Spectral assignments for the aldose reductase inhibitor 4(S)-2,3-dihydro-6-fluoro-2(R)-methylspiro[chroman-4,4′-imidazoline] -2′,5′-dione and its synthetic intermediates

Article abstract of DOI:10.1002/mrc.1697

The ¹H and ¹³C NMR chemical shifts of the aldose reductase inhibitor 4(S)-2,3-dihydro-6-fluoro-2(R)-methylspiro[chroman-4, 4′-imidazoline]-2′,5′-dione, methylsorbinil, and its seven synthetic intermediates, have been completely assigned on the basis of DEPT, COSY, g-HSQC and g-HMBC. All C-F coupling constants from one-bond to four-bond in the ¹³C NMR spectra and H-F and H-H coupling constants from three-bond to four-bond in ¹H spectra were obtained. Copyright

Full text of DOI:10.1002/mrc.1697

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2005; 43: 1008–1011
Published online 19 September 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1697
Spectral assignments for the aldose reductase inhibitor
4(S)-2,3-dihydro-6-fluoro-2(R)-methylspiro[chroman-
4,4ꢀ-imidazoline]-2’,5ꢀ-dione and its synthetic
intermediates
Mingan Wang, Manoj K. Mishra, Wenjun Zhu and Peter F. Kador∗
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198-6025, USA
Received 12 January 2005; Revised 13 July 2005; Accepted 21 July 2005
The 1H and 13C NMR chemical shifts of the aldose reductase inhibitor 4(S)-2,3-dihydro-6-fluoro-2(R)-
methylspiro[chroman-4,4ꢀ-imidazoline]-2’,5ꢀ-dione, methylsorbinil, and its seven synthetic intermediates,
have been completely assigned on the basis of DEPT, COSY, g-HSQC and g-HMBC. All C–F coupling
constants from one-bond to four-bond in the 13C NMR spectra and H–F and H–H coupling constants from
three-bond to four-bond in 1H spectra were obtained. Copyright  2005 John Wiley & Sons, Ltd.
KEYWORDS: 1H NMR; 13C NMR; DEPT; 2D NMR; C–F and H–F coupling constants; methylsorbinil; aldose reductase
inhibitor
has been extensively used in long-term galactose-fed dogs
to establish that both sugar cataracts and retinopathy can be
dose-dependently inhibited.12–14 2-Methylsorbinil contains
two chiral centers, and the most potent of the four possible
isomers has been determined by X-ray diffraction to possess
the 2(R),4(S) configuration.2,15–17 To obtain adequate quan-
tities of this 2(R),4(S) isomer, a stereospecific synthesis has
been developed in which stereochemistry of the 2-methyl
substituent of the future chroman ring is first established by
reaction of (S)-ethyl lactate with p-fluorophenol. After ring
closure, this is followed by resolution of position 4 of the
chroman ring with ˛-chymotrypsin.18–21 In the synthesis of
2-methylsorbinil (2), seven chiral intermediates (3–9) as well
as the final product (2) have been isolated (Fig. 1). Using 500-
MHz NMR, we report here the first complete assignments
INTRODUCTION
Aldose reductase (E.C. 1.1.1.21; ALR2) is an enzyme in
the polyol pathway that utilizes the coenzyme NADPH
to reduce the aldehyde group of a number of simple
sugars to their respective alcohols. Extensive animal studies
indicate that the reduction of glucose and galactose to their
respective sugar alcohols, sorbitol and galactitol, by aldose
reductase are linked to the formation of long-term diabetic
complications which include neuropathy, nephropathy,
retinopathy, keratopathy and cataract.1–7 Animal studies
also demonstrate that the onset and progression of these
complications can be prevented or significantly delayed by
inhibition of this enzyme.4–7 Therefore, aldose reductase
inhibitors represent a pharmacological approach that is
independent of the control of blood sugar levels for
the treatment of diabetic complications. This premise is
supported by recent clinical findings that indicate that the
susceptibility of type 1 and type 2 diabetic patients to develop
nephropathy, retinopathy, diabetic neuropathy and cataract
is linked to the presence of certain aldose reductase allels.8,9
These findings have led to the development of a number
of structurally diverse aldose reductase inhibitors. The spiro-
hydantoin inhibitor sorbinil, 1, (2,3-dihydro-6-fluoro-2(R)-
methylspiro[chroman-4,40-imidazoline]-20,50-dione) and its
more potent two methyl analog 2-methylsorbinil, 2,
(4(S)-2,3-dihydro-6-fluoro-2(R)-methylspiro[chroman-4,40-
imidazoline]-20,50-dione, M79175) have been extensively
used to investigate and establish the role of aldose reductase
in long-term animal studies (Fig. 1).10–14 2-Methylsorbinil
1
of H and 13C NMR chemical shifts based on DEPT, COSY,
g-HSQC and g-HMBC spectra, and the F–C or F–H coupling
constants in the NMR spectra for the chiral compounds (2–9).
RESULTS AND DISCUSSION
The data obtained from the 1H and 13C NMR spectra of
methylsorbinil, 2, its 4(R)-isomer, 9, and the intermediates
(3–8) are summarized in Tables 1 and 2. All eight chiral
compounds examined showed complex 1H and 13C NMR
spectra because of the existence of F–H and F–C coupling.
However, with the assistance of DEPT, COSY, g-HSQC and
g-HMBC spectra, all of the 1H and 13C NMR signals for
compounds (2–9) were assigned. Using 4(R)-amino-6-fluoro-
2(R)-methylchroman-4-carboxylic acid, 5, as an example, the
protons at υ 7.02 (dd, J D 2.9, 8.8 Hz, 1H), 7.15 (ddd, J D 2.9,
8.8, 9.2 Hz, 1H) and 6.96 (dd, J D 4.8, 9.2 Hz, 1H) correlated
in the COSY spectrum and were assigned to be H-5, H-7 and
ŁCorrespondence to: Peter F. Kador, College of Pharmacy 986025
Nebraska Medical Center, Omaha, NE 68198-6025, USA.
E-mail: pkador@unmc.edu
Copyright  2005 John Wiley & Sons, Ltd.

Spectral assignments for the aldose reductase inhibitor 1009
Figure 1. Structures of sorbinil, 1, it more potent 2-methyl analog, 2 and the seven intermediates (3–9) obtained in the synthesis of 2.
Table 1. 1H NMR data of compounds
No.
H-2
3
4
5
6
7
8
2
9
4.41 m
2.09 dd
4.63 m
1.98 dd
4.29 m
2.45 dd
4.45 m
1.72 dd
4.36 m
2.25 dd
4.25 m
2.41 dd
4.76 m
1.82 dd
4.20 m
2.02 dd
H˛-3
Hˇ-3
2JHH D 13.9
3JHH D 11.5
2.33 d
2JHH D 14.2
3JHH D 11.7
2.46 d
2JHH D 15.1
3JHH D 12.2
2.35 d
2JHH D 13.7
3JHH D 11.8
2.41 d
2JHH D 13.7
3JHH D 11.8
1.80 d
2JHH D 14.2
3JHH D 12.0
2.83 d
2JHH D 13.7
3JHH D 12.0
2.27 d
2JHH D 14.1
3JHH D 12.2
2.13 d
2JHH D 13.9
2JHH D 14.2
2JHH D 15.1
2JHH D 13.7
2JHH D 13.7
2JHH D 14.2
2JHH D 13.7
2JHH D 14.1
6.95 dd
7.07 dd
7.02 dd
7.17 dd
6.81 dd
7.30 dd
6.93 dd
6.82 dd
H-5
H-7
4JHH D 3.0
3JFH D 8.3
7.14 ddd
4JHH D 3.0
3JHH D 9.0
3JFH D 8.3
6.96 dd
4JHH D 2.9
3JFH D 8.3
7.11 ddd
4JHH D 2.9
3JHH D 9.3
3JFH D 8.3
6.93 dd
4JHH D 2.9
3JFH D 8.8
7.15 ddd
4JHH D 2.9
3JHH D 9.2
3JFH D 8.8
6.96 dd
4JHH D 3.2
3JFH D 9.0
6.86 ddd
4JHH D 3.2
3JHH D 9.5
3JFH D 9.0
6.76 dd
4JHH D 3.0
3JFH D 8.6
6.87 ddd
4JHH D 3.0
3JHH D 8.8
3JFH D 8.6
6.78 dd
4JHH D 2.9
3JFH D 9.5
7.05 ddd
4JHH D 2.9
3JHH D 8.8
3JFH D 9.5
6.88 dd
4JHH D 3.0
3JFH D 9.0
7.09 ddd
4JHH D 3.2
3JHH D 9.3
3JFH D 9.0
6.89 dd
4JHH D 3.0
3JFH D 8.8
7.12 ddd
4JHH D 3.0
3JHH D 8.8
3JFH D 9.3
6.88 dd
H-8
3JHH D 9.0
4JFH D 4.7
1.39 d
3JHH D 9.3
4JFH D 4.9
1.40 d
3JHH D 6.4
3JHH D 9.2
4JFH D 4.8
1.48 d
3JHH D 6.4
3JHH D 9.5
4JFH D 4.6
1.39 d
3JHH D 8.8
4JFH D 4.8
1.42 d
3JHH D 8.8
4JFH D 4.9
1.45 d
3JHH D 6.4
3JHH D 9.3
4JFH D 4.9
1.32 d
3JHH D 9.3
4JFH D 4.9
1.37 d
H-11
Other
3JHH D 6.3
3JHH D 6.4
3JHH D 6.3
3JHH D 6.3
3JHH D 6.4
7.99 d
3.72 s
3.72 s
10.92 s
11.09 s
7.69 t
8.35 s
8.84 s
7.59 t
Compounds 4 and 5, 6 and 7 and 2 and 9 are diastereomers at C-4. NMR solvents: 2, 3, and 9 in DMSO-d6; 4 and 5 in D2O; 6 and 7 in
CDCl3; 8 in CD3OD.
H-8. The CH3 proton at υ 1.48 (d, J D 6.4 Hz, 3H) correlated
with the proton at υ 4.29 (m, 1H) and the proton at υ 4.29
correlated with two protons at υ 2.35 (d, J D 15.1 Hz, 1H) and
2.45 (dd, J D 15.1, 12.2 Hz, 1H). In the COSY spectrum of 5,
these two protons also exhibited cross peaks, which indicated
that H-2 is located at υ 4.29 and that the two protons at υ
2.35 and 2.45 are the geminal protons at C-3. In the g-HSQC
spectrum of 5, correlations between υ 4.63 and 68.69; 2.35, 2.45
and 37.38; 7.02 and υ 113.79; 7.15 and 118.64; 6.96 and 119.31;
and 1.48 and 20.23 indicated that they are the carbons at
C-2, C-3, C-5, C-7, C-8 and C-11, respectively. The remaining
upfield quaternary carbon at υ 59.45 was assigned to be C-4.
In the g-HMBC spectrum of 5, long-range correlations were
observed between the carbon at υ 157.01 and the protons at
υ 6.96, 7.07 and 7.15; the carbon at υ 151.06 and the protons
υ 6.96, 7.07 and 7.15; the carbon at υ 119.28 and the protons
υ 6.96 and 2.35, which showed these quaternary carbons at υ
157.01, 151.06 and 119.28 are C-6, C-9 and C-10 respectively.
In the g-HMBC spectrum of 5, long-range correlations were
also observed between the carbon at υ 174.69 and the two
C-3 protons at υ 2.35 and 2.45; the carbon at υ 59.45 and the
protons at υ 2.35, 2.45 and 4.29; the carbon at υ 68.69 and the
protons at υ 2.45 and 1.48; the carbon at υ 37.38 and the proton
at υ 1.48 and the carbon at υ 20.23 and the protons at υ 2.35,
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: 1008–1011

1010
M. Wang et al.
Table 2. 13C NMR data of compounds
No.
3
4
5
6
7
8
2
9
C-2
C-3
C-4
C-5
69.23
38.47
68.49
71.26
38.02
58.73
68.69
37.38
59.45
70.28
42.27
56.26
68.28
40.96
57.35
68.97
36.93
57.90
69.80
37.74
60.52
68.79
36.31
61.05
114.26
2J D 23.5
156.34
1J D 238.0
117.92
2J D 23.5
119.02
3J D 8.1
151.36
4J D 1.9
119.70
3J D 7.2
20.71
178.94
160.08
133.37
129.22
128.13
125.59
112.32
113.79
113.27
2J D 23.5
156.68
1J D 238.5
116.10
2J D 23.0
118.01
3J D 8.2
150.72
4J D 1.9
125.38
3J D 6.7
21.09
112.69
2J D 23.0
156.60
1J D 238.5
116.21
2J D 23.5
118.58
3J D 7.7
150.57
4J D 1.9
124.62
3J D 6.8
20.79
113.52
2J D 24.5
156.84
1J D 237.0
116.90
2J D 24.0
118.68
3J D 7.7
151.94
4J D 1.8
121.26
3J D 7.2
20.13
112.81
2J D 23.5
156.83
1J D 237.0
117.57
2J D 23.1
119.19
3J D 7.6
152.29
4J D 1.9
121.94
3J D 6.7
21.39
113.44
2J D 23.5
156.44
1J D 237.5
117.47
2J D 23.5
118.88
3J D 8.1
151.61
4J D 1.9
121.48
3J D 7.2
20.83
2J D 24.5
2J D 24.0
C-6
C-7
C-8
C-9
C-10
157.21
157.01
1J D 238.5
117.77
1J D 238.5
118.64
2J D 23.5
2J D 23.5
119.27
119.31
3J D 8.7
151.26
3J D 8.2
151.06
4J D 1.9
4J D 2.4
120.58
119.28
3J D 7.2
20.20
3J D 7.1
20.23
C-11
C-12
Other
175.05
174.69
174.49
52.72
176.38
52.79
174.12
159.07
177.35
157.26
176.62
156.06
1
HMBC were utilized to clearly distinguish the H and 13C
NMR signals of H-5, H-7, H-8 and C-5, C-7 and C-8 in
the benzene ring. This enabled us to obtain the coupling
constants between H and H, H and F and C and F.
These coupling constants data are consistent with that of
p-substituted fluorobenzenes reported by Miyajima22 and
Frank.23
2.45 and 4.29. On the basis of these long-range correlations
and the assignments for proton and carbon signals in the 1H
and 13C NMR of 5 as indicated above, the carbon at υ 174.69
was identified to be the C-12 carboxylic acid.
The F–H coupling constants with three-bond and four-
bond and F–C coupling constants with one-bond and four-
bond were also obtained from 1H and 13C NMR assignments.
The 1H NMR spectra obtained for p-fluorophenol in DMSO-
d6 showed that H-2(6) is a double doublet at υ 6.78 with
Compounds 4 and 5, 6 and 7 and 2 and 9 are
diastereomers. The NMR data of these pairs of diastereomers
confirmed that the chemical shifts observed are influenced
by the configuration of the chiral center. Changing the
configuration from (S) to (R) at C-4 resulted in distinct shifts
ranging between υ 0.10 and 0.56 for the protons connected
at C-2 and C-3 and shifts ranging from υ 0.53 to 2.57 for
the carbons at C-2, C-3 and C-4. For example, in 13C NMR,
C-2 is 71.26 for 4, C-2 is 68.69 for 5, C-4 is 58.73 for 4, C-4
is 59.45 for 5; the differences are 2.57 and 0.72 ppm. On
the basis of these observations, the optical purities of the
synthetic intermediates and final product in the synthesis of
2-methylsorbinil 2 can be assessed by NMR spectroscopy.
3
4
coupling constants of JHH D 8.3 Hz and JFH D 4.4 Hz;
H-3(H-5) is located at υ 6.94 with coupling constants of
3JHH D 8.3 Hz and JFH D 8.7 Hz. The 13C NMR of p-
3
fluorophenol indicated that the C-1 is located at υ 154.07
4
with a coupling constant of JFC D 2.0 Hz, C-2 is located
3
at υ 116.48 with a JFC D 7.7 Hz, C-3 is located at υ 115.90
2
with a JFC D 23.0 Hz and C-4 is located at υ 156.04 with a
1JFC D 237.5 Hz. Comparison of the spectra of 4-fluorophenol
with those of compounds (2–9) in Tables 1 and 2 indicate that
while the proton and carbon chemical shifts change upfield
or downfield in response to structure differences, the F–H
coupling constants with three-bond and four-bond and the
F–C coupling constants with one-bond and four-bond do
not significantly change.
EXPERIMENTAL
Using a 60-MHz spectrometer, Urban21 has previously
reported H NMR data for a mixture of compounds 4 and
All reagents and solvents were of commercial grade obtained
from Acros Organics and Fisher Chemicals (Fairlawn, NJ) or
Sigma-Aldrich (Milwaukee, WI) and were utilized without
further purification. All solvents were reagents or of HPLC
grade. All deuterated solvents for NMR were obtained
from Sigma-Aldrich. HPLC was conducted on an HP 1100
HPLC system equipped with a C18 reverse phase Luna
1
5, the ester 6, and the product (2R, 4S) 2-methylsorbinil, 2.
Using a 500-MHz NMR spectrometer, this paper extends the
data by reporting results obtained with pure 4 and 5 as well
as the ester 7, and the (2R, 4R) isomer 9 of 2-methylsorbinil in
addition to ester 6, and the product (2R, 4S) 2-methylsorbinil,
2. Two-dimensional NMR experiments were conducted so
that all signals could be assigned, and COSY, HMQC and
˚
(250 ð 4.6 mm, 5 µ, 100 A) using a CH3OH–H2O (75 : 25,
V/V) mobile phase at a flow rate of 0.9 ml/min and UV
Copyright  2005 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2005; 43: 1008–1011

Spectral assignments for the aldose reductase inhibitor 1011
detection at 215, 254 and 280 nm. All melting points are
uncorrected.
in the magnitude mode with 512 points in the F2 dimension
and 256 increments in the F1 dimension, a spectral width of
3700 Hz and a relaxation delay of 1.0 s. Data were zero-filled
once in the F1 dimension before 2D Fourier transformation
with 1024 ð 1024. The gradient HSQC spectra were obtained
in the states-TPPI mode with 512 points in the F2 dimension
and 256 increments in the F1 dimension, a spectral width of
3700 Hz for F2 dimension and 21 356 Hz for F1 dimension,
decoupling with Wurst 40 modulation and a relaxation delay
of 1.2 s. Data were zero-filled twice in the F1 dimension
before 2D Fourier transformation with 1024 ð 2048. For the
gradient HMBC experiment, the spectra were obtained in
the magnitude mode with 512 points in the F2 dimension
and 400 increments in the F1 dimension, a spectral width of
3700 Hz for F2 dimension and 30 143 Hz for F1 dimension.
A relaxation delay of 1.2 s was applied and a delay time
of 62.5 ms was used to obtain the long-range correlations
General synthetic and separation procedures
A mixture of (4R)-2,3-dihydro-6-fluoro-2(R)-methylspiro-
[4H-benzopyran-4,40-20-phenyloxazolidin]-50-one, 3, and its
(4S)-isomer was prepared in five steps from p-fluorophenol
and (S)-ethyl lactate according to Urban et al.20,21 The mixture
of isomers was obtained as an orange oil from which, after the
addition of methanol, crystals of 4(R)-2,3-dihydro-6-fluoro-
2(R)-methylspiro[4H-benzopyran-4,40-20-phenyloxazolidin]-
0
°
5 -one, 3 (m.p. 154–155 C) were obtained with 15% yield.
Hydrolysis of the orange oil of 4(R,S)-2,3-dihydro-6-fluoro-
2(R)-methylspiro[4H-benzopyran-4,40-20-phenyloxazolidin]-
50-one by refluxing with equal volumes of formic acid and
concentrated HCl gave a solid mixture of 4(S)-amino-6-
fluoro-2(R)-methylchroman-4-carboxylic acid, 4, and 4(R)-
4-amino-6-fluoro-2(R)-methylchroman-4-carboxylic acid, 5,
with 50% yield. Recrystallization of the solid mixture from
water gave pure 4(S),2(R) acid, 4, with 35% yield (m.p.
1
n
for J D 140 Hz and J D 8 Hz. In the HMBC experiments,
data were zero-filled once in the F1 dimension and twice
in the F2 dimension before 2D Fourier transformation with
2048 ð 4096.
°
228–229 C). Similar hydrolysis of the 4(R),2(R) isomer 3 gave
°
the 4(R),2(R) acid, 5 with 95% yield, (m.p. 230–231 C). Drop-
Acknowledgement
We thank Dr. Paul A. Keifer of University of Nebraska Medical
Center for his help in obtaining the 2D NMR spectra.
wise addition of thionyl chloride to a methanolic solution
of 4(S, R)-amino-6-fluoro-2(R)-methylchroman-4-carboxylic
acid, 4 and 5, gave a mixture of methyl-4(R,S)-amino-
6-fluoro-2(R)-methylchroman-4-carboxylate (6 and 7) with
51% yield as a light yellow oil. This oil was dissolved
in an aqueous NaCl solution at pH 5 and resolved with
˛-chymotrypsin. Following extraction, methyl-4(S)-amino-
6-fluoro-2(R)-methylchroman-4-carboxylate, 6, was obtained
as a colorless oil with 42% yield. The 4(S),2(R) ester 6 was
then cyclized with sodium cyanate in glacial acetic acid to
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of 4(R)-2,3-dihydro-6-fluoro-2(R)-methylspiro[chroman-4,40-
0
0
°
imidazoline]-2 ,5 -dione, 9, with 85% yield, m.p. 238–239 C.
NMR spectroscopy
1H NMR, 13C NMR, DEPT, COSY, g-HSQC and g-HMBC
spectra were recorded on a Varian Inova Unity 500
NMR spectrometer operating at 499.56 MHz for 1H and
125.63 MHz for 13C. Measurements were conducted at room
temperature in 5 mm NMR tubes with TMS as internal
1
standard. The H and 13C chemical shifts are expressed on
the υ scale and are referenced to TMS. The one-dimensional
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Copyright  2005 John Wiley & Sons, Ltd.
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