A simple approach to multifunctionalized N1-alkylated 7-amino-6-azaoxindole derivatives using their in situ stabilized tautomer form

Article abstract of DOI:10.1016/j.tet.2016.08.055

A simple approach for the synthesis of multifunctionalized N1-alkyl 7-amino-6-azaoxindole derivatives was developed and investigated. Formation of 5-amino- and 7-amino-6-aza-2-oxindoles 12a and 13a, respectively, was achieved using an intramolecular reductive cyclization as a key step. Subsequent alkylation of the pyrrole N1 atom in 12a led to the desired N1-alkylated compounds 22a–24 comprising different functionalities. Alkylation of 5-amino-substituted regioisomer 13a under the same conditions as used for 12a did not resulted in N1-alkylated products. To find a plausible explanation for the observed differences in reactivity, we investigated the possible tautomers of 12a and 13a and the distribution of their neutral and ionized forms in a gas phase. The relevant physicochemical properties of compounds 12a and 23 were determined.

Full text of DOI:10.1016/j.tet.2016.08.055

Tetrahedron xxx (2016) 1e12
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Tetrahedron
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A simple approach to multifunctionalized N1-alkylated 7-amino-6-
azaoxindole derivatives using their in situ stabilized tautomer form
,
b
b
c
Nikolay T. Tzvetkova , Beate Neumann , Hans-Georg Stammler , Liudmil Antonov
*
a Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
b
€
Department of Chemistry, University of Bielefeld, Universitatsstr. 25, 33615 Bielefeld, Germany
c Institute of Organic Chemistry, Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 9, Sofia 1113, Bulgaria
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple approach for the synthesis of multifunctionalized N1-alkyl 7-amino-6-azaoxindole derivatives
was developed and investigated. Formation of 5-amino- and 7-amino-6-aza-2-oxindoles 12a and 13a,
respectively, was achieved using an intramolecular reductive cyclization as a key step. Subsequent al-
kylation of the pyrrole N1 atom in 12a led to the desired N1-alkylated compounds 22ae24 comprising
different functionalities. Alkylation of 5-amino-substituted regioisomer 13a under the same conditions
as used for 12a did not resulted in N1-alkylated products. To find a plausible explanation for the observed
differences in reactivity, we investigated the possible tautomers of 12a and 13a and the distribution of
their neutral and ionized forms in a gas phase. The relevant physicochemical properties of compounds
12a and 23 were determined.
Received 20 June 2016
Received in revised form 16 August 2016
Accepted 18 August 2016
Available online xxx
Keywords:
Amino-azaindole
Reductive cyclization
N-Alkylation
Synthesis
Ó 2016 Elsevier Ltd. All rights reserved.
Tautomers
1. Introduction
benzamido-substituted 4-azaindole NTZ-2130 (5) has been recently
discovered as a promising monoamine oxidase B (MAO-B) inhibitor
Azaindoles represent a growing class of condensed heterobicycles
thatdisplayawiderangeofsignificantbiologicalandpharmacological
properties.1,2 In contrast to indoles, there are relatively few examples
of natural products such as variolin3 and grossularine4 alkaloids
comprising an azaindole core. However, the most azaindoles are
synthetic products or a fragment of synthetic analogues of naturally
occurring alkaloids.5 Due to their physicochemical features,6 azain-
doles are frequently exploited as bioisosteres of indoles and purines,7
and belong to the so-called ‘privileged structures’ in drug de-
velopment.2,8,9 The azaindolenucleusshows better solubilityinwater
than the indole scaffold due to the sp2-hybridized nitrogen of the
pyridine moiety by providing an additional position for protonation
and salt formation.7 Structurally, azaindoles consist of a fused [5,6]-
membered ring system, which arises from the different condensa-
for neuroprotective treatment of Parkinson’s disease (PD).10 The N1-
methyl-1-morpholin-4-ylmethanone-substituted
5-azaindole
GSK554418A (6) has been identified as a potent cannabinoid re-
ceptor type 2 (CB2) agonist for the therapy of chronic pain.11 An ex-
ample for advanced drug development represents the N1-
phosphonooxymethyl prodrug BMS-663749 (7) comprising 6-
azaindole core. This compound was profiled as an effective HIV-1
attachment inhibitor in a variety of preclinical in vitro and in vivo
models.12 Furthermore, highly functionalized 7-azaindole 8 was re-
ported as peroxisome proliferator-activated receptor gamma (PPAR
g)
modulator for potential treatment of type 2 diabetes (T2DM).13
As a result of the versatile biological and physicochemical
properties of azaindoles, several methodologies for their synthesis
including heterocyclisations,7,9,14 and organometallic-promoted
heteroannulations15 have been developed. The large number of
these methods has been deduced from the classical synthetic
strategies used for the formation of indole ring systems.7,9,14,16
However, the most common synthetic procedures were applied
for the construction of 7-azaindole derivatives,7b,14,17 whereas
considerably few have been developed for the synthesis of highly
functionalized 6-azaindole analogues.7b,17
tion of the p-electron-deficient pyridine ring and the p-electron-rich
pyrrole ring resulting in 4-, 5-, 6- or 7-azaindole (pyrrolopyridine)
scaffolds 1e4, respectively, differ in the location of the pyridine ni-
trogen atom (Fig. 1, panel A). Furthermore, the ring fusion in 1e4 is
consistent with the specific arrangement of the substitution pattern
and variety of biological activities of their N1-substituted analogues
5e8 (Fig. 1, panel B). For example, N1-3,4-difluorobenzoyl-
An explanation for this fact could be the unfavorable electron-
deficient constitution of the pyridine ring caused by an altered
electron delocalization during the heterocyclization step to form
p-
* Corresponding author. Fax: þ359 88 411 7458; e-mail address: ntzvetkov@gmx.
de (N.T. Tzvetkov).
http://dx.doi.org/10.1016/j.tet.2016.08.055
0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Tzvetkov, N. T.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.08.055

2
N.T. Tzvetkov et al. / Tetrahedron xxx (2016) 1e12
A)
NH
NH
NH
NH
N
N
N
N
1
2
3
4
4-azaindole scaffold
5-azaindole scaffold
6-azaindole scaffold
7-azaindole scaffold
pyrolo[3,2-b]pyridine
pyrolo[3,2-c]pyridine
pyrolo[2,3-c]pyridine
pyrolo[2,3-b]pyridine
F
Ph
N
B)
OMe
F
HO2C
O
H
N
Cl
N
O
Me
O
Cl
N
N
O
Me
N
O
N
N
O
O
O
HO
O
N
N
O
OH
H
P
N
MeO
F
O
N
F
5
6
NTZ-2130
(MAO-B inhibitor)
GSK554418A
(CB2 agonist)
OMe
N
OMe
7
8
BMS-663749
(HIV-1 inhibitor)
(PPARg modulator)
Fig. 1. Structures of azaindole scaffolds 1e4 (A) and biologically active compounds 5e8 with a 4-, 5-, 6- and 7-azaindole core (B).
the pyrrolo[2,3-c]pyridine ring system.7b Hence, many conven-
tional indole synthesis methods are not effective or have limited
application for the preparation of 6-azaindole scaffold.7b
In this contribution, a facile approach for the synthesis of drug-
like 5-amino- and 7-amino-6-azaoxindole derivatives 13a and 12a
using reductive cyclization as a key step has been recently de-
veloped.18 When starting from the commercially available 2-
amino-4-chloropyridine (9) both, 5-amino- or 7-amino-2-
hydroxy[2,3-c]pyridine (12a or 13a) are easily accessible in
a regioselective manner depending on the substitution pattern of
To continuously explore the substitution of the nitrogen N1, we
performed series of N-alkylation reactions with a variety of resi-
dues (R) in the very last step. While the regioselective N-alkylation
of 12a led to the formation of the desired N1-alkyl 7-amino-6-
azaoxindole derivative 14, synthesis of its 5-amino-substituted
isomer 13a failed due to the unfavorable tautomer forms under the
same reaction conditions (Fig. 2).
In this article we summarize an investigation of the influence of
the amino function and the reaction conditions on the regiose-
lective N1-alkylation of 7-amino- and 5-amino-6-azaoxindole de-
rivatives 12a and 13a. To explain the completely different
accessibility for substitution of the nitrogen atom N1 and prefer-
ences of N1 versus N6 alkylation, we systematically studied the
possible tautomer forms of structures 12 and 13 by considering the
energetics of the neutral and ionized species during the alkylation
step.
the
corresponding
3-nitro-
or
5-nitro-substituted
4-
chloropyridine-2-amine precursors 10 or 11, respectively (Fig. 2).
Consequently, we were interested in the feasible formation of N1-
substituted 6-azaindoles containing an amino group, either in the
5- or 7-position of the pyrrolo[2,3-c]pyridine framework, re-
spectively. In either case the favored structures would consist
a hydrogen bond donor (HBD, free NH2 group) and an adjacent
hydrogen bond acceptor (HBA, pyridine nitrogen atom) in a highly
functionalized pyrrolo[2,3-c]pyridine ring system. Such structures
may be considered as adrenomimetics interacting with many im-
portant drug targets such as protein kinases19 and ecto-nucleotid-
ases.20 Moreover, N1-alkylated 5-amino- and 7-amino-6-azaindole
derivatives may act as precursors for further modifications allowing
the introduction of diverse substitution pattern, e.g., substitution at
the pyridine nitrogen, CO2Et and/or the amino group.
2. Results and discussion
The synthesis of the 5- and 7-amino-substituted 6-azaoxindoles
12a and 13a was achieved following an adapted and optimized
reaction sequence.18,21 The advantage of this three-steps synthesis
consists in the avoiding of protection of the amino function, which
allows a regioselective formation of the desired products 12a and
This work
OH
N
EtO2C
N-alkylation
1
R
7
7-NH2
OH
NH
EtO2C
Cl
Cl
N
NH2
1
14
5
3
O2N
5
7
N
NH2
N
NH2
N
H2N
OH
EtO2C
9
10, 3-NO2
11, 5-NO2
12a, 7-NH2
13a, 5-NH2
5-NH2
1
N
R
5
H2N
N
15
Fig. 2. Feasible synthetic approach to multifunctionalized N1-alkylated 7-amino-6-aza-2-oxindole derivatives.
Please cite this article in press as: Tzvetkov, N. T.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.08.055

N.T. Tzvetkov et al. / Tetrahedron xxx (2016) 1e12
3
13a and their hydrochloride salts in high yields and purity even in
large scale (route A, Scheme 1).
product ratio, 10:11¼1.3:1) via 2-nitraminopyridine rearrange-
ment.18,22 Separation and isolation of the regioisomers was per-
formed following a modified three-step purification procedure (for
details, see Supplementary data).22b
Compounds 18 and 19 were obtained in 87 and 84% yield, re-
spectively, by a condensation of 10 and 11 with diethyl malonate in
the presence of sodium hydride as a base in DMF.18,21a Equally high
yields were obtained when the addition of 10 or 11 to the malonic
ester proceeds at a reaction temperature not higher than 52e55 ꢀC.
The conversion of 3-substituted pyridine was completed in shorter
reaction times than the condensation of its 5-substituted analogue
(40 min for 10 to 18 cf. 4 h for 11 to 19). This fact could be explained
by the faster mesomeric destabilization of the reactive in-
termediate 10 following by a rapid tautomeric stabilization of 18
under basic conditions. The X-ray structures of 18 and 19 provide
insight into their structural features (Fig. 3).23 Both form dimers
connected by NeH/N hydrogen bonds (Fig. S3, Supplementary
data). Compound 18 contains two independent molecules in the
asymmetric unit and shows a second H-bond intramolecular to an
oxygen atom of the ortho-nitro group. It reveals two rotamers A and
B, differing in the orientation of both ethyl groups (Fig. S4,
Supplementary data). The 5-amino-substituted derivative 19 con-
tains a second H-bond intermolecular to an oxygen atom of a CO2Et
group of a neighboring molecule and shows only one molecule per
asymmetric unit. The importance of the free amino function for the
stabilization of the in situ formed tautomers during the whole
synthetic approach was demonstrated by reaction of 10 and 11 with
DMF-DMA in methanol towards the corresponding amino-
protected compounds 16 and 17, respectively (route B, Scheme
1).24 However, their further condensation with various malonic
esters (R¼Me, Et, CH2Ph) failed to produce 20 and 21, respectively,
probably due to the rigid structure of the reactive intermediates 16
and 17. Therefore, an alternative three-step conversion of 17 to-
wards 5-amino-6-azaoxindole derivative 13a via protection of the
amino group was not feasible.25
Scheme 1. Synthesis of 5- and 7-amino-6-azaoxindole derivatives 12a and 13a (for
route A, see Ref. 18). Reagents and conditions: (i) NaH (3.6e4.2 equiv), malonic acid
diethyl ester (3.6e4.0 equiv), DMF, 40e55 ꢀC, 40e50 min (for 18) and 3e4 h (for 19);
Yield 87% (18) and 84% (19);21a (ii) DMFeDMA (1.0 mL per 1.0 mmol), MeOH/THF (2:1),
75 ꢀC, 16 h (for 10) and 20 h (for 11); Yield 87% (16) and 68% (17); (iii) 1) Zn dust
(20.0 equiv), AcOHeH2O (3:1), rt, 15 min (for 12a) and 20 min (for 13a), 2) NH4OH
(25%), rt, 5e20 min (method 1); Yield 78% (12a) and 96% (13a); (iv) 1) H2 (40 psi), Pd/C
(10%), EtOH, rt, 90 min, 2) HCl (18%), rt, over night (method 2); Yield 42% (12a) and 94%
(13a); (v) 1) SnCl2$2H2O (10.0 equiv), EtOH, 30 ꢀC, 2 h, 2) NH4OH (25%), rt, 30 min
(method 3); Yield 62% (12a).
Introduction of a nitro group at the 3- and 5-position of 2-
amino-4-chloropyridine (9) afforded a mixture of the respective
3- and 5-nitro-substituted regioisomers 10 and 11 (LC/ESI-MS
Fig. 3. X-ray crystal structures of 18 and 19. The thermal ellipsoids are drawn at 50% probability level.
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4
N.T. Tzvetkov et al. / Tetrahedron xxx (2016) 1e12
Subsequently, the desired 7-amino- and 5-amino-6-
as starting materials in series of N-alkylation experiments. Differ-
ent attempts were performed in order to access the desired N1-
substituted products and to optimize the reaction conditions.
Consequently, reaction time, temperature, type, and the amount of
bases and reagents were varied (Table 2).
azaoxindoles 12a and 13a were obtained by selective reduction of
the nitro group in 18 and 19 followed by an intramolecular heter-
ocyclization.18 This key step of the synthesis of amino-substituted
6-azaoxindole derivatives was performed applying different re-
action conditions, e.g., variation of the solvent, the reaction time,
and the amount of the reducing agent that was used (Table 1). We
found that fast reactions and highest yields of 12a (78%) and 13a
(96%) were obtained when the reductive cyclization was conducted
as an one-pot reaction with a large excess of zinc dust (20.0 equiv)
in AcOH/H2O (3:1) followed by addition of 25% aqueous ammonia
for a reaction time of 20e40 min at room temperature (method
1).18 Compound 13a was obtained in equally high yield (94%) when
the reductive step was performed in a Parr hydrogenator at 40 psi
using catalytic amount of palladium on charcoal (10%) in ethanol as
a solvent and subsequent heterocyclization by treatment of the
diamino intermediate with 18% hydrochloric acid (method 2).18
However, the reductive cyclization proceeds slower with reaction
times of up to 15 h. The proposed mechanism for the formation of
13a using Pd/C catalyst is illustrated in Fig. S5 (see Supplementary
data). There was no improvement towards the yield of 12a (20%)
when applying method 2.26 We observed that the isolation of the
products was difficult under these conditions. Depending of the
reaction conditions the synthesis of amino-substituted 6-
azaoxindoles is more complex when comparing with their unsub-
stituted analogues due to the formation of different tautomers and
the basicity of the free amino group. Thus, neutralization of the
reaction mixture to pH 6e7 after completed reaction is required in
order to obtain the products in high yields and purity. With the aim
to further improve the yield of 12a, conversion of 18 with a large
excess of SnCl2$H2O (20.0 equiv) in ethanol under ultrasonic irra-
diation27 at 30e35 ꢀC was conducted (method 3). Though, no sig-
nificant improvement for the formation of 12a (62% cf. 78% isolated
yield by method 1) was observed, even when the cyclization step
was performed at 70 ꢀC.
In general, regioselective N1-alkylation was performed with
1.0e1.5 equiv of the corresponding alkyl halides in DMF as a solvent
under basic reaction conditions.28 First, N1-substitution experi-
ments were conducted by reaction of 7-amino-substituted 6-
azaoxindole 12a with methyl methanesulfonate (MMS, 25),28b
methyl iodide (26), benzyl bromide (27), and 2-bromo-N-phene-
thylacetamide (28)29 as alkylation reagents. Substitution of the
thermodynamically preferred N1-position of 12a with 25 and 26
was achieved using potassium carbonate (1.1e1.25 equiv) as a base
at room temperature (method 1) affording N-methylated com-
pound 22a in moderate to good yields (Table 2, entries 1e4). The
reaction shows strong dependency of the amount of base and alkyl
halide that was used. The best yield (78%) was achieved when using
25 (1.35 equiv) as methylation reagent in the presence of 1.25 equiv
of potassium carbonate (entry 3), whereas shorter reaction time of
6 h was observed by methylation with methyl iodide (1.1 equiv)
leading to the formation of 22a in 52% isolated yield (entry 4). Al-
kylation of 12a with benzyl bromide was performed under different
conditions yielding N-benzylated product 23 in poor to high yields
(Table 2, entries 6e8). In the presence of sodium hydride (1.1 equiv)
at room temperature (method 2) the product was isolated in 36%
yield (entry 6). In this case when an excess of benzyl bromide
(1.5 equiv) was used, traces of bis-alkylated products were detected
by LC/ESI-MS analysis after a reaction time of up to 6 h. Moderate
yield of 23 (53%) was achieved when using method 1, however, the
alkylation took longer (entry 7). A significant improvement was
observed when the conversion was performed with stoichiometric
amount of 27 using both, sodium hydride (1.1 equiv) and K2CO3
(1.1 equiv) as bases starting the reaction at 0 ꢀC with gradually
increasing to room temperature for 6 h (method 3). Following this
Table 1
Methods and reaction conditions for the synthesis of 12a and 13a
Entry
Starting compd.
Reducing agent
Solvent
Cyclization
Methodb
Time (min)
Product
Yield (%)a
1
2
3
4
5
6
7
8
18 (3-NO2)
18 (3-NO2)
18 (3-NO2)
18 (3-NO2)
18 (3-NO2)
18 (3-NO2)
19 (5-NO2)
19 (5-NO2)
Zn (5.0 equiv)
Zn (10.0 equiv)
Zn (20.0 equiv)
AcOHeH2O (3:1)
AcOHeH2O (3:1)
AcOHeH2O (3:1)
EtOH
EtOH
EtOH
NH4OH (25%), H2O, rt
NH4OH (25%), H2O, rt
NH4OH (25%), H2O, rt
HCl (18%), EtOH, rt
HCl (18%), NH4OH (25%), H2O, 70 ꢀC
NH4OH (25%), H2O, 30 ꢀCc
NH4OH (25%), H2O, rt
HCl (18%), EtOH, rt
1
1
1
2
3
3
1
2
135
50
20
420
150
150
40
12a
12a
12a
12ae
12a
12a
13a
13a
65
74
78
20
48
62
96
94
H2 (40 psi), Pd/C (10%)c
SnCl2$2H2O (5.0 equiv)d
SnCl2$2H2O (10.0 equiv)d
Zn (20.0 equiv)
AcOHeH2O (3:1)
EtOH
H2 (40 psi), Pd/C (10%)
900
a
Isolated yields, unless otherwise noted.
Ref. 18 for method 1 and 2.
Hydrogenation was carried out in a Parr apparatus.
Under ultrasonic irradiation.
b
c
d
e
Product isolated as a hydrochloride salt.
Pure products 12a and 13a were isolated after repeated re-
method, compound 23 was isolated in 96% yield (entry 8). Con-
crystallization from different solvent mixtures. Structure de-
termination was carried out by NMR and LC/ESI-MS analysis.
Depending on the reaction conditions 6-azaoxindoles may exist as
a mixture of their keto and enol forms. However, based on NMR
spectral data in DMSO-d6 only the hydroxy tautomer of both
products was identified. The reason could be the stabilizing effect of
the ester group at the 3-position of 12a and 13a.18 An autoxidation
of the enol form was not detected (ESI-MS m/z 220 [MꢁH]e/222
[MþH]þ for 12a and 13a).21c
version of 12a with 2-bromo-N-phenethylacetamide (28) was
performed using method 1 and 3 with major modifications (Table 2,
entries 10e13). In this case, we found that the alkylation with 28
requires higher reaction temperatures of about 80 ꢀC than the re-
action with the above used alkylation reagents. The desired N-
alkylated product 24 was obtained in poor to moderate yields
(31e51%) when the alkylation was performed using method 1 at
room temperature (entries 10e12). An increasing of the reaction
temperature to 70 ꢀC was required. There was an improvement
towards the yield of 24 (51%) when the thermodynamic sub-
stitution took longer requiring a reaction time of up to 72 h (entry
12). Fast reaction and highest yield of 24 (73%) was obtained by
Following the proposed strategy for the formation of N1-
substituted amino-6-azaoxindole derivatives (14 or 15) possess-
ing an ester group at 3-position, we used compounds 12a and 13a
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N.T. Tzvetkov et al. / Tetrahedron xxx (2016) 1e12
5
Table 2
N-alkylation of amino-6-azaoxindoles 12a and 13a
O
OH
OH
EtO2C
EtO2C
Me
S OMe
Me–I
Ph
Br
R–X
Base, DMF
1
NH
N
R
O
25
26
27
5
7
R–X =
H
N
N
N
H2N
H2N
Ph
Br
12a, 7-HN2
13a, 5-NH2
O
28
Entry
Starting compd.
ReX (equiv)
Base (equiv)
Temp (ꢀC)
Method
Time (h)
Product
Yield (%)a
1
2
3
4
5
6
7
8
12a (7-NH2)
12a (7-NH2)
12a (7-NH2)
12a (7-NH2)
13a (5-NH2)
12a (7-NH2)
12a (7-NH2)
12a (7-NH2)
25 (1.1)
25 (1.3)
25 (1.35)
26 (1.1)
26 (1.1)
27 (1.5)
27 (1.1)
27 (1.0)
K2CO3 (1.1)
K2CO3 (1.2)
K2CO3 (1.25)
K2CO3 (1.2)
K2CO3 (1.1)
NaH (1.1)
rt
rt
rt
rt
rt
rt
rt
0/rt
1
1
1
1
1
2
1
3
19
24
11
6
24
6
22a
22a
22a
22a
d
49
74
78
52
NRb
36c
53
23
23
23
K2CO3 (1.1)
NaH (1.1)
29
6
96
K2CO3 (1.1)
NaH (1.1)
9
13a (5-NH2)
27 (1.2)
0/rt
3
12
d
NR
K2CO3 (1.1)
K2CO3 (1.1)
K2CO3 (1.2)
K2CO3 (1.2)
NaH (1.1)
10
11
12
13
12a (7-NH2)
12a (7-NH2)
12a (7-NH2)
12a (7-NH2)
28 (1.1)
28 (1.2)
28 (1.2)
28 (1.2)
rt/65
rt/65
rt/70
0/rt/70
1
1
1
3
24
48
72
12
24
24
24
24
31
38
51
73
K2CO3 (1.1)
K2CO3 (2.5)
NaH (1:1)
14
15
13a (5-NH2)
13a (5-NH2)
28 (1.2)
28 (2.0)
rt
1
3
4
192
d
d
NR
NR
0/rt/80
K2CO3 (1.1)
a
Isolated yields, unless otherwise noted.
b
c
NR¼no reaction, partly formation of by-products or recovery of starting material.
Product accompanied with <9% of bis-alkylated product.
applying method 3 with gradually increasing of the reaction tem-
perature from 0 to 70 ꢀC for 12 h (entry 13).
existence of the 2-OH- and 7-NH2ehydrogen atoms by proton ex-
change at 298 K (see Supplementary data). This observation ex-
cludes the formation of a 2-methoxy derivative. Compare to its
precursor 12a, the 1H signal for the 2-OH group in N1-methylated
product 22a is shifted from about 12 to 10 ppm, whereas no sig-
nificant changes could be observed for the 7-NH2 protons
(6.91 ppm for 12a vs. 6.97 ppm for 22a). The coupling constant
between C-4 and C-5 methine protons 4-H and H-5 of the pyridine
moiety in both compounds remains almost unchanged
(3J4He5Hw6.6e6.9), indicating of a non-substituted pyridine nitro-
gen atom. In the case of an N6-methylation of 12a, a shifting of the
hydrogen atoms at pyridine C-4 and C-5 positions as well as the 7-
NH2-group would be observed. Analysis of the 13C and DEPT spectra
of compound 22a confirms its preferable N1-substitution.
The regioselectivity of the N1-alkylation of ethyl 7-amino-2-
hydroxy-1H-pyrrolo[2,3-c]pyridine-3-carboxylate 12a was further
confirmed by re-synthesis of 22ae24 in large scale under optimized
conditions resulting in 85% yield in overage (Scheme 2). Overall, in
all these transformations, we did not find a non-selective formation
of pyridine N6-alkylated and/or bis-alkylated products. The final
products 22ae24 were isolated after recrystallization from meth-
anol/EtOAc/petroleum ether mixtures in high purity and charac-
terized by different NMR techniques and LC/ESI-MS analysis. The
NMR spectral data in DMSO-d6 reveal that only the hydroxy tau-
tomers of the N1-alkylated products 22ae24 are present. 1H NMR
analysis of 22a in deuterated methanol was used to provide the
Scheme 2. Synthesis of N-alkylated 7-amino-6-azaoxindoles 22ae24 using the optimized reaction conditions: (i) Entry 3 for 22a (method 1); (ii) Entry 8 and 13 for 23 and 24
(method 2). Isolated yields refer to 12a.
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6
N.T. Tzvetkov et al. / Tetrahedron xxx (2016) 1e12
Next, similar alkylation experiments were performed with 5-
excess of lithium chloride (2.0 equiv) in DMSO/water (1:1) under
thermal conditions (Scheme 3). However, no significant conversion
of the starting material towards 25 was observed even by a reaction
time of up to 18 h. In this case, we observed formation of by-
products when the reaction took place longer. We assume that
under these conditions the initially formed anion at 2-position of
amino-substituted 6-azaoxindole derivative 13a (Table 2, entries
5, 9, 14, and 15) using the same alkyl halides and reaction condi-
tions as applied for 12a. In Table 2 are outlined only a few repre-
sentative examples of these reactions. To achieve the
corresponding N-alkylated products of 13a, several attempts were
performed. However, the experiments were not successful to pro-
duce the desired structures. Furthermore, in the most cases a large
amount of the starting material was recovered and, depending on
the reaction time, side processes such as dimerization and tauto-
merization took place (ESI-MS analysis).
In order to find a plausible explanation of this observation and
to validate our hypothesis on the preferable N1-substitution for 7-
NH2- versus 5-NH2-substituted 6-azaoxindoles, theoretical calcu-
lations on the neutral and the charged (deprotonated) tautomer
forms of 12a and 13a in a gas phase were performed.
The possible tautomers of 12 and 13 are sketched in Figs. S6 and
S7, and the relative energies are collected in Tables S3 and S4, re-
spectively. Comparing the most stable tautomeric forms for 12 and
13 and their isomers (Fig. 4) it could be concluded that there is
almost no difference in the tautomeric behavior of 12 (panel A) and
13 (panel B) in a neutral state. In both cases, the a tautomer is
strongly stabilized and the most of the stable tautomers exhibit
a proton at N1, which does not allow a direct alkylation. At the same
time, the stability of these forms (a, b, i, and c), and their isomers
mostly related to the rotation of the CO2Et group, clearly indicates
that the OH proton in a is easily movable one and would be the first
to be lost during a deprotonation, giving an anion with an engaged
N1 position.
24 rapidly undergoes in situ tautomerization to form the stable b-
keto anion I (Scheme 3). Similarly, conversion of 24 under acidic
conditions, e.g., in the presence of 18% aqueous hydrochloric acid in
refluxing water was not successful to obtain 25 by saponification
and subsequent decarboxylation.31 Moreover, 24 was recovered
from the reaction mixture in form of its hydrochloride salt.
Finally, in order to access the physicochemical properties and to
evaluate the drug-likeness of 7-amino-2-hydroxy-1H-pyrrolo[2,3-
c]pyridines, we determined water solubility and logD7.4 values for
compound 12a and its N1-alkylated derivative 23 as representa-
tives of this class of 6-azaindoles. Additionally, the pKa and logP
values,32 and the HBA/HBD counts33 for these compounds were
calculated (for more details, see Table S8, Supplementary data).
Water solubility at physiological pH of 7.4 was found to be 3
(13.6 M) for 12a. The N1-benzylated compound 23 displayed
much better solubility of 20 g/mL (64.2 M) at pH 7.4, which is
mg/mL
m
m
m
a sufficient range for further development of peroral active drugs.
The logD values (octanol-buffer at pH 7.4, rt) were determined to be
0.08 (12a) and 1.50 (23), respectively, indicating that the nitrogen
N1 atom of the pyrrolo[2,3-c]pyridine core is important for in-
troducing of lipophilicity. The logD7.4 value for 23 is in the prefer-
able range for predictive oral bioavailability.34
Furthermore, the lipophilic character of the N1-substituted 6-
azaoxindoles can be fine-tuned for the purpose to obtain brain
penetrable CNS active drugs, e.g., by introducing of suitable sub-
stituents in the meta- and/or para-position of the benzyl residue of
23. The pKa values were evaluated to be 4.8 (acid)/10.2 (base) for
12a and 4.7 (acid)/10.2 (base) for 23, respectively. Both compounds
displayed similar pH profile with two pKa values revealing of their
almost amphoteric character (see Fig. S10, Supplementary data).
Thus, the molecules will be uncharged in the physiologically rele-
vant pH range of 6e8. The obtained data indicate that both, the
pyridine nitrogen N6 and the nitrogen N1 of the pyrrolo[2,3-c]
pyridine core can be protonated under physiological conditions in
the stomach (at pH<5). As aforementioned, under basic conditions
(pH>10), the compounds can be deprotonated due to the hydroxy
group at C2. The basicity of N1 in 12a can be modulated by an al-
kylation with a broad diversity of suitable substituents. In addition,
it appears to be indispensable to determine the physicochemical
properties of 6-azaindole derivatives, especially for their N1-
unsubstituted analogues at different physiologically relevant pH
ranges.
On the basis of above experimental data and the tautomer cal-
culations, further chemoselective transformations of highly func-
tionalized 6-azaoxindole derivatives can be achieved under basic
conditions,35 whereas treatment with selected inorganic and/or
organic acids leads to salts formation. Moreover, 7-amino-2-
hydroxy-1H-pyrrolo[2,3-c]pyridine 14 can be substituted with
a variety of residues (R1, R2 and/or R3). Depending on the target
molecules, one- or two-step modification of 14 are possible, e.g., by
reduction of the ester group at 3-position (route A), amide coupling
reaction at 7-position (route B) or combination of both (route C) to
achieve highly functionalized 6-azaindoles 26, 27 or 28, re-
spectively (Fig. 6).
The deprotonation under basic conditions (pH>10) sub-
stantially changes the tautomeric picture (Fig. 5). As seen, the most
stable deprotonated forms (ae and fe) in both 12 and 13 are very
similar. The structure ae allows alkylation only at N6, while the fe
tautomer gives such an option at both nitrogen atoms. However,
there is a substantial difference. In the case of 12, the 12fe anion is
slightly more stable allowing potential alkylation at N1, while 13ae
form is strongly dominating in the case of 13, giving no option for
N1 attack. This theoretical prediction matches very well the ob-
served experimental results. Returning back to 12fe, where both
nitrogens are available for alkylation, the total atomic charge of N1
is ꢁ0.194 against ꢁ0.176 at N6, which could give an idea about the
preferable obtainment of the N1-alkylated product (see Tables S5
and S6, Supplementary data). Furthermore, the analysis of the
tautomerism of 22 shows that it exists exclusively as 22a, i.e.,
preference for N1- versus N6-methylation of 12a (see Fig. S8 and S9,
Table S7, Supplementary data).
Summarizing our findings, we can conclude that (i) compounds
12 and 13 exist in their more stable enol form (tautomer a), (ii)
deprotonation of N1 proton in the 5-amino-substituted compound
13a is energetically unfavorable when comparing to its 7-amino-
substituted analogue 12a, i.e., the only deprotonated 12fe form is
accessible for an N1-alkylation (cf. 12fe and 13fe, Fig. 5), (iii) the
nitrogen N1 is preferable for an electrophilic substitution rather
than N6, (iv) the N-alkylation reaction of 12a proceeds via forma-
tion of stable enol intermediates (enolate anion and/or conjugate
base), (v) the N1-substitution (reaction time, temperature, amount
of base and reagent) depends on the substitutions pattern of the
alkylation reagent that is used, e.g., length and functionalities of the
side chain, and (vi) in the case that the N-alkylation takes longer
than expected, additionally amount of the corresponding base
should be added to the reaction to ensure the stabilization of the in
situ formed anion 12fe.
3. Conclusion
A decarboxylation of 24 to obtain the dealkoxycarbonylated
derivative 25 was attempted with a view to provide an indirect
evidence for the favorable N1-substitution of 12a. The reaction was
conducted in analogy to a method described by Krapcho30 using an
In summary, we have demonstrated that multifunctionalized
N1-alkyl-7-amino-6-azaoxindole derivatives with drug-like
properties can be successfully achieved by applying of a simple
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N.T. Tzvetkov et al. / Tetrahedron xxx (2016) 1e12
7
Fig. 4. Relative energies (in kcal/mol units) of the most stable tautomers and isomers of 12 (panel A) and 13 (panel B) in a gas phase (M06-2X/TZVP).
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8
N.T. Tzvetkov et al. / Tetrahedron xxx (2016) 1e12
Fig. 5. Relative energies (in kcal/mol units) of the most stable deprotonation forms of 12 (left) and 13 (right) in a gas phase (M06-2X/TZVP). The relative energies of the remaining
forms are more than 10 kcal/mol higher.
Scheme 3. Attempted decarboxylation of 24.
four-step synthetic approach. The convergent sequence includes
reductive cyclization as a key step leading to the regioselective
formation of the 5-amino- and 7-amino-substituted 6-
azaoxindoles 12a and 13a. The subsequent N1-alkylation of 12a
was conducted under various basic conditions providing high
tolerance of sensitive functionalities and high yields of the de-
sired N-alkylated amino-6-azaoxindoles 22ae24. The synthetic
procedure was optimized for all steps and will enable simple
scale-up in large scale. The N1-substitution of the amino-
substituted 6-azaoxindoles showed strong dependency on the
position of the amino group, i.e., preference for 7-NH2- versus 5-
NH2-substitution. An additional evidence for the proposed hy-
pothesis was obtained by means of quantum chemical calcula-
tions of the tautomers of 12, 13 and 22 in their ionized and
neutral forms. The experimentally determined and calculated
physicochemical properties of the representative compound 23
indicate of drug-like features in case of N1-alkylation of 7-amino-
6-azaindole derivatives. Regioselective alkylation in the final step
allows the introduction of broad structural modifications and an
easy access to compound libraries that can be applied for drug
design and development.
OH
N
R2O2C
4. Experimental section
4.1. General information
R1
N
NH2
All commercially available anhydrous solvents, reagent grade
solvents for chromatography, reagents and starting materials used
were obtained from various producers and used in reactions as
received without further purification or drying. Dry N,N-dime-
thylformamide (DMF. 99.8% extra dry over molecular sieves, Acro-
Seal, Acros) was used throughout the synthesis. Thin layer
chromatography (TLC) was performed on pre-coated Merck 60F254
14
R1 = R2 or R1 R2
1H-pyrrolo[2,3-c]pyridine
A
B
C
silica gel plates using UV light (
l
¼254 nm light source). Preparative
R2
OH
N
R2
OH
N
OH
R2O2C
column chromatography was performed on Acros Organics silica gel
ꢀ
3
3
7
60 A (0.060e0.200 mm). Hydrogenation was achieved by using
N
R1
R1
R1
a Hogen GC hydrogen generator (Proton Energy System, Inc.).
Sonication was performed in a Sonorex Super RK 100/H ultrasonic
cleaning bath at a frequency of 35 kHz, a nominal power of 80 Wand
a bath temperature at 30 ꢀC with explosion of the reactions to air in
standard glassware or glass sample vials. Solvents were removed in
7
N
NH
R3
N
NH2
26
N
NH
28
27
O
O
R3
€
Fig. 6. Possible functionalizations of N1-alkylated 7-amino-6-aza-2-oxindoles.
vacuo on a Buchi Rotavapor R-100 or R-300. Mass spectra were
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N.T. Tzvetkov et al. / Tetrahedron xxx (2016) 1e12
9
recorded on an API 2000 mass spectrometer (electron spray ion
4.3. Preparation of amidines 16 and 17
source ESI, Applied Biosystems) coupled with an Agilent 1100 HPLC
system. NMR spectra were recorded on a Bruker AV 500 MHz NMR
spectrometer at 500 MHz (1H), or 125 MHz (13C, DEPT) at 303 or
298 K on samples dissolved in an appropriate deuterated solvent
The compounds were prepared as described in the literature
with minor modifications.24 To a solution of the corresponding 4-
chloro-nitropyridin-2-amine (10 or 11, 1.0 mmol) in methanol
(10 mL per 1.0 mmol starting material) and dimethylformamide
(1.0 mL) was added dimethylformamide dimethyl acetal (DMF-
DMA, 1.0 mL) and heated to 75 ꢀC. After a reaction time of 16e20 h,
the solvent was evaporated and the residue was stirred under
waterepetroleum ether (50%, 10 mL) until full crystallization. The
product was filtered under reduced pressure, washed with petro-
leum ether (3ꢂ10 mL), and dried at 70 ꢀC to afford amidine 16 or 17.
(DMSO-d6 or CD3OD). Chemical shifts (
d) are reported in parts per
million (ppm) relative to the residual solvent peak in the respective
spectra: DMSO-d6 d 2.50 (1H) and 39.51 (13C); CD3OD
d
3.31 (1H) and
49.00 (13C). Coupling constants (J values) are reported in Hertz (Hz);
spin multiplicities are indicated by the following symbols: s (sin-
glet), br s (broad singlet), d (doublet), t (triplet), dd (doublet of
doublets), td (triplet of doublets), quartet (q), dq (doublet of quar-
tets), and m (multiplet). Melting points are uncorrected and were
€
obtained on a Buchi MP B-454 apparatus using open capillary tubes.
4.3.1. (E)-N0-(4-Chloro-3-nitropyridin-2-yl)-N,N-dimethylform-am-
ide (16).24 Yellow crystals (194.9 mg, 87%); mp 121e123 ꢀC; 1H
NMR (500 MHz, DMSO-d6) dH: 2.96 (3H, s, NMe2), 3.14 (3H, s,
NMe2), 7.21 (1H, d, J¼5.36 Hz, H-5), 8.28 (1H, d, J¼5.67 Hz, H-6),
8.61 (1H, s, NH¼NMe2); 13C NMR (125 MHz, DMSO-d6) dC: 34.5,
40.7, 116.8, 133.6, 138.9, 150.1, 154.6, 156.9; LC/ESI-MS calcd for
C8H9ClN4O2 m/z 228.1, found 228.9 [MþH]þ.
4.2. Optimized scale-up procedure for the preparation of
4-chloro-3-/5-nitropyridin-2-amines (10 and 11)
The compounds were prepared as described in the literature
with major modifications.22b To a solution of 4-chloropyridin-2-
amine 9 (2.60 g, 0.02 mol) in sulfuric acid (96%, 40 mL) was
slowly added fuming nitric acid (20 mL) under intensively stirring
at 0 ꢀC over a period of 6 h. The reaction mixture was allowed to
stay at 0e5 ꢀC for 3 days and then poured into ice water (120 g).
The mixture was adjusted to pH 3e5 with ammonium hydroxide
(25%, 160 mL) and the yellow precipitate filtered, washed with
cooled water, and dried at room temperature to give 4-chloro-2-
nitraminopyridine intermediate (3.46 g, 99%) as yellow crystals;
mp 204e205 ꢀC. The crude nitramine (2.73 g, 0.016 mol) was
carefully dissolved in small portions into cooled sulfuric acid (96%,
54 mL) under constant stirring. The solution was allowed to warm
to room temperature and stirred for further 3 h. After that, the
reaction mixture was poured into ice water (60 g) and adjusted to
pH 6e7 with ammonium hydroxide (25%, 150 mL) until a yellow
crystalline precipitate was formed. The cooled product (mixture of
3- and 5-nitro isomers) was filtered under normal pressure, dried
at room temperature over night, and washed several times with
ethyl acetate (170 mL) until the color of the crystalline precipitate
had turned to a slight yellow. The residue left after filtration was
dissolved in ethanol (100 mL), boiled for 15 min, filtered to remove
the insoluble precipitate, and recrystallized from dichloro-
methane/petroleum ether (50%, 50 mL) to give the pure product
11 (Fraction 1). The combined ethyl acetate layers were evaporated
to dryness and purified by repeated column chromatography on
silica gel using 50% dichloromethane/ethyl acetate as eluent.
Chromatographically purified products were additionally recrys-
tallized from dichloromethane/petroleum ether (50%, each 50 mL),
evaporated, and dried at 70 ꢀC to give pure product 10 (Fractions 2
and 4) and 11 (Fraction 3, for details, see Supplementary data,
Fig. S2).
4.3.2. (E)-N0-(4-Chloro-5-nitropyridin-2-yl)-N,N-dimethylform-am-
ide (17).24 Yellow crystals (180.2 mg, 68%); mp 144e145 ꢀC; 1H NMR
(500 MHz, DMSO-d6) dH: 3.08 (3H, s, NMe2), 3.18 (3H, s, NMe2), 6.99
(1H, s, H-3), 8.69 (1H, s, NH¼NMe2), 8.91 (1H, s, H-6); 13C NMR
(125 MHz, DMSO-d6) dC: 34.9, 40.9, 118.2, 137.1, 147.6, 158.0, 165.8;
LC/ESI-MS calcd for C8H9ClN4O2 m/z 228.1, found 229.1 [MþH]e.
4.4. Preparation of compounds 18 and 19
The compounds were prepared in analogy to a published pro-
cedure.18,21a A suspension of sodium hydride (60%, 1.5 mmol) in
mineral oil was washed with n-hexane (3ꢂ10 mL), the solvent was
pipetted out and traces were removed under reduced pressure, and
the remaining material was suspended in dry DMF (5 mL) under
argon atmosphere. The mixture was cooled to 0e5 ꢀC and a solution
of malonic acid dialkyl ester (1.0 mmol) in dry DMF (0.5 mL) was
added dropwise. During malonate addition the temperature was
not allowed to rise about 40 ꢀC. When the hydrogen evolution
ceased (about 5e10 min) the reaction was allowed to warm to 50 ꢀC
and stirred for further 30 min at the same temperature, after which
a solution of the corresponding 4-chloro-nitropyridine-2-amine
(10 or 11, 0.5 mmol) in DMF (1 mL) was added dropwise while
the temperature kept below 50e55 ꢀC. The mixture was stirred for
specified time at this temperature. After the starting material was
consumed as monitored by TLC control (90% dichloromethane/
methanol), the reaction mixture was evaporated under reduced
pressure, the residue diluted with ice water (5 mL), and neutralized
carefully with hydrochloric acid (2.0 N, 2 mL). The formed pre-
cipitate was filtered and washed with ice water (3ꢂ5 mL). The
crude product was purified by column chromatography on silica gel
using 50% dichloromethane/ethyl acetate as eluent to afford the
corresponding condensed product 18 or 19.
4.2.1. 4-Chloro-3-nitropyridin-2-amine (10).22b Slight yellow nee-
dles (1.47 g, 42% over two steps); mp 176e177 ꢀC; Rf (50% CH2Cl2/
EtOAc) 0.69; 1H NMR (500 MHz, DMSO-d6) dH: 6.85 (1H, d,
4.4.1. Diethyl 2-(2-amino-3-nitropyridin-4-yl)malonate (18).18 Yellow
crystals (240.3 mg, 87%); mp 117e118 ꢀC; Rf (50% CH2Cl2/EtOAc) 0.77;
1H NMR (500 MHz, DMSO-d6) dH: 1.17 (6H, t, J¼7.25 Hz, 2ꢂOCH2CH3),
4.17 (4H, dq, J¼1.58, 7.25 Hz, 2ꢂOCH2CH3), 5.20 (1H, s, CH), 6.58 (1H,
J¼5.36 Hz, H-5), 7.21 (2H, br s, NH2), 8.11 (1H, d, J¼5.36 Hz, H-6); 13
C
NMR (125 MHz, DMSO-d6) dC: 113.2 (C5), 129.5 (C3), 136.0 (C4),
152.1 (C6), 152.8 (C2); LC/ESI-MS calcd for C5H4ClN3O2 m/z 173.0,
found 171.9 [MꢁH]e, 174.9 [MþH]þ.
d, J¼5.36 Hz, H-5), 7.50 (2H, s, NH2), 8.27 (1H, d, J¼4.73 Hz, H-6); 13
C
NMR (125 MHz, DMSO-d6) dC: 13.9 (2ꢂOCH2CH3), 55.1 (CH), 66.9
(2ꢂOCH2CH3),114.3,128.2,139.4,153.8,153.9,166.3; LC/ESI-MS calcd
for C12H15N3O6 m/z 297.3, found 296.0 [MꢁH]e, 298.3 [MþH]þ.
4.2.2. 4-Chloro-5-nitropyridin-2-amine (11).22b Yellow crystals
(1.05 g, 30% over two steps); mp 260e261 ꢀC; Rf (50% CH2Cl2/EtOAc)
0.48; 1H NMR (500 MHz, DMSO-d6) dH: 6.58 (1H, s, H-3), 7.59 (2H,
br s, NH2), 8.79 (1H, s, H-6); 13C NMR (125 MHz, DMSO-d6) dC: 108.4
(C3), 133.5 (C5), 137.1 (C4), 149.7 (C6), 162.7 (C2); LC/ESI-MS calcd
for C5H4ClN3O2 m/z 173.0, found 171.9 [MꢁH]e.
4.4.2. Diethyl 2-(2-amino-5-nitropyridin-4-yl)malonate (19).18 Yellow
crystals (231.7 mg, 84%); mp 151e152 ꢀC; Rf (50% CH2Cl2/EtOAc) 0.81;
1H NMR (500 MHz, DMSO-d6) dH: 1.18 (6H, t, J¼7.25 Hz, 2ꢂOCH2CH3),
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10
N.T. Tzvetkov et al. / Tetrahedron xxx (2016) 1e12
4.18 (4H, dq, J¼2.83, 7.25 Hz, 2ꢂOCH2CH3), 5.35 (1H, s, CH), 6.34 (1H,
s, H-3), 7.60 (2H, s, NH2), 8.84 (1H, s, H-6); 13C NMR (125 MHz, DMSO-
d6) dC: 14.0 (2ꢂOCH2CH3), 55.2 (CH), 61.8 (2ꢂOCH2CH3), 108.3, 134.1,
138.8, 149.2, 162.9, 166.4; LC/ESI-MS calcd for C12H15N3O6 m/z 297.3,
found 296.3 [MꢁH]e, 298.4 [MþH]þ.
Alternatively, the work-up procedure was performed as described
for method 1 with small modifications.
4.5.4. Ethyl 7-amino-2-hydroxy-1H-pyrrolo[2,3-c]pyridine-3-
carboxylate (12a).18 Preparation according to method 1 afforded
12a as a white solid (401.3 mg, 95%); mp 302e303 ꢀC (dec); Rf (100%
MeOH) 0.29; 1H NMR (500 MHz, DMSO-d6) dH: 1.25 (3H, t,
J¼6.94 Hz, OCH2CH3), 4.13 (2H, q, J¼6.63 Hz, OCH2CH3), 6.91 (2H, br
s, NH2), 7.05 (1H, d, J¼6.62 Hz, H-4), 7.31 (1H, d, J¼6.62 Hz, H-5),
10.3 (1H, s, NH), 11.7 (1H, br s, OH); 13C NMR (125 MHz, DMSO-d6)
dC: 14.9 (OCH2CH3), 57.5 (OCH2CH3), 87.7, 104.5, 110.0, 126.0, 135.7,
4.5. Procedures for reductive cyclization of 18 and 19
4.5.1. Method 1 using Zn/AcOH.18 To a stirred suspension of the
corresponding diethyl malonate 18 or 19 (1.0 mmol) in glacial acetic
acid and water (4 mL, 3:1) was added zinc dust in small portions
(20.0 mmol). The mixture was stirred at room temperature until
a clear solution was formed and then filtered under reduced
pressure to remove the zinc residue. The filtrate was evaporated to
dryness, diluted with water (10 mL) and treated dropwise with
aqueous ammonia (25%) until an alkaline pH was reached. The
resulting mixture was stirred at room temperature for 5e10 min
and the formed precipitate was filtered under reduced pressure,
washed with water (2ꢂ10 mL), petroleum ether (2ꢂ10 mL), and
dried at 70 ꢀC to obtain pure products 12a or 13a as free amines.
Alternatively, the work-up was performed by extraction of the al-
kaline solution with n-butanol (3ꢂ10 mL). Then, the combined
organic extracts were washed with water (3ꢂ10 mL), dried over
sodium sulfate, filtered, and evaporated to dryness under reduced
pressure. The residue was recrystallized from ethyl acetate/petro-
leum ether and/or methanol mixtures (4:1 or 3:1:1, 15 mL).
137.9, 164.9, 166.5 (CO2Et); 13C-DEPT NMR (125 MHz, DMSO-d6) dC
:
14.9 (OCH2CH3), 57.5 (OCH2CH3), 104.5 (C4), 125.8 (C5); LC/ESI-MS
calcd for C10H11N3O3 m/z 221.1, found 220.1 [MꢁH]e, 222.3 [MþH]þ.
4.5.5. Ethyl 7-amino-2-hydroxy-1H-pyrrolo[2,3-c]pyridine-3-
carboxylate hydrochloride (12a$HCl). Following method 2, the hy-
drochloride salt of 12a was obtained as a white solid (18.2 mg, 20%);
mp 383e385 ꢀC (dec); 1H NMR (500 MHz, DMSO-d6) dH: 1.30 (3H, t,
J¼6.94 Hz, OCH2CH3), 4.25 (2H, q, J¼7.25 Hz, OCH2CH3), 7.21 (1H, d,
J¼6.93 Hz, H-4), 7.52 (1H, d, J¼6.94 Hz, H-5), 7.85 (2H, br s, NH2),
12.48 (1H, br s, OH), 12.88 (1H, br s, NH); 13C NMR (125 MHz,
DMSO-d6) dC: 14.6 (OCH2CH3), 59.3 (OCH2CH3), 89.4, 105.4, 110.5,
127.4, 134.4, 141.0, 159.1, 163.6 (CO2Et); LC/ESI-MS calcd for
C
10H11N3O3 m/z 221.1, found 222.3 [MþH]þ.
4.5.6. Ethyl 5-amino-2-hydroxy-1H-pyrrolo[2,3-c]pyridine-3-
carboxylate (13a).18 Preparation according to method 2 afforded
13a as an off-white solid (228.8 mg, 96%); mp 328e330 ꢀC (dec); Rf
(100% MeOH) 0.18; 1H NMR (500 MHz, DMSO-d6) dH: 1.27 (3H, t,
J¼7.25 Hz, OCH2CH3), 4.10 (2H, q, J¼7.25 Hz, OCH2CH3), 4.65 (2H, br
s, NH2), 6.43 (1H, s, H-4), 7.23 (1H, s, H-7), 9.13 (1H, s, NH), 11.1 (1H,
s, OH); 13C NMR (125 MHz, DMSO-d6) dC: 15.2 (OCH2CH3), 56.9
(OCH2CH3), 95.9,112.9,114.1,123.9,137.1, 152.8,159.7,166.9 (CO2Et);
LC/ESI-MS calcd for C10H11N3O3 m/z 221.1, found 219.9 [MꢁH]e,
222.3 [MþH]þ.
4.5.2. Method 2 using H2ePd/C.18 A mixture of the corresponding
diethyl malonate 18 or 19 (1.0 mmol) in absolute ethanol (5 mL)
was treated with palladium on charcoal (10 mol-%). The mixture
was subjected to hydrogenation using a Parr hydrogenator (40 psi)
ꢀ
at room temperature in the presence of molecular sieve (3 A) until
complete reduction of the staring material was detected. The re-
action was monitored by TLC analysis (50% ethyl acetate/dichloro-
methane). After complete reaction, the mixture was filtered
through Celite pad and the filtrate was evaporated to dryness. The
dark-red residue was treated with hydrochloric acid (2 N) and
stirred at room temperature for respective time (TLC control: 100%
methanol). When the reaction was complete, the reaction mixture
was concentrated under reduced pressure, treated with saturated
sodiumhydrogencarbonate solution (10 mL, pH>8), and extracted
with n-butanol (3ꢂ10 mL). The combined organic layers were
washed with water (3ꢂ10 mL), dried over sodium sulfate, filtered,
and evaporated to dryness under reduced pressure. The residue
was diluted with ethyl acetate/petroleum ether (20%, 20 mL) and
stirred at room temperature for 10 min. The formed slight green
precipitate was recrystallized from dichloromethane/petroleum
ether (50%, 20 mL) to afford 12a or 13a as white or off-white solids.
For the preparation of the corresponding hydrochloride of 12a, the
work-up procedure after complete cyclization is not necessary.
4.6. Procedures for alkylation of 18
4.6.1. Method 1 using K2CO3. A suspension of ethyl 7-amino-2-
hydroxy-1H-pyrrolo[2,3-c]pyridine-3-carboxylate (12a, 1.0 mmol)
and potassium carbonate (1.20e1.25 mmol) in dry DMF was treated
with a solution of the corresponding alkylating reagent (25e28,
1.1e1.7 mmol) in dry DMF (1 mL). The mixture was stirred at room
temperature for the appropriate period of time, or at 75e80 ꢀC (for
alkylation with 2-bromo-N-phenethylacetamide 28), respectively,
until complete conversion (TLC control: 90% dichloromethane/
methanol). After all of the starting material was dissipated, the
reaction mixture was evaporated to dryness under reduced pres-
sure, hydrolyzed with water (10 mL) and neutralized with hydro-
chloric acid (2 N). The precipitate was filtered, washed with water
(3ꢂ10 mL), dried at 70 ꢀC over night, and recrystallized from
methanol/ethyl acetate/petroleum ether (1:1:3) to afford products
22ae24 as free amines.
4.5.3. Method 3 using SnCl2$2H2O.27 To a stirred solution of 18
(1.0 mmol) in ethanol (5 mL) was added tin(II) chloride dihydrate
(10.0 mmol). The reaction mixture was then irradiated using ul-
trasonic bath for appropriate time at 30e35 ꢀC. The reaction was
monitored by TLC analysis (50% ethyl acetate/dichloromethane).
After complete reaction, the solution was evaporated to dryness
under reduced pressure and the residue was dissolved in water
(5 mL). The resulting mixture was made strong alkaline with
aqueous ammonia (25%, 5 mL) and then stirred at room tempera-
ture or under ice bath cooling until full precipitation. The pre-
cipitate was collected by filtration under reduced pressure, washed
with water (3ꢂ5 mL) and dried at 70 ꢀC. The residue was recrys-
tallized from dichloromethane/petroleum ether (50%, 20 mL) to
afford 12a or 13a as a white or off-white solid. Products were ob-
tained with small amounts of impurities (LC-MS analysis).
4.6.2. Method 2 using NaHeK2CO3. A suspension of sodium hydride
(60%, 1.1 mmol) in mineral oil was washed with n-hexane
(3ꢂ10 mL) under argon atmosphere, the solvent was pipetted out
and traces were removed under reduced pressure. The remaining
material was placed in a dry apparatus under argon atmosphere,
anhydrous DMF (5 mL) was added dropwise and the resulting
suspension was stirred at room temperature for 10 min until the
hydrogen evolution ceased. The reaction mixture was cooled to e2
to 0 ꢀC with ice/acetone bath and a solution of the appropriate
alkylating reagent (27 or 28, 1.0 mmol) in dry DMF (2 mL) was
added through a septum over 10e15 min. The reaction mixture was
Please cite this article in press as: Tzvetkov, N. T.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.08.055

N.T. Tzvetkov et al. / Tetrahedron xxx (2016) 1e12
11
stirred at ꢁ2 to 0 ꢀC for 30 min, then allowed to warm slowly to
room temperature and intensively stirred for 3e4 h. After the
mixture had been stirred for a definite time, potassium carbonate
(1.1 mmol) was added and the system was further stirred at am-
bient temperature until full conversion of the starting material. The
reaction was monitored either by TLC (90%, dichloromethane/
methanol) or by LC-MS analysis. The work-up procedure was per-
formed as described for method 1 yielding compounds 23 or 24 as
brownish solids.
precipitate was filtered, washed with petroleum ether (3ꢂ10 mL) to
yield 52.1 mg (87%) of a brown solid of the corresponding hydro-
chloride salt; mp>303 ꢀC (dec); 1H NMR (500 MHz, DMSO-d6) dH
:
1.31 (3H, t, J¼6.93 Hz, OCH2CH3), 2.75 (2H, t, J¼7.88 Hz, CH2CH2Ph),
3.34 (2H, q, J¼6.31 Hz, CH2CH2Ph), 4.27 (2H, q, J¼6.93 Hz,
OCH2CH3), 4.93 (2H, s, CH2), 7.17e7.24 (4H, m, Ar, H-4), 7.28 (2H, d,
J¼7.56 Hz, Ar), 7.53 (1H, d, J¼6.94 Hz, H-5), 8.23 (2H, br s, NH2), 8.42
(1H, t, J¼5.36 Hz, CONH), 12.58 (1H, s, OH); 13C NMR (125 MHz,
DMSO-d6) dC: 14.6 (OCH2CH3), 35.1, 40.8, 54.0 (CH2), 40.7, 53.5, 57.5
(OCH2CH3), 88.1, 105.2, 111.4, 126.3, 128.5 (2ꢂC, Ar), 128.7 (2ꢂC, Ar),
133.1, 133.2, 139.3, 141.9, 159.1, 163.4 (CONH), 164.9 (CO2Et); LC/ESI-
MS calcd for C20H22N4O4 m/z 382.2, found 381.1 [MꢁH]e, 383.3
[MþH]þ.
4.6.3. Ethyl
7-amino-2-hydroxy-1-methyl-1H-pyrrolo[2,3-c]pyri-
dine-3-carboxylate (22a). This compound was produced by method
1: 12a (222.2 mg, 1.0 mmol) was treated with methyl meth-
anesulfonate (MMS, 127.5 mg, 1.22 mmol) to afford 22a as a pale
brown solid (183.5 mg, 78%); mp 280e281 ꢀC (dec); 1H NMR
(500 MHz, DMSO-d6) dH: 1.24 (3H, t, J¼6.94 Hz, OCH2CH3), 3.70 (3H,
s, NMe), 4.12 (2H, q, J¼7.25 Hz, OCH2CH3), 6.97 (2H, br s, NH2), 7.03
(1H, d, J¼6.94 Hz, H-4), 7.42 (1H, d, J¼6.94 Hz, H-5), 10.08 (1H, br s,
OH); 1H NMR (500 MHz, CD3OD) dH: 1.44 (3H, t, J¼6.94 Hz,
OCH2CH3), 3.92 (3H, s, NMe), 4.42 (2H, q, J¼7.25 Hz, OCH2CH3), 7.32
(1H, d, J¼6.93 Hz, H-4), 7.61 (1H, d, J¼6.94 Hz, H-5); 13C NMR
(125 MHz, DMSO-d6) dC: 14.9 (OCH2CH3), 34.3 (NMe, signal over-
lapped with the residual signal from DMSO-d6), 57.7 (OCH2CH3),
87.7, 105.2, 111.2, 131.3, 136.2, 136.6, 164.8; 13C NMR (125 MHz,
CD3OD) dC: 15.1 (OCH2CH3), 41.3 (NMe), 61.6 (OCH2CH3), 90.8,108.2,
113.1, 134.1, 135.0, 143.4, 162.6, 166.7 (CO2Et); 13C-DEPT NMR
(125 MHz, CD3OD) dC: 15.1 (OCH2CH3), 41.1 (NMe), 61.6 (OCH2CH3),
108.2 (C4), 134.1 (C5); LC/ESI-MS calcd for C11H13N3O3 m/z 235.1,
found 234.4 [MꢁH]e, 236.3 [MþH]þ.
4.7. General procedure for the preparation of hydrochlorides
The corresponding free amine (1.0 mmol) was suspended in
hydrochloric acid (1 N, 5 mL, 5.0 mmol) and stirred under reflux for
3e5 h. Stirring was continued for further 3 h in which the mixture
was allowed to cool down to room temperature. The obtained slight
colored solution was evaporated to dryness. The residue was
treated with petroleum ether (20 mL) and stirred for 10 min under
ice bath cooling. The mixture was allowed to stay at 0e5 ꢀC over
night. The formed precipitate was filtered under atmospheric
pressure, washed with petroleum ether (3ꢂ10 mL) and dried at
70 ꢀC to obtain pure hydrochloride salts.
4.8. Theoretical calculations
Quantum-chemical calculations were performed using the
Gaussian 09 D.01 program suite.36 The M06-2X functional37 was
used with TZVP basis set.38 This fitted hybrid meta-GGA functional
with 54% HF exchange is specially developed to describe main-
group thermochemistry and non-covalent interactions, showing
very good results in prediction of the position of the tautomeric
equilibrium compounds possessing intramolecular hydrogen
bond.39 All structures were optimized in ground state without re-
strictions, using tight optimization criteria and ultrafine grid in the
computation of two-electron integrals and their derivatives. The
true minima were verified by performing frequency calculations.
4.6.4. Ethyl 7-amino-1-benzyl-2-hydroxy-1H-pyrrolo[2,3-c]pyridine-
3-carboxylate (23). This compound was produced by method 2:
12a (100.0 mg, 0.45 mmol) was treated with benzyl bromide (27,
77.0 mg, 0.45 mmol) to afford 22a as a brown solid (135 mg, 96%);
mp 287e289 ꢀC (dec); 1H NMR (500 MHz, DMSO-d6) dH: 1.21 (3H, t,
J¼7.25 Hz, OCH2CH3), 4.07 (2H, q, J¼7.25 Hz, OCH2CH3), 5.36 (2H, s,
CH2Ph), 6.70 (2H, br s, NH2), 7.11 (1H, d, J¼6.94 Hz, H-4), 7.16 (2H, d,
J¼7.25 Hz, Ar), 7.30 (1H, d, J¼7.25 Hz, Ar), 7.36 (2H, t, J¼6.94 Hz, Ar),
7.48 (1H, d, J¼6.94 Hz, H-5), 9.63 (1H, s, OH); 13C NMR (125 MHz,
DMSO-d6) dC: 14.9 (OCH2CH3), 53.9 (CH2Ph), 57.3 (OCH2CH3), 88.1,
105.5, 111.3, 126.6 (2ꢂC, Ar), 127.9, 128.8 (2ꢂC, Ar), 130.0 (Ar), 134.7,
135.8, 137.9, 164.6, 166.2 (CO2Et); LC/ESI-MS calcd for C17H17N3O3
m/z 311.1, found 310.1 [MꢁH]e, 312.5 [MþH]þ.
Acknowledgements
N.T.T. thanks Annette Reiner, Marion Schneider, and Sabine
Terhart-Krabe for their skillful technical assistance. Financial sup-
port by the German Federal Ministry of Education and Research
(BMBF) and UCB Pharma (Biopharma Neuroallianz project),
Bulgarian Science Fund (access to MADARA computer cluster by
project RNF01/0110) and the Swiss National Science Foundation
(SupraMedChem@Balkans.Net Institutional partnership project
under SCOPES Program) is gratefully acknowledged.
4.6.5. Ethyl 7-amino-2-hydroxy-1-(2-oxo-2-phenethylamino) ethyl-
1H-pyrrolo[2,3-c]pyridine-3-carboxylate (24). This compound was
produced by method 2: 12a (110.0 mg, 0.5 mmol) was treated with
2-bromo-N-phenethylacetamide (28, 145.0 mg, 0.45 mmol) to af-
ford 24 as a brown solid (135 mg, 96%); mp 191e193 ꢀC (dec); 1H
NMR (500 MHz, DMSO-d6) dH: 1.23 (3H, t, J¼6.94 Hz, OCH2CH3),
2.73 (2H, t, J¼7.57 Hz, CH2CH2Ph), 3.38 (2H, q, J¼6.51 Hz,
CH2CH2Ph), 4.08 (2H, q, J¼6.94 Hz, OCH2CH3), 4.77 (2H, s, CH2), 6.54
(2H, br s, NH2), 7.05 (1H, d, J¼6.94 Hz, H-4), 7.15e7.35 (6H, m, Ar, H-
5), 8.27 (1H, t, J¼5.68 Hz, CONH), 9.61 (1H, s, OH); 13C NMR
(125 MHz, DMSO-d6) dC: 14.9 (OCH2CH3), 35.1 (CH2), 40.7, 53.5, 57.3
(OCH2CH3), 88.1, 105.2, 111.4, 126.3, 128.5 (2ꢂC, Ar), 128.8 (2ꢂC, Ar),
131.5, 135.2, 137.9, 139.3, 164.7, 165.7 (CO2Et), 166.3 (CONH); LC/ESI-
MS calcd for C20H22N4O4 m/z 382.2, found 381.3 [MꢁH]e, 383.1
[MþH]þ.
Supplementary data
Supplementary data (synthetic and purification schemes for
compounds 10 and 11, proposed mechanism for the formation of
13a, all possible tautomeric forms for structures 12, 13 and 22,
physicochemical properties of 12a and 23, X-ray data of 18 and 19,
and NMR spectra of selected compounds) associated with this ar-
ticle can be found in the online version, at http://dx.doi.org/
10.1016/j.tet.2016.08.055.
4.6.6. Ethyl 7-amino-2-hydroxy-1-(2-oxo-2-phenethylamino) ethyl-
1H-pyrrolo[2,3-c]pyridine-3-carboxylate hydrochloride (24$2HCl).
This compound was produced by method 2 following by a conver-
sion with hydrochloric acid. The free amine 24 (50.0 mg, 0.13 mmol)
was refluxed with hydrochloric acid (2 N, 10 mL) for 3 h. Then, the
reaction mixture was cooled with ice/acetone bath and the formed
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