Synthesis and antitrypanosomal evaluation of derivatives of N-benzyl-1,2-dihydroquinolin-6-ols: Effect of core substitutions and salt formation

Article abstract of DOI:10.1016/j.bmc.2010.11.003

Analogs of the trypanocidal lead compound 1-benzyl-1,2-dihydro-2,2,4- trimethylquinolin-6-yl acetate were prepared to extend the structure-activity relationship in this series of molecules, improve the in vivo antitrypanosomal activity of the lead, and de

Full text of DOI:10.1016/j.bmc.2010.11.003

Bioorganic & Medicinal Chemistry 19 (2011) 513–523
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antitrypanosomal evaluation of derivatives
of N-benzyl-1,2-dihydroquinolin-6-ols: Effect of core substitutions and salt
formation
Carolyn S. Reid a, Donald A. Patrick b, Shanshan He a, Jean Fotie a, Kokku Premalatha a, Richard R. Tidwell b,
Michael Zhuo Wang c, Qiang Liu b, Pavel Gershkovich d, Kishor M. Wasan d, Tanja Wenzler e,f, Reto Brun e,f
,
Karl A. Werbovetz a,
⇑
a Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210, USA
b Department of Pathology and Laboratory Medicine, School of Medicine, The University of North Carolina, Chapel Hill, NC 27599, USA
c Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
d Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, BC, Canada V6T 1Z3
e Department of Medical Parasitology and Infection Biology, Swiss Tropical & Public Health Institute, CH-4002 Basel, Switzerland
f University of Basel, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Analogs of the trypanocidal lead compound 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate
were prepared to extend the structure–activity relationship in this series of molecules, improve the
in vivo antitrypanosomal activity of the lead, and determine whether ester prodrugs are needed to
overcome the instability of the dihydroquinolin-6-ols. Two of the most active compounds identified in
this study were 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride and 1-(2-methoxy)-
benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride. These stable solids possessed low
nanomolar IC50 values against Trypanosoma brucei rhodesiense STIB900 in vitro and provided cures in
an early treatment acute mouse model of African trypanosomiasis when given ip at 50 mg/kg/day for four
consecutive days.
Received 23 August 2010
Revised 26 October 2010
Accepted 1 November 2010
Available online 5 November 2010
Keywords:
Dihydroquinoline
African trypanosomiasis
Drug discovery
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Results from a phase III trial were reported in 2009 which demon-
strated that combination therapy consisting of eflornithine and the
There is a clear need for new drugs against human African
trypanosomiasis (HAT). The parenteral agents pentamidine and
suramin are used for treating the first stage of the disease when
the causative Trypanosoma brucei rhodesiense and Trypanosoma
brucei gambiense parasites, respectively, are limited to the hem-
olymphatic system of the human host. Since only parenteral drugs
are currently available, an oral treatment is desirable for this dis-
ease stage. New drugs for second stage HAT, where the parasites
invade the CNS, are more urgently needed. Pentamidine and sura-
min are not used against second stage HAT. Melarsoprol, an anti-
quated organoarsenical agent, has been used as the first line drug
for second stage disease since 1949 despite the occurrence of fatal
reactive encephalopathies in up to 5% of those who receive the
drug.1 Eflornithine was registered for the treatment of second stage
HAT caused by T. b. gambiense in 1990. In addition to its limited
spectrum of antitrypanosomal activity, eflornithine must also be
given in large intravenous doses four times per day for 2 weeks.2
anti-Chagas disease drug nifurtimox (the NECT regimen) showed
non-inferior clinical efficacy against second stage HAT caused by
T. b. gambiense compared to eflornithine monotherapy.3 This treat-
ment regimen against second stage HAT has been added to the
World Health Organization’s Essential Medicines List.4 The imple-
mentation of the NECT regimen is an important step forward in
the treatment of second stage HAT because it significantly reduces
the amount of eflornithine required for curing the infection. How-
ever, this regimen does not apply to patients with T. b. rhodesiense
infections, multiple infusions of eflornithine are still required, and
parasite resistance threatens the NECT regimen due to the increas-
ing use of eflornithine monotherapy.3 Several other classes of
compounds have recently shown promise against African trypano-
somes,5–7 but none of these agents has entered clinical trials to
date. Thus, the identification and development of new candidates
for the treatment of HAT should remain a high priority.
We recently reported the synthesis of a series of 2,2,4-tri-
methyl-1,2-dihydroquinolin-6-yl acyloxy compounds and related
congeners which possess potent and selective activity against Afri-
can trypanosomes in vitro.8 The ability of these compounds to form
⇑
Corresponding author. Tel.: +1 (614) 292 5499; fax: +1 (614) 292 2435.
E-mail address: werbovetz.1@osu.edu (K.A. Werbovetz).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.003

514
C. S. Reid et al. / Bioorg. Med. Chem. 19 (2011) 513–523
the corresponding dihydroquinolin-6-ol appeared to be essential
for activity against Trypanosoma brucei, as derivatives that pos-
sessed either an ether substitution at the 6-position of the dihydro-
quinoline ring system or lacked an oxygen atom at this position
were much less active against the parasites in vitro. We hypothe-
sized that antitrypanosomal activity in this series of molecules
required ester hydrolysis to provide the corresponding dihydro-
quinolin-6-ol, followed by oxidation to a quinone imine that could
undergo redox cycling in the parasite.8 Since quinones are known
to be substrates of the key trypanosome antioxidant enzyme trypa-
nothione reductase, in the process generating superoxide,9 the pro-
posed quinone imine may be the ultimate trypanotoxic species
produced through exposure of trypanosomes to 2,2,4-trimethyl-
1,2-dihydroquinolin-6-yl acyloxy compounds. We also demon-
strated that 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl
acetate (4a) significantly prolonged the lifespan of mice infected
with T. brucei brucei STIB795 parasites when this agent was given
at an ip dose of 4 Â 50 mg/kg/day. In the present work, we report
the synthesis and antitrypanosomal evaluation of additional dihy-
droquinolines in an attempt to provide a more complete under-
standing of the SAR of this series. In the process, three molecules
were identified that show in vivo antitrypanosomal activity supe-
rior to that of 4a.
those of the corresponding free bases 3a–c or acetate esters
4a–c. In contrast to the spectra of neutral compounds, spectra of
the salts recorded in DMSO-d6 or CD3OD gave extremely broad sig-
nals. Spectra of the salts recorded in CDCl3 were more discernible
although with some signal broadening. In the neutral molecules
the gem-dimethyl and benzylic protons were chemically equiva-
lent, giving rise to sharp singlets integrating for six and two pro-
tons, respectively. The corresponding protons were not
chemically equivalent in the hydrochloride salts, however, giving
rise to separate singlets for the methyl group and separate dou-
blets or multiplets for the benzylic protons.
BF3ÁOEt2 was a useful cyclization catalyst when 4-amino-2,6-
dichlorophenol (6) was reacted with excess acetone in toluene to
form the corresponding dihydroquinoline 7, although the reaction
proceeded in low yield. Benzylation followed by acetylation pro-
vided 8 as shown in Scheme 2.
The BF3ÁOEt2 promoted cyclization was unsuccessful when
other aminophenols were used, so 4-methoxy anilines 9a–c were
instead employed as starting materials for the synthesis of target
compounds containing substitutions at the 7 and 8 positions of
the dihydroquinoline ring system. These compounds were pre-
pared using the iodine-catalyzed Skraup cyclization8,10,11 as the
key step, although the yields of the reactions providing dihydro-
quinolines 10a–c remained poor. Aqueous HBr was then used to
demethylate the 6-methoxy group present in 10a–c, with the reac-
tion being selective in the case of dimethoxyquinoline 10b. Confir-
mation of the selective demethylation of 10b to give 11b was
provided through NOE difference spectroscopy. Irradiation of the
methoxy group of 11b at d 3.70 ppm resulted in enhancement of
only the aromatic proton H-7 at d 6.25 ppm. When the OH group
at d 8.44 ppm was irradiated, however, signals for both H-5 and
H-7 were enhanced. Benzylation of 11a–c at N1 followed by
O-acetylation provided 12a–c (Scheme 3).
Other target compounds substituted at the 7-position (Scheme 4)
were prepared from 3-bromo-4-methoxyaniline (13), which was
synthesized by reduction of 2-bromo-4-nitroanisole with SnCl2.12
Skraup cyclization of 13 provided 6-methoxy-7-bromodihydro-
quinoline 14 in modest yield. We observed only the 7-substituted
dihydroquinoline product in reactions between either 9c or 13
with acetone, consistent with literature reports of Skraup
cyclizations employing other m-substituted anilines as starting
2. Chemistry
The synthesis of target compounds 2a,b, 4a–d, and 5a–c is
shown in Scheme 1. 2,2,4-Trimethyldihydroquinolines possessing
6-alkyl ethers 2a,b were synthesized by N-benzylation of commer-
cially available ethoxyquin (1a). N1-Benzylated-6-O-acetyl dihy-
droquinolines 4a–d were prepared by the route described by
Fotie et al.8 Compound 1a was O-dealkylated to provide 1b, which
was N-benzylated to give dihydroquinolin-6-ols 3a–d. Acetylation
of 3a–d provided target compounds 4a–d, while treatment of 3a–c
with aqueous or methanolic HCl afforded hydrochloride salts 5a–c.
Unlike dihydroquinolin-6-ols 3a–c, which are oily materials that
turn black upon standing,8 their corresponding hydrochloride salts
5a–c are stable as crystalline materials. For example, 5a has been
stored at room temperature for over 1 year without any evidence
of decomposition as assessed by HPLC. The proton NMR spectra
of the hydrochloride salts 5a–c were substantially different from
RO
HO
R3
HO
e
c
H
N
N
H
N
Cl
R1
1a: R = Et
1b: R = H
a
R1
R3
R2
R2
5a-c
b
3a-d
d
O
N
2-5a: R1 = R2 = R3 = H
O
2-5b: R1 = H, R2 = R3 = F
O
3-5c: R1 = OCH3, R2 = R3 = H
3-4d: R1 = R2 = H, R3 = CH(CH3)2
N
R3
R1
R2
R3
R1
2a,b
R2
4a-d
Scheme 1. Reagents and conditions: (a) 48% HBr, reflux, overnight (53%); (b) ArCH2Br, NaH, DMF, overnight (87–89%); (c) ArCH2Br or ArCH2Cl, Et3N, toluene, reflux, overnight
(67–71%); (d) CH3COCl, Et3N, DCM, rt, overnight (66–71%); (e) aqueous or alcoholic HCl (35%).

C. S. Reid et al. / Bioorg. Med. Chem. 19 (2011) 513–523
515
bromobenzene according to the procedure described by Roesch
and Larock17 resulted in homo-coupling of the alkyne, but a similar
reaction between 19 and iodobenzene gave the desired product
20 in 81% yield. HPLC was used to monitor this reaction, as 19
and 20 were indistinguishable by TLC. CuCl-catalyzed ring closure
of 20 gave the expected product 21 in 59% yield. The latter
compound underwent the previously described dealkylation,
benzylation, and acetylation reactions to yield the 4-phenyl target
compound 22.
We also envisioned the synthesis of 1,2-dihydroquinolines
analogs lacking the 2,2-dimethyl substituents by the preparation
of 6-methoxy-1,2-dihydroquinolines followed by demethylation,
benzylation, and acetylation steps. However, these efforts proved
to be unsuccessful due to the instability of the methoxy dihydro-
quinolines. The CuCl-catalyzed cyclization of N-propargyl-p-anisi-
dine gave 6-methoxyquinoline rather than the expected
dihydroquinoline, analogous to the cyclization of N-propargyl-p-
toluidine to 6-methylquinoline.18 Lithium aluminum hydride
reduction of 6-methoxyquinoline in our lab gave not the reported
dihydroquinoline product19 but a 4:1 mixture of starting material
and tetrahydroquinoline product, although TLC analysis of the reac-
tion mixture showed a faster eluting major component that could
not be isolated by column chromatography. A similar reduction of
6-methoxy-4-methylquinoline (prepared from p-anisidine and
methyl vinyl ketone20), resulted only in the recovery of starting
O
Cl
Cl
Cl
HO
Cl
HO
Cl
O
a
b
NH2
N
H
Cl
N
6
7
8
Scheme 2. Reagents and conditions: (a) acetone, BF3ÁOEt2, toluene, reflux, 24 h
(23%); (b) 1-benzyl bromide, Et3N, reflux, overnight; 2-CH3COCl, Et3N, DCM, rt,
overnight (75% after two steps).
materials.13,14 Suzuki coupling between 14 and 4-(trifluoro-
methyl)phenylboronic acid in the presence of catalytic Pd(PPh3)4
in aqueous potassium carbonate/toluene gave the desired 7-aryldi-
hydroquinoline intermediate 15a in excellent yield, while the reac-
tion between 14 and methylboronic acid provided 15b in a more
modest yield. Compounds 15a and 15b were demethylated, benzy-
lated, and acetylated as described previously to yield the 7-substi-
tuted target compounds 16a and 16b (Scheme 4).
Synthesis of the 4-phenyl dihydroquinoline target compound
22 (Scheme 5) was accomplished using Sonogashira coupling15
and Cu-catalyzed cyclization16 as key steps. Reaction between
p-anisidine (17) and 3-chloro-3-methyl-1-butyne (18) gave the
N-propargyl adduct 19. A Sonogashira reaction between 19 and
O
H3CO
R2
HO
R2
H3CO
R2
O
c
b
a
a: R1 = CH3, R2 = H
b: R1 = OCH3, R2 = H
c: R1 = H, R2 = Cl
NH2
N
H
N
H
R2
N
R1
R1
R1
R1
12a-c
9a-c
10a-c
11a-c
Scheme 3. Reagents and conditions: (a) acetone, I2, toluene, reflux, 24 h (13–27%); (b) 48% HBr, reflux, overnight (31–53%); (c) 1-benzyl bromide, Et3N, reflux, overnight
(53–72%); 2-CH3COCl, Et3N, DCM, rt, overnight (84–90%).
O
H3CO
Br
O
R
H3CO
Br
H3CO
R
c
a
b
NH2
N
H
N
H
15a-c
N
13
14
a: R = 4-CF3Ph
b: R = CH3
16a,b
Scheme 4. Reagents and conditions: (a) acetone, I2, toluene, reflux, 24 h (48%); (b) ArB(OH)2, Pd(PPh3)4, aq K2CO3, toluene (90%) or CH3B(OH)2, Pd(PPh3)4, Cs2CO3, THF/H2O
(59%); (c) 1–48% HBr, reflux, 3 h (23–49%); 2-benzyl bromide, Et3N, reflux, overnight (75–88%); 3-CH3COCl, Et3N, DCM, rt, overnight (63–91%).
H3CO
H3CO
a
b
+
Cl
H3CO
NH
N
H
NH2
17
18
19
20
O
H3CO
d
c
O
N
H
N
21
22
Scheme 5. Reagents and conditions: (a) CuCl, Cu, Et3N, overnight (72%); (b) iodobenzene, PdCl2(PPh3)2, CuI, Et3N, 60 °C, 3 h (81%); (c) CuCl, toluene, reflux, overnight (59%);
(d) 1–48% HBr, reflux, 1 h (49%); 2-benzyl bromide, Et3N, toluene, reflux, overnight (99%); 3-acetyl chloride, Et3N, CH2Cl2 (87%).

516
C. S. Reid et al. / Bioorg. Med. Chem. 19 (2011) 513–523
material, despite TLC evidence of the formation of a more lipophilic
product. These findings demonstrate the importance of two methyl
(or other) substituents at the 2-position to protect the dihydroquin-
oline core from spontaneous oxidation to a quinoline system.
4c and hydrochloride salts 5a and 5c affected cures in this model,
however. The control compounds diminazene and pentamidine
gave cures when administered ip at daily doses of 10 mg/kg and
5 mg/kg, respectively, for four consecutive days.
3. Biological results
3.1. In vitro data
4. Discussion
N1-Benzylated-6-O-acyloxy dihydroquinolines emerged as a
promising new class of antitrypanosomal molecules based on their
potent and selective in vitro trypanocidal activity, the simplicity of
their structure, and the demonstration of the modest in vivo effi-
cacy of lead compound 4a.8 The goals of the studies reported here
were to further define the antiparasitic SAR of the dihydroquino-
lines, to improve the in vivo antitrypanosomal activity of lead 4a,
and to determine whether the instability of the dihydroquinolin-
6-ol free base demanded the use of acyloxy prodrugs. These studies
resulted in the identification of three new dihydroquinolines that
provide cures in the T. b. rhodesiense STIB900 murine trypanosomi-
asis model, including two that do not possess a metabolically labile
ester group.
Compounds 4b–d all possess substitutions on the aromatic ring
of the N1-benzyl group. As observed previously with 4a and other
N1-benzylated-6-O-acetyl dihydroquinolines,8 4b–d possess
potent activity against T. brucei in vitro, with IC50 values ranging
from 11 to 290 nM versus T. b. rhodesiense and from 41 to
970 nM versus T. b. brucei (the observed differences in sensitivity
between T. b. rhodesiense STIB900 and T. b. brucei s427 should
not be assigned to the taxonomic distinction but rather to the dif-
ferent assay protocols used in the two laboratories). Compound 4d,
which contains a p-isopropyl group on the N1-benzyl substituent,
is the least active against both parasites, consistent with our earlier
observation that bulky substitutions on the aryl ring of the
N1-benzyl group diminish in vitro antitrypanosomal potency.8 Like
the 6-benzyloxy dihydroquinolines reported earlier, 6-ethoxy
derivatives 2a and 2b display little in vitro antitrypanosomal
activity, as do congeners 12a and 12b, which bear 8-methyl and
8-methoxy substituents on the dihydroquinoline core, respec-
tively. In contrast, compounds possessing small substituents at
the 7-position of the dihydroquinoline core, as in the 7-chloro
dihydroquinoline 12c and the 7-methyl dihydroquinoline 16b,
exhibit potent in vitro antitrypanosomal activity. The 5,7-dichloro-
dihydroquinoline 8 also displays sub-micromolar antitrypanoso-
mal activity, although this compound is fivefold less potent than
4a against T. b. rhodesiense and over eightfold less potent than 4a
against T. b. brucei in vitro. When the substituent at the 7-position
of the dihydroquinoline ring becomes larger, as in the 7-aryl deriv-
ative 16a, in vitro potency against African trypanosomes decreases
dramatically compared to the 7-chloro and 7-methyl derivatives
12c and 16b. Replacement of the 4-methyl group present in 4a
with a phenyl group as in 22 results in an approximately 340-fold
decrease in potency against both T. b. rhodesiense and T. b. brucei.
However, dihydroquinolin-6-ol hydrochloride salts 5a–c all dis-
played in vitro antitrypanosomal potency similar to that of 4a.
Dihydroquinolines that showed low- to mid-nanomolar potency
against trypanosomes exhibited excellent selectivity for the para-
sites compared to L6 rat myoblasts, with compounds 4c, 5a, and
5c displaying IC50 values 1500, 850, and 860 times lower, respec-
tively, againstT. b. rhodesiense than against these mammalian cells.
A major focus of the current SAR investigation was to examine
the effect of dihydroquinoline core substitutions on in vitro anti-
trypanosomal potency. We previously showed that replacement
of the methyl group at the 4-position of 4a with a hydrogen de-
creased in vitro trypanocidal potency by 14-fold.8 Here, we demon-
strate that insertion of a phenyl group at the 4-position (compound
22) results in a more dramatic reduction in potency. When a chlo-
rine atom or a methyl group is placed at the 7-position (11c or 16b,
respectively), the molecules show in vitro trypanocidal potency
similar to that of 4a. However, 16a, which contains an aryl substi-
tuent at the 7-position, is over two orders of magnitude less potent
than 4a. The 5,7-dichloro compound (8) retains sub-micromolar
in vitro antitrypanosomal potency, but is approximately twofold
less potent than the 7-chloro compound 12c. While compounds
bearing small groups at the 7-position of the dihydroquinoline core
are potent trypanocides, congeners possessing a methyl or meth-
oxy group at the 8-position of the dihydroquinoline core (12a
and 12b, respectively) are over two orders of magnitude less po-
tent against T. b. brucei than 4a. Given that the 7-methyl derivative
16b is 300 times more potent against T. b. brucei in vitro than the
8-methyl derivative 12a despite the fact that these molecules dis-
play identical C log P values of 4.8 (as calculated by ChemDraw Pro
version 11.0.1), the 8-substituted compounds are likely to possess
steric or electronic features that interfere with antitrypanosomal
activity. Consistent with our previous results with 6-benzyloxy
compounds, 6-alkoxy dihydroquinolines 2a and 2b are essentially
inactive against trypanosomes in vitro. Interestingly, the dihydro-
quinolin-6-ol hydrochloride salts 5a–c, which are expected to gen-
erate the corresponding free bases at physiological pH, are all
comparable in potency to their corresponding 6-O-acetyl deriva-
tives 4a–c. This observation is consistent with the hypothesis that
the 6-acyloxy group present in the latter molecules is rapidly
cleaved in vitro to provide the corresponding dihydroquinolin-6-
ols. Earlier we proposed that the dihydroquinolin-6-ol is oxidized
to a trypanocidal quinone imine species, which is likely to partici-
pate in redox cycling within the parasite.8 Based on the compounds
that have been synthesized and tested in the present work and in
our previous study,8 a summary of the in vitro antitrypanosomal
SAR of these dihydroquinolines is presented in Figure 1.
3.2. In vivo data
Based on the in vitro antitrypanosomal data shown in Table 1,
4c, 5a, and 5c were selected for in vivo antitrypanosomal evalua-
tion and were compared to 4a, which had previously shown effi-
cacy against T. b. brucei STIB795 in vivo.8 Despite their lack of
in vitro potency, 2a and 2b were also selected for in vivo evaluation
in the hope that metabolic dealkylation would generate the active
dihydroquinolin-6-ol within the host animal. These agents were
administered to mice infected with T. b. rhodesiense STIB900 start-
ing 1 day after infection by ip injection at a daily dose of 50 mg/kg
given for four consecutive days (Table 2). Neither 2a nor 2b caused
any suppression of parasitemia compared to untreated controls as
assessed by microscopic examination of tail blood 5 days post
infection. While 4a suppressed parasitemia compared to untreated
controls 5 days after infection, animals that received this com-
pound relapsed on average 12.75 days post infection. Ester prodrug
Given their trypanocidal potency and stability, dihydroquino-
lin-6-ol hydrochloride salts may possess significant advantages
over their corresponding neutral 6-acyloxy esters as candidates
against HAT. We previously reported that the active dihydroquin-
olin-6-ol free bases we prepared quickly turned to dark, gummy
materials, most likely due to auto-oxidation, and that acylation

C. S. Reid et al. / Bioorg. Med. Chem. 19 (2011) 513–523
517
Table 1
Activity and toxicity of dihydroquinolines against African trypanosomes and L6 rat myoblasts in vitroa
Compound
IC50
(lM) versus T. b. rhodesiense
IC50
(l
M) versus T. b. brucei
IC50
(lM) versus L6 rat myoblasts
in vitro
in vitro
in vitro
2a
2b
4a
4b
4c
4d
5a
5b
5c
8
12a
12b
41 21
140 20
ND
ND
97
90
18
17
17
10
11
11
12
2
2
4
1
1
0
2
2
1
0.012 0.001
0.025 0.006
0.011 0.001
0.28 0.05
0.013 0.004
0.051 0.016
0.014 0.004
0.072 0.013
43 17
0.058 0.008
0.079 0.014b
0.041 0.008
0.97 0.15
0.038 0.007
0.068 0.030
0.045 0.006
0.49 0.17
9.2 2.8
110 30
48 13
ND
ND
>100
12c
16a
16b
0.042 0.008
9.0 2.9
ND
0.22 0.04
22
>190
ND
3
15
1
0.031 0.002
22
4.5 0.6
20
0
49
2
Melarsoprol
Diminazene
Pentamidine
Suramin
0.0099 0.0026
0.0058 0.0008
0.0013 0.0001
0.12 0.01
ND
ND
ND
5.0 1.6
25 11
ND
0.012 0.001
0.11 0.01
ND
Podophyllotoxin ND
0.012 0.001
a
Mean standard error of at least three independent measurements unless otherwise indicated.
Mean range of two independent measurements.
b
Table 2
In vivo activity of 2a, 2b, 4a, 4c, 5a, and 5c against T. b. rhodesiense STIB900
6-Acyloxy compounds may still offer advantages, however, such as
the opportunity to deliver a trypanocidal dihydroquinolin-6-ol
directly to the site of infection through selective ester hydrolysis
by a parasite esterase. Considering that both hydrochloride salt
5c and its corresponding 6-O-acetyl derivative 4c cure STIB900
infections at the same dose when given ip (Table 2), both classes
of dihydroquinolines deserve further study to assess their advanta-
ges and disadvantages as antitrypanosomal candidates.
Compound 4a served as a lead molecule in this series based on
its selective in vitro trypanocidal potency and its moderate efficacy
in the STIB795 murine model of T. b. brucei infection. We show in
the current study that 4a is only modestly effective against T. b.
rhodesiense strain STIB900 in vivo when given ip at a dose of
50 mg/kg/day  4 (see Table 2). However, 4a is the neutral 6-O-
acetyl derivative of hydrochloride salt 5a, which cures STIB900
infected mice when administered at the same dose as 4a. A preli-
minary iv pharmacokinetics study in rats also revealed that similar
levels of 3a were observed in plasma within 2 min after iv admin-
istration of either 4a or 5a,21 indicating that ester hydrolysis occurs
rapidly in the blood. Nevertheless, we offer two possible explana-
tions for the in vivo efficacy differences between compounds 4a
Compound
Dose (mg/kg)
Cured/treated
Mean relapse (days)
Control
2a
2b
4a
4c
5a
5c
Untreated
4 Â 50 ip
4 Â 50 ip
4 Â 50 ip
4 Â 50 ip
4 Â 50 ip
4 Â 50 ip
4 Â 10 ip
4 Â 5 ip
Mean survival: 12.25 0.95 daysa
0/4
0/4
0/4
4/4
4/4
4/4
4/4
4/4
Inactive
Inactive
12.75 1.75a
—
—
—
—
—
Diminazene
Pentamidine
a
Mean standard error.
of the oxygen atom at the 6-position was required to prevent the
color change and allow for a satisfactory elemental analysis.8
Fortunately, hydrochloride salts 5a–c are stable crystalline materi-
als. Compound 5a, for example, shows no sign of decomposition
after being stored at ambient room temperature for over a year.
Since 5a and 5c also show excellent in vivo antitrypanosomal
efficacy, it may be possible to avoid the difficulties that would re-
sult from attempting to develop an ester prodrug for treating HAT.
HO
N
Figure 1. Activity map for dihydroquinolin-6-ols against African trypanosomes in vitro.

518
C. S. Reid et al. / Bioorg. Med. Chem. 19 (2011) 513–523
and 5a despite their similar in vitro antitrypanosomal potencies.
First, compounds administered by the ip route are quickly deliv-
ered to the systemic circulation through the liver.22 Oxidative
metabolism of 4a by cytochrome P450 enzymes could occur in
the liver, which may result in conversion of 4a to an inactive form
before it appears in the bloodstream and is transformed by ester
hydrolysis to 3a. Second, examination of the stability of 4a, 4c,
5a, and 5c in a phosphate buffer system unexpectedly revealed
that esters 4a and 4c are less stable in this aqueous environment
than the corresponding alcohols 3a and 3c derived from hydro-
chloride salts 5a and 5c (see Supplementary Fig. 1). In this exper-
iment, the disappearance of 4a and 4c did not result in the
appearance of 3a and 3c. Thus, we cannot exclude the possibility
that degradation of 4a in the peritoneal cavity is responsible for
the lower in vivo antitrypanosomal efficacy of this compound com-
pared to 5a. Detailed pharmacokinetics studies and the investiga-
tion of in vivo trypanocidal action of 4c, 5a, and 5c at different
doses and by the oral route will be required to provide additional
clues regarding the classes of molecules that should be pursued
as HAT candidates.
The in vitro and in vivo results described here are consistent with
the mechanistic hypothesis for the dihydroquinolines that we out-
lined earlier.8 Both 6-O-acyloxy dihydroquinolines and dihydro-
quinolin-6-ol hydrochloride salts are capable of producing the
corresponding dihydroquinolin-6-ol free base in vitro and in vivo,
either by ester hydrolysis or by deprotonation, respectively. Consid-
ering that (1) the trypanotoxic N1-benzyl-dihydroquinolin-6-ols
darken spontaneously and (2) dihydroquinolines either lacking a
6-O atom or containing a 6-alkoxy or 6-benzyloxy substituent are
both stable and inactive against African trypanosomes, the produc-
tion of a trypanotoxic quinone imine species remains the most likely
explanation for the potent activity of these compounds against
T. brucei. Evidence for the production of such quinone imine species
has been obtained for several drugs including acetaminophen,23,24
diclofenac,25 thiabendazole,26 and lumiracoxib,27 but remains to
be demonstrated for the antitrypanosomal dihydroquinolines. Once
formed, the proposed quinone imine could display an antitrypanos-
omal effect and/or toxicity to the host either by acting as an electro-
phile and reacting with key cellular nucleophiles or by participating
in redox cycling and causing oxidative stress. Given the potential for
hepatotoxicity arising from the generation of a quinone imine spe-
cies in vivo, the host toxicity of candidate agents from this class
must be carefully monitored during the discovery and development
process.
spectrometer. Electrospray Ionization Mass Spectrometry
(ESI-MS) was performed on an Agilent Technologies 1100 Series
LC/MSD Trap spectrometer or by The Ohio State University Mass
Spectrometry and Proteomics facility, which also performed High
Resolution ESI-MS (HRESI-MS). Melting points were measured on
a Thomas Hoover capillary melting points apparatus and are
uncorrected. THF was distilled over Na metal and used immedi-
ately after collection or was dispensed directly from a Sure-sealÒ
container. Reaction mixtures were monitored using TLC silica gel
60 F254 plates from Dynamic Adsorbents, Inc. or Whatman Paper
Ltd or by reverse phase HPLC using a Zorbax SB-C8 column. Gravity
and flash column chromatography were performed using type 60A
silica gel (60–230 mesh) from Dynamic Adsorbents, Inc. or Fisher
Scientific. Select compounds were further purified using silica gel
GF preparative 1000 lm UV254 plates from Analtech. Elemental
analyzes were performed by Atlantic Microlab, Norcross, GA.
6.2. Materials
All chemicals and solvents were purchased from Aldrich Chem-
ical Co., Fisher Scientific or Acros Organics and were used without
further purification unless stated otherwise.
6.2.1. 1-Benzyl-6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline
(2a)
A mixture of 1a (500 mg, 2.3 mmol), benzyl bromide (410 lL,
3.4 mmol), and NaH (110 mg, 4.6 mmol) in DMF (15 mL) was al-
lowed to stir overnight under nitrogen. The solvent was then evap-
orated under reduced pressure. The residue was re-dissolved in
ethyl acetate, washed twice with distilled water, brine solution
and further purified on a silica gel column using hexanes–ethyl
acetate (9:1) as the mobile phase to yield 1-benzyl-6-ethoxy-1,2-
dihydro-2,2,4-trimethylquinoline (2a, 615 mg, 87%) as a brown
sticky oil that became solid after standing overnight, mp 50–
52 °C. 1H NMR (DMSO-d6) d 1.21–1.29 (9H, m), 1.94 (3H, s),
3.82–3.88 (2H, m), 4.41 (2H, s), 5.47 (1H, s), 6.10 (1H, d,
J = 8.7 Hz), 6.46 (1H, d, J = 8.7 Hz), 6.60 (1H, s), 7.17–7.35 (5H,
m); 13C NMR (DMSO-d6) d 14.8, 18.3, 27.3, 47.4, 56.3, 63.1, 110.7,
112.1, 113.4, 123.7, 126.1, 127.0, 128.2, 128.8, 130.9, 137.8,
140.1, 149.7; HRESI-MS: [M+H]+ m/z 308.2019 (calcd 308.2014)
corresponding to C21H25NO. Anal. (C21H25NO) Calcd C, 82.04; H,
8.20; N, 4.56. Found: C, 82.20; H, 8.16; N, 4.42.
6.2.2. 1-(3,4-Difluorobenzyl)-6-ethoxy-1,2-dihydro-2,2,4-
trimethylquinoline (2b)
5. Conclusions
The reaction was similar to the synthesis of 2a, using 1a
(500 mg, 2.3 mmol) and 3,4-difluorobenzyl bromide (714 mg,
3.4 mmol) to yield 1-(3,4-difluorobenzyl)-6-ethoxy-1,2-dihydro-
2,2,4-trimethylquinoline (2b, 702 mg, 89%). Purification on a silica
gel column using hexanes–ethyl acetate (9:1) provided the target
compound as white crystals, mp 75–77 °C. 1H NMR (DMSO-d6) d
1.24 (9H, m), 1.94 (3H, s), 3.82–3.89 (2H, m), 4.39 (2H, s), 5.47
(1H, s), 6.08 (1H, d, J = 8.7 Hz), 6.48 (1H, dd, J = 8.7, 2.9 Hz), 6.61
(1H, d, J = 2.9 Hz), 7.29–7.35 (3H, m); HRESI-MS: [M+H]+ m/z
344.1826 (calcd 344.1826). Anal. (C21H23F2NO) Calcd C, 73.45; H,
6.75; N, 4.08. Found: C, 73.32; H, 6.71; N, 4.11.
The work described here more clearly defines the antitrypanos-
omal SAR for dihydroquinolines and details the synthesis and
curative in vivo antitrypanosomal efficacy of three new dihydro-
quinolines, including two dihydroquinolin-6-ol hydrochloride
salts. Future studies must evaluate the oral bioavailability and
more clearly characterize the in vivo antitrypanosomal efficacy of
4c, 5a, and 5c, the potential of the trypanocidal dihydroquinolines
to generate a quinone imine intermediate, and more precisely de-
fine the mechanism of trypanocidal action of these compounds.
Such studies are either planned or in progress, and further results
concerning these intriguing candidates against HAT will be re-
ported in due course.
6.2.3. 1-(3,4-Difluorobenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-
6-ol (3b)
Compound 1b8 (420 mg, 2.22 mmol) was combined with 3,4-di-
6. Experimental
fluoro benzyl bromide (427 lL, 3.34 mmol), Et3N (463 lL,
3.33 mmol), and toluene (10 mL) and the mixture was stirred for
24 h under nitrogen. The solvent was evaporated and the residue
purified on silica gel using hexanes–dichloromethane (4:1) as the
mobile phase to yield 1-(3,4-difluorobenzyl)-1,2-dihydro-2,2,
4-trimethylquinolin-6-ol (3b, 487 mg, 70%) as a brown oil that
6.1. General experimental
1H NMR (300 MHz) and 13C NMR (75 MHz) spectral data were
recorded on a Bruker 300 UltraShield™ or a Varian Gemini 2000

C. S. Reid et al. / Bioorg. Med. Chem. 19 (2011) 513–523
519
solidified upon standing. 1H NMR (DMSO-d6) d 1.30 (6H, s), 2.00
(3H, s), 4.40 (2H, s), 5.49 (1H, s), 6.05 (1H, d, J = 8.7 Hz), 6.37 (1H,
dd, J = 8.7, 2.4 Hz), 6.58 (1H, s), 7.29–7.42 (3H, m), 8.52 (1H, s).
1-(4-isopropylbenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl
acetate (4d, 461 mg, 87%) as a yellow oil. 1H NMR (CDCl3) d 1.28
(6H, d, J = 6.9 Hz), 1.39 (6H, s), 2.03 (3H, s), 2.27 (3H, s), 2.84–
2.99 (1H, m), 4.49 (2H, s), 5.39 (1H, s), 6.25 (1H, d, J = 9.0 Hz),
6.65 (1H, dd, J = 2.6, 9 Hz), 6.80 (1H, d, J = 2.6 Hz), 7.19 (2H, d,
J = 8.1 Hz), 7.29 (2H, d, J = 7.8 Hz); 13C NMR (CDCl3) d 19.1, 21.5,
24.4, 29.0, 34.1, 48.4, 57.5, 112.4, 116.7, 121.1, 123.9, 126.5,
127.0, 127.8, 130.7, 137.1, 141.6, 142.5, 147.6, 170.6; ESI-MS:
[M+Na]+ m/z 386; HRESI-MS: [M+Na]+ m/z 386.2094 (calcd
386.2096). Anal. (C24H29NO2) Calcd C, 79.30; H, 8.04; N, 3.85.
Found: C, 79.31; H, 8.13; N, 3.81.
6.2.4. 1-(4-Isopropylbenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-
6-ol (3d)
Compound 1b (388 mg, 2.05 mmol) was combined with 4-iso-
propyl benzyl bromide (526 lL, 3.08 mmol), Et3N (427 lL,
3.08 mmol), and toluene (10 mL). The reaction mixture was stirred
for 24 h under nitrogen. The solvent was then evaporated and the
residue purified on silica gel using hexanes–dichloromethane (2:1)
as the mobile phase to yield 1-(4-isopropylbenzyl)-1,2-dihydro-
2,2,4-trimethylquinolin-6-ol (3d, 469 mg, 71%) as a brown oil that
solidified upon standing. 1H NMR (DMSO-d6) d 1.19 (6H, d,
J = 6.9 Hz), 1.28 (6H, s), 1.93 (3H, s), 2.78–2.92 (1H, m), 4.34 (2H,
s), 5.45 (1H, s), 6.07 (1H, d, J = 8.7 Hz), 6.33 (1H, d, J = 8.4 Hz),
6.52 (1H, d, J = 2.1 Hz), 7.15–7.26 (4H, m), 8.42 (1H, s).
6.2.8. 1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol
hydrochloride (5a)
Compound 3a8 (2.96 g, 10.6 mmol) was dissolved in THF
(50 mL) and treated with 1 N HCl (12 mL). The solution was evap-
orated under reduced pressure, dried overnight under high vac-
uum, then suspended in dry THF to give 5a as a white solid
(2.77 g, 83%), mp 185–187 °C. 1H NMR (300 MHz, CDCl3) d 1.50
(3H, s), 1.90 (3H, s), 2.11 (3H, d, J = 1.3 Hz), 4.15 (1H, dd, J = 11.4
and 8.2 Hz), 4.59 (1H, dd, J = 11.5 and 3.4 Hz), 5.57 (1H, d,
J = 1.0 Hz), 6.56 (2H, d, J = 1.2 Hz), 6.87 (1H, s), 7.33–7.03 (5H, m),
12.76 (1H, br s); EIMS m/z 208.9. Anal. (C19H21NOÁHCl) Calcd C,
72.25; H, 7.02; N, 4.43; Cl, 11.23. Found: C, 72.00; H, 6.96; N,
4.40; Cl, 11.09.
6.2.5. 1-(3,4-Difluorobenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-
6-yl acetate (4b)
Compound 3b (491 mg, 1.56 mmol), Et3N (325
lL, 2.34 mmol),
and acetyl chloride (166 L, 2.34 mmol) were stirred overnight
l
with dichloromethane (10 mL) under nitrogen. The solvent was
evaporated and the residue purified on silica gel with hexanes–
ethyl acetate (9:1) as the mobile phase to yield 1-(3,4-difluoroben-
zyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate (4b, 367 mg,
66%) as a yellow oil which solidified on standing. 1H NMR (CDCl3) d
1.37 (6H, s), 2.02 (3H, s), 2.27 (3H, s), 4.44 (2H, s), 5.41 (1H, s), 6.15
(1H, d, J = 8.7 Hz), 6.64 (1H, dd, J = 8.7, 2.4 Hz), 6.84 (1H, d,
J = 2.4 Hz), 7.08–7.20 (3H, m); 13C NMR (CDCl3) d 18.6, 21.0, 28.3,
47.3, 57.2, 115.1 (d, 2JCF = 24.75 Hz), 116.7, 117.3 (d, 2JCF = 16.5 Hz),
6.2.9. 1-(3,4-Difluorobenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-
6-ol hydrochloride (5b)
Compound 3b (186 mg, 0.59 mmol) was mixed with methanolic
HCl (7 mL) under nitrogen. After consumption of the starting mate-
rial, the solvent was evaporated and THF (10 mL) was added. The
mixture was kept at 0 °C overnight and the precipitate collected
and dried under vacuum to yield 5b (138 mg, 66%) as an off-white
solid, mp 174–175 °C. 1H NMR (CDCl3) d 1.49 (3H, br s), 1.86 (3H, br
s), 2.13 (3H, br s), 4.10 (1H, br s), 4.54 (1H, br s), 5.58 (1H, br s),
7.10–6.60 (6H, m), 8.75 (1H, br s); 13C NMR (methanol-d4) d 18.2,
4
120.8, 121.9 (dd, JCF = 3.75 Hz), 123.9, 127.5, 130.3, 136.6 (dd,
2
3
4JCF = 3.75 Hz), 141.6 (d, JCF = 16.5 Hz), 148.9 (d, JCF = 12.75 Hz),
1
1
149.0 (d, JCF = 244.5 Hz), 149.2 (d, JCF = 244.5 Hz), 152.3 (d,
3JCF = 12.75 Hz), 170.1. ESI-MS: [M+Na]+ m/z 380; HRESI-MS:
[M+Na]+ m/z 380.1439 (calcd 380.1438). Anal. (C21H21F2NO2) Calcd
C, 70.57; H, 5.92; N, 3.92. Found: C, 70.52; H, 5.96; N, 3.95.
2
24.4, 54.5, 65.3, 113.6, 115.5, 118.7 (d, JCF = 18 Hz), 122.3 (d,
2JCF = 17.3 Hz), 127.1, 129.0, 130.4, 131.1, 133.2, 151.0 (d,
6.2.6. 1-(2-Methoxybenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-
6-yl acetate (4c)
Compound 1b (300 mg, 1.6 mmol) and 2-methoxybenzyl chlo-
ride (376 mg, 2.4 mmol) were reacted with Et3N (335 lL,
2.4 mmol) in toluene (10 mL) to yield, after purification on a silica
gel column using hexanes–ethyl acetate (9:1), 1-(2-methoxyben-
zyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-ol (3c, 329 mg, 67%).
Compound 3c (329 mg, 1.06 mmol) was then stirred together with
3JCF = 12 Hz), 151.3 (d, 1JCF = 246.75 Hz), 151.4 (d, 1JCF = 246.75 Hz),
3
154.2 (d, JCF = 12 Hz), 160.9; ESI-MS: [MÀCl] m/z 316; HRESI-MS:
[MÀCl] m/z 316.1506 (calcd 316.1513). Anal. (C19H20ClF2NO) Calcd
C, 64.86; H, 5.73; Cl, 10.08; N, 3.98. Found: C, 64.42; H, 5.99; N,
3.83; Cl, 9.56.
6.2.10. 1-(2-Methoxybenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-
6-ol hydrochloride (5c)
acetyl chloride (500
l
L, 7.0 mmol) and Et3N (1 mL, 7.2 mmol) at
Compound 3c (332 mg, 1.07 mmol) was mixed with methanolic
HCl (10 mL). After consumption of the starting material, the sol-
vent was evaporated and THF (20 mL) was added. The mixture
was kept at 0 °C overnight and the precipitate collected and dried
under vacuum overnight to yield 5c as an off-white solid (267 mg,
72%), mp 195–198 °C. 1H NMR (methanol-d4) d 1.44 (3H, s), 1.85
(3H, s), 2.19 (3H, s), 3.54 (3H, s), 4.52 (1H, d, J = 11.9 Hz), 4.67
(1H, d, J = 11.9 Hz), 5.84 (1H, s), 6.34 (2H, m), 6.79 (1H, d,
J = 8.4 Hz), 6.88 (2H, m), 7.15 (1H, d, J = 7.5 Hz), 7.32 (1H, dd,
J = 8.1 Hz); 13C NMR (methanol-d4) d 18.3, 24.1, 24.7, 56.1, 65.1,
111.9, 112.9, 114.9, 119.6, 121.8, 124.1, 126.8, 128.6, 131.9,
133.3, 133.6, 134.6, 160.3, 160.7; ESI-MS: [MÀCl] m/z 310; HRES-
I-MS: [MÀCl] m/z 310.1802 (calcd 310.1807). Anal. (C20H24ClNO2)
Calcd C, 69.45; H, 6.99; Cl, 10.25; N, 4.05. Found: C, 68.68; H,
7.03; N, 3.98; Cl, 10.14.
room temperature overnight in DCM to yield, after purification
on a silica gel column using hexanes–DCM (3:1), 4c (325 mg,
87%) as a yellow oil. 1H NMR (CDCl3) d 1.37 (6H, s), 2.01 (3H, s),
2.26 (3H, s), 3.93 (3H, s), 4.46 (2H, s), 5.37 (1H, s), 6.11 (1H, d,
J = 8.7 Hz), 6.62 (1H, dd, J1 = 8.7 Hz and J2 = 2.4 Hz), 6.79 (1H, d,
J = 2.4 Hz), 6.87–6.91 (2H, m), 7.22–7.30 (2H, m); 13C NMR (CDCl3)
d 18.7, 21.1, 28.6, 43.0, 55.2, 57.2, 109.5, 111.5, 116.3, 120.6, 120.8,
123.1, 126.7, 127.2, 127.3, 127.4, 130.2, 141.0, 142.1, 156.6, 170.0;
ESI-MS: [M+H]+ m/z 352; HRESI-MS: [M+H]+ m/z 352.1912 (calcd
352.1913). Anal. (C22H25NO3) Calcd C, 75.19; H, 7.17; N, 3.99.
Found: C, 75.34; H, 7.42; N, 3.77.
6.2.7. 1-(4-Isopropylbenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-
6-yl acetate (4d)
A
solution of 3d (469 mg, 1.46 mmol), Et3N (304
lL,
2.19 mmol), acetyl chloride (155 L, 2.19 mmol), and dichloro-
l
6.2.11. 5,7-Dichloro-1,2-dihydro-2,2,4-trimethylquinolin-6-ol
(7)
A one step Lewis acid catalyzed reaction was used to prepare
this compound. A mixture of 4-amino-2,6-dichlorophenol (5.0 g,
methane (10 mL) were stirred overnight under nitrogen. The
solvent was evaporated and the residue purified on silica gel using
hexanes–dichloromethane (1:1) as the mobile phase to yield

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C. S. Reid et al. / Bioorg. Med. Chem. 19 (2011) 513–523
28 mmol), excess acetone (20 mL) and BF3ÁOEt2 (3.5 mL, 28 mmol)
in toluene (25 mL) was heated to reflux for 72 h. The reaction was
quenched with ice water and extracted with DCM, then the residue
was purified on silica gel column using hexanes–ethyl acetate
(85:15) to yield 5,7-dichloro-1,2-dihydro-2,2,4-trimethylquinolin-
6-ol (7, 1.64 g, 23%) as yellow oil. 1H NMR (DMSO-d6) d 1.13 (6H,
s), 2.18 (3H, s), 4.3 (1H, br s), 5.45 (1H, s), 6.62 (1H, s), 8.75 (1H,
br s).
1.18 (6H, s), 1.84 (3H, s), 1.98 (3H, s), 4.31 (1H, s), 5.29 (1H, s),
6.33 (2H, s), 8.25 (1H, s), mp 134–136 °C.
6.2.17. 1,2-Dihydro-8-methoxy-2,2,4-trimethylquinolin-6-ol
(11b)
Compound 10b (1.67 g, 7.16 mmol) was heated in 48% HBr
(30 mL) under nitrogen to reflux for 4 h. The mixture was neutral-
ized with 1 M Na2CO3 then extracted three times with chloroform.
The chloroform extract was washed with brine and the solvent re-
moved. The residue was purified on silica gel with hexanes–ethyl
acetate (2:1) as the mobile phase to yield the 6-O-demethylated
product, 1-benzyl-1,2-dihydro-8-methoxy-2,2,4-trimethylquino-
lin-6-ol (11b, 700 mg, 45%) as a brown solid. 1H NMR (DMSO-d6)
d 1.15 (6H, s), 1.83 (3H, s), 3.70 (3H, s), 4.27 (1H, s), 5.29 (1H, s),
6.13 (1H, br s), 6.24 (1H, br s), 8.44 (1H, s).
6.2.12. 1-Benzyl-5,7-dichloro-1,2-dihydro-2,2,4-trimethylquinolin-
6-yl acetate (8)
A mixture of 7 (500 mg, 1.94 mmol), benzyl bromide (497 mg,
2.91 mmol), and Et3N (404 lL, 2.90 mmol) were heated to reflux
overnight in toluene to yield 1-benzyl-5,7-dichloro-1,2-dihydro-
2,2,4-trimethylquinolin-6-ol that was further acetylated as de-
scribed above to yield, after purification on silica gel column using
hexanes–DCM (4:1), 8 (570 mg, 75%) as yellow oil that solidified
upon standing. 1H NMR (CDCl3) d 1.34 (6H, s), 2.35 (3H, s), 2.36
(3H, s), 4.50 (2H, s), 5.54 (1H, s), 6.41 (1H, s), 7.27–7.36 (5H, m);
13C NMR (CDCl3) d 20.3, 23.2, 27.2, 48.5, 56.0, 112.2, 121.8, 125.5,
126.1, 126.8, 127.1, 128.7, 128.8, 134.5, 135.0, 138.3, 144.8,
168.3; [M+Na]+ m/z 412; HRESI-MS: [M+Na]+ m/z 412.0830 (calcd
412.0847). Anal. (C21H21Cl2NO2) Calcd C, 64.62; H, 5.42; Cl,
18.17; N, 3.59. Found: C, 64.66; H, 5.46; Cl, 18.00; N, 3.61.
6.2.18. 7-Chloro-1,2-dihydro-2,2,4-trimethylquinolin-6-ol (11c)
Compound 10c (2.39 g, 10.05 mmol) was heated in 48% HBr
(20 mL) under nitrogen to reflux for 8 h. The mixture was neutral-
ized with Na2CO3 (1 M) then extracted three times with ethyl ace-
tate. The ethyl acetate extract was washed with brine and the
solvent removed, then the residue was purified on silica gel with
hexanes–ethyl acetate (10:1) as the mobile phase to yield the
demethylated compound (11c, 500 mg, 22%) as a white solid which
turned brown over time. 1H NMR (DMSO-d6) d 1.15 (6H, s), 1.84
(3H, s), 5.32 (1H, s), 5.46 (1H, s), 6.44 (1H, s), 6.60 (1H, s), 8.87
(1H, s), mp 138–140 °C.
6.2.13. 1,2-Dihydro-6-methoxy-2,2,4,8-tetramethylquinoline
(10a)
4-Methoxy-2-methyl aniline (9a, 5 g, 36.4 mmol), acetone
(5 mL), and iodine (922 mg, 3.64 mmol) were heated to reflux in
toluene (25 mL) for 24 h. The solvent was evaporated and the res-
idue purified on silica gel using hexanes–ethyl acetate (9:1) as the
mobile phase to yield 10a (1 g, 13%) as a yellow oil. 1H NMR
(DMSO-d6) d 1.22 (6H, s), 1.90 (3H, s), 2.06 (3H, s), 3.65 (3H, s),
4.53 (1H, s), 5.35 (1H, s), 6.46–6.53 (2H, m).
6.2.19. 1-Benzyl-1,2-dihydro-2,2,4,8-tetramethylquinolin-6-yl
acetate (12a)
Compound 11a (932 mg, 4.6 mmol) was added to benzyl bro-
mide (821 lL, 6.9 mmol), Et3N (960 lL, 6.9 mmol), and toluene
(15 mL). The reaction mixture was heated to reflux for 24 h, the
solvent was evaporated and the residue purified on silica gel using
hexanes–ethyl acetate (9:1) as the mobile phase to yield the N1
benzylated product, 1-benzyl-1,2-dihydro-2,2,4,8-tetramethyl-
quinolin-6-ol (950 mg, 70%), as a yellow oil. 1H NMR (DMSO-d6)
d 1.06 (6H, s), 1.88 (3H, s), 2.07 (3H, s), 3.90 (2H, s), 5.40, (1H, s),
6.46 (1H, d, J = 2.7 Hz), 6.52 (1H, d, J = 2.7 Hz), 7.10–7.23 (5H, m),
8.94 (1H, s). 1-Benzyl-1,2-dihydro-2,2,4,8-tetramethylquinolin-6-
ol (950 mg, 3.2 mmol), acetyl chloride (382 mg, 4.86 mmol), and
6.2.14. 1,2-Dihydro-6,8-dimethoxy-2,2,4-trimethylquinoline
(10b)
2,4-Dimethoxy aniline (9b) (2.0 g, 13.06 mmol), acetone (5 mL),
and iodine (330 mg, 1.31 mmol) were heated to reflux in toluene
(25 mL) for 24 h. The solvent was evaporated and the residue puri-
fied on silica gel using hexanes–ethyl acetate (10:1) as the mobile
phase to yield 1,2-dihydro-6,8-dimethoxy-2,2,4-trimethylquino-
line (10b, 722 mg, 24%) as a yellow oil. 1H NMR (DMSO-d6) d
1.20 (6H, s), 1.92 (3H, s), 3.68 (3H, s), 3.76 (3H, s), 4.47 (1H, s),
5.33 (1H, s), 6.28 (1H, d, J = 1.8 Hz), 6.42 (1H, d, J = 1.8 Hz).
Et3N (676 lL, 4.86 mmol) in dichloromethane (25 mL) were stirred
overnight at room temperature. The solvent was evaporated and
the residue was purified on a silica gel column using hexanes–ethyl
acetate (10:1) as the mobile phase to yield 1-benzyl-1,2-dihydro-
2,2,4,8-tetramethylquinolin-6-yl acetate (12a, 921 mg, 84%) as a
colorless oil which solidified on standing. 1H NMR (DMSO-d6) d
1.10 (6H, s), 1.90 (3H, s), 2.19 (3H, s), 2.25 (3H, s), 4.00 (2H, s),
5.50, (1H, s), 6.81–6.85 (2H, m), 7.12–7.28 (5H, m); 13C NMR
(DMSO-d6) d 18.2, 18.5, 21.1, 27.5, 53.5, 55.0, 114.0, 122.9, 126.4,
127.7, 127.9, 128.9, 131.3, 132.9, 134.1, 141.6, 143.4, 146.1,
169.6; ESI-MS: [M+Na]+ m/z 358; HRESI-MS: [M+Na]+ m/z
358.1783 (calcd 358.1784). Anal. (C22H25NO2) Calcd C, 78.77; H,
7.51; N, 4.18. Found: C, 78.57; H, 7.54; N, 4.22.
6.2.15. 7-Chloro-1,2-dihydro-6-methoxy-2,2,4-
trimethylquinoline (10c)
3-Chloro-p-anisidine 9c (5.0 g, 31.7 mmol), acetone (6 mL), and
iodine (803 mg, 3.17 mmol) were heated to reflux in toluene
(30 mL) for 24 h under nitrogen. The solvent was evaporated and
the residue purified on silica gel using hexanes–diethyl ether
(9:1) as the mobile phase to yield 10c (2.33 g, 31%) as a yellow
oil. 1H NMR (DMSO-d6) d 1.17 (6H, s), 1.91 (3H, s), 3.73 (3H, s),
5.32 (1H, s), 5.67 (1H, s), 6.52 (1H, s), 6.72 (1H, s).
6.2.20. 1-Benzyl-1,2-dihydro-8-methoxy-2,2,4-trimethylquinolin-
6-yl acetate (12b)
6.2.16. 1,2-Dihydro-2,2,4,8-tetramethylquinolin-6-ol (11a)
Compound 10a (0.966 mg, 4.44 mmol) was heated in 48% HBr
(30 mL) under nitrogen to reflux overnight. The mixture was neu-
tralized with aqueous Na2CO3 (1 M) then extracted three times
with chloroform. The chloroform extract was washed with brine,
the solvent removed and the residue purified on silica gel with
hexanes–ethyl acetate (10:1) as the mobile phase to yield the
demethylated product, 1,2-dihydro-2,2,4,8-tetramethylquinolin-
6-ol (11a, 481 mg, 53%), as a yellow solid. 1H NMR (DMSO-d6) d
A
mixture of 11b (533 mg, 2.43 mmol), Et3N (507
lL,
3.45 mmol), benzyl bromide (462 L, 3.9 mmol), and toluene
l
(15 mL) were heated to reflux overnight. The solvent was evapo-
rated and the residue purified on silica gel using hexanes–ethyl
acetate (9:1) as the mobile phase to yield the N1 benzylated
product, 1-benzyl-1,2-dihydro-8-methoxy-2,2,4-trimethylquino-
lin-6-ol (496 mg, 66%) as a brown solid. 1H NMR (DMSO-d6) d
1.06 (6H, s), 1.86 (3H, s), 3.69 (3H, s), 4.05 (2H, s), 5.39 (1H, s),
6.29 (1H, d, J = 2.4 Hz), 6.35 (1H, d, J = 2.4 Hz), 7.23–7.13 (3H,

C. S. Reid et al. / Bioorg. Med. Chem. 19 (2011) 513–523
521
m), 7.31–7.33 (2H, m), 9.01 (1H, s). 1-Benzyl-1,2-dihydro-8-meth-
oxy-2,2,4-trimethylquinolin-6-ol (409 mg, 1.32 mmol) was com-
bined with Et3N (276 L, 1.98 mmol), acetyl chloride (141 L,
6.2.24. 1,2-Dihydro-6-methoxy-2,2,4,7-tetramethylquinoline
(15b)
l
l
Compound 14 (639 mg, 2.26 mmol), methyl boronic acid
(163 mg, 2.72 mmol), Pd(PPh3)4 (159 mg, 6 mol %), Cs2CO3 (2.2 g,
6.79 mmol) and a 10:1 mixture of THF/water (22 mL) were heated
to reflux for 24 h. The solvent was removed and the residue puri-
fied on silica gel with hexanes–dichloromethane (1:1) as the mo-
bile phase to yield 15c (292 mg, 59%) as a brown oil that
solidified on standing. 1H NMR (DMSO-d6) d 1.14 (6H, S), 1.89
(3H, s), 2.02 (3H,s), 3.67 (3H, S), 5.22 (1H, s), 5.29 (1H, s), 6.28
(1H, s), 6.54 (1H, s).
1.98 mmol), and dichloromethane. The reaction mixture was stir-
red overnight under nitrogen to yield 1-benzyl-1,2-dihydro-8-
methoxy-2,2,4-trimethylquinolin-6-yl acetate (12b, 410 mg, 88%)
as a colorless oil which solidified on standing. 1H NMR (DMSO-
d6) d 1.33 (6H, s), 2.14 (3H, s), 2.77 (3H, s), 3.95 (3H, s), 4.46
(2H, s), 5.71 (1H, s), 6.87 (1H, d, J = 2.3 Hz), 6.93 (1H, d,
J = 2.3 Hz), 7.36–7.49 (3H, m), 7.56–7.59 (2H, m); 13C NMR
(DMSO-d6) d 18.4, 21.1, 27.5, 52.9, 54.5, 55.9, 105.6, 108.5,
126.2, 127.4, 127.8, 128.4, 130.1, 132.5, 132.9, 142.8, 145.3,
153.0, 169.5; ESI-MS: [M+Na]+ m/z 374; HRESI-MS: [M+Na]+ m/z
374.1731 (calcd 374.1732). Anal. (C22H25NO3) Calcd C, 75.19; H,
7.17; N, 3.99. Found: C, 74.55; H, 7.25; N, 3.90.
6.2.25. 1-Benzyl-7-(4-(trifluoromethyl)phenyl)-1,2-dihydro-
2,2,4-trimethylquinolin-6-yl acetate (16a)
Compound 15a (289 mg, 0.83 mmol) was heated in 48% HBr
(25 mL) under nitrogen for 7 h. The mixture was cooled, neutral-
ized with 1 M Na2CO3 (150 mL) and extracted with ethyl acetate.
The solvent was removed and the residue purified on silica gel
using dichloromethane as the mobile phase to yield the deme-
thylated product, 7-(4-(trifluoromethyl)phenyl)-1,2-dihydro-2,2,
4-trimethylquinolin-6-ol (63 mg, 23%), a yellow solid. 1H NMR
6.2.21. 1-Benzyl-7-chloro-1,2-dihydro-2,2,4-trimethylquinolin-
6-yl acetate (12c)
A
mixture of 11c (333 mg, 1.49 mmol), Et3N (310
lL,
2.23 mmol), and benzyl bromide (265 L, 2.23 mmol) were dis-
l
solved in toluene (10 mL) and stirred overnight under nitrogen.
The solvent was evaporated and the residue purified on silica gel
with hexanes–ethyl acetate (10:1) as the mobile phase to yield
the N1-benzylated product, 1-benzyl-7-chloro-1,2-dihydro-2,2,4-
trimethylquinolin-6-ol, as a brown oil (248 mg, 53%). 1H NMR
(DMSO-d6) d 1.28 (6H, s), 1.91 (3H, s), 4.38 (2H, s), 5.48 (1H, s),
6.09 (1H, s), 6.68 (1H, s), 7.16–7.34, (5H, m), 9.07 (1H, br s). 1-Ben-
(DMSO-d6)
d 1.18 (6H, s), 1.89 (3H, s), 5.37 (2H, s), 6.48
(1H, s), 6.65 (1H, s), 7.72 (4H, s), 8.69 (1H, s). 7-(4-(Trifluoro-
methyl)phenyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-ol (250 mg,
0.75 mmol), Et3N (156 lL, 1.12 mmol), benzyl bromide (134 lL,
1.12 mmol), and toluene (15 mL) were then heated to reflux over-
night under nitrogen. The solvent was evaporated and the residue
purified on silica gel with hexanes–dichloromethane (1:1) as the
mobile phase to yield the N1-benzylated product, 1-benzyl-7-(4-
(trifluoromethyl)phenyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-ol
(280 mg, 88%), as a yellow sticky oil. 1H NMR (DMSO-d6) d 1.33
(6H, s), 1.95 (3H, s), 4.44 (2H, s), 5.53 (1H, s), 6.19 (1H, s),
6.71 (1H, s), 7.15–7.22 (1H, m), 7.27–7.37 (4H, m), 7.42–7.47
(2H, m), 7.57–7.62 (2H, m), 8.85 (1H, s). 1-Benzyl-7-(4-(trifluoro-
methyl)phenyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-ol (280 mg,
zyl-7-chloro-1,2-dihydro-2,2,4-trimethylquinolin-6-ol
(248 mg,
L,
0.79 mmol), Et3N (165 L, 1.18 mmol), and acetyl chloride (84
l
l
1.18 mmol) were stirred overnight with dichloromethane (15 mL)
under nitrogen. The solvent was evaporated and the residue puri-
fied on silica gel with hexanes–dichloromethane (2:1) as the
mobile phase to yield 1-benzyl-7-chloro-1,2-dihydro-2,2,4-trim-
ethylquinolin-6-yl acetate (12c, 260 mg, 93%) as a white solid. 1H
NMR (CDCl3) d 1.28 (6H, s), 1.89 (3H, s), 2.22 (3H, s), 4.38 (2 H,
s), 5.29 (1H, s), 6.16 (1H, s), 6.69 (1H, s), 7.10–7.22 (5H, m); 13C
NMR (CDCl3) d 18.8, 20.8, 28.9, 48.4, 57.7, 112.5, 118.2, 122.7,
125.8, 126.3, 127.0, 127.1, 128.9, 130.7, 137.2, 138.8, 143.2,
169.6; ESI-MS: [M+Na]+ m/z 378; HRESI-MS: [M+Na]+ m/z
378.1241 (calcd 378.1237). Anal. (C21H22ClNO2) Calcd C, 70.88;
H, 6.23; N, 3.94. Found: C, 70.78; H, 6.16; N, 3.83, mp 147–149 °C.
0.66 mmol), Et3N (137 lL, 0.99 mmol), and acetyl chloride (70 lL,
0.99 mmol) were then stirred overnight with dichloromethane
(10 mL) under nitrogen. The solvent was evaporated and the
residue purified on silica gel with hexanes–dichloromethane
(1:1) as the mobile phase to yield 1-benzyl-7-(4-(trifluoromethyl)
phenyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate (16a,
280 mg, 91%) as a yellow oil that solidified on standing. 1H NMR
(CDCl3) d 1.32 (6H, s), 1.93 (3H, s), 1.97 (3H, s), 4.42 (2H, s), 5.33
(1H, s), 6.11 (1H, s), 6.72 (1H, s), 7.28–7.10 (7H, m), 7.43 (2H, d,
J = 8.1 Hz); 13C NMR (CDCl3) d 18.6, 20.8, 28.7, 48.3, 57.4, 113.3,
6.2.22. 7-Bromo-1,2-dihydro-6-methoxy-2,2,4-
trimethylquinoline (14)
A solution of 3-bromo-p-anisidine (13, 2.14 g, 10.6 mmol), ace-
tone (8 mL), and iodine (402 mg, 1.59 mmol) was heated to reflux
in toluene (30 mL) for 36 h. The reaction mixture was cooled,
washed with brine and the solvent removed. The residue was puri-
fied on silica gel using hexanes–dichloromethane (1:1) as the mo-
bile phase to yield 14 (1.43 g, 48%) as a brown oil that solidified on
standing. 1H NMR (DMSO-d6): d 1.16 (6H, s), 1.91 (3H, s), 3.72 (3H,
s), 5.35 (1H, s), 5.68 (1H, s), 6.67 (1H, s), 6.69 (1H, s).
1
3
117.7, 123.9, 124.2 (q, JCF = 270.8 Hz), 125.0 (q, JCF = 3.75 Hz),
2
126.2, 126.8, 127.0, 128.6, 128.9, 129.5 (q, JCF = 32.3 Hz), 130.9,
132.4, 138.0, 139.1, 142.1, 142.2, 170.0; ESI-MS: [M+H]+ m/z 466;
HRESI-MS: [M+H]+ m/z 466.1977 (calcd 466.1994). Anal. (C28H26
F3NO2) Calcd C, 72.24; H, 5.63; N, 3.01. Found: C, 72.18; H,
5.69; N 3.00.
6.2.26. 1-Benzyl-1,2-dihydro-2,2,4,7-tetramethylquinolin-6-yl
acetate (16b)
6.2.23. 7-(4-(Trifluoromethyl)phenyl)-1,2-dihydro-6-methoxy-
2,2,4-trimethylquinoline (15a)
Compound 15b (148 mg, 0.68 mmol) was heated in 48% HBr
(20 mL) under nitrogen for 2 h. The mixture was cooled, neutral-
ized with 1 M Na2CO3 (100 mL) and extracted with ethyl acetate.
The solvent was removed and the residue purified on silica gel with
hexanes–ethyl acetate (10:1) as the mobile phase to yield the
demethylated product, 1,2-dihydro-2,2,4,7-tetramethylquinolin-
6-ol (67 mg, 49%), as a white solid that turned brown on standing.
1H NMR (DMSO-d6) d 1.13 (6H, s), 1.82 (3H, s), 1.99 (3H, s), 5.06
(1H, s), 5.20 (1H, s), 6.22 (1H, s), 6.44 (1H, s), 8.14 (1H, s). 1,2-Dihy-
dro-2,2,4,7-tetramethylquinolin-6-ol (225 mg, 1.11 mmol), Et3N
To a solution of 14 (530 mg, 1.88 mmol) dissolved in toluene
(10 mL) were added a solution of 4-(trifluoromethyl)phenyl boro-
nic acid (535 mg, 2.82 mmol) in ethanol (2 mL), a 2 M solution of
K2CO3 (2 mL), and Pd(PPh3)4 (108 mg, 5 mol %) at room tempera-
ture and the mixture was heated to reflux for 3 h. The solvent
was removed and the residue purified on silica gel with hexane–
dichloromethane (1:1) as mobile phase to yield 15b (586 mg,
90%) as a yellow solid. 1H NMR (DMSO-d6) d 1.21 (6H, s), 1.97
(3H, s), 3.68 (3H, s), 5.38 (1H, s), 5.60 (1H, s), 6.51 (1H, s), 6.77
(1H, s), 7.63–7.77 (4H, m).

522
C. S. Reid et al. / Bioorg. Med. Chem. 19 (2011) 513–523
(231
l
L, 1.66 mmol), benzyl bromide (197
lL, 1.66 mmol), and tol-
6.2.30. 1-Benzyl-1,2-dihydro-2,2-dimethyl-4-phenylquinolin-6-
uene (10 mL) were then heated to reflux overnight under nitrogen.
The solvent was evaporated and the residue purified on silica gel
with hexanes–dichloromethane (1:1) as the mobile phase to yield
the N1 benzylated product, 1-benzyl-1,2-dihydro-2,2,4,7-tetram-
yl acetate (22)
A mixture of 21 (5.02 g, 18.9 mmol) in 48% HBr (60 mL) was
stirred at reflux for 1 h. The mixture was cautiously poured over
a mixture of ice and an excess of NaHCO3. This mixture was ex-
tracted with ethyl acetate followed by CH2Cl2. The extracts were
washed with brine, dried (MgSO4), concentrated, and chromato-
graphed on silica, eluting with hexanes–ethyl acetate (2:1). The
crude product was recrystallized from hexanes–ethyl acetate to
give 1,2-dihydro-2,2-dimethyl-4-phenylquinolin-6-ol (2.37 g,
49%) as a white solid, mp 179–180 °C. 1H NMR (DMSO-d6) d 1.23
(6H, s), 5.36 (1H, br s), 5.42 (1H, d, J = 1.3 Hz), 6.21 (1H, d,
J = 2.2 Hz), 6.42 (2H, m), 7.25 (2H, m), 7.38 (3H, m), 8.29 (1H, s);
ESI(+) MS m/z 252.8. Anal. (C17H17NOÁ0.1H2O) Calcd C, 80.66; H,
6.80; N, 5.53. Found: C, 80.76; H, 6.80; N, 5.54. A mixture of 1,2-
dihydro-2,2-dimethyl-4-phenylquinolin-6-ol (2.30 g, 9.15 mmol),
benzyl bromide (2.0 mL, 16.8 mmol), and triethyl amine (2.0 mL,
14.3 mmol) in toluene (90 mL) was then stirred at reflux overnight.
The reaction mixture was diluted with water. Layers were sepa-
rated and the aqueous layer was extracted with ethyl acetate.
Combined organic layers were washed with brine, dried (MgSO4),
concentrated, and chromatographed on a column of silica, eluting
with hexanes–ethyl acetate (4:1) to give 1-benzyl-1,2-dihydro-
2,2-dimethyl-4-phenylquinolin-6-ol as an oil that solidified to a
glass (3.23 g, 104%). 1H NMR (DMSO-d6) d 1.37 (6H, s), 4.43 (2H,
s), 5.57 (1H, s), 6.17 (1H, d, J = 8.7 Hz), 6.27 (1H, d, J = 2.8 Hz),
6.33 (1H, dd, J = 8.6 and 2.8 Hz), 7.20 (t, J = 7.2 Hz, 1H), 7.32 (m,
4H), 7.42 (m, 5H), 8.41 (s, 1H). Anal. (C24H23NOÁ0.25H2O) Calcd C,
83.32; H, 6.85; N, 4.05. Found: C, 83.07; H, 6.95; N, 3.97. A
mixture of 1-benzyl-1,2-dihydro-2,2-dimethyl-4-phenylquinolin-
6-ol (2.02 g, 5.92 mmol) and acetyl chloride (1.0 mL, 14.01 mmol)
in dichloromethane (50 mL) maintained at 0 °C was then treated
with triethylamine (1.5 mL, 10.8 mmol). The mixture was stirred
overnight at room temperature, then concentrated and chromato-
graphed on a column of silica, eluting with hexanes–ethyl acetate
(10:1) to give 22 as a glass (1.97 g, 86%), mp 57–59 °C. 1H NMR
(DMSO-d6) d 1.43 (6H, s), 2.10 (3H, s), 4.55 (2H, s), 5.61 (1H, s),
6.28 (1H, d, J = 8.9 Hz), 6.42 (1H, d, J = 2.8 Hz), 6.64 (1H, dd,
J = 8.8 and 2.7 Hz), 7.24 (1H, m), 7.38 (9H, m); ESI(+) MS m/z
384.6. Anal. (C26H25NO2Á0.2H2O) Calcd C, 80.67; H, 6.61; N, 3.62.
Found: C, 80.40; H, 6.67; N, 3.51.
ethylquinolin-6-ol (243 mg, 75%), as
a
brown oil. 1H NMR
(DMSO-d6) d 1.26 (6H, s), 1.87 (3H, s), 1.90 (3H, s), 4.38 (2H, s),
5.37 (1H, s), 6.00 (1H, s), 6.53 (1H, s), 7.14–7.35 (5H, m), 8.28
(1H, s). 1-Benzyl-1,2-dihydro-2,2,4,7-tetramethylquinolin-6-ol
(235 mg, 0.80 mmol), Et3N (167
lL, 1.20 mmol), and acetyl chlo-
ride (85 L, 1.20 mmol) were then stirred overnight with dichloro-
l
methane (10 mL) under nitrogen. The solvent was evaporated and
the residue purified on silica gel using hexanes–ethyl acetate
(10:1) as the mobile phase to yield 1-benzyl-1,2-dihydro-2,2,4,7-
tetramethylquinolin-6-yl acetate (16b, 168 mg, 63%) as a yellow
oil that solidified on standing. 1H NMR (DMSO-d6) d 1.31 (6H, s),
1.81 (3H, s), 1.90 (3H, s), 2.21 (3H, s), 4.48 (2H, s), 5.42 (1H, s),
6.08, (1H, s), 6.65 (1H, s), 7.14–7.35 (5H, s); 13C NMR (DMSO-d6)
d 16.4, 18.4, 20.7, 28.2, 47.5, 57.0, 113.4, 116.9, 121.3, 126.3,
126.6, 128.6, 128.9, 130.1, 139.8, 140.1, 141.8, 169.6; ESI-MS:
[M+H]+ m/z 336; HRESI-MS: [M+H]+ m/z 336.1964 (calcd
336.1964). Anal. (C22H25NO2) Calcd C, 78.77; H, 7.51; N, 4.18.
Found: C, 78.04; H, 7.52; N, 4.13.
6.2.27. 4-Methoxy-N-(2-methylbut-3-yn-2-yl)benzenamine (19)
A mixture of p-anisidine (17, 4.95 g, 40.2 mmol), 3-chloro-3-
methylbut-1-yne (18, 4.59 g, 44.8 mmol), copper(I) chloride
(403 mg, 4.08 mmol), and copper powder (316 mg, 4.98 mmol) in
triethylamine (80 mL) was stirred overnight. The mixture was fil-
tered through Celite and the Celite pad was washed with ethyl ace-
tate (300 mL). Combined filtrates were washed twice with
concentrated NH4OH solution followed by saturated NaCl solution,
dried (MgSO4), concentrated under reduced pressure, and chro-
matographed on silica eluting with hexanes/ethyl acetate (4:1) to
give an orange oil (5.99 g, 79%). 1H NMR (DMSO-d6) d 1.45 (6H,
s), 3.18 (1H, s), 3.65 (3H, s), 5.06 (1H, s), 6.73 (2H, d, J = 8.9 Hz),
6.87 (2H, d, J = 8.9 Hz).
6.2.28. 4-Methoxy-N-(2-methyl-4-phenylbut-3-yn-2-yl)
benzenamine (20)
A mixture of 19 (2.17 g, 10.1 mmol), 4-iodobenzene (2.47 g,
12.1 mmol), bis-(triphenylphosphine)palladium(II) chloride (212 mg,
0.302 mmol), and copper(I) chloride (75.9 mg, 0.399 mmol) in trieth-
ylamine (30 mL) was stirred at 60 °C until the reaction was complete
by HPLC (about 3 h). The reaction mixture was filtered through silica
gel and the silica pad was washed with ethyl acetate. Filtrates were
washed with saturated NaCl solution, dried (MgSO4), concentrated,
and chromatographed on silica eluting with hexanes–ethyl acetate
6.3. Preparation of compounds for testing
Compounds were dissolved in 100% dimethylsulfoxide (DMSO)
and finally diluted in culture medium prior to the in vitro assay.
The DMSO concentration never exceeded 1% in such assays. For
in vivo experiments, the compounds were dissolved in DMSO
and further diluted with distilled H2O to a final DMSO concentra-
tion of 10% prior to each injection into the animals.
1
(4:1) to give an oil (2.15 g, 81%). H NMR (DMSO-d6) d 1.55 (6H, s),
3.75 (3H, s), 5.17 (1H, br s), 6.76 (2H, d, J = 9.0 Hz), 6.94 (2H, d,
J = 9.0 Hz), 7.34 (5H, s).
6.4. In vitro growth inhibition assay of T. b. rhodesiense
(STIB900)
IC50 values were determined using the Alamar Blue assay and
were carried out three times independently and in duplicate.
Briefly, the compounds were tested in Minimum Essential Medium
with Earle’s salts, supplemented as previously described28 with the
following modifications: 2-mercaptoethanol 0.2 mM, sodium
pyruvate 1 mM, hypoxanthine 0.5 mM, and 15% heat-inactivated
horse serum. Serial drug dilutions were prepared in 96-well micro-
titer plates and each well inoculated with 2000 bloodstream forms
and incubated for 70 h at 37 °C under a humidified 5% CO2 atmo-
sphere. The viability marker Alamar Blue (12.5 mg resazurin
(Sigma) dissolved in 100 mL of phosphate buffered saline)
6.2.29. 1,2-Dihydro-6-methoxy-2,2-dimethyl-4-
phenylquinoline (21)
A mixture of 20 (8.46 g, 32.1 mmol) and copper(I) chloride
(3.20 g, 32.3 mmol) in toluene (120 mL) was stirred at reflux over-
night. The mixture was filtered through Celite and the Celite pad
was washed with ethyl acetate. Combined filtrates were washed
with NH4OH, followed by 10% NaCN, then by brine. The extract
was dried (MgSO4), concentrated, and purified on a column of silica
eluting with hexanes–ethyl acetate (4:1) to give an oil (5.02 g,
59%). 1H NMR (DMSO-d6) d 1.25 (6H, s), 3.52 (3H, s), 5.47 (1H, s),
5.59 (1H, br s), 6.28 (1H, d, J = 2.6 Hz), 6.53 (1H, d, J = 8.5 Hz),
6.61 (1H, dd, J = 8.6 and 2.7 Hz), 7.27 (2H, m), 7.39 (3H, m).
(10 lL) was then added to each well and the plate was incubated

C. S. Reid et al. / Bioorg. Med. Chem. 19 (2011) 513–523
523
for additional 2À5 h. The plates were read in a Spectramax Gemini
Acknowledgements
XS microplate fluorescence scanner (Molecular Devices) using an
excitation wavelength of 536 nm and an emission wavelength of
588 nm. The IC50 values were calculated from the sigmoidal inhibi-
tion curves using the SOFTMAXPRO software.
This work was supported by the Bill and Melinda Gates
Foundation, WHO/TDR through Grant A50866, and the Neglected
Global Diseases Initiative at the University of British Columbia.
Supplementary data
6.5. In vitro growth inhibition assay of T. b. brucei (s427)
The potency of compounds against bloodstream form T. b. brucei
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.11.003.
(s427) was assessed as outlined previously.29 In brief, 100
lL of
late log phase parasites cultured in HMI-9 medium30 were incu-
bated in the presence or absence of compounds in 96-well plates
at an initial density of 105 cells/mL at 37 °C in a humidified 5%
CO2 atmosphere for 72 h. Twenty-five microliters of 5 mg/mL solu-
tion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) was then added to each well, followed by incubation
of the plate at 37 °C as described for an additional 2 h. One hundred
microliters of 20% SDS lysis buffer in 50% aqueous dimethyl form-
amide was added to each well and plates were re-incubated as be-
fore for 3–6 h. Optical densities were measured at 570 nm using a
SpectraMax Plus microplate reader (Amersham Biosciences, Piscat-
away, NJ). IC50 values were determined with the aid of the software
program SOFTMAXPRO (Amersham Biosciences, dose–response equa-
tion y = [(a À d)/(1 + (x/c)b)] + d, where x = the drug concentration,
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Experiments were performed as previously reported5 with
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