Synthesis of N-aryl-3-(indol-3-yl)propanamides and their immunosuppressive activities

Article abstract of DOI:10.1016/j.bmcl.2010.07.001

N-Aryl-3-(indol-3-yl)propanamides were synthesized and their immunosuppressive activities were evaluated. This study highlighted the promising potency of 3-[1-(4-chlorobenzyl)-1H-indol-3-yl]-N-(4-nitrophenyl) propanamide 15 which exhibited a significant i

Full text of DOI:10.1016/j.bmcl.2010.07.001

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5203–5206
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of N-aryl-3-(indol-3-yl)propanamides and their
immunosuppressive activities
b
a
b
Francis Giraud a, Pascal Marchand a, , Delphine Carbonnelle , Michael Sartor , François Lang ,
*
Muriel Duflos a
a Université de Nantes, Nantes Atlantique Universités, Laboratoire de Chimie Thérapeutique, Cibles et Médicaments des Infections, de l’Immunité et du Cancer, IICiMed UPRES EA 1155,
Faculté de Pharmacie, 1 rue Gaston Veil, 44035 Nantes, France
b Université de Nantes, Nantes Atlantique Universités, Département de Pharmacologie, Cibles et Médicaments des Infections, de l’Immunité et du Cancer, IICiMed UPRES EA 1155,
Faculté de Pharmacie, 1 rue Gaston Veil, 44035 Nantes, France
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Aryl-3-(indol-3-yl)propanamides were synthesized and their immunosuppressive activities were
evaluated. This study highlighted the promising potency of 3-[1-(4-chlorobenzyl)-1H-indol-3-yl]-N-(4-
nitrophenyl)propanamide 15 which exhibited a significant inhibitory activity on murine splenocytes
proliferation assay in vitro and on mice delayed-type hypersensitivity (DTH) assay in vivo.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 15 May 2010
Revised 30 June 2010
Accepted 1 July 2010
Available online 6 July 2010
Keywords:
N-Aryl-3-(indol-3-yl)propanamides
Immunosuppressive activity
Cyclosporin A (CsA), tacrolimus (FK506), and sirolimus (rapamy-
cin) are known to be potent immunosuppressive agents. They act by
inhibiting T lymphocyte proliferation during immune response.1 All
three compounds are current chemotherapeutic agents, limiting
chronic rejection after organ transplantation and improving lifetime
of grafts receivers, and successfully used for treatment of autoim-
mune diseases in clinic.2 However, these molecules suffer from sev-
eral side effects such as nephrotoxicity, neurotoxicity, infection,
cancer, hyperlipidemia, and hypertension.3 Thus, the search for
new immunosuppressants with a comparable efficacy but with low-
er toxicity is an important task for medicinal chemistry.
We previously described a series of N-pyridinyl(methyl)-(indol-
3-yl)propanamides with a promising immunosuppressive activ-
ity.4,5 Our initial structure–activity investigations showed the
importance of the benzyl moiety and the nature of the pyridine ring
for immunosuppressive potential and this study disclosed com-
pound 1 as a promising lead (Fig. 1). This compound exerted a potent
inhibitory activity on murine splenocytes proliferation (87% inhibi-
Previously, compound 1 was found to decrease IL2-induced T
lymphocyte proliferation by inhibiting preferentially JAK3 kinase
over JAK2 (IC50 = 52.0 lM and 133.6 l
M, respectively).6 Moreover,
compound 1 significantly prolonged rat heart allograft survival
demonstrating its in vivo immunosuppressive potential.
The Janus kinases (JAKs), consisting of JAK1, JAK2, JAK3, and
TYK2, are an important family of cytoplasmic tyrosine kinases as
a consequence of their essential role in cytokine signal transduc-
tion.7 Janus Kinase 3 is a particularly attractive target for therapeu-
tic intervention in the treatment of autoimmune disorders,
inflammatory diseases, and organ transplant rejection because, un-
like other JAK family members that are widespread, JAK3 expres-
sion is restricted to haematopoietic cells.8 A number of inhibitors
of JAK3 have already been described.9
Herein, we will report our continuous work consisting of syn-
thesis and immunosuppressive activity evaluation of 3-[1-(4-chlo-
O
NH
tion at 90
obtained with CsA at 0.5
antiproliferative activity in vitro on T lymphocytes (IC50 = 17
l
M, 19% inhibition at 30
M). Previous experiments5 highlighted its
M)
lM, compared to 90% inhibition
l
l
N
and a significant immunosuppressive effect in vivo in a model of de-
layed-type hypersensitivity (DTH) in mouse. We also showed its
non-toxicity and its oral bioavailability.
N
Cl
* Corresponding author. Tel.: +33 240 412 874; fax: +33 240 412 876.
E-mail address: pascal.marchand@univ-nantes.fr (P. Marchand).
Figure 1. Chemical structure of lead compound 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.001

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F. Giraud et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5203–5206
Table 1
robenzyl)-1H-indol-3-yl]propanamide derivatives in order to
explore the effect of N-aryl substitution with compound 1 as the
initial lead.
Splenocytes proliferation assay data for the N-aryl-3-(indol-3-yl)propanamide com-
pounds 1, 6–16
Synthesis of N-aryl-3-(indol-3-yl)propanamides 6–15 was
performed, as previously described,4,5 in four or in five steps
from commercially available 3-(1H-indol-3-yl)propanoic acid 2
(Scheme 1).
O
NHAr
To obtain the target amides, the first step was the esterification
of 3-(1H-indol-3-yl)propanoic acid 2 in ethanol–hydrogen chloride
medium at reflux. Ester 3 was further reacted with 4-chlorobenzyl
chloride in the presence of Cs2CO3 in anhydrous acetonitrile to
afford the corresponding N-(4-chlorobenzyl)indole derivative 4.
Subsequent hydrolysis under basic conditions followed by the ami-
dation of the resulting propanoic acid 5 with Mukaiyama reagent
in refluxing CH2Cl2 gave the N-aryl-3-(indol-3-yl)propanamides
6–15 in low to moderate yields (17–58%) depending on the nature
of the aromatic amines.
N
Cl
Compounds Ar
Splenocytes proliferation
Inhibition% SEM Inhibition% SEM
at 90
l
Ma
at 30 l
Ma
All starting amines were commercially available except 4-ami-
noquinoline that was prepared by treatment of the 4-quinolone
with POCl3 at reflux, in a first step, to afford 4-chloroquinoline in
quantitative yield.10 Nucleophilic displacement of the chlorine
atom was performed in the presence of ammonia in phenol by fol-
lowing a literature procedure.11
Moreover, catalytic reduction of the nitro functional group of
compound 15, using palladium charcoal in THF at room tempera-
ture, yielded amino derivative 16.
1
6
7
8
9
10
11
12
13
14
15
16
Pyridin-4-yl
87 1.7
40 1.6
26 1.4
37 1.6
na
86 1.2
17 2.1
na
19 3.2
28 2.8
na
20 2.0
na
na
na
na
30 1.2
nd
Pyridin-3-yl
Pyridin-2-yl
Quinolin-4-yl
Quinolin-5-yl
Quinolin-6-yl
Isoquinolin-5-yl
Phenyl
4-Cyanophenyl
4-Trifluoromethyl-phenyl 18 1.7
4-Nitrophenyl
4-Aminophenyl
78 1.0
92 1.5
na
87 1.7
na
The new N-aryl-3-(indol-3-yl)propanamides 6–16 were tested
in vitro for their inhibitory activity on concanavalin A (ConA)-in-
duced T cell proliferation with compound 1 as the standard.
na = not active.
nd = not determined.
Freshly isolated murine spleen cells were stimulated with 1 lg/
a
Cell assay results of one representative experiment out of three performed.
mL ConA for 72 h in the presence of two different doses of the
tested compound.12 The pharmacological results are summarized
in Table 1.
To understand the incidence of the N-aryl substitution in the
pharmacological activity, diverse alternatives to the 4-pyridyl
group (compound 1) were studied. Replacement of the heteroaro-
matic ring by the phenyl ring (compound 12) or by 4-substituted
phenyls revealed that the presence of a polar and electro-with-
drawing group (compounds 13 and 15) was a more suitable mod-
ification for getting an active entity except for 14 bearing a
trifluoromethyl moiety. Indeed, the amino analog 16 displayed
no activity but stimulated the proliferation response of cells (data
not shown).
O
O
OEt
OH
O
O
OH
OEt
i
ii
iii
N
N
N
H
N
H
Cl
Cl
2
3
4
5
O
O
NHAr
NH
iv
v
NH2
N
N
Ar = 4-NO2Ph
Cl
Cl
6-15
16
Scheme 1. Reagents and conditions: (i) EtOH, HCl, reflux, 95%; (ii) Cs2CO3, 4-ClPhCH2Cl, CH3CN, reflux, 81%; (iii) NaOH 1 N, EtOH, reflux, 94%; (iv) Et3N, CNMPI, Ar-NH2,
CH2Cl2, reflux, 17–58%; (v) H2 (10 bars), Pd/C 10%, THF, rt, 45%.

F. Giraud et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5203–5206
5205
Introduction of fused-pyridine heterocyclic scaffolds such as
quinoline (compounds 8 and 9) or isoquinoline (compound 11)
had deleterious impact on inhibition of splenocytes proliferation,
even for 8, the direct analog of 1. Incorporation of bulky heteroar-
omatic amines led to a large decrease of potency. We suspected
gen interaction with the protein kinase target. Although there is
ample crystallographic, physicochemical, and theoretical evidence
that the nitrile group is a reasonably strong hydrogen-bond accep-
tor, there are few examples where it is known to act as the con-
served acceptor in a kinase ligand.13 In the literature, it was also
hypothesized that farnesyltransferase inhibitors might benefit
from the presence of a polar moiety with hydrogen-bond acceptor
properties like nitro group.14
The results showed that, among the tested compounds, N-(4-
nitrophenyl)propanamide 15 exhibited the highest bioactivity.
The antiproliferative effect of 15 was not due to direct toxicity
since 95% of the cells were still viable after 48 h incubation with
that the activity of derivative 10 (86% inhibition at 90
lM) was
the consequence of cellular toxicity since it was inactive at 30
lM.
Interestingly, shifting the nitrogen atom of the pyridine ring
from the para- to the meta- or ortho-position (compounds 6 and
7, respectively) resulted in a significant loss of activity. In the case
of ATP-competitive JAK3 inhibitor 1, the orientation of the pyridine
ring would be favorable for a hydrogen-bond between the pyridine
nitrogen atom and a suitable amino acid in the hinge region of the
ATP-binding site of the kinase.
90
l
M of 15 (data not shown).
Based upon previous data concerning the mechanism of action
In addition, the lack of activity of phenyl derivative 12 would
confirm a possible interaction between the heteroatom or the sub-
stituent at the 4-position and an amino acid residue in the hinge
region of the target enzyme. Very low activity of quinoline or iso-
quinoline derivatives would be rationalized by a steric hindrance
at the level of the binding site.
of compound 1 involving JAK3 inhibition, compound 15 was sub-
mitted to JAK 3 and JAK 2 kinases inhibition assay. Interestingly,
4-nitrophenyl derivative exhibited better activity than compound
1 against JAK3 enzyme with an IC50 of 34.2
for 1) and showed a threefold selectivity in favor of JAK3 relative
to JAK2 (IC50 = 106.7 M). These results were consistent with pre-
lM (IC50 = 52.0 lM
l
One possible explanation for keeping activity with compounds
13 and 15 would be that cyano or nitro groups may display hydro-
gen-bond acceptor properties and may mimic the pyridine nitro-
vious conclusion reporting the correlation between JAK3 inhibition
and immunosuppressive activity.
On the basis of screening data, compound 15 was subjected to
more in-depth characterization. We first investigated its effect on
proliferation of murine splenocytes using a large range of doses
in order to confirm its antiproliferative activity on T cells. As shown
in Figure 2, we found that it inhibited mitogen-dependent T cell
proliferation in a dose-dependent manner on murine splenocytes
120
100
80
60
40
20
0
with an IC50 of 19.8 1.9
lM, similar to compound 1 (IC50 =
17.0
l
M).5 These data confirmed its immunosuppressive potential.
Finally, compound 15 has been selected for in vivo evaluation
on delayed-type hypersensitivity reaction (DTH) in mice. Experi-
ments were performed as previously described with slight modifi-
cations.5 Animals were sensitized with 5 Â 106 sheep red blood
cells (SRBC) by iv and challenged four days later by injection of
SRBC into the hind footpad. Compounds were administrated orally
from the day of priming until the day of challenge. Figure 3 shows
the inhibition of DTH footpad swelling in mice treated with com-
pound 15 at 50 mg/kg, 80 mg/kg, and 120 mg/kg.15
Derivative 15 significantly inhibited the DTH response in a
dose-dependent manner. At the highest dose (120 mg/kg), inhibi-
tion of DTH (75.5%) was comparable to compound 1 (69%, Fig. 3)
and CsA (73.9%, data not shown). Moreover during the 5-days
treatment, no acute toxicity was observable in treated mice
compared to untreated control mice. These results thus demon-
0
20
40
60
80
100
concentration (
µ
Figure 2. Effect of compound 15 on T cell proliferation. Murine splenocytes were
stimulated with ConA in the presence of increasing concentrations of compound 15
(filled squares) or CsA at 1
experiments is presented.
lM (filled triangle). Mean SEM of three independent
Compound 15
(120mg/kg)
Compound 1
(120mg/kg)
n=15
n=15
**
**
Compound 15
(80mg/kg)
Compound 1
(80mg/kg)
n=15
n=15
**
**
Compound 15
(50mg/kg)
Compound 1
(50mg/kg)
n=14
n=14
**
**
n=15
n=15
Control
Control
0
2
4
6
0
2
4
6
Footpad swelling (mm)
Footpad swelling (mm)
Figure 3. Effect of compounds 15 and 1 on DTH response. Each bar represents the mean difference SEM in swelling between the two hind footpads, one injected with saline,
the other with SRBC, measured 24 h after challenge. Compounds were administered daily per os from the time of priming until SRBC challenge. The number of mice (n) in
**
each group is indicated. p <0.01 compared to the control group.

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F. Giraud et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5203–5206
Table 2
Kinase selectivity of compounds 1 and 15—IC50 values (lM)
JAK3
JAK2
Erk
HIPK1
Aurora-A
Pim1
c-Abl
KDR
TrkA
Yes
1
15
52.0
34.2
133.6
106.7
>100
>100
>100
>100
>100
>100
>100
>100
>100.0
>100.0
26.0
33.2
>100.0
>100.0
30.0
38.1
11. Abeywickrama, C.; Rotenberg, S. A.; Baker, A. D. Bioorg. Med. Chem. 2006, 14,
7796.
12. Splenocytes proliferation assay: All compounds were solubilized in DMSO and
further diluted in RPMI medium (Sigma, St Quentin Fallavier, France)
strated that compound 15 exerted significant immunosuppressive
effects in vivo.
Compound 1, the reference analog, and the most active com-
pound 15 were tested for selectivity against several kinases, and
these results are summarized in Table 2.16
Kinase profiling demonstrated that, in addition to their JAK3
micromolar activity, compounds 1 and 15 were also inhibitors of
tyrosine kinases KDR and Yes in the micromolar range. Compounds
1 and 15 were inactive for other kinases, including Erk, HIPK1, Aur-
ora-A, Pim1, c-Abl, and TrkA.
complemented with 1% L-glutamine (Gibco BRL, Paisley, Scotland) and 10%
heat inactivated FCS (Sigma) referred as complete medium. Splenocytes were
isolated from two spleens of 8-week-old female C57BL/6 (Janvier, Laval,
France) mice. Spleens were aseptically harvested and homogenized in a Petri
dish containing HBSS medium (Sigma) and splenocytes suspension was
hemolysed by buffer containing 20 mM Tris–HCl and 140 mM NH4Cl. Cells
were washed twice with RPMI, subsequently suspended in complete RPMI
medium and seeded at densities of 1.5 Â 105/well in U-bottom 96-well culture
plates (Falcon). Cells were incubated with 1 lg/mL concanavalin A (Sigma) in
the presence of several concentrations of studied compounds and cultured at
37 °C in 5% CO2 in a final volume of 150 of complete RPMI medium
supplemented with 50 M mercaptoethanol. Cell proliferation was assessed in
sextuplicate after 72 h of culture, by colorimetric detection. Briefly, cells were
incubated with 12.5 g/well of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) for 4 h at 37 °C. Formazan products
were solubilized by 100 L of lysis buffer (dimethylformamide (1 V), SDS 20%
In conclusion, among the tested compounds, 3-[1-(4-chloroben-
zyl)-1H-indol-3-yl]-N-(4-nitrophenyl)propanamide 15 showed
promising in vitro and in vivo immunosuppressive activity. Never-
theless, JAK kinase docking study needs to be performed to confirm
our SAR hypothesis. Further modification of compounds 1 and 15,
namely by introducing various substituents on the benzene ring of
the indole core, in order to improve their potency is currently in
progress. In addition, promising activity towards KDR and Yes ki-
nases will be also studied more in-depth.
lL
l
l
l
(2 V), pH 4.7) and overnight incubation at 37 °C. Cell growth was assessed
using a MRX microplate reader (Dynex Technologies, Chantilly, USA) with the
test wavelength at 570 nm and expressed as optical density (OD) values. The
inhibition of splenocytes proliferation was expressed as inhibitory rate [(OD
value of control untreated cells À OD value of treated cells)/OD value of control
untreated cells group] Â 100.
13. Angell, R. M.; Atkinson, F. L.; Brown, M. J.; Chuang, T. T.; Christopher, J. A.;
Cichy-Knight, M.; Dunn, A. K.; Hightower, K. E.; Malkakorpi, S.; Musgrave, J. R.;
Neu, M.; Rowland, P.; Shea, R. L.; Smith, J. L.; Somers, D. O.; Thomas, S. A.;
Thompson, G.; Wang, R. Bioorg. Med. Chem. Lett. 2007, 17, 1296.
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15. Delayed-type hypersensitivity assay: Compound 15 was solubilized in olive oil
and CsA was dissolved in olive oil solution containing 3% absolute ethanol.
Groups of five female BALB/C mice (Janvier, Laval, France), 8–9 weeks of age,
were injected with 5 Â 106 sheep red blood cells (SRBC) (BioMérieux, Marcy-
l’Etoile, France) in 200 lL PBS into the tail vein. Four days after immunisation,
mice were tested for DTH with a subinflammatory challenging dose (3–4 Â 108
SRBC suspended in 40 lL) injected into the right hind footpad. The left footpad
was used as a control and injected with PBS alone. The size of both hind
footpads was measured with an ‘Oditest’ (Kroeplin, Germany) 24 h after
challenge. DTH reaction was determined by the difference in size between the
SRBC- and PBS-injected footpads. Mice were daily treated orally for 5 days with
different doses of compound 15, from the time of priming until the SRBC
challenge. The DTH inhibition index was calculated as the ratio of DTH reaction
in the treated animal versus the DTH reaction in control mice. Results are
expressed as mean SEM. All results were compared statistically using
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16. Kinase inhibition assay: Recombinant kinases were purchased from Millipore or
ProQinase. AlphaScreen Protein-A-Detection Kit from Perkin–Elmer was used
to quantify the kinase activity. For the assessment of IC50 values, compounds
were tested at 10 final concentrations between 3.16 nM and 100
10 M ATP, kinase substrate and the test compound were incubated for 1 h on
a 384-well Optiplate in a final volume of 15 L. The kinase reaction was
stopped by adding 10 L ALPHA-Beadmix. The read out was done on the next
lM. Kinase,
l
l
l
morning using an Envision reader (Perkin–Elmer). IC50 values were calculated
using Graph Pad Prism software.
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