Low molecular weight antagonists of the myelin-associated glycoprotein: Synthesis, docking, and biological evaluation

J. Med. Chem. 2010, 53, 1597–1615 1597

DOI: 10.1021/jm901517k

Low Molecular Weight Antagonists of the Myelin-Associated Glycoprotein: Synthesis, Docking,

and Biological Evaluation

Stefanie Mesch,^†Delia Moser,^†Daniel S. Strasser,^†Antje Kelm,^‡Brian Cutting,^†Gianluca Rossato,^†Angelo Vedani,^†

Hendrik Koliwer-Brandl,^‡Matthias Wittwer,^†Said Rabbani,^†Oliver Schwardt,^†Soerge Kelm,^‡and Beat Ernst*^,†

^†Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland and ^‡Department of

Physiological Biochemistry, University of Bremen, D-28334 Bremen, Germany

Received August 10, 2009

The injured adult mammalian central nervous system is an inhibitory environment for axon regenera-

tion due to specific inhibitors, among them the myelin-associated glycoprotein (MAG), a member of the

Siglec family (sialic-acid binding immunoglobulin-like lectin). In earlier studies, we identified the lead

structure 5, which shows a 250-fold improved in vitro affinity for MAG compared to the tetrasaccharide

binding epitope of GQ1bR (1), the best physiological MAG ligand described so far. By modifying the

2- and 5-position, the affinity of 5 could be further improved to the nanomolar range (f19a). Docking

studies to a homology model of MAG allowed the rationalization of the experimental binding

properties. Finally, pharmacokinetic parameters (stability in the cerebrospinal fluid, logD and

permeation through the BBB) indicate the drug-like properties of the high-affinity antagonist 19a.

Introduction

To reduce the structural complexity of tetrasaccharide 1 and, at

the same time, improve its pharmacodynamic and pharmaco-

kinetic properties, numerous MAG antagonists have been

prepared. Because the affinity of a series of gangliosides

indicated that not only the terminal Neu5AcR(2-3)Gal struc-

ture is essential for MAG binding but also the internal sialic

acids, the corresponding sialylated and sulfated analogues were

synthesized.^21,22Furthermore, structural information obtained

by trNOE NMR²³and STD NMR²⁴suggested that the

Galβ(1-3)GalNAc core contributes to binding mainly by

hydrophobic contacts. This was confirmed by the successful

replacement of this disaccharide substructure by noncarbo-

hydrate linkers.²⁵In addition, the R(2-6)-linked sialic acid

could also be replaced by lipophilic substituents.²²Finally, a

pivotal simplification was reported by Kelm and Brossmer

when they found that sialic acid derivatives modified in the

2- (e.g., 2),^26,275- (e.g., 3),^26,27or 9-position^26,28(e.g., 4) exhi-

bited enhanced antagonistic activity.²⁶Combining the best

modifications found for the 2²⁹- and 9-position³⁰in one mole-

cule led to antagonists, e.g., 5, with affinities in the low

micromolar range.³⁰

In this communication, we report a small library of MAG

antagonists based on lead compound 5. The binding proper-

ties, evaluated by a hapten binding assay, surface plasmon

resonance (SPR), and competitive NMR experiments were

rationalized by docking studies to a homology model of

MAG. Finally, according to pharmacokinetic parameters,

e.g., stability in the cerebrospinal fluid, the drug-likeness of

the identified high-affinity antagonists could be demon-

strated.

The injured adult mammalian central nervous system (CNS)

lacks the ability for axon regeneration,^1,2predominantly due to

specific inhibitors expressed on residual myelin and on astro-

cytes recruited to the site of injury.^3-7Several inhibitor

proteins have been identified, one of them being the myelin-

associated glycoprotein (MAG).⁸MAG is a transmembrane

glycoprotein⁹belonging to the family of the sialic acid-binding

immunoglobulin like lectins, the so-called Siglecs.^10,11On the

surface of neurons, MAG interacts with two classes of targets:

proteins of the family of Nogo receptors (NgR^a)^12,13and the

gangliosides GD1a and GT1b.^11,14-16Although the relative

roles of gangliosides and NgRs as MAG ligands have yet to be

resolved,^8,17in some systems, MAG inhibition is completely

reversed by sialidase treatment, suggesting that MAG uses

sialylated glycans as its major axonal ligands.¹⁸Therefore,

blocking MAG with potent antagonists may be a valuable

therapeutic approach to enhance axon regeneration.

So far, the native carbohydrate ligand with the highest

affinity to MAG is the ganglioside GQ1bR (Figure 1).¹⁹

Recently, MAG affinity of a partial structure of GQ1bR, the

tetrasaccharide 1, could clearly be correlated with its ability

to reverse MAG-mediated inhibition of axonal outgrowth.²⁰

*To whom correspondence should be addressed. Phone: 0041 267 15

51. Fax: 0041 267 15 52. E-mail: beat.ernst@unibas.ch.

^aBBB, blood-brain barrier; CHO, Chinese hamster ovary; ClAc, 2-

chloroacetyl; DCE, 1,2-dichloroethane; DCM, dichloromethane; DMAP,

4-dimethylamino-pyridine; DMF, N,N-dimethylformamide; FAc, 2-fluoro-

acetyl; Gal, galactose; GalNAc, N-acetylgalactosamine; IgG, immuno-

globulin G; K_D, dissociation constant; MS, molecular sieve; Neu5Ac,

N-acetylneuraminic acid; NgR, Nogo receptor; NIS, N-iodosuccini-

mide; NMR, nuclear magnetic resonance; nosyl, 2-nitrobenzylsulfonyl;

PAMPA, parallel artificial membrane permeation assay; PDB, Protein

Data Bank; iPrOH, 2-propanol; RP, reversed phase; STD NMR,

saturation transfer difference NMR; TfOH, trifluoroacetic acid; THF,

tetrahydrofurane; TMS, trimethylsilyl; trNOE, transfer nuclear Over-

hauser enhancement; p-Ts, p-tolylsulfonyl.

Results and Discussion

The sialic acid derivatives reported by Kelm and Brossmer²⁶

and our group^29,30exhibit MAG affinities in the low μM

range. An example is the neuraminic acid (Neu5Ac) derivative

r

2010 American Chemical Society

Published on Web 01/22/2010

pubs.acs.org/jmc

1598 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4

Mesch et al.

Figure 1. MAG antagonists; GQ1bR, the partial structure 1,^19,20and sialic acid derivatives 2-5.^26,30

5³⁰with a K_Dof 17 μM. Because a broad optimization

effort for the 9-position had led to the identification of

p-chlorobenzamide as the best substitutent,³⁰this position

was not further investigated. The changeover from a methyl

substituent in the 2-position to a benzyl group was rewarded

by a 10-fold gain in affinity.²⁶This effect can be rationalized

by the results of STD-NMR investigations,²³indicating a

hydrophobic interaction of the R-face of ^D-galactose (see 1 in

Figure 1) with MAG. When the hydrophobic contact was

further extended by replacing the benzyl group with phenoxy-

benzyl or biphenyl substituents, only marginally improved

affinities were detected.²⁹Because the sheer enlargement of

the hydrophobic group did not exhibit improved affinities, the

electron density of the aglycone was altered in a next step.

Finally, with the substituents in the 2- and 9-position set, a

further optimization of the acyl group in the 5-position was

conducted.^26,27

Synthesis of Sialosides 19a-g. As reported earlier, halo-

genation of the N-acetyl group at the 5-position increases the

binding affinity toward MAG by a factor of 10-20.²⁶There-

fore, both the N-fluoroacetyl and N-chloroacetyl derivatives

19a and 19b were prepared. As sulfonamides adapt a differ-

ent geometry³⁶compared to the corresponding amides, 19c

and 19d allow a further exploration of the binding site.

Finally, the cyclopropyl and cyclobutyl derivatives 19f and

19g were synthesized in order to explore the possibility for

extended hydrophobic contacts.

For the cleavage of the N-acetate group, 10 was treated

with hydrazine in the presence of (Boc)₂O^37,38and subse-

quently reacetylated (f14). Deprotection with TMSCl and

PhOH³⁹(f15), followed by acylation with carboxylic or

sulfonic acid derivatives, yielded 16a-d. Glycosylation using

2,3-difluorobenzyl alcohol (f17a-d), amidation using mod-

ified Staudinger conditions³⁵(f18a-d) and final deprotec-

tion gave the test compounds 19a-d. When methanolic

NaOH was used in the final deprotection step of 18b, a

nucleophilic substitution occurred, leading to a 1:1 mixture

of the desired 19b and the methoxyacetamide derivative 19e

(Scheme 2).

For the synthesis of the two remaining test compounds 19f

and 19g, an analogue approach starting from the thiosialo-

side 10 was accomplished. However, in contrast to the

synthesis of 19a-e, a different sequence of the modifications

conducted at the 2-, 5-, and 9-position was performed

(Scheme 3). Cleavage of the N-acetate (f14) and amidation

under modified Staudinger conditions yielded compound 20.

Next, N-deprotection followed by N-acylation (f22f and

22g) and benzylation (f23f and 23g) yielded, after final

Synthesis of Sialosides 13a-f. Starting from Neu5Ac,

thioglycoside 6 was synthesized according to a reported

procedure.³¹After deprotection by Zemplen conditions

ꢀ

(f7), the hydroxy group in the 9-position was selectively

tosylated (f8).³²Substitution of the tosylate using sodium

azide and 15-crown-5 in DMF (f9)³³followed by acetyla-

tion yielded the sialyl donor 10. For the introduction of the

aglycone, 10 was then reacted with various benzyl alcohols

(see Table 1, entries 2-9) in the presence of the promotors

NIS/TfOH.³⁴The sialosides 11a-f were obtained as separ-

able anomeric mixtures (R/β = 7/1 to 9/1). Amidation with

p-chlorobenzoylchloride under modified Staudinger condi-

tions³⁵(f12a-f) and final deprotection gave the amides

13a-f in good yields (Scheme 1, Table 1).

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1599

Scheme 1^a

^a(a) NaOMe, MeOH (61%); (b) p-TsCl, pyridine (66%); (c) NaN₃, 15-crown-5, DMF (78%); (d) Ac₂O, DMAP, pyridine (73%); (e) R¹OH, NIS,

TfOH, MeCN (11a-f, R-isomers: 54-76%; β-isomers: 8-11%); (f) PPh₃, p-chlorobenzoylchloride, DCE, rt (12a-f, 45-60%); (g) 10% aq NaOH;

Dowex 50 ꢀ 8, Na^þform (13a-f, 39-70%).

Scheme 2^a

^a(a) (i) Boc₂O, DMAP, THF, 60 °C, 4 h, (ii) N₂H₄H₂O, MeOH, rt, 24 h, (iii) Ac₂O, pyridine (76%); (b) 4 M PhOH, 4 M TMSCl in abs DCM (70%);³⁹

3

(c) acylation agent, NEt₃, DMAP, abs DCM, rt, 4 h or [ClCH₂C(dO)]₂O, NEt₃, dioxane/H₂O, rt (16a-d, 66-85%); (d) 2,3-difluorobenzyl alcohol, NIS,

TfOH, MeCN (17a-d, R-isomers: 56-68%; β-isomers: 8-11%); (e) PPh₃, p-chlorobenzoylchloride, DCE, rt (18a-d, 48-58%); (f) 10% aq LiOH,

THF/H₂O; Dowex 50 ꢀ 8, Na^þform (19a-d, 30-60%); (g) 18b, 10% aq NaOH; Dowex 50 ꢀ 8, Na^þform (19b, 21%, 19e, 19%).

deprotection, the desired cycloalkylacetic acid derivatives

19f and 19g in excellent overall yields.

were performed for each compound tested with seven or

eight concentrations in triplicates. The affinities were mea-

sured relative to the reference compound 5 (rIC₅₀of 1,

Table 1, entry 2). For the K_Ddetermination in the Biacore

assay, MAG_d1-3-Fc was immobilized on a dextran chip

containing a surface of covalently bound protein A. A

reference cell providing only protein A was used to compen-

sate unspecific binding to the matrix.

By analyzing the affinities of 13a-f, a substantial increase

in affinity was achieved when the aromatic aglycone was

halogenated in ortho- and meta-positions (entries 8 and 9).

Less effective were halogens in the para-position (entries 4

and 5). In addition, with fluorine instead of chlorine, con-

sistently slightly higher affinities (entries 4 and 8 vs entries

5 and 9) were obtained. With the symmetric pentafluoro

benzyl group in 13c (entry 6), an increase in affinity

was expected, caused by an improved preorganization of

the aglycone in the bioactive conformation. The observed

Biological Evaluation. For the evaluation of the binding

properties of the sialosides 13a-f and 19a-g, two previously

reported assay formats were applied: a fluorescent hapten

binding assay⁴⁰and a SPR based biosensor (Biacore) experi-

ment²²(Table 1). For the hapten inhibition assay, a recom-

binant protein consisting of the three N-terminal domains of

MAG and the Fc part of human IgG (MAG_d1-3-Fc) was

produced by expression in CHO cells and affinity purifica-

tion on protein A-agarose.⁴⁰The relative inhibitory concen-

trations (rIC₅₀) of the test compounds as competitive ligands

were determined in microtiter plates coated with fetuin as

the binding target for MAG_d1-3-Fc. By complexing the Fc-

part with alkaline phosphatase-labeled anti-Fc antibodies

and measuring the initial velocity of fluorescein release from

fluorescein diphosphate, the amount of bound MAG_d1-3-Fc

could be determined. At least three independent titrations

1600 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4

Mesch et al.

Scheme 3^a

^a(a) (i) Boc₂O, DMAP, THF, 60 °C, 4 h, (ii) N₂H₄H₂O, MeOH, rt, 24 h, (iii) Ac₂O, pyridine (76%); (b) PPh₃, p-chlorobenzoylchloride, DCE,

3

rt (48%); (c) 4 M PhOH, 4 M TMSCl in abs DCM (63%);³⁹(d) R²COCl, NEt₃, DMAP, abs DCM, rt, 4 h (22f, 75%, 22g, 39%); (e) 2,3-difluoro-

benzyl alcohol, NIS, TfOH, MeCN (23f, R: 73%, β: 8%; 23g, R: 72%, β: 8%); (f) 10% aq LiOH, THF/H₂O; Dowex 50 ꢀ 8, Na^þform (19f, 44%;

19g, 50%).

diminished affinity, i.e., a loss of a factor 3 compared to 13f,

may be the result of steric hindrance. Finally, when a

2-naphthylmethyl substituent was introduced (13d), a slight

improvement of affinity compared to the benzyl substituent

as present in 5 was obtained, presumably due to a favorable

π-π interaction of the extended aromatic system. In a

next step, substituent R²was optimized based on the so

far most active antagonist 13f. Kelm and Brossmer have

demonstrated that halogenated acetamides in the 5-position

of sialic acid derivatives strongly improve binding of MAG

antagonists.²⁶When this observation was applied to our

series, the nanomolar fluoroacetamide 19a (entry 10) was

obtained. For chloroacetamide derivative 19b (entry 11), the

effect was less pronounced. Interestingly, an equally potent

antagonist was achieved with the phenylsulfone substituent

(19c, entry 12), while sulfone 19d (entry 13) suffers from a

drastic loss in activity. This is quite unexpected because an

increase in the size of the acyl substituent was reported to

lead to a reduction in affinity,²⁷an observation that was

confirmed by compounds 19e-g (entries 14-16).

Stability in Cerebrospinal Fluid. For nerve regeneration,

MAG antagonists will most likely be applied to the CNS by

a local infusion. We therefore tested the stability of the

fluoroacetate 19a and the corresponding acetate 13f in

artificial cerebrospinal fluid (aCSF)⁴¹and, as a control, in

buffer solution for 19 h at 37 °C. According to LC-MS

analysis, more than 95% of the initial concentrations of

both antagonists were recovered from both media, predict-

ing a high stability in the CNS, the target compartment of an

in vivo application. Furthermore, logD_{octanol/water}values

from -0.27 to 0.87 (see Table 1) might be beneficial for an

intrathecal application because these distribution coeffi-

cients suggest a loss from the CNS compartment by a passive

transport mechanism to be unlikely. This hypothesis is

further supported by the results of the BBB-PAMPA assay

showing log P_evalues for 13f and 19a in the range of -10.

For values below -5.7,⁴²no passive permeation through the

BBB is expected.

immobilized on protein A, which on its part was covalently

linked to a carboxymethyl dextrane surface of the chip.

Whereas earlier Biacore investigations with MAG anta-

gonists produced traditional sensorgrams,²²consistently

negative sensorgrams, i.e., a net decrease in resonance

units, were obtained with the compound series reported

in Table 1. As an example, the sensorgram of 13f is shown

in Figure 2a. When fitted to a binding isotherm, these

negative sensorgrams appear to clearly result from specific

receptor-ligand interactions. To elucidate the origin of

this unusual result, numerous factors such as the buffer

capacity, the ion strength of the buffer, type, and matrix of

the sensor chip as well as the applied type of immobiliza-

tion of MAG_d1-3-Fc were analyzed.

As a result of ionizable functional groups of the analytes,

pH variabilities could occur, a phenomenon previously

reported by Mannen et al.⁴⁶To avoid this effect, a sufficient

buffer capacity, 50 mM instead of 10 mM HEPES, was

applied. Furthermore, to exclude ionic repulsion effects,

measurements were carried out at increased salt concentra-

tions (150-500 mM NaCl). In addition, the effect of poten-

tial nonspecific binding to the dextran matrix or to protein A

was analyzed by adding either dextran (1 mg/mL) to the

buffer system or by conducting the experiment on a regen-

erated protein A surface. Because all these modifications of

assay parameters did not provide an indication for the cause

of the negative sensorgrams, different dextran biosensor

chips, varying in carboxylate density (CM5 vs CM4), were

analyzed as well. Although the reduction in signal intensity

correlated well with the degree of functionalization of the

chip surface, no influence on the sign of the sensorgrams

could be detected (see Table S1 in Supporting Information).

A further explanation for the negative sensorgrams could

be a ligand-induced conformational change of the immobi-

lized receptor leading to a decrease of its hydrodynamic

radius and, as a consequence, yielding a negative refraction

index.^47,48Because the negative refraction index correlates

with the analyte concentration, we mirrored the negative

sensorgrams, for an example see the sensorgram of 13f in

Figure 2b, to obtain SPR-determined equilibrium dissocia-

tion constants (K_Ds, see Table 1).

Surface Plasmon Resonance (SPR). The interaction bet-

ween MAG and MAG antagonists was analyzed by SPR

experiments.^43-45For this purpose, MAG_d1-3-Fc was

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1601

Table 1. Relative Inhibitory Concentrations (rIC₅₀) Relative to Reference Compound 5, K_DValues, and logD_7.3of Sialosides 13a-f and 19a-g

^arIC₅₀is the concentration when 50% of the protein are inhibited, measured relative to reference compound 5. ^bThe affinity of compound 4 was

measured using different protein batches, resulting in K_Ds of 137 μM and 105 μM (Table 2). All affinity data given in Table 1 were obtained with the

protein batch showing a K_Dof 137 μM for compound 4; ^cn.d. not determined

To justify this procedure, we analyzed whether K_Ds ob-

tained by mirroring the negative sensorgrams can be corre-

lated with rIC₅₀values determined by the fluorescent hapten

binding assay⁴⁰(Figure 3). K_Ds from previously reported

compounds^25,30(see Table S2, Supporting Information),

which exhibit much higher molecular weights and therefore

1602 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4

Mesch et al.

Figure 2. (a) Biacore sensorgrams for 13f after subtraction of the reference; (b) mirrored sensorgrams of 13f.

between selective inversion recovery of the para-hydrogen

of the benzamide substituent in the presence or absence of

MAG_d1-3-Fc (Figure 4a). For a quantitative evaluation of

the relative affinities of 13f and 25, a titration curve describ-

ing the concentration dependence of the selective inversion

recovery time of 4 was required (Figure 4b). The selective

inversion time constants (sT1) were fit to a one-site binding

model.

On the basis of the titration curve shown in Figure 4b, the

determination of the relative affinities of 13f and 25 became

possible. With a NMR sample consisting of MAG_d1-3-Fc

and compound 4 (500 μM), sT1 for the initial point of

the titration curve was remeasured. The obtained sT1 of

1.03 ( 0.08 s compared to 1.05 ( 0.06 s for the first sample

indicated a high degree of reproducibility. In a second step,

25 μM of either compound 13f or 25 were added. Then, the

sT1s were measured and the apparent concentration of

compound 4 determined. When 25 μM of compound 13f

were added, the sT1 increased to 1.84 ( 0.06 s, indicating

an apparent concentration of compound 4 of 1.36 mM.

Subtracting the actual concentration of compound 4 from

its apparent concentration and dividing the result by the

concentration of the inhibitor yields a relative affinity of

34.4 ( 2.1 for compound 13f with respect to the reference

compound 4 with the relative affinity of 1. With the identical

procedure, a relative affinity of 17.6 ( 1.0 for compound 25

with respect to compound 4 was found (Figure 5).

The competitive NMR assay demonstrates that com-

pounds 13f and 25 both bind to MAG_d1-3-Fc with an

affinity more than 1 order of magnitude greater than refer-

ence compound 4. The relative affinities displayed in Figure 5

are in good agreement with the corresponding values deter-

mined by Biacore and the hapten inhibition assay.⁴⁰Allow-

ing a factor of 2 in the estimation of a compound’s affinity

with a single technology implies that the comparison of

affinities between two assay formats may differ by as much

as a factor of 4. Because for any of the three assays employed

the affinities of 13f and 25 to MAG_d1-3-Fc are within this

range, the mirroring of the negative sensorgrams for the

determination of K_Dvalues is further justified.

Figure 3. Correlation of K_Dvalues obtained by SPR measurements

with rIC₅₀values determined in the competitive inhibition assay;⁴⁰

Compounds 5, 13a-f, 19a-g, and the compounds 25-29 from

Table S2 (Supporting Information) were included.

generate positive sensorgrams, were also included in this

correlation. The obtained correlation factor R²of 0.93

clearly suggests that the mirroring procedure does not falsify

the binding information.

Determination of Relative Affinities by NMR. A further

argument for the acceptance of the above-described mirror-

ing of the sensorgrams was accomplished by competitive

NMR experiments.⁴⁹The approach is based on the mole-

cular weight dependence of selective inversion recovery

experiments (sT1). In the absence of binding, a selectively

inverted NMR signal of a ligand requires a relatively long

time to recover back to equilibrium. By contrast, if the ligand

binds to a receptor, the time required to recover back to

equilibrium is reduced. As a result, the binding of a ligand to

a receptor is detectable through sT1 experiments. Further-

more, these sT1 experiments can be used for the ranking of

the affinities of ligands relative to a reference compound.

For our purpose, the binding of antagonist 4 to

MAG_d1-3-Fc was used as reference (K_Ddetermined by

Biacore is 137 μM), whereas compounds 13f and 25²²(for

the structure, see Supporting Information) were chosen as

competitors because they fulfill two criteria. First, SPR

experiments with both 13f and 25 gave comparable K_D

values, 2.4 and 2.8 μM, respectively, and therefore require

comparable concentrations for the observation of competi-

tive binding. Second, the observed sensorgrams of com-

pounds 13f and 25 are of opposite sign, negative and

positive, respectively.

Determination of Enthalpic and Entropic Contributions to

Binding. For the elucidation of the thermodynamic para-

meters of the MAG/antagonist interaction, K_Ds were mea-

sured in the Biacore assay at different temperatures. The

analysis of a library of ligands containing structural modi-

fications at the 2-, 5-, and 9-position of the neuraminic acid

scaffold should allow the assignment of the enthalpic and

entropic contributions to the various structural elements.

The K_Dvalues were determined at six different tempera-

tures, starting at 5 °C and elevating the temperature by 5 °C

In a first NMR experiment, it was demonstrated that 4

binds to MAG_d1-3-Fc according to the large differences

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1603

Figure 4. (a) Selective inversion recovery (sT1) of the para-hydrogen of the benzamide in compound 4, either in the presence of MAG_d1-3-Fc

(filled triangles) or in the absence of MAG_d1-3-Fc (filled squares). The normalized NMR signal represents the intensity of the para-hydrogen at

a particular time divided by its intensity after 60 s of relaxation. (b) Titration of MAG_d1-3-Fc with compound 4, and the observed sT1 (filled

circles), observed sT1 when 500 μM of 4 were mixed with 25 μM 13f (hollow circle), and observed sT1 when 500 μM of 4 were mixed with 25 μM

25 (hollow square). Vertical and horizontal arrows indicate the extent of attenuated relaxation of 4 when mixed with 25 μM or competitor 13f or

25 and the apparent concentration of 4, respectively.

Table 2. Weighting of ΔH and ΔS with Respect to ΔG

entry compd ΔH [kJ/mol] -T^aΔS* [kJ/mol] ΔG [kJ/mol] K_D[μM]

17

18

19

20

21

4³⁰

-20.09

-26.85

-28.13

-30.31

-37.33

-2.52

0.25

-22.61

-26.60

-30.1

-30.52

-34.91

106^b

22

24³⁰

13c

13f

19a

-1.98

-0.20

2.42

6.1

2.4

0.5

^aT=298.13 K. ^bThe affinity of compound 4 was measured using

different protein batches, resulting in K_Ds of 137 μM and 106 μM. For

the data given in Table 2, the protein batch showing a K_Dof 106 μM for

compound 4 was used.

position 2 in 4 by a benzyloxy group (24, entry 18)³⁰increased

binding enthalpy by more than 6 kJ/mol and at the same time

caused substantial entropy costs upon binding. With the

pentafluorobenzyloxy substituent (13c, entry 19), both ΔH

and ΔS were improved. Apparently, the interaction of the

reducing end substituent can be optimized with an electron-

poor aromatic ring. When the 2,3-difluorinated benzyloxy

substituent (13f, entry 20) was introduced, ΔG could be

further improved, mainly by a favorable enthalpy change.

On the other hand, entropy costs increased as a result of

the asymmetric substitution. The binding energy of the most

active compound 19a (entry 21, N-acetate replaced by

N-fluoroacetate) is mainly based on a further enthalpic

improvement. Unfortunately, in this case entropy costs of

more than 2 kJ/mol have to be compensated, probably due to

a specific conformational orientation requested for an opti-

mal interaction of the FAc group.

Figure 5. Affinity of compounds 13f (black) and 25 (gray), relative

to the affinity of compound 4 (= relative affinity of 1). The relative

affinities are determined by NMR, Biacore, and the fluorescent

hapten assay.⁴⁰

Structure-Affinity Relationships. To interpret the binding

affinities at the molecular level, we performed molecular

docking studies. In the absence of a crystal structure, we used

a homology model of the ligand-binding domain of MAG,

which was recently applied successfully to a series of MAG

antagonists (Figure 7).²²The compounds were manually

docked using the salt bridge between the carboxylate of the

sialosides with Arg118 and the hydrogen bond of C(9)-NH

to the backbone nitrogen of Thr128 as anchor points.

Finally, the protein-ligand complexes were fully minimized

in aqueous solution.

For validation purposes, 12 compounds were docked and

their binding strength quantified. Because these compounds

bind at the protein surface, the contribution of solvation and

entropy are difficult to estimate from thermodynamic dock-

ing studies. We therefore performed a series of molecular-

dynamical simulations (4.0 ꢀ 10^-9s at 300 K) to elucidate the

Figure 6. Van’t Hoff plot. Measured data (dots) and correspond-

ing linear fits according to eq 1.

ΔH ΔS

(1Þ

ln K_D

¼

-

RT

R

steps up to 30 °C. The values were fitted according to eq 1

(Figure 6) leading to ΔH and ΔS⁵⁰(Table 2).

Theanalysis (Table2) revealedthat theimprovement of the

binding energies ΔG resulted mainly from enhanced binding

enthalpies ΔH. The substitution of the methoxy group at

1604 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4

Mesch et al.

Some of the compounds modified at the 5-position seem

to undergo a favorable σ-π interaction with Trp22. In case

of 19a and 19b, we assume that the positively polarized

hydrogens of the FAc or ClAc, respectively, stick favorably

into the aromatic ring.⁵⁴As fluorine is more electronegative,

the polarization of the hydrogens is stronger and therefore

the interaction is more favorable. For compounds 19f and

19g, additional hydrophobic interactions are possible, but

the binding site seems to be spatially limited.²⁷The reduced

affinity of 19d could be a consequence of the different bond

angle for the sulfonamide substituent compared to an acetate

in the same position (13f, entry 9), leading to different spatial

requirements. Whereas methylsulfonamide 19d shows a

decrease in binding affinity, the nosyl substituent (19c) shows

an opposite behavior. This might be due to the formation of a

charge transfer complex with Trp22. To summarize, mod-

ifications at the reducing end improved binding affinity by a

factor of 7 (5f13f). Combined with the best modification at

the 5-position, the high affinity ligand 19a was obtained.

Figure 7. Homology model of MAG²²complexed with 19a (most

active compound of the series). Amino acids in white are forming

hydrogen bonds and amino acids in black contribute to hydropho-

bic pockets. The salt bridge formed by the carboxylate group of 19a

with Arg118 and a hydrogen bond by C(9)-NH and the backbone

nitrogen of Thr128 were used as anchor points for the docking.

Hydrophobic interactions are established by the fluoroacetamido

group and the side chains of Trp22 and Tyr124, the p-chlorobenza-

mide, and the side chains of Ser130 and Glu131, and the reducing

end substituent, the 2,3-difluorobenzyl and the side chains of Trp59,

Tyr60, and Tyr69, lining the main hydrophobic pocket. The image

has been generated using VMD.⁵¹

Conclusion

In conclusion, the nanomolar affinity of the sialic acid

derivative 19a containing a difluorobenzyl substituent at the

2-, a fluoroacetate at the 5-, and p-chlorobenzamide at the

9-position clearly indicates the additivity of the beneficial effect

of the various modifications. In addition, the thermodynamic

analysis reveals that the improved affinity of 19a predominantly

results from an increased binding enthalpy and not from an

entropy gain. The beneficial pharmacokinetic properties, e.g., a

high stability in the cerebrospinal fluid, also support the drug-like

properties of the newly identified MAG antagonist. However,

due to the shallow binding site generally responsible for short

half-lifes of carbohydrate-protein complexes,^22,55-58it remains

to be shown whether the complex formed by 19a and MAG

exhibits sufficient kinetic stability for in vivo applications.

kinetic aspects of binding and to quantify the contribution

of hydrogen bonding over time. Details are given in the

Supporting Information.

Upon analyzing the docking studies, the binding affinity

could be associated to hydrophilic as well as hydrophobic

interactions. The most important contribution is the salt

bridge between the carboxylic acid and Arg118.^26,52,53

Additionally, hydrogen-bond formations between 5-NH

and the backbone carbonyl of Gln126, the carboxylate and

the OH of Thr128, 8-OH and the backbone NH of Thr128

and 9-NH and the backbone carbonyl of Thr128 are ob-

served. This latter finding is in good agreement with previous

studies, where the abolishment of a hydrogen bond donor at

position 9 resulted in a reduced binding affinity.²⁶A con-

siderable contribution to the binding affinity results from

hydrophobic interactions. Thus, the p-chlorobenzamide is

shown to point into a hydrophobic pocket, built by Ser130

and Glu131. A second hydrophobic pocket, which hosts the

aglycone substituents, is defined by the side chains of Trp59,

Tyr60, and Tyr69. With respect to different substitution

patterns, the dichloro compound 13e (Table 1, entry 8)

shows a 4-fold and the difluoro compound 13f (see Table 1,

entry 9), a 7-fold enhancement in affinity compared to

reference compound 5, indicating a charge transfer complex

with the electron rich aromatic ring of Tyr60. The only

moderate improvement in binding affinity for the haloge-

nated compounds 13a and 13b could be due to a steric clash

of the p-substitutent with the protein. Compound 13c was

synthesized as a symmetric analogon of compound 13f in

order to compensate entropy loss due to the orientation of

the 2,3-difluorobenzyl ring. Again, that there is no improve-

ment of the binding affinity in comparison to compound

13f may be the consequence of a steric clash based on the

p-substitutent. Finally, the improved binding of 13d might

result from favorable π-π interactions of the naphthalene

with Tyr69.

Experimental Section

Chemistry. NMR spectra were recorded on a Bruker Avance

DMX-500 (500 MHz) spectrometer. Assignment of ¹H and ¹³

C

NMR spectra was achieved using 2D methods (COSY, HSQC,

TOCSY). Chemical shifts are expressed in ppm using residual

CHCl₃, CHD₂OD, and HDO as references. Optical rotations

were measured using Perkin-Elmer polarimeters 241 and 341.

MS analyses were carried out using a Waters Micromass ZQ

Detector system. The spectra were recorded in positive or

negative ESI mode. The HPLC/HRMS analyses were carried

out using a Agilent 1100 equipped with a photodiode array

detector and a Micromass QTOF I equipped with a 4 GHz

digital-time converter. All target compounds exhibit a purity

of g95%. Reactions were monitored by TLC using glass plates

coated with silica gel 60 F₂₅₄(Merck) and visualized by using

UV light and/or by charring with a molybdate solution (a

0.02 M solution of ammonium cerium sulfate dihydrate and

ammonium molybdate tetrahydrate in aqueous 10% H₂SO₄).

Column chromatography was performed on silica gel (Uetikon,

40-60 mesh). Methanol was dried by refluxing with sodium

methoxide and distilled immediately before use. Pyridine was

freshly distilled under argon over CaH₂. Dichloromethane

(DCM), dichloroethane (DCE), acetonitrile (MeCN), toluene,

and benzene were dried by filtration over Al₂O₃(Fluka, type

˚

5016 A basic). Molecular sieves (3 A) were activated under

vacuum at 500 °C for 2 h immediately before use. Compound 6

was prepared according to a published procedure.³¹HPLC

chromatograms and ¹H NMR spectra of the target compounds

can be found in the Supporting Information.

Article

Methyl (Methyl 5-Acetamido-3,5-dideoxy-2-thio-D-glycero-r-

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1605

1H, NHAc), 5.24 (m, 2H, H-7, H-8). ¹³C NMR (CDCl₃) δ 12.1

(SMe), 20.9, 21.1 (3C, 3 OAc), 23.2 (NHAc), 37.8 (C-3), 49.4

(C-5), 50.6 (C-9), 53.0 (OMe), 68.1 (C-4), 69.7 (C-7), 72.3 (C-6),

74.6 (C-8), 88.4 (C-2), 170.2, 170.3, 171.0 (5C, CO). ESI-MS calcd

for C₁₉H₂₈N₄O₁₀S [M þ Na]^þ527.40; found m/z 527.21.

D-galacto-2-nonulopyranosid)onate (7). Compound 6 (217 mg,

42.0 mmol) was dissolved in dry MeOH (8.0 mL) and treated

with NaOMe (1 M, 1.0 mL) for 2 h. The reaction mixture was

neutralized with Amberlyst 15, filtered over a pad of celite, and

the celite washed thoroughly with MeOH. The solvent was

evaporated under reduced pressure, and the crude product

was purified by chromatography on silica gel (1% gradient

MeOH in DCM) to yield 7 as a white foam (90.0 mg, 61%).

¹H NMR (500 MHz, CD₃OD) δ 1.97 (dd, J = 11.5, 13.9 Hz, 1H,

H-3a), 2.03 (s, 3H, SMe), 2.09 (s, 3H, NHAc), 2.47 (dd, J = 4.9,

13.9 Hz, 1H, H-3b), 3.54 (d, J = 9.4 Hz, 1H, H-7), 3.67 (dd, J =

5.6, 11.6 Hz, 1H, H-9a), 3.81-3.86 (m, 6H, H-5, H-8, H-9b,

OMe). 4.11 (m, 2H, H-4, H-6). ¹³C NMR (CD₃OD) δ 11.2

(SMe), 22.7 (NHAc), 41.2 (C-3), 53.1 (OMe), 54.1 (C-5), 65.2

(C-9), 68.3 (C-4), 70.2 (C-7), 71.2 (C-8), 72.6 (C-6), 84.6 (C-2),

170.8, 175.0 (2 CO). ESI-MS calcd for C₁₃H₂₃NO₈S [M þ Na]^þ

376.10; found m/z 376.10.

Methyl (Methyl 5-Acetamido-3,5-dideoxy-2-thio-9-tosyl-^D-

glycero-r-^D-galacto-2-nonulopyranosid)onate (8). To a solution

of 7 (3.20 g, 9.07 mmol) in freshly distilled pyridine (80 mL)

p-toluenesulfonyl chloride (1.90 g, 10.0 mmol) was added at 0 °C

and the mixture was stirred for 2 h at 0 °C. Afterward tosyl

chloride (0.70 g, 3.68 mmol) was added and the solution stirred

continuously for 16 h at 5 °C. The reaction mixture was warmed

to rt, methanol (20 mL) was added and stirring continued for

30 min. After evaporation of the solvents, the crude product was

purified by chromatography on silica gel (DCM:MeOH, 19:1)

to give 8 as a foam (3.00 g, 66%). ¹H NMR (500 MHz, CDCl₃)

δ 1.79 (dd, J = 11.2, 13.4 Hz, 1H, H-3a), 1.96 (s, 3H, SMe), 1.99

(s, 3H, NHAc), 2.37 (s, 3H, CH₃), 2.74 (dd, J = 3.7, 13.2 Hz, 1H,

H-3b), 3.25 (d, J = 9.5 Hz, 1H, H-6), 3.41 (d, J = 9.7 Hz, 1H, H-

7), 3.66 (s, 3H, OMe), 3.98 (m, 1H, H-8), 4.10 (m, 1H, H-9a), 4.24

(m, 1H, H-9b), 6.94 (d, J = 7.2 Hz, 1H, NHAc), 7.28, 7.72 (AA⁰,

BB⁰of AA⁰BB⁰, J = 8.2 Hz, 4H, CH_ar). ¹³C NMR (CDCl₃) δ 11.9

(SMe), 21.7 (CH₃), 23.0 (NHAc), 40.4 (C-3), 52.7 (C-5), 53.7

(OMe), 67.8, 68.6, 69.1 (C-4, C-7, C-8), 72.0 (C-6), 82.4 (C-2),

128.0, 130.0, 132.4, 145.1 (6 C-Ar), 170.4, 174.2 (2 CO). ESI-MS

calcd for C₂₀H₂₉NO₁₀S₂[M þ Na]^þ530.11; found m/z 530.19.

Methyl (Methyl 5-Acetamido-9-azido-3,5,9-trideoxy-2-thio-^D-

glycero-r-^D-galacto-2-nonulopyranosid)onate (9). Compound 8

(160 mg, 0.32 mmol) was dissolved in dry DMF (5 mL). NaN₃

(103 mg, 1.58 mmol) and 15-crown-5 (28.6 mg, 0.13 mmol) were

added successively and the reaction mixture was stirred at 60 °C

for 24 h. After filtration through a pad of celite, the solvent

was evaporated and the residue was purified by chromatogra-

phy on silica gel (gradient 1% MeOH in DCM) to yield 9

(96.0 mg, 78%). ¹H NMR (500 MHz, CD₃OD) δ 1.79 (m, 1H,

H-3a), 2.03 (s, 3H, SMe), 2.17 (s, 3H, NHAc), 2.77 (dd, J = 4.5,

12.8 Hz, 1H, H-3b), 3.39 (dd, J = 6.2, 12.7 Hz, 1H, H-9a), 3.48

(m, 2H, H-6, H-7), 3.57 (dd, J = 2.3, 12.7 Hz, 1H, H-9b), 3.69

(m, 1H, H-4), 3.79 (m, 1H, H-5), 3.86 (s, 3H, OMe), 3.93 (ddd,

J = 2.6, 5.7, 8.5 Hz, 1H, H-8), 4.01 (m, 1H, H-6). ¹³C NMR

(CD₃OD) δ 12.0 (SMe), 22.7 (NHAc), 41.8 (C-3), 53.6 (C-5),

53.8 (OMe), 55.2 (C-9), 69.0 (C-4), 71.0 (C-7), 71.7 (C-8), 76.8

(C-6), 171.7, 175.2 (2 CO). ESI-MS calcd for C₁₃H₂₂N₄O₇S

[M þ Na]^þ401.11; found m/z 401.15.

General Procedure for the Synthesis of Compounds 11a-f,

17a-d, 18f,g. Compound 10 (0.12 mmol) was dissolved in dry

acetonitrile (2.0 mL) under argon. The alcohol (0.26 mmol) and

˚

powdered MS 3 A (80.0 mg) were added. The mixture was

stirred at rt for 1.5 h. Then the suspension was cooled to -40 °C

and subsequently treated with N-iodosuccinimide (0.60 mmol)

and triflic acid (0.06 mmol in 0.2 mL MeCN). After 30 min, the

reaction mixture was warmed to -30 °C and stirring continued

for 16 h. The mixture was then warmed to rt, stirred for another

2 h, and filtered through a pad of celite. The celite was washed

with DCM (10 mL), and the filtrate was subsequently washed

with 20% aqueous Na₂S₂O₃(1 mL) and saturated aqueous

NaHCO₃(3 ꢀ 5 mL). The organic phase was dried over Na₂SO₄,

filtered, and concentrated under reduced pressure. The crude

product was purified by chromatography on silica gel.

General Procedure for the Synthesis of Compounds 12a-f, 20,

18a-d. Compounds 11a-f (14 or 17a-d) (0.09 mmol) and

p-chlorobenzoyl chloride (0.36 mmol) were dissolved in dry

DCE (3.0 mL) under argon. Triphenylphosphine (0.18 mmol)

in dry DCE (1.5 mL) was added after 5 min, and the solution was

stirred at rt for 24 h. The reaction mixture was diluted

with DCM (10 mL) and washed with saturated aqueous NaH-

CO₃(3 ꢀ 10 mL). The organic phase was dried over Na₂SO₄,

filtered, and concentrated under reduced pressure. The residue

was purified by chromatography on silica gel.

General Procedure for the Deprotection of 12a-f, 18b. Com-

pound 12a-f (18b) (0.06 mmol) was dissolved in MeOH (1.5 mL)

and treated with 10% aqueous NaOH (0.3 mL). The reaction

mixture was stirred at rt for 3 h. Then the reaction mixture was

neutralized with 7% aqueous HCl (0.2 mL). The solvent was

evaporated, and the crude product was purified by chromato-

graphy on RP-18.

General Procedure of the Deprotection of 18a-d,f-g. Com-

pound 18a-d,f-g (0.03 mmol) was dissolved in THF/H₂O

(2 mL/0.5 mL) and was reacted with LiOH (0.28 mmol). The

crude product was purified on RP-18 (10% gradient MeOH in

water) followed by ion exchange chromatography (Dowex-50)

and P2 size exclusion chromatography to yield 19.

Methyl (4-Chlorobenzyl 5-Acetamido-4,7,8-tri-O-acetyl-9-azido-

3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyranosid)onate (11a).

Compound 10 (61.0 mg, 0.12 mmol) was reacted with 4-chloroben-

zyl alcohol (38.0 mg, 0.26 mmol), N-iodosuccinimide (134 mg,

0.601 mmol), and triflic acid (6.00 μL, 9.00 mg, 0.06 mmol). The

crude product was purified by chromatography on silica gel (0.5%

gradient iPrOH in petrol ether/DCM 8:4) to yield 11a (55.0 mg,

1

76%) as a colorless oil. [R]²⁰-0.02 (c 0.28, CH₂Cl₂). H NMR

D

(500 MHz, CDCl₃) δ 1.90 (s, 3H, NHAc), 2.03 (m, 4H, OAc, H-3a),

2.17, 2.20 (2 s, 6H, 2 OAc), 2.65 (dd, J = 4.6, 12.9 Hz, 1H, H-3b),

3.26 (dd, J= 5.7, 13.4 Hz, 1H, H-9a), 3.55 (dd, J= 2.8, 13.4 Hz, 1H,

H-9b), 3.71(s, 3H, OMe), 4.12(m, 2H, H-5, H-6), 4.41, 4.76(A, Bof

AB, J = 12.3 Hz, 2H, CH₂Ar), 4.87 (m, 1H, H-4), 5.15 (d, J = 9.8

Hz, 1H, NH), 5.35 (m, 2H, H-7, H-8) 7.30 (m, 4H, CH_ar). ¹³CNMR

(CDCl₃) δ 20.8, 20.9, 21.1 (3 OAc), 23.3 (NHAc), 38.0 (C-3), 49.4

(C-5), 51.0 (C-9), 52.8 (OMe), 66.1 (CH₂Ar), 67.9 (C-7), 68.9 (C-4),

69.4 (C-8), 72.8 (C-6), 98.6 (C-2), 128.4, 129.1, 133.5, 135.7 (6

C-Ar), 168.3, 170.3, 170.3, 171.0 (5C, 5 CO). ESI-MS calcd for

C₂₅H₃₁ClN₄O₁₁[M þ Na]^þ621.17; found m/z 621.24.

Methyl (Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-

trideoxy-2-thio-^D-glycero-r-D-galacto-2-nonulopyranosid)onate

(10). Compound 9 (87.0 mg, 0.23 mmol) was dissolved in dry

pyridine (1.0 mL) and cooled to 0 °C. After 15 min, DMAP

(4.47 mg, 0.04 mmol) and Ac₂O (0.5 mL) were added succes-

sively. The reaction mixture was warmed to rt and stirred for

14 h. The solvent was evaporated and the residue purified by

chromatography on silica gel (toluene/ethylacetate, 1:3) to afford

10 (85.0 mg, 73%). ¹H NMR (500 MHz, CDCl₃) δ 1.81 (s, 3H,

NHAc), 1.91 (m, 1H, H-3a), 1.97, 2.05, 2.10 (3 s, 9H, 3 OAc), 2.13

(s, 3H, SMe), 2.67 (dd, J = 4.4, 12.5 Hz, 1H, H-3b), 3.19 (dd, J =

5.7, 13.4 Hz, 1H, H-9a), 3.59 (m, 1H, H-9b), 3.76 (m, 4H, H-6,

OMe), 4.01 (m, 1H, H-5), 4.81 (m, 1H, H-4), 4.91 (d, J = 7.7 Hz,

Methyl (4-Fluorobenzyl 5-Acetamido-4,7,8-tri-O-acetyl-9-

azido-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyranosid)-

onate (11b). Compound 10 (62.0 mg, 0.12 mmol) was reacted

with 4-fluorobenzyl alcohol (38.0 mg, 0.26 mmol), N-iodosuc-

cinimide (134 mg, 0.601 mmol), and triflic acid (6.00 μL, 9.00

mg, 0.06 mmol). The crude product was purified by chroma-

tography on silica gel (0.5% gradient iPrOH in petrol ether/

DCM 8:4) to yield 11b (53.0 mg, 76%) as a colorless oil. [R]²⁰

D

1606 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4

Mesch et al.

1

-0.01, (c 2.6, CH₂Cl₂). H NMR (500 MHz, CDCl₃) δ 1.89

(C-8), 69.3 (C-4), 70.5 (C-7), 73.5 (C-6), 109.6 (C-2), 127.6, 127.7,

130.0 (6C, 6 C-Ar), 168.5, 170.5, 170.6, 170.7, 171.4 (5 CO). ESI-

MS calcd for C₂₅H₃₀Cl₂N₄O₁₁[M þ Na]^þ655.13; found m/z

655.07.

(s, 3H, NHAc), 2.02 (m, 1H, H-3a), 2.03, 2.17, 2.20 (3 s, 9H, 3

OAc), 2.65 (dd, J = 4.5, 12.8 Hz, 1H, H-3b), 3.29 (dd, J = 5.4,

13.3 Hz, 1H, H-9a), 3.58 (dd, J = 2.0, 13.1 Hz, 1H, H-9b), 3.71

(s, 3H, OMe), 4.13 (m, 2H, H-5, H-6), 4.41, 4.76 (A, B of AB,

J = 11.9 Hz, 2H, CH₂Ar), 4.85 (m, 1H, H-4), 5.35 (m, 2H, H-8,

H-7), 7.02 (t, J = 8.6 Hz, 2H, CH_ar), 7.31 (dd, J = 5.6, 8.2 Hz,

2H, CH_ar). ¹³C NMR (CDCl₃) δ 20.8, 20.9, 21.1 (3 OAc), 23.2

(NHAc), 49.3 (C-3), 51.0 (C-5), 52.8 (C-9), 53.5 (OMe), 66.1

(CH₂Ar), 68.0 (C-7), 68.9 (C-4), 69.7 (C-8), 72.8 (C-6), 98.5

(C-2), 115.2 (J = 21.7 Hz), 129.6 (J = 8.4 Hz), 132.9 (J = 2.9

Hz), 162.4 (J = 246.0 Hz) (6 C-Ar), 168.4, 170.2, 170.3, 170.4,

171.0 (6C, 6 CO). ESI-MS calcd for C₂₅H₃₁FN₄O₁₁[M þ Na]^þ

605.20; found m/z 605.22.

Methyl (2,3-Difluorobenzyl 5-Acetamido-4,7,8-tri-O-acetyl-9-

azido-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyranosid)-

onate (11f). Compound 10 (86.0 mg, 0.17 mmol) was reacted

with 2,3-difluorobenzyl alcohol (29.0 μL, 37.0 mg, 0.26 mmol),

N-iodosuccinimide (46.0 mg, 0.21 mmol), and triflic acid

(6.00 μL, 10.0 mg, 0.07 mmol). The crude product was purified

by chromatography on silica gel (0.5% gradient iPrOH in petrol

ether/DCM 8:4) to yield 11f (67.0 mg, 66%) as a yellow oil. ¹H

NMR (500 MHz, CDCl₃) δ 1.82 (s, 3H, NHAc), 1.95 (m, 1H,

H-3a), 1.96, 2.10, 2.13 (3 s, 9H, 3 OAc), 2.58 (m, 1H, H-3b), 3.20

(m, 1H, H-9a), 3.50 (m, 1H, H-9b), 3.72 (s, 3H, OMe), 4.06 (m,

2H, H-5, H-6), 4.46, 4.75 (A, B of AB, J = 12.0 Hz, 2H, CH₂Ar),

4.82 (m, 1H, H-4), 5.29 (m, 2H, H-8, H-7), 7.05 (m, 3H, CH_ar).

¹³C NMR (CDCl₃) δ 21.3, 21.6 (3C, 3 OAc), 23.6 (NHAc), 38.2

(C-3), 49.8 (C-5), 51.3 (C-9), 53.3 (OMe), 60.8 (CH₂Ar), 68.4

(C-7), 69.3 (C-8), 70.3 (C-4), 73.4 (C-6), 99.1 (C-2), 117.4, 119.1

(J=17.0 Hz), 124.3, 125.5 (6 C-Ar), 168.5, 170.5, 170.6, 170.8,

171.4 (5 CO). ESI-MS calcd for C₂₅H₃₀F₂N₄O₁₁[M þ 2Na]^þ

645.99; found m/z 645.26.

Methyl (Pentafluorobenzyl 5-Acetamido-4,7,8-tri-O-acetyl-9-

azido-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyranosid)-

onate (11c). Compound 10 (60.0 mg, 0.12 mmol) was reacted

with pentafluorobenzyl alcohol (60.0 mg, 0.30 mmol), N-iodo-

succinimide (32.0 mg, 0.14 mmol), and triflic acid (4.00 μL,

7.00 mg, 0.04 mmol). The crude product was purified by chro-

matography on silica gel (0.5% gradient iPrOH in petrol ether/

DCM 8:4) to yield 11c (47.0 mg, 66%) as a colorless oil. [R]²⁰

D

-0.03 (c 0.5, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ 1.90 (s,

3H, NHAc), 1.94 (dd, J = 10.4, 12.6 Hz, 1H, H-3a), 2.03, 2.19,

2.21 (3 s, 9H, 3 OAc), 2.59 (dd, J = 4.6, 12.8 Hz, 1H, H-3b), 3.30

(dd, J = 5.6, 13.5 Hz, 1H, H-9a), 3.59 (dd, J = 2.9, 13.5 Hz, 1H,

H-9b), 3.86 (s, 3H, OMe), 4.12 (m, 2H, H-5, H-6), 4.43 (A of AB,

J = 11.0 Hz, 1H, CH₂Ar), 4.87 (m, 1H, H-4), 4.90 (B of AB, J =

10.7 Hz, 1H, CH₂Ar), 5.19 (d, J = 9.3 Hz, 1H, NH), 5.39 (m,

2H, H-7, H-8). ¹³C NMR (CDCl₃) δ 20.8, 20.9, 21.1 (3 OAc),

23.2 (NHAc), 37.7 (C-3), 49.4 (C-5), 51.0 (C-9), 53.0 (OMe),

54.0 (CH₂Ar), 67.8, 68.8, 69.3 (C-4, C-7, C-8), 72.9 (C-6),

98.7 (C-2), 167.8, 170.3 (5C, 5 CO). ESI-MS calcd for C₂₅H₂₇-

F₅N₄O₁₁[M þ Na]^þ677.16; found m/z 677.32.

Methyl (4-Chlorobenzyl 5-Acetamido-4,7,8-tri-O-acetyl-9-(4-

chlorobenzamido)-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulo-

pyranosid)onate (12a). Compound 11a (55.0 mg, 0.09 mmol)

was reacted with p-chlorobenzoyl chloride (46.0 μL, 63.0 mg,

0.36 mmol) and triphenylphosphine (47.0 mg, 0.18 mmol). The

crude product was purified by chromatography on silica gel

(0.5% gradient of MeOH in DCM) to yield 12a (35.0 mg, 59%)

1

as a yellow solid. [R]²⁰-0.01 (c 2.9, CH₂Cl₂). H NMR (500

D

MHz, CDCl₃) δ 1.89 (s, 3H, NHAc), 2.05 (m, 4H, H-3a, OAc),

2.14, 2.27 (2 s, 6H, 2 OAc), 2.67 (dd, J = 4.6, 12.8 Hz, 1H, H-3b),

2.92 (dt, J = 3.3, 14.9 Hz, 1H, H-9a), 3.65 (s, 3H, OMe), 4.05 (dd,

J = 2.0, 10.7 Hz, 1H, H-6), 4.21 (q, J = 10.4 Hz, 1H, H-5), 4.36

(ddd, J = 3.0, 8.7, 15.1Hz, 1H, H-9b), 4.41, 4.78 (A, B ofAB, J =

12.3 Hz, 2H, CH₂Ar), 4.84 (m, 1H, H-4), 5.15 (dd, J = 2.0, 10.0

Hz, 1H, H-7), 5.31(m, 2H, NHAc, H-8), 7.10 (dd, J = 4.0, 8.5 Hz,

1H, NH), 7.29 (m, 4H, CH_ar), 7.41, 7.77 (AA⁰, BB⁰of AA⁰BB⁰,

J = 8.5 Hz, 4H, CH_ar). ¹³C NMR (CDCl₃) δ 20.9, 21.2, 21.3

(3 OAc), 23.2 (NHAc), 38.1 (C-3), 38.4 (C-9), 49.5 (C-5), 52.7

(OMe), 66.1 (CH₂Ar), 67.9 (C-7), 68.2 (C-8), 69.0 (C-4), 72.2

(C-6), 98.4 (C-2), 128.4, 128.5, 128.8, 129.2, 132.0, 132.6, 133.5,

135.7, 137.7 (12 C-Ar), 168.1, 170.4, 171.2, 172.6 (6C, 6 CO).

ESI-MS calcd for C₃₂H₃₆Cl₂N₂O₁₂[M þ Na]^þ733.16; found m/z

733.25.

Methyl (2-Naphthyl 5-Acetamido-4,7,8-tri-O-acetyl-9-azido-

3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyranosid)onate

(11d). Compound 10 (50.0 mg, 0.10 mmol) was reacted with

2-naphthalenemethanol (24.0mg, 0.15 mmol), N-iodosuccinimide

(27.0 mg, 0.12 mmol), and triflic acid (4.00 μL, 7.00 mg, 0.04

mmol). The crude product was purified by chromatography on

silica gel (0.5% gradient iPrOH in petrol ether/DCM 8:4) to yield

11d (37.0mg, 61%) as a colorlessoil. ¹H NMR (500 MHz, CDCl₃)

δ 1.85 (s, 3H, NHAc), 2.09 (m, 1H, H-3a), 2.03, 2.15, 2.21 (3 s, 9H,

3 OAc), 2.26 (dd, J = 6.0, 13.5 Hz, 1H, H-9a), 3.59 (dd, J = 2.9,

13.5 Hz, 1H, H-9b), 3.67 (s, 3H, OMe), 4.14 (m, 2H, H-5, H-6),

4.63 (A of AB, J = 12.2 Hz, 1H, CH₂Ar), 4.89 (m, 1H, H-4), 4.96

(B of AB, J = 12.2 Hz, 1H, CH₂Ar), 5.34 (m, 2H, H-7, NH), 5.38

(m, 1H, H-8), 7.45-7.48 (m, 3H, CH_ar), 7.79-7.83 (m, 4H, CH_ar).

¹³C NMR (CDCl₃) δ 21.3, 21.5 (3C, 3 OAc), 23.6 (NHAc), 38.5

(C-3), 49.9 (C-5), 51.3 (C-9), 53.1 (OMe), 67.4 (CH₂Ar), 68.4

(C-7), 69.4 (C-4), 70.2 (C-8), 73.3 (C-6), 126.1, 126.4, 126.5, 126.9,

128.1, 128.3, 128.4 (10 C-Ar),170.7 (6C, 6 CO). ESI-MScalcdfor

C₂₉H₃₄N₄O₁₁[M þ Na]^þ637.33; found m/z 637.20.

Methyl (4-Fluorobenzyl 5-Acetamido-4,7,8-tri-O-acetyl-9-(4-

chlorobenzamido)-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulo-

pyranosid)onate (12b). Compound 11b (52.0 mg, 0.09 mmol)

was reacted with p-chlorobenzoyl chloride (46.0 μL, 63.0 mg,

0.36 mmol) and triphenylphosphine (52.0 mg, 0.19 mmol). The

crude product was purified by chromatography on silica gel (0.5%

gradient of MeOH in DCM) to yield 12b (37.0 mg, 60%) as a

yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 1.85 (s, 3H, NHAc),

2.00 (m, 1H, H-3a), 2.03, 2.11, 2.26 (3 s, 9H, 3 OAc), 2.68 (dd, J =

4.5, 12.7 Hz, 1H, H-3b), 2.96 (dt, J = 3.5, 15.0 Hz, 1H, H-9a), 3.61

(s,3H, OMe), 4.12 (d, J = 10.7 Hz, 1H, H-6), 4.23 (q, J = 10.2Hz,

1H, H-5), 4.36 (ddd, J = 3.0, 8.7, 15.3 Hz, 1H, H-9b), 4.40, 4.78

(A, B of AB, J = 11.9 Hz, 2H, CH₂Ar), 4.87 (m, 1H, H-4), 5.20

(dd, J = 1.1, 9.9 Hz, 1H, H-7), 5.35 (dt, J = 2.9, 9.9 Hz, 1H, H-8),

5.99 (m, 1H, NHAc), 7.00 (t, J = 8.7 Hz, 2H, CH_ar), 7.17 (dd, J =

3.8 Hz, 8.1 Hz, 1H, NH), 7.49 (dd, J = 5.4 Hz, 8.5 Hz, 1H, CH_ar),

7.38, 7.76 (AA⁰, BB⁰of AA⁰BB⁰, J = 8.4 Hz, 4H, CH_ar). ¹³C NMR

(CDCl₃) δ 21.2, 21.3 (3 OAc), 23.1 (NHAc), 38.3 (C-3), 38.5 (C-9),

49.4 (C-5), 52.6 (OMe), 66.1 (CH₂Ar), 68.0 (C-7), 68.2 (C-8), 69.2

(C-4), 72.2 (C-6), 98.4 (C-2), 115.0, 115.2 (J = 21.3 Hz), 128.6,

129.6, 129.7 (J = 7.5 Hz), 132.0, 132.8, 132.9, 137.7, 161.3, 163.5

(J = 275.1 Hz), 168.1, 166.3 (J = 237.5 Hz) (12 C-Ar), 170.39,

170.4, 171.0, 172.5 (6C, 6 CO). ESI-MS calcd for C₃₂H₃₆-

ClFN₂O₁₂[M þ Na]^þ717.19; found m/z 717.34.

Methyl (2,3-Dichlorobenzyl 5-Acetamido-4,7,8-tri-O-acetyl-

9-azido-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyranosid)-

onate (11e). Compound 10 (45.0 mg, 0.09 mmol) was reacted with

2,3-dichlorobenzyl alcohol (24.0 mg, 0.13 mmol), N-iodosuccini-

mide (24.0 mg, 0.12 mmol), and triflic acid (3.00 μL, 6.00 mg, 0.04

mmol). The crude product was purified by chromatography on

silica gel (0.5% gradient iPrOH in petrol ether/DCM 8:4) to yield

11e (30.0 mg, 54%) as a yellow oil. ¹H NMR(500 MHz, CDCl₃) δ

1.83 (s, 3H, NHAc), 2.01 (t, J = 12.9 Hz, 1H, H-3a), 1.97, 2.08,

2.11 (3 s, 9H, 3 OAc), 2.63 (dd, J = 4.7, 12.9 Hz, 1H, H-3b), 3.19

(dd, J = 6.0, 13.6 Hz, 1H, H-9a), 3.50 (dd, J = 3.0, 13.6 Hz, 1H,

H-9b), 3.72 (s, 3H, OMe), 4.05 (m, 2H, H-5, H-6), 4.50, 4.82 (A, B

of AB, J = 13.0 Hz, 2H, CH₂Ar), 4.85 (m, 1H, H-4), 5.13 (d,

J = 8.0 Hz, 1H, NHAc), 5.24 (m, 2H, H-7, H-8), 7.14 (t, J =

8.0 Hz, 1H, CH_ar), 7.34 (d, J = 8.1 Hz, 2H, CH_ar). ¹³C

NMR (CDCl₃) δ 21.3, 21.5 (3C, 3 OAc), 23.6 (NHAc), 38.2

(C-3), 49.9 (C-5), 51.2 (C-9), 53.4 (OMe), 64.7 (CH₂Ar), 68.4

Article

Methyl (Pentafluorobenzyl 5-Acetamido-4,7,8-tri-O-acetyl-

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1607

wasreactedwithp-chlorobenzoylchloride(57.0μL, 78.0mg, 0.45

mmol) and triphenylphosphine (65.0 mg, 0.25 mmol). The crude

product was purified by chromatography on silica gel (0.5%

gradient of MeOH in DCM) to yield 12f (44.0 mg, 55%) as a

yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 1.80 (s, 3H, NHAc),

2.04 (m, 4H, H-3a, OAc), 2.14, 2.27 (2 s, 6H, 2 OAc), 2.65 (dd,

J = 4.6, 12.8 Hz, 1H, H-3b), 2.97 (dt, J = 3.5, 15.0 Hz, 1H, H-

9a), 3.74 (s, 3H, OMe), 4.08 (m, 1H, H-6), 4.23 (q, J = 10.4 Hz,

1H, H-5), 4.33 (m, 1H, H-9b), 4.52, 4.84 (A, B of AB, J = 12.0

Hz, 2H, CH₂Ar), 5.17(dd, J =1.9, 9.8Hz, 1H, H-7), 5.31 (m, 1H,

H-8), 5.43 (m, 1H, NHAc), 7.75 (m, 4H, NH, CH_ar), 7.39, 7.77

(AA⁰, BB⁰of AA⁰BB⁰, J = 8.5 Hz, 4H, CH_ar). ¹³C NMR (CDCl₃)

δ 21.3, 21.5, 21.6 (3 OAc), 23.5 (NHAc), 38.3 (C-3), 38.9 (C-9),

49.9 (C-5), 53.2 (OMe), 60.8 (CH₂Ar), 68.3 (C-8), 68.7 (C-7), 69.4

(C-4), 72.7 (C-6), 98.0 (C-2), 117.3, 117.4 (J = 17.0 Hz), 125.7,

128.8, 129.2, 132.4, 132.5 (J = 9.0 Hz), 133.0, 138.1 (12 C-Ar),

166.7, 168.4, 170.7, 170.9, 171.6, 172.9 (6 CO). ESI-MS calcd for

C₃₂H₃₅ClF₂N₂O₁₂[M þ Na]^þ735.18; found m/z 735.15.

9-(4-chlorobenzamido)-3,5,9-trideoxy-D-glycero-r-D-galacto-2-

nonulopyranosid)onate (12c). Compound 11c (47.0 mg, 0.07

mmol) was reacted with p-chlorobenzoyl chloride (36.0 μL,

49.0 mg, 0.28 mmol) and triphenylphosphine (41.0 mg, 0.16

mmol). The crude product was purified by chromatography on

silica gel (0.5% gradient of MeOHin DCM) to yield12c (25.0 mg,

45%). ¹H NMR (500 MHz, CDCl₃) δ 1.86 (s, 3H, NHAc), 1.94

(m, 1H, H-3a), 2.02, 2.16, 2.19 (3 s, 9H, 3 OAc), 2.60 (dd, J = 4.5,

12.8 Hz, 1H, H-3b), 3.55 (dd, J = 6.3, 12.3 Hz, 1H, H-9a), 3.81

(m, 4H, OMe, H-9b), 4.07 (d, J = 10.3 Hz, 1H, H-6), 4.20 (dd,

J = 2.0, 10.8 Hz, 1H, H-5), 4.45, 4.93 (A, B of AB, J = 11.3 Hz,

2H, CH₂Ar), 5.37 (dd, J = 2.1, 8.1 Hz, 1H, H-7), 5.46 (m, 1H,

H-8), 5.68 (d, J = 9.6 Hz, 1H, NHAc), 7.38, 8.00 (AA⁰, BB⁰of

AA⁰BB⁰, J = 8.4 Hz, 4H, CH_ar). ¹³C NMR (CDCl₃) δ 21.0, 21.2,

21.3(3 OAc), 23.3 (NHAc), 37.8(C-3), 43.7 (C-9), 49.5(C-5), 53.1

(OMe), 54.1 (CH₂Ar), 68.3, 69.0 (C-4, C-7), 70.0 (C-8), 72.9

(C-6), 98.8 (C-2), 128.8, 128.9, 129.1, 131.5, 132.9, 133.2, 133.3

(12 C-Ar), 167.6, 167.9, 168.7, 170.3, 170.4, 170.6 (6 CO). ESI-

MS calcd for C₃₂H₃₂ClF₅N₂O₁₂[M þ Cl]^-801.45; found m/z

801.36.

Sodium (4-Chlorobenzyl 5-Acetamido-9-(4-chlorobenzamido)-

3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyranosid)onate

(13a). Compound 12a (25.0 mg, 0.03 mmol) was treated with

10% aqueous NaOH (0.2 mL) in MeOH (1.0 mL). The crude

product was purified by chromatography on RP-18 (5% gradient

of MeOH in H₂O) to yield 13a as a white solid (15.0 mg, 50%).

[R]²⁰_D-0.25 (c 0.41, H₂O). ¹H NMR (500 MHz, CD₃OD) δ 1.55

(dd, J = 3.3, 11.9 Hz, 1H, H-3a), 1.90 (s, 3H, NHAc), 2.81 (dd,

J = 2.0, 12.2 Hz, 1H, H-3b), 3.34 (dd, J = 1.8, 9.0 Hz, 1H, H-7),

3.41 (dd, J = 7.8, 13.6 Hz, 1H, H-9a), 3.55-3.63 (m, 3H, H-4, H-

5, H-6), 3.67 (dd, J = 3.1 Hz, 13.6 Hz, 1H, H-9b), 3.94 (m, 1H,

H-8), 4.40, 4.70 (A, B of AB, J = 11.6 Hz, 2H, CH₂Ar), 7.18, 7.25

(AA⁰, BB⁰of AA⁰BB⁰, J =8.4 Hz, 4H, CH_ar), 7.36, 7.72 (AA⁰, BB⁰

of AA⁰BB⁰, J = 8.6 Hz, 4H, CH_ar). ¹³C NMR (CD₃OD) δ 22.6

(NHAc), 42.7 (C-3), 44.6 (C-9), 54.2 (C-5), 66.5 (CH₂Ar), 69.6

(C-4), 71.2 (C-8), 72.5 (C-7), 74.4 (C-6), 102.1 (C-2), 129.2, 129.7,

130.1, 130.6, 134.0, 134.5, 138.6, 138.8 (12 C-Ar), 169.2, 174.3,

175.5 (3 CO). HRMS calcd for C₂₅H₂₈Cl₂N₂O₉[M - H]^-

615.0891; found 615.0888.

Methyl (2-Naphthyl 5-Acetamido-4,7,8-tri-O-acetyl-9-(4-chloro-

benzamido)-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyra-

nosid)onate (12d). Compound 11d (50.0 mg, 0.08 mmol) was

reacted with p-chlorobenzoyl chloride (42.0 μL, 57.0 mg, 0.32

mmol) and triphenylphosphine (47.0 mg, 0.18 mmol). The crude

product was purified by chromatography on silica gel (0.5%

1

gradient of MeOH in DCM) to yield 12d (25.0 mg, 47%). H

NMR (500 MHz, CDCl₃) δ 1.87 (s, 3H, NHAc), 2.04 (s, 3H,

OAc), 2.10 (t, J = 12.4 Hz, 1H, H-3a), 2.14, 2.24 (2 s, 6H, 2 OAc),

2.72 (dd, J = 4.5, 12.8 Hz, 1H, H-3b), 2.92 (m, 1H, H-9a), 3.59

(s, 3H, OMe), 4.11 (m, 1H, H-6), 4.23 (q, J = 10.3 Hz, 1H, H-5),

4.34 (ddd, J = 3.0, 8.5, 15.2 Hz, 1H, H-9b), 4.63 (A of AB, J =

12.3Hz, 1H, CH₂Ar), 4.87(dt, J = 4.6, 12.3 Hz, 1H, H-4), 4.98 (B

of AB, J = 12.3 Hz, 1H, CH₂Ar), 5.17 (d, J = 9.8 Hz, 1H, H-7),

5.34 (d, J = 9.9 Hz, 1H, H-8), 5.50 (m, 1H, NHAc), 7.11 (m, 1H,

NH), 7.39 (AA⁰of AA⁰BB⁰, J = 8.5Hz, 2H, CH_ar), 7.44-7.47 (m,

3H, CH_ar), 7.76 (BB⁰of AA⁰BB⁰, J = 8.5 Hz, 2H, CH_ar), 7.75-

7.83 (m, 4H, CH_ar). ¹³C NMR (CDCl₃) δ 21.3, 21.5, 21.6 (3 OAc),

23.5 (NHAc), 38.6 (C-3), 38.9 (C-9), 49.9 (C-5), 53.0 (OMe), 67.4

(CH₂Ar), 68.4 (C-7), 68.7 (C-8), 69.5 (C-4), 72.6 (C-6), 98.9 (C-2),

126.2, 126.3, 126.5, 127.0, 128.0, 128.3, 128.9, 129.0, 129.2, 132.4,

133.1, 133.3, 133.5, 135.1 (14 C-Ar), 166.6, 168.7, 170.8, 171.5,

172.9 (6C, 6 CO). ESI-MS calcd for C₃₆H₃₉ClN₂O₁₂[M þ Na]^þ

749.22; found m/z 749.25.

Methyl (2,3-Dichlorobenzyl 5-Acetamido-4,7,8-tri-O-acetyl-9-

(4-chlorobenzamido)-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-non-

ulopyranosid)onate (12e). Compound 11e (47.0 mg, 0.07 mmol)

was reacted with p-chlorobenzoyl chloride (38.0 μL, 52.0 mg,

0.30 mmol) and triphenylphosphine (43.0 mg, 0.16 mmol). The

crude product was purified by chromatography on silica gel (0.5%

gradient of MeOH in DCM) to yield 12e (25.0 mg, 47%) as a

yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 1.78 (t, J = 12.4 Hz,

1H, H-3a), 1.80 (s, 3H, NHAc), 1.97, 2.04, 2.17 (3 s, 9H, 3 OAc),

2.63 (dd, J = 4.8, 12.8 Hz, 1H, H-3b), 2.90 (ddd, J = 3.3, 3.7, 15.1

Hz, 1H, H-9a), 3.64 (s, 3H, OMe), 4.01 (d, J = 10.8 Hz, 1H, H-6),

4.15 (q, J = 10.4 Hz, 1H, H-5), 4.23 (m, 1H, H-9b), 4.49 (A of AB,

J = 13.0 Hz, 1H, CH₂Ar), 4.30 (m, 2H, H-4, CH₂Ar), 5.11 (d, J =

1.6 Hz, 1H, H-7), 5.20 (m, 1H, H-8), 5.55 (d, J = 8.8 Hz, 1H,

NHAc), 7.03 (dd, J = 3.8 Hz, 1H, NH), 7.13 (t, J = 7.8 Hz, 1H,

CH_ar), 7.39 (m, 2H, CH_ar), 7.68, 7.93 (AA⁰, BB⁰of AA⁰BB⁰, J =

8.5 Hz, 4H, CH_ar). ¹³C NMR (CDCl₃) δ 21.3, 21.5, 21.6 (3 OAc),

23.5 (NHAc), 38.2 (C-3), 39.0 (C-9), 49.9 (C-5), 53.2 (OMe), 64.8

(CH₂Ar), 68.4 (C-8), 68.8 (C-7), 69.5 (C-4), 72.7 (C-6), 110.0

(C-2), 127.6, 128.1, 130.0, 131.8, 133.0, 136.0, 138.1 (12 C-Ar),

160.0, 168.0, 170.6, 170.9, 171.5, 172.8 (6 CO). ESI-MS calcd for

C₃₂H₃₅Cl₃N₂O₁₂[M þ Na]^þ767.13; found m/z: 767.12.

Sodium (4-Fluorobenzyl 5-Acetamido-9-(4-chlorobenzamido)-

3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyranosid)onate

(13b). Compound 12b (37.0mg, 0.05 mmol) was treated with 10%

aqueous NaOH (0.3 mL) in MeOH (1.2 mL). The crude product

was purified by chromatography on RP-18 (5% gradient of

MeOH in H₂O) to yield 13b as a white solid (21.1 mg, 70%).

1

[R]²⁰-0.22 (c 0.33, H₂O). H NMR (500 MHz, CD₃OD) δ

D

1.54 (m, 1H, H-3a), 1.90 (s, 3H, NHAc), 2.80 (m, 1H, H-3b), 3.35

(m, 1H, H-7), 3.42 (m, 1H, H-9a), 3.58-3.69 (m, 4H, H-4, H-5,

H-6, H-9b), 3.96 (m, 1H, H-8), 4.39, 4.69 (A, B of AB, J =

11.2 Hz, 2H, CH₂Ar), 6.90 (t, J = 8.8 Hz, 2H, CH_ar), 7.27 (m, 2H,

CH_ar), 7.35 (m, 2H, CH_ar), 7.72 (BB⁰of AA⁰BB⁰, J = 8.5 Hz, 2H,

CH_ar). ¹³CNMR(CD₃OD) δ22.7(NHAc), 42.7(C-3), 44.5 (C-9),

54.2 (C-5), 66.7 (CH₂Ar), 69.6 (C-4), 71.4 (C-8), 72.5 (C-7), 74.4

(C-6), 102.1 (C-2), 115.8 (J = 12.5 Hz), 130.1, 129.7, 131.1 (J =

21.3 Hz), 138.6, 162.0 (12 C-Ar), 164.6, 169.3, 174.4 (3 CO).

HRMS calcd for C₂₅H₂₈ClFN₂O₉[M - H]^-577.1367; found

577.1369.

Sodium (Pentafluorobenzyl 5-Acetamido-9-(4-chlorobenzamido)-

3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyranosid)onate (13c).

Compound 12c (25.0 mg, 0.03 mmol) was treated with 10%

aqueous NaOH (0.2 mL) in MeOH (1.0 mL). The crude product

was purified by chromatography on RP-18 (5% gradient of

MeOH in H₂O) to yield 13c as a white solid (8.00 mg, 39%).

[R]²⁰_D-0.02 (c 0.32, H₂O). ¹H NMR (500 MHz, D₂O) δ 1.63 (t,

J = 12.1 Hz, 1H, H-3a), 1.97 (s, 3H, NHAc), 2.71 (dd, J = 4.7,

12.4 Hz, 1H, H-3b), 3.50 (m, 2H, H-7, H-9a), 3.65 (ddd, J = 4.7,

9.5, 11.9 Hz, 1H, H-4), 3.74-3.79 (m, 3H, H-5, H-6, H-9b), 3.88

(ddd, J = 2.9, 7.8, 8.8 Hz, 1H, H-8), 4.66, 4.86 (A, B of AB, J =

11.7 Hz, 2H, CH₂Ar), 7.50, 7.73 (AA⁰, BB⁰of AA⁰BB⁰, J =8.6Hz,

4H, CH_ar). ¹³C NMR (D₂O) δ 21.9 (NHAc), 40.3 (C-3), 42.6

(C-9), 51.8 (C-5), 54.1 (CH₂Ar), 68.1 (C-4), 69.8 (C-7), 70.3 (C-8),

72.8 (C-6), 101.0 (C-2), 128.7, 128.7, 132.2, 136.2, 137.5, 139.1

Methyl (2,3-Difluorobenzyl 5-Acetamido-4,7,8-tri-O-acetyl-9-

(4-chlorobenzamido)-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-non-

ulopyranosid)onate (12f). Compound 11f (67.0 mg, 0.11 mmol)

1608 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4

Mesch et al.

(12 C-Ar), 170.1, 172.8, 175.0 (3 CO). HRMS calcd for

C₂₅H₂₃ClF₅N₂NaO₉[M - H]^-625.1018; found 625.1015.

Sodium (2-Naphthyl 5-Acetamido-9-(4-chlorobenzamido)-3,5,9-

trideoxy-^D-glycero-r-D-galacto-2-nonulopyranosid)onate (13d). Com-

pound 12d (25.0 mg, 0.03 mmol) was treated with 10% aqueous

NaOH (0.2 mL) in MeOH (1.0 mL). The crude product was

purified by chromatography on RP-18 (5% gradient of MeOH

in H₂O) to yield 13d as a white solid (14.0 mg, 70%). [R]²⁰_D-0.37

reduced pressure to give a yellow oil. The crude product was re-

acted with acetic anhydride (0.9 mL) in dry pyridine (1.7 mL). A

catalytic amount of DMAP was added, and stirring was continued

at rt. After 18 h, the reaction mixture was washed with CuSO₄(0.5

M, 4 ꢀ 5 mL), saturated aqueous NaHCO₃(1 ꢀ 5 mL), and H₂O

(1 ꢀ 5 mL). The organic layer was dried over Na₂SO₄, filtered, and

the solvent was evaporated. The crude product was purified by

chromatography on silica gel (EA:PE, gradient 1:1 to 2:1) to yield

14 (72 mg, 76%) as a white foam. [R]²⁰_D0.31 (c 1.68, CH₂Cl₂). ¹H

NMR (500 MHz, CDCl₃) δ 1.37 (s, 9H, t-Butyl), 1.95 (m, 1H,

H-3a), 2.02 (s, 3H, SMe), 2.10, 2.14, 2.17 (3 s, 9H, 3 OAc), 2.73

(dd, J = 4.6, 12.7 Hz, 1H, H-3b), 3.25 (dd, J = 6.0, 13.5 Hz, 1H,

H-9a), 3.61 (dd, J = 3.2, 13.5 Hz, 1H, H-9b), 3.73 (m, 1H, H-6),

3.80 (m, 4H, OMe, H-5), 4.25 (d, J = 10.4 Hz, 1H, NH), 4.78 (m,

1H, H-4), 5.29 (m, 1H, H-8), 5.41 (m, 1H, H-7). ¹³C NMR

(CDCl₃) δ 12.1 (SMe), 20.8, 21.0, 21.3 (3 OAc), 27.9 (3C, C-

(CH₃)₃), 38.0 (C-3), 50.4 (C-9), 50.6 (C-5), 52.9 (OMe), 67.6 (C-7),

68.1 (C-8), 70.0 (C-4), 73.7 (C-6), 80.2 (C-2), 83.0 (C(CH₃)₃), 155.2

(CONH), 168.2, 170.0, 170.6, 174.0 (4 CO). ESI-MS calcd for

C₂₂H₃₄N₄O₁₁S [M þ Na]^þ585.19; found m/z 585.15.

1

(c 0.46, H₂O). H NMR (500 MHz, CD₃OD) δ 1.60 (m, 1H,

H-3a), 1.90 (s, 3H, NHAc), 2.84 (d, J = 11.8 Hz, H-3b), 3.35

(d, J = 8.9 Hz, H-7), 3.42 (dd, J = 7.7, 13.7 Hz, H-9a), 3.60-3.68

(m, 4H, H-4, H-5, H-6, H-9b), 4.60 (A of AB, J=11.5 Hz, 1H,

CH₂Ar), 3.96 (m, 1H, H-8), 4.89 (B of AB, J = 11.5 Hz, 1H,

CH₂Ar), 7.31-7.36 (m, 4H, CH_ar), 7.40 (dd, J=1.5, 8.4 Hz, 1H,

CH_ar), 7.67-7.72 (m, 6H, CH_ar). ¹³C NMR (CD₃OD) δ 21.7

(NHAc), 43.3 (C-3), 43.4 (C-9), 53.1 (C-5), 68.6 (CH₂Ar), 71.3

(C-7), 72.0 (C-4), 73.3 (C-6), 110.0 (C-2), 125.7, 125.9, 126.3,

126.4, 127.6, 127.7, 127.9, 128.6, 129.1 (16 C-Ar), 174.6, 175.3

(3C, CO). HRMS calcd for C₂₉H₃₀ClN₂NaO₉[M þ Na]^þ

631.1438; found 631.1435.

Sodium (2,3-Dichlorobenzyl 5-Acetamido-9-(4-chlorobenzamido)-

3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyranosid)onate (13e).

Compound 12e (25.0 mg, 0.03 mmol) was treated with 10%

aqueous NaOH (0.2 mL) in MeOH (1.0 mL). The crude product

was purified by chromatography on RP-18 (5% gradient of

MeOH in H₂O) to yield 13e as a white solid (10.0 mg, 50%).

[R]²⁰_D-0.19 (c 0.53, H₂O). ¹H NMR (500 MHz, CD₃OD) δ 1.69

(t, J = 11.8 Hz, 1H, H-3a), 1.93 (s, 3H, NHAc), 2.01 (dd, J = 9.1,

11.8 Hz, 1H, H-3b), 3.36 (dd, J = 1.7, 9.0 Hz, 1H, H-7), 3.45 (dd,

J = 7.6, 13.6Hz, 1H, H-9a), 3.59 (m, 1H, H-6), 3.65-3.70 (m, 3H,

H-4, H-5, H-9b), 3.92 (m, 1H, H-8), 4.65, 4.89 (A, B of AB, J =

13.6 Hz, 2H, CH₂Ar), 7.18 (t, J = 7.9 Hz, 1H, CH_ar), 7.32 (dd,

J = 1.5, 8.0 Hz, 1H, CH_ar), 7.39 (AA⁰of AA⁰BB⁰, J = 8.5 Hz, 2H,

CH_ar), 7.49 (dd, J = 1.1, 7.7 Hz, 1H, CH_ar), 7.74 (BB⁰of AA⁰BB⁰,

J = 8.5 Hz, 2H, CH_ar). ¹³C NMR (CD₃OD) δ 21.6 (NHAc), 41.5

(C-3), 43.4 (C-9), 53.1 (C-5), 63.5 (CH₂Ar), 68.6 (C-4), 70.2 (C-8),

71.5 (C-7), 73.4 (C-6), 102.5 (C-2), 113.3, 117.0, 118.4, 127.4,

127.5, 128.6, 128.9, 129.0, 139.6 (12 C-Ar), 163.8, 174.5 (3C, 3

CO). HRMS calcd for C₂₅H₂₇Cl₃N₂O₉[M þ Na]^þ627.0682;

found 627.0683.

Sodium (2,3-Difluorobenzyl 5-Acetamido-9-(4-chlorobenzamido)-

3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyranosid)onate (13f).

Compound 12f (44.0 mg, 0.06 mmol) was treated with 10% aqueous

NaOH (0.3 mL) in MeOH (1.5 mL). The crude product was purified

by chromatography on RP-18 (5% gradient of MeOH in H₂O) to

yield 13f as a white solid (23.0 mg, 64%). [R]²⁰_D-0.18 (c1.13, H₂O).

¹H NMR (500 MHz, CD₃OD) δ 1.56 (m, 1H, H-3a), 1.90 (s, 3H,

NHAc), 2.84 (dd, J = 3.0, 12.2 Hz, 1H, H-3b), 3.37 (d, J = 9.0 Hz,

1H, H-7), 3.46 (dd, J = 7.7, 13.6 Hz, 1H, H-9a), 3.59 (m, 1H, H-6),

3.63-3.70 (m, 3H, H-4, H-5, H-9b), 3.96 (m, 1H, H-8), 4.60, 4.85

(A, B of AB, J = 12.2 Hz, 2H, CH₂Ar), 7.04 (m, 2H, CH_ar), 7.24

(t, J = 6.5 Hz, 1H, CH_ar), 7.38, 7.75 (AA⁰, BB⁰of AA⁰BB⁰, J = 8.5

Hz, 4H, CH_ar). ¹³C NMR (CD₃OD) δ 21.6 (NHAc), 41.5 (C-3),

43.5 (C-9), 53.1 (C-5), 59.2 (CH₂Ar), 68.6 (C-4), 70.3 (C-8), 71.4

(C-7), 73.4 (C-6), 101.0 (C-2), 115.9, 116.1 (J = 17.3 Hz), 124.2,

125.3, 128.6, 129.1, 133.5, 137.6 (12 C-Ar), 168.3, 173.0, 174.5

(3 CO). HRMS calcd for C₂₅H₂₇ClF₂N₂O₉[M þ Na]^þ595.1273;

found 595.1272.

Methyl (Methyl 5-Amino-4,7,8-tri-O-acetyl-9-azido-3,5,9-tri-

deoxy-2-thio-^D-glycero-r-D-galacto-2-nonulopyranosid)onate (15).

Compound 14 (120 mg, 0.21 mmol) was dissolved in 4 M PhOH

(in abs DCM; 7.5 mL) and 4 M TMSCl (in abs DCM; 1.5 mL).

The reaction mixture was stirred at rt for 2 h. The reaction mix-

ture was washed with saturated aqueous NaHCO₃(2 ꢀ 5 mL) and

H₂O (1 ꢀ 5 mL). The organic layer was dried over Na₂SO₄, fil-

tered, and the solvent was evaporated. The crude product was

purified by chromatography on silica gel (EA:PE, gradient 1:1

to 8:1) to yield 15 as a white foam (74 mg, 70%). ¹H NMR (500

MHz, CDCl₃) δ 2.01 (s, 3H, SMe), 2.04 (m, 1H, H-3a), 2.10, 2.11,

2.19 (3 s, 9H, 3 OAc), 2.71 (dd, J = 4.7, 12.8 Hz, 1H, H-3b), 3.38

(d, J = 10.5 Hz, 1H, H-6), 3.66 (dd, J = 3.3, 13.3 Hz, 1H, H-9a),

3.70 (dd, J = 2.8, 13.3 Hz, 1H, H-9b), 3.76 (dd, J = 1.3, 9.4 Hz,

1H, H-7), 3.78 (s, 3H, OMe), 3.98 (dt, J = 8.0, 10.5 Hz, 1H, H-5),

4.93 (dd, J = 6.9, 10.7 Hz, 1H, H-4), 5.26 (t, J = 3.9, 9.4 Hz, 1H,

H-8), 5.94 (d, J = 7.8 Hz, 2H, NH₂). ¹³C NMR (CDCl₃) δ 12.0

(SMe), 21.1, 21.2, 23.2 (3 OAc), 37.7 (C-3), 51.2, 52.1, 52.9 (3C, C-

5 C-9, OMe), 66.8, 69.3, 70.0 (3C, C-4, C-7, C-8), 75.3 (C-6), 82.2

(C-2), 167.9, 170.2, 172.4, 172.9 (4 CO). ESI-MS calcd for

C₁₇H₂₆N₄O₉S [M þ Na]^þ485.14; found m/z 485.13.

Methyl (Methyl 5-Fluoroacetamido-4,7,8-tri-O-acetyl-9-azi-

do-3,5,9-trideoxy-2-thio-^D-glycero-r-D-galacto-2-nonulopyra-

nosid)onate (16a). Compound 15 (74.0 mg, 0.16 mmol) was

dissolved in dry DCM (1.8 mL) and cooled to 0 °C. Then, mono-

fluoroacetic chloride was added dropwise (30.0 mg, 31.0 μL, 0.32

mmol), followed by the addition of NEt₃(324 mg, 0.45 mL, 3.2

mmol) and DMAP (9.8 mg, 0.08 mmol). Stirring was continued

overnight, and the reaction mixture was allowed to come to rt.

The brown solution was washed with saturated aqueous NaHCO₃

(3 ꢀ 5 mL), saturated aqueous NaCl (1 ꢀ 5 mL), and H₂O (1 ꢀ 5

mL). The organic phase was dried over Na₂SO₄, filtered, and

concentrated under reduced pressure. The crude product was

purified by chromatography on silica gel (EA:PE, gradient 1:1 to

1

2:1) to yield 16a (71 mg, 85%). [R]²⁰0.34 (c 1.2, CH₂Cl₂). H

D

NMR (500 MHz, CDCl₃) δ 1.98 (m, 1H, H-3a), 2.03 (s, 3H, SMe),

2.12, 2.15, 2.21 (3 s, 9H, 3 OAc), 2.79 (dd, J = 4.7, 12.8 Hz, 1H,

H-3b), 3.24 (dd, J = 4.8, 13.6 Hz, 1H, H-9a), 3.64 (dd, J = 2.8,

13.6 Hz, 1H, H-9b), 3.84 (s, 3H, OMe), 3.90 (dd, J = 1.9, 10.7 Hz,

1H, H-6), 4.14 (m, 1H, H-5), 4.71 (m, 2H, CH₂F), 4.92 (td, J = 4.7,

11.6 Hz, 1H, H-4), 5.32 (m, 2H, H-7, H-8), 6.09 (dd, J = 3.3, 10.3

Hz, 1H, NH). ¹³C NMR (CDCl₃) δ 12.2 (SMe), 20.9 (3C, OAc),

37.8 (C-3), 48.6 (C-5), 50.6 (C-9), 53.1 (OMe), 67.8 (C-7), 69.4, 69.6

(2C, C-4, C-8), 74.1 (C-6), 80.8 (J = 186.1 Hz, CH₂F), 83.1 (C-2),

168.2, 170.2, 170.6 (5C, CO). ESI-MS calcd for C₁₉H₂₇FN₄O₁₀S

[M þ Na]^þ545.14; found m/z 545.15.

Methyl (Methyl 5-tert-Butyloxycarbonylamino-4,7,8-tri-O-

acetyl-9-azido-3,5,9-trideoxy-2-thio-^D-glycero-r-D-galacto-2-

nonulopyranosid)onate (14). Compound 10 (85.0 mg, 0.17

mmol) was dissolved in dry THF (0.7 mL) under argon. Boc₂O

(74.0 mg, 0.34 mmol) was added to the reaction mixture, followed

by DMAP (4.50 mg, 0.02 mmol). The reaction mixture was heated

up to 60 °C for 5 h. After cooling to rt, MeOH (0.7 mL) and

N₂H₄H₂O (52 μL, 1.1 mmol) were added and stirring was

3

continued for 16 h. The reaction mixture was washed successively

with 0.1 M HCl (1 ꢀ 5 mL), 0.5 M CuSO₄(1 ꢀ 5 mL), saturated

aqueous NaHCO₃(2 ꢀ 5 mL), and H₂O (1 ꢀ 5 mL). The organic

layer was dried over Na₂SO₄, filtered, and concentrated under

Methyl (Methyl 5-Chloroacetamido-4,7,8-tri-O-acetyl-9-azido-

3,5,9-trideoxy-2-thio-^D-glycero-r-D-galacto-2-nonulopyranosid)-

onate (16b). Compound 15 (55.0 mg, 0.12 mmol) was dissolved

in dioxane/water (0.5 mL/0.1 mL), treated with triethylamine

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1609

˚

dissolved in dry acetonitrile (2 mL). Powdered MS 3 A (50 mg)

(48.6 mg, 34 μL, 0.48 mmol), and cooled to 0 °C. Then,

chloroacetic anhydride (41.0 mg, 0.48 mmol) was added, and

stirring was continued at rt for 3 h. The reaction mixture was

diluted with CHCl₃(10.0 mL) and washed successively with

saturated aqueous NaHCO₃(3 ꢀ 5 mL) and H₂O (1 ꢀ 5 mL).

The organic layer was dried over Na₂SO₄, filtered, and concen-

trated under reduced pressure. The crude product was purified by

chromatography on silica gel (gradient, PE:EA; 1:1 to 1:2) to yield

16b as a white foam (42 mg, 66%). [R]²⁰_D0.33 (c 0.95, CH₂Cl₂). ¹H

NMR (500 MHz, CDCl₃) δ 1.91 (t, J = 12.2 Hz, 1H, H-3a), 1.96

(s, 3H, SMe), 2.06, 2.08, 2.13 (3 s, 9H, 3 OAc), 2.72 (dd, J = 4.7

Hz, 12.8 Hz, 1H, H-3b), 3.18 (m, 1H, H-9a), 3.59 (m, 1H, H-9b),

3.78 (s, 3H, OMe), 3.85 (A of AB, J = 15.0 Hz, 1H, CH₂Cl), 3.86

(m, 1H, H-6), 3.93 (B of AB, J = 15.0 Hz, 1H, CH₂Cl), 4.03 (m,

1H, H-5), 4.90 (dt, J = 4.7 Hz, 11.5 Hz, 1H, H-4), 5.24 (m, 2H,

H-7, H-8), 6.28 (d, J = 10.1 Hz, 1H, NH).¹³C NMR (CDCl₃)

δ 12.1 (SMe), 20.8, 20.9, 21.1 (3 OAc), 37.9 (C-3), 42.5 (CH₂Cl),

49.7 (C-9), 50.6 (C-5), 53.1 (OMe), 67.1, 67.9, 69.2 (3C, C-4, C-7,

C-8), 74.2 (C-6), 83.1 (C-2), 166.6, 167.8, 170.3, 170.6 (5C, CO).

ESI-MS calcd for C₁₉H₂₇ClN₄O₁₀S [M þ Na]^þ561.10; found m/z

561.37.

and 2,3-difluorobenzyl alcohol (35.0 μL, 42.0 mg, 0.29 mmol)

were added. The reaction mixture was stirred at rt for 1.5 h. Then

the suspension was cooled to -40 °C and was subsequently treated

with N-iodosuccinimide (35.0 mg, 0.16 mmol) and triflic acid

(8.00 μL, 13.0 mg, 0.09 mmol). After 30 min, the reaction mixture

was warmed to -30 °C and stirring was continued for 20 h. After

warming to rt, the mixture was filtered through a pad of celite

and washed with 20% Na₂S₂O₃(1 ꢀ 2 mL), saturated aqueous

NaHCO₃(3 ꢀ 5 mL), and H₂O (1 ꢀ 5 mL). The organic phase was

dried over Na₂SO₄, filtered, and concentrated under reduced

pressure. The crude product was purified by chromatography

on silica gel (1% gradient iPrOH in petrol ether/DCM 2:1) to yield

16a (43 mg, 66%) as a colorless oil. [R]²⁰_D-0.04 (c 0.73, CH₂Cl₂).

¹H NMR (500 MHz, CDCl₃) δ 2.03 (s, 3H, OAc), 2.04 (m, 1H,

H-3a), 2.16, 2.21 (2 s, 6H, 2 OAc), 2.69 (dd, J = 4.7, 12.9 Hz, 1H,

H-3b), 3.26 (dd, J = 5.3, 13.5 Hz, 1H, H-9a), 3.58 (dd, J = 2.8,

13.5 Hz, 1H, H-9b), 3.79 (s, 3H, OMe), 4.21 (m, 2H, H-5, H-6),

4.53 (A of AB, J = 12.0 Hz, 1H, CH₂Ar), 4.63 (m, 2H, CH₂F),

4.83 (B of AB, J = 12.3 Hz, 1H, CH₂Ar), 4.95 (ddd, J = 4.7, 10.0,

12.1 Hz, 1H, H-4), 5.35 (m, 2H, H-7, H-8), 6.18 (dd, J = 3.3, 9.0

Hz, 1H, NHFAc), 7.11 (m, 3H, CH_ar). ¹³C NMR (CDCl₃) δ 20.9

(3C, OAc), 37.9 (C-3), 48.6 (C-5), 50.9 (C-9), 53.0 (OMe), 60.5

(CH₂Ar), 67.8 (C-7), 68.7 (C-8), 69.3 (C-4), 72.6 (C-6), 80.1 (J =

186.1 Hz, CH₂F), 98.7 (C-2), 116.9, 117.1, 125.1, 126.6 (6C,

C-Ar), 168.0, 170.2, 170.7(5C, CO). ESI-MScalcd for C₂₅H₂₉F₃-

N₄O₁₁[M þ Na]^þ641.17; found m/z 641.14.

Methyl (Methyl 5-o-Nitrotoluenesulfonamido-4,7,8-tri-O-acetyl-

9-azido-3,5,9-trideoxy-2-thio-^D-glycero-r-D-galacto-2-nonulopyra-

nosid)onate (16c). Compound 15 (73.0 mg, 0.16 mmol) was

dissolved in dry DCM (3.0 mL), and it was cooled to 0 °C. Nosyl-

chloride (105 mg, 0.47 mmol), NEt₃(34.0 μL, 48.0 mg, 0.47

mmol), and DMAP (10.0 mg, 0.08 mmol) were added succes-

sively. Thereactionmixture wasstirredatrtovernight.Thenitwas

washed with saturated aqueous NaHCO₃(2 ꢀ 5 mL) and H₂O

(1 ꢀ 5 mL). The organic phase was dried over Na₂SO₄, filtered,

and concentrated under reduced pressure. The crude product was

purified by chromatography on silica gel (EA:PE, gradient 1:1 to

2:1) to yield 16c (81 mg, 78%). ¹H NMR(500 MHz, CDCl₃) δ1.85

(m, 1H, H-3a), 2.02 (s, 3H, SMe), 2.10, 2.13, 2.21 (3 s, 9H, 3 OAc),

2.80 (m, 1H, H-3b), 3.32 (dd, J = 6.2, 13.4 Hz, 1H, H-9a), 3.57

(dd, J = 3.2, 13.4 Hz, 1H, H-9b), 3.80 (m, 1H, H-5), 3.82 (s, 3H,

OMe), 3.91 (d, J = 10.5 Hz, 1H, H-6), 4.97 (td, J = 4.7, 11.4 Hz,

1H, H-4), 5.30 (m, 2H, H-7, H-8), 5.75 (d, J = 9.4 Hz, 1H, NH),

7.70 (m, 2H, CH_ar), 7.90 (d, J = 7.9, 1H, CH_ar), 8.10 (d, J = 6.5,

1H, CH_ar). ¹³C NMR (CDCl₃) δ 12.1 (SMe), 20.5, 21.1, 21.1

(3 OAc), 38.1 (C-3), 50.8 (C-9), 53.2, 53.5 (2C, C-5, OMe), 68.8

(C-7), 69.7 (C-4), 70.4 (C-8), 74.7 (C-6), 82.8 (C-2), 125.5, 130.4,

133.5, 135.5, 147.5 (6C, C-Ar), 167.8, 170.4 (4C, CO). ESI-MS

calcd for C₂₃H₂₉N₅O₁₃S₂[M - H]^-646.12; found m/z 646.56.

Methyl (Methyl 5-Methylsulfonamido-4,7,8-tri-O-acetyl-9-

azido-3,5,9-trideoxy-2-thio-^D-glycero-r-D-galacto-2-nonulopyra-

nosid)onate (16d). Compound 15 (50.0 mg, 0.11 mmol) was

dissolved in dry DCM (2.0 mL) under argon atmosphere and

subsequently cooled to 0 °C. Methanesulfonylchloride (25.0 μL,

37.0 mg, 0.32 mmol), NEt₃(45.0 μL, 33.0 mg, 0.32 mmol), and a

catalytic amount of DMAP were added successively. The reaction

mixture was stirred at 0 °C overnight. Then it was washed with

saturated aqueous NaHCO₃(2 ꢀ 5 mL) and H₂O (1 ꢀ 5 mL). The

organic phase was dried over Na₂SO₄, filtered, and concentrated

under reduced pressure. The crude product was purified by

chromatography on silica gel (EA:PE, gradient 1:1 to 2:1) to yield

16d (30 mg, 66%). ¹H NMR (500 MHz, CDCl₃) δ1.98 (t, J = 12.3

Hz, 1H, H-3a), 2.03 (s, 3H, SMe), 2.13, 2.17 (3 s, 9H, 3 OAc), 2.68

(dd, J = 4.7, 12.7 Hz, 1H, H-3b), 3.14 (s, 3H, CH₃), 3.29 (dd, J =

6.1, 13.4, 1H, H-9a), 3.66 (m, 2H, H-5, H-9b), 3.77 (s, 3H, OMe),

3.88 (dd, J =1.6, 10.6 Hz, 1H, H-6), 4.55 (d, J = 9.8 Hz, 1H, NH),

5.16 (td, J = 4.7, 11.5 Hz, 1H, H-4), 5.26 (m, 1H, H-8), 5.48 (dd,

J = 1.6, 7.2 Hz, 1H, H-7). ¹³C NMR (CDCl₃) δ 14.3 (SMe), 20.9,

21.1, 21.4 (3 OAc), 31.7 (CH₃), 38.2 (C-3), 50.7 (C-9), 52.5 (C-5),

53.0 (OMe), 68.3, 69.6 (2C, C-7, C-8), 70.4 (C-4), 74.5 (C-6), 82.8

(C-2), 167.8, 170.3, 171.7, 172.2 (4 CO). ESI-MS calcd for

C₁₈H₂₈N₄O₁₁S₂[M þ Na]^þ563.12; found m/z 563.18.

Methyl ((2,3-Difluorobenzyl) 5-Chloroacetamido-4,7,8-tri-O-

acetyl-9-azido-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulo-

pyranosid)onate (17b). Compound 16b (63.0 mg, 0.12 mmol) was

reacted with 2,3-difluorobenzyl alcohol (41.0 μL, 0.36 mmol),

N-iodosuccinimide (42.0 mg, 0.19 mmol), and triflic acid (8.0 μL,

14.4 mg, 0.1 mmol). The crude product was purified by chroma-

tography on silica gel (0.5% gradient iPrOH in PE:DCM, 8:4) to

yield 17b (50 mg, 68%) as a colorless oil. [R]²⁰-0.01,

D

1

(c 1.05, CH₂Cl₂). H NMR (500 MHz, CDCl₃) δ 2.02 (m, 1H,

H-3a), 2.03, 2.16, 2.21 (3 s, 9H, 3 OAc), 2.70 (dd, J = 4.6, 12.9 Hz,

1H, H-3b), 3.28 (dd, J = 5.8, 13.5 Hz, 1H, H-9a), 3.59 (dd, J =

3.0, 13.5 Hz, 1H, H-9b), 3.81 (s, 3H, OMe), 3.93, 4.01 (A, B ofAB,

J = 15.0 Hz, 2H, CH₂Cl), 4.11 (m, 1H, H-5), 4.24 (dd, J = 2.1,

10.7 Hz, 1H, H-6), 4.54, 4.83 (A, B of AB, J = 12.0 Hz, 2H,

CH₂Ar), 4.99 (m, 1H, H-4), 5.32 (dd, J = 2.1, 7.8 Hz, 1H, H-7),

5.36 (m, 1H, H-8), 6.41 (d, J = 10.1 Hz, 1H, NH), 7.06-7.17

(m, 3H, CH_ar). ¹³C NMR (CDCl₃) δ 20.8, 20.9, 21.1 (3 OAc), 37.9

(C-3) 42.4 (CH₂Cl), 49.7 (C-5), 50.9 (C-9), 60.5 (CH₂Ar), 67.9,

68.1, 68.4 (3C, C-4, C-7, C-8), 72.6 (C-6), 98.7 (C-2), 117.0, 123.9,

125.1, 126.5, 149.5, 151.4 (6 C-Ar), 166.7, 168.0, 170.2, 170.3,

170.7 (5 CO). ESI-MS calcd for C₂₅H₂₉Cl₂FN₄O₁₁[M þ Na]^þ

657.14; found m/z 657.29.

Methyl ((2,3-Difluorobenzyl) 5-(o-Nitrotoluenesulfonamido)-

4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-^D-glycero-r-D-galacto-

2-nonulopyranosid)onate (17c). Compound 16c (62.0 mg, 0.10

mmol) was reacted with 2,3-difluorobenzyl alcohol (30.0 μL,

38.0 mg, 0.29 mmol), N-iodosuccinimide (32.0 mg, 0.14 mmol),

and triflic acid (7.00 μL, 12.0 mg, 0.08 mmol). The crude product

was purified by chromatography on silica gel (toluene/EA,

1

gradient 1:1 to 2:1) to yield 17c (44 mg, 61%). H NMR (500

MHz, CDCl₃) δ 1.84 (t, J = 12.4 Hz, 1H, H-3a), 2.08, 2.17, 2.28

(3 s, 9H, 3 OAc), 2.64 (dd, J = 4.6, 12.8 Hz, 1H, H-3b), 3.29

(m, 1H, H-9a), 3.45 (m, 1H, H-9b), 3.73 (s, 3H, OMe), 3.79

(m, 2H, H-5, H-7), 4.12 (d, J = 8.8 Hz, 1H, H-6), 4.46, 4.77 (A, B

of AB, J = 12.0 Hz, 2H, CH₂Ar), 4.91 (td, J = 4.6, 11.5 Hz, 1H,

H-4), 5.25 (d, J = 7.8 Hz, 1H, H-8), 5.60 (d, J = 9.4 Hz, 1H, NH),

7.09 (m, 3H, CH_ar), 7.69 (m, 2H, CH_ar), 7.85 (d, J = 7.9 Hz, 1H,

CH_ar), 8.07 (d, J = 7.8 Hz, 1H, CH_ar). ¹³C NMR (CDCl₃) δ 21.1

(3C, OAc), 38.1 (C-3), 51.1 (C-5), 53.1 (C-9), 53.6 (OMe), 60.5

(CH₂Ar), 68.7, 68.8 (2C, C-4, C-7), 69.8 (C-8), 73.2 (C-6), 125.5,

130.4, 133.5, 147.6 (12C, C-Ar), 168.0, 170.1, 170.3, 170.5 (4 CO).

ESI-MS calcd for C₂₉H₃₁F₂N₅O₁₄S [M þ Na]^þ766.16; found m/z

766.14.

Methyl ((2,3-Difluorobenzyl) 5-Fluoroacetamido-4,7,8-tri-O-

acetyl-9-azido-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulo-

pyranosid)onate (17a). Compound 16a (55.0 mg, 0.16 mmol) was

1610 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4

Mesch et al.

Methyl ((2,3-Difluorobenzyl) 5-Methylsulfonamido-4,7,8-tri-

O-acetyl-9-azido-3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonulo-

pyranosid)onate (17d). Compound 16d (56.0 mg, 0.10 mmol) was

reacted with 2,3-difluorobenzyl alcohol (33.0 μL, 42.0 mg, 0.29

mmol), N-iodosuccinimide (35.0 mg, 0.16 mmol), and triflic acid

(8.00 μL, 13.0 mg, 0.09 mmol). The crude product was purified

by chromatography on silica gel (1% gradient iPrOH in petrol

ether/DCM 2:1) to yield 17d (37 mg, 56%). ¹H NMR (500 MHz,

CDCl₃) δ 1.95 (t, J = 12.5, 1H, H-3a), 2.13, 2.18(2 s, 9H, 3 OAc),

2.61 (dd, J = 4.6, 12.7 Hz, 1H, H-3b), 3.02 (s, 3H, CH₃), 3.31

(dd, J = 6.3, 13.5 Hz, 1H, H-9a), 3.60 (dd, J = 3.3, 13.5 Hz, 1H,

H-9b), 3.71 (m, 4H, H-5, OMe), 4.12 (dd, J = 1.7, 10.7 Hz, 1H,

H-6), 4.54, 4.81 (A, B of AB, J = 11.8 Hz, 2H, CH₂Ar), 5.09 (m,

1H, H-4), 5.31 (m, 1H, H-8), 5.49 (dd, J = 1.7, 7.5 Hz, 1H, H-7),

7.11 (m, 3H, CH_ar). ¹³C NMR (CDCl₃) δ 20.9, 21.1, 21.4

(3 OAc), 38.1 (C-3), 42.3 (CH₃), 50.8 (C-9), 52.6, 53.0 (2C,

OMe, C-5), 60.4 (CH₂Ar), 68.1 (C-4), 68.8 (C-7), 70.0 (C-8),

72.8 (C-6), 98.5 (C-2), 116.9, 117.0, 124.0, 125.1 (6C, C-Ar),

167.7, 170.1, 170.7, 172.1 (4 CO). ESI-MS calcd for C₂₄H₃₀-

F₂N₄O₁₂S [M þ Na]^þ659.15; found m/z 659.24.

17c (44.0 mg, 0.06 mmol) was dissolved in dry DCE (2 mL).

p-Chlorobenzoyl chloride (30.0 μL, 40.0 mg, 0.23 mmol) and

triphenylphosphine (31.0 mg, 0.12 mmol) were added. The reac-

tion mixture was stirred at rt overnight. The crude product was

purified by chromatography on silica gel (EA:PE, gradient 1:1 to

1

2:1) to yield 18c (29 mg, 58%) as a white foam. H NMR (500

MHz, CDCl₃) δ 1.90 (m, 1H, H-3a), 2.04, 2.14, 2.32 (3 s, 9H, 3

OAc), 2.69 (dd, J = 4.5, 12.7 Hz, 1H, H-3b), 2.92 (d, J = 15.2 Hz,

1H, H-9a), 3.75 (s, 3H, OMe), 3.91 (m, 1H, H-5), 4.12 (m, 1H,

H-6), 4.38 (m, 1H, H-9b), 4.52 (A of AB, J = 11.9 Hz, 1H,

CH₂Ar), 4.84 (m, 2H, H-4, CH₂Ar), 5.31 (m, 2H, H-7, H-8), 5.64

(d, J = 9.4 Hz, 1H, NH), 7.13 (m, 4H, CH_ar), 7.21 (m, 1H, NH),

7.40 (m, 3H, CH_ar), 7.72 (m, 4H, CH_ar). ¹³C NMR (CDCl₃) δ

20.3, 21.2, 21.3 (3 OAc), 38.2, 38.3 (2C, C-3, C-9), 53.0 (OMe),

53.7 (C-5), 60.6 (CH₂Ar), 68.2, 68.4 (2C, C-7, C-8), 69.0 (C-4),

72.6 (C-6), 98.2 (C-2), 117.0, 123.9, 125.6, 128.8, 130.3, 131.5,

132.1, 132.8, 133.3 (18C, C-Ar), 166.6, 167.7, 169.9, 170.4, 172.4

(5 CO). ESI-MS calcd for C₃₆H₃₆ClF₂N₃O₁₅S [M þ Na]^þ878.15;

found m/z 878.28.

Methyl ((2,3-Difluorobenzyl) 5-Methylsulfonamido-4,7,8-tri-

O-acetyl-9-(4-chlorobenzamido)-3,5,9-trideoxy-^D-glycero-r-D-

galacto-2-nonulopyranosid)onate (18d). Compound 17d (35.0

mg, 0.06 mmol) was dissolved in dry DCM (2 mL). p-Chloro-

benzoyl chloride(28.0μL, 38.0 mg, 0.22 mmol) andtriphenylpho-

sphine (29.0 mg, 0.11 mmol) were added. The reaction mixture

was stirred at rt overnight. The crude product was purified by

chromatography on silica gel (EA:PE, gradient 1:1 to 2:1) to yield

18d (21 mg, 52%) as a white foam. ¹H NMR (500 MHz, CDCl₃)

δ 1.98 (t, J = 13.6 Hz, 1H, H-3a), 2.05, 2.10, 2.13 (3 s, 9H, 3 OAc),

2.67 (dd, J = 4.6, 12.7 Hz, 1H, H-3b), 2.99 (s, 3H, CH₃), 3.05 (m,

1H, H-9a), 3.70 (s, 3H, OMe), 3.77 (m, 1H, H-5), 4.09 (d, J = 1.5

Hz, 1H, H-6), 4.30 (ddd, J = 3.0, 8.2, 15.1 Hz, 1H, H-9b), 4.54 (A

of AB, J = 12.1 Hz, 1H, CH₂Ar), 4.59 (d, J = 9.6 Hz, 1H, NH),

4.83(B of AB, J = 11.9 Hz, 1H, CH₂Ar), 4.99(ddd, J = 4.6, 10.4,

12.2 Hz, 1H, H-4), 5.30 (m, 1H, H-8), 5.37 (dd, J = 1.7, 9.6 Hz,

1H, H-7), 7.13 (m, 4H, CH_ar, NH), 7.40, 7.75 (AA⁰, BB⁰of

AA⁰BB⁰, J = 8.5 Hz, 4H, CH_ar). ¹³C NMR (CDCl₃) δ 21.2, 21.2,

21.3 (3 OAc), 29.7 (C-3), 38.2, 38.8 (2C, C-9, CH₃), 42.3 (C-5),

52.8 (OMe), 60.5 (CH₂Ar), 68.1, 68.5, 69.1 (3C, C-4, C-7, C-8),

72.4 (C-6), 98.4 (C-2), 116.9, 117.1, 125.3, 128.4, 128.9, 132.1,

132.7, 137.8 (12C, C-Ar), 166.4, 167.7, 170.5, 171.6, 172.6 (5 CO).

ESI-MS calcd for C₃₁H₃₅ClF₂N₂O₁₃S [M þ Na]^þ771.15; found

m/z 771.29.

Methyl ((2,3-Difluorobenzyl) 5-Fluoroacetamido-4,7,8-tri-O-

acetyl-9-(4-chlorobenzamido)-3,5,9-trideoxy-^D-glycero-r-D-galacto-

2-nonulopyranosid)onate (18a). Compound 17a (55.0 mg, 0.09

mmol) was dissolved in dry DCM (2 mL). p-Chlorobenzoyl

chloride (45.0 μL, 62.0 mg, 0.36 mmol) and triphenylphos-

phine (47.0 mg, 0.18 mmol) were added. The reaction mixture

was stirred at rt overnight. Afterward, the reaction mixture

was washed with saturated aqueous NaHCO₃(3 ꢀ 5 mL) and

H₂O (1 ꢀ 5 mL). The organic phase was dried over Na₂SO₄, fil-

tered, and concentrated under reduced pressure. The crude

product was purified by chromatography on silica gel (EA:PE,

gradient 1:1 to 2:1) to yield 18a (31 mg, 48%) as a white foam. ¹H

NMR (500 MHz, CDCl₃) δ 1.97 (m, 1H, H-3a), 1.97, 2.09, 2.18

(3 s, 9H, 3 OAc), 2.64 (dd, J = 4.6, 12.8 Hz, 1H, H-3b), 2.94 (dd,

J = 3.9, 15.0 Hz, 1H, H-9a), 3.69 (s, 3H, OMe), 4.09 (m, 1H,

H-6), 4.22 (m, 2H, H-5, H-9b), 4.46 (A of AB, J = 12.0 Hz, 1H,

CH₂Ar), 4.64 (m, 2H, CH₂F), 4.78 (B of AB, J = 12.0 Hz, 1H,

CH₂Ar), 4.86 (m, 1H, H-4), 5.11 (dd, J = 2.0, 9.8 Hz, 1H, H-7),

5.27 (m, 1H, H-8), 6.12 (dd, J = 3.3, 10.0 Hz, 1H, NHFAc), 6.89

(dd, J = 4.3, 7.9 Hz, 1H, NH), 7.03 (m, 3H, CH_ar), 7.34, 7.67 (AA⁰,

BB⁰of AA⁰BB⁰, J = 8.5 Hz, 4H, CH_ar). ¹³C NMR (CDCl₃) δ 20.8,

21.1 (3C, OAc), 37.9 (C-3), 38.7 (C-9), 48.7 (C-5), 52.9 (OMe), 60.5

(CH₂Ar), 67.8 (C-7), 68.2 (C-8), 68.7 (C-4), 72.0 (C-6), 80.0 (J =

186.0 Hz, CH₂F), 98.6 (C-2), 117.1, 124.0, 125.3, 126.6, 128.4,

128.8, 131.5, 132.6, 137.8 (12C, C-Ar), 166.4, 167.8, 168.3, 170.5,

170.7, 172.3 (6 CO). ESI-MS calcd for C₃₂H₃₄ClF₃N₂O₁₂[M þ

Na]^þ753.18; found m/z 753.19.

Sodium ((2,3-Difluorobenzyl) 5-Fluoroacetamido-9-(4-chloro-

benzamido)-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyr-

anosid)onate (19a). Compound 18a (28.0 mg, 0.04 mmol) was

dissolved in THF/water (2.0 mL/0.5 mL) and was reacted

with LiOH (9.00 mg, 0.38 mmol). The reaction mixture was

stirred at rt for 4 h. 7% HCl (aq) was added to adjust the pH to

7. The crude product was purified by reversed-phase chroma-

tography (RP-18, 10% gradient MeOH in H₂O) followed by

ion exchange chromatography (Dowex-50) and P2 size exclu-

Methyl ((2,3-Difluorobenzyl) 5-Chloroacetamido-4,7,8-tri-O-

acetyl-9-(4-chloro-benzamido)-3,5,9-trideoxy-^D-glycero-r-D-galacto-

2-nonulopyranosid)onate (18b). Compound 17b (52.2 mg, 0.08

mmol) was reacted with p-chlorobenzoyl chloride (23.0 μL, 31.0

mg, 0.18 mmol) and triphenylphosphine (47.0 mg, 0.18 mmol).

The crude product was purified by chromatography on silica gel

(0.5% gradient of MeOH in DCM) to yield in 18b (25.0 mg, 48%).

[R]²⁰_D0.04 (c 1.06, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ 2.00

(m, 1H, H-3a), 2.03, 2.16, 2.25 (3 s, 9H, 3 OAc), 2.71 (m, 1H,

H-3b), 3.01 (m, 1H, H-9a), 3.77 (s, 3H, OMe), 3.97 (m, 2H, CH₂Cl),

4.11 (m, 1H, H-6), 4.20 (m, 1H, H-5), 4.29 (m, 1H, H-9b), 4.54, 4.85

(A, B of AB, J = 11.9 Hz, 2H, CH₂Ar), 4.95 (m, 1H, H-4), 5.18 (m,

1H, H-7), 5.31 (m, 1H, H-8), 6.39 (m, 1H, NHAc), 6.97 (m, 1H,

NH), 7.06-7.15 (m, 3H, CH_ar), 7.40 (m, 2H, CH_ar), 7.74 (m, 2H,

CH_ar). ¹³C NMR (CDCl₃) δ 20.8, 21.2 (3C, OAc), 38.0 (C-3), 38.6

(C-9), 42.4 (CH₂Cl), 49.8 (C-5), 52.9 (OMe), 60.4 (CH₂Ar), 67.8,

68.3, 68.5 (3C, C-4, C-7, C-8), 72.1 (C-6), 98.6 (C-2), 117.0 (J = 17.5

Hz), 124.0, 125.3, 126.5, 128.4, 128.9, 132.7, 137.8 (12 C-Ar), 166.4,

166.7, 167.9, 170.5, 170.7, 172.3 (6 CO). ESI-MS calcd for

C₃₂H₃₄Cl₂F₂N₂O₁₂[M þ Na]^þ769.14; found m/z 769.34.

sion chromatography to yield 19a (7.0 mg, 30%) as a white

1

foam. [R]²⁰-0.73 (c 0.08, H₂O). H NMR (500 MHz, D₂O)

D

δ 1.59 (t, J = 12.2, 1H, H-3a), 2.65 (dd, J = 4.7, 12.4, 1H, H-3b),

3.31 (dd, J = 7.7, 14.0 Hz, 1H, H-9a), 3.41 (dd, J = 1.8, 8.8 Hz,

1H, H-5), 3.64(m,3H, H-4, H-8, H-9b), 3.77(dd,J =1.9, 10.5 Hz,

1H, H-6), 3.84 (m, 1H, H-7), 4.52 (A of AB, J = 11.7 Hz, 1H,

CH₂Ar), 4.70 (m, 2H, CH₂Ar, CH₂F), 4.81 (d, J = 5.1 Hz, 1H,

CH₂F), 7.02 (m, 3H, CH_ar), 7.39, 7.62 (AA⁰, BB⁰of AA⁰BB⁰, J =

8.7 Hz, 4H, CH_ar). ¹³C NMR (CDCl₃) δ 40.4, 42.6 (2C, C-3, C-9),

57.2 (C-5), 60.6 (CH₂Ar), 68.0 (C-4), 69.7 (C-7), 70.3 (C-8), 72.3

(C-6), 87.0 (CH₂F), 101.3 (C-2), 117.2, 125.8, 128.6, 128.8, 132.1,

137.5, 141.7 (12C, C-Ar), 173.1, 175.3, 189.7 (3 CO). HRMS

calcd for C₂₅H₂₆ClF₃N₂O₉[M - H]^-589.1206; found 589.1191.

Sodium ((2,3-Difluorobenzyl) 5-Chloroacetamido-9-(4-chloro-

benzamido)-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyrano-

sid)onate (19b). Compound 18b (21.0 mg, 0.03 mmol) was treated

with LiOH (6.7 mg, 0.3 mmol) in THF/water (2.0 mL/0.5 mL).

The crude product was purified by chromatography on silica gel

Methyl ((2,3-Difluorobenzyl) 5-(o-Nitrotoluenesulfonamido)-

4,7,8-tri-O-acetyl-9-(4-chlorobenzamido)-3,5,9-trideoxy-^D-gly-

cero-r-^D-galacto-2-nonulopyranosid)onate (18c). Compound

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1611

(0.1% gradient of H₂O in DCM/MeOH; 2:1) followed by ion

exchange chromatography (Dowex 50) and P2 size exclusion

chromatography to yield 19b as a white solid (10 mg, 60%). ¹H

NMR (500 MHz, MeOD) δ 1.58 (dd, J = 10.7, 12.1 Hz, 1H,

H-3a), 2.82 (m, 1H, H-3b), 3.35 (m, 1H, H-7), 3.46 (m, 1H, H-9a),

3.68 (m, 4H, H-4, H-5, H-6, H-9b), 3.93 (m, 2H, CH₂Cl), 4.57,

4.84 (A, B of AB, J = 12.1 Hz, 2H, CH₂Ar), 7.01 (m, 3H, CH_ar),

7.35, 7.72 (AA⁰, BB⁰of AA⁰BB⁰, J = 8.5 Hz, 4H, CH_ar). ¹³C NMR

(MeOD) δ 43.0 (C-3), 44.8 (C-9), 45.0 (CH₂Cl), 54.0 (C-5), 63.1

(CH₂Ar), 70.5, 72.2, 72.9 (3C, C-4, C-7, C-8), 74.9 (C-6), 103.8

(C-2), 119.7, 126.9, 128.4, 128.9, 131.2, 131.3, 140.1 (10 C-Ar),

170.0, 170.5, 173.11 (3 CO). HRMS calcd for C₂₅H₂₅Cl₂F₂N₂-

NaO₉[M þ Na]^þ651.0701; found m/z 651.0700.

173.5 (3C, CO). HRMS calcd for C₂₆H₂₈ClF₂N₂O₁₀Na [M þ

Na]^þ647.1195; found m/z 647.5573.

Sodium ((2,3-Difluorobenzyl) 5-Cyclopropylamido-9-(4-chloro-

benzamido)-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyra-

nosid)onate (19f). Compound 23f (31.0 mg, 0.04 mmol) was

dissolved in THF/water (2.0 mL/0.5 mL) and was reacted with

LiOH (10.0 mg, 0.42 mmol). The crude product was purified

on RP-18 (10% gradient MeOH in H₂O) followed by ion

exchange chromatography (Dowex-50) and P2 size exclusion

chromatography to yield 19f (11 mg, 44%). [R]²⁰_D-0.1 (c 0.26,

H₂O). ¹H NMR (500 MHz, D₂O) δ 0.59 (m, 2H, CH₂), 0.70 (m,

2H, CH₂), 1.44 (m, 1H, CH), 1.56 (t, J = 11.8 Hz, 1H, H-3a),

2.64 (dd, J = 3.5, 11.8 Hz, 1H, H-3b), 3.37 (d, J = 8.2 Hz, 1H,

H-7), 3.47 (m, 1H, H-9a), 3.63 (m, 4H, H-5, H-6, H-8, H-9b),

4.52, 4.69 (A, B of AB, J = 11.6 Hz, 2H, CH₂Ar), 7.04 (m, 3H,

CH_ar), 7.40, 7.63 (AA⁰, BB⁰of AA⁰BB⁰, J = 8.1 Hz, 4H, CH_ar).

¹³C NMR (CDCl₃) δ 6.8, 7.0 (2C, CH₂), 14.1 (CH), 40.5, 42.3

(2C, C-3, C-9), 51.9 (C-5), 60.5 (CH₂Ar), 68.0 (C-4), 69.4 (C-7),

70.1 (C-8), 72.8 (C-6), 101.2 (C-2), 117.1, 124.4, 125.9, 128.7,

132.1, 137.6 (12C, C-Ar), 170.0, 173.1, 178.3 (3 CO). HRMS

calcd for C₂₇H₂₈ClF₂N₂O₉[M - H]^-597.1457; found 597.1454.

Sodium ((2,3-Difluorobenzyl) 5-Cyclobutylamido-9-(4-chloro-

benzamido)-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyra-

nosid)onate (19g). Compound 23g (44.0 mg, 0.06 mmol) was

dissolved in THF/water (2.0 mL/0.5 mL) and was reacted with

LiOH (14.0 mg, 0.58 mmol). The crude product was purified on

RP-18 (10% gradient MeOH in H₂O) followed by ion exchange

chromatography (Dowex-50) and P2 size exclusion chromatog-

raphy to yield 19g (17 mg, 49%). [R]²⁰_D-0.2 (c 0.11, H₂O). ¹H

NMR (500 MHz, D₂O) δ 1.55 (m, 1H, H-3a), 1.85 (m, 6H, CH₂),

2.63 (dd, J = 3.9, 12.2 Hz, 1H, H-3b), 2.99 (quint, J = 8.8 Hz, 1H,

CH), 3.32 (d, J = 8.6 Hz, 1H, H-9a), 3.61 (m, 6H, H-4, H-5, H-6,

H-7, H-8, H-9b), 4.52, 4.68 (A, B of AB, J = 11.4 Hz, 2H,

CH₂Ar), 7.05 (m, 3H, CH_ar), 7.40, 7.63 (AA⁰, BB⁰of AA⁰BB⁰, J =

7.5 Hz, 4H, CH_ar). ¹³C NMR (D₂O) δ 17.4, 24.5 (3C, CH₂), 39.2

(CH), 40.5 (C-3), 42.1 (C-9), 51.6 (C-5), 60.5 (CH₂Ar), 67.8 (C-4),

69.3 (C-7), 70.1 (C-8), 72.7 (C-6), 110.0 (C-2), 115.6, 123.3, 125.9,

128.8, 150.9 (12C, C-Ar), 170.0, 175.8. 179.7 (3 CO). ESI-MS

calcd for C₂₈H₃₀ClF₂N₂NaO₉[M þ Na]^þ657.1403; found m/z

657.1401.

Methyl (Methyl 5-tert-Butyloxycarbonylamino-4,7,8-tri-O-

acetyl-9-(4-chlorobenzamido)-3,5,9-tride-oxy-2-thio-^D-glycero-r-

D-galacto-2-nonulopyranosid)onate (20). Compound 14 (167 mg,

0.30 mmol) was dissolved in dry DCE (5 mL). p-Chlorobenzoyl

chloride (150 μL, 210 mg, 1.19 mmol) and triphenylphosphine

(156 mg, 0.59 mmol) were added. The reaction mixture was

stirred at rt overnight. Afterward, the reaction mixture was

washed with saturated aqueous NaHCO₃(3 ꢀ 5 mL) and H₂O

(1 ꢀ 5 mL). The organic phase was dried over Na₂SO₄, filtered,

and concentrated under reduced pressure. The crude product

was purified by chromatography on silica gel (EA:PE, gradient

1:1 to 2:1) to yield 20 (95 mg, 48%) as a white foam. ¹H NMR

(500 MHz, CD₂Cl₂) δ 1.39 (s, 9H, C(CH₃)₃), 1.92 (s, 3H, SMe),

2.01 (s, 6H, 2 OAc), 2.12 (m, 1H, H-3a), 2.23 (s, 3H, OAc), 2.56

(dd, J = 4.7, 13.8 Hz, 1H, H-3b), 3.00 (td, J = 3.2, 15.2 Hz, 1H,

H-9a), 3.79 (m, 4H, OMe, H-5), 4.23 (d, J = 10.6 Hz, 1H, H-6),

4.41 (ddd, J = 3.1, 8.8, 14.4 Hz, 1H, H-9b), 4.52 (dd, J = 2.9,

10.0 Hz, 1H, NHAc), 5.09 (m, 1H, H-8), 5.16 (td, J = 4.6, 11.0

Hz, 1H, H-4), 5.33 (m, 1H, H-7), 7.23 (d, J = 3.9 Hz, 1H, NH),

7.54, 8.08 (AA⁰, BB⁰of AA⁰BB⁰, J = 8.5 Hz, 4H, CH_ar). ¹³C

NMR (CD₂Cl₂) δ 11.7 (SMe), 21.2, 21.3, 21.4 (3 OAc), 28.5

(C(CH₃)₃), 37.8 (C-3), 38.4 (C-9), 51.5 (C-5), 53.3 (OMe), 68.9

(C-7), 70.0 (C-4), 70.6 (C-8), 71.5 (C-6), 80.5 (C-2), 85.3

(C(CH₃)₃), 129.3, 132.4, 138.0, 141.8 (6C, C-Ar), 155.7

(CONH), 166.7, 168.6, 170.5, 170.7, 172.4 (5 CO). ESI-MS calcd

for C₂₉H₃₉ClN₂O₁₂S [M þ Na]^þ697.18; found m/z 697.25.

Methyl (Methyl 5-Amino-4,7,8-tri-O-acetyl-9-(4-chlorobenza-

mido)-3,5,9-trideoxy-2-thio-^D-glycero-r-D-galacto-2-nonulopyra-

nosid)onate (21). Compound 20 (95.0 mg, 0.14 mmol) was

dissolved in 4 M PhOH (in DCM; 4 mL) and 4 M TMSCl

Sodium ((2,3-Difluorobenzyl) 5-(o-Nitrotoluenesulfonamido)-

9-(4-chlorobenzamido)-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-

nonulopyranosid)onate (19c). Compound 18c (29.0 mg, 0.03

mmol) was dissolved in THF/H₂O (2 mL/0.5 mL) and was reacted

with LiOH (8.00 mg, 0.33 mmol). The crude product was purified

on RP-18 (10% gradient MeOH in water) followed by ion

exchange chromatography (Dowex-50) and P2 size exclusion chro-

matography to yield 19c (10 mg, 40%). [R]²⁰_D-0.35 (c 0.10, H₂O).

¹H NMR (500 MHz, D₂O) δ 1.45 (t, J = 12.2 Hz, 1H, H-3a), 2.52

(dd, J = 4.5, 12.3 Hz, 1H, H-3b), 3.16 (t, J = 9.8 Hz, 1H, H-5), 3.43

(m, 2H, H-4, H-9a), 3.60 (dd, J = 2.9, 14.2 Hz, 1H, H-9b), 3.67 (m,

2H, H-6, H-8), 3.74 (dd, J = 1.1, 8.9 Hz, 1H, H-7), 4.50 (A of AB,

J = 11.7 Hz, 1H, CH₂Ar), 4.65 (m, 1H, CH₂Ar), 7.04 (m, 3H,

CH_ar), 7.42 (AA⁰of AA⁰BB⁰, J = 8.5 Hz, 2H, CH_ar), 7.60 (m, 4H,

CH_ar), 7.98 (BB⁰of AA⁰BB⁰, J = 7.6 Hz, 2H, CH_ar). ¹³C NMR

(D₂O) δ40.4 (C-3), 42.4 (C-9), 56.5 (C-5), 60.5 (CH₂Ar), 69.4 (C-7),

69.5 (C-4), 70.3 (C-8), 73.8 (C-6), 101.2 (C-2), 117.1, 124.2, 125.8,

128.7, 130.2, 132.1, 132.5, 137.5, 146.8 (18C, C-Ar), 170.0, 173.3

(2 CO). HRMS calcd for C₂₉H₂₇ClF₂N₃NaO₁₂S [M þ Na]^þ

760.0767; found m/z 760.0775.

Sodium ((2,3-Difluorobenzyl) 5-Methylsulfonamido-9-(4-chloro-

benzamido)-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyrano-

sid)onate (19d). Compound 18d (21.0 mg, 0.03 mmol) was dis-

solved in THF/H₂O (2 mL/0.5 mL) and was reacted with LiOH

(7.00 mg, 0.28 mmol). The crude product was purified on RP-18

(10% gradient MeOH in water) followed by ion exchange chro-

matography (Dowex-50) and P2 size exclusion chromatography to

yield 19d (10 mg, 59%). [R]²⁰_D-0.44 (c 0.07, H₂O). ¹H NMR (500

MHz, D₂O) δ 1.56 (t, J = 12.2 Hz, 1H, H-3a), 2.62 (dd, J = 4.6,

12.4 Hz, 1H, H-3b), 3.02 (s, 3H, CH₃), 3.23 (t, J = 10.1 Hz, 1H, H-

5), 3.40 (dd, J = 7.8, 14.7 Hz, 1H, H-9a), 3.48 (td, J = 4.6, 11.9 Hz,

1H, H-4), 3.66 (m, 3H, H-6, H-8, H-9b), 3.75 (d, J = 8.7 Hz, 1H,

H-7), 4.51 (A of AB, J = 11.8 Hz, 1H, CH₂Ar), 4.66 (m, 1H,

CH₂Ar), 7.02 (m, 3H, CH_ar), 7.40, 7.63 (AA⁰, BB⁰of AA⁰BB⁰, J =

8.6 Hz, 4H, CH_ar). ¹³C NMR (D₂O) δ 40.9, 41.3 (2C, C-3, C-9),

42.5 (CH₃), 55.5 (C-5), 60.6 (CH₂Ar), 68.6 (C-7), 69.3 (C-4),

70.3 (C-8), 73.2 (C-6), 101.2 (C-2), 112.4, 117.2, 124.4, 125.8,

128.7, 128.8, 137.5 (12C, C-Ar), 173.1, 187.7 (2 CO). HRMS

calcd for C₂₄H₂₆ClF₂N₂NaO₁₀S [M þ Na]^þ653.0760; found m/z

653.0759.

Sodium ((2,3-Difluorobenzyl) 5-Methoxyacetamido-9-(4-chloro-

benzamido)-3,5,9-trideoxy-^D-glycero-r-D-galacto-2-nonulopyrano-

sid)onate (19e). Compound 18b (10 mg, 0.01 mmol) was treated

with 10% aqueous NaOH (0.3 mL) in MeOH (1.5 mL) at rt for

4 h. The crude product was purified by chromatography on RP-18

(5% gradient of MeOH in H₂O) to yield 19e (2.3 mg, 19%) and

19b (1.7 mg, 21%) as white solids. [R]²⁰_D-19.1 (c 0.46, MeOH).

¹H NMR (500 MHz, D₂O) δ 1.67 (t, J = 12.2 Hz, 1H, H-3a), 2.73

(dd, J = 4.6, 12.4 Hz, 1H, H-3b), 3.28 (s, 3H, OMe), 3.41-3.56

(m, 2H, H-7, H-9a), 3.62-3.79 (m, 3H, H-4, H-8, H-9b),

3.82-3.89 (m, 2H, H-5, H-6), 3.95, 3.98 (A, B of AB, J = 15.6

Hz, 2H, CH₂OMe), 4.62, 4.78 (A, B of AB, J = 11.7 Hz, 2H,

CH₂Ar), 7.00-7.22 (m, 3H, CH_ar), 7.49, 7.71 (AA⁰, BB⁰of

AA⁰BB⁰, J = 8.5 Hz, 4H, CH_ar). ¹³C NMR (D₂O) δ 40.4 (C-3),

42.4 (C-9), 51.5 (C-5), 58.9 (OMe), 60.6 (CH₂Ar), 67.9 (C-4), 69.6

(C-7), 70.2 (C-8), 70.8 (CH₂OMe), 72.4 (C-6), 101.2 (C-2), 110.0,

117.1, 117.2, 124.4, 125.9, 128.7, 128.8, 132.1 (12C, C-Ar), 173.0,

1612 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4

Mesch et al.

(in DCM; 2 mL). The reaction mixture was stirred at rt for 2 h.

The crude product was purified by chromatography on silica gel

(EA:PE, gradient 1:1 to 8:1) to yield 21 as a white foam (51 mg,

63%). ¹H NMR (500 MHz, CD₂Cl₂) δ 1.95 (s, 3H, SMe), 1.99

(dd, J = 5.7, 14.0Hz, 1H, H-3a), 2.04, 2.06, 2.19 (3 s, 9H, 3 OAc),

2.57 (dd, J = 4.7, 13.7 Hz, 1H, H-3b), 2.62 (t, J = 10.0 Hz, 1H,

H-5), 3.34-3.42 (m, 1H, H-9a), 3.76 (s, 3H, OMe), 4.06 (dd,

J = 1.4, 10.0 Hz, 1H, H-6), 4.17 (ddd, J = 3.3, 7.0, 15.0 Hz,

1H, H-9b), 4.98 (td, J = 4.7, 11.4 Hz, 1H, H-4), 5.18 (dt, J = 3.7,

7.5 Hz, 1H, H-8), 5.61 (dd, J = 1.5, 7.5 Hz, 1H, H-7), 6.90 (m,

2H, NH₂), 7.43, 7.74 (AA⁰, BB⁰of AA⁰BB⁰, J = 8.6 Hz, 4H,

CH_ar). ¹³C NMR (CD₂Cl₂) δ 11.8 (SMe), 21.3, 21.4, 21.4

(3 OAc), 37.0 (C-3), 39.4 (C-9), 52.2 (C-5), 53.3 (OMe), 69.5

(C-7), 71.3 (C-8), 73.0 (C-4), 73.8 (C-6), 84.7 (C-2), 129.1, 129.3,

130.1, 133.5 (6C, C-Ar), 166.3, 168.2, 170.3, 170.4, 171.4 (5 CO).

ESI-MS calcd for C₂₄H₃₁ClN₂O₁₀S [M þ Na]^þ597.13; found

m/z 597.14.

Methyl ((2,3-Difluorobenzyl) 5-Cyclopropylamido-4,7,8-tri-O-

acetyl-9-(4-chlorobenzamido)-3,5,9-trideoxy-^D-glycero-r-D-galac-

to-2-nonulopyranosid)onate (23f). Compound 22f (37.0 mg, 0.06

mmol) was reacted with 2,3-difluorobenzyl alcohol (18.0 μL,

23.0 mg, 0.16 mmol), N-iodosuccinimide (20.0 mg, 0.09 mmol),

and triflic acid (4.00 μL, 7.00 mg, 0.05 mmol). The crude product

was purified by chromatography on silica gel (1% gradient

iPrOH in petrol ether/DCM 2:1) to yield 23f (31 mg, 72%).

[R]²⁰_D0.23 (c 1.73, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ 0.79

(m, 2H, CH₂), 1.00 (m, 2H, CH₂), 1.29 (m, 1H, CH), 2.08 (m, 1H,

H-3a), 2.09, 2.21, 2.29 (3 s, 9H, 3 OAc), 2.72 (dd, J = 4.5, 12.8 Hz,

1H, H-3b), 2.98 (m, 1H, H-9a), 3.82 (s, 3H, OMe), 4.11 (m, 1H,

H-6), 4.34 (m, 1H, H-5), 4.41 (m, 1H, H-9b), 4.59, 4.89 (A, B of

AB, J = 12.0 Hz, 2H, CH₂Ar), 4.95 (m, 1H, H-4), 5.21 (dd, J =

1.8, 10.0 Hz, 1H, H-7), 5.36 (m, 1H, H-8), 5.49 (d, J = 10.2 Hz,

1H, 5-NH), 7.19(m, 4H, NH, CH_ar), 7.47, 7.84(AA⁰, BB⁰, J = 8.5

Hz, 4H, CH_ar). ¹³C NMR (CDCl₃) δ 7.1, 7.4 (2C, 2 CH₂), 14.6

(SMe), 20.8, 21.2, 21.3 (3 OAc), 25.4 (CH), 38.0, 38.4 (2C, C-3,

C-9), 49.5 (C-5), 52.8 (OMe), 60.5 (CH₂Ar), 67.9, 68.4, 68.9 (3C,

C-4, C-7, C-8), 72.5 (C-6), 98.6 (C-2), 115.8, 117.1, 123.9, 125.4,

126.7, 128.5, 132.7, 137.7 (12C, C-Ar), 166.2, 168.0, 170.3, 171.2,

172.4, 173.8 (6 CO). ESI-MS calcd for C₃₄H₃₇ClF₂N₂O₁₂[M þ

Na]^þ761.20; found m/z 761.16.

Methyl ((2,3-Difluorobenzyl) 5-Cyclobutylamido-4,7,8-tri-O-

acetyl-9-(4-chlorobenzamido)-3,5,9-trideoxy-^D-glycero-r-D-gal-

acto-2-nonulopyranosid)onate (23g). Compound 22g (53.0 mg,

0.08 mmol) was reacted with 2,3-difluorobenzyl alcohol (25.0 μL,

33.0 mg, 0.23 mmol), N-iodosuccinimide (27.0 mg, 0.12 mmol),

and triflic acid (6.00 μL, 10.0 mg, 0.07 mmol). The crude product

was purified by chromatography on silica gel (1% gradient

iPrOH in petrol ether/DCM 2:1) to yield 23g (44 mg, 72%).

[R]²⁰_D0.23 (c 1.7, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ 1.85

(m, 1H, H-3a), 1.99 (s, 3H, OAc), 2.04 (m, 4H, 2 CH₂), 2.13

(s, 3H, OAc), 2.18 (m, 2H, CH₂), 2.27 (s, 3H, OAc), 2.63 (dd, J =

4.5, 12.7 Hz, 1H, H-3b), 2.84 (quint, J = 10.0 Hz, 1H, CH), 2.91

(dt, J = 3.5, 15.0 Hz, 1H, H-9a), 3.73 (s, 3H, OMe), 4.03 (d, J =

12.5 Hz, 1H, H-6), 4.22 (q, J = 10.4 Hz, 1H, H-5), 4.33 (ddd, J =

2.8, 8.6, 15.1 Hz, 1H, H-9b), 4.51 (A of AB, J = 11.8 Hz, 1H,

CH₂Ar), 4.82 (m, 2H, H-4, CH₂Ar), 5.10 (dd, J = 1.7, 9.9 Hz, 1H,

H-7), 5.17 (d, J = 10.4 Hz, 1H, 5-NH), 5.26 (m, 1H, H-8), 5.36 (d,

J = 10.2 Hz, 1H, 5-NH), 7.09 (m, 3H, CH_ar), 7.19 (dd, J = 4.9,

8.3 Hz, 1H, NH), 7.37, 7.73 (AA⁰, BB⁰of AA⁰BB⁰, J = 8.4Hz, 4H,

CH_ar). ¹³C NMR (CDCl₃) δ 18.1 (2C, CH₂), 21.2 (3C, OAc), 25.3

(CH₂), 37.9 (C-3), 38.3 (C-9), 39.7 (CH), 49.1 (C-5), 52.8 (OMe),

60.3 (CH-Ar), 67.8, 68.2, 68.8 (3C, C-4, C-7, C-8), 72.3 (C-6),

98.5 (C-2), 116.4, 117.1, 123.7, 125.3, 128.7, 132.5, 137.6 (12C,

C-Ar), 167.9, 171.1, 172.4, 175.0 (6C, CO). ESI-MS calcd for

C₃₅H₃₉ClF₂N₂O₁₂[M þ Na]^þ775.22; found m/z 775.25.

Methyl (Methyl 5-Cyclopropylamido-4,7,8-tri-O-acetyl-9-(4-

chlorobenzamido)-3,5,9-trideoxy-2-thio-^D-glycero-r-D-galacto-2-

nonulopyranosid)onate (22f). Compound 21 (74.0 mg, 0.16

mmol) was dissolved in dry DCM (2.0 mL) under argon atmo-

sphere. Cyclopropanoyl chloride (24.0 μL, 28.0 mg, 0.27 mmol),

NEt₃(37.0 μL, 27.0 mg, 0.27 mmol), and a catalytic amount of

DMAP were added successively. The reaction mixture was

stirred at rt for 3.5 h. Then it was washed with saturated aqueous

NaHCO₃(3 ꢀ 5 mL) and H₂O (1 ꢀ 5 mL). The organic phase was

dried over Na₂SO₄, filtered, and concentrated under reduced

pressure. The crude product was purified by chromatography on

silica gel (EA:PE, gradient 1:1 to 2:1) to yield 22f (43 mg, 75%).

[R]²⁰_D0.48 (c 2.16, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ 0.70

(m, 2H, CH₂), 0.91 (m, 2H, CH₂), 1.22 (m, 1H, CH), 2.01 (m, 4H,

H-3a, SMe), 2.10, 2.12, 2.20 (3 s, 9H, 3 OAc), 2.71 (dd, J = 4.6,

12.7 Hz, 1H, H-3b), 2.86 (m, 1H, H-9a), 3.73 (dd, J = 2.0, 10.7

Hz, 1H, H-6), 3.76 (s, 3H, OMe), 4.24 (q, J = 10.5 Hz, 1H, H-5),

4.36 (ddd, J = 2.9, 8.7, 11.6 Hz, 1H, H-9b), 4.84(td, J = 4.6, 11.6

Hz, 1H, H-4), 5.12 (dd, J = 2.0, 10.1 Hz, 1H, H-7), 5.25 (dt, J =

2.7, 10.1Hz, 1H, H-8), 5.42 (d, J = 10.3Hz, 1H, 5-NH), 7.18(dd,

J = 4.0, 8.7 Hz, 1H, NH), 7.39, 7.75 (AA⁰, BB⁰of AA⁰BB⁰, J =

8.6 Hz, 4H, CH_ar). ¹³C NMR (CDCl₃) δ 7.4 (2C, CH₂), 12.1

(CH), 14.6 (SMe), 21.1 (3C, 3 OAc), 37.8, 38.2 (2C, C-3, C-9),

49.4 (C-5), 52.9 (OMe), 67.9, 68.2 (2C, C-7, C-8), 69.7 (C-4), 73.9

(C-6), 82.8 (C-2), 128.8, 129.4, 132.7, 137.7 (6C, C-Ar), 166.1,

167.7, 170.3, 171.2, 172.4, 173.7 (6 CO). ESI-MS calcd for

C₂₈H₃₅ClN₂O₁₁S [M þ Na]^þ665.17; found m/z 665.06.

Methyl (Methyl 5-Cyclobutylamido-4,7,8-tri-O-acetyl-9-(4-

chlorobenzamido)-3,5,9-trideoxy-2-thio-^D-glycero-r-D-galacto-2-

nonulopyranosid)onate (22g). Compound 21 (60 mg, 0.1 mmol)

was dissolved in dry DCM (2.4 mL). Cyclobutanecarbonyl

chloride (36 μL, 37 mg, 0.3 mmol), NEt₃(44 μL, 32 mg, 0.3

mmol), and a catalytic amount of DMAP were added succes-

sively. The reaction mixture was stirred at rt overnight. Then it

was washed with saturated aqueous NaHCO₃(3 ꢀ 5 mL) and

H₂O (1 ꢀ 5 mL). The organic phase was dried over Na₂SO₄,

filtered, and concentrated under reduced pressure. The crude

product was purified by chromatography on silica gel (EA:PE,

Hapten Inhibition Assays with MAG_d1-3-Fc. Murine

MAG_d1-3-Fc was affinity purified from CHO-Lec 3.2.8.1 cell

culture supernatant as described before,⁴⁰dialyzed against

10 mM phosphate buffer pH 7.4, sterile filtered, and stored at

4 °C. The purified protein is stable for several months. The

protein was analyzed by an ELISA and binding assay with

immobilized fetuin. Inhibition assays for MAG were performed

as described previously.^26,30,40In brief, fetuin was immobilized

in microtiter plates and binding of MAG-Fc was determined in

the presence of seven to eight different concentrations for each

inhibitor using alkaline phosphatase-labeled anti-Fc antibodies.

The half-maximal inhibitory concentrations were determined

from corresponding binding curves and used to calculate rela-

tive inhibitory concentrations (rIC₅₀).

SPR Analysis. The SPR measurements were performed on a

Biacore 3000 surface plasmon resonance based optical biosensor

(Biacore AB, Sweden). Sensor chips (CM5 and CM4), immobi-

lization kits, maintenance supply and HBS-EP (10 mM HEPES

pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% v/v surfactant

P20) were purchased from Biacore AB (HBS-EP ready-to-

use; degassed and filtered). CM5 (CM4, respectively) chips

were preconditioned prior to usage by injecting a series of

1

gradient 1:1 to 2:1) to yield 22g (27 mg, 39%). H NMR (500

MHz, CDCl₃) δ 2.00 (m, 4H, SMe, H-3a), 2.03 (s, 3H, OAc), 2.08

(m, 2H, CH₂), 2.13 (s, 3H, OAc), 2.18 (m, 4H, CH₂), 2.27 (s, 3H,

OAc), 2.71 (dd, J = 4.6, 12.7 Hz, 1H, H-3b), 2.86 (m, 2H, CH, H-

9a), 3.71 (dd, J = 2.1, 10.8 Hz, 1H, NH), 3.77 (s, 3H, OMe), 4.20

(t, J = 10.4 Hz, 1H, H-5), 4.37 (ddd, J = 2.9, 8.8, 15.2 Hz, 1H, H-

9b), 4.82 (td, J = 4.6, 11.6 Hz, 1H, H-4), 5.08 (d, J = 10.3 Hz, 1H,

H-6), 5.27 (m, 2H, H-7, H-8), 7.13 (dd, J = 4.0, 8.6 Hz, 1H, 5-

NH), 7.38, 7.74 (AA⁰, BB⁰of AA⁰BB⁰, J = 8.6 Hz, 4H, CH_ar). ¹³C

NMR (CDCl₃) δ 12.1 (SMe), 18.2 (CH₂), 20.9 (3C, OAc), 24.9

(2C, CH₂), 37.8, 38.2 (2C, C-3, C-9), 39.8 (CH), 49.2 (C-5), 53.0

(OMe), 68.0, 68.1, 69.6 (3C, C-4, C-7, C-8), 73.8 (C-4), 82.8 (C-2),

128.4, 132.7, 137.7 (6C, C-Ar), 166.1, 167.7, 170.3, 172.6, 175.0

(6C, CO). ESI-MS calcd for C₂₉H₃₇ClN₂O₁₁S [M þ Na]^þ679.18;

found m/z 679.11.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1613

conditioning solutions. A flow rate of 50 μL/min was used and 2

ꢀ 20 μL of 50 mM NaOH, 10 mM HCl, 0.1% SDS, and 100 mM

H₃PO₄were injected. The carboxy groups on the CM5 (CM4)

chip were activated for 10 min with a 1:1 mixture of 0.1 M

N-hydroxysuccinimide (NHS) and 0.1 M 3-(N,N-dimethyl-

amino)propyl-N-ethylcarbodiimide (EDC) at a flow rate of

10 μL/min. Protein A (P6031) was purchased from Sigma. A

sample and a reference surface were prepared sequentially or in

parallel. For immobilizing protein A, a stock solution (1 mg/mL

in 50 mM phosphate buffer, pH 7.0) was diluted in 10 mM

sodium acetate, pH 5.0 to obtain a concentration of 30 μg/mL.

This solution was then injected over the activated surface for

10 min at a flow rate of 10 μL/min. Protein A densities around

4000 RU and 5000 RU were achieved. Flow cells were blocked

with a 10 min injection of 1 M ethanolamine, pH 8.0. For

capturing, MAG_d1-3-Fc solution (expressed and purified as

described⁴⁰) was diluted to a 30-40 μg/mL concentration using

HBS-EP. Afterward, MAG_d1-3-Fc was injected at a flow rate of

1 μL/min for 10 min. The surface was equilibrated overnight at a

flow rate of 5 μL/min, achieving densities around 2000 RU.

10-fold dilution series were freshly prepared in eluent buffer

immediately before use. All binding experiments were conducted

at 25 °C (except thermodynamic measurements) at a flow rate of

20 μL/min. The samples were injected over 1 min, followed by 1

min dissociation. Each sample was measured with a duplicate of

one concentration, using a randomized concentration order.

Several buffer samples were injected before the first concentra-

tion, and one blank between each concentration, which were used

for the double blank referencing during data processing. Double

referencing was applied to correct for bulk effects and other

systematic artifacts. Data processing and equilibrium binding

constant determinations were accomplished with Scrubber

(BioLogic Software, Version 1.1 g or 2.0a). Kinetic data were

simultaneously fit using the nonlinear regression program Clamp

or Scrubber 2.0a.

Stability Test. To dermine the stability of a compound in the

central nervous system, an artificial cerebrospinal fluid (aCSF)

was prepared based on published data.⁴¹The following con-

centrations (all in mM/L) were used: sodium 140, chlorine 125,

hydrogen carbonate 22.5, potassium 2.9, calcium 1.15, magne-

sium 1, urea 4.16, and glucose 3.2. Because the composition of

proteins in the CSF is comparable to the serum but at a lower

concentration (the ratio of liquor protein to serum protein is

4 ꢀ 10^-3),⁵⁹0.4% v/v of human plasma (Sigma-Aldrich) was

added and the pH was adjusted to 7.3. Then 100 μM solutions of

the compound were prepared and shaken at 37 °C and 300 rpm

on an Eppendorf-Thermomixer Comfort. Samples were with-

drawn after 0, 30, 60, 120, 180 min and 20 h, respectively. The

value assigned to every time point was the average of a triplicate

measurement. The quantification of the samples was performed

on a Agilent 1100 series HPLC instrument with a UV-DAD

spectrometer using the ChemStation software.

P/N 110 212) were purchased from pION. Each donor compart-

ment of the preloaded PAMPA plate was filled with 200 μL of

pION’s system solution at pH 7.4, containing the analytes at a

concentration of 50 μM. Then 150 μL of the same solution were

transferred to an UV-plate (UV-Star, Greiner Bio-one) and UV

spectra were recorded as reference on a SpectraMax instrument

(Molecular Devices). The filter membranes of the acceptor

compartments were impregnated with 5 μL of BBB-1 lipid

solution and each compartment was filled with 200 μL of brain

sink buffer. The system was assembled and individual stirring of

the wells was induced by pION’s GutBox to yield an unstirred

water layer thickness of 40 μm. After 30 min, the UV data of the

acceptor and the donor plate were acquired on a SpectraMax

instrument (Molecular Devices) and analyzed by the PAMPA

Evolution command software (version 3.4, pION).

NMR. Shigemi NMR tubes were used to reduce the sample

volume needed for measurement to 250μL. MAG_d1-3-Fc protein

was diluted from a stock solution of 1 mg/mL by a factor of

2 using 99.8% D₂O (Armar Chemicals). Following dilution, the

0.5 mg/mL MAG _d1-3-Fc was in a solvent of 50% D₂O and 50%

H₂O, with 0.01% NaN₃with a buffer of 5 mM PBS. Stock

solutions of 4 were prepared in D₂O at 100, 50, and 20 mM and

added tothe NMR samples containing MAG_d1-3-Fc for both the

titration curve and competition experiments. Stock solutions of

13f and 25 were prepared in D₂O at 5 mM to add to the NMR

samples containing MAG_d1-3-Fc for the competition experi-

ments. All NMR experiments were carried out at 300 K on a

Bruker DRX500 spectrometer, equipped with Z-gradient SEI

probe. The pulse sequence used for the selective inversion recov-

ery experiments began with a selective 25 ms I-Burp-1⁶⁰180°

pulse applied to the para-hydrogen of the benzamide group of

compound 4. This proton does not overlap with any other

resonances of 13f and 25. A further benefit of the para-hydrogen

of the benzamide group of compound 4 was that its resonance

frequency was sufficiently different from the water resonance,

thus avoiding complications due to radiation damping.⁶¹Follow-

ing the selective inversion pulse, a 1 ms gradient pulse was applied

to dephase any residual transverse magnetization. The gradient

pulse was followed by a variable delay to allow for the recovery of

longitudinal magnetization. The delay was followed by a

DPFGSE water suppression sequence to suppress the magnetiza-

tion from the 50% H₂O.⁶¹

For each selective inversion recovery time measurement (sT1),

10 experiments were performed. These experiments consisted of

increasing delays following the selective inversion pulse and

gradient of 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 5, and 10 s. Then 32

scans, preceded by eight dummy scans, were measured for the

concentrations of compound 4 of 500 μM and 1 mM. Sixteen

scans, preceded by eight dummy scans, were measured for the

concentrations of compound 4 of 2, 4, and 7 mM. For the

competitive experiments with 25 μM of either 13f or 25 added

to 500 μM of 5, 32 scans, preceded by eight dummy scans, were

measured. A delay of 20 s following the measurement of each

transient was inserted to allow the magnetization to return to

equilibrium. Prior tothe measurement upon addition of either 13f

or 25, a 1 h incubation time for equilibration was allowed. The

NMR data were analyzed using XWINNMR version 3.5 operat-

ing on a PC running under Linux OS. The spectra were apodized

with an exponential decay function with 2 Hz line broadening.

The inversion recovery data, as well as the one-site binding model,

were fit using Prism 4 (GraphPad Software Inc., San Diego, CA).

logD_7.3Determination. Two similar ratios of octanol to buffer

were chosen according to the expected logD value, whereas

every ratio was measured as a triplicate. Phosphate buffer at pH

7.3 was prepared and shaken overnight together with octanol in

order to mutually saturate the two phases. Upon separation of

the two layers, the buffer phase was withdrawn and mixed with

an analyte stock solution in DMSO to yield a final concentra-

tion of 10^-4M. Both phases were transferred to a PCR plate,

which was covered with aluminum foil (Axygen PCR-AS-200)

and shaken for 1 h at 1200 rpm and 25 °C on a PHMP-4

instrument (Grant-bio). After 2.5 h at room temperature, the

aqueous phases were transferred to microvials, centrifuged for

30 s, and analyzed by HPLC (Agilent 1100 series). The values

were accepted if the mean values of the two ratios did not differ

by more than 0.1 unit.

Acknowledgment. We thank the Volkswagen Foundation,

the Swiss National Science Foundation, the German Federal

Ministry for Education and Research (BMBF, project

€

031632A), and the Tonjes-Vagt Foundation (project XXI)

for their support of this work.

BBB-PAMPA.⁴²Consumables (system solution, P/N

110151; brain sink buffer, P/N 110674; BBB-1 lipid solution,

P/N 110672; preloaded PAMPA sandwich with stirring devices,

Supporting Information Available: Surface plasmon reso-

nance assay, structure of compounds implemented additionally

1614 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4

Mesch et al.

in the Biacore-Hapten assay correlation, HRMS data, HPLC

traces, and ¹H spectra of target compounds 13a-f, 19a-g. This

material is available free of charge via the Internet at http://

pubs.acs.org.

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Article Doi

Low molecular weight antagonists of the myelin-associated glycoprotein: Synthesis, docking, and biological evaluation

DOI: 10.1021/jm901517k

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