Synthesis, antimalarial activity, and cellular toxicity of new arylpyrrolylaminoquinolines

Article abstract of DOI:10.1016/j.bmc.2010.08.001

A set of nine new arylpyrrolyl derivatives of 7-chloro-4-aminoquinoline, characterized by different substituents on the phenyl ring or different distance between the pyrrolic nitrogen and the 4-aminoquinoline, has been synthesized and tested for their activity against D-10 (CQ-S) and W-2 (CQ-R) strains of Plasmodium falciparum. All compounds exhibited activity against the CQ-S strain in the low nM range, comparable to that of chloroquine. Some of them were also highly active against the CQ-R strain and not toxic against normal cells. The antimalarial activity of this new class of compounds seems to be related to the inhibition of heme detoxification process of parasites, as in the case of chloroquine.

Full text of DOI:10.1016/j.bmc.2010.08.001

Bioorganic & Medicinal Chemistry 18 (2010) 6625–6633
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, antimalarial activity, and cellular toxicity of new
arylpyrrolylaminoquinolines
Manolo Casagrande a, Nicoletta Basilico b, Chiara Rusconi a, Donatella Taramelli b, Anna Sparatore a,
*
a Dipartimento di Scienze Farmaceutiche ‘Pietro Pratesi’, Università degli Studi di Milano, Via Mangiagalli, 25, 20133 Milan, Italy
b Dipartimento di Sanità Pubblica-Microbiologia-Virologia, Università degli Studi di Milano, Via Pascal, 36, 20133 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of nine new arylpyrrolyl derivatives of 7-chloro-4-aminoquinoline, characterized by different sub-
stituents on the phenyl ring or different distance between the pyrrolic nitrogen and the 4-aminoquino-
line, has been synthesized and tested for their activity against D-10 (CQ-S) and W-2 (CQ-R) strains of
Plasmodium falciparum. All compounds exhibited activity against the CQ-S strain in the low nM range,
comparable to that of chloroquine. Some of them were also highly active against the CQ-R strain and
not toxic against normal cells. The antimalarial activity of this new class of compounds seems to be
related to the inhibition of heme detoxification process of parasites, as in the case of chloroquine.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 14 May 2010
Revised 29 July 2010
Accepted 2 August 2010
Available online 6 August 2010
Keywords:
4-Aminoquinoline derivatives
Antimalarial agents
Chloroquine
1. Introduction
was advantageous within the 2,5-dimethylpyrrole subset of
compounds.4
Malaria is estimated to kill more than 1 million people annually
and most of these are children under the age of five.1
Starting from these first evidences, we explored more deeply the
electronic and lipophilic requirements for the antimalarial activity
of this class of compounds. In particular, the role of the substituent
in the para position of the phenyl ring, as well as the role of the dis-
tance between the pyrrolic nitrogen and the 4-aminoquinoline was
investigated. A set of new 7-chloro-4-[N-[2-methyl-[5-(4-R-phe-
nyl)]-1H-pyrrol-1-yl]amino]quinolines (4–7) and 7-chloro-4-[N-[3-
[5-(4-chlorophenyl)-2-methyl-1H-pyrrol-1-yl]propyl]amino]quino-
lines (8a, 8b) bearing a diethylaminomethyl or a pyrrolidinomethyl
moiety as basic head was synthesized (Fig. 2).
Presently, the most promising and successful strategy in fighting
malaria is artemisinin-based combination therapy (ACT), including
artemisininor one ofits derivatives, whichare all rapidly eliminated,
combined with a more slowly eliminated drug such as lumefantrine,
amodiaquine, mefloquine, or sulfadoxine-pyrimetamine.2 However
the recent reports of ACT failure in South East Asia and the potential
emergence of artemisinin resistance3 indicate that the search for
new drugs or new combinations is still highly necessary.
In order to develop new classes of antimalarial agents, we recently
explored the possibility of replacing the phenolic ring of amodiaquine
andtebuquinewithotherkindsofaromaticringssuchasapyrrole nu-
cleus, still linked to the quinoline moiety through the usual NH. We
showed that this new class of compounds (Fig. 1) is associated with
a good antimalarial activity against both chloroquine sensitive
(CQ-S) and chloroquine-resistant (CQ-R) strains of Plasmodium falci-
parum. In particular, we noted that the activity increased with the
increasing of lipophilicity as shown by 2-methyl-5-(4-chlorophenyl)
and 2-methyl-5-phenyl derivatives (3a, 3b, and 2a–2f, respectively)
which were more active than the corresponding 2,5-dimethyl deriv-
atives (1a–1f). We observed also that small basic heads such as the
diethylamino and pyrrolidino moieties were more profitable when
a phenyl substituent was present on the pyrrole ring compared to
bulkier ones, such as the quinolizidinylmethylamino group which
Experiments were conducted to evaluate the b hematin inhibi-
tory activity and the lipophilicity of these compounds and thus to
get some insights on their mechanism of action.
2. Chemistry
Compounds 4–6 and 8 were synthesised through the condensa-
tion of the appropriate
a-(4-substituted-phenacyl)-acetoacetate
(11–15) with 7-chloro-4-hydrazinoquinoline 9 (Scheme 1) and
the obtained pyrrole derivatives (16–18 and 20) were converted
to amides (21a,b–24a,b) by the action of relevant amines in pres-
ence of trimethylaluminium in toluene5,6 heating at 150 °C in a
microwaves synthesiser system. The use of microwaves allowed
us to shorten heating time from the 24 h previously required4 to
only 35 min and to improve the yield, especially of compound
22b (49% vs <10% with the traditional method).
Amides were then reduced to the expected aminocompounds
4–6, 8 by the means of diphenylsilane in the presence of tris(tri-
* Corresponding author. Tel.: +39 02 50319365; fax: +39 02 50319359.
E-mail address: anna.sparatore@unimi.it (A. Sparatore).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.001

6626
M. Casagrande et al. / Bioorg. Med. Chem. 18 (2010) 6625–6633
R
H3C
R'
R'
N
N
N
R''
N
R''
HN
HN
N
CH3
CH3
Cl
N
Cl
1a-d, f
R = H 2a-c, f, g
R = Cl 3a, b
H
N
H
N
N
R'
;
;
N
H
N
H
N
;
;
;
N
=
;
N
H
HN
N
N
H
N
R''
e
a
b
c
d
g
f
Figure 1. Structures of 4-[(pyrrol-1-yl)amino]quinoline derivatives with antimalarial activity.
ably due to the electronic effect of nitro group. Thus a different
synthetic pathway was set up (Scheme 2): compound 19 was
hydrolyzed9,10 to the corresponding carboxylic acid 25 which
was coupled with the pertinent amine in presence of DCC and
HOBt.11 Nitro- and amidogroups were than reduced with iron in
acetic acid and with LiAlH4 in THF,12,13 respectively, in order to
obtain compounds 7a and 7b.
Starting 4-[N-(3-aminopropyl)amino]-7-chloroquinoline 10 was
obtained as described by Solomon et al.14 with small modifications.
Finally, the required diketoesters of Scheme 1 were prepared by
reacting 4-substitutedphenacyl bromide with sodium ethyl aceto-
acetate.15–17
3. Results and discussion
Figure 2. Structures of the investigated compounds.
The compounds of Figure 2 were tested in vitro against D-10
(CQ-S) and W-2 (CQ-R) strains of P. falciparum. Their antimalarial
activity was quantified as inhibition of parasite growth18 and
was compared to the cytotoxicity19 on mammalian cell lines both
of human and murine origin (Table 1). All tested compounds exhib-
phenylphosphine)rhodium(I)carbonylhydride,7,8 thus preventing
the dehalogenation of the phenyl ring. Compounds 26a and 26b
could not be obtained through the above described method, prob-
R
NH2
n
R
HN
N
O
N
b
a
n
+
HN
N
COOEt
O
COOEt
Cl
Cl
R= F
11
n=0 R= F
16
n= 0 9
n= 3 10
R= CF3 12
R= Me 13
R= NO2 14
R= CF3 17
R= Me 18
R= NO2 19
R=Cl
15
n=3 R= Cl
20
R
R
R'
R'
c
N
N
n
N
n
N
HN
N
HN
N
R"
R"
O
Cl
Cl
n=0 R= F
4a, b
n=0 R= F
21a, b
R= CF3 5b
R= CF3 22b
R= Me 6a, b
R= Me 23a, b
n=3 R= Cl 8a, b
n=3 R= Cl 24a, b
Scheme 1. Reagents and conditions: (a) AcOH, reflux; (b) AlMe3, HNR’R’’, PhMe, m.w.; (c) Ph2SiH2, (PPh3)3(CO)HRh, THF.

M. Casagrande et al. / Bioorg. Med. Chem. 18 (2010) 6625–6633
6627
O2N
O2N
O2N
R'
R''
N
c
a
N
b
COOEt
N
N
COOH
HN
N
HN
N
HN
N
O
Cl
Cl
Cl
19
25
26a, b
H2N
H2N
R'
R'
R''
N
N
d
N
N
R''
HN
N
HN
N
O
Cl
Cl
7a, b
27a, b
Scheme 2. Reagents and conditions: (a) LiOH, EtOH, H2O, reflux; (b) HNR0R00, DCC, HOBt, DMF, m.w., 55 °C; (c) Fe, AcOH, EtOH, H2O, 80 °C; (d) LiAlH4, THF, reflux.
Table 1
In vitro antimalarial activities, b-hematin inhibitory activity (BHIA assay) and cellular cytotoxicity of the compounds under study
d
a,f
Compound D-10 (CQ-S) Ratio
W-2 (CQ-R) Ratio
Ratio
HMEC-1
K562 CC50
(nM)
WEHI-13
CC50 (nM)
HDF
BHIA IC50
IC50 (nM) IC50 CQ/compdb IC50 (nM)
IC50 CQ/compdb IC50 CQ-R/CQ-Sc CC50 (nM)
CC50 (nM) (molar equivalents
to hemin)
a
a
d
d
d
4a
61.6 7.2
32.6 2.7
58.0 13.0 0.5
32.7 2.8
30.7 2.0
53.4 20.0 0.5
43.1 11.9 0.5
0.5
0.8
79.8 18.0
78.1 28.0
59.0 16.6
55.4 16.8
65.8 12.7
5.0
5.7
6.7
6.4
5.4
1.3
2.4
1.0
1.7
2.1
5.8
10.6
6.9
10.7
1.4
3.3
1.6
1.6
17.8
18606 1252 2357 641
10070 1356 1655 368
3021 595 n.t.
2713 414 n.t.
n.t.
n.t.
n.t.
n.t.
0.45 0.07
n.t.
n.t.
n.t.
0.82 0.09
n.t.
4b
5b
6a
6b
7a
4701 2928
7051e
7151e
n.t.
1217 454
n.t.
n.t.
2680 206
3653 700
n.t.
0.6
0.6
10265e
4480 988 13185e
313.4 66.0 0.9
455.1 57.2 0.5
179.6 45.9 2.3
243.4 52.4 1.7
59.2 27.4g 9.9
139.5 56.0g 4.2
42.5 19.1g 18.5
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
7b
8a
n.t.
n.t.
26.0 6.9
22.7 5.1
0.7
0.8
n.t.
n.t.
8b
2ag
2bg
3ag
3bg
CQ
n.t.
n.t.
42.7 8.9g 0.7
42.9 13.0g 0.7
26.1 8.3g 0.9
30.7 4.3g 0.8
16694 5919g 3616 1268g 4380 2105g n.t.
12874 2824g 2920 866g 3878 638g n.t.
0.54 0.16
n.t.
0.36 0.07
1.27 0.13
5812 1511g n.t.
7176 1383g n.t.
1446 670g n.t.
49.6 5.2g
440.7 171.4 -
15.8
2060 125g
>38000
n.t.
>38000
24.8 4.1
-
>32000
>25000
n.t., not tested.
a
The results are expressed as IC50 SD of at least three different experiments each performed in duplicate or triplicate.
b
c
d
e
f
Mean of ratios between the IC50 of chloroquine and that of each compound against D-10 or W-2 strains of P. falciparum calculated for each single experiment.
Ratios between the IC50 values of each compound against the two strains of P. falciparum.
The cytotoxic activity was assayed in vitro on different cell line using the MTT assay.
Mean of two experiments each performed in duplicate.
The IC50 indicate the moles of compounds, relative to hemin, required to inhibit beta-hematin formation by 50%.
From Ref. 4.
g
ited high activity on the CQ-S (D-10) strain, with IC50 ranging from
22.7 to 61.6 nM. The IC50 of CQ was 24.7 nM (range 19.5–29.6 nM),
thus the tested compounds were from 0.5- to 0.8-fold as active as
CQ (Table 1). Five of the new compounds exhibited also a strong
activity against the CQ-R (W-2) strain, resulting from 5 to 6.7 times
more active than CQ, with IC50 values as low as 55.4–79.8 nM
(compounds 4a, 4b, 5b, 6a, and 6b) compared to 440.7 nM of CQ
(range 252–784 nM). Compounds 8a, 8b, 7a, and 7b, although in
some case more active than CQ on CQ-R strain, showed evident
cross resistance. In fact, the resistance factor, calculated as the ra-
tios between the IC50 of each compound against the two strains of
P. falciparum was significantly higher (range 5.8–10.7) for com-
pounds8a, 8b, 7a, and 7b, compared to4a, 4b, 5b, 6a, 6b, for which
the resistance factor ranged from 1.0 to 2.4, resulting about 7–17
times lower than that of CQ (17.8). This suggests that the latter
compounds were not (or only poorly) affected by the resistance
mechanisms.
The most active compounds exhibit low or moderate toxicity
against three different human or mouse cell lines with a therapeu-
tic index, calculated on W-2 strain, ranging between 21 and 233.
In addition, the ability of some representative compounds (2b,
3b, 6b, and 8b; Table 1) to inhibit b-hematin formation was evalu-
ated using the BHIA (b-hematin inhibitory activity) assay.20 Com-
pared to CQ, all of them showed a stronger ability to interfere
with the process of heme crystallization, with IC50 significantly
lower than that of CQ.
From these results it is evident that the hydrophilic amino
group of compounds 7a and 7b is not a suitable substituent for
the activity on CQ-R strains of P. falciparum. With the aim to better
understand the influence of lipophilicity (of the substituent in para
on the phenyl ring), we measured pKa and log P of the compounds
4b, 5b, 6b, and 7b, as well as those of the previously synthesized
compounds 2b and 3b (with R = H and Cl, respectively) all bearing
a pyrrolidine moiety as basic head. The results are collected in
Table 2. The determined pKa1, pKa2, log P and log D values obtained
for CQ are in good agreement with the data previously reported.21
Table 1 also shows the data related to the previously synthe-
sized compounds 2a, 2b, 3a, and 3b4 for a easier comparison.

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M. Casagrande et al. / Bioorg. Med. Chem. 18 (2010) 6625–6633
Table 2
Physicochemical characteristics of compounds bearing a pyrrolidine moiety as basic head.a
Compound
pKa1
pKa2
pKa3
Log P
Log D7.4
Calc.b
LogD5.2
VAR
LAR
c
Obs.
Calc.b
Obs.d
Calc.e
pH 5.2
4b
5b
6b
7b
8b
2b
3b
CQ
8.84 0.02
7.89 0.02
8.68 0.03
9.78 0.03
8.46 0.01f
8.38 0.02
8.52 0.03
10.20 0.03
6.20 0.02
6.05 0.02
6.28 0.03
—
—
—
4.86 0.02
5.28 0.18
5.19 0.06
4.38 0.02
4.38 0.04
4.60 0.01
5.17 0.06
4.73 0.01
3.45
4.72
3.91
2.03
2.35
3.28
4.06
0.90
3.37
4.65
3.86
1.95
2.22
3.55
3.99
0.88
0.18
1.69
0.59
ꢀ1.50
ꢀ2.21
0.32
1548.8
912.0
2818.4
52480.8
8128.3
107.1
2344.2
44668.4
7244.4
89.1
166.0
3548.1
9772.4
7.4
1862.1
16982.4
24547.1
1698.2
1513.6
22908.7
6.47 0.03
3.79 0.03
f
—
—
—
—
223.9
6.26 0.03
6.20 0.03
8.41 0.03
1905.5
11481.5
7.9
0.81
ꢀ3.49
a
pKa, log P, and log D values were calculated as described in Section 5. The experiments were conducted at 25 °C. Obs, observed; Calc, calculated; log D7.4 and log D5.2, log D
values at pHs 7.4 and 5.2, respectively.
b
Calculated from the following equation: log D = log P ꢀ log[1 + 10(pKa1ꢀpH) + 10(pKa1+pKa2ꢀ2pH)].
c
VAR, antilog (calculated log D7.4 ꢀ calculated log D5.2).
d
Antilog for observed log D7.4.
e
Antilog for calculated log D7.4
.
f
The pKa values of the two basic moieties are practically superimposed, since the presence of the trimethylene spacer restored the basicity of the 4-aminoquinoline moiety
to the level of CQ.
As shown, log P values of the novel compounds and CQ are very
close to each other (4.38–5.28). On the contrary, the distribution
coefficients at pH 7.4 are quite different with log D values spread
from 4.72 (5b, R = CF3) to 2.03 (7b, R = NH2), while log D of CQ
was still lower (0.90). The lower basicity of compounds 4b, 5b,
6b (pKa1 = 8.84, 7.89, and 8.68, respectively) compared to CQ
(pKa1 = 10.20), implies that at physiological pH they are less pro-
tonated. These features might improve the cellular permeation of
these compounds at physiological pH. At pH 5.2 (which is sup-
posed to reflect the pH of the digestive vacuole),22 compounds
2b-6b display a stronger lipophilic character than compounds 7b
and 8b and even more than CQ (log D5.2 = ꢀ3.49) (Table 2). This
means that, whereas CQ concentrates predominantly in the aque-
ous regions, compounds 2b–6b should concentrate in the lipid
regions or at the interface between lipids and water. This may
facilitate their interaction with heme molecules during the crystal-
lization process to hemozoin which is reported to occur in vitro at
the lipid–water interface or in vivo within neutral lipid nano-
sphere.23,24 This is partly confirmed by the strong inhibition of
b-hematin formation shown by compounds 6b, 8b and 2b, 3b
(Table 1).
In agreement, LAR (lipid accumulation ratio) values are signifi-
cantly higher for compounds 4b, 5b, 6b, and 3b compared to com-
pounds 7b, 8b and CQ. This finding may indicate the ability of the
former compounds to block drug efflux by hydrophobic interaction
with PfCRT (Plasmodium falciparum Chloroquine Resistance Trans-
porter) and explain the lower RI (resistance index) and the higher
efficacy toward CQ-R strains of P. falciparum.25
It has been reported that a high VAR (vacuolar accumulation
ratio) value in addition to the ability of inhibiting in vitro the for-
mation of b-hematin is important for activity against CQ-S para-
sites,25 however in our set of compounds, VAR values do not
seem well correlated to the strong activity against the CQ-S strain
of P. falciparum, suggesting that the interaction of hematin with
uncharged drug species may be more important than previously
suspected, as also observed by O’Neill et al.26
Figure 3. Relationship between log D5.2 and IC50 on W-2 strain of P. falciparum of
the compounds 2b-7b.
at pH 5.2 was lower. A multivariate analysis (Minitab 14 software)
of the results concerning compounds 2b–7b suggests a good corre-
lation (R2 = 0.975) between activity and lipophilicity. However, the
synthesis and the evaluation of additional compounds with log D5.2
in the region below 0.0 would be needed to provide a more sound
relation.
Anyway the present results are in agreement with the
data previously reported for other classes of aminoquinoline
derivatives.25,26,28,29
4. Conclusions
A set of new 7-chloro-4-[N-[2-methyl-[5-(4-R-phenyl)]pyrrol-
1-yl]amino]quinolines (4a,4b–7a,7b) and 7-chloro-4-[N-[3-[5-(4-
chlorophenyl)-2-methylpyrrol-1-yl]propyl]amino]quinolines (8a,
8b) bearing a diethylaminomethyl or a pyrrolidinomethyl moiety
as basic head was synthesized, in order to explore the best elec-
tronic and lipophilic requirements for the antimalarial activity of
this class of compounds. In particular, the role of the substituent
in the para position of the phenyl ring, as well as the role of the dis-
tance between the pyrrolic nitrogen and the 4-aminoquinoline was
investigated. Indeed, the most lipophilic compounds 5b, 3b, and 6b
were also the most active on W2 (CQ-R) strain of P. falciparum,
while the presence of a hydrophilic substituent on the phenyl ring,
although maintains the activity against CQ-S strain, was detrimen-
As shown in Figure 3, among the homogeneous set of com-
pounds bearing a pyrrolidine ring as basic head, the most lipophilic
compounds 5b, 3b, and 6b (R = CF3 and Cl and CH3, respectively),
were also the most active against CQ-R strain of P. falciparum
whereas the least lipophilic 4-amino substituted 7b was the least
active. It is worth noting that conforming to expectation,27 the
4-fluorosubstituted derivative 4b exhibited higher log P and
log D7.4 values in respect to the unsubstituted compound 2b, and
the activity of 4b was higher than that of 2b, however the log D

M. Casagrande et al. / Bioorg. Med. Chem. 18 (2010) 6625–6633
6629
tal for the activity on CQ-R strain. The increase of the distance be-
tween the pyrrolic nitrogen and the 4-aminoquinoline was not
profitable for the resistance index.
Similarly to other aminoquinoline derivatives, this new class of
compounds seems able to interfere with the heme detoxification
process of parasites.
1H); 3.49 (dd, J = 18.43, 5.50 Hz, 1H); 2.44 (s, 3H); 1.30 (t,
J = 7.16, 3H).
5.2.5. Ethyl 2-acetyl-4-(4-chlorophenyl)-4-oxobutanoate (15)16
CC (cyclohexane/AcOEt; 60:40); solid rinsed with petroleum
ether/diethyl ether (80:20). Yield: 81%. Mp 64.8–66.6 °C. 1H NMR
(CDCl3): d 7.92 (dd, J = 1.93, 6.88 Hz, 2H); 7.43 (dd, J = 1.93,
6.88 Hz, 2H); 4.25–4.18 (m, 3H); 3.67 (dd, J = 18.40, 8.25 Hz, 1H);
3.43 (dd, J = 18.40, 8.25 Hz, 1H); 2.43 (s, 3H); 1.30 (t, J = 6.82 Hz,
3H).
5. Experimental
5.1. General
5.3. N-(3-Aminopropyl)-4-amino-7-chloroquinoline (10)14
All commercially available solvents and reagents were used
without further purification, unless otherwise stated. CC = flash
column chromatography. Mps: Büchi apparatus, uncorrected. 1H
NMR spectra: Varian Mercury 300VX spectrometer; CDCl3 or
DMSO-d6; d in ppm, J in Hertz. High-resolution mass spectra
(HRMS) on a APEX II ICR-FTMS Bruker Daltonics mass spectrometer
in positive electro spray ionization (ESI). Elemental analyses were
performed on a Carlo Erba EA-1110 CHNS-O instrument in the
Microanalysis Laboratory of the Department of Pharmaceutical Sci-
ences of Genoa University. Where indicated, reactions were per-
formed with a Biotage Iniziator Microwave Synthesis System.
In a round bottom flask propane-1,3-diamine (10 ml, 120 mmol)
and 4,7-dichloroquinoline (2 g, 10.1 mmol) were added. The mix-
ture was heated to reflux under N2 atmosphere for 3.5 h and then
cooled in ice-bath, diluted with water and added with 2 M NaOH un-
til complete precipitation. Precipitate was filtered, washed with
water, diethyl ether, and dried over anhydrous Na2SO4. The crude
product was chromatographed on a column of silica gel (CH2Cl2/
MeOH/concd NH3; from 80:18:2 to 75:22.5:2.5). The resulted solid
was washed with diethyl ether. Yield: 78%. Mp 80–83 °C (lit.14 mp
96–98 °C). 1H NMR (DMSO-d6): d 8.30 (d, J = 5.50 Hz, 1H); 8.12 (d,
J = 9.07 Hz, 1H); 7.74 (d, J = 2.20 Hz, 1H); 7.50 (br s, 1H); 7.38 (dd,
J = 2.20, 9.08 Hz, 1H); 6.45 (d, J = 5.50 Hz, 1H); 3.50–3.00 (m, 4H);
2.57 (t, J = 6.88 Hz, 2H); 1.74–1.64 (m, 2H).
5.2. Ethyl 2-acetyl-4-oxo-4-[(4-substituted)phenyl]butanoate
(11–15): general method
According to literature,15–17ethyl acetoacetate (1.56 g, 12 mmol)
was added dropwise to a stirred suspension of NaH 60% p/p
(10 mmol) in 15 ml of anhydrous diethyl ether at rt under N2. After
10 min a solution of 4-substituted phenacylbromide (10 mmol) in
26 ml of anhydrous diethyl ether was added dropwise, and the
resulting mixture was heated at reflux for 2 h. After cooling, the NaBr
was filtered, washed twice with diethyl ether, and the joined organic
phases were evaporated to dryness and the crude product purified
by CC (silica gel; elution conditions as indicated for each compound).
5.4. Ethyl 1-[(7-chloroquinolin-4-yl)amino]-2-methyl-5-[(4-
substituted)phenyl]-1H-pyrrole-3-carboxylate (16–19) and
ethyl 1-[3-((7-chloroquinolin-4-yl)amino)propyl]-2-methyl-5-
(4-chlorophenyl)-1H-pyrrole-3-carboxylate (20): general
method
The suitable diketoester (11–15; 6.2 mmol) was added to a stir-
red solution of 7-chloro-4-hydrazinoquinoline (9) or N-(3-amino-
propyl)-4-amino-7-chloroquinoline (10; 6.2 mmol) in 10 ml of
glacial acetic acid. The mixture was refluxed under N2 for 2 h to
give a violet solution, which, after cooling, was alkalized with cold
2 N NaOH and extracted three times with CH2Cl2. The joined organ-
ic layers were dried (Na2SO4); the solvent was removed and the
crude solid was purified by CC (silica gel; different ratio of ethyl
acetate/cyclohexane or CH2Cl2/MeOH as indicated for each
compound).
5.2.1. Ethyl 2-acetyl-4-(4-fluorophenyl)-4-oxobutanoate (11)17
CC (cyclohexane/AcOEt; 80:20); solid crystallized with diethyl
ether and rinsed with petroleum ether. Yield: 59%. Mp 56.5–
58 °C. 1H NMR (CDCl3): d 8.03–7.98 (m, 2H); 7.16–7.10 (m, 2H);
4.26–4.19 (m, 3H); 3.68 (dd, J = 8.25, 18.43 Hz, 1H); 3.47 (dd,
J = 5.78, 18.43 Hz, 1H); 2,44 (s, 3H); 1.29 (t, J = 7.15 Hz, 3H).
5.4.1. Ethyl 1-[(7-chloroquinolin-4-yl)amino]-2-methyl-5-(4-
fluorophenyl)-1H-pyrrole-3-carboxylate (16)
5.2.2. Ethyl 2-acetyl-4-[4-(trifluoromethyl)phenyl]-4-
oxobutanoate (12)
CC (AcOEt/cyclohexane; 20:80); solid crystallized with diethyl
ether and rinsed with petroleum ether/diethyl ether (70:30). Yield
76%. Mp 186.4–188.7 °C. 1H NMR (CDCl3): d 8.17–8.14 (m, 2H);
7.87 (s, 1H); 7.44 (dd, J = 1.93, 9.08 Hz, 1H); 7.39–7.34 (m, 2H);
6.90–6.85 (m, 2H); 6.71 (s, 1H); 5.83 (d, J = 5.77 Hz, 1H); 4.29 (q,
J = 7.15 Hz, 2H); 2.40 (s, 3H); 1.36 (t, J = 7.15 Hz, 3H).
CC (cyclohexane/AcOEt; 93:7); solid crystallized with diethyl
ether and rinsed with petroleum ether. Yield: 78%. Mp 63.8–
66.7 °C. 1H NMR (CDCl3): d 8.08 (d, J = 7.98 Hz, 2H); 7.73 (d,
J = 8.26 Hz, 2H); 4.27–4.20 (m, 3H); 3.72 (dd, J = 8.25, 18.43 Hz,
1H); 3,50 (dd, J = 5.77, 18.47 Hz, 1H); 2.45 (s, 3H); 1.30 (t,
J = 7.15 Hz, 3H).
5.4.2. Ethyl 1-[(7-chloroquinolin-4-yl)amino]-2-methyl-5-[4-
(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxylate (17)
CC (AcOEt/cyclohexane; 15:85); solid rinsed with petroleum
ether/diethyl ether (50:50). Yield 69%. Mp 191–193 °C. 1H NMR
(CDCl3): d 8.31 (d, J = 8.81 Hz, 1H); 7.94 (br s, 1H); 7.80 (s, 1H);
7.61 (d, J = 7.97 Hz, 2H); 7.47–7.40 (m, 3H); 6.86 (s, 1H); 5.73 (d,
J = 6.06 Hz, 1H); 4.30 (q, J = 6.88 Hz, 2H); 2.41 (s, 3H); 1.37 (t,
J = 6.88 Hz, 3H).
5.2.3. Ethyl 2-acetyl-4-p-tolyl-4-oxobutanoate (13)
CC (cyclohexane/AcOEt; 70:30); solid rinsed with petroleum
ether/diethyl ether (90:10). Yield: 70%. Mp 39.5–42 °C. 1H NMR
(CDCl3): d 7.87 (d, J = 7.97 Hz, 2H); 7.25 (d, J = 7.7 Hz, 2H); 4.25–
4.18 (m, 3H); 3.69 (dd, J = 8.26, 18.43 Hz, 1H); 3.49 (dd, J = 5.77,
18.43 Hz, 1H); 2.43 (s, 3H); 2.41 (s, 3H); 1.29 (t, J = 7.15 Hz, 3H).
5.2.4. Ethyl 2-acetyl-4-(4-nitrophenyl)-4-oxobutanoate (14)
CC (cyclohexane/AcOEt; 85:15); solid rinsed with a mixture of
petroleum ether/diethyl ether (60:40). Yield: 61%. Mp 47.8–
48.7 °C. 1H NMR (CDCl3): d 8.32 (d, J = 9.08 Hz, 2H); 8.13 (d,
J = 9.08 Hz, 2H); 4.28–4.20 (m, 3H); 3.73 (dd, J = 18.70, 8.25 Hz,
5.4.3. Ethyl 1-[(7-chloroquinolin-4-yl)amino]-2-methyl-5-p-
tolyl-1H-pyrrole-3-carboxylate (18)
CC (AcOEt/cyclohexane; 20:80); resulting solid rinsed with
petroleum ether/diethyl ether (50:50). Yield 78%. Mp 180–181 °C.

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M. Casagrande et al. / Bioorg. Med. Chem. 18 (2010) 6625–6633
1H NMR (CDCl3): d 8.22–8.13 (m, 2H); 7.97 (s, 1H); 7.46 (dd, J = 2.2,
9.07 Hz, 1H); 7.27–7.25 (m, 2H); 6.98 (d, J = 7.97 Hz, 2H); 6.68 (s,
1H); 5.86 (d, J = 5.77 Hz, 1H); 4.29 (d, J = 6.87 Hz, 2H); 2.41 (s,
3H); 2.22 (s, 3H); 1.36 (t, J = 7.1 Hz, 3H).
joined to the product obtained at the end of the general procedure.
CC (CH2Cl2/MeOH; 97.5:2.5); solid rinsed with diethyl ether. Yield:
49%. Mp 318–320 °C (dec). 1H NMR (CF3COOD):
d 8.50 (d,
J = 6.07 Hz, 1H); 8.38 (d, J = 9.36 Hz, 1H); 8.02 (s, 1H); 7.85 (d,
J = 9.62 Hz, 1H); 7.56 (s, 4H); 6.80 (s, 1H); 6.27 (d, J = 6.05 Hz,
1H); 3.99 (br s, 4H); 2.41 (s, 3H); 2.23 (br s, 4H). HRMS (ESI) m/z
calcd for C26H23ClF3N4O [M+H]+: 521.13264; found: 521.13199.
5.4.4. Ethyl 1-[(7-chloroquinolin-4-yl)amino]-2-methyl-5-(4-
nitrophenyl)-1H-pyrrole-3-carboxylate (19)
CC (AcOEt/cyclohexane; 30:70); solid rinsed with petroleum
ether/diethyl ether (50:50). Yield 73%. Mp 242.3–244.5 °C. 1H
NMR (CD3OD): d 8.50–8.20 (br s, 2H); 8.06 (d, J = 8.25, 2H); 7.81
(d, J = 7.42 Hz, 2H); 7.65–7.30 (m, 2H); 7.07 (s, 1H); 4.33 (q,
J = 6.60 Hz, 2H); 2.41 (s, 3H); 1.37 (t, J = 7.15 Hz, 3H).
5.5.4. 1-[(7-Chloroquinolin-4-yl)amino]-2-methyl-5-p-tolyl-1H-
pyrrole-3-(N,N-diethyl)carboxamide (23a)
CC (CH2Cl2/MeOH; 90:10); solid rinsed with petroleum ether/
diethyl ether (80:20). Yield: 82%. Mp 255.1–256 °C. 1H NMR
(DMSO-d6): d = 10.65 (br s, 1H); 8.58 (s, 1H); 8.30 (s, 1H); 7.88 (s,
1H); 7.60 (s, 1H); 7.42 (d, J = 7.70 Hz, 2H); 7.02 (d, J = 7.15 Hz,
2H); 6.41 (s, 1H); 5.65 (s, 1H); 3.42 (q, J = 6.85 Hz, 4H); 2.48 (s,
3H); 2.16 (s, 3H); 1.13 (t, J = 6.87 Hz, 6H). HRMS (ESI) m/z calcd
for C26H28ClN4O [M+H]+: 447.19462; found: 447.19450.
5.4.5. Ethyl 1-[3-((7-chloroquinolin-4-yl)amino)propyl]-2-
methyl-5-(4-chlorophenyl)-1H-pyrrole-3-carboxylate (20)
CC (CH2Cl2/MeOH; 98:2); solid crystallized with a mixture of
CH2Cl2/diethyl ether and rinsed with diethyl ether. Yield 75%. Mp
148.5–151.5 °C. 1H NMR (CDCl3): d 8.40 (d, J = 5.50 Hz, 1H); 7.94
(s, 1H); 7.34–7.20 (m, 7H); 6,55 (s, 1H); 6.16 (d, J = 5.50 Hz, 1H);
4.27 (q, J = 6.88 Hz, 2H); 4.15 (t, J = 6.60 Hz, 2H); 3.14–3.08 (m,
2H); 2.62 (s, 3H); 1.98–1.90 (m, 2H); 1.34 (t, J = 6.88 Hz, 3H).
5.5.5. [1-((7-Chloroquinolin-4-yl)amino)-2-methyl-5-p-tolyl-
1H-pyrrol-3-yl](pyrrolidin-1-yl)methanone (23b)
During the reaction, a suspension was formed and a first portion
of 23b was filtered, washed with diethyl ether and then joined to
the product obtained at the end of the general procedure. CC
(CH2Cl2/MeOH; 97:3); solid rinsed with diethyl ether. Yield: 91%.
Mp 190–191 °C. 1H NMR (DMSO-d6): d 10.67 (s, 1H); 8.41 (d,
J = 3.85 Hz, 1H); 8.29 (d, J = 8.53 Hz, 1H); 7.90 (s, 1H); 7.62 (d,
J = 8.53 Hz, 1H); 7.41 (d, J = 7.98 Hz, 2H); 7.03 (d, J = 7.7 Hz, 2H);
6.6 (s, 1H); 5.66 (d, J = 4.4 Hz, 1H); 3.64 (br s, 2H); 3.44 (br s,
2H); 2.18–2.13 (m, 6H); 1.85 (s, 4H). HRMS (ESI) m/z calcd for
5.5. 1-[(7-Chloroquinolin-4-yl)amino]-2-methyl-5-[(4-substi-
tuted)phenyl]-1H-pyrrole-3-(N-substituted)carboxamide
(21a,b–23a,b, 24a,b): general method
In a microwaves vial, 2 M Al(CH3)3 in toluene (0.725 ml,
1.5 mmol) was added dropwise to an ice-cooled stirred solution
of the appropriate amine (1.5 mmol) in 3 ml of anhydrous toluene.
The resulting mixture was stirred under N2 at rt for 30 min, then
the relevant ester derivative (13–15, 17; 1 mmol) was added. The
solution was heated with microwaves (150 °C, 35 min), cooled
and then treated with 2 N NaOH solution. The precipitate was fil-
tered and washed four times with AcOEt, the organic phase was
separated, dried (Na2SO4) and evaporated. The solid was purified
by CC (silica gel; different ratio of CH2Cl2 and MeOH as indicated
for each compound).
C
26H26ClN4O [M+H]+: 445.17897; found: 445.17874.
5.5.6. 1-[3-((7-Chloroquinolin-4-yl)amino)propyl]-2-methyl-5-
(4-chlorophenyl)-1H-pyrrole-3-(N,N-diethyl)carboxamide (24a)
CC (CH2Cl2/MeOH; 97:3); solid rinsed with diethyl ether. Yield:
85%. Mp 176.5–177.5 °C. 1H NMR (DMSO-d6): d 8.33 (d, J = 5.23 Hz,
1H); 8.08 (d, J = 9.35 Hz, 1H); 7.76 (d, J = 1.92 Hz, 1H); 7.39 (dd,
J = 1.93, 9.08 Hz, 1H); 7.25 (d, J = 8.53 Hz, 2H); 7.20–7.03 (m, 3H);
6.23 (d, J = 5.50 Hz, 1H); 6.08 (s, 1H); 4.10–4.00 (m, 2H); 3.32 (q,
J = 6.90 Hz, 4H); 3.10–3.00 (m, 2H); 2.25 (s, 3H); 1.95–1.75 (m,
2H); 1.05 (t, J = 6.90 Hz, 6H). HRMS (ESI) m/z calcd for
5.5.1. 1-[(7-Chloroquinolin-4-yl)amino]-5-(4-fluorophenyl)-2-
methyl-1H-pyrrole-3-(N,N-diethyl)carboxamide (21a)
CC (CH2Cl2/MeOH; 98.5:1.5); solid rinsed with diethyl ether.
Yield: 62%. Mp 222–223 °C. 1H NMR (DMSO-d6): d 10.69 (s, 1H);
8.44 (d, J = 4.67 Hz, 1H); 8.27 (d, J = 9.36 Hz, 1H); 7.91 (s, 1H);
7.64–7.35 (m, 3H); 7.12–6.98 (m, 2H); 6.49 (s, 1H); 5.69 (d,
J = 5.23 Hz, 1H); 3.40 (q, J = 6.60 Hz, 4H); 2.20 (s, 3H); 1.13 (q,
J = 6.60 Hz, 6H). HRMS (ESI) m/z calcd for C25H25ClFN4O [M+H]+:
451.16954; found: 451.16926.
C
28H31Cl2N4O [M+H]+: 509.18694; found: 509.18650.
5.5.7. [5-(4-chlorophenyl)-1-(3-((7-chloroquinolin-4-yl)amino)-
propyl)-2-methyl-1H-pyrrol-3-yl](pyrrolidin-1-yl)methanone
(24b)
During the reaction, a suspension was formed and a first portion
of 24b was filtered, washed with diethyl ether and then joined to
the product obtained at the end of the general procedure. CC
(CH2Cl2/MeOH; 96:4); solid rinsed with diethyl ether. Yield: 78%.
Mp 235.7–237 °C. 1H NMR (DMSO-d6): d 8.33 (d, J = 5.22 Hz, 1H);
8.08 (d, J = 9.07 Hz, 1H); 7.42 (d, J = 2.20 Hz, 1H); 7.40 (dd,
J = 2.20, 9.07 Hz, 1H); 7.25 (d, J = 8,53 Hz, 2H); 7.17 (br s, 1H);
7.09 (d, J = 8.52 Hz, 2H); 6.28 (s, 1H); 6.24 (d, J = 5.50 Hz, 1H);
4.04 (t, J = 7.15 Hz, 2H); 3.60–3.30 (m, 4H); 3.15–3.00 (m, 2H);
2.38 (s, 2H); 1.77 (s, 4H). HRMS (ESI) m/z calcd for C28H29Cl2N4O
[M+H]+: 507.17129; found: 507.17078.
5.5.2. [1-((7-Chloroquinolin-4-yl)amino)-5-(4-fluorophenyl)-2-
methyl-1H-pyrrol-3-yl](pyrrolidin-1-yl)methanone (21b)
During the reaction, a suspension was formed and a first portion
of compound 21b was filtered, washed with diethyl ether and then
joined to the product obtained at the end of the general procedure.
CC (CH2Cl2/MeOH; 97:3); solid rinsed with diethyl ether. Yield:
45%. Mp 310–313 °C. 1H NMR (DMSO-d6): d 10.69 (s, 1H); 8.43
(d, J = 5.23 Hz, 1H); 8.27 (d, J = 8.81 Hz, 1H); 7.91 (s, 1H); 7.64–
7.35 (m, 3H); 7.11–6.98 (m, 2H); 6.70 (s, 1H); 5.70 (d, J = 4.96 Hz,
1H); 3.65 (br s, 2H); 3.44 (br s, 2H); 2.18 (s, 3H); 1.85 (br s, 4H).
HRMS (ESI) m/z calcd for C25H23ClFN4O [M+H]+: 449.15389; found:
449.15353.
5.6. 7-Chloro-4-[N-[5-(4-substituted-phenyl)-3-[(N-substituted-
amino)-methyl]-2-methyl-1H-pyrrol-1-yl]amino]quinoline
(4a,b-6a,b-) and 7-chloro-4-[N-[3-[5-(4-chlorophenyl)-3-[(N-
substituted-amino)methyl]-2-methyl-1H-pyrrol-1-
5.5.3. [1-((7-Chloroquinolin-4-yl)amino)-5-(4-(trifluoromethyl)-
phenyl)-2-methyl-1H-pyrrol-3-yl](pyrrolidin-1-yl)methanone
(22b)
During the reaction, a suspension was formed and a first portion
of compound 22b was filtered, washed with diethyl ether and then
yl]propyl]amine]quinoline (8a,b): general method
To a stirred suspension of corresponding amide (1 mmol) in
25 ml of anhydrous THF heated at reflux under N2, tris(triphenyl-
phosphine)rhodium(I) carbonyl hydride (45.94 mg, 0.05 mmol)

M. Casagrande et al. / Bioorg. Med. Chem. 18 (2010) 6625–6633
6631
and diphenylsilane (3.3 ml, 18 mmol) were added in five portions
during 2 h and the mixture was further stirred for 3 h, until the
amide was completely reduced. After cooling, THF was evaporated,
and the residue was partitioned between CH2Cl2 and 0.5 N HCl. The
acid phase was alkalized with a 2 N NaOH solution and extracted
with CH2Cl2. The organic solution was dried (Na2SO4) and evapo-
rated to yield the crude product that was purified by CC (silica
gel; different ratio of CH2Cl2 and MeOH as indicated for each
compound).
6.31 (s, 1H); 5.57 (d, J = 4.95 Hz, 1H); 3.45 (s, 2H); 2.48 (s, 3H);
2.47 (s, 3H); 1.67 (s, 4H); four protons are missing because covered
by the DMSO signal. HRMS (ESI) m/z calcd for C26H28ClN4 [M+H]+:
431.19970; found: 431.20047. Anal. Calcd for C26H27ClN4: C, 72.46;
H, 6.31; N, 13.00. Found: C, 72.40; H, 6.44; N, 12.64.
5.6.6. 7-Chloro-4-[N-[3-(5-(4-chlorophenyl)-3-((diethylamino)-
methyl)-2-methyl-1H-pyrrol-1-yl)propyl]amino]quinoline (8a)
CC (CH2Cl2/MeOH; 93.5:6.5); solid rinsed with diethyl ether.
Yield: 35%. Mp 99.3–101.2 °C. 1H NMR (DMSO-d6): d 8.32 (d,
J = 5.22 Hz, 1H); 8.08 (d, J = 9.07 Hz, 1H); 7.76 (d, J = 2.20 Hz, 1H);
7.39 (dd, J = 2.20, 9.07 Hz, 1H); 7.22 (d, J = 8.26 Hz, 2H); 7.15 (br
s, 1H); 7.08 (d, J = 8.25 Hz, 2H); 6.20 (d, J = 5.50 Hz, 1H); 5.97 (s,
1H); 4.03 (t, J = 7.43 Hz, 2H); 3.30 (s, 2H); 3.10–2.95 (m, 2H);
2.39 (q, J = 6.88 Hz, 4H); 2.18 (s, 3H); 1.81 (t, J = 6.61 Hz, 2H);
0.92 (t, J = 6.88 Hz, 6H). HRMS (ESI) m/z calcd for C28H33Cl2N4
[M+H]+: 495.20768; found: 495.20794. Anal. Calcd for
5.6.1. 7-Chloro-4-[N-[2-methyl-3-[(diethylamino)methyl]-5-(4-
fluorophenyl)-1H-pyrrol-1-yl]amino] quinoline (4a)
CC (CH2Cl2/MeOH; 93.5:6.5); solid rinsed with diethyl ether.
Yield: 10%. Mp 225–229 °C (dec). 1H NMR (DMSO-d6): d 10.62 (s,
1H); 8.39 (d, J = 4.95 Hz, 1H); 8.28 (d, J = 8.80 Hz, 1H); 7.89 (s,
1H); 7.59 (d, J = 8.80 Hz, 1H); 7.53–7.42 (m, 2H); 7.04 (t,
J = 8.80 Hz, 2H); 6.36 (s, 1H); 5.59 (d, J = 4.95 Hz, 1H); 3.44 (s,
2H); 2.01 (s, 3H); 1.00 (t, J = 6.60 Hz, 6H); four protons are missing
because covered by the DMSO signal. HRMS (ESI) m/z calcd for
C28H32Cl2N4: C, 67.87; H, 6.50; N, 11.31. Found: C, 67.61; H, 6.85;
N, 11.26.
C
25H27ClFN4 [M+H]+: 437.19028; found: 437.19192. Anal. Calcd
for C25H26ClFN4: C, 68.72; H, 6.00; N, 12.82. Found: C, 68.57; H,
6.31; N, 12.94.
5.6.7. 7-Chloro-4-[N-[3-(5-(4-chlorophenyl)-3-(pyrrolidin-1-yl-
methyl)-2-methyl-1H-pyrrol-1-yl)propyl]amino]quinoline (8b)
CC (CH2Cl2/MeOH; 95:5); solid rinsed with diethyl ether. Yield:
38%. Mp 157–158.5 °C. 1H NMR (DMSO-d6): d 8.32 (d, J = 5.22 Hz,
1H); 8.08 (d, J = 9.07 Hz, 1H); 7.76 (s, 1H); 7.40 (d, J = 9.07 Hz,
1H); 7.22 (d, J = 8.26 Hz, 2H); 7.13 (br s, 1H); 7.08 (d, J = 8.25 Hz,
2H); 6.21 (d, J = 5.50 Hz, 1H); 5.97 (s, 1H); 4.10–3.90 (m, 2H);
3.33 (s, 2H); 3.10–2.95 (m, 2H); 2.37 (s, 4H); 2.18 (s, 3H);
1.90–1.75 (m, 2H); 1.61 (s, 4H). HRMS (ESI) m/z calcd for
5.6.2. 7-Chloro-4-[N-[2-methyl-3-(pyrrolidin-1-ylmethyl)-5-(4-
fluorophenyl)-1H-pyrrol-1-yl]amino]quinoline (4b)
CC (CH2Cl2/MeOH; 94:6); solid rinsed with diethyl ether. Yield:
26%. Mp 170.5–173.5 °C. 1H NMR (DMSO-d6): d 10.60 (s, 1H); 8.40
(d, J = 5.23 Hz, 1H); 8.28 (d, J = 9.07 Hz, 1H); 7.89 (s, 1H); 7.60 (d,
J = 9.08 Hz, 1H); 7.53–7.48 (m, 2H); 7.04 (t, J = 9.08 Hz, 2H); 6.36
(s, 1H); 5.59 (d, J = 5.23 Hz, 1H); 3.46 (s, 2H); 2.01 (s, 3H); 1.68
(s, 4H); four protons are missing because covered by the DMSO sig-
nal. HRMS (ESI) m/z calcd for C25H25ClFN4 [M+H]+: 437.17463;
found: 437.17569. Anal. Calcd for C25H24ClFN4: C, 69.04; H, 5.56;
N, 12.88. Found: C, 68.98; H, 5.85; N, 12.89.
C
28H31Cl2N4 [M+H]+: 493.19203; found: 437.19274. Anal. Calcd
for C28H30Cl2N4: C, 68.15; H, 6.13; N, 11.35. Found: C, 68.15; H,
6.48; N, 11.29.
5.7. 1-((7-Chloroquinolin-4-yl)amino)-2-methyl-5-(4-nitro-
phenyl)-1H-pyrrole-3-carboxylic acid (25)
5.6.3. 7-Chloro-4-[N-[2-methyl-3-(pyrrolidin-1-ylmethyl)-5-(4-
(trifluoromethyl)phenyl)-1H-pyrrol-1-yl]amino]quinoline (5b)
Solid obtained from reduction was not purified by CC but only
rinsed with diethyl ether. Yield: 20%. Mp 187.5–190.2 °C. 1H
NMR (DMSO-d6): d 10.70 (s, 1H); 8.40 (br s, 1H); 8.32 (d,
J = 6.60 Hz, 1H); 7.90 (s, 1H); 7.73 (d, J = 7.98 Hz, 2H); 7.60–7.53
(m, 3H); 6.58 (s, 1H); 5.60 (br s, 1H); 3.47 (s, 2H); 2.02 (s, 3H);
1.68 (s, 4H); four protons are missing because covered by the
DMSO signal. HRMS (ESI) m/z calcd for C26H25ClF3N4 [M+H]+:
485.17144; found: 485.17221. Anal. Calcd for C26H24ClF3N4: C,
64.39; H, 4.99; N, 11.55. Found: C, 64.51; H, 5.29; N, 10.34.
In a round bottom flask, LiOH monohydrate (1.11 g, 26.4 mmol)
was dissolved in 24 ml of ethanol/water (2:1) and ester 19
(800 mg, 1.76 mmol) was added. The resulting mixture was heated
at reflux for 5 h. Then the ethanol was evaporated, water was
added and the resulting mixture was acidified with 1 M HCl to
pH 4. The pure acid (25) was filtered, washed with water and dried
overnight in vacuo (CaCl2) and than washed with diethyl ether.
Yield: 96%. Mp 205–207.5 °C (dec). 1H NMR (CD3OD): d 8.37 (d,
J = 8.8 Hz, 1H); 8.07 (d, J = 8.81 Hz, 2H); 7.93 (d, J = 6.33 Hz, 1H);
7.80 (d, J = 9.07 Hz, 2H); 7.69 (d, J = 1.65 Hz, 1H); 7.54 (dd,
J = 1.92, 9.07 Hz, 1H); 7.08 (s, 1H); 5.65 (d, J = 6.6 Hz, 1H); 2.43
(s, 3H).
5.6.4. 7-Chloro-4-[N-[2-methyl-3-((diethylamino)methyl)-5-p-
tolyl-1H-pyrrol-1-yl]amino]quinoline (6a)
CC (CH2Cl2/MeOH; 97.5:2.5); solid rinsed with a mixture of
diethyl ether/petroleum ether (50:50). Yield: 40%. Mp 219–
221 °C. 1H NMR (DMSO-d6): d 10.60 (s, 1H); 8.37 (d, J = 5.23 Hz,
1H); 8.3 (d, J = 9.07, 1H); 7.87 (s, 1H); 7.59 (d, J = 8.80 Hz, 1H);
7.39 (d, J = 7.98 Hz, 2H); 7.00 (d, J = 7.98 Hz, 2H); 6.32 (s, 1H);
5.56 (d, J = 4.95 Hz, 1H); 3.29 (s, 2H); 2.14 (s, 3H); 2.02 (s, 3H);
1.01 (t, J = 6.6 Hz, 6H); four protons are missing because covered
5.8. 1-[(7-Chloroquinolin-4-yl)amino]-2-methyl-5-(4-nitrophenyl)-
1H-pyrrole-3-(N-substituted)carboxamide (26a, 26b): general
method
In a microwave vial, acid 25 (1.83 g, 4.25 mmol), HOBt (781 mg,
5.10 mmol) and appropriate amine (5.10 mmol) were dissolved in
20 ml of dry DMF at room temperature. DCC (1.05 g, 5.10 mmol)
was added and the mixture was heated with a microwaves synthe-
sizer system at 60 °C for 120 min. After cooling, the precipitate of
dicyclohexylurea was filtered off and washed with DMF. The sol-
vent was removed under vacuum and the resulting sticky solid
was suspended in water, alkalinized with 2 N NaOH, filtered,
washed with water and dried in a desiccator over anhydrous CaCl2
(overnight under vacuum). The crude solid was purified by CC (sil-
ica gel; different ratio of CH2Cl2 and MeOH as indicated for each
compound).
by the DMSO signal. HRMS (ESI) m/z calcd for
C26H30ClN4
[M+H]+: 433.21535; found: 433.21637. Anal. Calcd for C26H29ClN4
H2O: C, 69.24; H, 6.93; N, 12.42. Found: C, 69.39; H,7.33; N, 12.43.
5.6.5. 7-Chloro-4-[N-[2-methyl-3-(pyrrolidin-1-ylmethyl)-5-p-
tolyl-1H-pyrrol-1-yl]amino]quinoline (6b)
CC (CH2Cl2/MeOH; 96:4); solid rinsed with diethyl ether. Yield:
20%. Mp 190–191 °C. 1H NMR (DMSO-d6): d 10.57 (s, 1H); 8.4 (d,
J = 4.95 Hz, 1H); 8.34 (d, J = 9.07 Hz, 1H); 7.87 (s, 1H); 7.6 (d,
J = 9.07 Hz, 1H); 7.38 (d, J = 7.98 Hz, 2H); 7.0 (d, J = 7.98 Hz, 2H);

6632
M. Casagrande et al. / Bioorg. Med. Chem. 18 (2010) 6625–6633
5.8.1. 1-((7-Chloroquinolin-4-yl)amino)-2-methyl-5-(4-
three times with CH2Cl2; organic layers were dried (Na2SO4) and
evaporated. The crude solid was purified by CC (silica gel; CH2Cl2
and MeOH as indicated for each single compound).
nitrophenyl)-1H-pyrrole-3-(N,N-diethyl)-carboxamide (26a)
CC (CH2Cl2/MeOH; 98:2); solid rinsed with diethyl ether. Yield:
74%. Mp 276–278.3 °C. 1H NMR (DMSO-d6): d 11.3 (s, 1H); 8.43 (d,
J = 7.7 Hz, 1H); 8.04 (d, J = 8.8 Hz, 2H); 7.89–7.86 (m, 3H); 7.86–
7.41 (m, 2H); 6.79 (s, 1H); 5.35 (s, 1H); 3.44 (d, J = 6.88 Hz, 4H);
2.05 (s, 3H); 1.13 (t, J = 6.87 Hz, 6H). HRMS (ESI) m/z calcd for
5.10.1. 7-Chloro-4-[N-[2-methyl-5-(4-aminophenyl)-3-((diethyl-
amino)methyl)-1H-pyrrol-1-yl]-amino]quinoline (7a)
CC (CH2Cl2/MeOH; 98:2); solid crystalizzed and rinsed with
CH2Cl2. Yield: 7%. Mp 250.5–254.5 °C. 1H NMR (CDCl3): d 8.49 (s,
1H); 8.00 (s, 2H); 7.81 (d, J = 8.25 Hz, 1H); 7.41 (d, J = 8.25 Hz,
1H); 7.11 (d, J = 7.97 Hz, 2H); 6.47 (d, J = 7.98 Hz, 2H); 6.25 (s,
1H); 5.99 (s, 1H); 3.58 (br s, 2H); 3.51 (s, 2H); 2.61 (d, J = 6.7 Hz,
4H); 1.11 (t, J = 6.7 Hz, 6H). HRMS (ESI) m/z calcd for C25H29ClN5
[M+H]+: 434.21060; found: 434.21080. Anal. Calcd for C25H28ClN5:
C, 69.19; H, 6.50; N, 16.14. Found: C, 69.26; H, 6.97; N, 16.20.
C
25H25ClN5O3 [M+H]+: 478.16404; found: 478.16410.
5.8.2. [1-((7-Chloroquinolin-4-yl)amino)-2-methyl-5-(4-nitro-
phenyl)-1H-pyrrol-3-yl](pyrrolidin-1-yl)methanone (26b)
CC (CH2Cl2/MeOH; 98:2); solid rinsed with diethyl ether. Yield:
84%. Mp 314.5–316.5 °C. 1H NMR (DMSO-d6): d 11.60 (s, 1H); 8.51–
8.44 (m, 1H); 8.31–7.85 (m, 4H); 7.67–7.39 (m, 3H); 7.06–6.99 (m,
1H); 5.69–5.15 (m, 1H); 3.44 (br s, 4H); 2.15 (s, 3H); 1.85 (s, 4H).
HRMS (ESI) m/z calcd for C25H23ClN5O3 [M+H]+: 476.14839; found:
476.14847.
5.10.2. 7-Chloro-4-[N-[2-methyl-5-(4-aminophenyl)-3-
(pyrrolidin-1-ylmethyl)-1H-pyrrol-1-yl]-amino]quinoline (7b)
CC performed twice (CH2Cl2/MeOH; 96:4); solid crystalizzed
and rinsed with CH2Cl2. Yield: 7%. Mp 216–218.5 °C (dec). 1H
NMR (DMSO-d6): d 10.46 (s, 1H); 8.38 (d, J = 4.95 Hz, 1H); 8.30
(d, J = 9.07 Hz, 1H); 7.87 (s, 1H); 7.6 (d, J = 8.8 Hz, 1H); 7.13 (d,
J = 8.26 Hz, 2H); 6.35 (d, J = 8.28 Hz, 2H); 6.10 (s, 1H); 5.6 (s, 1H);
4.95 (s, 2H); 3.41 (s, 2H); 2.43 (s, 4H); 1.98 (s, 3H); 1.67 (s, 4H).
HRMS (ESI) m/z calcd for C25H27ClN5 [M+H]+: 432.19495; found:
432.19501. Anal. Calcd for C25H26ClN5ꢁ0.75H2O: C, 67.40; H, 6.22;
N, 15.72. Found: C, 67.49; H, 6.80; N, 15.60.
5.9. 1-[(7-Chloroquinolin-4-yl)amino]-2-methyl-5-(4-amino-
phenyl)-1H-pyrrole-3-(N-substituted)carboxamide (27a, 27b):
general method
Iron powder (223 mg, 4 mmol), freshly washed with 1 N HCl
and distilled water, was added to a stirred solution of the corre-
sponding nitroderivative (26a and 26b; 1 mmol) in 11 ml of etha-
nol/water (10:1), and then 0.3 ml of glacial acetic acid was added.
The suspension was heated at 80 °C with vigorous stirring under N2
atmosphere until reaction was completed. After cooling, iron was
filtered off through a pad of CeliteÒ and washed with ethanol. After
evaporation of the solvent, the residue was dissolved in a solution
of CH2Cl2/MeOH (10:1), washed with cold 2 N NaOH and filtered
off. Organic phase was dried with anhydrous Na2SO4, evaporated
and resulting crude solid was purified by CC (silica gel; different ra-
tio of CH2Cl2 and MeOH as indicated for each compound).
5.11. Parasite cultures and drug susceptibility assay
P. falciparum cultures were carried out according to Trager and
Jensen with slight modifications.30 The CQ-sensitive, strain D10
and the CQ-resistant, strain W2 were maintained at 5% hematocrit
(human type A-positive red blood cells) in RPMI 1640 (EuroClone,
Celbio) medium with the addition of 1% AlbuMax (Invitrogen, Mi-
lan, Italy), 0.01% hypoxantine, 20 mM Hepes, and 2 mM glutam-
mine. All the cultures were maintained at 37 °C in a standard gas
mixture consisting of 1% O2, 5% CO2, and 94% N2. Compounds were
dissolved in either water or DMSO and then diluted with medium
to achieve the required concentrations (final DMSO concentration
<1%, which is non-toxic to the parasite). Drugs were placed in
96-well flat-bottomed microplates (COSTAR) and serial dilutions
made. Asynchronous cultures with parasitaemia of 1–1.5% and
1% final hematocrit were aliquoted into the plates and incubated
for 72 h at 37 °C. Parasite growth was determined spectrophoto-
metrically (OD650) by measuring the activity of the parasite lac-
tate dehydrogenase (pLDH), according to a modified version of
the method of Makler in control and drug-treated cultures.18 The
antimalarial activity is expressed as 50% inhibitory concentrations
(IC50); each IC50 value is the mean and standard deviation of at
least three separate experiments performed in duplicate.31
5.9.1. 5-(4-Aminophenyl)-1-((7-chloroquinolin-4-yl)amino)-2-
methyl-1H-pyrrole-3-(N,N-diethyl) carboxamide (27a)
CC (CH2Cl2/MeOH; 97.5:2.5); solid rinsed with diethyl ether.
Yield: 71%. Mp 243.5–245.5 °C. 1H NMR (DMSO-d6): d 10.56 (s,
1H); 8.41 (s, 1H); 8.28 (d, J = 7.7 Hz, 1H); 7.89 (s, 1H); 7.6 (d,
J = 6.87 Hz, 1H); 7.15 (d, J = 8.26 Hz, 2H); 6.37 (d, J = 8.25 Hz, 2H);
6.20 (s, 1H); 5.64 (s, 1H); 5.03 (s, 2H); 3.45–3.41 (m, 4H); 3.47 (s,
3H); 1.12 (t, J = 6.6 Hz, 6H). HRMS (ESI) m/z calcd for C25H27ClN5O
[M+H]+: 448.18986; found: 448.18964.
5.9.2. [5-(4-Aminophenyl)-1-[(7-chloroquinolin-4-yl)amino]-2-
methyl-1H-pyrrol-3-yl](pyrrolidin-1-yl)methanone (27b)
CC (CH2Cl2/MeOH; 97:3); solid rinsed with diethyl ether. Yield:
72%. Mp 293.8–295.3 °C. 1H NMR (DMSO-d6): d 10.56 (s, 1H); 8.40
(d, J = 5.22 Hz, 1H); 8.27 (d, J = 9.36 Hz, 1H); 7.89 (s, 1H); 7.6 (d,
J = 8.80 Hz, 1H); 7.15 (d, J = 8.53 Hz, 2H); 6.42 (s, 1H); 6.36 (d,
J = 8.25 Hz, 2H); 5.65 (d, J = 4.95 Hz, 1H); 5.03 (s, 2H); 3.43 (s,
2H); 3.37 (s, 2H); 2.16 (s, 3H); 1.84 (s, 4H). HRMS (ESI) m/z calcd
for C25H25ClN5O [M+H]+: 446.17421; found: 446.17407.
5.12. Cell cytotoxicity assays
The long-term human microvascular endothelial cell line
(HMEC-1) immortalized by SV 40 large T antigen32 was maintained
in MCDB 131 medium (Invitrogen, Milan, Italy) supplemented with
10% fetal calf serum (HyClone, Celbio, Milan, Italy), 10 ng/ml of epi-
5.10. 7-Chloro-4-[N-[2-methyl-5-(4-aminophenyl)-3-[(N-
substituted-amino)-methyl]-1H-pyrrol-1-yl]-amino]quinoline
(7a and 7b): general method
dermal growth factor (Chemicon), 1
2 mM glutamine, 100 U/ml of penicillin, 100 l
l
g/ml of hydrocortisone,
g/ml of streptomy-
l
Each amido compounds 27a and 27b (1 mmol) were suspended
in 30 ml of anhydrous THF under N2 and LiAlH4 (296 mg,
7.8 mmol) was carefully added; the mixture was heated at reflux
and stirred for 8 h and then left at 40 °C overnight. After cooling,
water and 1 N NaOH was added carefully, the suspension filtrated
and the residue was washed twice with THF. After evaporation of
the organic solvent, the residual aqueous mixture was extracted
cin, and 20 mM Hepes buffer (EuroClone). K562 human erythroleu-
kemia cells and WEHI Clone 13 murine fibrosarcoma line were
cultured in RPMI 1640 supplemented with 2 mM glutamine,
100 U/ml of penicillin, 100 lg/ml of streptomycin, and 10% fetal
calf serum. Human dermal fibroblasts (HDF) were maintained in
DMEM medium (EuroClone) supplemented with 10% fetal calf ser-
um, 2 mM glutamine, 100 units/ml of penicillin, 100 lg/ml of

M. Casagrande et al. / Bioorg. Med. Chem. 18 (2010) 6625–6633
6633
streptomycin. Unless stated otherwise, all reagents were from Sig-
References and notes
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Acknowledgments
Part of this manuscript was generated in the context of the
AntiMal project, funded under the 6th Framework Programme of
the European Community (Contract No. IP-018834). The authors
are solely responsible for its content, it does not represent the
opinion of the European Community, and the Community is not
responsible for any use that might be made of the information con-
tained therein. The financial support from the University of Milan
(PUR 2008) is also acknowledged.
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Taramelli, D. Bioorg. Med. Chem. 2005, 13, 5338.
We thank Dr. F. Ravagnani from the Blood Unit, National Cancer
Institute, Milan, Italy, and the Associazione Volontari Italiani San-
gue (AVIS Comunale Milano) for providing fresh red blood cells
for P. falciparum growth.
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