Conjugates of plumbagin and phenyl-2-amino-1-thioglucoside inhibit MshB, a deacetylase involved in the biosynthesis of mycothiol

Article abstract of DOI:10.1016/j.bmc.2010.02.049

N-Acetylglucosaminylinositol (GlcNAc-Ins)-deacetylase (MshB) and mycothiol-S-conjugate amidase (Mca), structurally related amidases present in mycobacteria and other Actinomycetes, are involved in the biosynthesis of mycothiol and in the detoxification of

Full text of DOI:10.1016/j.bmc.2010.02.049

Bioorganic & Medicinal Chemistry 18 (2010) 2501–2514
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Conjugates of plumbagin and phenyl-2-amino-1-thioglucoside inhibit MshB,
a deacetylase involved in the biosynthesis of mycothiol
b
b
b
David W. Gammon a, , Daniel J. Steenkamp , Vuyo Mavumengwana , Mohlopheni J. Marakalala ,
*
Theophilus T. Mudzunga a, , Roger Hunter a, Muganza Munyololo a
a Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
b Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Observatory 7925, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Acetylglucosaminylinositol (GlcNAc-Ins)-deacetylase (MshB) and mycothiol-S-conjugate amidase
(Mca), structurally related amidases present in mycobacteria and other Actinomycetes, are involved in
the biosynthesis of mycothiol and in the detoxification of xenobiotics as their mycothiol-S-conjugates,
respectively. With substrate analogs of GlcNAc-Ins, MshB showed a marked preference for inositol as
the aglycon present in GlcNAc-Ins. The inhibition of MshB and Mca by 10 thioglycosides, 7 cyclohexyl-
2-deoxy-2-C-alkylglucosides, and 4 redox cyclers was evaluated. The latter contained plumbagin tethered
Received 7 November 2009
Revised 21 February 2010
Accepted 23 February 2010
Available online 1 March 2010
Keywords:
Mycothiol
Enzyme inhibitors
Thioglucosides
Glucosamine derivatives
via 2 to 5 methylene carbons and an amide linkage to phenyl-2-deoxy-2-amino-1-thio-a-D-glucopyran-
oside. These proved to be the most potent amongst the 21 compounds tested as inhibitors of MshB. Their
inhibitory potency varied with the length of the spacer, with the compound with longest spacer being the
most effective.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
mycobacterial metabolism mirrors that of glutathione in other
organisms.
Tuberculosis is presently recognized as a worldwide cause of
mortality in underprivileged communities. Owing to the develop-
ment of strains of Mycobacterium tuberculosis that are resistant to
various combinations of the drugs currently used in the treatment
of tuberculosis, studies on the biochemistry of the mycobacteria
have assumed great urgency, since metabolic processes that are
present in pathogens, but not in their mammalian hosts, can be ex-
plored as potential targets for the development of new drugs.
While all eukaryotes and many bacteria produce glutathione as
a major metabolite, members of the Actinomycetes, the phylum to
which mycobacteria belong, uniquely produce mycothiol.1 The dis-
covery of mycothiol in the mycobacteria2 has prompted detailed
investigations of its biosynthetic enzymes and of enzymatic reac-
tions that involve mycothiol (reviewed in Refs. 3–6). While the full
spectrum of activities which utilize mycothiol has undoubtedly not
yet been explored, the available information suggest that its role in
Early attempts to exploit mycothiol as a potential drug target
focused on the enzyme mycothiol-S-conjugate amidase (Mca), a
Zn-dependent amidase thought to play a role in the detoxification
of alkylating agents and antibiotics such as rifampicin and cerule-
nin.7 A number of bromotyrosine alkaloids of marine origin are as
yet the most potent inhibitors of Mca that have been described.8
Research subsequently moved on to the design of substrate-based
inhibitors of Mca and of the related amidase, MshB, which cata-
lyzes the third step in the biosynthesis of mycothiol.9 To facilitate
the synthesis of such inhibitors a viable alternative was sought
which would obviate the necessity for costly preparation and res-
olution of the partially-protected myo-inositol unit, and its trouble-
some stereospecific glycosylation reaction, required during
synthesis of the pseudodisaccharide moiety present in mycothiol
and its precursors.10 It had indeed earlier been found that the pres-
ence of inositol is not an absolute requirement for substrate recog-
nition by the mycothioldisulfide reductase (mtr).11 Knapp et al.
introduced a S-linked cyclohexane as ‘spacefilling’ substitute for
the inositol moiety present in mycothiol and its specursors.12 As
an alternative, Metaferia et al. replaced inositol with a six-mem-
bered ring derived from quinic acid.13 This allowed retention of
at least some of the hydrophilic character of inositol, while afford-
ing the advantage that only a single dicyclohexylidene derivative is
obtained upon reaction with ethoxycyclohexene as a means of
selective protection of its hydroxyl groups. Either approach
Abbreviations: Mycothiol, 1-
glucopyranoside; MshB, 1- -myo-inosityl-2-acetamido-2-deoxy-
side N-deacetylase; GlcNAc-Ins, 1- -myo-inosityl-2-acetamido-2-deoxy-
pyranoside; MSSNaph, 2-S-mycothiolyl-6-hydroxynaphthyl-disulfide;
D
-myo-inosityl-2-(
L
-cysteinyl)amido-2-deoxy-
a-D-
D
a-D-glucopyrano-
D
a-
D-gluco-
Mca,
mycothiol-S-conjugate amidase; mtr, mycothioldisulfide reductase.
* Corresponding author. Tel.: +27 21 6502547; fax: +27 21 6505195.
E-mail address: david.gammon@uct.ac.za (D.W. Gammon).
Present address: Sasol Technology Research and Development, PO Box 1,
Sasolburg 1947, South Africa.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.049

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D. W. Gammon et al. / Bioorg. Med. Chem. 18 (2010) 2501–2514
afforded compounds with activity as substrates or inhibitors of
Mca, but there seemed to be little advantage in using the quinic
acid derived ring rather than cyclohexane as an inositol surrogate.
HO
HO
HO
O
Following identification of the Mca homolog, 1-
D-myo-inosityl-2-
Y
acetamido-2-deoxy- -glucopyranoside (GlcNAc-Ins) deacetylase
D
X
R2
R1
(MshB), as a mycothiol biosynthetic enzyme,9,14 the latter enjoyed
temporary prominence as a potential drug target, and its three-
dimensional structure was reported independently by two groups
during 2003 and 2004.15,16 It was, however, contemporaneously
found that MshB deficient mutants still produced significant
amounts of mycothiol.17 The continued production of low levels
of mycothiol in such mutants was ascribed to deacetylation of
N-acetyl-glucosaminylinositol (GlcNAc-Ins) by Mca or an alternate,
as yet unidentified amidase.16,18 This meant that complete block-
age of the N-deacetylation of GlcNAc-Ins, and therefore of mycoth-
iol synthesis, can only be achieved by inhibition of both MshB and
the alternate amidase activity responsible for GlcN-Ins formation
in MshB-mutants.
I
X = O or S
Y = NH or CH2
In previous studies, substituent X in compound I has mostly
been sulfur12,19 and R1 cyclohexane, but no comparative analysis
of the suitability of different aglycons as surrogates of the inositol
moiety has as yet been undertaken. We were able to obtain a num-
ber of substrate analogs of GlcNAc-Ins in which a limited range of
sulfur- or oxygen-linked six-membered rings had been introduced
in lieu of inositol (see below in Table 1.)
2.2. Synthesis of mycothiol analogs in which naphthoquinolyl
The most important determinant of the substrate specificities
of Mca and MshB appears to be the presence or absence of a
substituent on the amino group of the cysteine moiety, which
is present in mycothiol. Thus the bimane derivative of desa-
cetylmycothiol proved to be a better substrate of MshB than
the natural substrate, GlcNAc-Ins, which implies that MshB, like
Mca, has a binding site for aromatic groups distal from the bind-
ing pocket that accommodates the pseudodisaccharide moiety.14
It therefore, seemed a reasonable assumption that inhibitors
which simultaneously inhibit both enzymes can be developed.
Such compounds were, indeed, recently reported by Metaferia
et al.19 in an exhaustive study of a series of cyclohexylthioglyco-
sides bearing various substituents on the amino group of the
glucosamine moiety. The most potent inhibitor of both MshB
and Mca was a compound in which the amino group substituent
was 2-carbonyl-5-(p-chlorophenyl)-furan. Variations in the struc-
ture of this aromatic substituent impacted significantly on the
inhibitory potency of this series of inhibitors. The study by
Metaferia et al.19 thus provided further confirmation of the pres-
ence of an apolar binding pocket in proximity to the substrate
binding site of MshB.
In this study we evaluate the substrate specificity of MshB for a
limited series of substrates in which different six-membered rings
substitute for the inositol moiety present in GlcNAc-Ins. We report
the synthesis and evaluation of a number of substrate-mimic
inhibitors, including potential transition state analogs, as inhibi-
tors of Mca and MshB. It was also of interest to establish whether
the range of aromatic substituents in MshB inhibitors can be ex-
tended to include functional groups with known cytotoxicity, such
as the quinone redox cyclers. A limited range of 2,4-naphthoqui-
nones, tethered via spacers of varying length to the amino group
of 1-S-thiophenyl-2-deoxy-2-aminoglucopyranose, were synthe-
sized as potential inhibitors of MshB using a strategy similar to
that reported by Salmon-Chemin et al.20 Some of these com-
pounds were amongst the most potent inhibitors of MshB yet
found. The substrate specificity of Mca was found to overlap not
only with that of MshB, as previously reported, but also with that
of the disulfide reductase, mtr, since both mtr and Mca utilized the
heterodisulfide 2-S-mycothiolyl-6-hydroxynaphthyldisulfide21 as
a substrate.
carboxylic acids replaces N-acetylcysteine
Previous efforts to obtain mycothiol deficient mutants of M.
tuberculosis, by targeted disruption of the mshC22 and mshA23
genes, have been unsuccessful and, seen together with the results
of high density mutagenesis,24 indicated that mycothiol biosynthe-
sis is essential for the growth of M. tuberculosis in culture. The
recent isolation of mycothiol deficient, clinical isolates of M. tuber-
culosis that were resistant to ethionamide and isoniazid, however,
caste doubt on the validity of the earlier studies.25 In view of these
contradictory findings it seemed prudent to attempt the synthesis
of compounds that would not only inhibit MshB, thus lowering the
levels of mycothiol, but which would also serve as subversive sub-
strates of mtr. Subversive substrates are substrate analogs bearing
groups that facilitate the transfer of single electrons between the
flavin present in flavoenzymes and oxygen, with the resultant for-
mation of superoxide. That such an approach could be successful
was suggested by the finding that the mtr reduces various naph-
thoquinones at measurable rates comparable to those reported
for other disulfide reductases.26
In a previous study of the substrate specificity of mtr the suit-
ability of a few different aglycons in lieu of inositol in mycothiol
analogs was explored.27 The benzyl group was found to be the
most suitable. This finding was rationalized on the basis that ino-
sitol has a hydrophobic ’patch’ on the surface of the six-membered
ring which displays three axial hydrogens. In the present study we
found that thiophenylglycosides were also the most active sub-
strate analogs in the reaction catalyzed by MshB. Therefore, a set
of compounds 1a–d (Scheme 1) was designed and prepared, in
which the phenylthioglycoside is tethered to the naphthoquinone,
plumbagin (5, Scheme 1). The choice of plumbagin as the redox cy-
cling naphthoquinone was suggested by its high activity when
used in subversive substrates of trypanothione reductase, and from
the point of view of its synthesis, the possibility of selective mono-
alkylation of the naphthoquinone due to the presence of the
methyl group at the 2-position.
The strategy chosen for preparation of 1a–d envisaged initial
alkylation at the 3-position of plumbagin to form a series of naph-
thyl-substituted alkanoic acids, followed by amide-coupling to a
free amine on the sugar unit. Thus, as illustrated in Scheme 1, thi-
ophenylglycoside 4 was first prepared by treatment of the readily
available acetylated 2-azido glucose 228 with thiophenol under
BF3 catalysis, followed by hydrogenation of the azide group. The
naphthoquinonyl carboxylic acids 7 were then prepared by treat-
ment of plumbagin (5) with a series of di-carboxylic acids 6 in a sil-
ver-mediated oxidative decarboxylation.20 Amine 4 and plumbagin
derivatives 7 were then combined under standard peptide-coupling
2. Rationale and chemical synthesis
2.1. Alternate substrates of MshB
All inhibitors in this study, designed as substrate analogs (I) of
either Mca or MshB, incorporated glucosamine or glucose as a
substructure.

D. W. Gammon et al. / Bioorg. Med. Chem. 18 (2010) 2501–2514
2503
Table 1
Evaluation of a range of GlcNAc glycosides as substrates of MshB
HO
HO
HO
O
HN
X
R
O
Compound
RX
Substrate?
Yes
Activity at 250
106
lM (nmol/min/mg)
OH
OH
OH
O
HO
12
HO
11
13
Yes
No
27
S
O2N
NO2
S
O
14
15
Yes
Yes
6.07
6.11
S
S
OH
O
16
No
OMe
RO
RO
RO
HO
HO
HO
AcO
O
O
O
AcO
AcO
(i)
(iv)
R1
H2N
OAc
S
S
N3
9
2
R = Ac, R1 = N3
R = Ac, R1 = NH2
3
(ii)
4
RO
(iii)
(iv)
O
RO
RO
R = Ac, R1 = NHAc
R = H, R1 = NHAc
10
11
OH
O
(vi)
HN
S
O
HO
n
O
OH
O
OH
(v)
O
O
n
5
O
O
8a d
-
R = Ac, n = 2 - 5
R = H, n = 2 - 5
(iv)
O
O
1a d
-
7a-d (
n = 2 - 5)
HO
OH
n
6 (
n = 2 - 5)
Scheme 1. Reaction conditions: (i) PhSH, BF3ꢀEt2O, 55 °C, 12 h (61%); (ii) H2, Pd/C, rt, 5 h (93%); (iii) Ac2O, pyridine (88%); (iv) cat. NaOMe, MeOH, rt, 1 h (yields of 11, 1a, 1b,
1c, 1d were 98%; 34%, 46%, 60%, 52%, respectively); (v) HO2C(CH2)nCO2H, 30% aq CH3CN, AgNO3, NH4(S2O8)2, 70 °C, 8 h (yields of 7a, 7b, 7c, 7d were 98%; 32%, 40%, 40%, 50%,
respectively); (vi) EDC, HOBt, CH2Cl2/THF, rt, 18 h (yields of 8a, 8b, 8c, 8d were 57%; 84%, 64%, 78%, respectively).
conditions to give the desired amides 1a–d after de acetylation of
the intermediate acetates 8a–d.
ies of cyclohexyl-1-thioglycosides (22–26) with variable substitu-
ents at C-2, and (d) series of C-2-alkylated cyclohexyl
a
glycosides (29, 30, 31, 33, 35, 37, 38), potential substrate or transi-
2.3. Synthesis of substrates analogs and substrate-based
inhibitors of Mca and MshB
tion state isosteres of the natural substrate of MshB.
Substrate analog phenyl-2-acetamido-1-thio-a-glucoside 11
was prepared by N-acetylation and then de-O-acetylation of 4,
which could also be converted directly to the unprotected 2-ami-
noglucoside 9 (Scheme 1). The analogous b-thioglucosides 20 and
21 were prepared from acetylated glucosamine 17 via documented
procedures29,30 (Scheme 2).
In addition to the potential redox cyclers described above a total
of 21 compounds were evaluated as substrates or inhibitors of Mca
and MshB. These included (a) the natural substrate, GlcNAc-Ins
(129) of MshB, (b) a set of substrate analogs retaining the N-acetyl-
glucosamine unit, but with the inositol unit replaced by a range of
O- and S-linked alternatives (11, 13–16 (Table 1), 20, 21), (c) a ser-
The set of cyclohexyl-2-C-alkylglucosides was prepared as
shown in Scheme 3. Acetylated 2-C-allylglucosyl fluoride 2731

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D. W. Gammon et al. / Bioorg. Med. Chem. 18 (2010) 2501–2514
was treated with cyclohexanol in the presence of BF3ꢀEt2O to give
the anomeric mixture of cyclohexyl glycosides 28a and 28b, which
could be readily separated and deacetylated to provide cyclohexyl-
2-C-allylglucosides 29 and 30, respectively. Hydrogenation of 29
gave 2-C-propylglucoside 31, while Wacker oxidation of the allyl
group in 28a yielded ketone 32 which was deacetylated to give
the 2-C-oxopropylglucoside 33 or reduced with NaBH4 to give,
after deprotection, the inseparable mixture of diastereomeric alco-
hols 35. The latter was also obtained by LiAlH4 reduction of the
mixture of epoxides 36, available by treatment of 2-C-allylgluco-
side 28a with m-chloroperbenzoic acid. Deacetylation of 36 using
catalytic sodium methoxide in methanol gave a moderate yield
of epoxide 37 accompanied by a bicyclic product 38, presumed
to arise from attack by the intermediate C-3 alkoxide formed upon
deacetylation of the glucose unit, on the terminal carbon of the
epoxypropyl unit.
3. Results and discussion
3.1. Alternate substrates of MshB
Compounds evaluated as substrates of MshB are presented in
Table 1. The Km value for all compounds other than GlcNAc-Ins
12 and phenyl-2-acetamido-2-deoxy-1-thio-a-D-glucopyranoside
(11) could not be established, because of limited availability and
poor solubility (Fig. 1), but a comparison of their rate of cleavage,
when present at a similar concentration of 250
in Table 1.
lM, is presented
RO
RO
RO
AcO
AcO
AcO
RO
(ii)
O
O
(i)
O
RO
RO
S
OAc
NHAc
S
NHAc
19
NHAc
17
R = Ac
21 R = H
18
(iii)
R = Ac
(iii)
20 R = H
Scheme 2. Reaction conditions: (i) PhSH, SnCl4, CH2Cl2, reflux, 24 h (75%); (ii) (NH2)2CS, CH3CN, 80 °C, 15 min, then PhCH2Br, Et3N, rt, 3 h (75%); (iii) cat. NaOMe, MeOH, rt
(yields of 20, 21 were 91%, 89%, respectively).
HO
HO
HO
HO
HO
HO
O
O
(iii)
O
O
29
(ii)
31
AcO
AcO
AcO
AcO
AcO
AcO
RO
RO
RO
O
(i)
O
O
O
+
F
O
28b R = Ac
30
(ii)
27
R = H
28a
(iv)
(vi)
AcO
AcO
O
O
AcO
AcO
AcO
AcO
O
O
O
O
32
36
(vii)
(v)
(ii)
(ii)
HO
HO
HO
RO
RO
RO
HO
HO
HO
HO
HO
O
O
O
O
O
+
O
O
O
HO
34
O
O
OH
38
O
R = Ac
33
37
(ii)
35 R = H
Scheme 3. Reagents and conditions: (i) cyclohexanol, BF3ꢀEt2O, CH2Cl2, rt, 1 h (yields of 28a, 28b were 42%, 36%, respectively); (ii) NaOMe, MeOH. rt (yields of 29, 30, 33, 35,
37, 38 were 99%, 98%, 84%,89%, 50%, 42%, respectively); (iii) H2, Pd/C, EtOAc–MeOH (97%); (iv) O2, PdCl2, CuCl2, DMF–H2O (50%); (v) NaBH4, THF–MeOH, ꢁ20 °C to 0 °C, 2 h
(88%); (vi) m-CPBA, CH2Cl2 (91%).

D. W. Gammon et al. / Bioorg. Med. Chem. 18 (2010) 2501–2514
2505
500
400
300
200
100
0
For cleavage of GlcNAc-Ins 12 by MshB the kinetic constants
were estimated to be, Km = 347.7 27.2 M, Vmax = 436.6 148
nmol/min/mg, kcat = 0.24 0.08 sꢁ1 and kcat/Km = 690.3 Mꢁ1 sꢁ1
For the deacetylation of phenylthioglycoside 11 the corresponding
values were Km = 1695 151 M, Vmax = 214 57 nmol/min/mg,
kcat = 0.120 0.03 sꢁ1 and kcat/Km = 69.4 Mꢁ1 sꢁ1
From the results presented in Table 1 and Figure 1 it is evident
that the choice of aglycon is an important determinant of the rec-
ognition of substrate analogs of GlcNAc-Ins by MshB, the planar
phenyl ring in a thiophenyl aglycon, as in phenylthioglycoside
11, being better tolerated than the thiocyclohexyl group. By con-
trast the corresponding b-phenylthioglycoside was not a substrate
(Table 2) The introduction of an electron withdrawing substituent,
even though located in proximity to the scissile bond in cyclo-
l
.
l
.
hexyl-2-chloroacetamido-2-deoxy-1-thio-a-D-glucopyranoside 22
(Table 2), did not seem to alter the rate of amidase cleavage by
MshB significantly.
0
500
1000
1500
2000
2500
substrate (μΜ)
Figure 1. Dependence of the rate of amidase cleavage of substrate analogs on the
nature of the aglycon in N-acetylglucosamine glycosides. The curves represent the
dependence of GlcN-Ins formation with the natural substrate GlcNAc-Ins 12 (d),
3.2. Evaluation of a series of thioglycosides as inhibitors of
MshB and Mca
phenyl-2-acetamido-2-deoxy-1-thio-
acetamido-2-deoxy-1-thio- -glucopyranoside 15 (j), cyclohexyl-2-acetamido-
2-deoxy- -glucopyranoside 14 (.) and cyclohexyl-2-chloroacetamido-2-deoxy-
1-thio- -glucopyranoside 22 ( ).
a-D-glucopyranoside 11 (s), cyclohexyl-2-
a-D
Thioglycosides tested as inhibitors of MshB and Mca (Table 2)
included compounds that were not substrates, despite the
a-D
a-
D
r
Table 2
Inhibition of Mca and MshB by thioglycosides: percentage inhibition was determined at substrate and inhibitor concentrations of 250
l
M each for Mca and 500
lM each for MshB
HO
O
HO
HO
R2
R1
Compound
R1
R2
Mca (%)
0
MshB (%)
29.4
H
N
S
NO2
13
O
O2N
H
N
S
OH
O
16
27
0
0
OMe
O
O
O
S
H
N
20 (b-linked)
15.1
H
N
21 (b-linked)
0
13.3
6.1
S
9
–NH2
17
S
H
N
S
22
23
24
Cl
0
0
0
14.9
16.4
0
O
S
S
S
–NH2
H
N
CH3
O
O
H
N
S
S
CH3
25
26
6.8
15
0
NH2
H
N
H
N
17
O

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D. W. Gammon et al. / Bioorg. Med. Chem. 18 (2010) 2501–2514
Table 4
presence of a cleavable N-acetyl group as in 13, 16, 20 and 21.
These compounds were also poor inhibitors, which confirms again
the importance of the linkage to the glucosamine and of the struc-
ture of the aglycon for binding to the active sites of these enzymes.
These observations are in agreement with those of Metaferia
et al.19 in that it was found that inhibitors lacking an aglycon were
much poorer inhibitors than the corresponding structures in which
a spacefilling, thiocyclohexyl moiety served as inositol surrogate.
Thioglycosides 9 and 23, analogs of the product Glc-Ins, were also
poor inhibitors.
Inhibition of Mca and MshB by substituted naphthoquinones: percentage inhibition
was determined at substrate and inhibitor concentrations of 250 M each for Mca and
500 M each for MshB
l
l
HO
HO
HO
O
HN
S
O
HO
n
O
3.3. Evaluation of cyclohexyl-2-deoxy-2-C-alkylglucosides as
inhibitors of MshB and Mca
O
1a n = 2
1b
n = 3
1c n = 4
Amongst the cyclohexyl-2-deoxy-2-C-alkylglucosides listed in
Table 3 compound 29 stood out as the only inhibitor with reason-
able potency, and then only against Mca. This group of inhibitors
included the transition state analog 35 and 33 in which an isosteric
2-oxo-propyl group substituted for the acetamide group present in
the substrate. While it may be inferred from the results presented
in Figure 1 and Table 1 that the cyclohexyl moiety present in the
compounds listed in Table 3 is not the preferred aglycon for inter-
action with MshB, the results presented in Table 3 do suggest that
transition state analogs bearing the hydroxyethylene isostere, as in
35, or a noncleavable mimic of the amide bond, as in 33, hold little
promise for further development into effective inhibitors of Mca or
MshB.
1d
n = 5
Substrate
Mca
MshB
1a
1b
1c
1d
28.8
37.8
23.2
44.5
57.4
81.6
81.4
94.8
substrate of trypanothione reductase than plumbagin alone.20 It
should be noted that the presence and nature of a substituent on
the cysteine amino group present in mycothiol was found to be
an important determinant of the utilization of substrates not only
by Mca, but also by mtr. Thus desacetylmycothiol or N-succinylat-
ed mycothiol (succ-Cys-GlcN-Ins) were surprisingly poor sub-
strates of the enzyme and the current findings are, therefore,
consistent with previous studies on the substrate specificity of
mtr.32 It should, therefore, be possible to overcome this difficulty
by appropriate modification of the spacers used in 1a–d.
3.4. Thiophenylglycosides linked to plumbagin are potent
inhibitors of MshB
The naphthoquinone glycosides were amongst the most potent
inhibitors of MshB yet described and also inhibited Mca, but to a
lesser extent (Table 4 and Fig. 2). The less potent inhibition of
Mca by 1a–d is consistent with earlier reports14,25 that the binding
of substrates to Mca is influenced to a major extent by the presence
of the acetyl group in the N-acetylcysteine substructure of mycoth-
iol. When tested as subversive substrates compounds 1a–d were
also much poorer substrates of mtr than plumbagin itself. By con-
M. tuberculosis mtr reduced the heterodisulfide, 2-S-mycothiol-
yl-6-hydroxynaphthyldisulfide (MSSNaph), with a Vmax that, under
the conditions of the assay, was similar to that observed for the
natural substrate, mycothioldisulfide, but with a Km,app value of
23 lM, which is 3–5-fold lower than values previously reported
for the reduction of mycothioldisulfide,33 thus indicating the pres-
ence of an apolar binding site distal from the site that accommo-
dates the more hydrophilic mycothiol. As a result MSSNaph is a
particularly useful substrate for the assay of mtr, because it allows
a more economical use mycothiol, which is available only in
limited amounts when isolated from natural sources. 6,60-
dihydroxynaphthyldisulfide was, however, not reduced by mtr.
MSSNaph was also an efficient substrate of Mca with a Km value
trast plumbagin tethered to spermidine was
a much better
Table 3
Inhibition of Mca and MshB by cyclohexyl-2-deoxy-2-C-alkylglucosides: percentage
inhibition was determined at substrate and inhibitor concentrations of 250 M each
l
for Mca and 500
l
M each for MshB
HO
of 328 22 lM and a Vmax that exceeded the rate of cleavage of
MSmB by a factor of about 3.34 Seen together it may be concluded
that the substrate binding sites of MshB, Mca and mtr all interact
favorably with molecules bearing aromatic groups distal from the
more polar GlcN-Ins pseudodisaccharide.
O
HO
HO
R
O
Compound
R
Mca
MshB
More detailed studies of the inhibition of MshB by the series of
naphthoquinone derivatives were undertaken to determine inhibi-
tion constants, and the results obtained in the case of 1d are shown
in Figure 3. Inhibition by the series of naphthoquinones seems to
have been competitive. Inhibition constants for 1a, 1c and 1d were
29
30
31
73.8
9.3
0
0
0
22.0
estimated to be 167 15
respectively. These values may be compared to the Km values of
348 27 M for the natural substrate, GlcNAc-Ins (12), and, more
appropriately, with an estimated value of 1.695 0.151 mM for
the deacetylation of phenyl-2-acetamido-2-deoxy-1-thio-
glucopyranoside 9, which has the same aglycon that is present in
the naphthoquinone derivatives.
The presence of the aromatic naphthoquinone, therefore, has a
pronounced effect on the affinity with which these compounds are
bound to the active site of MshB. The Ki values of the naphthoqui-
lM, 94 11 lM and 16.8 1.9 lM,
35
33
20.5
17.2
11.4
6.7
l
OH
a-D-
O
O
37
38
19.4
35.0
19.7
6.6
See structure (Scheme 3)

D. W. Gammon et al. / Bioorg. Med. Chem. 18 (2010) 2501–2514
2507
100
90
80
70
60
50
40
30
20
10
0
30 29 31 35 33 37 38
13 16 20 21 22 23 15 24 25 26
1a 1b 1c 1d
100
90
80
70
60
50
40
30
20
10
0
30 29 31 35 33 37 38
13 16 20 21 22 23 15 24 25 26
1a 1b 1c 1d
Figure 2. Comparison of inhibition of MshB (Panel A) and Mca (Panel B) by cyclohexyl-2-deoxy-2-C-alkylglycosides, various thioglycosides and compounds having
plumbagin tethered via 2 to 5 methylene carbons by amide linkage to phenyl-2-deoxy-2-amino-1-thio- -glucopyranoside. In the case of MshB the substrate was 500
M MSmB was the substrate for Mca and inhibitors were included at 250 M. The product GlcN-Ins was
a-
D
lM
GlcNAc-Ins and inhibitors were present at 500
detected by hplc of the AccQ-fluor derivative.
lM, whereas 250
l
l
nones indicated that their binding affinity increases with the
length of the spacer. To understand this phenomenon, the naph-
thoquinones with 2 carbons and 5 carbon spacers, 1a and 1d, were
docked to the crystal structure of MshB previously published by
Maynes et al.15 Preliminary results showed that the longer carbon
chain of 1d extends the molecule such as to allow interaction of the
naphthoquinone group with a hydrophobic dipeptide of Val 184
and Leu 185, when the carbonyl group of the potentially cleavable
amide link is positioned such as to interact with the active site Zn
atom. These studies are still continuing. A similar pattern of inhibi-
tion was observed for Mca in that naphthoquinones with longer
spacers were also better inhibitors of Mca.
contained 50 mM NaCl and 1.0 mM each of the protease inhibitors,
TPCK, TLCK and PMSF in 50 mM Hepes, pH 7.5 and disrupted by
sonication. The mixture was clarified by centrifugation at 15,000g
for 30 min and the supernatant was dialyzed overnight against
25 mM Tris–Cl, pH 8.0. The preparation was applied to a DEAE–Se-
pharose column (45 ꢂ 4.5 cm) which was then eluted with eluted
with 350 ml of 25 mM NaCl in 25 mM Tris–Cl, pH 8.0 and then with
a 1 l gradient from 25 mM to 600 mM NaCl in 25 mM Tris–Cl, pH
8.0. Active fractions were pooled, concentrated by ultrafiltration
and dialyzed overnight against 1 l of 50 mM phosphate buffer,
pH 7.0 containing 0.5 M NaCl. The preparation was applied to a
Zn-IMAC column (1.6 ꢂ 12.5 cm) which was then eluted with
75 ml of 1 mM imidazole in 50 mM K-phosphate buffer, pH 7.0
containing 0.5 M NaCl and then with a gradient to 20 mM imidaz-
ole in the same buffer. Active fractions were concentrated by ultra-
filtration and chromatographed on Sephacryl-S300 (95 ꢂ 0.9 cm)
using 50 mM K-phosphate pH 7.0 as eluent. The resultant prepara-
tion was electrophoretically homogeneous and was stored in 20%
glycerol at ꢁ20 °C.
4. Experimental
4.1. Isolation and assays of enzymes
4.1.1. Expression and isolation of MshB
The gene encoding MshB, Rv1170, was amplified from M. tuber-
culosis DNA using pfu DNA polymerase and ligated into pET28
using the Nde1 and BamH1 restriction sites. The forward primer
was 50-gcccatatggtgtctgagacgccgcg-30 and the reverse primer
50-gttgggatcctacgtgccggacgcg-30. The resultant construct was
transformed into Escherichia coli BL21(DE3) pLysS and MshB
expression was induced by addition of 0.4 mM IPTG at 18 °C. The
induced cells (3.5 g) were suspended in 20 ml of lysis buffer, which
4.1.2. Isolation of Mca
Recombinant mca had previously been purified from the E. coli
host cells by a three step procedure and the last chromatographic
step on Phenyl-Sepharose, in agreement with our own results,
entailed considerable loss of activity.34 A theoretical basis for
partial denaturation of proteins during hydrophobic interaction

2508
D. W. Gammon et al. / Bioorg. Med. Chem. 18 (2010) 2501–2514
50
40
30
20
10
0
4.1.4. Enzyme assays
4.1.4.1. Mycothiol-S-conjugate amidase (Mca). Mca activity was
2500
2000
1500
1000
500
determined by hplc analysis of the bimane derivative of N-acetyl-
cysteine derived from the cleavage of mycothiol–bimane, MSmB.
Assay mixtures contained 8.0 nmol MSmB, where required,
8.0 nmol of an inhibitor and 1.0
pH 7.5, and NaCl and 7.7 g Mca in a final volume of 40
reaction mixtures were incubated for 10 min at 37 °C. The reaction
was stopped by adding 20 l acetonitrile and 20 l 5.0% TFA fol-
lowed by heating at 60 °C for 10 min. Samples were stored at
ꢁ80 °C until analyzed. Aliquots of the assay mixtures were injected
onto a Phenomenex C-18 hplc column (250 ꢂ 4.6 mm). The column
was eluted (Method 1) at a rate of 0.8 ml/min for 5 min with 5% B,
then with a linear gradient to 90%B with a 5 min plateau at 90%B, fol-
lowedby a return to initialconditions(A: 0.1%trifluoro acetic acid, B:
100% acetonitrile). The excitation and emission wavelengths on the
fluorescence detector were 396 nm and 482 nm, respectively and
the results were analyzed using Chromperfect software.
l
mol each of Na–Hepes buffer,
l
l
l. The
0
0
20
40
60
80 100 120
l
l
Vu5 (μΜ)
4.1.4.2. GlcNAc-Ins deacetylase, MshB. Assay mixtures con-
tained, in a final volume of 40
pH 7.5, 1.0 mol of NaCl, 3.26
a concentration range of 50–2000
bated for 10 min at 37 °C. The reaction was terminated by the addi-
tion of 20 l acetonitrile and 20 l 0.1% TFA followed by heating for
l
l, 1.0
g of MshB and GlcNAc-Ins within
M The mixtures were incu-
lmol of Na–Hepes buffer
l
l
0.000
0.005
0.010
GlcNAc-Ins-1
0.015
0.020
0.025
l
l
l
Figure 3. Inhibition of MshB by 1d. The substrate GlcNAc-Ins was varied in the
range 50–2000 M and 1d concentrations as follows: no inhibitor (d), 10 M (j),
25 M (s), 50 M (N) and 100 M (.). Assays were as described in Section 4 and
the product GlcN-Ins was determined by hplc as the AccQ-fluor derivative.
10 min at 60 °C. Seventy microliters of the clarified supernatant
were derivatized with AccQ-Fluor. The samples were then ana-
lyzed by hplc for GlcN-Ins, as the AccQ-fluor derivative, using a
Phenomenex C-18 column (250 ꢂ 4.6 mm).
l
l
l
l
l
The column was eluted at a rate of 0.8 ml/min for 5 min with 5%
B, then with a 30 min linear gradient to 90%B with a 5 min plateau
at 90%B followed by a return to initial conditions (A: 0.1% TFA, B:
acetonitrile).
chromatography had earlier been provided by Jungbauer et al.35
Mca was therefore isolated from Mycobacterium smegmatis using
(NH4)2SO4 precipitation and chromatography on DEAE cellulose,
hydroxyapatite and a Biosep SEC2000 hplc column to obtain an
electrophoretically homogeneous preparation. 50 grams M.
smegmatis was suspended in 160 ml of 50 mM Hepes buffer, pH
7.5, which contained 3 mM 2-mercaptoethanol and 0.1 mM each
of the protease inhibitors, TPCK, TLCK and PMSF. The cells were
disrupted by sonication for 10 min on ice and the mixture was clar-
ified by centrifugation. The supernatant was fractionated with
(NH4)2SO4 and the fraction precipitated between 15% and 50%
was collected and dialyzed against 25 mM Tris–Cl buffer, pH 8.0.
The dialyzed preparation was applied to a DEAE–Sepharose col-
umn that had been equilibrated against the same buffer and was
eluted using a linear gradient to 600 mM NaCl. Active fractions
from DEAE–cellulose chromatography were pooled and concen-
trated by ultrafiltration using a membrane with 30 kDa exclusion
limit. The concentrated enzyme preparation was dialyzed over-
night against 10 mM potassium phosphate (KPi), pH 7.2 and was
then loaded onto a hydroxyapatite column (2.2 ꢂ 30 cm) that
had been equilibrated with 6 volumes of the starting buffer. The
column was eluted with a 1000 ml gradient from 10 mM to
400 mM KPi, pH 7.2. Further purification of aliquots of this prepa-
ration by chromatography on Biosep Sec-S2000 was used to obtain
the electrophoretically homogeneous enzyme.
In order to construct calibration curves that would allow quanti-
tation of the amount of product formed, amidase cleavage of known
amounts of alternate substrates by MshB were allowed to go com-
pletion. Compounds having a phenyl group as aglycon could be de-
tected in the ultraviolet region and thus quantified by hplc without
further derivatization, while compounds having a cyclohexyl agly-
con were dansylated prior to analysis by hplc. The assay mixtures
contained 1
and 8.15 g of MshB in a total volume of 40
incubated for 10 min at 37 °C. The reactions were then terminated
by addition of 40 l of a 1:1 mixture of a TEA–acetic acid buffer
lmol NaCl, 1
l
mol Hepes buffer, pH 7.5, the substrate
l
ll. The mixtures were
l
(0.088%:0.57%) and acetonitrile and the samples were injected onto
a Phenomenex luna C-18 hplc column (250 ꢂ 4.6 mm). The column
was eluted using Method 1 described above, but substituting TEA–
acetic acid (0.088%:0.57%) for 0.1% TFA.
4.1.4.3. Mycothioldisulfide reductase (Mtr). Assay mixtures
contained 0.150
hydroxynaphthyl disulfide (MSSNaph), 50
(25 °C) and 2 mol EDTA in 1 ml. Reaction rates were determined
lmol of NADPH, 44 nmol of 2-S-mycothiolyl-6-
lmol Tris–Cl, pH 7.6
l
at 30 °C. Assays were started by addition of the enzyme and the de-
crease in absorbance at 340 nm was followed by means of an
Ocean Optics Mini-diode array spectrometer. Absorbance traces
were imported into Sigma plot and the rates determined by linear
regression.
4.1.3. Mycothiol disulfide reductase
The gene encoding M. tuberculosis mtr was expressed in M.
smegmatis as described,33 with minor modifications: The forward
primer was 50-CCC CTA CAA GTT TAA ACG TAC GAC-30. To lower
the level of constitutive expression of Rv2855 the transformed cells
were cultured at 30 °C and expression was induced by raising the
temperature to 42 °C. Under these conditions a ꢃ160-fold increase
in the level of mtr specific activity was achieved as compared to
M. smegmatis lacking the pMV261 plasmid with Rv2855 insert.
5. Synthesis of substrate analogs and inhibitors
5.1. General
Cyclohexyl-1-thio-glycosides 22, 23, 24, 25, 26 and 15 were
kindly provided12 by Professor Spencer Knapp from the Department

D. W. Gammon et al. / Bioorg. Med. Chem. 18 (2010) 2501–2514
2509
of Chemistry & Chemical Biology, Rutgers University, Piscataway,
New Jersey, USA. Thioglycosides 13 and 16 were provided Dr M. An-
war Jardine from the Department of Chemistry, University of Cape
Town, Rondebosch, South Africa. All other compounds were synthe-
sized as described below.
74.72, 69.00, 62.30, 55.15, 20.68. [M+H] calcd for C18H23NO7S:
398.1268; found 398.1283.
5.4. Phenyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-a-D-
glucopyranoside (10)
All reactions solvents were dried and distilled before use. Com-
mercially available reagents were used without purification unless
otherwise stated. All reactions were performed under an inert
atmosphere of nitrogen in flame dried glassware. Reactions were
monitored by thin-layer chromatography (TLC) carried out on
Merck F254 silica plates and products visualized under UV light
and by wetting the plate with a solution of anisaldehyde and sul-
furic acid in ethanol, followed by heating. Column chromatography
was performed on glass columns loaded with Merck Silica Gel 60
(70–230 mesh) eluting with mixtures of light petroleum and ethyl
acetate. Nuclear magnetic resonance spectra (1H and 13C) were re-
corded on either a Varian Mercury (300 MHz) or Varian Unity
(400 MHz) with CDCl3 as the solvent and TMS (d = 0 ppm) as the
internal standard. All chemical shifts are reported in ppm. Melting
points were determined using a Reichert-Jung Thermovar hot plate
microscope. The authenticity and purity of compounds that had
not previously been reported in the literature were verified by ele-
mental analysis.
A solution of 4 (37 mg, 0.093 mmol) in pyridine (2 ml) was trea-
ted at room temperature with acetic anhydride (0.9 ml, 9.52 mmol)
for 2 h. The reaction mixture was then diluted with CH2Cl2 (10 ml),
then the organic phase washed with 1 M HCl (2 ꢂ 10 ml), aqueous
NaHCO3 (2 ꢂ 10 ml) and brine (10 ml), before drying over MgSO4.
Compound 10 was obtained as a white solid (36 mg, 88%), mp
124–126 °C (lit.37 mp 204–205 °C); [
a]D = +160.1 (c 1.25, CHCl3);
lit.37
[a]
D = ꢁ22.1 (c 1.26, CHCl3); 1H NMR (CDCl3): d 7.44–7.42
(m, 5H, phenyl), 5.88 (d, 1H, J = 8.79, N–H), 5.69 (d, 1H, J = 5.13,
H-1), 5.15 (m, 2H, H-3, H-4), 4.59 (m, 1H, H-2), 4.50 (ddd, 1H, J =
5.1, 7.3, 12.5, H-5), 4.27 (dd, 1H, J = 2.56, 12.45, H-6a), 4.08 (dd,
1H, J = 2.56, 12.45, H-6b), 2.05, 2.04, 1.98 (s, 12H, 4ꢂ–COCH3).
[M+H] calcd for C20H25NO8S: 440.1374; found: 440.1393.
5.5. Phenyl-2-acetamido-2-deoxy-1-thio-a-D-glucopyranoside
(11)
A suspension of 10 (30 mg, 0.07 mmol 0.65 g, 1.48 mmol) in
methanol (2 ml) was treatment with a catalytic amount of sodium
methoxide in methanol at room temperature. The reaction was
complete after 2 h, whereupon the solution was treated with
methanol-washed Amberlite H+ resin, then filtered and the filtrate
concentrated under vacuum to give 11 as a white solid (22 mg,
5.2. Phenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-a-D-
glucopyranoside (3)à
BF3ꢀOEt2 (2.6 ml, 18.1 mmol,) was added to a solution of 228
(1.5 g, 4.0 mmol) and thiophenol (0.862 ml, 8.0 mmol) in CH2Cl2
(30 ml) at 0 °C. The reaction mixture was stirred at 55 °C for
12 h. On completion, the reaction mixture was diluted with CH2Cl2,
washed with brine and the organic layer separated. The aqueous
layer was extracted with CH2Cl2. The combined organic layers were
dried over MgSO4 and concentrated under reduced pressure. Silica
gel chromatography (EtOAc–petroleum ether 10:90) of the crude
98%); mp 220–222 °C (lit.37 mp 230 °C); [
a]D = +108.6 (c 0.5,
MeOH) (lit.37 D = +5.7 (c 0.51, MeOH)); 1H NMR (400 MHz,
[a]
CD3OD) d 7.52–7.22 (m, 5H, phenyl), 5.68 (d, 1H, J = 5.25 Hz, H-
1), 4.15–3.99 (m, 2H), 3.77 (dq, 2H, J = 11.94, 11.89, 11.89,
3.66 Hz,), 3.64 (dd, 1H, J = 11.02, 8.71 Hz), 3.43 (dd, 1H, J = 9.84,
8.70 Hz), 3.36–3.18 (m, 1H), 3.36–3.07 (m, 1H), 2.00 (s, 3H, 1ꢂ–
COCH3); 13C NMR (CD3OD): d 172.53, 134.84, 131.94, 131.00,
128.64, 127.24, 88.26, 73.85, 71.52, 71.26, 61.25, 55.06, 21.34.
Anal. Calcd for C14H19NO5S: C, 53.66; H, 6.11; N, 4.47; S, 10.23.
Found: C, 52.72; H, 5.98; N, 4.28; S, 9.90. [M+H] calcd for
C14H19NO5S: 314.1062; found: 314.1085.
mixture afforded 3 as an a/b mixture (1.03 g, 61%). The a-anomer
was obtained as white crystals by crystallization from absolute
ethanol: mp 93–97 °C; 1H NMR (400 MHz) d 7.51–7.47 (m, 2H,
Ar-H), 7.34–7.29 (m, 3H, Ar-H), 5.61 (d, 1H, H-1, J = 5.6 Hz), 5.31
(dd, 1H, H-3, J = 9.2 Hz, J = 10.5 Hz), 5.01 (dd, 1H, H-4, J = 9.2 Hz,
J = 10.3 Hz), 4.56 (ddd, 1H, H-5, J = 2.3 Hz, J = 5.1 Hz, J = 10.3 Hz),
4.26 (dd, 1H, H-6a, J = 5.1 Hz, J = 12.4 Hz), 4.04 (dd, 1H, H-6b,
J = 6.1 Hz, J = 11.1 Hz), 2.07 (s, 3H, CH3), 2.02 (s, 3H, CH3), 1.99 (s,
3H, CH3). 13C NMR (CDCl3) d 170.4, 169.7, 132.4, 132.2, 129.2,
128.0, 86.5 (C-1), 76.6, 72.0, 68.7, 68.5, 61. 9, 61.5, 20.6, 20.5. Anal.
Calcd for C18H21N3O7S: C, 51.06; H, 5.19; N, 9.92; S, 7.57. Found: C,
51.64; H, 5.11; N, 9.94; S, 7.23.
5.6. Preparation of acid derivatives 7a–d
The naphthalenyl carboxylic acids 7a–d were prepared by com-
bining plumbagin 5 and dicarboxylic acids 6 following the proce-
dure of Salmon-Chemin et al., with analytical data corresponding
to that reported.20
5.6.1. 2-Carboxymethyl-8-hydroxy-3-methyl-1,4-
naphthoquinone (7a)
5.3. Phenyl-3,4,6-tri-O-acetyl-2-amino-2-deoxy-1-thio-a-D-
Obtained as an orange solid (32%); 1H NMR (acetone-d6) d
(300 MHz) 12.09 (OH, 1H), 7.70 (dd, 1H, H-6, J = 7.5 Hz,
J = 8.4 Hz), 7.57 (dd, 1H, H-7, J = 1.1 Hz, J = 7.5 Hz), 7.26 (dd, 1H,
H-5, J = 1.2 Hz, J = 8.4 Hz), 2.96 (t, J = 8.2, 2H, CH2–COOH), 2.58 (t,
2H, CH2–CH2–COOH, J = 7.9 Hz), 2.22 (s, 3H, CH3). 13C NMR (ace-
tone-d6) d 190.4, 184, 173, 162, 145.7, 145, 136.4, 133, 123.5,
118.6, 114.9, 31.9, 22.1, 12.
glucopyranoside (4)
A suspension of 3 (2.0 mmol, 0.844 g) and palladium on carbon
(0.424 g) in ethanol (48 ml) was stirred under hydrogen (1 atm) for
5 h at room temperature. The reaction mixture was then filtered
through Celite and further purified by silica column chromatogra-
phy (EtOAc–petroleum ether 60:40) to yield 4 as a white solid
(0.739 g, 93%), mp 83–85 °C. 1H NMR (CDCl3): d 7.42–7.23 (m,
5H, phenyl), 5.56 (d, 1H, J = 5.2 Hz, H-1), 5.05 (dd, 2H, J = 2.2,
10.3 Hz, H-4, H-6a), 4.6 (m, 1H, H-5), 4.32 (dd, 1H, J = 5.1,
12.3 Hz, H-2), 4.07 (dd, 1H, J = 2.3, 12.3 Hz, H-3), 3.32 (dd, 1H,
J = 5.2, 10.2 Hz, H-6b), 2.1 (s, 3H), 2.04 (s, 6H). 13C NMR (CDCl3):
d 170.70, 170.57, 169.78, 133.50, 131.88, 129.13, 127.74, 91.23,
5.6.2. 2-Carboxyethyl-8-hydroxy-3-methyl-1,4-
naphthoquinone (7b)
Obtained as an orange solid (40%). 1H NMR (acetone-d6) d
(400 MHz) ppm 12.09 (OH, 1H), 7.69 (dd, 1H, H-6, J = 7.5 Hz,
J = 8.4 Hz), 7.57 (dd, 1H, H-7, J = 1.1 Hz, J = 7.5 Hz),7.25 (dd, 1H,
H-5, J = 1.1 Hz, J = 8.4 Hz), 2.74 (t, J = 7.9, 2H, CH2–COOH), 2.44 (t,
J = 7.2, 2H, CH2–(CH2)2–COOH), 2.21 (s, 3H, CH3), 1.84 (m, 2H,
CH2–CH2–COOH). 13C NMR 190.4, 184, 173.4, 161, 146, 145, 136,
132, 123, 118, 115, 32.9, 25.5, 23.3, 12.
à
We note that this compound has been reported as a 2.1:1
/b mixture,36b but to our knowledge this is the first reported isolation and
characterization of the pure -anomer.
a
/b mixture36a and as a
3:1
a
a

2510
D. W. Gammon et al. / Bioorg. Med. Chem. 18 (2010) 2501–2514
5.6.3. 2-Carboxypropyl-8-hydroxy-3-methyl-1,4-naphthoqui-
none (7c)
123.9, 118.9, 114.9, 87.5 (C-1), 71.2, 68.9, 68.2, 62, 52.7, 35.9,
25.6, 24.1, 20.7, 20.6, 20.5, 12.7. Anal. Calcd for C33H35NO11S: C,
60.63; H, 5.4; N, 2.14; S, 4.91. Found: C, 59.78; H, 5.75; N, 1.71;
S, 4.0. [MꢁH]+ calcd for C33H35NO11S: 652.1852; found 652.1981.
Obtained as an orange solid (40%). 1H NMR (acetone-d6) d
(400 MHz) ppm 12.1 (OH, 1H), 7.69 (dd, 1H, H-6, J = 7.5 Hz,
J = 8.4 Hz), 7.56 (dd, 1H, H-7, J = 1.1 Hz, J = 7.5 Hz), 7.25 (dd, 1H,
H-5, J = 1.1 Hz, J = 8.4 Hz), 2.69 (t, J = 7.8, 2H, CH2–(CH2)3–COOH),
2.36 (t, J = 7.3, 2H, CH2–COOH), 2.2 (s, 3H, CH3), 1.73 (td, 2H,
CH2–CH2–COOH, J = 7.1 Hz, J = 14.5 Hz), 1.60 (m, 2H, CH2–(CH2)2–
COOH). 13C NMR 190.4, 184, 174, 162, 146.5, 144.7, 136, 133,
124, 119, 115, 33, 25.9, 25, 11.9.
5.7.3. Phenyl-3,4,6-tri-O-acetyl-2-deoxy-2-[30-(800-hydroxy-300-
methyl-100,400-dioxo-100,400-dihydronaphthalen-200-yl)pentanami-
do]-1-thio-a-D-glucopyranoside (8c)
Obtained as a red solid (64%, mp 127–130 °C). 1H NMR (CDCl3) d
(300 MHz) 12.0 (s, 1H, OH), 7.6–7.56 (m, 2H, Ar-H), 7.44–7.41 (m,
2H, Ar-H), 7.30–7.20 (m, 5H, Ar-H), 5.9 (d, 1H, NH, J = 8.5 Hz), 5.76
(d, 1H, H-1, J = 5.4 Hz), 5.17–5.13 (m, 2H, H-3&4), 4.65–4.48 (m,
2H, H-2&5), 4.28 (dd, 1H, H-6a, J = 4.9 Hz, J = 12.4 Hz), 4.09 (dd,
1H, H-6b, J = 2.3 Hz, J = 12.3 Hz), 2.63 (dt, 1H, J = 1.5 Hz, J = 7.1 Hz,
CH2–CONH), 2.25 (dt, 1H, J = 2.3 Hz, J = 7.2 Hz, –CH2–(CH2)3–
CONH), 2.15 (3H, CH3), 2.05, 2.04, 2.03 (s, 9H, 3ꢂ–COCH3), 1.7–
1.6 (m, 2H, CH2–CH2–CONH), 1.5–1.4 (m, 2H, CH2–(CH2)2–CONH).
13C NMR 190, 184, 172, 171.6, 170.5, 169.2, 161.2, 146.4, 144.8,
136.8, 135.9, 132.8, 132.1, 131.4, 129.2, 127.8, 123.8, 118.8,
114.9, 87.6 (C-1), 71.3, 68.9, 68.1, 62, 52.7, 36.1, 27.9, 25.9, 25.5,
20.7, 20.66, 20.6, 12.7. Anal. Calcd for C34H37NO11S: C, 61.16; H,
5.59; N, 2.1; S, 4.8. Found: C, 60.27; H, 5.67; N, 1.63; S, 3.91.
[MꢁH]+ calcd for C34H37NO11S: 666.2008; found 665.8366.
5.6.4. 2-Carboxybutyl-8-hydroxy-3-methyl-1,4-naphthoqui-
none (7d)
Obtained as a red solid (50%). 1H NMR (acetone-d6) d (400 MHz)
ppm 12.1 (OH, 1H), 7.69 (dd, 1H, H-6, J = 7.5 Hz, J = 8.4 Hz), 7.56
(dd, 1H, H-7, J = 1.1 Hz, J = 7.5 Hz), 7.25 (dd, 1H, H-5, J = 1.1 Hz,
J = 8.4 Hz), 2.67 (t, J = 7.9, 2H, CH2–COOH), 2.31 (t, J = 7.3, 2H,
CH2–(CH2)4–COOH), 2.2 (s, 3H, CH3), 1.67 (td, 2H, CH2–CH2–COOH,
J = 7.2 Hz, J = 14.6 Hz), 1.54 (m, 4H, (CH2)2–(CH2)2–COOH). 13C
NMR 190.2, 184.4, 173.8, 161, 146.6, 145, 136.3, 132.3, 12.43,
118.4, 114.9, 33.3, 25.9, 24.3, 11.9.
5.7. General procedure for coupling naphthoquinonyl carbox-
ylic acids 7 with phenylthioglycoside 4
5.7.4. Phenyl-3,4,6-tri-O-acetyl-2-deoxy-2-[30-(800-hydroxy-300-
Portions of acid 7 (1 equiv), phenylthioglycoside 4 (1.1 equiv),
EDC (1.2 equiv), and HOBt (1.2 equiv) were combined in a mixture
of CH2Cl2 and THF (1:2, 75 ml/g of 7) under nitrogen. After stirring
for 18 h at room temperature, the solvents were evaporated under
reduced pressure and the residue re-dissolved in CH2Cl2, washed
with 5% NaOH), 0.5 M HCl (2.5 ml), and then dried under reduced
pressure. The acetylated products 8a–d were each purified by pre-
parative TLC (MeOH–CH2Cl2 2.5:97.5).
methyl-100,400-dioxo-100,400-dihydronaphthalen-200-yl)hexanami-
do]-1-thio-a-D-glucopyranoside (8d)
Obtained as a red solid (78%, mp 55–58 °C). 1H NMR (CDCl3) d
(300 MHz) 12.0 (s, 1H, OH), 7.6–7.56 (m, 2H, Ar-H), 7.44–7.41
(m, 2H, Ar-H), 7.30–7.20 (m, 5H, Ar-H), 5.9 (d, 1H, NH, J = 8.5 Hz),
5.76 (d, 1H, H-1, J = 5.4 Hz), 5.17–5.13 (m, 2H, H-3&4), 4.65–4.48
(m, 2H, H-2&5), 4.28 (dd, 1H, H-6a, J = 4.9 Hz, J = 12.4 Hz), 4.09
(dd, 1H, H-6b, J = 2.3 Hz, J = 12.3 Hz), 2.6–2.5 (m, 2H, CH2–CONH),
2.2–2.18 (m, 2H, CH2–(CH2)4–CONH), 2.2 (3H, CH3), 2.058, 2.056,
2.04 (s, 9H, 3ꢂ–COCH3), 1.6–1.5 (m, 2H, CH2–CH2–CONH), 1.5–
1.4 (m, 4H, (CH2)2–CH2–CONH). 13C NMR 190, 184, 172, 171.6,
170.5, 169.2, 161.2, 146.4, 144.8, 136.8, 135.9, 132.8, 132.1,
131.4, 129.2, 127.8, 123.8, 118.8, 114.9, 87.6 (C-1), 71.3, 68.9,
68.1, 62, 52.7, 36.1, 27.9, 25.9, 25.5, 20.7, 20.66, 20.6, 12.7. Anal.
Calcd for C35H39NO11S: C, 61.66; H, 5.77; N, 2.05; S, 4.7. Found:
C, 63.9; H, 6.14; N, 1.6; S, 3.47. [MꢁH]+ calcd for C35H39NO11S:
680.2165; found 679.5982.
5.7.1. Phenyl-3,4,6-tri-O-acetyl-2-deoxy-2-[30-(800-hydroxy-300-
methyl-100,400-dioxo-100,400-dihydronaphthalen-200-yl)
propanamido]-1-thio-a-D-glucopyranoside (8a)
Obtained as an orange solid (57%, mp 163–165 °C). 1H NMR
(CDCl3) d (300 MHz) 12.0 (s, 1H, OH), 7.59–7.52 (m, 2H, Ar-H),
7.42–7.39 (m, 2H, Ar-H), 7.31–7.26 (m, 5H, Ar-H), 6.00 (d, 1H,
J = 8.5 Hz, NH), 5.72 (d, 1H, J = 5.2 Hz, H-1,), 5.18–5.14 (m, 2H, H-
3 & H-4), 4.61–4.47 (m, 2H, H-2 & H-5), 4.27 (dd, 1H, J = 5.0 Hz,
J = 12.4 Hz H-6a), 4.07 (dd, 1H, H-6b, J = 2.3 Hz, J = 12.3 Hz), 2.93
(dd, J = 6.6 Hz, J = 8.5 Hz, 2H, CH2–CONH), 2.41 (dd, 2H, CH2–CH2–
CONH, J = 6.6 Hz, J = 8.5 Hz), 2.2 (3H, CH3), 2.05, 2.04, 2.03 (s, 9H,
3ꢂ–COCH3). 13C NMR 190, 184, 171.8, 171.4, 170.7, 169.4, 161.5,
145.9, 145.1, 136.2, 132.9, 132.3, 131.7, 129.4, 128.09, 124.1,
119.2, 115.0, 87.6 (C-1), 71.5, 69.2, 68.3, 62.2, 53.0, 34.9, 22.7,
20.8, 20.8, 13.0. Anal. Calcd for C32H33NO11S: C, 60.08; H 5.2; N,
2.19; S, 5.01. Found: C, 59.21; H, 5.21; N, 1.65; S, 4.27. [M+H]+ calcd
for C32H33NO11S: [M+H]+ cacld for C32H33NO11S: 640.1847; found:
640.1843.
5.8. General procedure for deacetylations
A portion of 0.2 M NaOMe in MeOH was added to a stirred sus-
pension of acetylated glycoside 8 in MeOH at room temperature.
When TLC showed complete conversion to a single product with
lower polarity (typically after 1 h) Dowex-50WX-200(H+) ion ex-
change resin was added to the reaction mixture until a neutral pH
was reached. The dowex resin was then removed by filtration and
the clear filtrate concentrated in vacuo to give deacetylated product.
5.7.2. Phenyl-3,4,6-tri-O-acetyl-2-deoxy-2-[30-(800-hydroxy-300-me-
5.8.1. Phenyl-2-deoxy-2-[30-(800-hydroxy-300-methyl-100,400-
thyl-100,400-dioxo-100,400-dihydronaphthalen-200-yl)butanamido]-
dioxo-100,400-dihydronaphthalen-200-yl)propanamido]-1-thio-
a-
1-thio-
a
-
D
-glucopyranoside (8b)
D
-glucopyranoside (1a)
Obtained as a red/brown solid (34%, 238–241 °C). 1H NMR
(DMSO-d6) (400 MHz) 12.0 (s, 1H, OH),8.09 (d, 1H, NH,
Obtained as a yellow solid (84%, mp 157–158 °C). 1H NMR
(CDCl3) d (300 MHz) 12.0 (s, 1H, OH), 7.6–7.56 (m, 2H, Ar-H),
7.44–7.41 (m, 2H, Ar-H), 7.30–7.20 (m, 5H, Ar-H), 6.08 (d, 1H,
NH, J = 8.5 Hz), 5.76 (d, 1H, H-1, J = 5.4 Hz), 5.21–5.16 (m, 2H, H-
3&4), 4.65–4.48 (m, 2H, H-2&5), 4.28 (dd, 1H, H-6a, J = 4.9 Hz,
J = 12.4 Hz), 4.09 (dd, 1H, H-6b, J = 2.3 Hz, J = 12.3 Hz), 2.73–2.56
(m, 2H, CH2–CONH), 2.29 (t, 1H, J = 7.1 Hz, CH2–(CH2)2–CONH),
2.15 (3H, CH3), 2.06, 2.049, 2.048 (s, 9H, 3ꢂ–COCH3), 1.80 (m, 2H,
CH2–CH2–CONH). 13C NMR d 189.0, 184.0, 172, 171.5, 170.5,
169.2, 161.2, 145.9, 145.4, 136, 132.8, 132.1, 131.4, 129.2, 127.8,
d
J = 6.5 Hz), 7.69 (t, 1H, H-7, J = 8.0 Hz), 7.5-7.4 (m, 1H, H-5,1H),
7.38–7.35 (2H, m, Ar-H), 7.31–7.24 (4H, m, Ar-H), 5.60 (d, 1H, H-1,
J = 5.1 Hz), 5.12 (d, 1H, OH, J = 5.6 Hz), 4.85 (d, 1H, OH, J = 5.6 Hz),
4.51 (t, 1H, OH, J = 6.0 Hz), 3.87–3.80 (2H, m, H-2&3), 3.58–3.54
(2H, m, H-5&6a), 3.46–3.40 (1H, m,H-6b), 3.3–3.2 (1H, m, H-4),
2.8–2.7 (m, 2H, CH2–CONH), 2.38–2.34 (m, 2H, CH2–CH2–CONH),
2.1 (3H, CH3). 13C NMR 183.8, 172, 171.9, 159.7, 145.1, 136.2,
134.2, 131.6, 130.9, 129.08, 129.03, 128.9, 127, 123.3, 118.4, 114.5,

D. W. Gammon et al. / Bioorg. Med. Chem. 18 (2010) 2501–2514
2511
87 (C-1), 73.8, 70.37, 70.30, 70, 60, 54.5, 54.4, 33.52, 33.48, 22.2, 12.4.
[M+H] calcd for C26H27NO8S: 514.1536; found 514.1544.
CHCl3); 1H NMR (400 MHz, CDCl3): dH 7.52–7.50 (2H, m, Ph-H),
7.32–7.30 (3H, m, Ph-H), 5.60 (1H, d, J 9.3 Hz, NH), 5.23 (1H, t,
J = 9.8 Hz, H-3), 5.06 (1H, t, J = 9.7 Hz, H-4), 4.87 (1H, d,
J = 10.3 Hz, H-1), 4.20 (1H, dq, J = 4.2 &12.2 Hz, H-6a &b), 4.04
(1H, m, H-2), 3.73 (1H, ddd, J = 2.5, 5.2 & 10.0 Hz, H-5), 2.08 (3H,
s, CH3), 2.03 (3H, s, CH3), 2.02 (3H, s, CH3), 1.99 (3H, s, CH3);
(100 MHz, CDCl3): dC 170.9, 170.5,169.9, 169.2, 132.4 (2ꢂC),
128.8 (2ꢂC),128 (2ꢂC), 86.6 (C-1), 75.7, 73.7, 68.4, 62.3, 53.3,
23.2, 20.68, 20.62, 20.5. Anal. Cacld for C20H25NO8S: C, 54.66; H,
5.73; N, 3.19; S, 7.30. Found: C, 54.30; H, 5.74; N, 2.71; S, 6.89.
[M+H] calcd for C20H25NO8S: 440.1379; found: 440.1396.
5.8.2. Phenyl-2-deoxy-2-[30-(800-hydroxy-300-methyl-100,400-
dioxo-100,400-dihydronaphthalen-200-yl)butanamido]-1-thio-
glucopyranoside (1b)
a-D-
Obtained as a yellow/brown solid (46% mp 196–199 °C). 1H NMR
(DMSO-d6) d (300 MHz) 12.0 (s, 1H, OH), 7.86 (d, 1H, NH,J = 6.6 Hz),
7.69 (dd, 1H, J = 7.6 Hz, J = 8.3 Hz), 7.51 (dd, 1H, J = 1.1 Hz, J = 7.5 Hz),
7.40–7.37 (m, 2H, Ar-H), 7.29–7.18 (m, 4H, Ar-H), 5.64 (d, 1H, H-1,
J = 5.1 Hz), 5.01 (d, 1H, OH, J = 5.6 Hz), 4.76 (d, 1H, OH, J = 5.6 Hz),
4.39 (t, 1H, OH, J = 5.6 Hz), 3.9–3.8 (m, 2H, H-2&3), 3.6–3.5 (m, 2H,
H-5&6a), 3.49–3.41 (m, 1H, H-6b), 3.29–3.23 (m, 1H, H-4), 2.6–2.5
(2H, m, CH2–CONH), 2.3–2.2 (2H, m, CH2–(CH2)2–CONH), 2.0 (3H,
s, CH3), 1.7–1.6 (2H, m, CH2–CH2–CONH). 13C NMR 189, 184, 172,
160, 146, 145, 136, 135, 132, 131, 129, 127, 123, 118, 88 (C-1), 74,
71, 70, 61, 55, 34, 28, 25, 24, 12. [MꢁH]+ calcd for C27H29NO8S
526.1536; found 526.1526.
5.10. Phenyl-2-deoxy-2-acetamido-1-thio-b-D-glucopyranoside
(20)
NaOMe (0.2 M) in MeOH (10 ml) was added to a suspension of
18 (0.4 g, 0.911 mmol) in methanol (20 ml) with stirring at room
temperature. TLC showed complete conversion to a single more
polar product within 10 min. The reaction mixture was then stirred
with resin (Dowex-50WX-200(H+), then the resin removed by fil-
tration, and the filtrate concentrated under reduced pressure. The
product 20 was obtained as white powder (260 mg, 91%); mp
231–234 °C (lit.29b 222 °C; lit.29a 196–199 °C); 1H NMR (400 MHz,
D2O): dH 8.36 (1H, s, NH), 7.44 (2H, dd, J = 1.7 Hz and 7.8 Hz, Ph-
H), 7.34–7.29 (3H, m, Ph-H), 4.80 (1H, d, J = 10.4 Hz, H-1), 3.82
(1H, d, J = 12.0 Hz, H-6a), 3.71–3.66 (1H, m, H-5 & 6b), 3.51 (1H,
t, J 9.3 Hz, H-4), 3.41 (2H, m, H-2 & 3), 1.95 (3H, s, CH3);
(100 MHz, CDCl3): dC 171 (C@O), 132, 130 (2ꢂC), 128 (2ꢂC), 127,
87 (C-1), 81, 76, 71, 61.5, 53.7, 21.5 (–CH3). [M+H] calcd for
C14H19NO5S: 314.1062; found: 314.1079.
5.8.3. Phenyl-2-deoxy-2-[30-(800-hydroxy-300-methyl-100,400-
dioxo-100,400-dihydronaphthalen-200-yl)pentanamido]-1-thio-
a-
D
-glucopyranoside (1c)
Obtained as a brown solid (60%, mp 170–173 °C). 1H NMR
(DMSO-d6) d (300 MHz) 12.0 (s, 1H, OH), 7.86 (d, 1H, NH,
J = 6.5 Hz), 7.69 (dd, 1H, J = 7.6 Hz, J = 8.3 Hz), 7.51 (d, 1H,
J = 7.4 Hz), 7.40–7.3 (m, 2H, Ar-H), 7.26–7.16 (m, 4H, Ar-H), 5.6
(d, 1H, H-1, J = 5.1 Hz), 5.1 (d, 1H, OH, J = 5.5 Hz), 4.76 (d, 1H, OH,
J = 5.5 Hz), 4.39 (t, 1H, OH, J = 5.8 Hz), 3.9–3.8 (m, 2H, H-2&3),
3.6–3.5 (m, 2H, H-5&6a), 3.5–3.4 (m, 1H, H-6b), 3.3–3.2 (m, 1H,
H-4), 2.6–2.5 (2H, m, CH2–CONH), 2.2–2.1 (2H, m, –CH2–(CH2)3–
CONH), 2.0 (3H,s, CH3), 1.63–1.57 (2H, m, CH2–CH2–CONH), 1.5–
1.4 (2H, m, CH2–(CH2)2–CONH). 13C NMR 189, 184, 173, 160, 146,
144, 136, 135, 132, 131, 129, 128, 127, 123, 118, 114, 87 (C-1),
74, 70.4, 70.1, 61, 55, 35, 27, 26, 25,12. [MꢁH]+ calcd for
C28H31NO8S: 540.1692; found 540.1685.
5.11. Benzyl-3,4,6-tri-O-acetyl-2-deoxy-2-acetamido-1-thio-b-
D-glucopyranoside (19)
BF3ꢀEt2O (0.16 ml, 1.34 mmol) was added in one portion to a
cold suspension of 17 (0.5 g, 0.895 mmol). An exothermic reaction
was immediately observed and the reaction mixture was allowed
to stir for 50 min when TLC showed complete consumption of 17.
Anhydrous CH3CN (20 ml) was then added followed by thiourea
(0.136 g, 1.79 mmol) and the reaction mixture placed on a pre-
heated oil bath at 80 °C for 15 min. with constant stirring. After full
consumption of the starting material as judged by TLC, the reaction
mixture was cooled to room temperature and BnBr (0.458 g,
0.318 ml, 1.34 mmol) and Et3N (0.89 ml, 6.44 mmol) were added
in succession and the reaction allowed to stir for 3 h at ambient
temperature. After standard workup, the crude product was puri-
fied by flash column chromatography (EtOAc as eluent) to give
19 as a white powder (0.31 g, 75%), mp: 178–179 °C (lit.30b 199–
202 °C). 1H NMR (400 MHz, CDCl3): dH 7.33–7.28 (5H, m, Ph-H);
5.32 (1H, d, J = 9.3 Hz, NH), 5.10 (1H, t, J = 9.6 Hz, CH–), 5.02 (1H,
t, J = 9.5 Hz, CH–), 4.28–4.15 (4H, m, H-1,2,3 & 4), 3.96 (1H, d,
J = 13.1 Hz, H-6a), 3.96 (1H, d, J13.1 Hz, H-6b), 3.57 (1H, ddd,
J = 2.3, 4.9 & 9.6 Hz, H-5); (100 MHz, CDCl3): dC 171 (C@O), 170.9
(C@O), 170 (C@O), 169.5 (C@O), 137, 129 (2ꢂC), 128.6 (2ꢂC),127,
83 (C-1), 76, 74.1, 68.2, 62.1, 53.2, 34.2 (Ph–CH2–S), 23.2, 20.68,
20.62, 20.5. Anal. Calcd for C21H27NO8S: C, 55.62; H, 6.00; N,
3.09; S, 7.07. Found: C, 55.74; H, 5.90; N, 2.36; S, 7.01. [M+H] calcd
for C21H27NO8S: 454.1536; found: 454.1556.
5.8.4. Phenyl-2-deoxy-2-[30-(800-hydroxy-300-methyl-100,400-
dioxo-100,400-dihydronaphthalen-200-yl)hexanamido]-1-thio-
glucopyranoside (1d)
a-D-
Obtained as a brown/orange solid (52%, mp 157–159 °C). 1H
NMR (DMSO-d6) d (300 MHz) 12.0 (s, 1H, OH), 7.86 (d, 1H, NH,
J = 6.5 Hz), 7.7 (t, 1H, J = 8.3 Hz), 7.51 (d, 1H, J = 7.5 Hz), 7.4 (td,
2H, J = 1.2 & 8.2, Ar-H), 7.3–7.2 (m, 4H, Ar-H), 5.6 (d, 1H, H-1,
J = 4.8 Hz), 5.1 (d, 1H, OH, J = 5.7 Hz), 4.8 (d, 1H, OH, J = 5.8 Hz),
4.5 (t, 1H, OH, J = 6.1 Hz), 3.9–3.8 (m, 2H, H-2&3), 3.6–3.5 (m, 2H,
H-5&6a), 3.5–3.4 (m, 1H, H-6b), 3.3–3.2 (m, 1H, H-4), 2.6–2.5 (2H,
m, CH2–CONH), 2.2–2.1 (2H, m, CH2–(CH2)4–CONH), 2.1 (3H, s,
CH3), 1.63–1.57 (2H, m, CH2–CH2–CONH), 1.5–1.4 (4H, m, (CH2)2–
CH2–CONH). 13C NMR 189.7, 184, 173.5, 160, 146.8, 144.5, 136.6,
135, 132, 131.5, 129, 127, 123.6, 118, 114, 87.9 (C-1), 74, 70.8,
70.6, 60.7, 55, 35, 29, 27.9, 26, 25,12.6. [MꢁH]+ calcd for
C29H33NO8S: 554.1849; found 554.1843.
5.9. Phenyl-3,4,6-tri-O-acetyl-2-deoxy-2-acetamido-1-thio-b-D-
glucopyranoside (18)
Thiophenol (0.208 ml, 2.14 mmol) and SnCl4 (1.25 ml,
1,25 mmol) were added to stirred solution of 17 (1.0 g,
a
1.79 mmol) in CH2Cl2 (20 ml). The reaction mixture was heated
at reflux for 24 h, cooled to room temperature and quenched by
addition of saturated aqueous NaHCO3. The aqueous layer was sep-
arated and extracted with DCM. The combined organic layers were
washed with brine, dried over Na2SO4 and concentrated under re-
duced pressure, and the solid residue was recrystallized from
Et2O–hexane (10:1) to give 18 (0.59 g, 75%); mp 186–187 °C (lit.29b
5.12. Benzyl 2-deoxy-2-acetamido-1-thio-b-D-glucopyranoside
(21)
NaOMe (0.2 M) in MeOH (10 ml) was added to a suspension of
19 (0.4 g, 0.911 mmol) in methanol (20 ml) with stirring at room
temperature. TLC showed complete conversion to a single more
polar product within 10 min. The reaction mixture was then stirred
199 °C); [
a
]
D = ꢁ24.9 (c 1.0, CHCl3), lit.29b
[a]
D = ꢁ20.4 (c 1.0,

2512
D. W. Gammon et al. / Bioorg. Med. Chem. 18 (2010) 2501–2514
with resin (Dowex-50WX-200(H+), then the resin removed by fil-
tration, and the filtrate concentrated under reduced pressure to
give 21 as white powder (0.27 g, 89%), mp: 125–127 °C (lit.30b
225–227 °C) 1H NMR (400 MHz, D2O): dH 8.36 (1H, s, NH), 7.7–
7.6 (5H, m, Ph-H), 4.25 (1H, d, J = 10.2 Hz, H-1), 3.9 (1H, d,
J = 12.2 Hz, H-6a), 3.8 (2H, m, –CH2–), 3.65 (2H, m, H-5 & 6b), 3.5
(2H, m, H-3 & 4), 3.41 (2H, m, H-2 & 3), 3.3 (1H, m, H-2), 2.22
(3H, s, CH3); (100 MHz, CDCl3): dC 172.5 (C@O), 137, 128 (2ꢂC),
127.7 (2ꢂC), 126, 82 (C-1), 79, 74, 68, 59, 53.5, 23 (CH2–), 21.5
(–CH3). Anal. Calcd for C15H21NO5S : C, 55.03; H, 6.47; N, 4.28; S,
9.79. Found: C, 53.66; H, 6.11; N, 4.47; S, 10.23. [M+H] calcd for
C15H21NO5S: 328.1219; found: 328.1214.
the solvent evaporated and the crystalline residue chromato-
graphed on silica using methanol–ethyl acetate (5:95) as eluent
to afford 29 as colorless crystals (196 mg, 99%), mp 165–168 °C
(from ethyl acetate and petroleum ether); [a]D = +120.8 (c 2.3,
MeOH); dH (400 MHz; CD3OD) 5.86–5.76 (1H, m, H-200), 5.07–4.98
(2H, m, H-300a, H-300b), 4.87 (1H, d, J = 3.2 Hz, H-1), 3.77 (1H, m, H-
6a), 3.69–3.63 (2H, m, H-5, H-6b), 3.58 (1H, m, H-10), 3.49 (1H,
dd, J = 10.6, 9.1 Hz, H-3), 3.24 (1H, t, J = 9.1 Hz, H-4), 2.51 (1H, m,
H-100a), 2.14–2.05 (1H, m, H-100b), 1.90–1.21 (11H, m, H-2, 5 ꢂ
–CH2–, cyclohexyl); dC (100 MHz; CD3OD) 136.84 (C-200), 115.38
(C-300), 96.65 (C-1), 74.62 (C-10), 72.82 (C-3), 72.73 (C-5), 72.15
(C-4), 61.87 (C-6), 46.59 (C-2), 33.39 (C–CH2–, cyclohexyl), 31.56
(C-100), 31.45, 25.69, 23.88, 23.71 (4 ꢂ –CH2–, cyclohexyl);
HRFABMS: m/z 309.168001 (M+Na)+. Calcd for C15H26O5Na
309.167784 (M+Na)+.
5.13. Cyclohexyl 3,4,6-tri-O-acetyl-2-C-allyl-2-deoxy-
glucopyranoside (28a) and cyclohexyl 3,4,6-tri-O-acetyl-2-C-
allyl-2-deoxy-b- -glucopyranoside (28b)
a-D-
D
5.15. Cyclohexyl 2-C-allyl-2-deoxy-b-D-glucopyranoside (30)
A mixture of glucosyl fluoride 2731 (550 mg, 1.65 mmol), cyclo-
hexanol (2.62 ml, 24.84 mmol) and molecular sieves 4 Å (1 g) in
CH2Cl2 (13 ml) was stirred at room temperature for 1 h before
BF3ꢀEt2O (1.22 ml, 9.94 mmol) was added dropwise. The stirring
was continued for 18 h, then Et3N (1.1 ml) added and the solids re-
moved by filtration. The filtrate was diluted with dichloromethane,
and the CH2Cl2 phase washed sequentially with water, saturated
NaHCO3 and water, then dried over MgSO4 and concentrated. Puri-
fication by column chromatography using ethyl acetate–petroleum
ether (3:17) gave an inseparable mixture of anomers 28a and 28b
Methanolic NaOMe (0.2 M, 0.5 ml, 0.1 mmol) was added to a
solution of 28b (200 mg, 0.48 mmol) in CH2Cl2–MeOH (3:5 ml) at
room temperature. After 2 h, the solution was neutralized with
Amberlite IR-120H+ resin. The resin was removed by filtration,
the solvent evaporated and the crystalline residue was chromato-
graphed on silica using methanol–ethyl acetate (5:95) as eluent
to afford 2-C-allylglucoside 30 as white crystals (135 mg, 98%);
mp 136–138 °C (from ethyl acetate and petroleum ether);
[
a
]
D = ꢁ35.4 (c 2.2, MeOH); dH (400 MHz; CD3OD) 5.97–5.87 (1H,
(a
:b ꢄ 1:1 by 13C NMR) as well as excess acceptor (cyclohexanol).
m, H-200), 5.10–4.99 (2H, m, H-300a, H-300b), 4.41 (1H, d, J = 8.8 Hz,
H-1), 3.83 (1H, dd, J = 11.7, 2.4 Hz, H-6a), 3.71 (1H, m, H-10), 3.65
(1H, dd, J = 11.7, 5.4 Hz, H-6b), 3.33 (1H, dd, J = 10.8, 8.4 Hz, H-3),
3.22 (1H, dd, J = 9.6, 8.4 Hz, H-4), 3.18–3.13 (1H, m, H-5), 2.38
(2H, m, H-100a, H-100b), 1.93–1.21 (11H, m, H-2, 5 ꢂ –CH2–, cyclo-
hexyl); dC (100 MHz; CD3OD) 135.34 (C-200), 116.06 (C-300), 100.48
(C-1), 76.43 (C-5), 76.32 (C-10), 73.80 (C-3), 71.97 (C-4), 61.89 (C-
6), 47.32 (C-2), 33.59, 31.70 (2 ꢂ –CH2–, cyclohexyl), 30.39 (C-100),
25.65, 23.94, 23.84 (3 ꢂ –CH2–, cyclohexyl); HRFABMS: m/z
309.168001 (M+Na)+. Calcd for C15H26O5Na 309.167784 (M+Na)+.
The b-anomer 28b crystallized from pentane at low temperature
(244 mg, 36%). Separation of the mother liquor by column chroma-
tography using ethyl acetate–petroleum ether (7:93) as eluent
afforded
mixture of anomers (105 mg, 15%) (overall yield 636 mg, 93%).
-Anomer 28a: [ D = +115.6 (c 2.6, CHCl3); dH (400 MHz; CDCl3)
a-anomer 28a (287 mg, 42%) as an oil together with a
a
a]
5.73–5.63 (1H, m, H-200), 5.22 (1H, dd, J = 11.2, 9.2 Hz, H-3), 5.06–
4.91 (3H, m, H-4, H-300a, H-300b), 4.92 (1H, d, J = 3.6 Hz, H-1), 4.24
(1H, dd, J = 12.2, 4.6 Hz, H-6a), 4.09–4.02 (2H, m, H-5, H-6b),
3.54–3.47 (1H, m, H-10), 2.14–2.10 (2H, m, H-100a, H-100b), 2.06,
2.00, 1.99 (9H-3 ꢂ COOCH3), 1.99 (1H, m, H-2), 1.87–1.19 (10H,
m, 5 ꢂ –CH2–, cyclohexyl); dC (100 MHz; CDCl3) 170.59, 170.42,
169.89 (3 ꢂ COOCH3), 135.25 (C-200), 116.64 (C-300), 96.84 (C-1),
75.95 (C-10), 72.45 (C-3), 70.11 (C-4), 67.66 (C-5), 62.52 (C-6),
44.31 (C-2), 33.31 (C–CH2–, cyclohexyl), 31.73 (C-100), 31.47,
25.48, 24.02, 23.79 (4 ꢂ –CH2–, cyclohexyl), 20.70, 20.60, 20.59
(3 ꢂ COOCH3); HRFABMS: m/z 435.199633 (M+Na)+. Calcd for
C21H32O8Na 435.199477 (M+Na)+.
5.16. Cyclohexyl 2-deoxy-2-C-propyl-a-D-glucopyranoside (31)
A solution of 29 (279 mg, 0.50 mmol) in ethyl acetate–methanol
(5:6 ml) was treated with H2 (1 atm) at room temperature in the
presence of 10% palladium on charcoal (400 mg) for 18 h. TLC anal-
ysis (ethyl acetate–petroleum ether, 4:1) showed the complete
conversion of the starting material into the deprotected derivative.
The catalyst was removed by filtration through Celite, the Celite
washed with methanol and the combined filtrates evaporated to
dryness. The remaining residue was passed through a reverse
phase isoluteÒ C18 column using methanol as an eluent to afford
31 as colorless crystals (140 mg, 97%), mp 184–186 °C (from ethyl
b-Anomer 28b: mp 100 °C (from pentane); [a]D = +16.7 (c 1.7,
CHCl3); dH (300 MHz; CDCl3) 5.84–5.70 (1H, m, H-200), 5.06 (1H,
dd, J = 10.8, 9.0 Hz, H-3), 5.02–4.89 (3H, m, H-4, H-300a, H-300b),
4.39 (1H, d, J = 8.7 Hz, H-1), 4.25 (1H, dd, J = 12.1, 5.3 Hz, H-6a),
4.07 (1H, dd, J = 12.1, 2.6 Hz, H-6b), 3.65–3.55 (2H, m, H-5, H-10),
acetate and petroleum ether); [a]D = +82.8 (c 2.3, MeOH); dH
2.25–2.21 (2H, m, H-100
,
a
H-100b), 2.05, 1.99, 1.99 (9H,
3
s,
(400 MHz; CD3OD) 4.92 (1H, d, J = 3.2 Hz, H-1), 3.77 (1H, d (br),
J = 9.6 Hz, H-6a), 3.69–3.60 (3H, m, H-10, H-5, H-6b), 3.46 (1H, dd,
J = 10.4, 9.0 Hz, H-3), 3.22 (1H, t, J = 9.0 Hz, H-4), 1.87–1.25 (15H,
m, H-2, 5 ꢂ –CH2–, cyclohexyl, -(CH2)2CH3), 0.92 (3H, t, J = 7.2 Hz,
H-300); dC (100 MHz; CD3OD) 96.75 (C-1), 74.20 (C-10), 73.32 (C-
3), 72.54 (C-5), 72.14 (C-4), 61.91 (C-6), 46.23 (C-2), 33.34, 31.13
(2 ꢂ –CH2–, cyclohexyl), 29.44 (C-100), 25.70, 23.75, 23.55 (3 ꢂ
–CH2–, cyclohexyl), 19.89 (C-200), 13.59 (C-300); HRFABMS: m/z
311.182957 (M+Na)+. Calcd for C15H28O5Na 311.183433 (M+Na)+.
3 ꢂ COOCH3), 2.03–1.21 (11H, m, H-2, 5 ꢂ –CH2–, cyclohexyl); dC
(75 MHz; CDCl3) 170.70, 170.37, 169.84 (3 ꢂ COOCH3), 134.21 (C-
200), 117.20 (C-300), 100.58 (C-1), 77.55 (C-10), 72.90 (C-3), 71.34
(C-5), 70.34 (C-4), 62.68 (C-6), 45.24 (C-2), 33.57, 31.80 (2 ꢂ
–CH2–, cyclohexyl), 31.12 (C-100), 25.55, 24.08, 24.01 (3 ꢂ –CH2–,
cyclohexyl), 20.75, 20.72, 20.66 (3 ꢂ COOCH3); HRFABMS: m/z
435.199633 (M+Na)+. Calcd for C21H32O8Na 435.199477 (M+Na)+.
5.14. Cyclohexyl 2-C-allyl-2-deoxy-a-D-glucopyranoside (29)
5.17. Cyclohexyl 3,4,6-tri-O-acetyl-2-deoxy-2-C-(200-oxopropyl)-
Methanolic NaOMe (0.2 M, 0.7 ml, 0.14 mmol) was added to a
solution of 28a (287 mg, 0.69 mmol) in CH2Cl2–MeOH (4:6 ml) at
room temperature. After 4 h, the solution was neutralized with
Amberlite IR-120H+ resin. The resin was removed by filtration,
a-D-glucopyranoside (32)
A mixture of PdCl2 (1.79 g, 10.10 mmol) and CuCl2 (3.5 g,
26.03 mmol) in DMF–H2O (1:7, 15 ml) was stirred at room temper-

D. W. Gammon et al. / Bioorg. Med. Chem. 18 (2010) 2501–2514
2513
ature under an atmosphere of oxygen (balloon) for 2 h. A solution
of 28a (1.05 g, 2.54 mmol) in CH2Cl2–DMF (1:1, 6 ml) was added
dropwise and stirring was continued for 4 days under an atmo-
sphere of oxygen. The reaction was stopped by the addition of a
solution of 3 M HCl, extracted with CH2Cl2, dried over MgSO4 and
concentrated. Purification by column chromatography using ethyl
acetate–petroleum ether afforded compound 32 as colorless crys-
tals (550 mg, 50%); mp 72–73 °C (from petroleum ether);
(100 MHz; CDCl3) 170.61, 170.46, 169.91 (3 ꢂ COOCH3), 97.51 (C-
1), 75.90 (C-10), 72.97 (C-3), 70.14 (C-4), 67.47 (C-5), 66.74 (C-200),
62.49 (C-6), 42.44 (C-2), 36.94 (C-100), 33.30, 31.34, 25.48 (3 ꢂ
–CH2–, cyclohexyl), 24.03 (C-300), 24.03, 23.79 (2 ꢂ –CH2–, cyclo-
hexyl), 20.76, 20.61, 20.60 (3 ꢂ COOCH3); Diastereomer 34b: dH
(400 MHz; CDCl3) 5.22 (1H, dd, J = 11.2, 9.3 Hz, H-3), 5.08 (1H, d,
J = 3.6 Hz, H-1), 4.95 (1H, dd, J = 10.4, 9.3 Hz, H-4), 4.25 (1H, t,
J = 4.4 Hz, H-6a), 4.08–4.03 (2H, m, H-5, H-6b), 3.85–3.77 (1H, m,
H-200), 3.57–3.50 (1H, m, H-10), 2.27–2.19 (1H, m, H-2), 2.06, 2.01,
2.00 (9H, 3 s, 3 ꢂ COOCH3), 1.88–1.19 (12H, m, 5 ꢂ –CH2–, cyclo-
hexyl, H-100a, H-100b), 1.16 (3H, d, J = 6.0 Hz, H-300); dC (100 MHz;
CDCl3) 170.61, 170.46, 169.85 (3 ꢂ COOCH3), 96.61 (C-1), 75.67
(C-10), 72.43 (C-3), 70.14 (C-4), 67.57 (C-5), 64.49 (C-200), 62.49
(C-6), 40.89 (C-2), 36.28 (C-100), 33.30, 31.34, 25.48 (3 ꢂ –CH2–,
cyclohexyl), 24.49 (C-300), 24.03, 23.79 (2 ꢂ –CH2–, cyclohexyl),
20.76, 20.61, 20.60 (3 ꢂ COOCH3); HRFABMS: m/z 453.210259
(M+Na)+. Calcd for C21H34O9Na 453.210041 (M+Na)+.
[a]D = +131.9 (c 2.9, CHCl3); m
max/cmꢁ1 1744 (C@O); dH (300 MHz;
CDCl3) 5.14 (1H, dd, J = 11.0, 9.6 Hz, H-3), 5.08 (1H, d, J = 3.3 Hz,
H-1), 4.95 (1H, t, J = 9.6 Hz, H-4), 4.22 (1H, dd, J = 12.0, 5.0 Hz, H-
6a), 4.05 (1H, dd, J = 12.0, 2.6 Hz, H-6b), 4.02 (1H, m, H-5), 3.47
(1H, m, H-10), 2.60 (1H, dd, J = 17.0, 9.0 Hz, H-100a), 2.54–2.45 (1H,
m, H-2), 2.37 (1H, dd, J = 17.0, 3.4 Hz, H-100b), 2.09 (3H, s, H-300),
2.05, 1.98, 1.97 (9H, 3 s, 3 ꢂ COOCH3), 1.84–1.15 (10H, m, 5 ꢂ
–CH2–, cyclohexyl); dC (75 MHz; CDCl3) 206.43 (C-200), 170.68,
170.69, 169.78 (3 ꢂ COOCH3), 96.16 (C-1), 75.65 (C-10), 71.99 (C-
3), 69.85 (C-4), 67.48 (C-5), 62.43 (C-6), 41.36 (C-100), 40.03 (C-2),
33.28, 31.32 (2 ꢂ –CH2–, cyclohexyl), 30.13 (C-300), 25.48, 24.01,
23.76 (3 ꢂ –CH2–, cyclohexyl), 20.73, 20.66, 20.63 (3 ꢂ COOCH3);
HRFABMS: m/z 451.194600 (M+Na)+. Calcd for C21H32O9Na
451.194392 (M+Na)+.
5.20. Cyclohexyl 2-deoxy-2-C-(200-hydroxypropyl)-
a-D-gluco-
pyranoside (35)
Methanolic sodium methoxide (0.2 M, 0.3 ml, 0.60 mmol) was
added to a solution of 34 (155 mg, 0.36 mmol) in CH2Cl2–MeOH
(3:5 ml) at room temperature. After 18 h, the solution was neutral-
ized with Amberilite IR-120H+ resin, the solids were removed by
filtration, the filtrate concentrated and the residue recrystallized
from ethyl acetate–petroleum ether (4:1) to afford an inseparable
mixture of diastereomers 35 as a solid (98 mg, 89%, 1:1 by 1H
NMR). Signals for the two diastereomers could be distinguished
in the 1H NMR spectrum of the mixture, and assigned with the
aid of COSY and HSQC experiments: Diastereomer 35a: dH (300
MHz; CD3OD) 4.90 (1H, d, J = 3.3 Hz, H-1), 3.92 (1H, m, H-200),
3.81–3.57 (4H, m, H-10, H-5, H-6a, H-6b), 3.52 (1H, dd, J = 10.1,
9.0 Hz, H-3), 3.26 (1H, t, J = 9.0 Hz, H-4), 1.89–1.24 (13H, m, H-2,
5.18. Cyclohexyl 2-deoxy-2-C-(200-oxopropyl)-
a-D-glucopyran-
oside (33)
A methanolic sodium methoxide (0.2 M, 0.2 ml, 0.04 mmol) was
added to a solution of 32 (200 mg, 0.47 mmol) in CH2Cl2–MeOH
(4:5 ml) at room temperature. After 18 h, water (1.7 ml) was added
and the solvent evaporated. Co-evaporation with methanol (ꢂ2)
gave a solid residue that was recrystallized from ethyl acetate–
petroleum ether (4:1) to afford 33 as white crystals (120 mg,
84%); mp 151–154 °C (from ethyl acetate and petroleum ether);
[a]D = +128.8 (c 2.1, MeOH); dC (400 MHz; CD3OD) 4.97 (1H, d,
J = 3.6 Hz, H-1), 3.77 (1H, dd, J = 11.6, 2.4 Hz, H-6a), 3.67 (1H, dd,
J = 11.6, 5.6 Hz, H-6b), 3.65–3.60 (1H, m, H-5), 3.55 (1H, m, H-10),
3.44 (1H, dd, J = 10.8, 8.8 Hz, H-3), 3.26 (1H, dd, J = 10.0, 8.8 Hz,
H-4), 2.84 (1H, dd, J = 17.9, 4.2 Hz, H-100a), 2.58 (1H, dd, J = 17.9,
9.2 Hz, H-100b), 2.14 (3H, s, H-300), 2.12 (1H, m, H-2), 1.87–1.19
(10H, m, 5 ꢂ –CH2–, cyclohexyl); dC (100 MHz; CD3OD) 209.81
(C-200), 96.44 (C-1), 74.38 (C-10), 72.68 (C-5), 72.37 (C-3), 71.98
(C-4), 61.81 (C-6), 42.39 (C-2), 41.52 (C-100), 33.26, 31.09 (2 ꢂ
–CH2–, cyclohexyl), 29.12 (C-300), 25.63, 23.79, 23.59 (3 ꢂ –CH2–,
cyclohexyl); HRFABMS: m/z 325.162838 (M+Na)+. Calcd for
C15H26O6Na 325.162699 (M+Na)+.
5 ꢂ –CH2–, cyclohexyl, H-100
,
H-100b), 1.16 (3H, d, J = 6.0 Hz,
a
H-300); dC (75 MHz; CD3OD) 99.07 (C-1), 75.78 (C-10), 74.67 (C-3),
73.77 (C-5), 73.25 (C-4), 67.32 (C-200), 63.05 (C-6), 44.95 (C-2),
38.46 (C-100), 34.56, 32.33, 26.89, 25.06, 24.86 (5 ꢂ –CH2–, cyclo-
hexyl), 23.54 (C-300); Diastereomer 35b: dH (300 MHz; CD3OD)
4.96 (1H, d, J = 3.6 Hz, H-1), 3.83 (1H, m, H-200), 3.82–3.57 (4H, m,
H-10, H-5, H-6a, H-6b), 3.50 (1H, dd, J = 10.8, 9.1 Hz, H-3), 3.27
(1H, t, J = 9.1 Hz, H-4), 1.89–1.24 (13H, m, H-2, 5 ꢂ –CH2–, cyclo-
hexyl, H-100a, H-100b), 1.17 (3H, d, J = 6.3 Hz, H-300); dC (75 MHz;
CD3OD) 98.49 (C-1), 75.46 (C-10), 74.50 (C-3), 73.77 (C-5), 73.25
(C-4), 66.30 (C-200), 63.05 (C-6), 44.87 (C-2), 38.87 (C-100), 34.52,
32.33, 26.87, 24.98, 24.79 (5 ꢂ –CH2–, cyclohexyl), 24.61 (C-300);
HRFABMS: m/z 327.177488 (M+Na)+. Calcd for C15H28O6Na
327.178348 (M+Na)+.
5.19. Cyclohexyl 3,4,6-tri-O-acetyl-2-deoxy-2-C-(200-hydroxy-
propyl)-a-D-glucopyranoside (34)
NaBH4 (0.19 g, 5.02 mmol) was added portion-wise to a solu-
tion of compound 32 (200 mg, 0.47 mmol) in THF–MeOH
(2:5 ml) at ꢁ20 °C and the reaction allowed to warm to 0 °C over
2 h. The reaction was quenched with saturated NaHCO3, extracted
with CH2Cl2, and the CH2Cl2 solution dried over MgSO4 and con-
centrated. Purification by column chromatography using ethyl ace-
tate–petroleum ether (3:7) afforded an inseparable mixture of
5.21. Cyclohexyl 3,4,6-tri-O-acetyl-2-deoxy-2-C-(200,300-epoxy-
propyl)-a-D-glucopyranoside (36)
To a solution of 28a (308 mg, 0.75 mmol) in CH2Cl2 (10 ml) at
0 °C was slowly added solution of m-CPBA (70%, 0.32 g,
a
1.29 mmol) in CH2Cl2 (3.0 ml) (m- CPBA was dissolved in CH2Cl2,
dried with MgSO4, filtered and concentrated before used). The tem-
perature was raised to room temperature and after stirring for
18 h, a further portion of m-CPBA (70%, 0.1 g, 0.40 mmol) in CH2Cl2
(2 ml) was added slowly to the mixture at 0 °C. The reaction was
stirred for 6 h at room temperature, then saturated Na2S2O3 added,
the aqueous mixture extracted with CH2Cl2, and the CH2Cl2
washed with saturated NaHCO3, dried over MgSO4 and concen-
trated. The crude residue was purified by column chromatography
using ethyl acetate–petroleum ether (1:4) as eluent to afford an
inseparable mixture of diastereomers 36 (292 mg, 91%, 1:1 by
1H NMR) as an oil. Signals for the two diastereomers could be
diastereomers 34 as an oil (178 mg, 88%, 1:1 by 1H NMR); mmax
/
cmꢁ1 3467 (br) (OH). Signals for the two diastereomers could be
distinguished in the 1H NMR spectrum of the mixture, and as-
signed with the aid of COSY and HSQC experiments: Diastereomer
34a: dC (400 MHz; CDCl3) 5.19 (1H, dd, J = 11.2, 8.9 Hz, H-3), 5.05
(1H, d, J = 3.6 Hz, H-1), 4.93 (1H, dd, J = 10.2, 8.9 Hz, H-4), 4.22
(1H, t, J = 4.4 Hz, H-6a), 4.08–4.03 (2H, m, H-5, H-6b), 3.85–3.77
(1H, m, H-200), 3.57–3.50 (1H, m, H-10), 2.07 (1H, m, H-2), 2.06,
2.01, 2.00 (9H, 3 s, 3 ꢂ COOCH3), 1.88–1.19 (12H, m, 5 ꢂ –CH2–,
cyclohexyl, H-100
,
H-100b), 1.17 (3H, d, J = 6.0 Hz, H-300); dC
a

2514
D. W. Gammon et al. / Bioorg. Med. Chem. 18 (2010) 2501–2514
distinguished in the 1H NMR spectrum of the mixture, and as-
signed with the aid of COSY and HSQC experiments: Diastereomer
36a: dH (400 MHz; CDCl3) 5.15 (1H, dd, J = 11.0, 9.4 Hz, H-3), 5.00
(1H, d, J = 3.2 Hz, H-1), 4.86 (1H, t, J = 9.4 Hz, H-4), 4.18–4.14 (1H,
m, H-6a), 4.03–3.96 (2H, m, H-5, H-6b), 3.48 (1H, m, H-10), 2.82
(1H, m, H-200), 2.66 (1H, t, J = 5.0 Hz, H-300a), 2.36 (1H, dd, J = 5.0,
2.6 Hz, H-300b), 2.07 (1H, m, H-2), 1.98, 1.93, 1.92 (9H, 3 s,
(C-5), 72.01 (C-4), 61.84 (C-6), 50.42 (C-200), 47.51 (C-300), 44.07
(C-2), 33.40, 31.27 (2 ꢂ –CH2–, cyclohexyl), 30.56 (C-100), 25.68,
23.95, 23.77 (3 ꢂ –CH2–, cyclohexyl); HRFABMS: m/z 325.162227
(M+Na)+. Calcd for C15H26O6Na 325.162699 (M+Na)+.
Acknowledgments
3 ꢂ COOCH3), 1.82–1.10 (12H, m, 5 ꢂ –CH2–, cyclohexyl, H-100
,
We thank the University of Cape Town, the National Research
Foundation (South Africa) and the Medical Research Council of
South Africa for funding.
a
H-100b); dC (100 MHz; CDCl3) 170.85, 170.83, 170.06 (3 ꢂ COOCH3),
96.94 (C-1), 75.99 (C-10), 72.41 (C-3), 70.23 (C-4), 67.85 (C-5),
62.66 (C-6), 49.97 (C-200), 47.53 (C-300), 42.39 (C-2), 33.51 (C-100),
31.56, 30.69, 25.66, 24.27, 24.04 (5 ꢂ –CH2–, cyclohexyl), 20.94,
20.78, 20.77 (3 ꢂ COOCH3); Diastereomer 36b: dH (400 MHz; CDCl3)
5.15 (1H, dd, J = 11.0, 9.5 Hz, H-3), 5.04 (1H, d, J = 3.2 Hz, H-1), 4.87
(1H, t, J = 9.5 Hz, H-4), 4.18–4.14 (1H, m, H-6a), 4.03–3.96 (2H, m,
H-5, H-6b), 3.48 (1H, m, H-10), 2.82 (1H, m, H-200), 2.62 (1H, t,
J = 4.8 Hz, H-300a), 2.31 (1H, dd, J = 4.8, 2.6 Hz, H-300b), 2.07 (1H, m,
H-2), 1.98, 1.93, 1.92 (9H, 3 s, 3 ꢂ COOCH3), 1.82–1.10 (12H, m,
5 ꢂ –CH2–, cyclohexyl, H-100a, H-100b); dC (100 MHz; CDCl3) 170.75,
170.63, 170.11 (3 ꢂ COOCH3), 97.69 (C-1), 76.68 (C-10), 72.48 (C-
3), 70.29 (C-4), 67.79 (C-5), 62.68 (C-6), 51.06 (C-200), 46.82 (C-
300), 43.89 (C-2), 33.51 (C-100), 31.52, 30.88, 25.71, 24.22, 24.00
(5 ꢂ –CH2–, cyclohexyl), 20.94, 20.78, 20.77 (3 ꢂ COOCH3);
HRFABMS: m/z 451.195037 (M+Na)+. Calcd for C21H32O9Na
451.194392 (M+Na)+.
References and notes
1. Newton, G. L.; Arnold, K.; Price, M. S.; Sherrill, C.; Delcardayre, S. B.;
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5.22. Cyclohexyl 2-deoxy-2-C-(200,300-epoxypropyl)-
pyranoside (37) and cyclohexyl (200R),(200S)-3,300-anhydro-2-
deoxy-2-C-(200,300-dihydroxypropyl)-
-glucopyranoside (38)
a-D-gluco-
14. Newton, G. L.; Ko, M.;Ta, P.;Av-Gay, Y.; Fahey, R. C. Protein Exp. Purif. 2006, 47, 542.
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a
-D
Methanolic sodium methoxide (0.2 M, 0.3 ml, 0.06 mmol) was
added to a solution of 36 (200 mg, 0.47 mmol) in CH2Cl2–MeOH
(3:5 ml) at room temperature. After 6 h, water was added (1 ml)
and the solvent evaporated to dryness. TLC indicated the formation
of two products and separation was achieved by column chroma-
tography using methanol–ethyl acetate (3:97) to obtain a diaste-
reomeric mixture of epoxides 37 as an oil (71 mg, 50%, 4:1 by 1H
NMR) and a diastereomeric mixtures of alcohols 38 as crystals
(60 mg, 42%, 1:1 by 1H NMR). For compound 37 signals for each
diastereomer could be distinguished in the 1H NMR spectrum,
and assigned with the aid of COSY and HSQC experiments. Diaste-
reomer 37a: dH (300 MHz; CD3OD) 5.15 (1H, d, J = 3.0 Hz, H-1), 4.11
(1H, m, H-200), 3.84–3.42 (8H, m, H-10, H-3, H-300a, H-300b, H-4, H-5,
1341.
19. Metaferia, B. B.; Fetterolf, B. J.; Shazad-Ul-Hussan, S.; Moravec, M.; Smith, J. A.;
Ray, S.; Gutierrez-Lugo, M. T.; Bewley, C. A. J. Med. Chem. 2007, 50, 6326.
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29. (a) Buskas, T.; Garegg, P. J.; Konradsson, P.; Maloisel, J.-L. Tetrahedron:
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30. (a) Tiwari, P.; Agnihotri, G.; Misra, A. K. J. Carbohydr. Chem. 2005, 24, 723; (b)
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33. Patel, M. P.; Blanchard, J. S. Biochemistry 2001, 40, 5119.
H-6a, H-6b), 2.05–1.30 (13H, m, 5 ꢂ –CH2–, cyclohexyl, H-2, H-100
,
a
H-100b); dC (75 MHz; CD3OD) 97.75 (C-1), 81.05 (C-10), 80.40 (C-200),
76.33 (C-3), 74.93 (C-4), 72.63 (C-5), 65.66 (C-300), 62.32 (C-6),
47.92 (C-2), 34.50, 32.58 (2 ꢂ –CH2–, cyclohexyl), 29.71 (C-100),
26.82, 24.94, 24.76 (3 ꢂ –CH2–, cyclohexyl); Diastereomer 37b: dH
(300 MHz; CD3OD) 5.14 (1H, d, J = 3.0 Hz, H-1), 4.11 (1H, m, H-
200), 3.84–3.42 (8H, m, H-10, H-3, H-300a, H-300b, H-4, H-5, H-6a, H-
6b), 2.05–1.30 (13H, m, 5 ꢂ –CH2–, cyclohexyl, H-2, H-100a, H-100b);
dC (75 MHz; CD3OD) 97.87 (C-1), 81.96 (C-10), 80.15 (C-200), 76.33
(C-3), 74.83 (C-4), 72.52 (C-5), 65.84 (C-300), 62.35 (C-6), 46.65 (C-
2), 34.50, 32.58 (2 ꢂ –CH2–, cyclohexyl), 29.14 (C-100), 26.82,
24.94, 24.76 (3 ꢂ –CH2–, cyclohexyl); HRFABMS: m/z 325.162227
(M+Na)+. Calcd for C15H26O6Na 325.162699 (M+Na)+. Compound
38: dH (400 MHz; CD3OD) 5.04 (1H, d, J = 3.2 Hz, H-1), 3.80–3.60
(4H, m, H-10, H-5, H-6a, H-6b), 3.50 (1H, dd, J = 10.8, 8.8 Hz, H-3),
3.26 (1H, dd, J = 9.2, 8.8 Hz, H-4), 3.07–3.00 (1H, m, H-200), 2.77
(1H, dd, J = 5.0, 4.0 Hz, H-300a), 2.51 (1H, d, J = 5.0, 2.8 Hz, H-300b),
1.95–1.24 (13H, m, 5 ꢂ –CH2–, cyclohexyl, H-2, H-100a, H-100b); dC
(100 MHz; CD3OD) 96.88 (C-1), 74.51 (C-10), 72.77 (C-3), 72.71
34. Marakalala, M.J. Inhibition of
a Mycothiol Biosynthetic Enzyme and a
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