Parallel synthesis of 9-aminoacridines and their evaluation against chloroquine-resistant Plasmodium falciparum

Article abstract of DOI:10.1016/j.bmc.2005.08.017

A parallel synthetic strategy to the 9-aminoacridine scaffold of the classical anti-malarial drug quinacrine (2) is presented. The method features a new route to 9-chloroacridines that utilizes triflates of salicylic acid derivatives, which are commercially available in a variety of substitution patterns. The route allows ready variation of the two diversity elements present in this class of molecules: the tricyclic aromatic heterocyclic core, and the disubstituted diamine sidechain. In this study, a library of 175 compounds was designed, although only 93 of the final products had purities acceptable for screening. Impurity was generally due to incomplete removal of 9-acridones (18), a degradation product of the 9-chloroacridine synthetic intermediates. The library was screened against two strains of Plasmodium falciparum, including a model of the drug-resistant parasite, and six novel compounds were found to have IC₅₀ values in the low nanomolar range.

Full text of DOI:10.1016/j.bmc.2005.08.017

Bioorganic & Medicinal Chemistry 14 (2006) 334–343
Parallel synthesis of 9-aminoacridines and their evaluation
against chloroquine-resistant Plasmodium falciparum
Marc O. Anderson,a John Sherrill,a Peter B. Madrid,a Ally P. Liou,b
Jennifer L. Weisman,b,c Joseph L. DeRisib and R. Kiplin Guya,c,
*
aDepartment of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-2280, USA
bDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-2280, USA
cDepartment of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143-2280, USA
Received 1 July 2005; revised 4 August 2005; accepted 5 August 2005
Available online 10 October 2005
Abstract—A parallel synthetic strategy to the 9-aminoacridine scaffold of the classical anti-malarial drug quinacrine (2) is presented.
The method features a new route to 9-chloroacridines that utilizes triflates of salicylic acid derivatives, which are commercially avail-
able in a variety of substitution patterns. The route allows ready variation of the two diversity elements present in this class of mol-
ecules: the tricyclic aromatic heterocyclic core, and the disubstituted diamine sidechain. In this study, a library of 175 compounds
was designed, although only 93 of the final products had purities acceptable for screening. Impurity was generally due to incomplete
removal of 9-acridones (18), a degradation product of the 9-chloroacridine synthetic intermediates. The library was screened against
two strains of Plasmodium falciparum, including a model of the drug-resistant parasite, and six novel compounds were found to have
IC50 values in the low nanomolar range.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Malaria is a devastating infectious disease caused by the
protozoan parasite Plasmodium.1 Four species of Plas-
modium are known to infect humans: P. vivax, P. mala-
riae, P. ovale, and P. falciparum, the latter being the
most deadly. While the controversial use of polychlori-
nated biphenyl pesticides (e.g., DDT) has generally led
to the eradication of malaria in North America and
most European countries,2 the disease is still widespread
in Africa, Central and South America, and Southeast
Asia. Malaria continues to affect 200–500 million people
and cause 1–2 million fatalities annually.3 As a result,
malaria has produced devastating social and economic
burdens on the countries most afflicted by it.1
The development of anti-malarial compounds continues
to be a major focus of many laboratories,4 and as such, a
Figure 1. Structures of common quinoline and acridine anti-malarial
variety of compounds have been explored in the prophy-
compounds.
laxis and treatment of this disease (Fig. 1). The efficacy
of quinine (1), a derivative of cinchona-bark, as an
anti-malarial compound was recognized as early as the
1600 s. The earliest synthetic anti-malarial agent was
quinacrine (2). Quinacrine was supplemented by chloro-
quine (3),5 along with various other 4- and 8-substituted
quinolines, including mefloquine (4).6 Chloroquine was
Keywords: Malaria; Acridine; Parallel synthesis.
*
Corresponding author. Tel.: +1 415 502 7051; fax: +1 415 514
0689; e-mail: rguy@cgl.ucsf.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.017

M. O. Anderson et al. / Bioorg. Med. Chem. 14 (2006) 334–343
335
Figure 2. A parallel synthetic strategy to generate libraries of 9-aminoacridines.
found to be highly effective against all four human-in-
fecting species of Plasmodium, and its inexpensive
production led it to quickly become a standard anti-ma-
larial treatment.5 Unfortunately, over several decades
this resulted in the development of chloroquine-resistant
strains of Plasmodium, particularly of P. falciparum.7,8
Certain substituted quinolines, including mefloquine
(4), have been efficacious against chloroquine-resistant
P. falciparum (CRPF), but this drug is also associated
with a variety of neuropsychiatric side-effects.9 Another
promising class of compounds with activity against
CRPF are the artemisinins.10–12 The continuing explora-
tion of compounds active against CRPF is an active pur-
suit of our laboratory.13,14
was done to generate the desired 9-aminoacridine chem-
set 10. A notable feature of this strategy is that it allows
quick access to variations of the tricyclic acridine hetero-
cycle as well as the amine sidechain, which will allow
probing of the cooperativity of these two structural
features.
2. Results
2.1. Chemistry
The synthesis of the 9-chloroacridine heterocycles is
illustrated in Scheme 1. The salicylic acid chemset (5)
was methylated and then activated for cross-coupling
as the corresponding aryl triflate chemset (11), generally
in near quantitative yield giving compounds of high
purity. These species were coupled to substituted aniline
chemset 6 to generate diarylamine chemset 12, using
conditions developed by Buchwald et al.,31 which pro-
ceeded smoothly. Next, hydrolysis of the methyl ester
groups was performed using non-aqueous conditions de-
signed for parallel synthesis,27 followed by Friedel–
Crafts cyclization with POCl3 to generate the 9-chloro-
acridine chemset (7). The transformation of 11 to 7
was accomplished in a parallel 3-step, one-pot proce-
dure, without intermediate workup, using a 12-place
reaction carrousel (Radleys Discovery Technologies).
The 9-chloroacridine products (7) were isolated in
In the context of exploring the 9-aminoacridine scaffold of
quinacrine (3) to target the propagation of prions,15–17 we
became interested in re-evaluating this class of molecules
for anti-malarial activity, particularly against CRPF.
Herein we report a parallel synthetic strategy to generate
libraries of 9-aminoacridines based on the general struc-
ture of quinacrine, along with in vitro cell-based screening
results against drug-sensitive and drug-resistant strains of
P. falciparum. In addition to having anti-prion and anti-
malarial properties, 9-aminoacridines are interesting for
other types of biological activity. For example, 9-amino-
acridines are known DNA intercalators,18 inhibitors of
mammalian topoisomerase I19 and acetylcholinester-
ase,20 and these compounds are also active against
African trypanosomes.21–23 Cancer chemotherapeutics
based on the 9-aminoacridine scaffold have been devel-
oped24 (e.g., Ledakrin and Amascrine). Additionally,
9-aminoacridines have been explored as photoaffinity
labels25 and as fluorescent probes used to detect cancer
cells.26
Our general synthetic strategy to 9-aminoacridines is
summarized in Figure 2. Derivatives of substituted sali-
cylic acid chemset 5 were coupled to aniline chemset 6 to
generate 9-chloroacridine chemset 7, as was previously
introduced.27 The use of salicylic acid precursors, acti-
vated as triflates, is novel and deviates from an earlier
approach to 9-chloroacridines based on the Ulmann
coupling of anilines with 2-bromoaryl carboxylic
acids,28–30 of which much fewer are commercially avail-
able. To approach the amine sidechain, 1,3-diaminopro-
pane (8) was dually functionalized to generate a library
of requisite diamine building block chemset 9. Finally,
parallel coupling of 9-chloroacridines with the diamines
Scheme 1. Synthesis of 9-chloroacridines. Reagents and conditions: (a)
MeI (1.05 equiv), Cs2CO3 (0.5 equiv) DMF (90–100% yield); (b) Tf2O
(1.1 equiv), Et3N (2.0 equiv), CH2Cl2, ꢀ78 ꢁC to rt (95–100% yield); (c)
substituted anilines 6 (1.05 equiv), Pd(OAc)2 (0.05 equiv), BINAP
(0.08 equiv), Cs2CO3 (1.4 equiv), toluene; (d) Ba(OH)2–8H2O (2.5
equiv), CH3OH, 80 ꢁC overnight; (e) POCl3 (neat) 120 ꢁC, 1 h (5–50%
yield).

336
M. O. Anderson et al. / Bioorg. Med. Chem. 14 (2006) 334–343
Scheme 2. Synthesis of diamines. Reagents and conditions: (a) 2-nitrobenzenesulfonyl chloride (0.1 equiv), CH2Cl2, ꢁC to rt, overnight (70 g scale,
85% yield); (b) Carboxylic anhydrides (14) (1.2 equiv), pyridine (1.1 equiv), THF (90–100% yields); (c) BH3dimethylsulfide complex (4.0 equiv), THF,
60 ꢁC, 30 min (67–77% yields); (d) Aldehydes 16 (1.5 equiv), sodium triacetoxyborohydride (1.5 equiv), THF, sonicated, 35 ꢁC, 1 h; (e) Benzenethiol
(5 equiv), Cs2CO3 (2.5 equiv), degassed CH3CN under argon.
acceptable yield and >95% purity after automated flash
column purification using a CombiFlash system (Isco).
The synthesis of the diamine building blocks is illus-
trated in Scheme 2. 1,3-diaminopropane (8) was
mono-sulfonylated under dilute conditions to attach
the thiolate-labile 2-nitrobenzenesulfonyl (nosyl = Ns)
protecting group32,33 to generate 13 in high yield and
purity. Amidation with carboxylic anhydride reagents
(14), followed by reduction with borane–dimethylsul-
fide complex,34 generated the corresponding secondary
amine chemset 15 in good yield and excellent purity.
An alternate acylation reaction using acid chloride re-
agents formed a byproduct with an additional acyl
group, apparently attached to the sulfonamide nitrogen
atom. Lastly, reductive amination with aldehyde chem-
set 16 and sodium triacetoxyborohydride,35 followed
Scheme 3. Coupling of 9-chloroacridines with diamines. Reagents and
˚
conditions: (a) phenol (15 equiv), Cs2CO3 (1 equiv), 3A molecular
by deprotection of nosyl groups by treatment with cesi-
um benzenethiolate,32,33 afforded the free amine chem-
sieves, DMSO, 100 ꢁC, 2 h; (b) Diamines (9) (4 equiv), DMSO, 100 ꢁC,
set 9. Steps a–c were carried out in large scale (>60 g
4 h; then methyl isocyanate polystyrene resin (NovaBiochem, 8 equiv),
initially) using traditional glassware, while steps d
overnight, rt.
and e (1.5 g/each) were done in parallel using the Rad-
leys carrousel apparatus. The final two heterogeneous
˚
by the addition of 3 A molecular sieves during the cou-
pling, although decomposition also occurred during
ambient storage of the precursor chloroacridines.
reactions proceeded slowly in the 12-place Radleys car-
rousel, presumably due to poor mixing; magnetic stir-
ring using the 6-place carrousel alleviated this
problem. The reductive amination reaction (step d)
could alternatively be performed using the smaller reac-
tion vessels of the 12-place carrousel, but agitated in a
sonicating wash bath rather than by magnetic stirring.
The products of the last two steps were isolated in high
purity, either by aqueous workup, or in parallel using
solid-phase extraction (SPE) with Dowex 50WX2-400
ion-exchange resin. Chromatographic purification was
not required in any of the steps of the sequence.
The coupling reactions are carried out in 48-position par-
allel synthesis blocks (Bohdan) and agitated with the Boh-
dan MiniBlock High Capacity Shaking and Washing
Station (600 RPM, 100 ꢁC). After coupling was achieved,
the remaining diamine was then scavenged by treatment
with electrophilic methyl isocyanate resin. Subsequent
workup was performed by SPE with Dowex 50WX2-
400 ion-exchange resin. A three-step parallel SPE proto-
col was devised to purify the 9-aminoacridine products:
first, the crude product was loaded on the column and
washed with TFA (5% in MeOH) to elute DMSO and
phenol; next, the resin was washed with pyridine (5% in
MeOH) to elute unreacted 9-phenoxyacridines (17) and
9-acridones (18); finally, the desired 9-aminoacridine
product chemset (10) was eluted using Et3N (5% in
MeOH).
The coupling of 9-chloroacridines with diamines to gen-
erate the desired 9-aminoacridine products is illustrated
in Scheme 3. 9-Chloroacridine chemset 7 was converted
to the reactive intermediate 9-phenoxyacridine chemset
17 by treatment with phenol in DMSO. Subsequent
reaction with the diamine chemset 9 afforded the
desired 9-aminoacridine products 10. Direct treatment
of 9-chloroacridines with diamines, that is, omitting
the activation step, afforded little or no product.
Furthermore, in some of the coupling reactions, the
formation of 9-acridone byproducts (chemset 18) was
observed, presumably due to hydrolysis of 9-chloroacri-
dines.36 This side reaction could be partially suppressed
2.2. Synthesis of 9-aminoacridine and diamine
components
A selection of 15 diversely substituted 9-chloroacridine
components and 16 diamine components was defined.

M. O. Anderson et al. / Bioorg. Med. Chem. 14 (2006) 334–343
337
Scheme 4. Composition of 9-chloroacridine (7) chemset.
The precursor components of the 9-chloroacridine
library are defined in Scheme 4. Various substituent
patterns were chosen to test the scope of the chemis-
try, specifically electronic effects in the Buchwald cou-
pling and cyclization steps. The synthesis results of the
9-chloroacridine component are outlined in Table 1.
The purified yields after the three-step sequence ran-
ged from moderate to fairly low. A degree of degrada-
tion was observed in nearly all of the 9-
chloroacridines synthesized, namely hydrolysis to form
9-acridones (18).
2.3. Proof-of-concept library
The coupling of 9-chloroacridines with disubstituted
diamines to generate a proof-of-concept library of 9-
aminoacridines (10) is outlined in Table 3. In the first set
of entries (1–15), the synthesized 9-chloroacridines were
coupled with commercially available sidechain 9{1,9},
whilein the secondsetofentries (16–31), the commercially
available 9-chloroacridine heterocycle 7{4,6} of quina-
crine was coupled with the set of synthesized diamines.
Yields and purities of the coupled products were highly
variable. Notable degradation of the synthesized 9-chlo-
roacridine starting materials to 9-acridones (18) accounts
for the overall lower yields in the first set of entries. Impu-
rity was generally accounted for by the presence of 9-acri-
dones not effectively separated during the SPE
purification. We suspect that the second set of entries
was lesspronetoimpurity problemsduetothe shelf stabil-
ity of the commercially available quinacrine 9-chloroacri-
dine 7{4,6} starting material. Products where final purity
was less than 50% by RP-HPLC analysis were indicated as
failed entries and constituted 20% of this library.
The design of the disubstituted diamine chemset (9) is
outlined in Figure 4. All amines in this library were
based on a 1,3-diaminopropane linker. Two carboxylic
anhydrides (acetic and propionic) were used to vary
R3, while the majority of variation was achieved with
a selection of aldehydes to vary R4. The results of the
synthesis of the diamines are outlined in Table 2. Of
the products, the volatile low molecular weight dial-
kyl-substituted diamines (entries 1–2, 9–10) were isolat-
ed as trifluoroacetate salts. Overall, yields were
moderate to very good in the reductive amination and
nosyl deprotection reactions. Each of the diamine com-
pounds was produced in very high purity, as assessed by
NMR.
2.4. Cross-coupled library
Having established our parallel synthesis methodology
in a proof-of-concept library, we then prepared a
cross-coupled library of synthesized 9-chloroacridines
with synthesized diamines. The proof-of-concept library
showed some classes of 9-chloroacridines achieving bet-
ter yields and purities than others during the coupling
reaction, with some being particularly more prone to
hydrolysis to the 9-acridone, possibly due to electron-
withdrawing substitutuents increasing electrophilicity
at C9. Thus, we designed a library utilizing a subset of
9-chloroacridines (7), specifically compounds 7{1,1},
7{1,2}, 7{1,4}, 7{2,1}, 7{2,2}, 7{2,4}, 7{4,1}, 7{4,2},
and 7{1,5}, that appeared be the most promising and
likely to generate compounds acceptable for screening.
These species were cross-coupled with the entire series
of synthesized amines (9), to generate a library of 144
compounds (Table S1, Supporting information). Using
the same criteria established for the proof of concept li-
brary, 53% of the compounds were marked as failed en-
tries, due to final purities being less than 50% after the
initial SPE purification. While the number of failed cou-
plings is significant, it is notable that the relative success
Table 1. 9-chloroacridine (7) chemset synthesis results
Entry
Salicylic acid
Aniline
Product
Yielda (%)
1
2
5{1}
5{1}
5{1}
5{1}
5{2}
5{2}
5{2}
5{2}
5{3}
5{3}
5{3}
5{4}
5{4}
5{4}
5{1}
6{1}
6{2}
6{3}
6{4}
6{1}
6{2}
6{3}
6{4}
6{1}
6{2}
6{3}
6{1}
6{2}
6{4}
6{5}
7{1,1}
7{1,2}
7{1,3}
7{1,4}
7{2,1}
7{2,2}
7{2,3}
7{2,4}
7{3,1}
7{3,2}
7{3,3}
7{4,1}
7{4,2}
7{4,4}
7{1,5}
48
26
21
44
42
37
6
3
4
5
6
7
8
31
25
30
31
18
40
23
49
9
10
11
12
13
14
15
a Purified yield of the final three-step one-pot sequence.

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M. O. Anderson et al. / Bioorg. Med. Chem. 14 (2006) 334–343
Table 2. Disubstituted diamine chemset (9) synthesis results
Entry
Anhydride
Aldehyde
Product
Yield: reductive amination (%)
Yield: nosyl deprotection (%)
1
2
14{1}
14{1}
14{1}
14{1}
14{1}
14{1}
14{1}
14{1}
14{2}
14{2}
14{2}
14{2}
14{2}
14{2}
14{2}
14{2}
16{1}
16{2}
16{3}
16{4}
16{5}
16{6}
16{7}
16{8}
16{1}
16{2}
16{3}
16{4}
16{5}
16{6}
16{7}
16{8}
9{1,1} Æ 2TFA
9{1,2} Æ 2TFA
9{1,3}
46
82
84
71
70
87
71
58
75
83
73
79
69
66
63
42
81
85
91
87
91
93
33
94
89
92
100
97
74
84
73
79
3
4
9{1,4}
9{1,5}
5
6
9{1,6}
9{1,7}
9{1,8}
7
8
9
9{2,1} Æ 2TFA
9{2,2} Æ 2TFA
9{2,3}
10
11
12
13
14
15
16
9{2,4}
9{2,5}
9{2,6}
9{2,7}
9{2,8}
Table 3. Proof library: coupling reactions to generate a library of 9-aminoacridines (10)
Entry
9-Chloroacridine component
Diamine component
Product
Yield (%) (purity, %)a
1
2
7{1,1}
7{1,2}
7{1,3}
7{1,4}
7{2,1}
7{2,2}
7{2,3}
7{2,4}
7{3,1}
7{3,2}
7{3,3}
7{4,1}
7{4,2}
7{4,4}
7{1,5}
7{4,6}
7{4,6}
7{4,6}
7{4,6}
7{4,6}
7{4,6}
7{4,6}
7{4,6}
7{4,6}
7{4,6}
7{4,6}
7{4,6}
7{4,6}
7{4,6}
7{4,6}
7{4,6}
9{1,9}
9{1,9}
10{1,1,1,9} Æ 2HCl
10{1,2,1,9} Æ 2HCl
10{1,3,1,9} Æ 2HCl
10{1,4,1,9} Æ 2HCl
10{2,1,1,9} Æ 2HCl
10{2,2,1,9} Æ 2HCl
10{2,3,1,9} Æ 2HCl
10{2,4,1,9} Æ 2HCl
10{3,1,1,9} Æ 2HCl
10{3,2,1,9} Æ 2HCl
10{3,3,1,9} Æ 2HCl
10{4,1,1,9} Æ 2HCl
10{4,2,1,9} Æ 2HCl
10{4,4,1,9} Æ 2HCl
10{1,5,1,9} Æ 2HCl
10{4,6,1,1} Æ 2HCl
10{4,6,1,2} Æ 2HCl
10{4,6,1,3} Æ 2HCl
10{4,6,1,4} Æ 2HCl
10{4,6,1,5} Æ 2HCl
10{4,6,1,6} Æ 2HCl
10{4,6,1,7} Æ 2HCl
10{4,6,1,8} Æ 2HCl
10{4,6,2,1} Æ 2HCl
10{4,6,2,2} Æ 2HCl
10{4,6,2,3} Æ 2HCl
10{4,6,2,4} Æ 2HCl
10{4,6,2,5} Æ 2HCl
10{4,6,2,6} Æ 2HCl
10{4,6,2,7} Æ 2HCl
10{4,6,2,8} Æ 2HCl
52 (100)
16 (83)
Failed
3
9{1,9}
9{1,9}
4
19 (100)
22 (95)
4 (81)
5
9{1,9}
9{1,9}
6
7
9{1,9}
9{1,9}
Failed
21 (92)
8 (56)
8
9
9{1,9}
9{1,9}
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
15 (50)
Failed
9{1,9}
9{1,9}
22 (81)
22 (73)
Failed
9{1,9}
9{1,9}
9{1,9}
10 (73)
32 (81)
18 (100)
48 (100)
34 (100)
52 (100)
32 (95)
Failed
9{1,1} Æ 2TFA
9{1,2} Æ 2TFA
9{1,3}
9{1,4}
9{1,5}
9{1,6}
9{1,7}
9{1,8}
19 (68)
34 (100)
60 (100)
69 (100)
62 (100)
73 (100)
47 (90)
Failed
9{2,1} Æ 2TFA
9{2,2} Æ 2TFA
9{2,3}
9{2,4}
9{2,5}
9{2,6}
9{2,7}
9{2,8}
51 (87)
a Purity determined by RP-HPLC-MS integration detected at 254 nm.
of certain 9-chloroacridines in the proof-of-concept li-
brary translated quite well to success in the cross-cou-
pled library. Specifically, certain 9-chloroacridines (7)
that worked relatively well in the proof library achieved
a reasonable degree of yield and purity in the cross-cou-
pled library: 7{1,1}, 7{2,1}, 7{2,4}, 7{4,1}, and 7{4,2}.

M. O. Anderson et al. / Bioorg. Med. Chem. 14 (2006) 334–343
339
2.5. Screening and biological evaluation
which are established models of chloroquine-sensitive
and chloroquine, mefloquine-resistant P. falciparum,
respectively. Initially, activity was screened at a fixed
concentration of 30 nM (Figure 3), with data represent-
ed as parasite growth relative to untreated controls.
Chloroquine (3) serves as a positive control for the
3D7 strain and a negative control for the W2 strain.
Quinacrine (2) also serves as a positive control for
The compounds of the proof-of-concept and cross-cou-
pled libraries were screened for in vitro anti-malarial
activity against P. falciparum cultured in human eryth-
rocytes. Growth inhibition of the parasite was measured
using a fluorescent-active cell sorting (FACS) assay.13,14
Two strains of the parasite were examined: 3D7 and W2,
Figure 3. Relative activity of the library defined in Table 3 against P. falciparum strains 3D7 and W2 cultured in human erythrocytes (measured at
fixed concentration: 30 nM). Data are represented in blue scale, as percent parasite growth relative to untreated controls, with darker squares
indicating a higher activity against the parasites. Omitted entries are due to failure during synthesis.
Figure 4. Composition of disubstituted diamine (9) chemset.

340
M. O. Anderson et al. / Bioorg. Med. Chem. 14 (2006) 334–343
3D7, while this compound also has partial activity
against the W2 strain. In this assay, several compounds
of the proof-of-concept library were found to be highly
active against both strains of the parasite, with notable
decrease in parasitic growth. The 9-aminoacridines in
the proof-of-concept library that appeared the most
interesting, particularly in inhibiting the growth of the
chloroquine-resistant W2 strain, were 10{1,4,1,9},
10{4,6,1,2}, 10{4,6,1,4}, 10{4,6,2,1}, 10{4,6,2,2}, and
10{4,6,2,4}. Of the products derived from novel 9-chlo-
roacridines, only 10{1,4,1,9}, derived from 7{1,4}, ap-
peared to have significant activity at 30 nM. Notably,
use of the commercially available acridine heterocycle
scaffold 7{4,6} with variation of the amine (Table 3, en-
tries 16–31) appears to be a promising strategy for opti-
mizing 9-aminoacridines for anti-malarial activity.
Unfortunately, fewer compounds in the cross-coupled li-
brary appear as interesting, as many entries in this li-
brary failed in synthesis and thus were not screened.
Several compounds were active against the 3D7 strain,
but only one, 10{1,1,1,8}, was found to have promising
activity against the chloroquine-resistant W2 strain.
potent, having sub-nanomolar EC50 values. The slightly
less potent compounds can be grouped by their amine
substitution patterns: 10{4,6,1,2} and 10{4,6,2,2} (both
containing an isobutyl substituent), as well as
10{4,6,1,4} and 10{4,6,2,4} (both containing a furylm-
ethyl substituent). The tolerance for fairly different styles
of amine substituents, for example, branched alkyl
groups and a furyl heterocyclic group, is an interesting
feature of the pharmacophore. EC50 values were also
determined for quinacrine (2) and chloroquine (3).
Notably all of the synthesized compounds had improved
potencies, when compared to 2 and 3, against both
strains of P. falciparum, and none of the compounds
have previously been described in the literature.
3. Conclusion
A parallel synthetic strategy to generate 9-aminoacri-
dines is described. The method features a new route to
9-chloroacridines that utilizes triflates of salicylic acid
derivatives, which are commercially available in fairly
diverse substitution patterns. Additionally, a procedure
for the synthesis of disubstituted diaminoalkanes was
presented. Two libraries were designed totaling 175
compounds, although only 93 of the final products
had purities suitable for in vitro screening. Low
purity was generally attributed to contamination by
9-acridones (18), a hydrolytic degradation product of
Structures and 50% effective inhibitory concentration
(EC50) values of the most promising 9-aminoacridines
10{1,4,1,9}, 10{4,6,1,2}, 10{4,6,1,4}, 10{4,6,2,1},
10{4,6,2,2}, and 10{4,6,2,4} from the initial screen are
presented in Figure 5. Of this set, compounds
10{1,4,1,9} and 10{4,6,2,1} appear to be the most
Figure 5. Structures and EC50 values of the most active compounds against P. falciparum strains 3D7 and W2 in cultured human erythrocytes, as
well as for quinacrine (2) and chloroquine (3). Activity at a series of 12 concentrations was determined as percent parasite growth relative to
untreated controls, EC50 values were determined with SigmaPlot.

M. O. Anderson et al. / Bioorg. Med. Chem. 14 (2006) 334–343
341
the 9-chloroacridine synthetic intermediates. The com-
pounds were screened initially at fixed concentration
(30 nM), with the most active hits re-analyzed to deter-
mine dose–response. Six previously undescribed com-
pounds were found to have nanomolar EC50 values and
were particularly interesting due to improved potency
against the W2 parasite strain, relative to quinacrine (2)
and chloroquine (3). Of this set, most of them were de-
rived from the commercially available acridine heterocy-
cle 7{4,6} with novel substituents on the amine
sidechain, suggesting that further modification of this
component may be a viable strategy for future
optimizations.
4.3. General procedure for the synthesis of salicyl triflate
chemset (11)
A solution of substituted salicylic acid 5 (11.7 mmol) in
DMF (100 ml) was treated with Cs2CO3 (0.5 equiv) and
iodomethane (1.05 equiv). The solution was stirred for
1.5 h, taken up in Et2O/EtOAc (2:1) (100 ml), and
washed with H2O (50 ml), NaHCO3 (10% aq, 50 ml),
and NaCl (satd aq) (25 ml). The organic product layer
was dried over MgSO4 and concentrated in vacuo to
yield methyl esters in excellent purity (80–100% yield).
The methyl esters (5.5 mmol) were dissolved in CH2Cl2
(50 ml), cooled to ꢀ78 ꢁC, and stirred under argon.
Treatment with Et3N (2 equiv) was followed by the
dropwise addition of trifluoromethanesulfonic anhy-
dride (1.1 equiv). The mixture was stirred at ꢀ78 ꢁC
for 30 min and then warmed to RT, whereupon TLC
indicated quantitative conversion. The solution was tak-
en up in Et2O (100 ml) and washed with HCl (1 M aq)
(25 ml). The aqueous layer was back-extracted with
additional Et2O (25 ml), and the combined organic
product layer was washed with NaCl (satd aq) (25 ml),
dried over MgSO4, and concentrated in vacuo to yield
salicyl triflate chemset (11) in excellent purity (95–
100% yield).
4. Experimental
4.1. General
NMR spectra were recorded on a Varian Model AS 400-
MHz machine. The following abbreviations are used to
describe peak splitting when appropriate: s, singlet, d,
doublet, t, triplet, q, quartet, br s, broad singlet, and
mult, multiplet. Reactions were carried out under an
atmosphere of argon. Reagents were of commercial
quality unless otherwise indicated. Analytical thin layer
chromatography (TLC) was carried out using plastic
plates coated with silica gel 60 F254 (Whatman Part
#4420 222). Developed plates were visualized using
short wave UV light (254 nm) and were typically stained
in an iodine/silica chamber and/or using a staining dip
(e.g., p-anisaldehyde or ceric ammonium molybdate)
followed by heating with a heat gun.
4.4. General procedure for the parallel synthesis of the
diarylamine chemset (12)
Following the general procedure of Buchwald,31 each of
the vessels of a Radleys 12-place carrousel was loaded
with
a
solution of salicyl triflate chemset 11
(0.728 mmol) in toluene (7 ml). The vessels were purged
under argon, and each one of them was then treated
with anilines (6) (1.2 equiv), Cs2CO3 (1.4 equiv), rac-
2,20-bis(diphenylphosphino)-1,10-binaphthalene (BIN-
AP) (0.08 equiv), followed by Pd(OAc)2 (0.05 equiv).
The carrousel was heated to 120 ꢁC for 16 h, after which
TLC of reaction mixtures generally indicated complete
conversion to the diarylamine products (12). The prod-
ucts were concentrated in vacuo using a Genevac HT-
4 parallel evaporation system and then used directly in
the next step without purification or workup.
4.2. Measurement of in vitro anti-malarial activity
Growth inhibition of P. falciparum cultured in human
erythrocytes was measured with flow cytometry.37 Syn-
chronized cultures of ring-stage parasites (0.8% parasite-
mia, 0.5% hematocrit) were grown in 96-well tissue
culture plates (Falcon) in the presence of compounds
for screening, at fixed concentration of 30 nM or serially
diluted for dose–response. Cultures were grown in atmo-
spherically regulated (6% CO2, 5% O2) incubators (Sa-
nyo) at 37 ꢁC for 72 h. Following drug incubation,
cultures were fixed with 1% paraformaldehyde for 1 h at
RT and subsequently stained with 50 nM YOYO-1
(Molecular Probes) in 1· PBS for 18 h at RT in the dark.
Fluorescence of each sample was then obtained on a Bec-
ton–Dickenson LSR2 flow cytometer. YOYO-1 is a DNA
intercalator that allows for distinction between parasit-
ized and unparasitized red blood cells (RBCs), as RBCs
lack DNA. Percent of parasitized RBCs was determined
from fluorescent signal due to YOYO-1, and growth inhi-
bition values were then calculated as the fraction of para-
sitized RBCs relative to cultures without drug. All
screening was done in triplicate. Compounds were
screened initially at a fixed concentration (30 nM). The
most promising compounds were then evaluated for
dose–response at 12 concentrations: 2000, 1000, 500,
250, 125, 62.5, 31.3, 15.6, 15.6, 7.8, 3.9, 2.0, and 0.9 nM.
Fifty percent effective inhibitory concentrations (EC50
values) were determined using SigmaPlot.
4.5. General procedure for the parallel synthesis of the
9-chloroacridine chemset (7)
The crude diarylamines (12) (ca. 0.728 mmol), loaded
into the vessels of a Radleys 12-place carrousel, were
redissolved in methanol (7 ml) and treated with barium
hydroxide octahydrate (1.5 equiv). The carrousel was
heated to 90 ꢁC overnight, after which TLC showed
complete ester hydrolysis. The crude carboxylic acid
species were concentrated in vacuo using a Genevac Par-
allel Evaporation System and used directly in the next
step without purification or workup. To effect cycliza-
tion, the species were treated with POCl3 (7 ml) and
heated to 120 ꢁC under air for 2 h. Parallel removal of
POCl3 was accomplished by heating the Radleys carrou-
sel to 160 ꢁC, while water (60 ꢁC) was pumped through
the Radleys upper water jacket using a Julabo MB-5
heated water circulator, and while house vacuum was

342
M. O. Anderson et al. / Bioorg. Med. Chem. 14 (2006) 334–343
applied to the carrousel (with an in-line dry ice/ethanol
vacuum trap). After the removal of POCl3, the crude 9-
chloroacridines were azeotropically dried with EtOAc
and then dry-loaded onto silica for flash column chroma-
tography. Automated flash column purification using the
CombiFlash system by Isco was done to yield the 9-chlo-
roacridine products (7) in high purity determined by
homogeneous 1H NMR spectra. A general solvent gradi-
ent for this class of compounds was devised: 5:95 ! 55:45
ethyl acetate/hexanes (2% Et3N) over 25 min.
complex (46 ml, 478 mmol, 4 equiv). The solution was
heated in an oil bath at 60 ꢁC for 30 min, with concom-
itant distillation of dimethylsulfide from the reaction
mixture. RP-HPLC analysis showed the reaction to be
complete. The distillation head was replaced with a con-
denser, and H2O was slowly and carefully added over
20 min., until major bubbling ceased. Next, HCl (1 M
aq, 100 ml) was added and the solution was heated to
60 ꢁC for 45 min. The solution was cooled to 0 ꢁC, treat-
ed with Et2O (500 ml), and extracted with HCl (1 M aq,
2· 300 ml). The combined aqueous layers were washed
with Et2O (200 ml), cooled to 0 ꢁC, treated directly with
solid NaOH until the mixture was visibly heterogeneous,
and then extracted with EtOAc (3 · 200 ml), dried over
Na2SO4, and concentrated in vacuo to yield the desired
secondary amines (15) in high purity (50–71% yield).
4.6. N-(3-Aminopropyl)-2-nitrobenzenesulfonamide (13)
1,3-Diaminopropane (236 ml, 2807 mmol) was cooled to
0 ꢁC under argon, and then a solution of 2-nitro-
benzenesulfonyl chloride (62.2 g, 280.7 mmol) in CH2Cl2
(2400 ml) was slowly added by pressure-equalizing drop-
ping funnel, over a period of ca. 2 h. The reaction mix-
ture was stirred overnight and then split into two equal
portions for workup. Each portion was cooled to 0 ꢁC,
followed by dropping funnel addition of H2O (300 ml)
and HCl (concd aq) (150 ml) (Caution: both additions
are exothermic!). The CH2Cl2 layers were extracted and
disposed. The aqueous layers were then cooled to 0 ꢁC,
treated directly with solid NaOH until the mixtures were
visibly heterogeneous, and then extracted with CH2Cl2
(6· 500 ml). The combined organic layers were dried
over Na2SO4 and concentrated in vacuo to yield the title
compound (13) (60.27 g, 83% yield), which can also be
purchased commercially (TCI America).
4.8. General procedure for parallel reductive amination
and nosyl deprotection (9)
Secondary amines (14) generated in the previous step
were loaded into the vessels of a Radleys 12-place reac-
tion carrousel as 0.3 M stock solutions in THF (12 ml
each, 3.5 mmol), followed by aldehydes (16) (1.5 equiv),
and sodium triacetoxyborohydride (1.5 equiv). The ves-
sels were purged under argon and then agitated by
placement in a Fisher Scientific FS20 sonicating water
bath for 30–60 min., after which RP-HPLC showed
the reductive amination reaction to be quantitative. Par-
allel solvent removal in vacuo was accomplished using a
GeneVac HT-4 instrument. Parallel workup by SPE: ini-
tially columns (Teledyne Isco Inc., # 69-3873-146) were
loaded with Dowex 50WX2-400 ion-exchange resin
(ꢁ15 g), and the resin was then pre-conditioned with
MeOH (1% CF3CO2H) (50 ml). The concentrated reac-
tion mixtures were redissolved in MeOH (5%
CF3CO2H) (50 ml) and loaded onto the columns. The
resin was washed with MeOH and then the products
were eluted with MeOH (10% Et3N) (50 ml). Finally,
the products of reductive amination were obtained after
parallel solvent removal in vacuo using the GeneVac
HT-4 instrument. Alternative aqueous workup: The
crude reaction mixture in THF was carefully quenched
with HCl (1 M aq) (25 ml), treated with Et2O (150 ml),
and the aqueous layer was extracted followed by an
additional aqueous extraction with HCl (1 M aq)
(25 ml). The aqueous layers were combined and washed
with Et2O (2 · 50 ml), basified with NaOH (1 M aq.) un-
til visibly heterogeneous, and the products were extract-
ed with Et2O (3 · 30 ml). The organic layer was dried
over Na2SO4 and concentrated in vacuo to yield the
products.
4.7. General procedure for amidation/reduction to
generate the secondary amine chemset (15)
A solution of N-(3-aminopropyl)-2-nitrobenzenesulf-
onamide (13) (31.0 g, 120 mmol) in THF (400 ml) was
stirred at 25 ꢁC under argon in an ambient water bath
and treated with pyridine (11 ml, 131 mmol, 1.1 equiv)
followed by carboxylic anhydride reagents (14) (1.2
equiv). The solution was stirred at 25 ꢁC for 1.5 h, after
which RP-HPLC analysis indicated quantitative conver-
sion to the amide. The reaction mixture was quenched
with ammonium hydroxide (30% aq) (70 ml, 598 mmol,
5 equiv), stirred an additional 15 min, and then concen-
trated in vacuo. The residue was treated with ammonium
chloride (satd aq): NaCl (satd aq) (2:1, 300 ml), and acid-
ified with HCl (1 M aq) to pH 1, and then the product
was extracted with Et2O/EtOAc (1:1) (2· 200 ml). The
organic layer was washed with HCl (1 M aq):NaCl (satd
aq) (1:1, 100 ml), dried over Na2SO4, and concentrated in
vacuo to yield the crude amide intermediate, used in the
next step without further purification.
The subsequent borane-methylsulfide reduction step re-
quired an apparatus to remove dimethylsulfide by distil-
lation:34 the reaction was carried out in a 2000 ml single-
neck round-bottomed flask attached to a 2-way Claisen
adaptor. The adaptor was equipped with a rubber sep-
tum for reagent addition and a vacuum distillation head
with a trap for distilled dimethylsulfide. The system was
charged with a solution of crude amide (ca. 120 mmol)
in anhydrous THF (400 ml), stirred at 25 ꢁC under ar-
gon, and treated dropwise with borane–dimethylsulfide
The crude tertiary amine products (ca. 3.5 mmol) were
dissolved in CH3CN (35 ml) and loaded into the vessels
of a Radleys 6-place reaction carrousel. The vessels were
purged of air by applying three iterations of house vac-
uum followed by positive pressure of argon. Each vessel
was then treated with Cs2CO3 (2.5 equiv) followed by
benzenethiol (5.0 equiv), and then the vessels were stir-
red vigorously under argon for 1 h at rt, after which
RP-HPLC showed near quantitative (>80%) conversion.
More benzenethiol (5.0 equiv) was added, and the

M. O. Anderson et al. / Bioorg. Med. Chem. 14 (2006) 334–343
343
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Supplementary data
Supplementary data associated with this article can be
found in the online version at doi:10.1016/
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