Spirotetrahydro β-carbolines (spiroindolones): A new class of potent and orally efficacious compounds for the treatment of malaria

Article abstract of DOI:10.1021/jm100410f

The antiplasmodial activity of a series of spirotetrahydro β-carbolines is described. Racemic spiroazepineindole (1) was identified from a phenotypic screen on wild type Plasmodium falciparum with an in vitro IC₅₀ of 90 nM. Structure-activity relationships for the optimization of 1 to compound 20a (IC₅₀ = 0.2 nM) including the identification of the active 1R,3S enantiomer and elimination of metabolic liabilities is presented. Improvement of the pharmacokinetic profile of the series translated to exceptional oral efficacy in the P. berghei infected malaria mouse model where full cure was achieved in four of five mice with three daily doses of 30 mg/kg.

Full text of DOI:10.1021/jm100410f

J. Med. Chem. 2010, 53, 5155–5164 5155
DOI: 10.1021/jm100410f
Spirotetrahydro β-Carbolines (Spiroindolones): A New Class of Potent and Orally Efficacious
Compounds for the Treatment of Malaria
Bryan K. S. Yeung,*,† Bin Zou,† Matthias Rottmann,‡,( Suresh B. Lakshminarayana,† Shi Hua Ang,† Seh Yong Leong,†
Jocelyn Tan,† Josephine Wong,† Sonja Keller-Maerki,‡,( Christoph Fischli,‡,( Anne Goh,† Esther K. Schmitt,§
Philipp Krastel,§ Eric Francotte, Kelli Kuhen,^ David Plouffe,^ Kerstin Henson,^ Trixie Wagner, Elizabeth A. Winzeler,^
Frank Petersen,§ Reto Brun,‡,( Veronique Dartois,† Thierry T. Diagana,† and Thomas H. Keller†
†Novartis Institute for Tropical Diseases, 10 Biopolis Road, no. 05-01 Chromos, Singapore 138670, ‡Swiss Tropical and Public Health Institute,
Parasite Chemotherapy, Socinstrasse 57, Basel, Switzerland CH-4002, §Natural Products Unit, Novartis Pharma AG, Basel, Switzerland
CH-4002, Novartis Institute for Biomedical Research, Basel, Switzerland CH-4056, ^Genomics Institute of the Novartis Research Foundation,
10675 John J. Hopkins Drive, San Diego, California 92121, and (University of Basel, Petersplatz 1, Basel, Switzerland CH-4003
Received April 1, 2010
The antiplasmodial activity of a series of spirotetrahydro β-carbolines is described. Racemic spiroaze-
pineindole (1) was identified from a phenotypic screen on wild type Plasmodium falciparum with an in
vitro IC50 of 90 nM. Structure-activity relationships for the optimization of 1 to compound 20a (IC50
=
0.2 nM) including the identification of the active 1R,3S enantiomer and elimination of metabolic
liabilities is presented. Improvement of the pharmacokinetic profile of the series translated to
exceptional oral efficacy in the P. berghei infected malaria mouse model where full cure was achieved
in four of five mice with three daily doses of 30 mg/kg.
Introduction
Chemistry
Malaria remains a persistent public health problem for
about 40% of the global population, mainly in sub-Saharan
Africa. Plasmodium falciparum, the most relevant malaria
parasite, is estimated to infect 300-500 million people per
year and result in over 863000 deaths in 2008 of which
approximately 90% were children.1 Malaria control pro-
grams relying on disease prevention and artemisinin combi-
nation therapies (ACTa) have been extremely effective in
reducing the disease burden. However, recent reports of
increased tolerance to artemisinin derivatives in Plasmodium
falciparum suggest that we may soon lose the last and only
widely effective antimalarial drugs.2
In an effort to discover new antimalarial leads, a high
throughput, whole cell screen based on the proliferation of
P. falciparum was performed and led to the identification of
racemic spiroazepineindole 1.3 The compound displayed
moderate potency against both wild type (NF54) and chloro-
quine resistant (K1) strains of the parasite, and when tested in
the Plasmodium berghei infected mouse model,4 a single
100 mg/kg dose of 1 resulted in a 96% reduction in para-
sitemia (activity). Encouraged by these preliminary results, an
optimization effort to increase potency and in vivo activity of
1 was initiated. The structure-activity relationships (SAR)
and in vivo efficacy of this new class of antimalarial is
described.5
To reconfirm the in vitro potency of the hit, racemic 1 was
prepared by reductive amination of ketone 2 followed by a
Pictet-Spengler cyclization6 with 5-bromoisatin, to provide
spiroazepineindole 1 in 66% yield (Scheme 1).7 The reaction
conditions were expected to provide an equal mixture of four
stereoisomers consisting of two pairs of the 1R,3S, 1S,3R, and
1S,3S, 1R,3R enantiomers. Instead, the reaction produced a
∼9:1 diastereomeric excess favoring one pair of enantiomers.8
To determine which pair of enantiomers were formed in
excess, chiral separation followed by X-ray crystallography
revealed that the more favored pair consisted of the 1R,3S and
1S,3R stereoisomers (data not shown). Interestingly, when
tested in vitro, the individual enantiomers displayed markedly
different potencies on the parasite where 1a was greater than
250-fold more potent than 1b (Figure 1) on strain NF54.
All spiroazepineindole derivatives (1, 4, and 6) were pre-
pared according to Scheme 1 starting from the appropriate
indoleamine, while the spirotetrahydro β-carboline deriva-
tives (17-20) were synthesized from the corresponding sub-
stituted indoles (Scheme 2).9 Briefly, Vilsmeyer-Haack
formylation of the indole followed by condensation with
Scheme 1. Synthesis of Racemic 1
*To whom correspondence should be addressed. Phone: þ65 6722
2923. Fax: þ65 6722 2918. E-mail: bryan.yeung@novartis.com.
aAbbreviations: ACT, artemisinin combination therapies; AS, arte-
sunate; CQ, chloroquine; CYP450, cytochrome P450; PAMPA, parallel
artificial permeability assay; hERG, human ether-a-go-go related gene.
Reagents and conditions: (a) NaBH3CN, NH4OAc, rt; (b) 5-bromo-
isatin, p-TsOH H2O (cat), 110 ꢀC.
3
r
2010 American Chemical Society
Published on Web 06/22/2010
pubs.acs.org/jmc

5156 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Yeung et al.
In general, the minor pair of diastereomers was not isolated
from the reaction mixture except in a few cases. For example,
in the case of 9, also prepared according to Scheme 2, all four
stereoisomers were isolated and tested individually for po-
tency. The minor 1S,3S and 1R,3R pair of stereoisomers were
found to be less potent than the corresponding active 1R,3S
stereoisomer (Figure 2). This trend was found to be consistent
across the class and in all cases where all four stereoisomers
wereisolated. From these results, weconcluded thatthe 1R,3S
configuration was the required stereochemistry for activity on
P. falciparum.
Diasteroselectivity in the Pictet-Spengler reaction is well
documented and has been studied by several groups.10 We
sought to take advantage of the high degree of diastereoselec-
tivityobservedinour system byusing enantiopureS-R-methyl
indoleamines instead of the racemic mixtures. This would
directly provide the corresponding 1R,3S enantiomer and
obviate the need for chiral resolution, as only a single pair
of diastereomers would be formed.11
Figure 1. In vitro activity of spiroindolone 1 and the readily
accessible stereoisomers.
Enantiopure 9a and 9b were obtained from the correspond-
ing S- and R-R-methyl indoleamines respectively. (S)-2-(1H-
Indol-3-yl)-1-methyl-ethylamine (22) was prepared from
D-tryptophanol after complete reduction of the hydroxymethyl
Figure 2. In vitro activity of spiroindolone 9 and its four stereo-
isomers.
nitroethane provided the corresponding nitroalkene. Next,
reduction of the nitroalkene with lithium aluminum hydride,
followed by condensation with 5-chloroisatin provided race-
mic 17-20 with a high degree of diastereoselectivity. The
individual 1R,3S and 1S,3R enantiomers were then resolved
by chiral chromatography and could be readily identified and
differentiated by optical rotation.
Figure 3. Crystal structure of the active 1R,3S enantiomer of 9a
(only one of the two independent molecules is shown). Atomic
displacement ellipsoids are drawn at the 50% probability level,
hydrogen atoms drawn as spheres of arbitrary radius. For clarity
only the stereogenic centers are labeled (C9R, C11S).
Scheme 2. Racemic Route to the Spirotetrahydro β-Carbolines Series
Reagents and conditions: (a) POCl3, DMF; (b) nitroethane, ammonium acetate, reflux; (c) LiAlH4, THF, reflux; (d) 5-chloroisatin, p-TsOH H2O
3
(cat), 110 ꢀC.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5157
Scheme 3. Synthesis of the Active Enantiomer of 9a from D-Tryptophanol
Reagents and conditions: (a) (1) CbzCl, Na2CO3, rt; (2) p-TsCl, TEA, rt; (b) H2, Pd(OH)2, rt; (c) 5-chloroisatin, p-TsOH H2O (cat), 110 ꢀC.
3
Table 1. In Vitro Antimalarial Activity and Structure-Activity Relationships of the Spiroindolones
Table 2. Key Physicochemical Properties of the Spiroindolonesa
or D-tryptophan derivatives. Because of this, we chose the
racemic route to compounds 17-20 utilizing a chiral resolution
solubility
(pH 6.8) (μg/mL)
2
compd
logP
clogD7.4
pKa1/pKa
step as the preferred synthesis to explore SAR. Upon identify-
ing biologically active racemates in vitro, the potency of a
particular compound could always be improved by isolation or
synthesis of the corresponding 1R,3S enantiomer.
1a
9a
79
194
140
8
3.9
3.4
3.6
4.8
4.0
4.0
3.5
3.8
4.4
3.9
5.2/nd
5.1/10.4
4.8/nd
5.2/10.6
4.9/nd
17a
18a
20a
170
Results and Discussion
a Data were experimentally determined except clogD7.4; nd, not
determined.
Structure-Activity Relationships. On the basis of the
difference in potencies between the enantiomers, we sur-
mised that the spirocenter was an essential feature of the
scaffold, and indeed its removal lead to an almost complete
loss of activity for the series (see Supporting Information).
Hence to readily access the spirocenter, we incorporated the
oxindole moiety via isatins in all of the derivatives described.
Replacement of the 50-bromide of 1 with other halogens
had negative effects except for the 50-chloro derivative (4).
Although a number of additional mono- and disubstituted
oxindole derivatives were prepared, only the monosubsti-
tuted, 50-chloro derivatives had the optimum balance be-
tween potency, favorable pharmacokinetics, and synthetic
accessibility.
to the corresponding methyl group (Scheme 3).12 Subsequent
cyclization with 5-chloroisatin in the presence of catalytic acid
provided enantiopure 9a after purification. The reduction of
the tosyl group could also be achieved with lithium aluminum
hydride with no observable racemization of the stereogenic
center. The opposite, 1S,3R enantiomer (9b) was obtained
from L-tryptophanol using the same procedure.
An X-ray crystal structure of 9a unambiguously assigned
the 1R,3S configuration to the active enantiomer (Figure 3)
and showed that both potency and the corresponding stereo-
chemistry of the spirotetrahydro β-carboline series was con-
sistent with that of the spiroazepineindole series (1a).
The only drawback of this potentially ideal route is the
limited availability of chiral, substituted R-methyl indoleamines
Simplifying the structure by removing the C3 stereogenic
center and increasing the size of the central ring azepine ring

5158 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Yeung et al.
Table 3. Comparison of the Pharmacokinetic Profile of the Spirotetrahydro β-Carbolinesc
a intrinsic clearance in liver microsomes, where high clearance corresponds to poor stability in the presence of liver enzymes. b Measured in mice at a
single 5 mg/kg iv dose. c nd, not determined.
by one carbon lead to an inactive compound (5: IC50 > 5000
nM); however, azepineindole (6) and tetrahydro-β-carboline
(7) derivatives retained moderate potency (Table 1). Inter-
estingly for the azepineindoles, the similar potency between 4
and 6 suggests that the C3 methyl is not essential in this
series. In contrast, the C3 methyl enhances potency in the
tetrahydro β-carboline series, where an almost five-fold
increase in activity is observed with the methyl group present
(7 vs 9). Substitutions at C3 were found to be extremely
limited. Only methyl or trifluoromethyl was tolerated be-
cause increasing the steric bulk to gem-dimethyl (8), hydro-
xymethyl, n-propyl, or methyl ester significantly decreased
activity even with the required stereochemistry (see Support-
ing Information).
Optimizing the Pharmacokinetic Profile. The spiroindo-
lones as a class proved to be an exceptionally high quality
scaffold from a medicinal chemistry perspective (Table 2).
Most derivatives prepared displayed favorable in vitro
solubility and permeability (PAMPA and Caco-2). Deriva-
tives generally did not show cytotoxicity across several
human cell lines nor was there significant binding in a panel
of human receptors, kinases, and ion channels (IC50 > 10
μM). Moreover the spiroindolones display low cardiotoxi-
city potential (hERG), and genotoxicity potential (minia-
turized Ames test and high content micronucleus), hence no
effort to improve these properties was required.13
In addition to in vitro potency, significant differences
between pairs of enantiomers were observed with respect to
metabolic stability and CYP450 inhibition. Compounds 9a
and 9b proved to be representative of the types of trends we
observed for most analogues in Table 3. More specifically,
the less active, or 1S,3R, enantiomers displayed superior
metabolic stability in the presence of liver microsomes, had
low to medium in vivo clearance, and showed little or no
propensity for drug-drug interactions (CYP450 liabilities).
In contrast, the biologically active 1R,3S enantiomers often
displayed high clearance with poor microsomal stability and
were found to inhibit CYP 2C9 preferentially although were
generally inactive against CYP 3A4 and CYP 2D6 isoforms
(Table 3).

Article
To address the metabolic stability of 9a and to reduce its
CYP 2C9 liabilities, we identified susceptible sites on the
molecule which could be modified or blocked by chemical
means and translate into a longer half-life.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5159
addition a three-fold improvement in potency on the parasite
and lower CYP 2C9 inhibition was achieved over 9a (Table 2).
Combining both C6 and C7 substitutions had an additive affect
on potency and further improved the pharmacokinetic proper-
ties. Compounds 19a and 20a now displayed subnanomolar
potency on the parasite with the desired in vitro stability and
decreased CYP 2C9 liabilities. Thus C7 substitutions were
critical in increasing metabolic stability, minimizing drug-drug
interaction, and increasing in vitro potency.
In Vivo Pharmacokinetic Profile of 9a and 20a. The marked
difference in the in vitro PK profile of unsubstituted (9a) and
substituted (20a) spiroindolones translated well in vivo
(Figure 4 and Table 4). Following intravenous administration,
9a displayed a moderate total systemic clearance (55% hepatic
blood flow), moderate volume of distribution (0.91 L/kg or 1.2
times the total volume of body water), and short half-life of
Focusing on the western part of the molecule, positions C6
and C7 stood out as susceptible metabolic sites prone to
oxidation and thus high clearance. The racemic 6-fluoro
derivative (17) showed a moderate improvement in meta-
bolic stability, however, upon separation of the two enantio-
mers (17a and 17b), only the less active 1S,3R enantiomer
displayed the desired stability profile in vitro (Table 2).
Moreover the 6-fluoro substitution had no effect on decreas-
ing CYP 2C9 inhibition although potency of the active ena-
ntiomer (17a) had improved slightly. Interestingly, despite
the high clearance and low stability predicted from in vitro
assays, 17a showed a nearly two-fold improvement in clear-
ance over 9a in vivo.
0.42 h. The relatively low oral exposure of 9a (3.88 μM h)
3
resulted in modest oral bioavailability (13%). In contrast, after
intravenous administration, 20a displayed a higher volume of
distribution, 2.7 times the total body water (1.60 L/kg). Similar
to the low in vitro clearance observed in liver microsomes, the
total systemic clearance was lower (∼10% hepatic blood flow)
and the compound had a markedly longer half-life than 9a.
Although the oral absorption (Tmax) of 20a was significantly
slower than 9a, the compound reached both higher peak
plasma concentrations and 18-fold greater exposure (AUC¥)
than 9a. The increased exposure translated to an apparent
bioavailability of 53%. Moreover the Cmax of 20a in mice is
about 40000-fold above the Plasmodium falciparum IC50 sug-
gesting lower doses for efficacy and decreased potential for
adverse toxicity.
In contrast to 17, when the individual enantiomers of 18
were profiled, the active 1R,3S enantiomer (18a) showed
improved microsomal stability that was similar to 18b. In
Table 5. Single Dose Efficacies of 9a and 20a in the P. berghei Mouse
Modela
1 ꢀ 30 mg/kg
1 ꢀ 100 mg/kg
compd
activity (%)
survival (d)
activity (%)
survival (d)
CQ
AS
9a
99.5
95.6
99.9
99.6
9.0
5.8
99.6
98.0
99.9
99.4
12.5
7.0
10.7
12.0
14.3
13.0
20a
a Control survival, 6-7 days; CQ, chloroquine; AS, artesunate; cure,
no parasites at day 30; compounds were formulated in 10% ethanol,
30% PEG400, 6% vitamin E TPGS.
Table 6. Multiple Dose Efficacies of 9a and 20a in the P. berghei Mouse
Modela
3 ꢀ 10 mg/kg
3 ꢀ 30 mg/kg
compd
activity (%)
survival (d)
activity (%)
survival (d)
CQ
AS
9a
99.8
96.0
99.9
99.8
13.6
8.1
99.8
99.0
99.9
99.8
14.3
12.2
23.8
29.4
14.6
17.2
20a
Figure 4. In vivo PK profile of 9a and 20a showing a plot of plasma
concentration vs time for 9a (]) and 20a (0) administered orally (A)
and intravenously (B) to mice. Plasma concentrations are indicated
in ng/mL ( standard deviation (n = 3).
a Control survival, 6-7 days; CQ, chloroquine; AS, artesunate; cure,
no parasites at day 30; compounds were formulated in 10% ethanol,
30% PEG400, 6% vitamin E TPGS.
Table 4. Pharmacokinetic Parameters for 9a and 20a Following Oral Dosing at 25 mg/kg and iv Dosing at 5 mg/kg in Micea
oral PK parameters
intravenous PK parameters
compd
C
max (μM)
Tmax (h)
AUC¥ (μM h)
T1/2 (h)
F (%)
Vss (L/kg)
CL (mL/min/kg)
T1/2 (h)
3
9a
20a
3.58
8.32
0.25
2.00
3.88
71.44
0.66
3.18
13
53
0.91
1.60
49.66
8.53
0.42
2.94
a Cmax, maximum concentration of drug in plasma; Tmax, time to maximum concentration of drug in plasma; AUC¥, area under the curve
extrapolated to infinity; Vss, volume of distribution at steady state; CL, clearance; T1/2, half-life; F, oral bioavailability.

5160 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Yeung et al.
Collectively, the data suggests that the excellent in vitro
potency, good bioavailability, long oral exposure, and half-
life (3 h) of 20a is amenable to a single or low dose oral
treatment for malaria. To evaluate the oral efficacy of 9a and
20a, the compounds were tested in the P. berghei infected
malaria mouse model.
Varian 300 MHz Mercury NMR or Bruker 500 MHz Ultrashield
spectrometer using CDCl3, DMSO-d6, or MeOD-d4 as solvents.
Preparative chiral separation was performed on a Chiracel OD-H
column (2 cm ꢀ 25 cm) with 90:10 hexanes:ethanol as the mobile
phase. Analytical chiral chromatography was carried out on a
Chiracel AD-H column and all single enantiomers reported were
greater than 98% e.e. Compound purity was determined by LC/
MS and HPLC and carried out on an Agilent LC110 HPLC
equipped with a Waters Symmetry Shield RP18, 3.5 μm, 4.6 mm ꢀ
150 mm column using a gradient (13 min) of 95:5 H2O (0.1%
formic acid):CH3CN to 5:95 H2O (0.1% formic acid):CH3CN.
The purity of all compounds reported were >95% measured at
254 nm. The specific rotation reported for all compounds was
measured on a Jasco P-1020 polarimeter at 25 ꢀC with sample
concentration and solvent noted where appropriate.
In Vivo Efficacy in the P. berghei Malaria Mouse Model.
Average mouse survival results using two different oral
dosing regimens of 9a and 20a are presented along with
chloroquine (CQ) and artesunate (AS) as comparisons
(Table 5). The spiroindolones show excellent in vivo activity.
A single oral dose of 9a (30 mg/kg) reduced parasitemia on
day three by more than 99% and prolonged average mouse
survival to 10.7 days. Increasing the dose to 100 mg/kg had
only a marginal effect on mouse survival (14.3 days) and
may be attributed to the high clearance of the compound.
At single doses, the increased absorption (high Cmax) and
greater exposure (AUC) of 20a did not lead to a significant
improvement in efficacy. A single oral dose of 30 mg/kg
reduced parasitemia by more than 99% and prolonged
mouse survival for 12 days while increasing the dose to
100 mg/kg lead to essentially no change in mouse survival.
However, under these conditions, both spiroindolones
were comparable to CQ (99.6%, 12.5 days) and outper-
formed AS (98.0% and 7.3 days) in terms of average mouse
survival.
X-ray Structure Determination. Intensity data collection for
9a was performed on the PX II beamline at the Swiss Light
˚
Source in Villigen at a wavelength of 0.71073 A, which corre-
sponds to Mo KR radiation. The structure was solved by dual-
space recycling methods and refined by full-matrix least-squares
on F2 using the SHELXTL program suite. The asymmetric unit
contains two independent molecules. Non-hydrogen atoms
were refined with anisotropic displacement parameters; hydro-
gen atoms were calculated in idealized positions and refined
using a riding model. The crystals are twinned (twin law -1 0 0 0
-1 0 0 0 1). CCDC 758814 contains the supplementary crystal-
lographic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
In a multiple oral dosing regimen, differences between 9a
and 20a became more apparent where, 20a proved to be
superior to 9a, CQ, and AS (Table 6). Three oral daily doses
of 10 mg/kg resulted in 17.2 days average mouse survival
while 29.4 days survival was achieved with 3 ꢀ 30 mg/kg oral
doses (full cure in four out of five mice). In addition, neither
toxicity nor behavioral changes after drug administration
was observed in any of the animals treated with 9a and 20a at
any dosing regimen.
The in vivo efficacy data suggests that despite the high
clearance and short half-life, 9a is a highly efficacious anti-
malarial when administered orally and comparable to
standard antimalarials in the P. berghei mouse model. Opti-
mizing the metabolic stability and improving potency re-
sulted in increased oral absorption, exposure, and efficacy in
mice. The in vivo profile and efficacy of 20a is consistent with
pharmacokinetic properties required for a single dose or low
dose cure for malaria.
The spiroindolones represents a promising new antimalarial
chemotype, displaying exceptional in vitro activity and para-
sitemia reduction at low doses in the P. berghei mouse model.
Although the mechanism of action (MoA) and molecular
target is currently unknown, preliminary evidence suggests
that the spiroindolones function by a different mechanism
than the heme detoxification pathway of 4-aminoquinolines
and instead may act by inhibiting protein synthesis within the
parasite.14 Moreover, the observation that only the 1R,3S
enantiomer is biologically active implies the presence of a
discrete molecular target. Further preclinical evaluation of this
series of compounds is currently ongoing.
Crystal data for 9a: colorless rod from chloroform/hexadecane,
size 0.04 ꢀ 0.02 ꢀ 0.01 mm3, C19H16ClN3O, Mr = 337.80, trigonal
˚
space group P32 (No. 145) with a = 14.834(2), c = 13.354(6) A,
V = 2544.8(7) A , Z = 6, Dc = 1.323 g cm-3, 19289 reflections
3
˚
3
measured, 5109 independent (Rint = 0.0429), 2.20ꢀ < θ < 24.10ꢀ,
T= 100 K, 444 parameters, 1 restraint, R1 = 0.0279, wR2 = 0.0637
for 4987 reflections with I > 2σ(I), R1 = 0.0294, wR2 = 0.0645 for
-3
˚
all 5109 data, GoF = 1.048, res el dens = þ0.13/-0.17 e A
.
3
Absolute structure parameter x = 0.08(6).
3-(1H-Indol-3-yl)-1-methyl-propylamine (3). Ammonium
acetate (4.45 g, 57.7 mmol) and sodium cyanoborohydride
(0.37 g, 5.9 mmol) were added to a solution of 4-(1H-indol-
3yl)-butan-2-one (2) (1.0 g, 5.3 mmol) in methanol (20 mL) and
stirred at room temperature. After 4 h, the reaction mixture was
quenched by slow addition of 1 N hydrochloric acid and
adjusted to pH ∼ 2. The mixture was concentrated in vacuo
and diluted with dichloromethane. The aqueous phase was
adjusted to pH ∼ 12 with 4 N NaOH and washed with
dichloromethane (3 ꢀ 50 mL). The combined organic phases
were dried over sodium sulfate and concentrated in vacuo. The
residue was purified by flashchromatography to afford3 (622 mg,
62%) as an oil. 1H NMR (300 MHz, DMSO-d6): δ 10.71 (s, 1H),
7.50 (d, J = 7.5 Hz, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.08 (d, J = 2.1
Hz, 1H), 7.04 (td, J = 8.1, 1.5 Hz, 1H), 6.95 (td, J = 7.2, 1.2 Hz,
1H), 2.81 (m, 1H), 2.70 (m, 2H), 1.61 (m, 2H), 1.02 (d, J = 6.3 Hz,
3H). MS (ESI) m/z 189.0 (M þ H)þ.
50-Bromo-3-methyl-3,4,5,10-tetrahydro-2H-spiro[azepino[3,4-b]-
indole-1,30-indol]-20(10H)-one (1). p-Toluenesulfonic acid (0.29 g,
1.51 mmol) was added to a flask charged with 3 (1.00 g, 5.32 mmol)
and 5-bromoisatin (1.20 g, 1.32 mmol) in n-butanol (11 mL) and
heated to 120 ꢀC. After 16 h, the reaction mixture was cooled to
room temperature and concentrated in vacuo. The residue was
dissolved in ethyl acetate and washed with 0.5 M NaOH and
saturated aqueous NaCl. The organic layer was separated and
dried over MgSO4, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography to provide racemic 1 as
dark-brown solid (1.4 g, 66%). 1H NMR (300 MHz, DMSO-d6): δ
10.43 (s, 1H), 9.96 (s, 1H), 8.14 (s, 1H), 7.45 (d, J = 8.1 Hz, 1H),
7.44 (d, J = 8.1 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.16 (dd, J =
6.3, 1.2 Hz, 1H), 6.99 (td, J = 6.9, 1.5 Hz, 1H), 6.94 (td, J = 6.9,
1.5 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 3.89 (m, 1H), 3.11 (ddd,
Experimental Section
General Methods. Reagents and solvents were purchased from
Aldrich, Acros, or other commercial sources and used without
further purification. Thin layer chromatography (TLC) was
performed on precoated silica gel 60 F254 plates from Merck.
Compounds were visualized under UV light, ninhydrin, or phos-
phomolybdic acid (PMA) stain. NMR spectra were obtained on a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5161
J = 12.0, 8.1, 4.5 Hz, 1H), 2.86 (ddd, J = 12.9, 8.7, 4.2 Hz, 1H),
2.79 (d, J = 5.7 Hz, 1H), 2.07 (m, 1H), 1.65 (dtd, J = 12.2, 8.4, 3.9
Hz, 1H), 1.05 (d, J = 6.6 Hz, 3H). MS (ESI) m/z 397.0 (M þ H)þ.
The racemate (1) was separated into its enantiomers by chiral
chromatography under the conditions described in General Meth-
ods. The absolute configuration was determined by X-ray crystal
analysis and characterized below.
heated to 100 ꢀC. After 23 h, the reaction mixture was cooled to
room temperature and concentrated in vacuo. The residue was
dissolved in ethyl acetate and washed with 0.5 M NaOH and
saturated aqueous NaCl. The organic layer was separated and
dried over Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography to provide 7 as an orange
solid (6 mg, 3%). 1H NMR (300 MHz, DMSO-d6): δ 10.57 (s, 1H),
10.48 (s, 1H), 7.47 (d, J = 7.2 Hz, 1H), 7.32 (dd, J = 8.1, 2.1 Hz,
1H), 7.19 (d, J = 7.8 Hz, 1H), 7.09 (d, J = 2.1 Hz, 1H), 7.02 (m,
2H), 6.95 (d, J = 8.1 Hz, 1H), 3.59 (m, 1H), 3.12 (m, 2H), 2.80 (m,
2H). MS (ESI) m/z 324.0 (M þ H)þ.
(1R,3S)-50-Bromo-3-methyl-3,4,5,10-tetrahydro-2H-spiro[azepino-
[3,4-b]indole-1,30-indol]-20(10H)-one (1a). 1H NMR (300 MHz,
DMSO-d6): δ 10.43 (s, 1H), 9.96 (s, 1H), 7.46 (d, J = 8.1 Hz,
1H), 7.45 (d, J = 8.7 Hz, 1H), 7.26 (d, J = 1.8 Hz, 1H), 7.16 (dd,
J = 7.0, 1.2 Hz, 1H), 6.99 (td, J = 6.9, 1.5, 1H), 6.94 (td, J = 6.9,
1.2 Hz, 1H), 6.86 (d, J = 8.4Hz, 1H), 3.88 (m, 1H), 3.11 (ddd, J =
12.0, 7.8, 4.2 Hz, 1H), 2.87 (ddd, J = 12.9, 8.7, 3.9 Hz, 1H), 2.79
(d, J = 6.0 Hz, 1H), 2.07 (m, 1H), 1.64 (dtd, J = 12.9, 8.7, 4.2 Hz,
1H), 1.04 (d, J = 6.6 Hz, 3H). MS (ESI) m/z 397.0 (M þ H)þ;
[R]2D5= þ226.9ꢀ (c = 1.0 g/L, methanol).
50-Chloro-3,3-dimethyl-2,3,4,9-tetrahydrospiro[β-carboline-1,30-
indol]-20(10H)-one (8). Sodium hydroxide (2.88 g, 72.0 mmol) was
added to a solution of gramine (12.0 g, 69.0 mmol) in 2-nitropro-
pane (44 mL, 490 mmol) and heated to reflux. After 18 h, the
reaction was cooled to room temperature and a 10% aqueous
acetic acid (60 mL) added. After stirring for an additional hour,
the mixture was diluted with water and extracted with diethyl
ether. The organic phase was separated and washed with water
then dried over MgSO4 to provide 3-(2-methyl-2-nitropropyl)-
1H-indole as a dark-brown oil (16.5 g, quantitative), which was
used in the next step without further purification.
(1S,3R)-50-Bromo-3-methyl-3,4,5,10-tetrahydro-2H-spiro[azepino-
[3,4-b]indole-1,30-indol]-20(10H)-one (1b). 1H NMR (300 MHz,
DMSO-d6): δ 10.43 (s, 1H), 9.96 (s, 1H), 7.46 (d, J = 8.1 Hz,
1H), 7.45 (d, J = 8.7 Hz, 1H), 7.26 (d, J = 1.5 Hz, 1H), 7.16 (bd,
J = 7.8 Hz, 1H), 6.98 (td, J = 6.9, 1.5 Hz, 1H), 6.94 (td, J = 7.2,
1.2 Hz, 1H), 6.86 (d, J = 8.4Hz, 1H), 3.89 (m, 1H), 3.11 (ddd, J =
11.7, 7.8, 4.2 Hz, 1H), 2.84 (ddd, J = 12.9, 8.7, 4.2 Hz, 1H), 2.79
(d, J = 6.0 Hz, 1H), 2.07 (m, 1H), 1.64 (dtd, J = 12.9, 8.7, 4.2 Hz,
1H), 1.04 (d, J = 6.4 Hz, 3H). MS (ESI) m/z 397.0 (M þ H)þ;
[R]2D5 = -254.9ꢀ (c = 1.0 g/L, methanol)
A solution of hydrazine hydrate (5.68 g, 114 mmol) in 95%
ethanol (6.5 mL) was added to a well stirred suspension of 3-(2-
methyl-2-nitropropyl)-1H-indole (8.5 g, 39.0 mmol) and Raney
Ni in 95% ethanol (75 mL) and heated to reflux. After 12 h, the
reaction was filtered through celite and concentrated in vacuo.
The residue was recrystallized from isopropanol to provide
1-(1H-indol-3-yl)-2-methylpropan-2-amine (2.8 g, 38%). 1H
NMR (300 MHz, DMSO-d6): δ 10.85 (s, 1H), 7.55 (d, J = 7.2
Hz, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.12 (d, J = 2.4 Hz, 1H), 7.03
(td, J = 6.9, 1.2 Hz, 1H), 6.95 (td, J = 7.2, 1.2 Hz, 1H), 2.68 (s,
2H), 1.02 (s, 6H). MS (ESI) m/z 189.0 (M þ H)þ.
50-Chloro-3-methyl-3,4,5,10-tetrahydro-2H-spiro[azepino[3,4-b]-
indole-1,30-indol]-20(10H)-one (4). Compound 4 was prepared
according to Scheme 1 with 5-chloroisatin. 1H NMR (300 MHz,
DMSO-d6): δ 10.43 (s, 1H), 9.95 (s, 1H), 8.25 (s, 1H), 7.46 (bd, J =
8.4 Hz, 1H), 7.32 (dd, J = 8.1, 2.4 Hz, 1H), 7.16 (m, 2H), 6.96 (m,
2H), 6.91 (d, J = 8.1 Hz, 1H), 3.89 (m, 1H), 3.11 (m, 1H), 2.86 (m,
1H), 2.07 (m, 1H), 1.64 (m, 1H), 1.04 (d, J = 6.6 Hz, 3H). MS
(ESI) m/z 352.0 (M þ H)þ.
p-Toluenesulfonic acid (0.022 g, 0.117 mmol) was added to
solution of 1-(1H-indol-3-yl)-2-methylpropan-2-amine (0.110 g,
0.59 mmol) and 5-chloroisatin (0.106 g, 0.59 mmol) in absolute
ethanol (6 mL) and heated to 130 ꢀC in a sealed tube. After 16 h,
the reaction was cooled to room temperature and concentrated
in vacuo. The residue was purified by flash chromatography to
50-Chloro-2,3,4,5,6,11-hexahydrospiro[azocino[3,4-b]indole-1,30-
indol]-20(10H)-one (5). p-Toluenesulfonic acid (0.020 g, 1.06 mmol)
was added to a flask charged with 4-(1H-indol-3-yl)-butylamine15
(0.10 g, 0.53 mmol) and 5-chloroisatin (0.11 g, 0.58 mmol) in
absolute ethanol (5 mL) and heated to 100 ꢀC. After 26 h, the
reaction mixture was cooled to room temperature and concen-
trated in vacuo. The residue was dissolved in ethyl acetate and
washed with 0.5 M NaOH and saturated aqueous NaCl. The
organic layer was separated and dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by reverse phase
chromatography to provide 5 as dark-brown solid (14 mg, 8%).
1H NMR (300 MHz, DMSO-d6): δ 10.54 (s, 1H), 8.45 (bs, 1H),
7.47 (d, J = 7.8 Hz, 1H), 7.43 (d, J = 2.1 Hz, 1H), 7.33 (m, 2H),
7.05 (m, 2H), 6.96 (m, 1H), 6.29 (bs, 1H), 2.54 (m, 1H), 2.40 (m,
3H), 1.39 (m, 4H). MS (ESI) m/z 352.0 (M þ H)þ.
1
provide 8 (98 mg, 43%). H NMR (300 MHz, DMSO-d6): δ
10.55 (s, 1H), 10.51 (s, 1H), 7.44 (d, J = 7.5 Hz, 1H), 7.28 (dd,
J = 8.4, 2.1 Hz, 1H), 7.17 (d, J = 8.1 Hz, 1H), 7.00 (m, 3H), 6.90
(d, J = 8.4 Hz, 1H), 2.76 (d, J = 14.7 Hz, 1H), 2.67 (d, J = 14.7
Hz, 1H), 1.84 (s, 1H), 1.30 (s, 3H), 1.29 (s, 3H). MS (ESI) m/z
352.0 (M þ H)þ.
50-Chloro-3-methyl-2,3,4,9-tetrahydrospiro[β-carboline-1,30-
indol-20(10H)-one (9). p-Toluene-sulfonic acid (0.113 g, 0.57 mmol)
was added to mixture of 1-(1H-indol-3-yl)propan-2-amine (0.100 g,
0.57 mmol) and 5-chloroisatin (0.115 g, 0.63 mmol) in absolute
ethanol (5.5 mL) and heated to 100ꢀC. After 18 h, the reaction was
cooled to room temperature and diluted with ethyl acetate and
then subsequently washed with 1 M HCl, water, and 1 M NaOH.
The organic phase was separated and dried over Na2SO4 and
concentrated in vacuo. The residue was redissolved in a minimun
amount of ethyl actate and triturated with methylene choride to
50-Chloro-3,4,5,10-tetrahydro-2H-spiro[azepino[3,4-b]indole-1,30-
indol]-20(10H)-one (6). p-Toluenesulfonic acid (0.11 g, 0.57 mmol)
was added to a flask charged with 3-(1H-indol-3-yl)-propylamine15
(0.10 g, 0.57 mmol) and 5-chloroisatin (0.12 g, 0.63 mmol) in
absolute ethanol (5 mL) and heated to 100 ꢀC. After 15 h, the
reaction mixture was cooled to room temperature and concentrated
in vacuo. The residue was dissolved in ethyl acetate and washed
with 0.5 M NaOH and saturated aqueous NaCl. The organic layer
was separated and dried over Na2SO4, filtered, and concentrated in
vacuo. The residue was purified by flash chromatography to
1
provide 9 (131 mg, 68%). H NMR (300 MHz, DMSO-d6): δ
10.45 (s, 1H), 10.42 (s, 1H), 7.43 (d, J = 7.2 Hz, 1H), 7.31 (dd, J =
8.4, 2.4 Hz, 1H), 7.16 (d, J = 7.2 Hz, 1H), 7.03 (d, J = 2.4 Hz, 1H),
6.99 (m, 1H), 6.92 (d, J = 8.4 Hz, 2H), 3.93 (m, 1H), 3.05 (d, J =
6.3 Hz, 1H), 2.79 (dd, J = 15.0, 3.6 Hz, 1H), 2.41 (dd, J =
15.0, 10.5 Hz, 1H), 1.18 (d, J = 6.3 Hz, 3H). MS (ESI) m/z 338.0
(M þ H)þ.
1
provide 6 as an orange solid (17 mg, 9%). H NMR (300 MHz,
DMSO-d6): δ 10.56 (s, 1H), 10.10 (s, 1H), 7.46 (dd, J = 7.0, 1.5 Hz,
1H), 7.32 (dd, J = 8.4, 2.2 Hz, 1H), 7.15 (m, 2H), 7.00 (dd, J = 6.9,
1.5 Hz, 1H), 6.97 (dd, J = 6.9, 1.8 Hz, 1H), 6.93 (d, J = 8.4 Hz,
1H), 3.47 (m, 2H), 3.02 (m, 3H), 1.98 (m, 2H). MS (ESI) m/z 338.0
(M þ H)þ.
Preparation of 9a from D-Tryptophanol. Benzyl chloroformate
(0.374 mL, 2.29 mmol) was added dropwise to a suspension of
sodium carbonate (482 mg, 3.92 mmol) and D-tryptophanol (500 mg,
2.27 mmol) in a 1:1 solution of water and acetone (22.6 mL) at 0 ꢀC.
After addition, the cooling bath was removed and the reaction stirred
at room temperature. After two hours, the reaction was carefully
acidified to pH 2 with concentrated HCl and diluted with water. The
aqueous layer was washed with ethyl acetate and the combined
50-Chloro-2,3,4,9-tetrahydrospiro[β-carboline-1,30-indol]-20(10H)-
one (7). p-Toluenesulfonic acid (0.12 g, 0.62 mmol) was added to a
flask charged with tryptamine (0.10 g, 0.62 mmol) and 5-chloroi-
satin (0.13 g, 0.69 mmol) in absolute ethanol (6 mL) and then

5162 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Yeung et al.
organic phases separated and dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by flash chroma-
tography column to give the corresponding N-Cbz-D-tryptophanol
(428 mg, 50%). 1H NMR (300 MHz, DMSO-d6): δ 10.76 (s, 1H),
7.58 (d, J = 7.5 Hz, 1H), 7.39-7.25 (m, 5H), 7.12-7.05 (m, 3H),
6.96 (t, J = 7.8 Hz, 1H), 4.99 (s, 2H), 4.68 (t, J = 5.4 Hz, 1H), 3.73
(m, 1H), 3.36 (m, 2H), 2.91 (dd, J = 14.4, 6.0 Hz, 1H), 2.73 (dd, J =
14.1, 6.0 Hz, 1H). MS (ESI) m/z 325.0 (M þ H)þ.
(s, 1H), 7.94 (dd, J = 8.2, 2.8 Hz, 1H), 7.57 (dd, J = 6.4, 4.4 Hz,
1H). MS (ESI) m/z 180.0 (M - H)þ.
5,6-Difluoro-3-(2-nitro-propenyl)-1H-indole (15d). A solution
of 5,6-difluoro-1H-indole-3-carbaldehyde (200 g, 1.11 mol) in
nitroethane (2 L) was refluxed with ammonium acetate (72.4 g,
0.94 mol). After 2 h, the reaction mixture was concentrated in
vacuo to remove nitroethane, diluted with ethyl acetate, and
washed with water and brine then dried over anhydrous
Na2SO4. The organic layer was concentrated in vacuo and
precipitated from 10% ethyl acetate in petroleum ether to
provide 15d (172 g, 65%) as a yellow solid. 1H NMR (500
MHz, CDCl3): δ 12.29 (bs, 1H), 8.39 (s, 1H), 8.05 (s,1H), 7.99
(dd, J = 8.2, 3.6 Hz, 1H), 7.52 (dd, J = 6.8, 4.0 Hz, 1H), 2.47 (s,
3H). MS (ESI) m/z 237.0 (M - H)þ.
p-Toluenesulfonyl chloride (199 mg, 1.05 mmol) was added to
a solution of N-Cbz-D-tryptophanol (320 mg, 0.988 mmol) and
triethylamine (267 μL, 1.93 mmol) in dry dichloromethane (2.8
mL) at 0 ꢀC and allowed to warm to room temperature. After
18 h, the reaction was concentrated in vacuo and the residue was
purified by flash chromatography to give the corresponding
1
tosylate (580 mg, 100%). H NMR (300 MHz, DMSO-d6): δ
2-(5,6-Difluoro-1H-indol-3-yl)-1-methyl-ethylamine (16d). A
solution of 15d (170 g, 0.714 mol) in dry THF (2 L) was added
dropwise to a suspension of lithium aluminum hydride (95 g, 2.5 mol)
in THF (1 L) at 0 ꢀC then heated to reflux. After two hours, the
reaction mixture was cooled to 0 ꢀC and quenched by slow
addition of brine then diluted with ethyl acetate and filtered
through celite. The filtrate was dried over anhydrous Na2SO4
and concentrated to give 16d (150 g crude) as a viscous brown
syrup. The residue was used in the next step without further
10.81 (s, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.50-7.25 (m, 10H), 7.08
(m, 2H), 6.95 (t, J = 7.2 Hz, 1H), 4.95 (s, 2H), 4.00 (m, 2H), 3.89
(m, 1H), 2.80 (d, J = 7.2 Hz, 2H), 2.39 (s, 3H). MS (ESI) m/z
479.0 (M þ H)þ.
Palladium(II) hydroxide (72.7 mg) was added to a solution of
the tosylate intermediate (580 mg, 1.21 mmol) in absolute
ethanol (36 mL) and stirred at 1 atm H2 atmosphere at room
temperature. After two hours, the reaction was filtered through
celite and concentrated in vacuo. The residue was dissolved in
ethyl acetate (50 mL) and washed with saturated aqueous
NaHCO3 (50 mL). The organic phase was dried over MgSO4,
filtered, and concentrated in vacuo to provide (S)-2-(1H-indol-
3-yl)-1-methyl-ethylamine (169 mg, 80%). 1H NMR (300 MHz,
DMSO-d6): δ 10.78 (s, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.32 (d,
J = 7.8 Hz, 1H), 7.11 (d, J= 2.4 Hz, 1H), 7.05 (td, J = 7.2, 1.2 Hz,
1H), 6.95 (td, J = 7.2, 1.2 Hz, 1H), 3.08 (m, 1H), 2.62 (d, J = 7.8
Hz, 2H), 0.98 (d, J = 6.3 Hz, 3H). MS (ESI) m/z 175.0 (M þ H)þ.
(1R,3S)-50-Chloro-3-methyl-2,3,4,9-tetrahydrospiro[β-carboline-
1,30-indol-20(10H)-one (9a). p-Toluenesulfonic acid (16.8 mg, 0.088
mmol) was added to solution of (S)-2-(1H-indol-3-yl)-1-methyl-
ethylamine (0.153 g, 0.881 mmol) and 5-chloroisatin (0.176 g,
0.969 mmol) in dry ethanol (3.1 mL) and heated to 110 ꢀC in a
sealed tube. After 16 h, the reaction was cooled to room tempera-
ture and concentrated in vacuo. The residue was purified by flash
1
purification. H NMR (500 MHz, CDCl3): δ 8.18 (bs, 1H), 7.3
(dd, J = 10.0, 4.0 Hz, 1H), 7.12 (dd, J = 8.6, 5.2 Hz, 1H), 7.05 (s,
1H), 3.25 (m, 1H), 2.8 (dd, J = 7.2, 11.6 Hz, 1H), 2.61 (dd, J =
10.6, 8.4 Hz, 1H), 1.16 (d, J = 8.0 Hz, 3H). MS (ESI) m/z 211.0
(M þ H)þ.
50-Chloro-6,7-difluoro-3-methyl-2,3,4,9-tetrahydrospiro[β-carbo-
line-1,30-indol]-20(10H)-one (20). p-Toluenesulfonic acid (13.6 g,
0.071 mol) was added to a mixture of 16d (150 g, 0.714 mol) and
5-chloroisatin (90.8 g, 0.5 mol) in absolute ethanol (1.5 L) and
heated to reflux. After 20 h, the reaction was cooled to room
temperature and concentrated in vacuo. The residue was diluted
with ethyl acetate and washed with 2N HCl. The organic phase was
separated and washed sequentially with saturated aqueous NaOH,
water, and brine. The organic layer was separated and dried over
anhydrous Na2SO4 and concentrated to provide a brown residue,
which was triturated with dichloromethane to provide racemic 20
(110 g, 41%) as a light-yellow solid. 1H NMR (500 MHz, DMSO-
d6): δ 10.65 (s, 1H), 10.48 (s, 1H), 7.43 (dd, J = 10.8, 4.0 Hz, 1H),
7.32 (dd, J= 8.8, 2.6 Hz, 1H), 7.1 (dd, J= 9.2, 6.0 Hz, 1H), 7.04 (d,
J = 2.4 Hz, 1H), 6.92 (d, J = 10.8 Hz, 1H), 3.90 (m, 1H), 3.08 (d,
J = 8.0 Hz, 1H), 2.76 (m, 1H), 2.33 (m, 1H), 1.16 (d, J = 8.8 Hz,
3H). MS (ESI) m/z 374.0 (M þ H)þ. The racemate (20) was
separated into its enantiomers by chiral chromatography under
the conditions described in General Methods.
1
chromatography to provide 9a (135 mg, 45%). H NMR (300
MHz, DMSO-d6): δ 10.45 (s, 1H), 10.42 (s, 1H), 7.43 (d, J = 7.2
Hz, 1H), 7.31 (dd, J = 8.4, 2.1 Hz, 1H), 7.16 (d, J = 7.2 Hz, 1H),
7.03 (m, 2H), 6.98 (m, 1H), 6.92 (d, J = 8.1 Hz, 1H), 3.92 (m, 1H),
3.05 (d, J = 6.0 Hz, 1H), 2.79 (dd, J = 14.9, 3.3 Hz, 1H), 2.41 (dd,
J = 4.5, 2.5 Hz, 1H), 1.18 (d, J = 6.0 Hz, 3H). MS (ESI) m/z 338.0
(M þ H)þ; [R]2D5 = þ253.6ꢀ (c = 0.104 g/L, methanol).
(1S,3R)-50-Chloro-3-methyl-2,3,4,9-tetrahydrospiro[β-carboline-
1,30-indol]-20(10H)-one (9b). Compound 9b was prepared using
the same procedure starting from L-tryptophanol. 1H NMR
(300 MHz, DMSO-d6): δ 10.46 (s, 1H), 10.42 (s, 1H), 7.43 (d,
J=7.5Hz, 1H), 7.31(dd,J=8.4, 2.1Hz, 1H), 7.16(d,J=7.2Hz,
1H), 7.03 (m, 2H), 6.98 (m, 1H), 6.92 (d, J = 8.1 Hz, 1H), 3.92 (m,
1H), 3.06 (d, J = 6.0 Hz, 1H), 2.78 (dd, J = 14.9, 3.6 Hz, 1H), 2.41
(dd, J = 4.5, 2.5 Hz, 1H), 1.18 (d, J = 6.3 Hz, 3H). MS (ESI) m/z
338.0 (M þ H)þ; [R]2D5 = -269.8ꢀ (c = 0.104 g/L, methanol).
The following procedure for racemic 20 was applied generally
for the preparation of racemic 17, 18, and 19 starting from the
corresponding halogenated indoles.
(1R,3S)-50-Chloro-6,7-difluoro-3-methyl-2,3,4,9-tetrahydrospiro-
[β-carboline-1,30-indol]-20(10H)-one (20a). 1H NMR (500 MHz,
DMSO-d6) δ 10.66 (bs, 1H), 10.49 (bs, 1H), 7.44 (m, 1H), 7.33
(dd, J = 8.5, 2.0 Hz, 1H), 7.11 (m, 1H), 7.04 (d, J = 2.0 Hz, 1H),
6.93 (d, J = 8.5 Hz, 1H), 3.91 (m, 1H), 3.10 (bd, J = 6.0 Hz, 1H),
2.76 (dd, J = 15.0, 3.5 Hz, 1H), 2.38 (dd, J = 15.5, 10.5 Hz, 1H),
1.17 (d, J = 6.5 Hz, 3H). MS (ESI) m/z 374.0 (M þ H)þ; [R]D25
=
þ220.8ꢀ (c = 0.100 g/L, methanol).
(1S,3R)-50-Chloro-6,7-difluoro-3-methyl-2,3,4,9-tetrahydrospiro-
[β-carboline-1,30-indol]-20(10H)-one (20b). 1H NMR (500 MHz,
DMSO-d6): δ 10.66 (bs, 1H), 10.49 (bs, 1H), 7.44 (m, 1H), 7.33
(dd, J = 8.0, 2.0 Hz, 1H), 7.11 (m, 1H), 7.04 (d, J = 1.5 Hz, 1H),
6.93 (d, J = 8.5 Hz, 1H), 3.91 (m, 1H), 3.10 (bd, J = 5.5 Hz, 1H),
2.76 (dd, J = 15.0, 3.5 Hz, 1H), 2.38 (dd, J = 15.0, 10.5 Hz, 1H),
5,6-Difluoro-1H-indole-3-carbaldehyde (14d). Phosphorus
oxychloride (180 mL, 1.96 mol) was added dropwise to dry
dimethylformamide (1.4 L) at -20 ꢀC over 30 min and stirred at
-5 ꢀC. After one hour, a solution of 5, 6-difluoroindole (200 g,
1.31 mol) in dry dimethylformamide (600 mL) was added
dropwise to the above mixture at -20 ꢀC. The cooling bath
was removed and the mixture allowed to warm to room tem-
perature. After one hour, the reaction was poured onto ice,
basified with solid KOH, and stirred for an additional 15 min.
The resulting precipitate was washed with 10% ethyl acetate in
petroleum ether to give 14d (180 g, 76%) as a pale-yellow solid.
1H NMR (500 MHz, CDCl3): δ 12.26 (bs, 1 H), 9.92 (s, 1H), 8.36
1.19 (d, J = 7.0 Hz, 3H). MS (ESI) m/z 374.0 (M þ H)þ; [R]D25
=
-230.9ꢀ (c = 0.100 g/L, methanol).
50-Chloro-6-fluoro-3-methyl-2,3,4,9-tetrahydrospiro[β-carboline-
1
1,30-indol]-20(10H)-one (17). H NMR (300 MHz, DMSO-d6): δ
10.53 (s, 1H), 10.47 (s, 1H), 7.32 (dd, J =8.4, 1.8 Hz, 1H), 7.19(dd,
J=9.9, 2.4Hz, 1H), 7.13(dd,J=8.9, 4.5Hz, 1H), 7.04(d,J=1.8
Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.85 (td, J = 8.9, 2.7 Hz, 1H),
3.91 (m, 1H), 3.35 (bs, 1H), 2.76 (dd, J = 14.9, 3.9 Hz, 1H), 2.38

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5163
(dd, J= 15.2, 10.2 Hz, 1H), 1.17 (d, J= 5.1 Hz, 3H). MS (ESI) m/z
356.0 (M þ H)þ.
Nicole Ye, and Peiting Zeng for pharmacology support and
Christophe Bury for chiral separation support. This work
was supported by a grant from the Medicines for Malaria
Venture and a translational research grant (WT078285) from
the Wellcome Trust to the Novartis Institute for Tropical
Diseases, the Genomics Institute of the Novartis Research
Foundation and the Swiss Tropical and Public Health
Institute.
(1R,3S)-50-Chloro-6-fluoro-3-methyl-2,3,4,9-tetrahydrospiro-
[β-carboline-1,30-indol]-20(10H)-one (17a). 1H NMR (500 MHz,
DMSO-d6): δ 10.53 (s, 1H), 10.48 (s, 1H), 7.32 (dd, J = 8.3, 2.2
Hz, 1H), 7.20 (dd, J = 9.8, 2.3 Hz, 1H), 7.16 (dd, J = 8.8, 4.6
Hz, 1H), 7.06 (d, J = 1.5 Hz, 1H), 6.93 (d, J = 6.0 Hz, 1H), 6.87
(dt, J = 6.9, 1.8 Hz, 1H), 3.95 (m, 1H), 3.35 (bs, 1H), 2.77 (dd,
J = 15.0, 3.7 Hz, 1H), 2.40 (dd, J = 15.0, 10.6 Hz, 1H), 1.18 (d,
J = 6.4 Hz, 3H). MS (ESI) m/z 356.1 (M þ H)þ; [R]D25
=
þ225.5ꢀ (c = 0.122 g/L, methanol).
Supporting Information Available: Additional experimental
details for in vitro and in vivo pharmacokinetic studies, anti-
malarial assays, and P. berghei mouse efficacy studies. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(1S,3R)-50-Chloro-6-fluoro-3-methyl-2,3,4,9-tetrahydrospiro-
[β-carboline-1,30-indol]-20(10H)-one (17b). 1H NMR (300 MHz,
DMSO-d6): δ 10.55 (s, 1H), 10.51 (s, 1H), 7.32 (dd, J = 8.4, 2.4
Hz, 1H), 7.20 (dd, J = 10.1, 2.4 Hz, 1H), 7.14 (dd, J = 8.8, 4.5
Hz, 1H), 7.05 (d, J = 2.4 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.86
(td, J = 9.3, 2.4 Hz, 1H), 3.93 (m, 1H), 3.36 (bs, 1H), 2.76 (dd,
J = 15.0, 3.6 Hz, 1H), 2.39 (dd, J = 15.2, 10.5 Hz, 1H), 1.17 (d,
References
J = 6.3 Hz, 3H). MS (ESI) m/z 357.0 (M þ H)þ; [R]D25
=
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-204.4ꢀ (c = 0.113 g/L, methanol).
50,7-Dichloro-3-methyl-2,3,4,9-tetrahydrospiro[β-carboline-1,30-
indol]-20(10H)-one (18). 1H NMR (300 MHz, DMSO-d6): δ 10.79
(s, 1H), 10.50 (s, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.32 (dd, J = 8.1,
2.4 Hz, 1H), 7.17 (d, J = 1.5 Hz, 1H), 7.04 (d, J = 2.1 Hz, 1H),
6.98 (dd, J = 8.4, 1.8 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 3.91 (m,
1H), 3.11 (bd, J = 5.5 Hz, 1H), 2.78 (dd, J = 15.2, 3.9 Hz, 1H),
2.39 (dd, J = 15.0, 10.8 Hz, 1H), 1.17 (d, J = 6.3 Hz, 3H). MS
(ESI) m/z 373.0 (M þ H)þ.
(1R,3S)-50,7-Dichloro-3-methyl-2,3,4,9-tetrahydrospiro[β-carbo-
line-1,30-indol]-20(10H)-one (18a). 1H NMR (500 MHz, CDCl3): δ
7.64 (bs, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.34 (bs, 1H), 7.29 (dd, J =
8.1, 2.4 Hz, 1H), 7.18 (d, J = 1.6 Hz, 1H), 7.15 (d, J = 2.0 Hz, 1H),
7.09 (dd, J = 8.4, 1.7 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 4.20 (m,
1H), 2.95 (dd, J = 15.4, 3.8 Hz, 1H), 2.53 (dd, J = 15.4, 10.4 Hz,
1H), 1.31 (d, J = 6.4 Hz, 3H), 1.21 (d, J = 6.2 Hz, 1H). MS (ESI)
m/z 372.1 (M)þ; [R]D25 = þ285.4ꢀ (c = 0.101 g/L, methanol).
(1S,3R)-50,7-Dichloro-3-methyl-2,3,4,9-tetrahydrospiro[β-carbo-
line-1,30-indol]-20(10H)-one (18b). 1H NMR (500 MHz, CDCl3): δ
7.91 (bs, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.35 (bs, 1H), 7.27 (dd, J =
8.4, 2.1 Hz, 1H), 7.17 (d, J = 1.6 Hz, 1H), 7.14 (d, J = 2.0 Hz, 1H),
7.09 (dd, J = 8.4, 1.7 Hz, 1H), 6.86 (d, J = 8.3 Hz, 1H), 4.21 (m,
1H), 2.95 (dd, J = 15.4, 3.8 Hz, 1H), 2.39 (dd, J = 15.4, 10.4 Hz,
1H), 1.31 (d, J = 6.4 Hz, 3H), 1.21 (d, J = 6.2 Hz, 1H). MS (ESI)
m/z 372.1 (M)þ; [R]D25 = -258.9ꢀ (c = 0.104 g/L, methanol).
50,7-Dichloro-6-fluoro-3-methyl-2,3,4,9-tetrahydrospiro[β-carbo-
line-1,30-indol]-20(10H)-one (19). 1H NMR (500 MHz, DMSO-d6):
δ 10.69 (s, 1H), 10.51 (s,1H), 7.43 (d, J = 10.0 Hz, 1H), 7.32 (bd,
J = 8.3 Hz, 1H), 7.26 (d, J = 6.5 Hz, 1H), 7.04 (bs, 1H), 6.93 (d,
J = 8.3 Hz, 1H), 3.91 (m, 1H), 3.12 (bd, J = 5.5 Hz, 1H), 2.77
(bd, J = 14.2 Hz, 1H), 2.38 (dd, J = 14.2, 10.9 Hz, 1H), 1.16 (d,
J = 6.5 Hz, 1H). MS (ESI) m/z 390.0 (M)þ.
(1R,3S)-50,7-Dichloro-6-fluoro-3-methyl-2,3,4,9-tetrahydrospiro-
[β-carboline-1,30-indol]-20(10H)-one (19a). 1H NMR (500 MHz,
DMSO-d6): δ 10.69 (s, 1H), 10.51 (s, 1H), 7.43 (d, J = 10.0 Hz,
1H), 7.33 (dd, J = 8.0, 2.2 Hz, 1H), 7.27 (d, J = 6.5 Hz, 1H), 7.05
(d, J = 2.3 Hz, 1H), 6.93 (d, J = 8.5 Hz, 1H), 3.91 (m, 1H), 3.13
(bd, J = 6.2 Hz, 1H), 2.74 (dd, J = 15.0, 3.0 Hz, 1H), 2.35 (dd, J =
15.0, 10.3 Hz, 1H), 1.15 (d, J = 6.0 Hz, 3H). MS (ESI) m/z 392.0
(M þ 2H)þ; [R]2D5 = þ255.4ꢀ (c = 0.102 g/L, methanol).
(1S,3R)-50,7-Dichloro-6-fluoro-3-methyl-2,3,4,9-tetrahydrospiro-
[β-carboline-1,30-indol]-20(10H)-one (19b). 1H NMR (500 MHz,
DMSO-d6): δ 10.70 (s, 1H), 10.51 (s, 1H), 7.44 (d, J = 9.7 Hz,
1H), 7.34 (dd, J = 8.6, 2.0 Hz, 1H), 7.27 (d, J = 6.0 Hz, 1H), 7.05
(d, J = 2.0 Hz, 1H), 6.94 (d, J = 8.5 Hz, 1H), 3.91 (m, 1H), 3.13
(bd, J = 6.2 Hz, 1H), 2.77 (dd, J = 15.0, 3.5 Hz, 1H), 2.39 (dd, J =
15.5, 10.5 Hz, 1H), 1.17 (d, J = 6.0 Hz, 3H). MS (ESI) m/z 392.0
(M þ 2H)þ; [R]2D5 = -238.9ꢀ (c = 0.134 g/L, methanol).
Acknowledgment. WethankHui Qing Ang, Kang MinLow,
Augustin Notedji, William Tan, Maria Tan, Jeanette Wu,

5164 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Yeung et al.
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