Synthesis of Pro-XylaneTM: A new biologically active C-glycoside in aqueous media

Article abstract of DOI:10.1016/j.bmcl.2008.12.037

The scope and limitation of Lubineau's reaction were evaluated for the synthesis of C-glycosides (compounds 1-13). Further transformation of side chain carbonyl was also achieved (compounds 16-23). Optimization of these two steps was investigated in xylos

Full text of DOI:10.1016/j.bmcl.2008.12.037

Bioorganic & Medicinal Chemistry Letters 19 (2009) 845–849
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of Pro-XylaneTM: A new biologically active C-glycoside
in aqueous media
Alexandre Cavezza a, Christophe Boulle a, Amélie Guéguiniat a, Patrick Pichaud a, Simon Trouille a,
Louis Ricard b, Maria Dalko-Csiba a,
*
a L’Oréal Recherche, Personal Care Chemistry, 93601 Aulnay sous Bois, Cedex, France
b Laboratoire ‘‘Hétéroéléments et Coordination”, Ecole Polytechnique, 91128 Palaiseau Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The scope and limitation of Lubineau’s reaction were evaluated for the synthesis of C-glycosides (com-
pounds 1–13). Further transformation of side chain carbonyl was also achieved (compounds 16–23).
Optimization of these two steps was investigated in xylose case. Some of the compounds were shown
to stimulate sulfated glycosaminoglycans (GAGs) synthesis. Compound 20 (called Pro-XylaneTM) was
identified as the best activator of GAGs biosynthesis. Pro-XylaneTM was developed using environmentally
friendly conditions relevant to ‘Green-Chemistry’ principles and launched on the market in September
2006. This compound is the first example of ‘Green’ chemical used in cosmetic.
Received 14 October 2008
Revised 28 November 2008
Accepted 2 December 2008
Available online 13 December 2008
Keywords:
Pro-Xylane
C-glycoside
Ó 2008 Elsevier Ltd. All rights reserved.
Glycosaminoglycan
The importance of carbohydrates in human biology is well
known.1 For example, cell-surface carbohydrates play a key role
in molecular recognition.2 They allow the cells to communicate
with their surrounding environment, and in particular they are
able to interact with other sugar derivatives such as lectins,3 glyco-
proteins4 or glycolipids,5 and thus with several hormones, en-
zymes, cancer cells, virus, bacteria, toxins and antibodies.
A class of carbohydrates of particular interest is represented by
proteoglycans (PGs) and glycosaminoglycans (GAGs). These poly-
saccharides are pivotal in dermal matrix structure that embeds
and sustains collagen fibers network.6 Recent studies have shown
that human aged skin contains less GAGs than young skin, which
contributes to the differences in their mechanical properties.7
These considerations raised the concept of a new class of potential
skin anti-aging molecules that could help maintaining unaltered
matrix structure through stimulating GAGs and PGs synthesis.
Recently, C-glycosides have received increasing interest as car-
bohydrate biomimetics.8 Indeed, the substitution of an O-glyco-
sidic position of native carbohydrate structures by a C-glycosidic
carbon–carbon linkage makes such analogs stable against acidic
or enzymatic hydrolysis. Therefore, C-glycosides have been shown
to be of interest as anti-tumor agents,9 antibiotics10 or anti-inflam-
matory agents.11
glycosylidene carbene formation,14 or substitution of the anomeric
hydroxyl with halogen and subsequent chemistry (Reformatsky,15
Grignard,16 or radical chemistry17). However, most of these C–C
linking reactions are burdened by the need for protecting-group
installation.
In 1986, Gonzalez et al. reported the first one-step procedure to
synthesize C-glycoside directly by reacting unprotected carbohy-
drates with barbituric acid derivatives in water.18 Later, Lubineau
et al. investigated this green reaction by reacting 1,3-diketone with
unprotected carbohydrates dissolved in alkaline aqueous media.19
In both cases, the reaction proceeded via a Knoevenagel reaction of
activated methylenes with aldose (onto sugars) followed by an
intramolecular Michael cyclization. Thus, the process is respectful
of ‘Green-Chemistry’ principles20: it is an eco-friendly synthesis
using renewable raw materials.
Here, we report the study of the scope and limitation of this
reaction for the preparation of several C-glycoside derivatives
and their subsequent transformation into compounds which stim-
ulate GAGs synthesis.
The original procedure described by Lubineau is shown to be
applicable to a great variety of sugars19,21 (Scheme 1).
Thus, various sugars were transformed with penta-2,4-dione
into b-keto-C-glycosides with moderate to good yields (Table 1,
compounds 1–7).22 From these results, it appears that the nature
of the sugar has an impact on the reaction yield (from 40% with
There are several methods commonly used to synthesize C-gly-
coside.12 These methods are usually based on nucleophilic attack at
the anomeric position,12a but also on Wittig-type reaction,13 or on
D-arabinose up to 100% with
D-glucose).
One major limitation of this method is the restricted availability
of other activated methylenes able to react with unprotected sug-
ars. Lubineau et al. succeeded in replacing pentane-2,4-dione by
* Corresponding author. Tel.: +33 148689905; fax: +33 158317076.
E-mail address: mdalko@rd.loreal.com (M. Dalko-Csiba).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.037

846
A. Cavezza et al. / Bioorg. Med. Chem. Lett. 19 (2009) 845–849
O
R
O
O
O
O
OH
+
+
O
R
ONa
R
R
n
n
HO
HO
n=0,1
1-15
Scheme 1. Formation of C-glycoside by reacting unprotected sugar with 1,3-diketone in water. Reactions and condition: NaHCO3, H2O, 90 °C.
Table 1
Table 2
Influence of the sugar or the diketone on the yield of the C-glycosides obtained using
Lubineau’s methodology
Effect of the base on Knoevenagel reaction of pentane-2,4-dione with D-xylose in
water
Compound
Starting sugar
R
Yield (%)
Entry
Base
Yield
Time
Temp. (°C)
1
2
3
4
5
6
7
8
D
D
D
D
D
D
-Glucose
-Xylose
-Lactose
-Galactose
-Fucose
Me
Me
Me
Me
Me
Me
Me
Ph
100a
87a
79a
98a
92a
40a
59a
58a
12a
6a
A
B
C
D
E
F
NaHCO3
NaHCO3
LiOH
NaOH
NaOH
NaOH
87%
Mixture
56%
88%
90%
18 h
1 h
18 h
18 h
1 h
90
90
90
90
90
50
-Arabinose
97%
45 min
3-Deoxy-
-Glucose
-Fucose
D
-arabinose
D
9
L
Ph
Ph
10
11
12
13
D
D
D
D
-Xylose
tion of furan by-products during the course of this Knoevenagel
reaction is based on such mechanistic considerations21
-Glucose
-Glucose
-Glucose
4-OBn-Ph
4-OMe-Ph
4-OH-Ph
37b
52c
34c
.
Compounds 14 and 15 (Fig. 1) were the only b-diketone deriv-
atives that reacted with unprotected sugars to give the desired C-
glycosides derivatives with low to moderate yield (Table 1, com-
pounds 8–13). Indeed, through modifying reaction conditions
using co-solvents such as dioxan or EtOH, we could produce C-gly-
cosides with acceptable yields contrary to the literature data.21
In order to improve the yield of the Knoevenagel reaction, the
effects of various bases were investigated. Xylose was condensed
with pentane-2,4-dione in water, using either sodium bicarbonate,
lithium hydroxide or sodium hydroxide (Table 2 and Scheme 2).
Surprisingly, the nature of the base had a tremendous effect on
reaction yield (Table 2, entries A, C and D). The base also impacted
reaction time, since the same yields were obtained with (NaHCO3,
18 h) and (NaOH, 1 h), whereas (NaHCO3, 1 h) led to a mixture of C-
glycoside diastereoisomers and starting materials (Table 2, entries
A, B and E). The best yield was obtained with NaOH at 50 °C for
45 min (Table 2, entry F). Decreasing reaction time and tempera-
ture are indeed two key points in green chemistry.
Solvent.
a
Water.
Dioxanne/H2O.
b
c
EtOH/H2O.
O
O
O
O
RO
OR
15
14
15a: R=H
15b: R=Bn
15c: R=CH3
Figure 1. Other b-diketone derivatives used.
Some of the keto C-glycosides described in Table 1 were then
subjected to further transformation. Reduction to alcohols 16–23
using aqueous sodium borohydride (NaBH4) yielded a mixture of
two diastereoisomers in good yield (Table 3 and Scheme 3).24
We noticed that reduction of C-xyloside 2 gave a slight diaste-
reomeric excess when performed in isopropanol (i-PrOH) com-
pared to water or methanol (Table 4, entries G–I and Scheme 4).
Adding acid could further improve the selectivity of reduction step
(Table 4, entries J and K). The reduction with NaBH4 in i-PrOH/
AcOH (9/2)25 yielded the most pure diastereoisomer with de ca.
90% as a crystalline product. Whereas all other conditions pro-
duced a sticky oily mixture of diastereoisomers. As shown in Figure
2, the structure of pure diastereoisomer obtained after re-crystalli-
zation was unambiguously determined by X-ray diffraction as the
S diastereoisomer.26
mixed diketone bearing a long alkyl chain.23 However, yields were
lower and water was replaced by a mixture of alcohol/water in
order to increase the solubility of the fatty diketones. Another
attempt was made in 2002 by Fessner et al. who unsuccessfully
tried to react acetoacetic acid tert-butyl ester with sugars.21 In
our study, malonates, malonamide, malononitrile, Meldrum’s acid,
hexafluoroacetylaceton, 1,3-indanedione, ethyl cyanoacetate
also failed to give any C-glycosides. This may be explained by
the hydrolytic instability of the esters under alkaline reaction
conditions.
However, it is surprising that these conditions are harsh enough
to decompose tert-butyl esters, and a possible neighboring hydro-
xyl group participation should not be excluded. Indeed, the forma-
O
O
O
OH
OH
O
O
+
HO
OH
HO
OH
OH
D-Xylose
2
Scheme 2. Formation of the keto C-xyloside 2. Reagents and condition: base, time, temperature, H2O.

A. Cavezza et al. / Bioorg. Med. Chem. Lett. 19 (2009) 845–849
847
27
Table 3
posed (Fig. 3). The first one is based on a Meerwein–Porndorf
type reduction, the second one is not.
C-glycosides from keto derivative reduction with NaBH4
Compound
Starting sugar
R0
Yield (%)
It is to note that we tried to directly use NaBH(OAc)3 that is
likely formed in situ.28 This reducing agent indeed gave the highest
purity of diastereoisomer (Table 4, entry L). On the other hand,
using other reported reagent combination gave either a far lower
rate (e.g., with NaBH4/CeCl329) or almost no reaction (e.g., with
NaBH4/ZnCl230): Table 4, entries M and N.
Although these reactions were performed in water and/or alco-
hols, the E-factor was not acceptable from an ecological viewpoint
due to a tedious procedure for borate salts removal.31 The E-factor
was much diminished using catalytic hydrogenation with Ru/C for
the reduction step.32 The reaction product was then a 50/50 diaste-
reoisomer mixture (de = 0) (Table 4, entry O).
The biological activities of some synthesized C-glycosides were
then studied on human fibroblast cultures in order to select the
best compound able to stimulate GAG synthesis and thereby have
potential anti-aging properties (Table 5). The best results were ob-
tained with compound 20, a C-xyloside (50/50 mixture of diastere-
oisomers). Sulfated GAG synthesis is often initiated by the same
monosaccharide, xylose.33 The GAG chains could then be linked
to a protein core via a b-O-glycosidic bond between xylose and
the hydroxyl group of a serine aminoacid of protein sequence.
The mechanism of GAGs synthesis stimulation by our C-xylosides
is still under investigation in our laboratory.
16
17
18
19
20
21
22
23
D
D
D
D
D
-Glucose
-Fucose
-Arabinose
-Lactose
-Xylose
Me
Me
Me
Me
Me
Me
88
65
90
65
98
L-Fucose
86
D
-Glucose
-Glucose
4-OMe-Ph
4-OH-Ph
100
75
D
O
O
O
OH
R'
R'
HO
HO
16-23
Scheme 3. Reduction of keto C-glycosides: NaBH4, H2O or alcohol.
Table 4
Effect of solvent, acid and reducing agent on the diastereoselectivity of the reduction
of compound 2
Entry
Reducing agent
Solvent
Acid
Yield (%)
de (%)
Some stereochemical features of compound 20 were important
for biological activity. Indeed, further studies indicated that the b-
C-glycosidic bond was essential to maintain activity compared to
G
H
I
J
K
L
M
N
O
NaBH4
NaBH4
NaBH4
NaBH4
H2O
None
None
None
HClaq
AcOH
None
None
None
None
99
78
95
98
98
90
0
6
0
MeOH
i-PrOH
i-PrOH
i-PrOH
i-PrOH
i-PrOH
i-PrOH
H2O
20
44
90
92
naa
40
0
the a-C-glycosidic. Furthermore, the stereochemistry of the hydro-
NaBH4
xyl group in the aglycon moiety has an impact on the activity level
of GAG synthesis, since different diastereomeric ratios led to differ-
ent activity level (Fig. 4).
NaBH(OAc)3
NaBH4/ZnCl2
NaBH4/CeCl3
Ru/C
100
82
In summary, we explored the scope and limitations of a very
powerful method to synthesize C-glycosides using environmen-
tally friendly conditions relevant to ‘Green-Chemistry’ principles.
The C-glycosides were further functionalized and tested for bio-
logical activity of potential interest in skin anti-aging applica-
tions in cosmetic. Some of these compounds were shown to
exhibit stimulating properties on GAGs synthesis. Further appli-
cations of these compounds and more functionalizations are still
under study in our laboratories. For example, aldol condensation,
reductive alkylation and oxime formation were achieved in good
yields in the same spirit of optimization of ‘Green-Chemistry’
conditions.
a
na, not applicable.
Compound 20 was identified as the best inducer of sulfated GAGs
synthesis in vitro and was also proved active in vivo in a clinical trial.
It was developed in cosmetic skincare products as Pro-XylaneTM 35
.
This compound is the first example of ‘‘Green” chemical in cosmetic.
It is made from a renewable feedstock (xylose is extracted from
beechwood), in only 2 steps in water and using a catalytic reduction
(E-factor 36). Furthermore, Pro-XylaneTM is an eco-friendly com-
pound. It is biodegradable (software BIOWIN36 + experiments),
Figure 2. X-ray diffraction of the C-xyloside diastereoisomer obtained from
selective reduction.
Since i-PrOH and acetic acid were crucial for diastereoselection
leading to S diastereoisomer, two transition states could be pro-
O
O
HO
HO
HO
HO
or
O
O
H
O
H
O
OiPr
O
B
B
OiPr
OiPr
Figure 3. Transition states proposed for the diastereoselective reduction of the C-xyloside.

848
A. Cavezza et al. / Bioorg. Med. Chem. Lett. 19 (2009) 845–849
O
O
O
OH
References and notes
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Effect of C-glycosides on the D-[6-H3]-glucosamine incorporation in the GAG fraction
by human fibroblasts34
Compound
None
[C]
%
P
—
100
348
—
Transforming growth factor-b (TGF-b)
(positive control)
10 ng/mL
<0.01
Xylose
0.5 mM
0.1 mM
0.02 mM
52
85
106
<0.01
>0.05
>0.05
Lyxose
2.0 mM
0.4 mM
0.08 mM
86
102
90
>0.05
>0.05
>0.05
Compound 2
Compound 4
Compound 20
Compound 10
10.0 mM
2.0 mM
0.4 mM
161
141
110
<0.01
<0.01
>0.05
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Bioorg. 2001, 4, 63.
10.0 mM
3.0 mM
1.0 mM
99
119
136
>0.05
>0.05
<0.01
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3.0 mM
1.0 mM
0.3 mM
218
169
139
<0.01
<0.01
>0.05
1.0 mM
0.3 mM
0.1 mM
95
102
120
>0.05
>0.05
>0.05
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350
300
250
200
150
100
50
Reference
22. General procedure: Monosaccharide (1 equiv), acetylaceton (1.2 equiv) and
sodium bicarbonate (1.5 equiv) were refluxed in water for 12 h. The aqueous
phase was cooled down to room temperature, washed with ethyl acetate,
acidified with the resin Dowex 50X-200, filtered and concentrated in vacuo, to
afford the pure product as a brown oil. The structures of the C-glycosides were
confirmed by NMR and mass spectroscopy.
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(1.2 equiv) in ethanol. After 12 h, the reaction mixture was quenched with
aqueous HCl (1 N), and the aqueous phase was extracted with butanol. The
organic phase was dried and concentrated in vacuo, to afford the compound.
The structures of the C-glycosides were confirmed by NMR and mass
spectroscopy.
dr = 50/50
dr = 70/30
0
control
TGF-b
1 mM
3 mM
10 mM
25. Trouille, S.; Cavezza, A.; Pichaud, P. Patent EP 1589010.
Figure 4. Effect of the diastereomeric ratio of compound 28 on the D-[6-H3]-
glucosamine incorporation in the GAG fraction by human fibroblasts (dr, diaste-
reomeric ratio; cpm, count per minute).
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mL) or compounds X–Y for 72 h at 37 °C in humid atmosphere of 95% air and
Acknowledgments
We thank our colleagues who contributed to the body of work
presented herein over the years.
Thanks for Pr Lubineau and Pr Sam Zard for helpful discussion.
Nicole Bonaventure, Valéria Manzin and Marie-Lise Chiron for ana-
lytical support.

A. Cavezza et al. / Bioorg. Med. Chem. Lett. 19 (2009) 845–849
849
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39. Unpublished data.