The synthesis and biological evaluation of lactose-based sialylmimetics as inhibitors of rotaviral infection

Bioorganic & Medicinal Chemistry 14 (2006) 739–757

The synthesis and biological evaluation of lactose-based

sialylmimetics as inhibitors of rotaviral infection

Angela Liakatos,^aMilton J. Kiefel,^aFiona Fleming,^b

Barbara Coulson^band Mark von Itzstein^a,*

^aInstitute for Glycomics, Griﬃth University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Qld 9726, Australia

^bDepartment of Microbiology, University of Melbourne, Parkville, Vic. 3010, Australia

Received 7 June 2005; revised 29 August 2005; accepted 29 August 2005

Available online 7 October 2005

Abstract—Rotaviruses are the most signiﬁcant cause of gastroenteritis in young children and are responsible for over 600,000 infant

deaths annually. The rotaviral haemagglutinin protein (VP8*) of some strains has been implicated in early recognition and binding

events of host cell-surface sialoglycoconjugates, and is therefore an attractive target for potential therapeutic intervention. Since N-

acetylneuraminic acid a(2,3)-linked to galactose is believed to be the minimum binding epitope of rotavirus to host cells, we report

here our development of an eﬃcient and ﬂexible synthetic route to a range of lactose-based sialylmimetics of a(2,3)-linked thiosi-

alosides. These compounds were biologically evaluated as inhibitors of rotaviral infection using an in vitro neutralisation assay. The

results suggest that these lactose-based sialylmimetics are not inhibitors of the rhesus rotavirus strain; however, they do exhibit

modest inhibition of the human (Wa) strain, presumably through inhibition of the rotaviral adhesion process.

1. Introduction

chemotherapeutic agent has yet been developed to treat

rotaviral infection.

Rotaviral infection is recognised as being responsible for

causing severe gastroenteritis in young children,¹with

almost all children, regardless of socioeconomic or envi-

ronmental conditions, infected by 5 years of age. It has

been estimated that over 600,000 infant deaths occur

annually, primarily in developing countries, due to diar-

rhea caused by rotavirus²Rotaviruses are also impor-

tant veterinary pathogens, infecting almost all species

of mammals.³Although there are several vaccine treat-

ments against rotaviral infection in clinical trials,^4,5the

safety and eﬃcacy of rotaviral vaccines in general has

been questioned.⁶The ﬁrst rotavirus vaccine licensed,

Rotashield^TM(a tetravalent rhesus-reassortment rotavi-

rus vaccine), was withdrawn in 1999 due to the increased

incidence of intussusception.^6–8More recently, Rota

Rix^ꢁ(GlaxoSmithKline) and RotaTeq^ꢁ(Merck) have

undergone extensive phase III clinical trials, with

RotaRix approved for use in Mexico mid-2004. No

Rotaviruses are double-stranded RNA viruses of the

family Reoviridae.¹Rotaviruses are highly host-cell spe-

ciﬁc and sites of mucosal infection are generally limited

to mature enterocytes in the mid and upper villous epi-

thelium of the small intestine.⁹Their structures can be

˚

simpliﬁed as ꢀ1000 A icosahedral particles consisting

of three concentric layers of protein: (1) the outer layer

proteins VP4 and VP7; (2) the intermediate layer protein

VP6, and (3) the inner core proteins VP1, VP2 and

VP3.^1,9Also known as the inner capsid protein, the core

contains the viral genome which consists of 11 segments

of AU-rich double-stranded DNA.^1,9VP4 consists of

˚

ꢀ100 A dimeric spikes that project from the outer core

of the virion, and also extend down and interact with

VP7 and VP6. The outer layer proteins VP4 and VP7

have been identiﬁed as the proteins responsible for

receptor binding and cell penetration,^10–14and indepen-

dently evoke a response from the immune system as well

as being determinants of virulence.¹⁵

Keywords: Sialylmimetics; Lactose derivatives; Rotavirus; Viral hae-

magglutinin; Sialic acid; Thiosialosides.

Although the functional properties of VP4 and VP7 have

been well deﬁned, the precise cellular recognition site(s)

for rotavirus still remains unknown. Elucidation of the

*

Corresponding author. Tel.: +617 5552 7025; fax: +617 5552 8098;

e-mail: m.vonitzstein@griﬃth.edu.au

doi:10.1016/j.bmc.2005.08.057

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A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

cellular receptor for rotavirus is complicated by the fact

that host cell entry is a multi-step process, requiring at

least three virus host-cell interactions.^{13,14,16–20}The gen-

erally accepted model is that rotavirus initially binds to

host cell-surface glycoconjugates that contain sialic acid,

followed by interactions independent of sialic acid, be-

lieved to be part of the lipid domains in which integrins

are thought to play an important role.^{11,13,16–18,21–24}De-

tails about the precise nature of the initial carbohydrate

epitope recognised by rotavirus are complicated by the

observations that diﬀerent rotavirus strains exhibit vary-

ing speciﬁcities for certain host cell-surface sialoglyco-

conjugate sequences, although some similarities do

exist between human and animal strains.^14,21,23,25De-

spite diﬀerent rotavirus strains recognising diﬀerent

cell-surface sialylglycoconjugates, they can be conve-

niently classiﬁed as being either sialidase-sensitive or sial-

idase insensitive, which is determined by pre-treatment

of cells with a sialidase and then monitoring the change,

if any, in the infectivity by rotavirus.^23,26,27Human

strains of rotavirus generally fall into the sialidase-insen-

sitive category, meaning that in vitro treatment of cells

with a sialidase has no eﬀect on the ability of human

rotaviruses to infect these cells.^23,28,29It has also been

suggested that the initial interaction of rotavirus strains

with sialic acids is linked to VP4 P genotype, rather than

to species of origin (VP7 G serotype).³⁰Several research-

ers have aimed to investigate the role of sialoglycoconju-

gates in rotavirus infectivity of host cells.^{19,26–28,30–32}In

an important investigation, the minimum binding

epitope of rotavirus was elucidated to be Neu5Ac-

a(2,3)-Gal or Neu5Gc-a(2,3)-Gal after examination of

rotavirus binding with 32 diﬀerent gangliosides.³²Fur-

ther studies with chemically modiﬁed glycoconjugates

determined that the carboxyl group of sialic acid plays

an important role in binding to rotavirus.³²In another

study, applied galectins (galactose-recognising proteins)

were also found to inhibit rotavirus infection, suggesting

that galactose is an important component of the host cell

receptor.²¹

from the same authors further demonstrated that the

rhesus VP8* core speciﬁcally binds to a-Neu5Ac with

a K_dof 1.2 mM.³⁸The binding aﬃnities of several a-

sialosides were examined by NMR spectroscopy, and

it was determined that the haemagglutinin protein re-

quires only a-linked Neu5Ac for binding, irrespective

of both the type of carbohydrate the Neu5Ac was linked

to and whether the linkage was a(2,3) or a(2,6).³⁸The

low binding aﬃnities and broad speciﬁcity shown by

sialic acid binding to the rhesus rotavirus haemaggluti-

nin are consistent with the hypothesis that sialic acid

mediates an initial interaction with the host cell that pre-

cedes more speciﬁc interactions determining rotavirus

cell type and host speciﬁcity.

The apparent important initial role that sialic acids play

in the infection process by rotavirus implies that this

may be a good target for therapeutic intervention. In

this regard, the highly sulfated sialoside 2 (NMSO3)

was found to inhibit four human strains of rotavirus

with IC₅₀values in the micromolar range.³⁹It was deter-

mined that NMSO3 (2) was blocking the binding of

rotavirus through the VP4 or VP7 proteins, and in an

in vitro study, prophylactic oral administration of

NMSO3 (2) to suckling mice prior to inoculation with

the human (MO) strain of rotavirus prevented the onset

of diarrhoea.³⁹We have previously reported the synthe-

sis and biological evaluation of a(2,6)-linked thiosialo-

sides, for example, 3–5,⁴⁰as well as the lactose-based

S-linked sialylmimetics of a(2,6)-thiosialosides 6–8⁴¹as

potential inhibitors of rotaviral infection. The partially

acetylated thiosialosides 3–5 showed modest rotaviral

inhibition, with IC₅₀values in the 10^ꢁ3M range, against

the bovine (NCDV) strain of rotavirus.⁴⁰These results

were in concordance with the literature, in which earlier

studies by Willoughby and Yolken found that partially

acetylated derivatives of Neu5Ac exhibited improved

inhibitory activity, in comparison to the non-acetylated

versions, against the bovine (NCDV) rotavirus strain.⁴²

The lactose-based S-linked sialylmimetics of a(2,6)-thi-

osialosides 6–8 were designed to mimic the biologically

relevant functionalities of thiosialosides like 3, in partic-

ular the carboxylate functionality of the Neu5Ac

residue. The ꢀmethylꢁ and ꢀethylꢁ lactose-based sialylmi-

metics 6 and 7, respectively, showed modest inhibition

against both the bovine (NCDV) and human (Wa)

strains of rotavirus, whilst the ꢀphenylꢁ sialylmimetic 8

was only active against the bovine (NCDV) strain.⁴¹

These results suggested that small alkyl groups are toler-

ated in the binding pocket of the viral adhesion proteins

of the bovine (NCDV) and human (Wa) strains, and

that aryl groups may be too sterically demanding (or

too hydrophobic) for the human (Wa) strain of rotavi-

rus. Although the level of inhibition is only moderate,

these were the ﬁrst examples of simple carbohydrate

derivatives exhibiting inhibition of a human strain of

rotavirus.⁴¹

Proteolytic treatment of VP4 results in an enhancement

of rotavirus infectivity, and leads to the generation of

VP8* (the C-terminal fragment responsible for haemag-

glutination), and VP5* (the N-terminal fragment which

contains an internal hydrophobic region and a putative

cell fusion region), both of which remain attached to the

virion.^33–36Detailed structural information of VP8*

from a rhesus rotavirus strain (RRV) was determined

by X-ray crystallography and NMR spectroscopy.^37,38

X-ray crystallography determined the three-dimensional

structure of rhesus VP8* in complex with 2-O-methyl a-

˚

D-N-acetylneuraminic acid (a-Me-Neu5Ac, 1) to 1.4 A

resolution.³⁷An interesting observation of the crystal

structure reveals a b-sandwich fold that is consistent

with the human galectins; however, the VP8* core and

the galectins show no signiﬁcant sequence homology.³⁷

Between the two b-sheets is a shallow groove that per-

mits Neu5Ac binding to VP8*, and predominant inter-

actions include hydrogen bonding and van der Waals

contacts, which are also common to some viral haemag-

glutinins, such as inﬂuenza and Newcastle disease virus-

N-Acetylneuraminic acid a(2,3)-linked to galactose is

not only believed to be the minimum binding epitope

of rotavirus to host cells,³²but is a commonly found epi-

tope in several important cell-surface glycoconjugates,

and is associated with diseases like cancer (through the

1

es. A subsequent H NMR spectroscopic investigation

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

741

H

OH

OSO₃Na

OH

CO₂Na

O

OC₂₂H₄₅

CO₂H

CO₂Na

HO

NaO₃SO

AcO

O

S

HO

OMe

R

H

R

H

O

HO

NaO₃SO

H

HO

OH

OSO₃Na

HO

R'

OMe

1 R = NHAc

2 R = NHAc

3 R = NHAc; R' = OH

4 R = R' = NHAc

H

R

CO₂Na

OH

CO₂Na

S

AcO

S

O

HO

R

H

OH

HO

AcO

O

HO

O

HO

O

OMe

OH

HO

OH

6 R = Me

7 R = Et

8 R = Ph

5 R = NHAc

upregulation of a(2,3)-sialyltransferases)⁴³and Chagasꢁ

disease (and the involvement of Trypanosoma cruzi

trans-sialidase).⁴⁴In our ongoing endeavour to develop

novel sialylmimetics as biological probes for sialic

acid-recognising proteins, this paper describes our ef-

forts towards the synthesis of Neu5Ac2-S-a(2,3)-

Lacb1Me 9 and lactose-based S-linked sialylmimetics,

represented by the general structure 10, that mimic the

key elements of a(2,3)-linked thiosialosides like 9. Sial-

ylmimetics of the general structure 10 do not need the

presence of sulfur to protect them from sialidase-cataly-

sed hydrolysis (as is the case with compound 9). Howev-

er, our continued interest in the preparation of sulfur

linked sialylmimetics, together with the fact that we

can introduce structural variability into our target com-

pounds late in the synthetic sequence (vide infra),

prompted us to prepare sialylmimetics like 10. The bio-

logical evaluation of compounds like 9 and 10 as poten-

tial inhibitors of rotaviral infection is also described.

Typically, transformations involving the introduction of

a sulfur functionality in pyranosides can be accom-

plished by displacement of a leaving group with an

appropriate nucleophile,⁴⁶through Mitsunobu chemis-

try⁴⁷or via the opening of epoxides.⁴⁸We have reported

elsewhere a double inversion sequence at the C-3⁰posi-

tion of a lactoside for the successful introduction of a

thiolacetate group to give a compound like 11.⁴⁹From

these investigations it was found that a non-participat-

ing functionality at C-4⁰, like a benzylidene acetal, was

critical for a double inversion sequence at C-3⁰to give

the key lactoside 11.⁴⁹For example, activation of the

C-3⁰hydroxyl in 12 with a triﬂate, followed by S_N2 dis-

placement using KNO₂, aﬀorded the desired gulo-con-

ﬁgured derivative 13 in 84% yield (Scheme 2).

However, subsequent formation of the C-3⁰triﬂate 14,

followed by treatment with KSAc, did not yield the de-

sired C-3⁰thiolacetylated derivative 15 but the galacto-

conﬁgured 3⁰-hydroxy compound 12 (Scheme 2).⁴⁹This

observation suggested the major reaction pathway to

proceed via neighbouring group participation of the

C-4⁰benzoate in 14, presumably via the cyclic interme-

diate 16 shown in Scheme 2. We also observed a similar

outcome when other acyl protecting groups at C-4⁰, like

2. Results and discussion

2.1. Synthesis of S-linked a(2,3)-sialyllactoside and

sialylmimetics

OH

H

OH

HO

S

NaO₂C

O

OH

O

HO

An important consideration in designing compounds

like 9 and 10 as probes for sialic acid-recognising pro-

teins is the metabolic stability of such compounds. In

this regard, it has been shown that the thioglycosidic

linkage in thiosialosides is resistant to hydrolysis by sia-

lidases.⁴⁵Accordingly, compound 9 was designed with a

thioglycosidic linkage to impart a degree of metabolic

stability in the ﬁnal target compound. The lactose-based

S-linked sialylmimetics represented by the general struc-

ture 10, wherein the entire Neu5Ac portion has been re-

placed by a carboxylate group with an appended

hydrophobic or hydrophilic moiety, can essentially be

considered as mimetics of the a(2,3)-linked thiosialoside

9. The inclusion of varying functionality in the mimetic

portion of 10 was designed to explore the eﬀects of

hydrophobic, hydrophilic and sterically demanding

groups upon their interaction with a given biomolecule

such as rotavirus VP8*. Our approach, as outlined in

retrosynthetic terms in Scheme 1, relies on both the thi-

osialoside 9 and the sialylmimetics of the general struc-

ture 10 being accessible by a common C-3⁰thiolacetyl

lactoside precursor such as 11.

O

HO

R

H

OMe

HO

OH

9 R = NHAc

HO

OH

OPG

PGO

OPG

O

AcS

O

OMe

PGO

OPG

11 PG = Protecting Group

OH

O

HO

S

OH

O

NaO₂C

OMe

HO

OH

R

10 R = alkyl, aryl

Scheme 1. Retrosynthetic approach towards the thiosialoside 9 and

the sialylmimetics 10.

742

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

OBz

O

BzO

OBz

O

BzO

HO

OBz

O

OBz

O

a,b

O

OMe

BzO

OBz

HO

OBz

12

13

a

OBz

O

OBz

O

BzO

Ph

OBz

O

OBz

O

Neighbouring

Group Participation

O

OMe

BzO

TfO

OBz

14

16

c

OBz

O

BzO

AcS

OBz

O

BzO

OBz

15

Scheme 2. Reagents and conditions: (a) Tf₂O, pyridine, CH₂Cl₂, ꢁ78 to 0 ꢂC, 1 h; (b) KNO₂, DMF, 50 ꢂC, 4 h, 84%; (c) KSAc, DMF, 50 ꢂC, 16 h.

an acetate group, were employed in this sequence of

reactions.⁴⁹Subsequent to our ﬁndings, there have been

other reports of the preparation of C-3⁰thiolacetylated

lactoside derivatives like 11 that support our ﬁndings in

relation to the importance of protecting group choice.^50,51

Ph

O

a,b

O

OMe

RO

HO

OH

A common transformation for preparing C-3⁰functional-

ised lactoside derivatives generally involves the use of

dibutylstannylidene mediated alkylation with p-methox-

y-benzyl chloride⁵²or allylbromide.⁵³In most cases, these

temporary C-3⁰alkyl protecting groups can be selectively

cleaved in the presence of most other protecting groups to

reveal the hydroxyl group for further elaboration.⁵⁴In

our hands, etheriﬁcations of this type had proved prob-

lematic due to the poor reactivities of the alkyl electro-

philes over extended reaction times, resulting in poor

yields. We required the synthesis of a key lactoside like

11 with 4⁰,6⁰-O-bezylidene acetal protection to be eﬃcient

in terms of overall chemical yield, to be reproducible on a

multi-gram scale, and to provide relatively quick access to

an advanced precursor for the preparation of a range of

compounds like 10. To this end, we felt it appropriate to

explore regioselective manipulations mediated by dibu-

tyltin oxide.^55,56using sulfonyl and acyl electrophiles on

the 4,6-O-benzylidenated galactoside 17 as a model sys-

tem. Accordingly, 17 was treated with Bu₂SnO in MeOH

followed by exposure of the intermediate dibutylstanny-

lidene acetal derivative to either MsCl, Tf₂O and ClAcCl

(Scheme 3). All attempts at selectively functionalising the

C-3 hydroxyl in 17 were successful. Mesylation was quan-

titative and aﬀorded exclusively the C-3 functionalised

product 18 (Scheme 3). Also, treatment of 17 with Tf₂O

via the 2,3-O-stannylidene acetal aﬀorded the corre-

sponding C-3 triﬂate 19 in 70% yield (Scheme 3). Addi-

tionally, chloroacetylation of 17 with ClAcCl also

yielded the C-3 derivatised product 20 (Scheme 3),

although 20% of the 2,3-di-O-chloroacetylated com-

pound was also isolated.

17

18 R = Ms (100%)

19 R = Tf (70%)

20 R = ClAc (79%)

Scheme 3. Reagents and conditions: (a) Bu₂SnO, MeOH, reﬂux, 2 h;

˚

(b) 4 A sieves, electrophile, toluene, 0 ꢂC, 30 min.

experiments was that they were not as promising as

those described for the galactoside model system 17.

The dibutylstannylidene mediated sulfonylations of the

4⁰,6⁰-O-benzylidenated lactoside 21 aﬀorded complex

mixtures of products, in which unreacted starting mate-

rial was always present. Fortunately, with ClAcCl as the

electrophile, the C-3⁰chloracetylated protected lactoside

22 was obtained in 70% yield, although a small amount

(ca. 19%) of the starting material was also always

recovered.

Ph

O

OH

O

OH

O

RO

OMe

HO

OH

21 R = H

22 R = ClAc

Starting with the C-3⁰chloroacetylated lactoside 22, the

successful synthesis of the desired C-3⁰thiolacetylated

lactoside 23⁴⁹is shown in Scheme 4. Brieﬂy, complete

benzoylation of the hydroxyl groups in the C3⁰-chloro-

acetate derivative 22 was achieved by using benzoyl

chloride and then BzOTf⁵⁸to give the tetra-O-benzoy-

lated lactoside 24. Dechloroacetylation of 24 with

hydrazinium acetate⁵⁹gave the C-3⁰hydroxyl derivative

25 in 78% yield. Inversion of conﬁguration at C-3⁰via

With such encouraging results on the galactoside 17, this

chemistry was explored on the known⁵⁷4⁰,6⁰-O-benzy-

lidenated lactoside 21. The initial observation from these

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

743

Ph

O

b

OR

O

OBz

O

ClAcO

O

HO

OMe

RO

BzO

OR

BzO

OBz

OR

22 R = H

24 R = Bz

25

a

c,d

Ph

O

c,e

OBz

O

OBz

O

AcS

OMe

BzO

HO

OBz

26

23

˚

Scheme 4. Reagents and conditions: (a) 4 A sieves, BzCl, pyridine, CH₂Cl₂, 0 ꢂC to rt, 2 h, and then 4 A sieves, BzOTf, pyridine, CH₂Cl₂, ꢁ78 ꢂC to

room temperature, 3 h, 85%; (b) H₂NNH₂ÆHOAc, DMF, 40 ꢂC, 2 h, 78%; (c) Tf₂O, pyridine, CH₂Cl₂, ꢁ78 to 0 ꢂC, 1–2 h; (d) KNO₂, DMF, 50 ꢂC,

16 h, 76%; (e) KSAc, DMF, 50 ꢂC, 16 h, 83%.

activation with a triﬂate and subsequent displacement

60

groups (NaOH in MeOH) followed by removal of the

4⁰,6⁰-O-benzylidene acetal (10% aqueous TFA) to aﬀord

the target compound 9 in good yield, after formation of

the sodium salt and HPLC puriﬁcation.

with KNO₂aﬀorded the gulo-conﬁgured derivative

26 in good yield. Formation of the C-3⁰triﬂate deriva-

tive of 26 and treatment with KSAc aﬀorded the desired

C-3⁰thiolacetylated lactoside derivative 23 in 83% yield

over the two steps.

The a(2,3)-linked lactose-based sialylmimetics of the

general structure 10 were prepared according to the

procedure we have successfully employed previously

in the synthesis of a variety of a(2,6)-linked galac-

tose-, glucose- and lactose-based sialylmimetics.^41,67

The general approach is shown in Scheme 6 and in-

volves the selective in situ de-S-acetylation of the C-

3⁰thiolacetate lactoside 23 with hydrazinium acetate⁶⁴

and subsequent reaction with commercially available

a-halo-esters in the presence of Et₃N to give the sial-

ylmimetics 30 in excellent yield (Scheme 6). Deprotec-

tion of sialylmimetics 30 was readily achieved under

the same conditions described for deprotection of the

thiosialoside 29, wherein saponiﬁcation of the ester

protecting groups (NaOH in MeOH) followed by

removal of the 4⁰,6⁰-O-benzylidene acetal (10% aqueous

TFA) gave the deprotected lactose-based sialylmimetics

10 in generally high yield after HPLC puriﬁcation

(Scheme 6).

Having prepared the key C-3⁰thiolacetylated lactoside

derivative 23, our attention was directed towards the syn-

thesis of Neu5Ac2-S-a(2,3)-Lacb1Me (9). Although the

ﬁrst reported synthesis of a lactose-based S-glycosidation

reaction to form an a(2,3)-linked thiosialoside was only

published recently,⁵¹the general concept is well estab-

lished. Our strategy towards the synthesis of 9 employs

the successful approach described by Schmidt and co-

workers for the synthesis of Neu5Ac2-S-a(2,3)-Gal deriv-

atives.^61–63Preparation of the glycosyl acceptor was

achieved by reaction of 23 with hydrazinium acetate⁶⁴

to liberate the thiol 27. Exposure of 27 to the sialosyl chlo-

ride 28^65,66under base (NaH) promoted S-glycosidation

conditions furnished the desired thiosialoside 29 in 57%

yield (Scheme 5). Deprotection of the a(2,3)-linked thios-

ialoside 29 was readily achieved via a two-step process

involving initial saponiﬁcation of the ester protecting

Ph

O

Ph

O

H

OAc

R

O

MeO₂C

O

OBz

O

AcO

b

O

BzO

O

S

RS

OMe

H

OAc

R

BzO

Cl

AcO

BzO

H

AcO

OBz

O

CO₂Me

29 R = NHAc

23 R = Ac

27 R = H

a

AcO

H AcO

28 R = NHAc

c,d

H

OH

O

HO

S

NaO₂C

OH

O

HO

O

HO

R

H

OMe

OH

HO

OH

9 R = NHAc

ꢁ

˚

Scheme 5. Reagents and conditions: (a) H₂NNH₂ÆHOAc, DMF, 3 h, 78%; (b) 4 A sieves, 28, NaH, Kryptoﬁx , THF, 0 ꢂC to rt, ꢀ2 h, 57%; (c) 1 M

NaOH, MeOH, rt, 24 h, quantitative; (d) 10% aq. TFA, 0 ꢂC, 8 h, 64%.

744

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

Ph

O

R

O

OBz

O

OBz

O

BzO

R'O₂C

S

O

OMe

O

BzO

S

OMe

BzO

R

OBz

BzO

OBz

O

31 R = H; R' = Et (90%)

36 R = H (85%)

37 R = Me (87%)

32 R = Me; R' = Et (84%)

33 R = Et; R' = Me (85%)

34 R = Pr; R' = Et (96%)

35 R = Ph; R' = Me (90%)

Ph

O

OH

O

HO

OH

O

OH

O

HO

NaO₂C

S

O

HO

HO₂C

S

OMe

OH

R

OH

R

45 R = H (67%)

46 R = Me (66%)

47 R = Et (65%)

48 R = Pr (69%)

49 R = Ph (69%)

38 R = H (100%)

39 R = Me (95%)

40 R = Et (97%)

41 R = Pr (100%)

42 R = Ph (100%)

50 R = CH₂CH₂OH (70%)

51 R = CH₂CH(OH)CH₃(68%)

43 R = CH₂CH₂OH (100%)

44 R = CH₂CH(OH)CH₃(88%)

The lactose-based sialylmimetics 31–37 were prepared

in this way.⁴⁹Since the a-halo-esters employed in

the coupling with 23 to give the products 32–37

were racemic, the products were obtained as mix-

tures of diastereomers. Interestingly however, it was

found that the majority of sialylmimetics 32–37 were

formed with one diastereomer predominating over

the other. Indeed, several of the sialylmimetics were

obtained as 2:1 mixtures. Whilst there is no clear

explanation for this outcome, the implication from

this may be that one face of the a-bromo-ester elec-

trophile is less accessible to the intermediate thiolate

nucleophile than the other, presumably due to

hydrogen bonding or unfavourable steric interactions

with the benzoyl protecting groups. In any event,

the high yielding nature of these coupling reactions,

together with the fact that racemisation of the unde-

ﬁned chiral centre in the mimetic portion will occur

during deprotection, means that this issue was not

further pursued. Saponiﬁcation of compounds 31–37

with dilute NaOH in MeOH gave the de-esteriﬁed

products 38–44 in excellent yield, and following de-

benzylidenation (90% aq. TFA) and HPLC puriﬁca-

tion, the target compounds 45–51 were obtained in

consistently high yields.

2.2. Biological evaluation of sialylmimetics as inhibitors of

rotaviral infection

Some of the synthesised sialylmimetics were evaluated as

potential inhibitors of rotaviral infection using a stan-

dard in vitro neutralisation assay^40,41as described in

Section 4. In summary, the assay involves preincubation

of the sialylmimetic to be evaluated with either a rhesus

(RRV) or human (Wa) strain of rotavirus, prior to incu-

bation onto MA104 cells (an African Green Monkey

kidney cell line). After incubation, virus neutralisation

was determined using indirect immunoﬂuorescent stain-

ing. The results from these biological evaluations are

shown in Table 1 and indicate the ability of the sialylmi-

metic at 10 mM to cause a decrease in rotavirus infectiv-

ity of MA104 cells (expressed as

inhibition), unless otherwise indicated.

a percentage

As the results in Table 1 indicate, none of the sial-

ylmimetics showed any signiﬁcant inhibition against

the rhesus (RRV) strain of rotavirus. The lack of

inhibition is perhaps a reﬂection on the apparent

preference for rhesus (RRV) VP8* to recognise a-

linked sialic acid only. This is based upon the obser-

vation by Dormitzer et al. that the haemagglutinin

Ph

O

OH

O

HO

S

O

OH

O

OBz

O

OBz

O

a

b,c

O

NaO₂C

O

AcS

R'O₂C

S

OMe

HO

BzO

OH

BzO

OH

R

OBz

R

23

10 R = alkyl, aryl

30 R = alkyl, aryl; R' = Me, Et

˚

Scheme 6. Reagents and conditions: (a) 4 A sieves, H₂NNH₂HOAc, a-halo-ester, Et₃N, DMF, rt, 2 h; (b) 1 M NaOH, MeOH, rt, 24 h; (c) 10% aq.

TFA, 0 ꢂC, 8 h.

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

Table 1. Inhibition of rotavirus by sialylmimetics^a

745

acid-recognising proteins that naturally recognise the

Neu5Ac-a(2,3)-Gal epitope in general, and has provided

a better understanding of the interactions between sialo-

sides or sialylmimetics and rotavirus. The preliminary

biological results obtained by evaluation of these com-

pounds against the human (Wa) strain of rotavirus pro-

vide some interesting data and clearly show that these

compounds interfere in the infectivity of rotavirus in vi-

tro. The albeit modest inhibition shown by these sial-

ylmimetics against the human (Wa) strain of rotavirus

provides a valuable starting point for the development

of sialylmimetics with improved binding aﬃnities and

may ultimately lead to the discovery of a potential che-

motherapeutic agent for rotaviral infection.

Compound

RRV (rhesus)

Wa (human)

9

45

46

47

48

49

50

31

10

n.t.^b

21 (5 mM)

15

37

30

25

0

<10

^aResults are expressed as a percentage inhibition and indicate the

decrease in rotavirus infectivity of MA104 cells at 10 mM concen-

tration of compound, unless otherwise indicated.

^bn.t. indicates the compound was not tested.

OH

O

HO

S

OH

O

(VP8*) of RRV required only a-linked Neu5Ac for

binding, and this was irrespective of both the type

of carbohydrate the Neu5Ac was linked to, and

whether the linkage was a(2,3) or a(2,6).³⁸Our previ-

ously published results with lactose-based sialylmimet-

ics of a(2,6)-linkages also support this notion with

respect to rhesus (RRV) rotavirus.⁴¹The results ob-

tained from the biological evaluation of the a(2,3)-

linked thiosialoside 9 (Table 1) are also consistent

with the known speciﬁcity of the rhesus (RRV) strain

of rotavirus. As expected, the sialoside 9 showed

modest inhibition against rhesus rotavirus and is sim-

ply an indication that the haemagglutinin protein

recognises the Neu5Ac unit of the thiosialoside 9.

O

HO

NaO₂C

OMe

OH

R

10 R = alkyl, aryl

4. Experimental section

4.1. General

Infrared spectra were recorded on a Bruker Optik Vec-

¨

tor 22 instrument using OPUS (version 3.01) software.

IR spectra were obtained in neat CHCl₃solutions be-

tween KBr plates. ¹H and ¹³C NMR spectra were

recorded using a Bruker Avance-300 spectrometer un-

¨

less indicated otherwise. Chemical shifts are expressed

The results shown in Table 1 for inhibition of the

human (Wa) strain of rotavirus by the lactose-based

sialylmimetics of a(2,3)-sialosides are somewhat more

encouraging. Most of these sialylmimetics appear to

be modest inhibitors of the human (Wa) strain of

rotavirus in comparison to the rhesus (RRV) strain.

The observation that the a(2,3)-linked mimetics

shown in Table 1 show modest inhibition of Wa (hu-

man) rotavirus is consistent with our earlier studies

into the inhibition of rotavirus by lactose-based

a(2,6)-linked mimetics.⁴¹These previous studies found

that the lactose-based sialylmimetic 7 exhibited 50%

inhibition of human rotavirus at 6.25 mM.⁴¹At this

stage it is not appropriate to draw too many conclu-

sions from the data presented here (Table 1), given

that no structural information from the human

(Wa) haemagglutinin is available. However, it could

be speculated that the mimetic portion (alkyl and

aryl groups) in these sialylmimetics is tolerated by

the human (Wa) strain of rotavirus, either through

binding to the haemagglutinin protein or by some

other unknown viral adhesion process.

in ppm (d) relative to the solvent used (CDCl₃: 7.27,

1

77.0 for H and ¹³C, respectively) or relative to external

Me₄Si for D₂O spectra. Where due reference is made,

chemical shifts indicated with a multiplication (·)

descriptor correspond to more than one signal of the

same functionality. Assignments indicated with an aster-

isk ( ) correspond to those resonances clearly due to the

*

corresponding diastereomer where such mixtures exist,

while (ꢂ) indicates overlapping signals. Two dimensional

1

¹H–¹H COSY and H–¹³C HSQC NMR spectra were

obtained where necessary, in order to assist with spectral

assignment. ESI low resolution mass spectra were

recorded on a Bruker Daltonics^ꢁEsquire 3000 Ion-Trap

¨

LC MS, using the positive mode with samples intro-

duced at 180 lL/h. High resolution mass spectrometry

was performed at the Department of Chemistry, Univer-

sity of Queensland, Australia, on a Finnigan MAT 900

XL Trap with a Finnigan AP1 III sprayer. Microanaly-

ses were performed at the Department of Chemistry,

University of Queensland, Australia, and were recorded

on a Carlo Erba Elemental Microanalyser, model 1106.

Reactions were monitored by TLC (Merck silica gel

plates GF₂₅₄, Cat. No. 1.05554) and products were gen-

erally puriﬁed by ﬂash chromatography using Merck sil-

ica gel 60 (0.040–0.063 mm, Cat. No. 1.09385) unless

otherwise stated. Deprotected products were puriﬁed

by HPLC using a Phenomenex C₁₈reversed-phase col-

umn (semi-preparative). Methyl b-^D-galactopyranoside

was purchased from Sigma–Aldrich. Methyl b-^D-lacto-

side was prepared by treating hepta-O-acetyl-a-lactos-

ylbromide with sodium methoxide.⁶⁸BzOTf was

prepared by reaction of BzCl with TfOH and puriﬁed

3. Conclusion

In conclusion, we have developed an eﬃcient synthesis

of Neu5Ac2-S-a(2,3)-Lacb1Me 9 and have developed

a ﬂexible route towards a range of novel lactose-based

sialylmimetics of the general structure 10 that mimic

the key elements of the thiosialoside 9. The work de-

scribed here has contributed towards the development

of biological probes and possible inhibitors for sialic

746

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

by distillation.⁵⁸All commercial solvents were distilled

prior to use. Dried solvents were distilled under N₂

according to Armarego and Perrin.⁶⁹

(75.5 MHz, CDCl₃): d 40.9 (OC(O)CH₂Cl), 57.3

(OMe), 66.3 (C-5), 68.4 (C-2), 69.0 (C-6), 73.1 (C-4),

75.4 (C-3), 101.0 (CHPh), 103.9 (C-1), 2· 126.2, 2·

128.2, 129.1 (CHPh), 137.4 (ipsoPh), 167.4

(OC(O)CH₂Cl); LRMS 381 (M+Na, 100%).

4.2. Synthesis of 3⁰-thiolacetyl-lactoside derivatives.

Methyl 4,6-O-benzylidene-3-O-methanesulfonyl-b-^D-

Galactopyranoside (18)

4.5. Methyl 4,6-O-benzylidene-3-O-chloroacetyl-b-^D-

galactopyranosyl-(1,4)-O-b-^D-glucopyranoside (22)

A suspension of 4,6-O-benylidenated galactoside 17⁵⁷

(200 mg, 0.71 mmol) and Bu₂SnO (194 mg, 0.78 mmol)

was stirred in reﬂuxing dry MeOH (4 mL) under Ar

for 2 h. The mixture was cooled and concentrated (co-

evaporation with toluene) under reduced pressure. To

the crude stannylidene in dry toluene (4 mL) containing

A suspension of 4⁰,6⁰-O-benylidenated lactoside 21⁵⁷

(200 mg, 0.45 mmol) and Bu₂SnO (123 mg, 0.50 mmol)

was stirred in reﬂuxing dry MeOH (4 mL) under Ar

for 2 h. The mixture was cooled and concentrated (co-

evaporation with toluene) under reduced pressure. To

the crude stannylidene in dry DMF (1 mL) and dry tol-

˚

4 A sieves was added MsCl (60 lL, 0.78 mmol) under Ar

˚

uene (3 mL) containing 4 A sieves was added ClAcCl

at 0 ꢂC. The reaction mixture was left to stir at 0 ꢂC for

30 min before being ﬁltered through Celite (MeOH as

eluant) and concentrated under reduced pressure. Col-

umn chromatography (EtOAc/hexane, 2:1 ! neat

EtOAc, R_f0.46) aﬀorded 18 (255 mg, quantitative) as

(39 lL, 0.50 mmol) under Ar at 0 ꢂC. The reaction mix-

ture was left to stir at 0 ꢂC for 30 min before being ﬁl-

tered through Celite (MeOH as eluant) and

concentrated under reduced pressure. Column chroma-

tography (neat EtOAc ! EtOAc/MeOH, 30:1 !

10:1 ! 4:1, R_f0.52) aﬀorded 22 [164 mg, 70% (82%

based on recovered 21)] as an amorphous mass: ¹H

NMR (CDCl₃): d 3.38–3.43 (2H, m, Glc H-2, Glc H-

5), 3.54 (3H, s, OMe), 3.66^ꢂ(1H, d, J_5,4= 0.9 Hz, Gal

H-5), 3.69^ꢂ(1H, t, J_4,3= J_4,5= 9.1 Hz, Glc H-4), 3.79^ꢂ

(1H, t, J_3,2= J_3,4= 9.1 Hz, Glc H-3), 3.83^ꢂ(1H, dd,

1

an amorphous mass: H NMR (CDCl₃): d 3.17 (3H, s,

OMs), 3.52 (1H, s, H-5), 3.61 (3H, s, OMe), 4.05–4.13

(2H, m, H-2, H-6), 4.28 (1H, d, J_1,2= 7.7 Hz, H-1),

4.38 (1H, dd, J_{6 ,6}= 12.6, J_{6 ,5}= 1.3 Hz, H-6⁰), 4.43

(1H, d, J_4,3= 3.6 Hz, H-4), 4.66 (1H, dd, J_3,2= 10.0,

J_3,4= 3.6 Hz, H-3), 5.59 (1H, s, CHPh), 7.35–7.37,

7.51–7.54 (5H, 2· m, CHPh); ¹³C NMR (75.5 MHz,

CDCl₃): d 38.6 (OMs), 57.4 (OMe), 66.2 (C-5), 68.7

(C-2), 68.8 (C-6), 74.9 (C-4), 80.3 (C-3), 101.0 (CHPh),

103.9 (C-1), 2· 126.3, 2· 128.1, 129.1 (CHPh), 137.3 (ip-

soPh); LRMS 383 (M+Na, 100%); HRMS Calcd for

C₁₅H₂₀O₈S (M⁺) 360.0878. Found 360.0879.

0

J_6,6= 12.5, J_6,5= 3.0 Hz, Glc H-6), 4.01 (1H, dd,

0

J_6,6= 12.5, J_6,5= 2.8 Hz, Glc H-6⁰), 4.05–4.15 (2H, m,

Gal H-2, Gal H-6), 4.18 (2H, d, OClAc), 4.22 (1H, d,

0

J_1,2= 7.8 Hz, Glc H-1), 4.31 (1H, dd, J_{6 ,6}= 12.8,

0

J_{6 ,5}= 1.4 Hz, Gal H-6⁰), 4.40 (1H, d, J_4,3= 3.5,

J_4,5= 0.9 Hz, Gal H-4), 4.63 (1H, d, J_1,2= 8.0 Hz, Gal

H-1), 4.99 (1H, dd, J_3,2= 10.2, J_3,4= 3.5 Hz, Gal H-3),

5.50 (1H, s, CHPh), 7.33–7.42, 7.44–7.54 (5H, 2· m,

CHPh); ¹³C NMR (75.5 MHz, CDCl₃): d 41.0 (OC(O)

CH₂Cl), 57.3 (OMe), 61.1 (Glc C-6), 66.5, 74.8 (Glc

C-3, Gal C-5), 67.3 (Gal C-2), 68.9 (Gal C-6), 73.2,

74.6 (Glc C-2, Glc C-5), 75.0 (Gal C-3), 77.8 (Glc C-

4), 101.0 (CHPh), 103.1 (Gal C-1), 103.6 (Glc C-1), 2·

126.2, 2· 128.3, 129.2 (CH Ph), 137.2 (ipsoPh), 167.6

(OC(O)CH₂Cl); LRMS 543 (M+Na, 100%).

The following were prepared in a similar manner:

4.3. Methyl 4,6-O-benzylidene-3-O-[(triﬂuoromethyl)sul-

fonyl]-b-^D-galactopyranoside (19)

In 70% yield (column chromatography EtOAc/hexane,

1:1 ! 2:1 ! neat EtOAc, R_f0.62) as a colourless sol-

id: ¹H NMR (CDCl₃): d 3.52 (1H, d, J_5,4= 1.2 Hz,

H-5), 3.60 (3H, s, OMe), 4.07–4.15 (2H, m, H-2,

H-6), 4.29 (1H, d, J_1,2= 7.7 Hz, H-1), 4.41 (1H, dd,

4.6. Methyl 2-O-benzoyl-4,6-O-benzylidene-3-O-chloro-

acetyl-b-^D-galactopyranosyl-(1,4)-O-2,3,6-tri-O-benzoyl-

b-^D-glucopyranoside (24)

J_{6 ,6}= 12.6, J_{6 ,5}= 1.6 Hz, H-6⁰), 4.45 (1H, dd,

J_4,3= 3.8, J_4,5= 1.2 Hz, H-4), 4.85 (1H, dd,

J_3,2= 10.0, J_3,4= 3.8 Hz, H-3), 5.60 (1H, s, CHPh),

7.35–7.40, 7.49–7.53 (5H, 2· m, CHPh); LRMS 437

(M+Na, 100%); HRMS Calcd for C₁₅H₁₇O₈F₃S

(M+Na) 437.0494. Found 437.0493.

0

To a stirred solution of 22 (3 g, 5.76 mmol) in dry

˚

CH₂Cl₂(60 mL) containing 4 A sieves were added dry

pyridine (11.18 mL, 0.14 mol), DMAP (cat.) and then

BzCl (8.02 mL, 0.07 mmol) under Ar at 0 ꢂC. The mix-

ture was warmed to room temperature and stirred for

2 h before being diluted with CH₂Cl₂(150 mL), washed

with 0.1 M HCl (3· 100 mL), dried (Na₂SO₄) and con-

centrated under reduced pressure. Column chromatog-

raphy (EtOAc/hexane, 1:2) gave the desired tetra-O-

benzoylated derivative 24 [2.70 g, 50% (EtOAc/hexane,

1:1, R_f0.42)] and the tri-O-benzoylated derivative

[1.92 g, 40% (EtOAc/hexane, 1:1, R_f0.40)]. BzOTf

(1.02 mL, 4.61 mmol) was added dropwise to a solution

of the tri-O-benzoylated derivative (1.92 g, 2.30 mmol)

in dry CH₂Cl₂(25 mL) and dry pyridine (559 lL,

4.4. Methyl 4,6-O-benzylidene-3-O-chloroacetyl-b-^D-

galactopyranoside (20)

In 79% yield (column chromatography EtOAc/hexane,

1:3 ! 1:2 ! 1:1, R_f0.12 ! neat EtOAc) as a colourless

1

solid: IR 1051, 1759, 3456 cm^ꢁ1; H NMR (CDCl₃): d

3.56 (1H, s, H-5), 3.61 (3H, s, OMe), 4.01–4.12 (2H,

m, H-2, H-6), 4.18 (2H, d, OClAc), 4.31 (1H, d,

J_1,2= 7.7 Hz, H-1), 4.38 (1H, dd, J_{6 ,6}= 13.7 Hz, H-6⁰),

0

4.45 (1H, d, J_4,3= 3.5 Hz, H-4), 4.95 (1H, dd,

J_3,2= 10.2, J_3,4= 3.5 Hz, H-3), 5.53 (1H, s, CHPh),

7.36–7.38, 7.49–7.52 (5H, 2· m, CHPh); ¹³C NMR

˚

6.91 mmol) containing 4 A sieves under Ar at ꢁ78 ꢂC.

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

747

The mixture was warmed to 0 ꢂC, left to stir for 1 h and

then allowed to warm to room temperature. The mix-

ture was stirred for a further 2 h at room temperature

before being diluted with CH₂Cl₂(100 mL), washed

with saturated aqueous NaHCO₃(3· 70 mL) and then

0.1 M HCl (3· 70 mL), dried (Na₂SO₄) and concentrat-

ed. Column chromatography (EtOAc/hexane, 2:3 !

1:1) aﬀorded 24 [1.89 g, 88% (overall yield from 22 is

85%)] which was crystallised from EtOAc/hexane to give

128.1, 128.2, 2· 128.3, 2· 129.1, 129.6, 2· 129.7, 129.8,

133.0, 2· 133.1 (5· Ph), 129.2, 129.3, 129.5, 129.7,

137.3 (5· ipsoPh), 165.0, 165.4, 165.7, 165.8 (4·

OC(O)Ph); LRMS 893 (M+Na, 100%).

4.8. Methyl 2-O-benzoyl-4,6-O-benzylidene-b-^D-gulopyr-

anosyl-(1,4)-O-2,3,6-tri-O-benzoyl-b-^D-glucopyranoside

(26)

1

colourless needles: IR 1071, 1316, 1726 cm^ꢁ1; H NMR

Tf₂O (218 lL, 1.30 mmol) was added dropwise to a stirred

solution of 25 (559 mg, 0.65 mmol) in dry CH₂Cl₂

(11 mL) and dry pyridine (210 lL, 2.60 mmol) under N₂

at ꢁ78 ꢂC. The mixture was then warmed to 0 ꢂC and left

to stir for 1 h before being diluted with CH₂Cl₂(40 mL),

washed with 0.1 M HCl (20 mL) and H₂O (20 mL), dried

(Na₂SO₄) and concentrated under reduced pressure. To

the crude triﬂate in dry DMF (11 mL) under N₂at room

temperature was added KNO₂(276 mg, 3.25 mmol). The

mixture was warmed to 50 ꢂC and left to stir for 16 h be-

fore being concentrated under reduced pressure. Column

chromatography (EtOAc/hexane, 1:2 ! 2:3 ! 1:1, R_f

0.34) gave 26 (426 mg, 76%) which was crystallised from

EtOAc/hexane to give colourless needles: IR 1116, 1272,

(CDCl₃): d 2.92 (1H, s, Gal H-5), 3.51 (3H, s, OMe),

0

3.56 (1H, appd, J_6,6= 12.4 Hz, Gal H-6), 3.74–3.97

(4H, m, Glc H-5, Gal H-6⁰, OClAc), 4.18–4.24 (2H, m,

0

Glc H-4, Gal H-4), 4.38 (1H, dd, J_6,6= 12.0,

J_6,5= 4.1 Hz, Glc H-6), 4.61^ꢂ(1H, d, J_1,2= 7.7 Hz, Glc

H-1), 4.59–4.63^ꢂ(1H, m, Glc H-6⁰), 4.78 (1H, d,

J_1,2= 7.9 Hz, Gal H-1), 5.03 (1H, dd, J_3,2= 10.4,

J_3,4= 3.6 Hz, Gal H-3), 5.30^ꢂ(1H, s, CHPh), 5.31^ꢂ

(1H, dd, J_2,3= 9.2, J_2,1= 7.7 Hz, Glc H-2), 5.62 (1H,

dd J_2,3= 10.4, J_2,1= 7.9 Hz, Gal H-2), 5.83 (1H, t,

J_3,2= J_3,4= 9.2 Hz, Glc H-3), 7.30–7.62, 7.87–8.04

(25H, 2· m, 5· Ph); ¹³C NMR (75.5 MHz, CDCl₃): d

40.5 (OC(O) CH₂Cl), 57.0 (OMe), 62.4 (Glc C-6), 66.2

(Gal C-5), 67.9 (Gal C-6), 69.2 (Gal C-2), 72.3 (C-4),

72.7 (Glc C-2, Glc C-3), 73.5 (Gal C-3), 74.0 (Glc

C-3), 76.8 (C-4), 100.8 (CHPh), 101.3 (C-1), 101.5

(C-1), 2· 128.3, 128.4, 128.5, 129.0, 129.6, 129.7,

129.9, 130.2, 133.0, 133.1, 133.4, 133.7, 137.2 (5· Ph),

128.6, 129.4, 129.5, 137.3 (5· ipsoPh), 2· 164.9, 165.4,

165.7 (4· OC(O)Ph), 167.2 (OC(O)CH₂Cl); LRMS 959

(M+Na, 100%); Anal. (C₅₀H₄₅O₁₆Cl) C, H.

1

1728, 3521 cm^ꢁ1; H NMR (CDCl₃): d 3.25 (1H, s, Gal

0

H-5), 3.39 (3H, s, OMe), 3.49 (1H, dd, J_6,6= 12.4,

0

J_6,5= 1.7 Hz, Gal H-6), 3.60 (1H, appd, J_{6 ,6}= 12.4 Hz,

Gal H-6⁰), 3.75–3.81 (2H, m, Glc H-5, Gal H-4), 4.12

(1H, t, J_4,3= J_4,5= 9.3 Hz, Glc H-4), 4.18 (1 H, t,

J_3,2= J_3,4= 3.2 Hz, Gal H-3), 4.50-4.60 (3H, m, Glc H-

1, Glc H-6, Glc H-6⁰), 5.04 (1H, d, J_1,2= 8.4 Hz, Gal H-

1), 5.24^ꢂ(1H, s, CHPh), 5.25^ꢂ(1H, dd, J_2,1= 8.4,

J_2,3= 3.2 Hz, Gal H-2), 5.31 (1H, dd, J_2,3= 9.3,

J_2,1= 7.8 Hz, Glc H-2), 5.73 (1H, t, J_3,2= J_3,4= 9.3 Hz,

Glc H-3), 7.18–7.55, 7.88–8.06 (25H, 2· m, 5· Ph). ¹³C

NMR (75.5 MHz, CDCl₃): d 57.0 (OMe), 62.6 (Glc C-

6), 65.6 (Gal C-6), 68.1 (Gal C-5), 68.8 (Gal C-3), 71.4

(Gal C-2), 72.1 (Glc C-2), 73.0, 75.8 (Glc C-5, Gal C-4),

73.6 (Glc C-3), 76.5 (Glc C-4), 98.6 (Gal C-1), 100.8

(CHPh), 101.6 (Glc C-1), 126.4, 128.0, 128.1, 128.2,

128.3, 128.5, 128.9, 2· 129.6, 129.7, 129.8, 132.8, 133.0,

133.1, 133.3 (5· Ph), 129.1, 129.3, 129.6, 137.5 (5·

ipsoPh), 164.7, 165.2, 165.4, 166.1 (4· OC(O)Ph);

LRMS 883 (M+Na, 100%); Anal. (C₄₈H₄₄O₁₅) C, H.

4.7. Methyl 2-O-benzoyl-4,6-O-benzylidene-b-^D-galacto-

pyranosyl-(1,4)-O-2,3,6-tri-O-benzoyl-b-^D-glucopyrano-

side (25)

To a stirred solution of 24 (2 g, 2.13 mmol) in dry DMF

(20 mL) was added hydrazinium acetate (393 mg,

4.27 mmol) under N₂at room temperature. The mixture

was warmed to 40 ꢂC and left to stir for 2 h before being

diluted with EtOAc (100 mL), washed with 0.1 M HCl

(3· 70 mL), dried (Na₂SO₄) and concentrated under re-

duced pressure. Column chromatography (EtOAc/hex-

ane, 1:2 ! 2:3 ! 1:1, R_f0.25) aﬀorded 25 (1.85 g,

1

78%) as a colourless solid: H NMR (CDCl₃): d 2.87

4.9. Methyl 3-S-acetyl-2-O-benzoyl-4,6-O-benzylidene-

b-^D-galactopyranosyl-(1,4)-O-2,3,6-tri-O-benzoyl-b-D-

glucopyranoside (23)

(1H, s, Gal H-5), 3.35 (3H, s, OMe), 3.50 (1H, dd,

0

J_6,6= 12.3, J_6,5= 1.6 Hz, Gal H-6), 3.61^ꢂ(1H, appd,

ꢂ

J_{6 ,6}= 12.3 Hz, Gal H-6⁰), 3.64 (1H, dd, J_3,2= 10.0,

J_3,4= 3.6 Hz, Gal H-3), 3.77 (1H, ddd, J_5,4= 9.5,

0

Tf₂O (397 lL, 2.36 mmol) was added dropwise to a stir-

red solution of 26 (678 mg, 0.79 mmol) in dry CH₂Cl₂

(13 mL) and dry pyridine (382 lL, 4.23 mmol) under

N₂at ꢁ78 ꢂC. The mixture was warmed to 0 ꢂC, left

to stir for 1 h and then allowed to warm to room tem-

perature. The mixture was stirred for a further 1 h at

room temperature before being diluted with CH₂Cl₂

(60 mL), washed with 0.1 M HCl (40 mL) and H₂O

(40 mL), dried (Na₂SO₄) and concentrated under re-

duced pressure. To a stirred solution of the crude triﬂate

in dry DMF (13 mL) was added KSAc (270 mg,

2.36 mmol) under N₂at room temperature. The mixture

was warmed to 50 ꢂC and left to stir for 16 h before

being concentrated under reduced pressure. Column

chromatography (EtOAc/hexane, 1:2 ! 2:3 ! 1:1, R_f

0

J_5,6= 4.4, J_5,6= 2.1 Hz, Glc H-5), 3.92 (1H, d,

J_4,3= 3.6 Hz, Gal H-4), 4.10 (1H, t, J_4,3= J_4,5

0

=

9.5 Hz, Glc H-4), 4.40 (1H, dd, J_6,6= 12.0,

J_6,5= 4.4 Hz, Glc H-6), 4.52^ꢂ(1H, d, J_1,2= 7.8 Hz, Glc

H-1), 4.55^ꢂ(1H, dd, J_{6 ,6}= 12.0, J_{6 ,5}= 2.1 Hz, Glc H-

6⁰), 4.59 (1H, d, J_1,2= 8.0 Hz, Gal H-1), 5.23^ꢂ(1H, dd,

J_2,3= 10.0, J_2,1= 8.0 Hz, Gal H-2), 5.25^ꢂ(1H, s, CHPh),

5.28 (1H, dd, J_2,3= 9.5, J_2,1= 7.8 Hz, Glc H-2), 5.73

(1H, t, J_3,2= J_3,4= 9.5 Hz, Glc H-3), 7.22–7.55, 7.86–

7.97 (25H, 2· m, 5· Ph); ¹³C NMR (75.5 MHz, CDCl₃):

d 56.9 (OMe), 62.5 (Glc C-6), 66.5 (Gal C-5), 67.9 (Gal

C-6), 71.6 (Gal C-3), 72.1 (Glc C-2), 2· 72.9 (Glc C-5,

Gal C-2), 73.6 (Glc C-3), 75.0 (Gal C-4), 76.6 (Glc C-

4), 2· 101.2 (Gal C-1, CHPh), 101.6 (Glc C-1), 126.5,

0

748

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

0.60) gave 23 (613 mg, 83%) which was crystallised from

EtOAc/hexane to give an oﬀ-white crystalline solid: H

NMR (CDCl₃): d 2.11 (3H, s, SAc), 2.98 (1H, s, Gal

0

H-5), 3.40 (3H, s, OMe), 3.53 (1H, dd, J_6,6= 12.4,

J_6,5= 1.7 Hz, Gal H-6), 3.78–3.86 (3H, m, Glc H-5,

Gal H-4, Gal H-6⁰), 4.08 (1H, dd, J_3,2= 11.5,

J_3,4= 3.3 Hz, Gal H-3), 4.22 (1H, t, J_4,3= J_4,5= 9.2 Hz,

0

Glc H-4), 4.30 (1H, dd, J_6,6= 12.1, J_6,5= 4.2 Hz, Glc

H-6), 4.59 (1H, d, J_1,2= 7.7 Hz, Glc H-1), 4.65 (1H,

4.11. Methyl [methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-

3,5-dideoxy-2-thio-^D-glycero-a-D-galacto-2-nonulopyr-

anosyl)onate]-(2,3)-S-(2-O-benzoyl-4,6-O-benzylidene-3-

thio-b-^D-galactopyranosyl)-(1,4)-O-2,3,6-tri-O-benzoyl-b-

D-glucopyranoside (29)

1

To a stirred solution of 27 (205 mg, 0.23 mmol) in dry

THF (8 mL) was added NaH (11 mg, 0.27 mmol) under

Ar at 0 ꢂC. After 10 min, Kryptoﬁx 21^ꢁ(10 mg,

0.05 mmol) and then a solution of 28^65,66(124 mg,

0.24 mmol) in dry THF (2 mL) were added to the reac-

tion before being warmed to room temperature. After

2 h, the mixture was diluted with EtOAc (40 mL),

washed with brine (3· 30 mL), dried (Na₂SO₄), and con-

centrated under reduced pressure. Column chromatogra-

phy (EtOAc/hexane, 1:1 ! 3:1, R_f0.13) gave 29 (180 mg,

57%) as an amorphous mass: IR 1113, 1269, 1734,

dd, J_{6 ,6}= 12.1, J_{6 ,5}= 2.0 Hz, Glc H-6⁰), 4.85 (1H, d,

J_1,2= 7.7 Hz, Gal H-1), 5.29 (1H, dd, J_2,3= 9.2,

J_2,1= 7.7 Hz, Glc H-2), 5.31 (1H, s, CHPh), 5.40 (1H,

dd, J_2,3= 11.5, J_2,1= 7.7 Hz, Gal H-2), 5.84 (1H, t,

J_3,2= J_3,4= 9.2 Hz, Glc H-3), 7.18–7.61, 7.82–8.01

(25H, 2· m, 5· Ph); ¹³C NMR (75.5 MHz, CDCl₃): d

30.4 (SC(O)CH₃), 46.7 (Gal C-3), 56.9 (OMe), 62.2

(Glc C-6), 67.0 (Glc C-6), 68.4 (Gal C-5), 69.5 (Gal C-

2), 72.3, 75.2 (Glc C-5, Gal C-4), 72,7 (Glc C-2), 74.3

(Gal C-5), 76.6 (Glc C-4), 100.9 (CHPh), 101.4 (Glc C-

1), 102.6 (Gal C-1), 126.2, 128.0, 128.1, 2· 128.3, 128.4,

128.9, 2· 129.6, 129.7, 129.9, 132.8, 2· 133.1 (5· Ph),

164.7, 165.1, 165.4, 165.5 (4· OC(O)Ph), 128.8, 129.3,

129.5, 137.3 (5· ipsoPh), 164.7, 165.1, 165.4, 165.5 (4·

OC(O)Ph), 194.8 (SC(O)CH₃); LRMS 941 (M+Na,

100%); Anal. (C₅₀H₄₆O₁₅S) C, H.

0

1

3018 cm ^ꢁ1; H NMR (CDCl₃): d 1.71 (3H, s, NAc),

1.83^ꢂ, 1.95, 1.99, 2.17 (12H, 4· s, 4· OAc), 1.81–1.87^ꢂ

(1H, m, Neu H-3a), 2.53 (1H, dd, J_3e,3a= 12.8,

J_3e,4= 4.7 Hz, Neu H-3e), 3.33 (1H, s, Gal H-5), 3.38

(3H, s, OMe), 3.45 (1H, d, J_4,3= 2.7 Hz, Gal H-4), 3.57

0

(1H, appd, J_6,6= 11.0 Hz, Gal H-6), 3.68 (1H, dd,

J_6,5= 10.8, J_6,7= 2.4 Hz, Neu H-6), 3.70 (3H, s,

CO₂Me), 3.72–3.86 (4H, m, Glc H-5, Gal H-3, Gal

H-6⁰, Neu H-5), 4.22 (1H, dd, J_9,9= 12.7,

J_9,8= 4.8 Hz, Neu H-9), 4.30-4.38 (2H, m, Glc H-4,

0

4.10. Synthesis of S-linked a(2,3)-sialyllactoside. Methyl

2-O-benzoyl-4,6-O-benzylidene-3-thio-b-^D-galactopyr-

anosyl-(1,4)-O-2,3,6-tri-O-benzoyl-b-^D-glucopyranoside

(27)

0

Glc H-6), 4.43 (1H, dd, J_{9 ,9}= 12.7, J_{9 ,8}= 2.4 Hz, Neu

H-9⁰), 4.55 (1H, d, J_1,2= 7.8 Hz, Glc H-1), 4.63 (1H,

dd, J_{6 ,6}= 12.0, J_{6 ,5}= 2.0 Hz, Glc H-6⁰), 4.80 (1H, td,

J_4,3a= J_4,5= 11.6, J_4,3e= 4.7 Hz, Neu H-4), 5.01 (1H,

d, J_NH,5= 9.8 Hz, NH), 5.19-5.27 (4H, m, Gal H-1,

Gal H-2, Neu H-7, CHPh), 5.30 (1H, dd, J_2,3= 9.5,

J_2,1= 7.8 Hz, Glc H-2), 5.69 (1H, ddd, J_8,7= 10.2,

0

To a stirred solution of 23 (300 mg, 0.32 mmol) in dry

DMF (3 mL) was added hydrazinium acetate (39 mg,

0.64 mmol) under N₂at room temperature. The mixture

was left to stir for 3 h before being diluted with EtOAc

(30 mL), washed with 0.1 M HCl (3· 20 mL), dried

(Na₂SO₄) and concentrated under reduced pressure.

Column chromatography (CHCl₃/hexane, 1:1 ! 1:2)

aﬀorded 27 [219 mg, 78% (neat CHCl₃, R_f0.25)] as a col-

0

J_8,9= 4.8, J_8,9= 2.4 Hz, Neu H-8), 5.78 (1H, t,

J_3,2= J_3,4= 9.5 Hz, Glc H-3), 7.20–7.62, 7.91–7.98,

8.13–8.16 (25H, 3· m, 5· Ph); ¹³C NMR (75.5 MHz,

CDCl₃): d 20.6, 20.7, 20.8, 21.3 (4· OC(O) Me), 23.1

(NC(O) Me), 37.0 (Neu C-3), 45.4, 49.0 (Gal C-3, Neu

C-5), 52.9 (CO₂Me), 56.8 (OMe), 62.2 (Neu H-9), 62.8

(Glc C-6), 66.6, 69.0 (Gal C-2, Neu C-7), 67.0 (Gal C-

5), 67.8 (Neu C-8), 68.1 (Gal C-6), 69.3 (Neu C-4),

72.3, 73.0, 73.4 (Glc C-2, Glc C-5, Neu C-6), 73.8, (Glc

C-3), 75.6 (Gal C-4), 75.9 (Glc C-4), 101.1, 101.4, 101.5

(Glc C-1, Gal C-1, CHPh), 126.3, 127.9, 128.0, 2·

128.2, 128.3, 128.6, 2· 129.6, 129.8, 130.0, 132.6, 132.8,

133.0 (5· Ph), 129.3, 129.6, 130.1, 137.5 (4· ipsoPh),

165.2, 165.4, 165.5, 165.6 (4· OC(O)Ph), 169.5, 169.9,

170.1, 170.2, 170.7, 170.8 (4· OC(O)Me, NC(O)Me,

CO₂Me); LRMS 1372 (M+Na, 100%); HRMS Calcd

for C₆₈H₇₁NO₂₆S (M+Na) 1372.3883. Found 1372.390.

ourless solid: IR 1114, 1268, 1728 cm^ꢁ^{1 1}H NMR

;

(CDCl₃): d 2.12 (1H, d, J_SH,3= 11.1 Hz, SH), 2.88

(1H, td, J_3,2= J_3,SH= 11.1, J_3,4= 2.5 Hz, Gal H-3),

2.88 (1H, s, Gal H-5), 3.42 (3H, s, OMe), 3.57 (1H,

0

dd, J_6,6= 12.4, J_6,5= 1.8 Hz, Gal H-6), 3.75 (1H, dd,

0

J_{6 ,6}= 12.4, J_{6 ,5}= 1.0 Hz, Gal H-6⁰), 3.83 (1H, ddd,

0

J_5,4= 9.5, J_5,6= 4.2, J_5,6= 2.0 Hz, Glc H-5), 3.89 (1H,

d, J_4,3= 2.5 Hz, Gal H-4), 4.18 (1H, t, J_4,3= J_4,5

0

=

9.5 Hz, Glc H-4), 4.41 (1H, dd, J_6,6= 12.1,

J_6,5= 4.2 Hz, Glc H-6), 4.59^ꢂ(1H, d, J_1,2= 7.8 Hz, Glc

H-1), 4.60^ꢂ(1H, dd, J_{6 ,6}= 12.1, J_{6 ,5}= 2.0 Hz, Glc H-

6⁰), 4.68 (1H, d, J_1,2= 7.8 Hz, Gal H-1), 5.26^ꢂ(1H, dd,

J_2,3= 11.1, J_2,1= 7.8 Hz, Gal H-2), 5.32^ꢂ(1H, dd,

J_2,3= 9.5, J_2,1= 7.8 Hz, Glc H-2), 5.35^ꢂ(1H, s, CHPh),

5.81 (1H, t, J_3,2= J_3,4= 9.5 Hz, Glc H-3), 7.61–7.62,

7.88–8.04 (25H, 2· m, 5· Ph); ¹³C NMR (75.5 MHz,

CDCl₃): d 43.5 (Gal C-3), 56.9 (OMe), 62.4 (Glc C-6),

67.9 (Gal C-6), 68.8 (Gal C-5), 72.1 (Glc C-2), 72.6,

72.7 (Glc C-5, Gal C-2), 73.7 (Glc C-3), 75.8 (Gal C-4),

76.3 (Glc C-4), 101.0 (CHPh), 101.4 (Glc C-1), 102.4

(Gal C-1), 126.2, 127.1, 127.9, 3· 128.2, 128.3, 128.8,

129.4, 129.5, 2· 129.6, 129.7, 132.9, 133.0, 133.1 (5·

Ph), 129.0, 129.2, 129.4 137.3 (4· ipsoPh), 164.8, 165.1,

165.3, 165.6 (4· OC(O)Ph); LRMS 899 (M+ Na, 100%).

0

4.12. Methyl (5-acetamido-3,5-dideoxy-2-thio-^D-glycero-

a-^D-galacto-2-nonulopyranosylonic acid)-(2,3)-S-(4,6-O-

benzylidene-3-thio-b-^D-galactopyranosyl)-(1,4)-O-b-D-

glucopyranoside

To a stirred solution of 29 (220 mg, 0.16 mmol) in

MeOH (5 mL) was added dropwise a 1 M solution of

NaOH (ꢀ1 mL) until a pH of ꢀ13 was attained. The

mixture was left to stir for 24 h before being neutralised

with Amberlyte IR-120 (H⁺), the resin ﬁltered, washed

with aqueous MeOH, and then the solvent was concen-

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

749

trated under reduced pressure. The residue was re-dis-

solved in H₂O (15 mL), the pH adjusted to ꢀ4 with

AcOH, washed with EtOAc (3· 30 mL) and the aqueous

phase freeze-dried to give the 4⁰,6⁰-O-benzylidenated

thiosialoside (122 mg, quantitative) as a colourless

solid: ¹H NMR (600 MHz, D₂O): d 1.76 (1H, dd,

J_3a,3e= 12.3, J_3a,4= 11.8 Hz, Neu H-3_a), 1.99 (3H, s,

NAc), 2.76 (1H, dd, J_3e,3a= 12.3, J_3e,4= 4.7 Hz, Neu

H-3e), 3.27 (1H, dd, J_2,3= 9.1, J_2,1= 8.2 Hz, Glc H-2),

3.49 (2H, m, Gal H-2, Gal H-3), 3.54 (3H, s, OMe),

3.55-3.58 (3H, m, Glc H-5, Neu H-6, Neu H-9), 3.61–

3.67 (4H, m, Glc H-3, Neu H-4, Neu H-5, Neu H-7),

3.80–3.87 (4H, m, Glc H-4, Glc H-6, Gal H-5, Neu H-

74.3, 74.8, 74.9 (Glc C-3, Glc C-5, Neu C-4), 77.7 (Gal

C-5), 78.0 (Neu C-6), 84.0 (Neu C-2), 103.0 (Glc C-1),

104.1 (Gal C-1), 174.5 (Neu C-1), 174.9 (NC(O)Me);

LRMS 686 (M+Na, 100%); Anal. (C₂₄H₄₀NNaO₁₈

S4H₂O) C, H.

4.14. Synthesis of sialylmimetics. Methyl 2-O-benzoyl-

4,6-O-benzylidene-3-thio-3-(ethoxycarbonylmethyl)-b-^D-

galactopyranosyl-(1,4)-O-2,3,6-tri-O-benzoyl-b-^D-gluco-

pyranoside (31)

A solution of 23 (300 mg, 0.32 mmol) in dry DMF

(6 mL) was thoroughly degassed (bubbling N₂) for

9⁰), 3.91 (1H, ddd, J_8,7= 8.8, J_8,9= 6.0, J_8,9= 2.3 Hz,

15 min before the addition of 4 A sieves and then hydra-

˚

0

Neu H-8), 4.01 (1H, dd, J_{6 ,6}= 12.0, J_{6 ,5}= 1.5 Hz, Glc

H-6⁰), 4.18–4.22 (3H, m, Gal H-4, Gal H-6, Gal H-6⁰),

4.37 (1H, d, J_1,2= 8.2 Hz, Glc H-1), 4.59 (1H, d,

J_1,2= 7.4 Hz, Gal H-1), 5.68 (1H, s, CHPh), 7.41–7.45,

7.48–7.51 (5H, 2· CHPh); ¹³C NMR (75.5 MHz,

CDCl₃): d 23.3 (NC(O) Me), 40.4 (Neu C-3), 48.2 (Glc

C-2), 51.6 (Neu C-5), 57.2 (OMe), 59.8 (Glc C-6), 62.5

(Neu C-9), 67.9 (Gal C-6), 68.1, 68.4, 68.7 (Glc C-5,

Gal C-2, Gal C-3), 71.9 (Glc C-2), 2· 72.7 (Neu C-4,

Neu C-7), 2· 74.1 (Glc C-3, Neu C-8), 74.8, 75.0 (Glc

C-4, Gal C-5), 76.3 (Gal C-4), 78.3 (Neu C-6), 84.1

(Neu C-2), 101.3 (CHPh), 103.0 (Glc C-1), 104.1 (Gal

C-1), 126.1, 128.6. 129.7 (CHPh), 136.6 (ipsoPh), 174.3

(Neu C-1), 174.9 (NC(O)Me); LRMS 797 (M+2Na,

100%).

zinium acetate (59 mg, 0.64 mmol) under N₂at room

temperature. The reaction mixture was left to stir for

1 h before the addition of ethyl bromoacetate (178 lL,

1.6 mmol) and then Et₃N (54 lL, 0.39 mmol). After

2 h, the mixture was diluted with EtOAc (15 mL),

washed with 0.1 M HCl (15 mL) and H₂O (15 mL),

dried (Na₂SO₄) and concentrated. Column chromatog-

raphy (CHCl₃/hexane, 1:2 ! 1:1) yielded 31 [277 mg,

90% (neat CHCl₃, R_f0.2)] which was crystallised from

EtOAc/hexane to give a colourless crystalline solid: IR

1

1071, 1269, 1729 cm^ꢁ1; H NMR (CDCl₃): d 1.13 (3H,

t, J_vic= 7.1 Hz, CO₂CH₂CH₃), 2.95 (1H, s, Gal H-5),

3.19^ꢂ(1H, dd, J_3,2= 11.2, J_3,4= 3.2 Hz, Gal H-3), 3.20^ꢂ

(2H, s, H-2⁰), 3.41 (3H, s, OMe), 3.58 (1H, appd,

0

J_6,6= 12.2 Hz, Gal H-6), 3.76 (1H, appd, J_{6 ,6}

=

12.2 Hz, Gal H-6⁰), 3.83 (1H, ddd, J_5,4= 9.4, J_5,6= 4.3,

0

J_5,6= 2.1 Hz, Glc H-5), 3.99 (2H, q, J_vic= 7.1 Hz, CO₂

4.13. Methyl (sodium 5-acetamido-3,5-dideoxy-2-thio-^D-

glycero-a-^D-galacto-2-nonulopyranosylonate)-(2,3)-S-(3-

thio-b-^D-galactopyranosyl)-(1,4)-O-b-D-glucopyranoside (9)

4 CH₂CH₃), 4.91 (1H, d, J_4,3= 3.2 Hz, Gal H-4), 4.19

(1H, t, J_4,3= J_4,5= 9.4 Hz, Glc H-4), 4.37 (1H, dd,

J_6,6= 12.0, J_6,5= 4.3 Hz, Glc H-6), 4.59^ꢂ(1H, d,

0

A solution of the 4⁰,6⁰-O-benzylidenated thiosialoside

(122 mg, 0.16 mmol) in 10% aqueous TFA (7 mL) was

stirred at 0 ꢂC for 8 h before being concentrated in vac-

uo (cold work-up). The residue was dissolved in H₂O

(5 mL) and the pH adjusted to ꢀ7.3 with 1 M NaOH be-

fore being freeze-dried. Size exclusion chromatography

[G10 Sephadex column, H₂O (EtOAc/MeOH/H₂O,

7:2:1, R_f0.17)] and then HPLC puriﬁcation (1% CH₃CN

in H₂O, 3 mL/min, R_t= 7.74 min) aﬀorded 9 (72 mg,

J_1,2= 7.7 Hz, Glc H-1), 4.61^ꢂ(1H, dd, J_{6 ,6}= 12.0,

0

J_{6 ,5}= 2.1 Hz, Glc H-6⁰), 4.73 (1H, d, J_1,2= 7.8 Hz, Gal

0

H-1), 5.31^ꢂ(1H, dd, J_2,3= 9.4, J_2,1= 7.7 Hz, Glc H-2),

5.33^ꢂ(1H, s, CHPh), 5.47 (1H, dd, J_2,3= 11.2, J_2,1

=

7.8 Hz, Gal H-2), 5.81 (1H, t, J_3,2= J_3,4= 9.4 Hz, Glc

H-3), 7.25–7.62, 7.88–8.04 (25H, 2· m, 5· Ph); ¹³C

NMR (75.5 MHz, CDCl₃): d 13.9 (CO₂CH₂CH₃), 33.4

(C-2⁰), 48.8 (Gal C-3), 56.9 (OMe), 61.2 (CO₂CH₂CH₃),

62.4 (Glc C-6), 67.9 (Gal C-6), 68.6 (Gal C-5), 71.1 (Gal

C-2), 72.2 (Glc C-2), 72.7 (Glc C-5), 73.9 (Glc C-3),

76.3 (Gal C-4), 100.9 (CHPh), 101.4 (Glc C-1), 102.4

(Gal C-1), 126.3, 127.9, 128.2, 128.3, 128.4, 128.7, 2·

129.6, 129.8, 132.9, 133.0, 133.2 (5· Ph), 128.9, 129.3,

129.5, 137.3 (5· ipsoPh), 2· 164.8, 165.4, 165.6 (4·

OC(O)Ph), 170.6 (C-1⁰); LRMS 985 (M+Na, 100%);

Anal. (C₅₂H₅₀O₁₆S) C, H.

1

64%) as a colourless solid: H NMR (600 MHz, D₂O):

d 1.79 (1H, dd, J_3a,3e= 12.3, J_3a,4= 11.7 Hz, Neu H-

3a), 1.99 (3H, s, NAc), 2.77 (1H, dd, J_3e,3a= 12.3,

J_3e,4= 4.7 Hz, Neu H-3e), 3.27 (1H, dd, J_2,3= 9.1,

J_2,1= 8.1 Hz, Glc H-2), 3.33 (1H, dd, J_3,2= 10.3,

J_3,4= 2.5 Hz, Gal H-3), 3.36 (1H, dd, J_2,3= 10.3,

J_2,1= 7.0 Hz, Gal H-2), 3.54 (3H, s, OMe), 3.55-3.58

(4H, m, Glc H-3, Glc H-5, Neu H-7, Neu H-9), 3.59–

3.66 (3H, m, Neu H-4, Neu H-5, Neu H-6), 3.68–3.69

(2H, m, Gal H-6, Gal H-6⁰), 3.74 (1H, dd, J_5,6= 6.3,

The following were prepared in a similar manner:

0

J_5,6= 5.8 Hz, Gal H-5), 3.80–3.85 (4H, m, Glc H-4,

4.15. Methyl 2-O-benzoyl-4,6-O-benzylidene-3-thio-3-[2⁰-

(ethyl propanoate)]-b-^D-galactopyranosyl-(1,4)-O-2,3,6-

tri-O-benzoyl-b-^D-glucopyranoside (32)

Glc H-6, Gal H-4, Neu H-9⁰), 3.89 (1H, ddd,

0

J_8,7= 8.8, J_8,9= 6.1, J_8,9= 2.4 Hz, Neu H-8), 3.98

(1H, dd, J_{6 ,6}= 12.3, J_{6 ,5}= 2.2 Hz, Glc H-6⁰), 4.37

(1H, d, J_1,2= 8.1 Hz, Glc H-1), 4.51 (1H, d,

J_1,2= 7.0 Hz, Gal H-1); ¹³C NMR (75.5 MHz, D₂O): d

22.0 (NC(O) Me), 40.5 (Neu C-3), 50.4 (Glc C-2), 51.5

(Neu C-5), 57.2 (OMe), 60.0 (Glc C-6), 61.2 (Gal C-6),

62.5 (Neu C-6), 68.1, 68.3, 68.4, 68.7 (Glc C-4, Gal C-

2, Gal C-4, Neu C-7), 71.9 (Neu C-8), 72.8 (Gal C-3),

0

In 84% yield (neat CHCl₃, R_f0.23) as a 2:1 mixture of

inseparable diastereomers by coupling between 23 and

ethyl ( ) 2-bromoproprionate to give a colourless crys-

talline solid: IR 1071, 1269, 1729 cm^ꢁ^{1 1}H NMR

;

(CDCl₃): d 1.03^ꢂ(1.32*) (2H, d, J_vic= 7.2 Hz, H-3⁰),

1.05^ꢂ(1.19*) (3H, t, J_vic= 6.9 Hz, CO₂CH₂CH₃), 2.90

750

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

(2.96*) (1H, s, Gal H-5), 3.25 (3.31*)^ꢂ(1H, dd,

J_3,2= 11.4, J_3,4= 3.2 Hz, Gal H-3), 3.32–3.38^ꢂ(1H, m,

H-2⁰), 3.41 (3H, s, OMe), 3.55^ꢂ(3.57*)^ꢂ(1H, dd,

C-1), 102.5 (102.6*) (Gal C-1), 126.3, 126.4, 127.9, 2·

128.3, 2· 128.4, 128.5, 128.8, 3· 129.7, 129.8, 129.9,

133.0, 2· 133.1, 133.3 (5· Ph), 128.9, 129.1, 129.4,

129.6, 137.3 (137.5*) (5· ipsoPh), 164.7, 164.9, 165.0,

165.5, 2· 165.7 (4· OC(O)Ph), 173.1 (173.6*) (C-1⁰);

LRMS 999 (M+Na, 100%); Anal. (C₅₃H₅₂O₁₆S) C, H.

0

J_6,6= 12.3, J_6,5= 1.7 Hz, Gal H-6), 3.75 (1H, appd,

0

J_{6 ,6}= 12.3 Hz, Gal H-6⁰), 3.83 (1H, ddd, J_5,4= 9.4,

J_5,6= 4.2, J_5,6= 1.8 Hz, Glc H-5), 3.90^ꢂ(4.11*) (2H,

0

q, J_vic= 7.2 Hz, CO₂CH₂CH₃), 3.94^ꢂ(3.95*) (1H, d,

J_4,3= 3.2 Hz, Gal H-4), 4.19^ꢂ(4.20*)^ꢂ(1H, t,

J_4,3= J_4,5= 9.4 Hz, Glc H-4), 4.38 (1H, dd,

4.17. Methyl 2-O-benzoyl-4,6-O-benzylidene-3-thio-3-[2⁰-

(ethyl valeroate)]-b-^D-galactopyranosyl-(1,4)-O-2,3,6-tri-

O-benzoyl-b-^D-glucopyranoside (34)

J_6,6= 12.0, J_6,5= 4.2 Hz, Glc H-6), 4.58^ꢂ(4.59*)^ꢂ(1H,

0

d, J_1,2= 7.5 Hz, Glc H-1), 4.60^ꢂ(1H, dd, J_{6 ,6}= 12.0,

0

J_{6 ,5}= 1.8 Hz, Glc H-6⁰), 4.73^ꢂ(4.75*)^ꢂ(1H, d,

In 96% yield (neat CHCl₃, R_f0.25) as a 2:1 mixture of

inseparable diastereomers by coupling between 23 and

ethyl ( ) 2-bromovalerate to give a colourless crystalline

0

J_1,2= 7.8 Hz, Gal H-1), 5.28–5.35^ꢂ(2H, m, Glc H-2,

CHPh),

5.36^ꢂ

(5.51*)

(1H,

dd,

J_2,3= 11.4,

J_2,1= 7.8 Hz, Gal H-2), 5.80^ꢂ(5.83*)^ꢂ(1H, t,

J_3,2= J_3,4= 9.4 Hz, Glc H-3), 7.19–7.62, 7.86–7.52

(25H, 2· m, 5· Ph); ¹³C NMR (75.5 MHz, CDCl₃): d

13.8 (16.5*) (CO₂CH₂CH₃), 14.0 (17.9*) (C-3⁰), 40.2

(42.5*) (C-2⁰), 47.2 (49.7*) (Gal C-3), 56.9 (OMe), 61.0

(61.1*) (CO₂CH₂CH₃), 62.4 (Glc C-6), 67.9 (Gal C-6),

68.5 (68.7*) (Gal C-5), 69.7 (72.2*) (Glc C-2), 72.2

(72.3*) (Gal C-2), 72.7 (72.8*) (Glc C-5), 73.8 (74.0*)

(Glc C-3), 75.3 (76.3*) (Gal C-4), 76.4 (Glc C-4), 100.8

(100.9*) (CHPh), 101.4 (Glc C-1), 102.4 (102.5*) (Gal

C-1), 126.2, 126.3, 127.9, 2· 128.2, 128.3, 128.4, 128.7,

129.5, 2· 129.6, 129.7, 129.8, 132.9, 133.0, 133.1, 133.2

(5· Ph), 128.8, 129.0, 129.3, 129.5, 132.3 (137.4*) (5·

ipsoPh), 164.7, 164.8, 164.9, 165.4, 165.6, 165.7 (4 ·

OC(O)Ph), 173.0 (173.4*) (C-1⁰); LRMS 999 (M+Na,

100%); Anal. (C₅₃H₅₂O₁₆S) C, H.

solid: IR 1150, 1342, 1729, 2961 cm^ꢁ^{1 1}H NMR

;

(CDCl₃): d 0.47 (0.74*) (3H, t, J_vic= 7.3 Hz, H-5⁰),

0.81–0.98 (1.14–1.31*)^ꢂ(2H, m, H-4⁰), 1.07 (1.19*)^ꢂ

(3H, t, J_vic= 7.1 Hz, CO₂CH₂CH₃), 1.14–1.31^ꢂ(1.40–

1.61*) (2H, m, H-3⁰), 2.90 (2.97*) (1H, s, Gal H-5),

3.11 (3.19–3.23*) (1H, dd, J_vic= 9.3, 6.0 Hz, H-2⁰),

3.23^ꢂ(3.31*) (1H, dd, J_3,2= 11.4, J_3,4= 3.1 Hz, Gal H-

3), 3.40 (3.41*) (3H, s, OMe), 3.51–3.60 (1H, m, Gal

H-6), 3.76 (1H, appd, J_{6 ,6}= 12.1 Hz, Gal H-6⁰), 3.82–

0

3.97 (4H, m, Glc H-5, Gal H-4, CO₂CH₂CH₃) [4.11*

(2H, q, J_vic= 7.1 Hz, CO₂CH₂CH₃)], 4.22 (1H, t,

J_4,3= J_4,5= 9.2 Hz, Glc H-4), 4.38 (1H, dd,

J_6,6= 12.0, J_6,5= 4.2 Hz, Glc H-6), 4.58^ꢂ(4.59*)^ꢂ(1H,

0

d, J_1,2= 7.8 Hz, Glc H-1), 4.66 (1H, dd, J_{6 ,6}= 12.0,

J_{6 ,5}= 2.0 Hz, Glc H-6⁰), 4.73 (1H, d, J_1,2= 7.8 Hz,

0

Gal H-1), 5.27–5.35^ꢂ(2H, m, Glc H-2, Gal H-2) [5.51*

(1H, dd, J_2,3= 11.4, J_2,1= 7.8 Hz, Gal H-2)], 5.32^ꢂ

(1H, s, CHPh), 5.83 (1H, t, J_3,2= J_3,4= 9.2 Hz, Glc H-

3), 7.18–7.61, 7.81–8.05 (25H, 2· m, 5· Ph); ¹³C NMR

(75.5 MHz, CDCl₃): d 13.2 (13.4*) (C-5⁰), 13.8 (14.0*)

(CO₂CH₂CH₃), 20.3 (20.5*) (C-4⁰), 33.1 (34.3*) (C-3⁰),

45.0 (47.8*) (C-2⁰), 47.1 (49.4*) (Gal C-3), 56.8 (OMe),

60.9 (61.0*) (CO₂CH₂CH₃), 62.3 (Glc C-6), 67.9 (Gal

C-6), 68.5 (68.6*) (Gal C-5), 69.4, 69.8, 72.2, 72.6,

72.7, 73.8, 74.0, 74.1, 75.1, 76.3, 76.4, 76.5 (Glc C-2,

Glc C-3, Glc C-4, Glc C-5, Gal C-2, Gal C-4), 100.8

(100.9*) (CHPh), 101.4 (Glc C-1), 102.3 (102.5*) (Gal

C-1), 2· 126.2, 127.2, 127.8, 127.9, 2· 128.1, 128.2, 2·

128.3, 128.6, 128.8, 129.5, 129.8, 132.8, 132.9, 2·

133.0, 133.1, 137.2 (5· Ph), 128.8, 129.0, 129.2, 129.4,

129.5, 137.3 (137.4*) (5· ipsoPh), 2· 164.7, 2· 164.8,

165.1, 165.3, 165.4, 165.5 (4· OC(O)Ph), 172.6

(173.0*) (C-1⁰); LRMS 1027 (M+Na, 100%); Anal.

(C₅₅H₅₆O₁₆S) C, H.

4.16. Methyl 2-O-benzoyl-4,6-O-benzylidene-3-thio-3-[2⁰-

(methyl butanoate)]-b-^D-galactopyranosyl-(1,4)-O-2,3,6-

tri-O-benzoyl-b-^D-glucopyranoside (33)

In 85% yield (neat CHCl₃, R_f0.23) as a 2:1 mixture of

inseparable diastereomers by coupling between 23 and

methyl ( ) 2-bromobutyrate to give a colourless crystal-

line solid: IR 1070, 1268, 1731 cm^ꢁ1; ¹H NMR (CDCl₃):

d 0.47 (0.77*) (3H, t, J_vic= 7.3 Hz, H-4⁰), 1.21–1.35

(1.45–1.65*)^ꢂ(1H, m, H-3⁰), 1.45–1.65^ꢂ, 1.74–1.89 (1H,

m, H-3⁰), 2.94 (2.96*) (1H, s, Gal H-5), 3.09 (3.17*)

(1H, dd, J_vic= 8.3, 6.6 Hz, H-2⁰), 3.25 (1H, dd,

J_3,2= 11.2, J_3,4= 3.1 Hz, Gal H-3), 3.41 (3H, s, OMe),

0

3.55 (3.58*) (1H, dd, J_6,6= 12.2, J_6,5= 1.5 Hz, Gal H-

0

6), 3.65 (3H, s, CO₂Me), 3.75 (1H, d, J_{6 ,6}= 12.2 Hz,

Gal H-6⁰), 3.84 (1H, ddd, J_5,4= 9.6, J_5,6= 4.2,

0

J_5,6= 1.9 Hz, Glc H-5), 3.88 (3.94*) (1H, d,

J_4,3= 3.1 Hz, Gal H-4), 4.18^ꢂ(4.20*)^ꢂ(1H, t,

J_4,3= J_4,5= 9.6 Hz, Glc H-4), 4.38 (1H, dd,

4.18. Methyl 2-O-benzoyl-4,6-O-benzylidene-3-thio-3-[2⁰-

(methyl 2-phenylacetate)]-b-^D-galactopyranosyl-(1,4)-O-

2,3,6-tri-O-benzoyl-b-^D-glucopyranoside (35)

0

J_6,6= 12.0, J_6,5= 4.2 Hz, Glc H-6), 4.56-4.63 (2H, m,

Glc H-1, Glc H-6⁰), 4.73^ꢂ(4.74*)^ꢂ(1H, d,

J_1,2= 7.8 Hz, Gal H-1), 5.28–5.38 (2H, m, Glc H-2,

Gal H-2, CHPh) [5.51* (1H, dd, J_2,3= 11.2,

J_2,1= 7.8 Hz, Gal H-2)], 5.80^ꢂ(5.83*)^ꢂ(1H, t,

J_3,2= J_3,4= 9.6 Hz, Glc H-3), 7.15–7.56, 7.76–8.00

(25H, 2· m, 5· Ph); ¹³C NMR (75.5 MHz, CDCl₃): d

11.5 (11.9*) (C-4⁰), 24.6 (25.7*) (C-3⁰), 47.3 (49.4*) (C-

2⁰), 47.7 (49.5*) (Gal C-3), 52.0 (52.3*) (CO₂Me), 57.0

(OMe), 62.5 (Glc C-6), 68.0 (Gal C-6), 68.6 (68.7*)

(Gal C-5), 69.9, 72.2, 72.3, 72.4, 2· 72.8 (Glc C-2, Glc

C-5, Gal C-2), 73.9 (74.1*) (Glc C-3), 75.4 (76.5*) (Gal

C-4), 76.4 (Glc C-4), 100.9 (101.0*) (CHPh), 101.5 (Glc

In 90% yield (neat CHCl₃, R_f0.23) as a 1:1 mixture of

inseparable diastereomers by coupling between 23 and

ethyl ( ) 2-bromovalerate to give a colourless crystalline

solid: IR 1113, 1269, 1732, 3022 cm^ꢁ1

;

¹H NMR

(CDCl₃): d 2.78^ꢂ(2.91*) (1H, s, Gal H-5), 2.80^ꢂ(3.14*)

(1H, dd, J_3,2= 11.2, J_3,4= 3.2 Hz, Gal H-3), 3.28

(3.68*) (3H, s, CO₂Me), 3.41 (3H, s, OMe), 3.49

0

(3.54*) (1H, dd, J_6,6= 12.4, J_6,5= 1.4 Hz, Gal H-6),

3.62–3.76 (2H, m, Gal H-4, Gal H-6⁰) [3.97* (1H, d,

J_4,3= 3.2 Hz, Gal H-4)], 3.83 (1H, ddd, J_5,4= 9.4,

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

751

J_5,6= 5.9, J_5,6= 2.0 Hz, Glc H-5), 4.15^ꢂ(4.20*)^ꢂ(1H, t,

0

J_5,6= 4.2, J_5,6= 2.2 Hz, Glc H-5), 4.01–4.24 (4H, m,

J_4,3= J_4,5= 9.4 Hz, Glc H-4), 4.38–4.48 (2H, m, Glc H-

6, Glc H-6⁰), 4.52–4.63 (3H, m, Glc H-1, Gal H-1, H-2⁰)

[4.72* (1H, d, J_1,2= 7.8 Hz, Gal H-1)], 5.22–5.34^ꢂ(1H,

m, Glc H-2), 5.31 (1H, s, CHPh), 5.51 (1H, dd,

J_2,3= 11.2, J_2,1= 7.8 Hz, Gal H-2), 5.77^ꢂ(5.81*^ꢂ(1H,

t, J_3,2= J_3,4= 9.4 Hz, Glc H-3), 6.79–6.82, 7.01–7.12,

7.28–7.63, 7.87–8.07 (30H, 4· m, 6· Ph); ¹³C NMR

(75.5 MHz, CDCl₃): d 48.3 (49.6*) (Gal C-3), 52.2

(52.8*(CO₂Me), 52.4 (C-2⁰), 57.0 (OMe), 62.5 (Glc C-

6), 67.9 (Gal C-6), 68.6 (Gal C-5), 71.0 (72.2*) (Gal C-

2), 72.1 (72.2*) (Glc C-2), 72.7 (72.8*) (Glc C-5), 73.7

(74.0*) (Glc C-3), 75.9 (76.0*) (Gal C-4), 76.2 (76.4*)

(Glc C-4), 100.7 (100.8*) (CHPh), 101.5 (101.6*) (Glc

C-1), 102.4 (Gal C-1), 126.3, 126.4, 127.9, 128.0, 128.1,

128.2, 2· 128.3, 128.4, 2· 128.5, 128.7, 128.8, 129.6,

129.7, 129.8, 129.9, 132.9, 133.0, 133.1, 133.2, 133.3 (6·

Ph), 128.9, 129.0, 129.3, 129.6, 134.8, 136.4, 137.3 (5·

ipsoPh), 164.6 (164.8*), 164.9 (165.0*), 165.4, 165.7 (4·

OC(O)Ph), 170.6 (171.8*) (CO₂Me); LRMS 1047

(M+Na, 100%); Anal. (C₅₇H₅₂O₁₆S) C, H.

Glc H-4, Gal H-4, H-4⁰), 4.34 (1H, dd, J_6,6= 12.0,

0

J_6,5= 4.2 Hz, Glc H-6), 4.59^ꢂ(1H, d, J_1,2= 7.7 Hz, Glc

H-1), 4.62^ꢂ(1H, dd, J_{6 ,6}= 12.0, J_{6 ,5}= 2.2 Hz, Glc H-

6⁰), 4.77 (1H, d, J_1,2= 7.7 Hz, Gal H-1), 5.30^ꢂ(1H, dd,

J_2,3= 9.5, J_2,1= 7.7 Hz, Glc H-2), 5.36^ꢂ(1H, dd,

J_2,3= 11.5, J_2,1= 7.7 Hz, Gal H-2), 5.38^ꢂ(1H, s, CHPh),

5.82 (1H, t, J_3,2= J_3,4= 9.5 Hz, Glc H-3), 7.34–7.62,

7.81–8.07 (25H, 2· m, 5· Ph); ¹³C NMR (75.5 MHz,

CDCl₃): d 30.0 (C-3⁰), 37.9 (C-2⁰), 47.5 (Gal C-3), 56.9

(OMe), 62.3 (Glc C-6), 66.7 (C-4⁰), 68.1 (Gal C-6),

68.7 (Gal C-5), 69.2 (Gal C-2), 72.3, 72.8 (Glc C-2,

Glc C-5), 74.1 (Glc C-3), 74.5 (Gal C-4), 76.6 (Glc C-

4), 101.1 (CHPh), 101.5 (Glc C-1), 102.6 (Gal C-1),

128.0, 2· 128.3, 2· 128.4, 128.9, 2· 129.7, 129.8, 129.9,

133.0, 2· 133.1, 133.2 (5· Ph), 129.4, 129.5, 137.5 (5· ip-

soPh), 164.9, 165.3, 165.4, 165.7 (4· OC(O)Ph), 175.4

(C-1⁰); LRMS 983 (M+Na, 100%).

0

4.20. Methyl 2-O-benzoyl-4,6-O-benzylidene-3-thio-3-[2⁰-

(c-valerolactone)]-b-^D-galactopyranosyl-(1,4)-O-2,3,6-tri-

O-benzoyl-b-^D-glucopyranoside (37)

4.19. Methyl 2-O-benzoyl-4,6-O-benzylidene-3-thio-3-[2⁰-

(c-butyrolactone)]-b-^D-galactopyranosyl-(1,4)-O-2,3,6-

tri-O-benzoyl-b-^D-glucopyranoside (36)

In 87% yield as a 2:1:1 mixture of separable diastereomers

by coupling between 23 and a-bromo-c-valerolactone

(chromatography EtOAc/hexane, 2:3 ! 1:1, R_f0.44,

0.39, 0.34). One diastereomer of 37 as a colourless crystal-

line solid: IR 1117, 1267, 1726 cm^ꢁ1; ¹H NMR (CDCl₃): d

1.23 (3H, d, J_vic= 6.2 Hz, H-5⁰), 1.74-1.94 (2H, m, H-3⁰),

2.93 (1H, s, Gal H-5), 3.40 (3H, s, OMe), 3.45 (1H, dd,

In 85% yield as a 2:1 mixture of separable diastereomers

by coupling between 23 and a-bromo-c-butyrolactone

(chromatography EtOAc/hexane, 2:3 ! 1:1, R_f0.30,

0.19). One diastereomer of 36 as a colourless crystalline

1

solid: IR 1113, 1268, 1731 cm^ꢁ1; H NMR (CDCl₃): d

J_vic= 8.2, 3.0 Hz, H-2⁰), 3.53 (1H, dd, J_6,6= 12.3,

0

1.69–1.78, 2.07–2.30 (2H, 2· m, H-3⁰), 2.89 (1H, s, Gal

J_6,5= 1.5 Hz, Gal H-6), 3.68 (1H, dd, J_3,2= 11.3,

0

J_3,4= 3.3 Hz, Gal H-3), 3.77 (1H, appd, J_{6 ,6}= 12.3, Gal

H-5), 3.32–3.36^ꢂ(1H, m, H-2⁰), 3.34^ꢂ(3H, s, OMe),

H-6⁰), 3.83 (1H, ddd, J_5,4= 9.6, J_5,6= 4.0, J_5,6= 1.8 Hz,

0

3.48 (1H, dd, J_6,6= 11.0, J_6,5= 1.3 Hz, Gal H-6), 3.61

0

(1H, dd, J_3,2= 10.3, J_3,4= 3.3 Hz, Gal H-3), 3.70 (1H,

appd, J_{6 ,6}= 11.0, Gal H-6⁰), 3.77 (1H, ddd, J_5,4= 9.7,

Glc H-5), 3.98 (1H, d, J_3,4= 3.3 Hz, Gal H-4), 4.22 (1H,

t, J_4,3= J_4,5= 9.6 Hz, Glc H-4), 4.32 (1H, dd,

0

J_6,6= 12.0, J_6,5= 4.0 Hz, Glc H-6), 4.59^ꢂ(1H, d,

0

J_5,6= 4.0, J_5,6= 1.7 Hz, Glc H-5), 3.96 (1H, d,

J_4,3= 3.3 Hz, Gal H-4), 4.02–4.23 (3H, m, Glc H-4, H-

0

J_1,2= 7.7 Hz, Glc H-1), 4.60-4.69^ꢂ(2H, m, Glc H-6⁰, H-

4⁰), 4.74 (1H, d, J_1,2= 7.8 Hz, Gal H-1), 5.29^ꢂ(1H, dd,

J_2,3= 9.6, J_2,1= 7.7 Hz, Glc H-1), 5.32^ꢂ(1H, s, CHPh),

5.55 (1H, dd, J_2,3= 11.3, J_2,1= 7.8 Hz, Gal H-2), 5.83

(1H, t, J_3,2= J_3,4= 9.6 Hz, Glc H-3), 7.32–7.62, 7.85–

8.02 (25H, 2· m, 5· Ph); ¹³C NMR (75.5 Hz, CDCl₃): d

20.4 (C-5⁰), 36.8 (C-3⁰), 40.7 (C-2⁰), 48.0 (Gal C-3), 56.9

(OMe), 62.3 (Glc C-6), 67.9 (Gal C-6), 68.4 (Gal C-5),

72.4 (Glc C-5), 72.7 (Glc C-2), 72.7 (Gal C-2), 74.2 (Glc

C-3), 75.8, 76.1 (Gal C-4, C-4⁰), 76.6 (Glc C-4), 100.8

(CHPh), 101.4 (Glc C-1), 102.4 (Gal C-1), 128.2, 128.3,

128.5, 128.6, 128.8, 129.6, 2· 129.7, 129.9, 133.0, 133.1,

133.2, 133.4 (5· Ph), 129.4, 129.5, 137.4 (5· ipsoPh), 2·

164.8, 165.4, 165.6 (4· OC(O)Ph), 176.0 (C-1⁰); LRMS

997 (M+Na, 100%); HRMS Calcd for C₅₃H₅₀O₁₆S

(M+Na) 997.2717. Found 997.2737. Other diastereomer

of 37 as a colourless crystalline solid: IR 1117, 1267,

4⁰), 4.28 (1H, dd, J_6,6= 12.0, J_6,5= 4.0 Hz, Glc H-6),

0

4.53^ꢂ(1H, d, J_1,2= 7.6, Glc H-1), 4.52–4.58^ꢂ(1H, m,

Glc H-6⁰), 4.68 (1H, d, J_1,2= 7.8 Hz, Gal H-1), 5.24^ꢂ

(1H, dd, J_2,3= 9.1, J_2,1= 7.6 Hz, Glc H-2), 5.26 (1H, s,

CHPh), 5.49 (1H, dd, J_2,3= 10.3, J_2,1= 7.8 Hz, Gal H-

2), 5.77 (1H, t, J_3,2= J_3,4= 9.1 Hz, Glc H-3), 7.13–

7.55, 7.79–7.99 (25H, 2· m, 5· Ph); ¹³C NMR

(75.5 MHz, CDCl₃): d 29.1 (C-3⁰), 39.1 (C-2⁰), 48.0

(Gal C-3), 56.9 (OMe), 62.3 (Glc C-6), 66.7 (C-4⁰),

67.9 (Gal C-6), 68.4 (Gal C-5), 72.3 (Glc C-5), 72.6

(Glc C-2), 72.7 (Gal C-2), 74.1 (Glc C-3), 76.1 (Gal

C-4), 76.5 (Glc C-4), 100.8 (CHPh), 101.4 (Glc C-1),

102.4 (Gal C-1), 126.0, 126.3, 127.9, 2· 128.2, 128.4,

128.6, 128.8, 2· 129.6, 129.9, 132.9, 2· 133.1, 133.3

(5· Ph), 128.8, 129.4, 129.5, 137.4 (5· ipsoPh), 164.7,

164.8, 165.4, 165.6 (4· OC(O)Ph), 176.6 (C-1⁰); LRMS

983 (M+Na, 100%); HRMS Calcd for C₅₂H₄₈O₁₆S

(M+Na) 983.2561. Found 983.2558. Other diastereomer

of 36 as a colourless crystalline solid: IR 1113, 1268,

1726 cm^ꢁ1

; d 1.29 (3H, d,

¹H NMR (CDCl₃):

J_vic= 6.3 Hz, H-5⁰), 1.35–1.45, 2.12–2.22 (2H, 2· m, H-

3⁰), 2.97 (1H, s, Gal H-5), 3.41 (3H, s, OMe), 3.52^ꢂ(1H,

1

ꢂ

1731 cm^ꢁ1; H NMR (CDCl₃): d 1.87–1.97, 2.42–2.55

t, J_vic= 9.2 Hz, H-2⁰), 3.56 (1H, dd, J_6,6= 12.4,

0

(2H, 2· m, H-3⁰), 2.95 (1H, s, Gal H-5), 3.40 (3H, s,

OMe), 3.43 (1H, dd, J_3,2= 11.5, J_3,4= 3.1 Hz, Gal H-

J_6,5= 1.7 Hz, Gal H-6), 3.72^ꢂ(1H, dd, J_3,2= 11.3,

J_3,4= 3.3 Hz, Gal H-3), 3.76^ꢂ(1H, dd, J_{6 ,6}= 12.4,

0

3), 3.56 (1H, dd, J_6,6= 12.2, J_6,5= 1.5 Hz, Gal H-6),

J_{6 ,5}= 1.0 Hz, Gal H-6⁰), 3.83 (1H, ddd, J_5,4= 9.6,

0

3.66 (1H, dd, J_vic= 8.3, 5.3 Hz, H-2⁰), 3.78^ꢂ(1H, appd,

J_5,6= 4.0, J_5,6= 2.1 Hz, Glc H-5), 4.15 (1H, d,

J_4,3= 3.3 Hz, Gal H-4), 4.21 (1H, t, J_4,3= J_4,5= 9.6 Hz,

0

ꢂ

J_{6 ,6}= 12.2 Hz, Gal H-6⁰), 3.82 (1H, ddd, J_5,4= 9.5,

0

752

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

Glc H-4), 4.26–4.33 (1H, m, H-4⁰), 4.63 (1H, dd,

m, Glc H-3, Glc H-4, Glc H-5, Gal H-2), 3.67 (1H, s,

0

Gal H-5), 3.68 (1H, dd, J_6,6= 12.0, J_6,5= 4.8 Hz, Glc

J_6,6= 12.0, J_6,5= 4.0 Hz, Glc H-6), 4.59^ꢂ(1H, d,

0

J_1,2= 7.7 Hz, Glc H-1), 4.58–4.63^ꢂ(1H, m, Glc H-6⁰),

4.72 (1H, d, J_1,2= 7.8 Hz, Gal H-1), 5.30 (1H, dd,

J_2,3= 9.6, J_2,1= 7.7 Hz, Glc H-2), 5.35 (1H, s, CHPh),

5.54 (1H, dd, J_2,3= 11.3, J_2,1= 7.8 Hz, Gal H-2), 5.82

(1H, t, J_3,2= J_3,4= 9.6 Hz, Glc H-3), 7.32–7.62, 7.86–

8.03 (25H, 2· m, 5· Ph); ¹³C NMR (75.5 MHz, CDCl₃):

d 21.3 (C-5⁰), 36.1 (C-3⁰), 41.2 (C-2⁰), 48.7 (Gal C-3),

57.0 (OMe), 62.4 (Glc C-6), 67.9 (Gal C-6), 68.5 (Gal C-

5), 72.3, 72.8 (Glc C-2, Glc C-5), 74.1 (Gal C-2), 75.4

(Glc C-3), 76.3, 76.5 (Glc C-4, Gal C-4, C-4⁰), 100.9

(CHPh), 101.5 (Glc C-1), 102.4 (Gal C-1), 126.3, 126.4,

2· 128.0, 128.3, 128.4, 128.6, 128.8, 129.6, 129.7, 129.9,

133.0, 133.1, 133.2, 133.4 (5· Ph), 128.9, 129.4, 137.5

(5· ipsoPh), 164.7, 164.9, 165.5, 165.7 (4· OC(O)Ph),

176.9 (C-1⁰); LRMS 997 (M+Na, 100%). Other two dia-

stereomers of 37 as a colourless crystalline solid: IR

H-6), 3.85 (1H, appd, J_{6 ,6}= 12.0 Hz, Glc H-6⁰), 4.08

0

(2H, apps, Gal H-6, Gal H-6⁰), 4.65^ꢂ(1H, d, J_4,3

=

3.0 Hz, Gal H-4), 4.68^ꢂ(1H, d, J_1,2= 8.1 Hz, Glc H-1),

4.87 (1H, d, J_1,2= 7.7 Hz, Gal H-1), 6.00 (1H, s, CHPh),

7.74–7.83, 8.15–8.18 (5H, 2· m, CHPh); ¹³C NMR

(75.5 MHz, D₂O): d 36.8 (C-2⁰), 50.3 (OMe), 59.8 (Glc

C-6), 68.6 (Gal C-5), 68.9 (Gal C-6), 69.9, 72.7, 74.1,

74.7, 75.8, 78.5 (Glc C-2, Glc C-3, Glc C-4, Glc C-5,

Gal C-2, Gal C-4), 101.2 (CHPh), 103.0 (Glc C-1),

103.8 (Gal C-1), 126.1, 128.3, 128.6, 128.7, 131.2 (CHPh),

136.6 (ipsoPh), 177.7 (C-1⁰); LRMS 525 (M+Na, 100%).

The following were prepared in a similar manner:

4.22. Methyl 4,6-O-benzylidene-3-thio-3-[2⁰-(propanoic

acid)]-b-^D-galactopyranosyl-(1,4)-O-b-D-glucopyranoside

(39)

1117, 1267, 1726 cm^ꢁ^{1 1}H NMR (CDCl₃): d 1.15

;

(1.23*) (3H, d, J_vic= 6.3 Hz, H-5⁰), 1.50–1.57, 1.94–

2.12, 2.49–2.58 (2H, 3· m, H-3⁰), 2.94 (2.95*(1H, s, Gal

H-5), 3.27 (3.50*) (1H, dd, J_3,2= 11.5, J_3,4= 3.0 Hz, Gal

H-3), 3.40 (3.42*) (3H, s, OMe), 3.58 (1H, dd,

In 95% yield as a 2:1 mixture of diastereomers: ¹H NMR

(D₂O): d 1.33 (1.37*) (3H, d, J_vic= 7.2 Hz, H-3⁰), 3.12

(3.19*) (1H, dd, J_3,2= 11.2, J_3,4= 3.2 Hz, Gal H-3),

3.25-3.31 (1H, m, Glc H-2), 3.54^ꢂ(3H, s, OMe), 3.54-

3.66^ꢂ(4H, m, Glc H-3, Glc H-4, Glc H-5, H-2⁰), 3.80–

3.85 (2H, m, Glc H-6, Gal H-5), 4.00 (1H, appd,

0

J_6,6= 11.3, J_6,5= 4.5 Hz, Gal H-6), 3.68 (1H, dd,

J_vic= 7.8, 3.7 Hz, H-2⁰) [3.80–3.89* (1H, m, H-2⁰)], 3.75

(1H, appd, J_{6 ,6}= 11.3 Hz, Gal H-6⁰), 3.80–3.89 (1H, m,

0

Glc H-5), 4.12–4.51 (4H, m, Glc H-4, Glc H-6, Gal H-4,

H-4⁰), 4.57–4.66 (2H, m, Glc H-1, Glc H-6⁰), 4.75

(4.78*) (1H, d, J_1,2= 7.7 Hz, Gal H-1), 5.26–5.40 (3H,

m, Glc H-2, Gal H-2, CHPh), 5.81, 5.82 (1H, t,

J_3,2= J_3,4= 9.1 Hz, Glc H-3), 7.23–7.62, 7.85–8.05

(25H, 2· m, 5· Ph); ¹³C NMR (75.5 MHz, CDCl₃): d

20.2 (20.8*) (C-5⁰), 37.1 (37.6*) (C-3⁰), 38.8 (40.3*) (C-

2⁰), 47.5 (47.6*) (Gal C-3), 56.9 (57.0*) (OMe), 62.3

(62.4*) (Glc C-6), 68.1 (Gal C-6), 68.6 (68.8*) (Gal C-5),

69.2 (69.4*) (Gal C-2), 72.3 (72.4*) (Glc C-5), 72.8

(72.9*) (Glc C-2), 73.9, 74.0, 74.2, 75.2, 75.8, 76.5 (Glc

C-3, Glc C-4, Gal C-4, C-4⁰), 101.0 (CHPh), 101.5

(101.6*) (Glc C-1), 102.6 (Gal C-1), 128.1, 128.2, 128.3,

128.4, 128.5, 128.6, 128.8, 128.9, 129.5, 2· 129.7, 2·

129.9, 132.9, 133.1, 133.2, 133.3, 133.4 (5· Ph), 128.9,

129.0, 129.4, 129.5, 137.5 (5· ispoPh), 164.9, 165.0,

165.2, 165.4, 165.6, 165.8 (4· OC(O)Ph), 174.8 (175.6*)

(C-1⁰); LRMS 997 (M+Na, 100%).

J_{6 ,6}= 12.5 Hz, Glc H-6⁰), 4.23 (2H, appts, Gal H-6,

Gal H-6⁰), 4.33^ꢂ(4.35*)^ꢂ(1H, d, J_4,3= 3.2 Hz, Gal H-

4), 4.38^ꢂ(1H, d, J_1,2= 8.0 Hz, Glc H-1), 4.59 (1H, d,

J_1,2= 7.8 Hz, Gal H-1), 5.72 (5.74*) (1H, s, CHPh),

7.43–7.47, 7.49–7.53 (5H, 2· m, CHPh); ¹³C NMR

(75.5 MHz, D₂O): d 18.0 (18.5*) (C-3⁰), 45.3 (46.2*)

(C-2⁰), 49.5 (50.0*) (Gal C-3), 57.2 (OMe), 59.9 (Glc

C-6), 68.6 (Gal C-5), 68.8 (68.9*) (Gal C-6), 69.5

(70.2*), 74.2, 74.7, 78.5 (Glc C-3, Glc C-4, Glc C-5,

Gal C-2), 72.7 (Glc C-2), 75.1 (76.3*) (Gal C-4), 101.2,

101.4 (CHPh), 103.0 (Glc C-1), 103.9 (Gal C-1), 2·

126.1 (126.2*), 2· 128.5 (128.6*), 129.6 (129.7*)

(CHPh), 136.6 (136.7*) (ipsoPh), 181.3 (181.4*) (C-1⁰);

LRMS 555 (M+Na, 100%).

4.23. Methyl 4,6-O-benzylidene-3-thio-3-[2⁰-(butanoic

acid)]-b-^D-galactopyranosyl-(1,4)-O-b-D-glucopyranoside

(40)

4.21. Methyl 4,6-O-benzylidene-3-thio-3-(carboxymeth-

yl)-b-^D-galactopyranosyl-(1,4)-O-b-D-glucopyranoside

(38)

In 97% yield as a 1:1 mixture of diastereomers: ¹H NMR

(D₂O): d 0.88 (0.90*) (3H, t, J_vic= 7.3 Hz, H-4⁰), 1.63–

1.70 (2H, m, H-3⁰), 3.08 (3.16*) (1H, dd, J_3,2= 11.1,

J_3,4= 3.1 Hz, Gal H-3), 3.24–3.41 (2H, m, Glc H-2, H-

2⁰), 3.51–3.64^ꢂ(4H, m, Glc H-3, Glc H-4, Glc H-5,

Gal H-2), 3.53^ꢂ(3H, s, OMe), 3.80^ꢂ(1H, s, Gal H-5),

To a stirred suspension of 31 (330 mg, 0.34 mmol) in

MeOH (7 mL) was added dropwise a 1 M solution of

NaOH (ꢀ1 mL) until a pH of ꢀ13 was attained. The

mixture was left to stir for 24 h before being neutralised

with Amberlyte IR-120 (H⁺), the resin ﬁltered, washed

with aqueous MeOH, and then the solvent was concen-

trated under reduced pressure. The residue was re-dis-

solved in H₂O (20 mL), the pH adjusted to ꢀ4 with

AcOH, washed with EtOAc (3· 30 mL) and the aqueous

phase freeze-dried to give 4⁰,6⁰-O-benzylidenated lacto-

side (116 mg, quantitative) as a colourless solid: ¹H

NMR (D₂O): d 2.97 (1H, dd, J_3,2= 11.1, J_3,4= 3.0 Hz,

Gal H-3), 3.13 (1H, t, J_2,1= J_2,3= 8.1 Hz, Glc H-2),

3.12 (2H, s, H-2⁰), 3.40^ꢂ(3H, s, OMe), 3.40–3.52^ꢂ(4H,

3.81^ꢂ(1H, dd, J_6,6= 12.1, J_6,5= 4.6 Hz, Glc H-6), 3.98

0

(1H, dd, J_{6 ,6}= 12.1, J_{6 ,5}= 1.7 Hz, Glc H-6⁰), 4.21

(2H, appd, Gal H-6, Gal H-6⁰), 4.31 (4.34*)^ꢂ(1H, d,

J_4,3= 3.1 Hz, Gal H-4), 4.37 (1H, d, J_1,2= 8.0 Hz, Glc

H-1), 4.57 (1H, d, J_1,2= 7.8 Hz, Gal H-1), 5.70 (5.73*)

(1H, s, CHPh), 7.41–7.45, 7.48–7.52 (5H, 2· m, CHPh);

¹³C NMR (75.5 MHz, D₂O): d 11.3 (11.4*) (C-4⁰), 25.8

(26.0*) (C-3⁰), 49.5 (50.0*) (C-2⁰), 51.9 (53.1*) (Gal C-

3), 57.1 (OMe), 59.9 (60.0*(Glc C-6), 68.6 (Gal C-5),

68.8 (68.9*) (Gal C-6), 69.3 (70.1*), 74.2, 74.7, 78.5

(Glc C-3, Glc C-4, Glc C-5, Gal C-2), 74.6 (76.4*)

(Gal C-4), 101.2 (101.5*) (CHPh), 103.0 (Glc C-1),

0

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

753

103.9 (Gal C-1), 2· 126.0 (126.1*), 2· 128.5 (128.6*),

129.6 (129.7*) (CHPh), 136.5 (136.6*) (ipsoPh), 180.1

(180.7*) (C-1⁰); LRMS 569 (M+Na, 100%).

(5H, m, Gal H-2, Glc H-3, Glc H-4, Glc H-5, H-2⁰),

3.54^ꢂ(3H, s, OMe), 3.81^ꢂ(1H, s, Gal H-5), 3.82^ꢂ(1H,

0

dd, J_6,6= 12.0, J_6,5= 4.6 Hz, Glc H-6), 4.0 (1H, appd,

0

ꢂ

J_{6 ,6}= 12.0 Hz, Glc H-6⁰), 4.21–4.39 (3H, m, Gal H-4,

4.24. Methyl 4,6-O-benzylidene-3-thio-3-[2⁰-(valeroic acid)]-

b-^D-galactopyranosyl-(1,4)-O-b-D-glucopyranoside (41)

Gal H-6, Gal H-6⁰), 4.38 (1H, d, J_1,2= 8.2 Hz, Glc H-

1), 4.59 (1H, d, J_1,2= 7.8 Hz, Gal H-1), 5.71 (5.74*)

(1H, s, CHPh), 7.43–7.45, 7.50–7.54 (5H, 2· m, CH

13

In quantitative yield as a 1:1 mixture of diastereomers:

¹H NMR (D₂O): d 0.83 (0.84*) (3H, t, J_vic= 7.3 Hz,

H-5⁰), 1.23–1.38 (2H, m, H-4⁰), 1.51–1.77 (2H, m, H-

3⁰), 3.08 (3.18*) (1H, dd, J_3,2= 11.2, J_3,4= 3.2 Hz, Gal

H-3), 3.24–3.30 (1H, m, Glc H-2), 3.41 (3.48*) (1H,

dd, J_vic= 9.2, 7.1, 3.0 Hz, H-2⁰), 3.53^ꢂ(3H, s, OMe),

3.53–3.64^ꢂ(4H, m, Glc H-3, Glc H-4, Glc H-5, Gal H-

Ph); C NMR (75.5 MHz, D₂O): d 34.8 (34.9*) (C-3⁰),

46.8 (49.8*) (C-2⁰), 48.1 (50.3*) (Gal C-3), 57.2 (OMe),

59.3 (59.4*) (C-4⁰), 59.9 (Glc C-6), 68.7 (Gal C-5), 68.9

(69.0*) (Gal C-6), 69.3 (70.2*), 74.2, 74.8, 78.6 (Gal C-

2, Glc C-3, Glc C-4, Glc C-5), 72.7 (Glc C-2), 74.8

(76.5*) (Gal C-4), 101.3 (101.5*) (CHPh), 103.1 (Glc

C-1), 103.9 (Gal C-1), 2· 126.1 (126.2*), 2· 128.6,

129.7 (129.8*) (CH Ph), 136.6 (136.7*) (ipsoPh), 182.3

(182.4*) (C-1⁰); LRMS 585 (M+Na, 100%).

2), 3.80^ꢂ(1H, s, Gal H-5), 3.81^ꢂ(1H, dd, J_6,6= 12.1,

0

J_6,5= 4.6 Hz, Glc H-6), 3.98 (1H, dd, J_{6 ,6}= 12.1,

0

J_{6 ,5}= 2.1 Hz, Glc H-6⁰), 4.21 (2H, appd, Gal H-6, Gal

H-6⁰), 4.31 (4.34*) (1H, d, J_4,3= 3.2 Hz, Gal H-4),

4.37 (1H, d, J_1,2= 8.0 Hz, Glc H-1), 4.57 (4.58*(1H, d,

J_1,2= 7.7 Hz, Gal H-1), 5.70 (5.74*) (1H, s, CHPh),

7.40–7.46, 7.48–7.53 (5H, 2· m, CHPh); ¹³C NMR

(75.5 MHz, D₂O): d 13.0 (C-5⁰), 20.2 (20.3*) (C-4⁰),

34.6 (34.7*) (C-3⁰), 49.5 (49.8*) (Gal C-3), 50.0 (51.2*)

(C-2⁰), 57.2 (OMe), 59.8 (59.9*(Glc C-6), 68.6 (Gal C-

5), 68.8 (68.9*) (Gal C-6), 69.2 (70.2*), 74.2, 74.7, 78.5

(Glc C-3, Glc C-4, Glc C-5, Gal C-2), 74.4 (76.5*(Gal

C-4), 101.2 (101.5*) (CHPh), 103.0 (Glc C-1), 103.9

(Gal C-1), 2· 126.1 (126.2*), 2· 128.5 (128.6*), 129.6

(129.7*) (CH Ph), 136.5 (136.6*) (ipsoPh), 180.4

(181.0*) (C-1⁰); LRMS 583 (M+Na, 100%).

4.27. Methyl 4,6-O-benzylidene-3-thio-3-[2⁰-(4⁰-hydroxy-

valeroic acid)]-b-^D-galactopyranosyl-(1,4)-O-b-D-gluco-

pyranoside (44)

1

In 88% yield as a mixture of diastereomers: H NMR

(D₂O): d 1.15–1.22 (3H, m, H-5⁰), 1.69–1.99 (2H, m,

H-3⁰), 3.11–3.25 (1H, m, Gal H-3), 3.32 (1H, t,

J_2,3= J_2,1= 8.5 Hz, Glc H-2), 3.48–3.70^ꢂ(5H, m, Glc

H-3, Glc H-4, Glc H-5, Gal H-2, H-2⁰), 3.57^ꢂ(3H, s,

OMe), 3.81^ꢂ(1H, s, Gal H-5), 3.81–3.95^ꢂ(1H, m, H-

ꢂ

4⁰), 3.86 (1H, dd, J_6,6= 12.3, J_6,5= 4.5 Hz, Glc H-6),

0

4.02 (1H, appd, J_{6 ,6}= 12.3 Hz, Glc H-6⁰), 4.23 (2H,

0

appd, Gal H-6, Gal H-6⁰), 4.34 (1H, apps, Gal H-4),

4.40 (1H, d, J_1,2= 8.0 Hz, Glc H-1), 4.61 (1H, d,

J_1,2= 7.7 Hz, Gal H-1), 5.71 (5.74*) (1H, s, CHPh),

7.46–7.48, 7.53–7.53 (5H, 2· m, CHPh); ¹³C NMR

(75.5 MHz, D₂O): d 22.4 (22.7*, 22.8*, 22.9*) (C-5⁰),

41.8 (41.9*) (C-3⁰), 47.5 (49.1*) (C-2⁰), 51.8 (Gal C-3),

57.9 (OMe), 60.6 (Glc C-6), 66.0 (66.1*, 66.6*, 66.7*)

(C-4⁰), 69.2 (69.3*) (Gal C-5), 69.4 (69.5*) (Gal C-6),

69.8 (71.0*), 74.9, 75.4, 79.2 (Glc C-3, Glc C-4, Glc C-

5, Gal C-2), 75.0 (77.8*) (Gal C-4), 101.9 (102.0*)

(CHPh), 103.7 (Glc C-1), 104.5 (Gal C-1), 2· 126.7

(126.8*), 2· 129.2 (129.3*), 130.3 (130.4*) (CHPh),

137.2, (137.3*, 137.4*) (ipsoPh), 182.0 (182.0) (C-1⁰).

LRMS 599 (M+Na, 100%).

4.25. Methyl 4,6-O-benzylidene-3-thio-3-[2⁰-(2⁰-phenyl-

acetic acid)]-b-^D-galactopyranosyl-(1,4)-O-b-D-glucopyr-

anoside (42)

In quantitative yield as a 1:1 mixture of diastereomers:

¹H NMR (D₂O): d 2.83 (2.91*) (1H, dd, J_3,2= 11.1,

J_3,4= 3.2 Hz, Gal H-3), 3.17 (1H, t, J_2,1= J_2,3= 8.0 Hz,

Glc H-2), 3.45^ꢂ(3H, s, OMe), 3.45–3.76^ꢂ(5H, m, Gal

H-2, Gal H-5, Glc H-3, Glc H-4, Glc H-5), 3.86–3.93

0

(1H, m, Glc H-6), 4.02 (1H, d, J_{6 ,6}= 12.1 Hz, Glc H-

6⁰), 4.13 (2H, appd, Gal H-6, Gal H-6⁰), 4.19 (1H, d,

J_4,3= 3.2 Hz, Gal H-4), 4.28 (1H, d, J_1,2= 8.0 Hz, Glc

H-1), 4.42 (1H, d, J_1,2= 7.8 Hz, Gal H-1), 4.74 (4.77*)

(1H, s, H-2⁰), 5.24 (5.64*) (1H, s, CHPh), 7.19–7.45

(5H, m, 2· Ph); ¹³C NMR (75.5 MHz, D₂O): d 49.7

(49.8*) (Gal C-3), 55.7 (57.2*)^ꢂ(C-2⁰), 57.2^ꢂ(OMe),

59.9 (Glc C-6), 68.5 (68.6*) (Gal C-5), 68.8 (68.9*) (Gal

C-6), 69.6 (69.7*), 74.2, 74.8, 78.5 (Gal C-2, Glc C-3,

Glc C-4, Glc C-5), 72.7 (Glc C-2), 75.2 (75.6*) (Gal C-

4), 101.3 (Gal C-1), 103.0 (Glc C-1), 103.8 (103.9*)

(CHPh), 126.2, 127.9, 128.1, 128.3, 2· 128.6, 128.8,

128.9, 129.7 (2· Ph), 136.5 (136.7*), 138.3 (139.6*) (2·

ipsoPh), 177.1 (177.6*) (C-1⁰); LRMS 617 (M+Na, 100%).

4.28. Methyl 3-thio-3-(sodium carboxymethyl)-b-^D-

galactopyranosyl-(1,4)-O-b-^D-glucopyranoside (45)

A solution of the 4⁰,6⁰-O-benzylidenated lactoside 38

(170 mg, 0.33 mmol) in 10% aqueous TFA (10 mL)

was stirred at 0 ꢂC for 8 h before being concentrated

in vacuo (cold work-up). The residue was dissolved in

H₂O (5 mL) and the pH adjusted to ꢀ7.3 with 1 M

NaOH before being freeze-dried. Size exclusion chroma-

tography [G10 Sephadex column, H₂O (EtOAc/MeOH/

H₂O, 7:2:1, R_f0.17)] and then HPLC puriﬁcation (5%

CH₃CN in H₂O, 3 mL/min, R_t= 4.34 min) aﬀorded 45

4.26. Methyl 4,6-O-benzylidene-3-thio-3-[2⁰-(4⁰-hydroxy-

butanoic acid)]-b-^D-galactopyranosyl-(1,4)-O-b-D-gluco-

pyranoside (43)

1

(99 mg, 67%) as a colourless solid: H NMR (D₂O): d

3.00 (1H, dd, J_3,2= 11.4, J_3,4= 2.7 Hz, Gal H-3), 3.31

(1H, t, J_2,3= J_2,1= 8.1 Hz, Glc H-2), 3.37 (2H, s, H-

2⁰), 3.53 (1H, dd, J_2,3= 11.4, J_2,1= 7.7 Hz, Gal H-2),

3.57 (3H, s, OMe), 3.59–3.68 (3H, m, Glc H-3, Glc H-

4, H-5), 3.71–3.85^ꢂ(3H, m, H-5, H-6, H-6⁰), 3.82^ꢂ(1H,

In quantitative yield as a 1:1 mixture of diastereomers:

¹H NMR (D₂O): d 1.77–2.09 (2H, m, H-3⁰), 3.13

(3.21*) (1H, dd, J_3,2= 11.5, J_3,4= 2.9 Hz, Gal H-3),

3.28 (1H, t, J_2,1= J_2,3= 8.2 Hz, Glc H-2), 3.47–3.70^ꢂ

0

dd, J_6,6= 12.3, J_6,5= 4.8 Hz, H-6), 3.98–4.02 (2H, m,

754

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

Gal H-4, H-6⁰), 4.41 (1H, d, J_1,2= 8.1 Hz, Glc H-1), 4.52

(1H, d, J_1,2= 7.7 Hz, Gal H-1); ¹³C NMR (75.5 MHz,

D₂O): d 36.1 (C-2⁰), 52.9 (Gal C-3), 57.2 (OMe), 60.0,

61.2 (Glc C-6, Gal C-6), 67.8, 74.7, 78.2 (Glc C-3, Glc

C-4, C-5), 70.0 (Gal C-4), 72.7 (Gal C-2), 74.3 (Glc

C-2), 77.5 (C-5), 103.0 (Glc C-1), 103.9 (Gal C-1),

178.1 (C-1⁰); LRMS 453 (M+Na, 100%); Anal.

(C₁₅H₂₅NaO₁₂S4H₂O) C, H.

H-2, H-2⁰), 3.59 (3H, s, OMe), 3.61–3.69 (3H, m, Glc

H-3, Glc H-4, H-5), 3.74–3.86^ꢂ(3H, m, H-5, H-6, H-

ꢂ

6⁰), 3.83 (1H, dd, J_6,6= 12.3, J_6,5= 4.9 Hz, H-6),

0

3.94–4.04 (2H, m, Gal H-4, H-6⁰), 4.42 (1H, d,

J_1,2= 8.2 Hz, Glc H-1), 4.53 (4.54*) (1H, d,

J_1,2= 7.7 Hz, Gal H-1); ¹³C NMR (75.5 MHz, D₂O): d

13.1 (C-5⁰), 20.3 (C-4⁰), 34.7 (34.8*) (C-3⁰), 49.5 (51.2*)

(C-2⁰), 52.0 (52.9*) (Gal C-3), 57.2 (OMe), 60.0

(60.1*), 61.2 (61.3*) (Glc C-6, Gal C-6), 67.4 (68.6*)

(Gal C-4), 69.4 (70.4*) (Gal C-2), 72.8 (Glc C-2), 74.4,

74.8, 78.3 (78.4*(Glc C-3, Glc C-4, C-5), 77.6 (C-5),

103.1 (Glc C-1), 104.0 (Gal C-1), 180.4 (181.2*) (C-1⁰);

LRMS 495 (M+Na, 100%); Anal. (C₁₈H₃₁NaO₁₂S1.

5H₂O) C, H.

In a similar manner were prepared:

4.29. Methyl 3-thio-3-[2⁰-(sodium propanoate)]-b-D-

galactopyranosyl-(1,4)-O-b-^D-glucopyranoside (46)

In 66% yield (HPLC, 5% CH₃CN in H₂O, 3 mL/min,

1

R_t= 5.86 min) as a colourless solid: H NMR (D₂O): d

4.32. Methyl 3-thio-3-[2⁰-(sodium 2⁰-phenylacetate)]-b-D-

galactopyranosyl-(1,4)-O-b-^D-glucopyranoside (49)

1.33 (1.33*) (3H, d, J_vic= 7.0 Hz, H-3⁰), 2.93 (2.98*)

(1H, dd, J_3,2= 11.3, J_3,4= 2.7 Hz, Gal H-3), 3.25 (1H,

t, J_2,3= J_2,1= 8.3 Hz, Glc H-2), 3.38–3.48 (2H, m, Gal

H-2, H-2⁰), 3.52 (3H, s, OMe), 3.53–3.62 (3H, m, Glc

In 69% yield (HPLC, 3% CH₃CN in H₂O, 3 mL/min,

R_t= 8.64, 9.20 min) as a mixture of separable diastereo-

H-3, Glc H-4, H-5), 3.65–3.79^ꢂ(3H, m, H-5, H-6, H-

1

mers. One diastereomer of 49 as a colourless solid: H

ꢂ

6⁰), 3.76 (1H, dd, J_6,6= 12.4, J_6,5= 4.8 Hz, H-6),

NMR (D₂O): d 2.77 (1H, dd, J_3,2= 11.2, J_3,4= 2.8 Hz,

Gal H-3), 3.31 (1H, t, J_2,3= J_2,1= 8.0 Hz, Glc H-2),

3.43 (1H, d, J_3,4= 2.8 Hz, Gal H-4), 3.54–3.67^ꢂ(7H, m,

0

3.87-3.96 (2H, m, Gal H-4, H-6⁰), 4.35 (1H, d,

J_1,2= 8.3 Hz, Glc H-1), 4.46 (1H, d, J_1,2= 7.6 Hz, Gal

H-1); ¹³C NMR (75.5 MHz, D₂O): d 18.1 (18.3*) (C-

3⁰), 44.3 (46.0*) (C-2⁰), 51.8 (52.7*) (Gal C-3), 57.1

(OMe), 60.0, 61.2 (61.3*) (Glc C-6, Gal C-6), 67.6

(68.3*) (Gal C-4), 69.4 (70.2*) (Gal C-2), 72.8 (Glc C-

2), 74.4, 74.7, 78.2 (78.3*) (Glc C-3, Glc C-4, C-5),

77.6 (C-5), 103.1 (Glc C-1), 103.9 (Gal C-1), 181.0

(181.6*) (C-1⁰); LRMS 467 (M+Na, 100%); Anal.

(C₁₆H₂₇NaO₁₂S2H₂O) C, H.

Glc H-3, Glc H-4, Glc H-5, Gal H-2, Gal H-5, H-6, H-

0

ꢂ

6⁰), 3.59 (3H, s, OMe), 3.84 (1H, dd, J_6,6= 12.4,

0

J_6,5= 4.4 Hz, H-6), 4.02 (1H, dd, J_{6 ,6}= 12.4,

0

J_{6 ,5}= 1.7 Hz, H-6⁰), 4.42 (1H, d, J_1,2= 8.0 Hz, Glc H-

1), 4.46 (1H, d, J_1,2= 7.7 Hz, Gal H-1), 4.87 (1H, s, H-

2⁰), 7.35– 7.46, 7.51–7.54 (5H, 2· m, Ph); ¹³C NMR

(75.5 MHz, D₂O): d 51.9 (Gal C-3), 57.1 (C-2⁰), 57.2

(OMe), 60.1, 61.1 (Glc C-6, Gal C-6), 68.4 (Gal C-4),

70.5, 74.4, 74.7, 77.5, 78.4 (Glc C-3, Glc C-4, Glc C-5,

Gal C-2, Gal C-5), 103.1 (Glc C-1), 104.0 (Gal C-1),

127.9, 2· 128.3, 2· 128.9 (Ph), 139.4 (ipsoPh), 177.3 (C-

1⁰); LRMS 529 (M+Na, 100%); Anal. (C₂₁H₂₉

NaO₁₂S3H₂O) C, H. Other diastereomer of 49 as a col-

ourless solid: ¹H NMR (D₂O): d 2.77 (1H, dd,

J_3,2= 11.3, J_3,4= 2.8 Hz, Gal H-3), 3.30 (1H, t,

J_2,3= J_2,1= 8.0 Hz, Glc H-2), 3.52 (1H, dd, J_2,3= 11.3,

J_2,1= 7.7 Hz, Gal H-2), 3.58 (3H, s, OMe), 3.60–3.82^ꢂ

(6H, Glc H-3, Glc H-4, Glc H-5, Gal H-5, H-6, H-6⁰),

4.30. Methyl 3-thio-3-[2⁰-(sodium butanoate)]-b-D-galac-

topyranosyl-(1,4)-O-b-^D-glucopyranoside (47)

In 65% yield (HPLC, 1% CH₃CN in H₂O, 3 mL/min,

1

R_t= 8.29 min) as a colourless solid: H NMR (D₂O): d

0.97^ꢂ(2.64*)^ꢂ(3H, t, J_vic= 7.3 Hz, H-4⁰), 1.64-1.87

(2H, m, H-3⁰), 2.97–3.06 (1H, m, Gal H-3), 3.30 (2H,

m, Glc H-2, H-2⁰), 3.44–3.52 (1H, m, Gal H-2), 3.59

(3H, s, OMe), 3.63–3.69 (3H, m, Glc H-3, Glc H-4, H-

5), 3.72–3.86^ꢂ(3H, m, H-5, H-6, H-6⁰), 3.83^ꢂ(1H, dd,

3.79^ꢂ(1H, dd, J_6,6= 12.5, J_6,5= 4.7 Hz, H-6), 3.95–

0

J_6,6= 12.3, J_6,5= 4.8 Hz, H-6), 3.95 (2H, m, Gal H-4,

4.00 (2H, m, Gal H-4, H-6⁰), 4.41^ꢂ(1H, d, J_1,2

=

0

H-6⁰), 4.42 (1H, d, J_1,2= 8.0 Hz, Glc H-1), 4.53 (4.54*)

(1H, d, J_1,2= 7.7 Hz, Gal H-1); ¹³C NMR (75.5 MHz,

D₂O): d 11.4 (C-4⁰), 26.0 (C-3⁰), 51.5 (51.9*) (C-2⁰),

52.9 (53.2*) (Gal C-3), 57.2 (OMe), 60.0 (60.1*), 61.2

(61.3*) (Glc C-6, Gal C-6), 67.5 (68.4*) (Gal C-4), 69.5

(70.3*) (Gal C-2), 72.8 (Glc C-2), 74.4, 74.8, 78.3

(78.4*) (Glc C-3, Glc C-4, C-5), 77.6 (C-5), 103.1 (Glc

C-1), 104.0 (Gal C-1), 180.3 (181.0*) (C-1⁰); LRMS

481 (M+Na, 100%); Anal. (C₁₇H₂₉NaO₁₂S3H₂O) C, H.

8.0 Hz, Glc H-1), 4.43^ꢂ(1H, d, J_1,2= 7.7 Hz, Gal H-1),

4.86 (1H, s, H-2⁰), 7.35–7.51 (5H, m, Ph); ¹³C NMR

(75.5 MHz, D₂O): d 52.2 (Gal C-3), 55.5 (C-2⁰), 57.2

(OMe), 60.0, 61.2 (Glc C-6, Gal C-6), 67.2 (Gal C-4),

69.7 (Gal C-2), 72.7 (Glc C-2), 74.4, 74.7, 77.5, 78.4

(Glc C-3, Glc C-4, Glc C-5, Gal C-5), 103.0, 103.9 (Glc

C-1, Gal C-1), 127.9, 2· 128.2, 2· 128.9 (Ph), 138.5 (ip-

soPh), 177.6 (C-1⁰). LRMS 529 (M+Na, 100%).

4.33. Methyl 3-thio-3-[2⁰-sodium (4⁰-hydroxy-butano-

ate)]-b-^D-galactopyranosyl-(1,4)-O-b-D-glucopyranoside

(50)

4.31. Methyl 3-thio-3-[2⁰-(sodium valeroate)]-b-D-galac-

topyranosyl-(1,4)-O-b-^D-glucopyranoside (48)

In 69% yield (HPLC, 3% CH₃CN in H₂O, 3 mL/min,

In 70% yield (HPLC, 1% CH₃CN in H₂O, 3 mL/min,

1

R_t= 6.87 min) as a colourless solid: H NMR (D₂O): d

R_t= 4.75 min) as a colourless solid: H NMR (D₂O): d

0.92 (3H, t, J_vic= 7.3 Hz, H-5⁰), 1.33–1.43 (2H, m, H-

4⁰), 1.58–1.84 (2H, m, H-3⁰), 3.01 (3.02*) (1H, dd,

J_3,2= 11.4, J_3,4= 2.5 Hz, Gal H-3), 3.33 (1H, t,

J_2,3= J_2,1= 8.2 Hz, Glc H-2), 3.41–3.52 (2H, m, Gal

1.81–2.08 (2H, m, H-3⁰), 3.03^ꢂ(3.05*)^ꢂ(1H, dd,

J_3,2= 11.6, J_3,4= 2.7 Hz, Gal H-3), 3.30 (1H, t,

J_2,3= J_2,1= 8.2 Hz, Glc H-2), 3.42–3.53 (2H, m, Glc

H-2, H-2⁰), 3.57 (3H, s, OMe), 3.59–3.83^ꢂ(8H, m, Glc

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

755

H-3, Glc H-4, Glc H-5, Gal H-5, H-6, H-6⁰, H-4⁰), 3.81^ꢂ

and the supernatants were stored at ꢁ70 ꢂC. Virus

stocks were activated prior to use by treatment of cell ly-

sates with 10 lg/mL porcine trypsin for 30 min at 37 ꢂC.

0

(1H, dd, J_6,6= 12.6, J_6,5= 4.9 Hz, H-6), 3.93–4.01 (2H,

m, Gal H-4, H-6⁰), 4.40 (1H, d, J_1,2= 8.2 Hz, Glc H-1),

4.51^ꢂ(4.52*)^ꢂ(1H, d, J_1,2= 7.6 Hz, Gal H-1); ¹³C NMR

(75.5 MHz, D₂O): d 34.7 (34.9*) (C-3⁰), 46.1 (48.1*) (C-

2⁰), 52.0 (53.0*) (Gal C-3), 57.2 (OMe), 59.3 (C-4⁰), 60.0

(60.1*), 61.2 (61.3*) (Glc C-6, Gal C-6), 67.4 (68.5*)

(Gal C-4), 69.3 (70.4*) (Gal C-2), 72.7 (Glc C-2), 74.4,

74.7, 77.6, 78.2 (78.3*) (Glc C-3, Glc C-4, Glc C-5,

Gal C-5), 103.0 (Glc C-1), 103.9 (Gal C-1), 179.7

(180.4*) (C-1⁰); LRMS 497 (M+Na, 100%); Anal.

(C₁₇H₂₉NaO₁₃S3. 5H₂O) C, H.

5.3. Neutralisation assays

For use in neutralisation assays, the virus was titrated

to determine a dilution which gave ꢀ200 ﬂuorescent fo-

cus forming units/well of the 96-well microtitre tray

(NUNC). Sialylmimetics were dissolved in DMEM,

brought to pH 7.5 by addition of a small volume of

sodium carbonate and incubated for 1 h at 37 ꢂC with

trypsin-activated virus. The sialylmimetic-virus mixture

was then added to conﬂuent monolayers of MA104

cells grown in 96-well microtitre trays and incubated

for 1 h at 37 ꢂC. The inoculum was then removed

and replaced with DMEM and the cells were incubated

for 16 h at 37 ꢂC in a 5% CO₂environment. Virus-in-

fected cells were detected by indirect immunoﬂuores-

cent staining. Rotavirus-infected cell monolayers were

ﬁxed and permeabilised in 80% acetone for 10 min.

The cells were then washed three times with PBS and

covered with 50 lL of hyper-immune rabbit anti-

RRV serum diluted 1 in 2000 in PBS and incubated

at 37 ꢂC for 30 min. Following this, cells were stained

with 30 lL of ﬂuorescein isothiocyanate-conjugated

sheep anti-rabbit immunoglobulin (Silenus, Australia)

diluted 1 in 100 in PBS. After 30 min at 37 ꢂC, cells

were washed three times with PBS and viewed through

a ﬂuorescent microscope. Inhibition of virus infection

was measured by determining the percentage reduction

in the number of rotavirus-infected cells at 10 mM con-

centration of the sialylmimetic, compared with the

number of infected cells in the absence of any sialylmi-

metic. For those sialylmimetics that showed inhibition

at 10 mM, the inhibition studies were repeated with

the compound at 5 mM, 2.5 mM, and 1.25 mM con-

centrations. The percentage inhibition of rotavirus

infectivity by the sialylmimetics at 5 mM is also record-

ed in the results if it were statistically signiﬁcant. Statis-

tical analysis was determined using the t test, with

signiﬁcance set at p = 0.05.

4.34. Methyl 3-thio-3-[2⁰-sodium (4⁰-hydroxy-valeroate)]-

b-^D-galactopyranosyl-(1,4)-O-b-D-glucopyranoside (51)

In 68% yield (HPLC, 1% CH₃CN in H₂O, 4 mL/min,

1

R_t= 5.49 min) as a colourless solid: H NMR (D₂O):

d 1.08–1.11 (3H, m, H-5⁰), 1.59–1.92 (2H, m, H-3⁰),

3.20 (1H, t, J_2,1= J_2,3= 8.3 Hz, Glc H-2), 3.32–3.43

(2H, m, Gal H-2, H-2⁰), 3.47^ꢂ(3H, s, OMe), 3.47–

3.57^ꢂ(3H, m, Glc H-3/Gal H-5, Glc H-4, Glc H-5),

3.60–3.74 (4H, m, Glc H-3/Gal H-5, 2· H-6, H-6⁰),

3.83–3.91 (3H, m, Gal H-4, H-6⁰, H-4⁰), 4.30 (1H,

d, J_1,2= 8.3 Hz, Glc H-1), 4.41 (4.42*) (1H, d,

J_1,2= 7.7 Hz, Gal H-1); ¹³C NMR (75.5 MHz,

D₂O):

d

21.7 (21.9*, 22.1*, 22.3*) (C-5⁰), 41.0

(41.3*, 41.4*, 41.5*) (C-3⁰), 46.0 (46.2*, 48.3*) (C-

2⁰), 52.0 (53.0*, 53.1*) (Gal C-3), 57.2 (OMe), 60.0

(60.1*), 61.2 (61.3*) (Gal C-6, Glc C-6), 65.3

(65.4*, 66.0*, 66.1*), 67.3 (67.4*, 68.5*, 68.6*) (Gal

C-4, C-4⁰), 69.1 (69.2*, 70.4*, 70.5*) (Gal C-2), 72.7

(Glc C-2), 74.4, 74.7, 77.6, 78.2 (78.3*, 78.4*) (Glc

C-3, Glc C-4, Glc C-5, Gal C-5), 103.0 (Glc C-1),

103.9 (Gal C-1), 179.7 (179.9*, 180.4*, 180.6*) (C-

1⁰);

(C₁₈H₃₁NaO₁₃S4H₂O) C, H.

LRMS

511

(M + Na,

100%);

Anal.

5. Biological evaluation

5.1. Cells

MA104 cells (an African Green Monkey kidney cell line)

were used to cultivate rotaviruses of two diﬀerent sero-

types, namely RRV (rhesus) and a strain that aﬀects hu-

mans (Wa). MA104 cells were grown at 37 ꢂC in

Dulbeccoꢁs modiﬁed Eagleꢁs medium (DMEM) supple-

mented with 10% foetal calf serum (FCS), 20 mM

Hepes, 26.6 lg/mL gentamicin and 2 lg/mL fungizone.

Acknowledgments

The authors thank the National Health and Medical Re-

search Council and the Australian Research Council for

ﬁnancial support. M.v.I. gratefully acknowledges the

award of an Australian Research Council Federation

Fellowship and the award of a von Humboldt For-

schungspreis from the Alexander von Humboldt Stif-

tung. A.L. acknowledges the Australian Government

for the award of an Australian Postgraduate Award

and the Institute for Glycomics, Griﬃth University,

for an Institute Postgraduate Award.

5.2. Virus

Virus infectivity was activated with 10 lg/mL porcine

trypsin (type IX; Sigma) for 30 min at 37 ꢂC before inoc-

ulation onto conﬂuent cell monolayers grown in 75 cm²

plastic cell culture ﬂasks (NUNC, Denmark) at a multi-

plicity of infection (m.o.i.) of 10 ﬂuorescent cell forming

units/cell. After 60 min at 37 ꢂC, the inoculum was re-

moved and infected cells were incubated in DMEM con-

taining 1 lg/mL porcine trypsin until extensive

cytopathic eﬀect (c.p.e.) was evident. Cell lysates were

frozen and thawed twice, centrifuged (3000g, 10 min),

Supplementary material

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/

j.bmc.2005.08.057.

756

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

32. Delorme, C.; Brussow, H.; Sidoti, J.; Roche, N.; Karlsson,

¨

K.-A.; Neeser, J.-R.; Teneberg, S. J. Virol. 2001, 75, 2276–

2287.

33. Fuentes-Panana, E. M.; Lopez, S.; Gorziglia, M.; Arias,

C. F. J. Virol. 1995, 69, 2629–2632.

References and notes

1. Estes, M. In Fundamental Virology; Fields, B. N., Knipe,

D. M., Howley, P. M., Eds.; Lippincott Raven Publishers:

Philadelphia, 1996, pp 731–761.

34. Fiore, L.; Greenberg, H. B.; Mackow, E. R. Virology

1991, 181, 553–563.

2. Parashar, U. D.; Bresee, J. S.; Gentsch, J. R.; Glass, R. I.

Emerg. Infect. Dis. 1998, 4, 561–570.

35. Crawford, S. E.; Mukherjee, S. K.; Estes, M. K.; Lawton,

J. A.; Shaw, A. L.; Ramig, R. F.; Prasad, B. V. V. J. Virol.

2001, 75, 6052–6061.

3. Paul, P. S.; Lyoo, Y. S. Vet. Microbiol. 1993, 37, 299–317.

4. Svennerholm, A.-M.; Steele, D. Best Practice Res. Clin.

Gastroenterol. 2004, 18, 421–445.

36. Dormitzer, P. R.; Greenberg, H. B.; Harrison, S. C. J.

Virol. 2001, 75, 7339–7350.

5. Fischer, T. K.; Bresee, J. S.; Glass, R. I. Vaccine 2004, 22,

S49–S54.

37. Dormitzer, P. R.; Sun, Z.-Y. J.; Wagner, G.; Harrison, S.

C. EMBO J. 2002, 21, 885–897.

6. Franco, M. A.; Greenberg, H. B. Virology 2001, 281,

153–155.

38. Dormitzer, P. R.; Sun, Z.-Y. J.; Blixt, O.; Paulson, J. C.;

Wagner, G.; Harrison, S. C. J. Virol. 2002, 76, 10512–10517.

39. Takahashi, K.; Ohashi, K.; Abe, Y.; Mori, S.; Taniguchi,

K.; Ebina, T.; Nakagomi, O.; Terada, M.; Shigeta, S.

Antimicrob. Agents Chemother. 2002, 46, 420–424.

40. Kiefel, M. J.; Beisner, B.; Bennett, S.; Holmes, I. D.; von

Itzstein, M. J. Med. Chem. 1996, 39, 1314–1320.

41. Fazli, A.; Bradley, S. J.; Kiefel, M. J.; Jolly, C.; Holmes, I.

H.; von Itzstein, M. J. Med. Chem. 2001, 44, 3292–3301.

42. Willoughby, R. E.; Yolken, R. H. J. Infect. Dis. 1990, 161,

116–119.

7. Murphy, T. V.; Gargiullo, P. M.; Massoudi, M. S.;

Nelson, D. B.; Jumaan, A. O.; Okoro, C. A.; Zanardi, L.

R.; Setia, S.; Fair, E.; LeBaron, C. W.; Wharton, M.;

Livingood, J. R. N. Engl. J. Med. 2001, 344, 564–572.

8. Glass, R. I.; Bresee, J. S.; Parashar, U. D.; Jiang, B.;

Gentsch, J. Lancet 2004, 363, 1547–1550.

9. Estes, M. K.; Cohen, J. Microbiol. Rev. 1989, 53, 410–449.

10. Ludert, J. E.; Feng, N.; Yu, J. H.; Broome, R. L.;

Hoshino, Y.; Greenberg, H. B. J. Virol. 1996, 70, 487–493.

11. Coulson, B. S.; Londrigan, S. L.; Lee, D. J. Proc. Natl.

Acad. Sci. U.S.A. 1997, 94, 5389–5394.

43. Zhou, Q.; Hakomori, S.-I.; Kitamura, K.; Igarashi, Y. J.

Biol. Chem. 1994, 269, 1959–1965.

´

12. Zarate, S.; Espinosa, R.; Romero, P.; Mendez, E.; Arias,

´

C. F.; Lopez, S. J. Virol. 2000, 74, 593–599.

´

13. Arias, C. F.; Isa, P.; Guerrero, C. A.; Mendez, E.; Zarate,

S.; Lopez, T.; Espinosa, R.; Romero, P.; Lopez, S. Arch.

´

44. Ferrero-Garcia, M. A.; Trombetta, S. E.; Sanchez, D. O.;

´

Reglero, A.; Frasch, A. C. C.; Parodi, A. J Eur. J.

Biochem. 1993, 213, 765–771.

´

Med. Res. 2002, 33, 356–361.

45. Wilson, J. C.; Kiefel, M. J.; Angus, D. I.; von Itzstein, M.

Org. Lett. 1999, 1, 443–446.

46. Turnbull, W. B.; Field, R. A. J. Chem. Soc. Perkin. Trans.

2000, 1, 1859–1866.

47. von Itzstein, M.; Jenkins, M. J.; Mocerino, M. Carbohydr.

Res. 1990, 208, 287–288.

14. Dormitzer, P. R.; Nason, E. B.; Prasad, B. V. V.;

Harrison, S. C. Nature 2004, 430, 1053–1058.

15. Greenberg, H. B.; Valdesuso, J.; Van Wyke, K.;

Midthun, K.; Walsh, M.; McAuliﬀe, V.; Wyatt, R. G.;

Kalica, A. R.; Flores, J.; Hoshino, Y. J. Virol. 1983, 47,

267–275.

48. Marcaurelle, L. A.; Bertozzi, C. R. J. Am. Chem. Soc.

2001, 123, 1587–1595.

49. Liakatos, A.; Kiefel, M. J.; von Itzstein, M. Org. Lett.

2003, 5, 4365–4368.

50. Rich, J. R.; Szpacenko, A.; Palcic, M. M.; Bundle, D. R.

Angew. Chem. Int. Ed. 2004, 43, 613–615.

51. Rich, J. R.; Bundle, D. R. Org. Lett. 2004, 6, 897–900.

52. Kameyama, A.; Ishida, H.; Kiso, M.; Hasegawa, A.

Carbohydr. Res. 1990, 200, 269–285.

´

16. Lopez, S.; Arias, C. F. Trends Microbiol. 2004, 12, 271–278.

17. Jayaram, H.; Estes, M. K.; Prasad, B. V. V. Virus Res.

2004, 101, 67–81.

´

18. Mendez, E.; Lopez, S.; Cuadras, M. A.; Romero, P.;

Arias, C. F. Virology 1999, 263, 450–459.

19. Guerrero, C. A.; Zarate, S.; Corkidi, G.; Lopez, S.; Arias,

C. F. J. Virol. 2000, 74, 9362–9371.

20. Tihova, M.; Dryden, K. A.; Bellamy, A. R.; Greenberg, H.

B.; Yeager, M. J. Mol. Biol. 2001, 314, 985–992.

21. Jolly, C. L.; Beisner, B. M.; Holmes, I. H. Virology 2000,

275, 89–97.

53. Jung, K. H.; Hoch, M.; Schmidt, R. R. Liebigs Ann.

Chem. 1989, 1099–1106.

54. Grindley, T. B. Protecting groups in oligosaccharide

synthesis. In Modern Methods in Carbohydrate Synthesis,

1996; Vol. 1, pp. 225–250.

55. David, S.; Hanessian, S. Tetrahedron 1985, 41, 643–663.

56. David, S. Selective O-substitution and oxidation using

stannylene acetals and stannyl ethers. In Preparative

Carbohydrate Chemistry; Hanessian, S., Ed.; Marcel

Dekker: New York, 1997, pp 69–86.

22. Hewish, M. J.; Takada, Y.; Coulson, B. S. J. Virol. 2000,

74, 228–236.

23. Kiefel, M. J.; von Itzstein, M. Methods Enzymol. 2003,

363, 395–412.

24. Ciarlet, M.; Crawford, S. E.; Cheng, E.; Blutt, S. E.; Rice,

D. A.; Bergelson, J. M.; Estes, M. K. J. Virol. 2002, 76,

1109–1123.

25. Ciarlet, M.; Crawford, S. E.; Estes, M. K. J. Virol. 2001,

75, 11834–11850.

57. Jansson, K.; Ahlfors, S.; Frejd, T.; Kihlberg, J.; Magnus-

son, G.; Dahmen, J.; Noori, G.; Stenvall, K. J. Org. Chem.

1988, 53, 5629–5647.

26. Fukudome, K.; Yoshie, O.; Konno, T. Virology 1989, 172,

196–205.

58. Brown, L.; Koreeda, M. J. Org. Chem. 1984, 49, 3875–

3880.

59. Bouhroum, S.; Vottero, P. J. A. Tetrahedron Lett. 1990,

31, 7441–7444.

27. Ciarlet, M.; Estes, M. K. J. Gen. Virol. 1999, 80, 943–948.

28. Guo, C. T.; Nakagomi, O.; Mochizuki, M.; Ishida, H.;

Kiso, M.; Ohta, Y.; Suzuki, T.; Miyamoto, D.; Hidari, K.

I.; Suzuki, Y. J. Biochem. 1999, 126, 683–688.

60. Raduchel, B. Synthesis 1980, 292–295.

¨

61. Eisele, T.; Toepfer, A.; Kretzschmar, G.; Schmidt, R. R.

´

29. Mendez, E.; Arias, C. F.; Lopez, S. J. Virol. 1993, 67,

5253–5259.

Tetrahedron Lett. 1996, 37, 1389–1392.

62. Eisele, T.; Schmidt, R. R. Liebigs Ann. Chem. 1997, 1303–

1313.

63. Eisele, T.; Schmidt, R. R. Liebigs Ann. Chem. 1997, 865–

872.

´

30. Ciarlet, M.; Ludert, J. E.; Iturriza-Gomara, M.; Liprandi,

F.; Gray, J. J.; Desselberger, U.; Estes, M. K. J. Virol.

2002, 76, 4087–4095.

31. Rolsma, M. D.; Kuhlenschmidt, T. B.; Gelberg, H. B.;

Kuhlenschmidt, M. S. J. Virol. 1998, 72, 9079–9091.

A. Liakatos et al. / Bioorg. Med. Chem. 14 (2006) 739–757

757

64. Park, W. K. C.; Meunier, S. J.; Zanini, D.; Roy, R.

Carbohydr. Lett. 1995, 1, 179–184.

65. Ogura, H.; Furuhata, K.; Itoh, M.; Shitori, Y. Carbohydr.

Res. 1986, 1, 137–158.

68. Furniss, B. S.; Hannaford, A. J.; Smith, P. W. G.;

Tatchell, A. R. Vogelꢁs Textbook of Practical Organic

Chemistry, 5th ed.; Longman Scientiﬁc and Technical:

England, 1989.

66. Eschenfelder, V.; Brossmer, R. Carbohydr. Res. 1987, 162–294.

67. Bradley, S. J.; Fazli, A.; Kiefel, M. J.; von Itzstein, M.

Bioorg. Med. Chem. Lett. 2001, 11, 1587–1590.

69. Armarego, W. L. F.; Perrin, D. D. Puriﬁcation of

Laboratory Chemicals, 4th ed.; Butterworth-Heinemann:

Oxford, 1997.

Article Doi

The synthesis and biological evaluation of lactose-based sialylmimetics as inhibitors of rotaviral infection

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