Preparation and antitubercular activities of alkylated amino alcohols and their glycosylated derivatives

Article abstract of DOI:10.1016/j.bmc.2007.08.045

A series of N- and C-alkylated amino alcohols and of their protected galactopyranosyl derivatives was synthesized and evaluated for antitubercular activity. Five of these compounds displayed good activity, with a MIC below 12.5 μg/mL. The presence of the carbohydrate slightly affected the antibacterial activity.

Full text of DOI:10.1016/j.bmc.2007.08.045

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7789–7794
Preparation and antitubercular activities of alkylated
amino alcohols and their glycosylated derivatives
a
Aline F. Taveira,a Mireille Le Hyaric,a Elaine F. C. Reis,a,b Debora P. Arau´jo,
´
b
b
b
´
Ana Paula Ferreira, Maria Aparecida de Souza, Lıvia L. Alves,
Maria C. S. Lourenc¸o,c Felipe Rodrigues C. Vicentec and Mauro V. de Almeidaa,*
aDepartamento de Qu´ımica, Universidade Federal de Juiz de Fora, Juiz de Fora-MG 36036-330, Brazil
bDepartamento de Parasitologia, Microbiologia e Imunologia, Universidade Federal de Juiz de Fora,
Juiz de Fora-MG 36036-330, Brazil
cInstituto de Pesquisa Clı´nica Evandro Chagas, IPEC, Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro-RJ 21041-250, Brazil
Received 4 May 2007; revised 20 August 2007; accepted 24 August 2007
Available online 29 August 2007
Abstract—A series of N- and C-alkylated amino alcohols and of their protected galactopyranosyl derivatives was synthesized and
evaluated for antitubercular activity. Five of these compounds displayed good activity, with a MIC below 12.5 lg/mL. The presence
of the carbohydrate slightly affected the antibacterial activity.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
synthesized.2 Among the targets for the development
of new anti-TB drugs, the mycobacterial cell membrane
is of great interest, since it is responsible for the imper-
meability of the bacterial cell to numerous drugs.3
Mycobacterial cell wall possesses in its structure com-
plex polysaccharides, lipoarabinomannan and arabino-
galactan, in which galactose and arabinose are
predominant.4–7 The inhibition of the glycosyltransfe-
rases evolved in their biosynthesis could result in a dis-
turbance of the cell wall biosynthesis.8–10 Good
candidates for this type of drugs are sugar-based glycos-
ylated amino alcohols and aminoesters.11–16 We recently
described the synthesis and anti-TB activity of some
aminoacylated derivatives of galactopyranose.17 In con-
tinuation to this work we report here the preparation
and evaluation of anti-TB activity of C- and N-alkylated
amino alcohols and of their D-galactose derivatives.
Tuberculosis (TB), a contagious disease which spreads
through air, is caused by Mycobacterium tuberculosis.
World Health Organization reports that nearly 2 mil-
lions deaths result from tuberculosis every year, and that
one-third of the world population is currently infected
with the TB bacillus.1 Tuberculosis is a leading cause
of death among people who are HIV positive, forming
with HIV a lethal combination, each one spreading the
other’s progress. Until 60 years ago there was no treat-
ment for tuberculosis. Since then several drugs have
been used, alone or in combination, and drug resistant
strains of mycobacterium have emerged. This resistance
is caused by partial treatment (patients stop taking the
medicine), by prescription of the wrong treatment regi-
men, or by an unreliable drug supply. MDR-TB (multi-
drug resistant TB) is a particularly dangerous form of
TB, being resistant to isoniazid and rifampicin, two of
the most powerful anti-TB drugs.
2. Results and discussion
During the last years, numerous new antitubercular
drugs having different mechanisms of action have been
For the obtention of N-alkylated compounds 3a–e18–20
and 721 alcohols 1a–e and 5 were first mesylated in pyr-
idine at 0 °C, leading to compounds 2a–e and 6 which
were used without purification (90–96% yield, Scheme
1). Mesylated derivatives were then treated with etha-
nolamine (1.2 equiv/mol) in ethanol at 90 °C, furnishing
the desired compounds 3a–e and 7 in moderate yields
Keywords: Mycobacterium tuberculosis; Amino alcohol; D-Galactose;
N-Alkylation.
*
Corresponding author. E-mail: mauro.almedia@ufjf.edu.br
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.045

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A. F. Taveira et al. / Bioorg. Med. Chem. 15 (2007) 7789–7794
b
a
CH3(CH2)nOH
CH3(CH2)nOMs
CH3(CH2)nNHCH2CH2OH
1a-e
2a-e
3a-e
a: n=7
+
b: n= 9
c: n=11
d: n=13
e: n=15
[CH3(CH2)n]2NCH2CH2OH
4a-e
a
b
CH3(CH2)3CHCH2OH
CH3(CH2)3CHCH2OMs
CH2CH3
CH3(CH2)3CHCH2NHCH2CH2OH
CH2CH3
CH2CH3
5
6
7
d
a, c
CH3(CH2)9CHCH2OH
OH
CH3(CH2)9CHCH2N3
CH3(CH2)9CHCH2NH2
OH
OH
10
8
9
Scheme 1. Preparation of N- and C-alkylated amino alcohols. Reagents and conditions: (a) MsCl, pyridine, 0 °C; (b) H2N(CH2)2OH, EtOH, 90 °C;
(c) NaN3, DMF, 120 °C; (d) H2, Pd/C, EtOH.
(50–52%) along with N,N0-dialkylated compounds 4a–e
(8–10% yield). After purification by column chromatog-
raphy amino alcohols 3a–e and 7 were characterized by
3. Biological activity
The activity of the compounds against M. tuberculosis
virulent strain H37Rv was determined in vitro as previ-
ously described,24 using rifampicin as a reference for
activity. The minimum inhibitory concentration
(MIC), concentration that inhibits the colony forming
ability of M. tuberculosis, was determined by incorporat-
ing decreasing concentrations of the test compound in
Middlebrook 7H9 agar medium. MIC values represent
means of three separate experiments and are reported
in Table 1.
1
1H NMR and 13C NMR spectroscopy. In the H NMR
spectra, the presence of signals in the regions of d 3.6
and 2.7 ppm referring to the ethanolamine moiety, along
with the disappearance of the singlet near 3.0 ppm cor-
responding to the methyl of the mesylate group, con-
firmed the conversion to amino alcohols. In the 13C
NMR spectra signals in the region of d 60–61 and d
49–53 ppm, corresponding to methylenic carbons
CH2N and CH2O, were observed.
Primary hydroxyl group of diol 8 was first converted to
mesylate and the resulting compound was treated with
sodium azide in DMF at 120 °C, leading to azide 9 in
quantitative yield. After a rapid purification, compound
9 was hydrogenated in the presence of palladium on
charcoal 10% in ethanol, furnishing C-alkyl amino alco-
hol 10 in 70% yield22 (Scheme 1).
It would appear that the antitubercular activity of the
free amino alcohols depends on the length of the alkyl
chain. The best results were obtained for the N-dode-
cyl-ethanolamine 3c (MIC = 0.027 mM) and C-decyl-
ethanolamine 10 (MIC = 0.015 mM). C-Alkylated com-
pound 10 is almost twice more active than its N-alkyl-
ated analogue 3c. Compounds 3d and 3e with longer
alkyl chain were twice less active than 3c, but still more
active than shorter compounds. The same effect was ob-
served for the glycosylated derivatives 13a–e.
No attempt was made to determine the stereochemistry
of compounds 7 and 10 as they were prepared from the
corresponding racemic alcohols (5 and 8, respectively).
Among the glycosylated derivatives, compound 13c was
the most active (MIC = 0.006 mM), but this time the N-
alkylated galactose derivative was four times more ac-
tive than its C-alkylated analogue.
D-Galactose 11 was converted into 6-deoxy-6-iodo-
1,2:3,4-di-O-isopropylidene-a-D-galactopyranose
12
according to the literature procedure23 and then treated
with amino alcohols 3a–e, 7, and 10 in DMSO at 90 °C
(Scheme 2). All the compounds were purified by column
chromatography (hexane:ethyl acetate) and were ob-
tained in 40–56% yields. The structure was assigned by
Glycosylation of N-alkylated amino alcohols increased
their activity, suggesting that the carbohydrate moiety
is important for the anti-TB activity of these com-
pounds. The mechanisms of action of these two classes
may be different. The glycosylated compounds could
act as inhibitors of the cell wall biosynthesis. The anti-
TB activity observed for compounds 3a–e could be a re-
sult of their action on the cytoplasmic membrane,
responsible for the antimicrobial action of some N-
alkylated amino alcohols against several organisms.25
N-Octyl-ethanolamine 3a is also able to inhibit the
growth of Mycobacteria smegmatis and Mycobacteria
marinum in metalworking coolant fluid.26
1H NMR, 13C NMR, and COSY experiments. All H
1
NMR spectra showed signals referring to the carbohy-
drate moiety (3.8–5.6 ppm), to the alkyl chain (0.8–
1.3 ppm), and, in the case of compounds 13a–e, to the
ethanolamine portion (2.7–3.6 ppm). 13C NMR spectra
showed characteristic signals near to d 96 ppm (C1)
and between d 66 and 73 ppm (C2–C5), corresponding
to the carbohydrate portion of the molecule, and two
signals corresponding to the isopropylidene groups were
observed between d 107 and 110 ppm. Three signals
between 53 and 56 ppm (CH2N) confirmed the presence
of the N-alkylated amino alcohol (compounds 13a–e
and 14).
Compound 7, containing a branched alkyl chain, and its
galactose derivative 14 did not exhibit antitubercular

A. F. Taveira et al. / Bioorg. Med. Chem. 15 (2007) 7789–7794
7791
HO OH
O I
HOCH2CH2
O N
(CH2)nCH3
O
O
O
HO
O
b
a
O
OH
O
O
11
12
13a-e
OH
O
O
d
c
HOCH2CH2
O
(CH2)3CH3
CH2CH3
(CH2)9CH3
H
N
O
N
CH2CH
CH2CH
OH
O
O
O
O
14
O
15
O
O
O
Scheme 2. Preparation of D-galactose derivatives. Reagents and conditions: (a) i—ZnCl2, H2SO4, acetone, t.a. (58%); ii—I2, PPh3, Imidazole,
toluene, reflux (90%); (b) 3a–e, DMSO, 90 °C, (40–56%); (c) 7, DMSO, 90 °C (56%); (d) 10, DMSO, 90 °C (41%).
data could be explained by the fact that, due to cell pro-
liferation, the higher number of cells inhibits the cellular
survival conditions by the lack of nutrients. Compound
13d stimulated cell proliferation at all the tested concen-
trations, and compound 10 displayed cell proliferation
over 100% at 0.05 lg/mL and at 0.005 lg/mL.
Table 1. Antitubercular activity
Compound
MIC (lg/mL)
MIC (mM)
3a
3b
100
50
0.578
0.248
0.027
0.051
0.046
>0.578
0.015
0.241
0.225
0.006
0.012
0.011
>0.240
0.028
—
3c
3d
6.25
12.5
12.5
>100
3.12
100
3e
7
10
4. Conclusion
13a
13b
100
A series of alkylated amino alcohols and their glycosyl-
ated derivatives were synthesized and evaluated for their
in vitro antitubercular activity. Five of these compounds
displayed good inhibition of M. tuberculosis growth,
with MIC values below 12.5 lg/mL, and a low toxicity.
This work may lead to further development of new effi-
cient antitubercular compounds.
13c
13d
3.12
6.25
6.25
>100
12.5
0.2
13e
14
15
Isoniazid
Ethambutol
3.25
—
Rifampicin 1.0 lg/mL was used as internal control.
5. Experimental
activity at the tested concentration, suggesting that the
alkyl chain would have to be linear for biological
activity.
5.1. General methods
´
Melting points were determined on a Microquımica
MQAPF apparatus and are uncorrected. IR spectra
were recorded using a BOMEM-FTIR MB102 spec-
trometer. Optical rotations were measured with a Per-
kin-Elmer 341 polarimeter, using a sodium lamp (k
Cell proliferation and cytotoxicity of the most active
compounds 3c, 10, 13c, 13d, and 13e were evaluated.
Both activities were determined in parallel on lineage
cell J744A.1 stimulated with interferon-gamma (IFN-
c) and evaluated by a colorimetric MTT assay. The
determination was based on the viability of the cells in
the presence of various concentrations of the tested
compounds. Results are displayed in Table 2. Com-
pounds 3c and 13e displayed low toxicity in all the tested
concentrations. Compound 10 was cytotoxic at 0.5 lg/mL.
Compounds 13c and 13d did not show any cytotoxicity
in all tested concentrations. Despite its toxicity, com-
pound 13e showed a statistically significant increase of
cell proliferation at 0.5 lg/mL and 0.05 lg/mL. These
1
589 nm) at 20 °C. H and 13C NMR spectra were re-
corded on Bruker Advance DRX300 spectrometer. Ele-
´
mental analyses were performed at the Central Analıtica
of Instituto de Qu´ımica of the Universidade de Sa˜o Pau-
lo, Brazil. Thin-layer chromatography (TLC) was per-
formed on glass plates and silica gel sheets (Silica Gel
F254, Merck), visualized with iodine vapor, and/or re-
vealed with ethanolic H2SO4 solution or with a 0.5%
ninhydrin solution. Column chromatography was car-
ried out on silica gel (E. Merck 230 and 400 mesh). Sol-
´
vents were purchased from Vetec Quımica and were

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A. F. Taveira et al. / Bioorg. Med. Chem. 15 (2007) 7789–7794
Table 2. Cell proliferation and citotoxicity
Compound
Concentration
0.5 lg/mL
0.05 lg/mL
0.005 lg/mL
Proliferation (%) Cytotoxicity (%)
Proliferation (%)
Cytotoxicity (%)
Proliferation (%)
Cytotoxicity (%)
3c
10
90.9
96.4
21.3
31.1
nc
77.6
112
98.9
8.4
nc
92.7
110.2
98.3
12.1
nc
13c
13d
13e
96.6
117.7
148.6*
nc
nc
nc
nc
nc
17.4
135.2*
112.8
104.3
110.4
16.3
10.6
nc, not cytotoxic.
* Statistically significant increase, p < 0.05 (ANOVA Bonferroni test).
distilled prior to use. Reagents were purchased from
Aldrich and used without further purification.
18H, CH2); 0.82 (t, 3H, J = 6.8 Hz, CH3); 13C NMR
(CDCl3, 75 MHz): 60.4 (CH2OH); 51.5
(NHCH2CH2OH); 49.8 (CH2NHCH2CH2 OH); 31.9–
d
5.2. General procedure for the preparation of N-alkylated
amino alcohols 3a–e and 7
22.7 (CH2); 14.0 (CH3).
5.2.4. N-Tetradecyl-amino ethanol (3d). White crystals;
mp, 53.1–53.4 °C; yield, 52%; 1H NMR (CDCl3,
300 MHz): d 3.66 (t, 2H, J = 5.0 Hz, CH2OH); 3.57
(m, 2H, NH and OH); 2.76 (t, 2H, J = 5.0 Hz,
To a solution of the alcohol 1a–e, 5 or 8 (50 mmol) in
CH2Cl2 (25 mL) was added a solution of methanesulfo-
nyl chloride (70 mmol) in CH2Cl2 (25 mL). The temper-
ature of the reaction mixture was lowered to 0 °C and
pyridine (5 mL) was added. The reaction mixture was
stirred at room temperature for 24 h and then concen-
trated under reduced pressure. The residue was dis-
solved in hexane and washed three times with water.
After concentration of the organic phase the crude mes-
ylate was dissolved in ethanol (30 mL) and slowly added
to a solution of 2-amino ethanol (100 mmol) in ethanol
(30 mL). The reaction mixture was stirred for 24 h at
90 °C and then concentrated under reduced pressure.
The residue was partitioned between CH2Cl2 and water.
After concentration of the organic phase the residue was
purified by column chromatography (CH2Cl2/MeOH)
to furnish the desired amino alcohols 3a–e and 7.
NHCH2CH2OH);
CH2NHCH2CH2O H); 1.48 (m, 2H, CH2); 1.22 (m,
2.62
(t,
2H,
J = 7.3 Hz,
22H, CH2); 0.85 (t, 3H, J = 6.5 Hz, CH3); 13C NMR
(CDCl3,
75 MHz):
d
60.4
(CH2OH);
51.2
(NHCH2CH2OH); 49.5 (CH2NHCH2CH2 OH); 32.0–
23.0 (CH2); 14.5 (CH3).
5.2.5. N-Hexadecyl amino ethanol (3e). White crystals;
mp 59.0–60.5 °C; yield, 50%; 1H NMR (CDCl3,
300 MHz): d 3.71 (t, 2H, J = 5.0 Hz, CH2OH); 2.80 (t,
2H, J = 5.0 Hz, NHCH2CH2OH); 2.66 (t, 2H,
J = 7.3 Hz, CH2NHCH2CH2O H); 1.55 (m, 2H, CH2);
1.28 (m, 26H, CH2); 0.89 (t, 3H, J = 6.6 Hz, CH3); 13C
NMR (CDCl3, 75 MHz): d 60.7 (CH2OH); 49.7
(NHCH2CH2OH); 49.5 (CH2NHCH2CH2 OH); 32.1–
22.9 (CH2); 14.2 (CH3).
1
5.2.1. N-Octyl-amino ethanol (3a). Oil; yield, 50%; H
NMR (CDCl3, 300 MHz): d 4.83 (br s, 1H, NH); 3.60
(t, 2H, J = 5.1 Hz, CH2OH); 3.20 (m, 1H, OH); 2.70
(t, 2H, J = 5.1 Hz, NHCH2CH2OH); 2.57 (t, 2H,
J = 7.3 Hz, CH2NHCH2CH2OH); 1.44 (m, 2H, CH2);
1.22 (m, 10 H, CH2); 0.82 (t, 3H, J = 6.7 Hz, CH3);
13C NMR (CDCl3, 75 MHz): d 60.6 (CH2OH); 51.4
(NHCH2CH2OH); 49.8 (CH2NHCH2CH2 OH); 31.9–
22.7 (CH2); 14.1 (CH3).
5.2.6. N-(2-Ethyl-hexyl) amino ethanol (7). Oil, yield,
51%; 1H NMR (CDCl3, 300 MHz): d 3.62 (t, 2H,
J = 5.2 Hz, CH2OH); 3.21 (m, 2H, NH and OH); 2.72
(t, 2H, J = 5.2 Hz, NHCH2CH2OH); 2.48 (d, 2H, J =
6.1 Hz, CH2NHCH2CH2O H); 1.21 (m, 9H, CH2aliph.,
CH2ethyl, CH(Et)CH2NH); 0.84 (t, 6H, J = 7.2 Hz,
CH3); 13C NMR (CDCl3, 75 MHz): d 60.4 (CH2OH);
52.8 (NHCH2CH2OH); 51.6 (CH3CH2CHCH2 NH);
39.0 (CH(Et)CH2NH); 31.2–23.1 (CH2); 14.2 and 10.7
(CH3).
1
5.2.2. N-Decyl-amino ethanol (3b). Oil; yield, 51%; H
NMR (CDCl3, 300 MHz): d 3.65 (t, 2H, J = 4.3 Hz,
CH2OH); 2.91 (m, 2H, NH and OH); 2.71 (t, 2H,
J = 4.3 Hz, NHCH2CH2OH); 2.57 (t, 2H, J = 7.2 Hz,
CH2NHCH2CH2O H); 1.47 (m, 2H, CH2); 1.27 (m,
14H, CH2); 0.87 (t, 3H, J = 6.8 Hz, CH3); 13C NMR
5.3. Preparation of C-alkylated amino alcohol (10)
The crude monomesylated precursor (1.47 g, 5.22 mmol),
obtained as described above, was dissolved in DMF
(10 mL) and sodium azide (0.75 g, 10.45 mmol) was
added. The reaction mixture was stirred at 120 °C for
24 h and concentrated under reduced pressure. The
crude product was dissolved in hexane and washed 3
times with water. After evaporation of the organic
phase, the residue was purified by column chromatogra-
phy leading to 1-azidododecan-2-ol 9 as an oil. Yield,
94%; 1H NMR (CDCl3, 300 MHz): d 3.77 (m, 1H,
(CDCl3,
(NHCH2CH2OH); 49.8 (CH2NHCH2CH2 OH); 32.0–
75 MHz):
d
60.8
(CH2OH);
51.4
22.8 (CH2); 14.2 (CH3).
1
5.2.3. N-Dodecyl-amino ethanol (3c). Oil; yield, 51%; H
NMR (CDCl3, 300 MHz): d 3.58 (t, 2H, J = 5.2 Hz,
CH2OH); 3.48 (m, 2H, NH and OH); 2.66 (t, 2H,
J = 5.2 Hz, NHCH2CH2OH); 2.54 (t, 2H, J = 7.3 Hz,
CH2NHCH2CH2OH); 1.41 (m, 2H, CH2); 1.18 (m,

A. F. Taveira et al. / Bioorg. Med. Chem. 15 (2007) 7789–7794
7793
CHOH); 3.36 (dd, 1H, J = 3.5 Hz, 12.5 Hz, CHH0N3);
3.22 (dd, 1H, J = 7.2 Hz, 12.5 Hz, CHH0N3); 2.90 (m,
1H, OH); 1.48 (m, 2H, CH2CHOH); 1.28 (m, 16H,
CH2); 0.87 (t, 3H, J = 6.6 Hz, CH3); 13C NMR (CDCl3,
75 MHz): d 71.0 (CHOH); 57.2 (CH2N3); 34.5
(CH2CHOH); 32.0–22.8 (CH2); 14.5 (CH3).
1.51; 1.43; 1.33; 1.31 (4s, CH3iPr); 1.24 (m, 16H, CH2a-
liph.); 0.88 (t, 3H, J = 6.7 Hz, CH3aliph.); 13C NMR
(CDCl3, 75 MHz): d 109.3 and 108.6 (CiPr); 96.1 (C1);
71.4; 71.0; 70.7 (C2, C3, C4); 66.1 (C5); 59.3 (CH2OH);
56.9; 55.5; 53.6 (CH2N); 32.0–22.8 (CH3iPr and CH2a-
liph.); 14.3 (CH3aliph.); Anal. Calcd for C24H45NO6:
C, 64.98; H, 10.22; N, 3.16. Found: C, 65.50; H, 10.44;
N. 2.71.
Compound 9 (0.681 g, 3 mmol) was dissolved in ethanol
(10 mL) and Pd/C 10% (20 mg) was added. The mixture
was stirred at room temperature for 48 h under an atmo-
sphere of H2. Catalyst was eliminated by filtration and
the mixture was concentrated under reduced pressure.
The crude residue was chromatographed on silica gel
(methylene chloride/methanol), furnishing amino alco-
hol 10 as an oil. Yield, 70%; IR (m, cmꢀ1, CsI): 3408,
5.4.3.
6-[(N-Dodecyl)-2-hydroxyethylamino]-6-deoxy-
1,2:3,4-di-O-isopropylidene-a-D-galactopyranose (13c).
Oil; yield, 53%; IR (m, cmꢀ1, CsI): 3488, 2925, 2853,
1072; 1H NMR (CDCl3, 300 MHz): d 5.54 (d. 1H,
J = 5.0 Hz, H1); 4.63 (dd, 1H, J = 2.0, 7.7 Hz, H3);
4.33 (dd, 1H, J = 2.0, 5.0 Hz, H2); 4.28 (dd, 1H,
J = 1.7, 7.9 Hz, H4); 3.88 (m, 1H, H5); 3.61 (m, 2H,
CH2OH); 3.26 (m, 1H, OH); 2.82 (m, 6H, CH2N);
1.54; 1.46; 1.35; 1.34 (4s, CH3iPr); 1.26 (m, 20H, CH2a-
liph.); 0.90 (t, 3H, J = 6.7 Hz, CH3aliph.); 13C NMR
(CDCl3, 75 MHz): d 109.3 and 108.5 (CiPr); 96.6 (C1);
72.0; 70.8; 70.4 (C2, C3, C4); 66.0 (C5); 60.3 (CH2OH);
56.1; 55.0 (CH2N); 31.9–22.7 (CH3iPr and CH2aliph.);
14.2 (CH3aliph.); Anal. Calcd for C26H49NO6: C,
66.21; H, 10.47; N, 2.97. Found C, 65.82; H, 10.93; N,
2.92.
1
2914; H NMR (DMSO-d6, 300 MHz): d 3.30 (m, 1H,
CHOH); 2.42 (dd, 1H, J = 3.5 Hz, 12.7 Hz, CHH0NH2);
2.30 (dd, 1H,J = 7.3 Hz, 12.7 Hz, CHH0NH2); 1.20 (m,
18H, CH2.); 0.81 (t, 3H, J = 6.6 Hz, CH3); 13C NMR
(CDCl3, 75 MHz): d 72.2 (CHOH); 47.5 (CH2NH2);
35.0–22.9 (CH2); 14.3 (CH3); Anal. Calcd for
C12H27NO: C, 71.58; H, 13.52; N, 6.96. Found C,
71.98; H, 13.64; N, 6.73.
5.4. General procedure for the preparation of the glycosy-
lated derivatives 13a–e, 14 and 15
5.4.4. 6-[(N-Tetradecyl)-2-hydroxyethylamino]-6-deoxy-
1,2:3,4-di-O-isopropylidene-a-D-galactopyranose (13d).
To a solution of 12 (1 mmol) in DMSO (5 mL) was
added a solution of the amino alcohol (1.2 mmol) in
DMSO (5 mL). The mixture was stirred at 90 °C for
48 h and the solution was concentrated under reduced
pressure. The crude product was dissolved in methylene
chloride and washed 3 times with water. After drying
with sodium sulfate the organic phase was concentrated
under reduced pressure. The residue was chromato-
graphed on silica gel (methylene chloride/methanol) to
furnish the desired compounds 13a–e, 14, and 15.
Oil; yield, 40%; [a]D: ꢀ21.9 (c 0.5, CHCl3); IR (m, cmꢀ1
,
CsI): 3488, 2928, 2856, 1070; 1H NMR (CDCl3,
300 MHz): d 5.54 (d, 1H, J = 5.0 Hz, H1); 4.62 (dd, 1H,
J = 2.4, 7.9 Hz, H3); 4.32 (dd, 1H, J = 2.4, 5.0 Hz, H2);
4.27 (dd, 1H, J = 2.2, 7.9 Hz, H4); 3.90 (m, 1H, H5);
3.61 (m, 2H, CH2OH); 2.64 (m, 6H, CH2N); 1.53; 1.45;
1.34; 1.33 (4s, CH3iPr); 1.26 (m, 24H, CH2aliph.); 0.88
(t, 3H, J = 6.7 Hz, CH3aliph.); 13C NMR (CDCl3,
75 MHz): d 109.3 and 108.7 (CiPr); 96.8 (C1); 72.2; 70.9;
70.7 (C2, C3, C4); 66.1 (C5); 60.5 (CH2OH); 56.2; 55.9;
55.5 (CH2N); 32.0–22.8 (CH3iPr and CH2aliph.); 14.3
(CH3aliph.); Anal. Calcd for C28H53NO6: C, 67.30; H,
10.69; N, 2.80. Found: C, 66.93; H, 10.78; N, 2.63.
5.4.1.
6-[(N-Octyl)-2-hydroxyethylamino]-6-deoxy-
1,2:3,4-di-O-isopropylidene-a-D-galactopyranose (13a).
Oil; yield, 45%; [a]D: ꢀ2.3 (c 0.5, CHCl3); IR (m, cmꢀ1
,
CsI): 3488, 2928, 2856, 1070; 1H NMR (CDCl3,
300 MHz): d 5.53 (d, 1H, J = 5.0 Hz, H1); 4.60 (dd,
1H, J = 2.4, 7.9 Hz, H3); 4.30 (dd, 1H, J = 2.4, 5.0 Hz,
H2); 4.25 (dd, 1H, J = 1.7, 7.9 Hz, H4); 3.90 (t, 1H,
J = 6.6 Hz, H5); 3.58 (m, 2H, CH2OH); 2.77 (m, 6H,
CH2N); 1.52; 1.44; 1.33; 1.32 (4s, CH3iPr); 1.24 (m,
12H, CH2aliph.); 0.89 (t, 3H, J = 6.7 Hz, CH3aliph.);
13C NMR (CDCl3, 75 MHz): d 109.3 and 108.6 (CiPr);
96.7 (C1); 72.1; 71.0; 70.7 (C2, C3, C4); 66.1 (C5); 59.3
(CH2OH); 56.0; 55.5; 53.5 (CH2N); 32.0–22.8 (CH3iPr
and CH2aliph.); 14.2 (CH3aliph.); Anal. Calcd for
C22H41NO6: C, 63.58; H, 9.94; N, 3.37. Found: C,
63.02; H, 9.91; N, 3.17.
5.4.5. 6-[(N-Hexadecyl)-2-hydroxyethylamino]-6-deoxy-
1,2:3,4-di-O-isopropylidene-a-D-galactopyranose (13e).
Oil; yield, 44%; [a]D: ꢀ133.5 (c 0.5, CHCl3); IR (m,
1
cmꢀ1, CsI): 3494, 2928, 2856, 1071; H NMR (CDCl3,
300 MHz): d 5.55 (d, 1H, J = 4.8 Hz, H1); 4.63 (m,
1H, H3); 4.34 (dd, 1H, J = 2.4, 5.0 Hz, H2); 4.29 (m,
1H, H4); 3.93 (t, 1H, J = 6.2 Hz, H5); 3.65 (m, 2H,
CH2OH); 2.95 (m, 6H, CH2N); 1.55; 1.47; 1.36; 1.34
(4s, CH3iPr); 1.27 (m, 28H, CH2aliph.); 0.89 (t, 3H,
J = 6.7 Hz, CH3aliph.); 13C NMR (CDCl3, 75 MHz): d
109.4 and 108.7 (CiPr); 96.8 (C1); 72.2; 71.0; 70.7 (C2,
C3, C4); 66.2 (C5); 59.2 (CH2OH); 56.9; 55.5; 53.6
(CH2N); 32.1–22.9 (CH3iPr and CH2aliph.); 14.4
(CH3aliph.); Anal. Calcd for C30H57NO6:C, 68.27; H,
10.89; N, 2.65. Found: C, 59.90; H, 10.91; N, 2.63.
5.4.2.
6-[(N-Decyl)-2-hydroxyethylamino]-6-deoxy-
1,2:3,4-di-O-isopropylidene-a-D-galactopyranose (13b).
Oil; yield, 40%; [a]D: ꢀ76.2 (c 0.5, CHCl3); IR (m,
1
cmꢀ1, CsI): 3480, 2914, 2849, 1070; H NMR (CDCl3,
5.4.6.
6-{[N-(2-Ethyl-hexyl)]-2-hydroxyethylamino}-6-
300 MHz): d 5.52 (d, 1H, J = 5.0 Hz, H1); 4.61 (dd,
1H, J = 2.4, 7.9 Hz, H3); 4.30 (dd, 1H, J = 2.4, 5.0 Hz,
H2); 4.25 (dd, 1H,J = 1.7, 7.9 Hz, H4); 3.88 (m, 1H,
H5); 3.55 (m, 2H, CH2OH); 2.76 (m, 6H, CH2N);
deoxy-1,2: 3,4-di-O-isopropylidene-a-D-galactopyranose
(14). Oil; yield, 56%; [a]D: ꢀ35.1 (c 0.5, CHCl3); IR (m,
1
cmꢀ1, CsI): 3494, 2928, 2858, 1071; H NMR (CDCl3,
300 MHz): d 5.47 (d, 1H, J = 5.0 Hz, H1); 4.54 (m,

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A. F. Taveira et al. / Bioorg. Med. Chem. 15 (2007) 7789–7794
1H, H3); 4.25 (dd, 1H, J = 2.4, 5.0 Hz, H3); 4.20 (m, 1H,
H2); 3.84 (t, 1H, J = 5.8 Hz, H5); 3.52 (m, 2H, CH2OH);
2.75 (m, 4H, HOCH2CH2 and CH2N); 2.27 (m, 2H,
EtCHCH2N); 1.46; 1.38; 1.28; 1.26 (4s, CH3iPr); 1.19
(m, 9H, [(CH2)3CHCH2CH3]; 0.84 (m, 6H, CH3aliph).
13C NMR (CDCl3, 75 MHz): d 109.4 and 108.8 (CiPr);
96.7 (C1); 72.2; 71.0; 70.6 (C2, C3, C4); 66.0 (C5); 60.4
(CH2OH); 59.2; 57.5; 54.5 (CH2N); 37.5 [(CH2)3CHCH2
CH3]; 31.4–23.1 (CH3iPr and CH2aliph.); 14.2 and 11.0
(CH3aliph.); Anal. Calcd for C22H41NO6: C, 63.58; H,
9.94; N, 3.37. Found: C, 63.97; H, 9.83; N, 4.76.
References and notes
1. World Health Organisation, http://www.who.int, 2007.
2. Janin, Y. L. Bioorg. Med. Chem. 2007, 15, 2479.
3. Katz, A. H.; Caufield, C. E. Curr. Pharm. Des. 2003, 9,
857.
4. Mc Neil, M.; Brennan, P. J. Res. Microbiol. 1991, 142,
451.
5. Besra, G. S.; Khoo, K.-H.; Mc Neil, M. R.; Dell, A.;
Morris, H. R.; Brennan, P. J. Biochem. 1995, 34,
4257.
6. Chatterjee, D. Curr. Opin. Chem. Biol. 1997, 1, 579.
7. Daffe, M.; Draper, P. Adv. Microb. Physiol. 1998, 39,
131.
5.4.7. 6-[N-2-Hydroxy-dodecylamino]-6-deoxy-1,2:34-di-
O-isopropylidene-a-D-galactopyranose (15). Oil; yield,
41%; IR (m, cmꢀ1, CsI): 3488, 2928, 2856, 1070; 1H
NMR (CDCl3, 300 MHz): d 5.50 (d, 1H, J = 4.8 Hz,
H1); 4.58 (dd, 1H, J = 2.4, 7.9 Hz, H3); 4.28 (dd, 1H,
J = 2.4, 4.8 Hz, H2); 4.16 (dd, 1H, J = 2.4, 7.9 Hz,
H4); 3.85 (m, 1H, H5); 2.94 (m, 1H, H6); 2.86 (m, 1H,
CHOH); 2.73 (m, 6H, CH2N); 1.49; 1.41; 1.29 (4s,
CH3iPr); 1.22 (m, 18H, CH2aliph.); 0.84 (t, J = 6.6 Hz,
CH3); 13C NMR (CDCl3, 75 MHz): d 109.3 and 108.6
(CiPr); 96.5 (C1); 72.0; 70.9; 70.7 (C2, C3, C4, CHOH);
66.8 (C5); 55.2 and 49.3 (CH2N); 35.1–22.7 (CH3iPr and
CH2aliph.); 14.2 (CH3aliph.); Anal. Calcd for
C24H44NO6: C, 64.98; H, 10.22; N, 3.16. Found: C,
64.45; H, 10.61; N, 3.18.
8. Pathak, A. K.; Pathak, V.; Seitz, L.; Maddry, J. A.;
Gurcha, S. S.; Besra, G. S.; Suling, W. J.; Reynolds, R. C.
Bioorg. Med. Chem. 2001, 9, 3129.
9. Pathak, A. K.; Pathak, V.; Suling, W. J.; Gurcha, S. S.;
Morehouse, C. B.; Besra, G. S.; Maddry, J. A.; Reynolds,
R. C. Bioorg. Med. Chem. 2002, 10, 923.
10. Wen, X.; Crick, D. C.; Brennan, P. J.; Hultin, P. G. .
Bioorg. Med. Chem. 2003, 1, 3579.
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N.; Orme, I. M.; Suling, W. J. Bioorg. Med. Chem. Lett.
1998, 8, 237.
12. Tripathi, R. P.; Tripathi, R.; Tiwari, V. K.; Bala, L.;
Sinha, S.; Srivastava, A.; Srivastava, R.; Srivastava, B. S.
Eur. J. Med. Chem. 2002, 37, 773.
13. Tewari, N.; Tiwari, V. K.; Mishra, R. C.; Tripathi, R. P.;
Srivastava, A. K.; Ahemad, R.; Srivastava, R.; Srivastava,
B. S. Bioorg. Med. Chem. 2003, 11, 2911.
14. Tewari, N.; Tiwari, V. K.; Tripathi, R. P.; Gaikwad, A.;
Sinha, S.; Shukla, P. K.; Srivastava, R.; Srivastava, B. S.
Bioorg. Med. Chem. Lett. 2004, 12, 329.
15. Katiyar, D.; Tiwari, V. K.; Tewari, N.; Verma, S. S.;
Sinha, S.; Gaikwad, A.; Srivastava, A.; Chaturvedi, V.;
Srivastava, R.; Srivastava, B. S.; Tripathi, R. P. Eur. J.
Med. Chem. 2005, 40, 351.
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F.; Prado, M. A.; Alves, R. B.; Silva, C. L. Pharmazie
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5.5. Antitubercular activity
The antitubercular activity of the tested compounds was
determined by incorporating decreasing concentrations
to a culture of M. tuberculosis H37Rv (ATCC 27294)
in Middlebrook 7H9 agar medium using the Microplate
Alamar Blue Assay (MABA).24 MIC values represent
means of three separate experiments. Rifampicin was
used as the reference compound.
17. Almeida, M. V.; Le Hyaric, M.; Amarante, G.; Silva
Lourenc¸o, M. C.; Lima Branda˜o, M. L. Eur. J. Med.
Chem. 2007, 42, 1076.
5.6. MTT colorimetric assay
Cell proliferation was measured using the MTT [3-(4,5-
dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide]
test. Culture plates without supernatants were incubated
with 100 lL of supplemented RPMI-medium and 10 lL
of MTT (5 mg/mL) for 4 h at 37 °C in 5% CO2. After
this time, the reaction was stopped by adding to each
weel 100 lL of an acidic isopropanol solution (100 mL
of isopropyl alcohol/0.4 mL of HCl 10 N). Following
10 min incubation at room temperature, the optical den-
sity (OD) values at 570 nm were determined (Spectra-
max 190—Molecular Devices—US). For determination
of cell proliferation and cytotoxicity the following for-
mulae were used:
18. Piłakowska-Pietras, D.; Lunkenheimer, K.; Piasecki, A.
Langmuir 2004, 20, 1572.
19. Edebo, L.; Sandin, M. US Patent 5,013,146, 1992.
20. Reasenberg, J. R.; Goldberg, S. D. J. Am. Chem. Soc.
1945, 67, 933.
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1503.
22. Foglia, T. A.; Swern, D. J. Org. Chem. 1967, 32, 75.
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24. Collins, L.; Franzblau, S. G. Antimicrob. Agents Chemo-
ther. 1997, 41, 1004.
25. Shepherd, J. M.; Large, P. J.; Midgley, M.; Ratle-
edge, C. World J. Microbiol. Biotechnol. 1998, 14,
535.
Cell proliferation ð%Þ ¼ ðOD
Cytotoxicity ð%Þ ¼ ½1ꢀðOD
=ODðJ774A:1=IFNÞÞꢁ100
ðJ774A:1=IFN=compoundÞ
26. Gernon, M. D.; Dowling, C. M.; Trumpfheller, C. M. US
Patent 0107270, 2005.
=ODðJ774A:1ÞÞꢂꢁ100:
ðJ774A:1=compoundÞ