Design, synthesis and biological evaluation of novel 4- alkapolyenylpyrrolo[1,2-a]quinoxalines as antileishmanial agents - Part III

Article abstract of DOI:10.1016/j.ejmech.2014.05.037

A series of new 4-alkapolyenylpyrrolo[1,2-a]quinoxaline derivatives, original and structural analogues of alkaloid chimanine B and of previously described 4-alkenylpyrrolo[1,2-a]quinoxalines, was synthesized in good yields using efficient palladium-catalyzed Suzuki-Miyaura cross-coupling reactions. These new compounds were tested for in vitro antiparasitic activity upon three Leishmania spp. strains. Biological results showed activity against the promastigote forms of L. major, L. mexicana and L. donovani with IC₅₀ ranging from 1.2 to 14.7 μM. In attempting to investigate if our pyrrolo[1,2-a]quinoxaline derivatives are broad-spectrum antiprotozoal compounds activities toward one Trypanosoma brucei brucei strain and the W2 and 3D7 Plasmodium falciparum strains were also investigated. In parallel, the in vitro cytotoxicity of these molecules was assessed on the murine J774 and human HepG2 cell lines. Structure-activity relationships of these new synthetic compounds are here discussed.

Full text of DOI:10.1016/j.ejmech.2014.05.037

Accepted Manuscript
Design, Synthesis and Biological Evaluation of Novel 4-
Alkapolyenylpyrrolo[1,2-a]quinoxalines as Antileishmanial Agents – Part III
Luisa Ronga , Marco Del Favero , Anita Cohen , Claire Soum , Patrice Le Pape ,
Solène Savrimoutou , Noël Pinaud , Catherine Mullié , Sylvie Daulouede , Philippe
Vincendeau , Natacha Farvacques , Patrice Agnamey , Fabrice Pagniez , Sébastien
Hutter , Nadine Azas , Pascal Sonnet , Jean Guillon
PII:
S0223-5234(14)00451-6
10.1016/j.ejmech.2014.05.037
EJMECH 6993
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 April 2014
Revised Date: 9 May 2014
Accepted Date: 11 May 2014
Please cite this article as: L. Ronga, M. Del Favero, A. Cohen, C. Soum, P. Le Pape, S. Savrimoutou,
N. Pinaud, C. Mullié, S. Daulouede, P. Vincendeau, N. Farvacques, P. Agnamey, F. Pagniez, S.
Hutter, N. Azas, P. Sonnet, J. Guillon, Design, Synthesis and Biological Evaluation of Novel 4-
Alkapolyenylpyrrolo[1,2-a]quinoxalines as Antileishmanial Agents – Part III, European Journal of
Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.05.037.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Design, Synthesis and Biological Evaluation of Novel 4-Alkapolyenylpyrrolo[1,2-a]quinoxalines as
Antileishmanial Agents – Part III
Luisa Ronga, Marco Del Favero, Anita Cohen, Claire Soum, Patrice Le Pape, Solène Savrimoutou, Noël Pinaud,
Catherine Mullié, Sylvie Daulouede, Philippe Vincendeau, Natacha Farvacques, Patrice Agnamey, Fabrice Pagniez,
Sébastien Hutter, Nadine Azas, Pascal Sonnet and Jean Guillon*
N
R1
CH3
(
)
n
N
N
N
N
R1
R1
CH3
(
)
n
N
CH3
N
N
N
R2
R1- = H-, CH3O-
n = 1-5
R1
chimanine B
R1- = H-, CH3O-
R2- = Alkyl-, Alkenyl-,
CH3
Series II
Alkynyl-, Aryl- etc ...
R1- = H-, CH3O-
n = 0-3
Series I
IC50 = 0.5-7 µM (IS = 3-16)
L. amazonensis or L. infantum
IC50 = 1-7 µM (IS = 7-40)
Series III
L. amazonensis or L. infantum
Research Highlights
> Synthesis of novel 4-alkapolyenylpyrrolo[1,2-a]quinoxaline derivatives.
> Compounds were tested for antiparasitic activity upon Leishmania spp. strains.
> Compounds were tested in vitro upon T. b. brucei. and Plasmodium falciparum strains.
> New compounds with IC50 in the micromolar range were identified.
1

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Novel 4-Alkapolyenylpyrrolo[1,2-a]quinoxalines as
Antileishmanial Agents – Part III
Luisa Ronga,a,b Marco Del Favero,a,b Anita Cohen,c Claire Soum,a,b Patrice Le Pape,d Solène Savrimoutou,a,b Noël Pinaud,e Catherine
Mullié,f Sylvie Daulouede,g Philippe Vincendeau,g Natacha Farvacques,f Patrice Agnamey,f,h Fabrice Pagniez,d Sébastien Hutter,c
Nadine Azas,c Pascal Sonnetf and Jean Guillona,b*
a Univ. Bordeaux, UFR des Sciences Pharmaceutiques, ARNA Laboratory, F-33076 Bordeaux cedex, France
b INSERM U869, ARNA Laboratory, F-33000 Bordeaux, France
c
Aix-Marseille Univ., Laboratory of Parasitology, UMR-MD3, Faculty of Pharmacy, 27 Bd Jean Moulin, CS30064, F-13385
Marseille cedex 5, France
d
Université de Nantes, Département de Parasitologie et Mycologie Médicale, IICiMed, EA1155, UFR des Sciences
Pharmaceutiques, F-44000 Nantes, France
e Univ. Bordeaux, ISM – CNRS UMR 5255, 351 cours de la Libération, F-33405 Talence cedex, France
f
Université de Picardie Jules Verne, Laboratoire de Glycochimie, des Antimicrobiens et des Agroressouces, CNRS FRE 3517,
UFR de Pharmacie, 1 rue des Louvels, F-80037 Amiens cedex 01, France
g UMR 177 IRD CIRAD, Université de Bordeaux, Laboratoire de Parasitologie, F-33076 Bordeaux Cedex, France
h CHU Amiens, Laboratoire de Parasitologie-Mycologie, Avenue Laënnec, 80054 Amiens, France
Corresponding author, Tel.: +33 5 57 57 16 52; fax: +33 5 57 57 13 52
E-mail address: jean.guillon@u-bordeaux.fr
ABSTRACT
A series of new 4-alkapolyenylpyrrolo[1,2-a]quinoxaline derivatives, original and structural analogues of alkaloid chimanine B
and of previously described 4-alkenylpyrrolo[1,2-a]quinoxalines, was synthesized in good yields using efficient palladium-
catalyzed Suzuki-Miyaura cross-coupling reactions. These new compounds were tested for in vitro antiparasitic activity upon
three Leishmania spp. Strains. Biological results showed activity against the promastigote forms of L. major, L. mexicana and L.
donovani with IC50 ranging from 1.2 to 14.7 µM. In attempting to investigate if our pyrrolo[1,2-a]quinoxaline derivatives are
broad-spectrum antiprotozoal compounds activities toward one Trypanosoma brucei brucei strain and the W2 and 3D7
Plasmodium falciparum strains were also investigated. In parallel, the in vitro cytotoxicity of these molecules was assessed on the
murine J774 and human HepG2 cell lines. Structure-activity relationships of these new synthetic compounds are here discussed.
Keywords
Pyrrolo[1,2-a]quinoxaline
Antileishmanial activity
Antitrypanosomal activity
Antimalarial activity
Synthesis
Suzuki reaction
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1. Introduction
Over three billion people are estimated to be at risk for the parasitic diseases such as malaria, trypanosomiasis and
leishmaniasis.1 Leishmaniasis is a widespread parasitic disease that is caused by protozoan parasites of the genus Leishmania in
tropical and subtropical areas in both the old and new worlds. According to recent World Health Organization reports, 88
countries are affected, comprehending 12 million infected people worldwide, with approximately 350 million people at risk. The
incidence is increasing worldwide, with 1-2 million new cases registered annually, despite all efforts being made to fight the
disease.1-4 It occurs in three major forms; cutaneous (CL), muco-cutaneous (MCL) and visceral leishmaniasis (VL), depending
upon parasite species. L. donovani and L. infantum are major causative agents of VL, while L. major, L. tropica, L. aethiopica, L.
braziliensis, L. panamensis, L. amazonensis and L. mexicana cause CL.5 L. braziliensis, L. panamensis also are agents of muco-
cutaneous leishmaniasis. Therapy of patients with leishmaniasis is still a serious problem. Historically, leishmaniasis
chemotherapy has been based on the use of toxic heavy metals, particularly antimony compounds. In fact, the drugs for
leishmaniasis treatment of all clinical forms are meglumine antimoniate (Glucantime®) and sodium stibogluconate (Pentostam®)
(Figure 1), despite their renal and cardiac toxicities.6-8 When this treatment is not effective, alternative drugs include pentamidine
(Pentacarinat®) and amphotericin B (Fungizone®, Ambisome®), also toxic with serious side effects.7,8 The liposomal form of
amphotericin B (Ambisome®), is less toxic but much more expensive. Miltefosine, a phosphocholine analogue originally
developed as an anticancer agent, has been found to be highly effective against leishmaniasis in vitro and in vivo. Now, this
compound is the only oral agent (Impavido®) against cutaneous and visceral leishmaniasis, although presenting severe
gastrointestinal side effects.7,8 As the leishmaniasis chemotherapy is still inefficient, there is an urgent need for the development of
new, efficient, and safe drugs.5,9,10 Traditional/ethnic medicine is often a good source for researchers looking for bioactive
substances. Leishmaniasis is not an exception, and plants have been used for the treatment of people living far from modern
medicine.7,10-15 Such compounds belong to the following groups: alkaloids, terpenes, quinines, lactones, coumarins, acetogenins of
annonaceae, chalcones, tetralones, lignans and saponins.11-16 As an example, some 2-substituted quinoline derivatives active
against leishmaniasis were isolated from Galipea longiflora, through a bioactivity-guided fractionation process from an
ethnopharmacological investigation in Bolivia.17,18 A series of various 2-substituted quinoline alkaloids were extracted from the
bark, root and leaves of Galipea longiflora Krause (Rutaceae).17-22 Hence, chimanine B (Figure 2) isolated from Galipea species
was claimed as the lead molecule useful for further pharmacomodulations.23,24
The pyrrolo[1,2-a]quinoxaline heterocyclic framework constitutes the basis of an important class of compounds possessing
interesting biological activities. These compounds have been reported to serve as key intermediates for the assembly of several
heterocycles including antipsychotic agent,25 anti-HIV agent,26 adenosine A3 receptor modulator,27 antiparasitic agents,28-32 and
antitumor agents.33-38
In the course of our work devoted to discover new compounds in the leishmaniasis chemotherapy, we previously identified a
series of 4-substituted pyrrolo[1,2-a]quinoxaline derivatives (Series I, Figure 2) designed as interesting bioactive isosteres of
Galipea species quinoline alkaloids.29 From these preliminary results, it appeared that 4-alkenylpyrrolo[1,2-a]quinoxalines
endowed with lipophilic unsaturated carbon chain, such as the 4-(E)-pentenyl derivatives, could initiate new valuable
antileishmanial chemistry scaffolding. Thus, taking into account our experience in the field of the synthesis of new bioactive
heterocyclic compounds of the type pyrrolo[1,2-a]quinoxaline,27-32,35-38 we designed, synthesized and evaluated new 4-(E)-
alkenylpyrrolo[1,2-a]quinoxalines correlated with a particular lipophilic behaviour (Series II, Figure 2).30 In this new work, we
present preliminary results concerning the design and the synthesis of a third series (Series III, Figure 2) of new 4-[(1E,3E)-alka-
1,3-dienyl]pyrrolo[1,2-a]quinoxalines 1a-l,1n and 4-[(1E,3E,5E)-alka-1,3,5-trienyl]pyrrolo[1,2-a]quinoxaline 1m via Suzuki-
Miyaura cross-coupling reactions, and initial in vitro antileishmanial activity upon Leishmania spp. strains. This third series was
designed in analogy with the best antileishmanial results obtained with the previously synthesized Series II by introducing a
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conjugated polyene chain to determine the importance of such polyene moiety in the putative biological activity. Such a
conjugated polyene chain was also observed in the chemical structure of amphotericin B.
Finally, all compounds were tested against L. major, L. mexicana and L. donovani protozoa with pentamidine as the
reference drug.
As these new compounds were designed as quinoline-like bio-isosters, and as the quinoline skeleton is the fundamental unit
of many antimalarial drugs, these pyrrolo[1,2-a]quinoxaline derivatives 1 were also investigated on other medically important
protozoans, Trypanosoma brucei brucei and Plasmodium falciparum strains in order to evaluate the specificity of their
antiparasitic activity. Plasmodium falciparum is the causative agent of the most severe type of malaria: cerebral malaria which
infects about 225 million people and causes nearly 800000 deaths, mostly in children annually.5 Human African Trypanosomiasis
(HAT) or sleeping sickness occurs in Sub-Saharan Africa and it is caused by two subspecies of the Trypanosoma brucei group.
HAT threatens about 50 million people living in Sub-Saharan Africa and causes an estimated 25000 deaths per year.5
In parallel, the in vitro cytotoxicity of these molecules was assessed on the murine J774A.1 and human HepG2 cell lines, in
order to determine their selectivity.
These new biological results were discussed in terms of structure-activity relationships studies to enlighten the influence of
the 4-substituted alkadienyl or alkatrienyl side chain on the pyrrolo[1,2-a]quinoxaline moiety for the modulation of anti-parasitic
activity.
2. Chemistry
All reported 4-[(E,E)-alkadienyl]pyrrolo[1,2-a]quinoxaline derivatives 1a-l were obtained from 1-(2-nitrophenyl)pyrroles 2a-d
(Scheme 1), prepared in acetic acid according to the Clauson-Kaas reaction, starting from 2-nitroanilines and 2,5-
dimethoxytetrahydrofuran (DMTHF). This pathway partially involved synthetic methodologies already described by our group.28-
32
The preparation of compounds 2a-d was performed under microwave irradiation. The resulting 1-(2-nitrophenyl)pyrroles
intermediates 2a-d were subsequently reduced into the attempted 1-(2-aminophenyl)pyrroles 3a-d using a sodium borohydride-
copper(II) sulfate in ethanol at room temperature according to the conditions described by Yoo and Lee.39 This NaBH4-CuSO4
system was found to be quite powerful in reducing our aromatic nitro groups with excellent yields (85-94%).28-32 The reaction of
3a-d with triphosgene in toluene gave the lactams 4a-d, which were subsequently chlorodehydroxylated with phosphorous
oxychloride, leading to the 4-chloroquinoxalines 5a-d.38 The 3D spatial structure of new pyrroloquinoxaline 5d was established
1
by X-ray crystallography (Figure 3) and confirmed the structure as anticipated on the basis of IR and H-NMR data. 4-[(1E,3E)-
alka-1,3-dienyl]pyrrolo[1,2-a]quinoxalines 1a-l were easily prepared in quite good yields (60-83%) by a direct palladium-
catalyzed Suzuki-Miyaura cross-coupling reaction of 4-chloropyrroloquinoxalines 5a-d with alka-dienyl N-methyliminodiacetic
acid MIDA boronates 6a-c (Table 1). The (1E,3E)-dienyl boronates 6a-c were synthesized by reacting the commercially available
trans-(2-bromovinyl) MIDA boronate with various (E)-alkenylboronic acids under Suzuki cross-coupling conditions [Pd(OAc)2,
SPhos, Cs2CO3 or KF] with complete stereoretention (Scheme 1).40-42 The first method (Method A) involved a one-pot MIDA
boronates hydrolysis under mild aqueous basic conditions (1N aq. NaOH) and subsequent cross-coupling with chlorides 5a-d
using Pd(OAc)2 and S-Phos in THF at room temperature leading to compounds 1e-g (Table 1).40-42 The moderate yield (41%)
obtained for the preparation of compound 1h (entry 8) led us to investigate the use of Pd(PPh3)2NBS as catalyst (Method B) as it
has recently been used successfully for the Suzuki coupling of alkenyl halides with MIDA boronates.43,44 Pleasingly, using this
catalyst, successful coupling was achieved with chloropyrroloquinoxaline 5d affording the coupled product 1h in 80% yield (entry
9). Consequently, the synthesis of 4-[(1E,3E)-alka-1,3-dienyl]pyrrolo[1,2-a]quinoxalines 1a-d and 1i-l was achieved using this
Pd(PPh3)2NBS catalyst through an efficient coupling of chloro derivatives 5a-d with a variety of MIDA boronates 6. The
reactions were essentially complete after 6 h but overnight heating could be employed without loss of yield.
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From NMR assignations, it appeared that the stereochemistry of the alkadienyl groups in derivatives 1a-l were retained affording
products as the E,E-isomers. Thus, their 1H NMR spectra showed the characteristic chemical shifts of a conjugated (E,E)-
alkadienyl chain in the olefinic region [δ 7.73-7.62 ppm (1H, dd, J = 15.80-14.90 and 11.25-10.80 Hz), 6.89-6.85 ppm (1H, d, J =
15.80-14.90 Hz), 6.42-6.38 ppm (1H, dd, J = 15.15-14.50 and 11.25-10.80 Hz), and 6.20-6.13 ppm (1H, dt, J = 15.15-14.50 and
7.35-6.90 Hz)].
As 5-methoxy-2-nitroaniline 7 is not commercially available, this nitroaniline was prepared according to the literature.45,46 This
methoxy-nitroaniline 7 was then converted into the nitrophenylpyrrole 2c by the previously described Clauson-Kaas reaction
(Scheme 2). In this work, we have also demonstrated that the 1-(5-methoxy-2-nitrophenyl)pyrrole 2c could be synthesized using a
Buchwald palladium-catalyzed intermolecular coupling of methanol and 5-chloro-2-nitrophenylpyrrole 2e (Method A),47 or by
reaction of sodium methanoate on the chloro-aryl derivative 2e using CuCl as catalyst through an Ullman reaction (Method B).48
In order to introduce a triene chain on our pyrroloquinoxaline skeleton, we investigated the first following pathway: the Corey-
Fuchs reaction of the ylide generated from CBr4 and triphenylphosphine on the pyrrolo[1,2-a]quinoxaline-4-carboxaldehyde 8,
previously described by our group,29 gave the 1,1-dibromoethene-pyrroloquinoxaline 9 (Scheme 3).49 Iron catalyzed
hydrodebromination50 of the dibromo derivative 9 with a Grignard reagent failed and did not afford the desired (E)-vinyl bromide,
a key intermediate in the synthesis of the 4-[(1E,3E,5E)-alka-1,3,5-trienyl]pyrrolo[1,2-a]quinoxaline 1m. As this first pathway did
not lead to the desired triene compound 1m, we investigated a second one. As shown in Scheme 3, hydrolysis of cross-coupling
product 6a with aqueous base followed by coupling of the (E,E)-dienylboronic acid without further purification to the trans-(2-
bromovinyl) MIDA boronate under anhydrous conditions yielded the desired (E,E,E)-triene MIDA boronate 6d. A final cross-
coupling of this polyene 6d with 4-chloropyrroloquinoxaline 5a led to the 4-[(1E,3E,5E)-alka-1,3,5-trienyl]pyrrolo[1,2-
a]quinoxaline 1m by using Pd(PPh3)2NBS as the catalyst, and sodium carbonate as the base.
In this new series, we also investigated the introduction of an alkoxy group on the pyrrole moiety of our heterocyclic
pyrroloquinoxaline skeleton. The starting material ethyl 3-ethoxypyrrole-2-carboxylate 10 was prepared by silver-catalyzed
cycloaddition of commercialy available ethoxyacetylene with ethylisocyanoacetate in 1,4-dioxane at 80°C.51,52 The preparation of
the N-aryl pyrrole 11 was obtained by nucleophilic substitution of the ethyl 3-ethoxypyrrole-2-carboxylate 10 with 2-fluoro-
nitrobenzene using cesium carbonate as the base in refluxing DMF solution (Scheme 4).36-38 Reduction of the nitro moiety of 11
with iron in hot glacial acetic acid produced the spontaneous ring closure onto the ester to afford the desired tricyclic pyrrolo[1,2-
a]quinoxaline 12 through a one-pot reduction-cyclization step. This lactam 12 was subsequently chlorodehydroxylated with
phosphorous oxychloride, leading to the 4-chloropyrrolo[1,2-a]quinoxaline 13. Then the 4-[(1E,3E)-alka-1,3,5-dienyl]pyrrolo[1,2-
a]quinoxaline 1n was synthesized via the Suzuki palladium-catalyzed cross-coupling reaction of 13 with (1E,3E)-dienyl boronates
6a in the presence of Pd(PPh3)2NBS as the catalyst, and sodium carbonate.
3. Biological evaluation
3.1. In vitro antileishmanial activity against promastigotes
All compounds 1a-n were evaluated for their in vitro antileishmanial activity upon the L. major (MHOM/IL/81/BNI), L.
mexicana (MHOM/MX/95/NAN1) and L. donovani (MHOM/IN/00/DEVI) strains. Pentamidine was used as the reference drug.
The screening data are presented in Table 2.
All the 4-alkapolyenylpyrrolo[1,2-a]quinoxalines 1a-n were found active against the promastigote forms of L. major with
IC50 ranging from 1.2 to 7.4 µM. It must be noticed that a majority of these new 4-alkapolyenylpyrrolo[1,2-a]quinoxaline
derivatives 1 (compounds 1b-f, 1h-j, 1m-n) with IC50 from 1.2 to 3.9 µM were found slightly more potent than pentamidine, the
reference compound (IC50 = 4.3 µM). In particular, 1m, the 4-[(1E,3E,5E)-alka-1,3,5-trienyl]pyrrolo[1,2-a]quinoxaline, was found
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to be 3.6 times more active than the reference drug (IC50 = 1.2 µM versus IC50 = 4.3 µM). In our previously described series I and
II, we have noticed that introduction of a methoxy group in positions 7 and 8 of the pyrrolo[1,2-a]quinoxaline moiety generally
increased the antileishmanial activity. In this new series in which we have introduced a (E,E)-alkadienyl chain in position 4, we
also decided to introduce a methoxy to evaluate the influence of such a group for their in vitro antileishmanial activity upon the L.
major, L. mexicana and L. donovani strains. Meanwhile, the presence of this methoxy group in the 7, 8 or 9 position seems not to
be detrimental for the activity against L. major as illustrated by results obtained for compounds 1b-d, 1f-h and 1j-l (IC50 = 2.5-7.4
µM) in comparison with unsubstituted compounds 1a, 1e and 1i (IC50 = 3.7-4.7 µM). The same observation could be done for the
length of the (E,E)-unsaturated lateral chain at position 4 of the pyrroloquinoxaline moiety that seemed also not to be crucial.
However, the introduction of an ethoxy in position 3 of the pyrrolo[1,2-a]quinoxaline skeleton (compound 1n) was found more
beneficial for the antiparasitic activity with an IC50 of 1.5 µM in comparison with the other compounds 1. Moreover, the presence
of a (E,E,E)-trienyl chain in the C-4 position of compound 1m conferred the best antileishmanial activity to our series (IC50 = 1.2
µM) on the L. major strain isolated from a localized cutaneous leishmaniasis.
A second evaluation was achieved against the promastigote forms of L. mexicana, always with pentamidine as the reference
(Table 2). Compounds 1a-n showed almost the same level of activity as those observed upon the L. major strain with IC50 ranging
from 3.2 to 12.1 µM. It must be noticed that all these new 4-alkapolyenylpyrroloquinoxaline derivatives 1a-m were found slightly
less potent than pentamidine, the reference drug (IC50 = 2.4 µM).
As a general rule, the introduction of a methoxy substituent in position 7 or 8 of the 4-alkadienylpyrrolo[1,2-a]quinoxaline
skeleton seemed to slightly increase the antileishmanial activity in comparison with their respective unsubstituted 4-
alkadienylpyrrolo[1,2-a]quinoxaline homologues (i.e. 1a compared to 1b and 1c; 1e to 1f and 1g; and 1i to 1k), with the exception
of 1j, which showed a quite similar antileishmanial activity compared to its unsubstituted analogue 1i (IC50 = 6.1 µM versus 6.6
µM for 1j) upon the L. mexicana strain. Surprisingly, in this same series, comparison of 7- and 8-methoxy substituted compounds
1b-c, 1f-g and 1k with their corresponding 9-methoxy substituted derivatives 1d, 1h and 1l respectively, showed that the 9-
methoxy substitution led to a slight decrease in the antileishmanial activity.
Compound 1n bearing the ethoxy group in position 3 of the pyrroloquinoxaline moiety showed modest antileishmanial
activity against this L. mexicana strain in comparison to the one observed against the L. major strain (IC50 = 9.6 µM upon L.
mexicana strain versus IC50 = 1.5 µM upon L. major strain). Moreover, the pyrrolo[1,2-a]quinoxaline 1m substituted by a (E,E,E)-
trienyl chain in the C-4 position exhibited a quite similar antileishmanial activity upon L. mexicana strain (IC50 = 4.4 µM) to that
observed for the 4-(E,E)-dienylpyrrolo[1,2-a]quinoxalines substituted by a methoxy group.
In comparison with our previously synthesized series, it could be noticed that new compounds 1a-n with IC50 from 3.2 to 12.1
µM against L. mexicana were found less potent than the previously reported 4-(E)-alkenylpyrrolo[1,2-a]quinoxaline derivatives
(Series II, Figure 2) (IC50= 0.5-7 µM) against the phylogenetically closer species L. amazonensis.
All molecules 1a-n were also screened in vitro on the promastigote stage of L. donovani by determining their 50% inhibitory
concentrations (IC50) and comparing them to pentamidine, chosen as the antileishmanial reference drug. Compounds 1a-n showed
almost the same level of activity as those observed against L. major and L. mexicana with IC50 ranging from 4.0 to 14.7 µM on
this visceral L. donovani strain. The C7 dienyl chain replacement by a C8 dienyl one in compounds 1i-l induced an activity loss,
i.e. IC50 = 7.6-9.9 µM for 1e-h versus IC50 = 11.6-14.7 µM for 1i-l. It must be noticed that three compounds 1b, 1c and 1n showed
an IC50 < 5.5 µM, more potent than the reference pentamidine (IC50 = 5.5 µM). Among them, 1n was found the most active
compound against the promastigote forms of L. donovani with an IC50 of 4.0 µM. As a general rule, the introduction of a methoxy
substituent in position 7, 8 or 9 of the 4-alkadienylpyrrolo[1,2-a]quinoxaline skeleton did not seem to be detrimental for the
antileishmanial activity upon L. donovani in comparison with their respective unsubstituted 4-alkadienylpyrrolo[1,2-a]quinoxaline
homologues. However, introduction of an ethoxy substituent in position C-3 of the 4-alkadienylpyrrolo[1,2-a]quinoxaline moiety
resulted in the increase of the antileishmanial activity.
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When compared to the previously reported 4-(E)-alkenylpyrrolo[1,2-a]quinoxaline derivatives (Series II, Figure 2), it could
be noticed that new compounds 1a-n with IC50 from 4.0 to 14.7 µM against L. donovani were found less active than the ones of
Series II showing IC50 of 0.5-5 µM against L. infantum, a closely related strain.
3.2. Activity against Trypanosoma brucei brucei
All these compounds 1a-n were also evaluated against T. b. brucei. All drugs were active on this T. b. brucei strain.
However, the efficacy of barely soluble chemicals was difficult to assess. The highest concentration of DMSO (0.4% in culture
medium) had no trypanocidal activity. The best efficacy was obtained with compounds 1a-d, 1f, 1j-1k, 1m. For these, the
concentration inhibiting parasite growth by 50% (IC50) was around 0.6 µg.mL-1 leading to a concentration ranging from 1 to 2.4
µM (Table 4). The pyrrolo[1,2-a]quinoxaline 1j substituted by a methoxy group in position 7 and by a (1E,3E)-octa-1,3-dienyl
chain in position 4 was noticed the most active compound against the T. b. brucei strain (IC50 = 1.1 µM).
3.3. In vitro antimalarial activity
All new 4-substituted pyrrolo[1,2-a]quinoxaline derivatives 1a-n were evaluated for their antimalarial activity in vitro on
P. falciparum CQ-resistant strain W2 (IC50 CQ = 0.198 µM) and the CQ-sensitive strain 3D7 (IC50 CQ = 0.037 µM, respectively).
As shown in Table 5, they were found to have IC50s between 0.8-19.8 µM on W2, and 1.5-10.4 µM on 3D7 P. falciparum strains,
respectively.
As seen in Table 5, almost all compounds 1 (except 1g) showed IC50 values in the same order for growth inhibition of
CQ-resistant strain W2 and for growth inhibition of CQ-sensitive strain 3D7. The three pyrrolo[1,2-a]quinoxalines 1e-f and 1h
substituted by a (1E,3E)-hepta-1,3-dienyl chain in position 4 were found the most active compounds against the CQ-resistant W2
strain (IC50 = 0.8-1.3 µM). Surprisingly, their 8-methoxy analogue 1g exhibited modest potency against the W2 strain (IC50 = 11.0
µM). On the other hand, the lengthening of the alkadienyl chain in C-4 of the pyrrolo[1,2-a]quinoxaline heterocycle (compounds
1i-l in comparison with compounds 1e-h) decreased the antiparasitic activity upon the P. falciparum W2 strain with IC50 values of
6.7 to 19.8 µM. In these same 4-(1E,3E)-octa-1,3-dienyl series, comparison of methoxy substituted 1j, 1k and 1l with their
corresponding unsubstituted derivative 1i, showed that the methoxy substitution led to a decrease in the antimalarial activity (IC50
= 13.3-19.8 µM for 1j-l versus 6.7 µM for 1i). Moreover, the 4-(1E,3E)-penta-1,3-dienyl substituted derivatives 1a-d were also
found to have a significant activity against the W2 strain (IC50 = 2.4-3.4 µM). However, the introduction of an ethoxy function in
position C-3 of the 4-[(1E,3E)-penta-1,3-dienyl]pyrroloquinoxaline skeleton (derivative 1n) slightly decreased the antimalarial
activity upon the CQ-resistant W2 strain (IC50 = 9.6 µM). In addition, it appears that the introduction of a C7 trienyl lateral chain
(compound 1m) instead of a C5 or C7 dienyl chain (compounds 1a-d and 1e-f,h) did not improve the activity against malaria
parasite (i.e. IC50 = 8.8 µM for 1m versus 0.8-3.4 µM for 1a-d and 1e-f,h against W2).
Against the CQ-sensitive strain (3D7), the various methoxy substituted or unsubstituted compounds bearing a C5 or C7
(E,E)-dienyl chain (pyrroloquinoxalines 1a-h) showed the same level of antimalarial activity with IC50 ranging from 1.5 to 3.3
µM. The replacement of the methoxy function in position 7, 8 or 9 of the 4-[(1E,3E)-penta-1,3-dienyl]pyrrolo[1,2-a]quinoxaline
moiety (compounds 1b-d) by an ethoxy in C-3 (1n) led to a slight decrease in the antimalarial activity (IC50 = 1.4-3.3 µM for 1b-
d versus 7.7 µM for 1n). Moreover, the replacement of the lateral C7 dienyl chain substituted in the position 4 (compounds 1e-h)
by a C7 trienyl chain (compound 1m) also decreased the anti P. falciparum activity against the 3D7 strain (IC50 = 10.1 µM).
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Preliminary antimalarial results could be also discussed in terms of physicochemical behaviour through the partitioning
theory, determined here by the fragment-based partition coefficients (Clog P). Consequently, Clog P was calculated by the
molinspiration web services.53 All compounds 1a-n were found lipophilic with Clog P values between 4.312 for 1a and 6.15 for
1j-k. A plot of antimalarial IC50 versus Clog P values is presented in Figure 4, allowing us to classify compounds 1a-n in various
subsets. In this new series, the less lipophilic compounds (1a-d) with Clog P values from 4.312 to 4.345 exhibited the best
antimalarial activities (IC50 = 2.4-3.4 µM on W2, and 1.5-3.3 µM on 3D7). When the lipophilicity slightly increased (Clog P =
4.673-4.832), as noticed in compounds 1m-n, the antimalarial activity decreased: i.e. IC50 = 8.8-9.6 µM on W2, and 7.7-10.1 µM
on 3D7. The three pyrrolo[1,2-a]quinoxalines (1e, 1f and 1h) with Clog P values between 5.612 and 5.644 were found to be the
most active compounds (IC50 = 0.8-1.3 µM and 1.6-2.5 µM against W2 and 3D7 strains, respectively). Compound 1g, showing the
same range of lipophilic value (Clog P = 5.644), was surprisingly found less active with an IC50 of 11.0 µM on the W2 strain, but
this derivative showed a better antimalarial activity on the CQ-sensitive strain 3D7 (IC50 = 3.3 µM). The more lipophilic
compounds (1i-l) were found less active (IC50 = 6.3-19.8 µM) on both P. falciparum strains.
3.4. Cytotoxicity and selectivity index
In order to assess their selectivity of action, the cytotoxicity of 1a-n was also evaluated in vitro upon two complementary
cell lines. The cytotoxic concentrations 50% (CC50) on the murine J774A.1 macrophage and metabolizing human hepatocyte
HepG2 cell lines allowed access to the corresponding selectivity indexes (SI) defined as ratio of cytotoxic and antiparasitic
activities (SI = CC50/IC50). The SI could validate their real potential as selective antiparasitic agents. The results concerning the
cytotoxicity and SI data are presented in Tables 2, 3 and 5. As expected, most of the active pyrrolo[1,2-a]quinoxalines 1a-m
showed significant level of cytotoxicity against the J774A.1 and HepG2 cell lines (IC50 = 3.5-10.0 µM, and 4.5-22.9 µM,
respectively). Among these new synthesized derivatives displaying significant antileishmanial activities, compounds 1m and 1k
showed interesting selectivity indexes of 8.8 and 6 on L. major and L. mexicana, respectively (considering the HepG2 human cell
line). Because of its metabolic activation potential, the HepG2 human cell line is one of the most frequently used cell line for
conducting in vitro toxicological screenings. Moreover pyrroloquinoxaline 1m reached a HepG2 selectivity index > 8, much better
than the one of pentamidine, the reference drug. In most of the cases, the SI of ours pyrrolo[1,2-a]quinoxaline compounds were
found higher than pentamidine: i.e. SI ranging from 0.4 to 8.8 for compounds 1a-n versus 0.1 to 1 for pentamidine.
The selectivity index for T. b. brucei varied from 2.8 to 17.6, suggesting that they might represent valuable compounds to
new therapeutic approach. Thus, interesting SI of 16.6 and 17.6 were found for compounds 1j and 1k, respectively.
Considering the W2 P. falciparum strain, the selectivity indexes varied between 0.5 and 13.4, and SI ranged from 0.6 to
10.9 by taking into account the 3D7 strain. This SI led to the identification of compound 1h with SI of 13.4 on W2 strain, and 10.9
on 3D7 strain. On the other hand, the pyrrolo[1,2-a]quinoxaline 1f is also interesting with SI = 11, on the W2 malaria strain. These
two molecules would constitute suitable pharmacophores for the design of new candidates in forthcoming pharmacological
investigations.
4. Conclusion
In the present report, a new series of new 4-alkapolyenylpyrrolo[1,2-a]quinoxaline derivatives, original and structural
analogues of alkaloid chimanine B and of previously described 4-alkenylpyrrolo[1,2-a]quinoxalines, was synthesized in good
yields using efficient palladium-catalyzed Suzuki-Miyaura cross-coupling reactions. These original compounds were then tested
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for their in vitro antiparasitic activity on i) three Leishmania spp. (L. major, L. mexicana and L. donovani) strains; ii) one
Trypanosoma brucei brucei strain; and iii) two Plasmodium falciparum strains (the CQ-resistant W2 and CQ-sensitive 3D7).
Biological results showed activity against the promastigote forms of L. major, L. mexicana and L. donovani with IC50 ranging
from 1.2 to 14.7 µM. In particular, 4-alkatrienylpyrrolo[1,2-a]quinoxaline 1m and 4-alkadienylpyrrolo[1,2-a]quinoxaline 1n were
found to exhibit significant activity against promastigotes forms of L. major (IC50 = 1.2 to 1.5 µM, respectively) and also against
L. donovani for 1n (IC50 = 4.0 µM). The studies of their antiprotozoal activity spectrum using a Trypanasoma brucei brucei strain
were also investigated leading to IC50 of the 1.1 to 2.4 µM range, which deserves further in vivo investigation. Moreover, among
these new synthesized molecules, few of them were identified as potential in vitro antiplasmodial hits, displaying IC50 ranging
from 0.8 to 3.4 µM on the W2 and 3D7 strains of P. falciparum. In addition, the antimalarial results have been discussed in terms
of lipophilic behavior for a preliminary SAR study.
These biological results show that the pyrrolo[1,2-a]quinoxalines 1 exhibit effects on both kinetoplastids and
haemosporidians. By taking into account the biological results and the structure-activity relationships of the previously described
Series II with the ones of this new Series III, our overall results could be now considered as a promising starting point for the
further development and optimization of new and potent antiprotozoan compounds.
5. Experimental
5.1. Chemistry
Commercially reagents were used as received without additional purification. Melting points were determined with an
SM-LUX-POL Leitz hot-stage microscope and are uncorrected. IR spectra were recorded on an NICOLET 380FT-IR
spectrophotometer. NMR spectra were recorded with tetramethylsilane as an internal standard using a BRUKER AVANCE 300
spectrometer. Splitting patterns have been designated as follows: s = singlet; bs = broad singlet; d = doublet; t = triplet; q =
quartet; dd = double doublet; ddd = double double doublet; dt = double triplet; m = multiplet. Analytical TLC were carried out on
0.25 precoated silica gel plates (POLYGRAM SIL G/UV254) and visualization of compounds after UV light irradiation. Silica gel
60 (70-230 mesh) was used for column chromatography. Microwave experiments were carried out at atmospheric pressure using a
focused microwave reactor (CEM Discover). High resolution mass spectra (electrospray in positive mode, ESI+) were recorded on
a Waters Q-TOF Ultima apparatus. Elemental analyses were found within ±0.4% of the theoretical values.
5.1.1. 1-(6-Methoxy-2-nitropenyl)pyrrole (2d)
A mixture of commercial 6-methoxy-2-nitroaniline (12 mmol) and 2,5-dimethoxytetrahydrofuran (13.2 mmol) in acetic
acid (13 mL) was refluxed for 10 min by microwave with vigorous stirring was irradiated for 10 min. The irradiation was
programmed to maintain a constant temperature (120°C) with a maximal output power of 200 W. After cooling, the reaction
mixture was poured into water 0°C. The mixture was then extracted twice with diethyl ether. The organic layer was dried over
Na2SO4 and evaporated to dryness under reduced pressure to give 2d. Orange crystals (78%); mp 71°C. 1H-NMR (CDCl3) δ: 7.49
(t, 1H, J=8.20 Hz, H-4), 7.41 (dd, 1H, J=8.20 and 1.60 Hz, H-3), 7.24 (dd, 1H, J=8.20 and 1.60 Hz, H-5), 6.73 (dd, 2H, J=2.20
and 2.20 Hz, 2H-α), 6.37 (dd, 2H, J=2.20 and 2.20 Hz, 2H-β), 3.88 (s, 3H, CH3O). Anal. Calcd. for C11H10N2O3 : C, 60.55; H,
4.62; N, 12.84. Found: C, 60.69; H, 4.48; N, 13.03.
5.1.2. 1-(2-Amino-6-methoxy)phenyl-pyrrole (3d)
To a solution of 1-(6-methoxy-2-nitrophenyl)pyrrole (2d) (28 mmol) in ethanol (120 mL) was added 19 mL of a 2M
aqueous solution of CuSO4. Sodium borohydride (138 mmol) was added portion-wise at 0°C to the reaction mixture which was
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then stirred at room temperature for 1 h. The reaction mixture was then diluted with ethyl acetate and filtered. The organic layer
was separated, dried over Na2SO4 and evaporated to dryness under reduced pressure to give 3d. Orange crystals (89%); mp 79°C.
1
IR (KBr) 3460, 3375 (NH2). H-NMR (CDCl3) δ: 7.15 (d, 1H, J=8.30 Hz, H-4), 6.75 (dd, 2H, J=2.10 and 2.10 Hz, 2H-α), 6.46
(dd, 1H, J=8.30 and 1.20 Hz, H-3), 6.42 (dd, 1H, J=8.30 and 1.20 Hz, H-5), 6.40 (dd, 2H, J=2.10 and 2.10 Hz, 2H-β), 3.76 (s, 3H,
CH3O), 3.65 (bs, 2H, NH2) . 13C-NMR (CDCl3) δ: 159.9 (C-4), 143.4 (C-2), 128.1 (C-1), 122.1 (C-α), 121.2 (C-6), 109.2 (C-β),
103.6 (C-5), 101.1 (C-3), 55.4 (CH3O). Anal. Calcd. for C11H12N2O : C, 70.19; H, 6.43; N, 14.88. Found: C, 70.27; H, 6.52; N,
15.05.
5.1.3. 9-Methoxy-5H-pyrrolo[1,2-a]quinoxalin-4-one (4d)
To a solution of compound 3d (24.5 mmol) in toluene (55 mL) was added triphosgene (8.17 mmol). The reaction mixture
was refluxed for 4 h, and nitrogen was bubbled in to drive off excess of phosgene. The solution was then set aside for 10 min. The
heavy crystalline precipitate was filtered off and washed with diethyl ether to give 4d. Beige crystals (81%), mp 260°C. IR (KBr)
1
3200, 2700 (NH), 1685 (CO). H-NMR (DMSO-d6) δ: 11.28 (s, 1H, NH), 8.45 (dd, 1H, J=2.80 and 1.50 Hz, H-1), 7.22 (t, 1H,
J=8.30 Hz, H-7), 7.06 (dd, 1H, J=3.90 and 1.50 Hz, H-3), 6.93 (d, 2H, J=8.30 Hz, H-6 and H-8), 6.62 (dd, 1H, J=3.90 and 2.80
Hz, H-2), 4.01 (s, 3H, CH3). 13C-NMR (DMSO-d6) δ: 157.0 (C-4), 155.2 (C-7), 129.7 (C-9a), 122.6 (C-5a), 117.7 (C-1), 116.8 (C-
3a), 116.0 (C-6), 112.3 (C-3), 111.0 (C-2), 108.9 (C-9), 100.8 (C-8), 55.4 (CH3O). Anal. Calcd. for C12H10N2O2 : C, 67.28; H,
4.71; N, 13.08. Found: C, 67.40; H, 4.81; N, 12.94.
5.1.4. 4-Chloro-9-methoxy-pyrrolo[1,2-a]quinoxaline (5d)
A solution of 5H-pyrrolo[1,2-a]quinoxalin-4-one (4d) (0.03 mol) in POCl3 (60 mL) was refluxed for 4 h. After removing
excess of reactive under vacuum, the residue was carefully dissolved in water at 0 °C and the resulting solution was made basic
with 30% aqueous ammonium hydroxide solution (pH= 9-10). The precipitate was filtered, solubilized in methylene chloride, the
1
organic layer was then dried over Na2SO4, filtered and evaporated to dryness to give 5d as white crystals (71%), mp 146°C. H-
NMR (CDCl3) δ: 8.75 (dd, 1H, J=2.80 and 1.20 Hz, H-1), 7.50 (dd, 1H, J=8.20 and 1.20 Hz, H-8), 7.35 (t, 1H, J=8.20 Hz, H-7),
7.08 (dd, 1H, J=4.00 and 1.20 Hz, H-3), 7.05 (dd, 1H, J=8.20 and 1.20, H-6), 6.83 (dd, 1H, J=4.00 and 2.80 Hz, H-2), 4.08 (s, 3H,
CH3O). Anal. Calcd. for C12H9ClN2O : C, 61.95; H, 3.90; N, 12.04. Found: C, 62.06; H, 3.95; N, 12.13.
5.1.5. General procedure for (1E,3E)-1,3-alkadienyl MIDA boronate esters (6a-c)
A solution of the Pd/S-Phos catalyst was prepared by mixing Pd(OAc)2 (0.03 mmol) and S-Phos (0.052 mmol) in
anhydrous toluene (3 mL). The resulting solution was stirred at room temperature for 45 min and used in the Suzuki reaction, as
follows: Method A: A mixture of (E)-1-propenylboronic acid (3.5 mmol), trans-(2-bromovinyl) MIDA boronate ester (2.33
mmol), Cs2CO3 (7.00 mmol) and catalyst solution in 22 mL of anhydrous THF was stirred at room temperature for 5 days under
an argon positive pressure. The resulting heterogeneous mixture was diluted with MeCN and filtered through a short pad of Celite.
The filtrate was concentrated in vacuum. The crude product was triturated in PE and filtered then washed with PE to give the
MIDA boronate esters 6a-c. Method B: A mixture of (E)-1-pentenylboronic acid (2.73 mmol), trans-(2-bromovinyl) MIDA
boronate ester (1.82 mmol), KF (3.6 mmol) and the catalyst solution 15 mL of dry toluene was stirred at 25°C for 5 days under an
argon positive pressure, then the slurry was diluted with acetonitrile (15 mL) and filtered through a short pad of Celite. The filtrate
was concentrated in vacuo, then the crude is triturated in PE, until the appearance of the crystals which were filtered and washed
with PE to give the MIDA boronate esters 6a-c.
5.1.5.1. (1E,3E)-Penta-1,3-dienyl MIDA boronate ester (6a)
1
White crystals (67%), mp 89°C, Rf =0.48 (AcOEt/ KMnO4). H-NMR (CDCl3) δ: 6.61 (dd, 1H, J=17.30 and 10.30 Hz,
H-2), 6.13 (dd, 1H, J= 15.00 and 10.30 Hz, H-3), 5.82 (dt, 1H, J=15.00 and 7.00 Hz, H-4), 5.42 (d, 1H, J=17.30 Hz, H-1), 4.02 (d,
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2H, J=16.60 Hz, N-CH2), 3.73 (d, 2H, J=16.60 Hz, N-CH2), 2.84 (s, 3H, N-CH3), 1.78 (d, 3H, J=7.00 Hz, CH3). 13C-NMR
(CDCl3) δ: 168.9 (C=O), 145.6 (C-1’), 134.5 (C-2’/ C-3’), 133.3 (C-4’), 62.5 (C-2), 47.8 (CH3-N), 19.2 (C-5’). Anal. Calcd. for
C10H14BNO4 : C, 53.85; H, 6.33; N, 6.28. Found: C, 54.10; H, 6.45; N, 6.42.
5.1.5.2. (1E,3E)-Hepta-1,3-dienyl MIDA boronate ester (6b)
40,42
Pale yellow crystals (79%); mp 96°C, Rf =0.47 visualized by UV lamp (λ=254 nm) or with KMnO4.
5.1.5.3. (1E,3E)-Octa-1,3-dienyl MIDA boronate ester (6c)
Pale yellow crystals (50%), mp 74°C, Rf =0.42 visualized by UV lamp (λ=254 nm) or with KMnO4. 1H-NMR (CDCl3) δ:
6.62 (dd, 1H, J=17.30 and 10.20 Hz, H-2), 6.11 (dd, 1H, J=15.3 and 10.20 Hz, H-3), 5.82 (dt, 1H, J=15.30 and 7.30 Hz, H-4),
5.43 (d, 1H, J=17.30 Hz, H-1), 3.99 (d, 2H, J=16.65 Hz, N-CH2), 3.72 (d, 2H, J=16.65 Hz, N-CH2), 2.85 (s, 3H, N-CH3), 2.10 (q,
2H, J=6.90 Hz, CH2), 1.41-1.29 (m, 4H, 2CH2), 0.91 (t, 3H, J=6.90 Hz, CH3). Anal. Calcd. for C13H20BNO4 : C, 58.90; H, 7.60;
N, 5.28. Found: C, 59.04; H, 7.68; N, 5.36.
5.1.6. (1E,3E,5E)-Hepta-1,3,5-trienyl MIDA boronate ester (6d)
To a stirred mixture of 6a (2.2 mmol) in THF (20 mL) at room temperature was added 1N NaOH aqueous solution (6.6
mmol, 3 eq.). The reaction mixture was stirred at room temperature for 15 minutes. The resulting mixture was treated with 1N
phosphate buffer solution (pH=7, 10 mL) and diluted with Et2O (15 mL). The organic layer was separated and the aqueous layer
was extracted with a mixture of Et2O/THF 1:1 (3ꢀ25 mL). The combined organic layers were dried over anhydrous Na2SO4, then
they were concentrated to 5 mL volume of THF in vacuo. This solution was immediately used in the next step without further
purification.
A solution of the Pd/S-Phos catalyst was prepared by mixing Pd(OAc)2 (0.03 mmol) and S-Phos (0.052 mmol) in anhydrous
toluene (3 mL). The resulting solution was stirred at room temperature for 45 min and used in the Suzuki reaction.
A mixture of trans-(2-bromovinyl) MIDA boronate ester (2.33 mmol), Cs2CO3 (2.6 mmol), boronic acid previously prepared in
THF solution, and catalyst solution in toluene (25 mL) was stirred 18 hours at 45°C. The resulting solution was diluted with
AcOEt (10 mL) and filtered through a short ad of Celite. The filtrate was concentrated in vacuo to provide an orange solid, then
the crude product was triturated with diethyl ether, filtered and dried to give 6d. Orange crystals (63%), mp 97°C, Rf =0.41
(AcOEt). 1H-NMR (CDCl3) δ: 6.67 (dd, 1H, J=17.40 and 9,90 Hz, H-2), 6.26 (dd, 1H, J=14.40 and 10.20 Hz, H-3), 6.21-6.05 (m,
2H, H-4 and H-5), 5.81 (dt, 1H, J=14.40 and 6.60 Hz, H-6), 5.53 (d, 1H, J=17.40 Hz, H-1), 3.92 (d, 2H, J=16.35 Hz, N-CH2),
3.71 (d, 2H, J=16.35 Hz, N-CH2), 2.85 (s, 3H, N-CH3), 1.80 (d, 3H, J=6.60 Hz, CH3). Anal. Calcd. for C12H16BNO4 : C, 57.87; H,
6.47; N, 5.62. Found: C, 58.12; H, 6.41; N, 5.79.
5.1.7. General procedure for 4-[(1E,3E)-penta-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1a-d, 1n), 4-[(1E,3E)-hepta-1,3-
dienyl]pyrrolo[1,2-a]quinoxaline (1e-h), 4-[(1E,3E)-octa-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1i-l) and 4-[(1E,3E,5E)-hepta-
1,3,5-trienyl]pyrrolo[1,2-a]quinoxaline (1m)
Method A: A solution of the Pd/S-Phos catalyst was prepared by mixing Pd(OAc)2 (0.037 mmol) and S-Phos (0.074
mmol) in anhydrous toluene (4 mL). The resulting solution was stirred at room temperature for 45 min and used directly in the
Suzuki reaction. To a mixture of 4-chloropyrrolo[1,2-a]quinoxaline (5a-d) (0.74 mmol), MIDA boronate esters (6a-d) (1.11
mmol), and 20 mL of degassed THF was added the solution of the Pd/S-Phos catalyst then an aqueous solution of NaOH (5
mmol) in 3.5 mL of degassed water. The solution was stirred at room temperature for 24 h. The resulting solution was diluted with
AcOEt and H2O was added. The organic layer was separated, filtered through a short ad of Celite and evaporated under reduced
pressure. The crude product was purified by flash chromatography (eluent 1:3 Et2O/petroleum ether) to give 1e-h. Method B:
MIDA boronate esters (6a-d) (0.838 mmol), 4-chloropyrrolo[1,2-a]quinoxaline (5a-d or 17) (0.65 mmol) and Pd(PPh3)2NBS
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(0.0193 mmol) were added into a two necked flask containing a reflux condenser with nitrogen inlet. 15 mL of degassed THF
(nitrogen bubbled through the solvent for 1 h under sonication) were added to the reaction flask. The solution was stirred at room
temperature for 10 min, then a 3M aqueous solution of Na2CO3 (1.7 mL) was added and the resulting solution was heated to reflux
overnight. After cooling the mixture was poured onto brine (15 mL), then AcOEt (20 mL) and water (5mL) were added. The
aqueous layer was extracted twice with AcOEt (2 ꢀ 20mL) and dried over Na2SO4 and filtered. Solvent was removed under
reduced pressure and the crude product was purified by flash chromatography (eluent 1:3 Et2O/petroleum ether) to give 1a-d,1h-
n.
5.1.7.1. 4-[(1E,3E)-Penta-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1a)
1
Yellow crystals (60%), mp 79°C, Rf =0.56 (Et2O/petroleum ether). H-NMR (CDCl3) δ: 7.97-7.75 (m, 1H, H-9), 7.93
(dd, 1H, J=2.80 and 1.30 Hz, H-1), 7.84-7.81 (m, 1H, H-6), 7.68 (dd, 1H, J=15.40 and 11.00 Hz, H-2’), 7.46-7.42 (m, 2H, H-7
and H-8), 7.01 (dd, 1H, J=3.95 and 2.80 Hz , H-3), 6.90-6.87 (m, 1H, H-2), 6.85 (d, 1H, J=15.40 Hz, H-1’), 6.42 (dd, 1H, J=14.50
and 11.00 Hz, H-3’), 6.18 (dt, 1H, J= 14.5 and 6.90 Hz, H-4’), 1.92 (d, 3H, J=6.90 Hz, CH3). 13C-NMR (CDCl3) δ: 151.0 (C-4),
138.3 (C-2’), 137.2 (C-5a), 136.9 (C-4’), 132.4 (C-3’), 130.4 (C-9), 128.0 (C-3a), 127.7 (C-7), 126.8 (C-9a), 126.2 (C-8), 125.0
(C-1’), 115.3 (C-1), 114.7 (C-2), 114.5 (C-6), 106.9 (C-3), 19.7 (C-5’). HRMS-ESI m/z [M+H]+ Calcd for C16H15N2: 235.1235,
Found: 235.1230.
5.1.7.2. 7-Methoxy-4-[(1E,3E)-penta-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1b)
1
Yellow crystals (71%), mp 92°C, Rf =0.37 (Et2O/petroleum ether). H-NMR (CDCl3) δ: 7.87 (dd, 1H, J=2.70 and 1.20
Hz, H-1), 7.75 (d, 1H, J= 8.90 Hz, H-9), 7.69 (dd, 1H, J=15.50 and 10.80 Hz, H-2’), 7.43 (d, 1H, J=2.75 Hz, H-6), 7.08 (dd, 1H,
J=8.90 and 2.75 Hz, H-8), 7.00 (dd, 1H, J= 4.10 and 1.20 Hz, H-3), 6.87 (d, 1H, J=15.50 Hz, H-1’), 6.86 (dd, 1H, J=4.10 and
2.70, H-2), 6.42 (dd, 1H, J=15.00 and 10.80 Hz, H-3’), 6.18 (dt, 1H, J=15.00 and 7.70 Hz, H-4’), 3.95 (s, 3H, O-CH3), 1.93 (d,
3H, J= 7.00 Hz, CH3). 13C-NMR (CDCl3) δ: 158.2 (C-7), 151.4 (C-4), 138.4 (C-4’), 138.3 (C-3’), 135.9 (C-5a), 132.4 (C-9),
126.7 (C-3a), 125.1 (C-2’), 122.5 (C-9a), 117.1 (C-8), 115.5 (C-3), 115.0 (C-1’), 114.4 (C-1), 111.6 (C-6), 106.6 (C-2), 56.7
(OCH3), 19.7 (C-5’). HRMS-ESI m/z [M+H]+ Calcd for C17H17N2O: 265.1341, Found: 265.1354.
5.1.7.3. 8-Methoxy-4-[(1E,3E)-penta-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1c)
1
Yellow crystals (65%), mp 97°C, Rf =0.29 (Et2O/petroleum ether). H-NMR (CDCl3) δ: 7.89 (d, 1H, J=9.00 Hz, H-6),
7.85 (dd, 1H, J=2.70 and 1.20 Hz, H-1), 7.62 (dd, 1H, J=15.30 and 10.80 Hz, H-2’), 7.26 (d, 1H, J= 2.60 Hz, H-9), 7.05 (dd, 1H,
J=9.00 and 2.60 Hz, H-7), 6.99 (dd, 1H, J=3.95 and 1.20 Hz, H-3), 6.89 (dd, 1H, J=3.95 and 2.70 Hz, H-2), 6.85 (d, 1H, J=15.30
Hz, H-1’), 6.41 (dd, 1H, J=14.85 and 10.80 Hz, H-3’), 6.14 (dt, 1H, J= 14.85 and 6.90 Hz, H-4’), 3.97 (s, 3H, O-CH3), 1.91 (d,
3H, J=6.90 Hz, CH3). 13C-NMR (CDCl3) δ: 159.6 (C-8), 148.6 (C-4), 137.2 (C-4’), 136.0 (C-3’), 132.4 (C-9), 130.1 (C-5a), 128.7
(C-3a), 126.7 (C-9a), 125.3 (C-2’), 116.5 (C-3), 114.6 (C-1’), 113.7 (C-1), 109.1 (C-6), 106.3 (C-2), 98.7 (C-7), 56.6 (OCH3),
16.5 (C-5’). HRMS-ESI m/z [M+H]+ Calcd for C17H17N2O: 265.1341, Found: 265.1340.
5.1.7.4. 9-Methoxy-4-[(1E,3E)-penta-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1d)
1
Yellow crystals (76%), mp 102°C, Rf =0.31 (Et2O/petroleum ether). H-NMR (CDCl3) δ: 8.77 (dd, 1H, J=2.70 and 1.35
Hz, H-1), 7.70 (dd, 1H, J=15.80 and 10.95 Hz, H-2’), 7.60 (dd, 1H, J=8.25 and 1.20 Hz, H-8), 7.35 (t, 1H, J=8.25 Hz, H-7), 7.04
(dd, 1H, J=4.20 and 1.35 Hz, H-3), 7.00 (dd, 1H, J=8.25 and 1.20 Hz, H-6), 6.88 (d, 1H, J=15.80 Hz, H-1’), 6.84 (dd, 1H, J=4.20
and 2.70 Hz, H-2), 6.42 (dd, 1H, J=14.90 and 10.95 Hz, H-3’), 6.18 (dt, 1H, J=14.90 and 6.90 Hz, H-4’), 4.07 (s, 3H, CH3O), 1.92
(d, 3H, J=6.90 Hz, CH3). 13C-NMR (CDCl3) δ: 150.2 (C-9), 149.7 (C-4), 138.2 (C-5a), 137.3 (C-2’), 135.7 (C-4’), 131.4 (C-7),
126.4 (C-3a), 124.3 (C-8), 123.9 (C-3’), 122.1 (C-3), 121.6 (C-1’), 118.4 (C-9a), 112.6 (C-1), 108.4 (C-6), 105.2 (C-2), 56.1
(OCH3), 18.7 (C-5’). HRMS-ESI m/z [M+Na]+ Calcd for C17H16N2ONa: 287.1160, Found: 287.1149.
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5.1.7.5. 4-[(1E,3E)-Hepta-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1e)
1
Yellow crystals (82%), mp 97°C, Rf =0.56 (Et2O/petroleum ether). H-NMR (CDCl3) δ: 7.97 (dd, 1H, J=8.70 and 2.10
Hz, H-9), 7.95 (dd, 1H, J=2.70 and 1.20 Hz, H-1), 7.84 (dd, 1H, J=8.70 and 2.10 Hz, H-6), 7.72 (dd, 1H, J=14.90 and 10.95 Hz,
H-2’), 7.51-7.41 (m, 2H, H-7 and H-8), 7.02 (dd, 1H, J=4.20 and 1.20 Hz, H-3), 6.91 (dd, 1H, J=4.20 and 2.70 Hz, H-2), 6.89 (d,
1H, J=14.90 Hz, H-1’), 6.41 (dd, 1H, J=14.80 and 10.95 Hz, H-3’), 6.16 (dt, 1H, J=14.80 and 7.20 Hz, H-4’), 2.22 (q, 2H, J=7.20
Hz, CH2), 1.52 (st, 2H, J=7.20 Hz, CH2), 0.97 (t, 3H, J=7.20 Hz, CH3). 13C-NMR (CDCl3) δ: 151.1 (C-4), 139.4 (C-2’), 137.2 (C-
5a), 131.2 (C-4’), 131.0 (C-3’), 129.4 (C-9), 128.1 (C-3a), 127.7 (C-7), 126.9 (C-9a), 126.9 (C-8), 121.5 (C-1’), 114.9 (C-1),
114.6 (C-2), 114.4 (C-6), 106.8 (C-3), 36.0 (C-5’), 23.2 (C-6’), 15.1 (C-7’). HRMS-ESI m/z [M+H]+ Calcd for C18H18N2:
263.1548, Found: 263.1547.
5.1.7.6. 7-Methoxy-4-[(1E,3E)-hepta-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1f)
Yellow oil (69%), Rf =0.44 (Et2O/petroleum ether). 1H-NMR (CDCl3) δ: 7.87 (dd, 1H, J=2.70 and 1.20 Hz, H-1), 7.75 (d,
1H, J=9.00 Hz, H-9), 7.69 (dd, 1H, J=15.45 and 10.95 Hz, H-2’), 7.43 (d, 1H, J=2.80 Hz, H-6), 7.08 (dd, 1H, J=9.80 and 2.80 Hz,
H-8), 7.00 (dd, 1H, J=4.20 and 1.20 Hz, H-3), 6.88 (d, 1H, J= 15.45 Hz, H-1’), 6.86 (dd, 1H, J=4.20 and 2.70 Hz, H-2), 6.40 (dd,
1H, J= 15.0 and 10.95 Hz, H-3’), 6.16 (dt, 1H, J=15.00 and 7.30 Hz, H-4’), 3.94 (s, 3H, CH3O), 2.22 (q, 2H, J=7.30 Hz, CH2),
1.50 (st, 2H, J=7.30 Hz, CH2), 0.97 (t, 3H, J=7.30 Hz, CH3). 13C-NMR (CDCl3) δ: 158.2 (C-7), 151.5 (C-4), 142.3 (C-4’), 138.4
(C-3’), 137.3 (C-5a), 131.2 (C-9), 126.8 (C-3a), 125.4 (C-2’), 122.6 (C-9a), 117.0 (C-8), 115.5 (C-3), 114.9 (C-1’), 114.3 (C-1),
111.7 (C-6), 106.4 (C-2), 56.7 (OCH3), 36.2 (C-5’), 23.2 (C-6’), 14.7 (C-7’). HRMS-ESI m/z [M+Na]+ Calcd for C19H20N2ONa:
315.1473, Found: 315.1483.
5.1.7.7. 8-Methoxy-4-[(1E,3E)-hepta-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1g)
1
Yellow crystals (80%), mp 81°C, Rf =0.18 (Et2O/petroleum ether). H-NMR (CDCl3) δ: 7.90 (d, 1H, J=9.00 Hz, H-6),
7.84 (dd, 1H, J=2.75 and 1.25 Hz, H-1), 7.64 (d, 1H, J=15.30 and 10.80 Hz, H-2’), 7.26 (d, 1H, J=2.40 Hz, H-9), 7.05 (dd, 1H,
J=9.00 and 2.40 Hz, H-7), 6.99 (dd, 1H, J=4.00 and 1,25 Hz, H-3), 6.89 (dd, 1H, J=4.00 and 2.75 Hz, H-2), 6.87 (d, 1H, J=15.30
Hz, H’-1), 6.38 (dd, 1H, J=14.85 and 10.80 Hz, H-3’), 6.13 (dt, 1H, J=14.75 and 7.35 Hz, H-4’), 3.97 (s, 3H, CH3O), 2.21 (q, 2H,
J=7.35 Hz, CH2), 1.52 (st, 2H, J=7.35 Hz, CH2), 0.97 (t, 3H, J=7.35 Hz, CH3). 13C-NMR (CDCl3) δ: 159.7 (C-8), 148.8 (C-4),
138.5 (C-4’), 134.4 (C-5a), 131.8 (C-3’), 131.2 (C-9), 128.9 (C-3a), 126.9 (C-9a), 125.4 (C-2’), 114.7 (C-3), 114.6 (C-1’), 113.8
(C-6), 113.7 (C-1), 106.3 (C-2), 98.5 (C-7), 56.8 (OCH3), 36.1 (C-5’), 23.3 (C-6’), 14.7 (C-7’). HRMS-ESI m/z [M+H]+ Calcd for
C19H21N2O: 293.1654, Found: 293.1651.
5.1.7.8. 9-Methoxy-4-[(1E,3E)-hepta-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1h)
1
Yellow crystals (82%), mp 66°C, Rf =0.42 (Et2O/petroleum ether). H-NMR (CDCl3) δ: 8.79-8.78 (m, 1H, H-1), 7.73
(dd, 1H, J=15.6 and 11.25 Hz, H-2’), 7.61 (d, 1H, J=8.10 Hz, H-8), 7.36 (t, 1H, J=8.10 Hz, H-7), 7.05 (d, 1H, J=3.30 Hz, H-3),
7.01 (d, 1H, J= 8.10 Hz, H-6), 6.89 (d, 1H, J=15.6 Hz, H-1’), 6.85 (t, 1H, J=3.30 Hz, H-2), 6.41 (dd, 1H, J=14.55 and 11.25 Hz,
H-3’), 6.15 (dt, 1H, J= 14H-2), 6.41 (dd, 1H, J=14.55 and 11.25 Hz, H-3’), 6.15 (dt, 1H, J=14.55 and 7.20 Hz, H-4’), 4.08 (s, 3H,
CH3O), 2.21 (q, 2H, J=7.20 Hz, CH2), 1.51 (st, 2H, J=7.20 Hz, CH2), 0.97 (t, 2H, J=7.20 Hz, CH3). 13C-NMR (CDCl3) δ: 151.9
(C-9), 150.7 (C-4), 142.3 (C-2’), 139.1 (C-5a), 139.0 (C-4’), 131.1 (C-3’), 127.3 (C-3a), 125.3 (C-7), 124.8 (C-1’), 123.2 (C-9a),
122.5 (C-8), 119.3 (C-1), 113.6 (C-2), 109.3 (C-6), 106.3 (C-3), 57.1 (OCH3), 36.2 (C-5’), 23.3 (C-6’), 14.7 (C-7’). HRMS-ESI
m/z [M+Na]+ Calcd for C19H20N2ONa: 315.1473, Found: 315.1473.
5.1.7.9. 4-[(1E,3E)-Octa-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1i)
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Yellow crystals (83%), mp 67°C, Rf =0.63 (Et2O/petroleum ether). 1H-NMR (CDCl3) δ: 7.98-7.94 (m, 2H, H-1 and H-9),
7.84 (dd, 1H, J=8.70 and 2.40 Hz, H-6), 7.71 (dd, 1H, J=15.30 and 10.80 Hz, H-2’), 7.51-7.41 (m, 2H, H-7 and H-8), 7.03 (dd,
1H, J=4.00 and 1.80 Hz, H-3), 6.90 (dd, 1H, J=4.00 and 2.65 Hz, H-2), 6.87 (d, 1H, J=15.30 Hz, H-1’), 6.40 (dd, 1H, J=15.00,
10.80 Hz, H-3’), 6.18 (dt, 1H, J=15.00 and 7.20 Hz, H-4’), 2.24 (q, 2H, J=7.20 Hz, CH2), 1.53-1.34 (m, 4H, 2CH2), 0.95 (t, 3H,
J=7.20 Hz, CH3). 13C-NMR (CDCl3) δ: 151.5 (C-4), 140.9 (C-5a), 140.6 (C-2’), 132.0 (C-4’), 130.9 (C-9), 130.6 (C-3’), 129.9 (C-
7), 128.1 (C-3a), 127.9 (C-9a), 126.3 (C-8), 121.4 (C-1’), 115.1 (C-1), 114.7 (C-2), 114.5 (C-6), 106.9 (C-3), 32.8 (C-5’), 31.1 (C-
6’), 22.2 (C-7’), 13.9 (C-8’). HRMS-ESI m/z [M+H]+ Calcd for C19H21N2: 277.1705, Found: 277.1718.
5.1.7.10. 7-Methoxy-4-[(1E,3E)-octa-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1j)
Yellow oil (82%), Rf =0.50 (Et2O/petroleum ether). 1H-NMR (CDCl3) δ: 7.90-7.87 (m, 1H, H-1), 7.76 (d, 1H, J=9.00 Hz,
H-9), 7.74-7.67 (m, 1H, H-2’), 7.48-7.46 (m, 1H, H-6), 7.09 (dd, 1H, J=9.00 and 3.00 Hz, H-8), 7.03-7.01 (m, 1H, H-3), 6.88 (d,
1H, J=15.30 Hz, H-1’), 6.87-6.86 (m, 1H, H-2), 6.40 (dd, 1H, J=15.15 and 10.65 Hz, H-3’), 6.22-6.18 (m, 1H, H-4’), 3.95 (s, 3H,
CH3O), 2.26 (q, 2H, J=7.20 Hz, CH2), 1.47-1.37 (m, 4H, 2CH2), 0.95 (t, 3H, J=7.20 Hz, CH3). 13C-NMR (CDCl3) δ: 158.2 (C-7),
151.3 (C-4), 142.6 (C-4’), 138.6 (C-3’), 138.1 (C-5a), 131.0 (C-9), 126.6 (C-3a), 125.4 (C-2’), 122.4 (C-9a), 116.9 (C-3), 115.4
(C-1’), 114.3 (C-1), 111.6 (C-6), 106.6 (C-2), 56.6 (OCH3), 32.1 (C-5’), 30.5 (C-6’), 23.2 (C-7’), 14.9 (C-8’). HRMS-ESI m/z
[M+Na]+ Calcd for C20H22N2ONa: 329.1630, Found: 329.1637.
5.1.7.11. 8-Methoxy-4-[(1E,3E)-octa-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1k)
1
Yellow crystals (66%), mp 54°C, Rf =0.32 (Et2O/petroleum ether). H-NMR (CDCl3) δ: 7.90 (d, 1H, J=8.95 Hz, H-6),
7.83 (dd, 1H, J=2.60 and 1.35 Hz, H-1), 7.64 (dd, 1H, J=15.30 and 10.80 Hz, h-2’), 7.25 (d, 1H, J=2.60 Hz, H-9), 7.04 (dd, 1H,
J=8.95 and 2.60 Hz, H-7), 6.99 (dd, 1H, J=3.90 and 1.35 Hz, H-3), 6.89 (dd, 1H, J=3.90 and 2.60 Hz, H-2), 6.86 (d, 1H, J=15.30
Hz, H-1’) 6.39 (dd, 1H, J=15.00 and 10.80 Hz, H-3’), 6.13 (dt, 1H, J=15.00 and 7.30 Hz, H-4’), 3.97 (s, 3H, CH3O), 2.23 (q, 2H,
J=7.30 Hz, CH2), 1.52-1.34 (m, 4H, 2CH2), 0.94 (t, 3H, J=7.30 Hz, CH3). 13C-NMR (CDCl3) δ: 159.6 (C-8), 148.7 (C-4), 141.9
(C-4’), 137.6 (C-3’), 131.6 (C-2’), 131.5 (C-5a), 131.0 (C-9), 128.8 (C-3a), 126.8 (C-9a), 125.3 (C-6), 114.8 (C-1’), 114.7 (C-3),
113.8 (C-1), 106.4 (C-2), 98.5 (C-7), 56.7 (OCH3), 33.7 (C-5’), 32.2 (C-6’), 23.2 (C-7’), 14.9 (C-8’). HRMS-ESI m/z [M+Na]+
Calcd for C20H22N2ONa: 329.1630, Found: 329.1639.
5.1.7.12. 9-Methoxy-4-[(1E,3E)-octa-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1l)
1
Yellow crystals (63%), mp 67°C, Rf =0.45 (Et2O/petroleum ether). H-NMR (CDCl3) δ: 7.90 8.78 (dd, 1H, J=2.85 and
1.20 Hz, H-1), 7.72-7.61 (m, 2H, H-2’ and H-8), 7.36 (t, 1H, J=8.10 Hz, H-7), 7.05 (dd, 1H, J=4.05 and 1.20 Hz, H-3), 7.01 (dd,
1H, J=8.10 and 1.20 Hz, H-6), 6.89 (d, 1H, J=15.30 Hz, H-1’), 6.85 (dd, 1H, J=4.05 and 2.85 Hz, H-2), 6.41 (dd, 1H, J=15.10 and
10.90 Hz, H-3’), 6.17 (dt, 1H, J=15.1 and 6.90 Hz, H-4’), 4.09 (s, 3H, CH3O), 2.24 (q, 2H, J=6.90 Hz, CH2), 1.50-1.34 (m, 4H,
2CH2), 0.96 (t, 3H, J=6.90 Hz, CH3). 13C-NMR (CDCl3) δ: 151.8 (C-9), 150.7 (C-4), 144.8 (C-5a), 143.9 (C-4’), 140.6 (C-3’),
135.3 (C-2’), 133.1 (C-3a), 132.2 (C-7), 130.9 (C-8), 129.1 (C-1’), 124.1 (C-9a), 123.3 (C-3), 121.5 (C-1), 109.9 (C-6), 108.5 (C-
2), 57.3 (OCH3), 33.8 (C-5’), 32.0 (C-6’), 23.6 (C-7’), 14.9 (C-8’). HRMS-ESI m/z [M+H]+ Calcd for C20H23N2O: 307.1810,
Found: 307.1800.
5.1.7.13. 4-[(1E,3E,5E)-Hepta-1,3,5-trienyl]pyrrolo[1,2-a]quinoxaline (1m)
1
Yellow crystals (87%), mp 139°C, Rf =0.47 (Et2O/petroleum ether). H-NMR (CDCl3) δ: 8.02-7.99 (m, 1H, H-9), 7.95
(dd, 1H, J=2.80 and 1.50 Hz, H-1), 7.84 (dd, 1H, J=8.20 and 2.60 Hz, H-6), 7.78-7.71 (m, 1H, H-2), 7.50-7.41 (m, 2H, H-7 and
H-8), 7.04 (dd, 1H, J=3.75 and 1.50 Hz, H-3), 6.95 (d, 1H, J=15.00 Hz, H-1’), 6.89 (dd, 1H, J=3.75 and 2.80 Hz, H-2), 6.63 (dd,
1H, J=15.00 and 11.10 Hz, H-3’), 6.45 (dd, 1H, J=15.00 and 11.10 Hz, H-4’), 6.26-6.20 (m, 1H, H-5’), 5.93 (dt, 1H, J=14.40 and
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7.70 Hz, H-6’), 1.87 (d, 1H, J=7.00 Hz, CH3). 13C-NMR (CDCl3) δ: 151.1 (C-4), 139.6 (C-4’), 138.2 (C-3’), 132.9 (C-6’), 132.2
(C-5’), 132.0 (C-5a), 131.7 (C-3a), 130.0 (C-9), 128.4 (C-2’), 127.1 (C-7), 126.9 (C-9a), 125.2 (C-8), 121.5 (C-2), 113.9 (C-1),
113.8 (C-1’), 113.6 (C-6), 105.7 (C-3), 18.50 (C-7’). HRMS-ESI m/z [M+H]+ Calcd for C18H17N2: 261.1392, Found: 261.1404.
5.1.7.14. 3-Ethoxy-4-[(1E,3E)-penta-1,3-dienyl]pyrrolo[1,2-a]quinoxaline (1n)
1
Orange crystals (53%), mp 103°C, Rf =0.34 (Et2O/petroleum ether). H-NMR (CDCl3) δ: 7.87-7.83 (m, 1H, H-9), 7.71-
7.67 (m, 2H, H-6 and H-2’), 7.68 (d, 1H, J=3.00 Hz, H-1), 7.45 (d, 1H, J=15.30 Hz, H-1’), 7.38-7.34 (m, 2H, H-7 and H-8), 6.49
(d, 1H, J=3.00 Hz, H-2), 6.41 (dd, 1H, J=15.00 and 11.00 Hz, H-3’), 6.16 (dt, 1H, J=15.00 and 6.90 Hz, H-4’), 4.19 (q, 2H,
J=7.00 Hz, O-CH2), 1.92 (d, 3H, J=6.90 Hz, CH3), 1.53 (t, 3H, J=7.00 Hz, CH3). 13C-NMR (CDCl3) δ: 151.7 (C-4), 145.2 (C-9),
135.1 (C-5a), 132.9 (C-4’), 132.8 (C-3’ and 2’), 130.0 (C-9), 127.6 (C-3a), 127.2 (C-7 and C-8), 126.5 (C-9a), 126.1 (C-1’), 113.6
(C-1 and C-6), 101.0 (C-2), 68.2 (C-1’’), 19.7 (C-2’’), 16.0 (C-5’). HRMS-ESI m/z [M+H]+ Calcd for C18H19N2O: 279.1497,
Found: 279.1505.
5.1.8. 1-(5-Methoxy-2-nitrophenyl)pyrrole (2c)
Method A: A mixture of 1-(5-chloro-2-nitrophenyl)pyrrole (2e) (4.5 mmol), Pd(OAc)2 (0.09 mmol), S-Phos (0.11 mmol),
MeOH (10 mmol), Cs2CO3 (6.7 mmol) in 10 mL of toluene was heated at 70°C under stirring until completion of the reaction
(monitoring by TLC with an eluent of 1:9 cyclohexane/AcOEt), then cooled at room temperature. After cooling, it was diluted
with 2mL of Et2O, filtered through a pad of celite and concentrated on reduced pressure. The crude product was purified by flash
chromatography on silica gel (eluent 1:9 AcOEt/cyclohexane) to give 2c. Yellow crystals (61%).28 Method B: A mixture of 1-(5-
chloro-2-nitrophenyl)pyrrole (3.60 mmol), MeONa (10.8 mmol), CuCl (0.054 mmol) in a mixture of 10mL of MeOH and 1mL of
DMF was warmed to gentle reflux under nitrogen flow overnight, then cooled to room temperature. The slurry is poured into a
rapidly stirring mixture of NH4Cl(aq) (5%) and 50 mL of Et2O. The aqueous phase was extracted twice with 50mL portions of
Et2O. The combined organic phases were dried over Na2SO4, filtered and concentrated on reduced pressure. The crude was
purified by flash chromatography on silica gel (eluent 1:9 AcOEt/cyclohexane). Yellow crystals (73%), mp 48°C.28
5.1.9. 4-[(2,2-Dibromovinyl)]pyrrolo[1,2-a]quinoxaline (9)
To a solution of CBr4 (4.08 mmol) in dry CH2Cl2 (5 mL) at 0°C was added dropwise a solution of PPh3 (8.16 mmol) in
CH2Cl2 (5 mL). The resulting slurry was stirred in a ice bath for 10 min. Then, was added dropwise a solution of pyrrolo[1,2-
a]quinoxaline-4-carboxyaldehyde 8 (2.04 mmol) in dry CH2Cl2 (7 ml). The reaction mixture was stirring at 0°C for 2 hours under
a N2 positive pressure. Then, the solution was diluted with water (10 mL). The aqueous layer was extracted twice with CH2Cl2
(2ꢀ25 mL), the organic layers were collected and were washed with a brine solution. The resulting solution was dried over
Na2SO4, filtered and concentrated under reduced pressure. The crude was triturated with Et2O, filtered, concentrated under
reduced pressure, then the residue was purified by flash chromatography (elution with AcOEt/Cyclohexane 1:9) to give 9 as pale
1
yellow crystals (17%). mp 96°C, Rf =0.79. H-NMR (CDCl3) δ: 8.06-7.98 (m, 2H, H-1 and H-9), 7.87 (dd, 1H, J=8.20 and 2.65
Hz, H-6), 7.60-7.44 (m, 2H, H-7 and H-8), 7.24 (s, 1H, HC=), 6.93-6.90 (m, 2H, H-2 and H-3). Anal. Calcd. for C13H8Br2N2 : C,
44.36; H, 2.29; N, 7.96. Found: C, 44.52; H, 2.10; N, 8.13.
5.1.10. 3-Ethoxypyrrole-2-carboxylic acid ethyl ester (10)
A mixture of ethoxyacetylene (17.8 mmol) and Ag2CO3 (1.19 mmol) in 1,4-dioxane (40 ml) was heated at 80°C for 5
min, then the mixture was cooling with an ice bath. To this resulting solution cooled at 0°C, ethyl isocyanoacetate (12 mmol) was
added dropwise. The reaction mixture was stirred for 10 min at room temperature, then heated 30 min at 80°C. The resulting
slurry was concentrated under reduced pressure and taken up with dichloromethane. The organic layer was washed with brine,
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dried with Na2SO4, filtered and evaporated under vacuum. The crude was purified on silica gel (eluent 3:7 AcOEt/Cyclohexane)
to give 10 as beige crystals (30%), mp 91°C.51
5.1.11. 3-Ethoxy-1-(2-nitrophenyl)pyrrole-2-carboxylic acid ethyl ester (11)
A mixture of 3-ethoxypyrrole-2-carboxylic acid ethyl ester (10) (7.10 mmol), Cs2CO3 (8.52 mmol) and 2-
fluoronitrobenzene (10.6 mmol) in 20 ml of DMF was refluxed for 90 min, then it was cooled and diluted with AcOEt. The
organic layer was washed with water, then brine, dried with Na2SO4 and concentrated under reduced pressure. Then, the crude
residue was cooling at 0°C, triturated into petroleum ether, the solvent was separated and the product dried under vacuum to give
1
11. Orange oil (94%), Rf=0.27 (2:8 AcOEt/PE) H-NMR (CDCl3) δ: 8.05 (dd, 1H, J=7.90 and 1.55 Hz, H-3’); 7.67 (ddd, 1H,
J=7.90, 7.30 and 1.55 Hz, H-4’); 7.57 (ddd, 1H, J=7.90, 7.30 and 1.55 Hz, H-5’); 7.39 (dd, 1H, J=7.90 and 1.55 Hz, H-6’); 6.74
(d, 1H, J=3.30 Hz, H-Pyrrole); 6.10 (d, 1H, J=3.30 Hz, H-Pyrrole); 4.20-4.06 (m, 4H, 2CH2); 1.48 (t, 3H, J=7.05 Hz, CH3); 1.14
(t, 3H, J=7.05 Hz, CH3). Anal. Calcd. for C15H16N2O5 : C, 59.21; H, 5.30; N, 9.21. Found: C, 58.89; H, 5.42; N, 9.26.
5.1.12. 3-Ethoxy-5H-pyrrolo[1,2-a]quinoxalin-4-one (12)
A mixture of 3-ethoxy-1-(2-nitrophenyl)pyrrole-2-carboxylic acid ethyl ester (11) (6.90 mmol), and iron powder (27.6
mmol) in 25 ml of acetic acid was refluxed for 90 min, then it was cooled at room temperature and poured in an aqueous solution
of HCl 1M (60 ml). The solution was stirred for 10 min, then the precipitate was filtered, washed with an 1M aqueous solution of
HCl, water and diethyl ether. The crude was dried under reduced pressure to give 12. Beige crystals (96%) mp 210°C. 1H-NMR
(DMSO-d6) δ: 10.75 (s, 1H, NH); 7.95 (d, 1H, J=2.7 Hz, H-1); 7.90 (d, 1H, J=8.10 Hz, H-9); 7.18-7.06 (m, 3H, H-6,H-7,H-8);
6.44 (d, 1H, J=2.70 Hz, H-2); 4.08 (q, 2H, J=6.90 Hz, CH2); 1.31 (t, 3H, J= 6.90 Hz, CH3). Anal. Calcd. for C13H12N2O2 : C,
68.41; H, 5.30; N, 12.27. Found: C, 68.54; H, 5.36; N, 12.10.
5.1.13. 4-Chloro-3-ethoxypyrrolo[1,2-a]quinoxaline (13)
A solution of 5H-pyrrolo[1,2-a]quinoxalin-4-one (12) (0.0066 mol) in POCl3 (12 mL) was refluxed for 2 h. After
removing excess of reactive under vacuum, the residue was carefully dissolved in water at 0°C and the resulting solution was
made basic sodium carbonate. The precipitate was filtered, solubilized in methylene chloride, the organic layer was then dried
over Na2SO4, filtered and evaporated to dryness to give 13. Beige crystals (80%), mp 118°C, Rf= 0.69. 1H-NMR (CDCl3) δ: 7.79
(dd, 1H, J=8.05 and 1.55 Hz, H-9); 7.73 (d, 1H, J=3.30 Hz, H-1); 7.71 (dd, 1H, J=8.05 and 1.55 Hz, H-6); 7.46 (ddd, 1H, J=8.05,
7.50 and 1.55 Hz, H-7); 7.38 (ddd, 1H, J=8.05, 7.50 and 1.55 Hz, H-8); 6.52 (d, 1H, J=3.30 Hz, H-2); 4.20 (q, 2H, J=7.00 Hz,
CH2); 1.51 (t, 3H, J=7.00 Hz, CH3). 13C-NMR (CDCl3) δ: 146.6 (C-4), 145.2 (C-3), 135.8 (C-5a), 129.8 (C-9), 128.6 (C-7), 127.6
(C-3a), 126.5 (C-8), 114.0 (C-6), 113.7 (C-1), 111.2 (C-9a), 101.8 (C-2), 68.6 (C-1’), 15.9 (C-2’). Anal. Calcd. for C13H11ClN2O :
C, 63.29; H, 4.49; N, 11.36. Found: C, 63.42; H, 4.67; N, 11.39.
5.2. X-ray Data
The structure of compound 5d has been established by X-ray crystallography (Figure 3). Colorless single crystal of 5d
was obtained by slow evaporation from methanol/dichloromethane (20/80) solution: monoclinic, space group P21/c, a=7.9325(7)
Å, b=10.9755(9) Å, c=12.3847(9) Å, α=90°, β=108.488(7)°, γ=90°, V=1022.60(15) Å3, Z=4, δ(calcd)= 1.511 Mg.m-3,
FW=232.66 for C12H9ClN2O, F(000)=480. Full crystallographic results have been deposited at the Cambridge Crystallographic
Data Centre (CCDC-966984), UK, as supplementary material.54 The data were corrected for Lorentz and polarization effects and
for empirical absorption correction.55 The structure was solved by direct methods Shelx 201356 and refined using Shelx 201356
suite of programs.
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5.3. Biological assays
5.3.1. In vitro antileishmanial activity
5.3.1.1. Strains.
L. major (MHOM/IL/81/BNI) and L. mexicana (MHOM/MX/95/NAN1) promastigotes were maintained in Schneider's
insect medium (Sigma-Aldrich, France) supplemented with 13% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich),
penicillin (100 UI/mL) and streptomycin (100 mg/mL) at 26°C by passage every 7 days. L. donovani (MHOM/IN/00/DEVI) used
in this study was provided by the CNR Leishmania (Montpellier, France).
5.3.1.2. Antileishmanial activity against promastigote stage.
Susceptibility testing was performed on L. major (MHOM/IL/81/BNI) and L. mexicana (MHOM/MX/95/NAN1)
promastigote with a spectrofluorimetric micromethod previously described.57 Briefly, 100 µL of a 106 promastigotes/mL
suspension were placed into wells of a 96-well microplate (Nunc®). Cultures were exposed for 96 hours at 26 °C to the drugs.
Four hours before measurement, 10 µL of resazurin solution (700 µM) were added. Then fluorescence was measured at 590 nm
with an excitation at 550 nm.
The effects of the tested compounds on the growth of L. donovani (MHOM/IN/00/DEVI) promastigotes were assessed by
MTT assay.58 Briefly, promastigotes in log-phase in Schneider’s medium supplemented with 20% fetal calf serum (FCS), 2 mM
L-glutamine and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin), were incubated at an average density of
106 parasites/mL in sterile 96-well plates with various concentrations of compounds dissolved in DMSO (final concentration less
than 0.5% v/v), in duplicate. Appropriate controls treated by DMSO, pentamidine or amphotericin B (reference drugs purchased
from Sigma Aldrich) were added to each set of experiments. After a 72 h incubation period at 27 °C, parasite metabolic activity
was determined. Each plate-well was then microscope-examined for detecting possible precipitate formation. 10 µL of MTT
solution (10 mg/mL) was added to each well followed by incubation for another 4 hours. The enzyme reaction was then stopped
by addition of 100 µL of 50% isopropanol – 10% sodium dodecyl sulfate. Plates were shaken vigorously for a few minutes and
the absorbance measured in a plate reader at 570 nm. Inhibitory concentration 50% (IC50) was defined as the concentration of drug
required to inhibit by 50% the metabolic activity of L. donovani promastigotes compared to the control. IC50 were calculated by
non-linear regression analysis processed on dose–response curves, using TableCurve 2D V5 software. IC50 values represent the
mean value calculated from three independent experiments.
5.3.2. In vitro antitrypanosomal activity
5.3.2.1. Parasites
Female mice 3 month old (Charles River, L’arbresle, France) were housed and handled according to University
procedures approved by European Community guidelines. Experiments on animals complied with guidelines of the European
Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (CETS n° 123).
Experiments were approved by the Department for the protection of animals and plants of the Préfecture de la Gironde (January
2012) (Identification number A33-063-324).
T. b. brucei (Antat 1.1) were kindly provided by N. Van Meirvenne and D. Le Ray from the Institute of Tropical
Medicine (Antwerp, Belgium). These parasites were adapted and maintained in vivo by passages in normal mice.59 Mice were
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injected intraperitoneally with 5 × 103 parasites. Mice with high parasitemia were bled by cardiac puncture. Parasites were
purified from rodent blood using DEAE-cellulose.
5.3.2.2. In vitro assessment of drugs
Each well of a 96-well plates (Falcon Plastics, Oxnard, CA, USA) was filled with 100 µL of culture medium McCoy 5A
modified medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 25 mM HEPES, 0.1 mM 2-mercaptoethanol, 2
mM sodium pyruvate, 0.2 mM L-cysteine and 10% foetal calf serum.60-62 Products were first dissolved in 100 % dimethyl
sulfoxide (DMSO). Adequate dilutions in culture medium of each compound were added into each well in triplicate while control
wells contained medium alone or with DMSO (0.4%). Then 100 µL of a suspension containing 105 blood-purified parasites were
added in each well. Cultures were maintained at 37°C in 5% CO2 incubator for 24 h. Parasite count was performed using a
haemocytometer. The activity was expressed in concentration inhibiting parasite growth by 50% (IC50). All experiments were
performed twice, with each drug concentration in triplicate. The mean of IC90 and standard deviation obtained for the 6 cultures of
each drug were calculated.
5.3.3. In vitro antiplasmodial activity
The in vitro antiplasmodial activities were tested over a concentration range of 39 nM-40 µM against culture-adapted
Plasmodium falciparum reference strains 3D7 and W2. The former strain is susceptible to chloroquine but displays a decreased
susceptibility to mefloquine while the latter is considered as resistant to chloroquine. The parasites were cultivated in RPMI
medium (Sigma–Aldrich, Lyon, France) supplemented with 0.5% Albumax I (Invitrogen-Life sciences, Saint-Aubin, France),
hypoxanthine (Sigma–Aldrich), gentamicin (Sigma–Aldrich), and human red blood cells, as described previously63 and were
incubated at 37°C in a candle jar. The P. falciparum drug susceptibility test was carried out in 96-well flat bottom sterile plates
under a final volume of 250 µL. After a 48-h incubation with the drugs, quantities of DNA in treated and control cultures of
parasites in human erythrocytes were compared according to the SYBR Green I (Sigma-Aldrich) fluorescence-based method.63,64
Briefly, after incubation, plates were frozen at -20°C until use. They were then left to thaw for 2 hours at room temperature and
100 µL of the homogenized culture were transferred to 96-well flat bottom sterile black plates (Nunc Inc) already containing 100
µl of the SYBR Green I lysis buffer (2XSYBR Green, 20 mM Tris base pH 7.5, 5 mM EDTA, 0.008% w/v saponin, 0.08% w/v
Triton X-100). A negative control, controls treated by solvents (DMSO and H2O, typically) and positive controls (chloroquine and
mefloquine) were added to each set of experiments. Plates were incubated for 1h at room temperature and then read on a
fluorescence plate reader (Tecan, Austria) using excitation and emission wavelengths of 485 and 535 nm, respectively.
Concentrations inhibiting 50% of the parasite’s growth (half maximal inhibitory concentration or IC50 values) were then
calculated from the obtained experimental results using a regression program available on line.65
5.3.4. Cytotoxicity Evaluation
A cytotoxicity evaluation was realized employing the MTT method58 to determine the cytotoxic concentrations of 50% (CC50)
and using doxorubicin as a cytotoxic reference-compound. These assays were performed toward two complementary cell lines: murine
J774A.1 macrophages (J774A.1 CC50) and human HepG2 (HepG2 CC50). J774A.1 macrophages (mouse macrophage cell line
ECACC, Salisbury UK) are commonly used as in vitro host cell model for screening antileishmanial drugs allowing intracellular stage
amastigote Leishamnia species to develop.66 HepG2 (hepatocarcinoma cell line purchased from ATCC, ref HB-8065) is a commonly
used human-derived hepatocarcinoma cell line that has shown characteristics similar to those of primary hepatocytes. These cells express
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many of the hepatocyte-specific metabolic enzymes, thus enabling the cytotoxicity of tested product metabolites to be evaluated. Briefly,
cells in 100 µL of complete medium, [RPMI supplemented with 10% FCS, 1% L-glutamine (200 mM) and penicillin (100 U/mL) /
streptomycin (100 µg/mL)] were inoculated into each well of 96-well plates and incubated at 37 °C in a humidified 6% CO2 with 94%
air atmosphere. After a 24 h incubation, 100 µL of medium with various product concentrations were added and the plates were
incubated for 72 h at 37 °C. Each plate-well was then microscope-examined for detecting possible precipitate formation before the
medium was aspirated from the wells. 100 µL of MTT solution (0.5 mg/mL in PBS) were then added to each well with 100 µl of
medium without FCS. Cells were incubated for 2 h at 37 °C. After this time, the MTT solution was removed and DMSO (100 µL) was
added to dissolve the resulting blue formazan crystals. Plates were shaken vigorously (300 rpm) for 5 min. The absorbance was
measured at 570 nm with 630 nm as reference wavelength with a microplate spectrophotometer. DMSO was used as blank and
doxorubicin (purchased from Sigma Aldrich) as positive control. Cell viability was calculated as percentage of control (cells incubated
without compound). The 50% cytotoxic concentration was determined from the dose–response curve by using the TableCurve 2D V5
software.
Acknowledgments
We thank the DGA and ANR (project ANR-12-ASTR-003) for financial support of this study. Financial supports from
the" Région Picardie" (France) and "Fonds Européens pour le Développement Régional" (Europe) are gratefully acknowledged
for the doctoral fellowships to Natacha Farvacques.
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J.W. Albright, J.F. Albright, International Journal for Parasitology 10 (1980) 137-142.
P. Vincendeau, B. Guillemain, S. Daulouede, C. Ripert, International Journal for Parasitology 16 (1986) 387-390.
P. Vincendeau, M. Daëron, S. Daulouede, Infection & Immunity 53 (1986) 600-605.
T. N. Bennett, M. Paguio, B. Gligorijevic, C. Seudieu, A. D. Kosar, E. Davidson, P. D. Roepe, Antimicrobial Agents &
Chemotherapy 48 (2004) 1807-1810.
[64]
D. J. Bacon, C. Latour, C. Lucas, O. Colina, P. Ringwald, S. Picot, Antimicrobial Agents & Chemotherapy 51 (2007)
1172-1178.
[65]
[66]
H. Kaddouri, S. Nakache, S. Houzé, F. Mentré, J. Le Bras, Antimicrobial Agents & Chemotherapy 50 (2006) 3343-3349.
K. Seifert, P. Escobar, S.L. Croft, Journal of Antimicrobial Chemotherapy 65 (2010) 508-511.
21

ACCEPTED MANUSCRIPT
OH
HO
HO
OH
OH
OH
-
H3C
OH
O
O
O
O
O
N
H
O
O
O
O
O
Sb
OH
OH
+
Sb
Sb
3 Na
OH
-
OH
COO
-
Meglumine antimoniate
OOC
Sodium stibogluconate
NH
NH
H2N
NH2
O
O
Pentamidine
OH
OH
OH
H3C
HO
O
O
OH
OH
OH
OH
O
CH3
COOH
H
H3C
CH3
OH
O
O
Amphotericin B
NH2
OH
O
+
CH2-CH2-N (CH3)3
H3C-(CH2)14-CH2
O
P
O
-
O
Miltefosine
Figure 1. Structure of meglumine antimoniate (Glucantime®), sodium stibogluconate (Pentostam®), amphotericin B (Fungizone®,
Ambisome®), pentamidine (Pentacarinat®), and miltefosine (Impavido®).
22

ACCEPTED MANUSCRIPT
N
N
R1
R1
N
CH3
N
CH3
N
R2
R1- = H-, CH3O-
chimanine B
R1- = H-, CH3O-
R2- = Alkyl-, Alkenyl-,
IC50 = 1-7 µM (IS = 7-40)
Alkynyl-, Aryl- etc ...
L. amazonensis or L. infantum
Series I
2
1
9
3
N
8
7
R1
N
N
CH3
( )n
4
5N
6
9
R1
2
1
CH3
( )n
3
N
R1- = H-, CH3O-
n = 1-5
8
7
R1
4
Series II
5N
CH3
6
R1- = H-, CH3O-
n = 0-3
IC50 = 0.5-7 µM (IS = 3-16)
L. amazonensis or L. infantum
Series III
Figure 2. Structure of natural product Chimanine B, general structure of Series I-II and design of Series III.
23

ACCEPTED MANUSCRIPT
Figure 3. The ORTEP drawing of pyrrolo[1,2-a]quinoxaline 5d with thermal ellipsoids at 30% level.
24

ACCEPTED MANUSCRIPT
20
18
16
14
12
10
8
6.5
6
5.5
5
4.5
4
IC50
(µM)
Clog P
3.5
3
6
4
2.5
2
2
W2
0
3D7
1a 1d 1b 1c 1n 1m 1e 1h 1f 1g 1i 1l 1j 1k
Clog P
Figure 4. CLog P / activity relationship for pyrrolo[1,2-a]quinoxalines 1a-n on P. falciparum strains.
25

ACCEPTED MANUSCRIPT
R3
R3
R3
R2
R1
NH2
NO2
R2
R1
N
R2
R1
N
ii
i
NH2
NO2
2a-d
3a-d
R3
R3
R2
R1
N
N
R2
R1
N
iv
iii
Cl
N
H
O
4a-d
5a-d
R3
R2
N
v
CH3
( )n
R1
N
1a-l
CH3
CH3
OH
B
N
N
vi
CH3
O
O
CH3
B
O
O
+
( )n
HO
B
O
O
O
( )n
O
Br
6a-c
-
-
-
R1
R2
R3
n
-
-
-
0
0
0
1a
H-
H-
R1
H-
R2
H-
R3
H-
H-
1b CH3O- H-
H-
H-
H-
2-5a
1c
1d
1e
H-
H- CH3O-
2-5b
2-5c
CH3O- H-
H- CH3O-
H-
H-
CH3O-
H-
0
2
2
2
H-
H-
2-5d
H-
CH3O-
H-
H-
1f CH3O- H-
1g
1h
1i
H-
H- CH3O-
n
H-
H-
CH3O-
H-
2
3
H-
H-
0
6a
3
3
H-
1j CH3O- H-
6b
6c
2
3
H-
1k
1l
H- CH3O-
H-
CH3O-
3
H-
Scheme 1. Synthesis of new 4-(alka-1,3-dienyl)pyrrolo[1,2-a]quinoxalines 1a-l; Reagents and conditions: (i) 2,5-diMeOTHF,
AcOH, ∆; (ii) CuSO4/NaBH4, EtOH, RT; (iii) (Cl3CO)2CO, toluene, ∆; (iv) POCl3, ∆; (v) method A) Pd(OAc)2/SPhos, NaOH,
RT; method B) Pd(PPh3)2NBS, Na2CO3, THF, ∆; (vi) method A) Pd(OAc)2/SPhos, Cs2CO3, THF, RT; method B)
Pd(OAc)2/SPhos, KF, toluene, RT.
26

ACCEPTED MANUSCRIPT
H3CO
NH2
Cl
NH2
NO2
NO2
7
ii
i
H3CO
N
Cl
N
iii
NO2
NO2
2c
2e
Scheme 2. Different strategies used in the synthesis of 1-(5-methoxy-2-nitrophenyl)pyrrole 2c; Reagents and conditions: (i) or
(ii) 2,5-diMeOTHF, AcOH, ∆; (iii) method A) CH3OH, Pd(OAc)2/SPhos, Cs2CO3, toluene, 70°C; method B) CH3ONa, CuCl,
CH3OH/DMF, ∆.
27

ACCEPTED MANUSCRIPT
N
N
N
Br
ii
N
N
i
CHO
N
Br
Br
8
9
CH3
CH3
N
N
N
N
O
iv
iii
B
O
O
O
B
O
O
O
CH3
O
CH3
CH3
6a
6d
1m
N
N
Cl
5a
Scheme 3. Synthesis of new 4-(alka-1,3,5-trienyl)pyrrolo[1,2-a]quinoxaline 1m; Reagents and conditions: (i) CBr4, PPh3, 0°C;
(ii) i-PrMgCl, Fe(acac)3, -10°C; (iii) 1) NaOH, THF/H2O, RT; 2) trans-(2-bromovinyl) MIDA boronate, Pd(OAc)2/SPhos,
Cs2CO3, toluene, 45°C; (iv) Pd(PPh3)2NBS, Na2CO3, THF, ∆.
28

ACCEPTED MANUSCRIPT
N
N
OC2H5
OC2H5
COOC2H5
i
ii
HN
OC2H5
COOC2H5
N
H
O
NO2
10
12
11
N
N
N
N
OC2H5
OC2H5
iii
iv
Cl
CH3
1n
13
CH3
N
O
O
B
O
O
CH3
6a
Scheme 4. New 4-(alka-1,3-dienyl)pyrrolo[1,2-a]quinoxaline 1n; Reagents and conditions: (i) 2-fluoro-nitrobenzene, Cs2CO3,
DMF, ∆; (ii) Fe, CH3COOH, ∆; (iii) POCl3, ∆; (iv) Pd(PPh3)2NBS, Na2CO3, THF, ∆.
29