A prodrug approach for improving antituberculosis activity of potent mycobacterium tuberculosis type II dehydroquinase inhibitors

ARTICLE

pubs.acs.org/jmc

A Prodrug Approach for Improving Antituberculosis Activity of Potent

Mycobacterium tuberculosis Type II Dehydroquinase Inhibitors^†,‡

Lorena Tizꢀon,^§Josꢀe M. Otero,^||Verꢀonica F. V. Prazeres,^§Antonio L. Llamas-Saiz,^{^}Gavin C. Fox,^||

Mark J. van Raaij,^#Heather Lamb,³Alastair R. Hawkins,³Josꢀe A. Ainsa,^OLuis Castedo,^[and

Concepciꢀon Gonzꢀalez-Bello*^,§

^§Centro Singular de Investigaciꢀon en Química Biolꢀogica y Materiales Moleculares, Universidad de Santiago de Compostela,

calle Jenaro de la Fuente s/n, 15782 Santiago de Compostela, Spain

Laboratoire des Proteines Membranaires, Institut de Biologie Structurale J. P. Ebel, 38027 Grenoble, France

^{^}Unidad de Rayos X, RIAIDT, Ediﬁcio CACTUS, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain

^#Departamento de Estructura de Macromolꢀeculas, Centro Nacional de Biotecnología (CSIC), Campus Cantoblanco, 28049 Madrid,

Spain

³Institute of Cell and Molecular Biosciences, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, U.K.

^OGrupo de Genꢀetica de Micobacterias, Departamento de Microbiología, Medicina Preventiva y Salud Pꢀublica, Facultad de Medicina,

50009 Zaragoza, Spain, and CIBER Enfermedades Respiratorias, Spain

^[Departamento de Química Orgꢀanica, Facultad de Química, Universidad de Santiago de Compostela, Avenida de las Ciencias s/n,

15782 Santiago de Compostela, Spain

S Supporting Information

b

ABSTRACT: The synthesis of high-aﬃnity reversible compe-

titive inhibitors of Mycobacterium tuberculosis type II dehydro-

quinase, an essential enzyme in Mycobacterium tuberculosis

bacteria, is reported. The inhibitors reported here are mimics

of the enol intermediate and the eﬀect of substitution on C2 was

studied. The crystal structures of Mycobacterium tuberculosis

type II dehydroquinase in complex with three of the reported

inhibitors are also described. The results show that an aromatic

substituent on C2 prevents the closure of the active site by impeding the hydrogen-bonding interaction of Arg108 with the essential

Tyr24 of the ﬂexible loop, the residue that initiates catalysis. Chemical modiﬁcations of the reported acids were also carried out to

improve internalization into Mycobacterium tuberculosis through an ester prodrug approach. Propyl esters proved to be the most

eﬃcient in achieving optimal in vitro activities.

’ INTRODUCTION

to ﬁrst-line and second-line TB antibiotics.^3,4MDR and XDR

forms of TB are extraordinarily diﬃcult to treat, particularly in

patients with a compromised immune system.⁵

Tuberculosis (TB), which is caused by Mycobacterium tuber-

culosis, remains one of the deadliest infectious diseases for

humans, and it has been identiﬁed by the World Health

Organization (WHO) as one of the three priority diseases for

drug research and development.¹Thanks to global control

eﬀorts over the past decade, the estimated worldwide incidence

rate fell to 137 cases per 100000 people in 2009, after peaking in

2004 at 142 cases per 100000.¹However, the rate is still falling

too slowly.

The concomitant emergence of HIV and the surge of multidrug-

resistant isolates of M. tuberculosis (MDR-TB) have reaﬃrmed

tuberculosis as a primary public health threat.²In 2010,

the largest WHO MDR-TB survey reported the highest-ever

rates of MDR-TB, with peaks of up to 28% of new TB cases in

some regions of the former Soviet Union. The continued

evolution and aggravation of the drug resistance problem has

led to extensively drug-resistant TB (XDR-TB), which is resistant

TB therapy requires long periods of treatment (around 6À24

months) with multiple drugs. This extended treatment leads to

poor compliance, a factor that promotes resistance development.

The various antibiotics that constitute the ﬁrst- and second-line

drugs for TB therapy target only a small number of essential

functions of the bacterium. It is therefore necessary discover

further pathways that are required for bacterial growth and to

develop compounds that target these pathways.^6À8This ap-

proach should provide novel targets for the rational design of

more eﬀective treatments for tuberculosis, which should also

reduce the duration of therapy necessary to eradicate M. tuber-

culosis from the patient.

Received: May 13, 2011

Published: July 22, 2011

r

2011 American Chemical Society

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Scheme 1. Proposed E1CB Mechanism for the Enzymatic

Conversion of 3-Dehydroquinic Acid (1) to 3-Dehydroshi-

kimic Acid (2) Catalyzed by DHQ2^a

Figure 1. Relevant examples of competitive reversible inhibitors of

DHQ2-Mt.

^aThe reaction proceeds via the enol intermediate 3. Relevant residues

are indicated.

The shikimate pathway is an attractive target for antibiotic

development because it is present in bacteria, fungi, plants, and in

apicomplexan parasites such as Plasmodium falciparum (malaria),⁹

Toxoplasma gondii, and Cryptosporidium parvum, but it is absent

in mammals. This route involves seven enzymes that catalyze the

sequential conversion of erythrose-4-phosphate and phosphoe-

nol pyruvate to chorismic acid, an essential precursor in the

synthesis of aromatic amino acids and other metabolites, includ-

ing folate cofactors, ubiquinone, and vitamins E and K.¹⁰The

absence of the shikimate pathway in mammals, combined with its

essential nature in M. tuberculosis,¹¹makes it an attractive target

for the development of new anti-TB agents.

Dehydroquinase (3-dehydroquinate dehydratase, DHQ, EC

4.2.1.10) is the third enzyme of the shikimate pathway, and it

catalyzes the reversible dehydration of 3-dehydroquinic acid (1)

to form 3-dehydroshikimic acid (2) (Scheme 1). This reaction is

part of two metabolic pathways: the biosynthetic shikimate

pathway and the catabolic quinate pathway. There are two

distinct types of enzymes, known as type I (DHQ1) and type

II (DHQ2), which have diﬀerent biochemical and biophysical

properties and do not show any sequence similarity. DHQ1,

which is found in plants, fungi, and many bacterial species (for

example Salmonella typhi and Escherichia coli), is exclusively

biosynthetic, whereas DHQ2 (found in Streptomyces coelicolor,

Mycobacterium tuberculosis, Aspergillius nidulans, Helicobacter

pylori, and Neurospora crassa) has both biosynthetic and catabolic

roles. DHQ1 and DHQ2 utilize completely diﬀerent mechan-

isms to catalyze the same overall reaction.¹²The type I mechan-

ism involves covalent imine intermediates between the enzyme

and the substrate and proceeds with syn stereochemistry.¹³In

contrast, the type II reaction proceeds through an enol inter-

mediate with overall anti stereochemistry (Scheme 1).¹⁴The

reaction is initiated by an essential tyrosine of the active site,

which removes the pro-S hydrogen from C2 of 1. The ﬁnal step is

the acid-catalyzed elimination of the C-1 hydroxyl group, a

reaction mediated by His101 acting as a proton donor. Two

residues, Arg19 and Tyr24, have been identiﬁed by chemical

modiﬁcation and site-directed mutagenesis studies as being

essential for enzyme activity.¹⁵Both residues reside in a ﬂexible

loop that closes over the active site upon substrate binding. The

essential arginine of the loop is presumed to orient the tyrosine in

Figure 2. 3-Methoxybenzothiophenyl derivatives that are inhibitors of

DHQ2-Hp.

an appropriate manner for proton abstraction.^16,17The proximity

of both Arg108 and Arg19 lowers the pK_aof the catalytic

tyrosine. It has also been suggested that the essential Arg19

and a conserved water molecule close to the carbonyl group of

Asn12 stabilize the enol intermediate.

In recent years, we and other groups have designed inhibitors

of DHQ2, especially from M. tuberculosis (DHQ2-Mt) and H.

pylori (DHQ2-Hp), two pathogenic bacteria in which the

enzyme is essential (the respective genes are AroQ and AroD).

Most of the reported inhibitors are mimics of the enol inter-

mediate 3 (compounds 4, Figure 1).^18À31Some examples of

high-aﬃnity inhibitors of DHQ2-Mt include 2-thienyl com-

pound 4a²⁷(K_iof 250 nM), alkenyl derivative 4b²⁶(K_iof 120

nM), 3-nitrophenyl derivative 4d^22,31(K_iof 54 nM), and triazole

4c³⁰(K_iof 39 nM). On the other hand, we recently showed that

3-methoxyaryl derivatives 5a (K_iof 132 nM) and 5b (K_iof 130

nM) are potent competitive inhibitors of DHQ2-Hp and the

crystal structure of DHQ2-Hp in complex with compound 5b has

been solved at 2.95 Å (Figure 2).²⁸

The resolution of this crystal structure has been very impor-

tant to clarify the role of the aromatic rings on C3 for inhibition.

These rings expel the essential arginine side chain from the active

site. In fact, this structure, along with others solved later, reveals

an important change in the conformation and ﬂexibility of the

loop that closes over the substrate binding site.^17,28,29Molecular

dynamics simulation studies suggest that the aromatic ring

prevents appropriate orientation of the catalytic tyrosine of the

loop for proton abstraction and disrupts its basicity.¹⁷The

synthesis of compounds 5a and 5b involved O-alkylation, but

some C-alkylation at C2 was also obtained. It was found that the

resulting disubstituted compounds are also potent inhibitors of

DHQ2. To investigate in more detail the eﬀect of substitution of

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Scheme 3^a

Figure 3. Target compounds 5À11.

Scheme 2^a

^aReagents and conditions: (a) (1) KHMDS, DMF/PhMe, À78 ꢀC, (2)

ArCH₂Br; (b) (1) LiHMDS, THF, RT, (2) ²RCH₂I; (c) (1) KHMDS,

DMF/PhMe, À78 ꢀC, (2) ¹RCH₂Br; (d) TBAF, THF, 0 ꢀC; (e) NaOH,

THF, RT.

compounds incorporate the same aromatic ring in both positions

2 and 3. The effect of substitution with naphthyl, thienyl, and

benzo[b]thiophenyl rings was investigated.^23,27,28In the case of

the benzo[b]thiophenyl substituent, we further studied the effect

of the incorporation of a methyl group and a chloro-substituent.

The synthesis of compounds 5aÀf and 9aÀf was achieved by

alkylation of ketone 12²²(Scheme 2). Treatment of ketone 12

with KHMDS at À78 ꢀC followed by reaction with the corre-

sponding bromide afforded a chromatographically separable

mixture of O-alkylated 13aÀf and the dialkyl products 14aÀf.

Finally, deprotection of the TBS groups in lactones 13aÀf and

14aÀf with TBAF, followed by basic hydrolysis of the corre-

sponding lactones 15aÀf and 16aÀf, gave the desired enol

mimics 5aÀf and 9aÀf, respectively.

^aReagents and conditions: (a) (1) KHMDS, DMF/PhMe, À78 ꢀC, (2)

ArCH₂Br; (b) TBAF, THF, 0 ꢀC; (c) NaOH, THF, RT.

C2, and therefore the potential for the development of new

enzyme inhibitors, we report the synthesis of several 3-substi-

tuted and 2,3-disubstituted derivatives, compounds 5 and 9,

respectively (Figure 3). It was found that these compounds are

new high aﬃnity inhibitors of DHQ2-Mt. The crystal structure of

DHQ2-Mt in complex with compound 5b and with the doubly

substituted enol mimics 9d and 9h is described along with the

results of inhibition studies on compounds 5 and 9 against

DHQ2-Mt and DHQ2-Hp. In addition, chemical modiﬁcation

of the reported acids was carried out with the aim of obtaining

improved internalization into M. tuberculosis. This resulted in the

synthesis of esters 6À8 and 10À11. The results of inhibition

studies of these componds against DHQ2-Mt and DHQ2-Hp

and the molecular docking studies using GOLD 5.0 are also

described.

Synthesis of Disubstituted Derivatives 9gÀj. To study the

relative effect of the type and size of the substituent in position 2,

we synthesized compounds 9gÀj (Scheme 3). Another reason to

synthesize allyl derivative 9g was to understand the role of an

aromatic group on C2.

2-Allyl compound 9g was synthesized in a three-step reaction

sequence from our previously reported (2S)-2-allyl ketone 17.³²

First, alkylation of ketone 17 by treatment with KHMDS followed

by reaction with 2-(bromomethyl)benzo[b]thiophene³²aﬀorded

ether 18g in 45% yield. Finally, compound 18g was eﬃciently

converted into the 2-allyl derivative 9g as for compounds 5.

The synthesis of compounds 9hÀj involved the sequential C-

alkylation and O-alkylation of ketone 12 with two diﬀerent

alkylating agents. First, C-alkylation of ketone 12 was achieved

by treatment of 12 with LiHMDS at room temperature followed

by reaction with the appropriate iodide to aﬀord the correspond-

ing 2-alkyl ketones as a mixture of diastereoisomers in C2.

Subsequent treatment of the resulting 2-alkyl ketones with

KHMDS at À78 ꢀC followed by reaction with the corresponding

’ RESULTS AND DISCUSSION

Synthesis of O-Alkylaryl Derivatives 5aÀf and Disubsti-

tuted Compounds 9aÀf. The synthesis of O-alkylaryl deriva-

tives 5aÀf and dialkyl analogues 9aÀf was carried out first. These

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Table 1. K_i(nM) of Compounds 5 and 9 against DHQ2-Mt and DHQ2-Hp

^a50 mM Tris.HOAc, pH = 7.0, 25 ꢀC. ^b50 mM Tris.HCl, pH = 7.0, 25 ꢀC.

bromide gave the desired disubstituted compounds 18hÀj in low

to moderate overall yield. Finally, compounds 18hÀj were con-

verted into the desired derivatives 9hÀj as for allyl derivative 9g.

Inhibition Assay Results. Enol mimics 5 and 9 were assayed

in the presence of 3-dehydroquinic acid (1) for their inhibitory

properties of DHQ2-Mt. All of the compounds proved to be

competitive reversible inhibitors of the enzyme. The inhibition

data are summarized in Table 1.

In general, the monosubstituted compounds 5 proved to be

more potent than the disubstituted series 9 (Table 1, entries 1À6

vs 7À12). Among compounds 5, signiﬁcant diﬀerences were not

found between the diﬀerent aromatic moieties, although the

presence of a larger ring provides slightly more active compounds

(Table 1, entries 1À5 vs 6). With the exception of thiophene 5f,

compounds 5 have inhibition constants in the low nanomolar

range (28À43 nM), with benzothiophene 5a being the most

potent in the series with a K_iof 28 nM.

In general, substitution of C2 in 5 leads to a loss of activity.

However, with some exceptions, the disubstituted compounds 9

still have inhibition constants in the nanomolar range. Although

the disubstituted compounds 9 do not follow an absolutely

clear pattern, certain trends can be identiﬁed. In general, the

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Table 2. Crystallographic Data Collection and Reﬁnement Statistics for the DHQ2-Mt Complex with Inhibitors 5b, 9d, and 9h

ARTICLE

data processing^a

DHQ2-Mt/5b

DHQ2-Mt/9d

DHQ2-Mt/9h

space group

F23

cell parameters (Å)^b

wavelength (Å)

detector

observed reﬂections^c

resolution range (Å)

Wilson B (Å²)

multiplicity

a = b = c = 126.63

0.9724

a = b = c = 126.43

0.8726

a = b = c = 125.80

0.9921

ADSC Q315r CCD

26866 (3925)^d

29.06À1.50 (1.58À1.50)

17.0

MarMosaic225 CCD

5918 (858)^d

ADSC Q210r CCD

30571 (3015)^d

31.45À1.40 (1.48À1.40)

16.8

38.00À2.50 (2.64À2.50)

28.6

7.8 (4.0)

14.5 (14.6)

7.5 (1.6)

completeness

R_merge

0.995 (0.998)

0.062 (0.367)

1.000 (1.000)

0.120 (0.388)

0.943 (0.649)

0.062 (0.397)

Reﬁnement^e

21.41À1.50 (1.58À1.50)

25423 (3691)

resolution range (Å)

reﬂections used in reﬁnement^d

reﬂections used for R_free

R factor^f

20.00À2.50 (2.63À2.50)

5397 (775)

20.00À1.50 (1.58À1.50)

24982 (3601)

1330 (192)

1443 (223)

508 (83)

0.127 (0.174)

0.162 (0.173)

0.211 (0.220)

0.012/1.5

0.162 (0.221)

0.184 (0.262)

0.014/1.5

g

R_free

0.161 (0.215)

rmsd (bonds (Å)/angles (deg))

0.014/1.5

Final Model

1091/24/2/25/1/129

protein/inhibitor/sodium/sulfate/water atoms

average B protein (Å²)/inhibitor (Å²)/

sulfate (Å²)/sodium (Å²)/water (Å²)

Ramachandran statistics^h(%)

1039/43/-/15/52

1079/46/-/20/107

16.7/21.4/34.3/40.7/36.3

14.5/31.2/-/36.21/15.2

14.0/16.6/-/21.4/28.5

98.5/100.0

2Y71

98.5/100.0

2Y76

98.5/100.0

2Y77

PDB accession code

^aResults from SCALA.^{45 b}One Ångstrom (Å) is 0.1 nm. ^cNo σ cutoﬀ or other restrictions were used for inclusion of reﬂections. ^dValues in parentheses

are for the highest resolution bin, where applicable. ^eResults from REFMAC.^{44 f}R-factor = ∑ F_obs(hkl)| À |F_calc(hkl) /∑|F_obs(hkl)|. ^gAccording to

Brunger.^{42 h}According to the program MOLPROBITY.⁴⁷The percentages indicated are for residues in favored and total allowed regions, respectively.

substitution of position 2 with an allyl group reduces the binding

aﬃnity vs a benzyl group (Table 1, entries 7 and 14 vs 13). For

compounds 9aÀf, which are substituted with the same aromatic

ring in both positions 2 and 3, the 2-benzothiophene moiety

gives higher binding aﬃnities to the M. tuberculosis enzyme.

Benzothiophene 9a proved to be the most potent compound in

the series with a K_iof 40 nM. In the case of compounds 9hÀj,

which are susbstituted with diﬀerent aromatic rings in positions 2

and 3, compound 9j proved to be the most potent one with a K_i

of 59 nM. The inhibition results for the disubstituted compounds

9 suggest that, in addition to the size and the type of aromatic

ring, the relative disposition of the two substituents in the active

site might also aﬀect the binding aﬃnity. Structural and molec-

ular modeling studies on these compounds were therefore

required and will be discussed in the next section.

Compounds 5 and 9 also proved to be competitive reversible

inhibitors of the H. pylori enzyme but with lower K_ivalues

(Table 1). Among the monosubstituted compounds 5, ben-

zothiophenes 5a and 5b proved to be the most potent inhibitors,

with inhibition constants of 132 and 130 nM, respectively. As far

as the M. tuberculosis enzyme is concerned, among compounds

9aÀf, which are substituted with the same aromatic ring in both

positions 2 and 3, compounds 9a and 9b proved to be the most

potent, with inhibition constants of 97 and 50 nM, respectively.

With the exception of compound 9c, the aﬃnity remained almost

unchanged when a benzyl substituent was present on C2

(Table 1, entries 7À8 and 10À11 vs 1À2 and 4À5). For com-

pounds 9hÀj, with diﬀerent aromatic rings in positions 2 and 3,

the presence of an aromatic moiety on C2 is clearly more

favorable than an allyl group (i.e., compound 9g). Compound

9j also proved to be the most potent of this series, with a K_iof

100 nM.

Structural Studies. To obtain structural information on the

binding mechanism of these inhibitors, crystal structures of the

methylbenzothiophene 5b and disubstituted compounds 9d and

9h in complex with DHQ2-Mt were solved at 1.5, 2.5, and 1.5 Å,

respectively. DHQ2-Mt/9d and DHQ2-Mt/9h binary com-

plexes were obtained by cocrystallization, and the DHQ2-

Mt/5b binary complex crystals were obtained by soaking apo-

DHQ2-Mt crystals. Data were collected from cryocooled crystals

using synchrotron radiation and were processed (Table 2). The

structures were solved by molecular replacement, using the

crystal structure of DHQ2-Mt bound to an oxime solved by

Lapthorn et al. (PDB entry 1H0S³³) as a search model and then

refined (Figure 4). All three complexes contain a single DHQ2-

Mt molecule in the crystallographic asymmetric unit.

Comparison of DHQ2-Mt/5b and our recently solved crystal

structure of DHQ2-Mt in complex with (2R)-2-(4-methox-

y)benzyl-3-dehydroquinic acid (PDB entry 2XB8¹⁷) shows that

both structures are virtually identical (0.11 Å root-mean-square

diﬀerence after superposition of 121 R-carbon atoms) with the

exception of three amino acids located on the loop, residues

18À20, which are not visible in the structure reported here

(Figure 5a). In PDB entry 2XB8, amino acids belonging to the

loop (residues 18À25) also showed more disorder than the rest

of the protein. The cyclohexene core of 5b occupies appro-

ximately the same site as the cyclohexane ring of (2R)-2-

(4-methoxy)benzyl-3-dehydroquinic acid in PDB entry 2XB8.

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Figure 4. Unbiased electron density for inhibitors: (a) 5b; (b) 9d; (c) 9h. From the model obtained by molecular replacement, reﬁnement was

performed to obtain unbiased density for the inhibitor molecule and other model changes. A maximum-likelihood weighted 2F_oÀ F_cmap⁴⁴contoured

at 1σ is shown up to 1.6 Å around the inhibitor molecule (yellow). The ﬁnal model (gray), including the inhibitor molecule [5b (green); 9d (purple); 9h

(cyan)], is superposed onto the map. Inhibitors 9d and 9h both showed alternative conformations for their O-alkyl substituents.

Calculated maps showed clear high electron density for the

inhibitor molecule 5b. The benzothiophene ring of 5b is located

with its sulfur atom orientated at the same side as the oxygen of

C4, as shown in Figure 4a. The higher electron density obtained

at this side of the aromatic ring, as well as in its para position,

suggests that the orientation of the benzothiophene ring is as

indicated in Figure 4a and not the other putative rotamer around

the methylene group. This binding mode may be due to two

factors; ﬁrst, in this disposition the methyl group can interact

with the carbon side chain of Arg15 and, second, in the other

putative conformation the methyl group would be located too

close to Glu92 of a symmetry-related neighboring molecule.

Inhibitor 5b ﬁts snugly in the active site by establishing a large

number of polar interactions (carboxylate and hydroxyl groups),

including the essential water molecule involved in the enzymatic

reaction (Figure 5b). This crystal structure also shows that the

aromatic moiety of inhibitor 5b interacts with the apolar part of

the active site by establishing important lipophilic interactions,

i.e., it is in close contact with the side chain carbons of Arg15 and

Leu13 and the main chain of Leu13 and Leu16 (and with side

chain carbon atoms of Glu92 of the symmetry-related molecule).

The methylene group of the inhibitor side chain is in close

contact with the side chain of Leu13 (Figure 5c). Moreover, the

ﬁve-membered ring of the benzothiophene moiety interacts with

the essential Tyr24 through a CHÀπ interaction.

that the larger substituent on C2 (the benzo[b]thiophen-5-

ylmethoxy moiety of 9d vs the thien-2-ylmethyl moiety of 9h)

and/or the diﬀerent C3 substituent (the benzo[b]thiophen-5-

ylmethoxy moiety of 9d vs the benzo[b]thiophen-2-ylmethoxy

moiety of 9h) somehow lead to more disorder of the covering

loop and perhaps to disturbance of certain crystal contacts. In

both binary complexes, the benzyl moiety on C2 is located

approximately perpendicular to the position that should be

occupied by the side chain of the catalytic Tyr24. The substituent

on C2 seems to block the approach of the catalytic tyrosine to

Arg108, thus preventing the formation of the hydrogen-bonding

interaction required to close the active site upon substrate

binding. The proximity of both residues, together with the

essential Arg19, is responsible for lowering the pK_aof Tyr24.

In addition, as for methylbenzothiophene 5b, the substituent on

C3 should block the entrance of the catalytic arginine into the

active site. Therefore, the disubstituted inhibitors appear to

inhibit the enzyme by a double eﬀect. On the one hand, they

avoid the closure of the active site by preventing proper align-

ment of Arg108 and Tyr24 and, on the other hand, they block the

entry of Arg19 into the active site. The latter residue appro-

priately orients the catalytic tyrosine to initiate the enzymatic

reaction and is also responsible for the basicity of the tyrosine.

In Vitro Activity: Synthesis of Ester Prodrugs. The in vitro

antibacterial activity of our inhibitors was studied by determining

the minimum inhibitory concentration (MIC) against M. tuber-

culosis H37Rv by using the Alamar Blue Assay.³⁴In general,

activity was not observed for compounds 5and 9below 200 μg/mL.

We assume that this lack of activity could be due to the high

hydrophilicity of these compounds. It is known that hydrophilic

agents cross the mycobacterial cell wall slowly because the

mycobacterial porin is inefficient in allowing the permeation of

solutes and is only present in low amounts.³⁵To improve

permeability and therefore antibacterial activity, lipophilic pro-

drugs 6À8 and 10À11 were designed. We hypothesized that

these compounds would be slowly hydrolyzed to the carboxylate

active form after absorption by the bacterium. Three types of

esters with different stabilities against hydrolysis were synthe-

sized and evaluated. In addition, transformation of secondary

hydroxyl groups into butyryl esters was also studied.

As observed for PDB entry 2XB8 and the crystal structure of

methylbenzothiophene 5b in complex with DHQ2-Hp (PDB

entry 2WKS²⁸), the essential arginine of the loop, Arg19, is

probably located outside the active site, with its position occu-

pied by the aromatic moiety of the inhibitor. Molecular dynamics

calculations suggest that the required orientation of Tyr24 for

proton abstraction is induced by the proximity of Arg19.¹⁷

Therefore, the conformational changes in the loop that predo-

minantly aﬀect the inappropriate orientation of the catalytic

tyrosine and the inability of the essential arginine to be located

close to it are probably responsible for the high inhibition

potency of methylbenzothiophene 5b. Similar behavior is ex-

pected for the monosubstituted inhibitors 5aÀe.

There is no signiﬁcant diﬀerence between the protein struc-

tures in the DHQ2-Mt/9d and the DHQ2-Mt/9h binary com-

plexes (root-mean-square diﬀerence of 0.12 Å after superposition

of 128 R-carbon atoms). Therefore, an explanation for the

diﬀerent crystallographic resolutions obtained is not obvious

(2.5 and 1.5 Å, respectively) (Figure 6). We can only hypothesize

First, ester prodrugs of inhibitors 5, compounds 6À8, were

synthesized. Yields are shown in Table 3. Methyl esters 6 were

prepared from the corresponding lactones 15 by treatment with

sodium methoxide in methanol. Ethyl derivatives 7 and propyl

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Figure 6. Superposition of DHQ2-Mt structure in complex with (2R)-

2-(4-methoxy)benzyl-3-dehydroquinic acid (PDB entry 2XB8,¹⁷red)

with: (a) DHQ2-Mt/9d binary complex (PDB entry 2Y76, green); (b)

DHQ2-Mt/9h binary complex (PDB entry 2Y77, gray). The loop is

disordered in PDB entries 2Y76 and 2Y77.

esters 8 were synthesized by a nucleophilic substitution of ethyl

bromide and propyl bromide by sodium salts 5, respectively. A

similar strategy was employed for the synthesis of disubstituted

compounds 10 and 11 (Table 3).

The antibacterial activity of compounds 6À8 and 10À11

against M. tuberculosis H37Rv was determined, and the results are

summarized in Table 4. In general, the activity dramatically

increases with the stability of the ester, i.e., propyl esters 8 are

more active than the corresponding methyl esters 6 (Table 4,

entries 1 and 4 vs 12 and 14). The propyl esters 8a, 8f, and 8g

proved to be the most potent of the series with an MIC value of 5

μg/mL (Scheme 4). Furthermore, ethyl esters showed higher

MIC values than the corresponding propyl esters. However, the in

vitro activity of ethyl esters 7 can be increased by esteriﬁcation of

their secondary hydroxyl groups as butyryl esters (Table 4, entry 7 vs

9). Thus, ethyl esters 7e and 7f gave an MIC value of 5 μg/mL. It is

important to highlight that esteriﬁcation of all free hydroxyl groups

provides MIC values that are as high as those of the methyl ester

derivatives 6 (>160 μg/mL). In addition, esteriﬁcation to give less

stable esters, such as acetyl esters, is also less eﬃcient.

Figure 5. (a) Superposition of DHQ2-Mt structure in complex with oxime

(PDBentry1H0S,⁴¹gray),(2R)-2-(4-methoxy)benzyl-3-dehydroquinic acid

(PDB entry 2XB8,¹⁷cyan), and 5b (PDB entry 2Y71, magenta). Residues

18À20 are not solved in 2Y71. (b) Polar contacts (red dashes) between

ligand 5b and DHQ2-Mt. (c) Apolar contacts (black dashes) between ligand

5b and DHQ2-Mt. Only contacts less than 3.3 Å are indicated.

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Table 3. Synthesis of Esters 6À8 and 10À11

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Table 4. The Minimum Inhibitory Concentration (MIC, μg/mL) of Esters 6À11 against M. tuberculosis H37Rv

Furthermore, 2,3-disubstituted ester derivatives 10À11 are

less active than the corresponding monosubstituted compounds

6À7. This ﬁnding could be due to the higher volume of

compounds 10À11, a characteristic that hinders their entry into

the bacterium.

The cytotoxicity of esters 6À11 was evaluated against

human ﬁbroblast cells (MRC-5) using the crystal violet

toxicity assay protocol. No toxicity of cell growth was

observed up to the highest concentration tested, 300À

400 μg/mL.

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Scheme 4. Synthesis of Ethyl Esters 7eÀg and Propyl Esters

8eÀg^a

The crystal structures of binary complexes DHQ2-Mt/9d and

DHQ2-Mt/9h suggest that these 2,3-disubstituted compounds

might inhibit the enzyme by a double eﬀect. Thus, these

inhibitors avoid the closure of the active site by preventing

hydrogen-bonding interaction between the essential Tyr24 and

Arg108 and they also block the entry of Arg19 in the active site.

Therefore, compounds that are able to block the alignment

between Tyr24 and Arg108 would also be good competitive

inhibitors of DHQ2 enzymes.

An ester prodrug approach was used to improve permeability

of the mycobacterial cell. These studies showed that the stability

of the ester is crucial to achieve improved in vitro activities.

Propyl esters 8 were the most eﬃcient in achieving improved in

vitro activities, which are in the low micromolar range. Ethyl

esters 7 can also be used, but esteriﬁcation of the free secondary

hydroxyl groups as butyryl esters is required. In this way MIC

values similar to those of the propyl esters 8 could be achieved.

The results obtained highlight the possibility that inhibitors of

the shikimic acid pathway could be used as new anti-TB agents

and reveal the importance of improving inhibitor hydrophilicity

to traverse the mycobacteria wall.

’ EXPERIMENTAL SECTION

^aReagents and conditions: (a) nPrCOCl, Py, CH₃CN, RT.

General Procedures. All starting materials and reagents were

commercially available and were used without further purification. H

1

’ CONCLUSIONS AND FINAL REMARKS

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 carried out by a combination

of 1 D, COSY, and DEPT-135 experiments. FT-IR spectra were recorded as

NaCl plates or KBr discs. [R]²_D⁰values are given in 10^À1deg cm²g^À1. Purity

of compounds 5À11 were determined by a combination of ¹H NMR and

reverse-phase and normal-phase HPLC and were found to be >95%.

Compounds 5aÀb, 5d, 5f, 14aÀb, 14d, and14f were prepared as described

previously.²⁸

Several enol mimics, compounds 5 and 9, of the reaction

catalyzed by DHQ2, the third enzyme of the shikimic acid

pathway, have been synthesized and tested. All compounds

proved to be reversible competitive inhibitors of DHQ2 from

M. tuberculosis and H. pylori, which are essential enzymes for

these pathogenic bacteria. The eﬀect of the substituent at C2 of

enol mimics 5 was studied with the 2,3-disubstituted compounds 9.

The studies reported here showed that the introduction of a

benzyl group on C2 led to a decrease in the inhibition potency,

but in most cases the inhibition constants are also in the

nanomolar range. Benzothiophene derivative 5a proved to be

the most potent of the monosubstituted series with a K_iof 28 nM

against DHQ2-Mt. The corresponding disubstituted derivative

9a showed a K_iof 40 nM. The substitution of both C2 and C3

with diﬀerent benzyl moieties also provided good inhibition

potencies, with compound 9j having a K_ivalue of 59 nM.

Compounds 5 and 9 also proved to be good competitive

inhibitors of DHQ2-Hp. The disubstituted derivatives 9a and 9b

were the most potent compounds, with inhibition constants of

97 and 50 nM, respectively.

Monosubstituted benzothiophene derivative 5b and double

substituted compounds 9d and 9h were cocrystallized with

DHQ2-Mt, and their structures were solved at 1.5, 2.5, and 1.5 Å,

respectively. Compound 5b binds to DHQ2-Mt in a similar way

to the previously reported DHQ2-Hp/5b complex (PDB entry

2WKS²⁸). However, in the binary DHQ2-Mt/5b complex

reported here, the benzothiophene ring is rotated by 180ꢀ to

orient the methyl group of the aromatic ring in such a way that it

is in close contact with the carbon side chain of Arg15. The

aromatic moiety of inhibitor 5b interacts with the apolar part of

the active site (side chain of Arg15, Leu13, Leu16, etc.) and

expels the essential arginine of the loop from the active site. It has

been suggested that the reaction is initiated by the proximity of

this essential arginine, which appropriately orients the essential

tyrosine to initiate the enzymatic reaction.^16,17

2-Bromomethyl-6-chlorobenzo[b]thiophene. A stirred solu-

tion of 6-chlorobenzo[b]thiophen-2-ylmethanol (400 mg, 2.01 mmol)

and PPh₃(950 mg, 3.62 mmol) in dry dichloromethane (150 mL) was

treated with CBr₄(801 mg, 2.42 mmol) under argon. After stirring for

1 h, diethyl ether was added and the resulting precipitate was filtered off

and washed with diethyl ether. The filtrate was concentrated under

reduced pressure, and the resulting residue was purified by flash

chromatography eluting with (30:70) diethyl ether/hexanes to give

2-bromomethyl-6-chlorobenzo[b]thiophene (483 mg, 92%) as a white

amorphous solid; mp 79À81 ꢀC. ¹H NMR (250 MHz, CDCl₃) δ 7.74

(d, J = 1.5 Hz, 1H, H-7), 7.59 (d, J = 8.7 Hz, 1H, H-4), 7.30 (dd, J = 8.7

and 1.5 Hz, 1H, H-6), 7.24 (s, 1H, H-3) and 4.75 (s, 2H, CH₂) ppm. ¹³

C

NMR (63 MHz, CDCl₃) δ 141.6 (C), 141.4 (C), 137.5 (C), 130.9 (C),

125.4 (CH), 124.5 (CH), 123.8 (CH), 121.9 (CH), and 26.8 (CH₂)

ppm. MS (EI) m/z (%) 260 and 262 (M⁺). HRMS calcd for

C₉H₆ClSBr⁷⁹(M⁺): 259.9062; found, 259.9067.

(1R,4S,5R)-1,4-Di(tert-butyldimethylsilyloxy)-3-(6-chloroben-

zo[b]thiophen-5-yl)methoxycyclohex-2-en-1,5-carbolactone

(13c) and (1R,4S,5R)-1,4-Di(tert-butyldimethylsilyloxy)-3-(6-

chlorobenzo[b]thiophen-5-yl)methoxy-2-(6-chlorobenzo[b]-

thiophen-5-yl)methylcyclohex-2-en-1,5-carbolactone (14c).

A flame-dried round-bottomed flask was charged with KHMDS

(2.32 mL, 1.16 mmol, 0.5 M in toluene) and dry DMF (1.16 mL) under

an inert atmosphere. The resultant solution was cooled to À78 ꢀC, and a

solution of ketone 12²²(232 mg, 0.58 mmol) in a 1:1 mixture of DMF

and toluene (5.8 mL), both dry, was added. The mixture was stirred for

30 min, and a solution of 2-bromomethyl-6-chlorobenzo[b]thiophene

(300 mg, 1.42 mmol) in a 3:2 mixture of DMF and toluene (4.8 mL),

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both dry, was added. After 45 min, the reaction mixture was diluted

successivelywithdiethyl ether and water. The organic phase was separated

and the aqueous layer was extracted with diethyl ether (Â2). The

combined organic extracts were dried (anhyd Na₂SO₄), filtered, and

evaporated under reduced pressure. The residue was purified by flash

chromatography eluting with diethyl ether/hexanes [1) 0:100, 2) 5:95]

to afford O-alkyl derivative 13c (47 mg, 23%) and dialkyl derivative 14c

ppm. IR (KBr) 3436 (OÀH) and 1683 (CdO) cm^À1. MS (ESI) m/z

(%) 369 (M À H). HRMS calculated for C₁₆H₁₃O₆SClNa (M À H),

391.0014; found, 391.0027.

(1R,4S,5R)-2-(6-Chlorobenzo[b]thiophen-5-yl)methyl-3-

(6-chlorobenzo[b]thiophen-5-yl)methoxy-1,4-dihydroxy-

cyclohex-2-en-1,5-carbolactone (16c). The experimental pro-

cedure was the same as for compound 15c using silyl ether 14c (144

mg), TBAF (0.42 mL), and THF (2.7 mL). Yield = 67 mg (66%).

Yellow solid; mp 201À202 ꢀC. [R]²_D⁰= À152ꢀ (c 0.6, MeOH/acetone

(142 mg, 46%). Data for 14c: beige solid; mp 153À155 ꢀC. [R]_D²⁰

=

À116ꢀ (c1.2, CHCl₃). ¹H NMR(250 MHz, CDCl₃) δ 7.55 (d, J= 1.7 Hz,

1H, ArH), 7.50 (d, J = 1.7 Hz, 1H, ArH), 7.38 (d, J = 8.5 Hz, 1H, ArH),

7.26 (d, J = 8.5 Hz, 1H, ArH), 7.07 (m, 2H, 2 Â ArH), 6.83 (s, 1H, ArH),

6.72 (s, 1H, ArH), 4.91 (d, J = 12.5 Hz, 1H, OCHH), 4.84 (d, J = 12.5 Hz,

1H, OCHH), 4.44 (dd, J = 5.7 and 3.5 Hz, 1H, H-5), 4.33 (d, J = 3.5 Hz,

1H, H-4), 3.75 (d, J = 15.7 Hz, 1H, CHHAr), 3.62 (d, J = 15.7 Hz, 1H,

CHHAr), 2.41 (d, J = 11.0 Hz, 1H, H-6_eq), 2.30 (dd, J = 11.0 and 5.7 Hz,

1H, H-6_ax), 0.81 (s, 9H, C(CH₃)₃), 0.62 (s, 9H, C(CH₃)₃), 0.06 (s, 3H,

CH₃), 0.05 (s, 3H, CH₃), 0.01 (s, 3H, CH₃) and À0.08 (s, 3H, CH₃)

ppm. ¹³C NMR (63 MHz, CDCl₃) δ 175.1 (C), 148.5 (C), 144.6 (C),

141.0 (C), 140.2 (2 Â C), 138.4 (C), 137.5 (C), 130.5 (C), 129.0 (C),

128.6 (CH), 125.1 (CH), 124.5 (C), 124.3 (CH), 123.3 (CH), 121.8

(CH), 121.4 (CH), 120.7 (CH), 74.6 (C), 74.5 (CH), 67.9 (CH₂), 67.2

(CH), 37.4 (CH₂), 25.7 (C(CH₃)₃), 25.4 (CH₂+ C(CH₃)₃), 18.0

(C(CH₃)₃), 18.0 (C(CH₃)₃), À3.3 (CH₃), À3.5 (CH₃), À4.4 (CH₃)

and À4.5 (CH₃) ppm. IR (KBr) 1792 (CdO) cm^À1. MS (ESI) m/z (%)

761 (MH⁺). HRMS calculated for C₃₇H₄₇O₅S₂Si₂Cl₂(MH⁺), 761.1775;

found, 761.1754.

1

(2:1)). H NMR (250 MHz, DMSO-d₆) δ 8.08 (d, J = 2.0 Hz, 1H,

ArH), 7.93 (d, J = 2.0 Hz, 1H, ArH), 7.79 (d, J = 8.5 Hz, 1H, ArH),

7.61 (d, J = 8.5 Hz, 1H, ArH), 7.39 (dd, J = 8.5 and 2.0 Hz, 1H, ArH),

7.39 (s, 1H, ArH), 7.28 (dd, J = 8.5 and 2.0 Hz, 1H, ArH), 7.04 (s, 1H,

ArH), 6.39 (s, 1H, OH), 6.12 (d, J = 7.0 Hz, 1H, OH), 5.37 (d, J = 13.0

Hz, 1H, OCHH), 5.28 (d, J = 13.0 Hz, 1H, OCHH), 4.62 (m, 1H,

H-5), 4.49 (dd, J = 7.0 and 3.5 Hz, 1H, H-4), 3.81 (d, J = 14.7 Hz, 1H,

CHHAr), 3.67 (d, J = 14.7 Hz, 1H, CHHAr) and 2.29 (m, 2H, CH₂-6)

ppm. ¹³C NMR (63 MHz, DMSO-d₆) δ 176.2 (C), 147.8 (C), 145.5

(C), 142.1 (C), 140.6 (C), 140.2 (C), 138.4 (C), 137.7 (C), 129.2

(C), 127.9 (C), 125.0 (CH), 124.9 (CH), 124.3 (CH), 123.8 (CH),

122.3 (CH), 122.0 (CH), 121.5 (CH), 121.3 (C), 120.8 (CH), 74.6

(CH), 71.7 (C), 64.8 (CH₂), 64.3 (CH), 36.8 (CH₂) and 24.5 (CH₂)

ppm. IR (KBr) 3408 (OÀH) and 1749 (CdO) cm^À1. MS (ESI) m/z

(%) 555 (MNa⁺). HRMS calcd for C₂₅H₁₈O₅S₂Cl₂Na (MNa⁺),

554.9865; found, 554.9865.

Sodium (1R,4S,5R)-3-(6-Chlorobenzo[b]thiophen-5-yl)-

methoxy-2-(6-chlorobenzo[b]thiophen-5-yl)methyl-1,4,5-

trihydroxycyclohex-2-en-1-carboxylate (9c). The experimen-

tal procedure was the same as for compound 5c using lactone 16c

(8 mg), NaOH (30 μL) and THF (0.1 mL). Yield = 8 mg (93%). Beige

solid; mp 189 ꢀC (dec). [R]_D²⁰= À42ꢀ (c 0.8, (2:1) DMSO/MeOH).

¹H NMR (500 MHz, DMSO-d₆) δ 8.65 (d, J = 8.0 Hz, 1H, ArH), 7.71

(d, J = 9.0 Hz, 1H, ArH), 7.53 (d, J = 8.5 Hz, 1H, ArH), 7.35 (dd, J = 8.5

and 2.0 Hz, 1H, ArH), 7.23 (m, 2H, 2 Â ArH), 7.02 (s, 1H, ArH), 5.21

(s, 1H, OH), 5.16 (d, J = 13.0 Hz, 1H, CHHO), 5.12 (d, J = 13.0 Hz,

1H, CHHO), 4.08 (s, 1H, H-4), 3.63 (m, 1H, H-5), 3.60 (d, J =

16.0 Hz, 1H, CHHAr), 3.21 (d, J = 16.0 Hz, 1H, CHHAr), 2.10 (dd, J =

14.0 and 3.0 Hz, 1H, CHH-6) and 1.68 (dd, J = 14.0 and 3.5 Hz, 1H,

CHH-6) ppm. ¹³C NMR (125 MHz, DMSO-d₆) δ 176.9 (C), 150.1

(C), 147.6 (C), 143.1 (C), 140.5 (C), 140.3 (C), 138.4 (C), 137.7 (C),

128.8 (C), 127.3 (CH), 124.7 (2 Â CH), 123.9 (CH), 123.4 (CH),

121.8 (CH), 121.4 (CH), 121.2 (CH), 120.5 (C), 120.4 (CH), 74.1

(C), 69.8 (CH), 67.9 (CH), 64.0 (OCH₂), 34.8 (CH₂) and 26.2

(CH₂) ppm. IR (KBr) 3444 (OÀH) and 1684 (CdO) cm^À1. MS

(ESI) m/z (%) 573 (MNa⁺). HRMS calcd for C₂₅H₂₀O₆S₂Cl₂Na

(MNa⁺), 572.9971; found, 572.9973.

(1R,4S,5R)-1,4-Dihydroxy-3-(6-chlorobenzo[b]thiophen-

5-yl)methoxycyclohex-2-en-1,5-carbolactone (15c). A stir-

red solution of silyl ether 13c (164 mg, 0.28 mmol) in dry THF

(4.0 mL) was treated with tetrabutylammonium fluoride (0.62 mL,

0.62 mmol, ca. 1.0 M in THF). After 1 h, ethyl acetate and water were

added. The organic layer was separated, and the aqueous phase was

extracted with ethyl acetate (Â3). The combined organic extracts were

dried (anhyd Na₂SO₄), filtered, and concentrated under reduced

pressure. The resulting residue was purified by flash chromatography

eluting with ethyl acetate/hexane (50:50) to give diol 15c (31 mg,

31%) as a white foam; mp 79À81 ꢀC. [R]²_D⁰= À116ꢀ (c 1.6, MeOH).

¹H NMR (250 MHz, CD₃OD) δ 7.88 (d, J = 1.7 Hz, 1H, ArH), 7.73

(d, J = 8.5 Hz, 1H, ArH), 7.37 (d, J = 0.7 Hz, 1H, ArH), 7.33 (dd, J = 8.5

and 1.7 Hz, 1H, ArH), 5.19 (s, 1H, H-2), 5.12 (d, J = 13.0 Hz, 1H,

OCHH), 5.06 (d, J = 13.0 Hz, 1H, OCHH), 4.63 (m, 1H, H-5), 4.12

(d, J = 3.5 Hz, 1H, H-4) and 2.32 (m, 2H, CH₂-6) ppm. ¹³C NMR

(63 MHz, CD₃OD) δ 179.1 (C), 155.3 (C), 142.8 (C), 142.0 (C),

139.4 (C), 131.6 (C), 126.2 (CH), 125.8 (CH), 124.1 (CH), 122.9

(CH), 105.6 (CH), 77.0 (CH), 73.0 (C), 67.7 (CH), 66.2 (CH₂) and

38.3 (CH₂) ppm. IR (KBr) 3398 (OÀH) and 1770 (CdO) cm^À1. MS

(ESI) m/z (%) 375 (MNa⁺). HRMS calcd for C₁₆H₁₃O₅SClNa

(MNa⁺), 375.0064; found, 375.0062.

Sodium (1R,4S,5R)-3-(6-Chlorobenzo[b]thiophen-5-yl)-

methoxy-1,4,5-trihydroxycyclohex-2-ene-1-carboxylate (5c).

A solution of lactone 15c (8 mg, 0.02 mmol) in THF (0.2 mL) and

aqueous sodium hydroxide (45 μL, 0.02 mmol, 0.5M) was stirred at room

temperature for 15 min. Water was added, and THF was removed under

reduced pressure. The resulting aqueous solution was washed with diethyl

ether (Â2), and the aqueous extract was lyophilized to afford derivative 5c

(9 mg, 99%) as a white solid; mp 200À202 ꢀC (dec). [R]²_D⁰= À10ꢀ (c 0.5,

H₂O). ¹H NMR (400 MHz, D₂O/CD₃CN (2:1)) δ 8.40 (d, J = 2.0 Hz,

1H, ArH), 8.26 (d, J = 8.4 Hz, 1H, ArH), 7.86 (s, 1H, ArH), 7.85 (dd, J =

8.4 and 2.0 Hz, 1H, ArH), 5.54 (d, J = 12.4 Hz, 1H, OCHH), 5.47 (d, J =

12.4 Hz, 1H, OCHH), 5.34 (s, 1H, H-2), 4.43 (d, J = 6.0 Hz, 1H, H-4),

4.35 (m, 1H, H-5) and 2.47 (m, 2H, CH₂-6) ppm. ¹³C NMR (100 MHz,

D₂O/CD₃CN(2:1)) δ 180.9 (C), 155.6 (C), 141.3 (C), 138.3 (C), 130.2

(C), 125.4 (CH), 125.3 (CH), 123.3 (CH), 122.2 (CH + C), 102.0

(CH), 73.7 (C), 71.4 (CH), 69.9 (CH), 65.0 (CH₂) and 37.2 (CH₂)

(1R,4S,5R)-3-(Benzo[b]thiophen-2-yl)methoxy-2-(benzo-

[b]thiophen-2-yl)methyl-1,4-dihydroxycyclohex-2-en-1,5-

carbolactone (16a). The experimental procedure was the same as

for compound 15c using silyl ether 14a²⁸(75 mg), TBAF (0.29 mL),

and THF (1.6 mL). Yield = 50 mg (98%). Light-yellow oil. [R]_D²⁰

=

1

À228.5ꢀ (c 1.0, acetone). H NMR (250 MHz, acetone-d₆) δ 7.89

(m, 1H, ArH), 7.76 (m, 2H, 2 Â ArH), 7.61 (m, 1H, ArH), 7.39À7.19

(m, 5H, 5 Â ArH), 7.12 (m, 1H, ArH), 5.50À5.34 (m, 4H, OCH₂+

2 Â OH), 4.70 (m, 2H, H-4 + H-5), 4.01 (d, J = 14.8 Hz, 1H, CHHAr),

3.82 (d, J = 14.8 Hz, 1H, CHHAr), 2.52 (dd, J = 11.0 and 2.8 Hz, 1H,

CHH-6) and 2.42 (dd, J = 11.0 and 5.8 Hz, 1H, CHH-6) ppm. ¹³C NMR

(63 MHz, acetone-d₆) δ 177.7 (C), 149.8 (C), 146.4 (C), 143.1 (C),

142.2 (C), 141.9 (C), 141.5 (C), 141.3 (CH), 126.3 (CH), 126.2 (CH),

125.6 (CH), 125.5 (CH), 125.0 (CH), 124.9 (C), 124.6 (CH), 124.5

(CH), 124.2 (CH), 123.7 (CH), 123.3 (CH), 76.9 (CH), 74.3 (C), 67.4

(CH₂), 67.2 (CH), 39.1 (CH₂) and 26.6 (CH₂) ppm. IR (film): 3415

(OÀH) and 1788 (CdO) cm^À1. MS (ESI) m/z (%) 487 (MNa⁺).

HRMS calcd for C₂₅H₂₀O₅S₂Na (MNa⁺), 487.0644; found, 487.0644.

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Sodium (1R,4S,5R)-3-(Benzo[b]thiophen-2-yl)methoxy-

2-(benzo[b]thiophen-2-yl)methyl-1,4,5-trihydroxycyclohex-

2-en-1-carboxylate (9a). The experimental procedure was the same

as for compound 5c using lactone 16a (52 mg), NaOH (220 μL), and

THF (1 mL). Yield = 54 mg (97%). Beige solid. [R]²_D⁰= À62.7ꢀ (c 1.5,

(1R,4S,5R)-3-(Benzo[b]thiophen-5-yl)methoxy-2-(benzo-

[b]thiophen-5-yl)methyl-1,4-dihydroxycyclohex-2-en-1,5-

carbolactone (16d). The experimental procedure was the same as

for compound 5c using ether 14d²⁸(97 mg, 0.14 mmol), TBAF

(280 μL), and THF (2.0 mL). Yield = 56 mg (86%). White solid; mp

1

MeOH). H NMR (400 MHz, DMSO-d₆) δ 8.62 (d, J = 8.0 Hz, 1H,

123À125 ꢀC. [R]²_D⁰= À163ꢀ (c 1.5, acetone). H NMR (250 MHz,

ArH), 7.87 (m, 1H, ArH), 7.75 (m, 1H, ArH), 7.69 (m, 1H, ArH), 7.55

(m, 1H, ArH), 7.35À7.27 (m, 2H, 2 Â ArH), 7.25À7.16 (m, 3H, 3 Â

ArH), 7.04 (s, 1H, OH), 5.24 (br s, 1H, OH), 5.19À5.12 (m, 3H, OCH₂

+ OH), 4.10 (br s, 1H, OH), 3.63 (m, 2H, H-5 + CHHAr), 3.23 (d, J =

15.2 Hz, 1H, CHHAr), 2.12 (dd, J = 14.0 and 3.2 Hz, 1H, CHH-6) and

1.70 (dd, J = 14.0 and 3.2 Hz, 1H, CHH-6) ppm. ¹³C NMR (63 MHz,

DMSO-d₆) δ 177.3 (C), 150.1 (C), 146.3 (C), 142.1 (C), 139.8 (C),

139.2 (C), 139.0 (C), 138.9 (C), 124.2 (CH), 124.1 (CH), 123.6 (CH),

123.4 (CH), 122.7 (CH), 122.4 (CH), 122.2 (CH), 121.9 (CH), 121.8

(CH), 120.8 (CH), 120.5 (C), 74.2 (C), 69.8 (CH), 68.0 (CH), 64.2

(CH₂), 34.9 (CH₂) and 26.2 (CH₂) ppm. IR (KBr): 3398 (OÀH) and

1601 (CdO) cm^À1. MS (ESI) m/z (%) 505 (MH⁺). HRMS calcd for

C₂₅H₂₂O₆S₂Na (MH⁺), 505.0750; found, 505.0751.

CD₃OD) δ 7.74 (d, J = 8.2 Hz, 1H, ArH), 7.61 (m, 3H, 3 Â ArH), 7.49

(d, J = 5.5 Hz, 1H, ArH), 7.39 (d, J = 5.5 Hz, 1H, ArH), 7.19 (m, 3H, 3

Â ArH), 7.10 (d, J = 5.5 Hz, 1H, ArH), 5.13 (d, J = 11.5 Hz, 1H,

OCHH), 4.92 (d, J = 11.5 Hz, 1H, OCHH), 4.57 (m, 1H, H-5), 4.51

(d, J = 3.2 Hz, 1H, H-4), 3.73 (d, J = 14.2 Hz, 1H, CHHAr), 3.56 (d, J =

14.2 Hz, 1H, CHHAr) and 2.30 (m, 2H, CH₂-6) ppm. ¹³C NMR

(63 MHz, acetone-d₆) δ 177.8 (C), 149.6 (C), 141.4 (C), 140.6 (C),

138.7 (C), 138.4 (C), 135.8 (C), 128.7 (CH), 127.7 (CH), 127.5

(CH), 125.8 (CH), 125.4 (CH), 125.3 (C), 125.3 (CH), 125.2 (CH),

125.1 (C), 124.2 (CH), 123.8 (CH), 123.1 (CH), 76.6 (CH), 74.2

(C), 71.4 (CH₂), 66.8 (CH), 38.9 (CH₂) and 30.9 (CH₂) ppm. IR

(KBr) 3452 (OÀH), 3363 (OÀH) and 1770 (CdO) cm^À1. MS (CI)

m/z (%) 465 (MH⁺). HRMS calcd for C₂₅H₂₁O₅S₂(MH⁺), 465.0830;

found, 465.0831.

(1R,4S,5R)-1,4,5-Trihydroxy-3-(5-methylbenzo[b]thiophen-

2-yl)methoxy-2-(5-methylbenzo[b]thiophen-2-yl)methylcyclo-

hex-2-en-1,5-carbolactone (16b). The experimental procedure was

the same as for compound 15c using silyl ether 14b²⁸(119 mg), TBAF

(0.44 mL), and THF(2.4 mL). Yield = 70mg (84%). Beige solid. [R]²_D⁰=À

232.4ꢀ (c1.7, acetone). ¹H NMR (250 MHz, acetone-d₆) δ 7.74 (d, J = 8.3

Hz, 1H, ArH), 7.62 (d, J = 8.3 Hz, 1H, ArH), 7.52 (s, 1H, ArH), 7.35 (s, 1H,

ArH), 7.24 (s, 1H, ArH), 7.17 (dd, J = 8.0 and 0.8 Hz, 1H, ArH), 7.06 (dd,

J = 8.0 and 0.8 Hz, 1H, ArH), 6.99 (s, 1H, ArH), 5.43 (d, J = 12.5 Hz, 1H,

OCHH), 5.35 (d, J = 12.5 Hz, 1H, OCHH), 4.68 (m, 2H, H-4+H-5), 3.97

(d, J=14.8Hz, 1H, CHHAr), 3.79 (d, J=14.8Hz, 1H, CHHAr), 2.51(d, J=

12.5 Hz, 1H, CHH-6), 2.45À2.38 (m, 1H, CHH-6), 2.41 (s, 3H, Me) and

2.38 (s, 3H, Me) ppm. ¹³C NMR (63 MHz, acetone-d₆) δ 177.8 (C), 149.8

(C), 146.5 (C), 143.1 (C), 142.5 (C), 141.7 (C), 139.2 (C), 138.7 (C),

135.7 (C), 135.1 (C), 127.9 (CH), 126.6 (CH), 125.4 (CH), 125.0 (CH),

124.5 (CH), 124.4 (CH), 123.8 (CH), 123.3 (CH), 123.1 (CH), 76.9

(CH), 74.3 (C), 67.4 (OCH₂), 67.2 (CH), 39.1 (CH₂), 26.6 (CH₂) and

22.3 (2 Â CH₃) ppm. IR (film): 3471 (OÀH), 3344 (OÀH) and 1770

(CdO) cm^À1. MS (CI) m/z (%) 493 (MH⁺). HRMS calcd for

C₂₇H₂₅O₅S₂(MH⁺), 493.1143; found, 493.1131.

Sodium (1R,4S,5R)-3-(Benzo[b]thiophen-5-yl)methoxy-

2-(benzo[b]thiophen-5-yl)methyl-1,4,5-trihydroxycyclohex-

2-en-1-carboxylate (9d). The experimental procedure was the same

as for compound 5c using lactone 16d (25 mg), NaOH (0.1 mL), and

THF (0.4 mL). Yield = 25 mg (99%). White solid; mp 197À200 ꢀC

(dec). [R]²_D⁰= À67ꢀ (c 1.3, 50% MeOH/H₂O). ¹H NMR (250 MHz,

50% CD₃OD/D₂O) δ 7.66 (m, 3H, 3 Â ArH), 7.47 (m, 2H, 2 Â ArH),

7.26 (d, J = 8.2 Hz, 1H, ArH), 7.14 (m, 2H, 2 Â ArH), 7.05 (d, J = 5.5 Hz,

1H, ArH), 6.98 (d, J = 8.2 Hz, 1H, ArH), 4.84 (d, J = 10.5 Hz, 1H,

OCHH), 4.58(d, J = 10.5Hz, 1H, OCHH), 4.41(d, J = 5.0Hz, 1H, H-4),

3.98 (m, 1H, H-5), 3.57 (d, J = 15.7 Hz, 1H, CHHAr), 3.28 (d, J = 15.7

Hz, 1H, CHHAr) and 2.20 (m, 2H, CH₂-6) ppm. ¹³C NMR (63 MHz,

50% CD₃OD/D₂O) δ 181.9 (C), 153.1 (C), 141.9 (C), 141.6 (C), 141.0

(C), 139.7 (C), 138.8 (C), 135.3 (C), 128.9 (CH), 128.2 (CH), 127.6

(CH), 126.4 (CH), 125.8 (CH), 125.7 (C), 125.3 (CH), 125.0 (CH),

124.3(CH), 123.9 (CH), 123.5 (CH), 78.5(C), 72.2 (CH), 72.0 (CH₂),

70.9 (CH), 38.8 (CH₂) and 33.8 (CH₂) ppm. IR (KBr) 3410 (OÀH)

and 1595 (CdO) cm^À1. MS (ESI) m/z (%) 481 (M^À). HRMS calcd for

C₂₅H₂₁O₆S₂Na (M^À), 481.0774; found, 481.0776.

Sodium (1R,4S,5R)-1,4,5-Trihydroxy-3-(5-methylbenzo-

[b]thiophen-2-yl)methoxy-2-(5-methylbenzo[b]thiophen-

2-yl)methylcyclohex-2-en-1-carboxylate (9b). The experi-

mental procedure was the same as for compound 5c using carbolac-

tone 16b (35 mg), NaOH (126 μL), and THF (0.65 mL). Yield =

37 mg (98%). Beige solid. [R]²_D⁰= À61.3ꢀ (c 1.5, MeOH). ¹H NMR

(250 MHz, DMSO-d₆) δ 8.63 (d, J = 8.0 Hz, 1H, ArH), 7.73 (d, J =

8.0 Hz, 1H, ArH), 7.61 (d, J = 8.0 Hz, 1H, ArH), 7.45 (br s, 1H, ArH),

7.29 (br s, 1H, ArH), 7.13 (dd, J = 8.0 and 1.6 Hz, 1H, ArH), 7.08 (br s,

1H, ArH), 7.01 (dd, J = 8.0 and 1.6 Hz, 1H, ArH), 6.92 (s, 1H, OH),

5.21 (br s, 1H, OH), 5.15 (br s, 1H, OH), 5.14 (d, J = 13.2 Hz, 1H,

OCHH), 5.10 (d, J = 13.2 Hz, 1H, OCHH), 4.08 (br s, 1H, H-4), 3.64

(m, 1H, H-5), 3.60 (d, J = 15.2 Hz, 1H, CHHAr), 3.19 (d, J = 15.2 Hz,

1H, CHHAr), 2.38 (s, 3H, Me), 2.35 (s, 3H, Me), 2.11 (dd, J = 14.0

and 3.2 Hz, 1H, CHH-6) and 1.68 (dd, J = 14.0 and 3.2 Hz, 1H, CHH-6)

ppm. ¹³C NMR (100 MHz, DMSO-d₆) δ 177.2 (C), 150.1 (C),

146.4 (C), 142.1 (C), 140.1 (C), 139.4 (C), 136.4 (C), 136.1 (C),

133.3 (C), 132.5 (C), 125.7 (CH), 124.3 (CH), 123.3 (CH), 122.2

(CH), 122.0 (CH), 121.7 (CH), 121.4 (CH), 120.6 (CH), 120.5 (C),

74.3 (C), 69.8 (CH), 68.0 (CH), 64.2 (OCH₂), 34.9 (CH₂), 26.3

(CH₂), 21.0 (CH₃) and 20.9 (CH₃) ppm. IR (KBr): 3435 (OÀH) and

1599 (CdO) cm^À1. MS (ESI) m/z (%) 533 (MH⁺). HRMS calcd for

C₂₇H₂₆O₆S₂Na (MH⁺), 533.1068; found, 533.1072. Elemental anal-

(1R,4S,5R)-1,4-Di(tert-butyldimethylsilyloxy)-3-(naphth-

2-yl)methoxycyclohex-2-en-1,5-carbolactone (13e) and

(1R,4S,5R)-1,4-Di(tert-butyldimethylsilyloxy)-3-(naphth-2-

yl)methoxy-2-(naphth-2-yl)methylcyclohex-2-en-1,5-car-

bolactone (14e). The experimental procedure was the same as for

compounds 13c and 14c using ketone 12²²(522 mg, 1.30 mmol),

KHMDS (5.2 mL), and 2-(bromomethyl)naphtalene (577 mg).

Purification by flash chromatography on silica gel eluting with diethyl

ether/hexanes [(1) 5:95; (2) 10:90] gave ethers 13e (302 mg, 43%)

and 14e (259 mg, 29%), both as colorless oils.

Data for 13e: [R]²_D⁰= À106ꢀ (c 1.0, CHCl₃). ¹H NMR (250 MHz,

CDCl₃) δ 7.85 (m, 4H, 4 Â ArH), 7.49 (m, 3H, 3 Â ArH), 5.11 (s, 1H,

H-2), 4.93 (d, 1H, J = 11.5 Hz, CHHO), 4.86 (d, 1H, J = 11.5 Hz,

CHHO), 4.54 (dd, 1H, J = 5.0 and 3.5 Hz, H-5), 4.27 (d, 1H, J = 3.5 Hz,

H-4), 2.47 (d, 1H, J = 10.7 Hz, H-6_eq), 2.38 (dd, 1H, J = 10.7 and 5.0 Hz,

H-6_ax), 0.99 (s, 9H, C(CH₃)₃), 0.95 (s, 9H, C(CH₃)₃), 0.21 (s, 3H,

SiCH₃), 0.16 (s, 3H, SiCH₃), 0.14 (s, 3H, SiCH₃) and 0.12 (s, 3H,

SiCH₃) ppm. ¹³C NMR (63 MHz, CDCl₃) δ 176.0 (C), 153.4 (C),

133.2 (C), 133.0 (C), 132.9 (C), 128.1 (CH), 127.7 (CH), 127.6 (CH),

126.5 (CH), 126.1 (CH), 126.0 (CH), 125.3 (CH), 104.9 (CH), 75.2

(CH), 73.7 (C), 69.6 (OCH₂), 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), À3.2 (2 Â CH₃),

À4.6 (CH₃) and À5.2 (CH₃) ppm. IR (ﬁlm) 1803 (CdO) cm^À1. MS

(ESI) m/z (%) 563 (MNa⁺). HRMS calcd for C₃₀H₄₄O₅Si₂Na (MNa⁺),

563.2619; found, 563.2622.

ysis C₂₇H₂₅O₆S₂Na 2H₂O Calcd: C, 57.03; H, 5.14; S, 11.28. Found:

3

C, 56.69; H, 5.19; S, 10.91.

6074

dx.doi.org/10.1021/jm2006063 |J. Med. Chem. 2011, 54, 6063–6084

Journal of Medicinal Chemistry

ARTICLE

Data for 14e: [R]²_D⁰= À151ꢀ (c 1.0, CHCl₃). ¹H NMR (250 MHz,

CDCl₃) δ 7.84À7.60 (m, 7H, 7 Â ArH), 7.51À7.26 (m, 7H, 7 Â ArH),

4.98 (d, 1H, J = 11.5 Hz, CHHO), 4.87 (d, 1H, J = 11.5 Hz, CHHO),

4.65 (dd, 1H, J = 5.7 and 3.5 Hz, H-5), 4.56 (d, 1H, J = 3.5 Hz, H-4), 3.94

(d, 1H, J = 15.0 Hz, CHHAr), 3.80 (d, 1H, J = 15.0 Hz, CHHAr), 2.67 (d,

1H, J = 10.7 Hz, H-6_eq), 2.52 (dd, 1H, J = 10.7 and 6.0 Hz, H-6_ax), 1.00

(s, 9H, C(CH₃)₃), 0.76 (s, 9H, C(CH₃)₃), 0.22 (s, 3H, SiCH₃), 0.21 (s,

3H, SiCH₃), 0.19 (s, 3H, SiCH₃) and 0.08 (s, 9H, SiCH₃) ppm. ¹³C

NMR (63 MHz, CDCl₃) δ 175.6 (C), 149.0 (C), 137.6 (C), 134.3 (C),

133.4 (C), 133.1 (C), 132.8 (C), 131.9 (C), 129.1 (C), 128.0 (CH),

127.9 (CH), 127.6 (CH), 127.5 (CH), 127.4 (CH), 127.4 (CH), 127.3

(CH), 126.6 (CH), 126.0 (CH), 126.0 (CH), 125.9 (CH), 125.5 (CH),

125.0 (CH), 124.8 (CH), 75.0 (C), 74.8 (CH), 72.7 (OCH₂), 67.3

(CH), 37.6 (CH₂), 30.4 (CH₂), 25.7 (C(CH₃)₃), 25.4 (C(CH₃)₃), 18.0

(2 Â C(CH₃)₃), À3.3 (CH₃), À3.5 (CH₃), À4.5 (CH₃) and À4.5

(CH₃) ppm. IR (Film) 1799 (CdO) cm^À1. MS (ESI) m/z (%) 703

(MNa⁺). HRMS calcd for C₄₁H₅₂O₅Si₂Na (MNa⁺), 703.3245; found,

703.3248.

Sodium (1R,4S,5R)-1,4,5-Trihydroxy-3-(naphth-2-yl)-

methoxy-2-(naphth-2-yl)methylcyclohex-2-en-1-carboxylate

(9e). The experimental procedure was the same as for compound 5c

using carbolactone 16e (29 mg), NaOH (120 μL), and THF (0.6 mL).

Yield = 28 mg (95%). White solid; mp 184À187 ꢀC [R]²_D⁰= À4ꢀ (c1.1,

H₂O). ¹H NMR (250 MHz, 50% D₂O/CD₃CN) δ 7.76À7.57 (m, 6H, 6

Â ArH), 7.46À7.29 (m, 7H, 7 Â ArH), 7.15 (br d, J = 8.2 Hz, 1H, ArH),

4.92 (d, J = 11.2 Hz, 1H, OCHH), 4.71 (d, J = 11.2 Hz, 1H, OCHH), 4.33

(d, J = 3.0 Hz, 1H, H-4), 3.88 (m, 1H, H-5), 3.60 (d, J = 15.7 Hz, 1H,

CHHAr), 3.22 (d, J = 15.7 Hz, 1H, CHHAr) and 2.10 (m, 2H, CH₂-6)

ppm. ¹³C NMR (63 MHz, 50% D₂O/CD₃CN) δ 180.5 (C), 151.9 (C),

140.1 (C), 136.0 (C), 134.2 (C), 133.7 (C), 133.5 (C), 132.5 (C), 128.7

(CH), 128.6 (CH), 128.5 (CH), 128.3 (CH), 128.2 (2 Â CH), 127.9

(CH), 127.3 (CH), 127.1 (CH), 126.9 (CH), 126.8 (CH), 126.8 (CH),

126.5 (CH), 125.7 (CH), 122.3 (C), 76.9 (C), 70.8 (CH), 70.4 (CH₂),

69.2 (CH), 36.8 (CH₂) and 32.7 (CH₂) ppm. IR (KBr) 3435 (OÀH)

and 1660(CdO) cm^À1. MS(ESI) m/z (%) 493 (MH⁺). HRMS calcd for

C₂₉H₂₆O₆Na (MH⁺), 493.1622; found, 493.1620.

(1R,4S,5R)-1,4-Dihydroxy-3-(thien-3-yl)methoxy-2-(thien-3-

yl)methylcyclohex-2-en-1,5-carbolactone (16f). The experimen-

tal procedure was the same as for compound 15c using silyl ether 14f²⁸

(145 mg), TBAF (0.54 mL), and THF (3.5 mL). Yield = 75 mg (86%).

White solid; mp 95À97 ꢀC. [R]²_D⁰= À189ꢀ (c 1.2, MeOH). ¹H NMR (300

MHz, CD₃OD) δ 7.36 (d, J = 4.8 Hz, 1H, ArH), 7.07 (m, 1H, ArH), 7.02

(s, 1H, ArH), 6.96 (m, 1H, ArH), 6.81 (s, 2H, 2 Â ArH), 5.22 (d, J = 12.3

Hz, 1H, OCHH), 5.11 (d, J = 12.3 Hz, 1H, OCHH), 4.61 (m, 1H, H-5),

4.51 (d, J = 2.4 Hz, 1H, H-4), 3.76 (d, J = 14.4 Hz, 1H, CHHAr), 3.64 (d,

J = 14.4 Hz, 1H, CHHAr), 2.38(d, J= 11.1 Hz, 1H, H-6_ax) and2.32(dd, J=

11.1 and 6.0 Hz, 1H, H-6_eq) ppm. ¹³C NMR (75 MHz, CD₃OD) δ 178.8

(C), 148.5 (C), 144.1 (C), 141.0 (C), 127.9 (CH), 127.7 (CH), 127.3

(CH), 127.3 (CH), 126.2 (CH + C), 124.0 (CH), 76.8 (CH), 73.6 (C),

66.1 (CH), 65.9 (CH₂), 38.4 (CH₂) and 24.9 (CH₂) ppm. IR (KBr) 3498

(OÀH), 3413 (OÀH) and 1774 (CdO) cm^À1. MS (CI) m/z (%) 365

(MH⁺). HRMS calcd for C₁₇H₁₆O₅S₂(MH⁺), 365.0517; found, 365.0517.

Sodium (1R,4S,5R)-1,4,5-Trihydroxy-3-(thien-3-yl)methoxy-

2-(thien-3-yl)methylcyclohex-2-en-1-carboxylate (9f). The ex-

perimental procedure was the same as for compound 5c using lactone

16f (25 mg), NaOH (140 μL), and THF (0.6 mL). Yield = 25 mg (97%).

Beige solid; mp 178À181 ꢀC. [R]²_D⁰= À89ꢀ (c 1.2, H₂O). ¹H NMR (300

MHz, D₂O) δ 7.46 (d, J = 4.8 Hz, 1H, ArH), 7.19 (dt, J = 5.1 and 1.2 Hz,

1H, ArH), 7.13 (br d, J = 2.7 Hz, 1H, ArH), 7.05 (ddd, J = 5.1, 3.6, and 0.6

Hz, 1H, ArH), 6.92 (dt, J = 3.6 and 0.9 Hz, 1H, ArH), 6.85 (m, 1H, ArH),

5.18 (d, J = 12.0 Hz, 1H, CHHO), 4.92 (d, J = 12.0 Hz, 1H, CHHO), 4.37

(d, J = 6.6 Hz, 1H, H-4), 3.93 (m, 1H, H-5), 3.68 (d, J = 15.6 Hz, 1H,

CHHAr), 3.28 (d, J= 15.6 Hz, 1H, CHHAr), 2.16 (dd, J= 13.8 and 11.4 Hz,

1H, H-6_ax) and 2.03 (dd, J = 13.8 and 3.3 Hz, 1H, H-6_eq) ppm. ¹³C NMR

(75 MHz, D₂O) δ 182.9 (C), 153.8 (C), 146.8 (C), 142.0 (C), 131.2

(CH), 130.3 (CH), 130.2 (CH), 129.8 (CH), 128.4 (CH), 126.8 (CH),

126.7 (C), 79.8 (C), 73.0 (CH), 72.9 (CH), 68.4 (CH₂), 41.7 (CH₂) and

29.8 (CH₂) ppm. IR (KBr) 3390 (OÀH) and 1597 (CdO) cm^À1. EM

(ESI) m/z (%) 381 (M^À). HRMS calcd for C₁₇H₁₇O₆S₂(M^À), 381.0461;

found, 381.0461.

(1R,4S,5R)-1,4-Dihydroxy-3-(naphth-2-yl)methoxycyclohex-

2-en-1,5-carbolactone (15e). The experimental procedure was the

same as for compound 15c using lactone 13e (220 mg), TBAF (0.9 mL),

and THF (5.8 mL). Yield = 88 mg (69%). Colorless oil. [R]²_D⁰= À112ꢀ

(c 1.0, MeOH). ¹H NMR (250 MHz, CD₃OD) δ 7.79 (m, 4H, 4 Â ArH),

7.44 (m, 3H, 3 Â ArH), 5.01 (s, 1H, H-2), 4.91 (s, 2H, OCH₂Ar), 4.59 (m,

1H, H-5), 4.11 (d, J = 3.5 Hz, 1H, H-4) and 2.27 (m, 2H, CH₂-6) ppm. ¹³

C

NMR (63 MHz, CD₃OD) δ 179.3 (C), 155.8 (C), 135.3 (C), 134.8 (C),

134.6 (C), 129.2 (CH), 129.0 (CH), 128.7 (CH), 127.6 (CH), 127.3

(CH), 127.2 (CH), 126.6 (CH), 105.1 (CH), 77.0 (CH), 73.0 (C), 70.9

(CH₂), 67.8 (CH) and 38.4 (CH₂) ppm. IR (film) 3446 (OÀH) and 1770

(CdO) cm^À1. MS (ESI) m/z (%) 335 (MNa⁺). HRMS calcd for

C₁₈H₁₆O₅Na (MNa⁺), 335.0890; found, 335.0889.

(1R,4S,5R)-1,4-Dihydroxy-3-(naphth-2-yl)methoxy-2-

(naphth-2-yl)methylcyclohex-2-en-1,5-carbolactone (16e).

The experimental procedure was the same as for compound 15c using

lactone 14e (73 mg), TBAF (0.24 mL), and THF (1.6 mL). Yield =

32 mg (64%). White solid; mp 203À207 ꢀC. [R]²_D⁰= À190ꢀ (c 1.4,

acetone). ¹H NMR (250 MHz, acetone-d₆) δ 7.89À7.65 (m, 8H, 8 Â

ArH), 7.53À7.37 (m, 6H, 6 Â ArH), 5.33 (d, J = 11.8 Hz, 1H, OCHH),

5.15 (d, J = 11.8 Hz, 1H, OCHH),4.69 (m, 2H, H-4 + H-5), 3.92 (d, J =

14.5 Hz, 1H, CHHAr), 3.74 (d, J = 14.5 Hz, 1H, CHHAr), 2.53 (d, J =

11.0 Hz, 1H, H-6_eq) and 2.41 (dd, 1J = 11.0 and 5.7 Hz, 1H, H-6_ax) ppm.

¹³C NMR (63 MHz, acetone-d₆) δ 177.8 (C), 149.8 (C), 140.1 (C),

140.1 (C), 137.1 (C), 135.2 (C), 134.9 (C), 134.6 (C), 133.6 (C), 129.5

(CH), 129.5 (CH), 129.4 (CH), 129.1 (CH), 129.0 (CH), 128.9 (CH),

128.7 (CH), 128.4 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH), 127.2

(CH), 127.0 (CH), 126.4 (CH), 76.7 (CH), 74.3 (C), 71.4 (CH₂), 66.9

(CH), 38.9 (CH₂) and 31.2 (CH₂) ppm. IR (KBr) 3460 (OÀH), 3346

(OÀH) and 1768 (CdO) cm^À1. MS (ESI) m/z (%) 475 (MNa⁺).

HRMS calcd for C₂₉H₂₄O₅Na (MNa⁺), 475.1516; found, 475.1511.

Sodium (1R,4S,5R)-1,4-Trihydroxy-3-(naphth-2-yl)methoxy-

cyclohex-2-en-1-carboxylate (5e). The experimental procedure was

the same as for compound 5c using carbolactone 15e (8 mg, 0.02 mmol),

NaOH (51 μL), and THF (0.2 mL). Yield = 9 mg (99%). Beige solid; mp

(1R,4S,5R)-2-Allyl-1,4-di(tert-butyldimethylsilyloxy)-3-(benzo-

[b]thiophen-2-yl)methoxycyclohex-2-en-1,5-carbolactone

(18g). To a solution of KHMDS (1.8 mL, 0.91 mmol, 0.5 M in

toluene) in dry DMF (3 mL), under argon and at À78 ꢀC, was added a

solution of (2S)-2-allyl ketone 17³²(200 mg, 0.45 mmol) in dry DMF

(3 mL) and dry toluene (1.9 mL). The resultant solution was stirred at

this temperature for 30 min. A solution of 2-(bromomethyl)benzo-

[b]thiophene²⁷(206 mg, 0.91 mmol) in DMF (1.8 mL) and toluene

(1.2 mL), both dry, was then added. After 1 h, water and brine

were added. The aqueous phase was extracted with diethyl ether

(3 Â 2 mL). The combined organic extracts were dried (anhyd

MgSO₄), filtered, and concentrated. The resulting residue was purified

1

54 ꢀC (dec). [R]²_D⁰= À12ꢀ (c 1.1, H₂O). H NMR (300 MHz, 50%

CD₃OD/D₂O) δ 7.93 (m, 4H, 4 Â ArH), 7.57 (m, 3H, 3 Â ArH), 5.04

(d, J = 11.7 Hz, 1H, OCHH), 4.97 (d, J = 11.7 Hz, 1H, OCHH), 4.95 (s,

1H, H-2), 4.09 (d, J = 6.0 Hz, 1H, H-4), 4.01 (m, 1H, H-5), 2.16 (dd, J =

13.8 and 8.1 Hz, 1H, H-6_ax) and 2.06 (dd, J = 13.8 and 3.9 Hz, 1H, H-6_eq)

ppm. ¹³C NMR (75 MHz, 50% CD₃OD/D₂O) δ 182.1 (C), 157.0 (C),

135.4 (C), 134.3 (C), 134.1 (C), 129.2 (CH), 128.9 (CH), 128.6 (CH),

127.5 (CH), 127.4 (CH), 127.3 (CH), 126.8 (CH), 102.2 (CH), 74.8

(CH), 72.8 (C), 70.9 (CH), 70.6 (CH₂) and 38.7 (CH₂) ppm. IR (KBr)

3435 (OÀH) and 1660 (CdO) cm^À1. MS (ESI) m/z (%) 375 (MNa⁺).

HRMS calcd for C₁₈H₁₇O₆Na₂(MNa⁺), 375.0815; found, 375.0817.

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by flash chromatography on silica gel, eluting with diethyl ether/

hexanes [1) (0:100), 2) (20:80)] to afford ether 18g (118 mg, 45%) as

a pale-yellow oil. [R]_D²⁰= À100ꢀ (c 1.0, CHCl₃). ¹H NMR (250 MHz,

CDCl₃) δ 7.66 (dd, J = 6.5 and 2.2 Hz, 1H, ArH), 7.59 (dd, J = 6.5 and

2.5 Hz, 1H, ArH), 7.17 (m, 2H, 2 Â ArH), 7.03 (s, 1H, ArH),

with a solution of KHMDS (0.4 mL, 0.5 M in toluene) in dry DMF

(0.2 mL). After 20 min, there was a solution of 2-(bromomethyl)-

benzo[b]thiophene²⁸(46 mg, 0.20 mmol) in dry DMF (0.5 mL) and

dry toluene (0.3 mL). After 40 min, water and diethyl ether were added,

the organic layer was separated, and the aqueous layer was extracted

with diethyl ether (Â3). The combined organic extracts were dried

(anhyd Na₂SO₄), filtered, and concentrated under reduced pressure.

The resulting residue was purified by flash chromatography, eluting

with a gradient of diethyl ether/hexanes [(1) 0:100, (2) 5:95] to afford

compound 18h (28 mg, 44%) as a yellow oil. [R]²_D⁰= À132ꢀ (c 1.2,

CHCl₃). ¹H NMR (250 MHz, CD₃OD) δ 7.80 (m, 1H, ArH), 7.72 (m,

1H, ArH), 7.32 (m, 2H, 2 Â ArH), 7.07 (m, 2H, 2 Â ArH), 6.86 (dd, J =

3.5 and 5.0 Hz, 1H, ArH), 6.79 (d, J = 3.5 Hz, 1H, ArH), 5.03 (s, 2H,

OCH₂), 4.54 (dd, J = 5.8 and 3.5 Hz, 1H, H-5), 4.44 (d, J = 3.5 Hz, 1H,

H-4), 3.87 (d, J = 15.2 Hz, 1H, CHHAr), 3.74 (d, J = 15.2 Hz, 1H,

CHHAr), 2.53 (d, J = 10.8 Hz, 1H, H-6_ax), 2.42 (dd, J = 10.8 and 5.8 Hz,

1H, H-6_eq), 0.95 (s, 9H, C(CH₃)₃), 0.81 (s, 9H, C(CH₃)₃), 0.20 (s, 3H,

CH₃), 0.18 (s, 3H, CH₃), 0.16 (s, 3H, CH₃) and 0.06 (s, 3H, CH₃)

ppm. ¹³C NMR (63 MHz, CDCl₃) δ 175.2 (C), 148.2 (C), 142.7 (C),

140.0 (C), 139.9 (C), 139.1 (C), 129.7 (C), 126.4 (CH), 124.6 (CH),

124.3 (CH), 124.2 (CH), 123.6 (CH), 122.8 (CH), 122.6 (CH), 122.3

(CH), 74.6 (C), 74.5 (CH), 68.5 (CH₂), 67.3 (CH), 37.4 (CH₂), 25.6

(C(CH₃)₃), 24.7 (C(CH₃)₃), 18.0 (C(CH₃)₃), 17.9 (C(CH₃)₃), À3.4

(CH₃), À3.5 (CH₃), À4.5 (CH₃) and À4.6 (CH₃) ppm. IR (film)

1797 (CdO) cm^À1. MS (ESI) m/z (%) 665 (MNa⁺). HRMS calcd for

C₃₃H₄₆O₅S₂Si₂Na(MNa⁺), 665.2217; found, 665.2225.

(1R,4S,5R)-3-(Benzo[b]thiophen-2-yl)methoxy-1,4-dihy-

droxy-2-(thien-2-yl)methylcyclohex-2-en-1,5-carbolactone

(19h). The experimental procedure was the same as for compound 15c

using silyl ether 18h (15 mg), TBAF (50 μL), and THF (0.4 mL).

Yield = 7 mg (84%). White solid. [R]²_D⁰= À155ꢀ (c 1.2, MeOH). ¹H NMR

(250 MHz, CD₃OD) δ 7.71 (m, 2H, 2 Â ArH), 7.25 (m, 3H, 3 Â ArH),

7.18 (s, 1H, ArH), 7.00 (dd, J = 5.0 and 1.5 Hz, 1H, ArH), 6.74 (m, 1H,

ArH), 5.27 (d, J= 12.5 Hz, 1H, OCHH), 5.17 (d, J = 12.5 Hz, 1H, OCHH),

4.55 (m, 1H, H-5), 4.47 (d, J = 3.2 Hz, 1H, H-4), 3.78 (d, J = 14.8 Hz, 1H,

CHHAr), 3.63 (d, J = 14.8 Hz, 1H, CHHAr) and 2.29 (m, 2H, CH₂-6)

ppm. ¹³C NMR (63 MHz, CD₃OD) δ 178.8 (C), 148.6 (C), 144.2 (C),

142.3 (C), 141.6 (C), 140.9 (C), 128.0 (C), 127.3 (CH), 126.3 (CH),

125.6 (CH), 125.4 (CH), 124.8 (CH), 124.1 (CH), 124.0 (CH), 123.3

(CH), 76.9 (CH), 73.7 (C), 66.8 (CH₂), 66.3 (CH), 38.4 (CH₂) and 24.9

(CH₂) ppm. IR (KBr) 3452 (OÀH) and 1770 (CdO) cm^À1. MS(CI) m/

z (%) 415 (MH⁺). HRMS calcd for C₂₁H₁₉O₅S₂(MH⁺), 415.0674; found,

415.0674.

Sodium (1R,4S,5R)-3-(Benzo[b]thiophen-2-yl)methoxy-

1,4,5-trihydroxy-2-(thien-2-yl)methylcyclohex-2-en-1-car-

boxylate (9h). The experimental procedure was the same as for

compound 5c using carbolactone 19h (20 mg), NaOH (97 μL), and

THF (0.5 mL). Yield = 22 mg (99%). White solid; mp 184 ꢀC (dec).

[R]²_D⁰= À24ꢀ (c 1.5, H₂O). ¹H NMR (250 MHz, 50% CD₃OD/D₂O)

δ 7.82 (m, 1H, ArH), 7.77 (m, 1H, ArH), 7.35 (m, 2H, 2 Â ArH), 7.26

(s, 1H, ArH), 7.10 (dd, J = 3.3 and 3.0 Hz, 1H, ArH), 6.82 (d, J =

3.8 Hz, 2H, 2 Â ArH), 5.25 (d, J = 12.0 Hz, 1H, OCHH), 5.04 (d, J =

12.0 Hz, 1H, OCHH), 4.36 (d, J = 5.2 Hz, 1H, H-4), 3.92 (m, 1H,

H-5), 3.73 (d, J = 15.3 Hz, 1H, CHHAr), 3.31 (m, J = 15.3 Hz, 1H,

CHHAr) and 2.10 (m, 2H, CH₂-6) ppm. ¹³C NMR (63 MHz, 50%

CD₃OD/D₂O) δ 180.8 (C), 152.0 (C), 145.4 (C), 142.7 (C), 141.5

(C), 140.9 (C), 127.1 (CH), 126.3 (CH), 125.4 (CH), 125.3 (CH),

124.7 (CH), 123.7 (2 Â CH + C), 123.3 (CH), 77.1 (C), 71.6 (CH),

70.0 (CH), 66.5 (CH₂), 37.4 (CH₂) and 27.2 (CH₂) ppm. IR (KBr)

3442 (OÀH) and 1668 (CdO) cm^À1. MS (ESI) m/z (%) 431 (M^À).

HRMS calcd for C₂₁H₁₉O₆S₂(M^À), 431.0618; found, 431.0602.

(1R,4S,5R)-1,4-Di(tert-butyldimethylsilyloxy)-2-(5-methyl-

benzo[b]thiophen-2-yl)methyl-3-(thien-2-yl)methoxycyclo-

hex-2-en-1,5-carbolactone (18i). A solution of ketone 12²²(350mg,

5.87À5.71 (m, 1H, CHdCH₂), 4.95À4.80 (m, 4H, OCH₂

+

CHdCH₂), 4.34 (dd, J = 4.7 and 3.5 Hz, 1H, H-5), 4.17 (d, J = 3.5

Hz, 1H, H-4), 2.96 (d, J = 6.2 Hz, 2H, CH₂-CHdCH₂), 2.26 (m, 2H,

CH₂-6), 0.78 (s, 9H, C(CH₃)₃), 0.77 (s, 9H, C(CH₃)₃), 0.08 (s, 3H,

SiCH₃), 0.02 (s, 3H, SiCH₃), 0.00 (s, 3H, SiCH₃) and À0.03 (s, 3H,

SiCH₃) ppm. ¹³C NMR (75 MHz, CDCl₃) δ 176.3 (C), 148.3 (C),

140.9 (C), 139.2 (C), 136.5 (CH), 131.2 (C), 125.1 (CH), 125.0

(CH), 124.3 (CH), 123.1 (CH), 123.0 (CH), 122.9 (C), 116.1 (CH₂),

77.3 (C), 75.4 (CH), 69.8 (CH₂), 68.1 (CH), 38.0 (CH₂), 30.1 (CH₂),

26.4 (C(CH₃)₃), 26.2 (C(CH₃)₃), 18.9 (C(CH₃)₃), 18.7 (C(CH₃)₃),

À2.5 (SiCH₃), À2.6 (SiCH₃), À3.8 (SiCH₃) and À4.0 (SiCH₃) ppm.

IR (film): 1799 (CdO) cm^À1. MS (CI) m/z (%) 587 (MH⁺). HRMS

calcd for C₃₁H₄₇O₅SSi₂(MH⁺), 587.2683; found, 587.2682.

(1R,4S,5R)-2-Allyl-1,4-dihydroxy-3-(benzo[b]thiophen-2-

yl)methoxycyclohex-2-en-1,5-carbolactone (19g). The ex-

perimental procedure was the same as for compound 15c using silyl

ether 18g (78 mg), TBAF (0.29 mL), and THF (0.9 mL). Yield = 38 mg

(83%). Beige solid; mp 122À125 ꢀC. [R]²_D⁰= À143ꢀ (c 1.5, MeOH).

¹H NMR (250 MHz, CD₃OD) δ 7.76 (m, 1H, ArH), 7.69 (m, 1H,

ArH), 7.26 (m, 2H, 2 Â ArH), 7.22 (s, 1H, ArH), 5.78 (m, 1H,

CHdCH₂), 5.22 (d, J = 12.5 Hz, 1H, OCHH), 5.12 (d, J = 12.5 Hz, 1H,

OCHH), 4.97 (dq, J = 17.0 and 1.7 Hz, 1H, CHdCHH), 4.81 (m, 1H,

CHdCHH), 4.55 (m, 1H, H-5), 4.40 (d, J = 3.5 Hz, 1H, H-4), 3.00 (d,

J = 6.5 Hz, 2H, CH₂CHdCH₂) and 2.29 (m, 2H, CH₂-6) ppm. ¹³C

NMR (63 MHz, CD₃OD) δ 178.8 (C), 148.1 (C), 142.3 (C), 141.5

(C), 140.8 (C), 137.1 (CH), 126.5 (C), 125.5 (CH), 125.3 (CH), 124.7

(CH), 123.8 (CH), 123.2 (CH), 115.5 (CH₂), 76.8 (CH), 73.8 (C),

67.2 (CH₂), 66.4 (CH₂), 38.3 (CH₂) and 29.5 (CH₂) ppm. IR (KBr):

3482 (OÀH), 3369 (OÀH) and 1780 (CdO) cm^À1. MS (ESI) m/z

(%) 381 (MNa⁺). HRMS calcd for C₁₉H₁₈O₅SNa (MNa⁺), 381.0751;

found, 381.0758.

Sodium (1R,4S,5R)-2-Allyl-3-(benzo[b]thiophen-2-yl)-

methoxy-1,4,5-trihydroxycyclohex-2-en-1-carboxylate (9g).

The experimental procedure used was the same as for compound 5c

using carbolactone 19g (30 mg), NaOH (160 μL), and THF (0.75 mL).

Yield = 33 mg (99%). Beige solid. [R]²_D⁰= À49ꢀ (c 1.1, H₂O). ¹H NMR

(250 MHz, D₂O) δ 7.80 (m, 2H, 2 Â ArH), 7.34 (m, 2H, 2 Â ArH), 7.30

(s, 1H, ArH), 5.79 (m, 1H, CHdCH₂), 5.09 (d, J = 11.8 Hz, 1H,

OCHH), 4.98 (m, 3H, OCHH + CHdCH₂), 4.32 (d, J = 6.7 Hz, 1H,

H-4), 3.93 (m, 1H, H-5), 2.91 (dd, J = 15.0 and 6.5 Hz, 1H,

CHHCHdCH₂), 2.66 (dd, J = 15.0 and 6.5 Hz, 1H, CHHCHdCH₂)

and 2.05 (m, 2H, CH₂-6) ppm. ¹³C NMR (63 MHz, D₂O) δ 180.5 (C),

150.9 (C), 140.8 (C), 140.7 (C), 139.8 (C), 137.4 (CH), 125.4 (CH),

125.2 (CH), 124.6 (CH), 123.9 (C), 123.1 (CH), 115.9 (CH₂), 77.1

(C), 70.7 (CH), 70.6 (CH), 39.4 (CH₂) and 31.5 (CH₂) ppm. IR (KBr):

3427 (OÀH) and 1597 (CdO) cm^À1. MS (ESI) m/z (%) 399 (MH⁺).

HRMS calcd for C₁₉H₂₀O₆SNa (MH⁺), 399.0873; found, 399.0887.

(1R,4S,5R)-3-(Benzo[b]thiophen-2-yl)methoxy-1,4-di(tert-

butyldimethylsilyloxy)-2-(thien-2-yl)methylcyclohex-2-en-

1,5-carbolactone (18h). A solution of ketone 12²²(500 mg,

1.25 mmol) in dry THF (12.5 mL), at room temperature and under

argon, was treated with a solution of LHMDS (1.9 mL, 1.87 mmol, 1 M

in THF). The resulting mixture was stirred for 1 h and was then treated

with a solution of 2-iodomethylthiophene²⁸(560 mg, 2.5 mmol) in dry

THF (4 mL). After 30 min, the solvent was removed and the residue

was purified by flash chromatography on silica gel, eluting with diethyl

ether/hexanes (5:95) to yield a diastereomeric mixture of C-alkylated

products (206 mg, 33%). A solution of 50 mg (0.10 mmol) of the latter

mixture in DMF (0.5 mL) and toluene (0.5 mL), both dry, was treated

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0.87mmol) indry THF (17.5mL), atroomtemperature and under argon,

was treated with a solution of LHMDS (1.3 mL, 1.30 mmol, 1 M in THF).

The resulting mixture was stirred for 1 h, and it was then treated with

2-iodomethyl-5-methylbenzo[b]thiophene²⁸(480 mg, 1.75 mmol). After

40 min, water and diethyl ether were added, the organic layer was

separated, and the aqueous phase was extracted with diethyl ether. The

combined organic extracts were dried (anhyd Na₂SO₄), filtered, and

concentrated under reduced pressure. The resulting residue was purified

by flash chromatography, eluting with hexane and with diethyl ether/

hexanes (10:90) to yield a diastereomeric mixture of C-alkyl products

(158 mg, 33%). The resulting ketone was converted into compound 18i

by following the same experimental procedure as for compound 18g using

KHMDS (1.2 mL) in DMF (0.6 mL), 2-iodomethylthiophene (102 mg)

inDMF (1.4 mL), and toluene (1.0 mL). Yield = 54 mg (29%). Yellow oil.

(CH₂), 28.3 (CH₂) and 21.1 (CH₃) ppm. IR (KBr) 3408 (OÀH), 1660

(CdO) and 1605 (CdC) cm^À1. MS (ESI) m/z (%) 469 (MH⁺).

HRMS calcd for C₂₂H₂₂O₆S₂Na (MH⁺), 469.0750; found, 469.0747.

(1R,4S,5R)-1,4-Di(tert-butyldimethylsilyloxy)-2-(benzo[b]-

thiophen-5-yl)methyl-3-(thien-2-yl)methoxycyclohex-2-en-

1,5-carbolactone (18j). A solution of ketone 12²²(350 mg, 0.87

mmol) in dry THF (17.5 mL) was treated with LHMDS (1.3 mL, 1.31

mmol) at room temperature. After 1 h, 5-(iodomethyl)benzothio-

phene²⁸(480 mg, 1.75 mmol) was added and the resultant mixture

was stirred for 40 min. The reaction mixture was diluted with water and

diethyl ether. The organic layer was separated, and the aqueous phase

was extracted with diethyl ether (3 Â 25 mL). The combined organic

extracts were dried (anhyd Na₂SO₄), filtered, and concentrated. The

residue was purified by flash chromatography on silica gel eluting with

diethyl ether/hexanes [1) 0:100; 2) 10:90] to give the corresponding

2-alkyl ketone (158 mg, 33%). To a stirred solution of KHMDS (1.2 mL,

0.58 mmol, 0.5 M in toluene) in dry DMF (1.4 mL), under argon and at

À78 ꢀC, was added a solution of the previously obtained ketone (158

mg) in 2.8 mL of a mixture of dry DMF and dry toluene (1:1). The

resultant solution was stirred at this temperature for 30 min. A solution

of 2-(iodomethyl)thiophene (102 mg, 0.58 mmol) in a mixture of DMF

and toluene (1.4:1, 2.4 mL), both dry, was then added. After 1 h, water

and brine were added. The aqueous phase was extracted with diethyl

ether (3 Â 2 mL). The combined organic extracts were dried (anhyd

MgSO₄), filtered, and concentrated. The residue was purified by flash

chromatography on silica gel, eluting with diethyl ether/hexanes [(1)

5:95; (2) 10:90] to give compound 18j (54 mg, 29%) as a colorless oil.

¹H NMR (250 MHz, CDCl₃) δ 7.55 (d, J = 8.2 Hz, 1H, ArH), 7.43

(s, 1H, ArH), 7.20 (d, J = 5.5 Hz, 1H, ArH), 7.11À6.97 (m, 5H, 5 Â

ArH), 4.73 (d, J = 12.2 Hz, 1H, OCHH), 4.68 (d, J = 12.2 Hz, 1H,

OCHH), 4.39 (dd, J = 5.5 and 3.5 Hz, 1H, H-5), 4.26 (d, J = 3.5 Hz, 1H,

H-4), 3.63 (d, J = 15.0 Hz, 1H, CHHAr), 3.49 (d, J = 15.0 Hz, 1H,

CHHAr), 2.37 (d, J = 11.0 Hz, 1H, H-6_ax), 2.25 (dd, J = 11.0 and 6.0 Hz,

1H, H-6_eq), 0.79 (s, 9H, C(CH₃)₃), 0.54 (s, 9H, C(CH₃)₃), 0.04 (s, 3H,

SiCH₃), 0.02 (s, 3H, SiCH₃), À0.05 (s, 3H, SiCH₃) and À0.15 (s, 3H,

SiCH₃) ppm. ¹³C NMR (63 MHz, CDCl₃) δ 175.5 (C), 148.3 (C),

139.7 (C), 138.8 (C), 136.0 (C), 130.3 (C), 127.8 (CH), 126.8 (CH),

126.6 (CH), 126.0 (CH), 125.4 (CH), 124.5 (C), 123.7 (CH), 123.1

(CH), 121.8 (CH), 75.0 (C), 74.7 (CH), 67.4 (CH₂), 67.3 (CH), 37.6

(CH₂), 30.1 (CH₂), 25.7 (C(CH₃)₃), 25.4 (C(CH₃)₃), 18.0 (2 Â

C(CH₃)₃), À3.3 (SiCH₃), À3.5 (SiCH₃) and À4.5 (2 Â SiCH₃)

ppm. MS (ESI) m/z (%) 643 (MH⁺). HRMS calcd for C₃₃H₄₇O₅S₂Si₂

(MH⁺), 643.2398; found, 643.2393.

(1R,4S,5R)-2-(Benzo[b]thiophen-5-yl)methyl-1,4-dihydroxy-

3-(thien-2-yl)methoxycyclohex-2-en-1,5-carbolactone (19j).

The experimental procedure was the same as for compound 15c using

silyl ether 18j (40 mg), TBAF (140 μL), and THF (0.9 mL). Yield = 22

mg (88%). Yellow solid; mp 140À143 ꢀC. ¹H NMR (250 MHz,

CD₃OD) δ 7.67 (d, J = 8.2 Hz, 2H, 2 Â ArH), 7.44 (d, J = 5.5 Hz,

1H, ArH), 7.36 (dd, J = 5.0 and 1.2 Hz, 1H, ArH), 7.26 (d, J = 8.2 Hz, 1H,

ArH), 7.22 (d, J = 5.5 Hz, 1H, ArH), 6.97 (m, 2H, 2 Â ArH), 5.21 (d, J =

12.2 Hz, 1H, OCHH), 5.09 (d, J = 12.2 Hz, 1H, OCHH), 4.62 (m, 1H,

H-5), 4.55 (d, J = 3.5 Hz, 1H, H-4), 3.74 (d, J = 14.0 Hz, 1H, CHHAr),

3.57 (d, J = 14.0 Hz, 1H, CHHAr), 2.38 (d, J = 11.0 Hz, 1H, H-6_ax) and

2.31 (dd, J = 11.0 and 5.2 Hz, 1H, H-6_eq) ppm. ¹³C NMR (63 MHz,

CD₃OD) δ 178.9 (C), 148.4 (C), 141.2 (C), 141.1 (C), 138.5 (C),

138.0 (C), 128.1 (CH), 127.7 (CH), 127.4 (CH), 127.1 (CH), 127.0

(CH + C), 124.8 (CH), 124.7 (CH), 122.6 (CH), 76.9 (CH), 73.9 (C),

66.2 (CH), 65.8 (CH₂), 38.5 (CH₂) and 30.6 (CH₂) ppm. MS (ESI)

m/z (%) 437 (MNa⁺). HRMS calcd for C₂₁H₁₈O₅S₂Na (MNa⁺),

437.0488; found, 437.0481.

1

[R]²_D⁰= À111ꢀ (c 1.3, CHCl₃). H NMR (300 MHz, CDCl₃) δ 7.60

(d, J = 8.1 Hz, 1H, ArH), 7.42 (s, 1H, ArH), 7.27 (m, 1H, ArH), 7.05

(dd, J = 7.8 and 0.6 Hz, 1H, ArH), 6.92 (m, 3H, 3 Â ArH), 4.97 (s, 2H,

OCH₂), 4.56 (dd, J = 6.0 and 3.3 Hz, 1H, H-5), 4.43 (d, J = 3.3 Hz, 1H,

H-4), 3.86 (d, J = 15.6 Hz, 1H, CHHAr), 3.74 (d, J = 15.6 Hz, 1H,

CHHAr), 2.56 (d, J = 10.8 Hz, 1H, H-6_ax), 2.44 (m, 4H, CH₃+ H-6_eq),

0.97 (s, 9H, C(CH₃)₃), 0.78 (s, 9H, C(CH₃)₃), 0.21 (s, 3H, SiCH₃), 0.20

(s, 3H, SiCH₃), 0.17 (s, 3H, SiCH₃) and 0.06 (s, 3H, SiCH₃) ppm. ¹³

C

NMR (75 MHz, CDCl₃) δ 175.3 (C), 148.6 (C), 144.1 (C), 140.5 (C),

138.7 (C), 136.5 (C), 133.3 (C), 129.1 (C), 127.0 (CH), 126.7 (CH),

126.3 (CH), 124.8 (CH), 122.7 (CH), 121.5 (CH), 120.8 (CH), 77.4 (C),

74.7 (CH), 67.7 (CH₂), 67.3 (CH), 37.5 (2 Â CH₂), 25.7 (C(CH₃)₃),

25.5 (C(CH₃)₃), 21.4 (CH₃), 18.1 (C(CH₃)₃), 18.0 (C(CH₃)₃), À3.3

(SiCH₃), À3.4 (SiCH₃) and À4.5 (2 Â SiCH₃) ppm. IR (film) 1799

(CdO) cm^À1. MS (CI) m/z (%) 657 (MH⁺).

(1R,4S,5R)-1,4-Dihydroxy-2-(5-methylbenzo[b]thiophen-

2-yl)methyl-3-(thien-2-yl)methoxy cyclohex-2-en-1,5-car-

bolactone (19i). The experimental procedure was the same as for

compound 15c using silyl ether 18i (40 mg), TBAF (0.14 μL), and

THF (0.9 mL). Yield = 22 mg (88%). Yellow solid; mp 140À143 ꢀC.

[R]²_D⁰= À148ꢀ (c 1.2, CH₃OH). ¹H NMR (250 MHz, CD₃OD) δ 7.56

(d, J = 8.2 Hz, 1H, ArH), 7.39 (s, 1H, ArH), 7.36 (d, J = 5.0 Hz, 1H,

ArH), 7.03 (m, 2H, 2 Â ArH), 6.94 (m, 2H, 2 Â ArH), 6.97 (m, 2H, 2 Â

ArH), 5.25 (d, J = 12.2 Hz, 1H, OCHH), 5.15 (d, J = 12.2 Hz, 1H,

OCHH), 4.63 (m, 1H, H-5), 4.54 (d, J = 3.2 Hz, 1H, H-4), 3.82 (d, J =

14.7 Hz, 1H, CHHAr), 3.69 (d, J = 14.7 Hz, 1H, CHHAr) and

2.43À2.31 (m, 5H, CH₂-6 + CH₃) ppm. ¹³C NMR (63 MHz, CD₃OD)

δ 178.8 (C), 149.0 (C), 145.6 (C), 142.0 (C), 141.0 (C), 138.3 (C),

134.5 (C), 128.1 (CH), 127.7 (CH), 127.4 (CH), 125.9 (CH), 125.4

(C), 123.7 (CH), 122.5 (CH), 122.4 (CH), 76.9 (CH), 73.7 (C), 66.2

(CH₂), 65.9 (CH), 38.4 (CH₂), 25.9 (CH₂) and 21.5 (CH₂) ppm. IR

(KBr) 3483 (OÀH), 3429 (OÀH) and 1729 (CdO) cm^À1. MS (ESI)

m/z (%) 541 (MNa⁺). HRMS calcd for C₂₂H₂₀O₅S₂Na (MNa⁺),

451.0644; found, 451.0646.

Sodium (1R,4S,5R)-1,4,5-Trihydroxy-2-(5-methylbenzo[b]-

thiophen-2-yl)methyl-3-(thien-2-yl)methoxycyclohex-2-en-

1-carboxylate (9i). The experimental procedure was the same as for

compound 5c using carbolactone 19i (20 mg), NaOH (100 μL), and

THF (0.5 mL). Yield = 22 mg (97%). Beige solid; mp 191À194 ꢀC.

[R]²_D⁰= À55ꢀ (c 1.2, H₂O). ¹H NMR (250 MHz, D₂O) δ 7.59 (d, J =

8.0 Hz, 1H, ArH), 7.41 (s, 1H, ArH), 7.22 (d, J = 4.7 Hz, 1H, ArH), 7.02

(d, J = 8.0 Hz, 1H, ArH), 6.94 (s, 1H, ArH), 6.85 (m, 2H, 2 Â ArH), 5.08

(d, J = 11.5 Hz, 1H, OCHH), 4.84 (d, J = 11.5 Hz, 1H, OCHH), 4.38 (d,

J = 6.2 Hz, 1H, H-4), 3.98 (m, 1H, H-5), 3.68 (d, J = 16.0 Hz, 1H,

CHHAr), 3.37 (d, J = 16.0 Hz, 1H, CHHAr), 2.30 (s, 3H, CH₃), 2.21

(dd, J = 13.7 and 9.7 Hz, 1H, H-6_ax) and 2.09 (dd, J = 13.7 and 3.7 Hz,

1H, H-6_eq) ppm. ¹³C NMR (63 MHz, D₂O) δ 180.3 (C), 151.7 (C),

146.0 (C), 140.9 (C), 139.5 (C), 137.0 (C), 134.8 (C), 128.5 (CH),

127.7 (CH), 127.5 (CH), 125.7 (CH), 123.3 (CH), 123.3 (C), 122.5

(CH), 122.0 (CH), 77.2 (C), 70.6 (CH), 70.3 (CH), 65.8 (OCH₂), 38.8

Sodium (1R,4S,5R)-1,4,5-Trihydroxy-3-(thien-2-yl)methoxy-

2-(benzo[b]thiophen-5-yl)methylcyclohex-2-en-1-carboxy-

late (9j). The experimental procedure was the same as for compound

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5c using carbolactone 19j (20 mg), NaOH (97 μL), and THF (0.5 mL).

Yield = 22 mg (97%). White solid; mp 199À201 ꢀC. [R]²_D⁰= +57ꢀ (c 1.0,

H₂O). ¹H NMR (250 MHz, 50% D₂O/CD₃CN) δ 8.27 (d, J = 8.5 Hz,

1H, ArH), 8.21 (br s, 1H, ArH), 8.01 (d, J = 5.5 Hz, 1H, ArH), 7.82 (m,

3H, 3 Â ArH), 7.44 (m, 2H, 2 Â ArH), 6.85 (m, 3H, 3 Â ArH), 5.51 (d,

J = 11.5 Hz, 1H, OCHH), 5.35 (d, J = 11.5 Hz, 1H, OCHH), 4.84 (m,

1H, H-4), 4.41 (m, 1H, H-5), 4.16 (d, J = 15.2 Hz, 1H, CHHAr), 3.66

(d, J = 15.7 Hz, 1H, CHHAr) and 2.61 (m, 2H, CH₂-6) ppm. ¹³C NMR

(63 MHz, 50% D₂O/CD₃CN) δ 180.5 (C), 151.4 (C), 140.7 (C), 140.5

(C), 138.5 (C), 137.4 (C), 127.9 (CH), 127.6 (CH), 127.2 (CH), 127.2

(CH), 126.7 (CH), 124.8 (CH), 124.2 (CH), 123.7 (C), 122.4 (CH),

76.9 (C), 70.7 (CH), 69.5 (CH), 65.3 (CH₂), 37.2 (CH₂) and 32.4

(CH₂) ppm. IR (KBr) 3433 (OÀH), 3290 (OÀH) and 1687

(CdO) cm^À1. MS (ESI) m/z (%) 455 (MH⁺). HRMS calcd for

C₂₁H₂₀O₆S₂Na (MH⁺), 455.0594; found, 455.0591.

Methyl (1R,4S,5R)-3-(Benzo[b]thiophen-2-yl)methoxy-1,4,

5-trihydroxycyclohex-2-en-1-carboxylate (6a). A solution of

the lactone 15a (26 mg, 0.08 mmol) in dry methanol (0.9 mL) was

treated with sodium methoxide (3.4 mg, 0.09 mmol). The resultant

mixture was stirred at room temperature for 30 min, and the reaction

mixture was diluted with ethyl acetate and water. The organic layer was

separated, and the aqueous phase was extracted with ethyl acetate (Â2).

The combined organic extracts were dried (anhyd Na₂SO₄), filtered, and

concentrated under reduced pressure. The obtained residue was purified

by flash chromatography eluting with (50:50) ethyl acetateÀhexanes to

give methyl ester 6a (15 mg, 53%) as a white solid; mp 152À154 ꢀC.

[R]²_D⁰= À40ꢀ (c 1.1, MeOH). ¹H NMR (250 MHz, CD₃OD) δ 7.83 (m,

1H, ArH), 7.76 (m, 1H, ArH), 7.34 (m, 3H, 3 Â ArH), 5.10 (s, 2H,

OCH₂), 5.02 (s, 1H, H-2), 4.00 (m, 2H, H-4 + H-5), 3.66 (s, 3H, OMe)

and 2.05 (m, 2H, CH₂-6) ppm. ¹³C NMR (63 MHz, CD₃OD) δ 176.5

(C), 158.5 (C), 141.6 (C), 141.2 (C), 140.9 (C), 125.6 (CH), 125.4

(CH), 124.7 (CH), 124.5 (CH), 123.3 (CH), 99.9 (CH), 74.1 (CH),

73.9(C), 70.4 (CH), 66.3 (CH₂), 53.1 (OCH₃), and 40.2 (CH₂) ppm. IR

(KBr): 3435 (OÀH) and 1726 (CdO) cm^À1. MS (ESI) m/z (%) 373

(MNa⁺). HRMS calcd for C₁₇H₁₈O₆S₂Na (MNa⁺), 373.0716; found,

373.0716.

Methyl (1R,4S,5R)-1,4,5-Trihydroxy-3-(5-methylbenzo[b]-

thiophen-2-yl)methoxycyclohex-2-ene-1-carboxylate (6b).

The experimental procedure was the same as for compound 6a using

lactone 15b (36 mg), NaOMe (6 mg), and MeOH (1.2 mL). Yield =

25 mg (62%). White solid; mp 183À185 ꢀC. [R]²_D⁰= À31ꢀ (c 1.2, in

MeOH). ¹H NMR (400 MHz, CD₃OD) δ 7.69 (d, J = 8.4 Hz, 1H, ArH),

7.56 (s, 1H, ArH), 7.28 (s, 1H, ArH), 7.16 (dd, J = 8.0 and 1.6 Hz, 1H,

ArH), 5.07 (s, 2H, OCH₂Ar), 5.01 (s, 1H, H-2), 4.04À3.96 (m, 2H, H-4

+ H-5), 3.67 (s, 3H, OCH₃), 2.43 (s, 3H, CH₃) and 2.05 (m, 2H, CH₂-6)

ppm. ¹³C NMR (100 MHz, CD₃OD) δ 176.5 (C), 158.5 (C), 141.2

(C), 141.2 (C), 138.8 (C), 135.3 (C), 127.3 (CH), 124.6 (CH), 124.3

(CH), 123.0 (CH), 99.9 (CH), 74.1 (CH), 73.9 (C), 70.5 (CH), 66.4

(CH₂), 53.1 (OCH₃), 40.2 (CH₂) and 21.4 (CH₃) ppm. IR (KBr) 3487

(OÀH), 3435 (OÀH) and 1718 (CdO) cm^À1. MS (ESI) m/z (%) 387

(MNa⁺). HRMS calcd for C₁₈H₂₀O₆SNa (MNa⁺), 387.0873; found,

387.0866.

Methyl (1R,4S,5R)-1,4,5-Trihydroxy-3-(6-chlorobenzo[b]-

thiophen-2-yl)methoxycyclohex-2-ene-1-carboxylate (6c).

The experimental procedure was the same as for compound 6a using

lactone 15c (16 mg), NaOMe (3 mg), and MeOH (0.4 mL). Yield =

7 mg (46%). Beige solid; mp 81À83 ꢀC. [R]²_D⁰= À16ꢀ (c 1.0, in MeOH).

¹H NMR (250 MHz, acetone-d₆) δ 8.00 (d, J = 2.0 Hz, 1H, ArH), 7.82

(d, J = 8.5 Hz, 1H, ArH), 7.46 (s, 1H, ArH), 7.38 (dd, J = 8.5 and 2.0 Hz,

1H, ArH), 5.11 (d, J = 12.5 Hz, 1H, OCHHAr), 5.04 (d, J = 12.5 Hz, 1H,

OCHHAr), 5.00 (s, 1H, H-2), 4.51 (s, 1H, OH), 4.42 (s, 1H, OH), 4.20

(s, 1H, OH), 4.02 (m, 2H, H-4 + H-5), 3.66 (s, 3H, OCH₃) and 2.05

(m, 2H, CH₂-6) ppm. ¹³C NMR (63 MHz, acetone-d₆) δ 177.0 (C), 159.0

(C), 143.3 (C), 140.1 (C), 131.8 (C), 126.9 (CH), 126.7 (CH), 124.6

(CH), 123.7 (CH + C), 100.8 (CH), 74.9 (CH), 74.4 (C), 71.2 (CH),

66.6 (CH₂), 53.7 (OCH₃) and 40.9 (CH₂) ppm. IR (KBr) 3436 (OÀH)

and 1739 (CdO) cm^À1. MS (ESI) m/z (%) 407 (MNa⁺). HRMS calcd

for C₁₇H₁₇O₆SClNa (MNa⁺), 407.0327; found, 407.0330.

Methyl (1R,4S,5R)-3-(Benzo[b]thiophen-5-yl)methoxy-1,4,

5-trihydroxycyclohex-2-en-1-carboxylate (6d). The experimen-

tal procedure was the same as for compound 6a using lactone 15d

(52 mg), NaOMe (10 mg), and MeOH (1.8 mL). Yield = 38 mg (68%).

White solid. [R]²_D⁰= À31ꢀ (c 1.4, MeOH). ¹H NMR (300 MHz,

CD₃OD) δ 7.89 (d, J = 8.4 Hz, 2H, 2 Â ArH), 7.58 (d, J = 5.7 Hz, 1H,

ArH), 7.40 (dd, J = 8.4 and 1.2 Hz, 1H, ArH), 7.37 (d, J = 5.7 Hz, 1H,

ArH), 4.98 (s, 1H, H-2), 4.94 (s, 2H, OCH₂), 4.02 (m, 2H, H-4 + H-5),

3.70 (s, 3H, OCH₃) and 2.04 (m, 2H, CH₂-6) ppm. ¹³C NMR (75 MHz,

CD₃OD) δ 176.7 (C), 158.9 (C), 141.3 (C), 134.3 (C), 128.2 (C), 128.1

(CH), 125.3 (CH), 124.9 (CH), 123.9 (CH), 123.4 (CH), 99.5 (CH),

74.3 (C), 74.0 (CH), 71.0 (OCH₂), 70.5 (CH), 53.1 (CH₃) and 40.3

(CH₂) ppm. IR (KBr) 3446 (OÀH), 3305 (OÀH) and 1732

(CdO) cm^À1. MS (ESI) m/z (%) 373 (MNa⁺). HRMS calcd for

C₁₇H₁₈O₆SNa (MNa⁺), 373.0716; found, 373.0711. Elemental anal-

ysis C₁₇H₁₈O₆S Calcd: C, 58.27; H, 5.18; S, 9.15. Found: C, 57.93; H,

5.36; S, 8.94.

Ethyl (1R,4S,5R)-3-(Benzo[b]thiophen-2-yl)methoxy-1,4,

5-trihydroxycyclohex-2-en-1-carboxylate (7a). A solution of

sodium carboxylate 5a (6 mg, 0.02 mmol) and potassium carbonate

(8 mg, 0.06 mmol) in DMF (0.3 mL) was stirred at room temperature for

30 min. Bromoethane (64 μL, 0.86 mmol) was added, and the reaction

mixture was stirred for 2 h. Ethyl acetate was added, and the precipitate

was filtered off. The resulting solution was washed with brine, dried

(Na₂SO₄anhyd), filtered, and concentrated under reduced pressure.

The residue was purified by flash chromatography, eluting with ethyl

acetate/hexanes (90:10) to give ester 7a (3 mg, 49%) as a colorless oil.

[R]²_D⁰= À13ꢀ (c 0.5, MeOH). ¹H NMR (500 MHz, CD₃OD) δ 7.83

(d, J = 7.5 Hz, 1H, ArH), 7.76 (dd, J = 7.0 and 1.5 Hz, 1H, ArH), 7.37

(s, 1H, ArH), 7.33 (m, 2H, 2 Â ArH), 5.13 (s, 2H, OCH₂Ar), 5.01

(s, 1H, H-2), 4.10 (m, 2H, OCH₂CH₃), 3.99 (m, 2H, H-4 + H-5), 2.03

(m, 2H, CH₂-6) and 1.16 (t, J = 7.0 Hz, 3H, CH₃) ppm. ¹³C NMR (125

MHz, CD₃OD) δ 176.1 (C), 158.4 (C), 141.6 (C), 141.4 (C), 140.9

(C), 125.6 (CH), 125.4 (CH), 124.7 (CH), 124.4 (CH), 123.3 (CH),

100.0 (CH), 74.2 (CH), 73.9 (C), 70.5 (CH), 66.3 (OCH₂), 62.7

(OCH₂), 40.3 (CH₂), and 14.4 (CH₃) ppm. IR (film): 3427 (OÀH)

and 1651 (CdO) cm^À1. MS (ESI) m/z (%) 387 (MNa⁺). HRMS calcd

for C₁₈H₂₀O₆SNa (MNa⁺), 387.0873; found, 387.0870. Elemental

analysis C₁₈H₂₀O₆S Calcd: C, 59.33; H, 5.53; S, 8.80. Found: C, 58.96;

H, 5.87; S, 8.81.

Ethyl (1R,4S,5R)-1,4,5-Trihydroxy-3-(6-chlorobenzo[b]-

thiophen-2-yl)methoxycyclohex-2-ene-1-carboxylate (7b).

The experimental procedure was the same as for compound 7a using

compound 5c (15 mg), potassium carbonate (16 mg), bromoethane

(80 μL), and DMF (0.4 mL). Yield = 7 mg (46%). Colorless oil. [R]_D²⁰

=

1

À16ꢀ (c 1.0, MeOH). H NMR (250 MHz, CD₃OD) δ 7.88 (s, 1H,

ArH), 7.73 (d, J = 8.5 Hz, 1H, ArH), 7.37 (s, 1H, ArH), 7.33 (dd, J = 8.5

and 2.0 Hz, 1H, ArH), 5.11 (s, 2H, OCH₂Ar), 5.00 (s, 1H, H-2), 4.11

(m, 2H, OCH₂CH₃), 3.99 (m, 2H, H-4 + H-5), 2.04 (m, 2H, CH₂-6)

and 1.14 (t, J = 8.0 Hz, 3H, CH₃) ppm. ¹³C NMR (63 MHz, CD₃OD)

δ 176.0 (C), 158.4 (C), 142.7 (C), 142.5 (C), 139.5 (C), 131.6 (C),

126.2 (CH), 125.8 (CH), 123.9 (CH), 122.9 (CH), 100.1 (CH), 74.1

(C), 73.8 (CH), 70.5 (CH), 66.1 (OCH₂), 62.7 (OCH₂), 40.3 (CH₂)

and 14.4 (CH₃) ppm. IR (KBr) 3419 (OÀH), 1728 (CdO) and 1651

(CdC) cm^À1. MS (ESI) mz/z (%) 421 (MNa⁺). HRMS calcd for

C₁₈H₁₉O₆SClNa (MNa⁺), 421.0483; found, 421.0470.

Ethyl (1R,4S,5R)-3-(Benzo[b]thiophen-5-yl)methoxyl-1,4,

5-trihydroxycyclohex-2-ene-1-carboxylate (7c). The experi-

mental procedure was the same as for compound 7a using compound

5d (63 mg), potassium carbonate (73 mg), bromoethane (400 μL), and

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DMF (1.8 mL). Yield = 37 mg (58%). Yellow oil. [R]²_D⁰= À20ꢀ (c 1.1,

MeOH). ¹H NMR (250 MHz, CD₃OD) δ 7.88 (m, 2H, 2 Â ArH), 7.57

(d, J = 5.5 Hz, 1H, ArH), 7.38 (m, 2H, 2 Â ArH), 4.95 (br s, 3H, H-2 +

OCH₂), 4.05 (m, 2H, OCH₂CH₃), 3.93 (m, 2H, H-4 + H-5), 2.05 (m,

2H, CH₂-6) and 1.19 (t, J = 7.2 Hz, 3H, OCH₂CH₃) ppm. ¹³C NMR (63

MHz, CD₃OD) δ 176.2 (C), 158.8 (C), 141.3 (C), 140.7 (C), 134.3

(C), 128.1 (CH), 125.2 (CH), 124.9 (CH), 123.8 (CH), 123.4 (CH),

99.5 (CH), 74.3 (CH), 73.9 (C), 70.9 (OCH₂), 70.5 (CH), 62.7

(OCH₂), 40.3 (CH₂) and 14.4 (CH₃) ppm. IR (film) 3400 (OÀH)

and 1728 (CdO) cm^À1. MS (ESI) m/z (%) 387 (MNa⁺). HRMS calcd

for C₁₈H₂₀O₆SNa (MNa⁺), 387.0873; found, 387.0870.

ArH), 7.34 (d, J = 5.6 Hz, 1H, ArH), 4.99 (d, J = 11.6 Hz, 1H, OCHHAr),

4.94 (m, 2H, OCHHAr + H-2), 4.02 (m, 4H, OCH₂CH₂CH₃+ H-4 +

H-5), 2.04 (m, 2H, CH₂-6), 1.58 (m, 2H, OCH₂CH₂CH₃) and0.89 (t, J =

7.2 Hz, 3H, OCH₂CH₂CH₃) ppm. ¹³C NMR (63 MHz, CD₃OD) δ

176.2(C), 158.8(C), 141.3(C), 140.7(C), 134.3 (C), 128.1 (CH), 125.2

(CH), 124.9 (CH), 123.8 (CH), 123.4 (CH), 99.5 (CH), 74.3 (CH),

73.9 (C), 70.9 (OCH₂), 70.5 (CH), 62.7 (OCH₂), 40.3 (CH₂) and 14.4

(CH₃) ppm. IR (film): 3425 (OÀH) and 1643 (CdO) cm^À1. MS (ESI)

m/z (%) 401 (MNa⁺). HRMS calcd for C₁₉H₂₂O₆SNa (MNa⁺),

401.1029; found, 401.1020.

Propyl (1R,4S,5R)-1,4,5-Trihydroxy-3-(naphth-2-yl)methoxy-

cyclohex-2-ene-1-carboxylate (8d). The experimental procedure

was the sameasfor compound 7ausing compound 5e(30mg), potassium

carbonate (35 mg), 1-bromopropane (0.22 mL), and DMF (0.8 mL).

Yield = 16 mg (50%). Orange oil. [R]²_D⁰= À36ꢀ (c 1.0, MeOH). ¹H NMR

(300 MHz, CD₃OD) δ 7.86 (m, 4H, 4 Â ArH), 7.52 (dd, J = 8.4 and 1.5

Hz, 1H, ArH), 7.49À7.46 (m, 2H, 2 Â ArH), 5.04 (d, J = 12.0 Hz, 1H,

CHHO), 4.98 (d, J = 12.0 Hz, 1H, CHHO), 4.97 (s, 1H, H-2), 4.21 (dd,

J = 5.4 and 0.9 Hz, 1H, H-5), 4.01 (m, 3H, OCH₂CH₂CH₃+ H-4), 2.06

(m, 2H, CH₂-6), 1.54 (m, 2H, OCH₂CH₂CH₃) and 0.86 (t, J = 7.5 Hz,

3H, OCH₂CH₂CH₃) ppm. ¹³C NMR (75 MHz, CD₃OD) δ 176.2 (C),

158.9 (C), 135.7 (C), 134.8 (C), 134.6 (C), 129.2 (CH), 129.0 (CH),

128.7 (CH), 127.4 (CH), 127.2 (CH), 127.1 (CH), 126.5 (CH), 99.7

(CH), 74.4 (CH), 74.0 (C), 70.9 (OCH₂), 70.5 (CH), 68.1 (OCH₂),

40.4 (CH₂), 22.9 (CH₂) and 10.6 (CH₃) ppm. IR (film) 3377 (OH) and

1730 (CdO) cm^À1. MS (ESI) m/z (%) 395 (MNa⁺). HRMS calcd for

C₂₁H₂₄O₆Na (MNa⁺), 395.1465; found, 395.1461.

Ethyl(1R,4S,5R)-1,4,5-Trihydroxy-3-(naphth-2-yl)methoxy-

cyclohex-2-enecarboxylate (7d). The experimental procedure was

the same as for compound 7a using compound 5e (7 mg), potassium

carbonate (8 mg), bromoethane (40 μL), and DMF(0.2 mL). Yield:3 mg

(42%). Yellow oil. [R]²_D⁰= À39ꢀ (c 1.2, MeOH). ¹H NMR (250 MHz,

CD₃OD) δ 7.88 (m, 4H, 4 Â ArH), 7.51 (m, 3H, 3 Â ArH), 5.01 (s, 2H,

CH₂O), 4.98 (s, 1H, H-2), 4.08 (m, 4H, OCH₂CH₃+ H-4 + H-5), 2.04

(m, 2H, CH₂-6) and 1.17 (t, J = 7.2 Hz, 3H, CH₃) ppm. ¹³C NMR

(63 MHz, CD₃OD) δ 176.2 (C), 158.9 (C), 135.7 (C), 134.8 (C), 134.6

(C), 129.2 (CH), 129.0 (CH), 128.7 (CH), 127.5 (CH), 127.3 (CH),

127.1 (CH), 126.6 (CH), 99.6 (CH), 74.3 (CH), 73.9 (C), 70.9(OCH₂),

70.5 (CH), 62.6 (CH₂), 40.3 (CH₂) and 14.4 (CH₃) ppm. IR (film):

3419 (OÀH) and 1649 (CdO) cm^À1. MS (ESI) m/z (%) 381 (MNa⁺).

HRMS calcd for C₂₀H₂₂O₆Na (MNa⁺), 381.1309; found, 381.1307.

Propyl (1R,4S,5R)-3-(Benzo[b]thiophen-2-yl)methoxy-1,4,

5-trihydroxycyclohex-2-en-1-carboxylate (8a). The experi-

mental procedure was the same as for compound 7a using compound

5a (15 mg), potassium carbonate (17 mg), 1-bromopropane (0.1 mL),

and DMF (0.4 mL). Yield = 6 mg (38%). Colorless oil. [R]²_D⁰= À27ꢀ

(c 1.0, MeOH). ¹H NMR (250 MHz, CD₃OD) δ 7.83 (m, 1H, ArH),

7.76 (m, 1H, ArH), 7.33 (m, 3H, 3 Â ArH), 5.13 (s, 2H, OCH₂Ar), 5.00

(s, 1H, H-2), 4.00 (m, 4H, OCH₂CH₂CH₃+ H-4 + H-5), 2.04 (m, 2H,

CH₂-6), 1.53 (m, 2H, OCH₂CH₂CH₃) and 0.85 (t, J = 7.2 Hz, 3H, CH₃)

ppm. ¹³C NMR (63 MHz, CD₃OD) δ 176.2 (C), 158.4 (C), 141.6 (C),

141.5 (C), 140.9 (C), 125.6 (CH), 125.4 (CH), 124.7 (CH), 124.3

(CH), 123.3 (CH), 100.1 (CH), 74.2 (CH), 73.9 (C), 70.5 (CH), 68.2

(OCH₂), 66.3 (OCH₂), 40.4 (CH₂), 22.9 (CH₂) and 10.6 (CH₃) ppm.

IR (film): 3375 (OÀH) and 1730 (CdO) cm^À1. MS (ESI) m/z (%)

401 (MNa⁺). HRMS calcd for C₁₉H₂₂O₆Na (MNa⁺), 401.1029; found,

401.1030.

Propyl (1R,4S,5R)-1,4,5-Trihydroxy-3-(6-chlorobenzo[b]-

thiophen-2-yl)methoxycyclohex-2-ene-1-carboxylate (8b).

The experimental procedure was the same as for compound 7a using

compound 5c (15 mg), potassium carbonate (16 mg), 1-bromopropane

(0.1 mL), and DMF (0.4 mL). Yield = 6 mg (38%). Yellow oil. ¹H NMR

(250 MHz, CD₃OD) δ 7.88 (d, J = 1.8 Hz, 1H, ArH), 7.73 (d, J = 8.5 Hz,

1H, ArH), 7.36 (s, 1H, ArH), 7.33 (dd, J = 8.5 and 2.0 Hz, 1H, ArH),

5.12 (s, 2H, OCH₂Ar), 4.99 (s, 1H, H-2), 4.11 (m, 2H, OCH₂Ar), 3.99

(m, 4H, OCH₂+ H-4 + H-5), 2.03 (m, 2H, CH₂-6), 1.53 (q, J = 6.7 Hz,

2H, CH₂CH₃) and 0.85 (t, J = 7.2 Hz, 3H, CH₃) ppm. ¹³C NMR (63

MHz, CD₃OD) δ 176.1 (C), 158.3 (C), 142.7 (C), 142.6 (C), 139.5

(C), 131.6 (C), 126.2 (CH), 125.8 (CH), 123.8 (CH), 122.9 (CH),

100.2 (CH), 74.2 (C), 73.9 (CH), 70.5 (CH), 68.2 (OCH₂), 66.1

(OCH₂), 40.4 (CH₂), 22.9 (CH₂) and 10.6 (CH₃) ppm. IR (film): 3421

(OÀH) and 1720 (CdO) cm^À1. MS (ESI) m/z (%) 435 (MNa⁺).

HRMS calcd for C₁₉H₂₁O₆ClNa (MNa⁺), 435.0640; found, 435.0638.

Propyl (1R,4S,5R)-3-(Benzo[b]thiophen-5-yl)methoxy-1,4,

5-trihydroxycyclohex-2-ene-1-carboxylate (8c). The experi-

mental procedure was the same as for compound 7a using compound

5d (12 mg), potassium carbonate (14 mg), 1-bromopropane (85 μL),

and DMF (0.3 mL). Yield = 4 mg (32%). Colorless oil. [R]²_D⁰= À46ꢀ

(c 1.2, MeOH). ¹H NMR (250 MHz, CD₃OD) δ δ 7.86 (m, 2H, 2 Â

ArH), 7.55 (d, J = 5.6 Hz, 1H, ArH), 7.37 (dd, J = 8.8 and 1.2 Hz, 1H,

Methyl (1R,4S,5R)-3-(5-Methylbenzo[b]thiophen-2-yl)-

methoxy-2-(5-methylbenzo[b]thiophen-2-yl)methyl-1,4,5-tri-

hydroxycyclohex-2-en-1-carboxylate (10a). The experimental

procedure was the same as for compound 2a using carbolactone 16b

(123 mg), NaOMe (15 mg), and MeOH (2.8 mL). Yield = 32 mg (24%).

1

Beige solid; mp 168À170 ꢀC. [R]²_D⁰= +48ꢀ (c 1.2, MeOH). H NMR

(250 MHz, acetone-d₆) δ 7.72 (d, J = 8.2 Hz, 1H, ArH), 7.60 (d, J = 8.2 Hz,

1H, ArH), 7.53 (s, 1H, ArH), 7.31 (s, 1H, ArH), 7.22 (s, 1H, ArH), 7.16

(dd, J = 8.2 and 1.2 Hz, 1H, ArH), 7.05 (dd, J = 8.2 and 1.2 Hz, 1H, ArH),

6.83 (s, 1H, ArH), 5.51 (d, J = 12.2 Hz, 1H, OCHHAr), 5.27 (d, J = 12.2

Hz, 1H, OCHHAr), 4.60 (d, J = 6.5 Hz, 1H, OH), 4.49 (s, 1H, OH), 4.38

(t, J = 6.5 Hz, 1H, H-4), 4.05 (m, 1H, H-5), 3.62 (s, 2H, CH₂Ar), 3.27

(s, 3H, OMe), 2.41 (s, 3H, Me), 2.36 (s, 3H, Me), 2.16 (dd, J = 13.0 and

11.3 Hz, 1H, H-6_ax) and 1.99 (m, 1H, H-6_eq) ppm. ¹³C NMR (63 MHz,

acetone-d₆) δ 177.1 (C), 154.7 (C), 146.6 (C), 143.4 (C), 142.2 (C),

141.7 (C), 139.3 (C), 138.7 (C), 135.6 (C), 135.1 (C), 127.9 (CH), 126.7

(CH), 125.4 (CH), 124.7 (CH), 124.4 (CH), 123.8 (CH), 123.3 (CH),

123.2 (CH), 120.1 (C), 77.0 (C), 73.6 (CH), 71.9 (CH), 67.9 (OCH₂),

53.6 (OCH₃), 42.1 (CH₂), 29.0 (CH₃), 22.4 (CH₃) and 22.3 (CH₃) ppm.

IR (KBr): 3502 (OÀH), 3435 (OÀH) and 1730 (CdO) cm^À1. MS

(ESI) m/z (%) 547 (MNa⁺). HRMS calcd for C₂₈H₂₈O₆S₂Na (MNa⁺),

547.1220; found, 547.1215. Elemental analysis C₂₈H₂₈O₆S₂H₂O Calcd:

3

C, 61.97; H, 5.57; S, 11.82. Found: C, 61.40; H, 5.30; S, 11.52.

Methyl (1R,4S,5R)-3-(6-Chlorobenzo[b]thiophen-5-yl)-

methoxy-2-(6-chlorobenzo[b]thiophen-5-yl)methyl-1,4,5-tri-

hydroxycyclohex-2-en-1-carboxylate (10b). The experimental

procedure was the same as for compound 2a using lactone 16c (42 mg),

NaOMe (5 mg), and MeOH (0.9 mL). Yield = 22 mg (49%). Beige solid;

1

mp 154À156 ꢀC. [R]²_D⁰= +74ꢀ (c 1.1, MeOH). H NMR (300 MHz,

acetone-d₆) δ 7.90 (d, J = 1.8 Hz, 1H, ArH), 7.77 (d, J = 2.1 Hz, 1H, ArH),

7.74 (d, J = 8.4 Hz, 1H, ArH), 7.55 (d, J = 8.4 Hz, 1H, ArH), 7.34 (dd, J =

8.4 and 1.8 Hz, 1H, ArH), 7.31 (d, J = 0.9 Hz, 1H, ArH), 7.24 (dd, J = 8.4

and 2.1 Hz, 1H, ArH), 6.97 (d, J = 0.9Hz, 1H, ArH), 5.52 (dd, J = 12.6 and

0.9 Hz, 1H, OCHHAr), 5.27 (dd, J = 12.6 and 0.9 Hz, 1H, OCHHAr),

4.69 (d, J = 6.9 Hz, 1H, OH), 4.58 (s, 1H, OH), 4.55 (d, J = 3.9 Hz, 1H,

OH), 4.38 (t, J = 6.6Hz, 1H, H-4), 4.05 (m, 1H, H-5), 3.66(d, J = 15.6 Hz,

1H, CHHAr), 3.59 (d, J = 15.6 Hz, 1H, CHHAr), 3.34 (s, 3H, OMe), 2.16

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(dd, J = 13.2 and 11.1 Hz, 1H, H-6_ax) and 2.01 (m, 1H, H-6_eq) ppm. ¹³

C

(33 mg), potassium carbonate (24 mg), bromoethane (120 μL), and

DMF (0.6 mL). Yield = 7 mg (21%). Yellow oil. [R]²_D⁰= +32ꢀ (c 0.7,

MeOH). ¹H NMR (250 MHz, CD₃OD) δ 7.77 (d, J = 2.0 Hz, 1H, ArH),

7.65 (m, 2H, 2 Â ArH), 7.39 (d, J = 8.5Hz, 1H, ArH), 7.30 (dd, J =8.5 and

2.0 Hz, 1H, ArH), 7.18 (m, 2H, 2 Â ArH), 6.84 (s, 1H, ArH), 5.42 (d, J =

12.5 Hz, 1H, OCHHAr), 5.22 (d, J = 12.5 Hz, 1H, OCHHAr), 4.33 (d, J =

7.5 Hz, 1H, H-4), 3.99 (m, 1H, H-5), 3.79 (m, 2H, CH₂CH₃), 3.88À3.58

(m, 4H, OCH₂CH₃+ CH₂Ar), 2.18 (t, J = 13.0 Hz, 1H, H-6_ax), 1.98

(dd, J = 13.0 and 4.0 Hz, 1H, H-6_eq) and 0.89 (t, J = 7.2 Hz, 3H, CH₃)

ppm. ¹³C NMR (63 MHz, CD₃OD) δ 175.9 (C), 154.1 (C), 146.7 (C),

143.3 (C), 143.0 (C), 142.2 (C), 139.9 (C), 139.4 (C), 131.5 (C), 130.3

(C), 126.1 (CH), 125.8 (CH), 125.6 (CH), 124.6 (CH), 124.1 (CH),

122.9 (CH), 122.4 (CH), 122.3 (CH), 119.9 (C), 76.6 (C), 72.7 (CH),

71.2 (CH), 67.0 (OCH₂), 62.9 (CH₂), 41.4 (CH₂), 28.5 (CH₂) and 14.0

(CH₃) ppm. IR (film) 3419 (OÀH) and 1643 (CdO) cm^À1. MS (ESI)

m/z (%) 601 (MNa⁺). HRMS calcd for C₂₇H₂₄O₆S₂Cl₂Na (MNa⁺),

601.0284; found, 601.0283.

Ethyl (1R,4S,5R)-3-(Benzo[b]thiophen-5-yl)methoxy-2-

(benzo[b]thiophen-5-yl)methyl-1,4,5-trihydroxycyclohex-2-

en-1-carboxylate (11b). The experimental procedure was the same

as for compound 7a using compound 9d (8 mg), potassium carbonate

(6 mg), bromoethane (33 μL), and DMF (0.2 mL). Yield = 4 mg (49%).

Colorless oil. [R]²_D⁰= +4ꢀ (c 0.5, MeOH). ¹H NMR (400 MHz,

CD₃OD) δ 7.77 (d, J = 8.4 Hz, 1H, ArH), 7.63 (m, 2H, 2 Â ArH),

7.56 (s, 1H, ArH), 7.53 (d, J = 5.2 Hz, 1H, ArH), 7.45 (d, J = 5.2 Hz, 1H,

ArH), 7.26 (d, J = 8.4 Hz, 1H, ArH), 7.21 (d, J = 5.2 Hz, 1H, ArH), 7.15

(m, 2H, 2 Â ArH), 5.27 (d, J = 11.2 Hz, 1H, OCHHAr), 4.91 (d, J =

11.2 Hz, 1H, OCHHAr), 4.37 (d, J = 7.6 Hz, 1H, H-4), 4.01 (m, 1H,

H-5), 3.77 (m, 2H, OCH₂CH₃), 3.41 (m, 2H, CH₂Ar), 2.17 (t, J = 13.2

Hz, 1H, H-6_ax), 1.97 (dd, J = 13.2 and 4.0 Hz, 1H, H-6_eq) and 0.91 (t, J =

7.2 Hz, 3H, CH₃) ppm. ¹³C NMR (100 MHz, CD₃OD) δ 176.2 (C),

154.0 (C), 141.2 (C), 141.1 (C), 138.5 (C), 137.6 (C), 135.2 (C), 127.9

(CH), 127.2 (CH), 127.1 (CH), 126.0 (CH), 125.0 (CH), 124.7 (CH),

124.7 (CH), 124.5 (CH), 124.5 (C), 123.2 (CH), 122.5 (CH), 120.9

(C), 77.0 (C), 73.0 (CH), 72.2 (OCH₂), 71.4 (CH), 62.7 (CH₂), 41.6

(CH₂), 33.2 (CH₂) and 14.0 (CH₃) ppm. IR (film) 3421 (OÀH) and

1639 (CdO) cm^À1. MS (ESI) m/z (%) 533 (MNa⁺). HRMS calcd for

C₂₇H₂₆O₆S₂Na (MNa⁺), 533.1063; found, 533.1058.

NMR (75 MHz, acetone-d₆) δ 177.1 (C), 154.8 (C), 147.9 (C), 144.4

(C), 143.3 (C), 142.8 (C), 140.5 (C), 140.0 (C), 131.7 (C), 130.5 (C),

126.7 (CH), 126.6 (CH), 126.2 (CH), 125.5 (CH), 124.3 (CH), 123.6

(CH), 123.1 (CH), 123.0 (CH), 119.9 (C), 77.1 (C), 73.4 (CH), 71.8

(CH), 67.7 (OCH₂), 53.7 (OCH₃), 42.1 (CH₂) and 29.2 (CH₂) ppm. IR

(KBr) 3419 (OÀH) and 1730 (CdO) cm^À1. MS (ESI) m/z (%) 587

(MNa⁺). HRMS calcd for C₂₆H₂₂O₆S₂Cl₂Na (MNa⁺), 587.0127; found,

587.0123. Elemental analysis C₂₆H₂₂O₆S₂Cl₂.H₂O Calcd: C, 53.52; H,

4.15; S, 10.99. Found: C, 53.72; H, 4.36; S, 11.34.

Methyl (1R,4S,5R)-3-(Benzo[b]thiophen-5-yl)methoxy-2-

(benzo[b]thiophen-5-yl)methyl-1,4,5-trihydroxycyclohex-

2-enecarboxylate (10c). A solution of carbolactone 16d (57 mg,

0.12 mmol) in dry methanol (0.7 mL) and acetonitrile (0.7 mL) was

treated with sodium methoxide (7 mg, 0.13 mmol). The resultant

mixture was stirred at room temperature for 2 h and was diluted with

ethyl acetate and water. The organic layer was separated, and the aqueous

phase was extracted with ethyl acetate (Â3). The combined organic

extracts were filtered (anhyd Na₂SO₄) and concentrated under

reduced pressure. The residue was purified by flash chromatography

on silica gel, eluting with (1:1) ethyl acetateÀhexanes to afford methyl

ester 10c (24 mg, 40%) as a white solid; mp 148À152 ꢀC. [R]²_D⁰= +9ꢀ

(c 1.2, MeOH). ¹H NMR (300 MHz, CD₃OD) δ 7.72 (d, J = 8.4 Hz,

1H, ArH), 7.60 (m, 2H, 2 Â ArH), 7.52 (br s, 1H, ArH), 7.48 (d, J = 5.4

Hz, 1H, ArH), 7.42 (d, J = 5.4 Hz, 1H, ArH), 7.23 (dd, J = 8.1 and 1.5

Hz, 1H, ArH), 7.17 (dd, J = 5.7 and 0.6 Hz, 1H, ArH), 7.11 (m, 2H, 2 Â

ArH), 5.26 (d, J = 11.1 Hz, 1H, OCHHAr), 4.87 (d, J = 11.1 Hz, 1H,

OCHHAr), 4.38 (d, J = 7.5 Hz, 1H, H-4), 4.03 (m, 1H, H-5), 3.55 (d,

1H, J = 15.3 Hz, CHHAr), 3.38 (d, 1H, J = 15.3 Hz, CHHAr), 3.14 (s,

3H, OCH₃), 2.18 (dd, J = 13.2 and 12.0 Hz, 1H, H-6_ax) and 1.99 (dd,

J = 13.2 and 3.9 Hz, 1H, H-6_eq) ppm. ¹³C NMR (75 MHz, CD₃OD) δ

176.6 (C), 154.0 (C), 141.2 (2 Â C), 140.6 (C), 138.5 (C), 137.5 (C),

135.1 (C), 127.9 (CH), 127.2 (CH), 127.0 (CH), 125.9 (CH), 124.9

(CH), 124.7 (2 Â CH), 124.4 (CH), 123.2 (CH), 122.5 (CH), 120.8

(C), 77.0 (C), 72.9 (CH), 72.2 (CH₂), 71.3 (CH), 52.7 (OCH₃), 41.5

(CH₂) and 33.1 (CH₂) ppm. IR (KBr) 3410 (OÀH) and 1734

(CdO) cm^À1. MS (ESI) m/z (%) 519 (MNa⁺). HRMS calcd for

C₂₆H₂₄O₆S₂Na (MNa⁺), 519.0907; found, 519.0901. Elemental anal-

ysis C₂₆H₂₄O₆S₂1/2H₂O Calcd: C, 61.76; H, 4.98; S, 12.68. Found:

C, 61.47; H, 5.28; S, 12.30.

Ethyl (1R,4S,5R)-1,4,5-Trihydroxy-3-(thien-2-yl)methoxy-

2-(5-methylbenzo[b]thiophen-2-yl)methylcyclohex-2-en-1-

carboxylate (11c). The experimental procedure was the same as for

compound 7a using compound 9i (33 mg), potassium carbonate (15 mg),

bromoethane (80 μL), and DMF (0.4 mL). Yield = 6 mg (37%).

Yellow oil. [R]²_D⁰= +24ꢀ (c 0.8, MeOH). ¹H NMR (400 MHz, CD₃OD)

δ 7.54 (d, J = 8.4 Hz, 1H, ArH), 7.37 (s, 1H, ArH), 7.31 (dd, J = 4.8 and

0.8 Hz, 1H, ArH), 7.01 (m, 2H, 2 Â ArH), 6.91 (dd, J = 5.2 and 3.6 Hz,

1H, ArH), 6.78 (s, 1H, ArH), 5.36 (d, J = 11.6 Hz, 1H, OCHHAr), 5.08

(d, J = 11.6 Hz, 1H, OCHHAr), 4.26 (d, J = 7.6 Hz, 1H, H-4), 3.94 (m,

1H, H-5), 3.83 (m, 1H, CH₃CHHO), 3.62 (d, J = 15.2 Hz, 1H, CHHAr),

3.48 (d, J = 15.2 Hz, 1H, CHHAr), 3.78 (m, 1H, CH₃CHHO), 2.37 (s,

3H, CH₃), 2.14 (dd, J = 12.4 and 12.8 Hz, 1H, H-6_ax), 1.94 (dd, J = 12.8

and 4.0 Hz, 1H, H-6_eq) and 0.89 (t, J = 7.2 Hz, 3H, CH₃CH₂O) ppm.

¹³C NMR (100 MHz, CD₃OD) δ 176.0 (C), 154.1 (C), 145.6 (C),

141.7 (C), 141.2 (C), 138.4 (C), 134.7 (C), 128.3 (CH), 127.6 (CH),

127.4 (CH), 126.1 (CH), 123.7 (CH), 122.7 (CH), 122.5 (CH), 120.2

(C), 76.5 (C), 72.8 (CH), 71.2 (CH), 66.5 (OCH₂), 62.9 (OCH₂), 41.5

(CH₂), 28.3 (CH₂), 21.5 (CH₃) and 14.0 (CH₃) ppm. IR (film) 3435

(OÀH), 1722 (CdO) and 1651 (CdC) cm^À1. MS (ESI) m/z (%) 497

(MNa⁺). HRMS calcd for C₂₄H₂₆O₆S₂Na (MNa⁺), 497.1063; found,

497.1054.

3

Methyl (1R,4S,5R)-1,4-Dihydroxy-3-(thien-2-yl)methoxy-

2-(thien-2-yl)methylcyclohex-2-en-1-carboxylate (10d). The

experimental procedure was the same as for compound 2a using lactone

16f (40 mg), NaOMe (6 mg), and MeOH (1.2 mL). Yield = 22 mg

(51%). Beige solid; mp 55À57 ꢀC. [R]²_D⁰= +7ꢀ (c 1.1, MeOH). H

1

NMR (300 MHz, CD₃OD) δ 7.34 (dd, J = 5.1 and 1.2 Hz, 1H, ArH),

7.10 (dd, J = 5.1 and 1.2 Hz, 1H, ArH), 7.04 (m, 1H, ArH), 6.95 (dd, J =

5.1 and 3.3 Hz, 1H, ArH), 6.80 (dd, J = 5.1 and 3.3 Hz, 1H, ArH), 6.67

(m, 1H, ArH), 5.37 (d, J = 11.4 Hz, 1H, OCHHAr), 5.01 (d, J = 11.4 Hz,

1H, OCHHAr), 4.25 (d, J = 7.8 Hz, 1H, H-4), 3.93 (m, 1H, H-5), 3.61

(d, J = 15.3 Hz, 1H, CHHAr), 3.43 (d, J = 15.3 Hz, 1H, CHHAr), 3.33 (s,

3H, OCH₃), 2.14 (dd, J = 12.9 and 12.0 Hz, 1H, H-6_ax) and 1.95 (dd, J =

12.9 and 3.9 Hz, 1H, H-6_eq) ppm. ¹³C NMR (75 MHz, CD₃OD)

δ 176.2 (C), 153.7 (C), 144.1 (C), 141.1 (C), 128.1 (CH), 127.4 (CH),

127.2 (CH), 126.9 (CH), 126.4 (CH), 124.3 (CH), 120.9 (C), 76.3 (C),

72.7 (CH), 71.1 (CH), 66.5 (CH₂), 52.8 (CH₃), 41.3 (CH₂) and 27.2

(CH₂) ppm. IR (KBr) 3435 (OÀH) and 1734 (CdO) cm^À1. MS (ESI)

m/z (%) 419 (MNa⁺). HRMS calcd for C₁₈H₂₀O₆S₂Na (MNa⁺),

419.0594; found, 419.0581. Elemental analysis C₁₈H₂₀O₆S₂1/4H₂O

3

Calcd; C, 53.92; H, 5.15; S, 15.99. Found: C, 54.15; H, 5.46; S, 15.65.

Ethyl (1R,4S,5R)-3-(6-Chlorobenzo[b]thiophen-5-yl)-

methoxy-2-(6-chlorobenzo[b]thiophen-5-yl)methyl-1,4,5-tri-

hydroxycyclohex-2-en-1-carboxylate (11a). The experimental

procedure was the same as for compound 7a using compound 9c

Ethyl (1R,4S,5R)-4,5-Dibutyroxy-3-(benzo[b]thiophen-5-

yl)methoxy-1-hydroxycyclohex-2-enecarboxylate (7e). To a

stirred solution of ethyl ester 7c (22 mg, 0.06 mmol) and pyridine

(17 μL, 0.21 mmol) in dry acetonitrile (1.5 mL) at 0 ꢀC was added

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butyryl chloride (22 μL, 0.21 mmol). The resulting solution was stirred

at room temperature for 10 h, and it was then partitioned into water and

ethyl acetate. The organic layer was separated, and the aqueous phase

was extracted with ethyl acetate (Â3). The combined organic extracts

were dried (anhyd MgSO₄), filtered, and concentrated under reduced

pressure. The residue was purified by flash chromatography eluting with

(50:50) ethyl acetate/hexane to afford ester 7e (11 mg, 36%) as a yellow

oil. [R]²_D⁰= À26ꢀ (c 1.0, MeOH). ¹H NMR (250 MHz, CD₃OD) δ 7.88

(d, J = 8.5 Hz, 1H, ArH), 7.81 (br s, 1H, ArH), 7.59 (d, J = 5.5 Hz, 1H,

ArH), 7.35 (d, J = 5.5 Hz, 1H, ArH), 7.31 (br d, J = 8.5 Hz, 1H, ArH),

5.63 (d, J = 7.8 Hz, 1H, H-4), 5.44 (m, 1H, H-5), 5.18 (s, 1H, H-2), 4.94

(s, 2H, OCH₂Ar), 4.16 (m, 2H, OCH₂CH₃), 2.26 (m, 2H, CH₂-6),

1.70À1.48 (m, 4H, 2 Â CH₂), 1.36À1.11 (m, 4H, 2 Â CH₂), 0.97 (m,

6H, 2 Â CH₃) and 0.79 (t, J = 7.2 Hz, 3H, OCH₂CH₃) ppm. ¹³C NMR

(63 MHz, CD₃OD) δ 175.1 (C), 174.7 (C), 174.1 (C), 154.8 (C), 141.3

(C), 140.8 (C), 134.0 (C), 128.3 (CH), 125.0 (CH), 124.9 (CH), 123.6

(CH), 123.5 (CH), 101.8 (CH), 73.4 (C), 71.6 (CH), 71.1 (OCH₂),

70.6 (CH), 62.9 (OCH₂), 37.4 (CH₂), 37.0 (CH₂), 37.0 (CH₂), 19.5

(CH₂), 19.4 (CH₂), 14.4 (CH₃), 13.9 (CH₃) and 13.9 (CH₃) ppm. IR

(film) 3398 (OÀH) and 1736 (CdO) cm^À1. MS (ESI) m/z (%) 527

(MNa⁺). HRMS calcd for C₂₆H₃₂O₈SNa (MNa⁺), 527.1710; found,

527.1695.

Propyl (1R,4S,5R)-3-(Benzo[b]thiophen-5-ylmethoxy)-4,

5-dibutyroxy-1-hydroxycyclohex-2-enecarboxylate (8e). A

stirred solution of ethyl ester 8c (3.4 mg, 8.99 μmol) in dry acetonitrile

(0.2 mL) at 0 ꢀC was treated with a solution of dry pyridine (30 μL, 1 M

in acetonitrile, 0.03 mmol). After 10 min, a solution of butyryl chloride

(30 μL, 1 M in acetonitrile, 0.03 mmol) was added. The resuting

solution was stirred at room temperature for 10 h and then partitioned

into water and ethyl acetate. The organic layer was separated, and the

aqueous phase was extracted with ethyl acetate (Â3). The combined

organic extracts were dried (anhyd MgSO₄), filtered, and concentrated

under reduced pressure. The residue was purufied by flash chroma-

tography on silica gel, eluting with ethyl acetate/hexane (35:65) to give

ester 8e (3 mg, 68%) as a yellow oil. ¹H NMR (500 MHz, CD₃OD) δ

7.88 (d, J = 8.0 Hz, 1H, ArH), 7.80 (s, 1H, ArH), 7.59 (d, J = 5.5 Hz, 1H,

ArH), 7.36 (d, J = 5.5 Hz, 1H, ArH), 7.31 (br d, J = 8.0 Hz, 1H, ArH),

5.63 (d, J = 8.0 Hz, 1H, H-4), 5.45 (m, 1H, H-5), 5.18 (s, 1H, H-2), 4.94

(s, 2H, OCH₂Ar), 4.06 (t, J = 6.5 Hz, 2H, OCH₂CH₂CH₃), 2.32À2.12

(m, 5H, 2 Â OCOCH₂+ H-6_ax), 2.14 (dd, J = 13.0 and 3.5 Hz, H-6_eq),

1.61 (m, 4H, 2 Â CH₂), 1.53 (m, 2H, CH₂), 0.91 (m, 6H, 2 Â CH₃)

and 0.80 (t, J = 7.5 Hz, 3H, OCH₂CH₂CH₃) ppm. ¹³C NMR (125

MHz, CD₃OD) δ 175.1 (C), 174.7 (C), 174.1 (C), 154.8 (C), 141.3

(C), 140.8 (C), 134.0 (C), 128.3 (CH), 124.9 (CH), 124.9 (CH),

123.6 (CH), 123.5 (CH), 101.9 (CH), 73.5 (C), 71.6 (CH), 71.1

(OCH₂), 70.6 (CH), 68.3 (OCH₂), 37.4 (CH₂), 37.0 (CH₂), 37.0

(CH₂), 23.0 (CH₂), 19.5 (CH₂), 19.4 (CH₂), 13.9 (CH₃), 13.9 (CH₃)

and 10.7 (CH₃) ppm. MS (ESI) m/z (%) 541 (MNa⁺). HRMS calcd

for C₂₇H₃₄O₈SNa (MNa⁺), 541.1867; found, 541.1853.

123.4 (CH), 101.1 (CH), 75.0 (CH), 73.8 (C), 71.0 (OCH₂), 68.3

(OCH₂), 68.0 (CH), 40.8 (CH₂), 37.2 (CH₂), 23.0 (CH₂), 19.5 (CH₂),

13.9 (CH₃) and 10.7 (CH₃) ppm. IR (film) 3435 (OÀH) and 1651

(CdO) cm^À1. MS (ESI) m/z (%) 471 (MNa⁺). HRMS calcd for

C₂₃H₂₈O₇SNa (MNa⁺), 471.1448; found, 471.1447.

Esterification of Ethyl Ester 7d: Preparation of Ethyl

(1R,4S,5R)-4,5-Dibutyroxy-1-hydroxy-3-(naphth-2-yl)methoxy-

cyclohex-2-ene-1-carboxylate (7f) and Ethyl (1R,4S,5R)-4-Bu-

tyroxy-1,5-dihydroxy-3-(naphth-2-yl)methoxycyclohex-2-ene-

1-carboxylate (7g). A stirred solution of ethyl ester 7d (12 mg,

0.06 mmol) in dry acetonitrile (0.8 mL) at 0 ꢀC was treated with a

solution of dry pyridine (0.12 mL, 1 M in acetonitrile, 0.12 mmol).

After 10 min, a solution of butyryl chloride (0.12 mL, 1 M in aceto-

nitrile, 0.12 mmol) was added. The resulting solution was stirred at

room temperature for 10 h and was then partitioned into water and

ethyl acetate. The organic layer was separated and the aqueous phase

was extracted with ethyl acetate (Â3). The combined organic extracts

were dried (anhyd MgSO₄), filtered, and concentrated under reduced

pressure. The residue was purufied by flash chromatography eluting

with ethyl acetate/hexane (35:65) to afford ester 7f (7.3 mg, 42%) and

ester 7g (6.6 mg, 46%), both as colorless oils.

Data for 7f: [R]²_D⁰= À29ꢀ (c 1.0, in MeOH). ¹H NMR (250 MHz,

CD₃OD) δ 7.84 (m, 4H, 4 Â ArH), 7.48 (m, 2H, 2 Â ArH), 7.44 (dd, J =

8.5 and 1.5 Hz, 1H, ArH), 5.65 (dd, J = 7.7 and 1.1 Hz, 1H, H-4), 5.46

(m, 1H, H-5), 5.21 (s, 1H, H-2), 4.98 (s, 2H, OCH₂Ar), 4.14 (q, J = 7.2

Hz, 2H, OCH₂CH₃), 2.23À2.21 (m, 5H, 2 Â CH₂CO + H-6_ax), 2.14

(dd, J = 13.3 and 4.0 Hz, 1H, H-6_eq), 1.65À1.50 (m, 4H, 2 Â

COCH₂CH₂CH₃), 1.21 (t, J = 7.2 Hz, 3H, OCH₂CH₃), 0.93 (t, J =

7.2 Hz, 3H, COCH₂CH₂CH₃) and 0.79 (t, J = 7.2 Hz, 3H,

COCH₂CH₂CH₃) ppm. ¹³C NMR (63 MHz, CD₃OD) δ 175.0 (C),

174.6 (C), 174.1 (C), 154.8 (C), 135.2 (C), 134.7 (C), 134.6 (C), 129.3

(CH), 128.9 (CH), 128.8 (CH), 127.3 (CH), 127.3 (CH), 127.2 (CH),

126.3 (CH), 101.9 (CH), 73.4 (C), 71.6 (CH), 71.0 (OCH₂), 70.6

(CH), 62.8 (OCH₂), 37.4 (CH₂), 37.0 (CH₂), 37.0 (CH₂), 19.5 (CH₂),

19.4 (CH₂), 14.4 (CH₃), 13.9 (CH₃) and 13.9 (CH₃) ppm. IR (ﬁlm)

3419 (OÀH) and 1739 (CdO) cm^À1. MS (ESI) m/z (%) 521 (MNa⁺).

HRMS calcd for C₂₈H₃₄O₈Na (MNa⁺), 521.2146; found, 521.2136.

Data for 7g: [R]²_D⁰= À33ꢀ (c 0.5, MeOH). H NMR (250 MHz,

1

CD₃OD) δ 7.83 (m, 4H, 4 Â ArH), 7.45 (m, 3H, 3 Â ArH), 5.49 (d, J =

8.2 Hz, 1H, H-4), 5.10 (s, 1H, H-2), 4.94 (s, 2H, OCH₂Ar), 4.25À4.11

(m, 3H, OCH₂CH₃+ H-5), 2.32 (m, 2H, COCH₂CH₂CH₃), 2.21À2.11

(m, 2H, CH₂-6), 1.55 (q, J = 7.2 Hz, 2H, COCH₂CH₂CH₃), 1.21 (t, J =

7.0 Hz, 3H, OCH₂CH₃) and 0.78 (t, J = 7.2 Hz, 3H, COCH₂CH₂CH₃)

ppm. ¹³C NMR (63 MHz, CD₃OD) δ 175.7 (C), 175.3 (C), 155.7 (C),

135.3 (C), 134.7 (C), 134.6 (C), 129.2 (CH), 128.9 (CH), 128.7 (CH),

127.3 (CH), 127.2 (CH), 127.1 (CH), 126.3 (CH), 101.1 (CH), 75.0

(CH), 73.7 (C), 70.9 (OCH₂), 68.0 (CH), 62.8 (OCH₂), 40.7 (CH₂),

37.1 (CH₂), 19.5 (CH₂), 14.4 (CH₃) and 13.9 (CH₃) ppm. IR (ﬁlm)

3446 (OÀH) and 1651 (CdO) cm^À1. MS (ESI) m/z (%) 451 (MNa⁺).

HRMS calcd for C₂₄H₂₈O₇Na (MNa⁺), 451.1727; found, 451.1732.

Propyl (1R,4S,5R)-4,5-Dibutyroxy-1-hydroxy-3-(naphth-

2-yl)methoxycyclohex-2-ene-1-carboxylate (8g). The experi-

mental procedure was the same as for compound 7f using compound 8d

(13 mg), 1 M pyridine in acetonitrile (0.12 mL), 1 M butyryl chloride in

acetonitrile (0.12 mL), and acetonitrile (0.9 mL). Yield = 11 mg (62%).

Colorless oil [R]²_D⁰= À33ꢀ (c 1.0, MeOH). ¹H NMR (250 MHz,

CD₃OD) δ 7.83 (m, 4H, 4 Â ArH), 7.45 (m, 3H, 3 Â ArH), 5.65 (dd, J =

7.7 and 0.7 Hz, 1H, H-4), 5.46 (m, 1H, H-5), 5.20 (s, 1H, H-2), 4.98 (s,

2H, OCH₂Ar), 4.04 (t, J = 6.5 Hz, 2H, OCH₂CH₂CH₃), 2.35À2.24 (m,

5H, 2 Â COCH₂CH₂CH₃+ H-6_ax), 2.14 (dd, J = 13.0 and 3.7 Hz, 1H,

H-6_eq), 1.65À1.50 (m, 6H, 2 Â COCH₂CH₂CH₃+ OCH₂CH₂CH₃),

0.93 (t, J = 7.2 Hz, 3H, OCH₂CH₂CH₃), 0.88 (t, J = 7.5 Hz, 3H,

COCH₂CH₂CH₃) and 0.80 (t, J = 7.2 Hz, 3H, COCH₂CH₂CH₃) ppm.

¹³C NMR (63 MHz, CD₃OD) δ 175.1 (C), 174.6 (C), 174.1 (C), 154.8

Propyl (1R,4S,5R)-3-(Benzo[b]thiophen-5-ylmethoxy)-4-

butyroxy-1,5-dihydroxycyclohex-2-enecarboxylate (8f). The ex-

perimental procedure was the same as for compound 8e using compound

8c (10 mg), pyridine solution (45 μL), butyryl chloride solution (45 μL),

and acetonitrile (0.7 mL). Yield = 3.5 mg (29%). Colorless oil. [R]_D²⁰

=

À27ꢀ (c 0.5, MeOH). ¹H NMR (400 MHz, CD₃OD) δ 7.87 (d, J = 8.4 Hz,

1H, ArH), 7.80 (br s, 1H, ArH), 7.58 (d, J = 5.6 Hz, 1H, ArH), 7.35 (d, J =

5.6 Hz, 1H, ArH), 7.30 (dd, J = 8.4 and 1.6 Hz, 1H, ArH), 5.47 (dd, J = 8.0

and 0.8 Hz, 1H, H-4), 5.07 (s, 1H, H-2), 4.90 (s, 2H, OCH₂Ar), 4.19 (m,

1H, H-5), 4.07 (m, 2H, OCH₂CH₂CH₃), 2.31 (q, J = 7.2 Hz, 2H,

COCH₂), 2.16 (dd, J = 13.2 and 11.2 Hz, 1H, H-6_ax), 2.10 (dd, J = 13.2

and 4.4 Hz, 1H, H-6_eq), 1.62 (m, 2H, CH₂), 1.55 (m, 2H, CH₂), 0.92 (t, J =

7.6 Hz, 3H, CH₃) and 0.79 (t, J = 7.6 Hz, 3H, OCH₂CH₂CH₃) ppm. ¹³

C

NMR (100 MHz, CD₃OD) δ 175.9 (C), 175.3 (C), 155.7 (C), 141.3 (C),

140.8 (C), 134.1 (C), 128.2 (CH), 124.9 (CH), 124.9 (CH), 123.5 (CH),

6081

dx.doi.org/10.1021/jm2006063 |J. Med. Chem. 2011, 54, 6063–6084

Journal of Medicinal Chemistry

ARTICLE

(C), 135.2 (C), 134.7 (C), 134.6 (C), 129.3 (CH), 128.9 (CH), 128.8

(CH), 127.3 (CH), 127.3 (CH), 127.2 (CH), 126.2 (CH), 102.0 (CH),

73.4 (C), 71.6 (CH), 71.0 (OCH₂), 70.6 (CH), 68.3 (OCH₂), 37.4

(CH₂), 37.0 (2 Â CH₂), 22.9 (CH₂), 19.5 (CH₂), 19.4 (CH₂), 13.9

(CH₃), 13.9 (CH₃) and 10.7 (CH₃) ppm. IR (film) 3433 (OÀH) and

1739 (CdO) cm^À1. MS (ESI) m/z (%) 535 (MNa⁺). HRMS calcd for

C₂₉H₃₆O₈Na (MNa⁺), 535.2302; found, 535.2292.

’ AUTHOR INFORMATION

Corresponding Author

*Phone: +34 881815726. Fax: +34 881815704. E-mail:

concepcion.gonzalez.bello@usc.es.

’ ACKNOWLEDGMENT

Dehydroquinase Assays. The enzyme was purified and assayed

as described previously.^27,36

Financial support from the Xunta de Galicia (10PXIB2200-

122PR and GRC2010/12) and the Spanish Ministry of Science

and Innovation (SAF2010-15076 to C.G.B. and BFU2008-01-

588/BMC to M.J.v.R.) is gratefully acknowledged. L.T. and V.F.

V.P. thank the Spanish Ministry of Science and Innovation and

the Portuguese Func-ac~ao para a Ci^encia e a Tecnología for FPU

and FCT fellowships, respectively. J.M.O. thanks the Xunta de

Galicia and the Spanish Ministry of Science and Innovation for

Crystallization of DHQ2/9d and DHQ2/9h Binary Com-

plexes. First, DHQ2-Mt was concentrated to 20 mg mL^À1in 50 mM

Tris-HCl pH 7.5, 1 mM 2-mercaptoethanol, 1 mM ethylenediami-

netetraacetic acid, and 200 mM sodium chloride. A 250 mM solution

of inhibitor 9d or 9h in methanol was then added at 1:20 (v/v) to give

solutions of approximately 10 equiv of inhibitor per protein mono-

mer. Bipyramidal crystals of up to 0.2 mm Â 0.2 mm of the DHQ2-

Mt/9d and 9h binary complexes were obtained after 3 weeks of vapor

diffusion in sitting drops comprised of 2.0 μL of protein/inhibitor

solution mixed with 2.0 μL of reservoir solution and equilibrated

against 0.15 mL reservoirs containing 33% (v/v) 2-methyl-2,4-

pentanediol, 0.3 M ammonium sulfate, and 0.1 M 4-(2-hydro-

xyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES so-

dium salt), pH 7.5.

ꢀ

“Angeles Alvarin~o” and “Josꢀe Castillejo” fellowships, respectively.

We are also thankful to the ESRF for the provison of beam time on

beamlines ID23-1, ID23-2 and BM16.

’ DEDICATION

^†In memory of Professor Rafael Suau.

Crystallization of DHQ2-Mt/5b Binary Complex. Apo-

DHQ2-Mt crystals^12cwere soaked in a 10 mM solution of inhibitor

5b in the crystallization mixture (32% (v/v) 2-methyl-2,4-pentane-

diol, 0.3 M ammonium sulfate and 0.1 M 4-(2-hydroxyethyl)-

piperazine-1-ethanesulfonic acid sodium salt (HEPES sodium salt),

pH 7.5) for 24 h.

’ ABBREVIATIONS USED

TB, tuberculosis; MDR-TB, multidrug-resistant isolates of My-

cobacterium tuberculosis; XDR-TB, drug-resistant isolates of My-

cobacterium tuberculosis; DHQ, 3-dehydroquinate dehydratase or

dehydroquinase; DHQ1, type I dehydroquinase; DHQ2, type II

dehydroquinase; DHQ2-Mt, type II dehydroquinase from Myco-

bacterium tuberculosis; DHQ2-Hp, type II dehydroquinase from

Helicobacter pylori; PDB, Protein Data Bank; KHMDS, potassium

hexamethyldisilazane; LHMDS, lithium hexamethyldisilazane;

TBS, tert-butyldimethylsilyl;TBAF, tetrabutylammonium ﬂuor-

ide; DMF, N,N-dimethylformamide; THF, tetrahydrofuran; DTT,

1,4-dithiothreitol; Tris, tris(hydroxymethyl)aminomethane

Structure Determination of Binary Complexes. Crystals

were flash-frozen directly from the crystallization mixtures by rapid

immersion in liquid nitrogen. Data were collected on beamline ID23À1,

ID23À2, and BM16 (ESRF, Grenoble, France) from crystals maintained

at 100 K. The data were processed, scaled, and analyzed using

MOSFLM,³⁷SCALA,³⁸and other programs of the CCP4 software

suite.³⁹The structure was solved by molecular replacement using the

program MOLREP⁴⁰with a search model generated from PDB entry

1H0S.⁴¹Reflections for calculating R_freewere selected randomly,

42

and model building and refinement were carried out with COOT⁴³

and REFMAC,⁴⁴respectively. Structure validation was performed

using MOLPROBITY.⁴⁵

’ REFERENCES

(1) Tuberculosis; World Health Organization: Geneva, 2011; http://

www.who.int/tb/research/en/.

The data collection, reﬁnement, and model statistics are summarized

in Table 2. Coordinates and structure factors are available from the

Protein Data Bank with accession codes 2Y71, 2Y76, and 2Y77 for

DHQ2-Mt/5b, DHQ2-Mt/9d, and DHQ2-Mt/9h complexes, respec-

tively. Figures were prepared using PyMOL.⁴⁶

In Vitro Antibacterial Activity Assay. The in vitro antibacterial

activity of acids 5 and 9 and their ester derivatives 6À11 was studied by

determining their minimum inhibitory concentrations (MICs μg/mL)

against M. tuberculosis H37Rv, using the Alamar Blue Assay.³⁴MICs

were defined as the lowest concentration at which bacterial growth was

no longer evident.

(2) (a) Espinal, M. A. The global situation of MDR-TB. Tuberculosis

2003, 83, 44–51. (b) Dye, C.; Williams, B. G. The population dynamics

and control of tuberculosis. Science 2010, 328, 856–861.

(3) Centers for Disease Control Prevention. Emergence of Myco-

bacterium tuberculosis with extensive resistance to second-line drugs-

worldwide, 2000À2004. MMWR Recomm. Rep. 2006, 55, 301–305.

(4) Raviglione, M. C.; Smith, I. XDR tuberculosis-implications for

global public health. New Engl. J. Med. 2007, 356, 656–659.

(5) Haydel, S. E. Extensively Drug-Resistant Tuberculosis: A Sign of

the Times and an Impetus for Antimicrobial Discovery. Pharmaceuticals

2010, 3, 2268–2290.

(6) Haydel, S. E. Extensively Drug-Resistant Tuberculosis: A Sign of

the Times and an Impetus for Antimicrobial Discovery. Pharmaceuticals

2010, 3, 2268–2290.

(7) Zhang, Y. The magic bullets and tuberculosis drug targets. Annu.

Rev. Pharmacol. Toxicol. 2005, 45, 529–564.

(8) Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K. The

challenge of new drug discovery for tuberculosis. Nature 2011, 469,

483–490.

(9) (a) Roberts, F.; Roberts, C. W.; Johnson, J. J.; Kyle, D. E.; Krell,

T.; Coggins, J. R.; Coombs, G. H.; Milhous, W. K.; Tzipori, S.; Ferguson,

D. J.; Chakrabarti, D.; McLeod, R. Evidence for the shikimate pathway in

apicomplexan parasites. Nature 1998, 393, 801–805. (b) Campbell,

S. A.; Richards, T. A.; Mui, E. J.; Samuel, B. U.; Coggins, J. R.; McLeod,

’ ASSOCIATED CONTENT

1

Supporting Information. Copies of H NMR, ¹³C NMR,

S

b

and DEPT spectra for compounds 5À11 and Dixon plots for

compounds 5 and 9. This material is available free of charge via

the Internet at http://pubs.acs.org.

Accession Codes

^‡Coordinates and structure factors are available from the Protein

Data Bank with accession codes 2Y71, 2Y76, and 2Y77.

6082

dx.doi.org/10.1021/jm2006063 |J. Med. Chem. 2011, 54, 6063–6084

Journal of Medicinal Chemistry

ARTICLE

R.; Roberts, C. W. A complete shikimate pathway in Toxoplasma gondii:

an ancient eukaryotic innovation. Int. J. Parasit. 2004, 34, 5–13.

(10) Abell, C. Enzymology and Molecular Biology of the Shikimate

Pathway. In Comprehensive Natural Products Chemistry; Sankawa, U.,

Ed.; Pergamon, Elsevier Science Ltd.: Oxford, 1999; p 573.

(11) (a) Parish, T.; Stoker, N. G. The common aromatic amino acid

biosynthesis pathway is essential in Mycobacterium tuberculosis. Micro-

biology 2002, 148, 3069–3077.(b) Data base for essential genes in

bacteria, see www.essentialgene.org. (c) Zhang, R.; Lin, Y. DEG 5.0, a

database of essential genes in both prokaryotes and eukaryotes. Nucleic

Acids Res. 2009, 37, D455–D458. (d) Zhang, R.; Ou, H.-Y.; Zhang, C.-T.

DEG, a database of essential genes. Nucleic Acids Res. 2004,

32, D271–D272. (e) Sassetti, C. M.; Boyd, D. H.; Rubin, E. J. Genes

required for mycobacterial growth deﬁned by high density mutagenesis.

Mol. Microbiol. 2003, 48, 77–84.

reaction catalyzed by type II dehydroquinases. Org. Biomol. Chem. 2004,

2, 1592–1596.

(22) Sꢀanchez-Sixto, C.; Prazeres, V. F. V.; Castedo, L.; Lamb, H.;

Hawkins, A. R.; Gonzꢀalez-Bello, C. Structure-based design, synthesis

and biological evaluation of inhibitors of Mycobacterium tuberculosis type

II dehydroquinase. J. Med. Chem. 2005, 48, 4871–4881.

(23) Prazeres, V. F. V.; Sꢀanchez-Sixto, C.; Castedo, L.; Lamb, H.;

Hawkins, A. R.; Riboldi-Tunnicliﬀe, A.; Coggins, J. R.; Lapthorn, A. J.;

Gonzꢀalez-Bello, C. Nanomolar mechanism-based inhibitors of Myco-

bacterium tuberculosis and Streptomyces coelicolor type II dehydroquinase.

ChemMedChem 2007, 2, 194–207.

(24) Payne, R. J.; Riboldi-Tunnicliﬀe, A. L.; Kerbarh, V. V. V.; Abell,

A. D.; Lapthorn, A. J.; Abell, C. Design, synthesis, and structural studies

on potent biaryl inhibitors of type II dehydroquinases. ChemMedChem

2007, 2, 1010–1013.

(12) (a) Shneier, A.; Harris, J.; Kleanthous, C.; Coggins, J. R.;

Hawkins, A. R.; Abell, C. Evidence for opposite stereochemical courses

for the reactions catalyzed by type I and type II dehydroquinases. Bioorg.

Med. Chem. Lett. 1993, 3, 1399–1402. (b) Harris, J.; Kleanthous, C.;

Coggins, J. R.; Hawkins, A. R.; Abell, C. Diﬀerent mechanistic and

stereochemical courses for the reactions catalysed by type I and type II

dehydroquinases. J. Chem. Soc., Chem. Commun. 1993, 13, 1080–1081.

(c) Gourley, D. G.; Shrive, A. K.; Polikarpov, I.; Krell, T.; Coggins, J. R.;

Hawkins, A. R.; Issacs, N. W.; Sawyer, L. The two types of 3-dehydro-

quinase have distint structures but catalyze the same overall reaction.

Nature Struct. Biol. 1999, 6, 521–525.

(25) Toscano, M. D.; Payne, R. J.; Chiba, A.; Kerbarh, O.; Abell, C.

Nanomolar inhibition of type II dehydroquinase based on the enolate

reaction mechanism. ChemMedChem 2007, 2, 101–112.

(26) Payne, R. J.; Peyrot, F.; Kerbarh, O.; Abell, A. D.; Abell, C.

Rational design, synthesis, and evaluation of nanomolar type II dehy-

droquinase inhibitors. ChemMedChem 2007, 2, 1015–1029.

(27) Prazeres, V. F. V.; Sꢀanchez-Sixto, C.; Castedo, L.; Shuh, S. W.;

Lamb, H.; Hawkins, A. R.; Ca~nada, F. J.; Jimꢀenez-Barbero, J.; Gonzꢀalez-

Bello, C. Competitive inhibitors of Helicobacter pylori type II dehydro-

quinase: synthesis, biological evaluation, and NMR studies. ChemMed-

Chem 2008, 3, 756–770.

(13) (a) Chauduri, C.; Ducan, K.; Graham, L. D.; Coggins, J. R.

Identiﬁcation of the active-site lysine residues of two biosynthetic

3-dehydroquinases. Biochem. J. 1990, 275, 1–6. (b) Shneier, A.;

Kleanthous, C.; Deka, R.; Coggins, J. R.; Abell, C. Observation of an

imine intermediate on dehydroquinase by electrospray mass spectro-

metry. J. Am. Chem. Soc. 1991, 113, 9416–9418.

(28) Prazeres, V. F. V.; Tizꢀon, L.; Otero, J. M.; Guardado-Calvo, P.;

Llamas-Saíz, A. L.; van Raaij, M. J.; Castedo, L.; Lamb, H.; Hawkins,

A. R.; Gonzꢀalez-Bello, C. Synthesis and biological evaluation of new

nanomolar competitive inhibitors of Helicobacter pylori type II dehy-

droquinase. Structural details of the role of the aromatic moieties with

essential residues. J. Med. Chem. 2010, 53, 191–200.

(14) (a) Harris, J.; Kleanthous, C.; Coggins, J. R.; Hawkins, J. R.;

Abell, C. Diﬀerent mechanistic and stereochemical courses for the

reactions catalysed by type I and type II dehydroquinases. J. Chem.

Soc., Chem. Commun. 1993, 1080–1081. (b) Shneier, A.; Harris, J.;

Kleanthous, C.; Coggins, J. R.; Hawkins, A. R.; Abell, C. Evidence for

opposite stereochemical courses for the reaction catalysed by type I and

type II dehydroquinases. Bioorg. Med. Chem. Lett. 1993, 3, 1399–1402.

(15) (a) Krell, T.; Pitt, A. R.; Coggins, J. R. The use of electrospray

mass spectrometry to identify an essential arginine residue in type II

dehydroquinases. FEBS Lett. 1995, 360, 93–96. (b) Krell, T.; Horsburgh,

M. J.; Cooper, A.; Kelly, S. M.; Coggins, J. R. Localization of the active

site of type II dehydroquinase. Identiﬁcation of a common arginine-

containing motif in the two classes of dehydroquinases. J. Biol. Chem.

1996, 271, 24492–24497.

(29) Paz, S.; Tizꢀon, L.; Otero, J. M.; Llamas-Saiz, A. L.; Fox, G. C.;

van Raaij, H. J.; Lamb, H.; Hawkins, A. R.; Lapthorn, A. J.; Castedo, L.;

Gonzꢀalez-Bello, C. Tetrahydrobenzothiophene derivatives: conforma-

tionally restricted inhibitors of type II dehydroquinase. ChemMedChem

2011, 6, 266–272.

(30) Tran, A. T.; Cergol, K. M.; Britton, W. J.; Bokhari, S. A. I.;

Ibraim, M.; Lapthorn, A. J.; Payne, R. J. Rapid assembly of potent type II

dehydroquinase inhibitors via click chemistry. Med. Chem. Commun.

2010, 1, 271–275.

(31) Prazeres, V. F. V.; Sꢀanchez-Sixto, C.; Castedo, L.; Canales, A.;

Ca~nada, F. J.; Jimꢀenez-Barbero, J.; Lamb, H.; Hawkins, A. R.; Gonzꢀalez-

Bello, C. Determination of the bound conformation of a competitive

nanomolar inhibitor of Mycobacterium tuberculosis type II dehydroqui-

nase by NMR spectroscopy. ChemMedChem 2006, 1, 990–996.

(32) Prazeres, V. F. V.; Castedo, L.; Lamb, H.; Hawkins, A. R.;

Gonzꢀalez-Bello, C. 2-Substituted-3-dehydroquinic acids as potent com-

petitive inhibitors of type II dehydroquinase. ChemMedChem 2009,

4, 1980–1984.

(16) Roszak, A. W.; Robinson, D. A.; Krell, T.; Hunter, I. S.;

Frederickson, M.; Abell, C.; Coggins, J. R.; Lapthorn, A. J. The structure

and mechanism of the type II dehydroquinase from Streptomyces

coelicolor. Structure 2002, 10, 493–503.

(17) Peꢀon, A.; Otero, J. M.; Tizꢀon, L.; Prazeres, V. F. V.; Llamas-Saiz,

A. L.; Fox, G. C.; van Raaij, M. J.; Lamb, H.; Hawkins, A. R.; Gago, F.;

Castedo, L.; Gonzꢀalez-Bello, C. Understanding the key factors that

control the inhibition of type II dehydroquinase by (2R)-2-benzyl-3-

dehydroquinic acids. ChemMedChem 2010, 5, 1726–1733.

(18) Gonzalez-Bello, C.; Castedo, L. Progress in type II dehydro-

quinase inhibitors: from concept to practice. Med. Res. Rev. 2007,

27, 177–208.

(19) Frederickson, M.; Parker, E. J.; Hawkins, A. R.; Coggins, J. R.;

Abell, C. Selective inhibition of type II dehydroquinases. J. Org. Chem.

1999, 64, 2612–2613.

(20) Gonzꢀalez-Bello, C.; Lence, E.; Toscano, M. D.; Castedo, L.;

Coggins, J. R.; Abell, C. Parallel solid-phase synthesis and evaluation of

inhibitors of Streptomyces coelicolor type II dehydroquinase. J. Med. Chem.

2003, 46, 5735–5744.

(33) The X-ray crystal structure is available from the Protein Data

Bank (PBD: 1H0S): Robinson, D. A.; Roszak, A. W.; Frederickson, M.;

Abell, C.; Coggins, J. R.; Lapthorn, A. J. Structural basis for speciﬁcity of

oxime based inhibitors towards type II dehydroquinase from M.

tuberculosis. (to be published) Residues 20À25 are not visible, including

the essential residue Tyr24.

(34) Palomino, J. C.; Martin, A.; Camacho, M.; Guerra, H.; Swings,

J.; Portaels, F. Resazurin microtiter assay plate: simple and inexpensive

method for detection of drug resistance in Mycobacterium tuberculosis.

Antimicrob. Agents Chemother. 2002, 46, 2720–2722.

(35) Charman, W. N.; Porter, C. J. H. Lipophilic prodrugs designed

for intestinal lymphatic transport. Adv. Drug Delivery Rev. 1996, 19,

149–169.

(36) Gourley, D. G.; Coggins, J. R.; Isaacs, N. W.; Moore, J. D.;

Charles, I. G.; Hawkins, A. R. Crystallization of a type II dehydroquinase

from Mycobacterium tuberculosis. J. Mol. Biol. 1994, 241, 488–491.

(37) Leslie, A. G. The integration of macromolecular diﬀraction

data. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 48–57.

(21) Frederickson, M.; Roszak, A. W.; Coggins, J. R.; Lapthorn, A. J.;

Abell, C. (1R,4S,5R)-3-Fluoro-1,4,5-trihydroxy-2-cyclohexene-1-car-

boxylic acid: the ﬂuoro analogue of the enolate intermediate in the

6083

dx.doi.org/10.1021/jm2006063 |J. Med. Chem. 2011, 54, 6063–6084

Journal of Medicinal Chemistry

ARTICLE

(38) Evans, P. Scaling and assessment of data quality. Acta Crystal-

logr., Sect. D: Biol. Crystallogr. 2006, 62, 72–82.

(39) Winn, M. D. An overview of the CCP4 project in protein

crystallography: an example of a collaborative project. J. Synchrotron

Radiat. 2003, 10, 23–25.

(40) Vagin, A.; Teplyakov, A. MOLREP: an automated program for

molecular replacement. J. Appl. Crystallogr. 1997, 30, 1022–1025.

(41) The X-ray crystal structure is available from the Protein Data

Bank (PBD: 1H0S): Robinson, D. A.; Roszak, A. W.; Frederickson, M.;

Abell, C.; Coggins, J. R.; Lapthorn, A. J. Structural basis for speciﬁcity of

oxime based inhibitors towards type II dehydroquinase from M.

tuberculosis. (to be published) Residues 20À25 are not visible, including

the essential residue Tyr24.

(42) Brunger, A. T. Free R value: cross-validation in crystallography.

Methods Enzymol. 1997, 277, 366–396.

(43) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular

graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126–2132.

(44) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Reﬁnement of

macromolecular structures by the maximum-likelihood method. Acta

Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240–255.

(45) Chen, V. B.; Arendall, W. B., III; Headd, J. J.; Keedy, D. A.;

Immormino, R. M.; Kapral, G. J.; Murray, L. W.; Richardson, J. S.;

Richardson, D. C. MolProbity: all-atom structure validation for macro-

molecular crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr.

2010, D66, 12–21.

(46) DeLano, W. L. The PyMOL Molecular Graphics System; DeLano

Scientiﬁc LLC: Palo Alto, CA, 2008; http://www.pymol.org.

(47) Davis, I. W.; Leaver-Fay, A.; Chen, V. B.; Block, J. N.; Kapral,

G. J.; Wang, X.; Murray, L. W.; Arendall, W. B., 3rd; Snoeyink, J.;

Richardson, J. S.; Ricchardson, D. C. MolProbity: all-atom contacts and

structure validation for proteins and nucleic acids. Nucleic Acids Res.

2007, 35, W375–W383.

6084

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A prodrug approach for improving antituberculosis activity of potent mycobacterium tuberculosis type II dehydroquinase inhibitors

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