Bicyclic (galacto)nojirimycin analogues as glycosidase inhibitors: Effect of structural modifications in their pharmacological chaperone potential towards β-glucocerebrosidase

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PAPER

Bicyclic (galacto)nojirimycin analogues as glycosidase inhibitors: Effect of

structural modiﬁcations in their pharmacological chaperone potential towards

b-glucocerebrosidase†

Matilde Aguilar-Moncayo,^aM. Isabel Garc´ıa-Moreno,^aAna Trapero,^bMeritxell Egido-Gaba´s,^b

Amadeu Llebaria,^bJose´ M. Garc´ıa Ferna´ndez*^cand Carmen Ortiz Mellet*^a

Received 14th February 2011, Accepted 23rd February 2011

DOI: 10.1039/c1ob05234a

A molecular-diversity-oriented approach for the preparation of bicyclic sp²-iminosugar glycomimetics

related to nojirimycin and galactonojirimycin is reported. The synthetic strategy takes advantage of the

ability of endocyclic pseudoamide-type atoms in ﬁve-membered cyclic iso(thio)ureas and guanidines to

undergo intramolecular nucleophilic addition to the masked carbonyl group of monosaccharides. The

stereochemistry of the resulting hemiaminal stereocenter is governed by the anomeric effect, with a

large preference for the axial (pseudo-a) orientation. A library of compounds differing in the

stereochemistry at the position equivalent to C-4 in monosaccharides (D-gluco and D-galacto), the

heterocyclic core (cyclic isourea, isothiourea or guanidine) and the nature of the exocyclic nitrogen

substituent (apolar, polar, linear or branched) has been thus prepared and the glycosidase inhibitory

activity evaluated against commercial glycosidases. Compounds bearing lipophilic substituents behaved

as potent and very selective inhibitors of b-glucosidases. They further proved to be good competitive

inhibitors of the recombinant human b-glucocerebrosidase (imiglucerase) used in enzyme replacement

therapy (ERT) for Gaucher disease. The potential of these compounds as pharmacological chaperones

was assessed by measuring their ability to inhibit thermal-induced denaturation of the enzyme in

comparison with N-nonyl-1-deoxynojirimycin (NNDNJ). The results indicated that amphiphilic

sp²-iminosugars within this series are more efﬁcient than NNDNJ at stabilizing b-glucocerebrosidase

and have a strong potential in pharmacological chaperone (PC) and ERT-PC combined therapies.

Introduction

Sugar mimics bearing endocyclic nitrogen atoms (iminosugars and

azasugars), such as deoxynojirimycin (DNJ) 1 and castanosper-

mine 2, have been shown to behave as potent glycosidase inhibitors

(Fig. 1). Given the broad range of biological and pathological

processes in which glycosidases are involved, from the catabolism

of sugars to the biosynthesis of the complex oligosaccharide

chains in glycoproteins and glycolipids, speciﬁc inhibitors of

^aDepartamento de Qu´ımica Orga´nica, Facultad de Qu´ımica, Universidad

de Sevilla, Apartado 553, E-41012, Sevilla, Spain. E-mail: mellet@us.es;

Fax: +34 954624960; Tel: +34 954559806

^bResearch Unit on Bioactive Molecules (RUBAM), Departament de

Quı´ımica Biome`dica, Institut de Quı´ımica Avanc¸ada de Catalunya (IQAC -

CSIC), Jordi Girona 18-26, E-08034, Barcelona, Spain

^cInstituto de Investigaciones Qu´ımicas (IIQ), CSIC - Universidad de Sevilla,

Ame´rico Vespucio 49, Isla de la Cartuja, E-41092, Sevilla, Spain. E-mail:

jogarcia@iiq.csic.es; Fax: +34 954460565; Tel: + 34 954489553

† Electronic supplementary information (ESI) available: Experimental

procedures for the preparation of isothiocyanato precursors 16–21, plots

of Imiglucerase activity after thermal denaturation (48 ^◦C) in the presence

of increasing concentrations of compounds 5–7, 9, 37, 52, 55, Lineweaver–

Burk plots for K_ideterminations and NMR spectra of all new compounds.

See DOI: 10.1039/c1ob05234a

Fig. 1 Structures of DNJ (1), castanospermine (2), and the bicyclic

nojirimycin analogues 3–10.

these enzymes bear strong potential for the development of

new pharmaceuticals¹e.g., in the treatment of type II diabetes

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mellitus,²viral infections,³cancer⁴or hereditary enzyme deﬁciency

diseases.⁵

proﬁling also evidenced differences in their chaperoning activity

that were mutation-dependent.¹⁰

Despite their promise, neither 1 or 2 nor other members of this

family of glycomimetics have realized their full clinical potential.

This is largely because of a lack of commercially viable syntheses

and difﬁculties in preparing a comprehensive palette of variant

structures. Moreover, in most cases the lack of speciﬁcity between

different isoenzymes represents an important problem for clinical

applications. In order to tackle the problems of enzyme selectivity,

several years ago we developed a new family of glycomimetics

in which the amine-type nitrogen characteristic of iminosugars

was replaced by a pseudoamide-type nitrogen atom with high sp²-

character (sp²-iminosugars; e.g. compounds 3 and 4).⁶This struc-

turalmodiﬁcationprofoundlyalters the stereoelectronic properties

at the pseudoanomeric region, rendering compounds that are

chemically, conformationally and conﬁgurationally stable in water

solution even when incorporating oxygen (hemiaminals), nitrogen

(gem-diamines) or sulfur substituents (aminothioacetals) at the

position equivalent to C-1 in monosaccharides.⁷For instance, the

pseudoanomeric hydroxyl group in the cyclic carbamate nojir-

imycin derivative 3 was found exclusively in the axial orientation,

as determined by the generalized anomeric effect, matching the

conﬁgurational pattern of a-glucopyranosides.⁸Accordingly, it

behaved as a potent and selective inhibitor of yeast a-glucosidase

(K_i2.2 mM), being inactive against several b-glucosidases.

Notwithstanding, a dramatic shift in the a/b-glucosidase selectiv-

ity was observed for cyclic isourea analogues bearing aliphatic sub-

stituents at the exocyclic nitrogen atom, e.g. 5 (K_i1.7 mM against

almond b-glucosidase; no inhibition of yeast a-glucosidase) even

though they likewise existed in the a-like conﬁguration in water

solution.⁹Compound 5 was also a potent and selective inhibitor

of human lysosomal b-glucosidase (b-glucocerebrosidase, Glc-

Cerase). Most interestingly, it promoted the correct folding in

GlcCerase mutants associated with Gaucher disease and their

trafﬁcking to the lysosome, being currently under study as

pharmacological chaperone for the treatment of this pathology.¹⁰

X-Ray crystallographic studies showed that isourea 5 is actually

present in the b-conﬁguration in the active site of b-glucosidases.¹¹

This enzyme-dependent induced ﬁt process is promoted by the sub-

stituent at the imine functionality, which locates in a hydrophobic

pocket at the vicinity of the active site of b-glucosidases resulting in

a remarkably high favourable entropic contribution to binding.¹¹

Interestingly, replacement of the endocyclic oxygen atom by sulfur

or nitrogen increased the inhibitory potency for b-glucosidases

while preserving the anomeric selectivity (K_i0.76 and 0.42 mM

against almond b-glucosidase for isothiourea 6 and guanidine

7, respectively).¹²Inversion of the conﬁguration at the position

equivalent to C-4 in monosaccharides, thereby shifting from a

hydroxylation pattern of structural complementarity to D-glucose

to a D-galactose-type proﬁle, further enhanced the inhibitory

potential against b-glucosidases (e.g., K_i0.023 mM against almond

b-glucosidase for isothiourea 9 and K_i0.04 mM against bovine

liver b-glucosidase for guanidine 10). Actually, compounds 6–

10 also showed chaperone activity in Gaucher ﬁbroblasts with

several mutations, with isothiourea derivatives 6 and 9 being

the most active candidates followed by guanidine 7. Contrary

to classical sp³-iminosugars such as 1 or the corresponding N-

alkyl derivatives, the sp²-iminosugars were particularly active for

mutations leading to neuronopathic types of the disease. Mutation

The above commented results illustrate how structural modiﬁca-

tions in the skeleton and substitution proﬁle of sp²-iminosugar gly-

comimetics can be exploited to ﬁnely tune their enzyme inhibition

and pharmacological chaperone selectivity and potency even for

closely similar mutant enzymes. The implementation of synthetic

strategies allowing elaboration of a collection of derivatives for

structure–activity relationship (SAR) studies seemed therefore

very appealing. Given the promising results obtained with the

isothiourea and guanidine-type bicyclic cores both of the D-

gluco and D-galacto isosteric series in pharmacological chaperone

therapy approaches, a systematic investigation of the effect of a

variety of N¢-substituents in their biological activity has now been

undertaken.

Results and discussion

Synthesis

In principle, the target bicyclic skeleton containing the isoth-

iourea or guanidine functionality I (X = S or NH) can be

obtained by intramolecular furanose→piperidine rearrangement

of 2-aminothiazoline or 2-aminoimidazoline pseudo-C-nucleoside

precursors (IIa), respectively, after liberating the aldehyde func-

tionality (IIb) of the monosaccharide. For iminothiazolidine

derivatives, the key thiazoline synthetic intermediate (II, X =

S) can be prepared from vicinal hydroxythiourea precursors III

followingactivationofthe primaryhydroxylgroup for nucleophilic

displacement by the thiocarbonyl sulfur atom. This retrosynthetic

scheme is particularly appealing for generating molecular diversity

in an efﬁcient manner since thioureas can be readily prepared by

coupling of isothiocyanates and amines, which generally proceed

with total chemoselectivity and high yield in the presence of OH

functionalities.¹³The corresponding imidazoline derivatives (II,

X = NH) could be obtained from 5,6-(cyclic isothiouronium) salt

intermediates IV by nucleophilic displacement of the alkylthio

group by amine (Fig. 2).¹²

Fig.

2

Retrosynthesis of bicyclic isothiourea and guanidine-type

sp²-iminosugars.

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For practical reasons, the already reported 5-azido-5-deoxy-1,2-

O-isopropylidene-6-O-tetrahydropyranyl-a-D-glucofuranose and

5-azido-5-deoxy-1,2-O-isopropylidene-a-D-galactofuranose 11¹⁴

and 12¹⁵were chosen as starting materials (Fig. 3). The cor-

responding 5-amino-5-deoxy-sugars can be readily generated

by catalytic hydrogenation and were used in the subsequent

coupling reactions without further puriﬁcation. For the synthesis

of thiourea intermediates III, we ﬁrst selected n-butyl isothio-

cyanate (13), 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl isothio-

cyanate (14)¹⁶and phenyl isothiocyanate (15) as representative

examples of alkyl, glycosyl and aryl substituted electrophile

counterparts, respectively. Since a preliminary screening revealed

the superiority of alkyl-bearing amphiphilic sp²-iminosugars as

b-glucosidase binders, a broader battery of alkyl isothiocyanate

building blocks was incorporated, including branched and lin-

ear derivatives bearing different chemical functionalities (16–21;

Fig. 3).

(1-adamantylcarboxamido)butyl isothiocyanate (19) was carried

out using a two-step process that involved condensation of N-(tert-

butoxycarbonyl)butanediamine (26) and adamantane-1-carbonyl

chloride (→27) followed by carbamate hydrolysis and isothio-

cyanation. A similar strategy was followed for the preparation

of compound 20, incorporating an oligoethylene segment, from

11-azido-3,6,9-trioxaundecylamine (28)¹⁹but in this case the

azido group in the amine adduct 29 was directly transformed

into isothiocyanate by reaction with triphenylphosphine–carbon

disulﬁde.²⁰Finally, the azidoalkyl isothiocyanate 21 was obtained

from 28 (Scheme 1) following isothiocyanation with thiophosgene.

Fig. 3 Building block precursors for the synthesis of bicyclic isothiourea–

type sp²-iminosugars.

Scheme 1 Synthesis of the isothiocyanate precursors 16, 17, and 19–21.

The selected isothiocyanate precursors were commercially

available (13, 15 and 18) or were obtained in a limited num-

ber of steps by isothiocyanation of the corresponding amines.

Thus, the glucopyranosyl derivative 14 was prepared from D-

glucose as reported in the literature.¹⁶The branched isoth-

iocyanate 16 was synthesized from [bis(2-aminoethyl)-2-(tert-

butoxycarbonylamino)ethyl]amine (22)¹⁷by N-hexanoylation of

the free primary amino groups (→23), subsequent deprotection of

the carbamate group (→24) and isothiocyanation of the generated

primary amine by treatment with thiophosgene. Compound 17

was obtained in good yield by isothiocyanation of the corre-

sponding amine 25¹⁸using thiophosgene. The preparation of 4-

The synthesis of the 5-N,6-S-(N¢-butyl-, N¢-b-D-glucopy-

ranosyl- and N¢-phenyl-iminomethylidene)-6-thionojirimycin

derivatives starts with reduction of the azido group in 11 by

catalytic hydrogenation, coupling of the resulting amine with

the corresponding isothiocyanate (13–15), acetylation and ﬁnal

hydrolysis of the tetrahydropyranyl group to afford the N¢-

thiourea adducts 30–32 (Scheme 2). Temporary protection of

the secondary hydroxyl as the corresponding acetate was consid-

ered advantageous in order to prevent regioselectivity problems

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Scheme 2 Reagents and conditions: (a) 1. H₂, Pd/C 10%, MeOH, r.t., 1 h. 2. 13–15, Et₃N, pyridine, r.t., 18 h. 3. (1 : 1) Ac₂O–pyridine, -15 ^◦C, 5 h. 4.

p-TsOH, CH₂Cl₂–MeOH (1 : 1), r.t., 2 h; (b) MsCl, pyridine, -20 ^◦C → 10 ^◦C, 7 h; (c) 1. NaOMe, MeOH, r.t., 30 min, solid CO₂. 2. 90% TFA–H₂O, r.t.,

30 min. 3. Amberlite IRA 68 (OH^-); (d) 1. MsCl, pyridine, -20 ^◦C → 10 ^◦C, 7 h. 2. NaOMe, MeOH, r.t., 30 min, solid CO₂; (e) 1. 90% TFA–H₂O, r.t.,

30 min. 2. Amberlite IRA 68 (OH^-).

during the subsequent thiazoline ring formation. In the case

of the N¢-phenylthioureido derivative concomitant N-acetylation

occurred,²¹leading to a 2 : 1 mixture of 32 and 33. This is not

detrimental for the overall yield of the reaction sequence as

discussed hereinafter. Attempts to activate the primary hydroxyl

as leaving group by triﬂuoromethanesulfonylation (triﬂation)²²

in 30–33 were unsuccessful. Nevertheless, treatment with mesyl

chloride and pyridine at low temperature proceeded with spon-

taneous nucleophilic displacement of the transient mesylate by

the vicinal thiocarbonyl sulfur atom to afford the requested 2-

aminothiazolines with total selectivity in good yield (60–86%).

The thiourea sulfur is reported to react faster than the nitrogen

atoms in nucleophilic displacements, which is consistent with

the obtained result.²³In the case of the N¢-butyl and N¢-b-

D-glucopyranosyl derivatives the 3-O-acetyl derivatives 34 and

35 were thus characterized, whereas in the case of the of N¢-

phenylthioureas 32 and 33 the cyclization product was subjected

to subsequent deacetylation to yield the 2-aminothiazoline 36 as

the only reaction product (67%). Further removal of the protecting

groups afforded the target bicyclic nojirimycin derivatives 37–39

in 70–80% yield.

galactofuranose (12), available from commercial a-D-galactose,¹⁵

was used as starting material for accessing the corresponding

thioureas 58 and 59, which were subjected to cyclization (→60

and 61) and ﬁnal pyranose→sp²-iminosugar rearrangement.

5-Deoxy-5-guanidinosugars can be accessed from the corre-

sponding 5-azido-5-deoxysugars via carbodiimide intermediates;²⁴

however, their use as precursors of cyclic guanidines via nu-

cleophilic displacement of a vicinal sulfonate ester is not ap-

propriate due to the high basicity and low nucleophilicity of

the guanidine functionality. An alternative strategy was thus

developed for the preparation of 6-amino-6-deoxy-5,6-di-N¢-(N¢-

iminomethylidene)nojirimycin (70, 71) and galactonojirimycin

derivatives (72, 73) starting from the 5,6-(cyclic thiourea) interme-

diates 64 and 65.^10,12Transformation of the thioureas into S-methyl

isothiouronium salts followed by reaction with n-butylamine or

benzylamine afforded the corresponding cyclic guanidines 66–69

in good yields, which were characterized as the corresponding

hydrochlorides. Deprotection of the acetal functionality and

treatment with sodium hydroxide gave the target sp²-iminosugar

glycomimetics 70–73, likewise characterized as the corresponding

hydrochloride salts (Scheme 5).

The synthesis of the 5-N,6-S-(N¢-iminomethylidene)-6-

thionojirimycin derivatives 52–57 followed a similar approach,

starting from azide 11 as a common intermediate. Protection of

the secondary hydroxyl group OH-3 in the thiourea derivatives

40–45 was found to be not necessary since the mesyl-promoted

cyclization proceeded with total regioselectivity to give the desired

thiazolines 46–51 (Scheme 3). This general synthetic strategy

was next applied to the preparation of the bicyclic galactono-

jirimycin derivatives 62 and 63, the C-4 epimers of compounds 37

and 39 (Scheme 4). 5-Azido-5-deoxy-1,2-O-isopropylidene-b-D-

The structure and purity of all the prepared compounds were

conﬁrmed by spectroscopic and analytical techniques. The ¹³C

NMR spectra of the 2-aminothiazoline derivatives (34–36, 46–

51, 60, 61) showed a characteristic signal at 166.2–161.3 ppm

corresponding to the double bonded carbon and a high ﬁeld

shift of the C-6 resonance (37.9–33.3 ppm) with respect to the

thiourea precursors (30–33, 40–45, 58 and 59), conﬁrming the

replacement of oxygen by sulfur at this position. In the case

of the 2-aminoimidazoline derivatives (66–69) a signal at 159.9–

155.9 ppm conﬁrmed the presence of the imino functionality.

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Scheme 4 Reagents and conditions: (a) 1. H₂, Pd/C 10%, MeOH, r.t., 1 h.

2. 13 or 15, Et₃N, pyridine, r.t., 18 h, 69–72%; (b) MsCl, pyridine, -20 ^◦

C

→ 10 ^◦C, 7 h, 52–60%; (c) 1. 90% TFA–H₂O, r.t., 30 min. 2. Amberlite

IRA 68 (OH^-), 60–86%.

Scheme 5 Reagents and conditions: (a) 1. MeI, MeOH, 70 ^◦C, 2 h, 90%. 2.

nBuNH₂or PhCH₂NH₂, DMF, 70 ^◦C, 18 h, 55–76%; (b) 1. 90% TFA–H₂O,

^◦C, 1 h. 2. NaOH 0.1 N, 60–80%.

Scheme 3 Reagents and conditions: (a) 1. H₂, Pd/C 10%, MeOH, r.t.,

1 h. 2. 16–21, Et₃N, pyridine, r.t., 18 h. 3. p-TsOH, CH₂Cl₂–MeOH (1 : 1),

r.t., 2 h, 47–85%; (b) MsCl, pyridine, -20 ^◦C → 10 ^◦C, 7 h, 46–78%; (c) 1.

90% TFA-H₂O, r.t., 30 min. 2. Amberlite IRA 68 (OH^-), 82–99%.

0

glucosidase (bovine liver, cytosolic), a-mannosidase (Jack bean),

b-mannosidase (Helix pomatia), trehalase (pig kidney), amy-

loglucosidase (Aspergillus niger), naringinase (b-glucosidase/a-

L-rhamnosidase; Penicillium decumbens),²⁵a-galactosidase (green

coffee beans), isomaltase (yeast), and a-L-fucosidase (pig kidney).

The corresponding inhibition constants (K_i) are collected in

Tables 1 (for bicyclic nojirimycin derivatives) and 2 (for bicyclic

galactonojirimycin derivatives). The corresponding data for the

previously synthesized derivatives 5–10 have been also included

for comparative purposes.

In agreement with previous results on related N-substituted imi-

nomethylydene derivatives,⁹no or weak inhibition of the glycosi-

dases acting on a-glucopyranosides, namely yeast a-glucosidase,

isomaltase or amyloglucosidase, was observed in all cases in spite

of the matching conﬁguration at the (pseudo)anomeric centre. It

must be noted, however, that except compounds 38 and 52 all

compounds with D-gluco conﬁgurational pattern were weak to

moderate inhibitors of pig kidney trehalase, an enzyme known

to be strongly inhibited by isourea and isothiourea derivatives

(Table 1).²⁶The isothiourea-type nojirimycin derivatives also

The ¹H and ¹³C NMR spectra of the sp²-iminosugars (37–

39, 52–57, 62, 63, 70–73) registered in D₂O solution conﬁrmed

their bicyclic character. Thus, the high ﬁeld resonance of the

pseudoanomeric carbon (C-1) resonance (76.7–74.9 ppm) was

in agreement with the presence of the hemiaminal functionality,

whereas the vicinal ³J_H,Hvalues about the six-membered ring were

indicative of the ⁴C₁chair conformation with the pseudoanomeric

groups in axial (a-like) orientation (J_1,2= 3.2–4.0 Hz) ﬁtting the

anomeric effect, a distinctive feature of sp²-iminosugars. In the

case of compounds with aliphatic substituents, the presence of the

b-like anomer in the solution was detected by NMR, but with a

large preference (>95%) for the a-like diastereomer.

Evaluation of the glycosidase inhibitory activity

All the new sp²-iminosugars synthesised were ﬁrst tested

as inhibitors against

a series of commercial glycosidases

including a-glucosidase (yeast), b-glucosidase (almonds), b-

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Table 1 K_ivalues (mM) for the bicyclic nojirimycin derivatives 5–7, 37–39, 52–57, 70 and 71 against a panel of glycosidases^a

Enzyme

5

6

7

37

38

39

52

53

54

55

56

57

70

71

a-glucosidase (baker yeast)

b-glucosidase (almond)

168

1.9

2.7

182

n.d.^c

n.d.

3.44

n.i.^b

0.76

3.7

13.4

0.23

18

n.i.

0.42 24

35

40

0.18 1.1

260

3.8

n.i.

654

251

n.i.

110

48

n.i.

n.i

383

226

14

2.4

n.d.

n.i.

19

24

n.i.

1.3

151

5.9

n.i.

13

9.3

24

0.23 1.8

2.6

n.i.

13

256

116

292

0.45

68

103

0.10

7.1

n.i.

28

165

433

0.35

17.6

173

n.i.

33

126

398

0.67

4.4

n.i.

144

508

87

1.9

514

101

n.i.

23

85

154

0.68

529

150

b-gluco/b-galactosidase (bovine liver)

58

20.1

trehalase (pig kidney)

naringinase (Penicillium decumbens)

a-L-fucosidase (pig kidney)

6.2

24.1

10.8

n.d.

d

R

Imiglucerase (Cerezymeꢀ, Genzyme)

0.69

n.d.

5.5

22.3

^aInhibition was competitive in all cases. No inhibition was observed for any compound at 2 mM concentration on green coffee bean a-galactosidase,

Aspergillus niger amyloglucosidase, Jack bean a-mannosidase or Helix pomatia b-mannosidase. ^bn.i., no inhibition observed at 2 mM concentration of

the inhibitor. ^cn.d., not determined. ^dIC₅₀values [mM].

inhibited pig kidney a-L-fucosidase in the low micromolar range

(Table 1).

detrimental for the inhibitory activity in the cyclic guanidine-type

bicyclic nojirimycin series.

Most interestingly, all the N¢-alkyl-substituted iminothiazoli-

dine derivatives (6, 37, 52–57) behaved as micromolar to nanomo-

lar inhibitors of the b-glucosidases from almonds and bovine liver

as well as of naringinase from Penicillium decumbens, an enzyme

having b-glucosidase (E.C. 3.2.1.21) and a-L-rhamnosidase (E.C.

3.2.1.40) activities, in spite of the mismatching conﬁguration at the

pseudoanomeric centre. Compounds exhibiting strong inhibition

for the two ﬁrst b-glucosidases have been shown previously

to be good candidates as pharmacological chaperones for the

treatment of Gaucher disease. Therefore, this assay was used

to select compounds for further evaluation against human b-

glucocerebrosidase. The inhibition potency seems to be related

to the hydrophobicity of the N¢-substituent. Thus, incorporation

of a terminal protonatable amino group (i.e., 53 and 54) resulted

in a signiﬁcant increase in the corresponding K_ivalues (2.5 and

4.4-fold in the case of the bovine liver enzyme) as compared

with the parent N¢-alkyl derivative (i.e., 6 and 37). Similarly,

compounds 56 and 57, incorporating a triethylene glycol moiety,

were one to two orders of magnitude weaker inhibitors than

compounds having hydrophobic substituents. This substantially

worse inhibition might have its origin in the high desolvation

energy of the polyethylene glycol-based arm, an effect that

increases with length.²⁷

Comparison of the inhibition data for D-gluco (Table 1) and

D-galacto-conﬁgured compounds (Table 2) indicated that the later

tend to be more potent inhibitors against the b-glucosidases

from almonds and bovine liver (i.e., 7, 37, 39, and 70 versus 10,

62, 63, and 72, respectively). It is known that iminosugars with

hydroxylation patterns of stereochemical complementarity to D-

galactose are often moderate to good inhibitors of family 1 b-

glucosidases,²⁸which are not very selective enzymes regarding the

conﬁguration at C-4 in the substrates.

Compounds 5–10, 37, 52, 55–57, 62, 70, and 71were further eval-

uated as inhibitors of the recombinant GlcCerase (imiglucerase,

R

Cerezymeꢀ from Genzyme) (Tables 1 and 2). Activity data

reﬂect a clear dependence of GlcCerase inhibition on the nature

of the exocyclic substituent. In agreement with the correlation

between amphiphilicity and inhibitory activity observed in other

glycomimetic families,²⁹the inhibitory potency decreased when

decreasing the hydrophobicity of the N¢-substituent, irrespective

of the nature of the endocyclic heteroatom at the ﬁve-membered

ring. Thus, the presence of polar groups (e.g. 56 and 57 versus 55)

or shortening the aliphatic alkyl chain (i.e. 37, 62 and 70 versus

6, 9 and 7, respectively) involved a signiﬁcant diminution of the

inhibitory activity.

The mammalian b-glucosidase seems to be more sensitive to

modiﬁcations at the exocyclic nitrogen substituent of the inhibitor.

Thus, the adamantylcarboxamidobutyl derivative 55 was up to

1.7-fold a more potent inhibitor that the N¢-octyl derivative 6

against almond b-glucosidase but 18.4-fold weaker against bovine

liver b-glucosidase. Replacement of the octyl chain (i.e., in 7) into

butyl and phenyl (i.e., in 70 and 71, respectively) was also strongly

Evaluation of the potential for chaperoning activity

Enzyme stabilization under thermal denaturation conditions was

used as an indication of the potential of the target compounds to

behave as pharmacological _◦chaperones.³⁰Recovery of GlcCerase

activity after heating at 48 C was measured in the absence and

in the presence of increasing concentrations (50, 100 and 150 mM)

Table 2 K_ivalues (mM) for 8, 9, 10, 62, 63, 72, and 73 against a panel of glycosidases^a

Enzyme

8

9

10

62

63

72

73

b-glucosidase (almond)

0.019

n.i.^b

0.052

n.d.^c

n.d.

0.023

n.i.

0.042

37

11

4.0

0.16

n.i.

0.04

44

n.d.

0.9

0.49

458

1.2

144

n.i.

83

69

n.i.

55

n.i.

n.d.

5.7

n.i.

2.5

n.d.

n.i.

n.d.

n.i.

63

n.d.

a-galactosidase (green coffee bean)

b-gluco/b-galactosidase (bovine liver)

naringinase (Penicillium decumbens)

a-L-fucosidase (pig kidney)

d

R

Imiglucerase (Cerezymeꢀ, Genzyme)

10.6

^aInhibition was competitive in all cases. No inhibition was observed for any compound at 2 mM concentration on baker’s yeast a-glucosidase, pig

kidney trehalase, yeast isomaltase, Jack bean a-mannosidase or Helix pomatia b-mannosidase. ^bn.i., no inhibition observed at 2 mM concentration of

the inhibitor. ^cn.d., not determined. ^dIC₅₀values [mM].

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Fig. 4 Relative enzymatic activity after thermal denaturation (48 ^◦C) for 20 min, 40 min, and 60 min at the indicated inhibitor concentrations (mM)

compared to the corresponding assay at 37 ^◦C. Data for control (C) are obtained as above except that no inhibitor is present.

of the selected sp²-iminosugars at different incubation times

(Fig. 4). Untreated GlcCerase lost most of its activity after

Conclusions

In summary, a general strategy for the preparation of bicyclic

1 h of denaturation under these conditions (less than 10%

activity remained). Compounds 5, 6, 7, 9, 52, and 55 exhibited a

remarkable stabilization effect, whereas compounds 8, 37, 62, 10,

56, 57, 71 did not protect the enzyme against thermal inactivation.

Accordingly, this protective effect increased gradually with the

lipophilicity.

nojirimycin and galactonojirimycin related sp²-iminosugars ana-

logues adapted to molecular diversity-oriented schemes has been

disclosed and applied to the synthesis of a library of glycomimet-

ics differing in C-4 conﬁguration (D-gluco and D-galacto), the

structure of the ﬁve-membered ring (cyclic isourea, isothiourea

The stabilization activity of the sp²-iminosugars was challenged

against that of N-(n-nonyl)deoxynojirimycin (NNDNJ),²⁹an

amphiphilic iminosugar currently under investigation as pharma-

cological chaperone for Gaucher disease. Compound 5 behaved

similarly to NNDNJ, whereas compounds 6, 7, 9, 52, and 55

were signiﬁcantly more efﬁcient. For example, the recombinant

enzyme incubated with 50 mM 6 retained over 40% of the initial

activity after 1 h, which is eight-fold higher than the untreated

enzyme and about twice as compared with 50 mM NNDNJ-treated

enzyme.³²The protective effect was strongly dose-dependent for

the D-gluco conﬁgured derivatives bearing the n-octyl substituent

(5–7), a feature also observed for NNDNJ, but was almost dose-

independent in the 50–150 mM range in the case of the branched

derivative 52, the adamantane derivative 55 and, especially, for the

bicyclic galactonojirimycin derivative 9. This might reﬂect that

those compounds have already reached the maximum stabilizing

activity at 50 mM, about 80% imiglucerase retained activity after

20 min at 48 ^◦C. Although this compares unfavourably with 6 or

7 (about 100 and 90% retained activity at 150 mM M after 20 min,

respectively), it should be noted that 9, 52 and 55 are much more

efﬁcient at protecting the enzyme over longer period of times (e.g.,

60% activity after 60 min in the presence of 50 mM of 9 or 70%

after 60 min in the presence of 150 mM of 52; to be compared

with only 50% for 6 or NNDNJ after 60 min at 150 mM),³⁰

which makes them very attractive candidates for further biological

studies.

or guanidine) and the nature of the N¢-substituent (apolar,

polar, linear or branched). Glycosyl hydrolase inhibition studies

evidenced a remarkable inﬂuence of the nature of the exocyclic

substituent on the potency and selectivity of binding towards a

range of commercial glycosidases, although selectivity towards

b-glucosidases was generally observed. Amphiphilic derivatives

bearing hydrophobic N¢-substituents were much more potent than

derivatives bearing polar substituents, which also correlated with

potent inhibition of the recombinant human b-glucocerebrosidase

(imiglucerase) used in enzyme replacement therapy for Gaucher

disease. We have further tested the ability of these compounds

to increase imiglucerase stability against thermal denaturation,

as an in vitro assessment of their potential as pharmacological

chaperones. Analysis of the results in comparison with data for the

iminosugar derivative NNDNJ evidenced the higher chaperoning

potency of sp²-iminosugars and stresses their potential in phar-

macological chaperone therapy and combined therapy strategies.

Experimental

General methods

Reagents and solvents were purchased from commercial sources

and used without further puriﬁcation. Optical rotations were

measured with a JASCO P-2000 polarimeter, using a sodium

lamp (l = 589 nm) at 22 ^◦C in 1 cm or 1 dm tubes. IR

spectra were recorded on a JASCO FTIR-410 instrument. UV

spectra were recorded on Philips PU-8710 instrument, unit for

e values: mm^-1cm^-1. NMR experiments were performed at 300

(75.5), 400 (100.6), and 500 (125.7) MHz using Bruker DMX300,

DRX400, and DRX500. 1-D TOCSY as well as 2-D COSY

and HMQC experiments were carried out to assist in signal

assignment. In the FABMS spectra, the primary beam consisted

It is worth noting that the ensemble of results above dis-

cussed evidences that the inhibitory activity is not necessarily

correlated with the enzyme stabilization effect. Thus, compounds

5–7 and 8–10 very similarly inhibited imiglucerase (Tables 1

and 2) but behaved quite differently in the thermal-denaturation

protection assay, with compounds 8 and 10 being actually

inactive.

3704 | Org. Biomol. Chem., 2011, 9, 3698–3713

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of Xe atoms with maximum energy of 8 keV. The samples were

dissolved in m-nitrobenzyl alcohol or thioglycerol as the matrices

and the positive ions were separated and accelerated over a

potential of 7 keV. NaI was added as cationizing agent. Thin-

layer chromatography was performed on E. Merck precoated

TLC plates, silica gel 30F-245, with visualization by UV light

and by charring with 10% H₂SO₄or 0.2% w/v cerium(IV) sulfate-

5% ammonium molybdate in 2 M H₂SO₄or 0.1% ninhydrin in

EtOH. Column chromatography was performed on Chromagel

(SDS silica 60 AC.C, 70–200 mm). Elemental analyses were

performed at the Servicio de Microana´lisis del Instituto de In-

vestigaciones Qu´ımicas de Sevilla, Spain. 5-Azido-5-deoxy-1,2-O-

isopropylidene-6-O-tetrahydropyranyl-a-D-glucofuranose 11 was

prepared from commercial 6,3-D-glucuronolactone following

the procedure already reported.¹⁴2,3,4,6-Tetra-O-acetyl-b-D-

glucopyranosyl isothiocyanate (14) was synthesized from the

corresponding per O-acetyl glucopyranosyl bromide following

the procedure of Camarasa et al.¹⁶5-Azido-5-deoxy-1,2-di-O-

isopropylidene-a-D-galactofuranose 12 was obtained from D-

galactose using the reported route.¹⁵The cyclic thiourea a-D-

glucofuranose derivative 64 was prepared from commercial 6,3-

D-glucuronolactone following the procedure already reported.¹²

The cyclic thiourea a-D-galactofuranose derivative 65 was syn-

thesized from 5-azido-3-O-benzoyl-5-deoxy-a-D-galactofuranose

following the reported route.¹⁰

lutions (25 mL from a stock solution containing 0.1 mg of

protein/mL) in the presence of 0.2% (w/v) sodium taurocholate

and 0.1% (v/v) TritonX-100 in McIlvaine buffer (pH 5.2)

were incubated at 37 ^◦C without (control) or with inhibitor

during 30 min, and after addition of corresponding substrate

solution (60 mL), incubations were maintained at 37 ^◦C for

10 min. Enzymatic reactions were stopped by the addition of

150 mL of 100 mm glycine/NaOH buffer (pH 10.6). The amount

of 4-methylumbelliferone formed was determined with a 1420

VICTOR²Multilabel Counter (Wallac) ﬂuorometer at 355 nm

(excitation) and 460 nm (emission). IC₅₀values were determined

by plotting percent activity versus log [I], using at least ﬁve different

inhibitor concentrations.

Thermal stabilization assay

Following a modiﬁcation of a reported method,³¹Imiglucerase

aliquots (48 mL, 2 mg ml^-1) were incubated at pH 7.4 with 0

(control), 150, 100, or 150 mm test compound at 48 ^◦C. Subse-

quently, 150 mL of 0.1 m acetate–phosphate buffer (pH 5.0) and

100 mL of substrate (4 mM 4-methyllumbelliferyl b-D-glucoside)

in McIlvaine buffer (pH 5.2) were added at different times and

◦

incubated for 10 min at 37 C, in the presence of 0.l% Triton X-

100 and 0.2% taurodeoxycholic acid. Then an amount of 300 mL

of glycine/NaOH buffer (100 mM, pH 10.6) was added and

liberated 4-methylumbelliferone was measured. Enzyme activity

was reported relative to that of the enzyme at 37 ^◦C.

General procedure for the inhibition assay against the commercial

enzymes

General procedure for the synthesis of 5-thioureido-a-D-

glucofuranose derivatives 30–33 and 40–45. A solution of

5-azido-5-desoxy-1,2-O-isopropylidene-6-O-tetrahydropyranyl-

a-D-glucofuranose¹⁴11 (300 mg, 0.91 mmol) in MeOH (5 mL)

was hydrogenated at atmospheric pressure for 1 h using 10%

Pd/C (90 mg) as catalyst. The suspension was ﬁltered through

Celite and concentrated. To a solution of the resulting residue

in pyridine (6 mL), Et₃N (0.71 mL, 5.1 mmol, 5.6 eq) and the

corresponding isothiocyanate (1.1 mmol, 1.2 eq) were added and

the mixture was stirred at room temperature for 18 h. In the case

of 30–33 the reaction mixture was cooled at -15 ^◦C, Ac₂O (3 mL)

was added dropwise, and the mixture was further stirred for 5 h.

Then, the solvent was removed under reduced pressure and the

resulting residue coevaporated several times with toluene. The

crude product was dissolved in CH₂Cl₂–MeOH (1 : 1, 12 mL) and

p-toluenesulfonic acid (50 mg, 0.26 mmol, 0.3 eq) was added. The

reaction mixture was stirred for 2 h at room temperature, and

then diluted with CH₂Cl₂(8 mL), washed with saturated aqueous

NaHCO₃(2 ¥ 8 mL), dried (MgSO₄), and concentrated. The

resulting residue was puriﬁed by column chromatography using

the eluent indicated in each case.

Inhibition constant (K_i) values were determined by spectropho-

tometrically measuring the residual hydrolytic activities of the

glycosidases against the respective o- (for b-glucosidase from

bovine liver) or p-nitrophenyl a- or b-D-glycopyranoside (for other

glycosidases) or a,a¢-trehalose (for trehalase) in the presence of

compounds 37–39, 52–57, 62, 63, and 70–73. Each assay was

performed in phosphate buffer or phosphate-citrate buffer (for

a- or b-mannosidase and amyloglucosidase) at the optimal pH

for the enzymes. The reactions were initiated by addition of

enzyme to a solution of the substrate in the absence or presence of

various concentrations of inhibitor. The mixture was incubated

for 10–30 min at 37 ^◦C or 55 ^◦C (for amyloglucosidase) and

the reaction was quenched by addition of 1 M Na₂CO₃or a

solution of GLC-Trinder (Sigma, for trehalase). Reaction times

were appropriate to obtain 10–20% conversion of the substrate

in order to achieve linear rates. The absorbance of the resulting

mixture was determined at 405 nm or 505 nm (for trehalase).

Approximate values of K_iwere determined using a ﬁxed con-

centration of substrate (around the K_Mvalue for the different

glycosidases) and various concentrations of inhibitor. Full K_i

determinations and enzyme inhibition mode were determined

from the slope of Lineweaver–Burk plots and double reciprocal

analysis. Representative examples of the Lineweaver–Burk plots,

with typical proﬁle for competitive inhibition mode, are shown in

the Electronic Supplementary Information.†

3-O-Acetyl-5-(N¢-butylthioureido)-5-deoxy-1,2-O-isopropyli-

dene-a-D-glucofuranose (30). Column chromatography, eluent

50 : 1 CH₂Cl₂–MeOH. Yield: 220 mg (64%). [a]_D-24.7 (c 1.0,

CH₂Cl₂). R_f0.32 (20 : 1 CH₂Cl₂–MeOH). IR (KBr) n_max2929,

1747, 1375, 1070 cm^-1. UV (CH₂Cl₂) 251 nm (e_mM15.1). ¹H NMR

(500 MHz, CDCl₃, 323 K) d 6.26 (bs, 1 H, N¢H), 6.16 (bd, 1 H,

J_5,NH= 7.9 Hz, NH), 5.88 (d, 1 H, J_1,2= 3.7 Hz, H-1), 5.28 (d,

1 H, J_3,4= 3.1 Hz, H-3), 4.79 (m, 1 H, H-5), 4.46 (d, 1 H, H-2),

4.44 (dd, 1 H, J_4,5= 7.9 Hz, H-4), 3.91 (dd, 1 H, J_6a,6b= 11.3 Hz,

J_5,6a= 3.5 Hz, H-6a), 3.82 (dd, 1 H, J_5,6b= 3.3 Hz, H-6b), 3.33 (m,

Imiglucerase inhibition assay

In vitro activity was determined with 4 mM 4-methylumbelliferyl-

b-D-glucopyranoside in McIlvaine buffer (pH 5.2). Enzyme so-

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2 H, CH₂N), 2.11 (s, 3 H, MeCO), 1.54 (m, 2 H, CH₂CH₂N), 1.48,

1.28 (2 s, 6 H, CMe₂), 1.36 (m, 2 H, CH₂CH₃), 0.92 (t, 3 H, ³J_H,H

2 H, H-6a, H-6b), 2.07 (s, 3 H, MeCO), 1.92 (s, 3 H, MeCONH),

1.53, 1.30 (2 s, 6 H, CMe₂). ¹³C NMR (125.7 MHz, CDCl₃, 313

K) d 184.6 (CS), 175.1 (CO amide), 169.8 (CO ester), 142.3–128.9

(Ph), 112.5 (CMe₂), 105.0 (C-1), 83.5 (C-2), 77.9 (C-4), 75.3 (C-

3), 62.1 (C-6), 55.5 (C-5), 28.0 (MeCONH), 26.8, 26.2 (CMe₂),

21.6 (MeCO). FABMS: m/z 461 (15, [M + Na]⁺), 439 (30, [M +

H]⁺). FABMSHR m/z 461.136042. Calcd for C₂₀H₂₆N₂O₇NaS:

461.135843.

=

7.4 Hz, CH₃). ¹³C NMR (125.7 MHz, CDCl₃, 313 K) d 182.1 (CS),

169.9 (CO), 112.3 (CMe₂), 104.8 (C-1), 83.4 (C-2), 78.4 (C-4), 75.7

(C-3), 62.7 (C-6), 53.8 (C-5), 43.9 (CH₂N), 30.9 (CH₂CH₂N), 26.6,

26.1 (CMe₂), 21.3 (MeCO), 20.0 (CH₂CH₃), 13.6 (CH₃). ESIMS:

m/z 375 [M - H]⁺. Anal. Calcd for C₁₆H₂₈N₂O₆S: C, 51.05; H,

7.50; N, 7.44. Found: C, 50.88; H, 7.36; N, 7.33%.

3-O-Acetyl-5-deoxy-1,2-O-isopropylidene-5-[N¢-(2,3,4,6-tetra-

O-acetyl-b-D-glycopyranosyl)thioureido]-a-D-glucofuranose (31).

Column chromatography, eluent 1 : 1 → 2 : 1 EtOAc–petroleum

ether. Yield: 456 mg (77%). [a]_D-6.9 (c 1.0, CH₂Cl₂). R_f0.46

(2 : 1 EtOAc-petroleum ether). UV (CH₂Cl₂) 255 nm (e_mM12.6).

IR (KBr) n_max2942, 1749, 1375, 1038 cm^-1. ¹H NMR (300 MHz,

CDCl₃, 313 K) d 6.84 (d, 1 H, J_NH,5= 8.7 Hz, NH), 6.76 (bd, 1 H,

5-[N¢-(N,N -Bis(2-hexanamidoethyl)aminoethyl)thioureido]-5-

deoxy-1,2-O-isopropylidene-a-D-glucofuranose

(40). Column

chromatography, eluent 40 : 1 → 20 : 1 CH₂Cl₂–MeOH. Yield:

204 mg (47%). [a]_D+35.4 (c 1.0, CH₂Cl₂). R_f0.49 (10 : 1 CH₂Cl₂–

MeOH). IR (KBr) n_max3296, 2956, 1646, 1374, 1074 cm^-1. UV

1

(CH₂Cl₂) 241 nm (e_mM16.0). H NMR (300 MHz, CDCl₃, 313

K) d 7.82 (bs, 1 H, N¢H), 7.35 (d, 1 H, J_5,NH= 8.7 Hz, NH),

6.22 (bs, 2 H, NHCO), 5.92 (d, 1 H, J_1,2= 3.6 Hz, H-1), 5.17

(bs, 1 H, OH), 4.63 (m, 1 H, H-5), 4.55 (d, 1 H, H-2), 4.17 (d,

1 H, J_3,4= 1.8 Hz, H-3), 4.10 (dd, 1 H, J_4,5= 10.1 Hz, H-4),

3.99 (dd, 1 H, J_6a,6b= 11.3 Hz, J_5,6a= 3.4 Hz, H-6a), 3.80 (dd, 1

H, J_5,6b= 3.2 Hz, H-6b), 3.49 (m, 2 H, CH₂NH), 3.49 (m, 4 H,

N¢H), 5.93 (d, 1 H, J_1,2= 3.7 Hz, H-1), 5.55 (t, 1 H, J_1¢,2¢= J_1¢,N¢H

=

9.4 Hz, H-1¢), 5.34 (t, 1 H, J_2¢,3¢= J_3¢,4¢= 9.4 Hz, H-3¢), 5.29 (d,

1 H, J_3,4= 3.0 Hz, H-3), 5.07 (t, 1 H, J_4¢,5¢= 9.4 Hz, H-4¢), 5.00

(t, 1 H, H-2¢), 4.78 (m, 1 H, H-5), 4.50 (d, 1 H, H-2), 4.46 (dd,

1 H, J_4,5= 7.8 Hz, H-4), 4.27 (dd, 1 H, J_6¢a,6¢b= 12.4 Hz, J_5¢,6¢a

=

3

4.7 Hz, H-6¢a), 4.15 (dd, 1 H, J_5¢,6¢b= 5.2 Hz, H-6¢b), 3.98 (dd, 1 H,

J_6a,6b= 11.4 Hz, J_5,6a= 2.9 Hz, H-6a), 3.85 (dd, 1 H, J_5,6b= 2.9 Hz,

H-6b), 3.84 (m, 1 H, H-5¢), 2.15, 2.09, 2.08, 2.04, 2.03 (s, 15 H,

MeCO), 1.51, 1.32 (2 s, 6 H, CMe₂). ¹³C NMR (75.5 MHz, CDCl₃,

313 K) d 184.0 (CS), 171.4, 170.7, 169.9, 169.8, 169.5 (MeCO),

112.3 (CMe₂), 104.7 (C-1), 83.4 (C-2), 82.6 (C-1¢), 77.9 (C-4), 75.7

(C-3), 73.3 (C-3¢), 73.0 (C-5¢), 71.0 (C-2¢), 68.5 (C-4¢), 62.1 (C-6),

61.9 (C-6¢), 53.8 (C-5), 26.6, 26.0 (CMe₂), 21.2, 20.7, 20.6, 20.5

(MeCO). FABMS: m/z 674 (100, [M + Na]⁺), 652 (15, [M + H]⁺).

Anal. Calcd for C₂₆H₃₈N₂O₁₅S: C, 47.99; H, 5.89; N, 4.31. Found:

C, 47.84; H, 5.66; N, 4.24%.

CH₂NHCO), 2.66 (t, 2 H, J_H,H= 5.1 Hz, CH₂CH₂NH), 2.55 (t,

3

4 H, J_H,H= 5.3 Hz, CH₂CONH), 2.18 (t, 4 H, J_H,H= 7.3 Hz,

CH₂CH₂NHCO), 1.61 (m, 4 H, CH₂CH₂CONH), 1.48, 1.31 (2 s,

6 H, CMe₂), 1.29 (m, 8 H, CH₂), 0.88 (t, 6 H, ³J_H,H= 6.7 Hz, CH₃).

¹³C NMR (75.5 MHz, CDCl₃) d 182.5 (CS), 174.5 (CO), 111.5

(CMe₂), 105.1 (C-1), 84.6 (C-2), 80.5 (C-4), 73.9 (C-3), 63.0 (C-6),

54.9 (CH₂CH₂NHCO), 53.6 (C-5), 53.4 (CH₂CH₂NH), 42.7

(CH₂NH), 38.0 (CH₂NHCO), 36.7 (CH₂CONH), 31.4 (CH₂),

26.8, 26.1 (CMe₂), 25.3 (CH₂CH₂CONH), 22.3 (CH₂CH₃), 13.8

(CH₃). FABMS: m/z 626 (50, [M + Na]⁺), 604 (35, [M + H]⁺).

Anal. Calcd for C₂₈H₅₃N₅O₇S: C, 55.70; H, 8.85; N, 11.60. Found:

C, 55.75; H, 8.68; N, 11.52%.

3-O-Acetyl-5-deoxy-1,2-O-isopropylidene-5-(N¢-phenylthio-

ureido)-a-D-glucofuranose (32). Column chromatography, eluent

100 : 1 → 30 : 1 CH₂Cl₂–MeOH. Yield: 202 mg (56%). [a]_D-51.0 (c

1.0, CH₂Cl₂). R_f0.27 (20 : 1 CH₂Cl₂–MeOH). UV (CH₂Cl₂) 268 nm

5-[N¢-(8-tert-Butoxycarbonylaminooctyl)thioureido]-5-deoxy-

1,2-O-isopropylidene-a-D-glucofuranose (41). Column chroma-

tography, eluent 40 : 1 → 30 : 1 CH₂Cl₂–MeOH. Yield: 219 mg

(67%). [a]_D+42.6 (c 1.0, CH₂Cl₂). R_f0.20 (20 : 1 CH₂Cl₂–MeOH).

IR (KBr) n_max3336, 2977, 1683, 1368, 1074 cm^-1. UV (CH₂Cl₂)

246 nm (e_mM14.1). ¹H NMR (400 MHz, CDCl₃, 313 K) d 6.58 (bs,

2 H, NH), 5.93 (d, 1 H, J_1,2= 3.6 Hz, H-1), 5.07 (bs, 1 H, OH),

4.65 (m, 1 H, H-5), 4.59 (d, 1 H, H-2), 4.20 (s, 1 H, H-3), 4.11

(m, 1 H, H-4, H-6a), 3.78 (dd, 1 H, J_6a,6b= 10.5 Hz, J_5,6b= 1.7 Hz,

H-6b), 3.45 (m, 2 H, CH₂NHCS), 3.20 (bs, 1 H, NH), 3.10 (m, 2 H,

CH₂NHCO), 2.07 (bs, 1 H, OH), 1.60 (m, 2 H, CH₂CH₂NHCS),

1.51, 1.33 (2 s, 6 H, CMe₂), 1.48 (m, 2 H, CH₂CH₂NHCO), 1.46 (s,

9 H, CMe₃), 1.33 (m, 8 H, CH₂). ¹³C NMR (100.6 MHz, CDCl₃,

313 K) d 181.4 (CS), 156.5 (CO), 111.6 (CMe₂), 104.9 (C-1), 84.6

(C-2), 79.7 (C-4), 79.2 (CMe₃), 73.8 (C-3), 62.3 (C-6), 53.7 (C-

5), 44.3 (CH₂NHCS), 40.6 (CH₂NHCO), 29.8, 28.8, 28.7, 28.6

(CH₂), 28.4 (CMe₃), 26.7, 26.0 (CMe₂), 26.5, 26.4 (CH₂). FABMS:

m/z 528 (100, [M + Na]⁺), 506 (35, [M + H]⁺). Anal. Calcd for

C₂₃H₄₃N₃O₇S: C, 54.63; H, 8.57; N, 8.31. Found: C, 54.44; H, 8.42;

N, 8.24%.

1

(e_mM13.3). IR (KBr) n_max3052, 1741, 1524, 1370, 1076 cm^-1. H

NMR (500 MHz, CDCl₃, 313 K) d 7.97 (bs, 1 H, N¢H), 7.42–7.22

(m, 5 H, Ph), 6.54 (d, 1 H, J_5,NH= 9.0 Hz, NH), 5.88 (d, 1 H, J_1,2

=

3.7 Hz, H-1), 5.25 (d, 1 H, J_3,4= 3.0 Hz, H-3), 4.94 (m, 1 H, H-5),

4.45 (d, 1 H, H-2), 4.38 (dd, 1 H, J_4,5= 7.8 Hz, H-4), 3.90 (dd, 1 H,

J_6a,6b= 11.3 Hz, J_5,6a= 3.1 Hz, H-6a), 3.80 (dd, 1 H, J_5,6b= 3.4 Hz,

H-6b), 2.02 (s, 3 H, MeCO), 1.46, 1.27 (2 s, 6 H, CMe₂). ¹³C NMR

(125.7 MHz, CDCl₃, 313 K) d 180.7 (CS), 169.8 (CO), 136.0–125.2

(Ph), 112.4 (CMe₂), 104.6 (C-1), 83.3 (C-2), 78.0 (C-4), 75.7 (C-3),

62.3 (C-6), 54.5 (C-5), 26.7, 26.1 (CMe₂), 21.3 (MeCO). FABMS:

m/z 419 (75, [M + Na]⁺), 397 (100, [M + H]⁺). Anal. Calcd for

C₁₈H₂₄N₂O₆S: C, 54.53; H, 6.10; N, 7.07. Found: C, 54.36; H, 5.99;

N, 6.88%.

3-O-Acetyl-5-(N¢-acetyl-N¢-phenylthioureido)-5-deoxy-1,2-O-

isopropylidene-a-D-glucofuranose (33). Column chromatogra-

phy, eluent 100 : 1 → 30 : 1 CH₂Cl₂–MeOH. Yield: 136 mg (34%).

[a]_D+37.4 (c 1.1, CH₂Cl₂). R_f0.53 (20 : 1 CH₂Cl₂–MeOH). UV

(CH₂Cl₂) 277 nm (e_mM9.1), 230 nm (e_mM10.3). IR (KBr) n_max3479,

5-[N¢-(4-tert-Butoxycarbonylaminobutyl)thioureido]-5-deoxy-

1,2-O-isopropylidene-a-D-glucofuranose (42). Column chroma-

tography, eluent 30 : 1 → 15 : 1 CH₂Cl₂–MeOH. Yield: 204 mg

(70%). [a]_D+45.5 (c 1.0, CH₂Cl₂). R_f0.33 (15 : 1 CH₂Cl₂–MeOH).

IR (KBr) n_max2933, 1682, 1367, 1075 cm^-1. UV (CH₂Cl₂) 247 nm

1

3090, 2935, 1747, 1677, 1523, 1370, 1217, 1070 cm^-1. H NMR

(500 MHz, CDCl₃, 313 K) d 7.46–7.20 (m, 5 H, Ph), 5.93 (d, 1 H,

J_1,2= 3.7 Hz, H-1), 5.42 (d, 1 H, J_3,4= 2.9 Hz, H-3), 4.96 (m, 1 H,

H-5), 4.57 (dd, 1 H, J_4,5= 9.1 Hz, H-4), 4.49 (d, 1 H, H-2), 3.96 (m,

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(e_mM12.3). H NMR (300 MHz, CDCl₃, 313 K) d 6.96 (bs, 1 H,

1076 cm^-1. UV (CH₂Cl₂) 243 nm (e_mM16.3). ¹H NMR (300 MHz,

CDCl₃, 313 K) d 7.25 (bs, 2 H, NH), 6.17 (bs, 1 H, NH), 5.93 (d,

1 H, J_1,2= 3.6 Hz, H-1), 4.63 (m, 1 H, H-5), 4.57 (d, 1 H, H-2),

4.19 (d, 1 H, J_3,4= 1.7 Hz, H-3), 4.10 (dd, 1 H, J_4,5= 10.0 Hz,

H-4), 4.02 (dd, 1 H, J_6a,6b= 11.3 Hz, J_5,6a= 3.2 Hz, H-6a), 3.82

(dd, 1 H, J_5,6b= 3.2 Hz, H-6b), 3.64 (m, 14 H, OCH₂, CH₂NHCS),

3.45 (m, 2 H, CH₂NHCO), 2.06 (bs, 3 H, CH), 1.86 (m, 6 H,

CCH₂), 1.74 (m, 6 H, CH₂), 1.49, 1.32 (2 s, 6 H, CMe₂). ¹³C

NMR (75.5 MHz, CDCl₃, 313 K) d 182.5 (CS), 178.8 (CO), 111.5

(CMe₂), 105.0 (C-1), 84.7 (C-2), 80.0 (C-4), 73.8 (C-3), 70.7, 70.5,

70.3, 70.1, 69.9 (OCH₂), 62.5 (C-6), 53.7 (C-5), 44.9 (CH₂NHCS),

40.7 (CCONH), 39.3 (CH₂NHCO), 39.2 (CH), 36.5 (CCH₂), 28.1

(CH₂), 26.8, 26.1 (CMe₂). FABMS: m/z 638 (100, [M + Na]⁺).

Anal. Calcd for C₂₉H₄₉N₃O₉S: C, 56.56; H, 8.02; N, 6.82. Found:

C, 56.45; H, 7.90; N, 6.72%.

1

N¢H), 6.96 (bd, 1 H, J_NH,5= 7.8 Hz, NH), 5.92 (d, 1 H, J_1,2= 3.6 Hz,

H-1), 5.07 (bs, 1 H, OH), 4.82 (bs, 1 H, NH), 4.58 (d, 1 H, H-2),

4.54 (m, 1 H, H-5), 4.19 (d, 1 H, J_3,4= 1.9 Hz, H-3), 4.09 (dd, 1 H,

J_4,5= 9.8 Hz, H-4), 4.03 (dd, 1 H, J_6a,6b= 11.3 Hz, J_5,6a= 3.1 Hz, H-

6a), 3.80 (dd, 1 H, J_5,6b= 3.0 Hz, H-6b), 3.47 (m, 2 H, CH₂NHCS),

3.11 (m, 2 H, CH₂NHCO), 1.60 (m, 4 H, CH₂), 1.49, 1.31 (2 s, 6 H,

CMe₂), 1.49 (s, 9 H, CMe₃). ¹³C NMR (75.5 MHz, CDCl₃, 313 K)

d 181.8 (CS), 156.7 (CO), 111.6 (CMe₂), 104.9 (C-1), 84.7 (C-2),

79.8 (CMe₃), 79.7 (C-4), 73.8 (C-3), 62.4 (C-6), 53.7 (C-5), 44.1

(CH₂NHCS), 40.2 (CH₂NHCO), 28.4 (CMe₃), 27.4 (CH₂), 26.7,

26.0 (CMe₂), 25.7 (CH₂). FABMS: m/z 472 (100, [M + Na]⁺), 450

(15, [M + H]⁺). Anal. Calcd for C₁₉H₃₅N₃O₇S: C, 50.76; H, 7.85;

N, 9.35, S, 7.13. Found: C, 50.78; H, 7.93; N, 9.28; S, 6.96%.

5-[N¢-(4-Adamantan-1-ylcarbonylaminobutyl)thioureido]-5-

deoxy-1,2-O-isopropylidene-a-D-glucofuranose

(43). Column

General procedure for the synthesis of (4R)-4-(L-treofuranos-

4¢-yl)-2-amino-2-thiazoline derivatives 34–36 and 46–51. To a

solution of the corresponding thioureido derivative 30–33 and

40–45 (0.86 mmol) in pyridine (23 mL) at -20 ^◦C under Ar,

methanesulfonic chloride (80 mL, 1.03 mmol, 1.2 eq) was added.

The reaction mixture was stirred for 7 h and allowed to warm

to 10 ^◦C. Then, ice-water (30 mL) was added and the solution

was extracted with CH₂Cl₂(3 ¥ 25 mL). The combined extracts

were washed with iced saturated aqueous NaHCO₃(20 mL),

dried (MgSO₄), and concentrated. In the case of the mixture of

thioureido derivatives 32 and 33 the reaction was deacetylated by

the Zemplen method. The resulting residue was puriﬁed by column

chromatography using the eluent indicated in each case.

chromatography, eluent 1 : 1 acetone–cyclohexane. Yield: 236 mg

(71%). [a]_D+29.5 (c 1.0, CH₂Cl₂). R_f0.44 (1 : 1 acetone-

cyclohexane). IR (KBr) n_max2909, 1627, 1374, 1074 cm^-1. UV

(CH₂Cl₂) 246 nm (e_mM9.8). ¹H NMR (300 MHz, CDCl₃, 313 K)

d 6.75 (bd, 1 H, ³J_NH,H-5= 8.6 Hz, NH), 5.92 (d, 1 H, J_1,2= 3.6 Hz,

H-1), 5.91 (bs, 1 H, N¢H), 5.29 (bs, 1 H, OH), 4.60 (d, 1 H, H-2),

4.52 (m, 1 H, H-5), 4.18 (s, 1 H, H-3), 4.12 (d, 1 H, J_4,5= 10.0 Hz,

H-4), 4.02 (dd, 1 H, J_6a,6b= 11.2 Hz, J_5,6a= 3.3 Hz, H-6a), 3.83 (dd,

1 H, J_5,6b= 3.1 Hz, H-6b), 3.48 (m, 2 H, CH₂NHCS), 3.26 (m, 2

H, CH₂NHCO), 2.05 (bs, 3 H, CH), 1.83 (m, 6 H, CCH₂) 1.73 (m,

6 H, CHCH₂), 1.62 (m, 4 H, CH₂), 1.49, 1.26 (2 s, 6 H, CMe₂).

¹³C NMR (75.5 MHz, CDCl₃, 313 K) d 182.1 (CO), 179.3 (CO),

111.6 (CMe₂), 105.0 (C-1), 84.7 (C-2), 79.8 (C-4), 73.8 (C-3),

62.6 (C-6), 53.7 (C-5), 44.0 (CH₂NHCS), 40.7 (CCONH), 39.3

(CH), 38.8 (CH₂NHCO), 36.4 (CCH₂), 28.1 (CHCH₂), 27.3, 26.8

(CH₂), 26.1, 25.3 (CMe₂). FABMS: m/z 534 (100, [M + Na]⁺),

512 (25, [M + H]⁺). Anal. Calcd for C₂₅H₄₁N₃O₆S: C, 58.68; H,

8.08; N, 8.21; S, 6.27. Found: C, 58.84; H, 7.91; N, 8.12, S, 5.94%.

(4R)-4-[(4¢R)-3¢-O-Acetyl-1¢,2¢-O-isopropylidene-b-L-threofu-

ranos-4¢-yl]-2-butylamino-2-thiazoline (34). Column chromatog-

raphy, eluent 1 : 4 acetone–cyclohexane, recovering 35% of the

starting material. Yield: 185 mg (60%). [a]_D-45.4 (c 1.0, CH₂Cl₂).

R_f0.76 (1 : 2 acetone–cyclohexane, 2 elutions). IR (KBr) n_max3383,

2931, 1747, 1624, 1374, 1078 cm^-1. ¹H NMR (500 MHz, CDCl₃)

d 5.87 (d, 1 H, J_1,2= 3.8 Hz, H-1), 5.23 (d, 1 H, J_3,4= 3.0 Hz,

5-[N¢-(11-Azido-3,6,9-trioxaundecyl)thioureido]-5-deoxy-1,2-O-

isopropylidene-a-D-glucofuranose (44). Column chromatogra-

phy, eluent 40 : 1 → 30 : 1 CH₂Cl₂–MeOH. Yield: 150 mg (48%).

[a]_D+43.7 (c 1.0, CH₂Cl₂). R_f0.49 (10 : 1 CH₂Cl₂–MeOH). IR

(KBr) n_max2932, 2108, 1375, 1075 cm^-1. UV (CH₂Cl₂) 246 nm (e_mM

14.4). ¹H NMR (500 MHz, CDCl₃, 313 K) d 6.90 (bs, 2 H, NH),

5.94 (d, 1 H, J_1,2= 3.6 Hz, H-1), 4.97 (bs, 1 H, OH), 4.58 (d, 1 H, H-

2), 4.56 (m, 1 H, H-5), 4.20 (s, 1 H, H-3), 4.10 (d, 1 H, J_4,5= 10.0 Hz,

H-3), 4.48 (d, 1 H, H-2), 4.41 (m, 1 H, H-5), 4.13 (dd, 1 H, J_4,5

=

9.2 Hz, H-4), 3.45 (dd, 1 H, J_6a,6b= 11.0 Hz, J_5,6a= 7.3 Hz, H-6a),

3

3.39 (dd, 1 H, J_5,6b= 5.1 Hz, H-6b), 3.20 (t, 2 H, J_H,H= 7.3 Hz,

CH₂N), 2.08 (s, 3 H, MeCO), 1.49 (m, 2 H, CH₂CH₂N), 1.48, 1.28

3

(2 s, 6 H, CMe₂), 1.33 (m, 2 H, CH₂CH₃), 0.87 (t, 3 H, J_H,H

=

7.4 Hz, CH₃). ¹³C NMR (125.7 MHz, CDCl₃) d 169.8 (CO), 163.0

(CN), 112.1 (CMe₂), 104.9 (C-1), 83.5 (C-2), 80.0 (C-4), 76.5 (C-3),

69.9 (C-5), 44.9 (CH₂N), 37.6 (C-6), 31.9 (CH₂CH₂N), 26.7, 26.2

(CMe₂), 21.0 (MeCO), 20.0 (CH₂CH₃), 13.7 (CH₃). FABMS: m/z

359 (100, [M + H]⁺). Anal. Calcd for C₁₆H₂₆N₂O₅S: C, 53.61; H,

7.31; N, 7.82. Found: C, 53.56; H, 7.33; N, 7.67%.

H-4), 4.06 (bd, 1 H, J_6a,6b= 10.1 Hz, H-6a), 3.82(dd, 1 H, J_5,6b

=

2.4 Hz, H-6b), 3.79 (m, 2 H, CH₂NH), 3.69 (m, 12 H, OCH₂), 3.44

(t, 1 H, ³J_H,H= 5.2 Hz, CH₂N₃), 2.94 (bs, 1 H, NH), 2.94 (bs, 1 H,

OH), 1.51, 1.33 (2 s, 6 H, CMe₂). ¹³C NMR (125.7 MHz, CDCl₃,

313 K) d 182.4 (CS), 111.6 (CMe₂), 105.0 (C-1), 84.7 (C-2), 79.8

(C-4), 73.7 (C-3), 71.4, 70.6, 70.5, 70.2, 69.9 (OCH₂), 62.4 (C-6),

53.7 (C-5), 50.8 (CH₂N₃), 44.9 (CH₂NH), 42.6 (CH₂CH₂N₃), 26.7,

26.1 (CMe₂). FABMS: m/z 502 (100, [M + Na]⁺), 480 (45, [M +

H]⁺). Anal. Calcd for C₁₈H₃₃N₅O₈S: C, 45.08; H, 6.94; N, 14.60.

Found: C, 44.85; H, 6.76; N, 14.46%.

(4R)-4-[(4¢R)-3¢-O-Acetyl-1¢,2¢-O-isopropylidene-b-L-threofu-

ranos-4¢-yl]-2-(2,3,4,6-tetra-O-acetyl-b-D-glycopyranosyl)amino-

2-thiazoline (35). Column chromatography, eluent 1 : 1 EtOAc–

petroleum ether. Yield: 468 mg (86%). [a]_D-13.2 (c 1.0, CH₂Cl₂).

R_f0.59 (2 : 1 EtOAc-petroleum ether). IR (KBr) n_max3462, 2942,

1749, 1631, 1374, 1036 cm^-1. ¹H NMR (300 MHz, CDCl₃) d 5.92

(d, 1 H, J_1,2= 3.7 Hz, H-1), 5.31 (d, 1 H, J_3,4= 3.0 Hz, H-3), 5.30 (t,

1 H, J_2¢,3¢= J_3¢,4¢= 9.5 Hz, H-3¢), 5.07 (t, 1 H, J_4¢,5¢= 9.5 Hz, H-4¢),

5.02 (d, 1 H, J_1¢,2¢= 9.5 Hz, H-1¢), 4.93 (t, 1 H, H-2¢), 4.49 (d, 1 H,

5-[N¢-(11-Adamantan-1-ylcarbonylamino-3,6,9-trioxaundecyl)-

thioureido] - 5 - deoxy - 1,2 - O - isopropylidene - a - D - glucofuranose

(45). Column chromatography, eluent 1 : 2 → 1 : 1 acetone–

cyclohexane. Yield: 340 mg (85%). [a]_D+30.4 (c 1.0, CH₂Cl₂). R_f

0.36 (1 : 1 acetone-cyclohexane). IR (KBr) n_max2906, 1634, 1350,

H-2), 4.37 (m, 1 H, H-5), 4.36 (dd, 1 H, J_6¢a,6¢b= 12.5 Hz, J_5¢,6¢a

=

4.4 Hz, H-6¢a), 4.18 (dd, 1 H, J_5¢,6¢b= 1.9 Hz, H-6¢b), 4.13 (dd,

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1 H, J_4,5= 4.5 Hz, H-4), 3.76 (ddd, 1 H, H-5¢), 3.53 (dd, 1 H,

J_6a,6b= 11.1 Hz, J_5,6a= 7.3 Hz, H-6a), 3.45 (dd, 1 H, J_5,6b= 5.0 Hz,

H-6b), 2.05, 2.03, 1.99, 1.96, 1.94 (s, 15 H, MeCO), 1.42, 1.23

(2 s, 6 H, CMe₂). ¹³C NMR (75.5 MHz, CDCl₃, 313 K) d 170.8,

170.0, 169.6, 169.5 (MeCO), 161.3 (CN), 112.2 (CMe₂), 105.1 (C-

1), 83.5 (C-1¢, C-2), 79.9 (C-4), 75.9 (C-3), 73.2 (C-3¢), 73.0 (C-5¢),

70.7 (C-2¢), 69.6 (C-5), 68.2 (C-4¢), 61.8 (C-6¢), 37.7 (C-6), 26.7,

26.1 (CMe₂), 21.4, 21.0, 20.7, 20.6 (MeCO). FABMS: m/z 655 (25,

[M + Na]⁺), 633 (100, [M + H]⁺). Anal. Calcd for C₂₆H₃₆N₂O₁₄S:

C, 49.36; H, 5.74; N, 4.43. Found: C, 49.16; H, 5.56; N, 4.36%.

1 H, J_6a,6b= 10.9 Hz, J_5,6a= 7.4 Hz, H-6a), 3.41 (dd, 1 H, J_5,6b=

5.5 Hz, H-6b), 3.25 (m, 2 H, CH₂N), 3.13 (m, 2 H, CH₂NHCO),

1.52, 1.35 (2 s, 6 H, CMe₂), 1.56 (m, 2 H, CH₂CH₂N), 1.47 (s, 9

H, CMe₃), 1.49 (m, 2 H, CH₂CH₂NHCO), 1.46 (s, 9 H, CMe₃),

1.33 (m, 8 H, CH₂). ¹³C NMR (75.5 MHz, CDCl₃) d 164.3 (CN),

156.0 (CO), 111.5 (CMe₂), 105.2 (C-1), 85.2 (C-2), 81.9 (C-4), 79.0

(CMe₃), 75.3 (C-3), 70.5 (C-5), 45.3 (CH₂N), 40.6 (CH₂NHCO),

37.7 (C-6), 30.0, 29.7, 29.0 (CH₂), 28.4 (CMe₃), 26.9, 26.2 (CMe₂),

26.7 (CH₂). FABMS: m/z 510 (10, [M + Na]⁺), 488 (100, [M +

H]⁺). Anal. Calcd for C₂₃H₄₁N₃O₆S: C, 56.65; H, 8.47; N, 8.62.

Found: C, 56.25; H, 8.34; N, 8.45%.

(4R)-4-[(4¢R)-1¢,2¢-O-Isopropylidene-b-L-threofuranos-4¢-yl]-2-

phenylamino-2-thiazoline (36). Column chromatography, eluent

60 : 1 → 30 : 1 CH₂Cl₂–MeOH. Yield: 257 mg (67%). [a]_D+23.1 (c

0.9, MeOH). R_f0.30 (20 : 1 CH₂Cl₂–MeOH). IR (KBr) n_max3324,

2929, 1629, 1588, 1370, 1070 cm^-1. ¹H NMR (300 MHz, CD₃OD)

d 7.34–6.96 (m, 5 H, Ph), 5.89 (d, 1 H, J_1,2= 3.6 Hz, H-1), 4.52 (d,

1 H, H-2), 4.43 (m, 1 H, H-5), 4.18 (d, 1 H, J_3,4= 2.6 Hz, H-3),

4.08 (dd, 1 H, J_4,5= 9.0 Hz, H-4), 3.45 (dd, 1 H, J_6a,6b= 11.0 Hz,

J_5,6a= 7.2 Hz, H-6a), 3.35 (dd, 1 H, J_5,6b= 5.3 Hz, H-6b), 1.44, 1.29

(2 s, 6 H, CMe₂). ¹³C NMR (75.5 MHz, CD₃OD) d 162.4 (CN),

144.5–121.0 (Ph), 112.8 (CMe₂), 106.4 (C-1), 86.8 (C-2), 82.6 (C-

4), 75.6 (C-3), 69.5 (C-5), 35.9 (C-6), 27.1, 26.4 (CMe₂). FABMS:

m/z 359 (45, [M + Na]⁺), 337 (100, [M + H]⁺). Anal. Calcd for

C₁₆H₂₀N₂O₄S: C, 57.12; H, 5.99; N, 8.33. Found: C, 57.08; H, 5.89;

N, 8.27%.

(4R)-2-(4-tert-Butoxycarbonylamino)butylamino-4-[(4¢R)-1¢,2¢-

O-isopropylidene-b-L-threofuranos-4¢-yl]-2-thiazoline (48). Colu-

mn chromatography, eluent 20 : 1 → 10 : 1 CH₂Cl₂–MeOH. Yield:

291 mg (78%). [a]_D-7.3 (c 1.0, CH₂Cl₂). R_f0.46 (7 : 1 CH₂Cl₂–

1

MeOH). IR (KBr) n_max3372, 2934, 1696, 1367, 1166 cm^-1. H

NMR (300 MHz, CDCl₃) d 5.93 (d, 1 H, J_1,2= 3.6 Hz, H-1), 4.79

(bs, 1 H, NH), 4.54 (d, 1 H, H-2), 4.42 (m, 1 H, H-5), 4.25 (bs,

2 H, OH, NH), 4.23 (d, 1 H, J_3,4= 2.5 Hz, H-3), 4.03 (dd, 1 H,

J_4,5= 8.4 Hz, H-4), 3.51 (dd, 1 H, J_6a,6b= 10.9 Hz, J_5,6a= 7.4 Hz,

H-6a), 3.39 (dd, 1 H, J_5,6b= 4.9 Hz, H-6b), 3.24 (m, 2 H, CH₂N),

3.11 (m, 2 H, CH₂NHCO), 1.53 (m, 4 H, CH₂), 1.48, 1.30 (2 s,

6 H, CMe₂), 1.33 (s, 9 H, CMe₃). ¹³C NMR (75.5 MHz, CDCl₃)

d 164.6 (CN), 156.1 (CO), 111.5 (CMe₂), 105.1 (C-1), 85.2 (C-2),

81.6 (C-4), 79.3 (CMe₃), 74.9 (C-3), 69.8 (C-5), 44.9 (CH₂N), 40.0

(CH₂NHCO), 37.4 (C-6), 28.4 (CMe₃), 27.3, 26.8 (CH₂), 26.8, 26.1

(CMe₂). FABMS: m/z 454 (50, [M + Na]⁺), 432 (100, [M + H]⁺).

Anal. Calcd for C₁₉H₃₃N₃O₆S: C, 52.88; H, 7.71; N, 9.74; S, 7.43.

Found: C, 52.66; H, 7.65; N, 9.48; S, 7.19%.

(4R)-2-[2¢-[N,N -Bis(2-hexanamidoethyl)aminoethyl]amino]-4-

[(4¢R)-1¢,2¢-O-isopropylidene-b-L-threofuranos-4¢-yl]-2-thiazoline

(46). Column chromatography, eluent 9 : 1 : 0 → 7 : 1 : 0 →

70 : 10 : 1 → 40 : 10 : 1 CH₂Cl₂–MeOH–H₂O, recovering 35% of the

starting material. Yield: 344 mg (70%). [a]_D-9.1 (c 1.0, CH₂Cl₂). R_f

0.41 (70 : 10 : 1 CH₂Cl₂–MeOH–H₂O). IR (KBr) n_max3286, 2953,

(4R)-2-(4-Adamantan-1-ylcarbonylamino)butylamino-4-[(4¢R)-

1¢,2¢-O-isopropylidene-b-L-threofuranos-4¢-yl]-2-thiazoline

(49).

1

1645, 1374, 1070 cm^-1. H NMR (300 MHz, CDCl₃) d 6.69 (bs,

Column chromatography, eluent 20 : 1 → 15 : 1 → 7 : 1 CH₂Cl₂–

1 H, NH), 5.96 (d, 1 H, J_1,2= 3.6 Hz, H-1), 4.57 (d, 1 H, H-2),

4.45 (m, 1 H, H-5), 4.29 (d, 1 H, J_3,4= 2.8 Hz, H-3), 4.11 (dd,

MeOH. Yield: 315 mg (74%). [a]_D-6.0 (c 1.0, CH₂Cl₂). R_f0.45

(7 : 1 CH₂Cl₂–MeOH). IR (KBr) n_max3344, 2907, 1620, 1373,

1

1 H, J_4,5= 8.0 Hz, H-4), 3.54 (dd, 1 H, J_6a,6b= 11.0 Hz, J_5,6a

=

1075 cm^-1. H NMR (300 MHz, CDCl₃) d 5.93 (d, 1 H, J_1,2

=

7.5 Hz, H-6a), 3.42 (dd, 1 H, J_5,6b= 5.6 Hz, H-6b), 3.28 (m, 6

H, CH₂NH, CH₂NHCO), 2.62 (m, 2 H, CH₂CH₂NH), 2.54 (t,

3.6 Hz, H-1), 5.86 (t, 1 H, ³J_NH,H= 5.5 Hz, NH), 4.52 (d, 1 H, H-2),

4.42 (m, 1 H, H-5), 4.24 (d, 1 H, J_3,4= 2.5 Hz, H-3), 4.05 (dd, 1 H,

J_4,5= 8.5 Hz, H-4), 3.51 (dd, 1 H, J_6a,6b= 11.0 Hz, J_5,6a= 7.4 Hz,

H-6a), 3.39 (dd, 1 H, J_5,6b= 5.1 Hz, H-6b), 3.25 (m, 4 H, CH₂N,

CH₂NHCO), 2.02 (bs, 3 H, CH), 1.82 (m, 6 H, CCH₂) 1.69(m,

6 H, CHCH₂), 1.54 (m, 4 H, CH₂), 1.47, 1.29 (2 s, 6 H, CMe₂).

¹³C NMR (75.5 MHz, CDCl₃) d 178.4 (CO), 165.0 (CN), 111.5

(CMe₂), 105.2 (C-1), 85.3 (C-2), 81.6 (C-4), 74.9 (C-3), 69.2 (C-5),

45.1 (CH₂N), 40.6 (CCONH), 39.3 (CH), 38.7 (CH₂NHCO), 37.2

(C-6), 36.5 (CCH₂), 28.1 (CHCH₂), 27.1, 26.7 (CH₂), 26.9, 26.2

(CMe₂). FABMS: m/z 516 (90, [M + Na]⁺), 494 (100, [M + H]⁺).

Anal. Calcd for C₂₅H₃₉N₃O₅S: C, 60.82; H, 7.96; N, 8.51; S, 6.50.

Found: C, 60.63; H, 7.79; N, 8.37, S, 6.29%.

3

4 H, J_H,H= 5.4 Hz, CH₂CONH), 2.22 (t, 4 H, J_H,H= 7.4 Hz,

CH₂CH₂NHCO), 1.63 (m, 4 H, CH₂CH₂CONH), 1.48, 1.31 (2 s,

6 H, CMe₂), 1.29 (m, 8 H, CH₂), 0.88 (t, 6 H, ³J_H,H= 6.8 Hz, CH₃).

¹³C NMR (75.5 MHz, CDCl₃) d 174.3 (CO), 163.0 (CN), 111.5

(CMe₂), 105.3 (C-1), 85.3 (C-2), 81.7 (C-4), 75.1 (C-3), 68.2 (C-5),

54.6 (CH₂CH₂NHCO), 52.6 (CH₂CH₂NH), 43.1 (CH₂NH), 37.6

(CH₂NHCO), 37.0 (C-6), 36.5 (CH₂CONH), 31.6 (CH₂), 26.9,

26.2 (CMe₂), 25.5 (CH₂CH₂CONH), 22.4 (CH₂CH₃), 14.0 (CH₃).

FABMS: m/z 608 (75, [M + Na]⁺), 586 (90, [M + H]⁺). Anal. Calcd

for C₂₈H₅₁N₅O₆S: C, 57.41; H, 8.78; N, 11.96. Found: C, 57.33; H,

8.71; N, 11.84%.

(4R)-2-(8-tert-Butoxycarbonylamino)octylamino-4-[(4¢R)-1¢,2¢-

O-isopropylidene-b-L-threofuranos-4¢-yl]-2-thiazoline (47). Colu-

mn chromatography, eluent 30 : 1 → 20 : 1 CH₂Cl₂–MeOH, recov-

ering 20% of the starting material. Yield: 319 mg (76%). [a]_D-9.0

(c 1.0, CH₂Cl₂). R_f0.47 (10 : 1 CH₂Cl₂–MeOH). IR (KBr) n_max

3356, 2971, 1697, 1367, 1076 cm^-1. ¹H NMR (500 MHz, CDCl₃)

d 5.99 (d, 1 H, J_1,2= 3.7 Hz, H-1), 4.59 (d, 1 H, H-2), 4.58 (bs,

1 H, NH), 4.45 (m, 1 H, H-5), 4.28 (d, 1 H, J_3,4= 2.5 Hz, H-3),

4.12 (dd, 1 H, J_4,5= 8.1 Hz, H-4), 3.78 (bs, 1 H, OH), 3.56 (dd,

(4R)-2-(11-Azido-3,6,9-trioxaundecyl)amino-4-[(4¢R)-1¢,2¢-O-

isopropylidene-b-L-threofuranos-4¢-yl]-2-thiazoline (50). Column

chromatography, eluent 1 : 1 → 2 : 1 acetone–cyclohexane. Yield:

279 mg (70%). [a]_D-7.6 (c 1.0, CH₂Cl₂). R_f0.26 (1 : 1 acetone–

cyclohexane). IR (KBr) n_max3379, 2930, 2107, 1620, 1075 cm^-1.

¹H NMR (300 MHz, CDCl₃) d 5.97 (d, 1 H, J_1,2= 3.7 Hz, H-1),

4.57 (d, 1 H, H-2), 4.43 (m, 1 H, H-5), 4.25 (d, 1 H, J_3,4= 2.8 Hz,

H-3), 4.08 (dd, 1 H, J_4,5= 8.2 Hz, H-4), 3.65 (m, 12 H, OCH₂), 3.54

(dd, 1 H, J_6a,6b= 10.9 Hz, J_5,6a= 7.4 Hz, H-6a), 3.42(m, 5 H, H-6b,

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CH₂N₃, CH₂N), 1.50, 1.29 (2 s, 6 H, CMe₂). ¹³C NMR (75.5 MHz,

CDCl₃) d 163.7 (CN), 111.4 (CMe₂), 105.2 (C-1), 85.1 (C-2), 82.0

(C-4), 75.4 (C-3), 71.4, 71.1 (OCH₂), 70.7 (C-5), 70.6, 70.0, 69.6

(OCH₂), 50.7 (CH₂N₃), 44.6 (CH₂N), 42.7 (CH₂CH₂N₃), 37.9 (C-

6), 26.9, 26.2 (CMe₂). FABMS: m/z 484 (65, [M + Na]⁺), 462 (80,

[M + H]⁺). Anal. Calcd for C₁₈H₃₁N₅O₇S: C, 46.84; H, 6.77; N,

15.17. Found: C, 46.65; H, 6.48; N, 14.99%.

CH₃). ¹³C NMR (125.7 MHz, D₂O) d 162.9 (CN), 76.0 (C-1),

74.3 (C-4), 72.8 (C-3), 71.4 (C-2), 59.8 (C-5), 53.9 (CH₂N), 32.0

(C-6), 30.4 (CH₂CH₂N), 19.8 (CH₂CH₃), 13.2 (CH₃). FABMS:

m/z 299 (40, [M + Na]⁺), 277 (100, [M + H]⁺). Anal. Calcd for

C₁₁H₂₀N₂O₄S: C, 47.81; H, 7.29; N, 10.14. Found: C, 47.65; H,

7.14; N, 9.99%.

5-N,6-S-(N¢-b-D-Glucopyranosyliminomethylidene)-6-thiono-

jirimycin (38). Column chromatography, eluent 6 : 1 : 1 → 6 : 3 : 1

CH₃CN–H₂O–NH₄OH. Yield: 104 mg (70%). [a]_D-15.5 (c

(4R)-2-(11-Adamantan-1-ylcarbonylamino-3,6,9-trioxaunde-

cyl)amino-4-[(4¢R)-1¢,2¢-O-isopropylidene-b-L-threofuranos-4¢-yl]-

2-thiazoline (51). Column chromatography, eluent 20 : 1 → 7 : 1

CH₂Cl₂–MeOH. Yield: 385 mg (46%). [a]_D-9.5 (c 1.0, CH₂Cl₂).

R_f0.55 (7 : 1 CH₂Cl₂–MeOH). IR (KBr) n_max3356, 2905, 1623,

1

0.97, H₂O). R_f0.39 (6 : 3 : 1 CH₃CN–H₂O–NH₄OH). H NMR

(500 MHz, D₂O) d 5.70 (d, 1 H, J_1,2= 3.8 Hz, H-1), 4.37 (d, 1

H, J_1¢,2¢= 8.3 Hz, H-1¢), 3.87 (m, 2 H, H-5, H-6¢a), 3.74 (t, 1 H,

1

1375, 1075 cm^-1. H NMR (300 MHz, CDCl₃) d 6.14 (bs, 1 H,

J_2,3= J_3,4= 9.9 Hz, H-3), 3.72 (dd, 1 H, J_6¢a,6¢b= 12.5 Hz, J_5¢,6¢b

=

5.4 Hz, H-6¢b), 3.60 (dd, 1 H, H-2), 3.52 (m, 3 H, H-6a, H-3¢,

NH), 5.95 (d, 1 H, J_1,2= 3.6 Hz, H-1), 4.56 (d, 1 H, H-2), 4.43 (m,

H-5¢), 3.46 (t, 1 H, J_4,5= 9.9 Hz, H-4), 3.44 (t, 1 H, J_3¢,4¢= J_4¢,5¢

9.9 Hz, H-4¢), 3.36 (t, 1 H, J_2¢,3¢= 8.3 Hz, H-2¢), 3.17 (t, 1 H, J_6a,6b

=

1 H, H-5), 4.25 (d, 1 H, J_3,4= 2.8 Hz, H-3), 4.09 (dd, 1 H, J_4,5

=

8.1 Hz, H-4), 3.63 (m, 12 H, OCH₂), 3.57 (dd, 1 H, J_6a,6b= 11.0 Hz,

J_5,6a= 5.3 Hz, H-6a), 3.46 (m, 1 H, H-6b), 3.42 (m, 4 H, CH₂N,

CH₂NHCO), 2.02 (bs, 3 H, CH), 1.84 (m, 6 H, CCH₂), 1.70 (m, 6

H, CH₂), 1.48, 1.31 (2 s, 6 H, CMe₂). ¹³C NMR (75.5 MHz, CDCl₃)

d 178.0 (CO), 163.9 (CN), 111.3 (CMe₂), 105.1 (C-1), 85.0 (C-2),

81.8 (C-4), 75.2 (C-3), 70.4, 70.3, 70.2, 70.0, 69.8 (OCH₂), 69.2 (C-

5), 44.6 (CH₂N), 40.4 (CCONH), 39.0 (CH), 38.9 (CH₂NHCO),

37.5 (C-6), 36.4 (CCH₂), 28.0 (CH₂), 26.7, 26.0 (CMe₂). FABMS:

m/z 620 (100, [M + Na]⁺), 598 (20, [M + H]⁺). Anal. Calcd for

C₂₉H₄₇N₃O₈S: C, 58.27; H, 7.93; N, 7.03. Found: C, 57.98; H, 7.77;

N, 6.81%.

J_5,6b= 10.3 Hz, H-6b). ¹³C NMR (125.7 MHz, D₂O) d 168.2 (CN),

93.2 (C-1¢), 77.6 (C-5¢), 76.1 (C-3¢), 75.8 (C-1), 74.4 (C-2¢), 74.0

(C-4), 72.6 (C-3), 71.0 (C-2), 69.6 (C-4¢), 60.8 (C-6¢), 59.9 (C-5),

30.7 (C-6). FABMS: m/z 405 (15, [M + Na]⁺). Anal. Calcd for

C₁₃H₂₂N₂O₉S: C, 40.83; H, 5.80; N, 7.33. Found: C, 4.83; H, 5.83;

N, 7.32%.

5-N,6-S-(N¢-Phenyliminomethylidene)-6-thionojirimycin (39).

Column chromatography, eluent 80 : 10 : 1 → 40 : 10 : 1 CH₂Cl₂–

MeOH–H₂O. Yield: 81 mg (70%). [a]_D-38.0 (c 0.5, H₂O). R_f0.20

1

(9 : 1 CH₂Cl₂–MeOH). H NMR (500 MHz, CD₃OD) d 7.22 (t,

2 H, Ph), 7.00 (t, 1 H, Ph), 6.88 (d, 2 H, Ph), 5.72 (d, 1 H, J_1,2

=

General procedure for the synthesis of 5-N,6-S-(N¢-

iminomethylidene)-6-thionojirimycin derivatives 37–39 and 52–57.

To a solution of the corresponding 2-amino-2-thiazoline precursor

34 and 35 (0.39 mmol) in dry MeOH (3.5 mL), methanolic

NaMeO (1 m, 0.1 equiv per mol of acetate) was added. The

reaction mixture was stirred at room temperature for 30 min,

then neutralized with solid CO₂, and concentrated. The resulting

deacetylated product was treated with TFA-H₂O (9 : 1, 2.4 mL)

for 30 min, concentrated under reduced pressure, coevaporated

several times with water, neutralized with Amberlite IRA-68 (OH^-)

ion-exchange resin, and subjected to column chromatography with

the eluent indicated in each case to obtain the isothioureas 16

and 17. An analogous TFA-catalyzed reaction starting from 36

afforded the phenyl derivative 18. The corresponding 2-amino-

2-thiazoline precursor 46–51 (0.13 mmol), after treatment with

TFA-H₂O (9 : 1, 1 mL) for 30 min at 0 ^◦C followed by concentration

under reduced pressure, coevaporation several times with water,

neutralization with Amberlite IRA-68 (OH-) ion-exchange resin

and column chromatography with the eluent indicated in each

case afforded the corresponding isothioureas 52–57. Compounds

53 and 54 were obtained as the corresponding hydrochloride salts

by freeze-drying from a solution of hydrochloric acid (pH 5).

3.7 Hz, H-1), 3.82 (td, 1 H, J_4,5= J_5,6b= 9.5 Hz, J_5,6a= 6.5 Hz,

H-5), 3.71 (t, 1 H, J_2,3= J_3,4= 9.5 Hz, H-3), 3.45 (dd, 1 H, H-2),

3.35 (dd, 1 H, J_6a,6b= 10.9 Hz, H-6a), 3.29 (t, 1 H, H-4), 3.00

(dd, 1 H, H-6b). ¹³C NMR (125.7 MHz, CD₃OD) d 162.3 (CN),

152.6–123.0 (Ph), 77.8 (C-1), 76.3 (C-4), 74.8 (C-3), 73.4 (C-2),

61.1 (C-5), 31.8 (C-6). FABMS: m/z 319 (75, [M + Na]⁺). Anal.

Calcd for C₁₃H₁₆N₂O₄S: C, 52.69; H, 5.44; N, 9.45. Found: C,

52.50; H, 5.13; N, 9.29%.

5-N,6-S -(N¢(N,N -Bis(2-hexanamidoethyl)aminoethyl)imino-

methylidene)-6-thionojirimycin (52). Column chromatography,

eluent 60 : 10 : 1 → 40 : 10 : 1 CH₂Cl₂–MeOH–H₂O. Yield: 64 mg

(90%). [a]_D-15.5 (c 1.0, H₂O). R_f0.41 (40 : 10 : 1 CH₂Cl₂–MeOH–

H₂O). ¹H NMR (500 MHz, D₂O) d 5.62 (d, 1 H, J_1,2= 3.8 Hz, H-1),

3.85 (m, 1 H, H-5), 3.74 (t, 1 H, J_2,3= J_3,4= 9.5 Hz, H-3), 3.57 (dd,

1 H, H-2), 3.56 (dd, 1 H, J_6a,6b= 10.5 Hz, J_5,6a= 6.5 Hz, H-6a), 3.45

(m, 6 H, CH₂NCS, CH₂NHCO), 3.44 (t, 1 H, J_4,5= 9.5 Hz, H-4),

3.25 (m, 2 H, CH₂NCS), 3.18 (m, 4 H, CH₂CH₂NHCO), 3.17 (t, 1

H, J_5,6b= 10.5 Hz, H-6b), 2.16 (t, 4 H, ³J_H,H= 7.5 Hz, CH₂CONH),

1.47 (m, 4 H, CH₂CH₂CONH), 1.18 (m, 8 H, CH₂), 0.76 (t, 6 H,

³J_H,H= 7.0 Hz, CH₃). ¹³C NMR (125.7 MHz, D₂O) d 178.3 (CO),

162.9 (CN), 75.9 (C-1), 74.3 (C-4), 72.6 (C-3), 71.3 (C-2), 60.5 (C-

5), 54.4 (CH₂CH₂NCS), 54.0 (CH₂CH₂NHCO), 48.0 (CH₂NCS),

35.7 (CH₂NHCO), 35.6 (CH₂CONH), 31.1 (C-6), 30.6 (CH₂), 25.0

(CH₂CH₂CONH), 21.7 (CH₂CH₃), 13.2 (CH₃). FABMS: m/z 568

(30, [M + Na]⁺), 546 (40, [M + H]⁺). Anal. Calcd for C₂₅H₄₇N₅O₆S:

C, 55.02; H, 8.68; N, 12.83. Found: C, 55.09; H, 8.66; N, 12.86%.

5-N,6-S-(N¢-Butyliminomethylidene)-6-thionojirimycin

(37).

Column chromatography, eluent 70 : 10 : 1 → 40 : 10 : 1 CH₂Cl₂–

MeOH–H₂O. Yield: 86 mg (80%). [a]_D-22.9 (c 0.7, H₂O). R_f0.26

1

(40 : 10 : 1 CH₂Cl₂–MeOH–H₂O). H NMR (500 MHz, D₂O) d

5.52 (d, 1 H, J_1,2= 3.7 Hz, H-1), 3.75 (td, 1 H, J_4,5= J_5,6b= 9.5 Hz,

J_5,6a= 6.4 Hz, H-5), 3.71 (t, 1 H, J_2,3= J_3,4= 9.5 Hz, H-3), 3.55

(dd, 1 H, H-2), 3.45 (dd, 1 H, J_6a,6b= 10.5 Hz, H-6a), 3.40 (t, 1 H,

H-4), 3.18 (m, 2 H, CH₂N), 3.07 (dd, 1 H, H-6b), 1.52 (m, 2 H,

CH₂CH₂N), 1.30 (m, 2 H, CH₂CH₃), 0.87 (t, 3 H, ³J_H,H= 7.4 Hz,

5-N,6-S-(N¢-8¢-Aminooctyliminomethylidene)-6-thionojirimycin

hydrochloride (53). Column chromatography, eluent 6 : 1 : 1

CH₃CN–H₂O–NH₄OH. Yield: 41 mg (91%). [a]_D-7.0 (c 1.0,

H₂O). R_f0.49 (6 : 3 : 1 CH₃CN–H₂O–NH₄OH). ¹H NMR

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(500 MHz, D₂O, 313 K) d 5.76 (d, 1 H, J_1,2= 4.0 Hz, H-1), 4.46

(m, 1 H, H-5), 3.92 (m, 2 H, H-3, H-6a), 3.78 (dd, 1 H, J_2,3

444.152890. Anal. Calcd for C₁₅H₂₇N₅O₇S: C, 42.75; H, 6.46; N,

16.62; S, 7.61. Found: C, 42.51; H, 6.55; N, 16.46; S, 7.39%.

=

9.5 Hz, H-2), 3.71 (t, 1 H, J_3,4= J_4,5= 9.5 Hz, H-4), 3.61 (t, 1 H,

5-N,6-S-(N¢-11-Adamantane-1-carbonylamino-3,6,9-trioxaun-

decyliminomethylidene)-6-thionojirimycin (57). Column chro-

J_6a,6b= J_5,6b= 10.0 Hz, H-6b), 3.57 (t, 2 H, ³J_H,H= 7.0 Hz, CH₂N),

3

3.12 (t, 2 H, J_H,H= 7.5 Hz, CH₂NH₂), 1.79 (m, 4 H, CH₂), 1.48

matography, eluent 80 : 10 : 1

→

40 : 10 : 1 CH₂Cl₂–H₂O–

(m, 8 H, CH₂). ¹³C NMR (125.7 MHz, D₂O, 313 K) d 173.0 (CN),

76.6 (C-1), 73.3 (C-4), 71.9 (C-3), 70.8 (C-2), 63.6 (C-5), 49.2

(CH₂N), 39.8 (CH₂NH₂), 31.4 (C-6), 28.2, 28.1, 28.0, 26.9, 27.8,

25.7 (CH₂). FABMS: m/z 348 (30, [M + H]⁺). HRFABMS: m/z

348.194640; calcd. for C₁₅H₃₀N₃O₄S: 348.195704. Anal. Calcd for

C₁₅H₃₀ClN₃O₄S: C, 46.92; H, 7.88; N, 10.94; S, 8.35. Found: C,

46.57; H, 7.69; N, 10.61; S, 8.04%.

MeOH. Yield: 72 mg (99%). [a]_D-6.2 (c 1.0, H₂O). R_f0.51

(40 : 10 : 1 CH₂Cl₂–H₂O–MeOH). ¹H NMR (500 MHz, D₂O, 313

K) d 5.76 (d, 1 H, J_1,2= 3.7 Hz, H-1), 4.21 (m, 1 H, H-5), 3.90 (t,

1 H, J_2,3= J_3,4= 9.5 Hz, H-3), 3.84 (m, 12 H, OCH₂), 3.79 (dd,

1 H, J_6a,6b= 11.2 Hz, J_5,6a= 7.1 Hz, H-6a), 3.73 (dd, 1 H, H-2),

3.67 (m, 2 H, CH₂N), 3.63 (t, 1 H, J_4,5= 9.5 Hz, H-4), 3.52 (t,

3

2 H, J_H,H= 5.5 Hz, CH₂NHCO), 3.42 (t, 1 H, J_5,6b= 11.2 Hz,

H-6b), 2.15 (bs, 3 H, CH), 1.94 (m, 6 H, CCH₂) 1.85 (m, 6 H,

CH₂). ¹³C NMR (125.7 MHz, D₂O, 313 K) d 182.0 (CO), 163.0

(CN), 76.4 (C-1), 74.0 (C-4), 72.5 (C-3), 71.2 (C-2), 69.9, 69.8, 69.7,

69.3, 69.2 (OCH₂), 61.9 (C-5), 51.1 (CH₂N), 40.8 (CCONH), 39.1

(CH₂NHCO), 38.7 (CH), 36.1 (CCH₂), 31.2 (C-6), 27.9 (CH₂).

FABMS: m/z 580 (100, [M + Na]⁺), 558 (5, [M + H]⁺). Anal.

Calcd for C₂₆H₄₃N₃O₈S: C, 55.99; H, 7.77; N, 7.53; S, 5.75. Found:

C, 55.68; H, 7.58; N, 7.32; S, 5.39%.

5-N,6-S-(N¢-4¢-Aminobutyliminomethylidene)-6-thionojirimycin

hydrochloride (54). Column chromatography, eluent 10 : 1 : 1 →

6 : 1 : 1 → 6 : 3 : 1 CH₃CN–H₂O–NH₄OH. Yield: 35 mg (82%).

[a]_D-11.3 (c 1.0, H₂O). R_f0.22 (6 : 3 : 1 CH₃CN–H₂O–NH₄OH).

¹H NMR (500 MHz, D₂O) d 5.61 (d, 1 H, J_1,2= 3.7 Hz, H-1),

4.32 (m, 1 H, H-5), 3.76 (m, 2 H, H-3, H-6a), 3.62 (dd, 1 H, J_2,3

=

9.5 Hz, H-2), 3.56 (t, 1 H, J_3,4= J_{4, 5}= 9.6 Hz, H-4), 3.46 (m, 3

3

H, H-6b, CH₂N), 3.00 (t, 2 H, J_H,H= 7.1 Hz, CH₂NH₂), 1.73

(m, 4 H, CH₂). ¹³C NMR (125.7 MHz, D₂O) d 175.7 (CN), 79.0

(C-1), 75.7 (C-4), 74.2 (C-3), 73.1 (C-2), 66.0 (C-5), 50.6 (CH₂N),

41.5 (CH₂NH₂), 33.9 (C-6), 27.7, 26.5 (CH₂). FABMS: m/z 314

(40, [M + Na - HCl]⁺), 292 (90, [M + H - Cl]⁺). Anal. Calcd for

C₁₁H₂₂ClN₃O₄S: C, 40.30; H, 6.76; N, 12.82; S, 9.78. Found: C,

39.95; H, 6.47; N, 12.49; S, 9.41%.

General procedure for the synthesis of 5-thioureido-a-D-

galactofuranose derivatives 58 and 59. A solution of 5-azido-

5-deoxy-1,2-O-isopropylidene-a-D-galactofuranose¹⁶12 (350 mg,

1.43 mmol) in MeOH (7 mL) was hydrogenated at atmospheric

pressure for 1 h using 10% Pd/C (123 mg) as catalyst. The suspen-

sion was ﬁltered through Celite and concentrated. The resulting

residue was dissolved in pyridine (8 mL), Et₃N (0.96 mL, 6.9 mmol,

5.6 eq) and the corresponding isothiocyanate (1.5 mmol, 1.2 eq)

were added and the mixture was stirred at room temperature for

18 h. Then, the solvent was removed under reduced pressure and

the resulting residue coevaporated several times with toluene and

puriﬁed by column chromatography using the eluent indicated in

each case.

5-N,6-S -(N¢-4¢-(Adamantane-1-carbonylamino)butylimino-

methylidene)-6-thionojirimycin (55). Column chromatography,

eluent 40 : 10 : 1 CH₂Cl₂–H₂O–MeOH. Yield: 58 mg (99%). [a]_D

1

-7.1 (c 1.0, H₂O). R_f0.28 (40 : 10 : 1 CH₂Cl₂–H₂O–MeOH). H

NMR (500 MHz, D₂O, 313 K) d 5.73 (d, 1 H, J_1,2= 3.7 Hz, H-1),

4.38 (m, 1 H, H-5), 3.91 (t, 1 H, J_2,3= J_3,4= 9.5 Hz, H-3), 3.87 (dd, 1

H, J_6a,6b= 11.3 Hz, J_5,6a= 7.5 Hz, H-6a), 3.74 (dd, 1 H, J_2,3= 9.5 Hz,

H-2), 3.67 (t, 1 H, J_4,5= 9.5 Hz, H-4), 3.53 (m, 3 H, H-6b, CH₂N),

3.33 (t, 2 H, ³J_H,H= 6.7 Hz, CH₂NHCO), 2.14 (bs, 3 H, CH), 1.92

5-(N¢-Butylthioureido)-5-deoxy-1,2-O-isopropylidene-a-D -

galactofuranose (58). Column chromatography, eluent 20 : 1

CH₂Cl₂–MeOH. Yield: 344 mg (72%). [a]_D+21.9 (c 1.0, CH₂Cl₂).

R_f0.46 (10 : 1 CH₂Cl₂–MeOH). IR (KBr) n_max3356, 2933, 1375,

1066 cm^-1. UV (CH₂Cl₂) 250 nm (e_mM15.7). ¹H NMR (300 MHz,

CDCl₃, 313 K) d 6.59 (bs, 2 H, NH), 5.80 (d, 1 H, J_1,2= 4.4 Hz, H-

1), 4.69 (m, 1 H, H-5), 4.66 (dd, 1 H, J_2,3= 2.8 Hz, H-2), 4.23 (dd,

(m, 6 H, CCH₂) 1.83 (m, 6 H, CHCH₂), 1.67 (m, 2 H, CH₂). ¹³

C

NMR (125.7 MHz, D₂O, 313 K) d 182.0 (CO), 173.0 (CN), 76.6

(C-1), 73.5 (C-4), 72.0 (C-3), 70.9 (C-2), 63.1 (C-5), 49.3 (CH₂N),

40.8 (CCONH), 38.8 (CH), 38.6 (CH₂NHCO), 36.1 (CCH₂), 31.4

(C-6), 28.0 (CHCH₂), 25.9, 25.6 (CH₂). FABMS: m/z 476 (20,

[M + Na]⁺), 454 (10, [M + H]⁺). Anal. Calcd for C₂₂H₃₅ClN₃O₅S:

C, 58.25; H, 7.78; N, 9.26; S, 7.07. Found: C, 58.17; H, 7.66; N,

9.05; S, 6.84%.

1 H, J_3,4= 6.6 Hz, H-3), 3.96 (m, 1 H, H-4), 3.95 (dd, 1 H, J_6a,6b

=

11.8 Hz, J_5,6a= 3.6 Hz, H-6a), 3.83 (dd, 1 H, J_5,6b= 4.4 Hz, H-6b),

3.42 (m, 2 H, CH₂N), 1.62 (m, 2 H, CH₂CH₂N), 1.52, 1.39 (2 s, 6

3

H, CMe₂), 1.40 (m, 2 H, CH₂CH₃), 0.96 (t, 3 H, J_H,H= 7.3 Hz,

CH₃). ¹³C NMR (75.5 MHz, CDCl₃, 313 K) d 182.5 (CS), 113.4

(CMe₂), 104.6 (C-1), 86.6 (C-2), 77.2 (C-4), 75.8 (C-3), 63.9 (C-

6), 55.0 (C-5), 44.2 (CH₂N), 30.8 (CH₂CH₂N), 27.7, 27.0 (CMe₂),

20.0 (CH₂CH₃), 13.6 (CH₃). FABMS: m/z 357 (52, [M + Na]⁺),

335 (100, [M + H]⁺). Anal. Calcd for C₁₄H₂₆N₂O₅S: C, 50.28; H,

7.84; N, 8.38. Found: C, 50.14; H, 7.63; N, 8.25%.

5-N,6-S-(N¢-11-Azido-3,6,9-trioxaundecyliminomethylidene)-6-

thionojirimycin (56). Column chromatography, eluent 70 : 10 : 1

→ 40 : 10 : 1 CH₂Cl₂–MeOH–H₂O. Yield: 43 mg (78%). [a]_D-13.0

(c 1.0, H₂O). R_f0.57 (40 : 10 : 1 CH₂Cl₂–MeOH–H₂O). ¹H NMR

(500 MHz, D₂O) d 5.55 (d, 1 H, J_1,2= 3.7 Hz, H-1), 3.73 (m, 14

H, H-3, H-5, OCH₂), 3.54 (dd, 1 H, J_2,3= 9.8 Hz, H-2), 3.45 (m,

2 H, CH₂N), 3.45 (dd, 1 H, J_6a,6b= 10.7 Hz, J_5,6a= 6.3 Hz, H-6a),

3.39 (t, 1 H, J_4,5= 9.7 Hz, H-4), 3.35 (m, 2 H, CH₂N₃), 3.05 (t, 1

H, J_5,6b= 10.7 Hz, H-6b). ¹³C NMR (125.7 MHz, D₂O) d 166.1

(CN), 78.3 (C-1), 77.0 (C-4), 75.3 (C-3), 73.9 (C-2), 73.4, 73.1,

72.1, 72.0, 71.8 (OCH₂), 62.1 (C-5), 56.0 (CH₂N₃), 52.7 (CH₂N),

45.7 (CH₂CH₂N₃), 33.1 (C-1). FABMS: m/z 444 (15, [M +

Na]⁺). HRFABMS: m/z 444.154344; calcd. for C₁₅H₂₇N₅O₇NaS:

5-Deoxy-1,2-O-isopropylidene-5-(N¢-phenylthioureido)-a-D-

galactofuranose (59). Column chromatography, eluent 40 : 1→

20 : 1 CH₂Cl₂–MeOH. Yield: 350 mg (69%). [a]_D+4.0 (c 0.53,

CH₂Cl₂). R_f0.28 (20 : 1 CH₂Cl₂–MeOH). UV (CH₂Cl₂) 264 nm

(e_mM12.7). IR (KBr) n_max3370, 2929, 1383, 1064 cm^-1. ¹H NMR

(300 MHz, CDCl₃, 313 K) d 8.10 (bs, 1 H, N¢H), 7.46–7.24 (m, 5

H, Ph), 6.92 (d, 1 H, J_5,NH= 7.7 Hz, NH), 5.71 (d, 1 H, J_1,2= 4.4 Hz,

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H-1), 4.93 (m, 1 H, H-5), 4.64 (dd, 1 H, J_2,3= 3.1 Hz, H-2), 4.21

(dd, 1 H, J_3,4= 7.7 Hz, H-3), 3.96 (dd, 1 H, J_6a,6b= 11.8 Hz, J_5,6a

J_2,3= 10.2 Hz, J_3,4= 1.8 Hz, H-3), 3.83 (dd, 1 H, H-2), 3.49 (d, 2

H, H-6a, H-6b), 3.34 (t, 2 H, ³J_H,H= 6.8 Hz, CH₂N), 1.61 (m, 2 H,

CH₂CH₂N), 1.33 (m, 2 H, CH₂CH₃), 0.89 (t, 3 H, ³J_H,H= 7.3 Hz,

CH₃). ¹³C NMR (125.7 MHz, D₂O) d 170.4 (CN), 76.0 (C-1),

69.1 (C-3), 68.9 (C-4), 67.4 (C-3), 61.8 (C-5), 50.0 (CH₂N), 30.6

(CH₂CH₂N), 27.4 (C-6), 19.3 (CH₂CH₃), 12.9 (CH₃). FABMS:

m/z 299 (55, [M + Na]⁺), 277 (100, [M + H]⁺). Anal. Calcd for

C₁₁H₂₀N₂O₄S: C, 47.81; H, 7.29; N, 10.14. Found: C, 47.69; H,

7.17; N, 9.94%.

=

3.0 Hz, H-6a), 3.85 (m, 2 H, H-6b, H-4), 2.67, 1.84 (2 bs, 2 H,

OH), 1.35, 1.27 (2 s, 6 H, CMe₂). ¹³C NMR (125.7 MHz, CDCl₃,

313 K) d 181.3 (CS), 136.1–124.9 (Ph), 114.1 (CMe₂), 104.3 (C-1),

86.9 (C-2), 84.7 (C-4), 75.8 (C-3), 63.6 (C-6), 55.4 (C-5), 27.7, 27.0

(CMe₂). FABMS: m/z 377 (100, [M + Na]⁺), 355 (15, [M + H]⁺).

Anal. Calcd for C₁₆H₂₂N₂O₅S: C, 54.22; H, 6.26; N, 7.90. Found:

C, 54.21; H, 5.98; N, 7.75%.

5-N,6-S-(N¢-Phenyliminomethylidene)-6-thiogalactonojirimycin

(63). Column chromatography, eluent 80 : 10 : 1 CH₂Cl₂–

MeOH–H₂O. Yield: 23 mg (60%). [a]_D-25.5 (c 0.5, H₂O). R_f0.55

General procedure for the synthesis of (4S)-4-(L-threofuranos-

4¢-yl)-2-amino-2-thiazoline derivatives 60 and 61. The 2-amino-

2-thiazolines 60 and 61 were prepared from the corresponding

thioureido derivatives 58 and 59 following the general procedure

above described.

1

(40 : 10 : 1 CH₂Cl₂–MeOH–H₂O). H NMR (500 MHz, D₂O) d

7.38–7.01 (m, 5 H, Ph), 5.74 (bs, 1 H, H-1), 4.23 (bt, 1 H, J_5,6a

=

J_5,6b= 9.0 Hz, 6.5 Hz, H-5), 4.04 (bs, 1 H, H-4), 3.95 (bd, 1 H,

(4S)-2-Butylamino-4-[(4¢S)-1¢,2¢-O-isopropylidene-b-L-threofur-

anos-4¢-yl]-2-thiazoline (60). Column chromatography, eluent

80 : 10 : 1 CH₂Cl₂–MeOH–H₂O. Yield: 163 mg (60%). [a]_D-29.6 (c

1.0, CH₂Cl₂). R_f0.50 (70 : 10 : 1 CH₂Cl₂–MeOH–H₂O). IR (KBr)

n_max3361, 2924, 1615, 1375, 1063 cm^-1. ¹H NMR (300 MHz,

CDCl₃) d 5.82 (d, 1 H, J_1,2= 3.8 Hz, H-1), 4.87 (bs, 1 H, NH),

4.56 (dd, 1 H, J_2,3= 1.3 Hz, H-2), 4.53 (m, 1 H, H-5), 4.25 (dd,

1 H, J_3,4= 5.2 Hz, H-3), 4.06 (t, 1 H, J_4,5= 5.2 Hz, H-4), 3.41

(d, 2 H, J_5,6= 7.8 Hz, H-6), 3.21 (m, 2 H, CH₂N), 1.54 (m, 2 H,

CH₂CH₂N), 1.51, 1.24 (2 s, 6 H, CMe₂), 1.35 (m, 2 H, CH₂CH₃),

0.91 (t, 3 H, ³J_H,H= 7.3 Hz, CH₃). ¹³C NMR (75.5 MHz, CDCl₃) d

166.2 (CN), 113.4 (CMe₂), 104.6 (C-1), 87.6 (C-2), 86.6 (C-4), 74.8

(C-3), 68.7 (C-5), 46.0 (CH₂N), 34.0 (C-6), 31.6 (CH₂CH₂N), 27.4,

26.5 (CMe₂), 19.8 (CH₂CH₃), 13.5 (CH₃). FABMS: m/z 339 (30,

[M + Na]⁺), 317 (100, [M + H]⁺). Anal. Calcd for C₁₄H₂₄N₂O₄S:

C, 53.14; H, 7.65; N, 8.85. Found: C, 53.16; H, 7.56; N, 8.78%.

J_2,3= 9.3 Hz, H-3), 3.88 (bd, 1 H, H-2), 3.30 (bt, 1 H, J_6a,6b

=

9.0 Hz, H-6a), 3.20 (bt, 1 H, H-6b). ¹³C NMR (125.7 MHz, D₂O)

d 173.9 (CN), 149.0–122.5 (Ph), 75.8 (C-1), 69.7 (C-3), 68.8 (C-4),

67.9 (C-2), 59.2 (C-5), 26.7 (C-6). FABMS: m/z 322 (100, [M +

Na + 3H]⁺), 297 (10, [M + H]⁺). Anal. Calcd for C₁₃H₁₆N₂O₄S: C,

52.69; H, 5.44; N, 9.45. Found: C, 52.69; H, 5.38; N, 9.38%.

General procedure for the synthesis of 5,6-di-N-(N¢-imino-

methylidene)-a-D-(galacto)glucofuranose derivatives 66–69. A so-

lution of 64¹or 65²(0.73 mmol) and the corresponding amine

(1.5 eq) in DMF (15 mL) was heated at 70 ^◦C under Ar for 18 h

and concentrated. The resulting residue was puriﬁed by column

chromatography using the eluent indicated in each case. Except-

ing 66, the 5,6-di-N-(N¢-iminomethylidene)-a-D-glycofuranose

derivatives were obtained as the corresponding hydrochloride salt

by freeze-drying from a solution of hydrochloric acid (pH 5).

5,6-Diamino-5,6-di-N-(N¢-butyliminomethylidene)-5,6-dideoxy-

1,2-O-isopropylidene-a-D-glucofuranose (66). Column chro-

matography, eluent 80 : 10 : 1 CH₂Cl₂–MeOH–H₂O. Yield: 166 mg

(76%). [a]_D-45.4 (c 1.0, CH₂Cl₂). R_f0.49 (70 : 10 : 1 CH₂Cl₂–

MeOH–H₂O). IR (KBr) n_max3284, 2959, 1673, 1375, 1075 cm^-1.

¹H NMR (300 MHz, CD₃OD) d 5.95 (d, 1 H, J_1,2= 3.6 Hz, H-1),

4.53 (d, 1 H, H-2), 4.35 (m, 1 H, H-5), 4.26 (dd, 1 H, J_4,5= 4.9 Hz,

(4S)-4-[(4¢S)-1¢,2¢-O-Isopropylidene-b-L-threofuranos-4¢-yl]-2-

phenylamino-2-thiazoline (61). Column chromatography, eluent

50 : 1 → 20 : 1 CH₂Cl₂–MeOH. Yield: 150 mg (52%). [a]_D-86.3

(c 1.0, CH₂Cl₂). R_f0.30 (20 : 1 CH₂Cl₂–MeOH). IR (KBr) n_max

3361, 2962, 1621, 1375, 1015 cm^-1. ¹H NMR (300 MHz, CD₃OD)

d 7.29–6.99 (m, 5 H, Ph), 5.90 (d, 1 H, J_1,2= 3.9 Hz, H-1), 4.56

(dd, 1 H, J_2,3= 0.75 Hz, H-2), 4.36 (m, 1 H, H-5), 4.22 (dd, 1 H,

J_3,4= 3.3 Hz, H-3), 3.94 (dd, 1 H, J_4,5= 8.1 Hz, H-4), 3.43 (dd,

J_3,4= 3.0 Hz, H-4), 4.20 (d, 1 H, H-3), 3.82 (dd, 1 H, J_6a,6b

=

9.9 Hz, J_5,6a= 7.0 Hz, H-6a), 3.78 (t, 1 H, J_5,6b= 9.9 Hz, H-6b),

1 H, J_6a,6b= 11.0 Hz, J_5,6a= 7.5 Hz, H-6a), 3.19 (dd, 1 H, J_5,6b

=

3

3.23 (t, 2 H, J_H,H= 7.0 Hz, CH₂N), 1.60 (m, 2 H, CH₂CH₂N),

6.8 Hz, H-6b), 1.51, 1.33 (2 s, 6 H, CMe₂). ¹³C NMR (75.5 MHz,

CD₃OD) d 163.0 (CN), 147.7-122.0 (Ph), 114.1 (CMe₂), 106.6 (C-

1), 89.5 (C-2), 88.9 (C-4), 77.1 (C-3), 66.6 (C-5), 33.3 (C-6), 27.5,

26.5 (CMe₂). FABMS: m/z 359 (50, [M + Na]⁺), 337 (100, [M +

H]⁺). Anal. Calcd for C₁₆H₂₀N₂O₄S: C, 57.12; H, 5.99; N, 8.33.

Found: C, 56.93; H, 5.75; N, 8.21%.

1.48, 1.32 (2 s, 6 H, CMe₂), 1.42 (m, 2 H, CH₂CH₃), 0.98 (t, 3

H, ³J_H,H= 7.3 Hz, CH₃). ¹³C NMR (75.5 MHz, CD₃OD) d 160.2

(CN), 113.0 (CMe₂), 106.6 (C-1), 86.8 (C-2), 82.5 (C-4), 75.4

(C-3), 55.6 (C-5), 46.1 (C-6), 43.6 (CH₂N), 32.2 (CH₂CH₂N),

27.2, 26.4 (CMe₂), 20.8 (CH₂CH₃), 13.9 (CH₃). FABMS: m/z 300

(100, [M + H]⁺). Anal. Calcd for C₁₄H₂₅N₃O₄: C, 56.17; H, 8.42;

N, 14.04. Found: C, 55.96; H, 8.65; N, 13.94%.

Synthesis of 5-N,6-S-(N¢-iminomethylidene)-6-thiogalactono-

jirimycin derivatives 62 and 63. The isothioureas 60 and 61 were

prepared from the corresponding 2-amino-2-thiazoline precursor

(60 and 61) following the method B in general procedure above

described.

5,6-Diamino-5,6-di-N-(N¢-benzyliminomethylidene)-5,6-dideo-

xy-1,2-O-isopropylidene-a-D-glucofuranose hydrochloride (67).

Column chromatography, eluent 90 : 10 : 1 → 40 : 10 : 1 CH₂Cl₂–

MeOH–H₂O. Yield: 175 mg (65%). [a]_D-15.4 (c 1.0, CH₂Cl₂). R_f

0.63 (40 : 10 : 1 CH₂Cl₂–MeOH–H₂O). IR (KBr) n_max3292, 2985,

1669, 1378, 1074 cm^-1. ¹H NMR (300 MHz, CD₃OD) d 7.43–7.33

(m, 5 H, Ph), 5.95 (d, 1 H, J_1,2= 3.6 Hz, H-1), 4.52 (d, 1 H, H-2),

4.45 (s, 2 H, CH₂Ph), 4.38 (ddd, 1 H, J_5,6b= 10.1 Hz, J_5,6a= 6.7 Hz,

J_4,5= 4.8 Hz, H-5), 4.26 (dd, 1 H, J_3,4= 3.0 Hz, H-4), 4.17 (d,

1 H, H-3), 3.85 (dd, 1 H, J_6a,6b= 10.2 Hz, H-6a), 3.80 (t, 1 H,

5-N,6-S-(N¢-Butyliminomethylidene)-6-thiogalactonojirimycin

(62). Column chromatography, eluent 60 : 10 : 1 → 40 : 10 : 1

CH₂Cl₂–MeOH–H₂O. Yield: 31 mg (86%). [a]_D-5.5 (c 0.6, H₂O).

[a]₅₄₆-7.3 (c 0.6, H₂O). R_f0.21 (40 : 10 : 1 CH₂Cl₂–MeOH–H₂O).

¹H NMR (500 MHz, D₂O) d 5.61 (d, 1 H, J_1,2= 3.2 Hz, H-1),

4.51 (t, 1 H, J_5,6= 9.0 Hz, H-5), 4.05 (bs, 1 H, H-4), 3.93 (dd, 1 H,

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9.5 Hz, H-4), 3.50 (t, 1 H, J_5,6b= 9.5 Hz, H-6b), 3.23 (t, 2 H, ³J_H,H

H-6b), 1.48, 1.32 (2 s, 6 H, CMe₂). ¹³C NMR (75.5 MHz, CD₃OD)

d 160.3 (CN), 137.5–128.4 (Ph), 113.0 (CMe₂), 106.6 (C-1), 86.9

(C-2), 82.5 (C-4), 75.5 (C-3), 55.8 (C-5), 47.3 (CH₂Ph), 46.2 (C-6),

27.2, 26.4 (CMe₂). FABMS: m/z 334 (100, [M + H - Cl]⁺). Anal.

Calcd for C₁₇H₂₄ClN₃O₄: C, 55.21; H, 6.54; N, 11.36. Found: C,

55.15; H, 6.56; N, 11.29%.

=

7.1 Hz, CH₂N), 1.57 (m, 2 H, CH₂CH₂N), 1.35 (m, 2 H, CH₂CH₃),

3

0.92 (t, 3 H, J_H,H= 7.3 Hz, CH₃). ¹³C NMR (75.5 MHz, D₂O)

d 156.6 (CN), 74.6 (C-1), 73.5 (C-4), 72.6 (C-3), 71.7 (C-2), 56.3

(C-5), 46.3 (C-6), 42.8 (CH₂N), 30.3 (CH₂CH₂N), 19.3 (CH₂CH₃),

12.9 (CH₃). FABMS: m/z 260 (100, [M + Cl]⁺). Anal. Calcd for

C₁₁H₂₄ClN₃O₅: C, 42.11; H, 7.71; N, 13.39. Found: C, 42.21; H,

7.81; N, 13.31%.

5,6-Diamino-5,6-di-N-(N¢-butyliminomethylidene)-5,6-dideoxy-

1,2-O-isopropylidene-a-D-galactofuranose hydrochloride (68).

Column chromatography, eluent 100 : 10 : 1 → 80 : 10 : 1 CH₂Cl₂–

MeOH–H₂O. Yield: 297 mg (75%). [a]_D-31.6 (c 1.0, MeOH). R_f

0.49 (40 : 10 : 1 CH₂Cl₂–MeOH–H₂O). IR (KBr) n_max3285, 2954,

6-Amino-5,6-di-N-(N¢-benzyliminomethylidene)-6-deoxynojiri-

mycin hydrochloride (71). Column chromatography, eluent

4 : 1 : 1 CH₃CN–H₂O–NH₄OH. Yield: 93 mg (65%). [a]_D+4.0

1

1673, 1379, 1068 cm^-1. H NMR (300 MHz, CD₃OD) d 5.93 (d,

1

(c 1.0, H₂O). R_f0.29 (4 : 1 : 1 CH₃CN–H₂O–NH₄OH). H NMR

1 H, J_1,2= 3.7 Hz, H-1), 4.57 (d, 1 H, H-2), 4.31 (m, 1 H, H-5),

4.07 (d, 1 H, J_3,4= 2.6 Hz, H-3), 3.89 (dd, 1 H, J_4,5= 8.9 Hz, H-4),

(400 MHz, D₂O) d 7.34 (m, 5 H, Ph), 5.40 (d, 1 H, J_1,2= 3.8 Hz,

H-1), 4.40 (s, 2 H, CH₂Ph), 3.93 (m, 1 H, H-5), 3.76 (t, 1 H, J_6a,6b

=

3.87 (t, 1 H, J_6a,6b= J_5,6a= 10.0 Hz, H-6a), 3.55 (dd, 1 H, J_5,6b

=

J_5,6a= 9.5 Hz, H-6a), 3.66 (t, 1 H, J_2,3= J_3,4= 9.5 Hz, H-3), 3.57

(dd, 1 H, H-2), 3.46 (t, 1 H, J_4,5= 9.5 Hz, H-4), 3.42 (dd, 1 H,

J_5,6b= 9.5 Hz, H-6b). ¹³C NMR (75.5 MHz, D₂O) d 156.5 (CN),

136.3-127.0 (Ph), 74.4 (C-1), 73.3 (C-4), 72.4 (C-3), 71.4 (C-2),

56.2 (C-5), 46.1 (C-6, CH₂Ph). FABMS: m/z 294 (60, [M + Cl]⁺).

Anal. Calcd for C₁₄H₂₂ClN₃O₅: C, 48.35; H, 6.38; N, 12.08. Found:

C, 48.19; H, 6.20; N, 11.92%.

5.9 Hz, H-6b), 3.24 (t, 2 H, ³J_H,H= 7.0 Hz, CH₂N), 1.60 (m, 2 H,

CH₂CH₂N), 1.53, 1.34 (2 s, 6 H, CMe₂), 1.41 (m, 2 H, CH₂CH₃),

0.98 (t, 3 H, ³J_H,H= 7.3 Hz, CH₃). ¹³C NMR (75.5 MHz, CD₃OD)

d 160.5 (CN), 114.0 (CMe₂), 107.1 (C-1), 89.9 (C-4), 88.4 (C-2),

76.3 (C-3), 58.2 (C-5), 46.1 (C-6), 43.7 (CH₂N), 32.2 (CH₂CH₂N),

27.4, 26.2 (CMe₂), 20.7 (CH₂CH₃), 13.9 (CH₃). FABMS: m/z 300

(100, [M + H - Cl]⁺). Anal. Calcd for C₁₄H₂₆ClN₃O₄: C, 50.07; H,

7.80; N, 12.51. Found: C, 50.1; H, 8.03; N, 12.30%.

6-Amino-5,6-di-N-(N¢-butyliminomethylidene)-6-deoxygalacto-

nojirimycin hydrochloride (72). Column chromatography, eluent

100 : 10 : 0.5 → 100 : 10 : 1 CH₃CN–H₂O–AcOH. Yield: 73 mg

(60%). [a]_D+8.0 (c 1.0, H₂O). R_f0.61 (20 : 2 : 1 CH₃CN–H₂O–

AcOH). ¹H NMR (500 MHz, D₂O) d 5.40 (d, 1 H, J_1,2= 4.0 Hz,

H-1), 4.25 (t, 1 H, J_5,6a= J_5,6b= 9.7 Hz, H-5), 3.98 (m, 1 H, H-4),

3.82 (dd, 1 H, J_2,3= 9.8 Hz, J_3,4= 2.5 Hz, H-3), 3.74 (dd, 1 H,

H-2), 3.69 (t, 1 H, J_6a,6b= 9.7 Hz, H-6a), 3.56 (t, 1 H, H-6b), 3.15

5,6-Diamino-5,6-di-N-(N¢-benzyliminomethylidene)-5,6-dideo-

xy-1,2-O-isopropylidene-a-D-galactofuranose hydrochloride (69).

Column chromatography, eluent 100 : 10 : 1 → 70 : 10 : 1 CH₂Cl₂–

MeOH–H₂O. Yield: 179 mg (55%). [a]_D-62.0 (c 1.0, MeOH). R_f

0.43 (40 : 10 : 1 CH₂Cl₂–MeOH–H₂O). IR (KBr) n_max3389, 2931,

1674, 1367, 1067 cm^-1. ¹H NMR (500 MHz, CD₃OD) d 7.41–7.33

(m, 5 H, Ph), 5.94 (d, 1 H, J_1,2= 3.8 Hz, H-1), 4.58 (d, 1 H, H-

3

(t, 2 H, J_H,H= 7.0 Hz, CH₂N), 1.48 (m, 2 H, CH₂CH₂N), 1.26

(m, 2 H, CH₂CH₃), 0.81 (t, 3 H, ³J_H,H= 7.3 Hz, CH₃). ¹³C NMR

(125.7 MHz, D₂O) d 155.9 (CN), 74.0 (C-1), 69.2 (C-3), 68.1 (C-4),

67.8 (C-2), 55.7 (C-5), 42.7 (CH₂N), 41.7 (C-6), 30.1 (CH₂CH₂N),

19.2 (CH₂CH₃), 12.9 (CH₃). FABMS: m/z 260 (100, [M - Cl]⁺).

HRFABMS: m/z 260.160864; calcd. for C₁₁H₂₂N₃O₄: 260.161031.

Anal. Calcd for C₁₁H₂₂ClN₃O₄: C, 44.67; H, 7.50; N, 14.21. Found:

C, 44.29; H, 7.35; N, 13.93%.

2), 4.47 (s, 2 H, CH₂Ph), 4.36 (m, 1 H, H-5), 4.09 (d, 1 H, J_3,4

2.6 Hz, H-3), 3.91 (dd, 1 H, J_4,5= 9.1 Hz, H-4), 3.90 (t, 1 H, J_6a,6b

=

J_5,6a= 9.9 Hz, H-6a), 3.58 (dd, 1 H, J_5,6b= 5.9 Hz, H-6b), 1.53,

1.34 (2 s, 6 H, CMe₂). ¹³C NMR (125.7 MHz, CD₃OD) d 160.5

(CN), 137.5–128.4 (Ph), 114.0 (CMe₂), 107.1 (C-1), 89.9 (C-4),

88.4 (C-2), 76.3 (C-3), 58.3 (C-5), 47.3 (CH₂Ph), 46.2 (C-6), 27.4,

26.2 (CMe₂). FABMS: m/z 334 (100, [M + HCl]⁺). HRFABMS:

m/z 334.176627; calcd. for C₁₇H₂₄N₃O₄: 334.176682. Anal. Calcd

for C₁₇H₂₆ClN₃O₅: C, 52.64; H, 6.76; N, 10.83. Found: C, 52.64;

H, 6.64; N, 10.71%.

6-Amino-5,6-di-N-(N¢-benzyliminomethylidene)-6-deoxygalac-

tonojirimycin hydrochloride (73). Column chromatography, elu-

ent 50 : 5 : 1 → 20 : 2 : 1 CH₃CN–H₂O–AcOH. Yield: 92 mg (68%).

[a]_D+7.0 (c 0.8, H₂O). R_f0.32 (20 : 2 : 1 CH₃CN–H₂O–AcOH).

¹H NMR (300 MHz, D₂O) d 7.39–7.27 (m, 5 H, Ph), 5.45(d, 1 H,

General procedure for the synthesis of 6-amino-6-deoxy-5,6-

di-N-(N¢-iminomethylidene)(galacto)nojirimycin derivatives 70–

73. The corresponding 2-amino-2-imidazoline precursor 66–

69 (0.41 _◦mmol) was treated with TFA-H₂O (9 : 1, 1.7 mL) for

1 h at 0 C, concentrated under reduced pressure, coevaporated

several times with water, treated with NaOH 0.1 N until pH 8, and

subjected to column chromatography with the eluent indicated

in each case. The corresponding guanidines were isolated as

hydrochloride salt, puriﬁed by GPC (Sephadex G-10, 1 : 1 MeOH–

H₂O), concentrated, and freeze-dried.

J_1,2= 3.8 Hz, H-1), 4.41 (s, 2 H, CH₂Ph), 4.28 (td, 1 H, J_5,6a= J_5,6b

=

9.8 Hz, J_4,5= 1.4 Hz, H-8a), 3.99 (dd, 1 H, J_3,4= 2.7 Hz, H-4),

3.84(dd, 1 H, J_2,3= 10.2 Hz, H-3), 3.77 (dd, 1 H, H-2), 3.68 (t, 1 H,

J_6a,6b= 9.8 Hz, H-6a), 3.55 (t, 1 H, H-6b). ¹³C NMR (75.5 MHz,

D₂O) d 156.7 (CN), 136.1–127.6 (Ph), 74.8 (C-1), 69.7 (C-3), 68.7

(C-4), 68.4 (C-2), 56.4 (C-5), 46.4 (CH₂Ph), 42.5 (C-6). FABMS:

m/z 294 (100, [M - Cl]⁺). HRFABMS: m/z 294.144663; calcd. for

C₁₄H₂₀N₃O₄: 294.145381. Anal. Calcd for C₁₄H₂₂ClN₃O₅: C, 48.35;

H, 6.38; N, 12.08. Found: C, 48.02; H, 6.16; N, 11.76%.

6-Amino-5,6-di-N -(N¢-butyliminomethylidene)-6-deoxynojiri-

mycin hydrochloride (70). Column chromatography, eluent

4 : 1 : 1 CH₃CN–H₂O–NH₄OH. Yield: 103 mg (80%). [a]_D+7.7

Acknowledgements

1

(c 0.8, H₂O). R_f0.53 (6 : 3 : 1 CH₃CN–H₂O–AcOH). H NMR

(300 MHz, D₂O) d 5.42 (d, 1 H, J_1,2= 3.7 Hz, H-1), 3.95 (m, 1

H, H-5), 3.86 (t, 1 H, J_6a,6b= J_5,6a= 9.5 Hz, H-6a), 3.72 (t, 1 H,

The Spanish Ministerio de Ciencia e Innovacio´n (contract num-

bers CTQ2006-15515-CO2-01, CTQ2009-14551-C02-01, CTQ-

2010-15848, CTQ2008-01426/BQU and SAF2010-15670;

J_2,3= J_3,8= 9.5 Hz, H-3), 3.60 (dd, 1 H, H-2), 3.52 (t, 1 H, J_4,5

=

3712 | Org. Biomol. Chem., 2011, 9, 3698–3713

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coﬁnanced with the Fondo Europeo de Desarrollo Regional

FEDER), the Fundacio´n Ramo´n Areces, and the Junta

de Andaluc´ıa (P08-FQM-03711) are thanked for funding.

Imiglucerase was generously supplied by Genzyme Corporation.

12 B. Brumshtein, M. Aguilar-Moncayo, M. I. Garc´ıa-Moreno, C. Ortiz

Mellet, J. M. Garc´ıa Ferna´ndez, I. Silman, Y. Shaaltiel, D. Aviezer, J. L.

Sussman and A. H. Futerman, ChemBioChem, 2009, 10, 1480.

13 J. M. Garc´ıa Ferna´ndez and C. Ortiz Mellet, Adv. Carbohydr. Chem.

Biochem., 1999, 55, 35.

14 K. Dax, B. Gaigg, V. Grassberger, B. Kolbringer and A. E. Stu¨tz,

J. Carbohydr. Chem., 1990, 9, 479.

15 P. D´ıaz Pe´rez, M. I. Garc´ıa-Moreno, C. Ortiz Mellet and J. M. Garc´ıa

Ferna´dez, Eur. J. Org. Chem., 2005, 2903.

16 M. J. Camarasa, P. Ferna´ndez-Resa, M. T. Garc´ıa-Lo´pez, F. G. de

las Heras, P. P. Me´ndez-Castrillo´n and A. San Fe´lix, Synthesis, 1984,

509.

17 J. M. Benito, M. Go´mez-Garc´ıa, C. Ortiz Mellet, I. Baussanne, J. Defaye

and J. M. Garc´ıa Ferna´ndez, J. Am. Chem. Soc., 2004, 126, 10355.

18 C. Dardonville, C. Ferna´ndez-Ferna´ndez, S-L. Gibbons, G. J. Ryan, N.

Jagerovic, A. M. Gabilondo, J. L. Fauche`re, F. Lamaty, A. Molla and

R. Lazaro, Eur. J. Org. Chem., 1998, 853.

19 S. S. Iyer, A. S. Anderson, S. Reed, B. Swanson and J. G. Schmidt,

Tetrahedron Lett., 2004, 45, 4285.

20 M. I. Garc´ıa-Moreno, P. D´ıaz-Pe´rez, J. M. Benito, C. Ortiz Mellet,

J. Defaye and J. M. Garc´ıa Ferna´ndez, Carbohydr. Res., 2002, 337,

2329.

21 M. I. Garc´ıa-Moreno, J. M. Benito, C. Ortiz Mellet and J. M. Garc´ıa

Ferna´ndez, Tetrahedron: Asymmetry, 2000, 11, 1331.

22 J. L. Chiara, I. Storch de Gracia, A. Garc´ıa, A. Bastida, S. Bobo and

M. D. Mart´ın-Ortega, ChemBioChem, 2005, 6, 186.

23 M. Avalos Gonza´lez, R. Babiano Caballero, P. Cintas Moreno, J.

Fuentes Mota, J. L. Jime´nez Requejo and J. C. Palacios Albarra´n,

Heterocycles, 1989, 29, 1; M. Avalos Gonza´lez, R. Babiano Caballero,

P. Cintas Moreno, J. L. Jime´nez Requejo and J. C. Palacios Albarra´n,

Carbohydr. Res., 1990, 198, 247.

Notes and references

1 B. G. Winchester, Tetrahedron: Asymmetry, 2009, 20, 645; Iminosugars:

From Synthesis to Therapeutic Applications, ed. P. Compain and

O. R. Martin, Wiley-VCH, Weinheim, Germany, 2007; Iminosugars as

Glycosidase Inhibitors: Nojirimycin and Beyond, ed. A. E. Stu¨tz, Wiley-

VCH, Weinheim, Germany, 1999; T. D. Heightman and A. T. Vasella,

Angew. Chem., Int. Ed., 1999, 38, 750; M. Bols, Acc. Chem. Res., 1998,

31, 1; G. Legler, Adv. Carbohydr. Chem. Biochem., 1990, 48, 319.

2 M. C. Deshpande, V. Venkateswarlu, R. K. Babu and R. K. Trivedi,

Int. J. Pharm., 2009, 380, 16.

3 D. Durantel, C. Alotte and F. Zoulim, Curr. Opin. Investig. Drugs, 2007,

8, 125; P. Greimel, J. Spreitz, A. E. Stu¨tz and T. M. Wrodnigg, Curr.

Top. Med. Chem., 2003, 3, 513.

4 T. M. Wrodnigg, A. J. Steiner and B. J. Ueberbacher, Anti-Cancer

Agents Med. Chem., 2008, 8, 77; Y. Nishimura, in Iminosugars: From

Synthesis to Therapeutic Applications, ed. P. Compain and O. R. Martin,

Wiley-VCH, Weinheim, Germany, 2007, p 269.

5 J. Q. Fan, Biol. Chem., 2008, 389, 1; T. D. Butters, Expert Opin.

Pharmacother., 2007, 8, 427; Z. Yu, A. R. Sawkar, L. J. Whalen, C.-H.

Wong and J. W. Kelly, J. Med. Chem., 2007, 50, 94; J. Matsuda, O.

Suzuki, A. Oshima, Y. Yamamoto, A. Noguchi, K. Takimoto, M. Itoh,

Y. Matsuzaki, Y. Yasuda, S. Ogawa, Y. Sakata, E. Nanba, K. Higaki, Y.

Ogawa, L. Tominaga, K. Ohno, H. Iwasaki, H. Watanabe, R. O. Brady

and Y. Suzuki, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 15912; T. D.

Butters, H. R. Mellor, K. Narita, R. A. Dwek and F. M. Platt, Philos.

Trans. R. Soc. London, Ser. B, 2003, 358, 927.

6 J. L. Jime´nez Blanco, V. M. D´ıaz Pe´rez, C. Ortiz Mellet, J. Fuentes,

J. M. Garc´ıa Ferna´ndez, J. C. D´ıaz Arribas and F. J. Can˜ada, Chem.

Commun., 1997, 1969; M. I. Garc´ıa-Moreno, J. M. Benito, C. Ortiz

Mellet and J. M. Garc´ıa Ferna´dez, J. Org. Chem., 2001, 66, 7604; P.

D´ıaz Pe´rez, M. I. Garc´ıa-Moreno, C. Ortiz Mellet and J. M. Garc´ıa

Ferna´dez, Synlett, 2003, 3, 341; M. I. Garc´ıa-Moreno, D. Rodr´ıguez-

Lucena, C. Ortiz Mellet and J. M. Garc´ıa Ferna´dez, J. Org. Chem.,

2004, 69, 3578.

7 E. M. Sa´nchez-Ferna´ndez, R. R´ısquez-Cuadro, M. Aguilar-Moncayo,

M. I. Garc´ıa-Moreno, C. Ortiz Mellet and J. M. Garc´ıa Ferna´ndez, Org.

Lett., 2009, 11, 3306; E. M. Sa´nchez-Ferna´ndez, R. R´ısquez-Cuadro,

M. Chasseraud, A. Ahidouch, C. Ortiz Mellet, H. Ouadid-Ahidouch

and J. M. Garc´ıa Ferna´ndez, Chem. Commun., 2010, 46, 5328.

8 V. M. D´ıaz Pe´rez, M. I. Garc´ıa-Moreno, C. Ortiz Mellet, J. Fuentes,

J. C. D´ıaz Arribas, F. J. Can˜ada and J. M. Garc´ıa Ferna´dez, J. Org.

Chem., 2000, 65, 136.

9 M. I. Garc´ıa Moreno, P. D´ıaz-Pe´rez, C. Ortiz Mellet and J. M. Garc´ıa

Ferna´ndez, Chem. Commun., 2002, 848; M. I. Garc´ıa Moreno, P. D´ıaz-

Pe´rez, C. Ortiz Mellet and J. M. Garc´ıa Ferna´ndez, J. Org. Chem., 2003,

68, 8890.

24 M. Aguilar, P. D´ıaz-Pe´rez, M. I. Garc´ıa-Moreno, C. Ortiz Mellet and

J. M. Garc´ıa Ferna´ndez, J. Org. Chem., 2008, 73, 1995.

25 M. Puri and U. C. Banerjee, Biotechnol. Adv., 2000, 18, 207; F. Michon,

V. Pozsgay, J.-R. Brisson and H. J. Jennigs, Carbohydr. Res., 1989, 194,

321.

26 Y. Kobayashi, Carbohydr. Res., 1999, 315, 3; R. P. Gibson, T. M. Gloster,

S. Roberts, R. A. J. Warren, I. Storch de Gracia, A. Garcia, J. L. Chiara

and G. J. Davies, Angew. Chem., Int. Ed., 2007, 46, 4115.

27 I. Lundt, A. J. Steiner, A. E. Stu¨tz, C. A. Tarling, S. Ully, S. Withers

and T. M. Wrodnigg, Bioorg. Med. Chem., 2006, 14, 1737.

28 C. W. Ekhart, M. H. Fechter, P. Hadwiger, E. Mlaker, A. E. Stu¨tz, A.

Tauss and T. M. Wrodnigg, in Iminosugars as Glycosidase Inhibitors:

Nojirimycin and Beyond, ed. A. E. Stu¨tz, Wiley-VCH, Weinheim,

Germany, 1999, p 253; P. Compain, V. Desvergnes, V. Liautard, C.

Pillard and S. Toumieux, in Iminosugars: From Synthesis to Therapeutic

Applications, ed. P. Compain and O. R. Martin, Wiley-VCH, Weinheim,

Germany, 2007, p 327; G. Legler and S. Pohl, Carbohydr. Res., 1986,

155, 119.

29 S. Ogawa, M. Kanto and Y. Suzuki, Mini-Rev. Med. Chem., 2007, 7,

679; T. D. Butters, R. A. Dwek and F. M. Platt, Glycobiology, 2005,

15, 43R; M. Egido-Gaba´s, D. Canals, J. Casas, A. Llebaria and A.

Delgado, ChemMedChem, 2007, 2, 992.

30 The recovery of recombinant GCase activity after thermal denaturation

is currently used as an in vitro model assay for potential pharmacolog-

ical chaperones (see ref. 31).

10 Z. Luan, K. Higaki, M. Aguilar-Moncayo, H. Ninomiya, K. Ohno,

M. I. Garc´ıa-Moreno, C. Ortiz Mellet, J. M. Garc´ıa Ferna´ndez and Y.

Suzuki, ChemBioChem, 2009, 10, 2780.

11 M. Aguilar-Moncayo, T. M. Gloster, J. P. Turkenburg, M. I. Garc´ıa-

Moreno, C. Ortiz Mellet, G. J. Davies and J. M. Garc´ıa Ferna´ndez, Org.

Biomol. Chem., 2009, 7, 2738.

31 A. R. Sawkar, W. C. Cheng, E. Beutler, C. H. Wong, W. E. Balch and

J. W. Kelly, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 15428.

32 L. D´ıaz, J. Bujons, J. Casas, A. Llebaria and A. Delgado, J. Med. Chem.,

2010, 53, 5248.

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Article Doi

Bicyclic (galacto)nojirimycin analogues as glycosidase inhibitors: Effect of structural modifications in their pharmacological chaperone potential towards β-glucocerebrosidase

DOI: 10.1039/c1ob05234a

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Full text of DOI:10.1039/c1ob05234a

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