Efficient synthesis of a water-soluble glucoamide inhibitor against human aldose reductase by click chemistry

Article abstract of DOI:10.1080/07328303.2013.816852

While D-glucose is the natural substrate of aldose reductase (AR) in the polyol pathway, the K_m value of D-glucose against AR is large. A glucoamide 1 was designed as a tool to investigate whether AR has a strong affinity for the open form of D-glucose. Glucoamide 1 was synthesized in high yield by modification of the reaction condition for click chemistry. It was found that our modified condition was applicable for highly polar alkynes and gave coupling products in excellent yield (90% to 100%). Although weak inhibitory activity against AR was observed, kinetic studies showed that AR does not accept glucoamide 1 in its active site.

Full text of DOI:10.1080/07328303.2013.816852

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Efficient Synthesis of a Water-Soluble
Glucoamide Inhibitor Against Human
Aldose Reductase by Click Chemistry
Mikio Fujii a , Shinji Sudo a , Yasuyuki Kitagawa b & Keisuke Kato a
a Faculty of Pharmaceutical Sciences , Toho University , Funabashi ,
Chiba , 274-8510 , Japan
b School of Pharmacy , Showa University , Shinagawa-ku , Tokyo ,
142-8555 , Japan
Published online: 11 Oct 2013.
To cite this article: Mikio Fujii , Shinji Sudo , Yasuyuki Kitagawa & Keisuke Kato (2013) Efficient
Synthesis of a Water-Soluble Glucoamide Inhibitor Against Human Aldose Reductase by Click
Chemistry, Journal of Carbohydrate Chemistry, 32:7, 424-437
To link to this article: http://dx.doi.org/10.1080/07328303.2013.816852
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Journal of Carbohydrate Chemistry, 32:424–437, 2013
ꢀ
C
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2013.816852
Efficient Synthesis of a
Water-Soluble Glucoamide
Inhibitor Against Human
Aldose Reductase by Click
Chemistry
Mikio Fujii,1 Shinji Sudo,1 Yasuyuki Kitagawa,2 and Keisuke Kato1
1Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510,
Japan
2School of Pharmacy, Showa University, Shinagawa-ku, Tokyo 142-8555, Japan
While D-glucose is the natural substrate of aldose reductase (AR) in the polyol path-
way, the Km value of D-glucose against AR is large. A glucoamide 1 was designed as
a tool to investigate whether AR has a strong affinity for the open form of D-glucose.
Glucoamide 1 was synthesized in high yield by modification of the reaction condition
for click chemistry. It was found that our modified condition was applicable for highly
polar alkynes and gave coupling products in excellent yield (90% to 100%). Although
weak inhibitory activity against AR was observed, kinetic studies showed that AR does
not accept glucoamide 1 in its active site.
Keywords Click chemistry; Aldose reductase; Glycoside
INTRODUCTION
Aldose reductase (AR, EC 1.1.1.21) catalyzes the conversion of D-glucose to
D-sorbitol, which is the first step in the polyol pathway (Sch. 1) of glucose
metabolism and is implicated in the pathogenesis of a variety of diabetic com-
plications such as neuropathy.[1–3] Many AR inhibitors, such as epalrestat,
sorbinil, and fidarestat, have been developed to prevent diabetic complica-
tions.[4–7]
Received April 23, 2013; accepted June 15, 2013.
Address correspondence to Mikio Fujii, Faculty of Pharmaceutical Sciences, Toho
University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan. E-mail: mfujii@phar.
toho-u.ac.jp
424

Efficient Synthesis of a Water-Soluble Glucoamide Inhibitor
425
Scheme 1: Polyol pathway.
There are many reports investigating substrate specificities and charac-
teristics of AR.[8–10] It is an NADP-dependent enzyme and formally promotes
a hydride shift from NADPH to the aldehyde group of D-glucose (Fig. 1).
AR possesses broad substrate specificity. The apparent Km value of D-glucose
(55.0 mM) for AR was much larger than that of D,L-glyceraldehyde (0.1 mM).[9]
This phenomenon has also been reported by Grimshow.[10] We questioned
whether the large Km of D-glucose against AR arises from the nature of its
cyclic structure or its poor binding ability. D-Glucose, being in equilibrium be-
tween the aldehyde and the cyclic hemiacetal forms (Sch. 1), mainly forms
Figure 1: Reaction mechanism of AR and structure of inhibitor 1.

M. Fujii et al.
426
cyclic hemiacetals in water. AR accepts the aldehydic form of D-glucose to re-
duce its aldehyde group, but hemiacetals were not reduced. Normally, the ap-
parent Km value involves the equilibrium constant between hemiacetal and
aldehyde forms and is estimated to be larger than expected with the intrin-
sic binding affinity against the aldehyde form. Thus, a glucoamide inhibitor 1
was designed as a model of the open-chain structure of D-glucose (Fig. 1). If
a glucoamide inhibitor 1 is a strong inhibitor against AR, it becomes a highly
water-soluble inhibitor that differs from hydrophobic known inhibitors.[4–7]
Compound 1 was a coenzyme–substrate complex-type inhibitor possessing
ribofuranoside for the NADPH and glucoamide moieties and could be synthe-
sized by conjugation of glucoamide 2 and azide 3 with a ribosyl moiety added
by the Cu (I)-catalyzed Huisgen reaction (click chemistry).[11]
In this paper we report the synthesis of 1 and its inhibitory activity against
AR.
RESULTS AND DISCUSSION
Synthesis of 1
Compound 1 was synthesized as shown in Scheme 2. Glucoamide 2 was
synthesized by a reported method.[12] Azide 3 was synthesized by a modified
reported method.[13,14] Coupling of 2 and 3 under conditions using CuSO4 and
sodium ascorbate was tested,[11] but the reaction was not completed within
1
2 days, and separation of 4 (68% conversion on H NMR) from the remaining
Scheme 2: Synthesis of 1.

Efficient Synthesis of a Water-Soluble Glucoamide Inhibitor
427
2 and 3 by column chromatography and precipitation failed. While these cou-
pling conditions have commonly been used for water-soluble compounds, there
were several reports that gave products in low to moderate yields and required
long reaction times and heating.[15–17] Our compounds 1 and 4 were difficult to
separate from starting materials 2 and 3 and reagents such as sodium ascor-
bate. We tested several conditions and found that basic conditions using an
excess amount of 3 (1.2 eq. against 2) were required to complete the reaction.
Coupling of 2 and 3 in the presence of Cu(OAc)2, Na2CO3, and sodium ascor-
bate proceeded smoothly, and 2 disappeared on TLC after 1 h of reaction to give
4. Inorganic and organic salts were separated by ODS column chromatography
and the obtained 4 was washed with ethyl acetate to afford pure 4 in 96% iso-
lated yield. Treatment of 4 with aqueous TFA (30%) gave 1 quantitatively.
Modified Click Reaction of the Other Compounds
Table 1 shows the results of application of our modified condition (condi-
tion A) to other alkynes (5a–f). The standard condition[11] (condition B) was
also tested with the alkynes and the conversion is listed in Table 1 as well. The
modified condition was applied to acidic compounds (5a–c), neutral compounds
(5e, 5f, and 5h), phenolic compounds (5d), and basic compounds (5g and 5i).
The reactions proceeded smoothly in all cases and the starting alkynes disap-
peared within 1 h shown by TLC analysis, while the reactions under condition
B using copper sulfate were not completed within 1 h. Although the conver-
sion under condition B in Table 1 might have been increased when water was
removed by evaporation during the workup procedure, the starting materials
5 and 3 still remained after the evaporation in the case of neutral compounds
(5e and 5h) and basic compounds (5g and 5i). Improvement of the conversions
on the reaction was observed under condition A. The separation of the prod-
uct and the reactants became easier and the products 6a–i were obtained in
excellent isolated yield (90%–100%) by the basic conditions.
Inhibitory Activities of Glucoamides 1 and 2
Inhibitory activities of 1 and 2 against AR were tested. When epalrestat
was used as a control, 1 μM of epalrestat reduced the activity to 38%. While
10 mM of glucoamide 2 did not inhibit the activity of AR, 10 mM of glu-
coamide 1 reduced the activity to 82% (Fig. 2). Since weak inhibitory activ-
ity of 1 was observed, the inhibitory manner of 1 was further investigated
by Cornish-Bowden’s method as shown in Figures 3 and 4. The Km for D,L-
glyceraldehyde was estimated to be 0.14 mM, which is similar to the reported
value.[9] As shown in Figures 3 and 4, glucoamide 1 exhibited uncompetitive
inhibition against both glyceraldehyde and NADPH, and the Ki value was
14 mM. The binding affinity of 1 against AR was two orders lower than that

M. Fujii et al.
428
Table 1: The click reactions of 3 with various alkynes
Condition Aa
Condition Bb
Conv.c
Product
Isolated yield (%)
[Conv.c]
6a
6b
6c
6d
6e
6f
6g
6h
6i
98%
99%
[>98%]
[>98%]
[>98%]
[>98%]
[>98%]
[>98%]
[>98%]
[>98%]
[≥98%]
>98%
>98%
98%
95%
100%
90%
100%
100%
100%
90%
>98%
86%
>98%
85%
91%
91%
a3: 1.0 mmol; alkyne: 0.9 mmol; Cu(OAc)2: 0.25 mmol; Na2CO3: 0.2 mmol; sodium ascorbate:
1.0 mmol; water: 2 mL, rt, 2 h.
b3: 1.0 mmol; alkyne: 0.9 mmol; CuSO4: 0.25 mmol; sodium ascorbate: 1.0 mmol; water: 2 mL,
rt, 2 h.
cConversion was calculated based on 1H NMR spectra of reaction mixture.
Figure 2: Inhibitory activity of 1 and 2. Conditions: aldose reductase 15 mg/mL, NADPH
0.25 mM, glyceraldehyde 0.2 mM, 0.1% BSA, sodium phosphate buffer (0.1 M, pH 7.0).
Inhibitors: epalrestat: 1 μM, 1 and 2: 10 mM.

Efficient Synthesis of a Water-Soluble Glucoamide Inhibitor
429
Figure 3: Cornish-Bowden’s plot for inhibition by 1 against AR at various concentrations of
glyceraldehyde. Conditions: 15 μg/mL aldose reductase, 0.25 mM NADPH, 0.1% BSA, sodium
phosphate buffer (0.1 M, pH 7.0), [1] = 0, 2.5, 5, and 10 mM, [S: glyceraldehyde] = 0.2 (•), 0.1
(ꢀ), and 0.05(ꢁ) mM (color figure available online).
of D,L-glyceraldehyde. Thus, AR was not found to accept our glucoamide in-
hibitors 1 and 2 for its substrate binding site.
In conclusion, synthesis of glucoamide 1 was achieved by modifying the re-
action conditions of click chemistry. Although there are reports to activate the
click reaction by using organic base,[18] our method is useful for the synthesis
Figure 4: Cornish-Bowden’s plot for inhibition by 1 against AR at various concentrations of
NADPH. Conditions: 15 μg/mL aldose reductase, 0.1 mM glyceraldehyde, 0.1% BSA, sodium
phosphate buffer (0.1 M, pH 7.0), [1] = 2.5, 5, and 10 mM. [S: NADPH] = 0.25 (•), 0.13 (ꢀ), and
0.063 (ꢁ) mM (color figure available online).

M. Fujii et al.
430
of water-soluble nucleotides and nucleosides since inorganic salts were utilized
as activators of the reaction. Use of copper acetate and sodium carbonate are
considered to activate the generation of the acetylide ion in order to coordinate
to copper (I) under basic conditions. Inhibitory activities of glucoamides 1 and
2 against AR were investigated to determine whether the aldehyde form of
D-glucose bound to AR strongly. However, glucoamides 1 and 2 containing the
structure of the open form of D-glucose were not found to bind to the active site
of AR. Thus, D-glucose, known as the natural substrate on the polyol pathway,
is a poor substrate due to its low binding affinity against AR, but not due to its
cyclic hemiacetal structures.
EXPERIMENTAL
General
The NMR (1H, 13C, H-H COSY, HMQC, and HMBC) spectra were recorded
1
on JEOL-ECP-500 (500 MHz for H NMR and 125 MHz for 13C NMR) and
1
JEOL-ECS-400 spectrometers (400 MHz for H NMR and 100 MHz for 13C
NMR). Mass spectra were measured by a JEOL JMS-T100LP. IR spectra were
obtained on a JASCO FT/IR 4100 using the ATR method (neat). Specific rota-
tion was measured on a JASCO P-2200. Chemicals were purchased from Tokyo
Kasei Industry Co., Ltd., Wako Chemicals, or Aldrich Inc. unless otherwise in-
dicated. ODS column chromatography was performed on Chromatorex ODS
DM1020T (Fuji-Silysia Chemical Co. Ltd.). Inhibition studies were performed
on GeneQuant 1300 spectrophotometers. Human AR (1.0 U/μg, ALR2, AKR
1B1, recombinant from E. coli) was purchased from AT Gen Co., Ltd.
Synthesis of 1
2,3-O-Isopropylidene-1β-azido-D-ribofuranose (3)
To a solution of tetra-O-acetyl-β-D-ribofuranose (20.0 g, 63.0 mmol) and
TMSN3 (13.0 mL, 98.8 mmol) in CH2Cl2 (100 mL) was added boron trifluoride-
diethyl etherate (6.0 mL, 94.4 mmol) under Ar atmosphere at 0◦C. The re-
sulting mixture was stirred for 6 h, then poured into aq. NaHCO3 and ex-
tracted by CHCl3 twice. The organic layer was dried over Na2SO4 and evapo-
rated under reduced pressure. The residue was dissolved in methanolic NH3
(2 M, 300 mL). The solution was stirred for 12 h and then evaporated under
reduced pressure. The residue was dissolved in acetone (100 mL) containing
2,2-dimethoxypropane (12 mL, 97.9 mmol) and HClO4 (1.0 mL). The mixture
was stirred for 10 min and methanolic NH3 (2 M, 3 mL) was added to the
mixture. The residue was purified by silica gel column chromatography (200 g

Efficient Synthesis of a Water-Soluble Glucoamide Inhibitor
431
of silica gel) eluted with hexane/ethyl acetate (20/1 to 8/1) to afford 3 (12.1 g,
56.1 mmol, 89%).
N-(1-(2,3-O-Isopropylidene-β-D-ribofuranosyl)-triazol-4-ylmethyl)-D-
glucoamide (4)
To a solution of 2 (212 mg, 0.907 mmol) and Cu(OAc)2 monohydrate
(49.9 mg, 0.300 mmol) in water (2 mL) was added a solution of sodium L-
ascorbate (198.3 mg, 1.00 mmol), sodium carbonate (20 mg, 0.20 mmol), and
3 (219 mg, 1.01 mmol) in water (1 mL) at rt. The resulting mixture was stirred
for 2 h and then evaporated under reduced pressure. The residue was purified
by ODS column chromatography (30 g ODS gel) eluted with water to afford
crude 4 with unreacted 3. Removal of 3 from the crude 4 by washing with ethyl
acetate afforded 4 (390 mg, 0.871 mmol, 96%). [α]D = –23.0 (c 0.51, H2O). 1H
22
NMR (D2O, 500 MHz): δ 1.32 (3H, s, CH3), 1.50 (3H, s, CH3), 3.36 (1H, dd, J =
12.1, 6.6 Hz, rib-5ꢁꢁ), 3.48 (1H, dd, J = 12.4, 4.6 Hz, rib-5ꢁꢁ), 3.53 (1H, dd, J =
11.7, 4.8 Hz, glc-6), 3.62 (2H, s, glc-4, glc-5), 3.69 (1H, d, J = 11.7 Hz, glc-6),
3.98 (1H, d, J = 2.3 Hz, glc-3), 4.24 (1H, d, J = 2.3 Hz, glc-2), 4.37 (1H, s, rib-
4ꢁꢁ), 4.44 (2H, s, CH2), 4.92 (1H, d, J = 5.0 Hz, rib-3ꢁꢁ), 5.44 (1H, d, J = 5.0 Hz,
rib-2ꢁꢁ), 6.23 (1H, s, rib-1ꢁꢁ), 8.03 (1H, s, triazole-5’). 13C NMR (D2O, 125 MHz):
δ 24.2 (CH3), 25.8 (CH3), 34.3 (CH3), 61.4 (5ꢁꢁ), 62.7 (6), 70.5 (3), 71.1 (5), 72.1
(4), 73.5 (2), 81.3 (3ꢁꢁ), 84.0 (2ꢁꢁ), 88.1 (4ꢁꢁ), 93.3 (1ꢁꢁ), 114.5 (isopropylidene-C),
123 (br, 5’), 174.7(1). ESI-MS: Calcd. for C17H28N4O10Na [M+Na+]: 471.1703;
Found: 471.1710. IR: ν 3324, 1649, 1587, 1535, 1428, 1383 cm−1
.
N-(1-(β-D-Ribofuranosyl)-triazol-4-ylmethyl)-D-glucoamide (1)
Compound 4 (491 mg, 0.955 mmol) was dissolved in 30% aq. trifluoroacetic
acid (TFA) (2 mL). The mixture was stirred for 3 h and then evaporated under
reduced pressure to afford 1 (500 mg, partially TFA complex). To remove TFA
from the obtained 1, the TFA complex of 1 was dissolved in a small portion
of aq. NaHCO3 and subjected to ODS column chromatography (50 g ODS gel)
eluted with water to afford 1 (324 mg, 0.793 mmol, 83%) and recovered TFA
complex of 1 (1:1-TFA = 4:3 by 1H NMR analysis, 75.0 mg, 0.163 mmol, 17%).
[α]D = –17.2 (c = 1.04, H2O). 1H NMR (D2O, 500 MHz): δ 3.51 (1H, dd, J =
22
12.3, 5.6 Hz, glc-6), 3.60 (3H, m, glc-4, glc-5, rib-5ꢁꢁ), 3.68 (1H, dd, J = 12.3,
2.3 Hz, glc-6), 3.72 (1H, dd, J = 12.6, 3.2 Hz, rib-5ꢁꢁ), 3.97 (1H, t, J = 3.1 Hz,
glc-3), 4.10 (1H, dd, J = 8.3, 5.0 Hz, rib-4ꢁꢁ), 4.23 (1H, d, J = 3.1 Hz, glc-2), 4.29
(1H, t, J = 5.0 Hz, rib-3ꢁꢁ), 4.42 (2H, s, NCH2), 4.54 (1H, dd, J = 5.0, 4.5 Hz,
rib-2ꢁꢁ), 5.99 (1H, d, J = 4.5 Hz, rib-1ꢁꢁ), 7.99 (1H, s, triazole-5’). 13C NMR (D2O,
125 MHz): δ 34.3 (CH2), 61.2 (5ꢁꢁ), 62.7 (6), 70.2 (3ꢁꢁ), 70.5 (3), 71.2 (5), 72.1 (4),
73.5 (2), 74.9 (2ꢁꢁ), 85.3 (4ꢁꢁ), 92.1 (1ꢁꢁ), 122.8 (5’), 145.2 (br, 4’), 174.7 (1). ESI-MS
m/z: Calcd. for C14H24N4O10Na [M + Na+]: 431.1390; Found: 431.1361. IR: ν
3314, 1687, 1650, 1642, 1535 cm−1
.

M. Fujii et al.
432
General Procedure (Condition A) of Click Reactions for Table 1
To a solution of alkyne (0.90 mmol) and Cu(OAc)2 monohydrate (40 mg,
0.25 mmol) in water (2 mL) was added a solution of sodium L-ascorbate
(198 mg, 1.00 mmol), sodium carbonate (20 mg, 0.20 mmol), and 3 (215 mg,
1.0 mmol) in water (1 mL) at rt. In the case of condition B, copper (II) sulfate
monohydrate (50 mg, 0.25 mmol) was used instead of Cu(OAc)2 monohydrate
and sodium carbonate. The resulting mixture was stirred for 2 h and evapo-
rated under reduced pressure. The residue was purified by silica gel column
chromatography (10 g silica gel) eluted with CH3Cl/CH3OH (30/1∼10/1) to af-
ford 5.
N-(1-(2,3-O-Isopropylidene-β-D-ribofuranosyl)-triazol-4-ylmethyl)succinamide
(6a)
6a (329 mg, 0.889 mmol, 98%) from 5a[18] (140 mg, 0.903 mmol) and 3
21
1
(215 mg, 1.00 mmol). [α]D
= –50.5 (c = 0.88, CH3OH). H NMR (CD3OD,
500 MHz): δ 1.38 (3H, s, CH3), 1.56 (3H, s, CH3), 2.51 (2H, t, J = 6.9 Hz, CH2),
2.62 (2H, t, J = 6.9 Hz, CH2), 3.49 (1H, dd, J = 12.0, 4.4 Hz, rib-5’), 3.52 (1H,
dd, J = 12.0, 4.0 Hz, rib-5ꢁ), 4.33–4.37 (1H, m, rib-4ꢁ), 4.45 (2H, d, NHCH2),
4.95 (1H, d, J = 4.8 Hz, rib-3ꢁ), 5.35 (1H, d, J = 4.8 Hz, rib-2ꢁ), 6.21 (1H, s, rib-
1ꢁ), 8.08 (1H, s, triazole-5ꢁ); 13C NMR (125 MHz, CD3OD): δ 24.2 (CH3), 26.0
(CH3), 28.9 (CH2), 30.1 (CH2), 34.5 (CH2), 61.8 (5ꢁ), 82.0 (3ꢁ), 84.8 (2ꢁ), 88.6 (4ꢁ),
94.3 (1ꢁ), 113.5 (isopropylidene-C), 122.5 (5), 145.6 (4), 173.3 (CO), 175.0 (CO);
ESI- MS: Calcd. for C15H22N4O7Na (M+Na+): 393.1386; Found: 393.1297; IR:
ν 3335, 1716, 1652,1541 cm−1
.
N-(1-(2,3-O-Isopropylidene-β-D-ribofuranosyl)-triazol-4-ylmethyl)glutaramide
(6b)
6b (380 mg, 0.959 mmol, 99%) from 5b[19] (163 mg, 0.964 mmol) and 3
25
1
(230 mg, 1.07 mmol). [α]D
= –37.1 (c = 1.26, CH3OH). H NMR (CD3OD,
500 MHz): δ 1.39 (3H, s, CH3), 1.57 (3H, s, CH3), 1.90 (2H, quintet, J = 6.0 Hz,
CH2), 2.29 (2H, t, J = 6.0 Hz, CH2), 2.31 (2H, t, J = 6.0 Hz, CH2), 3.50 (1H,
dd, J = 12.0, 4.2 Hz, rib-5ꢁ), 3.53 (1H, dd, J = 12.0, 4.4 Hz, rib-5ꢁ), 4.34–4.38
(1H, m, rib-4ꢁ), 4.45 (2H, s, NHCH2), 4.90–4.96 (1H, m, rib-3ꢁ), 5.34 (1H, d, J =
1.3 Hz, rib-2ꢁ), 6.21 (1H, d, J = 1.3 Hz rib-1ꢁ), 8.11 (1H, s, triazole-5ꢁ); 13C
NMR (125 MHz, CD3OD): δ 20.9 (CH2), 24.1 (CH3), 26.0 (CH3), 33.5 (CH2),
34.3 (CH2), 34.7 (CH2), 61.8 (5ꢁ), 82.0 (3ꢁ), 84.8 (2ꢁ), 88.6 (4ꢁ), 94.8 (1ꢁ), 113.6
(isopropylidene-C), 122.5 (5), 145.1 (4), 174.2 (CO), 175.9 (CO); ESI- MS: Calcd.
for C16H24N4O7Na (M+Na+): 407.1543; Found: 407.1533; IR: ν 3396, 1646,
1558, 1541 cm−1
.

Efficient Synthesis of a Water-Soluble Glucoamide Inhibitor
433
N-(1-(2,3-O-Isopropylidene-β-D-ribofuranosyl)-triazol-4-ylmethyl)-4-
methylbenzene sulfonamide (6c)
6e (369 mg, 0.870 mmol, 95%) from 5e (191 mg, 0.918 mmol) and 3 (215 mg,
26
1
1.00 mmol). [α]D
= –43.2 (c 1.02, CH3OH). H NMR (CDCl3, 500 MHz): δ
1.34 (3H, s, CH3), 1.55 (3H, s, CH3), 2.38, (3H, s, CH3), 3.52 (1H, dd, J = 12.0,
2.9 Hz, rib-5ꢁ), 3.65 (1H, dd, J = 12.0, 3.3 Hz, rib-5ꢁ), 4.15 (2H, d, NHCH2),
4.41–4.43 (1H, m, rib-4ꢁ), 4.90 (1H, d, J = 6.0, 1.1 Hz, rib-3ꢁ), 5.15 (1H, d, J =
6.0, 2.0 Hz, rib-2ꢁ), 6.01 (1H, d, J = 2.0 Hz, rib-1ꢁ), 7.25 (2H, d, J = 8.3 Hz, Ts-
3,5), 7.67 (2H, d, J = 8.3 Hz, Ts-2,6), 7.77 (1H, s, triazole-5ꢁ); 13C NMR (CDCl3,
125 MHz): δ 21.5 (CH3), 25.1 (CH3), 27.0 (CH3), 28.2 (CH2), 62.6 (5ꢁ), 81.8 (3ꢁ),
85.3 (2ꢁ), 88.7 (4ꢁ), 95.1 (1ꢁ), 113.9 (isopropylidene-C), 122.6 (5), 127.1 (2C,Ts-
3,5), 129.8 (2C,Ts-2,6), 136.7 (Ts-4), 143.7 (Ts-1), 145.5 (4); ESI- MS: Calcd. for
C18H22\4N4O6SNa (M + Na+): 447.1314; Found: 393.1268; IR: ν 3258, 1457,
1319 cm−1
.
4-Hydroxy-N-(1-(2,3-O-isopropylidene-β-D-ribofuranosyl)-triazol-4-
ylmethyl)benzamide (6d)
6d (361 mg, 0.926 mmol, 100%) from 5d[20] (162 mg, 0.926 mmol) and 3
26
1
(215 mg, 1.00 mmol). [α]D
= –59.5 (c = 1.11, CH3OH). H NMR (CD3OD,
500 MHz): δ 1.37 (3H, s, CH3), 1.56 (3H, s, CH3), 3.49 (1H, dd, J = 12.4, 5.5 Hz,
rib-5ꢁ), 3.54 (1H, dd, J = 12.4, 5.5 Hz, rib-5ꢁ), 4.35 (1H, dt, J = 1.6, 5.5 Hz,
rib-4ꢁ), 4.63 (2H, s, NHCH2), 4.94 (1H, dd, J = 1.6, 6.1 Hz, rib-3ꢁ), 5.34 (1H,
dd, J = 1.9, 6.1 Hz, rib-2ꢁ), 6.20 (1H, d, J = 1.9 Hz, rib-1ꢁ), 6.82 (2H, d, J
= 8.6 Hz, PhOH-H-3,5), 7.73 (2H, d, J = 8.6 Hz, PhOH-H-2,6), 8.11 (1H, s,
triazole-5ꢁ). 13C NMR (125 MHz, CD3OD): δ 24.2 (CH3), 26.0 (CH3), 34.8 (CH2),
61.8 (5ꢁ), 82.0 (3ꢁ), 84.8 (2ꢁ), 88.6 (4ꢁ), 94.3 (1ꢁ), 113.5 (isopropylidene-C), 114.8
(2C, PhOH-3,5), 122.6 (5), 124.7 (PhOH-1), 129.1 (2C, PhOH-2,6), 145.6 (4),
160.9 (C-O), 168.7 (C = O); ESI- MS: Calcd. for C18H22N4O6Na (M + Na+):
413.1437; Found: 417.1372; IR: ν 3336, 1352, 1607, 1507 cm−1
.
N-(1-(2,3-O-Isopropylidene-β-D-ribofuranosyl)-triazol-4-ylmethyl)acetamide
(6e)
6e (253 mg, 0.811 mmol, 90%) from 5e (87.1 mg, 0.896 mmol) and 3
21
1
(215 mg, 1.00 mmol). [α]D
= –72.1 (c = 1.03, CH3OH). H NMR (400 MHz,
CDCl3): δ 1.38 (3H, s, CH3), 1.59 (3H, s, CH3), 1.98 (3H, s, CH3), 2.03 (1H, s,
OH), 3.64 (1H, dd, J = 12.4, 4.4 Hz, rib-5ꢁ), 3.77 (1H, dd, J = 12.4, 3.6 Hz,
rib-5ꢁ), 4.44 (2H, d, NHCH2), 4.49–4.52 (1H, m, rib-4ꢁ), 4.98 (1H, dd, J = 5.8,
1.8 Hz, rib-3ꢁ), 5.23 (1H, dd, J = 5.8, 2.0 Hz, rib-2ꢁ), 6.11 (1H, d, J = 2.0 Hz,
rib-1ꢁ), 7.19 (1H, s, NH), 7.91 (1H, s, triazole-5ꢁ); 13C NMR (100 MHz, CDCl3):
δ 22.8 (CH3), 25.0 (CH3), 26.9 (CH3), 34.6 (CH2), 62.8 (5ꢁ), 81.6 (3ꢁ), 85.3 (2ꢁ),
88.6 (4ꢁ), 95.1 (1ꢁ), 113.7 (isopropylidene-C), 122.6 (5), 144.7 (4), 170.9 (CO).

M. Fujii et al.
434
ESI- MS: Calcd. for C13H20N4O5Na (M + Na+): 335.1331; Found: 335.1193; IR:
ν 3297, 1658, 1550 cm−1
.
4-(Ethoxycarbamoylmethyl)-triazol-1-yl 2,3-O-isopropylidene-β-D-
ribofuranoside (6f)
6f (351 mg, 1.02 mmol, 100%) from 5f[21] (141 mg, 1.11 mmol) and 3
26
1
(219 mg, 1.02 mmol). [α]D
= –57.8 (c = 0.52, CH3OH). H NMR (CDCl3,
500 MHz): δ 1.10 (3H, t, J = 7.0 Hz, CH3), 1.26 (3H, s, CH3), 1.48 (3H, s, CH3),
3.40–3.70 (2H, m, rib-5ꢁ), 3.98 (3H, t, OCH2), 4.29 (2H, s, NCH2), 4.34 (1H, s,
rib-4ꢁ), 4.90 (1H, d, J = 5.0 Hz, rib-3ꢁ), 5.20 (1H, d, J = 5.0 Hz, rib-2ꢁ), 6.03 (1H,
s, rib-1ꢁ), 6.16 (1H, brs, OH), 7.68 (1H, brs, NH), 7.81 (1H, s, triazole-5ꢁ); 13C
NMR (125 MHz, CDCl3): δ 14.6 (CH3), 25.1 (CH3), 27.0 (CH3), 36.2 (CH2), 61.0
(CH2), 62.5, (5ꢁ), 81.9 (3ꢁ), 85.2 (2ꢁ), 88.8 (4ꢁ), 94.7 (1ꢁ), 113.6 (isopropylidene-C),
122.4 (5), 148.7 (4), 156.9 (CO); ESI- MS: Calcd. for C14H22N4O6Na (M+Na+):
365.1437; Found: 365.1320; IR: ν 3340, 1698, 1540 cm−1
.
N-(1-(2,3-O-Isopropylidene-β-D-ribofuranosyl)-triazol-4-ylmethyl)-pyridin-3-
ylcarbamide (6e)
6e (270 mg, 0.750 mmol, 100%) from 5e[22] (120 mg, 0.750 mmol) and 3
27
1
(192 mg, 0.89 mmol). [α]D
= –62.6 (c = 1.41, CH3OH). H NMR (CDCl3,
500 MHz): δ 1.35 (3H, s, CH3), 1.50 (3H, s, CH3), 3.50 (1H, dd, J = 12.2, 4.9 Hz,
rib-5ꢁ), 3.62 (1H, dd, J = 12.2, 3.9 Hz, rib-5ꢁ), 4.39 (1H, ddd, J = 4.9, 3.9, 1.8 Hz,
rib-4ꢁ), 4.54 (1H, d, J = 12.0 Hz, NHCH2), 4.57 (1H, d, J = 12.0 Hz, NHCH2),
4.87 (1H, dd, J = 6.0, 1.8 Hz, rib-3ꢁ), 5.17 (1H, dd, J = 2.0, 6.0 Hz, rib-2ꢁ), 6.21
(1H, d, J = 2.0 Hz, rib-1ꢁ), 7.34 (1H, br, pyr-5), 8.00 (1H, s, triazole-5ꢁ), 8.12 (1H,
br, pyr-4), 8.58 (1H, br, pyr-6), 8.95 (1H, br, pyr-2); 13C NMR (CDCl3, 125 MHz):
δ 25.1 (CH3), 26.9 (CH3), 35.1 (CH2), 62.4 (5ꢁ), 81.7 (3ꢁ), 85.3 (2ꢁ), 88.5 (4ꢁ),
95.0 (1ꢁ), 113.8 (isopropylidene-C), 122.9 (5), 123.8 (pyr-5), 130.0 (pyr-4), 144.7
(pyr-6) 148.1 (4), 151.7 (pyr-2), 166.1 (CO); ESI- MS: Calcd. for C17H21N5O5Na
(M+Na+): 398.1440; Found: 398.1401; IR: ν 3309, 1646, 1541 cm−1
.
4-Hydroxymethyltriazol-1-yl 2,3-O-isopropylidene-β-D-ribofuranoside (6h)
6h (278 mg, 1.02 mmol, 100%) from 5h (63.0 mg, 1.13 mmol) and 3 (221 mg,
1.02 mmol). [α]D = –72.7 (c = 0.99, CH3OH); 1H NMR (CD3OD, 400 MHz): δ
21
1.28 (3H, s, CH3), 1.48 (3H, s, CH3), 3.42 (1H, dd, J = 12.4, 4.4 Hz, rib-5ꢁ), 3.51
(1H, dd, J = 12.4, 3.6 Hz, rib-5ꢁ), 4.29–4.33 (1H, m, rib-4ꢁ), 4.60 (2H, s, OCH2),
4.84 (1H, d, J = 5.8 Hz, rib-3ꢁ), 5.23 (1H, d, J = 5.8 Hz, rib-2ꢁ), 6.08 (1H, s,
rib-1ꢁ), 7.89 (1H, s, triazole-5ꢁ); 13C NMR (100 MHz, CD3OD): δ 24.0 (CH3),
25.8 (CH3), 54.7 (CH2), 61.3 (5ꢁ), 81.2 (3ꢁ), 84.2 (2ꢁ), 87.8 (4ꢁ), 93.8 (1ꢁ), 113.1
(isopropylidene-C), 121.5 (5), 147.5 (4); ESI-MS: Calcd. for C13H20N4O5Na
(M+Na+): 294.1066; Found: 194.0752; IR: ν 3352, 1682 cm−1
.

Efficient Synthesis of a Water-Soluble Glucoamide Inhibitor
435
4-Aminomethyltriazol-1-yl 2,3-O-isopropylidene-β-D-ribofuranoside (6i)
6i (242 mg, 0.898 mmol, 90%) from 5i (61.0 mg, 1.11 mmol) and 3 (215 mg,
1.00 mmol) (purified by silica gel chromatography using Cromatorex NH pur-
21
1
chased from Fuji Silisia Chemical Ltd.) [α]D
= –0.4 (c = 0.15, CH3OH); H
NMR (CD3OD, 500 MHz): δ 1.38 (3H, s, CH3), 1.57 (3H, s, CH3), 3.42–3.60
(2H, m, rib-5ꢁ), 3.78 (2H, s, NCH2), 4.30–4.35 (1H, m, rib-4ꢁ), 4.94 (1H, d, J =
5.0 Hz, rib-3ꢁ), 5.34 (1H, d, J = 5.0 Hz, rib-2ꢁ), 6.22 (1H, s, rib-1ꢁ), 8.10 (1H, s,
triazole-5ꢁ); 13C NMR (100 MHz, CD3OD): δ 25.2 (CH3), 27.0 (CH3), 52.7 (CH2),
62.3 (5ꢁ), 81.6 (3ꢁ), 85.2 (2ꢁ), 88.7 (4ꢁ), 94.8 (1ꢁ), 113.2 (isopropylidene-C), 125.7
(5), 147.4 (4); ESI-MS: Calcd. for C11H18N4O4Na (M+Na+): 293.1226; Found:
293.1485; IR: ν 3287, 1652, 1285 cm−1
.
Kinetics and Inhibitory Activity of Glucoamide
Enzyme activity was determined by measuring the change in absorbance
due to NADPH at 340 nm (ε = 6300 M−1cm−1). The activity was recorded for
4 min after 30 s from mixing an enzyme solution and a substrate solution. A
standard reaction mixture contained 15 μg/mL AR, 0.25 mM NADPH, 0.2 mM
glyceraldehyde, and 0.1% BSA in sodium phosphate buffer (0.1 M, pH 7.0). Ini-
tial velocities were fitted to appropriate equations (1/v vs. [I] plot for competi-
tive inhibition or [S]/v vs. [I] plot for uncompetitive inhibition) with graphical
methods to determine the inhibitory manner.[23] The Km value for glyceralde-
hyde against AR was calculated from the data in the absence of inhibitor in
Figure 3.
ACKNOWLEDGMENT
This work was supported by KAKENHI No. 23790103 [Grant-in-Aid for Young
Scientists (B)].
REFERENCES
1. Judzewitsch, R.G.; Jaspan, J.B.; Polonsky, K.S.; Weinberg, C.R.; Halter, J.B.; Halar,
E.; Pfeifer, M.A.; Vukadinovic, C.; Bernstein, L.; Schneider, M.; Liang, K.Y.; Gabbay,
K.H.; Rubenstein, A.H.; Porte, D. Jr. Aldose reductase inhibition improves nerve con-
duction velocity in diabetic patients. N. Engl. J. Med. 1983, 308, 119–125.
2. Gabbay, K.H. The sorbitol pathway and the complications of diabetes. N. Engl. J.
Med. 1973, 288, 831–836.
3. Jaspan, J. Pharmacological inhibition of aldose reductase in human diabetic neu-
ropathy. Drugs 1986, 32, 23–29.

M. Fujii et al.
436
4. Oka, M.; Kato, N. Aldose reductase inhibitors. J Enzyme Inhib. 2001, 16, 465–473.
5. Sato, N.; Kashima, K.; Uehara, Y.; Ohtani, K.; Shimizu, H.; Mori, M. Epalrestat,
an aldose reductase inhibitor, improves an impaired generation of oxygen-derived free
radicals by neutrophils from poorly controlled NIDDM patients. Diabetes Care 1997,
20, 995–998.
6. Giannoukakis, N.; Fidarestat. Sanwa Kagaku/NC Curex/Sankyo. Curr. Opin. In-
vestig. Drugs 2003, 4, 1233–1239.
7. Uchida, K.; Kigoshi, T.; Nakano, S.; Ishii, T.; Kitazawa, M.; Morimoto, S. Effect
of 24 weeks of treatment with epalrestat, an aldose reductase inhibitor, on peripheral
neuropathy in patients with non-insulin-dependent diabetes mellitus. Clin. Ther. 1995,
17, 460–466.
8. Balendiran, G.K.; Sawaya, M.R.; Schwarz, F.P.; Ponniah, G.; Cuckovich, R.; Verma,
M.; Cascio, D. The role of Cys-298 in aldose reductase function. J. Biol. Chem. 2011, 286,
6336–6344.
9. Grimshaw, C.E.; Shahbaz, M.; Putney C.G. Mechanistic basis for nonlinear ki-
netics of aldehyde reduction catalyzed by aldose reductase. Biochemistry. 1990, 29,
9947–9955.
10. Grimshaw, C.E. Aldose reductase: model for a new paradigm of enzymic perfection
in detoxification catalysts. Biochemistry 1992, 31, 10139–10145.
11. Rostovtsev, V.V.; Green, L.G.; Fokin, V.V.; Sharpless, K.B. A stepwise Huisgen cy-
cloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal
alkynes. Angew. Chem. Int. Ed. 2002, 41, 2596–2599.
12. Ryu, E.-H.; Zhao Y. Efficient synthesis of water-soluble calixarenes using click
chemistry. Org. Lett. 2005, 7, 1035–1037.
13. Deranieh, R.M.; Greenberg, M.L.; LeCalvez, P.-B.; Mooney, M.C.; Migaud, M.E.
Probing myo-inositol 1-phosphate synthase with multisubstrate adducts. Org. Biomol.
Chem. 2012, 10, 9601–9619.
14. Li, L.; Lin, B.; Yang, Z.; Zhang, L.; Zhang, L. A concise route for the preparation
of nucleobase-simplified cADPR mimics by click chemistry. Tetrahedron Lett. 2008, 49,
4491–4493.
15. Bossu, I.; Berthet, N.; Dumy, P.; Renaudet, O. Synthesis of glycocyclopeptides
by click chemistry and inhibition assays with lectins. J. Carbohydr. Chem. 2011, 30,
458–468.
16. Dedola, S.; Hughes, D.L.; Nepogodiev, S.A.; Rejzek, M.; Field, R.A. Synthesis of
α- and β-D-glucopyranosyl triazoles by CuAAC click chemistry: reactant tolerance, re-
action rate, product structure and glucosidase inhibitory properties. Carbohydr. Res.
2010, 345, 1123–1134.
17. Baron, A.; Bleriot, Y.; Sollogoub, M.; Vauzeilles, B. Phenylenediamine catalysis of
“click glycosylations” in water: practical and direct access to unprotected neoglycocon-
jugates. Org. Biomol. Chem. 2008, 6, 1898–1901.
18. Loison, S.; Cottet, M.; Orcel, H.; Adihou, H.; Rahmeh, R.; Lamarque, L.; Trinquet,
E.; Kellenberger, E.; Hibert, M.; Durroux, T.; Mouillac, B.; Bonnet, D. Selective fluores-
cent nonpeptidic antagonists for vasopressin V2 GPCR: application to ligand screening
and oligomerization assays. J. Med. Chem. 2012, 55, 8588–8602.
19. Wang, Q.; Chan, T.R.; Hilgraf, R.; Fokin, V.V.; Sharpless, K.B.; Finn, M.G. Biocon-
jugation by copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc.
2003, 125, 3192–3193.

Efficient Synthesis of a Water-Soluble Glucoamide Inhibitor
437
20. Punna, S.; Meunier, S.; Finn, M.G. A hierarchy of aryloxide deprotection by boron
tribromide. Org. Lett. 2004, 6, 2777–2779.
21. Wipf, P.; Aoyama, Y.; Benedum, T.E. A practical method for oxazole synthesis by
cycloisomerization of propargyl amides. Org. Lett. 2004, 6, 3593–3595.
22. Mohammad, A.; Peiming, Z.; Donald, E.B. A comparative study of the thermal sta-
bility of oligodeoxyribonucleotides containing 5-substituted 2’-deoxyuridines. Nucleic
Acids Res. 1998, 26, 3127–3135.
23. Dixon, M.; Webb, E.C. Enzymes, 3rd ed, Longman: London, 1979; pp. 332–467.