Synthesis of natural product-inspired inhibitors of Mycobacterium tuberculosis mycothiol-associated enzymes: The first inhibitors of GlcNAc-Ins deacetylase

Article abstract of DOI:10.1021/jm070669h

Synthesis and evaluation of a chemical library of inhibitors of the mycothiol biosynthesis enzyme GlcNAc-Ins deacetylase (MshB) and the mycothiol-dependent detoxification enzyme mycothiol-S-conjugate amidase (MCA) from Mycobacterium tuberculosis are repor

Full text of DOI:10.1021/jm070669h

6326
J. Med. Chem. 2007, 50, 6326–6336
Synthesis of Natural Product-Inspired Inhibitors of Mycobacterium tuberculosis
Mycothiol-Associated Enzymes: The First Inhibitors of GlcNAc-Ins Deacetylase
Belhu B. Metaferia, Brandon J. Fetterolf, Syed Shazad-ul-Hussan, Matthew Moravec, Jeremy A. Smith, Satyajit Ray,
Maria-Teresa Gutierrez-Lugo, and Carole A. Bewley*
Laboratory of Bioorganic Chemistry, National Institute of Diabetes and DigestiVe and Kidney Diseases, National Institutes of Health,
Bethesda, Maryland 20892
ReceiVed June 9, 2007
Synthesis and evaluation of a chemical library of inhibitors of the mycothiol biosynthesis enzyme GlcNAc-
Ins deacetylase (MshB) and the mycothiol-dependent detoxification enzyme mycothiol-S-conjugate amidase
(MCA) from Mycobacterium tuberculosis are reported. The library was biased to include structural features
of a group of natural products previously shown to competitively inhibit MCA. Molecular docking studies
that reproducibly placed the inhibitors in the active site of the enzyme MshB reveal the mode of binding
and are consistent with observed biological activity.
Introduction
cell.7 Independent studies have demonstrated that chemically
or genetically altered mycobacterial strains that are deficient in
MSH production become hypersensitive to most currently used
antitubercular antibiotics,8,9 that exposure to these antibiotics
results in upregulation of MSH biosynthesis genes in Myco-
bacterium tuberculosis, BCG,10 and that MSH is indeed essential
for viability of M. tuberculosis,11–13 the strain that causes
tuberculosis. The persisting need for extensive treatment
regimens, which currently consist of 6–12 months of multiple
drug therapy, combined with the now common place emergence
of drug resistant mycobacterial strains in tuberculosis patients
emphasizes the continuing need for discovery of new classes
of antibiotics and new antituberculars.14 Collectively, the above
observations suggest that small molecules that can interfere with
MSH biosynthesis or MSH-assisted detoxification may have
therapeutic potential, and this hypothesis is being explored by
an increasing number of researchers in the way of identification
and synthesis of inhibitors of MSH-associated enzymes.15
Mycothiol (MSH,a 1) is a small molecular weight thiol1–3
that is found exclusively in a subset of Gram-positive bacteria,
or actinomycetes, that encompass mycobacteria.4 Like its
eukaryotic and Gram-negative counterpart, glutathione, MSH
is present in reduced (1) and oxidized forms (2, MSSM),5 and
plays a critical role in maintaining a reductive intracellular
environment by functioning as a redox reagent in its own right,
by functioning as a cofactor for enzymes involved in redox
reactions, and by facilitating detoxification of electrophiles and
toxins via nucleophilic addition to the sulfhydryl group of
cysteine (exemplified by the S-conjugate MSH bimane, 3,
MsmB).6
In previous studies we identified several novel and known
natural products16–18 and synthetic compounds19,20 that inhibit
the mycobacterial detoxification enzyme MCA with sub- to low
micromolar IC50 values and demonstrated that a series of
bromotyrosine-derived molecules, exemplified by natural prod-
ucts 4-6, are competitive inhibitors of MCA.17 MCA has been
shown to be a zinc metalloenzyme.17,21 On the basis of NMR
structural studies of competitive inhibitors of MCA of the
substrate mycothiol-S-bimane (3, MsmB) and biosynthetic
intermediates such as GlcN-Ins, we proposed that competitive
inhibitors such as 4-6, each of which possess amide bonds and
moieties known to chelate metals such as oximes and oxazolines,
mimic conformations of natural substrates when bound to
MCA.22 These cumulative findings led us to speculate that a
small molecule incorporating features of the natural substrates,
namely, the pseudo disaccharide of MSH, together with those
of the competitive natural product inhibitors, may be able to
inhibit not only MCA but also the homologous biosynthetic
enzyme 1-D-myo-inosityl 2-acetamido-2-deoxy-R-D-glucopyra-
noside (GlcNAc-Ins) deacetylase (MshB). Here we describe the
synthesis and characterization of a new set of inhibitors that
MSH comprises one unit each of 1-myo-D-inositol (myo-D-
Ins), glucosamine (GlcN), and N-acetyl-cysteine. MSH biosyn-
thesis is carried out by four biosynthetic enzymes, MshA-D,
as depicted in Scheme 1. MSH-assisted detoxification is
facilitated by a fifth enzyme known as mycothiol-S-conjugate
amidase (MCA), which cleaves the scissile N-AcCys-GlcN
amide bond of mycothiol-S-conjugates to yield mercapturic
acids, or AcCys-S-conjugates, that can be excreted from the
* To whom correspondence should be addressed. Tel.: (301) 594-5187.
Fax (301) 402-0008. E-mail: caroleb@mail.nih.gov.
a
Abbreviations: TFA, trifluoroacetic acid; MSH, mycothiol; MSmB,
mycothiol-S-bimane; GlcNAc-Ins, N-acetyl-1-D-myo-inosityl-2-amino-2-
deoxy-R-D-glucopyranoside; MCA, mycothiol-S-conjugate amidase; MshB,
N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-R-D-glucopyranoside deacetylase.
10.1021/jm070669h This article not subject to U.S. Copyright. Published 2007 by the American Chemical Society
Published on Web 11/17/2007

Inhibitors of GlcNAc-Ins Deacetylase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25 6327
Scheme 1. (a) MSH Biosynthesis and (b) MSH-Mediated Detoxification
Scheme 2. Preparation of Triacetyl Thioglycosidea
meet these criteria and are the first reported inhibitors of a MSH
biosynthetic enzyme, namely, MshB.
a Reagents and conditions: (a) DMAP, Boc2O, THF, heat, 6 h; (b) MeOH,
H2NNH2, 0 °C, 2 h; (c) TMSOTf, CH2Cl2, 0 °C, 4 h, 65%; (d) H2NNH2
(neat), reflux, 24 h, 78%.
and DMAP followed by hydrazine afforded N-Boc protected
amine 9,25 and free amine triacetate 10a was obtained in
quantitative yield upon treatment with TMSOTf in methylene
chloride.
Sulfonamide analogs shown in Scheme 3 were prepared by
the addition of the appropriate sulfonyl chloride to 10a in the
presence of triethylamine, followed by deprotection with sodium
methoxide in methanol. Thus, treatment of 10a with mesyl or
tosyl chloride furnished triacetoxy sulfonamides 11 and 12 in
greater than a 90% yield, and quantitative deprotection gave
triols 13 and 14. Horner-Wadsworth-Emmons olefination of
benzaldehye (15a) or 3-benzyl propanal (15b) with sulfonyl
phosphonate 1626 gave exclusively (E)-olefins 17a and 17b, and
activation with thionyl chloride, TBAI, and triphenylphosphine
provided sulfonyl chlorides 18a,b for coupling to amine 10a to
give 19a and 19b. Deprotection of a portion of 19a and 19b
provided R,ꢀ-unsaturated sulfonamides 20 and 21, and reduction
of the remaining material with palladium on carbon followed
by hydrolytic deprotection gave saturated sulfonamides 22 and
23.
To test the effect of a trifluoroacetate group on the amide
suspected to be near to the active site, amine 10a was treated
with trifluroacetic anhydride to give analog 24. In a previous
study on a library of synthetic psammaplin analogs, we showed
that compounds containing a phenyl ring separated by two to
four carbon atoms from the cyclic portion of the structure were
among the best inhibitors.19 As an extension of this finding,
we sought to generate a few analogs with this moiety. Coupling
of N-Boc-L-homophenylalanine and 10a gave protected inter-
mediate 25, and removal of the acetates afforded compound
26. Intermediate 25 was in turn treated with TFA followed by
acetylation, trifluoroacetylation, or sulfonylation of the amine
to give triacetates 27-29; global deprotection of each provided
triols 30-32, respectively (Scheme 4).
Results and Discussion
Chemical Synthesis and Rationale. MSH is composed of
one unit each of 1-myo-D-inositol (myo-D-Ins), GlcN, and
N-acetyl-cysteine and is assembled as shown in Scheme 1.
Earlier, Knapp and co-workers synthesized an S-bimane deriva-
tive of a simplified MSH analog, namely, the cyclohexyl
thioglycoside analog 7, which was shown to be a practical
substrate for the detoxification enzyme MCA with rates about
2-fold less than those measured for a well-studied substrate
MSmB (3).23 More recently, we reported a series of inhibitors
wherein the cyclitol is replaced by a quinic acid moiety.20
Together, our results indicate that the presence of the cyclitol
myo-D-inositol, at least for the amidase MCA, is not strictly
required and that MCA likely binds or tolerates the cyclohexyl
and quinic acid derived groups. In terms of meeting the criteria
for our notion of the ideal inhibitor for MCA and homologues
described above, incorporation of the cyclohexyl thioglycoside
unit was most attractive. Building on this scaffold, we synthe-
sized 23 compounds that incorporated a selection of substruc-
tures that would allow us to explore the effects on enzyme
inhibition of substituents coupled to the amino group of GlcN.
Mindful of the information gleaned from our earlier studies of
natural product and synthetic inhibitors, we chose to include
phenyl and sulfonyl groups, oxazole or oxazine units, and
cysteine isosteres such as aspartic acid and homoserine.
Inspired by the activity of a series of bromotyrosine-derived
natural products containing spiroisooxazoline moieties, we were
especially interested in analogs containing similar units. Syn-
thesis of such analogs began with D/L-serine (33a) or D/L-
homoserine (33b; Scheme 5). Conversion of the primary
hydroxyl to its silyl ether followed by esterification using
trimethylsilyldiazomethane27 in THF gave the desired methyl
esters 34a,b in acceptable yields (49% over two steps).
Amidation with 3-fluorobenzoic acid through activation by
diethyl cyanophosphonate at 0 °C gave amides 35a,b. Efficient
cyclization of 35a to oxazole 36a and of 35b to oxazine 36b
Synthesis of the analogs began with the preparation of
cyclohexyl thioglycoside tetraacetate 8 (Scheme 2) following
the route published by Knapp et al.24 Conversion of 8 to
2-amino-3,4,6-triacetoxy cyclohexyl thioglycoside 10a, which
allows coupling to the thioglycoside scaffold under a variety
of conditions compared to unprotected aminotriol 10b, was
accomplished in two steps. Treatment of 8 with Boc-anhydride

6328 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25
Scheme 3. Preparation of Sulfonamidesa
Metaferia et al.
a Reagents and conditions: (a) MsCl, Et3N, CH2Cl2, rt, 1 h; (b) TosCl, Et3N, CH2Cl2, rt, 1 h; (c) NaOMe, MeOH; (d) n-BuLi, THF, -78 °C to rt, 18 h;
(e) TBAI, acetone, heat, 24 h; (f) SOCl2, triphenylphosphine, CH2Cl2, 0 °C to rt, 1 h; (g) 10a, Et3N, CH2Cl2, rt, 4 h; (h) H2, Pd-C, EtOH.
Scheme 4. Preparation of Homophenylalanine Analogsa
another type of analog, 42, in which the thioether sulfur atom
has been oxidized to a sulfone.
At this point, preliminary screening results indicated that the
heterocycle-containing analogs 39 and 40, generated from
racemic serine and homoserine, were among the most potent
of the analogs described above. With the protected amine 10a
in hand and the methodology now optimized, we proceeded to
synthesize the optically pure diastereomers of 39 and 40 starting
with D- or L-serine (to give 39D and 39L) and D- or L-
homoserine (to give 40D and 40L). In addition, three other
related and hydrolytically stable analogs were prepared using
commercially available acids to give analogs 43-45 in good
yields (Scheme 7).
Last, to test whether the cyclohexyl group is necessary for
inhibition and binding to MCA or MshB, three analogs built
upon GlcN alone were prepared (Scheme 6). 1,3,4,6-Tetraac-
etoxy GlcN 46 was prepared according to a literature
procedure and EDCI/HOBt coupling to acids used for
inhibitors 43-45, followed by deacetylation, provided ana-
a Reagents and conditions: (a) 10a, DIEA, EDCI/HOBt, DMF, 0 °C-rt,
3 h; (b) NaOMe/MeOH; (c) TFA, CH2Cl2; (d) acetic anhydride, pyridine;
logs 47-49 (Scheme 8).
Biological Characterization. A total of 23 analogs, 18 of
which are built on the thioglycoside scaffold and three upon
GlcN, were tested for their ability to inhibit two homologous,
MSH-associated enzymes from M. tuberculosis, namely, the
MSH biosynthetic enzyme MshB and the detoxification enzyme
MCA. Previously described fluorescence-detected HPLC assays7,17
were used for screening against both enzymes. The results of
initial screening at two concentrations (200 and 50 µM) are
presented in Figure 1. Dose–response curves were subsequently
generated for compounds that reproducibly inhibited one or both
of the enzymes by at least 50% when tested at 200 µM and
20% when tested at 50 µM (Table 1).
(e) trifluoroacetic anhydride, pyridine; (f) mesyl chloride, pyridine.
was accomplished by treatment with DAST ((diethylamino)sul-
fur trifluoride).28a,b Basic hydrolysis of the methyl esters gave
acids 37a and 37b that were in turn coupled to 10a to provide
thioglycoside amides 38a and 38b. Deacetylation of 38a and
38b gave the desired target compounds 39 and 40 in quantitative
yields. Compounds in which the cysteine unit is replaced by a
similar amino acid were obtained by coupling N-Boc-L-
homoserine to 10a using the same coupling and deprotection
protocols as described above to give analog 41 (Scheme 6). A
portion of this homoserine coupled triacetate intermediate was
subjected to RuCl3 oxidation and deacetylation to provide yet
Sulfonamides 21 and 23, homophenylalanine trifluoroacetate
31, homoserine analog 41, and heterocycles 39, 40, 43, and 44

Inhibitors of GlcNAc-Ins Deacetylase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25 6329
Scheme 5. Preparation of Oxazole and Oxazine Heterocylcesa
a Reagents and conditions: (a) TBDMSCl, DBU, CH3CN, rt; (b) TMSCHN2, MeOH/benzene; (c) 3-fluorobenzoic acid, DIEA, DECP, DMF, 0 °C, 3 h;
(d) DAST, CH2Cl2; (e) LiOH (3:1, MeOH/water); (f)10a, DIEA, DEPC, 0 °C-rt; (g) NaOMe, MeOH, rt.
Scheme 6. Preparation of 41 and 42a
a Reagents and conditions: (a) TFA; (b) 10a, DIEA, EDCI/HOBt, DMF,
0 °C-rt, 3 h; (c) NaOMe/MeOH; (d) aq NaIO(a), CH3CN/CCl4, RuCl3 (2
mol%).
were the strongest inhibitors of the biosynthetic enzyme MshB.
For MCA, a similar pattern emerged where sulfonamides
20-23, trifluoroacetate 31 and its N-Boc analog 26, and the
same set of heterocycles (39, 40, 43, 44) stood out as the best
inhibitors, while analog 41 was only weakly inhibitory at 50
µM. Somewhat surprisingly, the optically pure forms of
compounds 39 and 40, incorporating D- and L-serine and D- and
L-homoserine, respectively, (namely, 39D and 39L and 40D and
40L) were found to inhibit MshB with very similar IC50 values
as the diastereomeric mixtures first synthesized.
Modeling. Crystal structures of the deacetylase MshB were
solved contemporaneously by the groups of James29 and Baker30
in 2003. In the structure solved by McCarthy et al.,30 ꢀ-octyl
Figure 1. Inhibition of MshB and MCA. A total of 23 compounds
were tested at 200 µM and 50 µM for inhibition of (A) MshB and (B)
glucoside, a component of the crystallization buffer cocrystal-
lized in the active site giving rise to a reliable model of the
true substrate GlcNAc-Ins bound to MshB. To gain insight into
MCA using fluorescence-detected HPLC assays. Grey bars indicate %
inhibition at 200 µM in both graphs and blue and pink bars indicate %
inhibition at 50 µM against MshB and MCA, respectively. Bars
the mode of binding of our inhibitors to MshB, docking
calculations were performed for three of the most potent
inhibitors (Autodock 4.031). We first tested whether docking
the substrate GlcNAc-Ins to MshB would duplicate the model
generated from the crystal structure. Starting from random initial
positions and orientations, GlcNAc-Ins was docked to MshB
in two steps (see Supporting Information). Multiple runs defining
the entire surface of the protein with Zn2+ in the active site32
produced a highly populated cluster of solutions (1.0 Å cutoffs)
that also exhibited the lowest binding energies and occupied a
cleft-like binding site formed by 10 residues, including His13,
Asp15, Asp16, Glu45, Arg68, Asp95, His144, His147, Gln247,
and Ser260. The refinement step (defining a region slightly larger
represent the average of at least two independent assays run in duplicate
or triplicate; standard errors averaged (15% of indicated values for
both enzymes.
than and centered around the binding site) likewise produced a
low-energy cluster of solutions that showed nearly identical
docked geometries relative to one another and to the X-ray
model, with the same atoms participating in hydrogen bonding
interactions with MshB and Zn2+ (Figure 2a).
The same protocol was used to dock heterocycles 39, 40,
and 43 to MshB, with results for 40L and 43 shown in Figure
2b and Figure 2c, respectively. For all three inhibitors, the most
highly populated cluster of conformers occupied the same low-

6330 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25
Metaferia et al.
Figure 2. Results of docking GlcNAc-Ins (A) and inhibitors 40L (B) and 43 (C) to MshB. MshB residues in contact with the ligands are displayed
as rods with N and O atoms colored blue and red, respectively. Ligands are shown in yellow, the Zn2+ ion is shown as a green sphere, and
hydrophobic residues and their surfaces are colored purple.
Scheme 7. Heterocyclic Analogs and Yields from Couplingsa
Scheme 8a
a Reagents and conditions: (a) DIEA, EDCI/HOBt, RCOOH, CH2Cl2;
(b) NaOMe, MeOH.
Table 1. IC50 Values for the most Potent Inhibitors of MshB or MCA
IC50 (µM)
cmpd
MshB
MCA
21
23
26
39
40
43
105 ( 27
101 ( 14
NDa
28 ( 6
10 ( 2
7 ( 1
90 ( 17
120 ( 26
145 ( 14
NDa
NDa
33 ( 4
a Not determined; IC50 values greater than 200 µM.
whose walls are formed by Leu259 on one side and Tyr142
and Gly143 on the other accommodates the aromatic and hetero-
cyclic portion of the inhibitors. The docked positions and hydrogen
bonding patterns were the same for the D- and L-isomers of 39
and 40 (not shown). The lowest energy conformation of furan
43 makes an additional hydrogen bond between the furan
oxygen and His144 (Figure 2c), which is absent in 39 and 40.
Concluding Remarks. In recent years, MSH has been shown
to be essential for growth of Mtb, supporting the validity of
targeting MSH biosynthetic enzymes for the development of new
classes of antimycobacterials.11–13 To date, inhibitors of MSH-
associated enzymes have been limited to those of the detoxi-
fication enzyme MCA and have included natural products,
natural product-like libraries, and substrate mimics. Interestingly,
although we have tested many of these compounds for their
ability to inhibit MshB (unpublished data, C.A.B., S.R., Lisa
Eckman), none of our previously reported MCA inhibitors have
displayed notable activity toward that biosynthetic enzyme. This
was surprising given that the active sites of these two enzymes
are predicted to be nearly identical on the basis of sequence
alignments, and it prompted us to attempt to design and
synthesize a small molecule that would inhibit MshB, or ideally,
both MshB and MCA.
a Reagents and conditions: (a) 10b, DIEA, EDCI/HOBt, DMF, 0 °C-rt,
3 h.
energy binding site as that of GlcNAc-Ins, located at the active
site of the enzyme, and the inhibitors fit well into the cleft
formed by residues lining the active site. The amide O atom of
each inhibitor ligates the zinc (average distance of 2.25 Å), and
in each complex, the cyclohexyl ring packs against the
hydrophobic residues Met98 and Gly46 (Figure 2b,c). Although
the average positions of the GlcN rings in the docked structures
are shifted slightly relative to one another, each is positioned
to allow formation of at least two hydrogen bonds between either
the C3, C4, or C6 hydroxyl groups of the ligand and the Nη
atoms of Arg68 or the Oδ atoms of Asp 15 or Asp95. In
addition, the C-3 hydroxyls in 39 (not shown) and 40 are bound
to zinc (average distance of 2.10 Å). The hydrophobic groove
In this study, we have constructed a small library of
compounds built upon GlcN or a thioglycoside scaffold shown

Inhibitors of GlcNAc-Ins Deacetylase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25 6331
(2R,3S,4R,5R,6R)-2-(Acetoxymethyl)-6-(cyclohexylthio)-5-(4-
methylphenylsulfonamido)-tetrahydro-2H-pyran-3,4-diyl Diac-
etate (12). The title compound 12 was obtained following the
standard coupling procedure described above. Purification of the
crude product by preparative TLC (15/85 MeOH/CH2Cl2) afforded
12 as a white solid (93%). 1H NMR (300 MHz, CDCl3) δ 7.75 (d,
J ) 8.1 Hz, 2H), 7.31 (d, J ) 8.4 Hz, 2H), 5.15 (d, J ) 5.1 Hz,
1H), 4.87–5.01 (m, 3H), 4.23–4.34 (m, 2H), 4.01 (dd, J ) 1.8, 4.2
Hz, 1H), 3.76 (dt, J ) 10.2, 5.4 Hz, 1H), 2.59–2.66 (m, 1H), 2.42
(s, 3H), 2.08 (s, 3H), 1.99 (s, 3H), 1.56–1.93 (m, 9H), 1.23–1.40
(m, 6H).
General Procedure for Global Deacetylation. In the final
deprotection step, 1 mL of sodium methoxide (1 M solution in
MeOH) was added to each triacetate (ca. 0.074 mmol in 1 mL of
MeOH), and the mixtures were stirred for 1 h at rt. The solutions
were concentrated in vacuo prior to purification by preparative TLC
or HPLC.
previously to bind to MCA and modestly inhibit the growth of
M. tuberculosis.33 The diversity of structures appended to these
scaffolds was selected in part on the basis of structures of
competitive, natural product inhibitors. Although inhibitory
concentrations remain in the low micromolar range, this
approach has been successful in that it has allowed us to
construct the first reported inhibitors of a MSH biosynthetic
enzyme, namely, MshB, and supports the notion that natural
product-substrate chimeras might act as dual inhibitors toward
MshB and MCA. These results should provide the basis for
further development of more potent inhibitors of MSH
biosynthesis.
Experimental Section
Materials and Reagents. Recombinant forms of MshB34 and
MCA7 were expressed and prepared using published protocols,7,34
and fluorescent-detected HPLC assays were carried out as described
previously.17 All chemicals were of analytical grade and used
without further purification.
N-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihydroxy-6-(hy-
droxymethyl)-tetrahydro-2H-pyran-3-yl)methanesulfonamide
(13). The title compound 13 was obtained following the deacety-
lation procedure described above. Purification of the crude product
by preparative TLC (15/85 MeOH/CH2Cl2) afforded 13 as a white
(2R,3S,4R,5R,6R)-2-(Acetoxymethyl)-5-(tert-butoxycarbonyl-
amino)-6-(cyclohexylthio)-tetrahydro-2H-pyran-3,4-diyl Diac-
etate (9). To a solution of tetracetate 8 (1.5 g, 3.37 mmol) and
DMAP (83 mg, 0.67 mmol) in 6 mL of THF was added Boc2O
(1.46 g, 6.73 mmol), and the solution was refluxed at 60 °C for
6 h. The mixture was cooled to 0 °C, diluted with MeOH (10 mL),
and treated with hydrazine (0.42 mL, 13.5 mmol) for 2 h. The
mixture was diluted with CH2Cl2, washed with 0.5 N HCl, CuSO4,
and NaHCO3, dried over MgSO4, and concentrated in vacuo, and
the residue was purified over Si gel (25/75 EtOAc/CH2Cl2) to give
the desired carbamate 9 in 70% yield. 1H NMR (300 MHz, CDCl3)
δ 5.43 (d, J ) 5.4 Hz, 1H), 4.99 (m, 2H), 4.71 (d, J ) 9.6 Hz,
1H), 4.37 (m, 1H), 4.21 (dt, J ) 4.8, 7.5 Hz, 1H), 4.06 (dd, J )
1.8, 10.2 Hz, 1H), 2.83 (m, 1H), 2.09 (s, 3H), 2.02 (s, 6H), 1.75
(m, 2H), 1.56 (m, 2H), 1.42 (s, 9H), 1.30–1.42 (m, 7H); 13C NMR
(75 MHz, CDCl3) δ 171.1, 170.8, 169.6, 84.4, 80.2, 71.9, 68.7,
62.3, 53.5, 45.2, 34.4, 33.8, 28.4, 26.0, 25.7, 20.8; LRMS
C23H38NO9S (M + H+) 504.2.
(2R,3S,4R,5R,6R)-2-(Acetoxymethyl)-5-amino-6-(cyclohexyl-
thio)-tetrahydro-2H-pyran-3,4-diyl Diacetate (10a). To a solution
of carbamate 9 (0.20 g, 0.40 mmol) in CH2Cl2 (7 mL) at 0 °C was
added TMSOTf (137 µL, 0.80 mmol), and the mixture was stirred
for 4 h, after which time the mixture was quenched with cold
saturated aq NaHCO3 and extracted with CH2Cl2 (3 × 15 mL).
The combined organic layers were dried over MgSO4, the solvent
was removed in vacuo, and the crude product was purified on a
short Si gel column (5/95 MeOH/CH2Cl2) to give the free amine
10a in 65% yield. 1H NMR (300 MHz, CDCl3) δ 5.40 (d, J ) 5.4
Hz, 1H), 4.94 (m, 2H), 4.46 (m, 1H), 4.30 (dd, J ) 5.1, 7.2 Hz,
2H), 4.05 (dd, J ) 2.1, 10.2 Hz, 2H), 3.22 (br, 2H), 2.85 (m, 1H),
2.08 (s, 3H), 2.06 (s, 3H), 2.02 (s, 3H), 1.75 (m, 2H), 1.59 (m,
1H), 1.27–1.47 (m, 8H); LRMS C18H30NO7S (M + H+) 404.2.
General Procedure for Coupling Sulfonyls and Amine 10a
(Scheme 3). To a solution of 10a (0.31 mmol) and triethylamine
(0.15 mL, 1.11 mmol) in CH2Cl2 (2.2 mL) at 0 °C was added the
desired sulfonyl chloride (0.63 mL of a 0.47 M solution in CH2Cl2,
0.28 mmol). The reaction was allowed to go to completion (2–4
h), as determined by consumption of starting material 10a by TLC
(50/50 EtOAc/CH2Cl2).
1
solid (93%). H NMR (300 MHz, CD3OD/CDCl3) δ 5.17 (d, J )
4.5 Hz, 1H), 3.97 (m, 1H), 3.68 (m, 2H), 3.29 (m, 1H), 3.18 (m,
1H), 2.81 (s, 3H), 2.59 (m, 1H), 1.75 (m, 2H), 1.49 (m, 2H), 1.34
(m, 1H), 1.09 (m, 6H); 13C NMR (75 MHz, CDCl3) δ 85.5, 72.6,
72.2, 70.9, 61.2, 57.7, 55.9, 44.4, 41.1, 34.0, 33.4, 25.7, 25.4; IR
(cm-1) 3373 (br), 2927, 1729, 1455, 1311, 1154; solvent A, 11.9;
solvent B, 15.6 min; HRMS (ES+) m/z calcd for C13H25NO6S2Na
([M + Na]+), 378.1021; found, 378.1013; ∆ ) -2.1 ppm.
N-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihydroxy-6-(hy-
droxymethyl)-tetrahydro-2H-pyran-3-yl)-4-methylbenzenesulfona-
mide (14). The title compound 14 was obtained following the
deacetylation procedure described above. Purification of the crude
product by preparative TLC (15/85 MeOH/CH2Cl2) afforded 14 as
1
a white solid (95%). H NMR (300 MHz, CD3OD/CDCl3) δ 7.82
(d, J ) 8.1 Hz, 2H), 7.37 (d, J ) 8.1 Hz, 2H), 5.02 (d, J ) 4.8 Hz,
1H), 3.91 (m, 1H), 3.74 (m, 1H), 3.69 (dd, J ) 5.4, 6.7 Hz, 1H),
3.47 (dd, J ) 5.4, 6.0 Hz, 1H), 3.40 (m, 1H), 3.31 (m, 1H), 2.44
(s, 3H), 2.41 (m, 1H), 1.87 (m, 1H), 1.71 (m, 3H), 1.58 (m, 1H),
1.26 (m, 3H), 1.18 (m, 2H); 13C NMR (125 MHz, CD3OD) 144.6,
140.6, 130.7 (2C), 128.2 (2C), 85.9, 74.3, 72.9, 72.3, 62.4, 59.3,
45.4, 35.5, 34.5, 27.0, 26.9, 21.5; IR (cm-1) 3280, 2923, 2615,
1597, 1451, 1315, 1158; solvent A, 24.4; solvent B, 26.0 min;
HRMS (ES+) m/z calcd for C19H29NO6NaS2 ([M + Na]+),
454.1334; found, 454.1334; ∆ ) 0.0 ppm.
General Procedure for Horner-Wadsworth-Emmons Ole-
fination. To a solution of triethyl-R-phosphonomethanesulfonate
(16; 0.26 g, 1.01 mmol) in THF (10.0 mL) at -78 °C, n-BuLi
(0.40 mL of a 2.5 M solution in hexanes, 1.01 mmol) was added
dropwise. The resulting pale yellow solution was maintained for
15 min, after which benzaldehyde (15a) or 3-phenyl-propionalde-
hyde (15b; 1.0 mmol) was added and the mixture was allowed to
warm to rt in 1 h. After 4 h, the solution was concentrated in vacuo,
the crude residue was suspended in water, the aqueous layer was
extracted with CH2Cl2 (3 × 10 mL), and the combined organic
layers were dried over MgSO4, concentrated in vacuo, and purified
by column chromatography (Si Gel, 15/85 EtOAc/hexanes) to give
17a (78%) and 17b (82%) as white solids.
(E)-Ethyl 2-Phenylethenesulfonate (17a). 1H NMR (300 MHz,
CDCl3) δ 7.64 (s, 1H), 7.58 (s, 1H), 7.50–7.54 (m, 2H), 7.43–7.47
(m, 2H), 6.74 (d, J ) 15.3, 1H), 4.24 (q, J ) 7.2 Hz, 2H), 1.41 (t,
J ) 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 144.7, 132.0, 131.5,
129.3, 128.6, 121.3, 67.0, 15.0.
(2R,3S,4R,5R,6R)-2-(Acetoxymethyl)-6-(cyclohexylthio)-5-
(methylsulfonamido)-tetrahydro-2H-pyran-3,4-diyl Diacetate
(11). The title compound 11 was obtained following the standard
coupling procedure described above. Purification of the crude
product by preparative TLC (1/4 MeOH/CH2Cl2) afforded 11 as a
white solid (93%). 1H NMR (300 MHz, CDCl3) δ 5.44 (d, J ) 5.1
Hz, 1H), 5.00 (m, 2H), 4.82 (d, J ) 10.2 Hz, 1H), 4.35–4.41 (m,
1H), 4.28 (dd, J ) 4.5, 12.3 Hz, 1H), 4.06 (dd, J ) 2.1, 12.3 Hz,
1H), 3.86 (dt, J ) 10.5, 5.4 Hz, 1H), 2.96 (s, 3H), 2.84–2.92 (m,
1H), 2.08 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H), 1.73–1.80 (m, 2H),
1.56–1.60 (m, 1H), 1.22–1.49 (m, 5H).
(E)-Ethyl 4-Phenylbut-1-ene-1-sulfonate (17b). 1H NMR (300
MHz, CDCl3) δ 7.16–7.35 (m, 5H), 6.91 (dt, J ) 6.9, 11.5 Hz,
1H), 6.17 (d, J ) 11.5 Hz, 1H), 3.97–4.09 (m, 2H), 2.79–2.86 (m,
2H), 2.62 (m, 2H), 1.32 (m, 3H).
General Procedure for the Synthesis of Thionyl Chlorides.
To a solution of sulfone ethylester 17a or 17b (0.38 mmol) in 3.8
mL of acetone, tetrabutylammonium iodide (0.23 g, 0.62 mmol)

6332 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25
Metaferia et al.
was added, and the mixture was heated at reflux and monitored by
TLC for complete consumption of starting material, after which
the solution was cooled to rt, concentrated in vacuo, and the crude
product was used directly in the next step. To this crude product
in CH2Cl2 (3 mL) at 0 °C was added triphenylphosphine (0.2 g,
0.76 mmol). The mixture was stirred for 10 min and thionyl chloride
(0.84 mL of a 1.0 M solution in CH2Cl2, 0.84 mmol) was added
slowly. The mixture was allowed to warm to rt, stirred for 1 h, and
concentrated in vacuo, and the crude sulfonyl chlorides 18a and
18b were used directly in the next coupling step.
141.4, 137.6, 129.8 (2C), 127.3, 120.4, 118.8, 87.1, 74.6, 72.6, 62.3,
59.6, 53.4, 45.3, 39.8, 35.7, 34.6, 27.0; IR (cm-1) 3318, 2971, 2927,
1739, 1449, 1321, 1217; solvent A, 31.5; solvent B, 33.6 min;
HRMS (ES+) m/z calcd for C22H33NO6NaS2 ([M + Na]+),
494.1647; found, 494.1632; ∆ ) -3.0 ppm.
General Procedure for Hydrogenation of Sulfonamide Tri-
acetates. An aliquot of each of the R,ꢀ-unsaturated sulfonamides
19a or 19b (0.12 mmol) in EtOH (1.2 mL) was subjected to
hydrogenation (1 atm) in the presence of 10% Pd/C for 8 h. The
heterogeneous suspension was filtered through a pad of Celite,
washed with EtOAc (40 mL), and concentrated in vacuo. The crude
products were purified by preparative TLC (SiO2, 40/60 EtOAc/
hexanes) to provide quantitatively the corresponding reduction
products 22 (88%) and 23 (90%) as white solids.
N-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihydroxy-6-(hy-
droxymethyl)-tetrahydro-2H-pyran-3-yl)-2-phenylethanesulfona-
mide (22). 1H NMR (300 MHz, CDCl3) δ 7.31–7.18 (m, 5H), 5.42
(d, J ) 5.7 Hz, 1H), 3.96 (t, J ) 12.0 Hz, 2H), 3.75–2.69 (m, 2H),
3.59–3.28 (m, 3H), 3.17–3.10 (m, 2H), 2.79–2.72 (m, 1H), 1.91
(bs, 2H), 1.68–1.55 (m, 3H), 1.36–1.18 (m, 5H); HRMS (ES) m/z
calcd for C20H31NO6NaS2 ([M + Na]+), 468.1491; found, 468.1483;
∆ ) -1.6 ppm.
2-Phenyl-ethene-1-sulfonyl Chloride (Crude 18a). Yield, 74%
(two steps); 1H NMR (300 MHz, CDCl3) δ 7.58 (s, 1H), 7.15–7.31
(m, 5H), 6.98 (s, 1H).
4-Phenyl-but-1E-ene-1-sulfonyl Chloride (Crude 18b). Yield,
1
69% (two steps); H NMR (300 MHz, CDCl3) δ 7.25–7.45 (m,
5H), 7.08 (m, J ) 15 Hz, 1H), 6.78 (d, J ) 15 Hz, 1H), 2.95 (t, J
) 7.8 Hz, 2H), 2.76 (q, J ) 7.2 Hz, 2H).
(2R,3S,4R,5R,6R)-2-(Acetoxymethyl)-6-(cyclohexylthio)-5-
((E)-2-phenylvinylsulfonamido)-tetrahydro-2H-pyran-3,4-diyl Di-
acetate (19a). The title compound 19a was obtained by following
the standard sulfonyl coupling procedure described above. Purifica-
tion of the crude product by preparative TLC (Si gel, 2/3 EtOAC/
hexanes) provided 19a as a white solid (83%). 1H NMR (300 MHz,
CDCl3) δ 7.40–7.51 (m, 5H), 6.74 (d, J ) 15.6 Hz, 1H), 5.45 (d,
J ) 5.4 Hz, 1H), 5.01 (m, 2H), 4.86 (d, J ) 10.2 Hz, 1H), 4.35–4.40
(m, 1H), 4.28 (dd, J ) 4.8, 12.3 Hz, 1H), 4.04 (dd, J ) 2.4, 12 Hz,
1H), 3.80–3.89 (m, 1H), 2.77–2.86 (m, 1H), 2.08 (s, 3H), 2.02 (s,
3H), 1.90 (s, 3H), 1.69–1.70 (m, 2H), 1.57 (bs, 1H), 1.23–1.46 (m,
7H).
N-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihydroxy-6-(hy-
droxymethyl)-tetrahydro-2H-pyran-3-yl)-4-phenylbutane-1-sul-
1
fonamide (23). H NMR (300 MHz, CD3OD/CDCl3) δ 7.26 (m,
2H), 7.20 (m, 2H), 5.39 (d, J ) 5.2 Hz, 1H), 3.99 (m, 1H), 3.80
(d, J ) 2.0, 9.6 Hz, 1H), 3.73 (d, J ) 5.3, 6.6 Hz, 1H), 3.47 (m,
1H), 3.17 (m, 2H), 2.89 (m, 1H), 2.66 (m, 2H), 2.02 (m, 2H), 1.89
(m, 2H), 1.75 (m, 5H), 1.59 (m, 2H), 1.35 (m, 6H); 13C NMR (125
MHz, CD3OD) δ 142.4, 129.6 (2C), 129.5 (2C), 127.0, 87.0, 74.6,
73.5, 72.9, 62.7, 59.4, 54.5, 45.3, 36.6, 35.6, 35.0, 31.5, 27.8, 27.4,
27.0, 24.6; solvent A, 32.4; solvent B, 34.7 min; IR (cm-1) 3342
(br), 3026, 2927, 1738, 1447, 1366, 1217; HRMS (ES+) m/z calcd
for C22H35NO6NaS2 ([M + Na]+), 496.1804; found, 496.1796; ∆
) -1.5 ppm.
(2R,3S,4R,5R,6R)-2-(Acetoxymethyl)-6-(cyclohexylthio)-5-
((E)-4-phenylbut-1-enylsulfonamido)-tetrahydro-2H-pyran-3,4-
diyl Diacetate (19b). The title compound 19b was obtained by
following the standard sulfonyl coupling procedure described above.
Purification of the crude product by preparative TLC (Si gel, 2/3
1
EtOAC/hexanes) provided 19b as a white solid (79%). H NMR
(300 MHz, CDCl3) δ 7.33–7.17 (m, 5H), 5.96–6.09 (m, 1H),
5.58–5.64 (m, 1H), 5.48 (d, J ) 5.4 Hz, 1H), 5.03 (q, J ) 7.8 Hz,
2H), 4.71 (d, J ) 102 Hz, 1H), 4.38–4.40 (m, 1H), 4.92 (dd, J )
4.5, 12.6 Hz, 1H), 4.08–4.16 (m, 2H), 3.81–3.96 (m, 2H), 3.49 (d,
7.2 Hz, 2H), 2.82–2.95 (m, 1H), 2.09 (s, 3H), 2.02–2.04 (m, 6H),
1.74 (bs, 2H), 1.37 (bs, 1), 1.23–1.43 (m, 6H); 13C NMR (75 MHz,
CDCl3) δ 170.7, 170.6, 169.6, 137.6, 128.7, 128.6, 128.5, 126.5,
116.5, 85.8, 71.2, 68., 68.6, 62.0, 56.0, 52.9, 45.6, 34.3, 33.9, 33.6,
25.9, 25.6, 20.9, 20.8, 20.7.
N-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihydroxy-6-(hy-
droxymethyl)-tetrahydro-2H-pyran-3-yl)-2,2,2-trifluoroaceta-
mide (24). To a solution of 10a (0.077 mmol) in CH2Cl2 (0.77
mL) was added pyridine (0.31 mmol), and the mixture was cooled
to 0 °C. Trifluoroacetic anhydride (0.092 mmol) was added to the
solution and the mixture was allowed to warm to rt and stirred
overnight. The mixture was concentrated in vacuo and the crude
oil was purified via prep TLC (40/60 EtOAc/Hex) to provide 24
1
as a white solid (88%). H NMR (600 MHz, CD3OD/CDCl3), δ
5.56 (d, J ) 5.5 Hz, 1H), 4.04 (dd, J ) 5.4, 5.9, 1H), 4.00 (m,
1H), 3.82 (d, J ) 9.9 Hz, 1H), 3.75 (dd, J ) 5.3, 6.7 Hz, 1H), 3.69
(t, J ) 8.9 Hz, 1H), 3.39 (t, J ) 9.2 Hz, 1H), 2.82 (m, 1H), 1.98
(m, 5H), 1.33 (m, 6H); 13C NMR (125 MHz, CD3OD) δ 159.3,
118.5, 83.4, 74.6, 72.7, 72.2, 62.6, 56.1, 45.1, 35.4, 35.1, 27.0, 26.5,
26.4; solvent A, 20.4; solvent B, 24.3 min; IR (cm-1) 3456, 3016,
2977, 1739, 1442, 1366, 1228; HRMS (ES-) m/z calcd for
C14H21NO5F3S ([M - H]-), 372.1093; found, 372.1091; ∆ ) -0.4
ppm.
General Procedure for Coupling of Acids to Amine 10a
(Schemes 4, 6, 7). To a stirred solution of carboxylic acid (0.19
mmol) and 10a (0.21 mmol) at 0 °C in DMF (2.0 mL) was added
sequentially DIEA (40 µL, 0.23 mmol) and DEPC (40 µL, 0.26
mmol). The mixture was warmed to rt over a 1 h period and
maintained for an additional 1 h. The now light yellow solution
was diluted with H2O (20 mL) and extracted with CH2Cl2 (3 × 20
mL). Purification by preparative TLC (SiO2, 40/60 EtOAc/hexanes)
provided the UV-active products.
(E)-N-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihydroxy-6-
(hydroxymethyl)-tetrahydro-2H-pyran-3-yl)-2-phenylethene-
sulfonamide (20). The title compound 20 was obtained by
following the standard deacetylation procedure described above.
Purification of the crude product by preparative TLC (Si gel, 10/
1
90 MeOH/CH2Cl2) provided 20 as a white solid (96%). H NMR
(300 MHz, CD3OD/CDCl3) δ 7.61 (m, 1H), 7.48 (d, J ) 16.3 Hz,
1H), 7.44 (m, 4H), 5.45 (d, J ) 5.1 Hz, 1H), 3.98 (m, 1H), 3.79
(dd, J ) 2.5, 9.4 Hz, 1H), 3.73 (dd, J ) 5.4, 6.8 Hz, 1H), 3.53 (dd,
J ) 5.3, 5.5 Hz, 1H), 3.48 (dd, J ) 8.6, 8.5 Hz, 1H), 3.37 (m, 1H),
3.33 (m, 1H), 2.79 (m, 1H), 1.97 (m, 6H), 1.70 (m, 2H), 1.57 (m,
1H), 1.26 (m, 1H); 13C NMR (125 MHz, CD3OD), 140.7, 134.7,
131.7, 130.3(2C), 129.4(2C), 128.9, 86.3, 74.3, 73.2, 72.4, 62.7,
59.4, 45.3, 35.4, 34.9, 27.1, 26.9; IR (cm-1) 3456, 3023, 2929,
1740, 1366, 1217, 1149; solvent A, 26.3; solvent B, 27.3 min;
HRMS (ES+) m/z calcd for C20H29NO6NaS2 ([M + Na]+),
466.1334; found, 466.1320; ∆ ) -3.0 ppm.
(E)-N-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihydroxy-6-
(hydroxymethyl)-tetrahydro-2H-pyran-3-yl)-4-phenylbut-1-ene-
1-sulfonamide (21). The title compound 21 was obtained by
following the standard deacetylation procedure described above.
Purification of the crude product by preparative TLC (Si gel, 10/
(2R,3S,4R,5R,6R)-2-(Acetoxymethyl)-5-(2-(tert-butoxycarbo-
nylamino)-4-phenylbutanamido)-6-(cyclohexylthio)-tetrahydro-
2H-pyran-3,4-diyl Diacetate (25). The title compound 25 was
obtained by following the standard acid coupling procedure
1
1
90 MeOH/CH2Cl2) provided 21 as a white solid (91%). H NMR
described above to provide 25 as a white solid (74%). H NMR
(300 MHz, CD3OD/CDCl3) δ 7.27 (m, 5H), 5.95 (m, 1H), 5.74
(m, 1H), 5.44 (d, J ) 5.2 Hz, 1H), 4.17 (m, 1H), 4.02 (m, 2H),
3.79 (m, 2H), 3.60 (m, 1H), 2.89 (m, 1H), 2.03 (m, 5H), 1.77 (m,
2H), 1.65 (m, 1H), 1.47 (m, 6H), 13C NMR (125 MHz, CD3OD) δ
(300 MHz, CDCl3) δ 7.26–7.31 (m, 3H), 7.15–7.19 (m, 2H), 6.27
(d, J ) 7.8 Hz, 1H), 5.50 (d, J ) 5.1 Hz, 1H), 5.12 (m, 2H), 4.90
(m, 1H), 4.42 (m, 2H), 4.27 (dd, J ) 4.8, 12.3 Hz, 1H), 4.09 (m,
1H), 2.81 (m, 1H), 2.63 (t, J ) 7.5 Hz, 1H), 2.09 (s, 3H), 2.02 (s,

Inhibitors of GlcNAc-Ins Deacetylase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25 6333
3H), 1.92 (s, 3H), 1.70 (m, 2H), 1.58 (m, 1H), 1.46 (s, 9H),
1.24–1.40 (m, 10H); LRMS C33H49N2O10S (M + H+) 665.3.
tert-Butyl 1-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihy-
droxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-3-ylamino)-1-
oxo-4-phenylbutan-2-ylcarbamate (26). The title compound 26
was obtained by following the standard deacetylation procedure
10.8 Hz, 1H), 3.90 (m, 1H), 2.91 (s, 3H), 2.72 (m, 3H), 2.08 (s,
3H), 2.04 (s, 3H), 1.93 (s, 3H), 1.92 (m, 2H), 1.70 (m, 3H), 1.56
(m, 1H), 1.25–1.28 (m, 6H); LRMS C29H43N2O10S2 (M + H+)
643.3.
N′-acetyl-N-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihy-
droxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-3-yl)-4-phenyl-
butanehydrazide (30). Title compound 30 was obtained following
the standard deacetylation procedure described above. Purification
of the crude product by preparative TLC (10/90 MeOH/CH2Cl2)
provided 30 as a white solid (95%). 1H NMR (600 MHz, CD3OD)
δ 7.27 (m, 2H), 7.21 (d, J ) 7.5 Hz, 2H), 7.17 (t, J ) 7.1 Hz, 1H),
5.53 (d, J ) 5.3 Hz, 1H), 4.41 (dd, J ) 5.7, 3.0 Hz, 1H), 4.00 (m,
2H), 3.80 (m, 1H), 3.73 (dd, J ) 5.3 Hz, J ) 6.7 Hz, 1H), 3.54
(m, 1H), 3.36 (t, J ) 9.5 Hz, 1H), 2.82 (m, 1H), 2.69 (m, 4H),
2.09 (m, 2H), 2.06 (s, 3H), 1.95 (m, 3H), 1.33 (m, 6H); 13C NMR
(125 MHz, CD3OD/CDCl3), 174.5, 173.4, 142.8, 138.2, 130.0,
127.2, 84.3, 74.7, 73.1, 72.7, 62.7, 56.0, 54.6, 45.2, 35.5, 35.1, 35.0,
1
described above to provide 26 as a white solid (97%). H NMR
(600 MHz, CD3OD/CDCl3), δ 7.31 (m, 2H), 7.26 (d, J ) 7.0 Hz,
2H), 7.22 (t, J ) 8.2 Hz, 1H), 5.62 (d, J ) 4.7 Hz, 1H), 4.14 (t, J
) 5.6 Hz, 1H), 4.07 (m, 2H), 3.86 (m, 2H), 3.59 (t, J ) 8.3 Hz,
1H), 3.48 (t, J ) 9.0 Hz, 1H), 2.86 (m, 1H), 2.78 (m, 1H), 2.72
(m, 2H), 2.13 (m, 2H), 1.99 (m, 4H), 1.56 (s, 9H), 1.28–1.45 (m,
5H); 13C NMR (125 MHz, CD3OD) δ 175.3, 170.8, 158.0, 142.7,
129.8, 129.7 (2C), 127.4, 84.6, 81.2, 74.5, 73.4, 72.6, 62.7, 56.0,
45.5, 35.6, 35.4, 35.2, 33.2, 29.2 (3C), 27.1; solvent A, 35.0; solvent
B, 40.2 min; IR (cm-1) 3342, 3016, 2970, 1739, 1447, 1366, 1217;
HRMS (ES+) m/z calcd for C27H42N2O7NaS ([M + Na]+),
561.2610; found, 561.2615; ∆ ) 0.8 ppm.
33.2, 27.0, 22.7; solvent A, 21.6; solvent B, 27.3 min; IR (cm-1
)
3457, 3026, 2971, 1739, 1435, 1366, 1229; HRMS (ES+) m/z calcd
for C24H36N2O6NaS ([M + Na]+), 503.2192; found, 503.2177; ∆
) -2.9 ppm.
General Procedure for Removal of the t-Boc Group with
TFA. Cold anhydrous TFA (2.5 mL) was added to N-Boc protected
amine (0.054 mmol) at 0 °C. The yellow mixture was stirred for 5
min, warmed to rt with stirring, and concentrated in vacuo. The
acid was quenched with pyridine (2 mL), stirred for 30 min, and
concentrated in vacuo. The crude products were passed through a
plug of Si gel (10/90 MeOH/ CH2Cl2), concentrated in vacuo, and
treated with desired acid, anhydride, or acid chloride.
(2R,3S,4R,5R,6R)-5-(2-Acetamido-4-phenylbutanamido)-2-
(acetoxymethyl)-6-(cyclohexylthio)-tetrahydro-2H-pyran-3,4-
diyl Diacetate (27). Title compound 27 was obtained following
treatment with TFA, as desribed above. To a solution of 10a (0.077
mmol) in CH2Cl2 (0.77 mL) was added pyridine (0.31 mmol), and
the mixture was cooled to 0 °C. Acetic anhydride (0.092 mmol)
was added to the solution and the mixture was allowed to warm to
rt and stirred overnight. The mixture was concentrated in vacuo
and the crude oil was purified via prep TLC (10/90 MeOH/CH2Cl2)
to provide 27 as a white solid (93%). 1H NMR (300 MHz, CDCl3)
δ 7.20–7.32 (m, 5H), 6.37 (d, J ) 8.1 Hz, 1H), 5.98 (d, J ) 7.5
Hz, 1H), 5.58 (d, J ) 5.4 Hz, 1H), 5.12 (m, 2H), 4.43–4.49 (m,
3H), 4.32 (dd, J ) 4.8, 7.5 Hz, 1H), 4.13 (m, 1H), 2.84 (m, 1H),
2.67 (m, 2H), 2.14 (s, 3H), 2.08 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H),
1.92 (m, 2H), 1.75 (m, 2H), 1.61 (m, 1H), 1.28–1.46 (m, 8H);
LRMS C30H43N2O9S (M + H+) 607.3.
N-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihydroxy-6-(hy-
droxymethyl)-tetrahydro-2H-pyran-3-yl)-4-phenyl-2-(2,2,2-tri-
fluoroacetamido)butanamide (31). Title compound 31 was ob-
tained following the standard deacetylation procedure described
above. Purification of the crude product by preparative TLC (10/
1
90 MeOH/CH2Cl2) provided 31 as a white solid (91%). H NMR
(600 MHz, CD3OD/CDCl3) δ 6.97 (m, 3H), 6.88 (m, 2H), 5.20 (d,
J ) 5.3, 1H), 4.30 (bs, 1H), 4.18 (m, 1H), 3.67 (dt, J ) 10.0, 2.9
Hz, 1H), 3.49 (d, J ) 3.4 Hz, 2H), 3.22 (dd, J ) 3.4, 8.9 Hz, 1H),
3.14 (t, J ) 9.7 Hz, 1H), 2.47 (m, 1H), 2.39 (m, 2H), 1.85 (m,
2H), 1.75 (m, 2H), 1.63 (m, 6H), 1.41 (m, 2H), 1.27 (m, 1H), 0.95
(m, 1H); 13C NMR (125 MHz, CD3OD), 172.6, 142.1, 129.9, 129.8
(2C), 127.6, 84.5, 74.2, 73.1, 72.6, 62.8, 56.0, 54.9, 45.7, 35.6,
35.1, 34.8, 33.1, 27.0; solvent A, 31.4; solvent B, 35.5 min; IR
(cm-1) 3456, 3026, 2971, 1739, 1447, 1366, 1229; HRMS (ES+)
m/z calcd for C24H33N2O6F3NaS ([M + Na]+), 557.1909; found,
557.1911; ∆ ) 0.3 ppm.
N-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihydroxy-6-(hy-
droxymethyl)-tetrahydro-2H-pyran-3-yl)-2-(methylsulfonamido)-
4-phenylbutanamide (32). Title compound 32 was obtained
following the standard deacetylation procedure described above.
Purification of the crude product by preparative TLC (10/90 MeOH/
CH2Cl2) provided 32 as a white solid (89%). 1H NMR (600 MHz,
CD3OD/CDCl3), 7.27 (m, 2H), 7.21 (d, J ) 7.5 Hz, 2H), 7.17 (t,
J ) 7.1 Hz, 1H), 5.58 (d, J ) 5.3 Hz, 1H), 3.99 (m, 2H), 3.80 (m,
2H), 3.59 (dd, J ) 3.2, 8.8 Hz, 1H), 3.42 (t, J ) 3.6 Hz, 1H), 2.97
(s, 3H), 2.78 (m, 2H), 2.71 (m, 1H), 2.06 (m, 3H), 1.94 (m, 5H),
1.69 (m, 2H), 1.57 (m, 1H), 1.26 (m, 3H); 13C NMR (125 MHz,
CD3OD), 174.0, 142.1, 129.2, 126.9, 83.6, 78.9, 73.6, 72.4, 72.0,
62.2, 57.9, 55.4, 44.5, 41.2, 36.2, 35.1, 34.6, 32.6, 26.5; solvent A,
23.7; solvent B, 26.8 min; IR (cm-1) 3456, 3017, 2971, 1739, 1456,
1366, 1229; HRMS (ES+) m/z calcd for C23H36N2O7NaS2 ([M +
Na]+), 539.1862; found, 539.1866; ∆ ) 0.8 ppm.
General Procedure for Preparation of tert-Butyldimethylsilyl
Methyl Esters. To a stirred solution of tert-butyldimethylsilyl
chloride (3.80 g, 25.2 mmol) in CH3CN (16 mL) was added 33a
or 33b.27 Upon cooling to 0 °C, DBU (3.8 mL, 25.2 mmol) was
added to the mixture, and stirring was maintained at rt overnight.
The white heterogeneous mixture was filtered, and the precipitate
was crystallized from MeOH/CH3CN to give silyl ethers 34a or
34b as white powders in 70–85% yield. To a stirred solution of
34a or 34b (8.55 mmol) in benzene (80 mL) and MeOH (24 mL)
at rt was added trimethylsilyldiazomethane (2.0 M in hexanes, 22
mL, 44 mmol), and the mixture was stirred overnight at rt. The
yellowish solution was concentrated in vacuo to give the desired
O-tert-butyldimethylsilyl methyl ester quantitatively to be used in
the next step without further purification.
(2R,3S,4R,5R,6R)-2-(Acetoxymethyl)-6-(cyclohexylthio)-5-(4-
phenyl-2-(2,2,2-trifluoro-acetamido)butanamido)-tetrahydro-
2H-pyran-3,4-diyl Diacetate (28). Title compound 28 was obtained
following treatment with TFA, as described above. To a solution
of 10a (0.077 mmol) in CH2Cl2 (0.77 mL) was added pyridine (0.31
mmol), and the mixture was cooled to 0 °C. Trifluoroacetic
anhydride (0.092 mmol) was added to the solution and the mixture
was allowed to warm to rt and stirred overnight. The mixture was
concentrated in vacuo and the crude oil was purified via prep TLC
1
(10/90 MeOH/CH2Cl2) to provide 28 as a white solid (90%). H
NMR (300 MHz, CDCl3) δ 7.14-7.32 (m, 5H), 7.05 (d, J ) 6.6
Hz, 1H), 6.18 (d, J ) 7.8 Hz, 1H), 5.50 (d, J ) 5.4, 1H), 5.09 (m,
2H), 4.39–4.51 (m, 3H), 4.28 (dd, J ) 4.8, 7.8 Hz, 1H), 4.08 (dd,
J ) 2.4, 9.9 Hz, 1H), 2.81 (m, 1H), 2.58–2.65 (m, 2H), 2.18 (m,
1H), 2.09 (s, 3H), 2.03 (s, 3H), 1.90 (s, 3H), 1.70 (m, 2H), 1.25–1.30
(m, 10H); LRMS C30H40F3N2O9S (M + H+) 661.3.
(2R,3S,4R,5R,6R)-2-(Acetoxymethyl)-6-(cyclohexylthio)-5-(2-
(methylsulfonamido)-4-phenylbutanamido)-tetrahydro-2H-py-
ran-3,4-diyl Diacetate (29). Title compound 29 was obtained
following treatment with TFA, as described above. To a solution
of 10a (0.077 mmol) in CH2Cl2 (0.77 mL) was added pyridine (0.31
mmol), and the mixture was cooled to 0 °C. Methanesulfonic acid
anhydride (0.1 mmol) was added to the solution and the mixture
was allowed to warm to rt and stirred overnight. The mixture was
concentrated in vacuo and the crude oil was purified via prep TLC
1
(10/90 MeOH/CH2Cl2) to provide a white solid (88%). H NMR
(300 MHz, CDCl3) δ 7.16–7.29 (m, 5H), 6.41 (d, J ) 8.4 Hz, 1H),
5.58 (d, J ) 5.1 Hz, 1H), 5.23 (d, J ) 8.1 Hz, 1H), 5.10 (m, 2H),
4.45 (m, 2H), 4.27 (dd, J ) 5.1, 12.3 Hz, 1H), 4.07 (dd, J ) 2.1,
3-(tert-Butyl-dimethyl-silanyloxy)-2-(3-fluoro-benzoylamino)-
propionic Acid (34b). H NMR (300 MHz, CD3OD) δ 4.89 (bs,
2H), 3.85 (t, J ) 3.7 Hz, 2H), 3.68 (dd, J ) 4.5, 7.8 Hz, 1H), 3.29
1

6334 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25
Metaferia et al.
(m, 2H), 2.15 (m, 1H), 1.97 (m, 1H), 0.93 (s, 9H), 0.13 (s, 6H);
HRMS (APCI+) m/z calcd for C10H24NO3Si ([M + H]+), 234.1525;
found, 234.1517; ∆ ) -3.6 ppm.
2-(3-Fluorophenyl)-5,6-dihydro-4H-1,3-oxazine-4-carboxylic
Acid (37b). 1H NMR (300 MHz, CD3OD) δ 7.71 (bd, J ) 7.8 Hz,
1H), 7.62 (bd, J ) 9.6 Hz, 1H), 7.37–7.47 (m, 1H), 7.20–7.27 (m,
1H), 4.48 (t, J ) 5.7 Hz, 2H), 4.30 (t, J ) 5.4 Hz, 1H), 2.24–2.35
(m, 1H), 2.07–2.18 (m, 1H).
General Procedure for Coupling Benzoic Acids to Amines.
To a stirred solution containing 3-fluorobenzoic acid (0.64 g, 4.62
mmol), HOBt (0.70 g, 4.62 mmol), EDCI (0.89 g, 4.62 mmol),
and DIEA (1.7 mL, 9.62 mmol) in anhydrous DMF (20 mL) was
added 34a or 34b (3.85 mmol), and the mixture was maintained at
rt for 12 h with stirring. Upon dilution with ethyl acetate (60 mL)
and water (40 mL), the organic layer was separated and washed
with saturated sodium bicarbonate solution (3 × 35 mL) and brine
(35 mL), dried over MgSO4, and concentrated in vacuo. The crude
products were purified by column chromatography (Si gel, gradient
15–45% EtOAc/hexanes) to give the amides 35a (71%) and 35b
(69%).
(2R,3S,4R,5R,6R)-2-(Acetoxymethyl)-6-(cyclohexylthio)-5-(2-
(3-fluorophenyl)-4,5-dihydrooxazole-4-carboxamido)-tetrahydro-
2H-pyran-3,4-diyl Diacetate (38a). The title compound 38a was
obtained following the standard coupling procedure described above.
Purification of the crude product by preparative TLC (40/60EtOAc/
hexanes) afforded 38a as a white solid (79%). 1H NMR (300 MHz,
CDCl3) δ 7.80 (d, J ) 8.1 Hz, 1H), 7.71 (dt, J ) 2.4, 9.3, 1H),
7.42 (dt, J ) 5.7, 8.1 Hz, 1H), 7.23 (dd, 2.4, 8.4 Hz, 1H), 7.05 (d,
J ) 9 Hz, 1H), 5.35 (d, J ) 5.7 Hz, 1H), 5.04–5.21 (m, 2H), 4.78
(dd, J ) 6.9, 11.1 Hz, 1H), 4.59–4.71 (m, 2H), 4.45–4.54 (m, 1H),
4.34–4.38 (m, 1H), 4.27 (dd, J ) 4.8, 12.6 Hz, 1H), 4.04–4.16 (m,
1H), 2.08 (s, 3H), 2.03–2.05 (m, 6H), 1.05–1.86 (m, 11H).
(2R,3S,4R,5R,6R)-2-(Acetoxymethyl)-6-(cyclohexylthio)-5-(2-
(3-fluorophenyl)-5,6-dihydro-4H-1,3-oxazine -4-carboxamido)-
tetrahydro-2H-pyran-3,4-diyl Diacetate (38b). The title com-
pound 38b was obtained following the standard coupling procedure
described above. Purification of the crude product by preparative
TLC (40/60EtOAc/hexanes) afforded 38b as a white solid (71%).
1H NMR (300 MHz, CDCl3) δ 7.76 (m, 2H), 7.36 (m, 1H), 7.15
(m, 1H), 5.56 (d, J) 5.4 Hz, 0.5H), 5.48 (d, J) 5.4 Hz, 0.5H),
5.18 (m, 2H), 5.10 (m, 1H), 4.54–4.25 (m, 5H), 4.13–4.00 (m, 2H),
2.95–2.88 (m, 1H), 2.80–2.73 (m, 1H), 2.48 (m, 1H), 2.08 (m, 9H),
1.35–1.95 (m, 10H).
Methyl 3-(tert-Butyldimethylsilyloxy)-2-(3-fluorobenzami-
1
do)propanoate (35a).. H NMR (300 MHz, CDCl3) δ 7.51–7.57
(m, 2H), 7.42 (m, 1H), 7.22 (m, 1H), 6.95 (d, J ) 7.8 Hz, 1H),
4.83 (dt, J ) 2.4, 2.7 Hz, 1H), 4.14 (dd, J ) 2.4, 7.8 Hz, 1H), 3.95
(dd, J ) 3.0, 7.2 Hz, 1H), 3.78 (s, 3H), 0.86 (s, 9H), 0.03 (s, 6H);
HRMS (APCI+) m/z calcd for C17H27NO4SiF (M + H+), 356.1693;
found, 356.1682; ∆ ) -3.2 ppm.
Methyl 4-(tert-Butyldimethylsilyloxy)-2-(3-fluorobenzami-
do)butanoate (35b). 1H NMR (300 MHz, CDCl3) δ 7.47–7.56 (m,
3H), 7.33–7.40 (m, 1H), 7.13–7.20 (m, 1H), 4.81 (m, 1H), 3.80
(m, 2H), 3.74 (s, 3H), 2.13 (m, 2H), 0.83 (s, 9H), 0.05 (s, 6H); 13
C
NMR (75 MHz, CDCl3) δ 172.5, 166.1, (164.5, 161.3 F-C
coupling), 130.3, 136.5, 122.8, 118.7, 114.7, 60.9, 52.5, 52.2, 33.5,
26.1 (3C), -5.3 (2C); HRMS (APCI+) m/z calcd for C18H29NO4SiF
([M + H]+), 370.1850; found, 370.1848; ∆ ) -0.5 ppm.
(R)-N-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihydroxy-6-
(hydroxymethyl)-tetrahydro-2H-pyran-3-yl)-2-(3-fluorophenyl)-
4,5-dihydrooxazole-4-carboxamide (39D). Title compound 39D
was obtained following the standard deacetylation protocol de-
scribed above. Purification of triol by preparative TLC (10/90
Methyl 2-(3-Fluorophenyl)-4,5-dihydrooxazole-4-carboxylate
(36a). To a stirred solution containing amide 35a (0.81 mmol) in
CH2Cl2 (3.5 mL) was added DAST (0.6 mL, 3.24 mmol) at rt, and
the solution was maintained for 7 h after which it was quenched
slowly with saturated aq NaHCO3. The aqueous layer was extracted
three times with CH2Cl2, and the organic layer was dried over
MgSO4, filtered, and concentrated in vacuo. Purification by column
chromatography (Si gel, 15–30% EtOAc/hexanes) provided oxazole
methyl ester 36a as a clear oil (94%). 1H NMR (300 MHz, CDCl3)
δ 7.75–7.79 (m, 1H), 7.65–7.70 (m, 1H), 7.35–7.42 (m, 1H), 7.20
(m, 1H), 4.96 (dd, J ) 2.7, 8.1 Hz, 1H), 4.71 (dd, J ) 0.6, 8.1 Hz,
1H), 4.61 (dd, J ) 2.1, 8.7 Hz, 1H), 3.82 (s, 3H); 13C NMR (75
MHz, CDCl3) δ Hrms (APCI+) m/z calcd for C11H11NO3F (M +
H+), 224.0723; found, 224.0728; ∆ ) 2.2 ppm.
Methyl 2-(3-Fluorophenyl)-5,6-dihydro-4H-1,3-oxazine-4-car-
boxylate (36b). Title compound 36 was obtained following the
same procedure as described above for 36a. Purification by column
chromatography (Si gel, 15–30% EtOAc/hexanes) provided oxazine
methyl ester 36b as a clear oil (70%). 1H NMR (300 MHz, CDCl3)
δ 7.71–7.74 (m, 1H), 7.61–7.66 (m, 1H), 7.26–7.35 (m, 1H),
7.07–7.14 (m, 1H), 4.30–4.47 (m, 3H), 3.78 (s, 3H), 2.12–2.22 (m,
2H); 13C NMR (75 MHz, CDCl3) δ 172.8, 164.4, 161.1, 155.9,
135.9, 129.8, 123.2, 117.9, 114.6, 63.7, 54.6, 52.6, 24.2; HRMS
(APCI+) m/z calcd for C12H13NO3F (M + H+), 238.0879; found,
238.0886; ∆ ) 2.7 ppm.
1
MeOH/CH2Cl2) provided 39D as a white solid (90%). H NMR
(300 MHz, CD3OD/CDCl3) δ 7.75 (d, J ) 5.1 Hz, 1H), 7.76 (m,
1H), 7.43 (m, 1H), 7.25 (m, 1H) 5.51 (d, J ) 5.4 Hz, 1H), 4.70
(m, 2H), 4.88 (dd, J ) 8.4, 2.4 Hz, 1H), 4.07 (dd, J ) 5.1, 5.4 Hz,
1H), 3.95 (m, 1H), 3.77 (m, 2H), 3.46 (m, 2H), 2.82 (m, 1H), 1.96
(m, 2H), 1.74 (m, 2H), 1.58 (m, 1H), 1.22–1.42 (m, 5H); 13C NMR
(75 MHz, CD3OD) δ 172.9, 166.6, 164.8 (doublet due to 19F-13C
coupling), 131.0, 124.9, 119.9 (doublet), 116.2, 115.9 (doublet),
83.9, 73.5, 73.0, 71.5, 71.3, 69.6, 62.0, 54.8, 45.1, 34.9, 34.5, 26.5,
26.4, 26.3; solvent A, 26.7; solvent B, 33.9 min; HRMS (ES-)
m/z calcd for C22H28FN2O6S ([M - H]-), 467.1652; found,
467.1642; ∆ ) -2.2 ppm.
(S)-N-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihydroxy-6-
(hydroxymethyl)-tetrahydro-2H-pyran-3-yl)-2-(3-fluorophenyl)-
4,5-dihydrooxazole-4-carboxamide (39L). Title compound 39L
was obtained following the standard deacetylation protocol de-
scribed above. Purification of triol by preparative TLC (10/90
1
MeOH/CH2Cl2) provided 39L as a white solid (78%). H NMR
(300 MHz, CD3OD/CDCl3) δ 7.79 (d, J ) 5.8 Hz, 1H), 7.67 (d, J
) 6.0 Hz, 1H), 7.45 (m, 1H), 7.27 (m, 1H), 5.27 (d, J ) 5.4 Hz,
1H), 4.91 (dd, J ) 8.4, 1.5 Hz, 1H), 4.66 (m, 1H), 4.13 (dd, J )
5.1, 5.4 Hz, 1H), 3.91 (m, 1H), 3.77 (m, 2H), 3.45 (m, 2H), 2.67
(m, 1H), 1.84 (m, 1H), 1.67 (m, 1H), 1.58 (m, 1H), 1.47 (m, 2H)
1.03–1.21 (m, 6H); 13C NMR (75 MHz, CD3OD) δ 173.4, 166.7,
165.1, 161.8, 131.3, 125.2, 120.1, 116.3, 84.7, 74.2, 73.5, 71.6,
69.4, 62.2, 54.5, 45.3, 34.8, 34.5, 26.5, 26.4, 26.3; solvent A, 25.8;
solvent B, 32.4 min; HRMS (ES-) m/z calcd for C22H28FN2O6S
([M - H]-), 467.1652; found, 467.1653; ∆ ) 0.2 ppm.
General Procedure for Preparation of Acids. To an ice-cooled
solution of either oxazine methyl ester 36a or oxazoline methyl
ester 36b in MeOH/water (3:1) was added LiOH (2.3 equiv) with
stirring. The mixture was allowed to warm to rt in 1 h, concentrated
in vacuo, and the white solid was dissolved in water/CH2Cl2. The
layers were separated and the aqueous layer was acidified with 1
N HCl to pH 2.0 and extracted with CH2Cl2 (3 × 10 mL). The
combined organic layers were dried over MgSO4, filtered, and
concentrated in vacuo to provide the respective acids 37a and 37b
in quantitative yields.
(R)-N-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihydroxy-6-
(hydroxymethyl)-tetrahydro-2H-pyran-3-yl)-2-(3-fluorophenyl)-
5,6-dihydro-4H-1,3-oxazine-4-carboxamide (40D). Title com-
pound 40D was obtained following the standard deacetylation
protocol described above. Purification of triol by preparative TLC
1
(10/90 MeOH/CH2Cl2) provided 40D as a white solid (91%). H
2-(3-Fluorophenyl)-4,5-dihydrooxazole-4-carboxylic Acid (37a).
1H NMR (300 MHz, CDCl3) δ 7.78 (d, J ) 8.1 Hz, 1H), 7.65–7.69
(m, 1H), 7.49 (dt, J ) 5.7, 8.1 Hz, 1H), 7.31 (dt, 2.4, 8.7 Hz, 1H),
4.94 (q, J ) 9.0 Hz, 1H), 4.69 (d, J ) 8.7 Hz, 2H).
NMR (300 MHz, CD3OD/CDCl3) δ 8.02 (d, J ) 8.7 Hz, 1H), 7.76
(dd, J ) 7.8, 10.2 Hz, 1H), 7.35 (m, 1H), 7.16 (dt, J ) 2.7, 5.7
Hz, 1H), 5.45 (d, J ) 5.1 Hz, 1H), 4.51 (m, 1H), 4.41 (m, 1H),
4.39 (m, 1H), 4.30 (m, 1H), 4.21 (m, 1H), 3.97 (m, 1H), 3.76 (m,

Inhibitors of GlcNAc-Ins Deacetylase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25 6335
1H), 3.70 (t, J ) 9.0 Hz, 1H), 3.52 (m, 1H), 2.86 (m, 1H), 2.47
(m, 2H), 2.02 (m, 2H), 1.75 (m, 1H), 1.59 (m, 1H), 1.23–1.46 (m,
8H); solvent A, 31.7; solvent B, 37.6 min; HRMS (ES-) m/z calcd
for C23H30FN2O6S ([M - H]-), 481.1809; found, 481.1808; ∆ )
-0.1 ppm.
xazole-3-carboxamide (44). Title compound 44 was obtained
following the standard coupling procedure described above with
coupling to 10b. Purification of the crude product by RP-HPLC
(20–80% MeOH in 0.05% aq TFA) provided 44 as a white solid
1
(60%). H NMR (600 MHz, CD3OD/CDCl3) δ 7.91 (d, J ) 9.2
Hz, 2H), 7.60 (d, J ) 8.3 Hz, 2H), 7.15 (s, 1H), 5.71 (d, J ) 5.4
Hz, 1H), 4.12 (m, 1H), 3.92 (m, 2H), 3.75 (dd, J ) 3.2, 8.7 Hz,
1H), 3.59 (t, J ) 9.5 Hz, 1H), 2.93 (m, 1H), 2.06 (m, 2H), 1.84
(m, 2H), 1.67 (m, 1H), 1.30–1.53 (6H); 13C NMR (75 MHz,
CD3OD) δ 171.4, 159.7, 131.6, 130.2 (2C), 128.0 (2C), 126.0,
100.0, 84.0, 73.8, 72.3, 71.6, 62.1, 55.2, 45.2, 34.9, 34.5, 26.6, 26.5,
26.3; solvent A, 27.8; solvent B, 36.4 min; HRMS (ES-) m/z calcd
for C22H26N2O6ClS ([M - H]-), 481.1200; found, 481.1183; ∆ )
-3.6 ppm.
(S)-N-((2R,3R,4R,5S,6R)-2-(Cyclohexylthio)-4,5-dihydroxy-6-
(hydroxymethyl)-tetrahydro-2H-pyran-3-yl)-2-(3-fluorophenyl)-
5,6-dihydro-4H-1,3-oxazine-4-carboxamide (40L). Title com-
pound 40L was obtained following the standard deacetylation
protocol described above. Purification of triol by preparative TLC
1
(10/90 MeOH/CH2Cl2) provided 40L as a white solid (90%). H
NMR (300 MHz, CD3OD/CDCl3) δ 7.96 (d, J ) 9.0 Hz, 1H), 7.74
(dd, J ) 7.8, 10.2 Hz, 1H), 7.33 (m, 1H), 7.13 (dt, J ) 2.7, 6.0
Hz, 1H), 5.30 (d, J ) 5.1 Hz, 1H), 4.42 (m, 1H), 4.30 (m, 3H),
3.99 (m, 2H), 3.77 (m, 2H), 3.61 (m, 1H), 2.74 (m, 1H), 2.47 (m,
2H), 1.85 (m, 2H), 1.55 (m, 2H), 1.06–1.33 (m, 6H); solvent A,
29.2; solvent B, 35.7 min; HRMS (ES-) m/z calcd for
C23H30FN2O6S ([M - H]-), 481.1809; found, 481.1817; ∆ ) 1.7
ppm.
(2S)-2-Amino-N-((2R,3R,4R,5S,6R)-2-(cyclohexylthio)-tetrahy-
dro-4,5-dihydroxy-6-(hydroxymethyl)-2H-pyran-3-yl)-4-hydroxy-
butanamide (41). Title compound 41 was obtained by following
the standard procedures for removal of the N-Boc group with TFA,
coupling, and deacetylation described above. Purification of the
crude product by preparative TLC (15% MeOH/CH2Cl2) provided
41 as a white solid (69% yield over two steps). 1H NMR (300
MHz, CD3OD/CDCl3) δ 5.54 (d, J ) 5.1 Hz, 1H), 5.50 (t, J ) 4.2
Hz, 2H), 4.62 (m, 1H), 4.39 (m, 1H), 4.27 (m, 1H), 4.00 (m, 2H),
3.79 (m, 1H), 3.65 (m, 1H), 2.82 (m, 1H), 2.07 9m, 1H), 1.86 (m,
1H), 1.68–1.75 (m, 5H), 1.36–1.46 (m, 6H); solvent A, 16.2; solvent
B, 18.8 min; HRMS (ES+) m/z calcd for C16H31N2O6S ([M + H]+),
379.1916; found, 379.1913; ∆ ) -0.8 ppm.
(S)-3-((2R,3R,4R,5S,6R)-2-(Cyclohexylsulfonyl)-tetrahydro-
4,5-dihydroxy-6-(hydroxymethyl)-2H-pyran-3-ylcarbamoyl)-3-
aminopropanoic Acid (42). Title compound 42 was obtained in
four steps described previously. Following the standard procedures
for removal of the N-Boc group and acid coupling to 10a, as
described above, the intermediate was passed through a plug of Si
gel (15/85 MeOH/CH2Cl2) and dried in vacuo. To a stirred solution
of intermediate (0.11 mmol) in 1:1 CH3CN/CCl4 (2 mL) was added
NaIO4 (3 equiv) in water (1.5 mL), followed by RuCl3 (2 mol%).
The mixture was stirred for 2 h, filtered through a pad of Celite,
concentrated in vacuo, and purified by column chromatography (Si
gel, 1/1 MeOH/CH2CL2). Global deacetylation following the general
procedure described above afforded 42 as a white solid (35% yield in
four steps). 1H NMR (300 MHz, CD3OD/CDCl3) δ 5.29 (d, J ) 5.4
Hz, 1H), 4.29 (m, 2H), 4.13 (t, J ) 5.1 Hz, 1H), 3.92 (d, J ) 10 Hz,
1H), 3.65 (dd, J ) 5.7, 6.9 Hz, 1H), 3.49 (m, 2H), 3.10 (br, 2H), 2.87
(m, 1H), 2.66 (dd, J ) 2.8, 6.2 Hz, 2H), 2.13 (m, 2H), 1.93 (m, 1H),
1.83 (m, 1H), 1.72 (m, 1H), 1.21–1.48 (m, 6H); solvent A, 14.8; solvent
B, 16.4 min; HRMS (ES+) m/z calcd for C16H29N2O9S ([M + H]+),
425.1594; found, 425.1591; ∆ ) -0.7 ppm.
5-(5-Chloro-2-(trifluoromethyl)phenyl)-N-((2R,3R,4R,5S,6R)-
2-(cyclohexylthio)-tetrahydro-4,5-dihydroxy-6-(hydroxymethyl)-
2H-pyran-3-yl)furan-2-carboxamide (45). Title compound 45 was
obtained following the standard coupling procedure described above
with coupling to 10b. Purification of the crude product by RP-
HPLC (20–80% MeOH in 0.05% aq TFA) provided 45 as a white
solid (65%). 1H NMR (300 MHz, CD3OD/CDCl3) δ 8.02 (s, 1H),
7.29–7.41 (m, 2H), 7.05 (dd, J ) 3.6, 8.4 Hz, 2H), 5.30 (d, J )
5.1 Hz, 1H), 4.06 (dd, J ) 5.4, 5.3 Hz, 1H), 3.77 (m, 1H), 3.57
(m, 2H), 3.40 (dd, J ) 1.8, 9.0 Hz, 1H), 3.27 (dd, J ) 9.6 Hz,
1H), 2.58 (m, 1H), 1.72 (m, 2H), 1.45 (m, 2H), 1.29 (m, 1H),
0.96–1.17 (m, 6H); 13C NMR (75 MHz, CD3OD) δ 173.3, 158.7,
150.5, 146.7, 134.2, 131.5, 129.7, 129.3, 128.6, 125.2, 116.3, 113.8,
83.5, 72.7, 72.0, 70.7, 61.3, 53.8, 44.4, 33.9, 33.6, 25.6, 25.5, 25.3;
solvent A, 27.8; solvent B, 35.3 min; HRMS (APCI+) m/z calcd
for C24H28NO6F3SCl ([M + H]+), 550.1278; found, 550.1279; ∆
) 0.2 ppm.
General Procedure for Synthesis and Purification of GlcN
Analogs. GlcN amides were prepared in two consecutive steps by
following the standard coupling and global deacetylation procedures
described above. Crude products were purified by preparative TLC
(SiO2, 15/85 MeOH/CH2Cl2) to provide each analog as a white
solid in 60–65% yield in two steps.
5-(2-Chloro-5-(trifluoromethyl)phenyl)-N-((2R,3R,4R,5S,6R)-
tetrahydro-2,4,5-trihydroxy-6-(hydroxymethyl)-2H-pyran-3-yl)
furan-2-carboxamide (47). 1H NMR (600 MHz, CD3OD/CDCl3)
δ 8.46 (bs, 1H), 7.75 (d, J ) 8.3 Hz, 1H), 7.65 (d, J ) 8.3 Hz, 1H)
7.32 (d, J ) 3.8 Hz, 1H), 7.29 (d, J ) 3.8 Hz, 1H), 5.19 (br, 1H),
4.11 (m, 1H), 3.88 (m, 2H), 3.74 (m, 1H), 3.46 (t, J ) 9.5 Hz,
1H), 3.38 (t, J ) 8.4 Hz, 1H); solvent A, 20.8; solvent B, 22.4
min; HRMS (ES+) m/z calcd for C18H18NO7ClF3 ([M + H]+),
452.0724; found, 452.0705; ∆ ) -4.2 ppm.
5-(4-Chlorophenyl)-N-((2R,3R,4R,5S,6R)-tetrahydro-2,4,5-tri-
hydroxy-6-(hydroxymethyl)-2H-pyran-3-yl)isoxazole-3-carbox-
1
amide (48). H NMR (600 MHz, CD3OD/CDCl3) δ 7.89 (d, J )
9.1 Hz, 2H), 7.55 (d, J ) 8.4 Hz, 2H), 7.16 (s, 1H), 5.23 (d, J )
7.6 Hz, 1H), 4.08 (dd, J ) 4.2, 6.4 Hz, 1H), 3.85 (m, 2H), 3.74
(dd, J ) 4.3, 5.7 Hz, 1H), 3.44 (t, J ) 9.2 Hz, 1H), 3.37 (t, J ) 7.8
Hz, 1H); solvent A, 21.3; solvent B, 23.1 min; HRMS (ES+) m/z
calcd for C16H17N2O7ClNa ([M + Na]+), 407.0622; found, 407.0604;
∆ ) -4.4 ppm.
5-(4-Chlorophenyl)-N-((2R,3R,4R,5S,6R)-2-(cyclohexylthio)-
tetrahydro-4,5-dihydroxy-6-(hydroxymethyl)-2H-pyran-3-yl)fu-
ran-2-carboxamide (43). Title compound 43 was obtained fol-
lowing the standard coupling procedure described above with
coupling to 10b. Purification of the crude product by RP-HPLC
(20–80% MeOH in 0.05% aq TFA) provided 43 as a white solid
5-(4-Chlorophenyl)-N-((2R,3R,4R,5S,6R)-tetrahydro-2,4,5-tri-
hydroxy-6-(hydroxymethyl)-2H-pyran-3-yl)furan-2-carboxam-
1
ide (49). H NMR (600 MHz, CD3OD/CDCl3) δ 7.97 (d, J ) 7.5
1
Hz, 2H), 7.54 (d, J ) 6.6 Hz, 2H), 7.25 (d, J ) 7.3 Hz, 1H), 7.15
(d, J ) 8.4 Hz, 1H); solvent A, 20.3; solvent B, 21.2 min; HRMS
(ES+) m/z calcd for C17H18NO7ClNa ([M + Na]+), 406.0669;
found, 406.0680; ∆ ) 2.6 ppm.
(71%). H NMR (600 MHz, CD3OD/CDCl3) δ 7.94 (d, J ) 9.8
Hz, 2H), 7.54 (d, J ) 9.1 Hz, 2H), 7.35 (d, J ) 4.6 Hz, 1H), 7.04
(d, J ) 3.8 Hz, 1H), 5.74 (d, J ) 5.6 Hz, 1H), 4.34 (dd, J ) 4.9,
6.8 Hz, 1H), 4.13 (m, 1H), 3.94 (dd, J ) 2.6, 11.8 Hz, 1H), 3.87
(dd, J ) 3.7, 5.4 Hz, 1H), 3.84 (m, 1H), 3.55 (t, J ) 8.8 Hz, 1H),
2.95 (m, 1H), 2.06 (m, 1H), 1.82 (m, 2H), 1.65 (m, 1H), 1.30–1.54
(m, 6H); 13C NMR (75 MHz, CD3OD) δ 173.4, 159.4, 155.1, 146.2,
134.8, 129.3 (2C), 128.1, 125.9 (2C), 117.9, 108.0, 84.6, 73.9, 72.9,
70.8, 61.7, 53.9, 45.4, 34.6, 34.5, 26.6, 26.5, 26.3; solvent A, 26.8;
solvent B, 34.4 min; HRMS (ES+) m/z calcd for C23H29NO6ClS
([M + H]+), 482.1404; found, 482.1387; ∆ ) -3.6 ppm.
Acknowledgment. We thank J. Lloyd for HRMS data. This
work was supported in part by the National Institutes of Health
Intramural Research Program (NIDDK) and the Intramural
AIDS Targeted Antiviral Program, Office of the Director, NIH
(C.A.B.).
5-(4-Chlorophenyl)-N-((2R,3R,4R,5S,6R)-2-(cyclohexylthio)-
tetrahydro-4,5-dihydroxy-6-(hydroxymethyl)-2H-pyran-3-yl)iso-
Supporting Information Available: General experimental
methods, details of the docking protocols using AutoDock 4.0, and

6336 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 25
Metaferia et al.
(19) Nicholas, G. M.; Eckman, L. L.; Ray, S.; Hughes, R. O.; Pfefferkorn,
J. A.; Barluenga, S.; Nicolaou, K. C.; Bewley, C. A. Bromotyrosine-
derived natural and synthetic products as inhibitors of mycothiol-S-
conjugate amidase. Bioorg. Med. Chem. Lett. 2002, 12, 2487–2490.
(20) Metaferia, B.; Ray, S.; Smith, J. A.; Bewley, C. A. Design and
synthesis of substrate-mimic inhibitors of mycothiol-S-conjugate
amidase from Mycobacterium tuberculosis. Bioorg. Med. Chem. Lett.
2007, 17, 444–447.
a figure showing the overlay of docked inhibitors and substrate.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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