Synthesis, structure and in vitro anti-trypanosomal activity of non-toxic arylpyrrole-based chalcone derivatives

Article abstract of DOI:10.3390/molecules25071668

With an intention of identifying chalcone derivatives exhibiting anti-protozoal activity, a cohort of relatively unexplored arylpyrrole-based chalcone derivatives were synthesized in moderate to good yields. The resultant compounds were evaluated in vitro for their potential activity against a cultured Trypanosoma brucei brucei 427 strain. Several compounds displayed mostly modest in vitro anti-trypanosomal activity with compounds 10e and 10h emerging as active candidates with IC₅₀ values of 4.09 and 5.11 μM, respectively. More importantly, a concomitant assessment of their activity against a human cervix adenocarcinoma (HeLa) cell line revealed that these compounds are non-toxic.

Full text of DOI:10.3390/molecules25071668

molecules
Article
Synthesis, Structure and In Vitro Anti-Trypanosomal
Activity of Non-Toxic Arylpyrrole-Based
Chalcone Derivatives
Ayanda I. Zulu 1, Ogunyemi O. Oderinlo 1 , Cuan Kruger 1, Michelle Isaacs 2,
Heinrich C. Hoppe 2,3 , Vincent J. Smith 1,2 , Clinton G. L. Veale 4,5 and
Setshaba D. Khanye 1,2,5,
*
1
Department of Chemistry, Faculty of Science, Rhodes University, Grahamstown 6140, South Africa;
azulu50@gmail.com (A.I.Z.); oderinloyemi@yahoo.com (O.O.O.); g13k8200@campus.ru.ac.za (C.K.);
v.smith@ru.ac.za (V.J.S.)
2
3
4
5
Centre for Chemico and Biomedicinal Research, Rhodes University, Grahamstown 6140, South Africa;
m.isaacs@ru.ac.za (M.I.); h.hoppe@ru.ac.za (H.C.H.)
Department of Biochemistry and Microbiology, Faculty of Science, Rhodes University, Grahamstown 6140,
South Africa
School of Chemistry and Physics, Pietermaritzburg Campus, University of KwaZulu-Natal, Private Bag X01,
Scottsville 3209, South Africa; VealeC@ukzn.ac.za
Division of Pharmaceutical Chemistry, Faculty of Pharmacy, Rhodes University, Grahamstown 6140,
South Africa
*
Correspondence: s.khanye@ru.ac.za; Tel.: +27-46-603-8397
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Andrew Tsotinis
Received: 21 March 2020; Accepted: 2 April 2020; Published: 4 April 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: With an intention of identifying chalcone derivatives exhibiting anti-protozoal activity,
a cohort of relatively unexplored arylpyrrole-based chalcone derivatives were synthesized in moderate
to good yields. The resultant compounds were evaluated in vitro for their potential activity against a
cultured Trypanosoma brucei brucei 427 strain. Several compounds displayed mostly modest in vitro
anti-trypanosomal activity with compounds 10e and 10h emerging as active candidates with IC50
values of 4.09 and 5.11 µM, respectively. More importantly, a concomitant assessment of their activity
against a human cervix adenocarcinoma (HeLa) cell line revealed that these compounds are non-toxic.
Keywords: arylpyrrole; chalcones; trypanosomiasis; Trypanosoma brucei; molecular hybridization
1. Introduction
Infectious diseases caused by kinetoplastid parasites are a serious health threat worldwide.
Among these is human African trypanosomiasis (HAT), commonly referred to as sleeping sickness [
HAT has also been prioritized by the World Health Organization (WHO) in the list of neglected tropical
diseases (NTDs) in 149 endemic countries [ ]. A significantly large population mainly in poor
rural locations of sub-Saharan Africa remains at risk of contracting the disease [ ]. The causative
agents of HAT are protozoan trypanosomes of the genus Trypanosoma: T. brucei rhodesiense and T. brucei
gambiense [ ]. These are transmitted by the insect vector tsetse fly of the Glossina species, which injects
metacyclic trypomastigotes into the human host [ ]. T. b. gambiense, which is common to west and
1,2].
3–5
5,6
7,8
9
central Africa, is responsible for >98% of reported cases of HAT in the continent of Africa [10], whilst
T. b. rhodesiense is found in eastern and southern Africa and accounts for less than 2% of reported cases.
Despite their discovery almost a century ago, the most successful agents in clinical use for the
treatment of HAT are pentamidine, melarsoprol (Figure 1), suramin, and eflornithine [11]. However,
these drugs suffer from some limitations, including poor oral bioavailability and complex and
Molecules 2020, 25, 1668; doi:10.3390/molecules25071668
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Molecules 2020, 25, 1668
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time-consuming intravenous (IV) administration, which is costly and requires highly skilled health
practitioners and facilities that are limited in poverty-stricken rural areas [12 13], and thus impact the
implementation of treatment [14]. Additionally, currently available anti-trypanosomal drugs have
serious side effects, such as nephrotoxicity, hypertension, anemia, and neuropathy [15 16]. In the
absence of effective vaccines to prevent infections inflicted by HAT, treatment efforts are solely reliant
on chemotherapeutic intervention [ 17]. Treatment options are implemented such that for stage 1
,
,
3
,
of the disease pentamidine and suramin are utilized, despite their limited penetration of the central
nervous system (CNS) [18]. For stage 2 of the disease, melarsoprol and eflornithine are the preferred
treatment options, however, these drugs suffer from issues relating to toxicity and trypanosomal
resistance [19]. Considering the urgent need to develop therapeutic alternatives that target both
stages of trypanosomiasis, a partnership between Sanofi and Drug for Neglected Diseases initiative
(DNDi) has led to the approval of fexinidazole (Figure 1) as an alternative therapeutic armament for
tackling both stage 1 and 2 HAT [1,20]. However, the ever-growing concern regarding resistance
against clinically approved drugs, and the potential for an epidemic outbreak, has resulted in several
campaigns focused on identifying new compounds with novel modes of action to prevent and control
trypanosomal infections [21–23].
H
N
O
O
H2N
N
HN
NH
NH2
N
N
S
As
S
NH2
NH2
Melarsoprol
OH
Pentamidine
N
N
O
NO2
S
Fexinidazole
Figure 1. Examples of some drugs used for the treatment of human African sleeping sickness.
Chalcones are open-chain flavonoids, which feature a characteristic three carbon
α,β-unsaturated
carbonyl system [24 25]. More importantly, chalcones are appealing as key synthetic intermediates
,
and frameworks in the design of therapeutic tools for the treatment of various ailments including
antimicrobial, antibacterial, antimalarial, anticancer, anti-inflammatory, anti-HIV, anti-Alzheimer’s,
etc. [24–32]. The structural modification of chalcones where the B-ring (Figure 2) is substituted with
other bioactive fragments or units is a contemporary strategy that has been extensively utilized by
various research groups involved in designing bioactive compounds for the treatment of different
diseases [33]. A typical example is that of Zhou and co-workers, who prepared a series of novel
chalcones by incorporating the benzoxaborole motif to form hybrid molecules (Figure 2) showing
excellent in vivo activity in a murine model of T. b. brucei, with compound
active compound, with an IC50 value of 0.010 µg/mL [34].
1 emerging as the most
In an effort to identify compounds with desirable potency against methicillin-resistant
Staphylococcus aureus (MRSA), Os rio and co-workers reported a focused library of chalcone derivatives,
including compound , bearing an N-arylpyrrole moiety [35]. Thus far, compound remains unexplored
ó
2
2
for antiprotozoal activity, with no examples of analogues appearing in the literature. Importantly,
in our context, several studies have shown that compounds containing the arylpyrrole framework
display a broad spectrum of biological properties, including antiparasitic activities, as illustrated
by the anti-malarial lead compound 3 [36]. Furthermore, numerous chalcone derivatives, including

Molecules 2020, 25, 1668
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compounds
4
and
5
, have been reported as possessing anti-bacterial and anti-tuberculosis activity [37].
In a separate study, Dave et al. explored chalcone derivatives in which the B-ring was substituted for a
quinoline nucleus, for their potential as antiplasmodial agents. In that study, compound displayed
6
favorable anti-plasmodial potency, with the 5-bromo-2-hydroxy substitution pattern on ring A (portion
in blue) appearing to be a crucial element of the pharmacophore [38].
OEt
O
OH
B
O
F3C
O
O
H3CO
H2N
H3CO
O
N
N
N
OCH3
H
CF3
A
B
2
1
TDR32750 (3)
R
O
O
N
Br
Br
Br
OH
OH Cl
N
OH
Cl
6
4
5
O
Br
A
B
N
R
OH
Target compounds
Figure 2. Examples of some bioactive chalcone derivatives, arylpyrroles, and target compounds.
Molecular hybridization, which combines two or more dissimilar pharmacophoric subunits to
improve efficacy and other pharmaceutical profiles is becoming an attractive approach to design novel
biologically active compounds [39
develop new antiparasitic compounds [42
–
41]. Considering our ongoing research interests to identify and
44], we employed a molecular hybridization approach to
–
design a target series of chalcone derivatives (Figure 2) featuring A ring with hydroxyl and bromine
substituents at position 2 and 5, respectively, and arylpyrrole containing B ring system. Herein, we
wish to report the synthesis of a representative series of new and non-toxic arylpyrrole-based chalcone
derivatives, along with their in vitro anti-trypanosomal effects against the nagana T. b. brucei strain.
2. Results and Discussion
2.1. Chemical Synthesis
Reaction of commercially available 2,5-hexadione
7 with a variety of anilines under the
standard Paal-Knorr condensation reaction conditions (Scheme 1) afforded starting arylpyrrole
intermediates (see Supplementary Information) with yields varying between 38%–81% [45].
Subsequently, the Vilsmeier-Haack formylation of resultant arylpyrrole intermediates led to the
formation of the desired aldehydes 8a
commercially available 2,5-dimethyl-1H-pyrrole under similar Vilsmeier-Haack reaction conditions
yielded compound 8l as a white solid in good yield. Treatment of prepared aldehydes 8a
with the 2-hydroxy-5-bromoacetophenone under base-catalyzed Claisen-Schmidt condensation
conditions, afforded the desired arylpyrrole-chalcone derivatives 10a , which were isolated in yields
varying between 9%–71%. Similarly, compound 11a was obtained in a poor yield by treating
with 2,5-dimethyl-1H-imidazole-4-carbaldehyde, while compound 11b was synthesized from
–k in moderate to excellent yields [46]. Subjecting the
–
l
9
–
l
8
the 2-chloro-6-methoxyquinoline-3-carbaldehyde at a 39% yield, as previously described in the
literature [38]. Further preliminary structure-activity relationship (SAR) investigations were conducted

Molecules 2020, 25, 1668
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through structural modification of the ring A. The starting commercially available acetophenones
were reacted with selected arylpyrrolecarbaldehydes 8e 8h, and 8i under the same base-catalyzed
,
Claisen-Schmidt reaction conditions (Scheme 1), to form the desired compounds 12
–
19 with yields in
the range 5%–89%.
R1
R2
Ar
12
13
14
15
16
17
18
19
H
NO2
NO2
NO2
NO2
NO2
NO2
H
Ph
R1
O
H
4-Me-Ph
4-F-Ph
4-OMe-Ph
Ar
R2
N
H
H
H
2,4-diOMe-Ph
4-NO2-Ph
12 - 19
H
CF3
NO2
2-OH, 5-Cl-Ph
2-OH, 5-Cl-Ph
v
H
O
O
i, ii
O
Br
O
Ar
Br
R
N
iv
O
OH
7
8 a - l
OH
9
11a - b
Ar
iii
a
NH
N
O
R
O
Br
a = Ph
g = 2-CF3-Ph
h = 4-NO2-Ph
i = 3-NO2-Ph
j = 4-OMe-Ph
k = H
b
N
R
Cl
N
b = 4-F-Ph
OH
10a - l
c = 4-Cl-Ph
d = 4-Br-Ph
e = 3-CF3-Ph
f = 4-CF3-Ph
l = Cyclohexyl
Scheme 1. Reagents and conditions: (i) Anilines or cyclohexalamine, solvent free, 160 ◦C, 16 h;
(ii) POCl3, DMF, 0 ◦C–60 ◦C, 4 h; (iii) 60% NaOH, MeOH, rt, 12 h; (iv) Aldehydes, 60% NaOH, MeOH,
rt, 12 h.
All of the synthesized compounds were fully characterized using routine spectroscopic methods
(NMR, IR and MS). The coupling constant (J) of the α,β-unsaturated protons featured values in the
range 12–16 Hz, thus indicating a trans configuration [47]. This observation was corroborated by
the molecular structure of compound 10e, determined by single crystal X-ray diffraction analysis.
Crystal and experimental data are summarized in Table S1 (Supplementary Information). Compound
10e crystallizes in the orthorhombic crystal system, in the centrosymmetric space group Pbca with
eight molecules per unit cell, Z = 8. The crystal structure of compound 10e (Figure 3 and Figure S1)
confirmed unequivocally a trans configuration about the double bond (C(8A)-C(9A)).

Molecules 2020, 25, 1668
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Figure 3. Molecular structure of 10e depicting the numbering scheme and the intramolecular hydrogen
bond (shown here by the red dashed line). The hydrogen atoms on carbons were fixed in geometrically
calculated while those bound to oxygen were freely refined. The structure was solved and refined
to a final residual factor R1 = 0.0751. All non-hydrogen atoms were refined anisotropically, with the
ellipsoids drawn at the 50% probability level (see Figure S1 in the Supplementary Materials).
From the structure of 10e, bond lengths and bond angles are comparable to chalcone derivatives,
which have been reported in the literature [48]. The existence of an
α,β-unsaturated ketone is
further confirmed by short O(2A)-C(7A) and C(8A)-C(9A) bond lengths of 1.244(7) Å and 1.359(6) Å,
respectively; and bond angles O(2A)-C(7A)-C(8A) and C(7A)-C(8A)-C(9A) of 121.2(5)◦ and 120.8(4)◦,
respectively. More importantly, the structure exhibits intramolecular hydrogen bonding between
the hydroxyl oxygen of ring A and the ketone adjacent to ring A. Introduction of intramolecular
hydrogen bond (IMHB) into the bioactive compounds has been reported to have beneficial effects,
including biological activity [49]. For example, Attram and co-workers synthesised a novel class
of benzimidazoles containing an IMHB structural feature showing in vitro antiplasmodial activity
against chloroquine-sensitive (NF54) and multi-drug resistant (K1) strains of the malaria parasite
Plasmodium falciparum [50]. The aryl moiety on ring B of the chalcone is rotated about the N(1A)-C(16A)
bond with corresponding four torsion angles (Figure S2). The observed torsion angles suggest the
deviation from planarity of the aryl group attached to the pyrrole framework. The crystal packing
(Figure 4) of the molecule is stabilized by
between the phenyl and pyrrole rings ( ). The centroid-to-centroid distances (
3.927(3) Å and 3.731(2) Å, which further confirms the intermolecular stacking interactions between
π
-
π
stacking interactions between phenyl rings (
A) and
B
A,B) are approximately
π-π
chalcone molecules in the crystal structure [48]. Additionally, the supramolecular assembly is supported
by weak C-H···π and C-F···π interactions between the trifluorophenyl ring moieties of the arylpyrrole
(C) [C(21A)-H(21A)···π is 2.95 Å and C(22A)-F(2A)···π is 3.32(1) Å].

Molecules 2020, 25, 1668
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Figure 4. Packing diagram depicting π···π stacking and C-H···π interactions of 10e that run diagonally
across the bc plane. The C-F···π interactions have been omitted for clarity.
2.2. In Vitro Biological Assessment
A single concentration (20 µM) cytotoxicity evaluation of all compounds was conducted in
duplicates using a human cervix adenocarcinoma (HeLa) cell line, with emetine (EMT) included as a
reference drug and cell viability relative to untreated cells assessed using resazurin. Compounds 10a–l
possessed negligible cytotoxicity effects against the HeLa cell line, and in general, >80% HeLa cell
viability was observed (see Table S2). The anti-trypanosomal activity of prepared compounds against
nagana T. b. brucei (the subspecies responsible for African trypanosomiasis in cattle) parasite cultures
were determined using the T. b. brucei 427 strain. Pentamidine (PMD), which is an existing drug for
the treatment of trypanosomiasis, was included as a positive control drug. The trypanocidal activity of
each compound was initially assessed at a fixed concentration of 20 µM, using resazurin as a read-out
of cell viability after compound exposure. All of the compounds which inhibited cell viability below
25% relative to untreated controls at this concentration were taken forward for IC50 determination.
From Table 1, the structural modification was initially performed on the arylpyrrole moiety (ring
B) of the chalcones, while keeping the ring A unchanged. Compound 10a, with an unsubstituted
aromatic ring attached to the arylpyrrole moiety, showed modest activity against T. b. brucei, with an
IC50 value of 11.5
10b) appeared to enhance the activity against T. b. brucei; however, the activity diminished when
the fluorine was replaced by chlorine (10c). Despite 10d exhibiting similar activity to compound 10a
µM. Introduction of the fluorine, highly electronegative atom, at the para-position
(
,
inclusion of the bromine substituent (10d) slightly restored activity comparable to 10c. The presence of
the trifluoromethyl (CF3) functionality at the para- position resulted in compound 10e, whose activity
closely mirrored 10b. The positional isomer of 10e which features CF3 at the meta- position (10f
displayed reduced activity with an IC50 value of 11.4 M, while the ortho substituted analogue 10g
showed moderate activity in line with compounds 10a 10d, and 10f. Exchanging of CF3 with yet
another electron withdrawing substituent, NO2 at the para position (10h), also showed improved
biological activity with an IC50 < 6 M, while the meta isomer (10i) possessed modest activity, with an
M. Compound 10j, which features an electron donating methoxy moiety,
)
µ
,
µ
IC50 value slightly below 10
showed no activity.
µ

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Table 1. In vitro bioassay data for chalcone derivatives 10a–l, 11a–b, and 12–19 showing IC50 values
for inhibition of T. b. brucei 427.
T. b. brucei
T. b. brucei
Comp
Comp
IC50/µM
IC50/µM
10a
10b
10c
10d
10e
10f
10g
10h
10i
11.5
6.9
40.0
10.5
4.1
11.4
10.0
5.1
10l
11a
11b
12
13
14
15
16
17
18
19
-
11.5
na
9.1
na
44.3
36.5
12.3
10.6
na
9.3
11.7
-
9.3
na
na
10j
10k
PMD
0.68 nM
PMD = Pentamidine. na = not active (compounds producing a cell viability of
assay at 20 µM).
≥
50% in the preliminary single point
Compound 10k, which lacked an aromatic ring substituent on the pyrrole ring, showed a complete
loss of activity against the T. b. brucei strain, while compound 10l, with a saturated cyclohexyl ring
instead of either a substituted or non-substituted aromatic ring, showed no improvement in terms of
potency, and its IC50 value was comparable to that of compounds 10a and 10f. Compounds 11a and
11b, which featured aromatic replacements of the arylpyrrole moiety, were evaluated for trypanocidal
activity. While 11a showed no activity, compound 11b, with a quinoline heterocyclic unit, was found to
be moderately active against trypanosomes, with an IC50 value of 9.1 µM. Having identified compounds
10e and 10h as the most active compounds in our initial cohort, we proceeded to explore compounds
which maintained the arylpyrrole substituents of either compounds 10e and 10h, whilst replacing the
2-hydro-5-bromo-substituted aromatic moiety. These compounds (12
activity against T. b. brucei, with IC50 values ranging between 9.3–44.3
–
19) showed moderate inhibitory
M (Table 1). Despite moderate
µ
activity, the data suggest that a combination of the hydroxyl group at position 2 and the bromine at
position 5 in the ring A is necessary for activity against the T. b. brucei parasite.
3. Materials and Methods
3.1. General Information
All commercially available reagents were purchased from chemical suppliers, Sigma-Adrich
(Pty) Ltd. (Johannesburg, South Africa) and Merck (Pty) Ltd. (Johannesburg, South Africa), and
were used without further purification unless stated otherwise. Solvents were distilled before use.
The progress of reactions were monitored by thin layer chromatography (TLC) using Merck F254 silica
gel plates (Merck, Johannesburg, South Africa) supported on aluminium, and were visualized under
ultra-violet (UV 254 and 366 nm) light, and, where necessary, stained in an iodine tank. The crude
compounds were purified by a silica gel column chromatography using Merck Kieselgel 60 Å: 70–230
(0.068–0.2 mm) silica gel mesh. NMR (1H and 13C) spectra were recorded on Bruker Biospin 300, 400,
or 600 MHz spectrometer. Chemical shifts are reported in ppm and are referenced internally using
residual solvent signals (DMSO-d6 δH 2.50, δC 39.5 ppm; CDCl3 δH 7.26, δC 77.0 ppm). The coupling
constants are given in Hertz. High-resolution mass spectrometry (HRMS) was performed on a Waters
API Q-TOF Ultima spectrometer (Stellenbosch University, Stellenbosch, South Africa). The IR spectra
were recorded on PerkinElmer Spectrum 100 FT-IR Spectrometer (Johannesburg, South Africa) in the
mid-IR range (640–4000 cm−1). Melting points were measured using a Reichert hot-stage apparatus and
are uncorrected. Elemental microanalysis was performed on Elementar Analysensysteme varioMICRO
V1.6.2 GmbH analysis system. The calculated values were determined using an online Elemental

Molecules 2020, 25, 1668
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Composition Calculator from Microanalysis Laboratory, School of Chemical Sciences, University of
Illinois at Urbana, USA [51]. Herein, compound 11b was synthesized from the starting ketone and
8
2-chloro-6-methoxyquinoline-3-carbaldehyde, according to the method previously described in the
literature [38].
3.2. General Procedure for the Synthesis of Arylpyrrole-Chalcone Hybrids
A 60% NaOH (10 mL) solution was cooled in an ice bath while stirring, and 5-bromo-
2-hydroxyacetophenone (1 eq.) was slowly added and the reaction mixture allowed stirring for
20 min. Thereafter, an appropriate aldehyde (1 eq.) dissolved in MeOH (10 mL) was added, and
the resultant reaction mixture allowed to stir for 12 h at room temperature. The reaction progress
was monitored by TLC (Hex: EtOAc), which showed the formation of a new spot, while the traces
of starting material were still visible. After the completion of the reaction, the product precipitated
out of the solution, which was filtered and washed with ice cold water and methanol. In cases where
there was no precipitate formed, the product was poured onto water and extracted with ethyl acetate.
The organic layer was dried (Na2SO4), filtered, and evaporated the solvent in vacuo. The resulting
crude product was purified by a silica gel column chromatography to give products as solids.
(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)prop-2-en-1-one 10a, Yellow
◦
◦
1
crystalline solid. Yield = 24%. m.p. 148 C–150 C. H NMR (300 MHz, CDCl3): δH 13.3 (1H,
s, OH), 8.07 (1H, d, J = 14.7 Hz, Hαβ), 8.00 (1H, d, J = 2.1 Hz, ArH), 7.53–7.47 (4H, m, ArHs), 7.23–7.20
(2H, m, ArHs), 7.16 (1H, d, J = 14.7 Hz, Hαβ), 6.90 (1H, d, J = 9.0 Hz, ArH), 6.41 (1H, s, pyrrole-H),
2.18 (3H, s, CH3), 2.04 (3H, s, CH3). 13C NMR (75 MHz, CDCl3): δC 192.6, 162.50, 140.7, 138.0, 137.7,
136.9, 132.1, 131.7, 129.7, 128.9, 128.1, 121.9, 120.5, 118.0, 112.9, 110.2, 104.4,12.9, 11.2. HRMS (ESI+) m/z
(ESI-HRMS) found 396.0597, calcd for C21H19NO2Br: 396.0594 [M + H]+. Anal. Found: C, 62.79; H,
4.13; N, 3.09%. Anal. calcd. C21H18NO2Br·0.5H2O: C, 62.23; H, 4.73; N, 3.46%.
(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(1-(4-fluorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)prop-2-en-1-one 10b
,
◦
◦
1
Yellow solid. Yield = 39%. m.p. 135 C-138 C. H NMR (400 MHz, CDCl3): δH 13.3 (1H, s,
OH), 8.05 (1H, d, J = 14.8 Hz, Hαβ), 7.99 (1H, d, J = 2.4 Hz, ArH), 7.51 (1H, dd, J = 2.4 and 8.8 Hz,
ArH), 7.34–7.26 (4H, m, ArHs), 7.16 (1H, d, J = 14.8 Hz, Hαβ), 6.90 (1H, d, J = 9.2 Hz, ArH), 6.40 (1H,
s, pyrrole-H), 2.17 (3H, s, CH3), 2.03 (3H, s, CH3). 13C NMR (100 MHz, CDCl3): δC 192.5, 163.8 (2C),
161.2 (2C), 140.5, 138.1, 136.7, 133.6, 132.0, 131.6, 129.8, 121.8, 120.5, 118.1, 116.7, 113.1, 110.3, 104.5, 12.9,
11.2. m/z (ESI-HRMS) found 414.0505, calcd for C21H18BrFNO2: 414.0499 [M + H]+. Anal. Found: C,
56.92; H, 2.74; N, 2.87%. Anal. calcd. C21H17NO2BrF·2H2O: C, 56.01; H, 4.70; N, 3.11%.
(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)prop-2-en-1-one 10c
Yellow crystalline solid. Yield = 53%. m.p. 127 C–129 C. H NMR (300 MHz, CDCl3): δH
,
◦
◦
1
13.3 (1H, s, OH), 8.03 (1H, d, J = 14.4 Hz, Hαβ), 8.00 (1H, d, J = 2.4 Hz, ArH), 7.53–7.48 (3H, m, Hαβ
ArHs), 7.19–7.14 (3H, m, ArHs), 6.90 (1H, d, J = 9.0 Hz, ArH), 6.40 (1H, s, pyrrole-H), 2.17 (3H, s, CH3),
2.03 (3H, s, CH3). 13C NMR (75 MHz, CDCl3): δC 192.6, 162.5, 140.4, 138.1, 136.5, 136.2, 134.9, 131.9,
131.7, 130.0, 129.4, 121.9, 120.6, 118.3, 113.4, 110.3, 104.7, 12.9, 11.3. HRMS (ESI+) m/z (ESI-HRMS) found
430.0200, calcd for C21H18NO2ClBr: 430.0204 [M + H]+. Anal. Found: C, 58.15; H, 3.71; N, 3.19%. Anal.
calcd. C21H17NO2BrCl: C, 58.56; H, 3.98; N, 3.25%.
,
(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(1-(4-bromophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)prop-2-en-1-one 10d
,
Yellow crystalline solid. Yield = 45%. m.p. 141 ◦C–144 ◦C. 1H NMR (300 MHz, CDCl3): δH 13.3 (1H, s,
OH), 8.04 (1H, d, J = 15.0 Hz, Hαβ), 7.99 (1H, d, J = 2.4 Hz, ArH), 7.65 (2H, d, J = 8.7 Hz, ArHs), 7.52
(1H, dd, J = 2.4 and 9.0 Hz, ArH), 7.16 (1H, d, J = 14.7 Hz, Hαβ), 7.10 (2H, d, J = 8.7 Hz, ArHs), 6.90
(1H, d, J = 8.7 Hz, ArH), 6.40 (1H, s, pyrrole-H), 2.18 (3H, s, CH3), 2.03 (3H, s, CH3). 13C NMR (75 MHz,
CDCl3): δC 192.4, 162.4, 140.2, 138.0, 136.6, 136.2, 132.8, 131.6, 131.5, 129.6, 122.7, 121.7, 120.4, 118.1,
113.2, 110.1, 104.6, 12.3, 11.0. HRMS (ESI+) m/z (ESI-HRMS) found 473.9701, calcd for C21H18NO2Br2:

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473.9699, [M + H]+. Anal. Found: C, 52.65; H, 3.19; N, 2.89%. Anal. calcd. C21H17NO2Br2
52.08; H, 3.75; N, 2.89%.
·0.5H2O: C,
(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(2,5-dimethyl-1-(3-(trifluoromethyl)phenyl)-1H-pyrrol-3-yl)prop-2-en-1-one
10e, Yellow crystalline solid. Yield = 20%. m.p. 135 ◦C–137 ◦C. 1H NMR (300 MHz, CDCl3): δH 13.2
(1H, s, OH), 8.04 (1H, d, J = 15.0 Hz, Hαβ), 8.00 (1H, d, J = 2.4 Hz, ArH), 7.77–7.75 (1H, m, ArH), 7.67
(1H, d, J = 7.8 Hz, Ar), 7.54 – 7.50 (2H, m ArHs), 7.44 (1H, d, J = 7.8 Hz, ArH), 7.18 (1H, d, J = 14.7 Hz,
Hαβ), 6.91 (1H, d, J = 9.0 Hz, ArH), 6.43 (1H, s, pyrrole-H), 2.19 (3H, s, CH3), 2.05 (3H, s, CH3); 13
C
NMR (75 MHz, CDCl3): δC 192.4, 162.2, 140.2, 138.2, 138.0, 136.0, 131.6, 131.6, 131.4, 130.3, 125.5,
125.2, 124.9, 121.6, 120.4, 118.4, 114.9, 113.6, 110.1, 105.0, 12.8, 11.1. m/z (ESI-HRMS) found 464.0456
calcd for C22H16NO2F3Br: 464.0468 [M + H]+. Anal. Found: C, 57.24; H, 4.76; N, 2.86%. Anal. calcd.
C22H17NO2BrF3·0.5CH3OH: C, 56.27; H, 3.99.; N, 2.92%.
(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(2,5-dimethyl-1-(4-(trifluoromethyl)phenyl)-1H-pyrrol-3-yl)prop-2-en-1-one
10f, Yellow crystalline solid. Yield = 70%. m.p. 157 ◦C–159 ◦C. 1H NMR (400 MHz, CDCl3): δH 13.2
(1H, s, OH), 8.04 (1H, d, J = 14.8 Hz, Hαβ), 8.00 (1H, d, J = 2.4 Hz, ArH), 7.80 (2H, d, J = 8.4 Hz,
ArHs), 7.52 (1H, dd, J = 2.4 and 8.8 Hz, ArH), 7.37 (2H, d, J = 8.4 Hz, ArHs), 7.18 (1H, d, J = 14.8 Hz,
Hαβ), 6.91 (1H, d, J = 8.8 Hz, ArH), 6.43 (1H, s, pyrrole-H), 2.19 (3H, s, CH3), 2.05 (3H, s, CH3).
13C NMR (100 MHz, CDCl3): δC 192.6, 162.5, 140.1, 138.2, 136.1, 131.7, 131.3, 130.9, 128.8, 126.9,
125.1, 122.4, 121.81, 120.6, 118.6, 113.8, 110.3, 105.2, 12.9, 11.2. m/z (ESI-HRMS) found 464.0468, calcd
for C22H18NO2BrF3: 464.0468 [M + H]+. Anal. Found: C, 56.60; H, 3.88; N, 2.96%. Anal. calcd.
C22H17NO2BrF3·0.5CH3OH: C, 56.27; H, 3.99; N, 2.92%.
(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(2,5-dimethyl-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-3-yl)prop-2-en-1-one
10g, Yellow crystalline solid. Yield = 71%. m.p. 115 ◦C–118 ◦C. 1H NMR (400 MHz, CDCl3): δH 13.3
(1H, s, H13), 8.04 (1H, d, J = 14.7 Hz, Hαβ), 8.00 (1H, d, J = 2.4 Hz, ArH), 7.89–7.86 (1H, m, ArH),
7.76–7.71 (1H, m, ArH), 7.68–7.63 (1H, m, ArH), 7.51 (1H, dd, J = 2.4 and 9.0 Hz, ArH), 7.32–7.27 (1H,
m, ArH), 7.17 (1H, d, J = 14.7 Hz, Hαβ), 6.90 (1H, d, J = 8.7 Hz, ArH), 6.41 (1H, s, pyrrole-H), 2.09
(3H, s, CH3), 1.95 (3H, s, CH3). 13C NMR (75 MHz, CDCl3): δC 192.7, 162.5, 140.5, 138.1, 137.8, 135.8,
133.4, 132.9, 131.7, 131.3, 129.9, 129.0, 127.8, 121.8, 120.5, 118.3, 113.4, 110.4, 110.3, 104.4, 12.5, 10.9. m/z
(ESI-HRMS) found 462.0299, calcd for C22H16NO2F3Br: 462.0322 [M-H]+. Anal. Found: C, 57.20; H,
1.94; N, 2.95%. Anal. calcd. C22H17NO2BrF3: C, 56.91; H, 3.69; N, 3.02%.
(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(2,5-dimethyl-1-(4-nitrophenyl)-1H-pyrrol-3-yl)prop-2-en-1-one 10h
,
◦
◦
1
Yellow crystalline solid. Yield = 37%. m.p. 168 C–170 C. H NMR (300 MHz, CDCl3): δH
13.2 (1H, s, OH), 8. 41 (2H, d, J = 8.8 Hz, ArHs), 8.04–7.99 (2H, m, ArH, Hαβ), 7.53 (1H, dd, J = 2.4 and
8.8 Hz, ArH), 7.43 (2H, d, J = 8.8 Hz, ArHs), 7.19 (1H, d, J = 14.8 Hz, Hαβ), 6.91 (1H, d, J = 8.8 Hz,
ArH), 6.45 (1H, s, pyrrole-H), 2.21 (3H, s, CH3), 2.10 (3H, s, CH3). 13C NMR (75 MHz, CDCl3): δC 192.6,
162.5, 147.6, 143.3, 139.6, 138.3, 135.6, 131.7, 131.4, 129.1, 125.2, 121.7, 120.6, 119.0, 114.2, 110.3, 105.7,
13.0, 11.3. m/z found 441.0444, calcd for C21H18N2O4Br: 441.0444 [M + H]+.
(E)-1-(5-bromo-2-hydroxyphenyl)-3-(2,5-dimethyl-1-(3-nitrophenyl)-1H-pyrrol-3-yl)prop-2-en-1-one 10i
,
Yellow crystalline solid. Yield = 68%. m.p. 102 ◦C–104 ◦C. 1H NMR (300 MHz, CDCl3): δH 13.2 (1H,
s, OH), 8.39 – 8.35 (1H, m, ArH), 8.14 (1H, t, J = 2.1 Hz, ArH), 8.06–8.00 (2H, m, ArH, Hαβ), 7.75
(1H, t, J = 7.8 Hz, Ar), 7.62–7.58 (1H, m, ArH), 7.53 (1H, dd, J = 2.4 and 9.0 Hz, ArHs), 7.20 (1H, d,
J = 15.0 Hz, Hαβ), 6.91 (1H, d, J = 9.0 Hz, ArH), 6.45 (1H, s, pyrrole-H), 2.21 (3H, s, CH3), 2.07 (3H, s,
CH3); 13C NMR (75 MHz, CDCl3): δC 192.6, 162.5, 148.9, 139.9, 138.9, 138.3, 135.9, 134.3, 131.7, 131.6,
130.7, 123.8, 123.4, 121.7, 120.6, 118.7, 114.1, 110.3, 105.4, 12.9, 11.3. m/z (ESI-HRMS) found 441.0278,
calcd for C21H18N2O4Br: 441.0444 [M + H]+. Anal. Found: C, 57.66; H, 4.16; N, 6.10%. Anal. calcd.
C21H17N2O4Br: C, 57.16; H, 3.88; N, 6.35%.

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(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(1-(4-methoxyphenyl)-2,5-dimethyl-1H-pyrrol-3-yl)prop-2-en-1-one 10j
,
Yellow crystalline solid. Yield = 14%. m.p. 138 ◦C–140 ◦C. 1H NMR (400 MHz, CDCl3): δH 13.3 (1H, s,
OH), 8.07 (1H, d, J = 14.8 Hz, Hαβ), 8.00 (1H, d, J = 2.4 Hz, ArH), 7.51 (1H, dd, J = 2.4 and 8.8 Hz,
ArH), 7.15 (1H, d, J = 14.8 Hz, Hαβ), 7.12 (2H, d, J = 8.8 Hz, ArHs), 7.01 (2H, d, J = 8.8 Hz, ArHs),
6.90 (1H, d, J = 8.8 Hz, ArH), 6.38 (1H, s, pyrrole-H), 3.87 (3H, s, OCH3), 2.17 (3H, s, CH3), 2.02 (3H, s,
CH3). 13C NMR (100 MHz, CDCl3): δC 192.4, 162.5, 159.8, 140.9, 138.1, 137.3, 132.3, 131.7, 130.2, 129.1,
121.8, 120.6, 117.8, 114.9, 112.7, 110.0, 104.0, 55.6, 12.8, 11.1. m/z (ESI-HRMS) found 426.0698, calcd for
C22H21NO3Br: 426.0699 [M + H]+.
(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(2,5-dimethyl-1H-pyrrol-3-yl)prop-2-en-1-one 10k, Orange solid. Yield =
9%. m.p. 88 ◦C–89 ◦C. 1H NMR (400 MHz, CDCl3): δH 13.3 (1H, s, OH), 8.00–7.97 (3H, m, ArHs, Hαβ),
7.50 (1H, dd, J = 2.4 and 8.8 Hz, ArH), 7.08 (1H, d, J = 14.8 Hz, Hαβ), 6.89 (1H, d, J = 8.8 Hz, ArH), 6.22
(1H, s, pyrrole-H), 2.39 (3H, s, CH3), 2.25 (3H, s, CH3). 13C NMR (100 MHz, CDCl3): δC 192.7, 162.3,
140.6, 137.9, 134.6, 131.6, 128.9, 121.8, 120.6, 118.6, 112.9, 110.2, 104.2, 12.9, 11.5. m/z (ESI-HRMS) found
318.0144, calcd for C15H13NO2Br: 318.0130 [M-H]+. Anal. Found: C, 55.69; H, 2.48; N, 3.44%; Anal.
calcd. C15H14NO2Br·CH3OH: C, 55.37; H, 4.80; N, 4.17%.
(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(1-cyclohexyl-2,5-dimethyl-1H-pyrrol-3-yl)prop-2-en-1-one 10l, Yellow
solid. Yield = 42%. m.p. 73 ◦C–76 ◦C. 1H NMR (400 MHz, CDCl3): δH 13.4 (1H, s, OH), 8.03 (1H, d,
J = 14.4 Hz, Hαβ), 7.96 (1H, d, J = 2.4 Hz, ArH), 7.49 (1H, dd, J = 2.4 and 9.2 Hz, ArH), 7.07 (1H, d,
J = 14.4 Hz, Hαβ), 6.88 (1H, d, J = 8.8 Hz, ArH), 6.27 (1H, s, pyrrole-H), 4.00–3.92 (1H, m, cyclic-H),
2.44 (3H, s, CH3), 2.33 (3H, s, CH3), 2.00–1.75 (7H, m, cyclic-Hs), 1.45–1.20 (3H, m, cyclic-Hs). 13C
NMR (100 MHz, CDCl3): δC 192.6, 162.5, 140.7, 137.9, 136.2, 131.5, 131.1, 122.0, 120.4, 112.0, 110.2, 105.5,
57.4, 32.3 (2C), 26.4, 25.9, 14.3, 11.8. m/z (ESI-HRMS) found 402.1061, calcd for C21H25NO2Br: 402.1063
[M + H]+. Anal. Found: C, 62.08; H, 5.06; N, 3.44%; Anal. calcd. C21H24NO2Br: C, 62.69; H, 6.01;
N, 3.48%.
(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(1H-imidazol-4-yl)prop-2-en-1-one 11a. Yellow solid. Yield = 9%. m.p.
116 ◦C–120 ◦C. 1H NMR (600 MHz, DMSO-d6): δH 12.3 (1H, s, OH), 8.01 (1H, d, J = 2.4 Hz, ArH), 7.88
(1H, s, imidazole-H), 7.73 (1H, s, imidazole-H), 7.70 (1H, d, J = 15.0 Hz, Hαβ), 7.64–7.61 (2H, m, ArH,
Hαβ), 6.94 (1H, d, J = 8.4 Hz, ArH); 13C NMR (150 MHz, DMSO-d6): δC 191.9, 159.9, 138.5, 137.7, 136.7,
131.9 (2C), 123.4, 120.1 (2C), 117.8, 110.13. m/z (ESI-HRMS) found 292.9914, calcd for C12H10N2O2Br:
292.9920 [M + H]+. Anal. Found: C, 49.99; H, 3.30; N, 9.76. Anal. calcd. C12H9N2O2Br: C, 49.17; H,
3.09; N, 9.56%.
(E)-3-(2,5-Dimethyl-1-(4-nitrophenyl)-1H-pyrrol-3-yl)-1-phenylprop-2-en-1-one, 12, Yellow crystalline solid.
Yield = 65%. m.p. 169 ◦C–174 ◦C. 1H NMR (300 MHz, CDCl3): δH 8.39 (2H, d, J = 9.0 Hz, ArHs), 8.03 –
8.00 (2H, m, ArHs), 7.89 (1H, d, J = 15.3 Hz, Hαβ), 7.57–7.46 (3H, m, ArHs), 7.40 (2H, d, J = 8.7 Hz,
ArHs), 7.21 (1H, d, J = 15.0 Hz, Hαβ), 6.39 (1H, s, pyrrole-H), 2.18 (3H, s, CH3), 2.07 (3H, s, CH3). 13C
NMR (75 MHz, CDCl3): δC 190.7, 147.6, 143.6, 139.2, 137.9, 134.1, 132.4, 130.9, 129.2, 128.6, 128.4, 125.1,
118.9, 117.4, 105.7, 13.1, 11.3. m/z (ESI-HRMS) found 347.1392, calcd for C21H19N2O3: 347.1390 [M +
H]+. Anal. Found: C, 72.30; H, 6.22; N, 7.44%. Anal. calcd. C21H18N2O3: C, 72.82; H, 5.24; N, 8.09%.
(E)-3-(2,5-Dimethyl-1-(4-nitrophenyl)-1H-pyrrol-3-yl)-1-(p-tolyl)prop-2-en-1-one, 13, Yellow solid. Yield =
34%. m.p. 172 ◦C–173 ◦C. 1H NMR (400 MHz, CDCl3): δH 8.39 (2H, d, J = 8.8 Hz, ArHs), 7.92 (2H, d,
J = 8.4 Hz, ArHs), 7.88 (1H, d, J = 15.2 Hz, Hαβ), 7.41 (2H, d, J = 8.8 Hz, ArHs), 7.28 (2H, d, J = 8 Hz,
ArHs), 7.21 (1H, d, J = 15.2 Hz, Hαβ), 6.40 (1H, s, pyrrole-H), 2.43 (3H, s, CH3), 2.18 (3H, s, CH3),
2.06 (3H, s, CH3). 13C NMR (75 MHz, CDCl3): δC 190.2, 147.5, 143.7, 143.1, 137.4, 136.6, 133.9, 130.8,
129.3, 129.2, 128.6, 125.0, 118.9, 117.4, 105.7, 21.9, 13.1, 11.3. m/z (ESI-HRMS) found 361.1552, calcd for
C22H21N2O3: 361.1547 [M + H]+. Anal. Found: C, 73.08; H, 5.86; N, 7.43%. Anal. calcd. C22H20N2O3:
C, 73.32; H, 5.59; N, 7.77%.

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(E)-3-(2,5-Dimethyl-1-(4-nitrophenyl)-1H-pyrrol-3-yl)-1-(4-fluorophenyl)prop-2-en-1-one, 14, Yellow solid.
Yield = 86%. m.p. 160 ◦C–164 ◦C. 1H NMR (300 MHz, CDCl3): δH 8.39 (2H, d, J = 9.0 Hz, ArHs),
8.07–8.02 (2H, m, ArHs), 7.89 (1H, d, J = 15.3 Hz, Hαβ), 7.41 (2H, d, J = 9.0 Hz, ArHs), 7.205–7.12
(3H, m, ArHs, Hαβ), 6.39 (1H, s, pyrrole-H), 2.19 (3H, s, CH3), 2.07 (3H, s, CH3). 13C NMR (75 MHz,
CDCl3): δC 189.1, 147.7, 143.8, 138.3, 135.6, 134.5, 131.1, 131.0, 129.3, 125.2, 119.1, 117.0, 115.9, 115.7,
105.7, 13.2, 11.4. m/z (ESI-HRMS) found 365.1305, calcd for C21H18N2O3F: 365.1296 [M + H]+. Anal.
Found: C, 69.15; H, 5.24; N, 7.19%. Anal. calcd. C21H17N2O3F: C, 69.22; H, 4.70; N, 7.69%.
(E)-3-(2,5-Dimethyl-1-(4-nitrophenyl)-1H-pyrr◦ol-3-yl)-1-(4-methoxyphenyl)prop-2-en-1-one, 15, Yellow
◦
1
crystalline solid. Yield = 18%. m.p. 157 C–158 C. H NMR (300 MHz, CDCl3): δH 8.39 (2H,
d, J = 8.7 Hz, ArHs), 8.04 (2H, d, J = 9.0 Hz, ArHs), 7.88 (1H, d, J = 15.3 Hz, Hαβ), 7.41 (2H, d, J = 9.0 Hz,
ArHs), 7.23 (1H, d, J = 15.3 Hz, Hαβ), 6.97 (2H, d, J = 9.0 Hz, ArHs), 6.39 (1H, s, pyrrole-H), 3.89 (3H, s,
CH3), 2.18 (3H, s, CH3), 2.07 (3H, s, CH3). 13C NMR (75 MHz, CDCl3): δC 189.0, 163.1, 147.4, 143.7,
137.0, 133.8, 131.9, 130.7, 130.6, 129.1, 125.0, 118.9, 117.1, 113.8, 105.5, 55.5, 13.0, 11.5. m/z (ESI-HRMS)
found 377.1502, calcd for C22H21N2O4: 377.1496 [M + H]+. Anal. Found: C, 68.91; H, 5.73; N, 7.23%.
Anal. calcd. C22H20N2O4·0.5CH3OH: C, 68.86; H, 5.65; 7.14%.
(E)-1-(2,4-Dimethoxyphenyl)-3-(2,5-dimethyl-1-(4-nitrophenyl)-1H-pyrrol-3-yl)prop-2-en-1-one 16. Yellow
◦
◦
1
crystalline solid. Yield = 21%. m.p. 146 C–149 C. H NMR (600 MHz, CDCl3): δH 8.38 (2H, d,
J = 8.4 Hz, ArHs), 7.75–7.72 (2H, m, ArH, Hαβ), 7.40 (2H, d, J = 9.0 Hz, ArHs), 7.16 (1H, d, J = 15.6 Hz,
Hαβ), 6.55 (1H, dd, J = 2.4 and 9.0 Hz, ArH), 6.50 (1H, d, J = 2.4 Hz, ArH), 6.32 (1H, s, pyrrole-H), 3.90
(3H, s, CH3), 3.87 (3H, s, CH3), 2.15 (3H, s, CH3), 2.053 (3H, s, CH3). 13C NMR (75 MHz, CDCl3): δC
191.0, 163.8, 160.2, 147.5, 143.8, 135.6, 133.2, 132.7, 130.5, 129.2, 125.0, 123.2, 122.9, 119.1, 105.8, 105.1,
98.9, 55.9, 55.7, 13.1, 11.3. m/z (ESI-HRMS) found 407.1608, calcd for C23H23N2O5: 407.1601 [M + H]+.
Anal. Found C, 67.99; H, 5.84; N, 6.86%; Anal. calcd. C23H22N2O5: C, 67.41; H, 5.61; N, 6.61%.
(E)-3-(2,5-Dimethyl-1-(4-nitrophenyl)-1H-pyrrol-3-yl)-1-(4-nitrophenyl)prop-2-en-1-one, 17, Reddish solid.
Yield = 5%. m.p. 175 ◦C–178 ◦C. 1H NMR (400 MHz, CDCl3): δH 8.41 (2H, d, J = 8.8 Hz, ArHs), 8.33 (2H,
d, J = 8.4 Hz, ArHs), 8.13 (2H, d, J = 8.8 Hz, ArHs), 7.93 (1H, d, J = 15.2 Hz, Hαβ), 7.42 (2H, d, J = 8.8 Hz,
ArHs), 7.14 (1H, d, J = 15.2 Hz, Hαβ), 6.40 (1H, s, pyrrole-H), 2.20 (3H, s, CH3), 2.07 (3H, s, CH3). 13C
NMR (100 MHz, CDCl3): δC 188.9, 149.9, 147.7, 144.3, 143.4, 139.9, 135.3, 131.4 (2C), 129.3, 129.2, 125.2,
123.9, 118.9, 116.5, 105.6, 13.1, 11.4. m/z (ESI-HRMS) found 392.1246, calcd for C21H18N3O5: 392.1241
[M + H]+. Anal. Found: C, 63.30; H, 5.00; N, 9.29%. Anal. calcd. C21H17N3O5
0.5CH3COCH2CH3: C,
·
63.44; H, 4.86; N, 9.65%.
(E)-1-(5-Chloro-2-hydroxyphenyl)-3-(2,5-dimethyl-1-(3-(trifluoromethyl)phenyl)-1H-pyrrol-3-yl)prop-2-en-1-one
18. Yellow crystalline solid. Yield = 87%. m.p. 110 ◦C–112 ◦C. 1H NMR (400 MHz, CDCl3): δH 13.2
(1H, s, OH), 8.04 (1H, d, J = 2.4 Hz, ArH), 7.76 (1H, d, J = 7.6 Hz, ArH), 7.70–7.66 (1H, m, ArH),
7.52–7.49 (1H, m, ArH), 7.45–7.42 (1H, m, ArH), 7.40 (1H, dd, J = 2.4 and 9.2 Hz, ArH), 7.19 (1H, d,
J = 14.8 Hz, Hαβ), 6.95 (1H, d, J = 8.8 Hz, ArH), 6.42 (1H, s, pyrrole-H), 2.19 (3H, s, CH3), 2.05 (3H,
s, CH3). 13C NMR (100 MHz, CDCl3): δC 192.7, 162.1, 140.1, 138.4, 136.2, 135.9, 135.4, 132.3, 131.7,
131.6, 130.4, 128.7, 125.7, 125.1, 123.3, 121.2, 120.2, 118.5, 113.8, 105.1, 12.9, 11.2. m/z (ESI-HRMS) found
420.0945, calcd for C22H17NO2ClF3: 420.0948 [M + H]+. Anal. Found: C, 62.34; H, 4.28; N, 2.94%; Anal.
calcd. C22H17NO2ClF3·0.5CH3CH2OH: C, 62.38; H, 4.55; N, 3.16%.
(E)-1-(5-Chloro-2-hydroxyphenyl)-3-(2,5-dimethyl-1-(3-nitrophenyl)-1H-pyrrol-3-yl)prop-2-en-1-one 19
,
Yellow crystalline solid. Yield = 89%. m.p. 71 ◦C–74 ◦C. IR (cm−1): 1630 (C=O), 1527 (C=C). 1H NMR
(300 MHz, CDCl3): δH 13.2 (1H, s, OH), 8.38 – 8.34 (1H, m, ArH), 8.14 – 8.13 (1H, m, ArH), 8.03 (1H, d,
J = 15.0 Hz, Hαβ), 7.86 (1H, d, J = 2.7 Hz, ArH), 7.77–7.72 (1H, m, ArH), 7.61–7.58 (1H, m, ArH), 7.40
(1H, dd, J = 2.4 and 8.7 Hz, ArH), 7.20 (1H, d, J = 14.7 Hz, Hαβ), 6.96 (1H, d, J = 9.0 Hz, ArH), 6.44 (1H,
s, pyrrole-H), 2.19 (3H, s, CH3), 2.07 (3H, s, CH3). 13C NMR (75 MHz, CDCl3): δC 192.8, 162.1, 148.9,
139.8, 138.9, 135.9, 135.5, 134.3, 131.5, 130.7, 128.7, 123.7, 123.3 (2C), 121.1, 120.2, 118.7, 114.1, 105.3, 13.0,

Molecules 2020, 25, 1668
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11.3. m/z (ESI-HRMS) found 397.0947, calcd for C21H18N2O4Cl: 397.0955 [M + H]+. Anal. Found: C,
63.74; H, 5.23; N, 6.62%; Anal. calcd. C21H17N2O4Cl·0.4 CH3CH2OH: C, 63.06; H, 4.71; N, 6.75%.
3.3. X-ray Crystallographic Analysis
A single crystal was covered in a small amount of paratone N oil and mounted on a MiTeGen
microloop. X-ray intensity data were collected at 100 K on a Bruker DUO APEX CCD with graphite
monochromated Mo radiation (λ = 0.71073). The detector to crystal distance was 60 mm. Data were
collected using phi and omega scans and were scaled and reduced using the APEX III software suite
(Bruker SAINT). The structure was solved using SHELXT 2018/2 (Sheldrick, 2018) and refined using
SHELXL-2018/3 (Sheldrick, 2018) [52]. Hydrogen atoms were placed in calculated positions and refined
using the riding model. The program X-SEED [53], an interface to SHELX, was used during the
structure solution and refinement. The accession code for 10e is (CCDC 1991652). The supplementary
information contains crystallographic data for compound 10e. The data can be obtained free of charge
from The Cambridge Crystallographic Data Centre on http://www.ccdc.cam.ac.uk/conts/retrieving.
html or The Director, CCDC, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; or
deposit@ccdc.cam.ac.uk.
3.4. In Vitro Antitrypanosomal Assay
Trypanosoma brucei brucei 427 trypomastigotes were cultured in Iscove’s Modified Dulbecco’s
medium (IMDM, Lonza, Basel Switzerland) and supplemented with 10% fetal calf serum, HMI-9
supplement [54], hypoxanthine and penicillin/streptomycin at 37 ◦C in a 5% CO2 incubator. Serial
dilutions of test compounds were incubated with the parasites in 96-well plates for 24 h, and residual
parasite viability in the wells determined by adding 20 µL resazurin toxicology reagent (Sigma-Aldrich)
and incubating for an additional 24 h. Reduction of resazurin to resorufin by viable parasites was
assessed by fluorescence readings (excitation 560 nm, emission 590 nm) in a Spectramax M3 plate
reader (Molecular Devices, San Jose, CA, USA). Fluorescence readings were converted to % parasite
viability relative to the average readings obtained from untreated control wells. IC50 values were
determined by plotting % viability vs. log[compound] and performing non-linear regression using
GraphPad Prism (v. 5.02) software [55].
3.5. In Vitro Cytotoxicity Assay
HeLa cells (Cellonex, Johannesburg, South Africa) seeded in 96-well plates were incubated
with 20 µM test compounds and cell viability assessed with resazurin, as previously described [56].
Fluorescence readings (excitation 560 nm, emission 590 nm) obtained for the individual wells were
converted to % cell viability relative to the average readings obtained from untreated control wells.
Plots of % cell viability vs. log[compound] were used to determine IC50 values by non-linear regression
using GraphPad Prism (v. 5.02) [56].
4. Conclusions
In this study, we presented a series of arylpyrrole-chalcone derivatives, which were obtained in
yields ranging from poor to excellent. Analysis of the NMR and crystal structure of compound 10f
confirmed unequivocally that achieved compounds assumed a trans configuration about the double
bond of the
suggested that they are generally non-toxic, and displayed weak growth inhibition of HeLa cells
at 20 M, and none of the compounds inhibited cell viability to <80%. More importantly, these
compounds showed anti-trypanosomal activity, with most active compounds 10e and 10h displaying
IC50 values of < 6.0 M. Despite modest activity, a combination of electron donating groups on the
α,β-unsaturated carbonyl system. The in vitro bioassays data of resultant compounds
µ
µ
ring A and electron withdrawing groups on the B ring appeared to be required for the activity of these
compounds. Compounds lacking the aryl groups on the B ring side of the chalcone scaffold showed no
activity at the maximum tested concentration, suggesting that the arylpyrrole moiety is necessary for

Molecules 2020, 25, 1668
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activity, and it is worthy of further exploitation for the development of alternative compounds for the
treatment of infections caused by trypanosomes.
Supplementary Materials: The following are available online (Synthesis of arylpyrrole intermediates, compound
8a-l, X-ray crystallographic data of compound 10e and selected 1H and 13C NMR spectra of some compounds).
Author Contributions: A.I.Z. synthesized target chalcones and collection of experimental data. O.O.O. contributed
some of the starting arylpyrroles. C.K. contributed on some single crystal X-ray data. H.C.H. and M.I. coordinated
and performed the T. b. brucei and cytotoxicity assays. V.J.S. assisted and coordinated single crystal X-ray
diffraction analysis. C.G.L.V. and S.D.K. conceptualized and provided intellectual leadership of the project
including supervision. All authors have read and agreed to the published version of the manuscript
Funding: The research was funded by the Rhodes University Sandisa Imbewu (SDK, CGLV, and HCH) and
Thuthuka National Research Foundation (SDK), grant number 87894 and the APC was funded by Rhodes
University. The bioassay component of the project was funded by the South African Medical Research Council
(MRC) with funds from National Treasury under its Economic Competitiveness and Support Package.
Acknowledgments: AIZ gratefully acknowledged financial support from the National Research Foundation
Innovation. OOO and CK were supported by Rhodes University through Sandisa Imbewu. Our great appreciation
from the Stellenbosch University Central Analytical Facility (CAF) for mass spectrometric analysis.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).