Selective synthesis and comparative activity of olefinic isomers of 1,2-benzothiazine-1,1-dioxide carboxylates as aldose reductase inhibitors

Article abstract of DOI:10.1039/c4ra01016g

α,β- and β,γ-unsaturated carboxylate isomers of 1,2-benzothiazine-1,1-dioxide were selectively synthesized via the Wittig olefination reaction under various temperature conditions. At 40 °C, α,β-unsaturated esters with high Z-stereoselectivity (83-87%) we

Full text of DOI:10.1039/c4ra01016g

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: S. Parveen, S.
Hussain, S. Zhu, X. Qin, X. Hao, S. Zhang, J. Lu and C. Zhu, RSC Adv., 2014, DOI: 10.1039/C4RA01016G.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Page 1 of 9
RSC Advances
View Article Online
DOI: 10.1039/C4RA01016G
RSC Advances
RSCPublishing
ARTICLE
Selective synthesis and comparative activity of olefinic
isomers of 1,2ꢀbenzothiazineꢀ1,1ꢀdioxide carboxylates
as aldose reductase inhibitors
Cite this: DOI: 10.1039/x0xx00000x
Shagufta Parveen,a Saghir Hussain,a Shaojuan Zhu, a Xiangyu Qin,a Xin Hao,a
Shuzhen Zhang,a Jianglu Lu,a and Changjin Zhu (s)a,b
Received 00th January 2014,
Accepted 00th January 2014
α,βꢀ and β,γꢀunsaturated carboxylate isomers of 1,2ꢀbenzothiazineꢀ1,1ꢀdioxide were selectively
synthesized via Wittig olefination reaction under various temperature conditions. At 40 °C afforded α,βꢀ
unsaturated esters with high Zꢀstereoselectivity (83ꢀ87 %) while β,γꢀunsaturated esters preferentially
formed with moderate to excellent regioselectivity at 100ꢀ120 °C (77ꢀ96%). The acid isomers were
found to be active in aldose reductase inhibition in rank order of β,γꢀunsaturated > Zꢀα,βꢀunsaturated >
Eꢀα,βꢀunsaturated isomers. The β,γꢀunsaturated isomer 7b, 2ꢀ[2ꢀ(4ꢀbromoꢀ2ꢀfluorobenzyl)ꢀ1,1ꢀdioxidoꢀ
2Hꢀ1,2ꢀbenzothiazinꢀ4(3H)ꢀylidene]acetic acid, exhibited most potent inhibition activity with an IC50
value of 0.057 ꢁM. This was further supported by docking study.
DOI: 10.1039/x0xx00000x
www.rsc.org/
However, many failed to be advanced through late stage of
clinical trials or to lead to success on the market. This was
Introduction
The pathogenesis of diabetic complications such as retinopathy,
neuropathy, cataracts, and stroke, is attributed to multiple
biochemical pathway. Substantial evidences suggested a key role for
hyperglycaemiaꢀinitiated polyol pathway, advanced glycation end
products, and oxidative stress in diabetic complications.1, 2 Aldose
reductase (ALR2, EC 1.1.1.21), which catalyses NADPHꢀdependent
reduction of glucose to sorbitol, is the rateꢀdetermining enzyme of
polyol pathways. It has been identified as a key target for therapeutic
owing to their poor efficacy or side effects which were often
associated with deficiency of ARI selectivity. That deficiency
was measured by inhibitory activity against aldehyde reductase
(EC 1.1.1.2, ALR1). Both ALR1 and ALR2 are members of
aldoꢀketo reductase (AKR) superfamily. They share a high
degree of sequences and structural homology with majority of
differences at the Cꢀterminal end of the enzymes.11, 12
intervention to prevent and treat diabetic complications.3ꢀ5
Therefore, aldose reductase inhibitors (ARIs) are becoming
promising potential drugs. In the past few decades, a number of
compounds have been developed as ARIs. Some selected ARIs
are shown in Fig. 1.6ꢀ10
a Department of Applied Chemistry, Beijing Institute of Technology, No.
5, Zhongguancun South Street, 100081 Beijing, China
b Eꢀmail: zcj@bit.edu.cn Tel./fax: +86 10 68918506 (C. Zhu),
† Electronic supplementary information (ESI) available: Supplementary
table S1, copies of 1H, 13C, and NOESY NMR spectra and single Xꢀray
crystal structure data.
. See DOI: 10.1039/b000000x/
Fig. 1 Selective aldose reductase inhibitors
We have recently designed several series of potent ARIs based on
the scaffolds of 1,2,4ꢀbenzothiazineꢀ1,1ꢀdioxide,8 pyrido[2,3ꢀ
e][1,2,4]thiadiazineꢀ1,1ꢀdioxide,9, 131,2ꢀbenzothiazineꢀ1,1ꢀdioxide.14
The 1,2ꢀbenzothiazineꢀ1,1ꢀdioxide series including C4ꢀα,βꢀ
unsaturated and C4ꢀacetic acids (Fig. 1) were prepared. Nevertheless
the stereochemistry of the α,βꢀdouble bond and the C4 carbon
This journal is © The Royal Society of Chemistry 2014
RSC Advances, 2014, 00, 1-3 | 1

RSC Advances
Page 2 of 9
View Article Online
ARTICLE
RSC Advances
DOI: 10.1039/C4RA01016G
remained uncleared. Therefore, it is of importance to achieve stereoꢀ
controlled synthesis of 1,2ꢀbenzothiazineꢀ1,1ꢀdioxide acids.
Subsequently, to demonstrate the effect of orientation of carboxylic
acid group over the activity of aldose reductase inhibition.
Wittig reaction15, 16 proceeds in stereoselective fashion for numerous
18
substrates.17,
It is demonstrative of a collection of versatile
transformations in natural product synthesis19ꢀ21 and industrial
processes.22ꢀ24 Previously, we reported the formation of α,βꢀ
unsaturated (exocyclic olefine) esters of 1,2ꢀbenzothiazineꢀ1,1ꢀ
dioxide.14 More currently, we have found the formation of only β,γꢀ
unsaturated (endocyclic olefine) ester isomers at higher temperature.
The present study focuses on the regioꢀ and stereoꢀselective
synthesis of 1,2ꢀbenzothiazineꢀ1,1ꢀdioxide ester isomers via Wittig
olefination. Further, also deals with the behaviour of conformations
of the carboxylic acid group on the inhibitory activity against aldose
reductase.
Results and discussion
The starting materials 1aꢀb were prepared from commercially
available sodium saccharin. Which on reacting with methyl
bromoacetae under mild conditions produce methyl [1,1ꢀdioxidoꢀ3ꢀ
oxoꢀ1,2ꢀbenzothiazolꢀ2(3H)ꢀyl] acetate in excellent yield.25
GabrieleꢀColuman type ring expension from 5ꢀmembered isothiazol
ring to 6ꢀmembered thiazole ring yeilded methyl 4ꢀhydroxyꢀ2H,1,2ꢀ
benzothiazineꢀ3ꢀcarboxylate 1,1ꢀdioxide under inert atmoshphere.26,
27
Consequently, which resulted in the formation of 2Hꢀ1,2ꢀ
benzothiazineꢀ4(3H)ꢀone when refluxed with concentrated
hydrochloric acid. The resulted product provided Nꢀsubstituted
ketone 1aꢀb by alkylation with commercially available benzyl
28
bromide in three steps.14,
1aꢀb were allowed to react with the
stabilized ylide of methyl 2ꢀ(triphenylphosphoranylidene) acetate to
produce isomers of 1,2ꢀbenzothiazine 1,1ꢀdioxide carboxylates. The
Wittig olefination was carried out at different temperature (40ꢀ
120°C) in toluene under salt free conditions. Subsequently, afforded
Z, E, and endocyclic olefinic isomers with moderate to good stereoꢀ
and regioꢀselectivity. Wittig reaction of stabilized ylides (Ph3PCHR,
R=EWG) normally furnishes olefins with high E selectivity under
kinetic control condition.29 Anomalous Z selectivity depending on
substrate and solvent is also reported.30ꢀ32
Fig. 2 Thermal controlled olefination of 1a and 1b
.
O
O
O
O
O
High
Z
selectivity can be explained by considering [2+2]ꢀ
cycloaddition mechanism.33 Two stable half chair conformations A
and B are regularly suggested for heterocyclic haxene ring as shown
in Fig. 3. When methyl 2ꢀ(triphenylphosphoranylidene) acetate
approach A, oxaphosphetane transition state I is formed. Parallel
orientation of carbonyl and CꢀCO2Me dipole and high steric
repulsion between phenyl ring of benzothiazine and CꢀCO2Me cause
destability. No such destabilizing factors are involved in the
formation of II as shown in Fig. 3. As a result, stereospecific
decomposition of oxaphosphetane diastereomers leads to high Zꢀ
alkene with phosphine oxide as side product.
Ph3P=CHCOOCH3
PhCH3, 40 to 120°C
R
R
R
N
N
N
S
S
S
O
O
2a-b
3a-b
O
O
O
O
4a-b
1
1a R = 2,4,5-F3
1b
10M HCl,
10M HCl,
1, 4-dioxane, 60°C
R = 2-F,4-Br
1, 4-dioxane, 80°C
O
O
OH
HO
R
R
N
N
S
S
O
O
O
O
5a-b
6a-b
7a-b
Isomerization of exocyclic to endocyclic olefins 4aꢀb was also
observed with good to excellent regioselectivity at high temperature
(Fig. 2). The products from the reaction of 1a at 100 °C were
obtained in yield of 88.9% (2a:3a:4a = 17:6:77). However, those
from the reaction of 1b at 120 °C were in 84.5% (2b:3b:4b =
2:2:96). These results are in well agreement with previous
observations of Wittig olefination of monoketone and diketone with
stabilized phosphorus ylide.34 These results indicated that the
endocyclic olefins are thermodynamically stable.
Scheme 1 Synthesis of olefinic isomers of benzothiazineꢀ1,1ꢀdioxide
carboxylate
The reaction of 1aꢀb 40°C proceeded with high Z stereoselectivity
(76%; 2a:3a = 87:13) and (89%; 2b:3b = 83:17) respectively, (Fig.
2).
2 | RSC Advances, 2014, 00, 1-3
This journal is © The Royal Society of Chemistry 2014

Page 3 of 9
RSC Advances
RSC Advances
View Article Online
ARTICLE
DOI: 10.1039/C4RA01016G
Further, NMRꢀNOESY showed the NOE effect between the αꢀ
proton of 2aꢀb and the proton at phenyl ring of benzothiazine 1,1ꢀ
dioxide framework. This indicated that methyl ester group is on
opposite side to phenyl ring of benzothiazine 1,1ꢀdioxide, assigning
Zꢀconfiguration for 2aꢀb. The Eꢀconfiguration for 3aꢀb was also
assigned in the same way. In case of 4aꢀb, NOESY correlations of
the olefinic C3ꢀproton of benzothiazine 1,1ꢀdioxide framework were
observed with two benzyl ring protons and two αꢀprotons,
respectively. However, no such correlation was observed between αꢀ
protons and any phenyl ring proton of the benzothiazine 1,1ꢀdioxide
framework. This allows an assignment of endocyclic nonꢀconjugated
structure for 4aꢀb.35 In addition, the structures and stereochemistry
of compounds 2a and 4a were further confirmed by Xꢀray
crystallography (Fig. 4).
Biological Activity
The synthesized olefinic acid isomers of 1,2ꢀbenzothiazine 1,1ꢀ
dioxide were tested for their inhibitory activity on ALR2 isolated
from rat lenses. This is performed to identify the structure or
stereochemistry which is crucial for optimal performance. A great
difference in the activity between these isomers was observed as
shown in Table 1. The comparison of inhibitory ability in the aꢀ
series compounds showed that Zꢀisomer 5a was more active than the
Eꢀisomer 6a. Whereas, endocyclic olefine isomer 7a was the most
active in this series (Table 1). The comparison in bꢀseries
compounds gave the same results. Particularly, the endocyclic
olefine isomer 7b was found to be a potent ARI with an IC50 value of
0.057ꢁM.
Fig. 3 Proposed mechanisms for high Zꢀselectivity
However, the rapid fall in yield of 4a at 120°C (Fig. 2) may be
resulted from decomposition of the product at this temperature.
Similar result was also observed in case of 4b when temperature was
higher than 120 °C.
The ester isomers afforded their corresponding exocyclic and
endocyclic olefine acid isomers 5aꢀb, 6aꢀb, and 7aꢀb respectively,
by acid hydrolysis. However, base hydrolysis of all isomers gave
only 7aꢀb.
Compound structure and stereochemistry
All of the synthesized compounds gave satisfactory spectroscopic
data. The structures and stereochemistry of Z, E and endocyclic
olefine isomers were determined. Firstly, the stereochemistry of
geometrically pure isomers were determined. The differences in the
chemical shifts of protons present at α, γ, and methylene protons of
substituted Nꢀbenzyl group are present. For instance, chemical shift
(δ) values of single proton at α or γ position for 2a, 3a, and 4a are
6.76 ppm, 6.23 ppm, and 7.01 ppm respectively.
Table 1 Biological activity of olefinic isomers of 1,2ꢀbenzothiazine 1,1ꢀ
dioxide carboxylates
Compounds ALR2 IC50 (ꢁM)a
5a
6a
7a
5b
6b
7b
7.48 (0.25ꢀ6.18)
43.86 (4.5ꢀ59.3)
7.2 (0.23ꢀ5.9)
1.47 (0.21ꢀ3.36)
6.18 (0.22ꢀ7.16)
0.057 (0.002ꢀ0.071)
Epalrestate 0.12 (0.05–0.19)
a
IC50 (95% CL) values represent the concentration required to produce
Fig. 4 Xꢀray crystallography of 2a and 4a H= white, C= gray,
O = red, S = yellow, N = blue, F = cyan
50% enzyme inhibition.
This journal is © The Royal Society of Chemistry 2014
RSC Advances, 2014, 00, 1-3 | 3

RSC Advances
Page 4 of 9
View Article Online
ARTICLE
RSC Advances
DOI: 10.1039/C4RA01016G
Consequently, olefinic isomers of 1,2ꢀbenzothiazineꢀ1,1ꢀdioxide inhibitor as compared to Eꢀisomer 6a (Fig. 5B). Since carboxylate
carboxylic acid behaved differently with ranking order of head of 6a formed only two hydrogen bond interactions with OH of
endocyclic olefine isomers > Zꢀisomers > Eꢀisomers. The endocyclic Tyr48 (2.71 Å) and NE2 of His110 (2.32 Å). The endocyclic isomer
olefine isomers 7aꢀb showed the highest efficiency as compared to 7a appeared to reside in twisted conformation into the anion binding
the other respective isomers. This was probably because of the pocket by four hydrogen bonding interactions. OH group of
flexible orientation of C4ꢀacetic acid moiety, being made by the β,γꢀ carboxylic head formed two hydrogen bonds with NE2 of His110
double bond. This provided more favourable orientation for the (2.69 Å) and NE1 of Trp111 (3.44 Å) respectively. Whereas, the
interaction between the acid head and the anion binding site of carbonyl oxygen has hydrogen bond with OH of Tyr48 (2.77 Å).
ALR2.
One of oxygens of thionyl group twisted to form a hydrogen binding
with NE1 of Trp111 (3.21 Å). Benzyl ring of all ligands formed a
stacking interaction against the indole ring of Trp111 side chain.
Therefore, it is well placed into the specificity pocket (Fig. 5C).
In case of bꢀseries compounds, 5b and 6b showed similar patterns of
interactions as those of 5a and 6a, respectively. However, the
endocyclic olefine isomer 7b (Fig. 5D) behaved differently. Herein,
the OH of carboxylate head formed two hydrogen bonds. One
hydrogen bond with OH of Tyr48 (2.97 Å) and another with NE2 of
imidazole ring of His110 (2.91 Å). A hydrogen bond is formed
between a thionyl oxygen and NE1 of indole ring of Trp111 (2.99
Å). An extra strong hydrogen bond (2.39Å) between C=O of
carboxylate head and side chain of Cys298 is present. This bond may
be responsible for its highest activity.
Molecular modeling
Molecular docking of the two series of compounds were performed
on human ALR2 complexed with NADP+ and IDD594 (PDB code =
1US0). The docking results may explain the above mentioned
stereostructureꢀactivity relationship as shown in Fig. 5. All the three
isomers of each series revealed a significant different pattern of
binding interactions. In case of aꢀseries compounds, Zꢀisomer 5a
formed three hydrogen bonding interactions to occupy the active site
of enzyme. The OH group of carboxylate head formed Hꢀbonds with
Tyr48 (3.02 Å) and His110 (2.73 Å), in anion binding pocket. The
carbonyl oxygen atom also formed a hydrogen bond with the side
chain of Cys298 (3.09 Å) as shown in (Fig. 5A). These three
hydrogen bonding interactions might make the Zꢀisomer a stronger
Fig. 5 Molecular docking of different isomers of inhibitors, selected and labelled protein residues are presented in wireframe in medium violet red (C) with
docked molecules shown in stick and NADPH+ shown in ball and stick model in lime (C). Docked pose A presented docking for 5a in Cyan (C), B for 6a in
pink (C), C for 7a in olive (C), and D for 7b in olive (C), red (O), blue (N), yellow (S), gray (F), and maroon (Br). Hydrogen Bond interactions are shown in
yellow dashed lines.
4 | RSC Advances, 2014, 00, 1-3
This journal is © The Royal Society of Chemistry 2014

Page 5 of 9
RSC Advances
RSC Advances
View Article Online
ARTICLE
DOI: 10.1039/C4RA01016G
(KBr) √/cmꢀ1 4000ꢀ450, 3060, 1703, 1199, 1045, 798. MS m/z 396.1
[MꢀH]ꢀ, 420.1 [M+Na]+
Conclusions
Three olefinic carboxylate isomers of 1,2ꢀbenzothiazineꢀ1,1ꢀdioxide
were synthesized with excellent stereoꢀ and regioꢀ selectivity in high
yield under various temperature conditions. At 40°C only α,βꢀ
unsaturated esters were formed with high Zꢀselectivity. Whereas,
higher temperature 100ꢀ120 °C resulted in β,γꢀunsaturated esters
favorably with moderate to excellent regioselectivity under base free
condition. The stereochemistry was defined after successful
separation and recrystallization of isomers. Comparative ALR2
inhibitory activity of these isomers demonstrated a key role of
orientation of C4ꢀcarboxylic acid head of the compounds.
Eꢀ2ꢀ[2ꢀ(2,4,5ꢀTrifluorobenzyl)ꢀ1,1ꢀdioxidoꢀ2Hꢀ1,2ꢀbenzothiazinꢀ
4(3H)ꢀylidene]acetic acid methyl ester (3a)
Isolated yield: 16.1% (0.3 g) at 60°C (for 48 h), white solid, mp: 96ꢀ
99 °C. δH (400 MHz, DMSOꢀd6) 3.66 (3H, s), 4.18 (2H, s), 4.40 (2H,
s), 6.23 (1H, s), 7.48 (1H, dd, J = 6.8, 4.7 Hz), 7.56 – 7.51 (1H, m),
7.66 – 7.62 (2H, m), ), 7.68 (1H, d, J = 5.0 Hz), 7.82 (1H, dd, J =
6.2, 2.6 Hz). δC (100 MHz, DMSOꢀd6) 39.09, 39.30, 39.51, 39.72,
39.92, 46.45, 51.61, 53.98, 106.44, 118.87, 120.60, 123.61, 129.95,
130.46, 130.57, 132.07, 135.75, 135.77, 138.23, 165.59; IR (KBr)
√/cmꢀ1 4000ꢀ450, 3057, 1733, 1196, 1052, 791, 759. MS m/z 395.9
[MꢀH]ꢀ
Experimental
Endoꢀ2ꢀ[2ꢀ(2,4,5ꢀTrifluorobenzyl)ꢀ1,1ꢀdioxidoꢀ2Hꢀ1,2ꢀ
benzothiazinꢀ4(3H)ꢀylidene]acetic acid methyl ester (4a)
Melting points were recorded on a XT4A microscopic melting points
apparatus and uncorrected. Thin layer chromatography (TLC) was
performed on silica gel Merck 60F254. Nuclear Magnetic Resonance
(NMR) spectra were recorded using a 400 MHz spectrometer
(1HNMR at 400MHz and 13CNMR at 100MHz) in [d6]ꢀDMSO
using TMS as internal reference. 1HNMR data are reported as
follow: chemical shifts (δ, ppm), multiplicity (s=singlet, d= doublet,
dd= doublet of doublet, t=triplet, q=quartet, m=multiplet, br=broad),
integration, coupling constants (Hz). Data for 13C NMR are reported
in terms of chemical shifts (δ, ppm). Individual resonances were
assigned on the basis of their chemical shifts, signal intensities,
multiplicity of resonances and coupling constants. MS was
performed on varian 500ꢀms ion trap mass by the ESI method and
HRMS(ESIꢀTOF) was performed on Agilent 6210 TOF. FTIR
spectra was obtained from PerkinElmer Spectrum One FTIR
spectrometer.
Isolated yield: 68.4% (1.63 g) at 100°C (for 12 h), white crystalline
solid, mp: 91ꢀ95 °C. δH (400 MHz, DMSOꢀd6) 3.60 (3H, s), 3.73
(2H, s), 4.94 (2H, s), 7.01 (1H, s), 7.41 (1H, dd, J = 17.6, 8.8 Hz),
7.54 (1H, d, J = 8.1 Hz), 7.60 (2H, t, J = 8.1 Hz), 7.75 (1H, t, J = 7.7
Hz), 7.90 (1H, d, J = 7.8 Hz), δC (100 MHz, DMSOꢀd6) 40.02, 44.23,
44.44, 44.65, 44.86, 45.07, 45.28, 45.48, 49.19, 57.14, 114.55,
114.74, 117.09, 117.28, 126.71, 129.78, 133.23, 133.36, 134.01,
134.13, 136.05, 136.12, 136.75, 136.85, 136.96, 137.33, 137.83,
176.49; IR (KBr) √/cmꢀ1 4000ꢀ450, 3084, 1739, 1193, 1046, 780,
750. MS m/z 398.1 [M+H]+, 420.2 [M+Na]+
Zꢀ2ꢀ[2ꢀ(2ꢀbromo,4ꢀfluorobenzyl)ꢀ1,1ꢀdioxidoꢀ2Hꢀ1,2ꢀ
benzothiazinꢀ4(3H)ꢀylidene]acetic acid methyl ester (2b)
Isolated yield: 74% (1.95 g) at 40°C (for 48 h), white crystalline
solid, mp: 141ꢀ147 °C. δH (400 MHz, DMSOꢀd6) 3.70 (3H, s),4.17
(2H, s), 4.89 (2H, s), 6.78 (1H, s), 7.44 – 7.38 (2H, m), 7.56 – 7.51
(1H, m), 7.75 – 7.71 (2H, m), 7.90 – 7.86 (1H, m), 8.05 (1H, d, J
=7.5 Hz). δC (100 MHz, DMSOꢀd6) 39.07, 39.28, 39.49, 39.70,
39.70, 39.91, 45.53, 46.23, 46.66, 49.18, 49.45, 51.66, 53.44, 53.96,
118.24, 118.36, 118.50, 118.74, 118.99, 120.65, 123.14, 123.51,
123.66, 124.32, 124.44, 124.48, 126.41, 126.63, 126.73, 127.71,
129.72, 129.77, 129.98, 130.48, 130.54, 131.36, 132.08, 132.77,
133.34, 134.05, 141.83, 142.64, 165.52. IR (KBr) √/cmꢀ1 4000ꢀ450,
3096, 1720, 1193, 1067, 792, 758. MS m/z 461.9 [M+Na]+
General synthetic procedure for 2ꢀ4 (aꢀb)
Solution of triphenylphosphine (26.2 g, 0.1 mol) in benzene (300
mL) was stirred for 15 minuts. Which was followed by dropwise
addition of a solution of methyl bromoacetate (1.5 eq) in benzene
(100 mL) over 1 h. The solid was filtered and washed with
petroleum ether to provide a white solid phosphonium product. The
crude product was dissolved in water (800 mL) and made it alkaline
by adding Et3N dropwise till the solution became basic enough. The
reaction mixture was stirred for 1h. The precipitates thus obtained
was filtered and washed with water to provide the crude product,
Methyl 2ꢀ(triphenyl phosphoranylidene) acetate. The crude product
was purified by recrystallization from methanol.
A solution of an appropriate compound 1 (6 mmol) and methyl 2ꢀ
(triphenylphosphoranylidene) acetate (9 mmol) in toluene (30 mL)
was stirred at 40 °C to 120 °C till reaction completed. After
evaporation of the solvent, the residue was purified by silica gel
column chromatography (ethylacetate: petroleum ether; 1:15ꢀ1:20)
to acquire pure product.
Eꢀ2ꢀ[2ꢀ(2ꢀbromo,4ꢀfluorobenzyl)ꢀ1,1ꢀdioxidoꢀ2Hꢀ1,2ꢀ
benzothiazinꢀ4(3H)ꢀylidene]acetic acid methyl ester (3b)
Isolated yield: 15.3% (0.403 g) at 60°C (for 48 h), white solid. mp:
121.126 °C. δH (400 MHz, DMSOꢀd6) 3.62 (3H, s), 4.14 (2H, s),
4.32 (2H, s), 6.19 (1H, s), 7.36 – 7.32 (1H, m), 7.38 (1H, s), 7.49
(1H, d, J = 9.7 Hz), 7.62 (2H, d, J = 3.2 Hz), 7.64 (1H, d, J = 4.7 Hz,
7.80 – 7.77 (1H, m). δC (100 MHz, DMSOꢀd6) 38.94, 39.15, 39.36,
39.57, 39.78, 39.99, 46.73, 51.74, 54.07, 118.84, 119.09, 120.76,
123.73, 127.79, 130.04, 130.62, 132.16, 132.49, 135.80, 138.25,
165.76. IR (KBr) √/cmꢀ1 4000ꢀ450, 3035, 1721, 1216, 1068, 792,
763, 590. MS m/z 461.9 [M+Na]+
Zꢀ2ꢀ[2ꢀ(2,4,5ꢀTrifluorobenzyl)ꢀ1,1ꢀdioxidoꢀ2Hꢀ1,2ꢀbenzothiazinꢀ
4(3H)ꢀylidene]acetic acid methyl ester (2a)
Isolated yield: 66% (1.57 g) at 40°C (for 48 h), white crystalline
solid, mp: 132ꢀ136 °C. δH (400 MHz, DMSOꢀd6 ) 3.71 (3H, s), 4.20
(2H, s), 4.93 (2H, d, J =11.4 Hz), 6.76 (1H, s), 7.47 – 7.40 (1H, m),
7.55 – 7.48 (1H, m), 7.72 (2H, dd, J = 6.3, 2.7 Hz), 7.90 – 7.85 (1H,
m), 8.06 – 8.01 (1H, m). δC (100 MHz, DMSOꢀd6) 39.06, 39.27,
39.48, 39.68, 39.89, 46.04, 49.43, 51.60, 105.90, 106.13, 106.40,
106.66, 118.25, 119.19, 121.56, 124.42, 126.55, 126.55, 128.77,
131.29, 132.06, 132.06, 133.31, 134.82, 134.82, 142.62, 165.16; IR
Endoꢀ2ꢀ[2ꢀ(2ꢀbromo,4ꢀfluorobenzyl)ꢀ1,1ꢀdioxidoꢀ2Hꢀ1,2ꢀ
benzothiazinꢀ4(3H)ꢀylidene]acetic acid methyl ester (4b)
Isolated yield: 81% (2.13 g) at 120°C (for 12 h), white crystalline
solid, mp: 125ꢀ127 °C. δH (400 MHz, DMSOꢀd6) 3.59 (3H, s), 3.72
(2H, s), 4.94 (2H, s), 6.97 (1H, s), 7.27 (1H, t, J = 8.3 Hz), 7.42 (1H,
This journal is © The Royal Society of Chemistry 2014
RSC Advances, 2014, 00, 1-3 | 5

RSC Advances
Page 6 of 9
View Article Online
ARTICLE
RSC Advances
DOI: 10.1039/C4RA01016G
d, J = 8.2 Hz), 7.57 – 7.51 (2H, m), 7.62 – 7.58 (1H, m), 7.74 (1H, t, Hz), 7.48 (1H, s), 7.72 – 7.65 (2H, m), 7.86 (1H, d, J = 7.4 Hz), 8.00
J = 7.4 Hz), 7.89 (1H, d, J = 7.9 Hz), δC (100 MHz, DMSOꢀd6) (1H, d, J = 7.0 Hz). δC (100 MHz, DMSOꢀd6) 44.14, 44.35, 44.56,
34.78, 39.17, 39.38, 39.59, 39.79, 40.00, 44.16, 51.90, 111.78, 44.77, 44.98, 45.18, 45.39, 51.47, 54.68, 114.51, 124.00, 124.24,
118.90, 119.14, 121.45, 124.49, 128.06, 131.83, 132.54, 171.24. IR 125.27, 129.69, 131.73, 132.95, 136.34, 137.63, 137.99, 138.58,
(KBr) √/cmꢀ1 4000ꢀ450, 3071, 1730, 1198, 1045, 783, 764, 579. MS 139.07, 171.90. IR (KBr) √/cmꢀ1 4000ꢀ450, 2869, 1685, 1170, 1047,
m/z 461.9 [M+Na]+
772, 743. MS m/z 423.9 [MꢀH]ꢀ. HRMS (ESIꢀTOF) m/z: [MꢀH]−
Calcd for C17H12BrFNO4S 423.966; Found 423.9662.
General method for acid 5ꢀ7(aꢀb)
A mixture of the appropriate ester
Eꢀ2ꢀ[2ꢀ(4ꢀBromoꢀ2ꢀfluorobenzyl)ꢀ1,1ꢀdioxidoꢀ2Hꢀ1,2ꢀ
benzothiazinꢀ4(3H)ꢀylidene]acetic acid (6b)
2ꢀ4 (0.5 mmol), 1,4ꢀdioxane
(5 mL) and 10M HCl (8 mL) was refluxed at temperature range
of 60ꢀ80 °C. After completion of reaction, ice cold water (5 Isolated yield: 70% (0.157 g) at 60 °C (for 1h), white crystalline
mL) was added. The precipitates formed was then filtered and solid.mp: 94ꢀ98°C. δH (400 MHz, DMSOꢀd6) 4.17 (2H, s), 4.31 (2H,
washed with cold water. The crude product was purified by s), 6.17 (1H, s), 7.36 (1H, t, J = 8.0 Hz), 7.44 – 7.40 (1H, m), 7.54 –
flash chromatography and recrystallized from ethanol.
7.50 (1H, m), 7.67 – 7.62 (2H, m) 7.72 – 7.69 (1H, m), 7.82 – 7.79
(1H, m). δC (100 MHz, DMSOꢀd6). 43.99, 44.19, 44.40, 44.61,
44.82, 45.03, 45.23, 51.46, 59.06, 123.87, 124.12, 127.90, 128.86,
132.79, 134.82, 135.41, 135.92, 137.17, 137.50, 140.36, 140.67,
172.03. IR (KBr) √/cmꢀ1 4000ꢀ450, 2960, 1704, 1169, 1071, 769,
Zꢀ2ꢀ[2ꢀ(2,4,5ꢀTrifluorobenzyl)ꢀ1,1ꢀdioxidoꢀ2Hꢀ1,2ꢀbenzothiazinꢀ
4(3H)ꢀylidene]acetic acid (5a)
Isolated yield: 82% (0.157 g) at 80°C (for 12 h), white crystalline 743. MS m/z 423.9 [MꢀH]ꢀ. HRMS (ESIꢀTOF) m/z: [MꢀH]− Calcd
solid.mp: 192ꢀ195°C. δH (400 MHz, DMSOꢀd6) 4.19 (2H, s), 4.88 for C17H12BrFNO4S 423.966; Found 423.9664.
(2H, d, J = 21.0 Hz), 6.67 (1H, s), 7.56 – 7.36 (2H, m), 7.70 (2H, dd,
J = 6.7, 2.8 Hz), 7.86 (1H, dd, J = 6.6, 2.5 Hz), 8.08 – 7.92 (1H, m),
12.86 (1H, br, s). δC (100 MHz, DMSOꢀd6) 44.15, 44.35, 44.56,
44.77, 44.98, 45.19, 45.40, 51.25, 54.61, 111.34, 114.46, 124.46,
Endoꢀ2ꢀ[2ꢀ(4ꢀBromoꢀ2ꢀfluorobenzyl)ꢀ1,1ꢀdioxidoꢀ2Hꢀ1,2ꢀ
benzothiazinꢀ4(3H)ꢀylidene]acetic acid (7b)
125.14, 129.64, 131.63, 135.68, 136.25, 137.59, 138.52, 139.99, Isolated yield: 90% (0.192 g) at 80°C (for 1 h), white crystalline
146.62, 171.87. IR (KBr) √/cmꢀ1 4000ꢀ450, 2938, 1682, 1183, 1079, solid.mp: 105ꢀ109°C. δH (400 MHz, DMSOꢀd6) 3.61 (2H, s). 4.94
778, 766. MS m/z 382.0 [MꢀH]ꢀ. HRMS (ESIꢀTOF) m/z: [MꢀH]− (2H, s), 6.96 (1H, s), 7.29 (1H, t, J = 8.1 Hz), 7.42 (1H, d, J = 8.2
Calcd for C17H11F3NO4S 382.0366; Found 382.0372
Hz), 7.56 (1H, d, J = 7.6 Hz), 7.63 – 7.58 (2H, m), 7.75 (1H, t, J =
7.6 Hz), 7.90 (1H, d, J = 7.8 Hz,), 12.48 (1H, s). δC (100 MHz,
DMSOꢀd6) 35.22, 38.87, 39.08, 39.29, 39.50, 39.71, 39.92, 40.13,
44.06, 112.38, 118.81, 119.05, 121.32, 124.48, 127.79, 127.88,
130.71, 131.41, 131.76, 131.80, 132.38, 132.76, 172.17. IR (KBr)
Eꢀ2ꢀ[2ꢀ(2,4,5ꢀTrifluorobenzyl)ꢀ1,1ꢀdioxidoꢀ2Hꢀ1,2ꢀbenzothiazinꢀ
4(3H)ꢀylidene]acetic acid (6a)
Isolated yield: 79% (0.152 g) at 60°C (for 2 h), white crystalline √/cmꢀ1 4000ꢀ450, 2916, 1702, 1180, 1068, 789, 770. MS m/z: 423.9
solid. mp: 159ꢀ163°C. δH (400 MHz, DMSOꢀd6) 4.19 (2H, s), 4.36 [MꢀH]ꢀ. HRMS (ESIꢀTOF) m/z
:
[MꢀH]− Calcd for
(2H, d, J = 1.4 Hz), 6.18 (1H, t, J = 1.4 Hz), 7.57 – 7.44 (2H, m), C17H12BrFNO4S 423.966; Found 423.9659.
7.66ꢀ7.66 (2H, m), 7.74 – 7.69 (1H, m), 7.82ꢀ7.79 (1H, m), 12.85
(1H, br, s). δC (100 MHz, DMSOꢀd6) 18.60, 39.17, 39.38, 39.58,
39.79, 40.00, 46.29, 54.03, 56.09, 106.02, 106.30, 106.51, 118.71,
122.75, 123.77, 129.81, 130.30, 130.91, 132.12, 135.48, 135.66,
166.95. IR (KBr) √/cmꢀ1 4000ꢀ450, 2967, 1699, 1164, 1054, 768,
744. MS m/z 382.0 [MꢀH]ꢀ. HRMS (ESIꢀTOF) m/z: [MꢀH]− Calcd
for C17H11F3NO4S 382.0366; Found 382.0358
Biological assay
ALR2 was prepared by following method of Hayman and
Kinoshita36 and La Motta et. al.37 Enzyme was extracted from freshly
obtained rat lenses. Lenses were grinded and homogenized in three
volume of deionized water and centrifuged at 12000rpm for 30 min
at 0ꢀ4°C. The supernatant was skimmed off and precipitated with
(NH4)2SO4. The precipitates were dissolved in 0.05 M NaCl solution
and dialyzed overnight which was further used for inhibition assay.
NADP, D,LꢀGlyceraldehyde, and all other chemical of reagent grade
were purchase from Sigma–Aldrich. Epalrestat was prepared
Endoꢀ2ꢀ[2ꢀ(2,4,5ꢀTrifluorobenzyl)ꢀ1,1ꢀdioxidoꢀ2Hꢀ1,2ꢀ
benzothiazinꢀ4(3H)ꢀylidene]acetic acid (7a)
Isolated yield: 88% (0.169 g) at 80°C (for 1h), white crystalline solid.
mp: 172ꢀ176°C. δH (400 MHz, DMSO) 3.60 (2H, s), 4.93 (2H, s),
6.97 (1H, s), 7.44ꢀ7.33 (1H, m), 7.58 – 7.53 (2H, m), 7.60ꢀ7.62 (1H,
m), 7.73ꢀ 7.77 (1H, m), 7.89 (1H, dd, J =7.9, 1.1 Hz), 12.39 (1H, br,
s). δC (100 MHz, DMSOꢀd6) 35.21, 38.86, 39.06, 39.27, 39.48,
39.69, 39.90, 40.11, 43.81, 106.32, 112.63, 118.40, 121.35, 124.53,
125.25, 127.95, 130.72, 131.30, 132.45, 132.74, 172.23. IR (KBr)
√/cmꢀ1 4000ꢀ450, 2963, 1708, 1189, 1045, 769, 752. MS m/z 382.0
according to a reported method.
Enzyme inhibition activity was assayed on a Shimadzu UVꢀ1800
UV spectrophotometer. This assay was carried out by measuring the
decrease in absorption of NADPH at λ 340 nm, which accompanies
the oxidation of NADPH catalyzed by ALR2. ALR2 activity was
performed at 32 °C in a reaction mixture containing 0.25 mL of 0.10
mM NADPH, 0.25 mL of 0.1 M sodium phosphate buffer (pH 6.2),
0.1 mL of enzyme extract, 0.15 mL of deionized water, and 0.25 mL
of 10 mM D,Lꢀglyceraldehyde as substrate in a final volume of 1
mL. The reaction mixture except for D,Lꢀglyceraldehyde was
incubated at 32 °C for 10 min. The substrate was then added to start
the reaction, which was monitored for 4 min.
[MꢀH]ꢀ. HRMS (ESIꢀTOF) m/z
:
[MꢀH]− Calcd for
C17H11F3NO4S 382.0366; Found 382.036.
Zꢀ2ꢀ[2ꢀ(4ꢀBromoꢀ2ꢀfluorobenzyl)ꢀ1,1ꢀdioxidoꢀ2Hꢀ1,2ꢀ
benzothiazinꢀ4(3H)ꢀylidene]acetic acid (5b)
Isolated yield: 85% (0.181 g) at 80 °C (for 20 h), white crystalline
solid.mp: 160ꢀ166°C. δH (400 MHz, DMSOꢀd6) 4.15 (2H, s), 4.87
(2H, s), 6.68 (1H, s), 7.32 (1H, t, J = 8.0 Hz), 7.38 (1H, d, J = 8.3
The inhibitory activity of the newly synthesized compounds against
ALR2 was assayed adding 5 ꢁL of the inhibitor. The inhibitory
effect of the synthetic compounds was routinely estimated at a
6 | RSC Advances, 2014, 00, 1-3
This journal is © The Royal Society of Chemistry 2014

Page 7 of 9
RSC Advances
RSC Advances
View Article Online
ARTICLE
DOI: 10.1039/C4RA01016G
concentration of 10ꢀ4 M (the concentration refers to that of the
compound in the reaction mixture).
11. D. A. Carper, G. Wistow, C. Nishimura, C. Graham,
K. Watanabe, Y. Fujii, H. Hayashi and O. Hayaishi,
Exp. Eye Res, 1989, 49, 377ꢀ388.
12. O. A. Barski, K. H. Gabbay, C. E. Grimshaw and K.
M. Bohren, Biochemistry, 1995, 34, 11264ꢀ11275.
13. Y. C. Yang, S. Z. Zhang, B. B. Wu, M. M. Ma, X.
Chen, X. Y. Qin, M. L. He, S. Hussain, C. J. Jing, B.
The compounds found to be active were tested at additional
concentrations between 10ꢀ4 and 10ꢀ8 M. Each dose–response curve
was generated using at least four concentrations of compound with
inhibitory activity between 20% and 80 %, with three replicates at
each concentration. The 95% confidence limits (95% CL) were
calculated from t values for nꢀ2, where n is the total number of
determinations.
Ma and C. J. Zhu, ChemMedChem, 2012, 7, 823ꢀ835.
Docking studies were performed using Molegro Virtual Docker,
version 5.0. The crystal structure of human aldose reductase with
bound inhibitor IDD594 (PDB code: 1US0) retrieved from the
RCSB Protein Data Bank. All solvent molecules within the protein
structure were removed for the docking procedure. Five binding
cavities were detected to get the potential binding site in the protein.
The cavity around the anion binding site (volume of ~124 Å3) was
chosen using the gridꢀbased MolDock score (GRID) function with a
grid resolution of 0.30 Å. The lowest energy ligand poses were
chosen on the basis of the MolDock score and ReRank score. All
structural parameters of ligands such as bond orders, hybridization,
explicit hydrogen atoms, and charges were assigned when necessary
in the Molegro Virtual Docker software.
14. X. Chen, Y. C. Yang, B. Ma, S. Z. Zhang, M. L. He,
D. Q. Gui, S. Hussain, C. J. Jing, C. J. Zhu and Q. Yu,
Bioorg. Med. Chem, 2011, 19, 7262ꢀ7269.
15. Wittig G and G. G., Liebigs Ann. Chem, 1953, 580
,
44.
16. Wittig G and S. U., Chem. Ber., 1954, 87, 1318.
17. B. E. Maryanoff and A. B. Reitz, Chem. Rev., 1989,
89, 863.
18. E. Vedejs and M. Peterson, J. Top. Stereochem., 1994,
21, 1.
19. K. C. Nicolaou, M. W. Harter, J. L. Gunzner and A.
Nadin, Liebigs Ann./Recl., 1997,
7, 1281.
Acknowledgements
20. Zarbin P, Princival JL, de Lima E, dos Santos A,
Ambrogio B and d. O. A., Tetrahedron Lett., 2004, 45
239.
21. S. M. Mali, A. Bandyopadhyay, S. V. Jadhav, M. G.
,
This work was supported by the National Natural Science
Foundation of China (grant no. 21272025), the Research Fund for
the Doctoral Program of Higher Education of China (grant no.
20111101110042), and the Science and Technology Commission of
Beijing, China (grant no. Z131100004013003).
Kumar and H. N. Gopi, Organic & Biomolecular
Chemistry, 2011, 9, 6566ꢀ6574.
22. P. H., Angew. Chem., 1977, 89, 437.
23. P. H., Angew. Chem. Int. Ed., 1977, 16, 423.
24. Pommer H and T. C., Top. Curr. Chem., 1983, 109
165.
References:
,
1. K. H. Gabbay, M. L. O and F. R. A, Science, 1966,
25. J. G. Lombardino, E. H. Wiseman and W. Mclamore,
J. Med. Chem., 1971, 14
26. C. B. Schapira, I. A. Perillo and S. Lamdan, J.
Heterocycl. Chem., 1980, 17
151, 209ꢀ210.
.
2. D. M. Nathan, N. Engl. J. Med., 1988, 318, 1315ꢀ
1321.
.
3. S. S. Chung and S. K. Chung, Curr. Med. Chem.
2003, 10, 1375ꢀ1387.
,
27. M. ZiaꢀurꢀRehman, J. A. Choudary, M. R. J.
Elsegood, H. L. Siddiqui and K. M. Khan, Eur. J.
Med. Chem. , 2009, 44, 1311.
28. E. W. Brooke, S. G. Davies, A. W. Mulvaney, M.
Okada, F. Pompeo, E. Sim, R. J. Vickers and I. M.
4. J. H. Kinoshita and C. Nishimura, Diabetes Metab.
rev., 1988, , 323ꢀ337.
4
5. C. YabeꢀNishimura, Pharmacol. Rev. , 1998, 50, 21ꢀ
33.
Westwood, Bioorg. Med. Chem. Lett., 2003, 13
.
6. R. Kikkawa, I. Hatanaka, H. Yasuda, N. Kobayashi,
Y. Shigeta, H. Terashima, T. Morimura and M.
Tsuboshima, Diabetologia, 1983, 24, 290.
29. R. Robiette, J. Richardson, V. K. Aggarwal and J. N.
Harvey, Journal of the American Chemical Society
2005, 127, 13468ꢀ13469.
,
7. K. Sestanj, F. Bellini, S. Fung, N. Abraham, A.
Treasurywala, L. Humber, N. SimardꢀDuquesme and
D. Dvornik, J. Med. Chem., 1984, 27, 255.
8. X. Chen, C. J. Zhu, F. Guo, X. W. Qiu, Y. C. Yang, S.
Z. Zhang, M. L. He, S. Parveen, C. J. Jing, Y. Li and
B. Ma, J. Med. Chem., 2010, 153, 8330ꢀ8344.
9. S. Zhang, X. Chen, S. Parveen, S. Hussain, Y. Yang,
30. S. Valverde, M. MartinꢀLomas, B. Herradon and S.
GarciaꢀOchoa, Tetrahedron 1987, 43, 1895.
31. J. M. J. Tronchet and B. Gentile, Helu. Chim. Acta
1979, 62, 2091.
32. J. S. Brimacombe, R. Hanna, A. K. M. S. Kabir, F.
Bennett and I. D. Taylor, J. Chem. Soc., Perkin Trans.
1
, 1986, 815.
C. Jing and C. J. Zhu, ChemMedChem, 2013, 8, 603ꢀ
613.
33. P. A. Byrne and D. G. Gilheany, J. AM. Chem. Soc
2012, 134, 9225.
,
10. S. J. Ao, Y. Shingu, C. Kikuchi, Y. Notsu and I.
34. J. Wu, D. Li, H. Wu, L. Suna and W.ꢀM. Daia,
Yamaguchi, Metabolism, 1991, 40, 77.
Tetrahedron, 2006, 62, 4643.
35. see Supporting information
.
This journal is © The Royal Society of Chemistry 2014
RSC Advances, 2014, 00, 1-3 | 7

RSC Advances
Page 8 of 9
View Article Online
ARTICLE
RSC Advances
DOI: 10.1039/C4RA01016G
36. S. Hayman and J. H. Kinoshita, J. Biol. Chem, 1965,
240, 877ꢀ882.
37. C. L. Motta, S. Sartini, L. Mugnaini, F. Simorini, S.
Tliani, S. salerno, A. M. Marini, F. D. Settimo, A.
Lavecchia, E. Novellino, M. Cartore, P. Failli and M.
Ciuffi, J. Med. Chem., 2007, 50, 4917ꢀ4927.
8 | RSC Advances, 2014, 00, 1-3
This journal is © The Royal Society of Chemistry 2014

Page 9 of 9
RSC Advances
View Article Online
DOI: 10.1039/C4RA01016G
Graphic Abstract
α,β- and β,γ-unsaturated carboxylate isomers of 1,2-benzothiazine-1,1-dioxide were selectively synthesized via Wittig
olefination reaction under various temperature conditions. At 40 °C afforded α,β-unsaturated esters with high Z-
stereoselectivity (83-87 %) while β,γ-unsaturated esters preferentially formed with moderate to excellent regioselectivity at
100-120 °C (77-96%). The acid isomers were found to be active in aldose reductase inhibition in rank order of β,γ-
unsaturated
> Z-α,β-unsaturated > E-α,β-unsaturated isomers. The β,γ-unsaturated isomer 7b, 2-[2-(4-bromo-2-
fluorobenzyl)-1,1-dioxido-2H-1,2-benzothiazin-4(3H)-ylidene]acetic acid, exhibited most potent inhibition activity with an
IC50 value of 0.057 µM. This was further supported by docking study.
O
O
O
HO
HO
R1
Ph3P=CHCOOMe
R
R
+
N
N
N
S
S
S
O
O
O
O
O
O
Z>E