A new four-component reaction involving the Michael addition and the Gewald reaction, leading to diverse biologically active 2-aminothiophenes

Article abstract of DOI:10.1039/c7ob00707h

The Gewald three-component reaction yielding highly substituted 2-aminothiophene heterocycles has been known for a long time and holds an extraordinary potential in the pharmaceutical industry. Herein, we describe a four-component reaction initiated by the conjugate addition of different indole derivatives to α,β-unsaturated carbonyl compounds. This is followed by an in situ Gewald three-component reaction which results in the formation of a compound containing an indole and a 2-aminothiophene moiety separated by a methylene spacer. We also examined the impact of the use of other nucleophilic components such as pyrrole derivatives on this MG-4CR (Michael-Gewald four component reaction). All these synthesized compounds were tested for anti-proliferative activity and three of them showed activity in the nanomolar range.

Full text of DOI:10.1039/c7ob00707h

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Takeharu Haino et al.
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A New Four-Component Reaction Involving Michael Addition and Gewald Reaction,
Leading to Diverse Biologically Active 2-Aminothiophenes
a
a
a
a
b
Joice Thomas , Sampad Jana , Mahendra Sonawane , Bert Fiey , Jan Balzarini , Sandra
b
a
Liekens * and Wim Dehaen *
(a) Molecular Design and Synthesis, Department of Chemistry, KU Leuven, Celestijnenlaan
2
00F, 3001 Leuven, Belgium
wim.dehaen@chem.kuleuven.be
(b) Department of Microbiology and Immunology, Rega Institute for Medical Research,
KU Leuven, Bꢀ3000 Leuven, Belgium
sandra.liekens@kuleuven.be
TOC

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ABSTRACT
The Gewald threeꢀcomponent reaction yielding highly substituted 2ꢀaminothiophene
heterocycles has been known for a long time and holds an extraordinary potential in
pharmaceutical industry. Herein, we describe a fourꢀcomponent reaction initiated by the
conjugate addition of different indole derivatives to α, βꢀunsaturated carbonyl compounds.
This is followed by an in situ Gewald threeꢀcomponent reaction which results in the formation
of a compound containing an indole and a 2ꢀaminothiophene moiety separated by a methylene
spacer. We also examined the impact of the use of other nucleophilic components such as
pyrrole derivatives on this MGꢀ4CR (MichaelꢀGewald four component reaction). All these
synthesized compounds were tested for antiꢀproliferative activity and three of them showed
activity in the nanomolar range.
INTRODUCTION
One of the major challenges in pharmaceutical industries is the diversityꢀoriented construction
of molecular complexity from simple and readily available substrates. This is aimed at the
development of very powerful molecular libraries to fulfil the requirements of structureꢀ
activity relationship studies of drugꢀlike compounds. The chemistry of multicomponent
processes has gained wide acceptance in this regard and gives great opportunities for drug
1
discoveries. For instance, the application of the Gewald threeꢀcomponent reaction (Gꢀ3CR)
for the synthesis of 2ꢀaminothiophene derivatives has already proven to be rewarding for the
2
ꢀ5
pharmaceutical industries (Figure 1). These include a piperidine fused derivative of 2ꢀ
aminothiophene, Tinoridine, which is a nonꢀsteroidal antiꢀinflammatory drug possessing
antiperoxidative properties while the carbocycle fused 2ꢀaminoꢀ3ꢀbenzoylthiophene analog Tꢀ
3
6
2 is a selective allosteric enhancer of the adenosine A receptor. Further research on these
heterocyclic scaffolds generated key intermediates for the discovery of a number of promising

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pharmaceuticals that exhibit a broad range of biological properties. Some examples include
4
TPCAꢀ1, which contains a 2ꢀureidothiophene unit and was recently developed as a smallꢀ
molecule IκB kinase β (IKKβ) inhibitor, and the thienobenzodiazepine analog olanzepine,
which is an antipsychotic drug approved by the U.S. Food and Drug Administration (FDA)
5
for the treatment of schizophrenia and bipolar disorders. These examples illustrate that the
design and discovery of a new extension of the Gewald reaction will help to address scaffold
diversity in 2ꢀaminothiophene derivatives, leading to the discovery of new pharmaceutical
agents.
Figure 1. Pharmaceutical agents derived from Gewald reactions
In recent years, considerable attention has been focused on the development of efficient and
operationally simple protocols for carbonꢀcarbon and carbonꢀheteroatom bond formation,
aiming to construct valuable molecules. Michael addition of indoles, pyrroles, amines and
thiols (Michael donors) to α, βꢀunsaturated aldehydes (Michael acceptors) has received much
interest in this regard, due to the occurrence of a number of derivatives in Nature that exhibit
6
ꢀ7
interesting biological activities. The versatility and effectiveness of the resulting carbonyl
compounds as pivotal synthetic intermediates for the Gewald reaction has not been explored
yet (Scheme 1). It is therefore attractive to think that the combination of these two reactions
would result in the development of a new fourꢀcomponent reaction for the selective
construction of highly functionalized 2ꢀaminothiophene derivatives of high synthetic and
biological importance.

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Scheme 1. General scheme of the MGꢀ4CR
RESULTS AND DISCUSSION
Chemistry
In order to validate this synthetic design, we investigated the feasibility of MichaelꢀGewald
fourꢀcomponent reactions (MGꢀ4CRs) under basic conditions with as the starting components
(i) indoles, (ii) α, βꢀunsaturated carbonyl compounds, (iii) elemental sulfur and (iv) activated
nitriles containing an αꢀmethylene moiety. The first step is the Michael addition of the indole
to an α, βꢀunsaturated carbonyl compound using an iminium activation mode. This results in
8
the direct Cꢀ3 alkylation of different indole derivatives. The Michael adducts are
subsequently subjected in the same pot to an Knoevenagel condensation with the activated
nitrile, followed by a baseꢀpromoted reaction with elemental sulfur. Thus, this reaction results
in the formation of multisubstituted 2ꢀaminothiophene derivatives with an electronꢀ
withdrawing group in the 3ꢀposition and an indolemethyl moiety at the 5ꢀposition of the
thiophene ring.
Optimization of the reaction conditions was performed using readily available building blocks
such as indole, acrolein, methyl cyanoacetate and elemental sulfur (Table 1). Initial
experiments were conducted via the simultaneous addition of all four model components in
8
the presence of additives, such as morpholine.trifluoroacetic acid (Morph.TFA) salt as the
catalyst for the Michael addition reaction and NEt
3
as the base for the successive Gewald
reaction, using THF as the solvent at 60°C (Table 1, entry 1). However, only a small amount

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of the desired product was formed. Further investigations showed that a oneꢀpot fourꢀ
component reaction was more effective when the Michael addition was performed between 1a
8
and 2a in presence of Morph.TFA at 40°C over a period of 3 hours. This is followed by the
3
addition of one equivalent of methyl cyanoacetate 3a, elemental sulfur and NEt to this crude
reaction mixture and heating was continued under reflux temperature over a period of 16
hours which afforded 4a in 25% isolated yield (Table 1, entry 2). After these initial results,
catalysts, solvents, base, ratio of different starting components and other reaction parameters
were investigated (Table 1, entry 3ꢀ9). The best result was obtained when performing a oneꢀ
pot MGꢀ4CR using 1a, 2a, 3a and sulfur in a respective molar ratio of 1:1:0.7:0.7 using
methanol as solvent in the presence of 20 mol% Morph.TFA salt and one equivalent of NEt
as the base under argon atmosphere, affording 81 % of the desired product 4a (Table 1, entry
).
3
6
a
Table 1: Optimization of the Gꢀ4CR
b
Entry Solvent
Ratio
Catalyst
Base yield
1
a:2a:3a:S
8
(20 mol%)
Morph.TFA
1
2
3
4
THF
THF
1:1:1:1
1:1:1:1
NEt
3
4%
Morph.TFA
Morph.TFA
Morph.TFA
NEt3
NEt3
25%
33%
55%
CH CN 1:1:1:1
3
MeOH 1:1:1:1
NEt
3

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5
6
7
8
9
MeOH 1:1:0.5:0.5 Morph.TFA
NEt
3
69%
81%
78%
49%
MeOH 1:1:0.7:0.7 Morph.TFA NEt
3
MeOH 1:1:0.8:0.8 Morph.TFA
NEt
3
3
MeOH 1:1:0.7:0.7 Morph.TsOH NEt
MeOH 1:1:0.7:0.7 Morph.TFA
DIPEA 57%
a
Reaction conditions: 1a is always in 1.7 mmol amount and the molar ratio of 2a, 3a and S is
8
b
calculated on the basis of this, solvent (5 ml). Isolated yield after column chromatography.
Entry (1): simultaneous addition of all the reagents and reaction temperature of 60°C. Entry
8 3
(2ꢀ9): 3a, S and NEt were added after 3h of reaction, reaction temperature was 40°C for the
first 3h and reflux condition for the next 16h. morpholine.trifluoroacetic acid = Morph.TFA.
morpholine.pꢀtoluenesulfonic acid = Morph.TsOH.
With the optimized reaction conditions in hand, we explored the generality of this MGꢀ4CR to
a variety of indole derivatives, α, βꢀunsaturated aldehydes and activated nitriles (Table 2).
Different indoles having both electronꢀdonating and electronꢀwithdrawing groups smoothly
underwent the desired transformation, generating an indolemethyl moiety at the 5ꢀposition of
the 2ꢀamino thiophene ring 4 in good to excellent yields. It is important to note that the
alkylation reaction of indoles having an electron withdrawing group requires a longer reaction
time, as compared to 1a, whereas the rate of the reaction is faster in the case of an indole
having an electron donating group. Similarly, the steric effect of the phenyl substituent at the
2ꢀposition of indoles (1e-1h) diminishes the yields and slows down the synthesis of the C3
alkylated intermediate. It is worth mentioning that the reactivity of the Michael adducts 4’ in
the Gewald reaction has no direct effect from the substituent of the indole heterocycles. An

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initial investigation with 1ꢀmethylindole did not give the expected product 4i, most likely due
to the low reactivity of 1ꢀmethylindole in the conjugate addition reaction. We then examined
the scope of this reaction with respect to crotonaldehyde and cinnamaldehyde. Similar
reactivity as for acrolein was observed while using crotonaldehyde. However, the reactivity of
cinnamaldehyde towards this MGꢀ4CR was very low under the optimized conditions which
delivered the expected product 4l only in 12% isolated yield. After some further optimization,
the yield of 4l was improved to 32% but this required reflux condition in the initial Michael
addition reaction. A better yield (49 %) was observed in the case of 5ꢀmethoxyindole 1m,
which is attributable to the higher reactivity of the electron rich building block.

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Table 2. Scope of MGꢀ4CRsa
NC R3
R3
NH2
CHO
3
R2
Morph:TFA
20 mol%)
R1
+
+
R2
(
S
N
O
R1
S8
R1
H
N
4
0 °C, MeOH, 3h
H
NEt , reflux, 16 h
N
1
2
3
H
4
'
4
entry
1
1
2
3
4
COOMe
NH2
S
O
NC COOMe
N
N
4
a (81%)
H
H
COOMe
2
S
NH2
F
F
O
NC COOMe
NC COOMe
4b (69%)
N
N
H
H
COOMe
NH2
S
3
4
Br
Br
O
N
N
4c (82%)
COOMe
H
H
Br
Br
S
S
S
NH2
4d (59%)
O
O
NC COOMe
N
H
N
H
COOMe
NH2
5b
NC COOMe
NC COOMe
4e (78%)
N
H
N
H
COOMe
NH2
F
6b
O
F
N
H
N
H
4f (69%)
COOMe
NH2
7b
S
S
OMe
O
O
NC COOMe
NC COOMe
N
H
N
H
OMe
4
g (75%)
COOMe
OMe
OMe
OMe
8c
NH
2
OMe
N
H
N
H
OMe
MeO
4h (24%)
COOMe
S
NH2
9
O
N
NC COOMe
N
4i (0%)
COOMe
S
NH2
1
0
N
H
O
NC COOMe
NC COOMe
4j (71%)
N
H
COOMe
1
1
MeO
MeO
S
NH2
O
N
H
4k (78%)
N
H
COOMe
NH2
1
2d
S
NC COOMe
N
H
4
l (32%)
O
N
H
COOMe
S
NH2
3d
1
1
MeO
MeO
NC COOMe
N
O
N
H
4m (49%)
H
O
NHPh
4e
S
NH2
O
NC
O
N
H
N
H
NHPh
4
n (34%)
CN
S
NH2
4o (0%)
MeO
MeO
1
5
O
NC CN
N
N
H
H

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a
Reaction conditions: 1 (1.0 equiv.), 2 (1.0 equiv.), 3 (0.7 equiv.), S (0.7 equiv.), NEt (1.0
8
3
b
c
equiv.), Isolated yield. ꢁ, 40 °C for 5 h and reflux condition for another 16 h. ꢁ, 40 °C for 10
d
h and reflux condition for another 24 h. ꢁ, 60 °C for 24 h and reflux condition for another 48
e
h. . ꢁ, 40 °C for 3 h and reflux condition for another 24 h.
Next, we examined the impact of different nitrile components on these MGꢀ4CRs. As
expected, a slightly lower reactivity was observed in the case of 2ꢀcyanoacetanilide 3n which
furnished the amideꢀsubstituted Gewald product 4n in a moderate yield of 34%. On the other
hand, in the case of malonitrile 3o the reaction took another course (Scheme 2). Careful
analysis and separation of the crude reaction mixture revealed that 5o is the major product
formed under the present reaction circumstances. This was formed as a result of the conjugate
addition of the protic solvent methanol to the resulting Knoevenagel intermediate 4o’. Our
efforts to avoid this side reaction by using more sterically hindered protic solvents, such as
isopropanol or tertꢀbutanol, or even with an aprotic solvent, such as THF, did not deliver the
anticipated product 4o.
Scheme 2. Unexpected side reaction with malonitrile
The versatility of this MGꢀ4CR was further examined by changing the nucleophilic
component. For instance, an aromatic πꢀnucleophilic building block such as Nꢀmethylpyrrole
was investigated (Scheme 3). Gratifyingly, this reaction led to the formation of the expected
compound 7 which contains a pyrrole and a 2ꢀaminothiophene moiety separated by a
methylene spacer, albeit in lower yield. Unfortunately, a similar reaction with 1Hꢀpyrrole
resulted in a very complex reaction mixture. We assume that polymerization of pyrrole
occurred under the slightly acidic conditions generated by Morph:TFA catalyst used in the
initial Michael addition reaction.

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Scheme 3. Scope with respect to Nꢀmethylpyrrole
Biological Results
The synthesized compounds were first evaluated for their antiꢀproliferative activity in three
different tumor cell lines: mouse lymphocytic leukemia (L1210), human lymphoblastic
leukemia (CEM) and human cervical carcinoma (HeLa) cells. Data are expressed as IC50 (50%
inhibitory concentration) defined as the concentration at which the compounds reduce cell
9
proliferation by 50% and are shown in Table 3. Compound 4a caused a modest inhibition of
tumor cell proliferation (IC50 of 40ꢀ60 µM). Addition of electronꢀwithdrawing groups at the
indole did not improve (compounds 4b and 4c) or slightly improved (IC of 4d between 11
5
0
and 25 µM) the cytostatic activity. Interestingly, 4e-4g containing a phenyl substituent at the
ꢀposition of the indole group inhibited the proliferation of all cell lines tested in the lower
2
micromolar range. Addition of extra methoxy groups at the phenyl ring (4h versus 4g)
resulted in 10ꢀfold lower antiꢀproliferative activity.
Table 3. Cytostatic activity of tested compounds against L1210, CEM and HeLa cells
*
IC50 (µM)
Compound
L1210
43 ± 19
90 ± 4
CEM
66 ± 21
55 ± 19
39 ± 3
HeLa
65 ± 17
95 ± 21
81 ± 2
4
a
4b
4
c
70 ± 1
4d
25 ± 0
11 ± 4
13 ± 5
4e
1.0 ± 0.1
0.75 ± 0.11
0.91 ± 0.06

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4
f
1.6 ± 1.0
1.3 ± 0.2
20 ± 1
0.53 ± 0.33
0.80 ± 0.24
13 ± 7
0.90 ± 0.07
4g
1.6 ± 1.2
17 ± 3
4h
4j
20 ± 2
10 ± 3
11 ± 9
4
k
112 ± 6
27 ± 2
86 ± 50
27 ± 1
90 ± 6
4
l
34 ± 12
19 ± 2
4m
23 ± 0
21 ± 11
69 ± 19
105 ± 25
4.0 ± 0.0
28 ± 0
4n
90 ± 7
104 ± 5
101 ± 11
13 ± 2
7
95 ± 13
19 ± 3
8
9
106 ± 3
81 ± 36
To evaluate the importance of the amino and the ester groups on the thiophene ring system of
the active compound 4e towards cytostatic activity, compounds 8 and 9 were synthesized in
reasonable yields (Figure 1). Compound 8 was synthesized by using a classical amidation
10
reaction whereas compound 9 was obtained by following the previously reported procedure.
The cytostatic studies indicate that substitution of the amino (8) or deletion of the ester group
9) on the indole ring resulted in 10ꢀfold reduction or complete loss of activity, respectively.
(
Figure 1. Structures of 8 and 9
To further investigate the effect of compound 4e on cell proliferation, cell cycle analysis was
9
performed. Whereas control cells show a typical distribution in the different phases of the
cell cycle (Figure 2), compound 4e caused a doseꢀdependent increase in the G2/M phase

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population and an increase in subG1 cells at 0.8ꢀ20 µM. These data indicate that our
compounds prevent the treated cells from proceeding through mitosis, after which they
undergo apoptosis.
2a)
2b)
Control
4g (20 ꢀM)
4g (4 ꢀM)
4g (0.8 ꢀM)
Figure 2: Cell cycle distribution. a, b) HeLa cells were treated with compound 4e for 24 h.
Next, the cells were harvested, stained with propidium iodide, and cell cycle distribution was
evaluated by flow cytometry. b) Percentages of cells in the different phases of the cell cycle
are indicated.
CONCLUSION
In summary, we have developed a versatile Gewald fourꢀcomponent reaction from readily
available building blocks such as indole derivatives, α, βꢀunsaturated aldehydes, activated
nitriles and elemental sulfur that generates 2ꢀaminothiophene derivatives decorated with
useful functional groups in moderate to excellent yield. The impact of different nucleophilic
components such as pyrrole on this MGꢀ4CR was also investigated. This elegant strategy
offers an operationally simple procedure, metalꢀfree conditions and demonstrates a broad
substrate scope. These synthesized compounds were also tested for antiproliferative activity in
three different cell lines. Among these, compounds 4e, 4f and 4g, with a phenyl substitution at
2ꢀposition of indole heterocycle of 4 exhibited antiꢀproliferative activity in the low µM range.
We envisioned that the utility of this MGꢀ4CR towards the rapid synthesis of a focused

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combinatorial library of cytostatic compounds including the combination of biologically
relevant 2–aminothiophene and indole heterocycles will certainly provide ample opportunity
to discover new pharmaceutically active agents.
EXPERIMENTAL SECTION
General experimental methods
NMR spectra were acquired on commercial instruments (300 MHz and 400 MHz and 600
MHz) and chemical shifts (δ) are reported in parts per million (ppm) referenced to
1
1
13
3
tetramethylsilane ( H) or the internal (NMR) solvent (CDCl ) signal ( H & C). Mass spectra
were recorded on a HewlettꢀPackard 5989A mass spectrometer (EI mode and CI mode).
Highꢀresolution mass data were obtained with a Kratos MS50TC instrument. Melting points
(not corrected) were determined using a Reichert Thermovar apparatus. The compounds
prepared were obtained in a pure form after column chromatography and evaporation of the
fractions, and no further attempt was made to crystallize them. Therefore, the melting points
reflect this purity after chromatography. For column chromatography, 70@230 mesh silica 60
(E. M. Merck) was used as the stationary phase. Chemicals received from commercial sources
were used without further purification. Solvents used in reactions were used as received.
Experimental and characterization data
General procedure for the synthesis of 4 as shown in Table 1.
A solution of indole 1 (1.7 mmol), α, βꢀunsaturated carbonyl compound 2 (1.7 mmol) and
morpholine TFA salt (0.34 mmol) in MeOH (5 ml) was stirred (room temperature to 60°C)
for 3ꢀ12 hours under argon atmosphere. This reaction mixture was then cooled to room
temperature and to this was added activated nitrile (1.12 mmol), elemental sulfur (1.12 mmol)
and NEt
3
(1.7 mmol). The resulting reaction mixture was then refluxed for an additional 16ꢀ
48 hours. The MeOH was evaporated under reduced pressure and the residue was extracted

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with dichloromethane (3
solution of NaHCO , dried over magnesium sulfate and the solvent was evaporated under
reduced pressure. Purification by column chromatography (Silica, eluent heptaneꢀethyl acetate
0:40) afforded the final compound.
× 50ml). The combined organic phases were washed with a saturated
3
6
Methyl 5-((1H-indol-3-yl)methyl)-2-aminothiophene-3-carboxylate (4a)
Reaction time is 19 h (40°C for the first 3 h and reflux condition for the remaining 16 h after
8
adding methyl cyanoacetate and S ). The product was purified by flash column
chromatography (heptane/EtOAc = 1:1) afforded 4a (232 mg, 81% yield) as an offꢀwhite
1
solid; m.p. 47 – 48 ˚C; H NMR (300 MHz, CDCl
3
) δ 8.03 (s, 1H), 7.55 (d,
J = 7.9 Hz, 1H),
7
2
1
.32 (d,
J
= 8.1 Hz, 1H), 7.25 – 7.05 (m, 2H), 6.98 (d,
J
= 2.2 Hz, 1H), 6.73 (s, 1H), 5.73 (s,
1
3
H), 4.04 (s, 2H), 3.75 (s, 3H); C NMR (75 MHz, CDCl ) δ 165.9, 162.0, 136.4, 127.2,
3
26.2, 122.4, 122.2, 122.0, 119.5, 119.0, 114.5, 111.2, 106.8, 50.9, 25.9; MS (EI): 286;
14 2 2
HRMS: Calculated for C15H N O S: 286.0776, found 286.0771.
Methyl 2-amino-5-((5-fluoro-1H-indol-3-yl)methyl)thiophene-3-carboxylate (4b)
Reaction time is 19 h (40°C for the first 3 h and reflux condition for the remaining 16 h after
adding methyl cyanoacetate and S ). The product was purified by flash column
8
chromatography (heptane/EtOAc = 1:1) afforded 4b (235 mg, 69% yield) as an offꢀwhite
1
solid; m.p. 54 – 55 ˚C; H NMR (300 MHz, CDCl
3
) δ 8.07 (s, 1H), 7.32 – 7.13 (m, 2H), 7.07
(
s, 1H), 6.93 (td,
J
= 9.0, 2.4 Hz, 1H), 6.73 (s, 1H), 5.78 (s, 2H), 4.00 (s, 2H), 3.77 (s, 3H);
) δ 165.9, 162.0, 133.0, 125.8, 124.2, 122.2, 112.0, 111.9, 110.9,
1
3
C NMR (75 MHz, CDCl
3
110.5, 106.0, 104.2, 103.9, 51.0, 26.0; MS (EI): 304; HRMS: Calculated for C H FN O S:
15 13 2 2
304.0682, found 304.0686.
Methyl 2-amino-5-((5-bromo-1H-indol-3-yl)methyl)thiophene-3-carboxylate (4c)

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Reaction time is 19 h (40°C for the first 3 h and reflux condition for the remaining 16 h after
adding methyl cyanoacetate and S8). The product was purified by flash column
chromatography (heptane/EtOAc = 1:1) afforded 4c (333 mg, 82% yield) as an offꢀwhite solid;
1
m.p. 52 – 53 ˚C; H NMR (300 MHz, CDCl
3
) δ 8.06 (s, 1H), 7.67 (s, 1H), 7.32 – 7.18 (m,
13
2
H), 7.05 (s, 1H), 6.71 (s, 1H), 5.78 (s, 2H), 4.01 (s, 2H), 3.78 (s, 3H); C NMR (75 MHz,
CDCl ) δ 165.9, 162.0, 135.1, 129.0, 125.5, 125.3, 123.7, 122.3, 121.7, 114.5, 113.0, 112.7,
3
1
06.2, 51.0, 25.8; MS (EI): 364, 366; HRMS: Calculated for C H BrN O S: 363.9881,
15 13 2 2
found 363.9885.
Methyl 2-amino-5-((4-bromo-1H-indol-3-yl)methyl)thiophene-3-carboxylate (4d)
Reaction time is 19 h (40°C for the first 3 h and reflux condition for the remaining 16 h after
adding methyl cyanoacetate and S8). The product was purified by flash column
chromatography (heptane/EtOAc = 1:1) afforded 4d (220 mg, 59% yield) as an offꢀwhite
1
solid; m.p. 50 – 51˚C; H NMR (300 MHz, CDCl
3
) δ 8.20 (s, 1H), 7.27 (dd,
J
= 10.6, 5.7 Hz,
13
2
H), 7.05 – 6.96 (m, 2H), 6.65 (s, 1H), 5.78 (s, 2H), 4.35 (s, 2H), 3.76 (s, 3H); C NMR (75
MHz, CDCl
3
) δ 166.0, 162.0, 137.8, 126.6, 125.2, 124.6, 124.2, 123.1, 122.3, 115.8, 114.1,
S:
1
10.7, 106.2, 51.0, 26.7; MS (EI): 364, 366; HRMS: Calculated for C15
63.9881, found 363.9884.
2 2
H13BrN O
3
Methyl 2-amino-5-((2-phenyl-1H-indol-3-yl)methyl)thiophene-3-carboxylate (4e)
Reaction time is 21 h (40°C for the first 5 h and reflux condition for the remaining 16 h after
adding methyl cyanoacetate and S8). The product was purified by flash column
chromatography (heptane/EtOAc = 1:1) afforded 4e (290 mg, 78% yield) as an offꢀwhite
1
solid; m.p. 64 – 65 ˚C; H NMR (300 MHz, CDCl ) δ 8.14 (s, 1H), 7.60 – 7.52 (m, 3H), 7.45
3
(
t,
J
= 7.4 Hz, 2H), 7.37 (t,
J
= 5.9 Hz, 2H), 7.22 (dd,
J
= 12.4, 4.4 Hz, 1H), 7.12 (q,
J
= 7.0
) δ
1
3
Hz, 1H), 6.66 (s, 1H), 5.72 (s, 2H), 4.13 (s, 2H), 3.72 (s, 3H); C NMR (75 MHz, CDCl
3

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1
1
3
65.9, 161.9, 136.0, 135.4, 132.7, 129.1, 129.1, 129.0, 128.1, 126.8, 122.7, 121.7, 120.1,
19.5, 111.0 110.5, 106.2, 51.0, 25.2; MS (EI): 362; HRMS: Calculated for C H N O S:
21
18
2
2
62.1089, found 362.1086.
2-amino-5-((2-(4-fluorophenyl)-1H-indol-3-yl)methyl)thiophene-3-carboxylate
Methyl
4f)
(
Reaction time is 21 h (40°C for the first 5 h and reflux condition for the remaining 16 h after
adding methyl cyanoacetate and S8). The product was purified by flash column
chromatography (heptane/EtOAc = 1:1) afforded 4f (293 mg, 69% yield) as an offꢀwhite solid;
1
m.p. 68 – 69 ˚C; H NMR (300 MHz, CDCl
3
) δ 8.14 (s, 1H), 7.53 (m, 3H), 7.49 (m, 1H), 7.17
1
3
(
3
m, 5H), 6.63 (s, 1H), 5.75 (s, 2H) 4.08 (s, 2H), 3.72 (s, 3H); C NMR (75 MHz, CDCl ) δ
1
71.2, 169.4, 165.9, 164.2, 161.9, 135.9, 134.5, 129.8, 129.7, 128.8, 129.1, 126.5, 122.7,
21.7, 120.1, 119.4, 116.2, 110.1 115.9, 111.0, 106.23, 50.9, 25.0; MS (EI): 380; HRMS:
1
2 2
Calculated for C21H17FN O S: 380.0995, found 380.0993.
Methyl 2-amino-5-((2-(4-methoxyphenyl)-1H-indol-3-yl)methyl)thiophene-3-carboxylate
4g)
(
Reaction time is 21 h (40°C for the first 5 h and reflux condition for the remaining 16 h after
adding methyl cyanoacetate and S8). The product was purified by flash column
chromatography (heptane/EtOAc = 1:1) afforded 4g (329 mg, 75% yield) as an offꢀwhite
1
solid; m.p. 71 – 72 ˚C; H NMR (300 MHz, CDCl
3
) δ 8.13 (s, 1H), 7.56 (d,
J
= 7.9 Hz, 1H ),
= 8.9 Hz,
H), 6.65 (s, 1H), 5.78 (s, 2H), 4.09 (s, 2H); 3.83 (s, 3H), 3.72 (s, 3H); C NMR (75 MHz,
7.54 (d,
J
= 7.1 Hz, 2H ), 7.39 (d,
J
= 7.8 Hz, 1H ), 7.24 – 7.10 (m, 2H), 7.01 (d, J
13
2
CDCl ) δ 165.9, 161.8, 159.5, 135.7, 135.3, 129.2, 129.1, 126.9, 125.1, 122.2, 121.4, 119.9,
3
119.0, 114.5, 110.8 109.6, 106.1, 60.5, 55.3, 50.8, 25.1; MS (EI): 392; HRMS: Calculated for
22 20 2 3
C H N O
S: 392.1195, found 392.1198.

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Methyl
2-amino-5-((2-(3,4,5-trimethoxyphenyl)-1H-indol-3-yl)methyl)thiophene-3-
carboxylate (4h)
Reaction time is 34 h (40°C for the first 10 h and reflux condition for the remaining 24 h after
adding methyl cyanoacetate and S8). The product was purified by flash column
chromatography (heptane/EtOAc = 1:1) afforded 4h (121 mg, 24% yield) as an offꢀwhite
1
1
solid; m.p. 78– 79 ˚C; H NMR (300 MHz, CDCl
H), 7.60 (d, = 7.9 Hz, 1H), 7.42 (d, = 7.9 Hz, 1H), 7.28 – 7.13 (m, 2H), 6.79 (s, 2H), 6.70
t, = 1.3 Hz, 1H), 5.80 (s, 2H), 4.15 (s, 2H), 3.89 (s, 3H); 3.82 (s, 6H), 3.73 (s, 3H);
NMR (75 MHz, CDCl ) δ 171.2, 169.4, 165.9, 164.2, 161.9, 135.9, 134.5, 129.8, 129.2,
28.2, 126.9, 122.6, 121.7, 120.1, 119.2, 110.9 110.2, 105.1, 61.0, 56.2, 55.9, 50.9, 25.3; MS
EI): 452; HRMS: Calculated for C24 S: 452.1406, found 452.1401.
3 3
) δ H NMR (300 MHz, CDCl ) δ 8.27 (s,
1
J
J
13
(
J
C
3
1
(
24 2 5
H N O
Methyl 5-(1-(1H-indol-3-yl)ethyl)-2-aminothiophene-3-carboxylate (4j)
Reaction time is 19 h (40°C for the first 3 h and reflux condition for the remaining 16 h after
adding methyl cyanoacetate and S8). The product was purified by flash column
chromatography (heptane/EtOAc = 1:1) afforded 4j (238 mg, 71% yield) as an offꢀwhite solid;
1
m.p. 65 – 66 ˚C; H NMR (400 MHz, CDCl
3
) δ 8.06 (s, 1H), 7.53 (d,
= 7.5 Hz, 1H), 7.06 (t, = 7.5 Hz, 1H), 6.97 (s, 1H), 6.76 (s, 1H),
= 7.0 Hz, 1H), 3.76 (s, 3H), 1.68 (d,
) δ 166.0, 161.9, 136.6, 132.8, 126.5, 122.2, 121.2, 121.0, 120.5, 119.5, 119.5,
11.3, 105.7, 51.0, 32.2, 22.5; MS (EI): 300; HRMS: Calculated for C16 S: 300.0932,
found 300.0934.
J = 7.9 Hz, 1H), 7.31
(
d,
.71 (s, 2H), 4.36 (q,
MHz, CDCl
J
= 8.1 Hz, 1H), 7.16 (t,
J
J
13
5
J
J = 7.1 Hz, 3H); C NMR (101
3
1
16 2 2
H N O
Methyl 2-amino-5-(1-(5-methoxy-1H-indol-3-yl)ethyl)thiophene-3-carboxylate (4k)
Reaction time is 19 h (40°C for the first 3 h and reflux condition for the remaining 16 h after
adding methyl cyanoacetate and S8). The product was purified by flash column

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chromatography (heptane/EtOAc = 1:1) afforded 4k (288 mg, 78% yield) as an offꢀwhite
1
solid; m.p. 72 – 73 ˚C; H NMR (300 MHz, CDCl ) δ 7.96 (s, 1H), 7.22 (d,
J
= 8.8 Hz, 1H),
3
6.98 (t,
J
= 2.5 Hz, 2H), 6.84 (dd,
J
= 8.8, 2.3 Hz, 1H), 6.76 (s, 1H), 5.73 (s, 2H), 4.32 (q, J =
1
3
7.0 Hz, 1H), 3.78 (d,
J
3
= 9.1 Hz, 6H), 1.68 (d, J = 7.0 Hz, 3H); C NMR (75 MHz, CDCl ) δ
166.0, 161.9, 153.9, 132.7, 131.8, 126.95, 122.0, 120.6, 120.4, 112.2, 112.0, 105.8, 101.6,
5
6.1, 51.0, 32.1, 22.4; MS (EI): 330; HRMS: Calculated for for C H N O S: 330.1038,
17
18
2
3
found 330.1039.
Methyl 5-((1H-indol-3-yl)(phenyl)methyl)-2-aminothiophene-3-carboxylate (4l)
Reaction time is 72 h (60 °C for the first 24 h, reflux condition for 48 h after adding methyl
cyanoacetate and S
8
). The product was purified by flash column chromatography
(
˚
heptane/EtOAc = 1:1) afforded 4l (129mg, 32% yield) as an offꢀwhite solid; m.p. 131 – 132
1
C; H NMR (300 MHz, CDCl ) δ 8.09 (s, 1H), 7.36 (d,
J = 8.8 Hz, 1H), 7.33 – 7.29 (m,
3
2
1
1
1
3
H), 7.26 (t,
J = 5.6 Hz, 2H), 7.22 – 7.14 (m, 2H), 7.07 – 7.00 (m, 1H), 6.82 (s, 1H), 6.61 (s,
13
H), 5.81 (s, 1H), 5.60 (s, 1H), 5.30 (s, 2H), 3.73 (s, 3H). C NMR (75 MHz, CDCl
3
) δ
66.0, 162.4, 143.3 136.7, 129.5, 128.5, 126.9, 126.8, 123.7, 123.2, 122.3, 119.8, 119.7,
19.1, 111.3, 106.0, 51.0, 43.9; MS (EI): 362; HRMS: Calculated for for C H N O S:
21
18
2
2
62.1089, found 362.1084.
Methyl 2-amino-5-((5-methoxy-1H-indol-3-yl)(phenyl)methyl)thiophene-3-carboxylate
4m)
Reaction time is 72 h (60 °C for the first 24 h, reflux condition for 48 h after adding methyl
cyanoacetate and S ). The product was purified by flash column chromatography
heptane/EtOAc = 1:1) afforded 4m (215 mg, 49% yield) as an offꢀwhite solid; m.p. 138– 139
(
8
(
˚
1
C; H NMR (300 MHz, CDCl
3
) δ 8.00 (s, 1H), 7.34 – 7.27 (m, 4H), 7.26 – 7.13 (m, 2H),
6
.83 (dd,
H), 3.72 (s, 3H); C NMR (75 MHz, CDCl
J = 8.8, 2.4 Hz, 1H), 6.78 (s, 2H), 6.61 (s, 1H), 5.81 (s, 2H), 5.54 (s, 1H), 3.72 (s,
13
3
3
) δ 166.0, 162.4, 154.0, 143.2, 131.8, 129.4,

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128.5, 127.2, 126.8, 124.5, 123.2, 118.7, 112.3, 112.0, 105.9, 101.8, 56.0, 50.9, 43.9; MS
(
EI): 392; HRMS: Calculated for for C H N O S: 392.1195, found 392.1197.
22
20
2
3
5-((1H-indol-3-yl)methyl)-2-amino-N-phenylthiophene-3-carboxamide (4n)
Reaction time is 27 h (40 °C for the first 3 h, reflux condition for 24 h after adding methyl
cyanoacetate and S ). The product was purified by flash column chromatography
heptane/EtOAc = 1:1) afforded 4n (132 mg, 34% yield) as an offꢀwhite solid; m.p. 145– 146
8
(
1
˚
C; H NMR (300 MHz, CDCl ) δ 8.14 (s, 1H), 7.54 – 7.46 (m, 3H), 7.35– 7.19 (m, 8H),
3
1
3
6
.56 (s, 1H), 6.60 (s, 2H), 4.07 (s, 2H); C NMR (75 MHz, CDCl
36.5, 129.0, 127.1, 126.9, 124.1, 122.5, 122.4, 120.6, 119.7,119.1, 114.2, 111.4, 108.3, 26.0;
OS: 347.1092, found 347.1097.
3
) δ 164.1, 160.8, 138.1,
1
17 3
MS (EI): 347; HRMS: Calculated for C20H N
Synthesis of 2-(1-methoxy-3-(5-methoxy-1H-indol-3-yl)butyl)malononitrile (5o, mixture
of different diastereomers)
A solution of 5ꢀmethoxyindole 1 (1.7 mmol), crotonaldehyde 2 (1.7 mmol) and morpholine
TFA salt (0.34 mmol) in MeOH (5 ml) was stirred at 40 °C for 3 hours under argon
atmosphere. This reaction mixture is then cooled to room temperature and to this was added
malonitrile (1.12 mmol), elemental sulfur (1.12 mmol) and NEt (1.7 mmol). The resulting
3
reaction mixture is then refluxed for an additional 16 hours. The MeOH was evaporated under
reduced pressure and the residue was extracted with dichloromethane (3
×
50ml). The
, dried over
combined organic phases were washed with a saturated solution of NaHCO
3
magnesium sulfate and the solvent was evaporated under reduced pressure. Purification by
column chromatography (Silica, eluent heptaneꢀethyl acetate 80:20) afforded 5o in 31% yield
1
(
103 mg). Offꢀwhite solid; m.p. 101 – 102 ˚C; H NMR (300 MHz, CDCl
3
) δ ) δ 8.01 (s, 1H),
)
δ 7.91 (s, 1H), 7.23 (d,
J = 8.9 Hz, 2H), δ 7.11 (s, 1H), 7.00 – 6.83 (m, 4H), 6.48 (s, 1H),
4
3
.57 (s, 2H), 4.39 – 4.30 (m, 2H), 3.86 (s, 3H), 3.80 (s, 3H), 3.31 (s, 3H), 3.29 (s, 3H), 3.21–
1
3
.09 (m, 2H), 2.10– 1.88 (m, 4H), 1.368 (d,
J
= 7.2 Hz, 3H), 1.37 (d,
J
= 7.0 Hz, 3H);
C

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NMR (75 MHz, CDCl ) δ 170.5, 168.4, 156.9, 153.9, 146.3, 143.6, 140.9, 131.7, 121.4,
3
1
20.9, 115.2, 111.8, 111.5, 103.9, 101.4, 88.3, 88.0, 56.0, 52.3, 50.38, 40.3, 35.6, 26.8, 26.2,
4.2, 21.9; MS (EI): 297; HRMS: Calculated for for C17 : 297.1477, found 297.1479.
2
19 3 2
H N O
Synthesis of methyl 2-amino-5-((1-methyl-1H-pyrrol-2-yl)(phenyl)methyl)thiophene-3-
carboxylate (7)
A solution of 1ꢀmethylꢀ1Hꢀpyrrole 1 (1.7 mmol), cinnamaldehyde (1.7 mmol) and
morpholine TFA salt (0.34 mmol) in MeOH (10ml) was stirred at room temperature for 12
hours under argon atmosphere. This reaction mixture is then cooled to room temperature and
3
was added methyl cyanoacetate (0.85 mmol), elemental sulfur (0.85 mmol) and NEt (1.7
mmol). The resulting reaction mixture is then refluxed for additional 16 hours. The MeOH
was evaporated under reduced pressure and the residue was extracted with dichloromethane
(3
×
50ml). The combined organic phases were washed with a saturated solution of NaHCO
3
,
dried over magnesium sulfate and the solvent was evaporated under reduced pressure.
Purification by column chromatography (Silica, CH
yield (91 mg).
2 2
Cl ) afforded the final compound in 33%
1
Semiꢀsolid; H NMR (300 MHz, CDCl ) δ 7.35 – 7.23 (m, 3H), 7.19 (d,
J
= 7.4 Hz, 2H), 6.59
= 3.0 Hz, 1H), 5.84 (s, 2H), 5.76 (s, 1H), 5.32 (s, 1H), 3.73 (s,
H), 3.40 (s, 3H); C NMR (75 MHz, CDCl ) δ 165.9, 162.6, 141.8, 134.0, 128.6, 128.5,
27.8, 127.1, 123.6, 122.5, 109.1, 106.5, 105.9, 51.0, 44.4, 34.0; MS (EI): 326; HRMS:
S: 326.1089, found 326.1086.
3
(
s, 1H), 6.49 (s, 1H), 6.04 (t, J
13
3
1
3
18 2 2
Calculated for for C18H N O
Methyl 2-acetamido-5-((2-phenyl-1H-indol-3-yl)methyl)thiophene-3-carboxylate (8)
To a solution of 4d (200 mg, 0.55 mmol) dissolved in 10 ml acetonitrile was added acetyl
chloride (0.55 mmol), and triethyl amine (0.55 mmol). The resulting solution was stirred at
room temperature for 16 hrs. The solvent was evaporated to dryness, dissolved in ethyl

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acetate and washed with distilled water, dried over magnesium sulfate, filtered and dried to
obtain the crude product. Purification by column chromatography (Silica, eluent heptaneꢀethyl
acetate 70:30) afforded the corresponding pure compound as amorphous solid in 46 % yield
(102 mg).
1
Offꢀwhite solid; m.p. 115– 116 ˚C; H NMR (300 MHz, CDCl
.53 (m, 7H), 7.44 – 7.15 (m, 2H), 6.83 (s, 1H) 4.25 (s, 2H), 3.78 (s, 3H); C NMR (75 MHz,
CDCl ) δ 167.0, 166.2, 147.7, 136.1, 135.5, 135.1, 132.6, 129.1, 128.1, 128.0 122.6, 120.1,
3
) δ 10.88 (s, 1H), 8.19 (s, 1H),
13
7
3
119.4, 111.9, 110.9, 110.1; 51.6 , 25.0, 25.6; MS (EI): 404; HRMS: Calculated for for
23 20 2 3
C H N O
S: 404.1195, found 404.1191.
1
0
2-Phenyl-3-(thiophen-2-ylmethyl)-1H-indole (9)
2ꢀphenyl indole (100 mg, 0.51 mm), thiophenꢀ2ꢀylmethanol (177 mg, 1.5 mm) and potassium
hydroxide (38.34 mg, 0.63 mm) was dissolved in 3ml of methanol. The resulting solution was
heated at 60°C for 16 hrs. The reaction mixture was quenched with water (10ml). Ethyl
acetate 20 ml was added and the organic layer was washed with distilled water, dried over
magnesium sulfate, filtered and evaporated to dryness to obtain the crude product.
Purification by column chromatography (Silica, eluent heptaneꢀethyl acetate 80:20) afforded
the final compound as amorphous solid yield in 63% yield (100 mg).
1
Offꢀwhite solid; m.p. 110– 111 ˚C; H NMR (300 MHz, CDCl
3
) δ 8.13 (s, 1H), 7.57 (m, 3H),
7
1
1
.41 (m, 2H), 7.39 (m, 2H); 7.23 (m, 1H), 7.12 (m, 2H), 7.10 (s, 1H), 6.90 (m, 2H), 6.82 (m,
13
H); C NMR (75 MHz, CDCl
3
) 145.4, 136.0, 135.2, 132.7, 129.2, 129.0, 128.0, 126.9,
24.5, 123.4, 122.6, 120.0, 119.5, 111.2, 110.9, 25.4; MS (EI): 289; HRMS: Calculated for
for C H NS: 289.0925, found 289.0929.
19
15
Biological assays

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Cell proliferation. Human cervical carcinoma (HeLa) cells were seeded in 96ꢀwell plates at
5,000 cells/well in the presence of 5ꢀfold dilutions of the compounds. After 4 days of
1
incubation, the cells were trypsinized and counted by means of a Coulter counter (Analis,
Belgium). Suspension cells (Mouse leukemia L1210 and human lymphoid Cem cells) were
seeded in 96ꢀwell plates at 60,000 cells/well in the presence of the compounds. L1210 and
Cem cells were allowed to proliferate for 48 h or 96 h, respectively and then counted. The 50%
inhibitory concentration (IC ) was defined as the compound concentration required to reduce
5
0
9
cell proliferation by 50%.
Cell cycle analysis. HeLa cells were seeded in 6ꢀwell plates at 125,000 cells/well in DMEM
with 10% FBS. The following day, the cells were exposed to different concentrations of the
compounds for 24 h. Next, the DNA of the cells was stained with propidium iodide using the
CycleTEST PLUS DNA Reagent Kit (BD Biosciences, San Jose, CA). The DNA content of
the stained cells was assessed by flow cytometry on a FACSCalibur flow cytometer and
analyzed with CellQuest software (BD Biosciences) within 3h after staining. Cell debris and
clumps were excluded from the analysis by appropriate dot plot gating. Percentages of subꢀG1,
2
G1, S, and G /M cells were estimated using appropriate region markers.
Supporting information
†
1
13
Electronic supplementary information (ESI) available: H and C NMR spectra of the novel
precursors.
Acknowledgments
We thank the Fonds Wetenschappelijk Onderzoek (FWO) and the KU Leuven for financial
support. Erasmus Mundus Lot 13, EuroꢀIndia is acknowledged for the doctoral fellowship to
S.J. The research of JB and SL was supported by the KU Leuven (GOA 15/19 TBA). We also
thank Lizette Van Berckelaer and Eef Meyen for excellent technical assistance.
Notes and references

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DOI: 10.1039/C7OB00707H
1. a) J. Zhu, H. Bienayme, Eds. Multicomponent Reactions; WileyꢀVCH: Weinheim,
Germany, 2005. b) A. R. Katritzky, C. A. Ramsden, E. F. V. Scriven, R. J. K. Taylor, Eds.
Comprehensive Heterocyclic Chemistry III; Elsevier: New York, NY, USA, 2008. c) A.
Dömling, Chem. Rev. 2006, 106, 17. d) B. B. Touré and D.G. Hall, Chem. Rev. 2009, 109,
4
439. e) C. Kalinski, M. Umkehrer, L. Weber, J. Kolb, C. Burdack and G. Ross, Mol. Divers.
010, 14, 513. f) A. Dömling, W. Wang and K. Wang, Chem. Rev. 2012 112, 3083. g) I.
2
,
AkritopoulouꢀZanze, S. Djuric, In Synthesis of Heterocycles via Multicomponent Reactions II
;
Orru, R. V. A., Ruijter, E., Eds. Springer: Berlin/Heidelberg, 2010, 25, 231. h) C de. Graaff,
E. Ruijter and R. V.A. Orru, Chem. Soc. Rev. 2012, 41, 3969. i) B. Jiang, F. Shi and SꢀJ. Tu,
Curr. Org. Chem. 2010, 14, 357. j) V. Nair, R. S. Menon, A. T. Biju and K. G. Abhilash,
Chem. Soc. Rev. 2012, 41, 1050. k) L. A. Wessjohann, C. R. B. Rhoden, G. G. Rivera and O.
E. Vercillo, Top Heterocycl Chem. 2010, 23, 199. l) J. Thomas, J. John, N. Parekh, N and
Dehaen, W. Angew. Chem. Int. Ed., 2014, 53, 10155. m) R. C. Cioc, E. Ruijter, R. V. A.
Orru, : Green Chem. 2014, 16, 2958. n) J. John, Thomas, J and Dehaen, W. Chem. Commun.
,
,
2015, 51, 10797. o) J. Thomas, S. Jana, J. John, S. Liekens and W. Dehaen, Chem. Commun.
2
016, 52, 2885. p) D. G. Hall, T. Rybak and T. Verdelet, Acc. Chem. Res. 2016, 49, 2489. q) J.
9236.
Thomas, S. Jana, J. John, S. Liekens and W. Dehaen, Chem. Commun. 2016, 56
,
(
2) a) A. Dömling and Y. Huang, Mol. Divers. 2011, 15, 3. b) S. H. AbdelꢀHafez, Russ J Org
Chem. 2005, 41, 396. c) K. Wang, D. Kim and A. Dömling, . J. Comb. Chem.
010, 12, 111. d) C. Valant, L. Aurelio, S. M. Devine, T. D. Ashton, J. M. White, P.M.
2
Sexton, A. Christopoulos and P.J. Scammells, J. Med. Chem. 2012, 55, 2367. e) J. Balzarini,
W. Dehaen, J. Thomas, S. Liekens, R.Romagnoli and P.G. Baraldi, PCT Int. Appl. 2013,
WO 2013190137 A2 20131227. f) J. Balzarini, J. Thomas, S. Liekens, S. Noppen, W.
Dehaen, R. Romagnoli, Invest. New Drugs. 2014, 32, 200. g) J. Thomas, A. Jejcic, P.
Vervaeke, R.Romagnoli, S. Liekens, J. Balzarini and Dehaen, W. Chem Med Chem, 2014, 9,

Organic & Biomolecular Chemistry
Page 24 of 25
View Article Online
DOI: 10.1039/C7OB00707H
2
744. h) M. S. Abaee, A. H. Mohammad, M. Mojtahedi and M. R. Halvagar, Tetrahedron
Lett. 2017, 58, 1408.
3) G. Kazuhiro, H. Masao and I. Hiroshi, Biochem. Pharmacol. 1977
(
, 26, 11.
(4) P.L. Podolin, J. F.Callahan, B. J. Bolognese, Y. H. Li, K. Carlson, T. G. Davis, G. W.
Mellor, C. Evans and A. K. Roshak, J. Pharmacol. Exp. Ther. 2005, 312, 373.
(5) H. Y. Meltzer and H. C. Fibiger, Neuropsychopharmacology 1996, 14, 83.
(6) a) J. Franze´n and A. Fisher, Angew. Chem., Int. Ed. 2009, 48, 787. b) Y. Chi, S. T.
Scroggins and J.M.J. Frechet, J. Am. Chem. Soc. 2008, 130, 6322. c) P. Galzerano, F.
Pesciaioli, A. Mazzanti, G. Bartoli and P. Melchiorre, Angew. Chem., Int. Ed. 2009, 48, 7892.
d) T. Arai and N. Yokoyama, Angew. Chem., Int. Ed. 2008, 47, 4989. e) D. Enders, C.Wang,
M. Mukanova and A. Greb, Chem.Commun. 2010, 46, 2447 f) M. Shiri, Chem. Rev. 2012,
112, 3508.
(7) a) Khatik, G. L.; Sharma, G.; Kumar, R.; Chakraborti, A. K. Tetrahedron 2007, 63, 1200.
b) Chen, CꢀT.; Lin, YꢀD.; Liu, CꢀY. Tetrahedron 2009, 65, 10470. c) Lenardão, E. J.;
Ferreira, P. C.; Jacob, R. G.; Perina, G.; Leite, F. P. L. Tetrahedron Lett. 2007, 48, 6763.
(
8) Xiang, SꢀK.; Zhang, B.; Zhang, LꢀH.; Cui, Y.; Jioa, N. Chem.Commun. 2011, 47, 8097.
9) Canela, M.ꢀD.; PerezꢀPe rez, M.ꢀJ.; Noppen, S.; Sa ezꢀCalvo, G.; Díaz, J. F;, Camarasa,
M.ꢀJ.; Liekens, S.; and Priego, E.ꢀM. J. Med. Chem. 2014, 57, 3924−3938.
10) Ding, W.; Zhou, QꢀQ.; Xuan, J.; Li, TꢀR.; Lu, LꢀQ.; Xiao, WꢀJ. Tetrahedron Lett. 2014,
, 4648ꢀ4652.
(
́
́
́
(
5
5

Page 25 of 25
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB00707H