The green synthesis and molecular docking of novel N-substituted rhodanines as effective inhibitors for carbonic anhydrase and acetylcholinesterase enzymes

Article abstract of DOI:10.1016/j.bioorg.2019.103096

Recently, inhibition effects of enzymes such as acetylcholinesterase (AChE) and carbonic anhydrase (CA) has appeared as a promising approach for pharmacological intervention in a variety of disorders such as epilepsy, Alzheimer's disease and obesity. For this purpose, novel N-substituted rhodanine derivatives (RhAs) were synthesized by a green synthetic approach over one-pot reaction. Following synthesis the novel compounds, RhAs derivatives were tested against AChE and cytosolic carbonic anhydrase I, and II (hCAs I, and II) isoforms. As a result of this study, inhibition constant (Ki) were found in the range of 66.35 ± 8.35 to 141.92 ± 12.63 nM for AChE, 43.55 ± 14.20 to 89.44 ± 24.77 nM for hCA I, and 16.97 ± 1.42 to 64.57 ± 13.27 nM for hCA II, respectively. Binding energies were calculated with docking studies as ?5.969, ?5.981, and ?9.121 kcal/mol for hCA I, hCA II, and AChE, respectively.

Full text of DOI:10.1016/j.bioorg.2019.103096

Accepted Manuscript
The Green Synthesis and Molecular Docking of Novel N-substituted Rhoda-
nines as Effective Inhibitors for Carbonic Anhydrase and Acetylcholinesterase
Enzymes
Sinan Bayindir, Cuneyt Caglayan, Muhammet Karaman, İlhami Gülcin
PII:
S0045-2068(19)30849-1
https://doi.org/10.1016/j.bioorg.2019.103096
103096
DOI:
Article Number:
Reference:
YBIOO 103096
Bioorganic Chemistry
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24 May 2019
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27 June 2019
Please cite this article as: S. Bayindir, C. Caglayan, M. Karaman, I. Gülcin, The Green Synthesis and Molecular
Docking of Novel N-substituted Rhodanines as Effective Inhibitors for Carbonic Anhydrase and
Acetylcholinesterase Enzymes, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103096
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The Green Synthesis and Molecular Docking of Novel N-substituted
Rhodanines as Effective Inhibitors for Carbonic Anhydrase and
Acetylcholinesterase Enzymes
Sinan Bayindir*1, Cuneyt Caglayan2, Muhammet Karaman3, İlhami Gülcin4,*
1Department of Chemistry, Faculty of Sciences and Arts, Bingol University, 12000-Bingöl, Turkey
2Department of Biochemistry, Faculty of Veterinary Medicine, Bingol University, 1200-Bingöl,
Turkey
3Department of Molecular Biology and Genetics, Faculty of Arts and Science, Kilis 7 Aralik
University, 79000-Kilis, Turkey
4Department of Chemistry, Faculty of Sciences, Atatürk University, 25240-Erzurum, Turkey
*Corresponding author:
E-mail : igulcin@atauni.edu.tr, Tel:+9044223143375 (İ.G)
E-mail : sbayindir@bingol.edu.tr, Tel: +90 04262160031 (S.B)

ABSTRACT
Recently, inhibition effects of enzymes such as acetylcholinesterase (AChE) and carbonic
anhydrase (CA) has appeared as a promising approach for pharmacological intervention in a
variety of disorders such as epilepsy, Alzheimer’s disease and obesity. For this purpose, novel
N-substituted rhodanine derivatives (RhAs) were synthesized by a green synthetic approach
over one-pot reaction. Following synthesis the novel compounds, RhAs derivatives were
tested against AChE and cytosolic carbonic anhydrase I, and II (hCAs I, and II) isoforms. As
a result of this study, inhibition constant (Ki) were found in the range of 66.35±8.35 to
141.92±12.63 nM for AChE, 43.55±14.20 to 89.44±24.77 nM for hCA I, and 16.97±1.42 to
64.57±13.27 nM for hCA II, respectively. Binding energies were calculated with docking
studies as -5.969, -5.981, and -9.121 kcal/mol for hCA I, hCA II, and AChE, respectively.
Keywords: Rhodanine, aza-ylides, acetylcholinesterase, carbonic anhydrase, enzyme
inhibition, molecular docking
2

1. INTRODUCTION
In recent years, rhodanine (1, Rh) has become a very important group of heterocyclic
compounds in drug discovery [1-3]. The number of patents and scientific publications
describing an abundance of the different biological activities of rhodanine-based molecules
has been on the rise. Many natural and synthetic biological active compounds containing
rhodanine core are being used as therapeutic agents like DNA helicase and HIV-1 inhibitors
[4-6], antiviral agents [7] were shown to exhibit antibacterial and anticancer activities (Figure
1) [8].
Figure 1
Therefore, there has been an increasing interest in developing innovative synthetic
strategies for the decoration of rhodanine-cores [9,10]. Moreover, the rich chemistry of
rhodanine (1) and the importance of the heterocyclic and metal complexes being steadily
derived from this, encourage the further development of the green synthetic methods in this
field [11,12]. Furthermore, the studies over 3-amino-2-thioxothiazolidin-4-one (2) are still not
enough. While rhodanine derivatives with the exocyclic double bond at position five are
usually synthesized by base-catalyzed Knoevenagel condensation between the rhodanine and
aldehyde or ketone, it can be obtained aza-ylide (A) derivatives as well as Knoevenagel
reaction derivatives via reaction of amino-rhodanine (2) with aldehyde or ketone. The direct
synthesis of N-substituted rhodanine derivatives from 3-amino-2-thioxothiazolidin-4-one (2)
is limited. One of these studies is carried out by Al-Romaizan and co-workers [13]. In this
approach, the to N-substituted rhodanines were synthesized through ring closure reactions of
dithioic formic acid hydrazones with functional reagents in the different medium (Figure 2A).
On the other hand, a plurality of synthesized organic molecules including functional groups
such as alcohol, acid, ester and aldehyde groups have been reported as important enzyme
inhibitors [14].
Enzymes are biocatalyst and biomolecules that are targeted for drug discovery and
development due to their vital roles in many diseases [15]. Nearly half of the therapeutic
agents currently having a market share perform their therapeutic effects by activating or
inhibiting enzymes. Therefore, chemists and especially biochemists have focused on identify
and optimize drug candidates that act as enzyme activators or inhibitors that molecules that
bind to enzymes and decreases theirs activities [16,17]. Carbonic anhydrases (CAs,
E.C.4.2.1.1) catalyze the rapid conversion of water (H2O) and carbon dioxide (CO2) to a
−
proton (H+) and bicarbonate anion (HCO3 ) [18,19]. Seven different, genetically distinct CA
3

families are known to date: the ??-, ??-, ??-, ??-, ??-, n- and ??-CAs [20-22]. There are sixteen
distinct CA isoenzymes commonly recorded in mammals belongs to ??-CA family [23,24].
CO2 + H2O ⇌ H2CO3 ⇌ HCO 3‒ + H +
CA isoenzymes play an important role in multiple biochemical and physiological
processes such as pH homeostasis, acid-base regulation, calcification, gluconeogenesis,
ureagenesis, bone resorption, and tumorigenicity [25-27]. CA inhibitors (CAIs) are a class of
pharmaceuticals or chemicals that inhibit the CA isoenzymes activities. Thus, CAIs have
clinically usage as antiepileptics, diuretics and anti-glaucoma agents. They are also frequently
used in the treatment of duodenal and gastric ulcers, idiopathic intracranial hypertension,
osteoporosis and neurological disorders [28-30].
On the other hand, it was reported that Alzheimer's disease (AD) is the leading cause
of dementia and other neurodegenerative diseases [31]. Acetylcholinesterase (AChE,
E.C.3.1.1.7) is a specific cholinesterase and also is a hydrolytic enzyme of the serine class
(Figure 3). This enzyme is important for hydrolysis choline esters in the cholinergic synapses
of the somatic system and central nervous system [32-34]. The specific and potent inhibitors
of AChE cause an increase in ACh level in the brain, thereby increasing the transmission of
electrical impulses [35,36]. Therefore, diverse AChE inhibitors are used for treating AD
symptoms [37]. Recently, cholinesterase inhibitors compounds such as tacrine, donepezil,
rivastigmine and galantamine have been used for the clinical treatment of AD. Moreover the
use of these drugs is limited due to adverse effects such as gastrointestinal disturbance and
hepatotoxicity [38-40]. Therefore, the investigation of novel non-toxic inhibitor compounds
still remains important for the treatment of AD.
The synthesis of rhodanine derivatives and investigating their potential inhibitory
actions are very popular. Furthermore there is very little study over the synthesis of amino-
rhodanine derivatives, and also there is no any study investigating the effect of rhodanine
derivatives on hCA isoenzymes and AChE enzyme activity. For this purpose in the present
study, we synthesized of novel N-substituted rhodanine derivatives, which have differed
nature, by a green synthetic approach (Figure 3B) and investigated their inhibition effects on
hCA enzymes and AChE enzyme activity.
Figures 2 and 3
2. RESULTS AND DISCUSSION
4

2.1. Chemistry
As mentioned above, in this work we interested in the synthesis and enzyme activity of N-
substituted rhodanine (RhAs) derivatives starting from commercially available 3-amino-2-
thioxothiazolidin-4-one (2) and aldehydes (3a-3h). For this purpose, initially, the reaction
conditions were optimized by changing of parameters including temperature, time, catalysts
and solvents for identification favorable reaction conditions. It was determined that the most
favorable reaction conditions for the reactions were non-catalyzed reaction conditions in
ethanol at room temperature. As a result of optimization studies, the synthesis of other RhAs
derivatives was carried out under these conditions (Scheme 1). Moreover, since the synthesis
of RhAs derivative RhA-7 was synthesized at relatively a low yield (12%) at room
temperature, the synthesis of RhA-7 was carried out at the reflux temperature of ethanol.
O
H
Ar
S
S
Ar
3a-3h
S
S
NH2
N
N
N
rt, EtOH
O
2
O
RhAs
no-catalyst
OMe
OH
HO
Ar
:
OH
CH3
OMe
NO2
RhA-1
RhA-2
RhA-3
RhA-4
68%
76%
78%
71%
HO
Br
HN
NH
RhA-5
RhA-6
RhA-7
RhA-8
82%
69%
73%
84%
Scheme 1. Synthesis of N-substituted rhodanine derivatives
The bisindole alkaloids, which exhibit various interesting biological activities, are widely
found in nature, and their analogs exhibit antitumor and antifungal activities [41,42]. In
addition of mono-substituted rhodanine derivatives, we also synthesized bisindole-substituted
rhodanine derivative 4 (Scheme 2). For this purpose, firstly, we tried to obtain of target
5

compound bisindole alkaloid 4 from the reaction of two equivalents of 1H-indole-3-
carbaldehyde (3h) with one equivalent of 3-amino-2-thioxothiazolidin-4-one (2).
Unfortunately, the target bisindole-substituted rhodanine derivative 4 was obtained with a low
yield (42%) from the reaction that carried out in acid catalysis in ethanol. Upon this, the target
bisindole-substituted rhodanine derivative 4 was obtained through a synthesis approach
involving two easy and effective steps. The RhA-8 was synthesized as per the reported
procedure but without a catalyst in 84% yield. After synthesis of RhA-8, the target compound
4 was obtained from the reaction of RhA-8 with 3-amino-2-thioxothiazolidin-4-one (2)
without catalyst at the reflux temperature of ethanol in 87% yields.
O
H
N
H
S
S
3h
(2 equiv.)
S
S
N NH2
HN
NH
N N
AcOH (two drop),
reflux, EtOH
42%
O
O
2
(1 equiv.)
4
3h
(1 equiv.),
Without cat., rt, EtOH
84%
S
3h
(1 equiv.)
4
S
NH
N N
Without cat.,
reflux, EtOH
87%
O
RhA-8
Scheme 2. The synthesis of N, 5-bisindole-rhodanine derivative 4
A plausible mechanism for the reaction of one equivalent 3-amino-2-
thioxothiazolidin-4-one (2) with one equivalent of aldehydes (3a-3h) is shown in Scheme 3.
The mechanism for the formation of RhAs derivative involves classical condensation reaction
of 3-amino-2-thioxothiazolidin-4-one (2) with aldehydes (3a-3h), which have different nature
(Scheme 3A). It is supported in literature that RhA-8 is in balance with the tautomerization
product RhA-8B at high temperatures [43,44]. According to Scheme 3, we proposed a
Knoevenagel condensation reaction mechanism based on for the formation of the other
1
product 4 (Scheme 3B). The H NMR spectra of RhA-8 and 4, which have different nature,
6

are shown in Figures S3 and S4. When the 1H NMR spectra of RhA-8 and 4 are examined, it
is seen that the NH (of indole core) proton peaks are resonance at 11.00 (s, NH, 1H) ppm and
12.41 (s, NH, 1H), 12.08 (s, NH, 1H) ppm, respectively. At the same time, it is seen that
protons of -HC=N- (azaylide groups) in the structure of the target molecules RhA-8 and 4
gave resonances signals at 8.60 (s, N=CH, 1H) and 8.93 (s, N=CH, 1H) ppm, respectively.
Moreover, the most important evidence that bisindole-substituted rhodanine derivative 4 has
been obtained is the disappearance of peak, which is resonance 4.16 (s, CH2, 2H) ppm, in the
structure of RhA-8 and the presence of a new exogenous =CH peak in 8.15 (s, =CH, 1H)
ppm. Detailed procedures and characterization can be found in the experimental section.
(A)
RhAs
Proposed formation mechanism for
O
S
N
H
O
Ar
H
N
S
O H
H
O
H+
3a 3h
-
Ar
-H2O
RhAs
2
O
H2N N
Ar
S
S
(B)
4
Proposed formation mechanism for
O
O
H
O
H
O
H
N
H
N
N
N N
HN
HN
S
NH
S
-H2O
heat
4
RhA-8
S
S
RhA-8B
6
Scheme 3. Proposed formation mechanism for RhAs and 4
2.2. Biochemical Results
In addition to synthesis of new N-substituted rhodanine derivatives, we also investigated the
inhibition effects of RhAs on hCA I, and hCA II isoenzymes and AChE enzyme under in
vitro conditions. The hCAs isoenzymes have been the subject of many kinds of research since
the biological importance in many living organisms has been discovered [45]. Recently, these
isoenzymes have become an attractive target for the design of novel activator and/or
inhibitors compounds as previously described [46]. Thus, the inhibition act of the CA
isoforms is noteworthy aims for several clinical applications such as antitumor, antiobesity,
diuretics, antiglaucoma drugs and anticonvulsant factors/diagnostic tools [47,48].
7

The hCA I isoenzyme plays an important role in retinal and cerebral edema, and the
inhibition of this isoenzyme can be a worth factor to reduce these adverse side effects [49].
The hCA I isoenzyme was strongly inhibited by the rhodanine core 2 and novel rhodanine
derivatives (4 and RhAs) with Ki values were found between 43.55 ± 14.20 and 89.44 ±
24.77ꢀnM. Furthermore, RhA-8 showed the most effective hCA I isoenzyme inhibition
properties with Ki value of 43.55 ± 14.20 nM as competitive inhibition. The control and
clinically used drug acetazolamide (AZA) demonstrated a Ki value of 110.11±10.13 nM.
Thus, the investigated novel rhodanine derivatives showed better inhibitory profiles compared
to the AZA (Table 1 and Figure 4). Therewithal, the physiologically dominant isoform hCA II
is mostly associated with several diseases such as osteoporosis, epilepsy, glaucoma and renal
tubular acidosis [50]. The hCA II was also efficiently inhibited by the rhodanine core 2 and
novel rhodanine derivatives (4 and RhAs) investigated in this study. These compounds had
powerful hCA II inhibition with Ki values ranging from 16.97±1.42 to 64.57±13.27 nM.
These values are better than those of the clinically used drugs AZA (Ki of 116.97±13.34 nM).
The RhA-1 is a competitively inhibits cytosolic dominant hCA II isoenzyme and has seven
times more effective than AZA in inhibiting hCA II isoenzyme (Table 1 and Figure 4).
Table 1
AChE inhibitors such as tacrine, galantamine, donepezil and rivastigmine have been
approved by the Food and Drug Administration (FDA) for the treatment of AD [51,52].
Moreover, all of these drugs don't achieve significant improvement due to the side effects,
such as nausea, diarrhea, vomiting and anorexia [53, 54]. Therefore, it is urgent to find novel
compounds, which have low toxicity and high AChE inhibitory activity. In this study, the
AChE inhibition profiles of the rhodanine-based azaylide compounds evaluated here were
quite interesting. Most of these compounds effectively inhibited AChE enzyme activity at the
nanomolar level. The 3-amino-rhodanine (2) and novel rhodanine derivatives (4 and RhAs),
effectively inhibited AChE enzyme with Ki values in the range of 66.35±8.35 to
141.92±12.63 nM. Also, tacrine (TAC), used as a standard AChE inhibitor in this study,
demonstrated Ki value of 145.19±17.53ꢀnM toward AChE. Furthermore, it was found that the
most potent AChE inhibition observed in compound of RhA-5 as competitive inhibition. Ki
value of RhA-5 was found as 66.35±8.35 nM. On the other hand, Ki value was calculated as
145.19±17.53ꢀnM for TAC as clinical used AChE inhibitor (Table 1).
Figure 4
In order to understand hCAs inhibition mechanism by novel rhodanine derivatives
should be reminded that the active domain of CAIs contains an active Zn2+ ion site, which is a
strong Lewis acid that binds to and activates a substrate water molecule to catalyze the
8

reversible hydration reaction of carbon dioxide. According to this, the Zn2+ ion is situated at
the bottom of the active site, being coordinated by three histidine residues (His 94, His 96 and
His 119) and H2O molecule [55]. On the other hand, the sulfonamide derivatives are the most
important inhibitors of hCAs. According to researches, on the basis of X-ray crystallographic
structures of sulfonamide inhibitors, it has been reported that the deprotonated sulfonamide
was coordinated to the Zn2+ ion of the enzyme [56,57]. In this context, it was reported that the
novel organic ligands, which are containing similar groups with sulfonate amide, have also
shown similar inhibition effects. There are also a large number of heteroatoms in the structure
of the new rhodanine derivatives synthesized in this study. It is observed that studies between
new rhodanine derivatives and hCAs and AChE enzymes have a significant inhibitory effect
against enzymes compared to literature data. In the light of all these data, the mechanism of
the interaction between the RhA-1 and hCA II enzyme is proposed as in Figure 5. According
to this, its NH moiety of RhA-1 participates in an active Zn2+ ion site is engaged, and as a
result, the enzyme is inhibited. Molecular docking studies were conducted to confirm this
proposed mechanism, which is in the light of literature data and the obtained results confirm
the proposed mechanism (Figure 8).
Figure 5
In recent studies, it was reported that indole bearing thiadiazole analogs and indole base
oxadiazole derivatives had β-glucuronidase inhibition effects. Also, oxindole-based chalcones
demonstrated antiglycation potentials against glycation of proteins. Additionally, Schiff bases
of tryptamine act as potent inhibitors of nucleoside triphosphate diphosphohydrolases [58].
2.3. Molecular Docking Studies
Following in vitro studies, we carried out molecular docking studies to understand the
inhibition mechanism of most active RhAs on hCA I, hCA II, and AChE receptors. We firstly
prepared ligands and receptors for docking studies. Then, binding site of prepared receptors
was detected. SiteScores were calculated to determine whether binding sites are catalytic
active site. SiteScores of the receptors were 1.063, 0.971, and 1.090 for hCA I, hCA II, and
AChE receptors, respectively. The scores indicated that predicted binding sites exhibited
catalytic active sites properties. Moreover, Dscores were calculated to understand whether
catalytic active sites are druggable. Dscore of the receptors were 1.061, 0.945, and 1.113 for
hCA I, hCA II, and AChE receptors, respectively. According to the Dscore, catalytic active
sites of the receptors are druggable. We also used a catalytic active site to evaluate docking
hits as seen in Figure 6.
9

Figure 6
The ADME results of the selected RhAs were analyzed by comprising to properties of
95% of known drugs and demonstrated in Table 2. Molecular weight, partition coefficient,
hydrogen bond acceptors (AHB) and hydrogen bond donors (DHB) of the RhAs are
compatible with Lipinski's rule. Moreover, IC50 values for HERG K+ channel blockage
(logHERG) were below 25.0 for each selected RhAs. Brain/blood partition coefficient of
selected RhAs was noteworthy range. The selected RhAs can easily diffuse through the cell
membrane because of their MDCK values. Also, human serum albumin binding value was
slightly below an acceptable range. Lastly, the molecules can be easily absorbed by orally
because of their high percentage of human oral absorption.
We carried out docking process of co-crystallized ligands and most active RhAs into
the predicted catalytic active site of the receptors. We began by checking accuracy of the
docking methodology before selected RhAs were docked into receptors. Docking validation
results shown in Figure 7. When the figure was examined, co-crystallized ligands and re-
docked ligands very closely located into the catalytic active site of the receptors. The results
revealed the accuracy of the docking process.
Figure 7
Moreover, we calculated the binding score of selected RhAs on the receptors. The
binding scores are presented in Table 3. The scores have shown that selected RhAs, especially
RhA-5, have good binding affinity against their receptors compared to standard inhibitors’
binding affinity of the receptors. Best-scored ligand into catalytic active sites was approved as
best pose of the ligand. 2D interaction diagrams of best-posed ligands were illustrated in
Figure 8. Indole ring of RhA-8 was formed hydrogen bond with Pro 201 residue and
interacted through π-π interaction with Hie64 and His 200 residues of hCA I receptor (Figure
8a). Detailed binding mode of RhA-8 has shown that indole ring formed aromatic hydrogen
bound with Pro 202 residues (Figure 9a). 4-Hydroxy-3-methoxybenzylidene moiety of RhA-1
was formed hydrogen bond with Asn 62 and Thr 200 residues. Also, 2-thioxothiazolidin
moieties were interacted with Zn 265 of hCA II (Figure 8b). Moreover, benzyl ring formed
aromatic hydrogen bond as seen Figure 9b. 4-Bromo-2-hydroxybenzylidene moiety of RhA-5
was formed hydrogen bond with Tyr124 and Hip 447 residues and interacted through π-π
interaction with Tyr 337, Phe 338, and Tyr 341 residues of AChE receptor (Figure 8c). In our
previously studies, we detected similar interaction for each receptor [46,47,59]. We think that
His 64 and His 200 residues of hCA I, His94 and Thr 200 residues of hCA II and Tyr 124,
10

Phe 295, and Tyr 337 residues of AChE are key residues for inhibition for the enzymes. Also,
in the case of AChE, residues Tyr124 and Tyr337 are located at the peripheral anionic site
while Phe 295 belongs to the active site namely the acyl pocket [60].
3. CONCLUSION
The easy synthesis of new rhodanine derivatives containing important aromatic groups and
investigating their potential inhibitory actions is very popular. In the present study, we
synthesized of N-substituted rhodanine derivatives RhAs, which have differed nature, by a
green synthetic approach (Figure 3b) and investigated their inhibition effects on hCAs (hCA I
and hCA II) and AChE enzymes activity in vitro. Initially, novel RhAs derivatives, which are
the skeleton of natural and important medicinal chemicals, were synthesized via a green
approach without catalyst. Following synthesis, we investigated their hCA I, and hCA II
isoenzymes and AChE enzyme inhibition properties. As a result of this study, new rhodanine
derivatives showed inhibition at the nanomolar levels against these enzymes. The N-
substituted rhodanine derivatives RhA-8, RhA-1 and RhA-5 were good inhibitors for hCA I,
and hCA II isoenzymes and AChE enzyme, respectively. Moreover, we identified the binding
modes of these inhibitors with molecular docking studies. In this context, synthesized by a
green and easy synthetic approach candidates for anticholinergic and antiepileptic
applications. Furthermore, it is thought that there will be a need for clinical studies before
application is recommended.
4. EXPERIMENTAL SECTION
4.1.General Information
1
All chemicals and solvents were commercially available from Sigma-Aldrich or Merck. H
NMR and 13C NMR spectra were recorded on a 400 (100)-MHz Bruker spectrometer.
Infrared spectra were recorded on a Mattson 1000 FT-IR spectrophotometer. High-
resolution mass spectrometric analysis was carried out on an Agilent 1260 Infinity Series Q-
TOF LC/MS (ESI/MS). The reaction progress was monitored by thin-layer chromatography
(TLC, 0.25 mm-thick precoated silica plates).
4.2.Synthesis of Organic Compounds
General procedure for synthesis of N-substituted-rhodanine derivatives RhAs: To a solution
of aldehydes (3a-3h, 1.0 equiv.) in ethanol (10 mL) was added slowly to the solution of 3-
amino-2-thioxothiazolidin-4-one (2, 1.0 equiv.) in EtOH. The reaction mixture was stirred at
room temperature without a catalyst for between 4h and 12h, and was monitored by TLC.
11

After, the mixture product was recrystallized from EtOH. After recrystallization, N-
substituted-rhodanine derivatives (RhAs) were obtained as follows.
(E)-3-((4-hydroxy-3-methoxybenzylidene)amino)-2-thioxothiazolidin-4-one [61]: The product
o
1
RhA-1 was obtained as yellow solid (76% yield). Mp: 160.5-161.5 C. H-NMR (400 MHz,
DMSO-d6):  9.58 (s, OH, 1H), 8.48 (s, N=CH, 1H), 7.41 (d, J = 1.6 Hz, =CH, 1H), 7.25 (dd,
J = 7.7, 1.6 Hz, =CH, 1H), 7.06 (d, J = 7.7 Hz, =CH, 1H), 4.33 (s, CH2, 2H), 3.86 (s, OCH3,
3H); 13C-NMR (100 MHz, DMSO-d6):  196.8, 170.5, 169.8, 152.3, 147.0, 123.6, 113.0,
111.8, 55.7, 34.7 (Fig. S1); ESI-MS (m/z) [M+H]+ calcd. for C11H10N2O3S2 283.02, found:
283.12; IR (KBr, cm–1): 3112 cm−1 (=C-H, aromatic H), 1717 cm−1 (C=O), 1638 cm−1 (O=C-
N-C=S), 1544 cm−1 (C-C, stretch in ring), 1439, 1332 cm−1 (C=S).
(E)-3-((4-methylbenzylidene)amino)-2-thioxothiazolidin-4-one: The product RhA-2 was
o
1
obtained as pale yellow solid (78% yield). 139-141 C. H-NMR (400 MHz, DMSO-d6): 
8.66 (s, N=CH, 1H), 7.80 (d, J = 8.0 Hz, =CH, 2H), 7.37 (d, J = 8.0 Hz, =CH, 2H), 4.35 (s,
CH2, 2H), 2.39 (s, CH3, 3H); 13C-NMR (100 MHz, DMSO-d6):  196.8, 170.6, 169.7, 143.6,
129.8, 129.1, 128.9, 34.7, 21.3 (Fig. S2); ESI-MS (m/z) [M] calcd for C11H10N2OS2 250.02,
found: 250.11; IR (KBr, cm–1): 3161 cm−1 (=C-H, aromatic H), 1729 cm−1 (C=O), 1615 cm−1
(O=C-N-C=S), 1414, 1311 cm−1 (C=S), 1129 cm−1 (C-N, stretch peak).
(E)-3-((3-hydroxy-4-methoxybenzylidene)amino)-2-thioxothiazolidin-4-one: The product
o
1
RhA-3 was obtained as yellow solid (71% yield). Mp: 152-153 C. H-NMR (400 MHz,
DMSO-d6):  9.57 (s, OH, 1H), 8.47 (s, N=CH, 1H), 7.40 (s, =CH, 2H), 7.26 (d, J = 7.5 Hz,
13
=CH, 1H), 7.07 (d, J = 7.5 Hz, =CH, 1H), 4.34 (s, CH2, 2H), 3.85 (s, OCH3, 3H); C-NMR
(100 MHz, DMSO-d6):  197.2, 170.9, 170.2, 152.7, 147.4, 124.6, 124.0, 113.4, 112.2, 56.1,
35.1 (Fig. S3); ESI-MS (m/z) [M] calcd for C11H10N2O3S2 282.02, found: 282.03; IR (KBr,
cm–1): 3191 cm−1 (=C-H, aromatic H), 1726 cm−1 (C=O), 1617 cm−1 (O=C-N-C=S), 1452,
1321 cm−1 (C=S), 1125 cm−1 (C-N, stretch peak).
(E)-3-((2-hydroxy-4-nitrobenzylidene)amino)-2-thioxothiazolidin-4-one: The product RhA-4
was obtained as yellow solid (68% yield). Mp: 169-171 oC. 1H-NMR (400 MHz, DMSO-d6):
 11.01 (s, OH, 1H), 8.97 (s, N=CH, 1H), 7.98 (d, J = 1.6 Hz, =CH, 1H), 7.61 (d, J = 7.7 Hz,
13
=CH, 1H), 6.98 (d, J = 7.7 Hz, =CH, 1H), 4.32 (s, CH2, 2H); C-NMR (100 MHz, DMSO-
d6):  197.1, 169.7, 164.8, 157.8, 136.9, 130.4, 129.6, 119.8, 110.7, 34.7 (Fig. S4); ESI-MS
(m/z) [M+H]+ calcd for C10H7N3O4S2 298.31, found: 298.38; IR (KBr, cm–1): 3151 cm−1 (=C-
H, aromatic H), 1725 cm−1 (C=O), 1632 cm−1 (O=C-N-C=S), 1421, 1311 cm−1 (C=S).
(E)-3-((4-bromo-2-hydroxybenzylidene)amino)-2-thioxothiazolidin-4-one: The product RhA-5
was obtained as yellow solid (82% yield). Mp: 221-222 oC. 1H-NMR (400 MHz, DMSO-d6):
12

 11.00 (s, OH, 1H), 8.96 (s, N=CH, 1H), 7.97 (d, J = 2.6 Hz, =CH, 1H), 7.62 (dd, J = 8.8, 2.6
Hz, =CH, 1H), 6.98 (d, J = 8.8 Hz, =CH, 1H), 4.32 (s, CH2, 2H); 13C-NMR (100 MHz,
DMSO-d6):  197.1, 169.7, 164.8, 157.8, 136.9, 129.6, 119.8, 119.2, 110.7, 34.7 (Fig. S5);
ESI-MS (m/z) [M] calcd for C10H7BrN2O2S2 330.92, found: 330.95; IR (KBr, cm–1): 3191
cm−1 (=C-H, aromatic H), 1724 cm−1 (C=O), 1618 cm−1 (O=C-N-C=S), 1427, 1315 cm−1
(C=S), 1115 cm−1 (C-N, stretch peak).
(E)-3-((pyren-1-ylmethylene)amino)-2-thioxothiazolidin-4-one: The product RhA-6 was
obtained as orange solid (69% yield). Mp: > 300 oC. 1H-NMR (400 MHz, DMSO-d6):  9.76
(s, N=CH, 1H), 8.87 (d, J = 8.5 Hz, =CH, 1H), 8.67 (d, J = 8.5 Hz, =CH, 1H), 8.43-8.34 (m,
=CH, 5H), 8.25 (d, J = 8.5 Hz, =CH, 1H), 8.16 (t, J = 8.5 Hz, =CH, 1H), 4.47 (s, CH2, 2H);
13C-NMR (100 MHz, DMSO-d6):  197.0, 170.0, 169.8, 134.0, 130.6, 130.3, 129.9, 129.8,
129.7, 127.3, 126.8 (2C), 126.6 (2C), 125.1, 124.0, 123.8, 123.3, 122.5, 34.9 (Fig. S6); ESI-
MS (m/z) [M+H]+ calcd for C20H12N2OS2 365.05, found: 365.07; IR (KBr, cm–1): 3158 cm−1
(=C-H), 1712 cm−1 (C=O), 1627 cm−1 (O=C-N-C=S), 1424, 1317 cm−1 (C=S).
(E)-3-(((1H-pyrrol-2-yl)methylene)amino)-2-thioxothiazolidin-4-one: The product RhA-7 was
obtained as a black solid (73% yield) from reaction 2 with 1H-pyrrole-2-carbaldehyde (3g) at
o
1
reflux temperature of EtOH. Mp: 156-157 C. H-NMR (400 MHz, DMSO-d6):  12.06 (bs,
NH, 1H), 8.29 (s, N=CH, 1H), 7.16 (d, J = 1.4 Hz, =CH, 1H), 6.82 (d, J = 1.4 Hz, =CH, 1H),
6.28 (t, J = 1.4 Hz, =CH, 1H), 4.32 (s, CH2, 2H); 13C-NMR (100 MHz, DMSO-d6):  196.7,
170.3, 161.5, 126.2, 125.0, 119.3, 110.5, 34.5 (Fig. S7); ESI-MS (m/z) [M+H]+ calcd for
C8H7N3OS2 326.29, found: 326.32; IR (KBr, cm–1): 3142 cm−1 (=C-H), 1721 cm−1 (C=O),
1640 cm−1 (O=C-N-C=S), 1413, 1324 cm−1 (C=S).
(E)-3-(((1H-indol-3-yl)methylene)amino)-2-thioxothiazolidin-4-one [62]: The product RhA-8
o
1
was obtained as red solid (84% yield). Mp: 204-205 C. H-NMR (400 MHz, acetone-d6): 
11.09 (bs, NH, 1H), 8.73 (s, N=CH, 1H), 8.37 (d, J = 7.7 Hz, =CH, 1H), 8.04 (d, J = 3.0 Hz,
13
=CH, 1H), 7.55 (d, J = 7.7 Hz, =CH, 1H), 7.30-7.22 (m, =CH, 2H), 4.30 (s, CH2, 2H); C-
NMR (100 MHz, acetone-d6):  197.3, 170.8, 165.9, 141.3, 135.3, 130.8, 124.4, 123.8, 122.5,
120.2, 112.9, 34.6 (Fig. S8); ESI-MS (m/z) [M] calcd for C12H9N3OS2 275.34, found: 275.38;
IR (KBr, cm–1): 3202 cm−1 (=C-H), 1718 cm−1 (C=O), 1626 cm−1 (O=C-N-C=S), 1428, 1317
cm−1 (C=S).
The synthesis of (Z)-5-((1H-indol-3-yl)methylene)-3-(((E)-(1H-indol-3-yl)methylene)amino)-
2-thioxo thiazolidin-4-one (4):
13

Procedure A. To a solution of 1H-indole-3-carbaldehyde (3h, 489.7 mg, 3.37 mmol) in
ethanol (10 mL) was added slowly to the solution of 3-amino-2-thioxothiazolidin-4-one (2,
250 mg, 1.69 mmol) in ethanol and was added to acetic acid (2 drops) as a catalyst. The
reaction mixture was refluxed overnight, and the mixture was cooled to room temperature.
The red product formed was recrystallized from ethanol, filtered, and dried in vacuo.
Compound 4 (286 mg, 42%) was obtained as red solid after recrystallization.
Procedure B. To a solution of RhA-8 (300 mg, 1.09 mmol) in ethanol (10 mL) was added
slowly a solution of 3-amino-2-thioxothiazolidin-4-one (2, 158.2 mg, 1.09 mmol) in ethanol
and was added to acetic acid (2 drops) as a catalyst. The reaction mixture was refluxed for 8h,
and was monitored by TLC. After the completion of the reaction, the mixture was cooled to
room temperature. The red product formed was recrystallized from ethanol, filtered, and dried
o
in vacuo. After recrystallization, 4 (382 mg, 87%) was obtained as red solid (Mp: > 300 C).
1H-NMR (400 MHz, DMSO-d6):  12.41 (bs, NH, 1H), 12.08 (bs, NH, 1H), 8.82 (s, N=CH,
1H), 8.22 (d, J = 7.7 Hz, =CH, 1H), 8.14-8.13 (m, =CH, 2H), 8.00-7.97 (m, =CH, 2H), 7.54-
7.52 (m, =CH, 2H), 7.31-7.22 (m, =CH, 3H), 5.96 (s, =CH, 1H); 13C-NMR (100 MHz,
DMSO-d6):  206.9, 171.1, 165.7, 157.0, 147.8, 144.8, 138.2, 137.3, 136.4, 135.6, 135.1,
130.7, 126.7, 124.2, 123.4, 122.3, 121.6, 113.3, 112.6, 111.2, 110.3 (Fig. S9); IR (KBr, cm–1):
3242 cm−1 (=C-H), 1729 cm−1 (C=O), 1612 cm−1 (O=C-N-C=S), 1414, 1315 cm−1 (C=S); ESI-
MS (m/z) [M] calcd for C21H14N4OS2 402.49, found: 402.12.
4.3.Biochemical studies
hCA I and II isoenzymes purification and inhibition studies: In this study, hCA I and II
isoenzymes were purified from fresh human blood erythrocytes using by Sepharose-4B-L-
Tyrosine-sulfanilamide affinity chromatography [63] as described previously [64]. To control
the purity of both isoenzymes, sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was applied [65]. The measurement of hCA I and II isoenzymes was
investigated by the method of Verpoorte et al. [66]. One enzyme unit of CA esterase activity
is defined as hydrolysis of 1 ??mol PNA to p-nitrophenol and acetate in 1 min [67]. This
method is based on the hydrolysis of p-nitrophenylacetate (PNA) to p-nitrophenol by CA
isoenzymes. The p-nitrophenol has a maximum absorbance at 348 nm using a
spectrophotometer (UV-1800 Shimadzu, Kyoto, Japan). Bradford method was used to
measure the protein content at 595 nm [68]. Bovine serum albumin (BSA) was used as
standard protein. Acetazolamide (AZA) was used as a positive control for hCA I, and II
isoenzymes. The IC50 values were calculated from activity (%) versus compounds plots. To
14

obtain Ki values, three different concentrations were used. The Lineweaver-Burk graphs were
drawn and calculations were realized.
Determination of AChE activity and inhibition studies: AChE activities were measured
according to the spectrophotometric method of Ellman et al. [69]. AChE enzyme obtained
from Electrophorus electricus (C3389) was supplied from market (Sigma Aldrich chemical
Company, Germany). 5,5′-dithio-bis(2-nitro-benzoic) acid (DTNB) was utilized for the
measurement of the AChE activity. As the substrate of the enzyme AChE in the enzymatic
reaction, acetylcholine iodide (AChI) was used. Briefly, 100 µL of buffer solution (1.0 M
Tris/HCl pH 8.0) and different concentration of sample solutions (5-100 µL) dissolved in pure
water were added to 15 µL of AChE enzymes solutions (5.32×10−3 EU). Then 50 µL of
DTNB (0.5 mM) was added. Subsequently, the reaction was initiated by the addition of 50 µL
of AChI (10 mM). Activity of this enzyme was performed spectrophotometrically at a
wavelength of 412 nm. One AChE enzyme unit is the amount of enzyme that hydrolyzes 1.0
??mol of ACh to choline and acetate per minute at pH 8.0 at 37oC [70].
4.4.Molecular Docking Studies
Receptor and ligand were prepared using the Small Drug Discovery Suit Package of Maestro
11.9. The x-ray crystal structures of CAI, CAII, and AChE receptors (PDB ID: 4WR7,
5AML, and 4M0E, respectively) were downloaded from RCSB Protein Data Bank. The
crystal structures were chosen so that they have high resolution and best percentile rank. The
crystal structures were repaired and prepared in three steps. (I) Bond order and charges were
assigned and subsequently, all missing hydrogen atoms were attached to the crystal structure.
(II) Missing side chains on the structure were filled. (III) amino acids were ionized at pH
7.0±2.0, water molecules that were formed less than 3 contacts with the protein or ligand were
removed and energy was minimized. 2D structures the most active compounds were sketched
and following their 3D structures were created with Maestro 11.9. Their correct molecular
geometries and protonation state at 7.0±2.0 were prepared [47,59,71].
Potential binding site of the receptors was predicted and Sitescore and Dscore of the
binding site were calculated using SiteMap module at Maestro 11.9. Catalytic active site
properties of the binding site were designated using these score [46,59,72]. ADME analysis
was carried out using QikProp module in Maestro11.9. The analysis provides information
about pharmacokinetic properties including absorption, distribution, metabolism, and
excretion of the compounds. The drug-likeness of the compounds was evaluated on basis of
Lipinski’s rule of five (mol MW, logPo/w, donorHB, andaccptHB) [46,73,74].
15

Docking studies were carried out at three steps using Induced Fit Docking module at
Maestro 11.9. The same module was used for the accuracy of the docking process. (I) The
centroid box was generated around the selected co-crystallized ligand in the catalytic site. (II)
The side chains were automatically trimmed based on B-factor. (III) The closest residues to
the ligand were refined within 3.4ꢀÅ of ligand pose. The accuracy of the docking process was
tested by re docking co-crystallized ligand into the active site of the receptors [46,59,75].
Acknowledgement
We are grateful to the Department of Chemistry and Bingöl University for financial support
for this work.
Conflicts of Interest
The authors declare no conflict of interest.
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24

Table 1. The inhibition values of novel N-substituted rhodanines RhAs against human carbonic anhydrase isoenzymes I and II (hCA I
and II), and acetylcholinesterase (AChE)
IC50 (nM)
Ki (nM)
hCA II
Compound
hCA I
r2
hCA II
r2
AChE
r2
hCA I
AChE
s
2
73.00
57.75
63.50
64.35
64.26
62.06
69.30
60.56
53.30
64.35
118.32
-
0.9603
0.9851
0.9770
0.9755
0.9610
0.9706
0.9859
0.9793
0.9417
0.9742
0.9887
-
57.75
38.50
63.00
49.50
49.60
36.47
55.77
69.45
46.20
69.30
112.18
-
0.9743
0.9860
0.9877
0.9817
0.9795
0.9746
0.9840
0.9750
0.9994
0.9806
0.9763
-
99.00
86.46
118.32
115.50
113.65
86.62
115.53
98.45
103.65
98.34
-
0.9748
0.9828
0.9861
0.9773
0.9686
0.9796
0.9038
0.9912
0.9901
0.9822
-
47.78±10.21
50.06±5.45
83.35±5.98
50.43±12.94
76.71±11.55
51.96±6.35
78.06±13.17
89.44±24.77
43.55±14.20
44.73±6.66
110.11±10.13
-
52.56±14.88
16.97±1.42
56.58±2.86
54.38±5.48
56.81±7.14
31.86±7.02
64.57±13.27
54.73±7.75
46.61±10.28
55.57±8.26
116.97±13.34
-
80.30±6.92
98.70±13.78
141.92±12.63
94.98±5.75
90.20±10.90
66.35±8.35
96.15±3.15
86.26±11.25
79.55±4.80
80.04±4.21
-
RhA-1
RhA-2
RhA-3
RhA-4
RhA-5
RhA-6
RhA-7
RhA-8
4
AZA*
TAC**
162.56
0.9664
145.19±17.53
*AZA (acetazolamide) was used as a positive control for human carbonic anhydrase I and II isoforms (hCA I and II).
**TAC (tacrine) was used as a positive control for acetylcholinesterase (AChE).
25

Table 2. Pharmaceutically properties of the most active RhAs
Compounds
aMW
bDHB
cAHB dlogPo/w elogHERG flogBB gMDCK hlogKhsa i% Hum. Oral Abs.
RhA-1
RhA-5
RhA-8
282.332
331.201
275.343
1
1
1
5.50
4.75
4.00
2.051
2.552
2.872
-4.314
-4.505
-4.889
-0.505 1510.730
-0.215 4840.722
-0.174 2781.760
-0.330
-0.221
-0.009
90.778
94.648
100.000
a Molecular weight (<500 Da).
b Number of hydrogen bond donors (<5).
c Number of hydrogen bond acceptors (<10).
d Octanol/water partition coefficient (recommended range: -2.0 to 6.5).
e IC50 value for blockage of HERG K+ channels (acceptable range: above 25.0).
f B/blood partition coefficient (recommended range: –3.0 to 1.2)
g Cell permeability in nm/sec (<25 is poor and >500 is great).
h Binding to human serum albumin (acceptable range: 21.5 to 1.5).
i Percentage of human oral absorption (<25% is poor and >80% is high).
26

Table 3. Binding scores (kcal/mol) of the most active compounds in the catalytic sites of human carbonic
anhydrase isoenzymes I and II (hCA I and II), and acetylcholinesterase (AChE)
IFD Glide Score
Compounds
hCA I
hCA II
AChE
RhA-1
RhA-5
RhA-8
AZA*
TAC**
-
-5.981
-
-
-
-9.121
-5.969
-9.016
-
-
-9.560
-
-
-
-9.579
*Acetazolamide (AZA) was used as a standard inhibitor for human carbonic anhydrase isoenzymes I, and II (hCA I, and II).
** Tacrine (TAC) was used as a standard inhibitor for acetylcholinesterase (AChE) enzyme.
27

FIGURE LEGENDS
Figure 1. Structures of rhodanine (1), 3-amino-2-thioxothiazolidin-4-one (2) and some rhodanine-based
medicines
Figure 2. Strategies for the synthesis of N-substituted rhodanines
Figure 3. Hydrolyzation reaction of acetylcholine in the presence of acetylcholinesterase (AChE) enzyme
Figure 4. Determination of Lineweaver-Burk graphs for excellent inhibitors of hCAs (hCA I and hCA II) and
acetylcholinesterase (AChE)
Fig. 5. Proposed hCA I inhibition mechanism by RhA-1
Figure 6. Catalytic active sites; (a) RhA-8-hCA I, (b) RhA-1-hCA II, and (c) RhA-5-AChE. Catalytic active
sites are represented as grey mesh, hydrophilic site is represented as green surface, hydrophobic site is
represented as yellow surface, and metal binding site is represented as purple surface.
Figure 7. Docking methodology test. (a) 3TV-hCA I, (b) 51J-hCA II, and (c) 1YL-AChE. The poses of co-
crystallized ligands are represented in black color ball and stick modelling while that of docked
ligands is represented in pink color ball and stick mode.
Figure 8. 2D receptor-ligand interaction diagram; (a) RhA-8-hCA I, (b) RhA-1-hCA II, and (c) RhA-5-AChE.
Figure 9. 3D detailed binding mode of the most active RhAs into hCA I, hCA II, and AChE receptors. (a) Best-
pose of RhA-8 into catalytic active site of hCA I, (b) Best pose of RhA-1 into catalytic active site of
hCA II, and (c) Best-pose of RhA-5 into catalytic active site of AChE. Receptors are presented in the
ribbon model. RhAs are represented in ball and stick modelling which orange color carbon and
residues are represented in thick tube modelling which hCA I with cyan color, hCA II with purple
color, and AChE with grey color.
28

O
S
S
H3CO
HO
NO2
N
S
S
S
NH
N NH2
S
OCH3
O
O
1
2
DNA helicase inhibitor
N
HO
HN
N
N
NH
O
H3CO
H3CO
O
N
S
Br
N
N
O
S
S
OCH3
S
ATPase DDX3 inhibitor
HIV-1 gp41 inhibitor
Figure 1
29