Isoquine and Related Amodiaquine Analogues: A New Generation of Improved 4-Aminoquinoline Antimalarials

Article abstract of DOI:10.1021/jm030796n

Amodiaquine (AQ) (2) is a 4-aminoquinoline antimalarial that can cause adverse side effects including agranulocytosis and liver damage. The observed drug toxicity is believed to involve the formation of an electrophilic metabolite, amodiaquine quinoneimin

Full text of DOI:10.1021/jm030796n

J . Med. Chem. 2003, 46, 4933-4945
4933
Isoqu in e a n d Rela ted Am od ia qu in e An a logu es: A New Gen er a tion of Im p r oved
4-Am in oqu in olin e An tim a la r ia ls
Paul M. O’Neill,*,†,‡ Amira Mukhtar,† Paul A. Stocks,† Laura E. Randle,‡ Stephen Hindley,† Stephen A. Ward,*,§
Richard C. Storr,† J amie F. Bickley,† Ian A. O’Neil,† J ames L. Maggs,‡ Ruth H. Hughes,§ Peter A. Winstanley,‡
Patrick G. Bray,§ and B. Kevin Park*,‡
Department of Chemistry, The Robert Robinson Laboratories, University of Liverpool, Liverpool L69 7ZD, Department of
Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 3GE, UK, and Molecular and Biochemical
Parasitology Group, Liverpool School of Tropical Medicine, University of Liverpool, Pembroke Place, Liverpool L3 5QA, UK
Received February 3, 2003
Amodiaquine (AQ) (2) is a 4-aminoquinoline antimalarial that can cause adverse side effects
including agranulocytosis and liver damage. The observed drug toxicity is believed to involve
the formation of an electrophilic metabolite, amodiaquine quinoneimine (AQQI), which can
bind to cellular macromolecules and initiate hypersensitivity reactions. We proposed that
interchange of the 3′ hydroxyl and the 4′ Mannich side-chain function of amodiaquine would
provide a new series of analogues that cannot form toxic quinoneimine metabolites via
cytochrome P450-mediated metabolism. By a simple two-step procedure, 10 isomeric amodi-
aquine analogues were prepared and subsequently examined against the chloroquine resistant
K1 and sensitive HB3 strains of Plasmodium falciparum in vitro. Several analogues displayed
potent antimalarial activity against both strains. On the basis of the results of in vitro testing,
isoquine (ISQ1 (3a )) (IC50 ) 6.01 nM ( 8.0 versus K1 strain), the direct isomer of amodiaquine,
was selected for in vivo antimalarial assessment. The potent in vitro antimalarial activity of
isoquine was translated into excellent oral in vivo ED50 activity of 1.6 and 3.7 mg/kg against
the P. yoelii NS strain compared to 7.9 and 7.4 mg/kg for amodiaquine. Subsequent metabolism
studies in the rat model demonstrated that isoquine does not undergo in vivo bioactivation, as
evidenced by the complete lack of glutathione metabolites in bile. In sharp contrast to
amodiaquine, isoquine (and Phase I metabolites) undergoes clearance by Phase II glucuronida-
tion. On the basis of these promising initial studies, isoquine (ISQ1 (3a )) represents a new
second generation lead worthy of further investigation as a cost-effective and potentially safer
alternative to amodiaquine.
In tr od u ction
Resistance to chloroquine (1) (CQ) in Plasmodium
falciparum malaria has become a major health concern
of the developing world. This resistance has prompted
a reexamination of the pharmacology of alternative
antimalarials that may be effective against resistant
strains.1,2 Amodiaquine (2) (AQ) is a 4-aminoquinoline
antimalarial which is effective against many chloro-
quine-resistant strains of P. falciparum (Figure 1).
However, clinical use of AQ has been severely restricted
F igu r e 1. Structures of chloroquine and amodiaquine.
because of associations with hepatotoxicity and agranu-
locytosis.3,4
(glutathione and cysteinyl).5 This observation indicates
Paracetamol (4-hydroxyacetanilide) contains a p-
that the parent drug undergoes extensive bioactivation
in vivo to form amodiaquine quinoneimine (AQQI) or
hydroxyanilino moiety, which is believed to undergo
P-450-catalyzed oxidation to a chemically reactive quino-
neimine (Scheme 1). Amodiaquine also contains this
semiquinoneimine (AQSQI) with subsequent conjugate
6
addition of glutathione
functionality and might be expected to undergo enzymic
Formation of one of these reactive species in vivo and
oxidation to a reactive metabolite. Studies in this
subsequent binding to cellular macromolecules could
affect cell function either directly or by immunological
mechanisms. Indeed IgG antibodies, which recognize the
laboratory have shown that in the rat amodiaquine is
excreted in bile exclusively as the 5′ thioether conjugates
5′-cysteinyl group, have been detected in patients with
* Authors for correspondence. (P.M.O.) Phone: 0151-794-3553.
adverse reactions to amodiaquine.7 In the case of para-
Fax: 0151-794-8218. E-mail: P.M.oneill01@liv.ac.uk. (B.K.P.) Phone:
cetamol it has been shown that introduction of fluorine
into the aromatic nucleus increases the oxidation po-
tential of the molecule and thereby blocks the in vivo
oxidation of the molecule to a cytotoxic quinoneimine.8
0151-794-5559. E-mail: B.K.Park@liv.ac.uk. (S.A.W.). E-mail: saward@
liv.ac.uk.
† Department of Chemistry.
‡ Department of Pharmacology and Therapeutics.
§ Molecular and Biochemical Parasitology Group.
10.1021/jm030796n CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/30/2003

4934 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
O’Neill et al.
Sch em e 1. Bioactivation of Amodiaquine and Paracetamol to Toxic Quinoneimines by P450
Further studies by our group demonstrated that, in a
Sch em e 2. Redesign of Amodiaquine
manner similar to paracetamol, the incorporation of
fluorine atoms into the 4-hydroxyanilino side-chain of
amodiaquine produces compounds with greater oxida-
tive and metabolic stability.9 From this earlier work,
we demonstrated that the 4′-hydroxyl group could be
replaced with a 4′-fluorine atom to produce an amodi-
aquine analogue, fluoroamodiaquine, with antimalarial
activity in the low nanomolar range. Despite these
promising observations, activity at the level of the
parent drug amodiaquine was never achieved with the
fluorinated derivatives and, on the basis of cost consid-
erations, it was decided that an alternative approach
Ch a r t 1. Isoquine ISQ1 (3a ) and Analogues 4a -12a
to producing more metabolically robust analogues,
retaining the key pharmacophoric groups, should be
sought.
From our previous SAR work,10,11 we have noted that
in the amodiaquine and tebuquine series of 4-amino-
quinoline analogues, the presence of the 4′ hydroxyl
group within the aromatic ring imparts greater inherent
antimalarial activity against chloroquine resistant para-
sites than the corresponding deoxo analogues. Inter-
change of the hydroxyl group and the Mannich side-
chain provides a means of preventing oxidation to toxic
metabolites while retaining possible important bonding
interactions with the aromatic hydroxyl function. In this
paper, we will describe the synthesis, antimalarial
activity, and metabolism of the prototype isoquine (3a ,
ISQ 1), an amodiaquine regioisomer that cannot form
toxic metabolites by simple oxidation and which is
potent against chloroquine resistant parasites in vitro
(Scheme 2). The antimalarial activity of isoquine (3a ,
ISQ 1) will be compared with nine other analogues in
this series (Chart 1, 4a -12a ). Apart from an excellent
antiparasitic profile, isoquine and its side-chain ana-
logues are extremely cheap antimalarials to synthesize
and on the basis of initial data reported in this paper,
may represent new leads for development of a safe,
cheap, affordable, and effective antimalarial for both
prophylaxis and treatment of malaria.
ing materials according to a method originally utilized
by Burkhalter and co-workers (Scheme 3).12 Thus, step
1 involves a Mannich reaction of the commercially
available 3-hydroxyacetanilide to provide the Mannich
product in yields ranging from 50 to 90% (Table 1).
Stage 2 of the sequence involves hydrolysis of the
amide function to provide the corresponding Mannich-
substituted 3-aminophenol that is subsequently coupled
with 4,7-dichloroquinoline to provide target mole-
cules shown in Chart 1. The main difference between
the synthesis of this isomeric series and the amodi-
aquine analogues we have previously prepared is that
after amide hydrolysis, the reaction should not be
buffered to pH ) 6. The intermediate 3-aminophenols
have been shown to be quite unstable at neutral pH
but are sufficiently nucleophilic to couple with 4,7-
dichloroquinoline at lower pHs, i.e., after hydrolysis of
the amide, the sequential reaction can be carried out
in ethanolic solvent by addition of 4,7-dichloroquino-
Ch em istr y
The preparation of isoquine and its analogues involves
a two-step procedure from commercially available start-

Improved 4-Aminoquinoline Antimalarials
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4935
Sch em e 3. Synthesis of Analogues 3a -12a
Ta ble 1. Yields for the Synthesis of Isoquine (3a ) and
Analogues 4a -12a
yield,
%
yield,
%
intermediate amide
product
(3) R1 ) Et, R2 ) Et
(4) R1 ) H, R2 ) t-Bu
(5) R1, R2 ) Me
69 ISQ 1 (3a ) R1 ) Et, R2 ) Et
70 (4a ) R1 ) H, R2 ) t-Bu
74 (5a ) R1, R2 ) Me
71
69
69
61
58
80
(6) R1, R2 ) n-propyl
(7) R1, R2 ) n-butyl
(8) R1, R2 ) (CH2)4
63 (6a ) R1, R2 ) n-propyl
52 (7a ) R1, R2 ) n-butyl
89 (8a ) R1, R2 ) (CH2)4
(9) R1, R2 ) (CH2)2O(CH2)2 72 (9a ) R1, R2 ) (CH2)2O(CH2)2 60
(10) R1, R2 ) (CH2)5
(11) R1 ) H, R2 ) i-Pr
(12) R1 ) H, R2 ) Et
80 (10a ) R1, R2 ) (CH2)5
51 (11a ) R1 ) H, R2 ) i-Pr
55 (12a ) R1 ) H, R2 ) Et
71
67
68
Ta ble 2. In Vitro Antimalarial Activities of Chloroquine,
Amodiaquine, and Analogues 3a -12a
IC50 (nM) SD ( IC50 (nM) SD (
druga
HB3
mean
K1
mean
chloroquine (1)
amodiaquine (2)
isoquine (ISQ-1) (3a )
isoquine diphosphate (3b)
14.98 (6)
9.60 (9)
12.65 (9)
9.02 (3)
3.98 183.82 (6) 11.13
3.73
4.75
15.08 (9)
17.63 (9)
6.01 (3)
9.36
7.00
8.00
4.06
(4a )
(5a )
(6a )
(7a )
(8a )
(9a )
(10a )
(11a )
(12a )
30.03 (3)
14.76 (9)
19.78 (9)
51.88 (4)
28.37 (4)
97.20 (4)
9.07 (4)
17.67
12.30
15.30
19.77
9.03
32.75 (4) 13.16
18.65 (9) 9.08
30.63 (4) 16.53
37.21 (4) 12.86
21.75 (3)
2.65
15.31 112.37 (4) 36.99
0.30
4.34
20.28 (3)
26.22 (4)
4.99
8.03
20.22 (4)
16.24 (4)
11.24
32.42 (4) 14.70
a
Amodiaquine was tested as the hydrochloride salt, ISQ-1 and
3a -12a were all tested as free bases. Chloroquine was tested as
the diphosphate.
F igu r e 2. X-ray cystal structures of ISQ-1 (3a ) and 8a .
line.13 For purification, analogues were chromato-
graphed as their hydrochloride salts using methanol/
dichloromethane (10-20% MeOH/ dichloromethane) as
eluent. The free bases could be conveniently obtained
by dissolving pure columned solid hydrochloride product
in distilled water and adding saturated sodium bicar-
bonate solution. The precipitated free base could then
be dried and recrystallized from either 2-propanol or
methanol. Compounds were analyzed by HPLC and full
spectroscopic details are included in the Experimental
Section. For ISQ 1 and its pyrrolidinyl analogue (8a ),
X-ray crystallography studies demonstrate that there
is an internal hydrogen bond between the hydroxyl
function (OH as donor) and the side-chain nitrogen
(Figure 2). This may lead to a subtle effect on the pKa
of the side-chain Mannich nitrogen atom and a reduc-
tion in basicity.
phate salt (3b), and compounds 5a , 6a , 10a , and 12a
all express activity below 20 nM. In line with previous
SAR studies on 4-aminoquinoline analogues, the mor-
pholinyl analogue (9a ) is a poor antimalarial with
activity close to 100 nM.32 Clearly, the two most potent
compounds tested against the HB3 strain were isoquine
free base and the diphosphate salt (3b) and the pip-
eridinyl analogue (10a ). The most potent compound
against the chloroquine resistant strain was again the
diphosphate salt of isoquine although the free base is
also as potent as amodiaquine in this strain. Isoquine
and its salt are both about 20 times more potent than
chloroquine diphosphate. Compounds 5a , 8a , 10a , and
11a also express excellent activity against this resistant
strain. It is clear that in addition to isoquine, compounds
5a and 10a are additional leads worthy of further
investigation. Isoquine diphosphate was subsequently
tested in vivo against the murine Plasmodium yoelii NS
strain. Data recorded in Table 3 suggests that isoquine
has superior antimalarial activity to amodiaquine in
vivo. Indeed, by the oral route, 3a is almost three times
more potent than AQ.
An tim a la r ia l Activity. Analogues were initially
tested in vitro against the chloroquine sensitive HB3
strain and against the highly chloroquine-resistant K1
strain of P. falciparum (Table 2). Against the HB3
chloroquine sensitive isolate, isoquine (3a ), its diphos-

4936 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
O’Neill et al.
Ta ble 3. In Vivo Antimalarial Activity of Isoquine and
Amodiaquine versus P. yoelii NS Strain in the Peters 4-Day
Test
administered AQ. Evidence to support this metabolite
was found with the presence of a mercapturate metabo-
lite in urine. In direct contrast there was no evidence
to suggest the formation of an ISQ-glutathione conju-
gate in the bile of rats dosed with ISQ 1. This suggests
that bioactivation has been blocked; furthermore, and
in sharp contrast to amodiaquine, there is evidence to
suggest that relocation of the phenolic OH from the 4′
position in AQ to the 3′ position provides a route of
metabolic escape. LCMS analysis revealed the presence
of glucuronide conjugates in the bile and urine of
isoquine-dosed rats. Direct glucuronidation of parent
compound (ISQ 1) was also seen, suggesting that the
hydroxyl group is no longer restricting Phase II conju-
gation.
Scheme 4 summarizes the main metabolites from
isoquine that were identified in this initial study. Note
that amodiaquine or its metabolites do not form glucu-
ronides (Scheme 5) in sharp contrast to isoquine.
Results from this initial metabolism study have
demonstrated that interchange of the diethylamino side
chain with the hydroxyl group of AQ as in ISQ1 (3a )
can prevent the bioactivation of this compound in vivo
and can influence the critical balance between bioacti-
vation and detoxication. We propose that this compound
may not produce the adverse reactions seen with AQ
on the basis that no evidence of chemically reactive
metabolites could be obtained. Furthermore, inter-
change also provides altered routes of metabolism
whereby the hydroxyl group can facilitate “metabolic
escape”. Full details of metabolite identification, LCMS
traces, and graphs of tissue distribution have been
included as Supporting Information.
compound
isoquine
amodiaquine
ED50 (mg/kg)
SD
2.65
( 1.48
7.65
( 0.35
a
Groups of male CD-1 mice (n ) 5 per dose group) were
inoculated by ip injection with 106 parasitized erythrocytes in
phosphate buffered saline. Drug was administered by oral gavage
at time ) 0 (equivalent to 2 h post parasite inoculation) and on
days 1, 2, and 3. Blood films were prepared from tail snips on day
4 and stained with Giemsa, and parasite density was counted
microscopically. Drug doses used were 25, 10, 3, 1, 0.3, and 0.1
mg/kg.
Met a b olism St u d ies. To determine whether the
side chain modifications introduced would alter the
comparative metabolic fate of metabolism, studies were
carried out in the rat. Rearrangement of the hydroxyl
and diethylamino side chains to a 1,3-aminophenol
suggest that this compound would be unable to form a
quinonimine, as it is not chemically feasible to lose two
hydrogens via the same oxidative mechanism as AQ.14
In addition to being capable of forming toxic metabo-
lites, internal hydrogen bonding between the p-hy-
droxyanilino moiety and the nitrogen of the diethylami-
no side chain could be responsible for preventing AQ
from undergoing the O-sulfation and/or O-glucuronida-
tion experienced by other structurally related com-
pounds.15 This results in the failure of AQ to undergo
phase II detoxication. Removal of the diethylamino side
chain has been shown to allow AQ to undergo excessive
O-sulfation in the rat. In the context of the present
study, we were interested to see if the 3′-OH function
in 3 would be capable of undergoing metabolic phase II
conjugation reactions.
Discu ssion
Using radiolabeled3[H] isoquine, initial in vivo dis-
positional studies indicated that there is no alteration
in the rate of excretion of ISQ1 into bile and urine after
5 h compared to AQ. The main site for accumulation of
ISQ was found to be the liver, a primary target organ
for drug metabolism. 6.85% of the AQ dose remained
in the liver, 24 h after administration to rats compared
to 20.82% of the dose at 5 h. This was coupled with a
3% increase in radioactivity excreted into urine between
5 h and 24 h. Only 5.48% of the ISQ1 dose remained in
the liver after 24 h compared to 32.93% after 5 h,
suggesting almost complete clearance of the compound
at 24 h.
For isoquine (3a ), during the 5h experiment, a 3-fold
increase in plasma levels compared to AQ was wit-
nessed. This increase in plasma levels for isoquine
(0.130%, (0.018) was significantly different to amodi-
aquine levels (0.05%, (0.012 P > 0.05). This observation
may be important since the blood is the primary site of
action for 4-aminoquinoline antimalarials. Using LCMS
analysis, the main circulating metabolite for isoquine
in the plasma was the parent compound, in contrast to
amodiaquine where the only metabolite detected was
the desethyl metabolite.16
The hemoglobin degradation pathway in P. falci-
parum is a specialized parasite process with a proven
history as an exploitable therapeutic target as exempli-
fied by the 4-aminoquinolines and the endoperoxide
derivatives.17 Furthermore, unlike parasite encoded
enzymes18 and transporters,19 that are currently under
investigation, the parasite has difficulty in developing
resistance to these two classes of drug (compare the
speed of resistance development to chloroquine with
that for the antifolates or atovaquone).20 Resistance to
chloroquine (CQ) was first reported in the late 50’s, and
by the 70’s there were examples of culture adapted
strains with IC50s of 200-300 nM. Despite the continued
widespread exposure of parasite populations to CQ in
many parts of the world, resistance beyond this level is
rarely observed. Attempts to increase this level of
resistance in a laboratory setting have failed, suggesting
the parasite may have difficulty in developing resistance
strategies beyond this point. Based on this and our
understanding of 4-aminoquinoline action, the develop-
ment of a new 4-aminoquinoline derivative effective
against these highly CQ resistant isolates should pose
the parasite major difficulties in terms of resistance
acquisition and would therefore have an expected useful
therapeutic lifespan in excess of many of the other drugs
currently under development.
After 5 h, 87.17% ISQ1 dose was accounted for in the
tissues, bile plasma, and urine. Similarly, 78.56% of the
AQ dose could be accounted for after 5 h. This suggests
that there are other sites for the accumulation of the
compounds which were not analyzed during this study.
Glutathione conjugates were only detected in rats
For the past 15 years we have been involved in
research aimed at understanding the mechanism(s) of
action and toxicity of and the basis of parasite resistance

Improved 4-Aminoquinoline Antimalarials
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4937
Sch em e 4. Metabolic Scheme for Isoquine ISQ-1 (3a )
Sch em e 5. Metabolic Scheme for Amodiaquine (2)
observation that amodiaquine retains antimalarial ac-
tivity against chloroquine resistant parasites,27b our ini-
tial studies involved the design and synthesis of fluoro-
amodiaquine (13a ) as a safer alternative to AQ. On the
basis of metabolism studies, 13a was chemically modi-
fied to produce a new lead compound (13b) which ex-
pressed activity in vitro at about half the level of amo-
diaquine versus CQ-resistant strains, but with equiva-
lent oral in vivo potency versus Plasmodium berghei.9
While analogues in this series initially looked promising,
concern about cost led us to consider three other series
of synthetically more accessible analogues; the tebu-
quine series (14),11 the bis-Mannich series (15),29 and
the 5′-alkyl series class of 4-aminoquinoline.21 Com-
pounds in the tebuquine and bis-Mannich series are
considerably more potent than AQ or CQ in vitro and
in vivo but have subsequently been shown to have
unacceptable toxicity profiles and extremely long half-
lives.23 On the basis of initial toxicological evaluation
and the fact that all three sets of amodiaquine deriva-
tive retain the 4-aminophenol “structural alert” further
investigations were not pursued. Recent studies by the
Sergheraert group have also examined analogues where
the 4′-aminophenol alert has been modified by removal
of the 4′ hydroxyl function. Compounds of the general
class (13c) where shown to have excellent in vitro and
in vivo potencies.28e
Our most recent studies on 4-aminoquinoline SAR
have revealed, like others,30,31 that short chain two-car-
bon side-chain chloroquine analogues retain activity
against chloroquine resistant plasmodia. Our efforts32
were directed toward compounds less likely to undergo
metabolic N-terminal dealkylation, a process that pro-
duces N-desalkyl metabolites that are considerably less
potent against chloroquine resistant strains. Some of
these 2-C analogues, e.g. 16a , display good antiparasitic
profiles.30-32
to the 4-aminoquinolines.21,28a-d Integral to our studies
of the basic biology has been the synthesis of novel
4-aminoquinoline analogues that have been used as
chemical probes to investigate structure-activity and
structure-toxicity relationships (see Chart 2). This
subsequently resulted in the establishment of a rational
drug design program that has now generated more than
100 chemical entities.
Chart 2 summarizes the different classes of 4-ami-
noquinoline that we have investigated in our drug de-
velopment program. On the basis of the important
From our SAR studies, it was clear that the presence
of a 4-arylamino moiety provides analogues with supe-

4938 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
O’Neill et al.
Ch a r t 2. Approaches Leading to the Discovery of the Isoquine Class of 4-Aminoquinoline Antimalarial
Ta ble 4. Comparison of Calculated Log P (ClogP) versus
Antimalarial Activity
rior activity against CQ resistant strains, and it was
also apparent that the presence of an aromatic hydroxyl
druga
IC50 (nM) HB3
ClogPa
function appears to be important for additional levels
11a
chloroquine (1)
amodiaquine (2)
isoquine (ISQ-1) (3a )
isoquine diphosphate (3b)
14.98
9.60
12.65
9.02
5.04
4.51
4.51
NA
of antiparasitic activity.
It therefore seemed reason-
able that interchange of the 3′-Mannich side chain with
the 4′-OH function would provide a new template
capable of delivering a series of compounds chemically
incapable of forming potentially toxic quinoneimine
metabolites. Furthermore, it was proposed that ana-
logues in this series would not only be potent against
resistant strains, but would also be as cheap to prepare
as AQ on an industrial scale.
A potential drawback with any new 4-aminoquinoline
antimalarials is the possibility of cross-resistance with
chloroquine. Clearly from Table 2, the data presented
here demonstrates that for isoquine and several ana-
logues, there is minimal cross-resistance with chloro-
quine in the highly CQ resistant K1 strain (not shown
to be statistically significant by the Mann-Whitney U
test). These observations are supported by the recent
demonstration that mutations in the pfcrt gene have a
minimal effect on the activity and accumulation of
amodiaquine compared with chloroquine. Although the
results of in vitro testing are encouraging, it is clear that
further studies will be required to include a wider panel
of chloroquine resistant isolates to fully determine the
potential utility of this new class of 4-aminoquinoline.
(4a )
(5a )
(6a )
(7a )
(8a )
(9a )
(10a )
(11a )
(12)
30.03
14.76
19.78
51.88
28.37
97.20
9.07
4.22
3.45
5.57
6.62
4.08
3.36
4.63
3.82
3.51
20.22
16.24
a
ClogP values calculated using the Biobyte Mac log P
4
Program.
As shown in Table 3, isoquine is orally active in the
mouse model of malaria with a superior ED50 to
amodiaquine. From Table 4 there is no correlation
between the calculated log P’s (ClogPs)11b of these
derivatives and in vitro antimalarial activity against the
chloroquine sensitive HB3 strain. The same applies to
the chloroquine resistant strain, indicating that other
factors apart from lipophilicity have a role in determin-
ing the expression of in vitro antimalarial activity.
Chloroquine and to a lesser extent AQ are the only
4-aminoquinoline antimalarials with which we have any

Improved 4-Aminoquinoline Antimalarials
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4939
real clinical experience. CQ is considered a safe drug
when used as recommended, although cardiovascular
and CNS toxicities are seen in overdose and prolonged
treatment is associated with retinopathy. However, AQ
has been shown to carry an unacceptable risk of
agranulocytosis and hepatitis when used for prophyl-
axis, this has resulted in deaths.3,4 The toxicological
concerns are that as AQ is used more extensively,
possibly as a component of an artemisinin combination,
the incidence of these adverse drug reactions will
increase as pediatric African populations are exposed
to the drug on multiple occasions per year.
defined structural alerts in minor drug metabolites for
such immune-mediated toxicities.
The strategy we have adopted is one in which we have
designed the chemical alert out of the drug structure
while retaining antimalarial activity. A similar strategy
has been successfully employed in the redesign of
general anesthetics and â-blockers currently in clinical
use. This modification has resulted in a marked shift
in the pattern of metabolism in the rat, which carries
pharmacological benefits; thus in the rat model, it was
observed that for isoquine there is a complete absence
of glutathione conjugates in the bile. This clearly
illustrates the lack of bioactivation of the 4-amino
arylalkyl side-chain. Cleavage of the side-chain by P450
enzymes is the primary Phase 1 biotransformation. This
biotransformation reduces steric hindrance around the
phenolic hydroxyl group and enables efficient Phase II
glucuronidation. The O-glucuronide is rapidly excreted
in the bile and does not accumulate in tissues. This
metabolic pathway sharply contrasts with that of AQ
where the major dealkylated metabolite, desethyl AQ,
accumulates in the liver.
There are two chemical features of AQ that are
considered to be relevant to its toxicity. First, because
it is a lipophilic weak base, it is lysosomotropic and is
therefore readily taken up by specific white cell popula-
tions. Second, as described earlier, the side-chain con-
tains a 4-aminophenol group. This is a substructure now
widely recognized as a structural alert for toxicity by
medicinal chemists, because of metabolic oxidation to
quinonimines.6,8 For example, as described earlier,
paracetamol undergoes oxidation in the liver by cyto-
chrome P450 enzymes to N-acetyl p-benzoquinonimine,
the cause of massive irreversible hepatic necrosis when
the drug is taken in overdose. We have shown that AQ
readily undergoes oxidation to a quinoneimine.5 Oxida-
tion is more likely to occur with AQ than paracetamol
because of its lower oxidation potential.9 Extensive
metabolic studies, in a variety of model systems, have
shown that the oxidation of AQ can be catalyzed by
myeoloperoxidase, hypochlorous acid (both released by
activated white cells), and cytochrome P450 enzymes.
The quinoneimine has been trapped and characterized
as a glutathione conjugate which provides a biomarker
for the in vivo bioactivation of the drug. AQ undergoes
extensive bioactivation in the mouse and the rat.5-7 One
reason for the extensive bioactivation of AQ is that the
phenolic group is refractory to O-glucuronidation, the
normal pathway of biochemical detoxication that one
would anticipate for such a molecule. We attribute this
to a combination of the pKa of the group and strong
internal hydrogen bonding with the O-diethylamino
group. Removal of the Mannich side-chain permits
phase II detoxication reactions. AQ also undergoes
bioactivation to a quinoneimine in human neutrophils,
and a similar metabolic process was observed for pyr-
onaridine and amopyroquine.24
One of the primary biotransformations of ISQ1 in-
volves cleavage of the dialkylamino side-chain. We (and
others) have shown that this side chain is essential for
pharmacological activity. Therefore, we can propose that
the in vivo pharmacological response of isoquine will be
related to plasma/tissue concentrations of parent drug
or the N-desethyl metabolite (12a ), as is the case for
amodiaquine. This may be important in terms of cross-
resistance patterns with CQ since the desethyl metabo-
lite of AQ rather than parent drug is the main circu-
lating metabolite in man16b and this metabolite demon-
strates more cross resistance to CQ than the parent
drug which may have clinical consequences.16 This may
be a potential concern with isoquine, and a much more
detailed study on the identity and antimalarial activity
of main plasma metabolites will have to be conducted
in future work.16d
As with all new entities, it is true that until we
have clinical experience, toxicity cannot be ruled out.
However, we can be certain that the chemical rear-
rangement in isoquine and its analogues precludes the
formation of a reactive quinonimine. Furthermore,
initial studies described here in rodents indicate that
isoquine is eliminated at least as quickly as AQ;
therefore, we can anticipate that toxicity due to ac-
cumulation will not be an issue. The 4-aminoquinolines
as a drug class give no other obvious cause for con-
cern based on clinical experience. As outlined above, the
type of toxicity that has been associated with AQ is a
type II idiosyncratic response characterized, as de-
scribed above, as nonpredictable. Therefore, there will
always remain a real concern that AQ can illicit this
form of toxicity when used clinically. The more wide-
spread use of AQ both as monotherapy and in combina-
tion, the exposure to multiple doses in high transmission
areas, and the use of the drug in HIV +ve individuals
suggest it may be unwise to give a safety recommenda-
tion based on the earlier retrospective analysis of
prophylactic subjects. It is clear that the presence of this
metabolic alert in a new drug entity would terminate
its further development by the pharmaceutical industry
today.
What are the toxicological consequences of bioactiva-
tion? It should first be stated that the formation of
glutathione conjugates of AQ does represent detoxica-
tion. Nevertheless, at doses of AQ that are not acutely
toxic, covalent modification of hepatic proteins has been
demonstrated in the rat after only a single dose of the
drug. Furthermore we have demonstrated that AQ is
weakly immunogenic and AQ quinoneimine is extremely
immunogenic in an animal model.5 More importantly,
we have demonstrated the presence of specific antidrug
(metabolite) antibodies in a cohort of patients with
serious adverse drug reactions to AQ.7 Thus the mech-
anism is consistent with that of other drugs such as
penicillin and aminopyrine which cause type II hyper-
sensitivity reactions in man.15 There are no animal
models currently available to test for such reactions in
preclinical screens. Nevertheless, there are now clearly

4940 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
O’Neill et al.
1
(74%): mp ) 135-136 °C; H NMR (400 MHz, CDCl3) δ 7.06
(d, 1H, J ) 1.80 Hz, Ar-H), 6.95 (d, 1H, J ) 8.13 Hz, Ar-H),
6.91 (dd, 1H, J ) 8.13, 1.80 Hz, Ar-H), 3.58 (s, 2H, CH2), 2.30
(s, 6H, NCH3), 2.08 (s, 3H, COCH3); 13C NMR (100 MHz,
CDCl3) δ 168.05, 138.43, 128.66, 110.58, 107.47, 62.47, 53.41,
50.88, 44.43, 24.66; MS (CI) m/z 209 [M + H]+ (100), 164 (10),
122 (7), 58 (2); IR (Nujol mull): 3270, 1699, 1614, 1549, 1304,
In summary, our initial studies suggest that the
isomeric series of amodiaquine analogues presented in
this publication are worthy of further investigation as
potential, safer alternatives to amodiaquine. In particu-
lar, isoquine ISQ1 (3a ) appears to have many advan-
tages over the clinically used derivatives in this class.
As such, isoquine36 and other members of this isomeric
class are currently the subject of preclinical evaluation
in a partnership between the Malaria for Medicines
Venture (MMV) and Glaxo Smithkline Pharmaceuticals.
1271, 1196, 1123, 1015, 975, 870, 824, 802, 763, and 727 cm-1
;
HRMS m/z calcd for C11H17N2O2 [M+ + 1] 209.12901; found,
209.12938. Anal. (C11H16N2O2) C, H, N.
N-(4-Dip r op yla m in om et h yl-3-h yd r oxyp h en yl)a cet a -
m id e (6). Compound 6 was prepared in a manner similar to
Mannich base 3 to provide the product as a pale brown foamy
residue (63%). 1H NMR (200 MHz, CDCl3) δ 7.04 (dd, 1H, J )
7.97 Hz, 1.92, Ar-H), 6.86 (d, 1H, J ) 7.97 Hz, Ar-H), 6.79 (d,
1H, J ) 1.92 Hz, Ar-H), 6.58 (s, 1H, OH), 3.68 (s, 2H, CH2),
2.43 (t, 4H, J ) 7.68 Hz, NCH2CH2CH3), 2.12 (s, 3H, COCH3),
1.50 (m, 4H, NCH2CH2CH3), 0.85 (t, 6H, J ) 7.42 Hz, NCH2-
CH2CH3); 13C NMR (100 MHz, CDCl3) δ 170.96, 158.71, 140.25,
131.116, 119.692, 111.97, 108.77, 56.53, 49.66, 23.86, 20.60,
12.10; IR (neat) 3316, 2963, 2779, 1739, 1694, 1668, 1614,
1538, 1466, 1373, 1272, 1167, 1115, 1082, 1059, 978, 863 and
757 cm-1; MS (CI) m/z 265 [M + H]+(100), 164 (13), 102 (98)
72 (26); HRMS m/z calcd for C15H25N2O2 [M+ + 1] 265.19159,
found 265.19116. Anal. (C15H24N2O2) C, H, N.
Exp er im en ta l Section
Ch em istr y. Unless otherwise noted, all solvents and re-
agents were obtained from commercial suppliers and used
without further purification. The 3-hydroxyacetoamidophenol
and all of the corresponding amines used in the experiments
were purchased from Aldrich Chemical Co. Analytical thin-
layer chromatography (TLC) was performed on aluminum
sheets precoated with silica gel obtained from Merck. Visual-
ization was accomplished by UV light (254 nm). Column
chromatography was carried out on Merck 938S silica gel.
Infrared (IR) spectra were recorded in the range 4000-600
cm-1 using a Perkin-Elmer 298 infrared spectrometer. Solid
samples were run as Nujol mulls and liquids neat on sodium
chloride disks, as indicated in text. Proton NMR spectra were
recorded using Brucker (400, 250, and 200 MHz) NMR
spectrometers as clarified in text. Spectra were referenced to
the residual solvent peak and chemical shifts expressed in ppm
N -(4-Dib u t yla m in om e t h yl-3-h yd r oxyp h e n yl)a ce t a -
m id e (7). Compound 7 was prepared in a manner similar to
Mannich base 3 to provide the product as a pale brown
foamy residue (52%); 1H NMR (200 MHz, CDCl3) δ 7.33 (s,
1H, OH), 7.03 (d, 1H, J ) 7.90 Hz, Ar-H), 6.85 (d, 1H, J )
7.90 Hz, Ar-H), 6.78 (d, 1H, Ar-H), 3.67 (s, 2H, CH2), 2.45 (t,
4H, J ) 7.12 Hz, NCH2CH2CH2CH3), 2.10 (m, 4H, NCH2CH2-
CH2CH3), 1.47 (m, 4H, NCH2CH2CH2CH3), 0.86 (t, 6H, J )
1
from the internal reference peak. Significant HNMR data are
written in order: number of protons, multiplicity (b, broad; s,
singlet; d, doublet; t, triplet; q, quartet; m, multiple; bs, broad-
singlet, bm, broad-multiplet), coupling constants in hertz,
assignment. Mass spectra were recorded at 70 eV using a
VG7070E and/or micromass LCT mass spectrometers. The
molecular ion M+, with intensities in parentheses, is given
followed by peaks corresponding to major fragment losses.
Melting points were performed using a Gallemkamp melting
point apparatus and are reported uncorrected. Elemental
analyses were performed in the microanalysis laboratory in
the Department of Chemistry, University of Liverpool.
7.14 Hz, NCH2CH2CH2CH3
;
13C NMR (100 MHz, CDCl3) δ
170.85, 159.89, 140.71, 130.20, 119.98, 112.35, 109.15, 54.72,
50.08, 30.51, 21.99, 14.69; IR (Nujol mull): 3200, 1714, 1696,
1657, 1614, 1578, 1538, 1330, 1258, 1200, 1179, 1018, 980, 870,
and 760 cm-1); MS (CI) m/z 293 [M + H]+ (95), 130 (100), 86
(25); HRMS m/z calcd for C17H29N2O2 [M+ + 1] 293.12801,
found, 293.12837. Anal. (C17H28N2O2) C, H, N.
N-(3-H yd r oxy-4-p yr r olid in -1-ylm et h ylp h en yl)a cet a -
m id e (8). Compound 8 was prepared in a manner similar to
Mannich base 3 to provide the product as a brown oily resi-
due (80%). 1H NMR (400 MHz, CDCl3) δ 7.10 (bs, 1H, OH),
7.04 (dd, 1H, J ) 7.92, 2.00 Hz, Ar-H), 6.91 (d, 1H, J ) 7.92
Hz, Ar-H), 6.84 (d, 1H, J ) 2.00 Hz, Ar-H), 3.78 (s, 2H,-CH2),
2.62 (bs, 4H, pyrrolidinyl-H), 2.15 (s, 3H, COCH3), 1.84 (bm,
4H, pyrrolidinyl-H); 13C NMR (100 MHz, CDCl3) δ 168.49,
158.94, 138.60, 129.11, 118.18, 110.96, 107.88, 62.01, 54.20,
24.36, 23.01, 23.00; IR (Nujol mull): 3250, 1668,1606, 1167,
1116, 1013, 863, and 722 cm-1; MS (CI) m/z 235 [M + H]+ (94),
152 (18), 72 (100), 70 (19); HRMS m/z calcd for C13H19N2O2
[M+ + 1] 235.30200, found 235.30232. Anal. (C13H18N2O2) C,
H, N.
N -(4-Die t h yla m in om e t h yl-3-h yd r oxyp h e n yl)a ce t a -
m id e (3). 3-Hydroxyacetanilide (5 g, 33.1 mmol) was added
to 100 mL round-bottom flask followed by ethanol (23.6 mL).
One equivalent of diethylamine (3.42 mL, 33.1 mmol) and
aqueous formaldehyde (2.46 mL) was added and the solution
was allowed to heat under reflux for 24 h. After this reflux
period, the solvent was removed under reduced pressure and
the crude material was purified by silica gel flash column
chromatography using 20-80% MeOH/dichloromethane as
eluent. This gave the desired product as a pale brown oily
1
residue (5.31 g, 69%); H NMR (250 MHz, CDCl3) δ 7.15 (bs,
1H, OH), 7.05 (dd, 1H, J ) 8.2, 1.92 Hz, Ar-H), 6.88 (d, 1H, J
) 8.2 Hz, Ar-H), 6.80 (d, 1H, J ) 1.92 Hz, Ar-H), 3.71 (s, 2H,
CH2), 2.59 (q, 4H, J ) 7.20 Hz, NCH2CH3), 2.13 (s, 3H,
COCH3), 1.07 (t, 6H, J ) 7.20 Hz, NCH2CH3); 13C NMR (100
MHz, CDCl3) δ 172.03, 160.17, 140.76, 130.33, 119.83, 112.40,
109.35, 72.04, 57.48, 47.92, 24.28, 11.94; MS (CI) m/z 237 [M
+ H]+ (100), 164 (28), 122 (12), 74 (61), 58 (32); IR (neat):
3500-2800 (broad-OH band), 1668, 1614, 1538, 1454, 1386,
1273, 1194, 1166, 1114, 1032, 863, 773 and 736 cm-1; HRMS
m/z calcd for C13H21N2O2 [M+ + 1] 237.16029 found, 237.16042.
Anal. (C13H20N2O2) C, H, N.
N-(3-H yd r oxy-4-m or p h olin -4-ylm et h ylp h en yl)a cet a -
m id e (9). Compound 9 was prepared in a manner similar to
Mannich base 3 to provide the product as a white solid (72%):
1
mp 150 °C; H NMR (200 MHz, CDCl3) δ 7.25 (bs, 1H, OH),
6.99 (dd, 1H, J ) 8.24, 1.92 Hz, Ar-H), 6.87 (d, 1H, J ) 8.24
Hz, Ar-H), 6.82 (d, 1H, J ) 1.92 Hz, Ar-H), 3.71 (bt, 4H, J )
4.66 Hz, morpholinyl-H) 3.62 (s, 2H, CH2), 2.51 (bs, 4H,
morpholinyl-H), 2.11 (s, 3H, COCH3); 13C NMR (100 MHz,
CDCl3): δ 168.28, 157.99, 138.77, 129.17, 116.77, 110.99,
107.66, 66.80, 61.45, 52.91; IR (Nujol mull): 3334, 1692, 1654,
1627, 1604, 1534, 1512, 1307, 1278, 1245, 1167, 1109, 1067,
1004, 981, 863, 847, and 716 cm-1; MS (CI) m/z 250 [M + H]+
(68), 164 (91), 122 (100), 86 (95); HRMS m/z calcd for
N-[4-(ter t-Bu tyla m in om eth yl)-3-h yd r oxyp h en yl]a ceta -
m id e (4). Compound 4 was prepared in a manner similar to
Mannich base 3 to give the product as a white solid (70%
yield): 1H NMR (200 MHz, CDCl3) δ 8.15 (s, 1H, Ar-H), 6.95
(d, 1H, J ) 8.30 Hz, Ar-H), 6.51 (d, 1H, J ) 8.30 Hz, Ar-H),
3.72 (s, 2H, CH2), 2.17 (s, 3H), 1.18 (s, 9H, t-Bu). Anal.
(C13H20N2O2) C, H, N.
N-(4-Dim et h yla m in om et h yl-3-h yd r oxyp h en yl)a cet a -
m id e (5). Compound 5 was prepared in a manner similar to
Mannich base 3 to give the product as an off-white solid
C
13H19N2O3 [M+ + 1] 250.13174, found 250.13208. Anal.
(C13H18N2O2) C, H, N.
N -(3-H yd r oxy-4-p ip e r id in -1-ylm e t h ylp h e n yl)a ce t a -
m id e (10). Compound 10 was prepared in a manner similar
to Mannich base 3 to give the product as a white solid (89%):
mp 172-173 °C; H NMR (400 MHz, CDCl3) δ 7.09 (bs, 1H,
OH), 7.03 (dd, 1H, J ) 8.11, 1.90 Hz, Ar-H), 6.89 (d, 1H, J )
1

Improved 4-Aminoquinoline Antimalarials
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4941
8.11 Hz, Ar-H), 6.85 (d, 1H, J ) 1.90 Hz, Ar-H), 3.63 (s, 2H,
CH2), 2.49 (bs, 4H, piperidinyl-H), 2.14 (s, 3H, COCH3), 1.62
(bm, 4H, piperidinyl-H), 1.49 (bs, 2H, piperidinyl-H); 13C NMR
(100 MHz, CDCl3); δ 168.50, 158.95, 138.66, 129.12, 118.18,
110.18, 110.97, 107.88, 62.09, 54.21, 26.20, 24.96, 24.96, 24.37;
IR (Nujol mull): 3307, 1669, 1609, 1325, 1305, 1296, 1191,
1102, 1037, 980, 900, 861 and 815 cm-1; MS (CI) m/z 249 [M
+ H]+ (100), 166 (19), 86 (41), 84 (9); HRMS m/z calcd for
C14H21N2O2 [M+ + 1] 249.16029 found 249.16061. Anal.
(C14H20N2O2) C, H, N.
N -[3-H y d r o x y -4-(is o p r o p y la m in o m e t h y l)p h e n y l]-
a ceta m id e (11). Compound (11) was prepared in a manner
similar to Mannich base 3 to give the product as a pale brown
residue (51%); 1H NMR (250 MHz, CDCl3) δ 7.15 (bs, 1H, OH),
7.05 (dd, 1H, J ) 8.08 Hz, 2.05, Ar-H), 6.87 (d, 1H, J ) 8.08
Hz, Ar-H), 6.80 (d, 1H, J ) 2.05 Hz, Ar-H), 3.91 (s, 2H, CH2),
2.24 (m, 1H, isopropyl-H), 2.11 (s, 3H, COCH3), 1.11 (d, 6H
isopropyl); 13C NMR (100 MHz, CDCl3) δ 169.79, 151.99,
139.95, 121.18, 119.75, 112.93, 110.58, 49.79, 48.28, 22.52,
16.12; IR (neat): 2982, 1687, 1682, 1614, 1539, 1427, 1276,
1203, 1135, 1026, 981, 964, 834, 799, 720 and 660 cm-1; MS
(CI) m/z 223 [M + H]+ (100), 164 (20), 60 (90); HRMS m/z calcd
for C12H19N2O2 [M+ + 1] 223.14465 found 223.14532. Anal.
(C12H18N2O2) C, H, N.
(400 MHz, CDCl3) δ 8.52 (d, 1H, J ) 5.30 Hz, quinoline-H),
8.01 (d, 1H J ) 2.14 Hz, quinoline-H), 7.85 (d, 1H, J ) 8.90
Hz, quinoline-H), 7.43 (dd, 1H, J ) 8.90, 2.14 Hz, quinoline-
H), 7.02 (d, 1H, J ) 5.32 Hz, quinoline-H), 6.99 (d, 1H, J )
7.98 Hz, Ar-H), 6.76 (d, 1H, J ) 2.20 Hz, Ar-H), 6.68 (dd,
1H, J ) 7.96, 2.20 Hz, Ar-H), 6.63 (bs, 1H, OH), 3.99 (s, 2H,
CH2), 1.20 (s, 9H, t-Bu). Anal. (C20H22ClN3O) C, H, N.
5-(7-Ch lor oq u in olin -4-yla m in o)-2-d im e t h yla m in o-
m eth ylp h en ol (5a ). This compound was prepared in
a
manner similar to 3a to provide a pale yellow solid (69%). mp
1
176.6-177.8 °C; H NMR (400 MHz, CDCl3) δ 8.56 (d, 1H, J
) 5.40 Hz, quinoline-H), 8.04 (d, 1H, J ) 2.06 Hz, quinoline-
H), 7.84 (d, 1H, J ) 9.06 Hz, quinoline-H), 7.45 (dd, 1H, J )
9.06, 2.06 Hz quinoline-H), 7.05 (d, 1H, J ) 5.40 Hz, quinoline-
H), 6.98 (d, 1H, J ) 7.95, Ar-H), 6.76 (d, 1H, J ) 2.22 Hz,
Ar-H), 6.69 (dd, 1H, J ) 7.95, 2.22 Hz, Ar-H), 6.51 (bs, 1H,
OH), 3.67 (s, 2H, CH2), 2.37 (s, 6H, NCH3); 13C NMR (100 MHz,
CDCl3) δ 159.78, 152.37, 149.50, 149.4, 146.60, 134.60, 129.59,
129.47, 126.42, 121.50, 119.04, 113.18, 110.39, 103.35, 62.86,
44.83, 31.24; IR (Nujol mull) 3194, 2376, 2306, 1702, 1684,
1653, 1458, 1375, and 1107 cm-1; MS (CI) m/z 328 [M +
H]+ (76), 285 (100), 251 (20), 63 (20); HRMS m/z calcd for
C18H19ClN3O [M+ + 1] 328.12164 found, 328.12123. Anal.
(C18H18ClN3O) C, H, N.
N-(4-E t h yla m in om et h yl-3-h yd r oxyp h en yl)a cet a m id e
(12). Brown oily residue (55%); H NMR (400 MHz, CDCl3) δ
5-(7-Ch lor oqu in olin -4-yla m in o)-2-d ip r op yla m in om eth -
ylp h en ol (6a ). This compound was prepared in a manner
similar to 3a to provide a pale yellow solid (61%). mp
1
7.27 (d, 1H, J ) 1.89 Hz, Ar-H), 7.11 (dd, 1H, J ) 8.18 Hz,
Ar-H), 6.90 (dd, 1H, J ) 8.18, 1.89 Hz, Ar-H), 3.99 (s, 2H, CH2),
2.89 (q, 2H, NHCH2CH3), 2.07 (s, 3H, COCH3), 1.08 (t, 3H,
NHCH2CH3); 13C NMR (100 MHz, CDCl3): δ 171.73, 148.89,
141.67, 131.66, 117.21, 112.07, 111.86, 108.54, 43.47, 23.91,
12.69; MS (CI) m/z 209 [M + H]+ (72), 166 (100), 152 (80);
HRMS m/z calcd for C11H17N2O2 [M+ + 1] 209.12900 found
209.12918. Anal. (C11H16N2O2) C, H, N.
1
183-184 °C; H NMR (400 MHz, CDCl3) δ 8.56 (d, 1H, J )
5.40 Hz, quinoline-H), 8.03 (d, 1H, J ) 2.20 Hz, quinoline-H),
7.84 (d, 1H, J ) 8.90 Hz, quinoline-H), 7.45 (dd, 1H, J )
8.90, 2.22 Hz, quinoline-H), 7.06 (d, 1H, J ) 5.41 Hz, quinoline-
H), 6.98 (d, 1H, J ) 7.95 Hz, Ar-H), 6.74 (d, 1H, J ) 2.22
Hz, Ar-H), 6.69 (dd, 1H, J ) 7.95, 2.22 Hz, Ar-H), 6.55
(bs, 1H, OH), 3.79 (s, 2H, CH2), 2.60 (t, 4H, J ) 7.63 Hz,
NCH2CH2), 1.59 (m, 4H, NCH2CH2), 0.92 (t, 6H, J ) 7.31
Hz, CH2CH3); 13C NMR (100 MHz, CDCl3) δ 159.87, 152.37,
150.14, 149.4, 147.76, 140.24, 135.57, 129.57, 129.46, 126.39,
121.50, 119.41, 113.13, 110.42, 103.30, 58.28, 55.83; IR (Nujol
mull) 3150, 1733, 1699, 1657,1578, 1538, 1331, 1258, 1181,
1159, 980, 855, 807 and 721 cm-1; MS (ES+) m/z 384.2 [M +
H]+ (100), 283 (82), 192.6 (34); HRMS m/z calcd. for C22H27N3-
OCl (M+ + 1) 384.1843 found 384.1845. Anal. (C22H26ClN3O)
C, H, N.
5-(7-Ch lor oqu in olin -4-yla m in o)-2-d ieth yla m in om eth -
ylp h en ol (3a ). Aqueous hydrochloric acid (20%) (25 mL) was
added to a round-bottom flask containing the amide 2a (4.47
g, 18.9 mmol) and the solution heated under reflux for 6 h.
The solvent was then removed in vacuo and the resulting
residue coevaporated with ethanol to give the corresponding
hydrochloride salt. 4,7-Dichloroquinoline (4.12 g, 20.8 mmol)
and ethanol (30 mL) were added, and the reaction was heated
under reflux for 12 h until completion of the reaction (deter-
mined by TLC). A pale yellow solid was obtained upon
removing the solvent under reduced pressure; this was sub-
sequently purified by flash column chromatography using 20-
80% MeOH/dichloromethane as eluent to yield the quinoline
hydrochloride salt as a yellow foamy solid (6.47 g, 80%). To
liberate the free base compound, this solid was dissolved in
distilled water (18 mL) and the solution basified by careful
dropwise addition of saturated sodium bicarbonate (added
until no more precipitate formed). Dichloromethane (100 mL)
was added, and the free base was extracted into the organic
layer. Subsequent drying and removal of solvent in vacuo
afforded the desired product as a pale off-white solid (3.76 g,
5-(7-Ch lor oqu in olin -4-yla m in o)-2-d ibu tyla m in om eth -
ylp h en ol (7a ). This compound was prepared in a manner
similar to 3a to provide a pale brown solid (58%). mp 153 °C;
1H NMR (200 MHz, CDCl3) δ 8.53 (d, 1H, J ) 5.22 Hz,
quinoline-H), 8.00 (d, 1H, J ) 2.20 Hz, quinoline-H), 7.83 (d,
1H, J ) 9.08 Hz, quinoline-H), 7.42 (dd, 1H, J ) 9.0, 2.22 Hz,
quinoline-H), 7.03 (d, 1H, J ) 5.50 Hz, quinoline-H), 6.96 (d,
1H, J ) 7.98 Hz, Ar-H), 6.72 (d, 1H, J ) 2.00 Hz, Ar-H),
6.67 (dd, 1H, J ) 7.98, 2.00 Hz Ar-H), 6.54 (bs, 1H, OH), 3.75
(s, 2H, CH2), 2.52 (t, 4H, J ) 7.14 Hz, NCH2CH2), 1.50 (m,
4H, CH2CH2CH2), 1.30 (m, 4H, CH2CH2CH3) 0.90 (t, 6H, J )
7.31 Hz, CH2CH3); 13C NMR (100 MHz, CDCl3) δ 159.55,
152.03, 149.85, 149.79, 147.44, 135.21, 129.24, 129.11, 127.93,
126.02, 121.16, 118.19, 112.82, 110.11, 102.94, 57.85, 53.23,
28.45, 20.62, 13.96; IR (Nujol mull) 3190, 2354, 1739, 1733,
1714, 1699, 1696, 1657, 1654, 1614, 1578, 1538, 1330, 1258,
1
71%): mp 134-135 °C; H NMR (400 MHz, CDCl3) δ 8.55 (d,
1H, J ) 5.24 Hz, quinoline-H), 8.02 (d, 1H, J ) 2.20 Hz,
quinoline-H), 7.83 (d, 1H, J ) 8.92 Hz, quinoline-H), 7.44 (dd,
1H, J ) 8.96, 2.16 Hz, quinoline-H), 7.04 (d, 1H, J ) 5.24 Hz,
quinoline-H), 6.98 (d, 1H, J ) 7.96 Hz Ar-H), 6.74 (d, 1H, J
) 2.08 Hz, Ar-H), 6.68 (dd, 1H, J ) 7.96, 2.22 Hz, Ar-H),
6.53 (bs, 1H, OH), 3.79 (s, 2H, CH2), 2.65 (q, 4H, J ) 7.14 Hz,
NCH2CH3), 1.14 (t, 6H, J ) 7.14 Hz, NCH2CH3); 13C NMR (100
MHz, CDCl3) δ 160.44 152.35, 150.11, 147.84, 140.22, 135.57,
129.58, 129.41, 126.38, 121.54, 119.27, 118.53, 113.17, 110.54,
103.28, 56.99, 46.72, 11.57; IR (Nujol mull) 2930, 2858, 1668,
1612, 1575, 1529, 1459, 1424, 1378, 1327, 1277, 1192, 1178,
1200, 1179, 1159, 1118, 980, 909, 870, 856, 818 and 760 cm-1
;
MS (ES+) m/z 412.2 [M + H]+ (100), 283 (92), 206 (65); HRMS
m/z calcd for C24H31N3OCl [M+ + 1] 412.2156 found 412.2150.
Anal. (C24H30ClN3O) C, H, N.
5-(7-Ch lor oqu in olin -4-yla m in o)-2-p yr r olid in -1-ylm eth -
ylp h en ol (8a ). This compound was prepared in a manner
similar to 3a to provide an off-white solid (80%): mp 163.1
°C; 1H NMR (400 MHz, CDCl3) δ 8.55 (d, 1H, J ) 5.41 Hz,
quinoline-H), 8.02 (d, 1H, J ) 2.07 Hz, quinoline-H), 7.84 (d,
1H, J ) 9.06 Hz, quinoline-H), 7.44 (dd, 1H, J ) 9.06, 2.07
Hz, quinoline-H), 7.03 (d, 1H, J ) 5.41 Hz, quinoline-H), 6.99
(d, 1H, J ) 7.95 Hz, Ar-H), 6.75 (d, 1H, J ) 2.06 Hz, Ar-H),
6.68 (dd, 1H, J ) 7.95, 2.07 Hz, Ar-H), 6.59 (bs, 1H, OH),
3.85 (s, 2H, CH2), 2.65 (bm, 4H, pyrrolidinyl-H), 1.90 (bm,
4H, pyrrolidinyl-H); 13C NMR (100 MHz, CDCl3) δ 159.41,
1159, 1115, 1079, 992, 974, 907, 873, 855, 814 and 772 cm-1
;
MS (CI) m/z 356 [M + H]+ (100), 285 (24), 271 (92), 110 (100);
HRMS m/z calcd for C20H23ClN3O [M+ + 1] 356.15292 found,
356.15169. Anal. (C20H22ClN3O) C, H, N.
2-(ter t-Bu tylam in om eth yl)-5-(7-ch lor oqu in olin -4-ylam i-
n o)p h en ol (4a ). This compound was prepared in a manner
similar to 3a to provide a pale yellow solid (69%); 1H NMR

4942 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
O’Neill et al.
151.98, 149.75, 147.54, 139.92, 135.24, 129.04, 128.77, 126.03,
121.24, 119.31, 118.19, 112.84, 110.07, 102.92, 58.50, 53.55,
23.72; IR (Nujol mull) 3175, 1667, 1652, 1615, 1576, 1536,
1512, 1457, 1427, 1376 cm-1; MS (CI) m/z 354 [M + H]+ (65),
285 (100), 271 (35), 72 (93); HRMS m/z calcd for C20H21ClN3O
[M+ + 1] 354.13730 found 354.13713 Anal. (C20H20ClN3O) C,
H, N.
110.48, 102.92, 52.10, 43.08, 14.83; IR 3274, 1615, 1573, 1542,
1461, 1336, 1282, 1113, 1082, 886, 909, 818 and 764 cm-1; MS
m/z 328 [M + H]+ (10), 285 (29), 271 (100), 207 (89) 91 (22),
58 (14); HRMS (CI) m/z calcd for C18H19ClN3O [M+ + 1]
328.1216 found 328.12155. Anal. (C18H18ClN3O) C, H, N.
Biology. In Vitr o Testin g P r otocol. An tim a la r ia l Ac-
tivity. Two strains of P. falciparum were used in this study:
(a) The K1 strain which is known to be CQ resistant and (b)
the HB3 strain which is sensitive to all antimalarials. Para-
sites were maintained in continuous culture using the method
of J ensen and Trager.33 Cultures were grown in flasks contain-
ing human erythrocytes (2-5%) with parasitemia in the range
of 1% to 10% suspended in RPMI 1640 medium, supplemented
with 25 mM HEPES and 32 mM NaHCO3, and 10% human
5-(7-Ch lor oqu in olin -4-yla m in o)-2-m or p h olin -4-ylm eth -
ylp h en ol (9a ). This compound was prepared in a manner
similar to 3a to provide the desired product an off-white solid
(60%): mp 185-186°C; 1H NMR (400 MHz, CDCl3) δ 8.56 (d,
1H, J ) 5.25 Hz, quinoline-H), 8.04 (d, 1H, J ) 2.06 Hz,
quinoline-H), 7.84 (d, 1H, J ) 8.91 Hz, quinoline-H), 7.45 (dd,
1H, J ) 8.91, 2.06 Hz, quinoline-H), 7.04 (d, 1H, J ) 5.25 Hz,
quinoline-H), 7.01 (d, 1H, J ) 7.95 Hz, Ar-H), 6.77 (d, 1H, J
) 2.22 Hz, Ar-H), 6.72 (dd, 1H, J ) 7.95, 2.22 Hz, Ar-H),
6.52 (bs, 1H, OH), 3.78 (bm, 4H, morpholinyl-H), 3.73 (s, 2H
CH2), 2.60 (bm, 4H, morpholinyl-H); 13C NMR (100 MHz,
CDCl3) δ 159.19, 152.33, 150.13, 147.63, 140.80, 135.66, 130.16,
129.46, 126.50, 121.51, 117.64, 113.46, 110.34, 103.44, 102.23,
67.16, 61.87, 53.32; IR (Nujol mull) 3200, 1699, 1654, 1575,
1533, 1346, 1335, 1249, 1118, 862 and 811 cm-1; MS (CI) m/z
370 [M + H]+ (100), 285 (71), 251 (30), 208 (34) 164 (26); HRMS
m/z calcd for C20H21ClN3O2 [M+ + 1] 370.13223 found 370.13276.
Anal. (C20H20ClN3O2) C, H,N.
serum (complete medium). Cultures were gassed with
a
mixture of 3% O2, 4% CO2, and 93% N2. Antimalarial activity
was assessed with an adaption of the 48-h sensitivity assay of
Desjardins et al. using [3H]-hypoxanthine incorporation as an
assessment of parasite growth.34 Stock drug solutions were
prepared in 100% dimethyl sulfoxide (DMSO) and diluted to
the appropriate concentration using complete medium. Assays
were performed in sterile 96-well microtiter plates, and each
plate contained 200 µL of parasite culture (2% parasitemia,
0.5% haematocrit) with or without 10 µL drug dilutions. Each
drug was tested in triplicate and parasite growth compared
to control wells (which consituted 100% parasite growth). After
24-h incubation at 37 °C, 0.5 µCi hypoxanthine was added to
each well. Cultures were incubated for a further 24 h before
they were harvested onto filter-mats, dried for 1 h at 55 °C,
and counted using a Wallac 1450 Microbeta Trilux Liquid
scintillation and luminescence counter. IC50 values were
calculated by interpolation of the probit transformation of the
log dose-response curve.
In Vivo Testin g P r otocol.35 Male, random Swiss albino
mice weighing 18-22 g were inoculated intraperitoneally with
107 parasitized erythrocytes with P. yoelii NS strain. Animals
were then dosed daily via two routes (intraparential or oral)
for four consecutative days beginning on the day of infection.
Compounds were dissolved or suspended in the vechile solution
consisting of methanol, phosphate-buffered saline, and DMSO
(2:5:3 v/v). The parasitemia was determined on the day
following the last treatment and the ED50 (50% suppression
of parasites when compared to vehicle only treated controls)
calculated from a plot of log dose against parasitemia.
5-(7-Ch lor oqu in olin -4-yla m in o)-2-p ip er id in -1-ylm eth -
ylp h en ol (10a ). This compound was prepared in a manner
similar to 3a to provide the desired product as an off-white
1
solid (71%): mp 182.6; H NMR (200 MHz, CDCl3) δ 8.53 (d,
1H, J ) 5.22 Hz, quinoline-H), 8.00 (d, 1H, J ) 1.92 Hz,
quinoline-H), 7.83 (d, 1H, J ) 9.06 Hz, quinoline-H), 7.42 (dd,
1H, J ) 9.06, 1.92 Hz, quinoline-H), 7.03 (d,1H, J ) 5.22 Hz,
quinoline-H), 6.97 (d, 1H, J ) 7.98 Hz, Ar-H), 6.73 (d, 1H, J
) 2.06 Hz, Ar-H), 6.67 (dd, 1H, J ) 7.98, 2.06 Hz, Ar-H),
6.58 (bs, 1H, OH), 3.67 (s, 2H, CH2), 2.51 (bm, 4H, piperidinyl-
H), 1.64 (bm, 6H, pyrrolidinyl-H); 13C NMR (100 MHz, CDCl3)-
159.81, 152.31, 150.06, 147.85, 140.26, 135.61, 129.74, 129.39,
126.40, 121.53, 118.78, 118.52, 113.21, 110.48, 103.29, 62.14,
54.28, 26.22, 24.35; MS (CI) m/z 368 [M + H]+ (70), 285 (100),
271 (37), 86 (93); IR (Nujol mull) 3200, 1614, 1575, 1455, 1377,
1249, 1198 and 822 cm-1; HRMS m/z calcd for C21H23ClN3O
[M+ + 1] 368.15292 found 368.15403. Anal. (C21H22ClN3O) C,
H, N.
5-(7-Ch lor oq u in olin -4-yla m in o)-2-(isop r op yla m in o-
m eth yl)p h en ol (11a ). This compound was prepared in a
manner similar to 3a to provide the product as a pale yellow
Meta bolism Stu d ies. Ma ter ia ls. Opti-solv tissue solubi-
lizer was a product of Wallac (Loughborough U.K). Ultima-
gold liquid scintillation fluid was purchased from Packard
bioscience. Glacial acetic acid was a Merck product. [3H]AQ
and [3H]ISQ were synthesized by the University of Liverpool.
Heparin was a product of CP pharmaceuticals (Wrexham UK).
All other chemicals used were purchased from Sigma (Poole,
U.K.)
1
solid (67%): mp 157.6-158.2; H NMR (400 MHz, CDCl3) δ
8.55 (d, 1H J ) 5.32 Hz, quinoline-H), 8.03 (d, 1H, J ) 2.08
Hz, quinoline-H), 7.84 (d, 1H, J ) 8.90 Hz, quinoline-H),
7.49 (dd, 1H, J ) 8.90, 2.08 Hz, quinoline-H), 7.02 (d, 1H, J )
5.30 Hz, quinoline-H), 6.99 (d, 1H, J ) 7.92 Hz, Ar-H), 6.75
(d, 1H, J ) 2.24 Hz, Ar-H), 6.69 (dd,1H, J ) 7.92, 2.24 Hz,
Ar-H), 6.51 (bs, 1H, OH), 4.02 (s, 2H, CH2), 2.93 (septet, J )
6.36 Hz, 1H, isopropyl-H), 1.19 (d, 6H, J ) 6.36 Hz, isopropyl-
H); 13C NMR (100 MHz, CDCl3) δ 159.79, 151.99, 149.76,
147.49, 139.95, 135.24, 129.08, 128.98, 126.04, 121.18, 119.75,
118.18, 112.93, 110.58, 102.91, 49.79, 48.28, 22.52. IR (Nujol
mull) 3280, 1600, 1576, 1429, 1333, 1280, 1179, 1159, 1118,
814 and 761 cm-1; MS m/z 342.1 [M + H]+ (100), 283 (30), 171
(76); HRMS (ES+) m/z calcd for C19H21ClN3O [M+ + 1]
342.1373 found 342.1377. Anal. (C19H20Cl N3O) C, H, N.
Sta tistics. All values are given as the mean ( SEM. All
statistical analyses were carried out using a Mann-Whitney
test, and the differences were deemed significant at p < 0.05
In vestiga tion of Bilia r y a n d Ur in a r y Excr etion of [3H]
AQ a n d [3H] ISQ a fter Ad m in istr a tion to Ma le Wista r
Ra ts. Male Wistar rats (200-300 g) were anesthetized with
urethane (1.4 g/mL in 20 mL in 0.9% saline, 20 mL/kg) and
their state of consciousness determined, using the cornea reflex
test and the limb retraction test. The rats were carefully
monitored throughout the procedure to ensure that anesthesia
was maintained.
5-(7-Ch lor oq u in olin -4-yla m in o)-2-e t h yla m in om e t h -
ylp h en ol (12a ). This compound was prepared in a manner
similar to 3a to provide the product as a pale brown solid
1
A small incision was made in the throat, and the trachea
was located, via blunt dissection of the surrounding connective
tissue. A 1.57 mm (I.D.) polythene tube cannula was inserted
and securely fastened. The jugular vein was also cannulated
with 0.58 mm (I.D.) tubing to allow iv administration of the
compounds. A syringe containing saline was attached to the
jugular cannula to act as a seal preventing air bubbles from
entering the vein. An incision was made along the midline of
the abdomen. The common bile duct was located and allowed
to dilate before a 0.58 mm (I.D.) cannula was inserted. Control
(68%). mp 136.3-131.7 °C; H NMR (400 MHz, CDCl3) 8.54
(d, 1H, J ) 5.32 Hz, quinoline-H), 8.01 (d, 1H, J ) 2.14 Hz,
quinoline-H), 7.85 (d, 1H, J ) 8.95 Hz, quinoline-H), 7.43 (dd,
1H, J ) 8.95, 2.14 Hz, quinoline-H), 7.02 (d, 1H, J ) 5.32 Hz,
quinoline-H), 6.99 (d, 1H, J ) 7.98 Hz, Ar-H), 6.76 (d, 1H, J
) 2.20 Hz, Ar-H), 6.68 (dd, 1H, J ) 7.96, 2.20 Hz, Ar-H),
6.63 (bs, 1H, OH), 4.02 (s, 2H, CH2), 2.76 (q, 2H, J ) 7.17 Hz
NHCH2CH3), 1.19 (t, 3H, J ) 7.17 Hz, NHCH2CH3); 13C NMR
(100 MHz, CDCl3) 159.70, 151.97, 149.74, 147.53, 139.99,
135.23, 129.16, 129.01, 126.01, 121.26, 119.32, 118.20, 112.93,

Improved 4-Aminoquinoline Antimalarials
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4943
bile was obtained. The penis was ligated, to allow urine to be
collected during the experiment.
tivity (all volumes of chemicals to be added were doubled for
solubulizing the red blood cells).
An a lysis of Ur in a r y, Bilia r y, a n d P la sm a Meta bolites
of Ma le Wista r Ra ts Dosed w ith [3H]AQ or [3H]ISQ.
Aliquots (50-100 µL) of bile, urine, and plasma ether extracts
were eluted from a Zorbax SB-18 column with a slow aceto-
nitrile gradient (10-50% over 30 min) in ammonium acetate
(5.0 mM, pH 3.8) at 0.9 mL/min. Two J asco PU-980 pumps
were linked to a mixing module, allowing effluent to mix with
scintillation fluid prior to reaching the Flo-One A250 beta
radioactive flow detector or allowing the effluent to be deliv-
ered to the Quattro II mass spectrometer. Nebulizing and
drying gas were delivered at a rate of 13 L/h and 300 L/h,
respectively. The temperature of the LC/MS interface was 70
°C, and the capillary voltage was 3.7 × 10-2 V. The extent of
fragmentation was modulated via altering the cone voltage.
The radiolabeled compounds had been made up in a vehicle
composed of 50% dimethyl sulfoxide (DMSO)-49% water-1%
citric acid. DMSO replaced the saline in the jugular cannula
to prevent the drug precipitating out of solution during dosing.
A 500 µL Hamilton syringe was filled with either [3H]AQ
or [3H]ISQ (54 µmol/kg, 25 µCi/kg) and connected to the
cannula. The radiolabeled compounds were infused via the
jugular vein over a period of 30 min to prevent respiratory
depression caused by DMSO. Bile fractions were collected at
hourly intervals for 5 h from the start of dosing, All samples
were weighed and their weight recorded.
After 5 h, any remaining urine was aspirated from the
bladder, and blood was collected via cardiac puncture with a
heparinized needle into a heparinized tube. The sample was
centrifuged to allow the plasma and red blood cells to be
separated (4000 rpm for 5 min). The volume of plasma was
recorded. Tissues (brain, heart, kidney, liver, lung, spleen, and
skin) were removed from the animal, rinsed in saline before
being placed in vials, and weighed. All samples were stored
at -80 °C until they were required for analysis.
Ack n ow led gm en t. We thank the Medicines for
Malaria Venture (MMV), Geneva, the Wellcome Trust
(S.A.W., B.K.P.), the Leverhulme Trust (P.A.S.), Glaxo-
Smithkline Pharmaceuticals (S.A.W., PON, B.K.P.,
P.A.W.), and the EPSRC (A.M.) for funding this pro-
gram. The authors also thank the Wellcome Trust for a
grant for a mass spectrometer, the EPSRC for a single-
crystal diffractometer (GR/N36851 EPSRC), and NMR
facility (GR/M90801).
In vestiga tion of [3H] AQ a n d [3H] ISQ Rem a in in g in
th e P la sm a a fter Dosin g. The animal was anesthetized and
the jugular vein cannulated as described above. To allow blood
samples to be taken regularly, the carotid artery was cannu-
lated with 0.58 mm I.D. polythene tubing. The cannula was
attached to a heparinized saline syringe to prevent blood
escaping from the cannula and to prevent air bubbles from
entering the blood stream. The animals were dosed as de-
scribed above, and the clock was started at the end of the
dosing period. Blood samples (300 µL) were collected at 15 min,
30 min, and hourly postdosing. Heparinized saline was flushed
through the cannula after every blood collection to prevent
clotting within the tubing. The saline was allowed to drain
out of the cannula prior to every collection point.
Su p p or tin g In for m a tion Ava ila ble: X-ray crystallo-
graphic data and further details of metabolism studies on
isoquine. This material is available free of charge via the
Internet at http://pubs.acs.org.
Refer en ces
(1) World Health Organisation (WHO). Conquering, Suffering,
Enriching Humanity: The World Health Report; World Health
Organisation Publishers: Geneva, 1997.
After the fifth hour sample had been collected, all remaining
blood within the rat was allowed to drain from the cannula
into a heparinized tube. All samples were centrifuged (4000
rpm for 5 min) immediately after collection, and the pellet and
supernatant were separated. The volume of the plasma was
recorded. The tissues were also removed and weighed, and all
samples were stored at -80 °C until analyzed. The active
components of the plasma were removed via extraction with
ether for LC/MS analysis.
24 h Meta bolism Stu d y. [3H]AQ (54 µmol/kg) was admin-
istered ip to male Wistar rats (200-300 g). Each animal was
placed in a wire-bottom metabolism cage with access to food
and water. Urine was collected over 24 h, and the cage was
rinsed with distilled water (10 mL) at the end of the collection
period. After 24 h, the animals were anesthetized with
phenobarbitone (60 mg/kg in 0.9% saline) and their tissues
removed and blood collected via cardiac puncture with a
heparinized needle. All samples were stored at -80 °C until
analyzed. Previous ISQ data was obtained for comparison with
AQ. These data was produced following the same procedure
used for AQ.
An a lysis of th e Ra d ioa ctivity Excr eted in to Ur in e,
Bile, a n d P la sm a . Aliquot of bile (2 × 10 µL), urine (2 × 50
µL), and plasma (2 × 50 µL) from animals dosed with [3H]AQ
or [3H]ISQ were added to 4 mL of liquid scintillant and
vortexed thoroughly. Samples were left in darkness overnight
to prevent chemiluminescence. Radioactivity was then deter-
mined using a Packard 1500 Liquid Scintillation Analyzer.
In vestiga tion of th e Tissu e Distr ibu tion of [3H]AQ or
[3H]ISQ over 5 h . Portions of each tissue (50-100 mg) and
aliquots of red blood cells (50-60 mg) were taken in duplicate,
and tissue solubilizer (0.5 mL) was added to each sample and
left overnight at 50 °C. The samples were cooled to room
temperature before being decolorized with hydrogen peroxide
(200 µL) and left for 1 h. The mixture was then neutralized
with glacial acetic acid (30 µL) and 12 mL of scintillation fluid
was added. The mixture was mixed thoroughly and left
overnight in the dark. The samples were assayed for radioac-
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4944 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
O’Neill et al.
(11) (a) O’Neill, P. M.; Willock, D. J .; Hawley, S. R.; Bray, P. G.; Storr,
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(13) A byproduct in this process is the 4-ethoxy-7-chloroquinoline that
arises via nucleophilic aromatic substitution of the 4-chloro atom
by solvent. That 4-ethoxy-7-chloroquinoline is an intermediate
en route to the desired product remains to be established. By
analogy, it has been shown that for reactions involving 4-chlo-
roquinolines with alkylamines in phenol as solvent, the inter-
mediate 4-phenoxyquinoline is an intermdiate. For examples,
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