Synthesis of rigidified shikimic acid derivatives by ring-closing metathesis to imprint inhibitor efficacy against shikimate kinase enzyme

Abstract

Diverse rigidified shikimic acids derivatives, which are stable mimetics of the high-energy conformation of shikimic acid, have been synthesized to enhance inhibitor efficacy against shikimate kinase enzyme (SK), an attractive target for antibiotic drug discovery. The synthesis of the reported conformationally restricted shikimic acid derivatives was carried out by ring-closing metathesis of allyloxy vinyl derivatives as the key step. The rigidification of the ligand conformation was used to maximize the effectiveness of the substituents introduced in the ether carbon bridge of the scaffold by pre-orienting their interaction with key residues and enzyme domains that are essential for catalysis and enzyme motion. Molecular Dynamics simulation studies on the enzyme/ligand complexes revealed marked differences in the positioning of the ligand substituent in the active site of the two enzymes studied (SK from Mycobacterium tuberculosis and Helicobacter pylori) and this explains their greater efficacy against one of the enzymes. This enhancement is due to the distinct induced-fit motion of the two homologous enzymes. A 20-fold improvement against the H. pylori enzyme was achieved by the introduction of a CH₂OEt group in the rigid ether bridge of the reported shikimic acid analogs.

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Introduction

Flexible ligands upon binding to their biological target may suffer an entropic penalty due to the freezing of their rotatable bonds to achieve the active binding conformation.^1,2 In some cases these ligands may also adopt high-energy active conformations in order to maximize favorable interactions with the residues involved in the protein binding pocket.^3,4 Hence, pre-organization of the ligand conformation or stabilization of the required high-energy active arrangements through the introduction of conformational constraints is a very attractive strategy that is used in drug design.^5–9 The rigidification of the ligand conformation can also be considered as an ‘atom-efficient approach’, since it maximizes the efficiency of the functional groups introduced into the initial scaffold during the drug optimization process as the interactions of those groups with the binding pocket are well pre-oriented.^10–12

We became interested in using this appealing concept in the development of inhibitors of the fifth enzyme of the shikimic acid pathway, namely the shikimate kinase (SK) enzyme. SK is an attractive target for antibiotic drug discovery because (i) it has no counterpart in human cells; and (ii) it is essential in several very relevant pathogenic bacteria that nowadays show high levels of resistance to many antibiotics in clinical use. Specifically, SK is crucial for: (i) Mycobacterium tuberculosis, which is responsible for tuberculosis – a globally established Word Health Organization (WHO) priority; (ii) Helicobacter pylori, which is the causative agent of gastric and duodenal ulcers and has also been classified as a type I carcinogen; and (iii) Pseudomonas aeruginosa, which is one of the most common pathogens in healthcare-associated infections and a WHO critical pathogen for R&D of new antibiotics. SK catalyzes the stereospecific phosphorylation of the C3 hydroxyl group of shikimic acid (1) by transferring the γ-phosphate group of ATP to the hydroxyl group to provide shikimate 3-phosphate and ADP (Fig. 1A). This enzyme is an amazing example of how the specific transformation of only one of three hydroxyl groups of the ligand is achieved by an exquisitely designed stabilization of its high-energy conformation. By forcing the axial disposition of the C4 and C5 hydroxyl groups in 1, the enzyme achieves the equatorial arrangement of the C3 hydroxyl group for selective phosphorylation by ATP.

Fig. 1 A. Schematic representation of shikimic acid (1) recognition by SK. B. Previously reported reversible competitive inhibitors. C. Target compounds.

Based on the aforementioned recognition, we reported previously that the rigidified shikimic acid derivative 2, in which the conformation that the enzyme recognizes for catalysis is fixed by an unsaturated ether bridge between positions C3 and C5 in 1, is a reversible competitive inhibitor of SK from M. tuberculosis (Mt-SK) (Fig. 1B).¹³ Compound 2 proved to have an inhibition constant (K_i) of 62 μM, which is lower than the enzyme K_m (544 μM). The crystal structure of Mt-SK in complex with ADP and 2 (PDB entry 4BQS, 2.15 Å) revealed that the ligand occupies the active site with a similar arrangement and polar interactions (hydrogen bonding and electrostatic interactions) as 1. More importantly, the structure shows that the rigidification of the diaxial conformation of the C4 and C5 hydroxyl groups in 1 by a C3–C5 ether bridge causes a dramatic reduction in the flexibility of the lid and shikimic acid binding (SB) domains, the plasticity of which is essential for catalytic turnover. The SB domain, which involves several highly conserved lipophilic residues, isolates the substrate from the solvent environment to perform the reaction. Molecular Dynamics (MD) simulation studies also revealed that a closed form of the lid and SB domains are required for catalysis.¹³ Moreover, reduction of the double bond of the C3–C5 ether bridge in 2, to give compound 3, improves the ligand affinity a little more (K_i = 46 μM) by enhancing lipophilic interactions between the ether bridge and residues of the lid domain, thus sealing the active site even more.

Based on these results, we report herein the possible enhancement of the inhibitor efficiency of this scaffold, compound 2, by promoting favorable lipophilic interactions between the ligand and the lid (Fig. 1C). To this end, we carried out the synthesis of rigidified shikimic acid derivatives 4–5 in which the closest sp² carbon of the unsaturated bridge to the lid was substituted with diverse apolar groups that pre-orient their interaction with this important part of the enzyme. In addition, the relevance of the double bond to ligand affinity was studied with compounds 5. The results of inhibition studies with the SK from M. tuberculosis and from H. pylori, along with MD simulation studies on the enzyme/ligand complexes, allowed us to explain the higher efficacy of the reported compounds observed for the H. pylori enzyme.

Results and discussion

Synthesis of compounds 4–5

The synthesis of conformationally restricted shikimic acid derivatives 4–5 was carried out by ring-closing metathesis of the allyloxy vinyl derivatives 6 as the key step (Fig. 2).

Fig. 2 Synthetic approach.

In an effort to facilitate the formation of the seven-membered bridge ring in 6, the axial arrangement of the vinyl group in C3 was induced by protecting the hydroxyl groups in the C3 and C4 positions of the shikimic acid derivative as an acetal (Fig. S1†). The key compounds 6 were prepared by Trost allylation of previously reported alcohol 7 ¹³ using the appropriate allyl methyl carbonates 8.

Allyl methyl carbonates 8 were prepared from the corresponding alcohols 14–21 by treatment with methyl chloroformate and pyridine (Scheme 1). Alcohols 15–17 were synthesized by 1,2-reduction of α,β-unsaturated aldehydes 11 (commercially available) and 12–13, with the latter compounds readily prepared from pentanal (9) and hexanal (10), respectively. Alcohol 18 was obtained in four steps from ethyl malonate (22): (i) alkylation of 22 with cyclopropylmethyl bromide; (ii) decarboxylative hydrolysis; (iii) aldol condensation; and (iv) 1,2-reduction of α,β-unsaturated acid 23. Alcohols 20–21 were synthesized by alkylation of commercially available 2-methylene-1,3-propanodiol (19). Finally, carbonate 8i was obtained by TBS-protection of carbonate 8h.

Scheme 1 Synthesis of carbonates 8. Reagents and conditions. (a) HCHO, pyrrolidine, propionic acid, iPrOH, 45 °C. (b) NaBH₄, MeOH, Et₂O, 0 °C to RT. (c) 1. NaH, DMF, 0 °C; 2. EtBr, RT. (d) 1. NaH, THF, 0 °C; 2. BnBr, RT. (e) MeOCOCl, Py, DCM, 0 °C to RT. (f) 1. NaH, THF, 0 °C; 2. BrCH₂cPr, Δ. (g) NaOH (2 M), Δ. (h) piperidine, HCHO, EtOH, 80 °C. (i) BH₃·Me₂S, THF, 0 °C to RT. (j) TBSCl, DMAP, TBAI, Et₃N, DMF, 0 °C to RT.

Palladium-catalyzed Trost allylation of 7 with allyl methyl carbonates 8a–g and 8i gave the key intermediates 6a–g and 6i in yields ranging from 32–88% (Scheme 2 and Table 1). Ring-closing metathesis of 6a–g and 6i was achieved by using second-generation Grubbs’ catalyst in toluene at 90 °C to afford bicyclic derivatives 24a–g and 24i in yields ranging from 16–63%, and from 42–99% considering the recovered starting material. Bicyclic derivative 24h was efficiently prepared from 24i by TBS-deprotection with TBAF. As expected, the metathesis reaction proved to be quite sensitive to the presence of substitution in the allyl moiety, since: (i) when R = H the transformation took place at room temperature and in a higher yield (88%);¹³ and (ii) an increase in the steric hindrance of the substituent led to lower reaction yields and required higher reaction temperatures (Table 1). Derivatives 24g and 24f, which contained a CH₂OBn and a CH₂OEt group, respectively, gave the lowest yields. These compounds were alternatively prepared by alkylation of alcohol 24h in 64% and 31% (57%) yield, respectively. Deprotection of the acetal group in 24, followed by basic hydrolysis of the resulting esters 25a–h and subsequent protonation with Amberlite IR-120 (H⁺) ion-exchange resin efficiently afforded the target compounds 4. Finally, compounds 5, which have a flexible substituted ether bridge, were synthesized from methyl derivative 25a by catalytic hydrogenation using Rosenmund catalyst in the presence of pyridine, followed by hydrolysis of the methyl ester to give a 1 : 1 mixture of epimers in the C4 position, i.e., compounds 5S and 5R, which were separated by HPLC. The configuration of the new chiral center was determined by NOE experiments. Inversion of H10 in bicycles 5S and 5R led to enhancement of the signals for H4 (3.6%) and the methyl group (5.2%), respectively.

Scheme 2 Synthesis of compounds 4–5. Reagents and conditions. (a) 8a–g and 8i, Pd₂(dba)₃ (cat), dppb, THF, Δ. (b) 2^nd generation Grubbs’ catalyst, PhMe, 90 °C. (c) MeOH, HCl (6 M), 60 °C. (d) TBAF, THF, 0 °C. (e) 1. NaH, THF, 0 °C; 2. BnBr, RT. (f) 1. NaH, DMF, 0 °C; 2. EtBr, RT. (g) 1. LiOH (aq.), THF, RT. 2. Amberlite IR-120 (H⁺), RT. (h) H₂, Rosenmund catalyst, MeOH, Py. RT.

Table 1 Yields for the conversion 7 → 6 and 6 → 24

R	6	Yield^a (%)	24	Yield^a (%)
a Isolated yields. Corrected yields are shown in brackets.
Me	6a	88	24a	63 (77)
Et	6b	32 (74)	24b	47 (97)
nPr	6c	47	24c	63 (89)
nBu	6d	49	24d	54
cPr	6e	34	24e	45 (99)
CH2OEt	6f	54	24f	16 (78)
CH2OBn	6g	63	24g	26 (94)
CH2OTBS	6i	43	24i	42

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Inhibition studies

The inhibitory activity of the reported conformationally restricted shikimic acid derivatives 2–5 was assayed against SK from Helicobacter pylori (Hp-SK) and from M. tuberculosis (Mt-SK). All of the compounds proved to be competitive reversible inhibitors of shikimic acid for both enzymes. The inhibition data (K_i), which were obtained from Dixon plots (1/v vs. [I]), are summarized in Table 2.

Table 2 K _i (μM) values of compounds 2–5 against SK enzymes^a

Entry	Comp	R	H. pylori	M. tuberculosis
a Assay conditions: Tris·HCl (100 mM, pH 7.7), ATP (2.5 mM), NADH (0.2 mM), PEP (1 mM), MgCl2 (5 mM), KCl (100 mM), lactate dehydrogenase/pyruvate kinase (∼2.8 units), 25 °C. For Mt-SK: Km (1) = 544 ± 14 μM; kcat = 295 ± 8 s^−1. For Hp-SK: Km (1) = 39 ± 8 μM; kcat = 116 ± 4 ms^−1. ND = not determined.
1	2	H	104 ± 4	62 ± 1 (ref. 13)
2	3	—	47 ± 6	46 ± 2 (ref. 13)
3	4a	Me	54.5 ± 5.7	28 ± 1
4	4b	Et	15.5 ± 1.1	41 ± 2
5	4c	nPr	9.2 ± 1.0	72 ± 4
6	4d	nBu	12 ± 2	177 ± 3
7	4e	cPr	10.0 ± 0.6	101 ± 2
8	4f	CH2OEt	5.0 ± 0.3	170 ± 3
9	4g	CH2OBn	68 ± 3	121 ± 5
10	4h	CH2OH	38 ± 3	333 ± 10
11	5S	Me	465 ± 41	360 ± 7
12	5R	Me	ND	645 ± 16

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In general: (i) the ligands proved to be more potent against the H. pylori enzyme than the M. tuberculosis enzyme; (ii) a rigid ether bridge between the C3 and C5 positions of shikimic acid provided more potent inhibitors (Table 2, entries 3 vs. 11); (iii) for Hp-SK, the inhibition potency of the ligands increased with the length of the substituent chain (Table 2, entries 5 and 8 vs. 1 and 4), while for Mt-SK only the introduction of a methyl group in the rigid ether bridge improved the inhibitory activity (Table 2, entries 3 vs. 5); (iv) the presence of a hydroxyl or an ether group in the substituent only enhanced the inhibitory activity for Hp-SK (Table 2, entries 8 vs. 5).

For the H. pylori enzyme, the best inhibitor in the series was compound 4f, which has a CH₂OEt substituent in the ether bridge. This enhanced the inhibitory potency by up to 20-fold. For the M. tuberculosis enzyme, a 2-fold improvement in activity was achieved with compound 4a, which has a methyl group. Computational studies were performed in an effort to gain a better understanding at the atomic level of the differences observed experimentally in the inhibitory potency of the reported conformationally rigid shikimic acid analogs 4–5. The results of these studies are discussed below.

Computational studies

Molecular docking using the GOLD 5.2.2 ¹⁴ program and the protein coordinates found in the crystal structures of Hp-SK in complex with shikimate-3-phosphate and ADP (PDB entry 3MUF,¹⁵ 2.3 Å) and of Mt-SK in complex with 2 and ADP (PDB entry 4BQS,¹³ 2.15 Å) were carried out first. The highest score solutions obtained by docking were further analyzed by Molecular Dynamics (MD) simulation studies in order to assess the stability and therefore the reliability of the postulated binding. The monomer of the Hp-SK/ATP/Mg²⁺/ligand and Mt-SK/ATP/Mg²⁺/ligand complexes in a truncated octahedron of water molecules obtained with the molecular mechanics force field AMBER¹⁶ was employed and the system was then subjected to 100 ns of dynamic simulation. The latter was carried out with the most active ligands, compounds 4a–d and 4f, as well as the analogs with a flexible ether bridge, i.e., 5S and 5R (Fig. 3).

Fig. 3 Binding mode of compounds 4a (green), 4c (cyan) and 4f (yellow) obtained by docking and MD simulation studies in the active site of the Hp-SK (gray) and Mt-SK (dark blue) enzymes. A–C. Overall view of the Hp-SK/ATP/Mg²⁺/ligand complexes. Snapshots after 80 ns of simulation are shown. Mg²⁺ (sphere) and ligands (sticks) and ATP (sticks) are displayed. D–F. Close view of ligands 4a, 4c and 4f in the SB binding pocket of Hp-SK. Note how these ligands and the substituents of the ether bridge are perfectly surrounded by the enzyme, in particular for compounds 4c and 4f. G–H. Close view of ligands 4a and 4f in the Mt-SK/ATP/Mg²⁺/ligand complexes. Note how for ligand 4f the substituent of the ether bridge would not be embedded in the active site as for the H. pylori enzyme. I. Percentage of conformations of the ligands 4b–d and 4f during the 100 ns of simulation in which the substituent of the ether bridge (Et, nPr, nBu, CH₂OEt) would be located pointing towards the active site (inside).

The results of the computational studies revealed that, in all cases, the ligands would be stable in the shikimic acid active site, since significant variations were not observed during the whole simulation, both in the position of the ligand and in the protein backbone (Fig. S2 and S3†). As one would expect, the ligands would be anchored to the active site by the same electrostatic and polar interactions as the original compound 2 (Fig. S4†). More importantly, relevant differences were identified in the arrangement of the substituent of the ether bridge of the ligands for both enzymes and this would explain the experimentally obtained activity. Thus, for Hp-SK and during most of the simulation, these substituents were mainly embedded in the active site, with both the lid and the SB domain completely surrounding the entire ligand (Fig. 3A–F). For compounds 4b–d and 4f (R ≠ H, Me), the percentage of conformations with the substituent ‘inside’ the active site increased as the chain length increased, which is in good agreement with the observed improvement in the inhibitory potency (CH₂OEt > nBu > nPr > Et) (Fig. 3I). These values were calculated by analyzing the variation of the dihedral angle between the atoms C5 (CAF), C4 (CAE) and the first two atoms of the substituent, C (CAP) and C (CAR)/O (OAR), in 4b–d and 4f during the whole simulation (Fig. S5 and S6†). ‘Substituent inside conformations’ were considered for values of the dihedral angle between −50° and −150°. As a result of this arrangement, the shikimic acid active site remained neatly closed, thus avoiding the entrance of the natural substrate, because the ligands caused a dramatic reduction in the flexibility of the lid and SB domain by a series of favorable apolar interactions between the substituent and the residues in this pocket (Fig. 4A–D). It is worth highlighting that MD studies in the enzyme product complex, i.e., in the presence of ADP and shikimate-3-phosphate, revealed that the flexibility of the lid and the SB domain are key for the catalytic turnover.¹³ The lid is the substrate-covering loop that closes over the shikimic acid binding site for catalysis and it contains the essential residue Arg116/Arg117 (H. pylori and M. tuberculosis, respectively). NMR studies revealed that this residue might also be involved in the phosphoryl-transfer mechanism catalyzed by SK by activating and positioning the reaction intermediate for subsequent nucleophilic attack by the C3 hydroxyl group in 1.¹⁸ The aforementioned apolar interactions would be more numerous as the length of the chain increases, which would explain the enhancement in ligand affinity. In general, these interactions would involve the residues of the: (i) lid: Arg116 (essential), Pro117 (conserved) and Leu118; (ii) the SB domain: Val44 and Arg45; and (iii) the P-loop: Met10 (Fig. S7†). For the most potent inhibitor, compound 4f, an additional interaction was identified between the oxygen atom of the substituent and the amide main chain (carbonyl) of Val44 through a water network, and this could explain the higher affinity of 4f for Hp-SK than 4d, which has a CH₂ group in the same position (Fig. 4D and C, respectively).

Fig. 4 Detailed view of the interactions of the ether bridge substituent in 4a, 4c–d and 4f with Hp-SK (A–D) and in 4a and 4f with Mt-SK (E–F) in their respective enzyme complexes. Relevant side chain residues are shown and labeled. Apolar (magenta) and polar (blue) contacts are shown as dashed lines. The interaction of the oxygen atom of the substituent in 4f with the amide main chain (carbonyl) of Val44 through a water network is highlighted with blue shading.

Moreover, for ligands 5, the simulation studies revealed a different behavior of both compounds, mainly relative to the ether bridge. Thus, while for ligand 5R no significant conformational changes were observed during the dynamic simulation, this was not the case for ligand 5S. The ether bridge moiety in 5S underwent a conformational change to locate the methyl group in parallel to the cyclohexene ring. This conformation remained stable after ∼40 ns of simulation (Fig. S8†). As a consequence, for both ligands 5, an interaction of the methyl group with the carbon chain of the essential arginine was not identified, as observed with compound 4f and previously reported saturated derivative 3. This fact revealed how the rigidity of the ether bridge in the ligands would be crucial to fix the position and direction of the substituent towards the key residues of the lid.

In contrast to the above, for Mt-SK, as the length of the substituent increases (R = Et, nPr, nBu, CH₂OEt) the ligands would be located preferentially with the substituent pointing outside the active site (Fig. 3G–H). In this arrangement, the introduction of this type of substituent in 2 would not contribute to an improvement in ligand affinity since additional interactions with the residues of the active site could not be established (Fig. 4E–F).

For both enzymes, the substituent of the ligand interacts with a similar region of the lid and this is quite similar in terms of amino acid sequences. However, the results of the computational studies revealed a clear and markedly distinct induced fitting of the ligands by the two enzymes, which would explain the differences found. These are due to key differences in the folding of the lid over the active site – a situation that can be easily visualized by analysis of the vibrational modes of the two enzymes (Fig. 5).¹⁹

Fig. 5 Overall view of the Hp-SK and Mt-SK motion obtained by examination of the vibrational modes. The motion of the lid is highlighted with blue and yellow shading, respectively. Note how the folding of the lid and as a consequence the essential arginine that it contains is quite distinct in the two enzymes.

Conclusions

The functionalization of the double bond of previously identified scaffold 2, a stable mimetic of the high-energy conformation of shikimic acid and a competitive reversible inhibitor of the shikimate kinase enzyme, was carried out by ring-closing metathesis of allyloxy vinyl derivatives 6 as the key step. The latter compounds were prepared by Trost allylation of previously reported alcohol 7 with allyl methyl carbonates 8. The RCM approach required reaction temperatures of 90 °C and the use of second-generation Grubbs’ catalyst.

The results obtained for the rigidified shikimic acid derivatives reported, namely compounds 4–5, with the two enzymes studied, SK from M. tuberculosis and from H. pylori, revealed that: (i) the rigidification of the functionalized ether bridge between C3 and C5 of the shikimic acid is crucial for improving ligand affinity; (ii) this functionalization generally provides more potent analogs against the H. pylori enzyme than the M. tuberculosis enzyme. A 20-fold improvement against the H. pylori enzyme was achieved by the introduction of a CH₂OEt group in the rigid ether bridge of the reported shikimic acid analogs. For the M. tuberculosis enzyme, the introduction of a methyl group in 2 enhanced the ligand potency by a factor of two.

Computational studies revealed that the differences in affinity found with the two homologous enzymes are due to the distinct induced-fit conformation adopted by the two enzymes upon ligand binding, which mainly involves the substrate-covering loop (lid). For Hp-SK, the substituents (R = Et, nPr, nBu, CH₂OEt) would be embedded in the active site, with both the lid and the SB domain completely surrounding the entire ligand. As a result, the active site would be neatly closed because the ligands cause a dramatic reduction in the flexibility of the lid and SB domain through a series of favorable apolar interactions between the substituent and the residues in this pocket. In contrast, for Mt-SK, as the length of the substituent increases (R = Et, nPr, nBu, CH₂OEt) this moiety of the ligand would be pointing away from the active site and therefore they would not contribute to an improvement in ligand affinity. The results reported here can be considered as a good example of how the rigidification of a ligand is a useful strategy to enhance ligand affinity for a target due to the pre-orientation and maximization of the interactions of its substituents.

Experimental

General

All starting materials and reagents were commercially available and were used without further purification. ¹H NMR spectra (250, 300 and 500 MHz) and ¹³C NMR spectra (63, 75 and 125 MHz) were measured in deuterated solvents. J values are given in hertz. NMR assignments were carried out by a combination of 1 D, COSY, and DEPT-135 experiments. FT-IR spectra were recorded in a PerkinElmer two FTIR spectrometer with attenuated total reference. [α]²⁰_D = values are given in 10⁻¹ deg cm² g⁻¹. MilliQ deionized water was used in all the buffers. Melting points were measured in a Büchi M-560 apparatus. The experimental procedures for the synthesis of carbonates 8 are described in the ESI.†

General procedure for synthesis of compounds 6

To a stirred solution of Pd₂(dba)₃ (0.025 mmol) and dppb (0.1 mmol) in dry THF (0.8 mL), under argon and at room temperature, were added the alcohol 7 ¹³ (1 mmol), followed by a solution of the carbonates 8a–g and 8i (1.5 mmol) in dry THF (3 mL, 0.5 M). The resulting suspension was heated at 60 °C for 24 h and then cooled to room temperature. The mixture was filtered over Celite® and the residue was washed with diethyl ether. The filtrate and washings were evaporated under reduced pressure to yield an oil which was purified by flash chromatography to afford the allyl ethers 6.

Methyl (3R,4S,5R)-5-(2-methyl)allyloxy-3,4-O-isopropylidenedioxy-3-vinylcyclohex-1-ene-1-carboxylate (6a, R = Me). It was prepared following the general allylation procedure using 7 (49 mg), Pd₂(dba)₃ (4.9 mg), dppb (8.1 mg), methyl-2-methylallyl carbonate (8a)¹⁷ (30 mg) in dry THF (0.28 mL), THF (0.8 mL). Eluent for chromatography = (30 : 70) diethyl ether/hexane. Yield = 88% (52 mg). Colorless oil. [α]²⁰_D = +107.6° (c 3.5, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.64 (m, 1H, H2), 5.92 (dd, J = 17.3 and 10.7 Hz, 1H, CH=CH₂), 5.44 (dd, J = 17.3 and 1.5 Hz, 1H, CH=CH₂), 5.17 (dd, J = 10.7 and 1.5 Hz, 1H, CH=CH₂), 4.89 (d, J = 18.6 Hz, 2H, H₃CCH=CH₂), 4.09 (d, J = 4.0 Hz, 1H, H4), 4.02 (dd, J = 6.6 and 3.9 Hz, 1H, H5), 3.91 (d, J = 5.3 Hz, 2H, CH₂), 3.76 (s, 3H, OCH₃), 2.64 (dd, J = 18.0 and 2.6 Hz, 1H, CHH-6), 2.44 (ddd, J = 18.0, 3.9 and 2.7 Hz, 1H, CHH-6), 1.69 (s, 3H, CH₃), 1.41 (s, 3H, CH₃) and 1.34 (s, 3H, CH₃) ppm. ¹³C NMR (63 MHz, CDCl₃) δ: 167.4 (C), 141.9 (C), 137.8 (C), 137.6 (C), 126.2 (C), 115.0 (CH₂), 112.1 (CH₂), 109.1 (C), 80.1 (C), 77.3 (CH), 73.3 (CH₂), 73.2 (CH), 51.9 (CH), 27.9 (CH₃), 26.9 (CH₃), 24.3 (CH₂) and 19.4 (CH₃) ppm. FTIR (film) ν: 1719 (CO) cm⁻¹. MS (ESI) m/z = 331 (MNa⁺). HRMS calcd for C₁₇H₂₄O₅Na (MNa⁺): 331.1516; found, 331.1511.

Methyl (3R,4S,5R)-5-(2-ethylallyloxy)-3,4-O-isopropylidenedioxy-3-vinylcyclohex-1-ene-1-carboxylate (6b, R = Et). It was prepared following the general allylation procedure using 7 (530 mg), Pd₂(dba)₃ (48 mg), dppb (89 mg), methyl (2-methylenebutyl) carbonate (8b)¹⁷ (599 mg) in THF (8.3 mL), THF (9 mL). Eluent for chromatography = (50 : 50) diethyl ether/hexane. Yield = 32% (213 mg). It was recovered 221 mg of starting material. Corrected yield = 74%. Colorless oil. [α]²⁰_D = +99.1° (c 1.1, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.64 (t, J = 1.3 Hz, 1H, H2), 5.92 (dd, J = 10.7 and 17.3 Hz, 1H, CH=CH₂), 5.45 (dd, J = 1.5 and 17.3 Hz, 1H, CH=CHH), 5.18 (dd, J = 1.4 and 10.7 Hz, 1H, CH=CHH), 4.95 (m, 1H, C=CHH), 4.87 (m, 1H, C=CHH), 4.09 (m, 1H, H5), 4.02 (m, 1H, H4), 3.99 (d, J = 12.5 Hz, 1H, OCHH), 3.92 (d, J = 12.5 Hz, 1H, OCHH), 3.76 (s, 3H, OCH₃), 2.65 (dd, J = 2.5 and 17.9 Hz, 1H, CHH-6), 2.44 (ddd, J = 2.8, 3.8 and 18.0 Hz, 1H, CHH-6), 2.02 (q, J = 7.4 Hz, 2H, CH₂CH₃), 1.42 (s, 3H, CH₃), 1.34 (s, 3H, CH₃) and 1.02 (t, J = 7.4 Hz, 3H, CH₂CH₃) ppm. ¹³C NMR (63 MHz, CDCl₃) δ: 167.5 (C), 147.5 (C), 137.9 (CH), 137.7 (CH), 126.3 (C), 115.1 (CH₂), 110.3 (CH₂), 109.1 (C), 80.2 (C), 77.3 (CH), 73.2 (CH), 72.5 (CH₂), 52.0 (OCH₃), 28.0 (CH₃), 27.0 (CH₃), 25.9 (CH₂), 24.4 (CH₂) and 12.0 (CH₃) ppm. FTIR (film): 1710 (CO) cm⁻¹. MS (ESI) m/z = 345 (MNa⁺). HRMS calcd for C₁₈H₂₆O₅Na (MNa⁺): 345.1672; found, 345.1681.

Methyl (3R,4S,5R)-3,4-O-isopropylidenedioxy-5-(2-propylallyloxy)-3-vinylcyclohex-1-ene-1-carboxylate (6c, R = nPr). It was prepared following the general allylation procedure using 7 (198 mg), Pd₂(dba)₃ (20 mg), dppb (33 mg), methyl (2-methylenepentyl) carbonate (8c) (320 mg) in THF (2.3 mL), THF (3 mL). Eluent for chromatography = (20 : 80) diethyl ether/hexane. Yield = 47% (122 mg). Colorless oil. [α]²⁰_D = +92.8° (c 1.1, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.62 (t, J = 1.4 Hz, 1H, H2), 5.89 (dd, J = 10.7 and 17.4 Hz, 1H, CH=CH₂), 5.42 (dd, J = 1.5 and 17.3 Hz, 1H, CH=CHH), 5.14 (dd, J = 1.5 and 10.7 Hz, 1H, CH=CHH), 4.93 (br s, 1H, C=CHH), 4.83 (br s, 1H, C=CHH), 4.06 (br d, J = 4.0 Hz, 1H, H4), 4.00 (td, J = 2.6 and 3.9 Hz, 1H, H5), 3.94 (d, J = 12.6 Hz, 1H, OCHH), 3.87 (d, J = 12.6 Hz, 1H, OCHH), 3.73 (s, 3H, OCH₃), 2.63 (dd, J = 2.6 and 18.0 Hz, 1H, CHH-6), 2.41 (ddd, J = 2.8, 3.8 and 18.0 Hz, 1H, CHH-6), 1.95 (t, J = 7.5 Hz, 2H, CH₂CH₃), 1.47–1.35 (m, 5H, CH₂ + CH₃), 1.31 (s, 3H, CH₃) and 0.86 (t, J = 7.3 Hz, 3H, CH₂CH₃) ppm. ¹³C NMR (63 MHz, CDCl₃) δ: 167.5 (C), 145.8 (C), 137.9 (CH), 137.7 (CH), 126.3 (C), 115.0 (CH₂), 111.4 (CH₂), 109.1 (C), 80.2 (C), 77.4 (CH), 73.3 (CH), 72.3 (CH₂), 52.0 (OCH₃), 35.2 (CH₂), 28.0 (CH₃), 27.0 (CH₃), 24.4 (CH₂), 20.8 (CH₂) and 13.9 (CH₃) ppm. FTIR (film): 1711 (CO) cm⁻¹. MS (ESI) m/z = 359 (MNa⁺). HRMS calcd for C₁₉H₂₈O₅Na (MNa⁺): 359.1829; found, 359.1826.

Methyl (3R,4S,5R)-3,4-O-isopropylidenedioxy-5-(2-butylallyloxy)-3-vinylcyclohex-1-ene-1-carboxylate (6d, R = nBu). It was prepared following the general allylation procedure using 7 (373 mg), Pd₂(dba)₃ (38 mg), dppb (63 mg), methyl (2-methylenehexyl) carbonate (8d) (380 mg) in THF (4.4 mL), THF (6.4 mL). Eluent for chromatography = (10 : 90) diethyl ether/hexane. Yield = 49% (250 mg). Colorless oil. [α]²⁰_D = 105.9° (c 1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.64 (m, 1H, H2), 5.91 (dd, J = 10.7 and 17.3 Hz, 1H, CH=CH₂), 5.44 (dd, J = 1.5 and 17.3 Hz, 1H, CH=CHH), 5.17 (dd, J = 1.5 and 10.7 Hz, 1H, CH=CHH), 4.94 (m, 1H, C=CHH), 4.85 (m, 1H, C=CHH), 4.08 (td, J = 1.1 and 4.0 Hz, 1H, H4), 4.01 (dt, J = 2.5 and 3.8 Hz, 1H, H5), 3.96 (d, J = 12.8 Hz, 1H, OCHH), 3.89 (d, J = 12.8 Hz, 1H, OCHH), 3.75 (s, 3H, OCH₃), 2.64 (m, 1H, CHH-6), 2.43 (ddd, J = 2.8, 3.8 and 18.0 Hz, 1H, CHH-6), 1.99 (t, J = 6.9 Hz, 2H, CH₂(CH₂)₂CH₃), 1.43–1.23 (m, 4H, CH₂(CH₂)₂CH₃), 1.41 (s, 3H, CH₃), 1.33 (s, 3H, CH₃) and 0.88 (t, J = 7.1 Hz, 3H, CH₂(CH₂)₂CH₃) ppm. ¹³C NMR (63 MHz, CDCl₃) δ: 167.5 (C), 146.1 (C), 137.9 (CH), 137.7 (CH), 126.3 (C), 115.0 (CH₂), 111.2 (CH₂), 109.1 (C), 80.2 (C), 77.3 (CH), 73.2 (CH), 72.4 (CH₂), 52.0 (OCH₃), 32.8 (CH₂), 29.8 (CH₂), 28.0 (CH₃), 27.0 (CH₃), 24.4 (CH₂), 22.5 (CH₂) and 14.0 (CH₃) ppm. FTIR (film): 1717 (CO) cm⁻¹. MS (ESI) m/z = 373 (MNa⁺). HRMS calcd for C₂₀H₃₀O₅Na (MNa⁺): 373.1985; found, 373.1982.

Methyl (3R,4S,5R)-5-(2-cyclopropylmethylallyloxy)-3,4-O-isopropylidenedioxy-3-vinylcyclohex-1-ene-1-carboxylate (6e, R = CH₂cPr). It was prepared following the general allylation procedure using 7 (191 mg), Pd₂(dba)₃ (39 mg), dppb (64 mg), 2-(cyclopropylmethyl)allyl methyl carbonate (8e) (191 mg) in THF (2.2 mL), THF (3 mL). Eluent for chromatography = (20 : 80) diethyl ether/hexane. Yield = 34% (89 mg). Colorless oil. [α]²⁰_D = +93.8° (c 1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.64 (m, 1H, H2), 5.91 (dd, J = 10.7 and 17.3 Hz, 1H, CH=CH₂), 5.44 (dd, J = 1.4 and 17.3 Hz, 1H, CH=CHH), 5.18 (dd, J = 1.4 and 10.7 Hz, 1H, CH=CHH), 5.01 (br s, 1H, C=CHH), 4.97 (br s, 1H, C=CHH), 4.07 (m, 1H, H5), 4.04–4.02 (m, 2H, H4 + OCHH), 3.95 (br d, J = 12.5 Hz, 1H, OCHH), 3.76 (s, 3H, OCH₃), 2.65 (dd, J = 2.5 and 18.0 Hz, 1H, CHH-6), 2.44 (ddd, J = 2.8, 3.8 and 18.0 Hz, 1H, CHH-6), 1.90 (d, J = 6.8 Hz, 2H, CH₂CH(CH₂)₂), 1.42 (s, 3H, CH₃), 1.34 (s, 3H, CH₃), 0.84–0.70 (m, 1H, CH₂CH(CH₂)₂), 0.50–0.44 (m, 2H, CH₂CHCH₂CH₂) and 0.08–0.03 (m, 2H, CH₂CHCH₂CH₂) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.6 (C), 146.1 (C), 138.0 (CH), 137.8 (CH), 126.4 (C), 115.2 (CH₂), 111.6 (CH₂), 109.2 (C), 80.3 (C), 77.5 (CH), 73.3 (CH), 72.6 (CH₂), 52.1 (OCH₃), 38.2 (CH₂), 28.1 (CH₃), 27.1 (CH₃), 24.4 (CH₂), 9.3 (CH), 4.8 (CH₂) and 4.7 (CH₂) ppm. FTIR (film): 1717 (CO) cm⁻¹. MS (ESI) m/z = 371 (MNa⁺). HRMS calcd for C₂₀H₂₈O₅Na (MNa⁺): 371.1829; found, 371.1825.

Methyl (3R,4S,5R)-5-[(2-(ethoxymethyl)allyloxy]-3,4-O-isopropilidendioxi-3-vinylcyclohex-1-ene-1-carboxylate (6f, R = CH₂OEt). It was prepared following the general allylation procedure using 7 (255 mg), Pd₂(dba)₃ (26 mg), dppb (43 mg), 2-(ethoxymethyl)allyl methyl carbonate (8f) (209 mg) in tetrahydrofuran (2.4 mL), tetrahydrofuran (4.3 mL). Eluent for chromatography = (20 : 80) diethyl ether/hexane. Yield = 54% (190 mg). Colorless oil. [α]²⁰_D = +85.2° (c 1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.63 (t, J = 1.2 Hz, 1H, H2), 5.89 (dd, J = 10.7 and 17.3 Hz, 1H, CH=CH₂), 5.43 (dd, J = 1.4 and 17.3 Hz, 1H, CH=CHH), 5.16 (dd, J = 1.4 and 10.7 Hz, 1H, CH=CHH), 5.12 (m, 2H, C=CH₂), 4.08–3.96 (m, 4H, H4 + H5 + OCH₂), 3.91 (br s, 2H, CH₂OEt), 3.74 (s, 3H, OCH₃), 3.44 (q, J = 7.0 Hz, 2H, OCH₂CH₃), 2.64 (dd, J = 2.5 and 18.0 Hz, 1H, CHH-6), 2.43 (ddd, J = 2.8, 3.7 and 18.0 Hz, 1H, CHH-6), 1.40 (s, 3H, CH₃), 1.32 (s, 3H, CH₃) and 1.18 (t, J = 7.0 Hz, 3H, OCH₂CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.5 (C), 142.9 (C), 137.9 (CH), 137.7 (CH), 126.3 (C), 115.1 (CH₂), 113.9 (CH₂), 109.2 (C), 80.2 (C), 77.4 (CH), 73.7 (CH), 71.4 (CH₂), 70.3 (CH₂), 65.9 (CH₂), 52.0 (OCH₃), 28.0 (CH₃), 27.0 (CH₃), 24.5 (CH₂) and 15.3 (CH₃) ppm. FTIR (film): 1716 (CO) cm⁻¹. MS (ESI) m/z = 375 (MNa⁺). HRMS calcd for C₁₉H₂₈O₆Na (MNa⁺): 375.1778; found, 375.1777.

Methyl (3R,4S,5R)-5-[(2-(benzyloxymethyl)allyloxy]-3,4-O-isopropilidendioxi-3-vinylcyclohex-1-ene-1-carboxylate (6g, R = CH₂OBn). It was prepared following the general allylation procedure using 7 (217 mg), Pd₂(dba)₃ (22 mg), dppb (37 mg), 2-(benzyloxymethyl)allyl methyl carbonate (8g) (242 mg) in tetrahydrofuran (2.1 mL), tetrahydrofuran (3.7 mL). Eluent for chromatography = (50 : 50) diethyl ether/hexane. Yield = 63% (207 mg). Yellow oil. [α]²⁰_D = +85.9° (c 0.8, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 7.38–7.28 (m, 5H, 5 × ArH), 6.65 (t, J = 1.3 Hz, 1H, H2), 5.89 (dd, J = 10.7 and 17.3 Hz, 1H, CH=CH₂), 5.43 (dd, J = 1.4 and 17.3 Hz, 1H, CH=CHH), 5.18–5.14 (m, 3H, CH=CHH + C=CH₂), 4.49 (s, 2H, OCH₂), 4.12–4.02 (m, 4H, H4 + H5 + CH₂OBn), 3.99 (br s, 2H, OCH₂Ph), 3.76 (s, 3H, OCH₃), 2.65 (dd, J = 2.5 and 18.0 Hz, 1H, CHH-6), 2.45 (ddd, J = 2.8, 3.7 and 18.0 Hz, 1H, CHH-6), 1.42 (s, 3H, CH₃) and 1.34 (s, 3H, CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.5 (C), 142.6 (C), 138.3 (C), 137.9 (CH), 137.7 (CH), 128.5 (2 × CH), 127.8 (2 × CH), 127.8 (CH), 126.3 (C), 115.2 (CH₂), 114.3 (CH₂), 109.2 (C), 80.2 (C), 77.4 (CH), 73.7 (CH), 72.3 (CH₂), 70.9 (CH₂), 70.3 (CH₂), 52.0 (OCH₃), 28.0 (CH₃), 27.0 (CH₃) and 24.5 (CH₂) ppm. FTIR (film): 1709 (CO) cm⁻¹. MS (ESI) m/z = 437 (MNa⁺). HRMS calcd for C₂₄H₃₀O₆Na (MNa⁺): 437.1935; found, 437.1936.

Methyl (3R,4S,5R)-5-[(2-((tert-butyldimethylsilyloxy)methyl)allyloxy]-3,4-O-isopropilidendioxi-3-vinylcyclohex-1-ene-1-carboxylate (6i, R = CH₂OTBS). It was prepared following the general allylation procedure using 7 (96 mg), Pd₂(dba)₃ (10 mg), dppb (16 mg), 2-((tert-butyldimethylsilyloxy)methyl)allyl methyl carbonate (8i) (118 mg) in THF (1 mL), THF (1.6 mL). Eluent for chromatography = (25 : 75) diethyl ether/hexane. Yield = 43% (72 mg). Colorless oil. [α]²⁰_D = +63.2° (c 1.0, CHCl₃). ¹H NMR (250 MHz, CDCl₃) δ: 6.63 (br s, 1H, H2), 5.89 (dd, J = 10.7 and 17.3 Hz, 1H, HC=CH₂), 5.43 (dd, J = 1.4 and 17.3 Hz, 1H, HC=CHH), 5.18–5.14 (m, 2H, HC=CHH + C=CHH), 5.04 (br s, 1H, C=CHH), 4.10 (br s, 2H, OCH₂), 4.07–3.94 (m, 4H, H4 + H5 + CH₂OTBS), 3.75 (s, 3H, OCH₃), 2.63 (dd, J = 1.9 and 18.0 Hz, 1H, CHH-6), 2.42 (dt, J = 3.3 and 18.0 Hz, 1H, CHH-6), 1.40 (s, 3H, CH₃), 1.33 (s, 3H, CH₃), 0.89 (s, 9H, C(CH₃)₃) and 0.04 (s, 6H, 2 × CH₃) ppm. ¹³C NMR (63 MHz, CDCl₃) δ: 167.5 (C), 145.0 (C), 137.9 (CH), 137.7 (CH), 126.3 (C), 115.2 (CH₂), 112.0 (CH₂), 109.2 (C), 80.2 (C), 77.3 (CH), 73.3 (CH), 70.2 (CH₂), 63.9 (CH₂), 52.1 (OCH₃), 28.0 (CH₃), 27.0 (CH₃), 26.0 (C(CH₃)₃), 24.4 (CH₂), 18.5 (C(CH₃)₃) and −5.3 (2 × CH₃) ppm. FTIR (film): 1716 (CO) cm⁻¹. MS (ESI) m/z = 461 (MNa⁺). HRMS calcd for C₂₃H₃₈O₆SiNa (MNa⁺): 461.2330; found, 461.2330.

General procedure for ring-closing metathesis of compounds 6a–g and 6i

A solution of compounds 6a–g and 6i (1 mmol) and 2^nd generation Grubbs’ catalyst (0.02 mmol) in dry toluene (20 mM), under an inert atmosphere, was heated at 90 °C for 24–48 h. After cooling to room temperature, the mixture was filtered over Celite® and the residue was washed with diethyl ether. The filtrate and the washings were concentrated under reduced pressure and purified by flash chromatography to yield the bicycles 24a–g and 24i.

Methyl (1R,6S,10S)-6,10-O-isopropylidenedioxy-4-methyl-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (24a, R = Me). It was prepared following the general RCM procedure using 6a (100 mg), 2^nd generation Grubbs’ catalyst (5.4 mg), toluene (16 mL). Reaction time = 24 h. Eluent for chromatography = (50 : 50) diethyl ether/hexane. Yield = 63% (57 mg). It was also recovered 18 mg of starting material. Corrected yield = 77%. Colorless oil. [α]²⁰_D = +11.4° (c 1.1, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.70 (q, J = 1.7 Hz, 1H, H7), 5.68 (t, J = 1.7 Hz, 1H, H5), 4.50 (m, 1H, H1), 4.35 (dd, J = 5.0 and 1.7 Hz, 1H, H10), 4.28 (br d, J = 16.8 Hz, 1H, OCHH), 3.97 (d, J = 16.8 Hz, 1H, OCHH), 3.72 (s, 3H, OCH₃), 2.52 (m, 2H, CH₂-9), 1.66 (s, 3H, CH₃), 1.39 (s, 3H, CH₃) and 1.31 (s, 3H, CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.6 (C), 140.0 (C), 138.0 (CH), 125.2 (C), 123.9 (CH), 110.1 (C), 79.8 (C), 75.0 (CH), 70.3 (CH), 68.0 (OCH₂), 51.9 (OCH₃), 28.1 (CH₃), 27.3 (CH₃), 27.3 (CH₂) and 21.9 (CH₃) ppm. FTIR (film): 1798 (CO) cm⁻¹. MS (ESI) m/z = 303 (MNa⁺). HRMS calcd for C₁₅H₂₀O₅Na (MNa⁺): 303.1203; found, 303.1202.

Methyl (1R,6S,10S)-4-ethyl-6,10-O-isopropylidenedioxy-2-oxabiciclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (24b, R = Et). It was prepared following the general RCM procedure using 6b (190 mg), 2^nd generation Grubbs’ catalyst (7 mg), toluene (30 mL). Eluent for chromatography = (30 : 70) diethyl ether/hexane. Reaction time = 48 h. Yield = 47% (82 mg). It was also recovered 95 mg of starting material. Corrected yield = 97%. Colorless oil. [α]²⁰_D = +30.8° (c 1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.67 (m, 1H, H7), 5.63 (m, 1H, H5), 4.46 (m, 1H, H1), 4.35–4.26 (m, 2H, H10 + OCHH-3), 3.99 (d, J = 16.6 Hz, 1H, OCHH-3), 3.70 (m, 3H, OCH₃), 2.51 (m, 2H, CH₂-9), 1.93 (q, J = 7.4 Hz, 2H, CH₂CH₃), 1.37 (s, 3H, CH₃), 1.30 (s, 3H, CH₃) and 0.98 (t, J = 7.3 Hz, 3H, CH₂CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.5 (C), 145.4 (C), 138.0 (CH), 125.0 (C), 122.1 (CH), 110.0 (C), 80.0 (C), 75.0 (CH), 70.2 (CH), 67.3 (CH₂), 51.9 (OCH₃), 28.6 (CH₂), 28.1 (CH₃), 27.3 (CH₃), 27.2 (CH₂) and 12.0 (CH₃) ppm. FTIR (film): 1712 (CO) cm⁻¹. MS (ESI) m/z = 317 (MNa⁺). HRMS calcd for C₁₆H₂₂O₅Na (MNa⁺): 317.1359; found, 317.1359.

Methyl (1R,6S,10S)-6,10-O-isopropylidenedioxy-4-propyl-2-oxabiciclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (24c, R = nPr). It was prepared following the general RCM procedure using 6c (162 mg), 2^nd generation Grubbs’ catalyst (6 mg), toluene (24 mL). Eluent for chromatography = (20 : 80) diethyl ether/hexane. Reaction time = 48 h. Yield = 63% (94 mg). It was also recovered 43 mg of starting material. Corrected yield = 89%. Colorless oil. [α]²⁰_D = +32.0° (c 1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.69 (m, 1H, H7), 5.67 (m, 1H, H5), 4.48 (m, 1H, H1), 4.36 (dd, J = 1.6 and 6.6 Hz, 1H, H10), 4.29 (dd, J = 1.9 and 16.7 Hz, 1H, OCHH-3), 4.01 (d, J = 16.7 Hz, 1H, OCHH-3), 3.73 (m, 3H, OCH₃), 2.53 (m, 2H, CH₂-9), 1.92 (t, J = 7.5 Hz, 2H, CH₂CH₂CH₃), 1.44–1.36 (m, 5H, CH₂CH₂CH₃ + CH₃), 1.33 (s, 3H, CH₃) and 0.88 (t, J = 7.3 Hz, 3H, (CH₂)₂CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.5 (C), 143.8 (C), 137.9 (CH), 125.0 (C), 123.3 (CH), 110.0 (C), 79.9 (C), 74.9 (CH), 70.2 (CH), 67.3 (CH₂), 51.9 (OCH₃), 38.0 (CH₂), 28.1 (CH₃), 27.2 (CH₃), 27.2 (CH₂), 20.8 (CH₂) and 13.8 (CH₃) ppm. FTIR (film): 1717 (CO) cm⁻¹. MS (ESI) m/z = 331 (MNa⁺). HRMS calcd for C₁₇H₂₄O₅Na (MNa⁺): 331.1516; found, 331.1513.

Methyl (1R,6S,10S)-4-butyl-6,10-O-isopropylidenedioxy-2-oxabiciclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (24d, R = nBu). It was prepared following the general RCM procedure using 6d (110 mg), 2^nd generation Grubbs’ catalyst (4 mg), toluene (16 mL). Eluent for chromatography = (15 : 85) diethyl ether/hexane. Reaction time = 24 h. Yield = 54% (54 mg). Colorless oil. [α]²⁰_D = 37.5° (c 1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.68 (m, 1H, H7), 5.65 (br s, 1H, H5), 4.47 (m, 1H, H1), 4.34 (dd, J = 1.7 and 5.0 Hz, 1H, H10), 4.29 (dd, J = 2.2 and 16.7 Hz, 1H, OCHH-3), 4.00 (d, J = 16.7 Hz, 1H, OCHH-3), 3.72 (m, 3H, OCH₃), 2.52 (m, 2H, CH₂-9), 1.92 (t, J = 6.9 Hz, 2H, CH₂(CH₂)₂CH₃), 1.38 (s, 3H, CH₃), 1.36–1.21 (m, 4H, CH₂(CH₂)₂CH₃), 1.31 (s, 3H, CH₃) and 0.87 (t, J = 7.1 Hz, 3H, (CH₂)₃CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.6 (C), 144.1 (C), 138.0 (CH), 125.0 (C), 123.1 (CH), 110.1 (C), 79.9 (C), 75.0 (CH), 70.2 (CH), 67.4 (CH₂), 51.9 (OCH₃), 35.7 (CH₂), 29.8 (CH₂), 28.1 (CH₃), 27.3 (CH₃), 27.2 (CH₂), 22.5 (CH₂) and 14.0 (CH₃) ppm. FTIR (film): 1717 (CO) cm⁻¹. MS (ESI) m/z = 345 (MNa⁺). HRMS calcd for C₁₈H₂₆O₅Na (MNa⁺): 345.1672; found, 345.1670.

Methyl (1R,6S,10S)-4-cyclopropylmethyl-6,10-O-isopropylidenedioxy-2-oxabiciclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (24e, R = CH₂cPr). It was prepared following the general RCM procedure using 6e (105 mg), 2^nd generation Grubbs’ catalyst (19 mg), toluene (15 mL). Eluent for chromatography = (20 : 80) diethyl ether/hexane. Reaction time = 48 h. Yield = 45% (43 mg). It was also recovered 59 mg of starting material. Corrected yield = quant. Colorless oil. [α]²⁰_D = +30.5° (c 1.1, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.69 (m, 1H, H7), 5.82 (m, 1H, H5), 4.48 (m, 1H, H1), 4.38–4.30 (m, 2H, H10 + OCHH-3), 4.05 (d, J = 16.7 Hz, 1H, OCHH-3), 3.72 (m, 3H, OCH₃), 2.52 (m, 2H, CH₂-9), 1.90 (dd, J = 6.4 and 15.8 Hz, 1H, CHH), 1.77 (dd, J = 7.0 and 15.9 Hz, 1H, CHH), 1.40 (s, 3H, CH₃), 1.32 (s, 3H, CH₃), 0.71 (m, 1H, CH₂CH(CH₂)₂), 0.50–0.45 (m, 2H, CHCH₂CH₂) and 0.06–0.03 (m, 2H, CHCH₂CH₂) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.6 (C), 144.0 (C), 138.1 (CH), 125.1 (C), 123.2 (CH), 110.1 (C), 80.0 (C), 75.0 (CH), 70.3 (CH), 67.4 (CH₂), 52.0 (OCH₃), 40.5 (CH₂), 28.1 (CH₃), 27.3 (CH₃), 27.3 (CH₃), 9.0 (CH), 4.8 (CH₂) and 4.7 (CH₂) ppm. FTIR (film): 1718 (CO) cm⁻¹. MS (ESI) m/z = 343 (MNa⁺). HRMS calcd for C₁₈H₂₄O₅Na (MNa⁺): 343.1516; found, 343.1524.

Methyl (1R,6S,10S)-4-(ethoxymethyl)-6,10-O-isopropylidenedioxy-2-oxabiciclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (24f, R = CH₂OEt). It was prepared following the general RCM procedure using 6f (251 mg), 2^nd generation Grubbs’ catalyst (9 mg), toluene (23 mL). Reaction time = 48 h. Eluent for chromatography = (20 : 80) diethyl ether/hexane. Yield = 16% (36 mg). It was also recovered 156 mg of starting material. Corrected yield = 78%. Yellow oil. [α]²⁰_D = +36.9° (c 1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.68 (m, 1H, H7), 5.95 (m, 1H, H5), 4.51 (m, 1H, H1), 4.40 (dd, J = 1.4 and 4.8 Hz, 1H, H10), 4.32 (dd, J = 1.5 and 16.7 Hz, 1H, OCHH-3), 4.16 (d, J = 16.7 Hz, 1H, OCHH-3), 3.84 (sa, 2H, CH₂OEt), 3.74 (s, 3H, OCH₃), 3.43 (q, J = 7.0 Hz, 1H, OCH₂CH₃), 2.55 (m, 2H, CH₂-9), 1.40 (s, 3H, CH₃), 1.33 (s, 3H, CH₃) and 1.18 (t, J = 7.0 Hz, 3H, CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.5 (C), 140.6 (C), 137.4 (CH), 125.9 (CH), 125.6 (C), 110.3 (C), 79.6 (C), 74.8 (CH), 73.1 (CH₂), 70.3 (CH), 66.0 (CH₂), 65.0 (CH₂), 52.0 (OCH₃), 28.1 (CH₃), 27.3 (CH₃), 27.2 (CH₂) and 15.2 (CH₃) ppm. FTIR (film): 1695 (CO) cm⁻¹. MS (ESI) m/z = 347 (MNa⁺). HRMS calcd for C₁₇H₂₄O₆Na (MNa⁺): 347.1465; found, 347.1466.

Preparation of 24f from 24i

A solution of the alcohol 24i (34 mg, 0.10 mmol) in dry DMF (0.2 mL), at 0 °C and under argon, was treated with NaH (6.6 mg, 0.17 mmol, ca. 60% w/w in mineral oil). After 30 min stirring, bromoethane (20 μL, 0.17 mmol) was added, the ice bath was removed and the reaction mixture was stirred for 3 h at room temperature. The reaction mixture was diluted with a mixture of (4 : 1) water/ethyl acetate, the organic layer was separated and the aqueous layer was extracted with ethyl acetate keeping the same proportion. The combined organic extracts were dried (anh. Na₂SO₄), filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography, eluting with (50 : 50) ethyl acetate/hexane, to give compound 24f (12 mg, 31%). It was also recovered 4 mg of starting material. Corrected yield = 57%.

Methyl (1R,6S,10S)-4-(benzyloxymethyl)-6,10-O-isopropylidenedioxy-2-oxabiciclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (24g, R = CH₂OBn). It was prepared following the general RCM procedure using 6g (202 mg), 2^nd generation Grubbs’ catalyst (7 mg), toluene (27 mL). Reaction time = 48 h. Eluent for chromatography = (20 : 80) diethyl ether/hexane. Yield = 26% (53 mg). It was also recovered 138 mg of starting material. Corrected yield = 94%. Yellow oil. [α]²⁰_D = +22.3° (c 1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 7.37–7.26 (m, 5H, 5 × ArH), 6.69 (m, 1H, H7), 5.98 (br s, 1H, H5), 4.51 (m, 1H, H1), 4.46 (s, 2H, CH₂Ph), 4.39 (dd, J = 1.5 and 4.8 Hz, 1H, H10), 4.33 (dd, J = 1.4 and 16.8 Hz, 1H, OCHH-3), 4.18 (d, J = 16.8 Hz, 1H, OCHH-3), 3.89 (sa, 2H, CH₂OBn), 3.74 (s, 3H, OCH₃), 2.55 (m, 2H, CH₂-9), 1.41 (s, 3H, CH₃) and 1.34 (s, 3H, CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.5 (C), 140.4 (C), 137.9 (C), 137.3 (CH), 128.6 (2 × CH), 127.9 (3 × CH), 126.5 (CH), 125.7 (C), 110.3 (C), 79.6 (C), 74.8 (CH), 72.6 (CH₂), 72.5 (CH₂), 70.3 (CH), 65.1 (CH₂), 52.0 (OCH₃), 28.1 (CH₃), 27.3 (CH₃) and 27.2 (CH₂) ppm. FTIR (film): 1716 (CO) cm⁻¹. MS (ESI) m/z = 409 (MNa⁺). HRMS calcd for C₂₂H₂₆O₆Na (MNa⁺): 409.1622; found, 409.1619.

Preparation of 24g from 24i

A solution of the alcohol 24i (31 mg, 0.10 mmol) in dry THF (0.3 mL), at 0 °C and under argon, was treated with NaH (6 mg, 0.15 mmol, ca. 60% w/w in mineral oil). After 30 min stirring, benzyl bromide (20 μL, 0.15 mmol) was added, the ice bath was removed and the reaction mixture was stirred for 3 h at room temperature. Saturated NH₄Cl was added, the organic solvent was removed under reduced pressure and the aqueous solution was extracted with ethyl acetate (×3). The combined organic extracts were dried (anh. Na₂SO₄), filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography, eluting with (25 : 75) ethyl acetate/hexane, to give compound 24g (25 mg, 64%).

Methyl (1R,6S,10S)-4-((tert-butyldimethylsilyloxy)methyl)-6,10-O-isopropylidenedioxy-2-oxabiciclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (24i, R = CH₂OTBS). It was prepared following the general RCM procedure using 6i (133 mg), 2^nd generation Grubbs’ catalyst (9 mg), toluene (15 mL). Reaction time = 48 h. Eluent for chromatography = (20 : 80) diethyl ether/hexane. Yield = 42% (52 mg). Colorless oil. [α]²⁰_D = +33.9° (c 1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.68 (m, 1H, H7), 5.92 (m, 1H, H5), 4.51 (m, 1H, H1), 4.39 (dd, J = 1.6 and 4.8 Hz, 1H, H10), 4.28 (dd, J = 1.4 and 16.7 Hz, 1H, OCHH-3), 4.08 (d, J = 16.7 Hz, 1H, OCHH-3), 4.01 (dd, J = 1.3 and 4.6 Hz, 2H, CH₂), 3.73 (s, 3H, OCH₃), 2.54 (m, 2H, CH₂-9), 1.40 (s, 3H, CH₃), 1.33 (s, 3H, CH₃), 0.89 (s, 9H, C(CH₃)₃), 0.05 (s, 3H, CH₃) and 0.05 (s, 3H, CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.5 (C), 142.7 (C), 137.6 (CH), 125.4 (C), 123.3 (CH), 110.2 (C), 79.8 (C), 74.9 (CH), 70.3 (CH), 65.3 (CH₂), 64.7 (CH₂), 52.0 (OCH₃), 28.1 (CH₃), 27.2 (CH₃), 27.2 (CH₂), 26.0 (C(CH₃)₃), 18.5 (C(CH₃)₃), −5.2 (CH₃) and −5.2 (CH₃) ppm. FTIR (film): 1716 (CO) cm⁻¹. MS (ESI) m/z = 433 (MNa⁺). HRMS calcd for C₂₁H₃₄O₆SiNa (MNa⁺): 433.2017; found, 433.2013.

Methyl (1R,6S,10S)-4-(hydroxymethyl)-6,10-O-isopropylidenedioxy-2-oxabiciclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (24h, R = CH₂OH). A solution of silyl ether 24i (53 mg, 0.13 mmol) in dry THF (1.3 mL), at 0 °C and under inert atmosphere, was treated with TBAF (0.5 mL, ca. 1 M in THF). The reaction mixture was stirred for 50 min and then diluted with ethyl acetate. The organic solution was washed with water (×2), NaHCO₃ (sat) (×2), dried (anh. Na₂SO₄), filtered and concentrated under reduced pressure. The resulting residue was purified by flash chromatography, eluting with (50 : 50) diethyl ether/hexane, to give the alcohol 24h (31 mg, 81%) as a colorless oil. [α]²⁰_D = +37.1° (c 1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.68 (d, J = 1.0 Hz, 1H, H7), 5.97 (br s, 1H, H5), 4.52 (m, 1H, H1), 4.40–4.32 (m, 2H, H10 + OCHH-3), 4.18 (d, J = 16.7 Hz, 1H, OCHH), 4.02 (t, J = 4.3 Hz, 2H, CH₂), 3.74 (s, 3H, OCH₃), 2.55 (m, 2H, CH₂-9), 1.40 (s, 3H, CH₃) and 1.34 (s, 3H, CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.3 (C), 142.9 (C), 137.2 (CH), 125.5 (C), 124.4 (CH), 110.2 (C), 79.5 (C), 74.6 (CH), 70.2 (CH), 65.0 (CH₂), 64.5 (CH₂), 51.9 (CH₃), 28.0 (CH₃), 27.1 (CH₃) and 27.0 (CH₂) ppm. FTIR (film): 3441 (OH) and 1709 (CO) cm⁻¹. MS (ESI) m/z = 319 (MNa⁺). HRMS calcd for C₁₅H₂₀O₆Na (MNa⁺): 319.1152; found, 319.1152.

General procedure for the acetal deprotection in 24a–h

A solution of the acetals 24a–h (1 mmol) in methanol (7 mL) and aqueous HCl (8.6 mL, 6 M) was heated at 60 °C for 6 h. The mixture was cooled and concentrated under reduced pressure. The residue was purified by flash chromatography to yield the diols 25a–h.

Methyl (1R,6S,10S)-6,10-dihydroxy-4-methyl-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (25a, R = Me). It was prepared following the general deprotection procedure using 24a (39 mg), HCl (0.2 mL) and methanol (1 mL). Eluent for chromatography = (50 : 50) ethyl acetate/hexane. Yield = 88% (30 mg). Colorless oil. [α]²⁰_D = −86.6° (c 3.0, CH₃OH). ¹H NMR (300 MHz, CD₃OD) δ: 6.64 (m, 1H, H7), 5.46 (m, 1H, H5), 4.25 (m, 2H, H1 + OCHH), 4.12 (dd, J = 4.9 and 1.6 Hz, 1H, H10), 3.85 (d, J = 16.5 Hz, 1H, OCHH), 2.59 (dt, J = 17.9 and 2.9 Hz, 1H, CHH-9), 2.28 (dd, J = 17.9 and 2.3 Hz, 1H, CHH-9) and 1.68 (s, 3H, CH₃) ppm. ¹³C NMR (75 MHz, CD₃OD) δ: 168.3 (C), 144.0 (CH), 140.2 (C), 139.9 (CH), 129.4 (C), 128.7 (CH), 75.4 (CH), 72.6 (C), 70.6 (CH), 67.5 (CH₂), 28.3 (CH₂) and 22.2 (CH₃) ppm. FTIR (ATR): 3416 (OH) and 1698 (CO) cm⁻¹. MS (ESI) m/z = 263 (MNa⁺). HRMS calcd for C₁₂H₁₆O₅Na (MNa⁺): 263.0890; found, 263.0881.

Methyl (1R,6S,10S)-4-ethyl-6,10-dihydroxy-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (25b, R = Et). It was prepared following the general deprotection procedure using 24b (128 mg), HCl (0.7 mL) and methanol (2.9 mL). Eluent for chromatography = ethyl acetate. Yield = 91% (99 mg). Colorless oil. ¹H NMR (300 MHz, CDCl₃) δ: 6.69 (m, 1H, H7), 5.47 (dd, J = 1.6 and 3.5 Hz, 1H, H5), 4.33 (quint, J = 2.6 Hz, 1H, H1), 4.21–4.15 (m, 2H, H10 + OCHH-3), 3.92 (d, J = 16.4 Hz, 1H, OCHH-3), 3.72 (s, 3H, OCH₃), 3.00 (br s, 2H, 2 × OH), 2.61 (td, J = 2.9 and 18.3 Hz, 1H, CHH-9), 2.40 (dd, J = 2.0 and 18.3 Hz, 1H, CHH-9), 1.97 (qd, J = 1.2 and 7.4 Hz, 2H, CH₂CH₃) and 0.98 (t, J = 7.4 Hz, 3H, CH₃) ppm. ¹³C NMR (63 MHz, CDCl₃) δ: 167.8 (C), 145.3 (C), 138.8 (CH), 127.4 (C), 125.7 (CH), 73.3 (CH), 72.0 (C), 69.8 (CH), 66.3 (CH₂), 52.1 (OCH₃), 29.0 (CH₂), 27.2 (CH₂) and 12.2 (CH₃) ppm. FTIR (film): 3395 (OH) and 1701 (CO) cm⁻¹. MS (ESI) m/z = 277 (MNa⁺). HRMS calcd for C₁₃H₁₈O₅Na (MNa⁺): 277.1046; found, 277.1044.

Methyl (1R,6S,10S)-6,10-dihydroxy-4-propyl-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (25c, R = nPr). It was prepared following the general deprotection procedure using 24c (122 mg), HCl (0.7 mL) and methanol (2.6 mL). Reaction time = 18 h. Eluent for chromatography = (80 : 20) diethyl ether/hexane. Yield = 80% (87 mg). Colorless oil. [α]²⁰_D = −48.1° (c 1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.69 (br s, 1H, H7), 5.48 (d, J = 1.4 Hz, 1H, H5), 4.32 (m, 1H, H1), 4.20–4.14 (m, 2H, H10 + OCHH-3), 3.93 (d, J = 16.4 Hz, 1H, OCHH-3), 3.72 (m, 3H, OCH₃), 3.07 (br s, 2H, 2 × OH), 2.61 (dt, J = 2.9 and 18.3 Hz, 1H, CHH-9), 2.40 (dd, J = 1.7 and 18.2 Hz, 1H, CHH-9), 1.93 (t, J = 7.4 Hz, 2H, CH₂CH₂CH₃), 1.38 (m, 2H, CH₂CH₂CH₃) and 0.87 (t, J = 7.3 Hz, 3H, (CH₂)₂CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.8 (C), 143.6 (C), 138.8 (CH), 127.3 (C), 127.0 (CH), 73.3 (CH), 72.0 (C), 69.7 (CH), 66.2 (CH₂), 52.0 (OCH₃), 38.6 (CH₂), 27.2 (CH₂), 21.0 (CH₂) and 13.8 (CH₃) ppm. FTIR (film): 3412 (OH) and 1700 (CO) cm⁻¹. MS (ESI) m/z = 291 (MNa⁺). HRMS calcd for C₁₄H₂₀O₅Na (MNa⁺): 291.1203; found, 291.1202.

Methyl (1R,6S,10S)-4-butyl-6,10-dihydroxy-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (25d, R = nBu). It was prepared following the general deprotection procedure using 24d (124 mg), HCl (0.7 mL) and methanol (3 mL). Reaction time = 6 h. Eluent for chromatography = (90 : 10) diethyl ether/hexane. Yield = 89% (95 mg). Colorless oil. [α]²⁰_D = −42.3° (c 1.0, CH₃OH). ¹H NMR (300 MHz, CD₃OD) δ: 6.68 (br s, 1H, H7), 5.48 (br s, 1H, H5), 4.31–4.26 (m, 2H, H1 + OCHH-3), 4.16 (br d, J = 4.8 Hz, 1H, H10), 3.94 (d, J = 16.3 Hz, 1H, OCHH-3), 3.77 (s, 3H, OCH₃), 2.63 (td, J = 2.8 and 18.0 Hz, 1H, CHH-9), 2.32 (br d, J = 18.5 Hz, 1H, CHH-9), 2.02 (t, J = 6.5 Hz, 2H, CH₂(CH₂)₂CH₃), 1.44–1.34 (m, 4H, CH₂(CH₂)₂CH₃) and 0.95 (t, J = 6.8 Hz, 3H, (CH₂)₃CH₃) ppm. ¹³C NMR (75 MHz, CD₃OD) δ: 169.2 (C), 144.1 (C), 140.9 (CH), 128.9 (CH), 127.8 (C), 75.1 (CH), 72.5 (C), 70.5 (CH), 66.8 (CH₂), 52.3 (OCH₃), 37.2 (CH₂), 31.1 (CH₂), 28.2 (CH₂), 23.3 (CH₂) and 14.3 (CH₃) ppm. FTIR (film): 3419 (OH) and 1710 (CO) cm⁻¹. MS (ESI) m/z = 305 (MNa⁺). HRMS calcd for C₁₅H₂₂O₅Na (MNa⁺): 305.1359; found, 305.1357.

Methyl (1R,6S,10S)-4-cyclopropylmethyl-6,10-dihydroxy-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (25e, R = CH₂cPr). It was prepared following the general deprotection procedure using 24e (160 mg), HCl (0.8 mL) and methanol (3.3 mL). Reaction time = 18 h. Eluent for chromatography = (90 : 10) diethyl ether/hexane. Yield = 76% (107 mg). White solid. [α]²⁰_D = −44.4° (c 1.0, CHCl₃). Mp: 128.2–128.7 °C. ¹H NMR (300 MHz, CDCl₃) δ: 6.74 (m, 1H, H7), 5.66 (m, 1H, H5), 4.38 (m, 1H, H1), 4.28–4.22 (m, 2H, OCHH-3 + H10), 4.01 (d, J = 16.4 Hz, 1H, OCHH-3), 3.74 (s, 3H, OCH₃), 2.65 (dt, J = 3.0 and 18.3 Hz, 1H, CHH-9), 2.45 (dd, J = 2.1 and 18.3 Hz, 1H, CHH-9), 1.88 (m, 2H, CH₂), 0.78–0.65 (m, 1H, CH₂CH(CH₂)₂), 0.52–0.46 (m, 2H, CHCH₂CH₂), 0.07 (dd, J = 1.3 and 5.0 Hz, 1H, CHCH₂CHH) and 0.04 (dd, J = 1.4 and 4.7 Hz, 1H, CHCH₂CHH) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.8 (C), 143.7 (C), 138.8 (CH), 127.3 (C), 126.8 (CH), 73.3 (CH), 72.0 (C), 69.7 (CH), 66.3 (CH₂), 52.0 (OCH₃), 41.0 (CH₂), 27.2 (CH₂), 9.1 (CH), 4.8 (CH₂) and 4.7 (CH₂) ppm. FTIR (film): 3393 (OH) and 1685 (CO) cm⁻¹. MS (ESI) m/z = 303 (MNa⁺). HRMS calcd for C₁₅H₂₀O₅Na (MNa⁺): 303.1203; found, 303.1200.

Methyl (1R,6S,10S)-4-ethoxymethyl-6,10-dihydroxy-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (25f, R = CH₂OEt). It was prepared following the general deprotection procedure using 24f (49 mg), HCl (0.3 mL) and methanol (1 mL). Reaction time = 11 h. Eluent for chromatography = (70 : 30) ethyl acetate/hexane. Yield = 80% (34 mg). Brown oil. [α]²⁰_D = −35.1° (c 1.0, CHCl₃). ¹H NMR (300 MHz, CDCl₃) δ: 6.70 (br s, 1H, H7), 5.77 (br s, 1H, H5), 4.38 (q, J = 2.3 Hz, 1H, H1), 4.24–4.19 (m, 2H, H10 + OCHH-3), 4.08 (d, J = 16.5 Hz, 1H, OCHH-3), 3.85 (br s, 2H, CH₂OEt), 3.74 (s, 3H, OCH₃), 3.44 (q, J = 7.0 Hz, 2H, OCH₂CH₃), 2.65 (dt, J = 2.9 and 18.4 Hz, 1H, CHH-9), 2.45 (br d, J = 17.9 Hz, 1H, CHH-9) and 1.18 (t, J = 7.0 Hz, 3H, OCH₂CH₃) ppm. ¹³C NMR (75 MHz, CDCl₃) δ: 167.6 (C), 140.2 (C), 138.1 (CH), 129.7 (CH), 127.9 (C), 73.5 (CH), 73.4 (CH₂), 71.8 (C), 69.6 (CH), 66.1 (CH₂), 64.0 (CH₂), 52.1 (OCH₃), 27.2 (CH₂) and 15.2 (CH₃) ppm. FTIR (film): 3406 (OH) and 1709 (CO) cm⁻¹. MS (ESI) m/z = 307 (MNa⁺). HRMS calcd for C₁₄H₂₀O₆Na (MNa⁺): 307.1152; found, 307.1153.

Methyl (1R,6S,10S)-4-benzyloxymethyl-6,10-dihydroxy-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (25g, R = OBn). It was prepared following the general deprotection procedure using 24g (55 mg), HCl (0.2 mL) and methanol (0.9 mL). Reaction time = 12 h. Eluent for chromatography = diethyl ether. Yield = 85% (41 mg). Colorless oil. [α]²⁰_D = −24.2° (c 1.0, MeOH). ¹H NMR (300 MHz, CD₃OD) δ: 7.39–7.27 (m, 5H, 5 × ArH), 6.69 (m, 1H, H7), 5.77 (m, 1H, H5), 4.48 (br s, 2H, CH₂Ph), 4.33–4.27 (m, 2H, H1 + OCHH-3), 4.20 (dd, J = 1.4 and 4.9 Hz, 1H, H10), 4.08 (d, J = 16.4 Hz, 1H, OCHH-3), 3.94 (s, 2H, CH₂OBn), 3.76 (s, 3H, OCH₃), 2.65 (dt, J = 2.9 and 18.1 Hz, 1H, CHH-9) and 2.28 (m, 1H, CHH-9) ppm. ¹³C NMR (75 MHz, CD₃OD) δ: 169.2 (C), 140.6 (C), 140.3 (CH), 139.4 (C), 132.3 (CH), 129.4 (2 × CH), 129.0 (2 × CH), 128.7 (CH), 128.4 (C), 75.3 (CH), 73.8 (CH₂), 73.0 (CH₂), 72.4 (C), 70.3 (CH), 64.6 (CH₂), 52.3 (OCH₃) and 28.2 (CH₂) ppm. FTIR (film): 3406 (OH) and 1709 (CO) cm⁻¹. MS (ESI) m/z = 369 (MNa⁺). HRMS calcd for C₁₉H₂₂O₆Na (MNa⁺): 369.1309; found, 369.1307.

Methyl (1R,6S,10S)-4-hydroxymethyl-6,10-dihydroxy-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylate (25i, R = CH₂OH). It was prepared following the general deprotection procedure using 24i (81 mg), HCl (0.3 mL) and methanol (1.8 mL). Reaction time = 8 h. Eluent for chromatography = ethyl acetate. Yield = 95% (66 mg). Colorless oil. [α]²⁰_D = −147.5° (c 0.8, MeOH). ¹H NMR (300 MHz, CD₃OD) δ: 6.65 (br s, 1H, H7), 5.69 (br s, 1H, H5), 4.29–4.24 (m, 2H, H1 + OCHH-3), 4.16 (m, 1H, H10), 4.01 (d, J = 16.4 Hz, 1H, OCHH-3), 3.95 (d, J = 13.5 Hz, 1H, CHH-OH), 3.89 (d, J = 13.5 Hz, 1H, CHH-OH), 3.72 (s, 3H, OCH₃), 2.60 (dt, J = 2.8 and 18.0 Hz, 1H, CHH-9) and 2.29 (d, J = 18.0 Hz, 1H, CHH-9) ppm. ¹³C NMR (75 MHz, CD₃OD) δ: 169.2 (C), 143.4 (C), 140.5 (CH), 129.6 (CH), 128.2 (C), 75.2 (CH), 72.4 (C), 70.3 (CH), 65.5 (CH₂), 64.4 (CH₂), 52.3 (OCH₃) and 28.2 (CH₂) ppm. FTIR (film): 3372 (OH) and 1697 (CO) cm⁻¹. MS (ESI) m/z = 279 (MNa⁺). HRMS calcd for C₁₂H₁₆O₆Na (MNa⁺): 279.0839; found, 279.0838.

General procedure for the ester hydrolysis in 25a–h

A solution of the esters 25a–h (1 mmol) in THF (10 mL) was treated at room temperature with an aqueous solution of LiOH (1.2 mL, 2.5 M). After stirring for 1 h, water was added and THF was removed under reduced pressure. The aqueous layer was washed with ethyl acetate (×3) and then treated with Amberlite IR-120 (H⁺) until pH 6. The resin was filtered off and washed with MilliQ water. The filtrate and the washings were lyophilized to give acids 4.

(1R,6S,10S)-6,10-Dihydroxy-4-methyl-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylic acid (4a, R = Me). It was prepared following the general basic hydrolysis procedure using 25a (230 mg), LiOH (0.14 mL) and THF (1.2 mL). Yield = 86% (25 mg). White solid. Mp: 216 °C (dec.). [α]²⁰_D = −52° (c 2.5, CH₃OH). ¹H NMR (300 MHz, CD₃OD) δ: 6.63 (br s, 1H, H7), 5.47 (m, 1H, H5), 4.27–4.22 (m, 2H, H1 + OCHH), 4.12 (dd, J = 4.9 and 1.5 Hz, H10), 3.85 (d, J = 16.4 Hz, 1H, OCHH), 2.28 (dt, J = 18.0 and 2.8 Hz, 1H, CHH-9), 2.28 (dd, J = 18.0 and 2.0 Hz, 1H, CHH-9) and 1.68 (s, 3H, CH₃) ppm. ¹³C NMR (75 MHz, CD₃OD) δ: 171.2 (C), 140.2 (CH), 139.9 (C), 129.4 (CH), 128.7 (C), 75.3 (CH), 72.6 (C), 70.6 (CH), 67.5 (CH₂), 28.3 (CH₂) and 22.2 (CH₃) ppm. FTIR (ATR): 3325 (OH) and 1637 (CO) cm⁻¹. MS (ESI) m/z = 225 (M − H). HRMS calcd for C₁₁H₁₃O₅ (M − H): 225.0768; found, 225.0764.

(1R,6S,10S)-4-Ethyl-6,10-dihydroxy-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylic acid (4b, R = Et). It was prepared following the general basic hydrolysis procedure using 25b (39 mg), LiOH (0.18 mL) and THF (1.5 mL). Yield = 99% (35 mg). White solid. Mp: 216 °C (dec.). [α]²⁰_D = −24.7° (c 1.0, MeOH). ¹H NMR (300 MHz, D₂O) δ: 6.57 (s, 1H, H7), 5.36 (m, 1H, H5), 4.23–4.17 (m, 2H, H1 + OCHH), 4.07 (dd, J = 1.2 and 4.8 Hz, 1H, H10), 3.81 (d, J = 16.4 Hz, 1H, OCHH), 2.49 (dt, J = 2.8 and 18.1 Hz, 1H, CHH-9), 2.18 (dd, J = 1.3 and 18.0 Hz, 1H, CHH-9), 1.89 (q, J = 7.4 Hz, 2H, CH₂) and 0.91 (t, J = 7.4 Hz, 3H, CH₃) ppm. ¹³C NMR (75 MHz, D₂O) δ: 170.8 (C), 145.5 (C), 140.8 (CH), 128.1 (C), 127.8 (CH), 75.2 (CH), 72.6 (C), 70.4 (CH), 66.8 (CH₂), 29.9 (CH₂), 28.2 (CH₂) and 12.7 (CH₃) ppm. FTIR (film): 3365 (OH) and 1688 (CO) cm⁻¹. MS (ESI) m/z = 240 (M − H). HRMS calcd for C₁₂H₁₅O₅ (M − H): 239.0925; found, 239.0921.

(1R,6S,10S)-6,10-Dihydroxy-4-propyl-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylic acid (4c, R = nPr). It was prepared following the general basic hydrolysis procedure using 25c (58 mg), LiOH (0.9 mL) and THF (2.2 mL). Yield = 99% (55 mg). White foam. [α]²⁰_D = −38.1° (c 1.0, H₂O). ¹H NMR (300 MHz, D₂O) δ: 6.72 (br s, 1H, H7), 5.47 (br s, 1H, H5), 4.38–4.27 (m, 3H, H1 + H10 + OCHH-3), 3.93 (d, J = 16.6 Hz, 1H, OCHH-3), 2.56 (br d, J = 18.6 Hz, 1H, CHH-9), 2.29 (d, J = 18.5 Hz, 1H, CHH-9), 1.95 (t, J = 7.2 Hz, 2H, CH₂CH₂CH₃), 1.35 (m, 2H, CH₂CH₂CH₃) and 0.82 (t, J = 7.3 Hz, 3H, (CH₂)₂CH₃) ppm. ¹³C NMR (75 MHz, D₂O) δ: 173.2 (C), 146.4 (C), 141.9 (CH), 129.6 (C), 129.1 (CH), 75.8 (CH), 74.3 (C), 71.1 (CH), 68.0 (CH₂), 40.4 (CH₂), 29.2 (CH₂), 22.9 (CH₂) and 15.4 (CH₃) ppm. FTIR (film): 3392 (OH) and 1688 (CO) cm⁻¹. MS (ESI) m/z = 253 (M − H). HRMS calcd for C₁₃H₁₇O₅ (M − H): 253.1081; found, 253.1081.

(1R,6S,10S)-4-Butyl-6,10-dihydroxy-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylic acid (4d, R = nBu). It was prepared following the general basic hydrolysis procedure using 25d (87 mg), LiOH (1.2 mL) and THF (3 mL). Yield = 98% (82 mg). White solid. Mp: 129.1–131.0 °C. [α]²⁰_D = −20.3° (c 1.0, H₂O). ¹H NMR (300 MHz, D₂O) δ: 6.52 (br s, 1H, H7), 5.47 (br s, 1H, H5), 4.39–4.32 (m, 2H, H1 + OCHH-3), 4.25 (dd, J = 1.8 and 5.0 Hz, 1H, H10), 3.93 (br d, J = 16.5 Hz, 1H, OCHH-3), 2.57 (td, J = 3.0 and 18.6 Hz, 1H, CHH-9), 2.26 (br d, J = 18.8 Hz, 1H, CHH-9), 1.98 (t, J = 6.8 Hz, 2H, CH₂(CH₂)₂CH₃), 1.36–1.18 (m, 4H, CH₂(CH₂)₂CH₃) and 0.83 (t, J = 7.1 Hz, 3H, (CH₂)₃CH₃) ppm. ¹³C NMR (75 MHz, D₂O) δ: 172.7 (C), 143.1 (C), 136.6 (CH), 129.5 (C), 127.0 (CH), 73.3 (CH), 71.8 (C), 68.7 (CH), 65.3 (CH₂), 35.6 (CH₂), 29.3 (CH₂), 27.2 (CH₂), 21.7 (CH₂) and 13.2 (CH₃) ppm. FTIR (film): 3367 (OH) and 1691 (CO) cm⁻¹. MS (ESI) m/z = 267 (M − H). HRMS calcd for C₁₄H₁₉O₅ (M − H): 267.1238; found, 267.1235.

(1R,6S,10S)-4-Cyclopropylmethyl-6,10-dihydroxy-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylic acid (4e, R = CH₂cPr). It was prepared following the general basic hydrolysis procedure using 25e (44 mg), LiOH (0.5 mL) and THF (1.4 mL). Yield = 99% (37 mg). White foam. [α]²⁰_D = −14.8° (c 1.0, H₂O). ¹H NMR (300 MHz, D₂O) δ: 6.43 (m, 1H, H7), 5.62 (m, 1H, H5), 4.42–4.36 (m, 2H, H1 + OCHH-3), 4.26 (dd, J = 1.6 and 4.9 Hz, 1H, H10), 3.98 (d, J = 16.5 Hz, 1H, OCHH-3), 2.57 (td, J = 2.9 and 18.7 Hz, 1H, CHH-9), 2.27 (d, J = 18.5 Hz, 1H, CHH-9), 1.87 (d, J = 6.8 Hz, 2H, CH₂), 0.73 (m, 1H, CH₂CH(CH₂)₂), 0.48–0.42 (m, 2H, CHCH₂CH₂) and 0.04 (m, 2H, CH₂) ppm. ¹³C NMR (75 MHz, D₂O) δ: 174.3 (C), 143.1 (C), 134.7 (CH), 131.3 (C), 126.9 (CH), 73.5 (CH), 72.0 (C), 68.7 (CH), 65.5 (CH₂), 40.1 (CH₂), 27.6 (CH₂), 8.5 (CH), 3.8 (CH₂) and 3.7 (CH₂) ppm. FTIR (film): 3286 (OH) and 1680 (CO) cm⁻¹. MS (ESI) m/z = 265 (M − H). HRMS calcd for C₁₄H₁₇O₅ (M − H): 265.1081; found, 265.1079.

(1R,6S,10S)-4-Ethoxymethyl-6,10-dihydroxy-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylic acid (4f, R = CH₂OEt). It was prepared following the general basic hydrolysis procedure using 25f (39 mg), LiOH (0.6 mL) and THF (1.4 mL). Reaction time = 30 min. Yield = 45% (17 mg). White foam. [α]²⁰_D = −18.0° (c 1.0, H₂O). ¹H NMR (300 MHz, D₂O) δ: 6.71 (br s, 1H, H7), 5.82 (br s, 1H, H5), 4.45 (m, 1H, H1), 4.35 (m, 2H, H10 + OCHH-3), 4.07 (d, J = 16.5 Hz, 1H, OCHH-3), 3.96 (s, 2H, CH₂OEt), 3.53 (q, J = 7.0 Hz, 2H, OCH₂CH₃), 2.65 (dt, J = 2.6 and 18.8 Hz, 1H, CHH-9), 2.36 (br d, J = 18.5 Hz, 1H, CHH-9) and 1.18 (t, J = 7.0 Hz, 3H, OCH₂CH₃) ppm. ¹³C NMR (75 MHz, D₂O) δ: 171.0 (C), 139.0 (C), 137.7 (CH), 130.7 (CH), 128.4 (C), 73.4 (CH), 72.4 (CH₂), 71.5 (C), 68.2 (CH), 65.5 (CH₂), 63.3 (CH₂), 26.6 (CH₂) and 14.0 (CH₃) ppm. FTIR (film): 3361 (OH) and 1689 (CO) cm⁻¹. MS (ESI) m/z = 269 (M − H). HRMS calcd for C₁₃H₁₇O₆ (M − H): 269.1031; found, 269.1030.

(1R,6S,10S)-4-Benzyloxymethyl-6,10-dihydroxy-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylic acid (4g, R = CH₂OBn). It was prepared following the general basic hydrolysis procedure using 25g (58 mg), LiOH (0.7 mL) and THF (1.7 mL). Reaction time = 4 h. Yield = 99% (56 mg). White solid. Mp: 186 °C (dec.). [α]²⁰_D = −5.5° (c 1.0, H₂O). ¹H NMR (300 MHz, D₂O) δ: 7.38 (m, 5H, 5 × ArH), 6.35 (br s, 1H, H7), 5.80 (br s, 1H, H5), 4.48 (br s, 2H, CH₂Ph), 4.37 (m, 1H, H1), 4.29 (d, J = 16.5 Hz, 1H, OCHH-3), 4.23 (d, J = 4.3 Hz, 1H, H10), 4.02 (d, J = 16.5 Hz, 1H, OCHH-3), 3.97 (br s, 2H, CH₂OBn), 2.59 (dt, J = 2.7 and 18.7 Hz, 1H, CHH-9) and 2.28 (d, J = 18.5 Hz, 1H, CHH-9) ppm. ¹³C NMR (75 MHz, D₂O) δ: 175.0 (C), 137.8 (C), 137.1 (C), 133.1 (C), 132.7 (CH), 132.2 (CH), 128.6 (2 × CH), 128.5 (2 × CH), 128.2 (CH), 73.7 (CH), 72.5 (CH₂), 71.8 (C), 71.6 (CH₂), 68.4 (CH), 63.1 (CH₂) and 27.7 (CH₂) ppm. FTIR (film): 3286 (OH) and 1681 (CO) cm⁻¹. MS (ESI) m/z = 331 (M − H). HRMS calcd for C₁₈H₁₉O₆ (M − H): 331.1187; found, 331.1185.

C₁₄H₁₇O₅ (M − H): 265.1081; found, 265.1079.

(1R,6S,10S)-4-Hydroxylmethyl-6,10-dihydroxy-2-oxabicyclo[4.3.1]deca-4(Z),7-diene-8-carboxylic acid (4h, R = CH₂OH). It was prepared following the general basic hydrolysis procedure using 25h (58 mg), LiOH (0.9 mL) and THF (2.3 mL). Reaction time = 8 h. Yield = 99% (55 mg). White solid. Mp: 117.3–119.2 °C. [α]²⁰_D = −28.1° (c 1.0, H₂O). ¹H NMR (300 MHz, D₂O) δ: 6.36 (br s, 1H, H7), 5.59 (br s, 1H, H5), 4.26–4.19 (m, 2H, H1 + OCHH-3), 4.14 (m, 1H, H10), 3.89–3.77 (m, 3H, OCHH-3 + CH₂OH), 2.45 (dt, J = 2.8 and 18.8 Hz, 1H, CHH-9) and 2.15 (br d, J = 18.6 Hz, 1H, CHH-9) ppm. ¹³C NMR (75 MHz, D₂O) δ: 175.4 (C), 143.8 (C), 138.2 (CH), 132.8 (C), 131.0 (CH), 76.1 (CH), 74.2 (C), 70.9 (CH), 66.4 (CH₂), 65.6 (CH₂) and 29.7 (CH₂) ppm. FTIR (film): 3349 (OH) and 1688 (CO) cm⁻¹. MS (ESI) m/z = 241 (M − H). HRMS calcd for C₁₁H₁₃O₆ (M − H): 241.0718; found, 241.0719.

(1R,4S,6S,10S)-(5S) and (1R,4R,6S,10S)-6,10-dihydroxy-4-methyl-2-oxabicyclo[4.3.1]dec-7-ene-8-carboxylic acid (5R). A suspension of the alkene 25a (57 mg, 0.20 mmol), Rosenmund catalyst (10 mg, 5 wt% loading) and a few drops of pyridine in methanol (5 mL) was shaken under a hydrogen atmosphere at room temperature for 7 h. The mixture was filtered over Celite® and the residue was washed with methanol. The filtrate and washings were evaporated under reduced pressure. A solution of the resulting oil (63 mg) in ethanol (1.5 mL) and aqueous HCl (0.4 mL, 6 M) was heated at 60 °C for 6 h. The mixture was cooled and concentrated under reduced pressure. A solution of the resulting oil (50 mg) in THF (2 mL) was treated at room temperature with an aqueous solution of LiOH (1.3 mL, 0.63 mmol, 0.5 M). After stirring for 4 h, water was added and THF was removed under reduced pressure. The aqueous layer was washed with ethyl acetate (×3) and it was then treated with Amberlite IR-120 (H⁺) until pH 6. The resin was filtered off and washed with milliQ water. The filtrate and the washings were lyophilized to give acids 5S and 5R (48 mg, 99%) as a mixture of epimers in C4. Both compounds were separated by HPLC using semipreparative column (Phenomenex Luna5u, 250 × 10 mm, C18), eluting with a gradient of acetonitrile–water [(1) 0–5 min (5 : 95 → 10 : 90) CH₃CN/H₂O; (2) 5–20 min (10 : 90 → 20 : 80) CH₃CN/H₂O], at a flow rate of 3.5 mL min⁻¹.

Data for 5R. Yield = 45%. Retention time: 16.2 min. Mp: 192.1–193.2 °C. [α]²⁰_D = +7° (c 0.5, H₂O). ¹H NMR (500 MHz, D₂O) δ: 6.70 (br s, 1H, H7), 4.42 (m, 1H, H1), 4.24 (m, 1H, H10), 3.56 (dd, J = 12.9 and 10.1 Hz, 1H, OCHH), 3.47 (m, 1H, OCHH), 2.56 (dt, J = 18.3 and 3.0 Hz, 1H, CHH-9), 2.26 (dd, J = 18.1 and 2.0 Hz, 1H, CHH-9), 2.02 (ddd, J = 12.9, 4.3 and 1.6 Hz, 1H, CHH-5), 1.66–1.58 (m, 1H, H4), 1.43 (t, J = 12.9 Hz, 1H, CHH-5) and 0.83 (d, J = 6.9 Hz, 3H, CH₃) ppm. ¹³C NMR (100 MHz, D₂O) δ: 170.5 (C), 140.5 (CH), 129.2 (C), 73.4 (CH), 71.4 (C), 69.3 (OCH₂), 68.7 (CH), 45.3 (CH₂), 31.4 (CH), 27.2 (CH₂) and 16.8 (CH₃) ppm. FTIR (ATR): 3376 (OH) and 1686 (CO) cm⁻¹. MS (ESI) m/z = 227 (M − H). HRMS calcd for C₁₁H₁₅O₅ (M − H): 227.0925; found, 227.0922.

Data for 5S. Yield = 45%. Retention time: 16.6 min. Mp: 179.4–180.9 °C. [α]²⁰_D = −22° (c 0.6, H₂O). ¹H NMR (500 MHz, D₂O) δ: 6.77 (br s, 1H, H7), 4.36 (dt, J = 4.0 and 1.9 Hz, 1H, H1), 4.23 (dd, J = 4.6 and 1.7 Hz, 1H, H10), 3.82 (dd, J = 13.0 and 3.0 Hz, 1H, CHH-3), 3.42 (dd, J = 13.0 and 5.5 Hz, 1H, CHH-3), 2.62 (ddd, J = 18.9, 4.0 and 2.8 Hz, 1H, CHH-9), 2.32 (d, J = 18.9 Hz, 1H, CHH-9), 2.10–2.04 (m, 1H, H4), 1.96 (dd, J = 14.3 and 6.4 Hz, 1H, CHH-5), 1.81 (dd, J = 14.3 and 5.9 Hz, 1H, CHH-5) and 0.91 (d, J = 7.2 Hz, 3H, CH₃) ppm. ¹³C NMR (100 MHz, D₂O) δ: 170.5 (C), 142.8 (CH), 127.9 (C), 73.1 (CH), 71.5 (C), 69.3 (CH), 67.6 (OCH₂), 42.8 (CH₂), 30.9 (CH), 26.7 (CH₂) and 18.5 (CH₃) ppm. FTIR (ATR): 3396 (OH) and 1690 (CO) cm⁻¹. MS (ESI) m/z = 227 (M − H). HRMS calcd for C₁₁H₁₅O₅ (M − H): 227.0925; found, 227.0928.

Docking studies

They were carried out using the GOLD 5.2.2 program and the enzyme coordinates found in the crystal structures of Hp-SK in complex with shikimate-3-phosphate and ADP (PDB entry 3MUF,¹⁵ 2.3 Å) and of Mt-SK in complex with 2 and ADP (PDB entry 4BQS,¹³ 2.15 Å). Ligand geometries were minimized using the AM1 Hamiltonian as implemented in the program Gaussian 09 ²⁰ and used as MOL2 files. Each ligand was docked in 25 independent genetic algorithm (GA) runs, and for each of these a maximum number of 100 000 GA operations were performed on a single population of 50 individuals. Operator weights for crossover, mutation and migration in the entry box were used as default parameters (95, 95, and 10, respectively), as well as the hydrogen bonding (4.0 Å) and van der Waals (2.5 Å) parameters. The position of shikimate-3-phosphate and compound 2 present in the aforementioned PDB files were used to define the active-site and the radius was set to 8 Å. All crystallographic water molecules and the aforementioned ligands were removed for docking. The “flip ring corners” flag was switched on, while all the other flags were off. The GOLD scoring function was used to rank the ligands in order to fitness. The molecular graphics program PyMOL was employed for visualization and depicting ligand/protein structures.²¹

Molecular dynamics simulation studies

Ligand minimization. Ligand geometries were minimized using a restricted Hartree–Fock (RHF) method and a 6-31G(d) basis set, as implemented in the ab initio program Gaussian 09. The resulting wavefunctions were used to calculate electrostatic potential-derived (ESP) charges employing the restrained electrostatic potential (RESP)²² methodology, as implemented in the assisted model building with energy refinement (AMBER)²³ suite of programs. The missing bonded and non-bonded parameters were assigned, by analogy or through interpolation, from those already present in the AMBER database (GAFF).^20,24

Generation and minimization of ternary complexes. Simulations of SK/ATP/Mg²⁺/ligand complexes were carried out using the highest score solution obtained by docking and the enzyme geometries used in those docking studies, as described above. Computation of the protonation state of titratable groups at pH 7.0 was carried out using the H⁺⁺ Web server.²⁵ Addition of hydrogen and molecular mechanics parameters from the ff14SB and GAFF force fields, respectively, were assigned to the protein and the ligands using the LEaP module of AMBER Tools 17.^26,27 ATP and Mg²⁺ parameters used with the AMBER force field were included.^28,29 All systems were minimized in four stages: (a) initial minimization of the ligand and the closest residues of the SB domain (500 steps, first half using steepest descent and the rest using conjugate gradient); (b) minimization of the solvent and ions (5000 steps, first half using steepest descent and the rest using conjugate gradient); (c) minimization of the side chains, waters and ions (5000 steps, first half using steepest descent and the rest using conjugate gradient); (d) final minimization of the whole system (5000 steps, first half using steepest descent and the rest using conjugate gradient). A positional restraint force of 50 kcal mol⁻¹ Å⁻² was applied to those unminimized atoms during the first three stages (a–c). The complex was immersed in a truncated octahedron of ∼5200 TIP3P water molecules and neutralized by addition of chloride (Mt-SK) or sodium (Hp-SK) ions.^30,31

Simulations. MD simulations were performed using the pmemd.cuda_SPFP³² module from the AMBER 16 suite of programs. Periodic boundary conditions were applied and electrostatic interactions were treated using the smooth particle mesh Ewald method (PME)³³ with a grid spacing of 1 Å. The cutoff distance for the non-bonded interactions was 9 Å. The SHAKE algorithm³⁴ was applied to all bonds containing hydrogen, using a tolerance of 10⁻⁵ Å and an integration step of 2.0 fs. The minimized system was then heated at 300 K at 1 atm by increasing the temperature from 0 K to 300 K over 100 ps and by keeping the system at 300 K another 100 ps. A positional restraint force of 50 kcal mol⁻¹ Å⁻² was applied to all α carbons during the heating stage. An equilibration of the system at constant volume (100 ps with positional restraints of 5 kcal mol⁻¹ Å⁻² to α alpha carbons) and constant pressure (another 100 ps with positional restraints of 5 kcal mol⁻¹ Å⁻² to α alpha carbons) were performed. The positional restraints were gradually reduced from 5 to 1 mol⁻¹ Å⁻² (5 steps, 100 ps each), and the resulting systems were allowed to equilibrate further (100 ps). Unrestrained MD simulations were carried out for 100 ns. System coordinates were collected every 10 ps for further analysis.

Abbreviations

SK	Shikimate kinase
Hp-SK	Shikimate kinase from Helicobacter pylori
Mt-SK	Shikimate kinase from Mycobacterium tuberculosis
MD	Molecular dynamics
PDB	Protein data bank

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Financial support from the Spanish Ministry of Economy and Competiveness (SAF2016-75638-R), the Xunta de Galicia [Centro singular de investigación de Galicia accreditation 2016–2019 (ED431G/09) and ED431B 2018/04], and the European Union (European Regional Development Fund – ERDF) is gratefully acknowledged. MP and EL thank the Xunta de Galicia for their respective predoctoral and postdoctoral fellowships. We are grateful to the Centro de Supercomputación de Galicia (CESGA) for use of computational facilities.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Full details of the experimental procedures for the synthesis of carbonates 8, inhibition assays and extra figures. See DOI: 10.1039/c9qo00562e

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