Synthesis and biological evaluation of new nanomolar competitive inhibitors of Helicobacter pylori type II dehydroquinase. Structural details of the role of the aromatic moieties with essential residues

Article abstract of DOI:10.1021/jm9010466

The shikimic acid pathway is essential to many pathogens but absent in mammals. Enzymes in its pathway are therefore appropriate targets for the development of novel antibiotics. Dehydroquinase is the third enzyme of the pathway, catalyzing the reversible dehydratation of 3-dehydroquinic acid to form 3-dehydroshikimic acid. Here we present the synthesis of novel inhibitors with high affinity for Helicobacter pylori type II dehydroquinase and efficient inhibition characteristics. The structure of Helicobacter pylori type II dehydroquinase in complex with the most potent inhibitor shows that the aromatic functional group interacts with the catalytic Tyr22 by π-stacking, expelling the Arg17 side chain, which is essential for catalysis, from the active site. The structure therefore explains the favorable properties of the inhibitor and will aid in design of improved antibiotics.

Full text of DOI:10.1021/jm9010466

J. Med. Chem. 2010, 53, 191–200 191
DOI: 10.1021/jm9010466
Synthesis and Biological Evaluation of New Nanomolar Competitive Inhibitors of Helicobacter pylori
Type II Dehydroquinase. Structural Details of the Role of the Aromatic Moieties with Essential
Residues†,‡
§
§
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Veronica F. V. Prazeres, Lorena Tizon, Jose M. Ot¥ero, Pablo Guardado-Calvo, Antonio L. Llamas-Saiz,
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Mark J. van Raaij, Luis Castedo, Heather Lamb, Alastair R. Hawkins, and Concepcion Gonzalez-Bello*
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Laboratorio de Quımica Organica (CSIC) y Departamento de Quımica Organica, Facultad de Quımica, Universidad de Santiago de Compostela,
15782 Santiago de Compostela, Spain, Departamento de Bioquımica y Biologıa Molecular, Facultad de Farmacia, Universidad de Santiago de
ꢀ
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´
´
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Compostela, 15782 Santiago de Compostela, Spain, ^Unidad de Rayos X, Edificio CACTUS, Universidad de Santiago de Compostela, 15782
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Santiago de Compostela, Spain, Instituto de Biologıa Molecular de Barcelona, Consejo Superior de Investigaciones Cientıficas (IBMB-CSIC),
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Parc Cientıfic de Barcelona, Baldiri Reixach 10-12, E-08028 Barcelona, Spain, and Institute of Cell and Molecular Biosciences, Medical School,
´
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University, Newcastle upon Tyne, Catherine Cookson Building, Framlington Place, Newcastle upon Tyne NE2 4HH, U.K.
Received July 14, 2009
The shikimic acid pathway is essential to many pathogens but absent in mammals. Enzymes in its
pathway are therefore appropriate targets for the development of novel antibiotics. Dehydroquinase is
the third enzyme of the pathway, catalyzing the reversible dehydratation of 3-dehydroquinic acid to
form 3-dehydroshikimic acid. Here we present the synthesis of novel inhibitors with high affinity for
Helicobacter pylori type II dehydroquinase and efficient inhibition characteristics. The structure of
Helicobacter pylori type II dehydroquinase in complex with the most potent inhibitor shows that the
aromatic functional group interacts with the catalytic Tyr22 by π-stacking, expelling the Arg17 side
chain, which is essential for catalysis, from the active site. The structure therefore explains the favorable
properties of the inhibitor and will aid in design of improved antibiotics.
Scheme 1. Shikimic Acid Pathway
Introduction
The capacity of antibiotics to cure infectious diseases that in
the past were fatal, such as tuberculosis, pneumonia, bubonic
plague, leprosy, and cholera, has ended in the false idea that
antibiotics are miracle medicines with “powers”. However, in
most of the cases, this belief exceeds their real pharmacology
properties. The excessive use and in many cases misuse of
antibiotics in medicine, veterinary, and agriculture has given
rise to a fast increase of the prevalence of resistant micro-
organism to antibiotics.1,2 Therefore, the development of new
agents that are able to overcome existing resistance mechan-
isms or that have novel mechanisms of action is much needed.
The ulcer-causing Helicobacter pylori bacterium is a good
example, with strains resistant to clarithromycin becoming
more and more prevalent.3,4 Specifically, it has been estimated
that the widespread of H. pylori resistance to clarithromycin is
<5% for adults in northern Europe but as high as 20% in
southern Europe.5 It is present in approximately 13% of
patients in the U.S., but in selected regions such as Alaska,
it has been reported to have rates as high as 30%.4,6 In
addition, H. pylori infected persons are at increased risk for
mucosa-associated lymphoid tissue lymphoma and gastric
adenocarcinoma.7
In the past few years, our research group has been studying
the possible development of new antibiotics whose action is
based on the selective and effective inhibition of an essential
route in bacteria, the shikimic acid pathway.8 This route
consists of seven enzymes that catalyze the sequential conver-
sion of erythrose 4-phosphate (1) and phosphoenol pyruvate
(2) to chorismic acid (3), which is the precursor in the synthesis
of aromatic compounds, including the aromatic amino
acids L-phenylalanine, L-tyrosine, and L-tryptophan, folate
cofactors, ubiquinone, and vitamins E and K (Scheme 1).9,10
This pathway is present in bacteria, fungi, and higher
plants and has been discovered in apicomplexan parasites,
Plasmodium falciparum (which are the cause of malaria),
†Coordinates and structure factors are available from the Protein
Data Bank with accession code 2WKS.
‡Dedicated to Prof. Josep Font on the occasion of his 70th birthday.
*To whom correspondence should be addressed. Phone: þ34 981
563100 extension 14368. Fax: þ34 981 595012. E-mail: concepcion.
gonzalez.bello@usc.es.
r
2009 American Chemical Society
Published on Web 11/13/2009
pubs.acs.org/jmc

192 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Prazeres et al.
Scheme 2. Proposed E1CB Mechanism for the Enzymatic Conversion of 3-Dehydroquinic Acid (4) to 3-Dehydroshikimic Acid (6)
Catalyzed by DHQ2a
a The reaction proceeds via the enol intermediate 5. Relevant residues are indicated.
Toxoplasma gondii, and Cryptosporidium parvum.11 The ab-
sence of the shikimate pathway in mammals, combined with
its essential nature in certain microorganisms,12,13 makes it an
attractivetarget for the developmentofnew antimicrobialand
antiparasitic agents.
the mechanism is the elimination of the C1 hydroxyl group
catalyzed by active-site His102.
Recently, several enol mimics, compounds 8, have been
reported as competitive inhibitors of H. pylori type II dehy-
droquinase (DHQ2-Hp) (Figure 1).21,22 These compounds
have an aromaticringora heteroaryl grouponC3, which were
incorporated to establish π-stacking interactions with essen-
tial Tyr22. The biaryl derivative 8a21 and the 2-thienyl deri-
vative 8b22 proved to be the most potent ones, having a Ki of
490 and 540 nM, respectively. On the other hand, docking and
NMR studies also suggested strong lipophilic interactions of
the aromatic or the heterocyclic moieties with the enzyme
residues Pro9, Asn10, Ile11, Gly78, and Ala79.22
In the search for more potent inhibitors against this enzyme
as potential antimicrobial agents, we report here the synthesis
of four novel enol mimics, compounds 9, with high affinity
against Helicobacter pylori dehydroquinase (Figure 2). The
crystal structure of DHQ2-Hp in complex with the enol mimic
9d is also reported. The structure shows that the aromatic
moietyindeed interacts withthe catalytic Tyr22by π-stacking,
as designed. More importantly, it clarifies the possible inter-
action of the aromatic moiety with the essential Arg17.
Inhibition studies of these compounds against DHQ2-Hp
and docking studies using GOLD4.0 are also described.
Dehydroquinase (3-dehydroquinate dehydratase, DHQ,a
EC 4.2.1.10) is the third enzyme of the pathway, catalyzing the
reversible dehydration of 3-dehydroquinic acid (4) to form
3-dehydroshikimic acid (6) (Scheme 2). Biochemical and
genetic studies have shown that there are two different
dehydroquinases, known as type I and type II, which do not
have sequence homology and catalyze the same overall reac-
tion but utilizing completely different mechanisms.14,15 The
type I enzyme (DHQ1) is found in plants, fungi, archaebac-
teria, and eubacterial species like Escherichia coli and
Salmonella typhi.16 Type II dehydroquinase (DHQ2) is found
in bacteria like Streptomyces coelicolor, Mycobacterium
tuberculosis, and Helicobacter pylori.17
The proposed dehydration mechanism of DHQ218 involves
the abstraction of the pro-S hydrogen from C2 of the sub-
strate, the 3-dehydroquinic acid (4), by Tyr22, for which the
lower pKa is the result of a basic environment formed by
Arg17 and Arg109. Tyr22 and Arg17 have been identified by
chemical modification and site-directed mutagenesis studies
as being essential for enzyme activity.19,20 Both residues are on
the flexible loop that closes over the active site upon substrate
binding. The enol intermediate 5 is then generated, which is
stabilized by a conserved molecule of water close to the
carbonyl group side chain of Asn10, the carbonyl group of
Pro9, and the main-chain amide of Ala79. The final step of
Results
Synthesis of O-Alkylaryl Derivatives 9. The synthesis of
O-alkylaryl derivatives 9 was carried out by a three-step
reaction sequence from our previously reported ketone 1023
(Scheme 3). The alkylaryl moieties of compounds 9 were
introduced by O-alkylation of ketone 10. Various bases
(LDA, LHMDS, KHMDS), solvents (THF, DMF), leaving
groups (tosylates, bromides, and iodides) and reaction tem-
peratures were investigated. The best results were achieved
using bromides as alkylating agents, KHMDS at -78 ꢀC or
LHMDS at room temperature. Under these conditions a
chromatographically separable mixture of the O-alkylated
a Abbreviations: DHQ, 3-dehydroquinate dehydratase or dehydro-
quinase; DHQ1, type I dehydroquinase; DHQ2, type II dehydroquinase;
DHQ2-Hp, type II dehydroquinase from Helicobacter pylori; PDB,
Protein Data Bank; KHMDS, potassium hexamethyldisilazane;
LHMDS, lithium hexamethyldisilazane; TBS, tert-butyldimethylsilyl;
TBAF, tetrabutylammonium fluoride; DMF, N,N-dimethylformamide;
THF, tetrahydrofuran; DTT, 1,4-dithiothreitol; Tris, tris(hydroxy-
methyl)aminomethane.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 193
Scheme 3a
Figure 1. Enol mimics as competitive inhibitors of DHQ2-Hp.
Figure 2. New enol mimics.
11 and the dialkylated products 12 was obtained in moderate
yield. The required bromides 15 were prepared by bromina-
tion of the corresponding commercially available alcohols
14. Finally, deprotection of the TBS groups with TBAF
followed by basic hydrolysis of the corresponding lactone 13
gave the desired enol mimics 9.
Inhibition Assay Results. Compounds 9 were assayed in the
presence of 3-dehydroquinic acid (4) for their inhibitory
properties against DHQ2-Hp. The inhibition data, as
obtained from Dixon plots (1/v vs [I]),24 are summarized
in Table 1. The UV spectrophotometric assay was used
to measure the initial rate of product formation, detec-
ting the enone-carboxylate chromophore at 234 nm in
3-dehydroshikimic acid (6).
a Reagents and conditions: (a) (1) KHMDS, DMF, -78 ꢀC or
LHMDS, DMF, room temp, (2) 15; (b) TBAF, THF, 0 ꢀC; (c) NaOH,
THF, room temp; (d) PPh3, CBr4, DCM, room temp; (e) PBr3, Py, 0 ꢀC
to room temp.
Table 1. Ki (nM) of Compounds 9 against DHQ2-Hpa
All the compounds were shown to be reversible competi-
tive inhibitors of DHQ2-Hp, proving to have (with the
exception of thiophene 9a) higher affinity than the best
previously reported competitive inhibitors, compounds
8a21 and 8b.22 Substitution of the thienyl ring by a benzothio-
phenyl one improves more than 5 times the inhibition
potency of the corresponding analogues. There is a slight
difference in Ki among 5-benzothiophenyl 9b (Ki = 166 nM)
and 2-benzothiophenyl derivatives 9c (Ki = 132 nM), but
almost no affinity changes were found with the incorpora-
tion of a methyl group on the benzothiophene motif. The
methylbenzothiophene 9d proved to be the most potent of
the series with a Ki of 130 nM.
a 50 mM Tris-HCl, pH 7.0, 25 ꢀC.
Structural Studies. To obtain structural information on
the binding mechanism of these inhibitors, we cocrystallized
the most potent competitive inhibitor of the series, methyl-
benzothiophene 9d, with DHQ2-Hp. Data were collected
from cryocooled crystals using synchrotron radiation and
processed (Table 2). The structure was solved by molecular
replacement, using the crystal structure of dehydroquinase
bound to citrate25 as a search model. Difference density
analysis showed the two structures are virtually identical
(1.0 A root-mean-square difference after superimposition),
but amino acids 15-23 changed conformation upon inhibi-
tor binding. In the same region, calculated maps showed
clear electron density for the inhibitor molecule, which was
modeled at full occupancy in each of the six crystallographi-
cally independent monomers (Figure 3; see also Supporting
Information). Parallel refinements were performed with the
benzothiophene ring in each of two orientations, rotated

194 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Prazeres et al.
Table 2. Crystallographic Data Collection and Refinement Statistics of
DHQ2-Hp Complex with Inhibitor 9d
parametera
space group
cell parameters (A)
I41
a = b = 157.85,b
c = 99.78
0.8150
wavelength (A)
detector
MAR555 flat
panel detector
350.0
crystal-to-detector
distance (mm)
observed reflectionsc
resolution range (A)
25 927 (3794)d
31.0-2.95
(3.11-2.95)
74.7
Wilson B (A2)
multiplicity
completeness
Rmerge
6.5 (6.5)
0.999 (0.999)
0.103 (0.394)
Refinemente
reflections used
in refinementc
reflections used
for Rfree
24653 (3579)
1198 (155)
resolution
range (A)
20.0-2.95
(3.11-2.95)
0.206 (0.29)
0.236 (0.31)
0.014/1.5
Figure 3. Electron density for inhibitor 9d in monomer A of the
crystal structure of DHQ2-Hp. From the model obtained by
molecular replacement, amino acids 15-23 of each of the six
monomers were removed and refinement was performed with tight
noncrystallographic restraints to obtain unbiased density for the
inhibitor molecules and other model changes. A maximum-like-
lihood weighted 2Fo - Fc map33 contoured at 1σf is shown up to 3 A
around the inhibitor molecules. Onto the map, the final model, as
deposited into the database, including inhibitor molecules, is super-
posed.
R factorf
g
Rfree
rmsd (bonds (A)/angles
(deg))
Final Model
protein/inhibitor/water
atoms
average B protein (A2)/
inhibitor (A2)/
water (A2)
7092/144/21
52.2/58.2/37.0
with side chain atoms of Met13, Leu14, and Pro19, establish-
ing important lipophilic interactions with this part of the
active site (Figure 5c). In addition, hydrogen H-2 of the
cyclohexene ring and the methylene group of the inhibitor
side chain are in close contact with the side chain of Leu11.
The methyl group of the benzothiophene ring is close to the
side chain of Leu93 and Met92 of the neighbor subunit of the
DHQ2-Hp.
Ramachandran
statisticsh (%)
93.5/100.0
a Results from MOSFLM (ref 22). b 1 Å is 0.1 nm. c No σ cutoff or other
restrictions were used for inclusion of reflections. d Values in parentheses are
for the highest resolution bin, where applicable. e Results from REFMAC (ref
P
P
33). f R = ||Fobs(hkl)| - |Fcalc(hkl)||/ |Fobs(hkl)|. g According to ref 31.
h According to the program MOLPROBITY (ref 34). The percentages
indicated are for residues in favored and total allowed regions, respectively.
Docking Studies. In order to rationalize the inhibition
observations noted above, docking studies were carried out
using GOLD 4.026 and the enzyme geometries found in
crystal of DHQ2-Hp binding inhibitor 9d. A good correla-
tion between the experimental inhibition data and the scored
solution of ligands 9a-c were found. Ligands 9a-c docked
with its cyclohexene ring and carboxyl group occupying
approximately the same locations as those of the ligand 9d
in the crystal structure. The docking results also showed that
the ligands side chains are in a similar orientation to binding
inhibitor 9d (Figure 5). However, important differences were
found in the space occupied by the side chain of the ligands
and therefore in their proximity to side chain atoms of
Met13, Leu14, and Pro19 of the active site and Leu93 and
Met92 of the neighbor subunit of the enzyme. It can be seen
from Figure 5c that in the proposed binding mode of ligand
9a, its side chain occupies only part of the benzothiophene
pocket. In this case, the flexible loop including Arg17 is
probably closer to the active site than in the crystal structure
of DHQ2-Hp/9d complex. Docking studies also predict a
stronger affinity of the 2-benzothiophene derivative 9c than
its 5-isomer, derivative 9b. This fact could be a result of
the balance between the differences in the strength of the
π-stacking interactions between the benzothiophene moiety
180ꢀ around the bond that connects it to the CH2 group.
Refinement parameters were slightly better for the orienta-
tion shown (R-factor 0.1% improved, Rfree of 0.4%), and the
maps also suggested the more electron dense sulfur atom to
be located on the side of the ring as shown in Figure 3 (see
also Supporting Information). However, at the resolution of
the studies reported, we cannot exclude the presence of a
mixture of the two conformations. After refinement, tem-
perature factors for the carbasugar moiety were 30-50 A2,
comparable to those of surrounding amino acids (around 35
A2). The benzothiophene ring system has higher temperature
factors (around 75 A2) and is more poorly ordered; corre-
spondingly, electron density is weaker for this part of the
inhibitor (Figure 3). Surrounding amino acids (loop 15-23)
have similar temperature factors. Thus, this loop, in the
presence of inhibitor, is conformationally flexible. Despite
the disorder, the structure shows that the benzothiophene
moiety interacts with Tyr22 by π-stacking, expelling Arg17
from the active site (Figure 4). The benzothiophene ring
system takes the place of the side chain of Arg17 in the active
site, an essential residue for catalysis. This crystal structure
also shows that the benzothiophene moiety is in close contact

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 195
Figure 4. Superposition of DHQ2-Hp structure with (orange) and without inhibitor 9d (light-blue). The structure without inhibitor is PDB
entry 2C4V. Two different orientations are shown in panels a and b, zoomed-in on the active site of monomer A. While Tyr22 only slightly
adjusts its position, the side chain of Arg17 flips out of the active site as a result of the benzothiophene ring system taking its place. Arg109 (not
shown) does not change position significantly.
Figure 5. Comparison of the position of inhibitor 9d in the enzyme-inhibitor crystal structure of DHQ2-Hp with the docking results of the
highest score solution of ligands: (a) 9a (green); (b) 9b (blue); (c) selected view of GOLD-predicted binding of ligand 9a.
and the aromatic ring of Tyr22 and the lipophilic interactions
with the above-mentioned residues.
identified to act as the base in the enzymatic mechanism.
More importantly, this structure clarifies the possible inter-
action of the aromatic moiety with Arg17, a residue also
essential for catalysis that is responsible, together with
Arg109, for the low pKa of the Tyr22. The competitive
inhibitor 9d expels Arg17 from the active site, and the
benzothiophene moiety occupies the place of Arg17 side
chain. This fact is probably due to important lipophilic
interactions that the benzothiophene moiety can establish
with the side chain residues of this part of the active site,
in particular Met13, Leu14, Pro19, and Leu11, and also
with the side chain of Leu93 and Met92 of the neighbor
subunit.
Therefore, we have shown that the high affinity of this new
enol mimics is due to two effects with essential residues for the
catalysis, i.e., a π-stacking interaction between the benzothio-
phene ring and the aromatic ring of Tyr22 and, on the other
hand, the expulsion of the Arg17 from the active site. The
availability of this structure now opens the possibility for the
structural design of the next generation of competitive inhi-
bitors against this enzyme, which is in progress.
Conclusions and Final Remarks
Four new enol mimics, compounds 9, were synthesized and
tested as inhibitors of DHQ2-Hp, the third enzyme of the
shikimic acid pathway. All of these compounds proved to be
reversible competitive inhibitors of this enzyme. With the
exception of 2-thienyl derivative 9a, all the reported com-
pounds proved to be more potent than the best previously
reported compounds for this enzyme, having Ki ranging from
130 to 166 nM, representing an improved affinity of almost
4-fold. The 2-benzothiophenyl derivatives 9c and 9d proved to
be the most potent ones with Ki of 132 and 130 nM, respec-
tively.
The most potent competitive inhibitor of the series, methyl-
benzothiophene 9d was cocrystallized with DHQ2-Hp, and its
structure has been solved at 2.95 A. This crystal structure
proves that the benzothiophene moiety of 9d interacts with
Tyr22 by π-stacking as designed, a residue that has been

196 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Prazeres et al.
Experimental Section
toluene) and dry DMF (1.5 mL) under inert atmosphere. The
resultant solution was cooled to -78 ꢀC, and then a solution of
ketone 1023 (300 mg, 0.75 mmol) in a 1:1 mixture of DMF and
toluene (7.5 mL), both dry, was added. After the mixture was
stirred for 30 min, a solution of 2-(bromomethyl)thiophene (15a)
(265 mg, 1.50 mmol) in a 3:2 mixture of DMF and toluene (6.25
mL), both dry, was added. After 45 min, the reaction mixture was
diluted successively with diethyl ether and water. The organic
phase was separated, and the aqueous layer was extracted with
diethyl ether (ꢀ2). All the combined organic extracts were dried
(anhydrous Na2SO4), filtered, and evaporated under reduced
pressure. The obtained residue was purified by flash chromatog-
raphy, eluting with diethyl ether-hexanes [(1) 0:100, (2) 5:95] to
afford O-alkyl derivative 11a (67 mg, 18%) and dialkyl derivative
12a (153 mg, 35%).
General Procedures. All starting materials and reagents were
commercially available and used without further purification.
FT-IR spectra were recorded as NaCl plates, or KBr discs. [R]D
values are given in 10-1 deg cm2 g-1. 1H NMR spectra (250, 300,
400, and 500 MHz) and 13C NMR spectra (63, 75, 100, and 125
MHz) were measured in deuterated solvents. J values are given
in hertz. NMR assignments were made by a combination of 1D,
COSY, and DEPT-135 experiments. Purities of compounds 9
1
were determined by a combination of H NMR and reverse-
phase HPLC and were found to be >95%.
(2-Bromomethyl)thiophene (15a). A solution of thien-2-yl-
methanol (14a) (1 mL, 10.55 mmol), phosphorus tribromide
(1.5 mL, 15.83 mmol), and three drops of dry pyridine in dry
dichloromethane (40 mL), under argon and 0 ꢀC, was stirred for
1 h. During this period, the reaction mixture was allowed to
reach room temperature. Water was then added, and the organic
phase was separated. The aqueous layer was extracted with
dichloromethne (ꢀ2). All the combined organic extracts were
washed with saturated sodium bicarbonate, dried (anhydrous
Na2SO4), filtered, and concentrated under reduced pressure to
afford bromide 15a (1.5 g, 80%) as a light-yellow oil. 1H NMR
(250 MHz, CDCl3) δ 7.73 (dd, 1H, J = 5.0 and 1.0 Hz, H-5), 7.52
(d, 1H, J = 3.0 Hz, H-3), 7.35 (dd, 1H, J = 5.0 and 3.0 Hz, H-4),
and 5.16 (s, 2H, CH2Br) ppm. 13C NMR (63 MHz, CDCl3)
δ 141.9 (C), 131.8 (CH), 128.4 (CH), 128.2 (CH), and 26.7
(CH2) ppm.
(5-Bromomethyl)benzo[b]thiophene (15b). To a stirred solu-
tion of benzo[b]thiophen-5-ylmethanol (14b) (100 mg, 0.61
mmol) in dry dichloromethane (15 mL) and under argon,
PPh3 (290 mg, 1.09 mmol) and then CBr4 (240 mg, 0.73 mmol)
were added. After the mixture was stirred for 1 h, diethyl ether
was added and the resulting precipitate was filtered and washed
with diethyl ether. The solvents were removed under reduced
pressure and the crude residue was purified by flash chroma-
tography, eluting with (10:90) diethyl ether/hexanes to afford
Data for 11a. White solid. Mp: 65-67 ꢀC. [R]2D0 -133ꢀ (c 1.2,
CHCl3). 1H NMR (250 MHz, CDCl3) δ 7.31 (dd, 1H, J = 5.0
and 1.2 Hz, ArH), 7.03 (d, 1H, J = 2.8 Hz, ArH), 6.98 (dd, 1H,
J = 5.0 and 3.8 Hz, ArH), 5.01 (s, 1H, H-2), 4.90 (d, 1H, J =
11.5 Hz, CHHAr), 4.84 (d, 1H, J = 11.5 Hz, CHHAr), 4.47 (dd,
1H, J = 5.2 and 3.5 Hz, H-5), 4.14 (d, 1H, J = 3.5 Hz, H-4), 2.40
(d, 1H, J = 10.5 Hz, H-6ax), 2.33 (dd, 1H, J = 10.5 and 5.2 Hz,
H-6eq), 0.93 (s, 9H, C(CH3)3), 0.87 (s, 9H, C(CH3)3), 0.19 (s, 3H,
SiCH3), 0.15 (s, 3H, SiCH3), 0.08 (s, 3H, SiCH3), and 0.05 (s, 3H,
SiCH3) ppm. 13C NMR (63 MHz, CDCl3) δ 176.1 (C), 153.2 (C),
137.7 (C), 127.1 (CH), 126.5 (CH), 126.3 (CH), 104.8 (CH), 75.2
(CH), 73.6 (C), 67.3 (CH), 64.3 (CH2), 37.9 (CH2), 25.6
(C(CH3)3), 25.5 (C(CH3)3), 18.0 (2 ꢀ C(CH3)3), -3.1 (2 ꢀ
CH3), -4.6 (CH3), and -5.3 (CH3) ppm. IR (KBr): 1801
(CdO) cm-1. MS (CI) m/z (%) 497 (MHþ). HRMS calcd for
C24H41O5SSi2 (MHþ): 497.2213; found, 497.2214.
Data for 12a. Yellow oil. [R]2D0 -127ꢀ (c 1.0, CHCl3). 1H NMR
(300 MHz, CDCl3) δ 7.29 (d, 1H, J = 4.2 Hz, ArH), 7.06 (d, 1H,
J = 4.5 Hz, ArH), 6.92 (m, 3H, 3 ꢀ ArH), 6.77 (s, 1H, ArH), 4.96
(s, 2H, CH2O), 4.54 (m, 1H, H-5), 4.42 (br s, 1H, H-4), 3.79 (d,
1H, J = 15.0 Hz, CHHAr), 3.69 (d, 1H, J = 15.0 Hz, CHHAr),
2.53 (d, 1H, J = 10.8 Hz, H-6ax), 2.41 (dd, 1H, J = 10.8 and 5.7
Hz, H-6eq), 0.95 (s, 9H, C(CH3)3), 0.80 (s, 9H, C(CH3)3), 0.20 (s,
3H, CH3), 0.18 (s, 3H, CH3), 0.17 (s, 3H, CH3), and 0.06 (s, 3H,
CH3) ppm. 13C NMR (75 MHz, CDCl3) δ 175.3 (C), 148.2 (C),
142.8 (C), 138.8 (C), 129.7 (C), 126.9 (CH), 126.7 (CH), 126.4
(CH), 126.3 (CH), 124.5 (CH), 122.8 (CH), 74.6 (CH þ C), 67.5
(CH2), 67.2 (CH), 37.5 (CH2), 25.7 (C(CH3)3), 25.5 (C(CH3)3),
24.7 (CH2), 18.1 (C(CH3)3), 18.0 (C(CH3)3), -3.3 (CH3), -3.5
1
bromide 15b (129 mg, 93%) as a white amorphous solid. H
NMR (250 MHz, CDCl3) δ 7.86 (m, 2H, 2xArH), 7.49 (d, 1H,
J = 5.5 Hz, ArH), 7.39 (d, 1H, J = 8.7 Hz, ArH), 7.33 (d, 1H,
J = 5.5 Hz, ArH), and 4.66 (s, 2H, CH2O) ppm. 13C NMR (63
MHz, CDCl3) δ 139.7 (C), 139.7 (C), 133.8 (C), 127.4 (CH),
125.2 (CH), 123.9 (CH), 123.7 (CH), 122.8 (CH), and 34.1 (CH2)
ppm. MS (CI) m/z (%) 227 and 229 (MHþ). HRMS calcd for
C9H8S79Br (MHþ): 226.9530; found, 226.9532.
(2-Bromomethyl)benzo[b]thiophene (15c). The reaction was
carried out as for bromide 15b by using benzo[b]thiophen-2-
ylmethanol (14c) (1.0 g, 6.09 mmol) as starting material, 2.88 g
of PPh3, 2.42 g of CBr4, and 152 mL of dichloromethane. Yield:
(CH3), and -4.5 (2 ꢀ CH3) ppm. IR (film): 1799 (CdO) cm-1
.
MS (CI) m/z (%) 593 (MHþ). HRMS calcd for C29H45O5S2Si2
(MHþ): 593.2247; found, 593.2248.
(1R,4S,5R)-1,4-Di(tert-butyldimethylsilyloxy)-3-(benzo[b]ti-
ophen-5-yl)methoxycyclohex-2-en-1,5-carbolactone (11b) and (1R,-
4S,5R)-1,4-Di(tert-butyldimethylsilyloxy)-3-(benzo[b]tiophen-5-yl)-
methoxy-2-(benzo[b]tiophen-5-yl)methylcyclohex-2-en-1,5-carbolac-
tone (12b). The reaction was carried out as for compounds 11a and
12a by using 500 mg of ketone 10 and 570 mg of (5-bromo-
methyl)benzo[b]thiophene (15b) as starting materials. Yield: 173
mg (25%) of O-alkyl derivative 11b and 237 mg (27%) of dialkyl
derivative 12b.
1
1.08 g (79%). White amorphous solid. H NMR (250 MHz,
CDCl3) δ 7.82-7.70 (m, 2H, 2 ꢀ ArH), 7.40-7.33 (m, 3H, 3 ꢀ
ArH), and 4.80 (s, 2H, CH2) ppm. 13C NMR (63 MHz, CDCl3) δ
141.0 (C), 140.5 (C), 139.1 (C), 125.0 (C), 124.6 (CH), 124.5
(CH), 123.8 (CH), 122.4 (CH), and 27.3 (CH2) ppm.
2-(Bromomethyl)-5-methylbenzo[b]thiophene (15d). The reac-
tion was carried out as for bromide 15b by using (5-methylbenzo-
[b]thiophen-2-yl)methanol (14d) (50 mg, 0.28 mmol), 131 mg of
PPh3, 113 mg of CBr4, and 7.0 mL of dichloromethane. Yield: 59
Data for 11b. Colorless oil. [R]2D0 -108ꢀ (c 1.2, CHCl3). H
1
NMR (250 MHz, CDCl3) δ 7.79 (d, 1H, J = 8.2 Hz, ArH), 7.72
(s, 1H, ArH), 7.39 (d, 1H, J = 5.2 Hz, ArH), 7.24 (m, 2H, 2 ꢀ
ArH), 4.97 (s, 1H, H-2), 4.82 (d, 1H, J = 11.2 Hz, CHHO), 4.74
(d, 1H, J = 11.2 Hz, CHHO), 4.43 (dd, 1H, J = 5.0 and 3.5 Hz,
H-5), 4.13 (d, 1H, J = 3.5 Hz, H-4), 2.35 (d, 1H, J = 10.5 Hz, H-
6ax), 2.28 (m, 1H, H-6eq), 0.87 (s, 9H, C(CH3)3), 0.82 (s, 9H,
C(CH3)3), 0.09 (s, 3H, SiCH3), 0.04 (s, 3H, SiCH3), 0.02 (s, 3H,
SiCH3), and -0.01 (s, 3H, SiCH3) ppm. 13C NMR (63 MHz,
CDCl3) δ 176.1 (C), 153.4 (C), 139.6 (C), 139.3 (C), 131.9 (C),
127.0 (CH), 124.0 (CH), 123.7 (CH), 122.8 (CH), 122.4 (CH),
104.9 (CH), 75.2 (C), 73.7 (C), 69.6 (CH2), 67.4 (CH), 38.0
(CH2), 25.6 (C(CH3)3), 25.5 (C(CH3)3), 18.0 (C(CH3)3), 17.9
(C(CH3)3), -3.2 (2 ꢀ SiCH3), -4.6 (SiCH3), and -5.2 (SiCH3)
1
mg (88%). White amorphous solid. Mp 98-101 ꢀC. H NMR
(250 MHz, CDCl3) δ 7.67 (d, 1H, J = 8.3 Hz, H-4), 7.53 (br s, 1H,
H-7), 7.25 (s, 1H, H-3), 7.18 (d, 1H, J = 8.3 and 0.8 Hz, H-5),
4.79 (s, 2H, CH2), and 2.47 (s, 3H, CH3) ppm. 13C NMR (63
MHz, CDCl3) δ 141.0 (C), 139.5 (C), 137.7 (C), 134.3 (C), 126.7
(CH), 124.2 (CH), 123.7 (CH), 122.0 (CH), 27.5 (CH2), and 21.3
(CH3) ppm.
(1R,4S,5R)-1,4-Di(tert-butyldimethylsilyloxy)-3-(thien-3-yl)meth-
oxycyclohex-2-en-1,5-carbolactone (11a) and (1R,4S,5R)-1,4-di(tert-
butyldimethylsilyloxy)-3-(thien-3-yl)methoxy-2-(thien-3-yl)methyl-
cyclohex-2-en-1,5-carbolactone (12a). A flame-dried round-bot-
tom flask was charged with KHMDS (3 mL, 1.5 mmol, 0.5 M in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 197
ppm. IR (film): 1801 (CdO) cm-1. MS (CI) m/z (%) 547 (MHþ).
HRMS calcd for C28H43O5SSi2 (MHþ): 547.2370; found,
547.2375.
124.2 (CH), 123.8 (CH), 123.6 (CH), 123.1 (CH), 122.8 (CH),
122.6 (CH), 122.3 (CH), 121.9 (CH), 121.2 (CH), 74.7 (C), 74.6
(CH), 68.5 (CH2), 67.4 (CH), 37.5 (CH2), 25.7 (C(CH3)3), 25.5
(C(CH3)3), 18.1 (C(CH3)3), 18.0 (C(CH3)3), -3.3 (SiCH3), -3.4
(SiCH3), -4.4 (SiCH3), and -4.5 (SiCH3) ppm. IR (film): 1799
(CdO) cm-1. MS (CI) m/z (%) 693 (MHþ). HRMS calcd for
C37H49O5S2Si2 (MHþ): 693.2560; found, 693.2563.
Data for 12b. Colorless oil. [R]2D0 -87ꢀ (c 1.7, CHCl3). 1H
NMR (250 MHz, CDCl3) δ 7.77 (d, 1H, J = 8.5 Hz, ArH), 7.69
(d, 1H, J = 8.5 Hz, ArH), 7.57 (s, 1H, ArH), 7.55 (s, 1H, ArH),
7.43 (d, 1H, J = 5.5 Hz, ArH), 7.36 (d, 1H, J = 5.5 Hz, ArH),
7.23-7.12 (m, 4H, 4 ꢀ ArH), 4.89 (d, 1H, J = 11.2 Hz, OCHH),
4.77 (d, 1H, J = 11.2 Hz, OCHH), 4.53 (dd, 1H, J = 5.5 and 3.5
Hz, H-5), 4.48 (d, 1H, J = 3.5 Hz, H-4), 3.81 (d, 1H, J = 15.0
Hz, CHHAr), 3.66 (d, 1H, J = 15.0 Hz, CHHAr), 2.56 (d, 1H,
J = 10.8 Hz, H-6ax), 2.44 (dd, 1H, J = 10.8 and 5.5 Hz, H-6eq),
0.95 (s, 9H, C(CH3)3), 0.73 (s, 9H, C(CH3)3), 0.17 (s, 3H,
SiCH3), 0.16 (s, 3H, SiCH3), 0.13 (s, 3H, SiCH3), and 0.03 (s,
3H, SiCH3) ppm. 13C NMR (63 MHz, CDCl3) δ 175.6 (C), 148.7
(C), 139.6 (C), 139.5 (C), 136.9 (C), 136.1 (C), 132.9 (C), 129.2
(C), 128.5 (C), 126.7 (CH), 125.9 (CH), 125.3 (CH), 123.8 (CH),
123.6 (CH), 123.5 (CH), 123.1 (CH), 122.3 (CH), 122.2 (CH),
121.7 (CH), 74.9 (C), 72.6 (CH2), 67.1 (CH), 37.6 (CH2), 29.9
(CH2), 25.6 (C(CH3)3), 25.4 (C(CH3)3), 17.9 (2 ꢀ C(CH3)3),
-3.4 (SiCH3), -3.6 (SiCH3), and -4.5 (2 ꢀ SiCH3) ppm. IR
(film): 1799 (CdO) cm-1. MS (CI) m/z (%) 693 (MHþ). HRMS
calcd for C37H49O5S2Si2 (MHþ): 693.2560; found, 693.2464.
(1R,4S,5R)-1,4-Di(tert-butyldimethylsilyloxy)-3-(benzo[b]thio-
phen-2-yl)methoxycyclohexan-1,5-carbolactone (11c) and (1R,-
4S,5R)-1,4-Di(tert-butyldimethylsilyloxy)-2-(benzo[b]thiophen-2-yl)-
methyl-3-(benzo[b]thiophen-2-yl)methoxycyclohexan-1,5-carbol-
actone (12c). A flame-dried round-bottom flask under inert
atmosphere was charged with ketone 10 (250 mg, 0.63 mmol)
and then dissolved in dry DMF (17 mL). The resultant solution
was treated with LHMDS (1.3 mL, 1.26 mmol, 1.0 M in THF).
After the mixture was stirred for 20 min, a solution of (2-
bromomethyl)benzo[b]thiophene (15c) (215 mg, 0.95 mmol) in
dry DMF (1.6 mL) was added. After 30 min, the reaction
mixture was diluted successively with diethyl ether and water.
The organic phase was separated, and the aqueous layer was
extracted three times with diethyl ether. All combined organic
extracts were dried (anhydrous Na2SO4), filtered, and evapo-
rated under reduced pressure. The obtained residue was purified
by flash chromatography, eluting with diethyl ether-hexanes
(5:95) to afford O-alkyl derivative 11c (63 mg, 18%) and dialkyl
derivatives 12c (128 mg, 29%), both as light-yellow oils.
(1R,4S,5R)-1,4-Di(tert-butyldimethylsilyloxy)-3-(5-methylbenzo-
[b]thiophen-2-yl)methoxycyclohexan-1,5-carbolactone (11d) and
(1R,4S,5R)-1,4-Di(tert-butyldimethylsilyloxy)-3-(5-methylbenzo[b]-
thiophen-2-yl)methoxy-2-(5-methylbenzo[b]thiophen-2-yl)methylcycl-
ohex-2-en-1,5-carbolactone (12d). The reaction was carried out as for
compounds 11c and 12c by using 200 mg of ketone 10 and 180 mg of
2-bromomethyl-5-methylbenzo[b]thiophene (15d) as starting mate-
rials. Yield: 62 mg (22%) of O-alkyl derivative 11d and 99 mg (28%)
of dialkyl derivative 12d, both as light-yellow oils.
Data for 11d. [R]2D0 -120.6ꢀ (c 1.0, CHCl3). H NMR (250
1
MHz, CDCl3) δ 7.70 (d, 1H, J = 8.5 Hz, ArH), 7.54 (s, 1H,
ArH), 7.17 (m, 2H, 2 ꢀ ArH), 5.05 (s, 1H, H-2), 4.98 (d, 1H, J =
12.0 Hz, CHHAr), 4.92 (d, 1H, J = 12.0 Hz, CHHAr), 4.48 (dd,
1H, J = 5.3 and 3.5 Hz, H-5), 4.17 (d, 1H, J = 3.5 Hz, H-4), 2.46
(s, 3H, CH3), 2.44-2.30 (m, 2H, H-6), 0.92 (s, 9H, C(CH3)3),
0.87 (s, 9H, C(CH3)3), 0.17 (s, 3H, SiCH3), 0.12 (s, 3H, SiCH3),
0.10 (s, 3H, SiCH3), and 0.08 (s, 3H, SiCH3) ppm. 13C NMR (63
MHz, CDCl3) δ 176.1 (C), 153.3 (C), 139.4 (C), 138.9 (C), 137.4
(C), 134.0 (C), 126.3 (CH), 123.6 (CH), 123.0 (CH), 122.0 (CH),
105.2 (CH), 75.3 (CH), 73.7 (C), 67.4 (CH), 65.2 (OCH2), 38.0
(CH2), 25.7 (C(CH3)3), 25.6 (C(CH3)3), 21.3 (CH3), 18.1
(C(CH3)3), 18.0 (C(CH3)3), -3.1 (2 ꢀ SiCH3), -4.4 (SiCH3),
and -5.2 (SiCH3) ppm. IR (film): 1801 (CdO) cm-1. MS (CI)
m/z (%) 561 (MHþ). HRMS calcd for C29H45O5SSi2 (MHþ):
561.2526; found, 561.2530.
Data for 12d. [R]2D0 -75.4ꢀ (c 1.3, CHCl3). 1H NMR (250
MHz, CDCl3) δ 7.65 (d, 1H, J = 8.0 Hz, ArH), 7.59 (d, 1H, J =
8.0 Hz, ArH), 7.45 (s, 1H, ArH), 7.37 (s, 1H, ArH), 7.13 (d, 1H,
J = 8.3 Hz, ArH), 7.05 (d, 1H, J = 8.3 Hz, ArH), 6.95 (s, 1H,
ArH), 6.87 (s, 1H, ArH), 5.02 (s, 2H, CH2O), 4.57 (d, 1H, J =
5.8 and 3.3 Hz, H-5), 4.46 (d, 1H, J = 3.3 Hz, H-4), 3.93 (d, 1H,
J = 15.5 Hz, CHHAr), 3.79 (d, 1H, J = 15.5 Hz, CHHAr), 4.17
(d, 1H, J = 3.5 Hz, H-4), 2.56 (d, 1H, J = 10.8 Hz, H-6eq), 2.45
(m, 1H, H-6ax), 2.44 (s, 3H, CH3), 2.42 (s, 3H, CH3), 0.96 (s, 9H,
C(CH3)3), 0.78 (s, 9H, C(CH3)3), 0.21 (s, 3H, SiCH3), 0.19 (s,
3H, SiCH3), 0.16 (s, 3H, SiCH3), and 0.07 (s, 3H, SiCH3) ppm.
13C NMR (63 MHz, CDCl3) δ 175.2 (C), 148.6 (C), 144.0 (C),
140.4 (C), 139.8 (C), 139.5 (C), 137.3 (C), 136.5 (C), 133.9 (C),
133.3 (C), 129.0 (C), 126.1 (CH), 124.8 (CH), 123.6 (CH), 122.7
(CH), 122.6 (CH), 121.9 (CH), 121.5 (CH), 121.0 (CH), 74.7 (C),
74.6 (CH), 68.5 (2 ꢀ CH2), 67.4 (CH), 37.5 (CH2), 25.7
(C(CH3)3), 25.5 (C(CH3)3), 21.4 (CH3), 21.3 (CH3), 18.1
(C(CH3)3), 18.0 (C(CH3)3), -3.3 (SiCH3), -3.4 (SiCH3), and
-4.5 (2 ꢀ SiCH3) ppm. IR (film): 1799 (CdO) cm-1. MS (CI)
m/z (%) 721 (MHþ). HRMS calcd for C39H53O5S2Si2 (MHþ):
721.2873; found, 721.2878.
(1R,4S,5R)-1,4-Dihydroxy-3-(thien-3-yl)methoxycyclohex-2-
en-1,5-carbolactone (13a). A stirred solution of silyl ether 11a (65
mg, 0.13 mmol) in dry THF (1.9 mL) was treated with tetra-
butylammonium fluoride (0.29 mL, 0.29 mmol, ∼1.0 M in
THF). After 1 h, ethyl acetate and water were added. The
organic layer was separated, and the aqueous phase was ex-
tracted with ethyl acetate (ꢀ3). All the combined organic
extracts were dried (anhydrous Na2SO4), filtered, and concen-
trated under reduced pressure. The obtained residue was puri-
fied by flash chromatography, eluting with ethyl acetate-
hexane (50:50) to afford 30 mg (86%) of diol 13a as a white
foam. [R]2D0 -189ꢀ (c 1.5, MeOH). 1H NMR (300 MHz, CD3OD)
δ 7.40 (dd, 1H, J = 5.1 and 0.9 Hz, ArH), 7.12 (d, 1H, J =
2.7 Hz, ArH), 6.99 (dd, 1H, J = 5.1 and 3.6 Hz, ArH), 5.15 (s,
1H, H-2), 4.99 (d, 1H, J = 12.3 Hz, CHHAr), 4.93 (d, 1H,
J = 12.3 Hz, CHHAr), 4.61 (m, 1H, H-5), 4.08 (d, 1H, J =
3.3 Hz, H-4), and 2.32 (m, 2H, H-6) ppm. 13C NMR (75 MHz,
1
Data for 11c. [R]2D0 -103.3ꢀ (c 2.1, CHCl3). H NMR (250
MHz, CDCl3) δ 7.86-7.79 (m, 1H, ArH), 7.78-7.72 (m, 1H,
ArH), 7.35 (m, 2H, 2 ꢀ ArH), 7.25 (m, 1H, ArH), 5.06 (s, 1H, H-
2), 5.01 (d, 1H, J = 12.0 Hz, CHHAr), 4.95 (d, 1H, J = 12.0 Hz,
CHHAr), 4.49 (dd, 1H, J = 5.5 and 3.5 Hz, H-5), 4.19 (d, 1H,
J = 3.5 Hz, H-6ax), 2.42 (d, 1H, J = 10.8 Hz, H-6ax), 2.34 (ddd,
1H, J = 10.8, 5.5, and 1.1 Hz, H-6ax), 0.93 (s, 9H, C(CH3)3), 0.89
(s, 9H, C(CH3)3), 0.17 (s, 3H, SiCH3), 0.13 (s, 3H, SiCH3), 0.11
(s, 3H, SiCH3), and 0.10 (s, 3H, SiCH3) ppm. 13C NMR (63
MHz, CDCl3) δ 176.1 (C), 153.2 (C), 140.3 (C), 139.1 (C), 138.9
(C), 124.5 (CH), 124.3 (CH), 123.6 (CH), 123.3 (CH), 122.4
(CH), 105.3 (CH), 75.3 (CH), 73.7 (C), 67.4 (CH), 65.2 (CH2),
38.0 (CH2), 25.7 (C(CH3)3), 25.6 (C(CH3)3), 18.1 (C(CH3)3),
18.0 (C(CH3)3), -3.1 (2 ꢀ SiCH3), -4.4 (SiCH3), and -5.1
(SiCH3) ppm. IR (film): 1803 (CdO) cm-1. MS (CI) m/z (%) 547
(MHþ). HRMS calcd for C28H43O5SSi2 (MHþ): 547.2370;
found, 547.2372.
Data for 12c. [R]2D0 -148.6ꢀ (c 1.0, CHCl3). H NMR (250
1
MHz, CDCl3) δ 7.82-7.67 (m, 3H, 3 ꢀ ArH), 7.61 (m, 1H,
ArH), 7.38-7.20 (m, 4H, 4 ꢀ ArH), 7.06 (s, 1H, ArH), 7.00 (s,
1H, ArH), 5.07 (s, 2H, OCH2), 4.60 (dd, 1H, J = 5.5 and 3.3 Hz,
H-5), 4.50 (d, 1H, J = 3.3 Hz, H-4), 3.98 (d, 1H, J = 15.5 Hz,
CHHAr), 3.83 (d, 1H, J = 15.5 Hz, CHHAr), 2.60 (d, 1H, J =
10.8 Hz, H-6eq), 2.48 (dd, 1H, J = 10.8 and 5.8 Hz, H-6ax), 0.99
(s, 9H, C(CH3)3), 0.80 (s, 9H, C(CH3)3), 0.24 (s, 3H, SiCH3),
0.22 (s, 3H, SiCH3), 0.20 (s, 3H, SiCH3), and 0.10 (s, 3H, SiCH3)
ppm. 13C NMR (63 MHz, CDCl3) δ 175.2 (C), 148.7 (C), 144.0
(C), 140.1 (C), 139.4 (C), 139.1 (C), 128.9 (2 ꢀ C), 124.4 (CH),

198 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Prazeres et al.
CD3OD) δ 179.2 (C), 155.3 (C), 146.0 (C), 139.6 (CH), 128.6
(CH), 127.7 (CH), 127.5 (CH), 127.5 (CH), 105.1 (CH), 76.9
(CH), 72.9 (C), 67.6 (CH2), 65.5 (CH), and 38.3 (CH2) ppm. IR
(KBr) 3463 (O-H) and 1774 (CdO) cm-1. MS (CI) m/z (%) 269
(MHþ). HRMS calcd for C12H13O5S (MHþ): 269.0484; found,
269.0480.
(CH), 124.8 (CH), 124.6 (CH), 123.3 (CH), 105.4 (CH), 77.0
(CH), 73.0 (C), 67.6 (CH), 66.3 (CH2), and 38.3 (CH2) ppm. IR
(KBr): 3390 (O-H) and 1765 (CdO) cm-1. MS (ESI) m/z (%)
319 (MHþ). HRMS calcd for C16H15O5S (MHþ): 319.0635;
found, 319.0634.
Sodium (1R,4S,5R)-1,4,5-Trihydroxy-3-(benzo[b]thiophen-2-
yl)methoxycyclohexan-1-carboxylate (9c). The experimental
procedure used was the same as for compound 9a using 28 mg
(0.09 mmol) of lactone 13c, 176 μL of NaOH, and 0.8 mL of
THF. Yield = 31 mg (98%). Beige solid. [R]2D0 -52.0ꢀ (c 1.3,
Sodium (1R,4S,5R)-1,4-Dihydroxy-3-(thien-3-yl)methoxycyclo-
hex-2-en-1-carboxylate (9a). A solution of the lactone 13a (29
mg, 0.11 mmol) in THF (1.0 mL) and aqueous sodium hydroxide
(215 μL, 0.11 mmol, 0.5M) was stirred at room temperature for 15
min. Water was added, and THF was removed under reduced
pressure. The resultant aqueous solution was washed with diethyl
ether (ꢀ2), and the aqueous extract was liophilized to afford
derivative 9a (33 mg, 97%) as a beige solid. Mp: 125-128 ꢀC.
[R]2D0 -31ꢀ (c 1.2, H2O). 1H NMR (300 MHz, D2O) δ 7.48 (br d,
1H, J=5.1Hz, ArH), 7.19(d, 1H, J= 3.0 Hz, ArH), 7.07 (dd, 1H,
J = 5.1 and 3.6 Hz, ArH), 5.06 (d, 1H, J = 12.0 Hz, CHHAr), 4.99
(d, 1H, J = 12.0 Hz, CHHAr), 4.97 (s, 1H, H-2), 4.05 (d, 1H, J =
7.5 Hz, H-4), 3.96 (m, 1H, H-5), 2.08 (dd, 1H, J = 13.5 and 10.2
1
MeOH). H NMR (250 MHz, CD3OD) δ 7.74 (m, 1H, ArH),
7.68 (m, 1H, ArH), 7.24 (m, 3H, 3 ꢀ ArH), 5.01 (d, 1H, J = 12.5
Hz, CHHO), 4.94 (d, 1H, J = 12.5 Hz, CHHO), 4.82 (s, 1H, H-
C
2), 3.87 (m, 2H, H-5 þ H-4), and 2.05 (m, 2H, CH2-6) ppm. 13
NMR (63 MHz, CD3OD) δ 182.1 (C), 157.1 (C), 141.6 (2 ꢀ C),
140.9 (C), 125.5 (CH), 125.4 (CH), 124.7 (CH), 124.1 (CH),
123.3 (CH), 103.6 (CH), 74.7 (C), 72.4 (CH), 71.6 (CH), 65.9
(OCH2), and 37.4 (CH2) ppm. IR (KBr): 3435 (O-H), 1664
(CdO), 1610 (CdO), and 1585 (CdO) cm-1. MS (ESI) m/z (%)
359 (MHþ). HRMS calcd for C16H16O6SNa (MHþ): 359.0560;
found, 359.0560.
Hz, H-6ax), and2.00(dd, 1H, J= 13.5 and 4.5 Hz, H-6eq) ppm. 13
C
NMR (75 MHz, D2O) δ 184.5 (C), 158.3 (C), 141.4 (C), 131.1
(CH), 130.3 (CH), 130.2 (CH), 103.7 (CH), 76.9 (C), 75.0 (CH),
72.6 (CH), 67.3 (CH2), and 41.9 (CH2) ppm. IR (KBr): 3419
(O-H), 1653 and 1601 (CdO) cm-1. MS (ESI) m/z (%) 331
(MNaþ). HRMS calcd for C12H13O6SNa2 (MHþ): 331.0223;
found, 331.0219.
(1R,4S,5R)-1,4-Dihydroxy-3-(5-methylbenzo[b]thiophen-2-yl)-
methoxycyclohexan-1,5-carbolactone (13d). The same experi-
mental procedure as for compound 13a was used by using silyl
ether 11d (72 mg, 0.13 mmol) as starting material and tetrabu-
tylammonium fluoride (0.34 mL, 0.34 mmol) and THF (1.9 mL).
Yield: 34mg(79%). Beigesolid. [R]2D0 -135.1ꢀ (c 1.4, acetone). 1H
NMR (250 MHz, CD3OD) δ 7.63 (d, 1H, J = 8.3 Hz, ArH), 7.50
(m, 1H, ArH), 7.21 (m, 1H, ArH), 7.10 (m, 1H, ArH), 5.14 (s, 1H,
H-2), 5.00 (br s, 2H, OCH2), 4.60 (m, 1H, H-5), 4.08 (d, 1H, J =
3.3 Hz, H-4), 2.38 (s, 3H, CH3), and 2.28 (m, 2H, CH2-6) ppm.
13C NMR (63 MHz, CDCl3) δ 179.2 (C), 155.3 (C), 141.1 (C),
140.8 (C), 138.8 (C), 135.3 (C), 127.3 (CH), 124.7 (CH), 124.4
(CH), 123.0 (CH), 105.4 (CH), 77.0 (CH), 73.0 (C), 67.6 (CH),
66.4 (OCH2), 38.4 (CH2), and 21.4 (CH3) ppm. IR (KBr): 3444
(O-H), 3377 (O-H), and 1765 (CdO) cm-1. MS (CI) m/z (%)
333 (MHþ). HRMS calcd for C17H17O5S (MHþ): 333.0797;
found, 333.0786.
(1R,4S,5R)-3-(Benzo[b]tiophen-5-yl)methoxy-1,4-dihydroxy-
cyclohex-2-en-1,5-carbolactone (13b). The same experimental
procedure as for compound 13a was used by using silyl ether
11b (173 mg, 0.32 mmol) as starting material and tetrabutylam-
monium fluoride (0.70 mL, 0.70 mmol) and THF (4.5 mL).
Yield: 94 mg (92%). White solid. Mp: 177-179 ꢀC. [R]2D0 -153ꢀ
1
(c 1.0, MeOH). H NMR (250 MHz, CD3OD) δ 7.81 (m, 2H,
2 ꢀ ArH), 7.51 (d, 1H, J = 5.5 Hz, ArH), 7.31 (m, 2H, 2 ꢀ ArH),
5.08 (s, 1H, H-2), 4.84 (s, 2H, CH2O), 4.57 (m, 1H, H-5), 4.08 (d,
1H, J = 3.2 Hz, H-4), and 2.26 (m, 2H, H-6) ppm. 13C NMR (63
MHz, CD3OD) δ 179.2 (C), 155.6 (C), 141.2 (C), 140.7 (C), 133.8
(C), 128.1 (CH), 125.1 (CH), 124.8 (CH), 123.8 (CH), 123.3
(CH), 104.9 (CH), 76.9 (CH), 72.9 (C), 70.9 (CH2), 67.7 (CH),
and 38.3 (CH2) ppm. IR (film): 3431 (O-H) and 1763 (CdO)
cm-1. MS (CI) m/z (%) 319 (MHþ). HRMS calcd for
C16H15O5S (MHþ): 319.0640; found, 319.0640.
Sodium (1R,4S,5R)-1,4,5-Trihydroxy-3-[(benzo[b]tiophen-5-
yl)methoxy]cyclohex-2-en-1-carboxylate (9b). The experimental
procedure used was the same as for compound 9a using 20 mg
(0.06 mmol) of lactone 13b, 125 μL of NaOH, and 0.6 mL of
THF. Yield = 21 mg (98%). White solid. Mp: 157-160 ꢀC
(dec). [R]2D0 -26ꢀ (c 1.4, H2O). 1H NMR (250 MHz, D2O) δ 7.99
(d, 1H, J = 8.5 Hz, ArH), 7.92 (s, 1H, ArH), 7.63 (d, 1H, J = 5.2
Hz, ArH), 7.45 (s, 1H, ArH), 7.42 (m, 1H, ArH), 4.94 (m, 3H,
CH2O þ H-2), 4.09 (d, 1H, J = 7.2 Hz, H-4), 3.98 (m, 1H, H-5),
2.11 (dd, 1H, J = 13.5 and 10.2 Hz, H-6ax), and 2.00 (dd, 1H,
J = 13.5 and 4.2 Hz, H-6eq) ppm. 13C NMR (63 MHz, D2O) δ
182.1 (C), 156.3 (C), 140.3 (C), 139.9 (C), 133.3 (C), 128.5 (CH),
125.0 (CH), 124.5 (CH), 123.8 (CH), 123.4 (CH), 101.1 (CH),
74.4 (C), 72.6 (CH), 70.4 (CH2), 70.2 (CH), and 39.5 (CH2) ppm.
IR (KBr): 3419 (O-H), 1655 and 1597 (CdO) cm-1. MS (ESI)
m/z (%) 359 (MHþ). HRMS calcd for C16H16O6SNa (MHþ):
359.0560; found, 359.0560.
(1R,4S,5R)-1,4-Dihydroxy-3-(benzo[b]thiophen-2-yl)methoxy-
cyclohexan-1,5-carbolactone (13c). The same experimental pro-
cedure as for compound 13a was used by using silyl ether 11c (42
mg, 0.077 mmol) as starting material and tetrabutylammonium
fluoride (0.20 mL, 0.20 mmol) and THF (1.1 mL). Yield: 24 mg
(99%). Beige solid. [R]D20 -151.2ꢀ (c 1.1, MeOH). 1H NMR (250
MHz, CD3OD) δ 7.77 (m, 1H, ArH), 7.70 (m, 1H, ArH),
7.32-7.21 (m, 3H, 3 ꢀ ArH), 5.13 (s, 1H, H-2), 5.02 (br s, 2H,
CH2O), 4.57 (m, 1H, H-5), 4.07 (d, 1H, J = 3.3 Hz, H-4), and
2.27 (m, 2H, H-6) ppm. 13C NMR (63 MHz, CD3OD) δ 179.2
(C), 155.3 (C), 141.6 (C), 140.8 (C), 140.8 (C), 125.7 (CH), 125.5
Sodium (1R,4S,5R)-1,4,5-Trihydroxy-3-(5-methylbenzo[b]-
thiophen-2-yl)methoxycyclohexan-1-carboxylate (9d). The same
experimental procedure as for compound 9a was used by using
lactone 13d (21 mg, 0.063 mmol) as starting material and THF
(0.6 mL) and NaOH (126 μL). Yield: 22 mg (95%). Beige solid.
1
[R]2D0 -41.9ꢀ (c 1.5, MeOH). H NMR (250 MHz, CD3OD) δ
7.63 (d, 1H, J = 8.3 Hz, H-40), 7.50 (br s, 1H, H-70), 7.21 (s, 1H,
H-30), 7.09 (dd, 1H, J = 8.3 and 1.0 Hz, H-50), 5.01 (d, 1H, J =
12.3 Hz, CHH), 4.94 (d, 1H, J = 12.3 Hz, CHH), 4.84 (s, 1H, H-
2), 3.88 (m, 2H, H-5 þ H-4), 2.38 (s, 3H, CH3), and 2.06 (m, 2H,
CH2-6) ppm. 13C NMR (63 MHz, CD3OD) δ 182.1 (C), 157.2
(C), 141.7 (C), 141.3 (C), 138.9 (C), 135.2 (C), 127.1 (CH), 124.6
(CH), 123.9 (CH), 122.9 (CH), 103.5 (CH), 74.7 (C), 72.4 (CH),
71.6 (CH), 66.0 (OCH2), 37.4 (CH2), and 21.5 (CH3) ppm. IR
(KBr): 3435 (O-H), 1649 and 1618 (CdO) cm-1. MS (ESI) m/z
(%) 373 (MHþ). HRMS calcd for C17H18O6SNa (MHþ):
373.0716; found, 373.0729.
Dehydroquinase Assays. Helicobacter pylori type II dehydro-
quinase was expressed and purified as we described previously.22
A concentrated solution of H. pylori dehydroquinase (6.4 mg
mL-1) was stored in potassium phosphate buffer (50 mM, pH
7.2), DTT (1 mM), and NaCl (150 mM) . When required for
assays, aliquots of the enzyme stocks were diluted into water and
buffer and stored on ice. Dehydroquinase was assayed in the
forward direction by monitoring the increase in absorbance at
234 nm in the UV spectrum because ofthe absorbance of the
enone-carboxylate chromophore of 3-dehydroshikimic acid (6)
(ε = 12 000 M-1 cm-1). Standard assay conditions for DHQ2-
Hp were pH 7.0 at 25 ꢀC in Tris-HCl (50 mM). Each assay was
initiated by addition of the substrate. Solutions of 3-dehydro-
quinic acid (4) were calibrated by equilibration with DHQ2 and
measurement of the change in the UV absorbance at 234 nm due

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 199
to the formation of the enone-carboxylate chromophore of
3-dehydroshikimic acid (6). The Ki values of acids 9 against
DHQ2-Hp were obtained from Dixon plots (1/v vs [I])24 of assay
data. The initial rates at fixed enzyme and substrate concentra-
tions (0.25-1.4 Km) were measured in the absence and in the
presence of various inhibitor concentrations. A standard inhibi-
tion assay consists of (1) incubation of 10 μL of enzyme solution
(in buffer) and the tested inhibitor (in water) in 50 mM Tris-HCl,
pH 7, at 25 ꢀC for 10 min, (2) addition of substrate (in water) and
after 1 min measurement of the initial rate at 234 nm at 25 ꢀC.
Crystallization and Structure Determination. H. pylori type II
dehydroquinase was concentrated to 20 mg mL-1 in 50 mM
Tris-HCl, pH 7.5, 1 mM 2-mercaptoethanol, 1 mM ethylene-
diaminetetraacetic acid, and 200 mM sodium chloride. Com-
pound 9d was dissolved at 0.25 M in methanol and added at a
ratio of 1:20 (v/v) to the protein solution to give a solution of
approximately 10 equiv of 9d per protein monomer. Diamond-
shaped crystals of up to 0.3 mm ꢀ 0.2 mm were obtained after 3
weeks of sitting drop vapor diffusion of 2.5 μL of protein/
inhibitor solution mixed with 2.5 μL of reservoir solution
against 0.15 mL reservoirs containing 26% (w/v) polyethylene-
glycol 4000 and 0.1 M 2-(N-morpholino)ethanesulfonic acid-
sodium hydroxide, pH 5.0. After harvesting, crystals were
sequentially transferred to solutions containing 35% and 45%
(w/v) polyethylene glycol 4000 in 0.1 M 2-(N-morpholi-
no)ethanesulfonic acid-sodium hydroxide, pH 5.0, and flash-
frozen in liquid nitrogen. Data were collected at the EMBL X11
beamline at the DORIS storage ring (DESY, Hamburg,
Germany) from a crystal maintained at 100 K. Crystallographic
data were integrated using MOSFLM27 and further processed
using SCALA28 and other programs of the CCP4 software
suite.29 The structure was solved by molecular replacement,
using the program PHASER30 and PDB entry 2C4V.25 Reflec-
439–443. (b) Jayaraman, R. Bacterial persistence: some new insights
into an old phenomenon. J. Biosci. 2008, 33, 795–805.
(2) (a) Giamarellou, H. Treatment options for multidrug-resistant
bacteria. Expert Rev. Anti-Infect. Ther. 2006, 4, 601–618. (b)
Appelbaum, P. C.; Jacobs, M. R. Recently approved and investigational
antibiotics for treatment of severe infections caused by Gram-positive
bacteria. Curr. Opin. Microb. 2005, 8, 510–517. (c) Okeke, I. N.;
Klugman, K. P.; Bhutta, Z. A.; Duse, A. G.; Jenkins, P.; O'Brien, T. F.;
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31
tions for calculating Rfree were selected in thin resolution
shells. Model building was done with COOT32 and refinement
with REFMAC.33 Validation was performed with MOLPROB-
ITY.34 For data, refinement, and model statistics, see Table 2.
Coordinates and structure factors are available from the Protein
Data Bank with accession code 2WKS. Structure figures were
prepared using PYMOL35 and VMD.36
Acknowledgment. We thank Patricia Ferraces-Casais for
excellent technical assistance and EMBL-Hamburg scientists
Paul Tucker and Santosh Panjikar for help with synchrotron
data collection. This work was supported by the Spanish
Ministry of Science and Innovation (under Projects SAF2007-
63533 to C.G.B., BFU2005-02974/BMC and BFU2008-01588/
BMC to M.J.v.R.), CSIC (Grant 488IE7 to M.J.v.R.), and
the Xunta de Galicia (Grants PGIDT07PXIB209080PR and
GRC2006/132 to C.G.B.). V.F.V.P., J.M.O., L.T., and P.G.-C.
(12) (a) Salama, N. R.; Shepherd, B.; Falkow, S. Global transposon
mutagenesis and essential gene analysis of Helicobacter pylori. J.
Bacteriol. 2004, 186, 7926–7935. (b) Sassetti, C. M.; Boyd, D. H.;
Rubin, E. J. Genes required for mycobacterial growth defined by high
density mutagenesis. Mol. Microb. 2003, 48, 77–84. (c) de Berardinis,
V.; Vallenet, D.; Castelli, V.; Besnard, M.; Pinet, A.; Cruaud, C.; Samair,
S.; Lechaplais, C.; Gyapay, G.; Richez, C.; Durot, M.; Kreimeyer, A.;
~
^
thank the Portuguese Fundac-ao para a Ciencia e a Tecnologı
´
a
for a FCT fellowship, the Xunta de Galicia for a “Angeles
~
Alvarino” contract, and the Spanish Ministry of Science and
Innovation for FPU fellowships, respectively. Data collection at
EMBL/DESY was supported by the European Community
FP6 Research Infrastructure Action “Structuring the European
Research Area”, Contract No. RII3/CT/2004/5060008.
ꢁ
€
€
ꢀ
Le Fevre, F.; Schachter, V.; Pezo, V.; Doring, V.; Scarpelli, C.; Medigue,
ꢁ
C.; Cohen, G. N.; Marliere, P.; Salanoubat, M.; Weissenbach, J. A
complete collection of single-gene deletion mutants of Acinetobacter
baylyi ADP1. Mol. Syst. Biol. 2008, 4, 1–15.
(13) For a database of essential bacterial genes, see the following: (a)
www.essentialgene.org.(b) Zhang, R.; Lin, Y. DEG 5.0, a database
of essential genes in both prokaryotes and eukaryotes. Nucleic
Acids Res. 2009, 37, D455–D458. (c) Zhang, R.; Ou, H.-Y.; Zhang,
C.-T. DEG: a database of essential genes. Nucleic Acids Res. 2004, 32,
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(14) Kleanthous, C. K.; Deka, R.; Davis, K.; Kelly, S. M.; Cooper, A.;
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comparison of the enzymological and biophysical properties of two
distinct classes of dehydroquinase enzymes. Biochem. J. 1992, 282,
687–695.
Supporting Information Available: 1H NMR, 13C NMR, and
DEPT spectra, Dixon plots for compounds 9, a table of close
contacts between ligand 9b and DHQ2-Hp, and a figure with the
electron density for inhibitor 9d in crystal structure with the final
2Fo - Fc map contoured at 1σ. This material is available free of
charge via the Internet at http://pubs.acs.org.
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