Synthesis, Structure and In Vitro Anti-Trypanosomal Activity of Non-Toxic Arylpyrrole-Based Chalcone Derivatives

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.

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 [1,2]. HAT has also been prioritized by the World Health Organization (WHO) in the list of neglected tropical diseases (NTDs) in 149 endemic countries [3,4,5]. A significantly large population mainly in poor rural locations of sub-Saharan Africa remains at risk of contracting the disease [5,6]. The causative agents of HAT are protozoan trypanosomes of the genus Trypanosoma: T. brucei rhodesiense and T. brucei gambiense [7,8]. These are transmitted by the insect vector tsetse fly of the Glossina species, which injects metacyclic trypomastigotes into the human host [9]. T. b. gambiense, which is common to west and 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 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 [3,17]. Treatment options are implemented such that for stage 1 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 [21,22,23].

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. [24,25,26,27,28,29,30,31,32]. 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 1 emerging as the most active compound, with an IC₅₀ value of 0.010 µg/mL [34].

In an effort to identify compounds with desirable potency against methicillin-resistant Staphylococcus aureus (MRSA), Osório and co-workers reported a focused library of chalcone derivatives, including compound 2, bearing an N-arylpyrrole moiety [35]. Thus far, compound 2 remains unexplored 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 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 6 displayed 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].

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 [39,40,41]. Considering our ongoing research interests to identify and develop new antiparasitic compounds [42,43,44], 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–k in moderate to excellent yields [46]. Subjecting the 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–l with the 2-hydroxy-5-bromoacetophenone 9 under base-catalyzed Claisen-Schmidt condensation conditions, afforded the desired arylpyrrole-chalcone derivatives 10a–l, which were isolated in yields varying between 9%–71%. Similarly, compound 11a was obtained in a poor yield by treating 8 with 2,5-dimethyl-1H-imidazole-4-carbaldehyde, while compound 11b was synthesized from 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 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%.

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)).

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 π-π stacking interactions between phenyl rings (A) and between the phenyl and pyrrole rings (B). The centroid-to-centroid distances (A,B) are approximately 3.927(3) Å and 3.731(2) Å, which further confirms the intermolecular π-π stacking interactions between 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].

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. In vitro bioassay data for chalcone derivatives 10a–l, 11a–b, and 12–19 showing IC₅₀ values for inhibition of T. b. brucei 427.

Comp	T. b. brucei	Comp	T. b. brucei
	IC50/µM		IC50/µM
10a	11.5	10l	11.5
10b	6.9	11a	na
10c	40.0	11b	9.1
10d	10.5	12	na
10e	4.1	13	44.3
10f	11.4	14	36.5
10g	10.0	15	12.3
10h	5.1	16	10.6
10i	9.3	17	na
10j	na	18	9.3
10k	na	19	11.7
PMD	0.68 nM	-	-

PMD = Pentamidine. na = not active (compounds producing a cell viability of ≥50% in the preliminary single point assay at 20 μM).

, 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 μM. Introduction of the fluorine, highly electronegative atom, at the para-position (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, 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 μM, 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 μM, while the meta isomer (10i) possessed modest activity, with an IC₅₀ value slightly below 10 μM. Compound 10j, which features an electron donating methoxy moiety, showed no activity.

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 (12–19) showed moderate inhibitory activity against T. b. brucei, with IC₅₀ values ranging between 9.3–44.3 µM (Table 1). Despite moderate 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 δ_H 2.50, δ_C 39.5 ppm; CDCl₃ δ_H 7.26, δ_C 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 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 8 and 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-one10a, Yellow crystalline solid. Yield = 24%. m.p. 148 °C–150 °C. ¹H NMR (300 MHz, CDCl₃): δ_H 13.3 (1H, s, OH), 8.07 (1H, d, J = 14.7 Hz, H_αβ), 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, H_αβ), 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): δ_C 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-one10b, Yellow solid. Yield = 39%. m.p. 135 °C-138 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 13.3 (1H, s, OH), 8.05 (1H, d, J = 14.8 Hz, H_αβ), 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, H_αβ), 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): δ_C 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-one10c, Yellow crystalline solid. Yield = 53%. m.p. 127 °C–129 °C. ¹H NMR (300 MHz, CDCl₃): δ_H 13.3 (1H, s, OH), 8.03 (1H, d, J = 14.4 Hz, H_αβ), 8.00 (1H, d, J = 2.4 Hz, ArH), 7.53–7.48 (3H, m, H_αβ, 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): δ_C 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-one10d, Yellow crystalline solid. Yield = 45%. m.p. 141 °C–144 °C. ¹H NMR (300 MHz, CDCl₃): δ_H 13.3 (1H, s, OH), 8.04 (1H, d, J = 15.0 Hz, H_αβ), 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, H_αβ), 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): δ_C 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: 473.9699, [M + H]⁺. Anal. Found: C, 52.65; H, 3.19; N, 2.89%. Anal. calcd. C₂₁H₁₇NO₂Br₂·0.5H₂O: C, 52.08; H, 3.75; N, 2.89%.

(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(2,5-dimethyl-1-(3-(trifluoromethyl)phenyl)-1H-pyrrol-3-yl)prop-2-en-1-one10e, Yellow crystalline solid. Yield = 20%. m.p. 135 °C–137 °C. ¹H NMR (300 MHz, CDCl₃): δ_H 13.2 (1H, s, OH), 8.04 (1H, d, J = 15.0 Hz, H_αβ), 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, H_αβ), 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); ¹³C NMR (75 MHz, CDCl₃): δ_C 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-one10f, Yellow crystalline solid. Yield = 70%. m.p. 157 °C–159 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 13.2 (1H, s, OH), 8.04 (1H, d, J = 14.8 Hz, H_αβ), 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, H_αβ), 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): δ_C 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-one10g, Yellow crystalline solid. Yield = 71%. m.p. 115 °C–118 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 13.3 (1H, s, H₁₃), 8.04 (1H, d, J = 14.7 Hz, H_αβ), 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, H_αβ), 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): δ_C 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-one10h, Yellow crystalline solid. Yield = 37%. m.p. 168 °C–170 °C. ¹H NMR (300 MHz, CDCl₃): δ_H 13.2 (1H, s, OH), 8. 41 (2H, d, J = 8.8 Hz, ArHs), 8.04–7.99 (2H, m, ArH_, H_αβ), 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, H_αβ), 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): δ_C 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-one10i, Yellow crystalline solid. Yield = 68%. m.p. 102 °C–104 °C. ¹H NMR (300 MHz, CDCl₃): δ_H 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, H_αβ), 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, H_αβ), 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): δ_C 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%.

(E)-1-(5-Bromo-2-hydroxyphenyl)-3-(1-(4-methoxyphenyl)-2,5-dimethyl-1H-pyrrol-3-yl)prop-2-en-1-one10j, Yellow crystalline solid. Yield = 14%. m.p. 138 °C–140 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 13.3 (1H, s, OH), 8.07 (1H, d, J = 14.8 Hz, H_αβ), 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, H_αβ), 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): δ_C 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-one10k, Orange solid. Yield = 9%. m.p. 88 °C–89 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 13.3 (1H, s, OH), 8.00–7.97 (3H, m, ArHs, H_αβ), 7.50 (1H, dd, J = 2.4 and 8.8 Hz, ArH), 7.08 (1H, d, J = 14.8 Hz, H_αβ), 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): δ_C 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-one10l, Yellow solid. Yield = 42%. m.p. 73 °C–76 °C. ¹H NMR (400 MHz, CDCl3): δ_H 13.4 (1H, s, OH), 8.03 (1H, d, J = 14.4 Hz, H_αβ), 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, H_αβ), 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): δ_C 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-one11a. Yellow solid. Yield = 9%. m.p. 116 °C–120 °C. ¹H NMR (600 MHz, DMSO-d₆): δ_H 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, H_αβ), 7.64–7.61 (2H, m, ArH, H_αβ), 6.94 (1H, d, J = 8.4 Hz, ArH); ¹³C NMR (150 MHz, DMSO-d₆): δ_C 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): δ_H 8.39 (2H, d, J = 9.0 Hz, ArHs), 8.03 – 8.00 (2H, m, ArHs), 7.89 (1H, d, J = 15.3 Hz, H_αβ), 7.57–7.46 (3H, m, ArHs), 7.40 (2H, d, J = 8.7 Hz, ArHs), 7.21 (1H, d, J = 15.0 Hz, H_αβ), 6.39 (1H, s, pyrrole-H), 2.18 (3H, s, CH₃), 2.07 (3H, s, CH₃). ¹³C NMR (75 MHz, CDCl₃): δ_C 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): δ_H 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, H_αβ), 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, H_αβ), 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): δ_C 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%.

(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): δ_H 8.39 (2H, d, J = 9.0 Hz, ArHs), 8.07–8.02 (2H, m, ArHs), 7.89 (1H, d, J = 15.3 Hz, H_αβ), 7.41 (2H, d, J = 9.0 Hz, ArHs), 7.205–7.12 (3H, m, ArHs_, H_αβ), 6.39 (1H, s, pyrrole-H), 2.19 (3H, s, CH₃), 2.07 (3H, s, CH₃). ¹³C NMR (75 MHz, CDCl₃): δ_C 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-pyrrol-3-yl)-1-(4-methoxyphenyl)prop-2-en-1-one,15, Yellow crystalline solid. Yield = 18%. m.p. 157 °C–158 °C. ¹H NMR (300 MHz, CDCl₃): δ_H 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, H_αβ), 7.41 (2H, d, J = 9.0 Hz, ArHs), 7.23 (1H, d, J = 15.3 Hz, H_αβ), 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): δ_C 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-one16. Yellow crystalline solid. Yield = 21%. m.p. 146 °C–149 °C. ¹H NMR (600 MHz, CDCl₃): δ_H 8.38 (2H, d, J = 8.4 Hz, ArHs), 7.75–7.72 (2H, m, ArH_, H_αβ), 7.40 (2H, d, J = 9.0 Hz, ArHs), 7.16 (1H, d, J = 15.6 Hz, H_αβ), 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): δ_C 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): δ_H 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, H_αβ), 7.42 (2H, d, J = 8.8 Hz, ArHs), 7.14 (1H, d, J = 15.2 Hz, H_αβ), 6.40 (1H, s, pyrrole-H), 2.20 (3H, s, CH₃), 2.07 (3H, s, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ_C 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-one18. Yellow crystalline solid. Yield = 87%. m.p. 110 °C–112 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 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, H_αβ), 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): δ_C 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-one19, 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): δ_H 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, H_αβ), 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, H_αβ), 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): δ_C 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, 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 [email protected].

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 α,β-unsaturated carbonyl system. The in vitro bioassays data of resultant compounds suggested that they are generally non-toxic, and displayed weak growth inhibition of HeLa cells at 20 µM, 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 µM. Despite modest activity, a combination of electron donating groups on the 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 activity, and it is worthy of further exploitation for the development of alternative compounds for the treatment of infections caused by trypanosomes.