Synthesis of tartaric acid analogues of FR258900 and their evaluation as glycogen phosphorylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.01.042

Di-O-cinnamoylated, -p-coumaroylated, and -feruloylated d-, l- and meso-tartaric acids were synthesized as analogues of the natural product FR258900, a glycogen phosphorylase (GP) inhibitor with in vivo antihyperglycaemic activity. The new compounds inhib

Full text of DOI:10.1016/j.bmcl.2013.01.042

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1789–1792
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of tartaric acid analogues of FR258900 and their evaluation
as glycogen phosphorylase inhibitors
a,
Gergely Varga a, Tibor Docsa b, Pál Gergely b, László Juhász a, , László Somsák
⇑
⇑
a Department of Organic Chemistry, Faculty of Science and Technology, University of Debrecen, PO Box 20, H-4010 Debrecen, Hungary
b Department of Medical Chemistry, Medical and Health Science Centre, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Di-O-cinnamoylated, -p-coumaroylated, and -feruloylated D-, L- and meso-tartaric acids were synthesized
Received 4 December 2012
Revised 10 January 2013
Accepted 12 January 2013
Available online 22 January 2013
as analogues of the natural product FR258900, a glycogen phosphorylase (GP) inhibitor with in vivo anti-
hyperglycaemic activity. The new compounds inhibited rabbit muscle GP in the low micromolar range,
and bound to the allosteric site of the enzyme. The best inhibitor was 2,3-di-O-feruloyl meso-tartaric acid
and had Ki values of 2.0
l
M against AMP (competitive) and 3.36
lM against glucose-1-phosphate (non-
competitive).
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
FR258900
Tartaric acid derivatives
Diabetes
Glycogen phosphorylase
Inhibitor
The number of patients suffering from diabetes mellitus (DM) is
dramatically increasing. In 2011 the International Diabetes Feder-
ation (IDF) indicated that the number of diabetic patients was
more than 360 million worldwide.1 In 2001 this number was pre-
dicted to be reached in 2030 only.2 More than 90% of the diagnosed
cases belong to type 2 or non-insulin dependent diabetes mellitus
(T2DM or NIDDM) characterized by peripheral insulin resistance,
elevated hepatic glucose production, and defects in pancreatic
insulin secretion.3 Although several drugs are in clinical use for
symptomatic treatment of T2DM,4,5 these therapies are inadequate
for 30–40% of the patients.6
Among several investigational fields to diminish hepatic glu-
cose output in T2DM, glycogen phosphorylase (GP) as a main reg-
ulatory enzyme of glycogen metabolism has become a validated
target.2
Protein crystallographic studies have shown that endogenous
and synthetic modulators can bind to six major sites in GP: the
catalytic, the inhibitor, the allosteric (or AMP-binding), the glyco-
gen storage, and the new allosteric sites,7 as well as the newly
discovered benzimidazole site.8 A number of GP inhibitors for the
different binding sites have been disclosed and several of them
have considerable in vivo effects towards normalizing blood
glucose and liver glycogen levels.9,10
In recent years, the interaction of GP and glycogen targeting
subunit (GL) of protein phosphatase 1 (PP1) has been identified
as a novel molecular target for the treatment of T2DM.11–13 It
was demonstrated in an in vivo mouse model that disruption of
GL–GP interaction resulted in an increased glycogen synthase
activity and the mice had improved glucose tolerance.14 X-ray
crystallography showed that the GL–GP interaction took place by
binding the C-terminal region of GL to the allosteric site of GP.15
Thus, occupation of this site may prevent GL–GP interaction, there-
by enhancing glycogen synthesis which, on the other hand, dimin-
ishes hepatic glucose production. This effect could be achieved by
modulators that bind to the allosteric site of GP.
Several molecules of high structural diversity were reported to
bind to the allosteric site of GP, such as derivatives of acyl urea,
dihydropyridine dicarboxylic acid, pentanedioic acid, phthalic acid,
N,N0-diaryl-urea, and pentacyclic triterpenoids.9,16
HO
core unit
HOOC
O
O
O
O
COOH
OH
FR258900
1
IC50 = 2.5 μM (human liver GP)17
Ki = 0.46 μM (rabbit muscle GP)18
⇑
Corresponding authors. Tel.: +36 52 512900x22474; fax: +36 52 512744 (L.J.),
tel.: +36 52 512900x22348; fax: +36 52 512744 (L.S.).
FR258900,
a bis-O-(p-coumaroylated)2,3-dihydroxypentane-
dioic acid derivative 1, was isolated from the fermentation broth
E-mail addresses: juhasz.laszlo@science.unideb.hu (L. Juhász), somsak.laszlo@
science.unideb.hu, somsak@tigris.unideb.hu (L. Somsák).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.042

1790
G. Varga et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1789–1792
of fungi No. 138354.17 The compound was shown to inhibit
glycogen phosphorylases and to bind to the allosteric site of
GP.18 Compound 1 stimulated glycogen synthesis in primary rat
hepatocytes, and investigations on glucagon-induced hyperglyce-
mia in C57BL/6 mice suggested that 1 could suppress hepatic glu-
cose output in vivo.19
R1
R2
COOH
R1
R2
COOR3
a then b
or b
10 - 14
15 - 17
a: ClCOOCH3 in 50% aq. NaOH at 0 °C; b: SOCl2, reflux, 6 h.
R1
H
R2
H
R3 Yield (%)
Abbreviation
Cinn-OH
On the basis of the above information and as a continuation of
our research on the design and synthesis of small molecule inhib-
itors of GP we envisaged the preparation of structural analogues of
1. Since the synthesis of 2,3-dihydroxy-pentanedioic acid is un-
known in the literature, the core unit was planned to be replaced
by easily available tartaric acid, also allowing to study the influ-
ence of configurational isomerism on the biological activity. Fur-
thermore, the substitution pattern of the aromatic rings was also
modified.
10
11
H
OH
Coum-OH
12 OCH3 OH
13 OCOOCH3 OH 84
14 OCH3 OCOOCH3 OH 88
Feru-OH
H
4-MC-Coum-OH
4-MC-Feru-OH
4-MC-Cinn-Cl
4-MC-Coum-Cl
4-MC-Feru-Cl
A similar derivative to our target compounds is chicoric acid20
(2) with immunostimulator and HIV-1 integrase inhibitor activi-
ties.21
15
16
H
H
H
Cl 100*
OCOOCH3 Cl 100*
HO
17 OCH3 OCOOCH3 Cl 100*
HOOC
O
O
OH
OH
* Conversion of the starting material.
HO
O
COOH
O
Scheme 2. Preparation of acid chlorides 15–17.
2
Crucial points for both the syntheses of chicoric acid20–23 and
the preparation of our target compounds are the protections of
phenolic OH and COOH groups. Acylation of unprotected tartaric
acids with carbonylcaffeoyl chloride is feasible, but this carbonate
type protection can be used only for caffeic acid.20 Benzylic protec-
tion of both functionalities seemed very attractive, however, for
the removal an equimolar amount of Pd(OAc)2 for each protective
group was necessary rendering this method extremely expen-
sive.23 Orthogonal ester type protection (acetyl for OH and tert-
butyl for COOH22 or methoxycarbonyl for OH and diphenylmethyl
for COOH21) were also applied in the syntheses of 2 and their ana-
logues. Our comparative preliminary experiments showed that
introduction of the latter pair of protecting groups was more effi-
cient and easier to reproduce than that of the benzylic protection.
Acylations of 7–9 were carried out in dry toluene using
2.2 equiv of acid-chlorides 15–17 and 2.2 equiv dry pyridine as
base (Scheme 3). The fully protected 18–25 were isolated by col-
umn chromatography in acceptable yields.
Subsequent deprotections following the suggested protocol21
(removal of methoxycarbonyl groups with Na2CO3/aq THF and
cleavage of DPM esters with 70% aq AcOH) caused in our hands to-
tal decomposition of the molecules, irrespective of the order of the
deprotection steps.
Therefore, a new protocol for the cleavage of protecting groups
in 18–25 was developed. The DPM esters could be cleaved by using
dry anisole–TFA reagent in dry CH2Cl2 at room temperature.25 Puri-
fication of the crude products by column chromatography
(PhCH3:AcOH 3:1) gave 26–33 in good to excellent yields (Scheme
4). Hydrolysis of the methoxycarbonyl esters was achieved by
using an aq solution of NH3 in MeOH and the products 34–39 were
isolated in good to excellent yields. Reversing of the sequence re-
sulted in decomposition of the molecules in the first step. The
structure of the molecules was identified by NMR and MS
measurements.
Thus, the COOH groups of D-, L- and meso-tartaric acids (3–5)
were transformed into diphenylmethyl (DPM) esters (7–9) by
diphenyldiazomethane (DPDAM) generated in situ from benzophe-
24
none-hydrazone (6) by oxidation with activated MnO2 in CH2Cl2
(Scheme 1). DPM esters 7–9 were isolated in excellent yields as
white crystals and used further without any purification.
Next, acid-chlorides 15–17 were prepared from commercially
available cinnamic (10), p-coumaric (11), and ferulic acids (12),
respectively (Scheme 2), whereby phenolic OH groups of 11 and
12 were protected as methyl-carbonates 13 and 14, respectively.
Thionyl-chloride treatment of carboxylic acids 10, 13, and 14 gave
acid-chlorides 15–17 which were used for acylations without fur-
ther purification.
The synthesized derivatives were evaluated as inhibitors of rab-
bit muscle glycogen phosphorylase b (rmGPb), and the results are
summarized in Table 1. For comparison FR258900 (1) was also
tested under our conditions. Compound 1 proved a competitive
inhibitor against AMP and the obtained Ki of 0.2
agreement with the literature value. When tested against G1P, 1
appeared as a non-competitive inhibitor with a Ki of 5.47
lM showed a good
l
M
(please, see Fig. 1 in the Supplementary data for details of the mea-
surements and plots of the data). Similar conclusions could be
drawn from the kinetic studies of compounds 32–39, as well,
thereby indicating that in general the tartaric acid derivatives
bound to the same site as FR258900 (1).
As non-competitive inhibitors against G1P, the cinnamoyl
derivatives 32 and 33, lacking the 4-OH substituents characteristic
of the natural product 1, proved practically inefficient. In the
p-coumaroyl (34–36) and feruloyl (37–39) series the meso-
configured compounds 36 and 39 proved most efficient. The latter
demonstrated that introduction of an additional substituent in the
aromatic rings (3-CH3O) was very advantageous and 39 proved
equipotent with 1.
MnO2 - MgSO4
Ph2CNNH2
dry CH2Cl2
6
rt.
HOOC
HO
DPMOOC
HO
Ph2CN2
OH
OH
COOH
3 - 5
dry CH2Cl2
rt.
COODPM
7 - 9
Starting compound Configuration
Product Yield (%)
3
4
5
D
L
or (2S,3S)
or (2R,3R)
7
8
9
85
83
82
meso or (2R,3S)
As competitive inhibitors against AMP, beside the meso-config-
Scheme 1. Preparation of DPM esters 7–9.
ured 36 and 39, the L-configured 38 proved most efficient.

G. Varga et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1789–1792
1791
DPMOOC
HO
DPMOOC
acid-chloride (15 - 17)
O
R
OH
R
O
dry pyridine
COODPM
7 - 9
dry toluene, r.t.
COODPM
18 - 25
Starting compound
R
Product (Configuration)
Yield (%)
7
8
7
8
9
7
8
9
18 (
19 (
20 (
21 (
D)
63
52
54
48
83
48
50
43
Cinn
L
D
L)
)
)
4-MC-Coum
4-MC-Feru
22 (meso)
23 (
D)
24 (
L
)
25 (meso)
Scheme 3. Acylations with acid chlorides 15–17.
R2
R1
O
COOH
32, 33
to
DPMOOC
R1
R2
O
25 eq. CF3COOH
7 eq. dry anisole
dry CH2Cl2
O
R
O
R
O
HOOC
32 - 39
Products B
O
34-39
to
HOOC
COODPM
18 - 25
O
R
O
COOH
25% NH3 solution
MeOH
R
26 - 31
Products A
Starting compound
(Configuration)
R
Products A Yield (%) Products B
R1
R2 Yield (%)
18 (
19 (
20 (
21 (
D
)
-
-
32
33
34
35
36
37
38
39
H
H
H
H
H
H
83
86
85
89
30
81
69
88
Cinn
L
)
-
-
H
D
)
26
27
28
29
30
31
88
82
82
85
79
92
OH
OH
OH
4-MC-Coum
L
)
22 (meso)
23 (
D
)
CH3O OH
CH3O OH
CH3O OH
4-MC-Feru
24 (
L)
25 (meso)
Scheme 4. Cleavage of the protective groups.
activities. The syntheses were characterized by using methoxycar-
bonyl (MC) and diphenylmethyl (DPM) esters to protect phenolic
OH and COOH groups, respectively. New methods were applied
for the removal of these protective groups (anisole–TFA for the
cleavage of DPM, aq NH3–MeOH to hydrolyse MC). Some of the
new compounds proved inhibitors of rabbit muscle glycogen phos-
phorylase b (competitive inhibition against AMP, non-competitive
against G1P) in the low micromolar range. It was demonstrated by
this work that simple synthetic compounds can bind to rmGPb
similarly to the natural product, thereby opening up a new way
for further studies of allosteric site inhibitors. The synthetic avail-
ability of these analogs offers obvious advantages over FR258900.
Table 1
Inhibition (Ki
compounds
[lM]) of rabbit muscle glycogen phosphorylase b by the synthetic
Compound (configuration)
G1P dependence
5.47
AMP dependence
a
1 (L
)
0.20
0.4618
—
32 (
33 (
34 (
35 (
D
)
)
800b
No inh.
109
L
)
—
D
26.4
11.3
5.68
19.0
2.68
2.0
L
)
300b
71.4
29.1
28.5
3.36
36 (meso)
37 (D)
38 (
L
)
39 (meso)
Acknowledgments
a
Configuration of the natural product corresponds to that of
Calculated from the IC50 value by using a web-based tool.26
L-tartaric acid.
b
This work was supported by NKTH-OTKA (CK-77712), TÁMOP
4.2.1/B-09/1/KONV-2010-0007 and TÁMOP-4.2.2./B-10/1-2010-
0024 projects co-financed by the European Union and the
European SocialFund, as well as János Bolyai Research Scholarships
(to L.J. and T.D.) of the Hungarian Academy of Sciences. Astellas
Pharma Inc., Japan is gratefully thanked for kind provision of a
sample of FR258900.
Nevertheless, the efficiency of the inhibitors lagged behind that of
1. The presence of the 3-CH3O moieties in 39 rendered this com-
pound the best inhibitor under these test conditions, as well.
In order to determine the molecular basis of the efficiency of
these synthetic compounds, preparation of further analogues as
well as molecular dockings and X-ray crystallographic studies are
in progress, and will be reported in due course.
Supplementary data
In conclusion, three series of D-, L- and meso-tartaric acids di-O-
acylated by cinnamic, p-coumaric, and ferulic acids were prepared
as synthetic analogues of FR258900, a natural product with
glycogen phosphorylase inhibitory and in vivo antihyperglycemic
Supplementary data (kinetics of FR258900 inhibition of rmGPb)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2013.01.042.

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G. Varga et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1789–1792
13. Zhang, L.; Liu, H. Expert Opin. Ther. Pat. 2010, 20, 969.
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