Application of multicomponent reactions to antimalarial drug discovery. Part 2: New antiplasmodial and antitrypanosomal 4-aminoquinoline γ- and δ-lactams via a 'catch and release' protocol

Article abstract of DOI:10.1016/j.bmc.2006.04.035

A parallel synthesis of a new series of 4-aminoquinoline γ- and δ-lactams synthesized via the Ugi 3-component 4-centre multicomponent reaction is described. The basicity of the quinoline nitrogen was exploited in the purification of compounds via a 'catch and release' protocol. Yields ranging from 60% to 77% and purities as high as 96% were obtained. Compound 29, the most active against a chloroquine-resistant W2 strain of Plasmodium falciparum with an IC₅₀ of 0.096 μM, also inhibited recombinant falcipain-2 in vitro (IC₅₀ = 17.6 μM). Compound 17 inhibited the growth of Trypanosoma brucei with an ED₅₀ of 1.44 μM whilst exhibiting a favourable therapeutic index of 409 against a human KB cell line.

Full text of DOI:10.1016/j.bmc.2006.04.035

Bioorganic & Medicinal Chemistry 14 (2006) 5605–5615
Application of multicomponent reactions to antimalarial drug
discovery. Part 2: New antiplasmodial and antitrypanosomal
4-aminoquinoline c- and d-lactams via a ‘catch and release’ protocol
Chitalu C. Musonda,a Jiri Gut,b Philip J. Rosenthal,b Vanessa Yardley,c
Renata C. Carvalho de Souzad and Kelly Chibalea,e,
*
aDepartment of Chemistry, University of Cape Town, Rondebosch 7701, Cape Town, South Africa
bDepartment of Medicine, San Francisco General Hospital, University of California at San Francisco, CA, USA
cDepartment of Infectious and Tropical Diseases, LSHTM, London WC1E 7HT, UK
dCentro de Pesquisas Rene´ Rachou, FIOCRUZ, 30190-002 Belo Horizonte, Brazil
eInstitute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, Cape Town, South Africa
Received 20 March 2006; accepted 13 April 2006
Available online 11 May 2006
Abstract—A parallel synthesis of a new series of 4-aminoquinoline c- and d-lactams synthesized via the Ugi 3-component 4-centre
multicomponent reaction is described. The basicity of the quinoline nitrogen was exploited in the purification of compounds via a
‘catch and release’ protocol. Yields ranging from 60% to 77% and purities as high as 96% were obtained. Compound 29, the most
active against a chloroquine-resistant W2 strain of Plasmodium falciparum with an IC50 of 0.096 lM, also inhibited recombinant
falcipain-2 in vitro (IC50 = 17.6 lM). Compound 17 inhibited the growth of Trypanosoma brucei with an ED50 of 1.44 lM whilst
exhibiting a favourable therapeutic index of 409 against a human KB cell line.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
rings.4,5 However, easy access and availability of suit-
ably functionalized heterocyclic building blocks for the
synthesis of diverse libraries is rather limited. As a con-
sequence the development of new, efficient and clean
synthetic reactions remains a vital challenge to organic
and medicinal chemists.6
Modern drug discovery is faced with the challenge of
designing sequential chemical reactions that are highly
efficient in providing a maximum of the elements of
structural complexity and diversity with minimal syn-
thetic steps for the assemblage of compounds with inter-
esting properties.1 Combinatorial chemistry has in the
recent past emerged as a powerful tool for the rapid
identification, generation and optimization of lead com-
pounds in the drug discovery process.2,3 Thus, the major
driving force behind the increased interest in this field
has been the need to discover and develop new chemical
entities with desirable properties in a more efficient and
cost-effective manner, and most importantly within a
short period of time. Presently most drugs on the market
are small organic compounds that contain heterocyclic
Multicomponent reactions (MCRs),7,8 reactions involv-
ing at least three starting materials in a one-pot reaction,
remain the most efficient method of rapidly introducing
molecular diversity. As such they have found wide-
spread use in organic and diversity-oriented synthesis
by their ability to access highly functionalized molecules
in simple and straightforward one-step transforma-
tions.9 Compared to conventional multistep organic
syntheses MCRs are advantageous owing to their great-
er atom efficiency, accessibility to large numbers of com-
pounds and complex molecules, wide structural diversity
and simplicity of their one-pot procedures making them
amenable to combinatorial synthesis. Among the known
multicomponent reactions to date, the most valuable
reactions are those based on isocyanides. One such
reaction is the Ugi 4-component condensation (4CC)
reaction8 combining an amine, aldehyde (or ketone),
Keywords: Parallel synthesis; Multicomponent reaction; Ugi reaction;
Polymer-supported
Antimalarial activity; Falcipain-2; Antitrypanosomal activity.
reagents;
4-Aminoquinolines;
Lactams;
*
Corresponding author. Tel.: +27 21 650 2553; fax: +27 21 689
7499; e-mail: chibale@science.uct.ac.za
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.035

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C. C. Musonda et al. / Bioorg. Med. Chem. 14 (2006) 5605–5615
carboxylic acid and isocyanide in a single-stage reaction
to afford a-acylamino amides.
our previous efforts we targeted cyclic variants of the
Ugi reaction known as c- and d-lactams (Scheme 2) with
a view to lowering the levels of flexibility often associat-
ed with acyclic amides of type 1.
As part of our continued drug discovery programme in
search of new agents for the treatment of malaria and
sleeping sickness, we became interested in the synthesis
of heterocyclic compounds via multicomponent
reactions built around the 7-chloro-4-aminoquinoline
unit, a known antimalarial pharmacophore. Recently,
we reported on the synthesis and biological evaluation
of new aminoquinoline a-acylamino amides, exemplified
by 1 (Fig. 1) by way of the Ugi 4CC reaction.10 Howev-
er, despite its synthetic potential, the classical Ugi
reaction is limited by giving rise to products that are
flexible and peptidic in nature, often being described
as being ‘non-drug-like’ and therefore making them less
attractive targets in the search for new drugs. Impor-
tantly though, several novel intramolecular variations
and secondary transformations of the Ugi 4CC that
produce more constrained, biologically relevant hetero-
cyclic compounds including tetrazoles,11 diketopipera-
zines,12 benzodiazepines13 and morpholines14 have
been developed. In one variant of the Ugi 4CC, termed
the Ugi 3-component 4-centre (3C/4C) reaction, two of
the functional groups that take part in the reaction are
tethered onto one starting material, leading to
heterocyclic compounds. This strategy has been used
to synthesize b-lactams15 and d- and c-lactams.16 Com-
binatorial syntheses in which multicomponent reactions
have been employed have largely been dominated by
solid-phase methods.17 To the best of our knowledge
the use of polymer-supported reagents as purification
and/or isolation tools specifically in the Ugi reaction
has not been previously demonstrated.
2. Chemistry
The requisite 1,x-diamines 7–10 (where x represents the
position of the N atom in the alkyl chains) needed as
amine inputs for the multicomponent synthesis of our
exploratory library of c- and d-lactams were prepared
by way of Scheme 1.19 Thus, neat mixtures of commer-
cially available 4,7-dichloroquinoline 2 and the alkyl
diamines 3–6 were condensed at 80 ꢁC for 1 h and then
at 135 ꢁC for 3 h. All the diamines were recrystallised
from ethylacetate to afford 7–10 in excellent yields
(Table 1).
We designed d- and c-lactams as synthetic targets and
planned the synthetic route as shown in Scheme 2. The
first step involved the Ugi 3C/4C reaction of diamines
7–10, levulinic (11a, m = 1) or 4-acetylbutyric acid
(11b, m = 2) and tert-butylisocyanide 12a or cyclohexy-
lisocyanide 12b in MeOH to give the compounds
14–29 in solution in parallel array format.
We were then faced with the challenge of having to puri-
fy our library by means of conventional chromatogra-
phy. Concerned about the laborious efforts involved,
we sought a protocol wherein purification and isolation
would be achieved by simple filtration and washing.
Such an approach should greatly accelerate the genera-
tion of larger compound libraries with minimal inputs
in terms of time and effort, and yield compounds of high
purity that are ready for high-throughput screening.
Two strategies were thus available to us, viz., solid-
phase organic synthesis and use of polymer-assisted
solution-phase synthesis. Although the use of solid-
phase synthesis of c- and d-lactams has been achieved
on solid support16 and is an attractive approach towards
yielding high purity compounds, immobilization of the
reagent onto and cleavage from solid support introduces
two extra steps in the reaction sequence. As an alterna-
tive, the use of polymer-bound reagents renders itself as
We targeted synthesis of cyclic compounds via MCRs as
these are known to impart molecular rigidity and
improve the bioavailability of drugs and drug candi-
dates. Indeed, studies have revealed that the number
of rotatable bonds in a drug candidate is a major con-
tributor to good oral bioavailability.18 As a sequel to
H
O
N
N
HN
N
Table 1. Recrystallised yields of 1-(70-chloro-40-amino)-x-diamines
O
Diamine
n
Yield (%)
N
7
8
1
2
3
5
91
86
83
90
Cl
1
9
10
Figure 1. Structure of a first-generation aminoquinoline a-acylamino
amide.10
Cl
NHCH2(CH2)nNH2
7, n = 1
8, n = 2
9, n = 3;
10, n = 5
i
NH2CH2(CH2)nNH2
+
Cl
N
Cl
N
3, n = 1; 4, n = 2
5, n = 3; 6, n = 5
2
Scheme 1. Reagents and conditions: (i) 3–6, 1-x-diamine, 80 ꢁC, 1 h, then 135 ꢁC, 3 h.

C. C. Musonda et al. / Bioorg. Med. Chem. 14 (2006) 5605–5615
5607
O
NHCH2(CH2)nNH2
7 - 10
( )m
O
H
NHCH2(CH2)n N
N
i-iv
12a, R = tBu
12b, R = cHex
R
+
RNC
Cl
N
O
14 - 29
Cl
N
( )m
CO2H
R = tBu, 14 - 17 (m =1), 22 - 25 (m =2)
R = cHex, 18 - 21 (m =1), 26 - 29 (m = 2)
11a, m = 1
11b, m = 2
O
O
S
OH
13
Scheme 2. Reagents and conditions: (i) MeOH, rt, 12–18 h; (ii) 13, 1 h; (iii) filter, wash; (iv) 3% NH3/MeOH, filter, wash.
an excellent approach towards synthesis of combinatori-
al libraries. Several concepts are used in polymer-assist-
ed solution-phase synthesis,20 among which the ‘catch
and release’ principle clearly stands out. In this concept,
a target compound is selectively bound from solution
onto the resin by means of a covalent or ionic bond.
Excess of starting materials are then removed at the
end of the reaction by means of filtration and simple
washings and finally, the products are released from
the resin support. More recently, a one-pot combinato-
rial synthesis of functionalized thiazine libraries using an
in situ polymer-assisted synthesis/catch and release pro-
tocol has been disclosed.21
products 14–29 in solution thus dictated the use of the
former as the limiting reagents.
We found the acidic resin-bound macroporous para-tol-
uene sulfonic acid 13 (MP-TsOH)22 to be an effective
tool for achieving the dual purpose of purifying and iso-
lating our target compounds. Since the starting amines
from which the lactams derive their basicity are com-
pletely consumed in the reactions, we reasoned that
the addition of 13 at the end of reaction should achieve
selective capture of the target compounds as the strong
ion pairs 30 from solution, leaving behind excess of
ketoacid and isocyanide in solution, which can then be
removed by simple filtration and resin washing with
the reaction solvent (Scheme 3).
We chose to employ the ‘catch and release’ concept of
polymer-supported reagents as a means of purifying
our compound library. We reasoned that the basicity
(albeit weak) of the quinoline nitrogen could be exploit-
ed for the capture of the products followed by purifica-
tion and subsequent release of pure products. The
quinoline nitrogen is rendered basic due to the reso-
nance or mesomeric release mediated by the 4-amino
group, Figure 2.
Therefore, after the reactions were deemed complete as
was indicative of TLC, MP-TsOH was added to the
reaction mixtures to scavenge the products from solu-
tion. After 1 h following addition of MP-TsOH 13, all
reactions revealed complete scavenging of the products
from solution (TLC) and were thus filtered and washed
repeatedly to remove excess of ketoacids (11a and 11b)
and isocyanides (12a and 12b). Release of the products
was achieved by agitating the resins in anhydrous
methanolic ammonia for 30 min. On addition of metha-
nolic ammonia a base interchange should occur between
the ionic pairs 30 and NH3 to give the free lactams 14–29
and the resin-bound ammonium salt 31. Yields were
generally good to excellent and were comparable with
those from traditional solution-phase synthesis. More
interestingly the purities of compounds 14–29 were high
enough to permit their use in biological assays (Table 2).
The reactions were performed in parallel using excess of
ketoacids (11a and 11b) and isocyanides (12a and 12b),
while the diamines 7–10 were used as the limiting
reagents (Scheme 2). The ratio of reactants was carefully
determined in such a way as to minimize the quantity of
amines used in the reactions. Since the presence of the
tertiary nitrogen atom in the aminoquinoline ring ren-
ders the lactam products basic and, since the ‘catch
and release’ concept relies on the formation of an ionic
pair between a basic and an acidic compound, use of ex-
cess of the diamines would ultimately be a source of
impurities in the final products. The predictable inability
of 13 to discriminate between starting amines 7–10 and
The approach discussed here offers the obvious advan-
tage of providing access to high purity 4-aminoquino-
line-based c- and d-lactams via multicomponent Ugi
3C/4C reaction in a parallel array format, whilst avoid-
ing the tedious work involved in chromatography; it is
also economical in the sense that only a single and read-
ily available commercial reagent is required for the puri-
fication of large numbers of compounds.
R
R
HN
N
HN
N
In summary, we have extended the concept of ‘catch and
release’ as a purification and isolation protocol to the
Ugi 3C/4C multicomponent synthesis of conformation-
ally constrained -aminoquinoline-based c- and d-lactams
using a readily available polymer-supported reagent.
Cl
Cl
Figure 2. Increased basicity of quinoline nitrogen due to resonance
effects.

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C. C. Musonda et al. / Bioorg. Med. Chem. 14 (2006) 5605–5615
O
( )m
O
O
H
( )m
O
NHCH2(CH2)n N
O
N
O
H
NHCH2(CH2)n N
R
13
S
N
O
R
14 - 29
Cl
N
Cl
N
H
30
NH3/MeOH
O
O
+
. NH4
S
+
14 - 29
O
31
Scheme 3. Formation of ionic pair 30 between lactams 14–29 and MP-TsOH 13.
Table 2. Isolated yields and purities of lactams 14–29
Entry
Compound
m
n
R
Yielda (%)
Yieldb (%)
Purityc (%)
1
2
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
1
2
3
5
1
2
3
5
1
2
3
5
1
2
3
5
tBu
57
84
77
75
84
67
70
84
65
76
75
70
76
80
78
79
60
79
73
69
75
70
68
77
75
71
69
74
66
72
74
72
91
92
90
94
88
91
95
94
91
96
87
93
95
94
91
93
tBu
3
tBu
tBu
4
5
cHexd
cHexd
cHexd
cHexd
tBu
6
7
8
9
10
11
12
13
14
15
16
tBu
tBu
tBu
cHexd
cHexd
cHexd
cHexd
a Yields from solution-phase synthesis.
b Yields from polymer-assisted solution-phase synthesis.
c RP-HPLC purity was determined as area under a curve (k = 220 and 254 nm).
d cHex, cyclohexyl.
The methodology is general and works because of the
presence of the basic quinoline nitrogen site in the mole-
cule. The method has allowed us to quickly isolate and
purify biologically interesting compounds in our contin-
ued search for new antiprotozoal agents. The disclosure
of our simple and economic method for rapidly accessing
libraries of 4-aminoquinolines is timely given the recent
interest in libraries of 4-aminoquinolines, which have
so far been purified by high-throughput HPLC tech-
niques.23 For biological assay purposes, compounds
14–29 were resynthesized and purified by means of pre-
parative layer chromatography.
compounds containing the same N-alkyl substituent.
Thus, compounds containing the six-membered lactam
ring were in general more active than those with the
five-membered ring. From the modest library, com-
pound 29 with an IC50 of 0.096 lM exhibited the best
activity, being 2.5 times as potent in this strain as chlo-
roquine (Table 3).
Compounds 17 (IC50 = 0.18 lM), 21 (IC50 = 0.27 lM)
and 22 (IC50 = 0.21 lM) exhibited comparable activities
to chloroquine, while 18 (IC50 = 0.35 lM) and 24
(IC50 = 0.37 lM) exhibited slightly reduced activities.
The remaining compounds on the other hand were less
efficacious than the control drug. In terms of methylene
spacer length, compounds with the 6-carbon spacer were
generally more efficacious, while in the case of the five-
membered c-lactams, those containing the cyclohexyl
ring turned out to be more potent than those with the
tert-butyl group. On the contrary, the tert-butyl-con-
taining d-lactams were more efficacious than the cyclo-
hexyl counterparts.
3. Biological results and discussion
Initially all lactams 14–29 were tested for their activities
against parasite cultures of the chloroquine-resistant W2
strain of P. falciparum (IC50 chloroquine = 0.24 lM). It
was found that the activities of the compounds were
dependent upon the size of the lactam ring for

C. C. Musonda et al. / Bioorg. Med. Chem. 14 (2006) 5605–5615
5609
Table 3. In vitro antiplasmodial activities of c- and d-lactams 14–29
The generally modest activities of these compounds may
be due to their overall reduced basicities. Whereas chlo-
roquine accumulation in the acidic food vacuole of the
parasite is dependent upon presence of two protonatable
nitrogen atoms, the absence of a second protonatable
nitrogen is likely to have implications for accumulation
and hence antiplasmodial activity. Consequently, these
lactams may be accumulating to much lower concentra-
tions than those required to significantly inhibit plasmo-
dial growth. Moreover, studies have revealed that
chloroquine adopts a bioactive conformation in which
the inter-nitrogen separation (N-(quinoline)–N-(dieth-
yl)) of 0.83 A is equivalent to that of the central Fe
atom and the two oxygens in the carboxylate groups
of haem,24 strongly suggesting that ferriprotoporphyrin
(haem) is the ‘receptor’ target. Such similarity is unlikely
to be present in these series of compounds in the absence
of a second basic nitrogen in the side chain. Therefore, it
may be possible that the modest antiplasmodial activi-
ties displayed by these compounds may result from their
inability to interact with haem or from not being deliv-
ered to the requisite site of action within the acidic food
vacuole. However, the reduced basicities of the com-
pounds relative to chloroquine do not fully explain the
observed antiplasmodial activities. Some compounds
(such as 17, 21 and 22) have comparable activities to
chloroquine and yet they lack the terminal basic nitro-
gen, which is more critical for the accumulation in the
acidic food vacuole. The varying degrees of activity of
the compounds in W2, a chloroquine-resistant strain,
support the assertion that P. falciparum resistance is
compound specific. Indeed, modifications to the side
chain of chloroquine have led to derivatives which are
able to circumvent chloroquine resistance.25 The most
notable result from this limited series of compounds is
that for 29 (W2 IC50 = 0.096 lM, falcipain-2
IC50 = 17.7 lM). To our knowledge, this is the first re-
port of an aminoquinoline lactam (albeit weak) inhibitor
of falcipain-2. The antiplasmodial activity of 29 may in
part be due to the inhibition of falcipain-2, which may
be attacking a carbonyl group of one of the amide bonds
O
( )m
H
N
N
( )n
HN
R
O
Cl
N
Compound
m
n
R
IC50 (lM)
W2 P.
falciparum
Rec. F-2a
24
˚
Chloroquine
—
1
—
1
—
0.24
nd
14
0.67
0.98
1.11
0.18
0.35
0.63
0.52
0.27
>10
15
16
17
18
19
20
21
1
1
1
1
1
1
1
2
3
5
1
2
3
5
>10
>10
>10
>10
>10
>10
>10
22
23
2
2
1
2
0.21
0.31
>10
>10
of this compound (29), forming
intermediate.26
a
tetrahedral
24
25
26
27
28
29
2
2
2
2
2
2
3
5
1
2
3
5
0.37
0.46
0.87
0.83
0.87
0.096
>10
>10
>10
>10
>10
17.62
For comparative purposes, pentamidine, a drug used for
the treatment of African sleeping sickness, was em-
ployed as the control drug in the determination of the
ED50s of compounds 14–29. On the other hand, podo-
phyllotoxin was used as the standard in the cytotoxicity
assays against a human KB cell.
The data in Table 4 reveal that pentamidine exhibited
superior antitrypanosomal activity (ED50 = 0.0002 lM),
with the majority of compounds only being active in the
low micromolar range. The most active compound in
the series, 17, was several hundred-fold less efficacious
than the standard drug pentamidine. Despite these low
antitrypanosomal activities, however, compounds 15
and 18–23 were active at or below 20 lM and were all
several orders of magnitude less toxic to the mammalian
cells employed in the assay as is revealed by the
cytotoxicity ED50s of podophyllotoxin versus 14–29.
Since Trypanosoma brucei exists in the bloodstream as
nd, not determined.
a Rec. F-2, recombinant falcipain-2.

5610
C. C. Musonda et al. / Bioorg. Med. Chem. 14 (2006) 5605–5615
Table 4. In vitro antitrypanosomal activities of compounds 14–29
Compound
ED50 lg/ml
ED50 (lM)
Cytotoxicity lg/ml
Cytotoxicity ED50 (lM)
Therapeutic index
Pentamidine
Podophyllotoxin
0.00007
—
0.0002
—
nd
—
0.00024
nd
—
0.41
nd
—
—
nd
15
16
409
6
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
nda
3.32
18.5
0.66
5.28
9.04
7.57
5.56
7.74
6.1
8
43
48.6
>300
269.8
29.6
194.5
>300
44.7
44.7
63.1
41.4
65.2
186.5
241.8
166.8
nd
117
697
589
69
1.44
12.3
20.4
16.6
11.5
18.6
14.2
>67.5
>63.5
30
440
658
92
22
54
8
107
147
93
6
10
—
—
14
20
—
nd
>30
>30
13.25
12.32
>30
nd
138
420
528
353
nd
27
>64
nd
a nd, not determined.
extracellular trypomastigotes, the problems that are usu-
ally associated with drug penetration of the macrophage
are absent and the compounds are likely to reach their
intended sites of action.
under an ultra-violet lamp. HPLC was performed at
ambient temperature on a Thermo Separations Prod-
uctsꢂ instrument, fitted with an Xteraꢂ C18 5 lm,
4.6 · 150 mm column and operating on a UV detector.
HPLC-grade acetonitrile and ultrafiltered water were
used as the eluting solvents at a concentration of
30% CH3CN/70% H2O in the presence of a 25 mM
phosphate buffer.
In summary, we have disclosed a simple, convenient and
general protocol for the parallel synthesis of 4-amino-
quinolines. Although only one compound (29) was iden-
tified as being more potent than chloroquine in the
P. falciparum chloroquine-resistant strain (W2), overall,
the lactams described herein represent a new class of 4-
aminoquinolines possessing dual antiplasmodial and
antitrypanosomal activities.
4.1. General procedure for the synthesis of diamines 7–10
A mixture of 4,7-dichloroquinoline (0.98 g, 4.94 mmol)
and the 1,x-diamine (24.7 mmol, 5 equiv) was heated
at 80 ꢁC for 1 h without stirring and then at 135 ꢁC for
3 h with stirring. The reaction mixture was cooled to
room temperature, basified with 10% NaOH (30 ml),
extracted with hot EtOAc (3· 50 ml) and dried
(Na4SO4). The solvent was removed in vacuo and the
residue recrystallised from EtOAc to afford the diamine
in high yield.
4. Experimental
Proton nuclear magnetic resonance (1H NMR) spectra
were recorded at ambient temperature using a Varian
Mercury (300 MHz) or a Varian Unity Spectrometer
(400 MHz) and TMS was used as an internal stan-
dard. Chemical shift values (d) are given in ppm rela-
tive to TMS (d = 0.00). Carbon-13 nuclear magnetic
resonance (13C NMR) spectra were recorded at
75 MHz or 100 MHz with the same instruments and
internal standard. Deuterated methanol (CD3OD)
and chloroform (CDCl3) were used in the determina-
tion of spectra for the amines and lactams, respective-
ly. Mass spectra were recorded by means of a VG
micromass 16 F spectrometer at 70 eV with accelerat-
ing voltage 4 kV. Accurate masses were determined
using a VG-70E spectrometer and VG (Micromass)
70-SE magnetic sector mass spectrometer. Infrared
spectra were measured in solution form using chloro-
form on a satellite FT-IR spectrophotometer. Melting
points were determined on a Reichert-Jung Thermovar
(temperature range 0–350 ꢁC) on coverslips and are
uncorrected. Preparative layer chromatography (PLC)
was performed on silica 60 F254 coated glass plates.
Reactions were monitored by thin-layer chromatogra-
phy (TLC) using silica gel coated plates and visualized
4.2. N-(2-Aminoethyl)-7-chloroquinolin-4-amine (3)
Yellow crystals (1.0 g, 91%); mp 115–117 ꢁC; Rf (NH3/
MeOH 1:49) 0.20; dH (300 MHz, CD3OD) 8.35 (d, 1H,
J = 5.4, H-2), 8.1 (d, 1H, J = 9.0, H-5), 7.77 (d, 1H,
J = 2.1, H-8), 7.38 (dd, 1H, J = 2.1, 9.0, H-6), 6.6 (d,
1H, J = 5.4, H-3), 3.43 (t, 2H, J = 6.0, ArNCH2), 2.97
(t, 2H, J = 6.0, NCH2); dC (75 MHz, CDCl3); dC
(75 MHz, CD3OD) 152.9, 150.5, 134, 128.9, 126.1,
125.2, 122, 117, 98, 53.6, 46.1.
4.3. N-(3-Aminopropyl)-7-chloroquinolin-4-amine (4)
Yellow crystals (1.0 g, 86%), mp 124–127 ꢁC; Rf (NH3/
MeOH 1:49) 0.21; dH (300 MHz, CD3OD) 8.33 (d, 1H,
J = 5.4, H-2), 8.12 (d, 1H, J = 9.0, H-5), 7.75 (d, 1H,
J = 2.1, H-8), 7.42 (dd, 1H, J = 2.1, 9.0, H-6), 6.62 (d,
1H, J = 5.4, H-3), 3.44 (t, 2H, J = 6.0, ArNCH2), 2.98
(t, 2H, J = 6.0, NCH2), 1.89 (m, 2H, CH2); dC
(75 MHz, CDCl3); dC (75 MHz, CD3OD) 152.9, 150.5,

C. C. Musonda et al. / Bioorg. Med. Chem. 14 (2006) 5605–5615
5611
134, 128.9, 126.4, 125.1, 122.2, 117, 98.8, 43.6, 41.4,
30.1.
4.8. N-tert-Butyl-1-(3-(7-chloroquinolin-4-ylamino)pro-
pyl)-2-methyl-5-oxopyrrolidine-2-carboxamide (15)
4.4. N-(4-Aminobutyl)-7-chloroquinolin-4-amine (5)
Yellow oil (74 mg, 79%); Rf (MeOH/DCM 1:9) 0.21; IR
mmax (CHCl3)/cmꢀ1 33344 (NH), 3019 (ArCH), 2984
(CH), 1719 (C@O), 1648 (C@C), 1532 (C@N); dH
(400 MHz, CDCl3) 8.50 (d, 1H, J = 5.6, H-2), 7.92 (d,
1H, J = 2.0, H-8), 7.85 (d, 1H, J = 9.0, H-5), 7.44 (dd,
1H, J = 2.0, 9.0, H-6), 6.30 (d, 1H, J = 5.6, H-3), 3.66
(t, 2H, J = 6.0, ArNCH2 (CH2)2N), 3.41 (t, 2H,
J = 6.0, ArN(CH2)2CH2N), 2.54–2.50 (m, 2H, CH2-40),
2.33–2.21 (m, 1H, H-30a), 1.97–1.88 (m, 1H, H-30b),
1.84–1.80 (m, 2H, ArCH2CH2CH2N), 1.48 (s, 3H,
CCH3), 1.33 (s, 9H, (CH3)3); dC (100 MHz, CDCl3)
178.6, 173.3, 151.0, 150.7, 134.0, 129.0, 126.2, 125.4,
122.2, 117.3, 98.6, 69.7, 52.3, 44.2, 40.4, 33.2, 30.2,
29.3, 28.4 (3C), 22.3; HRMS (FAB) m/z 416.19790,
(M+ C22H29ClN4O2 requires 416.19837); HPLC purity:
Yellow crystals (1.02 g, 83%), mp 45–47 ꢁC; Rf (NH3/
MeOH 1:49) 0.22; dH (300 MHz, CD3OD) 8.48 (d, 1H,
J = 5.4, H-2), 7.94 (d, 1H, J = 9.0, H-5), 7.69 (d, 1H,
J = 2.1, H-8), 7.27 (dd, 1H, J = 2.1, 9.0, H-6), 6.33 (d,
1H, J = 5.4, H-3), 3.39 (t, 2H, J = 6.0, ArNCH2), 3.04
(t, 2H, J = 6.0, NCH2), 1.89 (m, 2H, CH2), 1.62 (m,
2H, NCH2CH2); dC (75 MHz, CD3OD) 152.9, 150.5,
134, 128.9, 126, 124.9, 122, 117, 98.8, 43.6, 41.4, 30.1,
28.9.
4.5. N-(6-Aminohexyl)-7-chloroquinolin-4-amine (6)
Yellow needles (1.23 g, 90%), mp 136–138 ꢁC; Rf (NH3/
MeOH 1:49) 0.22; dH (300 MHz, CD3OD) 8.39 (d, 1H,
J = 5.4, H-2), 8.18 (d, 1H, J = 9.0, H-5), 7.79 (d, 1H,
J = 2.1, H-8), 7.44 (dd, 1H, J = 2.1, 9.0, H-6), 6.68 (d,
1H, J = 5.4, H-3), 3.36 (t, 2H, J = 6.0, ArNCH2), 2.67
(t, 2H, J = 6.0, NCH2) 1.77 (m, 2H, CH2), 1.51 (m,
6H, 3· CH2); dC (75 MHz, CDCl3); dC (75 MHz,
CD3OD) 152.9, 150.5, 134, 128.9, 126.3, 124.9, 122,
117, 98.6, 42.6, 41.1, 31.8, 28.2, 26.7, 26.5.
92%, t0 ¼ 2:04 min.
R
4.9. N-tert-Butyl-1-(4-(7-chloroquinolin-4-ylamino)bu-
tyl)-2-methyl-5-oxopyrrolidine-2-carboxamide (16)
White amorphous powder (71 mg, 73%), mp
203–205 ꢁC; Rf (MeOH/DCM 1:9) 0.21; IR mmax
(CHCl3)/cmꢀ1 3323 (NH), 3011 (ArCH), 2965 (CH),
1726 (C@O), 1651 (C@C), 1545 (C@N); dH (400 MHz,
CDCl3) 8.46 (d, 1H, J = 5.6, H-2), 7.91 (d, 1H, J = 2.0,
H-8), 7.83 (d, 1H, J = 9.0, H-5), 7.43 (dd, 1H, J = 2.0,
9.0, H-6), 6.24 (d, 1H, J = 5.6, H-3), 3.38 (t, 2H,
J = 6.0, ArNCH2 (CH2)2CH2N), 3.00 (t, 2H, J = 6.0,
ArNCH2 (CH2)2CH2N), 2.51–2.38 (m, 2H, CH2-40),
2.27–2.18 (m, 1H, H-30a), 1.91–1.87 (m, 1H, H-30b),
1.83–1.70 (m, 4H, ArNCH2CH2CH2CH2N), 1.48 (s,
3H, CCH3), 1.39 (s, 9H, C(CH3)3); dC (100 MHz,
CDCl3) 178.4, 173.0, 151.0, 150.6, 134.0, 129.0, 126.0,
125.2, 122.0, 117.0, 98.8, 69.3, 52.4, 44.2, 40.3, 33.2,
30.3, 29.3, 29.0, 28.5 (3C), 22.6; HRMS (FAB) m/z
430.21355, (M+ C23H31ClN4O2 requires 430.21150);
4.6. General procedure for the synthesis of lactams 14–29
Amines 7–10 (0.23 mmol, 1.0 equiv) were dispensed in
16 separate vials and dissolved in MeOH (5 ml). A solu-
tion of levulinic acid or 4-acetylbutyric acid (0.27 mmol,
1.2 equiv) was added via micro syringe and the reaction
mixtures stirred at room temperature for 30 min. At this
point isocyanide was added (0.27 mmol, 1.2 equiv) and
stirring continued for an additional 18 h. After the reac-
tions were deemed complete (TLC), MP-TSOH with a
loading capacity of 1.46 mmol/g (0.46 g, 0.67 mmol)
was added to each of the vials which were transferred
to an orbital shaker. After 1 h, the resins were filtered
and washed with MeOH and suspended in a solution
of 3% NH3 in MeOH and filtered after 30 min of shak-
ing at room temperature. The solvent was removed in
vacuo and concentrated to afford the lactams.
HPLC purity: 90%, t0 ¼ 2:19 min.
R
4.10. N-tert-Butyl-1-(6-(7-chloroquinolin-4-ylamino)hex-
yl)-2-methyl-5-oxopyrrolidine-2-carboxamide (17)
White amorphous powder (71 mg, 73%), mp
203–205 ꢁC; Rf (MeOH/DCM 1:9) 0.21; IR mmax
(CHCl3)/cmꢀ1 3323 (NH), 3011 (ArCH), 2965 (CH),
1726 (C@O), 1651 (C@C), 1545 (C@N); dH
(400 MHz, CDCl3) 8.46 (d, 1H, J = 5.6, H-2), 7.91
(d, 1H, J = 2.0, H-8), 7.83 (d, 1H, J = 9.0, H-5),
7.43 (dd, 1H, J = 2.0, 9.0, H-6), 6.24 (d, 1H, J = 5.6,
H-3), 3.38 (t, 2H, J = 6.0, ArNCH2 (CH2)5N), 3.00
(t, 2H, J = 6.0, ArN(CH2)5CH2N), 2.51–2.38 (m, 2H,
CH2-40), 2.27–2.18 (m, 1H, H-30a), 1.91–1.87 (m,
1H, H-30b), 1.83–1.70 (m, 4H, ArNCH2CH2
(CH2)2CH2 (CH2)2N), 1.48 (s, 3H, CCH3), 1.39 (s,
9H, C(CH3)3), 1.23–1.01 (m, 4H, ArN(CH2)2CH2CH2
(CH2)2N); dC (100 MHz, CDCl3) 178.5, 172.8, 151.2,
150.7, 134.2, 129.1, 126.2, 125.4, 122.0, 117.0, 99.0,
69.5, 52.5, 44.4, 40.2, 33.4, 30.2, 29.4, 29.0, 28.4
(3C), 22.8; HRMS (FAB) m/z 430.24355, (M+
C25H35ClN4O2 requires 458.24485); HPLC purity:
4.7. N-tert-Butyl-1-(2-(7-chloroquinolin-4-ylamino)ethyl)-
2-methyl-5-oxopyrrolidine-2-carboxamide (14)
White amorphous powder (54 mg, 60%), mp
112–114 ꢁC; Rf (MeOH/DCM 1:9) 0.20; IR mmax
(CHCl3)/cmꢀ1 3304 (NH), 3031 (ArCH), 2934 (CH),
1712 (C@O), 1627 (C@C), 1580 (C@N); dH (400 MHz,
CDCl3) 8.51 (d, 1H, J = 5.6, H-2), 7.94 (d, 1H, J = 2.0,
H-8), 7.87 (d, 1H, J = 9.0, H-5), 7.40 (dd, 1H, J = 2.0,
9.0, H-6), 6.29 (d, 1H, J = 5.6, H-3), 3.68 (t, 2H,
J = 6.0, ArNCH2CH2N), 3.43 (t, 2H, J = 6.0,
ArNCH2CH2N), 2.55–2.49 (m, 2H, CH2-40), 2.32 (m,
1H, H-30a), 1.99 (m, 1H, H-30b), 1.56 (s, 3H, CCH3),
1.24 (s, 9H, C(CH3)3) dC (100 MHz, CDCl3) 178.4,
173.0, 151, 150.6, 134.0, 129.0, 126.0, 125.2, 122.0,
117.0, 98.2, 69.3, 52.4, 44.2, 40.3, 33.2, 29.3, 28.5 (3C),
22.6; HRMS (FAB) m/z 402.17884, (M+ C21H27ClN4O2
requires 402.17874); HPLC purity: 91%, t0R ¼ 1:49 min.
90%, t0 ¼ 2:19 min.
R

5612
C. C. Musonda et al. / Bioorg. Med. Chem. 14 (2006) 5605–5615
4.11. 1-(2-(7-Chloroquinolin-4-ylamino)ethyl)-N-cyclo-
hexyl-2-methyl-5-oxopyrrolidine-2-carboxamide (18)
(M+ C25H33ClN4O2 requires 456.22920); HPLC purity:
95%, t0 ¼ 2:90 min.
R
White amorphous powder (72 mg, 75%), mp
210–210 ꢁC; Rf (MeOH/DCM 1:9) 0.20; IR mmax
(CHCl3)/cmꢀ1 3334 (NH), 3013 (ArCH), 2988 (CH),
1725 (C@O), 1650(C@C), 1555 (C@N); dH (300 MHz,
CDCl3) 8.46 (d, 1H, J = 5.4, H-2), 7.91 (d, 1H, J = 2.1,
H-8), 7.86 (d, 1H, J = 9.0, H-5), 7.41 (dd, 1H, J = 2.1,
9.0, H-6), 6.33 (d, 1H, J = 5.4, H-3), 3.68 (m, 3H,
ArNCH2CH2N, H-100), 3.48 (t, 2H, J = 6.0,
ArNCH2CH2N), 2.52–2.50 (m, 2H, CH2-40), 2.40–2.36
(m, 1H, H-30a), 2.10–1.99 (m, 1H, H-30b), 1.78 (br t,
2H, J = 12.0, CH-200a, CH-600a), 1.59 (m, 5H, CH-200b,
CH-600b, CCH3), 1.26–0.99 (m, 6H, CH2-300, CH2-400,
CH2-500); dC (75 MHz, CDCl3) 178.7, 172.0, 151.1,
150.0, 146.8, 135.0, 126.7, 126.0, 122.5, 117.0, 98.5,
67.8, 48.9, 44.3, 40.2, 33.4, 32.7 (2C), 29.4, 25.2, 24.7
(2C), 22.6; HRMS (FAB) m/z 428.19404, (M+
C23H29ClN4O2 requires 428.19790); HPLC purity:
88%, t0 R2:02 ¼ min.
4.14. 1-(6-(7-Chloroquinolin-4-ylamino)hexyl)-N-cyclo-
hexyl-2-methyl-5-oxopyrrolidine-2-carboxamide (21)
Yellow foam (84 mg, 77%); Rf (MeOH/DCM 1:9) 0.23;
IR mmax (CHCl3)/cmꢀ1 3323 (NH), 3026 (ArCH), 2954
(CH), 1716 (C@O), 1652 (C@C), 1583 (C@N); dH
(300 MHz, CDCl3) 8.46 (d, 1H, J = 5.4, H-2), 7.92 (d,
1H, J = 2.0, H-8), 7.81 (d, 1H, J = 9.0, H-5), 7.41 (dd,
1H, J = 2.1, 9.0, H-6), 6.33 (d, 1H, J = 5.4, H-3), 3.43
(m, 3H, ArNCH2 (CH2)5N, NCH), 3.22 (t, 2H,
J = 6.0, ArN(CH2)5CH2N), 2.50–2.46 (m, 2H, CH2-40),
2.41–2.39 (m, 1H, H-30a), 2.00–1.98 (m, 3H, H-30b,
ArCH2CH2 (CH2)4N), 1.70–1.62 (m, 4H, CH-200a, CH-
600a, ArCH2 (CH2)4CH2CH2N), 1.59 (m, 5H, CH-200b,
CH-600b, CCH3), 1.25–0.98 (m, 10H, CH2-300, CH2-400,
CH2-500, ArN(CH2)2CH2CH2 (CH2)2N); dC (75 MHz,
CDCl3) 178.6, 172.0, 151.4, 150.0, 146.7, 135.0, 126.3,
125.4, 122.5, 117.0, 98.9, 67.8, 48.7, 44.3, 40.2, 33.4,
32.3 (2C), 31.5, 29.5, 28.2, 26.7, 26.5, 25.2, 24.7 (2C),
22.8; HRMS (FAB) m/z 484.26050, (M+ C27H37ClN4O2
4.12. 1-(3-(7-Chloroquinolin-4-ylamino)propyl)-N-cyclo-
hexyl-2-methyl-5-oxopyrrolidine-2-carboxamide (19)
requires 484.26024); HPLC purity: 94%, t0 ¼ 2:46 min.
R
Yellow oil (69 mg, 70%); Rf (MeOH/DCM 1:9) 0.20; IR
mmax (CHCl3)/cmꢀ1 3309 (NH), 3010 (ArCH), 2973
(CH), 1718 (C@O), 1649 (C@C), 1556 (C@N); dH
(300 MHz, CDCl3) 8.48 (d, 1H, J = 5.4, H-2), 7.89 (d,
1H, J = 2.0, H-8), 7.81 (d, 1H, J = 9.0, H-5), 7.44 (dd,
1H, J = 2.1, 9.0, H-6), 6.31 (d, 1H, J = 5.4, H-3), 3.58
(m, 3H, ArNCH2 (CH2)2N, H-100), 3.28 (t, 2H, J = 6.0,
ArN(CH2)2CH2N), 2.53–2.48 (m, 2H, CH2-20), 2.39–
2.26 (m, 1H, H-30a), 2.16–1.90 (m, 3H, H-30b,
ArNCH2CH2CH2N), 1.78 (br t, 2H, J = 12.0, CH-200a,
CH-600a), 1.60–1.40 (m, 5H, CH-200b, CH-600b, CCH3),
1.38–0.98 (m, 6H, CH2-300, CH2-400, CH2-500); dC
(75 MHz, CDCl3) 178.5, 172.1, 151.3, 150.0, 146.5,
135.2, 126.5, 125.2, 122.7, 117.0, 99.0, 67.6, 49, 44.2,
40.4, 33.5, 32.8 (2C), 30.3, 29.6, 25.1, 24.5 (2C), 22.8,
HRMS (FAB) m/z 442.21325, (M+ C24H31ClN4O2 re-
quires 442.21355); HPLC purity: 91%, t0R ¼ 2:35 min.
4.15. N-tert-Butyl-1-(2-(7-chloroquinolin-4-ylamino)eth-
yl)-2-methyl-6-oxopiperidine-2-carboxamide (22)
White gum (70 mg, 75%); Rf (MeOH/DCM 1:9) 0.20; IR
mmax (CHCl3)/cmꢀ1 3317 (NH), 3030 (ArCH), 2974
(CH), 1715 (C@O), 1646 (C@C), 1578 (C@N); dH
(300 MHz, CDCl3) 8.44 (d, 1H, J = 5.7, H-2), 8.08 (d,
1H, J = 9.0, H-5), 7.9 (d, 1H, J = 2.1, H-8), 7.40 (dd,
1H, J = 2.1, 9.0, H-6), 6.26 (d, 1H, J = 5.4, H-3), 3.25
(t, 2H, J = 6.0, ArNCH2CH2N), 3.0 (t, 2H, J = 6.0,
ArNCH2CH2N), 2.56–2.40 (m, 2H, CH2-50), 2.20–2.14
(m, 2H, CH2-30), 1.80–1.74 (m, 2H, CH2-40), 1.55 (s,
3H, CCH3), 1.32 (s, 9H, C(CH3)3); dC (75 MHz, CDCl3)
178.7, 172.4, 151.6, 150.4, 147.9, 134.8, 126.3, 125.7,
122.3, 117.4, 98.2, 66.5, 51.4, 42.7, 41.0, 36.1, 34.0,
29.0, 28.6 (3C), 17.4; HRMS (EI) 416.19785 (M+
C22H29ClN4O2 requires 416.19790); HPLC purity:
91%, t0 ¼ 2:12 min.
R
4.13. 1-(4-(7-Chloroquinolin-4-ylamino)butyl)-N-cyclo-
hexyl-2-methyl-5-oxopyrrolidine-2-carboxamide (20)
4.16. N-tert-Butyl-1-(3-(7-chloroquinolin-4-ylamino)pro-
pyl)-2-methyl-6-oxopiperidine-2-carboxamide (23)
White amorphous solid (70 mg, 68%), mp 182–184 ꢁC;
Rf (MeOH/DCM 1:9) 0.21; IR mmax (CHCl3)/cmꢀ1
3331 (NH), 3014 (ArCH), 2984 (CH), 1711 (C@O),
1647 (C@C), 1553 (C@C); dH (300 MHz, CDCl3) 8.46
(d, 1H, J = 5.4, H-2), 7.91 (d, 1H, J = 2.0, H-8), 7.86
(d, 1H, J = 9.0, H-5), 7.41 (dd, 1H, J = 2.1, 9.0, H-6),
6.33 (d, 1H, J = 5.4, H-3), 3.68 (m, 3H,
ArNCH2(CH2)3N, H-100), 3.27 (t, 2H, J = 6.0,
ArN(CH2)3CH2N), 2.51–2.48 (m, 2H, CH2-40), 2.40–
2.38 (m, 1H, H-30a), 2.00–1.98 (m, 3H, H-30b,
ArCH2CH2 (CH2)CH2N), 1.78 (m, 4H, CH-200a CH-
600a, ArCH2 (CH2)2CH2CH2N), 1.61–1.48, (m, 5H,
CH-200b, CH-600b, CCH3), 1.38–0.98 (m, 6H, CH2-300,
CH2-400, CH2-500); dC (75 MHz, CDCl3) 178.8, 172.3,
151.3, 150.2, 146.5, 135.2, 126.8, 125.7, 122.4, 117.2,
98.7, 67.7, 48.6, 44.4, 40.6, 33.5, 32.4 (2C), 30.1, 29.3,
29, 25.3, 24.5 (2C), 22.3; HRMS (FAB) m/z 456.23033,
Cream white paste (69 mg, 71%); Rf (MeOH/DCM 1:9)
0.20; IR mmax (CHCl3)/cmꢀ1 3322 (NH), 3035 (ArCH),
2956 (CH), 1721 (C@O), 1647 (C@C), 1583 (C@N);
dH (300 MHz, CDCl3) 8.38 (d, 1H, J = 5.7, H-2), 8.10
(d, 1H, J = 9.0, H-5), 7.88 (d, 1H, J = 2.1, H-8), 7.42
(dd, 1H, J = 2.1, 9.0, H-6), 6.34 (d, 1H, J = 5.7, H-3),
3.26 (t, 2H, J = 6.0, ArNCH2 (CH2)2N), 3.10 (t, 2H,
J = 6.0, ArN(CH2)2CH2N), 2.57–2.40 (m, 2H, CH2-50),
2.20–2.12 (m, 2H, CH2-30), 1.94–1.89 (m, 2H, CH2-40),
1.83–1.72 (m, 2H, ArNCH2CHCH2N), 1.49 (s, 3H,
CH3), 1.38 (s, 9H, C(CH3)3); dC (75 MHz, CDCl3)
178.8, 172.2, 151.5, 150.2, 147.6, 134.7, 126.4, 125.8,
122.6, 117.2, 98.6, 66.5, 51.7, 42.5, 42.1, 36.3, 34.2,
30.4, 29.2, 28.8 (3C), 17.7; HRMS (EI) 430. 21319
(M+ C23H31ClN4O2 requires 430.21355); HPLC purity:
96%, t0 ¼ 1:81 min.
R

C. C. Musonda et al. / Bioorg. Med. Chem. 14 (2006) 5605–5615
5613
4.17. N-tert-Butyl-1-(4-(7-chloroquinolin-4-ylamino)bu-
tyl)-2-methyl-6-oxopiperidine-2-carboxamide (24)
4.20. 1-(3-(7-Chloroquinolin-4-ylamino)propyl)-N-cyclo-
hexyl-2-methyl-6-oxopiperidine-2-carboxamide (27)
Cream white paste (69 mg, 69%); Rf (MeOH/DCM 1:9)
0.21 IR mmax (CHCl3)/cmꢀ1 3332 (NH), 3027 (ArCH),
2966 (CH), 1715 (C@O), 1636 (C@C), 1582 (C@C); dH
(300 MHz, CDCl3) 8.37 (d, 1H, J = 5.7, H-2), 8.06 (d,
1H, J = 9.0, H-5), 7.81 (d, 1H, J = 2.1, H-8), 7.38 (dd,
1H, J = 2.1, 9.0, H-6), 6.24 (d, 1H, J = 5.7, H-3), 3.22
(t, 2H, J = 6.0, ArNCH2 (CH2)3N), 3.11 (t, 2H,
J = 6.0, ArN(CH2)3CH2N), 2.53–2.41 (m, 2H, CH2-50),
2.22–2.13 (m, 2H, CH2-30), 1.97–1.95 (m, 2H, CH2-40),
1.85–1.74 (m, 4H, ArNCH2CH2CH2N), 1.52 (s, 3H,
CH3), 1.34 (s, 9H, C(CH3)3); dC (75 MHz, CDCl3)
178.5, 172.6, 151.3, 150.5, 147.7, 134.2, 126.4, 125.5,
122.4, 117.2, 98.3, 66.3, 51.5, 42.8, 41.2, 36.3, 34.2,
30.5, 29.1, 28.7, 28.8 (3C), 17.9; HRMS (EI) 444.23007
(M+ C24H33ClN4O2 requires 444.22920); HPLC purity:
Cream white paste (74 mg, 72%); Rf (MeOH/DCM 1:9)
0.20; IR mmax (CHCl3)/cmꢀ1 3327 (NH), 3027 (ArCH),
2988 (CH), 1718 (C@O), 1630 (C@C), 1580 (C@N);
dH (300 MHz, CDCl3) 8.43 (d, 1H, J = 5.4, H-2), 7.85
(d, 1H, J = 2.0, H-8), 7.78 (d, 1H, J = 9.0, H-5), 7.40
(dd, 1H, J = 2.1, 9.0, H-6), 6.30 (d, 1H, J = 5.4, H-3),
3.66 (m, 1H, H-100), 3.30 (t, 2H, J = 6.0, ArNCH2
(CH2)2N), 3.11 (t, 2H, J = 6.0, ArN(CH2)2CH2N),
2.56–2.43 (m, 2H, CH2-50), 2.20–2.12 (m, 2H, CH2-30),
1.99–1.97 (m, 2H, CH2-40), 1.84–1.71 (m, 4H, CH-200a,
CH-600a, CH2-40), 1.67–1.65 (m, 5H, CH-200b, CH-600b,
CCH3), 1.26–1.24 (m, 3H, CH-300a, CH-400a, CH-500a),
1.10–1.00 (m, 3H, CH-300b, CH-400b, CH-500b); dC
(75 MHz, CDCl3) 178.3, 172.2, 151.4, 150.3, 146.8,
135.1, 126.5, 125.6, 122.8, 117.5, 98.4, 66.7, 49.4, 44.8,
43.3, 36.2, 33.0 (2C), 32.4, 30.3, 26.5, 25.2, 24.7 (2C),
18.0; HRMS (EI) 456.22954 (M+ C25H33ClN4O2
87%, t0 ¼ 1:95 min.
R
4.18. N-tert-Butyl-1-(6-(7-chloroquinolin-4-ylamino)hex-
yl)-2-methyl-6-oxopiperidine-2-carboxamide (25)
requires 456.22920); HPLC purity: 94%, t0 ¼ 2:51 min.
R
4.21. 1-(4-(7-Chloroquinolin-4-ylamino)butyl)-N-cyclo-
hexyl-2-methyl-6-oxopiperidine-2-carboxamide (28)
White paste (79 mg, 74%); Rf (MeOH/DCM 1:9) 0.20;
IR mmax (CHCl3)/cmꢀ1 3319 (NH), 3031 (ArCH), 2987
(CH), 1707 (C@O), 1653 (C@C), 1567 (C@N); dH
(300 MHz, CDCl3) 8.35 (d, 1H, J = 5.7, H-2), 8.0 (d,
1H, J = 9.0, H-5), 7.88 (d, 1H, J = 2.1, H-8), 7.41 (dd,
1H, J = 2.1, 9.0, H-6), 6.28 (d, 1H, J = 5.7, H-3), 3.30
(t, 2H, J = 6.0, ArNCH2 (CH2)5N), 3.00 (t, 2H,
J = 6.0, ArN(CH2)5CH2N), 2.56–2.40 (m, 2H, CH2-50),
2.20–2.14 (m, 2H, CH2-30), 2.05–2.01 (m, 2H, CH2-40),
1.90–1.80 (m, 2H, ArNCH2CH2(CH2)4N), 1.82–1.70
(m, 4H, ArN(CH2)2CH2CH2 (CH2)2N), 1.62–1.53 (m,
5H, ArN(CH2)2CH2 (CH2)2N, CCH3), 1.30 (s, 9H,
C(CH3)3); dC (75 MHz, CDCl3) 178.8, 172.6, 151.7,
150.5, 147.8, 134.7, 126.5, 125.8, 122.4, 117.6, 99.0,
66.6, 51.3, 42.8, 41.3, 36.5, 34.3, 31.8, 29.3, 28.8 (3C),
28.1, 26.5, 26.6, 17.7; HRMS (EI) 472.26001 (M+
C26H37ClN4O2 requires 472.26050); HPLC purity:
Off-white gum (78 mg, 74%); Rf (MeOH/DCM 1:9) 0.21;
IR mmax (CHCl3)/cmꢀ1 3322 (NH), 3302 (ArCH), 2974
(CH), 1721 (C@O), 1627 (C@C), 1580 (C@N); dH
(300 MHz, CDCl3) 8.44 (d, 1H, J = 5.4, H-2), 7.90 (d,
1H, J = 2.0, H-8), 7.86 (d, 1H, J = 9.0, H-5), 7.39 (dd,
1H, J = 2.1, 9.0, H-6), 6.28 (d, 1H, J = 5.4, H-3), 3.68
(m, 1H, H-100), 3.33 (t, 2H, J = 6.0, ArNCH2
(CH2)3N), 3.11 (t, 2H, J = 6.0, ArN(CH2)3CH2N),
2.53–2.44 (m, 2H, CH2-50), 2.3–2.13 (m, 2H, CH2-30),
1.80–1.72 (m, 6H, CH-200a, CH-600a, CH2-40,
ArNCH2CH2 (CH2)2N), 1.66–1.63 (m, 7H, CH-200b,
CH-600b, ArN(CH2)2CH2CH2N, CCH3), 1.24–1.22 (m,
3H, CH-300a, CH-400a, CH-500a), 1.0–0.98 (m, 3H, CH-
300b, CH-400b, CH-500b); dC (75 MHz, CDCl3) 178.2,
172.1, 151.2, 150.3, 146.7, 135.3, 126.4, 125.5, 122.9,
117.5, 99.0, 66.3, 49.5, 44.4, 41.3, 36.3, 33.4 (2C), 32.2,
30.3, 27.3, 25.6, 25.2, 24.7 (2C), 18.3; HRMS (EI)
470.24466 (M+ C26H35ClN4O2 requires 470.24485);
92%, t0 ¼ 1:93 min.
R
4.19. 1-(2-(7-Chloroquinolin-4-ylamino)ethyl)-N-cyclo-
hexyl-2-methyl-6-oxopiperidine-2-carboxamide (26)
HPLC purity: 91%, t0 ¼ 2:08 min.
R
Off-white crystals (65 mg, 66%); Rf (MeOH/DCM 1:9)
0.20; IR mmax (CHCl3)/cmꢀ1 3331 (NH), 3015 (ArCH),
2976 (CH), 1722 (C@O), 1617 (C@C), 1559 (C@N);
dH (300 MHz, CDCl3) 8.46 (d, 1H, J = 5.4, H-2), 7.91
(d, 1H, J = 2.0, H-8), 7.81 (d, 1H, J = 9.0, H-5), 7.41
(dd, 1H, J = 2.1, 9.0, H-6), 6.33 (d, 1H, J = 5.4, H-3),
3.68 (m, 1H, H-100), 3.32 (t, 2H, J = 6.0,
ArNCH2CH2N), 3.10 (t, 2H, J = 6.0, ArNCH2CH2N),
2.57–2.42 (m, 2H, CH2-50), 2.20–2.12 (m, 2H, CH2-30),
1.81–1.70 (m, 4H, CH-200a, CH-600a, CH2-40), 1.64–1.60
(m, 5H, CH-200b CH-600b, CCH3), 1.25–1.22 (m, 3H,
CH-300a, CH-400a, CH-500a), 1.22–1.01 (m, 3H, CH-300b,
CH-400b, CH-500b); dC (75 MHz, CDCl3) 178.4, 172.0,
151.3, 150.2, 146.9, 135.2, 126.6, 125.5, 122.7, 117.3,
98.8, 66.4, 49.8, 44.6, 43.9, 36.0, 33 (2C), 32.6, 30.0,
25, 24.8 (2C), 17.8; HRMS (EI) 442.21343 (M+
C24H31ClN4O2 requires 442.21355); HPLC purity:
4.22. 1-(6-(7-Chloroquinolin-4-ylamino)hexyl)-N-cyclo-
hexyl-2-methyl-6-oxopiperidine-2-carboxamide (29)
Off-white hygroscopic solid (81 mg, 72%); Rf (MeOH/
DCM 1:9) 0.22; IR mmax (CHCl3)/cmꢀ1 3319 (NH),
3011 (ArCH), 2984 (CH), 1734 (C@O), 1622 (C@C),
1558 (C@N); dH (300 MHz, CDCl3) 8.44 (d, 1H,
J = 5.4, H-2), 7.93 (d, 1H, J = 2.0, H-8), 7.80 (d, 1H,
J = 9.0, H-5), 7.38 (dd, 1H, J = 2.1, 9.0, H-6), 6.30 (d,
1H, J = 5.4, H-3), 3.60 (m, 1H, H-100), 3.34 (t, 2H,
J = 6.0, ArNCH2 (CH2)5N), 3.12 (t, 2H, J = 6.0,
ArN(CH2)5CH2N), 2.57–2.42 (m, 2H, CH2-50), 2.20–
2.12 (m, 2H, CH2-30), 1.80–1.70 (m, 6H, CH-200a, CH-
600a, CH2-40, ArNCH2CH2 (CH2)4N), 1.65–1.63 (m,
5H, CH-200b, CH-600b, CCH3), 1.40–1.36 (m, 6H,
ArNCH2CH2 (CH2)3CH2N), 1.23–1.18 (m, 3H, CH-
300a, CH-400a, CH-500a), 1.10–0.99 (m, 3H, CH-300b,
CH-400b, CH-500b); dC (75 MHz, CDCl3) 178.1, 172.2,
95%, t0 ¼ 2:14 min.
R

5614
C. C. Musonda et al. / Bioorg. Med. Chem. 14 (2006) 5605–5615
151.1, 150.4, 146.7, 135.3, 126.5, 125.4, 122.6, 117.1,
98.6, 66.5, 49.6, 44.5, 43.7, 36.2, 33.1 (2C), 32.3, 30.6,
30.3, 27.7, 27.4, 26.4, 25.0, 24.5 (2C), 18.1; HRMS
Assays were performed in sterile 96-well microtitre
plates, each well containing 100 ll of parasite culture
(1 · 104 bloodstream forms) with or without serial drug
dilutions at 37 ꢁC for 72 h in 5% CO2. The highest con-
centration for the test compounds was 30 lg/ml. Each
drug was tested in triplicate. A 3-fold serial dilution
was performed down to a suitable concentration to ob-
tain an IC50 value. Initial testing was at 30, 10, 3 and
0.1 lg/ml. The control drug was Pentamidine and was
diluted down to 0.0001 lg/ml (12 dilutions). Control
wells were without drug, blanks were medium only.
After 72 h of incubation, the plates were inspected under
an inverted microscope to assure growth of the controls
and to determine the minimum inhibitory concentration
(MIC): this is the lowest drug concentration at which no
trypanosomes with normal morphology and motility as
compared to the control wells can be seen.
(EI)
498.27656
(M+
C28H39ClN4O2
requires
498.27612); HPLC purity: 93%, t0 ¼ 2:36 min.
R
4.23. In vitro activities of compounds against falcipain-2
IC50 values against the recombinant enzyme (falcipain-
2) were determined as described by Greenbaum and
coworkers.27 Thus, an equal amount of recombinant
protein (ꢁ1 nM) was incubated with different concentra-
tions of inhibitors (added from 100x stock solutions in
DMSO) in 100 mM sodium acetate (pH 5.5)-10 mM
dithiothreitol for 30 min at room temperature before
addition of the substrate benzoxycarbonyl-Leu-
Arg-7-amino-4-methyl-coumarin (final concentration =
25 lM). Fluorescence was continuously monitored for
30 min at room temperature in a Labsystems Fluoro-
scanꢂ Ascent spectrofluorometer. IC50 values were
determined from plots of activity over inhibitor concen-
tration with GraphPad Prismꢂ software. IC50 values for
rhodesain were determined similarly at 3 nM.
Twenty microliters of Alamar BlueTM was added to each
well and the plates incubated for another 2–4 h. Then
the plates were read on a Gemini Plate Reader (Molec-
ular Devices) using an excitation wavelength of 530 nm
and an emission wavelength of 580 nm (cut off
550 nm).28
4.24. In vitro activities of compounds against
W2 P. falciparum
4.27. Primary screen
W2-strain P. falciparum parasites (1% parasitaemia, 2%
hematocrit) were cultured in 0.5 ml of medium in
48-well culture dishes. Appropriate inhibitors from
10 mM stocks in DMSO were added to cultured para-
sites to a final concentration of 20 lM. From 48-well
plates, 125 lM of culture was transferred to two 96-well
plates (duplicates). Serial dilutions (1%) of inhibitors
were made to final concentrations of 10 lM, 2 lM,
0.4 lM, 80 nM, 16 nM and 3.2 nM. Cultures were main-
tained at 37 ꢁC for 2 days after which the parasites were
washed and fixed with 1% formaldehyde in PBS. After
two days, parasitaemia was measured by flow cytometry
using the DNA stain YOYO-1 as a marker for cell
survival.27
The preliminary screen uses the T. b. brucei strain. The
compounds were tested at 7 concentrations (drug con-
centration ranges from 30 lg/ml to 0.1 lg/ml in 3-fold
dilutions). In this assay pentamidine had an ED50 value
of 0.1–0.02 ng/ml.
4.28. Secondary screen
Active compounds (IC50 <0.2 lg/ml) were tested again
over the appropriate dose range against T. b. rhodesiense
STIB 900 to confirm IC50 and IC90 values in comparison
to standard pentamidine and internal chemical group
standard if available.
4.29. Toxicity tests
4.25. In vitro activities against T. b. brucei S427
4.29.1. Cell cultures. KB cells—a cell line derived from a
human carcinoma of the nasopharynx, typically used as
an assay for antineoplastic agents. KB cells were main-
tained as monolayers in RPMI 1640 + 10% HIFCS.
All cultures and assays were conducted at 37 ꢁC under
an atmosphere of 5% CO2/95% air mixture.
The bloodstream form trypomastigotes were maintained
in MEM with Earle’s salts supplemented with 25 mM
HEPES, 1 g/l additional glucose, 10 ml/l MEM non-es-
sential aminoacids (100·), 0.2 mM 2-mercaptoethanol,
2 mM Na-pyruvate, 0.1 mM hypoxanthine, 0.05 mM
bathocuprionedisulfonic acid, 0.15 mM L-cysteine and
15% heat-inactivated, foetal calf serum.
4.29.2. Drug toxicity assays. Stock drug solutions were
prepared in 100% DMSO unless otherwise suggested
by the supplier at 20 mg/ml, and ball milled or sonicated
if necessary. The stocks were kept at 4 ꢁC. For the as-
says, the compound was further diluted to the appropri-
ate concentration using complete medium.
All cultures and assays were conducted at 37 ꢁC under
an atmosphere of 5% CO2/95% air mixture.
4.26. Drug sensitivity assays
Stock drug solutions were prepared in 100% DMSO un-
less otherwise suggested by the supplier at 20 mg/ml, and
ball milled or sonicated if necessary. The stocks were
kept at 4 ꢁC. For the assays, the compound was further
diluted to the appropriate concentration using complete
medium.
Acknowledgments
This work was supported by the South African National
Research Foundation under Grant No. 2053362 (K.C.)
and by the National Institutes of Health (AI35707,

C. C. Musonda et al. / Bioorg. Med. Chem. 14 (2006) 5605–5615
5615
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P.J.R.). P.J.R. is a Doris Duke Distinguished Clinical
Scientist. The investigation also received financial sup-
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gramme for Research and Training in Tropical
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