Design and synthesis of chiral 2H-chromene-N-imidazolo-amino acid conjugates as aldose reductase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.08.070

Aldose reductase (ALR2) inhibitors provide a viable mode to fight against diabetic complications. ALR2 exhibit plasticity in the active site vicinities and possible shifts in the nearby two supporting alpha helices. Therefore, a novel series of amino acid conjugates of chromene-3-imidazoles (13–15) were designed and synthesized based on natural isoflavonoids. The compounds were identified on the basis of spectral (¹H NMR,¹³C NMR and MS) data and tested in vitro for ALR2 inhibitory activity with an IC₅₀value ranges from 0.031 ± 0.082 μM to 4.29 ± 0.55 μM. Our in silico and biochemical studies confirmed that 15e has the best inhibition activity among the synthesized compounds with a high selective index against the Aldehyde reductase (ALR1). Supplementation of 15e to STZ induced rats decreased the blood glucose levels and delayed the progression of cataract in a dose-dependent manner. The present study thus provides novel series of compounds with a promising inhibitor to prevent or delay the cataract progression.

Full text of DOI:10.1016/j.ejmech.2016.08.070

European Journal of Medicinal Chemistry 124 (2016) 750e762
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design and synthesis of chiral 2H-chromene-N-imidazolo-amino acid
conjugates as aldose reductase inhibitors
Gudipudi Gopinath a, Venu Sankeshi b, Shaym perugu c, Malini D. Alaparthi d,
,
*
Srinivas Bandaru d, Vijay K. Pasala a, Prasad Rao Chittineni a, G.L.David Krupadanam a
,
,
**
Someswar R. Sagurthi d
a Department of Chemistry, Osmania University, Hyderabad, 500 007, India
b Department of Zoology, Osmania University, Hyderabad, 500 007, India
c Department of Biochemistry, Osmania University, Hyderabad, 500 007, India
d Department of Genetics, Drug Design & Molecular Medicine Lab, Osmania University, Hyderabad, 500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Aldose reductase (ALR2) inhibitors provide a viable mode to fight against diabetic complications. ALR2
exhibit plasticity in the active site vicinities and possible shifts in the nearby two supporting alpha
helices. Therefore, a novel series of amino acid conjugates of chromene-3-imidazoles (13e15) were
designed and synthesized based on natural isoflavonoids. The compounds were identified on the basis of
spectral (1H NMR, 13C NMR and MS) data and tested in vitro for ALR2 inhibitory activity with an IC50 value
Received 4 April 2016
Received in revised form
29 August 2016
Accepted 31 August 2016
Available online 4 September 2016
ranges from 0.031 0.082 mM to 4.29 0.55 mM. Our in silico and biochemical studies confirmed that 15e
has the best inhibition activity among the synthesized compounds with a high selective index against the
Aldehyde reductase (ALR1). Supplementation of 15e to STZ induced rats decreased the blood glucose
levels and delayed the progression of cataract in a dose-dependent manner. The present study thus
provides novel series of compounds with a promising inhibitor to prevent or delay the cataract
progression.
Keywords:
2H-chromene-3-carboxylicacids
Amino acids
Imidazole
Diabetic complications
Aldose reductase inhibitor
Molecular docking
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
common mechanism in cataract pathogenesis. Multiple mecha-
nisms that manifest in the pathogenesis of diabetic cataract which
Diabetes mellitus has surfaced as a major health issue affecting
almost 300 million people across the countries [1]. A major fraction
of diabetic patients suffer from long-term secondary complications
such as neuropathy, nephropathy, retinopathy and cataract [2].
Diverse pathways and regulatory networks have been proposed in
diabetic patients, of particular relevance, the polyol pathway which
happens to first discovered, is still a generally accepted as the
mechanism of prime importance in the diabetic complication and
pathogenesis [1,3]. Seminal studies by Kinoshita et al., way back in
1965 demonstrated the involvement of ALR2 (polyol pathway) in
the development of cataractous lens [4] and till date forms the most
includes inhibition of aldose reductase (ALR2; EC: 1.1.1.21) has now
become a potential pharmacological target [5e7]. In normal phys-
iological conditions, the cellular glucose is metabolized through
glycolysis pathway, however, an investigation by Van Heyningen
1959, showed the dynamic involvement of pentose shunt pathway,
which is now observed as a crucial pathway regulating in human
cataract as well [8]. During hyperglycemic conditions, the increased
amount of glucose activates an ALR2 which forms the first rate-
limiting enzyme of polyol pathway. ALR2 utilizes a large amount
of intracellular NADPH to catalyze the reduction of glucose to sor-
bitol, which is the initial or the primary step in polyol pathway [9].
The downstream effects involving increased flux of glucose through
the polyol pathway trigger the accumulation of sorbitol. The
hyperosmotic effects of sorbitol accumulation owing to its polar
nature, eventually leads to lens swelling, damage and change in
membrane permeability. In addition, the drastic reduction of
NADPH forms an important cofactor for reviving critical intracel-
lular antioxidants and reduced glutathione levels. An emerging
* Corresponding author. Department of Chemistry, Natural Product lab, Osmania
University, Hyderabad, 500 007, India.
** Corresponding author. Department of Genetics, Drug Design
& Molecular
Medicine Lab, Osmania University, Hyderabad, 500 007, India.
E-mail addresses: gldkrupa@gmail.com (G.L.David Krupadanam), drsomeswar@
osmania.ac.in (S.R. Sagurthi).
http://dx.doi.org/10.1016/j.ejmech.2016.08.070
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

G. Gopinath et al. / European Journal of Medicinal Chemistry 124 (2016) 750e762
751
evidence suggests that by dropping the amount of reduced gluta-
thione in hyperglycemic conditions, the polyol pathway increases
susceptibility to intracellular oxidative stress, which results in
diabetic cataract by activation of enzyme aldose reductase [10e13].
Noticeably, most of the ALR2 inhibitors (ARIs) are pharmaco-
logically control the abnormal accumulation of sorbitol and may
indirectly inhibit oxidative stress. A large variety of structurally
diverse compounds have been identified as potent inhibitors to
ALR2. The well characterized ALR2 inhibitors from the earlier
studies has been classified into three main chemical classes: (i)
carboxylic acid derivatives, such as Epalrestat [14], Tolrestat [15],
Zopolrestat [16] and Zenarestat [17]; (ii) spiro-hydantoins, such as
Sorbinil [18,19], Fidarestat [20]; Ranirestat [21] (iii) natural phenolic
derivatives, such as Quercetin [22] and their methyl ethers as well
as glycosides [22] (Fig. 1). Nevertheless, many of them are lacking
specificity and sensitivity towards the human ALR2 and also
penetration issues with the target tissues [23]. To the date, there are
no any FDA-approved drugs against human ALR2. However, in the
recent years, Ranirestat progressed to the late phase of clinical trials
[24]. Unfortunately, none of the proven ARI's have not shown suf-
ficient efficacy in human clinical trials and also failed with unde-
sirable side effects. In the pursuit for better ARIs, there has been a
paradigm shift towards novel chemical moieties, which can
accommodate the anionic and specific binding pockets (Fig. 2) of
ALR2 [25]. In the first part of this study, the flexible and adaptable
active site properties of ALR2 were analyzed with the help of
multiple crystal structures. The binding pocket of aldose reductase
consists of a rigid anion binding pocket and a flexible specificity
pocket. The anion-binding pocket is formed mostly by His 110, Tyr
48, Trp 111 and the positively charged NADPþ. The binding pocket
may only be opened by inhibitors that offer the right interactions.
The amino acids, Phe 122 and Leu 300 which otherwise serve as
gatekeepers and can seal the pocket, however, the main flexibility is
conferred by the Ala 299-Leu 300 backbones and is apparent in
different conformations of the Leu 300 side chain which de-
termines the open or closed conformation of the pocket. It was
demonstrated that ALR2 with citrate (PDB code: 1X96) [26]
exhibited specificity pocket in the closed conformation and how-
ever open conformation in another crystal structure (PDB code:
1US0) [27]. The plasticity of an ALR2 enzyme was well character-
ized, with soaked co-crystal structures by using Zopolrestat and
Tolrestat as inhibitors. The peptide flip is accompanied by a rupture
of an H-bond formed to the bound ligand Zopolrestat (PDB code
2DUX) [28]. In the recent studies, it was shown that two molecules
of inhibitors (indole-1-acetic acid derivatives) were accommodated
in the binding site of ALR2 (PDB code: 4QX4) [29]. The AR crystals
soaked with Tolrestat, enzyme found to hold four molecules of
Tolrestat in the active site and binding site's proximity (PDB code:
2FZB) [28], which is possible by pronounced shifts concerning two
helices interacting with the additional ligands and crystal packing,
most likely originating from protein flexibility resulting in different
geometries in deviating environments. Protein adaptability is an
important prerequisite for biological function. Nevertheless, it also
provides a special challenge to inhibitor design as flexibility com-
plicates the prediction of the binding mode of a small molecule
ligand. In line with the above findings, we used a strategy to search
for novel ARIs with the hypothesis that the combination of two
proven moieties in one single compound may result in a molecule
of choice to treat diabetes and related diabetic secondary compli-
cations. In this study, taking consideration of the ALR2 enzyme
flexibility and dynamic properties we designed a new series of
compounds from proven AR inhibitors. Present novel series con-
sists of fragments from Sorbinil, Fidarestat and Quercetin (Fig. 3).
The synthesized compounds were subjected to docking studies
towards Human ALR2 enzyme to evaluate their affinity and binding
interaction in the active site. The compounds were evaluated in-
vitro and most potent inhibitor from this novel series was assessed
in-vivo for its ability to delay or prevent the progression of cataract.
2. Results and discussion
2.1. Synthesis
The salicylaldehyde and 3-Methoxysalicylaldehyde (o-vanillin)
(1a,b) reaction with acrylonitrile in the presence of 1,4-diazabicyclo
[2.2.2]octane (DABCO) gave substituted-2H-chromene-3-nitriles
(2a,b), these substituted-2H-chromene-3-nitriles (2a,b) were hy-
drolyzed to substituted-2H-chromene-3-carboxylic acids (3a,b)
[30], (Scheme 1). The tert-butyl(5-aryl-1H-imidazol-2-yl)alkyl car-
bamates (6a-e, 7a-b & 8aec) were synthesized by the reaction of
substituted phenacyl bromides (5a-f) [30] with N-Boc-amino acids
(4aec) (like N-Boc-glycine (4a), N-Boc-valine (4b) and N-Boc-pro-
line (4c) in the presence of TEA, followed by refluxed with
ammonium acetate in toluene [31] (Scheme 2). Substituted-2H-
chromene-3-carboxylic acids (3a-b) were reaction with SOCl2 to
get the corresponding acid chlorides (9a, b) and tert-butyl(5-aryl-
1H-imidazol-2-yl)alkyl carbamates (6a-e, 7a-b & 8aec) on treat-
ment with TFA gave the corresponding Boc deprotected amines (5-
aryl-1H-imidazol-2-yl)alkylamine) as their TFA salts (10aee, 11aeb
& 12aec). The targeted compounds N-((5-substituted phenyl-1H-
Fig. 1. Proven Aldose reductase inhibitors.

752
G. Gopinath et al. / European Journal of Medicinal Chemistry 124 (2016) 750e762
Fig. 2. Schematic view of the binding site of ALR2. The anion binding pocket is mainly formed by Tyr 48, His 110, Trp 111, and NADPþ. The specificity pocket is opened and closed
because of the conformational changes in the backbone of the Phe 122, Cys 296, Leu 300 and Cys 303. The hydrophobic pocket is formed by Trp 219 and Trp 20.
Fig. 3. Designed ALR2 inhibitors as hybrids of Sorbinil, Fidarestat and quercetin.
Scheme 1. Synthesis of substituted-2H-chromene-3-carboxylic acids (3a-b).
imidazol-2-yl)alkyl)-2H-chromene-3-carboxamides (13aej, 14aed
& 15aef) were obtained from the reaction of chromene-3-
carboxylic acid chlorides (9a, b) with appropriate aryl imidazole
amines (10aee, 11aeb & 12aec) in the presence of TEA (Scheme 3).
2.2.1. In vitro ALR1 and ALR2 activity
The N-(imidazole)-2H-chromene-3-carboxamides derivatives
(13aej, 14aed & 15aef) were tested in in-vitro against the inhibi-
tion of aldose reductase (ALR2) and aldehyde reductase (ALR1)
with Sorbinil [32] and Valproic acid [33] as positive controls. The
enzymes ALR1 and ALR2 share 65% similarity of amino acid resi-
dues with some differences in their respective active sites, which
makes it difficult in designing inhibitors with selectivity and ulti-
mately leading to toxicity issues [34]. Most of the synthesized
compounds (13aej, 14aed & 15aef) were having selectivity as
2.2. Biological studies
All newly synthesized derivatives of N-(imidazole)-2H-chromene-
3-carboxamides derivatives (13aej, 14aed & 15aef) were screened
for their effective inhibitory potential against ALR1 and ALR2 en-
zymes, extracted from rat kidney and lenses, respectively, and puri-
fied. All the compounds were subjected against both enzymes in
order to determine their selectivity. In addition, to know the inhibi-
tory effect of compound 15e was evaluated for in-vivo AR activity.
evidenced by IC50 values ranging from 0.031
0.082 mM to
4.29 0.55 M (Table 1). Among all the derivatives, 15e is found to
m
be the most potent with an 8-methoxy group on chromene nucleus,
4-chlorophenyl on imidazole and with pyrrolidine spacer between
chromene, imidazole moieties against ALR2 inhibition.

G. Gopinath et al. / European Journal of Medicinal Chemistry 124 (2016) 750e762
753
Scheme 2. Synthesis of tert-butyl (5-aryl-1H-imidazol-2-yl)alkylcarbamate (6a-e, 7a, b & 8aec).
Scheme 3. Synthesis of N-((5-substituted phenyl-1H-imidazol-2-yl)alkyl-2H-chromene-3-carboxamides (13aej, 14aed & 15aef).
Table 1
Inhibitory Effect of Compounds 13e15 on Rat ALR2 and ALR1and selectivity index.
Compound
IC50 values (
m
M) of ALR2a
IC50 values (
m
M) of ALR1a
Selectivity Indexb (ALR1 IC50 SD/ALR2 IC50 SD)
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
15a
15b
15c
15d
15e
15f
2.122 0.14
0.114 0.031
0.732 0.023
0.42 0.011
0.275 0.01
0.731 0.027
0.065 0.033
0.091 0.076
0.071 0.082
0.899 0.02
3.67 0.54
0.337 0.042
0.222 0.049
3.072 0.65
0.031 0.082
0.146 0.077
4.29 0.55
0.22 0.082
1.0 0.6
5.193 0.22
2.421 0.022
3.381 0.01
1.842 0.062
2.537 0.070
1.215 0.059
1.441 0.062
1.558 0.007
1.621 0.081
1.311 0.022
5.719 0.26
3.882 1.32
2.432 0.097
1.831 0.074
3.422 0.081
1.418 1.03
9.412 0.037
2.277 0.046
4.873 0.01
3.408 0.005
e
2.477
21.23
4.61
4.385
9.22
1.66
22.16
17.12
22.83
1.45
1.55
11.51
10.95
0.596
110.39
9.712
2.193
10.35
4.87
14a
14b
14c
14d
0.771 0.011
0.43 0.014
e
4.42
Sorbinilc
Valproic acidd
53.1 0.55
a
n ¼ 5.
b
Defined as IC50[ALR1]&IC50[ALR2].
c
Reported IC50 of 0.43
Reported IC50 of 53.1 0.55 mM [33].
m
M by Ref. [32].
d

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G. Gopinath et al. / European Journal of Medicinal Chemistry 124 (2016) 750e762
Among the series 13aej (glycine conjugates), compound 13g
2.3. Molecular docking studies
was the most potent inhibitor of ALR2, possessing an IC50 value of
0.065 0.033
m
M. Compound 13g has a chlorine atom attached to
Human ALR2 crystal complexes have suggested that the in-
hibitors bind to the enzyme active site and are held in place through
hydrogen bonding and van der Waals interactions formed within
the hydrophobic pockets [35]. These ligand-dependent conforma-
tions indicate a remarkable induced fit or flexibility of the active
site in the human ALR2 from the earlier studies (PDB codes: 1Z3N &
1US0). Three binding pockets were proposed at the active site of
ALR2 based on the X-ray crystallography and mutagenesis [36]. The
first site is usually occupied by the anion head of ligand and thus
named “anion binding pocket”. It consists of Tyr48, His110, Trp20,
and Trp111 side chains and the positively charged nicotinamide
moiety of the cofactor NADPþ [37]. The second is a hydrophobic
pocket, known as specificity pocket, and lined by the residues Leu
300, Cys 298, Cys 303, Trp 111, Cys 303, and Phe 122. The specificity
pocket displays a high degree of flexibility and the third is another
hydrophobic pocket formed by the residues Trp 20, Trp 111, Phe
122, and Trp 219 [38a]. By using above information we defined the
ligand binding cavities and carried out docking studies by Schro-
dinger. Similar information was obtained from the ALR1 crystal
structure (PDB code 3H4G) [38b] and defines the binding sites
carried out the comparative studies (Table 2). Molecular docking
studies were carried out to substantiate the interacting patterns
and selectivity of synthesized compounds with the ALR2 and ALR1.
the phenyl group over imidazole and methoxy on chromene both
separated by carboxamide. A slight decrease in activity was
observed when the chlorine atom was replaced with a bromine,
fluorine group in compound 13h & 13i (IC50 values: 0.091 0.076 &
0.079 0.082 mM), and without methoxy group on chromene nu-
cleus decrease in the activity compared methoxy containing ana-
logs, resulting in fewer interactions with the active site of aldose
reductase, the compounds without any substitution (13a) on the
phenyl ring of the imidazole fragment and chromene has exhibited
lesser activity. Whereas among the series 14aed derived from L-
valine, these compounds have exhibited lesser activity compared to
other compounds.
In the series 15aef, compound 15e with IC50 values of
0.337 0.042 mM respectively, were found more potent inhibitors
of ALR2 than the standard inhibitor, Sorbinil. The higher activity
may be assigned to the presence of imidazole ring with para-chloro
substituted phenyl ring pendant is separated by a pyrrolidin-2-yl
amide linkage from the chromene at 3rd position resulting in a
more potent structure that gives stronger interactions with the
active site for binding.
2.2.2. Selectivity
The test for selectivity over the closely related aldehyde reduc-
tase (EC.1.1.1.2, ALR1) was carried out. The ALR1 inhibitory potencies
(expressed as IC50) were calculated in the reduction of the substrate
2.3.1. Structural activity relationship
All the compounds were docked against ALR2 in which the
glycine spacer series (13aej) have lower affinity than valine spacer
series 14aed, whereas these two series exhibited lower affinity
towards the enzyme when compared to the proline spacer series
(15aef). In the 13aej, 13fej has a higher affinity towards ALR2,
owing to the presence of 8-methoxy group on the chromene ring
and substituted phenyl group on the imidazole. Among 13gei side
chain exhibited higher affinity in which para-fluoro phenyl group
(13i) and methoxy phenyl group on imidazole (13j) compounds
have shown maximum affinity with a docking score of ꢀ8.79
and ꢀ8.68, respectively. In the 14aed, 4-bromophenyl substituted
derivatives (14b & 14d) on imidazole has shown a slightly lower
affinity, but the presence of 8-methoxy group on the chromene ring
14c has improved the affinity with a docking score of ꢀ8.87. The
15aef showed a maximum affinity towards ALR2 with a docking
score ranging from ꢀ8.12 to ꢀ9.35. In the 15 series the 4-
chlorophenyl substituted imidazole derivatives 15b and 15e
exhibited higher affinity with docking scores of ꢀ8.97 and ꢀ9.35.
But, the 3-pyridinyl ring on the imidazole of 15c and 15f showed
decreased affinity towards ALR2 with a score of ꢀ8.12 and ꢀ8.81 in
comparison with their respective 4-chlorophenyl substituted
imidazole derivatives (15b & e). The addition of 8-methoxy group
D-glucuronate by partially purified ALR1 from rat kidney, are shown
in Table 1. For comparison, the reference Valproic acid was included.
All tested compounds (13aej, 14aed & 15aef) were shown less
inhibitory activity with ALR1 compared to ALR2. In the series of the
most efficient ARIs, the selectivity indices were found to be the
highest for the compound 15e. In all these analogs tested against
ALR2 chromene moiety is represented with a 3 atom amide linkage,
of which amide carbonyl originates from chromen-3-acid chloride
and the other part from L-amino acids (glycine, valine & proline).
Therefore, among the glycine derivatives (13aej), 13b & 13g com-
pounds with 4-chlorophenyl substituted on imidazole exhibited
highest IC50 values 0.11 0.03 & 0.07 0.03 respectively. In valine
derivatives (14aed), 4-bromophenyl substituted imidazole de-
rivatives 14b & 14d have shown IC50 values 0.22 0.08 & 0.77 0.01
respectively. In the entire group of compounds 15e with 8-OMe & 4-
chlorophenyl substituted has exhibited the highest activity, sug-
gesting that the most hydrophobic nature of the substrates has
helped in enhancing the activity. In proline derivatives, 15b with
4-chlorophenyl substituted on imidazole ring has shown better
activity with IC50 ¼ 0.34 0.04. The interesting compounds were
observed in 15c with a 3-pyridinyl ring on imidazole which has
shown further improvement in the activity (IC50 ¼ 0.22 0.05). But
this trend is lost with the presence of 8-OMe group on chromene
moiety (15e: IC50 0.03 0.08, 15f: IC50 ¼ 0.15 0.08). Nevertheless,
important functional considered from in-vitro studies on this scaf-
fold is the 4-chlorophenyl, which has enhanced the activity irre-
spective of the substitutions in the carboxamide linker. Hence, 15e is
more potent under in vitro studies over Sorbinil which requires
further exploration of treatment of diabetic secondary complica-
tions. But against the ALR1, even though the similar trends were
observed the IC50 values are higher, indicating the selective of their
compounds, towards ALR2 over ALR1. It should be noted that the
most selective compound 15e, with a selectivity factor above (110.4),
is also the most potent ARI in this series. It is an important feature of
selectivity over the other members of the aldo-keto reductase
family. The non-inhibition of structurally related physiologically
relevant oxidoreductases may minimize the undesired side effects.
Table 2
Molecular docking scores of compounds screened against ALR1 (PDB code: 3H4G)
and ALR2 (PDB code: 1Z3N).
S. No
Mol dock score
ALR1
S. No
Mol dock score
ALR1
ALR2
ALR2
13a
13b
13c
13d
13e
13f
13g
13h
13i
ꢀ4.13
ꢀ4.3
ꢀ4.12
ꢀ5.6
ꢀ4.8
ꢀ7.0
ꢀ5.9
ꢀ5.7
ꢀ6.03
ꢀ6.0
ꢀ7.79
ꢀ7.59
ꢀ6.97
ꢀ7.12
ꢀ7.86
ꢀ8.06
ꢀ8.71
ꢀ8.28
ꢀ8.79
ꢀ8.68
14a
14b
14c
14d
15a
15b
15c
15d
15e
15f
ꢀ4.35
ꢀ4.13
ꢀ4.54
ꢀ5.9
ꢀ8.57
ꢀ8.34
ꢀ8.87
ꢀ8.63
ꢀ8.75
ꢀ8.97
ꢀ8.12
ꢀ8.89
¡9.35
ꢀ8.81
ꢀ6.0
ꢀ6.6
ꢀ5.9
ꢀ6.3
¡6.38
ꢀ7.0
13j

G. Gopinath et al. / European Journal of Medicinal Chemistry 124 (2016) 750e762
755
on the chromene ring and 4-chlorophenyl on imidazole derivative
(15e) has shown an utmost affinity with the highest docking score
of ꢀ9.35. Further, a closer look at the active molecule indicates that
the presence of 8-methoxy group on the chromene ring and a
halogenated phenyl group on the imidazoles have a higher affinity
towards ALR2. C. Nantasenamat et al. demonstrated that halogens
are essential for the observed ALR2 inhibitory activity, which is
further supported by K. Ko & Y. Won and B. Vyas et al. In their
studies also halogenated compounds and specifically chlorine de-
rivatives had shown the highest inhibitory activity [39aec]. In our
results (Table 1) the constant and coefficient values used in are in
higher end, even in the earlier studies had shown that there have
been QSAR models for ARI's reported with larger constant values
than that in our results. In the example, Soni and Kaskhedikar re-
ported a QSAR model for 2,4dioxo5(naphtha2ylmethylene)3thia-
zolidinylacetic acids and 2thioxoanalogues as pIC50 ¼ 6.634 þ 0.504
Sp
þ 0.659 Ix (r2 ¼ 0.726) [39d]. More recently, Nantasenamat et al.
reported a QSAR model with a much larger constant value, 6.766,
and much smaller coefficients of explanatory valuables
(0.0970.332) to predict aldose reductase inhibitory activity of sul-
fonylpyridazinones [39b]. The 15e molecule which is foreseen to be
the best ARI also has a 4-chlorophenyl substitution. Drug likeliness
and Physico-chemical properties of the molecules were also
calculated and incorporated in the supplementary data.
2.3.2. Docking analysis of 15e and structural superposition
It was observed that 15e docked well in the active site of ALR2
with a better docking score (ꢀ9.3) compared to any other com-
pound. It interacts with ALR2 at active site residues Trp 20, Val 47,
His 110, Trp 111 and Lue 300 (hydrogen bond distance 2.5, 2.3, 3.0,
2.8, 2.9 Å, respectively) (Supplementary data). 15e compound has
several electrostatic (Trp 20, Val 47, Trp 111, Thr113, Pro 218) and
van der Waal interactions (Lys 21, Tyr 48, Gln 49, Trp 79, Cys 80, His
110, Phe 122, Trp 219, Cys 298, Cys 303, Thr 309 and Phe 311)
(Fig. 4a). Hence, it appears that 15e compound might bind to ALR2
in a both conformation (the open conformation figure is incorpo-
rated in supplementary data) these can be confirmed by co-
crystallization of 15e with ALR2. In the case of ALR1, hydrogen
bonding was observed between and amino acid residues Trp 22,
Trp 114 Met 302 and Arg (bond distance less than 3.20 Å) (Fig. 4b).
Since Trp-20/Arg 312, Trp 111/Trp 22, His 110/Asn 163 were
replaced in ALR1, 15e did not interact with His 110 and Trp 20. It is
interesting to note that unlike with ALR1, 15e interacted with Leu
300 and Leu 301 in ALR2 those are involved in imparting plasticity
to ALR2. We have performed docking with the open and closed
conformation of ALR2 crystal structures as augmented in supple-
mentary data. The compound 15e was docked in reference to a co-
crystallized compound in the PDB structures which shows least
Root mean square deviation (as determined by OPLS force-fields)
demonstrating it to fit best in the active site.
Fig. 4. a: 2D representation of 15e interactions with the ALR2 active site pocket in
closed conformation (PDB code 1Z3N). b: 2D representation of 15e interactions at the
active site of ALR1 (PDB code 3H4G).
ALR2 [39a,b]. These in-silico assumptions have to be proved by co-
crystallization studies.
2.4. Delayed cataract progression in STZ induced rats
In order to know the inhibitory effect of compound 15e in in-
vivo, the following experiment was performed by following earlier
standardized protocols [40]. Rats were divided into six groups (each
group n ¼ 12), Group-I: normal control rats, Group-II: diabetic
control rats, Group-III & Group-IV: diabetic rats were fed with 5 and
In addition, 15e shows the superior docking score in both open
(PDB-1US0, docking score ꢀ10.34) and closed conformations of the
protein which confirms its high binding affinity. In addition, the
high affinity of compounds can be further testified by its optimal
potential energy, columbic and van der Waals forces, interaction
profile as well as excellent hydrogen bonding efficiency in the
active site of the protein. The molecular alignment was performed
with the highest dock scored compound 15e was used as the
template and all the other proven (Fidarestat, Sorbinil and Zopol-
restat) on the common core (Fig. 5a). Occupancy and spacial
arrangement of the 15e compound were compared Tolrestat
soaked crystal (PDB code: 2FZB (Fig. 5b)). 15e molecule nearly
covered the vicinity of two molecules of Tolrestat (Fig. 5b). From the
binding analysis of the 15e molecule with open and closed con-
formations anticipated answering the induced-fit properties of the
50
m
M/kg body weight of the 15e compound, respectively. Group-V
M/kg) & Group-VI (Fidarestat 50 M/kg body weight)
(Sorbinil 50
m
m
diabetic rats were fed with known AR inhibitors. All rats (Group I-
IV) were fed with ad libitum and it was noticed that there was an
increase in food intake in all diabetic rats (Group II-VI). Despite
increased food intake, the body weight of diabetic rats was found
low (Group-II 186
6.91 g), in relation to the control (Group-I
350 6.33 g) and compound 15e fed rats (Group-III 309 10.1 g
and Group-IV 320 3.17 g, Fig. 6). Elevated blood glucose levels
have been attenuated by feeding with compound 15e (Group-III
138.86
261.53
noticed a decrease in AR activity in Group-III and IV rats in a dose-
dependent manner (Table 3). On an extended feeding of compound
15e to diabetic rats, we have noticed a delay in the progression of
6.20 mg/dl, Group-IV 129.80
3.56 mg/dl Vs Group-II
53 mg/dl, Fig. 7). At the end of 8 weeks, we have

756
G. Gopinath et al. / European Journal of Medicinal Chemistry 124 (2016) 750e762
Fig. 5. a & b: Structural alignment of known ARIs in comparison to 15e. (a) Inhibitors represented in stick model in the active site, 15e is shown in pink, Sorbinil (PDB code 1AHO),
Fidarestat (PDB code 1PWM) and Zopolrestat (PDB code 2DUX) are shown in yellow, magenta and marine blue, respectively. (b) The soaked crystals of ALR2 with tolrestat (marine
blue color) accommodated four inhibitor molecules (PDB code 2FZB) at the binding pockets. This PDB superposed with 15e (magenta color) docked ALR2 structure. (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Effect of 15e on the body weight of different groups (I-VI) in diabetic rat lens.
Experimental diabetes was induced with a single dose of Streptozotocin (IP 32 mg/kg
body. Wt) and diabetic rats were maintained for 8 weeks with or without dietary
supplementation of compound 15e. Body weights were measured weekly basis in
different groups (G- I to G-VI) of rats. The compound 15e supplemented (G- III & G -IV)
and Sorbini (G-V) & Fidarestat (G-VI) rats body weights were compared with control
(GeI) and diabetic rats (G- II).
Fig. 7. Estimation of blood glucose levels in control (Group I; uninduced) and STZ
induced diabetic rats (Group II to Group VI). Experimental diabetes was induced with a
single dose of Streptozotocin (STZ) (IP 32 mg/kg body weight) and all rats were
maintained for 8 weeks. In Group I e control uninduced rats, Group eII to VI all were
STZ induced rats, Group III & IV supplemented with 15e with the concentration of 5
& 50 M, respectively. In the groups V & VI, supplemented with Sorbinil and Fidarstat
with 50 M each. Blood glucose levels were measured each group on a weekly basis to
assess the diabetic conditions in different groups of rats (Group I to Group VI). The data
are the mean SD (n ¼ 3).
mM
m
cataract (Fig. 8a & b) due to decreased levels of sorbitol and fructose
(Fig. 9a & b) in STZ induced diabetic rat lens, and were compared
with two known ARI inhibitors as controls Sorbinil (Group-V) and
Fidarestat (Group-VI). From the above in-vivo experiments the
inhibitory activity of 15e was demonstrated, it could bring the STZ
rats body weights to normal state and it also helped in attenuating
the increase in blood glucose levels. In similar to two well-known
proven ARI inhibitors, 15e could also delay the progression of
cataract in STZ induced rats.
m
Table 3
Effect of 15e compound in STZ induced diabetic rats with reference to AR activity.
Groups
Parameter
Aldose reductase activity
(Group-I)
(Group-II)
(Group-III, 5
(Group-IV, 50
(Group eV, 50
(Group eVI, 50
Control (uninduced)
Diabetic control
34.2 2.50
3. Conclusion
44.66 2.70a
41.26 1.94a
39.54 0.53b
38.95 1.00b
36.28 2.14b
m
M)
M)
M)
M)
Diabeticþ15e compound
Diabeticþ15e compound
Diabetic þ Sorbinil
m
m
In this study, we have described the design, synthesis, biological
activities of the compounds, and protein-ligand interactions of
novel series of N-(imidazole)-2H-chromene-3-carboxamide ana-
logs (13aej, 14aed & 15aef). Molecular docking studies gave an
idea about the binding of these inhibitors, which in turn helped us
to understand the interactions of chromene, imidazole and phenyl
m
Diabetic þ Fidarestat
The data are the mean SD (n ¼ 3). SD: Standard deviation.
Aldose reductase activity.
a
Statistically significant from Group-I (analyzed by ANOVA: p ꢁ 0.05).
b
Statistically not significant from Group-I (analyzed by ANOVA: p ꢁ 0.05).

G. Gopinath et al. / European Journal of Medicinal Chemistry 124 (2016) 750e762
757
15e might aid in guiding the development of highly specific ALR2
inhibitors without any side effects.
4. Experimental section
4.1. General experimental methods
Compounds 2H-3-chromenecarbonitriles (2a, b), 2H-3-
chromenecarboxylic acids (3a, b) and Ary acyl bromides (4a-f)
are prepared by a repeated procedure [30]. Unless otherwise
specified, all solvents and reagents were obtained from commercial
suppliers. Solvents were purified as per the procedures are given in
the ‘'Text book of practical organic chemistry’’ by Vogel (6th Edition).
All reactions were performed under a nitrogen atmosphere unless
otherwise noted. Column chromatography was performed using
Merck silica gel 60e120 mesh. 1H NMR spectra were recorded on
Bruker spectrometer at 400 MHz spectrometer, 13C NMR spectra
were acquired on 100.6 MHz with tetramethylsilane as an internal
standard, chemical shift (d) are reported in ppm (d) Shift (multi-
plicity, coupling constant, proton count). Mass spectral analysis was
accomplished using electro spray ionization (ESI) techniques.
4.2. General procedure for the synthesis of compounds 6a-e, 7a, b
& 8aec
Fig. 8. a & b: Delayed diabetic cataract progression of 15e. (A) Photographs of lenses
from each group at the end of 12 weeks and grading of cataract progression were
performed by slit-lamp examination on a weekly basis. In Group I e control uninduced
rats, Group eII to VI all were STZ induced rats, Group II diabetic rats were not sup-
plemented with any inhibitory compounds, Group III & IV supplemented with 15e
A mixture of N-Boc-amino acids (5a-c, 1.0eq) and triethylamine
(2.5eq) in dichloromethane in an ice bath was added appropriate
phenacyl bromides (4a-f, 1.2eq), after addition the solution stirred
overnight at room temperature. The reaction mixture was diluted
with chloroform and washed with water and brine solution, suc-
cessively, and dried over Na2SO4, after removal of the solvent under
reduced pressure; the residue was washed with pet-ether and
dried under vacuum. The residue was dissolved in toluene and
treated with ammonium acetate (7.0eq) under a nitrogen atmo-
sphere. The reaction mixture was reflux for 12 h. The reaction
mixture was quenched with saturated sodium bicarbonate solu-
tion. The product was extracted with ethyl acetate (2 times), and
washed with water, followed by brine solution, successively and
dried over Na2SO4. After removal of the solvent under reduced
pressure, the crude product was purified by column chromatog-
raphy using petroleum ether/ethyl acetate to get desired com-
pounds (6a-e, 7a, b & 8aec).
with the concentration of 5
mM & 50
mM, respectively. In the groups V & VI, supple-
mented with Sorbinil and Fidarestat with 50
mM each, respectively. (B) Cataract for-
mation was scored biweekly according to the following classification: clear normal lens
(0), peripheral vesicles (1), peripheral vesicles and cortical opacities (2), diffuse central
opacities (3) and mature cataract (4). The data are the mean SD (n ¼ 3).
rings with ALR2 and ALR1. Among the several promising com-
pounds described here, methoxy group on chromene nucleus, 4-
chlorophenyl on imidazole and with pyrrolidine-2-yl spacer de-
rivative (15e) emerged for the excellent ALR2 inhibitory activity. In
conclusion, the results of the present study indicate that 15e in-
hibits ALR2 and ALR1with better selectivity index for ALR2. All the
analogs were evaluated for their competent biological assays and
15e ability to delay progression of cataract in STZ rats. The 15e
compound also shown better Physico-chemical and drug-likeness
properties in our in-silico analysis. Thus, data also suggest that
Fig. 9. a & b: Analysis of Sorbitol and Fructose accumulation in diabetic rat lenses. Compound 15e decreased the accumulation of Sorbitol and Fructose levels in the diabetic rat lens.
Eight weeks old rat lens was dissected and used to estimate the Sorbitol and Fructose levels in different groups. In Group I e control uninduced rats, Group eII to VI all were STZ
induced rats, Group II diabetic rats were not supplemented with any inhibitory compounds, Group III & IV supplemented with 15e with the concentration of 5
respectively. In the groups V & VI, supplemented with a known ARIs, Sorbinil and Fidarestat with 50 M each.
mM & 50 mM,
m

758
G. Gopinath et al. / European Journal of Medicinal Chemistry 124 (2016) 750e762
4.2.1. tert-Butyl(5-(phenyl)-1H-imidazol-2-yl)methylcarbamate
(d, J ¼ 6.8 Hz, 3H, CH3), 1.38 (s, 9H, 3xCH3), 2.07 (m, CH), 4.4 (t,
0
0
(6a)
J ¼ 8.4 Hz, NeCH), 6.98 (d, J ¼ 9.2 Hz, NH-Boc), 7.51 (m, 3H, H3 , H5
Yield 70%, 1H-NMR (DMSO-d6)
d: 1.4 (s, 9H, 3xCH3), 4.19 (d,
& H4), 7.7 (d, J ¼ 7.2 Hz, 2H, H2 & H6 ), 11.86 (s, NH). 13C-NMR
0
0
0
J ¼ 5.6 Hz, 2H, NeCH2), 7.16 (t, J ¼ 7.2 Hz, 7.6 Hz, H4 ), 7.28 (t,
(DMSO-d6)
d
: 19.18 (CH3), 19.72 (CH3), 28.66 (3xCH3), 32.88
0
0
0
J ¼ 4.8 Hz, NH-Boc), 7.33 (t, J ¼ 7.6 Hz, 2H, H3 & H5 ), 7.47 (s, H4),
(C(CH3)2), 55.13 (NeC), 78.43 (C(CH3)3), 113.27 (C4), 118.98 (C4 ),
7.73 (d, J ¼ 6.8 Hz, 2H, H2 & H6 ), 11.87 (s, NH). 13C-NMR (DMSO-d6)
126.61 (C2 & C6 ), 131.74 (C1 ), 134.76 (C3 & C5 ), 138.76 (C5), 149.43
(C2), 155.72 (C]O). MS (ESI, m/z) ¼ 395.1 [MþH]þ, 398.22
[M þ Hþ2]þ.
0
0
0
0
0
0
0
: 28.71 (3xCH3), 38.44 (NeCH2), 78.5 (tC), 113.15 (C4), 124.55 (C2
&
0
d
0
0
0
0
0
C6 ), 126.36 (C4 ), 128.88 (C3 & C5 ), 135.35 (C1 ), 140.13 (C2), 146.5
(C5), 156.1 (C]O). MS (ESI, m/z) ¼ 274.05 [MþH]þ.
4.2.8. (S)-tert-butyl 2-(5-phenyl-1H-imidazol-2-yl)pyrrolidine-1-
4.2.2. tert-Butyl(5-(4-chlorophenyl)-1H-imidazol-2-yl)
carboxylate (8a)
Yield 66%, 1H-NMR (DMSO-d6)
d: 1.15 (s, 6H, 2xCH3), 1.40 (s, 3H,
methylcarbamate (6b)
Yield 65%, 1H-NMR (DMSO-d6)
d: 1.4 (s, 9H, 3xCH3), 4.18 (d,
CH3), 1.90 (m, 3H, H3, CH2-4), 2.20 (m, H3), 3.36 (m, H5), 3.53 (m,
J ¼ 5.6 Hz, 2H, NeCH2), 7.28 (t, J ¼ 4.8 Hz, NH-Boc), 7.37 (d,
00
0
00
0
H5), 4.92 (m, H2), 7.16 (t, H4 ), 7.36 (m, H3 & H5 ), 7.46 (s, H4 ), 7.72
(d, 2H, H2 & H6 ), 11.88 (s, NH). 13C-NMR (DMSO-d6)
28.3 (3xCH3), 33.8 (C3), 46.7 (C5), 55.7 (C2), 78.6 ( C), 112.2 (C4 ),
d
: 23.5 (C4),
0
0
0
&
J ¼ 8.4 Hz, 2H, H3 & H5 ), 7.52 (s, H4), 7.75 (d, J ¼ 8.4 Hz, 2H, H2
0
00
H6 ), 11.95 (s, NH). 13C-NMR (DMSO-d6)
d: 28.7 (3xCH3), 38.42
0
t
0
(NeCH2), 78.52 (tC), 113.93 (C4), 126.19 (C2 & C6 ), 128.86 (C3 & C5 ),
0
0
0
0
0
00
00
00
00
000
124.6 (C2 & C6 ), 126.2 (C4 ), 128.8 (C3 & C5 ), 135.6 (C1 ), 139þ.9
0
0
130.57 (C1 ), 134.16 (C4 ), 137.81 (C2), 146.78 (C5), 156.1 (C]O). MS
0
0
(C5 ), 150.9 (C2 ), 153.8 (C]O). MS (ESI, m/z) ¼ 341.10 [MþH] ,
336.05 [MþNa]þ.
(ESI, m/z) ¼ 307.98 [M]þ, 309.95 [Mþ2]þ.
4.2.3. tert-Butyl(5-(4-bromophenyl)-1H-imidazol-2-yl)
4.2.9. (S)-tert-butyl-2-(5-(4-chlorophenyl)-1H-imidazol-2-yl)
methylcarbamate (6c)
pyrrolidine-1-carboxylate (8b)
Yield 70%, 1H-NMR (DMSO-d6)
d
: 1.4 (s, 9H, 3xCH3), 4.18 (d,
Yield 65%, 1H-NMR (DMSO-d6)
d: 1.14 (s, 6H, 2xCH3), 1.39 (s, 3H,
J ¼ 5.6 Hz, 2H, NeCH2), 7.29 (t, J ¼ 4.8 Hz, NH-Boc), 7.49 (s, H4), 7.52
CH3),1.90 (m, 3H, H3, CH2-4), 2.17 (m, H3), 3.36 (m, H5), 3.52 (m, H5),
0
0
0
0
(d, J ¼ 7.2 Hz, 2H, H2 & H6 ), 7.77 (d, J ¼ 8.0 Hz, 2H, H3 & H5 ), 12.0 (s,
00
00
0
4.79 (m, H2), 7.36 (t, J ¼ 8.4 Hz, H3 & H5 ), 7.52 (s, H4 ), 7.75 (d,
NH). 13C-NMR (DMSO-d6)
( C), 119.0 (C4), 126.56 (C4 ), 130.07 (C2 & C6 ), 131.7 (C1 ), 131.76 (C3
d
: 28.7 (3xCH3), 38.47 (NeCH2), 78.52
J ¼ 8.4 Hz, 2H, H2 & H6 ), 11.96 (s, NH). 13C-NMR (DMSO-d6)
d
: 23.5
00
00
t
0
0
0
0
0
t
0
(C4), 28.3 (3xCH3), 33.7 (C3), 46.7 (C5), 55.7 (C2), 78.6 ( C),112.8 (C4 ),
0
& C5 ), 133.86 (C2), 146.82 (C5), 156.1 (C]O). MS (ESI, m/z) ¼ 352.99
[MþH]þ, 353.95 [M þ Hþ2]þ.
00
00
00
00
00
0
0
126.2 (C2 & C6 ), 128.8 (C3 & C5 ), 130.4 (C1 ), 138.8 (C5 ), 151.1 (C2 ),
153.8 (C]O). MS (ESI, m/z) ¼ 348.03 [M]þ, m/z 350 [Mþ2]þ.
4.2.4. tert-Butyl(5-(4-fluorophenyl)-1H-imidazol-2-yl)
methylcarbamate (6d)
4.2.10. (S)-tert-butyl-2-(5-(pyridin-3-yl)-1H-imidazol-2-yl)
Yield 60%, 1H-NMR (DMSO-d6)
d: 1.4 (s, 9H, 3xCH3), 4.19 (d,
pyrrolidine-1-carboxylate (8c)
Yield 59%, 1H-NMR (DMSO-d6)
d: 1.15 (s, 6H, 2xCH3), 1.40 (s, 3H,
J ¼ 5.6 Hz, 2H, NeCH2), 7.28 (t, J ¼ 4.8 Hz, NH-Boc), 7.33 (t, J ¼ 7.6 Hz,
CH3), 1.92 (m, 3H, H3, CH2-4), 2.20 (m, H3), 3.36 (m, H5), 3.53 (m,
0
0
0
0
2H, H3 & H5 ), 7.47 (s, H4), 7.93 (d, J ¼ 6.8 Hz, 2H, H2 & H6 ), 11.9 (s,
NH). 13C-NMR (DMSO-d6)
d: 28.71 (3xCH3), 38.44 (NeCH2), 78.5
00
H5), 4.80 (m, H2), 7.33 (dd, J ¼ 7.6, 4.8 Hz, H5 ), 7.62 (d, J ¼ 9.6 Hz,
(tC), 112.3 (C3 & C5 ), 114.5 (C4), 126.0 (C1 ), 128.8 (C2 & C6 ), 138.03
0
00
00
H4 ), 8.06 (dd, J ¼ 6.0, 6.4, 2.0, 1.6 Hz, H4 ), 8.35 (d, J ¼ 3.6 Hz, C6 ),
0
0
0
0
0
8.96 (d, J ¼ 1.2 Hz, H2 ), 12.0 (d, J ¼ 15.6 Hz, NH). 13C-NMR (DMSO-
00
0
(C2), 146.1 (C5), 156.0 (C]O), 159.0 (C4 ). MS (ESI, m/z) ¼ 278.2
d6) d
: 23.5 (C4), 28.3 (3xCH3), 33.7 (C3), 46.7 (C5), 55.7 (C2), 78.7 (tC),
[MþH]þ, 300.13 [MþNa]þ.
0
00
00
00
0
00
113.4 (C4 ), 124.0 (C5 ), 131.2 (C3 ), 131.5 (C4 ), 136.9 (C5 ), 146.2 (C6 þ),
0
00
147.2 (C2 ), 151.5 (C2 ), 153.8 (C]O). MS (ESI, m/z) ¼ 315.03 [MþH] ,
337.05 [MþNa]þ.
4.2.5. tert-Butyl(5-(4-methoxyphenyl)-1H-imidazol-2-yl)
methylcarbamate (6e)
Yield 55%, 1H-NMR (DMSO-d6)
d
: 1.4 (s, 9H, 3xCH3), 3.75 (s, 3H,
0
OCH3), 4.17 (d, J ¼ 5.6 Hz, 2H, NeCH2), 6.91 (d, J ¼ 8.8 Hz, 2H, H3
&
4.3. General procedure for the synthesis of compounds 13aej,
14aed & 15aef
0
H5 ), 7.26 (t, J ¼ 5.6 Hz, NH-Boc), 7.31 (s, H4), 7.64 (d, J ¼ 8.8 Hz, 2H,
H2 & H6 ), 11.9 (s, NH). 13C-NMR (DMSO-d6)
d
: 28.7 (3xCH3), 38.42
0
0
(NeCH2), 55.48 (OCH3), 78.48 (tC), 111.28 (C3 & C5 ), 114.35 (C4),
Step 1: In a RB flask, compounds (6a-e, 7a, b & 8aec; 1.0eq)
were dissolved in dichloromethane then added TFA. The solution
was stirred at room temperature for 6 h. Then the mixture was
evaporated to remove the excess of TFA under reduced pressure to
obtain the corresponding amines (9aee, 10a, b & 11aec).
Step 2: Substituted chromene-3-carboxylic acids (3a, b) was
carefully added to SOCl2 (5 vol) in a round bottom flask, the solution
was refluxed for 6 h, cooled and evaporated under reduced pres-
sure to obtain the corresponding acid chlorides (12a, b) as a sticky
gum.
Step 3: The acid chloride gum (step-2; 1.0eq) was dissolved into
dry dichloromethane cooled in ice-bath and the step-1 amines
(9aee, 10a, b & 11aec) (1.0eq) was slowly added followed by TEA
(3.0eq). The resulting mixture was stirred under N2 atmosphere at
rt for 12 h. The reaction mixture was washed with water, brine, and
then dried over anhydrous Na2SO4. The solvent was evaporated to
dryness and the resulted residue was purified by column chroma-
tography using petroleum ether/ethyl acetate to get required
compounds (13aej, 14aed & 15aef).
0
0
0
0
0
125.81(C1 ), 128.88 (C2 & C6 ), 138.03 (C2), 146.16 (C5), 156.1 (þC]O),
0
158.18 (C4 ). MS (ESI, m/z) ¼ 303.98 [MþH]þ, 326.0 [MþNa] .
4.2.6. tert-Butyl-(S)-2-methyl-1-(5-phenyl-1H-imidazol-2-yl)
propylcarbamate (7a)
Yield 67%, 1H-NMR (CDCl3)
d
: 0.76 (d, J ¼ 6.8 Hz, 3H, CH3), 0.9 (d,
J ¼ 6.4 Hz, 3H, CH3), 1.39 (s, 9H, 3xCH3), 2.08 (m, CH), 4.41 (t,
J ¼ 8.4 Hz, NeCH), 6.98 (d, J ¼ 9.2 Hz, NH-Boc), 7.17 (t, J ¼ 7.2 Hz,
0
0
0
H4 ), 7.33 (t, J ¼ 7.2 Hz, 2H, H3 & H5 ), 7.46 (s, H4), 7.72 (d, J ¼ 7.6 Hz,
2H, H2 & H6 ), 11.83 (s, NH). 13C-NMR (DMSO-d6)
d
: 19.29 (CH3),
0
0
19.72 (CH3), 28.62 (3xCH3), 32.98 (C(CH3)2), 55.15 (NeC), 78.42
0
0
0
0
(C(CH3)3), 113.56 (C4), 124.58 (C2 & C6 ), 126.41 (C4 ), 128.92 (C3 &
0
0
C5 ), 129.73 (C1 ), 144.02 (C5), 149.23 (C2), 155.71 (C]O). MS (ESI, m/
z) ¼ 316.2 [MþH]þ.
4.2.7. tert-Butyl-(S)-2-methyl-1-(5-(4-bromophenyl)-1H-imidazol-
2-yl)propylcarbamate (7b)
Yield 60%, 1H-NMR (CDCl3)
d
: 0.76 (d, J ¼ 6.8 Hz, 3H, CH3), 0.89

G. Gopinath et al. / European Journal of Medicinal Chemistry 124 (2016) 750e762
759
4.3.1. N-((5-phenyl-1H-imidazol-2-yl)methyl)-2H-chromene-3-
carboxamide (13a)
7.82 (m, 3H, H4 , H2 & H6 ), 9.04 (s, NHeC]O), 12.1 (s, NH). 13C-
NMR (DMSO-d6) : 35.9 (CeNH), 56.1 (OCH3), 64.6 (C2), 115.0 (C7),
115.4 (C4a), 120.7 (C4 & C5), 122.3 (C6), 125.3 (C2 & C6 ), 128.4 (C3
0
00
00
d
Off white solid, yield 60%, m.p: 138e140 ꢂC. 1H-NMR (DMSO-d6)
0
00
00
00
&
00
00
0
0
d
: 4.46 (d, J ¼ 5.6 Hz, 2H, CH2), 4.95 (s, 2H, OCH2), 6.86 (d, J ¼ 7.6 Hz,
C5 ), 128.6 (C1 ), 129.4 (C3), 134.9 (C4 & C2 ), 143.5 (C5 ),þ146.2 (C8a),
0
0
00
,
H8), 6.96 (t, J ¼ 7.2 Hz, H6), 7.2 (m, 4H, H7, H5 & H4 ), 7.36 (m, 3H, H3
148.1 (C8), 165.2 (C]O). MS (ESI) m/z ¼ 362.15 [MþH]
.
00
00
0
00
H4 & H5 ), 7.5 (s, H4), 7.74 (d, J ¼ 7.2 Hz, 2H, H2 & H6 ), 8.81 (t,
J ¼ 5.2 Hz, NHeC]O), 12.0 (s, NH). 13C-NMR (DMSO-d6)
d: 37.24
4.3.7. N-(5-(4-Chlorophenyl)-1H-imidazol-2-yl)methyl)-8-
methoxy-2H-chromene-3-carboxamide (13g)
0
(NeCH2), 64.9 (C2), 116.11 (C8 & C4a), 121.72C4 ), 122.29 (C6), 124.61
Off white solid, yield 68%, m.p: 180e185 ꢂC. 1H-NMR (DMSO-d6)
00
00
00
00
00
(C5), 126.49 (C2 & C6 ), 126.98 (C4 ), 127.77 (C3 & C5 ), 128.88 (C7),
00
0
128.94 (C1 ), 131.5 (C3), 136.0 (C4 & C2 ), 146.01 (C5), 154.58 (C8a),
d
: 3.79 (s, 3H, OCH3), 4.43 (d, J ¼ 5.6 Hz, 2H, CH2eN), 4.87 (s, 2H,
164.84 (C]O). MS (ESI, m/z) ¼ 332.1 [MþH]þ.
OCH2), 6.82 (d, J ¼ 7.6 Hz, H7), 6.9 (t, J ¼ 8.0, 7.6 Hz, H6), 6.98 (d,
0
00
00
J ¼ 6.8 Hz, H5), 7.32 (s, H4 ), 7.37 (d, J ¼ 8.4 Hz, 2H, H3 & H5 ), 7.58 (s,
00
00
4.3.2. N-((5-(4-Chlorophenyl)-1H-imidazol-2-yl)methyl)-2H-
chromene-3-carboxamide (13b)
H4), 7.77 (d, J ¼ 8.4 Hz, 2H, H2 & H6 ), 8.77 (t, J ¼ 4.8 Hz, NHeC]O),
12.0 (s, NH). 13C-NMR (DMSO-d6)
d: 37.2 (CH2eN), 56.1 (OCH3), 64.8
Light yellow solid, yield 60%, m.p: 150e155 ꢂC. 1H-NMR (DMSO-
(C2), 113.8 (C7), 114.8 (C4a), 120.6 (C4 ), 122.0 (C5), 122.4 (C6), 126.2
0
00 00 00 00 00 00
d6)
d
: 4.42 (d, J ¼ 5.6 Hz, 2H, CH2eN), 4.91 (s, 2H, OCH2), 6.81 (d,
(C2 & C6 ), 127.0 (C3 & C5 ), 127.9 (C1 ), 128.8 (C4 ), 130.5, (C3) 134.3
0
0 0
(C4), 138.9 (C2 ), 143.5 (C5 ), 148.1 (C8), 164.8 (C]O). MS (ESI) m/
J ¼ 7.6 Hz, H8), 7.15 (m, 3H, H4 , H5 & H6), 7.2 (t, J ¼ 7.4 Hz, H7), 7.38
z ¼ 396.15 [MþH] þ
.
00
00
00
&
(d, J ¼ 7.2 Hz, 2H, H3 & H5 ), 7.51 (s, H4), 7.7 (d, J ¼ 8.4 Hz, 2H, H2
H6 ), 8.78 (t, J ¼ 5.6, 5.2 Hz, NHeC]O), 12.0 (s, NH). 13C-NMR
00
(DMSO-d6)
d: 37.2 (CH2eN), 64.7 (C2), 113.8 (C8), 114.8 (C4a), 120.6
4.3.8. N-(5-(4-bromophenyl)-1H-imidazol-2-yl)methyl)-8-
methoxy-2H-chromene-3-carboxamide (13h)
0
00
00
00
00
(C4 ), 122.0 (C6), 126.2 (C6), 126.4 (C2 & C6 ), 127.0 (C3 & C5 ), 127.1
(C7), 127.9 (C1 ), 128.8 (C4 ), 130.6, (C3) 134.3 (C4), 138.9 (C2 ), 143.5
White solid, yield 60%, m.p: 241e245 ꢂC. 1H-NMR (DMSO-d6)
d:
00
00
0
0
(C5 ), 146.1 (C8a), 148.1 (C8), 164.8 (C]O). MS (ESI) m/z ¼ 366.1
3.76 (s, 3H, OCH3), 4.43 (d, J ¼ 5.6 Hz, 2H, CH2eN), 4.93 (s, 2H,
[MþH]þ.
OCH2), 6.83 (d, J ¼ 6.8 Hz, H7), 6.91 (t, J ¼ 7.6, 8.0 Hz, H6), 6.97 (d,
0
00
00
J ¼ 8.0 Hz, H5), 7.23 (s, H4 ), 7.44 (d, J ¼ 8.0 Hz, 2H, H3 & H5 ), 7.61 (s,
00
00
4.3.3. N-((5-(4-bromophenyl)-1H-imidazol-2-yl)methyl)-2H-
chromene-3-carboxamide (13c)
H4), 7.72 (d, J ¼ 7.2 Hz, 2H, H2 & H6 ), 8.25 (t, J ¼ 8.0 Hz, NHeC]O),
12.12 (s, NH). 13C-NMR (DMSO-d6)
d
: 37.2 (CeNH), 56.1 (OCH3), 64.7
Off white solid, yield 70%, m.p: 140e145 ꢂC. 1H-NMR (DMSO-d6)
(C2), 113.9 (C7), 114.8 (C4a), 120.6 (C4 ), 121.9 (C5), 122.4 (C6), 126.6
00
00 00 00 00 00 00
d
: 4.45 (d, J ¼ 5.6 Hz, 2H, CH2eN), 5.07 (s, 2H, OCH2), 6.86 (d,
(C4 ), 127.0 (C2 & C6 ), 127.9 (C1 ), 131.8 (C3 & C5 ), 133.3 (C3), 134.6
0
0
0
J ¼ 7.6 Hz, H8), 6.96 (t, J ¼ 7.6, 7.2 Hz, H6), 7.22 (m, 3H, H5, H7 & H4 ),
(C4 & C2 ), 143.5 (C5 ), 146.2 (C8a), 148.0 (C8þ),164.8 (C]O). MS (ESI)
m/z ¼ 440.05 [MþH]þ, 442.05 [M þ Hþ2]
.
00
00
7.53 (d, J ¼ 8.4 Hz, 2H, H3 & H5 ), 7.63 (s, H4), 7.7 (d, J ¼ 8.4 Hz, 2H,
H2 & H6 ), 10.04 (t, J ¼ 5.6, 4.4 Hz, NHeC]O), 12.24 (s, NH). 13C-
00
00
NMR (DMSO-d6)
d
: 37.7 (CeNH), 64.8 (C2), 113.9 (C8), 114.8 (C4a),
4.3.9. N-(5-(4-fluorophenyl)-1H-imidazol-2-yl)methyl)-8-
methoxy-2H-chromene-3-carboxamide (13i)
0
00
00
00
119.1 (C4 ), 120.9 (C4 ), 126.6 (C5), 127.1 (C7), 127.9 (C2 & C6 ), 131.8
Off white solid, yield 45%, m.p: 160e165 ꢂC. 1H-NMR (DMSO-d6)
00
00
00
0
0
(C3 & C5 ), 133.7 (C3 & C1 ), 139.0 (C2 ), 143.5 (C5 ), 148.1 (C8a), 164.8
(C]O). MS (ESI) m/z ¼ 411.0 [MþH]þ, 413.2 [M þ Hþ2]þ.
d
: 3.76 (s, 3H, OCH3), 4.6 (s, 2H, CH2eN), 4.92 (s, 2H, OCH2), 6.83 (d,
J ¼ 6.8 Hz, H7), 6.92 (t, J ¼ 8.4, 6.8 Hz, H6), 7.0 (d, J ¼ 7.2 Hz, H5), 7.12
0
00
00
00
00
4.3.4. N-(5-(4-fluorophenyl)-1H-imidazol-2-yl)methyl)-2H-
chromene-3-carboxamide (13d)
(s, H4 ), 7.27 (m, 2H, H3 & H5 ), 7.84 (m, 3H, H4, H2 & H6 ), 9.02 (s,
NHeC]O), 12.0 (s, NH). 13C-NMR (DMSO-d6)
: 35.9 (CeNH), 56.1
(OCH3), 64.6 (C2), 115.0 (C7), 115.4 (C4a), 117.7 (C3 & C5 ), 120.6 (C5 &
d
Brown solid, yield 55%, m.p: 150e154 ꢂC. 1H-NMR (DMSO-d6)
d:
00
00
0
0
0
0
0
0
0
4.12 (d, J ¼ 5.6 Hz, 2H, CH2), 4.62 (s, 2H, OCH2), 6.69 (d, J ¼ 8.0 Hz,
C4 ), 122.05 (C6), 125.3 (C1 ), 128.6 (C2 & C6 ), 129.5 (C3), 134.9 (C4 &
0
00
0
0
00
H8), 7.08 (s, H4 ), 6.97 (t, J ¼ 7.6 Hz, H6), 7.27 (m, 4H, H5, H7, H3
&
C2 ), 143.5 (C5 ), 146.2 (C8a), 148.0 (C8), 159.14 (C4 ), 165.2 (C]O). MS
00
00
00
H5 ), 7.66 (s, H4), 7.87 (m, 2H, H2 & H6 ), 9.6 (t, J ¼ 2.0 Hz, NHeC]
(ESI) m/z ¼ 381.3 [MþH]þ.
O), 11.83 (s, NH). 13C-NMR (DMSO-d6)
d
: 36.29 (NeCH2), 64.73 (C2),
00
00
0
116.17 (C8 & C4a), 117.67 (C3 & C5 ), 119.85 (C4 ), 122.38 (C6), 126.63
4.3.10. 8-Methoxy-N-((5-(4-methoxyphenyl)-1H-imidazol-2-yl)
methyl)-2H-chromene-3 carboxamide (13j)
00
00
00
(C1 ), 126.55 (C5), 127.29 (C7), 127.92 (C2 & C6 ), 129.02 (C3), 129.78
(C4), 141.84 (C2 ), 144.15 (C5 ), 154.78 (C8a), 157.38 (C4 ), 165.97 (C]
Off white solid, yield 45%, m.p: 145e150 ꢂC. 1H-NMR (DMSO-d6)
0
0
00
þ
O). 350.0 [MþH]
.
d
: 3.74 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 4.41 (d, J ¼ 5.6 Hz, 2H,
CH2eN), 4.91 (s, 2H, OCH2), 6.81 (d, J ¼ 7.6 Hz, H7), 6.9 (m, 3H, H6, H5
0
00
00
4.3.5. N-((5-(4-methoxyphenyl)-1H-imidazol-2-yl)methyl)-2H-
chromene-3-carboxamide (13e)
& H4 ), 6.98 (d, J ¼ 7.2 Hz, 2H, H3 & H5 ), 7.33 (s, H4), 7.66 (d,
00
00
J ¼ 8.8 Hz, 2H, H2 & H6 ), 8.78 (t, J ¼ 5.6, 5.2 Hz, NHeC]O), 11.85 (s,
Yellow solid, yield 50%, m.p: 160e167 ꢂC. 1H-NMR (DMSO-d6)
d:
NH).). 13C-NMR (DMSO-d6)
d: 35.8 (CH2eN), 56.2 (OCH3), 64.4 (C2),
00
00
0
3.77 9s, OCH3), 4.42 (d, J ¼ 5.6 Hz, CH2), 4.91 (s, OCH2), 6.85 (d,
115.0 (C7), 115.4 (C3 & C5 ), 116.5 (C4a), 120.0 (C5), 120.6 (C4 ), 122.5
00
00
00
00 00 00 0
(C6), 124.5 (C1 ), 128.6 (C2 & C6 ), 129.4 (C3), 134.9 (C4), 138.5 (C2 ),
143.5 (C5 ), 146.2 (C8a), 148.0 (C8), 159.5 (C4 ), 165.2 (C]O). MS (ESI)
m/z ¼ 392.15 [MþH]þ.
J ¼ 7.6 Hz, H8), 6.91 (m, 3H, H6, H3 & H5 ), 7.35 (d, J ¼ 7.2 Hz, H2
&
H6 ), 7.5 (s, H4), 8.79 (t, J ¼ 5.6, 5.2 Hz, NHeC]O), 11.85)s, NH). 13C-
00
0
00
NMR (DMSO-d6) d: 36.0 (NeCH2), 55.48 (OCH3), 64.7 (C2), 114.35
00
00
0
00
(C3 & C5 ), 116.2 (C4a & C8), 121.7 (C4 ), 122.2 (C6), 124.6 (C1 ), 126.5
00
00
0
(C5), 127.2 (C2 & C6 ), 127.9 (C7), 129.2 (C3), 136.5 (C4), 138.0 (C2 ),
4.3.11. N-(S)-2-Methyl-1-(5-phenyl-1H-imidazol-2-yl)propyl)-2H-
chromene-3- carboxamide (14a)
þ
0
00
141.5 (C5 ), 154.7 (C8a), 158.1 (C4 ), 165.0 (C]O). 362.15 [MþH]
.
Light green solid, yield 55%, m.p: 118e120 ꢂC. 1H-NMR (DMSO-
4.3.6. 8-Methoxy-N-((5-phenyl-1H-imidazol-2-yl)methyl)-2H-
d6)
d
: 0.81 (d, J ¼ 6.8 Hz, 3H, CH3), 0.98 (d, J ¼ 6.8 Hz, 3H, CH3), 2.3
chromene-3-carboxamide (13f)
(m, CH-(CH3)2), 4.9 (m, 3H, OCH2 & CHeN), 6.85 (d, J ¼ 8.0 Hz, H8),
Light yellow solid, yield 65%, m.p: 110e115 ꢂC. 1H-NMR (DMSO-
6.96 (t, J ¼ 7.6, 6.8 Hz, H6), 7.17 (t, J ¼ 7.2 Hz, H7), 7.23 (m, 3H, H4 , H4
00
0
00
00
d6)
d
: 3.76 (s, 3H, OCH3), 4.63 (d, J ¼ 5.6 Hz, 2H, CH2eN), 4.93 (s, 2H,
& H5), 7.34 (t, J ¼ 7.6 Hz, 2H, H3 & H5 ), 7.42 (s, H4), 7.74 (d,
0
00
00
00
00
00
OCH2), 6.92 (m, 3H, H7, H6 & H5), 7.36 (m, 4H, H4 , H3 , H5 & H4 ),
J ¼ 6.8 Hz, 2H, H2 & H6 ), 8.45 (d, J ¼ 8.8 Hz, NHeC]O), 12.0 (s,

760
G. Gopinath et al. / European Journal of Medicinal Chemistry 124 (2016) 750e762
NH). 13C-NMR (DMSO-d6)
(CH3)2), 53.58 (CeNH), 64.94 (C2), 116.11 (C4a), 121.75 (C4 ), 122.28
d
: 19.7 (CH3), 20.07 (CH3), 32.33 (C-
H4 ), 7.38 (d, J ¼ 8.0 Hz, 2H, H3 & H5 ), 7.53 (s, H4), 7.75 (m, 2H, H2
00
000
000
000
& H6 ), 12.15 (s, NH). 13C-NMR (DMSO-d6)
d: 25.32 (C4 ), 31.83 (C3 ),
0
000
0
0
00
00
00
00
00
0
0
0
(C6), 124.63 (C2 & C6 ), 126.92 (C4 ), 127.59 (C7), 128.87 (C3 & C5 ),
49.59 (C5 ), 55.56 (C2 ), 65.47 (C2), 116.05 (C8 & C4a), 121.88 (C4 ),
00
0
0
000
000
000
128.93 (C1 ), 131.44 (C3), 134.61 (C4), 142.92 (C5 ), 148.87 (C2 ),
122.17 (C6), 126.12 (C5), 128.15 (C2 & C6 ), 128.85 (C7), 129.36 (C3
154.61 (C8a), 164.61 (C]O). MS (ESI) m/z ¼ 374.2 ([MþH]þ.
& C5 ), 130.59 (C1 ), 131.12 (C4 & C3), 134.0 (C4), 140.0 (C5 ), 149.93
000
000
000
00
(C2 ), 154.3 (C8a), 166.34 (C]O). MS (ESI) m/z ¼ 406.15 [MþH]þ.
00
4.3.12. N-(S)-1-(5-(4-bromophenyl)-1H-imidazol-2-yl)-2-
methylpropyl)-2H-chromene-3-carboxamide (14b)
4.3.17. (2H-chromen-3-yl)(S)-2-(5-(pyridin-3-yl)-1H-imidazol-2-
White solid, yield 55%, m.p: 140e145 ꢂC. 1H-NMR (DMSO-d6)
d
:
yl)pyrrolidin-1-yl)methanone (15c)
0.82 (d, J ¼ 6.8 Hz, 3H, CH3), 0.98 (d, J ¼ 6.8 Hz, 3H. CH3), 2.3 (m, CH-
Brown solid, yield040%, m.p: 122e123 ꢂC. 1H-NMR (DMSO-d6)
d:
0
0
0
(CH3)2), 4.97 (m, 3H, OCH2 & CHeN), 6.85 (d, J ¼ 8.0 Hz, H8), 6.96 (t,
2.0 (m, 3H, H3 , CH2-4 ), 2.28 (m, H3 ), 3.8 (m, 2H, CH2-5 ), 5.0 (m, 3H,
0
0 00
OCH2 & H2 ), 6.9 (m, 2H, H8 & H6), 7.2 (m, 3H, H5, H7 & H4 ), 7.36 (s,
J ¼ 7.6, 6.4 Hz, H6), 7.23 (m, 2H, H5 & H7), 7.42 (s, H4 ), 7.51 (d,
00
00
00
000
000
000
000
J ¼ 8.0 Hz, 2H, H3 & H5 ), 7.58 (s, H4), 7.72 (d, J ¼ 7.6 Hz, 2H, H2
&
H4), 7.65 (m, H4 ), 8.08 (m, H5 ), 8.38 (m, H6 ), 8.96 (s, H2 )0, 12.4 (s,
H6 ), 8.45 (d, J ¼ 8.8 Hz, NHeC]O), 12.04 (s, NH). 13C-NMR (DMSO-
NH). 13C-NMR (DMSO-d6)
d: 23.49 (C4 ), 31.8 (C3 ), 33.76 (C5 ), 55.54
0
00
0
(C20), 65.77 (C2), 114.2 (C8), 116.06 (C4a), 119.1 (C4 ), 122.18 (C6),
00
d6)
d
: 19.6 (CH3), 20.1 (CH3), 32.2 (C-(CH3), 53.5 (CeNH), 64.9 (C2),
00
000 000 000
124.67 (C5 ), 126.4 (C5), 128.74 (C7), 131.67 (C1 ), 134.1 (C6 ), 136.6
113.3 (C8), 116.1 (C4a), 121.7 (C6), 122.3 (C4 ), 126.7 (C5), 126.9 (C7),
00
00
00
00
00
00
000
000
00
127.6 (C2 & C6 ), 128.8 (C1 ), 131.4 (C3 & C5 ), 131.7 (C3), 134.7 (C4),
(C4), 140.5 (C5 ), 146.66 (C4 ), 147.4 (C2 & C2 ), 157.72 (C8a), 164.11
(C]O). MS (ESI) m/z ¼ 373.2 [MþH]þ.
0
0
138.9 (C5 ), 149.1 (C2 ), 154.6 (C8a), 164.6 (C]O). MS (ESI) m/
z ¼ 452.1 [MþH] þ, 454.1 [M þ Hþ2] þ
.
4.3.18. (8-methoxy-2H-chromen-3-yl)(S)-2-(5-phenyl-1H-
4.3.13. 8-Methoxy-N-(S)-2-methyl-1-(5-phenyl-1H-imidazol-2-yl)
propyl)-2H-chromene-3-carboxamide (14c)
imidazol-2-yl)pyrrolidin-1-yl) methanone (15d)
Brown solid, yield 45%, m.p: 122e126 ꢂC. 1H-NMR (DMSO-d6)
d:
Off white solid, yield 50%, m.p: 155e157 ꢂC. 1H-NMR (DMSO-d6)
2.1 (m, 4H, CH2-30, CH2-40), 3.6 (m, 2H, CH2-50), 3.8 (s, 3H, OCH3),
0
d
: 0.82 (d, J ¼ 6.8 Hz, 3H, CH3), 0.98 (d, J ¼ 6.8 Hz, 3H, CH3), 2.33 (m,
4.92 (s, 2H, OCH2), 5.23 (m, H2 ), 6.83 (d, J ¼ 8.8 Hz, H7), 6.93 (t,
0
CH-(CH3)2), 3.75 (s, 3H, OCH3), 4.9 (m, 3H, OCH2 & CHeN), 6.85 ((d,
J ¼ 6.4 Hz, H7), 6.91 (t, J ¼ 7.6, 8.0 Hz, H6), 6.98 (d, J ¼ 6.8 Hz, H5), 7.17
J ¼ 5.6, 5.6 Hz, H6), 7.0 (t, J ¼ 8.0, 7.6 Hz, H5), 7.18 (s, H4 ), 7.25 (m, 2H,
00
00
00
00
00
H3 & H5 ), 7.34 (m, H4 ), 7.72 (m, 3H, H2 , H4 & H6 ), 12.1 (s, NH).
(t, J ¼ 7.2, 6.8 Hz, H4 ), 7.34 (t, J ¼ 7.2, 7.6 Hz, 2H, H3 & H5 ), 7.41 (s,
13C-NMR (DMSO-d6)
d: 25.3 (C4 ), 32.0 (C3 ), 42.5 (C5 ), 56.1 (OCH3),
00
00
00
0
0
0
0
00
00
0
0
H4 ), 7.48 (s, H4), 7.74 (d, J ¼ 7.2 Hz, 2H, H2 & H6 ), 8.46 (d, J ¼ 8.8 Hz,
NHeC]O), 12.0 (s, NH). 13C-NMR: 19.7 (CH3), 20.0 (CH3), 32.3 (C-
(CH3)2), 55.6 (OCH3), 56.1 (CeNH), 64.8 (C2), 114.7 (C7), 120.6 (C4a),
57.0 (C2 ), 65.3 (C2), 114.5 (C7), 118.5 (C4a), 120.6 (C4 ), 122.0 (C5),
122.7 (C6), 126.9 (C2 & C6 ), 127.6 (C4 ), 129.1 (C3 & C5 ), 131.5 (C3
000
000
000
000
000
000
00
00
& C1 ), 133.4 (C4), 142.52 (C5 ), 147.9þ(C8a & C2 ), 150.0 (C8), 172.8
0
00
00
00
00
121.9 (C5 & C4 ), 122.4 (C6), 124.6 (C2 & C6 ), 126.9 (C3 & C5 ), 126.4
(C]O). MS (ESI) m/z ¼ 402.2 [MþH]
.
00
00
0
(C4 ), 127.7 (C1 ), 128.9 (C3 & C4), 143.5 (C5 ), 148þ.0 (C8a), 148.9 (C8 &
0
C2 ), 164.6 (C]O). MS (ESI) m/z ¼ 404.2 [MþH] .
4.3.19. (S)-2-(5-(4-Chlorophenyl)-1H-imidazol-2-yl)pyrrolidin-1-
yl)(8-methoxy-2H-chromen-3-yl)methanone (15e)
4.3.14. N-(S)-1-(5-(4-bromophenyl)-1H-imidazol-2-yl)-2-
methylpropyl)-8-methoxy-2H-chromene-3-carboxamide (14d)
Off white solid, yield 50%, m.p: 185e187 ꢂC. 1H-NMR (DMSO-d6)
Light brown solid, yield 50%, m.p: 110e115 ꢂC. 1H-NMR (DMSO-
0
0 0
d6) d: 2.0 (m, 3H, H3 , CH2-4 ), 2.28 (m, H3 ), 3.76 (m, 4H, OCH3 &
0
H2 ), 5.2 (s, 2H, OCH2), 6.9 (d, J ¼ 8.0 Hz, 2H, H7 & H5), 7.2 (m, 2H. H6
0 00 00 00 00
& H4 ), 7.46 (m, 5H, H2 , H3 , H5 & H6 ), 7.75 (s, H4), 12.08 (s, NH),
d
: 0.82 (d, J ¼ 6.4 Hz, 3H, CH3), 0.97 (d, J ¼ 6.4 Hz, 3H, CH3), 2.3 (m,
CH-(CH3)2), 3.75 (s, 3H, OCH3), 4.86 (m, 3H, OCH2 & CHeN), 6.84 (d,
J ¼ 6.8 Hz, H7), 6.91 (t, J ¼ 7.6, 8.0 Hz, H6), 6.98 (d, J ¼ 8.0 Hz, H5), 7.4
13C-NMR (DMSO-d6)
d: 24.0 (C4 ), 31.9 (C3 ), 48.5 (C5 ), 56.3 (OCH3),
0
0
0
0
0
57.0 (C2 ), 64.5 (C2), 115.2 (C7), 119.8 (C4a), 120.5 (C4 ), 122.2 (C5),
000 000 000 000 000
122.5 (C6), 126.3 (C2 & C6 ), 128.8 (C3 & C5 ), 130.6 (C1 ), 131.1 (C3
0
00
00
(s, H4 ), 7.52 (d, J ¼ 8.0 Hz, 2H, H3 & H5 ), 7.56 (s, H4), 7.7 (d,
00
00
000
00
0
J ¼ 7.2 Hz, 2H, H2 & H6 ), 8.45 (d, J ¼ 8.4 Hz, NHeC]O), 120.3 (s,
NH). 13C-NMR (DMSO-d6)
: 19.9 (CH3), 20.1 (CH3), 32.3 (C-(CH3)2),
53.5 (OCH3), 56.1 (CeNH), 64.7 (C2), 114.8 (C7), 119.2 (C4a), 120.6
& C4 ), 135.2 (C4), 142.2 (C5 ), 145.4 (C8a & C2 ), 148.2 (C8), 167.6 (C]
d
O). MS (ESI) m/z ¼ 436.15 [MþH]þ.
0
00
00
00
00
(C4 ), 122.0 (C5), 122.3 (C4 ), 122.7 (C6), 124.1 (C2 & C6 ), 126.7 (C1 ),
4.3.20. (8-methoxy-2H-chromen-3-yl)(S)-2-(5-(pyridin-3-yl)-1H-
00
00
0
0
127.9 (C3 & C5 ), 131.8 (C3), 135.2 (C4), 143.5 (C5 ), 148.0 (C8a & C2 ),
imidazol-2-yl)pyrrolidin-1-yl)methanone (15f)
149.2 (C8), 164.7 (C]O). MS (ESI) m/z ¼ 484.1 [MþH] þ
.
Orange solid, yield 40%, m.p: 138e140 ꢂC. 1H-NMR (DMSO-d6)
d
0
:
0
0
0
0
1.92 (m, 3H, H3 , CH2-4 ), 2.20 (m, H3 ), 3.36 (m, H5 ), 3.53 (m, H5 ),
0
4.3.15. (2H-chromen-3-yl)(S)-2-(5-phenyl-1H-imidazol-2-yl)
pyrrolidin-1-yl)methanone (15a)
3.78 (s, OCH3), 4.80 (m, H2 ), 5.19 (s, OCH2), 6.8 (d, J ¼ 6 Hz, H7), 6.9
00
000
(m, 2H, H5 & H6), 7.19 (s, H4 ), 7.41 (t, J ¼ 4.8, 6.0 Hz, H5 ), 7.93 (s,
Light green solid, yield 50%, m.p: 130e132 ꢂC. 1H-NMR (DMSO-
H4), 8.15 (d, J ¼ 8.0 Hz, H6 ), 8.42 (d, J ¼ 3.2 Hz, H4 ), 9.03 (s, H2 ),
000
000
000
: 2.1 (m, 4H, CH2-30, CH2-40), 3.8 (m, 2H, CH2-50), 4.83 (s, 2H,
12.82 (s, NH). 13C-NMR (DMSO-d6): 23.4 (C4 ), 31.8 (C3 ), 47.0 (C5 ),
0
0
0
d6)
d
0
0 0
55.3 (OCH3), 56.5 (C2 ), 65.4 (C2), 114.2 (C7), 119.0 (C4a), 120.4 (C4 ),
000 000 000
122.0 (C5), 122.7 (C6), 124.6 (C5 ), 131.7 (C3 ), 134.5 (C6 & C3), 136.1
OCH2), 5.15 (m, H2 ), 6.86 (d, J ¼ 1.2 Hz, H8), 6.96 (d, J ¼ 2.8 Hz, H5),
00
000
000
000
7.01 (s, H4 ), 7.24 (m, 3H, H6 & H7), 7.34 (m, 3H, H3 , H4 & H5 ), 7.47
(s, H4), 12.14 (s, NH). 13C-NMR (DMSO-d6)
d
: 25.32 (C4 ), 31.83 (C3 ),
(C4), 140.5 (C5 ), 146.1 (C4 & C8a), 147.4 (C2 ), 148.0 (C2 & C8), 166.1
0
0
00
000
000
00
(C]O), MS (ESI) m/z ¼ 403.2 [MþH]þ.
0
0
0
49.83 (C5 ), 55.53 (C2 ), 65.47 (C2), 116.05 (C8 & C4a), 121.86 (C4 ),
122.2, 124.65 (C5), 126.47 (C2 & C6 ), 128.89 (C7), 128.99 (C3 &
000
000
000
000
000
00
00
C5 ), 131.95 (C1 ), 132.01(C3), 135.0 (C4), 142.87 (C5 ), 149.73 (C2 ),
5. Biological assay studies
160.5 (C]O). MS (ESI) m/z ¼ 372.15 [MþH]þ.
5.1. Materials
4.3.16. (S)-2-(5-(4-Chlorophenyl)-1H-imidazol-2-yl)pyrrolidin-1-
yl)(2H-chromen-3l)methanone (15b)
DL-glyceraldehyde, Lithium sulfate, b-mercaptoethanol, Butyl-
Light green solid, yield 50%, m.p: 140e142 ꢂC. 1H-NMR (DMSO-
ated hydroxytoluene, NADPH, Sorbinil, Fidarestat, were procured
from Sigma Chemicals (MO, USA). All other chemicals and solvents
were of analytical grade and obtained from SRL (Mumbai, India).
d6)
d
: 1.92 (m, 4H, CH2-30, CH2-40), 3.85 (m, 2H, CH2-50), 5.0 (m, 3H,
0
OCH2 & H2 ), 6.85 (d, J ¼ 8.0 Hz, 2H, H8 & H5), 7.2 (m, 3H, H6, H7 &

G. Gopinath et al. / European Journal of Medicinal Chemistry 124 (2016) 750e762
761
5.2. Aldose reductase inhibition
a single i.p injection of STZ (35 mg/kg) in 0.1 M citrate buffer pH 4.5.
After 72 h, fasting blood glucose levels was monitored and animals
having blood glucose levels >150 mg/dl were included in the
experiment. Diabetic rats were divided into five groups; one group
(Group-II) was fed on a regular diet and considered as diabetic
control group. Whereas the other four groups (Group-III & Group-
ALR2 activity was performed at 30 ꢂC in a reaction mixture
containing 0.25 mL of NADPH (0.10 mM), 0.25 ml of sodium
phosphate buffer (0.1 M, pH 6.2), 0.1 mL of enzyme extract, 0.15 mL
of deionized water, and 0.25 mL of DL-glyceraldehyde (10 mM) as
substrate in a final volume of 1 mL. The reaction mixture, except for
DL-glyceraldehyde, was incubated at 30 ꢂC for 10 min. The sub-
strate was then added to start the reaction, which was monitored
for 4 min. ALR1 activity assays were performed at 37 ꢂC in a reaction
mixture containing 0.25 mL of NADPH (0.12 mM), 0.1 mL of enzyme
extract, 0.25 mL of sodium phosphate buffer (0.1 M, pH 7.2), 0.15 mL
IV) of rats received 5 mM and 50 mM/kg body weight) of 15e com-
pound. 15e compound was suspended 2% Tween 80 in saline, is
administered p.o. consecutively for 8 weeks and diabetic controls
receive 2% Tween 80 in saline as vehicle during this period.
Whereas Group-V & Group-VI rats were received Fidarestat 50
mM
and Sorbinil 50 M/kg body weight, respectively. Blood glucose
m
of deionized water, and 0.25 mL of sodium
as a substrate in a final volume of 1 mL. The reaction mixture,
except for sodium
-glucuronate, was incubated at 37 ꢂC for 10 min.
The substrate was then added to start the reaction, which was
monitored for 4min.
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 H2O. To correct for the nonenzymatic oxidation of
NADPH, the rate of NADPH oxidation in the presence of all the re-
action mixture components except the substrate was subtracted
from each experimental rate. The inhibitory effect of the synthetic
compounds was routinely estimated in a concentration ranges from
D
-glucuronate (20 mM)
levels were recorded once a week for all the animals after an
overnight fasting. Cataract progression in experimental rats was
monitored on a weekly basis using slit-lamp microscope (VISIL 02
Excel) and scoring of cataract formation was performed as
described previously [41]. At the end of 8 weeks, the rats were
sacrificed by CO2 asphyxiation and the lenses were dissected by
posterior approach. Animal care and experimental protocols were
approved by the University Animal Ethical Committee.
D
5.5. Statistical analysis
The results were expressed as mean SD of 5 replicates and
were subjected to one way analysis of variance (ANOVA) followed
by Post Hoc Test. To analyze this data we have used the SPSS 15.0
version. Data was analyzed by Prof. V.V. Hara Gopal, Department of
Statistics, Osmania University, Hyderabad.
1 mM to 50 mM. Most dose-response curves were generated using at
least three 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 n ¼ 5, where n is the total number of determinations.
Acknowledgement
We thank Dr. P. R. Krishna (Asst. Prof. of Chemistry, IIT-Roorkee)
for helpful discussions. We also extend our thanks to UGC, Osmania
University & CFRD-OU facilities for providing infrastructure and
HeteroDrugs Pvt. Ltd. Hyderabad for financial assistance to G.
Gudipudi.
5.3. Molecular docking studies
We docked 20 molecules and conformational searches were
performed using version 2015-3 of the Schrodinger. The results are
compared with the co-crystal structures (PDB codes: 2FZB, 1Z3N,
2DUX, 1PWM & 1AH0). The PDB ID 1Z3N & 1US0 were used for the
docking studies. The protein was prepared by using the prot prep
option by treatment like adding hydrogen, adding missing residues,
refining the loop with prime and finally minimized by using OPLS
2005 force field. The search grid was generated by picking the co-
crystal ligand upto 15 Å search area. The hydroxyl groups of
search area were allowed to move.
All the molecules were minimized by using macromodule
application. We used 1000 iterations for minimization using OPLS
2005 force field and charges were also added from force field only.
All the molecules were docked by using glide SP (standard preci-
sion) dock application. We performed flexible docking by allowing
ring conformations and nitrogens to move to possible extensions.
10 poses were generated for each ligand. The docking results are
sorted out and analyzed based on docking scores and their in-
teractions with active site residues.
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