New ferrocenic pyrrolo[1,2-a]quinoxaline derivatives: Synthesis, and in vitro antimalarial activity

Article abstract of DOI:10.1016/j.bmc.2008.09.038

Following our search for antimalarial compounds, novel series of ferrocenic pyrrolo[1,2-a]quinoxaline derivatives 1-2 were synthesized from various substituted nitroanilines and tested for in vitro activity upon the erythrocytic development of Plasmodium falciparum strains with different chloroquine-resistance status. The pyrrolo[1,2-a]quinoxalines 1 were prepared in 6-8 steps through a regioselective palladium-catalyzed monoamination by coupling 4-chloropyrrolo[1,2-a]quinoxalines with 1,3-bis(aminopropyl)piperazine or -methylamine using Xantphos as the ligand. The ferrocenic bispyrrolo[1,2-a]quinoxalines 2 were prepared by reductive amination of previously described bispyrrolo[1,2-a]quinoxalines 9 with ferrocene-carboxaldehyde, by treatment with NaHB(OAc)₃. The best results were observed with ferrocenic pyrrolo[1,2-a]quinoxalines linked by a bis(3-aminopropyl)piperazine. Moreover, it was observed that a methoxy group on the pyrrolo[1,2-a]quinoxaline nucleus and no substitution on the terminal N-ferrocenylmethylamine function enhanced the pharmacological activity. Selected compounds 1b, 1f-h, 1l and 2a were tested for their ability to inhibit β-haematin formation, the synthetic equivalent of hemozoin, by using the HPIA (heme polymerization inhibitory activity) assay. Of the tested compounds, only 2a showed a β-haematin formation inhibition, but no inhibition of haem polymerization was observed with the other selected ferrocenic monopyrrolo[1,2-a]quinoxaline derivatives 1b, 1f-h and 1l, as the IC₅₀ values were superior to 10 equivalents.

Full text of DOI:10.1016/j.bmc.2008.09.038

Bioorganic & Medicinal Chemistry 16 (2008) 9133–9144
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New ferrocenic pyrrolo[1,2-a]quinoxaline derivatives: Synthesis,
and in vitro antimalarial activity
a
b
c
a
Jean Guillon a, , Stéphane Moreau , Elisabeth Mouray , Véronique Sinou , Isabelle Forfar ,
*
Solene Belisle Fabre a, Vanessa Desplat d, Pascal Millet d, Daniel Parzy c, Christian Jarry a, Philippe Grellier b
a EA 4138—Pharmacochimie, UFR des Sciences Pharmaceutiques, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France
b USM 0504 Biologie Fonctionnelle des Protozoaires, Département Régulations, Développement et Diversité Moléculaire, Muséum National d’Histoire Naturelle, 61 rue Buffon,
75231 Paris, France
c UMR-MD3 Relations Hôte-Parasites, Pharmacologie et Thérapeutique, Institut de Médecine Tropicale du Service de Santé des Armées (IMTSSA), Université de la Méditerranée,
56 boulevard Charles Livon, 13007 Marseille, France
d PPF Médicaments-Parasitologie, UFR des Sciences Pharmaceutiques, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 May 2008
Revised 1 September 2008
Accepted 10 September 2008
Available online 17 September 2008
Following our search for antimalarial compounds, novel series of ferrocenic pyrrolo[1,2-a]quinoxaline
derivatives 1–2 were synthesized from various substituted nitroanilines and tested for in vitro activity
upon the erythrocytic development of Plasmodium falciparum strains with different chloroquine-resis-
tance status. The pyrrolo[1,2-a]quinoxalines 1 were prepared in 6–8 steps through a regioselective
palladium-catalyzed monoamination by coupling 4-chloropyrrolo[1,2-a]quinoxalines with 1,3-bis(ami-
nopropyl)piperazine or -methylamine using Xantphos as the ligand. The ferrocenic bispyrrolo[1,
2-a]quinoxalines 2 were prepared by reductive amination of previously described bispyrrolo[1,2-a]quin-
oxalines 9 with ferrocene-carboxaldehyde, by treatment with NaHB(OAc)3. The best results were
observed with ferrocenic pyrrolo[1,2-a]quinoxalines linked by a bis(3-aminopropyl)piperazine. More-
over, it was observed that a methoxy group on the pyrrolo[1,2-a]quinoxaline nucleus and no substitution
on the terminal N-ferrocenylmethylamine function enhanced the pharmacological activity. Selected com-
pounds 1b, 1f–h, 1l and 2a were tested for their ability to inhibit b-haematin formation, the synthetic
equivalent of hemozoin, by using the HPIA (heme polymerization inhibitory activity) assay. Of the tested
compounds, only 2a showed a b-haematin formation inhibition, but no inhibition of haem polymeriza-
tion was observed with the other selected ferrocenic monopyrrolo[1,2-a]quinoxaline derivatives 1b,
1f–h and 1l, as the IC50 values were superior to 10 equivalents.
Keywords:
Ferrocenic pyrrolo[1,2-a]quinoxaline
derivatives
Synthesis
Antimalarial activity
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
urgent to replace ineffective drugs. This has led to the search for
novel antimalarial drugs that might circumvent the resistance
Parasitic infections caused by Plasmodium species are responsi-
ble for malaria, a severe disease causing 300–500 millions of cases
and 1.1 million deaths per years in tropical zones worldwide
according to World Health Organization estimations.1–3 The most
virulent protozoa, P. falciparum is the main cause of severe clinical
malaria and death and shows an increasing prevalence of resis-
tance to standard antimalarial drugs.4,5 Chloroquine (CQ)6 is, after
50 years, still a mainstream drug in the fight against malaria, but
its efficacy is being eroded by the emergence of resistant parasites.
This resistance to antimalarial drugs like CQ is widespread
throughout much of Africa and other parts of the developing world
where malaria transmission is high.7,8 The need of active antiplas-
modial drugs with new mode of action becomes more and more
mechanism of the parasite. Two series of compounds that show
promise in this regard are the bisquinoline and bisacridine antima-
larial drugs A and B (Fig. 1).9–12 These drugs show much lower
resistance indices than chloroquine, indicating that the bisquino-
line or bisacridine structures are less efficiently excluded by
drug-resistant parasites. An other method to overcome CQ efflux
consists of designing monoquinolines C linked by a 1,4-bis(3-ami-
nopropyl)piperazine substituted by a large variety of terminal
groups.13–16For several years, it was proposed a strategy for the
development of organometallic-based antimalarial drugs.17–19 Gi-
ven the avidity of Plasmodium for free iron, it was postulated that
an effective way of removing the chloroquine resistance of para-
sites might be the addition of iron to a chloroquine molecule and
that a ferrocene moiety will permit to vectorize the drug to the se-
lected target. Hence, some new organometallic compounds includ-
ing a ferrocene nucleus (dicyclopentadienyl iron) incorporated on
* Corresponding author. Fax: +33 (0) 5 57 57 13 52.
E-mail address: Jean.Guillon@chimphys.u-bordeaux2.fr (J. Guillon).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.038

9134
J. Guillon et al. / Bioorg. Med. Chem. 16 (2008) 9133–9144
CH3
CH3
CH3
*
Cl
Cl
N
HN
N
N
N
A
N
NH
HN
N
Cl
Chloroquine (CQ)
Cl
Cl
Cl
N
N
N
N
C
N
NH
N
N
NH
HN
N
B
R1
R1
R
= alkyl, aryl, cycloalkyl, benzyl ...
1
OCH3
CH3O
Fe
R1
Cl
N
Cl
HO
N
R1
CH3
*
HN
N
N
CH3
Fe
N
N
N
N
CF3
Cl
CF3
E
N
Ferroquine (FQ)
D
R1
R1
CH3
,
C2H5
,
N
N
=
N
,
N
N
N CH3
Fe
CH3
C2H5
N
N
N
N
F
NH
HN
R2
N
N
R2
R
R = H-, CH O-, Cl
2 3
1,
R1
R1
Figure 1. Structure of chloroquine (CQ), bisquinolines A, bisacridines B, quinolinylpiperazines C, ferroquine (FQ), ferrocenyl triazacyclononanes D, ferrocenic mefloquine
analogues E, and bispyrrolo[1,2-a]quinoxalines F.
chloroquine were designed, which led to the discovery of ferroqu-
ine (FQ) and of structural analogues.20–25 Then, new organometal-
lic antimalarial compounds were investigated, for example,
ferrocene triazacyclononane quinolines D and ferrocenic meflo-
quine analogues E.26,27
drawn, in relation to the chemical nature of both linker and sub-
stituents on the pyrroloquinoxaline moiety. Finally, pharmaco-
logical results will be discussed in terms of the lipophilic
behaviour of synthesized compounds quantified through the par-
titioning theory.
As we previously described
a novel synthetic approach
to pyrrolo[1,2-a]quinoxalines as analogues of quinoline
derivatives28–30 such as antimalarial bis{N-(pyrrolo[1,2-a]quinox-
alin-4-yl)-3-aminopropyl}piperazines F (Fig. 1)31 we used the
pyrrolo[1,2-a]quinoxaline moiety as a template for the design
of new antimalarial ferrocenic monoquinoline bioisostere
compounds. Hence, we report here the synthesis and in vitro
antiparasitic activity upon Plasmodium falciparum or a series of
ferrocenic pyrrolo[1,2-a]quinoxalines 1–2 in which the heterocy-
clic and ferrocenic moieties are linked through a bis(3-aminopro-
pyl)amine via a ‘‘pseudo-amidinic” bond (Fig. 2). The choice of
the linker was based, in particular, on previous results obtained
for bispyrrolo[1,2-a]quinoxalines F. From biological results ob-
tained for the new ferrocenic pyrrolo[1,2-a]quinoxaline deriva-
tives 1–2, preliminary structure-affinity relationships may be
2. Results and discussion
2.1. Chemistry
The new ferrocenic pyrrolo[1,2-a]quinoxaline derivatives 1a–
n, and 2a–b were synthesized from substituted 2-nitro-anilines
(Scheme 1). The Clauson–Kaas reaction achieved on anilines with
2,5-dimethoxytetrahydrofuran (DMTHF) in acetic acid gave the
pyrrolic derivatives 3a–b (75–81% yields), which were then re-
duced using a BiCl3–NaBH4 treatment to provide the attempted
1-(2-aminophenyl)pyrroles 4a–b (Scheme 1). Cyclization be-
tween the NH2 and the C-
by reacting 4a–b with triphosgene in toluene to give the lactams
5a–f, which were subsequently chlorodehydroxylated with
a of the pyrrole ring was performed

J. Guillon et al. / Bioorg. Med. Chem. 16 (2008) 9133–9144
9135
these secondary amines 9a–b with ferrocene-carboxaldehyde in
the presence of NaHB(OAc)3 (Scheme 3).
Compounds 1a–n and 2a–b were then converted into their oxa-
late salts in 63-86% yields by treatment with oxalic acid in reflux-
ing isopropanol (Table 1).
N
N
Fe
R1
NH
X
N
R2
1a-n
2.2. Pharmacology
N
N
N
N
All new ferrocenic pyrrolo[1,2-a]quinoxaline derivatives 1–2
were evaluated for their antimalarial activity in vitro upon the
P. falciparum CQ-sensitive strain F32 (IC50 CQ = 19.5 nM) and
the CQ-resistant strains FcB1 and PFB (IC50 CQ = 105.3 and
225.7 nM, respectively). As shown in Table 2, they were found
to have IC50s between 9.1–167.5 nM upon F32, 16.6–173.1 nM
upon FcB1, and 30.8-240.8 nM upon PFB P. falciparum strains,
respectively.
By comparing activities for compounds bearing one same sub-
stituent on the pyrroloquinoxaline moiety and on the N-ferroce-
nylmethylamino function, it appears that compounds with a
bis(3-aminopropyl)piperazine linker (1b, 1e–f, 1k–l) were gener-
ally more active (up to 2–8 times) than their counterparts with
bis(3-aminopropyl)methylamine linkage (compounds 1d, 1h–j,
1m–n), with the exception of 1a and 1c, that showed similar
antimalarial activities. The presence of a two proton-acceptor
site in the linker, such as a piperazine ring likely to increase vac-
uolar pH, was found to be favorable to antimalarial activity,
when compared with a mono proton-acceptor site such as a
methylamine function.
R1
NH
N
HN
R1
2a-b
Fe
N
R
= H-, CH O-
3
1
X = -NCH -,
N
3
R
= H-, -CH CH , -CH -C H ,
2 3 2 6 5
2
Fe
Figure 2. Structure of synthesized ferrocenyl pyrrolo[1,2-a]quinoxaline derivatives
1a–n and 2a–b.
phosphorous oxychloride to obtain the chloroquinoxalines 6a–
b.28,31 Instead of the previously adopted method for bis{N-(pyr-
rolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}piperazines F employ-
ing DMF and K2CO3,31 the key intermediates 7a–d, used as
precursors for synthesis of compounds 1, were obtained by cou-
pling 4-chloropyrrolo[1,2-a]quinoxalines 6a–b to 1,3-bis(amino-
propyl)piperazine or -methylamine in an efficient and
regioselective procedure using catalytic amounts of Pd(OAc)2
and the chelating ligand 9,9-dimethyl-4,6-bis(diphenylphos-
phino)xanthene (Xantphos). This Pd(OAc)2/Xantphos system be-
haved as a very efficient and selective catalyst for these mono
N-heteroarylations, giving 49–60% yields.32–34 The pyrrolo[1,2-
a]quinoxalines 7a–d were also prepared in 1-pentanol without
base by condensation of 4-chloropyrrolo[1,2-a]quinoxalines 6a–
b with 1,3-bis(aminopropyl)piperazine or -methylamine accord-
ing to an aromatic nucleophilic substitution mechanism.15,35
The excess of amine (3 equiv) led to a moderate yield (32–
45%) of the N-monosubstituted derivatives 7a–d. In both meth-
ods, formation of the dicoupling bis{N-(pyrrolo[1,2-a]quinoxa-
lin-4-yl)-3-aminopropyl} amines F31 was also observed as side-
products. The ferrocene-carboxaldehyde was then condensed
with amines 7a–d in refluxing ethanol giving the imines 8a–
d.21 Reduction of 8a–d by sodium borohydride in methanol gave
Antimalarial activity also clearly depended on the nature of
the substituent on the pyrrolo[1,2-a]quinoxaline nucleus. Never-
theless, whatever the nature of the linker, the introduction of a
methoxy substituent on C-7 carbon of the pyrroloquinoxaline
moiety (compounds 1b, 1d, 1g, 1j, 1l, 1n and 2b generally in-
creased the antimalarial activity, when compared to the non-
substituted counterparts (1a, 1c, 1e–f, 1h–i, 1k, 1m and 2a).
On the other hand, secondary ferrocenyl amines 1a–d were
found to be more active than corresponding tertiary ones (ferr-
ocenyl amines) 1e–n.
Concerning the introduction of an aliphatic ethyl group on
the terminal N-ferrocenylmethylamino moiety (compounds 1e
and 1h), an elongation of the chain tends to decrease com-
pounds activity, in comparison with 1a and 1c, respectively. A
similar behaviour was noticed among derivatives with to a ben-
zyl (1f–g and 1i–j) or ferrocenylmethyl (1k–n) substitution on
this terminal N-ferrocenylmethylamino function. For these com-
pounds
with a tertiary amine moiety, the isosteric replacement
of the benzyl group by a ferrocenylmethyl one was found to
be beneficial in terms of antimalarial activity (i.e., 1f compared
to 1k, 1g to 1l, 1i to 1m, and 1j to 1n, respectively). On the
other hand, compound 1b displayed
a remarkable activity
(IC50 = 9.1–22.2 nM) against the three P. falciparum strains show-
ing different degrees of CQ-resistance. This result seems to cor-
roborate the first preliminary structure–activity relationships in
terms of substitution on the pyrroloquinoxaline skeleton and
on the N-ferrocenylmethylamine function, and also in terms of
linker’s nature.
As seen in Table 2, almost all compounds (except 1f and 2a)
have lower IC50 values for growth inhibition of CQ-sensitive strain
F32 than for growth inhibition of CQ-resistant strains FcB1 and
PFB. A comparison of the IC50 values for the growth inhibition of
the resistant between sensitive strains of P. falciparum suggests rel-
atively low levels of cross-resistance to CQ. The resistance index
values (Table 2), calculated from the ratio between IC50 for the
resistant and IC50 for sensitive strains of P. falciparum, were lower
than those for CQ, in a 0.4–2.2 range (FcB1) and a 0.5–2.4 range
(PFB), excepted for 1a (4.3 on PFB). As it was already reported for
the
1-{N-(pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-4[N-
(ferrocenylmethyl)-3-aminopropyl]piperazines or methylamines
1a–d.36,37 These secondary amines 1a–d were then engaged in
a reductive amination with sodium triacetoxyborohydride and
acetaldehyde or benzaldehyde to give the ferrocenic pyrrol-
o[1,2-a]quinoxaline derivatives 1e–j.15 The diferrocenic pyrrol-
o[1,2-a]quinoxaline derivatives 1k–n were also obtained by
reductive amination between intermediates 7a–d and ferro-
cene-carboxaldehyde (3 equiv) using NaHB(OAc)3 in methylene
chloride at room temperature (Scheme 2).15
The bispyrrolo[1,2-a]quinoxalines 9a–b were synthesized by
reacting 3,3’-diamino-N-methyldipropylamine with a two equiva-
lents amount of 6a–b in DMF at 130 °C, in the presence of potas-
sium carbonate as an inorganic base.31 The ferrocenic
bispyrrolo[1,2-a]quinoxalines 2a–b were then prepared by treating

9136
J. Guillon et al. / Bioorg. Med. Chem. 16 (2008) 9133–9144
R1
R1
R1
i
ii
NO2
NO2
NH2
NH2
N
N
3a-b
4a-b
N
N
N
N
N
iii
iv
v
R1
N
H
O
R1
Cl
R1
NH
X
NH2
5a-b
7a-d
6a-b
R
X
R
2
1
N
N
vi
H-
-
Fe
N
N
N
7-8a, 1a
7-8b, 1b
R1
NH
X
N
CH O-
3
-
N
8a-d
H-
-NCH -
3
7-8c, 1c
7-8d, 1d
-
-
CH O-
3
-NCH -
3
N
N
vii
H-
H-
-CH
1e
1f
3
Fe
N
N
N
N
R1
NH
X
N
H
N
N
1a-d
1e-j
CH O-
3
1g
N
viii
-NCH -
-CH
3
H-
H-
1h
1i
3
Fe
R1
N
NH
X
N
-NCH -
3
R2
1j
CH O-
3
-NCH -
3
Scheme 1. Synthesis of 4-substituted pyrrolo[1,2-a]quinoxaline derivatives 1a–j. Reagents and conditions: (i) DMTHF,
D; (ii) BiCl3, NaBH4, EtOH; (iii) CO(OCCl3)2, toluene, D;
(iv) POCl3,
D
; (v) Method A: Xantphos, H2N–(CH2)3–X–(CH2)3–NH2, K2CO3, Pd(OAc)2, dioxane,
D
; Method B: H2N–(CH2)3–X–(CH2)3–NH2, 1-pentanol, D; (vi) ferrocene-
carboxaldehyde, EtOH,
D
; (vii) NaBH4, MeOH, rt; (viii) R2–CHO, NaHB(OAc)3, CH2Cl2, rt.
N
i
7a-d
Fe
R1
N
NH
X
N
1k-n
Fe
R
X
1
H-
N
N
N
1k
1l
CH O-
3
N
H-
-NCH -
3
1m
1n
CH O-
3
-NCH -
3
Scheme 2. Synthesis of 4-substituted pyrrolo[1,2-a]quinoxaline derivatives 1k–n. Reagents and conditions: (i) ferrocene-carboxaldehyde, NaHB(OAc)3, CH2Cl2, rt.
bisquinolines or bisacridines, these results could be related to the
bulky structure of studied bispyrroloquinoxalines with respect to
recognition difficulties by the parasite efflux proteins, involved in
CQ resistance.38,39

J. Guillon et al. / Bioorg. Med. Chem. 16 (2008) 9133–9144
9137
N
N
N
i
6a-b
R1
NH
N
H
HN
N
R1
9a-b
N
N
N
ii
R
1
H-
9a, 2a
9b, 2b
R1
N
NH
N
HN
R1
CH O-
3
2a-b
Fe
Scheme 3. Synthesis of bispyrrolo[1,2-a]quinoxaline derivatives 2a–b. Reagents and conditions: (i) ferrocene-carboxaldehyde, NaHB(OAc)3, CH2Cl2, rt.
Table 1
indicates that drugs act by inhibiting the formation of haemazoin,
thus preventing haem detoxification. Three mechanisms of action
can be proposed: (1) direct binding of the drug to haem monomers
or dimers in solution which interferes with the crystallization of
haemazoin42; (2) enzymatic inhibition of a protein that catalyses
haemazoin crystallization44; and (3) chemiabsorption of the drug
onto crystallised haemazoin, leading to inhibition of further haem
aggregation.45 Thus, a panel of selected active compounds 1b, 1f–
h, 1l and 2a, representing various substitutions on the pyrroloqui-
noxaline nucleus, was tested for its ability to inhibit b-haematin
formation (Table 3), the synthetic equivalent of hemozoin, by using
the HPIA (heme polymerization inhibitory activity) assay.46 Exper-
Physical properties of the final amines 1–2
Compound
Salta
Mp (°C)b
%Yieldc
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
2a
2b
4 (COOH)2, 4 H2O
4 (COOH)2, 4 H2O
3(COOH)2, 3H2O
3(COOH)2, 3H2O
4 (COOH)2, 4 H2O
4 (COOH)2, 4 H2O
4 (COOH)2, 4 H2O
3(COOH)2, 3H2O
3(COOH)2, 3H2O
3(COOH)2, 3H2O
4 (COOH)2, 4 H2O
4 (COOH)2, 4 H2O
3(COOH)2, 3H2O
3(COOH)2, 3H2O
3(COOH)2, 3H2O
3(COOH)2, 3H2O
191
203
174
216
>260
248
178
126
96
123
218
210
99
86
84
81
75
63
82
85
75
69
64
76
71
73
77
75
76
iments were conducted by mixing haematin (0.4 lmol/well) with
0–10 compounds equivalents, previously dissolved in the appro-
priate solvent, immediately before adding acetic acid. Plates were
then incubated for 20 h at 37 °C, and the b-haematin content was
determined by spectrophotometry at 405 nm. Artemisinin and
other endoperoxide antimalarials also act through their binding
with haem (Table 3). The binding of artemisinin to haem both
potentiates the redox activity of haem and inhibits hemoglobin
digestion as well as heme detoxification function.47 Using this
microassay, 50% inhibition of haem polymerization (IC50) was ob-
tained using 1.63 0.21 equivalent of chloroquine in aqueous solu-
tion and 1.50 0.28 equivalent of artemisinin in DMSO, data in
general agreement with previous reports.46,47 On the other hand,
sulfadimethoxine belonging to the antimalarial antifolates family,
inhibits dihydropteroate synthase (DHPS), a key enzyme in folate
biosynthesis pathway,48 and is also used as reference. As expected,
no inhibition of b-haematin formation was observed at any tested
dose for compounds 1b, 1f–h and 1l, as also for the reference
sulfadimethoxine. Of the tested compounds, only 2a showed a
b-haematin formation inhibition, with a 5.85 0.10 equivalents
IC50 value (for 1b, 1f–h and 1l IC50 values were superior to 10
equiv). Hence, it could be hypothesized that activity of 2a could
be related to the inhibition of b-haematin formation.
170
147
156
a
The amines 1–2 were dissolved in 30 mL of 2-propanol, heated to boiling, and
treated with oxalic acid (4 or 5 equiv, based on the amount of the starting material).
The oxalate salts crystallized upon cooling, were collected by filtration, and were
washed with 2-propanol and Et2O.
b
Crystallization solvent: 2-PrOH–H2O.
The yields included the conversions into the oxalates.
c
2.3. Cytotoxicity test on MRC-5 cells
All bispyrrolo[1,2-a]quinoxalines showed cytotoxicity upon the
MRC-5 cells in the micromolar range. The different substituent
modulations had less influence on the cytotoxicity IC50 values than
that observed for the antimalarial activity. Hence, for the pipera-
zine series IC50s on the MRC-5 cells varied only from 620 to 1480
nM (Table 2). The substitution of the N-ferrocenylmethylamine
function of compounds 1 by a benzyl group (compounds 1f, 1g,
1i–j) induced decreases in the cytotoxicity upon MRC-5 cells. In-
dexes of selectivity are defined as the ratio between the IC50 value
on the MRC-5 cells and the IC50 value on the CQ-sensitive or CQ-
resistant P. falciparum strains. Compounds that demonstrated high
selectivity (high indexes of selectivity) should offer a potential of
safer therapy. This led to identify only compound 2b, with selectiv-
ity indexes >86, that could constitute suitable candidate for further
pharmacological studies.
2.5. Lipophilicity
Preliminary pharmacological results of tested compounds could
be discussed in terms of their physicochemical behaviour through
the partitioning theory, evaluated here by the distribution
coefficient D (usually expressed as logD).49 Consequently, HPLC
determination of logD was achieved for ferrocenic pyrrolo[1,2-
a]quinoxaline derivatives 1–2 at two distinct pHs (5.0 considered
close to the probable pH of the digestive vacuole, and 7.4 assumed
to be the cytosol pH). A plot of antimalarial IC50 versus logD values
at pH 7.4 and 5.0 was presented in Figures 3 and 4, respectively,
2.4. Inhibition of b-haematin formation
In vitro experiment studies have previously established that
quinoline antimalarial drugs, such as CQ, are associated with the
crystallization of haemazoin.40–43 In fact, present evidence

9138
J. Guillon et al. / Bioorg. Med. Chem. 16 (2008) 9133–9144
Table 2
In vitro sensitivity of P. falciparum strains and in vitro cytotoxicity on MRC-5 cells
Compound
IC50 values (nM)a
P. falciparum strains
Index of selectivityb
FcB1
Resistance index
MRC-5 cells
F32
PFB
FcB1c
PFBd
F32
19.5 4.2
11.9 2.6
9.1 1.5
FcB1
PFB
CQ
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
2a
2b
105.3 16.2
26.4 3.5
16.6 1.2
23.0 0.8
52.5 11.0
28.8 7.9
61.9 13.1
52.3 14.4
59.1 19.3
173.1 16.8
132.0 16.4
46.7 10.3
24.9 8.2
133.0 24.8
77.4 2.5
58.9 12.9
27.2 10.3
225.7 52.6
51.4 18.7
22.2 10.5
50.2 13.1
68.8 13.7
30.8 6.6
28.5 29.0
78.4 19.8
67.7 16.7
240.8 51.4
133.7 24.4
48.7 15.1
30.9 8.5
50,000 6000
750 10
730 20
2564.1
63.0
80.2
55.0
42.1
30.5
26.1
40.5
22.0
16.2
16.7
19.2
26.8
14.4
22.7
16.4
86.6
474.8
28.4
44.0
57.0
29.5
23.6
22.3
28.3
20.6
15.7
10.8
14.1
24.9
10.1
19.0
39.9
93.0
221.5
14.6
32.9
26.1
22.5
22.1
48.4
18.9
18.0
11.3
10.7
13.6
20.1
7.4
5.4
2.2
1.8
0.9
1.4
1.3
1.2
1.4
1.0
1.0
1.5
1.3
1.1
1.4
1.2
0.4
0.9
11.6
4.3
2.4
2.1
1.9
1.4
0.5
2.1
1.2
1.4
1.5
1.4
1.3
1.9
1.3
0.7
2.1
23.8 4.1
36.8 10.0
22.3 3.2
52.9 10.5
36.5 5.9
55.4 13.3
167.5 17.7
85.8 8.3
34.3 8.9
23.1 2.8
93.2 10.0
64.7 16.6
143.6 42.2
29.2 7.6
1310 40
1550 130
680 30
1380 50
1480 40
1220 170
2720 100
1430 100
660 20
620 130
1340 40
1470 50
2350 60
2530 60
179.9 18.3
86.8 13.8
106.3 20.8
61.4 22.8
16.9
22.1
41.2
a
IC50 values were measured on the chloroquine-sensitive strain F32/Tanzania and the chloroquine-resistant strains FcB1/Colombia and PFB/Brazil. The IC50 (nM) values
correspond to the mean standard deviation from three independent experiments.
b
Index of selectivity (IS) was defined as the ratio between the IC50 value on the MRC-5 cells and the IC50 value against the P. falciparum F32, FcB1 and PFB strains.
IC50 (FcB1)/IC50 (F32).
IC50 (PFB)/IC50 (F32).
c
d
Table 3
These results clearly indicate that the choice and the substitu-
Haem polymerisation inhibitory activity (HPIA) of different compounds used in the
tion of the terminal N-ferrocenylmethylamino function in the ami-
microtitre assay
no linker of the pyrrolo[1,2-a]quinoxaline moiety correlated with a
particular lipophilic behaviour, and seem to be fundamental for the
antimalarial activity of these new compounds.
Compound
Inhibitiona
IC50 (drug:haematin molar ratio)b
CQ-diphosphate
Artemisinin
+
+
1.63 0.21
1.50 0.28
Sulfadimethoxine
1b
1f
1g
1h
1l
À
À
À
À
À
À
+
À
3. Conclusion
À
À
À
In conclusion, novel series of ferrocenic pyrrolo[1,2-a]quinox-
aline derivatives 1–2 were synthesized from various substituted
nitroanilines and tested for in vitro activity upon the erythro-
cytic development of Plasmodium falciparum strains with differ-
ent chloroquine-resistance status. Promising pharmacological
results were obtained, leading to the selection of 1b as the most
potent candidate, but further development was suspended for
toxicity purpose. Nevertheless, it appears that these new ferroce-
nic pyrrolo[1,2-a]quinoxaline derivatives could open the way to
original valuable medicinal chemistry scaffolding. Furthermore,
this work contributes to validate the choice of the ferrocene
moiety as a template useful for designing new antimalarial
compounds.
À
À
2a
5.85 0.10
a
Inhibition: +, positive; À, negative.
b
The IC50 represents the molar equivalents of test compounds, relative to
haematin, required to inhibit haem polymerisation by 50% (data are expressed as
mean standard deviation from at least three different experiments in triplicate).
permitting to classify compounds in various subsets. At pH 7.4, all of
studied compounds were found to be highly lipid-soluble. Introduc-
tionof aferrocenicmoietyinthelateralchainconsiderablyincreased
the lipophilicity, compared to the purely organic CQ reference drug.
The most active pyrrolo[1,2-a]quinoxaline derivatives without sub-
stituent on the terminal N-ferrocenylmethylamino function of the
amino linker (1a–d), or substituted by an ethyl group (1e and 1h)
have logD values between 2.58 and 4.25. An heterogenous behav-
iour was found for less active compounds 1i, 1j and 1m, with log D
between 4.88 and 5.08. In this last case, introduction of a bulky ben-
zyl or a second ferrocenic moiety on the terminal N-ferrocenylmeth-
ylamino function of the amino linker considerably increases
lipophilicity. Finally, a third group of compounds (1f, 1g, 1k and 1l)
with logD values in the 5.13–5.18 range showed appreciable po-
tency with IC50s < 80 nM. Comparatively with 1i, 1j and 1m, they in-
cluded a piperazine ring linker instead of an N-methyl function.
At pH 5.0, a larger dispersion of logD values can be noticed. The
most active compounds 1a–h and 1k–l were found the less lipo-
philic with logD values inferior to 0.5. Conversely, the slightly less
active five compounds (1i–j, 1m–n and 2a) are more lipophilic
with the exception of 2b: they were found slightly less active
(IC50 = 58.9–40.8 nM) on P. falciparum strains.
4. Experimental
4.1. Chemistry
Commercially, reagents were used as received without addi-
tional purification. Melting points were determinated with an
SM-LUX-POL Leitz hot-stage microscope and are uncorrected. IR
spectra were recorded on a BRUKER IFS-25 spectrophotometer.
NMR spectra were recorded with tetramethylsilane as an internal
standard using a BRUKER AVANCE 300 spectrometer (1H, 13C, 2D
COSY). Splitting patterns have been designated as follows: s, sin-
glet; bs, broad singlet; d, doublet; t, triplet; q, quartet; qt, quintu-
plet; sex, sextuplet; m, multiplet. Analytical TLC was carried out on
0.25 precoated silica gel plates (POLYGRAM SIL G/UV254) with visu-
alisation by irradiation with a UV lamp. Silica gel 60 (70–230
mesh) was used for column chromatography. Elemental analyses
(C, H, N) for new compounds were performed by CNRS

J. Guillon et al. / Bioorg. Med. Chem. 16 (2008) 9133–9144
9139
Figure 3. Log D/activity relationship for pyrrolo[1,2-a]quinoxalines 1–2 at pH 7.4.
Figure 4. Log D/activity relationship for pyrrolo[1,2-a]quinoxalines 1–2 at pH 5.0.
(Vernaison-France) and agreed with the proposed structures with-
in 0.3% of the theoretical values.
quinoxaline 6a–b (20 mmol, 1 equiv) and 1,4-bis(3-aminopro-
pyl)piperazine or 3,3’-diamino-N-methyldipropylamine (60 mmol,
3 equiv) in 33 mL of 1-pentanol was heated and held at reflux
(160 °C) for 24 h. After cooling to room temperature, the mixture
was diluted with 45 mL of CH2Cl2. The organic layer was washed
with 10% NaOH (3Â 45 mL), then with 45 mL of water, and dried
over Na2SO4. The solvents were evaporated, and the residue was
purified by flash column chromatography using CH2Cl2/metha-
nol/NH4OH (80:20:1 v/v/v) as eluent to afford 7a–d.
4.1.1. General procedure for 1-{N-(pyrrolo[1,2-a]quinoxalin-4-
yl)-3-aminopropyl}-4-(3-aminopropyl)piperazine (7a–b) and
{N-(pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-(3-
aminopropyl)methylamine (7c–d)
Method A: A three-necked flask was flushed with nitrogen and
charged with Xantphos (3.0 mmol, 1.2 equiv) and dry dioxane
(25 mL). After degassing, Pd(OAc)2 (0.5 mmol, 0.2 equiv) was
charged, and the mixture was stirred under nitrogen for 10 min.
In another three-necked round-bottom flask, 4-chloropyrrolo[1,2-
a]quinoxaline 6a–b (2.46 mmol), 1,4-bis(3-aminopropyl)pipera-
zine or 3,3’-diamino-N-methyldipropylamine (3.0 mmol, 1.2
equiv), and K2CO3 (49.3 mmol, 20 equiv) were poured into dry
dioxane (35 mL). Then, the Pd(OAc)2/Xantphos solution was added
with a double syringe. The resulting mixture was subsequently
heated to reflux under N2 with vigorous stirring for 3 h. After cool-
ing, the solid material was filtered-off and washed with CH2Cl2 and
methanol. The solvent was evaporated, and the resulting crude
product was purified by flash column chromatography using
CH2Cl2/methanol/NH4OH (80:20:1 v/v/v) as eluent to give 7a–d
as yellow oil. Method B: A solution of 4-chloropyrrolo[1,2-a]
4.1.1.1. 1-{N-(Pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-
4-(3-aminopropyl)piperazine (7a).
Yellow oil (Method A:
52%, Method B: 49%). IR (KBr) 3350, 3280 (NH2 and NH). 1H NMR
(CDCl3) d 7.78 (dd, 1H, J = 2.65 and 1.45 Hz, H-1), 7.70 (dd, 1H,
J = 8.05 and 1.40 Hz, H-9), 7.64 (dd, 1H, J = 8.05 and 1.40 Hz,
H-6), 7.30 (ddd, 1H, J = 8.05, 7.35 and 1.40 Hz, H-8), 7.19 (ddd,
1H, J = 8.05, 7.35 and 1.40 Hz, H-7), 6.91 (t, 1H, J = 5.40 Hz, NH),
6.74 (dd, 1H, J = 3.90 and 1.45 Hz, H-3), 6.72 (dd, 1H, J = 3.90 and
2.65 Hz, H-2), 3.79 (m, 2H, NCH2), 2.80 (t, 2 H, J = 6.60 Hz, NCH2),
2.60 (t, 8H, J = 6.00 Hz, CH2 pip.), 2.50 (t, 4H, J = 6.60 Hz, NCH2),
1.89 (qt, 2H, J = 6.60 Hz, CH2), 1.73 (br s, 2H, NH2), 1.69 (qt, 2H,
J = 6.60 Hz, CH2). Anal. Calcd for C21H30N6: C, 68.82; H, 8.25; N,
22.93. Found: C, 69.03; H, 8.39; N, 22.78.

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J. Guillon et al. / Bioorg. Med. Chem. 16 (2008) 9133–9144
4.1.1.2. 1-{N-(7-Methoxypyrrolo[1,2-a]quinoxalin-4-yl)-3-ami-
nopropyl}-4-(3-aminopropyl)piperazine (7b). Yellow crystals
(Method A: 60%, Method B: 43%); mp 72 °C. IR (KBr) 3385, 3290
(NH2 and NH). 1H NMR (CDCl3) d 7.71 (m, 1H, H-1), 7.60 (d, 1H,
J = 8.85 Hz, H-9), 7.15 (d, 1H, J = 2.45 Hz, H-6), 6.84 (m, 1H, NH),
6.81 (dd, 1H, J = 8.85 and 2.45 Hz, H-8), 6.70 (m, 2H, H-3 and H-
2), 3.89 (s, 3H, CH3O), 3.78 (m, 2H, NCH2), 3.76 (br s, 2H, NH2),
2.93 (t, 2H, J = 6.20 Hz, NCH2), 2.61 (t, 8H, J = 6.10 Hz, CH2 pip.),
2.56 (t, 4H, J = 6.20 Hz, NCH2), 1.90 (qt, 2H, J = 6.20 Hz, CH2), 1.78
(qt, 2H, J = 6.20 Hz, CH2). Anal. Calcd for C22H32N6O: C, 66.63; H,
8.13; N, 21.19. Found: C, 66.38; H, 8.05; N, 20.87.
4.1.2.2. 1-{N-(7-Methoxypyrrolo[1,2-a]quinoxalin-4-yl)-3-ami-
nopropyl}-4-[N-(ferrocenylmethylene)-3-aminopropyl]pipera-
zine (8b). Orange crystals (89%); mp 73 °C. IR (KBr) 3280 (NH),
1620 (C@N). 1H NMR (CDCl3) d 8.16 (s, 1H, CH@N), 7.71 (m, 1H,
H-1), 7.61 (d, 1H, J = 8.85 Hz, H-9), 7.17 (d, 1H, J = 2.40 Hz, H-6),
6.84 (br s, 1H, NH), 6.80 (dd, 1H, J = 8.85 and 2.40 Hz, H-8), 6.77
(m, 1H, H-3), 6.68 (m, 1H, H-2), 4.66 (m, 2H, Cp-ortho), 4.39 (m,
2H, Cp-meta), 4.18 (s, 5H, Cp’), 3.90 (s, 3H, CH3O), 3.81 (m, 2H,
NCH2), 3.53 (t, 2H, J = 6.95 Hz, NCH2), 2.69 (m, 8H, CH2 pip.), 2.52
(t, 4H, J = 6.95 Hz, NCH2), 1.92 (m, 4H, CH2). Anal. Calcd for
C33H40N6OFe: C, 66.89; H, 6.80; N, 14.18. Found: C, 66.97; H,
7.00; N, 14.33.
4.1.1.3. {N-(Pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-(3-
aminopropyl)methylamine (7c). Yellow oil (Method A: 54%,
Method B: 41%). IR (KBr) 3400, 3290 (NH2 and NH). 1H NMR
(CDCl3) d 7.77 (dd, 1H, J = 2.75 and 1.45 Hz, H-1), 7.70 (dd, 1H,
J = 8.00 and 1.35 Hz, H-9), 7.65 (dd, 1H, J = 8.00 and 1.35 Hz,
H-6), 7.31 (ddd, 1H, J = 8.00, 7.30 and 1.35 Hz, H-8), 7.27 (br s,
1H, NH), 7.20 (ddd, 1H, J = 8.00, 7.30 and 1.35 Hz, H-7), 6.71 (dd,
1H, J = 4.05 and 1.45 Hz, H-3), 6.58 (dd, 1H, J = 4.05 and 2.75 Hz,
H-2), 3.79 (m, 2H, NCH2), 2.78 (t, 2 H, J = 7.20 Hz, NCH2), 2.60 (t,
2H, J = 7.20 Hz, NCH2), 2.50 (t, 2H, J = 7.20 Hz, NCH2), 2.34 (s, H,
NCH3), 1.88 (qt, 2H, J = 7.20 Hz, CH2), 1.73 (qt, 2H, J = 7.20 Hz,
CH2), 1.37 (br s, 2H, NH2). Anal. Calcd for C18H25N5: C, 69.42; H,
8.09; N, 22.49. Found: C, 69.57; H, 8.01; N, 22.63.
4.1.2.3. {N-(Pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-[N-
(ferrocenylmethylene)-3-aminopropyl]methylamine (8c). Orange
oil (96%). IR (KBr) 3275 (NH), 1625 (C@N). 1H NMR (CDCl3) d 8.09 (s,
1H, CH@N), 7.74 (dd, 1H, J = 2.80 and 1.30 Hz, H-1), 7.69 (dd, 1 H,
J = 8.00 and 1.30 Hz, H-9), 7.65 (dd, 1H, J = 8.00 and 1.30 Hz, H-6),
7.31 (ddd, 1H, J = 8.00, 7.35 and 1.30 Hz, H-8), 7.22 (br s, 1H, NH),
7.20 (ddd, 1H, J = 8.00, 7.35 and 1.30 Hz, H-7), 6.70 (dd, 1H,
J = 3.95 and 1.30 Hz, H-3), 6.59 (dd, 1H, J = 3.95 and 2.80 Hz, H-2),
4.61 (t, 2H, J = 1.30 Hz, Cp-ortho), 4.35 (t, 2H, J = 1.30 Hz, Cp-meta),
4.15 (s, 5H, Cp’), 3.83 (m, 2H, NCH2), 3.52 (t, 2H, J = 6.95 Hz, NCH2),
2.64 (t, 2H, J = 6.95 Hz, NCH2), 2.54 (t, 2H, J = 6.95 Hz, NCH2), 2.38
(s, 3H, NCH3), 1.95 (m, 4H, CH2). Anal. Calcd for C29H33N5Fe: C,
68.64; H, 6.55; N, 13.80. Found: C, 68.51; H, 6.32; N, 13.96.
4.1.1.4. {N-(7-Methoxypyrrolo[1,2-a]quinoxalin-4-yl)-3-amino-
propyl}-(3-aminopropyl)methylamine
(7d).
Yellow
oil
4.1.2.4. {N-(7-Methoxypyrrolo[1,2-a]quinoxalin-4-yl)-3-amino-
propyl}-[N-(ferrocenylmethylene)-3-aminopropyl]methylamine
(8d).
(Method A: 49%, Method B: 40%). IR (KBr) 3390, 3280 (NH2 and
NH). 1H NMR (CDCl3) d 7.68 (dd, 1H, J = 2.70 and 1.35 Hz, H-1),
7.59 (d, 1H, J = 8.85 Hz, H-9), 7.33 (br s, 1H, NH), 7.15 (d, 1H,
J = 2.75 Hz, H-6), 6.80 (dd, 1H, J = 8.85 and 2.75 Hz, H-8), 6.66
(dd, 1H, J = 3.90 and 2.70 Hz, H-2), 6.55 (dd, 1H, J = 3.90 and
1.35 Hz, H-3), 3.89 (s, 3H, CH3O), 3.78 (m, 2H, NCH2), 2.77 (t, 2H,
J = 7.05 Hz, NCH2), 2.59 (t, 2H, J = 7.05 Hz, NH2), 2.49 (t, 2H,
J = 7.05 Hz, NCH2), 2.34 (s, 3H, NCH3), 1.88 (qt, 2H, J = 7.05 Hz,
CH2), 1.72 (qt, 2H, J = 7.05 Hz, CH2), 1.34 (br s, 2H, NH2). Anal. Calcd
for C19H27N5O: C, 66.83; H, 7.97; N, 20.51. Found: C, 67.05; H, 8.08;
N, 20.69.
Orange oil (97%). IR (KBr) 3275 (NH), 1620 (C@N). 1H
NMR (CDCl3) d 8.08 (s, 1H, CH@N), 7.68 (dd, 1H, J = 2.65 and
1.25 Hz, H-1), 7.59 (d, 1H, J = 8.90 Hz, H-9), 7.24 (t, 1H,
J = 5.35 Hz, NH), 7.17 (d, 1H, J = 2.70 Hz, H-6), 6.80 (dd, 1H,
J = 8.90 and 2.70 Hz, H-8), 6.66 (dd, 1H, J = 3.90 and 2.65 Hz,
H-2), 6.58 (dd, 1H, J = 3.90 and 1.25 Hz, H-3), 4.60 (t, 2H,
J = 1.75 Hz, Cp-ortho), 4.34 (t, 2H, J = 1.75 Hz, Cp-meta), 4.14 (s,
5H, Cp’), 3.89 (s, 3H, CH3O), 3.80 (m, 2H, NCH2), 3.51 (t, 2H,
J = 6.70 Hz, NCH2), 2.64 (t, 2H, J = 6.70 Hz, NCH2), 2.54 (t, 2H,
J = 6.70 Hz, NCH2), 2.38 (s, 3H, NCH3), 1.92 (m, 4H, CH2). Anal. Calcd
for C30H35N5OFe: C, 67.04; H, 6.56; N, 13.03. Found: C, 66.94; H,
6.45; N, 13.23.
4.1.2. General procedure for 1-{N-(pyrrolo[1,2-a]quinoxalin-4-
yl)-3-aminopropyl}-4-[N-(ferrocenylmethylene)-3-aminopropyl]-
piperazine (8a–b) and {N-(pyrrolo[1,2-a]quinoxalin-4-yl)-3-
aminopropyl}-[N-(ferrocenylmethylene)-3-
4.1.3. General procedure for 1-{N-(pyrrolo[1,2-a]quinoxalin-4-
yl)-3-aminopropyl}-4-[N-(ferrocenylmethyl)-3-aminopropyl]-
piperazine (1a–b) and {N-(pyrrolo[1,2-a]quinoxalin-4-yl)-3-ami-
nopropyl}-[N-(ferrocenylmethyl)-3-aminopropyl]methylamine
(1c–d)
To a solution of compound 8a–d (4.0 mmol) in methanol
(40 mL) at 0 °C was added NaBH4 (20 mmol) in small portions over
30 min. After the mixture was stirred at 0 °C for 1.5 h, the reaction
was quenched by addition of 40 mL of a 0.5 M NaOH aqueous solu-
tion. Then the mixture was extracted with dichloromethane (2x
50 mL). The organic layers were combined, washed with brine,
dried over Na2SO4, and the solvent was evaporated under reduced
pressure to give 1a–d.
aminopropyl]methylamine (8c–d)
To a solution of compound 7a–d (5.46 mmol) in ethanol
(40 mL) was added ferrocene-carboxaldehyde (5.46 mmol). The
reaction mixture was then refluxed for 4 hours. The mixture was
evaporated to dryness under reduced pressure. After cooling, the
residue was extracted with methylene chloride (150 mL). The or-
ganic layer was dried over Na2SO4 and evaporated to dryness.
The oily residue was used without further purification.
4.1.2.1. 1-{N-(Pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-4-
[N-(ferrocenylmethylene)-3-aminopropyl]piperazine (8a). Orange
oil (83%). IR (KBr) 3270 (NH), 1620 (C@N). 1H NMR (CDCl3) d 8.16
(s, 1H, CH@N), 7.77 (dd, 1H, J = 2.75 and 1.25 Hz, H-1), 7.70 (dd,
1H, J = 8.05 and 1.30 Hz, H-9), 7.65 (dd, 1H, J = 8.05 and 1.30 Hz,
H-6), 7.30 (ddd, 1H, J = 8.05, 7.40 and 1.30 Hz, H-8), 7.20 (ddd, 1H,
J = 8.05, 7.40 and 1.30 Hz, H-7), 6.92 (t, 1H, J = 5.35 Hz, NH), 6.76
(dd, 1H, J = 3.85 and 1.25 Hz, H-3), 6.71 (dd, 1H, J = 3.85 and
2.75 Hz, H-2), 4.65 (t, 2H, J = 1.85 Hz, Cp-ortho), 4.38 (t, 2H,
J = 1.85 Hz, Cp), 4.20 (s, 5H, Cp’), 3.80 (m, 2H, NCH2), 3.53 (t, 2H,
J = 7.20 Hz, NCH2), 2.63 (t, 8H, J = 5.90 Hz, CH2 pip.), 2.51 (t, 4H,
J = 7.20 Hz, NCH2), 1.91 (m, 4H, CH2). Anal. Calcd for C32H38N6Fe: C,
68.32; H, 6.81; N, 14.94. Found: C, 68.56; H, 7.03; N, 15.11.
4.1.3.1. 1-{N-(Pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-4-
[N-(ferrocenylmethyl)-3-aminopropyl]piperazine (1a). Yellow
oil (81%). IR (KBr) 3280 (NH). 1H NMR (CDCl3) d 7.78 (dd, 1H,
J = 2.75 and 1.30 Hz, H-1), 7.71 (dd, 1H, J = 8.05 and 1.25 Hz, H-9),
7.65 (dd, 1H, J = 8.05 and 1.25 Hz, H-6), 7.31 (ddd, 1H, J = 8.05, 7.35
and 1.25 Hz, H-8), 7.20 (ddd, 1H, J = 8.05, 7.35 and 1.25 Hz, H-7),
6.88 (t, 1H, J = 5.40 Hz, NH), 6.72 (m, 2H, H-3 and H-2), 4.22 (m, 2H,
Cp-ortho), 4.14 (s, 5H, Cp’), 4.12 (m, 2H, Cp-meta), 3.79 (m, 2H,
NCH2), 3.56 (s, 2H, CH2Fc), 2.73 (t, 2H, J = 6.80 Hz, NCH2), 2.61 (t,
8H, J = 5.95 Hz, CH2 pip.), 2.49 (t, 4H, J = 6.80 Hz, NCH2), 2.32 (br s,

J. Guillon et al. / Bioorg. Med. Chem. 16 (2008) 9133–9144
9141
1H, NH), 1.89 (qt, 2H, J = 6.80 Hz, CH2), 1.75 (qt, 2H, J = 6.80 Hz, CH2).
13C NMR (CDCl3) d 149.6 (C-4), 137.3 (C-5a), 126.5 (C-6), 125.2 (C-7),
125.1 (C-9a), 122.4 (C-8), 119.8 (C-3a), 114.1 (C-9), 113.3 (C-1), 112.0
(C-3), 102.6 (C-2), 86.2 (Cp-1), 68.5 (Cp’), 68.4 (Cp-ortho), 67.9 (Cp-
meta), 58.4 (NCH2), 57.0 (NCH2), 53.4 (CH2 pip.), 53.3 (CH2 pip.),
48.8 (NCH2), 48.0 (NCH2), 41.5 (NCH2), 26.4 (CH2), 24.6 (CH2). Anal.
Calcd for C32H40N6Fe: C, 68.08; H, 7.14; N, 14.88. Found: C, 67.97;
H, 7.26; N, 15.08.
ous layer was washed with CH2Cl2. Then the organic layers were
mixed and dried over Na2SO4, the solvent was evaporated, and
the residue was purified by column chromatography using
CH2Cl2/methanol/NH4OH (80:20:1 v/v/v) as eluent to yield 1e–j.
4.1.4.1. 1-{N-(Pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-
4-[N-alkyl-N-(ferrocenylmethyl)-3-aminopropyl]piperazine
(1e). Yellow oil (76%). IR (KBr) 3280 (NH). 1H NMR (CDCl3) d 7.79
(m, 1H, H-1), 7.70 (d, 1H, J = 7.75 Hz, H-9), 7.67 (d, 1H, J = 7.75 Hz, H-6),
7.31 (t, 1H, J = 7.75 Hz, H-8), 7.20 (t, 1H, J = 7.75 Hz, H-7), 6.82 (br s, 1H,
NH), 6.72 (m, 2H, H-3 and H-2), 4.18 (m, 4H, Cp-ortho and Cp-meta),
4.13 (s, 5H, Cp’), 3.81 (m, 2H, NCH2), 3.56 (s, 2H, CH2Fc), 2.60 (m,
10H, NCH2 and CH2 pip.), 2.40 (m, 6H, NCH2), 1.91 (m, 2H, CH2), 1.72
(m, 2H, CH2), 1.07 (t, 3H, J = 6.95 Hz, CH3). Anal. Calcd for C34H44N6Fe:
C, 68.91; H, 7.48; N, 14.18. Found: C, 69.06; H, 7.61; N, 13.94.
4.1.3.2. 1-{N-(7-Methoxypyrrolo[1,2-a]quinoxalin-4-yl)-3-ami-
nopropyl}-4-[N-(ferrocenylmethyl)-3-aminopropyl]piperazine
(1b).
Orange oil (88%). IR (KBr) 3275 (NH). 1H NMR (CDCl3) d
7.71 (dd, 1H, J = 2.80 and 1.30 Hz, H-1), 7.60 (d, 1H, J = 8.85 Hz,
H-9), 7.16 (d, 1H, J = 2.65 Hz, H-6), 6.89 (t, 1H, J = 5.35 Hz, NH),
6.81 (dd, 1H, J = 8.85 and 2.65 Hz, H-8), 6.69 (m, 2H, H-3 and
H-2), 4.21 (m, 2H, Cp-ortho), 4.14 (s, 5H, Cp’), 4.12 (m, 2H, Cp-
meta), 3.90 (s, 3H, CH3O), 3.80 (m, 2H, NCH2), 3.55 (s, 2H, CH2Fc),
2.73 (t, 2H, J = 6.85 Hz, NCH2), 2.52 (m, 8H, CH2 pip.), 2.50 (t, 4H,
J = 6.85 Hz, NCH2), 2.35 (br s, 1H, NH), 1909 (qt, 2H, J = 6.85 Hz,
CH2), 1.75 (qt, 2H, J = 6.85 Hz, CH2). Anal. Calcd for C33H42N6OFe:
C, 66.66; H, 7.12; N, 14.13. Found: C, 66.87; H, 7.01; N, 13.88.
4.1.4.2. 1-{N-(Pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-
4-[N-benzyl-N-(ferrocenylmethyl)-3-aminopropyl]piperazine
(1f). Yellow oil (71%). IR (KBr) 3280 (NH). 1H NMR (CDCl3) d 7.79
(m, 1H, H-1), 7.70 (d, 1H, J = 8.00 Hz, H-9), 7.66 (d, 1H, J = 8.00 Hz,
H-6), 7.36-7.27 (m, 6H, Ar-H and H-8), 7.21 (t, 1H, J = 8.00 Hz, H-7),
6.94 (t, 1H, J = 5.30 Hz, NH), 6.74 (m, 2H, H-3 and H-2), 4.17 (m,
2H, Cp-ortho), 4.13 (m, 2H, Cp-meta), 4.07 (s, 5H, Cp’), 3.80 (m, 2H,
NCH2), 3.52 (s, 4H, CH2Fc and CH2Ph), 2.59 (m, 10H, NCH2 and CH2
4.1.3.3. {N-(Pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-[N-
(ferrocenylmethyl)-3-aminopropyl]methylamine (1c). Yellow
crystals (85%); mp 31 °C. IR (KBr) 3280 (NH). 1H NMR (CDCl3) d
7.77 (dd, 1H, J = 2.85 and 1.35 Hz, H-1), 7.70 (d, 1H, J = 8.00 Hz,
H-9), 7.67 (d, 1H, J = 8.00 Hz, H-6), 7.31 (t, 1H, J = 8.00 Hz, H-8),
7.24 (br s, 1H, NH), 7.20 (t, 1H, J = 8.00 Hz, H-7), 6.71 (dd, 1H,
J = 4.00 and 2.85 Hz, H-2), 6.60 (dd, 1H, J = 4.00 and 1.35 Hz,
H-3), 4.14 (m, 2H, Cp-ortho), 4.08 (m, 7H, Cp-meta and Cp’), 3.77
(m, 2H, NCH2), 3.49 (s, 2H, CH2Fc), 2.72 (t, 2H, J = 6.50 Hz, NCH2),
2.57 (t, 2H, J = 6.50 Hz, NCH2), 2.49 (t, 2H, J = 6.50 Hz, NCH2),
2.37 (br s, 1H, NH), 2.33 (s, 3H, NCH3), 1.88 (qt, 2H, J = 6.50 Hz,
CH2), 1.77 (qt, 2H, J = 6.50 Hz, CH2). 13C NMR (CDCl3) d 149.6
(C-4), 137.4 (C-5a), 126.4 (C-6), 125.2 (C-7), 125.0 (C-9a), 122.2
(C-8), 119.9 (C-3a), 113.9 (C-9), 113.3 (C-1), 112.2 (C-3), 102.5
(C-2), 86.2 (Cp-1), 68.5 (Cp-ortho), 68.3 (Cp’), 67.8 (Cp-meta), 56.9
(NCH2), 56.8 (NCH2), 49.0 (NCH2), 48.1 (NCH2), 42.3 (NCH3), 40.9
(NCH2Fc), 27.4 (CH2), 25.3 (CH2). Anal. Calcd for C29H35N5Fe: C,
68.37; H, 6.92; N, 13.74. Found: C, 68.45; H, 7.12; N, 13.98.
pip.), 2.42 (m, 4H, NCH2), 1.89 (m, 2H, CH2), 1.72 (m, 2H, CH2). 13
C
NMR (CDCl3) d 149.5 (C-4), 139.9 (C-1’), 137.2 (C-5a), 128.7 (C-2’ and
C-6’), 128.1 (C-3’ and C-5’), 126.7 (C-4’), 126.5 (C-6), 125.2 (C-7),
125.1 (C-9a), 122.4 (C-8), 119.8 (C-3a), 114.0 (C-9), 113.2 (C-1),
112.0 (C-3), 102.6 (C-2), 82.9 (Cp-1), 70.0 (Cp-ortho), 68.4 (Cp’),
67.7 (Cp-meta), 58.5 (NCH2), 57.6 (NCH2), 56.6 (NCH2), 53.4 (CH2
pip.), 53.3 (CH2 pip.), 52.6 (NCH2), 50.7 (NCH2), 41.5 (NCH2), 24.5
(2CH2). Anal. Calcd for C39H46N6Fe: C, 71.55; H, 7.08; N, 12.84.
Found: C, 71.64; H, 7.20; N, 13.07.
4.1.4.3. 1-{N-(7-Methoxypyrrolo[1,2-a]quinoxalin-4-yl)-3-ami-
nopropyl}-4-[N-benzyl-N-(ferrocenylmethyl)-3-aminopropyl]-
piperazine (1g).
Yellow oil (73%). IR (KBr) 3275 (NH). 1H NMR
(CDCl3) d 7.71 (dd, 1H, J = 2.65 and 1.30 Hz, H-1), 7.61 (d, 1H,
J = 8.85 Hz, H-9), 7.36-7.25 (m, 5H, Ar-H), 7.18 (d, 1H, J = 2.75 Hz,
H-6), 6.94 (t, 1H, J = 5.35 Hz, NH), 6.82 (dd, 1H, J = 8.85 and 2.75
Hz, H-8), 6.74 (dd, 1H, J = 3.90 and 1.30 Hz, H-3), 6.69 (dd, 1H,
J = 3.90 and 2.65 Hz, H-2), 4.18 (t, 2H, J = 1.75 Hz, Cp-ortho), 4.14
(t, 2H, J = 1.75 Hz, Cp-meta), 4.08 (s, 5H, Cp’), 3.90 (s, 3H, CH3O),
3.81 (m, 2H, NCH2), 3.53 (s, 4H, CH2Fc and CH2Ph), 2.61 (m, 10H,
NCH2 and CH2 pip.), 2.42 (m, 4H, NCH2), 1.90 (m, 2H, CH2), 1.72
(m, 2H, CH2). Anal. Calcd for C40H48N6OFe: C, 70.17; H, 7.07; N,
12.27. Found: C, 70.22; H, 7.01; N, 12.35.
4.1.3.4. {N-(7-Methoxypyrrolo[1,2-a]quinoxalin-4-yl)-3-amino-
propyl}-[N-(ferrocenylmethyl)-3-aminopropyl]methylamine
(1d). Yellow oil (83%). IR (KBr) 3280 (NH). 1H NMR (CDCl3) d 7.70
(dd, 1H, J = 2.85 and 1.30 Hz, H-1), 7.60 (d, 1H, J = 8.90 Hz,
H-9), 7.26 (br s, 1H, NH), 7.17 (d, 1H, J = 2.75 Hz, H-6), 6.80 (dd, 1H,
J = 8.90 and 2.75 Hz, H-8), 6.67 (dd, 1H, J = 3.95 and 2.85 Hz, H-2),
6.56 (dd, 1H, J = 3.95 and 1.30 Hz, H-3), 4.14 (t, 2H, J = 1.70 Hz,
Cp-ortho), 4.08 (m, 7H, Cp-meta and Cp’), 3.90 (s, 3H, CH3O), 3.77
(m, 2H, NCH2), 3.48 (s, 2H, CH2Fc), 2.72 (t, 2H, J = 6.40 Hz, NCH2),
2.58 (t, 2H, J = 6.40 Hz, NCH2), 2.50 (t, 2H, J = 6.40 Hz, NCH2), 2.41
(br s, 1H, NH), 2.33 (s, 3H, NCH3), 1.88 (qt, 2H, J = 6.40 Hz, CH2),
1.77 (qt, 2H, J = 6.40 Hz, CH2). Anal. Calcd for C30H37N5OFe: C,
66.79; H, 6.91; N, 12.98. Found: C, 66.58; H, 6.83; N, 13.04.
4.1.4.4.
{N-(Pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-
[N-ethyl-N-(ferrocenylmethyl)-3-aminopropyl]methylamine
(1h). Yellow oil (78%). IR (KBr) 3285 (NH). 1H NMR (CDCl3) d 7.79(dd,
1H, J = 2.80 and 1.30 Hz, H-1), 7.70 (dd, 1H, J = 7.95 and 1.30 Hz, H-9),
7.66 (dd, 1H, J = 7.95 and 1.30 Hz, H-6), 7.43 (t, 1H, J = 5.40Hz, NH),
7.31 (ddd, 1H, J = 7.95, 7.30 and 1.30 Hz, H-8), 7.20 (ddd, 1H, J = 7.95,
7.30 and 1.30 Hz, H-7), 6.72 (dd, 1H, J = 3.85 and 2.80 Hz, H-2), 6.59
(dd, 1H, J = 3.85and 1.30Hz, H-3), 4.08 (m, 4H, Cp-ortho and Cp-meta),
4.04 (s, 5H, Cp’), 3.80 (m, 2H, NCH2), 3.48 (s, 2H, CH2Fc), 2.61 (t, 2H,
J = 6.40 Hz, NCH2), 2.41 (m, 6H, NCH2), 2.34 (s, 3H, NCH3), 1.88 (qt,
2H, J = 6.40 Hz, CH2), 1.75 (qt, 2H, J = 6.40 Hz, CH2), 1.01 (t, 3H,
J = 7.10 Hz, CH3). Anal. Calcd for C31H39N5Fe: C, 69.27; H, 7.31; N,
13.03. Found: C, 69.03; H, 7.18; N, 12.89.
4.1.4. General procedure for 1-{N-(pyrrolo[1,2-a]quinoxalin-4-
yl)-3-aminopropyl}-4-[N-alkyl-N-(ferrocenylmethyl)-3-amino-
propyl]piperazine (1e–g) and {N-(pyrrolo[1,2-a]quinoxalin-4-
yl)-3-aminopropyl}-[N-alkyl-N-(ferrocenylmethyl)-3-amino-
propyl]methylamine (1h–j)
To a solution of compounds 1a–d (0.8 mmol) in 10 mL of dry
CH2Cl2 were added acetaldehyde (2.40 mmol, 3 equiv) or benzalde-
hyde (1.7 mmol, 1.5 equiv) and NaHB(OAc)3 (1.2 mmol, 1.5 equiv).
After the mixture was stirred at room temperature for 24 h, a total
of 15 mL of aqueous 1 M NaOH solution was introduced. The mix-
ture left for 15 min, the organic layer was separated, and the aque-
4.1.4.5.
{N-(Pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-
[N-benzyl-N-(ferrocenylmethyl)-3-aminopropyl]methylamine
(1i). Yellow oil (87%). IR (KBr) 3275 (NH). 1H NMR (CDCl3) d 7.78
(dd, 1H, J = 2.85 and 1.30 Hz, H-1), 7.71 (d, 1H, J = 8.15 Hz, H-9),

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J. Guillon et al. / Bioorg. Med. Chem. 16 (2008) 9133–9144
7.67 (d, 1H, J = 8.15 Hz, H-6), 7.34–7.25 (m, 7H, Ar-H, H-8 and NH),
7.20 (t, 1H, J = 8.15 Hz, H-7), 6.71 (dd, 1H, J = 3.90 and 2.85 Hz,
H-2), 6.62 (dd, 1H, J = 3.90 and 1.30 Hz, H-3), 4.09 (m, 4H, Cp-ortho
and Cp-meta), 4.00 (s, 5H, Cp’), 3.80 (m, 2H, NCH2), 3.48 (s, 4H,
CH2Fc and CH2Ph), 2.61 (m, 2H, NCH2), 2.43 (m, 4H, NCH2), 2.35
(s, 3H, NCH3), 1.89 (m, 2H, CH2), 1.75 (m, 2H, CH2). Anal. Calcd
for C36H41N5Fe: C, 72.11 H, 6.89; N, 11.68. Found: C, 71.85; H,
6.95; N, 11.72.
4.1.5.3. {N-(Pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-[N,
N-bis(ferrocenylmethyl)-3-aminopropyl]methylamine (1m). Yellow
crystals (82%); mp 48 °C. IR (KBr) 3280 (NH). H NMR (CDCl3) d 7.79
(m, 1H, H-1), 7.71 (d, 1H, J = 7.90 Hz, H-9), 7.68 (d, 1H, J = 7.90 Hz,
H-6), 7.31 (m, 2H, H-8 and NH), 7.20 (t, 1H, J = 7.90 Hz, H-7), 6.74
(m, 1H, H-2), 6.60 (m, 1H, H-3), 4.13 (m, 4H, 2Cp-ortho), 4.10 (m,
4H, 2Cp-meta), 4.04 (s, 10H, 2Cp’), 3.80 (m, 2H, NCH2), 3.41 (s, 4H,
2CH2Fc), 2.59 (m, 2H, NCH2), 2.40 (m, 4H, NCH2), 2.34 (s, 3H, NCH3),
1.87 (m, 2H, CH2), 1.71 (m, 2H, CH2). Anal. Calcd for C40H45N5Fe2: C,
67.90; H, 6.41; N, 9.90. Found: C, 68.02; H, 6.46; N, 10.08.
1
4.1.4.6. {N-(7-Methoxypyrrolo[1,2-a]quinoxalin-4-yl)-3-amino-
propyl}-[N-benzyl-N-(ferrocenylmethyl)-3-aminopropyl]methyl-
amine (1j). Yellow oil (83%). IR (KBr) 3280 (NH). 1H NMR (CDCl3)
d 7.70 (dd, 1H, J = 2.70 and 1.30 Hz, H-1), 7.60 (d, 1H, J = 8.85 Hz,
H-9), 7.47 (t, 1H, J = 5.35 Hz, NH), 7.32-7.24 (m, 5H, Ar-H), 7.18
(d, 1H, J = 2.70 Hz, H-6), 6.81 (dd, 1H, J = 8.85 and 2.70 Hz, H-8),
6.67 (dd, 1H, J = 3.85 and 2.70 Hz, H-2), 6.57 (dd, 1H, J = 3.85 and
1.30 Hz, H-3), 4.12 (m, 4H, Cp-ortho and Cp-meta), 4.00 (s, 5H,
Cp’), 3.90 (s, 3H, CH3O), 3.78 (m, 2H, NCH2), 3.47 (s, 4H, CH2Fc
and CH2Ph), 2.61 (m, 2H, NCH2), 2.44 (m, 4H, NCH2), 2.34 (s, 3H,
NCH3), 1.88 (m, 2H, CH2), 1.76 (m, 2H, CH2). Anal. Calcd for
C37H43N5OFe: C, 70.58 H, 6.88; N, 11.12. Found: C, 70.54; H, 6.99;
N, 11.02.
4.1.5.4. {N-(7-Methoxypyrrolo[1,2-a]quinoxalin-4-yl)-3-amino-
propyl}-[N,N-bis(ferrocenylmethyl)-3-aminopropyl]methylamine
(1n). Yellow oil (83%). IR (KBr) 3275 (NH). 1H NMR (CDCl3) d 7.71
(dd, 1H, J = 2.70 and 1.30 Hz, H-1), 7.60 (d, 1H, J = 8.90 Hz, H-9),
7.38 (t, 1H, J = 5.40 Hz, NH), 7.19 (d, 1H, J = 2.80 Hz, H-6), 6.80
(dd, 1H, J = 8.90 and 2.80 Hz, H-8), 6.70 (dd, 1H, J = 3.90 and 2.70
Hz, H-2), 6.57 (dd, 1H, J = 3.90 and 1.30 Hz, H-3), 4.14 (t, 4H,
J = 1.75 Hz, 2Cp-ortho), 4.09 (t, 4H, J = 1.75 Hz, 2Cp-meta), 4.05 (s,
10H, 2Cp’), 3.92 (s, 3H, CH3O), 3.80 (m, 2H, NCH2), 3.40 (s, 4H,
2CH2Fc), 2.58 (t, 2H, J = 6.50 Hz, NCH2), 2.39 (m, 4H, NCH2), 2.33
(s, 3H, NCH3), 1.87 (qt, 2H, J = 6.50 Hz, CH2), 1.69 (qt, 2H, J = 6.50
Hz, CH2). Anal. Calcd for C41H47N5OFe2: C, 66.77; H, 6.42; N, 9.49.
Found: C, 67.04; H, 6.49; N, 9.55.
4.1.5. General procedure for 1-{N-(pyrrolo[1,2-a]quinoxalin-4-
yl)-3-aminopropyl}-4-[N,N-bis(ferrocenylmethyl)-3-aminopropyl]-
piperazine (1k–l) and {N-(pyrrolo[1,2-a]quinoxalin-4-yl)-3-amino-
propyl}-[N,N-bis(ferrocenylmethyl)-3-aminopropyl]methylamine
(1m–n)
To a solution of compound 7a–d (1.36 mmol) in 10 mL of dry
CH2Cl2 were added ferrocene-carboxaldehyde (4.08 mmol, 3 equiv)
and NaHB(OAc)3 (4.08 mmol, 3 equiv). After the mixture was stir-
red at room temperature for 24 hours, a total of 15 mL of aqueous
1 M NaOH solution was introduced. The mixture left for 15 min,
the organic layer was separated, and the aqueous layer was
washed with CH2Cl2. Then the organic layers were mixed and dried
over Na2SO4, the solvent was evaporated, and the residue was puri-
fied by column chromatography using CH2Cl2/methanol/NH4OH
(80:20:1 v/v/v) as eluent to yield 1k–n.
4.1.6. Bis{N-(pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-
amine (9a)
To a solution of 6a (7.4 mmol, 2 equiv) in dimethylformamide
(20 mL) were added K2CO3 (8.1 mmol, 2.2 equiv) then N-(3-amino-
propyl)-1,3-propanediamine (3.7 mmol, 1 equiv). The reaction
mixture was heated at 120–130 °C for 4 h and, after cooling, was
poured into water (100 mL). The precipitate was filtered, washed
with water and dissolved in dichloromethane. The organic layer
was washed with water (150 mL), dried over Na2SO4 and evapo-
rated to dryness to give 9a. Yellow crystals (81%), mp 89 °C. IR
(KBr) 3415 and 3300 (NH). 1H NMR (CDCl3) d 7.78 (dd, 2H,
J = 2.75 and 1.25 Hz, H-1), 7.72 (dd, 2H, J = 8.05 and 1.35 Hz,
H-9), 7.63 (dd, 2H, J = 8.05 and 1.35 Hz, H-6), 7.29 (ddd, 2H,
J = 8.05, 7.35 and 1.35 Hz, H-8), 7.24 (ddd, 2H, J = 8.05, 7.35 and
1.35 Hz, H-7), 6.69 (m, 4H, H-2 and H-3), 6.21 (m, 2H, NH), 3.86
(m, 4H, NCH2), 2.87 (t, 4H, J = 6.10 Hz, NCH2), 2.00 (qt, 4H,
J = 6.10 Hz, CH2), 1.95 (br s, 1H, NH). Anal. Calcd for C28H29N7: C,
72.54; H, 6.30; N, 21.15. Found: C, 72.61; H, 6.24; N, 21.20.
4.1.5.1. 1-{N-(Pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-4-
[N,N-bis(ferrocenylmethyl)-3-aminopropyl]piperazine (1k). Yellow
oil (85%). IR (KBr) 3270 (NH). 1H NMR (CDCl3) d 7.79 (m, 1H, H-1),
7.71 (d, 1H, J = 7.70 Hz, H-9), 7.66 (d, 1H, J = 7.70 Hz, H-6), 7.31 (t,
1H, J = 7.70 Hz, H-8), 7.21 (t, 1H, J = 7.70 Hz, H-7), 6.94 (t, 1H, J = 5.35
Hz, NH), 6.74 (m, 2H, H-3 and H-2), 4.20 (m, 4H, 2Cp-ortho), 4.14 (m,
4H, 2Cp-meta), 4.11 (s, 10H, 2Cp’), 3.80 (m, 2H, NCH2), 3.45 (s, 4H,
2CH2Fc), 2.60 (m, 10H, NCH2 and CH2 pip.), 2.37 (m, 4H, NCH2), 1.91
(m, 2H, CH2), 1.69 (m, 2H, CH2). 13C NMR (CDCl3) d 149.6 (C-4),
137.2 (C-5a), 126.5 (C-6), 125.2 (C-7), 125.1 (C-9a), 122.4 (C-8),
119.8 (C-3a), 114.0 (C-9), 113.3 (C-1), 112.0 (C-3), 102.6 (C-2), 83.2
(Cp-1), 70.1 (2Cp-ortho), 68.4 (2Cp’), 67.8 (2Cp-meta), 58.5 (NCH2),
56.7 (NCH2), 53.5 (CH2 pip.), 53.4 (CH2 pip.), 52.5 (NCH2), 49.9
(NCH2), 41.5 (NCH2), 24.6 (2CH2). Anal. Calcd for C43H50N6Fe2: C,
67.72; H, 6.61; N, 11.02. Found: C, 67.58; H, 6.75; N, 11.21.
4.1.7. General procedure for Bis{N-(pyrrolo[1,2-a]quinoxalin-4-
yl)-3-aminopropyl}-N-ferrocenylmethylamine (2a–b)
To a solution of compound 9a–b (1.00 mmol) in 10 mL of dry
CH2Cl2 were added ferrocene-carboxaldehyde (1.5 mmol, 1.5
equiv) and NaHB(OAc)3 (1.5 mmol, 1.5 equiv). After the mixture
was stirred at room temperature for 24 h, a total of 15 mL of aque-
ous 1 M NaOH solution was introduced. The mixture left for
15 min, the organic layer was separated, and the aqueous layer
was washed with CH2Cl2. Then the organic layers were mixed
and dried over Na2SO4, the solvent was evaporated, and the residue
was purified by column chromatography using CH2Cl2/methanol/
NH4OH (80:20:1 v/v/v) as eluent to yield 2a–b.
4.1.5.2. 1-{N-(7-Methoxypyrrolo[1,2-a]quinoxalin-4-yl)-3-ami-
nopropyl}-4-[N,N-bis(ferrocenylmethyl)-3-aminopropyl]piper-
azine (1l).
Yellow oil (83%). Yellow oil (88%). IR (KBr) 3280
4.1.7.1. Bis{N-(pyrrolo[1,2-a]quinoxalin-4-yl)-3-aminopropyl}-
N-ferrocenylmethylamine (2a). Pale-yellow crystals (76%); mp
94 °C. IR (KBr) 3300 (NH). 1H NMR (CDCl3) d 7.58 (dd, 2H, J = 2.80
and 1.25 Hz, H-1), 7.70 (dd, 2H, J = 8.00 and 1.40 Hz,
H-9), 7.66 (dd, 2H, J = 8.00 and 1.40 Hz, H-6), 7.30 (ddd, 2H,
J = 8.00, 7.40 and 1.40 Hz, H-8), 7.20 (ddd, 2H, J = 8.00, 7.40 and
1.40 Hz, H-7), 6.65 (dd, 2H, J = 3.85 and 2.80 Hz, H-2), 6.53 (dd,
2H, J = 3.85 and 1.25 Hz, H-3), 6.17 (t, 2H, J = 5.45 Hz, NH), 4.22
(t, 2H, J = 1.75 Hz, Cp-ortho), 4.13 (t, 2H, J = 1.75 Hz, Cp-meta),
(NH). 1H NMR (CDCl3) d 7.72 (m, 1H, H-1), 7.61 (d, 1H, J = 8.70
Hz, H-9), 7.16 (d, 1H, J = 2.15 Hz, H-6), 7.00 (t, 1H, J = 5.35 Hz,
NH), 6.81 (dd, 1H, J = 8.70 and 2.15 Hz, H-8), 6.71 (m, 2H, H-3
and H-2), 4.20 (m, 4H, 2Cp-ortho), 4.15 (m, 4H, 2Cp-meta), 4.11
(s, 10H, 2Cp’), 3.90 (s, 3H, CH3O), 3.80 (m, 2H, NCH2), 3.45 (s, 4H,
2CH2Fc), 2.61 (m, 10H, NCH2 and CH2 pip.), 2.38 (m, 4H, NCH2),
1.90 (m, 2H, CH2), 1.69 (m, 2H, CH2). Anal. Calcd for C44H52N6OFe2:
C, 66.67; H, 6.61; N, 10.60. Found: C, 66.77; H, 6.72; N, 10.47.

J. Guillon et al. / Bioorg. Med. Chem. 16 (2008) 9133–9144
9143
4.10 (s, 5H, Cp’), 3.75 (m, 4H, NCH2), 3.65 (s, 2H, CH2Fc), 2.65 (t, 4H,
J = 6.45 Hz, NCH2), 1.95 (qt, 4H, J = 6.45 Hz, CH2). Anal. Calcd for
C39H39N7Fe: C, 70.80; H, 5.94; N, 14.82. Found: C, 70.96; H, 5.88;
N, 14.75.
each fraction was transferred onto a new plate and serial four-fold
dilutions in 0.1 M NaOH were carried out. The amount of haematin
was determinated by measuring the absorbance at 405 nm using
an automatic plate reader (SAFIRE II, Tecan, Austria). The data are
expressed as the molar equivalents of test compounds relative to
haematin required to inhibit heme polymerization by 50%. Each
compound was tested in triplicate. Chloroquine diphosphate in
H2O and artemisinin in DMSO were used as positive controls. Sul-
fadimethoxine in DMSO was used as negative control.
4.1.7.2. Bis{N-(7-methoxypyrrolo[1,2-a]quinoxalin-4-yl)-3-ami-
nopropyl}-N-ferrocenylmethylamine (2b). Beige crystals (82%);
mp 108 °C. IR (KBr) 3295 (NH). 1H NMR (CDCl3) d 7.58 (m, 2H, H-1),
7.59 (d, 2H, J = 8.85 Hz, H-9), 7.14 (d, 2H, J = 2.80 Hz, H-6), 6.81 (dd,
2H, J = 8.85 and 2.80 Hz, H-8), 6.62-6.51 (m, 4H, H-2 and H-3), 6.26
(m, 2H, NH), 4.20 (m, 2H, Cp-ortho), 4.10 (m, 7H, Cp-meta and Cp’),
3.87 (m, 4H, NCH2), 3.85 (s, 6H, 2CH3O), 3.73 (s, 2H, CH2Fc), 2.65 (t,
4H, J = 6.15 Hz, NCH2), 1.94 (qt, 4H, J = 6.15 Hz, CH2). Anal. Calcd
for C41H43N7O2Fe: C, 68.23; H, 6.01; N, 13.58. Found: C, 68.10; H,
6.23; N, 13.71.
4.2.3. Cytotoxicity test upon human embryonic cells
A human diploid embryonic lung cell line (MRC-5, Bio-Whittak-
er 72211D) was used to assess the cytotoxic effects towards
eukaryotic host cells. MRC-5 cells were seeded at 5,000 cells per
well in 100
tions of the drug were added in 200
l
l. After 24 h, the cells were washed and twofold dilu-
l standard culture medium
l
4.2. Biological assays
(RPMI medium + 5% fetal calf serum) and maintained for five days
under 5% CO2 atmosphere. The final DMSO concentration in the
culture remained below 0.1%. Untreated cultures were included
as controls. The cytotoxicity was determined using the colorimetric
MTT assay according to the manufacturer’s recommendations (Cell
proliferation kit I, Roche Applied Science, France) and scored as a
percentage of reduction in absorption at 540 nm of treated cultures
versus untreated control cultures. IC50 values were obtained from
the drug concentration–response curve. The results were ex-
pressed as the mean the standard deviations determined from
several independent experiments. The index of selectivity was de-
fined as the ratio of the IC50 value on MRC-5 to that of P. falciparum.
4.2.1. In vitro P. falciparum culture and drug assays
Plasmodium falciparum strains were maintained continuously in
culture on human erythrocytes as described by Trager and Jen-
sen.50 In vitro antiplasmodial activity was determined using a
modification of the semi-automated microdilution technique of
Desjardins et al.51 P. falciparum CQ-sensitive (F32/Tanzania) and
CQ-resistant (FcB1R/Colombia and PFB/Brazil) strains were used
in sensitivity testing. Stock solutions of CQ diphosphate and test
compounds were prepared in sterile distilled water and DMSO,
respectively. Drug solutions were serially diluted with culture
medium and added to asynchronous parasite cultures (1% parasite-
mia and 1% final hematocrite) in 96-well plates for 24 h, at 37 °C,
4.3. Partition coefficients À logD (pH 7.4 and 5.0)
prior to the addition of 0.5 l
Ci of [3H]hypoxanthine (1–5 Ci/mmol;
Amersham, Les Ulis, France) per well, for 24 h. The growth inhibi-
tion for each drug concentration was determined by comparison
of the radioactivity incorporated into the treated culture with that
in the control culture (without drug) maintained on the same plate.
The concentration causing 50% inhibition (IC50) was obtained from
the drug concentration–response curve and the results were ex-
pressed as the mean the standard deviations determined from
several independent experiments. The DMSO concentration never
exceeded 0.1% and did not inhibit the parasite growth.
In this study, the relative logD (pH 7.4 and 5.0) were assessed at
pH7.4and5.0bythemicro-HPLCmethod. Determinationswere per-
formed with a chromatographic apparatus (Spectra Series, San Jose,
USA) equipped with a model P1000XR pump and a model SCM 1000
vacuum membrane degasser, a model UV 150 ultraviolet detector
(k = 210 nm) and a ChromJet data module integrator (ThermoFinni-
gan, San Jose, USA). The reversed phase column used, was a Stability
(C.I.L. Cluzeau) C18 (4.6 Â 150 mm; 5
lm particle size) with a mobile
phase consisting of acetonitrile—potassium dihydrogenophosphate
6.24 Â 10À2 M [KH2PO4/K2HPO4] (pH 5) (55:45, v/v (1h–j, 1m–n,
2a–b)), acetonitrile—phosphate buffer (pH 6) (35:65, v/v (1c)),
(40:60, v/v (1a–b, 1e)), (50:50, v/v (1f)), (55:45, v/v (1k–l)), (60:40,
v/v (1g)) acetonitrile—phosphate buffer (pH 7) (55:45, v/v (1d)).
The compounds were partitioned between n-octanol (HPLC grade)
and phosphate buffer (pH 7.4). Octanol was presatured with the ade-
quate phosphate buffer (1%), and conversely. An amount of 1 mg of
each compound was dissolved in an adequate volume of methanol
in order to achieve 1 mg/mL stock solutions. Then, an appropriate
aliquot of these methanolic solutions was dissolved in buffer to ob-
4.2.2. b-Haematin formation assay
Haematin (ferriprotoporphyrin IX hydroxide) (H-3281) and sul-
fadimethoxine (S-7385) were obtained from Sigma Co. (St. Louis,
MO, USA), chloroquine diphosphate (25745) and acetic acid were
from Fluka and dimethylsulfoxide (DMSO) and NaOH were from
Fisher Scientific (UK). Methanol was purchased from Elvetec
(Genas, France). Artemisinin was kindly provided by Pr Benakis
(Geneva, Switzerland).
The drugs effects upon b-haematin formation were assessed
according to Basilico et al. with minor modifications.46 One hun-
dred microlitres of a 4 mM solution of haematin, previously dis-
solved in 0.1 M NaOH, was distributed in 96-well microplates
tain final concentration of 50
chromatographic conditions, 50
into the chromatograph, leading to the determination of a peak area
before partitioning (W0). In screw-capped tubes, 4000 L of the
aqueous phase (Vaq) was then added to 10 L of n-octanol (Voct).
l
g/mL. Under the above-described
lL of aqueous phase was injected
(0.4
(20
l
mol/well). Drugs were added from DMSO stock solutions
l
ll) at a drug:haem ratio between 0:1 and 10:1. Controls con-
l
tained an equal amount of DMSO. Haematin polymerization was
initiated by adding 0.8 mmol of acetic acid (50 l) at a final pH of
3. The total volume of reaction was fitted to 200 l with 0.1 M
The mixture was shaken by mechanical rotation during 30 min, fol-
lowed by centrifugation achieved at 3000 rpm during 15 min. An
amount of 50 lL of the lower phase was injected into the chromato-
l
l
NaOH and the assay mixtures were incubated at 37 °C for 20 h.
Plates were then centrifuged at 3300 g for 15 min and the soluble
fraction of unprecipitated material collected. The remaining pellets
graph column. This led to the determination of a peak area after par-
titioning (W1). For each compound, the logD value was calculated
using the formula: logD = log[(W0 À W1)Vaq/W1Voct].
were resuspended with 200 ll of DMSO to remove unreacted
haematin. Plates were then centrifuged again at 3300g for
15 min. The DMSO-soluble fraction was collected and the pellets,
consisting of a pure precipitate of b-haematin, were dissolved in
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