Synthesis, Antimalarial Activity, and Molecular Modeling of New Pyrrolo[1,2-a]quinoxalines, Bispyrrolo[1,2-a]quinoxalines, Bispyrido[3,2-e]pyrrolo[1,2-a]pyrazines, and Bispyrrolo[1,2-a]thieno[3,2-e]pyrazines

Article abstract of DOI:10.1021/jm0310840

Three pyrrolo[1,2-a]quinoxalines, 15 bispyrrolo[1,2-a]quinoxalines, bispyrido[3,2-e]pyrrolo[1,2-a]pyrazines, and bispyrrolo[1,2-a]thieno[3,2-e]pyrazines were synthesized from various substituted nitroanilines or nitropyridines and tested for their in vitro activity upon the erythrocytic development of Plasmodium falciparum strains with different chloroquine-resistance status. Bispyrrolo[1,2-a]quinoxalines showed superior antimalarial activity with respect to monopyrrolo[1,2-a]quinoxalines. The best activity was observed with bispyrrolo[1,2-a]quinoxalines linked by a bis(3-aminopropyl)piperazine. Moreover, it was observed that the presence of a methoxy group on the pyrrolo[1,2-a]quinoxaline nucleus increased the pharmacological activity. Drug effects upon β-hematin formation were assayed and showed similar or higher inhibitory activities than CQ. A possible mechanism of interaction implicating binding of pyrroloquinoxalines to β-hematin was supported by molecular modeling.

Full text of DOI:10.1021/jm0310840

J . Med. Chem. 2004, 47, 1997-2009
1997
Syn th esis, An tim a la r ia l Activity, a n d Molecu la r Mod elin g of New
P yr r olo[1,2-a ]qu in oxa lin es, Bisp yr r olo[1,2-a ]qu in oxa lin es,
Bisp yr id o[3,2-e]p yr r olo[1,2-a ]p yr a zin es, a n d
Bisp yr r olo[1,2-a ]th ien o[3,2-e]p yr a zin es
J ean Guillon,† Philippe Grellier,‡ Mehdi Labaied,‡ Pascal Sonnet,§ J ean-Michel Le´ger,† Re´becca De´prez-Poulain,#
Isabelle Forfar-Bares,† Patrick Dallemagne,| Nicolas Lemaˆıtre,† Fabienne Pe´hourcq, J acques Rochette,§
Christian Sergheraert,# and Christian J arry*,†
EA 2962-Pharmacochimie, UFR des Sciences Pharmaceutiques, Universite´ Victor Segalen Bordeaux 2, 146 Rue Le´o Saignat,
33076 Bordeaux Cedex, France, USM 0504 Biologie Fonctionnelle des Protozoaires, De´partement Re´gulations, De´veloppement
et Diversite´ Mole´culaire, Muse´um National d’Histoire Naturelle, 61 Rue Buffon, 75231 Paris, France, GRBPD, UMR-INERIS,
Faculte´s de Me´decine et de Pharmacie, Universite´ de Picardie J ules Verne, 1 Rue des Louvels, 80037 Amiens Cedex 1, France,
UMR 8525 CNRS, Institut Pasteur de Lille, Faculte´ de Pharmacie, Universite´ de Lille II, 1 Rue du Pr Calmette,
59021 Lille, France, CERMN, UFR des Sciences Pharmaceutiques, 1 Rue Vaube´nard, 14032 Caen Cedex, France, and
EA 525-Distribution des Me´dicaments dans l’Organisme et Pharmacodynamie, UFR des Sciences Pharmaceutiques,
Universite´ Victor Segalen Bordeaux 2, 146 Rue Le´o Saignat, 33076 Bordeaux Cedex, France
Received November 3, 2003
Three pyrrolo[1,2-a]quinoxalines, 15 bispyrrolo[1,2-a]quinoxalines, bispyrido[3,2-e]pyrrolo[1,2-
a]pyrazines, and bispyrrolo[1,2-a]thieno[3,2-e]pyrazines were synthesized from various sub-
stituted nitroanilines or nitropyridines and tested for their in vitro activity upon the erythrocytic
development of Plasmodium falciparum strains with different chloroquine-resistance status.
Bispyrrolo[1,2-a]quinoxalines showed superior antimalarial activity with respect to monopyr-
rolo[1,2-a]quinoxalines. The best activity was observed with bispyrrolo[1,2-a]quinoxalines linked
by a bis(3-aminopropyl)piperazine. Moreover, it was observed that the presence of a methoxy
group on the pyrrolo[1,2-a]quinoxaline nucleus increased the pharmacological activity. Drug
effects upon â-hematin formation were assayed and showed similar or higher inhibitory
activities than CQ. A possible mechanism of interaction implicating binding of pyrroloquin-
oxalines to â-hematin was supported by molecular modeling.
In tr od u ction
this has been recently questioned by Ginsburg and co-
workers who have suggested that inhibition of the
degradation of ferriprotoporphyrin IX by glutathione-
dependent redox processes could be a second mode of
action of CQ.10 Biochemical studies indicate that isolates
of the CQ-resistant parasite accumulate less drug in the
food vacuole than their more sensitive counterparts.
However, opinion remains divided on the mechanistic
explanations for the reduction: (1) a rapid CQ efflux
mechanism by CQ-resistant parasites,11 (2) an elevated
pH in the food vacuole of the CQ-resistant parasites,12
(3) resistance being linked to a carrier-mediated CQ
uptake,13 (4) resistance being linked to a reduced CQ
affinity to ferriprotoporphyrin.14 Concerning the first
explanation, its reversal by molecules such as vera-
pamil, desipramine, and chlorpromazine suggests that
an enhanced CQ efflux by a multidrug-resistant mech-
anism may be implicated.11-15 A possibility of overcom-
ing this multidrug-resistant mechanism is to design new
quinoline-based drugs that will not be recognized by the
protein system involved in drug efflux. In this regard,
bulky bisquinoline antimalarial drugs are suggested to
be extruded with difficulty by a proteinaceous trans-
porter.16 Previous toxicity results have inhibited the
development of Ro 47-7737,17 and then further linker
modulations led to the promising A model of bisquino-
lines.16,18-23 These compounds show much lower resis-
tance indices than CQ, suggesting that the bisquinoline
structure is less efficiently excluded by drug-resistant
It is estimated that there are 300-500 million cases
of malaria annually resulting in 1-3 million deaths,
most of which are children under the age of 5. Malaria
is a particularly important disease in sub-Saharan
Africa, where about 90% of cases and deaths occur but
is also a serious public health problem in certain regions
of southeast Asia and South America.1,2 Human malaria
is caused by four species of Plasmodium (P. falciparum,
P. vivax, P. ovale, and P. malariae), which are transmit-
ted by female Anopheles mosquitoes. The majority of
cases of malaria and deaths are caused by P. falciparum.
Present chemotherapies are proving to be inadequate,
are toxic, or are becoming ineffective because of an
increase in resistance.3-5 Chloroquine (CQ)6 is, after 50
years, still a mainstream drug in the fight against
malaria, but its efficacy is being steadily eroded by the
development of resistant parasites.5,7 CQ is believed to
exert its activity by inhibiting hemozoin formation in
the digestive vacuole of the malaria parasite,8,9 though
* To whom correspondence should be addressed. Phone:
(33) 5 57 57 11 76. Fax: (33) 5 57 57 13 52. E-mail: Christian.J arry@
chimphys.u-bordeaux2.fr.
† EA 2962-Pharmacochimie, Universite´ Victor Segalen Bordeaux 2.
‡ Muse´um National d’Histoire Naturelle.
§ Universite´ de Picardie J ules Verne.
#
Universite´ de Lille II.
| CERMN, UFR des Sciences Pharmaceutiques.
EA 525-Distribution des Me´dicaments dans l’Organisme et Phar-
macodynamie, Universite´ Victor Segalen Bordeaux 2.
10.1021/jm0310840 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/16/2004

1998 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Guillon et al.
Ch a r t 1. Structure of Chloroquine, Ro 47-7737,
Bisquinolines A, Quinacrine, and Bisacridines B
Ch a r t 2. Structure of Synthesized
Pyrrolo[1,2-a]quinoxalines 1a -c,
Bispyrrolo[1,2-a]quinoxalines 2a -d , 4a -d , and 5,
Bispyrido[3,2-e]pyrrolo[1,2-a]pyrazines 4e,f, and
Bispyrrolo[1,2-a]thieno[3,2-e]pyrazines 6
of pyrroloquinoxalines to â-hematin by using X-ray
crystal structure results obtained for compounds 4b and
4e.
Ch em istr y
parasites. Moreover, the bisquinolines drugs were proved
to be active against CQ-resistant malaria, with a longer
duration of action and lower toxicity. On the other hand,
acridine derivatives have also been considered as po-
tential antimalarial drugs. For instance, quinacrine,24
the 9-amino-6-chloro-2-methoxyacridine analogue of CQ,
was used clinically before CQ.
The new pyrrolo[1,2-a]quinoxalines and pyrido-
[3,2-e]pyrrolo[1,2-a]pyrazines 1a -c, 2a -d , 3b-d , 4a -
f, and 5 or pyrrolo[1,2-a]thieno[3,2-e]pyrazines 6 were
synthesized from substituted 2-nitroanilines or py-
ridines 7a -f.28-30 Not commercialy available 5-methoxy-
2-nitroaniline 7d and 2-amino-6-methoxy-3-nitropyri-
dine 7f were prepared according to the literature.30-32
The Clauson-Kaas reaction of anilines 7a -f with 2,5-
dimethoxytetrahydrofuran (DMTHF) in acetic acid gave
the pyrrolic derivatives 8a -f in 75-81% yields, which
were then reduced using a BiCl3-NaBH4 treatment to
provide the attempted 1-(2-aminophenyl)pyrroles 9a -f
(Scheme 1).
Recently, bisacridines B with a symmetrical linker
-NH-(CH2)3-Y-(CH2)3-NH- (Y ) -NH-, -NR-, or
piperazine) were described as potent antimalarial drugs
(Chart 1).25,26
We previously described a novel synthetic approach
to pyrrolo[1,2-a]quinoxaline derivatives as interesting
bioactive analogues of quinoline or quinoxaline com-
pounds.27-30 Hence, the pyrrolo[1,2-a]quinoxaline moi-
ety could be developed as a template for the design of
bisheterocyclic bioactive compounds. We report here the
synthesis and in vitro antiparasitic activity study upon
Plasmodium falciparum of a series of pyrrolo[1,2-a]-
quinoxalines 1 and of bispyrrolo[1,2-a]quinoxalines,
bispyrido[3,2-e]pyrrolo[1,2-a]pyrazines 2-5, and bispyr-
rolo[1,2-a]thieno[3,2-e]pyrazines 6 in which aromatic
nuclei are joined by aliphatic polyamines linker (Chart
2). The choice of the linker was based on previous results
obtained for bisquinolines and bisacridines. All these
new derivatives have in common a bis- or tricyclic
aromatic moiety with the reference compounds but
joined by a bis(3-aminopropyl)amine linker via a “pseudo-
amidinic” bond. To examine the mechanism of action of
these bispyrrolo[1,2-a]quinoxalines, we have set up an
in vitro assay of â-hematin formation under conditions
that are designed to resemble the conditions within the
food vacuole. The lipophilicity behavior dependence of
new compounds was studied with respect to the parasite
vacuolar and cytosolic pHs.
The cyclization was then possible between the NH2
and the C-R of the pyrrole ring by reacting 9a -f with
triphosgene in toluene to give the lactams 10a -f, which
were subsequently chlorodehydroxylated with phospho-
rus oxychloride to obtain the chloroquinoxalines 11a -d
or the chloropyrazines 11e-f.28 Reaction of 10c with a
POCl3-PCl5 mixture led to the dichloroderivative 12c,
as previously described.33 The placement of the second
chlorine atom at position 1 was suggested by the AB
system due to H-2 and H-3 protons. Displacement of the
chlorine atom of 11a -c with 5-diethylamino-2-pentyl-
amine led to the pyrrolo[1,2-a]quinoxalines 1a -c (58-
79% yields), structural analogues of 2-aminoquinolines
(Scheme 2).
The bispyrrolo[1,2-a]quinoxalines and bispyrido-
[3,2-e]pyrrolo[1,2-a]pyrazines 2a -d , 3b-d , 4a -f, and
5 were synthesized by reacting 3,3′-diamino-N-methyl-
dipropylamine or N-(3-aminopropyl)-1,3-propanedi-
amine or 1,4-bis(3-aminopropyl)piperazine) with 2 equiv
of 11a -f or 12c in DMF at 130 °C in the presence of
potassium carbonate as an inorganic base (Schemes 3
and 4).
Finally, a molecular modeling study was carried out
to elucidate the mechanism of action implicating binding
A similar nucleophilic substitution with 1,4-bis(3-
aminopropyl)piperazine and 2 equiv of 5-chloropyrrolo-

Quinoxalines and Pyrazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 1999
Sch em e 1a
Sch em e 4a
a
(i) H2N-(CH2)3-piperazine-(CH2)3-NH2, K2CO3, DMF, ∆.
Sch em e 5a
a
(i) H2N-(CH2)3-piperazine-(CH2)3-NH2, K2CO3, DMF, ∆.
Ta ble 1. Physical Properties of the Final Amines (1-6)
a
(i) DMTHF, ∆; (ii) BiCl3, NaBH4, EtOH; (iii) CO(OCCl3)2,
compd
salta
mp (°C)b
% yieldc
toluene, ∆; (iv) POCl3, ∆; (v) POCl3, PCl5, ∆.
1a
1b
1c
2a
2b
2c
2d
3b
3c
3d
4a
4b
4c
4d
4e
4f
2 oxalate
2 oxalate
2 oxalate
3 oxalate
3 oxalate
3 oxalate
3 oxalate
3 oxalate
3 oxalate
3 oxalate
4 oxalate
4 oxalate
4 oxalate
4 oxalate
4 oxalate
4 oxalate
4 oxalate
4 oxalate
71-72
46
48
51
39
62
49
55
70
33
31
58
57
47
32
80
61
72
28
73-75
Sch em e 2a
72-74
144-146
210-211
125-126
87-89
158-159
108-110
186-188
203-204
209-211
221-223
197-199
244-246
231-233
187-189
210-212
a
(i) 5-Diethylamino-2-pentylamine, ∆.
5c
6
Sch em e 3a
a
Amines 1-6 were dissolved in 30 mL of 2-propanol, heated to
boiling, and treated with oxalic acid (4 equiv, based on the amount
of the starting material). The oxalate salts crystallized upon
cooling, were collected by filtration, and were washed with
b
2-propanol and Et2O. Crystallization solvent: 2-PrOH-H2O.
c The yields included the conversions into the oxalates.
falciparum CQ-sensitive strain Thai (IC50(CQ) ) 0.01
µM) and the CQ-resistant strains FcB1 and K1 (IC50-
(CQ) ) 0.13 and 0.24 µM, respectively). As shown in
Table 2, bispyrrolo[1,2-a]quinoxalines 2-6 were always
more active (IC50 values in the nanomolar range) than
their respective monomers 1a -c (IC50 values in the
micromolar range). For a same substitution on the
pyrroloquinoxaline moiety, compounds joined by a bis-
(3-aminopropyl)piperazine linker (compounds 4-6) were
generally more active (up to 3 times) than their coun-
terparts with bis(3-aminopropyl)methylamine (2) or bis-
(3-aminopropyl)amine linkages (3), with the exception
of 2c and 4c, joined by a bis(3-aminopropyl)methyl-
amine and a bis-(3-aminopropyl)piperazine linker re-
spectively, which showed similar antimalarial activities.
These new derivatives have a bis- or tricyclic aromatic
moiety in common with the reference compounds, but
they are linked to the bis(3-aminopropyl)amine linker
via a “pseudoamidinic” bond. Compounds 2 and 3
a
(i) H2N-(CH2)3-Y-(CH2)3-NH2, K2CO3, DMF, ∆.
[1,2-a]thieno[3,2-e]pyrazine 1334 furnished the bispyrrolo-
[1,2-a]thieno[3,2-e]pyrazines 6 (Scheme 5).
Compounds 1a -c, 2a -d , 3b-d , 4a -f, 5, and 6 were
then converted into their oxalate salts in 32-80% yields
by treatment with oxalic acid in refluxing 2-propanol
(Table 1).
P h a r m a cology
An tim a la r ia l Activity. All compounds were tested
for their antimalarial activity in vitro upon the P.

2000 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Guillon et al.
Ta ble 2. In Vitro Sensitivity of P. falciparum Strains and in Vitro Cytotoxicity on Mammalian L6 Cells
IC50 values (µM)a
P. falciparum strains
resistance index
mammalian L6
cells
index of
compd
Thai
FcB1
K1
selectivityb
FcB1d
K1e
CQ
1a
1b
1c
2a
2b
2c
2d
3b
3c
3d
4a
4b
4c
4d
4e
4f
0.01 ( 0.01
2.95 ( 0.13
1.94 ( 0.65
0.83 ( 0.04
0.12 ( 0.01
0.40 ( 0.12
0.05 ( 0.01
0.12 ( 0.03
0.47 ( 0.05
0.04 ( 0.01
0.14 ( 0.02
0.08 ( 0.01
0.28 ( 0.05
0.03 ( 0.01
0.04 ( 0.01
NDc
0.13 ( 0.04
4.97 ( 0.41
4.79 ( 0.09
2.47 ( 0.17
0.48 ( 0.01
1.01 ( 0.27
0.11 ( 0.01
0.32 ( 0.03
1.14 ( 0.06
0.19 ( 0.01
0.30 ( 0.04
0.29 ( 0.02
0.64 ( 0.04
0.13 ( 0.01
0.13 ( 0.01
1.09 ( 0.10
0.60 ( 0.02
0.72 ( 0.03
0.93 ( 0.03
0.24 ( 0.03
4.34 ( 0.27
3.06 ( 0.44
1.78 ( 0.25
0.28 ( 0.05
0.70 ( 0.01
0.05 ( 0.01
0.20 ( 0.01
0.79 ( 0.04
0.11 ( 0.02
0.33 (0.03
0.37 ( 0.15
0.48 ( 0.09
0.08 ( 0.02
0.08 ( 0.01
NDc
NDc
>20
17.31 ( 0.48
>20
NDc
>4.6
5.6
>11.2
22.1
7.1
106.3
19.2
6.1
26.6
11
13
24
1.7
2.5
3.0
4.0
2.5
2.2
2.7
2.4
4.7
2.1
3.6
2.3
4.3
3.2
NDc
NDc
2.9
NDc
1.5
1.6
2.1
2.3
1.8
1.0
1.7
1.7
2.7
2.4
4.6
1.7
2.7
2.0
NDc
NDc
1.32
NDc
6.2 ( 0.1
4.95 ( 0.96
5.74 ( 0.15
3.79 ( 0.63
4.81 ( 1.05
2.98 ( 0.06
3.62 ( 0.62
5.04 ( 0.19
4.01 ( 0.66
1.32 ( 0.09
4.06 ( 0.72
NDc
13.6
8.2
17.4
52.7
NDc
NDc
9.3
NDc
NDc
NDc
5
6
0.25 ( 0.05
0.33 ( 0.01
3.07 ( 0.69
NDc
NDc
NDc
NDc
a
IC50 values were measured on the chloroquine-sensitive strain Thai/Thailand and the chloroquine-resistant strains FcB1/Colombia
b
and K1/Thailand. The IC50 values correspond to the mean ( standard deviation from three independent experiments. Index of selectivity
is defined as the ratio of the IC50 value on the mammalian L6 cells to the IC50 value against the P. falciparum K1 strain. c ND: not
determined. IC50(FcB1)/IC50(Thai). e IC50(K1)/IC50(Thai).
d
bearing bis(3-aminopropyl)methylamine and an amine
linkage generally exhibit similar activities. Decrease of
the lipophilicity of molecules by hydroxymethylation
either on carbon C-7 (compounds 2c, 3c, and 4c) or on
carbon C-8 (compounds 2d , 3d , and 4d ) of the pyrrolo-
quinoxaline moiety increased the antimalarial activity
up to 5 times when compared to the nonsubstituted
counterparts (compounds 2a , 4a ). Finally, this hy-
droxymethyl substitution achieved on C-7 gave the most
active compounds 2c-4c.
In contrast, the addition of a chlorine atom on the
pyrroloquinoxaline moiety, in analogy with CQ and with
B, reduced the activity up to 10 times (i.e., 4a compared
to 4b, and 4c compared to 5). In the same way,
replacement of the pyrroloquinoxaline moiety by a
pyridopyrrolopyrazine or a pyrrolothienopyrazine moi-
ety, bioisosteres of the pyrroloquinoxaline nucleus,
reduced the antimalarial activity about 5 times (i.e., 4a
compared to 4e and 6), suggesting that introduction of
an electron-rich (or electronegative) atom, such as a
nitrogen or sulfur atom, decreases this activity. On the
other hand, introduction of a methoxy substituent on
position C-2 of the pyridopyrrolopyrazine moiety (com-
pound 4f) seems to increase the activity about 2 times
in comparison with derivative 4e, corroborating the
results obtained with 4c in comparison with 4a .
bulky structure of studied bispyrroloquinoxalines by the
efflux proteins involved in conferring CQ resistance.16,20
Cytotoxicity. All tested bispyrrolo[1,2-a]quinoxalines
showed cytotoxicity upon the mammalian L6 cells in the
micromolar range, and the different substitutions had
less influence on the IC50 values than that observed for
the antimalarial activity; e.g., for the piperazine series
where marked differences of cytotoxicity were observed,
IC50 values on the L6 cells varied from 1.3 to 5.0 µM
(Table 2). Index of selectivity was defined as the ratio
of the IC50 value on the mammalian cells to the IC50
value on the CQ-resistant P. falciparum strain K1.
Compounds that demonstrated high selectivity (high
index of selectivity) should offer the potential of safer
therapy. This led to the identification of compounds with
index of selectivity greater than 50 for compound 4d
and greater than 100 for compound 2c that could
constitute suitable candidates for further pharmacologi-
cal studies.
In h ibition of â-Hem a tin F or m a tion . In vitro ex-
periments have previously established that quinoline
antimalarial drugs such as CQ are associated with the
crystallization of haemazoin.35-38 In fact, current evi-
dence indicates that drugs act by inhibiting the forma-
tion of haemazoin and thus preventing haem detoxifi-
cation. 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
haemazoin;37 (2) enzymatic inhibition of a protein that
catalyzes haemazoin crystallization;39 and (3) chemiab-
sorption of the drug onto crystallized haemazoin, leading
to inhibition of further haem aggregation.40
As seen in Table 2, almost all compounds have lower
IC50 values for inhibition of growth of CQ-sensitive
strain Thai than for inhibition of growth of CQ-resistant
strains FcB1 and K1. A comparison of the IC50 values
for the inhibition of growth of the resistant and sensitive
strains of P. falciparum suggests relatively low levels
of cross-resistance to CQ. The resistance index values
calculated from the comparison of the IC50 values of the
resistant and sensitive strains of P. falciparum were
lower than those for CQ, in the ranges 1.7-4.7 (FcB1)
and 1.0-4.6 (K1) (Table 2). Because it was already
reported for bisquinolines, these results could be dis-
cussed in terms of recognition difficulties related to the
Given that methoxy substitution on C-7 resulted in
the most active compounds in precedent tests, the three
7-methoxy derivatives 2c, 3c, and 4c (only differing by
the nature of the linker) were tested and compared to
CQ in the ability to inhibit â-hematin formation (Figure
1), the main mechanism of action of CQ, according to
two selected protocols.8,9,35-37 By use of insoluble para-

Quinoxalines and Pyrazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2001
Ta ble 3. Vacuolar Accumulation Ratios (VARs) and log D
Values for Some Selected Compounds at pH 7.4 and pH 5
compd
VAR (×103)
log D7.4
log D5
CQ
2a
2b
2c
2d
3b
3c
3d
4a
4b
4c
4d
4e
4f
61.1
225.6
1.8
105.4
42.1
1.6
104.2
41.7
409.1
2.1
186.9
72.5
0.1
NDa
4.12
4.44
3.55
3.41
5.06
2.76
2.58
2.52
4.92
3.72
4.67
5.05
4.73
5.74
NDa
1.56
3.5
0.93
0.41
3.16
0.8
0.93
1.31
3.43
2.15
1.88
2.22
3.97
4.17
33.0
0.9
F igu r e 1. Percent inhibition of â-hematin formation versus
log(concentration of compound) for CQ and compounds 2a , 2c,
and 4c.
5
a
ND ) not determined.
site material as catalyst,41 compounds 2c, 3c, and 4c
at 100 µM inhibit 89%, 100%, and 59% â-hematin
formation, respectively. At the same concentration, 82%
of inhibition was measured for CQ. By use of 1-mo-
nooleoyl glycerol as catalyst,42,43 compounds 2a , 2c, 3c,
4c, and CQ showed a â-hematin formation inhibition
with IC50 values of 51, 60.5, 75, 47.9, and 59.9 µM,
respectively (Figure 1), while the IC50 values for 2b, 4a ,
and 4b were found to be superior at 75, 100 and 100
µM, respectively. These data could support the hypoth-
esis that bispyrroloquinoxalines inhibit parasite growth
via inhibition of â-hematin formation.
However, this approach also requires that these
symmetrical triprotic or tetraprotic bispyrroloquinoxa-
lines, like the diprotic weak base CQ, concentrate to
micromolar levels in the acidic food vacuole of the
parasite, the organelle in which â-hematin formation
takes place.44
2- to 5-fold), while the biological activity was twice less
important. On the other hand, for all compounds there
is a small difference in the range of lipophilic values at
pH 7.4, whereas there appears to be a larger difference
(more than 10-fold) at pH 5.0. The log D values of the
most active compounds c-d , determined at pH 5.0,
range between 0.41 and 0.93 for series 2 and 3 and
between 1.88 and 2.15 for series 4. Replacement of the
methoxy function with hydrogen has little effect on the
lipophilicity, whereas introduction of a chlorine group
increases it.
These preliminary results could lead to the hypothesis
that in addition to some specific mechanism of action,
the permeation of the tested compounds is certainly
implicated in their biological activity and also supports
that both pH trapping and â-hematin inhibition could
be some basis of antiplasmodial activity of these new
bisquinoline analogues.
The preliminary pharmacological results could be
discussed in terms of physicochemical behavior through
the partitioning theory,4 which involved the influence
of pKa and lipophilicity of studied compounds. Hence,
these two parameters determine the absorption, perme-
ability, and in vivo distribution of drugs. Consequently,
to evaluate the contribution to antimalarial activity of
the component accumulation in the food vacuole, HPLC
determination of log D at vacuolar and cytosolic pHs (5.0
and 7.4, respectively)45 and in silico vacuolar accumula-
tion ratios (VAR) based on a weak-base model were
carried out for the 14 bispyrrolo[1,2-a]quinoxalines 2a -
d , 3b-d , 4a -f, and 5.46
As shown in Table 3, methoxy-substituted compounds
2d -4d and 2c-4c would accumulate 30-70 times more
than the corresponding 8-chloro-substituted compounds
2b-4b, which correlates with an increase of the in vitro
antimalarial activity. For example, compound 4c (VAR
) 186.9 × 103, log D5 ) 2.15, log D7.4 ) 3.72) displayed
a greater accumulation than compound 4b (VAR ) 2.1
× 103, log D5 ) 3.43, log D7.4 ) 4.92) and is about 5-fold
more active. Hence, the introduction of a methoxy
substituent seems to considerably increase ion-trapping
of molecules. The best antimalarial activities were
obtained for the methoxy-substituted compounds 2c,d
and 3c,d displaying predicted VAR values in the range
of 41.7 × 103 to 186.9 × 103. Replacement of the
methoxy group in compounds c and d by a hydrogen
atom (a ) increases the predicted VAR values (more than
Molecu la r Mod elin g. Recently, Buller et al.37 have
described a theoretical growth of synthetic hemozoin
and proposed a noncovalent binding site for the quino-
line drug family at the end face of the fastest-growing
direction of synthetic hemozoin. Pagola47 has proposed
that the antimalarial quinolines act through growth
inhibition of hemozoin crystals as a result of absorption
onto its actively growing faces. With these studies in
mind and in order to evaluate the implication of spatial
conformation of synthesized bispyrroloquinoxalines, we
carried out a molecular modeling study based on the
crystallographic analysis of compounds 4b and 4e.
As shown in Figure 2,48 the pyrrolo[1,2-a]quinoxaline
ring systems are planar and are parallel to each other,
related by a center of symmetry. In the crystal, the
distances between the pyrrolo[1,2-a]quinoxaline rings
of 4b and 4e bound with symmetrical linker -NH-
(CH2)3-piperazine-(CH2)3-NH- is observed at 9.919
and 10.208 Å, respectively. The piperazine rings have
the chair conformation, and the atoms are alternately
displaced from the least-squares plane by 0.69 Å for 4b
and by 0.68 Å for 4e. In 4b and 4e, the distances
between the least-squares planes of the two pyrroloquin-
oxaline moieties were approximatively 2.40 and 2.05 Å,
respectively. By use of these experimental crystal-
lographic data, energy-minimized molecular conforma-
tions were generated for the bisquinoline analogues and
then were examined for their likely interaction with the
synthetic hemozoin described by Buller et al.37

2002 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Guillon et al.
al.37 (Figures 6 and 7). This model is in keeping with
NMR studies on the interaction between CQ and he-
matin.52 CQ and analogues form an (aromatic)N-HCd
C(vinyl) hydrogen bond, and a CCl-H3C interaction
with the host molecule helps to anchor the guest within
the crevice. It was proposed that aromatic interactions
of the pyrroloquinoxalines 4b and 4d are similar to
those of CQ and analogues (Figures 6 and 7). Indeed,
the size of the aromatic moiety is critical for crystal
inhibition,36 but the pyrroloquinoxaline aromatic nucleus
could be bound within the crevice as the quinoline
aromatic ring.
In the case of CQ, the exocyclic chain must twist the
C4-N(aniline) bond to form a salt bridge with the
carboxylate moiety and allows intercalation of the
aromatic moiety within the crevice. For studied com-
pounds, it is possible to define salt bridges of protonated
piperazine N atoms with the carboxylate moiety of
hematin, while the pyrroloquinoxaline nucleus could
destabilize the complex by a π-π intercalation between
two hematin molecular units (Figures 6 and 7).
Such a π-π interaction of the pyrroloquinoxaline ring
over the porphyrine could bring another noncovalent
binding site for the bispyrroloquinoxaline drug family
at the end face of the fastest-growing direction of
synthetic haemozoin. Finally, pyrroloquinoxalines de-
rivatives could also bind to and trap ferriprotoporphyrin
in a µ-oxo dimeric form and prevent the formation of
the â-hematin dimers that are required for haemozoin
formation.35,52,53
F igu r e 2. Side views of the crystal structures of 4b and 4e.
Displacement ellipsoids are drawn at the 30% probability level.
The “quinoline-like” antimalarial compounds are ac-
cumulated in the food vacuole of the parasite (pH 5.0),
so modeling studies were concentrated on the tetrapro-
tonated forms of the drugs. Simulation studies were
carried out on compounds 4b and 4d . Each molecule
was energy-minimized from the “as-constructed” con-
formation using the extensible systematic force field
(ESFF),49 the Discover program,50 and Gamess.51
From this procedure, 10 molecular conformations
were generated and three final structures were selected
that are in agreement with the nonprotonated ana-
logues from X-ray crystallography (Figure 2), for which
the aromatic pyrrolo[1,2-a]quinoxaline ring systems are
planar and parallel to each other (Figure 3). This result
corroborated the employment of the ESFF method in
this study.
The inter-nitrogen separation distances between the
first protonated nitrogen of the piperazine and the first
protonated nitrogen of the quinoxaline and between the
second protonated nitrogen of the piperazine and the
second protonated nitrogen of the quinoxaline are
between 6.94-7.43 and 7.36-7.38 Å for 4b and between
6.92-6.98 and 7.04-7.24 Å for 4d . In all cases, it was
observed that the inter-nitrogen atom separations in-
creased on protonation of the studied molecules, con-
trary to the values found for X-ray crystallography (5.12
Å for 4b).
After generation of a set of conformations for 4b and
4d , an investigation into their interactions with the
synthetic hemozoin was carried out. A model of the
hematin molecular unit linked through iron-carboxy-
late bonds to the centrosymmetric cyclic dimer was
constructed (Figure 4). This unit was then energy-
minimized with the ESFF potential set. Three MD runs
at 300 K for a simulation time of 5 ps were performed,
and the resulting structure was energy-minimized to
gain a low-energy conformation. The three obtained
molecular unit conformations are very closed and suc-
cessfully reproduce the expected hematin molecular unit
(Figure 4b).37 Four molecular units (Figure 4a) were
assemblied manually to obtain a “pseudo”-â-hematin
with respect to the distances observed between the
adjacent â-hematin in the crystal lattice (Figure 5).47
The conformation and docking surface site position and
orientation of the pyrroloquinoxalines 4b and 4d were
adjusted manually in this tetramer to yield acceptable
interatomic contact distances as described by Buller et
Finally, this molecular modeling study should be
considered as a preliminary hypothetical approach to
enlighten the antimalarial activity mechanism of bispyr-
roloquinoxaline derivatives. Nevertheless, further and
more elaborate modeling studies are required to confirm
the defined drug binding site on malarial pigment
crystal.
Con clu sion
In the present report, we have described the synthesis
and the antimalarial activity of new pyrrolo[1,2-a]-
quinoxalines 1 and bispyrrolo[1,2-a]quinoxalines 2-6
in which aromatic nuclei are joined by aliphatic
polyamines linker. Through this study, it was observed
that bispyrroloquinoxalines 2-6 showed superior anti-
malarial activity with respect to the monopyrrolo-
quinoxaline compounds, structural analogues of CQ.
Introduction of a methoxy substituent on the pyrroloquin-
oxaline nucleus had an influence on the activity, in
relation to the lipophilicity of described compounds. In
the bisheterocycles, a piperazine moiety was the best
linker to confer activity through a pseudosymmetry of
the molecule.
A preliminary molecular modeling study was carried
out to elucidate the action mechanism of 2-6 through
the implication of the molecules binding to â-hematin
crystal surface. On the basis of these findings, it is
possible to further identify new pyrrolo[1,2-a]quinoxa-
line derivatives that inhibit â-hematin formation by
semirational design.
Exp er im en ta l Section
Ch em istr y. Commercial reagents were used as received
without additional purification. Melting points were determi-
nated with an SM-LUX-POL Leitz hot-stage microscope and

Quinoxalines and Pyrazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2003
F igu r e 3. View of the backbone of the final selected superimposed structures.
F igu r e 4. (a) Model of hematin molecular unit with propionate oxygen-iron bonds. (b) View of the backbone of the final selected
superimposed structures.
F igu r e 5. “Pseudo”-â-hematin.
are uncorrected. IR spectra were recorded on an BRUKER IFS-
25 spectrophotometer. NMR spectra were recorded with tet-
ramethylsilane as an internal standard using a BRUKER AC
200 spectrometer (1H, 13C, 2D-COSY). Splitting patterns have
been designated as follows: s ) singlet; bs ) broad singlet; d
) doublet; t ) triplet; q ) quartet; m ) mutiplet. Analytical
TLC was carried out on 0.25 precoated silica gel plates
(POLYGRAM SIL G/UV254) with visualization 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 (Vernaison-France), and
the results agreed with the proposed structures within (0.3%
of the theoretical values.
F igu r e 6. Proposed interaction between compound 4b and
the tetramer. The following distances are between boldfaced
atoms: NH-HCdC(vinyl), 2.28 Å; H3C-CCl, 5.29 Å.
139.1 (C-5), 135.1 (C-1), 127.5 (C-4), 127.4 (C-R), 126.2 (C-6),
121.0 (C-3), 111.4 (C-â). Anal. (C10H7ClN2O2) C, H, N.
1-(4-Meth oxy-2-n itr op h en yl)p yr r ole (8c) was synthe-
sized from 7c according to the same conditions as those
described to obtain 8b. This gave 8c as orange crystals (75%),
1
mp 57 °C. H NMR (CDCl3) δ: 7.38 (d, 1H, J ) 8.85 Hz, H-6),
7.35 (d, 1H, J ) 2.75 Hz, H-3), 7.16 (dd, 1H, J ) 8.85 and 2.75
Hz, H-5), 6.75 (dd, 2H, J ) 2.00 and 2.00 Hz, H-R), 6.32 (dd,
2H, J ) 2.00 and 2.00 Hz, H-â), 3.88 (s, 3H, CH3). Anal.
(C11H10N2O3) C, H, N.
1-(5-Ch lor o-2-n itr op h en yl)p yr r ole (8b). A mixture of
2-nitroaniline 7b (0.07 mol) and 2,5-dimethoxytetrahydrofuran
(0.07 mol) in acetic acid (100 mL) was refluxed for 1 h with
vigorous stirring. After cooling, the reaction mixture was
poured into water (300 mL). The precipitate was filtered,
washed with water, dissolved in diethyl ether (150 mL), dried
over magnesium sulfate, and evaporated to dryness under
reduced pressure to give orange crystals, which were recrys-
tallized from petroleum ether. Orange crystals (79%), mp 73
1-(5-Meth oxy-2-n itr op h en yl)p yr r ole (8d ) was synthe-
sized from 7d according to the same conditions as those
described to obtain 8b. This gave 8d as orange crystals (78%),
1
mp 48 °C. H NMR (CDCl3) δ: 7.94 (d, 1H, J ) 8.85 Hz, H-3),
6.91 (dd, 1H, J ) 8.85 and 2.45 Hz, H-4), 6.89 (d, 1H, J ) 2.45
Hz, H-6), 6.77 (dd, 2H, J ) 2.15 and 2.15 Hz, H-R), 6.34 (dd,
2H, J ) 2.15 and 2.15 Hz, H-â), 3.89 (s, 3H, CH3). 13C NMR
(CDCl3) δ: 163.9 (C-5), 138.1 (C-1), 137.5 (C-3), 128.2 (C-2),
121.9 (C-R), 113.7 (C-4), 113.4 (C-6), 111.4 (C-â), 56.8 (CH3O).
Anal. (C11H10N2O3) C, H, N.
1
°C. H NMR (CDCl3) δ: 7.81 (d, 1H, J ) 8.45 Hz, H-3), 7.46
(d, 1H, J ) 2.10 Hz, H-6), 7.41 (dd, 1H, J ) 8.45 and 2.10 Hz,
H-4), 6.77 (dd, 2H, J ) 2.15 and 2.15 Hz, H-R), 6.37 (dd, 2H,
J ) 2.15 and 2.15 Hz, H-â). 13C NMR (CDCl3) δ: 143.0 (C-2),

2004 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Guillon et al.
7.25 (m, 2H, H-6 and H-7), 7.02 (dd, 1H, J ) 3.95 and 1.30
Hz, H-3), 6.65 (dd, 1H, J ) 3.95 and 2.60 Hz, H-2). 13C NMR
(DMSO-d6) δ: 158.2 (C-4), 131.0 (C-5a), 129.9 (C-3a), 128.8
(C-8), 127.0 (C-9a), 126.7 (C-1), 122.1 (C-7), 121.3 (C-3), 118.4
(C-9), 116.6 (C-6), 115.3 (C-2). Anal. (C11H7ClN2O) C, H, N.
7-Meth oxy-5H-pyr r olo[1,2-a ]qu in oxalin -4-on e (10c) was
synthesized from 9c according to the same conditions as those
described to obtain 10b. This gave 10c as orange crystals
(84%), mp 252 °C. IR (KBr) 3200, 2700 (NH), 1685 (CO) cm-1
.
1H NMR (DMSO-d6) δ: 11.15 (s, 1H, NH), 8.04 (dd, 1H, J )
2.45 and 0.80 Hz, H-1), 7.90 (d, 1H, J ) 8.85 Hz, H-9), 6.97
(dd, 1H, J ) 3.60 and 0.80 Hz, H-3), 6.82 (d, 1H, J ) 2.60 Hz,
H-6), 6.75 (dd, 1H, J ) 8.85 and 2.60 Hz, H-8), 6.60 (dd, 1H,
J ) 3.60 and 2.45 Hz, H-2), 3.75 (s, 3H, CH3O). 13C NMR
(DMSO-d6) δ: 157.0 (C-4), 155.2 (C-7), 129.7 (C-9a), 122.6 (C-
5a), 117.7 (C-1), 116.8 (C-3a), 116.0 (C-6), 112.3 (C-3), 111.0
(C-2), 108.9 (C-9), 100.8 (C-8), 55.4 (CH3O). Anal. (C12H10N2O2)
C, H, N.
8-Meth oxy-5H-pyr r olo[1,2-a ]qu in oxalin -4-on e (10d) was
synthesized from 9d according to the same conditions as those
described to obtain 10b. This gave 10d as pale-orange crystals
(53%), mp 265 °C. IR (KBr) 3200, 2800 (NH), 1655 (CO) cm-1
.
1H NMR (DMSO-d6) δ: 11.07 (s, 1H, NH), 8.20 (dd, 1H, J )
2.50 and 1.10 Hz, H-1), 7.61 (d, 1H, J ) 2.50 Hz, H-9), 7.21
(d, 1H, J ) 8.85 Hz, H-6), 6.98 (dd, 1H, J ) 3.65 and 1.10 Hz,
H-3), 6.89 (dd, 1H, J ) 8.85 and 2.50 Hz, H-7), 6.66 (dd, 1H,
J ) 3.65 and 2.50 Hz, H-2), 3.82 (s, 3H, CH3O). 13C NMR
(DMSO-d6) δ: 155.3 (C-4), 154.8 (C-8), 123.5 (C-9a), 123.3 (C-
5a), 122.3 (C-1), 118.4 (C-3a), 117.6 (C-6), 112.8 (C-3), 112.6
(C-2), 111.4 (C-9), 100.2 (C-7), 56.0 (CH3O). Anal. (C10H9ClN2)
C, H, N.
4,8-Dich lor op yr r olo[1,2-a ]qu in oxa lin e (11b). A solution
of 5H-pyrrolo[1,2-a]quinoxalin-4-one 10b (0.03 mol) in POCl3
(60 mL) was refluxed for 4 h. After removal of excess reactive
material under vacuum, the residue was carefully dissolved
in water at 0 °C and the resulting solution was made basic
with 30% aqueous ammonium hydroxide solution. The pre-
cipitate was filtered, dried, and recrystallized from ethyl
acetate to give 11b as white crystals (81%), mp 187 °C. 1H
NMR (DMSO-d6) δ: 8.57 (m, 1H, H-1), 8.42 (d, 1H, J ) 2.15
Hz, H-9), 7.74 (d, 1H, J ) 8.65 Hz, H-6), 7.45 (dd, 1H, J )
8.65 and 2.15 Hz, H-7), 7.00 (m, 1H, H-3), 6.95 (m, 1H, H-2).
Anal. (C11H6Cl2N2) C, H, N.
4-Ch lor o-7-m eth oxypyr r olo[1,2-a ]qu in oxalin e (11c) was
synthesized from 10c according to the same conditions as those
described to obtain 11b. This gave 11c as white crystals (92%),
mp 133 °C. 1H NMR (DMSO-d6) δ: 8.38 (m, 1H, H-1), 8.07 (d,
1H, J ) 8.90 Hz, H-9), 7.21 (d, 1H, J ) 2.10 Hz, H-6), 7.11
(dd, 1H, J ) 8.90 and 2.10 Hz, H-8), 6.89 (m, 1H, H-3), 6.85
(m, 1H, H-2), 3.81 (s, 3H, CH3O). 13C NMR (DMSO-d6) δ: 157.0
(C-4), 143.9 (C-7), 135.2 (C-9a), 122.5 (C-5a), 120.9 (C-9), 118.0
(C-3a), 116.7 (C-6), 115.8 (C-1), 114.0 (C-3), 110.5 (C-8), 107.9
(C-2), 55.5 (CH3O). Anal. (C12H9ClN2O) C, H, N.
4-Ch lor o-8-m eth oxypyr r olo[1,2-a ]qu in oxalin e (11d) was
synthesized from 10d according to the same conditions as those
described to obtain 11b. This gave 11d as beige crystals (83%),
mp 126 °C. 1H NMR (DMSO-d6) δ: 8.57 (m, 1H, H-1), 7.77 (d,
1H, J ) 2.60 Hz, H-9), 7.72 (d, 1H, J ) 8.65 Hz, H-6), 7.08
(dd, 1H, J ) 8.65 and 2.60 Hz, H-7), 6.98 (m, 2H, H-2 and
H-3), 3.93 (s, 3H, CH3O). 13C NMR (DMSO-d6) δ: 159.5 (C-4),
140.9 (C-8), 129.9 (C-9a), 128.2 (C-5a), 127.8 (C-3a), 122.8 (C-
6), 118.3 (C-1), 114.4 (C-3), 114.0 (C-2), 107.8 (C-9), 98.5 (C-
7), 56.1 (CH3O). Anal. (C12H9ClN2O) C, H, N.
F igu r e 7. Proposed interaction between compound 4d and
the tetramer. The following distances are between boldfaced
atoms: NH-HCdC(vinyl), 2.24 Å; H3C-COCH3, 5.34 Å.
1-(2-Am in o-5-ch lor op h en yl)p yr r ole (9b). To a solution
of 1-(2-nitrophenyl)pyrrole 8b (0.02 mol) in ethanol (130 mL)
was added BiCl3 (0.03 mol). Sodium borohydride (0.16 mol)
was added portionwise at 0 °C to the reaction mixture, which
was then stirred at room temperature for 2 h. The reaction
mixture was then poured into an aqueous hydrochloric acid
solution (1 N, 130 mL) and stirred for 1 h. Ethanol was
evaporated under reduced pressure. The residue was made
alkaline with concentrated aqueous ammonium hydroxide
solution and then extracted with ethyl acetate. The organic
layer was dried over MgSO4 and evaporated to dryness under
reduced pressure. The residue was then recrystallized from
petroleum ether to give 9b as pale-yellow crystals (63%), mp
87 °C. IR (KBr) 3375, 3300 (NH2) cm-1 1H NMR (CDCl3) δ:
.
7.14 (d, 1H, J ) 2.30 Hz, H-6), 7.12 (dd, 1H, J ) 8.05 and 2.30
Hz, H-4), 6.81 (dd, 2H, J ) 1.95 and 1.95 Hz, H-a), 6.70 (d,
1H, J ) 8.05 Hz, H-3), 6.35 (dd, 2H, J ) 1.95 and 1.95 Hz,
H-b), 3.72 (bs, 2H, NH2). 13C NMR (CDCl3) δ: 140.6 (C-2), 128.3
(C-4), 127.9 (C-1), 126.9 (C-6), 122.4 (C-5), 121.4 (C-R), 116.8
(C-3), 109.8 (C-â). Anal. (C10H9ClN2) C, H, N.
1-(2-Am in o-4-m eth oxyp h en yl)p yr r ole (9c) was synthe-
sized from 8c according to the same conditions as those
described to obtain 9b. This gave 9c as yellow oil (62%). IR
(KBr) 3460, 3375 (NH2) cm-1. 1H NMR (CDCl3) δ: 7.06 (d, 1H,
J ) 8.20 Hz, H-6), 6.78 (dd, 2H, J ) 2.05 and 2.05 Hz, H-R),
6.34 (m, 4H, H-3, H-5 and H-â), 3.79 (s, 3H, CH3O), 3.69 (bs,
2H, NH2). 13C NMR (CDCl3) δ: 159.9 (C-4), 143.4 (C-2), 128.1
(C-1), 122.1 (C-R), 121.2 (C-6), 109.2 (C-â), 103.6 (C-5), 101.1
(C-3), 55.4 (CH3O). Anal. (C11H12N2O) C, H, N.
1-(2-Am in o-5-m eth oxyp h en yl)p yr r ole (9d ) was synthe-
sized from 7d according to the same conditions as those
described to obtain 9b. This gave 9d as pale-yellow crystals
(47%), mp 43 °C. IR (KBr) 3435, 3355 (NH2) cm-1 1H NMR
.
(CDCl3) δ: 6.85 (m, 2H, H-3 and H-6), 6.76 (m, 3H, H-6 and
H-R), 6.34 (dd, 2H, J ) 2.10 and 2.10 Hz, H-â), 3.74 (s, 3H,
CH3O), 3.45 (bs, 2H, NH2). 13C NMR (CDCl3) δ: 153.1 (C-5),
136.0 (C-1), 128.8 (C-2), 122.2 (C-R), 117.9 (C-3), 115.2 (C-4),
112.8 (C-6), 110.2 (C-â), 56.4 (CH3O). Anal. (C11H12N2O) C, H,
N.
1,4-Dich lor o-7-m eth oxypyr r olo[1,2-a ]qu in oxalin e (12c).
A mixture of lactam 10c (0.006 mol) and phosphorus pen-
tachloride (0.0111 mol) in phosphorus oxychloride (8 mL) was
heated under reflux for 2.5 h. The reaction mixture was then
evaporated to dryness, the residue was dissolved in water at
0 °C, and the resulting solution was made alkaline with 30%
aqueous ammonium hydroxyde solution. The mixture was
extracted with methylene chloride. The organic layer was dried
over Na2SO4 and evaporated under reduced pressure to give
yellow crystals (77%), mp 126 °C. 1H NMR (DMSO-d6) δ: 8.72
(d, 2H, J ) 9.35 Hz, H-9), 7.20 (d, 2H, J ) 2.95 Hz, H-6), 6.95
8-Ch lor o-5H-p yr r olo[1,2-a ]qu in oxa lin -4-on e (10b). To
a solution of compound 9b (0.04 mol) in toluene (90 mL) was
added triphosgene (0.0134 mol). The reaction mixture was
refluxed for 4 h, and nitrogen was bubbled in to drive off the
excess of phosgene. The solution was then set aside for 30 min.
The heavy crystalline precipitate was filtered off and washed
with diethyl ether to give 10b as white crystals (90%), mp 270
°C. IR (KBr) 3300, 2700 (NH), 1690 (CO) cm-1
.
1H NMR
(DMSO-d6) δ: 11.33 (s, 1H, NH), 8.19 (m, 2H, H-1 and H-9),

Quinoxalines and Pyrazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2005
(dd, 2H, J ) 9.35 and 2.95 Hz, H-8), 6.90 (d, 2H, J ) 4.35 Hz,
H-3), 6.62 (d, 2H, J ) 4.35 Hz, H-2), 3.83 (s, 3H, CH3O). 13C
NMR (DMSO-d6) δ: 157.2 (C-4), 147.7 (C-7), 137.0 (C-9a),
123.3 (C-5a), 122.1 (C-9), 116.3 (C-3a), 115.9 (C-6), 114.5 (C-
1), 110.8 (C-3), 107.9 (C-8), 107.0 (C-2), 55.5 (CH3O). Anal.
(C12H8Cl2N2O) C, H, N.
CH2), 2.59 (t, 4H, J ) 6.45 Hz, CH2), 2.36 (s, 3H, CH3), 1.93
(qt, 4H, J ) 6.45 Hz, CH2). 13C NMR (CDCl3) δ: 149.5 (C-4),
137.2 (C-5a), 126.6 (C-6), 125.2 (C-7), 125.1 (C-9a), 122.5 (C-
8), 119.7 (C-3a), 114.1 (C-9), 113.3 (C-1), 112.2 (C-3), 102.3 (C-
2), 56.9 (CH2), 42.2 (CH2), 40.4 (CH2), 26.3 (CH2). Anal. (2a ‚
3 oxalate (C29H31N7‚3C2H2O4)) C, H, N.
N-(P yr r olo[1,2-a ]q u in oxa lin -4-yl)-N′,N′-d iet h yl-1-m e-
th ylbu ta n e-1,4-d ia m in e (1a ). To 11a (0.013 mol) was added
5 mL of 5-diethylamino-2-pentylamine. The reaction mixture
was heated at 150-160 °C for 4 h and, after cooling, was
poured into water (100 mL). The mixture was extracted with
diethyl ether. The organic layer was washed with water (150
mL), dried over Na2SO4, and evaporated to dryness to give 1a
B is {N -(8-c h lo r o p y r r o lo [1,2-a ]q u in o x a lin -4-y l)-3-
a m in op r op yl}m eth yla m in e (2b) was synthesized from 11b
according to the same conditions as those described to obtain
2a . This gave 2b as yellow crystals (72%), mp 107 °C. IR (KBr)
3435 (NH) cm-1 1H NMR (CDCl3) δ: 7.56 (m, 4H, H-1 and
.
H-9), 7.46 (d, 2H, J ) 8.55 Hz, H-6), 7.15 (dd, 2H, J ) 8.55
and 2.15 Hz, H-7), 6.55 (m, 4H, H-2 and H-3), 6.37 (t, 2H, J )
4.70 Hz, NH), 3.69 (m, 4H, CH2), 2.59 (t, 4H, J ) 6.30 Hz,
CH2), 2.33 (s, 3H, CH3), 1.88 (qt, 4H, J ) 6.30 Hz, CH2). 13C
NMR (CDCl3) δ: 149.4 (C-4), 135.9 (C-5a), 127.5 (C-6), 127.2
(C-7), 125.7 (C-9a), 125.2 (C-8), 119.5 (C-3a), 114.3 (C-9), 113.4
(C-1), 112.7 (C-3), 102.8 (C-2), 56.9 (CH2), 42.3 (CH2), 40.5
(CH2), 26.3 (CH2). Anal. (2b‚3 oxalate (C29H29Cl2N7‚3C2H2O4))
C, H, N.
as an orange oil (58%). IR (KBr) 3215 (NH) cm-1 1H NMR
.
(CDCl3) δ: 7.74 (dd, 1H, J ) 2.70 and 1.15 Hz, H-1), 7.65 (dd,
1H, J ) 7.85 and 1.35 Hz, H-9), 7.61 (dd, 1H, J ) 7.85 and
1.35 Hz, H-6), 7.23 (m, 2H, H-7 and H-8), 6.67 (dd, 1H, J )
3.85 and 2.70 Hz, H-2), 6.59 (dd, 1H, J ) 3.85 and 1.15 Hz,
H-3), 4.91 (d, 1H, J ) 8.15 Hz, NH), 4.52 (m, 1H, CH), 2.60 (t,
2H, J ) 7.25 Hz, CH2), 2.48 (q, 4H, J ) 7.30 Hz, CH2), 1.51
(m, 4H, CH2), 1.32 (d, 3H, J ) 6.55 Hz, CH3), 1.10 (t, 6H, J )
7.30 Hz, CH3). 13C NMR (CDCl3) δ: 148.7 (C-4), 137.3 (C-5a),
126.8 (C-6), 125.2 (C-7), 125.0 (C-9a), 122.5 (C-8), 119.6 (C-
3a), 114.1 (C-9), 113.2 (C-1), 112.1 (C-3), 101.9 (C-2), 52.8 (CH),
46.8 (CH2), 45.7 (CH2), 35.2 (CH2), 23.7 (CH3), 21.1 (CH2), 11.5
(CH3). Anal. (1a ‚2 oxalate (C20H28N4‚2C2H2O4)) C, H, N.
N-(8-Ch lor opyr r olo[1,2-a ]qu in oxalin -4-yl)-N′,N′-dieth yl-
1-m eth ylbu ta n e-1,4-d ia m in e (1b) was synthesized from 11b
according to the same conditions as those described to obtain
1a . This gave 1b as an orange oil (79%). IR (KBr) 3435 (NH)
cm-1. 1H NMR (CDCl3) δ: 7.64 (dd, 1H, J ) 2.90 and 1.25 Hz,
H-1), 7.62 (d, 1H, J ) 2.30 Hz, H-9), 7.50 (d, 1H, J ) 8.70 Hz,
H-6), 7.19 (dd, 1H, J ) 8.70 and 2.30 Hz, H-7), 6.67 (dd, 1H,
J ) 3.85 and 2.90 Hz, H-2), 6.58 (dd, 1H, J ) 3.85 and 1.25
Hz, H-3), 4.99 (d, 1H, J ) 8.10 Hz, NH), 4.48 (m, 1H, CH),
2.50 (t, 2H, J ) 7.20 Hz, CH2), 2.44 (q, 4H, J ) 7.30 Hz, CH2),
1.57 (m, 4H, CH2), 1.28 (d, 3H, J ) 6.50 Hz, CH3), 0.97 (t, 6H,
J ) 7.30 Hz, CH3). 13C NMR (CDCl3) δ: 148.7 (C-4), 136.0 (C-
5a), 127.7 (C-6), 127.2 (C-7), 125.7 (C-9a), 125.2 (C-8), 119.4
(C-3a), 114.4 (C-9), 113.4 (C-1), 112.7 (C-3), 102.5 (C-2), 52.8
(CH), 46.8 (CH2), 45.7 (CH2), 35.2 (CH2), 23.7 (CH3), 21.2 (CH2),
11.5 (CH3). Anal. (1b‚2 oxalate (C20H27ClN4‚2C2H2O4)) C, H,
N.
Bis{N -(7-m e t h oxyp yr r olo[1,2-a ]q u in oxa lin -4-yl)-3-
a m in op r op yl}m eth yla m in e (2c) was synthesized from 11c
according to the same conditions as those described to obtain
2a . This gave 2c as yellow crystals (70%), mp 63 °C. IR (KBr)
1
3240 (NH) cm-1. H NMR (CDCl3) δ: 7.61 (dd, 2H, J ) 2.60
and 1.35 Hz, H-1), 7.53 (d, 2H, J ) 8.85 Hz, H-9), 7.11 (d, 2H,
J ) 2.75 Hz, H-6), 6.76 (dd, 2H, J ) 8.85 and 2.75 Hz, H-8),
6.54 (dd, 2H, J ) 3.65 and 1.35 Hz, H-3), 6.52 (dd, 2H, J )
3.65 and 1.35 Hz, H-2), 6.31 (t, 2H, J ) 4.55 Hz, NH), 3.81 (s,
6H, CH3O), 3.74 (m, 4H, CH2), 2.57 (t, 4H, J ) 6.40 Hz, CH2),
2.34 (s, 3H, CH3), 1.91 (qt, 4H, J ) 6.40 Hz, CH2). 13C NMR
(CDCl3) δ: 157.2 (C-4), 149.9 (C-7), 138.4 (C-9a), 119.6 (C-5a),
119.2 (C-9), 114.1 (C-3a), 113.8 (C-6), 111.9 (C-9), 110.9 (C-3),
108.8 (C-8), 102.0 (C-2), 56.9 (CH2), 55.5 (CH3O), 42.2 (CH3O),
42.2 (CH3), 40.4 (CH2), 26.3 (CH2). Anal. (2c‚3 oxalate
(C31H35N7O2‚3C2H2O4)) C, H, N.
Bis{N -(8-m e t h oxyp yr r olo[1,2-a ]q u in oxa lin -4-yl)-3-
a m in op r op yl}m eth yla m in e (2d ) was synthesized from 11d
according to the same conditions as those described to obtain
2a . This gave 2d as yellow crystals (67%), mp 51 °C. IR (KBr)
1
3410 (NH) cm-1. H NMR (CDCl3) δ: 7.63 (m, 2H, H-1), 7.54
(d, 2H, J ) 8.70 Hz, H-6), 7.24 (d, 2H, J ) 2.65 Hz, H-9), 6.86
(dd, 2H, J ) 8.70 and 2.65 Hz, H-7), 6.60 (m, 2H, H-3), 6.51
(m, 2H, H-2), 6.01 (t, 2H, J ) 4.50 Hz, NH), 3.87 (s, 6H, CH3O),
3.74 (m, 4H, CH2), 2.53 (t, 4H, J ) 6.60 Hz, CH2), 2.36 (s, 3H,
CH3), 1.89 (qt, 4H, J ) 6.60 Hz, CH2). Anal. (2d ‚3 oxalate
(C31H35N7O2‚3C2H2O4)) C, H, N.
N-(7-Met h oxyp yr r olo[1,2-a ]q u in oxa lin -4-yl)-N′,N′-d i-
eth yl-1-m eth ylbu ta n e-1,4-d ia m in e (1c) was synthesized
from 11c according to the same conditions as those described
to obtain 1a . This gave 1c as an orange oil (65%). IR (KBr)
1
3340 (NH) cm-1. H NMR (CDCl3) δ: 7.64 (dd, 1H, J ) 2.60
B is {N -(8-c h lo r o p y r r o lo [1,2-a ]q u in o x a lin -4-y l)-3-
a m in op r op yl}a m in e (3b). To a solution of 11b (0.013 mol)
in dimethylformamide (35 mL) were added K2CO3 (0.0143 mol)
and then N-(3-aminopropyl)-1,3-propanediamine (0.0065 mol).
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 dichlo-
romethane. The organic layer was washed with water (150
mL), dried over Na2SO4, and evaporated to dryness to give 3b
as yellow crystals (65%), mp 44 °C. IR (KBr) 3420 and 3315
and 1.30 Hz, H-1), 7.53 (d, 1H, J ) 8.85 Hz, H-9), 7.08 (d, 1H,
J ) 2.85 Hz, H-6), 6.75 (dd, 1H, J ) 8.85 and 2.85 Hz, H-8),
6.68 (dd, 1H, J ) 3.90 and 1.30 Hz, H-3), 6.61 (dd, 1H, J )
3.90 and 2.60 Hz, H-2), 5.15 (d, 1H, J ) 7.95 Hz, NH), 4.51
(m, 1H, CH), 3.84 (s, 3H, CH3O), 2.63 (t, 2H, J ) 7.20 Hz,
CH2), 2.52 (q, 4H, J ) 7.30 Hz, CH2), 1.66 (m, 4H, CH2), 1.34
(d, 3H, J ) 6.60 Hz, CH3), 1.14 (t, 6H, J ) 7.30 Hz, CH3). 13C
NMR (CDCl3) δ: 157.9 (C-4), 149.9 (C-7), 139.0 (C-9a), 120.3
(C-5a), 119.8 (C-9), 114.7 (C-3a), 114.5 (C-6), 112.5 (C-1), 111.5
(C-2), 109.6 (C-8), 102.8 (C-2), 56.2 (CH3O), 53.2 (CH), 47.4
(CH2), 46.1 (CH2), 35.4 (CH2), 23.5 (CH3), 22.0 (CH2), 11.3
(CH3). Anal. (1c‚2 oxalate (C21H30N4O‚2C2H2O4)) C, H, N.
(NH) cm-1 1H NMR (CDCl3) δ: 7.63 (m, 4H, H-1 and H-9),
.
7.49 (d, 2H, J ) 8.60 Hz, H-6), 7.17 (dd, 2H, J ) 8.60 and 2.00
Hz, H-7), 6.62 (m, 4H, H-2 and H-3), 6.22 (m, 2H, NH), 3.78
(m, 4H, CH2), 2.80 (t, 4H, J ) 6.10 Hz, CH2), 1.91 (qt, 4H, J )
6.10 Hz, CH2). 13C NMR (CDCl3) δ: 149.5 (C-4), 135.8 (C-5a),
127.4 (C-6), 127.2 (C-7), 125.7 (C-9a), 125.2 (C-8), 119.4 (C-
3a), 114.4 (C-9), 113.4 (C-1), 112.7 (C-3), 103.0 (C-2), 48.1
(CH2), 39.8 (CH2), 29.1 (CH2). Anal. (3b‚3 oxalate (C28H27Cl2N7‚
3C2H2O4)) C, H, N.
Bis{N-(p yr r olo[1,2-a ]qu in oxa lin -4-yl)-3-a m in op r op yl}-
m eth yla m in e (2a ). To a solution of 11a (0.013 mol) in
dimethylformamide (35 mL) were added K2CO3 (0.0143 mol)
and then 3,3′-diamino-N-methyldipropylamine (0.0065 mol).
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 dichlo-
romethane. The organic layer was washed with water (150
mL), dried over Na2SO4, and evaporated to dryness to give 2a
Bis{N -(7-m e t h oxyp yr r olo[1,2-a ]q u in oxa lin -4-yl)-3-
a m in op r op yl}a m in e (3c) was synthesized from 11c accord-
ing to the same conditions as those described to obtain 3b.
This gave 3c as yellow crystals (71%), mp 56 °C. IR (KBr) 3395
as yellow crystals (64%), mp 48 °C. IR (KBr) 3255 (NH) cm-1
.
1H NMR (CDCl3) δ: 7.71 (m, 2H, H-1), 7.66 (d, 2H, J ) 7.75
Hz, H-9), 7.61 (d, 2H, J ) 7.75 Hz, H-6), 7.26 (t, 2H, J ) 7.75
Hz, H-8), 7.17 (t, 2H, J ) 7.75 Hz, H-7), 6.62 (m, 2H, H-3),
6.56 (m, 2H, H-2), 6.28 (t, 2H, J ) 4.90 Hz, NH), 3.75 (m, 4H,
1
and 3305 (NH) cm-1. H NMR (CDCl3) δ: 7.65 (m, 2H, H-1),
7.55 (d, 2H, J ) 8.85 Hz, H-9), 7.12 (d, 2H, J ) 2.80 Hz, H-6),
6.77 (dd, 2H, J ) 8.85 and 2.80 Hz, H-8), 6.60 (m, 4H, H-2

2006 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Guillon et al.
and H-3), 6.25 (m, 2H, NH), 3.85 (m, 4H, CH2), 3.82 (s, 6H,
CH3O), 2.78 (t, 4H, J ) 6.10 Hz, CH2), 1.90 (qt, 4H, J ) 6.10
Hz, CH2). 13C NMR (CDCl3) δ: 157.3 (C-4), 149.9 (C-7), 138.4
(C-9a), 119.6 (C-5a), 119.1 (C-9), 114.1 (C-3a), 113.9 (C-6),
111.9 (C-1), 110.9 (C-3), 108.8 (C-8), 102.1 (C-2), 55.5 (CH3O),
48.2 (CH2), 39.8 (CH2), 29.8 (CH2). Anal. (3c‚3 oxalate
(C30H33N7O2‚3C2H2O4)) C, H, N.
9a), 128.2 (C-5a), 126.3 (C-9), 120.7 (C-3a), 114.6 (C-6), 112.9
(C-1), 112.7 (C-3), 103.1 (C-7), 99.0 (C-2), 59.1 (CH2), 56.4
(CH3O), 54.3 (CH2), 42.1 (CH2), 25.7 (CH2). Anal. (4d ‚4 oxalate
(C34H40N8O2‚4C2H2O4)) C, H, N.
B is {N -(p y r id o [3,2-e]p y r r o lo [1,2-a ]p y r a zin -6-y l)-3-
a m in op r op yl}p ip er a zin e (4e) was synthesized from 11e
according to the same conditions as those described to obtain
4a . This gave 4e as pale-yellow crystals (34%), mp 215 °C. IR
Bis{N -(8-m e t h oxyp yr r olo[1,2-a ]q u in oxa lin -4-yl)-3-
a m in op r op yl}a m in e (3d ) was synthesized from 11d accord-
ing to the same conditions as those described to obtain 3b.
This gave 3d as yellow crystals (65%), mp 49 °C. IR (KBr) 3410
1
(KBr) 3240 (NH) cm-1. H NMR (CDCl3) δ: 8.20 (m, 4H, H-2
and H-9), 7.87 (dd, 2H, J ) 7.95 and 1.50 Hz, H-4), 7.25 (dd,
2H, J ) 7.95 and 4.70 Hz, H-3), 6.91 (t, 2H, J ) 4.20 Hz, NH),
6.75 (dd, 2H, J ) 3.75 and 1.45 Hz, H-7), 6.71 (m, 2H, H-8),
3.80 (m, 4H, CH2), 2.68 (m, 12H, CH2), 1.92 (qt, 4H, J ) 5.90
Hz, CH2). Anal. (4e‚4 oxalate (C30H34N10‚4C2H2O4)) C, H, N.
and 3350 (NH) cm-1 1H NMR (CDCl3) δ: 7.65 (dd, 2H, J )
.
2.60 and 1.15 Hz, H-1), 7.53 (d, 2H, J ) 8.75 Hz, H-6), 7.15
(d, 2H, J ) 2.80 Hz, H-9), 6.87 (dd, 2H, J ) 8.75 and 2.80 Hz,
H-7), 6.64 (dd, 2H, J ) 3.75 and 1.15 Hz, H-3), 6.60 (dd, 2H,
J ) 3.75 and 2.60 Hz, H-2), 5.94 (m, 2H, NH), 3.87 (s, 6H,
CH3O), 3.76 (m, 4H, CH2), 2.81 (t, 4H, J ) 6.15 Hz, CH2), 1.93
(qt, 4H, J ) 6.15 Hz, CH2). Anal. (3d ‚3 oxalate (C30H33N7O2‚
3C2H2O4)) C, H, N.
Bis{N-(2-m et h oxyp yr id o[3,2-e]p yr r olo[1,2-a ]p yr a zin -
6-yl)-3-a m in op r op yl}p ip er a zin e (4f) was synthesized from
11f according to the same conditions as those described to
obtain 4a . This gave 4f as yellow crystals (21%), mp 201 °C.
IR (KBr) 3230 (NH) cm-1 1H NMR (CDCl3) δ: 8.10 (m, 2H,
.
H-9), 7.83 (d, 2H, J ) 8.50 Hz, H-4), 6.73 (d, 2H, J ) 8.50 Hz,
H-3), 6.71 (m, 4H, H-7 and H-8), 6.59 (t, 2H, J ) 5.85 Hz, NH),
4.01 (s, 6H, OCH3), 3.73 (m, 4H, CH2), 2.63 (m, 12H, CH2),
1.92 (qt, 4H, J ) 5.85 Hz, CH2). Anal. (4f‚4 oxalate (C32H38N10O2‚
4C2H2O4)) C, H, N.
Bis{N-(p yr r olo[1,2-a ]qu in oxa lin -4-yl)-3-a m in op r op yl}-
p ip er a zin e (4a ). To a solution of 11a (0.013 mol) in dimeth-
ylformamide (35 mL) were added K2CO3 (0.0143 mol) and then
1,4-bis(3-aminopropyl)piperazine (0.0065 mol). 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 Na2-
SO4, and evaporated to dryness to give 4a as pale-yellow
crystals (61%), mp 203 °C. IR (KBr) 3230 (NH) cm-1. 1H NMR
(CDCl3) δ: 7.75 (m, 2H, H-1), 7.68 (d, 2H, J ) 7.75 Hz, H-9),
7.64 (d, 2H, J ) 7.75 Hz, H-6), 7.28 (t, 2H, J ) 7.75 Hz, H-8),
7.22 (t, 2H, J ) 7.75 Hz, H-7), 6.71 (m, 6H, NH), 3.79 (m, 4H,
Bis{N-(1-ch lor o-7-m eth oxyp yr r olo[1,2-a ]qu in oxa lin -4-
yl)-3-a m in op r op yl}p ip er a zin e (5) was synthesized from 12c
according to the same conditions as those described to obtain
4a . This gave 5 as yellow crystals (60%), mp 128 °C. IR (KBr)
3230 (NH) cm-1 1H NMR (CDCl3) δ: 8.77 (d, 2H, J ) 9.30
.
Hz, H-9), 7.12 (d, 2H, J ) 2.85 Hz, H-6), 6.76 (dd, 2H, J )
9.30 and 2.85 Hz, H-8), 6.70 (m, 2H, NH), 6.65 (d, 2H, J )
4.20 Hz, H-3), 6.54 (d, 2H, J ) 4.20 Hz, H-2), 3.86 (s, 6H,
CH3O), 3.75 (m, 4H, CH2), 2.64 (m, 12H, CH2), 1.89 (qt, 4H, J
) 5.80 Hz, CH2). 13C NMR (CDCl3) δ: 157.8 (C-4), 150.0 (C-
7), 138.4 (C-9a), 121.1 (C-5a), 120.1 (C-9), 116.8 (C-3a), 115.0
(C-6), 112.9 (C-1), 110.7 (C-3), 109.5 (C-8), 102.5 (C-2), 59.2
(CH2), 56.1 (CH3O), 54.2 (4CH2), 42.1 (CH2), 25.3 (CH2). Anal.
(5c‚4 oxalate (C34H38Cl2N8O2‚4C2H2O4)) C, H, N.
CH2), 3.65 (m, 12H, CH2), 1.90 (qt, 4H, J ) 5.85 Hz, CH2). 13
C
NMR (CDCl3) δ: 149.6 (C-4), 137.3 (C-5a), 126.6 (C-6), 125.2
(C-7), 125.1 (C-9a), 122.5 (C-8), 120.2 (C-3a), 114.1 (C-9), 113.3
(C-1), 112.1 (C-3), 102.6 (C-2), 58.5 (CH2), 53.6 (CH2), 41.4
(CH2), 24.8 (CH2). Anal. (4a ‚4 oxalate (C32H36N8‚4C2H2O4)) C,
H, N.
B is {N -(8-c h lo r o p y r r o lo [1,2-a ]q u in o x a lin -4-y l)-3-
a m in op r op yl}p ip er a zin e (4b) was synthesized from 11b
according to the same conditions as those described to obtain
4a . This gave 4b as yellow crystals (84%), mp 226 °C. IR (KBr)
B is {N -(p y r r o lo [1,2-a ]t h ie n o [3,2-e]p y r a zin -5-y l)-3-
a m in op r op yl}p ip er a zin e (6) was synthesized from 13 ac-
cording to the same conditions as those described to obtain
4a . This gave 6 as orange crystals (52%), mp 197 °C. IR (KBr)
1
3290 (NH) cm-1. H NMR (CDCl3) δ: 7.68 (m, 2H, H-1), 7.65
1
3320 (NH) cm-1. H NMR (CDCl3) δ: 7.31 (m, 2H, H-8), 7.20
(d, 2H, J ) 2.20 Hz, H-9), 7.53 (d, 2H, J ) 8.60 Hz, H-6), 7.21
(dd, 2H, J ) 8.60 and 2.20 Hz, H-7), 6.80 (t, 2H, J ) 4.90 Hz,
NH), 6.71 (m, 4H, H-2 and H-3), 3.77 (m, 4H, CH2), 2.66 (m,
12H, CH2), 1.91 (qt, 4H, J ) 5.90 Hz, CH2). 13C NMR (CDCl3)
δ: 149.5 (C-4), 135.8 (C-5a), 127.6 (C-6), 127.2 (C-7), 125.7 (C-
9a), 125.5 (C-8), 119.6 (C-3a), 114.3 (C-9), 113.4 (C-1), 112.6
(C-3), 103.0 (C-2), 58.5 (CH2), 53.6 (CH2), 41.4 (CH2), 24.7
(CH2). Anal. (4b‚4 oxalate (C32H34Cl2N8‚4C2H2O4)) C, H, N.
(d, 2H, J ) 5.60 Hz, H-2), 6.95 (d, 2H, J ) 5.60 Hz, H-3), 6.70
(m, 4H, H-6 and H-7), 6.52 (t, 2H, J ) 5.80 Hz, NH), 3.71 (m,
4H, CH2), 2.64 (m, 12H, CH2), 1.90 (qt, 4H, J ) 6.00 Hz, CH2).
Anal. (6‚4 oxalate (C28H32N8S2‚4C2H2O4)) C, H, N.
X-r a y Da ta . The structures of compounds 4b and 4e have
been established by X-ray crystallography (Figure 1).48 Color-
less single crystals (0.50 × 0.15 × 0.10 mm3) of 4b were
obtained by slow evaporation from a methanol/chloroform (40/
60) solution: triclinic, space group P1h, a ) 9.597(3) Å, b )
10.516(2) Å, c ) 10.846(1) Å, R ) 67.37(1)°, â ) 72.68(2)°, γ )
Bis{N -(7-m e t h oxyp yr r olo[1,2-a ]q u in oxa lin -4-yl)-3-
a m in op r op yl}p ip er a zin e (4c) was synthesized from 11c
according to the same conditions as those described to obtain
4a . This gave 4c as yellow crystals (63%), mp 199 °C. IR (KBr)
83.73(2)°, V ) 964.5(4) Å3, Z ) 1, F(calcd) ) 1.447 Mg‚m-3
,
FW ) 840.32 for C32H34Cl2N8‚2CHCl3, F(000) ) 432. Colorless
single crystals (0.37 × 0.15 × 0.01 mm3) of 4e were obtained
by slow evaporation from a methanol/chloroform (30/70) solu-
tion: monoclinic, space group P21/c, a ) 12.078(1) Å, b )
5.617(1) Å, c ) 19.647(1) Å, R ) 90.0°, â ) 93.65(2)°, γ ) 90.0°,
V ) 1330.2(3) Å3, Z ) 2, F(calcd) ) 1.335 Mg‚m-3, FW ) 534.68
for C30H34N10, F(000) ) 568. The unit cell dimensions were
determined using the least-squares fit from 25 reflections (25°
< θ < 35°). Intensities were collected with an Enraf-Nonius
CAD-4 diffactometer using the Cu KR radiation and a graphite
monochromator up to θ ) 55°. No intensity variation of two
standard reflections monitored every 90 min was observed. The
data were corrected for Lorentz and polarization effects and
for empirical absorption correction.54 The structure was solved
by direct methods Shelx 8655 and refined using the Shelx 9356
suite of programs.
1
3255 (NH) cm-1. H NMR (CDCl3) δ: 7.68 (m, 2H, H-1), 7.58
(d, 2H, J ) 8.85 Hz, H-9), 7.14 (d, 2H, J ) 2.80 Hz, H-6), 6.79
(m, 4H, H-8 and H-3), 6.68 (m, 4H, H-2 and NH), 3.86 (s, 6H,
CH3O), 3.79 (m, 4H, CH2), 2.66 (m, 12H, CH2), 1.91 (qt, 4H, J
) 5.95 Hz, CH2). 13C NMR (CDCl3) δ: 157.3 (C-4), 150.0 (C-
7), 138.5 (C-9a), 119.6 (C-5a), 119.3 (C-9), 114.1 (C-3a), 113.8
(C-6), 111.7 (C-1), 110.9 (C-3), 108.8 (C-8), 102.2 (C-2), 58.6
(CH2), 55.6 (CH3O), 53.6 (CH2), 41.5 (CH2), 24.7 (CH2). Anal.
(4c‚4 oxalate (C34H40N8O2‚4C2H2O4)) C, H, N.
Bis{N -(8-m e t h oxyp yr r olo[1,2-a ]q u in oxa lin -4-yl)-3-
a m in op r op yl}p ip er a zin e (4d ) was synthesized from 11d
according to the same conditions as those described to obtain
4a . This gave 4d as yellow crystals (61%), mp 73 °C. IR (KBr)
1
3325 (NH) cm-1. H NMR (CDCl3) δ: 7.67 (m, 2H, H-1), 7.56
(d, 2H, J ) 8.85 Hz, H-6), 7.16 (d, 2H, J ) 2.60 Hz, H-9), 6.90
(dd, 2H, J ) 8.85 and 2.60 Hz, H-7), 6.68 (m, 4H, H-2 and
H-3), 6.48 (t, 2H, J ) 5.05 Hz, NH), 3.88 (s, 6H, CH3O), 3.75
(m, 4H, CH2), 2.62 (m, 12H, CH2), 1.90 (qt, 4H, J ) 6.10 Hz,
CH2). 13C NMR (CDCl3) δ: 156.5 (C-4), 149.7 (C-8), 132.0 (C-
P a r tition Coefficien ts: log D (p H 7.4 or p H 5.0). The
relative log D at pH 7.4 and 5.0 of each compound in this study
was assessed by the micro-HPLC method.45 These determina-
tions were performed with
a chromatographic apparatus

Quinoxalines and Pyrazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2007
(Waters, Milford, MA) equipped with a model 501 constant flow
pump, a model 2487 ultraviolet detector (λ ) 254 nm), and a
746 data module integrator. A reversed-phase column was
used: a Waters Spherisorb S5C8 (4.6 mm × 150 mm, 5 µm
particle size) with a mobile phase consisting of acetonitrile-
acetate buffer (pH 4) (80:20, v/v). The column was maintained
at 60 °C.
The compounds were partitioned between 1-octanol (HPLC
grade) and phosphate buffer (pH 7.4) or acetate buffer (pH
5.0). Octanol was presaturated with buffer, and conversely,
the buffer was presaturated with octanol. 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 obtain final concentrations ranging from
50 to 250 µg/mL. Under the above-described chromatographic
conditions, 20 µL of this aqueous phase was injected into the
chromatograph, leading to the determination of a peak area
before partitioning (W0).
insoluble trophozoite material of the FcB1R strain (approxi-
mately equivalent to 4 × 107 parasites) was added to 900 µL
of a haem-acetate mixture (0.3 mM bovine hematin, 60 mM
sodium acetate, pH 5). Samples of 50 µL of drug solution at
different concentrations were mixed with the other compo-
nents. Samples without drug constituted controls. All of the
samples contained the same amount of DMSO (1%). Following
incubation for 4 h, at 37 °C, the samples were centrifuged at
27000g, 15 min, at 4 °C. The pellet was resuspended in 1 mL
of buffer A (68 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 5 mM
glucose, 50 mM sodium phosphate, pH 7.4) and repelleted. The
pellet was introduced to 2.5% SDS in buffer A and sonicated
for 10 min. The polymerized haem was collected by centrifuga-
tion at 27000g, 30 min, at 20 °C. The pellet was then washed
four times before being resuspended in 900 µL of 2.5% SDS in
buffer A and 100 µL of 1 M NaOH was added to dissolve the
polymerized haem. Following incubation for 1 h, the concen-
tration of haemozoin was determined by measuring the
absorbance at 404 nm.63 The amount of haemozoin formed
during the incubation period was corrected for the endogenous
haemozoin of the trophozoite preparation, and the percentage
of inhibition of â-hematin formation was determined by
comparison with samples maintained without inhibitor. Data
presented are the mean of triplicate experiments.
In scew-capped tubes, 1 mL of the aqueous phase (Vaq) was
then added to 5µL of n-octanol (Voct). The mixture was shaken
by mechanical rotation during 30 min. Then the centrifugation
was achieved at 3000 rpm in 10 min. An amount of 20 µL of
the lower phase was injected into the chromatograph column.
This led to the determination of a peak area after partitioning
(W1). The log D value was determined by the formula
Meth od B. Experiments were carried out in duplicate in
96 deep wells. In each well, samples of 250 µL of a solution of
700 µM of hemin in 25 mM NaOH were distributed, followed
by 250 µL of a suspension of 1 mM 1-monooleoylglycerol in 90
mM sodium acetate with 1% DMSO, at pH 5, extemporane-
ously prepared. Drugs were added from DMSO stock solutions
(5 µL). Microplates were incubated for 24 h at 37 °C. Controls
contained an equal amount of DMSO. Following incubation,
the samples were centrifuged at 4000 rpm at 4 °C for 30 min.
The pellet of â-hematin was washed with 10 mM sodium
phosphate, pH 7.4, containing 2.5% SDS and was vortexed for
10 min at 20 °C before repelleting until the supernatant was
colorless (five times). Dissolution of â-hematin was achieved
by addition of 450 µL of 10 mM sodium phosphate, pH 7.4,
containing 2.5% SDS and 25 µL of 1 M NaOH. Concentration
of residual haem was calculated from absorbance at 405 nm.
(W0 - W1)Vaq
log D ) log
[
]
W1Voct
Molecu la r Mod elin g. All molecular modeling was carried
out on a Indigo 2 R44OO silicon graphics workstation. In our
studies, we have employed the method of simulated annealing.
Molecular dynamics (MD) runs at 300 K for a simulation time
of 5 ps (time step of 1 fs) were performed for each drug
molecule, and the resulting structure was energy-minimized
to gain a low-energy conformation.57,58
This process was repeated until 10 structures per molecule
were generated. The rmsd values of the three final selected
conformations are close together, less than 1.129 638 Å
(2.150 644 Å) for 4b (4d ) and less than 2.859 583 Å for hematin
molecular unit model. For each MD calculation, the last
minimized structure in the set was used as the next starting
point. Each of the structures was finally minimized at the
restricted Hartee-Fock (RHF) level using the PM3 basis set
procedure implemented in GAMESS-US.59
Cytotoxicity Test on Ma m m a lia n L6 Cells. Samples of
104 L6 cells per well in 96-well plates were maintained in the
presence of different concentrations of inhibitors for up to 5
days. Cell proliferation was determined by using the XTT-
based colorimetric assay (Cell proliferation kit II, Boehringer
Mannheim S.A., Meylan, France). Control cultures received
an equivalent amount of DMSO instead of inhibitor. Inhibition
of proliferation was determined by comparison to control
cultures.
Biologica l Assa ys. In Vitr o P . fa lcipa r u m Cu ltu r e a n d
Dr u g Assa ys. P. falciparum strains were maintained continu-
ously in culture on human erythrocytes as described by Trager
and J ensen.60 In vitro antiplasmodial activity was determined
using a modification of the semiautomated microdilution
technique of Desjardins et al.61 P. falciparum CQ-sensitive
(Thai/Thailand) and CQ-resistant (FcB1R/Colombia and K1/
Thailand) strains were used in sensitivity testing. Stock
solutions of CQ diphosphate and test compounds were pre-
pared in sterile distilled water and DMSO, respectively. Drug
solutions were serially diluted with culture medium and added
to asynchronous parasite cultures (1% parasitemia and 1%
final hematocrite) in 96-well plates for 24 h, at 37 °C, prior to
the addition of 0.5 µCi of [3H]hypoxanthine (1-5 Ci/mmol;
Amersham, Les Ulis, France) per well, for 24 h. The growth
inhibition for each drug concentration was determined by
comparison of the radioactivity incorporated into the treated
culture with that in the control culture (without drug) main-
tained on the same plate. The concentration causing 50%
inhibition (IC50) was obtained from the drug concentration-
response curve, and the results were expressed as the mean
( the standard deviations determined from several indepen-
dent experiments. The DMSO concentration never exceeded
0.1% and did not inhibit the parasite growth.
Su p p or tin g In for m a tion Ava ila ble: Figures S1 and S2
showing the crystal structures of 4b and 4e and Tables S1-
S11 listing crystallographic results. This material is available
free of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Mishra, S. K.; Satpathy, S. K.; Mohanty, S. Survey of Malaria
Treatment and Deaths. Bull. W. H. O. 1999, 77, 1020.
(2) Bouland, P. B. Drug Resistance in Malaria; World Health
Organization: Geneva, Switzerland, 2001.
(3) Ridley, R. G. Medicinal need, scientific opportunity and the drive
for antimalarial drugs. Nature 2002, 415, 686-693.
(4) Ursos, L. M. B.; Roepe, P. D. Chloroquine Resistance in the
Malaria Parasite, Plasmodium falciparum. Med. Res. Rev. 2002,
22, 465-491.
(5) White, N. J . Antimalarial Drug Resistance: The Pace Quickens.
J . Antimicrob. Chemother. 1992, 30, 571-585.
(6) Surrey, A. R.; Hammer, H. F. Some 7-substituted 4-aminoquino-
line derivatives. J . Am. Chem. Soc. 1946, 68, 113-116.
(7) Van Est, H. G.; Skamene, G. E.; Schurr, E. Chemotherapy of
malaria: a battle against the odds? Clin. Invest. Med. 1993, 16,
285-293.
(8) Dorn, A.; Stoffel, R.; Matile, H.; Bubendorf, A.; Ridley, R. G.
Malrial Haemozoin/Beta-Haematin Supports Haem Polimeriza-
tion in the Absence of Protein. Nature 1995, 374, 269-271.
â-Hem a tin F or m a tion Assa y. Meth od A. The drug effects
upon â-hematin formation were assessed according to Raynes
et al.41 The haemozoin content was determined using the
procedure of Chou and Fitch.62 Briefly, a 50 µL aliquot of

2008 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Guillon et al.
(9) Dorn, A.; Vippagunta, S. R.; Matile, H.; J aquet, C.; Vennerstrom,
J . L.; Ridley, R. G. An Assessment of Drug-Haematin Binding
as Mechanism for Inhibition of Haematin Polimerization by
Quinoline Antimalarials. Biochem. Pharmacol. 1998, 55, 727-
736.
(10) Ginsburg, H.; Fatim, O.; Zhang, J .; Krugliak, M. Inhibition of
Glutathione-Dependent Degradation of Heme by Chloroquine
and Amodiaquine as a Possible Basis for Their Antimalarial
Mode of Action. Biochem. Pharmacol. 1998, 56, 1305-1313.
(11) Krogstad, D. J .; Gluzman, I. Y.; Kyle, D. E.; Oduola, A. M.;
Martin, S. K.; Milhous, W. K.; Schlesinger, P. H. Efflux of
chloroquine from Plasmodium falciparum: mechanism of chlo-
roquine resistance. Science 1987, 238, 1283-1285.
(12) Ginsburg, H.; Stein, W. D. Kinetic modeling of chloroquine
uptake by malaria-infected erythrocytes. Biochem. Pharmacol.
1991, 41, 1463-1470.
(13) Wuˆnsch, S.; Sanchez, C. P.; Gekle, M.; Grobe-Wortmann, L.;
Wiesner, J .; Lanzer, M. Differential stimulation of the Na+/H+
exchanger determines chloroquine uptake in Plasmodium fal-
ciparum. J . Cell Biol. 1998, 140, 335-345.
(14) Bray, P. G.; J anneh, O.; Raynes, K. J .; Mungthin, M.; Ginsburg,
H.; Ward, S. A. Cellular uptake of chloroquine is dependent on
binding to ferriprotoporphyrin IX and is dependent on NHE
activity in Plasmodium falciparum. J . Cell Biol. 1999, 145, 363-
376.
(15) Reed, M. B.; Saliba, K. J .; Caruana, S. R.; Kirk, K.; Cowman, A.
F. Pgh1 modulates sensitivity and resistance to multiple anti-
malarials in Plasmodium falciparum. Nature 2000, 403, 906-
909.
(16) Vennerstrom, J . L.; Ellis, W. Y.; Ager, A. L.; Andersen, S. L.;
Gerena, L.; Milhous, W. K. Bisquinolines. N,N-Bis(7-chloro-
quinolin-4-yl)alkanediamines with Potential against Chloro-
quine-Resistant Malaria. J . Med. Chem. 1992, 35, 2129-2134.
(17) Ridley, R. G.; Matile, H.; J aquet, C.; Dorn, A.; Hofheinz, W.;
Leupin, W.; Masciadri, R.; Theil, F.-P.; Richter, W. F.; Girometta,
M.-A.; Guenzi, A.; Urwyler, H.; Gocke, E.; Potthast, J .-M.; Csato,
M.; Thomas, A.; Peters, W. Antimalarial Activity of the Bis-
quinoline trans-N1,N2-Bis(7-chloroquinolin-4-yl)cyclohexane-1,2-
diamine: Comparison of Two Stereoisomers and Detailed Evalu-
ation of the S,S Enantiomer, Ro 47-7737. J . Antimicrob.
Chemother. 1997, 41, 677-686.
(18) Kalidas, P.; DeWitt Blanton, C. Synthesis of 1,4-Bis(6-methoxy-
8-quinolylaminoalkyl)piperazines as potential prophylactic an-
timalarial agents. J . Med. Chem. 1973, 16, 1391-1394.
(19) Shetty, R. V.; Dewitt Blanton, C. Synthesis and Antiprotozoan
Activity of N,N′-Disubstituted Piperazines. Eur. J . Med. Chem.
1979, 14, 353-356.
(20) Raynes, K.; Galatis, D.; Cowman, A. F.; Tilley, L.; Deady, L. W.
Synthesis and Activity of Some Antimalarial Bisquinolines. J .
Med. Chem. 1995, 38, 204-206.
(21) Raynes, K.; Foley, M.; Tilley, L.; Deady L. W. Novel Bisquinoline
Antimalarials. Biochem. Pharmacol. 1996, 52, 551-559.
(22) Vennerstrom, J . L.; Ager, A. L.; Dorn, A.; Andersen, S. L.;
Gerena, L.; Ridley, R. G.; Milhous, W. K. Bisquinolines. 2.
Antimalarial N,N-Bis(7-chloroquinolin-4-yl)heteroalkanediamines.
J . Med. Chem. 1998, 41, 4360-4364.
(23) Raynes, K. Bisquinoline Antimalarials: Their Role in Malaria
Chemotherapy. Int. J . Parasitol. 1999, 29, 367-379.
(24) Walls, L. P. Researches in the Phenanthridine Series. J . Chem.
Soc. 1935, 1405-1410.
(25) Girault, S.; Grellier, P.; Berecibar, A.; Maes, L.; Mouray, E.;
Lemie`re, P.; Debreu, M. A.; Davioud-Charvet, E.; Sergheraert,
C. Antimalarial, Antitrypanosomal, and Antileishmanial Activi-
ties and Cytotoxicity of Bis(9-amino-6-chloro-2-methoxyacridines):
influence of the linker. J . Med. Chem. 2000, 43, 2646-2654.
(26) Girault, S.; Delarue, S.; Grellier, P.; Berecibar, A.; Maes, L.;
Quirijnen, L.; Lemie`re, P.; Debreu-Fontaine, M. A.; Sergheraert,
C. Antimalarial in-vivo Activity of Bis(9-amino-6-chloro-2-meth-
oxyacridines). J . Pharm. Pharmacol. 2001, 53, 935-938.
(27) Guillon, J .; Dumoulin, H.; Dallemagne, P.; Reynolds, R.; Rault,
S. Synthesis and Antituberculosis Activity of New Phenylpyrrolo-
[1,2-a]quinoxalinylpyrrole Carboxylic Acid Derivatives. Pharm.
Pharmacol. Commun. 1998, 4, 33-38.
(28) Guillon, J .; Dallemagne, P.; Pfeiffer, B.; Renard, P.; Manechez,
D.; Kervran, A.; Rault, S. Synthesis of New Pyrrolo[1,2-a]-
quinoxalines: Potential Non-Peptide Glucagon Receptor Antago-
nists. Eur. J . Med. Chem. 1998, 33, 293-308.
(29) Guillon, J .; Boulouard, M.; Lisowski, V.; Stiebing, S.; Lelong,
V.; Dallemagne, P.; Rault, S. Synthesis of New 2-(Aminomethyl)-
4-phenylpyrrolo[1,2-a]quinoxalines and Their Preliminary In-
Vivo Central Dopamine Antagonist Activity Evaluation in Mice.
J . Pharm. Pharmacol. 2000, 52, 1369-1375.
Agonists of 5-HT3 Receptors. Synthesis and Biological Evaluation
of Piperazinopyridopyrrolopyrazines, Piperazinopyrroloquinoxa-
lines, and Piperazinopyridopyrroloquinoxaline. J . Med. Chem.
1997, 40, 1808-1819.
(31) Kauffman, J . M.; Litak, P. T.; Boyko, W. J . Syntheses and
Photophysical Properties of Fluorescent Dibenzofurans, a Diben-
zothiophene, and Carbazoles Substituted with Benzoxazole and
Hydroxyl Groups To Produce Excited State Intramolecular
Proton-Transfer. J . Heterocycl. Chem. 1995, 32, 1541-1555.
(32) Zhang, Z.; Tillekeratne, L. M. V.; Hudson, R. A. Synthesis of
Isomeric Analogues of Coenzyme Pyrroloquinoline Quinone
(PQQ). Synthesis 1996, 3, 377-382.
(33) Nagarajan, K.; Ranga Rao, V.; Venkateswarlu, A. Condensed
Heterotricycles: Pyrrolo[1,2-a]quinoxaline Derivatives. Indian
J . Chem. 1972, 10, 344-350.
(34) Rault, S.; Lancelot, J . C.; Prunier, H.; Robba, M.; Renard, P.;
Delagrange, P.; Pfeiffer, B.; Caignard, D. H.; Guardiola-Lemaˆı-
tre, B.; Hamon, M. Novel Selective and Partial Agonists of 5-HT3
Receptors. Synthesis and Biological Evaluation of Piperazinopyr-
rolothienopyrazines. J . Med. Chem. 1996, 39, 2068-2080.
(35) Egan, T. J .; Marques, H. M. The Role of Haem in the Activity of
Chloroquine and Related Antimalarial Drugs. Coord. Chem. Rev.
1999, 190-192, 493-517.
(36) Hawley, S. R.; Bray, P. G.; Mungthin, M.; Atkinson, J . D.;
O’Neill, P. M.; Ward, S. A. Relationship between Antimalarial
Drug Activity, Accumulation, and Inhibition of Heme Polymer-
ization in Plasmodium falciparum In Vitro. Antimicrob. Agents
Chemother. 1998, 42, 682-686.
(37) Buller, R.; Peterson, M. L.; Almarsson, O¨ .; Leiserowitz, L.
Quinoline Binding Site on Malaria Pigment Crystal: A Rational
Pathway for Antimalaria Drug Design. Cryst. Growth Des. 2002,
2, 553-562.
(38) Sullivan, D. J . Theories on Malarial Pigment Formation and
Quinoline Action. Int. J . Parasitol. 2002, 32, 1645-1653.
(39) Slater, A. F. G.; Cerami, A. Inhibition by chloroquine of a novel
haem polymerase enzyme activity in malaria trophozoites.
Nature 1992, 355, 167-169.
(40) Sullivan, D. J .; Matile, H.; Ridley, R. G.; Goldberg, D. E. A
common mechanism for blockade of heme polymerization by
antimalarial quinolines. J . Biol. Chem. 1998, 273, 31103-31107.
(41) Raynes, K.; Foley, M.; Tilley, L.; Deady, L. W. Novel Bisquinoline
Antimalarials. Biochem. Pharmacol. 1996, 52, 551-559.
(42) Fitch, C. D.; Cai, G.; Chen, Y.-F.; Shoemaker, J . D. Involvement
of lipids in ferriprotoporphyrin IX polymerization in malaria.
Biochim. Biophys. Acta 1999, 1454, 31-37.
(43) Ayad, F.; Tilley, L.; Deady, L.-W. Synthesis, Antimalarial
Activity and Inhibition of Haem Detoxification of Novel Bis-
quinolines. Bioorg. Med. Chem. Lett. 2001, 11, 2075-2077.
(44) Ferrari, V.; Cutler, D. Temperature Dependence of the Acid
Dissociation Constants of Chloroquine. J . Pharm. Sci. 1987, 76,
554-557.
(45) Pehourcq, F.; Thomas, J .; J arry, C. A microscale HPLC method
for the evaluation of octanol-water partition coefficients in a
series of new 2-amino-2-oxazolines. J . Liq. Chromatogr. Relat.
Technol. 2000, 23, 443-453.
(46) Vacuolar accumulation ratios (VARs) were calculated math-
ematically from the following equation: VAR ) [1 + 10(pKa1-pHv)
+10(pKa1+pKa2-2pHv)+10(pKa1+pKa2+pKa3-3pHv)+10(pKa1+pKa2+pKa3+pKa4-4pHv)]/
[1 + 10(pKa1-pHo) + 10(pKa1+pKa2-2pHo) + 10(pKa1+pKa2+pKa3-3pHo)
+
10(pKa1+pKa2+pKa3+pKa4-4pHo)], where pHv is the pH inside the
vacuole (assumed to be pH 5.0) and pHo is the external pH
(assumed to be pH 7.4). This equation is from a derivation of
the Henderson-Hasselbach equation, based on predicted values
of drug pKa according to previous studies of Hawey et al. (see
reference below). Values of pKa were calculated using ACD/pKa
dB software from Avanced Chemistry Development Inc., Toronto,
Canada. Hawley, S.; Bray, P. C.; O’Neill, P. M.; Park, B. K.;
Ward, S. A. The role of drug accumulation in 4-aminoquinoline
antimalarial potency. The influence of structural substitution
and physicochemical properties. Biochem. Pharmacol. 1996, 52,
723-733.
(47) Pagola, S.; Stephens, P. W.; Scott Bohle, D.; Kosar, A. D.;
Madsen, S. K. The Structure of Malaria Pigment â-Haematin.
Nature 2000, 404, 307-310.
(48) Crystallographic data (excluding structure factors) for the
structure reported in this paper have been deposited with the
Cambridge Crystallographic Data Centre as Supplementary
Publication Nos. CCDC-172281 and CCDC-177407. Copies of the
data can be obtained free of charge on application to CCDC,
University Chemical Lab, 12 Union Road, Cambridge, CB2 1EZ,
U.K. (fax, international code + 44-1223/336-033; e-mail, deposit@
ccdc.cam.ac.uk).
(49) Stewart, J .; Mopac, J . P. A Semiempirical Molecular-Orbital
Program. J . Comput.-Aided Mol. Des. 1990, 4, 1-45.
(50) Discover; Biosym Technologies Inc.: San Diego, CA, 1993.
(51) Hariharan, P. C.; Pople, J . A. Influence of polarization functions
on MO hydrogenation energies. Theor. Chim. Acta 1973, 28,
213-222.
(30) Prunier, H.; Rault, S.; Lancelot, J . C.; Robba, M.; Renard, P.;
Delagrange, P.; Pfeiffer, B.; Caignard, D. H.; Misslin, R.;
Guardiola-Lemaˆıtre, B.; Hamon, M. Novel and Selective Partial

Quinoxalines and Pyrazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2009
(52) Moreau, S.; Perly, B.; Biguet, J . Interaction of chloroquine with
ferriprotophorphyrin IX. Nuclear magnetic resonance study.
Biochimie 1982, 64, 1015-1025.
(53) Pandey, A. V.; Bisht, H.; Babbarwal, V. K.; Srivastav, J .; Pandey,
K. C.; Chauhan, V. S. Mechanism of Malaria Haem Detoxifica-
tion Inhibition by Chloroquine. Biochem. J . 2001, 355, 333-338.
(54) North, A. C. T.; Phillips, D. C.; Mathews, F. S. A Semi-empirical
Method of Absorption Correction. Acta Crystallogr. 1968, A24,
351-359.
(55) Sheldrick, G. M.; Kro¨ger, C.; Goddard, R. SHELX 86 in Crystal-
lographic Computing 3; Oxford University Press: New York,
1985; pp 175-189.
(56) Sheldrick, G. M. SHELX 93, Program for the Refinement of the
Crystal Structures; University of Go¨ttingen: Go¨ttingen, Ger-
many, 1993.
(57) O’Neill, P. M.; Willock, D. J .; Hawley, S. R.; Bray, P. G.; Storr,
R. C.; Ward, S. A.; Park, B. K. Synthesis, Antimalarial Activity,
and Molecular Modeling of Tebuquine Analogues. J . Med. Chem.
1997, 40, 437-448.
(58) Stocks, P. A.; Raynes, K. J .; Bray, P. G.; Park, B. K.; O’Neill, P.
M.; Ward, S. A. Novel Short Chain Chloroquine Analogues
Retain Activity against Chloroquine Resistant K1 Plasmodium
falciparum. J . Med. Chem. 2002, 45, 4975-4983.
(59) Schmidt, M. W.; Baldridge, K. K.; Boatz, J . A.; Elbert, S. T.;
Gordon, M. S.; J ensen, J . H.; Koseki, S.; Matsunaga, N.; Nguyen,
K.; Su, S.; Windus, T. L.; Dupuis, M.; Montgomery, J . A. General
Atomic and Molecular Electronic Structure System. J . Comput.
Chem. 1993, 14, 1347-1363.
(60) Trager, W.; J ensen, J . B. Human Malarial Parasites in Continu-
ous Culture. Science 1976, 193, 673-677.
(61) Desjardins, R. E.; Canfield, C. J .; Haynes, J . D.; Chulay, J . D.
Quantitative Assessment of Antimalarial Activity in vitro by a
Semiautomated Microdilution Technique. Antimicrob. Agents
Chemother. 1979, 16, 710-718.
(62) Chou, A. C.; Fitch, C. D. Control of Heme Polymerase by
Chloroquine and Other Quinoline Derivatives. Biochem. Biophys.
Res. Commun. 1993, 195, 422-428.
(63) Asakura, T.; Minakata, K.; Adachi, K.; Russel, M. O.; Schwartz,
E. Denatured Hemoglobin in Sickle Erythrocytes. J . Clin. Invest.
1977, 59, 633-640.
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