Design and synthesis of Mannich base-type derivatives containing imidazole and benzimidazole as lead compounds for drug discovery in Chagas Disease

European Journal of Medicinal Chemistry 223 (2021) 1 13646

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Design and synthesis of Mannich base-type derivatives containing

imidazole and benzimidazole as lead compounds for drug discovery in

Chagas Disease

a

,

b

c

a, b

ꢀ

Ivan Beltran-Hortelano , Richard L. Atherton , Mercedes Rubio-Hernandez

a,

a, b, **

ꢀ

Julen Sanz-Serrano , Veronica Alcolea , John M. Kelly , Silvia Perez-Silanes

,

Francisco Olmo ^c

,

*

^aUniversidad de Navarra, ISTUN Instituto de Salud Tropical, Irunlarrea 1, 31008, Pamplona, Spain

^bUniversidad de Navarra, Pharmacy and Nutrition Faculty, Department of Pharmaceutical Technology and Chemistry, Campus Universitario, 31080,

Pamplona, Spain

^cDepartment of Infection Biology, London School of Hygiene and Tropical Medicine, London, WC1 7HT, United Kingdom

^dUniversidad de Navarra, Pharmacy and Nutrition Faculty, Department of Pharmacology and Toxicology, Irunlarrea 1, 31008, Pamplona, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, the most important

parasitic infection in Latin America. The only treatments currently available are nitro-derivative drugs

that are characterised by high toxicity and limited efﬁcacy. Therefore, there is an urgent need for more

effective, less toxic therapeutic agents. We have previously identiﬁed the potential for Mannich base

derivatives as novel inhibitors of this parasite. To further explore this family of compounds, we syn-

thesised a panel of 69 new analogues, based on multi-parametric structure-activity relationships, which

allowed optimization of both anti-parasitic activity, physicochemical parameters and ADME properties.

Additionally, we optimized our in vitro screening approaches against all three developmental forms of

the parasite, allowing us to discard the least effective and trypanostatic derivatives at an early stage. We

ultimately identiﬁed derivative 3c, which demonstrated excellent trypanocidal properties, and a syn-

ergistic mode of action against trypomastigotes in combination with the reference drug benznidazole.

Both its druggability and low-cost production make this derivative a promising candidate for the pre-

clinical, in vivo assays of the Chagas disease drug-discovery pipeline.

Received 22 May 2021

Received in revised form

9 June 2021

Accepted 10 June 2021

Available online 16 June 2021

Keywords:

Mannich bases

Imidazole

Benzimidazole

Chagas disease

Neglected tropical diseases

Trypanosoma cruzi

1. Introduction

or drink), as well as global migratory movements, have spread CD

to previously unaffected regions and continents. To date, there are

Chagas disease (CD) is the main parasitic infection in Latin

America. It is caused by the protozoan Trypanosoma cruzi, which is

transmitted to humans by triatomine insect vectors [1]. About 6e8

million people are currently infected and more than 70 million

people are at risk of acquiring the disease. Unfortunately, less than

1% of people infected with T. cruzi have access to diagnosis and

treatment [2]. Other modes of transmission (congenital, blood

transfusions, organ transplants, and ingesting contaminated food

only two available treatments, benznidazole (BZ) and nifurtimox

(NFX). Both are close to 100% effective if given soon after infection,

but they are not approved for use against the symptomatic chronic

form of the disease. Additionally, both drugs have drawbacks, with

long treatment periods (60e90 days) and dose-dependent toxicity

leading to high drop-out rates amongst patients [2e4]. Novel

alternative approaches proposed and developed by groups such as

the Drugs for Neglected Diseases initiative (DNDi), include new

candidate compounds and modiﬁcations of existing drug regimens

(lower doses, shorter treatment durations and combinations). A

new BZ monotherapy regimen, BZ/fosravuconazole combination

therapy, and clinical trials of fexinidazole are examples of this [2].

However, despite these efforts, more effective new drugs that are

cheaper, safer and more effective than BZ and NFX, have yet to be

* Corresponding author.

** Corresponding author. Universidad de Navarra, ISTUN Instituto de Salud Trop-

ical, Irunlarrea 1, 31008, Pamplona, Spain.

ꢀ

E-mail addresses: sperez@unav.es (S. Perez-Silanes), Francisco.Olmo@lshtm.ac.

uk (F. Olmo).

https://doi.org/10.1016/j.ejmech.2021.113646

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I. Beltran-Hortelano, R.L. Atherton, M. Rubio-Hernandez et al.

European Journal of Medicinal Chemistry 223 (2021) 113646

found [5].

2. Results and discussion

Many compounds have been shown to have biological activity

against T. cruzi, as demonstrated in our previous research [6]. Of

note are the arylamine Mannich base-type derivatives, a family of

compounds with the ability to inhibit the iron-containing super-

oxide dismutase (Fe-SOD). The trypanosomatid isoform of this

metalloenzyme, which uses iron as cofactor, is not commonly found

in eukaryotes [6]. In addition, this group of compounds display

other interesting biological activities, including analgesic, anti-

2.1. Chemistry

Based on our group's previous experience in the synthesis of

trypanocidal compounds [1 7e20], we focused our attention on

Mannich base derivatives as their synthesis pathways are simple,

cost-effective and make use of commercially available chemical

reagents. The precise route of synthesis chosen is outlined in

Scheme 1, in which the vinyl ketone (i1) was obtained from the

corresponding methyl ketone (commercially available) using a

diisopropylammonium 2,2,2-triﬂuoroacetate catalyst and para-

formaldehyde, in tetrahydrofuran (THF), under reﬂux and acidic

conditions [26]. The following step describes the condensation of

the imidazole/benzimidazole ring (ii) with the previously syn-

thesised vinyl ketone, via a Michael addition reaction at room

temperature.

bacterial,

anti-cancer,

anti-convulsant,

anti-fungal, anti-

inﬂammatory, antioxidant and anti-viral properties, among others

[7,8]. Thus, Mannich bases represent promising alternatives to

current anti-parasitic agents, due to their potential biological ac-

tivity against parasites responsible for tropical diseases such as

trypanosomiasis [9], leishmaniasis [10e12] and malaria [13e16].

Our group has continued this line of research via the incorporation

of an array of amine fragments and substituents into the general

structure of the Mannich bases [1 7e20], in an attempt to enhance

their anti-chagasic and general anti-parasitic activity.

IR, ¹H NMR and ¹³C NMR spectroscopic data, elemental micro-

analysis (C, H, N) and electrospray ionisation mass spectra (MS-LC

TOF), were required for the full characterisation of all of these new

derivatives, as detailed in the Experimental section. In some cases,

for an accurate assignment of the signals in the ¹H and ¹³C NMR

spectra, which were registered in deuterated dimethylsulfoxide

(DMSO‑d₆), DEPT-135 and heteronuclear simple quantum coher-

ence (gHSQC) experiments were performed.

The discovery pipeline for the development of pre-clinical drug

candidates can be a lengthy process, with an urgent need to design

new, more rapid and efﬁcient methodologies. High-throughput

screening (HTS) approaches are being increasingly used in drug

discovery against parasitic diseases, such as CD, due to the rapidity

of the process, the highly informative data generated, and the

excellent reproducibility [21e24]. However, although HTS allows

many compounds to be tested simultaneously, they produce a high

number of ‘false-positive’ hits. This is mainly due to the lack of

detailed assays to properly assess the activity proﬁle of these

compounds, for example, discriminating between trypanocidal and

trypanostatic properties. Therefore, we have developed an opti-

mized strategy for testing small to medium series of compounds

(<100) that allows those with low activity to be discarded early in

the discovery cascade, the selection of pre-candidates with

conﬁrmed trypanocidal activity, and the synthesis of chemically

related compounds using the pre-candidate(s) as scaffolds. This

approach introduces a signiﬁcant reduction in the number of as-

says, time, materials and costs. The selection process ensures that

only promising compounds in vitro can be progressed to in vivo

assays. As the strategy progresses, the assays become more physi-

ologically relevant, providing valuable information on the kinetics

of the killing proﬁle of each candidate.

2.2. Killing proﬁle of the lead compounds: IC₅₀, SI determination

and kinetics of epimastigote killing

Preliminary screening allowed us to select 8 lead compounds

(7a, 18a, 19a, 21a, 28a, 15b, 17b and 3c) (see Supplementary sec-

tion), which showed activity against T. cruzi CL-Luc:Neon clone

epimastigotes [27] and limited toxicity to BSR mammalian cells,

from an initial library of 69 nitroheterocyclic derivatives. Their IC₅₀

and SI values against epimastigotes and BSR cells were then

determined (Table 1) according to the methodology described in

the Experimental section. Compound 3c was the most promising

candidate, with a lower IC₅₀value than BZ. We also followed the

growth rate of drug-exposed parasites in real time by monitoring

ﬂuorescence for 7 days (Experimental section). Reference drug BZ

is considered “fast-killing”, as a single dose can decrease the par-

asitaemia drastically in 24 h in animal models [28]. Consistent with

Heterocyclic compounds are a widely studied group that have

gained relevance in recent years in medicinal chemistry research.

Imidazole and benzimidazole rings, in particular, have occupied a

notable position in nitrogen-containing heterocyclic chemistry.

Benzimidazole is formed by the fusion of imidazole, a ﬁve-

membered aromatic framework containing two nitrogen atoms,

and benzene. Both heterocycles display amphoteric and highly

polar properties. Moreover, they can easily form diverse, weak

binding interactions with a variety of proteins, enzymes and re-

ceptors in biological systems, displaying broad pharmacological

properties and applications. These pharmacophores have gained

signiﬁcant importance and have been used for many years to treat

parasitic diseases including CD, with several imidazole and benz-

imidazole derivatives having potent anti-T. cruzi activity [25]. We

have therefore further explored the potential of these two hetero-

cyclic moieties in the design and development of new candidates

against T. cruzi. A total of 69 Mannich base-type derivatives con-

taining imidazole and benzimidazole components are presented in

this paper as novel candidates against T. cruzi. Our work also in-

troduces a new in vitro screening strategy, which has allowed rapid

and efﬁcient evaluation of this small library of compounds.

this, at all effective BZ concentrations (>2.5 mM), parasite growth

had dropped by day 3 of the treatment (Fig. 1A). A similar proﬁle

was shown by derivative 3c (Fig. 1B).

2.3. Drug-like properties

Employing the Molinspiration [29] and Osiris Data Warrior [30]

software, compounds (7a,19a, 28a and 3c) were analysed to predict

their physiochemical properties, in relation to absorption and

bioavailability (Table 2). None of the derivatives, including BZ,

violated the Lipinski's rule of ﬁve [31]. It has been well established

that optimal lipophilicity range, along with low logP (<5) and low

topological polar surface area (TPSA), are the major driving forces

that lead to good absorption, including intestinal absorption,

bioavailability, Caco-2 cell permeability, and blood-brain barrier

penetration. Molecules with a TPSA of <140 Å²are indicative of

excellent bioavailability [32]. The calculated logP and TPSA values

for derivatives 7a, 19a, 28a and 3c range from 0.12 to 2.51, sug-

gesting that these compounds should be able to cross cell mem-

branes. This was conﬁrmed by their ability to kill epimastigotes.

The drug score combines drug likeness, logP, molecular weight

and toxicity risks in one handy value that can be used to infer

therapeutic potential. A value of >0.5 is indicative of theoretical

2

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I. Beltran-Hortelano, R.L. Atherton, M. Rubio-Hernandez et al.

European Journal of Medicinal Chemistry 223 (2021) 113646

Scheme 1. Reagents and conditions for the synthesis of 69 Mannich base derivatives: (i) paraformaldehyde, diisopropylammonium 2,2,2-triﬂuoroacetate, triﬂuoroacetic acid,

THF, reﬂux, 8 h; (ii) THF, K₂CO₃, rt, from 24 to 72 h.

promise as a safe and efﬁcient drug [33]. Compound 3c had the

highest drug score (0.61) among the evaluated compounds. For

comparison, BZ displayed a drug score of 0.33.

values were still relatively modest, at 9.1 and 12.2 mM, respectively.

Non-replicative trypomastigotes are the parasite forms

responsible for cellular invasion, as well as the form taken up by the

insect vectors in a blood meal from an infected host. We tested the

selected compounds against this ﬂagellated extracellular form

(Experimental section). Compounds 28a and 3c were the most

2.4. IC₅₀and SI determination against amastigotes and

trypomastigotes

active derivatives, with IC₅₀values of 16.6 and 9.1

mM, respectively,

even more active than BZ (22.4 M). Compound 19a displayed

m

We also evaluated the ability of compounds 7a, 19a, 28a and 3c

to inhibit the replication of intracellular amastigotes (Table 3).

Amastigotes are the most clinically relevant life-cycle stage in the

mammalian host. Effective drugs must be able to cross the host cell

membrane to act against the parasite. The IC₅₀values, which were

calculated after 3 days exposure (see Experimental section),

revealed that derivatives 28a and 3c were similarly effective against

both epimastigotes and amastigotes. In contrast, compounds 7a

and 19a were more effective against amastigotes, although the IC₅₀

similar activity to the reference drug. In contrast, derivative 7a was

inactive against this parasitic form and discarded from future

assays.

2.5. Wash-out assays

To fully understand the activity proﬁle of compounds 19a, 28a

and 3c, we undertook wash-out assays, modifying a protocol

3

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European Journal of Medicinal Chemistry 223 (2021) 113646

Table 1

IC₅₀values (mM) for inhibition of the growth of T. cruzi CL-Luc:Neon clone epimastigotes and for the cytotoxicity toward BSR host cells of selected compounds. BZ has been

included as a reference drug. Data are presented as the mean SD of triplicates of three independent experiments after 72 h exposure. Yellow shading highlights compounds

selected for further evaluation.

Fig. 1. Kinetics of growth inhibition of T. cruzi CL-Luc:Neon clone epimastigotes over time in the presence of BZ (A) and compound 3c (B). Data are presented as units of ﬂuo-

rescence, obtained by real time ﬂuorescence readout for 5 days, by which time epimastigotes had reached the stationary phase.

Table 2

Theoretical molecular properties for selected compounds 7a, 19a, 28a and 3c.

Molecular Properties (Molinspiration and Osiris Calculations)

Compound

MW

cLogP

cLogS

TPSA

H

NV

Vol

Drug LK

Drug

Acep

Donor

Score

7a

245.24

268.29

275.26

260.25

0.12

2.51

0.42

0,12

ꢀ2.15

ꢀ3.61

ꢀ2.43

ꢀ2.15

80.72

34.89

89.95

92.75

6

3

7

0

1

0

212.58

238.17

238.13

224.99

ꢀ1.35

2.43

0.57

0.43

0.61

0.33

19a

28a

3c

ꢀ2.55

BZ

ꢀ1.71

Table 3

IC₅₀values (

of triplicates of three independent experiments after 72 h of incubation. nd, non-determined.

mM) against T. cruzi amastigotes and trypomastigotes of compounds 7a, 19a, 28a and 3c. BZ was included as a reference drug. Data are presented as the mean SD

Compound

IC₅₀(mean SD),

Amastigotes

m

M

Selectivity Index (SI)

Amastigotes

Trypomastigotes

BSR Cells

Trypomastigotes

7a

9.1 0.6

12.2 1.7

6.1 1.6

3.3 1.3

0.5 0.1

nd

93.9 1.9

177.4 1.8

72.2 1.2

84.7 1.2

267.3 1.3

10

15

12

28

504

nd

8

4

9

12

19a

28a

3c

23.0 1.0

16.6 0.3

9.1 2.3

22.4 3.5

BZ

described by Cal et al. [34] (Experimental section). The usefulness

of this assay has become more apparent since the advent of high-

throughput screening technologies. It allows viable persistent

parasites that survive treatment, because of their reduced drug

susceptibility, to be identiﬁed [35]. This assay provides an early

insight into the potential for compound failure, before their use in

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European Journal of Medicinal Chemistry 223 (2021) 113646

animal models. In the wash-out assay, parasite-infected cells were

3c could be inferred to have a strong trypanocidal effect when given

for 20 days at the 20 x IC₅₀concentration. By day 10 post-exposure,

this type of trypanocidal proﬁle was already visible, with appar-

ently destroyed parasites, some of which were broken up, and

others already taken into the lysosomal pathway of host cells for

degradation (Fig. 2F). Even 30 days after drug removal (Fig. 2I), the

BZ and compound 3c cultures remained clear of parasites. Collec-

tively, these results conﬁrm that 3c can eliminate T. cruzi intracel-

lular stages, without the survival of persistent parasites.

exposed to high test compound concentrations (10 and 20 x IC₅₀

)

for either 10 or 20 days, after which the compound was removed

from the culture medium. Infected and treated cultures were then

monitored by ﬂuorescent microscopy for a further 30 days after

removal of the drugs, to investigate parasite relapse (Fig. 2). By day

3 post-treatment, there was growth inhibition (Fig. 2A). However,

in all cases where less than 10 x IC₅₀treatment conditions were

used, renewed parasite growth was detectable, regardless of

whether initial exposure was for 10 or 20 days. In the case of de-

rivatives 19a and 28a, where due to host cell toxicity, we could only

use the 10 x IC₅₀concentration, intact parasites could still be seen

after 10 days treatment (Fig. 2BeD). Even with 20 days exposure,

dividing parasites were detectable 10 days after compound removal

(Fig. 2H), indicative of a trypanostatic mode of action for these

derivatives. By contrast, both the reference drug BZ and compound

2.6. Infectivity assay

Trypomastigotes are often more refractory to drug treatment

than the replicating amastigote form, which is consistent with the

obtained IC₅₀values (Table 3). For these assays, we determined the

drug concentrations that reduced trypomastigote infectivity by 50%

Fig. 2. Wash-out assays for BZ and derivatives 19a, 28a and 3c against T. cruzi CL-Luc:Neon clone amastigotes in BSR infected cells. A, Growth inhibition after 72 h treatment

measured as ﬂuorescent intensity compared to untreated group. B, Representative ﬁelds (of 30 captures per treatment) from the treated wells under epiﬂuorescence microscopy at

different timepoints post-treatment. C, D, E, F, G, H and I. Representative zoomed areas of interest under different treatments visualised by epiﬂuorescence microscopy. Cell dyes

(mitotracker and lysotracker) are used for a better location of parasites in the intracellular domain. dpTx, days post-treatment; dpWO, days post drug wash-out. N, mammalian cell

nuclei. White arrows indicate parasites. Scale bars ¼ 50

mm.

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European Journal of Medicinal Chemistry 223 (2021) 113646

compared to untreated control. We also sought to determine the

minimal concentration of drug required to produce a sterile

infection following 6 h incubation with trypomastigotes. Using

ﬂuorescence microscopy, we found that pre-exposure to 3c at

2.7. In vitro toxicity screening: comet assay

The formamide pyrimidine DNA glycosylase (Fpg)-modiﬁed

comet assay was performed to check the potential genotoxic effect

of derivative 3c on human cells. To do so, we used the human

lymphoblastoid TK-6 cell line, which is widely used in genotoxicity

testing since they are p53-competent (recommended by several

OECD guidelines for in vitro genotoxicity and mutagenicity assays).

This version of the assay detects both DNA strand breaks (SBs)

concentrations >50

tivity. In contrast, even at BZ concentrations >100

m

M completely blocked trypomastigote infec-

M, some

m

persistent parasites were observed 5 days post-infection (Fig. 3).

Whether the impact of 3c on infection was due to trypomastigotes

being killed directly by drug action, or by damage that prevents

them infecting host cells remains unknown.

and

oxidised/alkylated

bases

[36,37].

To

prevent

a

Fig. 3. Infectivity assays of T. cruzi CL-Luc:Neon trypomastigotes pre-treated with BZ or derivatives 19a, 28a and 3c for 6 h prior to infection of BSR cells (Experimental section).

Values were established from amastigote ﬂuorescence 5 days post-infection. Images of broad ﬁeld captures are shown at 20x under epiﬂuorescence microscopy for increasing drug

concentrations. These are representative of 30 images taken per treatment. Host-cell nuclei are shown in blue (DAPI staining), with parasites in green; scale bars ¼ 50

mm.

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European Journal of Medicinal Chemistry 223 (2021) 113646

misinterpretation of the comet assay readout due to widespread

DNA degradation induced by lethal doses, we simultaneously ran a

proliferation assay. The latter acts to conﬁrm cell integrity. DNA

damage associated with cells under conditions where proliferation

is inhibited by >25% is considered a false positive outcome with the

comet assay. Fig. 4 shows the results obtained in the Fpg-modiﬁed

and the proliferation assays after 3 h of treatment. A 46% decrease

3. Conclusion

In summary, we synthesised and characterised a total of 69

Mannich base-type derivatives bearing imidazole and

benzimidazole-functionalized cores. In vitro cell-based studies

demonstrated that, of these, compound 3c had the most promising

trypanocidal activity across the distinct life stages of the parasite. It

displayed fast-killing activity at low micromolar doses, lacked host

cell genotoxicity, and had a synergistic mode of action against

trypomastigotes in combination with the reference drug BZ.

in proliferation was observed in TK-6 cells treated with 40 mM 3c

(data not shown), and for the reasons explained, comet assays were

not performed at concentrations up to and beyond this limit. For all

the concentrations below, the levels of SBs and Fpg-sensitive sites

were not signiﬁcantly different (p > 0.05) from the untreated

4. Experimental section

controls. In contrast, analysis of cells treated with 20 mM of the DNA

alkylating agent MMS (methyl methanesulphonate), which were

included a positive control, revealed extensive genotoxic damage

(Fig. 4). Collectively, these results are consistent with a safe proﬁle

for compound 3c, in terms of DNA damage, over the concentration

range at which it is therapeutically active against the different

forms of T. cruzi.

4.1. Chemistry

Chemical reagents used for compound synthesis were pur-

chased from: E. Merck (Darmstadt, Germany); Panreac Química S.A.

(Montcada I Reixac, Barcelona, Spain); Sigma-Aldrich Química S.A.

(Alcobendas, Madrid, Spain); Acros Organics (Janssen Pharmaceu-

ticalaan, Geel, Belgium); abcr GmbH (Karlsruhe, Germany); Fluo-

rochem Ltd. (Hadﬁeld, Derbyshire, United Kingdom). The progress

of all reactions was monitored by thin layer chromatography (TLC)

using SIL G/UV₂₅₄, 0.2 mm thickness (ALUGRAM® Xtra SIL G,

Macherey-Nagel GmbH & Co. KG) as the stationary phase, and

solvent mixtures (CH₂Cl₂/methanol or hexane/ethyl acetate) as the

mobile phase; UV lamps (254 nm) were employed to spots detec-

tion. Puriﬁcation of compounds was carried out on a CombiFlash®

RF instrument (Teledyne Isco), employing Silica RediSep Rf Gold®

columns in a normal phase gradient. Infrared spectra (IR) were

recorded on a Thermo Nicolet FT-IR Nexus spectrophotometer us-

ing potassium bromide (KBr) pellets for solid samples. IR absorp-

tion pics signals (cm^ꢀ1) were expressed as strong (s), medium (m),

and weak (w). Proton (¹H) and carbon (¹³C) NMR spectra of every

compound were recorded on a Bruker Advance Neo 400 Ultra-

shield™ spectrometer (Rheinstetten, Germany) operating at 400/

100 MHz (¹H/¹³C) and using DMSO‑d₆as solvent and tetrame-

2.8. Drug combination assay

Whilst BZ is effective in long regimens, toxicity is a major reason

for low rates of compliance, which ultimately leads to treatment

failure. There is a strong rationale to search for compounds that in

combination with BZ can allow its dosage and/or treatment period

to be reduced, while obtaining similar curative rates. The trypa-

nocidal effect of derivative 3c against trypomastigotes, with activity

greater than BZ, encouraged us to undertake a combinatory assay.

The results (Fig. 5) showed a synergic effect of 3c on the BZ IC₅₀

.

While this effect was minimal against intracellular forms, it was

prominent against both extracellular forms. In the case of the epi-

mastigotes (insect vector forms), 3c decreased the BZ IC₅₀by almost

70%, in comparison with a single treatment of BZ. The synergic

effect was even greater in the case of trypomastigotes (mammalian

infective forms), where the IC₅₀value dropped to almost a quarter

of that obtained for BZ alone.

thylsilane (TMS) as reference. Chemical shifts (d) were reported in

parts per million (ppm), coupling constants (J) were given in hertz

(Hz) and multiplicities detected in ¹H-RMN. Elemental analysis was

performed on a LECO CHN-900 Elemental Analyzer (Leco, Tres

Cantos, Spain). Purity of compounds was conﬁrmed when

elemental analysis values were within the range of 0.4 with

respect to theoretical values. Melting points (Mp) were determined

on a Mettler FP82 þ FP80 apparatus (Greifensee, Switzerland).

High-resolution mass spectrometry data were obtained using G220

Accurate-Mass TOF LC/MS (time of ﬂight analyzer) and HPLC 1200

series Agilent® Technologies.

4.2. General procedure for the synthesis of compounds

4.2.1. Synthesis of the intermediate compounds (i1)

To a solution of the corresponding methyl ketone reagent (1eq)

in THF (10 mL), the paraformaldehyde (8eq), diisopropylammo-

nium 2,2,2-triﬂuoroacetate catalyst (1.5eq) and triﬂuoroacetic acid

(0.1eq) were added. The mixture was stirred at reﬂux for 8 h. New

8eq of paraformaldehyde were added to the mixture at 2 h and 5 h

of the reaction. The reaction was monitored by TLC (hexane/ethyl

acetate). Following this, the solvent was removed under vacuum by

rotatory evaporation. The residue was diluted in diethyl ether and

the corresponding vinyl ketone i1 was isolated by a triple extrac-

tion after addition of HCl 1 N (25 mL), NaOH 1 N (25 mL) and

saturated NaCl solution (25 mL). The organic phase was dried over

anhydrous Na₂SO₄, ﬁltered and the solvent was removed under

vacuum.

Fig. 4. Comet and proliferation assays performed on 3c-treated TK-6 cells after 3 h

exposure. SBs and Net-Fpg sensitive sites are represented as a percentage of the DNA in

the comet's tail relative to the DNA remaining in the comet. SBs and Fpg-sensitive sites

were not statistically different under all conditions tested when compared to control

valves (p > 0.05). For the proliferation assay, cells were washed after the treatment and

cultured for 48 h. Cells were then counted and the relative suspension growth (RSG)

was calculated (Experimental section) in relation to the untreated control. Results are

presented as the mean SD of triplicates of three independent experiments. MMS-

treated cells (20 mM) were included as a positive control for DNA damage.

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European Journal of Medicinal Chemistry 223 (2021) 113646

Fig. 5. Drug combination assay. A, isobolograms of concentration-effect curves of 3c and BZ and their combinations against different developmental forms of T. cruzi. B, table

showing the variation of BZ IC₅₀when the assays were performed in the presence of the IC₅₀concentrations of 3c; Ci (coefﬁcient of interaction) shows the synergic relation between

drugs (Ci < 1). Data are shown as mean SD (n ¼ 3). Orange lines link the IC₅₀values for drugs assayed independently (Da, BZ; Db, 3c), while blue lines show the shift of the IC₅₀for

BZ (da) assayed in the presence of 3c (db).

4.2.2. Synthesis of imidazole (1a-31a) and benzimidazole Mannich

bases-type derivatives (1be17b)

196.32 (CO); 165.16 (d, C₄, ¹J ¼ 252 Hz); 137.42 (C_2’); 133.00 (d, C₁,

⁴J ¼ 2.8 Hz); 130.99 (2C, d, C₂þC₆, ³J ¼ 9.5 Hz); 128.25 (C_4’); 119.41

(C_5’); 115.75 (2C, d, C₃þC₅, ²J ¼ 21.9 Hz); 41.13 (CH₂eN); 39.23

(CH₂eCO). Anal. Calc. for C₁₂H₁₁FN₂O: C 66.05%, H 5.08%, N 12.84%.

Found: C 65.67%, H 5.37%, N 12.77%.

The general procedure for the synthesis of all ﬁnal compounds

was carried out by Michael addition reaction between the previ-

ously synthesised vinyl ketones (i1) and imidazole/benzimidazole.

To a solution of i1 (1eq) in THF (20 mL) was added the corre-

sponding amine (1eq) and K₂CO₃(1.2eq), then stirred at room

temperature for 24e72 h. The reaction was monitored by TLC

(DCM/methanol). The residue was puriﬁed by automated ﬂash

chromatography using DCM/methanol gradient solvent. Next, the

solvent was removed under vacuum by rotatory evaporation. The

residue was diluted in DCM and the corresponding ﬁnal com-

pounds were isolated by an extraction after addition of water

(25 mL x 3 times). The organic phase was dried over anhydrous

Na₂SO₄, ﬁltered and the solvent was removed under vacuum. The

ﬁnal compounds were obtained by precipitation with n-hexane.

1-(4-chlorophenyl)-3-(1H-imidazole-1-yl)propan-1-one

Yield: 21%. Mp: 105.0e106.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1680 (s,

NMR (DMSO‑d₆, 400 MHz)

(3a).

n

_C]_O). ¹H

d

ppm: 7.98 (d, 2H, H₂þH₆, J_2-3¼ J_6-

¼ 8.6 Hz); 7.65 (s, 1H, H_2’); 7.59 (d, 2H, H₃þH₅, J_3-2¼ J_5-6¼ 8.6 Hz);

5

7.20 (s, 1H, H_5’); 6.86 (s, 1H, H_4’); 4.32 (t, 2H, CH₂eN, J_CH2-

¼ 6.8 Hz); 3.59 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

CH2

(DMSO‑d₆, 100 MHz)

d ppm: 196.77 (CO); 138.39 (C₄); 137.42 (C_2’);

134.89 (C₁); 129.88 (2C, C₂þC₆); 128.85 (2C, C₃þC₅); 128.26 (C_4’);

119.41 (C_5’); 41.08 (CH₂eN); 39.29 (CH₂eCO). Anal. Calc. for

C

₁₂H₁₁ClN₂O: C 61.42%, H 4.72%, N 11.94%. Found: C 61.38%, H 4.95%,

N 11.91%.

3-(1H-imidazole-1-yl)-1-(4-methoxyphenyl)propan-1-one (4a).

Yield: 32%. Mp: 76.5e77.5 ^ꢁC. IR (KBr) cm^ꢀ1: 1669 (s, _C]_O). ¹H

NMR (DMSO‑d₆, 400 MHz)

n

4.3. Compound characterisation

d

ppm: 7.95 (d, 2H, H₂þH₆, J_2-3¼ J_6-

¼ 8.9 Hz); 7.65 (s, 1H, H_2’); 7.20 (s, 1H, H_5’); 7.04 (d, 2H, H₃þH₅, J_3-

¼ J_5-6¼ 8.8 Hz); 6.85 (s, 1H, H_4’); 4.30 (t, 2H, CH₂eN, J_CH2-

¼ 6.8 Hz); 3.83 (s, 3H, OCH₃); 3.52 (t, 2H, CH₂eCO, J_CH2-

5

2

3-(1H-imidazole-1-yl)-1-phenylpropan-1-one (1a). Yield: 24%.

Mp: 96.5e97.5 ^ꢁC. IR (KBr)

n n

cm^ꢀ1: 1679 (s, _C]_O). ¹H NMR

CH2

(DMSO‑d₆, 400 MHz)

d

ppm: 7.97 (dd, 2H, H₂þH₆, J_2-3¼ J_6-

¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm: 195.98 (CO);

¼ 8.3 Hz, J_2-4¼ J_6-4¼ 1.2 Hz); 7.66 (s, 1H, H_2’); 7.64 (td, 1H, H₄, J_4-

5

3

163.32 (C₄); 137.41 (C_2’); 130.29 (2C, C₂þC₆); 129.24 (C₁); 128.23

(C_4’); 119.44 (C_5’); 113.92 (2C, C₃þC₅); 55.55 (OCH₃); 41.31

(CH₂eN); 38.89 (CH₂eCO). Anal. Calc. for C₁₃H₁₄N₂O₂: C 67.81%, H

6.13%, N 12.17%. Found: C 67.84%, H 6.31%, N 12.24%.

¼ J_4-5¼ 6.9 Hz, J_4-2¼ J_4-6¼ 1.6 Hz); 7.55e7.51 (m, 2H, H₃þH₅); 7.21

(s, 1H, H_5’); 6.86 (s, 1H, H_4’); 4,33 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz);

3.60 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆,

100 MHz) d ppm: 197.71 (CO); 137.44 (C_2’); 136.23 (C₁); 133.47 (C₄);

3-(1H-imidazole-1-yl)-1-(4-(triﬂuoromethyl)phenyl)propan-1-

128.74 (2C, C₂þC₆); 128.25 (C_4’); 127.94 (2C, C₃þC₅); 119.42 (C_5’);

41.17 (CH₂eN); 39.27 (CH₂eCO). Anal. Calc. for C₁₂H₁₂N₂O: C

71.98%, H 6.04%, N 13.99%. Found: C 71.78%, H 6.29%, N 13.86%.

1-(4-ﬂuorophenyl)-3-(1H-imidazole-1-yl)-1-phenylpropan-1-one

one (5a). Yield: 25%. Mp: 69.0e69.5 ^ꢁC. IR (KBr)

n n

cm^ꢀ1: 1683 (s,

_C]_O), 1325 (s,

(DMSO‑d₆, 400 MHz)

n

_Ce_F), 1211 (s,

ppm: 8.16 (d, 2H, H₂þH₆, J_2-3¼ J_6-

n _Ce_F), 1164 (s, n

_Ce_F). ¹H NMR

d

¼ 8.1 Hz); 7.90 (d, 2H, H₃þH₅, J_3-2¼ J_5-6¼ 8.3 Hz); 7.67 (s, 1H, H_2’);

5

(2a). Yield: 26%. Mp: 65.0e66.0 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1681 (s,

ppm: 8.06 (dd, 2H,

n _C]_O),

7.22 (s, 1H, H_5’); 6.87 (s, 1H, H_4’); 4.34 (t, 2H, CH₂eN, J_CH2-

1226 (s,

n

_Ce_F). ¹H NMR (DMSO‑d₆, 400 MHz)

d

¼ 6.8 Hz); 3.67 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

CH2

H₂þH₆, J_2-3¼ J_6-5¼ 8.8 Hz, J_2-F¼ J_6-F¼ 5.5 Hz); 7.65 (s, 1H, H_2’); 7.35

(t, 2H, H₃þH₅, J_3-2¼ J_5-6¼ J_3-F¼ J_5-F¼ 8.8 Hz); 7.20 (s, 1H, H_5’); 6.86

(s, 1H, H_4’); 4.32 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.59 (t, 2H,

(DMSO‑d₆, 100 MHz)

d ppm: 197.27 (CO); 139.32 (C₁); 137.45 (C_2’);

132.76 (q, C₄, ²J ¼ 32.1 Hz); 128.80 (2C, C₂þC₆), 128.31 (C_4’); 125.75

(2C, q, C₃þC₅, ³J ¼ 3.6 Hz); 123.75 (q, CF₃¹J ¼ 272.8 Hz); 119.43

CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm:

8

ꢀ

I. Beltran-Hortelano, R.L. Atherton, M. Rubio-Hernandez et al.

European Journal of Medicinal Chemistry 223 (2021) 113646

(C_5’); 41.02 (CH₂eN); 39.69 (CH₂eCO). Anal. Calc. for C₁₃H₁₁F₃N₂O:

C 58.21%, H 4.13%, N 10.44%. Found: C 58.60%, H 4.46%, N 10.46%.

3-(1H-imidazole-1-yl)-1-(p-tolyl)propan-1-one (6a). Yield: 24%.

129.60 (C₃); 128.76 (C₇); 128.33 (C_4’); 128.21 (C₈); 127.67 (C₉);

126.98 (C₄); 123.31 (C₆); 119.49 (C_5’); 41.35 (CH₂eN); 39.42

(CH₂eCO). Anal. Calc. for C₁₆H₁₄N₂O: C 76.78%, H 5.64%, N 11.19%.

Found: C 76.63%, 5.47H %, N 11.06%.

Mp: 55.0e56.0 ^ꢁC. IR (KBr)

(DMSO‑d₆, 400 MHz)

n n

cm^ꢀ1: 1681 (s, _C]_O). ¹H NMR

d

ppm: 7.87 (d, 2H, H₂þH₆, J_2-3¼ J_6-

1-(benzo[b]thiophen-3-yl)-3-(1H-imidazole-1-yl)propan-1-one

¼ 8.2 Hz); 7.65 (s, 1H, H_2’); 7.33 (d, 2H, H₃þH₅, J_3-2¼ J_5-6¼ 8.0 Hz);

(12a). Yield: 33%. Mp: 105.0e106.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1666 (s,

n n

5

7.20 (s, 1H, H_5’); 6.86 (s, 1H, H_4’); 4.31 (t, 2H, CH₂eN, J_CH2-

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d ppm: 9.00 (s, 1H, H₂); 8.61

¼ 6.8 Hz); 3.55 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz); 2.37 (s, 3H,

(dd, 1H, H₅, J_5-6¼ 8.0 Hz, J_5-8¼ 0.7 Hz); 8.08 (dd, 1H, H₈, J_8-

CH2

CH₃). ¹³C NMR (DMSO‑d₆,100 MHz)

d

ppm: 197.18 (CO); 143.87 (C₄);

¼ 7.9 Hz, J_8-5¼ 0.5 Hz); 7.68 (s, 1H, H_2’); 7.53e7.44 (m, 2H,

7

137.41 (C_2’); 133.80 (C₁); 129.27 (2C, C₃þC₅); 128.23 (C_4’); 128.05

(2C, C₂þC₆); 119.41 (C_5’); 41.22 (CH₂eN); 39.14 (CH₂eCO); 21.13

(CH₃). Anal. Calc. for C₁₃H₁₄N₂O: C 72.87%, H 6.59%, N 13.07%.

Found: C 72.72%, H 6.68%, N 13.24%.

H₆þH₇); 7.23 (s, 1H, H_5’); 6.87 (s, 1H, H_4’); 4.37 (t, 2H, CH₂eN, J_CH2-

¼ 6.8 Hz); 3.62 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.9 Hz). ¹³C NMR

CH2

(DMSO‑d₆, 100 MHz)

d ppm: 193.12 (CO); 140.21 (C₂); 139.35 (C₃);

137.42 (C_2’); 136.07 (C₄); 133.82 (C₉); 128.29 (C_4’); 125.82 (C₆);

125.40 (C₇); 124.61 (C₅); 122.91 (C₈); 119.43 (C_5’); 41.25 (CH₂eN);

40.67 (CH₂eCO). Anal. Calc. for C₁₄H₁₂N₂OS: C 65.60%, H 4.72%, N

10.93%. Found: C 65.75%, H 5.03%, N 10.86%.

3-(1H-imidazole-1-yl)-1-(4-nitrophenyl)-propan-1-one

Yield: 42%. Mp: 123.0e124.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1682 (s,

1520 (s, n _NO2), 1350 (s, n _NO2). ¹H NMR (DMSO‑d₆, 400 MHz)

(7a).

_C]_O),

ppm:

n

d

8.34 (d, 2H, H₃þH₅, J_3-2¼ J_5-6¼ 8.8 Hz); 8.20 (d, 2H, H₂þH₆, J_2-

1-(benzo[b]thiophen-2-yl)-3-(1H-imidazole-1-yl)propan-1-one

¼ J_6-5¼ 8.8 Hz); 7.67 (s, 1H, H_2’); 7.21 (s, 1H, H_5’); 6.87 (s, 1H, H_4’);

(13a). Yield: 31%. Mp: 149.0e150.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1660 (s,

n n

3

4.34 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.69 (t, 2H, CH₂eCO, J_CH2-

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d ppm: 8.40 (s, 1H, H₃); 8.05 (d,

¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d

ppm: 197.02 (CO);

1H, H₈, J_8-7¼ 8.1 Hz); 8.01 (d, 1H, H₅, J_5-6¼ 7.8 Hz); 7.67 (s, 1H, H_2’);

7.54 (t,1H, H₆, J_6-5¼ J_6-7¼ 7.1 Hz); 7.47 (t,1H, H₇, J_7-8¼ J_7-6¼ 7.2 Hz);

7.23 (s, 1H, H_5’); 6.87 (s, 1H, H_4’); 4.36 (t, 2H, CH₂eN, J_CH2-

CH2

150.07 (C₄); 140.71 (C₁); 137.43 (C_2’); 129.40 (2C, C₂þC₆); 128.31

(C_4’); 123.85 (2C, C₃þC₅); 119.41 (C_5’); 40.98 (CH₂eN); 39.65

(CH₂eCO). Anal. Calc. for C₁₂H₁₁N₃O₃: C 58.77%, H 4.52%, N 17.13%.

Found: C 58.74%, H % 4.71, N % 16.75.

¼ 6.8 Hz); 3.66 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

CH2

(DMSO‑d₆, 100 MHz)

d ppm: 192.42 (CO); 142.58 (C₉); 141.52 (C₄);

3-(1H-imidazole-1-yl)-1-(thiophen-3-yl)propan-1-one

Yield: 45%. Mp: 86.5e87.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1659 (s,

NMR (DMSO‑d₆, 400 MHz)

(8a).

139.04 (C₂); 137.42 (C_2’); 131.26 (C₃); 128.33 (C_4’); 127.84 (C₆);

126.35 (C₅); 125.30 (C₇); 123.15 (C₈); 119.39 (C_5’); 41.18 (CH₂eN);

39.61 (CH₂eCO). Anal. Calc. for C₁₄H₁₂N₂OS: C 65.60%, H 4.72%, N

10.93%. Found: C 65.66%, H 5.07%, N 10.74%.

n

_C]_O). ¹H

d

ppm: 8.53 (dd, 1H, H₂, J_2-5¼ 2.8 Hz, J_2-

¼ 1.3 Hz); 7.64 (s, 1H, H_2’); 7.63 (dd, 1H, H₅, J_5-4¼ 5.1 Hz, J_5-

¼ 2.8 Hz); 7.51 (dd, 1H, H₄, J_4-5¼ 5.1 Hz, J_4-2¼ 1.3 Hz); 7.19 (s, 1H,

4

2

1-(benzofuran-2-yl)-3-(1H-imidazole-1-yl)propan-1-one (14a).

H_5’); 6.86 (s, 1H, H_4’); 4.30 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.48 (t,

Yield: 26%. Mp: 131.0e132.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1677 (s, _C]_O). ¹H

n n

2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

NMR (DMSO‑d₆, 400 MHz)

(d, 1H, H₅, J_5-6¼ 7.8 Hz); 7.71 (dd,1H, H₈, J_8-7¼ 8.4 Hz, J_8-6¼ 0.6 Hz);

7.66 (s, 1H, H_2’); 7.54 (td, 1H, H₇, J_7-8¼ 8.4 Hz, J_7-6¼ 7.3 Hz, J_7-

d

ppm: 7.95 (d,1H, H₃, J_3-5¼ 0.7 Hz); 7.83

d

ppm: 192.01 (CO); 141.52 (C₃); 137.14 (C_2’); 134.12 (C₂); 128.27

(C_4’); 127.59 (C₅); 126.37 (C₄); 119.40 (C_5’); 41.07 (CH₂eN); 40.31

(CH₂eCO). Anal. Calc. for C₁₀H₁₀N₂OS: C 58.23%, H 4.89%, N 13.58%.

Found: C 57.93%, H 4.97%, N 13.64%.

¼ 1.2 Hz); 7.37 (t, 1H, H₆, J_6-5¼ J_6-7¼ 7.9 Hz); 7.21 (s, 1H, H_5’); 6.86

5

(s, 1H, H_4’); 4.36 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.54 (t, 2H,

3-(1H-imidazole-1-yl)-1-(thiophen-2-yl)propan-1-one

Yield: 45%. Mp: 75.5e76.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1673 (s,

NMR (DMSO‑d₆, 400 MHz)

(9a).

CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm:

n

_C]_O). ¹H

187.97 (CO); 155.00 (C₉); 151.62 (C₂); 137.43 (C_2’); 128.67 (C₇);

128.35 (C_4’); 126.68 (C₄); 124.11 (C₆); 123.75 (C₅); 119.39 (C_5’);

114.59 (C₃); 112.26 (C₈); 40.91 (CH₂eN); 39.46 (CH₂eCO). Anal.

Calc. for C₁₄H₁₂N₂O₂: C 69.99%, H 5.03%, N 11.66%. Found: C 70.35%,

H 5.25%, N 11.63%.

d

ppm: 8.02 (dd, 1H, H₅, J_5-4¼ 4.9 Hz, J_5-

¼ 1.1 Hz); 7.98 (dd, 1H, H₃, J_3-4¼ 3.8 Hz, J_3-5¼ 1.1 Hz); 7.64 (s, 1H,

3

H_2’); 7.24 (dd, 1H, H₄, J_4-5¼ 4.9 Hz, J_4-3¼ 3.8 Hz); 7.19 (s, 1H, H_5’);

6.85 (s, 1H, H_4’); 4.31 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.52 (t, 2H,

CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d

ppm:

1-(4-bromophenyl)-3-(1H-imidazole-1-yl)propan-1-one

Yield: 23%. Mp: 95.5e96.5 ^ꢁC. IR (KBr) cm^ꢀ1: 1686 (s,

NMR (DMSO‑d₆, 400 MHz)

(15a).

190.65 (CO); 143.30 (C₂); 137.40 (C_2’); 135.20 (C₅); 133.80 (C₃);

128.78 (C₄); 128.29 (C_4’); 119.39 (C_5’); 41.16 (CH₂eN); 39.61

(CH₂eCO). Anal. Calc. for C₁₀H₁₀N₂OS: C 58.23%, H 4.89%, N 13.58%.

Found: C 58.04%, H 5.07%, N 14.07%.

n

_C]_O). ¹H

d

ppm: 7.91 (d, 2H, H₂þH₆, J_2-3¼ J_6-

¼ 8.6 Hz); 7.74 (d, 2H, H₃þH₅, J_3-2¼ J_5-6¼ 8.6 Hz); 7.66 (s, 1H, H_2’);

5

7.21 (s, 1H, H_5’); 6.86 (s, 1H, H_4’); 4.32 (t, 2H, CH₂eN, J_CH2-

1-(furan-2-yl)-3-(1H-imidazole-1-yl)propan-1-one (10a). Yield:

¼ 6.8 Hz); 3.59 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

CH2

21%. Mp: 92.0e93.0 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1684 (s,

n

_C]_O). ¹H NMR

(DMSO‑d₆, 100 MHz) d ppm: 196.98 (CO); 137.43 (C_2’); 135.19 (C₁);

(DMSO‑d₆, 400 MHz)

d

ppm: 7.99 (dd, 1H, H₅, J_5-4¼ 1.6 Hz, J_5-

131.81 (2C, C₃þC₅); 129.98 (2C, C₂þC₆); 128.27 (C_4’); 127.61 (C₄);

¼ 0.6 Hz); 7.62 (s, 1H, H_2’); 7.50 (dd, 1H, H₃, J_3-4¼ 3.6 Hz, J_3-

¼ 0.5 Hz); 7.17 (s, 1H, H_5’); 6.85 (s, 1H, H_4’); 6.71 (dd, 1H, H₄, J_4-

¼ 3.6 Hz, J_4-5¼ 1.7 Hz); 4.30 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.37

119.42 (C_5’); 41.08 (CH₂eN); 39.31 (CH₂eCO). Anal. Calc. for

3

5

3

C

₁₂H₁₁BrN₂O: C 51.63%, H 3.97%, N 10.04%. Found: C 52.01%, H

4.01%, N 10.03%.

(t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

3-(1H-imidazole-1-yl)-1-(4-(triﬂuoromethoxy)phenyl)propan-1-

d

ppm: 185.78 (CO); 151.57 (C₂); 148.04 (C₅); 137.39 (C_2’); 128.31

one (16a). Yield: 23%. Mp: 45.0e46.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1691 (s,

n n

(C_4’); 119.35 (C_5’); 119.03 (C₃); 112.55 (C₄); 40.89 (CH₂eN); 38.96

(CH₂eCO). Anal. Calc. for C₁₀H₁₀N₂O₂: C 63.15%, H 5.30%, N 14.73%.

Found: C 63.35%, H 5.29%, N 15.11%.

_C]_O), 1265 (s,

(DMSO‑d₆, 400 MHz)

n

_Ce_F), 1213 (s,

ppm: 8.11 (d, 2H, H₂þH₆, J_2-3¼ J_6-

n

_Ce_F), 1161 (s, n_C-F). ¹H NMR

d

¼ 8.9 Hz); 7.66 (s, 1H, H_2’); 7.51 (d, 2H, H₃þH₅, J_3-2¼ J_5-6¼ 8.0 Hz);

5

3-(1H-imidazole-1-yl)-1-(naphthalene-2-yl)propan-1-one (11a).

7.20 (s, 1H, H_5’); 6.86 (s, 1H, H_4’); 4.32 (t, 2H, CH₂eN, J_CH2-

Yield: 24%. Mp: 110.5e111.5 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1682 (s,

n

_C]_O). ¹H

¼ 6.8 Hz); 3.62 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

CH2

NMR (DMSO‑d₆, 400 MHz)

d

ppm: 8.70 (s,1H, H₁); 8.12 (d,1H, H₃, J_3-

(DMSO‑d₆, 100 MHz) d ppm: 196.57 (CO); 151.72 (C₄); 137.45 (C_2’);

¼ 7.9 Hz); 8.03e7.97 (m, 3H, H₄þ H₆þ H₉); 7.71 (s, 1H, H_2’);

135.07 (C₁); 130.52 (2C, C₂þC₆); 128.29 (C_4’); 120.80 (2C, C₃þC₅);

119.93 (q, CF₃, ¹J ¼ 262.7 Hz); 119.43 (C_5’); 41.07 (CH₂eN); 39.40

(CH₂eCO). Anal. Calc. for C₁₃H₁₁F₃N₂O₂: C 54.93%, H 3.90%, N 9.86%.

Found: C 54.8%, H 3.85%, N 9.9%.

4

7.70e7.61 (m, 2H, H₈þ H₇); 7.25 (s,1H, H_5’); 6.88 (s,1H, H_4’); 4.39 (t,

2H, CH₂eN, J_CH2-CH2

¼

6.9 Hz); 3.74 (t, 2H, CH₂eCO, J_CH2-

¼ 6.9 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm: 197.62 (CO);

CH2

137.44 (C_2’); 135.14 (C₅); 133.50 (C₁₀); 132.15 (C₂); 130.14 (C₁);

4-(3-(1H-imidazole-1-yl)propanoyl)benzonitrile (17a). Yield:

9

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I. Beltran-Hortelano, R.L. Atherton, M. Rubio-Hernandez et al.

European Journal of Medicinal Chemistry 223 (2021) 113646

30%. Mp: 110.0e111.0 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 2230 (s,

ppm: 8.11 (d, 2H, H₂þH₆, J_2-

n

_C^_N), 1685 (s,

n

¼ 8.9 Hz); 7.20 (s, 1H, H_5’); 6.86 (s, 1H, H_4’); 4.31 (t, 2H, CH₂eN,

6

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d

J_CH2-CH2¼ 6.8 Hz); 3.62 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

¼ J_6-5¼ 8.6 Hz); 8.01 (d, 2H, H₃þH₅, J_3-2¼ J_5-6¼ 8.5 Hz); 7.66 (s,

(DMSO‑d₆, 100 MHz)

d

ppm: 195.64 (CO); 160.19 (d, C₄, ¹J ¼

3

1H, H_2’); 7.20 (s, 1H, H_5’); 6.86 (s, 1H, H_4’); 4.33 (t, 2H, CH₂eN, J_CH2-

254.0 Hz); 137.41 (C_2’); 133.94 (d, C₁, ⁴J ¼ 3.5 Hz); 130.75 (C₂);

129.30 (d, C₆, ³J ¼ 8.8 Hz); 128.25 (C_4’); 120.30 (d, C₃, ²J ¼ 18.2 Hz);

119.41 (C_5’); 117.36 (d, C₅, ²J ¼ 21.5 Hz); 41.03 (CH₂eN); 39.28

(CH₂eCO). Anal. Calc. for C₁₂H₁₀ClFN₂O: C 57.04%, H 3.99%, N

11.09%. Found: C 56.87%, H 4.14%, N 11.14%.

¼ 6.8 Hz); 3.66 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

CH2

(DMSO‑d₆, 100 MHz)

d ppm: 197.23 (CO); 139.28 (C₁); 137.43 (C_2’);

132.81 (2C, C₃þC₅); 128.58 (2C, C₂þC₆); 128.29 (C_4’); 119.41 (C_5’);

118.10 (CN); 115.39 (C₄); 40.98 (CH₂eN); 39.64 (CH₂eCO). Anal.

Calc. for C₁₃H₁₁N₃O: C 69.32%, H 4.92%, N 18.66%. Found: C 69.98%,

H 5.13%, N 18.31%.

1-(3-bromo-4-ﬂuorophenyl)-3-(1H-imidazole-1-yl)propan-1-one

(23a). Yield: 26%. Mp: 118.5e119.5 ^ꢁC. IR (KBr)

n n

cm^ꢀ1: 1686 (s,

3-(1H-imidazole-1-yl)-1-(o-tolyl)propan-1-one (18a). Yield: 30%.

_C]_O), 1245 (s, n d ppm: 8.29 (dd,

_Ce_F). ¹H NMR (DMSO‑d₆, 400 MHz)

Mp: 53.5e54.5 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1668 (s,

n

_C]_O). ¹H NMR

1H, H₂, J_2-F¼ 6.7 Hz, J_2-6¼ 2.1 Hz); 8.03 (ddd,1H, H₆, J_6-5¼ 8.6 Hz, J_6-

(DMSO‑d₆, 400 MHz)

d

ppm: 7.77 (d, 1H, H₆, J_6-5¼ 7.7 Hz); 7.65 (s,

¼ 4.9 Hz, J_6-2¼ 2.2 Hz); 7.65 (s, 1H, H_2’); 7.53 (t, 1H, H₅, J_5-F¼ J_5-

F

1H, H_2’); 7.44 (t, 1H, H₄, J_4-3¼ J_4-5¼ 7.5 Hz); 7.44 (m, 2H, H₃þH₅, J_3-

¼ 8.6 Hz); 7.20 (s, 1H, H_5’); 6.86 (s, 1H, H_4’); 4.31 (t, 2H, CH₂eN,

6

¼ J_5-4¼ 7.5 Hz); 7.20 (s, 1H, H_5’); 6.87 (s, 1H, H_4’); 4.31 (t, 2H,

J_CH2-CH2¼ 6.8 Hz); 3.62 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

4

CH₂eN, J_CH2-CH2¼ 6.7 Hz); 3.50 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.7 Hz);

(DMSO‑d₆, 100 MHz)

d

ppm: 195.56 (CO); 161.24 (d, C₄, ¹J ¼

2.36 (s, 3H, CH₃). ¹³C NMR (DMSO‑d₆, 100 MHz)

d

ppm: 201.51 (CO);

252.3 Hz); 137.41 (C_2’); 134.24 (d, C₁, ⁴J ¼ 3.4 Hz); 133.63 (d, C₂, ³J ¼

1.2 Hz); 129.95 (d, C₆, ³J ¼ 8.9 Hz); 128.25 (C_4’); 119.41 (C_5’); 117.14

(d, C₅, ²J ¼ 22.8 Hz); 108.74 (d, C₃, ²J ¼ 21.7 Hz); 41.03 (CH₂eN);

39.26 (CH₂eCO). Anal. Calc. for C₁₂H₁₀BrFN₂O: C 48.51%, H 3.39%, N

9.43%. Found: C 48.84%, H 3.70%, N 9.49%.

137.42 (C_2’); 137.20 (C₁); 137.13 (C₂); 131.66 (C₃); 131.57 (C₄); 128.79

(C₆); 128.28 (C_4’); 125.93 (C₅); 119.35 (C_5’); 41.93 (CH₂eN); 41.34

(CH₂eCO); 20.64 (CH₃). Anal. Calc. for C₁₃H₁₄N₂O: C 72.87%, H

6.59%, N 13.07%. Found: C 72.96%, H 6.37%, N 12.94%.

3-(1H-imidazole-1-yl)-1-(3-nitrophenyl)-propan-1-one

(19a).

1-(3,4-dimethoxyphenyl)3-(1H-imidazole-1-yl)propan-1-one

Yield: 44%. Mp: 83.5e84.5 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1686 (s,

n

_C]_O), 1526

ppm: 8.66

(24a). Yield: 23%. Mp: 149.0e150.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1671 (s,

n n

(s, n _NO2), 1359 (s, n _NO2). ¹H NMR (DMSO‑d₆, 400 MHz)

d

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d

ppm: 7.64 (m, 2H, H_2’þH₂);

(s, 1H, H₂); 8.47 (d, 1H, H₄, J_4-5¼ 8.2 Hz, J_4-2¼ J_4-6¼ 1.4 Hz); 8.39 (d,

1H, H₆, J_6-5¼ 7.8 Hz); 7.83 (t, 1H, H₅, J_5-4¼ J_5-6¼ 8.0 Hz) 7.68 (s, 1H,

H_2’); 7.22 (s, 1H, H_5’); 6.87 (s, 1H, H_4’); 4.36 (t, 2H, CH₂eN, J_CH2-

7.45 (d, 1H, H₆, J_6-2¼ 2.0 Hz); 7.20 (s, 1H, H_5’); 7.06 (d, 2H, H₅, J_5-

¼ 8.5 Hz); 6.85 (s, 1H, H_4’); 4.31 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz);

6

3.84 (s, 3H, OCH₃); 3.81 (s, 3H, OCH₃); 3.53 (t, 2H, CH₂eCO, J_CH2-

¼ 6.7 Hz); 3.73 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.7 Hz). ¹³C NMR

¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm: 196.07 (CO);

CH2

(DMSO‑d₆, 100 MHz)

d

ppm: 196.83 (CO); 148.53 (C₃); 137.93 (C_2’);

153.29 (C₄); 148.58 (C₃); 137.45 (C_2’); 129.21 (C₁); 128.22 (C_4’);

122.74 (C₂); 119.47 (C_5’); 110.89 (C₅); 110.16 (C₆); 55.76 (C₂); 55.52

(C₁); 41.38 (CH₂eN); 38.84 (CH₂eCO). Anal. Calc. for C₁₄H₁₆N₂O₃: C

64.60%, H 6.20%, N 10.76%. Found: C 64.33%, H 6.33%, N 10.74%.

1-(2,4-dimethoxyphenyl)3-(1H-imidazole-1-yl)propan-1-one

137.80 (C₁); 134.64 (C₆); 131.09 (C₅); 128.74 (C_4’); 128.11 (C₄);

122.81 (C₂); 119.93 (C_5’); 41.44 (CH₂eN); 40.07 (CH₂eCO). Anal.

Calc. for C₁₂H₁₁N₃O₃: C 58.77%, H 4.52%, N 17.13%. Found: C 58.49%,

H 4.61%, N 16.98%.

1-(3,4-diﬂuorophenyl)-3-(1H-imidazole-1-yl)propan-1-one

(25a). Yield: 25%. Mp: 58.0e59.0 ^ꢁC. IR (KBr)

n n

cm^ꢀ1: 1673 (s,

(20a). Yield: 24%. Mp: 95.5e96.5 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1682 (s,

n

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d

ppm: 7.67 (d, 1H, H₆, J_6-

_C]_O), 1284 (s,

n

_Ce_F). ¹H NMR (DMSO‑d₆, 400 MHz)

d

ppm: 8.02

¼ 8.7 Hz), 7.61 (s, 1H, H_2’); 7.15 (s, 1H, H_5’); 6.84 (s, 1H, H_4’);

5

(ddd,1H, H₂, J_2-F¼ 11.3 Hz, J_2-F’¼ 7.9 Hz, J_2-6¼ 2.1 Hz); 7.86e7.90 (m,

6.65e6.60 (m, 2H, H₅þH₃); 4.27 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz);

3.87 (s, 3H, OCH₃); 3.83 (s, 3H, OCH₃); 3.38 (t, 2H, CH₂eCO, J_CH2-

1H, H₆); 7.65 (s, 1H, H_2’); 7.60 (dt, 1H, H₅, J_5-F¼ 10.4 Hz, J_5-F’¼ J_5-

¼ 8.4 Hz); 7.20 (s, 1H, H_5’); 6.86 (s, 1H, H_4’); 4.31 (t, 2H, CH₂eN,

¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm: 196.19 (CO);

6

CH2

J_CH2-CH2¼ 6.8 Hz); 3.60 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

164.57 (C₄); 160.97 (C₂); 137.40 (C_2’); 131.91 (C₆); 128.21 (C_4’);

119.59 (C_5’); 119.43 (C₁); 106.16 (C₅); 98.45 (C₃); 55.91 (OCH₃);

55.65 (OCH₃); 44.42 (CH₂eN); 41.49 (CH₂eCO). Anal. Calc. for

(DMSO‑d₆, 100 MHz)

d

ppm: 195.67 (CO); 152.75 (dd, C₃, ¹J ¼

253.8 Hz, ²J ¼ 12.8 Hz); 149.47 (dd, C₄, 1J ¼ 247.9 Hz, ²J ¼ 13.1 Hz);

137.41 (C_2’); 133.72 (C₁); 128.27 (C_4’); 125.82 (dd, C₆, ³J ¼ 7.9 Hz, ⁴J ¼

C

₁₄H₁₆N₂O₃: C 64.60%, H 6.20%, N 10.76%. Found: C 64.78%, H 6.02%,

N 10.61%.

1-(3,5-dimethoxyphenyl)-3-(1H-imidazole-1-yl)-propan-1-one

(26a). Yield: 32%. Mp: 93.0e94.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1675 (s,

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

ppm: 7.65 (s, 1H, H_2’); 7.20 (s,

2

3.5 Hz); 119.40 (C_5’); 117.98 (d, C₅, J ¼ 17.9 Hz); 117.35 (d, C₂, J ¼

18.1 Hz); 41.04 (CH₂eN); 39.31 (CH₂eCO). Anal. Calc. for

C

₁₂H₁₀F₂N₂O: C 61.02%, H 4.27%, N 11.86%. Found: C 61.31%, H 4.4%,

N 11.84%.

1-(4-chloro-3-ﬂuorophenyl)-3-(1H-imidazole-1-yl)propan-1-one

(21a). Yield: 21%. Mp: 110.0e111.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1682 (s,

_C]_O), 1229 (s, ppm: 7.96 (dd,

_Ce_F). ¹H NMR (DMSO‑d₆, 400 MHz)

n

d

1H, H_5’); 7.08 (d, 2H, H₂þH₆, J_2-4¼ J_6-4¼ 2.3 Hz); 6.86 (s, 1H, H_4’);

n

6.77 (t, 1H, H₄, J_4-2¼ J_2-6¼ 2.3 Hz); 4.31 (t, 2H, CH₂eN, J_CH2-

n

d

¼ 6.7 Hz); 3.80 (s, 6H, OCH₃); 3.58 (t, 2H, CH₂eCO, J_CH2-

CH2

1H, H₂, J_2-F¼ 10.1 Hz, J_2-6¼ 1.9 Hz); 7.83 (dd, 1H, H₆, J_6-5¼ 8.5 Hz, J_6-

¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm: 197.40 (CO);

CH2

¼ 1.8 Hz); 7.77 (dd, 1H, H₅, J_5-6¼ 7.2 Hz, J_5-F¼ 5.5 Hz); 7.65 (s, 1H,

160.59 (2C, C₃þC₅); 138.27 (C₁); 137.44 (C_2’); 128.21 (C_4’); 119.45

(C_5’); 105.69 (2C, C₂þC₆); 105.35 (C₄); 55.52 (2C, C₁þC₂); 41.19

(CH₂eN); 39.39 (CH₂eCO). Anal. Calc. for C₁₄H₁₆N₂O₃: C 64.60%, H

6.20%, N 10.76%. Found: C 64.33%, H 6.33%, N 10.74%.

2

H_2’); 7.20 (s, 1H, H_5’); 6.86 (s, 1H, H_4’); 4.32 (t, 2H, CH₂eN, J_CH2-

¼ 6.8 Hz); 3.61 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

CH2

4

(DMSO‑d₆, 100 MHz)

d

ppm: 196.01 (d, CO, J ¼ 1.8 Hz); 157.25 (d,

C₃, ¹J ¼ 248.2 Hz); 137.41 (C_2’); 136.89 (d, C₁, ³J ¼ 5.6 Hz); 131.17 (C₅);

128.28 (C_4’); 125.17 (d, C₆, ⁴J ¼ 3.6 Hz); 124.99 (d, C₄, ²J ¼ 17.6 Hz);

119.40 (C_5’); 116.12 (d, C₂, ²J ¼ 21.9 Hz); 41.00 (CH₂eN); 39.42

(CH₂eCO). Anal. Calc. for C₁₂H₁₀ClFN₂O: C 57.04%, H 3.99%, N

11.09%. Found: C 57.18%, H 4.05%, N 11.16%.

3-(1H-imidazole-1-yl)-1-(3,4,5-trimethoxyphenyl)propan-1-one

(27a). Yield: 38%. Mp: 122.0e123.0 ^ꢁC. IR (KBr)

n n

cm^ꢀ1: 1669 (s,

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d ppm: 7.67 (s, 1H, H_2’); 7.26 (s,

2H, H₂þH₆); 7.21 (s, 1H, H_5’); 6.87 (s, 1H, H_4’); 4.32 (t, 2H, CH₂eN,

J_CH2-CH2¼ 6.7 Hz); 3.85 (s, 6H, OCH₃); 3.74 (s, 3H, OCH₃); 3.59 (t, 2H,

1-(3-chloro-4-ﬂuorophenyl)-3-(1H-imidazole-1-yl)propan-1-one

CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm:

(22a). Yield: 26%. Mp: 94.0e95.0 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1687 (s,

n

196.59 (CO); 152.78 (2C, C₃þC₅); 142.08 (C₄); 137.44 (C_2’); 131.57

(C₁); 128.16 (C_4’); 119.52 (C_5’); 105.57 (2C, C₂þC₆); 60.15 (C₂); 56.08

(2C, C₁þC₃); 41.33 (CH₂eN); 38.88 (CH₂eCO). Anal. Calc. for

_C]_O), 1248 (s, n d ppm: 8.18 (dd,

_Ce_F). ¹H NMR (DMSO‑d₆, 400 MHz)

1H, H₂, J_2-F¼ 7.2 Hz, J_2-6¼ 2.2 Hz); 8.00 (ddd, 1H, H₆, J_6-5¼ 8.6 Hz, J_6-

¼ 4.8 Hz, J_6-2¼ 2.2 Hz); 7.65 (s, 1H, H_2’); 7.58 (t, 1H, H₅, J_5-F¼ J_5-

C₁₅H₁₈N₂O₄: C 62.06%, H 6.25%, N 9.65%. Found: C 61.94%, H 6.14%, N

F

10

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I. Beltran-Hortelano, R.L. Atherton, M. Rubio-Hernandez et al.

European Journal of Medicinal Chemistry 223 (2021) 113646

9.57%.

C

₁₈H₁₆N₂O: C 78.24%, H 5.84%, N 10.14%. Found: C 77.99%, H 5.61%, N

1-(4-ﬂuoronaphthalen-1-yl)-3-(1H-imidazole-1-yl)-1-propan-1-

10.04%.

3-(1H-benzo[d]imidazole-1-yl)-1-phenylpropan-1-one

Yield: 19%. Mp: 90.5e91.5 ^ꢁC. IR (KBr) cm^ꢀ1: 1681 (s,

NMR (DMSO‑d₆, 400 MHz)

one (28a). Yield: 22%. Mp: 60.5e61.5 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1667 (s,

n

(1b).

_C]_O), 1233 (s,

n

_Ce_F). ¹H NMR (DMSO‑d₆, 400 MHz)

d

ppm: 8.62 (d,

n

_C]_O). ¹H

1H, H₉, J_9-8¼ 8.1 Hz); 8.20 (dd, 1H, H₂, J_2-3¼ 8.2 Hz, J_2-F¼ 5.6 Hz);

8.13 (d, 1H, H₆, J_6-7¼ 8.8 Hz); 7.75e7.67 (m, 3H, H₇þ H₈þ H_2’); 7.44

(dd, 1H, H₃, J_3-F¼ 10.3 Hz, J_3-2¼ 8.2 Hz); 7.24 (s, 1H, H_5’); 6.88 (s, 1H,

H_4’); 4.39 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.7 Hz); 3.69 (t, 2H, CH₂eCO,

d

ppm: 8.26 (s, 1H, H_2’); 7.97 (d, 2H, H₂þ

H₆, J_2-3¼ J_6-5¼ 7.2 Hz); 7.69 (d,1H, H_8’, J_8’-7’¼ 7.9 Hz); 7.63 (t, 2H, H_5’

þ H₄, J_5’-6’¼ J_4-3¼ J_4-5¼ 7.5 Hz); 7.50 (t, 2H, H₃þ H₅, J_3-2¼ J_3-4¼ J_5-

¼ J_5-6¼ 7.7 Hz); 7.26 (t, 1H, H_7’, J_7’-6’¼ J_7’-8’¼ 7.1 Hz); 7.20 (t, 1H,

4

J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d

ppm: 200.27

H_6’, J_6’-5’¼ J_6’-7’¼ 7.6 Hz); 4.62 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.69

(CO); 160.22 (d, C₄, ¹J ¼ 257.7 Hz); 137.47 (C_2’); 131.44 (d, C₁₀, J ¼

5.2 Hz); 130.97 (d, C₁, ⁴J ¼ 4.2 Hz); 130.29 (d, C₂, ³J ¼ 10.1 Hz); 129.17

(C₈); 128.32 (C_4’); 127.23 (d, C₇, ⁴J ¼ 1.2 Hz); 125.61 (d, C₉, ⁴J ¼

2.2 Hz); 123.12 (d, C₅, ²J ¼ 15.9 Hz); 120.29 (d, C₆, ³J ¼ 6.2 Hz);

119.42 (C_5’); 108.78 (d, C₃, ²J ¼ 20.5 Hz); 42.09 (CH₂eN); 41.51

(CH₂eCO). Anal. Calc. for C₁₆H₁₃FN₂O: C 71.63%, H 4.88%, N 10.44%.

Found: C 71.49%, H 4.72%, N 10.67%.

(t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

3

d

ppm: 197.77 (CO); 144.22 (C_2’); 143.39 (C_4’); 136.18 (C₁); 133.72

(C_9’); 133.45 (C₄); 128.71 (2C, C₃þC₅); 127.94 (2C, C₂þC₆); 122.23

(C_7’); 121.41 (C_6’); 119.35 (C_5’); 110.53 (C_8’); 39.40 (CH₂eN); 38.01

(CH₂eCO). HRMS calculated for C₁₆H₁₄N₂O: 250.111. Found:

250.123.

3-(1H-benzo[d]imidazole-1-yl)-1-(4-ﬂuorophenyl)propan-1-one

1-(5-ﬂuorobenzo[b]thiophen-3-yl)-3-(1H-imidazole-1-yl)propan-

(2b). Yield: 20%. Mp: 115.5e116.5 ^ꢁC. IR (KBr) cm^ꢀ1: 1684 (s,

n n

1-one (29a). Yield: 18%. Mp: 101.0e102.0 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1654

_C]_O), 1233 (s, n d ppm: 8.25 (s,

_Ce_F). ¹H NMR (DMSO‑d₆, 400 MHz)

(s,

n

_C]_O), 1185 (s,

n

_Ce_F). ¹H NMR (DMSO‑d₆, 400 MHz)

d

ppm: 9.10

1H, H_2’); 8.05 (dd, 2H, H₂þ H₆, J_2-3¼ J_6-5¼ 8.9 Hz, J_2-F¼ J_6-

(s, 1H, H₂); 8.29 (dd, 1H, H₅, J_5-F¼ 10.6 Hz, J_5-7¼ 2.6 Hz); 8.14 (dd,

1H, H₈, J_8-7¼ 8.9, Hz J_8-F¼ 5.1 Hz); 7.68 (s, 1H, H_2’); 7.37 (td, 1H, H₇,

J_7-F¼ J_7-8¼ 8.9 Hz, J_7-5¼ 2.6 Hz); 7.22 (s, 1H, H_5’); 6.86 (s, 1H, H_4’);

4.36 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.60 (t, 2H, CH₂eCO, J_CH2-

¼ 5.6 Hz); 7.68 (d, 1H, H_8’, J_8’-7’¼ 7.9 Hz); 7.64 (d, 1H, H_5’, J_5’-

F

¼ 7.8 Hz); 7.33 (t, 2H, H₃þ H₅, J_3-2¼ J_3-F¼ J_5-F¼ J_5-6¼ 8.9 Hz); 7.26

6’

(t, 1H, H_7’, J_7’-6’¼ J_7’-8’¼ 7.0 Hz); 7.19 (t, 1H, H_6’, J_6’-5’¼ J_6’-7’¼ 7.6 Hz);

4.60 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.68 (t, 2H, CH₂eCO, J_CH2-

¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d

ppm: 193.16 (CO);

¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm: 196.39 (CO);

CH2

161.15 (d, C₆, ¹J ¼ 240.4 Hz); 142.88 (C₂); 137.53 (C_2’); 137.40 (d, C₄,

³J ¼ 10.6 Hz); 135.22 (d, C₃, ⁴J ¼ 1.0 Hz); 133.40 (d, C₉, ⁴J ¼ 4.6 Hz);

128.33 (C_4’); 124.86 (d, C₈, ³J ¼ 9.7 Hz); 119.58 (C_5’); 114.21 (d, C₇,

²J ¼ 25.2 Hz); 110.03 (d, C₅, ²J ¼ 24.9 Hz); 41.28 (CH₂eN); 40.62

(CH₂eCO). Anal. Calc. for C₁₄H₁₁FN₂OS: C 61.30%, H 4.04%, N 10.21%.

Found: C 61.27%, H 4.25%, N 10.33%.

165.14 (d, C₄, J¹¼ 252.1 Hz); 144.20 (C_2’); 143.39 (C_4’); 133.72 (C_9’);

132.96 (d, C₁, J⁴¼ 2.7 Hz); 130.99 (2C, C₂þC₆, d, J³¼ 9.5 Hz); 122.21

(C_7’); 121.41 (C_6’); 119.34 (C_5’); 115.71 (d, C₃þC₅, J²¼ 21.9 Hz);

110.53 (C_8’); 39.37 (CH₂eN); 37.99 (CH₂eCO). HRMS calculated for

C

₁₆H₁₃FN₂O: 268.108. Found: 268.102.

3-(1H-benzo[d]imidazole-1-yl)-1-(4-chlorophenyl)propan-1-one

1-(3-benzo[d] [1,3]dioxol-5-yl)-3-(1H-imidazole-1-yl)propan-1-

(3b). Yield: 21%. Mp: 131.5e132.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1686 (s,

n n

one (30a). Yield: 25%. Mp: 146.0e147.0 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1663

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d ppm: 8.25 (s, 1H, H_2’); 7.98 (d,

(s,

n

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d

ppm: 7.65 (s, 1H, H_2’);

2H, H₂þ H₆, J_2-3¼ J_6-5¼ 8.6 Hz); 7.68 (d, 1H, H_8’, J_8’-7’¼ 7.9 Hz); 7.64

(d, 1H, H_5’, J_5’-6’¼ 7.8 Hz); 7.57 (d, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.6 Hz);

7.25 (t, 1H, H_7’, J_7’-6’¼ J_7’-8’¼ 7.6 Hz); 7.19 (t, 1H, H_6’, J_6’-5’¼ J_6’-

7.62 (dd, 1H, H₇, J_7-6¼ 8.2 Hz, J_7-2¼ 1.7 Hz); 7.45 (d, 1H, H₂, J_2-

¼ 1.6 Hz); 7.20 (s,1H, H_5’); 7.04 (d,1H, H₆, J_6-7¼ 8.2 Hz); 6.85 (s,1H,

7

H_4’); 6.13 (s, 2H, H₄); 4.29 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.51 (t,

¼ 7.6 Hz); 4.60 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.68 (t, 2H,

7’

2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm:

d

ppm: 195.67 (CO); 151.66 (C₅); 147.85 (C₃); 137.41 (C_2’); 130.95

197.04 (CO); 144.39 (C_2’); 143.60 (C_4’); 138.56 (C₁); 135.06 (C₄);

133.92 (C_9’); 130.08 (2C, C₂þC₆); 129.01 (2C, C₃þC₅); 122.42 (C_7’);

121.61 (C_6’); 119.55 (C_5’); 110.73 (C_8’); 39.52 (CH₂eN); 38.28

(CH₂eCO). HRMS calculated for C₁₆H₁₃ClN₂O: 285.079. Found:

285.095.

(C₁); 128.16 (C_4’); 124.52 (C₇); 119.46 (C_5’); 108.08 (C₂); 107.32 (C₆);

102.08 (C₄); 41.35 (CH₂eN); 38.98 (CH₂eCO). Anal. Calc. for

C

₁₃H₁₂N₂O₃: C 63.93%, H 4.95%, N 11.47%. Found: C 64.11%, H 5.26%,

N 11.69%.

1-(adamantan-1-yl)-3-(1H-imidazole-1-yl)propan-1-one (31a).

Yield: 16%. Mp: 103.0e104.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1696 (s, _C]_O). ¹H

NMR (DMSO‑d₆, 400 MHz) ppm: 7.55 (s, 1H, H_2’); 7.11 (s, 1H, H_5’);

3-(1H-benzo[d]imidazole-1-yl)-1-(4-methoxyphenyl)propan-1-

n

one (4b). Yield: 19%. Mp: 95.0e96.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1680 (s,

n n

d

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d ppm: 8.25 (s, 1H, H_2’); 7.94 (d,

6.83 (s, 1H, H_4’); 4.12 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.7 Hz); 3.00 (t, 2H,

CH₂eCO, J_CH2-CH2¼ 6.7 Hz); 1.95 (s, 3H, H₃þ H₅þ H₈); 1.68e1.60

(m, 12H, H₂þ H₄þ H₆þ H₇þ H₉þ H₁₀). ¹³C NMR (DMSO‑d₆,

2H, H₂þ H₆, J_2-3¼ J_6-5¼ 8.9 Hz); 7.68 (d, 1H, H_8’, J_8’-7’¼ 7.9 Hz); 7.63

(d, 1H, H_5’, J_5’-6’¼ 7.9 Hz); 7.25 (t, 1H, H_7’, J_7’-6’¼ J_7’-8’¼ 7.0 Hz); 7.19

(t, 1H, H_6’, J_6’-5’¼ J_6’-7’¼ 7.0 Hz); 7.01 (d, 2H, H₃þ H₅, J_3-2¼ J_5-

100 MHz)

d

ppm: 212.52 (CO); 137.34 (C_2’); 128.18 (C_4’); 119.28 (C_5’);

¼ 8.9 Hz); 4.59 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.82 (s, 3H,

6

45.51 (C₁); 41.03 (CH₂eN); 37.15 (3C, C₂þC₆þC₇); 36.99 (CH₂eCO);

OCH₃); 3.61 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆,

35.93 (3C, C₄þC₉þC₁₀); 27.26 (3C, C₃þC₅þC₈). Anal. Calc. for

100 MHz)

d ppm: 196.05 (CO); 163.31 (C₄); 144.22 (C_2’); 143.39

C

₁₆H₂₂N₂O: C 74.38%, H 8.58%, N 10.84%. Found: C 74.53%, H 8.41%,

(C_4’); 133.72 (C_9’); 130.29 (2C, C₂þC₆); 129.20 (C₁); 122.20 (C_7’);

121.38 (C_6’); 119.34 (C_5’); 113.88 (2C, C₃þC₅); 110.50 (C_8’); 55.53

(OCH₃); 39.25 (CH₂eN); 37.60 (CH₂eCO). HRMS calculated for

N 10.76%.

1-([1,1⁰-biphenyl]-4-yl)-3-(1H-imidazole-1-yl)propan-1-one

(32a). Yield: 18%. Mp: 104.0e104.5 ^ꢁC. IR (KBr) cm^ꢀ1: (s, _C]_O). ¹H

NMR (DMSO‑d₆, 400 MHz)

ppm: 8.05 (d, 2H, H₂þ H₆, J_2-3¼ J_6-

n

C

₁₇H₁₆N₂O₂: 281.128. Found: 281.141.

d

3-(1H-benzo[d]imidazole-1-yl)-1-(4-(triﬂuoromethyl)phenyl)

¼ 8.4 Hz); 7.82 (d, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.4 Hz); 7.74 (d, 2H,

propan-1-one (5b). Yield: 22%. Mp: 130.5e131.5 ^ꢁC. IR (KBr)

n

cm^ꢀ1

:

5

H_IIþ H_VI, J_II-III¼ J_VI-V¼ 7.2 Hz); 7.68 (s, 1H, H_2’); 7.50 (t, 2H, H_IIIþ H_V,

J_III-II¼ J_III-IV¼ J_V-IV¼ J_V-VI¼ 7.4 Hz); 7.43 (t, 2H, H_IV, J_IV-III¼ J_IV-

1689 (s,

(DMSO‑d₆, 400 MHz)

n _C]_O), 1335 (s, n _Ce_F), 1294 (s, n _Ce_F), 1160 (s, n

_Ce_F). ¹H NMR

d

ppm: 8.27 (s, 1H, H_2’); 8.16 (d, 2H, H₂þ H₆,

¼ 7.3 Hz); 7.23 (s, 1H, H_5’); 6.87 (s, 1H, H_4’); 4.35 (t, 2H, CH₂eN,

J_2-3¼ J_6-5¼ 8.1 Hz); 7.88 (d, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.2 Hz); 7.70 (d,

1H, H_8’, J_8’-7’¼ 7.8 Hz); 7.64 (d, 1H, H_5’, J_5’-6’¼ 7.7 Hz); 7.26 (t, 1H, H_7’,

J_7’-6’¼ J_7’-8’¼ 7.0 Hz); 7.20 (t, 1H, H_6’, J_6’-5’¼ J_6’-7’¼ 7.6 Hz); 4.63 (t,

V

J_CH2-CH2¼ 6.7 Hz); 3.62 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

(DMSO‑d₆, 100 MHz)

d ppm: 197.28 (CO); 144.81 (C₄); 138.81 (C_I);

137.48 (C_2’); 135.06 (C₁); 129.11 (2C, C_IIIþ C_V); 128.72 (2C, C₂þC₆);

128.46 (C_IV); 128.28 (C_4’); 127.01 (2C, C_IIþ C_VI); 126.92 (2C, C₃þC₅);

119.47 (C_5’); 41.23 (CH₂eN); 39.06 (CH₂eCO). Anal. Calc. for

2H, CH₂eN, J_CH2-CH2

¼

6.8 Hz); 3.76 (t, 2H, CH₂eCO, J_CH2-

¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm: 197.32 (CO);

CH2

144.18 (C_2’); 143.41 (C_4’); 139.29 (C₁); 133.72 (C_9’); 132.70 (q, C₄,

11

ꢀ

I. Beltran-Hortelano, R.L. Atherton, M. Rubio-Hernandez et al.

European Journal of Medicinal Chemistry 223 (2021) 113646

J²¼ 32.0 Hz); 128.79 (2C, C₂þC₆); 125.68 (2C, C₃þC₅, q, J³¼ 3.7 Hz);

123.72 (q, CF₃, J¹¼ 272.7 Hz); 122.22 (C_7’); 121.42 (C_6’); 119.35 (C_5’);

110.54 (C_8’); 39.25 (CH₂eN); 38.46 (CH₂eCO). HRMS calculated for

144.15 (C_2’); 143.36 (C_4’); 133.61 (C_9’); 122.27 (C_7’); 121.44 (C_6’);

119.37 (C_5’); 118.93 (C₃); 112.53 (C₄); 110.45 (C_8’); 38.85 (CH₂eN);

37.72 (CH₂eCO). HRMS calculated for C₁₄H₁₂N₂O₂: 241.097. Found:

241.113.

C

₁₇H₁₃F₃N₂O: 319.105. Found: 319.122.

3-(1H-benzo[d]imidazole-1-yl)-1-(4-methylphenyl)propan-1-one

3-(1H-benzo[d]imidazole-1-yl)-1-(naphthalene-2-yl)propan-1-

(6b). Yield: 19%. Mp: 109.0e110.0 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1678 (s,

n

one (11b). Yield: 27%. Mp: 159.0e160.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1685.13

n

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d

ppm: 8.25 (s, 1H, H_2’); 7.86 (d,

(s, n d ppm: 8.68 (s, 1H, H₁);

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

2H, H₂þ H₆, J_2-3¼ J_6-5¼ 8.2 Hz); 7.68 (d, 1H, H_8’, J_8’-7’¼ 7.8 Hz); 7.64

(d, 1H, H_5’, J_5’-6’¼ 7.7 Hz); 7.30 (d, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.0 Hz);

7.25 (t, 1H, H_7’, J_7’-6’¼ J_7’-8’¼ 7.0 Hz); 7.19 (t, 1H, H_6’, J_6’-5’¼ J_6’-

8.31 (s,1H, H_2’) 8.07 (d,1H, H₃, J_3-4¼ 7.9 Hz); 7.99e7.96 (m, 3H, H₄þ

H₆þ H₉); 7.73 (d, 1H, H_8’, J_8’-7’¼ 7.9 Hz); 7.67e7.58 (m, 3H, H₇þ H₈

þ H_5’); 7.27 (t,1H, H_7’, J_7’-6’¼ J_7’-8’¼ 7.1 Hz); 7.21 (t,1H, H_6’, J_6’-5’¼ J_6’-

¼ 7.6 Hz); 4.60 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.64 (t, 2H,

¼ 7.0 Hz); 4.68 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.9 Hz); 3.83 (t, 2H,

7’

CH₂eCO, J_CH2-CH2¼ 6.8 Hz); 2.35 (s, 3H, CH₃). ¹³C NMR (DMSO‑d₆,

CH₂eCO, J_CH2-CH2¼ 6.9 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm:

100 MHz)

d

ppm: 197.74 (CO); 144.70 (C_2’); 144.35 (C_4’); 143.88

197.70; 144.27; 143.46; 135.14; 133.77; 133.46; 132.15; 130.19;

129.59; 128.74; 128.30; 127.65; 126.95; 123.29; 122.27; 121.46;

119.39; 110.60; 39.57; 38.19. HRMS calculated for C₂₀H₁₆N₂O:

301.134. Found: 301.153.

(C₄); 134.25 (C₁); 134.20 (C_9’); 129.73 (2C, C₃þC₅); 128.54 (2C,

C₂þC₆); 122.70 (C_7’); 121.88 (C_6’); 119.83 (C_5’); 111.00 (C_8’); 39.93

(CH₂eN); 38.36 (CH₂eCO); 21.60 (CH₃). HRMS calculated for

C

₁₇H₁₆N₂O: 265.134. Found: 265.149.

3-(1H-benzo[d]imidazole-1-yl)-1-(4-nitrophenyl)propan-1-one

1-(benzo[b]thiophen-3-yl)-3-(1H-benzo[d]imidazole-1-yl)

propan-1-one (12b). Yield: 19%. Mp: 175.0e176.0 ^ꢁC. IR (KBr)

n

(7b). Yield: 22%. Mp: 153.0e154.0 ^ꢁC. IR (KBr)

_C]_O), 1521 (s,

n

cm^ꢀ1: 1689 (s,

n

cm^ꢀ1: 1663.89 (s, _C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

n d ppm: 8.98

n

_NO2), 1348 (s,

n

_NO2). ¹H NMR (DMSO‑d₆, 400 MHz)

(s, 1H, H₂); 8.62 (d, 1H, H₈, J_8-7¼ 7.7 Hz); 8.28 (s, 1H, H_2’); 8.07 (d,

d

ppm: 8.31 (d, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.9 Hz); 8.26 (s, 1H, H_2’);

1H, H₅, J_5-6¼ 7.8 Hz); 7.71 (d, 1H, H_8’, J_8’-7’¼ 8.0 Hz); 7.64 (d, 2H, H_5’,

8.19 (d, 2H, H₂þ H₆, J_2-3¼ J_6-5¼ 8.9 Hz); 7.70 (d, 1H, H_8’, J_8’-

J_5’-6’¼ 7.9 Hz); 7.53e7.43 (m, 2H, H₆þ H₇); 7.28e7.18 (m, 2H, H_6’þ

¼ 7.9 Hz); 7.64 (d, 1H, H_5’, J_5’-6’¼ 7.8 Hz); 7.26 (t, 1H, H_7’, J_7’-6’¼ J_7’-

H_7’); 4.66 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.9 Hz); 3.72 (t, 2H, CH₂eCO,

7’

¼ 7.0 Hz); 7.20 (t, 1H, H_6’, J_6’-5’¼ J_6’-7’¼ 7.0 Hz); 4.63 (t, 2H, CH₂eN,

J_CH2-CH2¼ 6.9 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm: 193.19

8’

J_CH2-CH2¼ 6.8 Hz); 3.78 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

(CO); 144.20 (C_2’); 143.41 (C_4’); 140.21 (C₂); 139.33 (C₃); 136.07 (C₄);

133.76 (C₉); 133.72 (C_9’); 125.81 (C₆); 125.38 (C₇); 124.61 (C₈);

122.89 (C₅); 122.25 (C_7’); 121.43 (C_6’); 119.37 (C_5’); 110.54 (C_8’);

39.45 (CH₂eN); 39.39 (CH₂eCO). HRMS calculated for C₁₈H₁₄N₂OS:

307.108. Found: 307.107.

(DMSO‑d₆, 100 MHz)

d ppm: 197.07 (CO); 150.02 (C₄); 144.17 (C_2’);

143.40 (C_4’); 140.68 (C₁); 133.71 (C_9’); 129.40 (2C, C₂þC₆); 123.79

(2C, C₃þC₅); 122.23 (C_7’); 121.44 (C_6’); 119.36 (C_5’); 110.55 (C_8’);

39.22 (CH₂eN); 38.71 (CH₂eCO). HRMS calculated for C₁₆H₁₃N₃O₃:

296.103. Found: 296.121.

1-(benzo[b]thiophen-2-yl)-3-(1H-benzo[d]imidazole-1-yl)

3-(1H-benzo[d]imidazole-1-yl)-1-(thiophen-3-yl)propan-1-one

propan-1-one (13b). Yield: 21%. Mp: 169.0e170.0 ^ꢁC. IR (KBr)

n

(8b). Yield: 24%. Mp: 125.5e126.5 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1674 (s,

n

cm^ꢀ1: 1665.26 (s, _C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

n d ppm: 8.38

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d

ppm: 8.51 (dd, 1H, H₂, J_2-

(s,1H, H₃); 8.26 (s,1H, H_2’); 8.04 (d,1H, H₅, J_5-6¼ 8.1 Hz); 7.98 (d,1H,

¼ 1.7 Hz); 8.24 (s, 1H, H_2’); 7.67 (d, 1H, H_8’, J_8’-7’¼ 7.9 Hz); 7.63 (d,

H₈, J_8-7¼ 7.9 Hz); 7.71 (d, 1H, H_8’, J_8’-7’¼ 8.0 Hz); 7.64 (d, 1H, H_5’, J_5’-

4

1H, H_5’, J_5’-6’¼ 8.1 Hz); 7.61 (dd, 1H, H₅, J_5-4¼ 5.3 Hz, J_5-2¼ 3 Hz);

7.50 (d, 1H, H₄, J_4-5¼ 5.0 Hz); 7.26 (t, 1H, H_7’, J_7’-6’¼ J_7’-8’¼ 7.3 Hz);

7.19 (t, 1H, H_6’, J_6’-5’¼ J_6’-7’¼ 7.4 Hz); 4.59 (t, 2H, CH₂eN, J_CH2-

¼ 7.9 Hz); 7.53 (t, 1H, H₆, J_6-5¼ J_6-7¼ 7.0 Hz); 7.46 (t, 1H, H₇, J_7-

6’

¼ J_7-6¼ 7.1 Hz); 7.27 (t, 1H, H_7’, J_7’-6’¼ J_7’-8’¼ 7.2 Hz); 7.20 (t, 1H,

8

H_6’, J_6’-5’¼ J_6’-7’¼ 7.1 Hz); 4.65 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.75

¼ 6.8 Hz); 3.58 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

(t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

CH2

(DMSO‑d₆, 100 MHz)

d

ppm: 192.08 (CO); 144.18 (C_2’); 143.38 (C_4’);

d ppm: 192.54 (CO); 144.22 (C_2’); 143.40 (C_4’); 142.54 (C₂); 141.50

141.46 (C₃); 134.11 (C₂); 133.67 (C_9’); 127.54 (C₅); 126.34 (C₄);

122.22 (C_7’); 121.40 (C_6’); 119.35 (C_5’); 110.50 (C_8’); 39.27 (CH₂eN);

39.04 (CH₂eCO). Anal. HRMS calculated for C₁₄H₁₂N₂OS: 257.092.

Found: 257.090.

(C₄); 139.06 (C₉); 133.68 (C_9’); 131.31 (C₃); 127.85 (C₆); 126.35 (C₈);

125.31 (C₇); 123.17 (C₅); 122.31 (C₇); 121.49 (C_6’); 119.39 (C_5’);

110.58 (C_8’); 39.41 (CH₂eN); 38.41 (CH₂eCO). HRMS calculated for

C

₁₈H₁₄N₂OS: 307.108. Found: 307.196.

3-(1H-benzo[d]imidazole-1-yl)-1-(thiophen-2-yl)propan-1-one

3-(1H-imidazole-1-yl)-1-(benzofuran-2-yl)propan-1-one (14b).

(9b). Yield: 23%. Mp: 115.0e116.0 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1666 (s,

n

Yield: 21%. Mp: 157.0e158.0 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1680.14 (s,

n

_C]_O).

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d

ppm: 8.23 (s, 1H, H_2’); 7.99

¹H NMR (DMSO‑d₆, 400 MHz)

d ppm: 8.26 (s, 1H, H_2’); 7.93 (s, 1H,

(dd,1H, H₅, J_5-4¼ 4.9 Hz, J_5-3¼ 1.1 Hz); 7.95 (dd,1H, H₃, J_3-4¼ 3.8 Hz,

H₃); 7.81 (d, 1H, H₅, J_5-6¼ 7.8 Hz); 7.73e7.67 (m, 2H, H₈þ H_8’); 7.64

(d, 1H, H_5’, J_5’-6’¼ 8.0 Hz); 7.54 (ddd, 1H, H₇, J_7-8¼ 8.4 Hz, J_7-

J_3-5¼ 1.1 Hz); 7.67 (d, 1H, H_8’, J_8’-7’¼ 7.9 Hz); 7.63 (d, 1H, H_5’, J_5’-

¼ 7.9 Hz); 7.26 (td, 1H, H_7’, J_7’-6’¼ J_7’-8’¼ 7.8 Hz, J_7’-5’¼ 1.1 Hz);

¼ 7.3 Hz, J_7-5¼ 1.2 Hz); 7.36 (t, 1H, H₆, J_6-5¼ J_6-7¼ 7.2 Hz); 7.28 (t,

6’

6

7.22e7.17 (m, 2H, H_6’þ H₄); 4.60 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz);

1H, H_7’, J_7’-6’¼ J_7’-8’¼ 7.1 Hz); 7.20 (t, 1H, H_6’, J_6’-5’¼ J_6’-7’¼ 7.0 Hz);

4.66 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.64 (t, 2H, CH₂eCO, J_CH2-

3.62 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆,

100 MHz)

d

ppm: 190.75 (CO); 144.18 (C_2’); 143.36 (C₂); 143.25

¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm: 188.11 (CO);

CH2

(C_4’); 135.17 (C₅); 133.79 (C_9’); 133.66 (C₃); 128.76 (C₄); 122.28 (C_7’);

121.45 (C_6’); 119.36 (C_5’); 110.51 (C_8’); 39.38 (CH₂eN); 38.39

(CH₂eCO). HRMS calculated for C₁₄H₁₂N₂OS: 257.092. Found:

257.093.

154.97 (C₉); 151.61 (C₂); 144.20 (C_2’); 143.40 (C_4’); 133.66 (C_9’);

128.65 (C₇); 126.69 (C₄); 124.11 (C₆); 123.74 (C₅); 122.32 (C_7’);

121.50 (C_6’); 119.41 (C_5’); 114.50 (C₃); 112.26 (C₈); 110.54 (C_8’); 38.88

(CH₂eN); 38.26 (CH₂eCO). HRMS calculated for C₁₈H₁₄N₂O₂:

291.113. Found: 291.131.

3-(1H-benzo[d]imidazole-1-yl)-1-(furan-2-yl)propan-1-one

(10b). Yield: 25%. Mp: 86.5e87.5 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1669.89 (s,

n

3-(1H-benzo[d]imidazole-1-yl)-1-(3,5-dimethoxyphenyl)propan-

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d

ppm: 8.21 (s, 1H, H_2’); 7.97 (d,

1-one (15b). Yield: 24%. Mp: 160.0e161.0 ^ꢁC. IR (KBr) cm^ꢀ1

n :

1H, H₅, J_5-4¼ 1.1 Hz); 7.66 (d, 1H, H_8’, J_8’-7’¼ 7.9 Hz); 7.63 (d, 1H, H_5’,

1685.35 (s, n d ppm: 8.25 (s, 1H,

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

J_5’-6’¼ 7.9 Hz); 7.47 (d, 1H, H₃, J_3-4¼ 3.3 Hz); 7.26 (td, 1H, H_7’, J_7’-

H_2’); 7.68 (d, 1H, H_8’, J_8’-7’¼ 7.9 Hz); 7.63 (d, 1H, H_5’, J_5’-6’¼ 7.9 Hz);

¼ J_7’-8’¼ 7.7 Hz, J_7’-5’¼ 1.2 Hz); 7.19 (t,1H, H_6’, J_6’-5’¼ J_6’-7’¼ 7.5 Hz);

7.26 (t, 1H, H_7’, J_7’-6’¼ J_7’-8’¼ 7.0 Hz); 7.19 (t, 1H, H_6’, J_6’-5’¼ J_6’-

6’

6.68 (dd,1H, H₄, J_4-3¼ 3.6 Hz, J_4-5¼ 1.7 Hz); 4.59 (t, 2H, CH₂eN, J_CH2-

¼ 7.0 Hz); 7.07 (d, 2H, H₂þ H₆, J_2-4¼ J_6-4¼ 2.3 Hz); 6.75 (t, 1H, H₄,

7’

¼ 6.8 Hz); 3.46 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

J_4-2¼ J_4-6¼ 2.2 Hz); 4.59 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.7 Hz); 3.77 (s,

CH2

(DMSO‑d₆, 100 MHz)

d ppm: 185.87 (CO); 151.52 (C₂); 147.98 (C₅);

6H, OCH₃); 3.67 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.7 Hz). ¹³C NMR

12

ꢀ

I. Beltran-Hortelano, R.L. Atherton, M. Rubio-Hernandez et al.

European Journal of Medicinal Chemistry 223 (2021) 113646

(DMSO‑d₆, 100 MHz)

d

ppm: 197.54 (CO); 160.59 (2C, C₃þC₅);

d

ppm: 8.99 (s, 1H, H₂); 8.59 (dt, 1H, H₅, J_5-6¼ 8.2 Hz, J_5-8¼ 0.9 Hz);

144.25 (C_2’); 143.38 (C_4’); 138.25 (C₁); 133.75 (C_9’); 122.24 (C_7’);

121.43 (C_6’); 119.35 (C_5’); 110.58 (C_8’); 105.67 (2C, C₂þC₆); 105.42

(C₄); 55.52 (2C, OCH₃); 39.45 (CH₂eN); 38.22 (CH₂eCO). HRMS

calculated for C₁₈H₁₈N₂O₃: 311.139. Found: 311.159.

8.09 (dt, 1H, H₈, J_8-7¼ 7.8 Hz, J_8-5¼ 0.9 Hz); 7.73 (d, 1H, H_5’, J_5’-

¼ 1.1 Hz); 7.54e7.17 (m, 2H, H₆þH₇); 7.17 (d, 1H, H_4’, J_4’-

4’

¼ 1.0 Hz); 4.79 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.73 (t, 2H,

5’

CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm:

1-(3-benzo[d]

propan-1-one (16b). Yield: 22%. Mp: 132.5e133.5 ^ꢁC. IR (KBr)

cm^ꢀ1: 1675.26 (s, _C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

ppm: 8.24

[1,3]dioxol-5-yl)-3-(1H-benzo[d]imidazole-1-yl)

192.82; 140.45; 139.37; 136.06; 133.58; 128.09; 127.71; 125.89;

125.46; 124.59; 122.98; 44.71; 39.82. Anal. Calc. for C₁₄H₁₁N₃O₃S: C

55.81%, H 3.68%, N 13.95%. Found: C 55.59%, H 3.93%, N 13.86%.

1-(4-ﬂuorophenyl)-3-(2-methyl-1H-imidazole-1-yl)propan-1-one

n

d

(s, 1H, H_2’); 7.68e7.59 (m, 3H, H₇þ H_5’þ H_8’); 7.45 (d, 1H, H₂, J_2-

¼ 1.6 Hz); 7.25 (t, 1H, H_7’, J_7’-6’¼ J_7’-8’¼ 7.1 Hz); 7.19 (t, 1H, H_6’, J_6’-

(1d). Yield: 21%. Mp: 117.0e117.5 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1673 (s,

ppm: 7.95e7.91 (m, 2H, H₂

n

7

¼ J_6’-7’¼ 7.0 Hz); 7.00 (d, 1H, H₆, J_6-7¼ 8.2 Hz); 6.12 (s, 2H, H₄);

_C]_O). ¹H NMR (CDCl₃, 400 MHz)

d

þ

5’

4.58 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.60 (t, 2H, CH₂eCO, J_CH2-

H₆); 7.13 (t, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.6 Hz); 6.87 (dd, 2H, H_4’þ H_5’,

J_4’-5’¼ J_5’-4’¼ 6.0 Hz, J_4’-CH3¼ J_5’-CH3¼ 1.2 Hz); 4.30 (t, 2H, CH₂eN,

J_CH2-CH2¼ 6.8 Hz); 3.35 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz); 2.42 (s,

¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm: 195.75 (CO);

CH2

151.66 (C₅); 147.84 (C₃); 144.23 (C_2’); 143.39 (C_4’); 133.73 (C_9’);

130.91 (C₁); 124.53 (C₇); 122.23 (C_7’); 121.42 (C_6’); 119.35 (C_5’);

110.55 (C_8’); 108.06 (C₆); 107.33 (C₂); 102.07 (C₄); 39.29 (CH₂eN);

37.76 (CH₂eCO). HRMS calculated for C₁₇H₁₄N₂O₃: 295.108. Found:

295.126.

3H, CH₃-Amine). ¹³C NMR (CDCl₃, 100 MHz)

d ppm: 195.18; 166.18

(d, ¹J ¼ 255.8 Hz); 144.60; 132.75 (d, ⁴J ¼ 2.9 Hz); 130.76 (2C, d, ³J ¼

9.4 Hz); 127.70; 119.18; 116.09 (2C, d, ²J ¼ 21.8 Hz); 40.70; 39.33;

13.20. Anal. Calc. for C₁₃H₁₃FN₂O: C 67.23%, H 5.64%, N 12.06%.

Found: C 67.58%, H 5.94%, N 12.09%.

1-(adamantan-1-yl)-3-(1H-benzo[d]imidazole-1-yl)propan-1-

one (17b). Yield: 20%. Mp: 114.5e115.5 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1695 (s,

n

1-(4-chlorophenyl)-3-(2-methyl-1H-imidazole-1-yl)propan-1-one

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d

ppm: 8.15 (s, 1H, H_2’);

(2d) Yield: 14%. Mp: 144.0e145.0 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1674 (s,

n

_C]_O).

ppm: 7.84 (d, 2H, H₂þ H_6,J_2-3¼ J_6-

¼ 8.6 Hz); 7.44 (d, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.6 Hz); 6.88 (d, 2H, H_4’

7.63e7.60 (m, 2H, H_5’þ H_8’); 7.25 (t, 1H, H_7’, J_7’-6’¼ J_7’-8’¼ 7.2 Hz);

¹H NMR (CDCl₃, 400 MHz)

d

7.18 (t, 1H, H_6’, J_6’-5’¼ J_6’-7’¼ 7.1 Hz); 4.41 (t, 2H, CH₂eN, J_CH2-

5

¼ 6.7 Hz); 3.10 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.7 Hz); 1.92 (s, 3H,

þ H_5’, J_4’-5’¼ J_5’-4’¼ 7.0 Hz); 4.31 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz);

CH2

H₃þ H₅þ H₈); 1.68e1.56 (m, 12H, H₂þ H₄þ H₆þ H₇þ H₉þ H₁₀).

3.35 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz); 2.42 (s, 3H, CH₃-Amine). ¹³

C

¹³C NMR (DMSO‑d₆, 100 MHz)

d

ppm: 213.18 (CO); 144.59 (C_2’);

NMR (CDCl₃, 100 MHz) d ppm: 195.60; 144.62; 140.42; 134.60;

143.81 (C_4’); 134.11 (C_9’); 122.67 (C_7’); 121.83 (C_6’); 119.82 (C_5’);

110.91 (C_8’); 46.02 (C₁); 39.72 (CH₂eN); 37.64 (3C, C₂þC₆þC₇);

36.37 (3C, C₄þC₉þC₁₀); 36.11 (CH₂eCO); 27.71 (3C, C₃þC₅þC₈).

HRMS calculated for C₂₀H₂₄N₂O: 309.196. Found: 309.215.

1-(4-ﬂuorophenyl)-3-(2-nitro-1H-imidazole-1-yl)propan-1-one

129.49 (2C); 129.29 (2C); 127.75; 119.18; 40.67; 39.42; 13.22. Anal.

Calc. for C₁₃H₁₃ClN₂O: C 62.78%, H 5.27%, N 11.26%. Found: C 62.55%,

H 5.41%, N 11.22%.

1-(4-methoxyphenyl)-3-(2-methyl-1H-imidazole-1-yl)propan-1-

one (3d) Yield: 20%. Mp: 104.0e105.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1666 (s,

n n

(1c). Yield: 21%. Mp: 92.5e93.5 ^ꢁC. IR (KBr)

1532 (s, _NO2), 1362 (s, _NO2), 1216 (s,

_Ce_F). ¹H NMR (DMSO‑d₆,

400 MHz)

n

cm^ꢀ1: 1679 (s,

n

_C]_O),

_C]_O). ¹H NMR (DMSO‑d₆, 400 MHz)

d

ppm: 7.95 (d, 2H, H₂þ H_6,J_2-

n

¼ J_6-5¼ 9.0 Hz); 7.07 (d, 1H, H_5’, J_5’-CH3¼ 1.3 Hz); 7.03 (d, 2H, H₃þ

3

d

ppm: 8.05 (dd, 2H, H₂þ H_6,J_2-3¼ J_6-5¼ 8.9 Hz, J_2-F¼ J_6-

H₅, J_3-2¼ J_5-6¼ 8.9 Hz); 6.68 (d, 1H, H_4’, J_4’-CH3¼ 1.3 Hz); 4.19 (t, 2H,

CH₂eN, J_CH2-CH2¼ 6.9 Hz); 3.84 (s, 3H, OCH₃); 3.46 (t, 2H, CH₂eCO,

J_CH2-CH2¼ 6.9 Hz); 2.31 (s, 3H, CH₃-Amine). ¹³C NMR (DMSO‑d₆,

¼ 5.5 Hz); 7.70 (d, 1H, H_5’, J_5’-4’¼ 0.9 Hz); 7.35 (t, 2H, H₃þ H₅, J_3-

F

¼ J_5-6¼ 8.9 Hz); 7.16 (d, 1H, H_4’, J_4’-5’¼ 0.9 Hz); 4.74 (t, 2H, CH₂eN,

2

J_CH2-CH2¼ 6.8 Hz); 3.70 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

100 MHz) d ppm: 196.07; 163.32; 143.84; 130.34 (2C); 129.28;

(DMSO‑d₆, 100 MHz)

d

ppm: 196.09; 165.21 (d, ¹J ¼ 252.1 Hz);

126.27; 119.38; 113.91 (2C); 55.57; 40.47; 38.52; 12.64. Anal. Calc.

for C₁₄H₁₆N₂O₂: C 68.83%, H 6.60%, N 11.47%. Found: C 68.73%, H

6.49%, N 11.86%.

144.85; 132.79 (d, ⁴J ¼ 2.8 Hz); 131.04 (2C, d, J ¼ 9.5 Hz); 127.99;

3

127.61; 115.75 (2C, d, ²J ¼ 21.9 Hz); 44.59; 39.49. Anal. Calc. for

C

₁₂H₁₀FN₃O₃: C 54.76%, H 3.83%, N 15.96%. Found: C 54.72%, H

1-(benzo[b]thiophen-3-yl)-3-(2-methyl-1H-imidazole-1-yl)

3.93%, N 15.69%.

propan-1-one (4d). Yield: 16%. Mp: 113.0e114.0 ^ꢁC. IR (KBr) cm^ꢀ1

n :

1-(4-chlorophenyl)-3-(2-nitro-1H-imidazole-1-yl)propan-1-one

1655 (s, n d ppm: 8.74 (d, 1H, H₅, J_5-

_C]_O). ¹H NMR (CDCl₃, 400 MHz)

(2c) Yield: 23%. Mp: 113.5e114.5 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1680 (s,

n

_C]_O),

ppm:

¼ 8.2 Hz); 8.21 (s, 1H, H₂); 7.87 (d, 1H, H₈, J_8-7¼ 8.0 Hz); 7.51 (t, 1H,

6

1540 (s, n _NO2), 1352 (s, n _NO2). ¹H NMR (DMSO‑d₆, 400 MHz)

d

H₆, J_6-7¼ 7.6 Hz); 7.44 (t, 1H, H₇, J_7-6¼ 7.6 Hz); 6.90 (d, 2H, H_4’þ H_5’,

J_4’-CH3¼ J_5’-CH3¼ 1.6 Hz); 4.36 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.41

(t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz); 2.44 (s, 3H, CH₃-Amine). ¹³C

7.98 (d, 2H, H₂þ H_6,J_2-3¼ J_6-5¼ 8.6 Hz); 7.70 (d, 1H, H_5’, J_5’-

¼ 0.9 Hz); 7.60 (d, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.6 Hz); 7.16 (d,1H, H_4’,

4’

J_4’-5’¼ 0.9 Hz); 4.74 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.7 Hz); 3.70 (t, 2H,

NMR (CDCl₃, 100 MHz) d ppm: 191.73; 144.64; 139.92; 137.43;

CH₂eCO, J_CH2-CH2¼ 6.7 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d

ppm:

136.39; 134.78; 127.73; 126.22; 125.87; 125.63; 122.47; 119.22;

40.85; 40.82; 13.23. Anal. Calc. for C₁₅H₁₄N₂OS: C 66.64%, H 5.22%, N

10.36%. Found: C 67.04%, H 5.34%, N 10.32%.

196.55; 144.86; 138.47; 134.70; 130.45; 129.92 (2C); 129.00; 128.85

(2C); 128.00; 127.61; 44.54; 38.55. Anal. Calc. for C₁₂H₁₀ClN₃O₃: C

51.53%, H 3.60%, N 15.02%. Found: C 51.55%, H 3.69%, N 14.91%.

1-(4-methoxyphenyl)-3-(2-nitro-1H-imidazole-1-yl)propan-1-

1-(4-ﬂuorophenyl)-3-(4-nitro-1H-imidazole-1-yl)propan-1-one

(1e). Yield: 22%. Mp: 137.5e138.5 ^ꢁC. IR (KBr)

n n

cm^ꢀ1: 1684 (s,

one (3c) Yield: 16%. Mp: 140.5e141.5 ^ꢁC. IR (KBr)

_C]_O), 1532 (s, _NO2), 1354 (s,

n

cm^ꢀ1: 1665 (s,

n

_C]_O), 1520 (s, n _NO2), 1335 (s,

n

_NO2). ¹H NMR (DMSO‑d₆, 400 MHz)

ppm: 8.46 (d, 1H, H_5’, J_5’-2’¼ 1.5 Hz); 8.07 (dd, 2H, H₂þ H_6,J_2-

n

_NO2). ¹H NMR (DMSO‑d₆, 400 MHz)

d

ppm: 7.95 (d, 2H, H₂þ H_6,J_2-3¼ J_6-5¼ 8.9 Hz); 7.70 (s, 1H, H_5’);

¼ J_6-5¼ 8.8 Hz, J_2-F¼ J_6-F¼ 5.6 Hz); 7.91 (d, 1H, H_2’, J_2’-5’¼ 1.4 Hz);

3

7.16 (s, 1H, H_4’); 7.04 (d, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.9 Hz); 4.73 (t, 2H,

CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.84 (s, 3H, OCH₃); 3.63 (t, 2H, CH₂eCO,

7.37 (t, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.8 Hz); 4.43 (t, 2H, CH₂eN, J_CH2-

¼ 6.8 Hz); 3.73 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR

CH2

J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d

ppm: 195.75;

(DMSO‑d₆, 100 MHz)

d

ppm: 195.99; 165.24 (d, ¹J ¼ 252.2 Hz);

163.41; 130.34 (2C); 129.01; 127.95; 127.62; 113.92 (2C); 55.57;

44.77; 38.16. Anal. Calc. for C₁₃H₁₃N₃O₄: C 56.72%, H 4.76%, N

15.27%. Found: C 56.55%, H 4.74%, N 15.72%.

163.99; 146.84; 137.71; 132.80 (d, ⁴J ¼ 3.2 Hz); 131.04 (2C, d, ³J ¼

9.5 Hz); 121.75; 115.79 (2C, d, ²J ¼ 21.9 Hz); 42.64; 38.44. Anal. Calc.

for C₁₂H₁₀FN₃O₃: C 54.76%, H 3.83%, N 15.96%. Found: C 54.98%, H

3.85%, N 15.82%.

1-(benzo[b]thiophen-3-yl)-3-(2-nitro-1H-imidazole-1-yl)propan-

1-one (4c). Yield: 23%. Mp: 138.0e139.0 ^ꢁC. IR (KBr)

n

cm^ꢀ1: 1663 (s,

1-(4-chlorophenyl)-3-(4-nitro-1H-imidazole-1-yl)propan-1-one

_C]_O), 1532 (s, n _NO2), 1351 (s, n _NO2). ¹H NMR (DMSO‑d₆, 400 MHz)

(2e) Yield: 26%. Mp: 147.0e148.0 ^ꢁC. IR (KBr) cm^ꢀ1: 1670 (s,

n n

13

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I. Beltran-Hortelano, R.L. Atherton, M. Rubio-Hernandez et al.

European Journal of Medicinal Chemistry 223 (2021) 113646

_C]_O), 1525 (s,

n

_NO2), 1336 (s,

n

_NO2). ¹H NMR (DMSO‑d₆, 400 MHz)

cm^ꢀ1: 1666 (s,

(DMSO‑d₆, 400 MHz) d

n

_C]_O), 1528 (s,

ppm: 9.03 (s, 1H, H₂); 8.60 (d, 1H, H₅, J_5-

n _NO2), 1392 (s, n

_NO2). ¹H NMR

d

ppm: 8.46 (d, 1H, H_5’, J_5’-2’¼ 1.5 Hz); 8.00 (d, 1H, H₂þ H_6,J_2-3¼ J_6-

¼ 8.6 Hz); 7.92 (d, 1H, H_2’, J_2’-5’¼ 1.4 Hz); 7.62 (d, 2H, H₃þ H₅, J_3-

¼ J_5-6¼ 8.5 Hz); 4.43 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.74 (t, 2H,

¼ 7.9 Hz); 8.39 (s, 1H, H_4’); 8.09 (d, 1H, H₈, J_8-7¼ 7.8 Hz); 7.49 (dt,

5

6

1H, H₆þ H₇, J_6-5¼ J_7-8¼ 21.5, Hz J_6-7¼ J_7-6¼ 7.1 Hz); 4.37 (t, 2H,

CH₂eN, J_CH2-CH2¼ 6.9 Hz); 3.72 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.9 Hz);

2

CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm:

196.43; 146.84; 138.52; 137.70; 134.69; 129.91 (2C); 128.89 (2C);

121.74; 42.58; 38.51. Anal. Calc. for C₁₂H₁₀ClN₃O₃: C 51.53%, H 3.60%,

N 15.02%. Found: C 51.37%, H 3.51%, N 15.18%.

2.45 (s, 3H, CH₃-Amine). ¹³C NMR (DMSO-d_6j, 100 MHz)

d ppm:

192.76; 145.36; 145.33; 140.46; 139.34; 136.02; 133.62; 125.89;

125.45; 124.56; 122.97; 122.11; 41.71; 39.45; 12.69. Anal. Calc. for

1-(4-methoxyphenyl)-3-(4-nitro-1H-imidazole-1-yl)propan-1-

C

₁₅H₁₃N₃O₃S: C 57.13%, H 4.16%, N 13.33%. Found: C 57.26%, H 4.02%,

N 10.32%.

1-(4-ﬂuorophenyl)-3-(5-methyl-4-nitro-1H-imidazole-1-yl)

propan-1-one (1g). Yield: 16%. Mp: 140.5e141.5 ^ꢁC. IR (KBr) cm^ꢀ1

1678 (s, _C]_O), 1535 (s, _NO2), 1353 (s,

_NO2). ¹H NMR (DMSO‑d₆,

400 MHz)

ppm: 8.08 (dd, 2H, H₂þ H_6,J_2-3¼ J_6-5¼ 8.2 Hz, J_2-F¼ J_6-

one (3e) Yield: 17%. Mp: 156.5e157.5 ^ꢁC. IR (KBr) cm^ꢀ1: 1676 (s,

n n

_C]_O), 1518 (s,

n _NO2), 1330 (s, n

_NO2). ¹H NMR (DMSO‑d₆, 400 MHz)

d

ppm: 8.46 (d, 1H, H_5’, J_5’-2’¼ 1.5 Hz); 7.96 (d, 2H, H₂þ H_6,J_2-3¼ J_6-

n

:

¼ 9.0 Hz); 7.91 (d, 1H, H_2’, J_2’-5’¼ 1.5k Hz); 7.05 (d, 2H, H₃þ H₅, J_3-

¼ J_5-6¼ 8.9 Hz); 4.41 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.84 (s, 3H,

n

5

d

2

OCH₃); 3.67 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz). ¹³C NMR (DMSO‑d₆,

¼ 5.7 Hz); 7.82 (s,1H, H_2’); 7.37 (t, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.8 Hz);

F

100 MHz)

d

ppm: 195.65; 163.44; 146.83; 137.71; 130.34 (2C);

4.37 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.7 Hz); 3.64 (t, 2H, CH₂eCO, J_CH2-

129.02; 121.76; 113.95 (2C); 55.58; 42.82; 38.11. Anal. Calc. for

₁₃H₁₃N₃O₄: C 56.72%, H 4.76%, N 15.27%. Found: C 56.55%, H 4.74%,

N 15.72%.

1-(benzo[b]thiophen-3-yl)-3-(4-nitro-1H-imidazole-1-yl)propan-

1-one (4e). Yield: 21%. Mp: 209.5e210.5 ^ꢁC. IR (KBr) cm^ꢀ1: 1664 (s,

_C]_O), 1521 (s, n _NO2), 1324 (s, n _NO2). ¹H NMR (DMSO‑d₆, 400 MHz)

¼ 6.8 Hz); 2.63 (s, 3H, CH₃-Amine). ¹³C NMR (DMSO‑d₆,

CH2

C

100 MHz)

d

ppm: 195.93; 165.22 (d, ¹J ¼ 252.2 Hz); 143.78; 135.93;

132.82 (d, ⁴J ¼ 2.9 Hz); 131.94; 131.07 (2C, d, ³J ¼ 9.5 Hz); 115.76 (2C,

d, ²J ¼ 21.8 Hz); 40.26; 38.04; 10.16. Anal. Calc. for C₁₃H₁₂FN₃O₃: C

56.32%, H 4.36%, N 15.16%. Found: C 56.31%, H 4.30%, N 15.22%.

1-(4-chlorophenyl)-3-(5-methyl-4-nitro-1H-imidazole-1-yl)

n

d

ppm: 9.02 (s, 1H, H₂); 8.59 (dd, 1H, H₅, J_5-6¼ 7.7 Hz, J_5-8¼ 1.3 Hz);

propan-1-one (2g) Yield: 15%. Mp: 164.5e165.5 ^ꢁC. IR (KBr)

1686 (s, _C]_O), 1566 (s, _NO2), 1342 (s,

400 MHz)

n

cm^ꢀ1

:

8.49 (d, 1H, H_5’, J_5’-2’¼ 1.4 Hz); 8.10 (d, 1H, H₈, J_8-7¼ 7.4 Hz); 7.94 (d,

1H, H_2’, J_2’-4’¼ 1.4 Hz); 7.54e7.45 (m, 2H, H₆þH₇); 4.48 (t, 2H,

CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.76 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz).

n

_NO2). ¹H NMR (DMSO‑d₆,

d

ppm: 7.99 (d, 2H, H₂þ H_6,J_2-3¼ J_6-5¼ 8.6 Hz); 7.81 (s,

1H, H_2’); 7.61 (d, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.6 Hz); 4.36 (t, 2H,

CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.64 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz);

¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm: 192.76; 140.48; 139.37;

137.75; 136.02; 133.60; 125.91; 125.48; 124.57; 122.99; 121.78;

42.74; 39.84. Anal. Calc. for C₁₄H₁₁N₃O₃S: C 55.81%, H 3.68%, N

13.95%. Found: C 56.03%, H 3.57%, N 14.15%.

2.63 (s, 3H, CH₃-Amine). ¹³C NMR (DMSO‑d₆, 100 MHz)

d ppm:

196.37; 143.78; 138.48; 135.93; 134.72; 131.94; 129.95 (2C); 128.84

(2C); 40.21; 38.12; 10.16. Anal. Calc. for C₁₃H₁₂ClN₃O₃: C 53.16%, H

4.12%, N 14.31%. Found: C 53.14%, H 4.27%, N 14.26%.

1-(4-ﬂuorophenyl)-3-(2-methyl-5-nitro-1H-imidazole-1-yl)

propan-1-one (1f). Yield: 16%. Mp: 197.0e198.0 ^ꢁC. IR (KBr)

n

cm^ꢀ1

:

1-(4-methoxyphenyl)-3-(5-methyl-4-nitro-1H-imidazole-1-yl)

1684 (s,

n

_C]_O), 1527 (s,

n

_NO2), 1388 (s,

n

_NO2). ¹H NMR (DMSO‑d₆,

propan-1-one (3g) Yield: 27%. Mp: 149.0e150.0 ^ꢁC. IR (KBr) cm^ꢀ1

n :

1682 (s,

400 MHz)

d

ppm: 8.36 (s, 1H, H_4’); 8.08 (dd, 2H, H₂þ H_6,J_2-3¼ J_6-

n _C]_O), 1565 (s, n _NO2), 1403 (s, n

_NO2). ¹H NMR (DMSO‑d₆,

¼ 8.5 Hz, J_2-F¼ J_6-F¼ 5.6 Hz); 7.37 (t, 2H, H₃þ H₅, J_3-2¼ J_5-

¼ 8.8 Hz); 4.32 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.69 (t, 2H,

d

ppm: 7.97 (d, 2H, H₂þ H_6,J_2-3¼ J_6-5¼ 8.9 Hz); 7.82 (s,

5

6

1H, H_2’); 7.05 (d, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.9 Hz); 4.35 (t, 2H,

CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.85 (s, 3H, OCH₃); 3.58 (t, 2H, CH₂eCO,

J_CH2-CH2¼ 6.8 Hz); 2.64 (s, 3H, CH₃-Amine). ¹³C NMR (DMSO‑d₆,

CH₂eCO, J_CH2-CH2¼ 6.8 Hz); 2.43 (s, 3H, CH₃-Amine). ¹³C NMR

(DMSO‑d₆, 100 MHz)

d

ppm: 196.01; 165.24 (d, ¹J ¼ 251.7 Hz);

145.36; 132.83 (d, ⁴J ¼ 2.9 Hz); 131.09 (2C, d, ³J ¼ 9.7 Hz); 122.13

100 MHz) d ppm: 195.60; 163.42; 143.76; 135.93; 131.92; 130.38

(2C); 115.78 (2C, d, ²J ¼ 21.8 Hz); 41.62; 38.13; 12.69. Anal. Calc. for

(2C); 129.03; 113.92 (2C); 55.59; 40.42; 37.68; 10.16. Anal. Calc. for

₁₄H₁₅N₃O₄: C 58.13%, H 5.23%, N 14.53%. Found: C 58.44%, H 5.40%,

N 14.49%.

1-(benzo[b]thiophen-3-yl)-3-(5-methyl-4-nitro-1H-imidazole-1-

yl)propan-1-one (4g). Yield: 16%. Mp: 180.0e181.0 ^ꢁC. IR (KBr)

cm^ꢀ1: 1662 (s, _NO2). ¹H NMR

_C]_O), 1568 (s, _NO2), 1342 (s,

(DMSO‑d₆, 400 MHz) ppm: 9.01 (s, 1H, H₂); 8.61 (d, 1H, H₅, J_5-

C

₁₃H₁₂FN₃O₃: C 56.32%, H 4.36%, N 15.16%. Found: C 56.39%, H

C

4.48%, N 15.18%.

1-(4-chlorophenyl)-3-(2-methyl-5-nitro-1H-imidazole-1-yl)

propan-1-one (2f) Yield: 13%. Mp: 194.5e195.5 ^ꢁC. IR (KBr)

n

cm^ꢀ1

:

n

1686 (s,

400 MHz)

¼ 8.0 Hz); 7.61 (d, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.2 Hz); 4.32 (t, 2H,

n

_C]_O), 1568 (s,

n

_NO2), 1387 (s,

n

_NO2). ¹H NMR (DMSO‑d₆,

n

d

ppm: 8.35 (s, 1H, H_4’); 8.01 (d, 2H, H₂þ H_6,J_2-3¼ J_6-

d

¼ 7.8 Hz); 8.09 (d, 1H, H₈, J_8-7¼ 7.8 Hz); 7.85 (s, 1H, H_2’); 7.49 (dtd,

5

6

CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.69 (t, 2H, CH₂eCO, J_CH2-CH2¼ 6.8 Hz);

1H, H₆þ H₇, J_6-5¼ J_7-8¼ 15.2, Hz J_6-7¼ J_7-6¼ 7.1 Hz, J_6-2¼ J_7-

2.43 (s, 3H, CH₃-Amine). ¹³C NMR (DMSO‑d₆, 100 MHz)

d

ppm:

¼ 5.8 Hz); 4.42 (t, 2H, CH₂eN, J_CH2-CH2¼ 6.9 Hz); 3.67 (t, 2H,

2

196.45; 145.34; 138.50; 134.73; 129.96 (2C); 128.86 (2C); 122.13;

41.56; 38.22; 12.68. Anal. Calc. for C₁₃H₁₂ClN₃O₃: C 53.16%, H 4.12%,

N 14.31%. Found: C 53.16%, H 4.12%, N 14.31%.

CH₂eCO, J_CH2-CH2¼ 6.9 Hz); 2.65 (s, 3H, CH₃-Amine). ¹³C NMR

(DMSO-d_6j, 100 MHz)

d ppm: 192.68; 143.81; 140.43; 139.33;

136.03; 135.95; 133.61; 131.87; 125.89; 125.45; 124.56; 122.97;

40.35; 40.19; 10.18. Anal. Calc. for C₁₅H₁₃N₃O₃S: C 57.13%, H 4.16%, N

13.33%. Found: C 57.53%, H 3.74%, N 13.12%.

1-(4-methoxyphenyl)-3-(2-methyl-5-nitro-1H-imidazole-1-yl)

propan-1-one (3f) Yield: 23%. Mp: 139.5e140.5 ^ꢁC. IR (KBr) cm^ꢀ1

n :

1678 (s,

400 MHz)

n

_C]_O), 1572 (s,

ppm: 8.36 (s, 1H, H_4’); 7.97 (d, 2H, H₂þ H_6,J_2-3¼ J_6-

n _NO2), 1391 (s, n

_NO2). ¹H NMR (DMSO‑d₆,

d

¼ 8.9 Hz); 7.05 (d, 2H, H₃þ H₅, J_3-2¼ J_5-6¼ 8.9 Hz); 4.30 (t, 2H,

4.4. Compound Handling

5

CH₂eN, J_CH2-CH2¼ 6.8 Hz); 3.84 (s, 3H, OCH₃); 3.62 (t, 2H, CH₂eCO,

J_CH2-CH2¼ 6.8 Hz); 2.43 (s, 3H, CH₃-Amine). ¹³C NMR (DMSO‑d₆,

Stocks of the synthesised compounds and the reference drug BZ

(Sigma-Aldrich) were prepared in 100% DMSO at 50 mM in

eppendorf tubes, and stored at 4 ^ꢁC. Working stocks were prepared

in culture medium on the same day as the assay. At ﬁnal concen-

tration, all the sample wells for the assay contained <0.5% of DMSO.

All plates were inspected microscopically to detect contamination

or precipitation of the compounds.

100 MHz)

d ppm: 195.69; 163.43; 145.34; 130.40 (2C); 129.05;

122.12; 113.93 (2C); 55.59; 41.79; 37.77; 12.69. Anal. Calc. for

₁₄H₁₅N₃O₄: C 58.13%, H 5.23%, N 14.53%. Found: C 58.30%, H 5.41%,

N 14.70%.

1-(benzo[b]thiophen-3-yl)-3-(2-methyl-5-nitro-1H-imidazole-1-

yl)propan-1-one (4d). Yield: 21%. Mp: 113.0e114.0 ^ꢁC. IR (KBr)

C

n

14

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I. Beltran-Hortelano, R.L. Atherton, M. Rubio-Hernandez et al.

European Journal of Medicinal Chemistry 223 (2021) 113646

4.5. Parasite and mammalian cell cultures

cells were seeded at 5 x 10⁴cells per well in 96 well plates in 100

of culture medium and allowed to adhere for 6 h at 37 ^ꢁC and 5%

CO₂. Then, another 100 L medium containing 80 M drug was

added for a ﬁnal concentration of 40 M in the assay. The plates

mL

T.

cruzi

CL-Luc:Neon

clone

parasites

(a

bio-

m

luminescent:ﬂuorescent derivative of the CL-Brener clone e DTU-VI)

were cultured in supplemented RPMI-1640 medium, as described

previously [27]. BSR cells (BHK-21 subclone) were cultured in Dul-

becco's Modiﬁed Eagle's Medium (DMEM, Sigma) supplemented

with 5% (v/v) Foetal Bovine Serum (FBS), 100 U/ml of penicillin, and

m

were then incubated for an additional 72 h before addition of AB.

4.6.3. In vitro screening and selectivity index

8-point potency curves were generated by serial dilution (2:1) of

the drugs in the corresponding culture medium. For epimastigote

and BSR cell screening, the seeding and incubation was performed

as above for the pre-screenings. For amastigote assays, BSR cells in

100 m

g/mL streptomycin at 37 ^ꢁC and 5% CO₂. For maintenance, BSR

cells were sub-cultured every 3 days at a ratio of 1:5. For the infec-

tion of cell monolayers, tissue culture trypomastigotes (TCTs) were

derived from previously infected BSR cells. Cell cultures were

exposed to TCTs for 18 h (overnight). Extracellular parasites were

then removed by washing with PBS, and the ﬂasks incubated with

fresh medium for a further 5e7 days. New extracellular TCTs were

isolated by collection and centrifugation of the culture medium at

1600 g. Pellets were re-suspended in DMEM with 10% of FBS and

kept at 37 ^ꢁC until use. Motile trypomastigotes were counted using a

haemocytometer.

100 mL growth medium were added to a black, clear-bottomed, 96-

well, lidded, polystyrene microplate at 5 ꢂ 10⁴cells/well. After 6 h

incubation to allow attachment, cells were infected overnight with

5 ꢂ 10⁵TCT/well, a multiplicity of infection (MOI) of 10. The try-

pomastigote to mammalian cell ratios used were determined

empirically, as the minimum ratio necessary to achieve optimal

infection levels is statistically distinguishable from background.

Next day, wells were washed 3 times with PBS to remove non-

internalised trypomastigotes, before adding 200

mL DMEM sup-

4.6. Biological assays

plemented with 1% FBS and containing the drugs at the different

concentrations. For assessment of activity against amastigote

replication, 72 h post-incubation, the plates were washed twice

with PBS and ﬁxed with 4% paraformaldehyde for 30 min. Then,

paraformaldehyde was removed, and wells washed with PBS,

before taking a ﬂuorescence readout in the BMG FLUOstar Omega

plate reader. For trypomastigote screening assays, infective TCTs

were isolated from a previously infected culture ﬂask and incu-

bated in eppendorf tubes with the different drug concentrations for

6 h in high-glucose DMEM, supplemented with 10% FBS, at 37 ^ꢁC.

Then, trypomastigotes were washed 3 times with PBS to remove

the drug from the medium, and used to infect a plate seeded with

BSR cells as above. Non-internalised parasites were removed from

the medium on the following day by washing the plates 3 times

4.6.1. Data analysis and deﬁnitions

Fluorescence intensities were determined using a BMG FLUOs-

tar Omega (with excitation 488 nm, emission 525 nm for the

ﬂuorescent parasites and excitation 530 nm, emission 595 nm for

the Alamar blue® assays). Dose-response curves were ﬁtted and

95% conﬁdence intervals were calculated using the sigmoidal dose-

response variable slope function from the Graph Pad Prism 8

software (La Jolla California USA, www.graphpad.com). In this

study: i) % inhibition of growth is the reduction in the replication

rate in the presence of the drug with respect to the untreated

controls; ii) IC₅₀is deﬁned as the compound concentration capable

of reducing the replication and/or infection by 50% as compared to

non-treated controls and values were determined by interpolation

from the ﬁtted dose-response curve. Values are expressed as

IC₅₀SD; iii) selectivity index (SI) is an indicator of the speciﬁcity

of growth inhibition of the parasite by the drug, in relation to its

inhibition of the host cell; it was calculated as the ratio of host

cell:parasite IC₅₀values. For the comet assay, the mean DNA in the

tail for each condition was compared with the vehicle control value

using the Kruskal-Wallis test followed, if needed, by the post-hoc

Bonferroni test. Statistical signiﬁcance was set at p < 0.05 in both

tests. The Statistics and Data Analysis (STATA) software v12.1 (TX,

USA) was used. All determinations were performed in 3 indepen-

dent experiments with 3 technical replicates per concentration,

unless stated otherwise.

with PBS, and 200 mL fresh growth medium supplemented with 1%

FBS was added. Infection was allowed to progress for an additional

72 h. For assessment of activity against trypomastigotes, we

measured the reduction of cell-invasion by quantifying ﬂuores-

cence generated by amastigote replication upon successful infec-

tion. IC₅₀values were calculated using the Graph Pad Prism 8

software as explained above.

4.6.4. Kinetics of killing

Changes in the ﬂuorescence intensity were assessed by daily

readouts using the plate reader. For this assay, epimastigotes were

seeded as above in 96-well microplates. Growth curves were

monitored during the 5 days of exponential growth in the presence

of the compounds, until the parasites reached stationary phase

(around day 5 after plating). BZ was included as a ‘fast-killing’ drug

control.

4.6.2. In vitro pre-screening assays

Single-point potency assays of 10

epimastigotes and BSR cells, respectively, were set for 72 h expo-

sure. The activity of the drugs was measured by adding 0.125 g/mL

mM and 40 mM of drug for

m

4.6.5. Wash-out assays

of Alamar Blue® (AB, Laboserv, Giessen, Germany) [38]. AB is

reduced by the organisms in a time-dependent process, and plates

were incubated for 24 h in the case of the epimastigotes and 6 h in

the case of the BSR cells. These incubation times were standardised

in our lab, as they depend on the initial plating concentration of the

untreated controls. After incubation, the plates were read at

540 nm. A linear relationship between cellular density and absor-

bance can be used to determine the growth.

In vitro infections were carried out as above. However, BSR cells

were seeded in 8-well, Ibidi m-slides with a polymer coverslip, (Cat.

No: 80,826) or in black, clear-bottomed, 24-well, lidded poly-

styrene microplates. The concentrations used in these assays were

10x and 20x the IC₅₀value obtained previously for the amastigote

form. Infections were divided into two groups for each concentra-

tion under study. One group was exposed to the drug for 10 days,

while the other group was exposure to the drug for 20 days.

Compounds were replaced every 4 days. Relapse day was deﬁned as

the ﬁrst day trypomastigotes could be observed by light microscopy

in culture after wash-out of the drug, or replication of amastigotes

was observed by ﬂuorescence microscopy. Three wells were pre-

pared per treatment and each individual well was inspected by

Epimastigotes of T. cruzi CL-Luc:Neon clone were seeded at 2.5 x

10⁵parasites per well in 96 well plates in 100

m

L of culture medium.

L of medium containing 20 M of

drug, to reach a ﬁnal concentration of 10 M in the assay. Plates

were then incubated at 27 ^ꢁC for 72 h before addition of AB. BSR

To these was added another 100

m

15

ꢀ

I. Beltran-Hortelano, R.L. Atherton, M. Rubio-Hernandez et al.

European Journal of Medicinal Chemistry 223 (2021) 113646

taking 30 captures per timepoint. These assays allow drugs to be

assessed as trypanocidal or trypanostatic.

48 h. The total suspension growth (TSG) was calculated by dividing

the number of cells after 48 h by the number of cells just before

treatment. Relative suspension growth (RSG) is the relation be-

tween the TSG of each treated cell suspension and the TSG of the

control cells, expressed as a percentage.

Images were acquired using an inverted Nikon Eclipse micro-

scope. The chamber/plate containing the infected cells was moved

along the x-y plane through a 580 nm LED illumination. Images

were collected using a 16-bit, 1-megapixel Pike AVT (Fe100B) CCD

camera set in the detector plane. An Olympus LMPlanFLN 40x/1.20

objective was used to collect the exit wave leaving the specimen.

Imaging was performed by placing the chamber slide/plate on a

microscope surrounded by an environmental chamber (OKOLab

cage incubator, USA) maintaining the cells and the microscope at

37 ^ꢁC and 5% CO₂. Sequences were created using the deconvolution

app in Nikon imaging software.

4.7.4. FPG-modiﬁed comet assay

The comet assay was carried out using the 12 minigels/slide

format and the 12-Gel Comet Assay Units (Norgenotech, Norway)

[40,41]. Treated cells were embedded in agarose by mixing 30

cell suspension with 140 L of 1% low-melting-point agarose in PBS

at 37 ^ꢁC. Six 5

L aliquots, 2 per condition tested, were placed on

mL of

m

agarose-pre-coated microscope slides. Slides were immersed in

lysis solution (2.5 M NaCl, 0.1 M Na₂EDTA, 0.1 M Tris, pH 10, 1%

Triton X-100) for 1 h at 4 ^ꢁC, and then three times, 5 min each, in

Fpg reaction buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/

mL BSA, pH 8.0). Each condition (two gels) involved incubation at

4.6.6. Infectivity assays

This assay is a variation of the trypomastigote screening pro-

cedure used to determine the efﬁcacy of drugs in preventing

infection, either by killing the parasite directly, or by affecting the

ﬁtness of the parasite, and blocking infection. Brieﬂy, trypomasti-

gotes are incubated for 6 h in serial drug concentrations, then

parasites were washed 3 times with PBS and used to infect the BSR

mammalian cells at a MOI of 10:1 (trypomastigote:cell) for 18 h.

This allows the concentration that prevents establishment of a

productive infection to be determined. Infected BSR cells were

readout 120 h post-infection, and chambers were inspected for

amastigote replication using an inverted Nikon Eclipse microscope,

as explained above.

37 ^ꢁC for 1 h with 30

mL of either lysis buffer (without Triton X-100),

enzyme reaction buffer, or Fpg. Slides were then immersed in the

electrophoresis solution (0.3 M NaOH, 1 mM Na₂EDTA, pH > 13) for

40 min at 4 ^ꢁC and electrophoresis was carried out at 1.2 V/cm for

an additional 20 min. Slides were then immersed sequentially in

DPBS and distilled water for 10 min each, and in 70% and 100%

ethanol, 15 min each, before drying overnight.

Comets were stained by adding a drop of 1

mg/mL 4,6-

diamidino-2-phenylindole (DAPI) to each mini-gel. The semi-

automated image analysis system Comet Assay IV (Perceptive In-

struments) was used to measure the percentage of tail DNA of 50

comets per gel (100 comets per condition). The median percentage

of DNA in the tail for 100 comets was the descriptor of each con-

dition; gels incubated with the lysis solution (without Triton X-100)

represents SBs and alkali labile sites (ALS), and net Fpg-sensitive

sites were calculated by subtracting the values in the enzyme re-

action buffer from those obtained after the Fpg incubation.

4.6.7. Drug combination assays

To test drug combinations, the Alamar Blue and ﬂuorescence

methods were used to determine the IC₅₀values. Parasites/cells

were seeded in the previously described conditions, but the BZ IC₅₀

values were re-evaluated in the presence of the IC₅₀of the drug

under study. Drug combinations were assessed in triplicate in each

plate and repeated at least twice. The combination index (CI) iso-

bologram method was used to analyse the nature of the interaction

[39]. A Ci value less than, equal to, or greater than 1 indicates

synergism, additivity, and antagonism, respectively.

Declaration of competing interest

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

4.7. Comet assay

4.7.1. Cell culture

Acknowledgements

TK-6 cells (human lymphoblastoid cell line) were obtained from

the American Type Culture Collection (ATCC). They were grown in

RPMI 1640 medium (ATCC modiﬁcation, ref. A1049101) supple-

mented with 10% FBS, 100 U/mL penicillin and 0.1 mg/mL strep-

tomycin (all from Gibco). Cells were maintained as a suspension

culture in continuous agitation at 37 ^ꢁC in a humidiﬁed atmosphere

with 5% CO₂for no longer than 60 days.

ꢀ

IB-H is grateful to Fundacion Caja Navarra, Obra Social la Caixa,

ꢀ

Fundacion Roviralta, Grupo Ubesol and Inversiones Garcilaso de la

Vega for their ﬁnancial support. Moreover, IB-H is grateful to the

ISTUN Instituto de Salud Tropical of the Universidad de Navarra for

their grant. SPS thanks to Fundacion La Caixa (LCF/PR/PR13/

11080005). FO acknowledges the support from the Wellcome's

Institutional Strategic Support Fund (ISSF grant: 204928/Z/16/Z).

ꢀ

4.7.2. Cell treatment

1 mL containing 6 x 10⁵TK-6 cell suspension was treated with

compound 3c in a 12-well plate, for 3 h. The treatment was per-

formed in the cell culture medium. After that, cells were washed

three times in, and then suspended in 1.5 mL of fresh cell culture

medium. 0.5 mL of cell suspension was used for analysis and 1 mL

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113646.

for the proliferation assay (below). Cells treated with 20 mM MMS

were used as a positive control for the Fpg-modiﬁed comet assay.

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17

Article Doi

Design and synthesis of Mannich base-type derivatives containing imidazole and benzimidazole as lead compounds for drug discovery in Chagas Disease

DOI: 10.1016/j.ejmech.2021.113646

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