Steroid and bile acids amide conjugates with D-glucosamine

Article abstract of DOI:10.1135/cccc2010105

New type of amide conjugates of steroid and bile acids with D-glucosamine 1 and 2 were prepared. Title compounds are prepared via acid chloride or using N-[({[(1E)-1-cyano- 2-ethoxy-2-oxoethylidene]amino}oxy)(dimethylamino) methylidene]-N-methylmethanaminium tetrafluoroborate as condensation agent. They were examined for gelation properties with negative results. Per-O-acetylated D-glucosamine hydrochloride was prepared in one step procedure from D-glucosamine hydrochloride by acetylation in a mixture of acetyl chloride and acetic acid.

Full text of DOI:10.1135/cccc2010105

D-Glucosamine Steroid Conjugates
65
STEROID AND BILE ACIDS AMIDE CONJUGATES WITH
D-GLUCOSAMINE
1
2,
Zdena NOVÁKOVÁ , Jana TOMANOVÁ, Lucie ŠTĚRBOVÁ and Pavel DRAŠAR *
Institute of Chemical Technology, Prague, Technická 5, 166 28 Prague 6, Czech Republic;
1
2
e-mail: novakovz@vscht.cz, pavel.drasar@vscht.cz
Received October 27, 2010
Accepted December 6, 2010
Published online January 5, 2011
New type of amide conjugates of steroid and bile acids with D-glucosamine 1 and 2 were
prepared. Title compounds are prepared via acid chloride or using N-[({[(1E)-1-cyano-
2-ethoxy-2-oxoethylidene]amino}oxy)(dimethylamino)methylidene]-N-methylmethan-
aminium tetrafluoroborate as condensation agent. They were examined for gelation proper-
ties with negative results. Per-O-acetylated D-glucosamine hydrochloride was prepared in one
step procedure from D-glucosamine hydrochloride by acetylation in a mixture of acetyl chlo-
ride and acetic acid.
Keywords: Steroids; Carbohydrates; Amides; Bile acids.
In the search of carbohydrate conjugated steroids and their supramolecular
properties we found interesting new properties1 as well as in the group of
steroids conjugated through amide function2. Some of the amide conju-
gates them are well known for their gelation properties3,4. The aim of prep-
aration of the new type of the title compounds was to enable their
utilization in supramolecular structures5. Similar types of compounds, de-
rived from 3β,7β-dihydroxy-5-cholen-24-oic acid and 3β-hydroxy-7-oxo-
5-cholen-24-oic acid were prepared as reference compounds for the study of
abnormalities of bile acid synthesis and of the metabolism of NP-C1 pa-
tients6. As gelators were synthesized amino acid conjugates of bile acids.
The reaction was carried out in the presence of diethyl phosphoryl cyanide
as condensation agent7. Similarly, as an organogelator conjugate of cholic
acid and 5-amino-1,10-phenanthroline was prepared2. As surface active
compound it was synthesized conjugate of dehydrocholic acid8, using
N-mercaptothiazoline active intermediate. The compounds were prepared
as surfactants, solubilizers of linoleic acid without denaturing soybean
lipoxygenase to have potential applications in membranology, enzymology
and extraction of proteins from biological systems. Such types of amide
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 1, pp. 65–74
© 2011 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2010105

66
Nováková, Tomanová, Štěrbová, Drašar:
conjugates are i.a. suitable for construction of synthetic immunomodula-
tors9, as liposome formation modulators, or as components of constructs
and products exhibiting transport and complexing properties to medicinal
substances. In some aspects they could be also considered as a part of the
group of compounds with a heterocycle linked to the side chain of steroid,
cf. e.g. ref.10.
In this study, presented preliminarily on a Czech conference11, we pre-
pared and studied two main types of sugar-steroid amide conjugates, one
derived from bile acids as cholic, lithocholic, deoxycholic (type A) and sec-
ond, derived from etienic acid (type B).
O
R4
H
O
R3
H
NH
R4
N
R4
R4
R4
H
R4
H
R4
O
H
O
H
H
R2
H
H
R4
R1
R1
H
type A
type B
1
2
3
4
1
4
Ia, R = R = R = R = OH
Id, R = R = OH
1
4
2
3
1
4
Ib, R = R = OH; R = R = H
IId, R = OCOH; R = OAc
1
2
3
4
1
4
Ic, R = R = R = O=; R = OH
IIe, R = ONO ; R = OAc
2
1
3
4
2
1
4
If, R = R = R = OH; R = H
IIh, R = OCOH; R = OH
1
2
3
4
1
4
IIa, R = R = R = OCOH; R = OAc
IIi, R = ONO ; R = OH
2
1
4
2
3
IIb, R = OCOH; R = OAc; R = R = H
1
2
3
4
IIc, R = R = R = O=; R = OAc
1
3
2
4
IIf, R = R = OCOH; R = H; R = OAc
1
3
4
2
IIg, R = R = R = OCOH; R = H
RESULTS AND DISCUSSION
Method of preparation of the amide conjugates consists of the reaction
of protected steroid acid chloride with protected D-glucosamine or of
the reaction of protected steroid acid with protected or unprotected
D-glucosamine with condensation agent TOTU (N-[({[(1E)-1-cyano-
2-ethoxy-2-oxoethylidene]amino}oxy)(dimethylamino)methylidene]-
N-methylmethanaminium tetrafluoroborate), both with reasonable results.
For all syntheses there was developed, however, one step preparation of
1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-β-D-glucopyranose hydrochloride
(3a) and a mixture of 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-α/β-D-gluco-
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D-Glucosamine Steroid Conjugates
67
pyranose hydrochlorides 3a and 3b by acetylation in a mixture of acetyl
chloride and acetic acid12. For the synthesis of steroid amide conjugates
protected glucosamine 3a was used. After deprotection we did not analyze
the anomeric purity of the conjugates as it was not the prime aim of the
study, the lipophility of both being almost identical.
OAc
OAc
OAc
OAc
OAc
OAc
Cl H3N
AcO
Cl H3N
AcO
O
O
3a
3b
The reaction procedure developed gives reasonable yield of the β-anomer
and enables simple work-up. The procedure given in Experimental gives the
best yields, change of the reaction conditions give either lower yield or con-
taminant α-anomer. Both were distinguished by the NMR spectra, where
for α-anomer we used as distinguishing marker peat at 6.45 d (1 H, J1,2
=
3.81 Hz, H-1) and for β-anomer similarly at 5.85 d (1 H, J1,2 = 8.79 Hz, H-1).
1,3,4,6-Tetra-O-acetyl-2-amino-2-deoxy-β-D-glucopyranose hydrochloride
and its anomeric partner are usually prepared by multistep reaction13 or
with special catalysts14.
O
R4
O
R3
H
R4
H
H
H
H
H
R2
H
H
R1
R1
H
type A
type B
1
2
3
4
1
4
IVa, R = R = R = OCOH; R = OH
IVd, R = OCOH; R = OH
1
4
2
3
1
4
IVb, R = OCOH; R = OH; R = R = H
Vd, R = OCOH; R = Cl
1
3
2
4
IVf, R = R = OCOH; R = H; R = OH
1
2
3
4
Va, R = R = R = OCOH; R = Cl
1
4
2
3
Vb, R = OCOH; R = Cl; R = R = H
1
2
3
4
Vc, R = R = R = O=, R = Cl
1
3
2
4
Vf, R = R = OCOH; R = H; R = Cl
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Nováková, Tomanová, Štěrbová, Drašar:
O
O
O
O
O
OH
OH
OH
H
O
Cl
O
H
H
H
H
O
H
O
H
H
H
H
O
H
H
O
HO
OH
O
O
O
O
H
O
H
O
H
4a
5a
O
O
O
O
O
O
O
OH
O
HN
O
O
OH
OH
HN
HO
OH
H
H
H
H
O
O
O
H
H
O
H
H
O
O
H
HO
OH
H
2a
1a
SCHEME 1
Example of synthetic pathway
Starting protected steroids were formylated by formic acid2, or nitrated by
a mixture of nitric acid–acetic acid. Steroid acid chlorides were prepared by
the reaction with oxalyl chloride. Deprotection of acyl protecting groups
on sugar and steroid part was done by alkaline hydrolysis. Cleavage of ni-
trates was done by the usage of zinc in protic media15 (Scheme 1).
Conjugates synthesized were examined (for concentrations 5 and 10
mg ml–1) for their gelation properties in methanol, chloroform, dichloro-
methane, acetone, toluene, ethanol, water and also their mixtures (1:1) at
room and lowered temperatures (+4 °C). There was also tried a method
from literature16 where the addition of SDS (sodium dodecyl sulfate) was
used to induce gelation. Different changes of temperature were tested for
the induction of gelation but in all cases we observed negative results.
Further properties are being investigated. These new types of steroidal de-
rivatives could be further simply derived to control their polarity as well as
noncovalent bonding (H-bond) capabilities.
EXPERIMENTAL
TLC was performed on HF254 plates (Merck), detection by UV light or by spraying with
a solution of 5 g of Ce(SO4)2(H2O)4 in 500 ml of 10% H2SO4 and subsequent heating. Flash
column chromatography was performed on silica gel (Merck, 100–160 µm) in solvents, dis-
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D-Glucosamine Steroid Conjugates
69
tilled prior to use. Optical rotations were measured in chloroform solutions on a Rudolph
Research Autopol IV polarimeter at 25 °C and [α]D values are given in 10–1 deg cm2 g–1 with
concentration in 10 g l–1. Routine UV spectra were recorded on a Varian Cary 50 UV-VIS
spectrometer; wavelengths are given in nm, molar absorptivity ε in m2 mol–1 (in parenthe-
ses). IR spectra (wavenumbers in cm–1) were recorded on a Perkin–Elmer PE 580 spectrome-
ter in CHCl3 solutions at 23 °C, unless stated otherwise. 1H and 13C NMR spectra were taken
in deuterochloroform (Aldrich, 99.8% D) on a Bruker AVANCE 500 (500.1 MHz for 1H)
FT NMR spectrometer at 300 K if not stated otherwise. As standard, the internal signal of
tetramethyl silane (δ 0.0) for 1H and central line of solvent (δ 77.0) for 13C spectra were
used. Chemical shifts are presented in ppm (δ-scale), coupling constants (J) in Hz. Mass spec-
tra were taken on a Q TOF micromass spectrometer with direct inlet (ESI) or on a ZAB-EQ
(VG Analytical) instrument (FAB) with Xe ionization, accelerating voltage 8 kV. Micro-
analysis was performed on an elemental analyzer Perkin–Elmer 2400 Series II CHNS/O.
All solvents used were of highest degree of purity and used as received. Steroids were pur-
chased from Steraloids, Inc. USA.
2-Deoxy-2-{[3α,7α,12α-trihydroxy-24-oxo-5α-cholan-24-yl]amino}-D-glucopyranose (1a)
Method A. To cholic acid triformate17 4a (1.7 g, 3.6 mmol) benzene (30 ml) and oxalyl
chloride (5.5 ml, 30 mmol) were added at room temperature. The reaction mixture was
stirred for 1 h, and after that evaporated to dryness. The obtained chloride of cholic acid
triformate2 5a was mixed with protected glucosamine 3a (1.4 g, 3.6 mmol), pyridine (50 ml)
and dichloromethane (50 ml), and the reaction mixture was stirred at room temperature for
5 h. Then, methanol (50 ml) was added and the mixture was stirred for another 1 h, and
after that evaporated to dryness and chromatographed on a column of silica (200 g) in
toluene–acetone. The obtained protected conjugate 2a (541 mg, 0.66 mmol, 18.5%) was a
20
yellowish amorphous powder of [α]D +65.2 (c 0.5, CHCl3). The conjugate 2a was dissolved
in methanol (30 ml), and 0.05 M MeONa (12 ml, methanolic solution) was added. The mix-
ture was stirred for 20 h. After all 2a was gone, the mixture was neutralized by Dowex 50.
Dowex 50 was filtered off and the filtrate evaporated to 416 mg yellowish solid which was
further purified on a column of silica (25 g) in chloroform–methanol. Obtained 1a (230 mg,
20
0.4 mmol, 61.3%) was a yellowish solid of [α]D +36.4 (c 0.25, CH3OH). 1H NMR: 0.38 s
(3 H, H-21); 0.55 s (3 H, H-18); 0.65 s (3 H, H-19); 0.70–2.10 m (23 H, H-steroid bundle);
2.95–4.8 (characteristic protons of saccharide). IR: ν(OH) + ν(H2O): 3401 vs, vbr; ν(C–OH):
1077 s, 1042 s; amide I: 1671 s, br; amide II: 1537 m, br; amide III: 1301 m, br; δs (CH3):
1377; βs (CH2) α to C=O: 1416 m. For C30H51NO9 (569.73) calculated: monoisotopic mass
569.3564, MS-ESI found: [M + Na+] 592.44; calculated: 63.24% C, 9.02% H, 2.46% N, found:
62.32% C, 8.86% H, 2.20% N.
Method B. Hydrochloride 3a (583 mg, 1.5 mmol), cholic acid triformate14 4a (536 mg,
1.09 mmol) and N-[({[(1E)-1-cyano-2-ethoxy-2-oxoethylidene]amino}oxy)(dimethylamino)-
methylidene]-N-methylmethanaminium tetrafluoroborate (433 mg, 1.32 mmol) were dis-
solved in dichloromethane (20 ml) and N,N′-dimethylformamide (5 ml), and N-ethyldiiso-
propyl amine (0.8 ml, 5.8 mmol) was added to the reaction mixture. Then the mixture was
stirred at room temperature for 30 h and finally distributed between chloroform and water
(50 ml each). The protected conjugate 2a was separated in organic phase, evaporated and
further purified on a column of silica (60 g) in chloroform–methanol. The obtained 2a
(560 mg, 0.68 mmol, 45%) was a yellowish substance. It was dissolved in methanol (30 ml)
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Nováková, Tomanová, Štěrbová, Drašar:
and treated with 0.05 M MeONa (12 ml, methanolic solution) for 20 h under stirring at
room temperature. The reaction mixture was neutralized by Dowex 50, filtered and evapo-
rated. The obtained yellowish material (416 mg) was purified on a column of silica (25 g) in
chloroform–methanol. It was obtained 1a (230 mg, 0.4 mmol, 61.3%) as a yellowish amor-
phous material of the same properties as under A).
2-Deoxy-2-{[3α-hydroxy-24-oxo-5β-cholan-24-yl]amino}-D-glucopyranose (1b)
Method A. Lithocholic acid formate18 4b (482 mg, 1.2 mmol) was mixed with benzene
(20 ml) and oxalyl chloride (1.5 ml, 17.2 mmol). The reaction was carried out during 1 h at
room temperature under stirring. Then it was evaporated to dryness and the obtained chlo-
ride 5b immediately mixed with pyridine (20 ml), dichloromethane (20 ml), carbohydrate
3a (435 mg, 1.1 mmol). The resulted mixture was stirred at room temperature for 5 h. Then
it was diluted with methanol (15 ml) and further stirred for
1 h, evaporated and
chromatographed on a column of silica (60 g) in toluene–acetone. It was obtained 2b
20
(250 mg, 0.32 mmol, 28%) as a white amorphous solid of [α]D +48 (c 1, CHCl3). 1H NMR:
0.60 s (3 H, H-21); 0.86 s (3 H, H-18); 0.92 s (3 H, H-19); 1.0–2.0 m (20 H, H-steroidal bun-
dle); 2.02 s (3 H, Ac-C2′); 2.08 s (3 H, Ac-C4′); 2.18 s (3 H, Ac-C3′); 2.30 s (3 H, Ac-C1′);
4.0–6.2 (characteristic protons of carbohydrate); 4.82 s (1 H, NH). For C39H59NO12 (733.89)
calculated: 63.83% C, 8.10% H, 1.91% N; found: 63.55% C, 8.30% H, 1.70% N.
Conjugate 2b (342 mg, 0.44 mmol) was dissolved in methanol (30 ml) and treated with
0.05 M MeONa (10 ml, methanolic solution) for 20 h under stirring at room temperature.
The reaction mixture was neutralized by Dowex 50, filtered and evaporated. The obtained
yellowish material was purified on a column of silica (25 g) in toluene–acetone. It was ob-
20
tained 1b (103 mg, 0.18 mmol, 41%) of [α]D + 41.2 (c 0.5, CH3OH). 1H NMR: 0.70 s (3 H,
H-21); 0.93 s (3 H, H-18); 0.95 s (3 H, H-19); 1.0–2.4 m (20 H, H-steroid); 3.2–5.1 (character-
istic range of carbohydrate protons). IR: ν(OH) + ν(H2O): 3421 vs, br; ν(C–OH): 1068 s,
1039 s; amide I: 1653 s, br; amide II: 1541 m, br; amide III: 1304 w, br; δs(CH3): 1376 m;
βs (CH2) α to C=O: 1419 m. For C30H51NO7 (537.73) calculated: monoisotopic mass
537.3666, MS-ESI found: [M + Na+] 560.40; calculated: 67.01% C, 9.56% H, 2.60% N, found:
62.35% C, 9.37% H, 2.18% N.
Method B. Carbohydrate 3a (583 mg, 1.5 mmol), lithochoic acid formate15 4b (485 mg,
1.2 mmol) and N-[({[(1E)-1-cyano-2-ethoxy-2-oxoethylidene]amino}oxy)(dimethylamino)-
methylidene]-N-methylmethanaminium tetrafluoroborate (492 mg) were dissolved in di-
chloromethane (20 ml) and N,N′-dimethyl formamide (5 ml) and N-ethyldiisopropyl amine
(0.8 ml, 5.8 mmol) and the resulting mixture was stirred at room temperature for 30 h. The
mixture was distributed between chloroform and water. The protected conjugate was taken
into organic phase which was evaporated and further purified on a column of silica (50 g) in
toluene–acetone with the yield of white amorphous 2b (400 mg (45%) of the same proper-
ties as under A).
2-Deoxy-2-{[3,7,12,24-tetraoxo-5β-cholan-24-yl]amino}-D-glucopyranose (1c)
Method A. Carbohydrate 3a (540 mg, 1.4 mmol), dehydrocholic acid (456 mg, 1.13 mmol)
and N-[({[(1E)-1-cyano-2-ethoxy-2-oxoethylidene]amino}oxy)(dimethylamino)methylidene]-
N-methylmethanaminium tetrafluoroborate (526 mg) were mixed in dichloromethane
(20 ml) and N-ethyldiisopropyl amine (0.8 ml, 5.8 mmol), and the mixture was stirred at
room temperature for 30 h. Then it was evaporated and purified on a column of silica (100 g)
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 1, pp. 65–74

D-Glucosamine Steroid Conjugates
71
in chloroform–methanol. There was obtained conjugate 2c as a white amorphous solid
(306 mg, 0.54 mmol, 38.3%) that was dissolved in methanol (20 ml) and treated with
0.05 M MeONa (8 ml, methanolic solution) for 2 h under stirring at room temperature. The
reaction mixture was neutralized by Dowex 50, filtered and evaporated. The obtained yel-
lowish material was purified on a column of silica (25 g) in chloroform–methanol to yield
20
yellowish amorphous conjugate 1c (68 mg, 0.12 mmol, 25%) of [α]D
+24.8 (c 0.25,
CH3OH). 1H NMR: 0.85 s (3 H, H-21); 1.10 s (3 H, H-18); 1.40 s (3 H, H-19); 1.5–2.55 m
(20 H, characteristic steroid bundle); 2.8–5.5 (characteristic saccharide protons). IR: ν(OH) +
ν(H2O): 3418 s, vbr; ν(C–OH): 1053 s; ν(C=O): 1710 vs; amide I: 1655 m, br; amide II:
1530 m, br – bonded, 1511 m – free; δs (CH3): 1382 m, 1365 w; βs (CH2) α to C=O: 1434 m.
For C38H45NO9 (563.68) calculated: monoisotopic mass 563.3094, MS-ESI found: [M] 564.3;
calculated: 63.92% C, 8.05% H, 2.42% N, found: 63.60% C, 7.95% H, 2.13% N.
Method B. Dehydrocholic acid (806 mg, 2 mmol) was dissolved in benzene (20 ml) and
oxalyl chloride (3 ml, 34.4 mmol) was added. The reaction mixture was stirred at room tem-
perature for 1 h. It was evaporated and chloride 5c immediately mixed with pyridine
(20 ml) and carbohydrate 3a (300 mg, 0.78 mmol). The mixture was stirred at room temper-
ature for 24 h. Then it was mixed with methanol (15 ml), stirred for 1 h and evaporated.
The residue was purified on a silica gel column (100 g) in toluene–acetone. There was ob-
tained 1c (195 mg, 34%) as a white amorphous material of identical properties as under A).
2-Deoxy-2-{[3α,12α-dihydroxy-24-oxo-5β-cholan-24-yl]amino}-D-glucopyranose (1f)
Method A. Steroid acid formate15 4f (538 mg, 1.2 mmol) was mixed with benzene (20 ml)
and oxalyl chloride (1.5 ml, 17.2 mmol). The reaction was carried out during 1 h at room
temperature under stirring. Then it was evaporated to dryness and the obtained chloride 5f
was immediately mixed with pyridine (50 ml) and carbohydrate 3a (400 mg, 1.04 mmol),
and stirred at room temperature for 5 h. Then the mixture was mixed with methanol
(15 ml), stirred for further 1 h and evaporated. The residue was purified on a silica column
20
(150 g) in toluene–acetone. It was obtained white amorphous 2f (517 mg, 64.9%) of [α]D
65.2 (c 0.5, CHCl3). 1H NMR: 0.72 s (3 H, H-18); 0.82 s (3 H, H-21); 0.92 s (3 H, H-19);
1.0–2.0 m (20 H, steroidal bundle); 2.02 s (3 H, Ac-C6´); 2.08 s (3 H, Ac-C4´); 2.19 s (3 H,
Ac-C3´); 2.35 s (3 H, Ac-C1´); 4.5 m (1 H, H-2´); 4.84 m (1 H, H-4´); 5.6 m (1 H, H-3´); 5.7 m
(1 H, H-3); 6.15 m (1 H, H-2´); 7.05 s (1 H, NH); 8.02 s (1 H, CHO-C12); 8.12 s (1 H,
CHO-C3). For C40H59NO14 (777.89) calculated: monoisotopic mass 777.3936, MS-ESI found:
[M + Na+] 800.3429.
Amide 2f (100 mg, 0.13 mmol) was dissolved in methanolic 0.03 M CH3ONa (60 ml).
After 1 h, the reaction was neutralized by adding Dowex H+ and filtered. After evaporation
the residues were purified on a silica gel column (40 g) in chloroform. It was obtained white
20
amorphous 1f (50 mg, 70%) of [α]D + 36.4 (c 0.25, CH3OH). 1H NMR: 0.38 s (3 H, H-21);
0.55 s (3 H, H-18); 0.65 s (3 H, H-19); 0.70–2.10 m (23 H, H-steroid); 2.95–4.8 (saccharide
part). For C30H51NO8 (553.72) calculated: monoisotopic mass 553.3615, MS-ESI found: [M +
Na+] 592.44; calculated: 65.07% C, 9.28% H, 2.53% N, found: 58.32% C, 8.56% H, 2.00% N.
Method B. Deoxycholic acid formate 4f (449 mg, 1 mmol), carbohydrate 3a (461 mg,
1.2 mmol) and O-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N′,N′-tetramethyluronium
tetrafluoroborate (394 mg, 1.2 mmol) were mixed with dichloromethane (20 ml), DMF
(8 ml) and N-ethyldiisopropylamine (0.6 ml) was added. The reaction was stirred under
argon for 24 h. The reaction mixture was extracted three times between CHCl3 and water.
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Nováková, Tomanová, Štěrbová, Drašar:
Organic phases were dried by MgSO4 and purified on a silica gel column (50 g) in toluene–
acetone. There was obtained white amorphous 2f (290 mg, 37%) of identical properties as
under A).
2-Deoxy-2-({[3α-(hydroxy)androst-5-en-17β-yl]carbonyl}amino)-D-glucopyranose (1d)
Method A. To etienic acid formate 4d (346 mg, 1 mmol, prepared by the known proce-
dure15 from etienic acid19), benzene (30 ml) and oxalyl chloride (1 ml, 5.4 mmol) were
added and the mixture was stirred at room temperature for 1 h and after that evaporated to
dryness. Chloride 5d was taken into dichloromethane (10 ml) and added dropwise to the so-
lution of carbohydrate 3a (210 mg, 0.4 mmol) in pyridine (30 ml). The mixture was stirred
at room temperature for 3 h. Then the reaction was quenched by addition of MeOH (15 ml)
and the mixture was evaporated to dryness. The residue was chromatographed on silica
(150 ml) in dichloromethane–methanol with the yield of conjugate 2d (113 mg, 46.2%) in
the form of yellowish amorphous solid. 1H NMR: 0.74 s (3 H, 3 × H-18); 1.02 s (3 H, 3 ×
H-19); 2.02 s (3 H, 3 × H-acetyl); 2.08 s (3 H, 3 × H-acetyl); 2.16 s (3 H, 3 × H-acetyl); 2.36 m
(3 H, H-4, H-16, H-17); 4.02 m (2 H, 2 × H-6′); 4.25 m (1 H, H-3); 5.38 m (1 H, H-6); 8.02 s
(1 H, H-formyl). Conjugate 2d was dissolved in methanol (20 ml) and treated with 0.05 M
MeONa (8 ml, methanolic solution) for 20 h under stirring at room temperature. The reac-
tion mixture was neutralized by Dowex 50, filtered and evaporated. After evaporation, the
residues were purified on a silica gel column (25 g) in chloroform–methanol. It was obtained
20
white amorphous 1d (88 mg, 93%) of [α]D + 0.7 (c 0.5, CH3OH). 1H NMR: 0.7 m (3 ×
H-18); 1.02 s (3 × H-19); 1.05–2.36 m (22 × H-steroid); 3.73 m (6 × H, C2′-C6′); 5.14 m
(1 H-1′), 5.35 m (1 × H-6). For C26H41NO7 (479.62) calculated: monoisotopic mass 479.2883,
MS-ESI found: [M + H+] 480.3.
Method B. Etienic acid formate 4d (346 mg, 1 mmol), nonprotected glucosamine hydro-
chloride (258 mg, 1.2 mmol) and O-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N′,N′-tetra-
methyluronium tetrafluoroborate (394 mg, 1.2 mmol) were mixed with DMF (20 ml) and
N-ethyldiisopropyl amine (0.5 ml) was added. The reaction mixture was stirred under argon
for 24 h. Then it was extracted three times between ether and water. Organic phases were
dried by MgSO4 and purified on a silica gel column (40 g) in chloroform–methanol. There
was obtained white amorphous 2h (220 mg, 43%). For C27H41NO8 (507.63) calculated:
monoisotopic mass 507.283217, MS-ESI found: [M + Na+] 508.2. This product was im-
mediately deprotected as given for amide 2d with the yield of 1d (88 mg, 93%) identical as
under A).
2-Deoxy-2-({[3α-(hydroxy)androst-5-en-17β-yl]carbonyl}amino)-D-glucopyranose (1d)
Method A. Etienic acid nitrate20 4e (365 mg, 1 mmol) was dissolved in benzene (30 ml)
and oxalyl chloride (2 ml, 10.8 mmol) was added. The mixture was stirred at room tempera-
ture for 1 h. Then the reaction mixture was evaporated to dryness. Crude 5e was dissolved
in dichloromethane (10 ml). 1,3,4,6-Tetra-O-acetyl-D-glucosamine hydrochloride (210 mg,
0.4 mmol) was dissolved in pyridine (30 ml) and to this solution the above chloride in di-
chloromethane was added. The mixture was stirred at room temperature for 3 h. Then meth-
anol (15 ml) was added, and the reaction mixture was evaporated to dryness and twice
co-evaporated with toluene. The residue was chromatographed on a silica gel column (50 g)
in toluene–ethanol. The chromatography afforded 2e (154 mg, 61%) as a yellowish amor-
phous solid. 1H NMR: 0.98 s (3 H, 3 × H-18); 1.02 s (3 H, 3 × H-19); 1.04–1.95 m (20 H, 20 ×
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 1, pp. 65–74

D-Glucosamine Steroid Conjugates
73
H-steroid); 1.98 m (3 H, 3 × H-acetyl); 2.01 m (3 H, 3 × H-acetyl); 4.21 m (2 H, 2 × H-6′);
4.90 m (1 H, H-3); 5.53 m (1 H, H-6); 5.91 m (1 H, H-1′). For C34H48N2O13 (692.77) cal-
culated: monoisotopic mass 692.3156, MS-ESI found: 693.5 [M + H+]. Amide 2e (350 mg,
0.5 mmol) was dissolved in THF (13.7 ml), acetic acid (3.4 ml) and distilled water (0.7 ml).
Zinc powder (511 mg, 7.8 mmol) was added in small portions during 3 h. The reaction mix-
ture was filtered and washed with chloroform. The filtrate was extracted successively with
water, NaHCO3 and water again. Organic phases were evaporated and dissolved in 0.1
M
MeONa (10 ml, methanolic solution) for 1 h under stirring at room temperature. Reaction
mixture was neutralized by Dowex 50, filtered and evaporated. After evaporation the resi-
dues were purified on a silica gel column (25 g) in chloroform–methanol. It was obtained
white amorphous 1d (188 mg, 57%) identical as under A).
Merthod B. Etienic acid nitrate 4e (182 mg, 0.5 mmol), nonprotected glucosamine hydro-
chloride (130 mg, 0.6 mmol) and O-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N′,N′-tetra-
methyluronium tetrafluoroborate (197 mg, 0.6 mmol) were mixed with DMF (10 ml) and
N-ethyldiisopropyl amine (0.3 ml) was added. The reaction was stirred under argon for 24 h.
The reaction mixture was extracted three times between ether and water. Organic phases were
dried by MgSO4 and purified on a silica gel column (35 g) in dichloromethane–methanol.
There was obtained white amorphous compound 2i (170 mg, 67%). For C26H40N2O9
(524.62) calculated: monoisotopic mass 524.27338, MS-ESI found: 525.2 [M + H+]. This prod-
uct was immediately deprotected as given for amide 2e with the yield of 1d (100 mg, 65%)
identical as under A).
1,3,4,6-Tetra-O-acetyl-2-amino-2-deoxy-b-D-glucopyranose hydrochloride (3a)
D-Glucosamine hydrochloride (5.2 g, 24.1 mmol) was mixed with acetic acid (47 ml) and
acetyl chloride (24 ml) and the mixture was stirred under argon at room temperature. After
3 h, chloroform (24 ml) was added and the mixture was further stirred for 24 h. Then it was
evaporated to dryness, the residue was treated with boiling ethanol (25 ml) and filtered. The
filtrate was cooled to yield 3a (3.53 g, 43%). Properties of compound prepared were identical
with those from literature21. The α- and β-anomers were distinguished by the NMR spectra,
where for α-anomer we used as distinguishing marker peak at 6.45 d (1 H, J1,2 = 3.81 Hz,
H-1) and for β-anomer similarly at 5.85 d (1 H, J1,2 = 8.79 Hz, H-1).
This work was supported by the NATO grant CBP.EAP.CLG.982972 (PD), by the Ministry of
Education, Youth and Sports of the Czech Republic with project No. MSM6046137305 (LŠ, JT), and
2B06024-SUPRAFYT of the National Research Program II (ZN). Elemental analyses were done by the
Analytical Department of the IOCB AS CR Prague.
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