−1. Solution concentrations (c) are given in the units of 10−2 g cm−3. Melting points are uncorrected and were determined on a Stuart SMP3 melting point apparatus. HPLC analysis was determined on an Agilent Technologies 1200 Series HPLC, using a ratio of HPLC grade hexanes and propan-2-ol as the eluent, using either a Chiralpak AD-H column (0.46 cm ∅ × 25 cm) or a Chiralcel OD column (0.46 cm ∅ × 25 cm), and detection by UV at 210 nm or 254 nm.

Synthetic procedures

1,2:3,4-Di-O-isopropylidine-β-D-psicofuranose 610. l,2:3,4-Di-O-isopropylidine-β-D-psicopiranose 5 (5.0 g, 19.2 mmol) was dissolved in acetone (50 mL). A15 amberlyst resin (250 mg) was added in one portion and the resulting suspension was stirred vigorously for 13 h, then the resin was filtered off, basified with triethylamine and washed with acetone. The filtrate was evaporated under reduced pressure to afford the crude residue as orange oil, which is pure enough to be used immediately for the next step. Purification of the crude product by column chromatography (silica gel, hexane/ethyl acetate 85/15) gave the pure protected β-D-psicofuranose 6 (4.4 g, 88% yield) as white solid. Data for 6: white solid, m.p. = 56–57 °C. [α]20D = −92.1 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.33 (3H, s, CH3), 1.41 (3H, s, CH3), 1.45 (3H, s, CH3), 1.53 (3H, s, CH3), 3.18 (1H, dd, J = 10.4, 2.8, OH), 3.65 (1H, td, J = 12.4, 3.6, H-6a), 3.77 (1H, dt, J = 12.4, 2.4, H-6b), 4.07 (1H, d, J = 10.0, H-1a), 4.30 (1H, t, J = 2.8, H-5), 4.34 (1H, d, J = 9.8, H-1b), 4.65 (1H, d, J = 6.0, H-3), 4.92 (1H, dd, J = 6.0, 1.2, H-4). 13C-NMR (100 MHz, CDCl3): 24.8 (CH3), 26.2 (CH3), 26.3 (CH3), 26.5 (CH3), 64.0 (C-6), 69.9 (C-1), 81.7 (C-4), 85.8 (C-3), 86.8 (C-5), 111.7 (C-2), 112.3 (C(CH3)2), 113.4 (C(CH3)2). IR (CHCl3, cm−1): 3480 (br), 2988 (w), 1372 (m), 1209 (m), 1062 (s), 1029 (s), 851 (s).
1,2:3,4-Di-O-isopropylidene-6-O-benzoyl-β-D-psicofuranose 710. To a stirring solution of l,2:3,4-di-O-isopropylidine-β-D-psicofuranose 6 (5.0 g, 19.2 mmol), triethylamine (13 mL, 96.0 mmol) and dimethylaminopyridine (230 mg, 1.92 mmol) in dichloromethane (60 mL) was added benzoyl chloride (2.45 mL, 21.1 mmol) dropwise at 0 °C. The resulting light yellow solution was allowed to warm to room temperature and stirred for 14 h. The mixture was partitioned between saturated aq. NaHCO3 and dichloromethane. The combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated to dryness under reduced pressure. Purification of the crude product by column chromatography (silica gel, hexane/ethyl acetate 9/1) gave the title compound 7 (5.6 g, 80% yield) as a white solid. Data for 7: white solid, m.p. = 72–73 °C. [α]20D = −64.4 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.34 (3H, s, CH3), 1.35 (3H, s, CH3), 1.43 (3H, s, CH3), 1.47 (3H, s, CH3), 4.08 (1H, d, J = 9.6, H-1a), 4.31 (1H, d, J = 9.6, H-1b), 4.36–4.48 (3H, m, H-3, H-4 and H-5), 4.68 (1H, d, J = 5.6, H-6a), 4.83 (1H, d, J = 6.0, H-6b), 7.44 (2H, t, J = 8.0, 2 × Hm), 7.57 (1H, tt, J = 7.6, 1.2, Hp), 8.08 (2H, dd, J = 8.4, 1.2, 2 × Ho). 13C-NMR (100 MHz, CDCl3): 25.1 (CH3), 26.2 (CH3), 26.3 (CH3), 26.4 (CH3), 64.8 (C-1), 66.1 (C-6), 82.2 (C-4), 83.0 (C-3), 85.2 (C-5), 111.7 (C-2), 112.8 (C(CH3)2), 113.7 (C(CH3)2), 128.2 (Cm), 129.7 (Cipso), 129.8 (Co), 133.1 (Cp), 166.1 (COPh). IR (CHCl3, cm−1): 2989 (w), 1720 (s), 1373 (m), 1268 (s), 1065 (s), 1024 (s), 854 (s), 709 (s).
1,2:3,4-Di-O-isopropylidene-6-O-benzyl-β-D-psicofuranose 811. Sodium hydride (60% dispersion in mineral oil, 5.2 g, 0.13 mol) was placed in a 1 L 3 neck round bottom flask equipped with a thermometer and pressure equalising dropping funnel and cooled to 0 °C. Anhydrous DMF (50 mL) was slowly added under inert atmosphere (N2), ensuring that the internal temperature remained less than 10 °C. A solution of protected D-psicofuranose 6 (28.66 g, 0.11 mol) in anhydrous DMF (200 mL) was then added dropwise over 30 min – again with control of the exotherm and stirred for a further 30 min at 0 °C. A solution of benzyl bromide (19.4 mL, 0.16 mol) in anhydrous DMF (100 mL) was then added dropwise at 0 °C and the resulting solution allowed to warm to room temperature. After this had been achieved, the mixture was stirred for a further 4 h. It was then treated with methanol, diluted with water and extracted with diethylether. The combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated to dryness under reduced pressure to afford the crude residue as orange oil. Purification of the crude product by column chromatography (silica gel, isohexane/ethyl acetate 9/1) gave the title compound 8 (32.26 g, 84% yield) as colourless syrup. Data for 8: pale yellow syrup. [α]20D = −65.6 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.29 (3H, s, CH3), 1.35 (3H, s, CH3), 1.41 (3H, s, 2 × CH3), 1.48–1.59 (2H, m, 2 × H-6), 4.03 (1H, d, J = 9.6, H-1a), 4.26–4.31 (1H, m, H-5), 4.28 (1H, d, J = 9.6, H-1b), 4.52 (2H, AB q, J = 12.4, CH2Ph), 4.36–4.57 (1H, d, J = 5.8, H-3), 4.72 (1H, d, J = 5.8, H-4), 7.24–7.32 (5H, m, 5 × Harom). 13C-NMR (100 MHz, CDCl3): 25.2 (CH3), 26.3 (CH3), 26.4 (CH3), 26.5 (CH3), 69.8 (C-1), 70.9 (C-6), 73.3 (C-1′), 82.6 (C-4), 83.8 (C-5), 85.2 (C-3), 111.4 (C-2), 112.5 (C(CH3)2), 113.6 (C(CH3)2), 127.6 (Cm), 129.6 (Cipso), 129.3 (Co), 138.2 (Cp). IR (CHCl3, cm−1): 2941 (w), 1450 (s), 1370 (s).
1-O-Trimethylsilyl-2-azido-2-deoxy-3,4-O-isopropylidene-6-O-benzoyl-β-D-psicofuranose 914. To a solution of spiroketal 7 (1.0 g, 2.74 mmol) and freshly distilled acetonitrile (30 mL) was added azidotrimethylsilane (750 μL, 5.48 mmol) at 0 °C under a nitrogen atmosphere in the presence of activated 4 Å molecular sieves. The solution was stirred for 5 min and trimethylsilyltriflate (146 μL, 0.82 mmol) added dropwise, and the stirring was continued at 0 °C for a further 5 min. The mixture was neutralized with triethylamine (228 μL, 1.64 mmol), diluted with ethyl acetate and warmed to room temperature. The resulting solution was partitioned between saturated aq. NaHCO3 and ethyl acetate. The organic layers were washed with brine, dried over MgSO4, filtered and concentrated to dryness under reduced pressure. Purification of the crude product by column chromatography (silica gel, hexane/ethyl acetate 95/5) gave the pure 9 (1.03 g, 89% yield) as colourless syrup. Data for 9: [α]20D = −89.6 (c = 0.01, CHCl3). 1H NMR (400 MHz, CDCl3): δ 0.01 (9H, s, Si(CH3)3), 1.13 (3H, s, CH3), 1.47 (3H, s, CH3), 3.81 (1H, d, J = 11.6, H-1a), 3.84 (1H, d, J = 11.2, H-1b), 4.24–4.28 (2H, m, H-4, H-6a), 4.33–4.37 (1H, m, H-6b), 4.42 (1H, td, J = 6.4, 1.6, H-5), 4.67 (1H, dd, J = 2.0, 6.0, H-3), 7.25 (2H, dd, J = 7.6, 7.2, 2 × Hm), 7.37 (1H, tt, J = 7.6, 1.2, Hp), 7.91(2H, d, J = 7.2, 2 × Ho). 13C NMR (100 MHz, CDCl3): 0.0 (Si(CH3)3), 25.7 (CH3), 27.1(CH3), 65.0 (C-6), 65.9 (C-1), 83.1 (C-3), 85.3 (C-5), 85.9 (C-4), 101.4 (C-2), 114.1 (C(CH3)2), 129.0 (Cm), 130.3 (Cipso), 130.4 (Co), 133.8 (Cp), 166.7 (COPh). IR (CHCl3, cm−1): 2956 (w), 2114 (m, N3), 1723 (s), 1247 (s), 1108 (m, Si–O), 839 (s), 709 (s). HRMS required for C19H27N3O6SiNa+ is 444.1561, found 444.1561.
2-Azido-2-deoxy-3,4-O-isopropylidene-6-O-benzoyl-β-D-psicofuranose 1014. To a solution of silyl ether derivative 9 (1.10 g, 2.61 mmol) in acetone (8 mL) were added methanol (10 mL) and glacial acetic acid (553 μL). The solution was stirred for 8 h at room temperature. Then, the reaction mixture was neutralized by triethylamine concentrated under reduced pressure. The resulting residue was partitioned between saturated aq. NaHCO3 and ethyl acetate. The organic layers were washed with brine, dried over MgSO4, filtered and concentrated to dryness under reduced pressure. Purification of the crude product by column chromatography (silica gel, hexane/ethyl acetate 8/2) gave the pure alcohol 10 (893 mg, 98% yield) as colourless syrup. Data for 10: [α]20D = −97.2 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.32 (3H, s, CH3), 1.51 (3H, s, CH3), 3.08 (1H, t, J = 6.4, OH), 4.00 (2H, d, J = 6.0, H-1a,b), 4.42-4.52 (1H, m, H-6a), 4.53–4.56 (2H, m, H-3 and H-6b), 4.63 (1H, td, J = 6.0, 1.2, H-5), 4.87 (1H, dd, J = 6.0, 1.6, H-4), 7.43 (2H, t, J = 7.6, 2 × Hm), 7.56 (1H, tt, J = 7.6, 1.2, Hp), 8.08 (2H, d, J = 7.2, 2 × Ho). 13C-NMR (100 MHz, CDCl3): 24.8 (CH3), 26.1 (CH3), 64.1 (C-1), 64.3 (C-6), 82.5 (C-4), 84.6 (C-5), 85.4 (C-3), 101.4 (C-2), 113.7 (C(CH3)2), 128.5 (Cm), 129.6 (Cipso), 129.7 (Co), 133.3(Cp), 166.2 (COPh). IR (CHCl3, cm−1): 3489 (br), 2956 (w), 2114 (m, N3), 1720 (s), 1270 (s), 709 (s). HRMS required for C16H19N3O6Na+ is 372.1166, found 372.1166.
6-O-Benzyl-3,4-O-isopropylidene-β-D-psicofuranosyl cyanide 1111. Spiroketal 8(38.0 g, 0.11 mol) was placed in a 1 L 3 neck round bottom flask equipped with a thermometer and pressure equalising dropping funnel and then cooled to −20 °C under an inert atmosphere of nitrogen. Trimethylsilyl cyanide (40.7 mL, 0.32 mol) was then added dropwise, followed by trimethylsilyl trifluoromethanesulfonate (22.9 mL, 0.16 mol). The reaction mixture was then stirred for 2 h at the same temperature (−20 °C). After this time, it was quenched with saturated sodium bicarbonate solution. The mixture was partitioned between saturated aq. NaHCO3 and dichloromethane and the aqueous layer treated with sodium hypochlorite. The combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated to dryness under reduced pressure. Purification of the crude product by column chromatography (silica gel, isohexane/ethyl acetate 75/25) gave the title compound 11 (20.4 g, 58% yield) as colourless syrup. Data for 11: oil. [α]20D= −29.0 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.34 (3H, s, CH3), 1.52 (3H, s, CH3), 2.61 (1H, br, OH), 3.54–3.65 (2H, m, 2 × H-6), 3.89 (2H, s, 2 × H-1), 4.42 (1H, m, H-5), 4.50 (1H, d, J = 12.1, H-7a), 4.69 (1H, d, J = 12.1, H-7b), 4.90 (1H, d, J = 5.9, H-3), 5.09 (1H, d, J = 5.8, H-4), 7.30–7.36 (5H, m, 5 × Harom). 13C-NMR (100 MHz, CDCl3): 24.5 (CH3), 25.7 (CH3), 64.9 (C-1), 70.1 (C-6), 73.5 (C-7), 82.9 (C-2), 83.3 (C-3), 84.9 (C-5), 85.2 (C-4), 113.9 (C(CH3)2), 120.0 (CN), 127.9 (Cp), 128.1 (Co), 128.5 (Cm), 137.3 (Cipso). IR (CHCl3, cm−1): 3298 (br), 2940 (w), 1714 (m), 1453 (m), 1360 (m).
((3aR,4R,6R,6aR)-6-Azido-2,2-dimethyl-6-((prop-2-yn-1-yloxy)methyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl benzoate 2a. Azido alcohol 10 (500 mg, 1.43 mmol) was dried by evaporation of freshly distilled acetonitrile and dissolved in the same solvent (8.4 mL). To the resulting solution were added BEMP (497 μL, 1.72 mmol) followed by the suitable propargyl bromide (2.15 mmol) dropwise at 0 °C under a nitrogen atmosphere. The reaction mixture was stirred at the same conditions until TLC indicated completion of the reaction (2–4 h). The resulting solution was partitioned between phosphate buffer solution pH 7.00 and ethyl acetate. The organic layers were washed with 10% NaCl aq. solution, dried over MgSO4, filtered and concentrated to dryness under reduced pressure to afford the crude residue as brown oil which in the majority of cases was used crude in the following cycloaddition step owing to spontaneous reactivity and in an effort to maximise overall yield. The characterisation for the purified intermediates is described below.

Chromatography of the crude residue over silica gel (hexane/ethyl acetate 8/2) gave the pure 2a (299 mg, 54% yield). Data for 2a: colourless oil, [α]20D = −86.5 (c = 0.02, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.21(3H, s, CH3), 1.49 (3H, s, CH3), 2.42 (1H, t, J = 2.4, H-3′), 3.89 (2H, s, H-1a,b), 4.25 (2H, d, J = 2.0, H-1′a,b), 4.33–4.49 (3H, m, H-3, H-6a,b), 4.54 (1H, td, J = 6.4, 1.6, H-5), 4.80 (1H, dd, J = 6.4, 1.6, H-4), 7.39 (2H, t, J = 8.0, 2 × Hm), 7.51 (1H, t, J = 7.2, Hp), 8.02 (2H, d, J = 7.6, 2 × Ho). 13C-NMR (100 MHz, CDCl3): 25.1 (CH3), 26.5 (CH3), 59.1 (C-1′), 64.2 (C-6), 70.4 (C-1), 75.2 (C-3′), 79.3 (C-2′), 82.5 (C-4), 85.0 (C-5), 85.5 (C-3), 100.7 (C-2), 114.0 (C(CH3)2), 128.5 (Cm), 129.7 (Cipso), 129.8 (Co), 133.4 (Cp), 166.3 (COPh). IR (CHCl3, cm−1): 3292 (w), 2927 (w), 2116 (m, N3), 1721 (s), 1270 (s), 1270 (s), 1068 (m), 711 (s). HRMS required for C19H22N3O6+ is 388.1503, found 388.1505.

2-Azido-2-deoxy-3,4-O-isopropylidene-β-D-psicofuranose 12. To a solution of compound 10 (75 mg, 0.21 mmol) in freshly distilled THF (1.5 mL), a solution of NaHDMS (47 mg, 0.26 mmol) was added dropwise at 0 °C under nitrogen atmosphere. After 5 min, propargyl bromide (36 μL, 0.32 mmol) was added and the reaction mixture was stirred for 4 h. Afterwards, saturated aq. NH4Cl was added and the resulting emulsion was extracted with ethyl acetate and washed with brine. The organic layers were dried over MgSO4, filtered and the solvent evaporated under reduced pressure. Chromatography of the crude residue over silica gel gave azidoalkyne 2a (hexane/ethyl acetate 8/2) as the minor product (9 mg, 11% yield) and debenzoylated psicofuranose 12 (hexane/ethyl acetate 6/4) as major product (36 mg, 70% yield). Data for 12: colourless syrup, 1H-NMR (400 MHz, CDCl3): δ 1.27 (3H, s, CH3), 1.46 (3H, s, CH3), 2.27 (2H, br, 2 × OH), 3.65-3.74 (2H, m, H-6a,b), 3.91 (2H, s, H-1a,b), 4.33 (1H, td, J = 6.0, 1.6, H-5), 4.46 (1H, d, J = 6.0, H-3), 4.75 (1H, dd, J = 6.0, 1.6, H-4). 13C-NMR (100 MHz, CDCl3): 23.4 (CH3), 25.2 (CH3), 62.4 (C-6), 63.4 (C-1), 81.1 (C-4), 84.7 (C-3), 86.6 (C-5), 100.3 (C-2), 112.6 (C(CH3)2).

General procedure for Sonogashira coupling

To a stirring suspension of aryl iodide (4.2 mmol), dichlorobis(triphenylphosphine)palladium(II) (30 mg, 0.042 mmol), copper(I) iodide (4 mg, 0.021 mmol) and freshly distilled diethylamine (50 mL) was added propargyl alcohol (4.2 mmol) dropwise under a N2 atmosphere.

The resulting reaction mixture was stirred for 5 h at room temperature. After this time, diethylamine was removed under reduced pressure and the crude product was partitioned between water and diethyl ether. The combined organic layers were washed with brine, dried (MgSO4), filtered and concentrated under reduced pressure.

General procedure for O-alkylation and intramolecular 1,3-dipolar cycloaddition14

Azido alcohol 10 (500 mg, 1.43 mmol) was dried by evaporation of freshly distilled acetonitrile and dissolved in the same solvent (8.4 mL). To the resulting solution BEMP (497 μL, 1.72 mmol) and the suitable propargyl bromide (2.15 mmol) were added drop-wise at 0 °C under a nitrogen atmosphere. The reaction mixture was stirred under these conditions until TLC indicated completion of the reaction (2–4 h). The resulting solution was partitioned between phosphate buffer solution pH 7 and ethyl acetate. The organic layers were washed with 10% NaCl aq. solution, dried over MgSO4, filtered, and concentrated under reduced pressure to afford the crude residue as brown oil which in the majority of cases was used as telescoped material in the following cycloaddition step owing to spontaneous reactivity and in an effort to maximise overall yield. Thus, the crude azido alkyne 2a–k was dissolved in toluene (28.6 mL) and refluxed until TLC analysis showed full conversion to corresponding triazolo-oxazine 16a–k (16–24 h). The solvent was removed under reduced pressure and the residue was purified on silica gel.
((3aR,4R,6R,6aR)-2,2-dimethyl-3a,6a-dihydro-4′H,6H,6′H-spiro[furo[3,4-d][1,3]dioxole-4,7′-[1,2,3]triazolo[5,1-c][1,4]oxazin]-6-yl)methyl benzoate 16a. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 6/4) gave the pure triazolooxazine 16a (282 mg, 95% yield). Data for 16a: colourless oil, [α]20D = −11.87 (c = 0.0016, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.37 (3H, s, CH3), 1.60 (3H, s, CH3), 4.19 (1H, d, J = 8.0, H-1a), 4.23 (1H, d, J = 8.2, H-1b), 4.55–4.69 (3H, m, H-5, H-6a,b), 4.83 (1H, d, J = 15.2, H-1′a), 5.04 (1H, d, J = 15.2, H-1′b), 5.16 (1H, d, J = 6.0, H-3), 5.30–5.31 (1H, m, H-4), 7.43 (2H, td, J = 7.8, 1.6, 2 × Hm), 7.52 (1H, s, H-3′), 7.55 (1H, tt, J = 7.3, 1.2, Hp), 8.06 (2H, dd, J = 8.0, 1.6, 2 × Ho). 13C-NMR (100 MHz, CDCl3): 25.3 (CH3), 26.8 (CH3), 62.5 (C-1′), 64.5 (C-6), 69.1 (C-1), 83.2 (C-4), 85.2 (C-3), 86.3 (C-5), 93.1 (C-2), 114.4 (C(CH3)2), 128.1 (C-3′), 128.3 (Cm), 129.7 (Cipso), 129.8 (Co), 131.4 (C-2′), 133.1 (Cp), 166.1 (COPh). IR (CHCl3, cm−1): 2923 (w), 1721 (s), 1269 (s), 1094 (s), 1068 (m), 713 (s). HRMS required for C19H22N3O6+ is 388.1503, found 388.1506.
((3aR,4R,6R,6aR)-2,2,3′-Trimethyl-3a,6a-dihydro-4′H,6H,6′H-spiro[furo[3,4-d][1,3]dioxole-4,7′-[1,2,3]triazolo[5,1-c][1,4]oxazin]-6-yl)methyl benzoate 16b. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 6/4) gave the pure triazolooxazine 16b (304 mg, 96% yield). Data for 16b: colourless oil, [α]20D = −24.82 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.38 (3H, s, CH3), 1.59 (3H, s, CH3), 2.27 (3H, s, 3 × H-4′), 4.15 (1H, d, J = 12.8, H-1a), 4.22 (1H, d, J = 12.4, H-1b), 4.53-4.67 (3H, m, H-5, H-6a,b), 4.73 (1H, d, J = 14.8, H-1′a), 4.95 (1H, d, J = 14.8, H-1′b), 5.15 (1H, d, J = 6.0, H-3), 5.28 (1H, dd, J = 6.0, 2.8, H-4), 7.43 (2H, t, J = 7.2, 2 × Hm), 7.55 (1H, t, J = 7.6, Hp), 8.06 (2H, d, J = 7.8, 2 × Ho). 13C-NMR (100 MHz, CDCl3): 10.0 (C-4′), 25.3 (CH3), 26.8 (CH3), 62.3 (C-1′), 64.6 (C-6), 68.9 (C-1), 83.2 (C-4), 85.1 (C-5), 86.1 (C-6), 93.0 (C-2), 114.4 (C(CH3)2), 127.8 (C-3′), 128.7 (Cm), 129.8 (Cipso), 129.9 (Co), 133.0 (Cp), 137.0 (C-2′), 166.1 (COPh). IR (CHCl3, cm−1): 2989 (w), 1719 (s), 1269 (s), 1096 (s), 1068 (m), 709 (s). HRMS required for C20H24N3O6+ is 402.1660, found 402.1658.
((3aR,4R,6R,6aR)-3′-ethyl-2,2-dimethyl-3a,6a-dihydro-4′H,6H,6′H-spiro[furo[3,4-d][1,3]dioxole-4,7′-[1,2,3]triazolo[5,1-c][1,4]oxazin]-6-yl)methyl benzoate 16c. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 6/4) gave the pure triazolooxazine 79c (255 mg, 82% yield). Data for 79c: colourless oil, [α]20Y = −27.26 (c = 0.015, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.26 (3H, t, J = 7.6, 3 × H-5′), 1.38 (3H, s, CH3), 1.59 (3H, s, CH3), 2.67 (2H, q, J = 7.6, 2 × H-4′), 4.17 (1H, d, J = 12.8, H-1a), 4.22 (1H, d, J = 12.8, H-1b), 4.54–4.67 (3H, m, H-5, H-6a,b), 4.76 (1H, d, J = 14.8, H-1′a), 4.97 (1H, d, J = 14.8, H-1′b), 5.15 (1H, d, J = 6.0, H-3), 5.29 (1H, dd, J = 5.6, 2.8, H-4), 7.43 (2H, t, J = 7.6, 2 × Hm), 7.55 (1H, t, J = 7.2, Hp), 8.05 (2H, d, J = 7.8, 2 × Ho). 13C-NMR (100 MHz, CDCl3): 13.2 (C-5′), 18.5 (C-4′), 25.3 (CH3), 26.8 (CH3), 62.4 (C-1′), 64.6 (C-6), 68.9 (C-1), 83.3 (C-4), 85.1 (C-5), 86.1 (C-6), 93.1 (C-2), 114.3 (C(CH3)2), 127.3 (C-3′), 128.3 (Cm), 129.8 (Cipso), 129.8 (Co), 133.1 (Cp), 142.8 (C-2′), 166.1 (COPh). IR (CHCl3, cm−1): 2978 (w), 1720 (s), 1271 (s), 1098 (s), 1069 (m), 712 (s). HRMS required for C21H26N3O6+ is 416.1803, found 416.1809.
((3aR,4R,6R,6aR)-2,2-Dimethyl-3′-(naphthalen-2-yl)-3a,6a-dihydro-4′H,6H,6′H-spiro[furo[3,4-d][1,3]dioxole-4,7′-[1,2,3]triazolo[5,1-c][1,4]oxazin]-6-yl)methyl benzoate 16d. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 8/2) gave the pure triazolooxazine 16d (433 mg, 94% yield). Data for 16d: colourless oil, [α]20D = −36.46 (c = 0.015, CHCl3). 1H-NMR (400 MHz, CDCl3): 1.42 (3H, s, CH3), 1.63 (3H, s, CH3), 4.29 (1H, d, J = 12.8, H-1a), 4.34 (1H, d, J = 12.4, H-1b), 4.70–4.77 (3H, m, H-5, H-6a,b), 4.81 (1H, d, J = 15.2, H-1′a), 4.97 (1H, d, J = 15.2, H-1′b), 5.29 (1H, d, J = 6.0, H-3), 5.39 (1H, dd, J = 5.6, 2.4, H-4), 7.40–7.46 (3H, m, H-Naph, 2 × Hm), 7.47–7.55 (4H, m, 3 × H-Naph, Hp), 7.89–7.91 (2H, m, 2 × H-Naph), 8.09–8.015 (3H, m, H-Naph, 2 × Ho). 13C-NMR (100 MHz, CDCl3): 25.4 (CH3), 26.9 (CH3), 63.2 (C-1′), 64.6 (C-6), 69.1 (C-1), 83.2 (C-4), 85.3 (C-5), 86.3 (C-6), 93.5 (C-2), 114.5 (C(CH3)2), 125.2, 125.5, 126.3, 126.6 (Naph), 127.4 (C-3′), 128.4 (Cm), 128.5,129.3 (Naph), 129.7 (Cipso), 129.8 (Co), 129.9 (Naph), 131.4 (C-2′), 133.1 (Cp), 133.9 (Naph), 141.0 (Cipso-Naph), 166.2 (COPh). IR (CHCl3, cm−1): 2990 (w), 1719 (s), 1271 (s), 1097 (s), 1069 (s), 1025 (m), 751 (s), 710 (s). HRMS required for C29H28N3O6+ is 514.1973, found 514.1970.
((3aR,4R,6R,6aR)-2,2-Dimethyl-3′-phenyl-3a,6a-dihydro-4′H,6H,6′H-spiro[furo[3,4-d][1,3]dioxole-4,7′-[1,2,3]triazolo[5,1-c][1,4]oxazin]-6-yl)methyl benzoate 16e. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 8/2) gave the pure triazolooxazine 16e (291 mg, 44% yield over two steps) as a white solid. Data for 16e: m.p. = 72–73 °C. [α]20D = −31.6 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): 1.40 (3H, s, CH3), 1.61 (3H, s, CH3), 4.25 (1H, d, J = 12.8, H-1a), 4.29 (1H, d, J = 12.8, H-1b), 4.58-4.72 (3H, m, H-5, H-6a,b), 5.01 (1H, d, J = 15.2, H-1′a), 5.21 (1H, d, J = 15.2, H-1′b), 5.21 (1H, d, J = 6.0, H-3), 5.33 (1H, dd, J = 5.8, 2.8, H-4), 7.35(1H, t, J = 7.2, H-7′), 7.40–7.47 (4H, m, 2 × H-6′, 2 × Hm), 7.55 (1H, t, J = 7.2, Hp), 7.62 (2H, d, J = 7.6, 2 × H-5′), 8.06 (2H, d, J = 7.2, 2 × Ho). 13C-NMR (100 MHz, CDCl3): 25.3 (CH3), 26.8 (CH3), 63.4 (C-1′), 64.6 (C-6), 68.8 (C-1), 83.3 (C-4), 85.2 (C-3), 86.3 (C-5), 93.3 (C-2), 114.4 (C(CH3)2), 126.2 (C-5′), 127.2 (C-2′), 128.2 (C-7′), 128.4 (Cm), 129.0 (C-6′), 129.8 (Cipso), 129.9 (Co), 130.3 (C-4′), 133.1 (Cp), 141.4 (C-3′), 166.2 (COPh). IR (CHCl3, cm−1): 2924 (w), 2358 (s), 1719 (s), 1270 (s), 1097 (s), 1069 (s),1025 (m), 749 (s), 709 (s). HRMS required for C25H26N3O6+ is 464.1816, found 464.1813.
((3aR,4R,6R,6aR)-3′-(4-chlorophenyl)-2,2-dimethyl-3a,6a-dihydro-4′H,6H,6′H-spiro[furo[3,4-d][1,3]dioxole-4,7′-[1,2,3]triazolo[5,1-c][1,4]oxazin]-6-yl)methyl benzoate 16f. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 8/2) gave the pure triazolooxazine 16f (320 mg, 45% yield over two steps) as white solid. Data for 16f: m.p. = 74–75 °C. [α]20D = −28.7 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): 1.39 (3H, s, CH3), 1.61 (3H, s, CH3), 4.25 (1H, d, J = 12.8, H-1a), 4.29 (1H, d, J = 12.8, H-1b), 4.57-4.72 (3H, m, H-5, H-6a,b), 4.99 (1H, d, J = 15.2, H-1′a), 5.19 (1H, d, J = 15.2, H-1′b), 5.21 (1H, d, J = 6.0, H-3), 5.32 (1H, dd, J = 6.0, 2.8, H-4), 7.41–7.45 (4H, m, 2 × H-6′, 2 × Hm), (2H, d, J = 7.2, 2 × H-5′), 8.06 (2H, d, J = 7.2, 2 × Ho). 13C-NMR (100 MHz, CDCl3): 25.3 (CH3), 26.8 (CH3), 63.3 (C-1′), 64.6 (C-6), 68.8 (C-1), 83.2 (C-4), 85.2 (C-3), 86.3 (C-5), 93.5 (C-2), 114.5 (C(CH3)2), 127.3 (C-5′), 127.9 (C-2′), 128.4 (Cm), 128.9 (C-4′), 129.3 (C-6′), 129.8 (Cipso), 129.9 (Co), 133.1 (Cp), 134.0 (C-3′), 166.1 (COPh). IR (CHCl3, cm−1): 2988 (w), 1718 (s), 1491 (m), 1271 (s), 1090 (s), 1069 (s), 1025 (m), 1001 (s), 752 (s), 711(s). HRMS required for C25H25N3O6Cl+ is 498.1432, found 498.1426.
((3aR,4R,6R,6aR)-3′-(4-Methoxyphenyl)-2,2-dimethyl-3a,6a-dihydro-4′H,6H,6′H-spiro[furo[3,4-d][1,3]dioxole-4,7′-[1,2,3]triazolo[5,1-c][1,4]oxazin]-6-yl)methyl benzoate 16g. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 7/3) gave the pure triazolooxazine 16g (303 mg, 43% yield over two steps) as white solid. Data for 16g: m.p. = 128–129 °C. [α]20D = −32.6 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): 1.39 (3H, s, CH3), 1.60 (3H, s, CH3), 3.84 (3H, s, OCH3), 4.23 (1H, d, J = 12.4, H-1a), 4.28 (1H, d, J = 12.8, H-1b), 4.58–4.71 (3H, m, H-5, H-6a,b), 4.97 (1H, d, J = 14.8, H-1′a), 5.17 (1H, d, J = 15.2, H-1′b), 5.20 (1H, d, J = 6.4, H-3), 5.32 (1H, dd, J = 5.8, 2.8, H-4), 6.98 (2H, d, J = 8.8, 2 × H-6′) 7.42 (2H, t, J = 7.6, 2 × Hm), 7.52–7.56 (1H, m, Hp), 7.52 (2H, d, J = 8.8, 2 × H-5′), 8.06 (2H, d, J = 7.2, 2 × Ho). 13C-NMR (100 MHz, CDCl3): 25.3 (CH3), 26.8 (CH3), 55.3 (OCH3), 63.4 (C-1′), 64.7 (C-6), 68.8 (C-1), 83.3 (C-4), 85.2 (C-3), 86.3 (C-5), 93.4 (C-2), 114.4 (C(CH3)2), 114.5 (C-6′), 123.0 (C-4′), 126.8 (C-2′), 127.5 (C-5′), 128.3 (Cm), 129.8 (Cipso), 129.9 (Co), 133.1 (Cp), 141.2 (C-3′), 160.0 (C-7′), 166.1 (COPh). IR (CHCl3, cm−1): 2938 (w), 1720 (s), 1507 (s), 1271 (s), 1248 (s), 1098 (s), 1069 (s), 1025 (m), 753 (s), 711(s). HRMS required for C26H28N3O7+ is 494.1922, found 494.1922.
((3aR,4R,6R,6aR)-3′-(4-Fluorophenyl)-2,2-dimethyl-3a,6a-dihydro-4′H,6H,6′H-spiro[furo[3,4-d][1,3]dioxole-4,7′-[1,2,3]triazolo[5,1-c][1,4]oxazin]-6-yl)methyl benzoate 16h. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 8/2) gave the pure triazolooxazine 16h (296 mg, 43% yield over two steps d) as colourless oil. Data for 16h: [α]20D = −32.6 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): 1.39 (3H, s, CH3), 1.61 (3H, s, CH3), 4.24 (1H, d, J = 12.4, H-1a), 4.29 (1H, d, J = 12.8, H-1b), 4.57–4.72 (3H, m, H-5, H-6a,b), 4.98 (1H, d, J = 15.2, H-1′a), 5.18 (1H, d, J = 15.2, H-1′b), 5.21 (1H, d, J = 6.0, H-3), 5.32 (1H, dd, J = 6.0, 2.8, H-4), 7.14 (2H, t, J = 8.8, 2 × H-6′), 7.42 (2H, app t, J = 8.0, 2 × Hm), 7.55 (1H, t, J = 7.2, Hp), 7.59 (2H, dd, J = 8.2, 5.2, 2 × H-5′), 8.06 (2H, d, J = 7.2, 2 × Ho). 13C-NMR (100 MHz, CDCl3): 25.3 (CH3), 26.8 (CH3), 63.3 (C-1′), 64.6 (C-6), 68.8 (C-1), 83.2 (C-4), 85.2 (C-3), 86.3 (C-5), 93.4 (C-2), 114.5 (C(CH3)2), 116.1 (d, 2JCF = 22, C-6′), 126.5 (d, 4JCF = 3, C-4′), 127.5 (C-2′), 127.9 (d, 3JCF = 8, C-5′), 128.4 (Cm), 129.7 (Cipso), 129.8 (Co), 133.1 (Cp), 140.5 (C-3′), 162.5 (d, 1JCF = 246, C-7′), 166.1 (COPh). IR (CHCl3, cm−1): 2933 (w), 1719 (s), 1505 (s), 1272 (s), 1099 (s), 1069 (s), 1026 (m), 759 (s), 711(s). HRMS required for C25H25N3O6F+ is 482.1722, found 482.1720.
((3aR,4R,6R,6aR)-3′-(3-fluorophenyl)-2,2-dimethyl-3a,6a-dihydro-4′H,6H,6′H-spiro[furo[3,4-d][1,3]dioxole-4,7′-[1,2,3]triazolo[5,1-c][1,4]oxazin]-6-yl)methyl benzoate 16i. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 8/2) gave the pure triazolooxazine 16i (268 mg, 39% yield over two steps) as colourless oil. Data for 16i: [α]20D = −40.2 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): 1.40 (3H, s, CH3), 1.61 (3H, s, CH3), 4.24 (1H, d, J = 12.8, H-1a), 4.30 (1H, d, J = 12.8, H-1b), 4.58–4.71 (3H, m, H-5, H-6a,b), 5.00 (1H, d, J = 15.2, H-1′a), 5.21 (1H, dd, J = 15.2, H-1′b), 5.22 (1H, d, J = 6.0, H-3), 5.32 (1H, dd, J = 6.0, 2.8, H-4), 7.05 (1H, td, J = 8.4, 1.6, H-7′), 7.32–7.45 (5H, m, H-5′, H-8′, H-9′, 2 × Hm), 7.35 (1H, dd, J = 13.6, 6.4, H-7′), 7.42 (2H, t, J = 8.0, Hm), 7.55 (1H, t, J = 6.8, Hp), 8.07 (2H, d, J = 7.6, 2 × Ho). 13C-NMR (100 MHz, CDCl3): 25.3 (CH3), 26.8 (CH3), 63.3 (C-1′), 64.6 (C-6), 68.8 (C-1), 83.2 (C-4), 85.2 (C-3), 86.3 (C-5), 93.5 (C-2), 113.1 (d, 2JCF = 23, C-5′), 114.5 (C(CH3)2), 115.0 (d, 2JCF = 22, C-7′), 121.7 (d, 4JCF = 3, C-9′), 128.2 (C-2′), 128.4 (Cm), 129.7 (Cipso), 129.9 (Co), 130.6 (d, 3JCF = 8, C-8′), 132.4 (d, 3JCF = 8, C-4′), 133.1 (Cp), 140.3 (d, 4JCF = 3, C-3′), 163.2 (d, 1JCF = 245, C-6′), 166.1 (COPh). IR (CHCl3, cm−1): 2987 (w), 1718 (s), 1270 (s), 1097 (s), 1070 (s), 1026 (m), 754 (s), 711(s). HRMS required for C25H25N3O6F+ is 482.1722, found 482.1722.
((3aR,4R,6R,6aR)-3′-(2-Fluorophenyl)-2,2-dimethyl-3a,6a-dihydro-4′H,6H,6′H-spiro[furo[3,4-d][1,3]dioxole-4,7′-[1,2,3]triazolo[5,1-c][1,4]oxazin]-6-yl)methyl benzoate spirocylic triazolo-oxazine 16j. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 8/2) gave the pure triazolooxazine 16j (310 mg, 45% yield over two steps) as colourless oil. Data for 16j: [α]20D = −36.5 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): 1.39 (3H, s, CH3), 1.60 (3H, s, CH3), 4.25 (1H, d, J = 14.0, H-1a), 4.29 (1H, d, J = 13.6, H-1b), 4.59–4.74 (3H, m, H-5, H-6a,b), 4.92 (1H, dd, J = 15.8, 2.4, H-1′a), 5.09 (1H, dd, J = 15.8, 2.4, H-1′b), 5.20 (1H, d, J = 6.0, H-3), 5.34 (1H, dd, J = 5.6, 2.8, H-4), 7.11 (1H, t, J = 8.4, H-6′), 7.26 (1H, t, J = 8.4, H-8′), 7.35 (1H, dd, J = 13.6, 6.4, H-7′), 7.42 (2H, t, J = 8.0, 2 × Hm), 7.55 (1H, t, J = 6.8, Hp), 7.98 (1H, td, J = 7.6, 1.6, H-9′), 8.07 (2H, d, J = 7.2, 2 × Ho). 13C-NMR (100 MHz, CDCl3): 25.3 (CH3), 26.8 (CH3), 63.8 (d, 5JCF = 18, C-1′), 64.7 (C-6), 68.8 (C-1), 83.2 (C-4), 85.2 (C-3), 86.3 (C-5), 93.6 (C-2), 114.4 (C(CH3)2), 115.7 (d, 2JCF = 21, C-6′), 118.1 (d, 2JCF = 15, C-4′), 124.8 (d, 4JCF = 3, C-8′), 128.3 (Cm), 129.6 (C-2′), 129.7 (Cipso), 129.8 (Co), 129.9 (d, 5JCF = 4, C-9′) 130.2 (d, 3JCF = 9, C-7′), 133.1 (Cp), 136.1 (C-3′), 162.5 (d, 1JCF = 244, C-5′), 166.1 (COPh). IR (CHCl3, cm−1): 2986 (w), 1720 (s), 1272 (s), 1097 (s), 1101 (s), 1069 (m), 757 (s), 711(s). HRMS required for C25H25N3O6F+ is 482.1722, found 482.1722.
((3aR,4R,6R,6aR)-2,2-Dimethyl-3′-(4-pentylphenyl)-3a,6a-dihydro-4′H,6H,6′H-spiro[furo[3,4-d][1,3]dioxole-4,7′-[1,2,3]triazolo[5,1-c][1,4]oxazin]-6-yl)methyl benzoate 16k. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 8/2) gave the pure triazolooxazine 16k (235 mg, 93% yield). Data for 16k: colourless oil, [α]20D = −28.5 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 0.89 (3H, t, J = 7.2, 3 × H-8′), 1.25–1.33 (4H, m, 2 × H-6′, 2 × H-7′), 1.33 (3H, s, CH3), 1.60 (3H, s, CH3), 1.64 (2H, quint, J = 7.2, 2 × H-5′), 2.63 (2H, td, J = 7.8, 2.4, 2 × H-4′), 4.17 (1H, d, J = 12.8, H-1a), 4.23 (1H, d, J = 12.8, H-1b), 4.54–4.67 (3H, m, H-5, H-6a,b), 4.74 (1H, d, J = 14.8, H-1′a), 4.95 (1H, d, J = 15.2, H-1′b), 5.14 (1H, d, J = 6.0, H-3), 5.298 (1H, dd, J = 6.0, 2.4, H-4), 7.43 (2H, t, J =7.2, 2 × Hm), 7.55 (1H, t, J = 7.2, Hp), 8.05 (2H, d, J = 7.2, 2 × Ho). 13C-NMR (100 MHz, CDCl3): 14.0 (C-8′), 22.4 (C-7′), 25.0 (C-4′), 25.3 (CH3), 26.8 (CH3), 28.6 (C-5′), 31.5 (C-6′), 62.5 (C-1′), 64.6 (C-6), 68.9 (C-1), 83.2 (C-4), 85.1 (C-5), 86.1 (C-6), 93.1 (C-2), 114.3 (C(CH3)2), 127.6 (C-3′), 128.3 (Cm), 129.8 (Cipso), 129.8 (Co), 133.1 (Cp), 141.7 (C-2′), 166.1 (COPh). IR (CHCl3, cm−1): 2931 (w), 1720 (s), 1450 (m), 1271 (s), 1097 (s), 1070 (m), 711 (s). HRMS required for C21H26N3O6+ is 416.1803, found 416.1809.
3,4-O-Isopropylidene spirocyclic triazolo-oxazine nucleoside 17a. Benzoate ester 16a was dissolved in a solution of 7 N NH3/MeOH (0.06 M concentration) and the resulting solution was stirred for 4 h. After evaporation of the solvent under reduced pressure, the crude product was purified on silica gel. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 1/1) gave the pure spironucleoside 17a (358 mg, 98% yield). Colourless needle crystals were obtained after recrystallisation with chloroform/hexane 1/3. Data for 17a: m.p. = 130–132 °C. [α]20D = −120.80 (c = 0.0025, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.39 (3H, s, CH3), 1.61 (3H, s, CH3), 3.84 (1H, ddd, J = 11.2, 9.6, 4.0, H-6a), 3.86–3.96 (1H, m, H-6b), 3.98 (1H, d, J = 12.4, H-1a), 3.21 (1H, bdd, J = 9.8, 2.4, OH), 4.31 (1H, d, J = 12.4, H-1b), 4.55 (1H, m, H-5), 4.88 (1H, d, J = 15.2, H-1′a), 4.97 (1H, d, J = 15.2, H-1′b), 5.20 (1H, d, J = 6.0, H-3), 5.29 (1H, dd, J = 6.0, 2.0, H-4), 7.54 (1H, s, H-3′). 13C-NMR (100 MHz, CDCl3): 24.6 (CH3), 26.1 (CH3), 62.4 (C-6), 63.8 (C-1′), 69.7 (C-1), 82.2 (C-4), 86.2 (C-3), 88.4 (C-5), 94.4 (C-2), 113.9 (C(CH3)2), 128.4 (C-3′), 132.3 (C-2′). IR (CHCl3, cm−1): 3352 (br), 2927 (w), 1095 (s), 1044 (m). HRMS required for C12H18N3O5 is 284.1241, found 284.1242.

General procedure for benzoyl and isopropyl group removal to give 18a–k14

Protected spirocyclic triazolo-oxazine nucleoside 16a–k (0.65 mmol) was dissolved in a solution of 7 N of NH3 in MeOH (10.8 mL) and the resulting solution was stirred at room temperature until TLC indicated the reaction was complete (ca. 4 h). After evaporation of all volatiles under reduced pressure, the crude residue was dissolved in a solution of methanol/water 8/2 (26 mL) and Dowex® 50WX8 hydrogen form (3.38 g) was added in one portion. The resulting suspension was stirred vigorously for 4–6 h at 50 °C, then the resin was filtered off and washed with methanol. The filtrate was concentrated to dryness under reduced pressure to afford a crude residue which was purified by column chromatography.
(2R,3R,4S,5R)-5-(Hydroxymethyl)-4,5-dihydro-3H,4′H,6′H-spiro[furan-2,7′-[1,2,3]triazolo[5,1-c][1,4]oxazine]-3,4-diol 18a. Chromatography of the crude residue over silica gel (dichloromethane/methanol 9/1) gave the pure anomeric spironucleoside 18a (88 mg, 56% yield) as colourless syrup. Data for 18a: [α]20D = −38.31 (c = 0.0065, CH3OH). 1H-NMR (400 MHz, CD3OD): δ 3.64 (1H, dd, J = 12.0, 6.4, H-6a), 3.73 (1H, dd, J = 12.0, 4.0, H-6b), 4.02 (1H, d, J = 12.4, H-1a), 4.03–4.07 (1H, m, H-5), 4.21 (1H, d, J = 12.8, H-1b), 4.44 (1H, d, J = 4.8, H-3), 4.62 (1H, dd, J = 6.6, 4.8, H-4), 4.73 (1H, d, J = 15.2, H-1′a), 4.89 (1H, d, J = 15.2, H-1′b), 7.46 (1H, s, H-3′). 13C-NMR (100 MHz, CD3OD): 63.1 (C-1′), 64.0 (C-6), 70.1 (C-1), 72.6 (C-4), 77.0 (C-3), 86.6 (C-5), 94.2 (C-2), 128.9 (C-3′), 134.4 (C-2′). IR (CH3OH, cm−1): 3360 (br), 2924 (m), 1615 (br), 1093 (s), 1020 (s). HRMS required for C9H14N3O5+ is 244.0928, found 244.0928.
(2R,3R,4S,5R)-5-(Hydroxymethyl)-3′-methyl-4,5-dihydro-3H,4′H,6′H-spiro[furan-2,7′-[1,2,3]triazolo[5,1-c][1,4]oxazine]-3,4-diol 18b. Chromatography of the crude residue over silica gel (dichloromethane/methanol 9/1) gave the pure spironucleoside 18b (78 mg, 47% yield) as colourless syrup. Data for 18b: [α]20Y = −57.1 (c = 0.01, CH3OH). 1H-NMR (500 MHz, CD3OD): δ 2.26 (3H, s, 3 × H-4′), 3.77 (1H, dd, J = 12.0, 6.0, H-6a), 3.86 (1H, dd, J = 12.0, 3.5, H-6b), 4.12 (1H, d, J = 12.5, H-1a), 4.15–4.20 (1H, m, H-5), 4.31 (1H, d, J = 12.5, H-1b), 4.56 (1H, d, J = 5.0, H-3), 4.73 (1H, dd, J = 6.5, 5.0, H-4), 4.80 (1H, d, J = 15.0, H-1′a), 4.94 (1H, d, J = 15.0, H-1′b). 13C-NMR (125 MHz, CD3OD): ppm 9.5 (C-4′), 62.8 (C-1′), 64.0 (C-6), 69.8 (C-1), 72.6 (C-4), 76.9 (C-3), 86.5 (C-5), 94.2 (C-2), 130.6 (C-3′), 137.8 (C-2′). IR (CH3OH, cm−1): 3321 (br), 2993 (m), 1099 (s), 1071 (m). HRMS required for C10H16N3O5+ is 258.1084, found 258.1086.
(2R,3R,4S,5R)-3′-Ethyl-5-(hydroxymethyl)-4,5-dihydro-3H,4′H,6′H-spiro[furan-2,7′-[1,2,3]triazolo[5,1-c][1,4]oxazine]-3,4-diol 18c. Chromatography of the crude residue over silica gel (dichloromethane/methanol 9/1) gave the pure spironucleoside 18c (90 mg, 51% yield) as colourless syrup. Data for 18c: [α]20Y = −34.6 (c = 0.01, CH3OH). 1H-NMR (400 MHz, CD3OD): δ 1.25 (3H, t, J = 7.6, 3 × H-4′), 2.67 (2H, q, J = 7.6, 3 × H-3′), 3.75 (1H, dd, J = 12.0, 6.4, H-6a), 3.84 (1H, dd, J = 12.0, 3.6, H-6b), 4.12 (1H, d, J = 12.4, H-1a), 4.13-4.18 (1H, m, H-5), 4.30 (1H, d, J = 12.4, H-1b), 4.55 (1H, d, J = 4.8, H-3), 4.72 (1H, dd, J = 6.4, 4.8, H-4), 4.81 (1H, d, J = 15.2, H-1′a), 4.96 (1H, d, J = 15.2, H-1′b). 13C-NMR (100 MHz, CD3OD): 13.6 (C-5′), 18.9 (C-4′), 63.0 (C-6), 64.0 (C-1′), 70.0 (C-1), 72.7 (C-4), 76.9 (C-3), 86.4 (C-5), 94.2 (C-2), 130.2 (C-2′), 143.6 (C-3′). IR (CH3OH, cm−1): 3392 (br), 2922 (m), 1642 (br), 1101 (s), 1033 (s), 940 (m). HRMS required for C11H18N3O5+ is 272.1241, found 272.1242.
(2R,3R,4S,5R)-5-(Hydroxymethyl)-3′-(naphthalen-2-yl)-4,5-dihydro-3H,4′H,6′H-spiro[furan-2,7′-[1,2,3]triazolo[5,1-c][1,4]oxazine]-3,4-diol 18d. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 15/85) gave the pure spironucleoside 18d (120 mg, 50% yield) as white solid. Data for 18d: m.p. = 101–102 °C. [α]20Y = −65.5 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 3.89 (1H, bd, J = 11.6, H-6a), 4.00 (1H, bd, J = 11.6, H-6b), 4.09 (1H, br, OH), 4.12 (1H, d, J = 12.8, H-1a), 4.25–4.28 (1H, m, H-4), 4.37 (1H, d, J = 12.4, H-1b), 4.55 (1H, br, OH), 4.66 (1H, d, J = 15.6, H-1′a), 4.75 (1H, br, H-3), 4.78 (1H, d, J = 15.2, H-1′b), 5.10 (1H, br, OH), 5.26 (1H, br, H-5), 7.30 (1H, d, J = 7.2, H-Naph), 7.34–7.51 (3H, m, 3 × H-Naph), 7.93 (3H, t, J = 8.4, 3 × H-Naph). 13C-NMR (100 MHz, CDCl3): 62.1 (C-6), 62.8 (C-1), 68.9 (C-1′), 70.9 (C-5), 76.4 (C-3), 85.2 (C-4), 93.4 (C-2), 124.9, 125.3, 126.4, 126.5, 126.9 (Naph), 127.5 (C-3′), 128.6 (Naph), 129.6 (C-2′), 130.8, 131.2, 133.9 (Naph), 140.6 (Cipso-Naph). IR (cm−1): 3381 (br), 2992 (w), 1211 (s), 1095 (s), 1076 (m), 777 (m), 751 (s). IR (CHCl3, cm−1): 3349 (br), 2926 (w), 1103 (s), 1035 (m), 777 (m), 757 (s). HRMS required for C19H20N3O5+ is 370.1397, found 370.1399.
(2R,3R,4S,5R)-5-(Hydroxymethyl)-3′-phenyl-4,5-dihydro-3H,4′H,6′H-spiro[furan-2,7′-[1,2,3]triazolo[5,1-c][1,4]oxazine]-3,4-diol 18e. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 1/9) gave the pure spironucleoside 18e (139 mg, 67% overall yield) as white foam. Data for 18e: [α]20D = −36.1 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CD3OD): δ 3.80 (1H, dd, J = 12.0, 6.4, H-6a), 3.80 (1H, dd, J = 12.4, 3.2, H-6b), 4.20 (1H, d, J = 12.0, H-1a), 4.19–4.23 (1H, m, H-5), 4.38 (1H, d, J = 12.4, H-1b), 4.64 (1H, d, J = 5.2, H-3), 4.73 (1H, dd, J = 6.4, 5.2, H-4), 5.02 (1H, d, J = 15.2, H-1′a), 5.17 (1H, d, J = 15.2, H-1′b), 7.38 (1H, t, J = 7.6, H-7′), 7.48 (2H, t, J = 7.6, 2 × H-6′), 7.62 (2H, d, J = 8.0, 2 × H-5′). 13C-NMR (100 MHz, CD3OD): 63.8 (C-6), 63.9 (C-1′), 69.8 (C-1), 72.7 (C-4), 77.0 (C-3), 86.6 (C-5), 94.5 (C-2), 127.3 (C-5′), 129.3 (C-2′), 130.1 (C-6′), 130.5 (C-7′), 131.5 (C-4′), 142.2 (C-3′). IR (CH3OH, cm−1): 3324 (br), 2922 (m), 1438 (s), 1101 (s), 1045 (s), 1006 (s), 943 (m). HRMS required for C25H26N3O6+ is 464.1816, found 464.1813.
(2R,3R,4S,5R)-3′-(4-Chlorophenyl)-5-(hydroxymethyl)-4,5-dihydro-3H,4′H,6′H-spiro[furan-2,7′-[1,2,3]triazolo[5,1-c][1,4]oxazine]-3,4-diol 18f. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 1/9) gave the pure spironucleoside 18f (145 mg, 63% overall yield) as colourless oil. Data for 18f: [α]20D = −31.0 (c = 0.01, CHCl3). 1H-NMR (500 MHz, CD3OD): δ 3.77 (1H, dd, J = 12.2, 6.0, H-6a), 3.85 (1H, dd, J = 12.2, 3.0, H-6b), 4.17 (1H, d, J = 12.5, H-1a), 4.15–4.19 (1H, m, H-5), 4.35 (1H, d, J = 12.5, H-1b), 4.61 (1H, d, J = 5.0, H-3), 4.75 (1H, dd, J = 7.0, 5.0, H-4), 5.03 (1H, d, J = 15.5, H-1′a), 5.15 (1H, d, J = 15.5, H-1′b), 7.47 (2H, d, J = 8.5, 2 × H-6′), 7.60 (2H, d, J = 8.5, 2 × H-5′). 13C-NMR (125 MHz, CD3OD): 62.4 (C-6), 63.5 (C-1′), 68.4 (C-1), 71.3 (C-4), 75.6 (C-3), 85.2 (C-5), 93.2 (C-2), 127.3 (C-5′), 128.9 (C-6′), 129.0 (C-2′), 129.4 (C-4′), 133.7 (C-7′), 139.7 (C-3′). IR (CH3OH, cm−1): 3361 (br), 2922 (m), 1636 (w), 1491 (s), 1090 (s), 1037 (s), 1002 (s), 943 (m), 831 (m). HRMS required for C15H17N3O5Cl+ is 354.0851, found 354.0851.
(2R,3R,4S,5R)-5-(Hydroxymethyl)-3′-(4-methoxyphenyl)-4,5-dihydro-3H,4′H,6′H-spiro[furan-2,7′-[1,2,3]triazolo[5,1-c][1,4]oxazine]-3,4-diol 18g. Chromatography of the crude residue over silica gel (dichloromethane/methanol 95/5) gave the pure spironucleoside 66g (118 mg, 52% overall yield). White needle crystals were obtained after recrystallisation with hexane/Et2O/CH3OH 6[thin space (1/6-em)]:[thin space (1/6-em)]3[thin space (1/6-em)]:[thin space (1/6-em)]1. Data for 66g: m.p. = 119–120 °C. [α]20D = −51.3 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CD3OD): δ 3.80 (1H, dd, J = 12.4, 6.4, H-6a), 3.84 (3H, s, OCH3), 3.88 (1H, dd, J = 12.0, 3.2, H-6b), 4.18 (1H, d, J = 12.4, H-1a), 4.18–4.22 (1H, m, H-5), 4.36 (1H, d, J = 12.8, H-1b), 4.63 (1H, d, J = 5.2, H-3), 4.78 (1H, dd, J = 6.4, 5.2, H-4), 5.00 (1H, d, J = 15.2, H-1′a), 5.12 (1H, d, J = 15.2, H-1′b), 7.02 (2H, d, J = 8.8, 2 × H-6′), 7.52 (2H, d, J = 8.8, 2 × H-5′). 13C-NMR (100 MHz, CD3OD): 55.8 (OCH3), 63.9 (C-6), 64.0 (C-1′), 69.8 (C-1), 72.7 (C-4), 77.0 (C-3), 86.6 (C-5), 94.5 (C-2), 115.5 (C-6′), 123.9 (C-3′), 128.7 (C-5′), 129.0 (C-2′), 142.2 (C-4′), 161.2 (C-7′). IR (CH3OH, cm−1): 3373 (br), 2925 (m), 1615 (m), 1508 (s), 1249 (s), 1097 (s), 1038 (s), 999 (s), 943 (m), 833 (m). HRMS required for C16H20N3O6+ is 494.1922, found 494.1922.
(2R,3R,4S,5R)-3′-(4-Fluorophenyl)-5-(hydroxymethyl)-4,5-dihydro-3H,4′H,6′H-spiro[furan-2,7′-[1,2,3]triazolo[5,1-c][1,4]oxazine]-3,4-diol 18h. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 1/9) gave the pure spironucleoside 18h (175 mg, 80% overall yield) as white solid. Data for 18h: [α]20D = −40.8 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CD3OD): δ 3.76 (1H, dd, J = 12.5, 6.3, H-6a), 3.86 (1H, dd, J = 12.5, 3.3, H-6b), 4.18 (1H, d, J = 12.2, H-1a), 4.16–4.20 (1H, m, H-5), 4.36 (1H, d, J = 12.6, H-1b), 4.62 (1H, d, J = 4.9, H-3), 4.76 (1H, t, J = 6.2, H-4), 5.03 (1H, d, J = 15.3, H-1′a), 5.15 (1H, d, J = 15.3, H-1′b), 7.21 (2H, t, J = 8.7, 2 × H-6′), 7.60 (2H, dd, J = 8.4, 5.4, 2 × H-5′). 13C-NMR (100 MHz, CD3OD): 63.8 (C-6), 63.9 (C-1′), 69.8 (C-1), 72.7 (C-4), 77.0 (C-3), 86.6 (C-5), 94.9 (C-2), 117.0 (d, 2JCF = 22, C-6′), 128.0 (C-4′),129.3 (d, 3JCF = 9, C-5′), 130.8 (C-2′), 141.5 (C-3′), 164.1 (d, 1JCF = 277, C-7′). IR (CH3OH, cm−1): 3363 (br), 2921 (m), 1646 (m), 1506 (s), 1230 (m), 1098 (s), 1038 (s), 1004 (s), 944 (m), 838 (m). HRMS required for C15H17N3O5F+ is 338.1147, found 338.1147.
(2R,3R,4S,5R)-3′-(3-Fluorophenyl)-5-(hydroxymethyl)-4,5-dihydro-3H,4′H,6′H-spiro[furan-2,7′-[1,2,3]triazolo[5,1-c][1,4]oxazine]-3,4-diol 18i. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 1/9) gave the pure spironucleoside 18i (162 mg, 74% overall yield) as colourless oil. Data for 18i: [α]20D = −46.6 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CD3OD): δ 3.79 (1H, dd, J = 12.0, 6.3, H-6a), 3.86 (1H, dd, J = 12.0, 3.2, H-6b), 4.17 (1H, d, J = 12.7, H-1a), 4.17–4.21 (1H, m, H-5), 4.35 (1H, d, J = 12.6, H-1b), 4.63 (1H, d, J = 4.9, H-3), 4.77 (1H, t, J = 6.3, H-4), 5.02 (1H, d, J = 15.3, H-1′a), 5.15 (1H, d, J = 15.3, H-1′b), 7.09 (1H, td, J = 8.3, 2.2, H-7′), 7.33–7.40 (2H, m, H-5′, H-9′), 7.46 (1H, m, H-8′). 13C-NMR (100 MHz, CD3OD): 63.8 (C-6), 63.9 (C-1′), 69.8 (C-1), 72.7 (C-4), 77.0 (C-3), 86.6 (C-5), 94.6 (C-2), 113.8 (d, 2JCF = 23, C-5′), 115.9 (d, 2JCF = 22, C-7′), 123.0 (d, 4JCF = 3, C-9′), 131.0 (C-2′), 132.0 (d, 3JCF = 9, C-8′), 133.9 (d, 3JCF = 8, C-4′), 141.0 (d, 4JCF = 2, C-3′), 164.6 (d, 2JCF = 243, C-6′). IR (CH3OH, cm−1): 3362 (br), 2921 (m), 1496 (m), 1102 (s), 1043 (s), 1005 (s), 943 (m), 821 (m). HRMS required for C15H17N3O5F+ is 338.1147, found 338.1147.
(2R,3R,4S,5R)-3′-(2-Fluorophenyl)-5-(hydroxymethyl)-4,5-dihydro-3H,4′H,6′H-spiro[furan-2,7′-[1,2,3]triazolo[5,1-c][1,4]oxazine]-3,4-diol 18j. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 1/9) gave the pure spironucleoside 18j (160 mg, 73% overall yield) as colourless oil. Data for 18j: [α]20D = −48.2 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CD3OD): δ 3.68 (1H, dd, J = 12.0, 6.3, H-6a), 3.75 (1H, dd, J = 12.0, 3.3, H-6b), 4.08 (1H, d, J = 12.4, H-1a), 4.06–4.10 (1H, m, H-5), 4.25 (1H, d, J = 12.6, H-1b), 4.52 (1H, d, J = 4.9, H-3), 4.66 (1H, t, J = 6.0, H-4), 4.78 (1H, d, J = 15.4, H-1′a), 4.90 (1H, d, J = 15.4, H-1′b), 7.11 (1H, t, J = 10.8, H-6′), 7.19 (1H, t, J = 7.6, H-8′), 7.33 (2H, dd, J = 14.3, 6.9, H-7′), 7.46 (1H, t, J = 7.2, H-8′). 13C-NMR (100 MHz, CD3OD): 63.9 (C-6), 64.0 (C-1′), 70.0 (C-1), 72.7 (C-4), 77.1 (C-3), 86.6 (C-5), 94.6 (C-2), 117.0 (d, 2JCF = 24, C-6′), 119.2 (d, 2JCF = 26, C-4′), 126.0 (d, 4JCF = 4, C-8′), 130.9 (d, 5JCF = 3, C-9′), 131.8 (d, 3JCF = 10, C-7′), 132.0 (C-2′), 137.3 (C-3′), 160.6 (d, 2JCF = 240, C-5′). IR (CH3OH, cm−1): 3381 (br), 2922 (m), 1496 (m), 1102 (s), 1043 (s), 1004 (s), 945 (m), 823 (m). HRMS required for C15H17N3O5F+ is 338.1147, found 338.1147.
(2R,3R,4S,5R)-5-(Hydroxymethyl)-3′-pentyl-4,5-dihydro-3H,4′H,6′H-spiro[furan-2,7′-[1,2,3]triazolo[5,1-c][1,4]oxazine]-3,4-diol 18k. Chromatography of the crude residue over silica gel (hexane/ethyl acetate 2/8) gave the pure spironucleoside 18k (140 mg, 69% yield) as colourless syrup. Data for 18k: [α]20D = −47.2 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CD3OD): δ 0.92 (3H, t, J = 6.6, 3 × H-8′), 1.26–1.39 (4H, m, 2 × H-6′, 2 × H-7′), 1.63 (2H, quint, J = 7.2, 2 × H-5′), 2.63 (2H, t, J = 7.6, 2 × H-4′), 3.74 (1H, dd, J = 12.0, 6.3, H-6a), 3.84 (1H, dd, J = 12.0, 3.2, H-6b), 4.11 (1H, d, J = 12.8, H-1a), 4.12-4.16 (1H, m, H-5), 4.29 (1H, d, J = 12.6, H-1b), 4.54 (1H, d, J = 4.9, H-3), 4.70 (1H, app t, J = 5.5, H-4), 4.78 (1H, d, J = 15.0, H-1′a), 4.89 (1H, d, J = 13.0, H-1′b). 13C-NMR (100 MHz, CD3OD): 14.3 (C-8′), 23.4 (C-7′), 25.5 (C-4′), 29.8 (C-5′), 32.5 (C-6′), 62.9 (C-1′), 64.0 (C-6), 70.1 (C-1), 72.6 (C-4), 77.0 (C-3), 86.2 (C-5), 94.2 (C-2), 130.4 (C-2′), 142.5 (C-3′). IR (CH3OH, cm−1): 3321 (br), 2922 (s), 1636 (br), 1456 (m), 1100 (s), 1036 (s), 941 (m). HRMS required for C14H24N3O5+ is 314.1716, found 314.1710.
(3aR,4R,6S,6aS)-2,2-Dimethyl-3a,6a-dihydro-4′H,6H,6′H-spiro[furo[3,4-d][1,3]dioxole-4,7′-[1,2,3]triazolo[5,1-c][1,4]oxazine]-6-carbaldehyde 19. Primary alcohol 17a (0.97 mmol) was dissolved in dichloromethane (10 mL). Sodium bicarbonate (2.91 mmol) and Dess–Martin periodinane (1.16 mmol) were added sequentially in one portion at 0 °C and the resulting mixture stirred vigorously at the same temperature until TLC analysis showed the reaction was complete (2 h). The reaction was quenched with 10% thiosulfate aq. solution (2 mL) and the mixture partitioned between saturated aq. NaHCO3 and dichloromethane. The organic layer was washed with brine, dried over MgSO4, filtered and concentrated to dryness under reduced pressure. Chromatography of the crude residue over silica gel (dichloromethane/methanol 95/5) gave the pure aldehyde 19 (217 mg, 80% yield) as white solid. Data for 19: m.p. = 74–75 °C. [α]25D = −60.0 (c = 0.01, l = 0.025 dm, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.38 (3H, s, CH3), 1.58 (3H, s, CH3), 4.26 (1H, d, J = 12.8, H-1a), 4.33 (1H, d, J = 12.8, H-1b), 4.63 (1H, d, J = 1.2, H-3), 4.84 (1H, d, J = 15.2, H-1′a), 4.98 (1H, d, J = 6.0, H-5), 5.08 (1H, d, J = 15.2, H-1′b), 5.50 (1H, dd, J = 5.8, 1.3, H-4), 7.55 (1H, s, H-3′), 9.31 (1H, s, H-6). 13C-NMR (100 MHz, CDCl3): 24.6 (CH3), 26.1 (CH3), 62.4 (C-6), 63.8 (C-1′), 69.7 (C-1), 82.2 (C-4), 86.2 (C-3), 88.4 (C-5), 94.4 (C-2), 113.9 (C(CH3)2), 128.4 (C-3′), 132.3 (C-2′). IR (CHCl3, cm−1): 2990 (w), 1732 (m, C[double bond, length as m-dash]O), 1377 (m), 1211 (m), 1096 (s), 864 (m), 732 (s). HRMS required for C12H18N3O5 is 284.1241, found 284.1242.
Diethyl(2-((3aR,4R,6R,6aR)-2,2-dimethyl-3a,6a-dihydro-4′H,6H,6′H-spiro[furo[3,4-d][1,3]dioxole-4,7′-[1,2,3]triazolo[5,1-c][1,4]oxazin]-6-yl)vinyl)phosphonate 20. Sodium hydride (60% dispersion in mineral oil, 59 mg, 1.47 mmol) was suspended in dry THF (1 mL) at 0 °C under an inert atmosphere of argon. A solution of tetraethyl methylenediphosphonate (443 mg, 1.54 mmol) in dry THF (0.5 mL) was then added dropwise. The suspension was allowed to warm to room temperature and stirred for 45 min. Reaction mixture cooled down to 0 °C and a solution of aldehyde 19 (197 mg, 0.70 mmol) in THF (1.5 mL) added dropwise. The resulting mixture was allowed to warm to room temperature and stirred for 2 h. The reaction mixture was treated with methanol, diluted with saturated NaHCO3 solution and extracted with ethyl acetate. The organic phase was then washed with brine, dried over MgSO4, filtered and concentrated to dryness under reduced pressure. Chromatography of the crude residue over silica gel (dichloromethane/methanol 96/4) gave 218 mg (80% yield) of 20 as an inseparable 1[thin space (1/6-em)]:[thin space (1/6-em)]4 mixture of E and Z alkene isomers. Data for Z-isomer 20: colourless syrup. [α]25D = +84.0 (c = 0.01, l = 0.025 dm, CHCl3).1H-NMR (400 MHz, CDCl3): δ 122–1.38 (6H, m, 6 × H-9), 1.40 (3H,s, CH3), 1.53 (3H,s, CH3), 3.97–4.07 (4H, m, 4 × H-8), 4.10–4.15 (2H, m, H-3, H-4), 4.22 (1H, d, J = 12.8, H-1a), 4.32 (1H, d, J = 13.2, H-1b), 4.82 (1H, d, J = 15.2, H-1′a), 4.90 (1H, q, J = 7.6, H-5), 4.96 (1H, d, J = 5.7, H-7), 5.09 (1H, d, J = 15.2, H-1′b), 5.63 (1H, dd, J = 5.7, 3.2, H-6), 7.53 (1H, s, H-3′). 13C-NMR (100 MHz, CDCl3): 16.3 (d, 3JCP = 5, C-9), 26.0 (CH3), 26.7 (CH3), 61.8 (d, 2JCP = 7, C-8a), 61.9 (d, 2JCP = 6, C-8b), 62.4 (C-1′), 68.1 (C-1), 80.2 (C-3), 82.6 (C-5), 91.8 (C-2), 94.5 (d, 3JCP = 11, C-5), 114.3 (C(CH3)2), 114.6 (C-7), 128.1 (C-3′), 131.1 (C-2′), 115.7 (d, 2JCP = 14, C-6). IR (cm−1): 2987 (w), 1251 (s, P[double bond, length as m-dash]O), 1099 (s), 1026 (s, P–O–C), 968 (s), 797 (m). HRMS required for C17H27N3O7P+ is 416.1587 found 416.1581.
Diethyl (2-((3aR,4R,6R,6aR)-2,2-dimethyl-3a,6a-dihydro-4′H,6H,6′H-spiro[furo[3,4-d][1,3]dioxole-4,7′-[1,2,3]triazolo[5,1-c][1,4]oxazin]-6-yl)ethyl)phosphonate 21. In a hydrogenation vessel, vinyl phosphonate 20 (218 mg 0.52 mmol) was dissolved in methanol (4 mL) and Pd/C 10% (22 mg) was added in one portion. The reactor was purged three times with N2 and the mixture was agitated for 5 min. After purging with H2 three times, the reactor was pressurised to 4 bar and the resulting suspension stirred vigorously for 6 h. The catalyst was filtered, and the filtrate was evaporated to dryness. Purification of the crude product by column chromatography (silica gel, dichloromethane/methanol 96/4) gave pure alkyl phosphonate 21 (214 mg, 94% yield) as colourless oil. Data for 21: colourless syrup. [α]25D = −380.0 (c = 0.01, l = 0.025 dm, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.31 (6H, td, J = 7.1, 1.9, 6 × H-9), 1.35 (3H,s, CH3), 1.53 (3H,s, CH3), 4.05–4.13 (4H, m, 4 × H-8), 4.09 (1H, d, J = 12.9, H-1a), 4.21 (1H, d, J = 12.8, H-1b), 4.61 (1H, t, J = 6.8, H-5), 4.80 (1H, d, J = 15.2, H-1′a), 5.05 (1H, d, J = 15.1, H-1′b), 5.08–5.11 (2H, m, H-3, H-4), 7.51 (1H, s, H-3′). 13C-NMR (100 MHz, CDCl3): 16.4 (d, 3JCP = 6, C-9), 22.0 (d, 1JCP = 142, C-7), 22.1 (d, 2JCP = 4, C-6), 24.5 (CH3), 25.9 (CH3), 61.5 (d, 2JCP = 6, C-8a), 61.6 (d, 2JCP = 7, C-8b), 62.4 (C-1′), 68.7 (C-1), 81.2 (C-3), 82.5 (d, 3JCP = 19, C-5), 85.5 (C-4), 91.5 (C-2), 113.6 (C(CH3)2), 127.8 (C-2′), 131.2 (C-3′). IR (CH3OH, cm−1): 2983 (br), 1376 (m), 1234 (s, P[double bond, length as m-dash]O), 1212 (s, P[double bond, length as m-dash]O), 1098 (s, P–O–C), 1029 (P–O–C), 963 (m). HRMS required for C17H29N3O7P+ is 418.1743 found 418.1736.
(2-((2R,3R,4S,5R)-3,4-Dihydroxy-4,5-dihydro-3H,4′H,6′H-spiro[furan-2,7′-[1,2,3]triazolo[5,1-c][1,4]oxazin]-5-yl)ethyl)phosphonic acid 23. Phosphonate nucleoside 21 (213 mg, 0.51 mmol) was dissolved in a solution of methanol/water 8/2 (20 mL) and Dowex® 50WX8 hydrogen form (2.7 g) was added in one portion. The resulting suspension was stirred vigorously for 4 h at 50 °C, then the resin was filtered off and washed with methanol. The filtrate was concentrated to dryness under reduced pressure to give diol 22 as yellow oil. This crude residue was dissolved in anhydrous dichloromethane (2 mL) in 10 mL round bottom flask. 2,6-Dimethylpyridine (672 μL, 6.12 mmol) was added under inert atmosphere, followed by bromo(trimethyl)silane (538 μL, 4.08 mmol). Reaction mixture was stirred for 2 h, then quenched with ammonium hydroxide aq. solution (5 mL). Aqueous phase was washed up with isohexane (1 mL), concentrated to dryness under vacuum to afford a crude residue which was purified by mass directed prep HPLC using a XBridge dC18 5μ OBD 30 × 100 mm prep column. RP-HPLC was conducted an elution gradient of 1–100% B over 11.40 min, where A is H2O in 10 mM ammonium acetate and B is CH3CN, to gave pure phosphonate nucleoside 23 (31 mg, 12% over two steps). Data for 23: colourless oil. [α]25D = −44.0 (c = 0.01, l = 0.025 dm, CHCl3). 1H-NMR (400 MHz, CD3OD): δ 1.69–1.91 (2H, m, 2 × H-7), 1.95–2.05 (2H, m, 2 × H-6), 4.13 (1H, d, J = 12.6, H-1a), 4.28 (1H, d, J = 12.6, H-1b), 4.41 (1H, dd, J = 4.6, 3.2, H-4), 4.46 (1H, td, J = 6.8, 3.1, H-5), 4.82 (1H, d, J = 15.1, H-1′a), 5.00 (1H, d, J = 15.1, H-1′b), 5.23 (1H, d, J = 4.3, H-3), 7.59 (1H, s, H-3′). 13C-NMR (100 MHz, CD3OD): 22.6 (d, 3JCP = 3, C-6), 23.1 (d, 1JCP = 85, C-7), 61.6 (C-1′), 69.9 (C-1), 71.8 (C-5), 77-3 (C-3), 81.8 (d, 4JCP = 15, C-4), 92.0 (C-2), 127.6 (C-2′), 133.5 (C-3′). 31P-NMR (200 MHz, CD3OD): 29.43. IR (CH3OH, cm−1): 3094 (br), 1628 (m), 1396 (s, P[double bond, length as m-dash]O), 1066 (s, P–O–C), 915 (m). HRMS required for C10H17N3O7P+ is 322.080 found 322.080.
6-O-Benzyl-2-deoxy-3,4-O-isopropylidene-2-aminomethyl-β-D-psicofuranose 24. Lithium aluminium hydride (2.38 g, 62.6 mmol) was placed in a three necked round bottom flask equipped with thermometer, pressure equalising dropping funnel and stirrer under a nitrogen atmosphere. Anhydrous diethyl ether (50 mL) was charged at 0 °C, maintaining an internal temperature of less than 25 °C. A solution of psicofuranosyl cyanide 11 (10.0 g, 31.3 mmol) in anhydrous diethyl ether (50 mL) was then added dropwise at 0 °C with careful control of the exotherm. The reaction mixture was allowed to warm to room temperature and stirred for 2 h. Excess LiAlH4 was carefully quenched with 7 mL of AcOEt at 0 °C added dropwise (CAUTION: violent reaction). The following treatment with 1.0 M K2CO3 gave a white precipitate that was removed under filtration over celite. The filtrate was evaporated under reduced pressure to afford the crude residue as colourless oil, which was pure enough to be used immediately for the next step. Purification of the crude product by column chromatography (silica gel, ethyl acetate/methanol/ammonium hydroxide 95/4/1) gave the pure amino alcohol 24 (9.9 g, 98% yield) as a colourless oil. Data for 24: oil. [α]25D = +7.2 (c = 0.01, l = 0.025, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.33 (3H, s, CH3), 1.53 (3H, s, CH3), 2.88 (1H, d, J = 13.0, H-1′a), 3.04 (1H, d, J = 13.0, H-1′b), 3.53 (1H, dd, J = 10.4, 4.3, H-6a), 3.60 (1H, dd, J = 10.4, 4.3, H-6b), 3.71 (1H, d, J = 11.8, H-1a), 3.81 (1H, d, J = 11.8, H-1b), 4.18 (1H, q, J = 4.1, H-5), 4.51 (2H, s, 2 × H-7), 4.53-4.60 (1H, m, H-3), 4.72 (1H, dd, J = 6.5, 4.4, H-4), 7.25–7.36 (5H, m, 5 × Harom). 13C-NMR (100 MHz, CDCl3): 24.5 (CH3), 26.6 (CH3), 45.3 (C-1′), 62.8 (C-1), 70.4 (C-6), 73.5 (C-7), 82.1 (C-4), 83.0 (C-2), 84.1 (C-5), 85.7 (C-3), 114.0 (C(CH3)2), 127.9 (Cp), 127.9 (Co), 128.5 (Cm), 137.6 (Cipso). IR (CHCl3, cm−1): 3200 (br), 2931 (m), 1559 (m), 1381 (m), 1210 (m), 1072 (s), 866 (m). HRMS required for C17H26NO5+ is 324.181, found 324.180.

General procedure for sulfonyl amido ester protection

To a stirred solution of telescoped amino alcohol 24 (1.0 eq.) in CH2Cl2 (0.1 M concentration), triethylamine (4.0 eq.), 4-dimethylaminopyridine (1.0 eq.) and an appropriate sulfonyl chloride (3.0 eq.) were added sequentially at 0 °C. The resulting mixture was allowed to warm to room temperature and stirred for 16 h. The reaction mixture was poured into water and the water layer was extracted with dichloromethane. The combined organic extracts were washed sequentially with 1.0 M HCl, water and brine. dried over MgSO4, filtered and concentrated to dryness under reduced pressure and purified by column chromatography.
6-O-Benzyl-2-deoxy-3,4-O-isopropylidene-1-O-mesyl-2-mesylaminomethyl-β-D-psicofuranose 25a16. Chromatography of the crude residue over silica gel (isohexane/ethyl acetate 1/1) gave the pure sulfonyl amidoester 25a (979 mg, 66% o.y.) as colourless oil. Data for 25a: oil. [α]20D = +3.1 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.32 (3H, s, CH3), 1.51 (3H, s, CH3), 2.74 (3H, s, NHSO2CH3), 3.05 (3H, s, SO3CH3), 3.29–3.30 (2H, m, 2 × H-1′), 3.59 (1H, dd, J = 10.4, 2.4, H-6a), 3.71 (1H, dd, J = 10.4, 2.4, H-6b), 4.10–4.16 (1H, m, H-5), 4.31 (1H, d, J = 10.8, H-1a), 4.36 (1H, J = 10.8, H-1b), 4.54 (1H, d, J = 12.0, H-7a), 4.59 (1H, d, J = 12.0, H-7b), 4.78 (1H, d, J = 6.0, H-3), 5.10 (1H, t, J = 5.2, H-4), 7.77 (1H, dd, J = 8.0, 4.4, NH), 7.28–7.36 (5H, m, 5 × Harom). 13C-NMR (100 MHz, CDCl3): 25.2 (CH3), 27.0 (CH3), 37.4 (SO3CH3), 40.0 (NHSO2CH3), 46.9 (C-1′), 69.0 (C-1), 69.6 (C-6), 73.7 (C-7), 81.6 (C-4), 83.4 (C-3), 84.4 (C-2), 84.5 (C-5), 114.1 (C(CH3)2), 128.0 (Cp), 128.2 (Co), 128.6 (Cm), 137.0 (Cipso). IR (CHCl3, cm−1): 3282 (br), 2936 (m), 2359 (w), 1559 (m), 1327 (m), 1175 (m), 1075 (s), 967 (m). HRMS required for C19H29NO9S2Na+ is 502.1181, found 502.1176.
6-O-Benzyl-2-deoxy-3,4-O-isopropylidene-1-O-tosyl-2-tosylaminomethyl-β-D-psicofuranose 25b. Chromatography of the crude residue over silica gel (isohexane/ethyl acetate 6/4) gave the pure sulfonyl amidoester 25b (1.22 g, 62% o.y.) as foamy gum. Data for 25b: [α]20D = +3.9 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.22 (3H, s, CH3), 1.32 (3H, s, CH3), 2.41 (3H, s, PhCH3), 2.42 (3H, s, PhCH3), 2.95 (1H, dd, J = 12.4, 2.8, H-1′a), 2.98 (1H, dd, J = 12.6, 10.0, H-1′b), 3.47 (1H, dd, J = 10.4, 2.0, H-6a), 3.59 (1H, dd, J = 10.4, 2.0, H-6b), 3.86–3.89 (1H, m, H-5), 4.01 (1H, d, J = 10.4, H-1a), 4.10 (1H, d, J = 10.4, H-1b), 4.56 (1H, d, J = 12.4, H-7a), 4.62 (1H, d, J = 12.4, H-7b), 4.70 (1H, d, J = 6.0, H-3), 4.86 (1H, t, J = 5.6, H-4), 6.01 (1H, dd, J = 9.6, 2.8, NH). 7.25 (2H, d, J = 8.0, 2 × Hm Tol), 7.30 (2H, d, J = 8.4, 2 × Hm Tol), 7.32–7.37 (5H, m, 5 × Harom Ph), 7.54 (2H, d, J = 8.0, 2 × Ho Tol), 7.54 (2H, d, J = 8.4, 2 × Ho Tol). 13C-NMR (100 MHz, CDCl3): 21.5 (PhCH3), 21.7 (PhCH3), 25.1 (CH3), 26.9 (CH3), 47.4 (C-1′), 69.0 (C-6), 69.2 (C-1), 73.6 (C-7), 81.2 (C-4), 83.5 (C-3), 84.2 (C-5), 84.3 (C-2), 113.9 (C(CH3)2), 126.8 (Co Tol), 127.8 (Co Tol), 128.3 (Cp Ph), 128.6 (Co Ph), 128.7 (Cm Ph), 129.8 (Cm Tol), 132.4 (Cipso Tol), 136.9 (Cipso Ph), 143.4 (Cp Tol), 145.0 (Cp Tol). IR (CHCl3, cm−1): 3270 (br), 2926 (w), 2359 (w), 1189 (s), 1176 (s), 1120 (m), 988 (m). HRMS required for C31H37NO9S2Na+ is 654.1807, found 654.1802.
6-O-Benzyl-2-deoxy-3,4-O-isopropylidene-1-O-nosyl-2-nosylaminomethyl-β-D-psicofuranose 25c. Chromatography of the crude residue over silica gel (isohexane/ethyl acetate 6/4) gave the pure sulfonyl amidoester 25c (2.15 g, 97% o.y.) as white solid. Data for 26c: m.p. = 96–97 °C. [α]25D = +2.1 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.29 (3H, s, CH3), 1.43 (3H, s, CH3), 3.52–3.61 (2H, m 2 × H-6), 4.00 (1H, d, J = 16.0, H-1′a), 4.11 (1H, d, J = 12.0, H-1a), 4.19 (1H, d, J = 16.0, H-1′b), 3.20-4.22 (1H, m, H-5), 4.35 (1H, d, J = 10.5, H-1b), 4.51 (1H, d, J = 11.6, H-7a), 4.55 (1H, d, J = 6.2, H-3), 4.57 (1H, d, J = 11.6, H-7b), 4.66 (1H, dd, J = 6.8, 4.1, H-4), 7.26-7.35 (5H, m, 5 × Harom Ph), 8.00 (2H, d, J = 8.0, 2 × Ho NHNs), 8.26–8.33 (5H, m, 4 × Harom ONs, NH), 8.31 (2H, d, J = 8.0, 2 × Hm NHNs). 13C-NMR (100 MHz, CDCl3): 24.6 (CH3), 25.8 (CH3), 53.3 (C-1′), 69.3 (C-1), 70.3 (C-6), 73.6 (C-7), 82.0 (C-2), 82.6 (C-4), 82.9 (C-5), 84.4 (C-3), 115.5 (C(CH3)3), 124.1 (Co NHNs), 124.2 (Cm ONs), 128.0 (Co Ph), 128.1 (Co ONs), 128.6 (Cp Ph), 129.5 (Co NHNs), 130.5 (Cm Ph), 137.4 (Cipso Ph), 141.2 (Cipso NHNs), 144.2 (Cipso ONs), 150.7 (Cp NHNs), 150.9 (Cp ONs). IR (CHCl3, cm−1): 3108 (w), 2357 (w), 1532 (s, N–O), 1350 (s, N–O), 1172 (m), 1082 (m), 978 (w). HRMS required for C23H27N2O8S+ is 491.1488, found 491.1482.

General procedure for sulfoazetidine spirocyclization

Sodium hydride (60% dispersion in mineral oil, 10 eq.) was placed in a three necked round bottom flask equipped with thermometer, stirrer bar and pressure equalising dropping funnel under a nitrogen atmosphere. It was then charged with anhydrous DMF (0.05 M) at 0 °C, maintaining an internal temperature less than 25 °C. A solution of suitable sulfonyl amidoester 25a–c in anhydrous DMF (0.05 M) was then added dropwise over 15 min and stirred for a further 15 min at 0 °C with careful control of exotherm. The reaction was allowed to warm to room temperature and stirred until TLC analysis showed complete reaction (16–48 h). The excess of NaH was quenched by careful dropwise addition of methanol at 0 °C maintaining an internal temperature of less than 25 °C. The suspension was then diluted with water and extracted with diethylether. The combined organic extracts were washed with saturated aq. NH4Cl solution, dried over MgSO4, filtered and concentrated to dryness under reduced pressure and purified by column chromatography.
(6R,7S,8R)-2-Aza-6-benzyloxymethyl-7,8-(dimethylmethylenedioxy)-2-N-mesyl-5 oxaspiro[3.4]-octane 26a16. Chromatography of the crude residue over silica gel (isohexane/ethyl acetate 7/3) gave the pure mesyl spiroazetidine 26a (485 mg, 63%) as colourless oil. Data for 26a: oily. [α]20D = −63.7 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.36 (3H, s, CH3), 1.42 (3H, s, CH3), 2.85 (3H, s, SO2CH3), 3.48 (1H, dd, J = 10.0, 2.5, H-6a), 3.52 (1H, dd, J = 10.0, 2.5, H-6b), 3.57 (1H, d, J = 8.8, H-1′a), 3.59 (1H, dd, J = 10.0, 2.5, H-6b), 3.67 (1H, d, J = 8.8, H-1′b), 3.92 (1H, d, J = 8.8, H-1a), 4.20–4.23 (1H, m. H-5), 4.21 (1H, d, J = 8.8, H-1b), 4.40 (1H, d, J = 12.0, H-7a), 4.50 (1H, d, J = 11.6, H-7b), 4.73 (1H, d, J = 6.0, H-3), 4.79 (1H, d, J = 6.0, H-4), 7.25-7.38 (5H, m, 5 × Harom). 13C-NMR (100 MHz, CDCl3): 25.2 (CH3), 27.0 (CH3), 37.4 (SO3CH3), 40.0 (NHSO2CH3), 46.9 (C-1′), 69.0 (C-1), 69.6 (C-6), 73.7 (C-7), 81.6 (C-4), 83.4 (C-3), 84.4 (C-2), 84.5 (C-5), 114.1 (C(CH3)2), 128.0 (Cp), 128.2 (Co), 128.6 (Cm), 137.0 (Cipso). IR (CHCl3, cm−1): 2935 (w), 2359 (w), 1336 (m), 1130 (s), 1070 (m), 960 (w). HRMS required for C18H25NO6SNa+ is 406.1295, found 406.1295.
(6R,7S,8R)-2-Aza-6-benzyloxymethyl-7,8-(dimethylmethylenedioxy)-2-N-tosyl-5 oxaspiro[3.4]-octane 26b. Chromatography of the crude residue over silica gel (isohexane/ethyl acetate 7/3) gave the pure tosyl spiroazetidine 26b (243 mg, 54%) as colourless oil. Data for 26b: oily. [α]20D = −70.4 (c = 0.01, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.29 (3H, s, CH3), 1.36 (3H, s, CH3), 2.45 (3H, s, PhCH3), 3.40 (2H, s, 2 × H-6), 3.54 (1H, d, J = 9.6, H-1′a), 3.57 (1H, d, J = 9.7, H-1′b), 3.67 (1H, d, J = 8.8, H-1a), 4.10–4.14 (2H, m, H-5, H-1b), 4.37 (1H, d, J = 11.7, H-7a), 4.43 (1H, d, J = 11.7, H-7b), 4.49 (1H, d, J = 5.9, H-3), 4.70 (1H, d, J = 5.9, H-4), 7.22–7.26 (2H, m, 5 × Hm Tol), 7.32–7.39 (5H, m, 2 × Harom Ph), 7.68 (2H, d, J = 7.9, Ho Tol). 13C-NMR (100 MHz, CDCl3): 21.6 (PhCH3), 25.0 (CH3), 26.4 (CH3), 57.3 (C-1), 64.0 (C-1′), 71.5 (C-6), 73.9 (C-7), 80.9 (C-2), 82.9 (C-4), 83.8 (C-5), 85.5 (C-3), 112.4 (C(CH3)2), 128.1 (Cp Ph), 128.2 (Co Ph), 128.4 (Co Tol), 128.6 (Cm Tol), 129.7 (Cm Ph), 131.7 (Cp Tol), 137.0 (Cipso Ph), 143.9 (Cipso Tol). IR (CHCl3, cm−1): 2937 (w), 2359 (w), 1344 (m), 1127 (s), 1090 (m). HRMS required for C24H29NO6SNa+ is 482.1613, found 482.1608.
(6R,7S,8R)-2-Aza-6-benzyloxymethyl-7,8-(dimethylmethylenedioxy)-2-N-nosyl-5 oxaspiro[3.4]-octane 26c. Chromatography of the crude residue over silica gel (isohexane/ethyl acetate 7/3) gave the pure nonyl spiroazetidine 26c (84 mg, 12%) as colourless oil. Data for 26c: oily. [α]25D = −67.2 (c = 0.01, l = 0.025 dm, CHCl3). 1H-NMR (400 MHz, CDCl3): δ 1.28 (3H, s, CH3), 1.36 (3H, s, CH3), 2.45 (3H, s, PhCH3), 3.41–3.47 (2H, m, 2 × H-6), 3.57 (1H, d, J = 9.3, H-1′a), 3.62 (1H, d, J = 9.3, H-1′b), 3.75 (1H, d, J = 9.1, H-1a), 4.12 (1H, t, J = 2.1, H-5), 4.20 (1H, d, J = 9.1, H-1b), 4.38 (1H, d, J = 12.0, H-7a), 4.42 (1H, d, J = 12.0, H-7b), 4.43 (1H, d, J = 5.9, H-3), 4.70 (1H, d, J = 6.0, H-4), 7.21–7.28 (2H, m, 2 × Ho Ph), 7.35–7.42 (3H, m, 2 × Hm, Hp Ph), 7.94 (2H, d, J = 7.9, Ho Ns), 8.37 (2H, d, J = 7.9, Ho Ns). 13C-NMR (100 MHz, CDCl3): 25.0 (CH3), 26.4 (CH3), 57.8 (C-1), 64.7 (C-1′), 71.6 (C-6), 74.0 (C-7), 81.2 (C-2), 83.1 (C-4), 83.8 (C-5), 85.5 (C-3), 112.6 (C(CH3)2), 124.2 (Cm Ns), 128.2 (Co Ph), 128.3 (Cp Ph), 128.6 (Cm Ph), 129.4 (Co Ns), 136.8 (Cipso Ph), 141.2 (Cipso Ns), 150.0 (Cp Ns). IR (CHCl3, cm−1): 2932 (w), 2157 (w), 1550 (s, N–O), 1349 (s, N–O), 1163 (s), 737 (s). HRMS required for C23H27N2O8S+ is 491.1488, found 491.1482.
(6R,7S,8R)-2-Aza-6-benzyloxymethyl-7,8-(dimethylmethylenedioxy)-5-oxa-2-azaspiro[3.4]-octane 28. To a stirred suspension of K2CO3 (56 mg, 0.41 mmol) in CH3CN (1 mL) was added a solution of N-nosyl spiroazetidine 26c (100 mg, 0.20 mmol) in CH3CN (1 mL) under N2 atmosphere followed by thiophenol (21 μL, 0.61 mmol). The reaction mixture was stirred for 18 h, diluted with ethyl acetate, washed with brine, dried over MgSO4, filtered and concentrated to dryness under reduced pressure. Purification was performed by mass directed prep HPLC using a XBridge dC18 5μ OBD 30 × 100 mm prep column. RP-HPLC was conducted with an elution gradient of 1–100% B over 11.40 min, where A is H2O in 10 mM ammonium acetate and B is CH3CN and gave pure azetidine spironucleoside 27 (29 mg, 48%). Data for 27: oily. [α]25D = −56.0 (c = 0.01, l = 0.025 dm, CH3OH). 1H-NMR (400 MHz, CDCl3): δ 1.35 (3H, s, CH3), 1.41 (3H, s, CH3), 3.44 (1H, dd, J = 10.2, 3.0, H-6a), 3.48 (1H, dd, J = 10.2, 3.0, H-6b), 3.59 (1H, d, J = 10.1, H-1′a), 3.76 (1H, d, J = 10.3, H-1′b), 3.87 (1H, d, J = 10.3, H-1a), 4.08 (1H, d, J = 10.2, H-1b), 4.19 (1H, t, J = 2.6, H-5), 4.39 (1H, d, J = 11.8, H-7a), 4.48 (1H, d, J = 11.8, H-7b), 4.76 (1H, d, J = 6.0, H-3), 4.82 (1H, d, J = 6.0, H-4), 5.04 (1H, br, NH), 7.22-7.38 (5H, m, 5 × Harom). 13C-NMR (100 MHz, CDCl3): 25.1 (CH3), 26.4 (CH3), 52.6 (C-1′), 59.2 (C-1), 71.5 (C-6), 73.9 (C-7), 83.1 (C-4), 83.9 (C-5), 84.5 (C-2), 85.4 (C-3), 112.5 (C(CH3)2), 128.0 (Co), 128.1 (Cp), 128.6 (Cm), 137.0 (Cipso). IR (CH3OH, cm−1): 2936 (m), 1556 (m), 1380 (s), 1280 (s), 1160 (s), 1051 (s), 871 (s). HRMS required for C7H14NO4+ is 306.170 found 306.170.

General procedure for O-deprotection

Isopropylidene sulfoazetidine spironucleoside 26a–b was dissolved in a solution of methanol/water 8/2 (0.025 M concentration). Dowex® 50WX8 hydrogen form (3.4 g mol−1) was added in one portion and the resulting suspension was stirred vigorously at 50 °C until total consumption of the starting material (4–6 h), then the resin was filtered off and washed with methanol. The filtrate was evaporated under reduced pressure to afford crude benzyl ether spironucleoside crude as pale yellow oil. Residue was dissolved in methanol (0.1 M) and charged into hydrogenation vessel. Palladium on activated charcoal 10 wt%, (10% of weight with respect of 26a–b) was added in one portion. The reactor was purged three times with N2 and the mixture was agitated for 5 min. After purging with H2 a further three times, the reactor was pressurised to 4 bar and the resulting suspension was stirred vigorously until total consumption of the starting material (ca. 22 h). The catalyst was filtered off, and the filtrate was evaporated to dryness.
(6R,7S,8R)-2-Aza-7,8-dihydroxy-6-hydroxymethyl-2-N-mesyl-5-oxaspiro[3.4]octane 27a. Purification of the crude product by column chromatography (silica gel, dichloromethane/methanol 94/6) gave the title compound 27a (120 mg, 94% yield) as colourless oil. [α]25D = −32.0 (c = 0.01, l = 0.025 dm, CH3OH). 1H-NMR (400 MHz, CD3OD): δ 2.94 (3H, s, SO2CH3), 3.54 (1H, dd, J = 12.0, 4.0, H-6a), 3.67 (1H, dd, J = 12.0, 3.5, H-6b), 3.80 (1H, dd, J = 9.0, 1.0, H-1a), 3.85-3.88 (1H, m, H-5), 3.90 (1H, dt, J = 9.0, 1.0, H-1′a), 3.96 (1H, dd, J = 9.0, 0.8, H-1′b), 3.98-4.06 (2H, m, H-3, H-4), 4.31 (1H, dd, J = 9.0, 1.0, H-1b). 13C-NMR (125 MHz, CD3OD): 34.3 (SO2CH3), 59.2 (C-1), 61.6 (C-1′), 62.9 (C-6), 72.4 (C-3), 75.7 (C-4), 80.1 (C-2), 85.6 (C-5). IR (CH3OH, cm−1): 3422 (br), 2963 (w), 1397 (w), 1262 (s), 1092 (s), 1024 (s), 801 (s).
(6R,7S,8R)-2-Aza-7,8-dihydroxy-6-hydroxymethyl-2-N-mesyl-5-oxaspiro[3.4]octane 27b. Purification of the crude product by column chromatography (silica gel, dichloromethane/methanol 95/5) gave the title compound 27b (215 mg, 52% yield) as amorphous solid. White needle crystals were obtained after recrystallisation with ethyl acetate. M.p. = 96–97 °C. [α]25D = −40.0 (c = 0.01, l = 0.025 dm, CH3OH). 1H-NMR (400 MHz, CD3OD): 1H-NMR (400 MHz, CDCl3): δ 2.46 (3H, s, PhCH3), 3.44 (1H, dd, J = 12.2, 4.1, H-6a), 3.53 (1H, d, J = 8.6, H-1a), 3.57 (1H, dd, J = 12.2, 3.1, H-6b), 3.67 (1H, d, J = 8.8, H-1′a), 3.71 (1H, d, J = 8.8, H-1′b), 3.73-3.77 (1H, m, H-5), 3.87 (1H, d, J = 4.7, H-3), 3.94 (1H, dd, J = 5.7, 4.7, H-4), 4.17 (1H, d, J = 8.6, H-1b), 7.46 (2H, d, J = 8.0, 2 × Hm), 7.72 (2H, d, J = 8.3, 2 × Ho). 13C-NMR (100 MHz, CD3OD): 22.1 (PhCH3), 60.0 (C-1), 62.7 (C-1′), 63.2 (C-6), 72.6 (C-4), 76.3 (C-3), 80.2 (C-2), 85.6 (C-5), 130.2 (Co), 131.5 (Cm), 132.8 (Cp), 146.3 (Cipso). IR (CH3OH, cm−1): 3347 (br), 2928 (w), 1332 (w), 1157 (m), 1090 (s), 1017 (s), 814 (m). HRMS required for C14H20NO6S+ is 330.101 found 330.100.

Crystallography

Crystal structures at 150 K for 17a and 27b were obtained using an Oxford Diffraction Gemini single-crystal diffractometer equipped with an Oxford Instruments Cryojet cooling device. Whilst a satisfactory solution and refinement of the structure of 17a was achieved, and the structure of 27b easily solved, refinement of the latter did not progress to a satisfactory conclusion. Diffraction data for 27b were therefore recollected at 150 K at the EPSRC UK National Crystallography Service at the University of Southampton and the structure then refined satisfactorily as a 4-component twin. Both crystal structures have been deposited with the CCDC as reference numbers 1840501 (17a) and 1840502 (27b).
Viruses and cells. The A59 strain of Mouse hepatitis virus was used in all experiments. 17Cl-1 mouse lung fibroblast cells were grown in complete growth medium consisting of Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% heat-inactivated foetal bovine serum (FBS; Invitrogen), penicillin and streptomycin (Invitrogen), and nonessential amino acids (NEAA; Invitrogen).
MTT toxicity assay. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays were used to measure cell viability of experimentally treated cells. 17Cl-1 cells in complete growth medium were treated with specified concentrations of test substance in a total volume of 2 mL and plated in 96-well plates at a density of about 50% confluence. After 72 h of treatment, 5 mg mL−1 MTT (Sigma) was added to each well in a six-well plate. Cells were then incubated at 37 °C for 1 h, when blue coloration appeared in the majority of untreated control cells indicating that the MTT had been reduced to the insoluble compound formazan. Assays were protected from light during MTT treatment. The medium was then aspirated and replaced with 1 mL of dimethyl sulfoxide to solubilize the cells. Absorbance at 540 nm was read in a Molecular Devices plate reader and analysed by the SOFTmax program.
Virus growth inhibition assay. 17Cl-1 cells were seeded at a density of 2 × 105 cells per well of a 12-well tissue culture plate and allowed to adhere overnight at 37 °C and 5% CO2. Cells were pre-treated for 3 h with 2 mL of complete medium containing 100 μL of test compounds dissolved in a vehicle of 90% water and 10% dimethyl sulfoxide or vehicle alone. After the pre-treatment, cells were inoculated at a multiplicity of about 10 PFU per cell with MHV-A59. After 1 h cells were rinsed three times with warm phosphate-buffered saline to remove any unbound virus, then culture medium containing the same treatment as before was reapplied. 14 h after inoculation, cell culture medium was collected and stored for virus quantification.
Virus quantification by plaque assay. 17Cl-1 cells were seeded in 12-well tissue culture plates at 2 × 105 cells per well and allowed to adhere overnight at 37 °C, 5% CO2. Culture medium was removed and replaced with 0.5 mL of serial dilutions of inoculum in culture medium, then incubated at 37 °C for 1 h. Inocula were left in place, and cells were overlaid with 0.7% agarose (Doc Frugal) in DMEM supplemented with 2% serum (final concentration) and incubated at 37 °C with 5% CO2 for 2 days. Cells were fixed with 25% formaldehyde in phosphate-buffered saline; agarose plugs were removed, and cells were stained with 0.1% crystal violet to visualize viral plaques.
Syncytium inhibition assay. 17Cl-1 cells were seeded in 25 cm2 flasks, pre-treated, and inoculated as before. Infection was allowed to proceed for 24 h before medium was removed, and cells were fixed and stained as before. Multinucleate cells, including virus-induced syncytia, were observed and photographed as described above. Multinucleate cells falling within a 4 mm2 window were counted manually for each flask. Statistical significance of differences between syncytium formation after control or experimental treatment was tested by using a two-sample t test in Instat 3.0 (Graphpad).

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We would like to thank the following for funding: University of Reading and Syngenta (to ADI), the Iraqi Ministry of Higher Education and Scientific Research, Al-Mustansiriya University and the University of Baghdad (to BOA and HMNA), the Society for General Microbiology (to WE), the Royal Society of Biology (to AWDB). Some of the viral work was supported by the German Center for Infection Research (DZIF), partner site Giessen, Germany (TTU Emerging Infections), for which we are grateful. We also thank the EPSRC UK National Crystallography Service at the University of Southampton for the collection of the crystallographic data from crystals of 27b, and in particular Dr James Orton for resolving the space group ambiguity for this material.

Notes and references

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Footnotes

Electronic supplementary information (ESI) available. CCDC 1840501 and 1840502. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c8nj02777c
Current Address: Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London SE1 1DB, UK. E-mail: andre.cobb@kcl.ac.uk
§ Current Address: Department of Biology, College of Science, Al-Mustansiriyah University, Baghdad, Iraq.
Current Address: Department of Biology, Texas A&M University-Texarkana, 7101 University Ave, Texarkana, TX 75503, USA.

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