Synthesis of 3-alkyl enol mimics inhibitors of type II dehydroquinase: Factors influencing their inhibition potency

View Online / Journal Homepage / Table of Contents for this issue

Organic &

Biomolecular

Chemistry

www.rsc.org/obc

Volume 10 | Number 18 | 14 May 2012 | Pages 3565–3768

ISSN 1477-0520

PAPER

Concepción González-Bello et al.

Synthesis of 3-alkyl enol mimics inhibitors of type II dehydroquinase:

factors inﬂuencing their inhibition potency

Dynamic Article Links

Organic &

View Online

Biomolecular

Chemistry

Cite this: Org. Biomol. Chem., 2012, 10, 3662

www.rsc.org/obc

PAPER

Synthesis of 3-alkyl enol mimics inhibitors of type II dehydroquinase:

factors inﬂuencing their inhibition potency†‡

Beatriz Blanco,^aAntía Sedes,^aAntonio Peón,^aHeather Lamb,^bAlastair R. Hawkins,^bLuis Castedo^cand

Concepción González-Bello*^a

Received 12th December 2011, Accepted 1st March 2012

DOI: 10.1039/c2ob07081b

Several 3-alkylaryl mimics of the enol intermediate in the reaction catalyzed by type II dehydroquinase

were synthesized to investigate the effect on the inhibition potency of replacing the oxygen atom in the

side chain by a carbon atom. The length and the rigidity of the spacer was also studied. The inhibitory

properties of the reported compounds against type II dehydroquinase from Mycobacterium tuberculosis

and Helicobacter pylori are also reported. The binding modes of these analogs in the active site of both

enzymes were studied by molecular docking using GOLD 5.0 and dynamic simulations studies.

Introduction

In recent years, we have been working on the development of

new antibiotics for the treatment of bacterial infections,¹by inhi-

bition of type II dehydroquinase (DHQ2), which catalyzes the

reversible dehydration of 3-dehydroquinic acid (1) to form 3-

dehydroshikimic acid (2) (Scheme 1).^2,3The reaction proceeds

through an enol intermediate 3, which is stabilized by a con-

served water molecule that interacts through hydrogen bonding

to Asn12, the carbonyl group of Pro11, and the main-chain

amide of Gly78. The ﬁnal step is the acid-catalyzed elimination

of the C-1 hydroxyl group – a reaction mediated by a histidine

residue, which acts as a proton donor.⁴

In particular, we have focused on the inhibition of two patho-

genic bacteria, Mycobacterium tuberculosis, the causative agent

of tuberculosis and Helicobacter pylori, the causative agent of

gastric and duodenal ulcers, which has also been classiﬁed as a

type I carcinogen. We recently showed that 3-methoxyaryl

derivatives 4a–c (Fig. 1), in which the aryl moiety is linked to

the cyclohexene core by a methoxy group, are potent competitive

Scheme 1 Enzymatic conversion of 3-dehydroquinic acid (1) to 3-

^aCentro Singular de Investigación en Química Biológica y Materiales

dehydroshikimic acid (2) catalyzed by DHQ2. The reaction proceeds via

Moleculares (CIQUS), Universidad de Santiago de Compostela, calle

an enol intermediate 3. Relevant residues are indicated (the numbering

corresponds to M. tuberculosis).

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

E-mail: concepcion.gonzalez.bello@usc.es; Fax: +34 881 815704;

Tel: +34 881 815726

^bInstitute of Cell and Molecular Biosciences, Medical School, University

of Newcastle upon Tyne, Catherine Cookson Building, Framlington

Place, Newcastle upon Tyne NE2 4HH, UK

inhibitors of DHQ2 from Helicobacter pylori (DHQ2-Hp) and

Mycobacterium tuberculosis (DHQ2-Mt).⁵

^cDepartamento de Química Orgánica, Facultad de Química,

Universidad de Santiago de Compostela, 15782 Santiago de

Compostela, Spain

The crystal structures of DHQ2-Hp and DHQ2-Mt in complex

with compound 4c have been solved at 2.95 Å and 1.5 Å,

respectively (Fig. 2).^5,6These crystal structures clariﬁed the role

of the aromatic rings on C3, which block the entrance of the

essential arginine side chain into the active site and cause an

important change in the conformation and ﬂexibility of the loop

†Electronic supplementary information (ESI) available: Plots for inhi-

bition data and extra data plots for Molecular dynamics simulations. See

DOI: 10.1039/c2ob07081b

‡This paper is dedicated to Prof. Miguel A. Miranda on the occasion of

his 60^thbirthday.

3662 | Org. Biomol. Chem., 2012, 10, 3662–3676

This journal is © The Royal Society of Chemistry 2012

View Online

Fig. 3 Target compounds.

Fig. 1 Selected examples of 3-methoxyaryl derivatives that are DHQ2

competitive inhibitors. Inhibition constants against DHQ2-Mt are

indicated.

Scheme 2 The synthesis of compounds 5. Reagents and conditions:

(a) HCCTMS, CuI, Pd(PPh₃)₂Cl₂, Et₃N, 40 °C; (b) TBAF, THF, RT; (c)

1. Catechol borane, THF, Δ; 2. Pinacol, THF, Δ; (d) Pd(PPh₃)₄, K₃PO₄

(aq.), dioxane, 80 °C; (e) 1. LiOH, THF, RT; 2. Amberlite IR-120 (H⁺).

abstraction and disrupts its basicity.⁷The crystal structure solved

at 1.5 Å shows that the oxygen atom of the methylenoxy spacer

of the inhibitor 4c is located 3.1 Å away from the conserved

water molecule involved in the catalysis (Fig. 2b). We assume

that an important contribution of the high potency of the inhibi-

tor, with K_ivalues of 42 nM^5band 130 nM^5aagainst DHQ2-Mt

and DHQ2-Hp, respectively, is due to the hydrogen-bonding

interaction between the oxygen atom of the methylenoxy spacer

with the conserved water molecule. In order to corroborate this

hypothesis, we decided to investigate the effect on the inhibition

potency of replacing the oxygen atom in the side chain of 4a–b

by a carbon atom. In addition, the length and the rigidity of the

alkylene spacer was also studied. To this end, 3-alkylaryl enol

mimics 5, 6 and 7, having a vinylene, ethylene and propylene

spacer, respectively, were designed (Fig. 3). The results of inhi-

bition studies of these compounds against DHQ2-Mt and

DHQ2-Hp, docking studies using GOLD 5.0 and dynamic simu-

lations studies are also described.

Fig. 2 Selected views of the crystal structures of the binary complex

of: (a) DHQ2-Hp/4c (PDB: 2WKS, 2.95 Å);^5a(b) DHQ2-Mt/4c (PDB:

2Y71, 1.5 Å).^5bRelevant residues are indicated.

Results and discussion

Synthesis of vinylene derivatives 5

that closes over the substrate binding site. Molecular dynamics

simulation studies suggest that the aromatic ring prevents appro-

priate orientation of the catalytic tyrosine of the loop for proton

The synthesis of the target compounds 5 was achieved by

Suzuki cross-coupling reactions between our previously reported

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 3662–3676 | 3663

View Online

Table 1 The synthesis of compounds 9–11, 13, 14 and 5^a

Table 2 Synthesis of compounds 15–18 and 6^a

Reaction

Comp

Yield (%)

Comp

Yield (%)

Reaction

Comp Yield (%) Comp Yield (%) Comp Yield (%)

8 → 9

9a

99

98

85

94

65

77

9b

98

87

94

87

43

79

13 → 15

13a → 15 15a

12 → 15

15 → 16

16 → 6

15a

75

78

80

79

85

—

15b

—

15b

16b

6b

56

—

80

60

83

96

15c

—

16c

6c

20

0

—

90

85

—

9 → 10

10 → 11

12 → 13

13 → 14

14 → 5

10a

11a

13a

14a

5a

10b

11b

13b

14b

5b

15a

16a

6a

8b → 17b

—

17b

—

^aa Ar = naphth-2-yl; b Ar = benzo[b]thiophen-2-yl.

^aa Ar = naphth-2-yl; b Ar = benzo[b]thiophen-2-yl; c Ar = 5,6,7,8-

tetrahydronaphth-2-yl.

and 15b in 75% and 56% yield, respectively. Surprisingly, the

reduction of naphthyl derivative 13a also afforded a 20% yield

of compound 15c resulting from a partial reduction of the

naphthyl moiety. However, this side reduction was avoided by

using RANEY-Ni® as catalyst to afford compound 15a as a

single product in 78% yield. The tetrahydronaphthyl derivative

15c was also transformed into its corresponding acid 6c to test

its biological activity.

The selective reduction of dienes 15 proved to be experimen-

tally problematic due to the difﬁculty in controlling and monitor-

ing the reduction. Because of that, we were particularly

interested in addressing the synthesis of the alkyl lactones 15 by

a

direct sp³–sp²cross-coupling reaction. After numerous

attempts using various sp³boronic acids or their corresponding

boronic acids pinacol esters, the cross-coupling was achieved by

using alkyl boranes 18 and PdCl₂(dppf) as catalyst in the pres-

ence of K₃PO₄in THF.⁸Alkyl boranes 18 were synthesized by

hydroboration with 9-BBN-H of vinyl derivatives 17. Non-com-

mercially available vinyl derivative 17b was prepared by Suzuki

cross-coupling of halide 8b and vinyl boronic acid pinacol ester.

Finally, compounds 15 were converted to the desired acids 6 in

the same way as acids 5 from lactones 13.

Scheme 3 Synthesis of acids 6. Reagents and conditions: (a) H₂,

Rosemund’s catalyst, 50% THF–MeOH, RT; (b) H₂, RANEY-Ni®, 50%

THF–MeOH, RT; (c) PdCl₂(dppf), K₃PO₄, THF, Δ; (d) TBAF, THF, RT;

(e) 1. LiOH, THF, RT; 2. Amberlite IR-120 (H⁺); (f) vinyl boronic acid

pinacol ester, Pd(PPh₃)₄, K₃PO₄(aq.), dioxane, 80 °C; (g) 9-BBN-H,

THF, 0 °C to RT.

Synthesis of propylene derivatives 7

Our initial attempts to synthesize compounds 7 involved as the

key step the Sonogashira cross-coupling between the triﬂate 12^2c

and the terminal alkynes 20, followed by selective reduction of

the resulting enynes (Scheme 4 and Table 3). The required

alkynes 20 were prepared by treatment of the Grignard derivative

of 8 with (3-bromoprop-2-ynyl)trimethylsilane followed by

deprotection. The latter reaction was achieved by treatment with

AgNO₃in ethanol as the usual TBAF or MeOH–K₂CO₃con-

ditions afforded allenes 21 in good yield.

A Sonogashira cross-coupling reaction between terminal

alkynes 20 and triﬂate 12^2cin the presence of piperidine, a cata-

lytic amount of copper iodide and Pd(PPh₃)₄catalyst provided

an excellent yield of the cross-coupling products 24. The selec-

tive reduction of enynes 24 by catalytic hydrogenation using

Rosemund’s catalyst gave saturated side chain derivatives 25 in

excellent yield. Alternatively, alkyl compounds 25 were syn-

thesized by B-alkyl Suzuki cross-coupling between triﬂate 12^2c

and alkyl boranes 23 using Pd(PPh₃)₄as catalyst and in the pres-

ence of K₃PO₄. Alkyl boranes 23 were prepared by reaction of

the Grignard derivative of 8 with allyl bromide followed by

hydroboration with 9-BBN-H of the corresponding allyl

vinyl triﬂate 12^2cand the appropriate boronic acid pinacol esters

11 (Scheme 2). Firstly, the Sonogashira cross-coupling reaction

of commercially available aryl bromides 8 with trimethylsilyla-

cetylene gave the protected alkynes 9, which, by deprotection

with TBAF, afforded terminal alkynes 10 (Scheme 2 and

Table 1). Finally, hydroboration of alkynes 10 with catechol

borane gave the required boronic acid pinacol esters 11 in good

yield. Suzuki cross-coupling between vinyl triﬂate 12^2cand

boronic acid pinacol esters 11 gave the corresponding cross-

coupling products 13, which were converted to the desired acids

5 by deprotection followed by basic hydrolysis of the corre-

sponding lactones 14 and protonation with an ion-exchange

resin.

Synthesis of ethylene derivatives 6

The synthesis of ethylene side-chain acids 6 was ﬁrst addressed

by selective reduction of the external double bond in dienes 13

(Scheme 3 and Table 2). Catalytic hydrogenation of 13 using

Rosemund’s catalyst gave the desired saturated derivatives 15a

3664 | Org. Biomol. Chem., 2012, 10, 3662–3676

This journal is © The Royal Society of Chemistry 2012

View Online

Table 4 K_i(nM) values for compounds 5–7 against DHQ2-Hp and

DHQ2-Mt

Entry

Comp

R

H. pylori^a

M. tuberculosis^b

1

2

3

4

5

6

7

8

9

5a

5b

6a

6b

6c

7a

7d

4a

4b

(E)CHvCH

(CH₂)₂

(CH₂)₃

OCH₂

1400 98

3110 249

790 29

2460 197

1150 115

243 19

780 94

520 31

436 13

254 20

274 16

180

73

9

4

295 10

310 46^5b

132 13^5a

35 2^5b

28 2^5b

^aAssay conditions: pH 7.0, 25 °C, 50 mM Tris·HCl. ^bAssay conditions:

pH 7.0, 25 °C, 50 mM Tris·HOAc.

The biological results show that, in general, the effects of

type, geometry and size of spacer were more pronounced in the

inhibition potency against the DHQ2-Hp enzyme and in all

cases the propylene spacer was the most potent of the series for

both enzymes. In general, compounds 6 and 7, having a ﬂexible

spacer, proved to be more potent than compounds 5 with a more

rigid one (Table 4, entry 6 vs. 1). Benzothiophene 7d, having a

propylene spacer, was the most potent compound in the series,

with K_ivalues of 73 nM and 295 nM against DHQ2-Mt and

DHQ2-Hp, respectively. Naphthyl derivative 7a also showed a

high afﬁnity against both enzymes, with K_ivalues of 180 nM

and 243 nM against DHQ2-Mt and DHQ2-Hp, respectively. In

addition, tetrahydronaphthalene 6c proved to have binding

afﬁnities against DHQ2 in the same range as the other unsatu-

rated analogs 6a–b (Table 4, entry 5 vs. 3). In order to get an

insight of the binding mode of these inhibitors, docking studies

using GOLD 5.0.1⁹were carried out, which are discussed below.

Scheme 4 Synthesis of compounds 7. Reagents and conditions: (a) (1)

Mg, I₂(cat), THF, Δ. (2) TMSCuCCH₂Br; (b) K₂CO₃, MeOH, 0 °C to

RT; (c) AgNO₃, EtOH (aq.), RT; (d) (1) Mg, I₂(cat), THF, Δ. (2)

AllylBr; (e) 9-BBN-H, THF, 0 °C to RT; (f) Pd(PPh₃)₄, piperidine, CuI,

THF, 40 °C; (g) H₂, Rosemund’s catalyst, 50% THF–MeOH, RT; (h)

PdCl₂(dppf), K₃PO₄, THF, Δ; (i) TBAF, THF, RT; ( j) 1. LiOH, THF,

RT; 2. Amberlite IR-120 (H⁺).

Docking studies

The binding modes of inhibitors 5–7 with DHQ2 enzymes were

studied using GOLD 5.0.1⁹with the enzyme geometries found

in crystals of DHQ2-Hp and DHQ2-Mt binding to 3-methox-

yaryl derivative 4c (PDB code: 2WKS^5aand 2Y71,^5b

respectively).

Table 3 Synthesis of compounds 19–26 and 7^a

Reaction

Comp

Yield (%)

Comp

Yield (%)

8 → 19

19a

21a

20a

22a

24a

25a

26a

7a

54

91

72

99

98

70

77

94

19d

—

69

—

61

89

95

98

42

67

87

19a → 21a

19 → 20

8 → 22

In general, 3-alkylaryl enol mimics with a three-carbon-atom

spacer, as in ligands 7, ﬁt more efﬁciently into the active site

than the corresponding ethylene ones (ligands 6) because they

locate the aromatic ring closer to the aliphatic residues of the

enzyme active site (leucine pocket). This fact may account for

the higher inhibition potency of propylene derivatives 7 relative

to inhibitors 5 and 6. The GOLD-predicted binding mode of one

of the most active ligands of the 3-alkylaryl series, compound

7d, in the active site of both enzymes is shown in Fig. 4. These

docking studies show that this inhibitor should have similar

polar interactions, through hydroxyl and carboxylate groups (not

shown), to other mimetics of the enol intermediate, such as the

ones present in the previously reported crystal structures (PDB

code: 2WKS^5aand 2Y71^5b), because the cyclohexene ring

occupies approximately the same position in the active site.

More importantly, in both cases, the benzothiophene ring and

the spacer are involved in a set of strong lipophilic interactions

in this part of the active site. The benzothiophene moiety

20d

22d

24d

25d

26d

7d

12 → 24

24 → 25

12 → 25

25 → 26

26 → 7

^aa Ar = naphth-2-yl; d Ar = benzo[b]thiophen-3-yl.

derivative 22. Finally, compounds 25 were converted to the

desired acids 7 in the same way as acids 5 from lactones 13.

Inhibition assay results

The inhibitory properties of compounds 5–7 against DHQ2-Hp

and DHQ2-Mt were tested. These compounds proved to be

reversible competitive inhibitors of both enzymes. The inhibition

data (K_i) are summarised in Table 4.

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 3662–3676 | 3665

View Online

Fig. 5 Comparison of the position of inhibitor 4c (green) in the

enzyme–inhibitor crystal structure of DHQ2-Hp (PDB code: 2WKS^5a

)

with the docking results of the highest score solution of ligands: 5a

(pale orange) and 6a (blue). Relevant residues are indicated. The hydro-

gen bonding interactions of hydroxyl groups on C-1 and C-5 with His82

and His102 are highlighted as dotted lines with the same color as the

corresponding ligand. Note how these contacts are much weaker for

ligand 5a than for compounds 6a and 4c.

chain ﬂexibility allows it to accommodate more adequately the

aromatic ring in the active site, thus maximizing interactions

(Fig. 5). In fact, the GOLD-predicted binding mode of ligand 5a

shows that the cyclohexene moiety is moved away from the

polar contacts of the active site that anchors the six-membered

ring of the substrate and the enol intermediate in the active site,

i.e. His82, His101, etc. (Fig. 5). Even assuming that in a

dynamic process the loop conformation and/or side chain resi-

dues might change, the ethylene spacer seems more suitable to

maintain the polar interactions that anchor the cyclohexene

moiety of the inhibitor.

Fig. 4 GOLD-predicted binding for ligand 7d to the active site of: (a)

DHQ2-Hp (PDB: 2WKS^5a); (b) DHQ2-Mt (PDB: 2Y71^5b). Relevant

residues are indicated. Symmetry-related neighboring chain close to the

active site is indicated in gray.

Molecular dynamics simulations

interacts with the essential tyrosine by π stacking in DHQ2-Hp

(Tyr22, Fig. 4a) and by an edge-face π–π interaction in DHQ2-

Mt (Tyr24, Fig. 4b). This aromatic ring is also in close contact

with the side chain of Leu14 and the ﬁve-membered ring of

Pro19 in DHQ2-Hp and the side chain of Leu16, the carbon side

chain of Arg15 and the essential Arg19 in DHQ2-Mt. The latter

residues are located in the ﬂexible loop that closes over the sub-

strate binding site. The benzothiophene ring also interacts with

some residues of a symmetry-related neighboring molecule

(speciﬁcally, the side chains of Leu93, Met92 and Asp89 for

DHQ2-Hp and the side chains of Ala91, Glu92 and Asp88 for

DHQ2-Mt). The propylene moiety of 7d interacts with the side

chain of Leu11/Leu14, the carbon side chain of Asn10/Asn12

and carbon main chain of Gly78/Gly77 for DHQ2-Hp and

DHQ2-Mt, respectively.

On the other hand, the inhibition data clearly show that the repla-

cement of the oxygen atom of the methylenoxy spacer by a

carbon atom affords less potent inhibitors. This fact suggests that

the oxygen atom of the spacer in compounds 4 is involved in a

strong binding interaction with the essential water involved in

the enzymatic mechanism, as described below. As shown in the

recently solved crystal structure of the binary complex DHQ2-

Mt/4c, the oxygen atom of the spacer is located 3.1 Å away from

the essential water molecule (Fig. 2b). Therefore, this interaction

should be lost on replacing the ether linkage by a methylene

group. In order to corroborate this hypothesis and further

analyze the binding mode of these inhibitors in the active site of

the DHQ2, we studied the binding mode of O-alkylaryl deriva-

tive 4c and the corresponding C-alkylaryl derivative 6e (Ar =

5-methylbenzo[b]thiophen-3-yl) by molecular dynamics simu-

lations (MD). The results show that the position in the active site

of 3-methoxybenzothiophenyl derivatives 4c, which has a

methylenoxy spacer, does not change signiﬁcantly during the

Comparison of saturated ligands 6 and the unsaturated ones 5

reveals that the saturated ones are predicted to be far more active

than the corresponding unsaturated derivatives 5, because the

3666 | Org. Biomol. Chem., 2012, 10, 3662–3676

This journal is © The Royal Society of Chemistry 2012

View Online

Fig. 6 Binding mode of ligand 4c (cyan) and ligand 6e (green) in the active site of DHQ2-Mt obtained by MD simulations: (a and c) after minimiz-

ation and previously to simulation; (b and d) after 10 ns of MD. Distance between the oxygen atom of the spacer of ligand 4c (O6), the corresponding

carbon atom in ligand 6e (C8) and essential water molecule is indicated. Only relevant residues are indicated.

simulation (10 ns) – including its position relative to the catalytic

water (Fig. 6a–b).

methylenoxy spacer by an alkylene one might cause the loss of a

favorable polar interaction between the ligand and the catalytic

water and this in turn causes a loss of inhibition potency.

For 3-ethylbenzothiophenyl ligand 6e, which contains an

ethylene spacer, relevant changes were not found in the position

of the cyclohexene moiety and therefore its polar contacts

through hydroxyl and carboxylate groups with residues of the

active site [His80, Arg111, Ser102, Asn74, His100, Asp89

(neighboring unit)]. However, an important change in the pos-

ition and conformation of the side chain and the aromatic ring

was observed. Both moieties are shifted signiﬁcantly after the

simulation, which causes a change in the position of the loop

because the volume occupied by the ligand 6e is now greater.

As shown in Fig. 6, while the distance between the oxygen

atom of the methylenoxy spacer in ligand 4c does not change

signiﬁcantly after 10 ns of simulation (from 3.1 Å to 2.8 Å), the

corresponding distance for ligand 6e increases from 3.2 Å to

4.2 Å (see also ESI†). Therefore, the substitution of the

Conclusions

Several 3-alkylaryl mimics of the enol intermediate in the reac-

tion catalyzed by the third enzyme of the shikimic acid pathway,

i.e. type II dehydroquinase – an essential enzyme in M. tubercu-

losis and H. pylori, were synthesized and tested as inhibitors of

these enzymes. Vinylene derivatives 5 were synthesized by

Suzuki cross-coupling reactions between previously reported

triﬂate 12^2cand boronic acids pinacol esters 11 as the key

step. 2- and 3-Alkylaryl enol mimics 6 and 7 were synthesized

by B-alkyl Suzuki cross-coupling reactions using alkyl boranes

18 and 23, respectively. Ethylene 6 and propylene side-

chain acids 7 were also synthesized by selective catalytic

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 3662–3676 | 3667

View Online

hydrogenation using Rosemund’s catalyst or RANEY-Ni® of the

external double and triple bond in dienes 13 and enynes 24,

respectively, which were obtained by Suzuki and Sonogashira

cross-coupling reactions.

obtained was puriﬁed by ﬂash chromatography on silica gel,

eluting with hexanes to give silane 9a (534 mg, 99%) as a brown

oil. δ_H(250 MHz; CDCl₃): 8.19 (1 H, br s, ArH), 7.90–7.83 (3

H, m, 3 × ArH), 7.70 (1 H, dd, J = 7.5 and 1.5 Hz, ArH), 7.56

(2 H, dd, J = 6.3 and 3.2 Hz, 2 × ArH) and 0.52 (9 H, s, 3 ×

SiCH₃); δ_C(63 MHz; CDCl₃): 133.0 (2 × C), 132.1 (CH), 128.6

(CH), 128.0 (2 × CH), 127.8 (CH), 126.8 (CH), 126.6 (CH),

120.5 (C), 106.8 (C), 95.6 (C) and 0.2 (3 × SiCH₃); ν_max(ﬁlm)/

cm⁻¹2152 (CuC).

The reported compounds were synthesized to evaluate the

contribution to the high potency of inhibitors 4 of the hydrogen-

bonding interaction between the oxygen atom of the methyle-

noxy spacer and the essential water involved in the catalysis, as

well as the length and the rigidity of the alkylene spacer. The

biological results show that the replacement of the oxygen atom

of the methylenoxy spacer of previously reported inhibitors 4a^5a

and 4c^5bby a carbon atom leads to a decrease in the inhibition

potency of up to 20-fold. The inhibition data together with the

molecular dynamics simulation studies performed show that this

hydrogen-bonding interaction has an important contribution on

the inhibition potency of inhibitors 4 and it should therefore be

considered in future designs. In general, effects of geometry and

size of the alkyl spacer were more pronounced in the inhibition

potency against the DHQ2-Hp enzyme and in all cases com-

pounds 6 and 7 having a ﬂexible spacer proved to be more

potent than compounds 5 with a more rigid one. Docking studies

using the program GOLD 5.0.1 suggest that compounds with a

three-carbon spacer ﬁt more efﬁciently into the active site

because they locate the aromatic ring closer to the aliphatic resi-

dues of the enzyme active site.

(Benzo[b]thiophen-2-ylethynyl)trimethylsilane (9b)

The experimental procedure used was the same as for alkyne 9a

utilizing 2-bromobenzo[b]thiophene (8b) (1 g, 4.69 mmol).

Yield = 1.05 g (98%). White solid. Mp: 57–58 °C; δ_H

(250 MHz; CDCl₃): 7.81–7.75 (2 H, m, 2 × ArH), 7.52 (1 H, br

s, ArH), 7.41–7.37 (2 H, m, 2 × ArH) and 0.37 (9 H, s, 3 ×

SiCH₃); δ_C(63 MHz; CDCl₃): 140.2 (C), 139.0 (C), 129.6

(CH), 125.6 (CH), 124.8 (CH), 124.0 (CH), 123.2 (C), 122.1

(CH), 101.1 (C), 98.0 (C), and 0.1 (3 × SiCH₃); ν_max(KBr)/

cm⁻¹2143 (CuC); MS (ESI) m/z 231 (MH⁺); HRMS (ESI)

calcd for C₁₃H₁₅SSi (MH⁺): 231.0658, found 231.0666.

2-Ethynylnaphthalene (10a)

Tetrabutylammonium ﬂuoride (2.9 mL, 2.87 mmol, ca. 1.0 M in

THF) was added to a stirred solution of the silyl ether 9a

(534 mg, 2.39 mmol) in dry THF (25 mL) under argon at room

temperature. After stirring for 1 h the solvent was removed under

reduced pressure and the residue was dissolved in ethyl acetate

and HCl (10%). The organic layer was separated and the

aqueous layer was extracted with ethyl acetate (×2). The com-

bined organic extracts were dried (anh. Na₂SO₄), ﬁltered and

concentrated under reduced pressure. The residue was puriﬁed

by ﬂash chromatography on silica gel, eluting with hexanes to

yield alkyne 10a (354 mg, 98%) as a colorless oil. δ_H

(250 MHz; CDCl₃): 8.19 (1 H, s, ArH), 7.92–7.85 (3 H, m, 3 ×

ArH), 7.71 (1 H, dd, J = 8.5 and 1.6 Hz, ArH), 7.60–7.57 (2 H,

m, ArH) and 3.36 (1 H, s, CH); δ_H(63 MHz; CDCl₃): 133.0

(C), 132.8 (C), 132.3 (CH), 128.5 (CH), 128.0 (CH), 127.7 (2 ×

CH), 126.9 (CH), 126.6 (CH), 119.4 (C), 84.1 (C) and 77.7

(CH); ν_max(KBr)/cm⁻¹2104 (CuC).

Experimental

General

All starting materials and reagents were commercially available

and were used without further puriﬁcation. ¹H NMR spectra

(250, 300, 400 and 500 MHz) and ¹³C NMR spectra (63, 75,

100 and 125 MHz) were measured in deuterated solvents. J

values are given in Hertz. NMR assignments were carried out by

a combination of 1 D, COSY, and DEPT-135 experiments. FT-IR

spectra were recorded as NaCl plates or KBr discs. [α]₂^D₀

=

values are given in 10⁻¹deg cm²g⁻¹. All procedures involving

the use of ion-exchange resins were carried out at room tempera-

ture using Milli-Q deionized water. Amberlite IR-120 (H⁺)

(cation exchanger) was washed alternately with water, 10%

NaOH, water, 10% HCl, and ﬁnally water before use. HPLC was

performed on a semipreparative column (Phenomenex Luna, 250

× 21.2 mm, C18), eluting with acetonitrile–water at a ﬂow rate

2-Ethynylbenzo[b]thiophene (10b)

of 7 mL min⁻¹

.

The experimental procedure used was the same as for 2-ethynyl-

naphthalene (10a) utilizing silyl ether 9b (1.05 g, 4.58 mmol).

Yield = 630 mg (87%). Red liquid. δ_H(250 MHz; CDCl₃):

7.84–7.78 (2 H, m, 2 × ArH), 7.58 (1 H, s, ArH), 7.45–7.41 (2

H, m, 2 × ArH) and 3.53 (1 H, s, CH); δ_C(63 MHz; CDCl₃):

140.1 (C), 138.7 (C), 130.1 (CH), 125.8 (CH), 124.8 (CH),

124.0 (CH), 122.0 (CH), 121.9 (C), 83.2 (C) and 77.3 (CH);

ν_max(ﬁlm)/cm⁻¹2100 (CuC).

Trimethyl(3-(naphthalen-2-yl)ethynyl)silane (9a)

A Schlenk tube was charged with 2-bromonaphthalene (8a)

(500 mg, 2.41 mmol), Pd(PPh₃)₂Cl₂(105 mg, 0.14 mmol), CuI

(25 mg, 0.14 mmol) and dry triethylamine (5 mL). The resulting

solution was deoxygenated and ethynyltrimethylsilane (0.5 mL,

3.62 mmol) was added dropwise. After addition of the ﬁrst drop,

the reaction color changed from yellow to black. The resulting

solution was heated at 40 °C for 5 h. After cooling to room

temperature, saturated ammonium chloride (0.5 mL) was added

and the reaction mixture was extracted with diethyl ether (×3).

The combined organic extracts were dried (anh. Na₂SO₄),

ﬁltered and concentrated under reduced pressure. The residue

(E)-4,4,5,5-Tetramethyl-2-(2-(naphth-2-yl)vinyl)-1,3,2-

dioxaborolane (11a)

A Schlenk tube was charged with 2-ethynylnaphthalene (10a)

(1.54 g, 10.14 mmol), catecholborane (1.28 mL, 11.15 mmol)

3668 | Org. Biomol. Chem., 2012, 10, 3662–3676

This journal is © The Royal Society of Chemistry 2012

View Online

and dry THF (2 mL). The resultant solution was heated under

reﬂux for 12 h. After cooling to room temperature, a solution of

pinacol (3.85 g, 32.57 mmol) in dry THF (20 mL) was added.

The reaction mixture was heated under reﬂux for 19 h. After

cooling to room temperature, the solvent was removed under

reduced pressure. The residue was puriﬁed by ﬂash chromato-

graphy on silica gel, preneutralized with (1 : 2 : 97) triethyl-

amine–diethyl ether–hexanes, using (3 : 97) diethyl ether–

hexanes as eluent, to give boronic acid pinacol ester 11a (2.43 g,

85%) as a yellow oil. δ_H(250 MHz; CDCl₃): 7.83 (4 H, m, 4 ×

ArH), 7.61 (1 H, d, J = 18.5 Hz, CHvCHB), 7.48 (3 H, m, 3 ×

ArH), 6.33 (1 H, d, J = 18.5 Hz, CHvCHB), 1.36 (9 H, s, 3 ×

CH₃) and 1.24 (3 H, s, CH₃); δ_C(63 MHz; CDCl₃): 149.5 (CH),

134.9 (C), 133.7 (C), 133.4 (C), 128.4 (CH), 128.2 (CH), 128.0

(CH), 127.6 (2 × CH), 126.4 (CH), 126.2 (CH), 124.9 (CH),

123.3 (CH), 83.3 (2 × C) and 24.8 (4 × CH₃).

25.7 (2 × C(CH₃)₃), 18.1 (2 × C(CH₃)₃), −2.9 (2 × CH₃), −3.9

(CH₃) and −4.1 (CH₃); ν_max(ﬁlm)/cm⁻¹1799 (CO) cm⁻¹; MS

(ESI) m/z = 559 (MNa⁺); HRMS (ESI) calcd for C₃₁H₄₄O₄SiNa

(MNa⁺): 559.2670, found 559.2670.

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

butyl-dimethylsilyloxy)cyclohex-2-en-1,5-carbolactone (13b)

The experimental procedure used was the same as for compound

13a utilizing triﬂate 12^2c(355 mg, 0.67 mmol) and dioxaboro-

lane 11b (382 mg, 1.34 mmol). White foam. Yield = 315 mg

(87%). [α]^D₂₀= −94.4° (c 1.0 in CHCl₃); δ_H(250 MHz; CDCl₃):

7.86–7.76 (2 H, m, 2 × ArH), 7.43–7.36 (2 H, m, 2 × ArH),

7.26 (1 H, s, ArH), 7.03 (1 H, d, J = 16.0 Hz, ArCHvCH), 6.54

(1 H, d, J = 16.0 Hz, ArCHvCH), 6.26 (1 H, s, H-2), 4.73 (1

H, dd, J = 3.3 and 5.2 Hz, H-5), 4.58 (1 H, d, J = 3.3 Hz, H-4),

2.60 (1 H, d, J = 10.6 Hz, H-6_ax), 2.55–2.42 (1 H, m, H-6_eq),

1.08 (9 H, s, C(CH₃)₃), 1.03 (9 H, s, C(CH₃)₃), 0.38 (3 H, s,

CH₃), 0.36 (3 H, s, CH₃), 0.33 (3 H, s, CH₃) and 0.31 (3 H, s,

CH₃); δ_C(63 MHz; CDCl₃): 175.2 (C), 142.2 (C), 140.1 (C),

139.1 (C), 136.0 (C), 134.5 (CH), 128.4 (CH), 125.0 (CH),

124.6 (C), 124.4 (CH), 123.8 (CH), 123.6 (CH), 122.3 (CH),

75.8 (CH), 75.2 (C), 66.3 (CH), 37.1 (CH₂), 25.7 (2 × C(CH₃)₃),

18.1 (2 × C(CH₃)₃), −2.9 (2 × CH₃), −3.9 (CH₃) and −4.3

(CH₃); ν_max(ﬁlm)/cm⁻¹1799 (CO); MS (ESI) m/z = 543

(MH⁺); HRMS (ESI) calcd for C₂₉H₄₃O₄SSi₂(MH⁺): 543.2415,

found 543.2404.

(E)-2-(2-(Benzo[b]thiophen-2-yl)vinyl)-4,4,5,5-tetramethyl-1,3,2-

dioxaborolane (11b)

The experimental procedure used was the same as for dioxaboro-

lane 11a utilizing alkyne 10b (600 mg, 3.79 mmol). Yield:

1.03 g (94%). Yellow oil. δ_H(250 MHz; CDCl₃): 7.84–7.76 (2

H, m, 2 × ArH), 7.69 (1 H, d, J = 18.0 Hz, CHvCHB),

7.35–7.32 (2 H, m, 2 × ArH), 7.29 (1 H, s, ArH), 6.16 (1 H, d, J

= 18.0 Hz, CHvCHB) and 1.38 (12 H, s, 4 × CH₃); δ_C

(63 MHz; CDCl₃): 143.8 (C), 142.3 (CH), 139.8 (C), 139.5 (C),

125.2 (CH), 125.0 (CH), 124.4 (CH), 123.9 (2 × CH), 122.2

(CH), 83.3 (2 × C) and 24.7 (4 × CH₃).

(1R,4R,5R)-1,4-Dihydroxy-3-((E)-2-(naphth-2-yl)vinyl)cyclohex-

2-en-1,5-carbolactone (14a)

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

2-yl)vinyl)cyclohex-2-en-1,5-carbolactone (13a)

The experimental procedure used was the same as for alkyne 9a

utilizing silyl ether 13a (290 mg, 0.54 mmol). Puriﬁcation by

ﬂash chromatography over silica gel, eluting with (1 : 1) ethyl

acetate–hexanes gave diol 14a (107 mg, 65%) as a colorless oil.

[α]^D₂₀= −95.7° (c 1.0 in MeOH); δ_H(250 MHz; CD₃OD):

7.80–7.75 (4 H, m, 4 × ArH), 7.64 (1 H, dd, J = 8.7 and 1.3 Hz,

ArH), 7.44–7.39 (2 H, m, 2 × ArH), 7.11 (1 H, d, J = 16.4 Hz,

ArCHvCH), 6.82 (1 H, d, J = 16.4 Hz, ArCHvCH), 6.16

(1 H, s, H-2), 4.73 (1 H, m, H-5), 4.54 (1 H, d, J = 3.2 Hz, H-4)

and 2.49–2.41 (2 H, m, CH₂); δ_C(63 MHz; CD₃OD): 178.4 (C),

138.4 (C), 135.8 (C), 135.1 (C), 134.8 (CH), 134.6 (C), 132.2

(CH), 129.3 (CH), 129.0 (CH), 128.6 (CH), 128.3 (CH), 127.8

(CH), 127.4 (CH), 127.1 (CH), 124.3 (CH), 78.0 (CH), 74.5 (C),

65.8 (CH₂) and 37.6 (CH₂); ν_max(ﬁlm)/cm⁻¹3381 (OH) and

1772 (CO); MS (ESI) m/z = 331 (MNa⁺); HRMS (ESI) calcd for

C₁₉H₁₆O₄Na (MNa⁺): 331.0941, found 331.0934.

A Schlenk tube was charged with triﬂate 12^2c(57 mg,

0.11 mmol), Pd(PPh₃)₄(4.1 mg, 0.035 mmol) and dry dioxane

(1 mL). K₃PO₄(0.18 mL, 0.18 mmol, 1 M) and dioxaborolane

11a (60 mg, 0.21 mmol) were added. The resultant solution was

deoxygenated and heated at 80 °C for 3.5 h under argon. After

cooling to room temperature, the solvent was removed under

reduced pressure. The residue was dissolved in a mixture of

dichloromethane and water. The organic layer was separated and

the aqueous phase was extracted with dichloromethane (×2). The

combined organic extracts were dried (Na₂SO₄), ﬁltered and con-

centrated under reduced pressure. The residue obtained was

puriﬁed by ﬂash chromatography on silica gel, eluting with a

gradient of dichloromethane–hexanes (15 : 85 to 25 : 75), to give

naphthyl derivative 13a (56 mg, 94%) as a white foam. [α]₂^D₀

=

−9.8° (c 1.0 in CHCl₃); δ_H(250 MHz; CDCl₃): 7.85–7.74 (4 H,

m, 4 × ArH), 7.50–7.41 (3 H, m, 3 × ArH), 6.89 (1 H, d, J =

16.3 Hz, ArCHvCH), 6.70 (1 H, d, J = 16.3 Hz, ArCHvCH),

6.18 (1 H, s, H-2), 4.66 (1 H, m, H-5), 4.58 (1 H, d, J = 3.2 Hz,

H-4), 2.55 (1 H, d, J = 10.6 Hz, H-6_ax), 2.41 (1 H, m, H-6_eq),

1.00 (9 H, s, C(CH₃)₃), 0.94 (9 H, s, C(CH₃)₃), 0.30 (3 H, s,

SiCH₃), 0.28 (3 H, s, SiCH₃), 0.26 (3 H, s, SiCH₃) and 0.24 (3

H, s, SiCH₃); δ_C(63 MHz; CDCl₃): 175.4 (C), 136.7 (C), 134.1

(C), 134.1 (CH), 133.7 (C), 133.2 (C), 130.6 (CH), 128.5 (CH),

128.1 (CH), 127.8 (CH), 126.8 (2 × CH), 126.5 (CH), 126.2

(CH), 123.2 (CH), 83.2 (C), 75.3 (CH), 66.2 (CH), 37.2 (CH₂),

(1R,4R,5R)-3-((E)-2-(Benzo[b]thiophen-2-yl)vinyl)-1,4-

dihydroxy-cyclohex-2-en-1,5-carbolactone (14b)

The experimental procedure used was the same as for diol 14a

utilizing silyl ether 13b (315 mg, 0.58 mmol). Yield = 79 mg

(43%). [α]^D₂₀= −82.4° (c 1.0 in MeOH); δ_H(250 MHz;

CD₃OD): 7.78–7.67 (2 H, m, 2 × ArH), 7.31–7.17 (3 H, m, 3 ×

ArH), 7.22 (1 H, d, J = 16.0 Hz, ArCHvCH), 6.56 (1 H, d, J =

16.0 Hz, ArCHvCH), 6.14 (1 H, s, H-2), 4.71 (1 H, m, H-5),

4.49 (1 H, d, J = 3.3 Hz, H-4) and 2.44–2.35 (2 H, m, CH₂); δ_C

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 3662–3676 | 3669

View Online

(63 MHz; CD₃OD): 178.2 (C), 143.7 (C), 141.6 (C), 140.4 (C),

138.0 (C), 135.6 (CH), 129.9 (CH), 126.1 (2 × CH), 125.7

(CH), 125.2 (CH), 124.6 (CH), 123.1 (CH), 78.0 (CH), 74.5 (C),

65.8 (CH) and 37.6 (CH₂); v_max(ﬁlm)/cm⁻¹3427 (OH) and

1780 (CO); MS (ESI) m/z = 337 (MNa⁺); HRMS (ESI) calcd for

C₁₇H₁₄O₄SNa (MNa⁺): 337.0505, found 337.0494.

atmosphere at room temperature for 12 h. The mixture was

ﬁltered over Celite and the residue was washed with methanol.

The ﬁltrate and washings were evaporated. The obtained residue

was puriﬁed by ﬂash chromatography on silica gel, eluting with

(1 : 2) dichloromethane–hexanes to yield naphthyl derivative 15a

(89 mg, 75%) and compound 15c (23 mg, 20%). Data for 15a:

White foam. [α]^D₂₀= −6.5° (c 1.0 in CHCl₃); δ_H(250 MHz;

CDCl₃): 7.86–7.80 (3 H, m, 3 × ArH), 7.62 (1 H, br s, ArH),

7.56–7.45 (2 H, m, 2 × ArH), 7.33 (1 H, dd, J = 8.4 and 1.6 Hz,

ArH), 5.81 (1 H, s, H-2), 4.53 (1 H, m, H-5), 4.11 (1H, d, J =

3.1 Hz, H-4), 3.00–2.75 (3 H, m, CH₂+ CHH), 2.52–2.35 (3 H,

m, CH₂+ CHH), 0.99 (9 H, s, C(CH₃)₃), 0.96 (9 H, s, C(CH₃)₃),

0.21 (3 H, s, CH₃), 0.18 (6 H, s, 2 × CH₃) and 0.15 (3 H, s,

CH₃); δ_C(63 MHz; CDCl₃): 176.2 (C), 138.6 (C), 138.4 (C),

133.7 (C), 132.1 (C), 131.4 (CH), 128.1 (CH), 127.6 (CH),

127.6 (CH), 127.0 (CH), 126.5 (CH), 126.0 (CH), 125.3 (CH),

76.0 (CH), 74.8 (C), 68.0 (CH), 37.3 (CH₂), 33.9 (CH₂), 33.5

(CH₂), 25.7 (2 × C(CH₃)₃), 18.1 (2 × C(CH₃)₃), −3.0 (2 × CH₃)

and −4.5 (2 × CH₃); ν_max(ﬁlm)/cm⁻¹1799 (CvO); MS (ESI)

m/z = 561 (MNa⁺); HRMS (ESI) calcd for C₃₁H₄₆O₄Si₂Na

(MNa⁺): 561.2829, found 561.2823. Data for 15c: Colorless oil.

[α]^D₂₀= −3.2° (c 1.0 in CHCl₃); δ_H(250 MHz; CDCl₃): 7.09

(1 H, s, ArH), 6.99 (1 H, d, J = 7.9 Hz, ArH), 6.86 (1 H, m,

ArH), 5.71 (1 H, s, H-2), 4.48 (1 H, m, H-5), 4.04 (1 H, d, J =

3.1 Hz, H-4), 2.74 (7 H, m, 3 × CH₂+ CHH), 2.33 (3 H, m,

CH₂+CHH), 1.79 (4 H, m, 2 × CH₂), 0.93 (9 H, s, C(CH₃)₃),

0.92 (9 H, s, C(CH₃)₃), 0.17 (3 H, s, CH₃), 0.16 (3 H, s, CH₃),

0.15 (3 H, s, CH₃) and 0.13 (3 H, s, CH₃); δ_C(63 MHz; CDCl₃):

176.2 (C), 138.7 (C), 138.1 (C), 137.2 (C), 134.9 (C), 131.3

(CH), 129.3 (CH), 129.2 (CH), 125.5 (CH), 76.1 (CH), 74.8 (C),

67.9 (CH), 37.4 (CH₂), 33.8 (CH₂), 33.5 (CH₂), 29.5 (CH₂),

29.2 (CH₂), 25.8 (C(CH₃)₃), 25.8 (C(CH₃)₃), 25.7 (3 × CH₃),

23.4 (CH₂), 23.4 (CH₂), 18.0 (2 × C(CH₃)₃), −2.9 (2 × CH₃),

−4.4 (CH₃) and −4.5 (CH₃); ν_max(ﬁlm)/cm⁻¹1799 (CO); MS

(ESI) m/z = 565 (MNa⁺); HRMS (ESI) calcd for C₃₁H₅₀O₄Si₂Na

(MNa⁺): 561.3140, found 561.3128.

(1R,4R,5R)-1,4,5-Trihydroxy-3-((E)-2-(naphth-2-yl)vinyl)

cyclohex-2-ene-1-carboxylic acid (5a)

A solution of the lactone 14a (30 mg, 0.10 mmol) in THF

(1 mL) and aqueous lithium hydroxide (0.5 mL, 0.25 mmol, 0.5

M) was stirred at room temperature for 10 min. Water was added

and THF was removed under reduced pressure. The resulting

aqueous solution was washed with diethyl ether (×2) and the

aqueous extract was treated with Amberlite IR-120 until pH 6.

The resin was ﬁltered off and washed with Milli-Q water. The

ﬁltrate and the washings were lyophilised to give acid 5a

(25 mg, 77%) as a yellow solid. Mp: 197–199 °C; [α]₂^D₀

=

−48.2° (c 1.0 in MeOH); δ_H(250 MHz; CD₃OD): 7.75–7.61 (5

H, m, 5 × ArH), 7.37 (2 H, m, 2 × ArH), 7.02 (1 H, d, J = 16.3

Hz, ArCHvCH), 6.89 (1 H, d, J = 16.3 Hz, ArCHvCH), 5.80

(1 H, s, H-2), 4.36 (1 H, d, J = 3.3 Hz, H-4), 3.96 (1 H, m, H-5)

and 2.17 (2 H, m, CH₂); δ_C(63 MHz; CD₃OD): 180.7 (C),

138.7 (C), 136.4 (C), 135.2 (C), 134.5 (C), 134.0 (CH), 130.9

(CH), 130.4 (CH), 129.2 (CH), 129.0 (CH), 128.6 (CH), 127.6

(CH), 127.3 (CH), 126.8 (CH), 124.5 (CH), 74.3 (C), 71.6 (CH),

68.8 (CH) and 35.9 (CH₂); ν_max(KBr)/cm⁻¹3410 (OH) and

1651 (CO); MS (ESI) m/z = 325 (M − H⁺); HRMS (ESI) calcd

for C₁₉H₁₇O₅(M − H⁺): 325.1071, found 325.1078.

(1R,4R,5R)-3-((E)-2-(Benzo[b]thiophen-2-yl)vinyl)-1,4,5-

trihydroxycyclohex-2-ene-1-carboxylic acid (5b)

The experimental procedure used was the same as for acid 5a

utilizing lactone 14b (22.8 mg, 0.07 mmol). Yield = 18.4 mg

(79%). Yellow solid. Mp: 184–185 °C; [α]^D₂₀= −41.6° (c 1.0 in

MeOH); δ_H(250 MHz; CD₃OD): 7.67 (2 H, m, 2 × ArH), 7.22

(4 H, m, 3 × ArH + ArCHvCH), 6.63 (1 H, d, J = 16.0 Hz,

ArCHvCH), 5.86 (1 H, s, H-2), 4.24 (1 H, d, J = 4.5 Hz, H-4),

3.99 (1 H, m, H-5) and 2.14 (2 H, m, CH₂); δ_C(63 MHz;

CD₃OD): 178.8 (C), 144.6 (C), 141.7 (CH), 140.3 (C), 140.2

(C), 131.5 (C), 131.1 (CH), 125.9 (CH), 125.5 (CH), 124.7

(CH), 124.5 (CH), 123.0 (CH), 123.1 (CH), 71.4 (C), 71.1 (CH),

37.9 (CH₂) and 30.7 (CH); v_max(KBr)/cm⁻¹3435 (OH), 1639

(CO); MS (ESI) m/z = 331 (M − H⁺); HRMS (ESI) calcd for

C₁₇H₁₅O₅S (M − H⁺): 331.0635, found 331.0634.

Reduction of 13a with RANEY-Ni®

A stirred solution of alkene 13a (932 mg, 1.74 mmol) in 50%

MeOH–THF (20 mL) was treated with an aqueous suspension of

RANEY-Ni® (approx. 0.24 equivalents). The resulting suspen-

sion was deoxygenated and was stirred under hydrogen atmos-

phere at room temperature for 2.5 h. The mixture was ﬁltered

over Celite and the residue was washed with methanol. The

ﬁltrate and washings were evaporated. The obtained residue was

puriﬁed by ﬂash chromatography on silica gel, eluting with

(5 : 95) acetone–hexanes to yield naphthyl derivative 15a

(731 mg, 78%).

Reduction of 13a with Rosemund’s catalyst: preparation of

(1R,4R,5R)-1,4-di(tert-butyldimethylsilyloxy)-3-(2-(naphthalen-

2-yl)ethyl)cyclohex-2-en-1,5-carbolactone (15a) and (1R,4R,5R)-

1,4-di(tert-butyldimethylsilyloxy)-3-(2-(5,6,7,8-

tetrahydronaphthalen-2-yl)ethyl)cyclohex-2-en-1,5-carbolactone

(15c)

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

dimethylsilyloxy)cyclohex-2-en-1,5-carbolactone (15b)

The experimental procedure used was the same as for compound

15a using Rosemund’s catalyst and utilizing alkene 13b

(115.8 mg, 0.21 mmol). Yield = 65.2 mg (56%). Colorless oil.

[α]^D₂₀= −88.3° (c 1.0 in CHCl₃); δ_H(250 MHz; CDCl₃):

7.77–7.65 (2 H, m, 2 × ArH), 7.34–7.22 (2 H, m, 2 × ArH),

A suspension of alkene 13a (115 mg, 0.22 mmol) and Rose-

mund’s catalyst (106 mg, 5% on weight) in a mixture of 50%

THF–methanol (10 mL) was stirred under

a hydrogen

3670 | Org. Biomol. Chem., 2012, 10, 3662–3676

This journal is © The Royal Society of Chemistry 2012

View Online

6.98 (1 H, s, ArH), 5.78 (1 H, s, 1H, H-2), 4.48 (1 H, m, H-5),

4.07 (1 H, d, J = 3.1 Hz, H-4), 3.07–3.00 (2 H, m, CH₂), 2.47 (2

H, m, CH₂), 2.33 (2 H, m, CH₂), 0.93 (9 H, s, C(CH₃)₃), 0.90 (9

H, s, C(CH₃)₃), 0.18 (3 H, s, SiCH₃), 0.15 (3 H, s, SiCH₃), 0.12

(3 H, s, SiCH₃) and 0.09 (3 H, s, SiCH₃); δ_C(63 MHz; CDCl₃):

176.0 (C), 144.9 (C), 137.7 (C), 131.7 (CH), 128.5 (C), 124.3

(CH), 123.7 (CH), 123.0 (CH), 122.3 (CH + C), 121.1 (CH),

76.1 (CH), 74.9 (C), 68.2 (CH), 37.3 (CH₂), 33.5 (CH₂), 28.9

(CH₂), 25.8 (2 × C(CH₃)₃), 18.1 (2 × C(CH₃)₃), −2.9 (2 × CH₃),

and −4.4 (2 × CH₃); ν_max(ﬁlm)/cm⁻¹1799 (CO); MS (ESI) m/z

= 567 (MNa⁺); HRMS (ESI) calcd for C₂₉H₄₄O₄SSi₂Na

(MNa⁺): 567.2391, found 567.2390.

CD₃OD): 179.0 (C), 140.7 (C), 139.4 (C), 137.8 (C), 135.4 (C),

131.1 (CH), 130.0 (2 × CH), 126.6 (CH), 77.9 (CH), 73.9 (C),

67.4 (CH), 37.4 (CH₂), 35.1 (CH₂), 34.3 (CH₂), 30.2 (CH₂),

30.0 (CH₂), 24.5 (CH₂) and 24.4 (CH₂); ν_max(ﬁlm)/cm⁻¹3409

(OH) and 1764 (CO); MS (ESI) m/z = 337 (MNa⁺); HRMS

(ESI) calcd for C₁₉H₂₂O₄Na (MNa⁺): 337.1410, found 337.1414.

(1R,4R,5R)-1,4-Dihydroxy-3-(2-(naphth-2-yl)ethyl)cyclohex-2-

ene-1-carboxylic acid (6a)

The experimental procedure used was the same as for acid 5a

utilizing lactone 16a (24.3 mg, 0.08 mmol). Yield = 22 mg

(85%). White solid. Mp: 120–122 °C; [α]^D₂₀= −8.3° (c 1.0 in

MeOH); δ_H(250 MHz; CD₃OD): 7.79–7.73 (3 H, m, 3 × ArH),

7.65 (1 H, br s, ArH), 7.43–7.35 (3 H, m, 3 × ArH), 5.52 (1 H,

s, H-2), 3.97–3.88 (2 H, m, H-4 + H-5), 3.06–3.98 (1 H, m,

CHH), 2.91–2.82 (1 H, m, CHH), 2.75–2.68 (1 H, m, CHH),

2.49–2.41 (1 H, m, CHH) and 2.07 (2 H, m, CH₂); δ_C(63 MHz;

CD₃OD): 178.3 (C), 145.0 (C), 140.8 (C), 135.1 (C), 133.5 (C),

128.8 (CH), 128.5 (2 × CH), 128.3 (CH), 127.4 (CH), 126.8

(CH), 126.1 (CH), 125.0 (CH), 74.9 (CH), 74.2 (C), 71.1 (CH),

40.3 (CH₂), 35.7 (CH₂) and 35.4 (CH₂); v_max(ﬁlm)/cm⁻¹3435

(OH) and 1720 (CO); MS (ESI) m/z = 327 (M − H⁺); HRMS

(ESI) calcd for C₁₉H₁₉O₅(M − H⁺): 327.1227, found 327.1243.

(1R,4R,5R)-1,4-Dihydroxy-3-(2-(naphth-2-yl)ethyl)cyclohex-2-

en-1,5-carbolactone (16a)

The experimental procedure used was the same as for diol 14a

using silyl ether 15a (89 mg, 0.16 mmol). Yield = 39 mg (79%).

White foam. [α]^D₂₀= −89.6° (c 1.0 in MeOH); δ_H(250 MHz;

CD₃OD): 7.76–7.69 (3 H, m, 3 × ArH), 7.53 (1 H, br s, ArH),

7.37 (2 H, m, 2 × ArH), 7.25 (1 H, dd, J = 8.4 and 1.6 Hz,

ArH), 5.71 (1 H, s, H-2), 4.59 (1 H, m, H-5), 4.05 (1 H, d, J =

2.9 Hz, H-4), 2.92–2.79 (2 H, m, CH₂), 2.55–2.42 (2 H, m,

CH₂) and 2.28 (2 H, m, CH₂); δ_C(63 MHz; CD₃OD): 179.0 (C),

140.6 (C), 140.1 (C), 135.1 (CH), 133.5 (C), 131.3 (CH), 128.9

(CH), 128.5 (2 × CH), 128.2 (CH), 127.5 (CH), 126.8 (CH),

126.2 (CH), 77.9 (CH), 73.9 (C), 67.6 (CH), 37.4 (CH₂) and

34.8 (2 × CH₂); ν_max(KBr)/cm⁻¹3448 (OH) and 1776, 1761

and 1726 (CO); MS (ESI) m/z = 333 (MNa⁺); HRMS (ESI)

calcd for C₁₉H₁₈O₄Na (MNa⁺): 333.1097, found 333.1226.

(1R,4R,5R)-3-(2-(Benzo[b]thiophen-2-yl)ethyl)-1,4-dihydroxy-

cyclohex-2-ene-1,5-carboxylic acid (6b)

The experimental procedure used was the same as for acid 5a

utilizing lactone 16b (40 mg, 0.13 mmol). Yield = 36 mg (83%).

White solid. Mp: 107–108 °C; [α]^D₂₀= −3.6° (c1.0 in MeOH);

δ_H(250 MHz; CD₃OD): 7.72 (1 H, d, J = 7.0 Hz, ArH), 7.63 (1

H, d, J = 7.0 Hz, ArH), 7.25–7.12 (3 H, m, 3 × ArH), 5.43 (1 H,

s, H-2), 3.87 (2 H, m, H-5 + H-4), 3.08 (1 H, m, CHH), 2.60 (1

H, m, CHH) and 2.09 (4 H, m, 2 × CH₂); δ_C(63 MHz;

CD₃OD): 186.1 (C), 146.7 (C), 141.7 (C), 140.7 (C), 140.0

(CH), 131.7 (C), 125.0 (CH), 124.5 (CH), 123.8 (CH), 122.9

(CH), 122.0 (CH), 78.0 (CH), 74.0 (C), 68.0 (CH), 40.6 (CH₂),

35.3 (CH₂) and 30.0 (CH₂); ν_max(KBr)/cm⁻¹3419 (OH) and

1778 (CO); MS (ESI) m/z = 333 (M − H⁺); HRMS (ESI) calcd

for C₁₇H₁₇O₅S (M − H⁺): 333.0791, found 333.0804.

(1R,4R,5R)-3-(2-(Benzo[b]thiophen-2-yl)ethyl)-1,4-dihydroxy-

cyclohex-2-en-1,5-carbolactone (16b)

The experimental procedure used was the same as for diol 14a

utilizing silyl ether 15b (65 mg, 0.12 mmol). Yield = 22 mg

(60%). White foam. [α]^D₂₀= −78.4° (c 1.0 in MeOH); δ_H

(250 MHz; CD₃OD): 7.72 (1 H, m, ArH), 7.64 (1 H, m, ArH),

7.11–7.28 (3 H, m, 3 × ArH), 5.76 (1 H, s, H-2), 4.60 (1 H, m,

H-5), 4.01 (1 H, d, J = 3.1 Hz, H-4), 3.10–3.02 (1 H, m, CHH),

2.62–2.52 (3 H, m, CH₂+ CHH) and 2.26 (2 H, m, CH₂); δ_C

(63 MHz; CD₃OD): 179.7 (C), 146.5 (C), 141.8 (C), 140.4 (C),

131.0 (CH), 129.8 (CH), 125.6 (CH), 125.0 (CH), 124.4 (CH),

123.4 (CH+C), 78.4 (CH), 74.4 (C), 68.0 (CH), 37.9 (CH₂),

35.0 (CH₂) and 29.9 (CH₂); ν_max(ﬁlm)/cm⁻¹3417 (OH), 1776

and 1770 (CO); MS (ESI) m/z = 339 (MNa⁺); HRMS (ESI)

calcd for C₁₇H₁₆O₄SNa (MNa⁺): 339.0662; found 339.0672.

(1R,4R,5R)-1,4,5-Trihydroxy-3-(2-(5,6,7,8-tetrahydronaphth-2-

yl)ethyl)cyclohex-2-ene-1-carboxylic acid (6c)

The experimental procedure used was the same as for acid 5a

utilizing lactone 16c (34.5 mg, 0.11 mmol). Yield = 31.2 mg

(85%). Mp: 127–128 °C; [α]^D₂₀= −1.2° (c 1.0 in MeOH); δ_H

(400 MHz; CD₃OD): 6.90 (2 H, m, 2 × ArH), 6.87 (1 H, m,

ArH), 5.45 (1 H, s, H-2), 3.89 (2 H, m, H-5 + H-4), 2.78–2.74

(1 H, m, CHH), 2.71 (4 H, m, 2 × CH₂), 2.63–2.57 (2 H, m,

CH₂), 2.29 (1H, m, CHH), 2.05 (2 H, m, CH₂), and 1.77 (4 H,

m, 2 × CH₂); δ_C(63 MHz; CD₃OD): 178.4 (C), 145.2 (C),

140.2 (C), 137.8 (C), 135.3 (C), 122.9 (2 × CH), 126.6 (CH),

124.7 (CH), 74.8 (CH), 74.3 (C), 71.0 (CH), 40.3 (CH₂), 36.0

(CH₂), 34.9 (CH₂), 30.4 (CH₂), 30.0 (CH₂), 24.6 (CH₂), and

24.5 (CH₂); v_max(KBr)/cm⁻¹3427 (OH), 1701 (CO); MS (ESI)

m/z = 331 (M − H⁺); HRMS (ESI) calcd for C₁₉H₂₃O₅(M −

H⁺): 331.1540, found 331.1543.

(1R,4R,5R)-1,4-Dihydroxy-3-(2-(5,6,7,8-tetrahydronaphth-2-yl)

ethyl)cyclohex-2-en-1,5-carbolactone (16c)

The experimental procedure used was the same as for diol 14a

utilizing ether 15c (118 mg, 0.23 mmol). Yield: 65 mg (90%).

White solid. Mp: 155.6–156.4 °C; [α]^D₂₀= −15.1° (c 1.0 in

MeOH); δ_H(250 MHz; CD₃OD): 7.00 (1 H, s, ArH), 6.90–6.76

(2 H, m, 2 × ArH), 5.67 (1 H, s, H-2), 4.59 (1 H, m, H-4), 4.03

(1 H, m, H-5), 2.68 (7 H, m, 3 × CH₂+ CHH), 2.29 (3 H, m, 2

× CH₂+ CHH) and 1.76–1.73 (4 H, m, 2 × CH₂); δ_C(63 MHz;

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 3662–3676 | 3671

View Online

2-Vinylbenzo[b]thiophene (17b)

(400 MHz; CDCl₃): 7.85–7.80 (4 H, m, 4 × ArH), 7.50–7.44 (3

H, m, 3 × ArH), 3.82 (2 H, s, CH₂) and 0.24 (9 H, s, 3 × CH₃);

δ_C(100 MHz; CDCl₃): 133.8 (C), 133.5 (C), 132.3 (C), 128.1

(CH), 127.6 (2 × CH), 126.4 (CH), 126.2 (CH), 126.1 (CH),

125.5 (CH), 104.2 (C), 87.2 (C), 26.4 (CH₂) and 0.11 (3 ×

CH₃); ν_max(KBr)/cm⁻¹2173 (CuC).

A Schlenk tube was charged with 2-bromobenzothiophene

(8b)¹⁰(250 mg, 1.17 mmol), vinylboronic acid pinacol ester

(0.3 mL, 1.73 mmol), Pd(PPh₃)₄(67 mg, 0.06 mmol), aqueous

K₂CO₃(3.45 mL, 1.1 M) and dioxane (10 mL). The resulting

solution was heated at 100 °C for 5 h. After cooling to room

temperature, the reaction mixture was diluted with diethyl ether

and water. The organic layer was separated and the aqueous

phase was extracted with diethyl ether (×3). The combined

organic extracts were dried (anh. Na₂SO₄), ﬁltered and concen-

trated under reduced pressure. The residue was puriﬁed by ﬂash

chromatography on silica gel, eluting with (3 : 97) diethyl ether–

hexanes to give 2-vinylbenzo[b]thiophene (17b)¹¹(180 mg,

96%).

(3-(Benzo[b]thiophen-3-yl)prop-1-ynyl)trimethylsilane (19d)

The experimental procedure used was the same as for alkyne

19a utilizing 3-bromobenzo[b]thiophene (8d) (1 g, 4.7 mmol)

and (3-bromoprop-1-ynyl)trimethylsilane (0.9 mL, 5.6 mmol).

Yield = 791 mg (69%). White solid. δ_H(250 MHz; CDCl₃):

7.90–7.86 (1 H, m, ArH), 7.76–7.74 (1 H, m, ArH), 7.45–7.37

(3 H, m, ArH), 3.81 (2 H, d, J = 1.25 Hz, CH₂) and 0.26 (9 H, s,

3 × CH₃); δ_C(63 MHz; CDCl₃): 140.6 (C), 137.9 (C), 130.6

(C), 124.3 (CH), 123.9 (CH), 123.0 (CH), 122.9 (CH), 121.3

(CH), 102.9 (C), 87.3 (C), 20.2 (CH₂) and 0.1 (3 × CH₃); ν_max

(KBr)/cm⁻¹2179 (CuC); MS (CI) m/z = 245 (MH⁺).

Preparation of 15a by B-alkyl Suzuki cross-coupling

(a) Preparation of borane 18a. A solution of 9-BBN-H

(5.7 mL, 2.85 mmol, ca. 0.5 M in THF) was added to a ﬂamed

round-bottom ﬂask under argon. After cooling to 0 °C, 2-vinyl-

naphthalene (17a) (200 mg, 1.29 mmol) was added. The mixture

was warmed up slowly to room temperature and stirred for 3 h to

give a solution of borane 18a.

2-(Propa-1,2-dienyl)naphthalene (21a)

A stirred solution of silyl ether 19a (30 mg, 0.13 mmol) in

methanol (1.5 mL) at 0 °C was treated with potassium carbonate

(17 mg, 0.13 mmol). The ice bath was removed and the resulting

mixture was stirred for 1 h. The reaction mixture was partitioned

in water and diethyl ether. The organic layer was separated and

the aqueous phase was extracted with diethyl ether (×2). The

combined organic extracts were dried (anh. Na₂SO₄), ﬁltered and

concentrated under reduced pressure. The residue was puriﬁed

by ﬂash chromatography on silica gel, eluting with (10 : 90)

diethyl ether–hexanes to give allene 21a (20 mg, 91%) as a

white solid. Mp: 55.7–56.3 °C; δ_H(250 MHz; CDCl₃):

7.43–7.36 (3 H, m, 3 × ArH), 7.26 (1 H, s, ArH), 7.13–7.02 (3

H, m, 3 × ArH), 5.96 (1 H, t, J = 6.25 Hz, CH) and 4.83 (2 H, d,

J = 5.0 Hz, CH₂); δ_C(75 MHz; CDCl₃): 210.5 (C), 133.8 (C),

132.7 (C), 131.5 (C), 128.4 (CH), 127.9 (CH), 127.8 (CH),

126.4 (CH), 125.8 (CH), 125.5 (CH), 124.8 (CH), 94.5 (CH)

and 79.2 (CH₂); MS (CI) m/z = 167 [MH⁺]; HRMS (CI) calcd

for C₁₃H₁₁(MH⁺): 167.0861, found 167.0860.

(b) B-alkyl Suzuki cross-coupling. To the borane solution

obtained above, a solution of triﬂate 12^2c(200 mg, 0.37 mmol)

in THF (4 mL), PdCl₂(dppf) (12.3 mg, 0.02 mmol) and aqueous

K₃PO₄(0.83 mL, 0.83 mmol, 1 M) were added. The resultant

solution was heated at 70 °C for 4 h under argon. After cooling

to room temperature, the solution was diluted with diethyl ether

and water. The organic layer was separated and the aqueous

phase was extracted with diethyl ether (×2). The combined

organic extracts were dried (Na₂SO₄), ﬁltered and concentrated

under reduced pressure. The residue was puriﬁed by ﬂash chrom-

atography on silica gel, eluting with (5 : 95) diethyl ether–

hexanes, to give compound 15a (156 mg, 80%).

Trimethyl(3-(naphth-2-yl)prop-1-ynyl)silane (19a)

A two necked round bottom ﬂask equipped with a condenser

and a pressure compensated addition funnel was charged with

magnesium turnings (141 mg, 5.82 mmol) and a few iodine

pellets. The system was ﬂamed under vacuum and cooled under

an argon atmosphere. Dry THF (3 mL) was added to the round

bottom ﬂask and the compensated addition funnel was charged

with a solution of 2-bromonaphthalene (8a) (1 g, 4.85 mmol) in

dry THF (5 mL). This solution was slowly added to the suspen-

sion, which was heated under reﬂux for 2 h. The reaction

mixture was cooled to room temperature and then it was treated

with a solution of 3-bromoprop-1-ynyl)trimethylsilane (0.9 mL,

7.2 mmol) in dry THF (3 mL). The reaction mixture was heated

under reﬂux for 2 h and then cooled to room temperature. Satu-

rated NH₄Cl was added and the organic layer was separated. The

aqueous phase was extracted with dichloromethane (×2). The

combined organic extracts were dried (anh. Na₂SO₄), ﬁltered and

concentrated under reduced pressure. Puriﬁcation by ﬂash

chromatography on silica gel, using hexanes as eluent, gave

alkyne 19a (621 mg, 54%) as a white solid. Mp: 61–63 °C; δ_H

2-(Prop-2-ynyl)naphthalene (20a)

A stirred solution of silyl silane 19a (600 mg, 2.5 mmol) in

ethanol (11 mL) was treated with a solution of AgNO₃in

(2.3 : 1) EtOH–H₂O (11 mL, 0.35 M). The resultant solution was

stirred in the dark at room temperature for 2 h during which time

a white solid was formed. An aqueous solution of potassium

cyanide (3.3 mL, 7.6 M) was then added and the reaction

mixture was stirred until disappearance of the white precipitate.

Diethyl ether was added and the aqueous layer was separated.

The organic extract was washed with brine, dried (anh. Na₂SO₄),

ﬁltered and concentrated under reduced pressure. The residue

was puriﬁed by ﬂash chromatography on silica gel, using

hexanes as eluent, to give alkyne 20a (297 mg, 72%) as a white

solid. Mp: 52–53 °C; δ_H(250 MHz; CDCl₃): 7.84–7.81 (4 H, m,

4 × ArH), 7.51–7.44 (3 H, m, 3 × ArH), 3.79 (2 H, d, J = 1.5

Hz, CH₂) and 2.27 (1 H, t, J = 1.8 Hz, CuCH); δ_C(63 MHz;

3672 | Org. Biomol. Chem., 2012, 10, 3662–3676

View Online

CDCl₃): 133.4 (2 × C), 132.3 (C), 128.2 (CH), 127.6 (2 × CH),

126.3 (CH), 126.2 (CH), 126.1 (CH), 125.6 (CH), 81.9 (C), 70.7

(CH) and 24.9 (CH₂); ν_max(KBr)/cm⁻¹3282 (CuC) cm⁻¹. MS

(CI) m/z = 167 (MH⁺); HRMS (CI) calcd for C₁₃H₁₁(MH⁺):

167.0861, found 167.0858.

extracts were washed with saturated solution of sodium bicarbon-

ate (×2), dried (anh. Na₂SO₄), ﬁltered and concentrated under

reduced pressure. The residue was puriﬁed by ﬂash chromato-

graphy on silica gel, eluting with a gradient of dichloromethane–

hexanes (5 : 95 to 35 : 65), to give naphthyl derivative 24a

(102 mg, 98%) as an orange foam. [α]^D₂₀= −136° (c 1.0 in

CHCl₃); δ_H(250 MHz; CDCl₃): 7.84–7.77 (4 H, m, 4 × ArH),

7.49–7.40 (3 H, m, 3 × ArH), 6.27 (1 H, s, H-2), 4.49 (1 H, m,

H-5), 4.17 (1 H, d, J = 3.3 Hz, H-4), 3.88 (2 H, s, CH₂Ar), 2.38

(2 H, m, CH₂-6), 0.92 (9 H, s, C(CH₃)₃), 0.89 (9 H, s, C(CH₃)₃),

0.20 (3 H, s, SiCH₃), 0.16 (3 H, s, SiCH₃) and 0.12 (6 H, s, 2 ×

SiCH₃); δ_C(63 MHz; CDCl₃): 175.0 (C), 140.7 (CH), 133.5

(C), 133.4 (C), 132.3 (C), 128.3 (CH), 127.6 (CH), 127.6 (CH),

126.4 (CH), 126.3 (CH), 126.2 (CH), 125.6 (CH), 127.8 (C),

89.5 (C), 80.4 (C), 75.8 (CH), 74.9 (C), 68.2 (CH), 36.8 (CH₂),

25.8 (CH₂), 25.6 (2 × C(CH₃)₃), 18.0 (2 × C(CH₃)₃), −3.1 (2 ×

SiCH₃), −4.6 (SiCH₃) and −4.9 (SiCH₃); v_max(KBr)/cm⁻¹2225

(CuC) and 1803 (CO) cm⁻¹; MS (ESI) m/z = 571 (MNa⁺);

HRMS (ESI) calcd for C₃₂H₄₄Si₂O₄Na (MNa⁺): 571.2670,

found 571.2664.

3-(Prop-2-ynyl)benzo[b]thiophene (20d)

The experimental procedure used was the same as for alkyne

20a utilizing silyl ether 19d (1.6 g, 6.5 mmol). Yield = 657 mg

(61%). Yellow oil. δ_H(250 MHz; CDCl₃): 8.00 (1 H, m, ArH),

7.91 (1 H, m, ArH), 7.79 (2 H, m, 2 × ArH), 7.58 (1 H, s, ArH),

3.79 (2 H, dd, J = 2.8 and 1.3 Hz, CH₂) and 2.29 (1 H, t, J = 2.8

Hz, CuCH); δ_C(63 MHz; CDCl₃): 140.1 (C), 138.5 (C), 131.3

(C), 124.6 (CH), 124.3 (CH), 123.2 (CH), 122.8 (CH), 121.2

(CH), 80.6 (CH), 70.6 (C) and 18.8 (CH₂); v_max(ﬁlm)/cm⁻¹

3293 (CuC); MS (CI) m/z = 173 (MH⁺); HRMS (CI) calcd for

C₁₁H₉S (MH⁺): 173.0425, found 173.0430.

2-Allylnaphthalene (22a)

The experimental procedure used was the same as for alkyne

19a utilizing 2-bromonaphthalene (8a) (200 mg, 0.96 mmol)

and allyl bromide (0.09 mL, 1 mmol). Yield = 158 mg (99%).

Colorless oil. δ_H(300 MHz; CDCl₃): 7.91–7.85 (3 H, m, 3 ×

ArH), 7.71 (1 H, s, ArH), 7.58–7.40 (3 H, m, 3 × ArH),

6.23–6.07 (1 H, m, CHvCH₂), 5.28–5.19 (2 H, m, CHvCH₂)

and 3.63 (2 H, d, J = 7.8 Hz, CH₂); δ_C(75 MHz; CDCl₃): 137.5

(C), 137.3 (CH), 133.6 (C), 132.1 (C), 127.9 (CH), 127.6 (CH),

127.4 (CH), 127.3 (CH), 126.6 (CH), 125.9 (CH), 125.2 (CH),

116.0 (CH₂) and 40.3 (CH₂); MS (CI) m/z = 169 (MH⁺); HRMS

(CI) calcd for C₁₃H₁₃(MH⁺): 169.1017, found 169.1023.

(1R,4R,5R)-3-(3-(Benzo[b]thiophen-3-yl)prop-1-ynyl)-1,4-di(tert-

butyldimethylsilyloxy)cyclohex-2-en-1,5-carbolactone (24d)

The experimental procedure used was the same as for naphthyl

derivative 24a using alkyne 20d (164 mg, 0.95 mmol) and

triﬂate 12^2c(100 mg, 0.19 mmol). Yield = 100 mg (95%).

Orange foam. [α]^D₂₀= −132° (c 1.0 in CHCl₃); δ_H(400 MHz;

CDCl₃): 7.87 (1 H, m, ArH), 7.75 (1 H, m, ArH), 7.43–7.37 (2

H, m, 2 × ArH), 7.35 (1 H, s, ArH), 6.26 (1 H, d, J = 1.6 Hz,

H-2), 4.48 (1 H, dd, J = 5.6 and 3.2 Hz, H-5), 4.14 (1 H, d, J =

3.2 Hz, H-4), 3.87 (2 H, s, CH₂Ar), 2.40 (1 H, d, J = 10.8 Hz,

H-6_ax), 2.36 (1 H, ddd, J = 10.8, 5.6 and 1.6 Hz, H-6_eq), 0.93 (9

H, s, C(CH₃)₃), 0.88 (9 H, s, C(CH₃)₃), 0.20 (3 H, s, SiCH₃),

0.16 (3 H, s, SiCH₃), 0.09 (3 H, s, SiCH₃) and 0.07 (3 H, s,

SiCH₃); δ_C(100 MHz; CDCl₃): 174.9 (C), 140.9 (CH), 140.6

(C), 137.9 (C), 130.1 (C), 124.5 (CH), 124.1 (CH), 123.2 (CH),

122.9 (CH), 122.6 (C), 121.3 (CH), 88.2 (C), 80.2 (C), 75.8

(CH), 74.9 (C), 68.2 (CH), 36.8 (CH₂), 25.6 (2 × C(CH₃)₃), 19.6

(CH₂), 18.0 (2 × C(CH₃)₃), −3.1 (2 × SiCH₃), −4.7 (SiCH₃) and

−4.9 (SiCH₃); ν_max(KBr)/cm⁻¹2227 (CuC) and 1803 (CO);

3-Allylbenzo[b]thiophene (22d)

The experimental procedure used was the same as for 2-allyl-

naphthalene (22a) utilizing 3-bromobenzo[b]thiophene (8d)

(300 mg, 1.4 mmol). Yield = 218 mg (89%). Colorless oil. δ_H

(250 MHz; CDCl₃): 8.05–7.86 (2 H, m, 2 × ArH), 7.58–7.38 (3

H, m, 3 × ArH), 6.28–6.15 (1 H, m, CHvCH₂), 5.36–5.27 (2 H,

m, CHvCH₂) and 3.75 (2 H, m, CH₂); δ_C(63 MHz; CDCl₃):

140.5 (C), 138.8 (C), 135.5 (CH), 134.5 (C), 124.2 (CH), 123.8

(CH), 122.8 (CH), 122.1 (CH), 121.8 (CH), 116.6 (CH₂) and

33.0 (CH₂); MS (CI) m/z = 175 (MH⁺); HRMS (CI) calcd for

C₁₁H₁₁S (MH⁺): 175.0581, found 175.0582.

MS (CI) m/z

=

555 (MH⁺); HRMS (CI) calcd for

C₃₀H₄₂O₄SSi₂Na (MNa⁺): 555.2408, found 555.2415.

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

propyl)cyclohex-2-en-1,5-carbolactone (25a)

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

prop-1-ynyl)cyclohex-2-en-1,5-carbolactone (24a)

The experimental procedure used was the same as for compound

15a using alkyne 24a (168 mg, 0.30 mmol), Rosemund’s cata-

lyst (150 mg) and 50% THF–methanol (6 mL). Puriﬁcation by

ﬂash chromatography on silica gel, eluting with (30 : 70) dichlor-

omethane–hexanes, gave saturated derivative 25a (166 mg, 98%)

as a colorless oil. [α]^D₂₀= −49.1° (c 1.0 in MeOH); δ_H

(400 MHz; CDCl₃): 7.76–7.58 (3 H, m, 3 × ArH), 7.47 (1 H, br

s, ArH), 7.45–7.39 (2 H, m, 2 × ArH), 7.29 (2 H, dd, J = 1.6

and 8.4 Hz, 2 × ArH), 5.73 (1 H, d, J = 1.6 Hz, H-2), 4.45 (1 H,

m, H-5), 4.00 (1 H, d, J = 3.2 Hz, H-4), 2.85 (2 H, td, J = 1.6

and 7.2 Hz, CH₂Ar), 2.31 (2 H, m, CH₂), 2.06 (2 H, m, CH₂),

A Schlenk tube was charged with triﬂate 12^2c(100 mg,

0.19 mmol) and dry THF (9.5 mL). CuI (7.6 mg, 0.04 mmol),

Pd(PPh₃)₄(45 mg, 0.04 mmol), 2-(prop-2-ynyl)naphthalene

(20a) (158 mg, 0.95 mmol) and piperidine (0.25 mL,

2.47 mmol) were added. The resultant solution was deoxyge-

nated and heated at 40 °C for 4 h. After cooling to room temp-

erature, the reaction mixture was diluted with diethyl ether and

water. The organic layer was separated and the aqueous phase

was extracted with diethyl ether (×2). The combined organic

Org. Biomol. Chem., 2012, 10, 3662–3676 | 3673

View Online

1.83 (2 H, m, CH₂), 0.92 (9 H, s, C(CH₃)₃), 0.85 (9 H, s, C

(CH₃)₃), 0.18 (3 H, s, SiCH₃), 0.14 (3 H, s, SiCH₃), 0.09 (3 H,

s, SiCH₃) and 0.04 (3 H, s, SiCH₃); δ_C(100 MHz; CDCl₃):

176.1 (C), 139.2 (C), 138.9 (C), 133.6 (C), 132.0 (C), 130.7

(CH), 128.0 (CH), 127.6 (CH), 127.4 (CH), 127.2 (CH), 126.5

(CH), 125.9 (CH), 125.1 (CH), 76.0 (CH), 74.7 (C), 67.7 (CH),

37.2 (CH₂), 35.6 (CH₂), 31.4 (CH₂), 28.5 (CH₂), 25.6 (2 × C

(CH₃)₃), 18.0 (C(CH₃)₃), −17.8 (C(CH₃)₃), −3.0 (2 × SiCH₃),

−4.6 (SiCH₃) and −4.8 (SiCH₃); v_max(ﬁlm)/cm⁻¹1799 (CO);

(67%). White solid. Mp: 156–160 °C. [α]^D₂₀= −128.7° (c 1.0 in

MeOH); δ_H(500 MHz; CD₃OD): 7.80 (1 H, d, J = 8.0 Hz,

ArH), 7.70 (1 H, d, J = 8.0 Hz, ArH), 7.35–7.27 (2 H, m, 2 ×

ArH), 7.16 (1 H, s, ArH), 5.79 (1 H, s, H-2), 4.59 (1 H, m, H-5),

4.01 (1 H, d, J = 3.5 Hz, H-4), 2.78 (2 H, m, CH₂Ar), 2.32–2.27

(2 H, m, CH₂-6), 2.25–2.21 (2 H, m, CH₂), 1.97 (1 H, m, CHH)

and 1.86–1.77 (1 H, m, CHH); δ_C(63 MHz; acetone-d₆): 178.6

(C), 141.3 (2 × C), 140.8 (C), 138.3 (C), 131.7 (CH), 126.0

(CH), 125.7 (CH), 124.5 (CH), 123.5 (CH), 123.2 (CH), 78.0

(CH), 74.6 (C), 68.4 (CH), 38.2 (CH₂), 33.4 (CH₂), 29.4 (CH₂)

and 28.4 (CH₂); v_max(KBr)/cm⁻¹2952 (OH), 2929 (OH) and

1799 (CO); MS (ESI) m/z = 353 (MNa⁺); HRMS (ESI) calcd for

C₁₈H₁₈O₄SNa (MNa⁺): 353.0823, found 353.0818.

MS (ESI) m/z

=

553 (MH⁺); HRMS (ESI) calcd for

C₃₂H₄₉Si₂O₄(MH⁺): 553.3164, found 553.3145.

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

butyldime-thylsilyloxy)cyclohex-2-en-1,5-carbolactone (25d)

(1R,4R,5R)-1,4,5-Trihydroxy-3-(3-(naphth-2-yl)propyl)cyclohex-

2-ene-1-carboxylic acid (7a)

The experimental procedure used was the same as for saturated

derivative 15a utilizing alkyne 24d (60 mg, 0.11 mmol). Yield =

60 mg (98%). Yellow oil. [α]^D₂₀= −86.4° (c 1.0 in CHCl₃); δ_H

(250 MHz; CDCl₃): 7.86 (1 H, d, J = 7.3 Hz, ArH), 7.72 (1 H,

d, J = 8.2 Hz, ArH), 7.38 (2 H, m, 2 × ArH), 7.07 (1 H, s, ArH),

5.76 (1 H, s, H-2), 4.48 (1 H, m, H-5), 4.01 (1 H, d, J = 3.0 Hz,

H-4), 2.85 (2 H, t, J = 7.3 Hz, CH₂Ar), 2.32 (2 H, m, CH₂-6),

2.12 (2 H, m, CH₂), 1.88 (2 H, m, CH₂), 0.93 (9 H, s, C(CH₃)₃),

0.87 (9 H, s, C(CH₃)₃), 0.19 (3 H, s, SiCH₃), 0.14 (3 H, s,

SiCH₃), 0.10 (3 H, s, SiCH₃) and 0.04 (3 H, s, SiCH₃); δ_C

(63 MHz; CDCl₃): 176.1 (C), 140.5 (C), 138.8 (C), 138.7 (C),

136.1 (C), 130.8 (CH), 124.1 (CH), 123.9 (CH), 122.9 (CH),

121.6 (CH), 121.4 (CH), 75.9 (CH), 74.7 (C), 67.7 (CH), 37.2

(CH₂), 31.2 (CH₂), 28.0 (CH₂), 26.6 (CH₂), 25.6 (2 × C(CH₃)₃),

18.0 (C(CH₃)₃), 17.9 (C(CH₃)₃), 1.0 (SiCH₃), −3.0 (SiCH₃), and

−4.6 (SiCH₃), −4.8 (SiCH₃); ν_max(ﬁlm)/cm⁻¹1799 (CO); MS

(CI) m/z = 559 (MH⁺).

The experimental procedure used was the same as for acid 5a

utilizing lactone 26a (30 mg, 0.09 mmol). Yield = 30 mg (94%).

White solid. [α]^D₂₀= −23.2° (c 1.0 in MeOH); Mp: 154–158 °C;

δ_H(400 MHz; CD₃OD): 7.67 (3 H m, 3 × ArH), 7.54 (1 H, br s,

ArH), 7.30 (3 H, m, 3 × ArH), 5.38 (1 H, s, H-2), 3.81 (2 H, m,

H-5 + H-4), 2.70 (2 H, m, CH₂Ar), 2.34 (1 H, m, CHH) and

2.06–1.75 (5 H, m, CHH+2 × CH₂); ¹³C NMR (100 MHz,

CD₃OD) δ: 178.4 (C), 145.1 (C), 141.1 (C), 135.2 (C), 133.5

(C), 128.8 (CH), 128.6 (CH), 128.4 (2 × CH), 127.6 (CH),

126.8 (CH), 126.1 (CH), 124.9 (CH), 74.7 (CH), 74.3 (C), 71.1

(CH), 40.5 (CH₂), 36.3 (CH₂), 33.1 (CH₂) and 30.1 (CH₂); ν_max

(KBr)/cm⁻¹3390 (OH) and 1718 (CO) cm⁻¹. MS (ESI) m/z =

341 [M − H]; HRMS (ESI) calcd for C₂₀H₂₁O₅[M − H]:

341.1384, found 341.1384.

(1R,4R,5R)-3-(3-(Benzo[b]thiophen-3-yl)propyl)-1,4,5-

trihydroxycyclohex-2-ene-1-carboxylic acid (7d)

(1R,4R,5R)-1,4-Dihydroxy-3-(3-(naphth-2-yl)propyl)cyclohex-2-

en-1,5-carbolactone (26a)

The experimental procedure used was the same as for acid 5a

utilizing diol 26d (30 mg, 0.09 mmol). Yield = 26 mg (87%).

White solid. Mp: 118–119 °C. [α]^D₂₀= −34.1° (c 1.0, MeOH).

¹H NMR (400 MHz, CD₃OD) δ: 7.82 (1 H, d, J = 8.0 Hz, ArH),

7.77 (1 H, d, J = 7.2 Hz, ArH), 7.36–7.27 (2 H, m, 2 × ArH),

7.21 (1 H, s, ArH), 5.47 (1 H, s, H-2), 3.91–3.84 (2 H, m, H-5 +

H-4), 2.95–2.79 (2 H, m, CH₂), 2.54–2.42 (1 H, m, CHH) and

2.18–1.80 (5 H, m, 2 × CH₂+ CHH); δ_C(100 MHz; CD₃OD):

178.8 (C), 144.7 (C), 141.9 (C), 140.4 (C), 137.9 (C), 125.3

(CH), 125.2 (CH), 124.9 (CH), 123.7 (CH), 122.8 (CH), 122.4

(CH), 74.6 (CH), 74.4 (C), 71.1 (CH), 40.3 (CH₂), 33.4 (CH₂),

28.8 (CH₂) and 28.2 (CH₂); ν_max(KBr)/cm⁻¹3367 (OH) and

1709 (CO); MS (ESI) m/z = 347 (M − H⁺); HRMS (ESI) calcd

for C₁₈H₁₉O₅S (M − H⁺): 347.0948, found 347.0955.

The experimental procedure used was the same as for diol 14a

utilizing silyl ether 25a (38 mg, 0.07 mmol). Puriﬁcation by

ﬂash chromatography on silica gel, eluting with (1 : 1 : 1) diethyl

ether–acetone–hexanes, gave diol 26a (17 mg, 77%) as a white

foam. [α]^D₂₀= −151.4° (c 1.0 in MeOH); Mp: 125–128 °C; δ_H

(400 MHz; CD₃OD): 7.71 (3 H, m, 3 × ArH), 7.53 (1 H, br s,

ArH), 7.36–7.23 (3 H, m, 3 × ArH), 5.74 (1 H, s, H-2), 4.56 (1

H, m, H-5), 3.98 (1 H, d, J = 4.0 Hz, H-4), 2.67 (2 H, t, J = 7.0

Hz, CH₂Ar), 2.27 (2 H, m, CH₂-6), 2.12 (2 H, m, CH₂) and 1.81

(2 H, m, CH₂); δ_C(100 MHz; CD₃OD): 179.3 (C), 141.2 (C),

140.7 (C), 135.1 (C), 133.5 (C), 130.8 (CH), 128.9 (CH), 128.6

(CH), 128.5 (CH), 128.3 (CH), 127.5 (CH), 126.9 (CH), 126.1

(CH), 78.0 (CH), 74.0 (C), 67.6 (CH), 37.5 (CH₂), 36.3 (CH₂),

32.5 (CH₂) and 29.7 (CH₂); v_max(KBr)/cm⁻¹3431 (OH), 3290

(OH) and 1757 (CO); MS (ESI) m/z = 323 (M − H⁺); HRMS

(ESI) calcd for C₂₀H₁₉O₄(M − H⁺): 323.1278, found 323.1287.

Preparation of 25a by B-alkyl Suzuki cross-coupling

(a) Preparation of borane 23a. To a solution of 9-BBN-H

(0.4 mL, 0.20 mmol, ca. 0.5 M in THF) in a ﬂamed round-

bottom ﬂask under argon 2-allylnaphthalene (22a) (63 mg,

0.37 mmol) was added. The mixture was stirred for 12 h to give

a solution of borane 23a.

(1R,4R,5R)-3-(3-(Benzo[b]thiophen-3-yl)propyl)-1,4-dihydroxy-

cyclohex-2-en-1,5-carbolactone (26d)

The experimental procedure used was the same as for diol 14a

utilizing silyl ether 25d (85 mg, 0.15 mmol). Yield: 33 mg

3674 | Org. Biomol. Chem., 2012, 10, 3662–3676

View Online

(b) B-alkyl Suzuki cross-coupling. To the borane solution

obtained above, K₃PO₄(63 mg, 0.28 mmol), Pd(PPh₃)₄(33 mg,

0.03 mmol), dioxane (0.8 mL) and triﬂate 12^2c(100 mg,

0.12 mmol) were added. The resultant solution was heated at

110 °C for 12 h under argon. After cooling to room temperature,

the solution was diluted with diethyl ether and water. The

organic layer was separated and the aqueous phase was extracted

with diethyl ether (×2). The combined organic extracts were

dried (Na₂SO₄), ﬁltered and concentrated under reduced

pressure. The residue was puriﬁed by ﬂash chromatography on

silica gel, eluting with a gradient of dichloromethane–hexanes

(10 : 90 to 40 : 60), to give compound 25a (73 mg, 70%).

migration in the entry box were used as default parameters (95,

95, and 10, respectively), as well as the hydrogen bonding (4.0

Å) and van der Waals (2.5 Å) parameters. The position of ligand

4c in both crystal structures was used to deﬁne the active-site

and the radius was set to 7 Å. The “ﬂip ring corners” ﬂag was

switched on, while all the other ﬂags were off. The GOLD

scoring function was used to rank the ligands in order of ﬁtness.

Molecular dynamics simulations

Ligand minimization. Ligand geometries were ﬁrst reﬁned by

means of the semi-empirical quantum mechanical program

MOPAC¹⁴using the AM1 Hamiltonian and PRECISE stopping

criteria, and further optimised using a restricted Hartree–Fock

(RHF) method and a 6-31G(d) basis set, as implemented in the

ab initio program Gaussian 09.¹³The resulting wavefunctions

were used to calculate electrostatic potential-derived (ESP)

charges employing the restrained electrostatic potential (RESP)¹⁵

methodology, as implemented in the assisted model building

with energy reﬁnement (AMBER)¹⁶suite of programs. The

missing bonded and non-bonded parameters were assigned, by

analogy or through interpolation from those already present in

the AMBER database (GAFF).^13,17

Preparation of 25d by B-alkyl Suzuki cross-coupling

(a) Preparation of borane 23d. To a solution of 9-BBN-H

(0.44 mL, 0.22 mmol, ca. 0.5 M in THF) in a ﬂamed round-

bottom ﬂask under argon 3-allylbenzo[b]thiophene (22d)

(65 mg, 0.37 mmol) was added. The mixture was stirred for 12 h

to give a solution of borane 23d.

(b) B-alkyl Suzuki cross-coupling. To the borane solution

obtained above, K₃PO₄(84 mg, 0.38 mmol), Pd(PPh₃)₄(32 mg,

0.03 mmol), dioxane (0.8 mL), KBr (25 mg, 0.21 mmol) and

triﬂate 12^2c(100 mg, 0.12 mmol) were added. The resultant sol-

ution was heated at 110 °C for 12 h under argon. After cooling

to room temperature, the solution was diluted with diethyl ether

and water. The organic layer was separated and the aqueous

phase was extracted with diethyl ether (×2). The combined

organic extracts were dried (Na₂SO₄), ﬁltered and concentrated

under reduced pressure. The residue was puriﬁed by ﬂash chrom-

atography on silica gel, eluting with a gradient of dichloro-

methane–hexanes (10 : 90 to 40 : 60), to give compound 25d

(44 mg, 42%).

Generation and minimization of the DHQ2–ligand complexes.

Simulations were carried out using the enzyme geometries found

in the crystal structure of DHQ2-Mt in complex 4c (PDB code

2Y71^5b). Not solved residues 18–20 were incorporated from the

crystal structure of the fully resolved crystal structure of DHQ2-

Mt in complex with (1R,2R,4S,5R)-1,4,5-trihydroxy-2-(4-meth-

oxybenzyl)-3-oxocyclohexanecarboxylic acid (PDB code:

2XB8⁷). Taking into account that unfolding and refolding studies

of DHQ2 have shown that the trimer¹⁸is the biological unit of

the enzyme and on the basis of preliminary simulations on the

monomer proving to be unstable under our simulation con-

ditions, the trimer was used for these studies. Hydrogens were

added to the protein using the web-based PROPKA3.1 server,¹⁹

which assigned protonation states to all titratable residues at the

chosen pH of 7.0. However, δ and/or ε protonation was manually

corrected for His102 (dual) of the active site due to the mechan-

istic considerations and on the basis of results from preliminary

MD simulations. Molecular mechanics parameters from the ff03

and GAFF force ﬁelds, respectively, were assigned to the protein

and the ligands using the LEaP module of AMBER 10.0.²⁰All

terminal hydrogens were ﬁrst minimized in vacuo (2000 steps,

half of them steepest descent, the other half conjugate gradient).

Then, energy minimization using the implicit solvent GB model

was carried out in stages, starting with ligand (1000 steps, half

of them steepest descent, the other half conjugate gradient),

protein side-chains (1000 steps, idem) and ﬁnally the entire

complex (1000 steps, idem). A positional restraint force constant

of 50 kcal mol⁻¹Å⁻²to those unminimized atoms in each step

was applied during all calculations. Thereafter each reﬁned

DHQ2–ligand complex was neutralized by addition of sodium

ions and immersed in a truncated octahedron of TIP3P water

molecules.^16,21,22

Dehydroquinase assays

The enzyme was puriﬁed and assayed as described

previously.^2d,12

Docking studies

They were carried out using the program GOLD 5.0.1⁹and the

enzyme geometries found in the crystal structure of the binary

complex DHQ2-Hp/4c (PDB code: 2WKS^5a) and DHQ2-Mt/4c

(PDB code: 2Y71^5b) In the latter case, not solved residues

18–20 were incorporated from the crystal structure of the fully

resolved crystal structure of DHQ2-Mt in complex with

(1R,2R,4S,5R)-1,4,5-trihydroxy-2-(4-methoxybenzyl)-3-oxocyclo-

hexanecarboxylic acid (PDB code: 2XB8⁷) The receptor was

used as a dimer. Water molecules were removed from all crystal

structures with the exception of the water involved in the mech-

anism, which is located close to the carbonyl group of C3.

Ligand geometries were minimized using the AM1 Hamiltonian

as implemented in the program Gaussian 09¹³and used as

MOL2 ﬁles. Each ligand was docked in 25 independent genetic

algorithm (GA) runs, and for each of these a maximum number

of 100 000 GA operations were performed on a single population

of 50 individuals. Operator weights for crossover, mutation and

Simulations. MD simulations were performed using the

AMBER 10.0 suite of programs and Amber ff03 force ﬁeld.

Org. Biomol. Chem., 2012, 10, 3662–3676 | 3675

View Online

5 (a) V. F. V. Prazeres, L. Tizón, J. M. Otero, P. Guardado-Calvo, A.

L. Llamas-Saíz, M. J. van Raaij, L. Castedo, H. Lamb, A. R. Hawkins

and C. González-Bello, J. Med. Chem., 2010, 53, 191–200; (b) L. Tizón,

J. M. Otero, V. F. V. Prazeres, A. L. Llamas-Saíz, M. J. van Raaij,

H. Lamb, A. R. Hawkins, J. A. Ainsa, L. Castedo and C. González-

Bello, J. Med. Chem., 2011, 54, 6063–6084.

6 This ﬁgure was prepared using PyMOL: W. L. DeLano, The PyMOL Mol-

ecular Graphics System, DeLano Scientiﬁc LLC, Palo Alto, CA, USA,

2008. http://www.pymol.org

7 A. Peón, J. M. Otero, L. Tizón, V. F. V. Prazeres, A. L. Llamas-Saiz, G.

C. Fox, M. J. van Raaij, H. Lamb, A. R. Hawkins, F. Gago, L. Castedo

and C. González-Bello, ChemMedChem, 2010, 5, 1726–1733.

8 S. R. Chemler, D. Trauner and S. J. Danishefsky, Angew. Chem., Int. Ed.,

2001, 40, 4544–4568; S. R. Chemler, D. Trauner and S. J. Danishefsky,

Angew. Chem., 2001, 113, 4676–4701.

Periodic boundary conditions were applied and electrostatic

interactions were treated using the smooth particle mesh Ewald

method (PME)²³with a grid spacing of 1 Å. The cutoff distance

for the non-bonded interactions was 9 Å. SHAKE algorithm²⁴

was applied to all bonds containing hydrogen, using a tolerance

of 10⁻⁵Å and an integration step of 2.0 fs. Minimization was

carried out in three steps, starting with the octahedron water

hydrogens, followed by solvent molecules and sodium counter-

ions and ﬁnally the entire system. The minimized system was

heated at 300 K (1 atm, 25 ps, a positional restraint force con-

stant of 50 kcal mol⁻¹Å⁻²). These initial harmonic restraints

were gradually reduced to 5 kcal mol⁻¹Å⁻²(10 steps) and the

resulting systems were allowed to equilibrate further. MD with

constraints of 5 kcal mol⁻¹Å⁻²were carried out to all protein

α-carbons of the two external subunits of the trimer and the beta

sheets and alpha helix of the central subunit of the trimer for 10

ns (500 steps). System coordinates were collected every 2 ps for

further analysis. Next, a slow-cooling MD simulation with con-

straints of 5 kcal mol⁻¹Å⁻²was performed (6 steps until

273 K). Finally, minimization of the entire complexes was per-

9 http://www.ccdc.cam.ac.uk/products/life_sciencies/gold/

10 J. Fournier dit Chabert, B. Márquez, L. Neville, L. Joucla, S. Broussous,

P. Bouhours, E. David, S. Pellet-Rostaing, B. Marquet, N. Moreau and

M. Lemaire, Bioorg. Med. Chem., 2007, 15, 4482–4497.

11 C. S. Bryan, J. A. Braunger and M. Lautens, Angew. Chem., Int. Ed.,

2009, 48, 7064–7068; C. S. Bryan, J. A. Braunger and M. Lautens,

Angew. Chem., 2009, 121, 7198–7202.

12 D. G. Gourley, J. R. Coggins, N. W. Isaacs, J. D. Moore, I. G. Charles

and A. R. Hawkins, J. Mol. Biol., 1994, 241, 488–491.

13 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb,

J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson,

H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov,

J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota,

R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,

H. Nakai, T. Vreven, J. A. Montgomery Jr., J. E. Peralta, F. Ogliaro,

M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov,

R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S.

S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J.

E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts,

R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J.

W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth,

P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas, J.

B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox, GAUSSIAN 09

(Revision A.2), Gaussian, Inc., Wallingford CT, 2009.

formed with constraints of 5 kcal mol⁻¹Å⁻²

.

Acknowledgements

Financial support from the Xunta de Galicia (10PXIB2200122PR

and GRC2010/12) and the Spanish Ministry of Science and

Innovation (SAF2010-15076) is gratefully acknowledged. BB,

AS and AP thank the Spanish Ministry of Science and

Innovation for FPU fellowships. We are also grateful to the

Centro de Supercomputación de Galicia (CESGA) for the use of

the Finis Terrae computer.

14 J. Stewart, J. Comput.-Aided Mol. Des., 1990, 4, 1–45.

15 (a) W. D. Cornell, P. Cieplak, C. I. Bayly, I. R. Gould, K. M. Merz, D.

M. Ferguson, D. C. Spellmeyer, T. Fox, J. W. Caldwell and P.

A. Kollman, J. Am. Chem. Soc., 1995, 117, 5179–5197; (b) http://q4md-

forcedﬁeldtools.org/RED/resp/

Notes and references

16 D. A. Case, T. E. Cheatham, T. Darden, H. Gohlke, R. Luo, K. M. Merz,

O. Onufriev, C. Simmerling, B. Wang and R. J. Woods, J. Comput.

Chem., 2005, 26, 1668–1688.

17 (a) J. Wang, R. M. Wolf, J. W. Caldwell, P. A. Kollman and D. A. Case,

J. Comput. Chem., 2004, 25, 1157–1174; (b) J. Wang, W. Wang, P.

A. Kollman and D. A. Case, J. Mol. Graphics Modell., 2006, 25, 247–260.

18 N. C. Price, D. J. Boam, S. M. Kelly, D. Duncan, T. Krell, D. G. Gourley,

J. R. Coggins, R. Virden and A. R. Hawkins, Biochem. J., 1999, 338,

195–202.

19 (a) H. Li, A. D. Robertson and J. H. Jensen, Proteins: Struct., Funct.,

Bioinf., 2005, 61, 704–721; (b) D. C. Bas, D. M. Rogers and J.

H. Jensen, Proteins: Struct., Funct., Bioinf., 2008, 73, 765–783;

(c) M. H. M. Olsson, C. R. Søndergard, M. Rostkowski and J. H. Jensen,

J. Chem. Theory Comput., 2011, 7, 525–537; (d) C. R. Søndergard,

M. H. M. Olsson, M. Rostkowski and J. H. Jensen, J. Chem. Theory

Comput., 2011, 7, 2284–2295.

20 D. A. Case, T. A. Darden, T. E. Cheatham III, C. L. Simmerling, J. Wang,

R. E. Duke, R. Luo, R. C. Walker, W. Zhang, K. M. Merz, B. Roberts,

B. Wang, S. Hayik, A. Roitberg, G. Seabra, I. Kolossvai, K. F. Wong,

F. Paesani, J. Vanicek, J. Liu, X. Wu, S. R. Brozell, T. Steinbrecher,

H. Gohlke, Q. Cai, X. Ye, J. Wang, M.-J. Hsieh, G. Cui, D. R. Roe, D.

H. Mathews, M. G. Seetin, C. Sagui, V. Babin, T. Luchko, S. Gusarov,

A. Kovalenko and P. A. Kollman, Amber Tools 1.5, AMBER 11, Univer-

sity of California, San Francisco, 2010.

1 A. Koul, E. Arnoult, N. Lounis, J. Guillemont and K. Andries, Nature,

2011, 469, 489–490.

2 (a) C. González-Bello and L. Castedo, Med. Res. Rev., 2007, 27, 177–

208; (b) C. González-Bello, E. Lence, M. D. Toscano, L. Castedo, J.

R. Coggins and C. Abell, J. Med. Chem., 2003, 46, 5735–5744;

(c) C. Sánchez-Sixto, V. F. V. Prazeres, L. Castedo, H. Lamb, A.

R. Hawkins and C. González-Bello, J. Med. Chem., 2005, 48, 4871–

4881; (d) V. F. V. Prazeres, C. Sánchez-Sixto, L. Castedo, H. Lamb, A.

R. Hawkins, A. Riboldi-Tunnicliffe, J. R. Coggins, A. J. Lapthorn and

C. González-Bello, ChemMedChem, 2007, 2, 194–207; (e) C. Sánchez-

Sixto, V. F. V. Prazeres, L. Castedo, S. W. Suh, H. Lamb, A. R. Hawkins,

F. J. Cañada, J. Jiménez-Barbero and C. González-Bello, ChemMed-

Chem, 2008, 3, 756–770.

3 See also: (a) M. Frederickson, E. J. Parker, A. R. Hawkins, J. R. Coggins

and C. Abell, J. Org. Chem., 1999, 64, 2612–2613; (b) M. Frederickson,

A. W. Roszak, J. R. Coggins, A. J. Lapthorn and C. Abell, Org. Biomol.

Chem., 2004, 2, 1592–1596; (c) M. D. Toscano, R. J. Payne, A. Chiba,

O. Kerbarh and C. Abell, ChemMedChem, 2007, 2, 101–112; (d) R.

J. Payne, F. Peyrot, O. Kerbarh, A. D. Abell and C. Abell, ChemMed-

Chem, 2007, 2, 1015–1029; (e) R. J. Payne, A. Riboldi-Tunnicliffe,

O. Kerbarh, A. D. Abell, A. J. Lapthorn and C. Abell, ChemMedChem,

2007, 2, 1010–1013; (f) A. T. Tran, K. M. Cergol, N. P. West, E.

J. Randall, W. J. Britton, S. A. I. Bokhari, M. Ibrahim, A. J. Lapthorn

and R. J. Payne, ChemMedChem, 2011, 6, 262–265; (g) M. V. B. Dias,

W. C. Snee, K. M. Bromﬁeld, E. J. Payne, S. K. Palaninathan, A. Ciulli,

N. I. Howard, C. Abell, J. C. Sacchettini and T. L. Blundell, Biochem. J.,

2011, 436, 729–739.

21 J. Aqvist, J. Phys. Chem., 1990, 94, 8021–8024.

22 W. L. Jorgensen, J. Chandrasekhar and J. D. Madura, J. Chem. Phys.,

1983, 79, 926–935.

23 T. A. Darden, D. York and L. G. Pedersen, J. Chem. Phys., 1993, 98,

10089–10092.

24 J.-P. Ryckaert, G. Ciccotti and H. J. C. Berendsen, J. Comput. Phys.,

1977, 23, 327–341.

4 (a) J. Harris, C. González-Bello, C. Kleanthous, J. R. Coggins, A.

R. Hawkins and C. Abell, Biochem. J., 1996, 319, 333–336; (b) A.

W. Roszak, D. A. Robinson, T. Krell, I. S. Hunter, M. Frederickson,

C. Abell, J. R. Coggins and A. J. Lapthorn, Structure, 2002, 10, 493–503.

3676 | Org. Biomol. Chem., 2012, 10, 3662–3676

Article Doi

Synthesis of 3-alkyl enol mimics inhibitors of type II dehydroquinase: Factors influencing their inhibition potency

DOI: 10.1039/c2ob07081b

Source and publish data:

Authors:

Article abstract of DOI:10.1039/c2ob07081b

Full text of DOI:10.1039/c2ob07081b

Products guided by the article

R&D Labs maybe for 1374000-00-6

Relevant to this article

Hot Product