Synthesis and evaluation of 7-substituted 4-aminoquinoline analogues for antimalarial activity

Article abstract of DOI:10.1021/jm200636z

We previously reported that substituted 4-aminoquinolines with a phenyl ether substituent at the 7-position of the quinoline ring and the capability of intramolecular hydrogen bonding between the protonated amine on the side chain and a hydrogen bond acceptor on the amine's alkyl substituents exhibited potent antimalarial activity against the multidrug resistant strain P. falciparum W2. We employed a parallel synthetic method to generate diaryl ether, biaryl, and alkylaryl 4-aminoquinoline analogues in the background of a limited number of side chain variations that had previously afforded potent 4-aminoquinolines. All subsets were evaluated for their antimalarial activity against the chloroquine-sensitive strain 3D7 and the chloroquine-resistant K1 strain as well as for cytotoxicity against mammalian cell lines. While all three arrays showed good antimalarial activity, only the biaryl-containing subset showed consistently good potency against the drug-resistant K1 strain and good selectivity with regard to mammalian cytotoxicity. Overall, our data indicate that the biaryl-containing series contains promising candidates for further study.

Full text of DOI:10.1021/jm200636z

ARTICLE
pubs.acs.org/jmc
Synthesis and Evaluation of 7-Substituted 4-Aminoquinoline
Analogues for Antimalarial Activity
Jong Yeon Hwang,† Takashi Kawasuji,†,‡ David J. Lowes,† Julie A. Clark,† Michele C. Connelly,† Fangyi Zhu,†
W. Armand Guiguemde,† Martina S. Sigal,† Emily B. Wilson,§ Joseph L. DeRisi,§ and R. Kiplin Guy*,†
†Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis,
Tennessee 38105, United States
‡Medicinal Research Laboratories, Shionogi & Co., Ltd., 12-4, Sagisu 5-Chome, Fukushima-ku, Osaka, Japan
§Department of Biochemistry and Biophysics, University of California at San Francisco, 600 16th Street, San Francisco, California 94143,
United States
S Supporting Information
b
ABSTRACT: We previously reported that substituted 4-aminoquinolines
with a phenyl ether substituent at the 7-position of the quinoline ring and
the capability of intramolecular hydrogen bonding between the protonated
amine on the side chain and a hydrogen bond acceptor on the amine’s alkyl
substituents exhibited potent antimalarial activity against the multidrug
resistant strain P. falciparum W2. We employed a parallel synthetic method
to generate diaryl ether, biaryl, and alkylaryl 4-aminoquinoline analogues in
the background of a limited number of side chain variations that had previously afforded potent 4-aminoquinolines. All subsets were
evaluated for their antimalarial activity against the chloroquine-sensitive strain 3D7 and the chloroquine-resistant K1 strain as well as
for cytotoxicity against mammalian cell lines. While all three arrays showed good antimalarial activity, only the biaryl-containing subset
showed consistently good potency against the drug-resistant K1 strain and good selectivity with regard to mammalian cytotoxicity.
Overall, our data indicate that the biaryl-containing series contains promising candidates for further study.
’ INTRODUCTION
substitution in the context of a shorter amino containing side chain,
which was generically more potent against MDR strains. In addition
other studies showed that exchanging the quinoline ring system with
an acridine ring system in the context of the same side chain gave
improved antimalarial potency against both CQ-sensitive and CQ-
resistant strains, suggesting that increased bulk and/or hydrophobi-
city might be advantageous.17 Given these previous SAR results, we
designed a new array of 4-aminoquinoline analogues (3) to explore
this hypothesis by using parallel medicinal chemistry. Here we report
the design, synthesis, and biological evaluation of this array.
Our strategy for the design of this compound array is shown in
Figure 1. We fixed the side chain portion using a propyl spacer
(three carbons) between two amines because such shorter side
chain variants have consistently given better antimalarial potency
than longer chains (more than four carbons).16 A fixed set of
substitutions on the terminal alkylamine was chosen to allow for
combinatorial interactions with the quinoline nucleus within the
highly potent “chemical space”. This diversity set was based on
previously obtained SAR for chloroquine analogues. The 7-posi-
tion of the quinoline ring system was varied through a range of
substituents that were accessible by the Ullmann coupling,18,19
Suzuki coupling,20,21 and Negishi coupling reactions,22 which
respectively generate diaryl ether, biaryl, and alkylaryl substitu-
tions. These three subseries were chosen to systematically vary
Malaria, a devastating infectious disease caused by five species
of Plasmodium, affects 200ꢀ500 million people and causes over
800 000 deaths annually.1 The most lethal form of malaria is
caused by Plasmodium falciparum.2 Since the development of
quinine, drugs containing the quinoline nucleus have been a
mainstay of antimalarial therapy.3,4 However, the spread of
chloroquine(CQ) and mefloquine resistant strainsof P. falciparum
has curbed the usefulness of this class of drugs.5,6 In the past
2 decades, a number of new potent quinolines that overcome
chloroquine resistance have been reported.7ꢀ9 Most contain the
7-chloroquinoline nucleus of chloroquine and vary in the length
and nature of their basic amine side chain.10ꢀ12
We have reported extensive studies on the role of side chains for
the antimalarial activity and efficacy.13,14 Additional work in our
laboratory systematically examined modification of the quinoline
ring and the basic side chain of 4-aminoquinolines (1)13ꢀ16
demonstrating that intramolecular hydrogen bonding (2) between
the protonated amine and the hydroxyl group on the tertiary basic
amine is crucial for the potency against CQ-resistant strains. In a
similar systematic survey we found that most modifications to the
quinoline nucleus resulted in lowered potency and had little effect
on relative potency in multidrug resistant (MDR) strains, such as
W2.15,16 This finding was in line with the consensus model from the
literature that requires a small, electron withdrawing group at this
position (chlorine or trifluoromethyl). From these studies, one
finding that did stand out was the high potency of the 7-PhO
Received: May 18, 2011
Published: September 12, 2011
r
2011 American Chemical Society
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the size, hydrogen bonding capabilities, electrostatics, and hydro-
phobicity at the 7-position. Overall this study was expected to
firmly establish which types of substitutions are tolerated at the
7-position and how this position interacts with the side chain in
defining potency in CQ-resistant Plasmodium species.
2:1 mixture that was inseparable by both flash column chromatog-
raphy and crystallization. Chlorination of the mixture with POCl3
allowed the successful separation of 7-bromo-4-chloroquinoline 7a,
the key intermediate, from its isomer 5-bromo-4-chloroquinoline 7b
by column chromatography. Condensing 7b with 20 equiv of 1,3-
propyldiamine afforded compound 8, which was used in the next
coupling reaction without purification.
’ CHEMISTRY
Intermediate 8 was coupled with three sets of building blocks
(diversity sets shown in Figure 2): phenols (x = 1, y = 1ꢀ8),
boronic acids (x = 2, y = 1ꢀ8), and alkyl/benzyl zinc halides
(x = 3, y = 1ꢀ12) in the presence of the corresponding metal
catalyst. Each diversity set was designed to sample widely in
sterics, electrostatics, and hydrophobicity. This parallel diversi-
fication step resulted in the production of the array of 7-sub-
stituted-4-aminoquinolines 9{x,y}. The final array was produced
by an orthogonal diversification at the terminal amine. Each
member of array 9{x,y} was treated in parallel with the four
aldehydes defined from our previous studies (z = 1ꢀ4) to give
4,7-disubstituted quinoline analogues 3{x,y,z}.
The general approach used to produce this compound array
followed our previously established route (Scheme 1). The main
challenge in this work was the development of a robust, general route
to 7-bromo-4-chloroquinoline 7a, which served as the key inter-
mediate for the diversification. Since chloro and bromo substituted
carbons in general have strongly differing reactivity, this intermediate
allowed for the selective synthesis of all targeted compounds. The
synthetic route to the key intermediate 7a followed a previously
reported method.16 Condensation of 3-bromoaniline with Mel-
drum’s acid 4 and trimethyl orthoformate gave enamine 5. Cycliza-
tion with microwave acceleration gave regioisomers 6a and 6b as a
The crude products were purified by flash chromatography
using a SP-1 (Biotage) purification system with dichloromethane
and methanol mixture (1% f 50% gradient condition). Each
purified product was then dissolved in a saturated methanolic
solution of hydrochloric acid and then evaporated to dryness to
afford the HCl salts. Purity and identity were verified for all
compounds by UPLC/MS and 1H NMR. The yield, purity, and
NMR data of final compounds are available in the Supporting
Information. All purified products were dissolved in DMSO to a
standard concentration of 10 mM for biological testing.
’ BIOLOGICAL AND PHARMACOLOGICAL TESTING
The compounds in array 3{x,y,z} were tested against CQ-
sensitive (3D7) and CQ-resistant (K1) strains of P. falciparum in
concentrationꢀresponse experiments in a 3-fold dilution schema
spanning 15 μM to 0.7 nM, using a previously established method.23
All experiments were carried out in triplicate, each triplicate ex-
periment being replicated on two separate days for a total of 9
replicates. Data are reported as mean EC50 values with 95% con-
fidence limits.
Figure 1. Rational design for 7-substituted 4-aminoquinolines. The
three-carbon spacer between two amines and the terminal secondary
amine was selected, since this pair consistently gave high potency in
previous work. To explore substitution on the quinoline ring at the
7-position, metal assisted-coupling reactions including the Ullmann,
Suzuki, and Negishi reactions were applied to generate diaryl ether,
biaryl, and alkylaryl subsets, respectively.
The compounds in array 3{x,y,z} were also tested for growth
inhibition against four human cell lines: Raji, a Burkett’s lymphoma
derived line; BJ, an immortalized foreskin fibroblast derived line;
Scheme 1. Synthesis of the Compound Array 3{x,y,z}a
a Reagents and conditions: (a) 3-bromoaniline, triethyl orthoformate, reflux, 1 h; (b) 225 °C, microwave, Ph2O, 5 min; (c) POCl3, toluene, 100 °C, 1 h; (d) 1,3-
aminopropane, K2CO3, TEA, NMP, 140 °C, 1 h. (e) For Ullmann reagent: phenols, CuI, Cs2CO3, ligand, 1,4-dioxane, 4 Å molecular sieves, reflux, 24 h. For
Suzuki reagents: boronic acids, PdCl2(dppf), Cs2CO3, toluene, 4 Å molecular sieves, 100 °C, 24 h. For Negishi reagents: zinc halides, Pd(PPh3)4,THF, reflux, 16 h.
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this testing schema are discussed below, broken out by subset of
the analogues at the 7-position of the quinoline nucleus.
Diaryl Ether Series. Thirty-two diaryl ethers derived from the
Ullmann reaction were synthesized and evaluated. The data are
summarized in the heat map in Table 1, which was produced by
using the EC50 values determined by fitting curves to the
concentrationꢀresponse experiments described above. The ta-
bulated experimental values and errors can be found in the
Supporting Information. Of 32 diaryl ethers, 21 compounds
showed reasonable potency according to the MMV lead criteria
(EC50 < 50 nM) against the CQ-sensitive 3D7 strain. However,
only two compounds (3{1,4,1} and 3{1,7,4}) exhibited highly
potent activity (20 and 31 nM EC50, respectively) against the
CQ-resistant strain K1. This is in stark contrast to the results with
the analogous chloro compounds, which are all equivalent com-
pounds that are roughly equipotent against the two strains.13
One set of compounds stands out: 3{1,1,4}, 3{1,3,4}, and
3{1,5,4}, all carrying the 3-F-6-MeO-Bn alkylamine substituent,
showed enhanced potency in strain K1. Generally the furfuryl
(z = 1) and 3-F-6-MeO-Bn (z = 4) substituted alkylamines in this
series exhibited better potency against strain K1 than the 2-HO-
3-MeO-Bn (z = 2) and piperonyl (z = 3) substituents. All of
2-HO-3-MeO-Bn (z = 2) substituted alkylamines had poor
antimalarial activity against strain K1, whereas they were highly
potent against strain 3D7. Electronic effects were not strongly
evident for the diaryl ethers, but electron-donating substituents
showed slightly better activity against strain 3D7.
The compounds were tested against a panel of four mamma-
lian cell lines (HepG2, HEK293, Raji, and BJ). In general HepG2
cells were the most sensitive, while other lines were less sensitive
against compounds (EC50 > 5 μM). Table 1 shows only HepG2
data, while all cytotoxicity data are summarized in the Supporting
Information. Most of the compounds active against strain 3D7
(<50 nM EC50) showed moderate cytotoxicity (EC50 of 3ꢀ15
μM) in HepG2, resulting in a reasonable average selectivity for
the parasite (>100-fold). Actives against strain K1 (<50 nM
EC50) also showed at least 100-fold selectivity. All compounds in
this series had reasonable solubility (14.5ꢀ46 μM) and good
permeability ((317ꢀ1309) ꢁ 10ꢀ6 cm/s) in pH 7.4 buffer
(Table 1 in Supporting Information).
Figure 2. Diversity sets used in generating the compound array.
Biaryl Series. Next we evaluated the biological activity of the
biaryl series derived from the Suzuki reaction. All data for this
series are summarized in the Supporting Information, and
selected data are shown in Table 2. Most compounds in this
series showed high potency against strain 3D7. Interestingly,
many of them were also highly potent (<50 nM EC50) against
strain K1. Thus, there is a stark contrast between the diaryl ether
and the biaryl series with respect to their activity against strain
K1, which expresses the PfCRT transporter, the primary medi-
ator of CQ resistance. However, compounds 3{2,1,2} and
3{2,5,2}, the most potent against 3D7 (both 9 nM EC50), only
showed weak potency against K1 (850 and 1950 nM EC50 values,
respectively), resulting in high selectivity for 3D7 (93- and 213-
fold). As was the case with the diaryl ether series, all 2-HO-3-
MeO-Bn substituted alkylamines showed very good activity in
strain 3D7.
HEK 293, an embryonic kidney fibroblast derived line; and
HepG2, a hepatocellular carcinoma derived line. The cytotoxicity
was determined by detection of total cellular ATP levels (Cell
Titer Glo). The same concentrationꢀresponse plates were used
for both antimalarial and growth proliferation experiments. All
experiments were carried out in triplicate with one replicate for a
total of three replicates. Data are reported as mean EC50 values
with 95% confidence limits.
Finally, the kinetic solubility and passive permeability of the
molecules in array 3(x,y,z) were investigated to provide guidance
in interpreting cell based assay results and to inform considera-
tions about the development potential of the compounds.
Solubility was determined using the pIon method by dilution
from DMSO stocks into PBS buffer containing citric acid at
neutral pH (7.4).24 Permeability was assessed using the pIon
implementation of the parallel artificial membrane permeation
assay (PAMPA) in PBS buffer containing citric acid at neutral pH
(7.4), thus reflecting the conditions required for activity in cell-
based assays.25 Both assays emulate the conditions used to
determine cytotoxicity and antimalarial activity. The results of
A notable trend was observed: increasing hydrophobicity in
this series was accompanied by decreasing antimalarial activity.
For example, all tert-Bu-Ph (y = 6) substituted, naphthyl substi-
tuted (y = 3), and 3,5-CF3-Ph substituted (y = 2) compounds
showed poor activity in both 3D7 and K1. In contrast to the
diaryl ether series, compounds with 2-HO-3-MeO-Bn (z = 2)
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Table 1. Biological and Pharmacological Evaluation of Diaryl Ethers: Antimalarial Activity and Cytotoxicityc
a Values are the mean of three independent experiments in triplicate. b Values are the mean of one experiment in triplicate. c Activity summary of the
compounds was sorted by EC50 value against K1 strain.
and piperonyl (z = 3) substituents on the terminal alkylamine
exhibited good activity against strain K1 (EC50 < 50 nM).
Although this series displayed moderate overall cytotoxicity
(0.7ꢀ15 μM EC50 in HepG2), all compounds with low nano-
molar EC50 values against 3D7 and K1 had a good selectivity for
the parasite relative to mammalian cells. All compounds in this
series also have good solubility (32ꢀ42 μM) and good perme-
ability ((237ꢀ2101) ꢁ 10ꢀ6 cm/s) in pH 7.4 buffer (Table 2 in
Supporting Information)
Alkylaryl Series. The alkylaryl series, derived from Negishi
couplings, was evaluated in the same test cascade (Table 3). This
set of compounds also showed good antimalarial activity against
strain 3D7. However, their activity against strain K1 was very
weak (EC50 > 200 nM). Most of the active compounds in this
series showed moderate to strong cytotoxicity (0.4ꢀ16 μM) in
HepG2, resulting in a reasonable selectivity relative to mamma-
lian cells (>100). However, given the poor potency on K1, there
was almost no selectivity relative to mammalian cells of that
strain. Interestingly, the most potent compound 3 with 4 nM
EC50 against 3D7 was quite toxic (403 nM EC50) to HepG2 cell,
but it still gave good selectivity (>100-fold) between parasite and
mammalian cells. Overall this series is the least promising with
respect to potential for development. In general the compounds
in this series tended to be less soluble and less permeable than the
other series studied. They had values ranging from modest to
good solubility (4.2ꢀ41 μM) and modest to good permeability
((23ꢀ2110) ꢁ 10ꢀ6 cm/s) in pH 7.4 buffer (Table 3 in
Supporting Information)
’ DISCUSSION
In earlier work we identified a previously unappreciated aspect
to the structureꢀactivity relationships of the 4-aminoquinoline
antimalarials in which the 7-chloro substituent of the quinolone
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Table 2. Biological and Pharmacological Evaluation for Biaryls: Antimalarial Activity and Cytotoxicityc
a Values are the mean of three independent experiments in triplicate. b Values are the mean of one experiment in triplicate. c Activity summary of the
compounds was sorted by EC50 value against K1 strain.
A-ring could be replaced by a hydrophobic substituent with
retention of potency. In order to more fully explore this obser-
vation, we applied three different coupling chemistries to a com-
mon synthetic intermediate to give access to fairly widely ranging
substituents at this position (R1 group). This array of compounds
provided a wide range of physicochemical properties for the
quinoline including variant pKa of the quinoline nitrogen; overall
electrostatics and sterics, based on the Topliss and Hammett
approaches; and overall lipophilicity.
It is known that the pKa of the ring and the basic side chain
affect the activity against both CQ-sensitive and CQ-resistant
strains of Plasmodium.12,26,27 We calculated overall lipophilicity
and pKa of quinoline nitrogens (data summarized in Supporting
Information).28 Surprisingly, the biaryl series (x = 2) and
alkylaryl series (x = 3) showed identical pKa values (9.47) on
the nitrogen of the quinoline ring, whereas the diaryl ether series
(x = 1) showed a slightly lower pKa value (9.28). Overall this did
not appear to correlate with activity trends. Interestingly, no
strong stereoelectronic effects were observed for substituents on
the R1 group. Unfortunately, we did not find a good correlation
between antimalarial activity and pKa.
Kaschula et al. reported that a more lipophilic group at the
7-position of 4-aminoquinoline enhanced antimalarial activity by
increasing hematin affinity.27 Therefore, correlations with ALogP
were explored to elucidate the relationship between activity and
lipophilicity (Supporting Information, Figure 2). Lipophilic
compounds in our tests exhibited slightly weaker antimalarial
activity in both the 3D7 and K1 strains when all series were
combined (Figure 3A). The same trend was observed in the
diaryl ether series alone (Figure 3B) and in the biaryl series
(Figure 3C). However, the trend was reversed in the alkylaryl
series (Figure 3D). We noted furthermore that in the diaryl ether
series the antimalarial activity in strain 3D7 was more sensitive to
lipophilicity than in strain K1. However, none of these trends
were strong enough to have any real interest or to warrant further
mechanistic studies with hematin. Overall, there were no strong
trends in activity that could be easily explained using any
conventional models for chloroquine mechanism of action.
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Table 3. Biological and Pharmacological Evaluation for Alkylaryls: Antimalarial Activity and Cytotoxicityc
a Values are the mean of three independent experiments in triplicate. b Values are the mean of one experiment in triplicate. c Activity summary of the
compounds was sorted by EC50 value against 3D7 strain.
Another possible explanation for variations in activity would
be variant cellular bioavailability of the compounds. The quino-
lines are well-known to accumulate in the acidic food vacuole of
the parasite, so permeability is usually a poor predictor of
potency, beyond a requirement that the compounds be able
to cross a membrane. As expected, we observed no strong
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Figure 3. Relationship between lipophilicity and antimalarial activity (3D7 strain (O) and K1 strain (2)): (A) ALogP vs log EC50, complete compound
array; (B) ALogP vs log EC50, diaryl ethers; (C) ALogP vs log EC50,biaryls; (D) ALogP vs log EC50-, alkylaryls.
correlations between permeability or solubility and potency. The
most striking finding was that the alkylaryl series had fairly
extreme cross-resistance with CQ-resistant strains. Presumably,
this substitution at the 7-position increases transport by PfCRT.
’ EXPERIMENTAL SECTION
General. All materials were obtained from commercial suppliers and
used without further purification. All solvents were dried using an
aluminum oxide column. Thin-layer chromatography was performed
on precoated silica gel 60 F254 plates. Purification of synthesized
compounds was carried out by normal phase column chromatography
(SP1 [Biotage], silica gel 230ꢀ400 mesh) followed by evaporation (HT-
4X evaporator [Genevac]). Initiator [Biotage] was used for microwave
’ CONCLUSIONS
A parallel synthetic strategy was employed to generate a
rationally designed array of 7-substituted 4-aminoquinolines.
Utilizing 7-bromo-4-chloroquinoline as a key intermediate en-
abled the exploration of two-dimensional diversity, using a fixed
propyl spacer between the quinoline and the distal basic center
and a small set of amine substituents previously shown to be
active in CQ-resistant Plasmodium strains when used with the
chloroquine nucleus. The diversity explored at the 7-position
included diaryl ethers, biaryls, and alkylaryls and was produced
using the Ullmann, Suzuki, and Negishi reactions, respectively.
The compounds were screened against CQ-sensitive and CQ-
resistant strains of P. falciparum and a panel of mammalian cell
line to establish antimalarial potency and selectivity for mam-
malian cells.
Physicochemical properties of 4-aminoquinolines such as pKa
and lipophilicity can be influenced by the substitution at the
7-position, resulting in changes of the potency against malaria.
However, in the series explored, the calculated pKa of the com-
pounds was not significantly changed by substitution at the
7-position, and no correlation was observed between pKa and
antimalarial activity. However, lipophilicity did weakly correlate
with antimalarial activity. Strikingly, less lipophilic compounds
were generally more potent against both the CQ-sensitive (3D7)
and CQ-resistant strain (K1), an effect that was especially strong
for the biaryl compounds.
1
reaction. H NMR spectra were recorded using a Bruker 400 MHz
spectrometer, in CDCl3 or DMSO-d6 solvent. Chemical shifts were
reported as parts per million (ppm) downfield from solvent reference.
Coupling constants (J) were measured in hertz (Hz). NMR peaks were
assigned by MestReNova (5.2.2). Identity of all compounds was
confirmed by proton and carbon NMR and by mass spectrometry.
The purity of all compounds was assessed using LC/MS/UV/ELSD/
CLND with the purity being assigned as the average determined by UV/
ELSD/CLND. All compounds used for subsequent studies had a
minimum purity of 95%.
Chemistry. 5-((3-Bromophenylamino)methylene)-2,2-di-
methyl-1,3-dioxane-4,6-dione (5). A solution of 2,2-dimethyl-1,3-
dioxane-4,6-dione (4) (21.6 g, 150 mmol) in triethyl orthoformate
(100 mL) was refluxed for 1 h. After the mixture was cooled to room
temperature, 3-bromoaniline (17.2 g, 100 mmol) was added to the solution.
The mixture was refluxed for 2 h, then cooled to room temperature.
The precipitates were collected and washed with ether to give compound
1
5 (32.8 g, quantitative yield) as pale yellow crystals. H NMR (CDCl3)
δ ppm 11.51ꢀ10.89 (m, 1H), 8.60 (d, J = 14.1 Hz, 1H), 7.47ꢀ7.37
(m, 2H), 7.31 (t, J = 8.0 Hz, 1H), 7.21ꢀ7.15 (m, 1H), 1.76 (s, 6H).
7-Bromoquinolin-4-ol (6a) and 5-Bromoquinolin-4-ol
(6b). Compound 5 (1.0 g, 3.1 mmol) and diphenyl ether (10 mL)
were irradiated by microwave at 225 °C for 5 min. Ether (10 mL) was
added to the mixture, and the resulting brown precipitates were collected
and washed with ether to give a mixture of compounds 6a and 6b.
Replicating this process 16 times in parallel gave 9.4 g (85% yield) of the
product mixture. Caution: The reaction vessel develops high pressure.
1H NMR (DMSO-d6) δ ppm 11.79 (brs, 1H), 7.99 (d, J = 8.6 Hz, 2/
Taken as a whole, this study highlights the potential for
developing novel 4-aminoquinolines including a biaryl at the
7-position of the quinoline. Further exploration in preclinical
studies is warranted.
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3H), 7.92 (d, J = 7.4 Hz, 2/3H), 7.82 (d, J = 7.4 Hz, 1/3H), 7.75 (d, J =
1.9 Hz, 2/3H), 7.52 (dd, J = 1.2, 8.0 Hz, 1/3H), 7.48 (dd, J = 1.4, 8.0 Hz,
1/3H), 7.45 (dd, J = 1.8, 8.6 Hz, 2/3H), 7.44 (dd, J = 8.0, 8.4 Hz, 1/3H),
6.05 (d, J = 7.5 Hz, 2/3H), 6.01 (d, J = 7.5 Hz, 1/3H).
MgSO4 and concentrated in vacuo. The crude product was purified by
flash chromatography (MeOH/TEA in CH2Cl2 = 10%/1% to 80%/8%
gradient) to give the corresponding compounds 9{x=3}. Benzylzinc
bromide solution was prepared as follows: To a suspension of Zn
powder (261 mg, 4 mmol) in THF (6 mL) was added benzyl bromide
(4 mmol). The solution was heated to 50 °C for 30 min. The solution
was used immediately for Negishi coupling.
Procedure for Parallel Synthesis of Compounds 3{x,y,z}:
Reductive Amination. To a solution of compound 9{x} (0.1ꢀ0.2
mmol) in methanol (1ꢀ2 mL) was added 1 M aldehyde stock solution
in methanol (1 equiv) After the mixture was stirred for 1 h, sodium
borohydride (0.5 equiv) was added to the reaction mixture and the
resulting mixture stirred for 30 min. The reaction was quenched by
addition of water (2 mL). The aqueous portion was extracted with
CH2Cl2, and the sample was concentrated in vacuo. The crude product
was purified by flash chromotography (1% methanol in CH2Cl2
(12 mL) and 30% methanol in CH2Cl2 (16 mL)). The solvent was
removed in vacuo, and the product was suspended in 1.25 M HCl in
methanol solution (1 mL). The solvent and excess of HCl were
evaporated in vacuo to give the corresponding HCl salt.
7-Bromo-4-chloroquinoline (7a). To a suspension of a mixture
of compounds 6a and 6b (9.4 g, 42 mmol) in toluene (70 mL) was added
phosphorus oxychloride (13 g, 84 mmol). The mixture was stirred for 1
h at 100 °C, then cooled to room temperature. Excess phosphorus
oxychloride was quenched by adding a small portion of iceꢀwater and
then stirring for 1 h. Additional water (100 mL) was added, and the
resulting aqueous portion was extracted with dichloromethane. The
organic phase was dried over MgSO4, and the solvent was removed in
vacuo. The crude product was purified by flash chromatography (EtOAc
in hexane = 5% f 25%) to give compound 7a (6.2 g, 61% yield) as white
crystals and compound 7b (3.0 g, 30% yield) as white crystals. 7a: 1H
NMR (CDCl3) δ ppm 8.79 (d, J = 4.8 Hz, 1H), 8.32 (d, J = 1.9 Hz, 1H),
8.12 (d, J = 9.0 Hz, 1H), 7.74 (dd, J = 9.0, 1.9 Hz, 1H), 7.51 (d, J = 4.7 Hz,
1H). 7b: 1H NMR (CDCl3) δ ppm 8.73 (d, J = 4.6 Hz, 1H), 8.12 (dd, J =
8.4, 1.2 Hz, 1H), 7.96 (dd, J = 7.6, 1.2 Hz, 1H), 7.56 (d, J = 4.4 Hz, 1H),
7.52 (t, J = 8.4, 7.6 Hz, 1H).
N1-(7-Bromoquinolin-4-yl)propane-1,3-diamine (8). To a
mixture of compound 7a (6.18 g, 25.6 mmol) and 1,3-diaminopropane
(37.9 g, 512 mmol) in N-methylpyrrolidinone (60 mL) was added
triethylamine (777 mg, 7.68 mmol) and K2CO3 (1.17 g, 8.45 mmol).
The resulting mixture was stirred at 140 °C for 1 h and then cooled to
room temperature. Water (200 mL) was added to the solution, and the
product was precipitated by cooling the mixture to 4 °C. The resulting
white precipitates were collected, washed with water, and dried to give
compound 8 (5.69 g, 80% yield) as white crystals. 1H NMR (DMSO-d6)
δ ppm 8.38 (d, J = 5.4 Hz, 1H), 8.15 (d, J = 9.0 Hz, 1H), 7.93 (d, J = 2.1
Hz, 1H), 7.54 (dd, J = 9.0, 2.1 Hz, 1H), 7.52 (brs, 1H), 6.48 (d, J = 5.5
Hz, 1H), 3.32 (brs, 2H), 2.68 (t, J = 6.5 Hz, 2H), 1.68ꢀ1.77 (m, 2H).
Procedure for Parallel Synthesis of Compounds 9{x=1}:
Ullmann Coupling. To a mixture of compound 8 (279 mg, 1 mmol)
in 1,4-dioxane (6 mL) were added the phenol reagent (4 mmol),
Cs2CO3 (1.3 g, 4 mmol), 4 Å powder molecular sieves (∼280 mg,
dried in oven), salicylaldoxime (55 mg, 0.4 mmol), and copper(I) iodide
(38 mg, 0.2 mmol). The mixture was heated to reflux and maintained at
that temperature for 24 h and then cooled to room temperature. A
solution of dichloromethane (10 mL) and methanol (1 mL) was added
to the mixture. After filtration the solvents were removed in vacuo. The
crude product was purified by flash chromatography (MeOH/TEA in
CH2Cl2 = 10%/1% to 40%/4% gradient) to give the corresponding
compound 9{x=1}.
Procedure for Parallel Synthesis of Compounds 9{x=2}:
Suzuki Coupling. To a mixture of compound 8 (279 mg, 1 mmol) in
toluene (6 mL) were added the boronic acid reagent (4 mmol), Cs2CO3
(1.3 g, 4 mmol), 4 Å powder molecular sieves (∼280 mg), and
PdCl2(dppf) (41 mg, 0.05 mmol). The mixture was heated to 100 °C
for 24 h and then cooled to room temperature. Water (50 mL) was
added and the resulting aqueous portion extracted with dichloro-
methane and 10% methanol. The organic phase was dried over MgSO4,
filtered, and dried in vacuo. The crude product was purified by flash
chromatography (MeOH/TEA in CH2Cl2 = 10%/1% to 80%/8%
gradient) to give the corresponding compounds 9{x=2}.
Antimalarial Activity. Two P. falciparum strains were used in this
study and were provided by the MR4 Unit of the American Type Culture
Collection (ATCC, Manassas, VA). Those two strains were the chloro-
quine sensitive strain 3D7 and the chloroquine resistant strain K1.
Asynchronous parasites were maintained in culture based on the
method of Trager.29 Parasites were grown in the presence of fresh group
O-positive erythrocytes (Lifeblood Memphis, TN) in Petri dishes at a
hematocrit of 4ꢀ6% in RPMI based medium (RPMI 1640 supplemen-
ted with 0.5% AlbuMAX II, 25 mM HEPES, 25 mM NaHCO3 (pH 7.3),
100 μg/mL hypoxanthine, and 5 μg/mL gentamycin). Cultures were
incubated at 37 °C in a gas mixture of 90% N2, 5% O2, 5% CO2. For IC50
determinations, 20 μL of RPMI 1640 with 5 μg/mL gentamycin were
dispensed per well in an assay plate (Corning 384-well microtiter plate,
clear bottom, tissue culture treated, catalog no. 8807BC). An amount of
40 nL of compound, previously serial diluted in a separate 384-well white
polypropylene plate (Corning, catalog no. 8748BC), was dispensed to
the assay plate by hydrodynamic pin transfer (FP1S50H, V&P Scientific
Pin Head) and then an amount of 20 μL of a synchronized culture
suspension (1% rings, 4% hematocrit) was added per well, thus making a
final hematocrit and parasitemia of 2% and 1%, respectively. Assay plates
were incubated for 72 h, and the parasitemia was determined by a
method previously described:30 Briefly, an amount of 10 μL of the
following solution in RPMI (10ꢁ Sybr Green I, 0.5% v/v Triton,
0.5 mg/mL saponin) was added per well. Assay plates were shaken for
30 s, incubated in the dark for 90 min, then read with the Envision
spectrophotometer at Ex/Em of 485 nm/535 nm. IC50 values were
calculated with the robust investigation of screening experiments (RISE)
with four-parameter logistic equation.
Cytotoxicity in Mammalian Cells. BJ, HEK293, Hep G2, and
Raji cell lines were purchased from the American Type Culture Collec-
tion (ATCC, Manassas, VA) and were cultured according to recom-
mendations. Cell culture media were purchased from ATCC. Cells were
routinely tested for mycoplasma contamination using the MycoAlert
mycoplasma detection kit (Lonza). Exponentially growing cells were
plated in Corning 384-well white custom assay plates and incubated
overnight at 37 °C in a humidified 5% CO2 incubator. DMSO
compound solutions were added the following day to a top final
Procedure for Parallel Synthesis of Compounds 9{x=3}:
Negishi Coupling. To a solution of compound 8 (279 mg, 1 mmol)
and Pd(PPh3)4 (58 mg, 0.05 mmol) was added the alkylzinc bromide
reagent (8 mL, 0.5 M in THF, 4 mmol). The mixture was refluxed for 16
h, then cooled to room temperature (1 h is sufficient for benzylzinc
halide). Then 1 N HCl (10 mL) was added to the mixture and stirred.
Additional water (40 mL) was added to the mixture, and the solution
was then neutralized by adding solid K2CO3. The mixture was extracted
with 10% methanol in dichloromethane, the organic phase dried over
1
concentration of 25 μM and then diluted /3 for a total of 10 testing
concentrations. Cytotoxicity was determined following a 72 h incuba-
tion using Promega Cell Titer Glo reagent according to the manufac-
turer’s recommendation. Luminescence was measured on an Envision
plate reader (Perkin-Elmer).
Solubility. The solubility assay was carried out on Biomek FX lab
automation workstation (Beckman Coulter, Inc.). An amount of 10 μL
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ARTICLE
of compound stock was added to 190 μL of 1-propanol to make a
reference stock plate. Then 5 μL from this reference stock plate was
mixed with 70 μL of 1-propanol and 75 μL of phosphate buffered saline
(PBS, pH 7.4) to make the reference plate, and the UV spectrum (250 ꢀ
500 nm) of the reference plate was measured using a SPECTRAmax
PLUS plate reader (Molecular Devices). An amount of 6 μL of 10 mM
test compound stock was added to 600 μL of PBS in a 96-well storage
plate and mixed. The storage plate was sealed and incubated at room
temperature for 18 h. The suspension was then filtered through a 96-well
filter plate (pION Inc.). Then 75 μL of filtrate was mixed with 75 μL of
1-propanol to make the sample plate for UV spectroscopic analysis. A
single experiment was performed in triplicate for each compound.
Solubility was calculated using μSOL Evolution software based on the
AUC (area under the curve) of the UV spectrum of the sample plate and
the reference plate.
Permeability Assay. The parallel artificial membrane permeability
assay (PAMPA) was carried out on a Biomek FX lab automation
workstation (Beckman Coulter, Inc.). An amount of 3 μL of test
compound stock (10 mM in DMSO) was mixed with 600 μL of SSB
(system solution buffer, pH 7.4 or 4, pION Inc.) to dilute the test
compound. Then 150 μL of diluted test compound in SSB was
transferred to a UV plate (pION Inc.) and the UV spectrum was
measured on a SPECTRAmax PLUS plate reader (Molecular Devices)
to establish a reference plate. The membrane on a preloaded PAMPA
sandwich (pION Inc.) was painted with 4 μL of GIT lipid (pION Inc.).
The acceptor chamber was then filled with 200 μL of ASB (acceptor
solution buffer, pION Inc.), and the donor chamber was filled with
180 μL of test compound diluted in SSB. The PAMPA sandwich (donor
and acceptor chambers) was assembled, placed on the Gut-box (pION
Inc.), and stirred for 30 min. The aqueous boundary layer was set to 40
μM for stirring, and the UV spectra (250ꢀ500 nm) of the donor and the
acceptor chambers were read. A single experiment was performed in
triplicate for each compound. The permeability coefficient was calcu-
lated using PAMPA Evolution 96 Command software (pION Inc.)
based on the AUC of the reference, donor, and acceptor plates.
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’ ASSOCIATED CONTENT
(13) Madrid, P. B.; Liou, A. P.; DeRisi, J. L.; Guy, R. K. Incorporation
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S
Supporting Information. Biological data (antimalarial,
b
cytotoxicity in mammalian cells, solubility, and permeability), 1H
NMR spectra, and purity of compounds. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (901) 595-5714. Fax: (901) 595-5715. E-mail: kip.guy@
stjude.org.
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’ ACKNOWLEDGMENT
This work was supported by NIH/NIAID (Grant Al075517),
the American Lebanese Syrian Associated Charities (ALSAC),
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CQ, chloroquine; MDR, multidrug resistant; PAMPA, parallel
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