Targeting the Motion of Shikimate Kinase: Development of Competitive Inhibitors that Stabilize an Inactive Open Conformation of the Enzyme

Article abstract of DOI:10.1021/acs.jmedchem.6b00483

The large conformational changes observed by Molecular Dynamics simulation studies on the product release in the LID and shikimic acid binding (SB) domains of the shikimate kinase (SK) enzyme have been exploited in the development of reversible competitive inhibitors against SK from Mycobacterium tuberculosis and Helicobacter pylori. This enzyme is a recognized target for antibiotic drug discovery. The reported C5-substituted shikimic acid analogues interact with the dynamic apolar pocket that surrounds the C4 and C5 hydroxyl groups of the natural substrate, cause the opening of the LID and SB domains, and capture the essential arginine far from the ATP binding site as required for catalysis. The 3-nitrobenzyl 3e and 5-benzothiophenyl derivatives 3i proved to be the most potent inhibitors. An ester prodrug of 3i was the most efficient derivative in achieving good in vitro activity against H. pylori, having a MIC value of 4 μg/mL.

Full text of DOI:10.1021/acs.jmedchem.6b00483

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Article
Targeting the Motion of Shikimate Kinase: Development of Competitive
Inhibitors that Stabilize an Inactive Open Conformation of the Enzyme
Verónica Prado, Emilio Lence, Maria Maneiro, Juan C. Vázquez-Ucha, Alejandro
Beceiro, Paul Thompson, Alastair R. Hawkins, and CONCEPCION GONZALEZ-BELLO
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00483 • Publication Date (Web): 11 May 2016
Downloaded from http://pubs.acs.org on May 14, 2016
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Targeting the Motion of Shikimate Kinase:
Development of Competitive Inhibitors that Stabilize
an Inactive Open Conformation of the Enzyme
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a
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Verónica Prado, Emilio Lence, María Maneiro, Juan C. Vázquez-Ucha, Alejandro Beceiro,
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Paul Thompson, Alastair R. Hawkins, and Concepción González-Bello*
a
Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS) and
Departamento de Química Orgánica, Universidade de Santiago de Compostela, 15782 Santiago de
Compostela, Spain.
b
Servicio de MicrobioloxíaꢀINIBIC, Complexo Hospitalario Universitario A Coruña (CHUAC),
1
5006 A Coruña, Spain.
c
Institute of Cell and Molecular Biosciences, Medical School, University of Newcastle upon Tyne,
Newcastle upon Tyne NE2 4HH, UK.
CORRESPONDING AUTHOR ADDRESS. Dr. Concepción GonzálezꢀBello, Centro Singular de
Investigación en Química Biolóxica e Materiais Moleculares (CIQUS), Universidade de Santiago de
Compostela, calle Jenaro de la Fuente s/n, 15782 Santiago de Compostela, Spain. FAX: +34 881
8
15704; Phone: +34 881 815726.
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ABSTRACT
The large conformational changes observed by Molecular Dynamics simulation studies on the
product release in the LID and shikimic acid binding (SB) domains of the shikimate kinase (SK)
enzyme have been exploited in the development of reversible competitive inhibitors against SK from
M. tuberculosis and H. pylori. This enzyme is is a recognized target for antibiotic drug discovery.
The reported C5ꢀsubstituted shikimic acid analogs interact with the dynamic apolar pocket that
surrounds the C4 and C5 hydroxyl groups of the natural substrate, cause the opening of the LID and
SB domains, and capture the essential arginine far from the ATP binding site as required for
catalysis. The 3ꢀnitrobenzyl 3e and 5ꢀbenzothiophenyl derivatives 3i proved to be the most potent
inhibitors. An ester prodrug of 3i was the most efficient derivative in achieving good in vitro activity
against H. pylori, having a MIC value of 4 µg/mL.
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INTRODUCTION
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Antibiotics are probably the most successful drugs to be developed in the history of Medicine. The
discovery of penicillin in 1928 and the wide therapeutic arsenal developed thereafter have saved the
lives of millions of people. Unfortunately, this enormous success was soon accompanied by the ever
increasing emergence of drug resistance in bacteria, a problem that has become one of the most
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important public health issues of the early 21st century. In a recent study it was demostrated that
antibiotic resistance is a natural phenomenon that predates the golden age of antibiotic therapy, but
the inappropriate and excessive use of these drugs in medicine, veterinary medicine and agriculture
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over the years has caused the antibiotic resistance problem to reach current levels. It is therefore an
urgent matter to develop novel and alternative therapies, to identify unexplored bacterial targets and
to gain a detailed knowledge of the mechanism of action and binding determinants of those targets in
order to develop effective inhibitors.
One of the most successful strategies for the development of new antibiotics consists on disambling
essential process in bacteria. However, most of the antibiotics in clinical use target a very small
number of key bacterial functions and resistance to them is widespread and well known. Therefore,
the search for unexplored bacterial functions appears to be a good option for the development of
novel antimicrobial agents with a new mechanism of action. In bacteria, there is a biosynthetic route,
9,10
namely the shikimic acid pathway, that have attracted much interest for antibiotic drug discovery.
By this route, the aromatic amino acids and other aromatic compounds (folates, ubiquinone and
vitamins E and K) are biosynthesized. The fact that the enzymes involved in this pathway are
essential in certain important microorganisms but absent in mammals makes them attractive targets
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for the development of antimicrobial agents. Our recent work has focused on the development of
inhibitors of shikimate kinase enzyme (SK, EC 2.7.1.71, aroK gene), which catalyzes the fifth step
9
of the shikimic acid pathway. SK is considered to be a good target for relevant pathogenic bacteria.
That is the case of the hard to treat infections caused by Mycobacterium tuberculosis, which is the
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causative agent of tuberculosis, Helicobacter pylori, which is responsible for gastric and duodenal
ulcers, and Pseudomonas aeruginosa, which is one of leading causes of nosocomial infections and
inherently resistant to most antibiotics. This enzyme is also essential in Acinetobacter baylyi,
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Escherichia coli, Haemophilus influenza, Campylobacter jejuni and Francisella novicida. By
inhibition of the SK enzyme, new antibiotics active against diverse and relevant bacterial diseases
would therefore be achieved. Diverse compounds that target the SK enzyme have been discovered by
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screening (Figure 1). For example, Simithy et al. used LCꢀMS and a library of about 400
antimycobacterial compounds, to identify several compounds with IC50 values in the micromolar
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range that inhibit SK from M. tuberculosis (MtꢀSK). Moreover, Han et al. discovered by highꢀ
throughput screening of a library of about 3000 compounds two inhibitors of SK from Helicobacter
pylori (HpꢀSK) with IC50 values also in the micromolar range.
SK catalyzes the conversion of shikimic acid (1) into shikimateꢀ3ꢀphosphate (2) by using ATP as a
cofactor (Scheme 1). NMR and computational studies have shown that γꢀphosphate of ATP is
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transferred to 1 by a dissociative mechanism via the formation of a metaphosphate intermediate.
The latter is then capturated by the C3 hydroxyl group in 1, which seems to be the rateꢀlimiting step
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of the reaction. Hartmann et al. also suggested that Asp33/Asp34 (in H. pylori and M. tuberculosis,
respectively) would be involved in the deprotonation of the C3 hydroxyl group in 1 for nucleophilic
attack. SK is a magnesiumꢀdependent enzyme that has two recognition centers, i.e., one for shikimic
acid (1) and another for the cofactor (ATP), with different key interactions in both cases. SK has
three important regions: (1) the ATP/ADP binding site, which is composed by the CORE domain and
the Pꢀloop; (2) the LID domain, which is the substrateꢀcovering loop that closes over the shikimic
acid binding site for catalysis and contains the essential Arg116/Arg117 (H. pylori and M.
tuberculosis, respectively); and (3) the substrate binding (SB) domain, which is involved in the
shikimic acid binding (Figure S1). The SK enzyme is amazingly designed to recognize an unstable
conformation for a cyclohexene ring with two of the three hydroxyl groups in an axial arrangement.
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The stereospecific phosphorylation is achieved by controlling the equatorial disposition of the C3
hydroxyl group in 1 through a strong control of the diaxial arrangement of the C4 and C5 hydroxyl
groups. The available crystal structures show that shikimic acid (1) is anchored to the active site (SB
domain) by a salt bridge with a conserved arginine (Arg132/Arg136 in H. pylori and M. tuberculosis,
respectively), a double hydrogen bond between the side chain of a conserved aspartate
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(Asp33/Asp34 in H. pylori and M. tuberculosis, respectively) and the C3 and C4 hydroxyl groups,
and a hydrogen bond between the C3 hydroxyl group and the NH main chain amide of a conserved
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glycine (Gly81/Gly80 in H. pylori and M. tuberculosis, respectively).
In addition, the axial
disposition of the C4 hydroxyl group is also controlled by hydrogen bonding with a structural water
molecule (WAT1), which is anchored in the bottom of the active site by three conserved residues
(Glu60/Glu61, Arg57/Arg58 and Gly81/Gly80 in H. pylori and M. tuberculosis, respectively). Two
water molecules that interact by hydrogen bonding with the residues located in the LID fix the
position of the C5 hydroxyl group. The substrate is surrounded by an apolar pocket generated by
several lipophilic residues located in αꢀhelices α2, α3 and α5 (SB domain) in such a way that it is
isolated from the solvent environment.
Structural and Molecular Dynamics (MD) simulation studies have shown that the catalysis only takes
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place if: (1) the LID and SB domains are tidily closed − under this arrangement, (a) the
guanidinium group of the essential arginine, which is located in the LID domain, is in close contact
of the γꢀphosphate of ATP for its activation and (b) the two substrates are located in close proximity
and isolated from the solvent environment for the reaction; and (2) the LID and SB domains are
opened for product release − the conformational changes of these domains allow that the essential
and conserved arginines be able to break the favorable interactions that anchor the product to the
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active site.
Reasoning that closed forms of the LID and SB domains are required for catalysis, we decided to
explore the possible inhibition of the SK enzyme by blocking the appropriate closed form of those
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domains. To this end, we report here the synthesis and inhibitory properties against MtꢀSK and Hpꢀ
SK of several shikimic acid analogs, namely compounds 3–6, that incorporate diverse aromatic
moieties at the C5 hydroxyl group of shikimic acid (1) (Figure 2). The design of the reported
inhibitors was based on: (1) the dynamic behavior of the SB and LID domains observed during
product release by MD simulations studies; (2) the role and the type of residues located in the SB
domain in order to achieve the interaction with this `dynamic apolar´ pocket; and (3) the crystal
structure of E114A HpꢀSK variant enzyme in complex with the naphthalene derivative 7 (PDB entry
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N2E, 2.53 Å) described by Cheng et al. (Figure 3). This structure reveals that this aromatic ligand,
which has an IC value of 4.9 µM against HpꢀSK, prevents closure of the active site for catalysis by
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causing a large conformational change in the LID domain with the subsequent inappropriate
disposition of the essential arginine. The shikimic acid derivatives described here incorporate diverse
Oꢀbenzyl groups (compounds 3), and 1,2,3ꢀtriazole groups (compounds 4–5). In addition,
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considering that (3S)ꢀ3ꢀaminoshikimic acid proved to be a competitive reversible inhibitor of Mtꢀ
SK with a K of 65 µM, we also studied the effect of replacing the C3 hydroxyl group in the Oꢀ
i
benzyl derivatives 3 by an amino group, namely compounds 6. The binding modes of the reported
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compounds with the two SK enzymes were initially studied using GOLD 5.2 and then further
analyzed by MD simulation studies. The results of inhibition studies, along with the computational
data, allowed us to understand the binding differences between the two enzymes. These studies also
reveal that the intrinsic motion of an enzyme is a key point to be considered in inhibitor design.
RESULTS AND DISCUSSION
In order to be selective for this kinase, we focused on the design of compounds that bind to the SB
binding site. The starting point for the design of the reported compounds was the study of the
structural changes required in the SB and LID binding domains for the release of shikimateꢀ3ꢀ
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phosphate (2) from the active site. This study was carried out by analyzing the dynamic behavior of
the SK/ADP/2 complex and the results are discussed below.
Dynamic Behavior of SK Enzymes: Molecular Dynamics Simulation Studies – For the MtꢀSK
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enzyme, we have shown previously that an opening of up to 10 Å of the SB domain and of up to 8 Å
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of the LID domain are required for product release (Figure 4). Overall, MtꢀSK undergoes opening
of the SB and LID domains in a similar way to the movement of an accordion, which is surprising
given that the SB domain is composed of three αꢀhelices α2, α3 and α5. This opening allows a rapid
conformational change of the product to its more stable cyclohexene conformation with most of the
substituents in a pseudoꢀequatorial disposition. This process is mainly carried out by three conserved
arginines, specifically Arg117, Arg136, and Arg58. The essential arginine triggers the process by
pulling on the phosphate group of the product, which in turn breaks the key interactions with the
active site.
In order to evaluate the possible differences between the two SK enzymes in relation to the structural
changes required for product release, MD simulation studies were also performed with HpꢀSK
(Figure 5). The dynamic behavior of HpꢀSK/ADP/2 was studied by using the enzyme geometries
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found in the crystal structures of HpꢀSK (PDB code 3MUF ) in complex with ADP and shikimateꢀ3ꢀ
phosphate (2). The monomer of HpꢀSK immersed in a truncated octahedron of water molecules
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obtained using the molecular mechanics force field AMBER was used. The results from 50 ns of
dynamic simulation showed significant differences between the two enzymes. In general, for the Hpꢀ
SK enzyme the release of 2 from the active site is a much slower process since it is not initiated until
around 7 ns of dynamic simulation and this process requires to be performed at 50 ºC. For the MtꢀSK
enzyme, a large motion of the SB domain was already observed after ∼20 ps of dynamic simulation
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at 25 ºC. Even bearing in mind that the computational time cannot be correlated with the velocity
of an enzyme, this finding is consistant with the huge velocity differences observed experimentally
between the two enzymes. Thus, under the same assay conditions, HpꢀSK has a kcat value that is
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2500ꢀfold lower than that for MtꢀSK. More importantly, the motion of the SB domain is also
different. Whereas for the MtꢀSK enzyme a synchronous and large opening of the three αꢀhelices α2
(∼7 Å), α3 (∼10 Å) and α5 (∼6 Å) was observed (Figure 5B), which is reminiscent of the movement
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of an accordion, for the HpꢀSK enzyme an asynchronous and more reduced opening was observed
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Figures S2ꢀS4). Thus, conformational changes were mainly focused on αꢀhelices α5 (∼4 Å) and α3
∼3 Å), which in both cases were less pronounced than in the M. tuberculosis enzyme (Figure 5B).
(
An opening of only a ∼2 Å was observed for the αꢀhelix α2. In contrast, significant differences were
not observed in the opening of the LID domain, which was very large in both cases (up to 10 Å).
Overall, the LID undergoes a large opening as for MtꢀSK and part of the SB domain twists to create
an additional gap in this region. These findings suggest that the ‘dynamic pocket’ in the SB domain
of HpꢀSK would be smaller and distinct from that in MtꢀSK. In both cases the three conserved
arginines are responsible for removing the product from the active site through strong electrostatic
interactions between their guanidinium groups and the phosphate group in 2. In addition, for the Hpꢀ
SK enzyme the presence of an arginine in αꢀhelix α3 (Arg45) instead of an alanine (Ala46) in Mtꢀ
SK, which is oriented towards the active site, allows this residue to also be involved in product
release (Figures 5C–5G).
Analysis of the available crystal structures and the amino acid sequence of various SK enzymes
reveals that the apolar pocket surrounding the C4 and C5 hydroxyl groups of the natural substrate
(SB domain), which undergoes the aforementioned motion, is quite conserved (Figures S5 and 6).
This pocket mainly involves the conserved residues Phe48/Phe49, Phe56/Phe57, Ile36/Ile37 and
Val44/Ile45 (in H. pylori and M. tuberculosis, respectively). In addition, further sealing from the bulk
water is achieved by several apolar residues in the vicinity (for HpꢀSK: Leu51, Leu42, Val40 and
Ile47; for MtꢀSK: Ile60 and Ile48) and this also prevents the entry of water from the environment.
Reasoning that the incorporation of benzyl groups in the C5 position of shikimic acid (1) would
allow favorable contacts to be established with the apolar residues within the pocket and would
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prevent the appropriate closure of the active site for catalysis, we decided to explore the possible
inhibition of the SK enzyme by compounds 3–6. A range of substituted benzene rings were
incorporated. Docking studies suggested that metaꢀsubstituted derivatives would lead to a more
favorable effect than the corresponding paraꢀsubstituted ones as the group would be pointing
towards the protein. In addition, considering that MtꢀSK appears to be able to achieve a wider
opening of the SB domain than HpꢀSK, we also studied the effect of larger aromatic moieties
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(naphthalene, benzothiophene, indole), compounds 3h–3i and 4–5. Finally, the effect of replacing the
C3 hydroxyl group in Oꢀbenzyl derivatives 3 by an amino group was also analyzed with compounds
6
.
Synthesis of Compounds 3 – The synthesis of Oꢀbenzyl shikimic acid derivatives 3 was carried out
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from the previously described alcohol 8, which is synthesized in two steps from commercially
available shikimic acid (1) (Scheme 2). Alkylation of alcohol 8 with various benzyl bromide
derivatives in the presence of sodium iodide and diisopropylethylamine at 150 ºC afforded benzyl
ethers 9. Removal of the acetal group in 9 with HCl followed by basic hydrolysis of the resulting
+
esters 10 and, in some cases, subsequent protonation with Amberlite IRꢀ120 (H ) ionꢀexchange resin
gave the Oꢀbenzyl shikimic acid derivatives 3 or their corresponding sodium salts. For compounds
9
d and 9f, treatment with HCl directly afforded the corresponding acids 3d and 3f, respectively.
Synthesis of Compounds 4 and 5 – The triazole moiety in 4–5 was introduced by a copper(I)ꢀ
catalyzed 1,3ꢀdipolar cycloaddition between azide 14 and 3ꢀphenylꢀ1ꢀpropyne or 3ꢀphenoxyꢀ1ꢀ
2
3
propyne, respectively (Scheme 3). Azide 14 was prepared in four steps from protected methyl
24
shikimate 11 following a modified procedure. First, treatment of alcohol 11 with mesyl chloride in
the presence of trimethylamine and subsequent acid hydrolysis of the acetal gave mesylate 12.
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Treatment of diol 12 with sodium methoxide and methanol afforded epoxide 13, which was treated
with sodium azide to give the desired azide 14. The cycloaddition reaction regioselectively afforded
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the 4ꢀsubstituted triazoles 15 and 16. Basic hydrolysis of the resulting methyl esters 15 and 16 and
protonation with an ionꢀexchange resin, led to the desired acids 4 and 5.
Synthesis of Compounds 6h–i – The C3 amino group in compounds 6h and 6i was introduced by
Mitsunobu reaction between diols 10h–i and freshly prepared hydrazoic acid followed by reduction
of the resulting azides (Scheme 4). Finally, basic hydrolysis of the amino methyl esters 17h–i gave
the desired amines 6h–i.
0
1
2
3
4
5
6
7
8
9
0
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0
Inhibitory Activity and Susceptibility Testing – The inhibitory properties of compounds 3–6
18
against both SK enzymes were tested as described before. All of the compounds that were found
not to be substrates for this enzyme were assayed in the presence of shikimic acid (1) and ATP for
their inhibitory properties against MtꢀSK and HpꢀSK. All compounds proved to be reversible
i
competitive inhibitors of both enzymes. The inhibition data (K ), which were obtained from Dixon
plots (1/v vs [I]), are provided in Table 1.
In general, the reported compounds proved to be more potent against HpꢀSK than MtꢀSK.
Compounds 4–6 were found to have K values above the K (Table 1, entries 10–13). In contrast,
i
m
compounds 3, which bear Oꢀbenzyl groups, were the best inhibitors for both enzymes with K values
i
below K
m
in most cases (Table 1, entries 1–9). For HpꢀSK, the latter compounds were found to have
K
i
values in the low micromolar or nanomolar range. The results show that the incorporation of
substituents in the metaꢀposition of the benzene ring and the use of large aromatic rings enhances the
inhibitory potency by up to 36ꢀfold. The most potent inhibitor in series 3 was the 3ꢀnitrobenzyl
derivative 3e, which gave K values of 0.46 µM and 10 µM against HpꢀSK and MtꢀSK, respectively
i
(Table 1, entry 5). 5ꢀBenzothiophenyl derivative 3i was also potent, with a K
against HpꢀSK. In view of the high inhibitory potency of the 3ꢀnitrobenzyl derivative 3e against both
enzymes, the high K values obtained with the perfluoro derivative 3g are surprising (Table 1, entry
). Compounds 3i and 3e proved to inhibit more efficiently HpꢀSK than previously reported ones.
i
value of 0.56 µM
i
19
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The in vitro antiꢀHelicobacter pylori activity of the most potent inhibitors of series 3, compounds 3e
and 3i, and compounds 4−6 was studied. Considering the high hydrophilicity of these compounds
due to the presence of a carboxylate group, which is required for enzyme recognition by the
conserved Arg132, their corresponding methyl ester prodrugs (compounds 10e, 10i, 15−16 and 17h-
i) were employed. We have previously used this strategy to achieve the internalization into
mycobacterial cell of diverse reversible competitive inhibitors of the type II dehydroquinase, the
10
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15
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26
third enzyme of the shikimic acid pathway. In the latter case, the free acids also proved to have low
in vitro activity. The agar dilution method was used to determine the Minimum Inhibitory
Concentration (MICs, µg/mL) of the compounds, according to the standard method recommended by
27
CLSI. A H. pylori clinical isolate obtained from a biopsy of a patient of the Guadalajara Hospital
(Guadalajara, Spain) was employed. MICs were defined as the lowest concentration of each
compound that completely inhibited visible growth on plates. MIC assays were performed at least
three times. The results are provided in Table 2. The methyl ester 10i, which is the ester prodrug of
the 5ꢀbenzothiophenyl derivative 3i with a K value of 560 nM, proved to be the most potent with an
i
MIC value of 4 µg/mL. Derivative 10e proved to have a 32ꢀfold higher MIC value, which might be
due to the lower stability of the nitrobenzyl moiety vs the benzothiophenyl one. As expected, higher
MICs values were obtained for methyl esters 15−16 and 17h-i.
In an effort to obtain further details of the binding interactions responsible for the inhibitory activity,
the binding modes of compounds 3–6 with MtꢀSK and HpꢀSK were studied. Molecular docking
20
using GOLD 5.2 was initially carried out with diverse inactive open forms of the SK active site.
The proposed binding modes were further analyzed by MD simulation studies and the results are
discussed below.
Binding Mode – Diverse inactive open conformations of the two enzymes, which were obtained by
MD simulation studies, were employed for docking studies. For the HpꢀSK enzyme, the first 15 ns of
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the MD simulation of the HpꢀSK products complex provided diverse poses of the LID and SB
domains in the open conformation. However, for the MtꢀSK enzyme, the MD simulation performed
with the MtꢀSK/ADP/2 complex showed that as soon as the simulation starts large displacements in
the positions of the conserved and essential active site residues occur. For instance, this is the case
for the conserved Arg136, which is the key residue for carboxylate recognition in the substrate.
Considering that under these circumstances the binding mode determined by GOLD might be quite
unrealistic, diverse poses from the MD simulation of MtꢀSK/ATP/(6R)ꢀ6ꢀhydroxyshikimic acid were
employed for docking studies (Figures 3C and 3D). The latter compound seemed to cause the
opening of the SB domain (4 Å) due to a conformational change to the most stable conformation of a
0
1
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8
cyclohexene ring but without large displacements of the conserved arginines.
In general, as one would expect from the incorporation of a large substituent in the C5 position of 1,
all of the ligands bind in the active site with the most stable conformation for a cyclohexene ring, i.e.,
with the C4 and C5 substituents in a pseudoꢀdiequatorial disposition. The rigidity that the triazole
moiety imparts to compounds 4 and 5 would disfavor their binding because the benzyl moiety would
be more restricted in terms of finding the optimal conformation to achieve good binding with the
apolar pocket – a situation that might explain their low inhibitory potency. The same limitation was
observed with the incorporation of an amino group in the C3 position (compounds 6h and 6i). This
group would cause the ligands to be in close contact with the γꢀphosphate of ATP due to attractive
electrostatic interactions between the phosphate group and the protonated amino group of the
ligands. Fortunately, Oꢀbenzyl derivatives 3 proved to bind in the active site of the two SK enzymes,
with the aromatic moiety interacting with the apolar pocket of the SB domain as desired. The highest
score solutions of the most potent inhibitors, specifically compound 3e for MtꢀSK and compounds
3
e, 3b, and 3i for HpꢀSK, were further evaluated by MD simulation studies in order to assess the
stability and therefore the reliability of the proposed binding mode. The results of the MD simulation
studies (50 ns) show that the aforementioned complexes are stable as relevant differences were not
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observed in the position of the ligand during most of the simulation. Overall, our computational
studies revealed that there are significant differences in the conformational changes caused by the
ligands in the LID and SB domains based on the size of the aromatic moiety incorporated on the C5
hydroxyl group as well as the type of SK enzyme. The results of these studies are discussed below.
10
11
12
13
14
15
16
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O-Benzyl derivative 3e – The binding mode of the most potent inhibitor of the monobenzyl series for
the two enzymes, namely the nitro derivative 3e, proved to be significantly different in the active
sites of MtꢀSK (Figures 7A and 7D) and HpꢀSK (Figures 7B and 7E). In the case of MtꢀSK, the
incorporation of the 3ꢀnitrobenzyl moiety at the C5 position of 1 would cause the opening of the LID
domain (9 Å) and αꢀhelix α3 of the SB domain (5 Å). This aromatic moiety would cause a large
conformational change in the position of the essential arginine and a reduction in the flexibility of the
LID and SB domains through a set of favorable lipophilic interactions with apolar residues of these
domains. The 3ꢀnitrobenzyl moiety would be trapped between the essential residues Phe49 and
Arg117 by establishing πꢀπ stacking and cationꢀπ interactions, respectively. The interaction with the
essential arginine seems to be crucial to enhance the binding affinity of the ligands in the active site
and therefore their inhibitory potency. The nitro group would be pointing towards αꢀhelix α3. In this
arrangement the benzyl group would interact with the side chains of Ile45 and Phe57 (αꢀhelix α3)
and with the side chain of Val116 and the carbon chain of Arg117 of the LID. In contrast, for the Hpꢀ
SK enzyme the 3ꢀnitrobenzyl moiety in 3e would stabilize an open conformation of the SB domain
(mainly αꢀhelices α2 and α5) by interaction with the apolar pocket between αꢀhelices α3 and α5,
instead of αꢀhelix α3 (Figures 7B and 7E). Significant changes were not observed in the position of
the essential arginine and the LID domain. The 3ꢀnitrobenzyl moiety would now be located between
the essential Phe48 and the carbon side chain of the conserved residue Glu53, with the nitro group
pointing towards the LID domain instead of towards αꢀhelix α3. In this arrangement the benzyl
group would establish πꢀπ stacking interactions with Phe48 and lipophilic interactions with the side
chains of Ile47, Leu51 and Glu53 (carbon chain). Moreover, the C5 CH group in 3e would interact
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5
5
6
with the side chain of Phe56 and the aromatic ring and the C6 methylene group would contact the
side chain of Leu118, which is located in the LID.
O-Benzyl derivative 3b – The introduction of a large and apolar methoxy group in the benzyl ring,
0
1
2
3
4
5
6
7
8
9
0
1
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0
namely compound 3b, which had a K of 1 µM against HpꢀSK, would also stabilize an open
i
conformation of the LID domain (Figures 7C and 7H). By establishing favorable lipophilic
interactions with Leu118, the ligand is more embedded in the active site and this allows apolar
interactions between the side chain of Met10 and the C3 and C4 CH bonds of the cyclohexene ring.
Moreover, this compound also locates its benzyl group in the apolar pocket between αꢀhelices α3
and α5 but causes the opening of αꢀhelix α5 only. The main difference with compound 3e with
respect to the lipophilic interactions with side chain residues Ile47, Phe56 and Phe48 is the lack of a
πꢀπ stacking interaction between the benzyl moiety in 3b and the essential Phe48, which might
account for the lower inhibitory potency.
In general, the results of our MD simulation studies revealed that the substituent in the metaꢀposition
in 3 would prefer to point towards the LID domain in the two SK enzymes. The exception to this
trend is the 3ꢀnitrobenzyl derivative 3e when it binds with MtꢀSK. In this case, the essential Arg117
seems to select the opposite arrangement to establish a strong cationꢀπ interaction with the ring – an
interaction that was not observed in the other analogs of the series or with the HpꢀSK enzyme. In
order to obtain further evidence for this trend, MD simulation studies with the 3ꢀnitro derivative 3e
and the 3ꢀmethoxybenzyl derivative 3b were carried out with the two possible orientations of the
substituent, i.e., pointing toward the LID or the SB domains. The binding free energies of these
28
ligands were calculated using the MM/PBSA approach in explicit water (generalized Born, GB) as
implemented in Amber. For MtꢀSK, the calculated binding energy of the 3ꢀnitrobenzyl derivative 3e
when the nitro group is pointing towards the SB domain is –23.1 kcal lower than for the opposite
pose. In addition, the 3ꢀmethoxybenzyl derivative 3b arranged with the methoxy group pointing
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towards the SB domain undergoes a rotation of 180° during the 50 ns of simulation to give the
opposite pose. For HpꢀSK, the calculated binding energy of 3b with the methoxy group pointing
towards the LID domain is –16.7 kcal lower than that for the opposite pose. A less marked difference
between the two poses was obtained for the nitro compound 3e (1.9 kcal). However, the
aforementioned binding mode provides a more stable complex during the whole simulation.
10
11
12
13
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i
O-Benzyl derivative 3i – The benzothiophene derivative 3i, which had a K value of 560 nM against
HpꢀSK, was predicted to have a similar binding mode to the 3ꢀmethoxy derivative 3b (Figures 7G
and 7H). Thus, this compound would also stabilize an open conformation of the LID (5 Å) and the
SB (3 Å) domains. As shown in Figure 7I, although both ligands interact with the same residues, the
arrangement and rigidity of the benzothiophene ring would probably enhance the binding, which
might account for the 2ꢀfold higher inhibitory potency.
CONCLUSIONS AND FINAL REMARKS
Several C5ꢀsubstituted shikimic acid analogs, compounds 3–6, have been synthesized and tested
against SK from M. tuberculosis and H. pylori, an essential enzyme in bacteria. These compounds
were designed to explore the possible inhibition of the SK enzyme by blocking the appropriate
closed form for catalysis. The two enzymes were found to undergo large conformational changes for
product release in the LID and SB domains but they were particularly distinct in the latter domain.
Specifically, the ‘dynamic apolar pocket’ that isolates the substrate for catalysis and involves a quite
conserved region of αꢀhelices α2, α3 and α5 is significantly smaller for the HpꢀSK enzyme and this
proved to have an intrinsically different motion for the two enzymes.
Compounds 3–6 are reversible competitive inhibitors of both SK enzymes but, in general, they are
i
more potent against the H. pylori, having K values in the low micromolar or nanomolar range.
Compounds 3, which contain an Oꢀbenzyl moiety, were the best inhibitors for both enzymes. Among
the Oꢀbenzyl series 3, the 3ꢀnitrobenzyl derivative 3e was found to be the most potent against the two
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2
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3
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5
5
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5
5
5
6
i
enzymes and this compound had K values of 460 nM and 10 µM against HpꢀSK and MtꢀSK,
respectively. The results of our MD simulation studies revealed that the 3ꢀnitrobenzyl derivative 3e
would cause the opening of the LID domain (9 Å) and αꢀhelix α3 (5 Å) of the SB domain of Mt-SK
and a large conformational change in the position of the essential arginine by establishing a cationꢀπ
interaction with the nitrobenzyl ring of the ligand, with the nitro group pointing towards the SB
domain. In contrast, for the HpꢀSK enzyme, compound 3e would mainly cause the opening of the
region between αꢀhelices α3 and α5 instead of αꢀhelix α3, an interaction with the essential arginine
would not be established and the nitro group would be oriented towards the LID domain.
0
1
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5ꢀBenzothiophenyl derivative 3i proved to be a potent reversible competitive inhibitor of HpꢀSK (Ki
=
560 nM). This compound would stabilize an open conformation of the LID (5 Å) and SB (3 Å)
domains through a set of favorable apolar interactions with this ‘dynamic apolar pocket’. Moreover,
compound 10i, a prodrug of inhibitor 3i, was the most efficient derivative in achieving good in vitro
activity against Helicobacter pylori, having a MIC value of 4 µg/mL.
The results from the studies described here have identified a good scaffold for the design of
reversible competitive inhibitors of the SK enzyme and have opened up new opportunities for the
development of novel inhibitors by targeting the dynamic apolar pocket that surrounds the C4 and
C5 hydroxyl groups of the natural substrate. Moreover, these studies also represent a good example
of the importance in drug design of taking into account the motion of an enzyme target. Thus,
stabilization of the inactive and inappropriate conformation for enzymatic catalysis seems to also be
a good approach for the development of inhibitors that can be used as drugs.
Experimental Section
General. All starting materials and reagents were commercially available and were used without
1
13
further purification unless is indicated. H NMR spectra (250, 300 and 500 MHz), C NMR spectra
31
19
(63, 75 and 125 MHz), P NMR spectra (202 MHz) and F NMR spectra (282 MHz) were
measured in deuterated solvents. J values are given in Hertz. NMR assignments were carried out by a
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combination of 1 D, COSY, and DEPTꢀ135 experiments. FTꢀIR spectra were recorded as NaCl
plates or KBr discs in a PerkinElmer Two FTIR spectrometer with attenuated total reference. [α]ꢁꢂ
=
ꢀ
−1
2
−1
values are given in 10 deg cm g . MilliQ deionized water was used in all the buffers. All
procedures involving the use of ionꢀexchange resins were carried out at room temperature using
10
11
12
13
14
15
16
17
18
19
20
21
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23
24
25
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28
29
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32
33
34
35
36
37
38
39
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
MilliꢀQ deionized water. Amberlite IRꢀ120 (H ) (cation exchanger) was washed alternately with
water, 10% NaOH, water, 10% HCl, and finally water before use. The spectroscopic measurements
were made on a Varian Cary 100 UVꢀVis spectrophotometer with a 1 cm pathlength cell fitted with a
Peltier temperature controller. The purity of compounds 3−6 was analyzed by HPLC and by NMR.
For compounds 3−5, HPLC was performed on a BioꢀRad Aminex ion exclusion HPXꢀ87H organic
acids column (300 mm × 16 mm), eluting with 100 mM aqueous formic acid at a flow rate of 0.6 mL
−1
min . For compounds 6, a Phenomenex Luna 5 µM C18 column (250 mm × 4.6 mm), eluting with a
–
1
gradient of acetonitrile/water [from (5:95) to (30:70)] at a flow rate of 1 mL min , was employed.
All tested compounds have a purity ≥95%.
22
General procedure for the synthesis of compounds 9 − A solution of alcohol 8 (1 equivalent),
N,Nꢀdiisopropylethylamine (1.6 equivalents), benzyl bromide (1.5 equivalents) and sodium iodide
(0.1 equivalents) under inert atmosphere, was heated at 150 °C for 1.5−4.5 h. After cooling to room
temperature, the resulting brown residue was dissolved in a mixture of ethyl acetate and saturated
solution of sodium bisulfate. The organic layer was separated and the aqueous layer was extracted
2 4
with ethyl acetate (×3). The combined organic extracts were dried (Na SO anh.), filtered and
concentrated under reduced pressure. The resulted reside was purified by flash chromatography to
give benzyl ethers 9.
Methyl (3R,4S,5R)-5-benzyloxy-3,4-(O-isopropiliden)cyclohex-1-ene-1-carboxylate (9a)
−
Alcohol 8 (100 mg, 0.44 mmol), N,Nꢀdiisopropylethylamine (0.12 mL, 0.70 mmol), benzyl bromide
(
78 ꢀL, 0.66 mmol) and sodium iodide (6.6 mg, 0.044 mmol). Reaction time = 4.5 h.
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6
2
D
0
Chromatographic eluent: (20:80) diethyl ether/hexane. 9a (47 mg, 39%) as a yellow oil. [α ] = –
1
3
9.0º (c2.0, CHCl
3
). H NMR (300 MHz, CDCl
3
) δ: 7.30 (m, 5H, 5×ArH), 6.88 (m, 1H, H2), 4.75
(
m, 1H, H3), 4.70 (d, J = 12.0 Hz, 1H, OCHH), 4.64 (d, J = 12.0 Hz, 1H, OCHH), 4.26 (t, J = 6.3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hz, 1H, H4), 3.77 (m, 1H, H5), 3.76 (s, 3H, OCH
), 2.70 (dd, J = 4.2 and 17.4 Hz, 1H, H6eq), 2.40
3
13
(
dd, J = 6.6 and 17.4 Hz, 1H, H6ax) and 1.39 (br s, 6H, 2×CH
3
) ppm. C NMR (75 MHz, CDCl
3
) δ:
1
66.7 (C), 138.1 (C), 134.6 (CH), 129.5 (C), 128.3 (2×CH), 127.6 (3×CH), 109.4 (C), 75.9 (CH),
74.6 (CH), 72.3 (CH), 71.5 (OCH
2
), 52.0 (OCH
3
), 27.8 (CH
3
), 26.1 (CH
2
) and 25.8 (CH
3
) ppm. IR
ꢀ1
+
+
(
film) ʋ: 1722 (CO) cm . MS (ESI) m/z = 341 (MNa ). HRMS calcd for C18
H
22
O
5
Na (MNa ):
3
41.1359, found 341.1352.
Methyl (3R,4S,5R)-5-(3-methoxybenzyloxy)-3,4-(O-isopropiliden)cyclohex-1-ene-1-carboxylate
9b) − Alcohol 8 (100 mg, 0.44 mmol), N,Nꢀdiisopropylethylamine (0.12 mL, 0.70 mmol), 3ꢀ
metoxybenzyl bromide (92 ꢀL, 0.66 mmol) and sodium iodide (6.6 mg, 0.044 mmol). Reaction time
1.5 h. Chromatographic eluent: (25:75) diethyl ether/hexane. 9b (47 mg, 31%) as a yellow oil.
(
=
2
D
0
1
[
α ] = –93.2º (c0.2, CHCl
3
). H NMR (300 MHz, CDCl
6.80 (m, 4H, 3×ArH+H2), 4.74 (m, 1H, H3), 4.66 (d, J = 12.6 Hz, 1H, OCHH), 4.62 (d, J = 12.6 Hz,
H, OCHH), 4.25 (t, J = 6.3 Hz, 1H, H4), 3.79 (s, 3H, OCH ), 3.75 (s, 3H, OCH ), 2.69 (dd, J = 3.6
and 17.4 Hz, 1H, H6eq), 2.39 (dd, J = 6.6 and 17.4 Hz, 1H, H6ax) and 1.38 (br s, 6H, 2×CH ) ppm.
) δ: 166.7 (C), 159.7 (C), 139.8 (C), 134.6 (CH), 129.5 (C), 129.3 (CH),
3
) δ: 7.23 (t, J = 7.8 Hz, 1H, ArH), 6.92–
1
3
3
3
1
3
C NMR (75 MHz, CDCl
3
1
19.9 (CH), 113.2 (CH), 113.0 (CH), 109.4 (C), 75.9 (CH), 74.6 (CH), 72.3 (CH), 71.4 (OCH
2
), 55.1
ꢀ1
(
OCH ), 52.0 (OCH ), 27.8 (CH ), 26.1 (CH ) and 25.8 (CH ) ppm. IR (film) ʋ: 1710 (CO) cm . MS
3
3
3
2
3
+
+
(
ESI) m/z = 371 (MNa ). HRMS calcd for C19
(3R,4S,5R)-5-(3-fluorobenzyloxy)-3,4-(O-isopropiliden)cyclohex-1-ene-1-carboxylate
9c) − Alcohol 8 (100 mg, 0.44 mmol), N,Nꢀdiisopropylethylamine (0.12 mL, 0.70 mmol), 3ꢀ
24 6
H O Na (MNa ): 371.1465, found 371.1469.
Methyl
(
fluorobenzyl bromide (81 ꢀL, 0.66 mmol) and sodium iodide (6.6 mg, 0.044 mmol). Reaction time =
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2 h. Chromatographic eluent: (30:70) diethyl ether/hexane. 9c (61 mg, 47%) as a yellow oil.
2
0
1
ꢃ
[
α
]
=ꢃ–63.4º (c0.8, CHCl
3
). H NMR (300 MHz, CDCl
2×ArH), 6.97–6.89 (m, 2H, ArH+H2), 4.75 (m, 1H, H3), 4.68 (d, J = 13.2 Hz, 1H, OCHH), 4.64 (d,
J = 13.2 Hz, 1H, OCHH), 4.25 (t, J = 6.6 Hz, 1H, H4), 3.76 (s, 3H, OCH ), 3.67 (m, 1H, H5), 2.72
dd, J = 3.6 and 17.4 Hz, 1H, H6eq), 2.36 (dd, J = 6.9 and 17.4 Hz, 1H, H6ax) and 1.39 (s, 6H,
3
) δ: 7.27 (m, 1H, ArH), 7.08 (m, 2H,
D
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
(
13
3 3
2×CH ) ppm. C NMR (75 MHz, CDCl ) δ: 166.6 (C), 162.9 (C, JC-F = 244 Hz), 140.9 (C, JC-F = 7
Hz), 134.5 (CH), 129.8 (CH, JC-F = 8 Hz), 129.6 (C), 122.8 (CH, JC-F = 3 Hz), 114.5 (CH, JC-F = 32
Hz), 114.2 (CH, JC-F = 34 Hz), 109.5 (C), 76.1 (CH), 75.0 (CH), 72.3 (CH), 70.7 (OCH ), 52.0
2
ꢀ1
(
OCH
3
), 27.7 (CH
3
), 26.2 (CH
2
) and 25.7 (CH
3
) ppm. IR (film) ʋ: 1710 (CO) cm . MS (ESI) m/z =
+
+
3
59 (MNa ). HRMS calcd for C H O FNa (MNa ): 359.1265, found 359.1269.
18 21
5
Methyl (3R,4S,5R)-5-(3-methylbenzyloxy)-3,4-(O-isopropiliden)cyclohex-1-ene-1-carboxylate
9d) − Alcohol 8 (284.5 mg, 2.25 mmol), N,Nꢀdiisopropylethylamine (0.35 mL, 2.0 mmol), 3ꢀ
(
methylbenzyl bromide (0.25 mL, 1.88 mmol) and sodium iodide (19 mg, 0.125 mmol). Reaction
time = 1.5 h. Chromatographic eluent: (40:60) diethyl ether/hexane. 9d (166.3 mg, 40%) as a yellow
2
0
1
oil. [α] = −55.7 º (c1.0, CHCl
3
). H NMR (250 MHz, CDCl
s, 2H, 2×ArH), 7.10 (t, J = 5.5 Hz, 1H, ArH), 6.89 (m, 1H, H2), 4.76 (m, 1H, H3), 4.67 (d, J = 12.0
Hz, 1H, OCHH), 4.61 (d, J = 12.0 Hz, 1H, OCHH), 4.26 (t, J = 6.3 Hz, 1H, H4), 3.79 (m, 1H, H5),
.77 (s, 3H, OCH ), 2.76−2.67 (m, 1H, H6ax), 2.40−2.35 (m, 1H, H6eq), 2.35 (s, 3H, CH ) and 1.40
(s, 6H, 2×CH ) ppm. C NMR (63 MHz, CDCl ) δ: 166.8 (C), 138.0 (2×C), 134.7 (CH), 129.6 (C),
28.5 (2×CH), 128.3 (CH), 124.9 (CH), 109.4 (C), 75.9 (CH), 74.5 (CH), 72.3 (OCH ), 71.5
3
) δ: 7.21 (d, J = 7.3 Hz, 1H, ArH), 7.16
D
(
3
3
3
13
3
3
1
3
(
OCH
2
), 52.1 (CH), 27.9 (CH
3
), 26.1 (CH
2
), 25.9 (CH
3
3
) and 21.4 (CH ) ppm. IR (ATR) ʋ: 1714
−
1
+
+
(
CO) cm . MS (ESI) m/z = 355 (MNa ). HRMS calcd for C19
H
24NaO
5
(MNa ): 355.1516; found,
3
55.1516.
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Methyl
(3R,4S,5R)-5-(3-nitrobenzyloxy)-3,4-(O-isopropyliden)cyclohex-1-eno-1-carboxylate
(
9e) − Alcohol 8 (115 mg, 0.50 mmol), N,Nꢀdiisopropylethylamine (0.14 mL, 0.80 mmol), 3ꢀ
nitrobenzyl bromide (162 mg, 0.75 mmol) and sodium iodide (7.5 mg, 0.05 mmol). Reaction time =
.5 h. Chromatographic eluent: (30:70) ethyl acetate/hexane. 9e (58.1 mg, 32%) as a yellow oil. ꢃ
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
D
0
1
[
3 3
α ] = –51.8º (c1.3, CHCl ). H NMR (300 MHz, CDCl ) δ: 8.22 (br s, 1H, ArH), 8.13 (d, J = 8.4
Hz, 1H, ArH), 7.68 (d, J = 7.6 Hz, 1H, ArH), 7.50 (t, J = 8.0 Hz, 1H, ArH), 6.91 (br s, 1H, H2), 4.77
(s, 2H, OCH ), 4.70 (br s, 1H, H3), 4.22 (t, J = 6.6 Hz, 1H, H4), 3.77 (s, 3H, OCH ), 3.72 (m, 1H,
2
3
H5), 2.78 (dd, J = 3.5 and 17.5 Hz, 1H, H6ax), 2.40−2.32 (m, 1H, H6eq) and 1.40 (s, 6H, 2×CH
3
)
13
ppm. C NMR (63 MHz, CDCl
3
) δ: 166.6 (C), 148.4 (C), 140.7 (C), 134.7 (CH), 133.4 (CH), 129.8
),
) ppm. IR (ATR) ʋ: 1710 (CO) and 1531 (NO)
(
C), 129.4 (CH), 122.7 (CH), 122.4 (CH), 109.7 (C), 76.5 (CH), 76.1 (CH), 72.5 (CH), 70.5 (OCH
2.3 (OCH ), 27.9 (CH ), 26.6 (CH ) and 25.8 (CH
cm . MS (ESI) m/z = 386 (MNa ). HRMS calcd for C H NO Na (MNa ): 386.1210; found,
2
5
3
3
2
3
–1
+
+
18
21
7
3
86.1216.
Methyl (3R,4S,5R)-5-(4-methylbenzyloxy)-3,4-(O-isopropiliden)cyclohex-1-ene-1-carboxylate
9f) − Alcohol 8 (250 mg, 1.10 mmol), N,Nꢀdiisopropylethylamine (0.30 mL, 1.76 mmol), 4ꢀ
methylbenzyl bromide (305 mg, 1.65 mmol) and sodium iodide (16 mg, 0.11 mmol). Reaction time
1.5 h. Chromatographic eluent: (40:60) diethyl ether/hexane. 9f (307 mg, 84%) as a yellow oil. ꢃ
(
=
2
D
0
1
[
α] = −50.2º (c1.1, CHCl
3
). H NMR (300 MHz, CDCl
d, J = 8.2 Hz, 2H, 2×ArH), 6.87 (m, 1H, H2), 4.73 (m, 1H, H3), 4.65 (d, J = 12.0 Hz, 1H, OCHH),
.59 (d, J = 12.0 Hz, 1H, OCHH), 4.24 (t, J = 6.2 Hz, 1H, H4), 3.77 (m, 1H, H5), 3.75 (s, 3H,
OCH ), 2.71−2.64 (m, 1H, H6ax), 2.45−2.36 (m, 1H, H6eq), 2.33 (s, 3H, CH ) and 1.38 (s, 6H,
2×CH ) ppm. C NMR (75 MHz, CDCl ) δ: 166.7 (C), 137.3 (C), 135.1 (C), 134.7 (CH), 129.5 (C),
3
) δ: 7.23 (d, J = 8.0 Hz, 2H, 2×ArH), 7.13
(
4
3
3
13
3
3
1
2
29.0 (2×CH), 127.8 (2×CH), 109.3 (C), 75.8 (CH), 74.3 (CH), 72.3 (CH), 71.3 (OCH ), 51.9
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9
−
1
(OCH
3
), 27.8 (CH
3
), 26.1 (CH
2
), 25.9 (CH
3
) and 21.1 (CH
3
) ppm. IR (ATR) ʋ: 1720 (CO) cm . MS
+
+
(ESI) m/z = 355 (MNa ). HRMS calcd for C19
H24NaO
5
(MNa ): 355.1516; found, 355.1517.
Methyl (3R,4S,5R)-3,4-(O-isopropiliden)-5-(perfluorobenzyl)cyclohex-1-ene-1-carboxylate (9g)
− Alcohol 8 (200 mg, 0.88 mmol), N,Nꢀdiisopropylethylamine (0.24 mL, 1.41 mmol),
perfluorobenzyl bromide (0.2 mL, 1.32 mmol) and sodium iodide (13.2 mg, 0.09 mmol). Reaction
time = 1.5 h. Chromatographic eluent: (10:90) diethyl ether/hexane. 9g (40 mg, 11%) as a yellow oil.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
D
0
1
ꢃ
[α ] =ꢃ−27.0° (c0.6, CHCl
3
). H NMR (500 MHz, CDCl
3
) δ: 6.89 (m, 1H, H2), 4.75 (dt, J = 2.0
), 3.73 (dt,
and 8.5 Hz, 1H, OCH
2
), 4.72 (m, 1H, H3), 4.19 (t, J = 6.5 Hz, 1H, H4), 3.77 (s, 3H, OCH
3
J = 4.5 and 7.0 Hz, 1H, H5), 2.73 (ddt, J = 17.5, 4.5 and 1.5 Hz, 1H, H6eq), 2.34 (ddt, J = 17.5, 2.9
13
and 7.5 Hz, 1H, H6ax), 1.40 (s, 3H, CH
66.5 (C), 146.6−136.4 (m, 5×C), 134.5 (CH), 129.5 (C), 111.4 (td, JC-F = 2 and 11 Hz, C), 109.6
C), 76.4 (CH), 76.1 (CH), 72.3 (CH), 58.8 (OCH ), 52.1 (OCH ), 27.7 (CH ), 26.4 (CH ) and 25.7
) δ: −142.9 (dd, J = 5 and 20 Hz, 2F), −153.9 (t, J = 20 Hz,
3 3 3
) and 1.39 (s, 3H, CH ) ppm. C NMR (125 MHz, CDCl ) δ:
1
(
(
2
3
3
2
19
CH
3 3
) ppm. F NMR (282 MHz, CDCl
ꢀ1
1
F) and −162.1 (dt, J = 7 and 20 Hz, 2F) ppm. IR (film) ʋ: 1722 (CO) cm . MS (ESI) m/z = 431
+
+
(
MNa ). HRMS calcd for C H F O Na (MNa ): 431.0888; found, 431.0889.
18 17 5 5
Methyl (3R,4S,5R)-5-(2-naphthyl)methoxy-3,4-(O-isopropiliden)cyclohex-1-ene-1-carboxylate
(9h) − Alcohol 8 (100 mg, 0.44 mmol), N,Nꢀdiisopropylethylamine (0.12 mL, 0.70 mmol), 2ꢀ
(
bromomethyl)naphatalene (150 mg, 0.66 mmol) and sodium iodide (6.6 mg, 0.044 mmol). Reaction
time = 3 h. Chromatographic eluent: gradient of diethyl ether/hexanes [(15:85) to (20:80)]. 9h (85
20
1
3 3
mg, 59%) as a yellow oil. ꢃ[α ] =ꢃ–52.1º (c0.9, CHCl ). H NMR (300 MHz, CDCl ) δ: 7.81 (m,
D
4
H, 4×ArH), 7.48 (m, 3H, 3×ArH), 6.91 (m, 1H, H2), 4.87 (d, J = 12.6 Hz, 1H, OCHH), 4.82 (d, J =
2.6 Hz, 1H, OCHH), 4.78 (m, 1H, H3), 4.31 (t, J = 6.3 Hz, 1H, H4), 3.84 (m, 1H, H5), 3.76 (s, 3H,
1
OCH
3
), 2.75 (dd, J = 4.2 and 17.4 Hz, 1H, H6eq), 2.45 (dd, J = 6.6 and 17.4 Hz, 1H, H6ax) and 1.41
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4
5
5
5
5
5
5
5
5
5
5
6
1
3
(br s, 6H, 2×CH
3
) ppm. C NMR (75 MHz, CDCl
3
) δ: 166.7 (C), 135.6 (C), 134.6 (CH), 133.2 (C),
1
1
2
33.0 (C), 129.5 (C), 128.1 (CH), 127.8 (CH), 127.6 (CH), 126.4 (CH), 126.0 (CH), 125.8 (CH),
25.6 (CH), 109.4 (C), 75.9 (CH), 74.6 (CH), 72.3 (CH), 71.6 (OCH
2
), 52.0 (OCH
3
3
), 27.8 (CH ),
ꢀ1
+
6.2 (CH ) and 25.8 (CH ) ppm. IR (film) ʋ: 1710 (CO) cm . MS (ESI) m/z = 391 (MNa ). HRMS
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
+
24 5
calcd for C22H O Na (MNa ): 391.1516; found, 391.1523.
Methyl (3R,4S,5R)-5-(benzo[b]thiophen-5-ylmethoxy)-3,4-(O-isopropiliden)cyclohex-1-ene-1-
carboxylate (9i) − Alcohol 8 (250 mg, 1.1 mmol), N,Nꢀdiisopropylethylamine (0.3 mL, 1.8 mmol),
5
ꢀ(bromomethyl)benzo[b]thiophene (373 mg, 1.6 mmol) and sodium iodide (16.5 mg, 0.11 mmol).
Reaction time = 1.5 h. Chromatographic eluent: (40:60) diethyl ether/hexane. 9i (137 mg, 33%) as a
2
D
0
1
yellow oil. ꢃ[α ] =ꢃ−42.2° (c1.6, CHCl ). H NMR (300 MHz, CDCl ) δ: 7.84 (d, J = 8.4 Hz, 1H,
3
3
ArH), 7.80 (s, 1H, ArH), 7.43 (d, J = 5.4 Hz, 1H, ArH), 7.32 (m, 2H, 2×ArH), 6.89 (m, 1H, H2),
4
.81 (d, J = 12.0 Hz, 1H, OCHH), 4.76 (m, 2H, OCHH+H3), 4.28 (t, J = 6.3 Hz, 1H, H4), 3.81 (m,
H, H5), 3.75 (s, 3H, OCH ), 2.73 (dd, J = 4.5 and 17.4 Hz, 1H, H6eq), 2.42 (dd, J = 6.6 and 17.4 Hz,
1
3
13
1H, H6ax), 1.40 (s, 3H, CH
39.6 (C), 139.0 (C), 134.5 (CH), 134.2 (C), 129.5 (C), 124.1 (CH), 123.7 (CH), 122.7 (CH), 122.5
CH), 122.3 (CH), 109.3 (C), 75.9 (CH), 74.4 (CH), 72.2 (CH), 71.5 (CH ), 51.9 (OCH ), 27.7
3 3 3
) and 1.39 (s, 3H, CH ) ppm. C NMR (75 MHz, CDCl ) δ: 166.6 (C),
1
(
(
2
3
ꢀ1
+
CH
3
), 26.1 (CH
2
) and 25.8 (CH
3
) ppm. IR (ATR) ʋ: 1710 (CO) cm . MS (ESI) m/z = 397 (MNa ).
+
HRMS calcd for C20
H
22
O
5
SNa(MNa ): 397.1080; found, 397.1091.
General procedure for the synthesis of compounds 10 − A solution of acetal 9 (1 equivalent) in
ethanol (0.15 M) and aqueous solution of HCl (0.16 mL/mmol, 6 M) was heated at 60 °C for 1.5−4
h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure
and the resulting residue was purified by flash chromatography to give diol 10.
Methyl (3R,4S,5R)-5-benzyloxy-3,4-dihydroxycyclohex-1-ene-1-carboxylate (10a) − Acetal 9a
(
45.8 mg, 0.15 mmol), ethanol (1 mL) and HCl (25 ꢀL, 6 M). Reaction time = 4 h. Chromatographic
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D
eluent: (75:25) diethyl ether/hexane. 10a (40 mg, 95%) as a yellow oil. ꢃ
[α
]
=ꢃ–133.9º (c3.2,
1
CHCl
3
). H NMR (300 MHz, CDCl
3
) δ: 7.34 (m, 5H, 5×ArH), 6.86 (m, 1H, H2), 4.71 (d, J = 11.4
Hz, 1H, OCHH), 4.51 (d, J = 11.4 Hz, 1H, OCHH), 4.48 (t, J = 4.2 Hz, 1H, H3), 3.88–3.78 (m, 2H,
H4+H5), 3.76 (s, 3H, OCH ), 2.93 (dd, J = 4.2 and 17.7 Hz, 1H, H6ax) and 2.27 (br dd, J = 6.9 and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
1
3
1
7.7 Hz, 1H, H6 ) ppm. C NMR (75 MHz, CDCl ) δ: 166.8 (C), 137.8 (C), 136.0 (CH), 130.2 (C),
ax 3
128.5 (CH), 127.9 (CH), 127.6 (CH ), 74.1 (CH), 71.4 (OCH
and 28.5 (CH ) ppm. IR (film) ʋ: 3423 (OH) and 1715 (CO) cm . MS (ESI) m/z = 301 (MNa ).
HRMS calcd for C15
2 3
), 70.9 (CH), 65.8 (CH), 52.0 (OCH )
ꢀ1
+
2
+
18 5
H O Na (MNa ): 301.1046; found, 301.1039.
Methyl (3R,4S,5R)-3,4-dihydroxy-5-(3-methoxybenzyloxy)cyclohex-1-ene-1-carboxylate (10b) −
Acetal 9b (37 mg, 0.11 mmol), ethanol (0.7 mL) and HCl (18 ꢀL, 6 M). Reaction time = 1.5 h.
Chromatographic eluent: (80:20) ethyl acetate/hexane. 10b (30 mg, 90%) as a colourless oil.
2
D
0
1
ꢃ
[
α
]
=ꢃ–114.8º (c2.9, CHCl
3
). H NMR (300 MHz, CDCl
m, 4H, 3×ArH+H2), 4.69 (d, J = 11.7 Hz, 1H, OCHH), 4.48 (m, 2H, OCHH+H3), 3.82 (m, 2H,
), 3.75 (s, 3H, OCH ), 2.93 (dd, J = 3.9 and 18.3 Hz, 1H, H6eq) and 2.26
(dd, J = 6.3 and 18.3 Hz, 1H, H6 ) ppm. C NMR (75 MHz, CDCl ) δ: 166.7 (C), 159.7 (C), 139.4
3
) δ: 7.26 (t, J = 7.8 Hz, 1H, ArH), 6.87
(
H4+H5), 3.80 (s, 3H, OCH
3
3
13
ax
3
(
(
(
C), 135.9 (CH), 130.3 (C), 129.6 (CH), 120.0 (CH), 113.3 (CH), 74.1 (CH ), 71.3 (OCH
2
), 71.0
CH), 65.8 (CH), 55.2 (OCH
3
), 52.1 (OCH ) and 28.6 (CH ) ppm. IR (film) ʋ: 3432 (OH) and 1715
3 2
ꢀ1
+
+
CO) cm . MS (ESI) m/z = 331 (MNa ). HRMS calcd for C16
H
20
O
6
Na (MNa ): 331.1152; found,
3
31.1156.
Methyl (3R,4S,5R)-5-(3-fluorobenzyloxy)-3,4-dihydroxycyclohex-1-ene-1-carboxylate (10c) −
Acetal 9c (47 mg, 0.14 mmol), ethanol (0.9 mL) and HCl (25 ꢀL, 6 M). Reaction time = 4 h.
2
0
Chromatographic eluent: (70:30) ethyl acetate/hexane. 10c (41 mg, 99%) as a yellow oil. [α ] = –
D
1
1
13.0º (c4.2, CHCl
3
). H NMR (300 MHz, CDCl
3
) δ: 7.30 (m, 1H, ArH), 7.09–6.95 (m, 3H, ArH),
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6.90 (m, 1H, H2), 4.69 (d, J = 12.0 Hz, 1H, CHH), 4.52 (d, J = 12.0 Hz, 1H, CHH), 4.49 (t, J = 3.9
Hz, 1H, H3), 3.83 (m, 2H, H4+H5), 3.75 (s, 3H, OCH ), 2.90 (m, 1H, H6 ) and 2.27 (m, 1H, H6 )
3
ax
eq
13
ppm. C NMR (75 MHz, CDCl
3
) δ: 166.7 (C), 162.9 (C, JC-F = 245 Hz), 140.5 (C, JC-F = 7 Hz),
1
36.1 (CH), 130.0 (CH, JC-F = 9 Hz), 123.0 (CH, JC-F = 3 Hz), 114.7 (CH, JC-F = 22 Hz), 114.4 (CH,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J
C-F = 22 Hz), 74.4 (CH), 70.9 (CH), 70.6 (OCH
2
), 65.9 (CH), 52.1 (OCH
3
2
) and 28.4 (CH ) ppm. IR
ꢀ1
+
(
film) ʋ: 3431 (OH) and 1716 (CO) cm . MS (ESI) m/z = 319 (MNa ). HRMS calcd for
+
C
15
H
17
O
5
FNa (MNa ): 319.0952; found, 319.0949.
Methyl (3R,4S,5R)-3,4-dihydroxy-5-(3-nitrobenciloxy)cyclohex-1-ene-carboxylate (10e)
−
Acetal 9e (56.2 mg, 0.15 mmol), ethanol (1 mL) and HCl (30 ꢀL, 6 M). Reaction time = 3 h.
20
Chromatographic eluent: (60:40) ethyl acetate/hexane. 10e (27.2 mg, 56%) as a yellow oil. [α ] = –
D
1
171.5º (c1.0, CHCl
3
). H NMR (300 MHz, CDCl
3
) δ: 8.20 (br s, 1H, ArH), 8.13 (d, J = 8.1 Hz, 1H,
ArH), 7.65 (d, J = 7.5 Hz, 1H, ArH), 7.51 (t, J = 7.9 Hz, 1H, ArH), 6.86 (br s, 1H, H2), 4.79 (d, J =
1
2.2 Hz, 1H, OCHH), 4.65 (d, J = 12.2 Hz, 1H, OCHH), 4.51 (br s, 1H, H3), 3.88 (m, 2H, H5+H4),
1
3
2
3
.95 (s, 1H, OH), 2.85 (m, 2H, OH+H6ax) and 2.29 (m, 1H, H6eq) ppm. C NMR (75 MHz, CDCl )
δ: 166.8 (C), 148.4 (C), 140.3 (C), 136.3 (CH), 133.4 (CH), 130.1 (C), 129.6 (CH), 122.9 (CH),
1
22.3 (CH), 75.1 (CH), 71.0 (CH), 70.3 (CH), 66.1 (OCH
2
), 52.3 (OCH
3 2
) and 28.5 (CH ) ppm. IR
ꢀ1
+
(
ATR): 3415 (OH), 1710 (CO) and 1525 (NO) cm . MS (ESI) m/z = 346 (MNa ). HRMS calcd for
+
C
15
H
17NO
7
Na (MNa ): 346.0897; found, 346.0907.
Methyl (3R,4R,5R)-3,4-dihydroxy-5-((perfluorophenyl)methoxy)cyclohex-1-ene-1-carboxylate
(10g) − Acetal 9g (73.5 mg, 0.18 mmol), ethanol (1.2 mL) and HCl (32 ꢀL, 6 M). Reaction time = 2
h. Chromatographic eluent: (25:75) diethyl ether/hexane. 10g (43.7 mg, 66%) as a colourless oil.
2
0
1
ꢃ
[
α
]
=ꢃ−92.6° (c1.3, CHCl
3
). H NMR (500 MHz, CDCl
Hz, 1H, OCHH), 4.65 (d, J = 11.0 Hz, 1H, OCHH), 4.46 (t, J = 4.0 Hz, 1H, H3), 3.84 (dt, J = 5.0 and
.0 Hz, 1H, H5), 3.77−3.74 (m, 4H, OCH +H4), 2.97 (dd, J = 5.0 and 18.0 Hz, 1H, H6ax) 2.75 (br s,
3
) δ: 6.86 (m, 1H, H2), 4.79 (d, J = 11.0
D
8
3
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1
3
2H, 2×OH) and 2.28−2.23 (m, 1H, H6eq) ppm. C NMR (125 MHz, CDCl
3
) δ: 166.6 (C),
1
46.6−136.5 (m, 5×C), 135.8 (CH), 130.0 (C), 111.0 (td, JC-F = 3 and 18 Hz, C), 75.1 (CH), 70.9
19
(
CH ), 65.8 (CH ), 58.4 (OCH ), 52.1 (OCH ) and 28.5 (CH ) ppm. F NMR (282 MHz, CDCl ) δ:
2 3 2 3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
−143.1 (dd, J = 7 and 21 Hz, 2F), −153.2 (t, J = 21 Hz, 1F) and −161.5 (dt, J = 8 and 21 Hz, 2F)
ꢀ1
+
ppm. IR (film): 3332 (OH) and 1716 (CO) cm . MS (ESI) m/z = 391 (MNa ). HRMS calcd for
+
C
15
H
13
F
5
O
5
Na (MNa ): 391.0575; found, 391.0575.
Methyl (3R,4S,5R)-3,4-dihydroxy-5-(2-naphthyl)methoxycyclohex-1-ene-1-carboxylate (10h) −
Acetal 9h (65 mg, 0.18 mmol), ethanol (1.2 mL) and HCl (30 ꢀL, 6 M). Reaction time = 3 h.
Chromatographic eluent: (60:40) ethyl acetate/hexane. 10h (56 mg, 95%) as a colourless oil.
2
0
1
ꢃ
[
α
]
=ꢃ–93.5º (c5.2, CHCl
3
). H NMR (300 MHz, CDCl
×ArH), 6.86 (t, J = 1.5 Hz, 1H, H2), 4.84 (d, J = 11.7 Hz, 1H, OCHH), 4.65 (d, J = 11.7 Hz, 1H,
), 3.00–2.90 (m, 3H,
×OH+CHH), and 2.31 (dd, J = 6.3 and 17.7 Hz, 1H, CHH) ppm. C NMR (75 MHz, CDCl ) δ:
66.8 (C), 136.1 (CH), 135.2 (C), 133.2 (C), 133.0 (C), 130.1 (C), 128.3 (CH), 127.8 (CH), 127.6
), 70.9
3
) δ: 7.80 (m, 4H, 4×ArH), 7.47 (m, 3H,
D
3
OCHH), 4.48 (m, 1H, H3), 3.85 (m, 2H, H4+H5), 3.75 (s, 3H, OCH
3
13
2
1
3
(
CH), 126.5 (CH), 126.3 (CH), 126.2 (CH), 126.0 (CH), 125.6 (CH), 74.1 (CH), 71.5 (OCH
2
ꢀ1
(CH ), 65.9 (CH ), 52.0 (OCH
3
) and 28.5 (CH
2
) ppm. IR (film) ʋ: 3427 (OH) and 1710 (CO) cm .
+
+
MS (ESI) m/z = 351 (MNa ). HRMS calcd for C19
H
20
O
5
Na (MNa ): 351.1203; found, 351.1196.
Methyl
(3R,4S,5R)-5-(benzo[b]thiophen-5-ylmethoxy)-3,4-dihydroxy-cyclohex-1-ene-1-
carboxylate (10f) − Acetal 9f (119.7 mg, 0.32 mmol), ethanol (2.1 mL) and HCl (57 ꢀL, 6 M).
Reaction time = 4 h. Chromatographic eluent: (75:25) diethyl ether/hexane. 10h (30.4 mg, 90%) as a
20
D
1
colourless oil.
[α
]
3 3
= −23.2° (c1.3, CHCl ). H NMR (300 MHz, CDCl ) δ: 7.84 (d, J = 8.1 Hz, 1H,
ArH), 7.76 (s, 1H, ArH), 7.44 (d, J = 5.4 Hz, 1H, ArH), 7.30 (m, 2H, 2×ArH), 6.84 (m, 1H, H2),
4
.78 (d, J = 11.4 Hz, 1H, OCHH), 4.55 (d, J = 11.4 Hz, 1H, OCHH), 4.47 (m, 1H, H3), 3.89−3.76
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(m, 2H, H4+H5), 3.74 (s, 3H, OCH
3
), 3.10 (br s, 2H, 2×OH), 2.91 (dd, J = 4.2 and 17.7 Hz, 1H,
13
H6eq) and 2.30 (dd, J = 6.6 and 17.7 Hz, 1H, H6ax) ppm. C NMR (75 MHz, CDCl
3
) δ: 166.8 (C),
1
39.6 (C), 139.2 (C), 136.2(CH), 133.9 (C), 130.0 (C), 127.0 (CH), 124.2 (CH), 123.7 (CH), 122.8
(CH), 122.5 (CH), 74.0 (CH), 71.4 (CH ), 70.8 (CH), 65.9 (CH), 52.0 (OCH ) and 28.4 (CH ) ppm.
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
2
ꢀ1
+
18 5
IR (ATR) ʋ: 3406 (OH) and 1707 (CO) cm . MS (ESI): 357 (MNa ). HRMS calcd for C17H O SNa
+
(
MNa ): 357.0767; found, 357.0772.
(3R,4R,5R)-3,4-dihydroxy-5-(3-methylbenzyloxy)cyclohex-1-ene-1-carboxylic acid (3d) − A
solution of compound 9d (166.3 mg, 0.50 mmol) in ethanol (3.4 mL) and aqueous solution of HCl
(3.2 mL, 6 M) was heated at 60 °C for 3 h. After cooling to room temperature, the reaction mixture
was concentrated under reduced pressure and the resulting residue was purified by flash
chromatography, eluting with (60:40) ethyl acetate/hexane, to give acid 3d (42.3 mg, 30%), as a
2
D
0
1
yellow oil.
×ArH), 7.02 (t, J = 7.0 Hz, 1H, ArH), 6.73 (br s, 1H, H2), 4.55 (d, J = 11.9 Hz, 1H, OCHH), 4.50
d, J = 11.9 Hz, 1H, OCHH), 4.35 (br s, 1H, H3), 3.79 (m, 2H, H4+H5), 2.58 (br d, J = 18.3 Hz, 1H,
[
α
]
= −90.6º (c1.0, CH
3 3
OH). H NMR (300 MHz, CD OD) δ: 7.17−7.07 (m, 3H,
3
(
13
3 3
H6ax), 2.29 (m, 1H, H6eq) and 2.26 (s, 3H, CH ) ppm. C NMR (75 MHz, CD OD) δ: 170.0 (C),
1
39.8 (C), 139.3 (CH), 139.0 (C), 130.3 (C), 129.4 (CH), 129.3 (CH), 129.2 (CH), 125.9 (CH), 76.4
(
CH), 72.5 (OCH ), 70.8 (CH), 67.4 (CH), 28.4 (CH ) and 21.4 (CH ) ppm. IR (ATR): 3409 (OH)
2
3
2
−
1
and 1695 (CO) cm . MS (ESI) m/z = 277 (M−H). HRMS calcd for C15
17 5
H O (M−H): 277.1081;
found, 277.1081.
(3R,4R,5R)-3,4-dihydroxy-5-(4-methylbenzyloxy)cyclohex-1-ene-1-carboxylic acid (3f) − A
solution of compound 9f (306.7 mg, 0.92 mmol) in ethanol (6.1 mL) and aqueous solution of HCl (6
mL, 6 M) was heated at 60 °C for 3 h. After cooling to room temperature, the reaction mixture was
concentrated under reduced pressure and the resulting residue was purified by flash chromatography,
20
eluting with (60:40) ethyl acetate/hexane, to give acid 3f (84.3 mg, 33%), as a white solid. [α]D
=
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.
1
−78.0 º (c1.0, CH
3
OH). Mp: 134.0−134.5 ˚C H NMR (300 MHz, CD
H, 2×ArH), 7.00 (d, J = 8.0 Hz, 2H, 2×ArH), 6.67 (m, 1H, H2), 4.47 (d, J = 11.8 Hz, 1H, OCHH),
.42 (d, J = 11.8 Hz, 1H, OCHH), 4.26 (m, 1H, H3), 3.72 (m, 2H, H4+H5), 2.56−2.48 (m, 1H, H6ax),
3
OD) δ: 7.09 (d, J = 8.0 Hz,
2
4
1
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.23−2.17 (m, 1H, H6eq) and 2.17 (s, 3H, CH
) ppm. C NMR (75 MHz, CD
3
OD) δ: 170.0 (C),
),
) ppm. IR (ATR) ʋ: 3460 (OH), 3371 (OH) and
(M−H): 277.1081; found,
3
1
39.2 (CH), 138.4 (C), 136.8 (C), 130.3 (C), 129.9 (2×CH), 128.9 (2×CH), 76.2 (CH), 72.4 (OCH
0.8 (CH), 67.5 (CH), 28.4 (CH ) and 21.2 (CH
2
7
3
2
−1
1688 (CO) cm . MS (ESI) m/z = 277 (M−H). HRMS calcd for C15
17 5
H O
2
77.1081.
General procedure for the synthesis of compounds 3 − A solution of ester 10 (1 equivalent) in
THF (0.1 M) was treated with an aqueous solution of lithium hydroxide (2.5 equivalents, 0.5 M).
The resulting mixture was stirred at room temperature for 30 min and then MilliQ water was added.
The organic solvent was removed under reduced pressure and the resulting aqueous solution was
+
washed with diethyl ether (×3). The aqueous layer was treated with Amberlite IRꢀ120 (H ) until pH
6
. The filtrate and the washings were lyophilized to give the acids 3.
(
3R,4R,5R)-5-benzyloxy-3,4-dihydroxycyclohex-1-ene-1-carboxylic acid (3a) − Ester 10a (34 mg,
2
0
0.12 mmol), THF (1.2 mL) and LiOH (0.6 mL, 0.5 M). 3a (24 mg, 76%) as a white solid. [α ]D
=
1
−
92.0° (c2.6, MeOH). Mp: 210.7–212.2 ºC. H NMR (300 MHz, CD
3
OD) δ: 7.24 (m, 5H, 5×ArH),
6
.60 (m, 1H, H2), 4.61 (d, J = 12 Hz, 1H, OCHH), 4.55 (d, J = 12 Hz, 1H, OCHH), 4.30 (m, 1H,
H3), 3.77 (m, 2H, H4+H5), 2.68 (dd, J = 3.9 and 18 Hz, 1H, H6ax) and 2.26 (dd, J = 2.2 and 19.7 Hz,
13
1
H, H6eq) ppm. C NMR (75 MHz, CD
3
OD) δ: 172.6 (C), 140.1 (C), 135.9 (CH), 133.6 (C), 129.3
2
), 71.8 (CH), 67.9 (CH) and 29.8 (CH )
(
2×CH), 128.9 (2×CH), 128.6 (CH ), 76.5 (CH), 72.5 (OCH
2
ꢀ1
ppm. IR (ATR) ʋ: 3382 (OH), 3299 (OH) and 1686 (CO) cm . MS (ESI): 263 (M−H). HRMS calcd
15 5
for C14H O
(M−H): 263.0925; found, 263.0918.
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(3R,4R,5R)-3,4-dihydroxy-5-(3-methoxybenzyloxy)cyclohex-1-ene-1-carboxylic acid (3b) − Ester
1
0a (27.6 mg, 0.09 mmol), THF (0.9 mL) and LiOH (0.46 mL, 0.5 M). 3b (20.7 mg, 78%), as a
2
0
1
white solid. ꢃ[α ] =ꢃ−83.3° (c1.8, MeOH). Mp: 130.3−132.1 ºC. H NMR (300 MHz, CD
3
OD) δ:
.19 (t, J = 7.5 Hz, 1H, ArH), 6.89 (m, 2H, 2×ArH), 6.78 (dd, J = 3.9 and 7.8 Hz, 1H, ArH), 6.63 (m,
H, H2), 4.62 (d, J = 12.3 Hz, 1H, OCHH), 4.56 (d, J = 12.0 Hz, 1H, OCHH), 4.33 (m, 1H, H3),
D
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
1
3.79 (m, 2H, H4+H5), 3.75 (s, 3H, OCH
3
), 2.70 (d, J = 17.7 Hz, 1H, H6ax) and 2.28 (dd, J = 3.9 and
OD) δ: 174.9 (C), 163.8 (C), 144.2 (2×C), 138.6
), 74.2 (CH), 70.3
13
1
7.7 Hz, 1H, H6eq) ppm. C RMN (75 MHz, CD
3
(
CH), 132.8 (CH), 123.5 (CH), 116.7 (CH ), 116.6 (CH), 79.0 (CH), 74.9 (OCH
(CH), 58.1 (OCH ) and 32.2 (CH ) ppm. IR (ATR) ʋ: 3375 (OH) and 1686 (CO) cm . MS (ESI):
93 (M−H). HRMS calcd for C15 (M−H): 293.1031; found, 293.1027.
2
ꢀ1
3
2
2
17 6
H O
(3R,4R,5R)-5-(3-fluorobenzyloxy)-3,4-dihydroxycyclohex-1-ene-1-carboxylic acid (3c) − Ester
1
0c (38.1 mg, 0.13 mmol), THF (1.3 mL) and LiOH (0.66 mL, 0.5 M). 3c (24.8 mg, 68%), as a white
2
D
0
1
solid.
[α
]
= −78.4° (c2.9, MeOH). Mp: 123.9−124.4 ºC. H NMR (300 MHz, CD
3
OD) δ: 7.26 (dt,
J = 6.9 and 9.0 Hz, 1H, 1×ArH), 7.07 (m, 2H, 2×ArH), 6.91 (dt, J = 2.7 and 10.5 Hz, 1H, 1×ArH),
6.66 (br s, 1H, H2), 4.62 (d, J = 15.0 Hz, 1H, OCHH), 4.56 (d, J = 15.0 Hz, 1H, OCHH), 4.31 (m,
13
1
H, H3), 3.78 (m, 2H, H4+H5), 2.66 (m, 1H, H6 ) and 2.25 (m, 1H, H6 ) ppm. C NMR (75 MHz,
ax
eq
3
CD OD) δ: 171.4 (C), 164.4 (C, J = 291 Hz), 143.9 (C, J = 9 Hz), 137.4 (CH), 132.1 (C), 131.1 (CH,
J = 10 Hz), 124.2 (CH, J = 3 Hz), 115.3 (CH, J = 3 Hz), 115.0 (CH, J = 1 Hz), 76.8 (CH), 71.7 (CH2),
ꢀ
1
71.5 (CH), 67.7 (CH) and 29.3 (CH
2
) ppm. IR (ATR) ʋ: 3407 (OH), 3312 (OH) and 1686 (CO) cm .
MS (ESI): 281 (M−H). HRMS calcd for C H FO (M−H): 281.0831; found, 281.0823.
14
14
5
Sodium (3R,4R,5R)-3,4-dihydroxy-5-((3-nitrobenzyloxy)cyclohex-1-ene-1-carboxylate (3e) − A
solution of ester 10e (30 mg, 0.09 mmol) in THF (0.9 mL) was treated with an aqueous solution of
sodium hydroxide (0.19 mL, 0.5 M). The resulting mixture was stirred at room temperature for 30
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min and then MilliQ water was added. The organic solvent was removed under reduced pressure and
the resulting aqueous solution was washed with diethyl ether (×2) and lyophilized to give the sodium
2
D
0
1
salt 3e (28 mg, 98%), as a brown foam.
[α
]
= −23.7º (c1.0, H O). H NMR (300 MHz, D O) δ:
2
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8.26 (br s, 1H, ArH), 8.18 (d, J = 7.6 Hz, 1H, ArH), 7.80 (d, J = 7.5 Hz, 1H, ArH), 7.60 (t, J = 7.9
Hz, 1H, ArH), 6.43 (br s, 1H, H2), 4.81 (d, J = 10.3 Hz, 1H, OCHH), 4.73 (d, J = 10.3 Hz, 1H,
OCHH), 4.42 (br s, 1H, H3), 3.91 (m, 2H, H5+H4), 2.83 (dd, J = 4.1 and 17.5 Hz, 1H, H6ax) and
13
2
.26 (dd, J = 5.3 and 17.5 Hz, 1H, H6eq) ppm. C NMR (125 MHz, D
2
O) δ: 175.1 (C), 147.8 (C),
139.7 (C), 135.4 (C), 134.7 (CH), 130.8 (CH), 129.7 (CH), 123.0 (CH), 122.8 (CH), 75.4 (CH), 70.4
(CH), 70.3 (OCH
2
), 66.3 (CH) and 30.0 (CH
2
) ppm. IR (ATR) ʋ: 3345 (OH), 1570 (CO) and 1531
ꢀ1
+
+
(
NO) cm . MS (ESI) m/z = 332 (MNa ). HRMS calcd for C H NO Na (MNa ): 332.0741; found,
14
15
7
3
32.0741.
Sodium (3R,4R,5R)-3,4-dihydroxy-5-((perfluorophenyl)methoxy)cyclohex-1-ene-1-carboxylate
(3g) − A solution of ester 10g (38.0 mg, 0.10 mmol) in THF (1.0 mL) was treated with an aqueous
solution of sodium hydroxide (0.2 mL, 0.10 mmol, 0.5 M). The resulting mixture was stirred at room
temperature for 6 h and then MilliQ water was added. The organic solvent was removed under
reduced pressure and the resulting aqueous solution was washed with diethyl ether (×3) and
2
0
[α ]
lyophilized to give the sodium salt 3e (38.5 mg, 99%), as a white solid. = −71.4º (c1.0, H O).
D
2
1
Mp: 225.2 ºC (dec.). H NMR (500 MHz, D
2
O) δ: 6.43 (br s, 1H, H2), 4.88 (d, J = 12.0 Hz, 1H,
OCHH), 4.86 (d, J = 12.0 Hz, 1H, OCHH), 4.39 (m, 1H, H3), 3.92 (m, 1H, H5), 3.84 (m, 1H, H4),
13
2.64 (dd, J = 4.0 and 18.0 Hz, 1H, H6 ) and 2.23 (dd, J = 6.5 and 18.0 Hz, 1H, H6 ) ppm. C NMR
eq
ax
(
125 MHz, D
2
O) δ: 175.0 (C), 146.4−136.3 (m, 5×C), 135.4 (C), 130.6 (CH), 110.6 (t, JC-F= 10 Hz,
19
C), 75.7 (CH), 70.2 (CH ), 66.2 (CH ), 58.5 (OCH
2
) and 30.0 (CH
2
) ppm. F NMR (282 MHz, D
2
O)
δ: −143.8 (d, J = 16 Hz, 2F), −154.3 (t, J = 21 Hz, 1F) and −162.8 (dt, J = 6 and 21 Hz, 2F) ppm. IR
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