Structural identification of 4-benzyl-voglibose hydrochloride monohydrate using NMR and single-crystal X-ray diffraction methods

Article abstract of DOI:10.1016/j.carres.2014.08.001

The chemical structure studies on an important related substance of voglibose have been carried out using NMR spectroscopy and single crystal X-ray crystallography. For the structure identification study, hydrochloride monohydrate of this compound was iso

Full text of DOI:10.1016/j.carres.2014.08.001

Carbohydrate Research 402 (2015) 81–86
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Structural identification of 4-benzyl-voglibose hydrochloride
monohydrate using NMR and single-crystal X-ray diffraction
methods
Fen-Fen Zhang a, Yu-Qin Zhao b, Jian-Ping Fan b, Li-Gang Liu b, Rui-Wen Li b, Wen-Bin Shen a,c,
,
⇑
Ya Ding a,
⇑
a Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of Education, Nanjing 210009, China
b Pharmaxyn Laboratories Ltd, Wuxi 214000, China
c Pharmaceutical Research Institute, China Pharmaceutical University, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 May 2014
Received in revised form 22 July 2014
Accepted 1 August 2014
Available online 12 August 2014
The chemical structure studies on an important related substance of voglibose have been carried out using
NMR spectroscopy and single crystal X-ray crystallography. For the structure identification study, hydro-
chloride monohydrate of this compound was isolated and purified. Its molecular structure was character-
ized and analyzed using 1D (1H NMR, 13C NMR, and DEPT) and 2D (1H,1H-COSY, HSQC, HMBC, TOCSY, and
ROESY) NMR spectra. The signal crystal structure was detected by X-ray crystallography. All results
indicated this related substance of voglibose was C17H27NO7ÁHClÁH2O, 5,6-dideoxy-4-benzyl-5-{[2-
Keywords:
hydroxy-1-(hydroxymethyl)ethy]amino}-1-C-(hydroxymethyl)-
D-epi-inositol hydrochloride monohydrate.
Structural identification
4-Benzyl-voglibose
NMR
Ó 2014 Elsevier Ltd. All rights reserved.
X-ray crystallography
1. Introduction
using method (a)9,10 as shown in Scheme 1, it has been found that
there are four isomeric cyclitols as by-products in the preparation
process of voglibose.12 However, few literature reports the quality
control of voglibose’s related substances except for analytical
method studies,13,14 although the existence of the related sub-
stance has an important impact on drug efficacy and stability.
In the study on the quality control of voglibose, we found these
four isomeric cyclitols all came from the final step of the synthesis
procedure (Scheme 1), the debenzylation and reduction of tetra-
benzyl voglibose by palladium carbon, formate, and H2.12 It is
interesting that among four benzyl groups, monitored by thin layer
chromatography (TLC) method, three of them are generated
gradually from tetra-benzyl voglibose with the reaction going.
Only one isomeric cyclitol with cyclohexylmethyl group is elimi-
nated slowly and could be mixed with target product when the
reaction was completed. Although clarifying the position of
cyclohexylmethyl groups on voglibose is helpful to interpret the
chemical structure of related substrates, it is difficult to obtain
the impurity as well as to characterize it. Till now, there is no
literature that reports the successful isolation and the exact
cyclohexylmethyl group position of this voglibose by-product.
Therefore, in this paper, method (b) shown in Scheme 1 has been
adopted as the reaction condition for the synthesis of voglibose
in the last step. In the absence of H2, only one by-product
(compound 4) was produced, although its chemical structure is
unknown. This voglibose by-product was isolated using silica gel
Diabetes is a group of metabolic diseases featuring with the
hyperglycemia and macro- and micro-vascular complications.
Among them, the prevalence of type 2 diabetes mellitus is
becoming a major concern worldwide. A number of drugs have
been designed for diabetes treatment including sulfonylureas,
biguanides, peroxisome proliferator-activated receptor agonists,
and so on.1 As a newly identified potent
a-glucosidase inhibitor
used for lowering post-prandial blood glucose levels in people
with diabetes mellitus, voglibose delays the absorption and
digestion of dietary polysaccharides by inhibiting several carbohy-
drate digestive enzymes2 and subsequently reduces the risk of
macro- and micro-vascular complications.3–7 Recent reports
indicate that voglibose has an inhibitory effect on melanogenesis
in a reconstructed skin mode,8 making it another significant role
for therapeutic applications.
Pharmaceutical quality control, especially the control of related
substances, is essential to guarantee the eligibility of drugs. The
synthesis procedure of voglibose (compound 2) has been reported
in the previous literatures.9–11 So far, due to the last synthesis step
⇑
Corresponding authors. Tel.: +86 025 83271233 (W.-B.S.), +86 025 83271090
(Y.D.).
E-mail addresses: cpunmrswb@163.com (W.-B. Shen), ayanju@163.com
(Y. Ding).
http://dx.doi.org/10.1016/j.carres.2014.08.001
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

82
F.-F. Zhang et al. / Carbohydrate Research 402 (2015) 81–86
Pc/C/HCOOH/H
MeOH
HOH2C
(a)
2
R
1OH2C
+
HO
R
2O
HO
R
3O
CH2OH
CH2OH
CH2OH
CH2OH
OH
OH
OR4
OH
HN
HN
BnOH2C
BnO
3
2
a
=
=
=
-
-
-
R2
R
R
R
H
C6 11CH2
,
,
,
,
,
,
,
,
,
voglibose
(yield=92%)
H,
H,
H,
H,
b R1 R3 R4
R1 C6H CH2
=
BnO
CH2OH
CH2OH
=
=
=
OBn
HN
,
,
R1 R3 R4
R2
H11
OH
c
C6 11CH2
,
,
,
d R1 R2 R4
R3 C6H CH2
=
- .
,
2
3
4
11
1
voglibose's related substances
HOH2C
HO
HOH2C
Pc/C/HCOOH
MeOH
(b)
+
HO
HO
HO
CH2OH
CH2OH
CH2OH
OH
OBn
OH
OH
HN
HN
CH2OH
4
2
voglibose
(yield=86%)
4-benzyl-voglibose
HCl ethanol
HOH2C
HO
HO
CH2OH
CH2OH
OBn
OH
H2N
Cl
H
2O
5
4-benzyl-voglibose hydrochloride monohydrate
!
R
R
Scheme 1. Illustration of synthesis procedure for voglibose and its by products Bn is
and C6H11CH2– is
.
column chromatography. Its chemical structure has been system-
atically studied via various nuclear magnetic resonance (NMR)
spectroscopic methods and X-ray crystallography. The NMR spec-
troscopic methods include 1D 1H NMR, 13C NMR, DEPT and 2D
1H–1H COSY, HSQC, HMBC, TOCSY, and ROESY spectra. Based on
the spectral data, it is proved that the related substrate of voglibose
by using method (b) is 4-benzyl-voglibose (compound 4 in
Scheme 1), and the structure analysis of its hydrochloride monohy-
drate (compound 5 in Scheme 1) is elucidated in detail in this
paper.
results, C-5 (54.4), C-4 (78.9), C-3 (70.4), C-2 (72.4) are identified.
Similarly, the benzyl group is assigned at C-8 (72.7), H-8
(4.81:4.69), C-10 (127.7), H-10 (7.48), C-11 (128.0), H-11 (7.34),
C-12 (127.3), H-12 (7.28). In addition, the residual 2-hydroxyl-1-
hydroxymethyl-ethylamine
group
contains
one
tertiary
carbon atom and two secondary carbon atoms, which can be
attributed to signals of C-14 (59.4), C-15 (58.2), and C-15 (56.7).
According to 1H/13C-HSQC data, theses carbon atoms correspond
to H-14 (3.43), H-15 (3.67:3.57), and H-15 (3.68:3.63),
respectively. Two protons at dH 8.16 and 7.47 ppm having
correlations with H5, H6, H14, and H15 identified by TOCSY should
be the –HN+H– group because these signals disappear when
deuteroxide is added.
2. Results and discussion
Furthermore, 1H, 1H-COSY, and TOCSY experiment (shown in
Figures 5 and 6) indicate another two separated proton spin sys-
tems. One system is 2-hydroxyl-1-hydroxymethyl-ethylamine
group system identified by H-14–H-15 correlations. The other is
benzyl group system. Revealed by ROESY data (Figure 7), both H-
10–H-11–H-12 and H-8–H-12 have correlations. 1H/13C-HMBC
results further indicate the replacement of benzyl located at the
4-position for C-8 (72.2) has long range correlations with H-4
(3.54). Detailed assignments are also listed in Table 1. Eight active
protons are omitted since they are exchanged when D2O is added
and cannot be assigned unambiguously. Therefore, the chemical
structure of 4-benzyl-voglibose has been identified by using NMR
methods, as shown in Figure 8 (A).
To further confirm the chemical configuration, a suitable crystal
was selected and analyzed on an Xcalibur, Eos, Gemini diffractom-
eter. Using Olex2,15 structure was solved with SheLXS16 structure
solution program using Direct methods and refined with SheLXL16
refinement package using least squares minimization. The crystal
structure and data are shown in Figure 8 (B) and Table 2, respec-
tively. This substance with the formula of C17H27NO7ÁHClÁH2O is
4-benzyl-voglibose hydrochloride monohydrate. The chemical
configuration is consistent with the NMR result. The benzyl moiety
is substituted on the hydroxyl group located at the 4-position with
To separate the target impurity from the product, the crude
product was purified by silica gel column chromatography. The
target impurity was collected, acidified, and concentrated. After
recrystallization in ethanol, the purified compound and its single
crystal were obtained, respectively.
To characterize the chemical structure of this mono-substituted
benzyl voglibose, 1D, and 2D NMR spectra of the purified com-
pound were detected. 1H and 13C NMR spectra are shown in Figures
1 and 2. The spectra and data (presented in Table 1) reveal that
there are 30 hydrogen atoms and 17 carbon atoms in this
compound. Among all hydrogen atoms, ten active protons are
contained, which are exchanged after the addition of D2O to
simplify the spectra and assignment. 17 carbon atoms in the struc-
ture include 2 quaternary, 10 tertiary, and 5 secondary carbon
atoms. According to the chemical shift, two quaternary carbon
atoms at 138.2 and 75.7 ppm can be attributed to C-9 and C-1,
respectively.
Since 1H/13C HMBC and 1H/13C HSQC spectra and data (shown
in Figures 3 and 4, and Table 1) indicate that four protons having
long range correlations with C-1 are H-7 (3.50:3.14) and H-6
(2.03:1.77), C-6 (28.0) and C-7 (64.3) are identified. 1H/1H-COSY
spectrum reveal a proton spin system: H-6 (2.03:1.77) -H-5
(4.04) -H-4 (3.54) -H-3 (3.78) -H-2 (2.32). Combining 1H/13C-HSQC

F.-F. Zhang et al. / Carbohydrate Research 402 (2015) 81–86
83
Figure 1. 1H NMR spectra of the compound 5 dissolved in (A) DMSO-d6 and (B) DMSO-d6 with the addition of D2O.
Figure 2. 13C NMR spectra of the compound 5 dissolved in DMSO-d6.

84
F.-F. Zhang et al. / Carbohydrate Research 402 (2015) 81–86
Table 1
1H and 13C NMR data and assignments of compound 5 (DMSO-d6)
Position
dH (ppm, J/Hz)
dC (ppm)
HMBC (1H to 13C), 3J
COSY (1H to 1H)
1
2
3
4
5
6
7
8
/
75.7
72.4
70.4
78.9
54.4
28.0
64.3
72.2
138.2
127.7
128.0
127.3
/
2, 6, 7
3, 6, 7
2, 4
2, 3, 6, 8, 13
4, 6
/
3
3.32 (1H, d, 9.68)
3.78 (1H, t, 9.68)
3.54 (1H, dd, 2.95, 9.68)
4.04 (1H, d, 2.95)
2.03, 1.77 (2H, dd, 2.95, 16.02)
3.14, 3.50 (2H, d, 10.58)
4.81, 4.69 (2H, d, 11.85)
/
7.48 (2H, d, 7.20)
7.34 (2H, t, 7.20)
7.28 (1H, t, 7.20)
8.16, 7.47 (2H, t/s, br)
3.43 (1H, m)
2, 4
3, 5
4, 6, 13
5, 6
7
7
/
4, 10
8, 11, 12
8, 11, 12
10, 12
10
/
15
14,15
8
/
9
10
11
12
13
14
15
11, 12
10, 12
11
13, 14
15
59.4
58.2, 56.7
3.68, 3.67, 3.63, 3.57 (4H, m)
14
Figure 3. 1H/13C HMBC spectrum of the compound 5 dissolved in DMSO-d6.
Figure 4. 1H/13C HSQC spectrum of the compound 5 dissolved in DMSO-d6.

F.-F. Zhang et al. / Carbohydrate Research 402 (2015) 81–86
85
Figure 5. 1H/1H COSY spectrum of the compound 5 dissolved in DMSO-d6.
Figure 7. ROESY spectrum of the compound 5 dissolved in DMSO-d6.
carried out on Bruker Avance 500 MHz spectrometer (Swiss).
DMSO-d6 and D2O were purchased from Sigma–Aldrich Chemical
Co (USA). Diffraction data were recorded on an Xcalibur, Eos,
Gemini diffractometer (USA).
3.2. Preparation of voglibose and 4-benzyl-voglibose
The synthesis of voglibose from the starting material, D-glucose,
to compound 1 referred to the previous reports.9–11 The last step of
the adopted method (b) is shown in Scheme 1. After stirring under
N2 overnight at room temperature, the solid in the solution of
crude compound 1 (44.0 g) was removed by filtration and washed
with MeOH/H2O (1:l) several times. The filtrates were combined
and concentrated. The residue was separated on a column of
Dowex 50WÂ8 (H+, 2.5 L), washed with H2O and then eluted with
0.5 N ammonium hydroxide. The eluate was concentrated and the
crystal of voglibose was obtained in MeOH (16.0 g, 86%). After
filtrating the voglibose crystal, the filtrate was concentrated and
the residue was separated on a column of silica gel (150 mL) with
n-propanol/HOAc/H2O (8:1:l) to give compound 4 (0.7 g, 2.8%) as a
pale yellow syrup.
Figure 6. TOCSY spectrum of the compound 5 dissolved in DMSO-d6.
3.3. Preparation of 4-benzyl-voglibose hydrochloride
monohydrate
lattice parameters a = 7.6341(2) Å, b = 9.8212(3) Å, c = 26.1037(8)
Å,
a = b = c
= 90°, v = 1957.2(10) Å3. As shown in Figure 8 (B), this
The syrup of compound 4 (0.7 g) was dissolved with etha-
nol (20 mL) and hydrochloride (1 mL) was added. The solution
was stirred for 10 min and concentrated. The residue was
heated and refluxed with 95% ethanol (15 mL) until completely
dissolved. And then the mixture was cooled at room tempera-
ture and deposited for 24 h to give 4-benzyl-voglibose
hydrochloride monohydrate (compound 5, 0.6 g, 73%) as white
crystals.
compound contains two different six-membered rings: cyclohex-
ane ring and benzyl ring bridged by O-4. The unit O-4–C-8–(C-9–
C14) connected with C-4 in equatorial bond, and the angles of
C-8–O-4–C-4 is 115.0(3)°, O-4–C-4–C-5 is 110.3(3)°, O-4–C-4–C-3
is 108.3(3)°. The 2-hydroxyl-1-hydroxymethyl-ethylamine group
aligned with C-5 in axial bond, and the angles of C-15–N–C-5 is
114.6(2)°, N–C-5–C-4 is 110.0(2)°, N–C-5–C-6 is 110.0(2)°.
3.4. Characterization
3. Experimental
3.1. General
3.4.1. NMR spectroscopy
NMR experiments were run on Bruker Avance 500 MHz spec-
trometer (1H NMR, 500.13 MHz; 13C NMR, 125.76 MHz) in
dimethyl sulfoxide (DMSO-d6), DMSO-d6 and deuteroxide (D2O)
mixed solution with tetramethyl-silane (TMS, dH = 0.00 ppm) or
All chemicals and reagents were purchased from Sinopharm
Chemical Reagent Co., Ltd and were of HPLC or analytical grade
and used without further purification. NMR experiments were

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F.-F. Zhang et al. / Carbohydrate Research 402 (2015) 81–86
A
B
OH
7
2
6
1
HO
4
H
2O
HO
5
OH
3
15
OBn
CH2OH
13
14
H2N
CH2OH
15
Cl
10
9
11
12
8
Bn=
11
10
Figure 8. (A) Chemical structure and (B) the crystal structure of compound 5.
Table 2
3.4.3. Supplementary data
Crystal data and structure refinement for compound 5
Complete crystallographic data for 4-benzyl-voglibose
hydrochloride monohydrate analysis have been deposited with
the Cambridge Crystallographic Data Centre, CCDC number
989802. These data may be obtained free of charge from The Direc-
tor, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: +44 1223
336033; e-mail: deposit@ccdc.cam.ac.uk).
Formula
C17H30ClNO8
411.8
291.2
Orthorhombic
P212121
7.6341 (2)
Fw (g/mol)
Temperature (K)
Crystal system
Space group
a (Å)
b (Å)
9.8212 (3)
c (Å)
26.1037 (8)
90.00
Acknowledgments
a
(°)
b (°)
90.00
90.00
1957.2 (10)
4
1.398
2.24
880.0
We thank Analysis and Testing Center of Zhengzhou University
for crystallographic data. This work was financially supported by
the Fundamental Research Funds for the Central Universities
(Nos. JKQ2009026, JKP2011008), the Program for New Century
Excellent Talents in University (No. NCET-10-0816), and the Open
Project Program of MOE Key Laboratory of Drug Quality Control
and Pharmacovigilance (No. MKLDP2013MS04).
c
(°)
V (Å)3
Z
qcalcd (mg/mm3)
m (mmÀ1
F (000)
)
Crystal size (mm3)
2h range (°)
0.23 Â 0.2 Â 0.2
6.78–134.4
Index ranges
À8 6 h 6 9, À7 6 k 6 11, À31 6 l 6 9
7402
3480 [R(int)] = 0.0237
3480/13/289
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
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3.4.2. X-ray crystallography
X-ray crystallography was performed by Xcalibur, Eos,
Gemini diffractometer at 291.2 K,
l
(Cu K ) = 2.124 mmÀ1, Dcalc =
a
1.398 g/cm3, 7402 reflections measured (6.78 6 2h 6 134.14), 3480
unique (Rint = 0.0237) which were used in all calculations. And
structure was solved with SheLXS16 structure solution program
using Direct methods and refined with SheLXL16 refinement
package using least squares minimization.
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