Indole-3-carbonitriles as DYRK1A inhibitors by fragment-based drug design

Article abstract of DOI:10.3390/molecules23020064

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a potential drug target because of its role in the development of Down syndrome and Alzheimer's disease. The selective DYRK1A inhibitor 10-iodo-11H-indolo[3, 2-c]quinoline-6-carboxy

Full text of DOI:10.3390/molecules23020064

molecules
Article
Indole-3-Carbonitriles as DYRK1A Inhibitors by
Fragment-Based Drug Design
Rosanna Meine 1,2, Walter Becker 3, Hannes Falke 1, Lutz Preu 1, Nadège Loaëc 4,
Laurent Meijer 4 and Conrad Kunick 1,2,
*
ID
ID
1
Institut für Medizinische und Pharmazeutische Chemie, Technische Universität Braunschweig,
Beethovenstraße 55, 38106 Braunschweig, Germany; r.meine@tu-bs.de (R.M.);
h.falke@tu-braunschweig.de (H.F.); l.preu@tu-bs.de (L.P.)
2
3
4
Zentrum für Pharmaverfahrenstechnik (PVZ), Technische Universität Braunschweig, Franz-Liszt-Straße 35A,
38106 Braunschweig, Germany
Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University,
Wendlingweg 2, 52074 Aachen, Germany; wbecker@ukaachen.de
ManRos Therapeutics, Perharidy Research Center, 29680 Roscoff, France; nadege.loaec@univ-brest.fr (N.L.);
meijer@manros-therapeutics.com (L.M.)
*
Correspondence: c.kunick@tu-bs.de; Tel.: +49-531-391-2754
Received: 22 December 2017; Accepted: 12 January 2018; Published: 24 January 2018
Abstract: Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a potential
drug target because of its role in the development of Down syndrome and Alzheimer’s disease.
The selective DYRK1A inhibitor 10-iodo-11H-indolo[3,2-c]quinoline-6-carboxylic acid (KuFal194),
a large, flat and lipophilic molecule, suffers from poor water solubility, limiting its use as
chemical probe in cellular assays and animal models. Based on the structure of KuFal194,
7-chloro-1H-indole-3-carbonitrile was selected as fragment template for the development of
smaller and less lipophilic DYRK1A inhibitors. By modification of this fragment, a series of
indole-3-carbonitriles was designed and evaluated as potential DYRK1A ligands by molecular
docking studies. Synthesis and in vitro assays on DYRK1A and related protein kinases identified
novel double-digit nanomolar inhibitors with submicromolar activity in cell culture assays.
Keywords: DYRK1A; fraction of saturation; fragment-based drug development; indole; lipophilicity;
molecular docking; protein kinase inhibitor; solubility
1. Introduction
Protein kinases are enzymes catalyzing the transfer of γ-phosphate from ATP to the hydroxyl
group of serine, threonine or tyrosine residues of their substrates. Since these substrates affect
important cellular processes such as differentiation, cell cycle regulation, proliferation and apoptosis,
the dysregulation of protein kinases is involved in numerous human diseases, e.g., cancer, diabetes,
inflammatory or neurodegenerative disorders [1].
Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) belongs to the CMGC
group of kinases which also includes the mitogen-activated protein kinases (MAPK), cyclin dependent
kinases (CDK), cdc2-like kinases (CLK) and glycogen synthase kinase-3 (GSK-3) [
DYRK1A activity is associated with Down syndrome (DS) and Alzheimer’s disease (AD) [
The localization of the DYRK1A gene in the Down syndrome critical region (DSCR) on chromosome
21 leads to a 1.5-fold overexpression in individuals with trisomy 21 [ ]. While DYRK1A is expressed
ubiquitously, high concentrations are only observed in certain brain areas [ ]. Because DYRK1A
2]. Increased
3,4].
5
6
plays an important role for the regulation of proliferation and differentiation of neuronal cells,
its overexpression was suspected to be linked to DS symptoms such as mental retardation and
Molecules 2018, 23, 64; doi:10.3390/molecules23020064
www.mdpi.com/journal/molecules

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reduced brain size [
6–
8]. The enhanced DYRK1A expression has been suggested as one of the reasons
for the early onset of AD-like neurodegeneration in DS individuals [5,9]. Amongst other proteins,
DYRK1A phosphorylates tau protein, the alternative splicing factor (ASF), amyloid precursor protein
and presenilin 1. These substrates are involved in formation of intracellular neurofibrillary tangles
and extracellular
β-amyloid plaques, the main morphological changes observed in the brains of
Alzheimer’s patients [10–14].
All kinases of the CMGC group share a high similarity regarding their ATP binding site. In this
respect, especially the closely related DYRK1B and CLK1 are very similar compared to DYRK1A.
While the catalytic domains of DYRK1A and DYRK1B have a sequence identity of 85%, their ATP
binding pockets only differ in one amino acid: the gatekeeper (gk) +2 amino acid Met240 of DYRK1A
is replaced by Leu192 in DYRK1B. Although DYRK1A and CLK1 feature an overall sequence identity
of only 30%, their ATP binding pockets show an analogy of 70% [15].
DYRK1A has been suggested as a potential drug target because of its involvement in
neurodegenerative disorders. Therefore, several inhibitors have been reported in recent years
(see reviews [
DYRK1A inhibitors are harmine (
3
,
4
,
9
,
16
,
17]) but it remains challenging to develop selective inhibitors. The most studied
) [18], INDY ( ) [19], and leucettine L41 ( ) [20], all of which act in
1
2
3
an ATP competitive manner (Figure 1). These inhibitors show submicromolar DYRK1A IC50 values
and activity in cell based assays. The major drawback of these compounds is lack of selectivity
compared to the related kinases DYRK1B and CLK1 [18
of monoamine oxidase-A (MAO-A) (IC50 = 5 nM), which limits its use as a chemical probe [26].
The methyl 9-anilinothiazolo[5,4-f]quinazoline-2-carbimidate EHT 5372 ( ) (Figure 1) inhibits
DYRK1A and DYRK1B at subnanomolar concentrations (IC50 DYRK1A 0.22 nM; IC50 DYRK1B
0.28 nM). Compound and closely related structures represent the most potent DYRK1A inhibitors
reported so far, with striking selectivity even compared to closely related kinases of the CMGC
group [27 29]. EHT 5372 also inhibits cellular DYRK1A-mediated tau phosphorylation and A
–25]. In addition, harmine is a potent inhibitor
4
4
–
β
production, albeit with significantly lower potency (IC50 1.06–1.17 µM) [30].
Figure 1. Structure of four dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A)
inhibitors mentioned in the literature: harmine (1); INDY (2); leucettine L41 (3); and EHT 5372 (4).
KuFal194 (5, compound 5j in the original report of Falke et al. [31]) has a good in vitro activity
(IC50 = 6 nM) against DYRK1A and considerable selectivity compared to DYRK1B and CLK1. However,
its cellular efficiency was significantly lower. For example, DYRK1A-mediated tau phosphorylation
was inhibited with an IC50 of 2.1 µM [31].

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Taken together, harmine (
1
), INDY (
2
) and leucettine L41 (
3
) show DYRK1A inhibitory activity in
cellular assays, but only low selectivity versus related kinases. In contrast, EHT 5372 (
4) and KuFal194
(5
) exhibit significant selectivity for DYRK1A. However, being lipophilic structures with large flat
aromatic molecular areas, their use as chemical probes in aqueous media for investigation in in vivo
models is probably limited.
In an attempt to develop new DYRK1A inhibitors with improved physicochemical properties,
the structure of KuFal194 (5) was downsized to 7-chloro-1H-indole-3-carbonitrile (6a) (Figure 2).
Figure 2. Fragment based drug design: The halogen-substituted indole element as starting point for
the development of DYRK1A inhibitors with decreased molecular mass and lipophilicity.
The resulting fragment (6a) was then used as a template for the design of a series of 2-substituted
indole-3-carbonitriles which were synthesized and evaluated for inhibitory activity against a panel of
kinases of the CMGC group.
2. Results and Discussion
2.1. Molecular Docking Studies
Docking experiments were performed to identify substitution-accessible positions of fragment 6a
We assumed a binding mode of the fragment in the ATP binding pocket of DYRK1A similar to KuFal194.
The ATP competitive inhibitor KuFal194 ( ) has been co-crystallized with DYRK1A (PDB: 4YLJ) [31].
.
5
In this ligand-protein complex, the deprotonated carboxylic acid is positioned in the back of the pocket
forming a salt bridge to the conserved Lys188. A hydrogen bond to a water molecule in the binding
pocket is observed which further interacts with the backbone atoms of Asp307 and Phe308 and the
sidechain of Glu203. The 10-iodo substituent is located near to the hinge region. ATP is bound to the
hinge region of protein kinases via hydrogen bonds to the adenine. In most cases, ATP mimetic kinase
inhibitors (Type I inhibitors) also form one or more hydrogen bonds to the hinge region [
is an exception to this rule but is nevertheless a potent DYRK1A inhibitor. On first sight, it might be
assumed that a halogen bond between the iodine atom of KuFal194 ( ) and the carbonyl oxygen of
Leu241 of the protein could substitute a hydrogen bond. However, the orientation of KuFal194 (
would in the present case not meet the geometric requirements for this kind of interaction, since in the
crystal structure the C-I–O
-hole angle is 132◦ which makes an attractive interaction improbable [32].
1]. KuFal194
5
5
)
σ
Instead of a direct halogen bond to the Leu241 carbonyl oxygen atom, an indirect connection to the
hinge region appears more probable. This interaction would comprise a halogen bond between the
ligand’s iodine atom and a water molecule which itself is attached to Leu241 via a hydrogen bond.
With a distance of 3.0 Å between iodine and oxygen and a C-I–O σ
-hole angle of 168◦, the indicated
interaction fulfils all characteristics of a classical halogen bond (Figure 3A). A recent PDB survey of
Xu et al. [33] demonstrated that a water molecule is the halogen bond acceptor in about 17% of all
halogen bonds observed in biomolecules.
For docking studies with newly designed congeners, the program GOLD [34] was employed with
the crystal structure of DYRK1A in complex with KuFal194 (PDB: 4YLJ). The water molecule in the back

Molecules 2018, 23, 64
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pocket was included in docking runs. An orientation of 6a analogous to KuFal194 (
5
) was generated
with the highest score. According to this pose, the indole scaffold of 6a is oriented in the same way
as in the original ligand KuFal194 ( ). The nitrile group of 6a forms a hydrogen bond to Lys188 and
to a water molecule in the back pocket. Concerning a possible halogen bond between the 7-chloro
substituent and the water molecule near the pocket entrance, the distance of 4.45 Å and a -hole angle
5
σ
of 148◦ are not optimal (Figure 3B). Because of the described highest-ranked predicted binding mode,
only positions 1 and 2 of the indole scaffold appeared accessible for insertion of substituents without
drastic alterations of the ligand orientation. The nitrile group was retained because it appeared to be
important for interactions with the protein. Guided by these structural requirements we designed
a set of new analogs. Within these structures, a further docking evaluation showed that a 2-phenyl
substituent, reminiscent of a corresponding benzene ring in KuFal194 (5), yielded the most promising
structures forming further hydrophobic interactions to the protein. A 7-iodo substituent appeared
favorable regarding the geometrical requirements for a halogen bond to the water molecule at the front
pocket (Figure 3C,D).
Figure 3. Results of molecular docking studies. The distance between the halogen atom and the
water molecule as well as the
KuFal194 ( yellow) in the ATP binding pocket of DYRK1A (blue, PDB: 4YLJ); (
mode of the starting fragment 6a orange); and ( ) docking poses of the most promising compounds
dark red) and 7-iodo-2-phenyl-1H- indole-3-carbonitrile
σ
-hole angle C-Hal–H2O are marked in red: (
A
) co-crystal structure of
5,
B
) predicted binding
(
C,D
7-chloro-2-phenyl-1H-indole-3-carbonitrile (6f
,
(6h, green).
2.2. Syntheses
The 2-substituted indoles
method published by Pei et al. [35
obtain the -chloro acetophenones
to yield the desired indoles 9.
9
were prepared starting from the appropriate anilines
36] (Scheme 1). The anilines were treated with chloroacetonitrile to
which subsequently were reacted with suitable Grignard reagents
7 according to a
,
7
α
8
The introduction of the nitrile group was accomplished by applying three alternative methods:
either by a two-step procedure via 2-(indol-3-yl)-2-oxocarboxylic acids (10) [37], or by reactions with
the cyanating agents chlorosulfonyl isocyanate (11) [38], or N-cyano-N-phenyl-p-toluenesulfonamide
(12, NCTS) [39] (Scheme 2).

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Scheme 1. Synthesis procedure for the preparation of 2-substituted indoles (
9
). Reagents and conditions:
◦
(
a
) chloroacetonitrile, AlCl3, BCl3, CH2Cl2, 0 C to reflux, 7–23 h; and (
b
) Grignard reagent R2-MgHal,
◦
THF or toluene, −10 C to rt, 60 min; for substituents R1 and R2 refer to Table 1.
Table 1. Structures of indole-3-carbonitriles 6.
Compound
R1
R2
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
Cl
Br
I
H
CH3
Cl
Br
I
Cl
Cl
Cl
Cl
I
I
I
I
I
I
I
H
H
H
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
4-Cl-Phenyl
4-OCH3-Phenyl
Allyl
2-(1,3-Dioxan-2-yl)ethyl
4-Cl-Phenyl
4-OCH3-Phenyl
3-OCH3-Phenyl
Pyridin-3-yl
Isopropyl
Cyclopropyl
Cyclopentyl
6s
Scheme 2. Three different synthesis procedures for the introduction of the nitrile group in position
3 of the indole core structure. Reagents and conditions: ( ) oxalyl chloride, diethyl ether, rt, 6 h;
) hydroxyl ammonium chloride, sodium acetate, ethanol/water, reflux, 6–9 h (compounds 6a 6k );
); and
a
(
(
(
b
–
c
r
,
–l
◦
c) 1. acetonitrile, 0 C to rt, 4 h; 2. dimethylformamide, acetonitrile, rt, 2 h (compounds 6d
,
6p–
◦
d
) BF3
·
OEt2, 1,2-dichloroethane or toluene, 100 C (bath temperature) or reflux, 15–48 h (compounds
6e–j,6m–o,6s).

Molecules 2018, 23, 64
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Furthermore, a series of N-alkylated indoles (13) (Table 2) was synthesized by treating the
unsubstituted indoles with benzyl bromide or methyl iodide as described in the literature [40] and
subsequent cyanation by NCTS (12) (Scheme 3).
Table 2. Structures of the synthesized N-alkylated indole-3-carbonitriles 13.
Compound
R1
R2
R3
13a
13b
13c
13d
13e
13f
Cl
Br
Cl
Br
Br
I
H
H
H
Benzyl
Benzyl
CH3
CH3
CH3
H
Phenyl
Phenyl
CH3
Scheme 3. Syntheses of N-alkylated indole-3-carbonitriles 13. Reagents and conditions: (
a
) methyl
iodide or benzyl bromide, NaH, THF or DMF, 0 ◦C to rt, 4.5–48 h; and (
b
) NCTS, BF3 OEt2,
·
◦
1,2-dichloroethane, 100 C (bath temperature), 22–46 h.
To design compounds with increased hydrophilicity, 7-chloro-2-(2,3-dihydroxypropyl)-1H-indole
-3-carbonitrile (14) was prepared from 2-allyl-7-chloro-1H-indole-3-carbonitrile (6k) via dihydroxylation
with osmium tetroxide according to Huchet et al. [41] (Scheme 4).
Scheme 4. Dihydroxylation of the allyl chain of 6k. Reagents and conditions: (a) OsO4, N-methylmorpholine
N-oxide, acetone/H2O, rt, 72 h.
2.3. Kinase Inhibitory Activity
The synthesized indole-3-carbonitriles 6a
GSK-3 for kinase inhibitory activity. First, the kinases were incubated with 10
–
s
,
13a
–
f
and 14 were tested on DYRKs, CLKs and
M solutions of the
µ
compounds and the residual activities compared to controls were measured. The IC50 values of
promising compounds were then determined from concentration-response curves (Table 3).
The starting fragment 6a inhibited DYRK1A with a micromolar IC50. CLK1 was the only off target
with comparable IC50. The fact that replacement of the 7-chloro substituent against bromine or iodine
caused an increase of DYRK1A inhibition underlines the important role of halogen bonds for ligand
binding. A similar observation was made in the series 6d–h. Introduction of a phenyl substituent
in position 2 led to the most potent DYRK1A inhibitors of this study with double-digit nanomolar
IC50 values. If the halogen at position 7 was removed or replaced by a methyl group, the activity was
at least 10-fold lower. As described in the literature for other DYRK 1A inhibitors, a considerable

Molecules 2018, 23, 64
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selectivity versus DYRK1B, CLK1 and CLK4 could not be achieved with the congeners of series
presented here.
6
The introduction of further residues at the 2-phenyl substituent provoked weaker activity
) or complete loss (6i and 6m). With the aim of solubility improvement, the phenyl
(6n
–
o
–j
substituent was replaced by hydrophilic or aliphatic substituents. Unfortunately, especially the
hydrophilic residues led to significantly decreased DYRK1A inhibitory activity (6l and 14), although
the docking experiments revealed three additional hydrogen bonds for the diol moiety of compound
14 (S1, Supplementary Materials). Polar compounds such as 14 probably are not well accepted in the
ATP binding pocket which is formed by nonpolar amino acids such as Phe170, Leu207, Val222 or
Val306. Only a pyridin-3-yl residue (6p) instead of a phenyl moiety (6h) was well tolerated.
The introduction of aliphatic residues into a flat aromatic drug molecule enhances the fraction of
saturation (Fsp3), a molecular manipulation suggested to improve physicochemical properties such
as aqueous solubility [42]. Regarding the inhibition of DYRK1A, such aliphatic residues (6q–s) were
significantly better tolerated then hydrophilic residues. Among the 2-alkyl-substituted congeners,
compound 6s with a 2-cyclopentyl residue turned out to be the most potent DYRK1A inhibitor with
an IC50 of 70 nM.
Substituents at the indole nitrogen were introduced to explore the structure-activity relationships
and to enhance the Fsp3. While a benzyl residue caused complete loss of inhibitory activity (13a
–b),
a smaller methyl group was better tolerated. Anyway, even a methyl group led to a significantly
decreased DYRK1A inhibitory activity. Thus, in the series reported here, an unsubstituted indole
nitrogen apparently is required for optimal protein-ligand interaction.
Table 3. Kinase inhibitory activity of indole-3-carbonitriles 6, 13 and 14 against DYRK1A and closely
related kinases of the CMGC group a.
IC50 [µM]/Residual Activity at 10 µM [%]
Compound
DYRK
1A
DYRK
1B
DYRK2 DYRK3
CLK1
CLK2
CLK3
CLK4
GSK3
6a
6b
6c
6d
6e
3.30
1.10
0.410
0.4
35%
0.040
0.025
0.010
60%
60%
0.690
3.9
42%
0.233
0.210
0.08
0.28
0.14
0.07
97%
81%
32%
1.9
>10
5.9
1.8
>10
>10
8
>10
>10
>10
-
3.33
2.9
1.9
>10
>10
8
>10
75%
>10
0.433
2
n.t.
n.t.
1.3
>10
92%
>10
3.3
>10
0.7
1
>10
>10
>10
-
>10
4
1.8
>10
>10
>10
-
6
-
0.800
56%
0.070
0.067
0.080
68%
47%
0.533
4.9
55%
0.333
0.633
0.200
0.1
0.110
0.12
102%
53%
56%
3.33
>10
2
74%
>10
0.120
0.570
n.t.
75%
>10
>10
>10
n.t.
n.t.
>10
>10
116%
>10
>10
-
>10
8
-
144%
n.t.
117%
>10
-
105%
>10
>10
>10
n.t.
n.t.
>10
>10
101%
>10
>10
-
>10
>10
-
95%
n.t.
95%
>10
-
124%
>10
>10
>10
n.t.
n.t.
>10
>10
130%
>10
>10
-
>10
-
-
127%
n.t.
97%
>10
-
43%
0.120
0.033
0.050
n.t.
n.t.
0.533
4
36%
0.220
0.290
>10
0.1
n.t.
>10
n.t.
>10
112%
118%
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
1.3
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
n.t.
1.3
>10
79%
>10
0.633
0.3
0.53
0.5
0.6
78%
n.t.
47%
>10
10
0.1
0.1
-
6s
1
>10
n.t.
114%
n.t.
>10
-
13a
13b
13c
13d
13e
13f
14
95%
n.t.
70%
>10
-
84%
n.t.
30%
>10
1
2.3
0.65
2
2.1
4.2
-
-
-
-
0.6
2
>10
10
-
-
1
2
-
2
a
The activity is indicated as IC50 (µM) or as residual activity (%) at 10 µM compared to controls. -: less than 20%
inhibition at 10 µM, IC50 was not determined; n.t.: not tested.

Molecules 2018, 23, 64
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2.4. Inhibition of DYRK1A Activity in Cell Culture
To evaluate the applicability of the 2-substituted indole-3-carbonitrile compounds in cell culture
experiments, we assessed toxicity of 6f 6h 6s and 13b on HeLa cells by viability assays (Table 4).
,
,
All test compounds exhibited minimal cytotoxicity up to a concentration of 3
their IC50 values for DYRK1A inhibition in biochemical assays.
µM, which is well above
Table 4. Cytotoxicity of selected compounds a.
Compound
1 µM
3 µM
10 µM
6f
6h
6s
94
97
99
94
78
20
98
97
100
94
85
87
82
66
45
13b
KuFal194 (5)
75
Staurosporine b
a
Viability of treated HeLa cells is given in percent relative to control cells treated with vehicle (means of three
b
experiments with duplicate measurement). Staurosporine is a known inducer of apoptosis and served as a
positive control.
To evaluate representative potent compounds for capacity of inhibiting DYRK1A in cell culture
experiments, we analyzed 6f, 6h, and 6s for their effects on the phosphorylation of the splicing factor
3b1 (SF3B1). Phosphorylation of T434 in SF3B1 depends on endogenous DYRK1A activity and can be
assessed with the help of a phosphospecific antibody [43]. Thus, the relative phosphorylation of T434
in HeLa cells provides a useful measure of the cellular efficacy of DYRK1A inhibitors [21
,31,44]. In this
assay, 6f 6h and 6s inhibited cellular DYRK1A activity in a concentration-dependent manner with
,
IC50 values well below the toxic concentrations (Figure 4). Thus, the new compounds are suitable to
inhibit DYRK1A in living cells with similar potency as other well characterized DYRK1A inhibitors
(e.g., harmine (1) and AnnH75 [21,44]). To control for the specificity of the assays, we took advantage of
a structurally related compound that did not inhibit DYRK1A in the biochemical assays (13b, Table 3).
Reassuringly, compound 13b did not affect the phosphorylation of SF3B1 on T434.
Figure 4. Cont.

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Figure 4. Inhibition of endogenous DYRK1A activity in HeLa cells by 6f, 6h, and 6s. HeLa cells
expressing GFP-SF3B1-NT were treated with the indicated compounds for 18 h before cells were lysed
with a denaturing lysis. The phosphorylation state of SF3B1 was determined by Western blot analysis of
total cellular lysates. The pT434 signals were normalized to the total amount of GFP immunoreactivity.
Compound 13b is an analog that does not inhibit DYRK1A and was used as negative control. The left
panels show representative western blots. The vertical lines indicate where irrelevant lanes were
deleted from the final image. The graphs present the quantitative evaluation of two (6s) or three
experiments. DYRK1A activity is expressed as normalized pT434 immunoreactivity relative to that in
cells treated with vehicle (means ± SEM).
2.5. Physicochemical Properties and Solubility Assays
All new compounds were evaluated based on their physicochemical properties regarding
Lipinski’s rule of five and the ligand-lipophilicity efficiency (LLE). Both concepts are used to assess
the druglikeness of drug development candidates. According to Lipinski et al. [45], drugs with oral
bioavailability should have a molecular weight <500 g/mol, logP <5 and the number of H-bond
acceptors and donors should not exceed 10 and 5, respectively. All biological active compounds of
the series met these requirements (S2, Supplementary Materials). The LLE is a parameter for the
estimation of druglikeness which takes into account binding affinity and lipophilicity of biological
active compounds. LLE is calculated from pIC50 minus calculated logP [46]. Oral available drugs
should have an LLE of 5 or higher. The LLE of the most potent compound in the series reported here
(6h) is 3.58 and thus is significantly lower than the suggested minimal value of 5 (Table 5). Replacement
of the 2-phenyl substituent for a more hydrophilic pyridin-3-yl substituent (6p) increased the LLE
from 3.58 to 3.89. The diol 14 reached the highest LLE of the series (4.59) and nearly met the target
value because of its low logP value (1.11).
Furthermore, the logS value (logarithm of molar aqueous solubility) was predicted for all
compounds. For selected compounds the predicted data was compared with the experimentally
determined solubility. The thermodynamic solubility was measured by a shake-flask method
and the kinetic solubility was quantified using nephelometry. In most cases, the experimentally
determined thermodynamic solubility was significantly lower than the solubility calculated with
MarvinSketch [47]. The starting compound 6a showed moderate water solubility. If the chlorine at
position 7 was replaced with bromine or iodine, the thermodynamic solubility decreased, as expected
because of increasing lipophilicity. Surprisingly, this effect was the other way around for the kinetic
solubility. Unfortunately, the most potent compound 6h retained poor aqueous solubility compared
to KuFal194 (5). The comparison of the kinetic solubility of 6h and 13f demonstrates that increasing
the Fsp3 by introduction of aliphatic residues improved solubility as expected. The same effect was
observed upon replacement of the phenyl substituent in 6h for aliphatic residues (compounds 6r
and 6s). Additional hydroxyl functions as part of this aliphatic side chain dramatically increased
the solubility, as is exemplified by compound 14. In contrast, the replacement of the 2-phenyl
substituent of 6h by a hydrophilic pyridin-3-yl substituent (compound 6p) improved the kinetic
solubility only slightly.

Molecules 2018, 23, 64
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Table 5. Calculated logP value, ligand-lipophilicity efficiency (LLE) and solubility data for selected
compounds a.
Sexp., therm., pH 7.4 [µM] b
Sexp., kin., pH 7.4 [µM] d
Scalc, pH 7.4 [µM]
Compound
logP
LLE
KuFal194 (5)
4.22
2.53
2.70
2.86
4.10
4.26
4.42
3.21
3.48
4.37
4.65
1.11
4.00
2.95
3.26
3.53
3.30
3.34
3.58
3.89
3.37
2.78
1.54
4.59
13183
525
224
< 0.5
143 ± 6.3c
136 ± 11
45.2 (44.8–45.6)
<0.5
5.28 (4.99–5.57)
116 ± 14
6a
6b
6c
142 ± 14
263
261 (254–267)
14.7 ± 4.5
6f
2.63
1.26
1.66
25.1
37.2
3.80
12.0
912
6g
6h
6p
6r
<0.5
<0.5
n.d.
<0.5
<0.5
<0.5
14.8 ± 0.26
4.57 (4.11–5.03)
10.0 (7.21–12.9)
38.1 (37.6–38.6)
16.2 (15.2–17.3)
37.5 (36.6–38.4)
n.d.
6s
13f
14
a
3447 ± 182 c
b
The logP und logS values were predicted using the program MarvinSketch [47]. Thermodynamic solubility:
Two or three independent experiments were performed. If the compounds caused signals lower than the limit of
quantification in two experiments, the thermodynamic solubility was indicated as <0.5 µM, which was the lowest
concentration of the calibration solutions. If two experiments were performed the mean value and range is indicated,
c
for three experiments the mean value ± SD is indicated. The solubility was determined after 24 h, 48 h, 72 h and
96 h. After 24 h the equilibrium was not reached. Indicated solubility is the mean value of the results after 48 h, 72 h
d
and 96 h. Kinetic solubility: Two or three independent experiments were performed. If two experiments were
performed, the mean value and range is indicated, for three experiments the mean value
not determined
±
SD is indicated. n.d.,
3. Materials and Methods
3.1. General Information
The starting materials and reagents were purchased from Acros Organics (Geel, Belgium),
Alfa Aesar (Karlsruhe, Germany) and Sigma-Aldrich (Steinheim, Germany). 7-Chloroindole was
purchased from Activate Scientific (Prien, Germany), 7-bromoindole was purchased from Maybridge
(Loughborough, United Kingdom). All reagents and solvents were used without further purification
unless otherwise stated. Anhydrous 1,2-dichloroethane, toluene, tetrahydrofuran, diethyl ether,
dichloromethane and acetonitrile were used if indicated and were dried according to published
methods [48]. Silica gel was used for purification by column chromatography. Reaction monitoring
was performed using thin layer chromatography (TLC): Polygram SIL G/UV254, 0.2 mm silica
gel 60, 40
×
80 mm (Macherey-Nagel, Düren, Germany), visualization by UV light (254 nm).
The melting points (m.p.) were detected in open-glass capillaries on an electric variable heater
(Electrothermal IA 9100, Bibby Scientific, Stone, UK). The infrared spectra were recorded on a
Thermo Nicolet FT-IR 200 spectrometer (Thermo Nicolet, Madison, WI, USA) using KBr pellets
or NaCl windows, respectively. 1H-NMR spectra and 13C-NMR spectra were recorded on Bruker
Avance III 400, Bruker Avance II 600 or Bruker Avance III HD 500 spectrometers (Bruker Biospin,
Rheinstetten, Germany) (at the NMR laboratories of the Chemical Institutes of the Technische
Universität Braunschweig) in DMSO-d6. Chemical shifts are presented as parts per million (ppm) in
relation to tetramethylsilane as internal standard (δ
= 0 ppm). Signals in 13C spectra were assigned
based on results of 13C-DEPT135 experiments. Electron ionization (EI) mass spectra were recorded
on a Finnigan-MAT 95 (Thermo Finnigan, Bremen, Germany), (EI) MS: ionization energy 70 eV.
Accurate measurements were performed according to the peakmatch method using perfluorokerosene
(PFK) as an internal mass reference (Department of mass spectrometry of the Chemical Institutes,
TU Braunschweig). Atmospheric pressure chemical ionization (APCI) spectra were determined
with an expressionL CMS spectrometer, the APCI source was coupled with ASAP (atmospheric
solids analysis probe) (Advion, Ltd., Harlow, UK). The elemental analyses were performed on a CE
Instruments Flash EA® 1112 Elemental Analyzer (Thermo Quest, San Jose, CA, USA). Purity was
determined using two independent high performance liquid chromatography (HPLC) methods
with isocratic or gradient elution. All compounds tested in biological systems had purity
≥
95%.

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The following devices and settings were used: system 1: Merck Hitachi Elite LaChrom system (Hitachi
High Technologies Corporation, Tokyo, Japan) (diode array detector (DAD): L-2450; pump: L-2130;
autosampler: L-2200; organizer box: L-2000); system 2: Merck Hitachi Elite LaChrom system (Hitachi
High Technologies Corporation, Tokyo, Japan) (UV detector: L-2400; pump: L-2130; autosampler:
L-2200; organizer box: L-2000); system 3: VWR Hitachi Chromaster system (Hitachi High Technologies
Corporation, Tokyo, Japan) (DAD detector: 5430; column oven: 5310; pump: 5110; autosampler: 5260);
column: Merck LiChroCART 125-4, LiChrospher 100 RP-18 (5 µm) (Merck, Darmstadt, Germany);
flow rate: 1.000 mL/min; detection wavelength: 254 nm and 280 nm (isocratic), 254 nm (gradient);
overall run time: 15 min (isocratic), 20 min (gradient); AUC, % method; ts = dead time related to
DMSO; tms = retention time). For gradient elution, an acetonitrile/water mixture was used (0–2 min:
10% ACN; 2–12 min: 10%
acetonitrile/water mixtures were used. Absorption maxima (
→
90% ACN (linear) 12–20 min: 90% ACN). For isocratic elution, different
max) were extracted from the UV spectra
λ
recorded by the DAD detector in the peak maxima during HPLC runs.
3.2. Synthesis and Characterization of 6, 13 and 14
The synthesis procedure and analytical characterization of the intermediates
listed in the Supplementary Materials.
8, 9, 10 and 12 are
3.2.1. General Procedure for the Synthesis of Indole-3-Carbonitriles (Procedure A)
Hydroxylammonium chloride (2 eq.) and sodium acetate (2 eq.) were dissolved in a mixture
of ethanol and water. The (indol-3-yl)-2-oxoacetic acid (10) (1 eq.) was added and the solution was
heated at reflux. The reaction was monitored by TLC and stopped after 6–9 h. After evaporation of the
solvent the crude product was purified by recrystallization or column chromatography.
3.2.2. General procedure for the synthesis of indole-3-carbonitriles (Procedure B)
The indole (9) (1 eq.) and NCTS (12) (1 eq.) were dissolved in anhydrous 1,2-dichloroethane
(1 mL) in an argon flushed microwave reaction vessel. After addition of boron trifluoride diethyl
etherate (1–2.5 eq.) the solution was stirred in a sealed vessel for 20–48 h in an oil bath heated to
100 ◦C. After cooling to room temperature, the solution was diluted with 1,2-dichloroethane (10 mL)
and was washed successively with aqueous sodium hydroxide (85 g/L), hydrochloric acid (73 g/L)
and water (10 mL each). After removal of the solvent under reduced pressure, the further purification
was performed as indicated in the specific synthesis procedure.
3.2.3. General procedure for the synthesis of indole-3-carbonitriles (Procedure C)
The synthesis was performed under nitrogen atmosphere. The indole (9) (1 eq.) was dissolved in
anhydrous acetonitrile (20–60 mL). The solution was cooled to 0 ◦C and a mixture of chlorosulfonyl
isocyanate (11) (1–7 eq.) and anhydrous acetonitrile (5 mL) was added dropwise. The solution was
allowed to warm to room temperature and was stirred for 4 h. Then, a mixture of dimethylformamide
(1 mL) and anhydrous acetonitrile (5 mL) was added and stirring was continued for 2 h. The reaction
was stopped by addition of water (30 mL). The reaction mixture was extracted with ethyl acetate
(3
×
30 mL), and subsequently the combined organic phases were dried over sodium sulfate and
the solvent was removed under reduced pressure. The resulting crude product was purified by
column chromatography.
7-Chloro-1H-indole-3-carbonitrile (6a): According to general procedure A with 2-(7-chloro-1H-indol-3-yl)
-2-oxoacetic acid (10a, 87 mg, 0.39 mmol), hydroxyl ammonium chloride (63 mg, 0.90 mmol) and
sodium acetate (74 mg, 0.90 mmol) in a mixture of ethanol (5 mL) and water (2 mL). After purification
by column chromatography (toluene-ethyl acetate-formic acid 10:1:1) and recrystallization from
n-hexane, a slightly yellow solid (34 mg, 49%) was obtained. m.p. 179–180 ◦C (lit.: 180–181 ◦C [49]);
IR (KBr): vemax 3256 (NH), 2220 cm-1 (C
≡
N); 1H-NMR (DMSO-d6, 400.4 MHz):
δ (ppm) = 7.26 (t, 1H,

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J = 7.5 Hz, H-5), 7.40 (dd, 1H, J = 7.7, 0.9 Hz, Ar-H), 7.64 (dd, 1H, J = 7.9, 0.9 Hz, Ar-H), 8.37 (s, 1H,
H-2), 12.62 (s, 1H, NH); 13C-NMR (DMSO-d6, 100.7 MHz):
(ppm) =117.5, 122.8, 123.0, 135.8 (CH),
δ
85.7, 115.6, 117.3, 128.5, 132.3 (C); C9H5ClN2 (176.60); HPLC (isocr.): 98.7% at 254 nm, 99.7% at
280 nm, tms = 3.2 min, tm = 1.1 min (ACN/H2O 50:50) (system 1); HPLC (gradient): 96.1% at 254 nm,
tms = 10.6 min, tm = 1.3 min (system 2); λmax 274 nm.
7-Bromo-1H-indole-3-carbonitrile (6b): According to general procedure A with 2-(7-bromo-1H-indol-3-yl)
-2-oxoacetic acid (10b, 135 mg, 0.504 mmol), hydroxyl ammonium chloride (69 mg, 0.99 mmol) and
sodium acetate (82 mg, 1.0 mmol) in a mixture of ethanol (8 mL) and water (3 mL). After purification
by column chromatography (toluene-ethyl acetate 4:1) and recrystallization from n-hexane-ethanol
20:1, a slightly yellow solid (50 mg, 45%) was obtained. m.p.: 148–151 ◦C; IR (KBr): vemax 3272 (NH),
1
2219 cm−1 (C
≡
N); H-NMR (DMSO-d6, 399.8 MHz):
δ
(ppm) = 7.19 (t, 1H, J = 7.8 Hz, H-5), 7.53
(dd, 1H, J = 7.8, 0.9 Hz, Ar-H), 7.67 (dd, 1H, J = 8.0, 0.9 Hz, Ar-H), 8.35 (s, 1H, H-2), 12.50 (s, 1H, NH);
13C-NMR (DMSO-d6, 100.5 MHz):
δ (ppm) = 118.0, 123.1, 126.1, 135.7 (CH), 85.8, 105.3, 115.6, 128.2,
133.7 (C); C9H5BrN2 (221.06) calc. C 48.90, H 2.28, N 12.67, found C 48.79, H 2.02, N 12.44; EIMS m/z
(%) 220 [M]+• (100), 141 [M+•-79] (48); HPLC (isocr.): 98.7% at 254 nm, 99.6% at 280 nm, tms = 3.5 min,
tm = 1.1 min (ACN/H2O 50:50) (system 1); HPLC (gradient): 98.5% at 254 nm, tms = 10.8 min,
t
m = 1.3 min (system 2); λmax 275 nm.
7-Iodo-1H-indole-3-carbonitrile (6c): According to general procedure A with 2-(7-iodo-1H-indol-3-yl)
-2-oxoacetic acid (10c, 181 mg, 0.575 mmol), hydroxyl ammonium chloride (79 mg, 1.1 mmol) and
sodium acetate (94 mg, 1.1 mmol) in a mixture of ethanol (15 mL) and water (3 mL). After purification
by column chromatography (toluene-ethyl acetate 9:1) and recrystallization from n-hexane-ethanol
10:1, slightly brown crystals (58 mg, 38%) were obtained. m.p.: 161–163 ◦C; IR (KBr): vemax 3233 (NH),
1
2229 cm−1 (C
≡
N); H-NMR (DMSO-d6, 400.4 MHz):
δ
(ppm) = 7.05 (t, 1H, J = 7.7 Hz, H-5), 7.66
(dd, 1H, J = 8.0, 1.0 Hz, Ar-H), 7.70 (dd, 1H, J = 7.5, 0.9 Hz, Ar-H), 8.30 (s, 1H, H-2), 12.21 (s, 1H, NH);
13C-NMR (DMSO-d6, 100.7 MHz):
δ (ppm) = 118.5, 123.5, 132.5, 135.3 (CH), 78.2, 85.9, 115.9, 127.1,
137.2 (C); C9H5IN2 (268.06) calc. C 40.33, H 1.88, N 10.45, found C 40.45, H 1.65, N 10.11; EIMS m/z (%)
268 [M]+• (100), 141 [M+•-127] (34); HPLC (isocr.): 99.4% at 254 nm, 100.0% at 280 nm, tms = 4.1 min,
tm = 1.1 min (ACN/H2O 50:50) (system 1); HPLC (gradient): 100.0% at 254 nm, tms = 11.1 min,
t
m = 1.3 min (system 2); λmax: 276 nm.
2-Phenyl-1H-indole-3-carbonitrile (6d): According to general procedure C from 2-phenyl-1H-indole
(400 mg, 2.07 mmol) and chlorosulfonyl isocyanate (200 L, 2.30 mmol) in acetonitrile (20 mL).
µ
Purification by column chromatography (petroleum ether-ethyl acetate 2:1) gave yellow crystals
(242 mg, 54%). m.p.: 238–241 ◦C; IR (KBr): vemax 3218 (NH), 2215 cm−1 (C
≡
N); 1H-NMR (DMSO-d6,
δ (ppm) = 7.28 (ddd, 1H, J = 8.1, 7.1, 1.1 Hz, Ar-H), 7.33 (ddd, 1H, J = 8.1, 7.1, 1.3 Hz, Ar-H),
400.4 MHz):
7.50–7.73 (m, 5H, Ar-H), 7.95–8.04 (m, 2H, Ar-H), 12.62 (s, 1H, NH); 13C-NMR (DMSO-d6, 100.7 MHz):
δ
(ppm) = 112.7, 118.4, 122.0, 123.9, 127.0 (2C), 129.32 (2C), 130.0 (CH), 81.4, 117.0, 128.3, 129.34, 135.5,
144.7 (C); C15H10N2 (218.26) calc. C 82.55, H 4.62, N 12.84, found C 82.67, H 4.37, N 12.73; APCI-MS m/z
(%) 219 [M + H]+ (100); HPLC (isocr.): 100.0% at 254 nm, 100.0% at 280 nm, tms = 3.1 min, tm = 1.1 min
(ACN/H2O 60:40) (system 3); HPLC (gradient): 99.2% at 254 nm, tms = 11.8 min, tm = 1.2 min (system 2);
λ
max 240, 307 nm.
7-Methyl-2-phenyl-1H-indole-3-carbonitrile
7-methyl-2-phenyl-1H-indole (9a, 300 mg, 1.45 mmol), NCTS (12, 394 mg, 1.45 mmol) and boron
trifluoride diethyl etherate (400 L, 3.16 mmol). The solution was heated for 24 h. During heating a
(
6e):
According to general procedure B from
µ
precipitate was formed, which was filtered off and recrystallized from methanol to yield a colorless
◦
1
powder (164 mg, 71%). m.p.: 285–286 C; IR (KBr): vemax 3184 (NH), 2226 cm−1 (C
≡
N); H-NMR
(DMSO-d6, 400.4 MHz):
δ (ppm) = 2.58 (s, 3H, CH3), 7.09–7.14 (m, 1H, Ar-H), 7.14–7.20 (m, 1H, Ar-H),
7.47 (dd, 1H, J = 7.8, 1.0 Hz, Ar-H), 7.50–7.75 (m, 3H, Ar-H), 7.94–8.03 (m, 2H, Ar-H), 12.22 (s, 1H, NH);
13C-NMR (DMSO-d6, 100.7 MHz):
δ (ppm) = 16.9 (CH3), 115.8, 122.2, 124.5, 127.7 (2C), 129.1 (2C),

Molecules 2018, 23, 64
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129.9 (CH), 82.2, 117.0, 122.5, 128.0, 129.4, 135.1, 145.2 (C); C16H12N2 (232.29) calc. C 82.73, H 5.21,
N 12.06, found C 82.50, H 4.97, N 11.87; EIMS m/z (%) 232 [M]+ (100), 116 [M+-116] (6); HPLC (isocr.):
99.8% at 254 nm, 99.9% at 280 nm, tms = 3.9 min, tm = 1.1 min (ACN/H2O 60:40) (system 1); HPLC
(gradient): 99.7% at 254 nm, tms = 12.5 min, tm = 1.2 min (system 2); λmax 242, 306 nm.
7-Chloro-2-phenyl-1H-indole-3-carbonitrile (6f): According to general procedure B from 7-chloro-2-phenyl
-1H-indole (9b, 154 mg, 0.659 mmol), NCTS (12, 179 mg, 0.661 mmol) and boron trifluoride
diethyl etherate (200
µL, 1.58 mmol). The solution was heated for 44 h. After purification by
column chromatography (petroleum ether-dichloromethane 1:2) and recrystallization from ethyl
◦
acetate-n-hexane 17:50, a colorless powder (31 mg, 19%) was obtained. m.p.: 266–268 C; IR (KBr): vemax
13C-NMR (DMSO-d6, 100.7 MHz):
−1
3209 (NH), 2200 cm (C
≡
N); 1H-NMR (DMSO-d6, 400.4 MHz):
δ
(ppm) = 7.28 (t, 1H, J = 7.8 Hz, H-5),
7.42 (dd, 1H, J = 7.7, 0.9 Hz, Ar-H), 7.54–7.69 (m, 4H, Ar-H), 7.94–8.04 (m, 2H, Ar-H), 12.77 (s, 1H, NH);
δ (ppm) = 117.4 , 123.1, 123.6, 128.1 (2C), 129.0 (2C), 130.3 (CH), 83.3,
116.2, 117.1, 128.7, 129.8, 132.7, 146.9 (C); C15H9ClN2 (252.70) calc. C 71.30, H 3.59, N 11.09, found C
71.21, H 3.45, N 10.81; EIMS m/z (%) 252 [M]+ (100), 217 [M+-35] (9); HPLC (isocr.): 98.3% at 254 nm,
99.3% at 280 nm, tms = 4.6 min, tm = 1.1 min (ACN/H2O 60:40) (system 1); HPLC (gradient): 98.7% at
254 nm, tms = 12.8 min, tm = 1.2 min (system 2); λ
243, 303 nm.
max
7-Bromo-2-phenyl-1H-indole-3-carbonitrile (6g): 7-Bromo-2-phenyl-1H-indole (9c, 203 mg, 0.746 mmol)
and NCTS (12, 204 mg, 0.749 mmol) were dissolved in anhydrous toluene (1 mL) in an argon flushed
round bottom flask. After addition of boron trifluoride diethyl etherate (0.20 mL, 1.6 mmol) the
solution was heated to reflux for 30 h under exclusion of moisture. The resulting suspension was
diluted with toluene (5 mL) and the precipitate was filtered off. The filtrate was washed successively
with sodium hydroxide solution (85 g/L, 5 mL), hydrochloride acid (73 g/L, 5 mL) and water (5
mL). After removal of the solvent, the residue was combined with the precipitate and recrystallized
◦
from methanol. Grey solid (30 mg, 14%). m.p.: 248–251 C; IR (KBr): vemax 3207 (NH), 2219 cm−1
(C δ (ppm) = 7.22 (t, 1H, J = 7.8 Hz, H-5), 7.56 (dd, 1H, J = 7.7,
N); 1H-NMR (DMSO-d6, 600.1 MHz):
≡
0.9 Hz, Ar-H), 7.57–7.65 (m, 3H, Ar-H), 7.67 (dd, 1H, J = 7.9, 0.9 Hz, Ar-H), 7.95–7.99 (m, 2H, Ar-H),
12.62 (s, 1H, NH); 13C-NMR (DMSO-d6, 150.9 MHz):
δ (ppm) = 117.9, 123.5, 126.8, 128.4 (2C), 128.9
(2C), 130.3 (CH), 83.6, 105.1, 116.3, 128.7, 129.6, 134.3, 147.1 (C); C15H9BrN2 (297.16); HR-EIMS m/z
calc. 295.99436, found 295.99473; EIMS m/z (%) 296 [M]+ (100), 217 [M+-79] (14); HPLC (isocr.): 99.0%
at 254 nm, 99.7% at 280 nm, tms = 4.8 min, tm = 1.1 min (ACN/H2O 60:40) (system 1); HPLC (gradient):
98.1% at 254 nm, tms = 13.0 min, tm = 1.2 min (system 2); λmax 242, 303 nm.
7-Iodo-2-phenyl-1H-indole-3-carbonitrile (6h): According to the general procedure from 7-iodo-2-phenyl
-1H-indole (9d, 123 mg, 0.385 mmol), NCTS (12, 105 mg, 0.386 mmol) and boron trifluoride diethyl
etherate (0.10 mL, 0.79 mmol). The solution was heated for 46 h. After purification by column
chromatography (petroleum ether-ethyl acetate 5:1) and recrystallization from methanol, colorless
crystals (40 mg, 30%) were obtained. m.p.: 223–224 ◦C; IR (KBr): vemax 3192 (NH), 2221 cm−1 (C
≡
N));
1H-NMR (DMSO-d6, 600.1 MHz):
δ (ppm) = 7.07 (t, 1H, J = 7.2 Hz, H-5), 7.55–7.65 (m, 3H, Ar-H), 7.67
(dd, 1H, J = 7.9, 0.9 Hz, Ar-H), 7.74 (dd, 1H, J = 7.5, 1.0 Hz, Ar-H), 7.90–7.96 (m, 2H, Ar-H), 12.32 (s, 1H,
NH); 13C-NMR (DMSO-d6, 150.9 MHz):
δ (ppm) = 118.4, 123.8, 128.54 (2C), 128.82 (2C), 130.2, 133.4
(CH), 77.7, 83.8, 116.4, 128.47, 128.77, 137.8, 146.9 (C); C15H9IN2 (344.16) calc. C 52.35, H 2.64, N 8.14,
found C 52.39, H 2.49, N 7.97; EIMS m/z (%) 344 [M]+ (100), 217 [M+-127] (22); HPLC (isocr.): 99.8% at
254 nm, 100.0% at 280 nm, tms = 5.9 min, tm = 1.1 min (ACN/H2O 60:40) (system 1); HPLC (gradient):
99.1% at 254 nm, tms = 13.3 min, tm = 1.2 min (system 2); λmax 242, 304 nm.
7-Chloro-2-(4-chlorophenyl)-1H-indole-3-carbonitrile (6i): According to general procedure B from
7-chloro-2-(4-chlorophenyl)-1H-indole (9e, 152 mg, 0.580 mmol), NCTS (12, 160 mg, 0.588 mmol)
and boron trifluoride diethyl etherate (0.10 mL, 0.79 mmol). The solution was heated for 46 h.
During each washing step a precipitate was formed at the phase interface which was filtered off.
The combined precipitates were recrystallized from methanol-toluene 6:1. Colorless solid (49 mg,

Molecules 2018, 23, 64
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◦
29%). m.p.: 306–308 C; IR (KBr): vemax 3221 (NH), 2216 cm−1 (C
≡
N); 1H-NMR (DMSO-d6, 600.1 MHz):
δ
(ppm) = 7.29 (t, 1H, J = 8.0 Hz, H-5), 7.43 (dd, 1H, J = 7.7, 0.9 Hz, Ar-H), 7.64 (dd, 1H, J = 8.0, 0.9 Hz,
Ar-H), 7.69–7.83 (m, 2H, Ar-H), 7.97–8.06 (m, 2H, Ar-H), 12.83 (s, 1H, NH); 13C-NMR (DMSO-d6,
150.9 MHz):
δ (ppm) = 117.5, 123.3, 123.8, 129.1 (2C), 129.9 (2C) (CH), 83.7, 116.1, 117.1, 127.6, 129.8,
132.8, 135.1, 145.5 (C); C15H8Cl2N2 (287.14) calc. C 62.74, H 2.81, N 9.76, found C 62.65, H 2.63,
N 9.62; EIMS m/z (%) 286 [M]+ (100), 251 [M+-35] (10); HPLC (isocr.): 99.5% at 254 nm, 99.7% at
280 nm, tms = 3.7 min, tm = 1.1 min (ACN/H2O 70:30) (system 1); HPLC (gradient): 98.2% at 254 nm,
tms = 13.7 min, tm = 1.2 min (system 2); λmax 249, 306 nm.
7-Chloro-2-(4-methoxyphenyl)-1H-indole-3-carbonitrile (6j): According to general procedure B from
7-chloro-2-(4-methoxyphenyl)-1H-indole (9f, 174 mg, 0.675 mmol), NCTS (12, 184 mg, 0.672 mmol)
and boron trifluoride diethyl etherate (0.10 mL, 0.79 mmol). The solution was heated for
46 h. During heating a precipitate was formed which was filtered off before the washing steps.
The precipitate was combined with the resulting residue after removal of the solvent and recrystallized
from methanol. A slightly brown powder was obtained (98 mg, 52%). m.p.: 255–257 ◦C; IR (KBr):
vemax 3204 (NH), 2218 cm−1 (C
≡
N); 1H-NMR (DMSO-d6, 600.1 MHz):
δ (ppm) = 3.87 (s, 3H, CH3),
7.15–7.22 (m, 2H, Ar-H), 7.25 (t, 1H, J = 7.8 Hz, H-5), 7.38 (dd, 1H, J = 7.7, 0.9 Hz, Ar-H), 7.59 (dd, 1H,
J = 7.9, 0.9 Hz, Ar-H), 7.93–8.22 (m, 2H, Ar-H), 12.61 (s, 1H, NH); 13C-NMR (DMSO-d6, 150.9 MHz):
δ
(ppm) = 55.5 (CH3), 114.5 (2C), 117.1, 123.0, 123.3, 129.7 (2C) (CH), 82.2, 116.6, 116.9, 121.1, 130.0,
132.6, 147.1, 160.9 (C); C16H11ClN2O (282.73) calc. C 67.97, H 3.92, N 9.91, found C 68.04, H 3.70,
N 9.65; EIMS m/z (%) 282 [M]+ (100), 267 [M+-15] (36); HPLC (isocr.): 99.9% at 254 nm, 100.0% at
280 nm, tms = 4.7 min, tm = 1.1 min (ACN/H2O 60:40) (system 1); HPLC (gradient): 99.6% at 254 nm,
tms = 12.9 min, tm = 1.2 min (system 2); λmax 256, 310 nm.
2-Allyl-7-chloro-1H-indole-3-carbonitrile (6k): According to general procedure A with 2-(2-allyl-7-chloro
-1H-indol-3-yl)-2-oxoacetic acid (10d, 245 mg, 0.929 mmol), hydroxyl ammonium chloride (129 mg,
1.86 mmol) and sodium acetate (152 mg, 1.85 mmol) in a mixture of ethanol (10 mL) and water
(5 mL). After purification by column chromatography (toluene-ethyl acetate 2:1), recrystallization
from n-hexane-ethyl acetate 20:1 and further column chromatography (n-hexane-ethyl acetate 3:1),
a yellow solid (72 mg, 36%) was obtained. m.p.: 121–123 ◦C; IR (KBr): vemax 3214 (NH), 2220 cm−1
(C δ (ppm) = 3.70 (dt, 2H, J = 6.4, 1.5 Hz, CH2), 5.06–5.25 (m, 2H,
N); 1H-NMR (DMSO-d6, 600.1 MHz):
≡
CH2,), 6.04 (ddt, 1H, J = 16.7, 10.2, 6.5 Hz, allyl-CH), 7.17–7.23 (m, 1H, Ar-H), 7.33 (dd, 1H, J = 7.7,
0.9 Hz, Ar-H), 7.53 (dt, 1H, J = 7.7, 0.8 Hz, Ar-H), 12.51 (s, 1H, NH); 13C-NMR (DMSO-d6, 150.9 MHz):
δ
(ppm) = 31.1, 117.8 (CH2), 117.0, 122.6, 122.7, 133.3 (CH), 84.1, 115.4, 116.6, 129.0, 131.8, 148.6 (C);
C12H9ClN2 (216.67) calc. C 66.52, H 4.19, N 12.93, found C 66.48, H 4.07, N 12.61; APCI-MS m/z (%)
217 [M + H]+ (100), 190 [M-26]+ (100), 189 [M-27]+ (50); HPLC (isocr.): 94.8% at 254 nm, 98.9% at
280 nm, tms = 5.6 min, tm = 1.1 min (ACN/H2O 50:50) (system 3); HPLC (gradient): 95.0% at 254 nm,
tms = 11.2 min, tm = 1.2 min (system 3); λmax 279 nm.
2-(2-(1,3-Dioxan-2-yl)ethyl)-7-chloro-1H-indole-3-carbonitrile (6l): According to general procedure A with
2-(2-(2-(1,3-dioxan-2-yl)ethyl)-7-chloro-1H-indol-3-yl)-2-oxoacetic acid (10e, 120 mg, 0.357 mmol),
hydroxyl ammonium chloride (53 mg, 0.76 mmol) and sodium acetate (60 mg, 0.73 mmol) in a mixture
of ethanol (10 mL) and water (5 mL). After purification by column chromatogra◦phy (toluene:ethyl
acetate 5:1), a yellow-orange solid (42 mg, 44%) was obtained. m.p.: 118–120 C; IR (KBr): vemax
3212 (NH), 2955, 2851, 2229 cm−1 (C N); 1H-NMR (DMSO-d6, 600.1 MHz):
δ (ppm) = 1.35 (dtt, 1H,
≡
J = 13.4, 2.7, 1.4 Hz), 1.89 (dtt, 1H, J = 13.4, 12.4, 5.0 Hz), 1.94–2.03 (m, 2H), 2.93–3.03 (m, 2H), 3.66–3.78
(m, 2H), 4.01 (ddt, 2H, J = 10.3, 5.0, 1.4 Hz), 4.56 (t, 1H, J = 5.0 Hz), 7.19 (t, 1H, J = 7.8 Hz, H-5),
7.31 (dd, 1H, J = 7.7, 0.9 Hz, Ar-H), 7.51 (dd, 1H, J = 8.0, 1.0 Hz, Ar-H), 12.42 (s, 1H, NH); 13C-NMR
(DMSO-d6, 150.9 MHz):
δ (ppm) = 21.6, 25.3, 33.9, 66.1 (2C) (CH2), 100.1, 116.9, 122.4, 122.6 (CH),
84.1, 115.6, 116.5, 128.9, 131.9, 150.8 (C); C15H15ClN2O2 (290.75) calc. C 61.97, H 5.20, N 9.64, found
C 62.26, H 4.90, N 9.24; EIMS m/z (%) 290 [M]+ (69), 231 [M+-59] (48), 216 [M+-74] (51), 203 [M+-87]
(100), 189 [M+-101] (50), 114 [M+-176] (68), 87 [M+-203] (30); HPLC (isocr.): 99.7% at 254 nm, 99.9% at

Molecules 2018, 23, 64
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280 nm, tms = 4.9 min, tm = 1.1 min (ACN/H2O 50:50) (system 1); HPLC (gradient): 98.7% at 254 nm,
tms = 11.6 min, tm = 1.2 min (system 2); λmax 278 nm.
7-Iodo-2-(4-chlorophenyl)-1H-indole-3-carbonitrile (6m): According to general procedure B from
7-iodo-2-(4-chlorophenyl)-1H-indole (9i, 75 mg, 0.21 mmol), NCTS (12, 58 mg, 0.21 mmol) and
boron trifluoride diethyl etherate (50 µL, 0.40 mmol). The solution was heated for 30 h. During
heating, a precipitate was formed, which was filtered off before the washing steps. The precipitate
was combined with the resulting residue after removal of the solvent and r◦ecrystallized twice from
methanol. A colorless solid was obtained (10 mg, 13%). m.p.: 261–263 C; IR (KBr): vemax 3200
(NH), 2213 cm−1 (C N); 1H-NMR (DMSO-d6, 600.1 MHz):
δ (ppm) = 7.07 (t, 1H, J = 7.7 Hz, H-5),
≡
7.67 (dd, 1H, J = 7.9, 0.9 Hz, Ar-H), 7.69–7.72 (m, 2H, Ar-H), 7.75 (dd, 1H, J = 7.5, 1.0 Hz, Ar-H),
7.92–8.01 (m, 2H, Ar-H), 12.37 (s, 1H, NH); 13C-NMR (DMSO-d6, 150.9 MHz):
δ (ppm) = 118.4, 123.9,
128.9 (2C), 130.3 (2C), 133.6 (CH), 77.8, 84.1, 116.2, 127.6, 128.4, 135.0, 137.8, 145.4 (C); C15H8ClIN2
(378.60); HR-EIMS m/z calc. 377.94152, found 377.94223; EIMS m/z 378 [M]+• (100), 216 [M+-162]
(19); HPLC (isocr.): 98.1% at 254 nm, 97.9% at 280 nm, tms = 4.3 min, tm = 1.1 min (ACN/H2O 70:30)
(system 1); HPLC (gradient): 97.3% at 254 nm, tms = 14.2 min, tm = 1.2 min (system 2); λmax 250, 307 nm.
7-Iodo-2-(4-methoxyphenyl)-1H-indole-3-carbonitrile (6n): According to general procedure B from
7-iodo-2-(4-methoxyphenyl)-1H-indole (9j, 79 mg, 0.23 mmol), NCTS (12, 63 mg, 0.23 mmol) and
boron trifluoride diethyl etherate (50 µL, 0.40 mmol). The solution was heated for 30 h. During
heating, a precipitate was formed, which was filtered off before the washing steps. The precipitate
was combined with the resulting residue after removal of the solvent and recrystallized twice from
◦
methanol. A slightly red solid was obtained (12 mg, 14%). m.p.: 229–230 C; IR (KBr): vemax 3198
(NH), 2219 cm−1 (C N); 1H-NMR (DMSO-d6, 600.1 MHz):
δ (ppm) = 3.87 (s, 3H, OCH3), 7.04 (t, 1H,
≡
J = 7.8 Hz, H-5), 7.14–7.20 (m, 2H, Ar-H), 7.62 (dd, 1H, J = 7.9, 1.0 Hz, Ar-H), 7.70 (dd, 1H, J = 7.5, 1.0 Hz,
Ar-H), 7.88–7.94 (m, 2H, Ar-H), 12.15 (s, 1H, NH); 13C-NMR (DMSO-d6, 150.9 MHz):
δ (ppm) = 55.5
(CH3), 114.3 (2C), 118.1, 123.7, 130.0 (2C), 133.1 (CH), 77.5, 85.8, 116.7, 121.1, 128.6, 137.6, 146.9, 160.8 (C);
C16H11IN2O (374.18): calc. C 51.36, H 2.96, N 7.49, found C 51.57, H 2.95, N 7.19; EIMS m/z (%) 374 [M]+
(100), 359 [M+-15] (23); HPLC (isocr.): 99.6% at 254 nm, 98.9% at 280 nm, tms = 3.3 min, tm = 1.1 min
(ACN/H2O 70:30) (system 1); HPLC (gradient): 99.3% at 254 nm, tms = 13.5 min, tm = 1.2 min (system 2);
λmax 257, 310 nm.
7-Iodo-2-(3-methoxyphenyl)-1H-indole-3-carbonitrile (6o): According to general procedure B from
7-iodo-2-(3-methoxyphenyl)-1H-indole (9k, 155 mg, 0.444 mmol), NCTS (12, 121 mg, 0.444 mmol) and
boron trifluoride diethyl etherate (200 µL, 1.58 mmol). The solution was heated for 24 h. After removal
of the solvent, ◦the residue was recrystallized from methanol to yield a beige powder (31 mg, 19%).
1
m.p.: 199–201 C; IR (KBr): vemax 3234 (NH), 2214 cm−1 (C
≡
N); H-NMR (DMSO-d6, 400.4 MHz):
δ
(ppm) = 3.88 (s, 3H, OCH3), 7.06 (t, 1H, J = 7.7 Hz, H-5), 7.11–7.19 (m, 1H, Ar-H), 7.45–7.60 (m, 3H,
Ar-H), 7.61–7.71 (m, 1H, Ar-H), 7.74 (dd, 1H, J = 7.5, 1.0 Hz, Ar-H), 12.27 (s, 1H, NH); 13C-NMR
(DMSO-d6, 150.9 MHz):
δ (ppm) = 55.4 (CH3), 114.0, 115.8, 118.4, 120.7, 123.9, 130.1, 133.5 (CH), 77.7,
83.9, 116.4, 128.5, 129.9, 137.7, 146.5, 159.3 (C); C16H11IN2O (374.18) calc. C 51.36, H 2.96, N 7.49, found
C 51.57, H 2.75, N 7.15; APCI-MS m/z (%) 375 [M + H]+ (100), 248 [M-126]+ (31); HPLC (isocr.): 99.5%
at 254 nm, 99.8% at 280 nm, tms = 3.6 min, tm = 1.1 min (ACN/H2O 70:30) (system 3); HPLC (gradient):
95.1% at 254 nm, tms = 12.7 min, tm = 1.2 min (system 3); λmax 236, 306 nm.
7-Iodo-2-(pyridin-3-yl)-1H-indole-3-carbonitrile (6p): According to general procedure C from
7-iodo-2-(pyridin-3-yl)-1H-indole (9l, 103 mg, 0.321 mmol) and chlorosulfonyl isocyanate (200 µL,
2.30 mmol) in acetonitrile (60 mL). Purification by column chromatography (ethyl acetate-petroleum
ether-triethylamine 4:1:1) gave a brown powder (23 mg, 23%). m.p.: 157–160 ◦C; IR (KBr): vemax 3193
(NH), 2209 cm−1 (C N); 1H-NMR (DMSO-d6, 600.1 MHz):
δ (ppm) = 7.09 (t, 1H, J = 7.8 Hz, H-5),
≡
7.65 (ddd, 1H, J = 8.0, 4.8, 1.0 Hz, Ar-H), 7.70 (dd, 1H, J = 7.9, 0.9 Hz, Ar-H), 7.77 (dd, 1H, J = 7.5, 1.0 Hz,
Ar-H), 8.31 (ddd, 1H, J = 8.0, 2.3, 1.6 Hz, Ar-H), 8.75 (dd, 1H, J = 4.9, 1.6 Hz, Ar-H), 9.09 (dd, 1H, J = 2.3,

Molecules 2018, 23, 64
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0.9 Hz, Ar-H), 12.52 (s, 1H, NH); 13C-NMR (DMSO-d6, 150.9 MHz):
δ
(ppm) = 118.5, 123.7, 124.0, 133.6,
136.1, 149.0, 150.7 (CH), 78.0, 84.7, 116.1, 125.2, 128.3, 138.1, 143.9 (C); C14H8IN3 (345.14); HR-EIMS
m/z calc. 344.97574, found 344.97588; EIMS m/z (%) 345 [M]+• (100), 218 [M+-127] (28); HPLC (isocr.):
99.9% at 254 nm, 100.0% at 280 nm, tms = 3.2 min, tm = 1.3 min (ACN/H2O 60:40) (system 3); HPLC
(gradient): 98.9% at 254 nm, tms = 11.1 min, tm = 1.2 min (system 3); λmax 306 nm.
7-Iodo-2-isopropyl-1H-indole-3-carbonitrile (6q): According to general procedure C from 7-iodo
-2-isopropyl-1H-indole (9m, 172 mg, 0.603 mmol) and chlorosulfonyl isocyanate (250 µL, 2.87 mmol) in
acetonitrile (20 mL). Purification by column chromatography (petroleum ether-ethyl acetate 4:1) gave
slightly yellow crystals (58 mg, 31%). m.p.: 175–176 ◦C; IR (KBr): vemax 3214 (NH), 2967, 2929, 2872,
2219 cm−1 (C
≡
N); 1H-NMR (DMSO-d6, 600.1 MHz):
δ
(ppm) = 1.42 (d, 6H, J = 7.0 Hz, 2
×
CH3), 3.40
(hept, 1H, J = 7.0 Hz, CH), 6.98 (t, 1H, J = 7.7 Hz, H-5), 7.54 (dt, 1H, J = 7.9, 0.8 Hz, Ar-H), 7.63 (dd, 1H,
J = 7.5, 1.0 Hz, Ar-H), 11.83 (s, 1H, NH); 13C-NMR (DMSO-d6, 150.9 MHz):
δ (ppm) = 21.6 (2C) (CH3),
27.7, 117.8, 123.3, 132.1 (CH), 77.3, 81.8, 116.1, 127.9, 136.6, 156.3 (C); C12H11IN2 (310.14); HR-EIMS
m/z calc. 309.99614, found 309.99563; EIMS m/z (%) 310 [M]+ (62), 295 [M+-15] (100), 168 [M+-142]
(28); HPLC (isocr.): 99.5% at 254 nm, 100.0% at 280 nm, tms = 4.7 min, tm = 1.3 min (ACN/H2O 60:40)
(system 3); HPLC (gradient): 98.5% at 254 nm, tms = 12.1 min, tm = 1.2 min (system 3); λmax 276 nm.
2-Cyclopropyl-7-iodo-1H-indole-3-carbonitrile (6r):
2-cyclopropyl-7-iodo-1H-indole (9n, 194 mg, 0.685 mmol) and chlorosulfonyl isocyanate (300
According to general procedure C from
L,
µ
3.45 mmol) in acetonitrile (20 mL). Purification by column chromatography (petroleum ether-ethyl
◦
acetate 2:1) gave a slightly brown powder (25 mg, 12%). m.p.: 157–160 C; IR (KBr): vemax 3236,
1
2215 cm−1 (C
≡
N); H-NMR (DMSO-d6, 600.1 MHz):
δ
(ppm) = 1.16–1.20 (m, 2H, CH2), 1.21–1.27
(m, 2H, CH2), 2.30 (tt, 1H, J = 8.6, 5.3 Hz, CH), 6.96 (t, 1H, J = 7.8 Hz, H-5), 7.48 (dt, 1H, J = 7.9,
0.8 Hz, Ar-H), 7.60 (dd, 1H, J = 7.6, 1.0 Hz, Ar-H), 11.85 (s, 1H, NH); 13C-NMR (DMSO-d6, 150.9 MHz):
δ
(ppm) = 8.8 (2C) (CH2), 9.3, 117.4, 123.3, 132.0 (CH), 76.9, 81.3, 116.0, 128.0, 136.7, 152.4 (C); C12H9IN2
(308.12); HR-EIMS m/z calc. 307.98049, found 307.98031; EIMS m/z (%) 308 [M]+ (100), 281 [M+-27] (20),
181 [M+-127] (45), 154 [M+-154] (36); HPLC (isocr.): 99.8% at 254 nm, 99.1% at 280 nm, tms = 4.0 min,
tm = 0.9 min (ACN/H2O 60:40) (system 3); HPLC (gradient): 98.7% at 254 nm, tms = 11.7 min,
t
m = 1.0 min (system 3); λmax 284 nm.
2-Cyclopentyl-7-iodo-1H-indole-3-carbonitrile
2-cyclopentyl-7-iodo-1H-indole (9o, 107 mg, 0.344 mmol), NCTS (12, 94 mg, 0.345 mmol) and
boron trifluoride diethyl etherate (100 L, 0.789 mmol). The solution was heated for 42 h.
(
6s):
According to general procedure B from
µ
After removal of the solvent, the residue was recrystallized from methanol to yield a brown powder
(35 mg, 30%). m.p.: 131–132 ◦C; IR (KBr): vemax 3267 (NH), 2964, 2865, 2210 cm−1 (C
≡
N); 1H-NMR
CH2), 2.05–2.15
(DMSO-d6, 600.1 MHz):
δ
(ppm) = 1.60–1.75 (m, 2H, CH2), 1.75–2.05 (m, 4H, 2
×
(m, 2H, CH2), 3.42 (tt, 1H, J = 9.9, 7.9 Hz, CH), 6.98 (t, 1H, J = 7.7 Hz, H-5), 7.53 (dt, 1H, J = 7.9, 0.8 Hz,
Ar-H), 7.62 (dd, 1H, J = 7.5, 0.9 Hz, Ar-H), 11.86 (s, 1H, NH); 13C-NMR (DMSO-d6, 150.9 MHz):
δ
(ppm) = 25.4 (2C), 32.8 (2C) (CH2), 38.5, 117.7, 123.2, 132.1 (CH), 77.2, 82.4, 116.1, 128.0, 136.7,
154.5 (C); C14H13IN2 (336.18): calc. C 50.02, H 3.90, N 8.33, found C 50.08, H 3.61, N 8.11; APCI-MS m/z
(%) 337 [M + H]+ (100); HPLC (isocr.): 97.4% at 254 nm, 99.2% at 280 nm, tms = 5.1 min, tm = 1.1 min
(ACN/H2O 65:35) (system 1); HPLC (gradient): 96.8% at 254 nm, tms = 13.7 min, tm = 1.2 min
(system 2); λmax 282 nm.
1-Benzyl-7-chloro-1H-indole-3-carbonitrile
(13a):
According to general procedure B from
1-benzyl-7-chloro-1H-indole (257 mg, 1.06 mmol), NCTS (12, 291 mg, 1.07 mmol) and boron
trifluoride diethyl etherate (0.15 mL, 1.2 mmol). The solution was heated for 27 h. Purification
by column chromatography (petroleum ether-ethyl acetate-triethylamine 5:1.1) gave a white solid
(122 mg, 43%). m.p.: 117–119 ◦C; IR (KBr): vemax 2218 cm−1 (C
≡
N); 1H-NMR (DMSO-d6, 400.4 MHz):
(ppm) = 5.84 (s, 2H, CH2), 6.98–7.09 (m, 2H, Ar-H), 7.20–7.40 (m, 5H, Ar-H), 7.67 (dd, 1H, J = 7.9,
δ
1.1 Hz, Ar- H), 8.56 (s, 1H, H-2); 13C-NMR (DMSO-d6, 100.7 MHz):
δ (ppm) = 51.9 (CH2), 118.3, 123.4,

Molecules 2018, 23, 64
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125.4, 125.8 (2C), 127.5, 128.7 (2C), 140.3 (CH), 85.2, 115.0, 117.1, 130.3, 130.4, 138.0 (C); C16H11ClN2
(266.73): calc. C 72.05, H 4.16, N 10.50, gef. C 72.21, H 4.22, N 10.10; EIMS m/z (%) 266 [M]+ (28),
91 [M+-175] (100); HPLC (isocr.): 99.2% at 254 nm, 99.7% at 280 nm, tms = 3.6 min, tm = 1.1 min
(ACN/H2O 70:30) (system 3); HPLC (gradient): 95.8% at 254 nm, tms = 13.5 min, tm = 1.2 min
(system 2); λmax 277 nm.
1-Benzyl-7-bromo-1H-indole-3-carbonitrile
(
13b):
According to general procedure B from
1-benzyl-7-bromo-1H-indole (300 mg, 1.05 mmol), NCTS (12, 285 mg, 1.05 mmol) and boron
trifluoride diethyl etherate (0.15 mL, 1.2 mmol). The solution was heated for 46 h. After purification
by column chromatography (petroleum ether-ethyl acetate-triethylamine 5:1:1) and recrystallization
◦
−1
from methanol, a brown solid (64 mg, 20%) was obtained. m.p.: 126–128 C; IR (KBr): vemax 2219 cm
1
(C
≡
N); H-NMR (DMSO-d6, 400.4 MHz):
δ
(ppm) = 5.89 (s, 2H, CH2), 6.93–7.07 (m, 2H, Ar-H),
7.21 (t, 1H, J = 7.8 Hz, H-5), 7.24–7.35 (m, 3H, Ar-H), 7.53 (dd, 1H, J = 7.7, 1.0 Hz, Ar-H), 7.72 (dd, 1H,
J = 8.0, 1.0 Hz, Ar-H), 8.55 (s, 1H, H-2); 13C-NMR (DMSO-d6, 100.7 MHz):
δ (ppm) = 51.4 (CH2),
118.8, 123.7, 125.8 (2C), 127.4, 128.7 (2C), 129.0, 140.5 (CH), 85.1, 104.3, 114.9, 130.3, 131.5, 138.0 (C);
C16H11BrN2 (311.18): calc. C 61.76, H 3.56, N 9.00, found C 62.11, H 3.45, N 8.86; EIMS m/z (%)
310 [M]+ (19), 91 [M+-219] (100); HPLC (isocr.): 97.2% at 254 nm, 97.9% at 280 nm, tms = 3.8 min,
tm = 1.1 min (ACN/H2O 70:30) (system 1); HPLC (gradient): 96.8% at 254 nm, tms = 13.5 min,
t
m = 1.2 min (system 2); λmax 279 nm.
7-Chloro-1-methyl-1H-indole-3-carbonitrile (13c): According to general procedure B from 7-chloro-1
-methyl-1H-indole (190 mg, 1.15 mmol), NCTS (12, 313 mg, 1.15 mmol) and boron trifluoride diethyl
etherate (0.20 mL, 1.58 mmol). The solution was heated for 22 h. After purification by column
chromatography (petroleum ether-ethyl acetate 5:1), the resulting material◦was recrystallized twice
from methanol to yield a yellow-red solid (25 mg, 11%). m.p.: 146–147 C; IR (KBr): vemax 3119,
2220 cm−1 (C N); 1H-NMR (DMSO-d6, 400.4 MHz):
δ (ppm) = 4.16 (s, 3H, CH3), 7.24 (t, 1H, J = 7.8 Hz,
≡
H-5), 7.37 (dd, 1H, J = 7.7, 1.0 Hz, Ar-H), 7.61 (dd, 1H, J = 7.9, 1.0 Hz, Ar-H), 8.31 (s, 1H, H-2); 13C-NMR
(DMSO-d6, 100.7 MHz):
δ (ppm) = 37.4 (CH3), 118.1, 123.1, 124.9, 140.3 (CH), 83.8, 115.2, 117.4, 130.2,
130.9 (C); C10H7ClN2 (190.03): calc. C 63.01, H 3.70, N 14.70, found C 62.89, H 3.59, N 14.48; EIMS
m/z (%) 190 [M]+ (100); HPLC (isocr.): 99.4% at 254 nm, 100.0% at 280 nm, tms = 5.5 min, tm = 1.1 min
(ACN/H2O 50:50) (system 1); HPLC (gradient): 98.2% at 254 nm, tms = 12.0 min, tm = 1.2 min (system 2);
λmax 287 nm.
7-Bromo-1-methyl-1H-indole-3-carbonitrile
(
13d):
According to general procedure B from
7-bromo-1-methyl-1H-indole (120 mg, 0.571 mmol), NCTS (12, 164 mg, 0.599 mmol) and boron
trifluoride diethyl etherate (0.10 mL, 0.789 mmol). The solution was heated for 22 h. After purification
by column chromatography (petroleum ether-dichloromethane 1:1.5) and recrystallization from ethyl
acetate-n-hexane 1:10, a slightly brown powder (30 mg, 22%) was yielded. m.p.: 158–159 ◦C; IR (KBr):
vemax 2212 cm−1 (C
≡
N); 1H-NMR (DMSO-d6, 400.4 MHz):
δ (ppm) = 4.17 (s, 3H, CH3), 7.17 (t, 1H,
J = 7.8 Hz, H-5), 7.54 (dd, 1H, J = 7.7, 1.0 Hz, Ar-H), 7.65 (dd, 1H, J = 8.0, 1.0 Hz, Ar-H), 8.32 (s, 1H,
H-2); 13C-NMR (d6-DMSO, 100.7 MHz):
δ (ppm) = 37.5 (CH3), 118.6, 123.4, 128.4, 140.5 (CH), 83.7,
104.5, 115.1, 130.1, 132.0 (C); C10H7BrN2 (235.08): calc. C 51.09, H 3.00, N 11.92, found C 51.15, H 3.01,
N 11.59; EIMS m/z (%) 234 [M]+ (98), 219 [M+ -15] (2), 155 [M+ -79] (11); HPLC (isocr.): 99.2% at
254 nm, 99.8% at 280 nm, tms = 6.3 min, tm = 1.1 min (ACN/H2O 50:50) (system 1); HPLC (gradient):
99.7% at 254 nm, tms = 12.1 min, tm = 1.2 min (system 2); λmax 287 nm.
7-Bromo-1-methyl-2-phenyl-1H-indole-3-carbonitrile (13e): According to general procedure B from
7-bromo-1-methyl-2-phenyl-1H-indole (121 mg, 0.423 mmol), NCTS (12, 115 mg, 0.423 mmol) and
boron trifluoride diethyl etherate (200 µL, 1.58 mmol). The solution was heated for 24 h. After removal
of the solvent, the residue was recrystallized from methanol to yield slightly green crystals (54 mg, 41%).
m.p.: 176–178 ◦C; IR (KBr): vemax 2212 (C
≡
N), 1556 cm−1 1H-NMR (DMSO-d6, 600.1 MHz):
;
δ (ppm) = 4.02 (s, 3H, CH3), 7.23 (t, 1H, J = 7.9 Hz, H-5), 7.61 (dd, 1H, J = 7.7, 1.0 Hz, Ar-H), 7.62–7.70

Molecules 2018, 23, 64
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(m, 6H, Ar-H); 13C-NMR (DMSO-d6, 150.9 MHz):
δ
(ppm) = 35.3 (CH3), 118.4, 123.8, 129.0 (2C), 129.1,
130.1 (2C), 130.4 (CH), 85.0, 104.6, 115.5, 128.0, 129.7, 132.9, 150.2 (C); C16H11BrN2 (311.18): calc. C
61.76, H 3.56, N 9.00, found C 61.79, H 3.47, N 8.87; APCI-MS m/z (%) 311 [M + H]+ (100); HPLC
(isocr.): 99.3% at 254 nm, 99.7% at 280 nm, tms = 4.7 min, tm = 1.3 min (ACN/H2O 70:30) (system 3);
HPLC (gradient): 98.7% at 254 nm, tms = 13.3 min, tm = 1.2 min (system 3); λmax 239, 297 nm.
7-Iodo-1-methyl-2-phenyl-1H-indole-3-carbonitrile (13f): According to general procedure B from
7-iodo-1-methyl-2-phenyl-1H-indole (202 mg, 0.605 mmol), NCTS (12, 165 mg, 0.606 mmol) and
boron trifluoride diethyl etherate (250 µL, 1.97 mmol). The solution was heated for 24 h. After removal
of the solvent, the residue was recrystallized from methanol to yield a beige powder (79 mg, 36%). m.p.:
154–157 ◦C; IR (KBr): vemax 2212 cm−1 (C
≡
N); 1H-NMR (DMSO-d6, 600.1 MHz):
δ (ppm) = 4.03 (s, 3H,
CH3), 7.06 (t, 1H, J = 7.7 Hz, H-5), 7.60–7.73 (m, 6H, Ar-H), 7.89 (dd, 1H, J = 7.5, 1.1 Hz, Ar-H); 13C-NMR
(DMSO-d6, 150.9 MHz):
δ (ppm) = 35.6 (CH3), 118.8, 124.2, 129.0 (2C), 130.1 (2C), 130.3, 136.4 (CH),
75.8, 84.7, 115.4, 128.2, 129.1, 135.6, 150.2 (C); C16H11IN2 (358.18): calc. C 53.65, H 3.10, N 7.82, found C
53.69, H 3.05, N 7.63; EIMS m/z (%) 358 [M]+ (100), 229 [M+ -129] (21), 204 [M+ -154] (13), 102 [M+ -256]
(11); HPLC (isocr.): 100.0% at 254 nm, 99.9% at 280 nm, tms = 5.0 min, tm = 1.1 min (ACN/H2O 70:30)
(system 3); HPLC (gradient): 98.9% at 254 nm, tms = 14.5 min, tm = 1.2 min (system 2); λmax 300 nm.
7-Chloro-2-(2,3-dihydroxypropyl)-1H-indole-3-carbonitrile (14): N-Methylmorpholine N-oxide (107 mg,
0.783 mmol) and osmiumtetroxide (2.5% solution in tert-butanol, 250 µL, 0.0199 mmol) were added
successively to a solution of 2-allyl-7-chloro-1H-indole-3-carbonitrile (6k, 55 mg, 0.254 mmol) in
acetone-water 4:1 (10 mL). The mixture was stirred for 72 h at room temperature. Saturated sodium
thiosulfate solution (20 mL) was added and the mixture was stirred for 2 h. The aqueous phase was
separated and extracted with ethyl acetate (3
×
20 mL). The combined organic phases were dried over
sodium sulfate and, after removal of the solvent under reduced pressure, a beige solid (56 mg, 88%)
was obtained. m.p.: 130–132 ◦C; IR (KBr): vemax 3379/3252 (NH/OH), 2221 cm−1 (C
≡
N); 1H-NMR
(DMSO-d6, 600.1 MHz):
δ
(ppm) = 2.87 (dd, 1H, J = 14.1, 9.0 Hz, H-10), 3.10 (dd, 1H, J = 14.1, 4.2 Hz,
H-10), 3.36 (dt, 1H, J = 11.0, 5.6 Hz, H-30), 3.41 (dt, 1H, J = 11.0, 5.6 Hz, H-30), 3.91–4.02 (m, 1H, H-20),
4.79 (t, 1H, J = 5.6 Hz, OH), 4.93 (d, 1H, J = 5.2 Hz, OH), 7.19 (t, 1H, J = 7.8 Hz, H-5), 7.30 (dd, 1H,
J = 7.7, 0.9 Hz, H-6), 7.51 (dd, 1H, J = 8.0, 0.9 Hz, H-4), 12.34 (s, 1H, NH); 13C-NMR (DMSO-d6,
150.9 MHz):
δ (ppm) = 32.1 (CH2), 65.7, 70.7, 116.8, 122.2, 122.4 (CH), 84.8, 115.9, 116.4, 129.1, 131.8,
149.5 (C); C12H11ClN2O2 (250.68); HREI-MS m/z calc. 250.05036, found 250.05021; APCI-MS m/z (%)
251 [M + H]+ (34), 233 [M-17]+ (39), 203 [M-48]+ (16), 189 [M-62]+ (100); HPLC (isocr.): 99.5% at 254 nm,
99.3% at 280 nm, tms = 4.7 min, tm = 1.1 min (ACN/H2O 30:70) (system 3); HPLC (gradient): 99.7% at
254 nm, tms = 8.4 min, tm = 1.2 min (system 3); λmax 278 nm.
3.3. Molecular Docking
The program GOLD [34] (version 5.2.2) on a Windows 7 system was used for docking studies.
The crystal structure 4YLJ [31] is available from the protein data bank (PDB). Chain A was used
as template structure. Protein preparation was performed using the LigX function of molecular
operating environment (MOE) [50] (version 2013.0801) and the prepared protein was saved as mol2 file.
The ligands were also created with MOE, energy minimized and saved as mol2 files. Docking runs were
performed using the wizard of GOLD in the HERMES interface (version 1.6.2) (CCDC Software Ltd.,
Cambridge, UK). Missing hydrogen atoms were added, and the ligands and all water molecules
except HOH683 were removed from the protein structure. The binding site was defined as a zone
of 10 Å around the co-crystallized inhibitor. The implemented scoring function chemscore_kinase
was used for evaluation and ranking of the docking results. Search efficiency was set to 200%,
10 GA runs were performed, the function “generate diverse solutions” was activated and the option
“allow early termination” was turned off. For the retained water molecule, the options “toggle” and
“spin” were specified. Results of docking experiments were analyzed and visualized using USCF

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Chimera, version 1.11.2 (Resource for Biocomputing, Visualization, and Informatics at the University
of California, San Francisco, USA (supported by NIGMS P41-GM103311)) [51].
3.4. Protein Kinase Assays
Kinase activities were assayed in buffer A (10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM
g heparin/mL) at 30 ◦C at a final ATP concentration of 15
Tris-HCl pH 7.5, 50 µ µmol/L. Blank values
were subtracted and activities were expressed in percent of the maximal activity, i.e., in the absence
of inhibitors. Controls were performed with appropriate dilutions of DMSO. The GS-1 and RS peptide
substrates were obtained from Proteogenix (Oberhausbergen, France).
The kinase activity of GSK-3 (porcine brain, native, affinity purified on axin1-sepharose beads)
was assayed in buffer A with 0.5 mg BSA /mL + 1 mM DTT and 1 mg/mL of a GSK-3 specific substrate
(GS-1: YRRAAVPPSPSLSRHSSPHQSpEDEEE, pS stands for phosphorylated serine) in the presence
of 15 µmol/L [γ µL. After 30 min
-33P]-ATP (3000 Ci/mmol; 10 mCi/mL) in a final volume of 30
incubation at 30 ◦C, the reaction was stopped by harvesting onto P81 phosphocellulose supernatant
(Whatman) using a FilterMate harvester (Packard) and washing in 1% phosphoric acid. Scintillation
fluid was added and the radioactivity measured in a Packard counter [52].
DYRK1A, 1B, 2, 3 (human, recombinant, expressed in E. coli as GST fusion proteins) and CLK1, 2,
3, and 4 (mouse, recombinant, expressed in E. coli as GST fusion proteins) were assayed as described
for GSK-3 in Buffer A (supplemented extemporaneously with 0.15 mg BSA/mL + 1 mM DTT) with
1 µg of RS peptide (GRSRSRSRSRSR) as a substrate.
All data points for construction of dose response curves were recorded in triplicate. Typically, the
standard deviation of single data points was below 10%.
3.5. Cell-Based Assays
The inhibitors were added to the cultured cells from stock solutions in DMSO to the desired final
concentration. For cytotoxicity assays, HeLa cells were grown in 96-well plates (20,000–30,000 cells
per well) and incubated with the test compounds for 3 days before cell viability was evaluated with
the help of a tetrazolium dye assay (XTT assay, AppliChem GmbH, Darmstadt, Germany).
Inhibitor assays of endogenous DYRK1A activity were performed with overexpressed
GFP-SF3B1-NT as described previously [21]. Briefly, HeLa cells were grown in 6-well plates and
treated with test compounds for 18 h before cells were lysed with 100 µL SDS lysis buffer (20 mM Tris
HCl pH 7.4, 1% SDS) at 96 ◦C and sonicated. Total cellular lysates were analysed by immunoblotting
with a custom-made rabbit antibody for detecting phosphorylated T434 [43] and a goat antibody
for GFP (no. 600-101-215, Rockland Immunochemicals, Gilbertsville, PA, USA). pT434 signals
were quantified using the AIDA Image Analyzer 5.0 program (Raytest, Straubenhardt, Germany).
Relative phosphorylation of SF3B1 was calculated by normalisation to total protein levels as determined
from GFP immunoreactivity. IC50 values were determined by non-linear curve fitting using the
GraphPad Prism 5.0 program (GraphPad Software, La Jolla, CA, USA).
3.6. Calculation of Physicochemical Properties
The predicted logP and logS values were calculated using the software MarvinSketch [47
]
(version 17.13). The theoretical solubility Scalc was generated from the logS value. The implemented
consensus method of MarvinSketch was used for calculation of the logP value. This method uses a
model which combines the method of Klopman et al. [53], the ChemAxon model based on the method
of Viswandhan et al. [54] and the PhysProp database [55]. The logS value was calculated using the
solubility plugin which uses a fragment based method according to Hou et al. [56].
3.7. Determination of Thermodynamic Solubility
For solubility assays an aqueous phosphate buffer pH 7.4 was used. The buffer was prepared by
dissolving Na2HPO4
×
2 H2O (298 mg), KH2PO4 (19 mg) and NaCl (800 mg) in water (100 mL). The pH

Molecules 2018, 23, 64
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was adjusted to 7.4 by addition of hydrochloric acid. The thermodynamic solubility was determined
using a shake-flask method with quantification by a HPLC method. The test compounds (500 g)
were incubated with aqueous phosphate buffer pH 7.4 (400 L) in sealed Whatman MiniUniPrep vials
µ
µ
(GE Healthcare, Freiburg, Germany). The mixtures were shaken at 25 ◦C and 400 rpm for 24–96 h
(IKA KS 3000 ic control, IKA-Werke, Staufen, Germany). After 24 h, 48 h and 72 h, the mixtures
were visually inspected for remaining solids and the solids were filtered off. The concentrations in
the resulting saturated solutions were quantified by isocratic HPLC using calibration with external
standard. If equilibrium was not reached after 72 h, solubility of a further sample was determined after
96 h. For calibration, a stock solution of 10 mM of the test compounds in DMSO was prepared and the
stock solution was diluted with ACN to suitable concentrations. The AUC at the wavelength maxima
was used for quantification. If the compounds caused signals lower than the limit of quantification,
the thermodynamic solubility was indicated as <0.5
calibration solutions.
µM, which was the lowest concentration of the
3.8. Determination of Kinetic Solubility
The kinetic solubility was determined using laser nephelometry. A stock solution of the test
compounds in DMSO was prepared and further diluted with DMSO to achieve a dilution series of 8–12
solutions with different concentrations. The DMSO solutions (2
plate and diluted with aqueous phosphate buffer pH 7.4 (198
µ
L) were placed in a row on a 96-well
µL). The well plate was scanned by a
nephelometer (Nephelostar Plus, BMG Labtech, Ortenberg, Germany). Unsolved particles scatter the
laser light which is detected by the nephelometer. The intensity of the scattered light is proportional to
the particle concentration in the suspension. The intensity of the scattered light was plotted versus
the concentration to obtain a kick off curve. The concentration at which the compounds began to
precipitate was directly read out from the kick off curve.
4. Conclusions
For the development of novel DYRK1A inhibitors, a fragment-based strategy was applied starting
from 7-chloro-1H-indole-3-carbonitrile. Five congeners of the so-designed series displayed IC50 values
at double-digit nanomolar concentrations. Docking experiments revealed that the halogen substituents
at position 7 of the indole core are likely to interact with the hinge region of the protein kinase by a
water-mediated halogen bond. At position 2 of the indole scaffold, only aromatic or lipophilic residues
were tolerated. The 2-phenyl-substituted derivative 6h was the most potent inhibitor of the series
and showed activity on the isolated enzyme (IC50 DYRK1A 10 nM) as well as on DYRK1A-mediated
phosphorylation of SF3B1 in HeLa cells (IC50 320 nM). However, 6h displayed only low selectivity
compared to related kinases of the CMGC group and poor aqueous solubility. To increase the solubility
of the compounds, hydrophilic or aliphatic residues at position 2 were introduced. Replacing the
2-phenyl substituent with pyridin-3-yl or cyclopentyl residues reduced the logP value and increased
the solubility while the DYRK1A activity was only slightly affected. Further modifications of the
7-halogenindole-3-carbonitrile parent structure are underway aiming at the development of potent,
highly selective and water-soluble DYRK1A inhibitors.
Supplementary Materials: The following are available online, Figure S1: Predicted binding mode of 14 in the
ATP binding site of DYRK1A, Table S2: Calculated physicochemical properties of all test compounds; synthesis
procedures of the intermediates 8, 9, 10 and 12; NMR, mass and IR spectra of compound 6h.
Acknowledgments: Rosanna Meine is grateful for financial support by the state of Lower Saxony, Germany,
within the graduate program “Processing of poorly soluble drugs at small scale”. Laurent Meijer was funded
by grants from the Fondation Jérôme Lejeune and Fonds Unique Interministériel (FUI-TRIAD). We thank
Simone Bamberg-Lemper for excellent technical assistance.
Author Contributions: Rosanna Meine, Walter Becker, Laurent Meijer, Hannes Falke and Conrad Kunick
conceived and designed the experiments; Rosanna Meine, Walter Becker, Hannes Falke and Nadège Loaëc
performed the experiments; Rosanna Meine, Walter Becker, Lutz Preu and Nadège Loaëc analyzed the data; and
Rosanna Meine, Walter Becker, Laurent Meijer, and Conrad Kunick wrote the paper.

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Conflicts of Interest: The authors declare no conflict of interest.
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