Design, synthesis and antimalarial activity of novel bis{N-[(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-aminopropyl}amine derivatives

Journal of Enzyme Inhibition and Medicinal Chemistry

ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20

Design, synthesis and antimalarial activity of novel

bis{N-[(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-

aminopropyl}amine derivatives

Jean Guillon, Anita Cohen, Nassima Meriem Gueddouda, Rabindra Nath Das,

Stéphane Moreau, Luisa Ronga, Solène Savrimoutou, Louise Basmaciyan,

Alix Monnier, Myriam Monget, Sandra Rubio, Timothée Garnerin, Nadine

Azas, Jean-Louis Mergny, Catherine Mullié & Pascal Sonnet

To cite this article: Jean Guillon, Anita Cohen, Nassima Meriem Gueddouda, Rabindra Nath

Das, Stéphane Moreau, Luisa Ronga, Solène Savrimoutou, Louise Basmaciyan, Alix Monnier,

Myriam Monget, Sandra Rubio, Timothée Garnerin, Nadine Azas, Jean-Louis Mergny, Catherine

Mullié & Pascal Sonnet (2017) Design, synthesis and antimalarial activity of novel bis{N-

[(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-aminopropyl}amine derivatives, Journal of Enzyme

Inhibition and Medicinal Chemistry, 32:1, 547-563, DOI: 10.1080/14756366.2016.1268608

To link to this article: http://dx.doi.org/10.1080/14756366.2016.1268608

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Date: 25 January 2017, At: 02:28

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017

VOL. 32, NO. 1, 547–563

http://dx.doi.org/10.1080/14756366.2016.1268608

RESEARCH ARTICLE

Design, synthesis and antimalarial activity of novel bis{N-[(pyrrolo[1,2-a]

quinoxalin-4-yl)benzyl]-3-aminopropyl}amine derivatives

a,b

Jean Guillon^a,b, Anita Cohen^c, Nassima Meriem Gueddouda^a,b, Rabindra Nath Das^a,b, Stephane Moreau

,

ꢀ

Luisa Ronga^a,b, Solene Savrimoutou^a,b, Louise Basmaciyan^c, Alix Monnier^a,b, Myriam Monget^a,b, Sandra Rubio^a,b

,

ꢁ

d

c

a,b

d

ꢀ

ꢀ^d

Timothee Garnerin , Nadine Azas , Jean-Louis Mergny , Catherine Mullie and Pascal Sonnet

^aARNA Laboratory, University Bordeaux, UFR des Sciences Pharmaceutiques, Bordeaux, France; INSERM U1212, UMR CNRS 5320, ARNA

Laboratory, Bordeaux, France; UMR-MD3, Faculty of Pharmacy, Aix-Marseille University, Laboratory of Parasitology, Marseille, France; Universite

b

c

d

ꢀ

de Picardie Jules Verne, Laboratoire de Glycochimie, des Antimicrobiens et des Agroressouces, UMR CNRS 7378, UFR de Pharmacie, Amiens,

France

ABSTRACT

ARTICLE HISTORY

Received 14 October 2016

Revised 29 November 2016

Accepted 1 December 2016

Novel series of bis- and tris-pyrrolo[1,2-a]quinoxaline derivatives 1 were synthesized and tested for in vitro

activity upon the intraerythrocytic stage of W2 and 3D7 Plasmodium falciparum strains. Biological results

showed good antimalarial activity with IC₅₀in the lM range. In attempting to investigate the large broad-

spectrum antiprotozoal activities of these new derivatives, their properties toward Leishmania donovani

were also investigated and revealed their selective antiplasmodial profile. In parallel, the in vitro cytotox-

icity of these molecules was assessed on the human HepG2 cell line. Structure–activity relationships of

these new synthetic compounds are discussed here. The bis-pyrrolo[1,2-a]quinoxalines 1n and 1p were

identified as the most potent antimalarial candidates with selectivity index (SI) of 40.6 on W2 strain, and

39.25 on 3D7 strain, respectively. As the telomeres of the parasite could constitute an attractive target, we

investigated the possibility of targeting Plasmodium telomeres by stabilizing the Plasmodium telomeric

G-quadruplexes through a FRET melting assay by our new compounds.

KEYWORDS

Antimalarial activity;

bis-pyrrolo[1,2-a]quinoxa-

line; Plasmodium falciparum;

G-quadruplex; antileishma-

nial activity

Introduction

until oral medication could be tolerated⁵. Artemisinin and its deriv-

atives are also used in combination with several different partner

drugs (including lumefantrine, mefloquine, amodiaquine, sulfadox-

ine/pyrimethamine and piperaquine) in artemisinin-based combin-

ation therapies (ACTs), now recommended as the first line of

treatment of uncomplicated Plasmodium falciparum malaria and as

the second part of treatment for 3 days of severe malaria in

endemic areas⁵. However, over the last decade evidence has

grown that artemisinin resistance has emerged and spread within

Malaria remains as one of the most devastating infectious diseases

of the developing world. Malaria remains a major cause of public

health problem in about 95 countries mainly located in the trop-

ical zone of the globe (notably Africa, South-East Asia and also

Eastern Mediterranean region)¹; while approximately 3.2 billion

people are at risk of being infected with malaria and developing

disease. The latest figures on the incidence and mortality of mal-

aria show that, despite progress in the implementation of prevent-

ive measures such as insecticide-treated mosquito nets and

intermittent preventive treatments, this parasitic disease is still

estimated to affect over 214 million people and to account for

438,000 deaths in 2015, of which approximately 80% are concen-

trated in just fifteen countries, mainly in Africa. The death toll is

particularly high in children under five and pregnant women of

the World Health Organization (WHO) African region¹. Five species

of protozoan parasites belonging to the Plasmodium genus,

namely, falciparum, malariae, vivax, ovale and knowlesi cause mal-

aria in human beings; from which, P. falciparum is the most dan-

Southeast Asia, first on the Cambodia-Thailand border in 2009^6,7

,

but now across a widening area of the Greater Mekong Subregion.

Rapid scientific advances in understanding of this problem have

taken place within the last five years^8–10and defined mutations in

“K13” gene of P. falciparum associated to reduced ring-stage sus-

ceptibility to artemisinins¹¹. Therefore, new antimalarial agents

with new mechanisms of action are required to overcome the

emergence of resistance and to control an ever-increasing number

of epidemics caused by the malaria parasite¹². Although current

efforts in antimalarial drug discovery are focused on identification

of new biological targets, continued research on new 4-aminoqui-

noline derivatives is still warranted. This is because the hemoglo-

bin degradation pathway in P. falciparum has a proven history as

an excellent therapeutic target to which the parasite has difficulty

gerous of these species^2–4

The increasing prevalence of multiple drug resistant strains of

P. falciparum in most malaria endemic areas has significantly

.

reduced the efficacy of the current antimalarial drugs. Nowadays, in evolving resistance. In spite of resistance to chloroquine, previ-

ous work has shown that modification of the lateral side chain of

chloroquine results in aminoquinoline derivatives that avoid the

the only fully effective antimalarial drugs utilize artemisinin and its

derivatives, notably parenteral artesunate now recommended as

the first line of treatment of severe malaria for at least 24 h and

chloroquine resistance mechanism^13,14

.

CONTACT Jean Guillon

jean.guillon@u-bordeaux.fr

Univ. Bordeaux, UFR des Sciences Pharmaceutiques, INSERM U1212/UMR CNRS 5320, ARNA Laboratory,

F-33076 Bordeaux cedex, France

ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-

bution, and reproduction in any medium, provided the original work is properly cited.

548

J. GUILLON ET AL.

A possibility to overcome this multidrug-resistant mechanism is

Materials and methods

to design new quinoline-based drugs which will not be recognized

by the protein system involved in drug efflux. In this regard,

two series of compounds that show promise in this regard are

the bisquinoline and bisacridine antimalarial drugs A and B

(Figure 1)^15–18. These drugs show much lower resistance indices

than chloroquine, indicating that the bisquinoline or bisacridine

structures are less efficiently excluded by drug-resistant parasites.

The pyrrolo[1,2-a]quinoxaline heterocyclic framework constitutes

the basis of an important class of compounds possessing interest-

ing biological activities. These compounds have been reported as

key intermediates for the assembly of several heterocycles including

antipsychotic agents, anti-HIV agents, adenosine A₃receptor modu-

lators¹⁹, antiparasitic agents^20–25and antitumor agents^26–30. In the

course of our work devoted to discover new compounds employed

in the antiparasitic chemotherapy, we previously identified some

series of substituted pyrrolo[1,2-a]quinoxaline derivatives designed

as interesting bioactive isosteres of quinoline derivatives^20–24.Thus,

taking into account our experience in the field of the synthesis of

new antimalarial heterocyclic compounds based on our pyrrolo[1,2-

a]quinoxaline heterocyclic core^{20–24,27–30}, we decided to incorporate

a benzyl group in position 4 of the heterocyclic skeletonof our pre-

viously described bispyrrolo[1,2-a]quinoxalines C²⁰to broaden the

structural diversity of these derivatives, and mainly to increase the

aromatic surface of these designed compounds (Figure 1).

Chemistry

Commercial reagents were used as received without additional

purification. Melting points were determined with an SM-LUX-POL

Leitz hot-stage microscope (Leitz GMBH, Midland, ON) and are

uncorrected. IR spectra were recorded on a NICOLET 380FT-IR

spectrophotometer (Thermo Electron Scientific Instruments LLC,

Madison, WI). NMR spectra were recorded with tetramethylsilane

as an internal standard using a BRUKER AVANCE 300 spectrometer

(Bruker BioSpin, Wissembourg, France). Splitting patterns have

been designated as follows: s ¼ singlet; bs ¼ broad singlet;

d ¼ doublet; t ¼ triplet; q ¼ quartet; qt ¼ quintet, dd ¼ double

doublet; ddd ¼ double double doublet; dt ¼ double triplet;

m ¼ multiplet. Analytical TLC were carried out on 0.25 precoated

silica gel plates (POLYGRAM SIL G/UV₂₅₄) and visualization of com-

pounds after UV light irradiation. Silica gel 60 (70–230 mesh) was

used for column chromatography. Microwave experiments were

carried out using a focused microwave reactor (CEM Discover,

Saclay, France). High resolution mass spectra (electrospray in posi-

tive mode, ESIþ) were recorded on a Waters Q-TOF Ultima appar-

atus. Mass spectra were recorded on an Ultraflex III TOF/TOF

system (Bruker Daltonics, Bremen, Germany), equipped with

200 Hz smartbeam laser (355 nm) and operating in reflectron posi-

tive ion mode. Mass spectra were acquired over the m/z range

300–5000 by accumulating data from 1000 laser shots for each

spectrum. The instrumental conditions employed to analyze

molecular species were the following: ion source 1: 25.08 kV; ion

source 2: 21.98 kV, lens: 11.03 kV, pulsed ion extraction: 30 ns,

reflector: 26.39 kV, reflector 2: 13.79 kV. Matrix suppression was

activated by deflection mode: suppression up to 450 Da. Mass cali-

bration was performed for each sample with a peptide calibration

mixture (8206195, Peptide Calibration Standard, Bruker Daltonics).

The instrument was controlled using Bruker's flexControl 3.4 soft-

ware and mass spectra were analyzed in Bruker's FlexAnalysis 3.4

software (Bruker Daltonics, Billerica, MA).

Hence, we described here the synthesis of new bis- or trispyr-

rolo[1,2-a]quinoxaline derivatives 1a–t (Figure 1) and reported on

their in vitro antiplasmodial activity against the chloroquine-sensitive

(3D7) and the chloroquine-resistant (W2) strains of the malaria para-

site Plasmodium falciparum. As these new compounds were

designed as quinoline-like bio-isosteres, and as the quinoline skel-

eton is the fundamental unit of many antiprotozoan drugs, these

pyrrolo[1,2-a]quinoxaline derivatives were also investigated on

another medically important protozoan, Leishmania donovani, in

order to evaluate the specificity of their antiparasitic activity.

Leishmaniasis is an infectious disease caused by protozoan parasites

belonging to the genus Leishmania. Leishmania parasites exist in

two major morphological stages: extracellular flagellated promasti-

gotes in the digestive tract of their sandfly vector, which is the

infective stage and immobile intracellular amastigotes in the cells of

their host’ mononuclear phagocytic system. Leishmaniasis presents

various clinical aspects including cutaneous leishmaniasis, the most

common form, muco-cutaneous leishmaniasis and visceral leishman-

iasis, the most severe form, lethal in untreated patients. Leishmania

donovani is one of the major causative agents of human visceral

leishmaniasis which represents a public health problem: 0.2 to 0.4

million visceral leishmaniasis cases occur each year and more than

90% of global visceral leishmaniasis cases occur in just five coun-

tries: India, Bangladesh, Sudan, Brazil and Ethiopa³¹. The current

treatment of the disease is based on a limited number of chemo-

therapeutic agents (meglumine antimoniate, sodium stibogluconate,

pentamidine, amphotericin B and miltefosine) which present many

limits, notably characterized by a high toxicity and cost^32,33. In add-

ition, the in vitro cytotoxicity of these new molecules was assessed

on the human HepG2 cell line, in order to determine their selectivity

index. Moreover, as the telomeres of the parasite could constitute

an attractive target, we also investigated the possibility of targeting

Plasmodium telomeres by stabilizing the Plasmodium telomeric G-

quadruplexes through a FRET melting assay by our new synthesized

compounds. Indeed, telomerase activity has been identified in

gametocytes and during the transition to erythrocytic stage of P. fal-

ciparum³⁴. The telomeric 3' G-overhang region of P. falciparum is

General procedure for the synthesis of 4-(pyrrolo

[1,2-a]quinoxalin-4-yl)benzaldehydes and 3-(pyrrolo

[1,2-a]quinoxalin-4-yl)benzaldehydes8

To suspension of 4-chloropyrrolo[1,2-a]quinoxaline 7 (3.3 mmol),

and Pd(PPh₃)₄(0.164 mmol) in a mixture of toluene/EtOH (50/3 mL)

under nitrogen were added K₂CO₃(3.6 mmol) and phenylboronic

acid (3.6 mmol). The reaction mixture was refluxed for 24 h, and

the cooled suspension was extracted with CH₂Cl₂(3 ꢀ 70 mL). The

organic layer was washed with a saturated solution of NaCl

(90 mL), and the combined organic extracts were dried over

sodium sulfate, filtered, and evaporated under reduced pressure.

The crude residue was triturated in ethanol. The resulting precipi-

tate was filtered, washed with ethanol, and purified by column

chromatography on silica gel using dichloromethane as eluent

gave the pure product 8.

4–(9-Methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzaldehyde (8d)

Yellow crystals, Yield: 85%, mp ¼162–164 ^ꢁC; IR m_max(KBr)/cm^ꢂ1

1705 (CHO); ¹H NMR d (300 MHz, CDCl₃) 10.15 (s, 1H, CHO), 8.90

(dd, 1H, J ¼ 2.70 and 1.20 Hz, H-1), 8.17 (d, 2H, J¼ 8.40 Hz, H-2⁰and

H-6⁰), 8.08 (d, 2H, J ¼ 8.40 Hz, H-3⁰and H-5⁰), 7.70 (dd, 1H, J ¼ 8.10

and 1.20 Hz, H-6), 7.42 (t, 1H, J¼ 8.10 Hz, H-7), 7.13 (dd, 1H,

J ¼ 8.10 and 1.20 Hz, H-8), 7.00 (dd, 1H, J ¼ 3.90 and 1.20 Hz, H-3),

6.90 (dd, 1H, J ¼ 3.90 and 2.70 Hz, H-2), 4.13 (s, 3H, OCH₃). MALDI-

TOF MS m/z [M þ H]^þCalcd for C₁₉H₁₅N₂O₂: 303.113, Found:

303.127.

0

comprised of repeated degenerate unit ⁵GGGTTYA³(where Y may

be T or C)³⁵which can fold into intramolecular G-quadruplex³⁶.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

549

CH₃

N

HN

N

Cl

Chloroquine (CQ)

Cl

N

NH

HN

N

B

N

NH

HN

N

A

OCH₃

H₃CO

X

N

NH

HN

C

R₂

N

R₂

R₁

R

R₁, R₂= H-, CH₃O-, Cl-

X = -NH-, -NCH₃-,

N

R₂

R₁

N

2

1

3

4

R₄

R₃

NH

R₃

R₂

R₁

N

R₅

2

N

R₂

R

R₅

1

3

N

⁴HN

X

NH

HN

1a-t

NH

N

R₁, R₂, R₃= H-, CH₃O-

N

R

₄= H-, C₆H₅-

₅= H-, EtO-

X = -NH-, -NCH₃-,

N

R₂

R₁

N

R₁

R₂

Figure 1. Structure of chloroquine, bisquinolines A, bisacridines B, bispyrrolo[1,2-a]quinoxalines C, and new synthesized substituted bis- and trispyrrolo[1,2-a]quinoxaline

derivatives 1a–t.

4–(3-Ethoxypyrrolo[1,2-a]quinoxalin-4-yl)benzaldehyde (8e)

(s, 1H, H-2⁰), 8.32 (d, 1H, J ¼ 7.80 Hz, H-6), 8.09 (dd, 1H, J ¼ 2.85

Yellow crystals, Yield: 68%, mp ¼170–172 ^ꢁC; IR m_max(KBr)/cm^ꢂ1and 1.30 Hz, H-1), 8.08–8.05 (m, 2H, H-4⁰and H-6⁰), 7.93 (d, 1H,

1700 (CHO); ¹H NMR d (300 MHz, CDCl₃) 10.14 (s, 1H, CHO), J ¼ 7.80 Hz, H-9), 7.75 (t, 1H, J ¼ 7.65 Hz, H-5⁰), 7.56 (t, 1H,

8.00–7.98 (m, 4H, H-2⁰, H-3⁰, H-5⁰and H-6⁰), 7.94 (dd, 1H, J ¼ 8.10 J ¼ 7.80 Hz, H-7), 7.53 (t, 1H, J ¼ 7.80 Hz, H-8), 7.02 (dd, 1H, J ¼ 3.90

and 1.40 Hz, H-6), 7.84 (d, 1H, J ¼ 3.15 Hz, H-1), 7.81 (dd, 1H, and 1.30 Hz, H-3), 6.96 (dd, 1H, J ¼ 3.90 and 2.85 Hz, H-2). MALDI-

J¼ 8.10 and 1.40 Hz, H-9), 7.50 (ddd, 1H, J ¼ 8.10, 8.10 and 1.40 Hz, TOF MS m/z [M þ H]^þCalcd for C₁₈H₁₃N₂O: 273.103, Found:

H-7), 7.43 (ddd, 1H, J ¼ 8.10, 8.10 and 1.40 Hz, H-8), 6.54(d, 1H, 273.127.

J ¼ 3.15 Hz, H-2), 3.97(q, 2H, J ¼ 6.90 Hz, OCH₂), 1.17 (t, 3H,

J ¼ 6.90 Hz, CH₃). MALDI-TOF MS m/z [M þ H]^þCalcd for

3–(8-Methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzaldehyde (8 h)

C₂₀H₁₇N₂O₂: 317.129, Found: 317.135.

Beige crystals, Yield: 87%, mp ¼135–138 ^ꢁC; IR m_max(KBr)/cm^ꢂ1

1705 (CHO); ¹H NMR d (300 MHz, CDCl₃) 10.16 (s, 1H, CHO), 8.54

(dd, 1H, J ¼ 1.50 and 1.50 Hz,H-2⁰), 8.30 (ddd, 1H, J ¼ 7.65, 1.50 and

3-(Pyrrolo[1,2-a]quinoxalin-4-yl)benzaldehyde (8 g)

Yellow crystals, Yield: 68%, mp ¼147–150 ^ꢁC; IR m_max(KBr)/cm^ꢂ11.50 Hz, H-4⁰), 8.05 (ddd, 1H, J ¼ 7.65, 1.50 and 1.50 Hz, H-6⁰), 8.02

1700 (CHO); ¹H NMR d (300 MHz, CDCl₃) 10.18 (s, 1H, CHO), 8.56 (d, 1H, J ¼ 9.15 Hz, H-6), 7.95 (dd, 1H, J ¼ 2.70 and 1.50 Hz, H-1),

550

J. GUILLON ET AL.

7.73 (t, 1H, J ¼ 7.65 Hz, H-5⁰),7.32 (d, 1H, J ¼ 2.70 Hz, H-9), 7.10(dd, 2 H-6⁰), 7.91 (dd, 2H, J ¼ 2.70 and 1.20 Hz, 2 H-1), 7.89 (d, 4H,

1H, J ¼ 9.15 and 2.70 Hz, H-7), 6.99 (dd, 1H, J ¼ 3.90 and 1.50 Hz, H- J ¼ 8.10 Hz, 2 H-3⁰and 2 H-5⁰), 7.78(d, 2H, J ¼ 9.00 Hz, 2 H-9), 7.49

3), 6.95 (dd, 1H, J ¼ 3.90 and 2.70 Hz, H-2), 4.01 (s, 3H, CH₃O). (d, 2H, J ¼ 2.90 Hz, 2 H-6), 7.14 (dd, 2H, J ¼ 9.00 and 2.90 Hz, 2 H-8),

MALDI-TOF MS m/z [M þ H]^þCalcd for C₁₉H₁₅N₂O₂: 303.113,

6.94 (dd, 2H, J ¼ 4.20 and 1.20 Hz, 2 H-3), 6.85 (dd, 2H, J ¼ 4.20 and

2.70 Hz, 2 H-2), 3.93 (s, 6H, 2 CH₃O), 3.74 (t, 4H, J ¼ 6.90 Hz, 2 CH₂),

2.52 (t, 4H, J ¼ 6.90 Hz, 2 CH₂), 2.31 (s, 3H, CH₃), 1.96 (qt, 4H,

J ¼ 6.90 Hz, 2 CH₂).

Found: 303.135.

General procedure for bis{N-[(pyrrolo[1,2-a]quinoxalin-4-

yl)benzylidene]-3-aminopropyl}methylamine and bis

{N-[(pyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-

aminopropyl}piperazine 9a-p, bis{N-[4-(indolo[1,2-a]quinoxalin-

6-yl)benzylidene]-3-aminopropyl}methylamine 9q and bis

{N-[(4-phenylpyrrolo[1,2-a]quinoxalin-2-yl)methylidene]-3-

aminopropyl}piperazine 9r

Bis{N-[4–(7-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-

aminopropyl}piperazine (9e)

Yellow crystals, Yield: 96%, mp ¼78–80 ^ꢁC; ¹H NMR d (300 MHz,

CDCl₃) 8.41 (s, 2H, 2 HC ¼ N), 8.07 (d, 4H, J ¼ 8.20 Hz, 2 H-2⁰and

2 H-6⁰), 7.97 (dd, 2H, J ¼ 2.70 and 1.20 Hz, 2 H-1), 7.91 (d, 4H,

J ¼ 8.20 Hz, 2 H-3⁰and 2 H-5⁰), 7.83 (d, 2H, J ¼ 9.00 Hz, 2 H-9), 7.53

(d, 2H, J ¼ 3.00 Hz, 2 H-6), 7.17 (dd, 2H, J ¼ 9.00 and 3.00 Hz, 2 H-8),

6.99 (dd, 2H, J ¼ 3.90 and 1.20 Hz, 2 H-3), 6.90 (dd, 2H, J ¼ 3.90 and

2.70 Hz, 2 H-2), 3.95 (s, 6H, 2 CH₃O), 3.73 (t, 4H, J ¼ 6.90 Hz, 2 CH₂),

2.71–2.45 (m, 8H, 4 CH₂pip.), 2.50 (t, 4H, J ¼ 6.90 Hz, 2 CH₂), 1.97

(qt, 4H, J ¼ 6.90 Hz, 2 CH₂).

To a solution of diamine (2.7 mmol) in ethanol (15 mL) was added

(pyrrolo[1,2-a]quinoxalin-4-yl)benzaldehyde 8a–h or (indolo[1,2-

a]quinoxalin-6-yl)benzaldehyde 8i or 4-phenylpyrrolo[1,2-a]qui-

noxaline-2-carboxaldehyde 8j (5.4 mmol). The reaction mixture was

then heated under reflux for 5 h and then evaporated to dryness

under reduced pressure. After cooling, the residue was extracted

with dichloromethane (40 mL). The organic layer was dried over

sodium sulfate and evaporated to dryness. The products were

then used without further purification.

Bis{N-[4–(8-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-

aminopropyl}methylamine (9f)

Yellow crystals, Yield: 96%, mp ¼61–63 ^ꢁC; ¹H NMR d (300 MHz,

CDCl₃) 8.40 (s, 2H, 2 HC ¼ N), 8.02 (d, 4H, J ¼ 8.20 Hz, 2 H-2⁰and

2 H-6⁰), 7.94 (d, 2H, J ¼ 9.00 Hz, 2 H-6), 7.88(d, 4H, J ¼ 8.20 Hz, 2 H-3⁰

and 2 H-5⁰), 7.87 (dd, 2H, J ¼ 2.75 and 1.20 Hz, 2 H-1), 7.26 (d, 2H,

J ¼ 2.70 Hz, 2 H-9), 7.05 (dd, 2H, J ¼ 9.00 and 2.70 Hz, 2 H-7), 6.95

(dd, 2H, J ¼ 3.90 and 1.20 Hz, 2 H-3), 6.86 (dd, 2H, J ¼ 3.90 and

2.75 Hz, 2 H-2), 3.99 (s, 6H, 2 CH₃O), 3.73 (t, 4H, J ¼ 6.90 Hz, 2 CH₂),

2.51 (t, 4H, J ¼ 6.90 Hz, 2 CH₂), 2.31 (s, 3H, CH₃), 1.96 (qt, 4H,

J ¼ 6.90 Hz, 2 CH₂).

Bis{N-[4-(pyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-aminopro-

pyl}amine (9a)

Yellow crystals, Yield: 93%, mp ¼107–109 ^ꢁC; ¹H NMR d (300 MHz,

CDCl₃) 8.31 (s, 2H, 2 HC ¼ N), 8.03–7.80 (m, 8H, 2 H-2⁰, 2 H-6⁰, 2 H-6

and 2 H-1), 7.63 (d, 4H, J ¼ 8.40 Hz, 2 H-3⁰and 2 H-5⁰), 7.54–7.34 (m,

4H, 2 H-7 and 2 H-8), 6.96 (dd, 2H, J ¼ 4.05 and 1.30 Hz, 2 H-3), 6.86

(dd, 2H, J ¼ 4.05 and 2.70 Hz, 2 H-2), 4.09 (s, 1H, NH), 3.73–3.20 (m,

4H, 2 CH₂), 2.88–2.31 (m, 4H, 2 CH₂), 2.11–1.85 (m, 4H, 2 CH₂).

Bis{N-[4-(pyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-aminopro-

pyl}methylamine (9b)

Bis{N-[4–(8-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-

aminopropyl}piperazine (9 g)

Yellow oil, Yield: 89%; ¹H NMR d (300 MHz, CDCl₃) 8.41 (s, 2H, 2

HC ¼ N), 8.05 (d, 4H, J ¼ 8.40 Hz, 2 H-2⁰and 2 H-6⁰), 8.02 (dd, 2H,

J ¼ 8.25 and 1.40 Hz, 2 H-6), 7.97 (dd, 2H, J ¼ 2.70 and 1.30 Hz, 2 H-

1), 7.90 (d, 4H, J ¼ 8.40 Hz, 2 H-3⁰and 2 H-5⁰), 7.85 (dd, 2H, J ¼ 8.25

and 1.40 Hz, 2 H-9), 7.52 (ddd, 2H, J ¼ 8.25, 8.25 and 1.40 Hz, 2 H-7),

7.44 (ddd, 2H, J ¼ 8.25, 8.25 and 1.40 Hz, 2 H-8),6.96 (dd, 2H,

J ¼ 4.05 and 1.30 Hz, 2 H-3), 6.87 (dd, 2H, J ¼ 4.05 and 2.70 Hz, 2 H-

2), 3.74 (t, 4H,J ¼ 7.10 Hz, 2 CH₂), 2.53 (t, 4H, J ¼ 7.10 Hz, 2 CH₂),

2.32 (s, 3H, CH₃), 1.96 (qt, 4H, J ¼ 7.10 Hz, 2 CH₂).

Pale-yellow crystals, Yield: 97%, mp ¼218–221 ^ꢁC; ¹H NMR d

(300 MHz, CDCl₃) 8.40 (s, 2H, 2 HC ¼ N), 8.06 (d, 4H, J ¼ 8.40 Hz,

2 H-2⁰and 2 H-6⁰), 7.99 (d, 2H, J ¼ 9.00 Hz, 2 H-6), 7.93 (dd, 2H,

J ¼ 2.70 and 1.20 Hz, 2 H-1), 7.90 (d, 4H, J¼ 8.40 Hz, 2 H-3⁰and 2 H-

5⁰), 7.31 (d, 2H, J ¼ 2.70 Hz, 2 H-9), 7.09 (dd, 2H, J ¼ 9.00 and

2.70 Hz, 2 H-7), 6.98 (dd, 2H, J ¼ 4.05 and 1.20 Hz, 2 H-3), 6.93 (dd,

2H, J ¼ 4.05 and 2.70 Hz, 2 H-2), 4.00 (s, 6H, 2 CH₃O), 3.73 (t, 4H,

J ¼ 6.90 Hz, 2 CH₂), 2.72–2.43 (m, 8H, 4 CH₂pip.), 2.53 (t, 4H,

J ¼ 6.90 Hz, 2 CH₂), 1.98 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂).

Bis{N-[4-(pyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-aminopro-

pyl}piperazine (9c)

Bis{N-[4–(9-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-

aminopropyl}methylamine (9 h)

Pale-yellow crystals, Yield: 78%, mp ¼177–180 ^ꢁC; ¹H NMR d

(300 MHz, CDCl₃) 8.41 (s, 2H, 2 HC ¼ N), 8.07 (d, 4H, J ¼ 8.40 Hz,

2 H-2⁰and 2 H-6⁰), 8.04 (dd, 2H, J ¼ 8.10 and 1.20 Hz, 2 H-6), 7.97

(dd, 2H, J ¼ 2.70 and 1.25 Hz, 2 H-1), 7.91 (d, 4H, J ¼ 8.40 Hz, 2 H-3⁰

and 2 H-5⁰), 7.90 (dd, 2H, J ¼ 8.10 and 1.20 Hz, 2 H-9), 7.55 (ddd,

2H, J ¼ 8.10, 8.10 and 1.20 Hz, 2 H-7), 7.49 (ddd, 2H, J ¼ 8.10, 8.10

and 1.20 Hz, 2 H-8), 7.01 (dd, 2H, J ¼ 3.90 and 1.25 Hz, 2 H-3), 6.93

(dd, 2H, J ¼ 3.90 and 2.70 Hz, 2 H-2), 3.73 (t, 4H, J ¼ 6.90 Hz, 2 CH₂),

2.78–2.50 (m, 8H, 4 CH₂pip.), 2.54 (t, 4H, J ¼ 6.90 Hz, 2 CH₂), 1.99

(qt, 4H, J ¼ 6.90 Hz, 2 CH₂).

Yellow crystals, Yield: 96%, mp ¼62–64 ^ꢁC; ¹H NMR d (300 MHz,

CDCl₃) 8.82 (dd, 2H, J ¼ 2.75 and 1.45 Hz, 2 H-1), 8.41 (s, 2H, 2

HC ¼ N), 8.03 (d, 4H, J ¼ 8.25 Hz, 2 H-2⁰and 2 H-6⁰), 7.90 (d, 4H,

J ¼ 8.25 Hz, 2 H-3⁰and 2 H-5⁰), 7.66 (dd, 2H, J ¼ 8.15 and 1.20 Hz,

2 H-8), 7.36 (t, 2H, J ¼ 8.15 Hz, 2 H-7), 7.05 (dd, 2H, J ¼ 8.15 and

1.20 Hz, 2 H-6), 6.98 (dd, 2H, J ¼ 4.10 and 1.45 Hz, 2 H-3), 6.83 (dd,

2H, J ¼ 4.10 and 2.75 Hz, 2 H-2), 4.09 (s, 6H, 2 CH₃O), 3.73 (t, 4H,

J ¼ 6.90 Hz, 2 CH₂), 2.51 (t, 4H, J ¼ 6.90 Hz, 2 CH₂), 2.31 (s, 3H, CH₃),

1.96 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂).

Bis{N-[4–(7-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-

Bis{N-[4–(9-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-

aminopropyl}methylamine (9d)

aminopropyl}piperazine (9i)

Yellow crystals, Yield: 96%, mp ¼67–69 ^ꢁC; ¹H NMR d (300 MHz, Pale-yellow crystals, Yield: 94%, mp ¼79–82 ^ꢁC; ¹H NMR

d

CDCl₃) 8.41 (s, 2H, 2 HC ¼ N), 8.03 (d, 4H, J ¼ 8.10 Hz, 2 H-2⁰and (300 MHz, CDCl₃) 8.87 (dd, 2H, J ¼ 2.70 and 1.35 Hz, 2 H-1), 8.40

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551

(s, 2H, 2 HC ¼ N), 8.05 (d, 4H, J ¼ 8.25 Hz, 2 H-2⁰and 2 H-6⁰), 7.90 2 H-1), 7.98–7.90 (m, 4H, 2 H-6⁰and 2 H-9), 7.63–7.46 (m, 4H, 2 H-5⁰,

(d, 4H, J ¼ 8.25 Hz, 2 H-3⁰and 2 H-5⁰), 7.69 (dd, 2H, J ¼ 8.10 and

2 H-7 and 2 H-8), 7.03–7.01 (m, 2H, 2 H-3), 6.93–6.91 (m, 2H, 2 H-2),

1.20 Hz, 2 H-8), 7.40 (t, 2H, J ¼ 8.10 Hz, 2 H-7), 7.09 (dd, 2H, J ¼ 8.10

and 1.20 Hz, 2 H-6), 7.00 (dd, 2H, J ¼ 4.20 and 1.35 Hz, 2 H-3), 6.87

(dd, 2H, J ¼ 4.20 and 2.70 Hz, 2 H-2), 4.12 (s, 6H, 2 CH₃O), 3.72 (t,

4H, J ¼ 6.90 Hz, 2 CH₂), 2.68–2.44 (m, 8H, 4 CH₂pip.), 2.52 (t, 4H,

J ¼ 6.90 Hz, 2 CH₂), 1.98 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂).

3.71 (t, 4H, J ¼ 6.90 Hz, 2 CH₂), 2.65–2.42 (m, 8H, 4 CH₂pip.), 2.48

(t, 4H, J ¼ 6.90 Hz, 2 CH₂), 1.95 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂).

Bis{N-[3–(8-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-

aminopropyl}methylamine (9o)

Yellow oil, Yield: 98%; ¹H NMR d (300 MHz, CDCl₃) 8.41 (s, 2H, 2

HC ¼ N), 8.29 (dd, 2H, J ¼ 1.50 and 1.50 Hz, 2 H-2⁰), 8.03 (ddd, 2H,

J ¼ 7.35, 1.50 and 1.50 Hz, 2 H-4⁰), 7.97 (d, 2H, J ¼ 9.00 Hz, 2 H-6),

7.93 (ddd, 2H, J ¼ 7.35, 1.50 and 1.50 Hz, 2 H-6⁰), 7.89 (dd, 2H,

J ¼ 2.85 and 1.35 Hz, 2 H-1), 7.56 (t, 2H, J ¼ 7.65 Hz, 2 H-5⁰), 7.30 (d,

2H, J ¼ 2.70 Hz, 2 H-9), 7.07 (dd, 2H, J ¼ 9.00 and 2.70 Hz, 2 H-7),

6.97 (dd, 2H, J ¼ 3.90 and 1.35 Hz, 2 H-3), 6.89 (dd, 2H, J ¼ 3.90 and

2.85 Hz, 2 H-2), 3.99 (s, 6H, 2 CH₃O),3.71 (t, 4H, J ¼ 7.05 Hz, 2 NCH₂),

2.50 (t, 4H, J ¼ 7.05 Hz, 2 NCH₂), 2.30 (s, 3H, NCH₃), 1.93 (qt, 4H,

J ¼ 7.05 Hz, 2 CH₂).

Bis{N-[4–(3-ethoxypyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-ami-

nopropyl}methylamine (9j)

Yellow crystals, Yield: 97%, mp ¼37–39 ^ꢁC; ¹H NMR d (300 MHz,

CDCl₃) 8.39 (s, 2H, 2 HC ¼ N), 7.91 (dd, 2H, J ¼ 7.80 and 1.50 Hz, 2 H-

6), 7.86 (d, 4H, J ¼ 8.40 Hz, 2 H-2⁰and 2 H-6⁰), 7.82(d, 4H, J ¼ 8.40 Hz,

2 H-3⁰and 2 H-5⁰), 7.76 (d, 2H, J ¼ 3.00 Hz, 2 H-1), 7.73 (dd, 2H,

J ¼ 7.80 and 1.50 Hz, 2 H-9), 7.43 (ddd, 2H, J ¼ 7.80, 7.80 and 1.50 Hz,

2 H-7), 7.35 (ddd, 2H, J ¼ 7.80, 7.80and 1.50 Hz, 2 H-8), 6.48 (d, 2H,

J ¼ 3.00 Hz, 2 H-2), 3.92 (q, 4H, J ¼ 6.90 Hz, 2 OCH₂), 3.72 (t, 4H,

J ¼ 6.90 Hz, 2 CH₂), 2.50 (t, 4H, J ¼ 6.90 Hz, 2 CH₂), 2.29 (s, 3H, CH₃),

1.95 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂), 1.17 (t, 6H, J ¼ 6.90 Hz, 2 CH₃).

Bis{N-[3–(8-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-

aminopropyl}piperazine (9p)

Bis{N-[4–(3-ethoxypyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-ami-

nopropyl}piperazine (9k)

Orange oil, Yield: 55%; ¹H NMR d (300 MHz, CDCl₃) 8.41 (s, 2H, 2

HC ¼ N), 8.30 (dd, 2H, J ¼ 1.50 and 1.50 Hz, 2 H-2⁰), 8.05 (ddd, 2H,

J ¼ 7.40, 1.50 and 1.50 Hz, 2 H-4⁰), 8.01 (d, 2H, J ¼ 9.00 Hz, 2 H-6),

7.94 (ddd, 2H, J ¼ 7.40, 1.50 and 1.50 Hz, 2 H-6⁰), 7.91 (dd, 2H,

J ¼ 2.70 and 1.35 Hz, 2 H-1), 7.59 (t, 2H, J ¼ 7.40 Hz, 2 H-5⁰), 7.29 (d,

2H, J ¼ 2.70 Hz, 2 H-9), 7.08 (dd, 2H, J ¼ 9.00 and 2.70 Hz, 2 H-7),

6.98 (dd, 2H, J ¼ 3.90 and 1.35 Hz, 2 H-3), 6.92 (dd, 2H, J ¼ 3.90 and

2.70 Hz, 2 H-2), 4.00 (s, 6H, 2 CH₃O),3.70 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂),

2.62–2.42 (m, 8H, 4 NCH₂pip.), 2.48 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂),

1.95 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂).

Yellow oil, Yield: 97%; ¹H NMR d (300 MHz, CDCl₃) 8.38 (s, 2H, 2

HC ¼ N), 7.93 (dd, 2H, J¼ 7.80 and 1.50 Hz, 2 H-6), 7.88 (d, 4H,

J ¼ 8.40 Hz, 2 H-2⁰and 2 H-6⁰), 7.83 (d, 4H, J ¼ 8.40 Hz, 2 H-3⁰and

2 H-5⁰), 7.80 (d, 2H, J ¼ 3.00 Hz, 2 H-1), 7.78 (dd, 2H, J¼ 7.80 and

1.50 Hz, 2 H-9), 7.47 (ddd, 2H, J ¼ 7.80, 7.80 and 1.50 Hz, 2 H-7),

7.41 (ddd, 2H, J ¼ 7.80, 7.80 and 1.50 Hz, 2 H-8), 6.53 (d, 2H,

J ¼ 3.00 Hz, 2 H-2), 3.96 (q, 4H, J ¼ 6.90 Hz, 2 OCH₂), 3.71 (t,

4H, J ¼ 7.05 Hz, 2 CH₂), 2.68–2.37 (m, 8H, 4 CH₂pip.), 2.48 (t, 4H,

J ¼ 7.05 Hz, 2 CH₂), 1.96 (qt, 4H, J ¼ 7.05 Hz, 2 CH₂), 1.19 (t, 6H,

J ¼ 6.90 Hz, 2 CH₃).

Bis{N-[4-(indolo[1,2-a]quinoxalin-6-yl)benzylidene]-3-aminopropyl}-

methylamine (9q)

Bis{N-[4–(2-phenylpyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-ami-

nopropyl}piperazine (9 l)

Yellow crystals, Yield: 84%, mp ¼226–229 ^ꢁC; ¹H NMR d (300 MHz,

CDCl₃) 8.57 (d, 2H, J ¼ 8.00 Hz, 2 H-11), 8.53 (d, 2H, J ¼ 8.10 Hz,

2 H-1), 8.44 (s, 2H, 2 HC ¼ N), 8.15–8.08 (m, 6H, 2 H-2⁰, 2 H-6⁰and

2 H-4), 7.97–7.92 (m, 6H, 2 H-3⁰, 2 H-5⁰and 2 H-8), 7.70–7.57 (m, 4H,

2 H-2 and 2 H-3), 7.51–7.45 (m, 4H, 2 H-9 and 2 H-10), 7.26 (s, 2H,

2 H-7), 3.76 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂), 2.87–2.55 (m, 12H, 4 NCH₂

pip. and 2 NCH₂), 2.09 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂).

1

Orange crystals, Yield: 95%, mp ¼116–119 ^ꢁC; H NMR d (300 MHz,

CDCl₃) 8.43 (s, 2H, 2 HC ¼ N), 8.29 (d, 2H, J ¼ 1.50 Hz, 2 H-1), 8.11

(d, 4H, J ¼ 8.40 Hz, 2 H-2⁰and 2 H-6⁰), 8.07 (dd, 2H, J ¼ 7.80 and

1.50 Hz, 2 H-6), 7.94 (dd, 2H, J ¼ 7.80 and 1.50 Hz, 2 H-9),7.92 (d, 4H,

J ¼ 8.40 Hz, 2 H-3⁰and 2 H-5⁰), 7.74–7.70 (m, 4H, 2 H-2” and 2 H-6”),

7.58 (ddd, 2H, J ¼ 7.80, 7.80 and 1.50 Hz, 2 H-7), 7.51 (ddd, 2H,

J ¼ 7.80, 7.80 and 1.50 Hz, 2 H-8), 7.43 (t, 4H, J ¼ 7.20 Hz, 2 H-3” and

2 H-5”), 7.36–7.30 (m, 2H, 2 H-4”), 7.25 (d, 2H, J ¼ 1.50 Hz, 2 H-3),

3.74(t, 4H, J ¼ 6.90 Hz, 2 CH₂), 2.67–2.40 (m, 8H, 4 CH₂pip.), 2.52 (t,

4H, J ¼ 6.90 Hz, 2 CH₂), 1.99 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂).

Bis{N-[(4-phenylpyrrolo[1,2-a]quinoxalin-2-yl)methylidene]-3-ami-

nopropyl}piperazine (9r)

1

Beige crystals, Yield: 60%, mp >250 ^ꢁC; H NMR d (300 MHz, CDCl₃)

8.44 (s, 2H, 2 HC ¼ N), 8.32 (d, 2H, J ¼ 1.10 Hz, 2 H-1), 8.06 (dd, 2H,

J ¼7.80 and 1.60 Hz, 2 H-6), 8.02–7.99 (m, 4H, 2 H-2⁰and 2 H-6⁰),

7.90 (dd, 2H, J ¼ 7.80 and 1.60 Hz, 2 H-9), 7.59–7.53 (m, 8H, 2 H-3⁰,

2 H-4⁰, 2 H-5⁰and 2 H-7), 7.50 (ddd, 2H, J ¼ 7.80, 7.80, 1.60 Hz, 2 H-

8), 7.29 (d, 2H, J ¼ 1.10 Hz, 2 H-3), 3.67 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂),

2.69–2.43 (m, 8H, 4 NCH₂pip.), 2.47 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂),

1.94 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂).

Bis{N-[3-(pyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-aminopro-

pyl}methylamine (9m)

Yellow oil, Yield: 97%; ¹H NMR d (300 MHz, CDCl₃) 8.43 (s, 2H, 2

HC ¼ N), 8.30 (t, 2H, J ¼ 1.50 Hz, 2 H-2⁰), 8.09–8.03 (m, 4H, 2 H-6 and

2 H-4⁰), 8.02 (dd, 2H, J ¼ 2.70 and 1.35 Hz, 2 H-1), 7.93–7.86 (m, 4H,

2 H-6⁰and 2 H-9), 7.63–7.44 (m, 4H, 2 H-5⁰, 2 H-7 and 2 H-8),7.03 (dd,

2H, J ¼ 3.90 and 1.35 Hz, 2 H-3), 6.90 (dd, 2H, J ¼ 3.90 and 2.70 Hz,

2 H-2), 3.71 (t, 4H, J ¼ 6.90 Hz, 2 CH₂), 2.47 (t, 4H, J ¼ 6.90 Hz, 2 CH₂),

2.29 (s, 3H, CH₃), 1.93 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂).

General procedure for tris{N-[(pyrrolo[1,2-a]quinoxalin-4-

yl)benzylidene]-3-aminopropyl}amines 9s-t

Bis{N-[3-(pyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-aminopro-

pyl}piperazine (9n)

To a solution of triamine (1.8 mmol) in ethanol (15 mL) was added

(pyrrolo[1,2-a]quinoxalin-4-yl)benzaldehyde 8i–j (5.4 mmol). The

reaction mixture was then heated under reflux for 5 h and then

Yellow oil, Yield: 98%; ¹H NMR d (300 MHz, CDCl₃) 8.42 (s, 2H, 2

HC ¼ N), 8.32 (s, 2H, 2 H-2⁰), 8.09–8.03 (m, 6H, 2 H-6, 2 H-4⁰and evaporated to dryness under reduced pressure. After cooling, the

552

J. GUILLON ET AL.

residue was extracted with dichloromethane (40 mL). The organic over sodium sulfate and evaporated to dryness. The residue were

layer was dried over sodium sulfate and evaporated to dryness. then purified by column chromatography on silica gel using

dichloromethane/methanol (90/10, v/v) as eluent to give the pure

Products were then used without further purification.

product 1.

Tris{N-[4–(7-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-

aminopropyl}amine (9s)

Bis{N-[4-(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-aminopropyl}-

amine (1a)

Orange oil, Yield: 82%; ¹H NMR d (300 MHz, CDCl₃) 8.42 (s, 2H, 3

HC ¼ N), 7.99 (d, 6H, J ¼ 8.40 Hz, 3 H-2⁰and 3 H-6⁰), 7.88 (d, 6H,

J ¼ 8.40 Hz, 3 H-3⁰and 3 H-5⁰), 7.86 (dd, 3H, J ¼ 2.70 and 1.20 Hz,

3 H-1), 7.73 (d, 3H, J ¼ 9.00 Hz, 3 H-9), 7.45 (d, 3H, J ¼ 2.80 Hz, 3 H-

6), 7.11 (dd, 3H, J ¼ 9.00 and 2.80 Hz, 3 H-8), 6.90 (dd, 3H, J ¼ 4.05

and 1.20 Hz, 3 H-3), 6.80 (dd, 3H, J ¼ 4.05 and 2.70 Hz, 3 H-2), 3.91

(s, 9H, 3 CH₃O), 3.76 (t, 6H, J ¼ 6.90 Hz, 3 CH₂), 2.64 (t, 6H,

J ¼ 6.90 Hz, 3NCH₂), 1.96 (qt, 6H, J ¼ 6.90 Hz, 3 CH₂).

Yellow oil, Yield: 91%; ¹H NMR d (300 MHz, CDCl₃) 8.02 (dd, 2H,

J ¼ 7.80 and 1.50 Hz, 2 H-6), 7.96 (d, 4H, J ¼ 8.20 Hz, 2 H-2⁰and 2 H-

6⁰), 7.93 (dd, 2H, J ¼ 2.70 and 1.20 Hz, 2 H-1), 7.82 (dd, 2H, J ¼ 7.80

and 1.50 Hz, 2 H-9), 7.48 (d, 4H, J ¼ 8.20 Hz, 2 H-3⁰and 2 H-5⁰),

7.47–7.39 (m, 4H, 2 H-7 and 2 H-8), 6.98 (dd, 2H, J ¼ 3.90 and

1.20 Hz, 2 H-3), 6.86 (dd, 2H, J ¼ 3.90 and 2.70 Hz, 2 H-2), 3.89 (s,

4H,

2

NCH₂), 3.73 (t, 4H, J ¼ 6.90 Hz,

2 NCH₂), 2.71(t, 4H,

J ¼ 6.90 Hz, 2 NCH₂), 1.84 (bs, 3H, 3 NH), 1.77 (qt, 4H, J ¼ 6.90 Hz, 2

CH₂) ¹³C NMR d (100 MHz, CDCl₃) 155.3 (C-4), 141.8 (C-3a), 139.0

(C-5a), 138.7 (C-4⁰), 137.6 (C-1⁰), 131.5 (C-8), 130.3 (C-2⁰and C-6⁰),

130.1 (C-3⁰and C-5⁰), 128.8 (C-7), 128.5 (C-9a), 126.6 (C-6), 116.0

(C-1), 115.4 (C-2), 115.0 (C-9), 110.0 (C-3), 54.5 (NCH₂), 50.0 (NCH₂),

48.9 (NCH₂), 27.7 (CH₂). MALDI-TOF MS m/z [M þ H]^þCalcd for

C₄₂H₄₂N₇: 644.350, Found: 644.342.

Tris{N-[3–(8-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzylidene]-3-

aminopropyl}amine (9t)

Orange oil, Yield: 71%; ¹H NMR d (300 MHz, CDCl₃) 8.40 (s, 3H, 3

HC ¼ N), 8.27 (dd, 3H, J ¼ 1.50 and 1.50 Hz, 3 H-2⁰), 7.99 (ddd, 3H,

J ¼ 7.80, 1.50 and 1.50 Hz, 3 H-4⁰), 7.98(d, 3H, J ¼ 9.00 Hz, 3H-6),

7.92 (ddd, 3H, J ¼ 7.80, 1.50 and 1.50 Hz, 3H-6⁰), 7.86 (dd, 3H,

J ¼ 2.85 and 1.35 Hz, 3 H-1), 7.52 (t, 3H, J ¼ 7.80 Hz, 3 H-5⁰), 7.25 (d,

3H, J ¼ 2.70 Hz, 3 H-9), 7.04 (dd, 3H, J ¼ 9.00 and 2.70 Hz, 3 H-7),

6.94 (dd, 3H, J ¼ 4.05 and 1.35 Hz, 3 H-3), 6.87 (dd, 3H, J ¼ 4.05 and

2.85 Hz, 3 H-2), 3.98 (s, 9H, 3 CH₃O),3.71 (t, 6H, J ¼ 6.90 Hz, 3 NCH₂),

2.61 (t, 6H, J ¼ 6.90 Hz, 3 NCH₂), 1.92 (qt, 6H, J ¼ 6.90 Hz, 3 CH₂).

Bis{N-[4-(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-aminopropyl}me-

thylamine (1b)

Orange oil, Yield: 94%; ¹H NMR d (300 MHz, CDCl₃) 8.00 (dd, 2H,

J ¼ 8.00 and 1.35 Hz, 2 H-6), 7.96 (d, 4H, J ¼ 8.40 Hz, 2 H-2⁰and 2 H-

6⁰), 7.95 (dd, 2H, J ¼ 2.70 and 1.30 Hz, 2 H-1), 7.83 (dd, 2H, J ¼ 8.00

and 1.35 Hz, 2 H-9), 7.49 (d, 4H, J ¼ 8.40 Hz, 2 H-3⁰and 2 H-5⁰),

7.47–7.39 (m, 4H, 2 H-7 and 2 H-8), 6.97 (dd, 2H, J ¼ 3.90 and

1.30 Hz, 2 H-3), 6.86 (dd, 2H, J ¼ 3.90 and 2.70 Hz, 2 H-2), 3.88 (s,

4H, 2 NCH₂), 2.71 (t, 4H, J ¼ 7.10 Hz, 2 NCH₂), 2.43 (t, 4H,

J ¼ 7.10 Hz, 2 NCH₂), 2.24 (s, 3H, NCH₃), 1.73 (qt, 4H, J ¼ 7.10 Hz, 2

CH₂). ¹³C NMR d (100 MHz, CDCl₃) 155.2 (C-4), 140.2 (C-3a), 139.3

(C-5a), 139.2 (C-4⁰), 137.5 (C-1⁰), 131.5 (C-8), 130.4 (C-2⁰and C-6⁰),

130.3 (C-3⁰and C-5⁰), 128.8 (C-7), 128.4 (C-9a), 126.6 (C-6), 116.0

(C-1), 115.4 (C-2), 115.0 (C-9), 110.0 (C-3), 57.3 (NCH₂), 54.1 (NCH₂),

48.6 (NCH₂), 43.4 (NCH₃), 27.0 (CH₂). MALDI-TOF MS m/z

[M þ H]^þCalcd for C₄₃H₄₄N₇: 658.366, Found: 658.362.

N, N⁰- [oxybis(2, 1-ethanediyloxy-2, 1-ethanediyl)] bis-3–(8-

methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzylimine 9u

To a solution of 1,11-diamino-3,6,9-trioxaundecane (0.56 mmol) in

ethanol (12 mL) was added 3-(pyrrolo[1,2-a]quinoxalin-4-yl)benzal-

dehyde 8j (1.12 mmol). The reaction mixture was then heated

under reflux for 5 h and then evaporated to dryness under

reduced pressure. After cooling, the residue was extracted with

dichloromethane (35 mL). The organic layer was dried over sodium

sulfate and evaporated to dryness. Product 9u was then used

without further purification. Yellow oil, Yield: 97%; ¹H NMR d

(300 MHz, CDCl₃) 8.39 (s, 2H, 2 HC ¼ N), 8.29 (dd, 2H, J ¼ 1.50 and

1.50 Hz, 2 H-2⁰), 8.05–7.87 (m, H, 2 H-4⁰, 2 H-6, 2 H-6⁰and 2 H-1),

7.59–7.55 (m, 2H, 2 H-5⁰),7.27 (d, 2H, J ¼ 2.70 Hz, 2 H-9), 7.06 (dd,

2H, J ¼ 9.00 and 2.70 Hz, 2 H-7), 6.96 (m, 2H, 2 H-3), 6.88 (m, 2H,

2 H-2), 3.95 (s, 6H, 2 CH₃O), 3.81–3.79 (m, 8H, 4OCH₂), 3.64–3.62

(m, 8H, 4 OCH₂).

Bis{N-[4-(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-aminopropyl}

piperazine (1c)

Colorless oil, Yield: 61%; ¹H NMR d (300 MHz, CDCl₃) 8.04 (dd, 2H,

J ¼ 8.25 and 1.35 Hz, 2 H-6), 8.00 (dd, 2H, J ¼ 2.70 and 1.20 Hz, 2 H-

1), 7.98 (d, 4H, J ¼ 8.40 Hz, 2 H-2⁰and 2 H-6⁰), 7.89 (dd, 2H, J ¼ 8.25

and 1.35 Hz, 2 H-9), 7.53 (ddd, 2H, J ¼ 8.25, 8.25 and 1.35 Hz, 2 H-7),

7.50 (d, 4H, J ¼ 8.40 Hz, 2 H-3⁰and 2 H-5⁰), 7.45 (ddd, 2H, J ¼ 8.25,

8.25 and 1.35 Hz, 2 H-8),7.01 (dd, 2H, J ¼ 3.90 and 1.20 Hz, 2 H-3),

6.91 (dd, 2H, J ¼ 3.90 and 2.70 Hz, 2 H-2), 3.91 (s, 4H, 2 NCH₂), 2.74

(t, 4H, J ¼ 6.90 Hz, 2 NCH₂), 2.58–2.39 (m, 8H, 4 NCH₂pip.), 2.44 (t,

4H, J ¼ 6.90 Hz, 2 NCH₂), 1.76 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂). ¹³C NMR

d (100 MHz, CDCl₃) 155.2 (C-4), 140.8 (C-3a), 139.3 (C-5a), 139.2 (C-

4⁰), 137.6 (C-1⁰), 131.6 (C-8), 130.3 (C-2⁰and C-6⁰), 130.2 (C-3⁰and

C-5⁰), 128.9 (C-7), 128.5 (C-9a), 126.7 (C-6), 116.1 (C-1), 115.4 (C-2),

115.0 (C-9), 110.0 (C-3), 58.5 (NCH₂), 54.4 (NCH₂), 54.2 (NCH₂), 49.5

(NCH₂), 26.5 (CH₂). MALDI-TOF MS m/z [M þ H]^þCalcd for

C₄₆H₄₉N₈: 713.408, Found: 713.412.

General procedure for bis{N-[(pyrrolo[1,2-a]quinoxalin-4-

yl)benzyl]-3-aminopropyl}methylamine and bis{N-[(pyrrolo[1,2-

a]quinoxalin-4-yl)benzyl]-3-aminopropyl}piperazine 1a-p, bis{N-

[4-(indolo[1,2-a]quinoxalin-6-yl)benzyl]-3-aminopropyl}

piperazine 1q and bis{N-[(4-phenylpyrrolo[1,2-a]quinoxalin-2-

yl)methyl]-3-aminopropyl}piperazine 1r and tris{N-[(pyrrolo[1,2-

a]quinoxalin-4-yl)benzyl]-3-aminopropyl}amine 1s-t and N,N⁰-

[oxybis(2,1-ethanediyloxy-2,1-ethanediyl)]bis-3–(8-

methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzylamine 1u

To a solution of compound 9 (1.26 mmol) in methanol (40 mL) was

added portion-wise at 0 ^ꢁC sodium borohydride (10.1 mmol; 8

equiv.). The reaction mixture was then heated under reflux for 4 h

Bis{N-[4–(7-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-amino-

and then evaporated to dryness under reduced pressure. After propyl}methylamine (1d)

cooling, the residue was triturated in water and extracted with Pale-yellow crystals, Yield: 88%, mp ¼65–67 ^ꢁC; ¹H NMR

d

dichloromethane (85 mL). The organic layer was separated, dried (300 MHz, CDCl₃) 7.95 (d, 4H, J ¼ 8.10 Hz, 2 H-2⁰and 2 H-6⁰), 7.90

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

553

(dd, 2H, J ¼ 2.70 and 1.20 Hz, 2 H-1), 7.76 (d, 2H, J ¼ 9.00 Hz, 2 H-9), (C-4), 143.3 (C-3a), 138.6 (C-5a), 132.7 (C-6), 132.0 (C-4⁰), 130.0 (C-2⁰

7.49 (d, 2H, J ¼ 2.70 Hz, 2 H-6), 7.48 (d, 4H, J ¼ 8.10 Hz, 2 H-3⁰and

and C-6⁰), 129.6 (C-3⁰and C-5⁰), 126.2 (C-1⁰), 126.7 (C-9a), 115.5 (C-

7), 115.4 (C-1), 114.2 (C-2), 109.5 (C-3), 98.9 (C-9), 58.4 (NCH₂), 57.2

2 H-5⁰), 7.11 (dd, 2H, J ¼ 9.00 and 2.70 Hz, 2 H-8), 6.96 (dd, 2H,

J ¼ 4.05 and 1.20 Hz, 2 H-3), 6.84 (dd, 2H, J ¼ 4.05 and 2.70 Hz, 2 H-

(OCH₃), 55.1 (NCH₂), 54.7 (NCH₂), 49.4 (NCH₂), 28.3 (CH₂). MALDI-

TOF MS m/z [M þ H]^þCalcd for C₄₈H₅₃N₈O₂: 773.429, Found:

2), 3.91 (s, 6H, 2 CH₃O), 3.89 (s, 4H, 2 NCH₂), 2.72 (t, 4H,

J ¼ 7.05 Hz, 2 NCH₂), 2.43 (t, 4H, J ¼ 7.05 Hz, 2 NCH₂), 1.73 (qt, 4H,

773.446.

J ¼ 7.05 Hz, 2 CH₂). ¹³C NMR d (100 MHz, CDCl₃) 158.5 (C-7), 155.9

(C-4), 143.7 (C-5a), 138.7 (C-3a), 138.5 (C-4⁰), 130.1 (C-2⁰and C-6⁰),

129.6 (C-3⁰and C-5⁰), 126.4 (C-1⁰), 122.8 (C-9a), 117.9 (C-9), 115.9

Bis{N-[4–(9-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-amino-

(C-8), 115.6 (C-1), 115.0 (C-2), 112.6 (C-3), 109.7 (C-6), 57.6 (NCH₂),

propyl}methylamine (1 h)

57.1 (OCH₃), 55.2 (NCH₂), 49.5 (NCH₂), 43.7 (NCH₃), 29.0 (CH₂).

Orange-pale oil, Yield: 92%; ¹H NMR d (300 MHz, CDCl₃) 8.83 (dd,

MALDI-TOF MS m/z [M þ H]^þCalcd for C₄₅H₄₈N₇O₂: 718.387,

2H, J ¼ 2.70 and 1.35 Hz, 2 H-1), 7.95 (d, 4H, J ¼ 8.10 Hz, 2 H-2⁰and

Found: 718.399.

2 H-6⁰), 7.66 (dd, 2H, J ¼ 8.10 and 1.20 Hz, 2 H-8), 7.48 (d, 4H,

J ¼ 8.10 Hz, 2 H-3⁰and 2 H-5⁰), 7.37 (t, 2H, J ¼ 8.10 Hz, 2 H-7), 7.04

Bis{N-[4–(7-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-amino-

propyl}piperazine (1e)

(dd, 2H, J ¼ 8.10 and 1.20 Hz, 2 H-6), 7.00 (dd, 2H, J ¼ 4.10 and

1.35 Hz, 2 H-3), 6.82 (dd, 2H, J ¼ 4.10 and 2.70 Hz, 2 H-2), 4.08 (s,

6H, 2 CH₃O), 3.89 (s, 4H, 2 NCH₂), 2.71 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂),

2.43 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂), 2.25 (s, 3H, NCH₃), 1.73 (qt, 4H,

J ¼ 6.90 Hz, 2 CH₂). ¹³C NMR d (100 MHz, CDCl₃) 155.9 (C-9), 151.1

(C-4), 143.5 (C-3a), 139.8 (C-5a), 138.5 (C-4⁰), 130.1 (C-2⁰and C-6⁰),

129.6 (C-3⁰and C-5⁰), 127.3 (C-9a), 125.7 (C-7), 123.9 (C-6), 123.7

(C-1), 119.8 (C-1⁰), 114.3 (C-2), 110.2 (C-8), 109.4 (C-3), 57.6 (OCH₃),

57.5 (NCH₂), 55.2 (NCH₂), 49.3 (NCH₂), 43.7 (NCH₃), 28.9 (CH₂).

MALDI-TOF MS m/z [M þ H]^þCalcd for C₄₅H₄₈N₇O₂: 718.387,

Found: 718.416.

Pale-yellow crystals, Yield: 93%, mp ¼51–53 ^ꢁC; ¹H NMR

d

(300 MHz, CDCl₃) 7.97 (d, 4H, J ¼ 8.20 Hz, 2 H-2⁰and 2 H-6⁰), 7.93

(dd, 2H, J¼ 2.75 and 1.25 Hz, 2 H-1), 7.79 (d, 2H, J ¼ 9.00 Hz, 2 H-9),

7.52 (d, 2H, J ¼ 2.70 Hz, 2 H-6), 7.49 (d, 4H, J ¼ 8.20 Hz, 2 H-3⁰and

2 H-5⁰), 7.13 (dd, 2H, J¼ 9.00 and 2.70 Hz, 2 H-8), 6.98 (dd, 2H,

J ¼ 3.90 and 1.25 Hz, 2 H-3), 6.87 (dd, 2H, J ¼ 3.90 and 2.75 Hz, 2 H-

2), 3.93 (s, 6H, 2 CH₃O), 3.89 (s, 4H, 2 NCH₂), 2.71 (t, 4H,

J ¼ 6.90 Hz, 2 NCH₂), 2.60–2.35 (m, 8H, 4 NCH₂pip.), 2.44 (t, 4H,

J ¼ 6.90 Hz, 2 NCH₂), 1.76 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂). ¹³C NMR d

(100 MHz, CDCl₃) 158.5 (C-7), 155.8 (C-4), 142.8 (C-5a), 138.8 (C-3a),

138.7 (C-4⁰), 130.1 (C-2⁰and C-6⁰), 129.8 (C-3⁰and C-5⁰), 126.4 (C-

1⁰), 122.8 (C-9a), 118.0 (C-9), 116.0 (C-8), 115.7 (C-1), 115.1 (C-2),

112.7 (C-3), 109.7 (C-6), 58.5 (NCH₂), 57.1 (OCH₃), 54.8 (NCH₂), 54.6

(NCH₂), 49.5 (NCH₂), 27.7 (CH₂). MALDI-TOF MS m/z [M þ H]^þCalcd

for C₄₈H₅₃N₈O₂: 773.429, Found: 773.428.

Bis{N-[4–(9-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-amino-

propyl}piperazine (1i)

Pale-yellow crystals, Yield: 94%, mp ¼66–68 ^ꢁC; ¹H NMR

d

(300 MHz, CDCl₃) 8.84 (dd, 2H, J ¼ 2.70 and 1.20 Hz, 2 H-1), 7.96 (d,

4H, J ¼ 8.10 Hz, 2 H-2⁰and 2 H-6⁰), 7.67 (dd, 2H, J ¼ 8.10 and

1.20 Hz, 2 H-8), 7.48 (d, 4H, J ¼ 8.10 Hz, 2 H-3⁰and 2 H-5⁰), 7.37 (t,

2H, J ¼ 8.10 Hz, 2 H-7), 7.05 (dd, 2H, J ¼ 8.10 and 1.20 Hz, 2 H-6),

7.00 (dd, 2H, J ¼ 4.00 and 1.20 Hz, 2 H-3), 6.85 (dd, 2H, J ¼ 4.00 and

Bis{N-[4–(8-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-amino-

propyl}methylamine (1f)

Yellow crystals, Yield: 71%, mp ¼71–73 ^ꢁC; ¹H NMR d (300 MHz,

CDCl₃) 7.95 (d, 2H, J ¼ 9.00 Hz, 2 H-6), 7.93 (d, 4H, J ¼ 8.10 Hz, 2 H- 2.70 Hz, 2 H-2), 4.09 (s, 6H, 2 CH₃O), 3.89 (s, 4H, 2 NCH₂), 2.71 (t,

2⁰and 2 H-6⁰), 7.86 (dd, 2H, J ¼ 2.70 and 1.20 Hz, 2 H-1), 7.47 (d,

4H, J ¼ 8.10 Hz, 2 H-3⁰and 2 H-5⁰), 7.26 (d, 2H, J ¼ 2.70 Hz, 2 H-9),

7.04 (dd, 2H, J ¼ 9.00 and 2.70 Hz, 2 H-7), 6.95 (dd, 2H, J ¼ 3.90 and

1.20 Hz, 2 H-3), 6.87 (dd, 2H, J ¼ 3.90 and 2.70 Hz, 2 H-2), 3.96 (s,

6H, 2 CH₃O), 3.88 (s, 4H, 2 NCH₂), 2.71 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂),

2.43 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂), 2.25 (s, 3H, NCH₃), 1.73 (qt, 4H,

J ¼ 6.90 Hz, 2 CH₂). ¹³C NMR d (100 MHz, CDCl₃) 160.4 (C-8), 153.0

(C-4), 143.3 (C-3a), 138.6 (C-5a), 132.7 (C-6), 132.0 (C-4⁰), 130.0 (C-2⁰

and C-6⁰), 129.6 (C-3⁰and C-5⁰), 126.2 (C-1⁰), 126.6 (C-9a), 115.4 (C-

7), 115.3 (C-1), 114.1 (C-2), 109.5 (C-3), 98.8 (C-9), 57.6 (NCH₂), 57.1

(OCH₃), 55.2 (NCH₂), 49.3 (NCH₂), 43.7 (NCH₃), 29.0 (CH₂). MALDI-

TOF MS m/z [M þ H]^þCalcd for C₄₅H₄₈N₇O₂: 718.387, Found:

718.399.

4H, J ¼ 6.90 Hz, 2 NCH₂), 2.57–2.31 (m, 8H, 4 NCH₂pip.), 2.43 (t,

4H, J ¼ 6.90 Hz, 2 NCH₂), 1.74 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂). ¹³C NMR

d (100 MHz, CDCl₃) 155.9 (C-9), 151.1 (C-4), 143.5 (C-3a), 139.8 (C-

5a), 138.5 (C-4⁰), 130.1 (C-2⁰and C-6⁰), 129.6 (C-3⁰and C-5⁰), 127.3

(C-9a), 125.7 (C-7), 123.7 (C-6), 123.5 (C-1), 119.8 (C-1⁰), 114.3 (C-2),

110.2 (C-8), 109.4 (C-3), 58.4 (NCH₂), 57.6 (OCH₃), 55.1 (NCH₂), 54.7

(NCH₂), 49.4 (NCH₂), 28.3 (CH₂). MALDI-TOF MS m/z [M þ H]^þCalcd

for C₄₈H₅₃N₈O₂: 773.429, Found: 773.446.

Bis{N-[4–(3-ethoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-amino-

propyl}methylamine (1j)

Yellow oil, Yield: 85%; ¹H NMR d (300 MHz, CDCl₃) 7.91 (dd, 2H,

J ¼ 7.80 and 1.50 Hz, 2 H-6), 7.79–7.74 (m, 8H, 2 H-2⁰, 2 H-6⁰, 2 H-1

and 2 H-9), 7.43–7.34 (m, 4H, 2 H-3⁰, 2 H-5⁰, 2 H-7 and 2 H-8), 6.48

(d, 2H, J ¼ 3.00 Hz, 2 H-2), 3.92 (q, 4H, J ¼ 6.90 Hz, 2 OCH₂), 3.89 (s,

4H, 2 NCH₂), 2.68 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂), 2.43 (t, 4H,

J ¼ 6.90 Hz, 2 NCH₂), 2.24 (s, 3H, NCH₃), 1.72 (qt, 4H, J ¼ 6.90 Hz, 2

CH₂), 1.16 (t, 6H, J ¼ 6.90 Hz, 2 CH₃). ¹³C NMR d (100 MHz, CDCl₃)

156.2(C-4), 145.7 (C-3a), 143.6 (C-5a), 138.2 (C-4⁰), 137.5 (C-1⁰),

131.1 (C-6), 130.9 (C-2⁰and C-6⁰), 128.5 (C-3⁰and C-5⁰), 128.3 (C-8),

128.0 (C-9a), 126.4 (C-7), 114.0 (C-1), 113.4 (C-3),113.1 (C-9), 102.2

(C-2), 68.7 (OCH₂), 57.6 (NCH₂), 55.2 (NCH₂), 49.0 (NCH₂), 43.6

(NCH₃), 28.8 (CH₂), 16.1 (CH₃). MALDI-TOF MS m/z [M þ H]^þCalcd

Bis{N-[4–(8-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-amino-

propyl}piperazine (1 g)

Yellow oil, Yield: 97%; ¹H NMR d (300 MHz, CDCl₃) 7.96 (d, 2H,

J ¼ 9.00 Hz, 2 H-6), 7.94 (d, 4H, J ¼ 8.10 Hz, 2 H-2⁰and 2 H-6⁰), 7.89

(dd, 2H, J ¼ 2.75 and 1.20 Hz, 2 H-1), 7.47 (d, 4H, J ¼ 8.10 Hz, 2 H-3⁰

and 2 H-5⁰), 7.29 (d, 2H, J ¼ 2.70 Hz, 2 H-9), 7.06 (dd, 2H, J ¼ 9.00

and 2.70 Hz, 2 H-7), 6.97 (dd, 2H, J ¼ 3.90 and 1.20 Hz, 2 H-3), 6.89

(dd, 2H, J ¼ 3.90 and 2.75 Hz, 2 H-2), 3.97 (s, 6H, 2 CH₃O), 3.88 (s,

4H, 2 NCH₂), 2.71 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂), 2.57–2.34 (m, 8H, 4

NCH₂pip.), 2.43 (t, 4H, J ¼ 6.90 Hz,

2 NCH₂), 1.74 (qt, 4H,

J ¼ 6.90 Hz, 2 CH₂). ¹³C NMR d (100 MHz, CDCl₃) 160.4 (C-8), 153.0 for C₄₇H₅₂N₇O₂: 746.418, Found: 746.472.

554

J. GUILLON ET AL.

Bis{N-[4–(3-ethoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-amino-

propyl}piperazine (1k)

2.58–2.34 (m, 8H, 4 NCH₂pip.), 2.34 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂),

1.95 (bs, 2H, 2 NH), 1.68 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂). ¹³C NMR d

(100 MHz, CDCl₃) 155.8 (C-4), 142.2 (C-3a), 139.9 (C-5a), 137.6 (C-3⁰),

131.5 (C-8), 131.0 (C-7), 130.0 (C-2⁰), 129.7 (C-5⁰), 128.9 (C-4⁰), 128.6

(C-6⁰), 128.5 (C-1⁰), 126.8 (C-9a), 126.7 (C-6), 116.0 (C-1), 115.4 (C-2),

Yellow oil, Yield: 76%; ¹H NMR d (300 MHz, CDCl₃) 7.93 (dd, 2H,

J ¼ 7.80 and 1.50 Hz, 2 H-6), 7.80–7.75 (m, 8H, 2 H-2⁰, 2 H-6⁰, 2 H-1

and 2 H-9), 7.47 (ddd, 2H, J ¼ 7.80, 7.80 and 1.50 Hz, 2 H-7), 7.42 (d,

4H, J ¼ 8.40 Hz, 2 H-3⁰and 2 H-5⁰), 7.36 (ddd, 2H, J ¼ 7.80, 7.80 and 115.0 (C-9), 110.1 (C-3), 58.4 (NCH₂), 55.2 (NCH₂), 54.6 (NCH₂), 49.5

(NCH₂), 28.2 (CH₂). MALDI-TOF MS m/z [M þ H]^þCalcd for

1.50 Hz, 2 H-8), 6.51 (d, 2H, J ¼ 3.00 Hz, 2 H-2), 3.94 (q, 4H,

J ¼ 6.90 Hz, 2 OCH₂), 3.88 (s, 4H, 2 NCH₂), 2.68 (t, 4H, J ¼ 6.90 Hz, 2

NCH₂), 2.56–2.31 (m, 8H, 4 NCH₂pip.), 2.42 (t, 4H, J ¼ 6.90 Hz,

2 NCH₂), 1.73 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂), 1.17 (t, 6H, J ¼ 6.90 Hz, 2

CH₃). ¹³C NMR d (100 MHz, CDCl₃) 156.2 (C-4), 145.7 (C-3a), 142.6

C₄₆H₄₉N₈: 713.408, Found: 713.447.

Bis{N-[3–(8-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-amino-

(C-5a), 138.2 (C-4⁰), 137.5 (C-1⁰), 131.2 (C-6), 130.9 (C-2⁰and C-6⁰), propyl}methylamine (1o)

Yellow oil, Yield: 97%; ¹H NMR d (300 MHz, CDCl₃) 7.95 (d, 2H,

J ¼ 8.70 Hz, 2 H-6), 7.90 (dd, 2H, J ¼ 1.50 and 1.50 Hz, 2 H-2⁰),

7.87–7.83 (m, 2H, 2 H-4⁰), 7.85 (dd, 2H, J ¼ 2.70 and 1.20 Hz, 2 H-1),

7.47–7.43 (m, 4H, 2 H-5⁰and 2 H-5⁰), 7.25 (d, 2H, J ¼ 2.70 Hz, 2 H-9),

7.03 (dd, 2H, J ¼ 8.85 and 2.70 Hz, 2 H-7), 6.94 (dd, 2H, J ¼ 3.90 and

1.20 Hz, 2 H-3), 6.86 (dd, 2H, J ¼ 3.90 and 2.70 Hz, 2 H-2), 3.94 (s,

6H, 2 CH₃O),3.86 (s, 4H, 2 NCH₂), 2.68 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂),

2.39 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂), 2.20 (s, 3H, NCH₃), 1.69 (qt, 4H,

J ¼ 6.90 Hz, 2 CH₂). ¹³C NMR d (100 MHz, CDCl₃) 160.4 (C-8), 153.1

(C-4), 141.8 (C-3a), 140.0 (C-5a), 132.7 (C-6), 131.9 (C-3⁰), 130.8

(C-2⁰), 129.9 (C-5⁰), 129.7 (C-4⁰), 129.2 (C-1⁰), 128.6 (C-6⁰), 126.6

(C-9a), 115.5 (C-7), 115.4 (C-1), 114.2 (C-2), 109.5 (C-3), 98.8 (C-9),

57.5 (NCH₂), 57.1 (OCH₃), 55.2 (NCH₂), 49.3 (NCH₂), 43.6 (NCH₃),

28.6 (CH₂). MALDI-TOF MS m/z [M þ H]^þCalcd for C₄₅H₄₈N₇O₂:

718.387, Found: 718.433.

128.5 (C-3⁰and C-5⁰), 128.4 (C-8), 128.0 (C-9a), 126.4 (C-7), 114.0

(C-1), 113.5 (C-3), 113.2 (C-9), 102.3 (C-2), 68.8 (OCH₂), 58.4 (NCH₂),

55.1 (NCH₂), 54.7 (NCH₂), 49.1 (NCH₂), 28.3 (CH₂), 16.1 (CH₃).

MALDI-TOF MS m/z [M þ H]^þCalcd for C₅₀H₅₇N₈O₂: 801.460,

Found: 801.505.

Bis{N-[4–(2-phenylpyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-amino-

propyl}piperazine (1 l)

Pale-yellow crystals, Yield: 79%, mp ¼81–83 ^ꢁC; ¹H NMR

d

(300 MHz, CDCl₃) 8.27 (d, 2H, J ¼ 1.20 Hz, 2 H-1), 8.07 (dd, 2H,

J ¼ 7.80 and 1.30 Hz, 2 H-6), 8.03 (d, 4H, J ¼ 8.10 Hz, 2 H-2⁰and 2 H-

6⁰), 7.94 (dd, 2H, J ¼ 7.80 and 1.30 Hz, 2 H-9),7.71 (d, 4H,

J ¼ 7.50 Hz, 2 H-2” and 2 H-6”), 7.58–7.41 (m, 8H, 2 H-3⁰, 2 H-5⁰, 2 H-

7 and 2 H-8), 7.45 (t, 4H, J ¼ 7.80 Hz, 2 H-3” and 2 H-5”),7.35–7.30

(m, 2H, 2 H-4”), 7.26 (d, 2H, J ¼ 1.20 Hz, 2 H-3), 2.73(t, 4H,

J ¼ 6.90 Hz, 2 NCH₂), 2.65–2.38 (m, 8H, 4 NCH₂pip.), 2.45 (t, 4H,

J ¼ 6.90 Hz, 2 NCH₂), 1.76 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂). ¹³C NMR d

(100 MHz, CDCl₃) 154.6 (C-4), 146.6 (C-3a), 140.0 (C-5a), 137.6 (C-4⁰),

135.4 (C-1”), 131.7 (C-8), 131.2 (C-1⁰), 130.6 (C-3” and C-5”), 130.4

(C-2⁰and C-6⁰), 129.7 (C-7), 129.2 (C-4”), 128.6 (C-3” and C-5”),

128.1 (C-9a), 127.6 (C-3⁰and C-5⁰), 126.9 (C-6), 118.7 (C-2), 115.0

(C-9), 112.8 (C-1), 107.2 (C-3), 58.5 (NCH₂), 54.0 (NCH₂), 53.2 (NCH₂),

49.5 (NCH₃), 31.1 (CH₂). MALDI-TOF MS m/z [M þ H]^þCalcd for

C₅₈H₅₇N₈: 865.470, Found: 865.454.

Bis{N-[3–(8-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-amino-

propyl}piperazine (1p)

Yellow oil, Yield: 75%; ¹H NMR d (300 MHz, CDCl₃) 7.97 (d, 2H,

J ¼ 9.00 Hz, 2 H-6), 7.93 (dd, 2H, J ¼ 1.50 and 1.50 Hz, 2 H-2⁰), 7.88

(dd, 2H, J ¼ 2.70 and 1.20 Hz, 2 H-1),7.88–7.84 (m, 2H, 2 H-4⁰),

7.50–7.46 (m, 4H, 2 H-5⁰and 2 H-5⁰), 7.28 (d, 2H, J ¼ 2.70 Hz, 2 H-9),

7.06 (dd, 2H, J ¼ 9.00 and 2.70 Hz, 2 H-7), 6.96 (dd, 2H, J ¼ 4.00 and

1.20 Hz, 2 H-3), 6.88 (dd, 2H, J ¼ 4.00 and 2.70 Hz, 2 H-2), 3.96 (s,

6H, 2 CH₃O),3.90 (s, 4H, 2 NCH₂), 2.69 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂),

2.53–2.34 (m, 8H, 4 NCH₂pip.), 2.33 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂),

2.20 (bs, 2H, 2 NH), 1.68 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂). ¹³C NMR d

(100 MHz, CDCl₃) 160.5 (C-8), 153.2 (C-4), 142.0 (C-3a), 140.0 (C-5a),

132.7 (C-6), 132.0 (C-3⁰), 130.7 (C-2⁰), 130.0 (C-5⁰), 129.6 (C-4⁰), 129.2

(C-1⁰), 128.6 (C-6⁰), 126.7 (C-9a), 115.5 (C-7), 115.4 (C-1), 114.2 (C-2),

109.5 (C-3), 98.9 (C-9), 58.4 (NCH₂), 57.2 (OCH₃), 55.2 (NCH₂), 54.6

(NCH₂), 49.5 (NCH₂), 28.1 (CH₂). MALDI-TOF MS m/z [M þ H]^þCalcd

for C₄₈H₅₃N₈O₂: 773.429, Found: 773.464.

Bis{N-[3-(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-aminopropyl}me-

thylamine (1m)

Pale-yellow oil, Yield: 83%; ¹H NMR d (300 MHz, CDCl₃) 8.04 (dd,

J ¼ 7.80 and 1.80 Hz, 2 H-6), 7.99 (dd, 2H, J ¼ 2.70 and 1.20 Hz, 2 H-

1), 7.94 (t, 2H, J ¼ 1.50 Hz, 2 H-2⁰), 7.89–7.85 (m, 4H, 2 H-6⁰and

2 H-9), 7.55–7.42 (m, 8H, 2 H-4⁰, 2 H-5⁰, 2 H-7 and 2 H-8), 6.99 (dd,

2H, J ¼ 3.90 and 1.20 Hz, 2 H-3), 6.88 (dd, 2H, J ¼ 3.90 and 2.70 Hz,

2 H-2), 3.88 (s, 4H, 2 NCH₂), 2.70 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂), 2.40 (t,

4H, J ¼ 6.90 Hz,

2 NCH₂), 2.22 (s, 3H, NCH₃), 1.72 (qt, 4H,

J ¼ 6.90 Hz, 2 CH₂). ¹³C NMR d (100 MHz, CDCl₃) 155.8 (C-4), 142.2

(C-3a), 139.8 (C-5a), 137.6 (C-3⁰), 131.5 (C-8), 131.0 (C-7), 130.0 (C-

2⁰), 129.7 (C-5⁰), 128.8 (C-4⁰),128.6 (C-6⁰), 128.5 (C-1⁰), 126.7 (C-9a),

126.6 (C-6), 116.0 (C-1), 115.4 (C-2), 115.0 (C-9), 110.1 (C-3), 57.4

(NCH₂), 55.3 (NCH₂), 49.4 (NCH₂), 43.6 (NCH₃), 28.8 (CH₂).MALDI-

TOF MS m/z [M þ H]^þCalcd for C₄₃H₄₄N₇: 658.366, Found: 658.395.

Bis{N-[4-(indolo[1,2-a]quinoxalin-6-yl)benzyl]-3-aminopropyl}

piperazine (1q)

Yellow oil, Yield: 86%; ¹H NMR d (300 MHz, CDCl₃) 8.55 (dd, 2H,

J ¼ 8.10 and 1.20 Hz, 2 H-11), 8.51 (dd, 2H, J ¼ 8.70 and 0.90 Hz,

2 H-1), 8.09 (dd, 2H, J ¼ 8.00 and 1.65 Hz, 2 H-4), 8.00 (d, 4H,

J ¼ 8.40 Hz, 2 H-2⁰and 2 H-6⁰), 7.95 (d, 2H, J ¼ 7.80 Hz, 2 H-8),

7.67–7.60 (m, 4H,2 H-2 and 2 H-3), 7.53 (d, 4H, J ¼ 8.40 Hz, 2 H-3⁰

and 2 H-5⁰), 7.48–7.43 (m, 4H, 2 H-9 and 2 H-10), 7.27 (s, 2H, 2 H-7),

3.92 (s, 4H, 2 NCH₂), 2.75 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂), 2.63–2.41 (m,

8H, 4 NCH₂pip.), 2.46 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂), 1.96 (bs, 2H, 2

NH), 1.77 (qt, 4H, J ¼ 6.90 Hz, 2 CH₂). ¹³C NMR d (100 MHz, CDCl₃)

157.3 (C-6), 142.8 (C-6a), 138.6 (C-11a), 137.7 (C-4a), 134.4 (C-4⁰),

131.9 (C-3), 131.6 (C-1⁰), 130.5 (C-8a), 130.2 (C-2⁰and C-6⁰), 129.9

(C-3⁰and C-5⁰), 129.7 (C-2), 125.8 (C-4), 125.6 (C-1), 124.1 (C-9),

Bis{N-[3-(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-aminopropyl}piper-

azine (1n)

Pale-yellow oil, Yield: 96%; ¹H NMR d (300 MHz, CDCl₃) 8.05 (dd,

J ¼ 7.80 and 1.80 Hz, 2 H-6), 8.01 (dd, 2H, J ¼ 2.70 and 1.20 Hz, 2 H-

1), 7.96 (t, 2H, J ¼ 1.50 Hz, 2 H-2⁰), 7.92–7.86 (m, 4H, 2 H-6⁰and

2 H-9), 7.56–7.43 (m, 8H, 2 H-4⁰, 2 H-5⁰, 2 H-7 and 2 H-8), 7.00 (dd,

2H, J ¼ 3.90 and 1.20 Hz, 2 H-3), 6.90 (dd, 2H, J ¼ 3.90 and 2.70 Hz,

2 H-2), 3.91 (s, 4H, 2 NCH₂), 2.69 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂), 124.0 (C-10), 116.0 (C-8 and C-11), 103.8 (C-7), 58.5 (NCH₂), 54.6

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

555

(NCH₂), 49.5 (NCH₂), 27.6 (CH₂). MALDI-TOF MS m/z [M þ H]^þCalcd 2 H-5⁰), 7.26 (d, 2H, J ¼ 2.70 Hz, 2 H-9), 7.05 (dd, 2H, J ¼ 9.00 and

for C₅₄H₅₃N₈: 813.439, Found: 813.432.

2.70 Hz, 2 H-7), 6.95 (dd, 2H, J ¼ 3.90 and 1.20 Hz, 2 H-3), 6.87 (dd,

2H, J ¼ 3.90 and 2.70 Hz, 2 H-2), 3.95 (s, 6H, 2 CH₃O), 3.91 (s, 4H, 2

NCH₂), 3.64–3.58 (m, 12H, 6 OCH₂), 2.83 (t, 4H, J ¼ 5.40 Hz, 2

NCH₂), 2.18 (bs, 2H, 2 NH). ¹³C NMR d (100 MHz, CDCl₃) 160.5 (C-8),

153.1 (C-4), 141.3 (C-3a), 140.0 (C-5a), 132.7 (C-6), 132.0 (C-3⁰),

131.0 (C-2⁰), 129.9 (C-5⁰and C-4⁰), 129.2 (C-1⁰), 128.7 (C-6⁰), 126.7

(C-9a), 115.4 (C-7), 115.3 (C-1), 114.2 (C-2), 109.6 (C-3), 98.9 (C-9),

71.8 (OCH₂), 71.6 (OCH₂), 71.5 (OCH₂), 56.7 (OCH₃), 54.8 (NCH₂),

49.8 (NCH₂). MALDI-TOF MS m/z [M þ H]^þCalcd for C₄₆H₄₉N₆O₅:

765.376, Found: 765.457.

Bis{N-[(4-phenylpyrrolo[1,2-a]quinoxalin-2-yl)methyl]-3-aminopro-

pyl}piperazine (1r)

Pale-yellow crystals, Yield: 70%, mp ¼62–64 ^ꢁC; ¹H NMR

d

(300 MHz, CDCl₃) 8.04 (dd, 2H, J ¼ 7.80 and 1.50 Hz, 2 H-6),

8.03–7.97 (m, 4H, 2 2 H-2⁰and 2 H-6⁰), 7.98 (d, 2H, J ¼ 1.10 Hz, 2 H-

1), 7.85 (dd, 2H, J ¼ 7.80 and 1.50 Hz, 2 H-9), 7.57–7.53 (m, 8H,

2 H-3⁰, 2 H-4⁰, 2 H-5⁰and 2 H-7), 7.47 (ddd, 2H, J ¼ 7.80, 7.80,

1.50 Hz, 2 H-8), 6.94 (d, 2H, J ¼ 1.10 Hz, 2 H-3), 3.95 (s, 4H, 2 NCH₂),

2.75 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂), 2.61–2.35 (m, 8H, 4 NCH₂pip.),

2.40 (t, 4H, J ¼ 6.90 Hz, 2 NCH₂), 1.93 (bs, 2H, 2 NH), 1.72 (qt, 4H,

J ¼ 6.90 Hz, 2 CH₂). ¹³C NMR d (100 MHz, CDCl₃) 155.2 (C-4), 139.5

(C-3a), 137.5 (C-5a), 131.6 (C-7), 131.4 (C-8), 130.1 (C-3⁰and C-5⁰),

130.0 (C-2⁰and C-6⁰), 129.2 (C-6), 128.0 (C-1⁰), 127.0 (C-9a), 126.7

(C-4⁰), 115.3 (C-9), 114.2 (C-2), 109.9 (C-1), 109.8 (C-3), 58.2 (NCH₂),

54.3 (NCH₂), 54.1 (NCH₂), 49.3 (NCH₂), 46.4 (NCH₂), 24.8 (CH₂).

MALDI-TOF MS m/z [M þ H]^þCalcd for C₄₆H₄₉N₈: 713.408, Found:

713.412.

In vitro antiplasmodial activity

The in vitro antiplasmodial activities were tested over concentra-

tions ranging from 39 nM to 40 lM against culture-adapted

Plasmodium falciparum reference strains 3D7 and W2. The former

strain is susceptible to chloroquine (CQ) but displays a decreased

susceptibility to mefloquine (MQ) 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

(Life Technologies corporation, Paisley, United

Kingdom), hypoxanthine (Sigma-Aldrich), gentamicin (Sigma-

Aldrich), and human erythrocytes and were incubated at 37 ^ꢁC in a

candle jar, as described previously³⁷. The P. falciparum drug sus-

ceptibility test was carried out in 96-well flat bottom sterile plates

under a final volume of 250 lL. After a 48 h incubation with the

drugs, quantities of DNA in treated and control cultures of para-

sites in human erythrocytes were compared according to the SYBR

Green I (Sigma-Aldrich) fluorescence-based method^38,39. Briefly,

after incubation, plates were frozen at -20 ^ꢁC until use. They were

then left to thaw for 2 h at room temperature and 100 lL of the

homogenized culture were transferred to 96-well flat bottom ster-

ile black plates (Nunc Inc) already containing 100 lL 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 1 h 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 para-

site⁰s growth (half maximal inhibitory concentration or IC₅₀values)

were then calculated from the obtained experimental results using

Tris{N-[4–(7-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-amino-

propyl}amine (1s)

Yellow oil, Yield: 94%; ¹H NMR d (300 MHz, CDCl₃) 7.94 (d, 6H,

J ¼ 8.40 Hz, 3 H-2⁰and 3 H-6⁰), 7.87 (dd, 3H, J ¼ 2.70 and 1.35 Hz,

3 H-1), 7.74 (d, 3H, J ¼ 9.00 Hz, 3 H-9), 7.48 (d, 6H, J ¼ 8.40 Hz, 3 H-3⁰

and 3 H-5⁰), 7.47 (d, 3H, J ¼ 2.70 Hz, 3 H-6), 7.09 (dd, 3H, J ¼ 9.00

and 2.70 Hz, 3 H-8), 6.93 (dd, 3H, J ¼ 4.05 and 1.35 Hz, 3 H-3), 6.81

(dd, 3H, J¼ 4.05 and 2.70 Hz, 3 H-2), 3.90 (s, 9H, 3 CH₃O), 3.88 (s,

6H, 3 NCH₂), 2.70 (t, 6H, J ¼ 6.90 Hz, 3 CH₂), 2.52 (t, 6H, J ¼ 6.90 Hz,

3 CH₂), 1.71 (qt, 6H, J ¼ 6.90 Hz, 6 CH₂). ¹³C NMR d (100 MHz,

CDCl₃) 158.5 (C-7), 155.8 (C-4), 143.1 (C-5a), 138.76 (C-3a and C-4⁰),

130.1 (C-2⁰and C-6⁰), 129.8 (C-3⁰and C-5⁰), 126.4 (C-1⁰), 122.8 (C-

9a), 117.9 (C-9), 115.9 (C-8), 115.6 (C-1), 115.0 (C-2), 112.6 (C-3),

109.7 (C-6), 57.1 (OCH₃), 55.1 (NCH₂), 53.7 (NCH₂), 49.2 (NCH₂), 28.4

(CH₂). MALDI-TOF MS m/z [M þ H]^þCalcd for C₆₆H₆₇N₁₀O₃:

1047.540, Found: 1047.613.

Tris{N-[3–(8-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-amino-

propyl}amine (1t)

Orange oil, Yield: 79%; ¹H NMR d (300 MHz, CDCl₃) 7.94 (d, 3H,

J ¼ 9.00 Hz, 3 H-6), 7.89 (dd, 3H, J ¼ 1.50 and 1.50 Hz, 3 H-2⁰),

7.86–7.80 (m, 6H, 3 H-4⁰and 3 H-1), 7.45–7.42 (m, 6H, 3 H-6⁰and

3 H-5⁰), 7.23 (d, 3H, J ¼ 2.70 Hz, 3 H-9), 7.03 (dd, 3H, J ¼ 9.00 and

2.70 Hz, 3 H-7), 6.93 (dd, 3H, J ¼ 3.90 and 1.20 Hz, 3 H-3), 6.85 (dd,

3H, J ¼ 3.90 and 2.70 Hz, 3 H-2), 3.96 (s, 9H, 3 CH₃O), 3.83 (s, 6H,

3NCH₂), 2.65 (t, 6H, J ¼ 6.90 Hz, 3 NCH₂), 2.47 (t, 6H, J ¼ 6.90 Hz, 3

NCH₂), 1.63 (qt, 6H, J ¼ 6.90 Hz, 3 CH₂). ¹³C NMR d (100 MHz,

CDCl₃) 160.4 (C-8), 153.1 (C-4), 141.9 (C-3a), 140.0 (C-5a), 132.7

(C-6), 132.0 (C-3⁰), 130.8 (C-2⁰), 129.9 (C-5⁰), 129.7 (C-4⁰), 129.2

(C-1⁰), 128.6 (C-6⁰), 126.6 (C-9a), 115.4 (C-7), 115.3 (C-1), 114.1 (C-2),

109.5 (C-3), 98.7 (C-9), 57.1 (OCH₃), 55.3 (NCH₂), 53.7 (NCH₂), 49.3

(NCH₂), 28.4 (CH₂). MALDI-TOF MS m/z [M þ H]^þCalcd for

C₆₆H₆₇N₁₀O₃: 1047.540, Found: 1047.598.

a regression program available on line⁴⁰

.

In vitro antileishmanial activity

L. donovani (MHOM/IN/00/DEVI) used in this study was provided

by the CNR Leishmania (Montpellier, France). The effects of the

tested compounds on the growth of L. donovani (MHOM/IN/00/

DEVI)promastigotes were assessed by MTT assay⁴¹.Briefly, promas-

tigotes in log-phase in Schneider⁰s medium supplemented with

20% fetal calf serum (FCS), 2 mM L-glutamine and antibiotics

(100 U/mL penicillin and 100 lg/mL streptomycin), were incubated

at an average density of 10⁶parasites/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 and pentamidine or amphotericin B (ref-

erence drugs purchased from Sigma-Aldrich) were added to each

N,N⁰-[oxybis(2,1-ethanediyloxy-2,1-ethanediyl)]bis-3–(8-methoxy-

pyrrolo[1,2-a]quinoxalin-4-yl)benzylamine (1u)

Yellow oil, Yield: 92%; ¹H NMR d (300 MHz, CDCl₃) 7.95 (d, 3H, set of experiments. After a 72 h incubation period at 27 ^ꢁC, para-

J ¼ 9.00 Hz, 2 H-6), 7.93 (dd, 3H, J ¼ 1.50 and 1.50 Hz, 2 H-2⁰), site metabolic activity was determined. Each plate-well was then

7.88–7.83 (m, 4H, 2 H-4⁰and 2 H-1), 7.50–7.45 (m, 4H, 2 H-6⁰and microscope-examined for detecting possible precipitate formation.

556

J. GUILLON ET AL.

20 lL of MTT 3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 1 ^ꢁC.min^ꢂ1. The data obtained were normalized between 0 and 1,

bromide) solution (5 mg/mL) were added to each well followed by

incubation for another 4 h. The enzyme reaction was then stopped

by addition of 100 lL of 50% isopropanol – 10% sodium dodecyl

sulfate⁴².Plates were shaken vigorously (300 rpm) for 10 minutes

and the absorbance measured in a plate reader at 570 nm in a

BIO-TEK ELx808 Absorbance Microplate Reader. Inhibitory concen-

tration 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. IC₅₀were calculated by

non-linear regression analysis processed on doseresponse curves,

using TableCurve 2D V5.0 software. IC₅₀values represent the

mean value calculated from three independent experiments.

and the temperature required for half-denaturationof oligonucleo-

tides corresponded to the emission value of 0.5 was calculated as

DT_m. Each experiment was performed in duplicate with 200 nM of

labeled oligonucleotide and 0, 1, 2, or 5 lM of compound under

K^þcondition. For each compound, two independent experiments

were carried out. The data were plotted using OriginPro 9.1 soft-

ware (OriginLab Corporation, Northampton, MA).

Results and discussion

Chemistry

The reported bis{N-[(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-amino-

propyl}amine or piperazine derivatives 1a–p were synthesized in

seven steps from 2-nitroaniline (Scheme 1). Preparation of 1–(2-

nitrophenyl)pyrroles 3a–d was performed according to the

Clauson-Kaas reaction run under micro-wave irradiation starting

from 2-nitroaniline and 2,5-dimethoxytetrahedrofuran in acetic

acid. This pathway partially involved synthetic methodologies

already described by our group^20–23,27. The resulting 1–(2-nitro-

phenyl)pyrrole intermediates 3a–d was subsequently reduced into

Cytotoxicity evaluation

A cytotoxicity evaluation was realized according to the method of

Mosmann⁴¹with slight modificationsto determine the cytotoxic

concentrations 50% (CC₅₀) and using doxorubicin as a cytotoxic

reference-compound. These assays were performed toward the

human HepG2 cell line (HepG2 CC₅₀). HepG2 (hepatocarcinoma

cell line purchased from ATCC, ref HB-8065) is a commonly used

human-derived hepatocarcinoma cell line that has shown charac- the attempted 1–(2-aminophenyl)pyrroles 4a–d using a sodium

teristics similar to those of primary hepatocytes. These cells

express many of the hepatocyte-specific metabolic enzymes, thus

enabling the cytotoxicity of tested product metabolites to be eval-

uated. Briefly, cells in 100 lL of complete RPMI medium, [RPMI

supplemented with 10% FCS, 1% ^L-glutamine (200 mM) and peni-

cillin (100 U/mL)/streptomycin (100 lg/mL)] were inoculated into

each well of 96-well plates and incubated at 37 ^ꢁC in a humidified

6% CO₂. After 24 h incubation, 100 lL of medium with various

product concentrations dissolved in DMSO (final concentration less

than 0.5% v/v) were added and the plates were incubated for 72 h

at 37 ^ꢁC. Duplicate assays were performed for each sample. Each

plate-well was then microscope-examined for detecting possible

precipitate formation before the medium was aspirated from the

wells. 100 lL of MTT solution (0.5 mg/mL in medium without FCS)

were then added to each well. Cells were incubated for 2 h at

37 ^ꢁC. After this time, the MTT solution was removed and DMSO

(100 lL) was added to dissolve the resulting blue formazan crys-

tals. Plates were shaken vigorously (300 rpm) for 5 minutes. The

absorbance was measured at 570 nm with 630 nm as reference

borohydride-copper (II) sulfate in ethanol at room temperature.

This NaBH₄-CuSO₄system was found to be quite powerful in

reducing our aromatic nitro group with excellent yield (73–85%).

The reaction of 4a–d with triphosgene in toluene gave the lac-

tams 5a–d²². Reduction of the nitro moiety of 6a–b with iron in

hot glacial acetic acid produced the spontaneous ring closure

onto the ester to afford the desired the lactams 5e–f through a

one-pot reduction-cyclization step^27,25

.

The lactams 5a–f were subsequently chlorodehydroxylated

with phosphorous oxychloride, leading to the 4-chloropyrrolo[1,2-

a]quinoxalines 7a–f.

Coupling chloro derivatives 7a–f with 3- or 4-formylphenylbor-

onic acid in the presence of Pd(PPh₃)₄as a catalyst under Suzuki-

Miyaura cross-coupling conditions proceeded to afford the substi-

tuted benzaldehydes 8a–h^22,27,28,30. Reaction of primary amines,

such as 3,3⁰-diamino-N-methyldipropylamine or N-(3-aminopropyl)-

1,3-propanediamine or 1,4-bis(3-aminopropyl)piperazine), with the

latter 7a–f gave the di-imines 9a–p, reduced into the bis{N-[(pyr-

rolo[1,2-a]quinoxalin-4-yl)benzyl]-3-aminopropyl}amines 1a–p using

sodium borohydride in methanol. The same pathway was used for

the synthesis of bisindoloquinoxaline 1q and bis(4-phenyl)pyrrolo-

quinoxaline 1r from aldehydes 8i and 8j, respectively (Schemes 2

and 3).

wavelength spectrophotometer using

a

BIO-TEK ELx808

Absorbance Microplate Reader. DMSO was used as blank and

doxorubicin (Sigma Aldrich) as positive control. Cell viability was

calculated as percentage of control (cells incubated without com-

pound). The 50% cytotoxic concentration was determined from

the dose-response curve by using the TableCurve 2D V5.0 software

(Systat Software, San Jose, CA).

This strategy was also used to prepare the tris{N-[(pyrrolo[1,2-

a]quinoxalin-4-yl)benzyl]-3-aminopropyl}amines 1s–t (Scheme 4)

and N,N⁰-[oxybis(2,1-ethanediyloxy-2,1-ethanediyl)]bis-(pyrrolo[1,2-

a]quinoxalin-4-yl)benzylamine 1u (Scheme 5).

All these quinoxaline compounds 1a–u were then converted

into their ammonium oxalate salts by treatment with oxalic acid in

refluxing isopropanol (Table 1).

FRET melting experiments

FRET melting experiments were performed with dual-labeled oli-

gonucleotides

mimicking

the

Plasmodium

telomeric

5

3

0

sequences FPf1T [FAM- (GGGTTTA)₃-GGG -TAMRA] and FPf8T

5

3

0

[FAM- (GGGTTCA)₃GGG -TAMRA] and the human telomeric

Biological activity

sequence F21T [FAM-(GGGTTA)₃-GGG - TAMRA]^36,43. The oligonu-

3

0

In vitro antimalarial activity

cleotides were prefolded in 10 mM lithium cacodylate buffer (pH

7.2), with 10 mM KCl and 90 mM LiCl (K^þcondition). The FAM emis-

sions were recorded at 516 nm using a 492-nm excitation wave-

length in the absence and presence of a single compound as a

function of temperature (25 to 95 ^ꢁC) in 96-well microplates by

All new substituted bis- and trispyrrolo[1,2-a]quinoxaline deriva-

tives 1a–u were evaluated for their antimalarial activity in vitro on

P. falciparum CQ-resistant strain W2 (IC₅₀CQ ¼0.20 lM) and the

CQ-sensitive and MQ decrease susceptibility strain 3D7 (IC₅₀CQ

using a Stratagene MX3000P real-time PCR device at a rate of ¼0.08 lM). As shown in Table 2, they were found to have IC₅₀

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

557

R₃

H₃CO

R₂

R₁

N

R₂

R₁

N

R₂

R₁

NH₂

NO₂

CuSO₄/ NaBH₄

RT

AcOH

Δ

+

O

NH₂

NO₂

OCH₃

4a-d

2a-d

3a-d

(Cl₃CO)₂CO

toluene

Δ

R₁

R₂

R₃

R₄

R₅

R₄

H-

H₃CO-

R₄

2-5a,7a

2-5b,7b

2-5c,7c

2-5d,7d

H-

H₃CO-

H-

R₃

H-

H₅C₂O-

H-

Fe / AcOH

R₂

R₁

N

R₅

N

R₅

Δ

H₃CO-

H-

6a,5e,7e H-

COOR

NO₂

N

H

O

C₆H₅- H-

H-

6b,5f,7f

5a-f

6a-b

POCl₃

Δ

R₄

R₃

HO

CHO

R₃

R₂

R₁

N

R₅

B

R₂

N

R₅

2

1

3

Pd[(PC₆H₅)₃]₄

R₁

Cl

CHO

4

K₂CO₃, toluene/EtOH

8a-h

7a-f

Δ

position

R₁

H-

R₂

R₃

R₄

R₅

H₂N

X

NH₂

H-

4

H-

H₃CO-

H-

8a

H₃CO-

8b

8c

8d

8e

8f

EtOH

Δ

H-

H₅C₂O-

4

H-

C₆H₅-

H-

H₃CO-

H-

R₄

H-

R₃

3

8g

H-

H₃CO-

H-

R₂

R₁

N

R₅

N

R₂

R

R₅

H-

8h

N

X

N

9a-p

R₄

R₃

R₂

R₁

N

R₅

N

R₂

R

R₅

NaBH₄

2

1

3

MeOH

Δ

N

4

HN

X

NH

1a-p

R₂

position

R₁

H-

position

R₃

R₄

R₅

X

R₁

R₂

R₃

R₄

R₅

X

-NH-

4

H-

9a,1a

4

H-

9i,1i

9j,1j

9k,1k

H₃CO- H-

H-

N

-NCH₃-

9b,1b

-NCH₃-

N

H-

H₅C₂O-

H-

4

N

H-

9c,1c

N

-NCH₃-

H₃CO-

9d,1d

N

4

H-

9l,1l

C₆H₅-

H₃CO-

H-

N

9e,1e

9f,1f

H₃CO- H-

-NCH₃-

3

H-

9m,1m

9n,1n

9o,1o

H-

4

9g,1g

9h,1h

N

H- H₃CO- H-

-NCH₃-

H-

H₃CO- H-

-NCH₃-

3

N

H-

9p,1p

Scheme 1. Synthesis of bispyrrolo[1,2-a]quinoxalines 1a–p.

between 0.05–4.20 lM on W2, and 0.04–8.96 lM on 3D7 P. falcip- bis-(3-aminopropyl)amine linker was found more active (up to 4 to

arum strains, respectively. 5 times) on the W2 strain than its counterparts with bis-(3-amino-

For the pyrroloquinoxalines unsubstituted on their propyl)methylamine (compounds 1b) or bis-(3-aminopropyl)pipera-

heterocyclic moiety (compounds 1a–c), compound 1a joined by a zine linkages (compounds 1c): i.e. IC₅₀¼0.85 lM for 1a versus 4.20

558

J. GUILLON ET AL.

H₂N

N

NH₂

N

EtOH

Δ

N

CHO

8i

9q

MeOH

Δ

NaBH₄

N

HN

N

NH

1q

Scheme 2. Synthesis of bisindolo[1,2-a]quinoxaline 1q.

CHO

N

H₂N

N

NH₂

N

EtOH

Δ

9r

8j

MeOH

Δ

NaBH₄

N

NH

N

HN

N

1r

Scheme 3. Synthesis of bispyrrolo[1,2-a]quinoxaline 1r.

and 3.40 lM for 1b and 1c, respectively.As a general rule against 4 (compounds 1b–c). Thus, compound 1n bearing a diaminopro-

the W2 strain, the introduction of a methoxy substituent in pos- pylpiperazine linker exhibited the most potent antiplasmodial

ition 7, 8 or 9 of the bis-pyrrolo[1,2-a]quinoxaline skeleton linked activity (IC₅₀¼0.05 lM) against the CQ-resistant strain (W2).

in position 4 of the benzyl ring (compounds 1d–i) strongly Nevertheless, in these subseries in which the polyaminoalkyl side

increased the antiparasitic activity in comparison with their chain was anchored in position 3 of the benzyl ring, the introduc-

respective unsubstituted bispyrrolo[1,2-a]quinoxaline homologs tion of a methoxy substituent on the pyrrolo[1,2-a]quinoxaline

1b–c: i.e. IC₅₀¼0.13–0.44 lM for 1d–i and 3.40–4.20 lM for 1b–c. heterocycles (compounds 1o–p) seemed to be less detrimental

Moreover, introduction of an ethoxy substituent in position C-3 of against drug-resistant strain W2 in comparison with their analogs

the pyrrolo[1,2-a]quinoxaline moieties joined by a bis-(3-amino- 1d–i. The bis-pyrrolo[1,2-a]quinoxaline 1r, structural isomer of

propyl) piperazine linker (compound 1k) was found more active derivative 1c, was also found to have a significant activity against

(up to 38 times) than its non-substituted homolog 1c the W2 strain (IC₅₀¼0.75 lM). In addition, the tris{N-[(pyrrolo[1,2-

(IC₅₀¼0.09 lM for 1k in comparison of 3.40 lM for 1c). In addition, a]quinoxalin-4-yl)benzyl]-3-aminopropyl}amines 1s–t showed simi-

1k was found to be 2.2 times more active than the reference drug lar antimalarial activities when compared with their bis-homologs

CQ (IC₅₀¼0.20 lM). In contrast, the addition of a phenyl in pos- 1d–i (IC₅₀¼0.19–0.29 lM for 1s–t versus 0.13–0.44 lM for 1d–i).

ition 2 of the pyrrolo[1,2-a]quinoxaline moiety (compound 1 l) Moreover, the replacement of the polyaminoalkyl linker by a 1,11-

reduced considerably the antimalarial activity on the W2 strain diamino-3,6,9-trioxaundecane chain (compound 1u) showed the

(IC₅₀¼1.39 lM).

same level of antimalarial activity against the W2 strain with

The bis-pyrrolo[1,2-a]quinoxalines 1m–n, which are linked with IC₅₀¼0.25 lM.

polyaminoalkyl chains on position 3 of the benzyl moieties,

Against the CQ-sensitive strain (3D7)and in the subseries

increased the antimalarial activity up to 35 and 68 times when of the pyrrolo[1,2-a]quinoxalines unsubstituted on their heterocyc-

compared to their non-substituted counterparts linked in position lic moieties (compounds 1a–c), compound 1c joined by

a

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

559

R₂

R₁

N

NH₂

R₂

R₁

N

H₂N

N

NH₂

N

2

1

3

4

EtOH

Δ

N

CHO

8i-j

9s-t

N

R₁

R₂

position

N

4

3

H₃CO- H-

H- H₃CO-

8i,9s,1s

8j,9t,1t

NaBH₄

N

R₂

MeOH

Δ

R₁

N

R₁

R₂

R₁

N

2

1

3

4

NH

N

1s-t

HN

NH

N

R₂

R₁

N

R₁

R₂

Scheme 4. Synthesis of trispyrrolo[1,2-a]quinoxalines 1s–t.

bis-(3-aminopropyl)piperazine linker was found more active than against 3D7 strain (IC₅₀¼1.00–7.22 lM). In the subseries in which

its structural analogs with bis-(3-aminopropyl)amine (compound

1a) or bis-(3-aminopropyl)methylamine linkages (compound 1b):

i.e. IC₅₀¼0.80 lM for 1c versus 3.42 and 1.20 lM for 1a and 1b,

respectively. Surprisingly, most of the pyrrolo[1,2-a]quinoxalines

1d–g and 1i bearing a methoxy substituent on the heterocyclic

skeleton exhibited moderate antimalarial activity against 3D7

strain with IC₅₀ranging from 1.28 to 5.85 lM, in the exception of

compound 1 h which showed an interesting IC₅₀of 0.25 lM

against this Plasmodium strain. As against the W2 strain, replace-

ment of the methoxy function by an ethoxy one in position 3 of

the pyrrolo[1,2-a]quinoxaline moieties (compounds 1j–k) increased

the anti P. falciparum activity against the 3D7 strain (IC₅₀¼0.26

and 0.19 lM for 1j and 1k, respectively). The introduction of a

phenyl ring in position 2 of the pyrrolo[1,2-a]quinoxaline moiety

(compound 1 l) reduced considerably the antimalarial activity on

the 3D7 strain (IC₅₀¼8.96 lM). Similar observations could be made

the polyaminoalkyl side chain was anchored in position 3 of the

benzyl ring, the piperazine substituted bis-pyrrolo[1,2-a]quinoxa-

line 1p was found to be two times more active than the reference

drugs chloroquine and mefloquine (IC₅₀¼0.040 lM for 1p versus

IC₅₀¼0.080 lM for the references). The tris-pyrrolo[1,2-a]quinoxa-

lines 1s–t showed similar antimalarial activities when compared

with

their

methoxy

substituted

bis-homologs

1o–p

(IC₅₀¼0.20–0.24 lM for 1s–t versus 0.04–0.18 lM for 1o–p).

Compound 1u bearing diaminotrioxaundecane linkage exhibited

similar activity against the 3D7 strain than those observed for

chloroquine and mefloquine (IC₅₀–0.08 lM).

In vitro antileishmanial activity against promastigote forms

In order to increase the biological profile of molecules 1a–u, com-

plementary analyses were performed. Notably, P. falciparum

with the structural analogs 1q–r which showed moderate activity belonging to coccidian protozoan parasites, their in vitro biological

560

J. GUILLON ET AL.

H CO

N

3

O

H CO

N

OCH

3

H N

2

O

NH

2

CHO

O

N

O

N

EtOH

Δ

8j

9u

MeOH

Δ

NaBH

4

H CO

3

N

OCH

3

O

NH

O

NH

1u

Scheme 5. Synthesis of bispyrrolo[1,2-a]quinoxaline 1u.

activity on flagellate protozoan parasites like Leishmania donovani

was evaluated (Table 2). In comparison with amphotericin B and

pentamidine used as reference drugs (IC₅₀¼0.1 lM and 5.5 lM,

respectively), most of the tested compounds 1 were found active

against the promastigote forms of L. donovani. It must be noticed 1c

that a majority of these bis-pyrrolo[1,2-a]quinoxaline derivatives 1

Table 1. Physical properties of the final amines 1a–t.

Compound

Salt^a

mp (^ꢁC)^b

% Yield^c

1a

1b

Yellow crystals

Pale-orange crystals

Yellow crystals

Oranges crystals

Pale-yellow crystals

Yellow crystals

Beige crystals

3 (COOH)₂

4 (COOH)₂

3 (COOH)₂

4 (COOH)₂

3 (COOH)₂

4 (COOH)₂

3 (COOH)₂

4 (COOH)₂

3 (COOH)₂

4 (COOH)₂

3 (COOH)₂

4 (COOH)₂

2 (COOH)₂

>260

245–248

>260

83

79

85

81

43

76

70

73

75

88

91

56

82

68

81

86

65

88

77

88

76

1d

1e

1f

1g

1h

1i

1j

1k

1l

224–226

246–249

240–242

236–239

160–162

>260

192–195

218–220

239–243

209–211

220–223

188–191

234–237

>260

(compounds 1a, 1c, 1d–j and 1m–q)with IC₅₀ranging from 1.0 to

4.26 lM were found slightly more potent than pentamidine, one

of the reference compounds (IC₅₀¼5.5 lM). In particular, bis{N-[4-

(indolo[1,2-a]quinoxalin-6-yl)benzyl]-3-aminopropyl}piperazine 1q

was found to be 9.65 times more active than pentamidine

(IC₅₀¼0.57 lM versus IC₅₀¼5.5 lM). In our previously described

antileishmanial series^21,22, we have noticed that introduction of a

1m

methoxy group in positions 7 and 8 of the pyrrolo[1,2-a]quinoxa- 1n

line moiety generally increased the antileishmanial activity. In this

new series, we also decided to introduce a methoxy or an ethoxy

to evaluate the influence of such groups for their in vitro antileish-

1o

1p

1q

1r

1s

1t

1u

>260

manial activity upon the L. donovani strain. Meanwhile, the pres-

ence of these alkoxy groups in the 7, 8, 9 or 3 position seems

not to be detrimental for the activity against L. donovani as illus-

trated by results obtained for compounds 1d–k and 1o–p

(IC₅₀¼1.07–4.26 lM) in comparison with unsubstituted compounds

1a, 1c and 1m–n (IC₅₀¼1.0–3.71 lM). The same observation could

be done for the nature of the polyalkylamine linker between the

two benzylpyrrolo[1,2-a]quinoxaline moieties that seemed also not

to be crucial, with the exception of piperazine substituted 1 g,

which showed a better antileishmanial activity in vitro compared

to its structural methylamine analog 1f (IC₅₀¼4.25 lM versus

1.07 lM for 1 g) upon the L. donovani strain.

Moreover, in the subseries in which the polyaminoalkyl side

chain was anchored in position 3 of the benzyl ring, the various

bis-pyrrolo[1,2-a]quinoxalines 1m–p were found less beneficial for

the in vitro antiparasitic activity with an IC₅₀of 2.80–3.71 lM in

comparison with their analogs 1a–c and 1f–g. In addition, the tri-

substitution of the tris(3-aminopropyl)amine linker by the pyr-

rolo[1,2-a]quinoxaline core (compounds 1s–t) led to a decrease in

the antileishmanial activity (IC₅₀>10 lM) in comparison with their

bis-pyrrolo[1,2-a]quinoxaline analogs 1d–e and 1o–p.

192–194

184–187

141–144

^aThe stoichiometry and composition of the salts were determined by elemental

analyses (within 0.4% of the theoretical values).

^bCrystallization solvent: 2-PrOH–H₂O.

^cThe yields only included the conversions into the ammonium oxalates.

trispyrrolo[1,2-a]quinoxalines 1 showed significant level of cytotox-

icity against the HepG2 cell line (IC₅₀¼1.25–3.38 lM). Considering

the W2 P. falciparum strain, the selectivity indexes varied between

0.37 and 40.6, and SI ranged from 0.53 to 39.25 by taking into

account the 3D7 strain. This SI led to the identification of com-

pound 1n with SI of 40.6 on W2 strain, and SI of 39.25 for deriva-

tive 1p on 3D7 strain. We could notice that these last bioactive

compounds 1n and 1p were bis-pyrrolo[1,2-a]quinoxalines linked

by a 1,4-bis(3-aminopropyl)piperazine moiety in position 3 of their

benzyl ring. On the other hand, the bis-pyrrolo[1,2-a]quinoxaline

1u is also interesting with SI ¼20.26, on the 3D7 malaria strain.

However, according to the SI values, these compounds appeared

to be toxic; thus, new modulations by replacing the benzyl substi-

tuted heterocyclic moiety to synthesize new candidates could be

developed for forthcoming pharmacological investigations.

Cytotoxicity and selectivity index

In order to assess their selectivity of action, the cytotoxicity of the

antimalarial compounds 1a-u was also evaluated in vitro upon one

human cell line. The cytotoxic concentrations 50% (CC₅₀) on the

metabolizing human hepatocyte HepG2 cell line allowed access to

the corresponding selectivity indexes (SI) defined as ratio of cyto-

toxic and antimalarial activities (SI ¼ CC₅₀/W2 or 3D7 IC₅₀). The SI

could validate their real potential as selective antiparasitic agents.

The results concerning the cytotoxicity and SI data are

FRET-melting experiments

As these new compounds presented the same requirements as

those required for G-4 stabilizing ligands, we have also investi-

gated their ability of targeting P. falciparum telomeres as a poten-

tial strategy to interfere with human protozoan parasite infections.

In fact, the telomeres of the parasite could constitute an attractive

target. They₀are composed of repetitions of a degenerate motif

0

presented in Table 2. As expected, most of the active bis- and (⁵GGGTTYA³, where Y could be T or C), which is different from

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

561

Table 2. In vitro sensitivity of compounds 1a-u on P. falciparum and L. donovani strains, and cytotoxicity on the HepG2 cell line.

P. falciparum strains IC₅₀

values (lM)^a

Selectivity Index^d

Cytotoxicity CC₅₀values (lM)^c

W2

3D7

L. donovani IC₅₀values (lM)^b

HepG2

HepG2/W2

HepG2/3D7

Chloroquine^e

0.20 0.03

0.032 0.001

n.d.^g

0.08 0.003

0.08 0.008

n.d.^g

30

150

300

Mefloquine^e

n.d.^g

2.13

0.37

7.56

6.95

26

n.d.^g

0.53

1.30

1.56

0.62

0.56

0.58

0.96

9.76

1.04

7.50

7.79

n.d.^g

1.81

5.49

8.33

39.25

n.d.^g

8.30

7.17

20.26

Pentamidine^f

5.5 0.8

0.1 0.04

1.0 0.2

n.d.^g

2.3 0.5

8.8 0.6

1.81 0.10

1.56 0.60

1.25 0.30

2.57 0.7

1.46 0.6

3.38 0.9

2.14 1.40

2.44 0.4

1.33 0.3

1.95 0.3

1.48 0.5

n.d.^g

1.94 1.0

2.03 1.2

1.50 0.9

1.57 0.8

1.56 0.70

n.d.^g

Amphotericin B^f

n.d.^g

1a

1b

1c

1d

1e

1f

1g

1h

1i

1j

1k

1l

1m

1n

1o

1p

1q

1r

0.85 0.15

4.20 0.65

3.40 0.74

0.34 0.04

0.21 0.03

0.13 0.01

0.36 0.04

0.44 0.11

0.32 0.04

0.49 0.16

0.09 0.01

1.39 0.13

0.12 0.01

0.05 0.01

0.206 0.03

0.17 0.03

n.d.^g

3.42 0.54

1.20 0.20

0.80 0.12

4.14 0.03

2.62 0.25

5.85 0.07

2.22 0.25

0.25 0.04

1.28 0.10

0.26 0.03

0.19 0.01

8.96 0.69

1.07 0.16

0.37 0.04

0.18 0.02

0.04 0.004

1.00 0.05

7.22 0.77

0.20 0.04

0.24 0.03

0.076 0.009

1.14 0.2

3.89 0.3

2.52 0.1

4.25 0.2

1.07 0.1

2.07 0.02

1.58 0.1

4.26 0.1

ꢃ10^h

5.94

5.54

4.16

3.98

16.44

n.d.^g

16.17

40.6

7.28

9.23

1.56

n.d.^g

5.72

9.05

6.16

n.d.^g

2.80 0.1

3.71 0.4

3.51 0.1

3.39 0.4

0.57 0.1

n.d.^g

0.75 0.10

0.29 0.05

0.19 0.02

0.25 0.08

1s

1t

ꢃ10^h

1.66 0.5

1.72 0.6

1.54 0.7

ꢃ10^h

1u

ꢃ10^h

^aIC₅₀values were measured on the chloroquine-resistant and mefloquine-sensitive strain W2 and the chloroquine-sensitive and mefloquine-resistant strain 3D7.

^bIC₅₀values were measured on the promastigotes of Leishmania donovani strain. The IC₅₀(lM) values correspond to the mean standard deviation from three inde-

pendent experiments.

^cCC₅₀values were measured on the HepG2 cell line. The CC₅₀(lM) values correspond to the mean standard deviation from three independent experiments.

^dSelectivity Index (SI) was defined as the ratio between the CC₅₀value on the HepG2 cells and the IC₅₀value against the P. falciparum W2 or 3D7 strain.

^eChloroquine and mefloquine were used as antiplasmodial drug-compounds of reference.

^fPentamidine and Amphotericin B were used as antileishmanial drug-compounds of reference.

^gn.d.: not done.

^hNo activity noted at the higher concentration tested.

0

the human one (⁵GGGTTA³). This antimalarial approach based on These last data could benow considered as a promising starting

targeting P. falciparum telomeres has been previously described

by our team³⁶. Thus, in this report, we investigated the stabiliza-

tion of Plasmodium telomeric G-quadruplexes by these new com-

pounds through a FRET melting assay. For this evaluation, the two

Plasmodium telomeric sequences (FPf1T and FPf8T) and the

human one (F21T), in potassium, were used (two different

Plasmodium telomeric repeats were used to fit the degenerate

consensus). Thus, we used this FRET melting assay to study the

interactions of some of our most bioactive compounds 1 and also

the reference drugs (chloroquine – CQ and mefloquine – MQ) with

these three telomeric sequences. For better visualization, a plot of

DT_mvalues induced on FPf1T or FPf8T versus the DT_minduced on

F21T is presented in Figure 2, allowing us to classify the more

active compounds 1 in various subsets. Almost all the tested com-

pounds displayed roughly the same stabilization profiles for the

two Plasmodium telomeric sequences FPf1T and FPf8T and slightly

better than that of the human one F21T, except 1s and 1t for

FPf1T and 1o and 1s for FPf8T.The reference compounds CQ and

MQ, never evaluated on these parasitic sequences before our

work, showed very slight stabilization on these three telomeric

sequences. These preliminary results mean that most of the tested

bis-pyrrolo[1,2-a]quinoxalines 1 have a moderate preference for

Plasmodium over human telomeric quadruplexes, excepted bispyr-

roloquinoxaline 1o. Surprisingly, the tris-pyrrolo[1,2-a]quinoxalines

1s–t were found more active on the human telomeric sequence

F21T in comparison with their bis-analogues1d–e and 1o–p. In

contrast, the bis{N-[4-(methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-

3-aminopropyl}piperazines 1e and 1i were found to be more

point for the further development and optimization of new and

potent antimalarial compounds as well as promising stabilizers for

the telomeric Plasmodium DNA sequences. As very little is known

about telomere regulation in P. falciparum, further studies should

be now investigated both on parasitic and human sequences

which presented different response or time-response to telomere

perturbation.

Conclusion

In the present report, we have described the synthesis and the

antimalarial activity of new bis- and trispyrrolo[1,2-a]quinoxaline

derivatives 1 in which aromatic nuclei are joined by various ali-

phatic polyamines linker. These new compounds were then tested

for their in vitro antiparasitic activity on (i) two P. falciparum strains

(the CQ-resistant W2 and CQ-sensitive 3D7); and (ii) the promasti-

gote forms of L. donovani strain. Among these new synthesized

molecules, few of them were identified as potential in vitro anti-

plasmodial hits, displaying IC₅₀ranging from 0.04 to 0.09 lM on

the W2 and 3D7 strains of P. falciparum. Thus, the most promising

antimalarial results were obtained for the two bis-pyrrolo[1,2-a]qui-

noxalines 1n and 1p linked by a 3-(aminopropyl)piperazine chain

in position 3 of their benzyl moieties. These compounds 1n and

1p were identified as the most potent antimalarial candidates with

selectivity index (SI) of 40.6 on W2 strain, and 39.25 on 3D7 strain,

respectively. In addition, biological results showed activity against

the promastigote forms of L. donovani with IC₅₀ranging from 0.57

active on the Plasmodium sequences with the higher DT_m. to 4.26 lM. In parallel, the in vitro cytotoxicity of these new

562

J. GUILLON ET AL.

Figure 2. (A and B) Thermal stabilization (DT_m) induced by different compounds 1 (at 5 lM) on the Plasmodium telomeric quadruplexes FPf1T and FPf8T versus DT_m

induced on the human telomeric quadruplex F21T.

molecules was assessed on the human HepG2 cell line. Structure-

activity relationships of these new synthetic compounds are here

also discussed, as well as their relative ability of targeting P. falcip-

arum telomeres as potential mechanism of action. Thus, as the

telomeres of the parasite could constitute an interesting target,

we have also established the possibility of targeting Plasmodium

telomeres by stabilizing the Plasmodium telomeric G-quadruplexes

through a FRET melting assay. These results led us to conclude

that the two bis{N-[4-(methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-

3-aminopropyl}piperazines 1e and 1i seemed to be able to dis-

criminate between Plasmodium and human telomeric quadru-

plexes. By taking into account the biological and biophysical

results and also the structure–activity relationships of these new

series and those previously described, our overall results could be

now considered as a promising starting point for the further

development and optimization of new and potent antiprotozoan

compounds.

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We thank the DGA and ANR (project ANR-12-ASTR-003) for the

financial support of this study.

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Disclosure statement

The authors report no conflicts of interest. The authors alone are

responsible for the content and writing of the paper.

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Article Doi

Design, synthesis and antimalarial activity of novel bis{N-[(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-aminopropyl}amine derivatives

DOI: 10.1080/14756366.2016.1268608

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Article abstract of DOI:10.1080/14756366.2016.1268608

Full text of DOI:10.1080/14756366.2016.1268608

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