Antimalarial activity of novel 5-aryl-8-aminoquinoline derivatives

Article abstract of DOI:10.1021/jm100911f

In an attempt to separate the antimalarial activity of tafenoquine (3) from its hemolytic side effects in glucose-6-phosphate dehydrogenase (G6PD) deficiency patients, a series of 5-aryl-8-aminoquinoline derivatives was prepared and assessed for antimalar

Full text of DOI:10.1021/jm100911f

J. Med. Chem. 2011, 54, 131–142 131
DOI: 10.1021/jm100911f
Antimalarial Activity of Novel 5-Aryl-8-Aminoquinoline Derivatives
Hiroaki Shiraki, Michael P. Kozar, Victor Melendez, Thomas H. Hudson, Colin Ohrt, Alan J. Magill, and Ai J. Lin*
Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue,
Silver Spring, Maryland 20910, United States
Received July 20, 2010
In an attempt to separate the antimalarial activity of tafenoquine (3) from its hemolytic side effects in
glucose-6-phosphate dehydrogenase (G6PD) deficiency patients, a series of 5-aryl-8-aminoquinoline
derivatives was prepared and assessed for antimalarial activities. The new compounds were found
metabolically stable in human and mouse microsomal preparations, with t1/2 > 60 min, and were equal
to or more potent than primaquine (2) and 3 against Plasmodium falciparum cell growth. The new agents
were more active against the chloroquine (CQ) resistant clone than to the CQ-sensitive clone. Analogues
with electron donating groups showed better activity than those with electron withdrawing substituents.
Compounds 4bc, 4bd, and 4be showed comparable therapeutic index (TI) to that of 2 and 3, with TI
ranging from 5 to 8 based on IC50 data. The new compounds showed no significant causal prophylactic
activity in mice infected with Plasmodium berghei sporozoites, but are substantially less toxic than 2 and
3 in mouse tests.
Introduction
researchers in recent years.9-13 However, the identities of the
toxic species and the mechanism underlying hemotoxicity
have remained unclear due to instability of the metabolites.
In general, putative 5-hydroxy-8-aminoquinolines are thought
to be responsible for the hemotoxic compounds.9-13 These
metabolites are capable of forming semiquinoneimine radical
and iminoquinolinone under physiological conditions, leading
to the subsequent generation of hydrogen peroxide and oxida-
tive stress in erythrocytes14 (Scheme 1). To avoid the quinonei-
mine metabolite formation and thus the hemolytic side effects,
a series of derivatives of 2 were reported over the years.3b,15-17
However, the new derivatives of 2 either lost antimalarial
activity or retained both activity and hemolytic toxicity.
Tafenoquine (3) is a 5-phenoxyl derivative of primaquine.
The drug is generally less toxic and has a longer plasma half-
life (2-3 weeks) than 2.18 In addition, 3 has been shown to be
at least 10 times more potent than 2.19-21 One of the reasons
by which 3 is less toxic than the other 8-AQ derivatives is
thought to be the resistance of the 5-(m-trifluoromethylphenyl)
group to enzymatic cleavage to generate the phenolic metabo-
lite which in turn forms the semiquinoneimine.22 However, 3
also exhibited the toxic side effects of hemolysis and methe-
moglobinemia, although to a lesser extent than 2.18,19 The
results suggest that the 3-trifluoromethylphenyloxyl group at
the 5-position of 3may be hydrolyzed to form an 8-iminoquino-
line derivative (3b and 3b0) in vivo as shown in Scheme 2.
Indeed, extensive metabolic conversion of 3 to putative meta-
bolites, 3b-c, 3b0-c0, in rat liver microsomes was reported.22
However, no information is available on how this transforma-
tion occurs.
Malaria is a life-threatening parasitic disease responsible
for 300-500 million acute illnesses and 1-3 million deaths
annually. The severity of the disease is illustrated by the fact
that malaria kills approximately one child under the age of 5
years every 30 s, or 3000 per day.1 Although many effective
antimalarials have been developed sofar, the extent ofmalaria
infection is worsening, mainly due to the rapid spread of drug
resistant malarial strains in many parts of the world.
The 8-aminoquinoline (8-AQa) antimalarials,2-4 such as
pamaquine (1) and primaquine (2), have attracted much
interest as chemotherapeutic and prophylactic agents against
the liver stages of Plasmodium vivax and Plasmodium falcipa-
rum malarias (Figure 1). The 8-AQs are the only FDA ap-
proved drugs for the treatment of relapses in Plasmodium
infections. However, the clinical value of this class is compro-
mised by the toxic side effects, namely, methemoglobinemia
and hemolytic anemia in patients with deficiency in glucose-
6-phosphate dehydrogenase (G6PD) activity.5,6
G6PD plays a crucial role in the oxidant defense system of
red blood cells. G6PD is required for the reduction of NADP
to NADPH, which, in turn, is needed for the maintenance of
glutathione in the reduced form (GSH). Because GSH works
to remove the oxidant stress which leads to cell damage,
the lack of G6PD activity enhances the sensitivity of the red
cells to the oxidant assault.7 It is generally believed that the
metabolites of 8-AQ are the toxic species to erythrocytes,
not parent compounds, at clinically relevant concentration.8
The hemolytic toxicity and toxic metabolites identification of
8-AQ antimalarials have attracted broad interest among
From the chemistry point of view, direct nucleophilic
displacement of the 3-trifluoromethylphenyloxyl group at
the 5-position of tafenoquine by water or other biological
substances is not likely to take place until tafenoquine is
metabolically oxidized to the corresponding 5-aryloxonium
*To whom correspondence should be addressed. Phone: 301-319-
9084. Fax: 301-319-9449. E-mail: ai.lin@us.army.mil.
a Abbreviations: CQ, chloroquine; TQ, tafenoquine; PQ, primaquine;
G6PD, glucose-6-phosphate dehydrogenase; 8-AQ, 8-aminoquinoline;
EE, exoerythrocytic; MQ, mefloquine.
r
2010 American Chemical Society
Published on Web 12/08/2010
pubs.acs.org/jmc

132 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shiraki et al.
Figure 1. Chemical structures of 8-aminoquinoline antimalarials.
of the new compounds were prepared with the substituent
at the 2-position of the 8-AQ being either a 2-methoxy or
2-trifluoromethyl group. The Suzuki coupling reaction was
used as the key step to introduce the aryl substituent at the
5-position of the 8-AQ ring, instead of displacement of the
5-chloro-8AQ with a phenolic nucleophile as in the synthesis of
5-aryloxy- analogues. Different intermediates, 8a or 8b, were
used to prepare the two subclasses of the final products 4aa-af
and 4ba-be. The intermediate 2,6-dimethoxy-4-methylquino-
line (8a) was obtained by treatment of the commercially avail-
able starting material, 2-chloro-6-methoxy-4-methylquinoline
(6), with sodium methoxide. The intermediate 8b was prepared
by annulations of commercially available p-anisidine (7) and
1,1,1-trifluoromethyl-2,4-pentanedione under the catalysis of
polyphosphoric acid.
Scheme 1. Proposed Pathways for Free Radical, Hydrogen
Peroxide, and Methemoglobin Production by PQ in the Ery-
throcyte (Adapted from ref 14)
Although the annulation reaction may yield two isomeric
products, 2-CF3- and 4-CF3-quinolines, the desired 2-CF3
isomer was the predominant product of the reaction. The
structure of 8b was established by 2-D NMR data. The cor-
relation between the 4-methyl-AQ singlet at 2.73 ppm and the
5-H-AQ singlet at 7.19 ppm as well as the correlation between
the 5-H-AQ singlet at 7.19 ppm and the 6-OCH3-AQ singlet
(3.98 ppm) on NOESY are shown in Figure 2. Chlorination
of 8a and 8b was achieved using sulfuryl chloride to give 9a
and 9b in 73% and 79% yields, respectively. Subsequent
nitration of 9a and 9b using phosphorus pentoxide and
potassium nitrate gave 5-chloro-2,6-dimethoxy-4-methyl-
8-nitroquinoline (10a) and 5-chloro-6-methoxy-4-methyl-8-ni-
tro-2-trifluoromethyl-quinoline (10b) in 73% and 47% yield,
respectively. Compound 10a was a known compound. The
newly synthesized10a was identicaltothe reported compound
in NMR and melting point.23,24 Compound 10b, however, is a
new compound. The position of the trifluoromethyl group at
the 2-position and the nitro group at the 8-position were
established by the correlation of the 4-methyl peak at 3.20
ppm and the carbon-8 carbon peak at 144.03 ppm on HMBC
as shown in Figure 2.
The 5-aryl-2,6-dimethoxy-4-methyl-8-nitroquinolines 11aa-af
was prepared in good to excellent yield from 2-methoxy-
5-chloro-8-aminoquinoline 10a by Suzuki coupling reactions
with a series of arylboronic acids.24,25 2-Trifluoromethylqui-
noline derivatives 10b, under the same Suzuki coupling reac-
tion conditions, gave slightly lower yields of 11ba-be. It was
noted that a phase transfer catalyst is essential for smooth
Suzuki coupling reactions between 10a and phenylboronic
acid.26 The yield of the reaction was poor and the majority of
the starting material 10a was recovered unchanged when no
phase transfer catalyst was used. Furthermore, separation
of the byproduct from the desired product by column chro-
matography was tedious and frustrating. However, when
the reaction was carried out with a phase transfer catalyst,
quinoneimine (3a) as shown in Scheme 2. 5-Aryloxonium
quinoneimine 3a is expected to be hydrolyzed readily to the
toxic quinoneimine 3b and/or 3b0. It is recognized that the
antimalarial efficacy as well as the toxicity of 8-AQ antima-
larials may be derived from the formation of quinoneimine
metabolites.8-14 However, there is no convincing evidence to
substantiate this contention.
In this study, a series of new 5-aryl substituted 8-AQ
derivatives 4 and 5 (Scheme 2) were designed, prepared, and
tested in an attempt to probe the possibility of separating the
antimalarial activity from the hemolytic toxicity of 8-AQ.
The chemical structure of the new compounds 4 and 5 are
almost identical to the corresponding 5-aryloxy analogues
of 3, except missing an oxygen atom at the 5-position of the
quinoline ring. It is anticipated that the new compounds will
retain the antimalarial activity of tafenoquine, if the parent
molecule is the active species, and not the metabolites. As far
as the new compounds are expected to be metabolically stable
and generate no toxic semiquinoimine metabolites like that
of 2 and 3, they may be devoid of hemolytic side effects, if
metabolites are responsible for the toxicity.
Chemistry
The new deoxo-tafenoquine analogues 4aa-4af, 4ba-4be,
and 5bb-5be were prepared as shown in Scheme 3. The
procedure for the preparation of 3 was adapted to prepare
the new compounds designed in this study.23,24 Two subgroups

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 133
Scheme 2. Proposed Metabolic Pathway of Tafenoquine (3) to Quinoneimines (3b and 3b0)
tetrabutylammonium bromide (TBAB),27,28 the yield of the
desired product 11aa improved and the byproduct formation
decreased greatly. Catalytic hydrogenation of 11aa-11af and
11ba-11be, using palladium on activated carbon or slurry
of Raney nickel in water, afforded the 8-aminoquinolines
12aa-12af or 12ba-12be in near quantitative yields. The
amines obtained were pure enough for the next step reactions
without further purification.
With the side chain terminal amino group of 4bb-be masked
by t-Boc, all 4 carbamates 5bb-be are crystalline, form no
hydrate, and gave good elemental analysis results. The struc-
tures of all compounds were characterized by LC/MS, infrared
1
(IR), H- and 13C- NMR spectrometry. Elemental analyses
were performed only on final products.
Experimental Section
Incorporation of the amino-alkyl side chain to the 8-amino
group of 12aa-af and 12ba-be was achieved by treatment of
the 8-amino-AQ with either (i) 2-(4-iodopentyl)-1,3-isoindo-
linedione (13) and diisopropylamine23 or (ii) 2-(4-oxopentyl)-
1,3-isoindolinedione (14) and borane-pyridine complex.24
Because the intermediate 13 and 14 were both unstable under
basic or acidic conditions, the coupling reactions gave poor
yields of 15aa-af or 15ba-be and numerous side products
when a 1:1 ratio of the starting material and the reagent was
used. However, moderate to excellent yields of the desired
products 15aa-af or 15ba-be was obtained when excess
iodide 13 or ketone 14 (>2 mol equiv) was added in small
portions until the starting materials 12aa-af or 12ba-be was
consumed.
The final products, 2-methoxyquinoline compounds 4aa-af,
were obtained by treatment of 15aa-af with hydrazine mono-
hydrate to yield 4aa-af as gummy oil which was converted to
succinic acid salts. The succinic acid salts were purified by
recrystallization from acetonitrile. Utilizing the same method,
2-trifluoromethylquinoline analogues 4ba-be were obtained as
a solid free base from 15ba-be. Like the 8-AQ derivatives, i.e., 2
and 3, the deoxo-TQ analogues, 4aa-af and 4ba-be, are
gummy and the succinic acid salts are hygroscopic. Thus, good
elemental analysis results were difficult to obtain without
adjustment for H2O or solvents, especially 4bb-bd. The gummy
and hygroscopic problems were solved by conversion of the
final products to the crystalline carbamate derivatives 5bb-be.
Melting points were determined in open capillary tubes on an
OptiMelt melting point apparatus (Standard Research Systems,
USA) and are uncorrected. 1H NMR and 13C NMR spectra were
recorded using Bruker Avance-300 and Bruker Avance 600
spectrometers (Bruker Instrument, Inc., Wilmington, DE). Chem-
ical shifts are given in ppm (δ) relative to tetramethylsilane(TMS)
as internal standard. Analytical thin-layer chromatography (TLC)
was performed using HPLC-HLF normal phase 150 μm silica gel
plates (Analtech, Newark, DE). Visualization of the developed
chromatogram was performed with UV absorbance. Flash chro-
˚
matography was conducted with silica gel 60 A (200-400 mesh)
from Sigma-Aldrich Co. Solvents and reagents obtained from
commercial sources were used without purification, unless noted.
Reactions were carried out under an inert atmosphere of nitrogen.
Elemental analysis was performed by Atlantic Microlab, Inc.
(Norcross, GA). Where analyses are indicated by symbols of the
elements, the analytical results obtained were within (0.4% of the
theoretical values. An LC/UV-vis/ion trap mass spectrometer was
employed for purity analysis and chromophore properties. The
system consisted of an Agilent 1100 series LC-UV/vis system in line
with a ThermoFinnigan (now Thermo Scientific; Waltham, MA)
LCQ MS equipped with electrospray ionization (ESI) source.
Samples were analyzed using shallow CH3CN: 1% HCOOH/
H2O gradients at analytical flow rates. The purity of the final
products was g 95%.
2, 6-Dimethoxy-4-methyl-quinoline (8a). A 25% NaOCH3/
CH3OH solution (55 mL, 240 mmol) was added to a solution
of 2-chloro-6-methoxy-4-methylquinoline (6) (10 g, 48 mmol)
in dry MeOH (120 mL). The resulting mixture was refluxed for

134 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shiraki et al.
Scheme 3. Synthetic Scheme of Compounds 4aa-4af, 4ba-4be, and 5bb-5be
Reagents and conditions: (i) NaOMe, MeOH, reflux, 42 h; (ii) 1,1,1-trifluoromethyl-2,4-pentanedione, polyphosphoric acid, 120 °C, 40 h; (iii)
SO2C12, AcOH, 60 °C, 10 min to 3 h; (iv) P2O5, KNO3, triethylphosphate, 35 °C, 30 min to 19 h; (v) Pd(OAc)2, ArB(OH)2, PPh3, Na2CO3,
tetrabutylammonium bromide, 1,2-dimethoxyethane, H2O, reflux, 14-39 h or Pd(PPh3)4, ArB(OH)2, Na2CO3, tetrabutylammonium bromide, 1,
2-dimethoxyethane, H2O, reflux, 5-20 h; (vi) H2, 10% Pd/C or Raney Ni, MeOH, or AcOEt, 2-24 h; (vii) 12aa-12af, (i-Pr)2NH, 27-49 h; (viii)
12ba-12be, BH3-pyridine, AcOH, 20-24 h; (ix) NH2NH2 H2O, EtOH, 1-3 h; (x) (t-Boc)2O, triethylamine, CHC13, 21-24 h.
3
heated to 120 °C. 1,1,1-Trifluoromethyl-2,4-pentanedione (19 g,
122 mmol) was added dropwise with stirring and heated at
120 °C for 40 h. On cooling, the solution was diluted with H2O
(150 mL) and the viscous suspension was basified with 20%
NaOHaqueous solution and extracted with CHCl3 (150 mL ꢀ 3).
The CHCl3 extracts were combined, washed with brine, dried
over Na2SO4, and concentrated in vacuo. The crude product
was purified with a silica gel column, eluted with hexane/CHCl3
(1: 3 v/v) mixed solvent, to give 14 g (69%) of the desired product
(8b) as a yellow solid. 1H NMR (CDCl3): δ 8.11 (d, J = 9.3 Hz,
1H), 7.54 (s, 1H), 7.45 (dd, J = 9.3 and 2.7 Hz, 1H), 7.19 (d, J =
2.7 Hz, 1H), 3.98 (s, 3H), 2.73 (s, 3H). 13C NMR (CDCl3): δ
159.0, 145.0, 142.8, 132.0, 130.0, 123.6, 123.0, 120.0, 117.6, 101.3,
55.5, 19.2. IR (neat): 1624, 1507, 1480, 1368, 1288, 1232, 1166,
1141, 1119, 1097, 1019, 927, 913, 871, and 841 cm-1. MS (ESI): m/z
242 [M þ 1]þ.
Figure 2. Determination of the structures of 8b and 10b.
19 h, followed by addition of another 22 mL of 25% CH3ONa/
CH3OH solution (96 mmol) and refluxed for an additional 24 h.
The reaction mixture was evaporated in vacuo to give a gum.
Water (100 mL) was added to the gummy residue, and the pH of
the suspension was adjusted to about 8 with 2N HCl. The
aqueous solution was extracted with CHCl3 (100 mL ꢀ 3),
and the CHCl3 extracts were combined, washed with brine,
dried over Na2SO4, and concentrated in vacuo to dryness. The
residue was purified with a silica gel column, eluted with 5%
EtOAc/hexane, to give 9.2 g (94%) of the desired product 8a as a
colorless solid. 1H NMR (CDCl3): δ 7.77 (d, J = 9.1 Hz, 1H),
7.27 (dd, J = 9.1 and 2.8 Hz, 1H), 7.15 (d, J = 2.8 Hz, 1H), 6.74
(s, 1H), 4.02 (s, 3H), 3.91 (s, 3H), 2.58 (s, 3H). IR (neat): 1611,
1577, 1514, 1465, 1448, 1430, 1413, 1344, 1240, 1198, 1185, 1169,
1125, 1033, 987, 955, 918, 875, and 832 cm-1. MS (ESI): m/z 204
[M þ 1]þ.
5-Chloro-2,6-dimethoxy-4-methyl-quinoline (9a). A solution
of compound 8a (9.0 g, 44 mmol) in glacial acetic acid (80 mL)
was heated to 60 °C. To the solution, sulfuryl chloride (9.0 g,
66 mmol) in glacial acetic acid (20 mL) was added dropwise over
20 min. The resulting solution was stirred for 10 min at 60 °C and
then poured into 50 mL of ice-water. The AcOH/H2O mixture
was evaporated to dryness under reduced pressure to give a
crude product which was dissolved in 100 mL of H2O, basified
with 20% NaOH, and extracted with CHCl3 (100 mL ꢀ 3). The
combined CHCl3 extracts were washed with brine, dried over
Na2SO4, and concentrated in vacuo to give 7.7 g (73%) of the
desired product 9a as a brown solid. 1H NMR (CDCl3): δ 7.76
(d, J = 9.2 Hz, 1H), 7.34 (d, J = 9.2 Hz, 1H), 6.71 (s, 1H), 4.01
6-Methoxy-4-methyl-2-trifluoromethyl-quinoline (8b). p-Ani-
sidine (7) (10 g, 81 mmol) in polyphosphoric acid (60 g) was

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Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 135
(s, 3H), 3.98 (s, 3H), 2.97 (s, 3H). IR (neat): 1648, 1603, 1585,
1517, 1473, 1378, 1366, 1353, 1339, 1326, 1280, 1269, 1227, 1199,
1183, 1096, 1067, 1057, 1041, 872, and 822 cm-1. MS (EI): m/z
237 [M]þ.
2,6-Dimethoxy-4-methyl-8-nitro-5-(3-trifluoromethylphenyl)-
quinoline (11ab). The same procedure was used to prepare 11ab,
using 3-trifluoromethylphenylboronic acid as the starting ma-
terial. Yield: 99%. 1H NMR (CDCl3): δ 7.80 (d, J = 7.7 Hz, 1H),
7.64 (s, 1H), 7.57 (t, J = 7.7 Hz, 1H), 7.51 (s, 1H), 7.44 (d, J = 7.7
Hz, 1H), 6.71 (s, 1H), 4.00 (s, 3H), 3.75 (s, 3H), 1.76 (s, 3H). MS
(EI): m/z 392 [M]þ.
2,6-Dimethoxy-4-methyl-8-nitro-5-(4-trifluoromethylphenyl)-
quinoline (11ac). The title compound was prepared by the same
procedure as described for the preparation of 11aa, using 4-tri-
fluoromethylphenylboronic acid as the starting material. Yield:
86%. 1H NMR (CDCl3): δ 7.71 (d, J = 8.0 Hz, 2H), 7.64 (s, 1H),
7.38 (d, J = 8.0 Hz, 2H), 6.71 (s, 1H), 3.99 (s, 3H), 3.78 (s, 3H),
1.79 (s, 3H). MS (EI): m/z 392 [M]þ.
5-Chloro-6-methoxy-4-methyl-2-trifluoromethyl-quinoline (9b).
Same method to prepare 9a was used to prepare 9b from 8b (22 g,
91 mmol) and sulfuryl chloride (19 g, 137 mmol) to give crude
solid product which was washed with hexane/CHCl3 (1:3 v/v)
mixed solvent several times to give 14 g (56%) of 9b as a pale-
yellow solid. The filtrate and the washing solutions were com-
bined, evaporated to dryness in vacuo, and applied to a silica gel
column. Upon elution with hexane and CHCl3 (1:3 v/v) mixed
solvent, additional desired product 9b (6 g, 23%) was obtained.
1H NMR (CDCl3): δ 8.14 (d, J = 9.3 Hz, 1H), 7.56 (d, J = 9.3
Hz, 1H), 7.48 (s, 1H), 4.07 (s, 3H), 3.13 (s, 3H). IR (neat): 1508,
1468, 1434, 1382, 1373, 1271, 1187, 1175, 1130, 1114, 1091, 1050,
942, 920, 873, and 831 cm-1. MS (EI): m/z 277 [M]þ.
5-Chloro-2,6-dimethoxy-4-methyl-8-nitro-quinoline (10a). Phos-
phorus pentoxide (15.7 g, 110 mmol) was added to a solution of
9a (7.7 g, 32 mmol) in triethylphosphate (90 mL). The pale-yellow
suspension was stirred for 1 h at rtand then heated to 35 °C. Tothe
suspension, KNO3 (6.6 g, 65 mmol) and MeOH (60 mL) were
added successively and the resulting mixture was stirred and
refluxed for 15 min. After cooling with an ice-bath, the crude
product was collected, washed successively with H2O (80 mL ꢀ 3)
and MeOH (80 mL ꢀ 1), and dried under vacuum at 56 °C to
give 7.4 g (73%) of the desired product, mp 200-201 °C (lit.23,24
199-200 °C). 1H NMR (CDCl3): δ 7.62 (s, 1H), 6.83 (s, 1H), 4.02
(s, 3H), 3.98 (s, 3H), 3.00 (s, 3H). IR(neat): 1606, 1534, 1518, 1467,
1375, 1352, 1321, 1272, 1193, 1174, 1063, 1054, 978, 895, 882, 860,
and 780 cm-1. MS (EI): m/z 282 [M]þ.
2, 6-Dimethoxy-5-(3-methoxyphenyl)-4-methyl-8-nitro-quino-
line (11ad). Compound 11ad was prepared by the same general
procedure using 3-methoxyphenylboronic acid as the start-
1
ing material. Yield: 62%. H NMR (CDCl3): δ 7.64 (s, 1H),
7.38-7.32 (m, 1H), 6.99-6.96 (m, 1H), 6.83-6.81 (m, 1H),
6.79-6.78 (m, 1H), 6.68 (s, 1H), 3.99 (s, 3H), 3.83 (s, 3H), 3.79
(s, 3H), 1.88 (s, 3H). IR (neat): 1606, 1533, 1519, 1473, 1455,
1437, 1376, 1292, 1266, 1193, 1158, 1059, 1046, 775, and
700 cm-1. MS (EI): m/z 354 [M]þ.
2, 6-Dimethoxy-4-methyl-5-(3-methylphenyl)-8-nitro-quino-
line (11ae). The same general procedure was used to prepare
11ae using 3-methylphenylboronic acid as the starting material.
Yield: 64%. 1H NMR (CDCl3): δ 7.65 (s, 1H), 7.35-7.24
(m, 2H), 7.06 (s, 1H), 7.04 (s, 1H), 6.69 (s, 1H), 4.01 (s, 3H),
3.76 (s, 3H), 2.41 (s, 3H), 1.83 (s, 3H). IR (neat): 1604, 1535, 1442,
1371, 1345, 1324, 1196, 1159, 1064, 1049, 773, and 704 cm-1. MS
(ESI): m/z 339 [M þ 1]þ.
5-(3,4-Dichlorophenyl)-2,6-dimethoxy-4-methyl-8-nitro-quin-
oline (11af). Compound 11af was prepared by the same proce-
dure using 3,4-dichlorophenylboronic acid as the starting
5-Chloro-6-methoxy-4-methyl-8-nitro-2-trifluoromethyl-quin-
oline (10b). Phosphorus pentoxide (23 g, 160 mmol) was added
to a solution consisting of 9b (13 g, 47 mmol) and triethylphos-
phate (100 mL). The pale-yellow suspension was stirred at rt for
1 h, heated to 35 °C, and followed by addition of KNO3 (9.5 g, 95
mmol). The reaction mixture was stirred at 35 °C for addition-
al 19 h and poured into an excess amount of ice-water (ca.
200 mL). The resulting slurry was kept at 4 °C for 1 h and the
precipitates were collected. The crude product was dissolved in
water (ca. 150 mL), basified with 20% NaOH, and extracted
with CHCl3 (100 mL ꢀ 3). The CHCl3 extracts were combined,
washed with brine, dried over Na2SO4, and concentrated in
vacuo. The crude product was purified by a silica gel column,
eluted with hexane and CHCl3 (1:3 v/v) mixed solvent, to give
1
material. Yield: 99%. H NMR (CDCl3): δ 7.63 (s, 1H), 7.53
(d, J = 8.2 Hz, 1H), 7.36 (d, J = 2.0 Hz, 1H), 7.10 (dd, J = 8.2
and 2.0 Hz, 1H), 6.73 (s, 1.0 H), 4.00 (s, 3H), 3.80 (s, 3H), 1.90 (s,
3H). IR (neat): 1606, 1532, 1373, 1273, 1193, 1166, 1068, 827,
793, and 697 cm-1. MS (EI): m/z 392 [M]þ.
General Procedure for the Preparation of 5-Aryl-6-methoxy-
4-methyl-8-nitro-2-trifluoro-methylquinoline (11ba-be). Suzuki
coupling reaction as described for the synthesis of 11aa-af was
used to prepare compounds 11ba-be from compound 10b (2.0
g, 6.2 mmol) and an appropriate arylboronic acid.
6-Methoxy-4-methyl-8-nitro-5-phenyl-2-trifluoromethyl-quin-
oline (11ba). Compound 11ba was obtained as dark-brown oil
by the general procedure using phenylboronic acid as reagent.
Yield: 73%. 1H NMR(CDCl3): δ 8.68 (s, 1H), 7.53-7.48 (m, 4H),
7.40-7.35 (m, 2H), 3.52 (s, 3H), 2.02 (s, 3H). IR (neat): 1539,
1431, 1362, 1269, 1201, 1187, 1139, 1038, 936, 847, 805, 762, and
754 cm-1. MS (EI): m/z 362 [M]þ.
1
7.2 g (47%) of the desired product 10b. H NMR (CDCl3): δ
8.57 (s, 1H), 7.68 (s, 1H), 4.13 (s, 3H), 3.20 (s, 3H). 13C NMR
(CDCl3): δ 148.8, 146.6, 144.0, 130.2, 127.4, 126.3, 122.9,
120.0, 115.9, 62.9, 25.9. IR (neat): 1542, 1424, 1365, 1262,
1183, 1128, 1096, 1036, 981, 935, 896, and 859 cm-1. MS (EI):
m/z 320 [M]þ.
General Synthetic Procedure for 5-Aryl-2,6-dimethoxy-
4-methyl-8-nitro-quinoline (11aa-af). Compound 10a (4.2 g,
15 mmol) was dissolved in dry dimethoxyethane (126 mL). To
the solution was added palladium acetate (0.25 g, 1.0 mmol),
phenylboronic acid (2.4 g, 19 mmol), triphenylphosphine (0.6 g,
2.0 mmol), tetrabutylammonium bromide (1.0 g, 3.0 mmol), and
Na2CO3 (3.2 g, 30 mmol) in H2O (10 mL). The suspension was
refluxed for 23 h, and additional tetrabutylammonium bromide
(38.4 g, 2.4 mmol) was added. The mixture was further refluxed
for 16 h and the solvents were evaporated to dryness in vacuo. A
saturated aqueous solution of NaHCO3 (50 mL) was added
to the dry residue and the mixture was extracted with CHCl3
(100 mL ꢀ 3). The combined organic layers were washed with
brine, dried over Na2SO4, and concentrated. The crude product
was purified using a silica gel column, eluted with CHCl3 and
hexane (1:1 v/v) mixed solvent, to give 4.4 g (91%) of the desired
product 11aa as a pale-brown solid. 1H NMR (CDCl3): δ 7.05
(s, 1H), 7.44-7.42 (m, 3H), 7.26-7.21 (m, 2H), 6.67 (s, 1H), 3.99
(s, 3H), 3.77 (s, 3H), 1.80 (s, 3H). MS (EI): m/z 324 [M]þ.
6-Methoxy-4-methyl-8-nitro-2-trifluoromethyl-5-(3-trifluoro-
methylphenyl)-quinoline (11bb). Compound 11bb was obtained
as a yellow solid by the same procedure using 3-trifluoromethyl-
phenylboronic acid as reagent. Yield: 44%. 1H NMR (CDCl3):
δ 8.75 (s, 1H), 7.83-7.81 (m, 1H), 7.71-7.53 (m, 4H), 3.53
(s, 3H), 1.99 (s, 3H). IR (neat): 1538, 1315, 1266, 1166, 1127,
1095, 1074, 1037, and 806 cm-1. MS (EI): m/z 430 [M]þ.
6-Methoxy-4-methyl-8-nitro-2-trifluoromethyl-5-(4-trifluoro-
methylphenyl)-quinoline (11bc). The title compound was ob-
tained as red oil by the same general procedure using 4-trifluoro-
1
methylphenyl-boronic acid as reagent. Yield: 62%. H NMR
(CDCl3):δ8.72 (s, 1H), 7.79 (d, J= 8.1 Hz, 2H), 7.55-7.50 (m, 3H),
3.53 (s, 3H), 2.01 (s, 3H). IR (neat): 1541, 1324, 1271, 1126, 1110,
1067, 1036, and 934 cm-1. MS (ESI): m/z 431 [M þ 1]þ.
6-Methoxy-5-(3-methoxyphenyl)-4-methyl-8-nitro-2-trifluoro-
methyl-quinoline (11bd). Compound 11bd was obtained as yel-
low solid by the same procedure for the preparation of 11be
using 3-methoxyphenylboronic acid as reagent. Yield: 64%. 1H
NMR (CDCl3): δ 8.67 (s, 1H), 7.53 (s, 1H), 7.44-7.39 (m, 1H),

136 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shiraki et al.
7.07-7.04 (m, 1H), 6.92-6.91 (m, 2H), 3.86 (s, 3H), 3.57 (s, 3H),
2.10 (s, 3H). IR (neat): 1548, 1468, 1430, 1274, 1254, 1150, 1136,
1115, 1033, 916, 791, and 648 cm-1. MS (EI): m/z 392 [M]þ.
6-Methoxy-4-methyl-5-(3-methylphenyl)-8-nitro-2-trifluoro-
methyl-quinoline (11be). Same method was used to prepare 11be,
20% EtOAc in hexane, to give 12be in a combined total of 1.4 g
(89%). 1H NMR (CDCl3): δ 7.44-7.15 (m, 6H), 4.38 (br s, 2H),
3.46 (s, 3H), 2.42 (s, 3H), 1.92 (s, 3H). IR (neat): 1636, 1471,
1385, 1370, 1257, 1164, 1139, 1107, 1005, 855, and 786 cm-1. MS
(ESI): m/z 347 [M þ 1]þ.
1
8-Amino-6-methoxy-4-methyl-5-phenyl-2-trifluoromethyl-quin-
oline (12ba). Compound 12ba was obtained by reduction of 11ba
according to the same procedure for the preparation of 12be.
Yield: 82%. 1H NMR (CDCl3): δ 7.47-7.45 (m, 4H), 7.37-7.36
(m, 2H), 7.16 (s, 1H), 3.44 (s, 3H), 1.91 (s, 3H). IR (neat): 1628,
1468, 1447, 1424, 1254, 1178, 1135, 1103, 1007, 936, 755, 739, and
704 cm-1. MS (EI): m/z 332 [M]þ.
using 3-methylphenylboronic acid as reagent. Yield: 78%. H
NMR (CDCl3): δ 8.65 (s, 1H), 7.52 (s, 1H), 7.39 -7.31 (m, 2H),
7.18-7.15 (m, 2H), 3.53 (s, 3H), 2.43 (s, 3H), 2.04 (s, 3H). IR
(neat): 1543, 1537, 1370, 1267, 1227, 1192, 1133, 1039, 1032, and
791 cm-1. MS (EI): m/z 376 [M]þ.
General Procedure for the Preparation of 8-Amino-5-aryl-2,
6-dimethoxy-4-methyl-quinoline (12aa-af). A solution of com-
pound 11aa (1.0 g, 3.1 mmol) in CH3OH (200 mL) with a cat-
alytic amount of 10% palladium on activated carbon (217 mg,
0.9 mmol) was hydrogenated under 50 psi pressure at rt for 2 h.
The reaction mixture was filtered through a layer of Celite, and
the yellow solution was evaporated in vacuo to give 0.9 g (99%)
of the crude product 12aa, which was pure enough for the next
step reaction without further purification. Yield: 99%. 1H NMR
(CDCl3): δ 7.35-7.31 (m, 3H), 7.27-7.24 (m, 2H), 6.77 (s, 1H),
6.59 (s, 1H), 4.92 (br s, 2H), 4.02 (s, 3H), 3.70 (s, 3H), 1.79
(s, 3H). MS (EI): m/z 294 [M]þ.
8-Amino-2,6-dimethoxy-4-methyl-5-(3-trifluoromethylphenyl)-
quinoline (12ab). Compound 12ab was prepared by catalytic re-
duction of 11ab as described for the preparation of 12aa except
that the reduction was conducted under atmospheric pressure.
Yield: 99%. 1H NMR (CDCl3): δ 7.61-7.45 (m, 4H), 6.75
(s, 1H), 6.52 (s, 1H), 4.97 (br s, 2H), 4.02 (s, 3H), 3.69 (s, 3H),
1.75 (s, 3H). MS (EI): m/z 362 [M]þ.
8-Amino-2,6-dimethoxy-4-methyl-5-(4-trifluoromethylphenyl)-
quinoline (12ac). The title compound was prepared by catalytic
reduction of 11ac. Yield: 94%. 1H NMR (CDCl3): δ 7.62 (d, J =
7.8 Hz, 2H), 7.38 (d, J = 7.8 Hz, 2H), 6.77 (s, 1H), 6.62 (s, 1H),
4.02 (s, 3H), 3.69 (s, 3H), 1.77(s, 3H). MS(ESI):m/z 363 [M þ 1]þ.
8-Amino-2,6-dimethoxy-5-(3-methoxyphenyl)-4-methyl-quin-
oline (12ad). Compound 12ad was prepared by the same pro-
cedure from 11ad, except EtOAc was used as solvent. Yield: 99%.
1H NMR (CDCl3): δ 7.30-7.25 (m, 1H), 6.91-6.81 (m, 3H), 6.77
(s, 1H), 6.60 (s, 1H), 4.92 (br s, 2H), 4.01 (s, 3H), 3.81 (s, 3H), 3.71
(s, 3H), 1.86 (s, 3H). MS (EI): m/z 324 [M]þ.
8-Amino-6-methoxy-4-methyl-5-(3-trifluoromethylphenyl)-2-tri-
fluoromethyl-quinoline (12bb). The title compound was obtained
by catalytic hydrogenation of 11bb. Yield: 63%. 1HNMR(CDCl3):
δ 7.75-7.73 (m, 1H), 7.65-7.53 (m, 3H), 7.49 (s, 1H), 7.18 (s, 1H),
4.36 (br s, 2H), 3.42 (s, 3H), 1.87 (s, 3H). IR (neat): 1628, 1471,
1351, 1324, 1313, 1252, 1156, 1127, 1103, 1001, 900, 856, and
808 cm-1. MS (ESI): m/z 401 [M þ 1]þ.
8-Amino-6-methoxy-4-methyl-5-(4-trifluoromethylphenyl)-2-tri-
fluoromethyl-quinoline (12bc). Title compound was obtained as
yellow solid from 11bc by the same reduction procedure. The
crude product was pure enough for the next reaction without fur-
ther purification. 1H NMR (CDCl3): δ 8.23 (s, 1H), 7.73 (d, J =
6.9 Hz, 2H), 7.50 (d, J = 6.9 Hz, 2H), 7.49 (s, 1H), 3.42 (s, 3H),
1.89 (s, 3H). IR (neat): 1324, 1150, 1123, 1109, 1104, 1067, 836,
784, 770, 748, and 718 cm-1. MS (ESI): m/z 401 [M þ 1]þ.
8-Amino-6-methoxy-4-methyl-5-(3-methoxyphenyl)-2-triflu-
oromethyl-quinoline (12bd). The title compound was obtained by
catalytic hydrogenation of 11bd. Yield: 99%. 1H NMR (CDCl3): δ
7.45 (s, 1H), 7.39-7.34 (m, 1H), 7.16 (s, 1H), 7.01-6.90 (m, 3H),
4.38 (br s, 1H), 3.84 (s, 3H), 3.49 (s, 3H), 1.98 (s, 3H). IR (neat):
1470, 1252, 1177, 1150, 1136, 1102, 1032, 916, 860, 790, and
728 cm-1. MS (ESI): m/z 363 [M þ 1]þ.
General Procedure for the Preparation of 8-[(1-Methyl-
4-phthalimido)-butyl]-amino-5-aryl-2,6-dimethoxy-4-methyl-quino-
line (15aa-af). Compound 13 (1.9 g, 5.5 mmol) in N-methyl-
pyrrolidinone (8 mL) was added dropwise a solution consist of
compound 12aa (1.6 g, 5.5 mmol) and diisopropylamine (0.4 g,
6.1 mmol) in 2 mL of N-methylpyrrolidinone. The resulting
mixture was heated for 20 h at 80 °C. Additional amount of
compound 13 (1.9 g, 5.5 mmol) and diisopropylamine (0.4 g,
6.1 mmol) were added slowly, and the reaction solution was
heated for 8 more hours. After cooling to rt, the mixture was
basified with 50 mL of 2N NaOH aqueous solution. The mixture
was extracted with diethyl ether (100 mL ꢀ 3), and the combined
ether extracts were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was purified by a
silica gel column, eluted with 9% hexane in CHCl3, to give 2.1 g
(57%) of the desired product 15aa as red oil.
8-Amino-2,6-dimethoxy-4-methyl-5-(3-methylphenyl)-quinoline
(12ae). Compound 12ae was prepared from 11ae by the same
procedure. Yield: 99%. 1H NMR (CDCl3): δ 7.29-7.24 (m, 1H),
7.17-7.14 (m, 1H), 7.09-7.07 (m, 2H), 6.78 (s, 1H), 6.60 (s, 1H),
4.77 (br s, 2H), 4.03 (s, 3H), 3.71 (s, 3H), 2.38 (s, 3H), 1.82 (s, 3H).
MS (ESI): m/z 309 [M þ 1]þ.
8-Amino-5-(3,4-dichlorophenyl)-2,6-dimethoxy-4-methyl-quin-
oline (12af). Compound 12af was prepared by hydrogenation
over a slurry of Raney nickel in water instead of palladium on
activated carbon. Yield: 95%. 1H NMR (CDCl3): δ 7.44 (d, J =
8.2 Hz, 1H), 7.37 (d, J = 2.0 Hz, 1H), 7.12 (dd, J = 8.2 and 2.0
Hz, 1H), 6.74 (s, 1H), 6.64 (s, 1H), 4.99 (br s, 1H), 4.03 (s, 3H),
3.72 (s, 3H), 1.88 (s, 3H). MS (EI): m/z 362 [M]þ.
8-[(1-Methyl-4-phthalimido)-butyl]-amino-2,6-dimethoxy-
4-methyl-5-phenyl-quinoline (15aa). Yield: 57%. 1H NMR (CDCl3):
δ 7.83-7.80 (m, 2H), 7.71-7.68 (m, 2H), 7.37-7.31 (m, 3H), 7.26-
7.23 (m, 2H), 6.57 (s, 1H), 6.49 (s, 1H), 5.95 (d, J=8.3Hz, 1H), 3.99
(s, 3H), 3.78-3.70 (m, 3H), 3.70 (s, 3H), 1.92-1.71 (m, 4H), 1.77
(s, 3H), 1.33 (d, J = 6.3 Hz, 3H). MS (ESI): m/z 510 [M þ 1]þ.
8-[(1-Methyl-4-phthalimido)-butyl]-amino-2,6-dimethoxy-
4-methyl-5-(3-trifluoromethyl-phenyl)-quinoline (15ab). Compound
15ab was prepared by the same procedure from 12ab and 13. Yield:
64%. 1H NMR (CDCl3): δ 7.85-7.79 (m, 2H), 7.73-7.69 (m, 2H),
7.60-7.58 (m, 1H), 7.51-7.46 (m, 3H), 6.60 (s, 1H), 6.47 (s, 1H),
6.01 (d, J = 8.5 Hz, 1H), 4.00 (s, 3H), 3.72 (s, 3H), 3.79-3.72
(m, 3H), 1.98-1.73 (m, 4H), 1.73 (s, 3H), 1.35 (d, J = 6.3 Hz, 3H).
MS (ESI): m/z 578 [M þ 1]þ.
General Procedure for the Preparation of 8-Amino-5-aryl-
6-methoxy-4-methyl-2-trifluoromethyl-quinoline (12ba-be). A
solution of compound 11be (1.7 g, 4.5 mmol) in EtOAc (80 mL)
was subjected to hydrogenation, using 10% palladium on acti-
vated carbon (300 mg, 1.3 mmol) as catalyst under atmospheric
pressure of H2 gas for 18 h. The reaction mixture was filtered
through a layer of Celite, and the yellow filtrate was evaporated
to dryness under the reduced pressure. The residue was sus-
pended in H2O (100 mL), and the pH of the suspension was
adjusted to 8 with NaOH aqueous solution. The mixture was
extracted with CHCl3 (100 mL ꢀ 3) and the combined extracts
were combined, washed with brine, dried over Na2SO4, and
concentrated in vacuo to give 1.6 g of the crude product, which
was washed with a solvent mixture of 9% EtOAc in hexane to
give 0.8 g (51%) of the desired product 12be after drying.
Additional amounts of compound 12be (0.6 g, 35%) was
recovered from the filtrate, using a silica gel column eluted with
8-[(1-Methyl-4-phthalimido)-butyl]-amino-2,6-dimethoxy-
4-methyl-5-(4-trifluoromethyl-phenyl)-quinoline (15ac). The title
compound was prepared by the same procedure from 12ac and
13. Yield: 85%. 1H NMR (CDCl3): δ 7.84-7.81 (m, 2H),
7.72-7.09 (m, 2H), 7.61 (d, J = 7.8 Hz, 2H), 7.38 (d, J = 7.8 Hz,
2H), 6.60 (s, 1H), 6.48 (s, 1H), 6.01 (d, J = 8.2 Hz, 1H), 4.00

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 137
(s, 3H), 3.79-0.68 (m, 3H), 3.72 (s, 3H), 2.03-1.62 (m, 4H), 1.72
(s, 3H), 1.35 (d, J = 6.3 Hz, 3H). MS (ESI): m/z 578 [M þ 1]þ.
8-[(1-Methyl-4-phthalimido)-butyl]-amino-2,6-dimethoxy-
4-methyl-5-(3-methoxyphenyl)-quinoline (15ad). The title compound
was prepared by the same procedure from 12ad and 13. Yield: 55%.
1H NMR (CDCl3): δ 7.84-7.81 (m, 2H), 7.73-7.69 (m, 2H),
7.30-7.25 (m, 1H), 6.91-6.83 (m, 3H), 6.59 (s, 1H), 6.50 (s, 1H),
5.97 (d, J = 8.0 Hz, 1H), 4.00 (s, 3H), 3.82 (s, 3H), 3.82-3.73
(m, 3H), 3.73 (s, 3H), 2.00-1.66 (m, 4H), 1.97 (s, 3H), 1.35 (d, J =
6.3 Hz, 3H). MS (ESI): m/z 540 [M þ 1]þ.
8-[(1-Methyl-4-phthalimido)-butyl]-amino-6-methoxy-4-meth-
yl-2-trifluoromethyl-5-(4-trifluoromethylphenyl)-quinoline (15bc).
Compound 15bc was prepared from 12bc and 14 by the same
procedure as described for the preparation of 15be. Difficulty was
encountered on purification of the title compound. The crude
product was partially purified by silica gel column and used
for the next synthesis without further purification. 1H NMR
(CDCl3): δ 7.84-7.80 (m, 2H), 7.73-7.67 (m, 2H), 7.67-7.23
(m, 4H), 7.23 (s, 1H), 7.12 (s, 1H), 4.57 (br d, J = 8.7 Hz, 1H),
3.76-3.72 (m, 3H), 3.34 (s, 3H), 1.86 (s, 3H), 1.86-1.69 (m, 4H),
1.29 (d, J = 6.2 Hz, 3H). IR (neat): 1713, 1609, 1397, 1371, 1324,
1255, 1168, 1126, 1105, 1066, 934, and 721 cm-1. MS (ESI): m/z
616 [M þ 1]þ.
8-[(1-Methyl-4-phthalimido)-butyl]-amino-2,6-dimethoxy-
4-methyl-5-(3-methylphenyl)-quinoline (15ae). The title compound
was likewise prepared by the same method from 12ae and 13.
Yield: 46%. 1H NMR (CDCl3): δ 7.85-7.80 (m, 2H), 7.73-7.69
(m, 2H), 7.29-7.06 (m, 4H), 6.58 (s, 1H), 6.50 (s, 1H), 5.95
(d, J = 7.6 Hz, 1H), 4.00 (s, 3H), 3.80-3.73 (m, 3H), 3.73
(s, 3H), 2.37 (s, 3H), 2.02-1.66 (m, 4H), 1.80 (s, 3H), 1.35 (d, J =
6.3 Hz, 3H). MS (ESI): m/z 524 [M þ 1]þ.
8-[(1-Methyl-4-phthalimido)-butyl]-amino-6-methoxy-5-(3-
methoxy)-phenyl-4-methyl-2-trifluoromethyl-quinoline (15bd).
The title compound 15bd was obtained from 12bd by the same
procedure. Yield: 76%, 1H NMR (CDCl3): δ 7.83-7.79 (m, 2H),
7.70-7.67 (m, 2H), 7.35-7.33 (m, 1H), 7.19 (s, 1H), 7.10 (s, 1H),
7.00-6.89 (m, 3H), 4.58 (br. d, J = 8.4 Hz, 1H), 3.85 (s, 1.5 H),
3.83 (s, 1.5 H), 3.76-3.71 (m, 3H), 3.40 (s, 3H), 1.95 (s, 3H),
1.95-1.70 (m, 4H), 1.28 (d, J = 6.3 Hz, 3H). IR (neat): 1709,
1607, 1397, 1370, 1254, 1172, 1131, 1104, 755, and 719 cm-1. MS
(ESI): m/z 578 [M þ 1]þ.
8-[(1-Methyl-4-phthalimido)-butyl]-amino-2,6-dimethoxy-
4-methyl-5-(3,4-dichlorophenyl)- quinoline (15af). The title com-
pound was prepared by the same procedure from 12af and 13.
Yield: 62%. 1H NMR (CDCl3): δ 7.84-7.81 (m, 2H), 7.71-7.68
(m, 2H), 7.45-7.42 (m, 1H), 7.38-7.36 (m, 1H), 7.14-7.10
(m, 1H), 6.62 (s, 1H), 6.48 (s, 1H), 6.04 (d, J = 8.4 Hz, 1H), 4.01
(s, 3H), 3.80-3.75 (m, 3H), 3.75 (s, 3H), 2.00-1.72 (m, 4H), 1.86
(s, 3H), 1.36 (d, J = 6.3 Hz, 3H). MS (EI): m/z 577 [M]þ.
General Procedure for the Preparation of 8-[(1-Methyl-
4-phthalimido)-butyl]-amino-5-aryl-6-methoxy-4-methyl-2-trifluoro-
methyl-quinoline (15ba-be). Compound 12be (0.8 g, 2.3 mmol)
and borane-pyridine complex (0.8 g, 1.4 mmol) was added to a
solution of compound 14 (0.8 g, 3.5 mmol) in glacial acetic acid
(10 mL). The resulting mixture was stirred at rt for 2 h, and
additional compound 14 (2.4 g, 11 mmol) was added. The so-
lution was stirred at ambient temperature for 19 h and evapo-
rated to dryness in vacuo. The residue was suspended in H2O
(100 mL), basified with NaOH aqueous solution and extracted
with CHCl3 (100 mL) 3 times. The combined extracts were
washed with brine, dried over Na2SO4, and evaporated to
dryness under reduced pressure. The residue was purified by a
silica gel column, eluted with 20% EtOAc in hexane, to give 1.5 g
(99%) of the desired product 15be as yellow oil.
General Synthetic Procedure for 8-[(4-Amino-1-methyl)-
butyl]-amino-5-aryl-2,6-dimethoxy-4-methyl-quinoline Succinate (4aa-
af ). A solution of compound 15aa (1.6 g, 3.1 mmol) in EtOH (12 mL)
was treated with an excess amount of hydrazine monohydrate (0.7 g,
13.2 mmol) and refluxed for 30 min. Upon cooling to rt, the pre-
cipitates were removed by suction filtration and washed with EtOH
(5 mL ꢀ 4). The filtrate and the EtOH washing solutions were
combined and concentrated to dryness. The crude product was
dissolved in CH2Cl2 (50 mL), washed twice with 25 mL of 25%
KOH, and subsequently once with water (25 mL). The organic layer
was dried over Na2SO4 andconcentratedinvacuotogiveanoilwhich
was purified by a silica gel column, eluted with CH2Cl2 containing 2%
of MeOH and 2% of triethylamine, to give 0.9 g of the desired product
as yellow oil. The oil (540 mg, 1.4 mmol) was dissolved in CH3CN
(5 mL) and treated with a solution of succinic acid (168 mg, 1.4 mmol)
in CH3OH (1 mL) and CH3CN (3 mL) mixed solvent to give 368 mg
(41%) of 4aa as a succinate salt.
8-[(4-Amino-1-methyl)-butyl]-amino-2,6-dimethoxy-4-methyl-
1
8-[(1-Methyl-4-phthalimido)-butyl]-amino-6-methoxy-4-meth-
yl-5-(3-methylphenyl)-2-trifluoromethyl-quinoline (15be). Yield:
5-phenyl-quinoline Succinate (4aa). Yield 41%; mp 156 °C. H
NMR (CD3OD): δ 7.36-7.30 (m, 3H), 7.22-7.15 (m, 2H), 6.63
(s, 1H), 6.61 (s, 1H), 3.98 (s, 3H), 3.85-3.79 (m, 1H), 3.68
(s, 3H), 3.01-2.96 (m, 2H), 2.50 (s, 4H), 1.90-1.76 (m, 4H), 1.79
(s, 3H), 1.37 (d, J = 6.4 Hz, 3H). 13C NMR (CD3OD): δ 181.6,
160.3, 156.1, 149.8, 145.3, 141.5, 133.1, 133.1, 128.6, 128.6,
127.7, 126.0, 116.2, 115.2, 96.2, 57.7, 53.3, 41.3, 35.2, 35.1,
28.9, 26.4, 24.7, 21.3. IR (neat): 1592, 1577, 1533, 1467, 1450,
1394, 1373, 1337, 1207, 1185, 1158, 1053, 1038, 891, 857, 777,
756, and 706 cm-1. MS (EI): m/z 379 [M]þ. Anal. (C27H35N3O6):
C, H, N.
1
99%. H NMR (CDCl3): δ 7.82-7.80 (m, 2H), 7.69-7.67 (m,
2H), 7.35-7.09 (m, 6H), 4.85 (br d, J = 7.0 Hz, 1H), 3.76-3.71
(m, 3H), 3.38 (s, 1.5 H), 3.37 (s, 1.5H), 2.42 (s, 1.5H), 2.41
(s, 1.5H), 1.89 (s, 3H), 1.89-1.62 (m, 4H), 1.28 (d, J = 6.0 Hz,
3H). IR (neat): 1711, 1609, 1450, 1397, 1370, 1253, 1173, 1132,
1105, 756, and 719 cm-1. MS (ESI): m/z 562 [M þ 1]þ.
8-[(1-Methyl-4-phthalimido)-butyl]-amino-6-methoxy-4-meth-
yl-5-phenyl-2-trifluoromethyl-quinoline (15ba). Compound 15ba
was obtained as yellow solid from 12ba by the same procedure as
1
8-[(4-Amino-1-methyl)-butyl]-amino-2,6-dimethoxy-4-methyl-
5-(3-trifluoromethylphenyl)-quinoline Succinate (4ab). Compound
4ab was prepared from 15ab by the same procedure except
crude product was used to prepare the succinate salt without
prior purification by column chromatography. Yield 46%; mp
154 °C. 1H NMR (CD3OD): δ 7.63-7.43 (m, 4H), 6.65 (s, 1H),
6.64 (s, 1H), 3.99 (s, 3H), 3.88-3.83 (m, 1H), 3.72 (s, 3H),
3.01-2.96 (m, 2H), 2.50 (s, 4H), 1.90-1.75 (m, 4H), 1.75
(s, 3H), 1.37 (d, J = 6.3 Hz, 3H). 13C NMR (CD3OD): δ 179.5,
160.3, 156.2, 148.9, 145.9, 142.7, 137.0, 132.9, 131.1, 130.7, 129.6,
129.4, 129.3, 127.8, 125.8, 124.4, 124.3, 116.6, 112.5, 95.0, 57.3,
53.4, 41.0, 34.9, 33.0, 25.7, 25.1, 21.3. IR (neat): 1640, 1598, 1536,
1474, 1430, 1394, 1374, 1338, 1325, 1308, 1230, 1210, 1162, 1121,
1084, 1073, 1051, 857, 838, 812, and 708 cm-1. MS (EI): m/z 447
described for the preparation of 15be. Yield: 99%. H NMR
(CDCl3): δ 7.83-7.80 (m, 2H), 7.69-7.67 (m, 2H), 7.46-7.44
(m, 3H), 7.35-7.34 (m, 2H), 7.19 (s, 1H), 7.10 (s, 1H), 4.59
(d, J = 8.4 Hz, 1H), 3.76-3.72 (m, 3H), 3.35 (s, 3H), 1.88
(s, 3H), 1.88-1.64 (m, 4H), 1.28 (d, J = 6.3 Hz, 3H). IR (neat):
1708, 1609, 1525, 1394, 1254, 1181, 1132, 1103, 757, and 717 cm-1
.
MS (ESI): m/z 548 [M þ 1]þ.
8-[(1-Methyl-4-phthalimido)-butyl]-amino-6-methoxy-4-meth-
yl-2-trifluoromethyl-5-(3-trifluoromethylphenyl)-quinoline (15bb).
Compound 15bb was prepared from 12bb and 14 by the same
procedure. Purification of the title compound was difficult. Thus,
the crude product was partially purified by silica gel column and
used for the next step synthesis. 1H NMR (CDCl3): δ 7.84-7.57
(m, 8H), 7.23 (s, 1H), 7.11 (s, 1H), 4.56 (br d, J = 7.1 Hz, 1H),
3.78-3.71 (m, 3H), 3.33 (s, 3H), 1.84 (s, 3H), 1.84-1.57 (m, 4H),
1.29 (d, J = 6.2 Hz, 3H). IR (neat): 1712, 1610, 1397, 1371, 1321,
1255, 1168, 1128, 1105, 1071, 758, and 720 cm-1. MS (ESI): m/z
616 [M þ 1]þ.
[M]þ. Anal. (C28H34F3N3O6 0.1H2O): C, H, N, F.
3
8-[(4-Amino-1-methyl)-butyl]-amino-2,6-dimethoxy-4-methyl-
5-(4-trifluoromethylphenyl)-quinoline Succinate (4ac). Compound
4ac was prepared from 15ac by the same procedure. Yield 32%;

138 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shiraki et al.
mp 144 °C. 1H NMR (CD3OD): δ 7.65-7.62 (m, 2H), 7.40-.35
(m, 2H), 6.64-6.63 (m, 2H), 3.98 (s, 3H), 3.88-3.82 (m, 1H), 3.71
(s, 3H), 3.01-2.96 (m, 2H), 2.50 (s, 4H), 1.91-1.78 (m, 4H), 1.78
(s, 3H), 1.37 (d, J = 6.3 Hz, 3H). 13C NMR (CD3OD): δ 178.9,
160.3, 156.1, 149.1, 146.2, 145.8, 133.8, 133.7, 132.9, 130.0, 129.6,
128.0, 125.7, 125.4, 125.3, 124.4, 116.5, 112.8, 95.1, 57.3, 53.4, 41.0,
34.9, 32.4, 25.7, 25.1, 21.3. IR (neat): 1715, 1590, 1532, 1475, 1450,
1394, 1372, 1338, 1322, 1248, 1206, 1152, 1125, 1106, 1068, 1049,
1022, 894, 841, 810, 801, 753, and 609 cm-1. MS (EI): m/z 447
1H), 3.74-3.67 (m, 1H), 3.38 (s, 3H), 2.77-2.72 (m, 2H), 2.41
(s, 3H), 1.90 (s, 3H), 1.76-1.52 (m, 4H), 1.30 (d, J = 5.9 Hz,
3H). 13C NMR (CDCl3): δ 147.9, 147.7, 147.4, 142.8, 137.9,
137.5, 131.2, 128.4, 127.8, 127.7, 127.6, 116.2, 106.0, 60.0, 47.9,
42.1, 34.1, 29.9, 24.0, 21.4, 20.5. IR (neat): 1609, 1496, 1452,
1399, 1384, 1371, 1254, 1230, 1173, 1131, 1105, 1006, 945, 907,
855, 785, 760, 748, and 689 cm-1. MS (ESI): m/z 432 [M þ 1]þ.
Anal. (C24H28F3N3O 0.9H2O): C, H, N, F.
3
8-[(4-Amino-1-methyl)-butyl]-amino-6-methoxy-4-methyl-
5-phenyl-2-trifluoromethyl-quinoline (4ba). Compound 4ba was ob-
tained as a brown oil from 15ba by the same procedure as de-
scribed for the preparation of 4be. Yield 99%; mp 99 °C. 1H NMR
(CDCl3): δ 7.46-7.44 (m, 3H), 7.36-7.33 (m, 2H), 7.23 (s, 1H),
7.11 (s, 1H), 4.65 (br d, J = 8.2 Hz, 1H), 3.74-3.68 (m, 1H), 3.36
(s, 3H), 2.77-2.73 (m, 2H), 1.88 (s, 3H), 1.70-1.55 (m, 4H), 1.30
(d, J = 6.3 Hz, 3H). 13C NMR (CDCl3): δ 147.9, 147.7, 147.3,
146.5, 146.0, 142.8, 138.0, 131.0, 130.5, 127.9, 127.7, 123.7, 120.9,
120.1, 116.2, 106.0, 59.9, 47.9, 42.1, 34.1, 30.1, 24.1, 20.6. IR
(neat): 1611, 1450, 1371, 1255, 1177, 1132, 1105, 1003, 935, 852,
768, 745, and 706 cm-1. MS (ESI): m/z 418 [M þ 1]þ. Anal.
(C23H26F3N3O): C, H, N, F.
[M]þ. Anal. (C28H34F3N3O6 0.8H2O): C, H, N, F.
3
8-[(4-Amino-1-methyl)-butyl]-amino-2,6-dimethoxy-5-(3-meth-
oxyphenyl)-4-methylquinoline Succinate (4ad). Compound 4ad
was prepared from 15ad by the same procedure. Yield 46%; mp
1
164 °C. H NMR (CD3OD): δ 7.28-7.22 (m, 1H), 6.91-6.88
(m, 1H), 6.80-6.74 (m, 2H), 6.63 (s, 1H), 6.61 (s, 1H), 3.98
(s, 3H), 3.85-3.79 (m, 1H), 3.79 (s, 3H), 3.70 (s, 3H), 3.01-2.96
(m, 2H), 2.50 (s, 4H), 1.86-1.80 (m, 4H), 1.86 (s, 3H), 1.37 (d,
J = 6.3 Hz, 3H). 13C NMR (CD3OD): δ 178.0, 159.1, 159.1,
158.8, 154.5, 148.3, 143.7, 141.2, 131.5, 128.0, 128.0, 124.4,
124.2, 124.2, 117.4, 117.4, 114.7, 113.5, 111.7, 111.5, 94.7,
56.2, 54.2, 51.8, 39.5, 33.4, 31.5, 24.1, 22.8, 19.8. IR (neat):
1638, 1597, 1535, 1468, 1449, 1393, 1371, 1337, 1283, 1218, 1154,
1081, 1051, 835, 806, 791, 788, 713, and 670 cm-1. MS (EI): m/z
8-[(4-Amino-1-methyl)-butyl]-amino-6-methoxy-4-methyl-2-tri-
fluoromethyl-5-(3-trifluoromethylphenyl)-quinoline (4bb). The
title compound was prepared from 15bb by the same procedure
409 [M]þ. Anal. (C28H37N3O7 0.8H2O): C, H, N.
3
1
8-[(4-Amino-1-methyl)-butyl]-amino-2,6-dimethoxy-4-methyl-
5-(3-methylphenyl)-quinoline Succinate (4ae). Compound 4ae was
prepared from 15ae by the same procedure as described for the
preparation of 4ab. Yield 73%; mp 157 °C. 1H NMR (CD3OD): δ
7.24-7.19 (m, 1H), 7.14-7.12 (m, 1H), 7.02-6.95 (m, 2H), 6.63
(s, 1H), 6.60 (s, 1H), 3.98 (s, 3H), 3.85-3.79 (m, 1H), 3.68 (s, 3H),
3.00-2.96 (m, 2H), 2.50 (s, 4H), 2.35 (s, 3H), 1.90-1.79 (m, 4H),
1.79 (s, 3H), 1.36 (d, J = 6.3 Hz, 3H). 13C NMR (CD3OD): δ
179.4, 160.2, 155.9, 149.8, 145.0, 141.2, 138.0, 138.0, 133.6, 133.5,
133.0, 130.0, 130.0, 128.4, 128.3, 128.2, 125.9, 116.0, 115.3, 96.1,
57.6, 53.1, 40.9, 34.8, 32.9, 25.5, 24.6, 21.5, 21.2. IR (neat): 1638,
1597, 1473, 1450, 1393, 1372, 1337, 1213, 1154, 1083, 1053,
856, 836, 807, 785, 754, 715, and 685 cm-1. MS (ESI): m/z 394
[M þ 1]þ. Anal. (C28H37N3O6): C, H, N.
as oil. Yield: 76%. H NMR (CDCl3): δ 7.73-7.57 (m, 4H),
7.26 (s, 1H), 7.13 (s, 1H), 4.64 (br d, J = 8.0 Hz, 1H), 3.73-3.71
(m, 1H), 3.34 (s, 3H), 2.79-2.74 (m, 2H), 1.92-1.57 (m, 4H),
1.85 (s, 3H), 1.31 (d, J = 6.3 Hz, 3H). 13C NMR (CDCl3): δ
148.1, 146.8, 142.9, 139.2, 134.1, 130.4, 129.5, 128.6, 127.6,
126.0, 124.8, 123.8, 122.4, 120.8, 120.2, 116.6, 107.0, 60.0, 48.1,
42.2, 34.3, 29.9, 24.6, 20.7. IR (neat): 1497, 1254, 1228, 1167,
1125, 1105, 1001, 806, and 749 cm-1. MS (ESI): m/z 486 [M þ 1]þ.
Compound 4bb is a gummy material that easily binds tightly with
water and solvents. Several attempts were made to obtain satisfac-
tory elemental analysis data but failed without adjustment for
solvent content. It was converted to a t-Boc derivative, 5bb, which
is a crystalline material and gave satisfactory elemental analytical
data.
8-[(4-Amino-1-methyl)-butyl]-amino-5-(3,4-dichlorophenyl)-
2,6-dimethoxy-4-methyl-quinoline succinate (4af). Compound
4af was prepared from 15af by the same procedure for 4ab
synthesis. Yield 37%; mp 115 °C. 1H NMR (DMSO-d6): δ
7.59-7.57 (m, 1H), 7.45-7.41 (m, 1H), 7.18-7.13 (m, 1H),
6.73 (s, 1H), 6.58 (s, 1H), 6.02 (br d, J = 8.8 Hz, 1H), 3.94
(s, 3H), 3.88-3.70 (m, 1H), 3.70 (s, 3H), 2.90-2.74 (m, 2H), 2.23
(s, 4H), 1.79 (s, 3H), 1.79-1.52 (m, 4H), 1.26 (d, J = 6.2 Hz,
3H). 13C NMR (DMSO-d6): δ 175.5, 158.3, 154.5, 147.3, 144.3,
140.7, 133.5, 133.4, 132.3, 132.3, 130.6, 130.2, 130.1, 129.6,
129.6, 129.3, 124.0, 115.3, 108.8, 93.3, 56.6, 52.8, 48.7, 47.1,
33.3, 32.6, 24.7, 24.4, 20.7. IR (neat): 1595, 1577, 1532, 1450,
1393, 1371, 1336, 1231, 1206, 1188, 1160, 1128, 1087, 1052, 1030,
828, 794, 757, 746, and 719 cm-1. MS (ESI): m/z 448 [M þ 1]þ.
8-[(4-Amino-1-methyl)-butyl]-amino-6-methoxy-4-methyl-2-tri-
fluoromethyl-5-(4-trifluoromethylphenyl)-quinoline (4bc). Com-
pound 4bc was prepared from 15bc by the same procedure.
Yield: 29% in 3 steps from 11bc, mp 145 °C. 1H NMR (CDCl3):
δ 7.73 (d, J = 7.9 Hz, 2H), 7.51 (d, J = 7.9 Hz, 2H), 7.26 (s, 1H),
7.13 (s, 1H), 4.64 (br d, J = 8.4 Hz, 1H), 3.72-3.70 (m, 1H),
3.34 (s, 3H), 2.78-2.73 (m, 2H), 1.87 (s, 3H), 1.75-1.55 (m, 4H),
1.31 (d, J = 6.3 Hz, 3H). 13C NMR (CDCl3): δ 148.1, 148.0,
146.9, 142.9, 142.3, 131.2, 130.6, 130.1, 129.7, 125.0, 122.6,
120.7, 116.5, 107.0, 60.1, 48.2, 42.3, 34.4, 30.3, 24.6, 20.7. IR
(neat): 1322, 1254, 1187, 1172, 1126, 1104, 1068, 1002, 934, 856,
and 833 cm-1. MS (ESI): m/z 486 [M þ 1]þ. The compound 4bc
was a gummy material, which failed to give satisfactory ele-
mental analysis results without adjustment for solvent content.
It was converted to the t-Boc derivative, 5bc, which is a crystal-
line material and gave good elemental analysis results after
purification.
8-[(4-Amino-1-methyl)-butyl]-amino-6-methoxy-5-(3-methoxy-
phenyl)-4-methyl-2-trifluoromethyl-quinoline (4bd). Compound
4bd was obtained as yellow oil from 15bd by the same procedure
described for the preparation of 4be. Yield: 77%. 1H NMR
(CDCl3): δ 7.39-7.34 (m, 1H), 7.27-7.12 (m, 2H), 7.01-6.90
(m, 3H), 4.65 (d, J = 8.2 Hz, 1H), 3.84 (s, 3H), 3.84-3.70
(m, 1H), 3.41 (s, 3H), 2.77-2.73 (m, 2H), 1.96 (s, 3H), 1.71-1.50
(m, 4H), 1.31 (d, J = 6.3 Hz, 3H). 13C NMR (CDCl3): δ 159.2,
147.3, 142.8, 139.4, 128.9, 123.2, 116.3, 116.2, 113.3, 106.2, 60.1,
55.3, 47.9, 42.2, 34.2, 30.1, 23.7, 20.5. IR (neat): 1608, 1585,
1498, 1479, 1451, 1371, 1254, 1174, 1131, 1105, 1047, 1003, 944,
858, 785, 753, and 689 cm-1. MS (EI): m/z 447 [M]þ. The
compound 4bd was determined by LC-MS/MS to be about
95% pure. It was converted to t-Boc 5bd which gave satisfactory
elemental analytical results.
Anal. (C27H33Cl2N3O6 0.5H2O): C, H, N, Cl.
3
General Synthetic Procedure for the Preparation of 8-[(4-
Amino-1-methyl)-butyl]-amino-5-aryl-6-methoxy-4-methyl-2-tri-
fluoromethyl-quinoline (4ba-be). A solution of compound 15be
(1.5 g, 2.7 mmol) in ethanol (15 mL) was treated with excess
amount of hydrazine monohydrate (0.6 g, 11 mmol) and refluxed
for 30 min. On cooling, the precipitates were removed by filtration
and washed with EtOH (5 mLꢀ4). The EtOH washing solutions
were combined and concentrated to dryness. The crude product
was dissolved in CHCl3 (100 mL), washed twice with 25 mL of
25% KOH aqueous solution, and followed twice with 25 mL of
H2O. The CHCl3 layer was dried over Na2SO4 and concentrated in
vacuo to give yellow oil which was purified by a silica gel column,
eluted with a CH2Cl2 solution containing 5% of CH3OH and 5%
of Et3N, to give 0.9 g (80%) of the desired product 4be as yellow oil.
8-[(4-Amino-1-methyl)-butyl]-amino-6-methoxy-4-methyl-
5-(3-methylphenyl)-2-trifluoromethyl-quinoline (4be). Yield: 80%.
1H NMR (CDCl3): δ 7.36-7.10 (m, 6H), 4.64 (br d, J = 7.5 Hz,

Article
General Synthetic Procedure for 8-[(4-tert-Butoxycarbonyla-
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 139
sulfadoxine, pyrimethamine, and quinine. Test compounds were
initially dissolved in DMSO and diluted 400-fold in RPMI 1640
culture medium supplemented with 25 mM Hepes, 32 mM
NaHCO3, and 10% Albumax I (Gibco BRL, Grand Island,
NY). These solutions were subsequently serial diluted 2-fold
with a Biomek 1000 (Beckman, Fullerton, CA) over 11 different
concentrations. The parasites were exposed to serial dilutions
of each compound for 48 h and incubated at 37 °C with 5% O2,
5% CO2, and 90% N2 prior to the addition of [3H]-hypox-
anthine. After a further incubation of 18 h, parasite DNA was
harvested from each microtiter well using Packard Filtermate
196 harvester (Meriden, CT) onto glass filters. Uptake of [3H]-
hypoxanthine was measured with a Packard Topcount scintilla-
tion counter. Concentration-response data were analyzed by a
nonlinear regression logistic dose response model, and the IC50
values (50% inhibitory concentrations) for each compound
were calculated.
ii. In Vitro Toxicity Assessment. Toxicity of the test com-
pounds were assessed in the macrophage cell line RAW 264.7,
which was obtained from the American Type Culture Collection
and was cultured as previously described.32 All steps of the
experimental procedure were performed using a Biomek 2000
robot. The stock solution of the test drugs were prepared by
dissolving in DMSO and serially diluted automatically by robot.
Ninety-six-well plates were seeded each with 2.5 ꢀ 104 cells per
well in 170 μL of culture media and incubated for approximately
12 h before the start of the assay. The following morning, cells
were exposed to different drug concentrations, ranging from
0.29-48 μg/mL/well, and were incubated for additional 24 h.
After incubation, the drugs were removed and 200 μL of fresh
media was added to each well and incubated again for 24 more h
to allow for recovery of the viable yet drug suppressed cells. The
viability of the cells was assessed using the MTT (thiazolyl blue
reduction) method as previously described.33 Each assay plate
includes the following 3 control wells: (a) background control,
no cells and no drugs were added; (b) target control, cells added
but no drugs; and (c) DMSO control, same as the target control,
except the culture media contains 0.6% DMSO, which is the
highest DMSO concentration among the wells with test drug
and is not harmful to the macrophages. In the background
control, the reading should be near zero while those in the target
and the 0.6% DMSO controls, 100% cell survival are antici-
pated. The experimental results are not considered valid unless
the results of all three controls are as expected. All experiments
were run in duplicate, and the 50% inhibitory concentrations
(IC50s) were determined using GraphPad Prism (sigmoidal
dose-response, variable slope).
mino-1-methyl)-butyl]-amino-5-aryl-6-methoxy-4-methyl-2-triflu-
oromethyl-quinoline (5bb-be). Di-tert-butyl-dicarbonate (607 mg,
2.78 mmol) and triethylamine (282 mg, 2.78 mmol) were added to a
solution of 4bb (450 mg, 0.93 mmol) in chloroform (40 mL). The
reaction mixture was stirred at rtfor 21 h and evaporated to dryness
in vacuo. The residue was purified by a silica gel column, eluted with
20% of EtOAc in hexane, to give 540 mg (99%) of the desired
product 5bb as yellow solid.
8-[(4-tert-Butoxycarbonylamino-1-methyl)-butyl]-amino-6-meth-
oxy-4-methyl-2-trifluoromethyl-5-(3-trifluoromethylphenyl)-quino-
line (5bb). Yield: 99%, mp 53-57 °C. 1H NMR (CDCl3): δ 7.73-
7.58 (m, 4H), 7.26 (s, 1H), 7.13 (s, 1H), 4.59-4.56 (m, 2H), 3.72
(m, 1H), 3.34 (s, 3H), 3.17-3.15 (m, 2H), 1.85 (s, 3H), 1.69-1.64
(m, 4H), 1.42 (s, 9H), 1.30 (d, J = 6.3 Hz, 3H). 13C NMR
(CDCl3): δ 156.2, 148.1, 146.8, 142.8, 139.2, 134.1, 129.6, 128.6,
127.6, 126.0, 124.8, 123.9, 122.4, 120.8, 120.2, 116.7, 107.1, 79.5,
60.0, 48.3, 40.8, 34.2, 28.6, 27.2, 24.6, 20.8. IR (neat): 1256, 1168,
1164, 1127, 1107, 1074, 1004, 837, 808, and 793 cm-1. MS (ESI):
m/z 586 [M þ 1]þ. Anal. (C29H33F6N3O3): C, H, N, F.
8-[(4-tert-Butoxycarbonylamino-1-methyl)-butyl]-amino-6-meth-
oxy-4-methyl-5-(4-trifluoro- methyl)-phenyl-2-trifluoromethyl-quino-
line (5bc). Compound 5bc was obtained as yellow solid from 4bc
by the same procedure as described for the preparation of 5bb.
Yield 99%; mp 53-58 °C. 1H NMR (CDCl3): δ 7.73 (d, J = 7.9
Hz, 2H), 7.50 (d, J = 7.9 Hz, 2H), 7.25 (s, 1H), 7.13 (s, 1H),
4.58-4.55 (m, 2H), 3.72-3.70 (m, 1H), 3.34 (s, 3H), 3.17-3.15
(m, 2H), 1.87 (s, 3H), 1.65-1.64 (m, 4H), 1.42 (s, 9H), 1.30 (d,
J = 6.3 Hz, 3H). 13C NMR (CDCl3): δ 156.0, 147.9, 147.8,
146.6, 142.6, 142.1, 131.0, 130.3, 129.9, 129.5, 124.8, 125.0,
122.3, 120.0, 116.4, 106.8, 79.2, 59.9, 47.9, 40.6, 33.9, 28.4,
26.9, 24.3, 20.5. IR (neat): 1708, 1609, 1521, 1498, 1452, 1253,
1172, 1135, 1105, 860, 852, 834, and 737 cm-1. MS (ESI): m/z
586 [M þ 1]þ. Anal. (C29H33F6N3O3): C, H, N, F.
8-[(4-tert-Butoxycarbonylamino-1-methyl)-butyl]-amino-6-meth-
oxy-5-(3-methoxyphenyl)-4-methyl-2-trifluoromethyl-quinoline
(5bd). Compound 5bd was obtained in 93% yield from 4bd as a
yellow solid by the same general procedure, mp 62-66 °C. 1H
NMR (CDCl3): δ 7.37-7.33 (m, 1H), 7.26-7.22 (m, 1H), 7.11
(s, 1H), 7.00-6.90 (m, 3H), 4.59-4.56 (m, 2H), 3.83 (s, 3H), 3.71
(m, 1H), 3.40 (s, 3H), 3.16 (m, 2H), 1.96 (s, 3H), 1.63-1.61 (m,
4H), 1.42 (s, 9H), 1.29 (d, J = 6.2 Hz, 3H). 13C NMR (CDCl3): δ
159.3, 156.2, 148.0, 147.8, 147.6, 142.9, 139.5, 131.0, 129.1,
123.9, 123.4, 121.1, 120.2, 116.4, 113.5, 106.3, 79.4, 60.4, 55.5,
48.0, 40.7, 34.1, 28.6, 27.1, 24.0, 20.8. IR (neat): 1499, 1286,
1238, 1155, 1113, 1084, 1048, 945, 790, 740, and 721 cm-1. MS
(ESI): m/z 548 [M þ 1]þ. Anal. (C29H36F3N3O4): C, H, N, F.
8-[(4-tert-Butoxycarbonylamino-1-methyl)-butyl]-amino-6-meth-
oxy-4-methyl-5-(3-methylphenyl)-2-trifluoromethyl-quinoline
(5be). Compound 5be was prepared from 4be in 69% yield as
iii. Causal Prophylactic Antimalarial Activity in Exoerythro-
cytic (EE) Mouse Model. The causal prophylactic activity of
the new compounds were assessed in mice infected with sporo-
zoites of P. berghei by intravenous inoculation, according to the
method described earlier.34
iv. Metabolic Stability Test. Metabolic stability of the new
compounds was assessed in the human and mouse microsomal
preparations according to the procedure described.34 Briefly,
the metabolic stability assay was performed in a 96-well plate on
a TECAN Genesis robotic sample processor following WRAIR
SOP SP 01-02. Samples were analyzed by LC-MS/MS using fast
LC gradient or isocratic methods. Parent drug was quantified
using external calibration and plots of parent drug response vs
amount.
1
yellow solid by the same procedure, mp 58-62 °C. H NMR
(CDCl3): δ 7.35-7.10 (m, 6H), 4.59-4.57 (m, 2H), 3.71-3.69
(m, 1H), 3.37 (s, 3H), 3.16-3.15 (m, 2H), 2.41 (s, 3H), 1.90
(s, 3H), 1.70-1.63 (m, 4H), 1.42 (s, 9H), 1.29 (d, J = 6.3 Hz,
3H). 13C NMR (CDCl3): δ 156.0, 147.9, 147.7, 147.4, 142.7,
137.9, 137.5, 137.5, 131.2, 128.4, 127.7, 127.6, 121.0, 120.1,
116.2, 106.1, 79.2, 60.0, 47.8, 40.6, 34.0, 28.4, 26.9, 24.0, 21.4,
20.6. IR (neat): 1699, 1610, 1520, 1498, 1455, 1397, 1384, 1370,
1254, 1170, 1134, 1106, 1007, 859, 841, and 784 cm-1. MS (ESI):
m/z 532 [M þ 1]þ. Anal. (C29H36F3N3O3): C, H, N, F.
Biological Studies. i. In Vitro Antimalarial Activity against P.
falciparum. The in vitro assays were conducted using a mod-
ification of the technique of Desjardins et al.29 and Chulay
et al.30 Two P. falciparum malaria parasite clones from CDC
Indochina III (W-2), and CDC Sierra Leone I (D-6) were
utilized in susceptibility testing. They were derived by direct
visualization and micromanipulation from patient isolates.31
The W-2 clone is susceptible to mefloquine (MQ) but resistant
to CQ, sulfadoxine, pyrimethamine, and quinine, whereas the
D-6 clone is naturally resistant to MQ but susceptible to CQ,
Results and Discussion. i. In Vitro Antimalarial Activity. All
new compounds prepared in this study (4aa-af, 4ba-be, and
5bb-be) were evaluated for their antimalarial activity against
both CQ-susceptible (D6) and CQ-resistant clones (W2) of P.
falciparum (Table 1). 8-Aminoquinoline antimalarials 2 and 3
were used as positive controls for the study. All new compounds
showed moderate activity against both CQ-susceptible and CQ-
resistant clones of P. falciparum with IC50 in the range of 1-3
μg/mL, which is comparable to or better than 2 and 3 (Table 1).

140 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shiraki et al.
Table 1. In Vitro Growth Inhibitory Activity in P falciparum and Toxicity against Macrophage Cell Line
metabolic stability t1/2 (min) EE P. berghei (mg/kg/day ꢀ 3) P. falciparum (IC50, μg/mL) macrophage cell line therapeutic indexa (TI)
compdc
human
mouse
MTD
MAD
D6
W2
(IC50, μg/mL)
D6
W2
4aa
4ab
4ac
4ad
4ae
4af
4ba
4bb
4bc
4bd
4be
5bb
5bc
5bd
5be
2
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
18
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
18
3.2
2.7
2.2
2.8
1.3
2.9
2.8
1.4
1.7
0.3
0.2
1.1
1.3
0.3
0.2
2.1
2.7
1.3
1.3
1.3
0.8
0.7
1.2
1.4
0.5
0.8
0.6
0.9
2.8
1.4
0.6
0.9
0.9
0.8
1.9
1.3
0.59
0.48
0.54
1.11
1.46
0.58
1.11
1.07
5.94
5.0
1.46
1.0
>480
>480
>480
960
>480
>480
>480
960
1.2
3.1
0.92
3.87
2.71
1.42
2.21
3.0
1.9
1.7
120
480
>480
>960
3.1
1.5
10.1
1.5
12.6
2.50
2.0
1.8
9.0
>960
>960
>960
>960
<120c
120c
>960
>960
>960
>960
60c
>48b
>48b
>80.2b
>48b
38.7
5.9
>39.7
>36
>267
>240
18.43
2.2
>17
>34
>134
>59
43
30
16
9
13
8
11
>60
>60
21
>60
3
7.5c
7.38
a TI, macrophage IC50/P. falciparum IC50; MTD, maximum tolerated dose; MAD, minimum active dose. b The compound was precipitated in media
at the highest concentration. c Historical data of WRAIR.
Structure-activity relationship studies indicated that the
antimalarial activity of the new compounds were affected sig-
nificantly by the substituent on the 5-aryl ring as well as at the
2-position of 8-AQ. The new agents appear more active against
the CQ-resistant clones W-2 than to the CQ-sensitive clone D-6.
Electronic effects of the substituent on the 5-aryl ring influenced
substantially the cell growth inhibitory activity. Compounds
with electron donating groups (m-methoxyl or m-methyl)
showed better activity than those with electron withdrawing
substituents (m- or p-CF3). Thus, compounds 4bd, 4be, 5bd, and
5be were more active than 4bb, 4bc, 5bb, and 5bc. Among the
compounds tested, 4ae, 4be, and 5be with m-methylphenyl
substituents at the 5-position of the quinoline ring showed the
best growth inhibitory activity in both clones of P. falciparum.
Furthermore, compounds with a trifluoromethyl (CF3) substit-
uent at the 2-position of 8-aminoquinoline ring possess supe-
rior activity over those with a 2-methoxy group. In general,
2-trifluoromethyl-8-AQ derivatives (4ba-be) are 2-6-fold
more active than the corresponding 2-methoxy-8-AQ analogues
(4aa-ae).
The structures of the new compounds closely resemble to that
of 3 and its analogues, except the oxygen atom at the 5-position
of TQ was removed in the new compounds. Theoretically, with
the 5-position blocked by a metabolically stable aryl group, the
new compounds are not expected to produce toxic metabolites,
quinone or quinoneimine. As hypothesized in the literature, if
the metabolites responsible for toxicity are also responsible for
the antimalarial activity then the new compounds would not
be expected to exhibit antimalarial activity or hemolytic side
effects. On the other hand, if the efficacy and toxicity of 3 are
the effects of the parent drug and reactive metabolites, respec-
tively, then the new compounds which showed antimalarial
activity may not necessarily show hemolytic toxicity. This con-
tention will be explored separately in other studies. The present
report focused mainly on chemical synthesis, assessment of
antimalarial efficacy in EE mouse model, P. falciparum cell
culture, and toxicity in the mammalian macrophage cell line
Raw 264.7.
compounds are about equal to 3 but more toxic than 2, based on
weight or molar concentration. However, compounds 4bc, 4bd,
and 4be, the most active compounds of the new series, showed a
comparable therapeutic index (TI) to that of 2 and 3, with TI
ranging from 5 to 8. The toxicity results are based on the growth
inhibition of macrophage cell growth which is not relevant to the
hemolytic toxicity but provide references to general toxicity of
the test compounds. In mice efficacy tests, the new compounds
are much less toxic and less active than 2 and 3, with oral
maximum tolerated dose (MTD) > 480-960 mg/kg, as com-
pared to that of 2 (∼90 mg/kg) and 3 (∼60 mg/kg) (Table 1).
Hemolysis side effects associated with G6PD deficiency
patients is the major concern of 8-AQ toxicity. However, there
is no validated animal model available that can be used to
predict this deadly toxicity. Currently, promising mice models
are under development in several laboratories, including Walter
Reed Army Institute of Research. Whether the new deoxo-TQ
analogues prepared in this study are less hemolytic or not will
have to wait until the validated model(s) are available.
iii. Causal Prophylactic Antimalarial Activity in EE Mouse
Model. The new compounds 4ab-af, 4ba, and 5bb-be were
further tested for causal prophylactic activity in P. berghei
sporozoites infected mouse model by oral administration and
were found to be inactive up to 320 mg/kg per day for 3 days,
except 4ae, which showed weak activity at 320 mg/kg/day ꢀ 3 by
oral, cured 1 out of 4 and delayed patency for 4 days 2 out of 4
mice. The weak activity of 8-AQ derivatives in exoerythrocytic
mouse model was well documented.21 Earlier efforts to develop
new drugs effective against tissue schizonts and hypnozoites
have been hampered by the difficulty in establishing an in vitro
cell based model or a mouse model predictive for the exoery-
throcytic stages of P. falciparum and P. vivax.21 Extrapolating
drug efficacy data from rodent models of malaria to primate
models can be difficult to interpret due to interspecies differ-
ences in absorption, distribution, metabolism, and excretion. In
addition, the rodent model may not be capable of distinguishing
EE activity from blood stage activity because compounds with
long half-life may result in a suppressive effect in the blood.
Furthermore, a series of 3 analogues which showed radical
curative activity in Rhesus were found inactive in causal pro-
phylactic test in mice.21 Consequently, developers of 8-AQ
antimalarials have relied heavily on Rhesus monkeys infected
with sporozoites of P. cynomolgi to confirm the antiparasitic
activity among the drug candidates. Efficacy in the monkey
model has a good correlation with efficacy against human P.
vivax infection.35,36 Similar difficulties were experienced in the
ii. In Vitro Toxicity Assessment in Macrophage Cell Line
RAW 264.7. Macrophage cell line Raw 264.7 was used to assess
the general toxicity of the new 8-AQ derivatives 4aa-af, 4ba-be,
and 5bb-be synthesized in this study (Table 1). The results
indicate that the concentration required for 50% growth inhibi-
tion (IC50) against the macrophage cell line were in the range
of 3-21 μmol/L for 4aa-af and 4ba-be vs 12.7 μmol/L
and 149 μmol/L for 3 and 2, respectively, indicating the new

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 141
lead optimization of a new class of imidazolinedione derivatives
which showed promising causal prophylactic and radical cure
activity in Rhesus but showed no or very weak activity in the EE
mouse model.34,37,38 Thus, although the new deoxo-TQ ana-
logues synthesized in this study showed poor efficacy in mice, it
cannot be ruled out that they do possess good causal prophy-
lactic and/or antihypnozoite activity in Rhesus monkey models.
iv. Metabolic Stability Test. Because metabolites were implied
to play a role in the efficacy and hemolytic toxicity of 8-AQ
antimalarials, metabolic stability of the new compounds were
assessed in human and mouse microsomal preparations accord-
ing to the published procedure.34 The results indicated that
compounds 4aa-af, and 4ba-be are, as expected, metabolically
stable with t1/2 > 60 min. Compounds 5bb-be, t-Boc of the
Supporting Information Available: Elemental analysis results
of 5-aryl-8-aminoquinoline derivatives. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
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Acknowledgment. Material has been reviewed by the Wal-
ter Reed Army Institute of Research. There is no objection to
its presentation and/or publications. The opinions or asser-
tions contained herein are the private views of the authors and
are not to be construed as official or as reflecting true views of
the Department of the Army or the Department of Defense.
This research was performed while Dr. Hiroaki Shiraki held a
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