Synthesis, antimicrobial study, and molecular docking simulation of 3,4-dimethoxy-β-nitrostyrene derivatives as candidate ptp1b inhibitor

Article abstract of DOI:10.3390/scipharm88030037

A derivative series of 3,4-dimethoxy-β-nitrostyrene was synthesized through nitroaldol reaction, including a new compound of 3,4-ethylenedioxy-β-bromo-β-nitrostyrene. The antimicrobial activity effect of 3,4-alkyloxy modification of β-nitrostyrene was investigated. A molecular docking study was also performed to obtain information about their interactions with protein tyrosine phosphatase 1B (PTP1B). The active residues of cysteine-215 and arginine-221 of PTP1B play a key role in signaling pathways that regulate various microorganism cell functions. It also acts as a negative regulator in signaling pathways of insulin that are involved in type 2 diabetes and other metabolic diseases. These derivatives exhibited potential antifungal activity. The studied compounds were also had potential as fragments to be PTP1B inhibitors by interacting with serine-216 and arginine-221 residues, according to their molecular docking. 3,4-Ethylenedioxy-β-methyl-β-nitrostyrene was the most successful potential candidate as a PTP1B inhibitor. However, further research is needed to investigate their potential for medicinal use.

Full text of DOI:10.3390/scipharm88030037

Scientia
Pharmaceutica
Article
Synthesis, Antimicrobial Study, and Molecular
Docking Simulation of 3,4-Dimethoxy-β-Nitrostyrene
Derivatives as Candidate PTP1B Inhibitor
Salman Alfarisi 1, Mardi Santoso 2, Alfinda Novi Kristanti 1, Imam Siswanto 1
and Ni Nyoman Tri Puspaningsih 1,3,
*
1
Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Kampus C-UNAIR,
Mulyorejo, Surabaya 60115, Indonesia; salman.alfarisi-2017@fst.unair.ac.id (S.A.);
alfinda-n-k@fst.unair.ac.id (A.N.K.); imamsiswanto@fst.unair.ac.id (I.S.)
Department of Chemistry, Faculty of Science and Data Analytics, Institut Teknologi Sepuluh Nopember,
Kampus ITS, Sukolilo, Surabaya 60111, Indonesia; tsv09@chem.its.ac.id
2
3
Proteomic laboratory, Research Center for Bio-Molecule Engineering, Universitas Airlangga,
Kampus C-UNAIR, Mulyorejo, Surabaya 60115, Indonesia
*
Correspondence: ni-nyoman-t-p@fst.unair.ac.id; Tel.: +62-0811-345-2009
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 19 July 2020; Accepted: 3 September 2020; Published: 7 September 2020
Abstract: A derivative series of 3,4-dimethoxy-
β-nitrostyrene was synthesized through nitroaldol
reaction, including a new compound of 3,4-ethylenedioxy-
β
-bromo- -nitrostyrene. The antimicrobial
β
activity effect of 3,4-alkyloxy modification of β-nitrostyrene was investigated. A molecular docking
study was also performed to obtain information about their interactions with protein tyrosine
phosphatase 1B (PTP1B). The active residues of cysteine-215 and arginine-221 of PTP1B play a key role
in signaling pathways that regulate various microorganism cell functions. It also acts as a negative
regulator in signaling pathways of insulin that are involved in type 2 diabetes and other metabolic
diseases. These derivatives exhibited potential antifungal activity. The studied compounds were also
had potential as fragments to be PTP1B inhibitors by interacting with serine-216 and arginine-221
residues, according to their molecular docking. 3,4-Ethylenedioxy-β-methyl-β-nitrostyrene was the
most successful potential candidate as a PTP1B inhibitor. However, further research is needed to
investigate their potential for medicinal use.
Keywords: 3,4-dimethoxy-β-nitrostyrene derivatives; antimicrobial agent; PTP1B; molecular docking
1. Introduction
The frequency of invasive, systemic fungal infections has increased dramatically, with worsening
disease severity and higher incidence rates worldwide. Unfortunately, few antifungal drugs that are
currently available for the treatment of systemic mycoses are ideal in terms of antifungal spectrum,
efficacy, and safety. Many classes of drug structures are currently used to fight fungal infections, such as
azoles, polyenes, pyrimidine analogs, and echinocandins (Figure 1). The increase in incidences of
fungal infections has been accompanied by an increase in antifungal drug resistance. This is exacerbated
by the fact that a new generation of antifungals is lagging behind the rate at which fungal infections
develop [1,2]. This encourages the widening of the hunt for the development of new antifungal
agents from several derivative compounds. One of which is the possibility of developing drugs from
β-nitrostyrene derivatives.
Compounds containing a
Several -nitrostyrene compounds have recently been examined as anticancer candidates. The identified
pharmacophore is the nitroethenyl side chain of the aromatic ring. Research on the antifungal and
β-nitrostyrene fragment have been described as having bioactivity [3].
β
Sci. Pharm. 2020, 88, 37; doi:10.3390/scipharm88030037
www.mdpi.com/journal/scipharm

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antibacterial activities of this frame of structures during the most recent decade is rather rare,
even though studies in this field have been conducted since the 1940s. In many cases, the -nitrostyrene
structures were known to be more active against Gram-positive bacteria than other types of bacteria [ ].
β
3
OH
NH2
OH
N
OH
N N
OH
H
N
HO
HO
F
N
N
O
O
O
O
N
N
O
F
OH
NH
HO
(b)
NH
O
O
OH
NH2
O
OH
OH
O
NH
OH
HO
O
OH OH OH
OH
O
(a)
OH
O
O
HO
OH
(c)
NH2
Figure 1. Representative antifungal drugs from several classes. (a) Caspofungin; (b) Fluconazole;
(c) Nystatin.
Park and Pei argued that β-nitrostyrene is a reversible tyrosine phosphatase inhibitor, inhibiting
and interfering with protein tyrosine phosphatases (PTPs) to restrain cell signaling in microorganisms.
Eukaryotic tyrosine phosphorylation controls normal cellular growth, cell-to-cell communication,
cell differentiation, cell migration, and gene transcription. Bacterial protein tyrosine phosphatases
have structural and sequential similarities to their eukaryotic counterparts but are not as specialized
as the protein tyrosine phosphatases of eukaryotes. The nitrovinyl chain of β-nitrostyrene acts as
a phosphotyrosine mimetic to inhibit protein tyrosine phosphatase 1B (PTP1B) through binding
interaction with Cys215 at the active site of the protein [4–6].
PTP1B, a member of the PTP family, is a major negative regulator of the signaling pathways they
are involved in [7]. PTP1B consists of 435 amino acids and is located at the endoplasmic reticulum,
with a molecular weight of 50 kDa and a PTP family-owned conservative sequence. The phosphatase
catalytic site of PTP1B is localized along the sequence of residues from histidine 214 to arginine 221 in
the p fold, in which the critical residues of its active sites are Cys215 and Arg221 [8].
Based on the diversity of PTP1B inhibitory mechanisms, several classes of synthetic compounds
and natural products have been characterized for development as therapeutic agents [9]. Even so,
inventing selective PTP1B inhibitors that possess good pharmacological aspects is difficult, which is
why there are no PTP1B-qualified inhibitors yet to be found in clinical trials. The basic challenge to
finding a new inhibitor structure for PTP1B involves its acting as a phosphotyrosine mimetic with
adequate selectivity, very low toxicity and high bioavailability [10]. Fragment-based drug design is a
new approach to determining the initial chemical starting point for drug discovery projects. The small
fragments make the optimization that follows, to create a molecule by exploring the chemical cavity in
the binding pocket, relatively easier [11]. For this reason, small molecules of PTP1B inhibitors could
be promising candidate drugs in the design of new therapeutic agents against bacterial or fungal
infections. Clearly, β-nitrostyrene derivatives can be considered as fragments for the design of drugs.

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Computer-aided docking is a significant tool for understanding binding interactions between
a ligand and its protein target. It is a sophisticated method for drug design, and small molecules
docking to the known protein structures has become an integral part of the drug discovery process [12].
In the last few years, various virtual screening approaches have been developed to predict the inhibitor
activity of PTP1B. Moreover, studies of molecular docking to representative inhibitors have been
conducted to investigate the binding pose of inhibitors at the active sites of PTP1B. The results of
docking studies indicate that the studied compounds form hydrogen bonds with residues such as
Arg24, Tyr46, Asp48, Asp181, Ser216, Gly220, Arg221, Arg254, and Gln262 [13,14]. To date, studies on
the molecular docking of β-nitrostyrene derivatives against PTP1B have not been carried out.
PTP1B also plays a key role as a negative regulator in the insulin-signaling pathways involved in
type 2 diabetes. The enzyme interacts with insulin receptors as well as substrate-1 of insulin receptors,
thereby dephosphorylating phosphotyrosine residues and effecting down-regulation of insulin action.
Type 2 drives the global pandemic, with an ever-increasing numbers of patients, accounting for 90%
of all diabetes cases [
of cancer, tumor and in inflammation processes [16
candidate for the therapy of type 2 diabetes and other metabolic diseases.
Several studies have reported that -nitrostyrene derivatives were successfully prepared for
biological purposes. However, only a few studies were related to the bioactivity effect of side chain
conformation of the aromatic ring of -nitrostyrene compounds, and a study of alkyloxy chains has
not yet been conducted. In the present work, several 3,4-dimethoxy- -nitrostyrene derivatives were
synthesized as antimicrobial agents that correlate with inhibition of PTP1B (Figure 2). In this series,
the alkyloxy chain at position 3,4 of -nitrostyrene was modified. Modifications of substituent at
-carbon of -nitrostyrene were also made to investigate the effect of structural changes on their
9
,15]. In addition, PTP1B overexpression has been implicated in the signaling
–
18]. Therefore, PTP1B is also an attractive drug
β
β
β
β
β
β
activity. The antimicrobial activity of the derivatives was assessed and docking simulations of them
into the catalytic site of PTP1B were also performed to compare the results.
H3CO
H3CO
NO2
H3CO
H3CO
NO2
H3CO
H3CO
NO2
NO2
NO2
Br
3
6
2
5
1
4
O
NO2
NO2
O
O
NO2
NO2
O
O
Br
Br
O
O
O
O
O
O
O
9
7
8
Figure 2. 3,4-Dimethoxy-β-nitrostyrene derivatives synthesized in the study. The number (1–9) was
used to represent the name of the compounds.
2. Materials and Methods
The major chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). Thin layer
chromatography and column chromatography were performed using Merck 60 F254 silica gel and
60 (70–230 mesh) silica gel, respectively (Darmstadt, Germany). FT-IR spectroscopy was conducted
using a Bruker ALPHA spectrometer with Ge-ATR (Billerica, MA, USA). 1H- and 13C-NMR analysis
was carried out using a JEOL JNM ECS-400 spectrometer (Tokyo, Japan) in deuterated chloroform
at 400 MHz. Mass spectroscopy was performed on an Agilent 5977B MSD-7890 GC System (Santa
Clara, CA, USA). High Resolution Mass Spectrometry (HRMS) was carried out using Waters Xevo

Sci. Pharm. 2020, 88, 37
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QToF MS (Milford, MA, USA). Melting points were measured using a Fisher-Johns apparatus and are
uncorrected. All commercial reagents and solvents used were of analytical grade.
2.1. Synthesis of 3,4-Dimethoxy-β-Nitrostyrene Derivatives
2.1.1. Synthesis of β-Nitrostyrene Series
The methods of Mee et al. [19], were slightly modified and adapted for the synthesis of the
compounds. The corresponding aldehyde was dissolved in stirred glacial acetic acid. Ammonium
acetate and nitromethane (p = 1.14 g/cm3) were added, then the mixture was refluxed while stirring
for 2 h at 50 ◦C (reaction progress monitored by TLC). The dark orange mixture was cooled to room
temperature, and distilled water was added. The crude orange product was filtered off, washed with
water, then extracted with dichloromethane. The phase of organic extract was dried over MgSO4,
filtered, and evaporated under a vacuum. The yellow solid was recrystallized twice from ethanol to yield
the product, with the melting point being determined and identified through spectrometric analysis.
Compound 1: A mixture of 3,4-dimethoxybenzaldehyde (2.29 g, 14 mmol) and nitromethane
(15.96 g, 256 mmol) with ammonium acetate (3.70 g, 48 mmol) in glacial acetic acid (5 mL) was reacted,
and light yellow crystals were obtained, 1.48 g, 50.5% yield, mp 139 ◦–141 ◦C. FT-IR (ATR;
1491 (asymmetric NO2), 1358 (symmetric NO2); H NMR (400 MHz, CDCl3): δH (ppm): 7.94 (1H,
υ
, cm−1):
1
d, J = 13.6 Hz, H-
J = 2.0 Hz, H-2), 6.89 (1H, d, J = 8.8 Hz, H-5), 3.92 (3H, s, C3-OCH3), 3.91 (3H, s, C4-OCH3); 13C NMR
(400 MHz, CDCl3) δC (ppm): 152.9 (C-3), 149.6 (C-4), 139.4 (C- ), 135.2 (C- ), 124.7 (C-1), 122.9 (C-6),
β), 7.52 (1H, d, J = 13.6 Hz, H-α), 7.16 (1H, dd, J = 2.0, 8.0 Hz, H-6), 6.99 (1H, d,
α
β
111.4 (C-5), 110.3 (C-2), 56.2 (C3-OCH3), 56.1 (C4-OCH3); GC/MS m/z (%): 209 (M, 100), 162 (51), 119
(14), 91 (16). HRMS: C10H11NO4Na [M+Na]+; calculated: 232.0586, found: 232.0604.
Compound
(9.12 g, 146 mmol) with ammonium acetate (2.11 g, 27.4 mmol) in glacial acetic acid (3 mL) was reacted,
and light yellow crystals were obtained, 1.20 g, 72.5% yield, mp 147 ◦–149 ◦C. FT-IR (ATR; , cm−1):
1509 (asymmetric NO2), 1338 (symmetric NO2); 1H NMR (400 MHz, CDCl3): δH (ppm): 7.89 (1H, d,
J = 13.2 Hz, H- ), 7.46 (1H, d, J = 13.6 Hz, H- ), 7.06 (1H, d, J = 2.4 Hz, H-2), 7.05 (1H, d, J = 6.8 Hz,
H-5), 6.90 (1H, dd, J = 2.4, 6.4 Hz, H-6), 4.33-4.27 (2H, m, C3-OCH2), 4.33–4.27 (2H, m, C4-OCH2);
13C NMR (400 MHz, CDCl3) δC (ppm): 147.5 (C-4), 144.1 (C-3), 139.1 (C-
), 135.6 (C- ), 123.6 (C-1),
4: A mixture of 3,4-ethylenedioxybenzaldehyde (1.29 g, 8 mmol) and nitromethane
υ
β
α
α
β
123.5 (C-6), 118.4 (C-5), 117.9 (C-2), 64.8 (C3-OCH2), 64.2 (C4-OCH2); GC/MS m/z (%): 207 (M, 100), 160
(66), 89 (26), 77 (19). HRMS: C10H9NO4Na [M+Na]+; calculated: 230.0429, found: 230.0439.
Compound
(9.12 g, 146 mmol) with ammonium acetate (2.11 g, 27.4 mmol) in glacial acetic acid (4 mL) was reacted,
and brownish yellow crystals were obtained, 0.80 g, 52% yield, mp 158 ◦–160 ◦C. FT-IR (ATR; , cm−1):
1492 (asymmetric NO2), 1332 (symmetric NO2); 1H NMR (400 MHz, CDCl3): δH (ppm): 7.91 (1H, d,
J = 13.6 Hz, H- ), 7.46 (1H, d, J = 13.6 Hz, H- ), 7.07 (1H, dd, J = 1.6, 7.6 Hz, H-6), 6.99 (1H, d, J = 1.6 Hz,
H-2), 6.86 (1H, d, J = 8.0 Hz, H-5), 6.05 (2H, s, C3-OCH2O-C4); 13C NMR (400 MHz, CDCl3) δC (ppm):
7: A mixture of 3,4-methylenedioxybenzaldehyde (1.18 g, 8 mmol) and nitromethane
υ
β
α
151.5 (C-3), 148.8 (C-4), 139.2 (C-α), 135.5 (C-β), 126.8 (C-1), 124.2 (C-6), 109.2 (C-2), 107.1 (C-5), 102.2
(C3-OCH2O-C4); GC/MS m/z (%): 193 (M, 92), 146 (100), 89 (61), 63 (31). HRMS: C9H7NO4Na [M+Na]+;
calculated: 216.0273, found: 216.0269.
2.1.2. Synthesis of β-Methyl-β-Nitrostyrene Series
The corresponding aldehyde was dissolved in stirred glacial acetic acid. Ammonium acetate
and nitroethane (p = 1.045 g/cm3) were added, then the mixture was refluxed while stirring for 2 h
at 50 ◦C (reaction progress monitored by TLC). The dark orange mixture was then cooled to room
temperature, extracted with dichloromethane, and washed with distilled water. The layer of organic
extract was dried over MgSO4 then filtered and concentrated under a vacuum. The crude orange solid
was recrystallized twice from ethanol to yield the product, with the melting point determined and
identified using spectrometric analysis.

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Compound
2: A mixture of 3,4-dimethoxybenzaldehyde (1.66 g, 10 mmol) and nitroethane (14.63 g,
200 mmol) with ammonium acetate (2.71 g, 35 mmol) in glacial acetic acid (5 mL) was reacted, and
light yellow crystals were obtained, 1.26 g, 56.5% yield, mp 66 ◦–68 ◦C. FT-IR (ATR; , cm−1): 1511
(asymmetric NO2), 1313 (symmetric NO2); 1H NMR (400 MHz, CDCl3): δH (ppm): 8.06 (1H, s, H-
),
7.08 (1H, dd, J = 2.0, 8.8 Hz, H-6), 6.94 (1H, d, J = 2.4 Hz, H-2), 6.93 (1H, d, J = 8.4 Hz, H-5), 3.93
(3H, s, C3-OCH3), 3.91 (3H, s, C4-OCH3), 2.48 (3H, s, H-
); 13C NMR (400 MHz, CDCl3) δC (ppm):
150.8 (C-3), 149.1 (C-4), 133.9 (C- ), 145.9 (C- ), 125.1 (C-1), 124.1 (C-6), 113.1 (C-5), 111.3 (C-2), 56.1
(C3-OCH3), 56.1 (C4-OCH3), 14.3 (C- ); GC/MS m/z (%): 223 (M, 100), 176 (51), 131 (25), 91 (16). HRMS:
C11H13NO4Na [M+Na]+; calculated: 246.0742, found: 246.0735.
Compound : A mixture of 3,4-ethylenedioxybenzaldehyde (0.70 g, 4.3 mmol) and nitroethane
(8.36 g, 100 mmol) with ammonium acetate (2.00 g, 26 mmol) in glacial acetic acid (5 mL) was reacted,
and light yellow crystals were obtained, 0.28 g, 30.3% yield, mp 73 ◦–75 ◦C. FT-IR (ATR; , cm−1): 1503
(asymmetric NO2), 1284 (symmetric NO2); 1H NMR (400 MHz, CDCl3): δH (ppm): 7.98 (1H, s, H-
),
6.98 (1H, d, J = 2.0 Hz, H-2), 6.96 (1H, dd, J = 2.0, 8.4 Hz, H-6), 6.91 (1H, d, J = 8.4 Hz, H-5), 4.32-4.27 (2H,
m, C3-OCH2), 4.32–4.27 (2H, m, C4-OCH2), 2.44 (3H, s, H-
); 13C NMR (400 MHz, CDCl3) δC (ppm):
146.2 (C-4), 145.5 (C-3), 133.6 (C- ), 143.7 (C- ), 125.7 (C-1), 124.4 (C-6), 119.2 (C-5), 117.9 (C-2), 64.7
(C3-OCH2), 64.3 (C4-OCH2), 14.2 (C- ); GC/MS m/z (%): 221 (M, 100), 174 (78), 103 (38), 91 (29). HRMS:
C11H11NO4Na [M+Na]+; calculated: 244.0586, found: 244.0575.
Compound : A mixture of 3,4-methylenedioxybenzaldehyde (1.50 g, 10 mmol) and nitroethane
(14.63 g, 200 mmol) with ammonium acetate (2.71 g, 35 mmol) in glacial acetic acid (5 mL) was reacted
and light yellow crystals were obtained, 0.99 g, 47.6% yield, mp 88 ◦–90 ◦C. FT-IR (ATR; , cm−1): 1508
(asymmetric NO2), 1319 (symmetric NO2); 1H NMR (400 MHz, CDCl3): δH(ppm): 8.01 (1H, s,H-
),
6.97 (1H, dd, J = 2.0, 8.0 Hz, H-6), 6.93 (1H, d, J = 1.2 Hz, H-2), 6.88 (1H, d, J = 8.0 Hz, H-5), 6.04 (2H, s,
C3-OCH2O-C4), 2.45 (3H, s, H-
); 13C NMR (400 MHz, CDCl3) δC (ppm): 149.4 (C-3), 148.3 (C-4), 133.8
(C- ), 146.2 (C-
υ
α
γ
α
β
γ
5
υ
α
γ
α
β
γ
8
υ
α
γ
α
β), 126.3 (C-1), 126.1 (C-6), 109.6 (C-2), 108.9 (C-5), 101.9 (C3-OCH2O-C4), 14.3 (C-γ);
GC/MS m/z (%): 207 (M, 83), 160 (87), 103 (100), 77 (43). HRMS: C10H9NO4Na [M+Na]+; calculated:
230.0429, found: 230.0431.
2.1.3. Synthesis of β-Bromo-β-Nitrostyrene Series
The corresponding aldehyde was dissolved in stirred glacial acetic acid. Ammonium acetate
and bromo-nitromethane (p = 2.007 g/cm3) were added, then the mixture was refluxed while stirring
for 2 h at 50 ◦C (reaction progress monitored by TLC). The dark orange mixture was cooled to room
temperature then extracted with dichloromethane and washed with distilled water. The organic
extracts were dried over MgSO4, filtered, and concentrated under a vacuum. The crude residue was
purified using column chromatography over silica gel with eluent of n-hexane-chloroform (1:4) to
yield the product with the melting point determined and identified through spectrometric analysis.
Compound
(2.01 g, 12.9 mmol) with ammonium acetate (0.70 g, 9 mmol) in glacial acetic acid (1.5 mL) was reacted,
and orange crystals were obtained, 0.31 g, 19.6% yield, mp 110 ◦–112 ◦C. FT-IR (ATR; , cm−1): 1517
(asymmetric NO2), 1307 (symmetric NO2); 1H NMR (400 MHz, CDCl3): δH (ppm): 8.63 (1H, s, H-
),
3: A mixture of 3,4-dimethoxybenzaldehyde (0.92 g, 5.5 mmol) and bromo-nitromethane
υ
α
7.59 (1H, d, J = 2.4 Hz, H-2), 7.51 (1H, dd, J = 2.0, 8.8 Hz, H-6), 6.96 (1H, d, J = 8.8 Hz, H-5), 3.95 (3H, s,
C3-OCH3), 3.93 (3H, s, C4-OCH3); 13C NMR (400 MHz, CDCl3) δC (ppm): 152.7 (C-3), 149.0 (C-4), 136.7
(C-α), 127.0 (C-1), 125.5 (C-6), 122.7 (C-β), 112.8 (C-5), 111.1 (C-2), 56.2 (C3-OCH3), 56.1 (C4-OCH3);
GC/MS m/z (%): 289 (M, 81Br, 55), 287 (M, 79Br, 56), 162 (100), 147 (45), 91 (29). HRMS, C10H10NO4NaBr
[M+Na]+; calculated: 309.9691, found: 309.9687.
Compound 6: A mixture of 3,4-ethylenedioxybenzaldehyde (0.90 g, 5.5 mmol) and bromo-
nitromethane (2.61 g, 17 mmol) with ammonium acetate (0.70 g, 9 mmol) in glacial acetic acid (2 mL)
was reacted, and light yellow crystals were obtained, 0.90 g, 57.3% yield, mp 129 ◦–131 ◦C. FT-IR (ATR;
υ
, cm−1): 1509 (asymmetric NO2), 1277 (symmetric NO2); 1H NMR (400 MHz, CDCl3): δH (ppm): 8.55
(1H, s, H-α), 7.59 (1H, d, J = 1.6 Hz, H-2), 7.39 (1H, dd, J = 2.4, 8.8 Hz, H-6), 6.95 (1H, d, J = 8.8 Hz,

Sci. Pharm. 2020, 88, 37
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H-5), 4.34–4.28 (2H, m, C3-OCH2), 4.34-4.28 (2H, m, C4-OCH2); 13C NMR (400 MHz, CDCl3) δC (ppm):
147.4 (C-4), 143.7 (C-3), 136.3 (C-α), 126.3 (C-1), 125.9 (C-β), 123.3 (C-6), 119.8 (C-5), 117.9 (C-2), 64.8
(C3-OCH2), 64.2 (C4-OCH2); GC/MS m/z (%): 287 (M, 81Br, 51), 285 (M, 79Br, 53), 160 (100), 104 (45), 76
(35). HRMS: C10H8NO4NaBr [M+Na]+; calculated: 307.9534, found: 307.9537.
Compound
nitromethane (4.82 g, 30 mmol) with ammonium acetate (1.4 g, 18 mmol) in glacial acetic acid (4 mL)
was reacted, and brown crystals were obtained, 0.61 g, 20.3% yield, mp 93 ◦–95 ◦C. FT-IR (ATR; , cm−1):
1495 (asymmetric NO2), 1295 (symmetric NO2); 1H NMR (400 MHz, CDCl3): δH (ppm): 8.58 (1H, s,
H- ), 7.62 (1H, d, J = 1.6 Hz, H-2), 7.34 (1H, dd, J = 1.2, 8.0 Hz, H-6), 6.91 (1H, d, J = 8.0 Hz, H-5), 6.08
(2H, s, C3-OCH2O-C4); 13C NMR (400 MHz, CDCl3) δC (ppm): 151.2 (C-3), 148.3 (C-4), 136.5 (C-
),
9: A mixture of 3,4-methylenedioxybenzaldehyde (1.66 g, 11 mmol) and bromo-
υ
α
α
129.1 (C-1), 125.8 (C-6), 124.1 (C-β), 109.4 (C-2), 108.9 (C-5), 102.2 (C3-OCH2O-C4); GC/MS m/z (%): 273
(M, 81Br, 32), 271 (M, 79Br, 32), 226 (81Br, 30), 224 (79Br, 30), 146 (100). HRMS: C9H6NO4NaBr [M+Na]+;
calculated: 293.9378, found: 293.9382.
2.2. Antimicrobial Assay
The minimum inhibitory concentration (MIC) for bacteria, yeast, and mold was set in
Mueller–Hinton Broth at 18 h, 24 h, and 5 d, respectively. The strains used for biological tests were:
Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger. The inoculum
was prepared by fitting a suspension to match 0.2 optical density (OD) at 600 nm. Broth microdilution
testing was adapting to National Committee for Clinical Laboratory Standards guidelines. The test
compounds were prepared in DMSO 2% at various concentrations. Microplate assays for bacterial and
yeast were performed in clear using 96-well plates. The test compounds were applied to the plates,
each containing 100 µL media, with a total volume of 200 µL per well. The microplates were incubated
24 h at 30 ◦C aerobically before the wells were read visually for turbidity. Disk diffusion assay for a
mold was performed. A swab dipped into the standardized inoculums was scratched evenly on plates
containing Mueller–Hinton (MH) agar. Suspension disks containing 20 uL of test compounds were
added to the surfaces of the inoculated plates. The plates were then incubated at 30 ◦C for 5 days to
allow for fungal growth. The zone diameter of inhibition was measured in millimeters. The positive
controls used in this assay were ciprofloxacin for bacteria, nystatin for yeast and griseofulvin for mold.
All assays were at least twice replicated. MIC results were reported as MIC (µg/mL) for standards.
The compounds were evaluated for activity. Statistical analyses were performed using MS Excel 2007.
2.3. Molecular Docking
Molecular docking was conducted using Autodock 4.2 which is supported by Autodock Tools
1.5.6 and MGL tools. The crystal structure of PTP1B (PDB ID 1XBO) was extracted from the PDB
server (http://www.rscb.org/pdb). The receptor native–ligand complex was pre-processed using UCSF
Chimera 1.14. Accelrys Discovery Studio 2.1 and Chem3D Ultra 10.0 were used for ligand preparation
and supported protein optimization. Determination of the residues in the active site was used to analyze
the dimensions of the grid box. Docking validation was undertaken by analyzing the root-mean-square
deviation (RMSD) between the native ligand structure and the residues in the active site of the protein
structure. 3,4-Dimethoxy-β-nitrostyrene derivatives were docked using Autodock 4.2 in a flexible
manner. Visualization of bonding interactions was generated using UCSF Chimera 1.14 and PyMol
2.0 [12,20].
2.4. Molecular Dynamic Simulation
Molecular dynamic simulation was performed using Amber Molecular Dynamic package program
for 100 ns simulation. A post-processing procedure was applied to calculate binding free energy
between receptor and each ligand calculation was carried out. Binding energy calculation was
carried out using MMPBSA.py module which is included in Amber Molecular Dynamic Package
Program [21,22]. Atomic partial charge was added to each atom of the receptor using molecular

Sci. Pharm. 2020, 88, 37
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mechanic (MM) methods with the AMBER ff14SB force field, while to each atom of the ligand using
semiempirical methods AM1-BCC [23].
3. Results and Discussion
3.1. Spectroscopic Analysis
3,4-Dimethoxy-β-nitrostyrene derivatives were synthesized through nitroaldol reaction (Scheme 1).
Spectroscopy analysis indicated that the spectral data from nuclear magnetic resonance and mass
spectrometry were compatible with the structures suggested. NMR and GCMS analysis of the
3,4-dimethoxy-β-nitrostyrene derivatives suggested that the β-E-nitrostyrene conformation was
predominant in most of the compounds synthesized. For example, for compound
2
the E/Z ratio is 45/2.
R1O
R2O
CHO
R1O
R2O
NO2
R3
NH4OAc, HOAc
reflux
R3
NO2
+
R1, R2 = CH3, -CH2-, -CH2-CH2-
R3 = H, CH3, Br
Scheme 1. General synthesis of 3,4-dimethoxy-β-nitrostyrene derivatives.
3.2. Antimicrobial Activity
Compound was used for the preliminary assay to investigate the antimicrobial potency of the
3,4-dimethoxy- -nitrostyrene derivatives by their activity against strains of bacteria, yeast, and mold.
The most inhibited strain was selected for the subsequent assay. The MIC result of compound is
displayed in Table 1. The second assay was used to evaluate the possible systemic anti-infective agents
of the 3,4-dimethoxy- -nitrostyrene derivatives (as the MIC for each compound), and the results are
displayed in the Table 2.
2
β
2
β
Table 1. Antimicrobial activity of compound 2 against several microbial strains.
Strain
Minimum Inhibitory Concentration (µg/mL)
Staphylococcus aureus
Pseudomonas aeruginosa
>128
>128
128
Candida albicans
Aspergillus niger
256
Ciprofloxacin (The positive control of Staphylococcus aureus)
Ciprofloxacin (The positive control of Pseudomonas aeruginosa)
Nystatin (The positive control of Candida albicans)
Griseofulvin (The positive control of Aspergillus niger)
>256
>256
20
>300
The antimicrobial assays (Table 1) indicated that compound
2
displayed potential broad
activity. Among the microbial strains, its highest activity was against Candida albicans (MIC of
128 g/mL). An analysis of the turbidity indicated that compound was also more potent against
µ
2
Staphylococcus aureus (gram-positive bacteria) than Pseudomonas auruginosa (gram-negative bacteria)
(data not shown). The MIC results of antimicrobial activity against Candida albicans for the
3,4-dimethoxy-
to 128 g/mL. Compounds
3,4-ethylenedioxy- -bromo-
Among compounds , and
β
-nitrostyrene derivatives are displayed in Table 2, with MICs ranging from 32
and were more active than compound . Between compounds , and
-nitrostyrene was the most active compound against Candida albicans.
, the 3,4-ethylenedioxy- -methyl- -nitrostyrene was clearly superior.
µ
4
7
1
3,
6
9,
β
β
2
,
5
8
β
β
Therefore, the ethylenedioxy group in position 3,4- of the benzene ring gave a stronger inhibitory
effect than the methylenedioxy or dimethoxy groups. The 3,4-dimethoxy series were the weakest

Sci. Pharm. 2020, 88, 37
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agents, and they showed almost identical results in activity against Candida albicans. Among the
3,4-ethylenedioxy series, and were more effective, but there was little difference in their activity.
Between compounds , and , 3,4-methylenedioxy- -nitrostyrene was the most active compound
against Candida albicans. Thus, the -methyl or -bromine group at the nitrovinyl chain induce the
decrease in the inhibitory effect. However, the fact that was superior to indicated that, in this case,
-methyl substitution leads to higher activity than -bromo substitution. Indeed, in previous studies,
4
5
7,
8
9
β
β
β
5
6
β
β
several antimicrobial assays were carried out with compounds
with varying MIC values [3–6].
1, 2, 7, and 8 against several strains
Table 2. Antimicrobial activity of the 3,4-dimethoxy-
β-nitrostyrene derivatives against Candida albicans.
Compound
Minimum Inhibitory Concentration (µg/mL)
3,4-Dimethoxy-β-nitrostyrene (1)
3,4-Dimethoxy-β-methyl-β-nitrostyrene (2)
3,4-Dimethoxy-β-bromo-β-nitrostyrene (3)
3,4-Ethylenedioxy-β-nitrostyrene (4)
128
128
128
32
3,4-Ethylenedioxy-β-methyl-β-nitrostyrene (5)
3,4-Ethylenedioxy-β-bromo-β-nitrostyrene (6)
3,4-Methylenedioxy-β-nitrostyrene (7)
3,4-Methylenedioxy-β-methyl-β-nitrostyrene (8)
3,4-Methylenedioxy-β-bromo-β-nitrostyrene (9)
Nystatin as positive control
32
64
32
128
128
20
The higher activity of some 3,4-dimethoxy-β-nitrostyrene derivatives might indicate that they
acted as tyrosine mimetics, interacting with tyrosine phosphatase and interrupting cell signaling in the
strains. Tyrosine phosphorylation in eukaryotes such as Candida albicans controls cell differentiation,
cell migration, and gene transcription. These enzymes are also involved in other cellular processes,
such as cell wall synthesis, the formation of hyphae, and maintenance of cellular integrity in stress
situations. Tyrosine phosphorylation are also known to be involved in mitogen-activated protein
kinase signaling cascades. The mitogen-activated protein kinase cascade in Candida albicans can trigger
the transition of budding yeast to form more invasive filamentous yeast [4,24,25].
Park and Pei proposed that the tyrosine phosphatase was PTP1B. They also argued that
β
-nitrostyrene compounds inhibit PTP1B through their interaction and formation of a covalent
complex with Cys215 residue at the active site [ ]. In order to learn more about the interaction of
3,4-dimethoxy- -nitrostyrene derivatives on their binding pocket with PTP1B, molecular docking with
4
β
conformational analysis was carried out.
3.3. Molecular Docking
Docking validation was performed to gain the best geometry of ligand–protein by re-docking the
original ligand to the receptor. The best result in RMSD analysis between the native ligand structures
and the residues of the receptor active site is 1.36 Å. The interaction between them is displayed in
Figure 3.
The results of docking analysis (Table 3) show that 3,4-dimethoxy-β-nitrostyrene derivatives form
H-bonds with the 1XBO amino acids Ser216 and Arg221 and that they were around the active site
residues of 1XBO (Figure 4).

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N
O
O
OH
O
OH
OH
O
O
(a)
(b)
Figure 3.
ligand; (
(
a
) protein tyrosine phosphatase 1B PTP1B (1XBO) amino acid residues around the original
b) the native ligand structure. The colors represent the element or atom (Red: Oxygen; Blue:
Nitrogen; Yellow: Sulphur; Orange: Phosphor; Green: Carbon of the receptor residues; Cyan: Carbon
of ligand; Dotted Yellow line: Interaction between the atoms).
Table 3. Binding energies calculated for the compounds by using Autodock 4.2.
Compound
Binding Energy (kcal/mol)
Inhibition Constant
Hydrogen Binding Interacting Residues
Native ligand
−10.35
−7.36
−8.13
−8.47
−7.77
−8.42
−8.81
−7.41
−8.02
−8.45
25.78 nM
4.04 µM
1.09 µM
615.90 nM
2.03 µM
672.70 nM
348.98 nM
3.71 µM
Arg24, Ser216, Arg221, Arg254, Gly262
Ser216, Arg221
1
2
3
4
5
6
7
8
9
Ser216, Arg221
Ser216, Arg221
Ser216, Arg221
Ser216, Arg221
Ser216, Arg221
Ser216, Arg221
Ser216, Arg221
Ser216, Arg221
1.33 µM
643.05 nM
Figure 4. Molecular docking related PTP1B inhibition by 3,4-dimethoxy-β-nitrostyrene derivatives.
Binding sites of compounds , and are represented by red, green, blue, yellow, magenta,
1,
2,
3,
4,
5
,
6,
7,
8
9
cyan, orange, tint, and gray structures, respectively. The figure was generated using PyMol 2.0.
The results of the binding energy calculation from the docking analysis of the 3,4-dimethoxy-β-
nitrostyrene derivative compounds on 1XBO showed that the new compound
structure, with a binding energy of 8.81 kcal/mol. The detailed binding site of compound
displayed in Figure 5. Four H-bonds were formed by compound with Arg221, while only one H-bond
was formed with Gly216. The nitro group of compound binds to the Arg221 residue via hydrogen
bonding interactions involving α-NH2, δ-NH, δ-NH, and ω-NH2 with bond distances of 1.915, 2.384,
6
was the best docking
−
6
is
6
6

Sci. Pharm. 2020, 88, 37
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1.837 and 1.629 Å, respectively. It also forms an H-bond with Gly216 residue involving
α
-NH2 and a
bond distance of 1.795 Å. To compare it with the inhibitory ability of compound
tyrosine, which acts as substrate of PTP1B in the living system, was also docked. The result is displayed
in Figure 6. The binding energy and inhibition constant of the substrate were 8.34 kcal/mol and 772.7
nM, respectively. It forms H-bonds with the 1XBO amino acids Ser216, Ala217, Gly220, and Arg221.
6, phosphorylated
−
(a)
(b)
in the PTP1B catalytic site. ( ) Key residues surrounding 6;
Figure 5. Docking result of compound
6
a
(b) Binding pose of 6. The colors represent the element or atom (Red: Oxygen; Blue: Nitrogen; Yellow:
Sulphur; Orange: Phosphor; Green: Carbon of the receptor residues; Cyan: Carbon of ligand; Dotted
Yellow line: Interaction between the atoms).
(a)
(b)
Figure 6. Docking result of phosphorylated tyrosine in the PTP1B catalytic site (
a) Key residues;
(
b) Binding pose. The colors represent the element or atom (Red: Oxygen; Blue: Nitrogen; Yellow:
Sulphur; White: Hydrogen; Orange: Phosphor; Green: Carbon of the receptor residues; Cyan: Carbon
of ligand; Dotted Yellow line: Interaction between the atoms).
The docking results indicate that all the docked compounds are around the active site residues of
1XBO (214–221 residues), the critical acids of catalytic sites are Cys215 and Arg221 [5]. They showed
interaction with Arg221 residue, whereas none was observed with Cys215 residue. An interaction
between a molecule with a Cys215 residue can occur via Michael addition, in which sulfhydryl groups
of cysteine act as attractive nucleophiles to the conjugated nitroalkene as acceptors [
distance between the sulfur of Cys215 and the α-carbon of 3,4-dimethoxy-β
suitable to making C-S bonds (i.e., <1.82 Å) [26
5
]. This occurs if the
-nitrostyrene derivatives is
,27]. The bond distances between them were determined
using PyMol 2.0 (Table 4), the distances are relatively large. Therefore, the compounds may not be
able to interact with Cys215. Even so, the
Cys215 than that of the others (Figure 7).
α-carbon of compounds 2 and 5 was closer to the sulfur of

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Table 4. Bond distance between
α
-carbon of all compounds and the sulfur of Cys215, calculated using PyMol 2.0.
Compound
Bond Distance (Å)
1
2
3
4
5
6
7
8
9
4.9
4.6
4.7
4.9
4.6
4.9
4.8
4.7
4.9
Figure 7. Bond distance between
β-carbon of compound 2 and thiol of Cys215, simulated using
PyMol 2.0. The colors represent the element or atom (Red: Oxygen; Blue: Nitrogen; Yellow: Sulphur;
Orange: Phosphor; Green: Carbon of the receptor residues; Cyan: Carbon of ligand; Dotted Yellow
line: Interaction between the atoms).
3.4. Molecular Dynamic Simulation
In order to validate the calculation which resulted from previous molecular docking procedure,
a molecular dynamic simulation was applied to all compound docked complexes to 100 ns. The results
of calculation of binding energies are displayed in the Table 5.
Table 5. Binding energies calculated for the compounds after 100 ns molecular dynamic simulation.
Compound
Binding Energy (kcal/mol)
1
2
3
4
5
6
7
8
9
−11.69
−20.21
−15.13
−20.52
−16.79
−12.97
−10.61
−12.60
−10.21
Compound
However, compounds
4
exhibited the best docking structure, with binding energy of
−
20.52 kcal/mol.
2
and were the promising most potential candidates for the PTP1B inhibitors
5
according to the result of binding energies from the two methods of calculation. The hydrogen bonds
of the two structures might stabilize the open shape of the enzyme and supply tighter binding to the
PTP1B active site, so that inhibition of PTP1B becomes more effective.

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3.5. Conformational Analysis
The conformational analysis showed that the side chain of the aromatic ring and substituent of the
-carbon of -nitrostyrene chain affect the direction of the nitro group so that it is in the appropriate
β
β
position to bind with hydrogen. The most stable geometries of molecular conformation before and
after molecular docking, are displayed in Figure 8. The change in direction of the nitro group relative
to the benzene was investigated by determining the dihedral angles between their planes, which are
relatively greater based on the order of the substituent in β-carbon: H < CH3 < Br (Table 6). Of course,
this corresponds to binding energy. The magnitude of the angle was calculated from the dihedral
angles of C6-C1-Cα-Cβ and Cα-Cβ-N-O.
Table 6. Dihedral angles of the nitro group relative to the benzene calculated using Chem3D Ultra 10.0.
Compound
Angle (◦)
1
2
3
4
5
6
7
8
9
41
54
37
31
49
66
34
45
61
(a)
(b)
Figure 8. (a) Conformation for each compound in the most stable geometries before molecular docking;
(b) After molecular docking. The colors represent the element or atom (Red: Oxygen; Blue: Nitrogen;
White: Hydrogen; Gray: Carbon; Dark red: Bromine)
In general, the ethylenedioxy group in position 3,4 of benzene ring provided relatively more
inhibitory effect than the methylenedioxy or methoxy groups. The results of this docking study support
the results regarding antimicrobial activity against Candida albicans. It is also possible to propose
that compound
compounds. The theoretical prediction of the toxicity of these derivatives supports the proposal that
compound is less toxic than the others. The predictions were processed using the pkCSM online tool
5 is the best candidate PTP1B inhibitor as an antimicrobial agent among the studied
5
available at http://biosig.unimelb.edu.au/pkcsm/prediction and the results are displayed in Table 7.
However, further study is required to prove this argument.

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Table 7. Toxicity data for 3,4-dmethoxy-β-nitrostyrene derivatives (acquired using pkCSM online tool).
AMES Toxicity
(Yes/No)
Max. Tolerated Dose
(Human) (Log mg/kg/day)
hERG Inhibitor
Oral Rat Acute Tox.
(LD50) (mol/kg)
Hepato- Toxicity
Skin Sensitization
Compound
(Yes/No)
(Yes/No)
(Yes/No)
1
2
3
4
5
6
7
8
9
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
0.847
0.889
0.845
0.546
0.342
0.597
0.614
0.405
0.664
No
No
No
No
No
No
No
No
No
2.170
2.211
2.464
2.289
2.382
2.316
2.352
2.432
2.345
No
No
No
No
No
No
No
No
No
Yes
Yes
No
Yes
No
No
Yes
Yes
No
4. Conclusions
The 3,4-dimethoxy-
activity, especially against Candida albicans. The molecular docking study also suggested that
3,4-dimethoxy- -methyl- -nitrostyrene and 3,4-ethylenedioxy- -methyl- -nitrostyrene have activity
as PTP1B inhibitors. Modification of the alkyloxy side chain at 3,4 position of the aromatic ring had
significant influence on antifungal activity of -nitrostyrenes. The most promising potential inhibitor
was 3,4-ethylenedioxy-β-methyl-β-nitrostyrene.
β-nitrostyrene derivatives were suggested to have potential antifungal
β
β
β
β
β
Author Contributions: Conceptualization, S.A., N.N.T.P., M.S. and A.N.K.; methodology, M.S., N.N.T.P., I.S.
and S.A.; investigation, S.A.; writing—original draft preparation, S.A.; validation, S.A., N.N.T.P., M.S., I.S.
and A.N.K.; writing—review and editing, N.N.T.P.; supervision, N.N.T.P. All authors have read and agreed to the
published version of the manuscript.
Funding: This study was funded by the Ministry of Religious Affairs (MORA), Indonesia.
Acknowledgments: Salman Alfarisi acknowledge to UNAIR Research University (Research Group Grant,
Universitas Airlangga, Rector Decree 347/UN3.14/PT/2020) and MORA, Indonesia for the PhD scholarship program.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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