The isolation and synthesis of two novel N-acetyl glucosamine derivatives from Dictyostelium cellular slime molds which exhibit neurite outgrowth activity

Article abstract of DOI:10.1016/S0040-4039(02)00038-2

To clarify the diversity of secondary metabolites of Dictyostelium cellular slime molds and explore any biologically active substances which they may hold, constituents of two species were investigated. From a methanol extract of their fruit bodies, two novel acylated amino sugars were isolated, dictyoglucosamine A (1) from D. purpureum and dictyoglucosamine B (2) from D. discoideum. For further analysis, these compounds were then synthesized from N-acetyl-D-glucosamine. Biological evaluation of 1 and 2 showed they induce neuronal differentiation of rat pheochromocytoma (PC-12) cells.

Full text of DOI:10.1016/S0040-4039(02)00038-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 1477–1480
The isolation and synthesis of two novel N-acetyl glucosamine
derivatives from Dictyostelium cellular slime molds which exhibit
neurite outgrowth activity
Haruhisa Kikuchi, Jun Komiya, Yoshinori Saito, Jun-ichi Sekiya, Shigeyoshi Honma,
Norimichi Nakahata and Yoshiteru Oshima*
Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama, Aoba-ku, Sendai 980-8578, Japan
Received 16 November 2001; accepted 21 December 2001
Abstract—To clarify the diversity of secondary metabolites of Dictyostelium cellular slime molds and explore any biologically
active substances which they may hold, constituents of two species were investigated. From a methanol extract of their fruit
bodies, two novel acylated amino sugars were isolated, dictyoglucosamine A (1) from D. purpureum and dictyoglucosamine B (2)
from D. discoideum. For further analysis, these compounds were then synthesized from N-acetyl-D-glucosamine. Biological
evaluation of 1 and 2 showed they induce neuronal differentiation of rat pheochromocytoma (PC-12) cells. © 2002 Elsevier
Science Ltd. All rights reserved.
Cellular slime molds are unique organisms with a two
stage life cycle: an initial unicellular animal-like feeding
stage (a vegetable amoeba stage) and a multicellular
plant-like fruit body stage (a reproductive fruit body
Fruit bodies of the cellular slime mold, D. purpureum,
were cultured on plates (wet weight: 189 g). They were
extracted three times with methanol at room tempera-
ture to yield an extract (7.3 g), which was partitioned
with ethyl acetate and water. The ethyl acetate soluble
fraction (2.0 g) was separated by conventional column
chromatography over silica gel and ODS to yield 1
(37.5 mg). In the same manner, 2 (0.5 mg) was afforded
from fruit bodies of D. discoideum (wet weight: 109 g).
1
stage). Because their fruit bodies are small (ca. 2–3 mm
in length), it is very difficult to obtain sufficient mate-
rial from natural sources for research on their natural
product chemistry. Fruit bodies of Dictyostelium cellu-
lar slime molds can also be supplied by the cultivation
of spores with Escherichia coli on A-medium, however,
the cultivation of a significant amount is tedious.
Therefore, little attention has been paid to studying
their secondary metabolites. Recently, we have cultured
ca. 200 g of cellular slime molds and isolated novel
secondary metabolites, such as biologically active a-
6
The molecular formula, C H NO , indicated for 1,
29
53
8
+
was established by HREI-MS (m/z 544.3872 [M+H] )
1
13
in addition to H and C NMR spectra (Table 1). The
13
C NMR spectrum of 1 revealed the presence of two
ester carbonyls, an amide carbonyl, an acetal, three
oxymethines, an oxymethylene, a nitrogen-adjacent
methine, seventeen methylenes, and three methyl
2–5
pyrones, aromatics and amino sugar analogues. The
obtained compounds suggested that cellular slime
molds may exhibit diversity in their secondary metabo-
lite biosyntheses, and prompted us to pursue a variety
of chemical constituents. In this paper, we describe the
structure elucidation and syntheses of two novel acyl
derivatives of N-acetyl glucosamine (1 and 2) as well as
their potential ability to induce neurite outgrowth in rat
pheochromocytoma (PC-12) cells.
1
1
1
groups. The H NMR spectrum and H– H COSY of 1
gave the sequence of protons as follows (l 5.24 (1H,
br.t, J=3.6 Hz, H-1), 4.21 (1H, ddd, J=10.8, 9.4, 3.6
Hz, H-2), 5.22 (1H, dd, J=10.8, 9.6 Hz, H-3), 3.59 (1H,
td, J=9.6, 5.6 Hz, H-4), 4.07 (1H, ddd, J=9.6, 3.9, 2.4
Hz, H-5), 4.46 (1H, dd, J=12.2, 3.9 Hz, H-6a) and 4.34
(
1H, dd, J=12.2, 2.4 Hz, H-6b)), suggesting 1 is an
analogue of an amino sugar. Coupling constants (J) of
these protons (J =3.6 Hz, J =10.8 Hz, J =9.6 Hz
1,2
2,3
3,4
and J =9.6 Hz) determined their spatial relationships
Keywords: cellular slime molds; amino sugars; differentiation; PC-12
4,5
from H-1 to H-5 as b, b, a, b and a, respectively. Thus,
1 is a derivative of 2-amino-2-deoxy-a-glucopyranose.
In the HMBC spectrum, correlations of an amide car-
cell.
*
Corresponding author. Tel.: +81-22-217-6822; fax: +81-22-217-6821;
e-mail: oshima@mail.pharm.tohoku.ac.jp
0
040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00038-2

1
478
H. Kikuchi et al. / Tetrahedron Letters 43 (2002) 1477–1480
13
1
a
Table 1. C and H NMR spectral data of dictyoglucosamine A (1) and B (2)
Dictyoglucosamine A (1)
Dictyoglucosamine B (2)
1H
Positions
13C
91.9
52.1
72.9
68.8
70.1
62.9
1H
13C
91.8
1
2
3
4
5
6
5.24 (1H, br.t, J=3.6 Hz)
5.23 (1H, br.t J=3.4 Hz)
4.21 (1H, ddd, J=10.8, 9.4, 3.6 Hz)
5.22 (1H, dd, J=10.8, 9.6 Hz)
3.59 (1H, td, J=9.6, 5.6 Hz)
4.07 (1H, ddd, J=9.6, 3.9, 2.4 Hz)
4.46 (1H, dd, J=12.2, 3.9 Hz)
52.1
73.0
68.8
70.1
62.7
4.19 (1H, ddd, J=10.8, 9.3, 3.4 Hz)
5.22 (1H, dd, J=10.8, 9.6 Hz)
3.59 (1H, td, J=9.6, 5.4 Hz)
4.07 (1H, ddd, J=9.6, 3.8, 2.4 Hz)
4.44 (1H, dd, J=12.2, 3.8 Hz)
4.34 (1H, dd, J=12.2, 2.4 Hz)
4.34 (1H, dd, J=12.2, 2.4 Hz)
1
2
1
2
%
%
¦
¦
170.4
23.1
175.3
34.3
170.4
23.1
175.3
34.3
1.95 (3H, s)
1.95 (3H, s)
b
2.35 (1H, dt, J=15.6, 7.8 Hz)
2.35 (1H, dt, J=15.6, 7.8 Hz)
2.34 (1H, dt, J=15.6, 7.8 Hz)
1.56–1.62 (2H, m)
1.20–1.32 (28H, m)
1.20–1.32 (28H, m)
2.34 (1H, dt, J=15.6, 7.8 Hz)
3
4
1
1
1
1
2
3
4
1
4
2
¦
25.0
1.56–1.62 (2H, m)
1.20–1.32 (28H, m)
1.20–1.32 (28H, m)
1.20–1.32 (28H, m)
0.88 (3H, t, J=6.8 Hz)
25.0
c
c
¦–15¦
6¦
7¦
8¦
§
§
§
§
31.9
22.7
14.1
175.2
27.4
9.0
31.9
22.7
14.1
174.0
43.2
25.7
22.4
1.20–1.32 (28H, m)
0.88 (3H, t, J=6.9 Hz)
b
2.41 (2H, q, J=7.5 Hz)
1.17 (3H, t, J=7.5 Hz)
2.26 (2H, d, J=7.1 Hz)
2.07–2.15 (1H, m)
0.98 (6H, d, J=6.7 Hz)
4.02 (1H, br.d, J=3.4 Hz)
3.05 (1H, d, J=5.4 Hz)
6.16 (1H, d, J=9.3 Hz)
-OH
-OH
-NH
3.93 (1H, br.d, J=3.6 Hz)
3.01 (1H, d, J=5.6 Hz)
6.13 (1H, d, J=9.4 Hz)
a
b
c
00 MHz for 1H and 125 MHz for 13C in CDCl3.
These signals were indistinguishable.
These carbon atoms were assigned to 12 signals: l 29.8, 29.7, 29.7, 29.7, 29.6, 29.6, 29.6, 29.5, 29.3, 29.3, 29.3, 29.1.
5
c
bonyl carbon (l 170.4) with the methyl protons (l 1.95
nearly identical to that of 1. However, signals due to
the propionyl group (l 2.41 (2H, q, J=7.5 Hz) and
1.17 (3H, t, J=7.5 Hz)) of 1 disappeared in the case of
2, while signals of two methyl groups (l 0.98 (6H, d,
J=6.7 Hz)), a methylene (l 2.26 (2H, d, J=7.0)) and a
methine (l 2.07–2.15 (1H, m)), which composed the
isovaleryl group, were instead observed. These findings
revealed that the proionyloxy group in the C-6 position
in compound 1 was replaced with an isovaleryloxy
group in compound 2.
c
(
3H, s)), the nitrogen-adjacent methine proton (l 4.21)
and the amide proton (l 6.13 (1H, d, J=9.4 Hz))
indicated the presence of an acetamide group at C-2.
1
1
H– H COSY pointed out the presence of a propiony-
loxy group. The presence of a stearoyloxy group was
deduced from the remaining groups including an ester
group, 16 methylene carbons and a methyl carbon. The
positions of two ester groups remained unresolved as
the correlational peaks of these carbonyl carbons (lc
175.3 and 175.2) were indistinguishable in the HMBC
spectrum.
To obtain sufficient samples for performing a biological
evaluation, we carried out syntheses of 1 and 2 (Scheme
Methanolysis of 1 at room temperature gave the par-
7
tially hydrolized product (3) (Scheme 1). The upfield-
shift of the signals of H-6 (l 3.89 (1H, dd, J=12.1, 3.0
HO
1
O
Hz) and 3.84 (1H, dd, J=12.1, 4.0 Hz)) in the H
a
1
HO
OH
NMR spectrum of 3 showed that the propionyloxy
group of 1 was connected at C-6. The stearoyloxy
group was thus located at C-3. Additionally, methanoly-
sis of 1 in refluxing conditions followed by acetylation
afforded the tetraacetyl derivative of methyl glycoside
CH3(CH2)16COO
NHAc
3
b, c
AcO
O
4
. The specific rotation of 4 ([h] +68.5 (c 0.11, CHCl ))
D
3
b, c
5
AcO
AcO
OMe
NHAc
was identical to synthetically produced 4 ([h] +83.5 (c
D
0.17, CHCl )) obtained from N-acetyl-D-glucosamine
3
(
5). From this result, the absolute configuration of 1
4
was determined to be identical to N-acetyl-D-
glucosamine.
Scheme 1. Reagent and conditions: (a) 2% H SO , MeOH, rt
2 4
(
25%); (b) 2% H SO , MeOH, reflux; (c) Ac O, pyridine, rt
2 4 2
6
The molecular formula of 2 was determined by HREI-
MS to be C H NO . Its H NMR spectrum was
(two steps, 25% from 1 and 38% from N-acetyl-D-glu-
cosamine).
1
3
1
57
8

H. Kikuchi et al. / Tetrahedron Letters 43 (2002) 1477–1480
1479
AcO
AcO
O
O
H3C
H3C
a, b
O
c, d
O
e, f
5
OAllyl
NHAc
OAllyl
NHAc
AcO
HO
6
7
HO
HO
RCOO
HO
RCOO
HO
O
O
O
g
h, i
OAllyl
NHAc
OAllyl
OH
NHAc
CH3(CH2)16COO
CH3(CH2)16COO
NHAc
CH3(CH2)16COO
1 : R = CH CH
2
8
9
9
a : R = CH3CH2
b : R = (CH3)2CHCH2
3
2 : R = (CH3)2CHCH2
Scheme 2. Reagent and conditions: (a) allyl alcohol, pTsOH, reflux; (b) Ac O, pyridine, rt (two steps, 64%); (c) MeONa, MeOH,
2
rt; (d) 2,2-dimethoxypropane, pTsOH, DMF, rt (two steps, 97%); (e) stearoyl chloride, Et N, DMAP, CH Cl , rt (71%); (f)
3
2
2
+
Dowex 50W (H ), MeOH, rt (80%); (g) propionyl chloride, pyridine, 0°C (93% for 9a) or isovaleric acid, EDCI, DMAP, CH Cl ,
2
2
0
°C (42% for 9b); (h) Rh(PPh ) Cl, DABCO, EtOH–H O (9:1), reflux; (i) microencapsulated OsO , NMO, H O–acetonitrile–ace-
3 3 2 4 2
tone (1:1:1), rt (32% for 1 and 55% for 2).
Figure 1. Effects of dictyoglucosamine A (1) and B (2) on morphological differentiation of PC-12 cells. PC-12 cells were cultivated
in the absence (A) or presence of 10 mM of 1 (B) or 2 (C). The concentration-dependency of 1 or 2 is shown in D. Each point
represents the mean with S.E. of three independent experiments.
2
). Treatment of N-acetyl-
D
-glucosamine (5) as a start-
cultivated in Dulbecco’s modified Eagle’s medium
(DMEM) containing 0.5% FCS with 1 or 2 at a concen-
tration of 0.5 to 10 mM for 4 days, after which the
neurite outgrowth of cells was observed under a phase-
contrast microscope (Fig. 1). Compounds 1 and 2
caused the morphological change of PC-12 cells to
extend neurites in a concentration-dependent manner.
ing material with p-toluenesulfonic acid in allyl alcohol
gave a mixture of the allyl glycoside and its deacety-
lated product. Acetylation of the mixture with acetic
anhydride in pyridine afforded a mixture of a- and
b-anomers of the tetraacetylated compound. Deaceyla-
tion of this a-anomer (6), using sodium methoxide and
subsequent acetonation, with 2,2-dimethoxypropane,
gave the 4,6-isopropylidene derivative (7). After esterifi-
cation of 7 with stearoyl chloride, deacetonation was
performed under acidic conditions to obtain stearate 8.
Regioselective esterification of 8 with propionyl chlo-
ride in pyridine at 0°C gave propionate 9a. The treat-
ment of 9a with Wilkinson’s catalyst in 10% aqueous
ethanol followed by oxidative cleavage of 1-propenyl
glycoside with microencapsulated osmium tetroxide
allowed us to complete the synthesis of 1. Isovalarate
It should be mentioned that these compounds are char-
acteristic in the point that the amino sugar part is
directly connected to the fatty acid. In addition to 1
and 2, furanodictine A and B were also isolated from
cellular slime molds. These compounds are known
acylated amino sugar derivatives that possess neuronal
4
differentiation activity. Thus, a structure–activity rela-
tionship study of amino sugar analogues may lead to
the discovery of novel nerve-rejuvenation drugs.
9
b, which was obtained from 8 with isovaleric acid in
the presence of EDCI and DMAP, was converted into
2
in the same manner as in the synthesis of 1. All of the
Acknowledgements
spectral data of synthetic 1 and 2 were identical with
those of natural compounds.
This work was supported in part by a Grant-in-Aid for
Scientific Research (No. 12470474) from the Ministry
of Education, Science, Sports, and Culture of Japan.
Biological evaluations of 1 and 2 on rat pheochromocy-
toma (PC-12) cells were performed. PC-12 cells were

1
480
H. Kikuchi et al. / Tetrahedron Letters 43 (2002) 1477–1480
6. Data for 1: colorless amorphous; [h] +11.9 (c 0.960,
References
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−
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1
2
2: colorless amorphous; [h] +18.2 (c 0.011, CHCl ); IR
D
3
−
1
1
13
(cm ) 3395, 2924, 2854, 1742, 1657. H and C NMR data
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1
1
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(1H, ddd, J=9.1, 4.0, 3.0 Hz, H-5), 3.89 (1H, dd, J=12.1,
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(1H, dt, J=15.5, 7.6 Hz, H-2¦), 1.96 (3H, s, H-2%), 1.55–1.61
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4
(2H, m, H-3¦), 1.20–1.35 (28H, m, H-4¦–H-17¦), 0.88 (3H,
+
6
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2
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5
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26 6