Synthesis and Spectroscopic Analysis of Piperine- And Piperlongumine-Inspired Natural Product Scaffolds and Their Molecular Docking with IL-1β and NF-κB Proteins

Article abstract of DOI:10.3390/molecules25122841

Inspired by the remarkable bioactivities exhibited by the natural products, piperine and piperlongumine, we synthesised eight natural product-inspired analogues to further investigate their structures. For the first time, we confirmed the structure of the key cyclised dihydropyrazolecarbothioamide piperine analogues including the use of two-dimensional (2D) ¹⁵N-based spectroscopy nuclear magnetic resonance (NMR) spectroscopy. Prior investigations demonstrated promising results from these scaffolds for the inhibition of inflammatory response via downregulation of the IL-1β and NF-κB pathway. However, the molecular interaction of these molecules with their protein targets remains unknown. Ab initio calculations revealed the electronic density function map of the molecules, showing the effects of structural modification in the electronic structure. Finally, molecular interactions between the synthesized molecules and the proteins IL-1β and NF-κB were achieved. Docking results showed that all the analogues interact in the DNA binding site of NF-κB with higher affinity compared to the natural products and, with the exception of 9a and 9b, have higher affinity than the natural products for the binding site of IL-1β. Specificity for the molecular recognition of 3a, 3c and 9b with IL-1β through cation–π interactions was determined. These results revealed 3a, 3c, 4a, 4c and 10 as the most promising molecules to be evaluated as IL-1β and NF-κB inhibitors.

Full text of DOI:10.3390/molecules25122841

Article
Synthesis and Spectroscopic Analysis of Piperine-
and Piperlongumine-Inspired Natural Product
Scaffolds and Their Molecular Docking with IL-1β
and NF-κB Proteins
Gabriel Zazeri 1,2 Ana Paula R. Povinelli 1,2, Cécile S. Le Duff , Bridget Tang ,
,
3
3
1
,
2,
Marinonio L. Cornelio * and Alan M. Jones *
1
Departamento de Física–IBILCE, Rua Cristovão Colombo, 2265 CEP 15054-000 São José do Rio Preto–São
Paulo, Brazil; gabriel.zazeri@unesp.br (G.Z.); ana.povinelli@unesp.br (A.P.R.P.)
2
School of Pharmacy, University of Birmingham, Edgbaston B15 2TT, UK
3
School of Chemistry, University of Birmingham, Edgbaston B15 2TT, UK;
C.S.LeDuff@bham.ac.uk (C.S.L.D); B.Tang@bham.ac.uk (B.T.)
*
Correspondence: m.cornelio@unesp.br (M.L.C.); a.m.jones.2@bham.ac.uk (A.M.J.)
Academic Editor: Maged Henary
Received: 19 May 2020; Accepted: 16 June 2020; Published: 19 June 2020
Abstract: Inspired by the remarkable bioactivities exhibited by the natural products, piperine and
piperlongumine, we synthesised eight natural product-inspired analogues to further investigate
their structures. For the first time, we confirmed the structure of the key cyclised
1
5
dihydropyrazolecarbothioamide piperine analogues including the use of two-dimensional (2D) N-
based spectroscopy nuclear magnetic resonance (NMR) spectroscopy. Prior investigations
demonstrated promising results from these scaffolds for the inhibition of inflammatory response
via downregulation of the IL-1β and NF-κB pathway. However, the molecular interaction of these
molecules with their protein targets remains unknown. Ab initio calculations revealed the electronic
density function map of the molecules, showing the effects of structural modification in the
electronic structure. Finally, molecular interactions between the synthesized molecules and the
proteins IL-1β and NF-κB were achieved. Docking results showed that all the analogues interact in
the DNA binding site of NF-κB with higher affinity compared to the natural products and, with the
exception of 9a and 9b, have higher affinity than the natural products for the binding site of IL-1β.
Specificity for the molecular recognition of 3a, 3c and 9b with IL-1β through cation–π interactions
was determined. These results revealed 3a, 3c, 4a, 4c and 10 as the most promising molecules to be
evaluated as IL-1β and NF-κB inhibitors.
Keywords: molecular docking; piperine; piperlongumine
1
. Introduction
Nature is a creative machine for the design of bioactive molecules with the potential to become
efficient drugs for the treatment of several diseases. In recent decades, such creative scaffolds have
driven the design of new molecules with pharmacological potential, including the concept of
diversity-oriented synthesis (DOS) [1,2]. In recent years, both piperine and piperlongumine have
been explored because of their wide spectrum of biological activity [3–8].
Piperine is an alkaloid found in piper species such as Piper nigrum (black pepper) and Piper
longum (long pepper). Piperine is not only used as a seasoning but also in various preparations of
Molecules 2020, 25, 2841; doi:10.3390/molecules25122841
www.mdpi.com/journal/molecules

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traditional medicine, including the oldest medical science, practiced in India since ancient times
Ayurveda) [9]. Piperine has been widely studied by the scientific community, because of its anti-
(
inflammatory, anti-carcinogenic, immunomodulatory and hepatoprotective activities [3]. As an anti-
inflammatory, piperine acts through the inhibition of IL-1β and NF-κB inflammation pathway,
leading to the downregulation of pro-inflammatory proteins, such as iNOS and COX-2 [10–12].
Piperine-inspired molecules investigated in the present work have had their anti-inflammatory
and anti-carcinogenic activity previously studied by Mathew et al. [13,14], with promising results. In
particular, 4a had a comparable anti-inflammatory effect to Diclofenac. Furthermore, 3c and 4c
showed inhibitory effects on the HCT116 colon cancer cell line. However, their interactions with the
IL-1β and NF-κB pathways have not been investigated.
Piperlongumine (PPL), is an alkaloid isolated from long pepper that is widely used in Indian
traditional medicine [15]. The biological activities of PPL and its analogues include anti-
inflammatory, anti-carcinogenic and anti-atherosclerotic activities, among others [6–8,16]. Similar to
piperine, studies indicated that anti-inflammatory and anti-carcinogenic activities of PPL are a
consequence of the inhibition of the NF-κB pathway [17–20].
PPL has been shown to bind to the unit p65 of NF-κB, inhibiting NF-κB/DNA binding activity
and blocking the transcription of cytokines and other pro-inflammatory proteins [18].
Despite the investigations reported in the literature that show promising results of these natural-
product-inspired analogues in the inhibition of inflammatory response via the downregulation of IL-
1
β and NF-κB pathway, the molecular interaction of these molecules with the protein targets are
unknown.
Herein, we explored the binding environment of the synthesised analogues in NF-κB and IL-1β
using molecular docking. For piperine-inspired molecules, we investigated the effect caused by the
substitution of the piperidine for an aromatic ring in the piperine scaffold (3a), a nitroarene group
(
3b) and an aminoarene group (3c). We investigated the effect of the cyclisation of the piperine
scaffold to form a dihydropyrazolecarbothioamide moiety (4a and 4c). For PPL-inspired molecules,
we investigated the effects of removing a Michael acceptor (9a), the conversion of a lactam to a ketone
(
10) and the influence of methoxy groups (9b).
We present an adapted synthesis of piperine- and PPL-inspired molecules including data from
1
15
2
D H- N HSQC and HMBC spectra, computational characterization of the electronic structure of
the analogues using ab initio calculations and molecular docking of the confirmed structures with the
proteins IL-1β and NF-κB.
2
. Results and Discussion
2
.1. Spectroscopic Characterization of Natural Product Inspired Analogues
The synthesis of the piperine-inspired analogues began with a MnO -mediated oxidation of
2
piperonyl alcohol to the corresponding aldehyde with a 97% yield (Scheme 1). Claisen–Schmidt
condensation of 2 with a variety of acetophenones afforded 3a–3c in excellent yields (80–90%) after
crystallisation. The initial synthetic route to 4c required the synthesis of the nitro derivative (3b)
followed by cyclisation with thiosemicarbazide and reduction, as the free amine may undergo
unwanted side reactions during the cyclisation step. Unfortunately, all attempts to react 3b with
thiosemicarbazide resulted in recovered starting material. However, for the parent unsubstituted
compound (3a) a cyclised compound (4a) was formed with 60% yield. Pleasingly, the unprotected
amine containing molecule 3c was smoothly converted to the cyclised molecule 4c with 60% yield,
mitigating the need to pursue 3b further.

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Scheme 1. Synthesis of piperine-inspired molecules.
The syntheses of piperine analogues have been reported by Mathew et al. [13] but with limited
1
spectroscopic characterization ( H NMR and IR spectroscopy) of molecules of type 4. We therefore
performed re-synthesis prior to the computational study to confirm the connectivity of the new ring
system. There is a potential for either a five-membered product (4a), or a seven-membered ring
1
13
1
15
system (5) to form (Scheme 2). Selected instructive 2D H- C HMBC and H- N HMBC NMR spectra
1
5
connectivities are shown in Scheme 2, along with the N NMR chemical shifts. Critically, the results
showed the correct connectivity for a five-membered dihydropyrazolecarbothioamide system
product (4a not 5). 4a was found to slowly degrade upon standing in CDCl at room temperature for
3
an extended period of time.
Scheme 2. Two potential cyclisation outcomes upon 3a and the selected instructive NMR correlations
1
13
1
15
for 4a in the 2D H- C HMBC and 2D H- N HMBC spectra.

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The synthesis of the PPL-inspired analogues (Scheme 3) used a one-pot Doebner-modified
Knoevenagel condensation and concomitant decarboxylation, to prepare the key acrylic acids (7a,b)
from the appropriate benzaldehydes. Reaction of the acrylic acids with thionyl chloride under
Vilsmeier–Haack conditions gave the short-lived vinylogous acyl chlorides (8a,b) which were reacted
with either sodium 2-oxopiperidin-1-ide (derived from δ-valerolactam) or with lithium cyclohex-1-
en-1-olate (derived from cyclohexanone), to afford the final products 9a,b and 10 with modest
isolated yields.
1
Although 10a has been reported in the literature, we identified a previously unobserved H NMR
spectroscopic signal at 15.76 ppm (Supplementary Material), indicative of an intramolecular
hydrogen bond occurring through tautomerization to give the stabilized structure, 10.
Scheme 3. Synthesis of piperlongumine-inspired molecules.

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2
.2. Electronic Structures of Piperine and PPL-Inspired Molecules
Ab initio calculations were performed to obtain the structural parameterisation to be used in the
molecular docking calculations. In addition, these calculations provided information about the
electronic structure that contributed to the characterisation of the compounds.
Figure 1 shows the structures resulting from ab initio calculations of their respective map of
electrostatic potential (MEP). 3a,b,c presented a planar structure due to the high number of
conjugated double bonds. A similar result was found for piperine previously [21]. 3a,b,c have the
most negative charge density around oxygen atoms, while the most positive charge densities are
concentrated on the extremities of the structures with the intermediate region being more neutral. As
a special case, the nitro group in 3b relocated the negative charges from the aromatic ring to the
oxygen atoms, consequently the aromatic ring of 3b became more positive when compared to 3a,c
and may explain its unreactivity to thiosemicarbazide. This relocation of negative charges caused 3b
to have high affinity for a microenvironment mainly composed by polar amino acids, which differs
from 3a,c, which preferred less polar microenvironments (see molecular docking). The MEP of 4a,c
indicated that the negative density charge was concentrated around the oxygen, nitrogen and the
sulfur atoms, while the positive density charge was concentrated on the dihydropyrazole and 1,3-
benzodioxole rings, with the neutral region being the aromatic ring.
Figure 1. Map of electronic potential (MEP) of piperine and PPL-inspired molecules, where the scale
is from negative density charge (represented by red) to positive density charge (represented by blue).
A minimal puckering of the five-membered ring system of 4a modelled in CDCl
3
revealed a
maximal torsional angle of +4.8° around C(10)-N(14)-N(13)-C(12) and a minimal torsional angle of
1
5
−
3.0° across the ring system. This deviation away from planarity may explain the low 15 ppm N
NMR signal observed in this ring system.
9
a, 10 and 9b presented a planar structure, except for methoxy groups (see S39, Supplementary
1
Material). H NMR of 10 (Figure S38, Supplementary Material) showed a resonance peak at 15.76
ppm, associated with the hydroxyl from cyclohexanone forming an intramolecular hydrogen bond
with the adjacent carbonyl oxygen; consequently, this hydrogen is not exchangeable and the signal
1
is observed in the H NMR spectrum. According to ab initio calculations for 10, the intramolecular
hydrogen bond length is 1.55 Å. The planarity of the three structures are quite similar to the
characteristics found in PPL [22]. According to MEP, the three structures presented a negative density
charge concentrated around oxygen atoms, while the positive density distributed on the extremity of

Molecules 2020, 25, 2841
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the molecule and the aromatic ring is a neutral part of the molecules. The negative density charge
concentrated on the atoms identified plays a crucial role in the interaction of 10 with NF-κB, as the
binding site is positively charged.
2
.3. Molecular Docking
According to molecular docking results with IL-1β (Figure 2), piperine and analogues 3a, 4a, 3c
and 4c bound to the same binding site with the binding energy scores of −6.08, −6.04, −7.18, −6.25 and
−
7.22 kcal/mol, respectively.
Figure 2. IL-1β (iceblue) binding environments to (A) piperine and analogues (Piperine-cyan, 3a-
orange, 4a-purple, 3b-yellow, 3c-red, 4c-green). (B) PPL and analogues (PPL-blue, 9a-pink, 10-lime,
9
b-iceblue).
Figure 3 and Table 1 show the amino acids that compose the microenvironment of each molecule
with IL-1β. According to the docking results 4a, 3c and 4c had a more negative binding energy than
piperine; having a higher affinity for this environment when compared to piperine (Table 2). Most of
the amino acids that composed the binding site are non-polar, with limited neutral and charged polar
amino acids participating. The higher affinity of the analogues for the binding site revealed that the
addition of an aromatic ring, and consequently the new electronic distribution, contributed to the
increase in affinity. Moreover, the aromatic rings of 3a,c exhibited cation–π interactions with the
protonated amine group of Lys77 amino acid (see Supplementary Material), which contributed to the
high negative binding energy of these analogues to the protein. The cation–π interaction is caused by
the molecular electron delocalization, reinforcing the importance of the increase in electron
delocalization for the affinity at the IL-1β binding site.

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Figure 3. IL-1β binding microenvironments details for piperine and PPL analogues visualized with
LigPlot.
Table 1. Amino acids that compose the microenvironment of the complex IL-1β/piperine and PPL
analogues, obtained by molecular docking.
Amino Acids
Molecule
Positively
Charged
Negatively
Charged
Non-Polar
Polar
Thr137, Thr79
Thr79
Leu80, Leu134, Val32,
Phe133, Pro131, Trp120
Leu82, Leu26, Leu80, Leu134, Phe133,
Val132 and Pro131
Piperine
Lys77
Lys77
-
-
3
4
a
a
Glu25
Leu80,Leu82, Leu134,Val132,
Phe133, Pro78
Thr79, Gln81,
Tyr24
-
Lys88, Lys92,
Lys93, Lys94
3
b
Pro2, Pro87, Pro91
-
Asn89, Tyr90,
Tyr24, Gln81
Leu80, Leu82, Leu134, Phe133,
Pro78, Trp120, Val132, Pro131
Leu26, Val132, Phe133, Pro78,
Leu134, Leu82 Pro131, Leu80,
Val132, Phe133, Gly135, Gly136,
Trp120, Leu134, Leu80
3
4
c
c
Lys77
-
Glu25
-
Gln81, Tyr24,
Thr79
PPL
Lys77
Asp142
Thr79, Thr137

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Leu80, Trp120, Gly136, Gly135,
Phe133, Leu134
9
1
a
0
Lys77
Lys77
Lys77
Asp142
Glu25
Thr79, Thr137
Tyr24, Gln81
Gln81, Tyr24
Trp120, Leu80, Leu82, Val32,
Leu134, Phe133
Leu82, Leu80, Phe133, Leu134,
Trp120, Val132
9
b
Glu25,
As a consequence of the new structural properties, 4a and 4c presented the best affinities to this
binding site. Analogue 3b bound with a binding energy of −7.44 kcal/mol to an orthosteric binding
site, where the majority of amino acids are positive and neutral polar amino acids. The differences
between 3a,c and 3b are due to the aromatic ring substitution, the nitro group in 3b drove the
interaction to a less hydrophobic environment, differently from the amino group (3c).
The binding site found by molecular docking for PPL and analogues was the same as the
piperine binding site. Table 2 shows that PPL, 9a, 10 and 9b had binding scores of −5.83, −5.38, −6.19
and −5.62 kcal/mol, respectively. In the case of PPL analogues, 10 presented the most negative binding
energy and highest affinity for the binding site. The results showed that the removal of two methoxy
groups (9b) limited the possibility of performing multiple hydrogen bonds. Furthermore, 9b
exhibited a cation–π interaction with the protonated amine of Lys77 (see supplementary material),
which contributed to the negative binding energy. Docking results also showed that the removal of
the double bond in the cyclic ring (9a) resulted in higher binding energy (less negative) than the other
molecules, which reinforced the importance of electron delocalization in the binding affinity.
Table 2. Binding energy scores obtained for the proteins IL-1β and NF-κB with Piperine, PPL and
analogues.
Molecules Energy Score kcal/mol (IL-1B) Energy Score kcal/mol (NF-KB)
Piperine
−6.08
−6.04
−7.44
−6.25
−7.18
−7.22
−5.83
−5.38
−6.19
−5.62
−6.08
−6.53
−6.24
−6.07
−6.64
−6.4
3
a
3
b
3
c
4
a
4
c
PPL
−5.36
−5.85
−5.72
−5.87
9
1
a
0
9
b
In the case of the interaction with NF-κB, all the piperine and piperlongumine analogues, with
the exception of 3c, presented a more negative binding energy (Table 2) than the natural products.
Piperine, 4a, 3b, 3c, 4c, PPL, 9a, 10 and 9b all bound to the DNA binding site of NF-κB (Figure 4) [23].
Table 3 and Figure 5 show that the microenvironments of interaction of these molecules in NF-κB
present a significant number of non-polar amino acids (except for 3b), similar to the IL-1β binding
site. However, in the case of the NF-κB binding site, there is also a greater number of positively
charged amino acids. The interaction of the analogues with the positively charged amino acids may
play an important role in the inhibition of the interaction NF-κB(p65)-DNA, as positively charged
amino acids (including Lys122 and Lys123) stabilize the NF-κB(p65)-DNA complex via interaction
with the negatively charged phosphates groups of the DNA [24].

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Figure 4. NF-κB (iceblue) binding environments to (A) Piperine and analogues (Piperine-cyan, 3a-
orange, 4a-purple, 3b-yellow, 3c-red, 4c-green). (B) PPL and analogues (PPL-blue, 9a-pink, 10-lime,
b-iceblue).
9
Chen Y.Q. et al. [24] showed that the phenol ring of Tyr36 is anchored by a hydrogen bond to
the phosphate backbone of DNA and makes van der Waals contacts with the methyl group of
thymine at positions 1 and 2 of DNA, which makes 3a, 4a, 3c, 4c and 9b good candidates to inhibit
this interaction. Our molecular docking results revealed that Tyr36 was part of the binding
microenvironment of these analogues and performed non-specific interactions.
Moreover, the side chain of Arg187 performs a hydrogen bond with the oxygen atom of thymine,
and the side chain of Arg33 performs hydrogen bonds to oxygen and nitrogen atoms of guanine [24].
In this case, our molecular docking results showed that 3b and 4c perform hydrogen bonds with
Arg33 and Arg187, indicating promising results for these compounds as inhibitors of this interaction
between the arginine and DNA bases thymine and guanine.
It has been shown that the specificity of the interaction p65-DNA is likely due to the interactions
between the G-C base pair and the side chains of Arg33 and Glu39 [24]. The amino acid Arg33,
highlighted as being fundamental to DNA molecular recognition, is present in the
microenvironments of the analogues 3b and 9a and performs a hydrogen bond with 3b, which
indicates that these analogues may shield the DNA recognition.

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Figure 5. NF-κB binding microenvironments details for piperine and PPL analogues visualized with
LigPlot.
Table 3. Amino acids that compose the microenvironment of the complex NF-κB/piperine and PPL
analogues, obtained by molecular docking.
Amino Acids
Negatively
Molecules
Non-Polar
Positively Charged
Lys195, Arg187, Lys218
Lys37, Lys122, Arg133
Polar
Asn186,
Charged
Glu193, Glu282,
Asp217
Piperine
Ala192, Leu194
Val121, Ala129, Cys38,
Cys120
3
4
a
a
Glu89
Asp185
Tyr36, Gln132
Tyr36, His88, Asn155
Asn186,
Ala188, Cys120, Leu154,
Val121, Pro189
Arg187, Lys122, Lys123,
His88
Arg33, Arg187, Lys195,
Lys218
3b
Ala192
Asp217, Glu193,
-
Cys120, Leu154, Val121,
Pro189, Ala188, Phe34
Pro189, Leu154, His88,
Val121, Cys120, Ala188
Cys120, Phe34, Leu154,
Ala188, Pro189
His88, Tyr36, Asn155,
Gln220, Asn190, Asp185
Asp185, Tyr36, His88,
Asn155,
3
4
c
c
His88, Lys123
Lys123, Lys122, Arg187
Lys218, Arg187, His88
-
PPL
Asp185,
Asp217, Glu193
Asp185
Tyr36, Asn155
Asn186,
Lys218, Lys195, Arg33,
Arg187, Lys194
9
1
a
0
Ala188, Ala192, Pro189
Ala188, Phe34, Cys120,
Leu154, Val121, Pro189
Lys218, Arg187, His88
Asn155, His88
Tyr36, Asn155
His88, Lys123, Lys122,
Arg124
9b
Leu154, Cys120, Val121,
Asp185

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3
. Materials and Methods
All reagents and solvents were obtained commercially from Sigma-Aldrich or Fischer Scientific
and were used as supplied, with the exception of tetrahydrofuran (THF) and dichloromethane (DCM)
which were treated with 3.0 Å molecular sieves (20% m/v) for a minimum of 48 h prior to use [25].
The progress of the syntheses was monitored by thin-layer chromatography (TLC). General
procedures for the syntheses of PPL-inspired molecules were adapted from Sun Lan-Di et al. [18] For
the syntheses of piperine-inspired molecules, the procedures were adapted from Mathew, A. et al.
[13].
1
13
1
H and C NMR spectra were recorded either on a Bruker AVIII operating at 300 MHz for H
1
and fitted with a 5mm BBFO probe or on a Bruker AVANCE NEO operating at 400 MHz for H fitted
1
1
1
13
1
15
1
13
with a 5mm “smart” BBFO probe, respectively. H- H COSY, H- C and H- N HSQC, H- C and
1
15
H- N HMBC NMR spectra were recorded on a Bruker AVANCE NEO console operating at 500 MHz
1
1
for H and fitted with a nitrogen-cooled BBFO probe. Chemical shift data for H are reported in parts
per million (ppm, δ scale) downfield from tetramethylsilane (TMS:δ 0.0) and referenced internally to
the residual proton in the solvent. The deuterated solvents used for NMR analysis were: chloroform
(CDCl
3: δH 7.26, δC 77.2), dimethyl sulfoxide (DMSO-d
6
: δH 2.50, δC 39.5), and methanol (CD OD:
3
δH 3.31, δC 49.0). Coupling constants (J) are given in hertz (Hz). The data are presented as follows:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiple, br
=
broad, app = apparent and combinations thereof), coupling constant and integration.
Mass spectra data were recorded using a Waters® Xevo G2-XS TOF using electro-spray
ionization in positive (ESI+) mode.
3
.1. Synthesis of Piperine-Inspired Molecules
Synthesis of Piperonal (2)
To a stirred solution of piperonyl alcohol (1) (152 mg, 1.0 mmol) in DCM (100 mL), manganese
dioxide (1739 mg, 20.0 mmol) was added at a rate of 1.0 mmol/h until completion. The reaction
®
mixture was filtered through a pad of Celite and the filtrate was concentrated in vacuo to afford the
1
desired compound as white crystals (148 mg, 97%). H NMR (300 MHz, CDCl
3) δ 9.81 (s, 1H), 7.41
1
3
(
dd, J = 7.9, 1.5 Hz, 1H), 7.33 (d, J = 1.5 Hz, 1H), 6.93 (d, J = 7.9 Hz, 1H), 6.08 (s, 2H); C NMR (101
MHz, CDCl3) δ 190.5, 153.3, 148.9, 132.1, 128.9, 108.5, 107.1, 102.3. Data were in accordance with the
literature [26].
Synthesis of (E)-3-(Benzo[d][1,3]dioxol-5-yl)-1-phenylprop-2-en-1-one (3a)
Acetophenone (240 mg, 2.0 mmol), 2 (300 mg, 2.0 mmol), ethanol (95%, 1.0 mL) and sodium
hydroxide solution (10% w/w in H O, 1.0 mL) were added to a round-bottom flask. The reaction
2
mixture was stirred at room temperature for 5 h. The reaction mixture was washed with ethanol (100
mL) and filtered under vacuum. The solid was dried in vacuo to afford the desired compound as a
1
yellow powder (270 mg, 90%). H NMR (300 MHz, CDCl
3
) δ 8.06–7.95 (m, 2H), 7.74 (d, J = 15.6 Hz,
1
H), 7.63–7.46 (m, 3H), 7.37 (d, J = 15.6 Hz, 1H), 7.19–7.08 (m, 2H), 6.85 (d, J = 8.0 Hz, 1H), 6.03 (s, 2H);
13C NMR (101 MHz, CDCl
) δ 190.6, 150.1, 148.6, 144.9, 138.6, 132.8, 129.5, 128.8, 128.6, 125.5, 120.3,
3
+
+
+
+
1
2
08.9, 106.8, 101.8; LRMS (ES ) m/z: 253.09 [M + H] ; HRMS (ES ) m/z calcd. C
6
H13
O [M + H] =
3
53.0865; observed 253.0860 (−2.0 ppm).
Synthesis of (E)-3-(Benzo[d][1,3]dioxol-5-yl)-1-(3-nitrophenyl)prop-2-en-1-one (3b)
A round bottom flask was charged with 3-nitroacetophenone (330.3 mg, 2.0 mmol), 2 (300.3mg,
.0 mmol), ethanol (95%, 1.0 mL), and sodium hydroxide solution (10% w/w in H O, 1.0 mL). The
2
2
mixture was stirred at room temperature for 5 h. The mixture was washed with ethanol (100 mL) and
filtered under vacuum. The powder was recrystallized from hot ethanol and the product was
1
afforded as orange crystals (240 mg, 80%). H NMR (400 MHz, CDCl
3
) δ 8.82 (t, J = 1.9 Hz, 1H), 8.43
(ddd, J = 8.2, 2.3, 1.1 Hz, 1H), 8.33 (ddd, J = 7.8, 1.7, 1.1 Hz, 1H), 7.81 (d, J = 15.5 Hz, 1H), 7.71 (t, J = 8.0

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1
3
Hz, 1H), 7.36 (d, J = 15.5 Hz, 1H), 7.21–7.14 (m, 2H), 6.87 (d, J = 8.0 Hz, 1H), 6.05 (s, 2H); C NMR (101
MHz, CDCl ) δ 188.0, 150.7, 148.7, 148.6, 146.8, 139.9, 134.2, 130.0, 128.9, 127.10, 126.1, 123.4, 118.7,
3
+
+
+
+
1
2
08.9, 106.9, 102.0; LRMS (ES ) m/z: 298.07 [M + H] ; HRMS (ES ) m/z: calcd. C16
98.0715; observed 298.0712 (1.0 ppm).
H
12NO [M + H] =
5
Synthesis of (E)-1-(3-Aminophenyl)-3-(benzo[d][1,3]dioxol-5-yl)prop-2-en-1-one (3c)
A round bottom flask was charged with 3-aminoacetophenone (270.3 mg, 2.0 mmol), 2 (300.3
mg, 2.0 mmol), ethanol (95%, 1.0 mL), and sodium hydroxide solution (10% w/w in H O, 1.0 mL). The
mixture was stirred at room temperature for 5 h. The mixture was washed with ethanol (100 mL) and
2
filtered under vacuum. The crude product was purified by column chromatography (SiO ;
2
1
EtOAc/hexane 1:1) to afford the product as a yellow powder (240.2 mg, 80%). H NMR (300 MHz,
CDCl
H), 6.90 (ddd, J = 7.9, 2.5, 1.1 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H), 6.04 (s, 2H), 3.85 (s, 2H); C NMR (101
MHz, CDCl ) δ 190.7, 150.01, 148.6, 147.0, 144.5, 139.7, 129.6, 129.6, 125.3, 120.6, 119.5, 118.9, 114.6,
3) δ 7.73 (d, J = 15.6 Hz, 1H), 7.39 (ddd, J = 7.7, 1.7, 1.1 Hz, 1H), 7.37–7.27 (m, 3H), 7.18–7.10 (m,
1
3
2
3
+
+
+
+
1
2
08.8, 106.8, 101.8; LRMS (ES ) m/z: 268.10 [M + H] ; HRMS (ES ) m/z: calcd. C16
68.0974; observed 268.0974 (0.0 ppm).
H
14NO [M + H] =
3
Synthesis of 5-(Benzo[d][1,3]dioxol-5-yl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (4a)
Ethanol (95%, 25 mL), NaOH (1.0 g, 0.025 mol), 3a (2.52 g, 0.01 mol) and thiosemicarbazide (911
mg, 0.01 mol) were added to a round-bottom flask. The reaction mixture was stirred for 3 h at reflux.
Upon completion, the reaction mixture was added to ice. The resulting precipitate was washed with
diethyl ether (100 mL) and water (100 mL) under vacuum filtration. The white powder was
1
recrystallized from hot ethanol to afford the desired product as white crystals (1.51 g, 60%). H NMR
(
300 MHz, CDCl ) δ 7.79–7.66 (m, 2H), 7.52–7.37 (m, 3H), 7.09 (s, 1H), 6.86–6.57 (m, 3H), 6.31–5.76 (m,
3
1
3
4
1
H), 3.81 (dd, J = 17.7, 11.4 Hz, 1H), 3.18 (dd, J = 17.8, 3.6 Hz, 1H); C NMR (126 MHz, CDCl
3
) δ 176.9,
1
5
56.2, 148.3, 147.2, 135.9, 131.3, 130.8, 129.1, 127.1, 119.2, 108.7, 106.1, 101.3, 63.5, 43.4; N NMR (51
+
+
+
MHz, CDCl
3
) δ 194.9; 101.3; 13.3; LRMS (ES ) m/z: 326.10 [M + H] ; HRMS (ES ) m/z: calcd. C17
H
16N
3
O S
2
+
[
M + H] = 326.0963; observed 326.0968 (1.5 ppm).
Synthesis of 3-(3-Aminophenyl)-5-(benzo[d][1,3]dioxol-5-yl)-4,5-dihydro-1H-pyrazole-1-
carbothioamide (4c)
A round-bottom flask was charged with ethanol (95%, 25 mL) NaOH (1000 mg, 0.025mol,), 3c
(2673 mg 0.01 mol) and thiosemicarbazide (911 mg, 0.01 mol). The reaction mixture was stirred for 3
h at reflux. Upon completion, the reaction mixture was added to ice. The resulting solid was washed
with diethyl ether and water, then filtered. The crude product was purified by column
chromatography (SiO
2
; EtOAc/hexane 7:3) to afford the desired compound as an off-yellow powder
) δ 7.98 (s, 1H), 7.67 (s, 1H), 7.08 (app t, J = 7.8 Hz, 1H),
1
(
1604 mg, 60%). H NMR (500 MHz, DMSO-d
6
7
2
1
.03 (d app t, J = 7.8, 1.1 Hz, 1H), 6.99 (app t, J = 2 Hz,1H), 6.83 (d, J = 8.0 Hz, 1H), 6.67 (ddd, J = 7.9,
.3, 1.1 Hz, 1H), 6.63 (d, J = 1.7 Hz, 1H), 6.59 (dd, J = 8.1, 1.8 Hz, 1H), 5.99–5.95 (m, 2H), 5.81 (dd, J =
1
3
1.2, 3.1 Hz, 1H), 5.18 (s, 2H), 3.81 (dd, J = 17.9, 11.3 Hz, 1H), 2.98 (dd, J = 17.8, 3.2 Hz, 1H); C NMR
(126 MHz, DMSO-d
6) δ 175.9, 155.8, 148.8, 147.2, 146.1, 136.9, 131.3, 129.1, 118.3, 116.2, 114.8, 112.0,
+
1
3
08.2, 105.8, 100.9, 62.4, 42.6; 15N NMR (51 MHz, CDCl
3
) δ 192.6; 108.6; 60.9; 14.6; LRMS (ES ) m/z:
+
+
+
41.11 [M + H] ; HRMS (ES ) m/z: calcd. C17
H
17N
4O2
S [M + H] = 341.1072; observed 341.1076 (1.2
ppm).
.2. Synthesis of PPL-Inspired Molecules
3
Synthesis of (E)-3-(3,4,5-Trimethoxyphenyl)acrylic acid (7a)
Malonic acid (1.24 g, 12mmol) was added to a stirred solution of 6a (1.94 g, 10 mmol), pyridine
(6.0 mL) and piperidine (0.6 mL). The reaction was stirred at reflux for 4 h. The mixture was then
cooled to room temperature and poured into a solution of HCl (10 M in H O, 80 mL) at 0 °C. The
2

Molecules 2020, 25, 2841
13 of 17
white solid that formed was recovered by filtration and washed with water (200 mL) to afford the
1
title compound as yellow powder (1.65g, 85%). H NMR (300 MHz, CDCl
3
) δ 7.71 (d, J = 15.8 Hz, 1H),
) δ 172.4,
53.6, 147.3, 140.7, 129.7, 116.6, 105.7, 61.2, 56.4; LRMS (ES ) m/z: 237.0763 [M-H] . Data was in
1
3
6
1
.78 (s, 2H), 6.36 (d, J = 15.9 Hz, 1H), 3.90 (s, 6H), 3.89 (s, 3H). C NMR (101 MHz, CDCl
3
-
+
accordance with the literature [27].
Synthesis of (E)-3-(4-Methoxyphenyl)acrylic acid (7b)
Malonic acid (1.24 g, 12 mmol) was added to a stirred solution of 6b (1.36 g, 10 mmol), pyridine
(6 mL) and piperidine (0.6 mL). The reaction was stirred at reflux for 4 h. The mixture was then cooled
to room temperature and poured into a solution of HCl (10 M in H O, 80 mL) at 0 °C. The white solid
2
that formed was recovered by filtration and washed with water (200 mL) to afford the title compound
1
as white powder (1.33g, 98%). H NMR (300 MHz, CD
3
OD) δ 7.62 (d, J = 16.0 Hz, 1H), 7.58–7.47 (m,
2
H), 7.01–6.88 (m, 2H), 6.33 (d, J = 15.9 Hz, 1H), 3.82 (s, 3H). Data were in accordance with the
literature [28].
Synthesis of (E)-3-(3,4,5-Trimethoxyphenyl)acryloyl chloride (8a)
To a stirred solution of 7a (714 mg, 3.0 mmol) in DCM (6.0 mL), thionyl chloride (0.65 mL, 9.0
mmol) and DMF (0.01 mL) were added. The reaction was stirred at reflux for 5 h under argon. The
solvent was removed under reduced pressure and dried in vacuo to afford the title compound as a
1
yellow powder (707 mg, 99%) and used immediately in the next step. H NMR (300 MHz, CDCl
3) δ
7
.75 (d, J = 15.4 Hz, 1H), 6.79 (s, 2H), 6.55 (d, J = 15.4 Hz, 1H), 3.91 (s, 3H), 3.91 (s, 6H).
Synthesis of (E)-3-(4-Methoxyphenyl)acryloyl chloride (8b)
To a stirred solution of 7b (534 mg, 3.0 mmol) in DCM (6.0 mL), thionyl chloride (0.65 mL, 9.0
mmol) and DMF (0.01 mL) were added. The reaction was stirred at reflux for 5 h under argon. The
solvent was removed under reduced pressure and dried in vacuo to afford the title compound as a
1
yellow powder (513 mg, 96%) and used immediately in the next step. H NMR (300 MHz, CD
3OD) δ
7
.62 (d, J = 16.0 Hz, 1H), 7.58–7.47 (m, 2H), 7.00–6.89 (m, 2H), 6.33 (d, J = 15.9 Hz, 1H), 3.82 (s, 3H).
Synthesis of (E)-1-(3-(3,4,5-Trimethoxyphenyl)acryloyl)piperidin-2-one (9a)
To a stirred solution of δ-valerolactam (278 mg, 2.8 mmol) in THF (12 mL) at 0 °C, NaH (112 mg,
2
.8 mmol, 60% w/w in oil) was added slowly. The reaction was warmed to room temperature and
stirred for 2 h. The mixture was cooled to 0°C and 8a (641 mg, 2.5mmol) was added. The reaction was
stirred for 1 h at 0 °C and for a further 24 h at room temperature under argon. The solution was
poured into ice water and stirred for 15 min. The organic phase was recovered by extraction with
ethyl acetate (15 mL), washed with brine (30 mL) and NaOH (2.0 M in H2O, 30 mL) and dried
(
MgSO ). The organic phase was concentrated by rotary evaporation under reduced pressure. The
4
crude product was purified by column chromatography (SiO
2
; EtOAc/petroleum ether 1:1) to afford
) δ 7.63 (d, J = 15.5
1
the title compound as a yellow powder (219 mg, 34%). H NMR (300 MHz, CDCl
3
Hz, 1H), 7.35 (d, J = 15.6 Hz, 1H), 6.78 (s, 2H), 3.88 (s, 6H), 3.86 (s, 3H), 3.79 (td, J = 5.1, 4.1, 2.7 Hz, 2H),
.68–2.53 (m, 2H), 1.95–1.80 (m, 4H). Data were in accordance with the literature [18].
2
Synthesis of (E)-1-(3-(4-Methoxyphenyl)acryloyl)piperidin-2-one (9b)
To a stirred solution of δ-valerolactam (278 mg, 2.8 mmol) in THF (12 mL) at 0 °C, NaH (112 mg,
2
.8 mmol, 60% w/w in oil) was added slowly. The reaction warmed to room temperature and stirred
for 2 h. The mixture was cooled to 0 °C and 8b (490 mg, 2.5mmol) was added. The reaction was stirred
for 1 h at 0 °C and for a further 24 h at room temperature under argon. The solution was poured into
ice water and stirred for 15 min. The organic phase was recovered by extraction with ethyl acetate
(
15 mL), washed with brine (30 mL) and NaOH (2.0 M in H
phase was concentrated by rotary evaporation under reduced pressure. The crude product was
purified by column chromatography (SiO ; EtOAc/petroleum ether 1:1) to afford the title compound
2
O, 30 mL) and dried (MgSO
4
). The organic
2

Molecules 2020, 25, 2841
14 of 17
1
as a yellow powder (114 mg, 23%). H NMR (300 MHz, CDCl
3) δ 7.69 (d, J = 15.6 Hz, 1H), 7.57–7.47
(m, 2H), 7.35 (d, J = 15.6 Hz, 1H), 6.95–6.81 (m, 2H), 3.83 (s, 3H), 3.79 (td, J = 5.2, 4.2, 2.3 Hz, 2H), 2.66–
2
.54 (m, 2H), 1.93–1.84 (m, 4H). Data were in accordance with the literature [29].
Synthesis (E)-6-(3-(3,4,5-Trimethoxyphenyl)acryloyl)cyclohex-2-en-1-one (10)
To a stirred solution of cyclohex-2-enone (577 mg, 6.0 mmol) in THF (15.0 mL) at −78 °C, lithium
diisopropylamide (0.6 mL, 6.0 mmol) was added dropwise. The solution was stirred at −78 °C for 45
min under argon. A solution of 8a (770 mg, 3.0 mmol) in THF (15 mL) was slowly added to the initial
solution and stirred for a further 1 h at −78 °C, then 1 h at 0 °C. The organic phase was recovered by
extraction with ethyl acetate (15 mL), washed with brine (30 mL), and dried (MgSO
4
)
.
The crude
; EtOAc/petroleum ether 1:1) to afford the title
) δ 15.76 (t, J = 1.1 Hz, 1H),
product was purified by column chromatography (SiO
2
1
compound as a yellow powder (228 mg, 40%). H NMR (300 MHz, CDCl
3
7
1
.55 (d, J = 15.5 Hz, 1H), 6.90–6.80 (m, 2H), 6.76 (s, 2H), 6.16 (dtd, J = 10.0, 2.0, 0.9 Hz, 1H), 3.90 (d, J =
.0 Hz, 6H), 3.88 (d, J = 0.9 Hz, 3H), 2.75 (t, J = 7.2 Hz, 2H), 2.42 (qd, J = 5.3, 4.7, 2.6 Hz, 2H). Data were
in accordance with the literature [29] except the signal at 15.76 ppm.
3
.3. Electronic Structure Characterization
The molecular structures of the analogues Piperine and PPL were built in Avogadro software
[30] and optimized by ab initio calculation. The calculations were performed using the Gamess2018
quantum mechanics package [31,32] with Hartree–Fock (HF) formalism [33] and density functional
theory (DFT) [34]. 6-311+G(1d,1p) was used as the set of the bases and B3LYP as the functional [35].
The optimized geometries were determined with a Polarizable Continuum Model (PCM) solvent
model [36] for H
2
O and 4a was also optimized in CHCl . The optimizations were followed by
3
harmonic frequency calculations to obtain the vibrational, the rotational, and the translational
contributions to the free energy. The electrostatic potential map and the partial charges were
determined using the geodesic method [37] along with the same functional base set and solvent
model used for the optimization of the structure. The structure and the normal frequency modes
analyses were visualized by wxMacMolPlt software [38] to assure that the result is not a structure in
a transition state.
3
.4. Molecular Docking
The optimized structures of each analogue were obtained from Section 3.3. IL-1β structure was
obtained from chain A of PDB-1ITB and p65 unit of NF-κB was obtained from the chain A of PDB-
O61.
AutoDockTools [39] software of the MGL program Tools 1.5.4 was used to prepare the proteins
2
by adding polar hydrogen atoms and Gasteiger charges. Blind docking was performed to explore the
whole IL-1β protein. The maps were generated by AutoGrid 4.2 program with a spacing of 0.4583 Å,
dimension of 126×126×108 points and grid center coordinates as being 41.028, −0.369 and 12.346 for
x, y and z coordinates, respectively. The NF-κB maps were generated by AutoGrid 4.2 program [39]
with a spacing of 0.375 Å, dimension of 90 × 82 × 92 points and grid center coordinates as being 21.573,
−
12.537 and −0.788 for x, y and z coordinates, respectively. AutoDock 4.2[39] was used to investigate
the protein binding site using the Lamarckian Genetic Algorithm (LGA) with a population size of
6
1
50, maximum number of generations of 27000 and energy evaluations equal to 2.5 × 10 . The other
parameters were selected as the software default. To generate different conformations, the total
numbers of runs were set to 100. The final energy scores were calculated following the equation
ꢇꢈꢇ
ꢂꢃꢄꢅꢆ
ꢇꢈꢇ
ꢊꢈꢊ
ꢂꢃꢄꢅꢆ
ꢊꢈꢊ
ꢊꢈꢇ
ꢂꢃꢄꢅꢆ
ꢊꢈꢇ
∆
ꢀ = (ꢁ
− ꢁ
) + (ꢁ
− ꢁ
) + (ꢁ
− ꢁ
)
ꢄꢉꢂꢃꢄꢅꢆ
ꢄꢉꢂꢃꢄꢅꢆ
ꢄꢉꢂꢃꢄꢅꢆ
(
1)
+
∆ꢋꢌꢃꢅꢍꢃꢎꢉꢏꢐꢑꢃꢅ
The potentials utilized to perform the calculation were the following:

Molecules 2020, 25, 2841
15 of 17
ꢝꢑꢞ ꢡꢑꢞ
ꢧꢑꢞ ꢨꢑꢞ
ꢟꢩꢣ + ꢪ. ꢛ
ꢫꢑ. ꢫꢞ
ꢠ
ꢬ. ꢔ
ꢑꢞ
ꢑ,ꢞ
ꢁ
(ꢒꢓꢒꢔꢕꢖ ꢗꢘꢙꢔꢒ) = ꢚ. ꢛ ꢜ
−
ꢣ + ꢤ. ꢛ ꢥ(ꢦ). ꢜ
−
+ ∆ꢭ
ꢟꢠ
ꢢ
ꢟꢠ
(2)
ꢔ
ꢔ
ꢔ
ꢔ
ꢑꢞ
ꢑꢞ
ꢑꢞ
ꢑꢞ
ꢑ,ꢞ
ꢑ,ꢞ
The weighting constants I, J, E(t) and K are those optimized to calibrate the empirical free energy
based on a set of experimentally characterized complexes. The first term is the Lenard–Jones
potential, in which parameters A and B are taken from the Amber force field. The second term refers
to the hydrogen bond in which parameters C and D are obtained to ensure a minimum energy of 5.0
kcal/mol in 1.9 Å for O-H and N-H and 1.0 kcal/mol in 2.5 Å for S-H [40]. The function E(t) provides
directionality based on the angle t of the geometry of an ideal hydrogen bond. The third term is a
shielded Coulomb potential for electrostatic interaction. The last term is the desolvation potential
based on the volume of the atoms surrounding a given atom and sheltering of the solvent [40]. The
final conformations were chosen among the most negative energies that belong to the most
representative cluster and visualized by visual molecular dynamics software (VMD) [41]. The
binding microenvironment was generated by LigPlot [42].
4
. Conclusions
In this study, we have confirmed the structure of some ambiguous complex natural product -
1
5
likescaffolds from the literature through de novo synthesis, spectroscopic analysis including N NMR
techniques, and ab initio calculations. With the definitive structures in hand, we used molecular
docking to investigate the rationale for the promising biological results these molecules exhibit in the
inhibition of the inflammatory response (via downregulation of IL-1β and NF-κB pathway). This
work revealed 3a,c, 4a,c and 10 as the most promising molecules to be evaluated as IL-1β and NF-κB
inhibitors in future studies.
Supplementary Materials: The following are available online, copies of H, 13C and 2D NMR spectra, and ab
1
initio calculations of selected dihedral angles.
Author Contributions: Conceptualization, M.L.C. and A.M.J.; methodology, M.L.C., A.M.J., A.P.R.P. and G.Z.;
software, A.P.R.P. and G.Z.; validation, B.T. and C.S.L.D., formal analysis, M.L.C., C.S.L.D., and A.M.J.;
investigation, A.P.R.P., G.Z., B.T. and C.S.L.D.; resources, A.M.J., B.T. and C.S.L.D.; data curation, A.P.R.P., G.Z.
and A.M.J.; writing—original draft preparation, A.P.R.P., G.Z. and A.M.J.; writing—review and editing, A.M.J.,
A.P.R.P., G.Z., M.L.C. and C.S.L.D.; visualization, A.P.R.P. and G.Z.; supervision, A.M.J. and M.L.C.; project
administration, A.M.J; funding acquisition, M.L.C, A.M.J, A.P.R.P. and G.Z.. All authors have read and agreed
to the published version of the manuscript.
Funding: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior–Brasil (CAPES)–Finance Code 001 in the form of research scholarships for A.P.R.P. and G.Z. The APC
was funded by Molecules, MDPI.
Acknowledgments: The computations described in this paper were performed using the University of
Birmingham’s BlueBEAR HPC service, which provides a High Performance Computing service to the
University’s research community. See http://www.birmingham.ac.uk/bear for more details.
Conflicts of Interest: The authors declare no conflict of interest.
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