Development of 2′-substituted (2S,1′ R,2′S)-2- (carboxycyclopropyl)glycine analogues as potent N-methyl-d-aspartic acid receptor agonists

Article abstract of DOI:10.1021/jm400346a

A series of 2′-substituted analogues of the selective NMDA receptor ligand (2S,1′R,2′S)-2-(carboxycyclopropyl)glycine ((S)-CCG-IV) have been designed, synthesized, and pharmacologically characterized. The design was based on a docking study hypothesizing that substituents in the 2′-position would protrude into a region where differences among the NMDA receptor GluN2 subunits exist. Various synthetic routes were explored, and two different routes provided a series of alkyl-substituted analogues. Pharmacological characterization revealed that these compounds are NMDA receptor agonists and that potency decreases with increasing size of the alkyl groups. Variations in agonist activity are observed at the different recombinant NMDA receptor subtypes. This study demonstrates that it is possible to introduce substituents in the 2′-position of (S)-CCG-IV while maintaining agonist activity and that variation among NMDA receptor subtypes may be achieved by probing this region of the receptor.

Full text of DOI:10.1021/jm400346a

Article
pubs.acs.org/jmc
Development of 2′-Substituted (2S,1′R,2′S)‑2-
(Carboxycyclopropyl)glycine Analogues as Potent
N‑Methyl‑D‑aspartic Acid Receptor Agonists
Rune Risgaard,† Simon D. Nielsen,† Kasper B. Hansen,†,‡ Christina M. Jensen,† Birgitte Nielsen,†
Stephen F. Traynelis,‡ and Rasmus P. Clausen*,†
†Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2
Universitetsparken, DK-2100 Copenhagen, Denmark
‡Department of Pharmacology, Emory University School of Medicine, Rollins Research Center, Atlanta, Georgia, United States
ABSTRACT: A series of 2′-substituted analogues of the
selective NMDA receptor ligand (2S,1′R,2′S)-2-
(carboxycyclopropyl)glycine ((S)-CCG-IV) have been de-
signed, synthesized, and pharmacologically characterized. The
design was based on a docking study hypothesizing that
substituents in the 2′-position would protrude into a region
where differences among the NMDA receptor GluN2 subunits
exist. Various synthetic routes were explored, and two different
routes provided a series of alkyl-substituted analogues.
Pharmacological characterization revealed that these com-
pounds are NMDA receptor agonists and that potency
decreases with increasing size of the alkyl groups. Variations
in agonist activity are observed at the different recombinant
NMDA receptor subtypes. This study demonstrates that it is possible to introduce substituents in the 2′-position of (S)-CCG-IV
while maintaining agonist activity and that variation among NMDA receptor subtypes may be achieved by probing this region of
the receptor.
INTRODUCTION
■
The glutamate receptors (GluRs) are a group of receptors with
a prominent role in neurotransmission because most of the
excitatory signals in the brain are mediated by glutamate
(Figure 1).1−3 These receptors are involved in key processes of
the central nervous system (CNS) such as learning and
memory4 but are also implicated in several neuropathological
conditions such as ischemia, epilepsy, schizophrenia, chronic
pain, and Alzheimer’s disease.5 Therefore, the GluRs have been
the target of extensive research for more than three decades,
and one of the goals has been to develop compounds that can
alter glutamatergic neurotransmission in a selective manner.
Such compounds have been and continue to be important tools
in discovering new therapeutic possibilities and to gain a
functional understanding of this group of receptors.6
Two different signaling pathways divide the GluRs:
metabotropic glutamate receptors (mGluRs) are G-protein-
Figure 1. Structures of glutamic acid (Glu), (2S,4R)-4-methylgluta-
mate (1b), and N-methyl-D-aspartic acid (NMDA, 2), and structures
coupled receptors that mediate slow modulatory responses, and
ionotropic glutamate receptors (iGluRs) are cation-conducting
ion channels that mediate fast neurotransmission via depola-
rization of the membrane potential. On the basis of activation
by selective ligands and sequence homology, the iGluRs are
divided into N-methyl-D-aspartic acid (NMDA), (S)-2-amino-
3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA),
and kainic acid (KA) receptors. The iGluRs are homo- or
of the NMDA receptor selective ligands 3a and 4a and a series of alkyl
substituted analogues.
Received: March 7, 2013
Published: April 24, 2013
© 2013 American Chemical Society
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Figure 2. (A) X-ray crystal structure (PDB: 2A5T) of the ABD of GluN2A (blue) containing Glu (1, blue), and 4a (pink) docked in a model of
GluN2A ABD created from the crystal structure using the protein preparation module and Glide in the Schrodinger Suite. (B) 3b (light blue) and 4d
(green) docked in GluN2A ABD, demonstrating the overlap of the alkyl groups in the 2′ position of 4a. (C) Broader view of GluN2A ABD showing
the orientation of 4d in the clamshell-like structure formed by the upper (S1) and lower (S2) lobes of the ABD.
heteromeric assemblies of four subunits. Seven NMDA
(GluN1, GluN2A−D, GluN3A−B), four AMPA (GluA1−4),
and five KA (GluK1−5) receptor subunits have been
identified.3 The majority of native NMDA receptors require
assembly of two GluN1 subunits together with two GluN2
subunits. GluN3 subunits can also assemble with GluN1 and
GluN2 subunits, but the function and physiological role of
these triheteromeric GluN1/GluN2/GluN3 receptors are less
well understood. NMDA receptors composed of GluN1 and
GluN2 subunits (i.e., GluN1/GluN2) are activated by the
simultaneous binding of the endogenous agonists glycine or
(R)-serine to GluN1 and glutamate to GluN2. Over the past
decade, an increasing number of crystal structures of the agonist
binding domains (ABDs) of the iGluRs have appeared (86
available structures to date, covering all the major mammalian
GluR groups as well as bacterial homologues), providing
detailed structural knowledge of agonist binding and the
molecular pharmacology of this group of receptors.2,7−11 These
crystallographic structures have shown that iGluR ABDs are
clamshell-like structures, where the agonist binds in the cleft
formed by two lobes. Upon agonist binding, the two lobes close
around the agonist, whereas binding of antagonists stabilizes an
open domain structure.
We have previously reported the synthesis and pharmaco-
logical characterization of a series of 4-substituted (RS)-2-(N-
hydroxypyrazol-5-yl)glycine ((RS)-NHP5G, 3a) analogues as
NMDA receptor ligands.12−14 This series demonstrated that
introduction of substituents at the 4-position of 3a led to
compounds that displayed different efficacies on different
GluN2 subtypes. Thus, 4-propyl substituted 3b appears to be a
functional antagonist at the GluN1/GluN2A subtype but a
partial agonist at the GluN1/GluN2D subtype. However, the
affinities and potencies of the compounds in this series were
weak.15 Furthermore, we have previously shown that the highly
potent (2S,4R)-4-methyl glutamate (SYM2081, 1b) shows a
significant difference in potency at GluN2 subunits.16 To
improve potency we exploited docking studies in the ABD of
the GluN2A receptor subunit to design, synthesize, and
characterize a series of compounds based on the most potent
NMDA receptor agonist (2S,1′R,2′S)-2-(carboxycyclopropyl)-
glycine ((S)-CCG-IV, 4a).16,17 CCGs are naturally occurring
glutamate analogues isolated from seeds of Aesculus parviflora
and Blighia sapida, belonging to the family Sapindaceae,18 and
seeds of Ephedra altissima belonging to the family Ephedra-
ceae.19 These have been important lead structures for the
development of a range of selective GluR agonists and
antagonists. Three of the stereoisomers are very potent
NMDA receptor agonists and may serve as lead structures for
obtaining subtype selective NMDA receptor ligands.
While a large number of 2-, 1′,- and 3′-substituted CCG
analogues have been synthesized, there are no analogues
described with substituents in the 2′-position, and therefore
synthetic routes are unknown. Herein, we describe our design
of the analogues, development of synthetic routes to the first
2′-substituted 4a analogues, as well as the pharmacological
characterization at iGluRs.
RESULTS
■
Design. Initially, the triionized state of 4d was minimized
using macromodel and docked using GLIDE. The grid was
prepared with GLIDE from the crystal structure of glutamate in
the ABD of GluN2A (2A5T:chain B) that had been prepared
and minimized following the standard settings in PRIME. This
gave a binding mode of 4d shown in Figure 2, overlapping
nicely with glutamate in the crystal structure. Comparing with
the presumed binding mode of 3b in the same model, we saw
that substituents in the 2′-position of 4a would overlap with the
4-position of 3a (Figure 2). The substituents in this position is
protruding into a region where nonconserved residues are
present. We therefore decided to probe the same region based
on the 4a scaffold as we have previously done with 3a by
synthesizing previously unreported 2′-substituted analogues of
4a.
Chemistry. Two distinct routes to the alkyl substituted
analogues of 4a were employed starting from commercially
available (R)-Garners aldehyde (GA) (Schemes 1 and 2).
Initially, Still−Genari reactions between GA and 5a−d were
explored (Scheme 1). The α-substituted bis(trifluoroethyl)-
phosphonoester 5a is commercially available, and 5b−d were
synthesized as previously described.20,21 The cis-α,β-unsatu-
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some instances.26 Compound 7c−e and 7g were prepared by
Negishi cross-couplings, whereas compound 7f and 7h was
prepared by Stille cross-couplings (Scheme 2).
a
Scheme 1
Shimamoto et al. have previously reported cyclopropanation
of 7a to obtain the two diastereoisomers in a 6:1 ratio (Scheme
2).27 In an attempt to improve the stereoselectivity and
suppress insertion of diazomethane into the NH bond,
additional bulk was introduced on the nitrogen by protection
with a second Boc group to afford the imide 9a. Cyclo-
propanation on this substrate resulted in isolation of 10a as a
single diastereoisomer with concomitant increased yield
compared to cyclopropanation of 7a. Several attempts were
performed to displace the highly toxic and potentially
dangerous diazomethane with other cyclopropanation reagents.
Among them the Simmon−Smith and Corey−Chaykovsky
cyclopropanation were attempted, however, without suc-
cess.28,29 Compound 7b−f were similarly bis-Boc protected
and subsequently underwent cyclopropanation to provide
10b−e in low yield (Scheme 3).
a
Reagents and conditions: (a) KHMDS, 5a−d, 18-crown-6, THF, −78
°C, 3 h (for 6a, 72%; for 6b, 89%; for 6c, 16%; for 6d, 10%); (b) TFA,
DCM, rt, 24 h for 7a; (c) p-TsOH, MeOH, rt, 24 h for 7b.
a
Scheme 2
a
Scheme 3
a
Reagents and conditions: (a) DMAP, Boc2O, ACN, rt, 2 h; (b)
Pd(OAc)2, CH2N2, Et2O, rt, 30 min (for 10b, 46%; for 10c, 31%; for
10d, 15%); (c) Pd2(dba)3, CH2N2, Et2O, rt, 30 min (for 10d, 37%; for
10e, 29%; for 10f, no reaction).
Compound 7h bearing a vinyl substituent decomposed under
the protection conditions and was only isolated in 21% yield.
Therefore, cyclopropanation was performed directly on 7h to
obtain 11 in 40% yield (Scheme 4). Interestingly, only
a
Reagents and conditions: (a) I2, pyridine, CCl4, 40 °C, 30 min; (b)
Et2Zn, Pd(PPh3)2Cl2, DMF, rt, 1.5 h (7c: 60%); (c) RZnX,
Pd(PPh3)2Cl2, DMF, rt, 1.5 h (X = Br or I; for 7d, 46%; for 7e,
35%; for 7g, 20%); (d) ZnBr2, Pd[(o-tolyl)3P)]2Cl2, Bu3SnPh or
tributyl(vinyl)tin, DMF, 65 °C, 1.5 h (for 7f, 89%; for 7h, 56%); (e)
DMAP, Boc2O, ACN, rt, 2 h; (f) Pd(OAc)2, CH2N2, Et2O, rt, 30 min.
a
Scheme 4
rated esters 6a and 6b were obtained in good yields under the
Still−Genari conditions, but the α-substituted bis-
(trifluoroethyl)phosphonoesters 5c and 5d resulted in no
reaction at −78 °C. Increasing temperature (−78 to 0 °C)
resulted in decomposition and isolation of the desired products
in very low yields (<15%). The intermediates 6a and 6b was
cyclized into the lactones 7a and 7b using TFA in DCM or p-
TsOH in MeOH as previously reported.22,23
To introduce larger alkyl substituents, to the unsubstituted
lactone 7a α-halogenation was attempted. Selective α-
iodination was performed using I2 in a mixture of CCl4 and
pyridine to afford 8.24
Negishi and Stille cross-couplings on this α-halo-lactone 8
introduced ethyl, n-propyl, n-butyl, benzyl, vinyl, and phenyl
substituents (Scheme 2). Increasing bulk of the Negishi reagent
resulted in diminished yield in the cross-coupling. The method
for preparation of the Negishi reagents was crucial for the yield
in the cross-coupling. Transmetalation with ZnBr2 from the
corresponding Grignard reagents resulted in very low yields in
the coupling compared to oxidative addition of highly reactive
Rieke zinc to alkyl halides.25 This was presumed to occur
because MgBr2 has been shown to inhibit cross-coupling in
a
Reagents and conditions: (a) Pd2(dba)3, CH2N2, Et2O, rt, 30 min.
cyclopropanation of the pendant double bond was observed
in an excess of diazomethane and none of the endocyclic
double bond was cyclopropanated, emphasizing how sensitive
the cyclopropanation is to steric bulk in this system. In the case
of the phenyl substituted compound 9f, no reaction was
observed in the cyclopropanation. The difficulty of cyclo-
propanation of highly substituted C−C double bonds or C−C
double bonds with cis-unsaturated esters or amides has been
noted by others.30,31 The low yields observed in the
cyclopropanation in this small series were not surprising
because these compounds both contain a trisubstituted double
bond and are α,β-unsaturated lactones. The cyclopropanated
products 10b−e were Boc-deprotected prior to hydrolysis to
obtain 12b−e in high yields (Scheme 5). Hydrolysis was
performed with NaOH in a mixture of water and THF to
obtain 13b−e. Subsequent oxidation of the alcohol with
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a
looking at the development in relative agonist efficacies of
compounds 4b−e, differences among the subtypes can be
observed. Thus, agonist efficacy relative to Glu increases from
83% to 94% at GluN2D when going from the methyl
substituted 4b to the n-butyl substituted 4e, whereas the
relative efficacy at GluN2A decreases from 94% to 65% (Figure
4).
Scheme 5
DISCUSSION
■
We have designed and synthesized a series of 2′-alkyl
substituted analogues of the highly potent NMDA receptor
agonist 4a in order to obtain NMDA receptor agonists that
display differences among the cloned GluN2 subtypes. The
design of this series was based on previous observations with a
series of 4-alkyl substituted analogues of the moderately potent
NMDA receptor agonist 3a that discriminated subtypes in
terms of agonist efficacy and that 1b discriminate subtypes
based on potency.14−16 Thus, we wanted to explore the
possibility of achieving more potent compounds with the ability
to discriminate the different subtypes either by potency or
efficacy by adding substituents to 4a that would protrude into
the same area as the 4-alkyl groups in 3a and 1b. Two different
synthetic routes were developed to yield this series of alkyl
substituted analogues of 4a.
a
Reagents and conditions: (a) TFA, DCM, rt, 10 min (for 12b, 98%;
for 12c, 95%; for 12d, 95%; for 12e, 94%); (b) 1M aq NaOH, THF,
40°C, 30 min (for 13b, 75%; for 13c, 79%; for 13d, 67%; for 13e,
62%); (c) 1M aq NaOH, KMnO4, rt, 4 h (for 14b, 71%; for 14c, 83%;
for 14d, 95%; for 14e, 75%); (d) 2M HCl in Et2O (for 4b, 71%; for
4c, 94%; for 4d, 71%; for 4e, 82%).
KMnO4 in basic solution, deprotection, and precipitation gave
the desired analogues 4b−e as HCl salts.
Pharmacology. The affinities of the compounds at the
major iGluR subgroups were investigated using radioligand
binding to rat cortical synaptosomes. As shown in Table 1, all
Pharmacological characterization of the new analogues
revealed that although some potency is lost when introducing
larger alkyl groups in the 2′-position, the analogues are indeed
more potent than the corresponding 4-substituted 3a and
previously published glutamate analogues.15,16 The increased
potency of 2′-substituted 4a compared to the corresponding 4-
substituted 3a may be due to a better orientation and thereby
receptor interaction of the distal acid group of the 4a analogues.
The variation in agonist efficacy of the new analogues of 4a at
the different subtypes is less pronounced than seen with the
series of 3a analogues. For example, 4-Et-NHP5G activates
GluN2A and GluN2D with 5% and 70% relative to Glu,
respectively, whereas ethyl-substituted 4c activates 83% and
91%, respectively. Nevertheless, propyl substituted 4d activates
GluN2A and GluN2B with 67% and 105%, respectively, thus
significant differences are achievable. Furthermore, differences
in potency is also observed, for example, compound 4c is 15-
fold more potent at GluN2D compared to GluN2A. While 1b
is slightly more selective compared to 4c, 1b is also a highly
potent agonist at kainate receptors and is less potent at
GluN2D,32 and thus 4c is one of the most potent and selective
agonists toward the GluN2D. This study shows that it is
possible to introduce substituents in the 2′ position of (S)-
CCG-IV, and we believe that more variation and introduction
of more polar substituents will lead to larger differences among
subtypes, however other synthetic routes needs to be explored
in order to facilitate the synthesis of such compounds.
Table 1. Receptor Binding Affinities of Compounds 4a−e at
Three Major Groups of iGluRs in Rat Cortical Synaptosome
a
Assay
Ki (μM)
compd
R
[3H]AMPA
[3H]KA
[3H]CGP39653
3a
3b
4a
4b
4c
4d
4e
H
>100
>100
>100
>100
10
14
n-Pr
H
1.1 [1.0;1.3] 0.31 [0.27;0.36] 0.0065 [0.0057;0.0074]
Me
Et
29 [26;32]
>100
0.11 [0.08;0.14] 0.0080 [0.0066;0.0098]
0.61 [0.58;0.63]
21 [19;22]
>100
0.061 [0.053;0.070]
1.2 [1.1;1.4]
n-Pr
n-Bu
>100
>100
1.3 [1.1;1.5]
a
The numbers in brackets [min, max] indicate mean SEM according
to a logarithmic distribution.44 CGP39653 is a competitive NMDA
receptor antagonist and AMPA and KA are agonists at AMPA and KA
receptors, respectively. Ki values are calculated using the Cheng−
Prusoff relationship. Data for 3a and 3b is from Clausen et al.14 ND
means not determined, and NA means no activity.
the analogues are selective NMDA receptor ligands and the
affinity decreases with increasing substituent size. Introducing a
methyl group in the 2′-position of 4a decreases the affinity at
AMPA receptors by more than an order of magnitude, while
the affinity at KA and NMDA receptors are maintained.
Extending the alkyl chain length to ethyl and n-propyl an order
of magnitude decrease in KA and NMDA receptor affinity is
observed for each additional carbon, and AMPA receptor
affinity is abolished. The n-butyl substituted analogue 4e is not
binding to AMPA and KA receptors at a concentration of 100
μM but displays weak affinity at NMDA receptors equipotent
to the propyl substituted analogue.
The affinity at native NMDA receptors prompted functional
characterization at recombinant NMDA receptors expressed in
Xenopus laevis oocytes to determine the activity at individual
receptor subtypes (Table 2, Figures 3 and 4).
The potencies of compounds 4b−e are decreasing at all four
subtypes with increasing substituent size as seen in the
synaptosomal binding assay (Figure 3 and Table 2). When
EXPERIMENTAL SECTION
■
Chemistry. General Methods. All reactions involving dry solvents
or air sensitive reagents were performed under an argon atmosphere,
and glassware was flame-dried prior to use under vacuum. Flash
chromatography (FC) was performed using silica Merck 60A (35−70
μm). TLC was carried out using Merck silica gel 60 F254 aluminum
sheets. The compounds were detected as single spots on TLC plates
and visualized using UV light (254 nm) and different spraying reagents
such as potassium permanganate, anisaldehyde, and ninhydrin. All
solvents and reagents were analytical grade purchased from Aldrich
and used without further purification. Dry THF and DMF were
obtained from a solvent purification system (SPS). 1H (400 MHz) and
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Table 2. Potency and Efficacy Relative to Glu of Compounds 4a−e at Recombinant GluN1/GluN2A−D Receptors Expressed in
a
Xenopus Oocytes
compd
R
subtype
EC50 (μM)
rel Imax
nHill
3a
H
GluN1/GluN2A
GluN1/GluN2B
GluN1/GluN2C
GluN1/GluN2D
82
48
0.45
0.58
0.52
ND
1.3
1.6
1.4
ND
54
ND
3b
4a
4b
4c
4d
4e
n-Pr
H
GluN1/GluN2A
GluN1/GluN2B
GluN1/GluN2C
GluN1/GluN2D
NA
105
429
153
NA
0.06
0.22
0.37
NA
1.9
1.1
1.6
GluN1/GluN2A
GluN1/GluN2B
GluN1/GluN2C
GluN1/GluN2D
0.262
0.083
0.110
0.036
0.99
1.23
0.90
1.11
1.1
1.1
1.1
1.3
Me
Et
GluN1/GluN2A
GluN1/GluN2B
GluN1/GluN2C
GluN1/GluN2D
0.277[0.261;0.292]
0.045[0.043;0.047]
0.124[0.120;0.129]
0.026[0.025;0.026]
0.94 0.01
1.14 0.02
0.81 0.04
0.83 0.02
1.2
1.7
1.3
1.9
GluN1/GluN2A
GluN1/GluN2B
GluN1/GluN2C
GluN1/GluN2D
1.18[0.673;1.40]
0.272[0.257;0.286]
0.540[0.519;0.559]
0.080[0.077;0.083]
0.83 0.03
1.02 0.04
0. 81 0.02
0. 91 0.01
1.3
1.5
1.5
1.6
n-Pr
n-Bu
GluN1/GluN2A
GluN1/GluN2B
GluN1/GluN2C
GluN1/GluN2D
46.3[45.2;47.4]
15.8[15.2;16.3]
29.9[29.3;30.5]
6.56 [6.15;6.90]
0.67 0.01
1.05 0.02
0. 70 0.02
0. 89 0.01
1.1
1.3
1.4
1.6
GluN1/GluN2A
GluN1/GluN2B
GluN1/GluN2C
GluN1/GluN2D
28.8[28.4;29.2]
10.2[9.82;10.5]
16.0[15.6;16.5]
3.30 [3.21;3.39]
0.65 0.02
1.07 0.01
0.85 0.02
0.94 0.01
1.4
1.6
1.5
1.7
a
Data are from 3−12 oocytes, and the numbers in brackets [min, max] indicate mean SEM according to a logarithmic distribution.44 The relative
maximal currents (relative Imax) are the maximal responses to the indicated agonists obtained by fitting the full concentration-response data
normalized to the maximal response activated by glutamate in the same recording (see Experimental Section). Data for 3a and 3b is from Clausen et
al14 and for 4a from Erreger et al.16 ND means not determined, and NA means no activity.
13C (100 MHz) NMR were recorded on a Bruker instrument using
BuOK (1.27 g, 11.3 mmol, 1.2 equiv) in THF (13 mL) was slowly
added methyl bis(2,2,2-trifluoroethyl)phosphonoacetate (2.0 mL, 3.0
g, 9.46 mmol, 1 equiv) at 0 °C under argon. The mixture was stirred at
0 °C for 30 min, and methyl iodide (2.96 mL, 47.3 mmol, 5 equiv) was
slowly added at 0 °C. The solution was allowed to warm up to room
temperature, stirred for 24 h, and treated with a saturated aq solution
of NH4Cl (20 mL). The reaction mixture was extracted with EtOAc (3
× 50 mL), dried over Na2SO4, filtered, and evaporated to dryness.
Purification by silica gel chromatography (eluent: 30% EtOAc in
TMS as internal standard. Chemical shifts (δ) are given in parts per
million (ppm), and coupling constants (J) are given in hertz (Hz). The
following abbreviations are used: bs = broad singlet, s = singlet, d =
doublet, dd = doublet of doublet, ddd = doublet of doublet of doublet,
dddd = doublet of doublet of doublet of doublet, t = triplet, dt =
doublet of triplet, dddt = doublet of doublet of doublet of triplet, tt =
triplet of triplet, q = quartet, dq = doublet of quartet, m = multiplet.
LC/MS(A) and LC/MS(B) were performed with an Agilent 6410
triple quadrupole mass spectrometer instrument using electron spray
coupled to an Agilent 1200 HPLC system (ESI-LC/MS) with a C18
reverse phase column (Zorbax Eclipse XBD-C18, 4.6 mm × 50 mm),
autosampler, and diode array detector using a linear gradient of the
binary solvent system of H2O/ACN/formic acid (A: 95/5/0.1, and B:
5/95/0.086) with a flow rate of 1 mL/min. During ESI-LC/MS
analysis, evaporative light scattering (ELS) traces were obtained with a
Sedere Sedex 85 light scattering detector. LC/MS(A): linear gradient
0−90% B over 4 min, then 0.5 min 100% B. LC/MS(B): linear
gradient 0−90% B over 3 min, then 1.5 min 100% B. Products were
more than 95% pure according to LC/MS.
1
heptane) afforded 5b (2.30 g, 73%) as a colorless oil. H NMR (300
MHz; CDCl3) δ: 4.49−4.38 (m, 4H), 3.78 (s, 3H), 3.20 (dq, J = 22.8,
7.4 Hz, 1H), 1.52 (dd, J = 19.3, 7.4 Hz, 3H). NMR identical to
literature.33
Methyl 2-(Bis(2,2,2-trifluoroethoxy)phosphoryl)butanoate (5c).
1
5c (2.69 g, 52%) as a colorless oil. H NMR (300 MHz; CDCl3): δ
4.53−4.34 (m, 4H), 3.78 (s, 3H), 3.06 (m, 1H), 2.08−1.90 (m, 2H),
1.04 (t, J = 6.9 Hz, 3H). NMR identical to literature.34
Methyl 2-(Bis(2,2,2-trifluoroethoxy)phosphoryl)pentanoate (5d).
1
5d (1.73 g, 32%). H NMR (300 MHz; CDCl3): δ 4.50−4.35 (m,
4H), 3.79 (s, 3H), 3.14 (ddd, J = 21.8, 10.3, 4.3 Hz, 1H), 2.09−1.78
(m, 2H), 1.53−1.27 (m, 2H), 0.96 (t, J = 7.3 Hz, 3H).
General Procedure for Alkylation Methyl Bis(2,2,2-
trifluoroethyl)phosphonoacetate. Methyl 2-(Bis(2,2,2-
trifluoroethoxy)phosphoryl)propanoate (5b). To a solution of t-
General Procedure for Still−Genari Reaction. (S,Z)-tert-Butyl
4-(3-Methoxy-3-oxoprop-1-en-1-yl)-2,2-dimethyloxazolidine-3-car-
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Figure 3. Mean concentration−response curves for compounds Me-, Et-, n-Pr-, and n-Bu-CCG (4b−e) determined using two-electrode voltage-
clamp recordings on Xenopus oocytes coexpressing GluN1 and GluN2A−D. The curves are normalized to the maximal current response (Imax) to the
agonist obtained by fitting the full concentration−response data to the Hill equation (see Experimental Section). Data points are represented as
mean SEM from 3−6 oocytes. All EC50 values are listed in Table 2.
Figure 4. (A) Comparison of maximal currents induced by 4a−e relative to Glu at NR1/NR2A−D subtypes. The relative efficacies are the maximal
current responses to the indicated agonists obtained by fitting the full concentration−response data normalized to the maximal response activated by
glutamate in the same recording (see Experimental Section). (B) Comparison of agonist potencies (pLogEC50) for 4a−e. Values are listed in Table
2. Data for (S)-CCG-IV (4a) are from Erreger et al., 2007.16
boxylate (6a). 18-Crown-6 (44 g, 123.3 mmol, 8.5 equiv) and 5a (8.1
g, 25.5 mmol, 1.3 equiv) were dissolved in dry THF (240 mL) and
cooled to −78 °C under an atmosphere of argon. Then 0.5 M
KHMDS (47.1 mL, 23.6 mmol, 1.2 equiv) was added dropwise, and
the solution was aged for 1 h. Garners aldehyde (4.5 g, 19.6 mmol, 1
equiv) in dry THF (13 mL) was slowly added dropwise. The solution
was stirred for 3 h at −78 °C. The reaction was quenched with
saturated aq NH4Cl (100 mL). The mixture was extracted with EtOAc
(3 × 150 mL), and the combined organic phases were dried over
Na2SO4, filtered, and evaporated to dryness. The crude product was
purified by silica gel chromatography (eluent: 20% EtOAc in heptane)
to afford 6a (4.0 g, 72%) as colorless oil. 1H NMR (400 MHz;
CDCl3): δ 6.36, 6.27 (dd, J = 10.7, 9.2 Hz, 1H, rotamers), 5.85 (m,
1H), 5.41 (bs, 1H), 4.33−4.25 (m, 1H), 3.79 (dd, J = 9.2, 3.1 Hz, 1H),
3.73 (s, 3H), 1.66−1.41 (m, 15H, rotamers). NMR identical to
literature.35
(S,Z)-tert-Butyl 4-(2-(Methoxycarbonyl)but-1-en-1-yl)-2,2-dime-
thyloxazolidine-3-carboxylate (6c). 6c (247 mg, 15.7%) as a pale-
1
yellow oil. H NMR (300 MHz; CD3OD): δ 5.94 (m, 1H), 5.03 (m,
1H), 4.20 (m, 1H), 3.73 (s, 3H), 2.42−2.21 (m, 2H), 1.48 (m, 15H),
1.05 (t, J = 7.1 Hz, 3H).
Methyl 2-(Bis(2,2,2-trifluoroethoxy)phosphoryl)pentanoate (6d).
6d (100 mg, 10.6%) as a pale-yellow oil. 1H NMR (300 MHz;
CD3OD): δ 5.94−5.91 (m, 1H), 5.06−5.00 (m, 1H), 4.22−4.17 (m,
1H), 3.72 (m, 4H), 2.37−2.15 (m, 2H), 1.58−1.37 (m, 17H), 0.91 (t,
J = 7.1 Hz, 3H).
(S)-tert-Butyl (6-Oxo-3,6-dihydro-2H-pyran-3-yl)carbamate (7a).
To a solution of 6a (5.1 g, 17.9 mmol) in DCM (100 mL) was added
TFA (2.87 mL, 38.6 mmol, 2.1 equiv), and the solution was stirred for
24 h at room temperature. The solvent was evaporated, and the crude
product was purified by silica gel chromatography (eluent: 60% EtOAc
1
in heptane) to afford 7a (2.5 g, 65%) as a white solid. H NMR (400
MHz; CDCl3): δ 6.88 (bs, 1H), 6.10 (dd, J = 9.8, 1.3 Hz, 1H), 4.94
(bs, 1H), 4.50−4.36 (m, 3H), 1.45 (s, 9H). NMR identical to
literature.27
(S)-tert-Butyl (5-Methyl-6-oxo-3,6-dihydro-2H-pyran-3-yl)-
carbamate (7b). 6b (2.36 g, 7.9 mmol, 1 equiv) was dissolved in
MeOH (43 mL). p-TsOH (75 mg, 0.40 mmol, 0.05 equiv) was added
(S,Z)-tert-Butyl 4-(3-Methoxy-2-methyl-3-oxoprop-1-en-1-yl)-2,2-
1
dimethyloxazolidine-3-carboxylate (6b). 6b (2.5 g, 89%). H NMR
(300 MHz; CDCl3): δ 6.10−5.95 (m, 1H), 5.14−5.12 (bs, 1H), 4.22
(m, 1H), 3.79−3.73 (m, 4H), 1.92 (s, 3H), 1.64−1.38 (m, 15H).
NMR identical to literature.23
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and the solution stirred at room temperature for 24 h. The solvent was
evaporated, and the crude product was purified by silica gel
chromatography (eluent: 20−50% EtOAc in heptane) to afford 7b
(1.1 g, 61%) as white solid ((460 mg, 1.54 mmol) starting material,
and (100 mg, 0.41 mmol) of the aminal opened intermediate
recovered) (76% yield based on recovered starting materiel). 1H NMR
(300 MHz; CDCl3): δ 6.59 (d, J = 4.8 Hz, 1H), 4.78 (d, J = 7.9 Hz,
1H), 4.48−4.34 (m, 3H), 1.96 (s, 3H), 1.46 (s, 9H). NMR identical to
literature.23
(S)-tert-Butyl (5-Iodo-6-oxo-3,6-dihydro-2H-pyran-3-yl)-
carbamate (8). 7a (1.4 g, 6.6 mmol, 1 equiv) was dissolved in CCl4
(10 mL) and pyridine (10 mL). A solution of iodine (8.38 g, 33 mmol,
5.0 equiv) dissolved in pyridine, and CCl4 (1:1, 45 mL) was added at
room temperature. The solution was heated at 40 °C for 30 min. The
reaction was quenched by addition of 1 M aq Na2S2O3 (50 mL) and
stirred for 10 min. The phases were separated and the water phase
extracted with DCM (2 × 50 mL). The combined organic phases were
dried over Na2SO4, filtered, and evaporated to dryness. The crude
product was purified by silica gel chromatography (eluent: 50% EtOAc
in heptane) to afford 8 (1.8 g, 80%) as off-white solid. 1H NMR (300
MHz; CD3OD): δ 7.62 (d, J = 4.9 Hz, 1H), 4.54 (dd, J = 10.7, 3.8 Hz,
1H), 4.36 (m, 2H), 1.47 (s, 9H). 13C NMR (75 MHz; CD3OD): δ
159.83 (s, 1C), 156.06 (s, 1C), 154.71 (s, 1C), 90.24 (s, 1C), 79.89 (s,
1C), 70.73 (s, 1C), 46.58 (s, 1C), 27.57 (s, 3C). LC/MS(A): RT 3.42.
ELSD: 100%, UV254 nm 100%, (M-Boc)H+ 240.0.
General Procedure for Negishi Cross-Coupling. (S)-tert-Butyl
(5-Ethyl-6-oxo-3,6-dihydro-2H-pyran-3-yl)carbamate (7c). A solu-
tion of 8 (310 mg, 0.91 mmol, 1 equiv) and Pd(PPh3)2Cl2 (63.8 mg,
0.091 mmol, 0.1 equiv) in dry degassed DMF (3.8 mL) was added
Et2Zn (0.59 mL, 0.59 mmol, 1 M in hexanes, 0.65 equiv) at room
temperature and stirred for 1.5 h. The reaction was quenched with
saturated aq NH4Cl (10 mL). Et2O (25 mL) was added, and the
phases separated. The water phase was extracted with Et2O (2 × 25
mL), and the combined organic phases were washed with brine (10
mL), dried over Na2SO4, filtered, and evaporated to dryness. The
crude product was purified by silica gel chromatography (eluent: 5%
acetone in DCM) to afford 7c (130 mg, 60%) as white solid. 1H NMR
(300 MHz; CD3OD): δ 6.62 (d, J = 2.2 Hz, 1H), 4.45−4.38 (m, 2H),
4.23 (m, 1H), 2.33 (q, J = 7.4 Hz, 2H), 1.48 (s, 9H), 1.11 (t, J = 7.4
Hz, 3H). 13C NMR (75 MHz; CD3OD): δ 166.05 (s, 1C), 157.36 (s,
1C), 139.54 (s, 1C), 135.94 (s, 1C), 80.69 (s, 1C), 71.16 (s, 1C),
44.88 (s, 1C), 28.69 (s, 3C), 24.86 (s, 1C), 12.71 (s, 1C). LC/MS(A):
RT 3.43. ELSD: 0%. UV254 nm 100%, (M-Boc)H+ 142.1.
alumina foil. A solution of butyl iodide (0.85 mL, 1.39 g, 7.54 mmol)
was added slowly at rt, and the mixture was stirred for 24 h.
(S)-tert-Butyl (5-Benzyl-6-oxo-3,6-dihydro-2H-pyran-3-yl)-
1
carbamate (7g). 7g (90 mg, 20%) as a colorless oil. H NMR (400
MHz; CD3OD): δ 7.36−7.19 (m, 5H), 6.48−6.46 (m, 1H), 4.39−4.35
(m, 2H), 4.21−4.16 (m, 1H), 3.59 (s, 2H), 1.43 (s, 9H). 13C NMR
(101 MHz; CD3OD): δ 166.03 (s, 1C), 157.58 (s, 1C), 142.04 (s,
1C), 139.44 (s, 1C), 134.53 (s, 1C), 130.26 (s, 2C), 129.60 (s, 2C),
127.63 (s, 1C), 79.87 (s, 1C), 71.23 (s, 1C), 45.06 (s, 1C), 37.69 (s,
1C), 28.72 (s, 3C). LC/MS(A): RT 3.99. ELSD: 0%. UV 95.6%, MH+
204.1.
Preparation of ∼0.5 M Benzylzinc(II) bromide. A dry flask was
charged with a highly reactive Rieke Zn suspension (4.96 mL, 0.76 M,
3.77 mmol). This suspension was cooled to 0 °C. A solution of benzyl
bromide (0.45 mL, 0.65 g, 3.77 mmol) in THF (2.13 mL) was added
dropwise, and the mixture was stirred at 0 °C for 3 h.
General Procedure for Stille Cross-Coupling. (S)-tert-Butyl (6-
Oxo-5-phenyl-3,6-dihydro-2H-pyran-3-yl)carbamate (7f). A flask
charged with ZnBr2 (36 mg, 0.16 mmol, 0.16 equiv) was flame-dried
and put under argon atmosphere. The flask was charged with Pd[(o-
tolyl)3P)]2Cl2 (40 mg, 0.05 mmol, 0.05 equiv) and 8 (339 mg, 1.0
mmol, 1 equiv), and DMF (2 mL) was added. Tributylphenyltin (0.35
mL, 400 mg, 1.1 mmol, 1.1 equiv) was added slowly at room
temperature and the solution stirred at 65 °C for 1.5 h. The reaction
was quenched by addition of saturated aq NH4Cl (10 mL), and Et2O
(20 mL) was added. The phases were separated and the organic phase
washed with brine, dried over Na2SO4, filtered, and evaporated to
dryness. The crude product was dissolved in ACN (30 mL) and
washed with heptane (4 × 20 mL). The ACN phase was evaporated to
dryness, and the crude product was purified by silica gel
chromatography (eluent: 30% EtOAc in heptane) to afford 7f (258
mg, 89%) as white solid. 1H NMR (400 MHz; CD3OD): δ 7.47−7.45
(m, 2H), 7.35 (m, 3H), 6.95 (d, J = 4.4 Hz, 1H), 4.54−4.52 (m, 2H),
4.34−4.30 (m, 1H), 1.47 (s, 9H). 13C NMR (101 MHz; CD3OD): δ
165.15 (s, 1C), 157.60 (s, 1C), 143.06 (s, 1C), 136.51 (s, 1C), 134.54
(s, 1C), 129.57 (s, 2C), 129.48 (s, 1C), 129.12 (s, 2C), 80.91 (s, 1C),
71.08 (s, 1C), 45.33 (s, 1C), 28.67 (s, 3C). LC/MS(A): RT 3.84.
ELSD: 100%. UV 100%, (M-Boc)H+ 190.1.
(S)-tert-Butyl (6-Oxo-5-vinyl-3,6-dihydro-2H-pyran-3-yl)-
1
carbamate (7h). 7h (1.17 g, 56%) as off-white solid. H NMR (400
MHz; CDCl3): δ 6.83 (d, J = 5.2 Hz, 1H), 6.53−6.46 (m, 1H), 5.87
(dd, J = 17.6, 1.2 Hz, 1H), 5.36 (dd, J = 11.2, 1.2 Hz, 1H), 4.94 (d, J =
4.6 Hz, 1H), 4.54 (dd, J = 7.7, 3.9 Hz, 1H), 4.46 (dd, J = 11.5, 4.0 Hz,
1H), 4.34 (dd, J = 11.4, 3.5 Hz, 1H), 1.47 (s, 9H). 13C NMR (101
MHz; CDCl3): δ 162.62 (s, 1C), 154.83 (d, J = 2.8 Hz, 1C), 137.19 (s,
1C), 131.03 (s, 1C), 130.27 (s, 1C), 119.34 (s, 1C), 80.66 (s, 1C),
70.03 (s, 1C), 43.48 (s, 1C), 28.27 (s, 3C). LC/MS(A): RT 2.87.
ELSD: 0%. UV254 nm 100%, MNa+ 262.1.
tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[(3S)-6-oxo-3,6-dihydro-
2H-pyran-3-yl]carbamate (9a). To a solution of 7a (260 mg, 1.22
mmol, 1 equiv) in ACN (5.6 mL), (Boc)2O (799 mg, 3.66 mmol, 3
equiv) and DMAP (29 mg, 0.24 mmol, 0.2 equiv) were added
successively. The solution was stirred for 2 h at room temperature, the
solvent evaporated off, and the crude product purified by silica gel
chromatography (eluent: 20% EtOAc in heptane) to afford 9a (375
mg, 98%) as white solid. 1H NMR (400 MHz; MeOD): δ 7.02 (ddd, J
= 10.1, 2.6, 1.6 Hz, 1H), 5.96 (dd, J = 10.1, 2.6 Hz, 1H), 5.28 (dddd, J
= 9.9, 6.1, 2.7, 2.7 Hz, 1H), 4.59 (dd, J = 10.4, 10.1 Hz, 1H), 4.44
(ddd, J = 10.6, 6.1, 1.6 Hz, 1H), 1.53 (s, 18H). 13C NMR (101 MHz;
MeOD): δ 165.07 (s, 1C), 153.49 (s, 2C), 150.45 (s, 1C), 119.74 (s,
1C), 85.39 (s, 2C), 69.12 (s, 1C), 50.93 (s, 1C), 28.40 (s, 6C). LC/
MS(A): RT 4.03. ELSD: 0%. UV 100%, MNa+ 336.1
(S)-tert-Butyl (6-Oxo-5-propyl-3,6-dihydro-2H-pyran-3-yl)-
1
carbamate (7d). 7d (415 mg, 46%) as white solid. H NMR (400
MHz; MeOD): δ 6.63 (d, J = 4.1 Hz, 1H), 4.45−4.38 (m, 2H), 4.20
(dd, J = 10.2, 4.8 Hz, 1H), 2.34−2.21 (m, 2H), 1.58−1.45 (m, 11H),
0.96 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz; MeOD): δ 166.28 (s,
1C), 157.61 (s, 1C), 140.98 (s, 1C), 134.54 (s, 1C), 80.80 (s, 1C),
71.23 (s, 1C), 44.97 (s, 1C), 33.84 (s, 1C), 28.65 (s, 3C), 22.35 (s,
1C), 13.95 (s, 1C). LC/MS(A): RT 3.74. ELSD: 0%. UV 100%, (M-
Boc)H+ 156.1.
Preparation of ∼0.7 M Propylzinc(II) Iodide. A dry flask was
charged with a highly reactive Rieke Zn suspension in THF (12.1 mL,
0.76 M, 9.2 mmol) under an atmosphere of argon and covered with
alumina foil. A solution of n-propyl iodide (0.90 mL, 1.56 g, 9.2 mmol)
was added at rt, and the mixture was stirred for 24 h.
(S)-tert-Butyl (5-Butyl-6-oxo-3,6-dihydro-2H-pyran-3-yl)-
1
carbamate (7e). 7e (273 mg, 35%) as a off-white solid. H NMR
(400 MHz; CDCl3): δ 6.61 (dd, J = 4.2, 1.0 Hz, 1H), 4.43−4.36 (m,
2H), 4.22−4.18 (m, 1H), 2.33−2.22 (m, 2H), 1.50−1.31 (m, 13H),
0.94 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz; CDCl3): δ 166.29 (s,
1C), 157.63 (s, 1C), 140.79 (s, 1C), 134.78 (s, 1C), 80.80 (s, 1C),
71.23 (s, 1C), 44.98 (s, 1C), 31.50 (s, 1C), 31.38 (s, 1C), 28.66 (s,
3C), 23.30 (s, 1C), 14.17 (s, 1C). LC/MS(A): RT 4.05. ELSD: 0%.
UV 100%, (M-Boc)H+ 170.1.
Preparation of ∼0.7 M Butylzinc(II) Iodide. A dry flask was
charged with a highly reactive Rieke Zn suspension in THF (9.92 mL,
0.76 M, 7.54 mmol) under an atmosphere of argon and covered with
tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[(1S,5S,6R)-2-oxo-3-
oxabicyclo[4.1.0]heptan-5-yl]carbamate (10a). Pd(OAc)2 (27 mg,
0.12 mmol, 0.05 equiv) was suspended in a solution of 9a (0.750 g, 2.4
mmol, 1 equiv) in Et2O (50 mL). A solution of CH2N2 (approximately
5.0 equiv) in Et2O was added dropwise over approximately 15 min.
The solution was stirred for another 15 min and quenched by addition
of glacial acetic acid (0.69 mL, 721 mg, 12 mmol), and the solvent was
evaporated off. The crude product was purified by silica gel
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chromatography (eluent: 20% EtOAc in heptane) to afford 10a (628
mg, 80%) as white solid. H NMR (400 MHz; CDCl3): δ 4.67 (m,
4.31−4.29 (m, 2H), 1.51 (s, 19H), 1.39 (s, 3H), 1.34 (dd, J = 5.8 Hz,
1H), 1.03 (dd, J = 8.2, 6.2 Hz, 1H). 13C NMR (151 MHz; CDCl3): δ
172.52 (s, 1C), 152.85 (s, 2C), 83.11 (s, 2C), 69.09 (s, 1C), 48.34 (s,
1C), 27.92 (s, 6C), 24.79 (s, 1C), 21.41 (s, 1C), 20.87 (s, 1C), 15.27
(s, 1C). LC/MS(A): RT 4.26. ELSD: 0%. UV 0%, (M-2Boc)H+ 142.1.
tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[(1S,5S,6R)-1-ethyl-2-oxo-
3-oxabicyclo[4.1.0]heptan-5-yl]carbamate (10c). 10c (70 mg, 31%,
57% based on recovered starting material). 1H NMR (300 MHz;
CD3OD): δ 4.67 (m, 1H), 4.45 (dd, J = 13.3, 4.6 Hz, 1H), 4.24 (ddd, J
= 13.4, 1.9, 1.2 Hz, 1H), 1.80 (dq, J = 14.3, 7.2 Hz, 1H), 1.64 (m, 1H),
1.46−1.36 (m, 20H), 1.10 (dd, J = 8.4, 6.2 Hz, 1H), 1.00 (t, J = 7.4 Hz,
3H). 13C NMR (75 MHz; CD3OD): δ 174.99 (s, 1C), 154.02 (s, 2C),
84.05 (s, 2C), 69.94 (s, 1C), 50.04 (s, 1C), 28.72 (s, 1C), 28.25 (s,
6C), 27.45 (s, 1C), 26.00 (s, 1C), 14.10 (s, 1C), 11.65 (s, 1C). LC/
MS(A): RT 4.55. ELSD: 100%. UV 0%, (M-2Boc)H+ 156.1.
1
1H), 4.40 (ddd, J = 13.3, 1.9, 1.6 Hz, 1H), 4.27 (dd, J = 13.3, 4.0 Hz,
1H), 2.03 (dddd, J = 9.4, 7.4, 4.9, 0.7 Hz, 1H), 1.91−1.85 (m, 1H),
1.53 (s, 18H), 1.32−1.23 (m, 2H). 13C NMR (101 MHz; CDCl3): δ
169.78 (s, 1C), 152.74 (s, 2C), 83.26 (s, 2C), 68.06 (s, 1C), 47.20 (s,
1C), 27.83 (s, 6C), 17.64 (s, 1C), 16.53 (s, 1C), 8.65 (s, 1C). LC/
MS(A): RT 3.91. ELSD: 0%. UV210 nm 0%, (2M)Na+ 677.4.
General Procedure for Boc Protection. tert-Butyl N-[(tert-
Butoxy)carbonyl]-N-[(3S)-5-methyl-6-oxo-3,6-dihydro-2H-pyran-3-
yl]carbamate (9b). A solution of 7b (1.1 g, 4.8 mmol, 1 equiv) in
ACN (22 mL) was added (Boc)2O (3.14 g, 14.4 mmol, 3 equiv) and
DMAP (117 mg, 0.96 mmol, 0.2 equiv) successively. The solution was
stirred for 2 h at room temperature, the solvent evaporated off, and the
crude product purified by silica gel chromatography (eluent: 20%
EtOAc in heptane) to afford 9b (1.54 g, 98%) as white solid. 1H NMR
(300 MHz; CDCl3): δ 6.53 (dq, J = 2.7, 1.4 Hz, 1H), 5.27−5.18 (m,
1H), 5.27−5.18 (m, 1H), 4.57 (td, J = 10.3, 1.2 Hz, 1H), 4.34 (ddt, J =
10.2, 6.0, 1.4 Hz, 1H), 1.95 (dt, J = 2.4, 1.3 Hz, 3H), 1.51 (s, 18H).
13C NMR (151 MHz; CDCl3): δ 164.39 (s, 1C), 151.95 (s, 2C),
tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[(1S,5S,6R)-2-oxo-1-prop-
yl-3-oxabicyclo[4.1.0]heptan-5-yl]carbamate (10d). 10d (105 mg,
1
15%, 48% based on recovered starting material) as white solid. H
NMR (400 MHz; MeOD): δ 4.68 (m, 1H), 4.46 (dd, J = 13.3, 4.7 Hz,
1H), 4.27 (ddd, J = 13.3, 2.1, 1.4 Hz, 1H), 1.72−1.35 (m, 24H), 1.12
(dd, J = 8.4, 6.2 Hz, 1H), 0.90 (t, J = 7.3 Hz, 3H). 13C NMR (101
MHz; MeOD): δ 175.22 (s, 1C), 154.26 (s, 2C), 84.20 (s, 2C), 69.93
(s, 1C), 50.20 (s, 1C), 38.14 (s, 1C), 28.22 (s, 6C), 26.51 (s, 1C),
26.19 (s, 1C), 21.22 (s, 1C), 14.74 (s, 1C), 14.58 (s, 1C). LC/MS(A):
RT 4.69. ELSD: 0%. UV210 nm 100%, (M-2Boc)H+ 170.1.
General Procedure for Cyclopropanation (Method B).
Pd2(dba)3 (52 mg, 0.057 mmol, 0.05 equiv) was suspended in a
solution of 9d (400 mg, 1.13 mmol, 1 equiv) in Et2O (22 mL). A
solution of CH2N2 (approximately 5 equiv) in Et2O was added
dropwise over approximately 15 min. The solution was stirred for
another 15 min and quenched by addition of glacial acetic acid (0.32
mL, 339 mg, 5.65 mmol), and the solvent was evaporated off. The
crude product was purified by silica gel chromatography (eluent: 20%
EtOAc in heptane) to afford 10d (155 mg, 37%, 63% based on
recovered starting material) as white solid. NMR and LC/MS identical
to method A.
142.00 (s, 1C), 126.42 (s, 1C), 83.98 (s, 2C), 67.80 (s, 1C), 49.80 (s,
1C), 28.08 (s, 6C), 17.38 (s, 1C). LC/MS(A): RT 4.28. ELSD: 0%.
UV 100%, (M-2Boc)H+ 128.1.
tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[(3S)-5-ethyl-6-oxo-3,6-di-
hydro-2H-pyran-3-yl]carbamate (9c). 9c (587 mg, 100%) as white
1
solid. H NMR (300 MHz; CD3OD): δ 6.65 (s, 1H), 5.24−5.19 (m,
1H), 4.54 (t, J = 9.8 Hz, 1H), 4.40 (dd, J = 10.6, 5.9 Hz, 1H), 2.33 (q,
J = 7.3 Hz, 2H), 1.53 (s, 18H), 1.13 (t, J = 7.4 Hz, 3H). LC/MS(A):
RT 4.51. ELSD: 100%. UV 100%, (M-2Boc)H+ 142.1.
tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[(3S)-6-oxo-5-propyl-3,6-
dihydro-2H-pyran-3-yl]carbamate (9d). 9d (500 mg, 100%) as
1
white solid. H NMR (400 MHz; CD3OD): δ 6.62 (m, 1H), 5.18
(dddt, J = 9.1, 5.9, 3.2, 1.6 Hz, 1H), 4.53 (dd, J = 10.7, 9.0 Hz, 1H),
4.38 (ddd, J = 10.7, 5.9, 1.3 Hz, 1H), 2.32−2.19 (m, 2H), 1.55−1.46
(m, 20H), 0.96 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz; CD3OD): δ
166.19 (s, 1C), 153.69 (s, 2C), 142.61 (s, 1C), 132.43 (s, 1C), 85.06
(s, 2C), 69.31 (s, 1C), 51.02 (s, 1C), 34.04 (s, 1C), 28.39 (s, 6C),
22.62 (s, 1C), 14.17 (s, 1C). LC/MS(A): RT 4.78. ELSD: 0%. UV
93%, (M-2Boc)H+ 156.1.
tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[(1S,5S,6R)-1-butyl-2-oxo-
3-oxabicyclo[4.1.0]heptan-5-yl]carbamate (10e). 10e (72 mg, 29%,
48% based on recovered starting material). 1H NMR (400 MHz;
MeOD): δ 4.67 (m, 1H), 4.46 (dd, J = 13.3, 4.7 Hz, 1H), 4.27 (ddd, J
= 13.3, 2.1, 1.4 Hz, 1H), 1.69−1.63 (m, 2H), 1.59−1.26 (m, 24H),
1.11 (dd, J = 8.5, 6.2 Hz, 1H), 0.91 (t, J = 7.3 Hz, 3H). 13C NMR (101
MHz; MeOD): δ 175.24 (s, 1C), 154.25 (s, 2C), 84.20 (s, 2C), 69.91
(s, 1C), 50.23 (s, 1C), 35.65 (s, 1C), 30.20 (s, 1C), 28.24 (s, 6C),
26.53 (s, 1C), 26.18 (s, 1C), 23.96 (s, 1C), 14.82 (s, 1C), 14.39 (s,
1C). LC/MS(B): RT 4.19. ELSD: 100%. UV 0%, (M-2Boc)H+ 184.1.
(S)-tert-Butyl (5-Cyclopropyl-6-oxo-3,6-dihydro-2H-pyran-3-yl)-
tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[(3S)-5-butyl-6-oxo-3,6-di-
1
hydro-2H-pyran-3-yl]carbamate (9e). 9e (340 mg, 100%). H NMR
(400 MHz; CD3OD): δ 6.62 (m, 1H), 5.17 (dddt, J = 9.1, 5.9, 3.2, 1.6
Hz, 1H), 4.53 (dd, J = 10.7, 9.0 Hz, 1H), 4.38 (ddd, J = 10.7, 5.9, 1.3
Hz, 1H), 2.35−2.22 (m, 2H), 1.50−1.34 (m, 22H), 0.93 (t, J = 7.2 Hz,
3H). 13C NMR (101 MHz; CD3OD): δ 165.97 (s, 1C), 153.47 (s,
2C), 142.20 (s, 1C), 132.44 (s, 1C), 84.83 (s, 2C), 69.10 (s, 1C),
50.80 (s, 1C), 31.44 (s, 1C), 31.41 (s, 1C), 28.19 (s, 6C), 23.23 (s,
1C), 14.18 (s, 1C). LC/MS(A): RT 4.95. ELSD: 0%. UV 100%, (M-
2Boc)H+ 170.1.
tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[(3S)-6-oxo-5-phenyl-3,6-
dihydro-2H-pyran-3-yl]carbamate (9f). 9f (300 mg, 88%). 1H
NMR (400 MHz; CDCl3): δ 7.47−7.45 (m, 2H), 7.37−7.29 (m,
3H), 6.89 (dd, J = 2.6, 1.8 Hz, 1H), 5.41 (ddd, J = 10.7, 6.0, 2.6 Hz,
1H), 4.69 (dd, J = 10.7, 10.1 Hz, 1H), 4.42 (ddd, J = 10.1, 6.0, 1.8 Hz,
1H), 1.49 (s, 18H). 13C NMR (101 MHz; CDCl3): δ 162.69 (s, 1C),
151.90 (s, 2C), 144.06 (s, 1C), 135.07 (s, 1C), 130.23 (s, 1C), 128.39
(s, 1C), 128.35 (s, 2C), 128.26 (s, 2C), 84.03 (s, 2C), 67.40 (s, 1C),
50.22 (s, 1C), 28.00 (s, 6C). LC/MS(A): RT 4.73. ELSD: 100%. UV
89%, MH+ 190.0.
1
carbamate (11). 11 (440 mg, 41%). H NMR (400 MHz; CDCl3):
δ 6.27 (d, J = 5.2 Hz, 1H), 4.79 (bs, 1H), 4.38 (d, J = 3.5 Hz, 1H),
4.36 (d, J = 3.8 Hz, 1H), 4.30−4.26 (m, 1H), 1.84−1.77 (m, 1H), 1.43
(s, 9H), 0.85 (m, 1H), 0.84−0.82 (m, 1H), 0.59−0.55 (m, 1H), 0.52−
0.48 (m, 1H).
General Procedure for Deprotection of a Single Boc Group.
tert-Butyl ((1S,5S,6R)-1-Methyl-2-oxo-3-oxabicyclo[4.1.0]heptan-5-
yl)carbamate (12b). 10b (300 mg, 0.88 mmol, 1 equiv) was dissolved
in DCM (5.85 mL), and TFA (67 μL, 0.88 mmol, 1 equiv) was added.
The solution was stirred for 10 min, and the solvent was evaporated
off. The compound was purified by silica gel chromatography (eluent:
40−70% EtOAc in heptane) to afford 12b (207 mg, 95%) as a white
General Procedure for Cyclopropanation (Method A). tert-
Butyl N-[(tert-Butoxy)carbonyl]-N-[(1S,5S,6R)-1-methyl-2-oxo-3-
oxabicyclo[4.1.0]heptan-5-yl]carbamate (10b). Pd(OAc)2 (11.2
mg, 0.05 mmol, 0.05 equiv) was suspended in a solution of 9b (330
mg, 1 mmol, 1 equiv) in Et2O (20 mL). A solution of CH2N2
(approximately 5 equiv) in Et2O was added dropwise over
approximately 15 min. The solution was stirred for another 15 min
and quenched by addition of glacial acetic acid (0.29 mL, 300 mg, 5
mmol), and the solvent was evaporated off. The crude product was
purified by silica gel chromatography (eluent: 0−50% EtOAc in
heptane) to afford 10b (157 mg, 46%, 72% based on recovered
1
solid. H NMR (300 MHz; CD3OD): δ 4.29−4.15 (m, 2H), 4.06 (s,
1H), 1.69−1.59 (m, 2H), 1.47 (s, 9H), 1.34 (s, 3H), 1.07 (dd, J = 7.7,
5.9 Hz, 1H). LC/MS(A): RT 3.04. ELSD: 0%. UV 0%, (M-Boc)H+
142.1.
tert-Butyl ((1S,5S,6R)-1-Ethyl-2-oxo-3-oxabicyclo[4.1.0]heptan-5-
yl)carbamate (12c). 12c (170 mg, 95%) of the desired product as a
1
white foam. H NMR (300 MHz; CD3OD): δ 4.32 (dd, J = 12.6, 2.9
Hz, 1H), 4.18 (ddd, J = 12.6, 1.6, 1.6 Hz, 1H), 4.08 (bs, 1H), 1.85−
1.69 (m, 2H), 1.60 (dd, J = 5.8, 5.8 Hz, 1H), 1.51−1.32 (m, 10H),
1.14 (dd, J = 8.1, 6.2 Hz, 1H), 0.98 (t, J = 7.0 Hz, 3H). 13C NMR (75
MHz; CD3OD): δ 174.74 (s, 1C), 157.60 (s, 1C), 80.43 (s, 1C), 69.83
1
starting material). H NMR (300 MHz; CDCl3): δ 4.71 (m, 1H),
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(s, 1C), 45.01 (s, 1C), 28.85 (s, 1C), 28.75 (s, 3C), 27.36 (s, 1C),
27.01 (s, 1C), 15.66 (s, 1C), 11.92 (s, 1C). LC/MS(A): RT 3.87.
ELSD: 100%. UV 0%, (M-Boc)H+ 156.0.
1C), 53.58 (s, 1C), 47.70 (s, 1C), 36.93 (s, 1C), 30.84 (s, 1C), 28.84
(s, 3C), 23.92 (s, 1C), 19.22 (s, 1C), 14.42 (s, 1C), 9.23 (s, 1C). LC/
MS(A): RT 4.04. ELSD: 100%. UV 0%, (M-Boc)H+ 202.0.
tert-Butyl ((1S,5S,6R)-2-Oxo-1-propyl-3-oxabicyclo[4.1.0]heptan-
General Procedure for Oxidation (Method A). (1S,2R)-2-((S)-
Amino(carboxy)methyl)-1-methylcyclopropanecarboxylic Acid Hy-
drochloride (14b and 4b·HCl). 13b (167 mg, 0.64 mmol, 1 equiv)
was dissolved in 1 M aq NaOH (3.2 mL), and KMnO4 (458 mg, 2.90
mmol, 4.5 equiv) was added. The mixture was stirred for 4 h at room
temperature. 2-Propanol (4 mL) was added and the solution stirred for
40 min. Mn2O was filtered off and the filter washed with 2-propanol.
The solvent was evaporated off and EtOAc (30 mL), and 1 M KHSO4
(10 mL) were added. The water phase was extracted with EtOAc (3 ×
30 mL). The combined organic phases were dried twice over Na2SO4,
filtered, and evaporated to dryness to afford 14b (123 mg, 71%) as a
white solid. LC/MS(A): RT 2.67. ELSD: 100%. UV 0%, (M-Boc)H+
174.0. 14b (123 mg, 0.45 mmol, 1 equiv) was dissolved in a minimum
amount of dioxane (∼1 mL). 4 M HCl in dioxane (1 mL) was added
dropwise. After 1 h, the precipitate was centrifuged and the solvent was
decanted off. The precipitate that was washed with Et2O (4 mL), the
mixture centrifuged, and the solvent decanted off. The white solid was
dried to afford 4b·HCl (81 mg, 86% (71% when corrected from 1,4-
dioxane)) as a white solid. 1H NMR (300 MHz; CD3OD): δ 4.22 (d, J
= 10.2 Hz, 1H), 1.50−1.42 (m, 2H), 1.37 (s, 3H), 1.19−1.12 (m, 1H).
13C NMR (75 MHz; CD3OD): δ 176.71 (s, 1C), 171.24 (s, 1C), 52.54
1
5-yl)carbamate (12d). 12d (130 mg, 95%) as white foam. H NMR
(300 MHz; CD3OD): δ 4.29 (dd, J = 12.6, 3.0 Hz, 1H), 4.15 (ddd, J =
12.6, 1.6, 1.6 Hz, 1H), 4.06 (ddd, J = 2.7, 2.7, 1.2 Hz, 1H), 1.82−1.71
(m, 2H), 1.58 (dd, J = 5.8, 5.8 Hz, 1H), 1.53−1.41 (m, 11H), 1.31
(ddd, J = 13.2, 9.5, 6.4 Hz, 1H), 1.12 (dd, J = 8.2, 6.1 Hz, 1H), 0.92 (t,
J = 7.3 Hz, 3H). 13C NMR (75 MHz; CD3OD): δ 174.78 (s, 1C),
157.61 (s, 1C), 80.44 (s, 1C), 69.82 (s, 1C), 45.04 (s, 1C), 38.14 (s,
1C), 28.76 (s, 3C), 27.54 (s, 1C), 25.98 (s, 1C), 21.60 (s, 1C), 16.07
(s, 1C), 14.55 (s, 1C). LC/MS(A): RT 4.03. ELSD: 100%. UV 0%,
(M-Boc)H+ 170.1.
tert-Butyl ((1S,5S,6R)-1-Butyl-2-oxo-3-oxabicyclo[4.1.0]heptan-5-
yl)carbamate (12e). 12e (90 mg, 94%) as white foam. 1H NMR (300
MHz; CD3OD): δ 4.30 (dd, J = 12.5, 3.0 Hz, 1H), 4.16 (ddd, J = 12.6,
1.9, 1.5 Hz, 1H), 4.06 (bs, 1H), 1.81 (m, J = 4.7 Hz, 2H), 1.58 (dd, J =
5.8, 5.8 Hz, 1H), 1.47−1.26 (m, 14H), 1.12 (dd, J = 8.1, 6.1 Hz, 1H),
0.92 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz; CD3OD): δ 174.79 (s,
1C), 157.69 (s, 1C), 80.44 (s, 1C), 69.81 (s, 1C), 45.05 (s, 1C), 35.69
(s, 1C), 30.65 (s, 1C), 28.79 (s, 3C), 27.54 (s, 1C), 26.05 (s, 1C),
23.83 (s, 1C), 16.19 (s, 1C), 14.50 (s, 1C). LC/MS(A): RT 4.04.
ELSD: 98.4%. UV 0%, (M-Boc)H+ 184.1.
General Procedure for Hydrolysis. (1S,2R)-2-((S)-1-((tert-
Butoxycarbonyl)amino)-2-hydroxyethyl)-1-methylcyclopropanecar-
boxylic Acid (13b). To a solution 12b (207 mg, 0.86 mmol, 1 equiv)
in THF (1.6 mL) was added 1 M aq NaOH (1.54 mL, 1.54 mmol, 1.8
equiv) at room temperature and stirred at 40 °C for 30 min. EtOAc
(50 mL) was added, then 1 M KHSO4 (10 mL). The phases were
separated and the water phase extracted with additional EtOAc (2 ×
50 mL). The combined organic phases were dried over Na2SO4,
filtered, and evaporated to dryness. The crude product was purified by
silica gel chromatography (eluent: 1% AcOH in EtOAc) to afford 13b
(167 mg, 75%) as a white solid. 1H NMR (300 MHz; CD3OD): δ 3.70
(ddd, J = 10.2, 5.8, 4.2 Hz, 1H), 3.51 (dd, J = 11.1, 4.2 Hz, 1H), 3.44
(dd, J = 11.1, 5.8 Hz, 1H), 1.44−1.37 (m, 10H), 1.27 (s, 3H), 1.24−
1.15 (m, 1H), 0.86 (dd, J = 8.5, 4.1 Hz, 1H). 13C NMR (75 MHz;
CD3OD): δ 177.68 (s, 1C), 158.06 (s, 1C), 79.95 (s, 1C), 65.33 (s,
1C), 53.25 (s, 1C), 32.10 (s, 1C), 28.88 (s, 3C), 25.04 (s, 1C), 21.69
(s, 1C), 20.87 (s, 1C). LC/MS(A): RT 2.64. ELSD: 100%. UV 0%,
(M-Boc)H+ 160.2.
(s, 1C), 29.54 (s, 1C), 25.61 (s, 1C), 21.35 (s, 1C), 20.92 (s, 1C). LC/
MS(A): RT 0.57. ELSD: 0%. UV 0%, MH+ 174.0.
General Procedure for Oxidation (Method B). (1S,2R)-2-((S)-
Amino(carboxy)methyl)-1-ethylcyclopropanecarboxylic Acid Hy-
drochloride (14c and 4c·HCl). 13c (145 mg, 0.53 mmol, 1 equiv)
was dissolved in 1 M NaOH (2.7 mL), and KMnO4 (376 mg, 2.39
mmol, 4.5 equiv) was added. The mixture was stirred for 4 h at room
temperature. 2-Propanol (4 mL) was added and the solution stirred for
40 min. Mn2O was filtered off and the filter washed with 2-propanol.
The solvent was evaporated off, and EtOAc (30 mL) and 1 M KHSO4
(10 mL) were added. The water phase was extracted with EtOAc (3 ×
30 mL). The combined organic phases were dried over Na2SO4,
filtered, and evaporated to dryness. The compound was purified by
silica gel chromatography (eluent: 1% CH3COOH in EtOAc) to afford
14c (125 mg, 0.44 mmol, 83%). 1H NMR (400 MHz; MeOD): δ 4.33
(d, J = 9.9 Hz, 1H), 1.72 (dq, J = 14.3, 7.1 Hz, 1H), 1.46−1.33 (m,
12H), 1.03 (t, J = 7.3 Hz, 3H), 0.93 (dd, J = 7.9, 3.8 Hz, 1H). LC/
MS(A): RT 2.90. ELSD: 100%. UV 0%, (M-Boc)H+ 188.1. 14c (125
mg, 0.44 mmol) was dissolved in a minimum amount of Et2O (0.3
mL). 2 M HCl in Et2O (2 mL) was added dropwise. The solution was
stirred for 1 h, the mixture centrifuged, and the solvent removed by
decantation. The precipitate was washed with Et2O (4 mL),
centrifuged, and the solvent removed by decantation. The white
(1S,2R)-2-((S)-1-((tert-Butoxycarbonyl)amino)-2-hydroxyethyl)-1-
ethylcyclopropanecarboxylic Acid (13c). 13c (145 mg, 79%) as white
1
foam. H NMR (300 MHz; CD3OD): δ 3.67 (ddd, J = 10.1, 6.0, 4.0
Hz, 1H), 3.56 (dd, J = 11.0, 3.8 Hz, 1H), 3.48 (dd, J = 11.1, 6.1 Hz,
1H), 1.86 (dq, J = 14.0, 7.1 Hz, 1H), 1.47 (s, 9H), 1.38 (dd, J = 5.9,
4.2 Hz, 1H), 1.32−1.19 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H), 0.86 (dd, J =
8.5, 4.1 Hz, 1H). 13C NMR (75 MHz; CD3OD): δ 176.91 (s, 1C),
157.90 (s, 1C), 79.81 (s, 1C), 65.16 (s, 1C), 53.24 (s, 1C), 31.56 (s,
1C), 30.79 (s, 1C), 29.92 (s, 1C), 28.80 (s, 3C), 19.22 (s, 1C), 12.08
(s, 1C). LC/MS(A): RT 2.91. ELSD: 100%. UV 0%, (M-Boc)H+
174.1
1
solid was dried on high vacuum to afford 4c·HCl (91 mg, 94%). H
NMR (300 MHz; CD3OD): δ 4.21 (d, J = 10.1 Hz, 1H), 1.70 (dq, J =
14.2, 7.2 Hz, 1H), 1.59 (dq, J = 14.2, 7.2 Hz, 1H), 1.49−1.40 (m, 2H),
1.15 (dd, J = 6.2, 2.4 Hz, 1H), 1.08 (t, J = 7.3 Hz, 3H). 13C NMR (101
MHz; CD3OD): δ 176.15 (s, 1C), 171.30 (s, 1C), 52.78 (s, 1C), 31.49
(s, 1C), 29.20 (s, 1C), 28.68 (s, 1C), 20.42 (s, 1C), 11.87 (s, 1C). LC/
MS(A): RT 0.89. ELSD: 100%. UV 0%, MH+ 188.1
(1S,2R)-2-((S)-Amino(carboxy)methyl)-1-propylcyclopropanecar-
boxylic Acid Hydrochloride (14d and 4d·HCl). 14d (92 mg, 0.31
mmol, 95%) as a white foam/solid. 1H NMR (300 MHz; CD3OD): δ
4.28 (d, J = 10.0 Hz, 1H), 1.73−1.22 (m, 15H), 0.92 (m, 4H). 13C
NMR (75 MHz; CD3OD): δ 176.64 (s, 1C), 175.30 (s, 1C), 157.70
(s, 1C), 80.29 (s, 1C), 53.51 (s, 1C), 39.02 (s, 1C), 31.41 (s, 1C),
30.48 (s, 1C), 28.77 (s, 3C), 21.66 (s, 1C), 20.23 (s, 1C), 14.67 (s,
1C). LC/MS(A): RT 3.20. ELSD: 100%. UV 0%, (M-Boc)H+ 202.1.
4d·HCl (52 mg, 71%). 1H NMR (400 MHz; CD3OD): δ 4.18 (d, J
= 9.8 Hz, 1H), 1.67−1.40 (m, 6H), 1.16−1.11 (m, 1H), 0.92 (t, J = 6.9
Hz, 3H). 13C NMR (101 MHz; CD3OD): δ 176.19 (s, 1C), 171.26 (s,
1C), 52.77 (s, 1C), 38.50 (s, 1C), 30.49 (s, 1C), 28.73 (s, 1C), 21.59
(s, 1C), 20.53 (s, 1C), 14.53 (s, 1C). LC/MS(A): RT 1.51. ELSD:
100%. UV 0%, MH+ 202.1.
(1S,2R)-2-((S)-1-((tert-Butoxycarbonyl)amino)-2-hydroxyethyl)-1-
1
propylcyclopropanecarboxylic Acid (13d). 13d (92 mg, 67%). H
NMR (300 MHz; CD3OD): δ 3.61 (ddd, J = 10.0, 6.1, 3.8 Hz, 1H),
3.53 (dd, J = 11.0, 3.8 Hz, 1H), 3.44 (dd, J = 11.0, 6.1 Hz, 1H), 1.82
(ddd, J = 13.5, 9.5, 6.1 Hz, 1H), 1.53−1.35 (m, 12H), 1.28−1.10 (m,
2H), 0.91−0.83 (m, 4H). 13C NMR (75 MHz; CD3OD): δ 176.82 (s,
1C), 157.88 (s, 1C), 79.80 (s, 1C), 65.14 (s, 1C), 53.34 (s, 1C), 39.24
(s, 1C), 30.73 (s, 1C), 30.48 (s, 1C), 28.81 (s, 3C), 21.77 (s, 1C),
19.36 (s, 1C), 14.62 (s, 1C). LC/MS(A): RT 3.22. ELSD: 100%. UV
0%, (M-Boc)H+ 188.1.
(1S,2R)-2-((S)-1-((tert-Butoxycarbonyl)amino)-2-hydroxyethyl)-1-
butylcyclopropanecarboxylic Acid (13e). 13e (52 mg, 62%) as white
solid. 1H NMR (400 MHz; MeOD): δ 3.64 (ddd, J = 10.1, 6.1, 3.9 Hz,
1H), 3.57 (dd, J = 11.1, 3.9 Hz, 1H), 3.48 (dd, J = 11.1, 6.1 Hz, 1H),
1.87 (ddd, J = 13.8, 9.2, 5.1 Hz, 1H), 1.50−1.17 (m, 16H), 0.92 (t, J =
7.3 Hz, 3H), 0.87 (dd, J = 8.6, 4.3 Hz, 1H). 13C NMR (101 MHz;
CD3OD): δ 177.30 (s, 1C), 158.15 (s, 1C), 79.96 (s, 1C), 65.30 (s,
(1S,2R)-2-((S)-Amino(carboxy)methyl)-1-butylcyclopropanecar-
boxylic Acid Hydrochloride (14e and 4e·HCl). 14e (40 mg, 0.13
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1
mmol, 75%). H NMR (400 MHz; MeOD): δ 4.28 (d, J = 9.8 Hz,
Notes
1H), 1.69 (ddd, J = 12.6, 10.6, 4.3 Hz, 1H), 1.52−1.25 (m, 16H), 0.91
(bs, 1H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz; CD3OD): δ
176.90 (s, 1C), 175.53 (s, 1C), 157.9 (s, 1C), 80.36 (s, 1C), 53.59 (s,
1C), 36.52 (s, 1C), 31.43 (s, 1C), 30.66 (s, 1C), 30.55 (s, 1C), 28.73
(s, 3C), 23.93 (s, 1C), 20.17 (s, 1C), 14.37 (s, 1C). 4e·HCl (26 mg,
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH-NIND (NS065371,
NS036654 SFT) and the University of Copenhagen Pro-
gramme of Excellence Glutarget.
1
82%). H NMR (400 MHz; CD3OD): δ 4.16 (d, J = 9.8 Hz, 1H),
1.65−1.40 (m, 6H), 1.32 (m, 2H), 1.11 (m, 1H), 0.91 (t, J = 7.3 Hz,
3H). 13C NMR (101 MHz; CD3OD): δ 176.22 (s, 1C), 171.32 (s,
1C), 52.77 (s, 1C), 36.06 (s, 1C), 30.66 (s, 1C), 30.57 (s, 1C), 28.80
(s, 1C), 23.90 (s, 1C), 20.58 (s, 1C), 14.34 (s, 1C). LC/MS(A): RT
1.73. ELSD: 100%. UV 0%, (M-Boc)H+ 216.1.
ABBREVIATIONS USED
■
NHP5G, N-hydroxypyrazol-5-ylglycine; NMDA, N-methyl-D-
aspartic acid; CCG, carboxycyclopropyl)glycine; GluRs, gluta-
mate receptors Glu; CNS, central nervous system; mGluRs,
metabotropic glutamate receptors; iGluRs, ionotropic gluta-
mate receptors; AMPA, (S)-2-amino-3-(3-hydroxy-5-methyl-4-
isoxazolyl)propionic acid; KA, kainic acid; ABD, agonist
binding domain
Molecular Modeling and Ligand−Protein Docking. The Schro-
̈
dinger 2011 suite was used for modeling and docking studies
(Schrodinger, Portland, OR). The crystal structure of the soluble
̈
GluN2A-ABD construct in complex with glutamate (PDB code
2A5T)36 was employed in the Protein Preparation Wizard in Maestro
(Schrodinger, Portland, OR) with standard parameters for assigning
̈
charges and protons and performing a minimization. None of the
missing amino acid side chains were modeled. This model was then
used to generate van der Waals and electrostatic grids with the docking
REFERENCES
■
module Glide using default parameters (Schrodinger, Portland, OR).
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(1) Brauner-Osborne, H.; Egebjerg, J.; Nielsen, E. O.; Madsen, U.;
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(2) Erreger, K.; Chen, P. E.; Wyllie, D. J.; Traynelis, S. F. Glutamate
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The CCG analogues 4a−e and (R)-Pr-NHP5G (3b) were
submitted to minimization in macromodel (Schrodinger, Portland,
̈
OR) in tri-ionized forms using default parameters. The best scoring
poses according to G-Score were used. Figures were created using
Pymol (ver. 0.99, Delano Scientific).
In Vitro Pharmacology. Receptor Binding Assays. Affinities for
native AMPA, KA, and NMDA receptors in rat cortical synaptosomes
were determined using 5 nM [3H]AMPA37 (Kd = 35 nM, 53.4 Ci/
mmol), 5 nM [3H]KA (Kd = 5.6 nM, 49.9 Ci/mmol),38 and 2 nM
[3H]CGP 39653 (Kd = 6 nM, 40.5 Ci/mmol),39 respectively, with
minor modifications as previously described.40 Rat brain membrane
preparations used in these receptor binding experiments were
prepared according to a method previously described.41
Two-Electrode Voltage-Clamp Electrophysiology. Wild-type
cDNAs for rat GluN1 (GluN1−1a; GenBank accession numbers
U11418), rat GluN2A (D13211), rat GluN2B (U11419), rat GluN2C
(M91563), and rat GluN2D (L31611) were provided by Drs. S.
Heinemann (Salk Institute, San Diego, CA), S. Nakanishi (Kyoto
University, Kyoto, Japan), and P. Seeburg (University of Heidelberg,
Heidelberg, Germany). For expression in Xenopus oocytes, DNA
constructs were linearized by restriction enzymes and used as
templates to synthesize cRNAs using the mMessage mMachine kit
(Ambion). Xenopus oocytes for injection were prepared as previously
described.42 Two-electrode voltage-clamp recordings were performed
essentially as previously described.43 During recordings, the oocytes
were voltage-clamped at −60 to −40 mV and continuously perfused
with extracellular solution containing (in mM) 90 NaCl, 0.5, BaCl2, 1
KCl, 0.01 EDTA, and 10 HEPES (pH 7.4). 50 μM glycine was
included in the extracellular solution at all times.
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Agonist concentration−response data for individual oocytes were
fitted to the Hill equation. The logEC50 and nH from the individual
oocytes were used to calculate the mean
SEM. For graphical
presentation, data sets from individual oocytes were normalized to the
fitted maximal response and the mean SEM was calculated for each
of the normalized data points. These averaged data points were then
fitted to the Hill equation and plotted together with the resulting
curve. Relative Imax was calculated from a full concentration−response
measurement as Imax, agonist/Imax, glutamate, where Imax, agonist is the fitted
value according to the Hill equation and Imax, glutamate is the maximal
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: +45 35 33 65 66. Fax: +45 35 33 60 40. E-mail: rac@
sund.ku.dk.
4080
dx.doi.org/10.1021/jm400346a | J. Med. Chem. 2013, 56, 4071−4081

Journal of Medicinal Chemistry
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