Chiral resolution, determination of absolute configuration, and biological evaluation of (1,2-benzothiazin-4-yl)acetic acid enantiomers as aldose reductase inhibitors

Article abstract of DOI:10.3109/14756366.2014.961447

A novel series of (1,2-benzothiazin-4-yl)acetic acid enantiomers was prepared by chiral resolution, and their absolute configurations were determined using the PGME method. The biological evaluation of the racemate and single enantiomers has shown a remar

Full text of DOI:10.3109/14756366.2014.961447

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–6
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.961447
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SHORT COMMUNICATION
Chiral resolution, determination of absolute configuration, and
biological evaluation of (1,2-benzothiazin-4-yl)acetic acid enantiomers
as aldose reductase inhibitors
Xin Hao, Xiangyu Qin, Saghir Hussain, Shagufta Parveen, Wei Zhang, Fengyan Fu, Bing Ma, and Changjin Zhu
Department of Applied Chemistry, Beijing Institute of Technology, Beijing, China
Abstract
Keywords
A novel series of (1,2-benzothiazin-4-yl)acetic acid enantiomers was prepared by chiral
resolution, and their absolute configurations were determined using the PGME method. The
biological evaluation of the racemate and single enantiomers has shown a remarkable
difference for the aldose reductase inhibitory activity and selectivity. The (R)-(ꢀ)-enantiomer
exhibited the strongest aldose reductase activity with an IC50 value of 0.120 mM, which was 35
times more active than the S-(+)-enantiomer. Thus, the stereocenter at the C4 position of this
scaffold was shown to have a major impact on the activity and selectivity.
Absolute configuration determination,
aldose reductase inhibitors, 1,2-benzothia-
zine 1,1-dioxide, chiral resolution
History
Received 3 August 2014
Accepted 31 August 2014
Published online 27 November 2014
Introduction
racemic form only, and the absolute configuration at the chiral
center of the single enantiomers remains to be established.
The interest in synthesizing biologically active products
containing stereogenic carbon atoms has notably increased
over the last decade, and chirality is currently of major
importance in drug discovery and development9. With the
development of new technologies, the preparation of enantio-
merically pure compounds is frequently requested because of the
different activities of drug enantiomers in pharmaceutical appli-
cations10–12. Consequently, current requirements from legal
regulation have largely increased the number of enantiomerically
pure drugs rather than racemates presented for approval13. As one
class of classic ARIs, spirohydantoins including sorbinil14,
fidarestat15 and ranirestat16, which also contain a stereogenic
center, were prepared in the enantiomerically pure forms as shown
in Figure 1.
Diabetes mellitus (DM) is a common metabolic disease caused
by a deficiency in production of insulin or by insulin resistance
in cells. Hyperglycemia accompanying the disease is the
primary cause of long-term diabetic complications, such as
neuropathy, retinopathy, nephropathy and atherosclerosis, because
a significant portion of glucose is forced to enter into the polyol
pathway1–3. Growing evidence reveals that diabetic complications
are related to an increased activity of the enzyme aldose reductase
(ALR2, EC 1.1.1.21) in the pathway4,5. ALR2 is the first enzyme
of this metabolic pathway to catalyze the NADPH-dependent
reduction of glucose to sorbitol2,6. Thus, inhibition of ALR2
represents an attractive approach to prevent or delay the
progression of diabetic complications. Various ALR2 inhibitors
(ARIs) have been reported but only epalrestat has been marketed
so far7, and many ARIs have been renounced either for their
undesirable effects or insufficient efficacy. Hence, development
of more ARIs is needed.
Therefore, as an extension of our previous work, we
have engaged in preparing the enantiomerically pure acids.
In the present study, the racemic 2-[2-(2,4,5-trifluorobenzyl)-1,
In our effort to design and synthesize new ARIs, we have
found a series of (1,1-dioxide-1,2-benzothiazin-4-yl) substituted
acetic acid derivatives (Figure 1) having good ALR2 inhibitory
activity8. Particularly, this class has a chiral carbon atom C4 to
which the acetate head, the key anion group for binding to ALR2,
is attached. The chirality of C4 provides a good handle not only to
study the structure–activity relationships (SAR) with regard to the
configuration of the anion head but also to gain further insight
into the structural profile of the active site of the enzyme.
However, until now this series of compounds is available in
1-dioxido-3,4-dihydro-2H-1,2-benzothiazin-4-yl]acetic
acid
(( )-1) having the highest inhibitory activity in this series was
chosen to resolve the corresponding enantiomers and to determine
their absolute configuration and stereostructure–activity
relationships.
Materials and methods
General
All reactions were routinely checked by TLC on silica gel
Merck 60F254.
1H NMR spectra were recorded at 400 MHz,
while 13C NMR spectra were recorded at 100 MHz. Chemical
shifts are given in ꢀ units (ppm) relative to internal standard
TMS and refer to CDCl3 solutions. MS was performed with a
Address for correspondence: Changjin Zhu, Department of Applied
Chemistry, Beijing Institute of Technology, No. 5, Zhongguancun South
Street, Beijing 100081, China. E-mail: zcj@bit.edu.cn

2
X. Hao et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
O
O
(dd, J ¼ 14.4, 6.4 Hz, 1H), 3.56–3.51 (m, 1H), 2.71–2.69
(m, 2H); 13C NMR (100 MHz, CDCl3) ꢀ: 175.94, 137.46,
136.85, 132.91, 128.61, 128.29, 124.77, 119.19, 118.99, 106.13,
105.93, 105.86, 105.65, 49.42, 43.50, 38.50, 32.31; MS m/z:
negative mode 384.1 ([M ꢀ H]ꢀ), positive mode 408.2
([M + Na]+).
O
NH
HN
N
NH
HN
O
O
O
HN
Br
O
F
O
F
N
NH2
O
O
O
F
Sorbinil
Fidarestat
Ranirestat
(ꢀ)-2-[2-(2,4,5-Trifluorobenzyl)-1,1-dioxido-3,4-dihydro-2H-
1,2-benzothiazin-4-yl]acetic acid ((ꢀ)-1). Pale yellow powder
S
N
CO2H
20
(0.82 g, 71%); m.p.: 80–82 ꢂC; ½ꢁꢃD ¼ ꢀ7.58 (c 1.214, CH2Cl2);
O
CO2H
HO
CO2H
1H NMR (400 MHz, CDCl3) ꢀ: 7.90 (d, J ¼ 6.8 Hz, 1H), 7.54
(t, J ¼ 6.4 Hz, 1H), 7.46 (t, J ¼ 7.6 Hz, 1H), 7.36–7.28 (m, 2H),
6.96–6.90 (m, 1H), 4.61 (d, J ¼ 14.4 Hz, 1H), 4.14 (d, J ¼ 14.8 Hz,
1H), 3.84 (dd, J ¼ 14.4, 4.8 Hz, 1H), 3.63 (dd, J ¼ 14.0,
6.0 Hz, 1H), 3.55–3.49 (m, 1H), 2.72–2.70 (m, 2H); 13C NMR
(100 MHz, CDCl3) ꢀ: 176.53, 137.25, 136.74, 132.83, 128.49,
128.23, 124.67, 119.07, 118.93, 106.03, 105.82, 105.75, 105.54,
49.18, 43.32, 38.31, 32.24; MS m/z: negative mode 384.1
([MꢀH]ꢀ), positive mode 386.2 ([M+H]+), 408.3 ([M+Na]+).
*
N
R
S
N
O
S
O
O
CF3
Tolrestat
Epalrestat
Benzothiazine1,1-dioxides
Figure 1. Chemical structures of aldose reductase inhibitors.
Varian 500-MS ion trap mass spectrometer equipped with an
ESI source. Optical rotations were measured at the indicated
concentration of g/100 mL. Melting points were recorded on an
X-4 microscopic melting point apparatus and were uncorrected.
Aldehyde reductase (ALR1, EC 1.1.1.2) and ALR2 were prepared
according to the method of Kinoshita17 and Concettina
La Motta18. Enzyme activity was assayed spectrophotometrically
on a Shimadzu UV-1800 UV spectrophotometer by measuring the
decrease in absorption of NADPH at l ¼ 340 nm.
Enzymatic assay
ALR1 activity was performed at 37 ꢂC in a reaction mixture
containing 0.12 mM NADPH (0.25 mL), enzyme extract (0.1 mL),
0.1 M sodium phosphate buffer (pH 7.2, 0.25 mL), deionized
water (0.15 mL) and 20 mM sodium D-glucuronate (0.25 mL) as
substrate in a final volume of 1 mL. The reaction mixture except
for sodium D-glucuronate was incubated at 37 ꢂC for 10 min.
The substrate was then added to start the reaction, which
was monitored for 4 min. ALR2 activity was performed at 30 ꢂC
in a reaction mixture containing 0.10 mM NADPH (0.25 mL),
0.1 M sodium phosphate buffer (pH 6.2, 0.25 mL), enzyme extract
(0.1 mL), deionized water (0.15 mL) and 10 mM D,L-glyceralde-
hyde (0.25 mL) as substrate in a final volume of 1 mL. The
reaction mixture except for D,L-glyceraldehyde was incubated at
30 ꢂC for 10 min. The substrate was then added to start the
reaction, which was monitored for 4 min.
The inhibitory activity of the newly synthesized compounds
against ALR2 and ALR1 was assayed by adding 5 mL of the
inhibitor solution to the reaction mixture described above. All
compounds were dissolved in dimethyl sulfoxide (DMSO) and
diluted with deionized water. To correct for the non-enzymatic
oxidation of NADPH, the rate of NADPH oxidation in the
presence of all of the reaction mixture components except
the substrate was subtracted from each experimental rate. The
inhibitory effect of the synthetic compounds was routinely
estimated at a concentration of 10ꢀ5 M (the concentration is
referred to that of the compound in the reaction mixture). Those
compounds found to be active were tested at additional concen-
trations between 10ꢀ5 and 10ꢀ7 M. Each dose–effect curve was
generated using at least three concentrations of inhibitor causing
an inhibition between 20 and 80% with three replicates at each
concentration.
Synthesis of racemic acid ( )-1
The racemic 2-[2-(2,4,5-trifluorobenzyl)-1,1-dioxido-3,4-dihydro-
2H-1,2-benzothiazin-4-yl]acetic acid (( )-1) was prepared accord-
ing to the synthetic pathway reported previously8.
Synthesis of diastereomeric amides (+)-5 and (ꢀ)-5
To a solution of ( )-1 (1 mmol) in DMF: chloroform (12 mL, 1:3)
was added HOBT (1.5 mmol) and EDCI (2.5 mmol) in sequence.
The reaction mixture was stirred at room temperature overnight.
After the formation of the activated ester, L-(ꢀ)-alpha-methyl-
benzylamine (1 mmol) and Et3N (0.3 mL) were added. The
reaction was allowed to stir for 6 h and then water (15 mL) was
added. The mixture was extracted with ethyl acetate (2 ꢁ 15 mL),
and the combined organic layers were washed with brine (10 mL),
dried over MgSO4, and concentrated in vacuo. Separation by
column chromatography using ethyl acetate/petroleum ether (1:4)
as the eluant afforded the title compounds (+)-5 (Rf ¼ 0.26, ethyl
acetate/petroleum ether, 1:1) and (ꢀ)-5 (Rf ¼ 0.29, ethyl acetate/
petroleum ether, 1:1).
Synthesis of the enantiomers (+)-1 and (ꢀ)-1
A solution of 5 (3 mmol) in dioxane (10 mL) was treated with
6 N HCl (8 mL) and refluxed for 10 h. The mixture was cooled,
basified to pH 14 with NaOH 32% in water, diluted with water
(25 mL) and extracted with dichloromethane (2 ꢁ 30 mL). The Results and discussion
resulting solution was acidified to pH 1 with 9 N HCl, extracted
Synthetic chemistry
with dichloromethane (3 ꢁ 30 mL). The combined organic layers
were dried over MgSO4 and evaporated in vacuum to give the The racemic compound ( )-1 was prepared according to the
desired compound. The crude product was purified by silica gel previously reported procedure depicted in Scheme 18, starting
chromatography (CH2Cl2/CH3OH; 100:1).
(+)-2-[2-(2,4,5-Trifluorobenzyl)-1,1-dioxido-3,4-dihydro-2H-
1,2-benzothiazin-4-yl]acetic acid ((+)-1). Pale yellow powder the N-substituted ketone 2 with methyl 2-(triphenylphosphorany-
with the preparation of the corresponding a,b-unsaturated ester as
precursor of the targeted C4-enantiomers. Wittig olefination of
20
(0.87 g, 75%); m.p.: 79–81 ꢂC; ½ꢁꢃD ¼ +7.26 (c 1.132, CH2Cl2); lidene) acetate provided a,b-unsaturated ester 319. Subsequent Pd/
1H NMR (400 MHz, CDCl3) ꢀ: 7.90 (d, J ¼ 8.0 Hz, 1H), 7.54 C-catalyzed hydrogenation gave the corresponding ester 420,
(t, J ¼ 7.6 Hz, 1H), 7.46 (t, J ¼ 7.6 Hz, 1H), 7.36–7.29 (m, 2H), which was converted to racemic acid ( )-1 by hydrolysis with
6.97–6.90 (m, 1H), 4.60 (d, J ¼ 14.8 Hz, 1H), 4.15 aqueous sodium hydroxide. Our next goal was the chiral
(d, J ¼ 14.8 Hz, 1H), 3.84 (dd, J ¼ 14.8, 4.8 Hz, 1H), 3.65 resolution of ( )-1. Efforts to resolve ( )-1 by conversion

DOI: 10.3109/14756366.2014.961447
(1,2-Benzothiazin-4-yl)acetic acid enantiomers
3
O
O
Scheme 1. Reagents and conditions:
(a) Ph3P¼CHCOOCH3, PhCH3, reflux;
(b) H2, 10% Pd/C, MeOH, AcOEt;
(c) 1,4-dioxane, NaOH; (d) HOBT,
EDCI, CHCl3, DMF, room temperature;
(e) L-(ꢀ)-alpha-methylbenzylamine, Et3N;
(f) 1,4-dioxane, 6 N HCl, reflux.
O
F
F
O
F
F
O
F
F
F
F
F
(a)
(b)
(c)
N
N
N
S
S
S
O
O
O
O
O
O
3
2
4
O
O
N
Ph
N
H
F
OH
F
F
F
O
N
(f)
N
O
OH
F
F
S
S
O
(−)−5
O
O
(−)−1
F
*
F
F
F
F
(d,e)
O
O
S
O
O
( )−1
Ph
N
H
OH
F
F
(f)
N
N
O
(+)−1
S
S
O
O
(+)−5
O
F
F
to diastereomeric esters were unsuccessful due to poor chroma-
tographic separation of the ester products. However, ( )-1
could be resolved by forming diastereomeric amides 5 with
L-(ꢀ)-alpha-methylbenzylamine via reaction with the N-hydro-
xybenzotriazole activated ester intermediate. The two amides
(+)-5 and (ꢀ)-5 were separated by column chromatography and
in turn converted into the free acids (+)-1 and (ꢀ)-1, respectively,
by acid hydrolysis21. The acids (+)-1 and (ꢀ)-1 were analyzed
for their enantiomeric purity using chiral HPLC: enantiomeric
excesses were 95.2 and 97.9%, respectively. This result con-
firmed the successful preparation of the single enantiomers of
compound 1.
H
N
R
1
R
CO Me
2
2
PGME
R
1
CO H
2
Ph---- (R)
H
H
O
H
R
2
(Ph) (H)---- (S)
Chiral propionic acid
PGME amide
O
PGME
C
H
H
CH
C
X
A
2
H
N
O
R (H
)
1
A-C
H
−Δδ
+Δδ
H
Y
B
R (H
)
2
X-Z
OMe
H
H
H
C
Z
H
H
O
Model
Δδ = δ -δ
PGME plane
(R) (S)
Determination of absolute configuration
Figure 2. The principle of the PGME method.
Following isolation of the separated enantiomers, it was essen-
tial to determine the absolute configurations of (+)-1 and (ꢀ)-1.
1H-NMR analysis of diastereomeric derivatives is one of the
few methods capable of establishing the absolute configuration
of chiral compounds. Reaction with a chiral agent turns the
enantiomers into diastereomeric derivatives, for which distinct
chemical shift changes can be correlated to their absolute
configurations. Phenylglycine methyl ester (PGME), a chiral
anisotropic reagent, has been developed for the elucidation of
the absolute configuration of chiral carboxylic acids by means of
1H-NMR22, and the technique also proved to be successful in the
present study.
The principle of the PGME method applied to (+)-1 and (ꢀ)-1
is outlined in Figure 2: a chiral b,b-disubstituted propionic acid is
condensed with (R)- and (S)-PGME, respectively, giving the
corresponding diastereomeric amides. In order for the PGME
benzene ring to exert a predictable diamagnetic field effect upon
R1 or R2, the backbone formed by the PGME amide and propionic
carbon chain needs to occur in one plane. In the most stable
conformation of the PGME amide of a b,b-disubstituted propionic
absolute configuration at the b-position of the chiral propionic
acid can be determined using the model [Dꢀ ¼ ꢀ(R) ꢀ ꢀ(S)] in
Figure 2. According to this principle, the 1H-NMR shift
differences Dꢀ between the diastereomeric (R)- and (S)-PGME
amides arising from the enantiomers (+)-1 and (ꢀ)-1 were
calculated, respectively, and the results are shown in Figure 3.
Without exception, the negative Dꢀ values were on the same side
of the molecule as the PGME benzene ring in (+)-6 and (ꢀ)-6,
and the positive Dꢀ values were on the opposite side. These results
allowed assignments of the S absolute configuration to (+)-1 and
the R one to (ꢀ)-1. Thus, the PGME method has been
successfully applied to determine the absolute configurations of
(+)-1 and (ꢀ)-1.
Aldose reductase inhibition and SAR studies
acid, this plane is fixed by the presence of an amide linkage and The single enantiomers were then tested for their potential
dipole–dipole interaction between the methoxycarbonyl group inhibitory effect on ALR2 isolated from rat lenses. The IC50
and the amide carbonyl22. If the conformation shown at the values were determined by linear regression analysis of the log
bottom of Figure 2 is correct for the PGME amide of a of the concentration–response curve8. The results of these
b,b-disubstituted propionic acid, the protons HX-Z would be biological evaluations are listed in Table 1, and the effectiveness
more shielded by the benzene ring of the (R)-PGME moiety than of the test compounds was evaluated with respect to the potent
those of the (S)-PGME amide. The reverse would be true for HA-C ALR2 inhibitor epalrestat used as a positive control. Compounds
which are more shielded in the (R)-PGME amide. Therefore, the (rac)-( )-1, (R)-(ꢀ)-1 and (S)-(+)-1 all showed significant

4
X. Hao et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
Table 1. Biological activity data for 1,2-benzothiazine 1,1-dioxide acetic
acid derivatives.
OC PGME
CH
OC PGME
F
F
CH
F
F
2
2
−0.16
+0.13
+0.01
−0.02
F
F
−0.05
+0.04
+0.04
O
+0.16
+0.05
−0.17
−0.08
−0.06
N
N
−0.13
+0.12
OH
F
F
S
S
+0.01 O
O
−0.03 O
−0.06
+0.04
O
+0.06
F
−0.10
(−)−6
(+)−6
N
O
S
O
Figure 3. Dꢀ values obtained for the PGME amides (ꢀ)-6 and (+)-6.
inhibitory activity against ALR2. Of these isomers, (R)-(ꢀ)-1 was
found to be the most active having an IC50 value of 0.120 mM
comparable to that of epalrestat (Table 1). In contrast, (S)-(+)-1
was less effective with an IC50 value of 4.174 mM. Here, for the
first time, we have demonstrated that the (R)- and (S)-enantiomers
of 1,2-benzothiazine-1,1-dioxide-based acetic acid ARIs
inhibit aldose reductase to a largely different extent and specified
the (R)-(ꢀ)-enantiomer as the stronger ARI.
ALR2
IC50* (mM)
ALR1
(% of inhibition)y
Compound
(rac)-( )-1
(R)-(ꢀ)-1
(S)-(+)-1
Epalrestat
0.676 (0.589–0.763)
0.120 (0.084–0.156)
4.174 (3.692–4.656)
0.086 (0.059–0.112)
14.7
7.5
11.8
73.6
*IC50 (95% CL) values represent the concentration required to produce
50% enzyme inhibition.
yThe inhibitory effect was estimated at a concentration of 10 mM.
In order to evaluate the selectivity of the ALR2 inhibition,
the isomers were also tested for the inhibition against ALR1
isolated from rat kidney. The enzyme ALR1 is most
closely related to ALR223 and plays a detoxification role24,25
.
Many failures of ARIs in clinic trail stages are believed to be while its benzyl group pointed into the deep direction of the
mainly due to a lack of selectivity26. Clearly, the isomer (R)-(ꢀ)-1 specificity pocket. This may explain the higher inhibitory
having the most potent activity in the ALR2 inhibition showed activity of (R)-1 compared to that of (S)-1, providing the basis
a significantly lower activity in the ALR1 inhibition compared for our surmise in the SAR studies.
with other isomers (Table 1), indicating an excellent selectivity
for (R)-(ꢀ)-1.
Conclusion
Therefore, our results suggest that the orientation of the acetic
The two key amines were employed throughout the chemistry
in the present study. First, racemic acid ( )-1 was coupled with
L-(ꢀ)-alpha-methylbenzylamine to produce diastereomeric
amides, which resulted in the chiral resolution of the 1,2-
benzothiazine-1,1-dioxide based racemic ARIs with excellent
enantiomeric purities. Then, the separated single enantiomers
(ꢀ)-1 and (+)-1 were coupled again with both of the enantiomeric
(R)- and (S)-phenylglycine methyl esters used as chiral aniso-
acid head at the C4 position of this class not only plays a key role
for the binding with ALR2 but also has an important function in
discriminating and excluding homologous enzymes. Actually,
there is often a large difference in biological activity between the
enantiomers of chiral drugs11.
Molecular modeling
Docking studies were performed in an attempt to investigate tropic reagents, to yield four diastereomeric amides; NMR
the difference in ALR2 inhibitory activity between the analysis of these coupling products enabled the assignment of
enantiomers and propose a binding mode that explains the the absolute configurations (R)-(ꢀ)-1 and (S)-(+)-1 to the
above-described SARs. (R)-1 and (S)-1 were individually enantiomers. These results allowed to study the stereostructure–
docked into the conformation of human ALR2 from the activity relationships and selectivity in the ALR2 inhibition, and
complex with NADP+/IDD594 (PDB code: 1US0) as shown to gain insight into the mechanism of binding of the key
in Figure 4.
Analysis of the docking results revealed that the carboxylic
carboxylate head group with the active site of ALR2.
Biological investigations of the single enantiomers showed
acid head of (R)-1 formed three tight hydrogen bonds with the that (R)-(ꢀ)-1 was approximately 35 times more active than
˚
OH of Tyr48 (2.73 A), the N"2 atom of His110 (2.83 A) and (S)-(+)-1 and also had much better selectivity in the ALR2
˚
˚
the N"1 of Trp111 (2.90 A), respectively, imbedded deeply inhibition. In agreement with the biological results, docking
into the anion binding pocket of the enzyme. Further, the studies indicated a more favored interaction for binding of
benzyl ring was deeply trapped in the hydrophobic cage of the (R)-(ꢀ)-1 with the active site of the enzyme than that for
specificity pocket and in turn ꢂ-stacked perfectly against the binding of (S)-(+)-1. Consequently, the stereogenic C4
indole moiety of Trp111. These interactions anchored (R)-1 position of these 1,2-benzothiazine-1,1-dioxide-based ARIs
tightly within the enzyme active site. In the docking of (S)-1, has a major impact on both the activity and selectivity of
it was found that one oxygen atom of the sulfur dioxide group the ALR2 inhibition; hence, chirality could be considered as a
˚
formed only one hydrogen bond with Trp111 (3.51 A), while useful tool in the lead optimization process, at least in the
the carboxylic acid head showed no binding interaction with case of this class of ARIs (Supplementary material).
the active site of the enzyme. Though the carboxylic acid side
chain residue was oriented away from the anion binding
Declaration of interest
pocket, the benzyl ring showed ꢂ-stacking with Trp111 and
was well placed into the specificity pocket. The docking
The authors report no conflicts of interest. This work was
supported by the National Natural Science Foundation of China
results suggested different binding modes for (R)-1 and (S)-1,
(grant no. 21272025), the Research Fund for the Doctoral Program
which extended their differently oriented C4-carboxylic acid
heads into different positions of the active site. The carboxylic
of Higher Education of China (grant no. 20111101110042), the
Science and Technology Commission of Beijing, China (grant no.
acid side chain of (R)-1 showed a favorable binding
interaction with the anion pocket of the enzyme ALR2,
Z131100004013003) and the Beijing Natural Science Foundation
(grant no. 3100021501401).

DOI: 10.3109/14756366.2014.961447
(1,2-Benzothiazin-4-yl)acetic acid enantiomers
5
Figure 4. Molecular docking of inhibitors (R)-1 and (S)-1 into the active site of ALR2. A and B presents docking for (R)-1, while C and D for (S)-1,
respectively. Ligands are shown as stick models, while the selected and labeled protein residues are presented (A and C) in line representation and
(B and D) in surface representation. Docked poses of compounds are shown in cyan (C), red (O), blue (N), yellow (S) and gray (F); hydrogen bonds are
shown as yellow dashed lines.
9. Fuji K. Asymmetric creation of quaternary carbon centers. Chem
Rev 1993;93:2037–66.
10. Mentel M, Blankenfeldt W, Breinbauer R. The active site of an
enzyme can host both enantiomers of a racemic ligand simultan-
eously. Angew Chem Int Ed 2009;48:9084–7.
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Supplementary material available online