Synthesis and inhibitory activities at mGluRs of 3-alkylated and N-alkylated cyclopentyl-glutamate analogues

Article abstract of DOI:10.1016/j.tet.2013.01.054

The conformationally restricted glutamate analogues, 3-alkyl-1-amino-2- cyclopentene-1,3-dicarboxylates and N-alkylated analogues have been prepared in a regioselective and diastereoselective manner. From the biological studies of the 3-alkylated analogues, compound 13b was found to be the most potent antagonist (IC₅₀ 7.7 μM) at mGluR2. Amongst the N-alkylated analogues, compound 20 was found to be a partial agonist (EC₅₀ 9.5 μM) and as well as an antagonist (IC₅₀ 47 μM) at mGluR2.

Full text of DOI:10.1016/j.tet.2013.01.054

Tetrahedron 69 (2013) 2577e2587
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and inhibitory activities at mGluRs of 3-alkylated and
N-alkylated cyclopentyl-glutamate analogues
,
b
c
c
Alison T. Ung a , Stephen G. Pyne , Franc¸ ois Bischoff , Anne S.J. Lesage ,
*
Brian W. Skelton d y, Allan H. White d
,
a School of Chemistry and Forensic Science, University of Technology Sydney, Broadway, NSW 2007, Australia
b School of Chemistry, University of Wollongong, Wollongong, NSW 2522, Australia
c CNS Discovery Research, Johnson and Johnson Pharmaceutical Research and Development, A Division of Janssen Pharmaceutica N.V., B-2340 Beerse,
Belgium
d School of Chemistry and Biochemistry, University of Western Australia, Crawley, WA 6009, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
The conformationally restricted glutamate analogues, 3-alkyl-1-amino-2-cyclopentene-1,3-
Received 12 September 2012
Received in revised form 28 December 2012
Accepted 21 January 2013
dicarboxylates and N-alkylated analogues have been prepared in
eoselective manner. From the biological studies of the 3-alkylated analogues, compound 13b was found
to be the most potent antagonist (IC50 7.7 M) at mGluR2. Amongst the N-alkylated analogues, com-
a regioselective and diaster-
m
Available online 29 January 2013
pound 20 was found to be a partial agonist (EC50 9.5
m
M) and as well as an antagonist (IC50 47
mM) at
mGluR2.
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Cyclopentyl-glutamate analogues
mGluR2
Agonists
Antagonists
Crystal structures
1. Introduction
(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD]
1, which selectively activates metabotropic glutamate receptors
(mGluRs) over the ionotropic type. (1S,3R)-ACPD 1 however is not
selective for the individual eight mGluR subtypes.5 While its di-
astereomer (1S,3S)-ACPD 2 is more potent and selective at mGluR2
L-Glutamate (Glu) is the principal excitatory amino acid (EAA)
neurotransmitter in the mammalian central nervous system (CNS)
and operates through two main types of glutamate receptors.1
Those which form ligand-gated cation channels (ionotropic gluta-
mate receptors, iGluRs) and those, which are coupled via G-protein
to intercellular enzyme systems, which influence the production of
second messengers (metabotropic glutamate receptors, mGluRs).2
Both iGluRs and mGluRs play crucial roles in the healthy, as well
as the diseased CNS and these receptors are therefore potential
targets for therapeutic intervention.3 Considerable research efforts
have been focused upon the development of selective agonists and
antagonists for iGluRs and mGluRs.4
(EC50 1.2 m
M) than at other subtypes6 (Fig. 1).
We previously reported the synthesis and biological activities of
the conformational restricted cyclopentenyl-glutamate compound
(S)-3, the dehydro-analogue of APCD. It was found to be an agonist
at mGluR5 (EC50 18 mM) and mGluR2 (EC50 45 m
M) receptors.7 We
also reported the synthesis of the 4-phenyl substituted derivative,
rac-4, and its aryl substituted derivatives. Compound rac-4 is a se-
lective antagonist at mGluR2 with modest activity with an IC50
value of 32 m
M.8
A variety of cyclic, conformationally restricted glutamate ana-
logues have been prepared in laboratories around the world but
only a few are highly potent and subtype selective. One such
compound is the conformationally restricted glutamate analogue,
In order to further understand the SAR of the derivatives of rac-
3, we aimed to synthesise its 3-alkylated and N-alkylated de-
rivatives and investigate their biological activities.
The synthesis of the 3-alkylated compounds can be achieved by
regioselective deprotonation at the
g-position of the a,b-un-
saturated ester moiety of protected derivatives of rac-3 by a strong
base to generate the corresponding extended enolate, which un-
* Corresponding author. Tel.: þ61 2 95141881; fax: þ61 2 95141460; e-mail ad-
dress: Alison.Ung@uts.edu.au (A.T. Ung).
y
dergoes regioselective alkylation at the
a-position to give 3-
Current address: Centre for Microscopy, Characterisation and Analysis M313,
alkylated products 5 (Scheme 1).9
University of Western Australia, Crawley, WA 6009, Australia.
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.01.054

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A.T. Ung et al. / Tetrahedron 69 (2013) 2577e2587
Fig. 1. Conformationally restricted glutamate analogues.
Scheme 1. Alkylation of a,b-unsaturated ester.
Scheme 2. Synthesis of racemic 9 and 10. Reagents and conditions: (a) KN(TMS)2,
THF, ꢀ78 ꢁC, RI or RBr; (b) 10% aq HCl, reflux.
Table 1
Isolated yields (%) of compounds 7 and 8
We report here the regioselective and diastereoselective syn-
thesis of 3-alkylated and N-alkylated derivatives of (S)-3. The bi-
ological activities of these compounds as agonists and antagonists
at mGluR2 will also be discussed.
Compounds
7 (%)
8 (%)
a (R¼Me)
48
45
77
36
6
6
11
1.6
b (R¼CH2CHCH2)
c (R¼Bn)
d [R¼(CH2)3Ph]
2. Results and discussion
2.1. Synthesis of compounds
analysis (Figs. 3e5).11 Suitable crystals for X-ray analysis of com-
pounds 8a, 8b and 7d were grown from a mixture of ethyl acetate
and hexane.
The stereoselectivity of compound 7 over 8 can be explained, via
the complexation of the carboxyl groups to the potassium ion. As
shown in intermediate A, addition of the alkyl group from the
upper face to intermediate A, would then give compound 7 as the
major product as shown in Fig. 6.
2.1.1. Cyclopentenyl derivatives. Racemates 9aed and their di-
astereoisomers 10aed were obtained, according to Scheme 2.
Treating the known racemate 67 with 2 equiv of KN(TMS)2 at
ꢀ78 ꢁC, followed by the addition of alkylating agents gave exclu-
sively the a-alkylated products 7 and 8 as the major and minor
products, respectively. These outcomes concurred with the works
reported by Katzenellenbogen et al., on the a-alkylation of lithium
dienolates derived from carbonyl esters.10 The ratio of the isolated
diastereoisomers 7 and 8 was determined by HPLC analysis, to be
within the range of 8:2 (Table 1).
Acid hydrolysis of 7aed and 8aed under similar conditions gave
the hydrochloride salts of 9aed and 10aed, respectively, in ac-
ceptable yields (Table 2).
1H NMR spectroscopic analysis of the two diastereomers, such
as 7a and 8a indicated that the major diastereomer 7a exhibited an
2.1.2. Cyclopentylderivatives. The racemic cyclopentyl derivatives,
13 and 14, were synthesised, according to Scheme 3. Removal of
diphenyl methylene group from 7 and 8 by a mild acid hydrolysis
followed by hydrogenation proceeded smoothly to give 11 and 12,
respectively, in good yields.
H-4 resonance (
J¼4.2 Hz). The CH3-3 resonance at
appeared more downfield than the corresponding resonance (
d
5.89, d, J¼4.2 Hz) and H-5 resonance (
d 5.71, d,
1.49 of the major isomer 7a
d
d
1.32) of the minor isomer 8a. The relative stereochemistry of 7a was
determined from NOESY experiments that showed significant
cross-peaks between the signal for He(Ar) and H-2a, H-2a and
Acid hydrolysis of 11aec and 12c gave the hydrochloride salts of
13aec and 14c, respectively (Table 3).
He(CH3-3) and that for He(Ar) and He(CH3-3). These cross-peaks
are consistent with the cis orientation of the two carboxyl groups
(Fig. 2). The relative stereochemistries of the compounds, 8a, 8b
and 7c were further unequivocally determined by X-ray structure
2.1.3. N,N-Dimethylated analogues. The synthesis of N-alkylated
compound 16 is shown in Scheme 4. Treatment of racemic 158 with
formaldehyde, NaBH3CN, acetic acid in dry methanol afforded 16 in

A.T. Ung et al. / Tetrahedron 69 (2013) 2577e2587
2579
Fig. 4. ORTEP diagram of the structure of compound 8b. Ellipsoids have been drawn at
the 30% probability level. Minor components of disordered atoms were omitted for
clarity.
Fig. 2. NOESY correlations for compound 7a.
Fig. 3. ORTEP diagram of the structure of compound 8a. Ellipsoids have been drawn at
the 20% probability level.
Fig. 5. ORTEP diagram of the structure of compound 7c. Ellipsoids have been drawn at
the 30% probability level.
70% yield, which was hydrolysed in aq 10% HCl to give the hydro-
chloride salt of 17 in 98% yield.
With compound 16 in hand, it was further reduced using Mg
turnings in dry methanol to give a mixture of diastereomers, cis 18
and the trans 19, as the major and minor diastereoisomers, re-
spectively, in the ratio of 4:1, respectively. The major di-
astereoisomer 18 was hydrolysed to afford 20 in a quantitative
yield.
1H NMR spectroscopic analysis of the two diastereomers 18 and
19 indicated that the major diastereomer 18 exhibited an H-3 res-
onance at
resonance (
d
2.92 (dq) more downfield than the corresponding
2.84, dq) of the minor isomer 19.
d
Fig. 6. Suggested mechanism leading to the stereochemistry of compound 7.

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A.T. Ung et al. / Tetrahedron 69 (2013) 2577e2587
Table 2
The relative stereochemistry of compound 18 was determined
by NOESY experiments that showed significant cross-peaks be-
tween the signal for H-3 and H-2 and that for H-2 and He(CH3).
Isolated yields (%) of compounds 9 and 10
a
a
a
Compounds
9 (%)
10 (%)
These confirmed the cis orientation of the two carboxyl groups
(Fig. 7).
a (R¼Me)
86
100
94
80
100
98
b (R¼CH2CHCH2)
c (R¼Bn)
d [R¼(CH2)3Ph]
63
76
Scheme 3. Synthesis of racemic 13 and 14. Reagents and conditions: (a) (i) 1 M HCl,
ether, rt, 16 h, (ii) Pd/C, H2, MeOH, rt; (b) 10% aq HCl, reflux.
Fig. 7. NOESY correlations for compound 18.
Table 3
Isolated yields (%) of compounds 11, 12, 13 and 14
Reduction of a,b-unsaturated esters and other electron deficient
alkenes using magnesium metal in dry methanol has been de-
scribed by us and others to be a selective and efficient method to
provide the desired cis 18 as the major product.8,12e14 The cis ori-
entation of the two carboxylic groups in 18 is considered to be
a significant structural feature for activities.6 The selectivity ob-
served in the magnesiumemethanol reduction of compound 16
might be due to the formation of the Mg(II) complex B, as shown in
Fig. 8, which upon stereoselective protonation would give com-
pound 18 as the major product.
Compounds
11
12
naa
93
13
14
a (R¼Me)
b (R¼Pr)
c (R¼Bn)
83
94
66
85
99
86
naa
naa
85
70
a
na: not attempted.
Fig. 8. Suggested mechanism leading to the stereochemistry of compound 18.
2.1.4. N-Alkylated analogues. The racemic N-alkylated compounds
22aee were obtained according to Scheme 5, the imine in-
termediates 21aee were obtained by treating the known 158 with
various aldehydes in dry THF in the presence of powdered molec-
ular sieves, then followed by the reduction in the presence of
NaBH3CN and acetic acid, in methanol to afford 21aee in acceptable
yields. Acid hydrolysis of 21aee provided the hydrochloride salts of
compounds 22aee in high yields (Table 4).
Scheme 4. Synthesis of racemic 17 and 20. Reagents and conditions: (a) HCHO,
NaBH3CN, acetic acid, CH3CN; (b) 10% aq HCl, reflux; (c) Mg turnings, dry CH3OH, rt.

A.T. Ung et al. / Tetrahedron 69 (2013) 2577e2587
2581
Only one compound in this series, namely 22c, was found to be
an inhibitor at the glycine site of the NMDA receptor with an IC50 of
1.9 mM (data is not shown), as measured in rat cortical membranes
by radioligand binding.
The other synthesised compounds in this series were found to
be inactive on the tested receptors.
By comparing the activities of the saturated and unsaturated
compound pairs, such as9band 13b, itwould appear thatreduction of
the double bond results in an improvement in the antagonist potency.
Compound 9b is not active in tested mGluR receptors, while 13b is
a selective antagonist at mGluR2. Within the structures of the satu-
rated active analogues, they were found to have the carboxylic groups
in the cis configuration. This was observed in the compound pair 17
and 20. The unsaturated analogue 17 showed no activity in the
Scheme 5. Synthesis of racemic 22. Reagents and conditions: (a) (i) RCHO, THF,
powdered molecular sieves (5 A), rt, (ii) NaBH3CN, acetic acid, MeOH; (b) 10% aq HCl,
reflux.
ꢀ
Table 4
Isolated yields (%) of compounds 21 and 22
Compounds
21 (%)
22 (%)
mGluR2 [35S]GTP
gS binding assay. Compound 20 was the most active
a (R¼Ph)
80
80
58
81
69
98
100
98
92
95
b (R¼2-pyridinyl)
c (R¼3-pyridinyl)
d [R¼3-(OH)C6H4]
e [R¼4-(OH)C6H4]
agonist as well as a partial antagonist. This structural feature was also
found important in rac-4 and its active analogues and thewell-known
(1S,3S)-ACPA.6 In summary, our results reveal that the ring flexibility
and the cis orientation of the two carboxylic groups appear to be
important structural features for activities at the mGluR2 receptor.
2.2. Biological studies: signal transduction at cloned metab-
otropic glutamate receptors
3. Conclusions
In conclusion, through this undertaking we have successfully
prepared 3-alkylated and N-alkylated derivatives of rac-3 in
a regioselective and diastereoselective manner. In this series of
compounds, we have discovered a number of novel mGluR2 ago-
Signal transduction experiments were performed with CHO
cells heterologously expressing human mGlu2 receptors. Signalling
at the mGlu2 receptor was measured by means of a [35S]GTP
gS
binding assay on membranes from these cells.5 The results for the
agonist and antagonist activities are summarised in Table 5.
nists and antagonists with potency in the low mM range. Although,
the conclusive structural activity relationship remains complex, we
have however gained a clearer understanding of some of the key
structure features required for a selective antagonist at mGluR2.
Table 5
Agonist and antagonist activities of synthesised compounds
Compounds
mGlu2-ago-AC,
mGlu2-ago-GTP,
mGlu2-antag-GTP,
EC50ꢃSD in
EC50ꢃSD in
IC50ꢃSD in
4. Experimental
4.1. Chemistry
m
M (Emax%)
m
M (Emax%)
mM (Emax%)
rac-3
(S)-3
73
55ꢃ14 (71%)a
45ꢃ10 (38%)a
Partial agoc
>1000
>300
ndb
60 (39%)
Partial antac
7.7ꢃ2.1 (71%)
200 (67%)
>100
47 (66%)
75ꢃ11 (100%)
>100
rac-9a
rac-13b
rac-14c
rac-17
rac-20
rac-22b
rac-22c
387 (53%)
Solvents and reagents were purchased from commercial sources
and used without further purification unless otherwise stated.
Unless specified, all NMR spectra were recorded at 300 MHz (1H
NMR) or 75 MHz (13C NMR) in CDCl3 solution. 13C NMR assignments
were based on DEPT experiments. Preparative HPLC was performed
using a Waters Delta prep 4000 HPLC, on a normal phase Pre Nova-
ndb
ndb
ndb
>100
3(51%)
ndb
9.7ꢃ0.5 (34%)
ndb
>100
ndb
>100
a
PakÒ HR Silica 6
m
m 60 (25ꢂ10 mm) column. All separations were
Values from Ref. 7.
nd: not determined.
No full dose response.
b
c
carried out by isocratic elution, using eluents A and B (A, in pe-
troleum spirit; B, ethyl acetate) in the ratio 98:2. The flow rate was
at 20 mL/min. Compounds were detected using a Waters 486
tunable UV absorbance detector.
Three compounds, namely 9a, 17 and 20 were found to be
mGlu2 agonists. Compound 17 was shown to exhibit mGluR2
agonism in the adenyl cyclase assay, with the potency of 3 M with
an Emax of 51% of the maximal glutamate response. However, the
agonist activity of 17 was not confirmed in the [35S]GTP
S binding
experiment, where this compound was expected to activate radi-
oligand binding. Compound 17 was also shown to have inhibitory
activity at the AMPA receptor in a glycine binding assay with an IC50
m
4.2. Biological testing
g
[ gS (specific activity 37 MBq/mL) was obtained from
35S]GTP
Amersham (Little Chalfort, UK). Dulbecco’s modified Eagle medium
(DMEM) and dialysed foetal calf serum were from Life technologies
(Gaithersburg, MD). Scintillation fluid Ultimaflo AF as well as the
Unifilter-96 GF/B plates were from Packard (Meriden, CT). Guanosine-
50-diphosphate dilithium salt (GDP) was from Boehringer Manheim
(Basel, Switzerland), Fluo 3-AM was from Molecular Probes (Leiden,
The Netherlands). Probenecid was from Sigma (St. Louis, MO). Black
96-well plates were from Costar (Merck, Overijse Belgium).
value of 6.8
The most active agonist in this series was 20 with a potency of
EC50 9.5 M and an Emax of 34% of the maximal glutamate response.
This compound also had inherent partial antagonist activity with
an IC50 of 47 M. The concentrationeresponse curves illustrated
mM (data is not shown).
m
m
that compound 20 is a partial agonist and as well as antagonist at
mGluR2 receptors (Fig. 9).
Full antagonistic activities were found amongst the saturated
(13b and 20), as well as unsaturated (22b) ring structures. The most
*
*
4.3. (1R ,3R )-3-Ethyl 1-methyl 1-(diphenylmethyleneamino)-
3-methylcyclopent-4-ene-1,3-dicarboxylate (7a)
potent antagonist was 13b with an IC50 of 7.7
trationeresponse curve revealed that 13b was an antagonist with
full inhibition of glutamate signalling in the [35S]GTP
S binding
assay, obtained from two independent experiments (Fig. 10).
mM. The concen-
To a solution of compound 6 (0.552 g, 1.46 mmol) in dry THF
(12 mL) at ꢀ78 ꢁC was added a solution of KN(TMS)2 (2.5 equiv,
7.3 mL, 3.66 mmol, 0.5 M in THF). After stirring for 1 h, a solution of
g

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A.T. Ung et al. / Tetrahedron 69 (2013) 2577e2587
Fig. 9. Effect of 20 on human mGluR2: partial agonist and antagonist activity in [35S]GTP
gS biding.
Fig. 10. Antagonist activity of 13b and antagonist activity of 22b at mGluR2.
methyl iodide (10 equiv, 0.9 mL, 14.6 mmol) in dry THF (2 mL) was
added drop wise to the mixture and the stirring continued for
another 1 h. The reaction mixture was allowed to warm to rt over
1 h then was diluted with a saturated aq solution of NaCl, and then
extracted with ether. The combined organic extracts were dried
over MgSO4 and the solvent was removed to give a dark yellow
residue, which was purified by column chromatography (ethyl ac-
etate/petroleum spirit, 1:9) to give a mixture of diastereomers
(54%) in the ratio of 8:1 as determined by 1H NMR spectroscopic
analysis. The two diastereomers were separated by HPLC (ethyl
acetate/petroleum spirit, 5:95).
4.4. (1R
*
,3S )-3-Ethyl 1-methyl 1-(diphenylmethyleneamino)-
*
3-methylcyclopent-4-ene-1,3-dicarboxylate (8a)
Minor isomer 8a, a yellow solid (34 mg, 6%). Rf (5% ethyl acetate/
petroleum spirit) 0.52. 1H NMR
d 7.60e7.12 (m, 10H), 5.90 (d,
J¼5.4 Hz, 1H), 5.71 (d, J¼5.4 Hz, 1H), 4.12e4.06 (m, 2H), 3.42 (s, 3H),
2.82 (d, J¼13.8 Hz, 1H), 2.50 (d, J¼13.8 Hz, 1H), 1.32 (s, 3H), 1.26 (t,
J¼6.9 Hz, 3H). 13C NMR
d 176.1 (C), 173.6 (C), 168.2 (C), 140.3 (C),
137.6 (C), 133.9 (CH), 130.2 (CH), 128.8 (2CH), 128.7 (2CH), 128.4
(4CH), 127.9 (2CH), 78.5 (C), 60.8 (CH3), 55.3 (C), 51.9 (CH2), 47.9
(CH2), 25.4 (CH3), 14.2 (CH3). MS (ESþ) m/z 392 ([MþH], 85%).
HRESIMS m/z 392.1815 (MHþ), calcd for C24H26NO4 (MHþ) 392.1817.
Major isomer 7a, a yellow solid (0.274 g, 48%). Rf (5% ethyl ac-
etate/petroleum spirit) 0.28. 1H NMR
d 7.60e7.12 (m, 10H), 5.89 (d,
J¼4.2 Hz, 1H), 5.71 (d, J¼4.2 Hz, 1H), 4.13e4.07 (m, 2H), 3.38 (s, 3H),
4.5. (1R
*
,3R )-1-Ethyl 3-methyl 1-allyl-3-(diphenylmethyle-
*
3.03 (d, J¼9.9 Hz, 1H), 2.31 (d, J¼9.9 Hz, 1H), 1.49 (s, 3H), 1.22 (t,
neamino)cyclopent-4-ene-1,3-dicarboxylate (7b)
J¼7.2 Hz, 3H). 13C NMR
d 175.8 (C), 173.2 (C), 168.3 (C), 140.3 (C),
137.7 (C), 133.5 (CH), 130.2 (CH), 128.7 (2CH), 128.6 (2CH), 128.4
(4CH), 127.9 (2CH), 78.3 (C), 60.7 (CH3), 55.2 (C), 51.7 (CH2), 49.3
(CH2), 26.1 (CH3), 14.1 (CH3). MS (ESþ) m/z 392 ([MHþ], 80%).
HRESIMS m/z 392.1817 (MHþ), calcd for C24H26NO4 (MHþ) 392.1817.
Major isomer 7b (0.274 g, 45%) yellow solid. Rf (5% ethyl acetate/
petroleum spirit) 0.36. 1H NMR
d 7.57e7.12 (m, 10H), 5.94 (d,
J¼5.4 Hz, 1H), 5.76 (d, J¼5.4 Hz, 1H), 5.82e5.68 (m, 1H), 5.76 (d,
J¼5.4 Hz, 1H), 5.14e5.10 (m, 1H), 5.09e5.07 (m, 1H) 4.15e4.05 (m,

A.T. Ung et al. / Tetrahedron 69 (2013) 2577e2587
2583
2H), 3.39 (s, 3H), 2.96 (d, J¼13.7 Hz, 1H), 2.67e2.52 (m, 2H), 2.40 (d,
4.7.3. (1R
*
,3R )-3-Ethyl 1-methyl 1-(diphenylmethyleneamino)-3-(3-
*
J¼13.7 Hz, 1H), 1.22 (t, J¼6.9 Hz, 3H). 13C NMR
d
174.4 (C), 173.1 (C),
phenylpropyl)cyclopent-4-ene-1,3-dicarboxylate (7d). Major isomer
7d, a yellow oil (0.260 g, 36%). Rf (5% ethyl acetate/petroleum
168.2 (C),140.2 (C),137.4 (C),136.0 (CH),134.2 (CH),133.7 (CH),132.3
(C), 130.2 (CH), 129.9 (CH),128.7 (CH), 128.5 (CH), 128.3 (2CH),128.2
(CH), 127.9 (2CH), 118.1 (CH2), 78.1 (C), 60.6 (CH2), 59.3 (C), 51.7
(CH3), 46.8 (CH2), 43.9 (CH2),14.1 (CH3). MS (ESþ) m/z 418.19 ([MHþ],
100.0%). HRESIMS m/z 418.2014 [MH]þ, calcd for C26H28NO4 (MHþ)
418.2012.
spirit) 0.36. 1H NMR
d
7.58e7.09 (m, 15H), 5.93 (d, J¼5.4 Hz, 1H),
5.71 (d, J¼5.4 Hz, 1H), 4.11e4.05 (m, 2H), 3.39 (s, 3H), 3.01 (d,
J¼13.6 Hz, 1H), 2.68e2.56 (m, 2H), 2.33 (d, J¼13.6 Hz, 1H),
1.94e1.79 (m, 2H), 1.68e1.60 (m, 2H), 1.20 (t, J¼6.8 Hz, 3H). 13C
NMR
d 174.8 (C), 173.0 (C), 168.1 (C), 141.7 (C), 140.0 (C), 137.2 (C),
136.1 (CH), 133.7 (CH), 130.1 (CH), 128.6 (CH), 128.4 (2CH), 128.2
(2CH), 128.1 (4CH), 127.8 (2CH), 127.7 (2CH), 125.6 (CH), 77.8 (C),
60.5 (CH2), 59.2 (C), 51.6 (CH3), 47.4 (CH2), 39.1 (CH2), 35.9 (CH2),
27.0 (CH2), 13.9 (CH3). MS (ESþ) m/z 496.0 ([MHþ], 100.0%).
HRESIMS m/z 496.2488 (MHþ), calcd for C32H34NO4 (MHþ)
496.2493.
4.6. (1R
*
,3R )-1-Ethyl 3-methyl 1-allyl-3-(diphenylmethyle-
*
neamino)cyclopent-4-ene-1,3-dicarboxylate (8b)
Minor isomer 8b, a yellow solid (36.6 mg, 6%). Rf (5% ethyl ac-
etate/petroleum spirit) 0.54. 1H NMR
d 7.57e7.12 (m, 10H), 5.93 (d,
J¼5.6 Hz, 1H), 5.77 (d, J¼5.6 Hz, 1H), 5.73e5.62 (m, 1H), 5.06e5.04
(br d, J¼5.6 Hz, 1H), 5.02 (br s, 1H), 4.21e4.09 (m, 2H), 3.43 (s, 3H),
2.81 (d, J¼14.4 Hz,1H), 2.58 (d, J¼14.4 Hz,1H) 2.42 (d, J¼5.6 Hz, 2H),
4.7.4. (1R
*
,3S )-3-Ethyl 1-methyl 1-(diphenylmethyleneamino)-3-(3-
*
phenylpropyl)cyclopent-4-ene-1,3-dicarboxylate (8d). Minor isomer
8d, a yellow oil (11.6 mg,1.6%). Rf (5% ethyl acetate/petroleum spirit)
1.25 (t, J¼6.8 Hz, 3H). 13C NMR
d 174.7 (C), 173.5 (C), 168.1 (C), 140.2
(C), 137.5 (C), 136.1 (CH), 134.5 (CH), 133.6 (CH), 130.2 (CH), 128.8
(CH), 128.6 (CH), 128.3 (2CH), 127.9 (3CH), 118.1 (CH2), 78.1 (C), 60.8
(CH2), 59.4 (C), 51.9 (CH3), 45.8 (CH2), 42.2 (CH2), 14.3 (CH3). MS
(ESþ) m/z 418.19 ([MHþ], 100.0%). HRESIMS m/z 418.2013 (MHþ),
calcd for C26H28NO4 (MHþ) 418.2012.
0.54. 1H NMR
d
7.56e7.11 (m, 15H), 5.93 (d, J¼5.4 Hz, 1H), 5.73 (d,
J¼5.4 Hz, 1H), 4.18e4.07 (m, 2H), 3.39 (s, 3H), 2.83 (d, J¼13.5 Hz,
1H), 2.57e2.54 (m, 2H), 2.52 (d, J¼13.5 Hz, 1H), 1.76e1.67 (m, 2H),
1.61e1.51 (m, 2H), 1.22 (t, J¼6.9 Hz, 3H). 13C NMR
d 175.2 (C), 173.5
(C), 168.0 (C), 142.0 (C), 140.2 (C), 137.5 (C), 136.4 (CH), 134.0 (CH),
130.2 (CH), 128.7 (CH), 128.6 (2CH), 128.3 (4CH), 128.2 (2CH), 127.8
(4CH), 125.7 (CH), 78.1 (C), 60.6 (CH2), 59.4 (C), 51.8 (CH3), 46.6
(CH2), 38.7 (CH2), 35.9 (CH2), 27.2 (CH2), 14.2 (CH3). MS (ESþ) m/z
496.0 ([MHþ], 100.0%). HRESIMS m/z 496.2488 (MHþ), calcd for
C32H34NO4 (MHþ) 496.2493.
4.7. General procedure for alkylation of 6
To a solution of compound 6 (0.552 g, 1.46 mmol) in dry THF
(12 mL) at ꢀ78 ꢁC was added a solution of KN(TMS)2 (2.5 equiv,
7.3 mL, 3.66 mmol, 0.5 M in THF). After stirring for 1 h, a solution of
alkyl bromide (2.5 equiv, 3.7 mmol) in dry THF (2 mL) was added
drop wise to the mixture and the stirring continued for another 1 h.
The reaction mixture was allowed to warm to rt over 1 h then was
diluted with a saturated aq solution of NaCl, and then extracted with
ether. The combined organic extracts were dried over MgSO4 and
the solvent was removed to give a dark yellow residue, which was
purified bycolumn chromatography (ethyl acetate/petroleum spirit,
1:9) to give a mixture of diastereomers. The two diastereomers were
separated by HPLC (ethyl acetate/petroleum spirit, 5:95).
4.8. General hydrolysis procedure for 7 and 8
Compound 7a (0.270 g, 0.70 mmol) in 10% aq HCl (3 mL) was
heated at 80 ꢁC for 16 h. After cooling, the reaction mixture was
diluted with water (2 mL) and washed with ether (5 mL). The water
was removed under reduced pressure to give the HCl salt of 9a as
a white solid (0.133 g, 86%).
4.8.1. (1R
*
,3R
*
)-1-Amino-3-methylcyclopent-4-ene-1,3-dicarboxylic
6.14 (d,
acid (9a). A white solid (0.133 g, 86%). 1H NMR (D2O)
d
4.7.1. (1R
*
,3R
*
)-1-Ethyl 3-methyl 1-benzyl-3-(diphenylmethylenea-
J¼5.4 Hz, 1H), 5.65 (d, J¼5.4 Hz, 1H), 3.01 (d, J¼14.7 Hz, 1H), 1.86 (d,
mino)cyclopent-4-ene-1,3-dicarboxylate (7c). Major isomer 7c,
a yellow solid (0.525 g, 77%). Rf (5% ethyl acetate/petroleum spirit)
J¼14.7 Hz, 1H), 1.30 (s, 3H). 13C NMR (D2O)
d 176.2 (C), 169.9 (C),
141.1 (CH), 124.0 (CH), 66.3 (C), 53.8 (C), 40.9 (CH2), 22.0 (CH3). MS
(ESþ) m/z 186.0 ([MHþ], 60%). HRESIMS m/z 186.0766 (MHþ) calcd
for C8H12NO4 (MHþ) 186.0761.
0.35. 1H NMR
d 7.62e7.59 (m, 2H), 7.43e7.12 (m, 13H), 5.93 (d,
J¼5.7 Hz, 1H), 5.75 (d, J¼5.7 Hz, 1H), 4.02 (q, J¼7.2 Hz, 2H), 3.38 (s,
3H), 3.28 (d, J¼13.2 Hz, 1H), 3.13 (d, J¼13.2 Hz, 1H), 2.91 (d,
J¼13.5 Hz, 1H), 2.56 (d, J¼13.5 Hz, 1H), 1.14 (t, J¼7.2 Hz, 3H). 13C
4.8.2. (1R
*
,3S
*
)-1-Amino-3-methylcyclopent-4-ene-1,3-dicarboxylic
6.18 (d,
NMR
d
174.2 (C), 173.8 (C), 168.2 (C), 144.1 (C), 140.0 (C), 137.4 (C),
acid (10a). A white solid (0.124 g, 80%). 1H NMR (D2O)
d
136.2 (CH), 133.8 (CH), 130.2 (2CH), 129.7 (2CH), 128.7 (4CH), 128.5
(2CH), 128.2 (2CH), 128.0 (CH), 127.9 (CH), 126.5 (CH), 77.9 (C), 60.7
(CH2), 60.5 (C), 51.7 (CH3), 47.4 (CH2), 45.6 (CH2), 13.9 (CH3). MS
(ESþ) m/z 468.0 ([MþH], 100.0%). Anal. Calcd for C30H29NO4: C,
77.06; H, 6.25; N, 3.00. Found: C, 77.31; H, 6.38; N, 2.75.
J¼5.4 Hz, 1H), 5.72 (d, J¼5.4 Hz, 1H), 2.59 (d, J¼15.3 Hz, 1H), 2.35 (d,
J¼15.3 Hz, 1H), 1.32 (s, 3H). 13C NMR (D2O)
d 176.2 (C), 169.9 (C),
141.1 (CH), 124.0 (CH), 66.5 (C), 53.8 (C), 40.9 (CH2), 22.0 (CH3). MS
(ESþ) m/z 186 ([MHþ], 60%). HRESIMS m/z 186.0763 (MHþ), calcd
for C8H12NO4 (MHþ) 186.0761.
4.7.2. (1R
*
,3S
*
)-1-Ethyl 3-methyl 1-benzyl-3-(diphenylmethylenea-
4.8.3. (1R
*
,3R
*
)-1-Allyl-3-aminocyclopent-4-ene-1,3-dicarboxylic
6.31
mino)cyclopent-4-ene-1,3-dicarboxylate (8c). Minor isomer 8c,
a yellow solid (75.0 mg, 11%). Rf (5% ethyl acetate/petroleum spirit)
acid (9b). A pale yellow solid (0.170 g, 100%). 1H NMR (D2O)
d
(d, J¼5.7 Hz, 1H), 5.80 (d, J¼5.7 Hz, 1H), 5.78e5.64 (m, 1H), 5.12 (br
0.65. 1H NMR
d 7.82e7.80 (m, 2H), 7.60e7.08 (m, 13H), 5.92 (d,
d, J¼4.8 Hz, 1H), 5.08 (br s, 1H), 3.03 (d, J¼15.0 Hz, 1H), 2.49 (br d,
J¼5.4 Hz, 1H), 5.80 (d, J¼5.4 Hz, 1H), 4.10e4.02 (m, 2H), 3.45 (s, 3H),
J¼4.8 Hz, 2H), 2.04 (d, J¼15.0 Hz, 1H). 13C NMR (D2O)
d 174.7 (C),
3.06 (d, J¼13.2 Hz, 1H), 2.96 (d, J¼13.2 Hz, 1H), 2.74 (s, 2H), 1.14 (t,
169.3 (C), 139.1 (CH), 129.4 (CH), 124.5 (CH), 116.4 (CH2), 66.0 (C),
57.5 (C), 39.6 (CH2), 38.4 (CH2). MS (ESþ) m/z 212 (MþH). HRESIMS
m/z 212.0919 (MHþ), calcd for C10H14NO4 (MHþ) 212.0917.
J¼7.2 Hz, 3H). 13C NMR
d 174.1 (C), 173.6 (C), 168.3 (C), 144.3 (C),
139.8 (C), 137.3 (C), 136.2 (CH), 133.6 (CH), 130.1 (2CH), 129.4 (2CH),
129.0 (4CH), 128.8 (2CH), 128.3 (2CH), 128.1 (CH), 127.5 (CH), 126.1
(CH), 77.6 (C), 61.0 (C), 60.3 (CH2), 52.0 (CH3), 48.3 (CH2), 45.8 (CH2),
14.0 (CH3). MS (ESþ) m/z 468.0 ([MHþ], 100.0%). Anal. Calcd for
C30H29NO4: C, 77.06; H, 6.25; N, 3.00. Found: C, 76.97; H, 6.47;
N, 2.81.
4.8.4. (1R
*
,3S
*
)-1-Allyl-3-aminocyclopent-4-ene-1,3-dicarboxylic
6.29
acid (10b). A pale yellow solid (22.1 mg,100%). 1H NMR (D2O)
d
(d, J¼4.3 Hz, 1H), 5,81 (d, J¼4.3 Hz, 1H), 5.74e5.64 (m, 1H),
5.11e5.04 (m, 2H), 2.65e2.58 (m, 1H), 2.55 (dd, J¼15.0 Hz, 1H), 2.52

2584
A.T. Ung et al. / Tetrahedron 69 (2013) 2577e2587
(d, J¼15.0 Hz, 2H), 2.47e2.41 (m, 1H). 13C NMR (D2O)
d
177.6 (C),
2.01 (ddd, J¼4.5, 10.5, 12.0 Hz, 1H), 1.86 (d, J¼15.0 Hz, 1H) 1.54 (ddd,
J¼4.5, 10.5, 12.0 Hz, 1H), 1.39 (s, 3H), 1.19 (t, J¼Hz, 3H). 13C NMR
172.9 (C), 143.5 (CH), 132.8 (CH), 127.9 (CH), 119.0 (CH2), 69.5 (C),
60.4 (C), 41.5 (CH2), 40.4 (CH2). MS (ESþ) m/z 212 ([MHþ], 100%).
HRESIMS m/z 212.0932 (MHþ), calcd for C10H14NO4 (MHþ)
212.0917.
(D2O) d 175.0 (C), 169.5 (C), 61.1 (C), 59.1 (CH2), 52.3 (C), 50.8 (CH3),
42.2 (CH2), 38.8 (CH2), 31.8 (CH2), 26.6 (CH3), 10.62 (CH3). MS (ESþ)
m/z 230.1 (MþH, 100%). HRESIMS m/z 230.1389 (MHþ), calcd for
C11H20NO4 (MHþ) 230.1387.
4.8.5. (1R
acid (9c). A pale yellow solid (43.4 mg, 94%). 1H NMR (D2O)
7.27e7.13 (m, 5H), 6.29 (d, J¼5.7 Hz, 1H), 5.74 (d, J¼5.7 Hz, 1H),
*
,3R**)-1-Amino-3-benzylcyclopent-4-ene-1,3-dicarboxylic
4.9.2. (1S
*
,3S
*
)-3-Ethyl 1-methyl 1-amino-3-propylcyclopentane-
d
1,3-dicarboxylate (11b). A yellow oil (97.5 mg, 94%). 1H NMR (D2O)
3.09 (d, J¼13.5 Hz, 1H), 3.01 (d, J¼14.7 Hz, 1H), 2.98 (d, J¼13.5 Hz,
d
4.14e4.07 (m, 2H), 3.71 (s, 3H), 2.94 (d, J¼15.0 Hz, 1H), 2.48e2.40
1H), 2.11 (d, J¼14.7 Hz, 1H). 13C NMR (D2O)
d
174.5 (C), 169.0 (C),
(m, 1H), 2.31 (ddd, J¼4.5, 10.5, 14.4 Hz, 1H), 2.00 (ddd, J¼4.5, 10.5,
14.4 Hz, 1H), 1.86 (d, J¼15.0 Hz, 1H) 1.79e1.66 (m, 2H), 1.54 (ddd,
J¼4.5, 10.5, 14.4 Hz, 1H), 1.18 (t, J¼6.9 Hz, 3H), 1.13e1.00 (m, 2H),
138.8 (CH), 132.9 (C), 126.7 (2CH), 125.4 (2CH), 124.4 (CH), 124.2
(CH), 66.1 (C), 58.7 (C), 41.2 (CH2), 38.6 (CH2). MS (ESþ) m/z 262
(MþH, 30%). HRESIMS m/z 262.1077 (MHþ), calcd for C14H16NO4
(MHþ) 262.1074.
0.78 (t, J¼7.2 Hz, 3H). 13C NMR (D2O)
d 175.0 (C), 169.5 (C), 61.1 (C),
59.1 (CH2), 52.3 (C), 50.8 (CH3), 42.2 (CH2), 38.8 (CH2), 31.8 (CH2),
30.3 (CH2), 15.8 (CH2), 10.6 (CH3), 10.4 (CH3). MS (ESþ) m/z 258.0
(MþH, 100%). HRESIMS m/z 258.1707 (MHþ), calcd for C13H24NO4
(MHþ) 258.1700.
4.8.6. (1R
*
,3S
*
)-1-Amino-3-benzylcyclopent-4-ene-1,3-dicarboxylic
7.26e7.13
acid (10c). A white solid (29.0 mg, 98%). 1H NMR (D2O)
d
(m, 5H), 6.31 (d, J¼5.7 Hz, 1H), 5.79 (d, J¼5.4 Hz, 1H), 3.18 (d,
J¼13.5 Hz, 1H), 3.03 (d, J¼13.5 Hz, 1H), 2.64 (d, J¼15.0 Hz, 1H), 2.49
4.9.3. (1S
1,3-dicarboxylate (11c). A yellow oil (79.0 mg, 66%). 1H NMR
(CD3OD) 7.29e7.11 (m, 5H), 4.12e4.04 (m, 2H), 3.78 (s, 3H), 3.13 (d,
*
,3R
*
)-3-Ethyl 1-methyl 1-amino-3-benzylcyclopentane-
(d, J¼15.0 Hz, 1H). 13C NMR (D2O)
d 174.5 (C), 169.1 (C), 138.9 (CH),
132.2 (C), 126.6 (2CH), 125.2 (2CH), 124.5 (CH), 124.3 (CH), 66.1 (C),
58.6 (C), 40.9 (CH2), 38.8 (CH2). HRESIMS m/z 262.1075 (MHþ), calcd
for C14H16NO4 (MHþ) 262.1074.
d
J¼13.2 Hz, 1H), 2.99 (d, J¼15.0 Hz, 1H), 2.97 (d, J¼13.2 Hz, 1H),
2.42e2.35 (m, 2H), 2.06e2.01 (m, 3H), 1.19 (t, J¼7.2 Hz, 3H). 13C
NMR (D2O) d 174.3 (C), 169.3 (C), 134.1 (C), 126.5 (2CH), 125.3 (2CH),
4.8.7. (1R
dicarboxylic acid (9d). A white solid (37.3 mg, 63%). 1H NMR (D2O)
7.25e7.15 (m, 5H), 6.25 (d, J¼5.7 Hz, 1H), 5.72 (d, J¼5.7 Hz, 1H),
3.02 (d, J¼14.4 Hz, 1H), 2.57e2.52 (m, 2H), 1.92 (d, J¼14.4 Hz, 1H),
1.78e1.20 (m, 4H). 13C NMR (D2O)
175.1 (C), 169.5 (C), 139.4 (C),
*
,3R
*
)-1-Amino-3-(3-phenylpropyl)cyclopent-4-ene-1,3-
123.9 (CH), 61.5 (C), 59.9 (CH2), 52.7 (CH3), 51.0 (C), 51.0 (CH2), 40.0
(CH2), 32.1 (CH2), 32.0 (CH2), 10.4 (CH3). MS (ESþ) m/z 306 (MþH,
100%). HRESIMS m/z 306.1705 (MHþ), calcd for C17H24NO4 (MHþ)
306.1700.
d
d
139.1 (CH), 125.4 (4CH), 124.2 (CH), 122.9 (CH), 66.0 (C), 57.5 (C),
39.1 (CH2), 35.1 (CH2), 31.7 (CH2), 23.2 (CH2). MS (ESþ) m/z 290
([MHþ], 100%). HRESIMS m/z 290.1389 (MHþ), calcd for C16H20NO4
(MHþ) 290.1387.
4.9.4. (1S
*
,3R
*
)-3-Ethyl 1-methyl 1-amino-3-propylcyclopentane-
1,3-dicarboxylate (12b). A yellow oil (6.2 mg, 93%). 1H NMR (D2O)
d
4.12e4.09 (m, 2H), 3.71 (s, 3H), 2.94 (d, J¼15.0 Hz, 1H), 2.46e2.26
(m, 2H), 2.08e1.95 (m, 1H), 1.85 (d, J¼15.0 Hz, 1H), 1.78e1.64 (m,
2H), 1.65e1.55 (m, 1H), 1.21 (t, J¼6.9 Hz, 3H), 1.0 (m, 2H), 0.80 (t,
4.8.8. (1R
*
,3S
*
)-1-Amino-3-(3-phenylpropyl)cyclopent-4-ene-1,3-
J¼7.2 Hz, 3H). 13C NMR (D2O)
d 174.6 (C), 169.2 (C), 61.5 (C), 59.0
dicarboxylic acid (10d). A white solid (22.7 mg, 76%). 1H NMR (D2O)
(CH2), 51.5 (C), 50.9 (CH3), 40.4 (CH2), 33.0 (CH2), 32.5 (CH2), 32.2
(CH2), 19.0 (CH2), 10.7 (CH3), 10.4 (CH3). MS (ESþ) m/z 258.0 (MþH,
100%). HRESIMS m/z 258.1707 (MHþ), calcd for C13H24NO4 (MHþ)
258.1700.
d
6.95e6.80 (m, 5H), 5.94 (d, J¼5.4 Hz, 1H), 5.45 (d, J¼5.4 Hz, 1H),
2.30 (d, J¼15.3 Hz, 1H), 2.21 (t, J¼7.5 Hz, 2H), 2.12 (d, J¼15.3 Hz, 1H),
1.58e1.48 (m, 1H), 1.35e1.28 (m, 1H), 1.25e1.14 (m, 2H). 13C NMR
(D2O) d 174.2 (C), 168.7 (C), 140.7 (CH), 138.5 (C), 124.9 (4CH), 123.9
(CH), 122.4 (CH), 66.0 (C), 57.0 (C), 37.4 (CH2), 33.1 (CH2), 31.4 (CH2),
22.7 (CH2). MS (ESþ) m/z 290 (MþH, 100%). HRESIMS m/z 290.1388
(MHþ), calcd for C16H20NO4 (MHþ) 290.1387.
4.9.5. (1S
1,3-dicarboxylate (12c). A yellow oil (43.3 mg, 70%). 1H NMR (D2O)
7.23 (br s, 3H), 7.08 (br s, 2H), 4.09e4.07 (m, 2H), 3.8 (s, 3H), 3.11
*
,3S
*
)-3-Ethyl 1-methyl 1-amino-3-benzylcyclopentane-
d
(d, J¼12.6 Hz, 1H), 2.89 (d, J¼12.6 Hz, 1H), 2.51e2.26 (m, 4H),
4.9. Representative procedure for preparation of rac-11 or
rac-12
2.04e19.4 (m, 2H), 1.16 (t, J¼7.2 Hz, 3H). 13C NMR (D2O)
d 174.3 (C),
169.3 (C), 134.0 (C), 126.5 (2CH), 125.3 (2CH), 123.8 (CH), 61.4 (C),
59.3 (CH2), 52.9 (CH3), 51.0 (C), 50.9 (CH2), 39.9 (CH2), 32.1 (CH2),
32.0 (CH2),10.3 (CH3). MS (ESþ) m/z 306 (MþH,100%). HRESIMS m/z
306.1705 (MHþ), calcd for C17H24NO4 (MHþ) 306.1700.
To a stirred solution of compound 7a (0.165 g, 0.422 mmol) in
diethyl ether (2 mL) at 0 ꢁC, was slowly added a solution of 1 M aq
HCl (0.5 mL). The reaction mixture was stirred at 0 ꢁC for 2 h and
then at rt overnight. The reaction mixture was diluted with water
(2 mL). The layers were separated and the aqueous layer was
extracted with diethyl ether. Water was removed to give the crude
intermediate. The crude intermediate was dissolved in methanol
(3 mL). To the resulting solution was added Pd/C (23 mg,
0.042 mmol). The reaction mixture was stirred at rt, under a H2
atmosphere (approx. 1 atm, balloon) for 24 h. The reaction mixture
was filtered through a pad of Celite and washed with methanol.
Methanol was removed to give a yellow oil, which was purified by
column chromatography (flash silica, ethyl acetate/hexane (60:40)
as eluent) to give 11a as a yellow oil (80.3 mg, 83%).
4.10. Representative procedure hydrolysis of rac-11 and rac-12
Compound 11a (80.0 mg, 0.35 mmol) in 10% aq HCl (2 mL) was
heated at 80 ꢁC for 16 h. After cooling, the reaction mixture was
diluted with water (2 mL) and washed with ether (5 mL). Water
was removed under reduced pressure to give the HCl salt of 13a as
a white solid (84.4 mg, 96%).
4.10.1. (1S
acid (13a). 1H NMR (D2O)
2H), 2.03e1.93 (m, 1H), 1.74 (d, J¼15.0 Hz, 1H) 1.66e1.57 (m, 1H),
*
,3S
*
)-1-Amino-3-methylcyclopentane-1,3-dicarboxylic
d
2.81 (d, J¼15.0 Hz, 1H), 2.85e2.16 (m,
1.21 (s, 3H). 13C NMR (D2O)
d 180.0 (C), 173.0 (C), 61.1 (C), 49.8 (C),
4.9.1. (1S
1,3-dicarboxylate (11a). 1H NMR (D2O)
3H), 2.95 (d, J¼15.0 Hz, 1H), 2.48e2.4 (m, 1H), 2.41e2.33 (m, 1H),
*
,3S
*
)-3-Ethyl 1-methyl 1-amino-3-methylcyclopentane-
43.4 (CH2), 35.0 (CH2), 33.3 (CH2), 25.6 (CH3). MS (ESþ) m/z 188
(Mþ1, 100%). HRMSESI m/z 188.0921 (MHþ), calcd for C8H14NO4
(MHþ) 188.0917.
d
4.15e4.06 (m, 2H), 3.78 (s,

A.T. Ung et al. / Tetrahedron 69 (2013) 2577e2587
2585
4.10.2. (1S
*
,3S
*
)-1-Amino-3-propylcyclopentane-1,3-dicarboxylic
2.91 (d,
4.13. Reduction of compound 16
acid (13b). A white solid (77.6 mg, 99%). 1H NMR (D2O)
d
J¼15.0 Hz, 1H), 2.44e2.36 (m, 1H), 2.29 (dd, J¼8.2, 12.0 Hz, 1H), 1.99
(ddd, J¼4.5, 8.2, 12.0 Hz, 1H), 1.83 (d, J¼15.0 Hz, 1H) 1.78e1.64 (m,
2H), 1.54 (td, J¼4.5, 12.0 Hz, 1H), 1.24e1.03 (m, 2H), 0.78 (t, J¼7.5 Hz,
To a solution of 16 (0.406 g, 1.62 mmol) in dry CH3OH (30 mL)
under a nitrogen atmosphere at 0 ꢁC was added Mg turnings
(0.393 g, 16.24 mmol). The reaction mixture was left to stir at 0 ꢁC
for 2 h, then at rt overnight. Methanol was removed under reduced
pressure. Water (30 mL) was added to the reaction mixture and the
resulting mixture was extracted with ethyl acetate (3ꢂ40 mL). The
combined organic extracted were washed with a saturated aq so-
lution of NaCl and dried over K2CO3. The solvent was removed
under reduced pressure to give an oil, which was purified by PTLC
(ethyl aceate/hexane (1:1)) to give compounds 18 (0.107 g, 27%) and
19 (35.5 mg, 9%).
3H). 13C NMR (D2O)
d 177.1 (C),171.0 (C), 61.1 (C), 51.9 (C), 41.9 (CH2),
38.7 (CH2), 31.9 (CH2), 30.3 (CH2), 15.7 (CH2), 10.5 (CH3). MS (ESþ)
m/z 216 (Mþ1, 100%). HRMSESI m/z 216.1235 (MHþ), calcd for
C10H18NO4 (MHþ) 216.1230.
4.10.3. (1S
*
,3R
*
)-1-Amino-3-benzylcyclopentane-1,3-dicarboxylic
7.26e7.11
acid (13c). A white solid (42.0 mg, 86%). 1H NMR (D2O)
d
(m, 5H), 3.05 (d, J¼13.2 Hz, 1H), 2.90 (d, J¼15 Hz, 1H), 2.87 (d,
J¼15 Hz, 1H), 2.33e2.20 (m, 2H), 2.03e1.94 (m, 2H), 1.90e1.83 (m,
1H).13C NMR (D2O)
d
175.9 (C), 170.7 (C), 133.9 (C), 126.3 (2CH),
4.13.1. (1S
*
,3S
*
)-3-Ethyl 1-methyl 1-(dimethylamino)cyclopentane-
4.20
125.3 (2CH),124.0 (CH), 61.0 (C), 53.4 (C), 41.7 (CH2), 41.3 (CH2), 31.6
(CH2), 29.8 (CH2). MS (ESþ) m/z 264 (Mþ1, 100%). HRMSESI m/z
264.1235 (MHþ), calcd for C14H18NO4 (MHþ) 264.1230.
1,3-dicarboxylate (18). A yellow solid (0.107 g, 27%). 1H NMR
d
(q, J¼7.2 Hz, 2H), 3.70 (s, 3H), 2.92 (ddd, J¼8.2, 8.2,16.8 Hz,1H), 2.47
(dd, J¼8.2, 14.0 Hz, 1H), 2.29 (s, 6H), 2.11 (dd, J¼8.2, 14.0 Hz, 1H),
2.09e1.98 (m, 1H), 1.94e1.71 (m, 3H), 1.29 (t, J¼7.2 Hz, 3H). 13C NMR
4.10.4. (1S
*
,3S
*
)-1-Amino-3-benzylcyclopentane-1,3-dicarboxylic
7.25e7.12
d 175.7 (C), 172.7 (C), 73.7 (C), 60.1 (CH2), 51.6 (CH3), 41.7 (CH), 40.3
acid (14c). A white solid (33.5 mg, 85%). 1H NMR (D2O)
d
(2CH3), 37.6 (CH2), 34.4 (CH2), 27.5 (CH2), 14.1 (CH3). MS (ESþ) m/z
244 (Mþ1, 100%). HRMSESI m/z 244.1547 (MHþ), calcd for
C12H22NO4 (MHþ) 244.1543.
(m, 5H), 3.14 (d, J¼13.5 Hz, 1H), 2.84 (d, J¼13.5 Hz, 1H), 2.43e2.35
(m, 2H), 2.30e2.20 (m, 2H), 2.09e1.97 (m, 1H), 1.94e1.84 (m, 1H).
13C NMR (D2O)
d 176.8 (C), 171.0 (C), 134.1 (C), 126.4 (2CH), 125.4
(2CH), 123.9 (CH), 61.3 (C), 52.9 (C), 39.7 (CH2), 39.6 (CH2), 32.8
(CH2), 32.0 (CH2). MS (ESþ) m/z 264 (Mþ1, 100%). HRMSESI m/z
264.1233 (MHþ), calcd for C14H18NO4 (MHþ) 264.1230.
4.13.2. (1S
*
,3R
*
)-3-Ethyl 1-methyl 1-(dimethylamino)cyclopentane-
4.20
1,3-dicarboxylate (19). A yellow solid (35.5 mg, 9%). 1H NMR
d
(q, J¼7.2 Hz, 2H), 3.71 (s, 3H), 2.84 (ddd, J¼7.6,17.2,17.2 Hz,1H), 2.50
(dd, J¼7.6, 17.2, 1H), 2.29 (s, 6H), 2.31e2.22 (m, 1H), 2.06e1.78 (m,
4.11. Rac-3-Ethyl 1-methyl 1-(dimethylamino)cyclopent-3-
ene-1,3-dicarboxylate (16)
4H), 1.29 (t, J¼7.2 Hz, 3H). 13C NMR
d 175.7 (C), 172.7 (C), 73.7 (C),
60.1 (CH2), 51.6 (CH3), 41.7 (CH), 40.3 (2CH3), 37.6 (CH2), 34.4 (CH2),
27.5 (CH2), 14.1 (CH3).
To a solution of compound 15 (1.17 g, 5.48 mmol) in CH3CN
(30 mL) was added a solution of formaldehyde (40% aq, 11.5 mL,
13.7 mmol) and NaCNBH3 (0.92 g, 14.6 mmol). After 20 min of
stirring, the pH of the reaction mixture was adjusted to pH¼4,
by addition of acetic acid. The reaction mixture was left to stir
at rt overnight. The mixture was diluted with a saturated aq
solution of Na2CO3 (30 mL). The resulting solution was extrac-
ted twice with ethyl acetate (2ꢂ30 mL). The combined extracts
were washed with a saturated solution of NaCl (30 mL) and
dried over K2CO3. The solvent was removed under reduced
pressure to give a yellow oil, which was purified by column
chromatography (flash silica, ethyl acetate/light petroleum
(1:1)) to give compound 16 (1.14 g, 86%) as a yellow oil. 1H NMR
4.14. (1S
*
,3S )-1-(Dimethylamino)cyclopentane-1,
*
3-dicarboxylic acid (20)
To a solution of compound 18 (92.4 mg, 0.38 mmol) in THF
(1.5 mL) was added 1 M aq NaOH (1.5 mL) and the resulting mixture
was left to stir at rt overnight. THF was removed and the crude
product was purified by ion-exchange column chromatography,
using DowexÒ 1X8 (chloride form, 50e100 mesh) basic exchange
resin. The crude product was added as a solution in 1 M aq NH3, and
after washing with demineralized water (120 mL), the product was
eluted from the column with 1 M HCl solution to give the HCl salt of
20 as a white solid (87.5 mg, 97%). 1H NMR (D 2O)
8.4, 16.4 Hz, 1H), 2.74 (s, 3H), 2.71 (s, 3H), 2.45 (dd, J¼8.4, 15.2 Hz,
1H), 2.32e2.25 (m, 2H), 2.13e1.83 (m, 3H). 13C NMR (D2O)
181.6
(C), 178.0 (C), 70.2 (C), 43.2 (CH), 40.0 (CH3), 39.5 (CH3), 35.2 (CH2),
31.8 (CH2), 28.9 (CH2). MS m/z 202 (Mþ1, 100%). HRMSESI m/z
202.1077 (MHþ), calcd for C9H16NO4 (MHþ) 202.1077.
d
3.00 (ddd, J¼8.4,
d
6.66 (dd, J¼1.2, 2.4, 4.5 Hz, 1H), 4.19 (q, J¼7.2 Hz, 2H), 3.75 (s,
3H), 3.18 (dd, J¼1.2, 17.4 Hz, 2H), 3.17 (dd, J¼1.2, 17.4 Hz, 1H),
2.76 (ddd, J¼2.4, 4.5, 17.4 Hz, 1H), 2.67 (ddd, J¼2.4, 4.5, 17.4 Hz,
d
1H), 2.64 (s, 6H), 1.29 (t, J¼7.2 Hz, 3H). 13C NMR
d 173.1 (C),
164.2 (C), 140.0 (CH), 133.9 (C), 74.3 (C), 60.2 (CH2), 51.7 (CH3),
40.2 (2CH3), 40.4 (CH2), 38.8 (CH2), 14.1 (CH3). MS (CI) m/z 242
(Mþ1, 100%). HRMSESI m/z 242.1392 (MHþ), calcd for
C12H20NO4 (MHþ) 242.1387.
4.15. General procedure for preparation of compound 21
To a solution of compound 15 (0.470 g, 2.22 mmol) in dry THF
4.12. rac-1-(Dimethylamino)cyclopent-3-ene-1,3-dicarboxylic
acid (17)
(10 mL) was added the appropriate aldehyde (2.22 mmol) and
powdered 5 A molecular sieves (2e3 g). The reaction mixture was
ꢀ
left to stir at rt. Upon completion, the reaction mixture was filtered
through a pad of Celite. THF was removed under reduced pressure
to give a yellow oil, which was diluted with dry methanol (12 mL).
To the resulting solution was added NaCNBH3 (0.280 g, 4.44 mmol).
The pH of the reaction mixture was adjusted to pH¼4, by the ad-
dition of acetic acid. The reaction mixture was left to stir at rt for
20 h. The mixture was diluted with H2O (20 mL) and made basic by
adding solid Na2CO3. The aqueous solution was extracted with
CH2Cl2 and the combined extracts were dried over K2CO3. The
solvent was removed to give a yellow oil, which was purified by
column chromatography (MeOH/ethyl acetate, 1:10).
Compound 16 (0.100 g, 0.415 mmol) in 10% aq HCl (2 mL) was
heated at 80 ꢁC for 16 h. After cooling, the reaction mixture was
diluted with water (2 mL) and washed with ether (5 mL). The water
was removed under reduced pressure to give the HCl salt of 17 as
a white solid (95.8 mg, 96%). 1H NMR (D2O)
d
6.71e6.69 (br m, 1H),
3.27 (br d, J¼13.8 Hz, 2H), 3.08e3.05 (br m, 1H), 3.02e3.00 (br m,
1H), 2.77 (s, 3H), 2.77 (s, 3H). 13C NMR (D2O)
168.7 (C), 163.4 (C),
d
137.9 (CH), 129.7 (C), 72.2 (C), 36.1 (2CH3), 36.1 (CH2), 34.3 (CH2).
MS (CI) m/z 200 (Mþ1, 100%). HRMSESI m/z 200.0923 (MHþ), calcd
for C9H14NO4 (MHþ) 200.0917.

2586
A.T. Ung et al. / Tetrahedron 69 (2013) 2577e2587
4.15.1. rac-3-ethyl 1-methyl 1-(benzylamino)cyclopent-3-ene-1,3-
4.16.1. rac-1-(Benzylamino)cyclopent-3-ene-1,3-dicarboxylic
acid
dicarboxylate (21a). A yellow oil (0.538 g, 80%). 1H NMR
(22a). A white solid (0.102 g, 98%). 1H NMR (D2O) 7.38 (s, 5H), 6.74
(br s, 1H), 4.11 (s, 2H), 3.31 (d, J¼18.0 Hz, 2H), 3.04 (d, J¼18.0 Hz,
d
7.34e7.26 (m, 5H), 6.76 (dd, 2.4, 1.8 Hz, 1H), 4.21 (q, J¼6.9 Hz, 2H),
3.78 (s, 3H), 3.66 (s, 2H), 3.19 (dd, J¼2.4, 14.7 Hz, 1H), 3.14 (dd, J¼1.8,
16.5 Hz, 1H), 2.81 (dd, J¼2.4, 14.7 Hz, 1H), 2.70 (dd, J¼1.8, 16.5 Hz,
2H). 13C NMR (D2O)
d 169.7 (C), 163.7 (C), 137.8 (CH), 129.5 (C), 127.4
(C), 126.6 (2CH), 126.5 (CH), 126.1 (2CH), 65.6 (C), 44.5 (CH2), 38.5
(CH2), 36.6 (CH2). MS (CI) m/z 262 (Mþ1, 100%). HRMSESI m/z
262.1078 (MHþ), calcd for C14H16NO4 (MHþ) 262.1074.
1H), 2.0 (br s, 1H, NH), 1.31 (t, J¼6.9 Hz, 3H). 13C NMR
d 175.9 (C),
164.0 (C), 139.6 (CH), 139.4 (C), 133.4 (C), 128.0 (2CH), 127.8 (2CH),
126.71 (CH), 68.8 (C), 59.9 (CH2), 51.9 (CH3), 48.5 (CH2), 43.2 (CH2),
41.8 (CH2), 13.8 (CH3). MS (ESþ) m/z 304 (Mþ1, 100%). HRMSESI m/z
304.1545 (MHþ), calcd for C17H22NO4 (MHþ) 304.1543.
4.16.2. rac-1-(Pyridin-2-ylmethylamino)cyclopent-3-ene-1,3-
dicarboxylic acid (22b). A white solid (0.202 g, 100%). 1H NMR
(D2O)
d
8.65 (dd, J¼1.5, 7.8 Hz, 1H), 8.42 (ddd, J¼1.5, 7.8, 7.8 Hz,
4.15.2. rac-3-Ethyl 1-methyl 1-(pyridin-2-ylmethylamino)cyclopent-
1H), 7.98 (d, J¼7.8 Hz, 1H), 7.88 (ddd, J¼1.5, 7.8, 7.8 Hz, 1H), 6.63 (br
3-ene-1,3-dicarboxylate (21b). A yellow oil (0.436 g, 80%). 1H NMR
s, 1H), 4.51 (s, 2H), 3.24 (br d, J¼17.4 Hz, 2H), 3.01e2.92 (m, 2H).
d
8.58e8.50 (m, H), 7.71e7.59 (m, 2H), 7.30e7.18 (m, 2H), 6.68 (br s,
13C NMR (D2O)
d 172.3 (C), 166.6 (C), 147.9 (CH), 144.5 (C), 143.1
1H), 4.18 (q, J¼7.2 Hz, 2H), 3.8 (s, 2H), 3.73 (s, 3H), 3.18 (ddd, J¼2.1,
(CH), 140.8 (CH), 132.62 (C), 128.8 (CH), 128.2 (CH), 69.9 (C), 43.8
(CH2), 41.8 (CH2), 40.0 (CH2). MS (ESþ) m/z 263 (Mþ1, 100%).
HRMSESI m/z 263.1033 (MHþ), calcd for C13H15N2O4 (MHþ)
263.1032.
16.5, 16.5 Hz, 2H), 2.82 (dd, J¼1.5, 16.5 Hz, H), 2.72 (dd, J¼1.5,
16.5 Hz, H), 1.28 (t, J¼7.2 Hz, 3H). 13C NMR
d 175.7 (C), 164.5 (C),
159.0 (C), 149.2 (CH), 140.0 (CH), 136.7 (CH), 133.9 (C), 122.4 (CH),
122.2 (CH), 68.9 (C), 60.4 (CH2), 52.5 (CH3), 50.0 (CH2), 43.8 (CH2),
42.3 (CH2), 14.4 (CH3). MS (ESþ) m/z 305.35 (Mþ1, 100%). HRMSESI
m/z 305.1496 (MHþ), calcd for C16H21N2O4 (MHþ) 305.1496.
4.16.3. rac-1-(Pyridin-3-ylmethylamino)cyclopent-3-ene-1,3-
dicarboxylic acid (22c). A white solid (0.208 g, 98%). 1H NMR (D2O)
d
8.76 (s, 1H), 8.65 (d, J¼5.7 Hz, 1H), 8.55 (dd, J¼1.5, 8.1 Hz, 1H), 7.93
4.15.3. rac-3-Ethyl 1-methyl 1-(pyridin-3-ylmethylamino)cyclopent-
(dd, J¼5.7, 8.1 Hz, 1H), 6.58 (br s, 1H), 4.35 (s, 2H), 3.19 (d, J¼16.8 Hz,
3-ene-1,3-dicarboxylate (21c). A yellow oil (0.267 g, 58%). 1H NMR
2H), 2.96 (d, J¼17.7 Hz, 1H), 2.91 (d, J¼17.7 Hz, 1H). 13C NMR (D2O)
d
8.49 (d, J¼1.5 Hz, 1H), 8.43 (dd, J¼1.5, 4.8 Hz, 1H), 7.63 (dd, J¼1.5,
d 172.1 (C), 166.4 (C), 148.8 (CH), 142.5 (CH), 142.3 (CH), 140.8 (CH),
7.8 Hz, 1H), 7.19 (dd, J¼4.8, 7.8 Hz, 1H), 6.62 (t, 1.5 Hz, 1H), 4.14 (q,
J¼6.9 Hz, 2H), 3.72 (s, 3H), 3.61 (s, 2H), 3.10 (ddd, J¼1.5, 16.5,
16.5 Hz, 2H), 2.72 (dd, J¼1.5, 16.5 Hz, 1H), 2.60 (dd, J¼1.5, 16.5 Hz,
132.5 (C), 131.1 (C), 128.0 (CH), 69.4 (C), 43.8 (CH2), 41.6 (CH2), 39.8
(CH2). MS (ESþ) m/z 263 (Mþ1, 100%). HRMSESI m/z 263.1033
(MHþ), calcd for C13H15N2O4 (MHþ) 263.1032.
1H), 2.22 (br s, 1H, NH), 1.23 (t, J¼6.9 Hz, 3H). 13C NMR
d 175.7 (C),
164.4 (C), 149.7 (CH), 148.5 (CH), 139.8 (CH), 136.0 (CH), 135.5 (C),
133.9 (C), 123.4 (CH), 69.0 (C), 60.4 (CH2), 52.5 (CH3), 46.3 (CH2),
43.7 (CH2), 42.3 (CH2), 14.3 (CH3). MS (ESþ) m/z 305 (Mþ1, 100%).
HRMSESI m/z 305.1497 (MHþ), calcd for C16H21N2O4 (MHþ)
305.1496.
4.16.4. rac-1-(3-Hydroxybenzylamino)cyclopent-3-ene-1,3-
dicarboxylic acid (22d). A yellow solid (0.113 g, 92%). 1H NMR (D2O)
d
7.20 (t, J¼7.5 Hz, 1H), 6.86 (d, J¼7.5 Hz, 1H), 6.80 (d, J¼7.5 Hz, 1H),
6.79 (s, 1H), 6.69 (br s, 1H), 3.98 (s, 2H), 3.26 (d, J¼17.6 Hz, 2H), 2.96
(d, J¼16.8 Hz, 2H). 13C NMR (D2O)
d 172.4 (C), 166.7 (C), 156.0 (C),
140.9 (CH), 132.6 (C), 132.1 (C), 130.8 (CH), 121.8 (CH), 116.7 (CH),
116.6 (CH), 68.4 (C), 47.4 (CH2), 41.6 (CH2), 39.7 (CH2). MS (CI) m/z
277.10 (M, 100%). HRMSESI m/z 278.1025 (MHþ), calcd for
C14H15NO5 (MHþ) 278.1023.
4.15.4. rac-3-Ethyl 1-methyl 1-(3-hydroxybenzylamino)cyclopent-3-
ene-1,3-dicarboxylate (21d). A yellow oil (0.163 g, 81%). 1H NMR
d
7.12 (t, J¼7.8 Hz, 1H), 6.80 (d, J¼7.8 Hz, 1H), 6.73 (s, 1H), 6.68 (d,
J¼7.8 Hz, 1H), 6.67 (t, J¼2.1 Hz, 1H), 4.17 (q, J¼6.9 Hz, 2H), 3.75 (s,
3H), 3.52 (s, 2H), 3.13 (dd, J¼2.1,16.9 Hz, 2H), 2.79 (d, J¼16.2 Hz,1H),
4.16.5. rac-1-(4-Hydroxybenzylamino)cyclopent-3-ene-1,3-di-
2.69 (dd, J¼2.1, 16.9 Hz, 1H), 1.27 (t, J¼6.9 Hz, 3H). 13C NMR
d
176.1
carboxylic acid (22e). A yellow solid (72.0 mg, 95%). 1H NMR (D2O)
(C), 165.0 (C), 156.8 (C), 140.8 (C), 140.5 (CH), 133.8 (C), 129.9 (CH),
120.2 (CH), 115.8.0 (CH), 114.9 (CH), 69.2 (C), 60.1 (CH2), 52.8 (CH3),
49.1 (CH2), 43.7 (CH2), 42.2 (CH2), 14.4 (CH3). MS (CI) m/z 319.36 (M,
100%). HRMSESI m/z 320.1499 (MHþ), calcd for C17H22NO5 (MHþ)
320.1498.
d
7.36 (d, J¼8.1 Hz, 2H), 6.64 (d, J¼8.1 Hz, 2H), 6.73 (br s, 1H), 4.13 (s,
2H), 3.36e3.30 (m, 2H), 3.20e3.11 (m, 2H). 13C NMR (CD3OD)
172.5 (C), 166.5 (C), 159.5 (C), 140.5 (CH), 134.6 (C), 132.8 (2CH),
d
122.8 (C), 116.7 (2CH), 69.5 (C), 48.1 (CH2), 42.8 (CH2), 41.3 (CH2).
MS (CI) m/z 277.10 (M, 100%). HRMSESI m/z 278.1026 (MHþ), calcd
for C14H15NO5 (MHþ) 278.1023.
4.15.5. rac-3-Ethyl 1-methyl 1-(4-hydroxybenzylamino)cyclopent-3-
ene-1,3-dicarboxylate (21e). A yellow oil (0.104 g, 69%), 1H NMR
4.17. Signal transduction at mGlu2 receptors in CHO cells
d
7.10 (d, J¼8.4 Hz, 2H), 6.68 (t, J¼2.1 Hz, 1H), 6.67 (d, J¼8.4 Hz, 2H),
4.17 (q, J¼7.2 Hz, 2H), 3.75 (s, 3H), 3.52 (s, 2H), 3.13 (ddd, J¼2.1,
16.6 Hz, 2H), 2.79 (d, J¼16.6 Hz, 1H), 2.69 (dd, J¼2.1, 16.6 Hz, 1H),
Human mGluR2 (cloned and expressed in house) were grown in
DMEM/Glutamax-I to which 2 mM glutamine, 46 mg/L proline, and
10% dialysed foetal calf serum were added.
1.27 (t, J¼7.2 Hz, 3H). 13C NMR
d 174.1 (C), 164.9 (C), 160.3 (C), 140.5
(CH), 133.8 (C), 130.8 (2CH), 127.6 (C), 116.0 (2CH), 75.0 (C), 68.1
(CH2), 60.9 (CH2), 53.0 (CH3), 44.6 (CH2), 42.6 (CH2), 14.4 (CH3). MS
(ESþ) m/z 320 (Mþ1, 100%). HRMSESI m/z 320.1498 (MHþ), calcd for
C17H22NO5 (MHþ) 320.1498.
4.18. [35S]GTP
gS radioligand binding assay for human mGlu2
4.18.1. Membrane preparation. Cells were grown to confluence.
Cells were washed twice with ice-cold PBS without Ca2þ and Mg2þ
,
4.16. Representative procedure for hydrolysis of
compound 21
scraped off and homogenized in buffer (EDTA 20 mM, Hepes
20 mM). After centrifugation (18,000 rpm, 15 min, 4 ꢁC), the pellet
was washed with 0.1 mM EDTA, 20 mM Hepes, and resuspended in
the same buffer for protein determination with the Biorad assay.
Membrane aliquots were stored at ꢀ70 ꢁC.
Compound 21a (0.106 g, 0.35 mmol) in 10% aq HCl (3 mL) was
heated at 80 ꢁC for 16 h. After cooling, the reaction mixture was
diluted with water (2 mL) and washed with ether (5 mL). The water
was removed under reduced pressure to give the HCl salt of 22a as
a white solid (102 mg, 98%).
4.18.2. [35S]GTP
10 g of membrane protein in 250
gS radioligand binding. Each incubate contained
m
m
L of binding buffer (HEPES

A.T. Ung et al. / Tetrahedron 69 (2013) 2577e2587
2587
20 mM, NaCl 100 mM, MgCl2 3 mM, GDP 3
m
M, pH 7.4). The in-
4.19.1.2. Compound 8b. C26H27NO4: monoclinic, space group
¼117.079(4)ꢁ,
ꢀ
b
cubation was started by addition of an appropriate concentration
of agonist and/or antagonist. Compounds were incubated with
the membranes at 37 ꢁC for 30 min. Subsequently, 0.1 nM [35S]
P21/c, a¼16.035(4), b¼9.114(2), c¼16.769(4) A,
3
V¼2182.0(9) A , Z¼4, Dc¼1.271 g cmꢀ3. 24,889 reflections collected,
ꢀ
3750 independent (Rint¼0.056). Final R indices, R1¼0.0930 (for
GTP
g
S (approximately 2ꢂ105 DPM) was added in the presence or
I>2
s
(I)), wR2¼0.2330 (all reflections), S¼1.157. The allyl group was
absence of 30 mM glutamate. The mixture was further incubated
for 30 min at 37 ꢁC. The reactions were terminated by separating
free and bound radioactivity by rapid vacuum filtration using
a Packard filtration manifold through GF/B pre-wetted glass fibre
filters.
modelled as being disordered over two sets of sites with occu-
pancies refined to 0.773(13) and its complement.
4.19.1.3. Compound 7c. C30H29NO4: triclinic, space group P1,
ꢀ
a¼6.0737(16), b¼12.605(3), c¼17.285(5) A,
a
¼70.961(4),
3
ꢀ
ꢀ3
ꢁ
Filters were rapidly washed two times with cold 10 mM
NaH2PO4/10 mM Na2HPO4 buffer, pH 7.4. Filters were transferred to
vials for subsequent counting in a scintillation counter. Results are
expressed as % of glutamate-induced response, the latter being
defined as 100%. Glutamate and amino acids were dissolved and
diluted in water. Concentrationeresponse curves were drawn on
a logarithmic scale. Sigmoidal curves of best fit were calculated by
nonlinear regression analysis using GraphPad software (San Diego,
CA). The pIC50-value referred to the concentration of a compound
producing half the maximum response.
b
¼81.126(4),
g
¼84.888(4) , V¼1234.9(6) A , Z¼2, Dc¼1.257 g cm
.
14,317 reflections collected, 6006 independent (Rint¼0.029). Final R
indices, R1¼0.0475 (for I>2
S¼0.947.
s
(I)), wR2¼0.1245 (all reflections),
Acknowledgements
We thank Johnson and Johnson Research Pty Limited (Sydney,
Australia) for financial support and Prof. Susan Pond (Johnson &
Johnson Research), Dr. Vic Sipido, and Dr. John Stuart Andrews
(Jannsen Pharmaceutica, Belgium) for their encouragement and
support.
4.19. X-ray crystallographic data
References and notes
Single crystal X-ray diffraction data were collected at 150 K
(8b,7c) or 298 K (8a) on a Bruker Smart diffractometer using Mo K
a
1. Leeson, P. D.; Iversen, L. L. J. Med. Chem. 1994, 34, 4053e4067.
2. Ornstein, P. L.; Schoepp, D. D.; Monn, J. A. Curr. Pharm. Des. 1995, 1,
355e362.
ꢀ
radiation (l¼0.71073 A). Following solution by direct methods, the
structures were refined against F2 with full-matrix least-squares
using the program SHELXL-97.15 All hydrogen atoms were added at
calculated positions and refined by use of riding models with iso-
tropic displacement parameters based on those of the parent
atoms. Except for the minor components of the disordered atoms in
(8b), anisotropic displacement parameters were employed
throughout for the non-hydrogen atoms. Crystallographic data
(excluding structure factors) for the structures in this paper have
been deposited with the Cambridge Crystallographic Data Centre as
supplementary publication nos. CCDC 899273e899275. Copies of
the data can be obtained, free of charge, on application to CCDC, 12
Union Road, Cambridge CB2 1EZ, UK (fax: þ44 (0)1223 336033 or e-
mail: deposit@ccdc.cam.ac.uk).
3. Conn, P. J.; Pin, J.-P. Annu. Rev. Pharmacol. Toxicol. 1997, 37, 205e237.
€
4. Brauner-Osborne, H.; Egebjerg, J.; Nielsen, E. é.; Madsen, U.; Krogsgaard-Larsen,
P. J. Med. Chem. 2000, 43, 2609e2645.
5. (a) Jayaraman, V.; Keesey, R.; Madden, D. R. Biochemistry 2000, 39, 8693e8697;
(b) Bessis, A.-S.; Jullian, N.; Coudert, E.; Pin, J.-P.; Acher, F. Neuropharmacology
1999, 38, 1543e1551.
6. Acher, F. C.; Tellier, F. J.; Azerad, R.; Brabet, I. N.; Fagni, L.; Pin, J.-P. R. J. Med.
Chem. 1997, 40, 3119e3129.
7. Ung, A. T.; Schafer, K.; Linsay, K. B.; Pyne, S. G.; Amornraksa, K.; Wouters, R.; Van
der Linden, I.; Biesmans, I.; Lesage, A. S. J.; Skelton, B. W.; White, A. H. J. Org.
Chem. 2002, 67, 227e233.
8. Ung, A. T.; Pyne, S. G.; Batenburg-Nguyen, U.; Davis, A. S.; Sherif, A.; Bischoff, F.;
Lesage, A. S. J. Tetrahedron 2005, 61, 1803e1812.
9. Landsbury, P. T.; Erwin, R. W.; Jeffrey, D. A. J. Am. Chem. Soc. 1980, 102,
1602e1608.
10. Katzenellenbogen, J. A.; Crumrine, A. L. J. Am. Chem. Soc. 1976, 98, 4925e4935.
11. CCDC deposition numbers for compounds 7c, 8a and 8b are 899273, 899274
and 899275, respectively. Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: þ44 (0)1223
336033 or e-mail: deposit@ccdc.cam.ac.uk).
4.19.1. Crystal data
4.19.1.1. Compound 8a. C24H25NO4: monoclinic, space group
ꢀ
12. Hudlicky, T.; Sinai-Zingde, G.; Natchus, M. G. Tetrahedron Lett. 1987, 28,
5287e5290.
P21/c,
b
a¼16.9344(19),
b¼8.8127(10),
c¼16.1275(18)
A,
¼117.393(2) , V¼2137.0(4) A , Z¼4, Dc¼1.217 g cmꢀ3. 15,529 re-
3
ꢁ
ꢀ
13. Walkup, R. D.; Park, G. Tetrahedron Lett. 1988, 29, 5505e5508.
flections collected, 5280 independent (Rint¼0.034). Final R indices,
ꢁ
14. Domínguez, C.; Csaky, A. G.; Plumet, J. Tetrahedron Lett. 1991, 32, 4183e4184.
R1¼0.0428 (for I>2
s(I)), wR2¼0.1223 (all reflections), S¼0.876.
15. Sheldrick, G. M. Acta Crystallogr., Sect. A 2008, 64, 112e122.