A Facile Synthesis of AICAR from Inosine

Article abstract of DOI:10.1055/s-2003-42455

5-Amino-1-β-D-ribofuranosylimidazole-4-carboxamide (AICAR; 1) (Figure 1) was efficiently prepared from inosine through its 1-alkoxymethylderivatives. The stability of these derivatives enabled their purification by column chromatography (silica gel). The operation ensured the high quality of 1, which was obtained in good yield through alkaline hydrolysis of the derivatives.

Full text of DOI:10.1055/s-2003-42455

PAPER
2639
A Facile Synthesis of AICAR from Inosine
F
acile Synthesis of
a
A
ICA
R
fr
o
om Inosine ki Kohyama, Yukio Yamamoto*
Graduate School of Human and Environmental Studies, Kyoto University, Sakyo-ku, Yoshida, Kyoto 606-8501, Japan
Fax +81(75)7536833; E-mail: yukio@fischer.jinkan.kyoto-u.ac.jp
Received 28 July 2003; revised 1 September 2003
dure was troublesome. In this context, we started the ex-
ploration of a facile synthesis of AICAR in the laboratory
without using special equipment.
Abstract: 5-Amino-1- -D-ribofuranosylimidazole-4-carboxamide
(AICAR; 1) (Figure 1) was efficiently prepared from inosine
through its 1-alkoxymethylderivatives. The stability of these deriv-
atives enabled their purification by column chromatography (silica
gel). The operation ensured the high quality of 1, which was ob-
tained in good yield through alkaline hydrolysis of the derivatives.
At first, we examined the synthesis of AICAR through hy-
drogenolysis of N-benzyl AICAR 4,8 which was prepared
from inosine in 2 steps (Scheme 1).
Key words: furans, inosine, nucleosides, hydrolyses, ring opening
5-Amino-1- -D-ribofuranosylimidazole-4-carboxamide
(AICAR; 1) is a constituent of AICA-ribotide, which is a
biosynthetic precursor of purine nucleotide (AMP, GMP)
(Figure 1). In synthetic organic chemistry, AICAR also
has been utilized as a precursor of nucleoside and nucle-
otide derivatives.1
O
N
NH2
N
O
NH2
HO
HO
OH
AICAR; 1
Figure 1
Now, AICAR is receiving great attention since it is an ac-
tivator of AMP-activated protein kinase (AMPK). AMPK
is considered to be a master switch, regulating key pro-
teins in metabolic pathways, for example, hepatic fatty
acid oxidation, lipogenesis, triglyceride synthesis, and
skeletal muscle fatty acid oxidation.2 AMPK is also sup-
posed to be an important mediator of insulin-independent
glucose transport in skeletal muscle.3 Thus, activation of
AMPK is an important target for the treatment of type 2
diabetes.4
Scheme 1
However, the hydrogenolysis did not proceed at all in sev-
eral conditions. The NH2 group of 4 was suspected to in-
hibit the hydrogenation and we planned to place the
benzyl group away from the NH2 group. Actually, we in-
troduced the benzyloxymethyl group, a well-known pro-
tecting group of hydroxy groups, at the N-1 atom of
inosine.
The desired aminal 6a was obtained in 49% yield when
tri-O-acetylinosine 5 was reacted with benzyl chlorome-
thyl ether in the presence of N,N-diisopropylethylamine in
CH2Cl2 at 0 °C. In order to carry out the aqueous alkaline
hydrolysis of the heterocycle in homogeneous solution,
the acetyl groups of 6a were removed prior to the hydrol-
ysis. Actually, 6a was treated with aq NH3 in MeOH at
room temperature to afford triol 7a. When 7a was treated
with aq NaOH (0.2 M) under reflux, we obtained AICAR
in 58% yield from 6a. It was presumed that the ring-open-
ing reaction proceeded prior to the deprotection of the
benzyloxymethyl group since inosine was not detected
during this alkaline hydrolysis. The formamidine moiety
Whereas AICAR has been produced industrially from the
fermentation broth of glucose,5 the laboratory scale prep-
aration is rather difficult. AICAR was synthesized from
diaminomaleonitrile and 1-bromo-2,3,5-tri-O-benzoyl- -
D-ribofuranose through more than 5 steps only in mg scale
and the overall yield was very low (<5%).6 Although the
preparation through hydrolysis of inosine in a sealed tube
was reported,7 the yield was low and the isolation proce-
SYNTHESIS 2003, No. 17, pp 2639–2642
x
x.
x
x
.
2
0
0
3
Advanced online publication: 23.10.2003
DOI: 10.1055/s-2003-42455; Art ID: F06003SS
© Georg Thieme Verlag Stuttgart · New York

2640
N.Kohyama, Y. Yamamoto
PAPER
of 7a is considered to be hydrolyzed, followed by elimi- With the intention of improving the overall yield, we ex-
nation of benzyl alcohol, to imine intermediate 8, which is amined other aminal derivatives as the synthetic interme-
then converted to AICAR (Scheme 2).
diates for AICAR (Table 1, Scheme 3).
Table 1 Evaluation of Aminal Derivatives for AICAR Synthesis
O
N
Compound
R1
Yield (%)
NH
5
6
6
1
overall
28
Ac2O
N
O
N
Inosine(2)
AcO
pyridine
99%
a
b
c
d
e
Bn
49
77
85
86
53
58
39
49
66
41
AcO
OAc
Me
30
5
Et
42
O
(CH2)2OMe
(CH2)2TMS
57
N
N
OBn
22
N
Cl OBn
O
N
RO
iPr2NEt
CH2Cl2
0°C
The yields of 6a–e were moderate to high as shown in
Table 1. However, 6b was relatively unstable and seemed
to be unsuitable as a synthetic intermediate because a yel-
low-colored impurity developed gradually on standing.
Each deacetylation reaction of 6 proceeded very smooth-
ly. The alkaline hydrolysis of 7 to AICAR also proceeded
well and the overall yields of AICAR were satisfactory as
shown in Table 1.
RO
6a; R= Ac
OR
49%
NH3 aq./MeOH
O
7a; R= H
N
N
OBn
H
0.2N-NaOH aq.
N
O
N
O
OH-
H
HO
reflux
Among them, we can conclude that N-MEM inosine 6d is
the best synthetic intermediate which is very stable and
enables a high overall yield of AICAR. The purification of
6d can be performed easily by column chromatography
(silica gel), which ensures the high quality of AICAR.
MOM derivative 7b was proposed as a synthetic interme-
diate for AICAR;9 however, neither detailed description
of the reaction conditions nor the product characterization
was given in the report. Now, AICAR can be facilely pre-
pared with ordinary laboratory equipment, and further ap-
plication for AICAR analogues is in progress. The present
facile AICAR synthesis is considered to contribute to the
chemistry of purine–imidazole transformation as well as
to the biochemistry of glucose metabolism.
HO
OH
O
H
N
N
N
H
CHO
O
N
H
HO
HO
OH
8
AICAR; 1
58% from 6a
Mps were determined on a Yanaco MP-500 instrument. 1H and 13
C
NMR were recorded with a JEOL-JNM-EX-270 (at 270 MHz to 1H,
68 MHz to 13C) instrument. All J values are given in Hz. Optical ro-
tation was measured with a JASCO DIP-1000 instrument (with a 1
dm cell). HR-FAB-MS were done with a JEOL JMS-DX-300 in-
strument. TLC analysis were performed on silica gel 60-F254 (Mer-
ck). Column chromatography was performed on silica gel (Wako
Pure Chemical Industries, Ltd. Wakogel C-300).
Scheme 2
O
N
N
N
OR1
Cl OR1
O
N
5
RO
iPr2NEt
2 ,3 ,5 -Tri-O-acetyl-1-[(2-methoxyethoxy)methyl]inosine (6d)
To a solution of inosine triacetate 5 (5.0 g, 12.7 mmol) in CH2Cl2
(89 mL) was added i-Pr2NEt (3.31 mL, 19.0 mmol) at 0 °C. The re-
action mixture was treated with 2-methoxyethoxymethyl chloride
(1.74 mL, 15.2 mmol) at 0 °C and stirred at the same temperature
for 65 min. The reaction was quenched with H2O, stirred for 20 min,
and then diluted with CHCl3. The aq layer was extracted with
CHCl3. The combined organic layers were washed with H2O and
then brine, dried (MgSO4), and concentrated. Purification by col-
umn chromatography (silica gel; EtOAc–MeOH, 50:1) gave 6d.
CH2Cl2
0°C
RO
OR
6; R= Ac
7; R= H
NH3 aq./MeOH
0.2N-NaOH aq.
reflux
AICAR; 1
Scheme 3
Yield: 5.26 g (86%); [ ]D25 –27.6 (c 0.50, CHCl3).
Synthesis 2003, No. 17, 2639–2642 © Thieme Stuttgart · New York

PAPER
Facile Synthesis of AICAR from Inosine
2641
1H NMR (CDCl3): = 8.15 (s, 1 H, H-8), 7.94 (s, 1 H, H-2), 6.11 (d,
1 H, J = 5.3, H-1 ), 5.87 (t, 1 H, J = 5.3, H-2 ), 5.58 (dd, 1 H, J = 5.3,
5.0, H-3 ), 5.56 (s, 2 H, NCH2O), 4.46–4.41 (m, 2 H, H-4 , H-5 ),
4.37 (m, 1 H, H-5 ), 3.82 (m, 2 H, OCH2CH2O), 3.53 (m, 2 H,
OCH2CH2O), 3.35 (s, 3 H, OCH3), 2.15 (s, 3 H, acetyl), 2.13 (s, 3
H, acetyl), 2.10 (s, 3 H, acetyl).
13C NMR: = 170.2, 169.5, 169.2 (MeCO), 156.5 (C-6), 147.7 (C-
8), 147.2, 125.1 (C-4, C-5), 138.5 (C-2), 86.6 (C-1 ), 80.4 (C-5 ),
75.2 (NCH2O), 73.3 (C-2 ), 71.6, 69.4 (OCH2CH2O, OCH2CH2O),
70.5 (C-3 ), 63.1 (C-4 ), 59.1 (OCH3), 20.9, 20.7, 20.6 (CH3CO).
HR-FAB-MS: m/z calcd for C19H25N4O9 (M + H): 453.1621; found:
453.1608.
2 ,3 ,5 -Tri-O-acetyl-1-[(2-trimethylsilylethoxy)methyl]inosine
(6e)
Obtained according to the same procedure described above using 2-
(trimethylsilyl)ethoxymethyl chloride to give 6e.
Yield: 53%; [ ]D25 –29.0 (c 1.00, CHCl3).
1H NMR (CDCl3): = 8.04 (s, 1 H, H-8), 7.86 (s, 1 H, H-2), 6.00 (d,
1 H, J = 5.0, H-1 ), 5.75 (dd, 1 H, J = 5.6, 5.0, H-2 ), 5.49 (dd, 1 H,
J = 5.6, 4.6, H-3 ), 5.36 (s, 2 H, NCH2O), 4.32–4.24 (m, 2 H, H-4 ,
H-5 ), 4.22 (m, 1 H, H-5 ), 3.55 (t, 2 H, J = 8.2, OCH2CH2Si), 2.00
(s, 3 H, acetyl), 1.97 (s, 3 H, acetyl), 1.95 (s, 3 H, acetyl), 0.81 (t, 2
H, J = 8.2, OCH2CH2Si), –0.15 [s, 9 H, Si(CH3)3].
13C NMR: = 169.7, 169.0, 168.7 (MeCO), 155.9 (C-6), 147.2 (C-
8), 146.7, 124.6 (C-4, C-5), 138.2 (C-2), 86.1 (C-1 ), 79.8 (C-5 ),
74.0 (NCH2O), 72.9 (C-2 ), 70.0 (C-3 ), 67.1 (OCH2CH2Si), 62.7
(C-4 ), 20.4, 20.2, 20.1 (CH3CO), 17.7 (OCH2CH2Si), –1.7
[Si(CH3)3].
HR-FAB-MS: m/z calcd for C20H27N4O10 (M + H): 483.1727;
found: 483.1730.
2 ,3 ,5 -Tri-O-acetyl-1-[(benzyloxy)methyl]inosine (6a)
Obtained according to the same procedure described above using
benzyl chloromethyl ether to give 6a.
Yield: 49%; [ ]D25 –28.0 (c 0.35, CHCl3).
1H NMR (CDCl3): = 8.11 (s, 1 H, H-8), 7.94 (s, 1 H, H-2), 7.36–
7.27 (m, 5 H, Ar-H), 6.09 (d, 1 H, J = 4.9, H-1 ), 5.85 (t, 1 H,
J = 5.4, 4.9, H-2 ), 5.60 (dd, 1 H, J = 5.4, H-3 ), 5.59 (s, 2 H,
NCH2O), 4.68 (s, 2 H, benzyl), 4.45–4.42 (m, 2 H, H-4 , H-5 ), 4.37
(m, 1 H, H-5 ), 2.15 (s, 3 H, acetyl), 2.13 (s, 3 H, acetyl), 2.11 (s, 3
H, acetyl).
13C NMR: = 170.3, 169.5, 169.3 (MeCO), 156.5 (C-6), 147.6 (C-
8), 138.5 (C-2), 136.7, 125.2 (C-4, C-5), 128.5, 128.1, 128.0 (Ph),
86.6 (C-1 ), 80.3 (C-5 ), 74.4 (NCH2O), 73.3 (C-2 ), 72.0 (PhCH2),
70.4 (C-3 ), 63.0 (C-4 ), 20.8, 20.5, 20.4 (CH3CO).
5-Amino-1- -d-ribofuranosylimidazole-4-carboxamide (AIC-
AR; 1)
To a solution of aminal 6d (5.0 g, 10.4 mmol) in MeOH (41 mL)
was added aq NH3 solution (28%; 21 mL) at r.t. The reaction mix-
ture was stirred for 1 h at the same temperature and concentrated in
vacuo to give triol 7d (3.35 g, 91%). Compound 7d was used for the
next alkaline hydrolysis without further purification. A stirred mix-
ture of 7d (3.21 g, 9.01 mmol) and aq NaOH (0.2 M; 90 mL) was
heated under reflux for 1 h. After cooling, the reaction mixture was
neutralized with aq HCl (6 M), and then concentrated in vacuo. The
residue was extracted with hot EtOH, filtered and concentrated in
vacuo. Purification by column chromatography (silica gel; CHCl3–
MeOH, 3:1) gave AICAR.
HR-FAB-MS: m/z calcd for C24H27N4O9 (M + H): 515.1778; found:
515.1766.
2 ,3 ,5 -Tri-O-acetyl-1-[(methoxy)methyl]inosine (6b)
Obtained according to the same procedure described above using
chloromethyl methyl ether to give 6b.
Yield: 77%; [ ]D25 –30.5 (c 0.65, CHCl3).
Yield: 1.70g (73%); mp 215–216 °C (crystallized from MeOH);
[ ]D25 –59.1 (c 1.00, H2O).
1H NMR (DMSO-d6): = 7.30 (s, 1 H, H-2), 6.70 (br s, 2 H,
CONH2), 5.91 (s, 2 H, NH2), 5.45 (d, 1 H, J = 6.4, H-1 ), 5.35 (d, 1
H, J = 6.4, 2 -OH), 5.22 (dd, 1 H, J = 5.2, 4.9, 5 -OH), 5.14 (d, 1 H,
J = 4.6, 3 -OH), 4.28 (dd, 1 H, J = 6.3, 5.9, H-2 ), 4.09 (m, 1 H, H-
3 ), 3.90 (m, 1 H, H-4 ), 3.58 (m, 2 H, H-5 ).
1H NMR (CDCl3): = 8.11 (s, 1 H, H-8), 7.94 (s, 1 H, H-2), 6.10 (d,
1 H, J = 5.0, H-1 ), 5.88 (dd, 1 H, J = 5.3, 5.0, H-2 ), 5.60 (dd, 1 H,
J = 5.3, 5.0, H-3 ), 5.47 (s, 2 H, NCH2O), 4.46–4.41 (m, 2 H, H-4 ,
H-5 ), 4.25 (m, 1 H, H-5 ), 3.45 (s, 3 H, OCH3), 2.17 (s, 3 H, acetyl),
2.15 (s, 3 H, acetyl), 2.13 (s, 3 H, acetyl).
13C NMR: = 166.5 (CONH2), 142.7 (C-4), 128.4 (C-2), 112.6 (C-
5), 87.6 (C-1 ), 85.2 (C-4 ), 72.8 (C-2 ), 70.2 (C-3 ), 61.2 (C-5 ).
13C NMR: = 170.3, 169.6, 169.3 (MeCO), 156.6 (C-6), 147.5 (C-
8), 147.2, 125.2 (C-4, C-5), 138.6 (C-2), 86.6 (C-1 ), 80.3 (C-5 ),
76.2 (NCH2O), 73.3 (C-2 ), 70.4 (C-3 ), 63.0 (C-4 ), 57.5 (OCH3),
20.8, 20.6, 20.4 (CH3CO).
Anal. Calcd for C9H14N4O5 (258.2): C, 41.86; H, 5.46; N, 21.70.
Found: C, 41.96; H, 5.37; N, 21.80.
HR-FAB-MS: m/z calcd for C18H23N4O9 (M + H): 439.1465; found:
439.1484.
References
2 ,3 ,5 -Tri-O-acetyl-1-[(ethoxy)methyl]inosine (6c)
Obtained according to the same procedure described above using
chloromethyl ethyl ether to give 6c.
Yield: 85%; [ ]D25 –29.5 (c 1.00, CHCl3).
1H NMR (CDCl3): = 8.17 (s, 1 H, H-8), 7.98 (s, 1 H, H-2), 6.13 (d,
1 H, J = 4.9, H-1 ), 5.90 (dd, 1 H, J = 5.2, 4.9, H-2 ), 5.64 (dd, 1 H,
J = 5.2, 5.0, H-3 ), 5.52 (s, 2 H, NCH2O), 4.47–4.45 (m, 2 H, H-4 ,
H-5 ), 4.39 (m, 1 H, H-5 ), 3.67 (q, 2 H, J = 6.9, OCH2Me), 2.16 (s,
3 H, acetyl), 2.13 (s, 3 H, acetyl), 2.11 (s, 3 H, acetyl), 1.22 (t, 3 H,
J = 6.9, OCH2CH3).
13C NMR: = 169.9, 169.2, 168.9 (MeCO), 156.1 (C-6), 147.3 (C-
8), 146.8, 124.8 (C-4, C-5), 137.1 (C-2), 86.3 (C-1 ), 80.0 (C-5 ),
74.4 (NCH2O), 73.2 (C-2 ), 70.2 (C-3 ), 65.5 (OCH2Me), 62.8 (C-
4 ), 20.6, 20.4, 20.3 (CH3CO), 14.9 (OCH2CH3).
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Synthesis 2003, No. 17, 2639–2642 © Thieme Stuttgart · New York