Synthesis and evaluation of anti-tubercular activity of new dithiocarbamate sugar derivatives

Article abstract of DOI:10.1016/j.bmcl.2010.12.084

The present study was undertaken to optimize the anti-tubercular activity of 2-acetamido-2-deoxy-β-d-glucopyranosyl N,N-dimethyldithiocarbamate (OCT313, Glc-NAc-DMDC), a lead compound previously reported by us. Structural modifications of OCT313 included the replacements of the DMDC group at C-1 by pyrrolidine dithiocarbamate (PDTC) and the acetyl group at C-2 by either propyl, butyl, benzyl or oleic acid groups. The antimycobacterial activities of these derivatives were evaluated against Mycobacterium tuberculosis (MTB). Glc-NAc-pyrrolidine dithiocarbamate (OCT313HK, Glc-NAc-PDTC) exhibited the most potent anti-tubercular activity with the minimal inhibitory concentration (MIC) of 6.25-12.5 μg/ml. The antibacterial activity of OCT313HK was highly specific to MTB and Mycobacterium bovis BCG, but not against Mycobacterium avium, Mycobacterium smegmatis, Staphylococcus aureus or Escherichia coli. Importantly, OCT313HK was also effective against MTB clinical isolates, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. Interestingly, OCT313HK was exerted the primary bactericidal activity, and it was also exhibited the bacteriolytic activity at high concentrations. We next investigated whether the mycobacterial monooxygenase EthA, a common activator of thiocarbamide-containing anti-tubercular drugs, also activated OCT313HK. Contrary to our expectations, the anti-tubercular activity of dithiocarbamate sugar derivatives and dithiocarbamates were not dependent on ethA expression, in contrast to thiocarbamide-containing drugs. Overall, this study presents OCT313HK as a novel and potent compound against MTB, particularly promising to overcome drug resistance.

Full text of DOI:10.1016/j.bmcl.2010.12.084

Bioorganic & Medicinal Chemistry Letters 21 (2011) 899–903
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of anti-tubercular activity of new dithiocarbamate
sugar derivatives
Yasuhiro Horita a,c, Takemasa Takii a, , Ryuji Kuroishi a, Taku Chiba b, Kenji Ogawa c, Laurent Kremer d,e
,
⇑
Yasuo Sato f, YooSa Lee a, Tomohiro Hasegawa a, Kikuo Onozaki a
a Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan
b Department of Pharmacy, College of Pharmacy, Kinjo Gakuin University, Nagoya, Japan
c Department of Clinical Research, National Hospital Organization, Higashi Nagoya National Hospital, Nagoya, Japan
d Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, UMR 5235 CNRS, Université de Montpellier II et I, Place Eugène Bataillon,
34095 Montpellier Cedex 05, France
e INSERM, DIMNP, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France
f Meiji Seika Kaisha, Tokyo, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study was undertaken to optimize the anti-tubercular activity of 2-acetamido-2-deoxy-b-D-
Received 2 November 2010
Revised 9 December 2010
Accepted 16 December 2010
Available online 22 December 2010
glucopyranosyl N,N-dimethyldithiocarbamate (OCT313, Glc-NAc-DMDC), a lead compound previously
reported by us. Structural modifications of OCT313 included the replacements of the DMDC group at C-1
by pyrrolidine dithiocarbamate (PDTC) and the acetyl group at C-2 by either propyl, butyl, benzyl or oleic
acid groups. The antimycobacterial activities of these derivatives were evaluated against Mycobacterium
tuberculosis (MTB). Glc-NAc-pyrrolidine dithiocarbamate (OCT313HK, Glc-NAc-PDTC) exhibited the most
Keywords:
potent anti-tubercular activity with the minimal inhibitory concentration (MIC) of 6.25–12.5 lg/ml. The
Mycobacterium tuberculosis
N-Acetyl glucosamine
Dithiocarbamate
Pyrrolidine
antibacterial activity of OCT313HK was highly specific to MTB and Mycobacterium bovis BCG, but not
against Mycobacterium avium, Mycobacterium smegmatis, Staphylococcus aureus or Escherichia coli. Impor-
tantly, OCT313HK was also effective against MTB clinical isolates, including multidrug-resistant (MDR)
and extensively drug-resistant (XDR) strains. Interestingly, OCT313HK was exerted the primary bacterici-
dal activity, and it was also exhibited the bacteriolytic activity at high concentrations. We next investigated
whether the mycobacterial monooxygenase EthA, a common activator of thiocarbamide-containing anti-
tubercular drugs, also activated OCT313HK. Contrary to our expectations, the anti-tubercular activity of
dithiocarbamate sugar derivatives and dithiocarbamates were not dependent on ethA expression, in con-
trast to thiocarbamide-containing drugs. Overall, this study presents OCT313HK as a novel and potent
compound against MTB, particularly promising to overcome drug resistance.
Ó 2010 Elsevier Ltd. All rights reserved.
More than 9.4 million people develop tuberculosis (TB)
annually, and 1.7 million die each year.1 New case of TB is still
increasing all over the world, especially in low-income countries,
and TB infection including both multidrug-resistant tuberculosis
(MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) is
a leading cause of death worldwide. The spread of both MDR-TB
and XDR-TB, due to poor compliance of anti-TB drugs, becomes a
global health problem. Forty years have passed since the last
development of anti-TB drug and the development of novel and
innovative compounds is urgently needed.2 We have recently
C-1 position was critical to the bactericidal activity. In this study,
in order to improve the antimycobacterial activity of OCT313, we
synthesized the derivative of dithiocarbamate group at the C-1
position and its antimycobacterial activity was evaluated. We
first examined whether the elongation of alkyl side chain of
dimethyldithiocarbamate, for example, diethyl and dibutyl,
improved the antimycobacterial activity against Mycobacterium
tuberculosis (MTB) H37Rv. The elongation of carbon chain resulted
in decreasing of anti-tubercular activity (Table 1). Previously, the
antimycobacterial activity of pyrrolidine dithiocarbamate (PDTC)
and dialkyldithiocarbamate derivatives have been demon-
strated.4–6 Next, we investigated whether dithiocarbamates con-
taining heterocyclic ring, for example, 4-imidazodithiocarboxylic
acid (IMTC) and PDTC were effective against MTB. As a result, PDTC
was the most potent compound in our experiments, which was
similar to first-line drugs in vitro.
reported that 2-acetamido-2-deoxy-b-D-glucopyranosyl N,N-dim-
ethyldithiocarbamate (Glc-NAc-DMDC), named as OCT313,
exhibited the potent antimycobacterial activity.3
Studies on the structure–activity relationships (SAR) at C-1, C-4
and C-6 positions of OCT313 established that the DMDC group at
Based on these findings, we synthesized C-1 derivative of
OCT313, 2-acetamido-2-deoxy-b-D-glucopyranosyl pyrrolidine-1-
⇑
Corresponding author. Tel.: +81 52 836 3421; fax: +81 52 836 3419.
E-mail address: ttakii@phar.nagoya-cu.ac.jp (T. Takii).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.084

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Y. Horita et al. / Bioorg. Med. Chem. Lett. 21 (2011) 899–903
Table 1
Antimycobacterial activity of dithiocarbamate derivatives
Agent
n
M
R1
R2
MIC for (MIC, lg/ml)
M. tuberculosis H37Rv
M. bovis BCG str. Tokyo 172
M. smegmatis JATA 64-01
Carbon chain
DMDC
DDC
DDC
DBuDC
1
1
2
2
Na
Na
Zn
Zn
–CH3
–CH3CH3
–CH2CH3
–CH2CH2CH2CH3
—
—
—
—
1.56
3.13
1.56
12.5
1.56
3.13
1.56
12 5
>100
>100
>100
>I00
Aromatic ring
DBzDC
2
Zn
25
25
>100
Heterocyclic ring
IMTC
PDTC
1
Na
6.25
0.2
12 5
0.4
>I00
>I00
1
NH3
DMDC. Na, sodium dimethyldithiocarbamate; DDC. Na, sodium diethyldithiocarbamate; DDC. Zn, zinc bis (diethyldithiocarbamate); DBuDC. Zn, zinc bis(dibutyldithiocar-
bamate); DBzDC. Zn, zinc bis(dibenzyldithiocarbamate); IMTC. Na, sodium 4-imidazodithiocarboxylic acid; PDTC. NH3, ammonium 1-pyrrolidine dithiocarbamate.
carbodithioate (OCT313HK, Glc-NAc-PDTC),14,15 which is the substi-
tution of the DMDC group at C-1 position of OCT313 to the PDTC
group and was determined the antibacterial activity (Table 2).
OCT313HK exhibited the potent antimycobacterial activity against
both MTB and Mycobacterium bovis BCG Tokyo with MICs of
(MIC = 32 lg/ml), but not P. aeruginosa (Table 2 and Supplementary
Table 1). These data indicate that the antibacterial spectrum of
OCT313HK is narrow compared to PDTC and exhibit 2 to 4-fold high-
er anti-tubercular activity than OCT313 (MIC = 25 lg/ml, Table 2).
We next evaluated the antimycobacterial activities of
OCT313HK and PDTC against 40 MTB clinical isolates, including
drug-sensitive and drug-resistant strains. As shown in Table 3,
OCT313HK exhibited the anti-tubercular activity with the MIC of
6.25 lg/ml and 12.5 lg/ml, respectively (Table 2). However,
OCT313HK failed to inhibit the growth of Mycobacterium smegmatis,
Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Entero-
coccus faecium and Pseudomonas aeruginosa. (Table 2 and Supple-
mentary Table 1). Meanwhile, PDTC exhibited the anti-tubercular
6.25–12.5 lg/ml, whereas the MIC value of PDTC was 0.2–0.4 lg/
ml. Importantly, no cross resistance to almost currently used
anti-TB drugs was demonstrated, that OCT313HK and PDTC exhib-
ited comparable activities against all these strains, including five
activity (MIC = 0.2
eus (MIC = 8–12.5
l
l
g/ml) and antibacterial activities against S. aur-
g/ml), E. faecalis (MIC = 32 g/ml), E. faecium
l
Table 2
Antibacterial activity of OCT313HK in vitro (MIC,
l
g/ml)a
Compound
Organisms
M tuberculosis
37RV
M. bovis BCG
str. Tokyo 172
M. avium subsp.
hominissuis 104
M. avium subsp.
avium ATCC2529I
M. smegmatis
JATA 64-01
S. aureus
MRSA 873
E. coli DH5a
H
Synthetic derivative
OCT313HK
OCT313
6.25
25
12.5
31.3
100
>100
50
>100
>100
>100
>100
>100
>100
>100
>100
>100
Raw material
Glc-NAc free
DMDC. Na
>100
0.78
0.2
>100
1.56
0.4
>100
>100
3.13
>100
>100
3 13
>100
>100
100
>100
>100
12.5
>100
>100
12.5
>100
>100
12.5
PDTC. NHi
anti-IB drug
INH
RFP
SM
EB
0.04
0.004
0.39
2.5
1.56
0.39
0.04
0.004
0.2
1.5
0.3
1.56
0.25
1.56
1.6
3.13
0.39
3 13
<0.05
3,13
1.6
3.13
1.56
6.25
1.56
0.39
12.5
3.13
0.39
>100
0.002
50
>100
12.5
0.2
>100
0.004
>100
>100
>100
ne
>100
50
50
>100
12.5
0.2
KM
CPFX
0.1
b-Lactam antibiotics
PCG
ABPC
IPM
500
12.5
3.13
500
12.5
3.13
ne
ne
ne
ne
ne
ne
ne
ne
ne
31.3
50
0.1
>500
>100
0.1
25
>100
0.1
a
Broth dilution methods using MiddleBrook 7H9 broth containing albumin, dextrose, and catalase for derivatives (ne, not examined). For Staphylococcus aureus, we used
the LB broth. OCT313HK, Glc-NAc-PDTC; OCT313, Glc-NAc-DMDC, Glc-NAc free, N-acetyl glucosamine; DMDC. Na, sodium dimethyldithiocarbamate; PDTC. NH3, ammonium
1-pyrrolidine dithiocarbamate; INH, isoniazid; RFP, rifampicin, SM, streptomycin; EB, ethambutol; KM, kanamycin, PAS, para-aminosalicylic acid; CPFX, ciprofloxacin; PCG,
penicillin G; ABPC, aminobenzyl penicillin; IPM, imipenem.

Y. Horita et al. / Bioorg. Med. Chem. Lett. 21 (2011) 899–903
901
Table 3
whether the lytic activity was suppressed by the presence of either
dextran or sucrose, which was used to increase the extracellular
osmotic pressure.7 The lytic activity of OCT313HK and OCT313
against M. bovis BCG were inhibited in the presence of these
reagents from day 2 (Fig. 1A and B). Taken together, these results
suggest that OCT313HK and OCT313 exert bactericidal and bacte-
riolytic activities. Of note, this feature has not been observed with
the currently used anti-TB drugs.
Antimycobacterial activities of OCT313HK and PDTC against clinical isolates of M.
tuberculosis
Clinical
isolates
Resistance to
MIC for (
l
g/ml)
OCT313HK
PDTC
Drug sensitive strain
1
2
3
4
5
6
7
6.25
6.25
6.25
6.25
6.25
6.25
12.5
12.5
6.25
6.25
6.25
6.25
12.5
6.25
6.25
6.25
6.25
6.25
6.25
12.5
0.2
0.2
0.2
0.2
0.2
0.2
0.4
0.4
0.2
0.2
0.2
0.2
0.4
0.2
0.2
0.2
0.2
0.2
0.4
0.4
PDTC and DMDC belong to dithiocarbamates. Thiocarbamide-
containing drugs, for example, ETH, thiacetazone (TAC) or isoxyl
(ISO), which have been used as second line drugs are activated
by the monooxygenase EthA.8 Approximately, 50% of ETH-resistant
clinical isolates possessed mutations in the ethA gene.9 Thereby, we
further studied whether ethA expression was required for
antimycobacterial activity of dithiocarbamates and dithiocarba-
mate sugar derivatives. This was achieved by using M. bovis BCG
strains carrying either the pMV261-ethA or the pMV261-ethR,
designed to overexpress either EthA or EthR under the control of
the constitutive hsp60 promoter, respectively.8 The MICs against
these strains were compared to those of a BCG strain harboring
the empty construct (Table 5). The EthR-overexpressing strain
expressed high levels of resistance to ETH, whereas the EthA-
overexpressing strain was hypersusceptible to ETH, consistently
with previous reports.8,10 In contrast, the MICs of dithiocarba-
mate-containing agents OCT313HK, OCT313 and PDTC against
the EthA- or EthR-overexpressing strains were similar to those of
the control strain, indicating that the anti-tubercular activity of
these two compounds does not rely on EthA expression. In addi-
tion, OCT313HK and PDTC demonstrated no cross-resistance to
ETH, because they were effective against ETH-resistant clinical iso-
late No. 20 (Table 3). These results suggest that the mode of action
of both dithiocarbamate sugar derivatives and dithiocarbamates
are different from the currently used anti-tubercular drugs, includ-
ing ETH.
8
9
10
11
12
13
14
15
16
17
I8
19
20
Drug-resistant strain
1
2
3
4
5
6
7
INH, RFP, EB, LVFX, SPFX, CPFX
INH, RFP, EB, LVFX, SPFX, CPFX
RFP, EB
INH, RFP, EB, LVFX, SI’I’X, CPFX
INH, RFP, EB, KM, LVFX, SPFX, CPFX
INH, RFP, SM, EB, KM, LVFX, SPFX, CPFX
RFP
6.25
6.25
6.25
12.5
6.25
6.25
6.25
6.25
6.25
12.5
12.5
6.25
6.25
6.25
6.25
6.25
6.25
6.25
12.5
6.25
6.25–12.5
0.2
0.2
0.2
0.4
0.2
0.2
0.2
0.2
0.2
0.4
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.4
0.2
0.2–0.4
8
9
RFP
INH, RFP, KM, LVFX, SPFX, CPFX
RFP
INH, RFP, EB, KM, LVFX, SPFX, CPFX
INH, RFP, EB
INH, RFP, SM, EB
INH, RFP, EB
INH, RFP, EB, LVFX, SPFX, CPFX
INH, RFP, SM, EB, KM, PAS, EVM, LVFX
INH, RFP
INH, RFP, SM, EB, PAS
INH, RFP, SM, EB, PAS, LVFX
INH, RFP, ETH
10
11
12
13
14
15
16
17
18
19
20
H3-Rv
Finally, in order to study the SAR for sugar moieties of OCT313,
we further synthesized the chemically modified derivatives at C-2
position of OCT313 (Table 6).14,16 Previously, it was demonstrated
that C-4 isomers of OCT313 was more potent anti-tubercular activ-
ity compared to OCT313. Nevertheless, the anti-tubercular activity
of C-2 derivatives, which were modified to some other types of
functional groups namely propionamido (R2), butyramido (R3),
benzamido (R4) and oleamido (R5) were lower than original com-
OCT313HK, Glc-NAc-PDTC; PDTC, ammonium 1-pyrrolidine dithiocarbamate; INH,
isoniazid; RFP, rifampicin; SM, streptomycin; EB, ethambutol; KM, kanamycin; PAS,
para-aminosalicylic acid; ETH, ethionamide; EVM, emviomycin; LVFX, levofloxacin;
SPFX, sparfloxacin; CPFX, ciprofloxacin.
pound OCT313 (MIC = 50–100
amino derivative of OCT313 (Glc-NH2-DMDC) (R6) was synthesized
by de-O-acetylation of 3,4,6-tri-O-acetyl-2-amino-2-deoxy-b-
lg/ml). Contrary to the strategy, 2-
D
-
glucopyranosyl N,N-dimethyldithiocarbamate hydrochloride with
an anion exchange resin.17 The anti-tubercular activity of Glc-
NH2-DMDC was similar to C-2 derivatives (MIC = 50 lg/ml). These
XDR-TB strains. XDR-TB is defined as TB that is resistant to at least
rifampicin and isoniazid plus fluoroquinolones, and at least one of
three injectable second line anti-TB drugs, that is, amikacin, kana-
mycin or capreomycin.1 These results suggest that OCT313HK and
PDTC are effective against strains, resistant to fluoroquinolones,
e.g. levofloxacin (LVFX), sparfloxacin (SPFX), ciprofloxacin (CPFX),
which have been considered as candidate compounds for new
anti-TB therapy. Therefore, OCT313HK and PDTC may represent
attractive drug candidates to be included in future pharmacologi-
cal developments against XDR-TB.
Next, we investigated the primary mode of action of OCT313HK
and OCT313. Each compound reduced not only the colony forming
units, but also the optical density (OD) at both log-phase and
stationary phase in time-rather than dose-dependent manner
(Fig. 1 and Table 4). Both OCT313HK and OCT313 remarkably de-
creased the turbidity compared to other bactericidal drugs, that
is, isoniazid (INH), ethionamide (ETH), streptomycin (SM), and
kanamycin (KM) (data not shown). Consequently, we determined
results suggest that the acetyl group at C-2 position of OCT313 was
optimal for anti-tubercular activity.
Inconclusion,thisstudyhasunraveledthepotentialofOCT313HK
and OCT313 as valuable compounds for future pharmacological
developments against MDR-TB and XDR-TB. Interestingly,
OCT313HK exhibited unstained bacteriolytic activity compared to
OCT313. The lytic activity of dithiocarbamate sugar derivatives is
probably due to dithiocarbamate structure. Surprisingly, dithiocar-
bamate sugar-resistant colonies were unable to grow on 7H11 agar
plate whereas, both anti-TB drug- and dithiocarbamate-resistant
colonies were observed spontaneously (data not shown). The resis-
tant strains to anti-TB drug, for example, INH, RFP, SM, KM, PAS
and CPFX, can be subcultured in liquid medium, commonly. Never-
theless, we were notable to subculture the resistant strainsto dithio-
carbamate, for example, PDTC and DDC, in any broth containing each
agent. This phenomenon was caused by robust clumping of dithio-
carbamate-resistant bacilli compared to other strains. Actually, the
colonization and morphology of dithiocarbamate-resistant strains

902
Y. Horita et al. / Bioorg. Med. Chem. Lett. 21 (2011) 899–903
Figure 1. Bacteriolytic activity of each agent under the presence of either dextran or sucrose at log phase. Lysis of M. bovis BCG was determined by incubation with 125 mg/ml
OCT313HK (A), 313 mg/ml OCT313 (B), 4 mg/ml PDTC (C) and 15.6 mg/ml DMDC (D) with or without 150 mM sucrose or 2.5% dextran (molecular weight = >500,000) for
7 days. Lysis of dilute suspensions of cells was determined by following spectrophotometrically the loss of reading at O.D. 530 nm. Experiments were carried out more than
three times and representative data were shown. Error bars represent means SD (n = 3).
Table 4
Table 5
Bactericidal activity of each agent under in the presence of either dextran or sucrose
at both log phase and stationary phase
MICs of ethionamide and dithiocarbamate-containing drugs against M. bovis BCG
overexpressing either EthA or EthR in 7H11 agar supplemented with OADC
Agent and condition
Ave. log CFU/ml SDa
day 21
Strain
MIC for (MIC,
OCT313
lg/ml)
day 7
OCT313HK
PDTC
ETH
A
B
C
D
E
BCG pMV261
BCG pMV261 ::ethA
BCG pMV261::ethR
1–2.5
1–2.5
1–2.5
10–20
10–20
10–20
0.5
0.5
0.25–0.5
1–5
0.5
10–25
OCT313HK
OCT313HK + sucrose
OCT313HK + dextran
4.55 0.14
6.25 0.03
6.07 0.13
4.43 0.08
5.76 0.10
5.79 0.05
OCT313HK, Glc-NAc-PDTC; OCT313. Glc-NAc-DMDC; PDTC, ammonium 1-pyrroli-
dine dithiocarbamate; ETH. ethionamide.
OCT313
OCT313 + sucrose
OCT313 + dextran
5.91 0.07
6.41 0.08
6.44 0.08
5.20 0.14
5.78 0.04
5.91 0.03
Table 6
PDTC
PDTC + sucrose
PDTC + dextran
4.65 0.08
6.05 0.03
6.93 0.06
4.41 0.08
5.56 0.04
5.56 0.09
Anti-tubercular activity of C2-derivatives of OCT313 in vitroa
DMDC
DMDC + sucrose
DMDC + dextran
6.94 0.05
6.18 0.07
6.09 0.04
8.21 0.23
8.22 0.03
8.12 0.09
R
Compound
MIC for (MIC, lg/ml)
M. tuberculosis H37-Rv
No agent
Sucrose
Dextran
8.44 0.35
8.50 0.23
8 34 004
7.92 0.06
8.13 0.06
8.26 004
R1 = COCH3
R2 = COC2H5
R3 = COC3H5
R4 = COC6H6
R5 = COC17H33
R6=H
OCT313 (Glc-NAc-DMDC)
Glc-NPro-DMDC
Glc-NBt-DMDC
Glc-NBz-DMDC
Gk-NOle-DMDC
Glc-NH2-DMDC
25
50
50
100
100
50
a
Bactericidal activity against M. bovis BCG was determined by incubation with
g/ml OCT313HK (A), 313 g/ml OCT313 (B), 4 g/ml PDTC (C) and 15.6 g/ml
125
l
l
l
l
DMDC (D) with or without 150 nM sucrose or 2.5% dextran for 7 days or 21 days,
respectively. Values represent means SD. OCT313HK, Glc-NAc-PDTC; OCT313.
Glc-NAc-DMDC; PDTC, ammonium 1-pyrrolidine dithiocarbamate; DMDC, sodium
dimethyldithiocarbamate.
a
Broth dilution methods using MiddleBrook 7H9 broth containing albumin,
dextrose, and catalase for derivatives.
were remarkably different from anti-TB drug-resistant strains. These
findings suggested that the anti-tubercular activity of dithiocarba-
mate and dithiocarbamate sugar might be not caused due to some
common known mechanism. Therefore, further work is required to
clarify the specific targets of dithiocarbamate and dithiocarbamate
sugar.

Y. Horita et al. / Bioorg. Med. Chem. Lett. 21 (2011) 899–903
903
thioate) as colorless needles, mp 198–199o (decomp.) in 78% yields. ½a 2D3
ꢀ
+46.4° (c 1.04, H2O), IR(KBr) cmꢁ1: 3500–3300 (br OH, NH), 1639 (amide I), 1532
(amide II). 1H NMR (CD3OD) d: 1.94 (s, 3H, NCOCH3), 1.97, 2.07, 3.59, 3.70, 3.86
(m, 8H, methylene protons of pyrrolidine group), 3.38 (ddd, 1H, J4,5 = 9.7 Hz,
J5,6a = 5.0 Hz, J5,6b = 2.3 Hz, H-5), 3.42 (dd, 1H, J3,4 = 8.6 Hz, H-4), 3.54 (dd, 1H,
J2,3 = 9.8 Hz, H-3), 3.68 (dd, 1H, J6a,6b = 12.0 Hz, H-6a), 3.82 (dd, 1H, H-6b), 4.05
(dd, 1H, J1,2 = 11.0 Hz, H-2), and 5.72 (d, 1H, H-1). 13C NMR(CD3OD) d 22.9
(COCH3), 25.1, 26.9, 52.0, 56.3 (methylene carbons of pyrrolidine group), 54.5 (C-
2), 62.6 (C-6), 71.6 (C-4), 77.6 (C-3), 82.3 (C-5), 89.4 (C-1), 173.6 (C@O), and 195.4
(C@S). HR-FAB-MS: m/z 351.1046 [M+H]+ (calcd for C13H23O5N2S2: 351.1049). 2-
Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-glucopyranosyl pyrrolidine-1-car-
bodithio ate (3): ½a 2D5 +50.8° (c 1.06, CHCl3), IR (KBr) cmꢁ1: 3283 (NH), 1747
ꢀ
(C@O), 1667 (amide I), 1544 (amide II). 1H NMR(CDCl3) d: 1.92 (s, 3H, NCOCH3),
1.99, 2.05, 3.60, 3.73, 3.90 (m, 8H, methylene protons of pyrrolidine group), 2.05
(ꢂ2), 2.08 (s, 9H, COCH3ꢂ3), 3.86 (ddd, 1H, J4,5 = 9.8 Hz, J5,6a = 2.1 Hz, J5,6b
=
4.9 Hz, H-5), 4.13 (dd, 1H, J6a,6b = 12.5 Hz, H-6a), 4.25 (dd, 1H, H-6b), 4.55 (ddd,
1H, J1,2 = 11.0 Hz, J2,NH = J2,3 = 9.8 Hz, H-2), 5.16 (dd, 1H, J3,4 = 9.5 Hz, H-4), 5.21
(dd, 1H, H-3), 5.86 (d, 1H, H-1), and 6.25 (d, 1H, NH). 13CNMR(CDCl3) d 20.7 (ꢂ2),
20.8 (OCOCH3ꢂ3), 23.2 (NCOCH3), 24.2, 26.0, 51.1, 55.4 (methylene carbons of
pyrrolidine group), 52.0 (C-2), 62.0 (C-6), 68.0 (C-4), 74.6 (C-3), 76.5 (C-5), 88.0
(C-1), 169.3, 170.2, 170.8, 171.2 (NCOCH3, OCOCH3ꢂ3), and 189.2 (C@S).
16. Acetamido group of OCT313 was chemically modified to some other types of
functional groups namely propionamido (R2), butyramido (R3), and benzamido
(R4), oleamido (R5) (Table 6). Synthetic method was as follows. After acyl
chlorides or anhydride were reacted to literature known 1,3,4,6-tetra-O-acetyl-
Scheme 1. Synthesis of 2-acetamido-2-deoxy-2-deoxy-b-
idine-1-carbodithioate (OCT313HK, Glc-NAc-PDTC) (4).
D-glucopyranosyl pyrrol-
Acknowledgments
We thank Dr. Masami Mori (Kinjo Gakuin University, Japan) for
his valuable comments on this work. This work was supported in
part by Grant-in-Aid for Scientific Research on (C) from Japan Soci-
ety for the Promotion of Sciences, a Grant for Research on Publicly
Essential Drugs and Medical Devices, No. KHC1016, from the Japan
Health Sciences Foundation, a Grant-in-Aid for Scientific Research
on the U.S.–Japan Cooperative Medical Sciences Program, Ministry
of Health, Labour and Welfare, Japan, and Fugaku Foundation.
2-amino-2-deoxy-b-D
-glucopyranose hydrochloride12 resulting N-acyl-b-
acetates were chlorinated with HCl gas in acetic acid and anhydride, and
reacted with sodium N,N-dimethyldithiocarbamate in acetone. After de-O-
acetylation some N-acyl derivatives of OCT313 were obtained. 2-Deoxy-2-
propionamido-b-
D
-glucopyranosyl N,N-dimethyldithiocarbamate (R2): a D22
½ ꢀ
+53.9° (c 1.11, MeOH), IR (KBr) cmꢁ1: 3510–3100 (br OH, NH), 1641 (amide
I), 1571 (amide II). 1H NMR (CD3OD + D2O, 3:4, v/v) d: 1.11 (t, 3H, J = 7.6 Hz,
CH2CH3), 2.24 (m, 2H, CH2CH3), 3.38, 3.53 (s, 6H, NCH3ꢂ2), 3.49 (m, 1H, H-4),
3.50 (m, 1H, H-5), 3.64 (dd, 1H, J2,3 = 9.9 Hz, J3,4 = 8.7 Hz, H-3), 3.73 (dd, 1H,
J5,6a = 0.6 Hz, J6a,6b = 12.4 Hz, H-6a), 3.86 (dd, 1H, J5,6b = 4.5 Hz, H-6b), 4.08 (dd,
1H, J1,2 = 11.0 Hz, H-2), and 5.70 (d, 1H, H-1). 13C NMR (CD3OD + D2O, 3:4, v/v) d
10.4 (CH2CH3), 30.3 (CH2CH3), 42.5, 46.1 (NCH3ꢂ2), 54.0 (C-2), 61.9 (C-6), 70.8
(C-4), 76.5 (C-3), 81.7 (C-5), 90.0 (C-1), 178.5 (C@O), and 194.7 (C@S). HR-FAB-
MS: m/z 339.1053 [M+H]+ (calcd for C12H23O5N2S2:339.1049). 2-Butyramido-2-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.084.
deoxy-b-D
-glucopyranosyl N,N-dimethyldithiocarbamate (R3): mp 171–172 °C
(decomp.), ½a 2D2 +49.6° (c 1.40, MeOH), IR(KBr) cmꢁ1: 3500–3150 (br OH, NH),
ꢀ
1643 (amide I), 1535 (amide II). 1H NMR (CD3OD + D2O, 2.5:1, v/v) d 0.93 (t, 3H,
J = 7.4 Hz, CH2CH3), 1.62 (m, 2H, CH2CH3), 2.21 (m, 2H, NCOCH2), 3.38, 3.53 (s,
6H, NCH3ꢂ2), 3.46 (m, 1H, H-5), 3.48 (m, 1H, H-4), 3.62 (dd, 1H, J2,3 = 9.2 Hz,
J3,4 = 8.5 Hz, H-3), 3.73 (dd, 1H, J5,6a = 4.6 Hz, J6a,6b = 12.4 Hz, H-6a), 3.86 (dd, 1H,
References and notes
1. WHO Web site, <http://www.who.org/>.
2. Gandhi, N. R.; Nunn, P.; Dheda, K.; Schaaf, H. S.; Zignol, M.; Van Soolingen, D.;
Jensen, P.; Bayona, J. Lancet 2010, 375, 1830.
3. Horita, Y.; Takii, T.; Chiba, T.; Kuroishi, R.; Maeda, Y.; Kurono, Y.; Inagaki, E.;
Nishimura, K.; Yamamoto, Y.; Abe, C.; Mori, M.; Onozaki, K. Bioorg. Med. Chem.
Lett. 2009, 19, 6313.
4. Byrne, S. T.; Gu, P.; Zhou, J.; Denkin, S. M.; Chong, C.; Sullivan, D.; Liu, J. O.;
Zhang, Y. Antimicrob. Agents Chemother. 2007, 51, 4495.
J5,6b = 1.8 Hz, H-6b), 4.10 (dd, 1H, J1,2 = 11.0 Hz, H-2), and 5.70 (d, 1H, H-1). 13
C
NMR (CD3OD + D2O, 2.5:1, v/v) d 13.9 (CH2CH3), 20.1 (CH2CH3), 39.0 (NCOCH2),
42.3, 45.9 (NCH3ꢂ2), 54.2 (C-2), 62.2 (C-6), 71.1 (C-4), 76.9 (C-3), 82.0 (C-5),
90.2 (C-1), 172.1 (C@O), and 195.1 (C@S). HR-FAB-MS: m/z 353.1209 [M+H]+
(calcd for C13H25O5N2S2: 353.1205). 2-Benzamido-2-deoxy-b-
D
-glucopyranosyl
N,N-dimethyldithiocarbamate (R4): ½a D22
ꢀ
+85.1° (c 1.31, MeOH), IR(KBr) cmꢁ1
:
3500–3200 (br OH, NH), 1641 (amide I), 1534 (amide II). 1H NMR (D2O) d 3.25,
3.40 (s, 6H, NCH3ꢂ2), 3.63 (dd, 1H, J3,4 = 8.8 Hz, J4,5 = 9.9 Hz, H-4), 3.69 (ddd, 1H,
J5,6a = 5.1 Hz, J5,6b = 2.1 Hz, H-5), 3.82 (dd, 1H, J6a,6b = 12.5 Hz, H-6a), 3.93 (dd,
1H, J2,3 = 9.9 Hz, H-3), 3.95 (dd, 1H, H-6b), 4.39 (dd, 1H, J1,2 = 10.9 Hz, H-2), 5.96
(d, 1H, H-1), and 7.40–7.90 (m, 5H, aromatic protons). 13C NMR (D2O) d 44.8,
48.3 (NCH3ꢂ2), 56.8 (C-2), 63.6 (C-6), 72.5 (C-4), 78.0 (C-3), 83.4 (C-5), 91.8 (C-
1), 130.0, 131.7, 135.3, 136.1 (aromatic carbons), 174.0 (C@O), and 195.7 (C@S).
HR-FAB-MS: m/z 387.1041 [M+H]+ (calcd for C16H23O5N2S2:387.1049). 2-
5. Makarov, V.; Riabova, O. B.; Yuschenko, A.; Urlyapova, N.; Daudova, A.; Zipfel, P.
F.; Möllmann, U. J. Antimicrob. Chemother. 2006, 57, 1134.
6. Güzel, O.; Salman, A. Bioorg. Med. Chem. 2006, 14, 7804.
7. Agar, N. S.; Mahoney, J. R., Jr.; Eaton, J. W. Biochem. Pharmacol. 1991, 41, 985.
8. Baulard, A. R.; Betts, J. C.; Engohang-Ndong, J.; Quan, S.; McAdam, R. A.;
Brennan, P. J.; Locht, C.; Besra, G. S. J. Biol. Chem. 2000, 275, 28326.
9. Boonaiam, S.; Chaiprasert, A.; Prammananan, T.; Leechawengwongs, M. Clin.
Microbiol. Infect. 2010, 16, 396.
10. Dover, L. G.; Alahari, A.; Gratraud, P.; Gomes, J. M.; Bhowruth, V.; Reynolds, R.
C.; Besra, G. S.; Kremer, L. Antimicrob. Agents Chemother. 2007, 51, 1055.
11. Conchie, J.; Levvy, G. A. Methods Carbohydr. Chem. 1963, 2, 332.
12. Bergmann, M.; Zervas, L. Ber. Dfsch. Chem. Ges. 1931, 64, 975.
13. Zervas, L.; Konstas, S. Chem. Ber. 1960, 92, 435.
Deoxy-2-oleamido-b-
D
-glucopyranosyl N,N-dimethyldithiocarbamate (R5):
½ ꢀ
a 2D2
+40.9° (c 1.33, MeOH), IR (KBr) cmꢁ1: 3500–3200 (br OH, NH), 2924,
2853 (CH2), 1643 (amide I), 1536 (amide II). 1H NMR (CD3OD) d 0.90 (t, 3H,
J = 6.8 Hz, CH2CH3), 1.23–2.25, 3.90 (m, 30H, methine and methylene protons of
oleoyl group), 3.35, 3.50 (s, 6H, NCH3ꢂ2), 3.38 (ddd, 1H, J4,5 = 9.8 Hz,
J5,6a = 5.1 Hz, J5,6b = 2.1 Hz, H-5), 3.43 (dd, 1H, J3,4 = 8.6 Hz, H-4), 3.55 (dd, 1H,
J2,3 = 9.8 Hz, H-3), 3.68 (dd, 1H, J6a,6b = 12.0 Hz, H-6a), 3.82 (dd, 1H, H-6b), 4.09
(dd, 1H, J1,2 = 11.0 Hz, H-2), and 5.67 (d, 1H, H-1). 13C NMR (CD3OD) d 14.5
(CH2CH3), 23.7, 26.9, 27.5, 27.6, 30.1, 30.2, 30.3, 30.4, 30.6 (ꢂ2), 30.7, 33.0 (ꢂ2),
37.2, 39.6, 65.1 (methine and methylene carbons of oleoyl group), 41.9, 45.9
(NCH3ꢂ2), 54.4 (C-2), 62.6 (C-6), 71.6 (C-4), 77.5 (C-3), 82.3 (C-5), 90.6 (C-1),
176.5 (C@O), and 195.4 (C@S). MS: m/z 569 [M+Na]+.
14. General procedures: Melting points were determined with
MP-S2 micro melting point apparatus and uncorrected. Solutions were
concentrated in rotary evaporator below 50 °C under vacuum. Optical
a Yamagimoto
a
rotations were measured with a JASCO P-1020 automatic digital polarimeter in
a 0.1 dm tube. IR spectra were recorded with a JASCO FT/IR-4100 Spectrometer.
1H NMR spectra were recorded at 500 MHz with a JNM-
a
500 spectrometer and
JNM-ECA500/KJ, at 600 MHz with a BRUKER-AV600. 13C NMR spectra were
recorded at 125 MHz with a JNM- 500 spectrometer. Tetramethylsilane was
used as an internal standard. Chemical shift are given on the d scale. TLC was
performed on precoated silica gel plates 0.25 mm thick (Kieselgel 60F254
17. 2-Amino derivative of OCT313 was synthesized by de-O-acetylation of 3,4,6-tri-
O-acetyl-2-amino-2-deoxy-b-D-glucopyranosyl N,N-dimethyldithiocarbamate
a
hydrochloride with an anion exchange resin DOWEX 1-X4 (OHꢁ) in methanol
in 60.2% yields, which was obtained by the reaction of 2-N-anisilidene-3,4,6-tri-
,
O-acetyl-a-D
-glucopyranosyl bromide13 and sodium N,N-dimethyldithiocarba-
Merck). Detection was effected with H2SO4 or by UV irradiation at 254 nm.
Column chromatography was performed on Silica Gel BW-820MH (Fuji-Silysia
Chemical Ltd, Nagoya, Japan).
mate in acetone, followed by deanisilidenation with 5 M hydrochloric acid.
2-Amido-2-deoxy-b- -glucopyranosyl N,N-dimethyldithiocarbamate (R6): mp
161–162 (decomp.), ½DaꢀD23 ꢁ77.4° (c 1.04, H2O), IR(KBr) cmꢁ1: 3500–3100 (br OH,
NH). 1H NMR (D2O) d 3.02 (dd, 1H, J1,2 = 10.7 Hz, J2,3 = 9.0 Hz, H-2), 3.44, 3.55 (s,
6H, NCH3ꢂ2), 3.47 (dd, 1H, J3,4 = 9.2 Hz, J4,5 = 9.5 Hz, H-4), 3.50 (dd, 1H, H-3), 3.59
(ddd, 1H, J5,6a = 5.3 Hz, J5,6b = 2.2 Hz, H-5), 3.74 (dd, 1H, J6a,6b = 12.5 Hz, H-6a),
3.89 (dd, 1H, H-6b), and 5.62 (d, 1H, H-1). 13C NMR (D2O) d 44.9, 48.4 (NCH3ꢂ2),
57.3 (C-2), 63.5 (C-6), 72.2 (C-4), 80.4 (C-3), 83.3 (C-5), 93.2 (C-1), and 195.7
(C@S). HR-FAB-MS: m/z 283.0776 [M+H]+ (calcd for C9H19O4N2S2:
283.0786).
15. 2-Acetamido-2-deoxy-b-
(Scheme 1) was synthesized as follows. Reaction of literature known
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
-glucopyranosyl chloride11 and
D-glucopyranosyl pyrrolidine-1-carbodithioate (4)
a-D
ammonium 1-pyrrolidinecarbodithioate in acetone gave thioglycoside perace-
tate in 51.4% yields. In its 1H NMR spectrum one proton doublet of H-1 appeared
at d 5.72 (J1,2 = 11.0 Hz), indicative of b-configuration. De-O-acetylation of
thioglycoside peracetate with 0.5 M sodium methoxide-methanol gave
compound (2-acetamido-2-deoxy-b-D-glucopyranosyl pyrrolidine-1-carbodi-