Synthesis and biological evaluation of potential small molecule inhibitors of tumor necrosis factor

Article abstract of DOI:10.1039/c5md00023h

Inhibition of tumor necrosis factor (TNF) production or function by small molecules has become a major focus in the pharmaceutical industry for the treatment of rheumatoid arthritis. In this study, a series of 39 novel SPD-304 analogs were designed, synth

Full text of DOI:10.1039/c5md00023h

View Article Online
View Journal
MedChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: C.
Papaneophytou, P. Alexiou, A. Papakyriakou, E. Ntougkos, K. Tsiliouka, A. Maranti, F. Liepouri, A.
Strongilos, A. Mettou, E. Couladouros, E. Eliopoulos, E. Douni, G. Kollias and G. Kontopidis, Med. Chem.
Commun., 2015, DOI: 10.1039/C5MD00023H.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/medchemcomm

View Article Online
DOI: 10.1039/C5MD00023H
Page 1 of 13
MedChemComm
MedChemComm
RSC
CONCISE ARTICLE
Synthesis and biological evaluation of potential small
molecule inhibitors of Tumor Necrosis Factor
Christos Papaneophytoua,b, Polyxeni Alexiouc, Athanasios Papakyriakoud,
Evangelos Ntougkose, Katerina Tsilioukaf, Anna Marantif, Fotini Liepourif,
Alexandros Strongilosf, Anthi Mettoua,b, Elias Couladourosc, Elias Eliopoulosd,
Eleni Dounid,e, George Kolliase and George Kontopidisa,b*
Inhibition of Tumor Necrosis Factor (TNF) production or function by small molecules has
become a major focus in the pharmaceutical industry for the treatment of Rheumatoid Arthritis.
In this study, a series of 39 novel SPD-304 analogs were designed, synthesized and evaluated
as TNF inhibitors. Our results show that small structural changes produce ligands with similar
binding affinities (Kd) for TNF, but significantly different potencies in a L929 cell-based
assay. In addition, contrary to the high affinity of compounds 4e, 8c and 10e for TNF in vitro,
the potency of these compounds was determined to be low. We propose that these differences
can partly be explained by the physicochemical characteristics of the synthesized SPD-304
analogs. Our findings were supplemented by molecular docking studies on the TNF dimer.
These synthesized analogs may serve as a starting point for developing novel TNF inhibitors
.
Up to the present time, only a small number of TNF inhibitors
have been identified so far, and none of them have reached clinical
trials.7 Most of the small molecule inhibitors reported in the
literature target TNF indirectly, by down-regulating the expression
of TNF.8-12 In 2010, Bandgar et al.13 reported the synthesis and
evaluation of a series of 2-hydroxy-β chlorovinyl-chalcones as
inhibitors of TNF expression. In 2011, Wu et al.14 reported the
inhibition of TNF production from 6 chalcone derivatives, after
screening 54 new synthetic chalcone compounds. In contrast, direct
inhibition of TNF/TNFR interaction, by disrupting the active TNF
trimer, has been an important strategy in the design and screening for
small-molecule based TNF inhibitors.15 However, only a few small
molecules have been reported to directly disrupt TNF/TNFR
interaction such as the polysulfonated naphthylurea, suramin and its
analogs.16 Furthermore the low potency and poor selectivity of
suramin coupled with its tendency to cause adverse side effects
renders it unsuitable for anti-TNF therapies.17 In 2005, He et al.18
reported a novel compound, SPD-304, which inhibited TNF activity
by first promoting subunit disassembly of this trimeric cytokine and
then binding to the resulting dimer. This mechanism was further
confirmed by an X-ray structure of a single compound in complex
with the TNF dimer. SPD-304 contains though the toxic 3-
alkylindole moiety, which was found to be metabolized by
cytochrome P450 enzymes through a dehydrogenation pathway
similar to that of the potent pneumotoxin 3-methylindole, producing
reactive electrophilic iminium species capable of reacting with
protein and DNA targets.19 In 2010, Chan et al.20, 21 identified two
small molecule TNF inhibitors, quinuclidine and quinolizine, from a
natural product and natural-product-like chemical library using a
structure-based virtual screening. Interestingly, quinolizidine was
found to be more potent against TNF compared to SPD-304, while
Introduction
Rheumatoid arthritis (RA) is a chronic disease characterized by
synovial inflammation, as well as degeneration of cartilage and
erosion of juxta-articular bone. RA affects about 0.5–1% of the
general population causing increased morbidity and mortality mainly
due to accelerated atherosclerosis.1, The inhibition of cytokines,
2
particularly Tumor Necrosis Factor (TNF) that plays a pivotal role in
regulating the inflammatory response in RA, has been successful in
the treatment of RA.3
The synthetic therapeutic antibodies etanercept (Enbrel),
infliximab (Remicade), and adalimumab Humira), bind to TNF
directly, inhibiting the interaction between TNF and the tumor
necrosis factor receptor (TNFR).4 These agents have produced
significant advances in RA treatment and validated the extracellular
inhibition of this pro-inflammatory cytokine as an effective therapy.5
The major disadvantages of these drugs are that they cannot be taken
orally; they are expensive and could cause immunogenicity. This has
stimulated the development of alternative small molecule-based
therapies as inhibitors of TNF with improved efficacy, better
tolerability, and oral administration.5 An orally administered, small
molecule that regulates TNF biology could either replace the
injectable ones, or provide better disease control when used alone or
in conjunction with existing therapies.5 In addition, it is expected
that rationally designed small molecule drugs based on scientific
advancements and biotechnological improvements may reach, or
even exceed the efficacy of protein drugs.6 Thus, new therapeutic
alternatives could be presented, with an improved outcome in the
care of rheumatic patients.6
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1-3 | 1

MedChemComm
View ArticlPe Oanglinee 2 of 13
DOI: 10.1039/C5MD00023H
ARTICLE
Journal Name
quinuclidine displayed a comparable activity to SPD-304. Moreover, Structure Activity Relationship Studies
Kumar et al.22 reported the synthesis of a series of iso-indolo [2,1-
The inhibitory effect of the synthesized compounds was initially
examined in terms of their affinity for TNF by utilizing an in vitro
fluorescence ligand-binding assay (Experimental Section).26
Specifically, the change of fluorescence signal of three tyrosine
residues located at the ligand-binding interface (Tyr59, Tyr119,
Tyr151) is monitored upon titration of the ligands.26 Fluorescence
titration of TNF with the parent compound 1 (SPD-304; Fig. 1)
yielded a Kd value of 5.36 ± 0.21 μΜ.26 Titration of TNF with the
SPD-304 analogs gave hyperbolic plots (Supporting Fig. S1), while
Scatchard plot analysis gave linear plots indicating a single binding
site (data not shown).
a]quinazoline derivatives as potent TNF inhibitors. In addition, the
first metal-based direct inhibitor of TNF based on airidium (III)
biquinoline complex has been reported.23 Leung and co-workers24
discovered two direct TNF inhibitors, Darifenacin (Enablex) and
ezetimibe (Zetia), from a database of 3,000 US Food and Drug
Administration (FDA)-approved drugs by applying structure-based
virtual screening methods. Recently, Shen and co-workers7
identified a new direct inhibitor of TNF with higher inhibition
activity than SPD-304 in a cell based-assay, by performing a virtual
screen on the SPECS database for molecules that TNF dimer
structure.
To date, crystallographic structures of TNF with bound inhibitors
have been reported only for SPD-304,18 which is also one of the
most active small molecule inhibitors. Therefore, SPD-304 is the
more appropriate choice for structure-based design of TNF
inhibitors. Recently, we have reported the synthesis of less toxic
SPD-304 derivatives, by eliminating the 6´-methyl group of the 4-
chromone moiety, and by incorporating electron- withdrawing
substituents at the indole moiety.25 In this study, we report the design
and synthesis of 39 new SPD-304 analogs and their in vitro and cell-
based evaluation against TNF provided valuable structure-activity
relationships.
Our biological screening strategy was based on testing the
capacity of the compounds to inhibit TNF functionality by
employing the most widely used assay of TNF bioactivity.27 The
latter utilizes the ability of TNF to induce death in the murine
fibrosarcoma cell line L929 following sensitization by the
transcription inhibitor actinomycin D. If the compounds obstruct
TNF activity at a functional level, they should prevent it from being
cytotoxic in this setting. Initially, all compounds were tested using
this assay at a concentration of 20 μΜ. Compounds showing at least
a 25% inhibition of TNF-induced cell death were further tested to
determine their IC50 values using the same assay in a dose-response
way. It should be mentioned that as the basis of our screening is the
protection from TNF-induced death, any compound that exhibited
pronounced toxicity would not have passed our first line of testing.
Having established that a selection of the compounds can obstruct
TNF functioning, and given that TNF exerts its functions primarily
through interacting with the TNF-R128 receptor, an ELISA-based
test was devised to test their effects on this interaction.
A major problem experienced during this work was that a
significant fraction of the tested compounds exhibited low aqueous
solubility. This issue was overcome during binding affinity
evaluation, by the addition of a co-solvent (either DMSO or
PEG3350) in the reaction mixture, at a final concentration of 5%.26
In addition, we employed a solubilisation protocol for this assay as
previously described by our group.26 It should be noted that TNF can
tolerate both DMSO and PEG3350 up to 10% in the binding assay.
However, those two co-solvents could not be used at this
concentration in our cell-based assay, because they cause
interference. As a result, many tested compounds may not have
reached the desirable concentration for inhibition in this assay.
Fig. 1 summarizes the structures of the first series of compounds
(2a-2e, 3a-3d and 4a-4f) where we investigated the effect of the
diamine cyclization of the lead compound 1. Synthesis of these
analogs was based on our previous docking studies.25 The predicted
conformation of these potential inhibitors, as derived from the
docking studies, revealed that substitution by the more rigid
piperazine ring results to a non-bound ligand conformation very
similar to the bound one. Thus, compounds containing a coupled
diamine bridge (transformation to piperazine) exhibit less variability
with lower entropic cost due to the fixed conformation of the
piperazine ring. Based on this observation, our initial point of
modification was to reduce the binding energy of the parent
compound 1 (SPD-304) by coupling the diamine bridge, leading to
the compound 2a and by elimination of 7´-CH3 of the chromone
moiety, without or with coupling of diamine bridge, in compounds
2b and 2c respectively (Fig. 1).
Results and discussion
Chemistry
Following our previous results25, we have attempted to modify
SPD-304 (Scheme 1) in 4 domains: i) modification of the chromone
moiety (elimination of 6´-CH3 or both methyl groups or substitution
of the chromone moiety with other aromatic groups); ii)
modification of the aromatic ring on the indole moiety; iii)
conversion of the diamine to diamide; iv) coupling of the diamine
bridge.
Scheme 1 Structural modifications of SPD-304
In this work, we describe the synthesis of 39 novel SPD-304
analogs that derived from the above mentioned modifications and
are organized in 5 series (Tables 1-5).
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

View Article Online
DOI: 10.1039/C5MD00023H
Page 3 of 13
Journal Name
MedChemComm
ARTICLE
compare to initial lead 1. Compounds 4a-4f containing the m-
substituent phenylsulfonyl group regained a fraction of the potency.
Binding affinity results (Kd values) of 4a-4f revealed that coupling
of the diamine bridge had a negative impact. In addition, the Kd
values of 4c-4g indicate that the presence of an amino group
promotes the binding affinity to TNF, while a nitro group is not a
favorable substitution. Noticeably, the optimal binding affinity was
shown by compound 4e (Kd= 0.95 μΜ) which features a m-
aminophenylsulfonyl group at the indole moiety (Table 1). We
hypothesize that the markedly reduced activity of 4e in the cell-
based assay could be explained by its low bioavailability resulting
from: i) poor cellular uptake and/or low solubility of this compound
(data not shown) or ii) metabolic degradation of this compound.
Overall, the affinity values of compounds 3b, 3d, 4b, 4d and 4f
suggest that introduction of the piperazine ring had a negative effect
on their potency.
Table 1. Evaluation of the binding affinity and potency of the synthesized
SPD-304 derivatives 2a-e, 3a-d and 4a-f
Inhibition of
TNF binding
affinity[b]
Inhibition of
TNF activity[d]
(IC50, μΜ)[c]
TNF/TNFR1
interaction[e]
ID
clogP[a]
(Kd, μΜ)[c]
(IC50, μΜ)[c]
Fig. 1 Structures of SPD-304 derivatives 2a-e, 3a-d and 4a-f
1[f]
2a
8.0
7.9
7.5
7.4
5.9
5.7
3.9
3.8
3.1
3.1
3.3
3.6
3.1
3.0
2.5
2.4
5.36 ± 0.21
10.12 ± 0.67
18.64 ± 0.92
4.78 ± 0.45
2.51 ± 0.16
5.12 ± 0.61
12.22 ± 0.85
25.36 ± 1.25
35.23 ± 1.92
14.12 ± 0.69
7.00 ± 0.44
12.81 ± 0.78
3.16± 0.21
5.0 ± 1.5
10 ± 1.9
30 ± 2.2
30 ± 3.1
20 ±0.6
Inact
5.0 ± 1.5
15.0 ± 1.4
10 ± 1.5
15 ± 1.2
>60
The evaluation of the first series of compounds is presented in
Table 1 where it can be seen that, although compounds 2a and 2b
showed lower binding affinity compared to SPD-304, compound 2c
showed a Kd value close to that of SPD-304 (Table 1). Based on our
previous results25 we have also transformed the diamine parent
compound to diamide analogs (2d, 2e, Fig. 1), with the aim to
eliminate the toxic effect of the methylene group. Even though
compound 2d25 exhibited approximately two-fold gain in affinity
with respect to the parent compound 1, it was approximately 5 times
less potent. Compound 2e displayed a comparable binding activity to 3c
SPD-304; however it was completely inactive in the cell-based assay
(Table 1), probably due to its low solubility (data shown). It must be
noted that assay conditions in binding determination, due to the
presence of the organic solvent, allows the inhibitors to reach higher
concentrations, thus the maximum inhibition could be observed.
Subsequently, the effect of variations of the substituents in the
2b
2c
2d[f]
2e
n.d
3a
inact
n.d
3b
Inact
n.d
Inact
n.d
3d
10 ± 1.3
>60
20 ± 1.8
>60
4a[f]
4b
4c
4d
4e
4f
15 ± 1.7
20 ± 1.6
20 ± 2.1
25 ± 1.8
20 ± 1.9
40 ± 3.1
40 ± 2.9
40 ± 4.5
20 ± 1.3
40 ± 1.9
16.82 ± 0.51
0.95 ± 0.06
5.45 ± 0.62
indole moiety of the parent compound
1 was investigated.
Compounds 3a-3d and 4a-4f (Fig. 1) were synthesized in order to
explore the effect of different substitutions of m-
trifluoromethylphenyl moiety of the carbonylated derivatives of lead
compound 1, in combination with demethylation of the chromone
moiety. For each substituted compound the effect of the piperazine
ring was also investigated. Initially, the trifluoromethyl group or the
aromatic ring was substituted with either a nitro (3a, 3b) or an amino
group (3c, 3d). In addition, the m-trifluoromethylphenyl moiety was
substituted by the more electron-withdrawing phenylsulfonyl group
in 4a25 and 4b or with m-nitrophenylsulfonyl group in 4c and 4d or
with an m-aminophenylsufonyl group in 4e and 4f (Fig. 1).
Conversion of the diamine parent compound 1 to the diamide 3a-3d
and 4a-4f and substitution of m-trifluoromethylphenyl with more
electron withdrawing groups, led to a significant decrease of
lipophilicity (Table 1). As outlined in Table 1, substitution of the
trifluoromethyl group with either nitro (compounds 3a and 3b) or
amino group (compounds 3c and 3d) resulted in a 2.5 to 7-fold
decrease in binding affinity. Compounds 3a-3c were completely
inactive while compound 3d revealed a two-fold loss in potency
[a] Calculated using ChemDraw [b] Determination of binding affinity to
TNF by fluorescence assay. [c] Mean SE (n=3 independents
experiments); p<0.05. [d] Quantification of inhibition of TNF-induced
death in L929 cells. [e] Quantification by TNF/TNFR1 ELISA. [f] data
from25. “Inact”=inactive; “n.d.”=not determined.
±
For the second series of compounds (5a-5g and 6a-6c; Fig. 2) we
substituted the chromone moiety with other aromatic groups, in
order to investigate the effect of both shape and hydrophobicity on
their binding affinity and potency. In compounds 5a-5g, m-
trifluoromethylphenyl has been replaced by the more electron-
withdrawing phenylsulfonyl group (Fig. 2). The evaluation of the 2nd
series of SPD-304 derivatives is summarized in Table 2. As it can be
seen, replacement of the chromone moiety with naphthalene in the
secondary and tertiary amines 5a and 5b resulted in a decrease of
lipophilicity (cLogP 5.3 and 5.1, respectively). Introduction of the
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 3

MedChemComm
View ArticlPe Oanglinee 4 of 13
DOI: 10.1039/C5MD00023H
ARTICLE
Journal Name
naphthalene group resulted in a 7- and 5- fold decrease in the affinity in 6c resulted in a 3-fold decrease in binding affinity (Kd=15.17 μΜ)
of 5a and 5b with an increase in the Kd values compared to parent with respect to the parent compound. Most notably, compounds 5a-g
compound.
and 6a-6c lost their activity in the cell based-assay, indicating that
the chromone moiety is essential to achieve TNF inhibition (Table
2).
Subsequently, the third series of compounds (7a-7b, 8a-8d and
9a-9b; Fig. 3) was designed in order to investigate whether the
introduction of a nitro- or an amino- group at the 5΄- position of the
indole moiety could improve the ligands’ binding affinity and
potency for TNF.
Fig. 2 Structures of SPD-304 derivatives 5a-g and 6a-c
Substitution of the chromone moiety with a phenyl group in 5c
(cLogP=5.0) had also a negative impact on its binding affinity.
Substitution of the chromone moiety with 1H-imidazole and o-
fluorobenzene in compounds 5d and 5e resulted in a significant
reduction of their binding affinity. The presence of the smaller, but
less hydrophobic, thiophene ring in 5f led to a slight decrease (1.6-
fold) in the binding affinity compared to 1. Introduction of napthyl in
5g resulted in a complete loss of binding affinity for TNF.
Fig. 3 Structures of SPD-304 derivatives 7a-c, 8a-c and 9a-b.
Table 2. Evaluation of the binding affinity and potency of the
synthesized SPD-304 derivatives 5a-g, and 6a-c
In the first three members of this series (7a-7c), m-
trifluoromethylphenyl remained intact, while the H-bond
acceptor nitro group has been introduced in the 5΄- position of
the indole moiety.
Inhibition of
TNF binding
affinity[b]
Inhibition of
TNF activity[d]
(IC50, μΜ)[c]
TNF/TNFR1
interaction [e]
(IC50, μΜ)[c]
ID cLogP[a]
(Kd, μΜ)[c]
Table 3. Evaluation of the binding affinity and potency of the
synthesized SPD-304 derivatives 7a-c, 8a-c and 9a-b
5a
5b
5c
5d
5e
5f
5.3
5.1
5.0
2.8
4.2
3.1
3.9
6.8
4.3
5.6
>35
25.03 ± 1.88
17.27 ± 1.26
Inact
Inact
inact
Inact
Inact
Inact
Inact
inact
Inact
Inact
Inact
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
Inhibition of
TNF binding
affinity[b]
Inhibition of
TNF activity[d]
(IC50, μΜ)[c]
TNF/TNFR1
interaction [e]
(IC50, μΜ)[c]
ID
cLogP[a]
(Kd, μΜ)[c]
Inact
7a
7b
7c
8a
8b
8c
9a
9b
4.8
4.8
5.7
2.1
3.3
3.6
2.5
2.0
13.28 ± 1.15
Inact
Inact
Inact
n.d
n.d
8.22 ± 0.45
Inact
5g
6a
6b
6c
10.33 ± 1.16
15.61 ± 1.46
Inact
Inact
n.d
Inact
40 ± 3.8
Inact
40 ± 3.1
n.d
>35
15.17 ± 1.34
0.74 ± 0.07
17.85 ± 1.28
10.74 ± 1.32
15 ± 1.4
Inact
20 ± 1.9
n.d
[a] Calculated using ChemDraw. [b] Determination of binding affinity
to TNF by fluorescence assay. [c] Mean ± SE (n=3 independent
experiments); p<0.05. [d] Quantification of inhibition of TNF-induced
death in L929 cells. [e] Quantification by TNF/TNFR1 ELISA.
“Inact”=inactive; “n.d.” =not determined.
Inact
n.d
[a] Calculated using ChemDraw. [b] Determination of binding affinity
to TNF by fluorescence assay. [c] Mean ± SE (n=3 independent
experiments); p<0.05. [d] Quantification of inhibition of TNF-induced
death in L929 cells. [e] Quantification by TNF/TNFR1 ELISA.
“Inact”=inactive; “n.d.” =not determined.
To further explore the importance of the chromone moiety on
binding affinity and potency, we kept the m-trifluoromethylphenyl
moiety in the last three members of this series, with concurrent
coupling of the diamide bridge (Fig. 2). Substitution of the
chromone moiety with naphalene in 6a and imidazole in 6b resulted
in complete loss of affinity for TNF. Finally, introduction of benzene
The results of 7a, in which the chromone group has also
remained intact, revealed a decrease in binding affinity,
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

View Article Online
DOI: 10.1039/C5MD00023H
Page 5 of 13
Journal Name
MedChemComm
ARTICLE
compared to
resulted in
1
a
(Table 3). Coupling of the diamine bridge in 7b smaller benzene ring in 10e resulted in a 3-fold increase in
complete loss of binding affinity, while binding affinity compared to 10c that contains the naphthalene
replacement of the chromone moiety with o-fluorobenzene in ring.
7c displayed an increased Kd value (Table 3). Replacement of
the m-trifluoromethylphenyl by m-aminobenzene and
Table 4. Evaluation of the binding affinity and potency of the synthesized
SPD-304 derivatives 10a-e
introduction of an amino group at the 5΄ position of the indole
ring in 8a (the chromone moiety remained intact) and in 8b (the
chromone has been replaced by chroman-4-one) caused a
significant decrease in both binding affinity and potency.
Interestingly, substitution of the trifluoromethylene by the nitro
group in 8c resulted in a significant increase of affinity (Kd
=0.74 μΜ) without any gain in potency, compared to SPD-304.
This suggests that electron-withdrawing groups could enhance
binding affinity, which is not directly linked to enhanced
potency for TNF inhibition. Complete elimination of the m-
trifluoromethylphenyl moiety in compounds 9a and 9b, in
combination with substitution of the chromone moiety with o-
flurobenzene and 2-thiophene, respectively, resulted in a
predicted reduction of their lipophilicity (cLogP =2.5 and 2.0,
respectively), followed by a large decrease of their binding
affinity (Table 3). This supports our previous findings that
larger substituents, such as chromone and naphthalene, are
necessary to achieve significant inhibition.
Inhibition of
TNF/TNFR1
interaction [e]
(IC50, μΜ)[c]
TNF binding
Inhibition of
TNF activity[d]
(IC50, μΜ)[c]
affinity[b]
ID
cLogP[a]
(Kd, μΜ)[c]
10a
10b
10c
10d
10e
5.9
7.4
7.7
7.5
6.6
1.61 ± 0.15
Inact
10 ± 0.9
Inact
20 ± 2.2
n.d
7.82 ± 1.06
Inact
20 ± 1.8
Inact
40 ± 3.2
n.d
2.14 ± 0.16
15 ± 1.1
20 ± 1.7
[a] Calculated using ChemDraw. [b] Determination of binding affinity to
TNF by fluorescence assay. [c] Mean ± SE (n=3 independent experiments);
p<0.05. [d] Quantification of inhibition of TNF-induced death in L929 cells.
[e] Quantification by TNF/TNFR1 ELISA. “Inact”=inactive; n.d. =not
determined.
For the compounds of series 5 (11a-11c, Fig. 4), we have
introduced three changes compared to SPD-304: i) the m-
trifluoromethylphenyl substituent of the indole moiety was
replaced by a phenylsulfonyl group, ii) the size of the diamine
linker was modified, and iii) the chromone moiety was either
maintained in 11a, or was replaced by naphthalene in 11b and
benzene in 11c. No change in binding affinity was observed
when m-trifluoromethylphenyl was replaced by phenylsulfonyl
group in 11a, while this compound was significantly less potent
In the fourth series of compounds (10a-10e; Fig. 4) we
further investigated the importance of substitution in the 5´-
position of the indole moiety. For this, derivatives containing or
lacking a nitro group at this position were synthesized. In this
series of compounds, the diamine bridge was cyclized to
piperazine, and the amine conjugated to the chromone moiety
of the initial lead was transformed to amide.
than the lead compound 1. However, compounds 11b and 11c,
lacking the chromone moiety, were completely inactive. These
examples further support the observation that activity can be
significantly altered by changes in the size of the substituent.
Table 5. Evaluation of the binding affinity and potency of the
synthesized SPD-304 derivatives 11a-c
TNF
Inhibition of
TNF/TNFR1
interaction [d]
(IC50, μΜ)[b]
binding
Inhibition of
TNF activity[d]
(IC50, μΜ)[b]
affinity[b]
(Kd, μΜ)[b]
ID
cLogP[a]
11a
11b
11c
4.6
5.4
4.3
5.2 ± 0.65
18.75 ± 1.87
Inact
20 ± 1.9
Inact
40 ± 2.8
n.d
Fig. 4 Structures of SPD-304 derivatives 10a-e and 11a-c
Compound 10a that lacks the 5´-nitro substituent showed a
significant increase in binding affinity (Kd=1.6 μΜ), but a 2-
fold decrease in potency compared to SPD-304 (Table 4).
However, introduction of the electron-withdrawing nitro group
in the 5´-position of the indole moiety in 10b resulted in
complete loss of potency. The influence of the 5´-substitution is
also reflected on compounds 10c and 10d, in which the
chromone moiety has been replaced by naphthalene.
Specifically, compound 10c lacking substitution in the 5´-
position showed a 1.5-fold decrease in affinity and a 4-fold
decrease in potency, while compound 10d containing the 5´-
nitro group was inactive. The latter supports the observation
that both binding affinity and TNF activity can be altered
dramatically by adjusting the electronic nature of the indole
substituent. Interestingly, substitution of chromone by the
Inact
n.d
[a] Calculated using ChemDraw. [b] Determination of binding affinity to
TNF by fluorescence assay. [c] Mean SE (n=3 independent
experiments); p<0.05. [d] Quantification of inhibition of TNF-induced
death in L929 cells. [e] Quantification by TNF/TNFR1 ELISA.
“Inact”=inactive; “n.d.”=not determined.
±
Molecular Docking
In an effort to gain a better understanding of the structure-
activity relationships of the synthesized compounds we
employed in silico molecular docking calculations using the X-
ray crystal structure of SPD-304 complex with a dimer of TNF
subunits.18 This structure revealed that the inhibitor binds in a
shallow
pocket
within
the
subunit
interfaces,
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 5

MedChemComm
View ArticlPe Oanglinee 6 of 13
DOI: 10.1039/C5MD00023H
Fig. 5 Predicted conformations of the most active compounds in comparison with SPD-304 shown at exactly the same orientation. (A) The
major interacting residues of the SPD-304 binding site that are colored with bright orange or cyan carbons for each TNF monomer,
respectively. All other atoms are colored with blue for nitrogen, red for oxygen and green for fluorine. (C)–(F) Predicted conformations of
the most active compounds 2d (B), 10e (C), 11a (D), 4c (E), and 8c (F).
crystallographic pose of SPD-304 as much as possible. These
conformations are designated as best-matched (Supporting Table S1)
and exhibit a significantly better agreement between the estimated
and the experimental dissociation constants of the most active
analogs (i.e. Kdexp < 5.4 μΜ). With a few exceptions, their interaction
at the ligand-binding pocket of the TNF dimer is governed by
hydrophobic and aromatic contacts, as in the case of SPD-304. A
striking example is the best-matched conformation of 2d (Fig. 5B)
that displayed a two-fold improvement in the binding affinity with
respect to the parent compound, and which could be attributed to the
transformation of the diamine linker to the more rigid diamide
analog. On the other hand, this modification imposes a change in the
putative arrangement of the trifluoromethyphenyl indole and the
dimethyl chromone moieties with respect to SPD-304, which is
predicted to result in loss of interactions with the two Leu57 residues
from each TNF monomer. In the same way, compounds 2e and 2c,
which are even more conformationally restrained by the piperazine
linker, display equipotent affinity with respect to SPD-304.
an interaction that is largely hydrophobic and shape-driven
without any intermolecular hydrogen bonds or salt bridges.18
The compound adopts a compact conformation, with the
trifluoromethyphenyl indole and the dimethyl chromone
moieties in a parallel arrangement, and the diamine linker
exposed towards the bulk solvent (Fig. 5A and Fig. S2A of
Supporting Information).
With the aim to provide structure-activity relationships
between the synthesized analogs, we employed docking
calculations at the ligand binding pocket of the TNF dimer,
initially by performing re-docking of SPD-304. However, its
crystallographic configuration proved to be highly problematic
for molecular docking, exhibiting a large number of distinct
conformational clusters with large deviations from the X-ray
pose.25 These results were considerably improved by using a
more limited search space in combination with SMILES-
generated initial coordinates for the inhibitor (see Experimental
Section).
Examination of the predicted conformations that match the X-ray
pose of SPD-304 reveal that either the dimethyl chromone in the
case of 2e, or the trifluoromethylphenyl ring in the case of 2c might
not be accommodated inside the ligand-binding pocket, which
results in diminished interactions with Tyr151΄ and Leu57/57΄,
respectively (Supporting Figs. S2B and S2C). It is thus possible that
the expected energetic gain from the more rigid diamide and
piperazine linkers is counterbalanced by a loss of hydrophobic
interactions as a result of such conformational restriction. The
significance of shape complementarity is demonstrated by
Docking of the synthesized analogs within the SPD-304 binding
pocket revealed an equally high number of distinct conformational
clusters, except for compounds bearing the more conformationally
restricted piperazine linker. However, the top-ranked conformational
clusters overestimated the binding affinity systematically
(Supporting Table S1), as in the case of SPD-304. Considering that
SPD-304 promotes disassembly of the TNF trimer mainly through a
shape-driven interaction, we have also considered higher energy and
less-populated
conformational
clusters
that
match
the
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1-3 | 6

View Article Online
DOI: 10.1039/C5MD00023H
Page 7 of 13
Journal Name
MedChemComm
ARTICLE
compound 10e (Kd = 2.1 μΜ), which is the only potent inhibitor that been suggested to access the buried interior of the intact
lacks the chromone ring. In its place, the less bulky phenyl ring biologically active TNF trimer to form an intermediate complex
allows the ligand to be completely buried inside the pocket, albeit that undergoes subunit dissociation, it is plausible that a number
displaying fewer hydrophobic interactions (Fig. 5C). In contrast, the of the synthesized analogs are inactive inhibitors due to their
three-fold gain in the binding affinity of 10a (Kd = 1.6 μΜ), that is inability to be properly accommodated at the interior of the
not entirely buried inside the SPD-304 binding pocket, could be TNF trimeric form, which further complicates the structure-
attributed to a potential hydrogen bond between the chromone keto based design process. In contrast, substitutions that result in
group and the side chain of Tyr119 (Supporting Fig. S2D).
bulkier ligands (that could not tolerate any adjustment in the
With the aim to increase the potential hydrogen bonding binding pocket) will have such a profound effect on activity and
interactions of the synthesized analogs, we have introduced a report as inactive. Kd values higher than 40µM would be
sulfonyl group between the indole moiety and its phenyl substituent, reported as inactive since that was the upper limit of the
which comprise polar nitro and amine groups as well. Among the particular assay.
potent inhibitors of TNF trimerization, compound 11a is predicted to
bind inside the binding pocket in a very similar configuration with Conclusions
respect to SPD-304 (Fig. 5D). Such a conformation may account for
In this study we report the design, synthesis and biological
their equal binding affinities (~ 5 μΜ), since the polar amide and evaluation of 39 novel SPD-304 analogs. Their binding affinity for
sulfonyl groups of 11a are not engaged in any hydrogen bonding TNF was determined using a fluorescence binding assay, and all
interactions. In contrast, the bulkier nitrobenzene ring of 4c is compounds were also screened using a L929 cell-based assay at 20
predicted to force the indole moiety out of the binding pocket, so μΜ concentration. Compounds that displayed at least 25% inhibition
that a potential hydrogen bond might be formed between its sulfonyl of TNF-induced cell death were subsequently tested using the same
group and the phenolic group of Tyr151 (Fig. 5E). Conversion of the assay in a dose-response way to determine their IC50 values.
3-nitro group of 4c to the corresponding amine in 4e results in a 3- Compounds 2a, 3b, 4b, 4c, 4d, 4f, 8c, 9a and 9e that exhibited IC50
fold gain in binding affinity, which could be attributed to the better values in the range of 10–20 μΜ were also tested in an ELISA-based
accommodation of the indole ring inside the binding pocket, in assay. Taken together, the data from both assays indicated
addition to the potential hydrogen bonding interaction between one compound 2a as having the most favorable profile with IC50=10 μΜ
of the two amide carbonyl groups of the linker and the side chain of in the L929 assay and IC50=15 mM in the ELISA assay. To assist
Tyr119 (Supporting Fig. S2E). On the other hand, cyclization of the interpretation of our findings, a molecular docking study of this new
linker moiety to piperazine in the closely related analogue 4f has a class of ligands with TNF dimer was performed. Our results suggest
negative effect on the binding affinity, probably due to loss of that there is a correlation between shape complementarity of the
hydrophobic interactions as in the case of 2e (Supporting Fig. S2F). strongest inhibitors within the SPD-304 binding pocked and their
Finally, the lowest dissociation constant was displayed by the TNF activity. Furthermore, substitution of m-trifluoromethylphenyl with
inhibitor 8c (Kd=0.74 μM) that comprises two nitro substituents on the more electron- withdrawing m-amino-phenylsulfonyl (compound
the phenyl indole moiety and a piperazine diamide linker, a 4e) or with m-nitrophenyl (compound 8c) group at the indole moiety
combination that probably renders it too bulky to adopt the compact resulted in a considerable increase of their binding affinity.
conformation of SPD-304 inside the binding pocket. Most Therefore, incorporation of electron-withdrawing moieties can
importantly the experimental Kd value of 0.74 μΜ for compound 8c further improve the inhibitory activity of the SPD-304 derivatives.
is good in agreement with the predicted value of 0.32 μΜ
(Supporting Table S1).As shown by the selected docked Experimental
conformation of 8c (Fig. 5F), its chromone ring could be stacked
over the phenolic ring of Tyr59, while the 5-nitroindole moiety
occupies the position of the dimethylchromone moiety of SPD-304.
In such a configuration two potential hydrogen bonds with both
Tyr151/151’, in addition to the hydrophobic and aromatic
interactions with Tyr119/119’, Tyr59 and Leu57 may account for the
high affinity of 8c for the TNF dimer.
Although experimental data from X-ray crystallography are
required to gain insight into more detailed structure-activity
relationships of the synthesized compounds, our docking
Chemistry
Unless otherwise stated, all reagents and solvents were obtained
from commercial sources and used without further purification. Air
sensitive chemistries were performed in dry and inert conditions
under an atmosphere of argon. All reactions were routinely checked
by TLC on Silica gel Merck 60 F254 and compounds were purified by
column chromatography on silica gel using the appropriate solvent
systems or by preparative HPLC. The purity of the tested
compounds was determined by an analytical HPLC method and was
found to be greater than or equal to 90% for all compounds unless
otherwise stated. The purity analysis was performed on a HPLC
Shimadzu 2010EV, equipped with a SPD-20A UV/Vis detector.
Characterization of compounds was established by a combination of
MS and NMR spectrometry techniques.1H and 13C NMR spectra
were recorded on a Bruker Avance DRX spectrometer (500 MHz).
Chemical shifts were presented in ppm (δ) with internal TMS
standard. MS were obtained by the mass detector of the already
mentioned HPLC Shimadzu 2010EV.
calculations suggest that
a
fine tuning of shape
complementarity with the SPD-304 binding pocket and
incorporation of polar groups at the appropriate positions could
furnish more potent TNF inhibitors. More specifically,
conversion of the diamine linker of SPD-304 to the
corresponding diamide has a considerable contribution in the
binding affinity of the analogs that combine a properly
substituted phenylsulfonyl group on the indole moiety, such as
4e. Similarly, incorporation of the more rigid piperazine linker
may result in loss of the compact conformation as displayed by
the X-ray structure of SPD-304, which could be compensated
by additional polar groups that mediate hydrogen bonding
interactions as in the case of 8c. Considering that SPD-304 has
SPD-304 derivatives bearing the methylene moieties were
prepared by reductive amination (route A, Scheme 2) of in the
presence of a reducing agent. Different reported methods were
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 7

MedChemComm
View ArticlPe Oanglinee 8 of 13
DOI: 10.1039/C5MD00023H
ARTICLE
Journal Name
applied: A1) NaBH(OAc)3/MeOH or/and DCE with or without pH
adjustment with AcOH29, 30, and A2) TMOF, NaBH3CN.31,32 The
reductive amination reactions were complicated due to the likely
formation of symmetrical byproducts from the reaction of aldehydes
with diamines, as well as the extended reaction times and the
difficulty in isolating and purifying the final products.
coupling reaction in the presence of pyridine or thiethylamine in
THF. 34, 35
For the reduction of the nitro-substituent present in certain
synthesized compounds the following methods were used depending
on the substrate: C1) H2, cat. Pd/C, EtOH/EtOAc, room temperature
(rt), C2) SnCl2.2H2O, DMF, rt.36
For the synthesis of the derivatives bearing the carbonyl groups
the following typical amide coupling reactions (reaction B, Scheme
2) were applied: B1) N,N′-Carbonyldiimidazole-Mediated Amide
Coupling in THF33 and B2) Acyl-chloride formation from the
corresponding acid with catalytic amount of DMF in THF (in certain
cases the chloride was commercially available), followed by the
Starting materials (substituted indole-3-aldehydes as well as 3-
formyl-chromones) were synthesized according to reported
procedures when not commercially available (data not included). It
should be noted though that their acids were obtained by an
oxidation reaction utilizing NaClO2 / sulfamic acid in t-BuOH/H2O
and Jone’s reagent in acetone respectively.25
Scheme 2 General synthetic routes applied for the synthesis of key intermediates and target SPD-304 analogs
occasionally. MeOH was removed under reduced pressure and
always under inert conditions, replaced with dichloroethane
(2.5 mL) and treated with sodium triacetoxyborohydride (1.5
mmol). The reaction mixture was stirred until the limiting
reactants were consumed or no further conversion was detected
by TLC or IR. The solvent was concentrated in vacuo and the
residue was purified by flash chromatography (gradient system:
General procedures for the reductive amination reactions (Route
A):
Method A1. Substituted indole-3-carbaldehyde (1 mmol) and
access of the diamine ii (10 mmol) were mixed in MeOH (2.5
mL). The reaction pH was 6-7 and was carefully adjusted to
pH=4-5 by addition of AcOH. The resulting mixture was stirred
at rt for 24-48h and azeotropic removal of water was applied
2-10% MeOH/DCM) to provide the intermediate amines iii
.
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

View Article Online
DOI: 10.1039/C5MD00023H
Page 9 of 13
Journal Name
MedChemComm
ARTICLE
The latter intermediate amines iii (1 mmol) and the appropriate
The previously synthesized acid chloride or commercially
aromatic aldehyde (ex. 3-formyl-chromone) (1 mmol) were available one (1 mmol) was dissolved in THF (10 mL) and pyridine
mixed in MeOH (2.5mL). Similar experimental procedure as (3.55 mmol) as well as the intermediate amides v (1 mmol) were
above was applied and the target amine derivatives 2a and 2c added. The mixture was stirred for 20-24h and removal of the
were obtained.
solvent gave a viscous residue, which was dissolved in ether (20 mL)
and washed with saturated NaHCO3 (2x10 mL) , water (1×10 mL),
1N HCl (2x10 mL), water (1×10 mL) and brine (1× 10mL). The
2a.
6,7-dimethyl-3-((4-((1-(3-(trifluoromethyl)phenyl)-1H-
indol-3-yl)methyl)piperazin-1-yl)methyl)-4H-chromen-4-one.
1
Yield 40%. H NMR (500 MHz, CDCl3 ) δ= 7.95 (s, 1H), 7.80-7.78 organic portions were combined and dried over Na2SO4. Removal of
(m, 2H), 7.73-7.71 (m, 1H), 7.68-7.61 (m, 2H), 7.55 (d, J=8.1Hz, the solvents produced a residue, which was subjected to flash
1H), 7.39-7.36 (m, 2H), 7.31-7.28 (m, 2H), 7.25-7.21 (m, 1H), 3.85 chromatography (gradient system: 0-10% MeOH/DCM) to provide
(s, 2H), 3.56 (s, 2H), 2.7 (br s, 8H), 2.40 (s, 3H), 2.37 (s, 3H); ESI- the target amide derivatives 2e, 3a-b, 4b-d, 5a-g, 6a-c, 7a-c, 8c, 9a-
MS (m/z) calcd. for C32H31F3N3O2+ [M+H]+ 546.24, found 546.01.
b and 11a-c.
2c. 6-methyl-3-((4-((1-(3-(trifluoromethyl)phenyl)-1H-indol-3-
2e. 6,7-dimethyl-3-(4-(1-(3-(trifluoromethyl)phenyl)-1H-indole-
yl)methyl)piperazin-1-yl)methyl)-4H-chromen-4-one (2c). Yield 3-carbonyl)piperazine-1-carbonyl)-4H-chromen-4-one.
Yield
1
24%, Purity 84%. 1H NMR (500 MHz, CDCl3) δ= 8.02 (s, 1H), 77%. H NMR (500 MHz, CDCl3 ) δ=8.16 (s, 1H), 7.94 (s, 1H),
7.82-7.79 (m, 2H), 7.73-7.71 (m, 1H), 7.68-7.61 (m, 2H), 7.55 (d, 7.79 (s, 1H), 7.78 – 7.74 (m, 1H), 7.76 – 7.64 (m, 3H), 7.49 (d, J =
J=7.84Hz, 1H), 7.48 (d, J=8.19Hz, 1H), 7.39-7.37 (m, 3H), 7.30- 5.0 Hz, 1H), 7.31 (d, J = 7.3 Hz, 3H), 7.17 (d, J = 6.8 Hz, 1H), 3.90
7.28 (m, 1H), 7.25-7.23 (m, 1H), 3.86 (s, 2H), 3.57 (s, 2H), 2.6 (br s, (s, 4H), 3.86 (s, 2H), 3.43 (s, 2H), 2.40 (s, 3H), 2.36 (s,3H); 13C
+
8H), 2.47 (s, 3H) ; ESI-MS (m/z) calcd. for C31H29F3N3O2 [M+H]+ NMR (125.5 MHz, CDCl3) δ 173.97, 166.54, 163.73, 157.03,
532.22, found 532.00.
154.75, 145.40, 139.26, 135.77, 130.75, 130.17, 127.94, 126.56,
125.75, 124.34, 124.07, 122.53, 122.05, 121.61, 120.88, 118.46,
Method A2. To the substituted indole-3-carbaldehyde i (1 mmol) 112.99, 110.86, 47.78, 42.84, 20.56, 19.46; ESI-MS (m/z) calcd. for
dissolved in trimethyl orthoformate (20 mL) under argon was added C32H27F3N3O4+ [M+H]+ 573.19, found 573.63.
access of the diamine ii (2 mmol) and the solution was allowed to
stir at room temperature for 5h. Sodium cyanoborohydride (1 mmol)
was added and the solution was stirred for 24-48h. Water (0.5 mL)
and MeOH (0.5 mL) were added and the solvent was then
concentrated in vacuo. The residue was purified by flash
chromatography (gradient system: 0-20% MeOH/DCM, followed by
a mixture of 20% MeOH/DCM+5% NH4OH) to provide the
intermediate amines iii.
3a. N-methyl-N-(2-(N-methyl-4-oxo-4H-chromene-3-
carboxamido)ethyl)-1-(3-nitrophenyl)-1H-indole-3-carboxamide.
Yield 92%. 1H NMR (500 MHz, CDCl3) δ= 8.47 (s, 1H), 8.22 (dd, J
= 26.1, 7.8 Hz, 2H), 8.01 – 7.82 (m, 3H), 7.79 – 7.59 (m, 3H), 7.53
(t, J = 8.1 Hz, 1H), 7.44 (dd, J = 15.6, 8.0 Hz, 2H), 7.32 (d, J = 3.6
Hz, 2H), 4.02 – 3.74 (m, 4H), 3.38 (s, 3H), 3.08 (s, 3H); 13C NMR
(125.5 MHz, CDCl3) δ 173.73, 167.19, 165.26, 156.10, 149.15,
139.98, 135.45, 134.29, 130.96, 130.80, 130.18, 129.09, 126.02,
125.87, 124.26, 124.18, 124.08, 122.55, 122.47, 121.91, 121.80,
121.74, 119.32, 119.20, 118.35, 118.28, 110.27, 37.23, 33.57; ESI-
The latter intermediate amines iii (1 mmol) and the appropriate
aromatic aldehyde (ex. 3-formyl-chromone) (1 mmol) were mixed in
trimethyl orthoformate (20 mL). Similar experimental procedure as
above was applied and the target amine derivative 2b was obtained.
2b. 6-methyl-3-((methyl(2-(methyl((1-(3-trifluoromethyl)
phenyl)-1H-indol-3-yl)methyl)amino)ethyl)amino)methyl)-4H-
+
MS (m/z) calcd. for C29H25N4O6 [M+H]+ 524.17, found 524.36.
3b. 3-(4-(1-(3-nitrophenyl)-1H-indole-3-carbonyl)piperazine-
1
1-carbonyl)-4H-chromen-4-one. Yield 52%. H NMR (500 MHz,
CDCl3) δ= 8.42 (s, 1H), 8.29 (d, J = 8.0 Hz, 1H), 8.23 (s, 1H), 7.88
(d, J = 7.5 Hz, 1H), 7.80-7.68 (m, 4H), 7.52 (t, J = 8.3 Hz, 2H), 7.47
(t, J = 7.3 Hz, 2H), 7.37 – 7.31 (m, 2H), 3.91 (s, 8H); 13C NMR
(125.5 MHz, CDCl3) δ 173.97, 165.94, 163.21, 157.26, 156.18,
149.29, 139.92, 135.61, 134.54, 131.04, 130.23, 129.55, 126.87,
126.29, 126.17, 124.35, 122.75, 122.60, 122.08, 121.13, 119.46,
118.42, 114.07, 110.70, 47.75, 42.80; ESI-MS (m/z) calcd. for
C29H23N4O6+ [M+H]+ 523.16, found 522.75.
1
chromen-4-one. Yield 35 %. H NMR (500 MHz, CDCl3) δ= 8.02
(s, 1H), 7.79-7.65 (m, 7H), 7.53-7.51 (m, 2H), 7.41 (d, J=7.98 Hz,
1H), 7.04-6.98 (m, 2H), 4.61 (br s, 2H), 4.21 (br s, 2H), 3.47 (br s,
4H), 2.98 (s, 3H), 2.39 (s, 3H), 2.28 (s, 3H); ESI-MS (m/z) calcd. for
C31H31F3N3O2+ [M+H]+ 534.24, found 533.75 .
General Procedures for the amide coupling reactions (Route B):
4b. 3-(4-(1-(phenylsulfonyl)-1H-indole-3-carbonyl)piperazine-
1
1-carbonyl)-4H-chromen-4-one. Yield 67%. H NMR (500 MHz,
Method B1. A mixture of substituted indole-3-carboxylic acid
analogs iv (1 mmol) and N,N'-carbonyldiimidazole (CDI) (1.1
CDCl3) δ= 8.21 (s, 2H), 7.98 (d, J = 8.3 Hz, 1H), 7.91 (d, J = 7.8 Hz,
2H), 7.79 (s, 1H), 7.72 (t, J = 7.7 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H),
mmol) were dissolved in dry THF (12 mL) and stirred for 3-5h and 7.56 (t, J = 7.4 Hz, 1H), 7.52 – 7.41 (m, 4H), 7.36 (t, J = 7.7 Hz,
at that time access of the diamine ii (10 mmol) was added at one 1H), 7.29 (t, J = 7.5 Hz, 1H), 3.80 (s, 6H), 3.39 (s, 2H) ; ESI-MS
(m/z) calcd. for C29H24N3O6S+ [M+H]+ 542.14, found 541.65.
portion. The reaction was left under stirring at room temperature for
20-24h, the volatiles were removed under reduced pressure and the
4c. N-methyl-N-(2-(N-methyl-4-oxo-4H-chromene-3-
carboxamido)ethyl)-1-(3-nitrophenylsulfonyl)-1H-indole-3-
carboxamide.Yield 35%. 1H NMR (500 MHz, CDCl3) δ = 8.32 (s,
1H), 7.90 – 7.60 (m, 3H), 7.59 (m, 1H), 7.54 (d, J = 8.3 Hz, 1H),
7.38 – 7.03 (m, 3H), 7.01 – 6.86 (m, 3H), 6.84 (s, 1H), 6.77 (s, 1H),
4.41 (br s, 4H), 2.79 (s, 3H), 2.56 (s, 3H); 13C NMR (125.5 MHz,
CDCl3) δ 173.85, 165.63, 165.40, 156.18, 155.88, 148.35, 139.78,
134.49, 134.40, 132.46, 131.27, 131.13, 129.06, 128.81, 128.64,
126.72, 126.18, 126.14, 126.10, 124.84, 124.36, 122.36, 121.95,
118.48, 118.38, 113.37, 45.34, 38.51, 37.37; ESI-MS (m/z) calcd.
for C29H25N4O8S+ [M+H]+ 589.14, found 588.79.
residue was subjected to purification with flash chromatography
(gradient system: 0-30% MeOH/DCM) to provide the intermediate
amides v.
Method B2. To a solution of chromone-3-carboxylic acid or other
appropriate carboxylic acid analogue (1 mmol) in dry THF (10 mL)
was added an excess of oxalyl chloride (10 mmol), followed by
DMF (0. 1 mL). The resulting mixture was stirred at room
temperature for 1h and at that time the solvent was evaporated in
vacuum and co-evaporated three times. The residue was dried under
high vacuum for 10 min giving the respective acid chloride as a
solid. In certain cases the acid chlorides used were commercially
available.
4d. 3-(4-(1-(3-nitrophenylsulfonyl)-1H-indole-3-carbonyl)
piperazine-1-carbonyl)-4H-chromen-4-one. Yield 94%.1H NMR
(500 MHz, CDCl3) δ = 8.27 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.78 –
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 9

View Article Online
DOI: 10.1039/C5MD00023H
MedChemComm
Page 10 of 13
ARTICLE
Journal Name
7.65 (m, 3H), 7.52 (d, J = 8.1 Hz, 1H), 7.29 (s, 1H), 7.26 – 7.15 (m, 1H NMR (500 MHz, CDCl3) δ = 7.83 (s, 1H), 7.71 (s, 1H), 7.64 (dd,
2H), 7.16 (d, J = 7.8 Hz, 1H), 7.05 – 6.89 (m, 3H), 6.86 (t, J = 7.4 J = 13.2, 7.7 Hz, 4H), 7.43 (d, J = 8.3 Hz, 1H), 7.24 (t, J = 7.4 Hz,
Hz, 1H), 3.33 (s, 4H), 2.97 – 2.82 (m, 4H); 13C NMR (125.5 MHz, 2H), 7.16 (d, J = 19.2 Hz, 2H), 7.04 (s, 1H), 3.83 – 3.76 (m, 4H),
+
CDCl3) δ 174.00, 163.98, 163.23, 162.14, 157.42, 156.18, 148.47, 3.66 – 3.58 (m, 4H); ESI-MS (m/z) calcd. for C24H21F3N5O2
139.72, 134.59, 134.39, 132.35, 131.26, 128.86, 128.55, 126.40, [M+H]+ 468.16, found 467.79
126.30, 126.22, 126.07, 124.99, 124.34, 122.45, 122.37, 121.45,
118.43, 118.25, 113.51, 47.60, 42.64; ESI-MS (m/z) calcd. for 1H-indol-3-yl)methanone. Yield 76%. 1H NMR (500 MHz, CDCl3)
C29H23N4O8S+ [M+H]+ 587.12, found 586.65 .
δ= 7.79 (s, 1H), 7.76-7.67 (m, 5H), 7.62 (d, J = 6.6 Hz, 1H), 7.50 (d,
6c.
(4-benzoylpiperazin-1-yl)(1-(3-(trifluoromethyl)phenyl)-
5a. N-(2-(2-naphthamido)ethyl)-1-(phenylsulfonyl)-1H-indole- J = 6.8 Hz, 1H), 7.45-7.39 (m, 4H), 7.33 – 7.29 (m, 2H), 3.81 (br s,
3-carboxamide. Yield 20%. 1H NMR (500 MHz, CDCl3) δ= 8.38 (s, 8H); ESI-MS (m/z) calcd. for C27H23F3N3O2+ [M+H]+ 478.17, found
1H), 8.11 (s, 1H), 8.08 (d, J = 7.9 Hz, 2H), 7.99 – 7.91 (m, 2H), 7.91 477.79 .
– 7.85 (m, 4H), 7.60 – 7.46 (m, 3H), 7.39 – 7.28 (m, 3H), 6.99 (s,
7aN-methyl-N-(2-(N-methyl-4-oxo-4H-chromene-3-
1H), 3.80 (s, 4H), 3.66 (s, 2H) ; ESI-MS (m/z) calcd. for carboxamido)ethyl)-5-nitro-1-(3-(trifluoromethyl)phenyl)-1H-
1
C28H24N3O4S+ [M+H]+ 498.15, found 497.65.
indole-3-carboxamide.Yield 50%. H NMR (500 MHz, CDCl3) δ =
8.97 (s, 1H), 8.18 (dd, J = 15.4, 8.6 Hz, 2H), 8.01 (s, 1H), 7.85 –
7.63 (m, 5H), 7.53 – 7.39 (m, 4H), 3.97 (s, 2H), 3.92 (s, 2H), 3.40 (s,
3H), 3.10 (s, 3H); ESI-MS (m/z) calcd. for C30H24F3N4O6+ [M+H]+
592.16, found 592.53.
5b. N-methyl-N-(2-(N-methyl-2-naphthamido)ethyl)-1-
(phenylsulfonyl)-1H-indole-3-carboxamide. Yield 49%. 1H NMR
(500 MHz, CDCl3) δ= 7.99 (d, J = 8.2 Hz, 1H), 7.92 (d, J = 8.6 Hz,
3H), 7.88 (d, J = 6.3 Hz, 3H), 7.79 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H),
7.56 (dd, J = 10.4, 6.5 Hz, 3H), 7.48 (dd, J = 14.6, 7.4 Hz, 3H), 7.38
(t, J = 7.7 Hz, 1H), 7.31 (t, J = 7.4 Hz, 1H), 3.74 (br s, 10H); ESI-
MS (m/z) calcd. for C30H28N3O4S+ [M+H]+ 526.18, found 525.75 .
5c. N-methyl-N-(2-(N-methylbenzamido)ethyl)-1-
7b. 3-(4-(5-nitro-1-(3-(trifluoromethyl)phenyl)-1H-indole-3-
carbonyl)piperazine-1-carbonyl)-4H-chromen-4-one. Yield 41%.
1H NMR (500 MHz, CDCl3) δ = 8.77 (s, 1H), 8.29 – 8.16 (m, 3H),
7.81 – 7.69 (m, 4H), 7.56 – 7.41 (m, 5H), 3.93 (s, 4H), 3.89 (s, 2H),
(phenylsulfonyl)-1H-indole-3-carboxamide. Yield 39%. 1H NMR
3.49 (s, 2H) ; ESI-MS (m/z) calcd. for C30H22F3N4O6 [M+H]+
+
(500 MHz, CDCl3) δ= 7.98 (d, J = 8.2 Hz, 1H), 7.93 – 7.69 (m, 4H), 591.15, found 590.79.
7.53 (t, J = 8.7 Hz, 1H), 7.42 – 7.29 (m, 9H), 3.90 (br s, 3H), 3.34 – 7c. N-(2-(2-fluoro-N-methylbenzamido)ethyl)-N-methyl-5-
3.17 (m, 4H), 3.06 (br s, 3H); ESI-MS (m/z) calcd. for nitro-1-(3-(trifluoromethyl)phenyl)-1H-indole-3-carboxamide.
C26H26N3O4S+ [M+H]+ 476.16, found 475.79.
5d. N-methyl-N-(2-(N-methyl-1H-imidazole-1-carboxamido)
Yield 41%. 1H NMR (500 MHz, CDCl3) δ = 8.97 (s, 1H), 8.20 (d, J
= 6.9 Hz, 1H), 7.77-7.68 (m, 4H), 7.56 – 7.41 (m, 2H), 7.41 – 7.28
(m, 1H), 7.14 – 7.01 (m, 2H), 6.97 (d, J = 5.4 Hz, 1H), 3.99 (br s,
ethyl)-1-(phenylsulfonyl)-1H-indole-3-carboxamide. Yield 75%.
1H NMR (500 MHz, CDCl3) δ= 7.99 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 4H), 3.39 (s, 3H), 3.04 (s, 3H); ESI-MS (m/z) calcd. for
7.1 Hz, 2H), 7.66 (t, J = 8.2 Hz, 2H), 7.60 (d, J = 8.1 Hz, 1H), 7.55 – C27H23F4N4O4+ [M+H]+ 543.16, found 542.79 .
7.49 (m, 2H), 7.47 (s, 1H), 7.42 – 7.34 (m, 2H), 7.31 (d, J = 7.7 Hz,
2H), 3.94 (br s, 3H), 3.65 (br s, 2H), 3.24 (s, 3H), 3.20 (s, 2H); ESI-
MS (m/z) calcd. for C23H24N5O4S+ [M+H]+ 466.15, found 465.79.
8c. 3-(4-(5-nitro-1-(3-nitrophenyl)-1H-indole-3-carbonyl)
piperazine-1-carbonyl)-4H-chromen-4-one. Yield 66%. H NMR
(500 MHz, CDCl3) δ= 8.21 (s, 1H), 7.86 – 7.79 (m, 2H), 7.70 (s,
1
5e.
N-(2-(2-fluoro-N-methylbenzamido)ethyl)-N-methyl-1- 1H), 7.66 (d, J = 11.0 Hz, 2H), 7.31 (dd, J = 20.7, 8.0 Hz, 2H), 7.21
1
(phenylsulfonyl)-1H-indole-3-carboxamide. Yield 45%. H NMR – 7.13 (m, 1H), 7.01 – 6.94 (m, 2H), 6.93 – 6.88 (m, 1H), 6.71 (s,
(500 MHz, CDCl3) δ = 7.98 (d, J=8.10Hz, 1H), 7.88 (d, J=6.81Hz, 1H), 3.37 (s, 6H), 2.90 (s, 2H); ESI-MS (m/z) calcd. for
2H), 7.81-7.78 (m, 1H), 7.75-7.72 (m, 1H), 7.55-7.51 (m, 1H), 7.43- C29H22N5O8+ [M+H]+ 568.15, found 567.75.
7.30 (m, 4H), 7.28-7.20 (m, 2H), 7.12-7.02 (m, 2H), 3.88 (br s, 3H),
3.51 (br s, 1H), 3.24 (s, 3H), 2.97 (s, 2H), 2.87 (s, 1H) ; ESI-MS yl)methanone. Yield 25%, Purity 87%. 1H NMR (500 MHz, CDCl3)
(m/z) calcd. for C26H25FN3O4S+ [M+H]+ 494.15, found 493.75.
δ = 8.77 (s, 1H), 8.02 (t, J = 6.4 Hz, 1H), 7.70 (s, 1H), 7.58 (s, 2H),
9a. 4-(2-fluorobenzoyl)piperazin-1-yl)(5-nitro-1H-indol-3-
5f. N-methyl-N-(2-(N-methylthiophene-2-carboxamido)ethyl)- 7.53 – 7.33 (m, 4H), 3.95 – 3.64 (m, 8H) ; ESI-MS (m/z) calcd. for
1-(phenylsulfonyl)-1H-indole-3-carboxamide. Yield 58%. 1H C20H18FN4O4+ [M+H]+ 397.13, found 397.00.
NMR (500 MHz, CDCl3) δ= 7.94 (d, J=7.85Hz, 1H), 7.86-7.83 (m,
9b. (5-nitro-1H-indol-3-yl)(4-(thiophene-2-carbonyl)
2H), 7.69-7.67 (m, 1H), 7.62 (d, J=7.03Hz, 1H), 7.52 (d, J=6.59Hz, piperazin-1-yl)methanone. Yield 43%. 1H NMR (500 MHz,
1H), 7.43-7.40 (m, 3H), 7.35-7.29 (m, 2H), 7.23 (d, J=8.49Hz, 1H), CDCl3) δ =8.91 (s, 1H), 8.73 (s, 1H), 8.19 (d, J = 8.9 Hz, 1H), 7.69
6.98 (br s, 1H), 3.87 (br s, 3H), 3.35 (br s, 4H), 3.17 (br s, 3H); ESI- (s, 1H), 7.49 (s, 2H), 7.34 (s, 1H), 7.07 (d, J = 3.5 Hz, 1H), 3.84 (d, J
MS (m/z) calcd. for C24H24N3O4S2+ [M+H]+ 482.12, found 481.78.
= 8.8 Hz, 8H) ; ESI-MS (m/z) calcd. for C18H17N4O4S+ [M+H]+
5g.
(4-(2-naphthoyl)piperazin-1-yl)(1-(phenylsulfonyl)-1H- 385.10, found 385.00.
indol-3-yl)methanone (5g), Yield 62%. 1H NMR (500 MHz,
11a. N-(3-(4-oxo-4H-chromene-3-carboxamido)propyl)-1-
CDCl3) δ= 7.99 (d, J = 8.1 Hz, 1H), 7.94-7.91 (m, 3H), 7.89-7.85 (phenylsulfonyl)-1H-indole-3-carboxamide. Yield 95%. 1H NMR
(m, 3H), 7.80 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.59-7.52 (m, 3H), (500 MHz, CDCl3) δ =9.10 (s, 1H), 8.56 (s, 1H), 7.82 – 7.74 (m,
7.47 (q, J = 17.4, 9.3 Hz, 3H), 7.37 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 2H), 7.73 – 7.67 (m, 1H), 7.48 – 7.40 (m, 2H), 7.28 (t, J = 8.6 Hz,
7.4 Hz, 1H), 3.73 (br s, 8H); 13C NMR (125.5 MHz, CDCl3) δ 1H), 7.16 – 6.98 (m, 4H), 6.96 (t, J = 7.8 Hz, 2H), 6.85 – 6.80 (m,
170.93, 164.68, 137.92, 134.62, 134.07, 132.89, 132.52, 129.77, 2H), 6.77 (s, 1H), 3.13 (dd, J = 12.2, 6.3 Hz, 2H), 3.04 (dd, J = 11.9,
128.78, 128.65, 128.43, 128.06, 127.58, 127.39, 127.20, 127.13, 6.0 Hz, 2H), 1.41 (dd, J = 11.9, 6.1 Hz, 2H) ; ESI-MS (m/z) calcd.
126.79, 125.91, 124.47, 124.31, 121.15, 116.87, 113.82, 66.04, for C28H24N3O6S+ [M+H]+ 530,14 , found 529.75.
29.90, 15.48; ESI-MS (m/z) calcd. for C30H26N3O4S+ [M+H]+
524.16, found 523.76.
6a. (4-(2-naphthoyl)piperazin-1-yl)(1-(3-
11b. N-(4-(2-naphthamido)butyl)-1-(phenylsulfonyl)-1H-
indole-3-carboxamide. Yield 54%. 1H NMR (500 MHz, CDCl3) δ
=7.92 (s, 1H), 7.81 (s, 1H), 7.64 (d, J = 7.7 Hz, 1H), 7.51 – 7.34 (m,
6H), 7.10 – 6.98 (m, 3H), 6.93 – 6.75 (m, 5H), 6.51 (s, 1H), 6.30 (s,
(trifluoromethyl)phenyl)-1H-indol-3-yl)methanone. Yield 88%
1H NMR (500 MHz, CDCl3) δ= 7.96 – 7.84 (m, 5H), 7.82 – 7.71 (m, 1H), 3.15 – 3.05 (m, 4H), 1.36 – 1.23 (m, 4H); ESI-MS (m/z) calcd.
2H), 7.69 (d, J = 8.0 Hz, 2H), 7.63 (d, J=6.98, 1H), 7.59 – 7.46 (m, for C30H28N3O4S+ [M+H]+ 526.18, found 525.75.
5H), 7.33 – 7.29 (m, 1H), 3.83 (br s, 8H); ESI-MS (m/z) calcd. for
C31H25F3N3O2+ [M+H]+ 528.19, found 527.75.
6b. (4-(1H-imidazole-1-carbonyl)piperazin-1-yl)(1-(3-
(trifluoromethyl)phenyl)-1H-indol-3-yl)methanone. Yield 87%.
11c. N-(3-benzamidopropyl)-1-(phenylsulfonyl)-1H-indole-3-
carboxamide. Yield 85%. 1H NMR (500 MHz, CDCl3) δ= 8.20 (s,
1H), 8.13 (d, J = 7.4 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.90 (dd, J =
21.8, 7.4 Hz, 4H), 7.55 (t, J = 7.5 Hz, 1H), 7.50 (t, J=7.6 Hz, 1H),
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

View Article Online
DOI: 10.1039/C5MD00023H
Page 11 of 13
Journal Name
MedChemComm
ARTICLE
7.47-7.40 (m, 3H), 7.39 – 7.31 (m, 2H), 7.25 – 7.17 (m, 1H), 3.62 –
3.51 (m, 4H), 1.88 – 1.80 (m, 2H), 1.70 (s, 1H), 1.25 (s, 1H) ; ESI-
MS (m/z) calcd. for C25H24N3O4S+ [M+H]+ 462.15, found 461.75.
3.44 (s, 2H); 13C NMR (125.5 MHz, CDCl3) δ 174.00, 163.98,
163.23, 162.14, 157.42, 156.18, 148.47, 139.72, 134.59, 134.39,
132.35, 131.26, 128.86, 128.55, 126.40, 126.30, 126.22, 126.07,
124.99, 124.34, 122.45, 122.37, 121.45, 118.43, 118.25, 113.51,
47.60, 42.64; ESI-MS (m/z) calcd. for C29H25N4O4+ [M+H]+ 493.19,
found 492.75.
General procedures for the synthesis of the mixed analogs 10a-e:
Compounds 10a-e bearing both the amine and the amide moieties
were synthesized according to method A1 for the formation of the
intermediate amines iii and method B2 for the amide coupling.
Method C2. The nitro-compound 3a, 4c or 4d respectively (0.03
mmol) and Tin(II) chloride dehydrate SnCl2 2H2O 1M in DMF (1ml)
.
were mixed and stirred at room temperature for 24h. At that time
.
Tin(II) chloride dehydrate SnCl2 2H2O 1M in DMF (1 mL) was
10a. 3-(4-((1-(3-(trifluoromethyl)phenyl)-1H-indol-3-yl)
methyl)piperazine-1-carbonyl)-4H-chromen-4-one. Yield 79%
1H NMR (500 MHz, CDCl3) δ =8.22 (d, J = 9.1 Hz, 1H), 8.16 (s,
1H), 7.82 (d, J = 7.8 Hz, 1H), 7.76 (s, 1H), 7.72 – 7.56 (m, 4H), 7.54
– 7.41 (m, 3H), 7.33 – 7.17 (m, 3H), 3.83 (br s, 4H), 3.41 (br s, 2H),
additionally added and the reaction was stirred for another 24h at
room temperature. DCM (20 mL) was added and the organic phase
was treated with 1N NaOH (1x10 mL), H2O (2x 10 mL) and brine
(1x 10 mL). The organic phase was then dried over Na2SO4,
filtrated, and the filtrate was concentrated under reduced pressure.
The crude residue was purified by flash chromatography (gradient
system: 1-10% MeOH/DCM) to provide the amine compounds 3c,
4e and 4f.
+
2.65 (d, J = 24.4 Hz, 5H); ESI-MS (m/z) calcd. for C30H25F3N3O3
[M+H]+ 532.18, found 531.75.
10b. 3-(4-((5-nitro-1-(3-(trifluoromethyl)phenyl)-1H-indol-3-
yl)methyl)piperazine-1-carbonyl)-4H-chromen-4-one. Yield 47%.
1H NMR (500 MHz, CDCl3) δ = 8.80 (s, 1H), 8.21 (d, J = 7.9 Hz,
1H), 8.16-8.14 (m, 2H), 7.74-7.70 (m, 5H), 7.49 (d, J = 8.7 Hz, 2H),
7.44 (t, J = 7.5 Hz, 2H), 3.84 (br s, 4H), 3.42 (br s, 2H), 2.65 (br s,
4H); ESI-MS (m/z) calcd. for C30H24F3N4O5+ [M+H]+ 577.17, found
576.78.
10c. naphthalen-2-yl(4-((1-(3-(trifluoromethyl)phenyl)-1H-
indol-3-yl)methyl)piperazin-1-yl)methanone. Yield 43%. 1H NMR
(500 MHz, CDCl3) δ = 7.91 (s, 1H), 7.88 – 7.80 (m, 4H), 7.76 (s,
1H), 7.70 (d, J = 7.8 Hz, 1H), 7.62 (dd, J = 16.5, 7.7 Hz, 2H), 7.51
(dd, J = 14.6, 8.1 Hz, 4H), 7.32 – 7.19 (m, 3H), 3.88 (br s, 2H), 3.82
(s, 2H), 3.52 (br s, 2H), 2.60 (d, J = 82.7 Hz, 4H); ESI-MS (m/z)
calcd. for C31H27F3N3O+ [M+H]+ 514.21, found 514.00.
10d. naphthalen-2-yl(4-((5-nitro-1-(3- (trifluoromethyl)
3c. 1-(3-aminophenyl)-N-methyl-N-(2-(N-methyl-4-oxo-4H-
chromene-3-carboxamido)ethyl)-1H-indole-3-carboxamide.
1
Yield 74%. H NMR (500 MHz, CDCl3) δ= 8.16 (d, J = 7.1 Hz,
1H), 8.01 – 7.83 (m, 2H), 7.80 – 7.56 (m, 2H), 7.56 – 7.33 (m, 5H),
7.20 (s, 2H), 7.06 (s, 1H), 6.99 (s, 1H), 4.02 – 3.70 (m, 4H), 3.53 (br
s, 2H), 3.34 (s, 3H), 3.07 (s, 3H) ; ESI-MS (m/z) calcd. for
C29H27N4O4+ [M+H]+ 495.20, found 494.95.
4e. 1-(3-aminophenylsulfonyl)-N-methyl-N-(2-(N-methyl-4-
oxo-4H-chromene-3-carboxamido)ethyl)-1H-indole-3-
carboxamide. Yield 65%. 1H NMR (500 MHz, CDCl3) δ= δ 8.30 –
8.14 (m, 1H), 7.94 (s, 2H), 7.76 – 7.64 (m, 2H), 7.58 – 7.49 (m, 1H),
7.43 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 7.6 Hz, 3H), 7.13 (s, 1H), 6.97
(d, J = 8.3 Hz, 1H), 6.68 (s, 1H), 4.31 (br s, 2H), 3.92 (br s, 4H),
3.30 (s, 3H), 3.05 (s, 3H); ESI-MS (m/z) calcd. for C29H27N4O6S+
phenyl)-1H-indol-3-yl)methyl)piperazin-1-yl)methanone. Yield
1
=63%. H NMR (500 MHz, CDCl3) δ = 8.81 (s, 1H), 8.16 (d, J = [M+H]+ 559.16, found 558.65.
4f. 3-(4-(1-(3-aminophenylsulfonyl)-1H-indole-3-carbonyl)
piperazine-1-carbonyl)-4H-chromen-4-one. Yield 76%. H NMR
10.5 Hz, 1H), 7.95 – 7.78 (m, 4H), 7.73-7.70 (m, 4H), 7.56 – 7.38
(m, 5H), 4.00 – 3.39 (m, 6H), 2.64 (d, J = 116.7 Hz, 4H); ESI-MS
(m/z) calcd. for C31H26F3N4O3+ [M+H]+ 559.20, found 559.02.
10e. phenyl(4-((1-(3-(trifluoromethyl)phenyl)-1H-indol-3-
1
(500 MHz, CDCl3) δ = 7.71 (s, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.23 –
7.17 (m, 1H), 7.12 (d, J = 4.8 Hz, 1H), 7.03 – 6.92 (m, 3H), 6.84 (t, J
= 7.7 Hz, 1H), 6.79 (d, J = 7.4 Hz, 1H), 6.74 (s, 2H), 6.68 (t, J = 7.9
Hz, 1H), 6.60 (s, 1H), 6.27 (d, J = 7.9 Hz, 1H), 3.42 (s, 2H), 3.31 (s,
6H), 2.88 (s, 2H) ; ESI-MS (m/z) calcd. for C29H25N4O6S+ [M+H]+
557.15, found 556.85.
1
yl)methyl)piperazin-1-yl)methanone. Yield =86%. H NMR (500
MHz, CDCl3) δ = 7.81 (d, J = 7.7 Hz, 1H), 7.75 (s, 1H), 7.71-
7.69(m, 1H), 7.67 – 7.57 (m, 2H), 7.53 (d, J = 8.2 Hz, 1H), 7.40-7.37
(m, 5H), 7.31 – 7.17 (m, 3H), 3.80 (br s, 4H), 3.44 (s, 2H), 2.56 (d, J
= 79.9 Hz, 4H); ESI-MS (m/z) calcd. for C27H25F3N3O+ [M+H]+
464.19, found 464.05 .
Method C3. To solution of the nitro-compound 8c (1 mmol) in
absolute ethanol (3 mL) and EtOAc (3 mL) was added PtO2 (15 mg).
The reaction mixture was degassed with vacuum and set under
argon. Then argon was removed with vacuum and the reaction
mixture was hydrogenated at atmospheric pressure at room
temperature until the starting material disappeared according to LC-
MS analysis. The reaction mixture was shaken with air and filtered
through Celite, and evaporated under reduced pressure. The crude
residue was purified by flash chromatography (gradient system: 1-
10% MeOH/DCM) to provide the target amine compound 8a and 8b
as a side product.
General reduction procedures for the synthesis of 3c-d, 4e-f and
8a-b:
Method C1. To solution of the nitro-compound 3b (0.04 mmol) in
absolute ethanol (0.5 mL) and EtOAc (0.5 mL) was slowly added
10% palladium on charcoal (20 mg). The reaction mixture was
degassed with vacuum and set under argon. Then argon was
removed with vacuum and the reaction mixture was hydrogenated at
atmospheric pressure at room temperature until the starting material
disappeared according to LC-MS analysis. The reaction mixture was
shaken with air and filtered through Celite, and evaporated under
reduced pressure. The residue was purified by flash chromatography
(gradient system: 1-3% MeOH/DCM) to provide the amine
compound 3d.
8a. 3-(4-(5-amino-1-(3-aminophenyl)-1H-indole-3-carbonyl)
1
piperazine-1-carbonyl)-4H-chromen-4-one. Yield 39%. H NMR
(500 MHz, CDCl3) δ = 8.23 (d, J = 10.2 Hz, 2H), 7.73 (t, J = 10.0
Hz, 1H), 7.57 – 7.49 (m, 2H), 7.49 – 7.43 (m, 1H), 7.36 (d, J = 8.7
Hz, 1H), 7.04 (s, 1H), 6.97 (d, J = 9.0 Hz, 1H), 6.84 (d, J = 9.4 Hz,
1H), 6.75 (s, 1H), 6.72 – 6.63 (m, 2H), 3.89 (s, 4H), 3.85 (s, 2H),
3d. 3-(4-(1-(3-aminophenyl)-1H-indole-3-carbonyl)piperazine-
1-carbonyl)-4H-chromen-4-one. Yield 96%. 1H NMR (500 MHz,
CDCl3) δ= 8.22 (s, 1H), 7.81 – 7.68 (m, 2H), 7.65 (s, 1H), 7.59 –
7.38 (m, 3H), 7.34 – 7.15 (m, 4H), 6.87 (d, J = 7.6 Hz, 1H), 6.79 (s,
1H), 6.72 (d, J = 7.9 Hz, 1H), 5.33 (s, 2H), 3.90 (s, 4H), 3.85 (s, 2H),
+
3.64 (s, 2H), 3.42 (s, 2H); ESI-MS (m/z) calcd. for C29H26N5O4
[M+H]+ 508.20, found 507.89.
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 11

View Article Online
DOI: 10.1039/C5MD00023H
MedChemComm
Page 12 of 13
ARTICLE
Journal Name
8b. 3-(4-(5-amino-1-(3-aminophenyl)-1H-indole-3-carbonyl)
dilution of an anti-rabbit secondary antibody conjugated with HRP
(Vector Laboratories, CA) for 1 h at room temperature. After a final
round of washes, the signal was developed using the TMB Substrate
piperazine-1-carbonyl)chroman-4-one. Yield 31%. 1H NMR (500
MHz, CDCl3) δ= 7.89 (d, J = 8.0 Hz, 1H), 7.54-7.51(m, 2H), 7.36
(d, J = 8.6 Hz, 1H), 7.09-7.01 (m, 4H), 6.84 (d, J = 7.0 Hz, 2H), 6.76 Kit (Thermo Scientific, IL) and measured spectrophotometrically at
(s, 2H), 6.69-6.65 (m, 3H), 4.21 – 3.99 (m, 6H), 3.86 (s, 2H), 3.65
450 nm. All experiments were performed in triplicate.
+
(br s, 2H), 3.41 (br s, 2H); ESI-MS (m/z) calcd. for C29H28N5O4
[M+H]+ 510.21, found 509.85.
Molecular Docking
The crystallographic structure of TNF dimer was retrieved from the
Protein Data Bank (accession code 2AZ5). The protein atoms
comprising chains A and B were extracted from the PDB file and
were used without any further modification for the docking
calculations. The initial conformations of the designed scaffolds
were generated from SMILES representations using the program
Omega v2.3 (OpenEye Scientific Software, Santa Fe, NM.
http://www.eyesopen.com) with default parameters.39 For protein
and ligands, only polar hydrogen atoms were added and Gasteiger
charges were applied using AutoDockTools v1.5.6. The search space
was defined by a grid box centered on the ligand and comprised of
55 × 55 × 41 grid points of 0.375 Å spacing. For each complex, 100
docking rounds were calculated with AutoDock v4.2.3 using the
Lamarckian genetic algorithm with the default parameters from
AutoDock 3.40, 41 The maximum number of energy evaluations was
set to 10 million and the results were clustered using a tolerance of
2.0 Å. Visual inspection of the resulting complexes and rendering of
figures were carried out using VMD v1.9.1.42
Binding affinity and toxicity assay
Expression and purification of TNF
The extracellular domain of TNF was expressed in BL21(DE3)
pLysS strain of E. coli as a GST-fusion protein as previously
described.37 GST-TNF was purified as previously described 37 while
separation of TNF from its GST fusion partner was accomplished by
proteolytic cleavage with the type-14 human rhinovirus 3C protease
(America Pharmacia Biotech). The concentration of protein in the
samples was determined by the Bradford method38 using bovine
albumin as standard.
Fluorescence binding assay and determination of dissociation
constant (Kd)
Fluorescence intensity was measured with a Hitachi F-2500
fluorescence spectrophotometer in 1.0 x 4.5 cm quartz cuvettes at 25
oC as previously described.26 A detailed analysis of the development
of the fluorescence ligand-binding assay is given in ref.26
Differences in fluorescence intensity at 302 nm between the complex
(TNF/ligand) and free protein (excitation at 274 nm) were analyzed
as previously described26 in order to determine the dissociation
constant (Kd) of ΤΝF with various ligands. Experiments were
performed in 10 mM citrate- phosphate (pH 6.5) containing either
5% DMSO or 5% PEG3350. The slits were set at 5 and 20 nm in the
excitation and emission respectively. In order to determine dilution
effect of TNF (due to ligand addition) and any fluorescence effect by
unbound ligand, a blank sample containing Tyr with the same
fluorescence signal, was titrated with sequential ligand additions.26
The sample absorbance was kept below 0.1 to minimize the inner
filter effect. Data were analyzed using Prism V.5
(GraphPadSoftware, San Diego, CA).
Acknowledgements
This work was funded by the project TheRAlead (09SYN-21-784)
co-financed by the European Union (European Regional
Development Fund – ERDF) and Greek national funds through the
Operational Program “Competitiveness & Entrepreneurship”, NSRF
2007–2013 in the context of GSRT-National action “Cooperation”.
Notes and references
a
Department of Biochemistry Veterinary School, University of
Thessaly, Trikalon 224, Karditsa 43100, Greece
Institute for Research and Technology of Thessaly
b.
(I.RE.TE.TH.), The Centre for Research & Technology Hellas
(CE.R.TH.), Dimitriados 95 & Paulou Mela, Volos 38333,
Greece
Laboratory of General Chemistry, Department of Science,
Agricultural University of Athens, Iera Odos 75, Athens
11855, Greece
Laboratory of Genetics, Department of Biotechnology,
Agricultural University of Athens, Iera Odos 75, Athens
11855, Greece
Biomedical Sciences Research Center “Alexander Fleming”, Vari,
Greece
TNF-induced death assay in L929 cells
L929 cells were seeded onto a 96-well plate (3×104 cells/well). On
the following day, cells were treated with 0.25 ng/mL human TNF
(PeproTech, NJ) and 2 mg/mL actinomycin D (Sigma–Aldrich,
MO). To test inhibition by synthesized compounds, TNF was pre-
incubated for 30 min at room temperature with the compounds prior
to addition to cells. An actinomycin D treated-only control was
included to measure background death. After 18–24 h, dead cells
were removed by washing with PBS. The remaining live cells were
fixed with methanol, stained with crystal violet and quantified
spectrophotometrically at 570nm after solubilization of the stain
using acetic acid. Cytotoxicity is expressed relative to the
background death control as well as to the compounds’ toxicity. All
experiments were performed in triplicate.
c
d.
e.
.f
pro-ACTINA S.A., Archimidous Str. 59, P.O.Box 205, Koropi,
Athens 19400, Greece.
TNF/TNF-R1 ELISA assay
*Corresponding author: G.A Kontopidis,
Ninety-six-well plates were coated with 0.1mg/mL recombinant
soluble human TNR-R1 (PeproTech) in PBS overnight at 4°C.
Following four washes with PBS containing 0.05% Tween-
20,blocking was carried out using 1% BSA in PBS. 0.025mg/mL
recombinant human TNF (PeproTech) in PBS was added and the
plates were incubated for 1 h at room temperature. After another
round of washes, plates were incubated with a 1:5000 dilution of a
rabbit anti-human-TNF antibody (provided by Prof. W. A. Buurman,
University of Maastricht) for 1 h at room temperature. Following
another round of washes, plates were incubated with a 1:5000
Laboratory of Biochemistry, Veterinary School, University of Thessaly,
Trikalon 224, Karditsa 43100, Greece
Tel: +30 24410 66081; Fax: +30 24410 66041
e-mail: gkontopidis@vet.uth.gr
Electronic Supplementary Information (ESI) available: See
DOI: 10.1039/b000000x/
12 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

View Article Online
DOI: 10.1039/C5MD00023H
Page 13 of 13
Journal Name
MedChemComm
ARTICLE
30.
31.
J. W. Coe, M. G. Vetelino and M. J. Bradlee, Tetrahedron Lett.,
1996, 37, 6045-6048.
G. C. Look, M. M. Murphy, D. A. Campbell and M. A. Gallop,
Tetrahedron Lett., 1995, 36, 2937–2940.
J. Matthews and R. A. Rivero, J. Org. Chem., 1997, 62, 6090-6092.
E. K. Woodman, J. G. K. Chaffey, P. A. Hopes, D. R. J. Hose and J.
P. Gilday, Org. Process Res. Dev., 2008, 13, 106-113.
C. A. G. N. Montalbetti and V. Falque, Tetrahedron, 2005, 61,
10827-10852.
J. J. Mason, J. Bergman and T. Janosik, J. Nat. Prod., 2008, 71,
1447-1450.
G. P. Wei and G. B. Phillips, Tetrahedron Lett., 1998, 39, 179-182.
C. P. Papaneophytou and G. A. Kontopidis, Protein Expr. Purif.,
2012, 86, 35-44.
M. M. Bradford, Anal. Biochem., 1976, 72, 248-254.
P. C. D. Hawkins, A. G. Skillman, G. L. Warren, B. A. Ellingson and
M. T. Stahl, J .Chem. Inf. Model., 2010, 50, 572-584.
G. M. Morris, H. Ruth, W. Lindstrom, M. F. Sanner, R. K. Belew, D.
S. Goodsell and A. J. Olson, J. Comput. Chem., 2009, 30, 2785-
2791.
G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart,
R. K. Belew and A. J. Olson, J. Comput. Chem., 1998, 19, 1639-
1662.
1.
2.
3.
I. B. McInnes and G. Schett, New Engl. J. Med., 2011, 365, 2205-
2219.
H. Maradit-Kremers, P. J. Nicola, C. S. Crowson, K. V. Ballman and
S. E. Gabriel, Arthritis Rheum., 2005, 52, 722-732.
R. Atreya, J. Mudter, S. Finotto, J. Mullberg, T. Jostock, S. Wirtz,
M. Schutz, B. Bartsch, M. Holtmann, C. Becker, D. Strand, J. Czaja,
J. F. Schlaak, H. A. Lehr, F. Autschbach, G. Schurmann, N.
Nishimoto, K. Yoshizaki, H. Ito, T. Kishimoto, P. R. Galle, S. Rose-
John and M. F. Neurath, Nat. Med., 2000, 6, 583-588.
K. Chatzantoni and A. Mouzaki, Curr. Top. Med. Chem., 2006, 6,
1707-1714.
M. A. Palladino, F. R. Bahjat, E. A. Theodorakis and L. L.
Moldawer, Nat. Rev. Drug Discov., 2003, 2, 736-746.
J. Stanczyk, C. Ospelt and S. Gay, Curr. Opin. Rheumatol., 2008, 20,
257-262.
Q. Shen, J. Chen, Q. Wang, X. Deng, Y. Liu and L. Lai, Eur. J. Med.
Chem., 2014, 85, 119-126.
M. R. Lee and C. Dominguez, Curr. Med .Chem., 2005, 12, 2979-
2994.
H. S. Rasmussen and P. P. McCann, Pharmacol. Ther., 1997, 75, 69-
75.
S. Haraguchi, N. K. Day, W. Kamchaisatian, M. Beigier-Pompadre,
S. Stenger, N. Tangsinmankong, J. W. Sleasman, S. V. Pizzo and G.
J. Cianciolo, AIDS Res. Ther., 2006, 3, 8.
32.
33.
34.
35.
4.
5.
6.
7.
8.
9.
10.
36.
37.
38.
39.
40.
41.
42.
W. Humphrey, A. Dalke and K. Schulten, J. Mol. Graph. Model.,
1996, 14, 33-38.
11.
J. R. Burke, M. A. Pattoli, K. R. Gregor, P. J. Brassil, J. F.
MacMaster, K. W. McIntyre, X. Yang, V. S. Iotzova, W. Clarke, J.
Strnad, Y. Qiu and F. C. Zusi, J. Biol. Chem., 2003, 278, 1450-1456.
C. H. Leung, S. P. Grill, W. Lam, W. Gao, H. D. Sun and Y. C.
Cheng, Mol. Pharmacol., 2006, 70, 1946-1955.
B. P. Bandgar, S. A. Patil, B. L. Korbad, S. H. Nile and C. N.
Khobragade, Eur. J. Med. Chem., 2010, 45, 2629-2633.
J. Wu, J. Li, Y. Cai, Y. Pan, F. Ye, Y. Zhang, Y. Zhao, S. Yang, X.
Li and G. Liang, J. Med. Chem., 2011, 54, 8110-8123.
J. M. Davis and J. Colangelo, Future Med. Chem., 2013, 5, 69-79.
F. Mancini, C. M. Toro, M. Mabilia, M. Giannangeli, M. Pinza and
C. Milanese, Biochem. Pharmacol., 1999, 58, 851-859.
R. P. McGeary, A. J. Bennett, Q. B. Tran, K. L. Cosgrove and B. P.
Ross, Mini Rev. Med. Chem., 2008, 8, 1384-1394.
12.
13.
14.
15.
16.
17.
18.
M. M. He, A. S. Smith, J. D. Oslob, W. M. Flanagan, A. C. Braisted,
A. Whitty, M. T. Cancilla, J. Wang, A. A. Lugovskoy, J. C. Yoburn,
A. D. Fung, G. Farrington, J. K. Eldredge, E. S. Day, L. A. Cruz, T.
G. Cachero, S. K. Miller, J. E. Friedman, I. C. Choong and B. C.
Cunningham, Science, 2005, 310, 1022-1025.
19.
20.
H. Sun and G. S. Yost, Chem. Res. Toxicol., 2008, 21, 374-385.
D. S. Chan, H. M. Lee, F. Yang, C. M. Che, C. C. Wong, R.
Abagyan, C. H. Leung and D. L. Ma, Angew. Chem. Int. Ed. Engl.,
2010, 49, 2860-2864.
21.
22.
D.-L. Ma, D. S.-H. Chana and C. H. Leung, Chem. Sci., 2011, 2,
1656-1665.
K. S. Kumar, P. M. Kumar, K. A. Kumar, M. Sreenivasulu, A. A.
Jafar, D. Rambabu, G. R. Krishna, C. M. Reddy, R. Kapavarapu, K.
Shivakumar, K. K. Priya, K. V. Parsa and M. Pal, Chem. Commun.
(Camb), 2011, 47, 5010-5012.
23.
24.
25.
C. H. Leung, H. J. Zhong, H. Yang, Z. Cheng, D. S. Chan, V. P. Ma,
R. Abagyan, C. Y. Wong and D. L. Ma, Angew. Chem. Int. Ed.
Engl., 2012, 51, 9010-9014.
C. H. Leung, D. S. H. Chan, M. H. T. Kwan, Z. Cheng, C. Y. Wong,
G. Y. Zhu, W. F. Fong and D. L. Ma, Chemmedchem, 2011, 6, 765-
768.
P. Alexiou, A. Papakyriakou, E. Ntougkos, C. P. Papaneophytou, F.
Liepouri, A. Mettou, I. Katsoulis, A. Maranti, K. Tsiliouka, A.
Strongilos, S. Chaitidou, E. Douni, G. Kontopidis, G. Kollias, E.
Couladouros and E. Eliopoulos, Archiv der Pharmazie, 2014, 347, 1-
8.
26.
27.
C. P. Papaneophytou, A. K. Mettou, V. Rinotas, E. Douni and G. A.
Kontopidis, Med. Chem. Lett., 2013, 4, 137-141.
B. B. Aggarwal and W. J. Kohr, Methods enzymol, 1985, 116, 448-
456.
28.
29.
G. Chen and D. V. Goeddel, Science, 2002, 296, 1634-1635.
A. F. Abdel-Magid, K. G. Carson, B. D. Harris, C. A. Maryanoff and
R. D. Shah, J. Org. Chem., 1996, 61, 3849-3862.
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 13