Stereoselective Synthesis of New (2 S,3 R)-3-Carboxyphenyl)pyrrolidine-2-carboxylic Acid Analogues Utilizing a C(sp3)-H Activation Strategy and Structure-Activity Relationship Studies at the Ionotropic Glutamate Receptors

Article abstract of DOI:10.1021/acschemneuro.0c00003

Competitive antagonists for ionotropic glutamate receptors (iGluRs) are highly valuable tool compounds for studying health and disease states in the central nervous system. However, only few subtype selective tool compounds are available and the discovery of antagonists with novel iGluR subtype selectivity profiles remains a profound challenge. In this paper, we report an elaborate structure-activity relationship (SAR) study of the parental scaffold 2,3-trans-3-carboxy-3-phenyl-proline by the synthesis of 40 new analogues. Three synthetic strategies were employed with two new strategies of which one being a highly efficient and fully enantioselective strategy based on C(sp3)-H activation methodology. The SAR study led to the conclusion that selectivity for the NMDA receptors was a general trend when adding substituents in the 5′-position. Selective NMDA receptor antagonists were obtained with high potency (IC₅₀ values as low as 200 nM) and 3-34-fold preference for GluN1/GluN2A over GluN1/GluN2B-D NMDA receptors.

Full text of DOI:10.1021/acschemneuro.0c00003

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Research Article
Stereoselective Synthesis of New (2S,3R)‑3-
Carboxyphenyl)pyrrolidine-2-carboxylic Acid Analogues Utilizing a
C(sp3)−H Activation Strategy and Structure−Activity Relationship
Studies at the Ionotropic Glutamate Receptors
Silke Kayser, Jacob C. Hansen, Markus Staudt, Aleksandra Moroz, Younes Larsen, Piero Temperini,
Feng Yi, Jed T. Syrenne, Niels Krogsgaard-Larsen, Stylianos Iliadis, Birgitte Nielsen, Kasper B. Hansen,
Darryl S. Pickering, and Lennart Bunch*
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ABSTRACT: Competitive antagonists for ionotropic glutamate
receptors (iGluRs) are highly valuable tool compounds for
studying health and disease states in the central nervous system.
However, only few subtype selective tool compounds are available
and the discovery of antagonists with novel iGluR subtype
selectivity profiles remains a profound challenge. In this paper,
we report an elaborate structure−activity relationship (SAR) study of the parental scaffold 2,3-trans-3-carboxy-3-phenyl-proline by
the synthesis of 40 new analogues. Three synthetic strategies were employed with two new strategies of which one being a highly
efficient and fully enantioselective strategy based on C(sp3)−H activation methodology. The SAR study led to the conclusion that
selectivity for the NMDA receptors was a general trend when adding substituents in the 5′-position. Selective NMDA receptor
antagonists were obtained with high potency (IC50 values as low as 200 nM) and 3−34-fold preference for GluN1/GluN2A over
GluN1/GluN2B-D NMDA receptors.
KEYWORDS: Ionotropic glutamate receptors, NMDA receptor antagonist, Proline analogues, C(sp3)−H activation, Electrophysiology
disease,17 amyotrophic lateral sclerosis (ALS),18 cerebral
INTRODUCTION
■
stroke,19 and epilepsy.8,20
(S)-Glutamate (Glu) belongs to the pool of common amino
acids, but it is the major excitatory neurotransmitter in the
The iGluRs have been of long interest in academic and
industrial research with the aim to develop fully subtype
mammalian central nervous system (CNS).1 Upon axon firing,
selective antagonists, as such pharmacological tools are
Glu is released into the synaptic cleft where it binds to pre- and
attractive for studying and understanding of the role and
postsynaptic Glu receptors. These are divided into two main
function of these receptors in the healthy and diseased brain.
classes: the metabotropic Glu receptors (mGluRs) that
Different approaches have been taken from design and
produce a slower signal transduction through second
synthesis of noncompetitive channel blockers,21,22 negative
messenger systems, and ionotropic Glu receptors (iGluRs)
allosteric modulators,23 to orthosteric antagonists. With focus
that are responsible for fast signal transmission. Based on
here on the latter class, amino acid bioisosters such as
quinoxalinedione (1) and its congeners24−27 have been
ligand selectivity studies, iGluRs have been divided into the
three groups, named the α-amino-3-hydroxy-5-methyl-4-
investigated intensely, but most importantly as structurally
isoxazolepropinic acid (AMPA) receptors (subunits GluA1−
modified amino acid analogues. Representative selective iGluR
antagonist scaffolds are willardiines (2),28−30 cis-decahydroi-
soquinolines (3),7,31−34 2,4-trans-prolines,35 and the more
recently identified class by us 2,3-trans-3-aryl prolines (4).36
However, despite extensive synthetic efforts, and a vast number
4), kainic acid (KA) receptors (subunits GluK1−5), and N-
methyl-D-aspartate (NMDA) receptors (subunits GluN1,
GluN2A−D, and GluN3A,B).2 Imbalance of Glu signaling
has been correlated to a number of neurological and/or
psychiatric diseases like anxiety,3 depression,4,5 migraine,6,7
pain,8 and schizophrenia.9−11 Furthermore, it has been shown
that elevated Glu signaling is neurotoxic and ultimately leads to
neuronal death.12−14 Thus, iGluRs are also thought to be
involved in the disease mechanism of neurodegenerative
diseases such as Alzheimer’s disease,15,16 Huntington’s
Received: January 2, 2020
Accepted: January 27, 2020
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of X-ray crystal structures and modeling, only subtype selective
antagonists have been developed for GluK1 (e.g., UBP30428,37
and LY4661957). Thus, there is still a profound need for the
development of subtype-selective antagonists for the GluA1−4
and GluK2−5 subunits.
In previous studies, we have shown that introduction of a
substituent in the 4′ position of 4a significantly influences its
iGluR binding affinity profile.38 While a hydroxyl group (4b)
results in a high nanomolar affinity for GluK3 (Ki = 0.87 μM)
with 10−5-fold preference over GluK2/GluK1, a methyl group
(4c) eliminates all affinity for AMPA and KA receptors leaving
midrange micromolar affinity for native NMDA receptors (Ki =
17 μM). The halogen series (F, Cl, and Br (4d−f))39 unveiled
a clear shift toward selectivity for native NMDA receptors with
the 4′-chloro and 4′-bromo substituents being optimal for
binding (Ki = 0.63 and 0.62 μM, respectively). Subsequently,
4e was shown to be a full antagonist with preference for
GluN1/GluN2A and furthermore shown to enhance the
anticancer effect of sorafenib in vitro.39 For the 5′ position,
neither a hydroxyl group (5a) nor large lipophilic groups (not
shown) improved the binding affinity significantly or modified
the selectivity profile positively.38 Directly on the proline
skeleton the 2,4-cis-4-position was substituted with an n-propyl
group (9a) which resulted in high nanomolar affinity for
GluK1 (Ki = 0.62 μM) but only with a 4-fold preference over
GluK3.38
Herein, we report further structure−activity relationship
(SAR) investigations of 4a in our search for subtype selective
iGluR antagonists. Furthermore, a new and efficient route for
the stereoselective synthesis of 4a and its analogues is
described utilizing a C(sp3)−H activation-arylation strategy
as the key step.
Figure 1. (A) X-ray crystal structure of 5a (green) which holds a 5′-
OH substituent, in the LBD of GluA2 (PDB: 4YMA).38 Receptor
surface is displayed in gray. (B) X-ray crystal structure of 5a (green)
in GluA2 (PDB: 4YMA). Protein surface is colored in gray. The 5′-
position directs toward a cave-shaped space with a depth of 10−12 Å.
RESULTS AND DISCUSSION
■
From the X-ray crystal structure of 5a in GluA2-LBD (Figure
1A), is it seen that the 4′-position directs substituents into
close contact with receptor residue Leu671 (GluA2 number-
ing). Our previous SAR study (4a−f, Table 1) also shows that
different substituents in that position greatly influence the
binding affinity profile. We therefore sought to expand the SAR
study further by synthesis of the 4′-amino analogue 4g and the
4′-carboxylic acid analogue 4h. Along this line, we also decided
to reposition the vital 3′-carboxylic acid group to the 4′-
position (compound 7, Figure 2).
explore the described cavity in the receptor, in a diversity
oriented way. For simplicity, we decided to first synthesize
analogues with only one amino acid, series 8a−s. Depending
on the results, additional analogues should be prepared.
The last series of compounds (9b−e) focus on the
modification of the C4-side chain (R3) of 9a. Although the
2,4-cis stereochemistry is opposite to the observed 2,4-trans
stereochemistry of naturally occurring ligands (e.g., kainate,40
domoate41,42), the binding mode of 9a in LBD of GluK1
(PDB: 4YMB) indicated a good fit of the propyl chain in the
binding pocket (Figure 3). Therefore, the 2,4-cis stereo-
chemistry remained in our design of new C4-analogues. As
widely accepted in the field, the LBD of iGluRs adopt an open
conformation when antagonists are bound.43 As indicated in
Figure 3, on the right-hand site of the receptor, a cavity is
formed that can be reached by extending the propyl chain.
Additionally, the crystal structure demonstrated that the propyl
chain is in close proximity to the side chain of Glu441 (GluK1
numbering). In the agonist (closed) conformation of the LBD,
Glu441 stabilizes interdomain contact.44−46 However, due to
the open conformation of the LBD stabilized by binding of 9a
this interaction is lost.38 We envisioned that alkyl amines 9b,c
would be able to interact with Glu441 through electrostatic
interactions as well as hydrogen bonding interactions.
Additionally, 9c will be able to reach into the channel.
Furthermore, alcohol (9d) and carboxylic acid 9e were
designed to replace the role of Glu441 and engage with the
The 5′-position of 4a (series 5) directs substituents into a
10−12 Å deep hydrophilic cone-shaped cavity (Figure 1B). We
decided to explore further the SAR of substituents with
hydrogen bonding properties by the synthesis of analogues 5c
(COOH) and 5d (1,2,3-triazole) (Figure 2). In addition, the
hydrogen bonding capable 5′-aryl groups pyridines 5e−g and
pyrimidines 5h,i were included in the SAR study (Figure 2). In
addition to the aforementioned bisaryl analogues, we decided
to design conformationally relieved analogues by incorporation
of a tethering nitrogen atom (compound 5j), to be further
functionalized to sulfonamides 5k,l and benzamide 5m (Figure
2).
Finally, analogue 6b was designed which comprises
substituents in both the 4′- and 5′-positions, based on the
NMDA receptor-selective analogue 4f (4′-Cl). The 5′-bromo
analogue 6a was included in the SAR study as it was easily
accessible from a synthetic intermediate (discussed later).
At this stage, we recognized that the 5′-carboxylate group
could easily be coupled with an amino acid or short peptide to
B
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Table 1. Generalized iGluR Antagonist Compound Classes 1−4 and Binding Affinities of Selected Antagonists UBP304 (2a),
LY466195 (3a), and 4a−e at Native AMPA, KA, and NMDA Receptors (Rat Synaptosomes) and Cloned Rat Homomeric
a
Receptors GluK1-3
native iGluRs
KA (IC50)
homomeric GluK receptors
R1
R2
R3
AMPA (IC50)
NMDA (Ki)
GluK1 (Ki)
GluK2 (Ki)
GluK3 (Ki)
111
8.9
2a28,37
3a7
−
−
H
OH
Me
F
Cl
Br
H
−
−
H
H
H
H
H
H
−
−
H
H
H
H
H
H
0.012
0.05
4.3
>100
−
4a36
4b38
4c39
4d39
4e39
4f39
51
2
22
1.4
>100
59
>100
>100
6.7
6
1
17
4.6
0.63
0.62
4.1
25
>100
10−100
>100
>100
>100
>100
32
8.1
0.87
>100
11
131
4.8
>100
12
154
>100
>100
>100
>100
>100
6.3
>100
5a38
9a38
OH
H
H
n-Pr
3.7
0.62
2.1
2.2
H
48
22
81
a
All values in μM. −: Values not determined. Radioligands: AMPA, [3H]AMPA; KA, [3H]KA; NMDA, [3H]CGP-39653; GluK1, [3H]NF608;
GluK2 and GluK3, [3H]KA.
Figure 2. Overview of herein designed and synthesized analogues of iGluR antagonist 4a: series 4−6, 8, 9 and compound 7.
C
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a
Scheme 2. Synthesis of Boronic Acid 17
Figure 3. Compound 9a in the GluA1-LBD (PDB: 4YMB).
extensive hydrogen bonding network in this region and thereby
further stabilize the open, inactive conformation of the LBD.
Chemistry. 4′-Carboxylic acid analogue 7 was synthesized
stereoselectively in accordance with our previously described
route (Scheme 1).38 1,4-Addition of the corresponding arylic
a
Reagents and conditions: (a) MeOH, H2SO4, reflux; (b) pinacol,
Et2O; (c) LiAlH4, THF, 0−20 °C, (86% over three steps);
TBDMSCl, imidazole, DMAP, DMF (quantitative).
Scheme 1. Synthesis of Analogue 7 from Enantiopure Enone
10
a
a
Scheme 3. Synthesis of 5e−i
a
Reagent and conditions: (a) nBuLi, ((3-bromo-benzyl)oxy)(tert-
butyl)dimethylsilane, CuCN, thiophene, Et2O, −78 to −42 °C,
(84%); (b) BH3·Et2O, THF, 0 °C to reflux, 18 h, (59%); (c) 1N
TBAF, THF, rt, 18h, (88%); (d) NaIO4, RuCl3·H2O, H2O/MeCN/
EtOAc, 0 °C, 1.5 h, (86%); (e) TFA, DCM, rt, overnight, 99%.
cuprate to enantiopure enone 10 gave adduct 11 as one
diastereomer in 84% yield. Reduction of the lactam by borane
in tetrahydrofuran (THF) (to give 11a not shown) followed
by removal of the tert-butyldimethylsilyl (TBS) ethers afforded
diol 12 in 52% yield over two steps. Oxidation with RuCl3 gave
the corresponding diacid (12a not shown), and after removal
of the BOC group by TFA the target compound 7 was
obtained in 86% yield over two steps.
a
Reagents and conditions: (a) Boronic ester 17, Rh(I), CsCO3,
(86%); (b) DMS·BH3, THF, reflux, (62%); (c) TBAF, THF, (68%);
(d) Pd-catalyst, Ar−B(OR)2, solvent, (Xantphos), heating, (31−
87%); (e) RuCl3, NaIO4, H2O, MeCN, EtOAc; (f) TFA, DCM, then
1 M HCl, (6−98%); (g) BBr3, DCM, (4%).
Analogues 5e−i were synthesized in a similar fashion as 7,
with the only change being that the copper catalyzed 1,4-
addition was replaced by a rhodium(I) catalyzed 1,4-
addition.39 The appropriate boronic ester 17 was prepared
from 13 in four steps (Scheme 2).
With boronic ester 17 in hand, rhodium(I) catalyzed 1,4-
addition to enantiopure enone 10 gave adduct 18 in a full
diastereoselective fashion (Scheme 3). Subsequent reduction
of the lactam functionality of 18 was attempted using several
reported conditions,36 with the dimethylsulfide−borane
complex being superior (intermediate 18a, not shown). The
two silyl ethers were cleanly removed with tetra-n-butylammo-
nium fluoride (TBAF) to provide diol 19 in 42% yield over
two steps. Initially, we pursued direct oxidation of diol 12 to
warrant late stage differentiation followed by only a
deprotection step, but unfortunately all attempts thereto failed.
Consequently, aryl group insertion was performed on 19 by
standard Suzuki cross coupling chemistry in 63−87% yield.
Ruthenium-catalyzed oxidation of diols 20a−e to their
corresponding diacid was accompanied by side reactions
mostly undesired N-oxidation. After purification, removal of
the Boc group with trifluoroacetic acid (TFA) in dichloro-
methane (DCM) or BBr3 in DCM by pyridine analogues 5e−g
D
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a
Scheme 4. Synthesis of 5j−m
a
Reagents and conditions: (a) (i) Et3N, NCS; (ii) Pyr, CbzCl, (55%); (b) NBS, DABCO, (80%); (c) 23, PdCl2dppf, Cs2CO3, THF/H2O, 80 °C,
(85%); (d) H2 (g), Pd/C, MeOH, rt, (quantitative); (e) Boc2O, DCM, rt, (50% over two steps); (f) RCl, pyridine, rt, overnight, (85−92%); (g)
(i) (for 26a) LDA, THF, −78 to 0 °C, 10 min, (50%, d.r. 1.3:1) or (ii) (for 26b−d) 25 wt % NaOMe in MeOH, MeOH, reflux, 24 h,
(quantitative); (h) 4 N HCl, reflux, 24 h, (16−74%); (i) (i) LiOH, MeOH/H2O, (ii) TFA, DCM, (20%); (j) MeOH, SOCl2, (73%).
Scheme 5. Retrosynthetic Analysis for CH Activation Strategy
a
Scheme 6. Synthesis of Intermediate 28a
a
Reagents and conditions: (a) 8-Aminoquinoline, EDCI, HOBt, DCM, rt, 24 h, (85%); (b) methyl-3-bromo-5-iodobenzoate, 10 mol % Pd(OAc)2,
30 mol % PivOH, Ag2CO3, toluene, reflux, 3 days, (40−48%); (c) NaOH, EtOH, reflux, 24 h, (95%); (d) TBTA, DCM, rt, 24 h, (74%).
in quantitative yields, while purification of 5h and 5i proved
more difficult, thus resulting in low yields.
( )-cis-25 was treated with LDA and quenched with NH4Cl.
Deprotection of ( )-trans-27a−c was achieved in 4 N HCl to
afford the target analogues ( )-5j−l. Benzamide ( )-trans-
27d was deprotected to give 5m in a two-step procedure: first
ester hydrolysis with LiOH followed by N-Boc removal with
TFA in DCM (20% over three steps).
For the remaining target molecules 4g−i, 5b−d, and 6a,b
the two above-described synthetic routes were not easily
applicable due to functional group incompatibilities. Further-
more, the cuprate approach (Schemes 1 and 3) is troubled by a
high number of protection and deprotection steps, while the
second approach (Scheme 4) lacks enantioselective control.
Therefore, a CH activation approach was pursued to access 5a
and the remaining analogues designed (Scheme 5).
For the synthesis of analogues 5j−m, a racemic diaster-
eoselective approach was taken due to efficiency (Scheme 4).
First, (S)-O-methyl-proline was converted to the correspond-
ing enone 21 as described by Huy et al.47 Bromination with
NBS gave bromine 22 in 80% yield, which was coupled with
boronic acid 23 to give 24 in high yield. Reduction of the
alkene over Pd/C yielded racemic 2,3-cis-diamine which was
protected in situ with Boc anhydride to give aniline ( )-cis-25
in 50% yield over two steps. Derivatization to sulfonamides
( )-cis-26b,c and benzamide ( )-cis-26d proceeded smoothly,
and epimerization of the alpha center was performed with 1 N
NaOMe in (trans:cis 10:1 to >20:1). To obtain ( )-cis-27a,
E
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To secure a global and mild deprotection, the amino and
carboxylic acid functionalities of 6e were protected as their tert-
butyl carbamate and tert-butyl ester, respectively (Scheme 5).
Intermediate 28 should derive from 29 by a sequence of amide
hydrolysis, α carbon epimerization, and esterification. A similar
analogue to 29 was recently reported as obtained from 30 by a
stereoselective Pd-catalyzed C−H activation-arylation protocol
of amide 30 with the corresponding aryl iodide 31.48,49 Amide
30 originates from commercially available N-Boc-D-proline.
Starting from commercially available N-Boc-D-proline, amide
formation was performed with 8-aminoquinoline (8-AQ) in
the presence of EDCI and HOBt in 85% yield (Scheme 6).
The key C−H activation-arylation step was carried out
according to reported conditions to afford 29 in 45%
yield.48,49 The 8-AQ directing group was then removed
under basic conditions with 10 equiv of NaOH in EtOH at
100 °C.49 Fortunately, due to the high stability of the amide,
the alpha center fully epimerized under these forcing
conditions and gave 32 as a single enantiomer. The subsequent
simultaneous protection of both carboxylic acids as tert-butyl
esters turned out to be rather challenging. Various conditions
were attempted and resulted in tert-butyl 2,2,2-trichloroacedi-
midate (TBTA) as the superior reagent to afford 28a in 70%
yield. To summarize, the new strategy delivers key
intermediate 28 in only four steps (21% yield) and full
diastereoselectivity.
methyl-3-iodobenzoate to give 33. Subsequent epimerization
and amide hydrolysis with NaOH in EtOH was followed by
Boc deprotection with TFA in DCM to afford 4a in 74% over
2 steps. Its 1H NMR spectrum proved identical to an authentic
sample of 4a obtained by 1,4-cuprate strategy, thus proving the
2,3-trans relationship. The 2,3-cis-4a diastereomer was
obtained by amide hydrolysis under acidic conditions with
40% aqueous H2SO4, and the chemical shift of the alpha
proton of 2,3-cis-4a was shown to be significantly different
(4.15 ppm for 4a vs 4.45 ppm for 2,3-cis-4a; measured in
D2O).
For analogues 4g and 4h the C−H activation-arylation was
performed with aryl iodide 36 (Scheme 8). C−H activation-
arylation of 30 with 36 gave 34a in 61%. After epimerization
and amide hydrolysis, 35a was further converted to 35b with
NaN3 in the presence of Cs2CO3, CuI, and EtOH.
Furthermore, 35a was protected as dimethyl ester and
subjected to Pd-catalyzed oxidative carbonylation50 conditions
to install 4′-carboxylate. However, this approach only yielded
in dehalogenation of the 4′-position. The obtained inter-
mediates 35a,b were deprotected with TFA in DCM to afford
4g in 39% yield and 4h in 14% yield over two steps,
respectively. In an alternative approach to diacid analogue 4i,
the diacid was installed by preparation of the 38. As 38 is not
commercially available, it was synthesized from 37 in three
steps by esterification, reduction, and subsequent Sandmeyer51
reaction to afford 38 in 30% overall yield. C−H activation-
arylation with 38 afforded 34c in 37% yield which was further
treated with NaOH in EtOH, followed by deprotection with
TFA in DCM. The final compound was purified by preparative
HPLC and transformed into its trisodium salt with 1 N NaOH
to afford 4i in 72% yield. To ensure that cyclization to a cyclic
anhydride had not occurred during purification and isolation of
the compound, the final product was treated with benzyl
amine. Analysis by LCMS and HPLC confirmed that no amide
formation had occurred.
To confirm that the epimerization had indeed taken place,
4a was resynthesized (Scheme 7). First, 30 was reacted with
a
Scheme 7. Synthesis of 4a and 2,3-cis-4a
Analogue 5b (Scheme 9) was obtained by direct
deprotection of 28a, while 5′-carboxylic acid 5c was obtained
by first converting 28a to its corresponding 5′-carboxylate 28b
by a Pd-catalyzed oxidative carbonylation50 in 90% yield.
Deprotection of 28b was carried out with TFA in DCM and
cleanly afforded 5c.
Sonogashira coupling52 of 23a (Scheme 10) with
trimethylsilylacetylene (TMSA) alkyne and subsequent de-
protection with TBAF afforded alkyne 35a in 66% yield over
two steps. CuAAC53 with TMSN3 afforded triazole 36a in 65%
a
Reagents and conditions: (a) NaOH, EtOH, reflux, 24 h, (95%); (b)
TFA, DCM, rt, (74%); (c) 40% H2SO4, H2O, (27%).
a
Scheme 8. Synthesis of 4f−h
a
Reagents and conditions: (a) 36 or 38, 10 mol % Pd(OAc)2, 30 mol % PivOH, Ag2CO3, toluene, reflux, 3 days, (61% and 37%); (b) NaOH,
EtOH, reflux, 24 h, (86% and 86%); (c) NaN3, Cs2CO3, CuI, EtOH, reflux, 3 days, (d) TFA, DCM, (14−72%); (e) SOCl2, MeOH, reflux,
overnight, (70%); (f) Pd/C, H2, MeOH, (95%); (g) I2, tert-BuNO2, MeCN, toluene, 0 °C, then rt, 18 h, (46%).
F
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a
yielded free amino acids 6a,b in 96% and 93% yield,
respectively.
Scheme 9. Synthesis of 5b,c
The synthesis of the 19 amino acid conjugates 8a−s
commenced from 5′-carboxylic acid 23a and suitably protected
amino acid under coupling reaction conditions, followed by
suitable deprotection (Scheme 12). For details, see the
Supporting Information.
Scheme 12. Synthesis of Amino Acid Conjugates 8a−s from
a
Intermediate 23a
a
Reagents and conditions: (a) HCOOH, Ac2O, Et3N, Pd(OAc)2,
Xantphos, 80 °C, (90%); (b) TFA, DCM, (77% and 75%,
respectively).
a
Scheme 10. Synthesis of 5d
a
Reagents and conditions: (a) EDCI, HOBt, AA-OtBu, DMF, rt, 24
h, (65−99%); (b) TFA, DCM, rt, overnight (33−97%); (c) (i) 1 N
LiOH, MeOH, rt, overnight, (ii) TFA, DCM, rt, overnight, (46%).
For the synthesis of analogues 9b−e comprising a C4′-
substituent, the C−H activation-arylation strategy was applied,
but starting from methyl-Boc-D-pyroglutamate (Scheme 13).
Deprotonation with LHMDS and alkylation with allyl bromide
gave a 2:1 mixture of the trans:cis diastereomers, which were
separated on flash column chromatography to afford 47 in 36%
yield as a single diastereomer. Lactam reduction was performed
with superhydride, followed by treatment with BF3 and Et3SiH
to give 48 in 72% yield over two steps. Ester hydrolysis and
amide coupling with 8-AQ in the presence of EDCI and HOBt
proceeded in 72% yield over two steps. The following C−H
activation-arylation step with methyl-3-iodobenzoate provided
49 in 36%. Epimerization and amide hydrolysis with NaOH in
EtOH, followed by esterification with TBTA gave protected
alkene, which was converted to aldehyde 50 by treatment with
OsO4 and NaIO4 (19% yield over three steps). To summarize,
aldehyde 50 was obtained in nine steps in 12% overall yield
from commercially available methyl-Boc-D-pyroglutamate.
Reductive amination of key aldehyde 50 with MeNH2·HCl
or BnNH2·HCl in the presence of NaBH3CN gave secondary
amines 51a,b in 48% and 70% yield, respectively (Scheme 14).
Furthermore, aldehyde 50 was reduced to alcohol 51c with
a
Reagents and conditions: (a) TMSA, PdCl2(PPh3)2, CuI, Et3N, 60
°C, 12 h, (77%); (b) 1 N TBAF, THF, rt, 30 min (82%); (c) TMSN3,
CuI, DMF-MeOH, (65%); (d) TFA, DCM, (92%)
yield. Finally, deprotection with TFA in DCM afforded 5d in
92%.
The synthesis of 6a,b (Scheme 11) commenced with
iodination of commercially available 43 followed by
esterification to give tetrasubstituted aryl iodide 44 (69%
yield over two steps). Coupling of highly functionalized aryl
iodide 44 with 30 under CH activation conditions, gave 45 in
31% yield and underlines the power of this synthetic strategy.
By following the before established sequence of basic
hydrolysis, epimerization and esterification with TBTA
intermediate 46a was obtained in 46% yield over two steps.
Subsequent Pd-catalyzed oxidative carbonylation50 gave
carboxylate 46b. Global deprotection of 46a,b with TFA
a
Scheme 11. Synthesis of 6a,b
a
Reagents and conditions: (a) NIS, TfOH, (99%); (b) SOCl2, MeOH, reflux, ON, (70%); (c) 44, 10 mol % Pd(OAc)2, 30 mol % PivOH, Ag2CO3,
toluene, reflux, 3 days, (31%); (d) NaOH, EtOH, reflux, 24 h, (88%); (e) TBTA, DCM, (52%); (f) HCOOH, Ac2O, Et3N, Pd(OAc)2, Xantphos,
80 °C, (63%); (g) TFA, DCM, (96% and 93%, respectively).
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a
both carboxylate groups are required for the observed shift to
full NMDA receptor selectivity.
Scheme 13. Synthesis of Intermediate Aldehyde 50
With a bromine in the 5′-position (compound 5b), no
significant change in the pharmacological profile was observed,
and the 5′carboxylic acid analogues 5c showed a 9-fold
increase in affinity for the NMDA receptors (Ki = 0.48 μM).
Although mid-micromolar affinity was observed for both
AMPA and KA receptors, 5c displays a 75-fold selectivity for
NMDA receptors. Likewise, 5′-triazole 5d showed a 7-fold
higher affinity for native NMDA receptors (Ki = 0.83 μM)
compared to 4a (Ki = 6.0 μM) but only a 24-fold preference
for NMDA over KA (IC50 = 20 μM) and AMPA (IC50 = 21
μM) receptors.
The group of six membered 5′-heteroaryl-analogues, 5e−i,
all had no significant change in their profile among native
iGluRs homomeric GluK1−3 (Table 3). From docking studies
of the group, it became apparent that the heteroaromoatic
group is in close proximity to residues that are not conserved
in GluA1 and GluA2 (GluA1: Ala666, Glu665; GluA2: Ser673,
Asp672). For this reason, we decided to determine the binding
affinity of 5e−i at GluA1 and GluA2. A general trend of 3−4-
fold higher affinity for GluA1 over GluA2 was observed, which
regrettably could not motivate subsequent design and SAR of
additional analogues.
The installation of amine ( )-5j as well as sulfonamide
( )-5k,l and amide linker ( )-5m did not lead to a significant
change in binding affinity profile. Mid-micromolar affinities
persisted for ( )-5j−l at native AMPA and NMDA receptors,
while affinity for native and cloned KA receptors varied over
the micromolar range (IC50 = 16 to >100 μM and Ki = 4 to
>100 μM)).
a
Reagents and conditions: (a) LHMDS, then allyl bromide, THF,
DMPU, −78 °C to −40 °C, 4 h, (36%); (b) (i) LiEt3SiH, then H2O2;
(ii) BF3·Et2O, Et3SiH, (72%); (c) LiOH, H2O, THF, (quantitative);
(d) EDCI, HOBt, 8-aminoquinoline, DCM, rt, overnight, (72%); (e)
methyl-3-iodobenziate, Pd(OAc)2, PivOH, toluene, reflux, 3 days,
(35%); (f) (i) NaOH, EtOH, 100 °C, overnight, (ii) TBTA, DCM, rt,
overnight, (35% over two steps); (g) OsO4, NaIO4, THF, H2O, rt, 4
h, (53%).
a
Scheme 14. Synthesis of 9b−e from Aldehyde 50
Compounds 6a and 6b, which are hybrid structures of 4e
and 5b,c, maintained their selectivity for the NMDA receptors,
and notably 6b showed an additive effect of the 4′-chloro
substituent rendering it the most selective highest affinity
NMDA receptor ligand in this series (Ki = 160 nM).
a
Reagents and conditions: (a) MeNH2·HCl, NaBH3CN, MeOH, rt,
overnight, (48%); (b) BnNH2·HCl, NaBH3CN, MeOH, (70%); (c)
NaBH4, MeOH, rt, 30 min, (83%); (d) NaO2Cl, THF/H2O/tert-
BuOH 1:1:1, 2-methyl-butene, phosphate buffer, (94%); (e) TFA,
DCM, rt, overnight, (53−99%).
The 19 analogues 8a−s which comprise an amino acid
coupled in the 5′-position showed a general preference for
binding to native NMDA receptors (Ki = 0.5−10 μM) over
native AMPA (IC50 = 5−50 μM) and native KA (IC50 = 10−
100 μM) receptors (Table 4). In more detail, the SAR study
shows that introduction of an alkyl substituent, analogues 8a−
e, leads to a size-dependent decrease in binding affinity for
native NMDA receptors (8a, Ki = 0.60 μM to 8e, Ki = 5−10
μM). Perhaps unexpected, analogue 8k, which comprises Glu,
maintained high nanomolar affinity for native NMDA
receptors and in fact displayed higher selectivity over AMPA
and KA receptors compared to 8a. On the other hand,
installation of hydroxyl groups in analogues 8h and 8i showed
no significant effect. Histidine analogue 8q was found to be the
best GluK3-preferring ligand of this series (Ki = 6.23 2.69),
but also lysine analogue 8o which displayed low micromolar
affinity for both GluK1 and GluK3. Unfortunately, amide
analogues 8l and 8n were both unstable in the assay buffer, and
thus, a clear pharmacological profile could not be obtained.
In comparison to the 2,4-cis-4-propyl analogue 9a, N-methyl
amine analogue 9b showed a general decrease in affinity for all
the iGluRs (Table 5). But interestingly, with an increase of
size, the N-benzyl analogue 9c showed affinity for GluK1 and
GluK3 in the low micromolar range (Ki = 2.98 and 2.46 μM,
respectively). This could indicate that the phenyl ring can
occupy the cavity in the open conformation of the LBD, but
also the increased lipophilicity of the ligand serves to lower its
NaBH4 in MeOH in 83% yield, and acid 51d was obtained by
Pinnick oxidation of aldehyde 50 in 94% yield. Global
deprotection of 51a−d was achieved with TFA in DCM to
provide target compounds 9b−e in 53−99% yield, respectively.
To summarize, a total of 40 new analogues of 4a were
synthesized by three different strategies. On the comparison of
these, the C−H activation-arylation approach is by far the most
efficient and also allows for full enantiocontrol of the product.
Pharmacological Characterization. All analogues syn-
thesized were first characterized in radioligand binding assays
at native iGluRs and cloned rat homomeric subtypes, GluK1−
3 (Table 2). Furthermore, analogues 5e−i were characterized
at cloned homomeric GluA1 and GluA2, and analogues which
showed good to high affinity for the NMDA receptors (Ki ≤ 1
μM) were furthermore investigated in functional assays at the
four GluN1/GluN2A−D NMDA receptor subtypes.
Surprisingly, 4′-amino analogue 4g displayed significantly
reduced affinity for all iGluRs compared to the 4′-hydroxy
analogue 4b (Table 1). In contrast, introduction of a carboxylic
acid group in the 4′-position (compound 4h) resulted in a fully
selective, low micromolar affinity NMDA receptor ligand. In
comparison with compound 7, it is interesting to observe that
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Table 2. Chemical Structures and Binding Affinity Data for Analogues 4g,h, 5b−d, 6a,b, and 7 at Native iGluRs (Rat
a
Synaptosomes) and Homomeric Recombinant Rat iGluRs
b
c
native iGluRs (synaptosomes)
KA IC50
cloned homomeric iGluRs
AMPA IC50
>100
>100
NMDA Ki
>100
1.3 [5.88 0.06]
8.5
GluK1 Ki
GluK2 Ki
GluK3 Ki
d
d
d
4g
4h
7
R1 = NH2
R1 = CO2H
>100
>100
>100
(70 16)
>100
>100
(77 5)
>100
(64 4)
>100
>100
>100
>100
5b
5c
5d
6a
6b
R2 = Br
26 [4.60 0.04]
36 [4.45 0.04]
21 [4.69 0.04]
>100
10 [4.99 0.04]
63 [4.21 0.05]
20 [4.71 0.06]
>100
2.4 [5.63 0.05]
0.48 [6.32 0.03]
0.83 [6.08 0.05]
1−10
5.88 0.60
12.5 1.7
>50
≥100
≥100
>100
>100
>100
≥100
≥100
2.42 0.11
15.7 3.1
10−50
≥100
R2 = CO2H
R2 = triazole
R2 = Br
R2 = CO2H
>100
>100
0.16 [6.82 0.08]
>50
a
All values in μM. −: Values not determined. Radioligands used: AMPA, [3H]AMPA; KA, [3H]KA; [3H]CGP-39653; GluK1, [3H]NF608; GluK2
b
and GluK3, [3H]KA. Data are mean values of three to six individual experiments performed in triplicate. For AMPA and KA: pIC50 values with
c
SEM in brackets. For NMDA: pKi values with SEM in brackets. Mean values SEM of at least three experiments conducted in triplicate at 12−16
drug concentrations. Percent specific binding mean values SD of three experiments at 10 μM ligand concentration.
d
Table 3. Chemical Structures and Binding Affinities of Analogues 5e−m at Native iGluRs (Rat Synaptosomes) and Homomeric
a
Recombinant Rat iGluRs
a
All values in μM. −: Values not determined, Radioligands used: AMPA, [3H]AMPA; KA, [3H]KA; [3H]CGP-39653; GluA1, [3H]AMPA; GluA2,
b
[3H]AMPA]; GluK1, [3H]NF608; GluK2 and GluK3, [3H]KA. Data are mean values of three to six individual experiments performed in triplicate.
For AMPA and KA: pIC50 values with SEM in brackets. For NMDA: pKi values with SEM in brackets. Mean values SEM of at least three
experiments conducted in triplicate at 12−16 drug concentrations. n = 1.
c
d
desolvation energy. The 4-ethyl hydroxy analogue 9d led to a
reduction of affinity for GluK1 compared to 9a, and finally
carboxylic acid 9e led to complete depletion of affinity for any
of the iGluRs (IC50 > 100 μM, Ki > 100 μM). This might be
explained by charge−charge repulsion between the negatively
charged Glu465 residue, that is in close proximity to the acid
side chain of 9e. To summarize, for series 9, the positive effect
of favorable polar interactions of the ligand with the receptor
did not result in an increase of affinity. Furthermore, the data
does not indicate energetically favorable interactions of the
ligand with the water network in the receptor to stabilize the
receptor−ligand complex.
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Table 4. Chemical Structures and Binding Affinities of Analogues 9a−s at Native iGluRs (Rat Synaptosomes) and Homomeric
a
Recombinant Rat iGluRs
b
c
native iGluRs (synaptosomes)
KA IC50
cloned homomeric iGluRs
amino acid
AMPA IC50
NMDA Ki
GluK1 Ki
25.2 1.8
13.3 0.5
4.41 0.63
11.7 1.4
GluK2 Ki
GluK3 Ki
22.3 6.0
17.8 2.1
5.80 0.54
20.6 3.1
(51 6)
(66 7)
5.69 0.90 (40 7)
(63 6)
(55 10)
2.41 1.06 (18 4)
(86 3)
−
(83 6)
−
5.06 0.03 (37 2)
10−50 (60 4)
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
Gly
17 [4.76 0.04]
44 [4.37 0.06]
0.60 [6.23 0.05]
0.87 [6.06 0.02]
3.4 [5.47 0.04]
3.9 [5.41 0.03]
5−10
1−5
0.96 [6.02 0.01]
1−5
’100
’100
’100
’100
L-Ala
L-Val
L-Leu
L-Ile
32 [4.52 0.10]
54 [4.28 0.06]
27 [4.57 0.02]
27 [4.57 0.05]
66 [4.20 0.09]
10−50
>50
10−50
>50
33 [4.49 0.07]
10−50
10−50
10
d
d
d
(68 5)
(86 8)
(62 1)
(75 2)
(91 6)
d
d
d
L-Phe
L-Tyr
L-Ser
L-Thr
L-Pro
L-Glu
L-Gln
L-Asp
L-Asn
L-Lys
L-Cys
L-His
L-Trp
L-Arg
(103 10)
d
d
d
d
(84 9)
(89 14)
d
d
d
10−50
10−50
10−50
10−50
−
5−10
−
10−50
10−50
10−50
10
d
d
d
10−50
10−50
>100
5
(80 2)
(104 7)
d
d
1.1 [5.98 0.03]
0.83 [6.08 0.01]
1.44 0.18 (35 1)
(86 1)
d
d
d
(91 6)
(100 14)
−
−
−
−
d
d
d
>100
−
10−50
10−50
10−50
10−50
10
1−5
−
10−50
1−5
5
1−5
10−50
(86 7)
(102 5)
−
−
d
d
d
d
d
d
3.27 0.55 (50 5)
(79 4)
(88 6)
(66 1)
(90 6)
d
d
d
(80 2)
(76 1)
(72 3)
d
d
6.23 2.69 (44 3)
10−50 (62 5)
2.78 1.41 (29 9)
d
d
d
d
10
2.65 1.06 (40 1)
10−50 (66 1)
a
All values in μM. −: Analogue unstable in buffer, values not determined. Radioligands used: AMPA, [3H]AMPA; KA, [3H]KA; [3H]CGP-39653;
b
GluK1, [3H]SYM2081; GluK2 and GluK3, [3H]KA. Data are mean values of three to six individual experiments performed in triplicate. For
AMPA and KA: pIC50 values with SEM in brackets. For NMDA: pKi values with SEM in brackets. Mean values SEM of at least three
experiments conducted in triplicate at 12−16 drug concentrations. Percent specific binding mean values SD of three experiments performed at
10 μM ligand concentration.
c
d
Table 5. Chemical Structures and Binding Affinities of Analogues 9a−e at Native iGluRs (Rat Synaptosomes) and Homomeric
a
Recombinant Rat iGluRs
b
c
native iGluRs (synaptosomes)
KA IC50
cloned homomeric iGluRs
AMPA IC50
NMDA Ki
GluK1 Ki
0.62
∼10 (50 6)
2.98 0.96
>10 (60 6)
GluK2 Ki
GluK3 Ki
9a
9b
9c
9d
9e
48
22
>100
45 [4.72 0.03]
13 [4.89 0.3]
>100
25
82
2.2
d
d
d
d
69 [4.16 0.02]
15 [4.83 0.05]
35 [4.47 0.007]
>100
>100
>100
61 [4.22 0.03]
>100
>100 (99 6)
>100 (86 6)
>100 (93 7)
>100 (97 7)
>100 (83 6)
2.46 0.54
>10 (57 2)
d
d
d
d
d
d
>100 (93 3)
>100 (95 3)
a
All values in μM. −: Values not determined. Radioligands used: AMPA, [3H]AMPA; KA, [3H]KA; [3H]CGP-39653; GluK1, [3H]NF608; GluK2
b
and GluK3, [3H]KA Data are mean values of three to six individual experiments performed in triplicate. For AMPA and KA: pIC50 values with
SEM in brackets. For NMDA: pKi values with SEM in brackets. Mean values SEM of at least three experiments conducted in triplicate at 12−16
drug concentrations. % specific binding mean values SD of three experiments, at the indicated ligand concentration.
c
d
Pharmacological Characterization at the Four GluN1/
GluN2A−D NMDA Receptors. The SAR study had
identified a total of eight new analogues 4h, 5c, 5d, and
8a,b,g,j,k which display ∼1 μM binding affinity for native
NMDA receptors. Previously published 4′-bromo analogue 4g
falls within this range and was therefore also included here.
Furthermore, compound 7 was also of interest as this analogue
represents a new lead for NMDA receptor ligands. To
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a
Table 6. Chemical Structures and Functional Characterization at Rat GluN1/GluN2A−D NMDA Receptors
R
native NMDA (Ki)
GluN1/2A (IC50)
GluN1/2B (IC50)
GluN1/2C (IC50)
GluN1/2D (IC50)
4a
4e39
4g
4h
5c
5d
6b
7
8a
8b
8g
8j
R1 = H
R1 = Cl
R1 = Br
R1 = COOH
R2 = COOH
R2 = triazole
−
6.0
11.8 0.23
4.7
34.5 0.97
10
91.2 2.60
24
>100
41
0.63
0.6239
1.3
0.48
0.83
0.16
8.5
0.60
0.87
0.96
1.1
1.83 0.11
0.75 0.03
0.63 0.01
1.36 0.06
0.20 0.02
45 1.5
1.32 0.05
1.64 0.07
1.87 0.08
1.00 0.06
0.61 0.06
6.15 0.17
4.24 0.13
4.37 0.17
3.51 0.18
0.75 0.04
165 14.1
5.76 0.22
7.17 0.17
1.73 0.05
2.89 0.10
3.89 0.22
21.4 1.09
5.17 0.12
6.27 0.13
7.28 0.17
1.33 0.03
193 6.1
9.94 0.52
11.55 0.62
9.70 0.48
4.50 0.22
2.61 0.07
25.5 1.46
24.3 1.46
21.2 0.37
18.5 1.54
5.15 0.23
159 8.7
44.30 7.90
33.84 4.47
25.20 1.46
34.79 12.25
23.50 1.00
−
Gly
L-Ala
L-Tyr
L-Pro
8k
L-Glu
0.83
a
Inhibition of recombinant NMDA receptor subtypes measured using two-electrode voltage-clamp recordings. IC50 values for inhibition of current
responses activated by coapplication of 100 μM glycine and 1 μM Glu to Xenopus oocytes expressing recombinant rat GluN1/GluN2A−D NMDA
receptors were determined from five to six oocytes, and Ki values were estimated using the Cheng−Prusoff relationship54 and previously
determined Glu EC50 values.55 Ki values are mean SEM. All values in μM. −: Values not determined.
Figure 4. Concentration−inhibition data of 4h and 6b at NMDA receptor subtypes. (A) Representative two-electrode voltage-clamp recordings
showing inhibition of GluN1/2A and GluN1/2D by 4h. Receptors were activated by 1 μM glutamate in the continuous presence of 100 μM
glycine. (B) Concentration−inhibition data for 4h and 6b at different recombinant NMDA receptor subtypes (GluN1/2A−D) measured using
two-electrode voltage-clamp recordings. Data are mean SEM from five to six oocytes for each receptor subtype. The IC50 values were used to
estimate Ki values using the Cheng−Prusoff relationship54 (Table 6).
investigate the functional properties across the GluN1/
GluN2A−D NMDA receptor subtypes, they were subse-
quently characterized in a two-electrode voltage-clamp record-
ing assay (Xenopus oocytes) (Table 6, Figure 4). All analogues
were full antagonists at the four NMDA receptor subtypes with
Ki values in the high nanomolar to low micromolar range. In
general, all analogues displayed a preference for GluN1/2A
over the remaining three NMDA receptor subtypes. The
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with an XTerra MS C 18 3.5 μm, 4.6 mm × 150 mm column, using a
5 → 95% MeCN gradient in H2O containing 0.1% TFA. For HPLC
control, data collection and data handling, Chromeleon software ver.
6.80 was used. Preparative HPLC was carried out on an Agilent Prep
HPLC system with an Agilent 1100 series pump, Agilent 1200 series
diode array, multiple wavelength detector (G1365B), and Agilent
PrepHT High Performance Preparative Cartridge Column (Zorbax,
300 SB-C18 Prep HT, 21.2 × 250 mm, 7 μm). LC-MS spectra were
recorded using either an Agilent 1200 series solvent delivery system
equipped with an autoinjector coupled to an Agilent 6400 series triple
quadrupole mass spectrometer equipped with an electrospray
ionization source or a Waters Aquity UPLC-MS with dual wavelength
detection with electrospray ionization. Gradients of 5% aqueous
MeCN + 0.1% HCO2H (solvent A) and 95% aqueous MeCN +
0.05% HCO2H (solvent B) were employed. IR spectra were recorded
on a PerkinElmer 801 spectrophotometer. Optical rotation was
measured using a PerkinElmer 241 spectrometer with a Na lamp (D
line, 589 nm). Compounds were dried under high vacuum or freeze-
dried using a Holm & Halby, Heto LyoPro 6000 freeze drier. The
purity of compounds submitted for pharmacological characterization
was determined by HPLC to be >95%.
degree of selectivity for GluN1/2A was determined to be 3−6-
old over GluN1/2B and GluN1/2C, while enhanced
preference/selectivity over GluN1/2D was observed (4−33-
fold). The best selectivity profiles were demonstrated for
analogues 4h and 5c, which displayed a 32−34-fold selectivity
for GluN1/2A over GluN1/2D, but only 6−10-fold selectivity
over GluN1/2B and GluN1/2C. Regrettably, analogue 7 did
not display a novel selectivity profile, but analogue 6b is worth
paying attention to as it is the most potent analogue of the
series (Ki = 200 nM at GluN1/GluN2A). From the data, it is
also noticeable that the amino acid analogues 8a,b,g,j,k
followed the same trend as when increasing bulk size of the
lead structure 4a by addition of traditional (functional) groups
in the 5′-position.
CONCLUSION
■
In conclusion, we have reported the SAR study of 40 new 3-
aryl prolines as ligands for the iGluRs. Their synthesis was
achieved by via three different synthetic routes: The first is a
previously reported strategy based on a cuprate or ruthenium-
(I)-catalyzed 1,4 Michael addition which delivered analogues
5e−i and 7 as pure enantiomers. The second strategy was
disclosed herein and is a racemic approach to afford analogues
( )-5j−m in an expedient way starting from readily available
bromo enone 22. The third and newly developed strategy takes
advantage of C(sp3)−H activation, and it delivered
enantiopure analogues 4g−i, 5b−d, 6a,b, and 8a−s in a
concise and fully stereoselective fashion starting from (R)-
proline.
Synthesis 4′-Carboxy Analogue 7. (2S,3R)-tert-Butyl 2-(((tert-
Butyldimethylsilyl)oxy)methyl)-3-(4-(((tert-butyldimethylsilyl)oxy)-
methyl)phenyl)-5-oxopyrrolidine-1-carboxylate (11). Solution A:
Thiophen (487 mg, 5.79 mmol, 1.90 equiv) was dissolved in dry
Et2O (8 mL) in a dry vial under N2. The mixture was cooled to 0 °C,
and 2.03 M n-BuLi (2.85 mL, 5.79 mmol, 1.90 equiv) was dropwise
added over the course of 15 min. The mixture was left to stir at 0 °C
for 15 min before being removed from the ice bath and stirred at rt for
1 h (a white, colloid precipitate was formed). (3-Bromobenzyl)oxy)-
(tert-butyl)dimethylsilane (1.15 g, 3.82 mmol, 1.25 equiv) was
dissolved in dry Et2O (30 mL) in a dry flask under N2. The mixture
was cooled to −78 °C, and 1.55 M t-BuLi (4.95 mL, 7.67 mmol, 2.51
equiv) was dropwise added over the course of 20 min. The mixture
was left to stir at −78 °C for 60 min (the solution became yellow/
brownish colored and unclear) before a suspension of CuCN (342
mg, 3.82 mmol, 1.25 equiv) in dry Et2O (5 mL) was added dropwise
over the course of 5 min. The mixture was left to stir at −78 °C for 10
min, 5 min at rt, and then 10 min at −42 °C before being recooled to
−78 °C over the course of 10 min. The mixture was dropwise added
to Solution A (7.5 mL, 3.8 mmol, 1.3 equiv) over the course of 15 min
and left to stir at −78 °C for 10 min, at rt for 5 min, at −42 °C for 10
min before being recooled to −78 °C over the course of 10 min. A
solution of enone 10 (1.00 g, 3.05 mmol, 1.00 equiv) dissolved in
Et2O (4 mL) was dropwise added over the course of 20 min (the
mixture instantly turned bright yellow). The mixture was left to stir at
−78 °C for 15 min, at rt for 5 min, and at −42 °C for 60 min before
being quenched; the reaction was quenched with sat. NH4Cl (6 mL)
and stirred at −42 °C for additional 10 min. The mixture was
transferred to a separation funnel containing sat. NaHCO3 (75 mL)
and EtOAc (40 mL). The aqueous layer was extracted with EtOAc (2
× 50 mL), and the combined organic phases were washed with brine
(75 mL), dried over MgSO4, filtered, and concentrated in vacuo. The
mixture was purified using DCVC (diameter = 7, 75 mL fractions, 0−
4% EtOAc in heptanes) to yield the title compound as a colorless oil
With the work presented herein, the SAR of this class of
iGluR antagonists now shows that potent and selective NMDA
receptor subtype antagonists are obtained by introduction of
substituents in the 5′-position, and that an additional chloro or
bromo substituent in the 4′-position enhances potency. In
terms of the NMDA receptor subtype selectivity profile, the
GluN1/GluN2A subtype is generally favored, and most
interestingly the 5′-amino acid analogues (series 8) demon-
strate that large groups can be accommodated in this position,
fitting into the cavelike space in the receptor and maintaining
nanomolar binding affinity. Based on data disclosed herein, it is
reasonable to suggest that the observed selectivity for and
potency at the NMDA receptors can be further improved and/
or directed toward one of the other NMDA receptor subtypes,
by continuing the SAR studies of the 5′-position.
EXPERIMENTAL SECTION
■
Chemistry. All reactions involving dry solvents or sensitive agents
were performed under an argon atmosphere and glassware was flame-
dried under vacuum prior to use. Commercially available chemicals
were used without further purification. DCM, THF, and DMF were
dried using a SG WATER solvent purification system (commercial-
ized by Pure Process Technology). MeOH was dried by standing over
4 Å molecular sieves for a minimum of 48 h. Reactions were
monitored by analytical thin-layer chromatography (TLC, Merck
silica gel 60 F254 aluminum sheets) or by HPLC. Flash
chromatography was carried out using Merck silica gel 60A (40−63
μm). For dry column vacuum chromatography (DCVC), Merck silica
gel 60 (15−40 μm) and a standard setup were used. 1H NMR spectra
were recorded at 400 or 600 MHz and 13C NMR spectra at 100 or
150 MHz on a Bruker Avance III and Bruker Avance III HD,
respectively. Chemical shifts (δ) are reported in ppm relative to TMS.
For 13C NMR in D2O, 1% CD3OD was added as internal reference.
HPLC was performed using a Dionex UltiMate 3000 pump and
photodiode array detector (210 and 254 nm, respectively) installed
1
that solidified upon standing (1.41 g, 84%). H NMR (300 MHz,
CDCl3) δ 0.08 (3H, s), 0.10 (3H, s), 0.12 (6H, s), 0.92 (9H, s), 0.96
(9H, s), 1.54 (9H, s), 2.53 (1H, dd, J = 18, 3 Hz), 3.15 (1H, dd, J =
18, 10 Hz), 3.45 (1H, dm, J = 9 Hz), 3.80 (1H, dd, J = 11, 2 Hz), 4.00
(1H, dd, J = 11, 4 Hz), 4.06 (1H, m), 4.72 (2H, s), 7.15 (2H, d, J = 8
Hz), 7.28 (2H, d, J = 8 Hz). 13C NMR (75 MHz, CDCl3) δ −5.2,
−5.0, 18.4, 18.7, 26.1, 26.2, 28.3, 38.7, 40.2, 63.8, 64.7, 67.0, 83.1,
126.3, 126.8, 140.3, 142.9, 149.8, 174.2. LCMS m/z [M + H]+ calcd:
450.3, found: 450.3; [a]22D: −27.55 (c = 0.65 g/100 mL; EtOAc).
(2S,3R)-tert-Butyl 2-(((tert-Butyldimethylsilyl)oxy)methyl)-3-(4-
(((tert-butyldimethylsilyl)oxy)-methyl)phenyl)pyrrolidine-1-carbox-
ylate (11a). Lactam 11 (1.43 g, 2.35 mmol, 1.00 equiv) was dissolved
in dry THF (15 mL), and 1 M BH3·THF complex (30.0 mL, 30.0
mmol, 11.7 equiv) was added over the course of 5 min. The mixture
was refluxed under N2 for 18 h. The mixture was cooled to 0 °C, and
L
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Research Article
H2O (6 mL) was dropwise added over the course of 2 h, NaOH (2M,
30 mL) was dropwise added over the course of 40 min, and H2O2
(30%, 10 mL) was added over the course of 15 min (organic/aqueous
ratio important). After 5 min, the mixture was remove from the ice
bath and left to stir at rt for 1.5 h. The mixture was poured into sat.
NaHCO3 (100 mL) and EtOAc (50 mL). After separation, the
aqueous phase was reextracted with EtOAc (2 × 75 mL) and the
combined organic phases was washed with brine (100 mL), dried over
MgSO4, filtered, concentrated in vacuo, and purified using DCVC (dia
= 4 cm, 30 mL fractions, 0−4% EtOAc in PhMe) to yield the title
atmosphere. The mixture was concentrated in vacuo to yield a clear,
colorless foam. The mixture was dissolved in H2O (4 mL) and a
white, amorph solid precipitated. The solid was dried to yield 112 mg.
1H NMR (300 MHz, NaOD in D2O) δ 1.48 (1H, m), 1.80 (1H, m),
2.59 (1H, m), 2.67−2.80 (2H, m), 3.00 (1H, d, J = 9 Hz), 6.94 (2H,
d, J = 8 Hz), 7.39 (2H, d, J = 8 Hz). 13C NMR (75 MHz, NaOD in
D2O) δ 36.0, 46.9, 51.7, 70.7, 128.1, 130.0, 135.0, 147.3, 176.1, 181.7.
[a]22D: + 21.97 (c = 0.47 g/100 mL; 2 M NaOH).
Synthesis of Analogues 5e−i. 3-Bromo-5-(methoxycarbonyl)-
phenyl)boronic Acid (14). Commercially available 3-borono-5-
bromobenzoic acid 13 (500 mg, 2.04 mmol, 1 equiv) was dissolved
in anhydrous MeOH (20 mL, 0.1 M) to which H2SO4 (50 μL, 18 M)
was added slowly. Resulting mixture was refluxed 20 h, and after
cooling to room temperature it was concentrated to dryness in vacuo.
The crude product was used without further purification in the next
1
compound (809 mg, 59%) as a colorless oil. H NMR (300 MHz,
CDCl3) δ 0.06 (6H, s), 0.13 (6H, s), 0.92 (9H, s), 0.97 (9H, s),
1.23−1.38 (1H, m), 1.50 (9H, s), 1.83−1.98 (1H, m), 2.18−2.34
(1H, m), 3.25−3.45 (1H, m), 3.47−3.80 (4H, m), 3.85 (0.53H, m),
4.04 (0.41H, m), 4.73 (2H, s), 7.10−7.23 (2H, m), 7.23−7.30 (2H,
m). 13C NMR (75 MHz, CDCl3 δ −5.1, −5.0, 18.4, 18.7, 26.1, 26.2,
28.8, 31.8, 33.0, 45.4, 46.3, 46.5, 47.2, 61.4, 62.9, 64.9, 65.6, 65.8,
79.1, 79.5, 126.4, 127.1, 127.3, 139.6, 142.1, 142.7, 154.2, 154.3.
LCMS m/z [M + H]+ calcd: 436.3, found: 436.3 [a]22D: + 7.27 (c =
0.87 g/100 mL; EtOAc).
(2S,3R)-tert-Butyl 2-(Hydroxymethyl)-3-(4-(hydroxymethyl)-
phenyl)pyrrolidine-1-carboxylate (12). The protected intermediate
11a (722 mg, 1.34 mmol, 1.00 equiv) was dissolved in dry THF (10
mL), and 1 M TBAF (5 mL) was added. The mixture was left to stir
under nitrogen atmosphere for 18 h. The mixture was transferred to a
separation funnel containing EtOAc (50 mL) and sat. NaHCO3 (50
mL). The aqueous phase was reextracted with EtOAc (2 × 50 mL).
The pooled organic phases were washed with brine (75 mL), dried
over MgSO4, filtered, and concentrated in vacuo. The mixture was
purified by DCVC (dia = 3 cm, 30 mL fractions, 0−100% EtOAc in
heptanes) to yield 12 (364 mg, 88%) as a clear, colorless oil. 1H NMR
(300 MHz, CDCl3) δ 1.48 (9H, s), 1.91 (1H, m), 2.12 (1H, m), 2.91
(1H, m), 3.20−3.45 (2H, m), 3.51−3.80 (3H, m), 3.88 (1H, m), 4.57
(2H, s), 5.20 (1H, m), 7.15 (2H, d, J = 8 Hz), 7.25 (2H, d, J = 8 Hz).
13C NMR (75 MHz, CDCl3) δ 28.6, 32.9, 47.1, 47.4, 64.5, 65.5, 66.8,
1
step. H NMR (400 MHz, DMSO) δ 8.37 (dd, J = 1.5, 1.0 Hz, 1H),
8.18 (dd, J = 2.1, 1.0 Hz, 1H), 8.08 (dd, J = 2.1, 1.6 Hz, 1H), 3.87 (s,
3H). 13C NMR (101 MHz, DMSO) δ 165.2, 141.0, 133.6, 132.9,
131.2, 121.6, 52.4. (C-B(OH)2 absent).
Methyl 3-Bromo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzoate (15). Crude boronic acid 14 (9.69 g, 37.4 mmol, 1
equiv) was suspended in Et2O (80 mL, 0.46 M), and pinacol (5.31 g,
44.9 mmol, 1.2 equiv) was added. The reaction mixture was stirred at
rt until it became a clear solution (1.5h). The mixture was partitioned
between Et2O and brine. The aqueous layer was extracted with Et2O
and combined organic phase dried over MgSO4, filtered, and
concentrated in vacuo. All attempts to separate excess pinacol were
unsuccessful, and thus, the crude mixture was used in the next step.
1H NMR (400 MHz, DMSO) δ 8.19−8.17 (m, 1H), 8.17 (t, J = 1.8
Hz, 1H), 7.97 (dd, J = 2.1, 1.0 Hz, 1H), 3.88 (s, 3H), 1.31 (s, 12H).
3-Bromo-5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenyl)methanol (16). Crude methyl 3-bromo-5-(4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolan-2-yl)benzoate (15) (11.42 g, 37.4 mmol, 1
equiv) was dissolved in anhydrous THF (200 mL, 0.18 M) and
cooled to 0 °C. After 10 min, LiAlH4 (2.84 g, 74.8 mmol, 2 equiv)
was added in small portions. The reaction mixture was allowed to
slowly warm up to rt and stirred for 14 h. The reaction was quenched
by slow addition of 1 M HCl, and the aqueous later was extracted
three times with Et2O. The organic layers were combined, washed
with 1 M HCl, dried over MgSO4, filtered, and concentrated in vacuo
to yield the title compound as a white solid (8.93 g, 86% over three
steps). 1H NMR (400 MHz, CDCl3) δ 7.85 (dd, J = 2.0, 1.0 Hz, 1H),
7.70−7.67 (m, 1H), 7.64 (dd, J = 1.7, 0.8 Hz, 1H), 4.68 (s, 2H), 1.34
(s, 12H). 13C NMR (101 MHz, DMSO) δ 145.2, 134.7, 131.8, 131.1,
121.5, 84.1, 61.9, 24.6. C-B is absent.
80.6, 127.3, 127.5, 139.8, 140.0, 156.6. LCMS m/z [M + H]+ calcd:
252.1, found: 252.1 (-t-Bu); [a]22D: + 3.98 (c = 0.43 g/100 mL;
EtOAc).
(2S,3R)-3-(3-(Benzyloxy)-5-carboxyphenyl)-1-(tert-
butoxycarbonyl)pyrrolidine-2-carboxylic acid (12a). Suspension B:
NaIO4 (1.92 g, 8.98 mmol, 9.87 equiv) and RuCl3·H2O (8 mg, 0.037
mmol, 0.04 equiv) were suspended in H2O (6 mL) and stirred at rt
for 1 min prior to use.
Alcohol 12 (280 mg, 0.91 mmol, 1.00 equiv) was dissolved in
CH3CN (5 mL) and EtOAc (5 mL), cooled to 0 °C, and Suspension B
was dropwise added over the course of 15 min. The flask containing
Suspension A was washed with H2O (2 mL), which was added to the
mixture over the course of 5 min. The mixture was left to stir at 0 °C
for 1.5 h. The mixture was filtered into a separation funnel through a
plug of Celite, which was afterward washed with water (20 mL) and
EtOAc (2 × 20 mL). The pH was adjusted to 1−2 using a couple of
drops of conc. HCl and sat. NaCl (5 mL) added for separation of the
two phases. After separation, the aqueous phase was extracted with
EtOAc (2 × 20 mL). The pooled organic phases were washed with
brine (50 mL), dried over MgSO4, filtered, concentrated in vacuo, and
purified using DCVC (dia = 3 cm, 25 mL fractions, 0−50% EtOAc in
heptane containing 2% AcOH) to yield the title compound (263 mg,
86%) as a white foam. 1H NMR (300 MHz, DMSO) δ 1.35 (6H, s),
1.42 (3H, s), 2.00 (1H, m), 2.21 (1H, m), 3.33−3.50 (2H, m), 3.50−
3.62 (1H, m), 4.06 (0.66H, d, J = 8 Hz), 4.09 (0.33H, d, J = 7 Hz),
7.41 (0.66H, d, J = 8 Hz), 7.43 (1.33H, d, J = 8 Hz), 7.88 (2H, d, J =
8 Hz). 13C NMR (75 MHz, DMSO) δ 28.0, 28.2, 32.1, 32.7, 46.0,
46.1, 48.2, 49.3, 65.1, 65.4, 79.1, 127.3, 127.5, 129.4, 129.5, 145.8,
146.1, 152.7, 153.2, 166.9, 172.8, 173.3. LCMS m/z [M + H]+ calcd:
236.1, found: 236.1 (-Boc); [a]22D: + 81.90 (c = 0.33 g/100 mL;
EtOAc).
((3-Bromo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
benzyl)oxy)(tert-utyl)dimethylsilane (17). Alcohol (16) (26.2 g, 84
mmol, 1 equiv) was dissolved in anhydrous DMF (250 mL, 0.34 M).
To the clear solution was added tert-butyldimethylsilyl chloride
(TBDMSCl, 25.3 g, 168 mmol, 2 equiv), imidazole (17.1 g, 251
mmol, 3 equiv), and N,N-dimethylpyridine-4-amine (DMAP, 1.02 g,
8.3 mmol, 0.1 equiv). The reaction mixture was stirred at rt for 21 h.
Then, the reaction was quenched with 1 M HCl, and the aqueous
layer was extracted with EtOAc. The combined organic layers were
washed four times with 1 M HCl, dried over MgSO4, filtered, and
concentrated in vacuo to afford the title compound as yellowish syrup
used without further purification due to instability on silica column
1
chromatography (35.8 g, quantitative yield). H NMR (400 MHz,
CDCl3) δ 7.80 (d, J = 1.2 Hz, 1H), 7.63−7.61 (m, 1H), 7.59−7.58
(m, 1H), 4.71 (s, 2H), 1.34 (s, 12H), 0.94 (s, 9H), 0.10 (s, 6H). 13C
NMR (101 MHz, CDCl3) δ 143.3, 136.10, 132.1, 130.8, 122.7, 84.3,
64.3, 26.1, 25.8, 25.0, −5.11. C-B is absent.
(2S,3R)-tert-Butyl 3-(3-Bromo-5-(((tert-butyldimethylsilyl)oxy)-
methyl)phenyl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5-oxopyr-
rolidine-1-carboxylate (18). In an optimized procedure, enone (10)
(10.0 g, 30.5 mmol, 1 equiv), boronic ester (17) (20.9 g, 48.9 mmol,
1.6 equiv), [Rh(cod)Cl]2 (753 mg, 1.53 mmol, 0.05 equiv), and
Cs2CO3 (15.9 g, 48.9 mmol, 1.6 equiv) were charged to a dry round-
bottom flask with argon atmosphere. Contents were evacuated and
purged with argon four times followed by addition of anhydrous, Ar-
(2S,3R)-3-(4-Carboxyphenyl)pyrrolidine-2-carboxylic Acid Tri-
fluoroacetic Acid (7). The Boc protected diacid 12a (215 mg, 0.64
mmol, 1.00 equiv) was dissolved in DCM (5 mL), and TFA (4 mL)
was added. The mixture was stirred for 18 h at rt under argon
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degassed THF (292 mL, 0.1 M) and subsequently Ar-degassed H2O
(881 μL, 48.9 mmol, 1.6 equiv). The reaction mixture was stirred at rt
for 24 h, at which point it was partitioned between EtOAc and H2O
after cooling to rt. The aqueous layer was extracted with EtOAc, and
the combined organic layers were washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo. The obtained residue was
purified by flash column chromatography (heptane/EtOAc, 20:1) to
afford the title compound as colorless syrup (16.60 g, 86%). 1H NMR
(600 MHz, CDCl3) δ 7.39−7.36 (m, 1H), 7.20 (t, J = 1.7 Hz, 1H),
7.03 (t, J = 1.8 Hz, 1H), 4.67 (s, 2H), 4.04 (dt, J = 3.8, 1.9 Hz, 1H),
3.99 (dd, J = 10.5, 3.8 Hz, 1H), 3.78 (dd, J = 10.5, 2.1 Hz, 1H), 3.41
(dt, J = 9.6, 2.0 Hz, 1H), 3.12 (dd, J = 18.0, 9.7 Hz, 1H), 2.49 (dd, J =
18.0, 2.6 Hz, 1H), 1.52 (s, 9H), 0.93 (s, 9H), 0.90 (s, 9H), 0.09 (s,
6H), 0.07 (s, 3H), 0.06 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
173.6, 145.0, 146.4, 144.8, 128.4, 128.1, 123.2, 122.6, 83.4, 66.6, 64.3,
63.8, 39.9, 38.7, 28.3, 26.1, 26.0, 18.6, 18.4, −5.11, −5.12, −5.34,
−5.35.
4h and then cooled to rt. The contents were diluted with EtOAc,
dried over MgSO4, filtered, and concentrated in vacuo. The crude was
purified by flash column chromatography (EtOAc/MeOH, 24:1) to
afford the desired compound. Common for these compounds was a
strong tendency to form EtOAc containing solids.
(2S,3R)-tert-Butyl-2-(hydroxymethyl)-3-(3-(hydroxymethyl)-5-
(pyridin-2-yl)phenyl)-pyrrolidine-1-carboxylate (20a). Bromide 19
(257 mg, 665 μmol, 1 equiv), MIDA-boronate, 6-methyl-2-(pyridin-2-
yl)-1,3,6,2-dioxazaborocane-4,8-dione, (311 mg, 1.33 mmol, 2 equiv),
Pd(OAc)2 (7.5 mg, 33 μmol, 0.05 equiv), and Xantphos (39 mg, 66.5
μmol, 0.1 equiv) were charged to a round-bottom flask. The contents
were evacuated and backfilled with argon four times followed by
addition of degassed dioxane (8.3 mL, 0.08 M). Degassed K3PO4
solution (1.66 mL, 3 M, 7.5 equiv) was added, and the resulting
suspension was stirred at 50 °C for 20 h. After allowing the reaction
mixture to cool to rt, it was diluted with EtOAc and the organic layer
was washed with brine. The aqueous layer was extracted with EtOAc
three times. Subsequently, the organic layers were combined, dried
over MgSO4, filtered, and concentrated in vacuo. The resulting crude
was purified by flash column chromatography (EtOAc/MeOH, 19:1)
to afford the title compound as white foam (78 mg, 31%*, inseparable
(2S,3R)-tert-Butyl 3-(3-bromo-5-(((tert-butyldimethylsilyl)oxy)-
methyl)phenyl)-2-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidine-
1-carboxylate (18a). To a solution of pyrrolidinone (18) (236 mg,
375 μmol, 1 equiv) in anhydrous THF (3 mL, 0.12 M) was added
borane dimethylsulfide complex (107 μL, 10.5 M, equiv) at rt
followed by refluxing for 2h. The solution was diluted with Et2O and
quenched with saturated NH4Cl solution. The organic layer was
washed with H2O, brine, dried over MgSO4 and filtered through a
plug of Celite. The residue was purified using DCVC (0−17% EtOAc
in heptane) to afford the title compound as colorless syrup (143 mg,
1
from PPh3 byproduct, 13% compared to product). H NMR (400
MHz, CDCl3) δ 8.65 (ddd, J = 4.8, 1.6, 1.0 Hz, 1H), 7.79 (s, 1H),
7.77 (s, 1H), 7.75−7.61 (m, 2H), 7.29 (s, 1H), 7.25−7.20 (m, 1H),
4.71 (s, 2H), 4.01 (s, 1H), 3.86−3.69 (m, 2H), 3.63 (dd, J = 11.6, 6.5
Hz, 1H), 3.35 (ddd, J = 10.9, 9.9, 6.5 Hz, 1H), 3.06−2.93 (m, 1H),
2.22−2.11 (m, 1H), 2.02−1.94 (m, 1H), 1.49 (s, 9H).
(2S,3R)-tert-Butyl-2-(hydroxymethyl)-3-(3-(hydroxymethyl)-5-
(pyridin-3-yl)phenyl)-pyrrolidine-1-carboxylate (20b). General pro-
cedure A. White solid (184 mg, 63%). 1H NMR (600 MHz, CDCl3) δ
8.83 (s, 1H), 8.61 (d, J = 4.0 Hz, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.47
(s, 1H), 7.38 (dd, J = 7.7, 4.8 Hz, 1H), 7.36 (s, 1H), 7.32 (s, 1H),
4.78 (s, 2H), 4.01 (t, J = 8.0 Hz, 1H), 3.78 (m, 2H), 3.66 (dd, J =
11.0, 6.7 Hz, 1H), 3.39 (td, J = 10.6, 6.5 Hz, 1H), 2.99 (dd, J = 15.8,
7.2 Hz, 1H), 2.24−2.17 (m, 1H), 1.85−1.80 (m, 1H), 1.51 (s, 9H).
13C NMR (151 MHz, CDCl3) δ 148.8, 148.4, 142.6, 138.9, 138.9,
1
62%, mixture of rotamers). H NMR (400 MHz, CDCl3) δ 7.32 (s,
1H), 7.22 (s, 1H), 7.07 (s, 1H), 4.67 (s, 2H), 4.03−3.59 (m, 4H),
3.47 (dd, J = 12.1, 7.2 Hz, 1H), 3.40−3.27 (m, 1H), 2.25 (td, J = 12.6,
7.2 Hz, 1H), 1.94−1.81 (m, 1H), 1.48 (s, 9H), 0.94 (s, 9H), 0.88 (s,
9H), 0.10 (s, 6H), 0.04 (s, 3H), 0.04 (s, 3H). 13C NMR (151 MHz,
CDCl3) δ 154.3, (146.3 + 145.9), (144.1 + 144.1), (129.0 + 128.9),
127.4, (123.9 + 123.7), 122.7, (79.8 + 79.4), (65.5 + 65.4), 64.4,
(63.1 + 61.7), (47.1 + 46.6), (46.5 + 45.5), (32.8 + 31.8), 28.7, 26.1,
26.0, (18.5 + 18.3), −5.11, −5.25.
(2S,3R)-tert-Butyl 3-(3-bromo-5-(hydroxymethyl)phenyl)-2-
(hydroxymethyl)pyrrolidine-1-carboxylate (19). To a solution of
18a (13.45 g, 26.8 mmol) in THF (250 mL, 0.1 M) was added TBAF
(131 mL, 1 M, 5 equiv) and the solution was stirred at rt for 7h. The
reaction mixture was diluted with EtOAc, washed with saturated
NaHCO3 and brine and subsequently dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by flash
chromatography (heptane-EtOAc 1:2) to afford the title compound
136.5, 134.8, 126.0, 125.9, 124.8, 123.9, 123.8, 80.9, 67.4, 66.1, 65.2,
48.1, 47.3, 32.0, 28.6.
(2S,3R)-tert-Butyl-2-(hydroxymethyl)-3-(3-(hydroxymethyl)-5-
(pyridin-4-yl)phenyl)pyrroli-dine-1-carboxylate (20c). General pro-
cedure A. White solid (249 mg, 85%). 1H NMR (400 MHz, CDCl3) δ
8.49 (dd, J = 4.7, 1.4 Hz, 2H), 7.46 (s, 1H), 7.41 (dd, J = 4.6, 1.6 Hz,
2H), 7.32 (s, 1H), 7.29 (s, 1H), 4.69 (s, 2H), 3.99−3.90 (m, 1H),
3.75−3.66 (m, 2H), 3.61 (dd, J = 11.5, 5.9 Hz, 1H), 3.38−3.28 (m,
1H), 3.09−2.97 (m, 1H), 2.22−2.11 (m, 1H), 1.98−1.87 (m, 1H),
1.44 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 150.5, 149.8, 148.4,
143.3, 138.6, 126.7, 125.1, 124.2, 121.8, 121.5, 80.6, 66.9, 65.2, 64.3,
47.5, 47.1, 32.8, 28.5.
(2S,3R)-tert-Butyl-2-(hydroxymethyl)-3-(3-(hydroxymethyl)-5-
(pyrimidin-5-yl)phenyl)-pyrrolidine-1-carboxylate (20d). General
procedure B. White foam (81 mg, 81%). 1H NMR (400 MHz,
CDCl3) δ 9.15 (s, 1H), 8.86 (s, 2H), 7.43 (s, 1H), 7.34 (s, 1H), 7.30
(s, 1H), 5.05 (broad s, 1H), 4.74 (s, 2H), 3.94 (m, 1H), 3.81−3.66
(m, 2H), 3.62 (dd, J = 11.4, 6.2 Hz, 1H), 3.36 (ddd, J = 10.9, 9.7, 6.6
Hz, 1H), 3.32−3.26 (broad s, 1H), 3.02 (m, 1H), 2.18 (m, 1H),
2.00−1.94 (m, 1H), 1.47 (s, 9H). 13C NMR (101 MHz, CDCl3) δ
157.6, 156.7, 155.0, 143.5, 142.8, 135.0, 134.3, 126.6, 125.5, 124.3,
80.8, 67.1, 65.5, 64.5, 47.6, 47.2, 33.0, 28.6.
(2S,3R)-tert-Butyl-2-(hydroxymethyl)-3-(3-(hydroxymethyl)-5-(2-
methoxypyrimidin-5-yl)-phenyl)-pyrrolidine-1-carboxylate (20e).
General procedure B. White foam (90 mg 84%). 1H NMR (600
MHz, CDCl3) δ 8.70 (s, 2H), 7.41 (s, 1H), 7.31 (s, 1H), 7.28 (s, 1H),
4.95 (broad s, 1H), 4.78 (d, J = 5.4 Hz, 2H), 4.07 (s, 3H), 4.02−3.97
(m, 1H), 3.88−3.73 (m, 2H), 3.66 (dd, J = 10.5, 7.7 Hz, 1H), 3.39
(td, J = 10.6, 6.4 Hz, 1H), 2.99 (dd, J = 15.6, 8.1 Hz, 1H), 2.24−2.18
(m, 1H), 2.07−1.98 (m, 1H), 1.79 (t, J = 5.8 Hz, 1H), 1.51 (s, 9H).
13C NMR (151 MHz, CDCl3) δ 165.4, 157.6, 142.9, 135.6, 132.3,
1
as a colorless oil (7.04 g, 68%). H NMR (600 MHz, CDCl3) δ 7.35
(s, 1H), 7.26 (s, 1H), 7.13 (s, 1H), 4.58 (s, 2H), 3.91−3.83 (m, 1H),
3.74−3.68 (m, 1H), 3.64 (d, J = 8.6 Hz, 1H), 3.57 (dd, J = 11.5, 6.4
Hz, 1H), 3.32 (dd, J = 16.1, 9.6 Hz, 1H), 2.93−2.82 (m, 1H), 2.17−
2.08 (m, 1H), 1.96−1.85 (m, 1H), 1.47 (s, 9H). 13C NMR (151
MHz, CDCl3) δ 156.7, 144.0, 143.4, 129.6, 128.6, 124.6, 122.9, 80.8,
66.8, 65.5, 64.1, 47.4, 47.1, 32.8, 28.6.
Suzuki Cross Coupling: General Procedure A. Bromine 19 (294
mg, 761 μmoL, 1 equiv) and boronic acid (3.0 mmol, 4 equiv) were
charged to a two-necked flask and contents were purged with N2.
Pd(PPh3)4 (44 mg, 38 μmol), degassed PhMe (3.8 mL), degassed
EtOH (1.7 mL), and Na2CO3 (1.7 mL, 2 M) were added under a
stream of N2. The reaction mixture was stirred at 90 °C for 5.5 h and
then cooled to rt. The contents were transferred to brine and
extracted four times with EtOAc. The organic layers were
concentrated in vacuo and purified by flash column chromatography
(EtOAc/MeOH, 19:1) to give the desired product. Note: Common
for these compounds was a strong tendency to form EtOAc
containing solids regardless of drying duration.
Suzuki Cross Coupling: General Procedure B. Bromine 19 (100
mg, 259 μmol, 1 equiv), boronic acid (311 μmol, 1.2 equiv), and
Pd(PPh3)4 (15.0 mg, 13.0 μmol (0.05 equiv) was charged to an
argon-purged vial to which was added dioxane (1.7 mL). The
contents were degassed with argon for 2 min before degassed Na2CO3
(647 μL, 1.28 mmol, 2.1 equiv) was added and the vial was sealed
with a septum-fitted cap. The reaction mixture was stirred at 95 °C for
132.2, 132.1, 128.7, 128.6, 128.1, 80.9, 67.4, 66.1, 65.2, 55.3, 48.1,
47.3, 33.2, 28.6.
N
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ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Oxidation of Alcohols: General Procedure C. NaIO4 (1.13 g, 5.28
mmol, 8.2 equiv) and RuCl3·H2O (8.0 mg, 3.9 μmol, 0.006 equiv) in
H2O (8.0 mL, 0.08 M) was added to a stirred solution of the
corresponding diol (645 μmol, 1 equiv) in MeCN/EtOAc (4.5 mL/
4.5 mL (0.07 M) at 0 °C. The mixture was stirred at 0 °C for 2 h and
then diluted with EtOAc. MgSO4 was added at 0 °C and the
suspension was filtered through a plug of silica. The filtrate was
concentrated under reduced pressure, and the residue was purified by
flash column chromatography (EtOAc/MeOH 19:1 + 1% AcOH) to
afford the desired compound unless otherwise stated.
(2S,3R)-1-(tert-Butoxycarbonyl)-3-(3-carboxy-5-(pyridin-2-yl)-
phenyl)pyrrolidine-2-carboxylic Acid. General procedure C. White
solid (54%). Rotamer 1: 1H NMR (600 MHz, MeOD) δ 8.64 (d, J =
4.7 Hz, 1H), 8.50 (d, J = 1.4 Hz, 1H), 8.15 (s, 1H), 8.04 (s, 1H),
7.93−7.89 (m, 2H), 7.39 (ddd, J = 10.6, 7.0, 3.9 Hz, 1H), 4.31 (d, J =
6.9 Hz, 1H), 3.73 (ddt, J = 12.6, 8.6, 4.3 Hz, 1H), 3.69−3.63 (m, 1H),
3.63−3.57 (m, 1H), 2.44−2.35 (m, 1H), 2.21−2.13 (m, 1H), 1.45 (s,
9H). Rotamer 2: 1H NMR (600 MHz, MeOD) δ 8.64 (d, J = 4.7 Hz,
1H), 8.50 (d, J = 1.4 Hz, 1H), 8.14 (s, 1H), 8.03 (s, 1H), 7.93−7.89
(m, 2H), 7.39 (ddd, J = 10.6, 7.0, 3.9 Hz, 1H), 4.38 (d, J = 5.9 Hz,
1H), 3.73 (ddt, J = 12.6, 8.6, 4.3 Hz, 1H), 3.69−3.63 (m, 1H), 3.63−
3.57 (m, 1H), 2.44−2.35 (m, 1H), 2.21−2.13 (m, 1H), 1.50 (s, 9H).
13C NMR (151 MHz, MeOD) δ (176.2, 175.9), (169.6, 169.5),
to the stirred solution. The solution was allowed to warm to rt and
then stirred for 2 h at ambient temperature. The volatiles were
removed under reduced pressure and the residue was dissolved in 1 M
HCl and concentrated in vacuo. This procedure was repeated followed
by lyophilization to afford the title compound as an off-white solid
(30.6 mg, 98%). 1H NMR (400 MHz, D2O) δ 8.84 (ddd, J = 6.0, 1.6,
0.7 Hz, 1H), 8.70 (td, J = 8.0, 1.6 Hz, 1H), 8.41 (t, J = 1.6 Hz, 1H),
8.35 (dd, J = 8.1, 1.0 Hz, 1H), 8.32 (t, J = 1.6 Hz, 1H), 8.17 (t, J = 1.8
Hz, 1H), 8.09 (ddd, J = 7.4, 5.9, 1.2 Hz, 1H), 4.55 (d, J = 10.0 Hz,
1H), 3.91 (td, J = 10.3, 7.4 Hz, 1H), 3.77 (ddd, J = 11.6, 8.2, 3.2 Hz,
1H), 3.61 (ddd, J = 11.8, 10.2, 6.9 Hz, 1H), 2.68 (dtd, J = 13.9, 7.0,
3.2 Hz, 1H), 2.38 (dtd, J = 13.3, 10.4, 8.2 Hz, 1H). 13C NMR (151
MHz, D2O) δ 170.8, 168.6, 150.9, 147.4, 141.5, 141.0, 132.2, 132.1,
132.0, 131.9, 128.6, 126.5, 126.1, 64.9, 47.5, 45.8, 33.0. LC-MS (m/z)
calcd for C17H17N2O4 [M + H]+, 313.2; found, 313.3
(2S,3R)-3-(3-Carboxy-5-(pyridin-3-yl)phenyl)pyrrolidine-2-car-
boxylic Acid Hydrochloride (5f). (2S,3R)-1-(tert-Butoxycarbonyl)-3-
(3-carboxy-5-(pyridin-3-yl)phenyl)pyrrolidine-2-carboxylic acid (54
mg, 131 μmol, 1 equiv) was dissolved in anhydrous DCM (2.6
mL) and cooled to 0 °C. After 5 min, TFA (2.6 mL) was added
dropwise. The solution was allowed to warm up to rt and was stirred
for 2 h at ambient temperature. The volatiles were removed under
reduced pressure, and the residue was dissolved in 1 M HCl and
concentrated in vacuo. The brown solid was purified by preparative
HPLC with subsequent triplicate evaporation from 5 mL of 1 M HCl
followed by lyophilization to afford title compound as a white solid
(18 mg, 39%). 1H NMR (600 MHz, D2O) δ 9.11 (d, J = 2.1 Hz, 1H),
8.89 (dt, J = 8.3, 1.7 Hz, 1H), 8.83 (d, J = 5.8 Hz, 1H), 8.28 (t, J = 1.6
Hz, 1H), 8.21−8.17 (m, 2H), 8.04 (t, J = 1.6 Hz, 1H), 4.56 (d, J =
10.0 Hz, 1H), 3.88 (td, J = 10.3, 7.4 Hz, 1H), 3.76 (ddd, J = 11.6, 8.3,
3.2 Hz, 1H), 3.60 (ddd, J = 11.9, 10.2, 6.9 Hz, 1H), 2.66 (dtd, J =
14.0, 7.1, 3.2 Hz, 1H), 2.42−2.32 (m, 1H). 13C NMR (151 MHz,
D2O) δ 170.7, 169.1, 145.0, 140.5, 140.0, 139.4, 139.1, 134.7, 131.8,
131.7, 129.9, 128.0, 127.6, 64.8, 47.6, 45.8, 33.0. LC-MS (m/z) calcd
for C17H17N2O4 [M + H]+, 313.2; found, 313.3.
(156.1, 155.8), 150.5, (144.0, 143.5), (141.2, 139.1), (139.0, 138.9),
133.6, (131.3, 131.2), (130.0, 129.9), (129.8, 129.2), (128.2, 128.1),
126.3, (122.6, 122.6), (81.9, 81.5), (67.6, 67.0), (51.2, 50.1), (47.4,
47.1), (33.9, 33.4), (28.8, 28.6).
(2S,3R)-1-(tert-Butoxycarbonyl)-3-(3-carboxy-5-(pyridin-3-yl)-
phenyl)pyrrolidine-2-carboxylic Acid. General procedure C. White
solid (28%). Rotamer 1: 1H NMR (600 MHz, MeOD) δ 8.84 (s, 1H),
8.55 (d, J = 4.3 Hz, 1H), 8.20−8.16 (m, 1H), 8.13 (s, 1H), 8.02 (s,
1H), 7.83 (s, 1H), 7.54 (dd, J = 7.7, 5.0 Hz, 1H), 4.28 (d, J = 7.0 Hz,
1H), 3.75−3.68 (m, 1H), 3.65 (dd, J = 15.0, 7.1 Hz, 1H), 3.59 (dd, J
= 17.5, 8.5 Hz, 1H), 2.44−2.34 (m, 1H), 2.20−2.11 (m, 1H), 1.44 (s,
1
9H). Rotamer 2: H NMR (600 MHz, MeOD) δ 8.84 (s, 1H), 8.55
(2S,3R)-3-(3-Carboxy-5-(pyridin-4-yl)phenyl)pyrrolidine-2-car-
boxylic Acid Hydrochloride (5g). (2S,3R)-1-(tert-Butoxycarbonyl)-3-
(3-carboxy-5-(pyridin-4-yl)phenyl)pyrrolidine-2-carboxylic acid) (75
mg, 182 μmol) was dissolved in anhydrous DCM (3.6 mL) and
cooled to 0 °C. After 5 min, TFA (3.6 mL) was added dropwise to the
stirring solution. The solution was allowed to warm to rt and was
stirred for 2 h at ambient temperature. The volatiles were removed
under reduced pressure, and the residue was dissolved in 1 M HCl
and concentrated three times. The brown solid was purified by
preparative HPLC with subsequent triplicate evaporation from 5 mL
of 1 M HCl followed by lyophilization to afford the title compound as
a white solid (18 mg, 28%). 1H NMR (400 MHz, D2O) δ 8.87 (d, J =
6.8 Hz, 2H), 8.46 (s, 1H), 8.39 (d, J = 6.9 Hz, 2H), 8.29 (s, 1H), 8.20
(s, 1H), 4.54 (d, J = 9.9 Hz, 1H), 3.90 (td, J = 10.3, 7.4 Hz, 1H), 3.78
(ddd, J = 11.5, 8.2, 3.1 Hz, 1H), 3.62 (ddd, J = 11.6, 10.3, 6.9 Hz,
1H), 2.68 (dtd, J = 10.2, 7.0, 3.1 Hz, 1H), 2.46−2.34 (m, 1H). 13C
NMR (151 MHz, D2O) δ 171.0, 168.8, 156.6, 141.3, 140.8, 135.6,
132.2, 131.9, 131.4, 128.5, 124.7, 65.0, 47.6, 45.8, 33.0. LC-MS (m/z)
calcd for C17H17N2O4 [M + H]+, 313.2; found, 313.3.
(2S,3R)-3-(3-Carboxy-5-(pyrimidin-5-yl)phenyl)pyrrolidine-2-car-
boxylic Acid Hydrochloride (5h). (2S,3R)-1-(tert-Butoxycarbonyl)-3-
(3-carboxy-5-(pyrimidin-5-yl)phenyl)pyrrolidine-2-carboxylic acid
was dissolved in anhydrous DCM (6 mL) and was cooled to 0 °C.
After 5 min, TFA (4 mL) was added dropwise to the stirring solution.
The solution was allowed to warm to rt and stirred for 2.5 h at
ambient temperature. The volatiles were removed under reduced
pressure, and the residue was purified by preparative HPLC with
subsequent triplicate evaporation from 5 mL of 1 M HCl followed by
lyophilization to afford title compound as a white solid (16 mg, 4%
from 20d). 1H NMR (600 MHz, D2O) δ 9.32 (s, 1H), 9.22 (d, J = 1.4
Hz, 2H), 8.00−7.98 (m, 2H), 7.89 (t, J = 1.7 Hz, 1H), 4.47 (d, J = 9.9
Hz, 1H), 3.76 (td, J = 10.3, 7.5 Hz, 1H), 3.66 (ddd, J = 11.7, 8.2, 3.2
Hz, 1H), 3.50 (ddd, J = 12.0, 10.3, 6.8 Hz, 1H), 2.54 (dtd, J = 13.9,
7.1, 3.1 Hz, 1H), 2.23 (dtd, J = 13.5, 10.4, 8.2 Hz, 1H). 13C NMR
(151 MHz, D2O) δ 170.2, 168.5, 154.9, 152.3, 140.5, 134.0, 132.3,
(d, J = 4.3 Hz, 1H), 8.20−8.16 (m, 1H), 8.15−8.13 (m, 1H), 8.01 (s,
1H), 7.81 (s, 1H), 7.54 (dd, J = 7.7, 5.0 Hz, 1H), 4.36 (d, J = 5.9 Hz,
1H), 3.75−3.68 (m, 1H), 3.65 (dd, J = 15.0, 7.1 Hz, 1H), 3.59 (dd, J
= 17.5, 8.5 Hz, 1H), 2.44−2.34 (m, 1H), 2.20−2.11 (m, 1H), 1.49 (s,
9H). 13C NMR (151 MHz, MeOD) δ (176.7, 176.3), (169.9, 169.8),
(156.1, 155.8), (149.2, 148.4), (144.6, 143.9), (139.3, 137.7), (136.8
136.7), 134.48, (131.3, 131.2), (129.9, 129.2), (129.2, 129.1), (128.1,
128.0), 126.3, 125.5, (81.8, 81.5), (67.9, 67.3), (51.1, 50.0), (47.3,
47.1), (33.8, 33.3), (28.8, 28.6).
(2S,3R)-1-(tert-Butoxycarbonyl)-3-(3-carboxy-5-(pyridin-4-yl)-
phenyl)pyrrolidine-2-carboxylic Acid. General procedure C. Yellow
syrup (35%). Rotamer 1: 1H NMR (600 MHz, MeOD) δ 8.61 (d, J =
4.4 Hz, 2H), 8.26 (d, J = 1.3 Hz, 1H), 8.06 (s, 1H), 7.92 (s, 1H), 7.76
(d, J = 5.9 Hz, 2H), 4.28 (d, J = 7.1 Hz, 1H), 3.75−3.68 (m, 1H),
3.66 (dd, J = 15.3, 7.0 Hz, 1H), 3.59 (dt, J = 10.2, 7.8 Hz, 1H), 2.43−
2.35 (m, 1H), 2.20−2.12 (m, 1H), 1.44 (s, 9H). Rotamer 2: 1H NMR
(600 MHz, MeOD) δ 8.61 (d, J = 4.4 Hz, 2H), 8.26 (d, J = 1.3 Hz,
1H), 8.05 (s, 1H), 7.90 (s, 1H), 7.76 (d, J = 5.9 Hz, 2H), 4.36 (d, J =
6.0 Hz, 1H), 3.75−3.68 (m, 1H), 3.66 (dd, J = 15.3, 7.0 Hz, 1H), 3.59
(dt, J = 10.2, 7.8 Hz, 1H), 2.43−2.35 (m, 1H), 2.20−2.12 (m, 1H),
1.49 (s, 9H). 13C NMR (151 MHz, MeOD) δ (176.5, 176.1), (169.6,
169.5), (156.1, 155.8), (150.6, 149.8), (144.6, 144.0), 139.6, 134.5,
(131.3, 131.1), (130.2, 130.1), (128.1, 128.0), 123.3, (81.9, 81.5),
(67.8, 67.2), (51.1, 50.0), (47.4, 47.1), (33.8, 33.3), (28.8, 28.6).
(2S,3R)-1-(tert-Butoxycarbonyl)-3-(3-carboxy-5-(pyrimidin-5-yl)-
phenyl)pyrrolidine-2-carboxylic Acid. General procedure C. Used in
the next step without purification.
(2S,3R)-1-(tert-Butoxycarbonyl)-3-(3-carboxy-5-(2-methoxypyri-
midin-5-yl)phenyl)pyrrolidine-2-carboxylic Acid. General procedure
C. Used in the next step without purification.
(2S,3R)-3-(3-Carboxy-5-(pyridin-2-yl)phenyl)pyrrolidine-2-car-
boxylic acid hydrochloride (5e). (2S,3R)-1-(tert-Butoxycarbonyl)-3-
(3-carboxy-5-(pyridin-2-yl)phenyl)pyrrolidine-2-carboxylic acid (37
mg, 90 μmol, 1equiv) was dissolved in anhydrous DCM (1.8 mL)
and cooled to 0 °C. After 5 min, TFA (1.8 mL) was added dropwise
O
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Research Article
131.7, 131.7, 130.1, 127.6, 64.4, 47.2, 45.8, 32.9. LC-MS (m/z) calcd
for C16H16N3O4 [M + H]+, 314.4; found, 314.3.
extracted with was EtOAc (3 × 20 mL). The combined organic
layers were dried over MgSO4, filtered, and concentrated in vacuo.
The crude residue was purified by flash column chromatography
(heptane/EtOAc 3:1) to afford the title compound as yellow solid
(312 mg, 85%). 1H NMR (600 MHz, CDCl3) δ 8.69 (dd, J = 2.1, 1.4
Hz, 1H), 8.30 (t, J = 2.0 Hz, 1H), 8.23 (t, J = 1.6 Hz, 1H), 7.41−7.32
(m, 5H), 5.19 (s, 2H), 4.09 (t, J = 9.4 Hz, 2H), 3.98 (s, 3H), 3.91−
3.63 (bs, 3H), 3.14 (t, J = 9.4 Hz, 2H). 13C NMR (151 MHz, CDCl3)
δ 164.7, 163.4, 152.2, 148.5, 135.7, 132.5, 132.0, 128.6, 128.6, 128.5,
124.5, 122.8, 68.3, 52.9, 46.4, 31.5. LC-MS (m/z) calcd for
C22H20N2O8 [M + H]+, 441.1; found, 441.1.
1 - ( t e r t - B u t y l ) - 2 - m e t h y l ( 2 S , 3 S ) - 3 - ( 3 - a m i n o - 5 -
(methoxycarbonyl)phenyl)pyrrolidine-1,2-dicarboxylate (( )-cis-
25). To a flame-dried round-bottom flask containing 1-benzyl 2-
methyl 3-(3-(methoxycarbonyl)-5-nitrophenyl)-4,5-dihydro-1H-pyr-
role-1,2-dicarboxylate (24) (0.68 g, 1.53 mmol, 1 equiv) was added
Pd/C (68 mg, 10 wt %). The flask was evaporated and refilled with
hydrogen gas three times. Then anhydrous MeOH (5.5 mL) was
added, and the reaction mixture was stirred at rt overnight. The
reaction mixture was filtrated over Celite, rinsed with MeOH (100
mL), and concentrated to dryness to afford the free diamine as a
yellow oil (411 mg, 95%, mixture of rotamers). 1H NMR (600 MHz,
CDCl3) δ 7.24−7.23 (m, 1H), 7.20 (d, J = 1.9 Hz, 1H), 6.68 (t, J =
1.9 Hz, 1H), 4.03 (d, J = 8.5 Hz, 1H), 3.88 (d, J = 2.6 Hz, 4H), 3.74
(s, 2H), 3.56 (q, J = 8.2 Hz, 1H), 3.31 (s, 3H), 3.03 (ddd, J = 10.9,
8.5, 7.4 Hz, 1H), 2.24−2.17 (m, 1H), 2.14−2.07 (m, 1H). 13C NMR
(151 MHz, CDCl3) δ 173.5, 167.3, 146.6, 142.2, 131.1, 119.6, 118.8,
114.6, 66.0, 52.2, 51.6, 48.9, 46.9, 32.6.
(2S,3R)-3-(3-Carboxy-5-(2-hydroxypyrimidin-5-yl)phenyl)-
pyrrolidine-2-carboxylic Acid Hydrochloride (5i). (2S,3R)-1-(tert-
Butoxycarbonyl)-3-(3-carboxy-5-(2-methoxypyrimidin-5-yl)phenyl)-
pyrrolidine-2-carboxylic acid) was suspended in anhydrous DCM (5
mL). To the solution was added dropwise BBr3 (5 mL) while
maintaining rt. The mixture was stirred at rt for 2 h and quenched
with H2O. The resulting aqueous layer was concentrated under
reduced pressure and purified by preparative HPLC with subsequent
triplicate evaporation from 5 mL of 1 M HCl followed by
lyophilization. The obtained product was recrystallized from H2O/
acetone to afford the title compound as a white solid (18 mg, 4% from
20e). 1H NMR (600 MHz, D2O) δ 8.91 (s, 2H), 8.18 (t, J = 1.7 Hz,
1H), 8.14 (t, J = 1.5 Hz, 1H), 7.87 (t, J = 1.6 Hz, 1H), 4.41 (d, J = 9.7
Hz, 1H), 3.82−3.77 (m, 1H), 3.74 (ddd, J = 11.6, 8.1, 3.2 Hz, 1H),
3.59 (ddd, J = 12.3, 10.3, 6.8 Hz, 1H), 2.64 (dtd, J = 13.8, 7.0, 3.1 Hz,
1H), 2.36 (dtd, J = 13.5, 10.3, 8.1 Hz, 1H). 13C NMR (151 MHz,
D2O) δ 171.6, 169.5, 156.5, 153.9, 153.2, 140.6, 132.8, 131.7, 130.5,
128.4, 126.7, 65.5, 47.9, 45.7, 33.0. LC-MS (m/z) calcd for
C16H16N3O5 [M + H]+, 330.4; found, 330.3.
Synthesis of Analogues ( )-5j−m. 3-Methoxycarbonyl-5-nitro-
phenylboronic Acid (23). 3-Borono-5-nitrobenzoic acid (1 g, 4.74
mmol, 1 equiv) was loaded in a flask and 10 mL of MeOH (0.5 M)
was added. The clear solution was cooled on ice, and SOCl2 (0.41
mL, 5.67 mmol, 1.2 equiv) was added dropwise over 10 min. After 10
min, the ice bath was removed and the reaction was allowed to stir at
room temperature overnight. The reaction became a thick white
suspension. Water (60 mL) was added, followed by 60 mL of EtOAc.
The layers were separated and the aqueous layer was extracted with
EtOAc (3 × 60 mL). The combined organic layers were dried over
Mg2SO4, filtered, and concentrated in vacuo. The crude product was
pure enough and not further purified. The title compound was
obtained as a white solid (0.95 g, 89%). 1H NMR (600 MHz, DMSO)
δ 8.84 (dd, J = 2.2, 1.1 Hz, 1H), 8.76 (dd, J = 1.4, 1.1 Hz, 1H), 8.66
(dd, J = 2.2, 1.4 Hz, 1H), 3.94 (s, 4H). 13C NMR (151 MHz, DMSO)
δ 164.8, 147.6, 140.4, 132.4, 130.5, 125.0, 52.7.
The crude diamine intermediate (0.41 g, 1.48 mmol, 1 equiv) was
dissolved in 12 mL of anhydrous DCM. To this was added Boc2O
(0.325 g, 1.48 mmol, 1 equiv), and the reaction mixture was allowed
to stir at rt overnight. The mixture was concentrated to dryness under
reduced pressure and directly subjected to purification. The crude
residue was purified by flash column chromatography on silica gel
(heptane/EtOAc 1:1) to afford the title product as a white solid (0.47
1
g, 84%). H NMR (600 MHz, CDCl3) δ 7.26−7.19 (m, 2H), 6.69
(dt, J = 3.7, 2.1 Hz, 1H), 4.51 (d, J = 8.6 Hz, 0.26H), 4.43 (d, J = 8.7
Hz, 0.42H), 4.35 (d, J = 6.1 Hz, 0.11H), 4.21 (d, J = 6.8 Hz, 0.21H),
3.91−3.76 (m, 5H), 3.73 (m, 0.5H), 3.68 (s, 1H), 3.57 (m, 1.5H),
3.42 (m, 0.73H), 3.36−3.29 (m, 2.27H), 2.58−2.43 (m, 0.73H), 2.25
(m, 0.27H), 2.08 (dq, J = 12.0, 5.9 Hz, 0.73H), 2.04−1.95 (m,
0.27H), 1.45 (m, 3H), 1.38 (m, 6H). 13C NMR (151 MHz, CDCl3) δ
173.2, 171.8, 171.7, 167.2, 167.1, 154.4, 153.7, 147.1, 146.8, 146.7,
142.1, 138.4, 138.3, 131.7, 131.3, 131.2, 119.1, 119.1, 118.9, 118.7,
118.3, 117.9, 115.0, 114.9, 114.8, 80.4, 80.2, 80.2, 65.7, 65.1, 64.2,
63.8, 52.3, 52.2, 52.1, 51.6, 51.5, 49.9, 48.7, 47.9 47.0, 46.3, 46.2, 46.1,
45.8, 33.1, 32.4, 28.5, 28.4, 27.6. LC-MS (m/z) calcd for C19H26N2O6
[M + H]+, 379.2; found, 379.5 (-Boc).
1-Benzyl-2-methyl-4,5-dihydro-1H-pyrrole-1,2-dicarboxylate
(21). Synthesis was performed according to a known literature
procedure already reported.47
1-Benzyl-2-methyl-3-bromo-4,5-dihydro-1H-pyrrole-1,2-dicar-
boxylate (22). A flame-dried round-bottom flask with addition funnel
was charged with 1-benzyl 2-methyl-4,5-dihydro-1H-pyrrole-1,2-
dicarboxylate (21) (0.5 g, 1.91 mmol, 1 equiv). To this was added
4 mL of anhydrous MeCN, followed by DABCO (0.43 g, 3.82 mmol,
2 equiv), and the reaction mixture was cooled to −15 °C. A solution
of NBS in 6 mL of anhydrous MeCN was added slowly over 30 min,
and the reaction mixture was allowed to slowly warm up to room
temperature. After 16 h, the resulting mixture was concentrated in
vacuo to a volume of 1−2 mL and directly loaded on the column. The
crude mixture was purified by flash column chromatography on silica
gel (heptane/EtOAc 4:1) to provide the title compound as a colorless
oil (0.52 g, 80%). 1H NMR (600 MHz, CDCl3) δ 7.39−7.28 (m, 5H),
5.13 (s, 2H), 4.00 (dd, J = 9.7, 8.7 Hz, 2H), 3.77−3.54 (m, 3H), 2.94
(dd, J = 9.7, 8.7 Hz, 2H).13C NMR (151 MHz, CDCl3) δ 161.8,
152.5, 135.6, 128.7, 128.6, 128.5, 100.1, 68.3, 52.6, 47.5, 36.4. LC-MS
(m/z) calcd for C14H14BrNO4 [M + H]+, 340.0 and 342.0; found,
340.0 and 342.0.
1-Benzyl-2-methyl 3-(3-(methoxycarbonyl)-5-nitrophenyl)-4,5-
dihydro-1H-pyrrole-1,2-dicarboxylate (24). To a round-bottom
flask equipped with reflux condenser was added 1-benzyl 2-methyl
3-bromo-4,5-dihydro-1H-pyrrole-1,2-dicarboxylate (22) (0.29 g, 0.84
mmol, 1 equiv), Ar−B(OH)2 (23, 0.28 g, 1.3 mmol, 1.5 equiv),
Cs2CO3 (0.39 mg, 1.17 mmol, 1.4 equiv), and PdCl2dppf·CH2Cl2 (68
mg, 0.08 mmol, 0.10 equiv). The flask was evacuated and refilled with
argon three times. Then THF (16 mL) and water (16 mL) were
added, and the reaction mixture was stirred at 85 °C until full
consumption of the starting material (∼3h). The reaction mixture was
allowed to cool to down to room temperature and the organic layer
was removed under reduced pressure. The aqueous layer was
1-(tert-Butyl)-2-methyl (2S,3S)-3-(3-(methoxycarbonyl)-5-((4-
methylphenyl)sulfonamido)-phenyl)pyrrolidine-1,2-dicarboxylate
(( )-cis-26b). A flame-dried vial was loaded with ( )-cis-25 (150 mg,
0.4 mmol, 1 equiv) and TsCl (0.91 mg, 0.48 mmol, 1.2 equiv) Then
pyridine (0.5 mL) was added and the reaction mixture was stirred at
rt overnight. The resulting mixture was diluted in EtOAc (10 mL),
washed with water (3 × 15 mL), dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (heptane/EtOAc 2:1) to provide the
title compound as a white solid (188 mg, 92%, mixture of rotamers).
1H NMR (400 MHz, DMSO) δ 10.54 (s, 1H), 7.72−7.67 (m, 3H),
7.54 (s, 1H), 7.42−7.40 (m, 2H), 7.27 (m, 1H), 4.38 (m, 1H), 3.82
(s, 3H), 3.81−3.75 (m, 1H), 3.70−3.62 (m, 1H), 3.59−3.58 (m, 1H),
3.36−2.89 (m, 1H), 3.05−2.98 (m, 2H), 2.32 (s, 3H), 2.24−2.14 (m,
1H), 2.05−2.00 (0.72H), 1.93−1.87 (m, 0.28H), 1.43−1.41 (m, 3H),
1.34−1.31 (m, 6H). LC-MS (m/z) for C26H32N2O8S [M + H]+
533.2; found, 433.1 (-Boc).
(2S,3R)-3-(3-Carboxy-5-(pyridine-2-sulfonamido)phenyl)-
pyrrolidine-2-carboxylic Acid (( )-cis-26c). A flame-dried vial was
loaded with ( )-cis-25 (150 mg, 0.4 mmol, 1 equiv) and pyridine-2-
sulfonyl chloride (0.11 g, 0.59 mmol, 1.5 equiv). To this was added
pyridine (0.48 mL), and the reaction mixture was stirred at rt
P
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ACS Chemical Neuroscience
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Research Article
overnight. The reaction mixture was diluted with EtOAc (10 mL) and
washed with water (3 × 15 mL). The combined organic layers was
dried over MgSO4, filtered, and concentrated in vacuo. The crude
material was purified by flash column chromatography on silica gel
(heptane/EtOAc 2:1) to afford the title compounds as white solid
(174 mg, 85%, mixture of rotamers). 1H NMR (400 MHz, CDCl3) δ
8.72 (d, J = 4.8 Hz, 1H), 7.95 (m, 1H), 7.86 (td, J = 7.7, 1.7 Hz, 1H),
7.69−7.64 (m, 2H), 7.49 (m, 1H), 7.32 (s, 1H), 7.16 (d, J = 9.3 Hz,
1H), 4.49 (d, J = 8.6 Hz, 0.4H), 4.43 (d, J = 8.6 Hz, 1H), 3.86 (s,
3H), 3.85−3.78 (m, 1H), 3.67−3.56 (m, 1H), 3.48−3.39 (m, 1H),
3.14 (m, 3H), 2.49 (m, 1H), 2.13−2.03 (m, 1H), 1.45 (m, 4H), 1.38
(m, 5H).
1-(tert-Butyl)-2-methyl (2S,3S)-3-(3-benzamido-5-
(methoxycarbonyl)phenyl)pyrrolidine-1,2-dicarboxylate (( )-cis-
26d). To a flame-dried vial was added ( )-cis-25 (150 mg, 0.4
mmol, 1 equiv), followed by pyridine (0.48 mL) and benzoyl chloride
(51 μL, 0.44 mmol, 1.1 equiv). The reaction mixture was stirred at
room temperature overnight and then diluted with EtOAc (40 mL).
The organic layer was washed with water (3 × 15 mL) then dried
over MgSO4, filtered ,and concentrated in vacuo. The crude residue
was purified by flash column chromatography on silica gel (heptane/
EtOAc 2:1) to provide the title compound as a white solid (175 mg,
92%, mixture of rotamers). 1H NMR (400 MHz, CDCl3) δ 8.14−8.07
(m, 1H), 7.94−7.87 (m, 3H), 7.72−7.69 (m, 1H), 7.60−7.57 (m,
1H), 7.54−7.50 (m, 2H), 4.60 (d, J = 8.7 Hz, 0.26H), 4.53 (d, J = 8.6
Hz, 0.46H), 4.41 (d, J = 7.2 Hz, 0.1H), 4.28 (d, J = 7.2 Hz, 0.16H),
3.93 (s, 3H), 3.73 (s, 1H), 3.93−3.59 (m, 2H), 3.55−3.45 (m, 1H),
3.38−3.34 (m, 2H), 2.66−2.57 (m, 0.7H), 2.38−2.33 (m, 0.3H),
2.22−2.09 (m, 1H), 1.48 (s, 3H), 1.41 (m, 6H). LC-MS (m/z) for
C26H30N2O7 [M + H]+, 483.2; found, 383.1 (-Boc).
( )-2,3-trans-(3-Amino-5-carboxyphenyl)pyrrolidine-2-carbox-
ylic Acid Dihydrochloride (5j). Aniline ( )-cis-25 (50 mg, 0.1 mmol,
1 equiv) was dissolved in THF (1 mL, 0.1 M) and cooled to −78 °C.
After 10 min, LDA (0.2 mL, 0.2 mmol, 1 M) was added dropwise,
followed by stirring at 0 °C for 10 min. Saturated NH4Cl was added
to quench the reaction. Ten mL water was added and the aqueous
layer was extracted with EtOAc (3 × 10 mL). The combined organic
layers were dried over MgSO4, filtered, and concentrated in vacuo.
The crude product was purified by flash column chromatography
(heptane/EtOAc 1:1) to afford the title compound as white foam (25
mg, 50%, d.r. 1.3:1 (trans:cis)).
The intermediate was dissolved in dioxane (1.9 mL), and to the
solution was added 4 N HCl (1.9 mL). The reaction mixture was
refluxed for 20 h and then concentrated to dryness under reduced
pressure. The crude material was purified by preparative HPLC to
afford the title compound as an off-white solid (15 mg, 35%). Rt: 4.15
min; flow: 20 mL min−1; gradient 0−25% B (45 min); λ = 210 nm.
1H NMR (400 MHz, D2O) δ 8.14 (s, 1H), 7.96 (s, 1H), 7.66 (s, 1H),
140.3, 137.9, 133.9, 130.8, 130.8, 128.4, 125.7, 125.7, 122.4, 66.0,
47.1, 34.6, 31.0, 21.6. LC-MS (m/z) for C19H20N2O6S [M + H]+,
405.10; found, 405.1.
( )-2,3-trans-3-(3-Carboxy-5-(pyridine-2-sulfonamido)phenyl)-
pyrrolidine-2-carboxylic Acid Hydrochloride (5l). To a flame-dried 2
mL vial containing ( )-cis-26c (66 mg, 0.13 mmol, 1 equiv) was
added 25 wt % NaOMe in MeOH solution (26 μL) and MeOH (100
μL), and the reaction mixture was stirred at 65 °C for 24 h. To the
reaction mixture was added acetic acid (pH 4), and the solution was
concentrated under reduced pressure. The crude material was used in
the next step without further purification. The crude residue was
dissolved in dioxane (1.4 mL) and 4 M HCl (1.4 mL). The reaction
mixture was stirred at reflux overnight, and after cooling to rt it was
concentrated to dryness in vacuo. The crude material was purified by
preparative HPLC to provide the title compound as white solid (8
mg, 16%, d.r. 38:1). Rt: 26.25 min; flow: 20 mL min−1; gradient 0−
1
20% B (45 min); λ = 210 nm. H NMR (400 MHz, MeOD) δ 8.59
(dt, J = 4.7, 1.4 Hz, 1H), 7.94−7.88 (m, 2H), 7.69 (t, J = 1.6 Hz, 1H),
7.63 (dd, J = 2.1, 1.4 Hz, 1H), 7.53 (t, J = 1.9 Hz, 1H), 7.55−7.47 (m,
2H), 4.28 (d, J = 9.4 Hz, 1H), 3.63−3.52 (m, 2H), 3.46−3.36 (m,
1H), 2.47 (dtd, J = 14.1, 7.2, 3.3 Hz, 1H), 2.20−2.07 (m, 1H). 13C
NMR (600 MHz, MeOD) δ 170.5, 168.7, 158.8, 151.3, 141.8, 140.0,
139.9, 133.8, 128.7, 125.8, 125.7, 124.0, 122.6, 66.0, 49.7, 47.1, 34.6.
LC-MS (m/z) C17H17N3O6S [M-H]−, 390.1; found, 390.1.
( )-2,3-trans-3-(3-Benzamido-5-carboxyphenyl)pyrrolidine-2-
carboxylic Acid Hydrochloride (5m). To a flame-dried 2 mL vial was
added ( )-cis-26d (50 mg, 0.10 mmol) followed by 25 wt % NaOMe
in MeOH solution (26 μL) and MeOH (100 μL). The reaction
mixture was stirred at 65 °C for 24 h and subsequently concentrated
under reduced pressure. The crude material was used in the next step
without further purification. The crude intermediate (50 mg, 0.10
mmol, 1 equiv) was added anhydrous MeOH (1.8 mL) and water
(1.28 mL). The reaction was cooled to 0 °C and LiOH·H2O (44 mg,
1 mmol, 10 equiv) was added in portions. The reaction was allowed
to stir at room temperature until all starting material was consumed.
The reaction mixture was concentrated to dryness, and the residue
was redissolved in water (15 mL). The aqueous layer was washed with
DCM and then acidified to pH 3 with 1 N HCl. The aqueous layer
was extracted with EtOAc (3 × 15 mL). The combined organic layers
were dried over MgSO4, filtered, and concentrated in vacuo. The
residue (26 mg) was used in the next step without further purification.
The crude residue was dissolved in anhydrous DCM (810 μL). To
the solution was added TFA (650 μL), and the reaction was allowed
to stir for 8 h at room temperature. The resulting mixture was
concentrated to dryness and the residue was purified by preparative
HPLC to afford the title compound as a single diastereomer as a white
solid (8 mg, 20%). Rt: 19.58 min; flow: 20 mL min−1; gradient 0−
1
70% B (50 min); λ = 210 nm. H NMR (400 MHz, MeOD) δ 8.15
4.24 (m, 1H), 3.73 (m, 2H), 3.58 (m, 1H), 2.62 (m, 1H), 2.35 (m,
1H). 13C NMR (151 MHz, D2O) δ 172.0, 168.7, 142.0, 132.8, 131.3,
128.7, 127.3, 123.2, 65.9, 47.8, 45.6, 32.9. LC-MS (m/z) for
C12H14N2O4 [M + H]+, 251.1; found, 251.0
(dt, J = 5.2, 2.0 Hz, 2H), 7.93−7.89 (m, 2H), 7.81 (s, 1H), 7.58−7.52
(m, 1H), 7.51−7.45 (m, 2H), 4.41 (d, J = 9.3 Hz, 1H), 3.76−3.66 (m,
1H), 3.64−3.55 (m, 1H), 3.51−3.40 (m, 1H), 2.61−2.51 (m, 1H),
2.32−2.19 (m, 1H). 13C NMR (151 MHz, MeOD) δ 170.7, 169.3,
169.2, 141.5, 141.1, 136.1, 133.6, 133.4, 129.9, 129.9, 128.9, 128.9,
125.8, 125.7, 122.9, 66.2, 49.8, 47.2, 34.7. LC-MS (m/z) for
C19H18N2O5 [M + H]+, 355.1; found 355.0.
Synthesis: Key Intermediate 28a. Methyl 3-Bromo-5-iodoben-
zoate (31). 3-Bromo-5-iodobenzoic acid (3 g, 9.17 mmol, 1 equiv)
was dissolved in anhydrous MeOH (30 mL, 0.3 M) and cooled on an
ice bath. After 10 min, SOCl2 (0.8 mL, 11.0 mmol, 1.2 equiv) was
added dropwise over 10 min. After stirring the mixture for another 10
min, the ice bath was removed and the mixture was heated to reflux
for 16 h. The mixture was allowed to cool down to rt and was
subsequently concentrated to dryness. The residue was resuspended
in EtOAc and washed three times with 1 N NaOH. The organic layer
was dried over MgSO4, filtered, and concentrated to give the title
compound as a white solid (2.85 g, 91%). The product was used in
the next step without further purification. 1H NMR (600 MHz,
DMSO) δ: 8.27 (t, J = 1.7 Hz, 1H), 8.20 (t, J = 1.5 Hz, 1H), 8.04 (t, J
= 1.6 Hz, 1H), 3.86 (s, 3H). 13C NMR (151 MHz, DMSO−) δ 163.7,
143.3, 136.5, 133.1, 131.0, 122.7, 960.1, 52.8.
( )-2,3-trans-(3-Carboxy-5-((4-methylphenyl)sulfonamido)-
phenyl)pyrrolidine-2-carboxylic Acid Hydrochloride (5k). To a
flame-dried 2 mL vial containing ( )-cis-26b (50 mg, 0.1 mmol, 1
equiv) was added 25 wt % NaOMe in MeOH solution (20 μL) and
MeOH (80 μL). The reaction mixture was stirred at 65 °C for 24 h.
To the resulting mixture was added acetic acid, and the solution was
concentrated in vacuo. The obtained residue was directly used in the
next step without further purification. The crude residue was dissolved
in dioxane (1.9 mL), and to the solution was added 4 N HCl (1.9
mL). The reaction mixture was refluxed for 20 h and then
concentrated to dryness under reduced pressure. The crude material
was purified by preparative HPLC to afford the title compound (30
mg, 74%, d.r. 10:1). Rt: 18.82 min; flow: 20 mL min−1; gradient 0−
100% B (45 min); λ = 210 nm. 1H NMR (400 MHz, MeOD) δ 7.76
(s, 1H), 7.65 (m, 2H), 7.52 (t, J = 1.7 Hz, 1H), 7.51 (t, J = 1.9 Hz,
1H), 7.29 (d, J = 8.2 Hz, 2H), 4.33 (d, J = 9.6 Hz, 1H), 3.68−3.60
(m, 2H), 3.50−3.43 (m, 1H), 2.56−2.49 (m, 1H), 2.36 (s, 3H), 2.16
(m, 1H). 13C NMR (600 MHz, MeOD) δ 170.5, 168.7, 145.5, 141.9,
Q
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ACS Chemical Neuroscience
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Research Article
(R)-tert-Butyl 2-(Quinolin-8-ylcarbamoyl)pyrrolidine-1-carboxy-
late (30). (R)-1-(tert-Butoxycarbonyl)pyrrolidine-2-carboxylic acid
(1 g, 4.64 mmol, 1 equiv) was dissolved in anhydrous DCM (19
mL, 0.25 M) in a flame-dried flask with argon atmosphere. To this
was added HOBt (0.75g, 5.57 mmol, 1.2 equiv) and 8-aminoquino-
line (0.80 g, 5.57 mmol, 1.2 equiv) in one portion each. After stirring
the mixture for 5 min at rt EDC·HCl (1.07 g, 5.57 mmol, 1.2 equiv)
was added in portion. The reaction mixture became brownish and was
stirred for 24h at rt. The reaction mixture was diluted with DCM (50
mL) and saturated NaHCO3 (50 mL) was added. The aqueous layer
was extracted with DCM (3 × 50 mL). The combined organic layers
were dried over MgSO4, filtered, and concentrated in vacuo. The title
compound was purified by flash column chromatography on silica gel
(heptane/EtOAc 7:1 → 5:1) to afford the product as a light-yellow
solid (1.35 g, 85%, mixture of rotamers). 1H NMR (400 MHz,
DMSO) δ 10.42 (s, 0.45H), 10.27 (s, 0.55H), 8.64 (dd, J = 1.4 Hz,
1H), 8.42 dd, J = 8.3, 1.6 Hz, 1H), 7.70−7.67 (dd, J = 8.3, 1 Hz, 1H),
7.64 (dd, J = 8.3, 4.2 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 4.50 (dd, J =
8.5, 4.1 Hz, 1H), 3.54−3.40 (m, 2H), 2.31−2.17 (bm, 1H), 2.12−
1.99 (bm, 1H), 1.93−1.83 (m, 1H), 1.45 (s, 4 H), 1.23 (s, 5H). 13C
NMR (151 MHz, DMSO) δ 171.5, 170.9, 154.2, 153.4, 148.9, 137.9,
136.6, 134.0, 127.8, 126.9, 122.2, 121.9, 116.2, 115.9, 79.3, 78.9, 61.2,
60.9, 46.9, 46.7, 30.9, 29.7, 28.1, 27.8, 24.0, 23.4. LC-MS (m/z) calcd
for C19H23N3O3 [M + H]+, 342.2; found, 342.5.
(2S,3R)-Di-tert-butyl 3-(3-Bromo-5-(tert-butoxycarbonyl)-
phenyl)pyrrolidine-1,2-dicarboxylate (28a). In a flame-dried vial
with argon atmosphere was loaded 32 (0.5 g, 1.21 mmol, 1 equiv),
followed by anhydrous DCM (2.4 mL, 0.5 M). To the brown solution
was added TBTA (1.32 g, 6 mmol, 5 equiv) in one portion. The vial
was capped, and the reaction was stirred at rt for 24 h. During the
reaction time, a white precipitate was formed. After 24 h, the reaction
mixture was concentrated to dryness in vacuo. The title compound
was purified by flash column chromatography on silica gel (heptane/
EtOAc 7:1) to afford the title compound as a white solid (0.48 g,
1
73%, mixture of rotamers). H NMR (400 MHz, CDCl3) δ 7.99 (s,
1H), 7.80 (s, 1H), 7.54 (s, 1H), 4.18 (d, J = 6.6 Hz, 0.3H), 4.08 (d, J
= 6.6 Hz, 0.7H), 3.80−3.75 (m, 0.7H), 3.68−3.62 (m, 0.3H), 3.59−
3.53 (m, 1H), 3.40−3.35 (m, 1H), 2.33−2.24 (m, 1H), 2.07−1.98
(m, 1H), 1.59 (s, 9H), 1.49−1.43 (m, 18H); 13C NMR (151 MHz,
DMSO) δ 171.2, 164.3, 153.9, 143.6, 134.3, 131.4, 127.2, 122.7, 82.1,
81.7, 80.4, 66.2, 49.9, 48.5, 46.1, 32.1, 28.5, 28.3, 28.2, 22.8. LC-MS
(m/z) calcd for C25H36Br NO6 [M + H]+, 526.2 and 528.2; found,
314.3 and 316.3 (-Boc, −2 × tert-Bu).
Synthesis of 4a and 2,3-cis-4a. tert-Butyl (2R,3R)-3-(3-
(Methoxycarbonyl)phenyl)-2-(quinolin-8-ylcarbamoyl)pyrrolidine-
1-carboxylate (33). To a seal tube equipped with a stir bar and argon
atmosphere was loaded 30 (0.5 g, 1.46 mmol, 1 equiv), methyl-3-
iodobenzoate (0.77 g, 2.92 mmol, 2 equiv), Pd(OAc)2 (32 mg, 0.15
mmol, 0.1 equiv), Ag2CO3 (0.48 g, 1.75 mmol, 1.2 equiv), and PivOH
(45 mg, 0.44 mmol, 0.3 equiv). To this was added anhydrous toluene
(4.9 mL, 0.3 M) in one portion. The reaction mixture was placed in a
preheated oil bath at 110 °C and stirred for 3 days. The reaction
mixture became dark brown. The reaction mixture was allowed to
cool down to rt, filtered over a silica-Celite pad, and rinsed with 1:1
EtOAc/heptane. The filtrate was concentrated in vacuo. The title
compound was obtained after flash column chromatography
(heptane/EtOAc 5:1) as a white foam (0.43 g, 62% (mixture of
(2R,3R)-tert-Butyl 3-(3-Bromo-5-(methoxycarbonyl)phenyl)-2-
(quinolin-8-ylcarbamoyl)pyrro-lidine-1-carboxylate (29). To a
sealed tube equipped with a stir bar and argon atmosphere was
loaded 30 (0.6 g, 1.76 mmol, 1 equiv), 31 (1.2 g, 3.52 mmol, 2 equiv),
Pd(OAc)2 (40 mg, 0.17 mmol, 0.1 equiv), Ag2CO3 (0.49 g, 1.76
mmol, 1equiv), and PivOH (54 mg, 0.53 mmol, 0.3 equiv). To this
was added anhydrous toluene (6 mL, 0.3 M) in one portion. The
reaction mixture was placed in a preheated oil bath at 110 °C and
stirred for 3 days. The reaction mixture became dark brown. The
reaction mixture was allowed to cool down to rt, filtered over a silica-
Celite pad, and rinsed with 1:1 EtOAc-heptane. The filtrate was
concentrated in vacuo. The title compound was obtained after flash
column chromatography (heptane/EtOAc 5:1) as a light-yellow foam
1
rotamers)). H NMR (400 MHz, CDCl3) δ 9.62 (bs, 1H), 8.66 (bs,
1H), 8.45 (s, 1H), 8.08 (bs, 1H), 7.96 (t, J = 1.8 Hz, 1H), 7.60 (m,
1H), 7.49−7.34 (m, 4H), 7.14 (m, 1H), 4.81−4.58 (m, 1H), 4.13−
3.93 (m, 1H), 3.86−3.77 (m, 4H), 3.62 (m, 1H), 2.75 (m, 1H),
2.31−2.18 (m, 1H), 1.42 (m, 9H). 13C NMR (151 MHz, CDCl3) δ
168.9, 166.9, 154.3, 148.2, 138.4, 137.5, 136.2, 133.8, 132.5, 130.3,
129.6, 128.6, 128.5, 127.8, 127.3, 121.6, 116.5, 80.7, 66.7, 66.1, 52.1,
48.6, 46.3, 28.6, 28.4, 28.2. LC-MS (m/z) calcd for C27H29N3O5 [M +
H]+, 476.2; found, 476.6.
1
(0.43 g, 44% (yield range 40−48%), mixture of rotamers). H NMR
(600 MHz, DMSO) δ 9.75 (s, 0.4H), 9.73 (s, 0.6H), 8.76 (s, 1H), 8.3
(d, J = 8.25 Hz, 1H), 8.26 (d, J = 7.3 Hz, 1H), 7.85 (s, 0.6H), 7.82 (s,
0.4H), 7.79 (s, 0.6H), 7.76 (s, 0.4H), 7.60−7.57 (m, 2H), 7.51 (s,
1H), 7.48−7.45 (t, J = 7.9 Hz, 1H), 5.00 (d, J = 8.0 Hz, 0.6 Hz), 4.95
(d, J = 8.0 Hz, 0.4H), 3.93−3.86 (m, 1H), 3.81−3.78 (m, 1H), 3.70
(s, 3H), 3.49−3.39 (bm, 1H), 2.46 (m, 1H), 2.14 (m, 1H), 1.44 (s,
3H), 1.26 (s, 6H). 13C NMR (151 MHz, DMSO) δ 169.9, 165.0,
153.5, 149.0, 141.3, 138.3, 136.9, 136.2, 134.0, 131.6, 130.4, 128.2,
128.1, 127.1, 122.4, 121.9, 117.0, 79.4, 65.2, 52.8, 47.2, 46.2, 40.6,
31.7, 28.8, 28.6, 28.4, 27.7, 22.6. LC-MS (m/z) calcd for
C27H28BrN3O5 [M + H]+, 554.1 and 556.1; found, 554.6 and 556.6.
(2S,3R)-3-(3-Bromo-5-carboxyphenyl)-1-(tert-butoxycarbonyl)-
pyrrolidine-2-carboxylic Acid (32). Intermediate 29 (1 g, 1.80 mmol,
1 equiv) was dissolved in 18 mL (0.1 M) of EtOH. To the yellow
solution was added NaOH (0.72 g, 18.0 mmol, 10 equiv). The
reaction mixture was placed in a preheated oil bath at 100 °C and
stirred at reflux for 24 h. The reaction mixture became slurry and
brown. The mixture was allowed to cool down to rt and then was
concentrated to dryness in vacuo. The residue was resuspended in
EtOAc and water. The aqueous layer was extracted three times with
EtOAc to remove 8-aminoquinoline. Then the aqueous layer was
acidified with 1 N HCl to pH 2−3 and was again extracted with
EtOAc. The combined organic layers were dried over MgSO4 and
concentrated in vacuo to give the title compound as light-brown foam
(0.67 g, 90%, mixture of rotamers). The synthesis was continued
(2S,3R)-3-(3-Carboxyphenyl)pyrrolidine-2-carboxylic Acid Hy-
drochloride (4a). Intermediate 33 (25 mg, 0.05 mmol, 1 equiv)
was dissolved in EtOH (0.27 mL, 0.2 M). To this was added NaOH
(65 mg, 1.63 mmol, 30 equiv), and the vial was sealed. The reaction
mixture was placed in a preheated oil bath at 100 °C and stirred for 24
h. The reaction mixture became slurry and brown. The mixture was
allowed to cool down to room temperature and then concentrated to
dryness in vacuo. The residue was resuspended in EtOAc and 1 N
NaOH. The aqueous layer was extracted with 3 × EtOAc to remove
8-aminoquinoline. Then the aqueous layer was acidified with 1 N HCl
to pH 2−3 and extracted three times with EtOAc. The organic layers
of the second extraction were combined, dried over MgSO4, and
concentrated in vacuo to give the diacid intermediate as a light-brown
foam (10 mg, 55%). The obtained product was not purified further
and was used as the crude in the next step. The intermediate was
dissolved in DCM (1 mL), and TFA (1 mL) was added. The reaction
mixture was stirred for 5 h at rt and then concentrated to dryness. The
crude residue was purified by preparative HPLC to afford a white
solid. The solid was dissolved in 2 mL 1 N HCl and concentrated to
dryness under reduced pressure. This was repeated two more times
and after lyophilization the title compound was obtained as a white
solid (10 mg, 74%). Rt: 10.2 min (flow: 20 mL min−1; gradient 0−
1
without further purification. H NMR (600 MHz, DMSO) δ 13.38
1
(bs, 1H), 12.66 (bs, 1H), 7.93 (s, 1H), 7.86−7.83 (m, 2H), 4.10 (d, J
= 7.7 Hz, 0.36 H), 4.07 (d, J = 7.7 Hz, 0.64H), 3.60−3.54 (m, 1H),
3.53−3.48 (m, 1H), 3.42−3.35 (m, 1H), 2.26−2.19 (m, 1H), 2.07−
2.00 (m, 1H), 1.43 (s, 3 H), 1.36 (s, 6H). LC-MS (m/z) calcd for
C17H20BrNO6 [M + H]+, 414.1 and 416.1; found, 314.3 and 316.3
(-Boc).
50% B (25 min); H NMR (600 MHz, D2O) δ 8.07 (t, J = 1.7 Hz,
1H), 8.02 (dt, J = 7.9, 1.3 Hz, 1H), 7.71 (dt, J = 7.9, 1.5 Hz, 1H), 7.60
(dd, J = 8.3, 7.1 Hz, 1H), 4.27 (d, J = 9.4 Hz, 1H), 3.73−3.67 (m,
2H), 3.57 (ddd, J = 11.5, 10.2, 6.9 Hz, 1H), 2.58 (dtd, J = 13.8, 7.1,
3.4 Hz, 1H), 2.38−2.24 (m, 1H). Analytical data in agreement with
reported data.36
R
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ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
(2R,3R)-3-(3-Carboxyphenyl)pyrrolidine-2-carboxylic acid hydro-
chloride (2,3-cis-4a). Intermediate 33 (0.05 g, 0.1 mmol) was loaded
in a vial to which was added 3 mL of 40% aqueous H2SO4. The
reaction was heated to 120 °C and stirred for 36 h. After cooling
down to rt, 4 N NaOH was added to reach pH 12. The aqueous layer
was washes with DCM three times. After this, the aqueous layer was
acidified to pH 4 and concentrated to dryness under reduced pressure.
The crude residue was purified by preparative HPLC. Concentration
afforded a white solid which was dissolved in 2 mL of 1 N HCl and
concentrated to dryness under reduced pressure. This was repeated
two more times, and after lyophilization the title compound was
obtained as a white solid (7 mg, 27%). Rt 21.4 min (flow: 20 mL
resuspended in EtOAc. The organic layer was washed twice with
basic Na2S2O3 solution, followed by a wash with brine. The organic
layer was dried over MgSO4, filtered, and concentrated in vacuo. The
crude mixture was purified by flash column chromatography
(heptane/EtOAc 10:1) to give the title compound as a yellow oil
(440 mg, 46%). 1H NMR (600 MHz, CDCl3) δ 8.04 (s, 1H), 7.88 (d,
J = 8.0 Hz, 1H), 7.47 (d, J = 8.0 Hz, 2H), 3.91 (s, 3H), 3.90 (s, 3H).
13C NMR (151 MHz, CDCl3) δ 167.2, 166.6, 140.0, 137.6, 133.7,
130.9, 130.4, 52.9, 52.8. LC-MS (m/z) calcd for C10H9IO4 [M + H]+,
321.0; found, 321.3.
3-Bromo-2-chloro-5-iodobenzoic Acid. 3-Bromo-2-chlorobenzoic
acid 43 (2g, 8.55 mmol, 1 equiv) was dissolved in 7 mL TfOH and
then cooled on an ice bath. NIS (2.31 g, 10.3 mmol, 1.2 equiv) was
added in portions over 10 min and then stirred at rt overnight. To the
reaction mixture was added EtOAc, followed by water and saturated
Na2S2O3 solution. The water layer was extracted three times with
EtOAc. The combined organic layers were dried over MgSO4, filtered,
and concentrated in vacuo. The title compound was obtained as a
white solid (3.05 g, 99%). The product was used in the next step
without further purification. 1H NMR (600 MHz, DMSO) δ 8.29 (d,
J = 2.0 Hz, 1H), 8.02 (d, J = 2.0 Hz, 1H). 13C NMR (151 MHz,
DMSO) δ 165.1, 142.9, 137.2, 135.9, 130.8, 124.7, 93.3.
Methyl 3-Bromo-2-chloro-5-iodobenzoate (44). 3-Bromo-2-
chloro-5-iodobenzoic acid (2 g, 5.53 mmol, 1 equiv) was dissolved
in anhydrous MeOH (7.5 mL, 0.75 M) and cooled on an ice bath.
After 10 min, SOCl2 (0.5 mL, 6.64 mmol, 1.2 equiv) was added
dropwise over 10 min. After stirring the mixture for another 10 min,
the ice bath was removed and the mixture was heated to reflux for 16
h. The mixture was allowed to cool down to rt and subsequently
concentrated to dryness. The residue was resuspended in EtOAc and
washed three times with 1 N NaOH. The organic layer was dried over
MgSO4, filtered, and concentrated to give the title compound as an
off-white solid (1.46 g, 70%). The product was used in the next step
without further purification. 1H NMR (600 MHz, DMSO) δ 8.35 (d,
J = 2.1 Hz, 1H), 8.07 (d, J = 2.0 Hz, 1H), 3.86 (s, 3H). 13C NMR
(151 MHz, DMSO) δ 163.9, 143.6, 137.7, 134.2, 131.1, 124.8, 93.4,
53.1.
1
min−1; gradient 0−50% B (45 min). H NMR (400 MHz, D2O) δ
8.00 (dt, J = 7.6, 1.5 Hz, 1H), 7.96 (t, J = 1.8 Hz, 1H), 7.61 (dt, J =
7.8, 1.5 Hz, 1H), 7.58−7.52 (m, 1H), 4.53 (d, J = 9.4 Hz, 1H), 4.07
(td, J = 9.4, 7.3 Hz, 1H), 3.86 (ddd, J = 11.9, 7.9, 4.0 Hz, 1H), 3.52
(ddd, J = 11.9, 9.4, 7.6 Hz, 1H), 2.62−2.53 (m, 1H), 2.44 (dtd, J =
13.4, 9.4, 8.0 Hz, 1H). 13C NMR (151 MHz, D2O) δ 171.0, 170.3,
137.9, 133.3, 123.0, 129.1, 129.0, 129.0, 64.8, 45.6, 45.3, 29.9.
Synthesis of Analogues 4g−i, 5b−d, and 6a,b. Methyl 2-Bromo-
5-iodobenzoate (36). 2-Bromo-5-iodobenzoic acid (2.5 g, 7.65
mmol, 1 equiv) was dissolved in anhydrous MeOH (26 mL, 0.3
M) and cooled on an ice bath. After 10 min, SOCl2 (0.7 mL, 9.2
mmol, 1.2 equiv) was added dropwise over 10 min. After stirring the
mixture for another 10 min, the ice bath was removed and the mixture
was heated to reflux for 16 h. The mixture was allowed to cool down
to room temperature and subsequently concentrated to dryness. The
residue was resuspended in EtOAc and washed three times with 1 N
NaOH. The organic layer was dried over MgSO4, filtered, and
concentrated to give the title compound as a white solid (1.69 g,
66%). The product was used in the next step without further
purification. 1H NMR (600 MHz, DMSO) δ 8.06 (d, J = 2.2 Hz, 1H),
7.81 (dd, J = 8.4, 2.2 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 3.85 (s, 3H).
13C NMR (151 MHz, DMSO δ 164.8, 141.5, 138.8, 135.6, 134.2,
119.9, 93.3, 52.8.
Dimethyl 4-Nitrophthalate. 4-Nitrophthalate 37 (1 g, 4.73 mmol,
1 equiv) was dissolved in anhydrous MeOH (6.3 mL, 0.75 M) and
cooled on an ice bath. After 10 min, SOCl2 (0.8 mL, 10.6 mmol, 2.2
equiv) was added dropwise over 10 min. After stirring the mixture for
another 10 min the ice bath was removed and the mixture was heated
to reflux for 16 h. The mixture was allowed to cool down to rt and
subsequently concentrated to dryness. The residue was resuspended
in EtOAc and washed three times with 1 N NaOH. The organic layer
was dried over MgSO4, filtered, and concentrated to give the title
compound as a yellow solid (0.78 g, 69%). The product was used in
the next step without further purification. 1H NMR (600 MHz,
CDCl3) δ 8.63 (d, J = 2.3 Hz, 1H), 8.39 (dd, J = 8.4, 2.3 Hz, 1H),
7.85 (d, J = 8.3 Hz, 1H), 3.97 (s, 3H), 3.96 (s, 3H). 13C NMR (151
MHz, CDCl3) δ 166.8, 165.5, 148.9, 138.2, 132.8, 130.1, 126.2, 124.6,
53.4.
C−H Activation-Arylation: General Procedure D. To a seal tube
equipped with a stir bar and argon atmosphere was added 30 (1
equiv), ArI (2 equiv), Pd(OAc)2 (0.1 equiv), Ag2CO3 (1 equiv), and
PivOH (0.3 equiv). To this was added anhydrous toluene (0.3M) in
one portion. The reaction mixture was placed in a preheated oil bath
at 110 °C and stirred for 3 days. The reaction mixture became dark
brown. The reaction mixture was allowed to cool down to room
temperature and was filtered over a silica-Celite pad and rinsed with
EtOAc. The filtrate was concentrated in vacuo. The crude material was
purified as stated below.
tert-Butyl (2R,3R)-3-(4-Bromo-3-(methoxycarbonyl)phenyl)-2-
(quinolin-8-ylcarbamoyl)-pyrrolidine-1-carboxylate (34a). General
procedure D. The reaction was performed with 0.6 g (1.76 mmol) of
30 and aryl iodide 36. The title compound was obtained after flash
column chromatography (heptane/EtOAc 5:1) as a light-yellow foam
Dimethyl 4-Aminophthalate. Dimethyl 4-nitrophthalate (0.44 g,
1.85 mmol, 1 equiv) was dissolved in anhydrous MeOH (6 mL, 0.3
M). To this was added 10 wt % Pd/C (0.044 g), followed by a
hydrogen gas balloon. The reaction was stirred vigorously at rt
overnight. The mixture was filtered over a Celite-silica plug and rinsed
with EtOAc. The filtrate was concentrated in vacuo to yield the title
compound as a sticky, colorless oil which crystallized upon standing
1
(0.6 g, 61%). H NMR (400 MHz, DMSO) δ 9.80- 9.74 (m, 1H),
8.79 (s, 1H), 8.35 (d, J = 7.2 Hz, 1H), 8.26 (dd, J = 7.7 Hz, 1.3 Hz,
1H), 7.72−7.68 (m, 1H), 7.62−7.57 (m, 2H), 7.50−7.50 (m, 2H),
7.39−7.36 (m, 1H), 4.94−4.89 (m, 1H), 3.87−3.77 (m, 2H), 3.69 (s,
3H), 3.53−3.38 (m, 1H), 2.47−2.41 (m, 1H), 2.18−2.10 (m, 1H),
1.43 (s, 3H), 1.24 (s, 6H). 13C NMR (151 MHz, DMSO) δ 174.6,
174.1, 171.0, 158.7, 158.4, 153.9, 143.1, 143.0, 142.7, 142.6, 141.6,
138.7, 138.7, 138.1, 137.9, 136.8, 136.7, 136.0, 135.9, 132.8, 131.9,
127.2, 123.8, 121.8, 121.6, 84.4, 84.2, 69.8, 69.6, 57.5, 51.78 51.3,
51.0, 51.0, 33.6, 33.3, 33.1, 32.6. LC-MS (m/z) calcd for C27H28N3O5
[M + H]+, 554.1 and 556.01; found, 554.6 and 556.1.
1
(0.37 g, 95%). H NMR (600 MHz, CDCl3) δ: 7.72 (d, J = 8.4 Hz,
1H), 6.73 (d, J = 2.4 Hz, 1H), 6.68 (dd, J = 8.4, 2.4 Hz, 1H), 4.10 (bs,
2H), 3.90 (s, 3H), 3.84 (s, 3H). 13C NMR (151 MHz, CDCl3) δ:
169.8, 166.7, 149.9, 136.63, 132.1, 118.6, 115.2, 113.5, 52.8, 52.2. LC-
MS (m/z) calcd for C10H11NO4 [M + H]+, 210.1; found, 210.5.
Dimethyl 4-Iodophthalate (38). Dimethyl 4-aminophthalate (0.63
g, 3 mmol, 1 equiv) was dissolved in 1 mL of anhydrous acetonitrile
and 19 mL of anhydrous toluene. To the solution was added iodine
(0.34 g, 3.28 mmol, 1.1 equiv) and the dark red solution was cooled
to 0 °C. After 15 min, tBuNO2 (0.53 mL, 1.79 mmol, 1.1 equiv) was
added dropwise over 15 min. The reaction mixture was stirred at 0 °C
for 2 h and then left stirring while slowly reaching rt overnight. After
24 h, the reaction mixture was concentrated to dryness and
Dimethyl 4-((2R,3R)-1-(tert-Butoxycarbonyl)-2-(quinolin-8-
ylcarbamoyl)pyrrolidin-3-yl)-phthalate (34c). General procedure
D. The reaction was performed with 0.235 g (0.69 mmol) of 30
and aryl iodide 38. The title compound was obtained after flash
column chromatography (heptane/EtOAc 1:1) as a light-yellow foam
1
(0.13 g, 37%). H NMR (600 MHz, CDCl3) δ 9.86−9.80 (m, 1H),
8.78 (m, 1H), 8.33 (d, J = 8.1 Hz, 1H), 8.26 (d, J = 7.5 Hz),7.69−
S
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Research Article
7.64 (m, 1H), 7.61−7.57 (m, 3H), 7.46 (m 2H), 4.99−4.93 (m, 1H)
3.94−3.88 (m, 1H), 3.82−3.79 (m, 1H), 3.66 (s, 3H), 3.65 (s, 3H),
3.54−3.41 (m, 1H), 2.23−2.14 (m, 1H), 1.43 (s, 3H), 1.24 (s, 6H).
13C NMR (151 MHz, CDCl3) δ 169.2, 167.2, 166.8, 153.1, 148.7,
141.6, 137.9, 136.3, 133.5, 131.4, 131.16, 130.9, 129.6, 128.6, 128.65,
127.6, 126.6, 121.9, 116.5, 79.2, 79.0, 64.6, 52.3, 46.93, 45.8, 28.4,
28.1, 27.9, 27.3. LC-MS (m/z) calcd for C29H31lN3O7 [M + H]+,
534.2; found, 534.7.
(2S,3R)-3-(3-Bromophenyl)-1-(tert-butoxycarbonyl)pyrrolidine-
2-carboxylic Acid. General procedure E. The reaction was performed
with 0.44 g (0.89 mmol) of tert-butyl (2R,3R)-3-(3-bromophenyl)-2-
(quinolin-8-ylcarbamoyl)pyrrolidine-1-carboxylate. Brown foam (0.24
g, 68%, mixture of rotamers). Product was obtained with 32%
impurity. 1H NMR (600 MHz, MeOD) δ 7.50−7.48 (m, 1H), 7.45−
7.44 (m, 1H), 7.30−7.26 (m, 2H), 4.27−4.18 (m, 1H (1.49H)),
3.73−3.65 (m, 1H (1.43H)), 3.59−3.39 (m, 2H (4.12H)), 2.41−2.29
(m, 1.45H), 2.14−1.88 (m, 1H (3.24H)), 1.51−1.44 (m, 9H (13
H)). LC-MS (m/z) calcd for C16H20BrNO4 [M − H]−, 370.1 and
372.1; found, 270.3 and 272.3 (-Boc).
(2S,3R)-3-(3-Bromo-5-carboxy-4-chlorophenyl)-1-(tert-
butoxycarbonyl)pyrrolidine-2-carbo-xylic Acid. General procedure
E. The reaction was performed with 0.3 g (0.51 mmol) of 45. Brown
foam (0.2 g, 88%, mixture of rotamers). 1H NMR (600 MHz, MeOD)
δ 7.95 (d, J = 2.3 Hz, 0.7 Hz),7.93 (d, J = 2.2 Hz, 0.3 H), 7.8 (d, J =
2.3 Hz, 0.7H), 7.78 (d, J = 2.2 Hz, 0.3H), 4.38 (d, J = 6.1 Hz, 0.H),
4.30 (d, J = 7.3 Hz, 0.7H), 3.83−3.77 (m, 1H), 3.67 (m, 2H), 2.48−
2.42 (m, 1H), 2.23−2.17 (m, 1H), 1.61 (s, 3H), 1.56 (s, 6H). LC-MS
(m/z) calcd for C17H19BrClNO6 [M − H]−, 446.0 and 448.0; found,
348.4 and 350.2 (-Boc).
Di-tert-butyl (2S,3R)-3-(3-Bromo-5-(tert-butoxycarbonyl)-4-
chlorophenyl)pyrrolidine-1,2-dicarboxylate (46a). To a flame-
dried vial with argon atmosphere was added 0.2 g of (2S,3R)-3-(3-
Bromo-5-carboxy-4-chlorophenyl)-1-(tert-butoxycarbonyl)-
pyrrolidine-2-carbo-xylic acid followed by anhydrous DCM (0.3−0.5
M). To the brown solution was added TBTA (5 equiv) in one
portion. The vial was capped and the reaction was stirred at rt for 24h.
During the reaction time a white precipitate was formed. After 24 h,
the reaction mixture was concentrated to dryness in vacuo. The title
compound was purified by column chromatography on silica gel
(heptane/EtOAc 5:1). The product was obtained as white foam (0.13
g, 52%, mixture of rotamers). 1H NMR (400 MHz, CDCl3) δ 7.60 (d,
J = 2.2 Hz, 1H), 7.43 (d, J = 2.2 Hz, 1H), 4.18 (d, J = 6.2 Hz, 0.3H),
4.05 (d. J = 6.4 Hz, 0.7H), 3.78−3.74 (m, 0.7H), 3.65−3.61 (m,
0.3H), 3.58−3.51 (m, 1H), 3.36−3.32 (m, 1H), 2.33−2.26 (m, 1H),
2.00−1.96 (m, 1H), 1.60 (s, 9H), 1.56−1.44 (m, 18H). 13C NMR
(151 MHz, DMSO) δ 171.1, 164.9, 153.8, 141.4, 135.29, 134.3,
134.3, 131.3, 128.1, 127.9, 124.9, 83.6, 83.5, 81.9, 80.6, 80.3, 66.1,
65.7, 49.2, 47.8, 46.1, 46.0, 32.7, 31.9, 28.6, 28.5, 28.3, 28.2, 28.1. LC-
MS (m/z) calcd for C25H35BrClNO6 [M + H]+, 560.1 and 562.1;
found, 348.3 and 350.2 (-Boc, −2 × tert-butyl).
(2S,3R)-3-(4-Amino-3-carboxyphenyl)-1-(tert-butoxycarbonyl)-
pyrrolidine-2-carboxylic Acid (35b). 35a (0.1 g, 0.24 mmol, 1 equiv),
NaN3 (78 mg, 1.2 mmol, 5 equiv) Cs2CO3 (0.39 g, 1.2 mmol, 5
equiv) and CuI (9.2 mg, 0.05 mmol, 0.2 equiv) were loaded in a
flame-dried vial with argon atmosphere. To this was added degassed
and anhydrous EtOH (1.2 mL, 0.2 M). The vial was capped, and the
reaction was stirred at 95 °C for 3 days. The reaction was allowed to
cool down to rt and concentrated to dryness. Water was added and
the aqueous layer was carefully acidified to pH 2−3. Subsequently, the
aqueous layer was extracted with EtOAc three times and the
combined organic layers were dried over MgSO4, filtered, and
concentrated in vacuo. The crude reaction mixture was not further
purified and was used as such in the deprotection step. The reaction
yielded a 0.7:1 mixture of 35a to 35b (based on crude NMR). LC-MS
(m/z) calcd for C17H22N2O6 [M + H]+, 350.15; found, 351.1 and
251.1 (-Boc).
tert-Butyl (2R,3R)-3-(3-Bromophenyl)-2-(quinolin-8-
ylcarbamoyl)pyrrolidine-1-carboxylate. General procedure D. The
reaction was performed with 0.6 g (1.76 mmol) of 30 and 3-bromo-
iodobenzene. The title compound was obtained after flash column
chromatography (heptane/EtOAc 5:1) as inseparable mixture of the
title compound and 30 (0.44 g, 28% of 30). The mixture was not
1
further purified and used as such in the next step. H NMR (600
MHz, DMSO) δ 9.76−9.73 (m, 1H), 8.84 (s, 1H), 8.34 (d, J = 8.2
Hz, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.59 (m, 3H), 7.48 (t, J = 8.0 Hz,
1H), 7.29 (d, J = 7.8 Hz, 1H), 7.12 (d, 7.8 Hz, 1H), 7.02 (t, J = 7.8
Hz, 1H), 4.93−4.88 (m, 1H), 3.51−3.37 (m, 2H), 2.15−2.08 (m,
1H), 1.90−1.85 (m, 1H), 1.43 (3H), 1.23 (s, 6H). LC-MS (m/z)
calcd for C25H26BrN3O3 [M + H]+, 496.1 and 498.1; found, 496.6 and
498.6 (as well as 30 (342.6)).
tert-Butyl (2R,3R)-3-(3-Bromo-4-chloro-5-(methoxycarbonyl)-
phenyl)-2-(quinolin-8-ylcarba-moyl)-pyrrolidine-1-carboxylate
(45a). General procedure D. The reaction was performed with 0.6 g
(1.76 mmol) of 30 and aryl iodide 44. The title compound was
obtained after flash column chromatography (heptane/EtOAc,
1
gradient 3:1 → 2:1) as a light-yellow foam (0.32 g, 31%). H NMR
(600 MHz, CDCl3) δ 9.53 (s, 1H), 8.69 (s, 1H), 8.48 (d, J = 6.9 Hz,
1H), 8.12 (s, 1H), 7.65 (s, 1H), 7.57 (s, 1H), 7.49−7.42 (m, 3H),
4.73−4.59 (m, 1H), 4.09−3.93 (m, 1H), 3.72 (m, 4H), 3.59 (bs, 1H),
2.65−2.55 (m, 1H), 2.23−2.19 (m, 1H), 1.51 (s, 3H), 1.37 (s, 6H).
13C NMR (151 MHz, CDCl3) δ 168.6, 165.4, 154.1, 148.4, 138.4,
137.3, 136.3, 136.1, 133.4, 132.4, 132.3, 129.6, 127.9, 127.2, 125.1,
122.1, 121.8, 116.9, 80.9, 66.5, 52.7, 47.9, 46.2, 28.6, 28.4. LC-MS
(m/z) calcd for C27H27BrClN3O5 [M + H]+, 588.1 and 590.1; found,
588.6 and 590.6.
Epimerization and Cleavage of the Directing Group: General
Procedure E. The product of the C−H activation (1 equiv) was
dissolved in EtOH (0.1 M). To this was added NaOH (10 equiv), and
the vial was sealed. The reaction mixture was placed in a preheated oil
bath at 100 °C and stirred for 24 h. The reaction mixture became
slurry and brown. The mixture was allowed to cool down to rt and
then concentrated to dryness in vacuo. The residue was resuspended
in EtOAc and 1 N NaOH. The aqueous layer was extracted 3× with
EtOAc to remove 8-aminoquinoline. Then the aqueous layer was
acidified with 1 N HCl to pH 2−3 and extracted three times with
EtOAc. The organic layers of the second extraction were combined,
dried over MgSO4, and concentrated in vacuo to give the title
compound as a light-brown foam. The obtained product was not
further purified.
(2S,3R)-3-(4-Bromo-3-carboxyphenyl)-1-(tert-butoxycarbonyl)-
pyrrolidine-2-carboxylic Acid (35a). General procedure E. The
reaction was performed with 0.1 g (0.18 mmol) of 34a. Brown foam
1
(64 mg, 86%, mixture of rotamers). H NMR (600 MHz, MeOD) δ
7.73 (m, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.38−7.34 (m, 1H), 4.27 (d, J
= 6.1 Hz, 0.35 Hz), 4.25 (d, J = 7.0 Hz, 0.65 H), 3.71−3.64 (m, 1H),
3.58−3.48 (m, 2H), 2.37−2.29 (m, 1H), 2.12−2.02 (m,1H), 1.49 (s,
3H), 1.44 (s, 9H). LC-MS (m/z) calcd for C17H20BrNO6 [M − H]−,
414.1 and 416.1; found, 314.3 and 316.3 (-Boc).
3-((2S,3R)-1,2-Bis(tert-butoxycarbonyl)pyrrolidin-3-yl)-5-(tert-
butoxycarbonyl)benzoic Acid (28b). HCOOH (0.2 mL, 5.32 mmol,
2 equiv) and Ac2O (0.5 mL, 5.32 mmol, 2 equiv) were mixed and
stirred in a vial for 1.5 h at rt. (2S,3R)-di-tert-Butyl 3-(3-bromo-5-
(tert-butoxycarbonyl)phenyl)-pyrrolidine-1,2-dicarboxylate (1.4 g,
2.66 mmol, 1 equiv), Pd(OAc)2 (30 mg, 0.13 mmol, 0.05 equiv),
and Xantphos (77 mg, 0.13 mmol, 0.05 equiv) were loaded into a vial
equipped with a septa containing screw-cap. The vial was evaporated
and backfilled with argon three times. Then degassed, anhydrous
toluene (5.31 mL, 0.5 M) was added, followed by anhydrous tert-
BuOH (0.5 mL, 5.32 mmol, 2 equiv). To the orange reaction mixture
was added the mixture of HCOOH and Ac2O in a dropwise manner.
4-((2S,3R)-1-(tert-Butoxycarbonyl)-2-carboxypyrrolidin-3-yl)-
phthalic Acid (35c). General procedure E. The reaction was
performed with 0.13 g (0.25 mmol) of 34b. Brown foam (50 mg,
1
53%, mixture of rotamers). H NMR (600 MHz, MeOD) δ 7.77−
7.76 (m, 1H), 7.68−7.66 (m, 1H), 7.56−7.53 (m, 1H), 4.31 (d, J =
6.0 Hz, 0.3H), 4.24 (d, J = 7.0 Hz, 0.7H), 3.72−3.67 (m, 1H), 3.61−
3.54 (m, 1H), 2.40−2.33 (m, 1H), 2.14−2.08 (m, 1H), 1.49 (s, 3H),
1.44 (s, 6H). LC-MS (m/z) calcd for C18H21NO8 [M − H]−, 380.1;
found, 280.3 (-Boc).
T
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ACS Chemical Neuroscience
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Research Article
During the addition, the color changed to dark-green. Then Et3N (1.8
mL, 13.3 mmol, 5 equiv) was added dropwise. Instantly, gas evolution
was observed with a color change of the reaction mixture to dark red.
(Caution: Overpressure!) After the addition was completed, the
reaction was placed in a preheated oil bath at 80 °C and the reaction
was stirred overnight at 80 °C. The reaction mixture became black
and was allowed to cool down to room temperature. The pressure was
released carefully, followed by the addition of water and EtOAc. The
aqueous layer was carefully acidified to pH 2 with 1 N HCl. The
aqueous layer was extracted three times with EtOAc. The combined
organic layers were dried over MgSO4, filtered and concentrated in
vacuo. The crude product was purified by flash column chromatog-
raphy (2:1 heptane/EtOAc + 1 vol % AcOH) to afford the title
atmosphere. To this was added DMF (0.16 M), MeOH (1.3 M), and
TMSN3 (1.5 equiv) in one portion each. The vial was capped and
stirred at 100 °C for 16 h. After the reaction was allowed to cool to rt,
the mixture was filtered over Celite, rinsed with EtOAc, and
concentrated to dryness. Further purification is stated below.
Di-tert-butyl (2S,3R)-3-(3-(tert-Butoxycarbonyl)-5-(1H-1,2,3-tria-
zol-5-yl)phenyl)pyrrolidine-1,2-dicarboxylate (42a). General proce-
dure I. The reaction was performed with 0.15 g (0.32 mmol) of 41a.
The title compound was purified by flash column chromatography on
silica gel (heptane/EtOAc 1:1). The product was obtained as a white
foam (71 mg, 65%, mixture of rotamers). 1H NMR (400 MHz,
CDCl3) δ 11.73 (bs, 1H), 8.28 (s, 1H), 8.03 (s, 1H), 7.93 (s, 1H),
7.87 (s, 1H), 4.29 (d, J = 6.5 Hz,0.31H), 4.16 (d, J = 6.8 Hz, 0.7H),
3.84−3.81 (m, 0.7H), 3.72−3.68 (m, 0.3H), 3.62−3.57 (m, 1H),
3.51−3.47 (m, 1H), 2.38−2.30 (m, 1H), 2.16−2.09 (m, 1H), 1.63 (s,
9H), 1.50 (s, 3H), 1.45 (s, 6H), 1.42 (s, 9H). 13C NMR (151 MHz,
CDCl3) δ 171.3, 165.2, 153.8, 142.2, 133.1, 130.7, 128.7, 128.6, 128.5,
125.9, 125.9, 81.7, 81.4, 81.4, 80.2, 79.9, 66.3, 66.0, 50.2, 48.8, 46.3,
46.1, 32.8, 32.1, 29.7, 28.5, 28.4, 28.2, 28.0. LC-MS (m/z) calcd for
C27H38N4O6 [M + H]+, 515.3; found, 515.6 and 303.1 (-Boc, −2 ×
tert-Bu).
5-((2S,3R)-1,2-Bis(tert-butoxycarbonyl)pyrrolidin-3-yl)-3-(tert-
butoxycarbonyl)-2-chlorobenzoic Acid (46b). Formic acid (21 μL,
0.54 mmol, 2 equiv) and Ac2O (52 μL, 0.54 mmol, 2 equiv) were
stirred in a vial for 1.5 h at rt. Then, 45a (0.15 g, 0.27 mmol, 1equiv),
Pd(OAc)2 (6 mg, 0.03 mmol, 0.1 equiv), and Xantphos (16 mg, 0.03
mmol, 0.1 equiv) were loaded in a flask equipped with screw cap
containing a septa. The flask was evaporated and backfilled with argon
three times. Then anhydrous toluene (0.55 mL, 0.5 M) was added,
followed by anhydrous tBuOH (52 μL, 0.54 mmol, 2 equiv). To the
orange reaction mixture was added the mixture of HCOOH and Ac2O
in a dropwise manner. During the addition the color changed to dark-
green. Then Et3N (190 μL, 1.35 mmol, 5 equiv) was added dropwise.
Instantly, gas evolution was observed and a color change of the
reaction mixture to dark red. (Caution: Overpressure!) After the
addition was completed, the reaction was placed in a preheated oil
bath at 80 °C and the reaction was stirred overnight at 80 °C. The
reaction mixture became black and was allowed to cool down to rt.
The pressure was released carefully, followed by the addition of water
and EtOAc. The aqueous layer was carefully acidified to pH 2 with 1
N HCl. The aqueous layer was extracted three times with EtOAc. The
combined organic layers were dried over MgSO4, filtered, and
concentrated in vacuo. The crude product was purified by flash
column chromatography on silica gel (heptane/EtOAc 2:1 with 1 vol
% AcOH) to afford the title compound as a white foam (89 mg, 63%,
mixture of rotamers). 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 2.2
Hz, 1H), 7.43 (d, J = 2.2 Hz, 1H), 4.18 (d, J = 6.2 Hz, 0.35H), 4.05
(d, J = 6.3 Hz, 0.65H), 3.79−3.73 (m, 0.65H), 3.66−3.61 (m, 0.35H),
3.57−3.51 (m, 1H), 3.37−3.31 (m, 1H), 2.33−2.25 (m, 1H), 2.03−
1.94 (m, 1H), 1.60 (s, 9H), 1.48−1.44 (m, 9H). 13C NMR (151
MHz, CDCl3) δ 171.1, 165.0, 153.8, 141.4, 135.3, 134.3, 131.3, 128.1,
124.9, 83.6, 81.9, 80.6, 66.1, 49.2, 47.8, 46.0, 31.9, 28.6, 28.5, 28.3,
28.2, 28.1. LC-MS (m/z) calcd for C26H36ClNO8 [M + H]+, 526.2;
found, 314.4 (-Boc, −2 × tert-Bu).
1
compound as white solid (1.18 g, 90%, mixture of rotamers). H
NMR (600 MHz, CDCl3) δ 8.59 (s, 0.7H), 8.58 (s, 0.3H), 8.17 (s,
0.3 H), 8.16 (s, 0.7H), 8.12 (s, 0.7H), 8.11 (s, 0.3H), 4.26 (d, J = 7.0
Hz, 0.3H), 4.13 (d, J = 7.0 Hz, 0.7H), 3.85−3.81 (m, 0.7H), 3.73−
3.69 (m, 0.3H), 3.61−3.55 (m, 1H), 3.50−3.47 (m, 1H), 2.35−2.29
(m, 1H), 2.13−2.07 (m, 1H), 1.62 (s, 9H), 1.50−1.42 (m, 18 H). 13C
NMR (151 MHz, CDCl3) δ 171.1, 169.7, 164.5, 153.8, 141.9, 133.5,
133.1, 132.6, 130.0, 129.9, 82.0, 81.5, 80.4, 66.2, 49.9, 46.1, 32.2 31.9,
29.0, 28.4, 28.2, 28.0, 22.7, 14.1.; LC-MS (m/z) calcd for C26H37NO8
[M + H]+, 491.2; found, 280.4 (-Boc, −2 x tert-butyl).
Di-tert-butyl (2S,3R)-3-(3-(tert-Butoxycarbonyl)-5-
((trimethylsilyl)ethynyl)phenyl)pyrrolidine-1,2-dicarboxylate. To a
flame-dried vial with argon atmosphere was added aryl bromide 39a
(0.5 g, 0.95 mmol, 1 equiv), PdCl2(PPh3)2 (0.1 equiv), and CuI (0.1
equiv). The vial was evaporated and refilled with argon three times.
DMF (0.3 M) was added, followed by TMSA (1.5 equiv) and Et3N
(0.45 M). The reaction was placed into a preheated oil bath at 60 °C
and stirred for 12 h. The reaction was allowed to cool down to rt
before EtOAc was added. The organic layer was washed twice with
water and once with brine. The organic layer was dried over MgSO4,
filtered, and concentrated in vacuo. The crude mixture was purified by
flash column chromatography on silica gel (heptane/EtOAc 10:1) to
1
afford the title compound as a white foam (0.4 g, 77%). H NMR
(400 MHz, CDCl3) δ 7.95 (s, 1H), 7.80 (s, 1H), 7.50 (s, 1H), 4.21
(d, J = 6.2 Hz, 0.3H), 4.09 (d, J = 6.5 Hz, 0.7H), 3.79−3.75 (m,
0.7H), 3.66−3.63 (m, 0.3H), 3.59−3.54 (m, 1H), 3.40−3.37 (m,
1H), 2.31−2.24 (m, 1H), 2.06−2.00 (m, 1H), 1.59 (s, 9H), 1.49 (s,
3H), 1.45 (s, 6H), 1.43 (s, 9H), 0.26 (s, 9H). 13C NMR (151 MHz,
CDCl3) δ 171.4, 164.9, 153.9, 141.7, 134.3, 132.7, 131.8, 128.7, 123.9,
104.1, 95.6, 81.8, 81.7, 81.6, 81.6, 80.4, 80.0, 66.2, 50.0, 46.2, 32.0,
28.6, 28.5, 28.3, 28.3, 28.2, 28.1, 0.03. LC-MS (m/z) calcd for
C30H45NO4Si [M + H]+, 544.3; found, 444.8 (-Boc).
Di-tert-butyl (2S,3R)-3-(3-(tert-Butoxycarbonyl)-5-
ethynylphenyl)pyrrolidine-1,2-dicarboxylate (41a). TMS-acetylene
intermediate (0.39 g, 0.72 mmol, 1 equiv) was loaded in a flame-dried
vial with argon atmosphere. To this was added anhydrous THF (0.3
M) and the solution was cooled on an ice bath. After 15 min, a 1 M
solution of TBAF in THF (1.1 equiv) was added dropwise. After 5
min the ice bath was removed and the reaction was stirred at room
temperature for 30 min. The reaction was quenched with saturated
NH4Cl, and subsequently the aqueous layer was extracted with EtOAc
three times. The combined organic layers were dried over MgSO4,
filtered, and concentrated in vacuo. The title compound was purified
by flash column chromatography on silica gel (heptane/EtOAc 10:1).
The product was obtained as a light-yellow foam (0.28 g, 82%,
Final Compounds for Pharmacological Characterization.
Deprotection: General Procedure J. The protected compound was
dissolved in 1 mL of anhydrous DCM. To this was added dropwise 1
mL of TFA. The reaction mixture was stirred at room temperature
overnight and then concentrated in vacuo. The crude mixture was
purified by preparative HPLC if necessary. The obtained TFA salt was
treated with 2 mL of 1 N HCl and concentrated to dryness in vacuo.
This procedure was repeated three times. After lyophilization, the final
product was obtained as HCl salt if not otherwise stated-
(2S,3R)-3-(4-Bromo-3-carboxyphenyl)pyrrolidine-2-carboxylic
Acid Hydrochloride (4g). General procedure J. The reaction was
performed with 50 mg (0.09 mmol) of 35a. The crude product was
purified by preparative HPLC. Rt 12.8 min (flow: 20 mL min−1;
gradient 0−50% B (25 min); λ = 210 nm). The solid was treated with
1 M HCl (2 mL) and the volatiles were evaporated under reduced
pressure to afford the hydrochloride salt as a white solid (12.3 mg,
1
mixture of rotamers). H NMR (400 MHz, CDCl3) δ 7.99 (s, 1H),
7.85 (s, 1H), 7.52 (s, 1H), 4.20 (d, J = 6.2 Hz, 0.3H), 4.09 (d, J = 6.6
Hz, 0.7H), 3.79−3.76 (m, 0.7H), 3.67−3.62 (m, 0.3H), 3.59−3.53
(m, 1H), 3.41−3.37 (m, 1H), 3.12−3.10 (m, 1H), 2.33−2.25 (m,
1H), 2.06−2.00 (m, 1H), 1.59 (s, 9H), 1.49 (s, 3H), 1.45 (s, 6H),
1.42 (s, 9H). 13C NMR (151 MHz, CDCl3) δ 171.3, 164.8, 154.2,
153.9, 142.0, 134.6, 132.9, 132.0, 132.0, 128.9, 128.8, 122.9, 122.8,
82.8, 81.8, 81.6, 80.4, 80.1, 66.3, 66.1, 50.0, 48.6, 46.3, 46.2, 32.7,
32.1, 28.6, 28.5, 28.3, 28.2, 28.1. LC-MS (m/z) calcd for C27H37NO6
[M + H]+, 472.3; found, 372.8 (-Boc) and 260.5 (-Boc, −2 × tert-Bu).
CuAAC Reaction: General Procedure I. Acetylene (1 equiv) and
CuI (0.05 equiv) were loaded in a flame-dried flask with argon
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39%). 1H NMR (600 MHz, D2O) δ 7.76 (d, J = 8.3 Hz, 1H), 7.72 (d,
J = 2.3 Hz, 1H), 7.45 (dd, J = 8.3, 2.3 Hz, 1H), 4.27 (d, J = 9.4 Hz,
1H), 3.70−3.63 (m, 2H), 3.55 (ddd, J = 11.8, 10.2, 6.8 Hz, 1H), 2.57
(dtd, J = 13.9, 7.1, 3.4 Hz, 1H), 2.30−2.23 (m, 1H). 13C NMR (151
MHz, D2O) δ 171.4, 171.1, 138.5, 134.4, 134.0, 131.7, 129.2, 118.8,
65.3, 47.4, 45.7, 32.8. LC-MS (m/z) calcd for C12H12BrNO4 [M +
H]+, 314.0 and 316.0; found, 314.4 and 316.5.
(2S,3R)-3-(4-Amino-3-carboxyphenyl)pyrrolidine-2-carboxylic
Acid Dihydrochloride (4h). General procedure J. The crude product
was purified by preparative HPLC. Rt 3.72 min (flow: 20 mL min−1;
gradient 0−25% B (30 min); λ = 210 nm). The solid was treated with
1 M HCl (2 mL), and the volatiles were evaporated under reduced
pressure to afford the hydrochloride salt as a white solid (10 mg, 14%
over 2 steps). 1H NMR (600 MHz, D2O) δ 8.09, 7.64 (d, J = 8.4 Hz,
1H), 7.33 (dd, J = 8.4, 2.6 Hz, 1H), 4.23 (dd, J = 9.7, 2.6 Hz, 1H),
3.70−3.63 (m, 2H), 3.57−3.53−8m, 1H), 2.56 (ddt, J = 13.1, 6.3, 3.3
Hz, 1H), 2.28 (dtd, J = 13.5, 10.2, 7.9 Hz, 1H). 13C NMR (151 MHz,
D2O) δ 172.3, 170.1, 136.0, 133.4, 130.7, 122.8, 121.0, 66.1, 47.7,
45.6, 32.4. LC-MS (m/z) calcd for C12H14N2O4 [M + H]+, 251.1;
found, 251.5.
was performed with 50 mg (0.09 mmol) of 46a. No further
purification was required. The title compound was treated with 1 N
HCl (2 mL), and all volatiles were removed under reduced pressure
1
to afford the hydrochloride salt as a white solid (32 mg, 96%). H
NMR (600 MHz, D2O) δ 7.93 (d, J = 2.2 Hz, 1H), 7.71 (d, J = 2.2
Hz, 1H), 4.35 (d, J = 9.6 Hz, 1H), 3.71−3.66 (m, 2H), 3.58−3.53 (m
1H), 2.62−2.57 (m 1H), 2.30−2.23 (m, 1H). 13C NMR (151 MHz,
D2O) δ 171.2, 170.4, 139.2, 135.0, 135.0, 130.2, 127.7, 124.1, 65.1,
46.9, 45.6, 32.7. LC-MS (m/z) calcd for C12H11BrClNO4 [M + H]+,
348.0 and 350.0; found, 348.3 and 350.3.
5-((2S,3R)-2-Carboxypyrrolidin-3-yl)-2-chloroisophthalic Acid
Hydrochloride (6b). General procedure J. The reaction was
performed with 89 mg (0.17 mmol) of 46a. No further purification
required. The title compound was treated with 1 N HCl (2 mL), and
all volatiles were removed under reduced pressure to afford the
1
hydrochloride salt as a white solid (55 mg, 93%). H NMR (600
MHz, D2O) δ 7.84 (s, 1H), 4.38 (d, J = 9.6 Hz, 1H), 3.77−3.68 (m,
2H), 3.59−3.54 (m, 1H), 2.63−2.58 (m, 1H), 2.33−2.26 (m, 1H).
13C NMR (151 MHz, D2O) δ: 171.3, 170.6, 138.3, 134.8, 130.5,
127.3, 65.1, 47.0, 45.6, 32.7. LC-MS (m/z) calcd for C13H12ClNO6
[M + H]+, 314.0; found, 314.3.
4-((2S,3R)-2-Carboxypyrrolidin-3-yl)phthalic Acid Trisodium Salt
(4i). General procedure J. The reaction was performed with 30 mg
(0.08 mmol) of 35c. The crude product was purified by preparative
HPLC. Rt 8.72 min (flow: 20 mL min−1; gradient 0−50% B (25 min);
λ = 210 nm). After lyophilization, the solid was treated with 1 M
NaOH (4 equiv) and the volatiles were evaporated under reduced
pressure to afford the tri sodium salt as a white solid (20 mg, 72%) 1H
NMR (600 MHz, D2O) δ 7.79 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 1.9
Hz, 1H), 7.58 (dd, J = 8.0, 1.9 Hz, 1H), 4.21 (d, J = 8.8 Hz, 1H),
3.69−3.65 (m, 2H), 3.57−3.52 (m, 1H), 2.58−2.53 (m, 1H), 2.32−
2.25 (m, 1H). 13C NMR (151 MHz, D2O) δ 173.9, 173.4, 173.0,
142.5, 135.0, 132.4, 129.8, 129.6, 127.5, 66.5, 48.1, 45.6, 32.9. LC-MS
(m/z) calcd for C13H13NO6 [M + H]+, 280.1; found, 280.5.
(2S,3R)-3-(3-Bromo-5-carboxyphenyl)pyrrolidine-2-carboxylic
Acid Hydrochloride (5b). General procedure J. The reaction was
performed with 50 mg (0.09 mmol) of 28a. No further purification
was required. The title compound was treated with 1 N HCl (2 mL),
and all volatiles were removed under reduced pressure to afford the
Synthesis of Analogues 9b−e. 1-(tert-Butyl)-2-methyl (2R,4S)-4-
allyl-5-oxopyrrolidine-1,2-dicarboxylate (47). A solution of 1-(tert-
Butyl)-2-methyl (R)-5-oxopyrrolidine-1,2-dicarboxylate (3.9 g, 16.3
mmol, 1.0 equiv) in anhydrous THF (160 mL) was cooled to −78 °C,
and 1 M LiHMDS in THF (19.3 mL, 19.3 mmol, 1.2 equiv) was
added dropwise over 45 min. The mixture was left to stir at −78 °C
for 30 min after which a solution of 3-bromoprop-1-ene (4.2 mL, 5.8
g, 48.1 mmol, 3 equiv) with DMPU (21.5 mL, 160 mmol, 10 equiv)
in anhydrous THF (240 mL) was added dropwise over 1.5 h. After
the addition was completed, the reaction mixture was stirred for
another 2 h at −78 °C. The mixture was quenched with saturated
NH4Cl solution and allowed to reach rt. The mixture was transferred
to a separation funnel, and the aqueous layer was extracted three
times with EtOAc. The combined organic layers were dried over
MgSO4, filtered, and concentrated in vacuo. The crude mixture was
filtered over a short silica pad and rinsed with 1:1 EtOAc/heptane.
The filtrate was concentrated and the crude mixture was purified by
flash column chromatography (heptane/EtOAc 10:1) to afford the
title compound as a colorless oil (3.25 g, 36%). 1H NMR (600 MHz,
CDCl3) δ 5.76−5.69 (m, 1H), 5.11−5.06 (m, 2H), 4.56 (dd, J = 9.7,
1.7 Hz, 1H), 3.77 (s, 3H), 2.76 (m, 1H), 2.64−2.59 (m, 1H), 2.21−
2.15 (m, 2H), 2.03- 1.97 (m, 1H), 1.49 (s, 9H). 13C NMR (151 MHz,
CDCl3) δ 174.2, 171.8, 149.4, 134.3, 117.7, 83.59, 56.9, 52.5, 41.2,
34.4, 27.9, 27.7. LC-MS (m/z) calcd for C14H21NO5 [M + H]+,
284.1; found, 284.4 and 184.5 (-Boc). Analytical data is in agreement
with literature. Nakache et al., J. Org. Chem. 2006, 71 (7), 2760−
2778.
1-(tert-Butyl) 2-Methyl (2R)-4-allylpyrrolidine-1,2-dicarboxylate
(48). Lactam 47 (3.1 g, 11 mmol, 1.0 equiv) was dissolved in
anhydrous THF (57 mL) and cooled to −78 °C. After 10 min, a 1 M
solution of LiEt3BH (11 mL, 11 mmol, 1 equiv) was added dropwise
over 20 min. The mixture was left to stir at −78 °C for 20 min. Then
the reaction was quenched with saturated NaHCO3 (6.5 mL) and
allowed to warm up to 0 °C. Then 50% H2O2 (5.6 mL) was added
dropwise, and the reaction was stirred for another 20 min. The
organic solvent was evaporated, and the aqueous layer was extracted
with DCM. The organic layers were combined, dried over MgSO4,
and concentrated in vacuo. The resulting residue was used for the next
reaction without further purification. The flask with the residue was
evaporated and refilled with argon. The residue was dissolved in
anhydrous DCM (56 mL) and cooled to −78 °C. To the stirring
solution, triethylsilane (3.7 mL, 23 mmol, 2.1 equiv) and boron
trifluoride etherate (2.85 mL, 23 mmol, 2.1 equiv) were added
dropwise. The reaction mixture was left to stir at −78 °C for 3 h and
then quenched with saturated NaHCO3 solution. The resulting
mixture was extracted with DCM, and the organic layers were
combined, dried over MgSO4, and concentrated in vacuo. The crude
mixture was purified by flash column chromatography on silica gel
(heptane/EtOAc 10:1), to afford the title compound a colorless oil
1
hydrochloride salt as a white solid (25.7 mg, 77%). H NMR (600
MHz, D2O) δ 8.17 (s, 1H), 8.01 (s, 1H), 7.89 (s, 1H), 4.23 (d, J = 9.3
Hz, 1H), 3.70−3.64 (m, 2H), 3.59−3.48 (m, 1H), 2.58 (dtd, J = 13.8,
7.0, 3.4 Hz, 1H), 2.33−2.21 (m, 1H). 13C NMR (151 MHz, D2O) δ
172.3, 169.1, 141.8, 135.4, 132.6, 131.7, 127.4, 122.3, 66.1, 47.8, 45.5,
32.9. LC-MS (m/z) calcd for C12H12BrNO4 [M + H]+, 314.0 and
316.0; found, 314.3 and 316.3.
5-((2S,3R)-2-Carboxypyrrolidin-3-yl)isophthalic Acid Hydrochlor-
ide (5c). General procedure J. The reaction was performed with 52
mg (0.1 mmol) of 28b. No further purification was required. The title
compound was treated with 1 N HCl (2 mL), and all volatiles were
removed under reduced pressure to afford the hydrochloride salt as a
1
white solid (23.6 mg, 75%). H NMR (600 MHz, D2O) δ 8.54 (s,
1H), 8.29 (s, 2H), 4.43 (d, J = 9.6 Hz, 1H), 3.74 (ddd, J = 9.6 8.1, 3.3
Hz, 1H), 3.59 (td, J = 10.8, 6.8 Hz, 1H), 2.64 (dtd, J = 13.9, 7.0, 3.3
Hz, 1H), 2.35 (dtd, J = 13.9, 10.8, 8.1 Hz, 1H).; 13C NMR (151 MHz,
D2O) δ 171.4, 169.1, 139.9, 133.4, 131.1, 130.1, 65.3, 47.6, 45.7, 32.9.
LC-MS (m/z) calcd for C13H13NO6 [M + H]+, 280.1; found, 280.3.
(2S,3R)-3-(3-Carboxy-5-(1H-1,2,3-triazol-5-yl)phenyl)pyrrolidine-
2-carboxylic Acid Hydrochloride (5d). General procedure J. The
reaction was performed with 70 mg (0.14 mmol) of 42a. No further
purification was required. The title compound was treated with 1 N
HCl (2 mL), and all volatiles were removed under reduced pressure
1
to afford the hydrochloride salt as a yellow solid (44 mg, 92%). H
NMR (600 MHz, D2O) δ 8.26−8.25 (m, 2H), 8.04 (s, 1H), 8.02 (s,
1H), 4.46 (d, J = 9.4 Hz, 1H), 3.82−3.73 (m, 2H), 3.62−3.58 (m,
1H), 2.66−2.61 (m, 1H), 2.38−2.31 (m, 1H). 13C NMR (151 MHz,
D2O) δ 171.3, 169.4, 144.5, 140.1, 131.2, 130.3, 129.8, 128.6, 126.3,
125.0, 65.2, 47.7, 45.8, 32.9. LC-MS (m/z) calcd for C14H14N4O4 [M
+ H]+, 303.0; found, 303.5.
(2S,3R)-3-(3-Bromo-5-carboxy-4-chlorophenyl)pyrrolidine-2-car-
boxylic Acid Hydrochloride (6a). General procedure J. The reaction
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1
(2.1 g, 72%, mixture of rotamers). H NMR (600 MHz, CDCl3) δ
5.80−5.67 (m, 1H), 5.07−5 (m, 2H), 4.37 (dd, J = 2.5, 8.9 Hz,
0.36H), 4.27 (dd, J = 3.1, 8.9 Hz, 0.54H), 3.71−3.63 (m, 4H), 3.07−
2.96 (m, 1H), 2.45−5.32 (m, 1H), 2.14−2.03 (m, 3H), 1.94−1.80
(m, 1H), 1.46 (s, 4H), 1.40 (s, 5H). 13C NMR (151 MHz, CDCl3) δ
173.6, 173.4, 154.4, 153.7, 135.8, 116.5, 79.9, 58.9, 58.6, 52.1, 51.9,
51.5, 51.2, 37.1, 36.9, 36.1, 35.3, 28.4, 28.3. LC-MS (m/z) calcd for
C14H23NO4 [M + H]+, 270.2; found, 170.5 (-Boc).
(s, 3H), 3.61−3.55 (m, 1H), 3.19−3.10 (m, 1H), 2.96 (m, 1H), 2.08
(m, 1H), 1.97−1.92 (m, 1H), 1.43 (s, 3H), 1.22 (s, 6H). 13C NMR
(151 MHz, DMSO) δ 169.9, 169.3, 166.4, 153.9, 153.5, 149.4, 148.9,
138.2, 137.2, 136.8, 136.4, 133.9, 133.6, 130.0, 129.9, 128.9, 128.2,
128.0, 127.1, 122.4, 122.9, 116.9, 116.8, 79.6, 79.4, 66.4, 66.1, 52.7,
52.4, 51.3, 39.2, 35.2, 31.7, 28.6, 28.3, 22.6, 14.4. LC-MS (m/z) calcd
for C30H33N3O5 [M + H]+, 516.2; found, 516.8.
(2S,3R,4S)-4-Allyl-1-(tert-butoxycarbonyl)-3-(3-carboxyphenyl)-
pyrrolidine-2-carboxylic Acid. Intermediate 49 (0.34 g, 0.67 mmol,
1.0 equiv) was dissolved in EtOH (3.4 mL), in a microwave vial.
NaOH pellets (0.27 mg, 6.7 mmol, 10.0 equiv) were added, the vial
was sealed, and the reaction mixture was heated to 100 °C and stirred
for 20 h. The reaction mixture became brown and slurry over time.
The reaction was allowed to cool down to rt and then concentrated to
dryness in vacuo. The residue was dissolved in 1N NaOH and then
extracted three times with EtOAc to remove 8-aminoquinoline. The
aqueous layer was acidified with 1 N HCl to pH 2−3 and extracted
with EtOAc three times. The combined organic layers were dried over
MgSO4, filtered, and concentrated in vacuo. The resulting brown-ish
foam (0.15 g, 60−90%) was used in the next step without further
(2R,4S)-4-Allyl-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic
Acid. Intermediate 48 (2.1 g, 7.8 mmol, 1.0 equiv) was dissolved in
THF (20 mL) and MeOH (4 mL). A 1 M solution of aq. LiOH (12
mL, 12 mmol, 1.5 equiv) was added, and the reaction was stirred at rt
overnight. The reaction mixture was concentrated to dryness in vacuo,
and then water and DCM were added. The aqueous layer was
acidified with 1 N HCl to pH 3 and extracted with DCM. The
combined organic layers were dried over MgSO4 and concentrated in
vacuo to give the title compound as a white solid (1.8 g, 90%, mixture
1
of rotamers). H NMR (600 MHz, CDCl3) δ 5.76 (m, 1H), 5.08−
5.02 (m, 2H), 4.39 (d, J = 8.3 Hz, 0.7H), 4.29 (bs, 0.3H), 3.72 (bs,
0.3H), 3.51 (m, 0.7H), 3.05 (bs, 0.3H), 2.97−2.94 (m, 7H), 2.49−
2.47 (m, 0.7H), 2.37 (m, 1H), 2.18 (m, 2.7H), 1.99−1.92 (m, 0.3H),
1.72−1.64 (m, 0.7H), 1.49−1.42 (m, 9H). 13C NMR (151 MHz,
CDCl3) δ 177.9, 173.5 157.2, 153.7, 135.7, 116.6, 82.1, 80.3, 59.4,
58.7, 51.9, 51.3, 37.1, 36.8, 36.0, 33.4, 28.4. LC-MS (m/z) calcd for
C13H21NO4 [M + H]+, 256.2; found, 156.5 (-Boc).
1
purification (mixture of rotamers). H NMR (400 MHz, DMSO) δ
12.45 (bs, 2H), 7.86−7.84 (m, 2H), 7.62 (d, J = 7.9 Hz, 1H), 7.48
(m, 1H), 5.72−5.62 (m, 1H), 4.98 (d, J = 15.8 Hz, 1H), 4.94 (d, J =
10.3 Hz, 1H), 4.08 (d, J = 9.3 Hz, 1H), 3.77−3.72 (m, 1H), 3.11−
2.97 (m, 1H), 1.99−1.97 (m, 2H), 1.42 (s,13H), 1.34 (s, 6H). 13C
NMR (151 MHz, DMSO) δ 173.3, 172.7, 172.0, 167.3, 167.2, 153.2,
153.0, 152.7, 139.6, 139.4, 135.8, 135.8, 132.5, 132.4, 131.2, 130.1,
130.1, 129.7, 129.0, 128.9, 128.3, 127.8, 126.7, 116.6, 116.5, 79.2,
79.1, 66.5, 66.3, 58.6, 55.0, 54.2, 51.4, 51.2, 44.7, 43.9, 34.3, 34.2,
27.9, 27.9, 21.1.
Di-tert-butyl-(2S,3R,4S)-4-allyl-3-(3-(tert-butoxycarbonyl)-
phenyl)pyrrolidine-1,2-dicarboxlate. (2S,3R,4S)-4-Allyl-1-(tert-bu-
toxycarbonyl)-3-(3-carboxyphenyl)pyrrolidine-2-carboxylic acid (561
mg, 1.5 mmol, 1.0 equiv) was loaded in a vial and dissolved in
anhydrous DCM (3 mL, 0.5 M). To this was added in one portion
TBTA (1.6 g, 7.5 mmol, 5 equiv). The reaction was stirred for 24 h at
rt. The brownish suspension was concentrated to dryness, and the
crude mixture was purified by flash column chromatography on silica
gel (heptane/EtOAc 10:1) to afford the title compound as a yellow oil
(0.42 g, 57%, mixture of rotamers) with small impurities (∼10%). 1H
NMR (600 MHz, CDCl3) δ 7.90 (m, 1H), 7.84 (s, 1H) 7.42−7.36
(m, 2H), 5.69−5.59 (m, 1H), 5.01−4.94 (m, 2H), 4.18−4.11 (m,
0.4H), 4.08−4.06 (d, J = 9.1 Hz, 0.75H), 3.99 (m, 0.6H), 3.86−3.82
(m, 0.25H), 3.21−3.16 (m, 1H), 2.97−2.92 (dd, J = 11.0, 9.0 Hz,
1H), 2.46−2.37 (m, 1H), 2.19−2.10 (m,1H), 1.99−1.90 (m,1H),
1.60 (s, 9H), 1.43 (s, 9H), 1.34 (s, 9H). 13C NMR (151 MHz,
CDCl3) δ 171.1, 170.9, 165.6, 153.9, 153.6, 139.5, 135.4, 135.2, 132.4,
131.9, 131.8, 129.1, 128.6, 128.5, 128.1, 116.8, 81,3, 81.2, 81.1, 80.2,
79.9, 67.3, 60.4, 59.8 59.7, 56.5, 55.3, 51.9, 51.6, 45.4, 44.7, 36.3, 35.2,
35.1, 28.5, 28.4, 28.3, 28.3, 28.2, 28.0, 27.9,21.0, 14.2. LC-MS (m/z)
calcd for C28H41NO6 [M + H]+, 488.2; found, 388.6 (-Boc) and 276.6
(-Boc, −2 × tert-butyl).
Di-tert-butyl-(2S,3R,4S)-3-(3-(tert-butoxycarbonyl)phenyl)-4-(2-
oxoethyl)pyrrolidine-1,2- dicarboxylate (50). Di-tert-butyl-
(2S,3R,4S)-4-allyl-3-(3-(tert-butoxycarbonyl)phenyl)pyrrolidine-1,2-
dicarboxlate (0.42 g, 0.86 mmol, 1 equiv) was dissolved in 8.5 mL of
1:1 THF/H2O. To the colorless solution was added OsO4 (75 μL,
0.01 equiv., 4 wt % in H2O). The mixture was stirred at rt for 15 min
and became black. Then NaIO4 (732 mg, 3.4 mmol, 4 equiv) was
added in one portion and stirred for another 4 h at rt. The reaction
mixture became a white suspension over time. To the mixture was
added water, and the organic layer was extracted three with EtOAc.
The combined organic layers were dried over MgSO4, filtered, and
concentrated in vacuo. The crude mixture was purified by flash
column chromatography on silica gel (heptane/EtOAc 3:1) to yield
the tile compound as a white foam (0.22 g, 53%, mixture of rotamers).
1H NMR (600 MHz, CDCl3) δ 9.62 (s, 0.35H), 9.59 (s, 0.65H), 7.90
tert-Butyl-(2R,4S)-4-allyl-2-(quinolin-8-ylcarbamoyl)pyrrolidine-
1-carboxylate. (2R,4S)-4-Allyl-1-(tert-butoxycarbonyl)pyrrolidine-2-
carboxylic acid (1.8 g, 7.1 mmol, 1.0 equiv) and 8-aminoquinoline
(1.2 g, 8.5 mmol, 1.2 equiv) were dissolved in anhydrous DCM (14
mL, 0.5 M). EDC·HCl (1.6 g, 8.5 mmol, 1.2 equiv) and HOBt (1.14
g, 8.5 mmol, 1.2 equiv) were added to the solution, and the reaction
was left to stir at rt for 24 h. To the reaction was added saturated
NaHCO3 solution, and the aqueous layer was extracted three times
with DCM. The combined organic layers were dried over MgSO4,
filtered, and concentrated in vacuo. The crude mixture was purified by
flash column chromatography on silica gel (heptane/EtOAc, gradient
7:1 to 6:1) to afford the title compound as a white solid (2.1 g, 80%,
mixture of rotamers). 1H NMR (600 MHz, CDCl3) δ 10.65 (s, 0.4H),
10.40 (s, 0.6H), 8.79 (m, 2H), 8.15 (bs, 1H), 7.53 (bs, 2H), 7.44 (bs,
1H), 5.77 (ddt, J = 17.1, 10.2, 7.0 Hz, 1H), 5.06 (dd, J = 17.1, 1.6 Hz,
1H), 5.01 (dd, J = 10.2, 1.6 Hz, 1H), 4.64 (s, 0.4 H), 4.49 (s, 0.6H),
3.83 (m, 0.6H), 3.7 (m, 0.4H), 3.23 (m, 0.6H), 3.09 (m, 0.4H), 2.47
(m, 0.4H), 2.43 (m, 1.6H), 2.18 (m, 2H), 1.96 (m, 0.6H), 1.83 (m,
0.4H), 1.55 (s, 4H), 1.37 (s, 5H). 13C NMR (151 MHz, CDCl3) δ
171.4, 170.9, 155.4, 154.6, 148.5, 148.2, 138.8, 138.6, 136.2, 136.0,
135.8, 134.5, 134.0, 127.9, 127.3, 121.8, 121.7, 121.5, 116.6, 116.4,
80.7, 80.4, 62.3, 61.6, 52.2, 51.9, 37.4, 37.1, 36.6, 36.5, 35.1, 29.7,
28.5, 28.2. LC-MS (m/z) calcd for C22H27N3O3 [M + H]+, 382.2;
found, 382.6. Mp: 130.3−134.3 °C.
tert-Butyl-(2R,3R,4S)-4-allyl-3-(3-(methoxycarbonyl)phenyl)-2-
(quinolin-8-ylcarbamoyl)-pyrrolidine-1-carboxylate (49). tert-Butyl-
(2R,4S)-4-allyl-2-(quinolin-8-ylcarbamoyl)pyrrolidine-1-carboxylate
(0.5 g, 1.3 mmol, 1.0 equiv), methyl 3-iodobenzoate (686 mg, 2.6
mmol, 2.0 equiv), palladium acetate (30 mg, 0.13 mmol, 0.1 equiv),
pivalic acid (44 mg, 0.4 mmol, 0.3 equiv), and silver carbonate (361
mg, 1.3 mmol, 1.0 equiv) were placed in a flame-dried, argon-filled
microwave vial and dissolved in anhydrous toluene (6.5 mL, 0.2 M).
The microwave vial was sealed and heated to 110 °C under vigorous
stirring. The reaction was left to stir at 110 °C for 48 h. After the
reaction was allowed to cool down to rt, the mixture was filtered
through a Celite-silica pad and rinsed with EtOAc. The resulting
filtrate was concentrated in vacuo and then purified by column
chromatography on silica gel (heptane/EtOAc, gradient 10:1 → 5:1
→ 4:1) to afford the title compound as a white foam (244 mg, 37%,
57% brsm, mixture of rotamers). However, the product could not be
obtained in 100% purity. Nonetheless, the product was used in the
1
next step. H NMR (600 MHz, DMSO) (mixture of conformers) δ
9.65 (s, 0.4H), 9.59 (s,0.6H), 8.71 (m, 1H), 8.31 (s, 2H), 7.91 (m,
1H), 7.58−7.55 (m, 2H), 7.51−7.45 (m, 3H) 7.20 (td (J = 7.7, 1.8
Hz, 1H), 5.75 (m, 1H), 5.01−4.93 (m, 2H), 3.91−3.89 (m, 1H), 3.72
(d, J = 6.6 Hz, 1H), 7.82 (s, 1H), 7.38 (s, 1H), 4.20−4.06 (m, 2H),
3.16−3.09 (m, 1H), 2.98−2.91 (m, 1H), 2.80−2.75 (m, 1H), 2.50−
2.40 (m, 2H), 1.58 (s, 9H) 1.46 (s, 3H), 1.42 (s, 6H), 1.32 (s, 9H).
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Research Article
13C NMR (151 MHz, CDCl3) δ 199.8, 199.4, 170.9, 165.3, 153.4,
water was added. The aqueous layer was acidified with 1 N HCl to pH
3 and extracted with EtOAc three times. The combined organic layers
were dried over MgSO4, filtered, and concentrated in vacuo. The
crude mixture was purified by column chromatography on silica gel
(heptane/EtOAc 2:1 + 1 vol % AcOH) to afford the title compound
138.3, 132.6, 131.7, 129.0, 128.9, 128.8, 81.4, 80.3, 80.1, 66.5, 56.4,
55.2, 51.9, 51.5, 45.3, 45.0, 39.9, 39.3, 28.4, 28.3, 28.1, 27.9, 27.8. LC-
MS (m/z) calcd for C27H39NO7 [M + H]+, 489.3; found, 278.5 (-Boc,
−2 × tert-butyl).
1
as a white foam (29 mg, 94%, mixture of rotamers). H NMR (400
Di-tert-butyl-(2S,3R,4S)-3-(3-(tert-butoxycarbonyl)phenyl)-4-(2-
(methylamino)ethyl)pyrroli-dine-1,2-dicarboxylate (51a). Aldehyde
50 (20 mg, 0.04 mmol, 1 equiv) was dissolved in anhydrous MeOH
(0.8 mL, 0.05 M). To this was added MeH2N·HCl (14 mg, 0.2 mmol,
5 equiv) in one portion, followed by NaBH3CN (13 mg, 0.2 mmol, 5
equiv). The white suspension was stirred at rt overnight and then
concentrated to dryness. To the crude mixture was added 1 N NaOH,
and the aqueous layer was extracted three times with EtOAc. The
combined organic layer was dried over MgSO4, filtered, and
concentrated in vacuo. The crude product was used in the next step
without further purification (crude yield: 10 mg, mixture of rotamers).
1H NMR (600 MHz, CDCl3) δ 7.88 (bs, 1H), 7.80 (bs, 1H), 7.35 (s,
2H), 4.08−3.94 (m, 1.75H), 3.84−3.79 (m, 0.25H), 3.12−3.05 (m,
1H), 2.90−2.85 (m, 1H), 2.33−2.24 (m, 1H), 2.16−2.01 (m, 2H),
1.92 (s, 1H), 1.60 (s, 9H), 1.42 (s, 9H), 1.33 (s, 9H), 1.25 (s, 3H,
over integrates to 6H) LC-MS (m/z) calcd for C28H44N2O6 [M +
H]+, 505.3; found, 505.8.
MHz, CDCl3) (Mixture of conformers) δ 7.90 (s, 1H), 7.82 (s, 1H),
7.3 (s, 2H), 4.21−4.13 (m, 1H), 4.09−4.02 (m, 1H), 3.23−3.18 (m,
1H), 2.98−2.93 (m, 1H), 2.73 (m, 1H), 2.37−2.22 (m, 2H), 1.59 (s,
9H), 1.42 (s, 9H), 1.32 (s, 9H). 13C NMR (100 MHz, CDCl3) δ
176.7, 175.8, 170.8, 165.4, 153.7, 138.2, 132.5, 131.8, 129.2, 128.9,
128.8, 81.4, 81.4, 80.7, 76.7, 66.8, 56.2, 51.7, 41.3, 35.3, 28.4, 28.3,
28.2, 28.0, 27.9, 27.9. LC-MS (m/z) calcd for C27H39NO8 [M + H]+,
505.3; found, 294.5 (-Boc, −2 × tert-butyl).
Deprotection of 51a−d: General Deprotection M. For the
deprotection, the starting material was dissolved in 1 mL of anhydrous
DCM. To this was added 1 mL of TFA. The reaction mixture was
stirred at room temperature overnight and subsequently concentrated
to dryness to yield the final compound. Purification was performed as
described below.
(2S,3R,4S)-3-(3-Carboxyphenyl)-4-(2-(methylamino)ethyl)-
pyrrolidine-2-carboxylic Acid (9b). General deprotection M. The
crude product was purified by preparative HPLC and concentrated to
dryness by lyophilization to afford the title compound as its zwitterion
as a white solid (6.3 mg, 53% over two steps). Compound contains
small amounts of an unknown impurity (<5%). Rt: 3.68 min; flow: 20
Di-tert-butyl-(2S,3R,4S)-4-(2-(benzylamino)ethyl)-3-(3-(tert-
butoxycarbonyl)phenyl)pyroli-dine-1,2-dicarboxylate (51b). Alde-
hyde 50 (50 mg, 0.1 mmol, 1 equiv) was dissolved in anhydrous
MeOH (1 mL, 0.1 M). To this was added BnH2N·HCl (44 mg, 0.3
mmol, 3 equiv) in one portion, followed by NaBH3CN (10 mg, 0.15
mmol, 1.5 equiv). The colorless mixture was stirred at rt overnight
and then concentrated to dryness. The crude mixture was purified by
flash column chromatography (Et3N/MeOH/EtOAc/heptane
0.05:0.05:1:4) to afford the title compound as white foam (40 mg,
1
mL min−1; gradient 100% A (10 min); λ = 210 nm. H NMR (600
MHz, D2O) δ 7.95−7.93 (m, 2H), 7.58 (d, J = 7.7 Hz, 1H), 7.49 (t, J
= 7,7 Hz, 1H), 4.25 (d, J = 10.5 Hz, 1H), 3.74 (dd, J = 11.7, 7.3 Hz,
1H), 3.25−3.16 (m, 2H), 2.82−2.77 (m, 1H), 2.70−2.66 (m, 1H),
2.62−2.57 (m, 1H), 2.46 (s, 3H), 1.75−1.70 (m, 2H). 13C NMR (151
MHz, D2O) δ 171.3, 170.0, 137.3, 133.2, 130.5, 129.5, 129.5, 128.9,
65.9, 53.8, 49.4, 46.7, 43.4, 32.3. 26.31. LC-MS (m/z) calcd for
C15H20N2O4 [M + H]+, 293.1; found, 293.5.
(2S,3R,4S)-4-(2-(Benzylamino)ethyl)-3-(3-carboxyphenyl)-
pyrrolidine-2-carboxylic Acid (9c). General deprotection M. The
crude product was purified by preparative HPLC and concentrated to
dryness by lyophilization to afford the title compound as its zwitterion
as a white solid (21 mg, 84%). Rt: 10.02 min; flow: 20 mL min−1;
gradient 0−50% B (10 min); λ = 210 nm. 1H NMR (600 MHz, D2O)
δ 7.91 (d, J = 7.8 Hz, 1H) 7.79 (s, 1H), 7.52−7.47 (m, 1H), 7.44 (t, J
= 7.7 Hz, 1H)), 7.35−7.32 (m, 1H), 7−29−7.26 (m, 2H), 7.09−7.08
(m, 2H), 4.14−4.09 (m, 1H), 4.04−3.86 (m, 2H), 3.70−3.67 (m,
1H), 3.15−3.08 (m, 2H), 2.58−2.53 (m, 2H), 2.34 (dt, J = 12.3, 5.2
Hz, 1H), 1.83−1.78 (m, 1H), 1.70−1.63 (m, 1H). 13C NMR (151
MHz, D2O) δ 171.5, 169.9, 137.6, 132.9, 130.4, 129.6, 129.5, 129.0,
128.8, 66.6, 54.0, 50.4, 49.5, 44.0, 43.4, 26.7. LC-MS (m/z) calcd for
C21H24N2O4 [M + H]+, 369.2; found, 369.4.
1
70%, mixture of rotamers). H NMR (600 MHz, CDCl3) δ 7.88 (m,
1H), 7.83 (m, 1H), 7.37 (s, 2H), 7.28−7.26 (m, 2H), 7.22−7.19 (m,
3H), 4.12−4.01 (m, 1.8H), 3.90−3.87 (m, 0.2H), 3.64 (m, 2H),
3.17−3.13 (m, 1H), 2.93−2.89 (m, 1H), 2.55−2.38 (m, 3H), 1.60 (s,
9H), 1.43 (s, 9H), 1.32 (s, 9H). 13C NMR (151 MHz, CDCl3) δ
171.1, 171.0, 165.5, 153.8, 153.5, 139.3, 132.4, 131.9, 131.7, 129.2,
129.1, 128.6, 128.6, 128.5, 128.4, 128.2, 127.1, 81.2, 81.1, 80.2, 79.9,
67.2, 67.1, 61.1, 57.3, 56.2, 53.6, 53.5, 52.4, 51.9, 47.4, 43.9, 43.3, 42.2
34.0, 31.4, 29.7, 28.4, 28.3, 28.2, 27.9, 27.9. LC-MS (m/z) calcd for
C34H48N2O6 [M + H]+, 581.4; found, 581.9.
Di-tert-butyl-(2S,3R,4S)-3-(3-(tert-butoxycarbonyl)phenyl)-4-(2-
hydroxyethyl)pyrrolidine-1,2-dicarboxylate (51c). Aldehyde 50 (20
mg, 0.04 mmol, 1 equiv) was dissolved in anhydrous MeOH (0.4 mL,
0.01 M). To this was added NaBH4 (1.5 mg, 0.04 mmol, 1 equiv) in
one portion. The reaction was stirred at rt for 30 min after which
saturated NH4Cl solution was added. The reaction mixture was
concentrated to dryness. The residue was taken up in EtOAc and
water. The aqueous layer was extracted with EtOAc three times. The
combined organic layers were dried over MgSO4, filtered, and
concentrated in vacuo. The crude mixture was purified by column
chromatography on silica gel (heptane/EtOAc, gradient 2:1 → 1:1)
to afford the title compound as a colorless sticky oil (16.5 mg, 82%,
(2S,3R,4S)-3-(3-Carboxyphenyl)-4-(2-hydroxyethyl)pyrrolidine-2-
carboxylic Acid (9d). General deprotection M. The crude product
was purified by preparative HPLC and concentrated to dryness by
lyophilization to afford the title compound as its zwitterion as a white
solid (8 mg, 90%). Rt: 10.99 min; flow: 20 mL min−1; gradient 0−
1
1
20% B (20 min); λ = 210 nm. H NMR (600 MHz, D2O) δ 7.92 (s,
mixture of rotamers). H NMR (600 MHz, CDCl3) δ 7.89 (s, 1H),
1H), 7.9 (d, J = 7.8 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H)), 7.47 (t, J = 7.7
Hz, 1H), 4.14 (d, J = 10.5 Hz, 1H), 3.73 (dd, J = 11.7, 7.2 Hz, 1H),
3.39−3.31 (m, 2H), 3.17−3.09 (m, 1H), 2.59−2.52 (m, 1H), 1.57−
1.48 (m, 2H). 13C NMR (151 MHz, D2O) δ 172.3, 170.3, 138.3,
133.4, 130.5, 129.4, 129.2, 129.1, 66.4, 59.7, 54.5, 50.2, 43.8, 32.3. LC-
MS (m/z) calcd for C14H17NO5 [M + H]+, 280.1; found, 280.5.
(2S,3R,4S)-4-(Carboxymethyl)-3-(3-carboxyphenyl)pyrrolidine-2-
carboxylic Acid Hydrochloride (9e). General deprotection M. The
obtained product was not further purified. The salt was treated with 1
M aq. HCl (2 mL), and the volatiles were evaporated under reduced
pressure, repeating the same procedure three times to afford the title
compound as a hydrochloride salt as a white solid (18 mg,
7.84−7.82 (m, 1H), 7.40 (s,1H), 4.11−4.03 (m, 1.75H), 3.96−3.93
(m, 0.25H), 3.54−3.50 (m, 1H), 3.20−3.16 (m, 1H), 2.95−2.92 (m,
1H), 2.52−2.43 (m, 1H), 1.59 (s, 9H) 1.42 (s, 9H), 1.33 (s, 9H). 13C
NMR (151 MHz, CDCl3) δ 171.1, 165.5, 153.6, 139.4, 139.3, 132.4,
131.8, 131.7, 129.2, 128.7, 128.6, 81.3, 81.2, 81.2, 81.1, 80.2, 79.9,
67.2, 61.2, 61.1, 57.3, 56.1, 52.4, 52.0 42.9, 42.2, 34.1, 33.9, 29.7, 28.5,
28.4, 28.3, 28.2, 28.0, 27.9. LC-MS (m/z) calcd for C27H41NO7 [M +
H]+, 492.3; found, 280.5 (-Boc, −2 × tert-butyl).
2-(3S,4R,5S)-1,5-Bis(tert-butoxycarbonyl)-4-(3-(tert-
butoxycarbonyl)phenyl)pyrrolidin-3-yl)acetic Acid (51d). Aldehyde
50 (30 mg, 0.06 mmol, 1 equiv) was dissolved in a 1:1:1 mixture of
THF/tBuOH/H2O (1.22 mL, 0.05M). To the colorless solution was
added NaH2PO4 (7.4 mg, 0.06 mmol, 1 equiv) and 2-methyl-2-butene
(28 μL, 0.06 mmol, 4 equiv). Then, NaO2Cl (11 mg, 0.12 mmol, 2
equiv) was added and the reaction was allowed to stir at rt. The
reaction became light yellow. After 1 h, the reaction was colorless and
1
quantitative). H NMR (600 MHz, D2O) δ 7.93 (s, 1H), 7.91 (d, J
= 7.7 Hz, 1H), 7.56 (d, J = 7.7 Hz, 1H)), 7.46 (t, J = 7.7 Hz, 1H),
4.29−4.25 (m, 1H), 3.81 (dd, J = 11.8, 7.5 Hz, 1H), 3.23−3.19 (m,
2H), 2.89−2.81 (m, 1H), 2.46−2.35 (m, 2H). 13C NMR (151 MHz,
X
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ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Research Article
D2O) δ 175.3, 171.6, 170.2, 137.1, 133.4, 130.4, 129.5, 129.5, 129.2,
65.8, 53.5, 49.6, 42.5, 34.2. LC-MS (m/z) calcd for C14H15NO6 [M +
H]+, 294.1; found, 294.6. Analytical HPLC: Rt: 9.61 min; flow: 1 mL
min−1; gradient 0−25% B (20 min); λ = 210 nm.
Pharmacological Studies. Radioligand Binding. Affinities for
native AMPA, KA, and NMDA receptors in rat cortical synaptosomes
were determined using 5 nM [3H]AMPA, 5 nM [3H]KA, and
[3H]CGP39653, respectively, as previously detailed.56 Rat brain
membrane preparations used in these receptor-binding experiments
were prepared according to a method previously described.57 Cpm
values were fitted by nonlinear regression using GraphPad Prism
version 7.02 (GraphPad Software, La Jolla, CA).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00003.
■
sı
Experimental details for the synthesis of 8a−s (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Lennart Bunch − Department of Drug Design and
Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen 2100, Denmark;
orcid.org/0000-0002-0180-4639; Email: lebu@
sund.ku.dk
Ligand binding affinities at recombinant rat homomeric GluA1,
GluA2, and GluK1−3 were determined as previously detailed.58,59
Ligands were diluted in assay buffer (GluA1−2: 50 mM Tris-HCl, 0.1
M KSCN, 2.5 mM CaCl2 pH 7.2 at 4 °C; GluK1−3: 50 mM Tris-HCl
pH 7.1 at 4 °C), mixed with sf 9 insect cell membranes expressing the
respective receptors and radioligand (GluA1−2: [3H]-(S)-AMPA
(55.7 Ci/mmol; PerkinElmer), GluK1: [3H]-(S)-NF608 (16.3 Ci/
mmol) (Alcaide et al.58), and GluK2 and GluK3: [isopropenyl-3H]-
kainic acid (43.6−47.2 Ci/mmol; PerkinElmer) in a total volume of
0.25 mL followed by 2 h of equilibration at 4 °C. GluA1−2
membranes were filtered through 24 mm GF/C-type glass fiber filters
on a 12-well Millipore manifold and washed twice with ice-cold assay
buffer. Filters were added to pony vials with 3 mL of UltimaGold
scintillation fluid, and DPM was determined with a TriCarb 2900
scintillation counter (PerkinElmer). GluK1−3 membranes were
filtered through GF/B-type glass fiber filters in microtiter plate
format (UniFilter-96, PerkinElmer) and washed twice with ice-cold
assay buffer on a FilterMate manifold (PerkinElmer). The filters were
dried at 70 °C for 1 h, and 50 μL/well Microscint 20 (PerkinElmer)
was added. Radioactivity was detected with a TopCounter
(PerkinElmer). All competition curves (n ≥ 3 per ligand) were
determined in triplicate at 12−16 ligand concentrations. Data were
analyzed using GraphPad Prism 6 to determine ligand affinity (Ki)
and Hill coefficient (nH) using the one-site Ki and four-parameter
logistics equations, respectively.
Two-Electrode Voltage-Clamp Electrophysiology. DNA con-
structs with rat cDNAs encoding GluN1-1a (Genbank accession
number U11418 and U08261; hereafter GluN1), GluN2A (D13211),
GluN2B (U11419), GluN2C (M91563), and GluN2D (L31611)
were linearized using restriction enzymes and used as templates to
synthesize cRNA using the mMessage mMachine kit (Ambion, Life
Technologies, Paisley, UK) for expression in Xenopus oocytes. The
cDNA encoding rat GluN2B was modified without changing the
amino acid sequence to remove a T7 RNA polymerase termination
site located in the C-terminal domain.60 Xenopus oocytes obtained
from Rob Weymouth (Xenopus 1, Dexter, MI) were prepared as
previously described61 and injected with cRNAs encoding GluN1 and
GluN2 in a 1:2 ratio. The oocytes were maintained at 18 °C in Barth’s
solution containing (in mM) 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.82
MgSO4, 0.33 Ca(NO3)2, 0.91 CaCl2, 10 HEPES (pH 7.5 with
NaOH) supplemented with 100 IU/mL penicillin, 100 μg/mL
streptomycin, and 50 μg/mL gentamycin (Invitrogen, Life Tech-
nologies, Paisley, UK). Two-electrode voltage-clamp recordings were
performed at room temperature on Xenopus oocytes 2−6 days
postinjection with the extracellular recording solution being
composed of (in mM) 90 NaCl, 1 KCl, 10 HEPES, 0.5 BaCl2, and
0.01 EDTA (pH 7.4 with NaOH) at a holding potential of −40 mV
essentially as previously described.61 NMDA receptor ligands were
dissolved in extracellular recording solution and applied to the oocyte
by gravity-driven perfusion. Concentration−inhibition data were fitted
to the Hill equation to obtain IC50 values for individual oocytes using
GraphPad Prism software (GraphPad Software, San Diego, CA) as
previously described.61
Authors
Silke Kayser − Department of Drug Design and Pharmacology,
Faculty of Health and Medical Sciences, University of
Copenhagen, Copenhagen 2100, Denmark
Jacob C. Hansen − Department of Drug Design and
Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen 2100, Denmark
Markus Staudt − Department of Drug Design and
Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen 2100, Denmark
Aleksandra Moroz − Department of Drug Design and
Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen 2100, Denmark
Younes Larsen − Department of Drug Design and
Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen 2100, Denmark
Piero Temperini − Department of Drug Design and
Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen 2100, Denmark;
orcid.org/0000-0003-4951-6879
Feng Yi − Department of Biomedical and Pharmaceutical
Sciences, Center for Structural and Functional Neuroscience, and
Center for Biomolecular Structure and Dynamics, University of
Montana, Missoula, Montana 59812, United States
Jed T. Syrenne − Department of Biomedical and
Pharmaceutical Sciences, Center for Structural and Functional
Neuroscience, and Center for Biomolecular Structure and
Dynamics, University of Montana, Missoula, Montana 59812,
United States
Niels Krogsgaard-Larsen − Department of Drug Design and
Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen 2100, Denmark
Stylianos Iliadis − Department of Drug Design and
Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen 2100, Denmark
Birgitte Nielsen − Department of Drug Design and
Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen 2100, Denmark
Kasper B. Hansen − Department of Biomedical and
Pharmaceutical Sciences, Center for Structural and Functional
Neuroscience, and Center for Biomolecular Structure and
Dynamics, University of Montana, Missoula, Montana 59812,
United States; orcid.org/0000-0002-3303-4819
Darryl S. Pickering − Department of Drug Design and
Pharmacology, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen 2100, Denmark;
orcid.org/0000-0001-8687-6094
Complete contact information is available at:
Y
https://dx.doi.org/10.1021/acschemneuro.0c00003
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
Yoneda, Y. (2009) Possible Protection by Notoginsenoside R1 against
Glutamate Neurotoxicity Mediated by N-Methyl-D-Aspartate Re-
ceptors Composed of an NR1/NR2B Subunit Assembly. J. Neurosci.
Res. 87 (9), 2145−2156.
https://pubs.acs.org/10.1021/acschemneuro.0c00003
Author Contributions
S.K.: Medicinal chemistry and manuscript preparation. J.C.H.:
Medicinal chemistry. M.S.: Medicinal chemistry. A.M.:
Medicinal chemistry. Y.L.: Pharmacology. P.T.: Pharmacology.
F.Yi.: Pharmacology. J.T.S.: Pharmacology. N.K.-L.: Medicinal
chemistry. S.I.: Medicinal chemistry. B.N.: Pharmacology.
K.B.H.: Pharmacology and manuscript preparation. D.S.P.:
Pharmacology & manuscript preparation. L.B.: Medicinal
chemistry and manuscript preparation.
(14) Kumar, A., Singh, R. L., and Babu, G. N. (2010) Cell Death
Mechanisms in the Early Stages of Acute Glutamate Neurotoxicity.
Neurosci. Res. 66 (3), 271−278.
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Amyloid, Glutamate, NMDA Receptors and Memantine - Searching
for the Connections. Br. J. Pharmacol. 167 (2), 324−352.
(16) Francis, P. T. (2008) Glutamatergic Approaches to the
Treatment of Cognitive and Behavioural Symptoms of Alzheimer’s
Disease. Neurodegener. Dis. 5 (3−4), 241−243.
Funding
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Aspartate (NMDA) Receptor Function and Excitotoxicity in
Huntington’s Disease. Prog. Neurobiol. 81 (5−6), 272−293.
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We would like to thank the Danish Research Council, Health
and Medical Sciences for financial support. This work was
funded by the National Institutes of Health (P20GM103546
and R01NS097536).
Notes
The authors declare no competing financial interest.
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https://dx.doi.org/10.1021/acschemneuro.0c00003
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX