Design, Synthesis, and Biological Evaluation of New 1-(Aryl-1 H-pyrrolyl)(phenyl)methyl-1 H-imidazole Derivatives as Antiprotozoal Agents

Article abstract of DOI:10.1021/acs.jmedchem.8b01464

We have designed and synthesized a series of new imidazole-based compounds structurally related to an antiprotozoal agent with nanomolar activity which we identified recently. The new analogues possess micromolar activities against Trypanosoma brucei rhod

Full text of DOI:10.1021/acs.jmedchem.8b01464

Subscriber access provided by UNIV OF LOUISIANA
Article
Design, Synthesis, and Biological Evaluation of New 1-(Aryl-1H-pyrrolyl)
phenyl)methyl-1H-imidazole Derivatives as Antiprotozoal Agents
(
Francesco Saccoliti, Valentina Noemi Madia, Valeria Tudino, Alessandro De Leo, Luca Pescatori, Antonella
Messore, Daniela De Vita, Luigi Scipione, Reto Brun, Marcel Kaiser, Pascal Mäser, Claudia Magalhaes
Calvet Alvarez, Gareth K Jennings, Larissa M. Podust, Giacomo Pepe, Roberto Cirilli, Cristina Faggi,
Annalise Di Marco, Maria Rosaria Battista, Vincenzo Summa, Roberta Costi, and Roberto Di Santo
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 07 Jan 2019
Downloaded from http://pubs.acs.org on January 8, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 78
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Design, Synthesis, and Biological Evaluation of New 1-
(Aryl-1H-pyrrolyl)(phenyl)methyl-1H-imidazole
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Derivatives as Antiprotozoal Agents
a
a
a
a
Francesco Saccoliti, Valentina Noemi Madia, Valeria Tudino, Alessandro De Leo,
a
a
a
a
b
Luca Pescatori, Antonella Messore, Daniela De Vita, Luigi Scipione, Reto Brun,
Marcel Kaiser, Pascal Mäser, Claudia M. Calvet,
Podust, Giacomo Pepe, Roberto Cirilli, Cristina Faggi, Annalise Di Marco, Maria
b
b
c, d
c
Gareth K. Jennings, Larissa M.
c
e
f
g
h
h
h
a,*
a
Rosaria Battista, Vincenzo Summa, Roberta Costi, Roberto Di Santo.
a
Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie
del Farmaco, “Sapienza” Università di Roma, p. le Aldo Moro 5, I-00185 Rome, Italy.
b
Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel,
Switzerland
c
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California
San Diego, La Jolla, California 92093, USA
d
Laboratório de Ultraestrutura Celular, Instituto Oswaldo Cruz (IOC), FIOCRUZ, Rio
de Janeiro, Rio de Janeiro, Brazil, 21040-360
ACS Paragon Plus Environment
1

Page 3 of 78
Journal of Medicinal Chemistry
e
Dipartimento di Farmacia, Università di Salerno, Via Giovanni Paolo II 132, I-84084
1
2
3
4
5
6
7
8
9
Fisciano, Salerno, Italy
f
Centro Nazionale per il Controllo e la Valutazione dei Farmaci, Istituto Superiore di
Sanita', Viale Regina Elena 299, I-00161 Rome Italy
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
g
Dipartimento di Chimica, Università degli studi di Firenze, Via della Lastruccia 13, I-
5
0019 Sesto Fiorentino, Florence, Italy
h
Drug Discovery, IRBM Science Park, Via Pontina km 30,600, 00071 Pomezia,
Rome, Italy
ACS Paragon Plus Environment
2

Journal of Medicinal Chemistry
Page 4 of 78
ABSTRACT: We have designed and synthesized a series of new imidazole-based
compounds structurally related to an antiprotozoal agent with nanomolar activity which
we identified recently. The new analogs possess micromolar activities against T. b.
rhodesiense and L. donovani and nanomolar potency against P. falciparum. Most of
the analogs displayed the IC50 within the low nanomolar range against T. cruzi, with
very high selectivity towards the parasite. Discussion of structure-activity relationships
and in vitro biological data for the new compounds are provided against a number of
different protozoa. The mechanism of action for the most potent derivatives (5i, 6a-c,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
b) was assessed by a target-based assays using recombinant T. cruzi CYP51.
Bioavailability and efficacy of selected hits was assessed in a T. cruzi mouse model,
where 6a and 6b reduced parasitemia in animals >99% following intraperitoneal
administration of 25 mg/kg/day dose for four consecutive days.
ACS Paragon Plus Environment
3

Page 5 of 78
Journal of Medicinal Chemistry
INTRODUCTION
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Plasmodium and trypanosomatid parasites cause vector-borne infections producing a
great deal of chronic diseases and affecting hundreds of millions people mainly in
developing countries. However, in the last years, due to several factors including
vector and human migrations or co-infections in immunosuppressed patients, such
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
diseases are dramatically spreading worldwide. Malaria is the most challenging and
deadly parasitic disease since nearly half of the world’s population is at risk of being
infected with roughly 216 million cases and an estimated 445.000 deaths in 2016.
Among the five species of Plasmodium parasites causing human disease, P.
falciparum is the most deadly.2 Human trypanosomatid diseases, including
leishmaniasis, Chagas disease and human African trypanosomiasis (HAT), are
classified by WHO as the most challenging among the Neglected Tropical Diseases
since they cause morbidity and mortality mainly in tropical and sub-tropical countries
hindering their economic development.3,4 Due to economic reasons, they do not
represent an attractive market for pharmaceutical companies which sharply contrasts
with the disproportional number of people at risk, affected patients and related
fatalities.5,6 Human trypanosomatid infections are becoming a severe global health
7
concern affecting more than 20 million people. Parasites of the genus Leishmania
ACS Paragon Plus Environment
4

Journal of Medicinal Chemistry
Page 6 of 78
cause three main forms of leishmaniasis, with the visceral form (VL) being life-
threatening and the second most deadly parasitic disease. Estimated numbers of VL
cases are 700.000-1 million new cases per year, with approximately 30.000 deaths
each year, although it is not often recognized or reported.8,9 Chagas disease is
caused by Trypanosoma cruzi representing a major cause of morbidity and mortality
in Latin America with as many as 7 million people infected worldwide.10 T. brucei
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
gambiense and T. brucei rhodesiense are the causative agents of HAT or sleeping
sickness, one of the most complex endemic tropical diseases restricted to Africa.11
No vaccines are available and the current antiprotozoal therapies are unsatisfactory
due to their low efficacy (especially in the late stage disease), high toxicity and due
to the appearance of resistant parasitic strains. Therefore, there is an urgent need to
develop new effective, safe and affordable antiprotozoal drugs.1,3,9,12,13
Azole-based compounds are known effective antifungal agents targeting sterol 14α-
demethylase (CYP51), a pivotal enzyme involved in ergosterol biosynthesis representing
the major component of fungal cytoplasmic membranes and playing both structural
and functional roles.14,15 This enzyme is a member of the cytochrome P450
superfamily (CYP51), catalyzing oxidative removal of the 14α -methyl group from
post-squalene sterol precursors. Similar to fungi, trypanosomatid parasites also
ACS Paragon Plus Environment
5

Page 7 of 78
Journal of Medicinal Chemistry
produce ergosterol and ergosterol-like molecules which are essential for membrane
functioning in parasite growth, development and division.15,16 Via drug repurposing
strategy, antifungal azole drugs showed promising antileishmanial and anti-T. cruzi
activities with miconazole (1) being the first antifungal agent tested against T. cruzi
showing potent growth inhibition.15,17-20 Experimental CYP51 inhibitors have been
developed that demonstrated encouraging results in both in vitro and in vivo
models.7,15,17,21-31 Recently, the triazole drugs posaconazole (2) and ravuconazole (3)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Figure 1) were tested in clinical trials for Chagas disease.15,17,32a-c Based on the
positive PCR read-out, 70–80% treatment failure was reported.32d,e Inferiority of both
CYP51 inhibitors to benznidazole (Bz) in these clinical trials put on hold development
of the azole inhibitors for the treatment of diseases caused by protozoan
parasites.32e,f
In support of the CYP51 target, the limited efficacy and lack of translation from in
vitro and in vivo models was attributed to the short duration of treatment and sub-
optimal doses administered during human clinical trials. Particularly, it has been
argued that the dose employed in clinical trials corresponded to only the 10-20% of
the curative dose in mice. The short follow-up time did not permit the evaluation of
the long-term effects and assessment of clinical symptoms. Due to the pivotal role
ACS Paragon Plus Environment
6

Journal of Medicinal Chemistry
Page 8 of 78
played by CYP51 in T. cruzi and Leishmania spp. biology, it is critical not to reject
this drug target before investigating CYP51 utility for the treatment of both acute and
chronic Chagas disease in more details.15,23,25,32g-i These considerations drive our
search for new CYP51 inhibitors with pharmacological properties, high efficacy and
low toxicity, making them amenable for long-term administration.7,15,23-25
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Recently, we evaluated the anti-trypanosomatid and antiplasmodial activities of our
in-house antifungal 1-[(aryl)(4-aryl-1H-pyrrol-3-yl)methyl]-1H-imidazole and 1-(phenyl(1-
phenyl-1H-pyrrol-3-yl)methyl)-1H-imidazole derivatives, with the former compounds
being endowed with higher potency against T. cruzi and P. falciparum.33a-h,34
The most promising compounds proved to inhibit T. cruzi CYP51 (TcCYP51) in an
orthogonal target-based assay. On the other hand, given that P. falciparum lacks a steroidogenic
pathway, inhibition of P. falciparum was via yet unknown target. Among them, derivative RDS
416 (4) (Figure 2) proved to be one of the most promising compounds, showing
nanomolar anti-T. cruzi activity, submicromolar antiplasmodial activity and micromolar
activity against L. donovani and T. brucei. In vitro, 4 was two orders of magnitude
more active than the reference drug Bz and also displayed high selectivity towards T.
cruzi (SI = 275).
ACS Paragon Plus Environment
7

Page 9 of 78
Journal of Medicinal Chemistry
In this work, starting from the promising scaffold 4, we designed and synthesized
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the new imidazole derivatives 5a-l, 6a-c, 7a-f and 8a-c (Figure 2), with the aim of
improving the structure-activity relationships (SARs) within this new class of
antiprotozoal compounds. In particular, we designed 1-[(aryl)(4-aryl-1H-pyrrol-3-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
yl)methyl]-1H-imidazoles
5a-l
and
1-(phenyl(1-phenyl-1H-pyrrol-3-yl)methyl)-1H-
imidazoles 7a-f, as derivatives of the recently discovered 1-[(aryl)(1(or 4)-aryl-1H-
pyrrol-3-yl)methyl]-1H-imidazoles related to 4. Moreover, we decided to further study
the effect of the position of the phenyl substituent on the pyrrole ring by designing 1-
(
phenyl(5-phenyl-1H-pyrrol-3-yl)methyl)-1H-imidazoles 6a-c and 1-(phenyl(1-phenyl-1H-
pyrrol-2-yl)methyl)-1H-imidazoles 8a-c.
Furthermore, the racemic mixture of 4, that was one of the most potent
antiprotozoal compounds previously identified by us, was separated by HPLC on
chiral stationary phase to obtain single enantiomers (+)-4 and (-)-4. The enantiomers
were characterized and separately tested against protozoa to define the effect of the
chiral center on the antiprotozoal activities.
Thus, the newly synthesized derivatives 5a-l, 6a-c, 7a-f, 8a-c and the enantiomers
(
+)-4 and (-)-4 were tested against the kinetoplastids T. cruzi, L. donovani and T. b.
ACS Paragon Plus Environment
8

Journal of Medicinal Chemistry
Page 10 of 78
rhodesiense, and the apicomplexan P. falciparum, in order to evaluate their
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
antiprotozoal activities and analyze their SARs.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
N
Cl
N
F
S
Cl
N
N
O
Cl
OH
N
N
Cl
N
MICONAZOLE (1)
RAVUCONAZOLE (2)
N
N
N
O
N
OH
N
N
F
N
N
O
F
O
POSACONAZOLE (3)
Figure 1. Chemical structures of some azole antifungal agents displaying antiprotozoal activities.
Derivatives 5a-l, 6a-c, 7a-f, 8a-c and the enantiomers (+)-4 and (-)-4 displayed
potency against the tested parasites in a broad range of concentrations. The highest
potencies were achieved by (-)-4 and 6a-c against T. cruzi. In vivo activity of 6a,b in
animal model of T. cruzi infection was also demonstrated. The orthogonal target-
based assay using recombinant target suggested that activity of compounds toward T.
cruzi was via inhibition of CYP51.
ACS Paragon Plus Environment
9

Page 11 of 78
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
N
N
R
N
Cl
Cl
Cl
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
R2
N
Cl
Cl
R
R1
N
N
N
4
H
5a-h,k,l
5i,j
H
H
N
N
R1
N
N
Cl
R1
N
N
N
N
Cl
R
N
6a-c
7
a-f
8
a-c
R
R
Figure 2. Structures of the reference compound 4 and the new derivatives 5a-l, 6a-c, 7a-f and 8a-c. For
substituents, see Table 1.
RESULTS AND DISCUSSION
Chemistry. The synthesis of the new derivatives 5a-k is depicted in Schemes 1 and 2, while
3
3g
derivative 5l has been synthesized as previously reported . The aldehydes used as starting materials
were commercially available (9a-d,h) or were synthesized as reported in literature (9e,g,i)35-37 with the
exception of 9f that was obtained through a microwave assisted Suzuki reaction of 2,4-
dichlorobenzaldehyde with thien-2-yl-boronic acid (Scheme 2). Noteworthy, by means of this
procedure, we also obtained by-products 9j and 9k. Benzaldehyde 9k was separated by column
chromatography and a mixture of isomers 9f and 9j (4:1 ratio) was obtained and employed for the next
step.
ACS Paragon Plus Environment
1
0

Journal of Medicinal Chemistry
Page 12 of 78
Compounds 9a-j underwent a Claisen-Schmidt reaction with the properly substituted acetophenone in
the presence of NaOH as base furnishing chalcones 10a,d38,39 and 10b,c,e-j (Scheme 1). Such α,β-
unsatured derivatives underwent to ring closure by reacting with toluene-4-sulfonylmethylisocyanide
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(TosMIC) in the presence of sodium hydride to obtain the corresponding 3-aroylpyrroles 11a-j.
Reduction of the carbonyl group of 11a-j in the presence of lithium aluminum hydride furnished the
corresponding alcohols 12a-j, which were reacted with N,N’-carbonyldiimidazole (CDI) to afford the
final imidazoles 5a-j. Finally, the sulfone derivative 5k was obtained by oxidation with oxone of the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
already reported imidazole 5l33g,34
.
Scheme 1. Synthetic Route to 5a-k Derivativesa
O
O
R2
CHO
CHO
O
Ar
R2
ii
or
Ar
R
R1
i
N
R
R2
H
9a-g,j
9h,i
10a-j
11a-j
iii
O
O
N
N
H C S
N
H CS
3
Cl
3
Cl
HO
N
R2
N
N
R2
Ar
Cl
Cl
Ar
iv
v
N
N
N
H
H
N
H
5l
5k
5a-j
12a-j
H
a
Reagents and conditions: (i) NaOH, EtOH, room temp, 15 h, 50-100 % yield; (ii)
TosMIC, NaH, DMSO, Et O, room temp, 15-60 min, 18-85% yield; (iii) LiAlH , THF, 0
2
4
°
C to room temp, 30 min-3 h, 96-100 % yield; (iv) CDI, CH CN dry, room temp, 45
3
min-20 h, 24-100 % yield; (v) oxone, H O, MeOH, 0 °C to room temp, 18 h, 100 %
2
yield. For substituents, see Table S1 in Supporting Information.
Scheme 2. Synthetic Route to 9f Derivativea
ACS Paragon Plus Environment
1
1

Page 13 of 78
Journal of Medicinal Chemistry
1
2
S
Cl
S
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Cl
OH
B
i
H
H
H
OH
H
O
S
S
O
Cl
O
Cl
O
S
9f
9j
9k
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
.
Reagents and conditions: (i) Pd(dba) /PCy , K PO H O, DMF, 100 W, 100 psi, 80 °C, 1 h, 44 %
2
3
2
4
2
(
9f), 11 % (9j), 21 % (9k) yields.
Azoles 6a-c were synthesized as reported in Scheme 3. The preparation of the crucial intermediate 14
was achieved by cycloaddition of isocyanide 13 with 3-chloro-1-(4-chlorophenyl)propan-1-one in the
presence of NaH. The pyrrole 14 that formed was alkylated by reaction with the appropriate alkyl halide
in the presence of K CO to obtain N-alkylpyrroles 15b,c. The methanones 14 and 15b,c were then
2
3
reduced to the corresponding alcohols 16a-c, which underwent to reaction with CDI to give imidazoles
6a-c.
Scheme 3. Synthetic Route to 6a-c Derivativesa
O
O
O
O
S
N C
i
Cl
Cl
Cl
N
H
14
Cl
1
3
Cl
iii
ii
N
HO
O
N
Cl
Cl
Cl
iii
iv
N
R
N
R
N
R
Cl
Cl
6
a-c
16a-c
15b,c
Cl
a
Reagents and conditions: (i) NaH, DMSO, Et O,room temp, 50 min, 33 % yield; (ii) proper alkyl
halide, K CO , DMF, 90 °C, 3.5-54 h, 44-92 % yield; (iii) NaBH , THF, room temp or reflux, 4.5-23
h, 100 % yield; (iv) CDI, CH CN dry, room temp, 80 min-15 h, 67-82 % yield. For substituents,
2
2
3
4
3
see Table S1 in Supporting Information.
ACS Paragon Plus Environment
1
2

Journal of Medicinal Chemistry
Page 14 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The known intermediate 13 necessary as the starting material was prepared
according to Scheme 4 through a procedure modified with respect of the one already
reported in the literature40 achieving high yields in shorter reaction times. In particular,
compound 13 was prepared by a Mannich reaction of formamide with p-toluenesulfinic
acid and 4-chlorobenzaldeheyde in the presence of trimethylsilyl chloride (TMSCl) to
give N-[(4-chlorophenyl)(tosyl)methyl]formamide40 (17) that was then dehydrated by
reaction with POCl and Et N at -10 °C for 45 min.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
Scheme 4. Synthetic Route to 13 Derivativea
O
O
H
CHO
O
O
SO H
2
S
N
H
S
N C
i
ii
O
Cl
Cl
17
Cl
1
3
a
Reagents and conditions: (i) formamide, TMSCl, toluene, CH CN, 50 °C, 5 h, 80 % yield; (ii) POCl ,
3
3
Et N, DME, -10 °C, 45 min, 91 % yield.
3
The synthetic pathway to obtain derivatives 7a-f and 8a-c is reported in Scheme 5. The N-
phenylpyrroles 18a-e41-43 underwent a Friedel-Craft acylation in the presence of the properly substituted
benzoyl chloride to give mixture of - and -isomers 19a-c and 20a-f, respectively, that were separated
by column chromatography. After separation, ketones 19a-c and 20a-f were reduced with NaBH to
4
ACS Paragon Plus Environment
1
3

Page 15 of 78
Journal of Medicinal Chemistry
alcohols 21a-c and 22a-f, that were then reacted with CDI to obtain the imidazole derivatives 7a-f and
a-c.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
Scheme 5. Synthetic Route to 7a-f and 8a-c Derivativesa
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
R1
R1
N
N
R
N
i
O
R
ii
R 18a-e
1
9a-c
20a-f
ii
N
R1
HO
N
R1
N
R1
R1
iii
N
iii
N
N
OH
N
N
R
8a-c
R
21a-c
22a-f
7a-f
R
R
a
Reagents and conditions: (i) substituted benzoyl chloride, AlCl , DCM, room temp, 15 h, 4-21 %
3
yield; (ii) NaBH , THF, room temp or reflux, 2.5-48 h, 88-100 % yield; (iii) CDI, CH CN dry, room
4
3
temp 40 min-15 h, 20-100 % yield. For substituents, see Table S1 in Supporting
Information.
Enantiomeric Separation of Racemic Mixture of 4. Racemate 4 was chosen to perform an
enantiomeric separation of the single enantiomers generated by the stereogenic center that characterizes
these series of antiprotozoal agents. Accurate semipreparative enantioselective HPLC of racemate 4 was
carried out on the Chiralcel OD chiral stationary phase (CSP) and enabled us to isolate tens of mg of
enantiopure samples of (-)-4 and (+)-4, in 90% isolation yields. The enantiomeric purity of the collected
enantiomers (-)-4 and (+)-4 was checked and demonstrated both by analytical HPLC and polarimetric
ACS Paragon Plus Environment
1
4

Journal of Medicinal Chemistry
Page 16 of 78
and circular dichroism (CD) analysis. As shown in Figure 3, the chiroptical properties of the
enantiopure antipodes isolated on mg-scale were perfectly specular.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
1
1
0
5
0
5
0
5
(
(
+)-4
-)-4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
99 (c 0.1, EtOH)
-
-99 (c 0.1, EtOH)
-
-
-
10
15
20
2
00
220
240
260
280
300
320
340
Wavelength (nm)
Figure 3. CD spectra and specific rotations of the enantiomers (-)-4 and (+)-4
recorded in ethanol.
The absolute configuration of the (-)-4 (second eluted enantiomer on the Chiralcel OD CSP using the
mixture 70:30 n-hexane/EtOH as a mobile phase) was unequivocally determined by X-ray analysis.
Suitable crystals of the enantiomer were obtained by crystallization from methanol/water. An ORTEP
view of (R)-(-)-4 is illustrated in Figure 4.
ACS Paragon Plus Environment
1
5

Page 17 of 78
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. An ORTEP view of the molecular structure of (R)-(-)-4.
Evaluation of Biological Activities. In vitro phenotypic studies. All the newly designed
and synthesized derivatives 5a-k, 6a-c, 7a-f and 8a-c were tested against T. cruzi
amastigotes, L. donovani axenic amastigotes, T. b. rhodesiense trypomastigotes and
apicomplexan P. falciparum blood stage forms.
The results are reported in Table 1. The compounds showed anti-T. cruzi and
antiplasmodial activities within the submicromolar and nanomolar range, proving to be
also able in inhibiting L. donovani and T. b. rhodesiense at micromolar concentration.
ACS Paragon Plus Environment
1
6

Journal of Medicinal Chemistry
Page 18 of 78
Due to these different ranges, the in vitro activities as well as the SARs of the
imidazole-based compounds 5a-k, 6a-c, 7a-f and 8a-c will be discussed separately
for each parasite.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Although most compounds exhibit a certain cytotoxicity, many of them displayed
high to very high selectivity towards T. cruzi, showing IC50 values within the
nanomolar range and SI > 100. Additionally, four compounds showed selective
submicromolar-nanomolar inhibitory potencies against P. falciparum parasite.
In vitro activity against Trypanosoma brucei and Leishmania donovani. The tested imidazole
derivatives 5a-k, 6a-c, 7a-f and 8a-c displayed lower activities against T. b. rhodesiense and L.
donovani compared to their anti-T. cruzi and antiplasmodial activities, according to the trend previously
3
4
described by us for 3-arylpyrrole derivative 4 and congeners .
In fact, 5a-k, 6a-c, 7a-f and 8a-c showed micromolar IC values in the range 7.39 - 89.25 M on the
5
0
trypomastigote bloodstream form of T. b. rhodesiense with SI ranging from 1 to 13. Only two
compounds (5f, 6a) highlighted IC < 10M with the α-phenyl pyrrole compound 6a being the most
5
0
active (6a, IC = 7.39 M) but about 800 times less active than the reference drug (MEL).
5
0
Otherwise, the newly synthesized derivatives displayed higher inhibitory potencies against axenic L.
donovani amastigotes compared to T. b. rhodesiense, showing IC in the range 2.04 - 84.43 M and SI
5
0
ranging from 1 to 9. Notably, more than 50% of the tested compounds showed IC < 10 M, with the
5
0
tetrachloro derivative 8c being the most active (8c, IC = 2.04 M), about 4 times less potent than
5
0
reference drug MF.
ACS Paragon Plus Environment
1
7

Page 19 of 78
Journal of Medicinal Chemistry
In vitro activity against Plasmodium falciparum. Although P. falciparum does not have
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
sterol biosynthesis pathway neither CYP51, we investigated the antiplasmodial
activities of the compounds since some azole derivatives have been reported in the
literature as promising antimalarial agents.44-48 Moreover, according to this, we recently
reported a series of imidazole-based compounds endowed with notable antiplasmodial
potencies within the submicromolar range.34
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The biological evaluation on the erythrocytic stage of P. falciparum gave from good to excellent
results since the tested derivatives were active in the range 0.059-4.99 µM with SI ranging from 9 to
2
53.
Among the 23 newly synthesized compounds, 14 (61%) displayed submicromolar or nanomolar
antiplasmodial activities, whereas the remaining 9 showed IC in the low micromolar range (IC < 5
50
50
µM). In particular, 11 out of 14 derivatives (5a-d,f,i,j, 6a,c, 8a,b) resulted active at submicromolar
concentrations whereas 3 of them (5g,h, 8c) highlighted nanomolar potencies, proving to be up to 3
times more active than the reference drug chloroquine (CHQ). On the other hand, none of the new
compounds resulted more active than artemisinin (ART).
It is noteworthy that the antiplasmodial activities are well correlated with the intramolecular distance
between the benzyl-imidazolyl moiety and the phenyl ring bound to the pyrrole core. In fact, it seems
that the higher is the gap (1,3-relative positions) the lower are the inhibitory potencies. According to
this, derivatives of series 6 and 7, displaying micromolar or high submicromolar inhibitory potencies,
resulted less active than 1,2-substituted series 5 and 8 which are endowed with submicromolar up to
nanomolar
potencies.
Interestingly,
1-(phenyl(1-phenyl-1H-pyrrol-3-yl)methyl)-1H-imidazole
derivatives (7a-f) proved to be the less encouraging antiplasmodial compounds of the series, according
to the trend we described previously.34
ACS Paragon Plus Environment
1
8

Journal of Medicinal Chemistry
Page 20 of 78
The chlorine atom in 4-position of the phenyl ring linked to the pyrrole core of 4 was replaced with
alkyl, methoxy, thiomethyl and methylsulphonyl groups, leading to derivatives 5a-d,l. These
compounds were comparable or less active than the parent compound 4. In particular, we observed
activities decreasing in the following order: SCH > CH(CH ) > CH > C H > OCH > SO CH . The
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3 2
3
2
5
3
2
3
most active compounds 5a, 5b, 5c and 5l (IC = 0.29 μM; IC = 0.30 μM; IC = 0.28 μM; IC = 0.24
5
0
50
50
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
μM, respectively) were derivatives characterized by lipophilic groups. Conversely, the replacement of
methyl with methoxy or methylthio with methylsulphonyl moieties gave more polar derivatives
endowed with decreased activities (compare 5a with 5d and 5l with 5k, respectively). This trend is
respected comparing the more polar methoxy derivative 5d (IC = 0.46 μM) with the more lipophilic
50
isoster 5l (IC = 0.24 μM).
5
0
The introduction of an aryl or heteroaryl substituents in 2’-position of the 4-phenyl group linked to the
pyrrole ring of 4 proved to be a useful approach. In fact, although the introduction of a pyrrole group
gave compound 5e that was 28 times less potent than the parent compound (IC = 4.53 μM vs IC =
5
0
50
0
.16 μM, respectively), the introduction of a 2-thienyl or phenyl rings gave very good results. In fact,
the thienyl derivative 5f was active at submicromolar concentration (IC50 = 0.27 μM) resulting
comparable to 4, and even more, the phenyl derivative 5g (IC = 96 nM) was 3 times more potent than
50
5
e and also 2 times more active than CHQ and the reference compound 4.
Very interestingly, the removal of the 2-chloro substituent from the 3-(2,4-
dichlorophenylmethylimidazole) moiety of the trichloro derivative 5g led to the dichloro counterpart 5h
that showed an increased activity if compared to the parent compound (IC = 59 nM vs IC = 96 nM)
5
0
50
resulting 2 times more active than 5g and 3 times more potent than the reference azole 4. It is worthy to
note that compound 5h proved to be the best antiplasmodial compound of this series resulting 3 times
more active than CHQ (IC = 59 nM vs IC = 0.174 µM).
5
0
50
Finally, the replacement of the 4-phenyl ring linked to position 4 of the pyrrole core of 4 with
naphthyl groups was exploited. In particular, the introduction of a 2-naphthyl and 4-chloro-1-naphthyl
ACS Paragon Plus Environment
1
9

Page 21 of 78
Journal of Medicinal Chemistry
led to derivatives 5i, and 5j which showed IC = 0.34 μM and IC = 0.33 μM, respectively, that were 2
5
0
50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
times less active than 4.
Excellent antiplasmodial activities have been highlighted by N-phenyl pyrroles 8a-c. These
compounds are structurally related with derivatives of series 5 and reference compound 4. In fact, they
share a joint geometry between the benzylimidazole moiety and the phenylpyrrole portions, since the
last ones have both a 1,2-substitution pattern (Figure 5). Thus, compound 8b can be directly compared
to reference derivative 4, and very interestingly, these compounds showed similar activity (IC = 0.18
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
μM and IC = 0.16 μM, respectively). The replacement of the 2,4-dichloro substituent of 8b with a 4-
5
0
tert-butyl group gave derivative 8a that showed similar activity (IC = 0.19 μM). It is worthy to note
50
that the introduction of a further chlorine atom in 2’ position of the N-phenyl ring of the 4 isomer 8b
caused an increase of the inhibitory potency. In particular, the tetrachloro-compound 8c showed IC =
5
0
8
0 nM, proving to be 2 times more potent than the parent compound and CHQ.
R1
N
1
R
2
N
1
R2
N
N
N
2
R1
N
R
H
8a-c
4, 5a-h,k,l
1
,2-substitution pattern
Submicromolar-nanomolar antiplasmodial activities
Figure 5. 1,2-Substitution pattern on pyrrole ring and related antiplasmodial activity.
ACS Paragon Plus Environment
2
0

Journal of Medicinal Chemistry
Page 22 of 78
Notably, all the compounds (5g,h, 8c) active at nanomolar concentration displayed higher selectivity
towards the parasite (SI range, 149-253) compared to the reference compound 4 (SI=60) with the most
active derivative 5h being also the most selective (SI = 253).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Finally, the separation of enantiomers of 4 led to the conclusion that the eutomer in this series of
antiplasmodial agent is the R isomer (R)-(-)-4 that showed IC = 92 nM.
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In vitro activity against Trypanosoma cruzi. The biological outcomes on the amastigote
form of T. cruzi were excellent. In fact, with the exception of derivative 7d, the newly
designed and synthesized azole derivatives proved to be highly active showing IC50 in
the range 0.002-11.95 μM and SIs ranging from 6 to 4030. Notably, 19 derivatives,
representing 86% of the tested compounds, displayed interesting activities with IC50
ranging from micromolar to nanomolar concentrations.
In particular, with the exception of 7b, 7c and 7e that displayed micromolar activities within the same
order of magnitude of Bz used as the reference drug (IC = 3.60 μM, IC = 6.18 μM, IC = 3.90 μM,
5
0
50
50
respectively), the remaining 16 compounds were active at submicromolar and nanomolar
concentrations, resulting from one to three orders of magnitude more active than Bz. In particular, three
derivatives (5g, 5h and 8a) proved to be active within the submicromolar range resulting 4-12 times
more active than Bz (5g, IC = 0.142 μM; 5h, IC = 0.203 μM; 8a, IC = 0.390 μM), whereas the
5
0
50
50
remaining 13 compounds displayed inhibitory potencies at nanomolar concentrations. In particular, nine
derivatives proved to be 25-92 times more active than the reference drug Bz, with derivatives 5f,i,k
being up to 2 times more active than 4 (5f, IC = 27 nM; 5i, IC = 18 nM; 5k, IC = 29 nM). Finally,
5
0
50
50
the other four compounds (8b and 6a-c) showed excellent anti-T. cruzi activities within the low
nanomolar range, resulting not only 2-3 order of magnitude (330-825 times) more active than Bz but
ACS Paragon Plus Environment
2
1

Page 23 of 78
Journal of Medicinal Chemistry
also one order of magnitude (7-175 times) more active than the internal reference compound 4 (6a, IC50
3 nM; 6b, IC = 3 nM; 6c, IC = 2 nM; 8b, IC = 5 nM).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
=
5
0
50
50
Notably, all the submicromolar/nanomolar active compounds displayed high or very high selectivity
against the parasite (SI range, 74-4030). In particular, derivatives showing inhibitory potencies at low
nanomolar concentrations highlighted SI > 2000, thus being far more selective than 4.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
With the exception of derivative 5e, all the analogues of 4 (5a-d,f-l) displayed good submicrolar-
nanomolar activities highlighting also high selectivity against the parasite.
The replacement of the 4’-chlorine atom of 4 with methyl, ethyl, isopropyl, thiomethyl,
methylsulfonyl or methoxy substituents led to derivatives 5a-d,k,l active at nanomolar concentration. In
particular, 5a, 5b, 5c 5d, and 5l, showed IC in the range 43-66 nM, while 5k, was the most potent
5
0
within this group with IC = 9 nM and proved to be 57 times more potent than Bz. It is worthy to note
5
0
that, contrary to what has been observed for P. falciparum, the oxidation of the methylsulphide group of
5l into the corresponding methylsulphone led to the more potent derivative 5k, which resulted also more
active than reference 4. In the end, it is possible to observe that the activity can be described in the
following order SO Me > Cl > SMe > OMe > i-Pr > Et (5k > 4 > 5l > 5d > 5a > 5b,c).
2
The introduction of an aryl or heteroaryl group in position 2 of the phenyl ring linked to the pyrrole
core on the 4-phenyl ring of 4 led to derivatives 5e-h endowed with a wider range of activities. In fact,
depending on the nature of the (hetero)aryl substituent, the inhibitory potencies ranged from micromolar
to nanomolar concentrations.
In particular, the introduction of a 1-pyrrolyl group caused a substantial decrease of activity since
compound 5e resulted nearly 340 times less active than the reference compound 4 being also one order
of magnitude less active than Bz (IC = 11.95 μM). On the contrary, the introduction of a phenyl group
5
0
in that position led to compounds active at submicromolar concentration. In particular, both biphenyl
derivatives 5g,h proved one order of magnitude more active than Bz and 4-6 times less potent than the
reference compound 4 (5g, IC = 0.142 μM; 5h, IC = 0.203 μM). Notably, the trichloro derivative 5g
5
0
50
was more active than the dichloro counterpart 5g.
ACS Paragon Plus Environment
2
2

Journal of Medicinal Chemistry
Page 24 of 78
Finally, the introduction of a 2-thienyl substituent on the phenyl linked to the pyrrole moiety led to
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compound 5f that proved active at nanomolar concentration (IC = 27 nM), was 61 times more active
5
0
than Bz and also slightly more potent than 4.
Thus, concerning the nature of the 2’-substituent linked to the 4-phenyl ring of 4 analogues it is
possible to observe that the activity has the following order: 2-thienyl > H > Ph >> 1-pyrrolyl (5f > 4 >
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
g >> 5e).
Finally, the replacement of the 4-phenyl ring of 4 with naphthyl groups led to compounds active at
nanomolar concentrations. In particular, the introduction of a 4-chloro-1-naphthyl group (5j) caused a
slight decrease of the inhibitory potency (IC = 48 nM). On the contrary, the replacement with a 2-
50
naphthyl moiety led to derivative 5i, which showed very good activity (IC = 18 nM). Notably, this
5
0
compound resulted 2 times more active than 4, proving to be also the most active anti-T. cruzi derivative
of series 5.
The shift of the phenyl ring of compounds of series 5 from position 4 to the positions 5 and 1 of the
pyrrole core, led to phenyl pyrroles of series 6 and 7, respectively. Concerning the compounds of series
7
, we obtained derivatives endowed with micromolar inhibitory potencies against T. cruzi, with the
exception of 7d, which was inactive. In particular, 7a-c,e,f proved to be from 2 to 6 times less active
than Bz displaying SI values ranging from 6 to 44. Among them, the most active compound was the
dinitro derivative 7b, which showed a nearly 2 times lower anti-T. cruzi activity in respect of Bz
proving to be also the most selective compound of this group (IC = 3.60 μM; SI = 44).
50
Generally, among the newly designed and synthesized azoles, 1-(phenyl(1-phenyl-1H-pyrrol-3-
yl)methyl)-1H-imidazole derivatives 7a-e proved to be endowed with the lowest inhibitory potencies
and selectivity, confirming the trend previously described by us.34
Conversely, the α-phenyl pyrroles 6a-c, obtained by shifting the 4-phenyl ring of 4 from position 4- to
position 5- of the pyrrole moiety, showed excellent potencies against T. cruzi. In fact, derivatives 6a-c
proved to be active within the low nanomolar range and presented the best anti-T. cruzi activities among
the newly synthesized antiprotozoal agents. They are 550-825 times more active than Bz and 12-18
ACS Paragon Plus Environment
2
3

Page 25 of 78
Journal of Medicinal Chemistry
times more potent than the internal reference compound 4. The selectivity of these agents was also very
good since 6a-c showed SIs values ranging from 2217 to 4030, resulting 8-15 times more selective than
4.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The substituent on the pyrrole nitrogen seems not to influence the activity of these compounds. In
fact, derivatives 6a,b showed the same anti-T. cruzi activities (6a,b, IC = 3 nM), suggesting that the
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
presence of an hydrogen or a methyl group in such position does not affect the antiprotozoal activity.
Further, the N-alkylation of the nitrogen pyrrole with an allyl group gave 6c that was just slightly more
effective than compounds 6a and 6b, with a nearly 2-times improvement of the inhibitory potency.
However, it is worthy to note that among the azole compounds described, derivative 6c was the most
active one, highlighting an IC of 2 nM. Notably, this derivative proved to be 825 times more potent
5
0
than Bz, and 18 times more active than 4 showing also a very high selectivity towards the T. cruzi
parasite (SI = 3880).
A further shift of the phenyl ring was realized obtaining N-phenyl pyrroles 8a-c. These compounds
are isomers of derivatives 7 but are strictly related to azoles of series 5 since the relative position of the
benzylimidazole moiety and the phenyl ring is 1,2 in both cases.
Derivatives 8a-c highlighted inhibitory potencies at submicromolar up to nanomolar concentrations,
resulting from 4 to 330 times more active than Bz and showing also high selectivity towards the parasite
with SI values ranging from 119 to 2776.
Since 8b and 4 have the same substituents and also the same geometry, a direct comparison can be
addressed. Noteworthy 8a displayed excellent inhibitory potency and resulted the most active and
selective compound of the 8 series (IC = 5 nM, SI = 2776), proving to be not only 3 orders of
5
0
magnitude more active than Bz but also 7 times more active than 4. The replacement of the chlorine
atom linked to the N-phenyl group with a tert-butyl substituent, gave derivative 8a, which showed a
decrease in activity (IC = 0.39 μM) if compared to both the parent compound 8b and 4. In fact,
5
0
although it resulted 4 times more potent than Bz, it resulted 11 times less active than 4 being also
endowed with 2 order of magnitude lower potency in respect of derivative 8b. Conversely, the
ACS Paragon Plus Environment
2
4

Journal of Medicinal Chemistry
Page 26 of 78
introduction of a further chlorine atom in 2’ position of the N-phenyl moiety of 8b led to compound 8c,
which, although active at nanomolar concentration, resulted less active than the parent compound. In
particular, 8c (IC = 62 nM) was 27 times more active than Bz, 2 times less active than 4 and 12 times
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
0
less potent than 8b.
In vitro activity of enantiomers of compound 4. Finally, separated enantiomers of the potent reference
derivative 4 have been tested against the protozoa panel.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
S)-(+)-4 isomer displayed higher activity against T. b. rhodesiense and comparable antileishmanial
potency in respect of the racemate 4. Additionally, it displayed promising submicromolar antiplasmodial
activity, resulting slightly more active than 4 (4, IC = 0.16 μM; (S)-(+)-4, IC = 0.11 μM) and nearly 2
50
50
times more potent than CHQ. Further, it showed very good potency against T. cruzi, resulting 2 orders
of magnitude more potent than Bz and 2 times more potent than the racemate 4 ((S)-(+)-4, IC = 17 nM;
5
0
4
, IC = 35 nM).
50
However, the levorotatory isomer (R)-(-)-4 showed the highest inhibitory potency proving to be the
eutomer, being from 2 to 39 times more active than racemate 4 against all parasites. In particular, it
resulted 2 times more potent than 4 against T. b. rhodesiense and comparable to it against L. donovani.
Moreover, (R)-(-)-4 showed promising nanomolar potency against P. falciparum proving to be 2 times
more active than 4 ((R)-(-)-4, IC = 92 nM; 4, IC = 0.16 μM) and the reference drug CHQ. Finally, it
5
0
50
is worthy to note that the eutomer (R)-(-)-4, showed subnanomolar activity against T. cruzi, (IC = 0.9
5
0
nM), being 40 times more active than 4, 1833 times more potent than Bz and highly selective towards
the parasite with SI = 2844.
ACS Paragon Plus Environment
2
5

Page 27 of 78
1
Journal of Medicinal Chemistry
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
Table 1. Antiprotozoal activity (T. b. rhodesiense, T. cruzi, L. donovani, and P. falciparum) and cytotoxicity of the tested compounds 5a-l, 6a-c 7a-
f and 8a-c.
IC50a
M)
CC50b
(M)
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
Tbc
SId
Tce
SIf
Ldg
SIh
Pfi
SIj
Cpds
R
R1
H
R2
5
5
5
a
b
c
CH
CH
3
2
2,4-Cl
2
18.83
22.51
24.22
28.37
11.89
8.31
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
0.050
0.066
0.066
0.050
11.95
0.027
0.142
0.203
0.018
0.048
0.029
0.043
0.003
0.003
0.002
367
202
192
296
-
5.64
4.87
5.22
5.51
10.75
2.78
13.89
15.01
6.41
4.83
10.15
5.12
3.01
5.70
2.16
3
3
2
3
-
0.29
0.30
0.28
0.46
4.53
0.27
0.096
0.059
0.34
0.33
1.30
0.24
0.78
1.41
0.63
63
45
45
32
2
18.36
13.35
12.70
14.79
7.67
CH
3
H
2,4- Cl
2,4- Cl
2,4- Cl
2,4- Cl
2,4- Cl
2,4- Cl
4-Cl
2
2
2
2
2
2
(CH
3
)
2
CH
H
5d
OCH
Cl
3
H
5
5
5
5
e
f
Pyk
Tiophl
Phm
Cl
183
101
74
2
1
-
18
149
253
19
15
52
30
9
4.93
g
h
Cl
29.03
34.43
15.58
16.47
33.02
15.81
7.39
14.31
14.92
6.54
Cl
Phm
-
5i
2-Npn
4-Cl-1-Npn
2,4- Cl
2,4- Cl
2,4- Cl
2,4- Cl
-
2
2
2
2
363
102
2344
168
2217
4030
3880
1
1
7
1
2
2
4
5
5
5
6
j
-
4.90
k
l34
a
SO
2
CH
3
H
H
-
67.98
7.22
SCH
H
3
6.65
6b
CH
CH
3
2
-
-
40.02
38.69
9
12.09
7.76
6
c
CH=CH
2
-
-
12
2
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 78
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
7a
NO
NO
NO
2
2
2
H
-
14.52
11.84
40.65
31.11
35.67
27.76
89.25
37.0
4
13
2
1
1
5
-
10.25
3.60
6
31.07
17.72
25.16
21.28
30.38
84.43
9.54
2
9
3
2
1
2
5
3
5
3
4.99
2.33
4.57
1.81
2.18
4.09
0.19
0.18
0.080
0.16
12
68
18
22
19
34
244
77
116
60
60.40
158.97
82.36
40.07
42.36
139.10
46.42
13.88
9.26
7
7
7
b
c
NO
Cl
2
-
44
13
-
-
6.18
d
CF
Cl
3
Cl
-
>74.66
3.90
7e
7f
CN
-
11
-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
CN
CN
-
Dnpo
0.39
t
8
8
8
4
a
b
c
4-Cl
4-Cl
2,4- Cl
Cl
4- Bu
2,4- Cl
2,4- Cl
H
-
119
2776
149
275
2
2
-
-
0.005
0.062
0.035
5.34
2
-
31.11
17.33
-
2.04
2,4- Cl
2
-
3.77
9.61
MELp
Bzq
0.0088
3284.1
28.9
>385
137
1.65
>233.3
MFr
0.56
244.6
CHQs
0.174
0.007
533.3
92.8
>5000 >350
0
ARTt
PPTu
aConcentration
of compound
0.007
required
decrease
parasite
to
b
viability by 50% compared to the number of parasites grown in the absence of the test compound; Cytotoxicity measurement for L6 cells;
c
d
e
Trypanosoma brucei rhodesiense, STIB900 trypomastigote; Calculated as (CC )/(IC for T.b. rhodesiense); Trypanosoma cruzi, Tulahuen C2C4
50
50
f
g
h
amastigote; Calculated as (CC )/(IC for T. cruzi); Leishmania donovani, MHOM/ET/67/L82 axenic amastigote; Calculated as (CC )/(IC for
5
0
50
50
50
2
7
ACS Paragon Plus Environment

Page 29 of 78
1
Journal of Medicinal Chemistry
i
j
k
l
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
L. donovani); Plasmodium falciparum K1 erythrocytic stage; Calculated as (CC )/(IC for P. falciparum); Py = 1-pyrrolyl; Tioph = 2-thienyl;
Ph = phenyl; Np = naphtyl; Determination not possible; melarsoprol; Bz; miltefosine; chloroquine; artemisinin; podophyllotoxin.
5
0
50
m
n
o
p
q
r
s
t
u
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
2
8
ACS Paragon Plus Environment