Synthesis and antitubercular activity of novel Schiff bases derived from d-mannitol

Article abstract of DOI:10.1016/j.carres.2009.08.006

Six Schiff base derivatives of d-mannitol, 1,6-dideoxy-1,6-bis-{[(E)-arylmethylidene]amino}-d-mannitol (6: aryl = XC₆H₄: X = o-, m- and p- Cl or NO₂), have been synthesized and evaluated for their in vitro antibacterial ac

Full text of DOI:10.1016/j.carres.2009.08.006

Carbohydrate Research 344 (2009) 2042–2047
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and antitubercular activity of novel Schiff bases derived
from D-mannitol
Marcelle de L. Ferreira a,b, Thatyana R. A. Vasconcelos b, Erika M. de Carvalho a, Maria C. S. Lourenço c,
Solange M. S. V. Wardell a, James L. Wardell d,e, Vitor F. Ferreira b, Marcus V. N. de Souza a,
*
a Fundação Oswaldo Cruz, Instituto de Tecnologia em Fármacos-Far Manguinhos, Fundação Oswaldo Cruz, 21041-250, Rio de Janeiro, RJ, Brazil
b Universidade Federal Fluminense, Instituto de Química, Departamento de Química Orgânica, Outeiro de S. João Batista s/n° Centro 24020-150, Niterói, RJ, Brazil
c Fundação Oswaldo Cruz, Instituto de Pesquisas Clínicas Evandro Chagas, Departamento de Bacteriologia, Av. Brasil, 4365, Manguinhos, Rio de Janeiro, Brazil
d Department of Chemistry, University of Aberdeen, Meston Walk, Old Aberdeen, AB24 3UE Scotland, UK
e Centro de Desenvolvimento Tecnológico em Saúde (CDTS), Fundação Oswaldo Cruz (FIOCRUZ), Casa Amarela, Campus de Manguinhos, Av. Brasil 4365,
21040-900 Rio de Janeiro, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 July 2009
Received in revised form 4 August 2009
Accepted 6 August 2009
Available online 11 August 2009
Six Schiff base derivatives of D-mannitol, 1,6-dideoxy-1,6-bis-{[(E)-arylmethylidene]amino}-D-mannitol
(6: aryl = XC6H4: X = o-, m- and p- Cl or NO2), have been synthesized and evaluated for their in vitro anti-
bacterial activity against Mycobacterium tuberculosis H37Rv using the Alamar Blue susceptibility test and
the activity expressed as the minimum inhibitory concentration (MIC) in
tives exhibit significant activities: activities of (6d: X = o-NO2), (6e: X = m-NO2) and (6f: X = p-NO2) are
12.5, 25.0 and 25.0 g/mL, respectively. When compared with first line drugs, such as ethambutol, they
lg/mL. All three nitro deriva-
l
Keywords:
can be considered as a good starting point to develop new lead compounds for the treatment of multi-
drug-resistant tuberculosis. Characterization of the new compounds 6 is generally achieved spectroscop-
ically. The structure of compound 3 has been confirmed by X-ray crystallography.
Ó 2009 Elsevier Ltd. All rights reserved.
D-Mannitol
Schiff bases
Antitubercular activity
1. Introduction
There are significant reasons for investigating Schiff base deriv-
atives based on carbohydrates, for example, their functional simi-
Amino alcohols are very important and versatile compounds
with significant applications in many fields, such as in synthetic
and medicinal chemistry.1–6 For example, ethambutol is an impor-
tant amino alcohol (Fig. 1) used in the treatment of tuberculosis
(TB). This disease is again a serious problem world-wide, and is
responsible for almost 2 million deaths each year. Factors involved
in the resurgence of TB7 include the AIDS epidemic, which emerged
in the mid-1980s, and the rapid spread of the multidrug-resistant
(MDR) TB strain against all major anti-tuberculosis drugs in the
market.8 Particularly worrisome is the super bacterium XDR-TB
(extensively drug-resistant tuberculosis), which is resistant to all
first and second line anti-TB drugs.9 Thus there is an urgency to
develop new drugs and strategies to fight against tuberculosis or
a tragedy will happen.
larities with amino alcohols and hydrazones, which can exhibit
remarkable anti-TB activity.10–16 Other reasons that can be men-
tioned are their significant lipophilicities, which could facilitate
their entry into the intracellular environment and the carbohydrat-
ic nature of the cell wall of Mycobacterium tuberculosis, which has
led to the investigation of carbohydrate derivatives as potential
anti-TB agents.17–19 Based upon our previous work with other clas-
ses of antitubercular agents, we restricted the substituents used in
6, 1,6-dideoxy-1,6-bis-{[(E)-arylmethylidene]amino}-D-mannitol,
to chloro and nitro groups.20–26
2. Results and discussion
2.1. The synthesis of the key intermediate 1,6-diazido-1,6-
dideoxy- -mannitol (3)
Continuing our search for new antitubercular compounds, we
D
have investigated Schiff base derivatives derived from
(Fig. 1), namely 1,6-dideoxy-1,6-bis-{[(E)-arylmethylidene]amino}-
-mannitol (6: aryl = XC6H4: X = o-, m- and p- Cl or NO2), as possible
D-mannitol
The key intermediate 3 was synthesized from D-mannitol. The
D
use of this carbohydrate in organic synthesis has been highlighted
in several reviews.27–31 It has also other important applications in
areas such as polymer science, cosmetics, medicine and pharma-
ceuticals.32–34 Significant features of this carbohydrate are its func-
tionality, versatility, low price and abundance.
active agents.
* Corresponding author. Tel.: +55 2139772404; fax: +55 2125602518.
E-mail address: marcos_souza@far.fiocruz.br (M.V.N. de Souza).
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.08.006

Marcelle de L. Ferreira et al. / Carbohydrate Research 344 (2009) 2042–2047
2043
OH OH
HO
OH OH
R
N
H
N
N
OH
CH3
H3C
N
H
HO
OH OH
OH OH
OH
R
D-Mannitol
Shiff bases derivatives from D-mannitol (6a-f)
Ethambutol
Figure 1. Structures of ethambutol, D-mannitol and the proposed Schiff bases derivatives (6a–f).
N3
N3
hydrogen bonds and Figure
3
shows the supramolecular
OH
arrangement arising from the O–Hꢁ ꢁ ꢁO intermolecular hydrogen
HO
HO
HO
HO
O
O
OH
OH
OH
OH
bonds.
a-d
e
OH
OH
2.2. Synthesis of 1,6-dideoxy-1,6-bis-{[(E)-phenylmethylidene]-
amino}-D-mannitol derivatives (6a–f)
N3
N3
OH
2
D-mannitol
3
Compound 3 was reduced using H2–Pd (10% on C) in methanol
solution to amino alcohol 4 in 98% yield. As 4 had only limited
thermal stability, it was converted to the dihydrochloride salt, 5
(Scheme 2). The IR spectrum of 5 indicates the loss of the azido
Scheme 1. Reagents and conditions: (a) acetone, H2SO4, 2 h, 77%; (b) AcOH 40%,
45 °C, 2 h, 98%; (c) TsCl, Py, 0 °C, 4 h; (d) NaN3, DMF, 70 °C, 3,5 h, 70% (two steps);
(e) HCl 2.5 N, 20 h; 95%.
group, by the disappearance of the azido vibration at 2105 cmꢀ1
,
þ
and the formation of the NH3 derivative,
m
(3202 cmꢀ1).
1,6-Diazido-1,6-dideoxy-3,4-O-isopropylidene-D-mannitol 2 was
The 1,6-dideoxy-1,6-bis-{[(E)-arylmethylidene]amino}-
D-man-
obtained in four steps in 36.9% overall yield, using the methodology
developed by Le Merrer and co-workers.35,36 The key intermediate
3 was prepared by removal of the isopropylidene group from 2 under
nitol derivatives, 6, were obtained from reactions of 4 and substi-
tuted benzaldehydes at room temperature (0.8–4 h), in 78–92%
yields (Scheme 2 and Table 2).
acidic conditions. Thus overall, 1,6-diazido-1,6-dideoxy-D-mannitol
3 was prepared in five steps in 50.2% overall yield, as shown in
Scheme 1.
All the compounds were identified by the spectral data. In gen-
eral, IR spectra show the N@C stretching vibration at 1633–
1650 cmꢀ1. Specifically, in the 1H NMR spectra, the imine proton
(N@C–H) appears as a singlet in the range 8.83–8.33 ppm, while
the C@N signals in the 13C NMR spectra occur between 156.6 and
160.1 ppm.
A mixture of two geometric isomers at the imine site was found
for the (6: X = Cl) compounds: the E and Z ratios were in the
approximate ratio of 80:20. However for the (6: X = NO2) com-
pounds, only E isomers were isolated, as confirmed by NOE NMR
analysis of the N@CH and NCH2 protons.
Characterization of 3 was achieved spectroscopically and by
X-ray crystallography. The IR spectrum showed O–H and N3
stretching vibrations at 3334 and 2105 cmꢀ1, respectively. The 1H
NMR spectrum exhibited peaks for the OH protons (doublets at
4.33 ppm, J = 8.4 Hz and 5.07 ppm, J = 6.4 Hz) and four sets of peaks
for the C–H protons (between 3.26 and 3.65 ppm). The 13C NMR
spectrum showed three signals at 69.5 (C3 and C4), 69.9 (C2 and
C5) and 54.4 ppm (C1 and C6).
The asymmetric unit of 3, found in the X-ray crystallography
study37–43 comprised two molecules (Mol.A and Mol.B) having
slightly different conformations. The atom arrangements and num-
bering scheme of Mol.A are shown in Figure 2, those for Mol.B have
suffix B replacing A. Each molecule exhibits some disorder about
one of its azido groups: N14–N15–N16 in Mol.A and N24–N25–
N26 in Mol.B. A complex network of O–Hꢁ ꢁ ꢁO hydrogen bonds
involving all four hydroxyl groups of each independent molecule
is present. Table 1 lists out the geometric parameters for the main
2.3. Antimycobacterial activity
The antimycobacterial tests against M. tuberculosis ATTC 27294
of the derivatives 3, 5 and 6a–f were performed42 using the micro
plate Alamar Blue assay (MABA).43 This methodology is nontoxic,
uses thermally stable reagent and shows good correlation with
proportional and BACTEC radiometric methods.44,45 The results
are shown in Table 3. Compounds 6d, 6e and 6f exhibited a prom-
Figure 2. Atoms numbering scheme and atom arrangements for 3. Probability ellipsoids are drawn at the 50% level. Hydrogen atoms are drawn as spheres of arbitrary radius.

2044
Marcelle de L. Ferreira et al. / Carbohydrate Research 344 (2009) 2042–2047
Table 1
Table 2
Selected hydrogen-bond parameters (Å, °) for 3
Yields and reaction times of the Schiff bases derivatives 6
D–Hꢁ ꢁ ꢁA
D–H
Hꢁ ꢁ ꢁA
Dꢁ ꢁ ꢁA
D–Hꢁ ꢁ ꢁA
Number
Substituents
R3
Yield (%)
Time (h)
O(13)–H(13)ꢁ ꢁ ꢁO(24)i
O(15)–H(15)ꢁ ꢁ ꢁO(23)i
O(23)–H(23)ꢁ ꢁ ꢁO(12)ii
0.84
0.84
0.84
0.84
0.84
0.84
0.84
0.84
2.09
1.99
1.90
1.99
1.92
1.86
2.02
1.96
2.717(5)
2.795(6)
2.699(6)
2.794(5)
2.686(7)
2.675(5)
2.675(5)
2.686(7)
131
161
158
161
151
165
134
144
R1
R2
R4
R5
6a
6b
6c
6d
6e
6f
Cl
H
H
NO2
H
H
H
Cl
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
80
78
82
85
80
92
1
4
0.8
1
3
iii
O(24)–H(24)ꢁ ꢁ ꢁO(14)
O(12)–H(12)ꢁ ꢁ ꢁO(25)
O(14)–H(14)ꢁ ꢁ ꢁO(22)
O(25)–H(25)ꢁ ꢁ ꢁO(12)
O(22)–H(22)ꢁ ꢁ ꢁO(14)
NO2
H
H
NO2
0.6
Symmetry codes: (i) x, y + 1, z; (ii) 1 ꢀ x, ꢀ1/2 + y, 2 ꢀ z; (iii) 2 ꢀ x, ꢀ1/2 + y, 2 ꢀ z.
2.4. Cell viability assay
The cytotoxicity of the active compounds 6d, 6e and 6f was
examined by Mosmans’s MTT(3-(4,5-dimethylthylthiazol-2yl)-
2,5-dimethyl tetrazolium bromide; Merck) microcultured tetrazo-
lium assay,46 and the results were represented as percentage cell
viability (Table 4). All the compounds were non-cytotoxic at all
concentrations tested.
Compounds 6d, 6e and 6f exhibited activity of 12.5, 25.0 and
25.0
lg/mL, respectively. The antimycobacterial activities of
D-mannitol derivatives suggest that they may be selectively tar-
geted to M. tuberculosis growth. These compounds are not cyto-
toxic to host cells at the concentrations effective in inhibiting M.
tuberculosis infection. When compared with first line drugs such
as ethambutol (MIC = 3.12 lg/mL), these derivatives could be con-
sidered as a good starting point for the development of new lead
compounds in the fight against multidrug-resistant tuberculosis.
3. Experimental
3.1. General methods
Melting points were determined on a Buchi apparatus and are
uncorrected. Infrared spectra were recorded on a Thermo Nicolet
Nexus 670 spectrometer in KBr disks and frequencies are
expressed in cmꢀ1. Mass spectra (MS) were carried out using a
Waters model ZQ-LC/MS 2000. NMR spectra were recorded at
ambient temperature on a Bruker Avance 500 spectrometer oper-
ating at 400.00 MHz (1H) and 100.0 MHz (13C), in deuterated
dimethyl sulfoxide. Chemical shifts are reported in ppm (d) relative
to tetramethylsilane. Elemental analyses were performed at ICSN,
CNRS, Gif-sur-Yvette, France.
Figure 3. Supramolecular arrangement of the H-bonds network along b-axis for
compound 3.
ising inhibitory activity, when compared with ethambutol, the
drug used as the positive control.
N3
NH2
NH2
HO
HO
HO
HO
HO
OH
OH
OH
OH
OH
OH
a
b
2 HCl
HO
N3
NH2
NH2
5
4
3
c
R2
11
R1
R3
R4
10
9
R1
OH
OH
12
13
8 '
10 '
R2
R3
N
4
6
2
9 '
14 '
1
N
5
3
14
11 '
8
OH
OH
R5
12 '
R5
13 '
6a-f
R4
Scheme 2. Reagents and conditions: (a) MeOH, Pd–C, H2, 4 h, 98%; (b) HCl (g), 10 min, 100%; (c) EtOH–H2O, different benzaldehydes, 78–92%.

Marcelle de L. Ferreira et al. / Carbohydrate Research 344 (2009) 2042–2047
2045
Table 3
3.6. 1,6-Dideoxy-1,6-bis-{[(E)-2-chlorophenylmethylidene]-
amino}- -mannitol (6a)
The in vitro activity of compounds 3, 5 and 6a–f against M. tuberculosis H37Rv strain
(ATCC 27294, susceptible to both rifampin and isoniazid)
D
Yield: 80%; mp: 163–165 °C. IR (KBr; cmꢀ1): 3333
m
OH; 1633 mN@
Number
Compound
MIC (lg/mL)
C–H. 1H NMR (400 MHz; DMSO); d (ppm): 3.55 (2H, dd, J = 11.6 and
3
5
6a
6b
6c
6d
6e
6f
—
—
Resistant
Resistant
Resistant
Resistant
Resistant
12.5
25.0
25.0
3.12
0
0
7.2 Hz; H1 and H6 ); 3.66 (2H, dd, J = 9.2 and 9.2 Hz; H3 and H4);
3.79 (2H, dd, J = 7.2 and 6.0 Hz; H2 and H5); 4.09 (2H, d,
J = 11.6 Hz; H1 and H6); 4.28 (2H, d, J = 9.2 Hz; OH3 and OH4);
R1 = Cl and R2 = R3 = R4 = R5 = H
R2 = Cl and R1 = R3 = R4 = R5 = H
R3 = Cl and R1 = R2 = R4 = R5 = H
R1 = NO2 and R2 = R3 = R4 = R5 = H
R2 = NO2 and R1 = R3 = R4 = R5 = H
4.56 (2H, d, J = 6.0 Hz; OH2 and OH5); 7.40 (8H, m; H11, H12, H13
,
H14 and H11 , H12 , H13 , H14 ); 8.46 (2H, s; H8 and H8 ). 13C NMR
0
0
0
0
0
R
3 = NO2 and R1 = R2 = R4 = R5 = H
(100 MHz, DMSO); d (ppm): 64.9 (C1 and C6); 70.1 (C2 and C5);
Ethambutol
—
0
0
70.5 (C3 and C4); 127.2 (C12 and C12 or C13 and C13 ); 128.2 (C12
0
0
0
0
and C12 or C13 and C13 ); 129.7 (C11 and C11 or C14 and C14 );
0
0
0
131.8 (C11 and C11 or C14 and C14 ); 132.9 (C9 and C9 ); 133.8 (C10
+
0
0
and C10 ); 157.5 (C8 and C8 ). ESIMS: [M+H] : 425.3. Anal. Calcd
for C20H22Cl2N2O4ꢁ1.0H2O: C, 54.19; H, 5.46; N, 6.32. Found: C,
54.28; H, 5.04; N, 6.41.
Table 4
Data of cytotoxicity on murine macrophages cells 18 h after the treatment
Compound
% Cell viability/doses (lg/mL)
0.01
0.1
1.0
10
100
3.7. 1,6-Dideoxy-1,6-bis-{[(E)-3-chloro-phenylmethylidene]-
amino}-D-mannitol (6b)
6d
6e
6f
100
100
100
98
98
98
98
98
98
98
97
98
98
96
98
Yield: 78%; mp: 155–157 °C. IR (KBr; cmꢀ1): 3320 mOH; 1650 mN
@C–H. 1H NMR (400 MHz; DMSO; d (ppm): 3.49 (2H, dd, J = 11.0 and
0
0
3.2. Synthesis of 1,6-diazido-1,6-dideoxy-
D
-mannitol (3)
6.7 Hz; H1 and H6 ); 3.66 (2H, dd, J = 8.2 and 7.5 Hz; H3 and H4);
3.80 (2H, dd, J = 6.7 and 6.0 Hz; H2 and H5); 4.05 (2H, d,
J = 11.0 Hz; H1 and H6); 4.33 (2H, d, J = 7.5 Hz; OH3 and OH4);
A solution of 1,6-diazido-1,6-dideoxy- 3,4-O-isopropylidene-
-mannitol 235,36 (15 g, 55.1 mmol) in HCl 2.5 M (50 mL) was
0
D
4.55 (2H, d, J = 6,0 Hz; OH2 and OH5); 7.47 (4H, m; H12, H12 , H13
0
0
stirred at room temperature for 20 h, concentrated in vacuo, the
crude product was chromatographed (CHCl3/MeOH, 95:5) to yield
3 (12.1 g, 95%) as a white crystalline solid. Mp: 93 °C (91–93 °C).47
and H13 ); 7.69 (2H, d, J = 7.0 Hz; H11 and H11 ); 7.81 (2H, s; H10
and H10´ ); 8.31 (2H, s; H8 and H8 ). 13C NMR (100 MHz, DMSO/
Me4Si; d (ppm): 64.5 (C1 and C6); 70.2 (C2 and C5); 70.5 (C3 and
C4); 126.6 (C14 and C14 ); 127.0 (C10 and C10 ); 130.0 (C12 and C12
or C13 and C13 ); 130.4 (C12 and C12 or C13 and C13 ); 133.4 (C9
and C9 ); 138.4 (C11 and C11 ); 160.1 (C8 and C8 ). ESIMS: [M+H] :
425.4. Anal. Calcd for C20H22Cl2N2O4: C, 56.48; H, 5.21; N, 6.59.
Found: C, 56.46; H, 5.18; N, 6.52.
0
[
a
]
+17.8 (c 1.0, MeOH). IR (KBr pellets; cmꢀ1): 3334
m
OH; 2105
0
0
0
D
1H NMR (400 MHz; DMSO/Me4Si; d (ppm)): 3.26 (2H, dd,
0
0
0
mN3
.
+
0
0
0
0
0
J = 12.6 and 6.4 Hz; H1 and H6 ); 3.44 (2H, dd, J = 12.6 and 2.0 Hz;
H1 and H6); 3.55 (2H, dd, J = 8.4 and 8.4 Hz; H3 and H4); 3.65
(2H, dddd, J = 8.4; 6.4; 6.4 and 2.0 Hz; H2 and H5); 4.33 (2H, d,
J = 8.4 Hz; OH3 and OH4); 5.07 (2H, d, J = 6.4 Hz; OH2 and OH5).;
13C NMR (100 MHz, DMSO/Me4Si; d (ppm)): 54.36 (C1 and C6);
69.53 (C3 and C4); 69.87 (C2 and C5). ESIMS: [MꢀH]: 231.2.
3.8. 1,6-Dideoxy-1,6-bis-{[(E)-4-chloro-phenylmethylidene]-
amino}-D-mannitol (6c)
3.3. Synthesis of 1,6-diamino-1,6-dideoxy-
D
-mannitol (4)
Yield: 82%; mp: 186 °C. IR (KBr; cmꢀ1): 3417 mOH; 1650 mN@C–H
1H NMR (400 MHz; DMSO); d (ppm): 3.48 (2H, dd, J = 11.6 and
.
0
0
To a solution of 3 (200 mg, 0.86 mmol) in MeOH (10 mL) was
added 10% Pd–C (25 mg), and the mixture was treated with H2
for 4 h. The catalyst was filtered off and washed with MeOH
(5 mL), and the combined organic extracts were concentrated to
yield 4 as an oil (185 mg, 98%). The crude product was used in
the next step without further purification.
8.0 Hz; H1 and H6 ); 3.67 (2H, dd, J = 8.0 and 8.0 Hz; H3 and H4);
3.79 (2H, ddd, J = 8.0; 8.0 and 6.4 Hz; H2 and H5); 4.03 (2H, d,
J = 11.6 Hz; H1 and H6); 4.31 (2H, d, J = 8.0 Hz; OH3 and OH4);
4.52 (2H, d, J = 6.4 Hz; OH2 and OH5); 7.76 (4H, d, J = 8.4 Hz; H11
0
0
0
and H11 or H13 and H13 ); 7.50 (4H, d, J = 8.4 Hz; H10 and H10 or
H14 and H14 ); 8.31 (2H, s; H8 and H8 ). 13C NMR (100 MHz, DMSO);
0
0
d (ppm): 64.5 (C1 and C6); 70.2 (C2 and C5); 70.6 (C3 and C4); 128.6
0
0
0
0
3.4. Synthesis of 1,6-diamino-1,6-dideoxy-
D
-mannitol
(C10 and C10 ; C14 and C14 ); 129.4 (C11 and C11 ; C13 and C13 ); 134.9
0
0
0
0
dihydrochloride (5)
(C9 and C9 or C12 and C12 ); 135.1 (C9 and C9 or C12 and C12 );
160.3 (C8 and C8 ). ESIMS: [M+H]+: 425.5. Anal. Calcd for
0
Hydrogen chloride was bubbled into the solution of 4 (1.0 g,
5.5 mmol) in MeOH (50 mL) for 5 min. The formed white precipi-
tate was collected, filtered and finally washed with cold water
(1.3 g, 100%). Mp: 218–220 °C (217–220 °C).48
C20H22Cl2N2O4ꢁ2.0H2O: C, 52.07; H, 5.68; N, 6.07. Found: C,
52.04; H, 5.81; N, 5.98.
3.9. 1,6-Dideoxy-1,6-bis-{[(E)-2-nitro-phenylmethylidene]-
amino}-D-mannitol (6d)
3.5. General procedure for the preparation of 1,6-dideoxy-1,6-
bis-{[(E)-phenylmethylidene]amino}-
D-mannitol derivatives
Yield: 85%; mp: 167–168 °C (decomposition). IR (KBr; cmꢀ1):
1521 and 1348 NO2; 3415 mOH; 1634 mN@C–H
1H NMR (400 MHz;
DMSO); d (ppm): 3.52 (2H, dd, J = 11.6 and 8.0 Hz; H1 and H6 );
3.66 (2H, dd, J = 8.0 and 8.0 Hz; H3 and H4); 3.81 (2H, dd, J = 8.0
and 6.4 Hz; H2 and H5); 4.01 (2H, d, J = 11.6 Hz; H1 and H6); 4.35
(2H, d, J = 8.0 Hz; OH3 and OH4); 4.55 (2H, d, J = 6.4 Hz; OH2 and
(6a–f)
m
.
0
0
Solutions of the appropriate benzaldehyde (2.2 equiv) in EtOH
and 4 (0.15 g, 0.83 mmol, 1.0 equiv) in water were mixed. The
resulting mixture was stirred for 0.6–4 h, at room temperature
and was concentrated under reduced pressure. The residues were
purified by washing with distilled water (2 mL) and cold diethyl
ether (10 mL) to give 6.
0
OH5); 7.70 (2H, dd, J = 7.6 and 7.6 Hz; H12 and H12 ); 7.79 (2H, dd,
0
J = 7.6 and 7.6 Hz; H13 and H13 ); 8.02 (2H, d, J = 7.6 Hz; H14 and
0
0
H14 ); 8.06 (2H, d, J = 7.6 Hz; H11 and H11 ); 8.56 (2H, s; H8 and

2046
Marcelle de L. Ferreira et al. / Carbohydrate Research 344 (2009) 2042–2047
H8 ). 13C NMR (100 MHz, DMSO); d (ppm): 64.3 (C1 and C6); 69.6
Absorption coefficient: 0.123 mmꢀ1, F(0 0 0): 488, Crystal size:
0.20 ꢂ 0.20 ꢂ 0.20 mm, h range for data collection, 2.11–25.00°, In-
dex ranges: ꢀ11 6 h 6 11; ꢀ11 6 k 6 11; ꢀ13 6 l 6 13, Reflections
collected: 11084, Independent reflections: 3696, [R(int) = 0.0322],
0
0
(C2 and C5); 70.0 (C3 and C4); 123.5 (C11 and C11 ); 128.9 (C9 and
C9 ); 129.5 (C14 and C14 ); 130.5 (C12 and C12 ); 132.8 (C13 and
C13 ); 148.3 (C10 and C10 ); 156.6 (C8 and C8 ). ESIMS: [M+H]:
447.0. Anal. Calcd for C20H22N4O8: C, 53.81; H, 4.97; N, 12.55.
Found: C, 53.64; H, 5.12; N, 12.23.
0
0
0
0
0
0
Reflections observed (>2r): 3313, Data Completeness: 0.997,
Refinement method: Full-matrix least-squares on F2, Data/re-
straints/parameters: 3696/1/293, Goodness-of-fit on F2: 1.035, Fi-
3.10. 1,6-Dideoxy-1,6-bis-{[(E)-3-nitro-phenylmethylidene]-
nal R indices [I > 2r(I)]: R1 = 0.0518 wR2 = 0.1218, R indices (all
amino}-
D
-mannitol (6e)
data): R1 = 0.0604 wR2 = 1268, Largest diff. peak and hole, 0.785
and ꢀ0.492 e Åꢀ3. CCDC deposition no.: 727270.
Yield: 80%; mp: 152–153 °C (with decomposition). IR (KBr;
cmꢀ1): 1532 and 1342
m
NO2; 3488 mOH; 1650 mN@C–H
.
1H NMR
3.13. Biological activity assays
0
(400 MHz; DMSO); d (ppm): 3.55 (2H, dd, J = 11.6 and 8.0 Hz; H1
0
and H6 ); 3.69 (2H, dd, J = 8.0 and 8.0 Hz; H3 and H4); 3.84 (2H,
3.13.1. Antimycobacterial activity
dd, J = 8.0 and 4.4 Hz; H2 and H5); 4.09 (2H, d, J = 11.6 Hz; H1 and
H6); 4.37 (2H, d, J = 8,0 Hz; OH3 and OH4); 4.62 (2H, d, J = 4.4 Hz;
Briefly, 200 lL of sterile deionized water was added to all outer-
perimeter wells of sterile 96 well plates (Falcon, 3072: Becton
Dickinson, Lincoln Park NJ) to minimize evaporation of the med-
ium in the test wells during incubation. The 96 plates received
0
OH2 and OH5); 7.75 (2H, dd, J = 7.6 and 7.6 Hz; H13 and H13 );
0
8.17 (2H, d, J = 7.6 Hz; H14 and H14 ); 8.29 (2H, d, J = 7.6 Hz; H12
and H12 ); 8.46 (2H, s; H8 and H8 ); 8.58 (2H, s; H10 and H10 ). 13C
100 lL of the Middlebrook 7H9 broth (Difco laboratories, Detroit,
0
0
0
NMR (100 MHz, DMSO); d (ppm): 64,5 (C1 and C6); 70,1 (C2 and
MI, USA) and a serial dilution of the compounds 3, 5 and 6a–f
0
C5); 70,5 (C3 and C4); 121,7 (C10 and C10 ); 124,7 (C14 and
was made directly on the plate. The final drug concentrations
0
0
0
C14 );130,2 (C12 and C12 );134,2 (C13 and C13 ); 137,9 (C9 and
tested were 0.01–100.0
with parafilm and were incubated at 37 °C for 5 days. After this
time, 25 L of a freshly prepared 1:1 mixture of Alamar Blue (Acc-
umed International, Westlake Ohio) reagent and 10% Tween 80
was added to the plate and was incubated for 24 h. A blue colour
in the well was interpreted as no bacterial growth, and a pink col-
our was scored as growth. The MIC (Minimal Inhibition Concentra-
tion) was defined as the lowest drug concentration, which
prevented a colour change from blue to pink.
lL/mL. Plates were covered and sealed
+
0
0
0
C9 );148,1 (C11 and C11 ); 159,7 (C8 and C8 ). MS/ESI: [M+H] :
447.0. Anal. Calcd for C20H22N4O8ꢁ1.0.H2O: C, 51.72; H, 5.21; N,
12.06. Found: C, 51.53; H, 5.11; N, 11.98.
l
3.11. 1,6-Dideoxy-1,6-bis-{[(E)-4-nitro-phenylmethylidene]-
amino}-D-mannitol (6f)
Yield: 92%; mp: 213 °C (decomposition). IR (KBr; cmꢀ1): 1527
and 1344 NO2; 3445 OH; 1645
N@C–H. 1H NMR (400 MHz; DMSO);
m
m
m
0
0
d (ppm): 3.54 (2H, dd, J = 11.6 and 8.0 Hz; H1 and H6 ); 3.66 (2H,
dd, J = 8.0 and 8.0 Hz; H3 and H4); 3,85 (2H, dd, J = 8.0 and
6.0 Hz; H2 and H5); 4.11 (2H, d, J = 11.6 Hz; H1 and H6); 4.35 (2H,
d, J = 8.0 Hz; OH3 and OH4); 4.60 (2H, d, J = 6.0 Hz; OH2 and OH5);
3.13.2. Cell viability
The cells were plated in flat bottom 96 well plates
(2.5 ꢂ 106 cells/mL) cultured for 1 h in a controlled atmosphere
(CO2 5% at 37 °C), and non-adherent cells were washed by gentle
flushing with RPMI 1640. Adherent cells were cultured in the pres-
ence of medium alone, Tween 20 (3%) (live and dead controls,
respectively) or different concentrations of compounds (0.1, 1.0,
10.0 and 100 mg/mL) in a triplicate assay. After 18 h, stock MTT
solution (5 mg/mL of saline; 20 mL/well) was added to the culture
and 4 h later, the supernatant was discharged and DMSO (100 mL/
well) was added for formazan crystals solubilization and the absor-
bance was read at 540 nm in a plate reader (Biorad—450) relation-
ship and their in vivo antibacterial activity test is in progress. The
results were represented as percentage cell viability (Table 4).
0
0
8.01 (4H, d, J = 8.8 Hz; H11 and H11 ; H13 and H13 ); 8.30 (4H, d,
0
0
0
J = 8.8 Hz; H10 and H10 ; H14 and H14 ); 8.45 (2H, s; H8 and H8 ).
13C NMR (100 MHz, DMSO); d (ppm): 64.8 (C1 and C6); 70.1 (C2
0
0
and C5); 70.5 (C3 and C4); 123.8 (C10 and C10 ; C14 and C14 );
128.8 (C11 and C11 ; C13 and C13 ); 141.9 (C9 and C9 ); 148.3 (C12
and C12 ); 160.0 (C8 and C8 ). ESIMS: [M+H] : 447.2. Anal. Calcd
for C20H22N4O8ꢁ1.0.H2O: C, 51.72; H, 5.21; N, 12.06. Found: C,
51.48; H, 5.18; N, 12.21.
0
0
0
+
0
0
3.12. X-ray crystallographic study
Intensity data for 1,6-diazido-1,6-dideoxy-
D
-mannitol 3 were
Acknowledgements
collected at 120 K with Mo K radiation using the
a
j-goniostat Bru-
ker–Nonius CCD camera of the EPSRC crystallographic service,
based at the University of Southampton, UK. Data collection was
carried using the program COLLECT37 and data reduction and unit cell
refinement were achieved with the COLLECT37 and DENZO programs.38
No correction for absorption was applied. The program ORTEP-3 for
We are indebted to the EPSRC for the use of the X-ray service at
the University of Southampton, UK. J.L.W. thanks Capes for
support.
Windows39 was used in the preparation of Figure 3 and SHELXL
-
Appendix
41
9740 and PLATON in the calculation of molecular geometry. The
structure was solved by direct methods using SHELXS-97 and was
fully refined by means of the program SHELXL-97.40 Stereospecific
synthesis using a chiral reagent unambiguously produced a single
stereoisomer. All hydrogen atoms were placed in the calculated
positions and were refined with a riding model. Crystal data and
structure refinement details are listed below.
CCDC 727270 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from the
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
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Empirical formula: C6H12N6O4, Formula weight: 232.22, Tem-
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