3-(Aminomethyl)piperazine-2,5-dione as a novel NMDA glycine site inhibitor from the chemical universe database GDB

Article abstract of DOI:10.1016/j.bmcl.2009.04.021

Docking of randomly selected compounds from the chemical universe database GDB-11, which contains all organic molecules up to 11 atoms of C, N, O, F possible under consideration of simple chemical stability and synthetic feasibility rules, into the NMDA r

Full text of DOI:10.1016/j.bmcl.2009.04.021

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3832–3835
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-(Aminomethyl)piperazine-2,5-dione as a novel NMDA glycine site inhibitor
from the chemical universe database GDB
Kong Thong Nguyen a, Erika Luethi a, Salahuddin Syed a, Stephan Urwyler a, Sonia Bertrand b,
Daniel Bertrand b, Jean-Louis Reymond a,
*
a Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, 3012 Berne, Switzerland
b Department of Neuroscience, Medical Faculty, 1, rue Michel Servet CH-1211 Geneva 4, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 February 2009
Revised 1 April 2009
Accepted 3 April 2009
Available online 10 April 2009
Docking of randomly selected compounds from the chemical universe database GDB-11, which contains
all organic molecules up to 11 atoms of C, N, O, F possible under consideration of simple chemical stabil-
ity and synthetic feasibility rules, into the NMDA receptor glycine site (1pb7.pdb) lead to the identifica-
tion of 3-(aminomethyl)piperazine-2,5-dione 3 and its close analog 5-(aminomethyl)piperazine-2,3-
dione 4 as possible new ligands for this drug target, which is implicated in synaptic plasticity, neuronal
development, learning and memory. Synthesis of these compounds in 4 and 6 steps, respectively, and
testing by radioligand displacement assays and electrophysiological measurements in Xenopus oocytes
Keywords:
Virtual screening
Glutamate receptors
Diketopiperazine
Neurochemistry
Docking
show that while 4 is inactive, 3 is indeed an inhibitor of glycine, with an estimated KD of 50 lM.
Ó 2009 Elsevier Ltd. All rights reserved.
In small molecule drug discovery one attempts to find the best
possible ligands for a given biological target.1 Ideally it should be
possible to test the ensemble of all possible organic molecules to
find such ligands. In our efforts towards this goal, we recently
assembled the chemical universe database GDB-11, which contains
26.4 million molecules up to 11 atoms of C, N, O, and F possible un-
der consideration of simple chemical stability and synthetic feasi-
bility rules.2 We have shown that active compounds can be readily
found in GDB, as in the example of dipeptides 1 and 2 identified as
inhibitors of the NMDA receptor glycine site by virtual screening,
synthesis and testing.3 This receptor is an important drug target
implicated in synaptic plasticity, neuronal development, learning
and memory for which inhibitors might have clinical application.4
The discovery of 1 and 2 provided an important proof-of-princi-
ple for using GDB in drug discovery. However the ligands were
very similar to known inhibitors of the NMDA glycine site such
Arg131 is replaced by the diketopiperazine pharmacophore. Syn-
thesis and activity testing by radioligand displacement assays
and functional testing on the NMDA receptor expressed in Xenopus
oocytes shows that 3 indeed binds to the NMDA glycine site and
inhibits glycine with an estimated KD of 50 lM. This experiment
shows for the first time that ligand discovery from GDB-11 is pos-
sible on the basis of the target protein structure only without prior
knowledge of other small molecule ligands.
O
O
H
N
H
N
OH
H2N
OH
NH2
O
O
1 (IC50 = 65 µM)
2 (IC50 = 28 µM)
as D-alanine and D D
-serine,5 -cycloserine,6 and small cyclic amino
acids,7 which was to be expected since compound selection had
been guided by structural analogy to these molecules. Herein we
report the identification of 3-(aminomethyl)piperazine-2,5-dione
3 and its close analog 5-(aminomethyl)piperazine-2,3-dione 4 by
direct virtual screening of GDB-11 using docking to the NMDA
receptor glycine site (Fig. 1). In these compounds the key carbox-
ylic group present in amino acid inhibitors and interacting with
H
H
O
N
N
O
H2N
H2N
N
H
O
N
H
O
3
4
(IC50 = 370 µM, KD = 50 µM)
(inactive)
Figure 1. NMDA receptor glycine site virtual hits 1–2 (Ref. 3) and 3–4 (this work)
identified by virtual screening of GDB-11. 1, 2 and 3 bind and inhibit the receptor at
the glycine site, 4 is not active.
* Corresponding author. Fax: +41 31 631 80 57.
E-mail address: jean-louis.reymond@ioc.unibe.ch (J.-L. Reymond).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.021

K. T. Nguyen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3832–3835
3833
inactive compounds, and are computationally very fast and there-
fore applicable to screen large databases.11 141 of the top docking
hits were selected as actives to train the classifier, considering
structures that would be relatively straightforward to prepare, in
particular amides, while complex combinations of polyenes, ami-
dines and hydrazones were rejected. All compounds docking weak-
er than glycine were used as inactives. The classifier returned
35,810 additional compounds from GDB scoring higher than the
lowest Bayesian score for the active set, from which 182 com-
pounds docked stronger than glycine.
A
B
4000
2000
0
glycine
C
Analysis of functional group enrichment showed that docking
did not select for monoamines, but strongly enriched amino acids
and amides, which is similar to our previously reported study3
(Table 1). Amides were very frequent in the final hit set because
we chose this functional group for ease of synthesis in the selection
of top-docking compounds from the first round of docking. Dock-
ing also enriched hydrogen bonding donor (HBD) and acceptor
sites (HBA). The random GDB selection used in this study had few-
er HDB (2.08) than the compounds previously selected on the basis
of known actives (5.45), but significantly more HBA (3.17 vs 2.13).
The higher HBA in the GDB random set compared to the Bayesian
hit sets might explain its better mean docking energy reflecting
high-scoring interactions with donor sites on the proteins.
As in our previous study3 virtual screening favoured acyclic
compounds, with many virtual hits showing structures reminis-
cent of 1 and 2. On the other hand the hit set also contained a
few six-membered ring heterocycles, in particular the intriguing
diketopiperazine ligands (S)-3 and (S)-4 at rank #58 and #20,
and docking energies of À8.36 kcal/mol and À8.81 kcal/mol,
respectively. In their best docking pose, the key carboxylate phar-
macophore of known amino acid inhibitors was replaced by the
diketopiperazine group, and the primary amino group, although
placed quite differently from that of glycine, interacted with anio-
nic residues similarly to the amino group of dipeptides 1 and 2
(Fig. 3). Since 3 and 4 had never been reported previously, we set
out to synthesize and test these ligands against the NMDA glycine
site.
-10
-5
0
5
10
kcal/mol
Figure 2. Distribution of docking energies of (A, red line) the 31,121 stereoisomers
generated from 8000 randomly selected GDB structures; (B, blue line) the 81,877
stereoisomers generated from the 35,810 virtual hits from the Bayesian classifier
trained with the docking results of A; (C, grey line) the 69,367 stereoiosmers
generated from the 15,061 virtual hits from the Bayesian classifier trained with
known NMDA-receptor inhibitors, as described in Ref. 3. The black vertical line
indicates the DE of glycine (À7.82 kcal/mol). Stereoisomers were generated from
SMILES using CORINA and docked into the glycine binding site of the NMDA-
receptor (pdb: 1PB7)20 using AUTODOCK3.0.5 for 10 cycles. In each case the energy of
the most favourable pose was used for scoring.
In our search for NMDA glycine site inhibitors from GDB-11, we
hypothesized that innovative ligands might be found if virtual
screening would be guided from the protein structure only without
using information from existing ligands. With docking as our main
tool,8 we first screened a randomly selected subset of 8000 GDB
structures. The structures were expanded to 31,121 stereoisomers
using CORINA9 and evaluated by AUTODOCK3.0.5,10 resulting in a typ-
ical gaussian curve for the distribution of docking energies (Fig. 2).
There were 702 molecules docking better than glycine, among
which amino acids were significantly enriched, showing the natu-
ral tendency of the binding pocket to select for that class of
compounds.
Diketopiperazine 3 was prepared as the (S) enantiomer in
four steps and 33% overall yield (Scheme 1). Peptide coupling
between CBz-protected L-asparagine (5) and glycine methyl ester
gave dipeptide 6. Oxidative Hofmann degradation12 of the aspar-
agine carboxamide side-chain using I,I-bis(trifluoroacetoxy)iodo-
benzene13 and protection of the primary amine gave the Boc-
derivative 7.14 Hydrogenation of the CBz group of the terminal
A Bayesian classifier was applied next to extract further ligands
from GDB. Bayesian classifiers determine a bioactivity probability
score for any compound from the product of the relative frequency
of occurrence of all its substructures in known active versus
a-amino group and intramolecular cyclization under mild basic
Table 1
Enrichment statistics during virtual screening
Library
GDB-11 Random set
1st Rounda top docking
Selectedb
Bayesian hitsc
2nd rounda top docking
Ref. 3 Bayesian hitsd
Ref. 3 top dockinga
Size
8000
25.8
702
141
35,810
33.8
0.42
23.3
2.29
2.35
55.9
182
24.7
18.1 (150)
41.8
2.82
15,061
21.7
7.3 (1100)
3.6
5.45
2.13
712
20.8
22.9 (163)
10.3
5.25
Monoaminese
%
28.4
1.00 (7)
16.0
3.32
3.38
71.8
42.6
2.1 (3)
24.8
1.59
2.84
55.3
Aminoacidsf
%
0.36 (29)e
9.2
Amidesg
%
HBD averageh
HBA averagei
Acyclics %
2.08
3.17
27.5
4.94
91.2
2.63
86.2
28.3
a
Molecules of the random set (1st round) or from the Bayesian hits in this work (2nd round) or from Ref. 3 docking stronger than the crystallographic ligand glycine
(DE = À7.83 kcal/mol) in the NMDA glycine site binding pocket as estimated by AUTODOCK 3.0.5.
b
Subset of 1st round top docking with simple functional groups.
All compounds from GDB-11 scoring better than the worst of the selected compounds in a Bayesian classifier trained with the 141 selected compounds as actives and all
c
structures with DE >À7.82 kcal/mol as inactives.
d
Bayesian hits from Ref. 3, in this case the classifier was trained with known actives and an ACX random set as inactives.
Monoamines are molecules without carboxylic groups and exactly one nitrogen atom with only H or saturated C-atoms neighbours.
Amino acids are monoamines with exactly one carboxylic acid. The absolute number of molecules is indicated in parentheses.
Amides are molecules with at least one amide function.
HBD is the hydrogen bond donor site count, that is, the total number of NH and OH bonds.
HBA is the hydrogen bond acceptor site count, that is, the total number of lone pairs.
e
f
g
h
i

3834
K. T. Nguyen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3832–3835
Binding assays for the NMDA glycine site by radioactive ligand
displacement as described previously3 showed weak but detectable
binding (30% inhibition at 100 M) by the diketopiperazine 3, while
l
compound 4 was inactive. Compound 3 was further investigated by
functional assays on the NMDA receptor expressed in Xenopus oo-
cytes. The electrophysiological measurements showed that 3 acts
as an inhibitor of glycine (IC50 = 370
to approximately KD = 50
lM, Fig. 4), which corresponds
l
M when taking binding of the competing
ligand glycine into account.17,18 Considering the small size of ligand
3 (MW = 143 Dal), this inhibition corresponds to a ligand efficiency
of 0.58 kcal/mol.19
In summary, direct virtual screening of the chemical universe
database GDB for ligands of the NMDA receptor glycine site using
docking without information from existing ligands lead to the
identification of diketopiperazine 3 and 4 as possible new ligands.
Synthesis of the ligands and bioactivity assays showed that com-
pound 3 binds to the NMDA glycine site with high micromolar
affinity and acts as an inhibitor of glycine in electrophysiological
measurements. Although the docking scores and bioactivities ob-
tained are lower than those previously obtained from a GDB-subset
enriched by substructure similarity to known actives, the present
experiment shows for the first time that a ‘naive’ approach can
help to uncover new ligand types in GDB. The combination of vir-
tual screening with synthesis might provide a general strategy for
ligand discovery for a variety of targets for which structural infor-
mation is available.
Figure 3. Binding modes within the NMDA-glycine site (1PB7.pdb). Glycine
(green); compound (S)-3 (pink, À8.36 kcal/mol); compound (S)-4 (brown,
À8.81 kcal/mol). Residue numbering according to 1PB7.pdb. Arg-131 corresponds
to R-523 in Ref. 20. Note that the enantiomeric structure dock with significantly
higher energies (R)-3: À6.47 kcal/mol. (R)-4: À6.63 kcal/mol.
conditions15 lead to diketopiperazine 8. Finally, Boc removal by
treatment with pure trifluoroacetic acid gave 3 (96% ee from
Mosher’s amide) as the TFA salt. Diketopiperazine 4 was pre-
pared as the racemate in six steps and 17% overall yield. Boc
protection of 2-amino-1,3-propanediol (9) to 10, mesylation to
11, and azide displacement gave 12 following a published proce-
dure.16 Boc-deprotection of 12 followed by amide bond forma-
tion with ethyl chlorooxoacetate gave the key intermediate 13.
Hydrogenation of the azido groups using H2 and Pd/C was fol-
lowed by an in situ intramolecular cyclization, providing the de-
sired diketopiperazine 4 as the free amine.
O
O
NHCBz
b
NHCBz
NHBoc
X
MeO2C
N
H
O
NH2
7
5 (X = OH)
a
c
6 (X = NHCH2CO2Me)
O
HN
NH
NHR
O
NHR
OX
XO
R = X =
8 (R = Boc)
3 (R = H2+.TFA-)
d
9
H
H
H
e
f
10 Boc
11 Boc Ms
g
O
O
HN
NHR
N3
i
NH
N3
NH2
12 (R = Boc)
h
4
13 (R = CO-CO2Et)
Scheme 1. Synthesis of 3 and 4. (a) EDC, HOBt, NMM, DMF/CH2Cl2 (1:1), rt, 8 h
(98%); (b) (i) I,I-bis(trifluoroacetoxy)iodobenzene, pyridine, DMF/H2O, rt, 5 h; (ii)
Boc2O, Et3N, MeOH, rt, 8 h (64%); (c) (i) H2, Pd/C, MeOH, rt, 90 min; (ii) 5% aq
NaHCO3, rt, 20 h (52%); (d) CF3CO2H, 0 °C, 1 h (quant); (e) Boc2O, EtOH, rt, 3 h (91%);
(f) MsCl, Et3N, CH2Cl2, 0 °C—rt, 1.5 h (87%); (g) NaN3, DMF, 70 °C, 12 h (97%); (h) 1 N
HCl, rt, 8 h; then ethyl chlorooxoacetate, Et3N, CH2Cl2, 0 °C—rt, 12 h (67%); (i) H2,
Pd/C, MeOH, rt, 36 h (34%).
Figure 4. Inhibition of the electrophysiological responses of the NMDA receptor
NR1/NR2A in Xenopus oocyte membranes by diketopiperazine (S)-3. Glu: 10
Gly: 1 M. All experiments were carried out with rodent NMDA receptors as
described previously.3
lM,
l

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This work was supported financially by the University of Berne
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