Synthesis of new sugar derivatives and evaluation of their antibacterial activities against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmcl.2009.09.095

A series of sugar derivatives (1-13) were synthesized and evaluated for antibacterial activity against Mycobacterium tuberculosis (MTB), especially multi-drug resistant (MDR) MTB, and the structure-activity relationships of these compounds were studied. The results showed that the compound OCT313 (2-acetamido-2deoxy-β-d-glucopyranosyl N,N-dimethyldithiocarbamate) (4) exhibited significant in vitro bactericidal activity, and that the dithiocarbamate group at C-1 position of the glucopyranoside ring was requisite for the antibacterial activity.

Full text of DOI:10.1016/j.bmcl.2009.09.095

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6313–6316
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of new sugar derivatives and evaluation of their antibacterial
activities against Mycobacterium tuberculosis
b
a
c
c
Yasuhiro Horita a, Takemasa Takii a, , Taku Chiba , Ryuji Kuroishi , Yasuhiro Maeda , Yukihisa Kurono ,
*
Emi Inagaki a, Kenji Nishimura a, Yoshifumi Yamamoto a, Chiyoji Abe d, Masami Mori b, Kikuo Onozaki a
a Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan
b College of Pharmacy, Kinjo Gakuin University, Nagoya, Japan
c Department of Hospital Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan
d Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Kiyose, Tokyo, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 June 2009
Revised 4 September 2009
Accepted 24 September 2009
Available online 27 September 2009
A series of sugar derivatives (1–13) were synthesized and evaluated for antibacterial activity against
Mycobacterium tuberculosis (MTB), especially multi-drug resistant (MDR) MTB, and the structure–activity
relationships of these compounds were studied. The results showed that the compound OCT313 (2-acet-
amido-2deoxy-b-D-glucopyranosyl N,N-dimethyldithiocarbamate) (4) exhibited significant in vitro bacte-
ricidal activity, and that the dithiocarbamate group at C-1 position of the glucopyranoside ring was
requisite for the antibacterial activity.
Keywords:
Carbohydrate
Tuberculosis
Ó 2009 Elsevier Ltd. All rights reserved.
Antibacterial activity
Tuberculosis (TB) has become an important worldwide prob-
lem: about two million people die each year, particularly in devel-
oping countries. It is estimated that about one-third of the world
population is currently infected with the bacillus in its latent form
and that nearly nine million new cases develop each year.1 Accord-
ing to WHO, multi-drug resistant tuberculosis is responsible for
approximately 460 thousand new cases per year and for about
740 thousand new patients infected by both Mycobacterium tuber-
culosis and HIV/AIDS. Recent estimates show that 10% of all new TB
infections are resistant to at least one anti-TB drug.2 To treat an
infection, a cocktail of drugs including, for example, isoniazid,
rifampin, ethambutol and pyrazinamide are prescribed for two
months followed by a continuation phase in which isoniazid and
rifampin are taken. Long-term therapies lasting for between six
and nine months have frequently led to patients’ non-compliance
and, in turn, contributed to the emergence of multi-drug resistant
TB (MDR-TB).3 The ever-increasing drug resistance, toxicity, and
side effects of currently used anti-tuberculosis drugs, and the dis-
appearance of their bactericidal activity necessitate new, safer, and
more effective antimycobacterial compounds. In the last 10 years
the research on M. tuberculosis and possible drug candidates have
made much progress with the genome unrevealed and the discov-
ery of different biological targets.4,5
Over 200 sugar derivatives were investigated for antibacterial
activity by the broth dilution method. Two candidates were
obtained after this random screening. One of them, OCT359 (allyl
O-(2,3,4,6-tetra-O-acetyl-
tri-O-acetyl- -galactopyranosyl)-(1?6)-O-2,3,4-tri-O-acetyl-
b-
-glucopyranoside), has been reported previously.6 In this study
another compound, OCT313, 2-acetamido-2deoxy-b- -glucopyran-
a-D-galactopyranosyl)-(1?6)-O-(2,3,4-
a
-D
D
D
osyl N,N-dimethyldithiocarbamate (4), was investigated for struc-
ture–activity relationships and antibacterial activities against
MTB, including multi-drug resistant (MDR) MTB. OCT313 (4) con-
sists N-acetyl-D-glucosamine and dimethyldithiocarbamate. This
compound was prepared from acetylated glucosamine (1),
followed by chlorination,7 substitution of the dimethyldithiocarba-
mate group and de-O-acetylation (Scheme 1).8–10 OCT313 (4) was
white crystals, mp 184–185 °C, and obtained in a 22.5% yield from
(1). In its NMR spectrum one proton doublet of H-1 appeared at d
5.67 (J1,2 = 11.0 Hz), indicative of b-configuration. The N-acetyl
group appeared at d 1.95 as a three proton singlet. Dimethyl signals
of the dimethyldithiocarbonate group appeared at d 3.37, 3.51 as
each 3H singlet.
The isomers of OCT313 (4) were synthesized (Scheme 2). The
general methods of synthesis of 2-acetamido-2-deoxy-
pyranosyl N,N-dimethyldithiocarbamate (5) and 2-acetamido-2-
deoxy-b- -galactopyranosyl N,N-dimethyldithiocarbamate (6) are
as follows. Instead of N-acetyl- -glucosamine, N-acetyl- -manno-
samine or N-acetyl- -galactosamine was used as a starting mate-
rial. The reaction steps were the same as the synthesis of OCT313
a-D-manno-
D
D
D
D
* 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 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.095

6314
Y. Horita et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6313–6316
AcOH, Ac2O
HCl gas bubbling, 4 hr
CH2OAc
CH2OAc
O
O
AcO
AcO
AcO
AcO
OAc
NHAc
NHAc
Cl
2
1
DMDTCBNa,Me2CO
100 o ,reflux,15min,
C
0.5 M MeONa-MeOH
r. t., stirring, overnight
CH2OAc
CH2OH
S
S
O
O
AcO
AcO
HO
HO
CH3
CH3
CH3
CH3
S-C-N
S-C-N
NHAc
NHAc
3
4
Scheme 1. Synthesis of 2-acetamido-2-deoxy-b-
D-glucopyranosyl N,N-dimethyldithiocarbamate (OCT313) (4).
HO
CH2OH
general method of synthesis of 2-acetamido-6-N,N-dimethyldithio-
CH2OH
AcHN
S
O
O
HO
HO
carbamyl-2,6-dideoxy-b-
bamate (13) is as follows. N-Acetyl-
D
-glucopyranosyl N,N-dimethyldithiocar-
-glucosamine (7) was
CH3
HO
S-C-N
S
CH3
D
NHAc
6
CH3
CH3
S-C-N
selectively tosylated, and then acetylated to give the tosylated per-
acetate (8). Treatment of the tosylated peracetate with sodium
iodite in boiling acetone caused replacement of the sulfonyloxy
group by iodine and crystalline 2-acetamido-1,3,4-tri-O-acetyl-
5
Scheme 2. Isomer of OCT313 2-acetamido-2-deoxy-
N,N-dimethyldithiocarbamate (5) and 2-acetamido-2-deoxy-b-
a-
D
-mannopyranosyl
D-galactopyranosyl
2,6-dideoxy-6-iodo-a-D-glucopyranose (9) was obtained in a yield
N,N-dimethyldithiocarbamate (6).
of 87.8%. This indicated that the sulfonyl group in the tosylated per-
acetate was located on the primary alcohol group. A mixture of (9)
and sodium dimethyldithiocabamate in acetone was refluxed for
15 min. After work-up, 6-dimethyldithiocabamate (10) was ob-
tained as an amorphous powder in a 92.3% yield. Compound 10
was similarly treated as described for the preparation of 4 to afford
13 in a 30.9% yield from 9.
(4). Their yields were 53.6% and 29.7% from their peracetates,
respectively.
Two dimethyldithiocarbamate groups were substituted at C-1
and C-6 position of N-acetyl-
D
-glucosamine (13) (Scheme 3). The
The antibacterial activity of compounds 1–13 was investigated
by using M. tuberculosis H37Rv, Mycobacterium bovis BCG (Tokyo-
172), Mycobacterium avium 724S, M. avium SmO, Mycobacterium
smegmatis, Staphylococcus aureus, and Escherichia coli as target bac-
teria (Table 1). OCT313 (4) was specifically effective to slow the
grows of Mycobacterium species, such as M. tuberculosis and M. bovis
BCG (Table 1). The character of a narrow spectrum of anti-bacterial
activity is appropriate to anti-tuberculosis drugs, because the ther-
apeutic term should be at least six months. The MIC of OCT313 (4)
CH2OH
O
HO
HO
OH
Ac2O, AcOH,
HCl gas
NHAc
7
S
CH3
CH2 S-C-N
i) p-TsCl, pyridine
ii) Ac2O
CH3
O
AcO
AcO
NHAc
Cl
11
CH2OTs
O
to M. tuberculosis and M. bovis BCG was between 25 and 32
(Table 1). The MIC of C-4 isomer of OCT313 (4), 2-acetamido-2-
deoxy-b- -galactopyranosyl N,N-dimethyldithiocarbamate (6) was
2 times lower than that of OCT313 (4). The MIC of 2-acetamido-
6-N,N-dimethyldithiocarbamyl-2,6-dideoxy-b- -glucopyranosyl
lg/ml
AcO
AcO
Me2NC(=S)SNa,
acetone, refrex,
15 min
OAc
NHAc
D
8
S
D
CH3
CH3
NaI, acetone
reflux
CH2 S-C-N
O
N,N-dimethyldithiocarbamate (13), a derivative of OCT313 with two
dimethyldithiocarbamate groups at C-1 and C-6 position was the
same as that of OCT 313 (4). The acetylated compounds, 3, lost the
antibacterial activity. A finding worthy of note was that sodium dim-
ethyldithiocarbamate (DMSTCA.SS) exhibited strong anti-bacterial
activity (Table 1).
The dimethyldithiocarbamate group at C-1 position of OCT313
(4) was responsible for the bactericidal effect. Sodium dimethyl-
dithiocarbamate exhibited toxicity to human cell lines; however,
the sugar bound to the dimethyldithiocarbamate compound,
OCT313, reduced the cytotoxicity (Supplementary Fig. 1 and Table
1). The ratio of the toxic to effective dose of OCT 313 was from 28
to 242 in OCT313, however that of the sodium dimethyldithiocar-
bamate was from 14 to 318 (Supplementary Table 1). Degradation
of OCT 313 (4) in the assay medium of anti-bacterial activity was
not observed during assay period (data not shown).
AcO
AcO
CH3
CH3
S-C-N
S
NHAc
CH2I
12
O
AcO
AcO
NHAc
0.5 M MeONa-MeOH
OAc
9
S
CH3
Me2NC(=S)SNa,
CH2 S-C-N
O
CH3
acetone, reflex, 1 h
HO
HO
CH3
CH3
S-C-N
NHAc
13
S
S
CH3
CH3
CH2 S-C-N
O
AcO
AcO
NHAc
OAc
10
In conclusion, the dimethyldithiocarbamate group at C-1 of
N-acetyl-D-glucosamine is critical for antibacterial activity (Table 1).
Scheme 3. Synthesis of 2-acetamido-6-N,N-dimethyldithiocarbamyl-2,6-dideoxy-
b- -glucopyranosyl N,N-dimethyldithiocarbamate (13).
D

Y. Horita et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6313–6316
6315
Table 1
Antibacterial activities of glucosamine derivatives (MIC,
l
g/ml)a
Organisms
M. avium 724S
M. tuberculosis H37Rv
M. bovis BCG (Tokyo)
M. avium SmO
M. smegmatis
S. aureus
E. coli
Compounds
1 Glc-N-Ac free
>100
>100
25
>100
12.5
25
>100
>100
31.3
>100
25
ne
ne
>100
>100
>100
>100
>100
>100
ne
ne
3 OCT313-peracetate
4 OCT313 (Glc-N-Ac DMDTCB)
5 Man-N-Ac DMDTCB
6 Gal-N-Ac DMDTCB
13 Glc-N-Ac 1,6 DMDTCB
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Raw materials
DMDTCA.SS
Dimethylamine
Dimethylamine HCl
0.78
>100
>100
0.78
>100
>100
ne
ne
ne
ne
ne
ne
>100
>100
>100
>100
ne
ne
>100
ne
ne
anti-MTB Antibiotics
INH
RFP
SM
0.04
0.004
0.39
2.5
0.16
1.75
1.56
3.13
0.04
0.004
0.20
1.5
50
0.78
0.3
50
3.13
0.004
0.39
1.56
100
1.56
1.56
1.56
6.25
1.56
0.39
12.5
1000
1.25
3.13
1.56
>100
0.002
50
>100
>100
100
>100
50
50
>100
>100
50
12.5
6.25
<0.05
0.39
3.13
100
>100
>100
25
EB
PAS
AMK
KM
GM
12.5
25
1.56
a
Broth dilution methods using MiddleBrook 7H9 broth containing albumin, dextrose, and catalase for derivatives (ne, not examined). For Staphylococcus aureus, we used
the heat-infusion broth. INH, isoniazid; RFP, rifampicin; SM, streptomycin; EB, ethambutol; AMK, amikacin; KM, kanamycin; GM, gentamycin.
Table 2
Antimycobacterial activity of OCT313 on drug-sensitive and resistant clinical isolates
of M. tuberculosis
Compounds having dimethyldithiocarbamate groups have been
used for pesticides and their toxicity for humans was due to inhi-
bition of choline esterase. However, the inhibitory effect of OCT313
on choline esterase was undetectable (Supplementary Fig. 2). The
precise mechanism of the anti-tuberculosis effect of dim-
ethyldithiocarbamate is unknown; however, OCT313 (4) exhibits
bactericidal and lytic activities against M. tuberculosis and M. bovis
BCG (Supplementary Fig. 3), strongly suggesting that OCT313 (4)
exerts antibacterial activity by the mechanism distinct from that
of dimethyldithiocarbamate. Furthermore, 25 clinical isolates of
drug-resistant MTB and 19 drug-sensitive MTB were sensitive to
OCT313 (4) (Table 2). The MICs of OCT313 (4) to these clinical iso-
Clinical isolates
Resistance to
MIC for OCT313 (lg/ml)
Drug-susceptive strains
A-1-1
A-1-2
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
6.25
12.5
6.25
A-3-5
A-3-11
A-3-12
A-3-13
A-3-15
A-3-16
A-3-20
A-3-21
A-3-22
A-3-9
A-3-22
A-1-3
A-2-5
A-3-1
A-3-2
lates were from 1.56 to 12.5 lg/ml. Cross-resistance of OCT313 (4)
to currently used anti-TB drugs was not observed (Table 2). These
results strongly indicate that OCT359 possesses novel drug targets
and may be a useful lead compound for MDB, especially MDR-MTB.
Acknowledgments
A-3-6
A-3-17
A-3-19
This work was supported in part by Grant-in-Aid for Scientific
Research on (C) from Japan Society for the Promotion of Sciences,
a grant for Research on Publicly Essential Drugs and Medical De-
vices, No. KHC1021, from the Japan Health Sciences Foundation,
and a Grant-in-Aid for Scientific Research on the U.S.-Japan Coop-
erative Medical Sciences Program, Ministry of Health, Labour and
Welfare, Japan.
Drug-resistant strains
A-3-47
A-4-8
A-2-1
A-2-3
SM
SM
INH
INH, RFP
6.25
6.25
6.25
3.12
6.25
6.25
6.25
6.25
1.56
6.25
6.25
6.25
6.25
3.12
6.25
6.25
6.25
6.25
6.25
J-1-19
K-3-6
INH, RFP
INH, SM, EB
INH, RFP, SM, EB
INH, RFP, EB
INH, RFP, SM, EB
INH, RFP, SM, EB
INH, RFP, SM, EB
INH, RFP, SM, EB
INH, RFP, SM, EB
INH, RFP, SM, EB
INH, RFP, SM, EB
INH, RFP, SM, EB
RFP
M-1-32
N-4-11
N-5-2
P-1-50
P-4-11
Q-4-1
R-1-38
U-2-15
U-4-6
Z-1-4
A-2-4
A-2-6
A-4-25
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.09.095.
References and notes
1. Zhang, Y. Annu. Rev. Pharmacol. Toxicol. 2005, 45, 529.
2. WHO Web site, http://www.who.org/.
3. Bentrup, K. H.; Russell, D. G. Trends Microbiol. 2001, 9, 597.
4. Camus, J. C.; Pryor, M. J.; Medigue, C.; Cole, S. T. Microbiology 2002, 148, 2967.
5. Nayyar, A.; Jain, R. Curr. Med. Chem. 2005, 12, 1873.
INH, RFP, SM, EB
INH, SM
Proportion methods using Middlebrook 7H11 agar plates for INH, RFP, SM, EB and
7H9 broth for OCT313. Cut off concentrations of each antibiotic were 10, 10, 100
and 100
lg/ml, respectively.
6. Chiba, T.; Takii, T.; Nishimura, K.; Yamamoto, Y.; Morikawa, H.; Abe, C.;
Onozaki, K. Bioorg. Med. Chem. Lett. 2007, 17, 2487.

6316
Y. Horita et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6313–6316
7. Conchie, J.; Levvy, G. A. Methods Carbohydr. Chem. 1963, 2, 332.
8. General procedures—Melting points were determined with a Yamagimoto MP-
S2 micro melting point apparatus and uncorrected. Solutions were
of the solvent gave (1) (12.4 g, 37.6 %) as an amorphous powder, ½a D20
ꢀ
+49.6 (c
1.36, CHCl3), IR (KBr) cmꢁ1: 3282 (NH), 1749 (C@O), 1666 (amide I), 1547 (amide
II). 1H NMR (CDCl3) d : 1.92,2.05 (ꢂ2), 2.08 (s, 12H, Ac ꢂ 4), 3.37, 3.54 (each s, 6H,
NCH3 ꢂ 2), 3.85 (m, 1H, H-5), 4.13 (dd, 1H, J5,6a = 2.1 Hz, J6a,6b = 12.5 Hz, H-6a),
4.25 (dd, 1H, J5,6b = 4.6 Hz,,H-6b), 4.57 (ddd, 1H, J1,2 = 11.0 Hz, J2,NH = 9.8 Hz,
J2,3 = 9.8 Hz, H-2), 5.16 (dd, 1H, J3,4 = 9.2 Hz, J4,5 = 9.8 Hz, H-4),5.20 (dd, 1H, H-3),
5.79 (d, 1H, H-1), and 6.13 (d, 1H, NH). 13C NMR (CDCl3) d: 20.8, 20.9, 21.0, 23.3
(COCH3 ꢂ 4), 42.1, 45.8 (NCH3 ꢂ 2), 52.2 (C-2), 62.1 (C-6), 68.1 (C-4), 74.8 (C-3),
76.7 (C-5), 89.3 (C-1), 169.5, 170.3, 170.9, 171.4 (COCH3 ꢂ 4), and 193.7 (C@S).
concentrated in
a rotary evaporator below 50 °C under vacuum. Optical
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
a
10. 2-Acetamido-2-deoxy-b-D-glucopyranosyl N,N-dimethyldithiocarbamate [(4),
,
OCT313]—A 0.5 M methanolic MeONa (1 ml) was added to a suspension of
(3) (9.3 g, 20.6 mmol) in dry MeOH (93 ml), and the mixture was stirred at
room temperature for 2 h under exclusion of moisture. After neutralization
with Amberlite IR-120B(H+) resin and removal of the resin by filtration, the
filtrate was evaporated to syrup which was crystallized from EtOH.
Recrystallization from EtOH gave pure (4) (4.0 g, 59.7 %) as white crystals,
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).
9. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-
D-glucopyranosyl N,N-dimethyldithioc-
arbamate (3)—To solution of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-
a
glucopyranosyl chloride7 (26.77 g, 73.2 mmol) in dry acetone (200 ml) was
added sodium N,N-dimethyldithiocarbamate dihydrate (21 g, 146.6 mmol) and
the mixture was refluxed for 15 min. After checked the disappearance of the
starting materials by TLC (CHCl3/acetone 6:1, v/v), the reaction mixture was
evaporated to afford a syrup which was dissolved in water and CHCl3. The
organic layer was separated, washed with H2O, dried (MgSO4), and evaporated to
afford a syrup. The syrup was dissolved in a small amount of CHCl3 and
chromatographed on a column with CHCl3/acetone (10:1–3:1, v/v). Evaporation
mp 184–185°, ½a 2D4
ꢀ
+50.7 (c 1.16, H2O), IR(KBr) cmꢁ1: 3600–3100 (br OH), 1656
(amide I), 1562 (amide II). 1H NMR (CD3OD) d: 1.95 (s, 3H, NCOCH3), 3.37, 3.51
(each s, 6H, NCH3 ꢂ 2), 3.39 (m, 1H, H-5), 3.44 (dd, 1H, J3,4 = 8.6 Hz, J4,5 = 9.5 Hz,
H-4), 3.56 (dd, 1H, J2,3 = 9.8 Hz, H-3), 3.69 (dd, 1H, J5,6a = 2.1 Hz, J6a,6b = 12.2 Hz,
H-6a), 3.83 (dd, 1H, J5,6b = 4.9 Hz, H-6b), 4.07 (dd, 1H, J1,2 = 11.0 Hz,H-2), and
5.67 (d, 1H, H-1). 13C NMR (CD3OD) d: 22.9 (COCH3), 42.0, 45.7 (NCH3 ꢂ 2), 54.6
(C-2), 62.6 (C-6), 71.6 (C-4), 77.6 (C-3), 82.3 (C-5), 90.4 (C-1), 173.7 (C@O), and
195.4 (C@S).