Preparation of amino alcohols condensed with carbohydrates: Evaluation of cytotoxicity and inhibitory effect on NO production

Article abstract of DOI:10.1111/j.1747-0285.2010.01026.x

This work reports the preparation of several amino alcohols condensed with D-arabinose, D-glucose, and D-galactose derivatives. These compounds were evaluated in vitro for their cytotoxicity and ability to decrease nitric oxide production in J774A.1 cells

Full text of DOI:10.1111/j.1747-0285.2010.01026.x

ª 2010 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.01026.x
Chem Biol Drug Des 2010; 76: 451–456
Research Letter
Preparation of Amino Alcohols Condensed with
Carbohydrates: Evaluation of Cytotoxicity and
Inhibitory Effect on NO Production
1
1,2
for the biologic activity of the drugs (5). Zhang et al. (6,7) have
investigated the effects of N-alkylated iminosugars on immune sys-
tem, showing that these compounds hold potential as immunosup-
pressive agents. A large number of lipidic amino alcohols and
derivatives have been described in the literature and their apoptotic
and immunosuppressive activities tested (4,5,8–11). In this context,
this work describes the synthesis of several amino alcohols con-
densed with three different carbohydrates and the biologic evalua-
tion of their cytotoxic properties and their ability to decrease NO
production. The aim of this study was to evaluate the influence of
amino alcohol and carbohydrate moieties in this activity.
ˆ
´
Taı´s A. Correa , Elaine F. C. Reis , Lıvia L.
Alves2, Caio C. S. Alves2, Sandra B. R.
Castro2, Alyria T. Dias2, Aline F. Taveira1,
Mireille Le Hyaric1, Mara R. C. Couri1,
Ana P. Ferreira2 and Mauro V. de Almeida1,*
1Departamento de Quꢀmica, ICE, Universidade Federal de Juiz de
Fora, 36036-330 Juiz de Fora, MG, Brazil
2Departamento de Parasitologia, Microbiologia e Imunologia, ICB,
Universidade Federal de Juiz de Fora, 36036-330 Juiz de Fora, MG,
Brazil
*Corresponding author: Mauro V. de Almeida,
mauro.almeida@ufjf.edu.br
Experimental
This work reports the preparation of several amino
alcohols condensed with D-arabinose, D-glucose,
and D-galactose derivatives. These compounds
were evaluated in vitro for their cytotoxicity and
ability to decrease nitric oxide production in
J774A.1 cells. Arabinofuranoside derivatives 5a,
5b and 5c showed a significant inhibition of nitric
oxide production (>80% at 5 lg ⁄ mL), while the
galactopyranoside derivative 8d showed a nota-
ble nitric oxide inhibitory activity (126% at
0.5 lg ⁄ mL).
General methods
Melting points were determined on a Microquꢀmica MQAPF apparatus
(Microquꢀmica Ltda, Cotia, SP-Brazil) and are uncorrected. IR spectra
were recorded using a BOMEM-FTIR MB102 spectrometer (ABB
Bomem Inc., Quebec, Canada). Optical rotations were measured with
a Perkin-Elmer 341 polarimeter (Perkin Elmer, Waltham, MA, USA),
using a sodium lamp at 20 ꢀC. 1H and 13C NMR spectra were
recorded on Bruker Advance DRX300 spectrometer (Bruker Corpora-
tion, Billerica, MA, USA). Thin-layer chromatography (TLC) was per-
formed on glass plates and silica gel sheets (Silica Gel F254; Merck,
Whitehouse Station, NJ, USA), visualized with iodine vapor, and ⁄ or
revealed with ethanolic H2SO4 solution. Column chromatography was
carried out on silica gel 60 (E. Merck 70–230 mesh). Solvents were
purchased from Vetec Quꢀmica (Vetec, Xerem, RJ, Brazil) and were
distilled prior to use. Reagents were purchased from Aldrich (Sigma
Aldrich, Saint Louis, MI, USA) and used without further purification.
Key words: amino alcohols, amphiphilicity, carbohydrate, cytotoxic-
ity, nitric oxide production
Received 29 January 2010, revised 7 July 2010 and accepted for publi-
cation 20 August 2010
The immune system consists of the innate and the acquired
immune responses (1). The knowledge of the main immune defense
mechanisms allows an understanding of the body's immune
response against various diseases (1). Nitric oxide (NO) is an impor-
tant molecule in innate immunity, mediating diverse functions and
also regulating the adaptive response. NO acts as an essential
multifunctional mediator in various biologic systems (2). Immunosup-
pressive drugs are the principal compounds that can affect the
immune response, reducing the activation of the innate and the
acquired immunity (3). These drugs play an important role in clinical
transplant patients and in the treatment of autoimmune diseases
such as multiple sclerosis, rheumatoid arthritis, and systemic lupus
erythematosus (2,4). The sugar component participates in the molec-
ular recognition of the cellular target, in many cases being essential
Preparation of methyl 5-O-p-toluenesulfonyl-a-
D-arabinofuranoside 3
p-Toluenesulfonyl chloride (22 mmol) was added to a mixture of
alcohol 2 (20 mmol) in pyridine (15 mL) at 0 ꢀC. The mixture was
stirred at room temperature for 24 h and then concentrated under
reduced pressure. The residue was dissolved in methylene chloride
and washed three times with water. After evaporation of the
organic phase, the residue was purified by column chromatography
to furnish the desired compound 3 in 56% yield (11.2 mmol).
[a]D: +3.0 (c 0.69, CH2Cl2); IR (m, cm)1, KRS-5): 3435, 1396, 863,
1
665; H NMR (CDCl3, 300 MHz): d 7.80 (d, 2H, J2¢,3¢ 6.0 Hz, H2¢ and
451

ˆ
Correa et al.
H6¢); 7.35 (d, 2H, J3¢,2¢ 6.0 Hz, H3¢ and H5¢); 4.81 (s, 1H, H1); 4.22–
4.05 (m, 4H, H2, H3, H4, H5a); 3.95–3.92 (m, 1H, H5b); 3.34 (s, 3H,
OCH3); 2.44 (s, 3H, CH3); 13C NMR (CDCl3, 75 MHz): d 145.3 (C4¢);
132.5 (C1¢); 130.0 (C3¢ and C5¢); 128.1 (C2¢and C6¢); 108.8 (C1);
82.3, 80.7, 77.6 (C2, C3, C4); 69.4 (C5); 55.2 (OCH3); 21.7 (CH3).
(CDCl3, 300 MHz): d 4.73 (s, 1H, H1); 4.16 (s, 1H, H4); 3.90 (s, 1H,
H3 or H2); 3.85 (s, 1H, H3 or H2); 3.60 (s, 2H, CH2OH); 3.33 (s, 3H,
OCH3); 2.80–2.38 (m, 6H, H5, H5¢ and CH2N); 1.42 (s, 2H,
NCH2CH2CH2aliph); 1.19 (s, 18H, CH2aliph); 0.81 (t, 3H, J 6.6 Hz,
CH3); 13C NMR (CDCl3, 75 MHz): d 109.9 (C1); 87.6, 79.3, 77.8 (C4,
C2, C3); 60.4–56.7 (C5, CH2N and CH2OH); 55.2 (OCH3); 32.1–22.9
(CH2aliph); 14.3 (CH3).
General procedure for the preparation of amino
alcohols 4, 5a-c, and 6
Methyl
5-[N-2-hydroxy-dodecylamino]-5-deoxy-a-D-
Tosylate 3 (1 mmol) was dissolved in ethanol (3 mL) and slowly
added to a solution of the amino alcohols: ethanolamine, N-alkyl-
aminoethanols (N-decyl-aminoethanol, N-dodecyl-aminoethanol), or
1-amino-dodecan-2-ol (1.2–2 mmol). The mixture was stirred at
reflux for 24–48 h, and the solution was concentrated under
reduced pressure. The crude product was dissolved in methylene
chloride and washed three times with water. After drying with
sodium sulfate, the organic phase was concentrated under reduced
pressure. The residue was chromatographed on silica gel (methy-
lene chloride ⁄ methanol) to furnish the desired compounds 5a–c
and 6. To obtain amino alcohol 4, the crude product was chromato-
graphed directly on silica gel (CH2Cl2 ⁄ MeOH).
arabinofuranoside 6. yield, 30%; [a]D: +35.5 (c 0.39, CH3OH);
1
IR (m, cm)1, KRS-5): 3366, 2922, 1099; H NMR (CDCl3, 300 MHz): d
4.89 (d, 1H, J1,2 3.1 Hz, H1); 4.23 (s, 1H, H4); 3.98–3.71 (m, 5H, H2,
H3, CHOH, OH and NH); 3.38 (s, 3H, OCH3); 3.13–3.03 (m, 1H, H5);
2.94–2.80 (m, 2H, H5¢ and H6); 2.70–2.59 (m, 1H, H6¢) 1.40 (s, 2H,
NCH2CH2CH2aliph); 1.25 (s, 16H, CH2aliph); 0.88 (t, 3H, J 6.1 Hz,
CH3); 13C NMR (CDCl3, 75 MHz): d 110.1 (C1); 86.2, 79.9, 78.7 (C4,
C2, C3); 69.9 (CH(OH)CH2aliph); 56.0 and 56.2 (OCH3); 55.2, 49.7,
49.8 (C5, CH2N); 35.5–22.9 (CH2aliph); 14.3 (CH3).
General procedure for the preparation of
compounds 8a-e, 9, 11, and 12
A solution of the amino alcohol (3.0 mmol) in ethanol (5 mL) was
added to a solution of 7 or 10 (2 mmol) in ethanol (3 mL). The mix-
ture was stirred at reflux for 48–120 h, and the solution was con-
centrated under reduced pressure. The crude product was dissolved
in methylene chloride and washed three times with water. After
drying with sodium sulfate, the organic phase was concentrated
under reduced pressure. The residue was chromatographed on silica
gel (methylene chloride ⁄ methanol) to furnish the desired compounds
8a–e and 9. To obtain amino alcohols 11 and 12, the crude prod-
ucts were directly chromatographed on silica gel (CH2Cl2 ⁄ MeOH).
Spectral data
Methyl
5-(2-hydroxyethylamino)-5-deoxy-a-D-arabi-
nofuranoside 4. yield, 89%; [a]D: +37.3 (c 0.5, CH3OH); IR (m,
cm)1, KRS-5): 3390, 1624, 1067; H NMR (CD3OD, 300 MHz): d 4.69
1
(s, 1H, H1); 3.93 (s, 1H, H4); 3.85 (s, 1H, H2); 3.65–3.52 (m, 3H, H3
and CH2OH); 3.29 (s, 3H, OCH3); 2.86–2.82 (m, 1H, H5); 2.75–2.69
(m, 3H, H5¢ and CH2N); 13C NMR (CD3OD, 75 MHz): d 110.9 (C1);
83.4, 83.1, 81.1 (C4, C2, C3); 60.6 (CH2OH); 55.5 (OCH3); 52.2; 52.1
(C5 and CH2N).
Methyl 5-[(N-octyl)-2-hydroxyethylamino]-5-deoxy-a-D-
arabinofuranoside 5a. yield, 32%; [a]D: +39.7 (c 0.3, CH3OH); IR
(m, cm)1, KRS-5): 3379, 2924, 1466, 1020; 1H NMR (CDCl3, 300 MHz):
d 4.90 (s, 1H, H1); 4.22 (s, 1H, H4); 3.98 (s, 1H, H3); 3.92 (s, 1H, H2);
3.68 (t, 2H, J7,6 3.5 Hz, CH2OH); 3.39 (s, 3H, OCH3); 2.92–2.78 (m, 3H,
H5 and CH2N); 2.72–2.65 (m, 2H, CH2N); 2.63–2.47 (m, 1H, H5¢); 1.50
(s, 2H, NCH2CH2CH2); 1.28 (s, 10H, CH2aliph); 0.88 (t, 3H, J 6.8 Hz,
CH3); 13C NMR (CDCl3, 75 MHz): d 109.9 (C1); 87.5, 79.3, 78.0 (C4, C2,
C3); 60.4–56.7 (C5, CH2N and CH2OH); 55.2 (OCH3); 32.0–22.8
(CH2aliph); 14.3 (CH3).
Spectral data
6-(2-Hydroxypropylamino)-6-deoxy-1,2:3,4-di-O-iso-
propylidene-a-D-galactopyranose 8a. yield, 68%; [a]D:
)33.6 (c 0.5, CH2Cl2); IR (m, cm)1, KRS-5): 3305, 2985, 1064; 1H
NMR (CDCl3, 300 MHz): d 5.50 (d, 1H, J1,2 4.8 Hz, H1); 4.57 (d, 1H,
J3,4 7.9 Hz, H3); 4.28 (d, 1H, J2,1 4.8 Hz, H2); 4.15 (d, 1H, J4,3
7.9 Hz, H4); 3.87 (s, 1H, H5); 3.77 (m, 2H, CH2OH); 3.06 (m, 2H,
CH2N); 2.88–2.76 (m, 4H, H6 and OH ⁄ NH); 1.68 (s, 2H, CH2); 1.31;
1.43; 1.50 (3s, 12H, CH3); 13C NMR (CDCl3, 75 MHz): d 109.5 and
108.8 (CiPr); 96.5 (C1); 72.1, 71.0, 70.7 (C4, C3, C2); 66.8 (C5); 64,2
(CH2OH); 49.5 and 49.4 (CH2N); 30.7 (CH2); 26.2; 26.1; 25.1; 24.5
(CH3iPr).
Methyl 5-[(N-decyl)-2-hydroxyethylamino]-5-deoxy-a-D-
arabinofuranoside 5b. yield, 39%; [a]D: +17.7 (c 0.23, CH3OH);
IR (m, cm)1, KRS-5): 3375, 2922, 1464, 1024; 1H NMR (CDCl3,
300 MHz): d 4.91 (s, 1H, H1); 4.24 (s, 1H, H4); 3.97 (s, 1H, H3); 3.92 (s,
1H, H2); 3.69 (s, 3H, CH2OH and OH); 3.40 (s, 3H, OCH3); 2.92–2.76 (m,
3H, H5 and CH2N); 2.67–2.62 (m, 2H, CH2N); 2.55–2.50 (m, 1H, H5¢);
1.49 (s, 2H, NCH2CH2CH2); 1.26 (s, 14H, CH2aliph); 0.88 (t, 3H, J
6.4 Hz, CH3); 13C NMR (CDCl3, 75 MHz): d 109.9 (C1); 88.0, 79.2, 77.6
(C4, C2, C3); 60.6–56.7 (C5, CH2N and CH2OH); 55.2 (OCH3); 32.1–22.9
(CH2aliph); 14.3 (CH3).
6-[(2¢-Hydroxy-1¢,1¢-dimethyl)-ethylamino]-6-deoxy-
1,2:3,4-di-O-isopropylidene-a-D-galactopyranose 8b.
White crystals; mp, 78.5–79.6 ꢀC; yield, 74%; [a]D: )60.5 (c 0.6,
CH2Cl2); IR (m, cm)1, KRS-5): 3294, 3211, 2871, 1067; 1H NMR
(CDCl3, 300 MHz): d 5.52 (d, 1H, J1,2 3.9 Hz, H1); 4.59 (d, 1H, J3,4
7.7 Hz, H3); 4.31–4.23 (m, 2H, H2 and H4); 3.80 (s, 1H, H5); 3.34
(d, 1H, J 10.3 Hz, CH2OH); 3.23 (d, 1H, J 10.3 Hz, CH2OH); 2.82 (dd,
1H, J6,5 8.8 Hz, J6,6¢ 10.5 Hz, H6); 2.63–2.61 (m, 1H, H6¢); 2.28 (s,
2H, OH and NH); 1.53; 1.44; 1.31 (3s, 12H, CH3); 1.06 (s, 6H, CH3)
13C NMR (CDCl3, 75 MHz): d 109.5 and 108.8 (CiPr); 96.5 (C1);
72.0, 70.9, 70.7 (C4, C3, C2); 68.0 (C5); 67.9 (CH2OH); 54.0
Methyl 5-[(N-dodecyl)-2-hydroxyethylamino]-5-deoxy-
a-D-arabinofuranoside 5c. yield, 30%; [a]D: )25.9 (c 0.15,
CH3OH); IR (m, cm)1, KRS-5): 3385, 2923, 1466, 1028; 1H NMR
452
Chem Biol Drug Des 2010; 76: 451–456

Preparation of Amino Alcohols Condensed with Carbohydrates
(NC(CH3)2); 42.0 (CH2N); 26.3, 25.4, 25.1, 24.9 (CH3); 24.6 and 23.6
(CH3).
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
well 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 density (OD) values at 570 nm were deter-
mined (Spectramax 190; Molecular Devices, Sunnyvale, CA, USA).
N,N-bis-6-(deoxy-1,2:3,4-di-O-isopropylidene-a-D-
galactopyranose)-3-hydroxypropylamino 9. yield, 12%;
[a]D: )69.5 (c 0.5, CH2Cl2); IR (m, cm)1, KRS-5): 3365, 2983, 1068;
1H NMR (CDCl3, 300 MHz): d 5.49 (d, 2H, J1,2 4.8 Hz, H1); 4.54 (d,
2H, J3,4 4.5 Hz, H3); 4.23–4.20 (m, 4H, H2, and H4); 3.92 (s, 2H,
H5); 3.71 (s, 2H, CH2OH); 3.17 (s, 1H, OH); 2.83–2.63 (m, 6H, CH2N);
1.73–1.64 (s, 2H, CH2); 1.55; 1.44; 1.33 (3s, 24H, CH3); 13C NMR
(CDCl3, 75 MHz): d 109.1 and 108.5 (CiPr); 96.6 (C1); 72.0, 70.9,
70.6 (C4, C3, C2); 65.9 (C5); 62.4 (CH2OH); 54.7 and 53.9 (CH2N);
29.1 (CH2); 26.2, 25.2, 25.0, 24.4 (CH3).
Griess method
Supernatants of J774A.1 cells were analyzed for the quantification
of nitrites (NOꢀ2 ) through the Griess method. Aliquots of superna-
tants were plated with 1% of sulfanilamide and 0.1% of N-(1-naph-
thyl) ethylenediamine. NOꢀ2 production was quantified by
comparing with a standard curve, made with different concentra-
tions of NaNO2. The reading was made in the microplate reader
(Spectramax 190; Molecular Devices) at 540 -nm wavelength.
Methyl 6-(2-hydroxypropylamino)-6-deoxy-a-D-gluco-
pyranoside 11. yield, 62%; [a]D: +128.1 (c 0.5, CH3OH); IR (m,
1
cm)1, KRS-5): 3361, 2920, 1047; H NMR (CD3OD, 300 MHz): d 4.53
(d, 1H, J1,2 6.0 Hz, H1); 3.52–3.44 (m, 3H, H2, H3, H5) 3.28–3.21
(m, 5H, CH2OH and OCH3); 3.0 (t, 1H, J4,5 9.0 Hz, H4); 2.87–2.82
(dd, 1H, J6,5 3.0 Hz, J6,6¢ 12.0 Hz, H6); 2.61–2.52 (m, 3H, H6¢ and
CH2N); 2.01–1.99 (m, 1H, NH ⁄ OH); 1.63–1.58 (m, 2H, CH2); 13C
NMR (CD3OD, 75 MHz): d 101.4 (C1); 75.1, 74.4, 73.7 (C4, C3, C2);
71.1 (C5); 61.7 (CH2OH); 56.0 (OCH3); 52.2, 48.2 (C6 and CH2N); 33.1
(CH2).
Results and Discussion
Initially, arabinofuranoside amino alcohol derivatives were synthe-
sized (Scheme 1). D-arabinose 1 was first reacted with methanol
under acidic conditions (Amberlite IR-120, Sigma-Aldrich) leading
to methyl a-D-arabinofuranoside 2 (73% yield), which was sub-
sequently treated with p-toluenesulfonyl chloride in pyridine to
give compound 3 at 56% yield. This tosylated derivative was
then treated with different amino alcohols in ethanol under
reflux, furnishing the desired compounds 4, 5a–c, and 6 at
30–89% yields. The low yield in the preparation of the amino
alcohols 5a–c and 6 was mainly because of the formation of
the methyl 2,5-anydro-a-D-arabinofuranoside.
Methyl 6-[(2¢-hydroxy-1¢,1¢-dimethyl)-ethylamino]-6-
deoxy-a-D-glucopyranoside 12. White crystals; mp, 98–
99.6 ꢀC; yield, 50%; [a]D: +92.3 (c 0.33, CH3OH); IR (m, cm)1, KBr):
1
3541, 3263, 2970, 1043; H NMR (CD3OD, 300 MHz): d 4.64 (d, 1H,
J1,2 3.6 Hz, H1); 3.62–3.56 (m, 2H, H3 and H5) 3.40–3.31 (m, 5H,
H2, NH or OH and OCH3); 3.28 (d, 2H, J 9.9 Hz, CH2OH); 2.93 (t,
1H, J4,5 9.0 Hz, H4); 2.94–2.89 (dd, 1H, J6,5 2.4 Hz, J6,6¢ 11.6 Hz,
H6); 2.62 (dd, 1H, J6¢,5 2.4 Hz, H6¢); 1.04 (s, 6H, CH3); 13C NMR
(CD3OD, 75 MHz): d 101.4 (C1); 75.0, 74.8, 73.7 (C4, C3, C2); 72.0
(C5); 69.4 (CH2OH); 56.0 (OCH3); 54.8 (NC(CH3)2CH2OH); 45.0 (C6);
24.2; 23.1 (CH3).
D-Galactose derivatives were prepared using a method previously
described (10). D-galactose was first converted into 6-deoxy-6-iodo-
1,2:3,4-di-O-isopropylidene-a-D-galactopyranose 7 according to the
literature procedure (11–13) and then treated with five different
amino alcohols in ethanol under reflux (Scheme 2). Purification of
the crude products was carried out by column chromatography (hex-
ane:ethyl acetate) resulting in the isolation of amino alcohols 8a–e
at 40–80% yields. In this series, reaction of iodided derivative 7
with 1,3-propanolamine resulted in the formation of amino alcohol
9 at 12% yield.
Cytotoxicity and inhibition of NO production
MTT assay
Cell proliferation was measured using the MTT [3-(4,5-dimethyl-
thiazolyl-2)-2,5-diphenyltetrazolium bromide] test. Culture plates
CHO
HO
H
TsO
HO
H
H
N
O
O
O
HOCH2CH2
c
OH
OH
OH
a
OH
OCH3
b
OH
H
OCH3
OCH3
CH2OH
4
OH
2
OH
Scheme 1: Preparation of
D-arabinose derivatives. Reagents
and conditions: (a) Amberlite-
IR120, MeOH, 60 ꢀC (73%);
(b) TsCl, pyridine, 0 ꢀC-r.t. (56%);
(c) ethanolamine, MeOH, reflux
(89%); (d) CH3(CH2)nCH2NHCH2CH2-
OH, EtOH, reflux (30–39%); (e)
NH2CH2CH(OH)(CH2)9CH3, EtOH,
reflux (30%).
OH
3
1
d
e
CH2(CH2)nCH3
H
N
HOCH2CH2
N
CH3(CH2)9CHCH2
HO
O
HO
O
OH
OCH3
OCH3
5 a-c
6
OH
OH
a: n = 6
b: n = 8
c: n = 10
Chem Biol Drug Des 2010; 76: 451–456
453

ˆ
Correa et al.
O
O
O
R
O
I
O
O
O
O
O
O
O
O
N
CH2CH2CH2OH
O
a
O
Scheme 2: Preparation of
D-galactose derivatives. Reagents
and conditions: (a) 8a: NH2CH2-
CH2CH2OH, EtOH, reflux (68%);
8b: NH2C(CH3)2CH2OH, EtOH,
reflux (74%); 8c: NH2CH2CH2OH,
EtOH, reflux (80%); 8d–e:
O
O
O
O
O
9
7
8a-e
O
a: R= NHCH2CH2CH2OH
b: R= NHC(CH3)2CH2OH
c: R= NHCH2CH2OH
d: R= N(CH2CH2OH)CH2(CH2)8CH3
e: R= N(CH2CH2OH)CH2(CH2)10CH3
NH(CH2CH2OH)CH2(CH2)nCH3,
DMSO, 90 ꢀC (40–56%).
C(CH3)2CH2OH
O
CH2CH2CH2OH
OTs
HN
HN
O
O
a
b
HO
HO
HO
HO
HO
HO
OH
OH
OH
12
11
10
OCH3
OCH3
OCH3
Scheme 3: Preparation of D-glucose derivatives. Reagents and conditions: (a) NH2CH2CH2CH2OH, EtOH, reflux (62%); (b)
NH2C(CH3)2CH2OH, EtOH, reflux (50%).
Amino alcohol derivatives 11 and 12 were obtained from methyl
a-D-glucopyranoside, which was first converted into its tosylate 10.
The later compound was treated with 1,3-propanolamine and
2-amino-2-methylpropanol giving amino alcohols 11 and 12,
respectively (Scheme 3). All the compounds were purified by column
based on the cell viability in the presence of serial concentrations
of the tested compounds. The percentage of cytotoxicity was calcu-
lated from the mean of optical density (OD) measures according to
the formula CTX (%) = [1)(OD(J774A.1 + compound) ⁄ OD(J774A.1))] · 100.
None of the tested compounds demonstrated cytotoxic activity at
low concentrations (0.5–0.00005 lg ⁄ mL) (Table 1).
1
chromatography. The structure was assigned by Infrared, H NMR,
13C NMR, and COSY experiments.
The ability of the compounds to decrease NO production was evalu-
ated by the Griess method using J744A.1 cell culture stimulated
with interferon-gamma (IFNc) plus lipopolysaccharide (LPS).
NG-monomethyl-L-arginine (LNMA) was used as a positive standard
Cytotoxicity and inhibition of NO production
The cytotoxicity of the amino alcohols on J774A.1 cell culture was
determined by a colorimetric MTT assay. The determination was
Table 1: Inhibition of nitric oxide (NO) production and cytotoxicity (CTX) evaluation of glycosylated amino alcohols. J774A.1 cells were
stimulated by IFN-c ⁄ LPS and treated by serial concentrations of glycosylated amino alcohols per 48 h
Inhibition of NO, and cytotoxicity (%) by different compound concentrations
5 lg ⁄ mL
0.5 lg ⁄ mL
0.05 lg ⁄ mL
0.005 lg ⁄ mL
0.0005 lg ⁄ mL
0.00005 lg ⁄ mL
Compound
NOa
CTXb
NO
CTX
NO
CTX
NO
CTX
NO
CTX
NO
CTX
4
5a
5b
5c
6
8a
8d
9
53.9
81.2
27.9
nsc
ns
50.6
48.8
71.8
30.6
71.3
62.9
126
52.4
42.6
44.9
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
56.2
60.2
61.7
ns
60.1
62.0
17
60.1
34.3
41.7
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
51.4
59.2
54.4
ns
62.3
55.8
13
56.4
34.8
42.3
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
53.4
47.3
41.5
ns
63.7
53.0
–
47.6
21.8
46.3
ns
ns
ns
ns
ns
ns
–
ns
ns
ns
38.7
ns
ns
ns
ns
ns
ns
ns
ns
–
ns
ns
ns
106.6
121.0
132.2
67.5
ns
ns
36.3
24.9
–
ns
ns
42.0
25.0
–
30.3
ns
d
–
51.5
46.3
45.3
11
12
ns
ns
aInhibition of NO production in percentage NO (%) = [1) (NO(J774A.1 + stimulus + compound))NO(J774A.1 + LNMA) ⁄ NO(J774A.1 + stimulus))NO(J774A.1 + LNMA))] · 100.
bCytotoxicity percentage CTX (%) = [1) (OD(J774A.1 + compound) ⁄ OD(J774A.1))] · 100.
cNon-significant values.
dNon-tested dosages.
454
Chem Biol Drug Des 2010; 76: 451–456

Preparation of Amino Alcohols Condensed with Carbohydrates
for NO inhibition (100%). The percentage of NO inhibition was cal-
culated using the following formula, using the mean of NO results:
NO (%) = [1) (NO(J774A.1 + stimulus + compound))NO(J774A.1 + LNMA)
Table 2: Nitric oxide (NO) inhibition (%) and log (P) at a concen-
tration of 5 lg ⁄ mL
⁄
Compound
Inhibition of NO
Log (P)
NO(J774A.1 + stimulus))NO(J774A.1 + LNMA))] · 100. The highest inhibi-
tion of NO production was obtained by arabinofuranoside derivative
6 at 5 lg ⁄ mL. However, it was cytotoxic at this concentration. In
lower concentrations (0.5–0.0005 lg ⁄ mL), this compound was not
cytotoxic and inhibited macrophage NO production by more than
60%. Other arabinofuranoside derivatives 5a, 5b, and 5c also
showed a significant inhibitory effect on NO production, above
80%, at 5 lg ⁄ mL concentration. The derivative 8d showed a nota-
ble NO inhibition (126% at 0.5 lg ⁄ mL). Although not cytotoxic, the
glucopyranoside derivatives 11 and 12 did not display significant
NO inhibitory activity in relation to other substances tested. Sec-
ondary amines 4 (arabinofuranoside) and 8a (galactopyranoside)
were less active than tertiary amines 5a, 5b, 5c, and 8d, at the
concentration of 5 lg ⁄ mL. Compounds 4 and 8a showed elevated
cytotoxicity at this same concentration.
11
4
46.3
53.9
45.3
51.5
81.2
)2.39
)1.86
)1.75
)1.05
1.02
12
9
5a
5b
5c
106.6
121.0
1.85
2.68
Log (P) values were calculated using Chemdraw Ultra 12 software (trial ver-
sion).
These results confirm the existence of a relationship between lipo-
philicity and inhibition of NO production using a 5 lg ⁄ mL concen-
tration of the tested compounds.
The studies on detailed immunosuppressive mechanism of amino
alcohols condensed with carbohydrate are still under investigation.
All alkylated compounds containing an unprotected carbohydrate
(compounds 5a–c and 6) displayed a good inhibition of NO produc-
tion at 5 lg ⁄ mL, while non-alkylated compounds 11 and 12 or
alkylated amino alcohols with a protected carbohydrate were less
active. These results suggest that the carbohydrate moieties of the
molecule play a part in the biologic activity and that amphiphilicity
could be essential.
Conclusion
In summary, a series of amino alcohols condensed with D-arabi-
nose, D-glucose, and D-galactose derivatives were prepared and
evaluated in vitro for their cytotoxicity and ability to influence NO
production in J774A.1 cells. Arabinofuranoside derivatives 5a, 5b,
and 5c showed a significant inhibition of NO production (>80%
at 5 lg ⁄ mL concentration), while galactopyranoside derivative 8d
showed a notable inhibitory activity on NO production (126% at
0.5 lg ⁄ mL). It seems that amphiphilicity is important for the bio-
logic activity.
A correlation between biologic activity and hydrophobic character is
frequently observed (14). The partition coefficient P of a compound in
an n-octanol ⁄ water system represents the hydrophobic properties of
this compound and can be determined experimentally or calculated.
Considering the inhibition of NO production for the non-toxic com-
pounds at a concentration of 5 lg ⁄ mL, we could establish a linear
correlation between NO inhibition (%) and log (P) (Table 2, Figure 1).
Acknowledgments
Linear regression led to the following function:
The authors gratefully acknowledge CAPES and CNPq for fellow-
ships. This research was supported by FAPEMIG.
NO inhibition ¼ 14:94 log ðPÞ þ 75:45 with a correlation coefficient
of 95%
y
160
140
120
100
80
60
40
20
Figure 1: Linear correlation
x
between nitric oxide (NO) inhibition
(%) and log (P ) at a concentration
of 5 lg ⁄ mL.
–3.5 –3 –2.5 –2 –1.5 –1 –0.5
–20
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
Chem Biol Drug Des 2010; 76: 451–456
455

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