Synthesis and evaluation of bidentate ligands designed to interact with PDZ domains

Article abstract of DOI:10.1016/j.bmc.2011.05.036

We designed bidentate ligands to target PDZ domains through two binding sites: site S0, delimited by the GLGF loop, and site S1, a zone situated around loop β_B/β_C. A molecular docking study allowed us to design a generic S0 binder, to which was attached a variable size linker, itself linked to an amino acid aimed to interact with the S1 site of PDZ domains. A series of 15 novel bidentate ligands was prepared in 6-11 steps in good overall yield (24-43%). Some of these ligands showed an inhibitory activity against serotonin 5-HT2A receptor/PSD-95 interaction. This was assessed by pull-down assay using a synthetic decapeptide corresponding to the C-terminal residues of the receptor as a bait.

Full text of DOI:10.1016/j.bmc.2011.05.036

Bioorganic & Medicinal Chemistry 19 (2011) 4346–4354
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of bidentate ligands designed to interact with
PDZ domains
Benjamin Boucherle a, Alexandre Vogrig a, Hemantkumar Deokar a, Naoual Bouzidi a,
Isabelle Ripoche b, Isabelle Thomas b, Philippe Marin c, Sylvie Ducki b,
⇑
a Clermont Université, UBP, EA 987, LCHG, BP 10448, F-63000 Clermont-Ferrand, France
b Clermont Université, ENSCCF, EA 987, LCHG, BP 10448, F-63000 Clermont-Ferrand, France
c IGF, CNRS UMR5203, INSERM U661,Universités Montpellier 1 & 2, F-34094 Montpellier, France
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed bidentate ligands to target PDZ domains through two binding sites: site S0, delimited by the
GLGF loop, and site S1, a zone situated around loop bB/bC. A molecular docking study allowed us to design
a generic S0 binder, to which was attached a variable size linker, itself linked to an amino acid aimed to
interact with the S1 site of PDZ domains. A series of 15 novel bidentate ligands was prepared in 6–11
steps in good overall yield (24–43%). Some of these ligands showed an inhibitory activity against seroto-
nin 5-HT2A receptor/PSD-95 interaction. This was assessed by pull-down assay using a synthetic deca-
peptide corresponding to the C-terminal residues of the receptor as a bait.
Received 10 February 2011
Revised 16 May 2011
Accepted 19 May 2011
Available online 24 May 2011
Keywords:
PDZ domain
Protein–protein interaction
Indole
Ó 2011 Elsevier Ltd. All rights reserved.
Bidentate ligands
1. Introduction
Since their discovery two decades ago, 214 human proteins con-
taining Post-synaptic density 95/Disc-Large/Zona ocludens-1 (PDZ)
domains have been identified.1 PDZ domains are important pro-
tein–protein interactions domains; they are almost always associ-
ated with cell membrane proteins where they play an essential role
in the clustering of proteins and signal transduction.2
PDZ domains consist of ꢀ90 residues folded into a highly con-
served secondary structure which interacts with the C-terminus
of a PDZ ligand (Fig. 1). These interactions involve extreme car-
boxy-terminal of ligand proteins binding to a hydrophobic groove
between aB helix and bB strand (S0 pocket), ended by GLGF loop of
the PDZ domain3, which is the most conserved pocket of the PDZ
domains.
These PDZ ligands belong to one of three classes: Class I (-X-S/
T-X-V/I/F) have a Ser or Thr at position (ꢁ2) which interacts with a
conserved basic residue at aB1 of the PDZ domain; those PDZ
Figure 1. Cartoon representation of the backbone of the 3D structure of PDZ1
domain of PSD95.
Abbreviations: CDI, carbonyldiimidazole; Cyc, cyclohexane; DABCO, diazabicy-
clooctane; DCM, dichloromethane; DIEA, diisopropylethylamine; DMAP, 4-dimeth-
ylaminopyridine; DMF, N,N-dimethylformamide; EDC, 1-ethyl-3-(3-dimethylamino
propyl)carbodiimide; EtOAc, ethylacetate; EtOH, ethanol; FC, flash chromatogra-
phy; HOBT, hydroxybenzotriazole; MeOH, methanol; RP, reverse phase; RT, room
temperature; TFA, trifluoroacetic acid; THF, tetrahydrofuran.
domains that lack the basic residue at aB1 bind ligands belonging
to class II (-X-F/Y-X-V/I/F), where a hydrophobic residue, such as
Phe or Tyr, at position (ꢁ2) was found to interact with hydrophobic
⇑
Corresponding author. Tel.: +33 (0)473407132; fax: +33 (0)473407008.
E-mail address: sylvie.ducki@univ-bpclermont.fr (S. Ducki).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.036

B. Boucherle et al. / Bioorg. Med. Chem. 19 (2011) 4346–4354
4347
O
O
residues at
aB1, while the third class of ligands (-X-D/E/K/R-X-V/I/
O
R2
NH2
L/F) includes ligands which have a preference for negatively
charged residues (ꢁ2) and a Tyr at aB1 of the PDZ domain.4
Structural studies have recently identified a less conserved area
involved in interactions between PDZ domains and their ligand
proteins, situated upstream of the conserved groove. It seems that
binding specificity could be attributed to the bB/bC loop, herein
named S1 site.5,6
N
N
n
H
OH
O
O
H
NH
NH
HO
HO
D
S0
Figure 3. Design of bidentate ligands.
S1
Several PDZ domain-containing proteins (PDZ proteins) have
been associated with pathological states (cancer,7–9 cystic fibro-
sis,10,11 schizophrenia,12 Parkinson’s disease,13 Alzheimer’s disease,4
cerebral ischemia,14 pain15,16) and the therapeutic usefulness of
inhibiting PDZ-based protein-protein interactions has been clearly
demonstrated by using peptide and nonpeptide small molecules
order to simplify the synthesis, the aromatic methyl was removed,
the carboxylic acid was kept to interact with the GLGF loop (con-
served in all PDZ domains), a methyl group was added on position
3 to allow interactions with hydrophobic residues on aB and a sec-
.
14,16,17 Recently, reports have emerged on nonpeptide inhibition of
ond carboxylic acid function was added at position 2 of the indole
to allow H-bonding with residues on aB of PDZ domains. Both li-
gands were connected via a linker of variable length to allow spa-
tial exploration.
PDZ domain-based interactions with small organic molecules. In
2003, Fujii et al. identified indole A18 (Fig. 2) as a selective inhibitor
of the PDZ2 domain of MAGI3, an adaptor protein important in the
regulation of tissue organisation and differentiation.
2. Results and discussion
Docking experiments revealed that this indole overlapped the
C-terminal tetrapeptide of PTEN, a known PDZ ligand of MAGI3,
with its hydroxymethyl moiety covalently bound to a His on aB
and the carboxylic acid interacting with the GLGF loop. Fujii later
used the indole core to design other PDZ inhibitors.19 Indole B dis-
rupts the interaction between the Frizzled-7 Wnt receptor (Frz-7)
and Dvl while indole C targets the PDZ domain of NHERF1-
PDZ1,20 an adaptor protein crucial in the recycling and sorting of
several receptors including b2-adrenergic receptor (b2AD-R)21, cys-
tic fibrosis transmembrane conductance regulator (CFTR),11,22,23
platelet-derived (PDGFR)24 and epidermal growth factor receptors
(EGFR).25 The carboxylic acid (COOH) on the side-chain at position
2 was introduced to mimic an aspartate residue Asp(ꢁ3) in the PDZ
ligand, granting its affinity.
PSD-95 is a three PDZ-domains containing protein identified as
a major partner of various membrane receptors including 5-HT2A
receptor.26 Disrupting 5-HT2A receptor/PSD-95 interaction was
found to reduce hyperalgesia in a rodent model of neuropathic
pain.16 Thus, inhibiting the interaction between PSD-95 and 5-
HT2A receptor could lead to the development of a novel class of
analgesic agents.
2.1. Design of bidentate ligands
The purpose of the docking was to validate our concept of
bidendate ligand, particularly to evaluate the interactions of the in-
dole core into the S0 site and to simulate the possible interactions
with the S1 site.
The molecular docking study was carried out using Molecular
Operating Environment software.27 We docked compound 13b
possessing the indole moiety into the active site (Fig. 4). Compound
13b was found to dock in the same binding site as the cypin pep-
tide ligand. When the best docking pose was visualized, it was ob-
served that this compound fitted in well within the PDZ domain
and the whole molecule made interactions with the protein: H-
bonds interaction with Phe17, Ser18, Ile19 residues and other
interactions (hydrophobic, electrostatic, vdW and columbic inter-
actions) were found with the following amino acid residues
Gly14, Leu15, Gly16, Phe17, Ser18, Ile19, His70, Val74 and Leu77.
For the best pose, the total interaction energy is ꢁ7.73 kcal/mol.
Considering the spatial orientation of 13b within the S0 site, we
simulated the interactions of amino acids linked to the indole res-
idue with the S1 loop. Unfortunately due to the high flexibility of
the S1 loop, these simulations were considered of low predictabil-
ity value and are not presented here.
Our strategy aims at targetting selectively the PDZ domain via
the extended groove between bB and aB delimited by the GLGF
loop, which represents a challenge in terms of selectivity since this
is the most conserved pocket of the PDZ domains herein named S0
site.
2.2. Synthesis of bidentate ligands
We now wish to report our recent work in the design of PDZ
inhibitors using a bidentate ligand approach which would be able
to interact both with the highly conserved (S0) pocket and the less
conserved (S1) loop (Fig. 1). Our bidentate ligands were conceived
from an indole core D, inspired from Fujii’s work cited above, to
interact with the S0 site of the PDZ domain, and an amino acid,
chosen to probe specific interactions with the S1 site (Fig. 3). In
Based on the docking model, we synthesized 15 novel bidentate
ligands designed to explore whether it is possible to inhibit inter-
action between PSD-95 and 5-HT2A. These bidentate ligands were
prepared in 6–11 steps from 1. Iodination of 1 led to ethyl-4-ami-
no-3-iodobenzoate 2,28,29 which gave the desired indole 3a by a
Trp(-1)
Ph
Ph
Val(0)
Val(0)
O
O
COOH
H
n-Bu
N
n-Bu
OH
N
N
HO
HO
Asp(-3)
HO
Ile(-3)
O
HO
HO
Thr(-2)
Ph
Ser/Thr(-2)
C
A
B
Figure 2. Fujii’s designed indoles.

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B. Boucherle et al. / Bioorg. Med. Chem. 19 (2011) 4346–4354
the S1 site. These diamines N-Boc mono protected32–34 were linked
by amide bond formation to the 2-carboxylic acid moiety of indole
3a. Optimisation of the coupling conditions led us to use EDC and
HOBT to attach the monoprotected diamines to indole 3a (Scheme
2).35,36 Boc-deprotection was performed in the presence of trifluo-
roacetic (TFA)37 and gave 4a–c which were isolated with good
yields of 85–90% (over two steps).
Acid 3a and ammonium salt 4b were treated with sodium
hydroxide to give the generic ligand 5 and free C6-ligand 6 in good
yields. Boc deprotection gave 4a–c in 85–90% yield.
Amino acids with different physicochemical properties were se-
lected to graft onto amines 4b: Ile 18a (hydrophobic), Lys 18b (po-
lar, charged) Trp 18c (hydrophobic, steric hindrance), Phe 18d
(hydrophobic, steric hindrance), Glu 18e (polar, uncharged), in or-
der to probe potential interactions with the S1 site.
The protected amino acids 18a–e were coupled to the C6-amine
4b (Scheme 3).37,38 Bidentate ligands 7a–e were deprotected to af-
ford the desired deprotected C6-bidentate ligands 8a–e with good
yields. When this protocol was applied to the C8 analogue 4a, we
encountered solubility problems of the intermediates which forced
us to abandon the synthesis of this serie.
In the same manner, the protected amino acids 18a–e were cou-
pled to the C4-amine 4c to afford protected C4-bidentate ligands.
However, when the deprotection under basic conditions was at-
tempted on the latter, we mainly obtained the amine 4c. Even un-
der milder conditions (LiOH in THF)39 we were unable to obtain the
desired C4-bidentate ligands.
We therefore envisaged a new synthesis of the indole core with
a benzyl ester protection of the acid moiety. Aniline 2 (Scheme 4)
was Boc-protected and saponified to afford acid 9. The latter was
Bn-protected and Boc-deprotected to recover the free amine 11.40
Finally, amine 11 was reacted with 2-oxobutanoic acid to give
Bn-protected indole 3b with a overall good yield (65% over five
steps).
We pursued the synthesis of the C2- and C4-bidentate ligands
using the previously optimized coupling conditions (Scheme 5). In-
dole 3b was coupled to the N-Boc C2- and C4-diamines, followed
by amine deprotection with TFA to afford ammonium 12a and
12b. The ammonium 12a–b were then treated with hydrogen over
Pd/C catalyst to give C2- and C4-ligands 13a and 13b. To avoid
basic conditions to remove protecting group of the amino acids,
we envisaged to protect the amino acids with a Boc or CBz. To
Figure 4. Docking of compound 13b into PDZ1 domain of PSD-95 showing H-bond
interactions (red color) and other interactions (green color).
palladium-catalyzed annulation with an overall yield of 56% for
two steps (Scheme 1).20,30,31
Next, we use aliphatic diamines with variable chain lengths
(number of carbons n = 2, 4, 6 and 8), to allow spatial probing of
EtOOC
I
EtOOC
EtOOC
ii
i
COOH
N
NH2
NH2
H
1
2
3a
Scheme 1. Reagents and conditions: (a) I2, Ag2SO4, EtOH, RT, 30 min (85%); (b) 2-oxobutanoic acid, Pd(OAc)2, DABCO, DMF, 105 °C, 16 h (65%).
NH3
i, ii
EtOOC
HN
n
3a
TFA-
N
H
O
O
iii
NH2
6
4a
HN
n = 8
iii
O
4b n = 6
4c
n = 4
O
Na
N
O
H
O
O
Na
6
Na
N
O
H
5
Scheme 2. Reagents and conditions: (a) H2N–(CH2)n–NHBoc, EDC, HOBT, DMF, 0 °C to RT, overnight; (b) TFA, DCM, RT, 2 h (4a n = 8, 85%; 4b n = 6, 85%;,4c n = 4, 90%; over two
steps); (c) NaOH, EtOH/H2O/dioxane (5: 80%, 6: >99%).

B. Boucherle et al. / Bioorg. Med. Chem. 19 (2011) 4346–4354
4349
Fmoc
R
Na+
O
O
HN
HN
H2N
HN
R
H
ii
i
O
O
EtO
O
H
4b
N
N
HN
O
HN
O
H
6
H
6
7a
8a
, R = CH(CH3)CH2CH3 (Ile)
, R = CH(CH3)CH2CH3 (Ile)
7b, R = (CH2)4NH2 (Lys)
8b, R = (CH2)4NH2 (Lys)
7c, R = CH2Indole (Trp)
8c, R = CH2Indole (Trp)
7d
7e
8d
8e
, R = CH2Ph (Phe)
, R = CH2CH2CONH2 (Glu)
, R = CH2Ph (Phe)
, R = CH2CH2CONH2 (Glu)
Scheme 3. Reagents and conditions: (a) Fmoc-N-protected-amino acid 18a–e, CDI, DIEA, DMF, 0 °C to RT, 5 h; (b) NaOH, H2O/EtOH, RT, 24 h (8a: 58%, 8b: 51%, 8c: 50%, 8d:
51%, 8e: 46% over two steps).
O
O
O
Table 1
EtO
BnO
HO
Yields for reactions: Indole-derivatives 12a–b ? identate ligands 16a–e and 17a–e
iii
i, ii
Entry Indole
derivatives
n
Amino acids
Bidentate
ligands
Yield
(%)
I
NH2
I
N(Boc)2
I
N(Boc)2
1
2
12a
4
4
Isoleucine
(Boc)
Isoleucine
(CBz)
Lysine-N0Boc
Tryptophane
Phenylalanine
Glutamine
Isoleucine
Lysine-N’Boc
Tryptophane
Phenylalanine
Glutamine
16a
16a
35
15
9
10
2
iv
3
4
5
6
4
4
4
4
2
2
2
2
2
16b
16c
16d
16e
17a
17b
17c
17d
17e
27
30
35
30a
45
41
40
OBn
v.
BnO2C
I
O
7
12b
COOH
8
9
10
11
N
H
NH2
30
3b
11
b
Scheme 4. Reagents and conditions: (a) (Boc)2O, Et3N, DMAP, DCM, RT, 1 h (>99%);
(b) NaOH 10 M, EtOH/H2O, RT, 12 h; (c) CDI, BnOH, DMF, RT, 24 h (95%); (d) TFA,
DCM, RT, 4 h (>99%); (e) 2-oxobutanoic acid, Pd(OAc)2, DABCO, DMF, 105 °C, 16 h
(75%).
a
A cyclisation by transamidation of the amide side chain of glutamine was
observed.
b
Unsoluble
yields after a two-steps synthesis from 12a,b (Table 1, yields 27–
45%).
optimize the coupling/deprotection sequence, we prepared com-
pound 16a (Scheme 5) using N-Boc and N-CBz isoleucine. As shown
in Table 1 (entries 1 and 2), the best results were obtained with the
N-Boc protected isoleucine which afford the desired product 16a in
35% yield. The difference of yield observed for this coupling could
be due to the poor solubility or/and stability of the intermediate
compound 14a, as the starting material is totally consumed. The
desired free bidentate ligands 16a–d and 17a–e were obtained as
ammonium chloride salts with a high purity (>95%) and moderate
2.3. Biochemical evaluations
We evaluated the ability of compounds 5 (S0 binding motif), 6,
13a–b (S0 binding motif and linker), 8a–e, 16a–e and 17a–e
(bidentate ligands) to affect the association of the 5-HT2A receptor
C-terminus with PSD-95, one of the major PDZ proteins known to
TFA-
Cl
HN
O
O
NH3
n
NH3
iii
i, ii
HN
HO
n
BnO
3b
N
H
O
N
H
O
13a
12a
n = 4
n = 4
13b n = 2
12b n = 2
iv
R2
Cl
O
O
H3N
HN
R
H
R
v
O
O
HO
BnO
H
HN
HN
N
H
HN
O
N
H
HN
O
n
n
16a-e, n = 4
17a-e
14a-e, n = 4
15a-e
n = 2
, n = 2
Scheme 5. Reagents and conditions: (a) H2N–(CH2)n–NHBoc, EDC, HOBT, DMF, 0 °C to RT, overnight; (b) TFA, DCM, RT, 2 h (12a: 81%, 12b: 76% over two steps); (c) H2 60 psi,
Pd(OH)2/C, MeOH, HCl 1 M, RT, 24 h (13a: 30%, 13b: 40%); (d) CBz- or Boc-N-protected amino acids a–e, CDI, DIEA, DMF, RT, 6 h; (e) H2 (60 psi), Pd(OH)2/C, MeOH, HCl 1 M, RT,
24 h (yields over two steps reported in Table 1).

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B. Boucherle et al. / Bioorg. Med. Chem. 19 (2011) 4346–4354
at S0 using the triangle matcher placement module in MOE. Refine-
ment of the docked poses was carried out using the forcefield
refinement module and scored using Affinity dG scoring system.
Around 30 poses were returned for each docking run. The docked
poses were ranked by London dG and Affinity dG scoring functions.
All the poses were examined visually for docking accuracy, error
and fitness. The best pose was selected and presented in Figure 4
using Maestro.44
4.2. Chemical synthesis
4.2.1. Generalities
NMR spectra were recorded on a Brüker 400 MHz apparatus.
The chemical shifts are reported in ppm (d scale) and all J values
are in Hz. The following abbreviations are used: singlet (s), doublet
(d), doubled doublet (dd), triplet (t), multiplet (m). High resolution
mass spectra were performed by CRMP (Clermont-Ferrand,
France). Monitoring of the reactions was performed using silica
gel TLC plates (silica Merck 60 F254). Spots were visualized by UV
light at 254 nm. Flash chromatography columns were performed
Figure 5. Effects of compounds 5, 13b and 17c on recruitment of PSD-95 by 5-HT2A
receptor C-terminal peptide Input represents 1% of the amount of proteins used in
pull-downs.
interact with the receptor26,41 using an in vitro binding assay. A
decapeptide comprising the C-terminal residues of the 5-HT2A
receptor was coupled N-terminally to beads and incubated with
protein extracts from mice brains in the presence of increasing
concentrations of tested compounds. Proteins were resolved by
SDS–PAGE and PSD-95, on the beads, was assessed by immuno-
blotting. As previously described,26 the 5-HT2A receptor C-termi-
nal decapeptide recruited PSD-95 ( Fig. 5). This interaction was
inhibited in a concentration-dependent manner by compounds 5,
13b and 17c (Fig. 5), consitent with an inhibitory activity towards
5-HT2A receptor/PSD-95 interactions.
using silica gel 60 (70–230 mesh) or RP18 (25–40 lM) from Merck
Chimie SAS (France) on a Flash II apparatus from Armen instru-
ment (France). All commercial chemical were purchased from Al-
drich (Milwaukee, WI) or Alfa Aesar. Anhydrous DMF was
purchased from Alfa Aesar. THF was freshly distillated on so-
dium/benzophenone.
4.2.2. Preparation of indole 3a
4.2.2.1.
acid (3a ).
5-(Ethoxycarbonyl)-3-methyl-1H-indole-2-carboxylic
To solution of iodo compound (1.00 g,
a
2
3.44 mmol) in anhydrous DMF (10 mL) under argon, 2-oxobutyric
acid (1.75 g, 17.18 mmol) and DABCO (1.16 g, 10.31 mmol) were
added. The solution was stirred for 5 min before addition of palla-
dium(II) diacetate (0.04 g, 0.17 mmol). The mixture was warmed to
105 °C for 16 h. After cooling to room temperature, DMF was re-
moved by evaporation and the resulting solid was dissolved in
EtOAc (150 mL) and extracted with NaOH (1 M, 4 ꢂ 150 mL). The
aqueous layers were combined, neutralized with HCl (1 M) and ex-
tracted with EtOAc (3 ꢂ 150 mL). The combined organic layers
were dried over anhydrous Na2SO4 and concentrated in vacuo to
obtain a brown solid which was purified by trituration in diethyl
ether to give the desired indole 3a as pale brown powder (0.55 g,
2.24 mmol, 65%); mp: 265–266 °C; Rf = 0.32 (SiO2, Cyc/EtOAc,
7:3); 1H NMR (400 MHz, CD3OD) d 8.39 (d, J = 2.0, 1H, H-4), 7.90
(dd, J = 9.0, 2.0, 1H, H-6), 7.43 (d, J = 9.0, 1H, H-7), 4.38 (q, J = 7.0,
2H, CH2), 2.63 (s, 3H, CH3), 1.42 (t, J = 7.0, 3H, CH3); 13C NMR
(101 MHz, CD3OD) d 169.2 (C@O), 165.1 (C@O), 140.3 (C), 129.2
(C), 126.9 (C), 126.6 (CH), 124.5 (CH), 122.7 (C), 121.8 (C), 112.8
(CH), 61.9 (CH2), 14.7 (CH3), 9.9 (CH3); HR-ESIMS: (M+H)+ m/z
calcd for C13H14NO4 248.0923, found 248.0926.
3. Conclusion
We have designed an indole which, by molecular docking, was
found to interact efficiently with the S0 site of PDZ domains. This
generic PDZ binder was used to conceive a series of 15 novel biden-
tate ligands aimed to explore interactions with the S1 site of PDZ
domains. The bidentate ligands were prepared in 6–11 steps in
good overall yield (24–43%).
The generic PDZ binder and bidentate ligands were evaluated in
pull-down assay and three of them (5, 13b and 17c) inhibited the
interaction between PSD-95 and the 5-HT2A receptor.42 These pre-
liminary results encourage us to further evaluate binding affinity
and specificity of this series of bidentate ligands for PDZ domains.
4. Experimental
4.1. Computational study
The studies were performed using Molecular Operating Envi-
ronment software (MOE 2009.10).27
4.2.3. Preparation of indole 3b
4.1.1. Protein preparation
4.2.3.1. Benzyl 4-(bis(tert-butoxycarbonyl)amino)-3-iodobenzo-
ate (10). To a solution of 2 (4.00 g, 13.65 mmol) in DCM (100 mL)
DMAP (0.166 g, 1.36 mmol), triethylamine (1.9 mL, 13.65 mmol)
and (Boc)2O (6.260 g, 28.66 mmol) were added. The mixture was
stirred at room temperature for 1 h and washed with HCl (0.1 M).
The organic layer was concentrated and the resulting solid was
directly engaged into the next step with EtOH (200 mL) and NaOH
(1 M, 40 mL). The solution was stirred for 12 h at room temper-
ature and then concentrated to remove ethanol. The resulting solid
was dissolved in water, acidified to pH 3 and then extracted with
EtOAC. The organic layer was concentrated to give the intermedi-
ate product 9 which was directly engaged into next step. To a
solution of this intermediate in DMF (100 mL), CDI (2.43 g,
15.01 mmol) and benzylic alcohol (1.56 mL, 15.0 mmol) were
The 3D structure of the PDZ1 domain of PSD-95 (PDB 2KA9)43
was downloaded from the protein database and prepared with
MOE: partial charges were assigned using Gasteiger (POPE) meth-
od, protein structure was minimized to 0.01 kcal/mol, the cypin li-
gand was removed, the binding site was identified using the site
finder module in MOE. The ligands to dock were built using the
molecular builder module in MOE and energy-miniumized to its
local minima using the MMF94X forcefield to
(0.001 kcal/mol).
a constant
4.1.2. Molecular docking
The docking study was carried out using the dock module in
MOE. The docking was restricted to the active site pocket located

B. Boucherle et al. / Bioorg. Med. Chem. 19 (2011) 4346–4354
4351
added. The mixture was stirred for 24 h, then concentrated and
purified by FC (100% Cyc to 100% EtOAc) to give the desired
product 10 as pale yellow foam (7.17 g, 12.97 mmol, 95%); Rf = 0.60
(SiO2, Cyc/EtOAc, 9:1); 1H NMR (400 MHz, CDCl3) d 8.52 (s, 1H, H-
2), 8.06 (d, J = 8.0, 1H, H-6), 7.47–7.25 (m, 6H, H-5, 5 ꢂ CHar), 5.37
(s, 2H, CH2), 1.42–1.39 (m, 18H, 6 ꢂ CH3); 13C NMR (101 MHz,
CDCl3) d 164.6 (C@O), 149.8 (2ꢂ C@O), 146.4 (C), 140.4 (CH), 135.7
(C), 130.7 (C), 130.4 (2 ꢂ CH), 128.9 (CH), 128.8 (CH), 128.7 (CH),
128.6 (2 ꢂ CH), 99.7 (C), 83.51 (2 ꢂ C), 67.4 (CH2), 28.0–27.0 (6
ꢂ CH3); HR-ESIMS: (M+Na)+ m/z calcd for 576.0859, found
576.0879.
4.2.4.1. 8-(5-(Ethoxycarbonyl)-3-methyl-1H-indole-2-carboxa-
mido)octan-1-aminium2,2,2-trifluoroacetate (4a). Com-
pound 4a (336 mg, 0.69 mmol, 85% from 3a 200 mg, 0.81
mmol); 1H NMR (400 MHz, CD3OD) d 8.38 (d, J = 2.0, 1H, H-4),
7.92 (dd, J = 9.0, 2.0, 1H, H-6), 7.44 (d, J = 9.0, 1H, H-7), 4.40 (q,
J = 7.0, 2H, CH2), 3.48 (t, J = 8.0, 2H, CH2), 2.93 (t, J = 7.0, 2H,
CH2), 2.60 (s, 3H, CH3), 1.70–1.30 (m, 15H, 6 ꢂ CH2, CH3); 13C
NMR (101 MHz, CD3OD) d 169.2 (C@O), 164.7 (C@O), 139.9 (C),
130.3 (C), 129.4 (C), 126.0 (CH), 124.0 (CH), 122.8 (C), 116.8 (C),
112.6 (CH), 61.8 (CH2), 40.7 (CH2), 40.6 (CH2), 30.6 (CH2), 30.1
(CH2), 28.6 (CH2), 27.9 (CH2), 27.4 (CH2), 14.7 (CH3), 9.8 (CH3);
HR-ESIMS: (M-CF3COOꢁ+H)+ m/z calcd for C21H32N3O3 374.2444,
found 374.2443.
4.2.3.2. Benzyl 4-amino-3-iodobenzoate (11).
To a solution
of carbamate 10 (5.66 g, 10.23 mmol) in DCM (100 mL) TFA
(6 mL) was added. The mixture was stirred at room temperature
for 4 h and then NaOH (1 M) was added to reach basic pH (10–
12). The organic layer was concentrated to give the desired amine
11 as yellow powder in quantitative yield (3.6 g, 10.23 mmol); mp:
80–82 °C; Rf = 0.32 (SiO2, Cyc/EtOAc, 9:1); 1H NMR (400 MHz,
CDCl3) d 8.36 (s, 1H, H-2), 7.86 (d, J = 8.0, 1H, H-6), 7.44 (m, 1H,
Har), 7.26 (m, 3H, 3 ꢂ CHar), 6.71 (d, J = 8.0, 1H, H-5), 5.31 (s, 2H,
CH2), 4.53 (ls, 2H, NH2); 13C NMR (101 MHz, CDCl3) d 165.1
(C@O), 150.8 (C), 141.1 (CH), 136.2 (C), 131.4 (CH), 128.6
(2 ꢂ CH), 128.2 (2 ꢂ CH), 121.1 (2 ꢂ CH), 113.1 (CH), 82.1 (C),
66.4 (CH2); HR-ESIMS: (M+H)+ m/z calcd for 353.9811, found
353.9811.
4.2.4.2. 6-(5-(Ethoxycarbonyl)-3-methyl-1H-indole-2-carboxa-
mido)-hexan-1-aminium 2,2,2-trifluoroacetate (4b).
Com-
pound 4b (317 mg, 0.69 mmol, 85% from 3a 200 mg, 0.81 mmol);
1H NMR (400 MHz, CD3OD) d 8.36 (s, 1H, H-4), 7.90 (d, J = 8.5,
1H, H-6), 7.42 (d, J = 8.5, 1H, H-7), 4.38 (q, J = 7.0, 2H, CH2), 3.44
(t, J = 7.0, 2H, CH2), 2.93 (t, J = 7.5, 2H, CH2), 2.58 (s, 3H, CH3),
1.74–1.60 (m, 4H, 2 ꢂ CH2), 1.51–1.44 (m, 4H, 2 ꢂ CH2), 1.41 (t,
J = 7.0, 3H, CH3); 13C NMR (101 MHz, CD3OD) d 169.2 (C@O),
164.8 (C@O), 139.9 (C), 130.2 (C), 129.4 (C), 126.1 (CH), 124.0
(CH), 122.8 (C), 117.01 (C), 112.6 (CH), 61.9 (CH2), 40.7 (CH2),
40.4 (CH2), 30.4 (CH2), 28.5 (CH2), 27.4 (CH2), 27.0 (CH2), 14.7
(CH3), 9.8 (CH3); HR-ESIMS: (M+H)+ m/z calcd for C19H28N3O3
346.2125, found 346.2130.
4.2.3.3. 5-(Benzyloxycarbonyl)-3-methyl-1H-indole-2-carbox-
ylic acid (3b).
To a solution of amine 11 (1.00 g, 3.44 mmol,
4.2.4.3. 4-(5-(Ethoxycarbonyl)-3-methyl-1H-indole-2-carboxa-
1) in DMF (10 mL) 2-oxobutyric acid (1.75 g, 17.18 mmol) and
DABCO (1.16 g, 10.31 mmol) were added. The solution was stirred
for 5 min and then palladium(II) diacetate (0.04 g, 0.17 mmol, 0.05)
was added. The mixture was warmed to 105 °C, stirred for 16 h,
and then concentrated to remove DMF. The resulting solid was dis-
solved in EtOAc (150 mL) and washed with water (150 mL). The or-
ganic layer was dried over anhydrous Na2SO4, then concentrated in
vacuo to obtain a dark brown solid which was purified by FC (Silica,
100% Cyc to 100% EtOAc) to afford the desired indole 3b as a brown
powder (0.798 g, 2.58 mmol, 75%); mp: 190–194 °C; Rf = 0.29
(SiO2, Cyc/EtOAc, 5:5); 1H NMR (400 MHz, CDCl3) d 8.36 (s, 1H,
H-4), 7.89 (d, J = 8.5, 1H, H-6), 7.45–7.30 (m, 6H, H-7, 5 ꢂ CHar),
5.34 (s, 2H, CH2), 2.58 (s, 3H, CH3); 13C NMR (101 MHz, CDCl3) d
168.2 (C@O), 164.5 (C@O), 139.7 (C), 137.4 (C), 129.0 (2 ꢂ CH),
128.6 (CH), 128.6 (2 ꢂ CH), 126.1 (CH), 124.1 (CH), 121.9 (C),
121.2 (C), 112.3 (CH), 66.9 (CH2), 9.3 (CH3); HR-ESIMS: (M+Na)+
m/z calcd for 331.0820, found 331.0824.
mido)butan-1-aminium 2,2,2-trifluoroacetate (4c).
Com-
pound 4c (314 mg, 0.73 mmol, 90% from 3a 200 mg, 0.81 mmol);
1H NMR (400 MHz, CD3OD) d 8.38 (d, J = 1.0, 1H, H-4), 7.91 (dd,
J = 8.0, 1.0, 1H, H-6), 7.43 (d, J = 8.0, 1H, H-7), 4.39 (q, J = 8.0, 2H,
CH2), 3.49 (m, 2H, CH2), 3.01 (m, 2H, CH2), 2.60 (s, 3H, CH3),
1.76–1.74 (m, 4H, 2 ꢂ CH2), 1.42 (t, 2H, CH3); 13C NMR (101 MHz,
CDCl3) d 162.7 (C@O), 156.9 (C@O), 138.1 (C), 134.0 (C), 128.5
(C), 125.7 (CH), 123.9 (CH), 122.8 (C), 111.9 (CH), 61.8 (CH2), 37.8
(2 ꢂ CH2), 28.8 (2 ꢂ CH2), 14.9 (CH3) 10.6 (CH3); HR-ESIMS: (M-
CF3COOꢁ+H)+ m/z calcd for C17H24N3O3 318.1818, found 318.1826.
4.2.4.4.
boxamido)-butan-1-aminium
(12a). Compound 12a (648 mg, 1.31 mmol, 81% from 3b
4-(5-(Benzyloxycarbonyl)-3-methyl-1H-indole-2-car-
2,2,2-trifluoroacetate
400 mg, 1.62 mmol); 1H NMR (400 MHz, CD3OD) d 8.38 (s, 1H, H-
4), 7.91 (d, J = 12.0, 1H, H-6), 7.48–7.32 (m, 6H, H-7, 5 ꢂ CHar),
5.36 (s, 2H, CH2), 3.47 (m, 2H, CH2), 2.99 (m, 2H, CH2), 2.56 (s,
3H, CH3), 1.75 (m, 4H, 2 ꢂ CH2); 13C NMR (101 MHz, CD3OD) d
168.3 (C@O), 163.7 (C@O), 138.7 (C), 131.4 (C), 129.8 (2 ꢂ CH),
129.6 (CH), 129.4 (2 ꢂ CH), 126.6 (CH), 125.0 (CH), 123.5 (C),
113.1 (CH), 66.5 (CH2), 40.0 (CH2), 39.2 (CH2), 28.0 (2 ꢂ CH2),
10.1 (CH3); HR-ESIMS: (M-CF3COOꢁ+H)+ m/z calcd for 380.1969,
found 380.1972.
4.2.4. General Procedure for the preparation of protected
indole-Cn 4a–c and 12a–b
To a solution of protected indole 3a or 3b (1 equiv) in DMF
(1 mL for 0.1 mmol of 3a or 3b), HOBT (1.1 equiv) and EDC
(1.1 equiv) were added at 0 °C. After stirring the solution for 1 h
at 0 °C, a solution of N-Boc-diamine (2 equiv) in DMF (1 mL for
0.05 mmol of N-Boc-diamine) was added drop wise to the mixture.
The reaction was slowly allowed to warm up to room temperature
and stirred overnight at room temperature. After removal of DMF,
the resulting residue was dissolved in EtOAc (4 mL for 0.1 mmol of
3a or 3b) and washed with water (20 mL for 0.1 mmol of 3a or 3b).
The organic layer was concentrated in vacuo and the resulting solid
was purified by FC (Silica, gradient 100% Cyc to 100% EtOAc) to give
the desired product which was directly engaged into the next step
in DCM in the presence TFA (5 equiv) for 2 h at RT. The mixture was
concentrated in vacuo and the resulting solid was purified by trit-
uration in diethyl ether and filtration to obtain the desired salts
4a–c and 12a,b.
4.2.4.5.
boxamido)-ethanaminium
(12b). Compound 12b (573 mg, 1.23 mmol, 76% from 3b
2-(5-(Benzyloxycarbonyl)-3-methyl-1H-indole-2-car-
2,2,2-trifluoroacetate
400 mg, 1.62 mmol); 1H NMR (400 MHz, CD3OD) d 8.42 (s, 1H, H-
4), 7.90 (d, J = 8.0, 1H, H-6), 7.49–7.32 (m, 6H, H-7, 5 ꢂ CHar),
5.38 (s, 2H, CH2), 3.71 (t, J = 8.0, 2H, CH2), 3.20 (t, J = 8.0, 2H,
CH2), 2.61 (s, 3H, CH3); 13C NMR (101 MHz, CDCl3) d 168.8
(C@O), 165.7 (C@O), 140.0 (C), 138.0 (C), 129.6 (2 ꢂ CH), 129.4
(CH), 129.2 (2 ꢂ CH), 126.4 (CH), 124.3 (CH), 122.7 (C), 117.7
(CH), 112.8 (CH), 67.5 (CH2), 41.2 (CH2), 38.6 (CH2) 9.8 (CH3);
HR-ESIMS: (M-CF3COOꢁ+H)+ m/z calcd for 352.1656, found
352.1660.

4352
B. Boucherle et al. / Bioorg. Med. Chem. 19 (2011) 4346–4354
4.2.5. Preparation of ligands 5 and 6
4.2.6.1.2.
(S)-2-((2,6-Diaminohexanamido)buthylcarbamoyl)-3-
4.2.5.1.
(5).
Sodium
3-methyl-1H-indole-2,5-dicarboxylate
methyl-1H-indole-5-carboxylic acid (8b). Compound 8b (100 mg,
0.22 mmol, 51% from 4b 200 mg, 0.44 mmol). Before the alkaline
treatment, the N-Boc of the side chain was removed in presence
of TFA (excess) in DCM; 1H NMR (400 MHz, D2O/CF3COOD, 95:5)
d 8.00 (s, 1H, H-4), 7.59 (d, J = 8.5, 1H, H-6), 7.14 (d, J = 8.5, 1H,
H-7), 3.80 (t, J = 6.7, 1H, CH), 3.24–2.95 (m, 4H, 2 ꢂ CH2), 2.82 (t,
J = 7.6, 2H, CH2), 2.16 (s, 3H, CH3), 1.81–1.64 (m, 2H, CH2), 1.59–
1.48 (m, 2H, CH2), 1.44–1.23 (m, 6H, 3 ꢂ CH2), 1.21–1.08 (m, 4H,
2 ꢂ CH2); 13C NMR low solubility; HR-ESIMS: (M+H)+ m/z calcd
for C23H36N5O6 446.2762, found 446.2786.
Indole 3a (100 mg, 0.40 mmol) is engaged with MeOH
(10 mL) and NaOH (2 M, 2 mL) and heated under reflux for 24 h.
The mixture was concentrated and purified by RPFC (RP18 Silica,
100% H2O to 100% MeOH) to give the desired salt 5 as a white solid
(85 mg, 0.323 mmol, 80%); 1H NMR (400 MHz, CD3OD) 8.41 (d,
J = 1.0, 1H, H-4), 7.92 (dd, J = 8.0, 1.0, 1H, H-6), 7.43 (d, J = 8.0,
1H, H-7), 2.63 (s, 3H, CH3); 13C NMR (101 MHz, CD3OD) d 168.4
(C@O), 165.1 (C@O), 140.2 (C), 128.8 (C), 126.3 (C), 125.7 (CH),
123.5 (CH), 122.9 (C), 121.2 (C), 111.9 (CH), 9.9 (CH3); HR-ESIMS:
[M-H]- m/z calcd for C11H9NO4 218.0459, found 218.0455.
4.2.6.1.3. Sodium ( S)-2-((2-amino-3-(1H-indol-2-yl)propanami-
do)-hexylcarbamoyl)-3-methyl-1H-indole-5-carboxylate
(8c). Compound 8c (160 mg, 0.31 mmol, 50% from 4b 280 mg,
0.61 mmol); 1H NMR (400 MHz, CD3OD) d 8.34 (s, 1H, H-4), 7.92
(d, J = 8.5, 1H, H-6), 7.57 (d, J = 8.5, 1H, H-7), 7.36–7.27 (m, 2H,
2 ꢂ CHar), 7.09–7.01 (m, 2H, 2 ꢂ CHar), 6.98 (t, J = 7.5, 1H, CHar),
3.60 (t, J = 6.5, 1H, CH), 3.37 (t, J = 7.0, 2H, CH2), 3.18–3.07 (m,
2H, CH2), 3.07–2.94 (m, 2H, CH2), 2.58 (s, 3H, CH3), 1.63–1.49 (m,
2H, CH2), 1.36–1.24 (m, 4H, 2 ꢂ CH2), 1.19–1.07 (m, 2H, CH2); 13C
NMR (101 MHz, CD3OD) d 176.9 (C@O), 176.6 (C@O), 165.1
(C@O), 138.8 (C), 138.1 (C), 130.3 (C), 129.3 (C), 129.1 (C), 128.8
(C), 127.1 (CH), 124.6 (CH), 123.0 (CH), 122.4 (CH), 119.8 (CH),
119.5 (CH), 117.2 (C), 112.3 (CH), 111.6 (CH), 111.2 (C), 57.1
(CH), 40.5 (CH2), 40.2 (CH2), 32.3 (CH2), 30.5 (CH2), 30.0 (CH2),
27.6 (CH2), 27.4 (CH2), 10.0 (CH3); HR-ESIMS: (M-Na+2H)+ m/z
calcd for C28H34N5O4 504.2611, found 504.2628.
4.2.5.2. Sodium 2-(6-aminohexylcarbamoyl)-3-methyl-1 H-
indole-5-carboxylate (6).
To a solution of ethyl-indole-C6-
TFA 4b (50 mg, 0.109 mmol) was added NaOH (1 M) (0.55 mL,
0.55 mmol) in EtOH (5 mL). The solution was heated to reflux for
24 h. Then the mixture was concentrated in vacuo. The resulting
salt was purified by RPFC (RP18 Silica, 100% H2O to 100% MeOH)
to give the desired salt
6 in quantitative yield (37 mg,
0.109 mmol); 1H NMR (400 MHz, CD3OD) d 8.38 (s, 1H, H-4), 7.91
(d, J = 12.0, 1H, H-6), 7.41 (d, 1H, J = 12.0, H-7), 3.44 (t, J = 8.0, 2H,
CH2), 2.93 (t, J = 8.0, 2H, CH2), 2.58 (s, 3H, CH3), 1.71 (m, 4H,
2 ꢂ CH2), 1.69 (m, 4H, 2 ꢂ CH2); 13C NMR (101 MHz, CD3OD) d
140.0 (C), 130.0 (C), 128.7 (C), 126.2 (CH), 124.3 (CH), 117.4 (C),
112.5 (CH), 40.2 (CH2), 39.6 (CH2), 33.0 (CH2), 31.5 (CH2), 27.5
(CH2), 25.8 (CH2), 9.7 (CH3); HR-ESIMS: (M+H)+ m/z calcd for
4.2.6.1.4. Sodium (S)-2-((2-amino-3-phenylpropanamido)hexylc-
arbamoyl)-3-methyl-1H-indole-5-carboxylate (8d). Compound 8d
(124 mg, 0.26 mmol, 51% from 4b 230 mg, 0.50 mmol); 1H NMR
(400 MHz, CD3OD) d 8.35 (s, 1H, H-4), 7.93 (d, J = 8.5, 1H, H-6),
7.33 (d, J = 8.5, 1H, H-7), 7.28–7.15 (m, 5H, 5 ꢂ CHar), 3.51 (t,
J = 6.5, 1H, CH), 3.38 (t, J = 7.0, 2H, CH2), 3.22–3.08 (m, 1H, CH2),
3.08–2.97 (m, 1H, CH2), 2.97–2.87 (m, 1H, CH2), 2.87–2.73 (m,
1H, CH2), 2.59 (s, 3H, CH3), 1.64–1.51 (m, 2H, CH2), 1.39–1.26 (m,
4H, 2 ꢂ CH2), 1.24–1.13 (m, 2H, CH2); 13C NMR (101 MHz, CD3OD)
d 176.7 (C@O), 176.3 (C@O), 165.0 (C@O), 138.7 (C), 138.6 (C),
130.4 (CH), 130.2 (C), 129.5 (CH), 129.3 (C), 129.1 (C), 127.7 (CH),
127.0 (CH), 123.0 (CH), 117.3 (C), 111.6 (CH), 57.8 (CH), 42.6
(CH2), 40.5 (CH2), 40.1 (CH2), 30.5 (CH2), 30.2 (CH2), 27.7 (CH2),
27.5 (CH2), 10.1 (CH3); HR-ESIMS: (M-Na+2H)+ m/z calcd for
C
17H24N3O3 318.1818, found 318.1819.
4.2.6. General Procedure for the preparation of indole-Cn-
amino acids 8a–e, 16a–e and 17a–e
To a solution of salt 4a–c, 12a or 12b (1 equiv) in DMF (1 mL for
0.1 mmol of 4a–c, 12a or 12b) was added DIEA (1.1 equiv) at room
temperature and the reaction was stirred for 45 min to give solu-
tion A. To a solution of N-protected amino acid (1.1 equiv) in
DMF (1 mL for 0.05 mmol of N-protected amino acid), CDI
(1.2 equiv) was added at room temperature and the mixture was
stirred for 1 h to give solution B. Solution A was added drop wise
to solution B at room temperature and stirred for 4 h. The mixture
was concentrated in vacuo to remove DMF and the desired biden-
tate ligands 7a–e, 9a, 14a–e, 15a–e were obtained, and directly en-
gaged into the deprotection step (Method A or B below), after
verification of crude 1H NMR spectra.
C26H32N4O4 465.2496, found 465.2511.
4.2.6.1.5. Sodium ( S)-2-((2-amino-4-carboxybutanamido)hexylc-
arbamoyl)-3-methyl-1H-indole-5-carboxylate (8e). Compound 8e
(134 mg, 0.29 mmol, 46% from 4b 286 mg, 0.62 mmol); 1H NMR
(400 MHz, CD3OD) d 8.36 (d, J = 1.0, 1H, H-4), 7.95 (dd, J = 8.5,
1.0, 1H, H-6), 7.35 (d, J = 8.5, 1H, H-7), 3.42–3.16 (m, 5H,
2 ꢂ CH2 + CH), 2.61 (s, 3H, CH3), 2.27 (t, J = 8.0, 2H, CH2), 2.02–
1.93 (m, 1H, CH2), 1.88–1.81 (m, 1H, CH2), 1.69–1.62 (m, 2H,
CH2), 1.60–1.50 (m, 2H, CH2), 1.46–1.39 (m, 4H, 2 ꢂ CH2); 13C
NMR (101 MHz, CD3OD) d 181.8 (C@O), 177.1 (C@O), 176.7
(C@O), 165.0 (C@O), 138.8 (C), 130.2 (C), 129.3 (C), 129.0 (C),
127.0 (CH), 122.9 (CH), 117.4 (C), 111.6 (CH), 55.9 (CH), 40.5
(CH2), 40.2 (CH2), 35.4 (CH2), 33.6 (CH2), 30.5 (CH2), 30.3 (CH2),
27.7 (CH2), 27.6 (CH2), 10.0 (CH3); HR-ESIMS: (M-Na+2H)+ m/z
calcd for C22H31N4O6 447.2238, found 447.2264.
4.2.6.1. Method A for deprotection.
To a solution of 7a–e,
(1 equiv) in EtOH (1 mL for 0.1 mmol of 7a–e) and dioxane (1 mL
for 0.1 mmol of 7a–e), NaOH (5 N, 1 mL for 0.1 mmol of 7a–e)
was added and the reaction was stirred at room temperature for
24 h. The mixture was then concentrated in vacuo, the resulting
solid washed with Et2O (20 mL) and purified by RPFC (RP18 silica,
gradient H2O/MeOH) to give the desired salt 8a–e.
4.2.6.1.1. Sodium 2-(((2S,3 S)-2-amino-3-methylpentanamido)-
hexyl-carbamoyl)-3-methyl-1H-indole-5-carboxylate (8a) . Com-
pound 8a 160 mg, 0.35 mmol, 58% from 4b 280 mg, 0.61 mmol);
1H NMR (400 MHz, CD3OD) d 8.33 (d, J = 1.5, 1H, H-4), 7.91 (dd,
J = 8.5, 1.5, 1H, H-7), 7.32 (d, J = 8.5, 1H, H-6), 3.42 (t, J = 7.0, 2H,
CH2), 3.29–3.13 (m, 2H, CH2), 3.09 (d, J = 6.0, 1H, CH), 2.58 (s, 3H,
CH3), 1.70–1.37 (m, 10H, 5 ꢂ CH2), 1.20–1.07 (m, 1H, CH), 0.96–
0.85 (m, 6H, 2 ꢂ CH3); 13C NMR (101 MHz, CD3OD) d 176.9
(C@O), 176.6 (C@O), 165.1 (C@O), 138.8 (C), 130.4 (C), 129.3 (C),
129.1 (C), 127.1 (CH), 123.0 (CH), 117.1 (C), 111.5 (CH), 61.0
(CH), 40.5 (CH2), 40.4 (CH), 40.1 (CH2), 30.6 (CH2), 30.4 (CH2),
27.7 (CH2), 27.7 (CH2), 25.5 (CH2), 16.0 (CH3), 11.9 (CH3), 10.0
(CH3); HR-ESIMS: (M-Na+2H)+ m/z calcd for C23H35N4O4
431.2653, found 431.2670.
4.2.6.2. Method B for deprotection.
To a solution of 12a and
b, 14a–e or 15a–e (1 equiv) in MeOH (2 mL for 0.1 mmol of 12a
and b, 14a–e or 15a–e), Pd(OH)2/C (1 mg for 0.01 mmol) was
added. The mixture was stirred under H2 (60 psi). After 24 h, the
solution was filtrated over CeliteÒ and an aqueous solution of
HCl (1 N) is added. The mixture was concentrated and purified
by RPFC (RP18 Silica, gradient 100% H2O to 100% MeOH) to allow
isolation of the desired salt 13a and 13b, 16a–e, 17a-d.

B. Boucherle et al. / Bioorg. Med. Chem. 19 (2011) 4346–4354
4353
4.2.6.2.1.
4-(5-Carboxy-3-methyl-1
H-indole-2-carboxa-
CD3OD) d 169.2 (C@O), 164.2 (C@O), 139.2 (C), 135.3 (C), 130.1
(2 ꢂ CH), 129.5 (2 ꢂ CH), 129.3 (C), 128.9 (C), 128.2 (CH), 126.1
(CH), 123.7 (CH), 116.9 (C), 112.0 (CH), 55.5 (CH), 39.7 (2 ꢂ CH2),
38.4 (CH2), 27.4 (CH2), 27.0 (CH2), 9.5 (CH3); HR-ESIMS: (M+H)+
m/z calcd for C24H29N4O3 437.2189, found 437.2178.
mido)butan-1-aminium chloride (13a). Compound 13a (19 mg,
0.06 mmol, 30% from 12a 100 mg, 0.20 mmol); 1H NMR
(400 MHz, CD3OD) d 8.37 (s, 1H, H-4), 7.90 (d, J = 12.0, 1H, H-6),
7.40 (d, 1H, J = 12.0, H-7), 3.50 (t, J = 8.0, 2H, CH2), 3.00 (t, J = 8.0,
2H, CH2), 2.58 (s, 3H, CH3), 1.75 (m, 4H, 2 ꢂ CH2); 13C NMR
(101 MHz, CD3OD) d 170.3 (C@O), 165.0 (C@O), 139.7 (C), 129.7
(C), 129.2 (C), 126.3 (CH), 124.1 (CH), 117.0 (C), 112.3 (CH), 40.2
(CH2), 39.6 (CH2), 27.5 (CH2), 25.8 (CH2), 9.7 (CH3); HR-ESIMS:
(M+H)+ m/z calcd for C15H20N3O3 290.1505, found 290.1512.
4.2.6.2.2. 2-(5-Carboxy-3-methyl-1H-indole-2-carboxamido)ethan
aminium chloride (13b). Compound 13b 25 mg, 0.08 mmol, 40%
from 12b 100 mg, 0.21 mmol); 1H NMR (400 MHz, CD3OD) d 8.41
(s, 1H, H-4), 7.93 (d, J = 12.0, 1H, H-6), 7.44 (d, 1H, J = 12.0, H-7),
3.72 (t, J = 8.0, 2H, CH2), 3.19 (t, J = 8.0, 2H, CH2), 2.62 (s, 3H,
CH3); 13C NMR (101 MHz, CD3OD) d 139.4 (C), 129.4 (C), 128.2
(C), 125.9 (CH), 123.7 (CH), 116.4 (C), 111.9 (CH), 41.5 (CH2), 40.8
(CH2), 9.2 (CH3); HR-ESIMS: M+ m/z calcd for C13H16N3O3
262.1192, found 262.1202.
4.2.6.2.7.
3-Methyl-2-(4-(5-oxopyrrolidine-2-carboxamido)-
butylcarbamoyl)-1 H-indole-5-carboxylic acid (16e). Compound
16e (70 mg, 0.17 mmol, 30% from 12a 286 mg, 0.58 mmol); 1H
NMR (400 MHz, CD3OD) d 8.36 (s, 1H, H-4), 7.89 (d, J = 12.0, 1H,
H-6), 7.40 (d, 1H, J = 12.0, H-7), 4.16 (m, 1H, CH), 3.43 (t, J = 8.0,
2H, CH2), 3.27 (t, J = 8.0, 2H, CH2), 2.57 (s, 3H, CH3), 2.35–2.40
(m, 4H, 2 ꢂ CH2), 1.68–1.62 (m, 4H, 2 ꢂ CH2); 13C NMR
(101 MHz, CD3OD) d 175.2 (C@O), 140.0 (C), 130.1(C), 129.6 (C),
126.8 (CH), 124.3 (CH), 117.3 (C), 112.5 (CH), 58.5 (CH), 40.4
(CH2), 40.3 (CH2), 30.7 (CH2), 28.1 (CH2), 28.0 (CH2), 27.0 (CH2),
10.1 (CH3); HR-ESIMS: (M+Na)+ m/z calcd for C20H24N4O5Na
423.1644, found 423.1643.
4.2.6.2.8. (2S,3S)-1-(2-(5-Carboxy-3-methyl-1H-indole-2-carboxa-
mido)-ethylamino)-3-methyl-1-oxopentan-2-aminium
chloride
4.2.6.2.3. (2S,3 S)-1-((5-Carboxy-3-methyl-1H-indole-2-carboxa-
(17a). Compound 17a (118 mg, 0.29 mmol, 45% from 12b
300 mg, 0.64 mmol); 1H NMR (400 MHz, CD3OD) d 8.33 (s, 1H, H-
4), 7.90 (d, J = 8.0, 1H, H-6), 7.34 (d, 1H, J = 8.0, H-7), 3.67 (m, 1H,
CH), 3.58 (m, 4H, 2 ꢂ CH2), 2.58 (s, 3H, CH3), 1.85 (m, 1H, CH),
1.52 (m, 1H, CH2), 1.16 (m, 1H, CH2), 0.88–0.84 (m, 6H, 2 ꢂ CH3);
13C NMR (101 MHz, CD3OD) d 165.2 (C@O), 164.9 (C@O), 139.4
(C), 129.3 (C), 126.8 (CH), 125.5 (C), 125.8 (C), 123.9 (CH), 117.4
(C), 112.2 (CH), 59.62 (CH), 40.5 (CH2), 40.2 (CH2), 38.3 (CH), 25.5
(CH2), 15.2 (CH3), 11.6 (CH3), 10.0 (CH3); HR-ESIMS: (M+H)+ m/z
calcd for C19H27N4O4 375.2032, found 375.2030.
mido)-butylamino)-3-methyl-1-oxopentan-2-aminium
chloride
(16a). Compound 16a (99 mg, 0.22 mmol, 35% from 12a
316 mg, 0.64 mmol); 1H NMR (400 MHz, CD3OD) d 8.28 (s, 1H, H-
4), 7.85 (d, J = 12.0, 1H, H-6), 7.28 (d, 1H, J = 12.0, H-7), 3.48 (m,
1H, CH), 3.36 (t, J = 8.0, 2H, CH2), 3.22 (t, J = 8.0, 2H, CH2), 2.51 (s,
3H, CH3), 1.60–1.57 (m, 7H, 3 ꢂ CH2, CH), 0.93–0.85 (m, 6H,
2 ꢂ CH3); 13C NMR (101 MHz, CD3OD) d 171.0 (C@O), 164.3
(C@O), 138.5 (C), 128.6 (C), 126.2 (CH), 122.7 (CH), 116.6 (C),
111.3 (CH), 58.9 (CH), 39.5 (2 ꢂ CH2), 38.0 (CH), 27.4 (CH2), 27.1
(CH2), 25.0 (CH2), 14.7 (CH3), 11.0 (CH3), 9.4 (CH3); HR-ESIMS:
M+ m/z calcd for C21H31N4O4 403.2345, found 403.2328.
4.2.6.2.9. ((S)-6-(2-(5-(Benzyloxycarbonyl)-3-methyl-1H-indole-2-
carboxamido)-ethylamino)-6-oxohexane-1,5-diaminium
chloride
4.2.6.2.4.
(S)-6-(4-(5-Carboxy-3-methyl-1H-indole-2-carboxa-
chloride
(17b). Compound 17b (104 mg, 0.23 mmol, 41% from 12b
256 mg, 0.55 mmol); 1H NMR (400 MHz, CD3OD) d 8.37 (s, 1H, H-
4), 7.90 (d, J = 8 Hz, 1H, H-6), 7.46 (d, J = 8 Hz, 1H, H-7), 3.95 (m,
1H, CH), 3.65–3.48 (m, 4H, 2 ꢂ CH2), 2.89 (m, 2H, CH2), 2.60 (s,
3H, CH3), 1.93–1.86 (m, 2H, CH2), 1.67 (m, 2H, CH2), 1.50 (m, 2H,
CH2); 13C NMR (101 MHz, CD3OD) 171.2 (C@O), 170.6 (C@O),
165.1(C@O), 140.0 (C), 129.8 (C), 129.5 (C), 126.7 (CH), 124.5
(CH), 123.2 (C), 117.9 (C) 112.8 (CH), 54.5 (CH), 40.6 (CH2), 40.4
(CH2), 40.3 (CH2), 32.2 (CH2), 28.2 (CH2), 23.1 (CH2), 10.2 (CH3);
HR-ESIMS: (M+H)+ m/z calcd for C19H27N5O4 390.2141, found
390.2122.
4.2.6.3.0. (S)-1-(2-(5-(Benzyloxycarbonyl)-3-methyl-1H-indole-2-
carboxamido)ethylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-amini-
um chloride (17c). Compound 17c (82 mg, 0.13 mmol, 40% from
12b 149 mg, 0.32 mmol); 1H NMR (400 MHz, CD3OD) d 8.35 (s,
1H, H-4), 7.89 (d, J = 8.0, 1H, H-6), 7.60 (d, 1H, J = 8.0, H-7), 7.37–
7.00 (m, 5H, 5 ꢂ CHar), 4.00 (m, 1H, CH), 3.41–3.12 (m, 6H,
3 ꢂ CH2), 2.46 (s, 3H, CH3); 13C NMR (101 MHz, CD3OD) d 171.4
(C@O), 164.8 (C@O), 139.3 (C), 137.9 (C), 129.1 (C), 129.0 (C), 128
(C), 126.4 (C), 125.0 (CH), 123.8 (CH), 122.6 (CH), 119.9 (CH),
118.8 (CH), 117.1 (CH), 112.3 (CH), 112.1 (CH), 108.3 (C), 55.2
(CH), 40.3 (CH2), 39.9 (CH2), 29.1 (CH2), 9.7 (CH3); HR-ESIMS: M+
m/z calcd for C24H26N5O4 448.1985, found 448.1974.
mido)-butylamino)-6-oxohexane-1,5-diaminium
(16b). Compound 16b (85 mg, 0.17 mmol, 27% from 12a
316 mg, 0.64 mmol); 1H NMR (400 MHz, CD3OD) d 8.20 (d, J = 1.0,
1H, H-4), 7.79 (dd, J = 8.0, 1.0, 1H, H-6), 7.19 (d, J = 8.0, 1H, H-7),
3.29 (m, 2H, CH2), 3.11 (m, 3H, CH2, CH), 2.45 (s, 3H, CH3), 2.41
(m, 2H, CH2), 1.52–1.16 (m, 10H, 5 ꢂ CH2); 13C NMR (101 MHz,
CD3OD) d 177.6 (C@O), 176.7 (C@O), 165.1 (C@O), 138.8 (C),
130.3 (C), 129.8 (C), 129.1 (C), 127.0 (CH), 122.9 (CH), 117.4 (C),
111.7 (CH), 56.1 (CH), 42.3 (CH2), 40.2 (CH2), 39.9 (CH2), 36.3
(CH2), 33.7 (CH2), 28.0 (CH2), 27.9 (CH2), 24.1 (CH2), 10.1 (CH3);
HR-ESIMS: (M+H)+ m/z calcd for C21H32N5O4 418.2449, found
418.2446.
4.2.6.2.5.
(S)-1-(4-(5-Carboxy-3-methyl-1H-indole-2-carboxa-
mido)-butylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-aminium chlo-
ride (16c). Compound 16c (89 mg, 0.17 mmol, 30% from 12a
286 mg, 0.58 mmol); 1H NMR (400 MHz, CD3OD) d 8.37 (s, 1H, H-
4), 7.91 (d, J = 12.0, 1H, H-6), 7.40 (d, 1H, J = 12.0, H-7), 7.40–7.00
(m, 5H, 5 ꢂ CHar), 4.06 (m, 1H, CH), 3.63–3.57 (m, 3H, 2 ꢂ CH2),
3.38–3.14 (m, 3H, 2 ꢂ CH2), 2.57 (s, 3H, CH3), 1.15–1.40 (m, 4H,
2 ꢂ CH2); 13C NMR (101 MHz, CD3OD) d 170.7 (C@O), 165.2
(C@O), 140.1 (C), 138.6 (C), 136.4 (C), 130.3 (C), 129.8 (C), 128.8
(C), 127.0 (CH), 125.9 (CH), 123.3 (CH), 120.7 (CH), 119.6 (CH),
117.6 (CH), 113.0 (CH), 112.8 (CH), 108.7 (C), 55.8 (CH), 40.6
(CH2), 40.7 (CH2), 29.4 (CH2), 28.3 (CH2), 27.8 (CH2), 10.33 (CH3);
HR-ESIMS: (M+H)+ m/z calcd for C26H30N5O4 476.2298, found
476.2314.
4.2.6.3.1. (S)-1-(2-(5-carboxy-3-methyl-1 H-indole-2-carboxa-
mido)-ethylamino)-1-oxo-3-phenylpropan-2-aminium
chloride
(17d). Compound 17d (43 mg, 0.10 mmol, 30% from 12b
149 mg, 0.32 mmol); 1H NMR (400 MHz, CD3OD) d 8.27 (s, 1H, H-
4), 7.82 (d, J = 8.0, 1H, H-6), 7.38–7.07 (m, 6H, H-7, 5 ꢂ CHar),
3.98 (m, 1H, CH), 3.46–2.93 (m, 6H, 3 ꢂ CH2), 2.46 (s, 3H, CH3);
13C NMR (101 MHz, CD3OD) d 170.4 (C@O), 164.8 (C@O), 139.5
(C), 135.7 (C), 130.3 (2 ꢂ CH), 129.8 (2 ꢂ CH), 129.4 (C), 129.2 (C),
128.5 (CH), 126.4 (CH), 124.0 (CH), 117.5 (C), 112.3 (CH), 55.9
(CH), 40.2 (CH2), 40.0 (CH2), 38.6 (CH2), 9.8 (CH3); HR-ESIMS: M+
m/z calcd for C22H25N4O4 409.1876, found 409.1859.
4.2.6.2.6. (S)-1-((5-carboxy-3-methyl-1H-indole-2-carboxamido)-
buthylamino)-1-oxo-3-phenylpropan-2-aminium
chloride
(16d). Compound 16d (96 mg, 0.20 mmol, 35% from 12a
286 mg, 0.58 mmol); 1H NMR (400 MHz, CD3OD) d 8.36 (s, 1H, H-
4), 7.90 (d, J = 12.0, 1H, H-6), 7.40 (d, 1H, J = 12.0, H-7), 7.31–7.13
(m, 5H, 5 ꢂ CHar), 4.06 (m, 1H, CH), 3.37–3.09 (m, 6H, 3 ꢂ CH2),
2.56 (s, 3H, CH3), 1.52–1.63 (m, 4H, 2 ꢂ CH2); 13C NMR (101 MHz,

4354
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4.3.1. Pull-down assay
Synthetic peptide (>95% purity, Eurogentec, Seraing, Belgium)
encompassing the 10 C-terminal amino acids (ETVNEKVSCV) of
mouse 5-HT2A receptor was coupled via its N-terminal extremity
to activated CH-Sepharose 4B (GE Healthcare) according to the
manufacturer’s instructions. MALDI–TOF mass spectrometry anal-
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Immobilized peptides were stored at 4 °C in Tris–HCl (50 mM, pH
7.4) and dithiothreitol (DTT, 10 mM) to prevent cysteine oxidation
at the (ꢁ1) position in the peptide sequence. Brains of Swiss mice
(obtained from Janvier, Le Genest-St. Isle, France) were thoroughly
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30 min. The membrane pellets were resuspended in CHAPS extrac-
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samples were centrifuged for 1 h at 10,000ꢂg. Solubilized proteins
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immobilized peptide in absence or presence of increasing concen-
trations of synthesized compounds. Samples were washed twice
with 1 ml of extraction buffer and then four times with PBS. Pro-
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Proteins, resolved on 8% polyacrylamide gels, were transferred
electrophoretically onto nitrocellulose membranes (Hybond-C,
GE-Healthcare). Membranes were incubated in blocking buffer
(Tris–HCl, 50 mM, pH 7.5, 200 mM NaCl, Tween 20, 0.1% and 5%
skimmed dried milk) for 1 h at room temperature and incubated
overnight with primary antibodies (anti-PSD-95, clone C7E3,
SC32290 mouse monoclonal IgG1, Santa Cruz Biotechnology)
1:500 in blocking buffer at 4 °C. Blots were washed three times
with blocking buffer and incubated with a horseradish peroxi-
dase-conjugated anti-mouse antibody (1:2000 in blocking buffer)
for 1 h at room temperature. Immunoreactivity was detected with
an enhanced chemiluminescence method (Renaissance Plus, Perk-
inElmer Life Sciences).
Acknowledgments
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44. Maestro, version 9.1, Schrödinger, New York, NY, USA.
We would like to thank the French National Research Agency
(ANR) for funding of the project PDZ-CANPAIN and a postdoctoral
fellowship for B.B. and N.B.; the Regional Council of Auvergne
(Conseil Régional d’Auvergne) and the European Fund for Regional
Economical Development (FEDER) for a doctoral scholarship for
A.V.; and the PRES Clermont Université for a postdoctoral fellow-
ship for H.D.
References and notes
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