Indol-2-yl ethanones as novel indoleamine 2,3-dioxygenase (IDO) inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.12.032

Indoleamine 2,3-dioxygenase (IDO) is a heme dioxygenase which has been shown to be involved in the pathological immune escape of diseases such as cancer. The synthesis and structure-activity relationships (SAR) of a novel series of IDO inhibitors based on the indol-2-yl ethanone scaffold is described. In vitro and in vivo biological activities have been evaluated, leading to compounds with IC₅₀ values in the micromolar range in both tests. Introduction of small substituents in the 5- and 6-positions of the indole ring, indole N-methylation and variations of the aromatic side chain are all well tolerated. An iron coordinating group on the linker is a prerequisite for biological activity, thus corroborating the virtual screening results.

Full text of DOI:10.1016/j.bmc.2010.12.032

Bioorganic & Medicinal Chemistry 19 (2011) 1550–1561
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Indol-2-yl ethanones as novel indoleamine 2,3-dioxygenase (IDO) inhibitors
a
b
c
d
a
´
Eduard Dolušic , Pierre Larrieu , Sébastien Blanc , Frédéric Sapunaric , Bernadette Norberg ,
Laurence Moineaux a, Delphine Colette c, Vincent Stroobant b, Luc Pilotte b, Didier Colau b, Thierry Ferain c,
Graeme Fraser c, Moreno Galeni d, Jean-Marie Frère d, Bernard Masereel a, Benoît Van den Eynde b,
Johan Wouters a, Raphaël Frédérick a,
⇑
a Drug design and Discovery Center, University of Namur, 61 Rue de Bruxelles, 5000 Namur, Belgium
b Ludwig Institute for Cancer Research, Université Catholique de Louvain, 74 Avenue Hippocrate, 1200 Brussels, Belgium
c Euroscreen SA, 47 Rue Adrienne Boland, 6041 Gosselies, Belgium
d Centre d’Ingénierie des Protéines, Université de Liège, Allée du 6 août, 4000 Liège, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Indoleamine 2,3-dioxygenase (IDO) is a heme dioxygenase which has been shown to be involved in the
pathological immune escape of diseases such as cancer. The synthesis and structure–activity relationships
(SAR) of a novel series of IDO inhibitors based on the indol-2-yl ethanone scaffold is described. In vitro and
in vivo biological activities have been evaluated, leading to compounds with IC50 values in the micromolar
range in both tests. Introduction of small substituents in the 5- and 6-positions of the indole ring, indole
N-methylation and variations of the aromatic side chain are all well tolerated. An iron coordinating group
on the linker is a prerequisite for biological activity, thus corroborating the virtual screening results.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 1 October 2010
Revised 7 December 2010
Accepted 13 December 2010
Available online 16 December 2010
Keywords:
Indoleamine 2,3-dioxygenase
IDO
Anticancer
Immunotherapy
1. Introduction
By depleting Trp locally, IDO blocks the proliferation of T lym-
phocytes, which are sensitive to Trp shortage.8 The downstream
Immunotherapy is
a
promising novel strategy for cancer
catabolites of the kynurenine pathway exacerbate this immuno-
suppressive effect1 and are also involved in other pathological
conditions, particularly in the nervous system.9–11
treatment. It consists of therapeutic vaccination of cancer patients
in order to stimulate their (natural) immune system against cancer
cells. This approach, however, showed limited efficacy in vivo. In-
deed, cancer cells are able to develop enzymatic mechanisms
allowing tumors to resist or escape immune rejection. Among the
enzymes involved, the indoleamine 2,3-dioxygenase (IDO; EC
1.13.11.52) represents a crucial actor.1,2
Many human tumors constitutively express IDO.12 It has been
shown that an increased level of IDO expression in tumor cells is
correlated with poor prognosis for survival in cancer.13,14 This led
to the hypothesis that IDO inhibition might enhance the efficacy
of cancer treatments. Indeed, results from in vitro and in vivo stud-
ies have suggested an improvement of the efficacy of therapeutic
vaccination or chemotherapy by concomitant administration of
an IDO inhibitor thus highlighting IDO as an attractive tar-
get.12,13,15–17
IDO is a ꢀ45 kDa monomeric extrahepatic cytosolic heme
dioxygenase which catalyzes the initial and rate-limiting step in
the catabolism of the essential amino acid tryptophan (Trp) along
the kynurenine pathway, the de novo biosynthetic route leading to
nicotinamide adenine dinucleotide (NAD).3,4 IDO is
a
more
Until recently, the best known IDO inhibitors18 displayed
affinities in the micromolar range and comprised mainly Trp
promiscuous enzyme than the catalytically related hepatic enzyme
tryptophan 2,3-dioxygenase (TDO; EC 1.13.11.11). Apart from both
tryptophan enantiomers, IDO substrates are indoleamines such as
serotonin, melatonin, and tryptamine.5 In order for IDO to be catalyt-
ically active, it is essential to maintain the heme iron ion in the fer-
rous (Fe2+) state. As the enzyme is susceptible to autoxidation, a
reductant is necessary for preserving the enzymatic activity.
Cytochrome b5 has been demonstrated to play this role in vivo.6,7
derivatives such as 1-methyltryptophan (1MT)19 (Ki = 37
lM) or
b-carbolines (Ki ꢀ0.12 mM).20 Other indole-based IDO inhibi-
tors, such as brassinin21,22 and methylthiohydantoin-tryptophan
(MTH-Trp)13 have been described. The best inhibitor of the brassinin
class has a Ki of 12 lM but is not Lipinski-compliant due to high
hydrophobicity.22
In 2006, potent IDO inhibitors were isolated from marine inver-
tebrate extracts,23,24 leading to two new series of uncompetitive
nanomolar inhibitors.26,27 However, some of the most active
compounds of the original series revealed to be inactive in a
⇑
Corresponding author. Tel.: +32 81 72 42 90; fax: +32 81 72 42 38.
E-mail address: Raphael.frederick@fundp.ac.be (R. Frédérick).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.032

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E. Dolušic et al. / Bioorg. Med. Chem. 19 (2011) 1550–1561
yeast-based cellular assay, suggesting poor cell penetration.7 This
test nevertheless provided two new micromolar inhibitors with
unknown modes of action.
groups in order to explain substrate binding as well as interaction
of 1MT. In this position, the 3-pyridyl group is projected towards
the entrance of the active site and is stabilized in the aromatic
Pocket B through T-shape interactions with Phe163 and Phe226
(Fig. 1).
In 1989, 4-phenylimidazole (PIM) was identified as a modestly
potent IDO inhibitor.4,19 Despite the uncompetitive inhibition
kinetics, the authors showed through spectroscopic studies that
PIM binds into the active site of IDO. Moreover, they discovered
a preferred binding of PIM to the inactive ferric (Fe3+) form of the
enzyme, which may explain this apparent discrepancy. The first
crystal structure of human IDO complexed with the inhibitor
reported later25 confirmed the binding of PIM in the active site. A
recently undertaken structure-based study26 of PIM-derived mole-
cules prompted by Sugimoto’s work confirmed the antecedent
findings while providing access to novel inhibitors with low micro-
molar potencies.
Our structural modifications of 7a were focused on exploiting
three binding interactions with IDO: (1) the interior of the active
site, in particular interactions with C129 and S167 in Pocket A;
(2) the active site entrance region (Pocket B) occupied by the
N-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer molecule
in a crystal structure;25 (3) the heme iron binding group. According
to the binding orientation of 7a we hoped to achieve interactions in
the interior region of the active site (Pocket A) by introducing sub-
stituents on the indole ring. The substituents were chosen with a
view to balance on one side the lipophilic character of this Pocket
introducing groups such as fluorine, bromine, chlorine, methyl;
and, on the other side the possibility of forming H-bond interac-
tions with specific amino acids with such substituents as hydroxyl,
methoxy, nitro, or trifluoromethoxy. The interaction within Pocket
B would be probed by replacement of the 3-pyridyl group of 7a
with other (hetero)aromatics. The heme iron binding interaction
would be explored by replacement of the ethanone moiety with
other linkers between the indole and the aromatic side chain.
Recent developments in the field include hypoxic-neoplastic
cells-targeting 1MT-tirapazamine hybrids,27 novel inhibitors ob-
tained from marine sediments (IC50 ꢁ 2
l
M),28 potent competitive
inhibitors including a hydroxyamidine chelating motif (IC50 values
down to 60 nM),29 strong inhibition by modification of cysteine
residues by ebselen,30 sub-micromolar S-benzylisothiourea-based
inhibitors active in cells,31 various potent inhibitors (IC50 values
down to 0.2 lM) discovered by a combination of a docking-based
pharmacophore model development and fragment-based drug de-
sign (FBDD).32
2. Chemistry
Despite all these efforts, modestly potent 1MT (Ki = 34 l
M)19
remains the only compound to have been passed into clinical trials
as an IDO inhibitor so far.33 This prompted us to search for new
scaffolds that could be further optimized with the objective of
identifying potent and selective IDO inhibitors. Briefly, a collection
of ꢀ62,000 structures was downloaded from the ZINC website
using Lipinski-style rules for fragments (molecular weight ꢂ 250,
Log P ꢂ 2.5, number of rotatable bonds ꢂ 5).34 These compounds
were docked inside the IDO active site and ranked according to
their best potential binding. From the 39 compounds that were se-
lected, bought and assayed on IDO, six derivatives displayed an
Initial efforts to prepare analogues of compound 7a included at-
tempts to react indole-2-acyl chlorides (Scheme 1, 3, R00 = Cl) with
reagents derived from 3-bromomethylpyridine hydrobromide un-
der various conditions.36–38 In fact, all of these approaches revealed
to be unsatisfactory, the main difficulty likely being the intrinsic
instability of the in situ generated free base 3-bromomethylpyri-
dine which readily oligomerizes.39 The only strategy allowing ac-
cess to 7a analogues was the approach originally developed by
Sundberg et al., as outlined in Scheme 1. This method involves
the LDA effected deprotonation of alkylarene 1 followed by
quenching of the in situ generated carbanion with a reactive indole
carbonyl compound (3 and 6) at 0 °C. Stirring overnight while
slowly attaining room temperature followed by aqueous work-
up, chromatographic purification and crystallization provided the
targeted compounds.
inhibitory potency >30% at a concentration of 100 lM. Among
these, compound 7a, characterized by an indole nucleus substi-
tuted in the 2-position by a 2-pyridinyl-ethanone function, was
our best hit (IDO IC50 ꢀ65
lM), with an inhibitory potency in the
same range as the reference compound 1MT. (Table 1). Herein
we report the synthesis and structure–activity study of compound
7a.
An initial docking study of 7a within the IDO binding cleft was
performed by means of the automated GOLD program35 and re-
vealed essential features stabilizing 7a in the IDO binding cleft
and particularly that (i) the oxygen atom of the ketone function,
being situated ꢀ2 Å above the plane of the heme, coordinates to
the heme iron, (ii) the indole ring is found to be stabilized in the
so-called lipophilic A-pocket of IDO.32 Indeed, this stabilization of
the indole residue in Pocket A was already suggested by other
Table 1
Inhibition of IDO activity by 1MT and 7a identified using VS
Compound
Structure
IC50 (lM)
Me
N
O
1MT
100 20
OH
NH2
H
N
7a
65
7
N
Figure 1. View of the lead compound 7a inside the IDO binding cleft. (Picture made
O
with Pymol34).

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E. Dolušic et al. / Bioorg. Med. Chem. 19 (2011) 1550–1561
1552
Scheme 1. General synthetic scheme for indol-2-yl ethanones. Reagents and conditions: (i) LDA, THF/hexanes, ꢃ78–0 °C, 1 h; (ii) SOCl2,
rt, 16–24 h; (iv) N3CH2CO2Et, NaOEt, EtOH, ꢃ10–4 °C, 1.5–20 h; (v) hexane, 200 °C ( W), 5 min; (vi) MeI, K2CO3, DMF, 80 °C, 16 h to 5 days; (vii) AlCl3, CH2Cl2, 0 °C to rt, 24 h;
(viii) HCO2NH4, Pd black, MeOH, rt, 1 h; (ix) H2 (1 atm), 3% Pd/C, EtOH, rt, 45 min; (x) PhB(OH)2, Pd(PPh3)4, K2CO3, EtOH/toluene 1:1, , 20 h.
D, 15 min; (iii) THF/hexanes, 0 °C to
l
D
Accordingly, most of the compounds 7 as well as 8a, 9a, 10a,
and 11a were prepared from indole esters (3, R00 = OMe, OEt) or
acyl chlorides (3, R00 = Cl) when these starting material were com-
mercially available. Otherwise, the necessary indole acyl chloride
The catalytic hydrogenation of 7a afforded the hydroxyl
derivative 18 (Scheme 2). Compound 19 bearing an ethylene linker
between the two aromatic rings was prepared from 7a by a
3
was prepared by reaction of the commercially available
indole-2-carboxylic acid 2 in refluxing thionyl chloride. Non-com-
mercially available indole esters 3 were made using the Hemets-
berger–Knittel procedure starting from the corresponding
a-azidocinnamates 5, which were in turn made by condensation
of the substituted benzaldehydes 4 with ethyl azidoacetate under
Knoevenagel conditions (Scheme 1).40,41 It should be noted that
compound 11a (Scheme 1) was prepared in 37% yield using com-
mercial Grignard reagent in lieu of the LDA procedure. Moreover,
during the course of this reaction, the tertiary alcohol 16 (inactive
in the biological tests; data not shown) was also isolated in 17%
yield.
Palladium catalyzed reductions were used to convert benzyloxy-
substituted 2-ketoindoles 7n and 7r to their debenzylated counter-
parts 7o and 7s, respectively. Bis-alcohol 17 was also isolated as a
byproduct during the catalytic reduction of 7n. The 5-phenylindole
derivative 7l was prepared from the corresponding 5-bromo ana-
logue 7h by a Suzuki cross-coupling with phenylboronic acid
(Scheme 1). Finally, after initial unsuccessful attempts to demethy-
late 8d with boron tribromide (which seemed to cause product
decomposition), the 4-hydroxyindole 8e was obtained by reaction
of 8d with aluminum trichloride in dichloromethane.
Scheme 2. Synthesis of compounds bearing different linker groups. Reagents and
conditions: (i) HCO2NH4, Pd black, MeOH, rt, 3 days; (ii) H2NNH2, KOH, (CH2OH)2,
For the synthesis of the N-methylated analogue 12a, the required
N-methylated indole ester 6 was synthesized by dimethylation of
N-unsubstituted indole-2-carboxylic acid 2 (Scheme 1).42
l
W, 1 h then aq NH4Cl; (iii) NH2OHꢄHCl, pyridine, EtOH,
lW (120 °C), 30 min; (iv)
SOCl2, , 15 min then 3-aminopyridine, DIPEA, THF, 0 °C?rt, 30 min.
D

´
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E. Dolušic et al. / Bioorg. Med. Chem. 19 (2011) 1550–1561
Table 2
Wolff–Kishner reduction. Oxime 20 was obtained by reacting
IDO inhibitory potency of compounds 7a–u, 12a
ketone 7a with hydroxylamine. Finally, amide 21 was made from
acid 2 by reaction with thionyl chloride followed by nucleophilic
substitution with 3-aminopyridine (Scheme 2).
R
1
7
6
R
N
2
N
The structure of all compounds was confirmed by means of 1H,
13C and (where applicable) 19F NMR, LC–MS, and elemental analy-
sis (see Section 5). In addition, single crystal X-ray diffraction was
used to corroborate the structures obtained in these series. The
molecular structures of compounds 7a and 7m with displacement
ellipsoids drawn at the 50% probability levels are depicted in
Figure 2. It should be noted that for 7m thermal agitation of the
trifluoromethoxy side chain led to large displacement ellipsoids
for the fluorine atoms.
5
O
3
4
Compound
R
R1
IDO IC50
(
l
M)
IDO cell assay
inhibition % @ 20 lM
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7o
7p
7q
7r
H
3-Br
4-F
4-MeO
4-OH
5-F
5-Cl
5-Br
5-Me
5-OMe
5-NO2
5-Ph
5-CF3O
5-OH
6-F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
65
NI
153 23
58
83 18
36.0 0.1
24.6 0.1
18
7
13
NI*
12
NI
5
NI
24
24
NI
21
NT⁄⁄
NI
NI
12
NI
NI
6
NI
11
NI
1
3. Results
87 11
49
NI
96
1
1
3.1. IDO inhibition through interactions in the interior of the
active site (Pocket A)
13.1 0.1
37
43
82
NI
63
139 12
45
34
3
7
5
Detailed analysis of the binding conformation of 7a inside the
IDO binding cleft suggests that introduction of small substituents
around the indole scaffold could be beneficial for IDO inhibition.
7-MeO
4-BnO, 5-MeO
4-OH, 5-MeO
4,6-DiCl
4,6-DiMeO
H
Starting from our hit 7a (IC50 = 65
l
M), we first explored the
7s
7t
7u
12a
3
influence of substitution around the indole ring. From Table 2 it
can be observed that introduction of substituents in the 3- (7b),
4- (7c–e), and 7-position (7q) does not generally afford more
potent compounds than the hit 7a. Substitutions in the 5- and
6-positions are well tolerated and more potent compounds are
obtained. Particularly, introducing a trifluoromethoxy group on
the 5-position of the indole ring led to a fivefold improvement in
the IDO inhibitory potency. Regarding the disubstitution, introduc-
tions of substituents in the 4,5- or the 4,6-positions do not drasti-
cally affect IDO inhibition. Finally, N-methylation of the indole
nucleus seems to be tolerated as 12a is in vitro twofold more po-
tent than parent compound 7a.
8
2
Toxic
NI
Me
⁄⁄
*
NT = not tested.
NI = no inhibition.
Table 3
IDO inhibitory potency of compounds 7a–11a
H
N
Ar
O
Compound
Ar
IDO IC50
(
lM)
IDO cell assay
inhibition % @ 20 lM
3.2. IDO inhibition through interactions with Pocket B of the
active site
*
*
7a
65
7
13
We next explored the influence of modifying the fragment
interacting with Pocket B of the IDO active site. From Table 3, it
can be observed that introductions of an unsubstituted phenyl
(11a), a 2-pyridyl (8a) or a 2-pyrazinyl group (10a) in lieu of the
3-pyridyl moiety of 7a slightly improve the IDO inhibitory potency
N
8a
37 15
NI
10
NI*
NI
N
*
9a
N
N
(IC50’s of 29, 37, and 26 lM, respectively). On the contrary, intro-
duction of a 4-pyridyl moiety (9a) afforded an inactive compound.
*
10a
26.2 0.2
29 26
N
3.3. IDO inhibition through interaction with the heme iron
binding group
*
11a
NI
Finally we appraised the importance of the ketone moiety of
compound 7a in establishing an interaction with the heme iron. In-
deed, reduction of this group (18) or its removal altogether (19) led
to inactive compounds thus confirming the requirement for an iron
coordinating group (Table 4). Oxime 20 is endowed with an in vivo
*
NI = no inhibition.
inhibitory potency similar to that of ketone 7a but surprisingly,
this compound is not active in vitro. This might result from a poor
stability of the oxime moiety in the experimental conditions of the
assay. Introducing a methyl group in the ethanone linker (7v) or its
replacement with an amide (21) afforded less potent compounds.
4. Discussion and conclusion
About 30 original compounds were synthesized around the
indol-2-yl ethanone scaffold and their in vivo and in vitro
inhibitory potency against indoleamine 2,3-dioxygenase (IDO)
was evaluated. Starting from the hit (1H-indol-2-yl)-2-pyridin-3-
Figure 2. The molecular structures of (a) 7a and (b) 7m. Displacement ellipsoids
are drawn at the 50% probability levels. (Pictures made with MERCURY 2.3).

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E. Dolušic et al. / Bioorg. Med. Chem. 19 (2011) 1550–1561
1554
Table 4
IDO inhibitory potency of compounds 7a, 7v, 18–21
Compound
Structure
IDO IC50
(
l
M)
IDO cell assay inhibition % @ 20
13
lM
O
7a
65
7
N
H
N
N
O
7v
141
NI
4
NI
N
H
Me
OH
18
19
20
NI
NI
25
NI
N
H
N
N
NI
N
H
NOH
O
19.4% @ 100 lM
N
H
N
21
94
2
N
H
HN
N
NI = no inhibition.
yl-ethanone (7a) identified through a virtual screening procedure
and possessing an IC50 of 65 M in the in vitro test, a fivefold
improvement in potency (IC50 = 13 M) could be achieved by
ies on a Büchi B-540 capillary melting point apparatus and are
uncorrected.
Thin layer chromatography (TLC) was performed on silica gel
plates (60F254, 0.2 mm thick, Merck) eluting with gradients of ethyl
acetate in cyclohexane. The spots were visualized by ultraviolet
light (wavelengths 254 and 366 nm).
l
l
introducing a trifluoromethoxy group in the 5-position of the in-
dole nucleus. Most small substituents around the indole moiety
led to compounds of roughly equivalent potency, indicating room
for further modifications but also a probable lack of close interac-
tions of the inhibitor molecules with the IDO Pocket A interior as
described by Rohrig et al.32 A similar generalization can be applied
to the other aromatic moiety and its putative interactions with
Pocket B of the IDO. Altogether, it seems no strong H-bond interac-
tions with any of the hydrogen bonding residues in the IDO active
site could be established. The presence of an iron chelating group
seems to be of crucial importance, since its deletion leads to loss
of potency.
1H NMR spectra were recorded in DMSO-d6 solutions on a Jeol
JNM EX 400 machine at 400 MHz with tetramethylsilane (TMS) as
internal standard. 13C and 19F NMR spectra were recorded on the
same machine in DMSO-d6 solutions at 100 MHz and 376.2 MHz,
respectively. Chemical shifts (d) are reported in ppm downfield
from TMS.
Elemental analyses (C, H, N, S) were performed on a Thermo
Finnigan FlashEA 1112 apparatus.
Analytical LC–MS analyses were performed on an Agilent 1100
Series LC–MSD Trap system using UV detection at 254 and 366 nm.
MS data were recorded using electrospray ionization (ESI) operat-
ing in positive mode. The following two methods were applied:
A number of compounds investigated here possess a moderate
in vivo activity. The compounds 7f and 7g (with small halogen
atoms in the 5-position of the indole moiety) are both endowed
with 24% IDO inhibition at a 20
assay, which indicates reasonable cell penetration. A very similar
value was obtained for oxime 20.
In summary, a novel series of compounds with a moderate
inhibitory activity against IDO has been identified. Both in vitro
and in vivo assays show promising results. Further modifications
aiming to achieve more favorable interactions with the enzyme ac-
tive site are in due course.
l
M concentration in the cellular
method 1: injection of 10
onto a C18-3.5
using a gradient (flow rate: 0.5 mL/min) of acetonitrile in acetic
acid 0.1% (v/v) in water: 5–95% acetonitrile during 5 min, holding
for 3 min, then reversing to 5% acetonitrile during 0.1 min and
ll of a 20 lg/mL acetonitrile solution
m Zorbax SB column (100 ꢅ 3 mm); separation
l
holding for an additional 5.4 min. Method 2: injection of 10
ll of
a 2 g/mL acetonitrile solution onto a C18-5 m Agilent LiChro-
l
l
spher 100 column (250 ꢅ 4 mm); separation using a gradient (flow
rate: 1.0 mL/min) of acetonitrile in acetic acid 0.1% (v/v) in water:
20–80% acetonitrile during 20 min, holding for 20 min, then
reversing to 20% acetonitrile during 0.1 min and holding for an
additional 5 min.
Automated flash chromatography was performed on the Biotage
AB (Uppsala, Sweden) SP1 system equipped with pre-packed flash
KP-Sil silica cartridges eluting with gradients of ethyl acetate in
cyclohexane. The peaks were detected by UV absorption at 254
and 320 nm.
5. Experimental part
5.1. Chemistry
5.1.1. General experimental procedures
Chemicals were purchased from commercial suppliers (Acros,
Sigma–Aldrich, Maybridge, Apollo) and used without further
purification. All solvents were used as purchased from Acros,
Sigma–Aldrich, Fisher Scientific and Biosolve. All reactions were
performed under an inert argon (Alphagaz 2) atmosphere, unless
stated otherwise. Melting points were determined in open capillar-
The yields refer to combined yields of chromatographically and
spectroscopically pure (>95%) compounds and the crops obtained
by evaporation of mother liquors. All new compounds were deter-
mined to be >95% pure by LC–MS and NMR.

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E. Dolušic et al. / Bioorg. Med. Chem. 19 (2011) 1550–1561
The microwave-assisted syntheses were carried out in an Initi-
ator 16 single-mode microwave instrument producing controlled
irradiation at 2.450 GHz (Biotage AB). Reaction times refer to hold
times at the temperatures indicated, not to total irradiation times.
The temperature was measured with an IR sensor on the outside of
the reaction vessel.
propylamine (4.0 equiv). The solution was cooled to ꢃ78 °C in a dry
ice/isopropanol bath and n-butyl lithium, 1.6 M solution in
hexanes (4.0 equiv), was added to give a bright yellow solution.
The mixture was stirred for 30 min below ꢃ60 °C whereupon a
solution of picoline or another appropriate reagent with an ioniz-
able methyl(ene) group (4.0 equiv) in THF (2 mL/mmol of starting
indole 3 or 6) was added to give a yellow to red colored mixture.
During a further 30 min, the bath temperature was allowed to rise
to approx. 0 °C during which time the reaction mixture became a
yellow to red suspension. The temperature was then kept at 0 °C
(using an ice/water bath) for an additional 30 min. At this point,
a solution of the indole-2-carbonyl compound 3/6 (1.0 equiv) in
THF (4 mL/mmol) was added dropwise over the flask wall. The
reaction mixture darkened (dark purple to dark brown color in
most cases) and was then allowed to attain room temperature
overnight. At 18 h (unless otherwise stated) after the addition of
the indole component, the reaction was quenched with saturated
aqueous ammonium chloride solution (10 mL/mmol of starting
compound 3/6) and stirred for several minutes. Upon partition be-
tween ethyl acetate and water, the aqueous phase was extracted
twice with dichloromethane and the combined organic layers were
dried over MgSO4. The title products were purified by chromatog-
raphy (the products typically eluted with 70–80% of ethyl acetate).
The products were then precipitated by concentration of the
pooled column fractions combined with the addition of cyclohex-
ane. The precipitates formed were filtered off, washed twice with
cyclohexane and dried at 40 °C in vacuo to yield analytically pure
samples. The following compounds were prepared in this way:
5.1.2. Methods for preparation of indolic intermediates (3 and
6)
5.1.2.1. Indole-2-acyl chlorides (3, R00 = Cl). Commercial indole-
2-carboxylic acid was dissolved in thionyl chloride (ꢀ1.5 mL/
mmol) and the suspension was boiled for 15 min, during which
time typically a dark green solution was formed. Excess thionyl
chloride was removed under reduced pressure and the solid residue
was suspended in toluene and the suspension was evaporated to
dryness (toluene treatment was repeated three times). The green-
grayish solid residues were used without further purification.
5.1.2.2. Indolic esters by Hemetsberger–Knittel synthesis (3,
R00 = OEt)40,41. Sodium (4.0 equiv) was dissolved in absolute etha-
nol (0.5 mL/mmol) and the solution was cooled to ꢃ10 °C in an ice/
acetone bath. A corresponding benzaldehyde (1.0 equiv) was dis-
solved in a commercial 25% solution of ethyl azidoacetate in etha-
nol (2.1 g per mmol of starting benzaldehyde, 4.0 equiv) and this
solution was added dropwise to the sodium ethoxide solution dur-
ing 30 min while the bath temperature was kept at ꢃ10 °C. The
reaction mixtures typically turned into bright yellow suspensions.
The stirring was continued until TLC (developing with 15% EtOAc
in cyclohexane) indicated completion or the reaction (1.5–2.5 h).
If the reaction required more than 1.5 h, the temperature was al-
lowed to rise to 0 °C. In one case (5r), the reaction had to be run
overnight. The mixtures were partitioned between ethyl acetate
and water. The aqueous phase was extracted twice with ethyl ace-
tate and the organic layers were pooled, washed with brine, dried
over magnesium sulfate, and concentrated under reduced pressure
to provide crude azidoacrylic esters (5) as yellow to orange oils or
liquids in a 46–100% yield. These products were suspended in
n-hexane (6–9 mL/mmol of crude azidoacrylic ester) and subjected
to microwave heating at 200 °C for 5 min (P = 100–140 W, p = 12–
13 bar). The resulting residues were taken into ethyl acetate and
evaporated with silica gel. The corresponding indolic esters 3
(yields 19–93%) were purified by chromatography. In one case, a
mixture of isomers was obtained. 2-Azido-3-(3-methoxyphenyl)-
acrylic acid ethyl ester gave 5-methoxy-1H-indole-2-carboxylic
acid ethyl ester (3j) and 7-methoxy-1H-indole-2-carboxylic acid
ethyl ester (3q) in a 1:1 ratio (each in a 47% yield).
5.1.3.1. 1-(1H-Indol-2-yl)-2-pyridin-3-yl-ethanone (7a)43–45
.
Prepared from commercial 1H-indole-2-carboxylic acid ethyl ester
3a (986 mg, 5.05 mmol) and 3-picoline. Yellowish solid (699 mg,
59%), mp 177–180 °C. LC–MS Rt 5.2 min (method 1), 7.0 min
(method 2); m/z [MH+] 237. 1H NMR d 11.76 (1H, br s, indole-
H1), 8.53 (1H, d, J = 1.7 Hz, ArH), 8.43 (1H, dd, J = 4.8, 1.7 Hz,
ArH), 7.71 (1H, d, J = 7.8 Hz, ArH), 7.69 (1H, d, J = 7.8 Hz, ArH),
7.56 (1H, d, J = 1.8 Hz, ArH), 7.41 (1H, d, J = 8.2 Hz, ArH), 7.33
(1H, dd, J = 7.8, 4.8 Hz, ArH), 7.26 (1H, t, J = 7.6 Hz, ArH), 7.06
(1H, t, J = 7.6 Hz, ArH), 4.36 (2H, s, CH2). 13C NMR d 190.1 (C@O),
151.1 (ArC), 148.3 (ArC), 138.6 (ArC), 137.8 (ArC), 135.4 (ArC),
131.6 (ArC), 127.4 (ArC), 126.2 (ArC), 124.0 (ArC), 123.3 (ArC),
120.9 (ArC), 113.3 (ArC), 110.7 (ArC), 42.0 (CH2). Anal. Calcd for
C15H12N2O: C, 76.25; H, 5.12; N, 11.86. Found: C, 76.16; H, 5.13;
N, 11.87.
5.1.3.2. 1-(3-Bromo-1H-indol-2-yl)-2-pyridin-3-yl-ethanone (7b).
Prepared from commercial 3-bromo-1H-indole-2-carboxylic acid
(267 mg, 1.09 mmol) and 3-picoline via corresponding acyl chlo-
ride 3b (method A). Bright yellow solid (100 mg, 29%), mp 194–
195 °C (dec). LC–MS Rt 6.0 min (method 1), 12.8 min (method 2);
m/z [MH+] 315; 317. 1H NMR d 12.19 (1H, br s, indole-H1), 8.49
(1H, d, J = 1.8 Hz, ArH), 8.45 (1H, dd, J = 4.8 Hz, 1.6, ArH), 7.69
(1H, d, J = 7.8 Hz, ArH), 7.57 (1H, d, J = 8.0 Hz, ArH), 7.49 (1H, d,
J = 8.2 Hz, ArH), 7.38–7.33 (2H, overlapped, 2 ArH), 7.19 (1H, t,
J = 7.6 Hz, ArH), 4.53 (2H, s, CH2). 13C NMR d 189.3 (C@O), 151.4
(ArC), 148.4 (ArC), 138.2 (ArC), 136.5 (ArC), 132.1 (ArC), 130.8
(ArC), 127.6 (ArC), 127.3 (ArC), 123.9 (ArC), 122.0 (ArC), 121.2
(ArC), 113.7 (ArC), 96.5 (ArC), 44.4 (CH2). Anal. Calcd for
5.1.2.3. N-Methylated indolic ester; methyl 1-methyl-1H-indole-
2-carboxylate (6). Prepared according to a literature procedure.42
Methyl iodide (5.0 mL, 80.0 mmol) was added to a stirred suspen-
sion of anhydrous potassium carbonate (6.3 g, 45.6 mol) and 1H-
indole-2-carboxylic acid (2; 5.3 mmol) in dry DMF (6 mL) and
the mixture was stirred at 80 °C until the reaction was complete
as indicated by TLC and/or LC–MS (3–5 days). Upon cooling to
room temperature, the suspension was partitioned between ethyl
acetate and water. The aqueous phase was extracted with ethyl
acetate and the combined organic layers were dried over MgSO4,
concentrated under reduced pressure and dried to yield the de-
sired N-methylated indole ester (6) as a beige solid in a 96–99%
yield.
C15H11BrN2O: C, 57.16; H, 3.52; N, 8.89. Found: C, 58.26; H, 3.57;
N, 8.48.
5.1.3. General synthetic procedure for 1-(1H-indol-2-yl)-2-aryl-
ketones (7–10, 12)43
An oven-dried flask was purged with argon while hot, then
allowed to cool down to room temperature under argon and
charged with dry THF (2 mL/mmol of starting indole 3/6) and diiso-
5.1.3.3. 1-(4-Fluoro-1H-indol-2-yl)-2-pyridin-3-yl-ethanone (7c).
Prepared from 4-fluoro-1H-indole-2-carboxylic acid ethyl ester 3c
(method B; 184 mg, 0.89 mmol) and 3-picoline. Yellow half-solid
(15 mg, 7%). LC–MS Rt 5.4 min (method 1); m/z [MH+] 255. 1H
NMR d 12.10 (1H, br s, indole-H1), 8.53 (1H, br s, ArH), 8.43 (1H,

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E. Dolušic et al. / Bioorg. Med. Chem. 19 (2011) 1550–1561
1556
d, J = 4.1 Hz, ArH), 7.73–7.65 (2H, overlapped, 2 ArH), 7.33 (1H, dd,
J = 7.2, 5.1 Hz, ArH), 7.24 (2H, overlapped, 2 ArH), 6.85 (1H, m,
ArH), 4.40 (2H, s, CH2). 13C NMR d 190.2 (C@O), 167.5 (ArC),
149.8 (d, JCF = 278 Hz, indole-C4), 137.8 (ArC), 135.7 (ArC), 132.2
(d, overlapped, ArC), 132.1 (ArC), 129.2 (ArC), 126.8 (d, JCF = 10 Hz,
ArC), 124.0 (ArC), 116.9 (d, JCF = 22 Hz, ArC), 109.9 (ArC), 106.0
(ArC), 105.0 (d, JCF = 18 Hz, ArC), 42.0 (CH2).
5.1.3.7.
1-(5-Chloro-1H-indol-2-yl)-2-pyridin-3-yl-ethanone
(7g). Prepared from commercial 5-chloro-1H-indole-2-carboxylic
acid (355 mg, 1.78 mmol) and 3-picoline via corresponding acyl
chloride 3g (method A). Yellow solid (137 mg, 29%), mp 206–
208 °C (dec). LC–MS Rt 5.8 min (method 1); m/z [MH+] 271. 1H
NMR d 11.97 (1H, br s, indole-H1), 8.51 (1H, br s, ArH), 8.43 (1H,
d, J = 4.8 Hz, ArH), 7.77 (1H, br s, ArH), 7.70 (1H, d, J = 7.8 Hz,
ArH), 7.52 (1H, br s, ArH), 7.43 (1H, d, J = 8.7 Hz, ArH), 7.33 (1H,
dd, J = 7.8, 4.8 Hz, ArH), 7.26 (1H, dd, J = 8.7, 1.8 Hz, ArH), 4.38
(2H, s, CH2). 13C NMR d 190.3 (C@O), 151.1 (ArC), 148.4 (ArC),
137.9 (ArC), 136.8 (ArC), 136.6 (ArC), 131.3 (ArC), 128.4 (ArC),
126.3 (ArC), 125.3 (ArC), 124.0 (ArC), 122.2 (ArC), 115.0 (ArC),
109.8 (ArC), 42.1 (CH2). Anal. Calcd for C15H11ClN2O: C, 66.55; H,
4.10; N, 10.35. Found: C, 67.48; H, 4.11; N, 9.99.
5.1.3.4. 1-(4-Methoxy-1H-indol-2-yl)-2-pyridin-3-yl-ethanone
(7d). Prepared from commercial 4-methoxy-1H-indole-2-carbox-
ylic acid methyl ester 3d (268 mg, 1.29 mmol) and 3-picoline.
White solid (238 mg, 69%), mp 159–160 °C (dec). LC–MS Rt
5.3 min (method 1); m/z [MH+] 267. 1H NMR d 11.75 (1H, br s, in-
dole-H1), 8.52 (1H, br s, ArH), 8.42 (1H, d, J = 4.9 Hz, ArH), 7.70 (1H,
d, J = 7.8 Hz, ArH), 7.57 (1H, br s, ArH), 7.32 (1H, dd, J = 7.6, 4.9 Hz,
ArH), 7.17 (1H, t, J = 8.1 Hz, ArH), 6.97 (1H, d, J = 8.1 Hz, ArH), 6.51
(1H, d, J = 7.6 Hz, ArH), 4.33 (2H, s, CH2), 3.88 (3H, s, OCH3). 13C
NMR d 189.7 (C@O), 154.9 (ArC), 151.1 (ArC), 148.3 (ArC), 139.9
(ArC), 137.7 (ArC), 134.3 (ArC), 131.8 (ArC), 127.5 (ArC), 123.9
(ArC), 118.8 (ArC), 108.3 (ArC), 106.1 (ArC), 100.0 (ArC), 55.6
(OCH3), 41.8 (CH2). Anal. Calcd for C16H14N2O2: C, 72.16; H, 5.30;
N, 10.52. Found: C, 72.36; H, 5.30; N, 10.36.
5.1.3.8. 1-(5-Bromo-1H-indol-2-yl)-2-pyridin-3-yl-ethanone (7h).
Prepared from commercial 5-bromo-1H-indole-2-carboxylic acid
(260 mg, 1.08 mmol) and 3-picoline via corresponding acyl chlo-
ride 3h (method A). White solid (79 mg, 23%), decomposes without
melting at 224–226 °C. LC–MS Rt 6.0 min (method 1), 13.3 min
(method 2); m/z [MH+] 315; 317. 1H NMR d 11.99 (1H, br s, in-
dole-H1), 8.51 (1H, d, J = 1.8 Hz, ArH), 8.43 (1H, dd, J = 4.8, 1.2 Hz,
ArH), 7.92 (1H, br s, ArH), 7.70 (1H, d, J = 7.8 Hz, ArH), 7.52 (1H,
d, J = 1.6 Hz, ArH), 7.37 (2H, overlapped, 2 ArH), 7.33 (1H, dd,
J = 7.8, 4.8 Hz, ArH), 4.38 (2H, s, CH2). 13C NMR d 190.3 (C@O),
151.2 (ArC), 148.4 (ArC), 137.9 (ArC), 137.0 (ArC), 136.4 (ArC),
131.3 (ArC), 129.1 (ArC), 128.7 (ArC), 125.3 (ArC), 124.0 (ArC),
115.4 (ArC), 113.2 (ArC), 109.6 (ArC), 42.1 (CH2). Anal. Calcd for
5.1.3.5. 1-(4-Hydroxy-1H-indol-2-yl)-2-pyridin-3-yl-ethanone
(7e). A solution of methoxyindole 7d (143 mg, 0.54 mmol) in
dry dichloromethane (5 mL) was cooled to 0 °C and anhydrous alu-
minum chloride (247 mg, 1.84 mmol) was added to give a yellow
suspension. Room temperature was slowly attained while the color
of the reaction mixture gradually changed to dark red. After 19 h,
as TLC still indicated presence of the starting compound, another
aliquot of aluminum chloride and 2 mL of dichloromethane were
added. At 24 h of total reaction time (most of the starting com-
pound still unreacted), the mixture was partitioned between ethyl
acetate and water. The aqueous phase was extracted with ethyl
acetate and the combined organic layers were washed with brine
and dried over magnesium sulfate. Chromatographic purification
(the title product eluted with 95–100% ethyl acetate) yielded alco-
hol ed 140-3 as an off-white solid (11 mg, 8%). LC–MS Rt 4.3 min
(method 1); m/z [MH+] 253. 1H NMR d 11.59 (1H, br s, indole-
H1), 9.88 (1H, br s, OH), 8.52 (1H, d, J = 1.8 Hz, ArH), 8.42 (1H,
dd, J = 4.6, 1.4 Hz, ArH), 7.70 (1H, d, J = 7.6 Hz, ArH), 7.57 (1H, d,
J = 1.6 Hz, ArH), 7.32 (1H, dd, J = 7.3, 4.6 Hz, ArH), 7.03 (1H, t,
J = 8.0 Hz, ArH), 6.82 (1H, d, J = 8.3 Hz, ArH), 6.37 (1H, d,
J = 7.6 Hz, ArH), 4.32 (2H, s, CH2). 13C NMR d 189.5 (C@O), 153.1
(ArC), 151.1 (ArC), 148.3 (ArC), 140.5 (ArC), 137.7 (ArC), 134.0
(ArC), 131.8 (ArC), 127.6 (ArC), 123.9 (ArC), 118.7 (ArC), 108.7
(ArC), 104.1 (ArC), 103.9 (ArC), 41.9 (CH2).
C15H11BrN2O: C, 57.16; H, 3.52; N, 8.89. Found: C, 57.00; H, 3.31;
N, 8.40.
5.1.3.9. 1-(5-Methyl-1H-indol-2-yl)-2-pyridin-3-yl-ethanone (7i).
Prepared from commercial 5-methyl-1H-indole-2-carboxylic acid
(482 mg, 2.75 mmol) and 3-picoline via corresponding acyl chlo-
ride 3i (method A). Yellowish solid (114 mg, 17%), mp 181–
182 °C (dec). LC–MS Rt 5.7 min (method 1); m/z [MH+] 251. 1H
NMR d 11.64 (1H, br s, indole-H1), 8.52 (1H, br s, ArH), 8.42 (1H,
d, J = 3.9 Hz, ArH), 7.70 (1H, d, J = 7.8 Hz, ArH), 7.46 (2H, br s,
2ArH), 7.34–7.29 (2H, m, 2ArH), 7.09 (1H, d, J = 8.5 Hz, ArH), 4.33
(2H, s, CH2), 2.34 (3H, s, CH3). 13C NMR d 190.0 (C@O), 151.1
(ArC), 148.3 (ArC), 137.7 (ArC), 137.1 (ArC), 135.4 (ArC), 131.7
(ArC), 129.6 (ArC), 128.2 (ArC), 127.7 (ArC), 123.9 (ArC), 122.3
(ArC), 113.0 (ArC), 110.1 (ArC), 42.0 (CH2), 21.6 (CH3). Anal. Calcd
for C16H14N2O: C, 76.78; H, 5.64; N, 11.19. Found: C, 77.04; H,
5.52; N, 10.90.
5.1.3.10. 1-(5-Methoxy-1H-indol-2-yl)-2-pyridin-3-yl-ethanone
(7j) and 1-(7-methoxy-1H-indol-2-yl)-2-pyridin-3-yl-ethanone
5.1.3.6. 1-(5-Fluoro-1H-indol-2-yl)-2-pyridin-3-yl-ethanone (7f).
Prepared from commercial 5-fluoro-1H-indole-2-carboxylic acid
(186 mg, 1.02 mmol) and 3-picoline via corresponding acyl chlo-
ride 3f (method A). Yellowish solid (62 mg, 24%), mp 194–197 °C.
LC–MS Rt 5.3 min (method 1); m/z [MH+] 255. 1H NMR d 11.88
(1H, br s, indole-H1), 8.51 (1H, br s, ArH), 8.43 (1H, d, J = 4.8 Hz,
ArH), 7.70 (1H, d, J = 7.8 Hz, ArH), 7.53 (1H, br s, ArH), 7.46 (1H,
(7q)43. Prepared from
a
ꢀ1:1 mixture (method B; 208 mg,
0.95 mmol) of 5-methoxy-1H-indole-2-carboxylic acid ethyl ester
(3j) and 7-methoxy-1H-indole-2-carboxylic acid ethyl ester (3q)
and 3-picoline. The two final products were separated by chroma-
tography. 7j: (Sundberg 1978) eluted with ꢀ80% ethyl acetate. Yel-
lowish solid (88 mg, 35%). LC–MS Rt 5.1 min (method 1), 8.4 min
(method 2); m/z [MH+] 267. 1H NMR d 11.64 (1H, br s, indole-
H1), 8.52 (1H, d, J = 2.0 Hz, ArH), 8.43 (1H, dd, J = 4.7, 1.5 Hz,
ArH), 7.70 (1H, dt, J = 7.8, 1.8 Hz, ArH), 7.44 (1H, d,
J = 1.8 Hz, ArH), 7.34–7.29 (2H, overlapped, 2 ArH), 7.11 (1H, d,
J = 2.4 Hz, ArH), 6.92 (1H, dd, J = 8.9, 2.4 Hz, ArH), 4.33 (2H, s,
CH2), 3.74 (3H, s, OCH3). 13C NMR d 189.8 (C@O), 154.6 (ArC),
147.4 (ArC), 135.6 (ArC), 134.0 (ArC), 132.2 (ArC), 132.1 (2 ArC),
129.2 (ArC), 127.7 (ArC), 118.0 (ArC), 114.3 (ArC), 110.1 (ArC),
102.8 (ArC), 55.8 (OCH3), 42.0 (CH2). 7q: eluted with ꢀ70% ethyl
acetate. White solid (111 mg, 44%), mp 171–175 °C (dec). LC–MS
3
4
3
dd, JHF = 9.6 Hz, JHH = 2.1 Hz, ArH), 7.42 (1H, dd, JHH = 9.1 Hz,
4JHF = 4.7 Hz, ArH), 7.33 (1H, dd, J = 7.8, 4.8 Hz, ArH), 7.14 (1H, dt,
J = 9.2, 2.3 Hz, ArH), 4.36 (2H, s, CH2). 13C NMR d 190.2 (C@O),
157.8 (d, JCF = 233 Hz, indole-C5), 151.1 (ArC), 148.4 (ArC), 137.8
(ArC), 136.8 (ArC), 135.2 (ArC), 131.4 (ArC), 127.4 (d, JCF = 10 Hz,
ArC), 124.0 (ArC), 115.2 (d, JCF = 27 Hz, ArC), 114.7 (d, JCF = 10 Hz,
ArC), 110.3 (d, JCF = 5 Hz, indole-C7a), 107.1 (d, JCF = 23 Hz, ArC),
42.1 (CH2). 19F NMR d ꢃ122.4. Anal. Calcd for C15H11FN2O: C,
70.86; H, 4.36; N, 11.02. Found: C, 70.61; H, 4.36; N, 10.87.

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E. Dolušic et al. / Bioorg. Med. Chem. 19 (2011) 1550–1561
(method 1), 14.8 min (method 2); m/z [MH+] 343. 1H NMR d
11.65 (1H, br s, indole-H1), 8.52 (1H, br s, ArH), 8.42 (1H, d,
J = 4.8 Hz, ArH), 7.70 (1H, d, J = 7.8 Hz, ArH), 7.45–7.43 (3H, over-
lapped, 3ArH), 7.38–7.27 (5H, overlapped, 5ArH), 7.21 (1H, d,
J = 1.8 Hz, ArH), 7.00 (1H, dd, J = 8.9, 2.3 Hz, ArH), 5.08 (2H, s, ben-
zyl CH2), 4.32 (2H, s, C(O)CH2). 13C NMR d 189.9 (C@O), 153.5 (ArC),
151.1 (ArC), 148.3 (ArC), 137.9 (ArC), 137.7 (ArC), 135.8 (ArC),
Rt 5.3 min (method 1), 8.7 min (method 2); m/z [MH+] 267. 1H NMR
d 11.72 (1H, br s, indole-H1), 8.51 (1H, d, J = 1.6 Hz, ArH), 8.42 (1H,
d, J = 3.4 Hz, ArH), 7.69 (1H, d, J = 8.0 Hz, ArH), 7.50 (1H,
d, J = 1.4 Hz, ArH), 7.32 (1H, dd, J = 7.7, 4.7 Hz, ArH), 7.25 (1H, d,
J = 8.0 Hz, ArH), 6.99 (1H, t, J = 7.9 Hz, ArH), 6.79 (1H, d,
J = 7.6 Hz, ArH), 4.35 (2H, s, CH2), 3.87 (3H, s, OCH3). 13C NMR d
189.7 (C@O), 151.1 (ArC), 148.3 (ArC), 147.4 (ArC), 137.8 (ArC),
135.6 (ArC), 131.6 (ArC), 129.5 (ArC), 128.8 (ArC), 123.9 (ArC),
121.6 (ArC), 115.3 (ArC), 111.0 (ArC), 105.6 (ArC), 55.9 (OCH3),
42.2 (CH2). Anal. Calcd for C16H14N2O2: C, 72.16; H, 5.30; N,
10.52. Found: C, 72.90; H, 5.14; N, 10.20.
134.1 (ArC), 131.7 (ArC), 128.9 (2C,
2 phenyl C), 128.3
(ArC), 128.2 (2C, 2 phenyl C), 127.7 (ArC), 124.0 (ArC), 118.5
(ArC), 114.3 (ArC), 110.2 (ArC), 104.4 (ArC), 70.1 (benzyl CH2),
42.0 (C(O)CH2). Anal. Calcd for C22H18N2O2: C, 77.17; H, 5.30; N,
8.18. Found: C, 76.95; H, 5.01; N, 8.10.
5.1.3.11. 1-(5-Nitro-1H-indol-2-yl)-2-pyridin-3-yl-ethanone (7k).
Prepared from commercial 5-nitro-1H-indole-2-carboxylic acid
ethyl ester 3k (249 mg, 1.01 mmol) and 3-picoline. Reaction time:
48 h. Bright yellow solid (42 mg, 15%). LC–MS Rt 5.3 min (method
1); m/z [MH+] 282. 1H NMR d 12.46 (1H, br s, indole-H1), 8.75
(1H, br s, ArH), 8.52 (1H, br s, ArH), 8.44 (1H, d, J = 4.6 Hz, ArH),
8.12 (1H, d, J = 9.2 Hz, ArH), 7.82 (1H, s, ArH), 7.71 (1H, d,
J = 7.8 Hz, ArH), 7.57 (1H, d, J = 9.2 Hz, ArH), 7.34 (1H, dd, J = 8.0,
5.0 Hz, ArH), 4.44 (2H, s, CH2).
5.1.3.15. 1-(5-Hydroxy-1H-indol-2-yl)-2-pyridin-3-yl-ethanone
(7o)
and
2-(1-hydroxy-2-pyridin-3-ylethyl)-1H-indol-5-ol
(17). Ammonium formate (485 mg, 7.50 mmol) and Pd black (sur-
face area 40–60 m2/g, 110 mg, 1.03 mmol) were added to a stirred
suspension of benzyl ether 7n (166 mg, 0.13 mmol) in dry metha-
nol (55 mL). The blue-greenish suspension was stirred at room
temperature for 1 h and filtered through Celite 545. The filter
was extensively washed with methanol and the combined filtrate
and washings were concentrated under reduced pressure. Chro-
matographic purification yielded hydroxyindole 7o (eluted with
95% ethyl acetate; 38 mg, 31%) and byproduct 17 (eluted with
100% ethyl acetate; 69 mg, 56%). Shortening the reaction time to
30 min afforded 7o as the only product in a 58% yield. Analytical
data: 7o, yellow solid which decomposes without melting above
200 °C. LC–MS Rt 4.1 min (method 1); m/z [MH+] 253. 1H NMR d
11.49 (1H, br s, indole-H1), 8.97 (1H, s, OH), 8.51 (1H, br s, ArH),
5.1.3.12. 1-(5-Phenyl-1H-indol-2-yl)-2-pyridin-3-ylethanone (7l)46
Prepared according to a literature procedure. A suspension of tetra-
kis(triphenylphosphine)palladium(0) (7 mg, 6 mol) ina mixtureof
.
l
1 M potassium carbonate (0.8 mL), ethanol (0.8 mL) and toluene
(0.8 mL) was added to a stirred suspension of bromoindole 7h
(38 mg, 0.12 mmol) and phenylboronic acid (33 mg, 0.26 mmol) in
a 1:1 mixture of toluene and ethanol (0.8 mL). The (initially ochre
colored) suspension was heated at reflux temperature for 20 h, al-
lowed to cool down to room temperature and diluted with ethyl ace-
tate. Evaporation with silica and chromatographic purification
afforded the title product as a bright yellow solid (12 mg, 32%). LC–
MS Rt 6.5 min (method 1), 10.2 min (method 2); m/z [MH+] 313. 1H
NMR d 11.85 (1H, br s, indole-H1), 8.54 (1H, d, J = 1.8 Hz, ArH), 8.44
(1H, dd, J = 4.8, 1.6 Hz, ArH), 7.96 (1H, br s, ArH), 7.72 (1H, dt,
J = 7.8, 1.8 Hz, ArH), 7.67–7.49 (5H, overlapped, 5 ArH), 7.43 (2H, t,
J = 7.7 Hz, 2 ArH), 7.33 (1H, dd, J = 8.0, 2.7 Hz, ArH), 7.29 (1H, t,
J = 7.2 Hz, ArH), 4.39 (2H, s, CH2). 13C NMR d 190.1 (C@O), 151.1
(ArC), 148.3 (ArC), 141.6 (ArC), 138.1 (ArC), 137.8 (ArC), 136.1
(ArC), 134.6 (ArC), 132.1 (ArC), 132.0 (ArC), 129.42 (2 ArC), 129.36
(ArC), 127.3 (2 ArC), 125.9 (ArC), 124.0 (ArC), 121.0 (ArC), 113.8
(ArC), 111.0 (ArC), 42.1 (CH2). Anal. Calcd for C21H16N2O: C, 80.75;
H, 5.16; N, 8.97. Found: C, 80.01; H, 5.23; N, 8.49.
3
8.42 (1H, d, J = 3.6 Hz, ArH), 7.69 (1H, dt, JHH = 7.8 Hz,
4JHH = 1.8 Hz, ArH), 7.36 (1H, d, J = 1.8 Hz, ArH), 7.32 (1H, dd,
J = 7.8, 4.8 Hz, ArH), 7.22 (1H, d, J = 8.9 Hz, ArH), 6.93 (1H, d,
J = 2.3 Hz, ArH), 6.81 (1H, dd, J = 8.7, 2.3 Hz, ArH), 4.29 (2H, s,
CH2). 13C NMR d 189.8 (C@O), 152.0 (ArC), 151.1 (ArC), 148.3
(ArC), 137.7 (ArC), 135.6 (ArC), 133.5 (ArC), 131.7 (ArC), 128.1
(ArC), 123.9 (ArC), 118.0 (ArC), 113.9 (ArC), 109.6 (ArC), 105.3
(ArC), 41.9 (CH2). Compound 17, yellowish solid, mp 188–190 °C.
LC–MS Rt 3.1 min (method 1); m/z [MH+] 255. 1H NMR d 10.68
(1H, br s, indole-H1), 8.50 (1H, br s, ArH), 8.33–8.32 (2H, over-
3
lapped, ArH, OH on indole), 7.55 (1H, dd, JHH = 7.8 Hz,
4JHH = 1.8 Hz, ArH), 7.22 (1H, dd, J = 7.8, 4.8 Hz, ArH), 7.08 (1H, d,
J = 8.6 Hz, ArH), 6.70 (1H, d, J = 2.0 Hz, ArH), 6.50 (1H, dd,
4
3JHH = 8.6 Hz, JHH = 2.3 Hz, ArH), 6.01 (1H, d, J = 1.4 Hz, ArH), 5.44
(1H, d, J = 5.3 Hz, CHOH), 4.81 (1H, m, CHOH), 3.07 (1H, dd,
3
2
2JHH = 13.6 Hz, JHH = 5.4 Hz, CH2), 2.97 (1H, dd, JHH = 13.6 Hz,
3JHH = 8.0 Hz, CH2). 13C NMR d 151.0 (ArC), 150.9 (ArC), 147.7
(ArC), 143.4 (ArC), 137.3 (ArC), 134.9 (ArC), 131.0 (ArC), 128.9
(ArC), 123.6 (ArC), 111.9 (ArC), 111.2 (ArC), 104.2 (ArC), 97.7
(ArC), 68.4 (CHOH), 41.0 (CH2).
5.1.3.13. 2-Pyridin-3-yl-1-(5-trifluoromethoxy-1H-indol-2-yl)-
ethanone (7m). Prepared from commercial 5-trifluoromethoxy-
1H-indole-2-carboxylic acid (256 mg, 1.01 mmol) and 3-picoline
via corresponding acyl chloride 3m (method A). Yellowish solid
(90 mg, 28%), mp 184–186 °C. LC–MS Rt 6.2 min (method 1); m/z
[MH+] 321. 1H NMR d 12.04 (1H, br s, indole-H1), 8.52 (1H, d,
J = 1.6 Hz, ArH), 8.43 (1H, dd, J = 4.6, 1.4 Hz, ArH), 7.71–7.69 (2H,
overlapped, 2ArH), 7.60 (1H, br s, ArH), 7.50 (1H, d, J = 8.9 Hz,
ArH), 7.33 (1H, dd, J = 7.8, 4.8 Hz, ArH), 7.24 (1H, d, J = 8.9 Hz,
ArH), 4.38 (2H, s, CH2). 13C NMR d 190.3 (C@O), 151.1 (ArC),
148.4 (ArC), 142.9 (ArC), 137.8 (ArC), 137.1 (ArC), 136.8 (ArC),
131.3 (ArC), 127.3 (ArC), 124.0 (ArC), 120.9 (d, JCF = 254 Hz,
OCF3), 120.1 (ArC), 115.2 (ArC), 114.8 (ArC), 110.6 (ArC), 42.1
(CH2). 19F NMR d ꢃ56.9. Anal. Calcd for C16H11F3N2O2: C, 60.00;
H, 3.46; N, 8.75. Found: C, 59.95; H, 3.43; N, 8.45.
5.1.3.16.
1-(6-Fluoro-1H-indol-2-yl)-2-pyridin-3-yl-ethanone
(7p). Prepared from 6-fluoro-1H-indole-2-carboxylic acid ethyl
ester 3p (method B; 80 mg, 0.39 mmol) and 3-picoline. Bright yel-
low solid (59 mg, 60%), mp 192–193 °C (dec). LC–MS Rt 5.4 min
(method 1); m/z [MH+] 255. 1H NMR d 11.86 (1H, br s, indole-
H1), 8.52 (1H, d, J = 1.7 Hz, ArH), 8.43 (1H, dd, J = 4.9, 1.7 Hz,
ArH), 7.74 (1H, dd, overlapped, ArH), 7.71 (1H, d, overlapped,
ArH), 7.60 (1H, d, J = 1.4 Hz, ArH), 7.33 (1H, dd, J = 7.7, 4.9 Hz,
3
4
ArH), 7.11 (1H, dd, JHF = 9.9 Hz, JHH = 1.9 Hz, ArH), 6.95 (1H, dt,
3J = 9.3 Hz, 4J = 1.9 Hz, ArH), 4.34 (2H, s, CH2). 13C NMR d 189.8
(C@O), 161.6 (d, JCF = 219 Hz, indole-C6), 151.1 (ArC), 148.4 (ArC),
138.6 (d, JCF = 13 Hz, ArC), 137.8 (ArC), 136.3 (d, JCF = 3 Hz, indole-
C3a), 131.5 (ArC), 125.1 (d, JCF = 11 Hz, ArC), 124.4 (ArC), 124.0
(ArC), 110.9 (ArC), 110.4 (d, JCF = 26 Hz, ArC), 98.6 (d, JCF = 26 Hz,
ArC), 41.9 (CH2). 19F NMR d ꢃ115.0. Anal. Calcd for C15H11FN2O:
C, 70.86; H, 4.36; N, 11.02. Found: C, 71.12; H, 4.72; N, 10.81.
5.1.3.14. 1-(5-Benzyloxy-1H-indol-2-yl)-2-pyridin-3-yl-ethanone
(7n). Prepared from commercial 5-benzyloxy-1H-indole-2-car-
boxylic acid ethyl ester 3n (499 mg, 1.69 mmol) and 3-picoline.
Off-white solid (166 mg, 29%), mp 183–184 °C. LC–MS Rt 6.5 min

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1558
5.1.3.17. 1-(4-Benzyloxy-5-methoxy-1H-indol-2-yl)-2-pyridin-3-
yl-ethanone (7r). Prepared from 4-benzyloxy-5-methoxy-1H-in-
dole-2-carboxylic acid ethyl ester 3r (method B; 282 mg,
0.87 mmol) and 3-picoline. Yellow solid (84 mg, 26%), mp 137–
139 °C. LC–MS Rt 6.3 min (method 1), 15.7 min (method 2); m/z
[MH+] 373. 1H NMR d 11.65 (1H, br s, indole-H1), 8.51 (1H, br s,
ArH), 8.43 (1H, d, J = 4.1 Hz, ArH), 7.69 (1H, d, J = 7.8 Hz, ArH),
7.54–7.50 (3H, overlapped, 3ArH), 7.38–7.30 (4H, overlapped,
4ArH), 7.14 (1H, d, J = 8.8 Hz, ArH), 7.07 (1H, d, J = 8.8 Hz, ArH),
5.22 (2H, s, benzyl CH2), 4.36 (2H, s, C(O)CH2), 3.79 (3H s, OCH3).
13C NMR d 190.0 (C@O), 151.1 (ArC), 148.3 (ArC), 144.8 (ArC),
141.5 (ArC), 138.4 (ArC), 137.8 (ArC), 135.6 (ArC), 131.6 (ArC),
128.8 (2 ArC), 128.6 (2 ArC), 128.4 (ArC), 124.0 (ArC), 122.9
(ArC), 117.4 (ArC), 108.4 (ArC), 107.8 (ArC), 74.6 (benzyl CH2),
58.5 (OCH3), 41.9 (C(O)CH2). Anal. Calcd for C23H20N2O3: C,
74.18; H, 5.41; N, 7.52. Found: C, 74.98; H, 5.29; N, 7.42.
41.5 (CH2). Anal. Calcd for C17H16N2O3: C, 68.91; H, 5.44; N, 9.45.
Found: C, 69.65; H, 5.17; N, 9.40.
5.1.3.21. 1-(1H-Indol-2-yl)-2-pyridin-3-yl-propan-1-one (7v).
Prepared from commercial 1H-indole-2-carboxylic acid ethyl ester
3a (983 mg, 5.04 mmol) and 3-ethylpyridine. Yellow half-solid
(220 mg, 17%). LC–MS Rt 5.7 min (method 1), 7.3 min (method
2); m/z [MH+] 251. 1H NMR d 11.74 (1H, br s, indole-H1), 8.65
(1H, d, J = 1.7 Hz, ArH), 8.39 (1H, dd, J = 4.8, 1.7 Hz, ArH), 7.75
(1H, dt, J = 7.8, 1.7 Hz, ArH), 7.64 (1H, d, J = 8.2 Hz, ArH), 7.53
(1H, d, J = 1.4 Hz, ArH), 7.37 (1H, d, J = 8.5 Hz, ArH), 7.31 (1H, dd,
J = 7.9, 4.7 Hz, ArH), 7.23 (1H, t, J = 7.7 Hz, ArH), 7.03 (1H, t,
J = 7.4 Hz, ArH), 4.89 (1H, q, J = 7.0 Hz, CHCH3), 1.46 (3H, d,
J = 7.0 Hz, CH3). 13C NMR d 193.0 (C@O), 149.7 (ArC), 148.6 (ArC),
138.6 (ArC), 137.7 (ArC), 135.5 (ArC), 134.7 (ArC), 127.3
(ArC), 126.3 (ArC), 124.3 (ArC), 123.3 (ArC), 120.9 (ArC), 113.3
(ArC), 110.7 (ArC), 44.5 (CHCH3), 19.1 (CH3).
5.1.3.18. 1-(4-Hydroxy-5-methoxy-1H-indol-2-yl)-2-pyridin-3-
yl-ethanone (7s). Benzyl ester 7r (59 mg, 0.16 mmol) was dis-
solved in absolute ethanol (15 mL) to give a clear yellow solution
5.1.3.22. 1-(1H-Indol-2-yl)-2-pyridin-2-yl-ethanone (8a). Pre-
pared from commercial 1H-indole-2-carboxylic acid ethyl ester
3a (218 mg, 1.12 mmol) and 2-picoline. Yellow solid (237 mg,
90%), mp 140–141 °C. LC–MS Rt 5.5 min (method 1), 9.7 min
(method 2); m/z [MH+] 237. 1H NMR d 11.75 (1H, br s, indole-
H1), 8.45 (1H, dd, J = 4.8, 0.9 Hz, ArH), 7.72 (1H, dd, J = 7.6,
1.8 Hz, ArH), 7.67 (1H, d, J = 8.0 Hz, ArH), 7.44 (1H, d, J = 1.6 Hz,
ArH), 7.42–7.38 (2H, overlapped, 2 ArH), 7.26–7.21 (2H, over-
lapped, 2 ArH), 7.04 (1H, t, J = 7.7 Hz, ArH), 4.43 (2H, s, CH2). 13C
NMR d 190.0 (C@O), 156.4 (ArC), 149.7 (ArC), 138.4 (ArC), 137.2
(ArC), 135.8 (ArC), 127.4 (ArC), 126.1 (ArC), 124.9 (ArC), 123.3
(ArC), 122.4 (ArC), 120.8 (ArC), 113.3 (ArC), 110.8 (ArC), 48.0
(CH2). Anal. Calcd for C15H12N2O: C, 76.25; H, 5.12; N, 11.86.
Found: C, 76.09; H, 5.06; N, 11.74.
to which 3% Pd on activated carbon (60 mg, 17 lmol) was added.
The flask was evacuated and back-filled with hydrogen gas (re-
peated three times). The suspension was stirred at room tempera-
ture for 45 min (until TLC indicated reaction completion) and
filtered through Celite 545. The filter was extensively washed with
ethyl acetate and the combined filtrate and washings were concen-
trated under reduced pressure. Chromatographic purification (the
title product eluted with 80–90% ethyl acetate) afforded the title
hydroxyindole 7s as a light brown half-solid in a 53% (24 mg) yield.
LC–MS Rt 4.3 min (method 1); m/z [MH+] 283. 1H NMR d 11.47 (1H,
br s, indole-H1), 9.26 (1H, s, OH), 8.52 (1H, br s, ArH), 8.42 (1H, d,
J = 3.9 Hz, ArH), 7.70 (1H, d, J = 7.6 Hz, ArH), 7.54 (1H, d, J = 1.4 Hz,
ArH), 7.33 (1H, dd, J = 7.8, 4.8 Hz, ArH), 7.04 (1H, d, J = 8.7 Hz, ArH),
6.78 (1H, d, J = 8.7 Hz, ArH), 4.31 (2H, s, CH2), 3.72 (3H s, OCH3). 13
C
5.1.3.23. 1-(1H-Indol-2-yl)-2-pyridin-4-yl-ethanone (9a)47. Pre-
pared from commercial 1H-indole-2-carboxylic acid ethyl ester
3a (220 mg, 1.13 mmol) and 4-picoline. Bright yellow solid
(240 mg, 90%), mp 169–170 °C. LC–MS Rt 5.0 min (method 1),
8.1 min (method 2); m/z [MH+] 237. 1H NMR d 11.79 (1H, br s, in-
dole-H1), 8.48 (2H, d, J = 6.0 Hz, 2 ArH), 7.68 (1H, d, J = 8.0 Hz, ArH),
7.55 (1H, d, J = 1.1 Hz, ArH), 7.41 (1H, dd, J = 8.3, 0.8 Hz, ArH), 7.34
(2H, d, J = 6.0 Hz, 2 ArH), 7.26 (1H, dt, J = 7.7, 1.1 Hz, ArH), 7.06 (1H,
dt, J = 7.5, 0.8 Hz, ArH), 4.35 (2H, s, CH2). 13C NMR d 189.5 (C@O),
150.0 (2 ArC), 144.8 (ArC), 138.6 (ArC), 135.4 (ArC), 127.4 (ArC),
126.3 (2 ArC), 125.6 (ArC), 123.3 (ArC), 120.9 (ArC), 113.3 (ArC),
110.9 (ArC), 44.2 (CH2). Anal. Calcd for C15H12N2O: C, 76.25; H,
5.12; N, 11.86. Found: C, 75.68; H, 5.09; N, 11.73.
NMR d 189.6 (C@O), 151.1 (ArC), 148.3 (ArC), 141.2 (ArC), 139.5
(ArC), 137.7 (ArC), 136.0 (ArC), 134.9 (ArC), 131.8 (ArC), 124.0
(ArC), 119.8 (ArC), 117.8 (ArC), 108.4 (ArC), 103.4 (ArC), 59.0
(OCH3), 41.9 (CH2).
5.1.3.19. 1-(4,6-Dichloro-1H-indol-2-yl)-2-pyridin-3-yl-ethanone
(7t). Prepared from commercial 4,6-dichloro-1H-indole-2-carbonyl
chloride 3t (647 mg, 2.47 mmol) and 3-picoline. Off-white solid
(198 mg, 26%), mp 223–225 °C. LC–MS Rt 6.5 min (method 1); m/z
[MH+] 305. 1H NMR d 12.26 (1H, br s, indole-H1), 8.52 (1H, d,
J = 1.7 Hz, ArH), 8.44 (1H, dd, J = 4.8, 1.7 Hz, ArH), 7.70 (1H, dt,
J = 7.8, 1.7 Hz, ArH), 7.62 (1H, br s, ArH), 7.41 (1H, dd, J = 1.6, 0.9 Hz,
ArH), 7.33 (1H, dd, J = 7.8, 4.8 Hz, ArH), 7.27 (1H, d, J = 1.8 Hz, ArH),
4.44 (2H, s, CH2). 13C NMR d 190.3 (C@O), 151.2 (ArC), 148.4 (ArC),
138.7 (ArC), 137.9 (ArC), 136.8 (ArC), 131.2 (ArC), 130.4 (ArC),
128.2 (ArC), 125.2 (ArC), 124.0 (ArC), 120.7 (ArC), 112.0 (ArC), 108.1
(ArC), 42.0 (CH2). Anal. Calcd for C15H10Cl2N2O: C, 59.04; H, 3.30; N,
9.18. Found: C, 59.79; H, 3.26; N, 9.21.
5.1.3.24. 1-(1H-Indol-2-yl)-2-pyrazin-2-yl-ethanone (10a). Pre-
pared from commercial 1H-indole-2-carboxylic acid ethyl ester
3a (195 mg, 1.00 mmol) and 2-methylpyrazine. Yellow solid
(97 mg, 41%), mp 175–176 °C. LC–MS Rt 5.5 min (method 1),
11.0 min (method 2); m/z [MH+] 238. 1H NMR d 11.80 (1H, br s, in-
dole-H1), 8.69 (1H, d, J = 1.6 Hz, ArH), 8.56 (1H, dd, J = 2.5, 1.6 Hz,
ArH), 8.51 (1H, d, J = 2.5 Hz, ArH), 7.69 (1H, d, J = 8.0 Hz, ArH),
7.51 (1H, d, J = 1.6 Hz, ArH), 7.41 (1H, d, J = 8.3 Hz, ArH), 7.26
(1H, dt, J = 7.7, 0.9 Hz, ArH), 7.06 (1H, dt, J = 7.6, 0.7 Hz, ArH),
4.57 (2H, s, CH2). 13C NMR d 189.3 (C = O), 152.4 (ArC), 146.4
(ArC), 144.7 (ArC), 143.4 (ArC), 138.5 (ArC), 135.5 (ArC), 127.4
(ArC), 126.3 (ArC), 123.3 (ArC), 120.9 (ArC), 113.3 (ArC), 110.9
(ArC), 45.1 (CH2). Anal. Calcd for C14H11N3O: C, 70.87; H, 4.67; N,
17.71. Found: C, 70.03; H, 4.63; N, 17.63.
5.1.3.20. 1-(4,6-Dimethoxy-1H-indol-2-yl)-2-pyridin-3-yl-etha-
none (7u). Prepared from commercial 4,6-dimethoxy-1H-indole-
2-carboxylic acid methyl ester 3u (241 mg, 1.01 mmol) and 3-pic-
oline. Off-white solid (240 mg, 80%), mp 195–197 °C. LC–MS Rt
5.3 min (method 1); m/z [MH+] 297. 1H NMR d 11.60 (1H, s, in-
dole-H1), 8.52 (1H, d, J = 1.6 Hz, ArH), 8.41 (1H, dd, J = 4.8, 1.6 Hz,
ArH), 7.69 (1H, dt, J = 7.8, 0.9 Hz, ArH), 7.51 (1H, d,
J = 1.8 Hz, ArH), 7.31 (1H, dd, J = 7.8, 4.8 Hz, ArH), 6.40 (1H, d,
J = 1.5 Hz, ArH), 6.17 (1H, d, J = 1.5 Hz, ArH), 4.25 (2H, s, CH2),
3.84 (3H, s, OCH3), 3.73 (3H, s, OCH3). 13C NMR d 188.6 (C@O),
160.8 (ArC), 155.4 (ArC), 151.0 (ArC), 148.2 (ArC), 140.6 (ArC),
137.6 (ArC), 133.5 (ArC), 132.0 (ArC), 123.9 (ArC), 113.8 (ArC),
109.2 (ArC), 93.1 (ArC), 87.0 (ArC), 55.83 (OCH3), 55.76 (OCH3),
5.1.3.25. 1-(1H-Indol-2-yl)-2-phenylethanone (11a)36,48 and 2-
(1H-indol-2-yl)-1,3-diphenylpropan-2-ol (16). An oven-dried
flask was purged with argon while hot, then allowed to cool down
to room temperature under argon and charged with dry THF (1 mL)

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E. Dolušic et al. / Bioorg. Med. Chem. 19 (2011) 1550–1561
and benzyl magnesium chloride, 2.0 M solution in THF (1 mL,
2.0 mmol) to give a clear grayish–yellowish solution. The solution
was cooled in an ice/water bath and a solution of 1H-indole-2-car-
boxylic acid ethyl ester 3a (104 mg, 0.53 mmol) in dry THF (2 mL)
was added dropwise over the flask wall. Clear yellowish solution
was allowed to attain room temperature overnight. After 22 h of
total reaction time (the mixture turned greenish), the reaction
was quenched with saturated aqueous ammonium chloride solu-
tion (5 mL). Upon stirring for several minutes, the mixture was par-
titioned between ethyl acetate and water. The aqueous phase was
extracted with ethyl acetate and the combined organic layers were
washed with brine and dried over MgSO4. Chromatographic purifi-
cation yielded the two products: 11a: eluted with ꢀ7% ethyl ace-
tate. Yellowish solid (46 mg, 37%), mp 125–133 °C. LC–MS Rt
6.8 min (method 1), 16.6 min (method 2); m/z [MH+] 236. 1H
NMR d 11.73 (1H, br s, indole-H1), 7.67 (1H, d, J = 8.0 Hz, ArH),
7.53 (1H, m, ArH), 7.40 (1H, d, J = 8.2 Hz, ArH), 7.33–7.20 (6H, over-
lapped, 6 ArH), 7.05 (1H, dt, J = 7.6, 0.9 Hz, ArH), 4.25 (2H, s, CH2).
13C NMR d 190.8 (C@O), 138.5 (ArC), 136.0 (ArC), 135.6 (ArC), 130.0
(2 ArC), 128.9 (2 ArC), 127.4 (ArC), 127.1 (ArC), 126.1 (ArC), 123.2
(ArC), 120.8 (ArC), 113.3 (ArC), 110.7 (ArC), 45.1 (CH2). Anal. Calcd
for C16H13NO: C, 81.68; H, 5.57; N, 5.95. Found: C, 80.67; H, 6.16; N,
5.51. 16: eluted with ꢀ9% ethyl acetate. White solid (30 mg, 17%),
mp 160–163 °C. LC–MS Rt 7.2 min (method 1), 19.0 min (method
2); m/z [MH+] 328. 1H NMR d 10.75 (1H, br s, indole-H1), 7.31
(1H, d, J = 7.8 Hz, ArH), 7.27 (1H, d, J = 8.0 Hz, ArH), 7.09–7.03
(6H, overlapped, 6 ArH), 6.98–6.96 (4H, overlapped, 4 ArH), 6.92
(1H, dt, J = 7.5, 1.0 Hz, ArH), 6.84 (1H, dt, J = 7.3, 0.9 Hz, ArH),
6.04 (1H, d, J = 1.4 Hz, ArH), 5.18 (1H, s, OH), 3.17 (2H, d,
Jgem = 13.5 Hz, CH2), 3.04 (2H, d, Jgem = 13.5 Hz, CH2). 13C NMR d
144.7 (ArC), 138.1 (2C, ArC), 136.2 (ArC), 131.0 (4C, ArC), 128.3
(ArC), 127.9 (4C, ArC), 126.3 (2C, ArC), 120.5 (ArC), 119.9 (ArC),
118.9 (ArC), 111.6 (ArC), 98.9 (ArC), 75.0 (COH), 48.0 (2C, CH2).
Anal. Calcd for C23H21NO: C, 84.37; H, 6.46; N, 4.28. Found: C,
83.84; H, 6.82; N, 3.91.
(1H, t, J = 7.4 Hz, ArH), 6.89 (1H, t, J = 7.4 Hz, ArH), 6.18 (1H, br s,
ArH), 5.59 (1H, br s, OH), 4.88 (1H, m, CHOH), 3.10 (1H, dd,
3
2
2JHH = 13.7 Hz, JHH = 5.5 Hz, CH2), 3.01 (1H, dd, JHH = 13.7 Hz,
3JHH = 7.9 Hz, CH2). 13C NMR d 151.0 (ArC), 147.7 (ArC), 143.1
(ArC), 137.3 (ArC), 136.5 (ArC), 134.8 (ArC), 128.2 (ArC), 123.7
(ArC), 121.0 (ArC), 120.2 (ArC), 119.2 (ArC), 111.7 (ArC), 98.4
(ArC), 74.3 (CHOH), 41.0 (CH2).
5.1.3.28. 2-(2-Pyridin-3-ylethyl)-1H-indole (19)50. Prepared
according to a literature procedure.51 A suspension of indole
ketone 7a (111 mg, 0.47 mmol), hydrazine hydrate (47 ll,
0.97 mmol) and potassium hydroxide pellets (130 mg, 2.32 mmol)
in ethylene glycol (5 mL) was subjected to microwave heating at
195 °C for 1 h (P ꢀ40 W, p = 1–3 bar). The resulting clear dark or-
ange solution was partitioned between dichloromethane and satu-
rated aqueous ammonium chloride. The aqueous phase was
extracted twice with dichloromethane and the combined organic
layers were dried over MgSO4. Concentration of the filtered solu-
tion combined with the addition of cyclohexane yielded the title
product as an ochre solid (87 mg, 84%), mp 139–142 °C. LC–MS Rt
5.2 min (method 1); m/z [MH+] 223. 1H NMR d 10.95 (1H, br s, in-
dole-H1), 8.41 (1H, s, ArH), 8.35 (1H, d, J = 4.8 Hz, ArH), 7.61 (1H,
dd, J = 7.8, 1.6 Hz, ArH), 7.35 (1H, d, J = 7.8 Hz, ArH), 7.27–7.24
(2H, overlapped, 2 ArH), 6.95 (1H, t, J = 7.5 Hz, ArH), 6.88 (1H, t,
J = 7.5 Hz, ArH), 6.09 (1H, s, ArH), 3.00 (4H, overlapped, 2 CH2).
13C NMR d 150.1 (ArC), 147.8 (ArC), 139.4 (ArC), 137.2 (ArC),
136.5 (ArC), 136.3 (ArC), 128.8 (ArC), 123.9 (ArC), 120.7 (ArC),
119.7 (ArC), 119.1 (ArC), 111.2 (ArC), 99.2 (ArC), 32.2 (CH2), 29.6
(CH2). Anal. Calcd for C15H14N2: C, 81.05; H, 6.35; N, 12.60. Found:
C, 80.33; H, 6.13; N, 12.15.
5.1.3.29. 1-(1H -Indol-2-yl)-2-(pyridin-3-yl)ethanone oxime
(20). A suspension of ketone 7a (52 mg, 0.22 mmol), hydroxyl-
amine hydrochloride (17 mg, 0.24 mmol) and pyridine (20 ll,
0.25 mmol) in absolute ethanol (1 mL) was subjected to microwave
heating at 120 °C for 30 min. The resulting clear yellow solution
was evaporated with silica and oxime 20 was purified by auto-
mated flash chromatography to yield a white solid (13 mg, 24%),
mp 187–192 °C. LC–MS Rt 5.2 min (method 1); m/z [MH+] 252. 1H
NMR d 11.43 (1H, br s), 11.11 (1H, br s), 8.55 (1H, d, J = 1.8 Hz, ArH),
8.34 (1H, dd, J = 4.8, 1.6 Hz, ArH), 7.65 (1H, d, J = 7.8 Hz, ArH), 7.47
(1H, d, J = 8.0 Hz, ArH), 7.34 (1H, d, J = 8.2 Hz, ArH), 7.26 (1H, dd,
J = 7.9, 4.8 Hz, ArH), 7.07 (1H, t, J = 7.6 Hz, ArH), 6.93 (1H, t,
J = 7.6 Hz, ArH), 6.84 (1H, d, J = 2.1 Hz, ArH), 4.08 (2H, s, CH2). 13C
NMR d 150.3 (C), 149.5 (C), 147.9 (C), 137.7 (ArC), 136.5 (ArC),
134.2 (ArC), 134.0 (ArC), 128.1 (ArC), 124.1 (ArC), 123.2 (ArC),
120.9 (ArC), 119.7 (ArC), 112.2 (ArC), 103.7 (ArC), 28.8 (CH2).44
5.1.3.26.
1-(1-Methyl-1H-indol-2-yl)-2-pyridin-3-yl-ethanone
(12a)43. Prepared from 1-methyl-1H-indole-2-carboxylic acid
methyl ester 6a (method C; 544 mg, 2.88 mmol) and 3-picoline.
Bright yellow solid (243 mg, 34%), mp 75–77 °C. LC–MS Rt
5.9 min (method 1); m/z [MH+] 251. 1H NMR d 8.51 (1H, s, ArH),
8.43 (1H, d, J = 4.6 Hz, ArH), 7.74–7.69 (3H, overlapped,
3
ArH), 7.55 (1H, d, J = 8.5 Hz, ArH), 7.37–7.31 (2H, overlapped, 2
ArH), 7.13 (1H, t, J = 7.5 Hz, ArH), 4.39 (2H, s, CH2), 3.95 (3H, s,
CH3). 13C NMR d 191.2 (C@O), 151.1 (ArC), 148.3 (ArC), 140.4
(ArC), 137.8 (ArC), 134.5 (ArC), 131.7 (ArC), 126.5 (ArC), 125.9
(ArC), 123.9 (ArC), 123.4 (ArC), 121.3 (ArC), 113.0 (ArC),
111.5 (ArC), 43.3 (CH2), 32.5 (CH3). Anal. Calcd for C16H14N2O: C,
76.78; H, 5.64; N, 11.19. Found: C, 77.64; H, 5.68; N, 11.24.
5.1.3.30. 1H-Indole-2-carboxylic acid pyridin-3-ylamide (21). A
yellow suspension of 1H-indole-2-carboxylic acid (482 mg,
2.95 mmol) in thionyl chloride (5 mL) was boiled for 15 min, during
which time the mixture became a clear dark brown solution. This
was concentrated under reduced pressure and the solid residue
was evaporated three times with toluene. 3-Aminopyridine
5.1.3.27.
1-(1H-Indol-2-yl)-2-pyridin-3-ylethanol
(18)49
.
Ammonium formate (845 mg, 13.4 mmol) and Pd black (surface
area 40–60 m2/g, 38 mg, 0.36 mmol) were added to a stirred solu-
tion of ketone 7a (203 mg, 0.86 mmol) in dry methanol (100 mL).
As no significant amount of alcohol 18 was detected by TLC after
24 h, more Pd black (163 mg, 1.54 mmol) was added. Three days
later, as TLC indicated complete consumption of starting material,
the reaction mixture was filtered through Celite 545 and the filter
was extensively washed with methanol. Concentration under re-
duced pressure, dilution with a small volume of ethyl acetate
and precipitation with cyclohexane yielded the title product as
an dark orange solid (183 mg, 89%), mp 144–151 °C with decompo-
sition. LC–MS Rt 4.4 min (method 1); m/z [MH+] 239. 1H NMR d
11.06 (1H, br s, indole-H1), 8.35–8.32 (2H, overlapped, 2 ArH),
7.56 (1H, d, J = 7.8 Hz, ArH), 7.39 (1H, d, J = 7.8 Hz, ArH), 7.30
(1H, d, J = 8.0 Hz, ArH), 7.23 (1H, dd, J = 7.8, 4.8 Hz, ArH), 6.98
(306 mg, 3.22 mmol) and diisopropylethylamine (785 ll,
4.43 mmol) were dissolved in dry THF (2 mL) to give a clear yellow-
ish solution which was cooled to 0 °C. A solution of the above 1H-
indole-2-carbonyl chloride residue in THF (3 mL) was added care-
fully to give a red-brown mixture. The ice/water bath was removed
after 5 min and the reaction mixture was stirred for a further
25 min and then partitioned between ethyl acetate and water.
The aqueous layer is extracted twice with ethyl acetate and the
combined organic layers were washed with brine and dried over
MgSO4. Chromatographic purification (the title product eluted with
50–60% ethyl acetate) afforded amide 21 as a yellow solid (372 mg,
53%), decomposes without melting above 258 °C. LC–MS Rt 5.1 min

´
E. Dolušic et al. / Bioorg. Med. Chem. 19 (2011) 1550–1561
1560
(method 1); m/z [MH+] 238. 1H NMR d 11.80 (1H, br s, indole-H1),
10.40 (1H, s, CONH), 8.95 (1H, d, J = 2.3 Hz, ArH), 8.29 (1H, dd,
J = 4.6, 1.2 Hz, ArH), 8.19 (1H, dd, J = 8.4, 1.6 Hz, ArH), 7.66 (1H, d,
J = 8.0 Hz, ArH), 7.45–7.42 (2H, overlapped, 2 ArH), 7.38 (1H,
dd, J = 8.3, 4.8 Hz, ArH), 7.21 (1H, t, J = 7.5 Hz, ArH), 7.05 (1H, t,
J = 7.5 Hz, ArH). 13C NMR d 160.6 (CONH), 145.0 (ArC), 142.2
(ArC), 137.5 (ArC), 136.2 (ArC), 131.5 (ArC), 127.6 (ArC), 127.5
(ArC), 124.6 (ArC), 124.2 (ArC), 122.4 (ArC), 120.6 (ArC),
113.0 (ArC), 104.9 (ArC). Anal. Calcd for C14H11N3O: C, 70.87; H,
4.67; N, 17.71. Found: C, 70.66; H, 4.57; N, 17.76.
1-MT. Dose–response inhibition of IDO is analyzed using GraphPad
Prism 4 program (GraphPad Software Inc.).
5.3. Cellular tests
5.3.1. Cell line
A plasmid construct encoding murine IDO was transfected into
mouse mastocytoma line P815B. Clone P185B-mIDO clone 6 (Uyt-
tenhove et al, 2003), which overexpresses IDO, was selected and
used for the cellular assay. Mouse IDO shares a 62% sequence iden-
tity with human IDO, and the active site residues are 100%
conserved.
5.2. Biology
5.2.1. Protein expression and purification of recombinant
human IDO
5.3.2. Assay
The assay was performed in 96-well flat bottom plates seeded
The coding region for human IDO (Ala2-Gly403) was cloned into
a derivative of plasmid pET9 (Novagen). The recombinant plasmid,
pETIDO, encodes a histidine tag at the N-terminus of IDO. Bacterial
strain BL21 AI (Invitrogen) was used for overexpression of IDO and
transformed with the pETIDO plasmid. The transformed cells were
grown on a rotary shaker at 37 °C and 220 rpm to an OD600 of 1.2,
with 2 ꢅ 105 cells in a final volume of 200
ll. To determine
whether compounds were significant IDO, the cells were incubated
overnight at 37 °C in HBSS (Hanks Balanced Salt Solution, Invitro-
gen) supplemented with 80
compound. The plates were then centrifuged 10 min at 300 g,
and 150 l of the supernatant were collected. The supernatant
lM L-Tryptophan and 20 lM of the
l
in LB medium supplemented with 25
-tryptophan and 10 M bovine hemin (Sigma). The culture was
cooled in a water/ice bath and supplemented again with 50 g/
mL -tryptophan and 10 M bovine hemin. The expression of His-
l
g/mL kanamycin, 50
l
g/mL
was analyzed by HPLC to measure the concentration of tryptophan
and kynurenine, based on the retention time and the UV absorp-
tion (280 nm for tryptophan, 360 nm for kynurenine). For the HPLC
L
l
l
L
l
analysis, 50 ll of supernatant were mixed with 500 ll acetonitrile
tagged IDO was induced by the addition of 1% (w/v) arabinose. In-
duced cells were grown at 20 °C and 60 rpm for 20 h. Cells (1 L
culture) were collected by centrifugation, resuspended in 40 mL
of 25 mM Mes, 150 mM KCl, 10 mM imidazole, and protease inhib-
itors (complete EDTA free, Roche Applied Science) (pH 6.5), and
disrupted with a French press. The extract was clarified by centri-
to precipitate the proteins. After centrifugation, the supernatant
was collected, concentrated on a speedvac, resuspended in a final
volume of 100 ll water and injected in the HPLC (C18 column).
In those conditions, about 50% of the initial amount of tryptophan
was degraded in the absence of inhibitor, and an equimolar
amount of kynurenine was produced. The percentages of inhibition
of tryptophan degradation and kynurenine production by the com-
pounds were calculated in reference to this maximal activity. The
fugation and filtration on a 0.22 lm filter. The enzyme was purified
by IMAC using Ni2þ as a ligand and an IMAC HITRAP column (5 mL,
GE Healthcare). Briefly, the extract was loaded on the column with
25 mM Mes, 150 mM KCl, and 10 mM imidazole (pH 6.5). The col-
umn was washed with 50 mL of the same buffer with the imidazole
concentration adjusted to 100 mM. Finally, the protein was eluted
with 25 mM Mes, 150 mM KCl, and 50 mM EDTA (pH 6.5). The buf-
fer was then exchanged into 25 mM Mes and 150 mM KCl (pH 6.5)
using a HITRAP desalting column (GE Healthcare). The purity of the
enzyme was estimated to be >95% based on the SDS–PAGE gel and
Coomassie blue staining. The ratio of absorbance at 404 nm to that
at 280 nm of the protein was around 1.9.
initial wells containing the cells in the remaining volume of 50
were used to estimate cell viability in a classical MTT assay. To that
end, 50 l of culture medium (Iscove medium with 10% FCS and
amino acids) were added to the wells together with 50 l of
MTT. After 3–4 h of incubation at 37 °C, 100 l of SDS/DMF were
ll
l
l
l
added to dissolve the crystals of formazan blue and the absorbance
at 570/650 nm was measured after overnight incubation at 37 °C.
5.4. X-ray determination
X-ray measurements were performed on a Gemini Ultra R sys-
5.2.2. Assay buffer
tem (4-circle kappa platform, Ruby CCD detector) using MoKa
Assay buffer was composed of Phosphate Buffer 50 mM (pH
6.5), Methylene Blue 5 lM (Sigma–Aldrich, M9140), and Ascorbic
radiation (k = 0.71073 Å) for the two compounds. After mounting
and centering of the single crystals on the diffractometer, cell
parameters were estimated from a pre-experiment run and full
datasets collected at room temperature. Software for data reduc-
tion was CrysAliPro.52 Structures were solved by direct methods
from SHELXS-97 program53 and then refined on F2 using SHELXL-97
software. Non-hydrogen atoms were anisotropically refined and
the hydrogen atoms in the riding mode with isotropic temperature
factors fixed at 1.2 times U (equiv) of the parent atoms.
Acid 40 mM (Sigma–Aldrich, A5960).
L-Tryptophan substrate (Sig-
ma–Aldrich, T0254) is diluted at 100
lM in Assay Buffer. IDO en-
zyme was diluted in Phosphate Buffer 50 mM at 0.05 mg/ml.
Hydrogen peroxide solution (Sigma–Aldrich, H1009) is prepared
at a concentration of 10 mM in water.
5.2.3. Enzymatic assay
Enzymatic Assay was performed in 384 Wells microplates (Gre-
iner bio one, 781108). Ninety five microliters of reaction buffer
with L-Tryptophan substrate at 100 lM and compound to be tested
are added in the microplate. The optical density at 320 nm of this
mixture is first recorded as a baseline with a Spectramax plus spec-
CCDC 794450 and 794451 contain the supplementary crystallo-
graphic data for this paper and can be obtained free of charge via
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cam-
bridge Crystallographic Data Centre, 12, Union Road, Cambridge
CB2 1EZ, UK; fax: +44 1223 336033; or deposit@ccdc.cam.ac.uk).
trophotometer (Molecular Devices). The hIDO enzyme (0.375
lg in
5
ll) is then added to the mixture. After 60 min incubation at room
5.5. Molecular modeling
temperature, the reaction is stopped by the addition of hydrogen
peroxide (1 mM) and a second optical reading at 320 nm is per-
formed. The difference between the two density values reflects
the level of tryptophan degradation and is used to calculate the
percentage of IDO inhibition relative to the reference inhibitor
Molecular modeling studies were carried out on a Linux work-
station. The compounds were built using the SKETCH module
implemented in SYBYL (version 8.0).54 Docking was performed using
the 3D-coordinates of TDO (pdb code 2D0T) with the help of the

´
1561
E. Dolušic et al. / Bioorg. Med. Chem. 19 (2011) 1550–1561
automated GOLD program35 (active site definition: residues within
7 Å around PIM). In order to take protein flexibility into account,
the enzyme-inhibitor complexes were optimized using the MINIMIZE
module. The minimization process uses the Powell method with
the Tripos force field (dielectric constant 1r) to reach a final con-
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.
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Acknowledgments
This work is supported by the FNRS (Belgium) and Biowin
(CANTOL: convention n°5678). R.F. is greatly indebted to the
Belgian ‘Fonds de la Recherche Scientifique—FNRS’ for the award
of a postdoctoral research grant.
Supplementary data
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