Identification of selective norbornane-type aspartate analogue inhibitors of the glutamate transporter 1 (GLT-1) from the chemical universe generated database (GDB)

Article abstract of DOI:10.1021/jm100959g

A variety of conformationally constrained aspartate and glutamate analogues inhibit the glutamate transporter 1 (GLT-1, also known as EAAT2). To expand the search for such analogues, a virtual library of aliphatic aspartate and glutamate analogues was generated starting from the chemical universe database GDB-11, which contains 26.4 million possible molecules up to 11 atoms of C, N, O, F, resulting in 101026 aspartate analogues and 151285 glutamate analogues. Virtual screening was realized by high-throughput docking to the glutamate binding site of the glutamate transporter homologue from Pyrococcus horikoshii (PDB code: 1XFH) using Autodock. Norbornane-type aspartate analogues were selected from the top-scoring virtual hits and synthesized. Testing and optimization led to the identification of (1R*,2R*,3S*, 4R*,6R*)-2-amino-6-phenethyl-bicyclo[2.2.1]heptane-2,3-dicarboxylic acid as a new inhibitor of GLT-1 with IC₅₀ = 1.4 μM against GLT-1 and no inhibition of the related transporter EAAC1. The systematic diversification of known ligands by enumeration with help of GDB followed by virtual screening, synthesis, and testing as exemplified here provides a general strategy for drug discovery.

Full text of DOI:10.1021/jm100959g

7236 J. Med. Chem. 2010, 53, 7236–7250
DOI: 10.1021/jm100959g
Identification of Selective Norbornane-Type Aspartate Analogue Inhibitors of the Glutamate
Transporter 1 (GLT-1) from the Chemical Universe Generated Database (GDB)
†
†
‡
†
‡
Erika Luethi, Kong T. Nguyen, Marc Burzle, Lorenz C. Blum, Yoshiro Suzuki, Matthias Hediger,‡,§ and
€
Jean-Louis Reymond*,†,§
†Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, 3012 Berne, Switzerland,
‡Institute of Biochemistry and Molecular Medicine, University of Berne, Bu€hlstrasse 28, 3012 Berne, Switzerland, and
§Swiss National Center of Competence in Research, NCCR-TransCure, University of Berne, Switzerland
Received July 28, 2010
A variety of conformationally constrained aspartate and glutamate analogues inhibit the glutamate
transporter 1 (GLT-1, also known as EAAT2). To expand the search for such analogues, a virtual
library of aliphatic aspartate and glutamate analogues was generated starting from the chemical
universe database GDB-11, which contains 26.4 million possible molecules up to 11 atoms of C, N, O, F,
resulting in 101026 aspartate analogues and 151285 glutamate analogues. Virtual screening was realized
by high-throughput docking to the glutamate binding site of the glutamate transporter homologue from
Pyrococcus horikoshii (PDB code: 1XFH) using Autodock. Norbornane-type aspartate analogues were
selected from the top-scoring virtual hits and synthesized. Testing and optimization led to the
identification of (1R*,2R*,3S*,4R*,6R*)-2-amino-6-phenethyl-bicyclo[2.2.1]heptane-2,3-dicarboxylic
acid as a new inhibitor of GLT-1 with IC50 = 1.4 μM against GLT-1 and no inhibition of the related
transporter EAAC1. The systematic diversification of known ligands by enumeration with help of GDB
followed by virtual screening, synthesis, and testing as exemplified here provides a general strategy for
drug discovery.
Introduction
subtypes, remove glutamate from the synaptic cleft to termi-
nate signal transmission and to prevent neurotoxic effects.10,11
Dysfunction of glutamate transporters has been identified in a
number of neurodegenerative diseases (e.g., Alzheimer’s dis-
ease, amyotrophic lateral sclerosis), ischemic stroke injury, or
epilepsy, whichmakes them interesting targets for biologyand
medicine.11-13 There is a need for more, especially subtype-
selective ligands of glutamate transporters, which might serve
for further elucidation of their physiological roles and even-
tually as therapeutics.11,14,15 Various medicinal chemistry
programs have identified analogues of aspartate and gluta-
mate as favorable structures for binding the glutamate trans-
porter 1 (GLT-1), such as 1 (trans-2,3-PDC),16 2 (3-Me-L-2,3-
PDC),17 3 (L-CCG-III),18 4 (L-CBG-IV),19 5 (L-3,4-MPDC),20
6 (azabicyclo-ODD),21 and 7 (WAY-855)22 (Figure 1).
Herein we report the identification of inhibitors of GLT-1
by virtual screening of a library of aspartate and glutamate
analogues derived from GDB-11, followed by synthesis, test-
ing, and optimization of selected hits. The study led to a new
class of norbornane-type aspartate analogues, including in
particular rac-23a and its optimized derivative rac-25a, which
exhibit enhanced selectivity between GLT-1 and the excita-
tory amino acid carrier 1 (EAAC1) compared to previously
known ligands.
Small molecule drug discovery forms an essential part of
modern medicine.1 New discoveries are however increasingly
difficult, in part because a very large number of small mole-
cules have already been described in the scientific and patent
literature. Recently, we reported the chemical universe gener-
ated database up to 11 atoms (GDB-11a) and up to 13 atoms
(GDB-13), enumerating all organic molecules virtually possi-
ble under simple chemical stability and synthetic feasibility
rules.2-4 The databases contain at least 1000-fold more struc-
tures than data sets of known molecules of similar size and
thus provide a very large reservoir of yet unexploited chemical
diversity for drug discovery in the context of de novo drug
design approaches.5,6
In our effort to demonstrate the use of GDB for identifying
new drugs,7-9 we became interested in the case of aspartate
and glutamate analogues as ligands of various targets in the
central nervous system (CNS). Glutamate is the major ex-
citatory neurotransmitter in the mammalian CNS and is
linked to higher brain functions such as memory and learning.
Concomitantly, glutamate acts as an excitotoxin at higher
extracellular concentrations triggering neuronal cell death.
One family of proteins that glutamate and aspartate analo-
gues act on are Na-dependent high-affinity glutamate trans-
porters. These transporters, which exist in five different
Results and Discussion
Virtual Screening. We set out to explore the chemical space
of glutamate and aspartate analogues for new ligands by
exhaustive enumeration from the chemical universe database
GDB, followed by high-throughput virtual screening, synthesis,
*To whom correspondence should be addressed. Phone: þ41 31 631
43 25. Fax: þ41 31 631 80 57. E-mail: jean-louis.reymond@ioc.unibe.ch.
a Abbreviations: GDB: generated database; GLT-1: glutamate trans-
porter 1; EAAC1: excitatory amino acid carrier 1.
r
pubs.acs.org/jmc
Published on Web 09/02/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7237
Figure 3. Distribution of estimated binding energies of glutamate
and aspartate analogues. Docking studies were performed with
Autodock on the crystal structure of the glutamate transporter
homologue from Pyrococcus horikoshii 1XFH.
R-amino acids with a β-carboxyl group, corresponding to
225266 structures. Similarly, the glutamate analogues were
obtained by considering all R-amino acids with a γ-carboxyl
group, resulting in 334531 structures. These virtual libraries
were further reduced to conformationally constrained structures
(rotatable bond count Rbc e3), leaving 101026 aspartate ana-
logues and 151285 glutamate analogues (Figure 2, Supporting
Information Table S1, Figure S1).
Figure 1. Native ligands L-glutamate and L-aspartate23 and ligands
of glutamate transporters. The inhibitory activities at GLT-1 are
indicated.
Virtual screening of the analogue libraries was realized by
high-throughput docking to the glutamate binding site of the
glutamate transporter homologue from Pyrococcus hori-
koshii, for which a crystal structure has recently been re-
ported with an electron density compatible with bound
glutamate at the active site (1XFH).25 This bacterial homo-
logue shares 37% amino acid sequence identity with the
human transporter GLT-1. The libraries were expanded to
3D using CORINA,26 resulting in 810702 (aspartates) and
1253354 (glutamates) stereoisomers, which were docked
using Autodock 3.0.5.27 This docking program is suitable
for proper positioning of ligands as well as for delivering good
estimates of binding energies for various ligands.28 Docking
positioned the natural ligand glutamate in a pose compatible
with the crystallographically observed electron density, and with
good estimated binding free energy score (BE = -9.10 kcal/
mol). For each molecule docked, the lowest estimated binding
energy for 15 docking attempts was taken as the reference value.
Gaussian curves were observed for the distribution of binding
energies (Figure 3). The known GLT-1 ligands 1-7 (Figure 1)
had estimated binding energies in the upper 6% of all docked
stereoisomers, with 4 and 7 being even among the top 0.54% of
all stereoisomers.
The best scoring ligands of each library were inspected
visually to select compounds for synthesis. The top-scoring
glutamate analogues were tri- and tetracyclic structures similar
to 7 which were judged too challenging for synthesis. The
aspartate library by contrast contained many bicyclic struc-
tures clearly amenable to chemical synthesis. We focused our
attention on norbornane-type aspartate analogues, including
the best docking and yet unknown aspartate analogue 8 (rank
1, BE = -13.34 kcal/mol) and the parent norbornane lacking
the vinyl appendage 9d (rank 19, BE = -12.52 kcal/mol), for
Figure 2. Construction of aspartate and glutamate libraries from
the chemical universe database GDB. See Supporting Information
Table S1 and Figure S1 for details on the database composition.
and testing. The enumeration was realized from GDB-11,24
which contains 26.4 million possible molecules up to 11 atoms of
C, N, O, and F. Two virtual libraries of aspartate and glutamate
analogues were constructed from GDB-11 as shown in Figure 2.
Thus, the subset of 1.3 million primary and secondary mono-
amines (CxHyNHz, x e 10, z = 1 or 2, excluding aziridines) was
extracted and expanded by replacing all possible pairs of
nongeminal hydrogens on carbons by a pair of carboxyl groups,
resulting in a library of 13.7 million aminodicarboxylic acids.
The aspartate analogues were obtained by considering only

7238 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Luethi et al.
Scheme 1. Structure of 8 and 9d and Synthesis of All Diaster-
eoisomers rac-9a-da
Figure 4. Structure of selected virtual hits. The binding energy
estimated by Autodock to the glutamate binding site in GLT-1 is
indicated in kcal/mol.
which only two of the eight stereoisomers had been described
previously29,30 (Figure 4). The simplified monocyclic analo-
gues 10 (rank 5176, BE = -10.42 kcal/mol) and 11 (rank 4888,
BE = -10.46 kcal/mol) were also considered because their
synthesis has already been described31-33 in connection with
biological assays for vesicular glutamate transporters,34
tumor-growth inhibition (cyclopentane derivative),32,35 herpes
simplex virus ribonucleotide reductase inhibition,33 and up-
take in tumor cells (cyclohexane derivative).36 Their activity on
EAATs had not been determined but 1S,2R-amino-cyclopen-
tanedicarboxylic acid is an agonist of metabotropic glutamate
receptors.37
Synthesis. The norbornane aspartate analogue 9 was pre-
pared starting with the Diels-Alder reaction of cyclopenta-
diene with diethyl acetylene dicarboxylate to provide 12.38
The crude norbornadiene 12 was subjected to Michael addi-
tion of ammonia by heating in a saturated ethanolic solution
of ammonium acetate to give 13 as a mixture of diastereoi-
somers, which were separated by column chromatography as
the benzyloxycarbonyl (Z) protected derivatives 14a-d
(Scheme 1). The relative stereochemistry was assigned by
2D-NMR spectroscopy (COSY, ROESY). An NOE-effect
between Hb and Hf indicated the exo-position of Hb in rac-
14b and rac-14d. On the other hand, the endo-position of Hb
in rac-14a and rac-14c was indicated by a strong W-coupling
between Hb and Hg in COSY-NMR and no NOE-effect
a Conditions: (a) NH4OAc, EtOH, 80 °C, 18 h, then (b) BnOCOCl,
Na2CO3, CH2Cl2/H2O (1/1), rt, 12 h (43% over 3 steps from cyclopenta-
diene and diethyl acetylene dicarboxylate; 14a, 10%; 14b, 4%; 14c, 10%;
14d, 19%); (c) Na, EtOH, rt, 40 h; (d) LiOH, THF/MeOH/H2O, 70 °C,
24 h, then separation/purification by RP-HPLC (rac-15a, 47%; rac-15b,
64%); (e) H2, Pd/C, MeOH, rt, 90 min (rac-9a, 89%; rac-9b, 84%);
(f) H2, Pd/C, MeOH, rt, 90 min, then 3 N HCl, reflux, 30 h (88%); (g) H2,
Pd/C, MeOH, rt, 90 min, then 6 N HCl, reflux, 3 days, then BnOCOCl,
NaOH, H2O, 0 °C f rt, 5 h, then separation/purification by RP-HPLC
(3%); (h) H2, Pd/C, MeOH, rt, 90 min (quant). All compounds were
prepared as racemates.
The assignment of the relative configuration of the amino
acid in this series was made possible by the crystal structure
determination of rac-16d (Scheme 1).
3
between Hb and Hf. Additionally, the J(Hb, Hc) coupling
constants of the rac-14b and rac-14d diastereoisomers dis-
playing Hb in the exo-position (both 3.21 Hz) were larger
than that of the Hb-endo diastereoisomers rac-14a (1.5 Hz)
and rac-14c (1.95 Hz), consistent with the Karplus-relation
Because of the strong tendency of the cis-dicarboxylate
diastereoisomers to epimerize to the trans-dicarboxylate
under a variety of conditions, the synthesis of 8 was envi-
sioned only for the case of the more stable trans-dicarbox-
ylate diastereoisomers (Scheme 2). The synthesis started
from rac-14d and rac-14b, which were most easily obtained
in pure form as the first and last eluting fraction during the
chromatographic separation. Hydroformylation under 40
bar of syngas and unmodified Rh(CO)2acac as catalyst at 60 °C
in toluene39 gave exclusively the exo-formylated products
rac-18a,b and rac-18c,d, a selectivity well documented for
similar cases.39,40 These aldehydes proved rather unstable
and formed insoluble precipitates when kept in solution at
room temperature or upon storage in pure form at -18 °C,
presumably through the formation of cyclic trioxanes,31
although their formation could not be confirmed. All at-
tempts to epimerize the aldehyde to the corresponding endo-
diastereoisomer in any of rac-18a-d under basic conditions
resulted in slow decomposition.
3
predicting that J(Hb, Hc) with Hb in exo-position is larger
3
than J(Hb, Hc) with Hb in the endo-position because the
dihedral angle for the latter is closer to 90°.
Partial epimerization of the ester function of rac-14 was
observed under basic conditions and allowed the pairwise
assignment of diastereoisomers possessing the same relative
configuration at the amino acid R-carbon atom as rac-14a/
rac-14d and rac-14b/rac-14c. Complete epimerization was
observed upon hydrolysis of the diastereoisomeric mixture
rac-14 with LiOH in THF/MeOH/water at 70 °C for 24 h to
afford the Z-protected trans-dicarboxylates rac-15a and rac-
15b. The products were separated by preparative RP-HPLC
and subjected to hydrogenolysis to provide the free amino
acids rac-9a and rac-9b. Hydrogenation of the cis-dicar-
boxylic ester rac-14c followed by acidic hydrolysis in 3 N
HCl under reflux gave the cis-dicarboxylic amino acid rac-9c
in good yields without any epimerization. The same sequence
applied to rac-14d however resulted in a mixture of cis-
and trans-dicarboxylates. Therefore rac-14d was reprotected
to the benzyl carbamate rac-16d, separated from its trans-
dicarboxylate diastereoisomer rac-16a by preparative RP-
HPLC, and subjected to hydrogenation to yield rac-9d.
Wittig olefination of rac-18a-d with triphenyl methylene
phosphorane gave the corresponding vinyl products rac-
19a-d. Ester hydrolysis, accompanied by epimerization to
the trans-dicarboxylate in the case of rac-19a,b, provided the
Z-protected diacids rac-21a-d, which were finally depro-
tected by acidic hydrolysis to yield amino acids rac-23a-d

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7239
Scheme 3. Synthesis of Additional Analogues of rac-23aa
Scheme 2. Synthesis of rac-23a-d (Diastereoisomers of 8 with
exo-Vinyl Substituent) and Analogues, and Structure of the
Known GLT-1 Inhibitors 17a-ba
a Conditions: (a) EtPPh3Br (rac-26a), nPrPPh3Br (rac-26b), (2-hydro-
xybenzyl)triphenylphosphonium bromide (rac-26c) or (4-chlorobenzyl)-
triphenylphosphonium chloride (rac-26d), nBuLi, THF, -18 °C, 15 min
(rac-26a, 88%; rac-26c, 76%; rac-26d, 51%); (b) LiOH, THF/MeOH/
H2O, 70 °C, 48 h (rac-27a, 46%; rac-27b, 39% over 2 steps; rac-27d,
43%); (c) H2, Pd/C, MeOH, rt, 90 min, (rac-28a, 96%; rac-28b, quant;
rac-28c, 27% over 2 steps) or H2, Pt/C, EtOH, rt, 4 h, then AcOH/HCl
(2/1), 80 °C, 90 min (rac-28d, 67%).
Scheme 4. Synthesis of Monocyclic Aspartate Analogues 10
and 11a
a Conditions: (a) CO/H2 (1/1), Rh(CO)2acac, toluene, 60 °C, 24 h
(rac-18a, 35%; rac-18b, 30%; rac-18c, 30%; rac-18d, 50%); (b)
MePPh3Br, nBuLi, THF, -18 °C, 70-90 min (rac-19a, 72%; rac-19b,
64%; rac-19c, 43%; rac-19d, 65%); (c) BnPPh3Cl, nBuLi, THF, -18 °C,
60-90 min (rac-20a, quant; rac-20b, 70%; rac-20c, 52%; rac-20d, 72%);
(d) LiOH, THF/MeOH/H2O, 70 °C, 48 h (rac-21a, 59%; rac-21b, 41%;
rac-21c, 67%; rac-21d, 64%; rac-22a, 43%; rac-22b, 45%; rac-22c, 68%;
rac-22d, 57%); (e) AcOH/conc HCl (4/1), 80 °C, 2 h (rac-23a, 60%; rac-
23b, 65%; rac-23c, 56%; rac-23d, 62%); (f) H2, Pd/C, MeOH, rt, 90 min
(rac-24a-d, quant; rac-25a, 44%; rac-25b, 75%; rac-25c, 46%; rac-25d,
quant).
after preparative RP-HPLC purification. Alternatively, hy-
drogenation of rac-21a-d provided the corresponding satu-
rated ethyl analogues rac-24a-d. With the aim of probing
substitution of the vinyl group for activity improvements in
analogy to the known benzyl-sustituted GLT-1 inhibitor 17a
(L-TBOA),41 olefination of rac-18a-d with benzylidene
triphenyl phosphorane yielded rac-20a-d as E/Z mixtures
and finally the phenethyl substituted norbornanes rac-
25a-d upon hydrogenation. The assignment of the position
and exo-configuration of the aldehyde group and its deriva-
tives in the different products was obtained from COSY-
NMR data and confirmed by the X-ray crystal structure of
rac-21b.
In view of the higher GLT-1 inhibitory activity of rac-9a,
rac-23a, and in particular rac-25a, indicating favorable
effects of added substituents on inhibition in these diaster-
eoisomers (see below), additional derivatives were prepared
for this diastereoisomer only (Scheme 3). Thus Wittig olefi-
nation of rac-18a with triphenyl (ethylidene)phosphorane,
a Conditions: (a) (i) KCN, (NH4)2CO3, EtOH/H2O (1/1), 60 °C, 28 h,
(ii) 4 N HCl, 40 °C, 24 h; (b) 130 °C, 5 h, sealed tube (19% overall, dr: 58/
42); (c) (i) (NH4)2CO3, KCN, EtOH/H2O (1/1), 50 °C, 8 h, (ii) 4 N HCl,
50 °C, 2 d; (d) 6 N HCl, reflux, 7 days (31% overall, dr: 81/19).
triphenyl (propylidene)phosphorane, triphenyl (2-hydroxy-
benzylidene)phosphorane, and triphenyl (4-chlorobenzyli-
dene)phosphorane gave intermediates rac-26a-d as E/Z
mixtures, respectively. Ester hydrolysis under basic condi-
tions accompanied by epimerization to the trans-dicarbox-
ylates then gave rac-27a-d, which were finally subjected to
hydrogenation to yield the free amino acids rac-28a-d. The
structures were confirmed by the crystal structure determi-
nation of rac-E-26c.
The monocyclic aspartate analogues 10 and 11 were
prepared as mixtures of diastereoisomers by a simple two-
step procedure (Scheme 4). Bucherer-Bergs reaction of
2-oxocyclopentane carboxylic acid ethyl ester, followed by
acidic ester hydrolysis in 4 N HCl at 40 °C, gave hydantoin 29

7240 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Luethi et al.
Table 1. IC50 Values for Inhibition of [3H]-Glutamate Uptake by GLT-1
and EAAC1 Expressed in Xenopus laevis Oocytes
IC50 (GLT-1) (μM)
IC50 (EAAC1) (μM)
rac-23a (vinyl)
rac-25a (phenethyl)
rac-25d (phenethyl)
rac-28a (propyl)
rac-28b (butyl)
rac-28c (o-HOPh)
rac-28d (p-ClPh)
722
130 ( 70
1.4 ( 0.7
19 ( 5
25 ( 3
14 ( 8
21 ( 11
17 ( 11
1.3
no inhibition
no inhibition
no inhibition
no inhibition
nd
no inhibition
no inhibition
53
17a41
3.8
0.017
7.0
0.3
17b43
Figure 5. Inhibition of [3H]-glutamate uptake in GLT-1-trans-
fected HEK293 cells measured at 500 μM ligand concentration.
GLT-1-HEK293 cells were incubated for 10 min at room tempera-
ture in HBSS buffer containing 40 nM [3H]-glutamate and 10 μM
cold glutamate. % indicates the inhibition of glutamate uptake
compared to control uptakes without compounds.
as a nonseparable mixture of diastereoisomers, which was
treated with 6 N HCl at 130 °C for 5 h and purified by anion-
exchange chromatography to yield 11. Similarly, the Bucherer-
Bergs reaction with 2-oxocyclohexane carboxylic acid ethyl
ester and acidic hydrolysis provided hydantoin 30, which was
further processed to amino acid 10.
Inhibition of Glutamate Transporters. The inhibitory po-
tential of the aspartate analogues rac-9a-d, rac-23a-d, rac-
24a-d, rac-25a-d, 10, and 11 was first tested at 500 μM
ligand concentration for inhibition of [3H]-glutamate uptake
in GLT-1-transfected HEK293 cells or oocytes (Figure 5).42
The uptake of [3H]-glutamate was measured in presence and
absence of ligand and the activities are given in percent
inhibition, corresponding to the percentage of glutamate
blocked from being transported into the cell compared to
the control measurement without ligand. In the case of the
unsubstituted norbornane aspartates rac-9a-d, significant
inhibition was observed with rac-9a (exo-amino trans-
dicarboxylate) and rac-9c (endo-amino cis-dicarboxylate).
The higher epimerization tendency of cis-dicarboxylate dia-
stereoisomers and the higher activity of the exo-amino trans-
dicarboxylate rac-9a led to the choice to investigate further
analogues in the latter case only, which also seemed reason-
able considering the trans-stereochemistry of other known
aspartate analogue inhibitors of GLT-1 (Figure 1). The most
active stereoisomer in the vinyl-substituted norbornanes rac-
23a-d was again the exo-amino trans-dicarboxylate, ligand
rac-23a, which carried the vinyl substituent meta to the
amino acid function similarly to the second most active
ligand in this series rac-23c. A similar activity pattern was
observed in the benzyl appended analogues rac-25a-d,
which showed 93% inhibition as an equimolar mixture of
diastereoisomers. The rac-25a diastereoisomer showed es-
sentially complete inhibition of glutamate uptake. The ethyl
substituted series rac-24a-d behaved differently, with rac-
24d showing the strongest activity in this assay. In the
monocyclic analogues, only the cyclohexane compound 10
showed significant inhibition, while the cyclopentane com-
pound 11 was inactive.
Figure 6. Estimated binding energy by Autodock as a function of
the calculated solvent accessible surface area (sasa). Orange dots: all
best-docking stereoisomers from the libraries of virtual aspartate
respectively glutamate analogues. Blue line: linear fits for the orange
dots, for aspartates: y = -19.86 þ 0.045x, r2 = 0.24, for glutamates:
y = -19.25 þ 0.041x, r2 = 0.26. Blue dots: average estimated binding
energy as a function of sasa. Red dots: values for selected ligands
(structures in Figure 1 and 4, ent-9a is the enantiomer of 9a, ent-23a
is the enantiomer of 23a).
various exo-alkyl substituents (Scheme 3) and the known
GLT-1 inhibitor 17a (Scheme 2) as a positive control. The
values were measured for inhibition of [3H]-glutamate up-
take by GLT-1 and EAAC1 expressed in Xenopus laevis
oocytes. The results confirmed the initial activity assay with
the exception of the vinyl substituted derivative rac-23a,
which had much weaker activity. The compounds showed
inhibition in the micromolar range, with the phenethyl
derivative rac-25a confirming its strong activity with an
IC50 = 1.4 μM (Table 1). This value is comparable to the
values reported for the polycyclic amino acid 7 and for 17a,
which contains a phenyl group like rac-25a. Interestingly, all
of the norbornane inhibitors showed complete selectivity
toward GLT-1 and no measurable inhibition of EAAC1,
while the positive control 17a inhibited both GLT-1 and
EAAC1. It should be noted that the 17a analogue 17b (TFB-
TBOA),43 in which the position 3 of the benzyl group bears a
p-(trifluoromethyl)benzamido group, is 100-fold more po-
tent than 17a but also significantly cross-reacts with EAAC1.
Molecular Modeling. The libraries of virtual aspartate and
glutamate analogues derived from GDB provided an essen-
tial source of molecular diversity from which the norbornane
aspartic acid skeleton was suggested by docking as a possible
A detailed characterization was carried out by determina-
tion of the IC50 values for the three ligands showing over
90% inhibition at 500 μM, rac-23a, rac-25a, and rac-25d.
The study was extended to analogues rac-28a-d bearing

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7241
Figure 7. Lowest energy poses predicted by Autodock in the glutamate binding pocket of 1XFH (left) and the corresponding aspartate binding
pocket of 2NWL (right) shown from the same viewpoint for all compounds from Figure 1 and all diastereoisomers of the norbornane aspartate
9. Aspartate is shown in green. The R-amino acid carboxyl group is at upper left.
lead structure. Docking was used for ranking because the
scores consistently placed the known aspartate and gluta-
mate analogue inhibitors among the top-scoring structures.
However, in the case of 9 where all diastereisomers were
tested, the experimentally tested inhibitory activity on GLT-
1 was not found in the virtual hit itself (rac-9d) but rather in
its diastereoisomers rac-9a and rac-9c. A reanalysis of the
docking results was therefore carried out to better under-
stand the role played by docking in the compound selection
process, including a comparison of docking results with the
more recently reported crystal structures of the glutamate
transporter homologue from Pyrococcus horikoshii 2NWL
and 2NWW,44 which contain aspartate, respectively, the
inhibitor 17a at the glutamate binding site.
One striking feature of the glutamate binding site in both
1XFH and 2NWL is that the binding pocket is quite small.
While there was no significant correlation with the molecular
weight or the number of cycles in the molecules, the docking
scores of the virtual aspartate and glutamate analogue library
in 1XFH were strongly influenced by the solvent accessible
surface area (sasa), which measures the overall compactness of
the structure (Figure 6). This measure was also consistent with
the high ranking obtained for the known reference ligands. This
suggests that the docking scores guided compound selection
mainly toward structurally compact analogues.
Analysis of the docking poses of the various aspartate and
glutamate analogues within the glutamate binding site in
1XFH showed a conserved pattern for the position of the
R-amino carboxylic acid, while the side chain carboxyl group
occupied one of two possible positions (Figure 7). A similar
pattern was observed for redocking into 2NWL, with the
pose of aspartate corresponding to its crystallographic posi-
tion. These positions were compatible with a variety of
backbone stereochemistries such as the various diastereoi-
somers of the norbornane aspartate 9. It is therefore hardly
surprising that the observed stronger inhibition of the trans-
dicarboxylic ligands rac-9a and rac-9c was not predicted by
docking of 9 to 1XFH. Although in the case of 2NWL the
strongest docking diastereoisomer of 9 indeed corresponded
to one of the enantiomers of the most active diastereoisomer
rac-9c, the analysis of stereochemical preferences within such
a tight binding pocket probably goes beyond the predicting
capability of docking, in particular considering that model-
ing was performed on the structure of glutamate receptor
analogues and not the actual GLT-1.
The increased potency of the phenethyl substituted ligand
rac-25a compared to the nonsubstituted analogues is prob-
ably due to hydrophobic contacts between the benzyl group
and the protein. This increased binding could not be ratio-
nalized by docking because the ligand did not dock properly
into 1XFH, 2NWL, or even 2NWW, which contains a
binding site occupied by the benzylether reference 17a. In
the latter case, even 17a itself did not redock in its crystal-
lographic position using either Autodock or Glide. These
programs also failed to produce satifactory docking poses
for the various exo-substituted norbornane aspartates
(23a-d, 24a-d, 25a-d, 28a-d), which either reflects signifi-
cant structural differences between these proteins and GLT-1
or indicates induced-fit upon binding. It is therefore apparent
that exploratory synthetic chemistry played an essential role in
the optimization process once the initial norbornane aspartate
hits had been identified.
Conclusion
In summary, the experiments above demonstrate the use of
the chemical universe database GDB for exploring system-
atically the small molecule chemical space for drug discovery
in the case of aspartate and glutamate analogues. Virtual
screening by high-throughput docking identified norbornane
aspartates among the highest scoring hits. Synthesis and
testing led to the identification of the new GLT-1 inhibitors
rac-9a and rac-23a and, through exploratory chemical synthe-
sis, the optimized analogue rac-25a, which compares favor-
ably with previously known GLT-1 inhibitors in terms of
activity and selectivity. Further activity optimization might
include the synthesis of pure enantiomers and more extended
variations of the aromatic group in rac-25a because these may
allow to increase the affinity.
The systematic diversification of known ligands by enu-
meration with help of GDB followed by synthesis and testing
as exemplified here provides a general strategy for drug
discovery. Similarly to our recent report on scaffold enumera-
tion from GDB to discover R7-nicotinic receptor ligands,9 the
present library design preserved the pharmacophore of the
reference ligands while varying only the carbon scaffold. This
approach produced a focused library with a strong bias
toward the desired activity, thus increasing the probability
of a reward from the synthetic resources invested independent
of the quality of the scoring function used. This conservative

7242 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Luethi et al.
design, although limiting the exploratory chemistry to rela-
tively modest variations of the reference ligands, also allowed
us to capitalize on well-established synthetic methods for
amino acids for the synthetic part of the study.
for 1 h and for 5 h at room temperature to yield the crude
Diels-Alder adduct (16.67 g), which was used without further
purification. A saturated solution of NH4OAc (20 g) in absolute
ethanol (24 mL) was added to the crude (6 g) in a sealable
reactor. After stirring at 80 °C for 18 h in the closed vessel, the
mixture was evaporated to dryness and partitioned between
0.1 M aq Na2CO3 (100 mL) and EtOAc (100 mL). The aqueous
phase was extracted with EtOAc (3 ꢀ 100 mL) and the combined
organic phases were washed with brine, dried over MgSO4, and
evaporated to give the crude amine as a brown oil. The residue
was dissolved in CH2Cl2 (130 mL) and Na2CO3 (2.96 g, 27.94
mmol) in H2O (130 mL) followed by benzylchloroformate were
added. After stirring at room temperature under an N2 atmo-
sphere for 12 h, the reaction was partitioned. The aqueous phase
was extracted with CH2Cl2 (3 ꢀ 80 mL), and the combined organic
phases were washed with brine, dried over MgSO4, and evapo-
rated. Purification by flash chromatography (hexane/EtOAc: 85/
15 to 70/30) gave the products as slightly yellow oils in the order
rac-14b (0.381 g, 0.983 mmol, 4%), rac-14a, and rac-14c (1.99 g,
5.14 mmol, 20%, ratio 1/1 by 1H NMR)), rac-14d (1.831 g, 4.73
mmol, 19%), and recovered Diels-Alder adduct (1.547 g, 26%).
rac-14a and rac-14c (100 mg, 0.26 mmol) were separated by RP-
HPLC (A/B: 50/50 to 38/62 in 12 min, then 38/62 in isocratic mode
while eluting the products, λ= 214 nm, tR (rac-14a) = 15.8 min, tR
(rac-14c) = 17 min) to give rac-14a (34 mg, 0.088 mmol) and rac-
14c (20 mg, 0.052 mmol) as transparent oils.
In the perspective of using GDB as a source for drug
discovery, one can speculate that more general searches of
the entire GDB, or of a biogenically biased subset of GDB,45
might lead to innovative bioactive chemotypes otherwise
difficult to discover. Such general searches will require enrich-
ment schemes with less constraining ligand-based filters than
those used here, combined with reliable scoring functions
compatible with the size of GDB (millions to billions of
structures) and larger laboratory resources to tackle more
diverse and probably more challenging syntheses.
Experimental Section
Database Generation. The aspartate and glutamate analogues
libraries were constructed by selecting and modifying GDB
molecules (available from www.gdb.unibe.ch) using the ChemAxon
JChem package.
Docking Studies. The protonation state of all the compounds
was set at pH = 7.4, and the compounds were expanded to 3D
structures using CORINA. AutoDock Tools (ADT3) were
applied to prepare and parametrize the receptor protein and
ligands in batch mode. All structures were then docked into the
target receptor protein using a set of precalculated AutoDock
grid maps of interactions between each atom type of the ligands
and each atom type of amino acid residues in the binding pocket.
Fifteen Larmackian genetic algorithm runs were set to output
15 best docking conformations and 15 best estimated binding
energies for each structure. The most favorable docking con-
formation and its estimated binding energy value was taken to
be the final docking result for that input structure.
(1S*,2R*,3S*,4R*)-2-Benzyloxycarbonylamino-bicyclo[2.2.1]-
hept-5-ene-2,3-dicarboxylic Acid Diethyl Ester (rac-14a). 1H NMR
(300 MHz, CDCl3) δ 7.35-7.28 (m, 5H), 6.32 (dd, J = 5.6, 3.0 Hz,
1H), 6.07 (br s, 1H), 5.67 (br s, 1H), 5.04 (s, 2H), 4.30-3.84 (m,
4H), 3.23 (d, J = 1.9 Hz, 1H), 3.05 (s, 1H), 3.01 (s, 1H), 2.25 (d,
J= 9.2 Hz, 1H), 1.64 (dd, J=9.4, 1.6Hz, 1H), 1.28-1.10(m, 6H).
13C NMR (75 MHz, CDCl3) δ 173.06, 170.93, 155.20, 139.61,
136.39, 134.76, 128.54, 128.31, 128.22, 67.84, 67.02, 61.62, 60.98,
52.49, 49.99, 47.62, 46.86, 14.19. HRMS: calcd for C21H25NNaO6,
410.1574; found, 410.1576.
Synthesis. All reagents were obtained from commercial
sources (Sigma-Aldrich, Fluka, Acros Organics) and used with-
out further purification. For reactions, HPLC grade solvents or
dry solvents (obtained from a solvent drying system) were used.
Solvents used for extraction and flash chromatography were
distilled from technical quality. Milli-Q water was used for
reactions, ion-exchange chromatography, and crystallization,
and deionized water was used for extractions. Sensitive reac-
tions were carried out under argon. Flash chromatography was
performed on silica gel (Fluka) with a particle size of 0.04-0.063
mm if not otherwise noted. For thin-layer chromatography
(TLC), Macherey-Nagel SIL G-25 UV254 precoated plates were
used. Preparative RP-HPLC was performed with a Waters
Delta Prep 4000 system with a Waters Prepak cartridge (500
g) as column and Waters 486 tunable absorbance detector.
Eluents were: A, Milli-Q water with 0.1% TFA; B, Milli-Q
water/acetonitrile (40/60) with 0.1% TFA; C, Milli-Q water/
acetonitrile (10/90) with 0.1% TFA. NMR experiments were
conducted on Bruker AC 300 or Bruker DRX 400 instruments.
Chemical shifts (δ) are reported in ppm relative to CDCl3 (1H,
δ = 7.26 ppm; 13C, δ = 77.16 ppm), MeOD (1H, δ = 3.31 ppm;
13C,: δ = 49.00 ppm), DMSO-d6 (1H, δ = 2.50 ppm; 13C, δ =
39.52 ppm) or D2O (1H, δ = 4.79 ppm). External calibration of
13C-spectra in D2O was done with MeOH. MS spectra were
provided by the service of mass spectrometry of the Department
of Chemistry and Biochemistry, University of Berne. Crystal-
(1S*,2S*,3R*,4R*)-2-Benzyloxycarbonylamino-bicyclo[2.2.1]-
hept-5-ene-2,3-dicarboxylic Acid Diethyl Ester (rac-14b). 1H NMR
(300 MHz, CDCl3) δ 7.34-7.28 (m, 5H), 6.37 (dd, J = 5.6, 2.9 Hz,
1H), 6.19 (dd, J = 5.6, 3.2 Hz, 2H), 5.04 (s, 2H), 4.24-4.18 (m,
2H), 4.12-4.01 (m, 2H), 3.62 (s, 1H), 3.48 (d, J = 3.1 Hz, 1H), 3.17
(s, 1H), 1.79 (d, J = 9.3 Hz, 1H), 1.54 (d, J = 9.4 Hz, 1H),
1.25-1.19 (m, 6H). 13C NMR (75 MHz, CDCl3) δ 172.91, 172.32,
155.60, 138.44, 136.76, 135.03, 128.53, 128.17, 128.13, 77.36, 67.73,
66.74, 61.85, 61.11, 51.80, 51.39, 46.94, 14.22, 14.17. HRMS: calcd
for C21H25NNaO6, 410.1574; found, 410.1577.
(1S*,2S*,3S*,4R*)-2-Benzyloxycarbonylamino-bicyclo[2.2.1]-
hept-5-ene-2,3-dicarboxylic Acid Diethyl Ester (rac-14c). 1H NMR
(300 MHz, CDCl3) δ 7.38-7.28 (m, 5H), 6.34 (dd, J = 5.6, 3.1 Hz,
1H), 6.16 (dd, J = 5.4, 3.1 Hz, 1H), 5.19-4.94 (m, 3H), 4.18-4.02
(m, 4H), 3.74 (s, 1H), 2.99 (s, 1H), 2.44 (d, J = 9.7 Hz, 1H), 2.16 (d,
J = 2.6 Hz, 1H), 1.69 (d, J = 9.7 Hz, 1H), 1.27-1.16 (m, 6H). 13
C
NMR (75 MHz, CDCl3) δ 172.04, 171.66, 155.27, 138.60, 137.28,
136.38, 128.65, 128.34, 128.26, 69.11, 67.02, 61.78, 61.23, 58.61,
49.97, 47.65, 46.49, 14.23, 14.03. HRMS: calcd for C21H25NNaO6,
410.1574; found, 410.1576.
(1S*,2R*,3R*,4R*)-2-Benzyloxycarbonylamino-bicyclo[2.2.1]-
hept-5-ene-2,3-dicarboxylic Acid Diethyl Ester (rac-14d). 1H NMR
(300 MHz, CDCl3) δ 7.45-7.28 (m, 5H), 6.33 (dd, J = 5.1, 3.3 Hz,
1H), 6.26 (dd, J = 5.5, 2.9 Hz, 1H), 5.42 (s, 1H), 5.26-4.93 (m,
2H), 4.16-3.93 (m, 4H), 3.44 (s, 1H), 3.11 (s, 1H), 2.97 (d, J = 3.4
Hz, 1H), 1.75 (d, J = 9.1 Hz, 1H), 1.59 (dt, J = 9.0, 1.65 Hz, 1H),
1.21 (t, J = 7.1 Hz, 3H), 1.13 (t, J = 7.1 Hz, 3H). 13C NMR (75
MHz, CDCl3) δ 171.04, 170.32, 155.11, 136.30, 135.40, 134.92,
128.34, 128.10, 128.02, 69.96, 66.64, 60.97, 60.51, 58.53, 51.99,
47.16, 46.01, 14.03, 13.79. HRMS: calcd for C21H25NNaO6,
410.1574; found, 410.1577.
€
lographic measurements were conducted by Dr. Jurg Hauser,
University of Bern, and Dr. Antonia Neels, CSEM Neuchatel.
^
All compounds tested had >95% purity as determined by
analytical RP-HPLC.
2-Benzyloxycarbonylaminobicyclo[2.2.1]hept-5-ene-2,3-dicar-
boxylic Acid Diethyl Ester (rac-14a-d). Freshly distilled cyclo-
pentadiene (4.66 g, 70.52 mmol) was added dropwise to diethyl
acetylenedicarboxylate (12.00 g, 70.52 mmol) cooled to 0 °C in
an ice-water bath under stirring. The mixture was stirred at 0 °C
Procedure A. Epimerization of (1S*,2S*,3R*,4R*)-2-Benzyloxy-
carbonylamino-bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic Acid Diethyl
Ester (rac-14b). Sodium (35 mg, 1.50 mmol) was dissolved in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7243
˚
absolute ethanol (2 mL) over 3 A molecular sieves under an Ar
atmosphere and cooled to 0 °C in an ice-water bath. rac-14b (58
mg, 0.150 mmol) in absolute ethanol (2 mL) was slowly added,
and the reaction mixture was warmed to room temperature and
stirred for 40 h. After cooling back to 0 °C, the reaction was
neutralized with 1 N HCl and partitioned between water (15 mL)
and ethyl acetate (15 mL). The aqueous phase was washed with
ethyl acetate (2 times), and the combined organic phases were
washed with brine, dried over Na2SO4, and evaporated. The
mixture of diastereomers was separated by flash chromatography
(hexane/EtOAc: 85/15) to yield rac-14b (18 mg, 0.046 mmol,
31%) and rac-14c (20 mg, 0.052 mmol, 34%).
(300 MHz, D2O) δ 3.61 (d, J = 3.3 Hz, 1H), 2.76 (s, 1H), 2.71 (s,
1H), 1.98 (d, J = 10.9 Hz, 1H), 1.80-1.37 (m, 5H). 13C NMR
(75 MHz, D2O) δ 175.35, 175.20, 64.45, 49.32, 46.82, 40.68,
36.70, 22.77, 22.62. HRMS: calcd for C9H14NO4, 200.0917;
found, 200.0920.
(1S*,2S*,3S*,4R*)-2-Aminobicyclo[2.2.1]heptane-2,3-dicar-
boxylic Acid (rac-9c). The compound rac-14c (32 mg, 0.083
mmol) was reacted following Procedure C to give the crude
aminodiester, which was further heated with 3 N HCl (3 mL)
under reflux for 30 h. Evaporation to dryness under vacuum
afforded rac-9c (17 mg, 0.072 mmol, 88%) as a white solid. 1H
NMR (300 MHz, D2O) δ 2.68 (s, 3H), 2.33 (d, J = 10.3 Hz,
1H), 1.86-1.62 (m, 2H), 1.61-1.44 (m, 2H), 1.43-1.31 (m,
1H). 13C NMR (75 MHz, D2O) δ 174.97, 172.95, 68.60, 57.44,
46.35, 38.76, 37.99, 27.36, 22.97. HRMS: calcd for C9H14NO4,
200.0917; found, 200.0920.
(1S*,2R*,3R*,4R*)-2-Benzyloxycarbonylaminobicyclo[2.2.1]-
heptane-2,3-dicarboxylic Acid (rac-16d) and (1S*,2R*,3S*,4R*)-
2-Benzyloxycarbonylaminobicyclo[2.2.1]heptane-2,3-dicarboxylic
Acid (rac-16a). The compound rac-14d (505 mg, 1.30 mmol) was
reacted following Procedure C to give the crude aminodiester,
which was further heated with 6 N HCl (12 mL) under reflux for 3
days. After evaporation under vacuum, the partly epimerized
amino acid was dissolved in H2O (4.8 mL), cooled to 0 °C in an
ice-water bath, and 1 N NaOH (4.8 mL) followed by benzyl-
chloroformate (0.6 mL, 4.09 mmol) were added. After stirring at
0 °C for 1 h and at room temperature for 4 h, the reaction was
cooled to 0 °C, then was adjusted to pH 7 by dropwise addition of
1 N HCl, and lyophilized to dryness. The crude was purified by
RP-HPLC (A/B = 68/32 to A/B = 38/62 in 30 min, λ = 214 nm)
to yield rac-16a (tR = 8.4 min, 103 mg, 0.309 mmol, 24%) and
rac-16d (tR = 18 min, 12 mg, 0.036 mmol, 3%) as white solids.
rac-16a: 1H NMR (300 MHz, MeOD) δ 7.41-7.19 (m, 5H),
5.10-4.94 (m, 2H), 3.54 (d, J = 1.6 Hz, 1H), 2.40 (dd, J = 14.9,
3.0 Hz, 3H), 1.69-1.23 (m, 5H). 13C NMR (75 MHz, MeOD) δ
176.01, 175.06, 138.26, 129.36, 128.81, 128.66, 70.60, 67.38, 54.75,
47.55, 44.06, 38.59, 28.64, 25.11. HRMS: calcd for C17H20NO6,
334.1285; found, 334.1293. rac-16d: 1H NMR (300 MHz, MeOD)
δ 7.45-7.25 (m, 5H), 5.17 (q, J = 12.4 Hz, 2H), 2.84 (d, J = 3.2
Hz, 1H), 2.59 (s, 1H), 2.50 (s, 1H), 2.21-2.06 (m, 1H), 1.81 (d,
J = 10.1 Hz, 1H), 1.58-1.34 (m, 4H). 13C NMR (75 MHz,
MeOD) δ 174.37, 173.60, 137.56, 129.53, 129.25, 129.02, 68.92,
68.60, 60.29, 49.39, 41.89, 38.29, 25.65, 22.90; HRMS: calcd for
C17H19NNaO6, 356.1105; found, 356.1112.
Epimerization of (1S*,2R*,3R*,4R*)-2-Benzyloxycarbonyl-
amino-bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic Acid Diethyl Ester
(rac-14d). rac-14d (140 mg, 0.361 mmol) was treated following
Procedure A to yield after flash chromatography (hexane/EtOAc:
85/15 to 80/20) rac-14a (87 mg, 0.225 mmol, 62%) and rac-14d
(22 mg, 0.0565 mmol, 16%) in separate form.
Procedure B. (1R*,2R*,3S*,4S*)-2-Benzyloxycarbonylamino-
bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic Acid (rac-15a). The com-
pound rac-14d (415 mg, 1.07 mmol) in THF/MeOH (16.7 mL/5.6
mL) was treated with LiOH H2O (270 mg, 6.43 mmol) in H2O (5.6
mL). The reaction was stirred at 70 °C for 24 h. After evaporation
of the organic solvents, the reaction mixture was cooled to 0 °C in
an ice-water bath, acidified to pH 2 with 1 N HCl, and extracted
with EtOAc (4 times). The organic phases were combined, washed
with brine, dried over MgSO4, and evaporated. The crude was
purified by RP-HPLC (tR = 18.0 min (A/B = 85/15 to A/B = 55/
45 in30 min)) to give rac-15a as a white solid (167 mg, 0.504 mmol,
1
47%). H NMR (300 MHz, MeOD) δ 7.52-7.14 (m, 5H), 6.39
(dd, J = 5.6, 3.0 Hz, 1H), 6.06 (br s, 1H), 5.06 (s, 2H), 3.36 (d, J =
2.0 Hz, 1H, overlaid with MeOD), 3.07 (s, 1H), 3.03 (s, 1H), 2.44
(d, J = 8.9 Hz, 1H), 1.62 (dd, J = 9.2, 1.5 Hz, 1H). 13C NMR (75
MHz, MeOD) δ 176.23, 174.68, 157.88, 141.30, 138.10, 135.05,
129.33, 128.80, 128.63, 68.81, 67.48, 53.50, 50.17, 49.29, 48.15.
HRMS: calcd for C17H18NO6, 332.1129; found, 332.1137.
(1R*,2S*,3R*,4S*)-2-Benzyloxycarbonylamino-bicyclo[2.2.1]-
hept-5-ene-2,3-dicarboxylic Acid (rac-15b). The compound rac-
14b (183 mg, 0.472 mmol) was treated following Procedure B
(RP-HPLC: tR=11.8 min (A/B: 75/25 to 55/45 in 20 min)) to give
rac-15b as a white solid (100 mg, 0.302 mmol, 64%). Alterna-
tively, a mixture of rac-14a and rac-14c (569mg, 1.469mmol) was
treated following Procedure B to give rac-15a (67 mg, 0.202
mmol, 14%) andrac-15b (99 mg, 0.299mmol, 20%). (RP-HPLC:
tR (rac-15a) = 20 min, tR (rac-15b) = 29.2 min with A/B = 85/15
to A/B = 45/55 in 40 min). 1H NMR (300 MHz, MeOD)
δ 7.38-7.25 (m, 5H), 6.32 (dd, J = 5.6, 2.9 Hz, 1H), 6.17 (dd,
J = 5.4, 3.1 Hz, 1H), 3.56 (s, 1H), 3.49 (d, J = 3.2 Hz, 1H), 3.19
(s, 1H), 1.85 (d, J = 9.1 Hz, 1H), 1.49 (d, J = 9.1 Hz, 1H).
13C NMR (75 MHz, MeOD) δ 176.45, 175.60, 157.62, 138.66,
138.13, 136.36, 129.41, 128.93, 128.69, 68.27, 67.46, 52.22, 51.85,
47.49. HRMS: calcd for C17H17NNaO6, 354.0953; found,
354.0955.
(1S*,2R*,3R*,4R*)-2-Aminobicyclo[2.2.1]heptane-2,3-dicar-
boxylic Acid (rac-9d). The compound rac-16d (9 mg, 0.027
mmol) was treated following Procedure C to give rac-9d (5.4
mg, 0.027 mmol, quant) as a white solid. 1H NMR (300 MHz,
D2O) δ 2.72 (d, J = 3.4 Hz, 1H), 2.57 (s, 1H), 2.44 (s, 1H),
2.08-2.00 (m, 1H), 1.81-1.63 (m, 2H), 1.62-1.43 (m, 3H). 13
C
NMR (75 MHz, D2O) δ 176.35, 172.91, 69.11, 55.57, 46.98,
39.61, 37.05, 25.42, 21.19. HRMS: calcd for C9H14NO4,
200.0917; found, 200.0920.
Procedure C. (1S*,2R*,3S*,4R*)-2-Amino-bicyclo[2.2.1]heptane-
2,3-dicarboxylic Acid (rac-9a). A solution of rac-15a (20 mg, 0.060
mmol) and 10% palladium on charcoal (7 mg) in MeOH (3 mL) was
stirred under 1 atm H2 at room temperature for 90 min. The reaction
mixture was filtered through a pad of Celite, washing with MeOH,
evaporated to dryness, and redissolved in 0.05 N HCl and re-
evaporated to dryness (3ꢀ) to yield rac-9a as the HCl salt (13 mg,
0.054 mmol, 89%) as a white solid. 1H NMR (300 MHz, D2O) δ
3.43 (d, J = 1.2 Hz, 1H), 2.71 (d, J = 2.3 Hz, 1H), 2.59 (d, J = 2.3
Hz, 1H), 2.01 (d, J = 11.4 Hz, 1H), 1.80-1.51 (m, 3H), 1.49-1.35
(m, 2H). 13C NMR (75 MHz, D2O) δ 175.50, 172.76, 68.68, 52.54,
47.15, 43.18, 37.04, 27.60, 24.16. HRMS: calcd for C9H14NO4,
200.0922; found, 200.0917.
(1R*,2R*,3R*,4S*,6R*)-2-Benzyloxycarbonylamino-6-formyl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Diethyl Ester (rac-
18a) and (1R*,2R*,3R*,4S*,5S*)-2-Benzyloxycarbonylamino-5-
formyl-bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Diethyl Ester
(rac-18b). The compound rac-14d (1.085 g, 2.801 mmol) in
toluene (11 mL) was transferred to a 50 mL autoclave and
(acetylacetonato)dicarbonylrhodium(I) (9.4 mg, 0.036 mmol,
1.3 mol %) was added. A CO/H2 (1/1) gas pressure of 40 bar
was applied, and the reaction was stirred at 60 °C for 24 h. After
cooling the reaction, the solvent was evaporated and the oily
residue was purified by flash chromatography (hexane/EtOAc:
80/20 to 70/30) to yield rac-18a (406 mg, 0.973 mmol, 35%) and
rac-18b (345 mg, 0.826 mmol, 30%) as colorless oils in separated
form. rac-18a: 1H NMR (400 MHz, C6D6) δ 9.34 (s, 1H), 7.29-
7.21 (m, 2H), 7.14-7.01 (m, 3H), 5.10 (d, J = 12.3 Hz, 1H), 4.99
(d, J = 12.3 Hz, 1H), 4.78 (br s, 1H), 4.17-3.96 (m, 2H), 3.96-
3.78 (m, 2H), 3.70-3.62 (m, 1H), 3.40 (s, 1H), 2.25-2.17 (m, 2H),
(1S*,2S*,3R*,4R*)-2-Amino-bicyclo[2.2.1]heptane-2,3-dicar-
boxylic Acid (rac-9b). The compound rac-15b (20 mg, 0.060
mmol) was reacted following Procedure C to yield the HCl salt
of rac-9b (12 mg, 0.051 mmol, 84%) as a white solid. 1H NMR

7244 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Luethi et al.
1.95-1.69 (m, 2H), 1.20 (d, J = 11.0 Hz, 1H), 1.03-0.88 (m, 6H),
0.84 (d, J = 10.7 Hz, 1H). 13C NMR (101 MHz, C6D6) δ 200.87,
170.38, 169.89, 154.79, 137.14, 128.70, 128.59, 128.34, 66.92,
66.90, 61.30, 60.60, 57.45, 48.45, 46.95, 40.56, 35.11, 24.26,
14.21, 14.01. HRMS: calcd for C22H27NNaO7, 440.1680; found,
(1S*,2R*,3R*,4S*,5R*)-2-Benzyloxycarbonylamino-5-vinyl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Diethyl Ester (rac-
19b). The compound rac-18b (120 mg, 0.287 mmol) was treated
following Procedure D to yield after purification by flash
chromatography (hexane/EtOAc: 85/15) rac-19b (76 mg,
0.183 mmol, 64%) as a colorless oil. 1H NMR (300 MHz,
CDCl3) δ 7.41-7.27 (m, 5H), 5.78 (ddd, J = 17.3, 10.2, 7.2 Hz,
1H), 5.19 (br s, 1H), 5.16-4.88 (m, 4H), 4.27-3.93 (m, 4H), 2.96
(br s, 1H), 2.93-2.81 (m, 2H), 2.46 (dd, J = 3.8, 1.4 Hz, 1H), 1.98
(ddd, J = 13.2, 8.4, 2.6 Hz, 1H), 1.72 (dd, J = 10.4, 1.3 Hz, 1H),
1.52 (d, J = 10.4 Hz, 1H), 1.37-1.26 (m, 1H), 1.26-1.10 (m, 6H).
13C NMR (75 MHz, CDCl3) δ 170.53, 170.43, 154.92, 142.77,
136.53, 128.58, 128.27, 128.23, 113.14, 67.51, 66.73, 61.49, 60.48,
57.77, 48.03, 45.68, 37.94, 35.18, 31.65, 14.25, 14.04. HRMS:
calcd for C23H29NNaO6, 438.1887; found, 438.1872.
1
440.1686. rac-18b: H NMR (300 MHz, C6D6) δ 9.36 (s, 1H),
7.29-7.21 (m, 2H), 7.15-7.01 (m, 3H), 5.06 (dd, J = 28.2, 12.3
Hz, 2H), 4.83 (s, 1H), 4.13-3.80 (m, 4H), 3.44-3.23 (m, 1H), 2.76
(br s, 1H), 2.60 (dd, J = 3.9, 1.4 Hz, 1H), 2.44 (d, J = 4.1 Hz, 1H),
1.91-1.78 (m, 1H), 1.71 (ddd, J = 13.4, 6.2, 4.3 Hz, 1H), 1.25-
1.16 (m, 1H), 1.03 (t, J = 7.1 Hz, 3H), 0.99-0.86 (m, 4H). 13
C
NMR (101 MHz, C6D6) δ 201.25, 170.26, 170.16, 154.86, 137.27,
128.67, 128.59, 67.92, 66.78, 61.44, 60.47, 57.07, 47.92, 47.82,
41.16, 35.51, 25.08, 14.22, 13.98. HRMS: calcd for C22H27NNaO7,
440.1680; found, 440.1692.
(1R*,2S*,3R*,4S*,6R*)-2-Benzyloxycarbonylamino-6-formyl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Diethyl Ester (rac-
18c) and (1R*,2S*,3R*,4S*,5S*)-2-Benzyloxycarbonylamino-5-
formyl-bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Diethyl Ester
(rac-18d). The compound rac-14b (272 mg, 0.702 mmol) and
(acetylacetonato)dicarbonylrhodium(I) (2.4 mg, 0.0093 mmol,
1.3 mol %) in toluene (3 mL) were treated according to the
procedure for hydroformylation above. Purification by flash
chromatography (hexane/EtOAc: 80/20) afforded rac-18c (89
mg, 0.214 mmol, 30%) and rac-18d (146 mg, 0.350 mmol,
50%) as colorless oils in separated form. rac-18c: 1H NMR
(400 MHz, C6D6) δ 9.23 (s, 1H), 8.03 (br s, 1H), 7.27-7.21 (m,
2H), 7.11-6.94 (m, 3H), 5.11 (d, J = 12.4 Hz, 1H), 5.04 (d, J =
12.4 Hz, 1H), 4.12-3.82 (m, 2H), 3.79-3.61 (m, 2H), 2.76 (dd,
J = 3.7, 1.7Hz, 1H), 2.59(d, J = 3.3 Hz, 1H), 2.21(brs, 1H), 1.96
(d, J = 12.6 Hz, 1H), 1.70 (d, J = 11.0 Hz, 1H), 1.31 (ddd, J =
13.3, 8.8, 2.5 Hz, 1H), 1.04 (d, J = 11.1 Hz, 1H), 0.97-0.84 (m,
3H), 0.79 (t, J = 7.1 Hz, 3H), 0.59 (br s, 1H). 13C NMR (101
MHz, C6D6) δ 200.62, 172.62, 172.40, 137.28, 128.65, 128.50,
128.19, 66.93, 64.18, 61.58, 61.15, 50.98, 48.66, 46.47, 40.49,
35.13, 23.16, 14.02, 13.88. HRMS: calcd for C22H27NNaO7,
(1R*,2S*,3R*,4R*,6S*)-2-Benzyloxycarbonylamino-6-vinyl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Diethyl Ester (rac-
19c). The compound rac-18c (245 mg, 0.586 mmol) was treated
following Procedure D to yield after purification by flash chro-
matography (hexane/EtOAc: 85/15) rac-19c (106 mg, 0.255 mmol,
1
43%) as a colorless oil. H NMR (300 MHz, CDCl3) δ 7.51 (s,
1H), 7.41-7.26 (m, 5H), 5.76 (ddd, J = 16.7, 10.1, 6.8 Hz, 1H),
5.08 (s, 2H), 5.03-4.85 (m, 2H), 4.31-3.99 (m, 4H), 3.01 (s, 1H),
2.94 (d, J = 2.2 Hz, 1H), 2.58 (br s, 2H), 1.85-1.50 (m, 4H),
1.41-1.30 (m, 1H), 1.26 (t, J = 7.1 Hz, 3H), 1.17 (t, J = 6.9 Hz,
3H). 13C NMR (75 MHz, CDCl3) δ 173.16, 172.47, 155.79,
142.23, 128.53, 128.18, 128.09, 113.65, 66.69, 64.18, 61.59, 61.20,
50.26, 40.99, 37.82, 34.81, 30.07, 14.24, 14.12. HRMS: calcd for
C23H29NNaO6, 438.1887; found, 438.1880.
(1S*,2S*,3R*,4S*,5R*)-2-Benzyloxycarbonylamino-5-vinyl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Diethyl Ester (rac-
19d). The compound rac-18d (134 mg, 0.321 mmol) was treated
following Procedure D to yield after purification by flash chroma-
tography (hexane/EtOAc: 90/10 to 80/20) rac-19d (87 mg, 0.209
mmol, 65%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.59
(s, 1H), 7.44-7.24 (m, 5H), 5.72 (ddd, J = 17.3, 10.3, 7.3 Hz, 1H),
5.19-5.02 (m, 2H), 5.00-4.86 (m, 2H), 4.33-4.02 (m, 4H), 3.22 (s,
1H), 2.93 (d, J = 3.8 Hz, 1H), 2.45 (d, J = 2.3 Hz, 1H), 2.40-2.28
(d, J = 6.6 Hz, 1H), 2.02-1.90 (m, 1H), 1.84 (d, J = 8.8 Hz, 1H),
1.62 (d, J = 10.7 Hz, 1H), 1.28 (t, J = 7.1 Hz, 5H), 1.19 (t, J = 7.1
Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 173.28, 172.16, 155.97,
142.29, 136.77, 128.44, 128.04, 128.00, 113.22, 66.57, 63.51, 61.46,
61.17, 51.04, 46.02, 45.62, 38.88, 34.93, 31.34, 14.18, 14.04. HRMS:
calcd for C23H29NNaO6, 438.1887; found, 438.1904.
(1R*,2R*,3R*,4R*,6S*)-2-Benzyloxycarbonylamino-6-styryl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Diethyl Ester (rac-
20a). The compound rac-18a (150 mg, 0.359 mmol) and benzyl-
triphenylphosphonium chloride (349 mg, 0.898 mmol) were
treated following Procedure D to yield after purification by flash
chromatography (hexane/EtOAc: 85/15 to 70/30) a mixture of
E/Z-isomers (ratio: 54/46) of rac-20a (177 mg, 0.359 mmol, quant)
as a colorless oil. 1H NMR (300 MHz, C6D6) δ 7.39 (d, J = 7.3 Hz,
1H), 7.31-6.96 (m, 10H, overlaid with solvent peak), 6.36 (dd, J =
28.5, 13.7 Hz, 1H), 6.07 (dd, J = 15.8, 7.3 Hz, 1H ꢀ 0.46), 5.39 (dd,
J = 11.5, 9.6 Hz, 1H ꢀ 0.54), 5.14-4.97 (m, 2H), 4.92 (br s, 1H ꢀ
0.54), 4.85 (br s, 1H ꢀ 0.46), 4.23-3.77 (m, 4H), 3.69-3.57 (m, 1H
ꢀ 0.54), 3.53 (d, J= 5.9Hz, 1Hꢀ 0.46), 3.02(s, 1Hꢀ 0.46), 2.82 (s,
1H ꢀ 0.54), 2.66-2.50 (m, 1H), 2.48-2.23 (m, 2H), 1.49-1.13 (m,
4H), 1.10-0.94 (m, 4H), 0.80 (t, J = 7.1 Hz, 3H ꢀ 0.54). 13C NMR
(75 MHz, C6D6) δ 170.72, 170.61, 170.40, 170.08, 154.78, 154.71,
138.27, 137.80, 137.33, 137.31, 137.05, 134.93, 129.39, 129.25,
128.70, 128.67, 128.64, 128.59, 128.56, 128.50, 128.25, 128.19,
127.16, 126.93, 126.59, 67.95, 67.71, 66.79, 66.72, 61.36, 61.18,
60.45, 60.30, 57.12, 56.98, 53.71, 52.63, 41.12, 40.64, 37.94, 35.29,
34.99, 34.28, 33.33, 31.74, 14.31, 14.26, 14.13, 13.75. HRMS: calcd
for C29H33NNaO6, 514.2205; found, 514.2205.
1
440.1680; found, 440.1684. rac-18d: H NMR (300 MHz, C6D6)
δ 9.06 (s, 1H), 7.40 (br s, 1H), 7.30-7.21 (m, 2H), 7.13-6.97 (m,
3H), 5.07 (s, 2H), 4.09-3.87 (m, 2H), 3.81-3.65 (m, 2H), 3.34 (br s,
1H), 2.97 (d, J = 3.9 Hz, 1H), 2.59 (d, J = 2.4 Hz, 1H), 2.50-2.33
(m, 1H), 1.85-1.59 (m, 2H), 1.56-1.39 (m, 1H), 1.08 (d, J = 10.9
Hz, 1H), 0.91 (t, J = 7.0 Hz, 4H), 0.81 (t, J = 7.1 Hz, 3H). 13C
NMR (75 MHz, C6D6) δ 200.50, 172.88, 171.79, 156.15, 137.37,
128.63, 128.46, 128.14, 66.82, 64.19, 61.54, 61.18, 50.69, 48.01,
45.69, 41.65, 35.75, 24.82, 14.03, 13.92. HRMS: calcd for C22H27-
NNaO7, 440.1680; found, 440.1686.
Procedure D. (1R*,2R*,3R*,4R*,6S*)-2-Benzyloxycarbonyl-
amino-6-vinyl-bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Diethyl
Ester (rac-19a). Methyltriphenylphosphonium bromide (428 mg,
1.20 mmol) was suspended in anhydrous THF (12 mL) in an oven-
˚
dried flask over 4 A molecular sieves and under an Ar atmosphere.
After cooling to -18 °C in a salt-ice bath, 2.5 M n-butyllithium in
hexanes (0.43 mL, 1.08 mmol) was added dropwise under stirring.
The strongly yellow solution was stirred at 0 °C for 1 h and at room
temperature for 30 min. A solution of rac-18a (200 mg, 0.479 mmol)
in anhydrous THF (3.6 mL) was transferred dropwise to the ylid at
-78 °C. The reaction was stirred at -18 °C, monitored by TLC,
and quenched with glacial acetic acid (0.5 mL) after 70 min. The
suspension was filtered, evaporated, and coevaporated with toluene
to dryness under vacuum. The crude was purified by flash chro-
matography (hexane/EtOAc: 80/20) to obtain rac-19a (143 mg,
0.344 mmol, 72%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ
7.40-7.29 (m, 5H), 5.77 (ddd, J = 17.1, 10.3, 6.8 Hz, 1H), 5.30 (s,
1H), 5.19-4.84 (m, 4H), 4.29-3.95 (m, 4H), 2.95-2.84 (m, 1H),
2.83-2.74 (m, 2H), 2.61 (br s, 1H), 2.14-1.96 (m, 1H), 1.70 (d, J =
10.8 Hz, 1H), 1.62-1.45 (m, 1H), 1.38-1.26 (m, 1H), 1.26-1.12
(m, 6H). 13C NMR (75 MHz, CDCl3) δ 170.83, 170.15, 154.68,
142.35, 136.43, 128.50, 128.19, 128.16, 113.28, 67.60, 66.72, 61.20,
60.50, 56.98, 52.12, 40.59, 38.13, 34.65, 30.31, 14.20, 13.98. HRMS:
calcd for C23H29NNaO6, 438.1887; found, 438.1870.
(1S*,2R*,3R*,4S*,5R*)-2-Benzyloxycarbonylamino-5-styryl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Diethyl Ester (rac-
20b). The compound rac-18b (350 mg, 0.838 mmol) and benzyl-
triphenylphosphonium chloride (815 mg, 2.09 mmol) were treated

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7245
following Procedure D to yield after purification by flash chro-
matography (hexane/EtOAc: 85/15) a mixture of E/Z-isomers
(ratio: 57/43) of rac-20b (287 mg, 0.584 mmol, 70%) as a colorless
oil. 1H NMR (300 MHz, C6D6) δ 7.51-7.39 (m, 2H), 7.27 (s, 3H),
7.33-6.95 (m, 8H), 6.37 (dd, J = 26.7, 13.6 Hz, 1H), 6.07 (dd, J =
15.8, 7.9 Hz, 1H ꢀ 0.43), 5.40 (dd, J = 11.6, 10.1 Hz, 1H ꢀ 0.57),
5.14 (dd, J = 12.3, 7.5 Hz, 1H), 5.03 (dd, J = 12.3, 8.7 Hz, 1H),
4.87-4.68 (m, 1H), 4.19-3.91 (m, 3H), 3.84 (q, J = 7.1 Hz, 1H),
3.60 (m, 1H ꢀ 0.57), 3.35 (m, 1H ꢀ 0.43), 3.09 (dd, J = 20.4, 1.9
Hz, 1H), 2.66-2.23 (m, 2H), 1.52-1.12 (m, 4H), 0.95 (ddt, J =
40.5, 20.8, 7.1 Hz, 6H). 13C NMR (75 MHz, C6D6) δ 170.43,
170.40, 170.27, 170.16, 155.17, 155.08, 138.24, 137.85, 137.38,
137.35, 135.21, 128.72, 128.67, 128.64, 128.60, 128.57, 128.46,
128.24, 128.19, 127.18, 126.88, 126.56, 67.56, 67.29, 66.75, 66.74,
61.33, 61.25, 60.40, 60.37, 58.04, 57.77, 48.21, 47.66, 46.72, 46.60,
37.97, 35.56, 35.42, 34.20, 33.80, 32.65, 14.32, 14.22, 14.01, 13.98.
HRMS: calcd for C29H33NNaO6, 514.2200; found, 514.2198.
(1R*,2S*,3R*,4R*,6S*)-2-Benzyloxycarbonylamino-6-styryl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Diethyl Ester (rac-
20c). The compound rac-18c (70 mg, 0.168 mmol) and benzyl-
triphenylphosphonium chloride (163 mg, 0.419 mmol) were
treated following Procedure D to yield after purification by
flash chromatography (hexane/EtOAc: 85/15) a mixture of
E/Z-isomers (ratio: 53/47) of rac-20c (43 mg, 52%) as a colorless
(m, 5H), 5.75 (ddd, J = 17.4, 10.4, 7.1 Hz, 1H), 5.09-4.91 (m,
4H), 3.54 (s, 1H), 2.45 (d, J = 2.8 Hz, 1H), 2.36-2.15 (m, 3H),
1.69-1.51 (m, 2H), 1.42 (dt, J = 12.6, 4.5 Hz, 1H). 13C NMR
(75 MHz, MeOD) δ 176.02, 174.89, 157.80, 143.26, 138.22,
129.37, 128.82, 128.66, 113.90, 70.64, 67.41, 54.23, 53.07, 44.15,
41.02, 36.28, 35.80. HRMS: calcd for C19H21NNaO6, 382.1266;
found, 382.1278.
(1S*,2R*,3S*,4S*,5R*)-2-Benzyloxycarbonylamino-5-vinyl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid (rac-21b). The com-
pound rac-19b (99 mg, 0.238 mmol) was treated following
Procedure E to yield after purification by recrystallization in
EtOH/H2O rac-21b (35 mg, 0.097 mmol, 41%) as a white solid.
1H NMR (300 MHz, MeOD) δ 7.66-7.02 (m, 5H), 5.78 (ddd,
J = 17.4, 10.3, 7.4 Hz, 1H), 5.25-4.91 (m, 4H), 3.60 (d, J = 1.4
Hz, 1H), 2.40 (d, J = 3.5 Hz, 1H), 2.27 (s, 3H), 1.71-1.49 (m,
2H), 1.32 (dt, J = 9.5, 4.4 Hz, 1H). 13C NMR (75 MHz, MeOD)
δ 175.79, 174.98, 157.86, 143.57, 138.23, 129.36, 128.81, 128.65,
113.58, 70.30, 67.40, 54.84, 49.72, 47.85, 45.19, 35.95, 32.62.
HRMS: calcd for C19H22NO6, 360.1442; found, 360.1446.
(1R*,2S*,3R*,4R*,6S*)-2-Benzyloxycarbonylamino-6-vinyl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid (rac-21c). The com-
pound rac-19c (26 mg, 0.062 mmol) was treated following
Procedure E to yield rac-21c (15 mg, 0.042 mmol, 67%) as a
white solid after precipitating it from a slowly evaporating
solution in EtOH/H2O. 1H NMR (300 MHz, MeOD) δ 7.69-
6.99 (m, 5H), 5.78 (ddd, J = 17.1, 8.7, 6.9 Hz, 1H), 5.27-4.75
(m, 4H), 3.06 (d, J = 2.5 Hz, 1H), 2.88 (s, 1H), 2.68-2.46 (m,
2H), 1.87-1.66 (m, 2H), 1.56 (d, J = 10.5 Hz, 1H), 1.44-1.26
(m, 1H). 13C NMR (75 MHz, MeOD) δ 176.42, 175.68, 157.76,
143.59, 138.28, 129.45, 128.96, 128.70, 113.83, 67.47, 65.04,
51.44, 50.77, 42.15, 39.19, 35.21, 31.21. HRMS: calcd for
C19H22NO6, 360.1442; found, 360.1455.
1
oil. H NMR (300 MHz, CDCl3) δ 7.44 (d, J = 8.3 Hz, 1H),
7.34-6.93 (m, 10H), 6.20 (dd, J = 19.4, 13.8 Hz, 1H), 6.01 (dd,
J = 15.8, 6.2 Hz, 0.51H), 5.45 (dd, J = 11.2, 10.0 Hz, 0.46H),
5.10-4.92 (m, 1.48H), 4.79 (d, J = 12.3 Hz, 0.43H), 4.25-3.83
(m, 4H), 3.08 (br s, 0.45H), 2.98 (d, J = 13.7 Hz, 1H), 2.86 (d,
J = 2.5 Hz, 0.53H), 2.80 (d, J = 2.3 Hz, 0.52H), 2.67 (br s,
0.54H), 2.56-2.42 (m, 1H), 1.83-1.62 (m, 2H), 1.56 (d, J = 8.1
Hz, 0.69H), 1.38-1.24 (m, 0.62H), 1.22-0.95 (m, 7H). 13C
NMR (75 MHz, CDCl3) δ 173.12, 173.01, 172.47, 155.80,
137.68, 136.43, 134.33, 129.18, 128.83, 128.58, 128.54, 128.46,
128.21, 128.11, 128.00, 127.10, 126.71, 126.16, 66.72, 66.64, 64.16,
61.62, 61.52, 61.24, 50.25, 50.19, 41.01, 40.92, 37.40, 35.52, 35.12,
33.11, 32.97, 30.99, 14.24, 14.20, 14.12, 14.08. HRMS: calcd for
C29H33NNaO6, 514.2200; found, 514.2209.
(1S*,2S*,3R*,4S*,5R*)-2-Benzyloxycarbonylamino-5-vinyl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid (rac-21d). The com-
pound rac-19d (181 mg, 0.436 mmol) was treated following
Procedure E to yield after purification by flash chromatogra-
phy (CH2Cl2/MeOH: 98/2 with 0.5% acetic acid) rac-21d (100
1
mg, 0.278 mmol, 64%) as a white solid. H NMR (300 MHz,
MeOD) δ 7.52-7.08 (m, 5H), 5.76 (ddd, J = 17.4, 10.2, 7.3 Hz,
1H), 5.07 (s, 2H), 5.00-4.89 (m, 2H), 3.11-3.01 (m, 2H), 2.49-
2.30 (m, 2H), 1.97-1.77 (m, 2H), 1.59 (d, J = 10.4 Hz, 1H),
1.38-1.15 (m, 1H). 13C NMR (75 MHz, MeOD) δ 176.71, 175.49,
157.97, 143.66, 138.21, 129.50, 129.02, 128.74, 113.60, 67.56,
64.56, 51.67, 47.41, 46.75, 40.28, 35.47, 32.21. HRMS: calcd for
C19H22NO6, 360.1442; found, 360.1449.
(1S*,2S*,3R*,4S*,5R*)-2-Benzyloxycarbonylamino-5-styryl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Diethyl Ester (rac-
20d). The compound rac-18d (80 mg, 0.192 mmol) and benzyl-
triphenylphosphonium chloride (186 mg, 0.479 mmol) were
treated following Procedure D to yield after purification by
flash chromatography (hexane/EtOAc: 80/20) a mixture of
E/Z-isomers (ratio: 63/37) of rac-20d (68 mg, 0.138 mmol,
1
(1R*,2R*,3S*,4R*,6S*)-2-Benzyloxycarbonylamino-6-styryl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid (rac-22a). The com-
pound rac-20a (294 mg, 0.598 mmol) was treated following
Procedure E to yield after purification by preparative RP-HPLC
(A/C: 50/50 to 20/80 in 30 min, λ = 214 nm, tR = 12.0 min) a
mixture of E/Z-isomers (ratio: 56/44) of rac-22a (112 mg, 0.257
72%) as a colorless oil. H NMR (300 MHz, CDCl3) δ 7.51
(d, J = 11.5 Hz, 1H), 7.34-7.03 (m, 10H), 6.23 (dd, J = 13.7, 7.8
Hz, 1H), 5.99 (dd, J = 15.8, 7.8 Hz, 0.58H), 5.41 (dd, J = 11.2,
10.3 Hz, 0.34H), 5.16-4.87 (m, 2H), 4.30-3.73 (m, 4H), 3.16 (s,
1H), 2.92-2.75 (m, 1H), 2.50-2.29 (m, 1H), 2.05-1.89 (m, 1H),
1.87-1.72 (m, 1H), 1.64 (t, J = 12.5 Hz, 1H), 1.34-0.88 (m,
8H). 13C NMR (75 MHz, CDCl3) δ 173.32, 172.19, 156.03,
137.43, 136.52, 134.24, 128.92, 128.62, 128.50, 128.24, 128.10,
128.07, 127.23, 126.80, 126.11, 66.65, 63.52, 61.56, 61.29, 51.17,
50.77, 46.87, 46.44, 45.71, 45.46, 41.28, 38.52, 35.26, 33.70,
33.28, 32.20, 29.78, 14.27, 14.09, 13.85. HRMS: calcd for
C29H33NNaO6, 514.2200; found, 514.2217.
1
mmol, 43%) as a white solid. H NMR (300 MHz, MeOD) δ
7.44-7.08 (m, 10H), 6.40-6.26 (m, 1H), 6.15 (dd, J = 15.8, 7.5
Hz, 1H ꢀ 0.56), 5.55 (dd, J = 11.5, 9.8 Hz, 1H ꢀ 0.44), 5.11-
4.95 (m, 2H), 3.65-3.49 (m, 1H), 2.88 (br s, 1H ꢀ 0.44), 2.57-
2.26 (m, 4H), 1.83-1.63 (m, 2H), 1.51 (dt, J = 12.6, 4.4 Hz, 1H ꢀ
0.56), 1.38 (dt, J = 12.6, 4.3 Hz, 1H ꢀ 0.44). 13C NMR (75 MHz,
MeOD) δ 176.22, 175.29, 157.81, 138.85, 138.32, 137.08, 134.93,
130.35, 129.77, 129.50, 129.36, 129.27, 128.81, 128.68, 128.65,
128.09, 127.76, 127.09, 70.96, 70.59, 67.42, 54.52, 53.58, 44.11,
40.61, 39.13, 37.05, 36.38, 36.04, 35.80. HRMS: calcd for C25-
H25NNaO6, 458.1579; found, 458.1595.
(1S*,2R*,3S*,4S*,5R*)-2-Benzyloxycarbonylamino-5-styryl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid (rac-22b). The com-
pound rac-20b (0.263 mg, 0.535 mmol) was treated following
Procedure E to yield after purification by preparative RP-HPLC
(A/C: 50/50 to 20/80 in 30 min, λ = 214 nm, tR = 4.4 min) a
mixture of E/Z-isomers (ratio: 55/45) of rac-22b (104 mg, 0.239
mmol, 45%) as a white solid. 1H NMR (300 MHz, MeOD)
δ 7.49-7.00 (m, 10H), 6.46-6.27 (m, 1H), 6.17 (dd, J = 15.8,
Procedure E. (1R*,2R*,3S*,4R*,6S*)-2-Benzyloxycarbony-
lamino-6-vinyl-bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid (rac-
21a). The compound rac-19a (110 mg, 0.265 mmol) was dis-
solved in THF/MeOH (7.2 mL/1.8 mL) and LiOH monohy-
drate (44 mg, 1.06 mmol) in H2O (1.8 mL) was added. The
solution was stirred at 70 °C for 48 h. After evaporation of the
organic solvents, the reaction mixture was cooled to 0 °C and
acidified to pH 2 with 1 N HCl. The suspension was extracted
with EtOAc (4 ꢀ 15 mL), the combined organic phases were
washed with brine, dried over MgSO4, and evaporated. Purifi-
cation by flash chromatography (CH2Cl2/MeOH: 98/2 with
0.5% acetic acid) yielded rac-21a (56 mg, 0.156 mmol, 59%)
1
as a white solid. H NMR (300 MHz, MeOD) δ 7.39-7.22

7246 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Luethi et al.
7.9 Hz, 1H ꢀ 0.45), 5.58 (dd, J = 11.4, 10.0 Hz, 1H ꢀ 0.55),
5.10-4.95 (m, 2H), 3.70-3.50 (m, 1H), 2.83-2.69 (m, 1H ꢀ 0.55),
yield the TFA-salt of rac-23c (6.3 mg, 0.019 mmol, 56%) as a white
solid (tR (RP-HPLC) = 12.8 min). 1H NMR (300 MHz, DMSO)
δ 5.75 (ddd, J = 17.0, 10.3, 6.4 Hz, 1H), 4.99 (dd, J = 13.6, 12.2
Hz, 2H), 3.36 (d, J = 2.7 Hz, 1H), 2.59 (br s, 1H), 2.29 (s, 1H),
1.85-1.65 (m, 2H), 1.36-1.17 (m, 3H). 13C NMR (101 MHz,
DMSO) δ 173.22, 172.49, 142.22, 113.75, 62.63, 51.30, 47.51, 36.49,
32.83, 29.72. HRMS: calcd for C11H16NO4, 226.1079; found,
226.1076.
2.56-2.21 (m, 4H), 1.86-1.60 (m, 2H), 1.49-1.24 (m, 1H). 13
C
NMR (75 MHz, MeOD) δ 175.58, 174.87, 157.88, 138.95, 138.55,
138.22, 137.66, 135.15, 130.17, 129.73, 129.47, 129.36, 129.25,
129.19, 128.82, 128.66, 128.03, 127.82, 127.12, 70.27, 67.43, 54.86,
54.59, 50.77, 50.21, 47.93, 44.77, 40.13, 36.56, 36.23, 35.33, 33.36.
HRMS: calcd for C25H25NNaO6, 458.1579; found, 458.1578.
(1R*,2S*,3R*,4R*,6S*)-2-Benzyloxycarbonylamino-6-styryl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid (rac-22c). The com-
pound rac-20c (42 mg, 0.085 mmol) was treated following
Procedure E to yield after purification by preparative RP-HPLC
(A/C: 50/50 to 20/80 in 30 min, λ= 214 nm, tR =7.1 min) a
mixture of E/Z-isomers (ratio: approximately 50/50) of rac-22c
(25 mg, 0.057 mmol, 68%) as a white solid. 1H NMR (300 MHz,
MeOD) δ 7.55-6.98 (m, 10H), 6.42-6.08 (m, 1H þ 1H ꢀ 0.5),
5.63 (t, J = 10.8 Hz, 1H ꢀ 0.5), 5.10 (dd, J = 35.8, 12.8 Hz, 2H),
3.23-3.03 (m, 1H), 2.95 (br s, 1H), 2.81-2.55 (m, 2H), 1.99-
1.75 (m, 2H), 1.69 (d, J = 10.3 Hz, 1H), 1.43 (d, J = 13.7 Hz,
1H), 1.36-1.22 (m, 1H). 13C NMR (75 MHz, MeOD) δ 176.29,
175.73, 157.59, 139.05, 138.72, 138.41, 137.97, 137.47, 135.32,
130.34, 129.73, 129.49, 129.46, 129.42, 129.15, 129.01, 128.89,
128.76, 128.53, 127.97, 127.68, 127.09, 67.54, 65.09, 52.00, 51.78,
51.06, 50.82, 42.23, 42.02, 38.86, 36.01, 35.49, 34.27, 32.17.
HRMS: calcd for C25H25NNaO6, 458.1574; found, 458.1575.
(1S*,2S*,3R*,4S*,5R*)-2-Benzyloxycarbonylamino-5-styryl-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid (rac-22d). The com-
pound rac-20d (59 mg, 0.120 mmol) was treated following
Procedure E to yield after purification by preparative RP-HPLC
(A/C: 50/50 to 20/80 in 30 min, λ = 214 nm, tR = 7.0 min) a
mixture of E/Z-isomers (ratio: 57/43) of rac-22d (30 mg, 0.069
(1S*,2S*,3R*,4S*,5R*)-2-Amino-5-vinyl-bicyclo[2.2.1]heptane-
2,3-dicarboxylic Acid Trifluoroacetate (rac-23d). The compound
rac-21d (39 mg, 0.109 mmol) was treated following Procedure F to
yield the TFA-salt of rac-23d (23 mg, 0.068 mmol, 62%) as a white
solid (tR (RP-HPLC) = 9.9 min). 1H NMR (300 MHz, DMSO) δ
5.76 (ddd, J = 17.5, 10.6, 7.0 Hz, 1H), 5.10-4.80 (m, 2H), 3.44 (d,
J = 4.0 Hz, 1H), 2.50-2.42 (m, 2H, overlaid with solvent peak),
2.35 (d, J = 3.3 Hz, 1H), 2.05-1.86 (m, 1H), 1.79 (d, J = 9.9 Hz,
1H), 1.41-1.15 (m, 2H). 13C NMR (75 MHz, DMSO) δ 172.59,
172.46, 142.70, 113.06, 62.35, 48.93, 46.34, 45.01, 38.06, 33.31,
29.70. HRMS: calcd for C11H16NO4, 226.1079; found, 226.1081.
(1R*,2R*,3S*,4R*,6R*)-2-Amino-6-ethyl-bicyclo[2.2.1]heptane-
2,3-dicarboxylic Acid Trifluoroacetate (rac-24a). The compound
rac-21a (12 mg, 0.033 mmol) was treated following Procedure C
(using CH3CN/H2O (1/1) þ 0.5% TFA for washing) to yield the
TFA salt of rac-24a (12 mg, quant) as a white solid. 1H NMR (300
MHz, MeOD) δ 3.44 (s, 1H), 2.64 (s, 1H), 2.29 (s, 1H), 1.92 (d, J =
8.8 Hz, 1H), 1.81-1.52 (m, 3H), 1.25 (ddd, J = 20.5, 14.0, 7.3 Hz,
3H), 0.87 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz, MeOD) δ
173.45, 171.85, 67.39, 50.78, 41.21, 38.07, 37.24, 35.66, 33.42, 29.77,
12.05. HRMS: calcd for C11H18NO4, 228.1230; found, 228.1237.
(1S*,2R*,3S*,4S*,5S*)-2-Amino-5-ethyl-bicyclo[2.2.1]heptane-
2,3-dicarboxylic Acid Trifluoroacetate (rac-24b). The compound
rac-21b (13 mg, 0.036 mmol) was treated following Procedure C
(using CH3CN/H2O (1/1) þ 0.5% TFA for washing) to yield the
TFA salt of rac-24b (13 mg, quant) as a white solid. 1H NMR (300
MHz, MeOD) δ 3.43 (s, 1H), 2.46 (s, 1H), 1.95 (d, J = 10.0 Hz,
1H), 1.87-1.75 (m, 1H), 1.72-1.52 (m, 2H), 1.49-1.01 (m, 4H),
0.91 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, MeOD) δ 175.52,
172.88, 163.37 (TFA), 162.91 (TFA), 120.15 (TFA), 116.27
(TFA), 68.72, 53.71, 48.63, 43.85, 34.88, 33.28, 29.90, 12.48.
HRMS: calcd for C11H18NO4, 228.1230; found, 228.1235.
(1R*,2S*,3R*,4R*,6R*)-2-Amino-6-ethyl-bicyclo[2.2.1]heptane-
2,3-dicarboxylic Acid Trifluoroacetate (rac-24c). The compound
rac-21c (10 mg, 0.028 mmol) was treated following Procedure C
(using CH3CN/H2O (1/1) þ 0.5% TFA for washing) to yield the
TFA salt of rac-24c (10 mg, quant) as a white solid. 1H NMR (300
MHz, MeOD) δ 3.61 (s, 1H), 2.68 (s, 1H), 2.42 (s, 1H), 2.04-1.67
(m, 3H), 1.62-1.07 (m, 4H), 0.93 (t, J = 7.3 Hz, 3H). 13C NMR
(101 MHz, DMSO) δ 173.73, 172.81, 62.91, 50.37, 47.05, 40.47,
34.82, 32.64, 31.20, 28.17, 11.76. HRMS: calcd for C11H18NO4,
228.1230; found, 228.1235.
1
mmol, 57%) as a white solid. H NMR (300 MHz, MeOD) δ
7.50-7.07 (m, 10H), 6.42-6.28 (m, 1H), 6.17 (dd, J = 15.8, 7.7
Hz, 1H ꢀ 0.57), 5.66-5.48 (m, 1H ꢀ 0.43), 5.21-5.00 (m, 2H),
3.17-3.03 (m, 2H), 3.02-2.88 (m, 1H ꢀ 0.43), 2.73-2.31 (m,
2H), 2.09-1.84 (m, 2H), 1.74 (t, J = 11.6 Hz, 1H), 1.41-1.15
(m, 1H). 13C NMR (75 MHz, MeOD) δ 176.69, 175.45, 157.98,
138.95, 138.49, 137.62, 135.27, 130.06, 129.71, 129.47, 129.19,
128.99, 128.71, 128.02, 127.72, 127.10, 67.53, 64.55, 51.75, 51.30,
47.82, 46.83, 46.62, 39.86, 35.90, 35.68, 35.05, 34.57, 32.93.
HRMS: calcd for C25H25NNaO6, 458.1574; found, 458.1584.
Procedure F. (1R*,2R*,3S*,4R*,6S*)-2-Amino-6-vinyl-bicyclo-
[2.2.1]heptane-2,3-dicarboxylic Acid Trifluoroacetate (rac-23a).
The compound rac-21a (12 mg, 0.033 mmol) was heated to
80 °C in glacial acetic acid/36% HCl (2 mL/0.5 mL) for 2 h under
stirring. Cooling to room temperature and evaporation of the
solvent under vacuum gave the crude product. Purification by RP-
HPLC (A/B: 90/10 to 60/40 in 20 min, tR (RP-HPLC) = 13.4 min)
afforded the TFA-salt of rac-23a (6.8 mg, 0.020 mmol, 60%) as a
white solid. 1H NMR (300 MHz, MeOD) δ 5.77 (ddd, J = 17.2,
10.3, 7.0 Hz, 1H), 5.11-4.94 (m, 2H), 3.50 (d, J = 1.6 Hz, 1H),
2.69 (d, J = 2.6 Hz, 1H), 2.58 (br s, 1H), 2.33 (s, 1H), 1.97 (d, J =
11.4 Hz, 1H), 1.85-1.72 (m, 1H), 1.65 (d, J = 11.3 Hz, 1H), 1.50
(dt, J = 12.7, 4.4 Hz, 1H). 13C NMR (75 MHz, DMSO) δ 173.35,
171.95, 142.59, 113.00, 67.32, 52.07, 51.73, 41.06, 34.83, 33.75.
HRMS: calcd for C11H16NO4, 226.1079; found, 226.1082.
(1S*,2S*,3R*,4S*,5S*)-2-Amino-5-ethyl-bicyclo[2.2.1]heptane-
2,3-dicarboxylic Acid Trifluoroacetate (rac-24d). The compound
rac-21d (20 mg, 0.056 mmol) was treated following Procedure C
(using CH3CN/H2O (1/1) þ 0.5% TFA for washing) to yield the
TFA salt of rac-24d (19 mg, quant) as a white solid. 1H NMR (300
MHz, MeOD) δ 3.63 (d, J = 3.4 Hz, 1H), 2.63 (s, 1H), 2.51 (s,
1H), 2.05-1.85 (m, 2H), 1.79-1.63 (m, 1H), 1.52 (d, J = 10.3 Hz,
1H), 1.45-1.07 (m, 3H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR (75
MHz, MeOD) δ 174.11, 64.59, 50.43, 48.50, 45.90, 38.33, 34.34,
32.13, 30.01, 12.33. HRMS: calcd for C11H18NO4, 228.1230;
found, 228.1235.
(1S*,2R*,3S*,4S*,5R*)-2-Amino-5-vinyl-bicyclo[2.2.1]heptane-
2,3-dicarboxylic acid trifluoroacetate (rac-23b). The compound
rac-21b (14 mg, 0.039 mmol) was treated following Procedure F to
yield the TFA-salt of rac-23b (8.6 mg, 0.025 mmol, 65%) as a
1
white solid (tR (RP-HPLC) = 12.2 min). H NMR (300 MHz,
D2O) δ 5.80 (ddd, J = 17.4, 10.3, 7.2 Hz, 1H), 5.14-4.93 (m, 2H),
3.47 (d, J = 1.3 Hz, 1H), 2.64-2.50 (m, 2H), 2.45-2.28 (m, 1H),
1.90 (d, J = 11.4 Hz, 1H), 1.78-1.58 (m, 2H), 1.56-1.40 (m, 1H).
13C NMR (75 MHz, DMSO) δ 173.17, 172.02, 142.73, 112.99,
66.91, 52.54, 46.81, 46.70, 43.50, 34.01, 30.99. HRMS: calcd for
C11H16NO4, 226.1079; found, 226.1080.
(1R*,2S*,3R*,4R*,6S*)-2-Amino-6-vinyl-bicyclo[2.2.1]heptane-
2,3-dicarboxylic Acid Trifluoroacetate (rac-23c). The compound
rac-21c (12 mg, 0.033 mmol) was treated following Procedure F to
(1R*,2R*,3S*,4R*,6R*)-2-Amino-6-phenethyl-bicyclo[2.2.1]-
heptane-2,3-dicarboxylic Acid Trifluoroacetate (rac-25a). The
compound rac-22a (19 mg, 0.044 mmol) was treated following
Procedure C using CH3CN/H2O (1/1) þ 0.5% TFA for wash-
ing. The crude product (16 mg) was purified by RP-HPLC (A/B:
60/40 to 40/60 in 20 min, λ = 254 nm, tR = 9.0 min) to give the
TFA-salt of rac-25a (8 mg, 0.019 mmol, 44%) as a white solid.
1H NMR (300 MHz, MeOD) δ 7.37-7.00 (m, 5H), 3.46 (s, 1H),
2.64 (s, 1H), 2.56 (t, J = 7.9 Hz, 2H), 2.31 (s, 1H), 2.03-1.85

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7247
(m, 2H), 1.82-1.40 (m, 4H), 1.36-1.21 (m, 1H). 13C NMR (75
MHz, DMSO) δ 173.46, 172. 37, 142.09, 128.24, 128.18, 125.55,
67.57, 51.79, 51.25, 40.98, 37.79, 35.95, 35.15, 33.50. HRMS:
calcd for C17H22NO4, 304.1548; found, 304.1543.
(1S*,2R*,3S*,4S*,5S*)-2-Amino-5-phenethyl-bicyclo[2.2.1]-
heptane-2,3-dicarboxylic Acid (rac-25b). The compound rac-
22b (20 mg, 0.046 mmol) was treated following Procedure C to
afford rac-25b (11 mg, 0.035 mmol, 75%) as a white solid. 1H
NMR (300 MHz, DMSO) δ 7.39-7.03 (m, 5H), 3.18 (s, 1H),
2.58-2.52 (m, 2H, overlaid with solvent peak), 2.41 (s, 1H),
2.01 (d, J = 11.2 Hz, 1H), 1.69-1.00 (m, 7H). 13C NMR (75
MHz, DMSO) δ 173.39, 172.04, 142.19, 128.25, 128.18, 125.55,
66.83, 52.89, 46.72, 45.85, 37.65, 33.86, 33.46, 32.08. HRMS:
calcd for C17H22NO4, 304.1543; found, 304.1553.
154.87, 153.12, 136.38, 135.96, 128.60, 128.50, 128.36, 128.30,
128.24, 128.12, 127.19, 124.85, 123.63, 120.61, 115.94, 67.74,
66.95, 61.48, 60.72, 60.58, 57.15, 52.72, 40.74, 38.20, 35.02, 31.35,
29.78, 14.27, 14.06. HRMS: calcd for C29H33NNaO7, 530.2149;
found, 530.2154.
(1R*,2R*,3S*,4R*,6S*)-2-Benzyloxycarbonylamino-6-[2-(4-
chloro-phenyl)-vinyl]-bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Di-
ethyl Ester (rac-26d). The compound rac-18a (457 mg, 1.10 mmol)
and (4-chlorobenzyl)triphenylphosphonium chloride (1.159 g, 2.737
mmol) were treated following Procedure D to yield after purification
by flash chromatography (hexane/EtOAc: 80/20) a mixture of E/Z-
isomers (ratio: 64/36) of rac-26d (294 mg, 0.559 mmol, 51%) as a
colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.55-7.28 (m, 9H),
6.52-6.33(m, 1H), 6.24(dd, J= 15.8, 7.4 Hz, 0.66H), 5.67 (dd, J=
11.5, 9.6 Hz, 0.37H), 5.39 (d, J = 5.1 Hz, 1H), 5.32-5.10 (m, 2H),
4.42-3.99 (m, 4H), 3.41-3.19 (m, 1H), 3.05 (s, 0.51H), 3.01-2.86
(m, 1H), 2.79 (br s, 1H), 2.52-2.37 (m, 0.49H), 2.30-2.21 (m,
0.51H), 2.03-1.68 (m, 2H), 1.59-1.21 (m, 7H), 1.12 (t, J = 7.1 Hz,
1H). 13C NMR (75 MHz, CDCl3) δ 170.73, 170.26, 154.83, 137.28,
136.48, 135.82, 132.56, 130.16, 128.67, 128.43, 128.32, 127.38,
127.10, 67.91, 66.91, 61.66, 60.61, 57.14, 53.26, 40.29, 35.36, 33.95,
32.85, 14.30, 13.77. HRMS: calcd for C29H32ClNNaO6, 548.1810;
found, 548.1819.
(1R*,2S*,3R*,4R*,6R*)-2-Amino-6-phenethyl-bicyclo[2.2.1]-
heptane-2,3-dicarboxylic Acid Trifluoroacetate (rac-25c). The
compound rac-22c (9 mg, 0.021 mmol) was treated following
Procedure C to afford after purification by RP-HPLC (A/B:
60/40 to 40/60 in 20 min, λ = 254 nm, tR = 11.2 min) the TFA
1
salt of rac-25c (4 mg, 0.010 mmol, 46%) as a white solid. H
NMR (300 MHz, MeOD) δ 7.37-7.04 (m, 5H), 3.22 (d, J = 3.0
Hz, 1H), 2.73-2.46 (m, 3H), 2.40-2.25 (m, 2H), 1.97-1.58 (m,
3H), 1.46 (d, J = 10.6 Hz, 2H), 1.36-1.26 (m, 1H), 1.15 (d, J =
11.2 Hz, 1H). 13C NMR (75 MHz, DMSO) δ 173.33, 172.75,
158.05 (TFA), 157.64 (TFA), 142.29, 128.25, 128.18, 125.61,
119.21 (TFA), 115.24 (TFA), 62.96, 50.69, 47.12, 37.75, 33.34,
32.75, 31.37. HRMS: calcd for C17H22NO4, 304.1543; found,
304.1555.
(1S*,2S*,3R*,4S*,5S*)-2-Amino-5-phenethyl-bicyclo[2.2.1]-
heptane-2,3-dicarboxylic Acid (rac-25d). The compound rac-
22d (10 mg, 0.023 mmol) was treated following Procedure C to
afford rac-25d (7 mg, 0.023 mmol, quant) as a white solid. 1H
NMR (300 MHz, MeOD) δ 7.37-7.01 (m, 5H), 3.52 (s, 1H),
2.74-2.44 (m, 4H), 2.19 (d, J = 10.1 Hz, 1H), 1.98-1.40 (m,
5H), 1.17 (d, J = 12.0 Hz, 1H). 13C NMR (75 MHz, DMSO) δ
173.86, 173.53, 142.35, 128.28, 128.15, 125.48, 62.00, 49.23,
46.20, 44.56, 38.69, 38.31, 34.66, 33.50, 33.24, 31.40. HRMS:
calcd for C17H22NO4, 304.1543; found, 304.1551.
(1R*,2R*,3S*,4R*,6S*)-2-Benzyloxycarbonylamino-6-prope-
nyl-bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Diethyl Ester
(rac-26a). The compound rac-18a (150 mg, 0.359 mmol) and
ethyltriphenylphosphonium bromide (334 mg, 0.898 mmol)
were treated following Procedure D to yield after purification
by flash chromatography (hexane/EtOAc: 80/20) a mixture of
E/Z-isomers of rac-26a (136 mg, 0.317 mmol, 88%, purity
∼93% (NMR)) as a colorless oil. 1H NMR (400 MHz, CDCl3)
δ 7.42-7.28 (m, 5H), 5.43-5.17 (m, 2H), 5.16-4.92 (m, 3H),
4.23-4.01 (m, 4H), 3.14-3.00 (m, 1H), 2.79 (dd, J = 4.2, 1.5 Hz,
1H), 2.70-2.53 (m, 2H), 2.22 (ddd, J = 12.8, 8.6, 2.4 Hz, 1H),
1.79-1.48 (m, 6H), 1.22 (dt, J = 17.8, 7.1 Hz, 7H). 13C NMR
(101 MHz, CDCl3) δ 170.94, 170.26, 154.68, 136.52, 135.82,
128.65, 128.55, 128.31, 123.98, 67.77, 66.88, 61.41, 60.60, 57.16,
53.31, 40.69, 35.18, 32.69, 32.48, 14.33, 14.15, 13.42. HRMS:
calcd for C24H31NNaO6, 452.2044; found, 452.2049.
(1R*,2R*,3S*,4R*,6S*)-2-Benzyloxycarbonylamino-6-[2-(2-
hydroxy-phenyl)-vinyl]-bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid
Diethyl Ester (rac-26c). The compound rac-18a (100 mg, 0.240
mmol), (2-hydroxybenzyl)triphenylphosphonium bromide (270
mg, 0.598 mmol), and 2.5 M n-butyllithium in hexane (0.48 mL,
1.198 mmol) were treated following Procedure D to yield after
purification by flash chromatography (hexane/EtOAc: 80/20 to
50/50) a mixture of E/Z-isomers of rac-26c (92 mg, 0.181 mmol,
76%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 7.39-7.26
(m, 6H), 7.07 (ddd, J = 9.6, 5.6, 1.7 Hz, 1H), 6.88-6.76 (m, 2H),
6.60 (d, J = 16.0 Hz, 0.74H), 6.11 (dd, J = 15.9, 7.4 Hz, 0.77H),
5.25-4.95 (m, 3H), 4.33-3.95 (m, 4H), 3.16-2.99 (m, J = 12.7,
7.8 Hz, 0.71H), 2.88 (s, 0.77H), 2.81 (d, J = 3.2 Hz, 0.68H), 2.65 (s,
0.87H), 2.34-2.22 (m, 0.73H), 2.16 (ddd, J = 12.8, 8.6, 2.0 Hz,
0.77H), 1.69 (dd, J = 38.2, 10.7 Hz, 2H), 1.49-1.36 (m, 1H),
1.32-1.10 (m, 7H). 13C NMR (75 MHz, CDCl3) δ 170.98, 170.33,
(1R*,2R*,3S*,4R*,6S*)-2-Benzyloxycarbonylamino-6-prope-
nyl-bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid (rac-27a). The
compound rac-26a (123 mg, 0.317 mmol) was treated following
Procedure E to yield after purification by preparative RP-HPLC
(A/C: 60/40 to 30/70 in 30 min, λ = 214 nm, tR = 17.8 min) a
mixture of E/Z-isomers of rac-27a (49 mg, 0.131 mmol, 46%) as
a white solid. 1H NMR (300 MHz, MeOD) δ 7.68-7.00 (m, 5H),
5.51-5.19 (m, 2H), 5.08-4.96 (m, 2H), 3.55 (s, 1H), 2.66-2.53
(m, 1H), 2.46 (d, J = 2.8 Hz, 1H), 2.31 (d, J = 10.7 Hz, 1H), 2.15
(s, 1H), 1.79-1.65 (m, 1H), 1.59 (d, J = 5.9 Hz, 4H), 1.29 (dt,
J = 12.4, 4.4 Hz, 1H). 13C NMR (75 MHz, MeOD) δ 176.06,
174.97, 157.77, 138.20, 136.42, 129.35, 128.80, 128.64, 124.67,
70.60, 67.40, 54.08, 53.87, 44.17, 38.32, 36.10, 34.59, 13.19.
HRMS: calcd for C20H23NNaO6, 396.1418; found, 396.1421.
(1R*,2R*,3S*,4R*,6S*)-2-Benzyloxycarbonylamino-6-but-1-
enyl-bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid (rac-27b). The
compound rac-18a (200 mg, 0.479 mmol) and propyltriphenyl-
phosphonium bromide (461 mg, 1.198 mmol) were treated fol-
lowing Procedure D to yield after purification by flash chroma-
tography rac-26b as a colorless oil (169 mg, purity 65% (NMR)).
This product (140 mg) was treated as described above for
the synthesis of rac-21a to yield after purification by prepara-
tive RP-HPLC (A/C: 60/40 to 40/60 in 20 min, λ= 214 nm, tR =
12.0 min) a mixture of E/Z-isomers of rac-27b (60 mg, 0.155
1
mmol, 39%) as a white solid. H NMR (300 MHz, MeOD) δ
7.48-7.16 (m, 5H), 5.38-5.16 (m, 2H), 5.10-4.94 (m, 2H), 3.54
(d, J = 1.4 Hz, 1H), 2.64-2.51 (m, 1H), 2.45 (d, J = 2.7 Hz, 1H),
2.42-2.25 (m, 1H), 2.14 (s, 1H), 2.11-1.95 (m, 2H), 1.69 (ddd,
J = 12 Hz, 7.5 Hz, 2.2 Hz, 1H), 1.59 (dd, J = 10.7, 0.9 Hz, 1H),
1.34-1.23 (m, 1H), 0.94 (t, J = 7.5 Hz, 3H). 13C NMR (75 MHz,
MeOD) δ 176.05, 174.93, 157.74, 138.16, 134.83, 132.38, 129.34,
128.79, 128.62, 70.59, 67.39, 54.04, 44.14, 38.45, 36.05, 34.70,
21.74, 14.56. HRMS: calcd for C13H22NO4, 256.1543; found,
256.1549.
(1R*,2R*,3S*,4R*,6S*)-2-Benzyloxycarbonylamino-6-[2-(4-
chloro-phenyl)-vinyl]-bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid
(rac-27d). The compound rac-26d (96 mg, 0.182 mmol) was
treated following Procedure E to yield after purification by
preparative RP-HPLC (A/C: 50/50 to 30/70 in 20 min, λ = 214
nm, tR = 14.0 min) a mixtureof E/Z-isomers(ratio: 51/49) of rac-
27d (37 mg, 0.079 mmol, 43%) as a white solid. 1H NMR (300
MHz, MeOD) δ 7.46-7.07 (m, 9H), 6.38-6.25 (m, 1H), 6.18 (dd,
J = 15.8, 7.4 Hz, 0.49H), 5.59 (dd, J = 11.5, 10.0 Hz, 0.48H),
5.09-4.95 (m, 2H), 3.62-3.47 (m, 1H), 2.88-2.74 (m, 1H),
2.56-2.25 (m, 3.47H), 1.83-1.62 (dd, J = 20.4, 10.0 Hz, 2H),
1.51 (dt, J = 12.6, 4.4 Hz, 0.53H), 1.38 (dt, J = 12.5, 4.3 Hz,
0.64H). 13C NMR (75 MHz, MeOD) δ 175.99, 174.84, 157.80,

7248 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Luethi et al.
138.22, 137.87, 137.60, 136.98, 135.89, 133.66, 133.57, 131.26,
129.59, 129.41, 129.37, 129.09, 128.82, 128.68, 128.65, 128.55,
127.90, 70.71, 70.46, 67.42, 54.32, 53.48, 44.17, 40.62, 38.90,
36.94, 36.37, 36.05, 35.77. HRMS: calcd for C25H24ClNNaO6,
492.1184; found, 492.1186.
(1R*,2R*,3S*,4R*,6R*)-2-Amino-6-propyl-bicyclo[2.2.1]-
heptane-2,3-dicarboxylic Acid Trifluoroacetate (rac-28a). The
compound rac-27a (22 mg, 0.059 mmol) was treated following
ProcedureC(using CH3CN/H2O (1/1) þ 0.5% TFA for washing)
to yield rac-28a (20 mg, 0.056 mmol, 96%) as a pale-yellow solid.
1H NMR (300 MHz, MeOD) δ 3.41 (s, 1H), 2.69 (s, 1H), 2.37 (s,
1H), 2.09-1.90 (m, 1H), 1.81-1.58 (m, 3H), 1.45-1.15 (m, 5H),
0.90 (t, J = 6.7 Hz, 3H). 13C NMR (75 MHz, DMSO) δ 172.42,
170.48, 67.40, 51.26, 50.35, 41.27, 37.28, 35.84, 34.77, 33.26,
20.21, 13.72. HRMS: calcd for C12H20NO4, 242.1387; found,
242.1391.
with 1 N HCl and extracted with EtOAc (4ꢀ). The organic
phases were combined, washed with brine, dried over Na2SO4,
and evaporated to provide 1.57 g of a white solid. Purification of
300 mg of this product by flash chromatography (EtOAc/
hexane: 5/5 to 8/2) gave 2-cyano-2-hydroxycyclopentanecar-
boxylic acid (66 mg) and a mixture of 29 and its ethyl ester (161
mg). A 505 mg portion of the 1.57 above was stirred in 4 N HCl
(10 mL) at 40 °C for 24 h. The solvent was evaporated under
reduced pressure to give crude 29 (376 mg) as a white solid,
which was used without further purification in the next step.
Purification of an 80 mg portion by RP-HPLC (A/B: 100/0 to
70/30 in 30 min) gave pure 29 (50 mg, 0.252 mmol, 38%,
diastereomeric ratio: 84/16) as a white solid. 1H NMR (300
MHz, MeOD) δ 3.23 (dd, J = 10.4, 8.9 Hz, 1H ꢀ 0.84), 3.11 (dd,
J = 10.3, 9.3 Hz, 1H ꢀ 0.16), 2.34-1.69 (m, 6H). 13C NMR (75
MHz, DMSO) δ 177.47, 172.58, 171.62, 156.73, 69.87, 68.72,
52.18, 37.39, 36.90, 27.25, 25.78, 22.14, 21.85. HRMS: calcd for
C8H10N2NaO4, 221.0538; found, 221.0539.
1-Amino-cyclopentane-1,2-dicarboxylic Acid Hydrochloride
Salt (11). Crude 29 (70 mg, 0.353 mmol) was stirred in a sealed
tube at 130 °C for 5 h. The solvent was removed under reduced
pressure, and the yellow solid was purified by anion exchange
chromatography (Dowex 1 ꢀ 8, 100-200 mesh, AcO form). The
crude was dissolved in a minimum amount of H2O, adjusted to
pH 9 with 1 N NaOH and deposited on the column. After
washing the resin with H2O, the amino acid was eluted with 0.5
M AcOH and the ninhydrine positive fractions were evaporated
to dryness. After treatment with diluted HCl (3ꢀ), 11 (23 mg,
0.133 mmol, 19% overall yield, diastereomeric ratio: 58/42) was
obtained as a white solid. 1H NMR (300 MHz, D2O) δ 3.43 (t,
J=9.0 Hz, 1H ꢀ 0.58), 3.10 (t, J=9.4 Hz, 1H ꢀ 0.42), 2.43-
1.68 (m, 6H). 13C NMR (75 MHz, D2O) δ 174.82, 174.26,
173.49, 173.36, 66.38, 66.26, 52.96, 51.01, 36.21, 27.78, 26.63,
22.39, 21.64. HRMS: calcd for C7H12NO4, 174.0766; found,
174.0771.
2,4-Dioxo-1,3-diaza-spiro[4.5]decane-6-carboxylic Acid (30).
The synthesis was adapted from literature.32,33 Ammonium
carbonate (1.86 g, 19.4 mmol) and KCN (459 mg, 7.05 mmol)
were added to ethyl-2-oxocyclohexane carboxylate (0.94 mL, 5.9
mmol) in EtOH/H2O (6.3 mL/6.3 mL). The reaction was stirred at
50 °C for 8 h. After evaporation of EtOH, the suspension was
extracted with EtOAc (3 times). The combined organic phases were
washed with H2O and brine, dried over MgSO4, and evaporated to
yield crude ethyl ester (1.20 g) as a white solid. The crude product
(150 mg) was stirred in 4 N HCl (3 mL) at 50 °C for 2 days.
Evaporation of the solvent and purification of the residue by RP-
HPLC (A/B: 90/10 to 60/40 in 30 min) yielded 30 (81 mg, 0.382
mmol, 52%, diastereomeric ratio: 84/16) as a white solid. 1H NMR
(300 MHz, MeOD) δ 2.83 (dd, J = 13.0, 4.2 Hz, 1H ꢀ 0.84), 2.62
(dd, J = 12.9, 4.0 Hz, 1H ꢀ 0.16), 2.33-1.19 (m, 8H). 13C NMR
(75 MHz, MeOD) δ 180.73, 179.21, 175.63, 174.40, 159.75, 159.51,
65.44, 62.59, 52.20, 47.43, 36.75, 36.62, 27.09, 25.83, 25.53, 25.33,
22.01, 21.94. HRMS: calcd for C9H12N2NaO4, 235.0695; found,
235.0696.
1-Amino-cyclohexane-1,2-dicarboxylic Acid Hydrochloride
Salt (10). 30 (69 mg, 0.289 mmol) was stirred in 6 N HCl
(2 mL) at 110 °C for 7 days. After removal of the solvent under
vacuum, the crude was purified by cation exchange chromato-
graphy (Dowex 50 ꢀ 8, 20-50 mesh, Hþ form). The yellow solid
was loaded on the column at pH 2 in a minimum amount of
H2O, the resin was washed with MeOH and H2O, and the
product was eluted with 0.5 N NaOH. The ninhydrine active
fractions were combined and evaporated to dryness. The residue
was purified by anion exchange chromatography (Dowex 1 ꢀ 8,
100-200 mesh, AcO form). It was loaded at pH 10 on the
column, the resin was washed with MeOH and H2O, and the
product was eluted with 1 N AcOH. The ninhydrine active
fractions were evaporated and the product was treated with 1 N
HCl (3 times) to yield the HCl salt of 10 (23 mg, 0.104 mmol, 36%,
diastereomeric ratio 81/19) as a white solid. 1H NMR (300 MHz,
(1R*,2R*,3S*,4R*,6R*)-2-Amino-6-butyl-bicyclo[2.2.1]heptane-
2,3-dicarboxylic Acid Trifluoroacetate (rac-28b). The compound
rac-27b (35 mg, 0.090 mmol) was treated following Procedure C
(using CH3CN/H2O (1/1) þ 0.5% TFA for washing) to yield rac-
1
28b (33 mg, 0.089 mmol, quant) as a white solid. H NMR (300
MHz, MeOD) δ 2.61 (s, 1H), 2.16 (s, 1H), 1.98 (s, 1H), 1.77 (d, J =
11.4 Hz, 2H), 1.56 (d, J= 10.7 Hz, 1H), 1.46-1.03 (m, 8H), 0.88 (t,
J = 6.8 Hz, 3H). 13C NMR (75 MHz, DMSO) δ 172.51, 158.04
(TFA), 115.81 (TFA), 115.34 (TFA), 67.61, 51.55, 50.84, 41.15,
36.02, 35.12, 34.98, 33.33, 29.44, 21.97, 13.92. HRMS: calcd for
C13H22NO4, 256.1543; found, 256.1549.
(1R*,2R*,3S*,4R*,6R*)-2-Amino-6-[2-(2-hydroxy-phenyl)-
ethyl]-bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Trifluoroa-
cetate (rac-28c). The compound rac-26c (90 mg, 0.177 mmol)
was treated following Procedure E to yield after purification
by preparative RP-HPLC (A/C: 65/35 to 35/65 in 30 min, λ =
214 nm, tR = 16.2 min) rac-27c as a white solid (45 mg, di-
carboxylic acid with side product, purity ∼80%). The dicar-
boxylic acid (30 mg) was treated following Procedure C (using
CH3CN/H2O (1/1) þ 0.5% TFA for washing) to afford after
purification by RP-HPLC (A/B: 70/30 to 40/60 in 30 min, λ =
254 nm, tR = 26.2 min) the TFA salt of rac-28c (14 mg, 0.032
mmol, 27%) as a white solid. 1H NMR (300 MHz, MeOD) δ
6.94-6.80 (m, 2H), 6.72-6.48 (m, 2H), 3.35 (s, 1H), 2.61-2.51
(m, 1H), 2.44 (ddd, J = 8.7, 6.8, 2.2 Hz, 2H), 2.26 (s, 1H),
1.92-1.73 (m, 2H), 1.70-1.29 (m, 4H), 1.27-1.12 (m, 2H).
13C NMR (75 MHz, DMSO) δ 172.91, 171.40, 154.90, 129.63,
128.09, 126.64, 118.80, 114.81, 67.47, 51.54, 50.88, 41.09,
35.92, 35.81, 35.47, 33.45, 28.01. HRMS: calcd for C17H22
NO5, 320.1492; found, 320.1499.
-
(1R*,2R*,3S*,4R*,6R*)-2-Amino-6-[2-(4-chloro-phenyl)-ethyl]-
bicyclo[2.2.1]heptane-2,3-dicarboxylic Acid Trifluoroacetate (rac-
28d). The compound rac-27d (20 mg, 0.043 mmol) and 10% Pt on
charcoal in EtOH (2 mL) were stirred under a 1 atm H2 atmo-
sphere for 4 h. The reaction was filteredthroughapadofCelite, the
residue was washed with CH3CN/H2O (1/1) þ 0.5% TFA, and the
filtrate was evaporated to dryness under vacuum. AcOH/HCl (2/1,
5 mL) was added, and the reaction was stirred at 80 °C for 90 min.
After evaporation of the solvent, the crude product was purified by
RP-HPLC (A/B: 50/50 to 30/70 in 20 min, λ = 254 nm, tR = 5.2
min) to afford the TFA salt of rac-28d (13 mg, 0.013 mmol, 67%)
1
as a white solid. H NMR (300 MHz, MeOD) δ 7.37-7.04 (m,
4H), 3.45 (s, 1H), 2.71-2.62 (m, 1H), 2.56 (t, J = 7.7 Hz, 2H), 2.31
(s, 1H), 2.02-1.80 (m, 2H), 1.80-1.38 (m, 4H), 1.28-1.15 (m,
1H). 13C NMR (75 MHz, DMSO) δ 173.51, 141.07, 130.16,
128.07, 67.55, 51.87, 51.19, 40.93, 37.46, 35.90, 34.89, 33.53,
32.68. HRMS: calcd for C17H20ClNO4, 337.1132; found, 337.1138.
2,4-Dioxo-1,3-diaza-spiro[4.4]nonane-6-carboxylic Acid (29).
The synthesis was adapted from literature.31 2-Oxo-cyclopenta-
necarboxylic acid ethyl ester (1.39 mL, 9.60 mmol) was added to
a suspension of ammonium carbonate (3.97 g, 41.37 mmol) and
KCN (1.50 g, 23.06 mmol) in EtOH/H2O (10.2 mL/10.2 mL).
The mixture was heated to 60 °C and stirred for 2 days. After
evaporation of EtOH, the aqueous residue was acidified to pH 3

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7249
D2O) δ 3.27 (dd, J = 12.9, 4.2 Hz, 1H ꢀ 0.81), 2.83 (dd, J = 12.1,
5.2 Hz, 1H ꢀ 0.19), 2.31-1.28 (m, 8H). 13C NMR (75 MHz, D2O)
δ 176.03, 175.93, 174.25, 172.67, 60.89, 60.21, 47.03, 45.49, 33.32,
32.42, 31.10, 25.42, 23.56, 23.34, 20.66, 18.93. HRMS: calcd for
C8H14NO4, 188.0923; found, 188.0918.
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Activity Assays. All compounds tested had g95% purity and
were conditioned as 8-100 mM stock solutions in DMSO.
Glutamate Transporter Assays.46 [3H]-Glutamate Uptake in
Human GLT-1-Transfected HEK293 Cells. The human embryo-
nic kidney cell line (HEK293) was purchased from American
Type Culture Collection (ATCC) and was cultured following
the instruction of ATCC. Cells were placed in 12-well plates
(1.5 ꢀ 105 cells/well) with poly-D-lysine coating. The human GLT-1
cDNA in pcDNA3.1 mammalian expression vector (Invitrogen)
was introduced into the cells using FuGENE6 transfection
reagent (Roche) following the manufacturer’s instruction. After
1 day of transfection, the medium was removed and the cells
were treated with 450 μL of HEPES-buffered saline (HBSS:
140 mM NaCl, 2.5 mM KCl, 1.2 mM MgCl2, 1.2 mM CaCl2, 1.2
mM K2HPO4, 10 mM glucose, 5 mM Tris, 10 mM HEPES
(pH 7.4)) with or without glutamate or aspartate analogue.
The uptake assay was started by adding 50 μL glutamate solution
(100 μM) containing 0.2 μCi/well of [3H]-glutamate (PerkinElmer).
After 10 min uptake at room temperature, the medium was removed
and the cells were washed with ice-cold HBSS twice then lysed with
0.5 M NaOH overnight. The radioactivity was measured by a
β-counter using scintillation solution. To eliminate a possibility of
nonspecific effect of the compound to the endogenous transporters
in the HEK293 cells, HEK293 cells without vector transfection were
analyzed with or without compound as well.
[3H]-Glutamate Uptake in Human GLT-1-Expressed Xenopus
Oocytes. Xenopus laevis oocytes were prepared as described pre-
viously. (1) The capped RNA was synthesized using SP6 poly-
merase (mMessage mMachine SP6 kit, Ambion) and human
GLT-1 cDNA in pTLNII Xenopus expression vector linealized
by Mlu I. The synthesized cRNA was injected into the oocytes
(5 ng/oocyte) and were incubated with MBM (88 mM NaCl,
1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.66 mM
NaNO3, 0.75 mM CaCl2, 10 mM HEPES (pH 7.5 with Tris
base), 100 units/mL penicillin, 0.1 mg/mL streptomycin) for
2 days at 17 °C. The uptake assays of radiolabeled glutamate
([3H]-glutamate, PerkinElmer) were carried out as described
before (2) in Naþ transport medium (100 mM NaCl, 2 mM KCl,
1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES (pH 7.5 with Tris
base), 10 μM cold glutamate) in the presence of 0.5 μCi of [3H]-
glutamate for each group with or without the glutamate or
aspartate analogue. To eliminate a possibility of nonspecific
effect of the compound to the endogenous transporters in the
oocytes, H2O-injected oocytes were analyzed with or without
compound as well. After 10 min uptake at room temperature,
the oocytes were washed with ice-cold Naþ transport medium,
and 200 μL of this solution containing a single oocyte was placed
into a new vial and treated with 200 μL of 10% SDS overnight.
The radioactivity of this lysate was measured by a β-counter
after the treatment of 5 mL of scintillation liquid overnight.
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sion and pharmacology of the human excitatory amino acid
transporter 2 subtype of L-glutamate transporter. Br. J. Pharmacol.
1999, 128, 1485–1490.
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D. J.; Bolte, J.; Gefflaut, T.; Bunch, L. Stereoselective chemoenzy-
matic synthesis of the four stereoisomers of l-2-(2-carboxycyclo-
butyl)glycine and pharmacological characterization at human
excitatory amino acid transporter subtypes 1, 2, and 3. J. Med.
Chem. 2006, 49, 6532–6538.
(20) Esslinger, C. S.; Koch, H. P.; Kavanaugh, M. P.; Philips, D. P.;
Chamberlin, A. R.; Thompson, C. M.; Bridges, R. J. Structural
determinants of substrates and inhibitors: probing glutamate
transporters with 2,4-methanopyrrolidine-2,4-dicarboxylate. Bioorg.
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Acknowledgment. This work was supported financially by
the University of Berne and the Swiss National Science
Foundation.
Supporting Information Available: Statistics of virtual aspar-
tate and glutamate library composition, images of 1H and 13
C
NMR spectra for all compounds, crystallography. This material
is available free of charge via the Internet at http://pubs.acs.org.
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at human glutamatetransporter subtypes 1, 2, and 3. J. Med. Chem.
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