2,3-Ethylene- and 2,3-trimethylene-bridged analogues of the group III metabotropic glutamate receptor ligand 2-amino-4-phosphonobutanoic acid

Article abstract of DOI:10.1016/j.bmcl.2004.10.040

The racemic trans- and cis-isomers of 1-amino-2-phosphonomethyl- cyclobutanecarboxylic acid (5 and 6) and 1-amino-2-phosphonomethyl- cyclopentanecarboxylic acid (7 and 8) were synthesized as extensions of the mGluR4 agonists trans- and cis-1-amino-2-phosphonomethyl-cyclopropanecarboxylic acid (3 and 4). Although the methylene bridge in 3 and 4 allows for retention of affinity toward the mGluR4 receptor, increasing the bridging unit to the ethylene group as in 5 and 6 or to the trimethylene group as in 7 and 8 introduces sufficient steric hindrance to eliminate affinity for the mGluR4 receptor.

Full text of DOI:10.1016/j.bmcl.2004.10.040

Bioorganic & Medicinal Chemistry Letters 15 (2005) 57–60
2,3-Ethylene- and 2,3-trimethylene-bridged analogues of the
group III metabotropic glutamate receptor ligand
2-amino-4-phosphonobutanoic acid
Rodney L. Johnson* and Kolluri S. S. P. Rao
Department of Medicinal Chemistry, University of Minnesota, 308 Harvard Street, SE, Minneapolis, MN 55455, USA
Received 27 August 2004; revised 12 October 2004; accepted 12 October 2004
Available online 2 November 2004
Abstract—The racemic trans- and cis-isomers of 1-amino-2-phosphonomethyl-cyclobutanecarboxylic acid (5 and 6) and 1-amino-2-
phosphonomethyl-cyclopentanecarboxylic acid (7 and 8) were synthesized as extensions of the mGluR4 agonists trans- and cis-1-
amino-2-phosphonomethyl-cyclopropanecarboxylic acid (3 and 4). Although the methylene bridge in 3 and 4 allows for retention
of affinity toward the mGluR4 receptor, increasing the bridging unit to the ethylene group as in 5 and 6 or to the trimethylene group
as in 7 and 8 introduces sufficient steric hindrance to eliminate affinity for the mGluR4 receptor.
Ó 2004 Elsevier Ltd. All rights reserved.
Glutamic acid is the principal excitatory neurotransmit-
ter in the central nervous system. It exerts its effects
through both ligand-gated ion channels and G-protein
coupled receptors. The latter are referred to as metabo-
tropic glutamate receptors (mGluR) of which eight sub-
types have been identified based upon their signal
transduction mechanisms and upon their reaction to-
ward different glutamic acid analogues.1 Group I is
made up of mGluR1 and mGluR5, while mGluR2
and mGluR3 make up Group II. Group III consists
of mGluR4, mGluR6, mGluR7, and mGluR8. A
distinctive characteristic of Group III mGluRs is their
activation by the glutamic acid analogue L-2-amino-4-
phosphonobutanoic acid (1, L-AP4).2–6 The a-methyl
derivative of 1, a-methyl-2-amino-4-phosphonobutanoic
acid (2), has been shown to be an antagonist at some
Group III mGluRs, in particular mGluR4a.7
Several analogues of 1 have been synthesized to investi-
gate the structure–activity relationships of this mole-
cule.8–12 Two of the most potent analogues of 1 are
the cyclopropyl derivatives 3 and 4.10 Both cyclopropyl
analogues show potent activity at the mGluR4 receptor
with the cis-isomer (4) being the most active.13,14 Since
2–4 all can be viewed as a-substituted analogues of 1,
the question that arises is why 3 and 4 exhibit agonist
activity, while 2 is an antagonist at mGluR4 receptors.
Clearly, there are electronic differences between the
2,3-methano group of 3 and 4 and the a-methyl group
of 2. Also, the CH2–Ca–Cb bond angle of ꢀ60° seen in
3 and 4 versus the CH3–Ca–Cb bond angle of ꢀ109°
found in 2 results in the methano group of 3 and 4 being
projected into a different area of space than the a-methyl
group of 2. To test the possible effect that such a differ-
ence might have on the different activity seen for 2–4, the
synthesis of analogues of 1 possessing either an ethylene
or trimethylene bridge between the a- and b-carbon
atoms was undertaken. This gave rise to the cyclobut-
yl-AP4 (5 and 6) and cyclopentyl-AP4 (7 and 8)
H2N
CO2H
H
H2N
CO2H
CH3
PO(OH)2
PO(OH)2
2
1
CO2H
CO2H
H2N
60˚
CO2H
H2N
88˚
H2N
108˚
PO(OH)2
PO(OH)2
PO(OH)2
3 (trans-isomer)
4 (cis-isomer)
5 (trans-isomer)
6 (cis-isomer)
7 (trans-isomer)
8 (cis-isomer)
Keywords: AP4; Conformational analogues; mGluR4.
*
Corresponding author. Tel.: +1 612 624 7997; fax: +1 612 624
0139; e-mail: johns022@umn.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.040

58
R. L. Johnson, K. S. S. P. Rao / Bioorg. Med. Chem. Lett. 15 (2005) 57–60
analogues with expected CH2–Ca–Cb bond angles of
ꢀ88° and ꢀ108°, respectively.
The relative stereochemistry of the cyclopentyl ana-
logues was determined at the N-benzoylamino nitrile
stage. Compound 21 was successfully crystallized from
hexane/CH2Cl2 (1:1) to give crystals from which a crys-
tal structure was obtained (Fig. 1) that showed it was the
trans-isomer.19 Crystals suitable for X-ray analysis,
however, could not be obtained for either of the two iso-
mers in the cyclobutane series.
Synthesis of the racemic trans- and cis-isomers of 1-ami-
no-2-phosphonomethyl-cyclobutanecarboxylic acid (5
and 6) and 1-amino-2-phosphonomethyl-cyclopentane-
carboxylic acid (7 and 8) was carried out as shown in
Scheme 1. A Mannich reaction on either cyclobutanone
(9) or cyclopentanone (10) with dimethylamine hydro-
chloride and aqueous formaldehyde gave the 2-dimethyl-
aminomethyl-cycloalkanones 11 and 12, respectively.15
Each of these two compounds was converted quantita-
tively to the corresponding methiodide salts 13 and 14
by treatment with methyl iodide. Reaction of 13 and 14
with triethylphosphite in benzene under refluxing condi-
tions yielded the ketophosphonates 15 and 16, respec-
tively, in good yield. A Strecker reaction on each
ketophosphonate gave the corresponding amino nitriles
17 and 18, each as a mixture of diastereoisomers.9 The
amino nitriles were not purified, but immediately benzo-
ylated with benzoyl chloride in pyridine at 0°C. The
trans- and cis-isomers of the cyclobutyl N-benzoylamino
nitriles, 19 and 20,16 were separated from one another by
preparative TLC using EtOAc as the eluting solvent. In
the case of the cyclopentyl N-benzoylamino nitriles 21
and 22,17 separation was accomplished by silica gel col-
umn chromatography eluting with hexanes/EtOAC
(1:1) initially, followed by EtOAc. Conversion of 19–22
to 5–8, respectively, was accomplished through hydroly-
sis with 6N HCl. Purification of 5–8 was carried out on
an AG WX8 cation exchange resin (50–100 mesh).18
The assignment of the stereochemistry for 19 and 20 was
made by comparing the relative NMR chemical shifts of
selected atoms in these cyclobutyl derivatives with
those seen for the same atoms in the corresponding
1
cyclopentyl derivatives. Analysis of the H NMR spec-
trum of 19 and 20 showed that the chemical shift of
Figure 1. X-ray crystal structure of 21.
O
O
(CH3)2NH • HCl
CH3I
n
n
aq. CH2O, ∆
Et2O, 0 ˚C
N(CH3)2
9: n = 1
10: n = 2
11: n = 1
12: n = 2
NaCN, NH4Cl
NH4OH
O
O
P(OEt)3
n
n
MeOH/H2O
(1:1)
Benzene, ∆
PO(OEt)2
N(CH3)3
I
13: n = 1
14: n = 2
15: n = 1
16: n = 2
NH2
NHCOPh
CN
CN
6N HCl, ∆
PhCOCl
5–8
n
n
Pyridine
0 ˚C
PO(OEt)2
PO(OEt)2
17: n = 1
18: n = 2
19: n = 1, trans-isomer
20: n = 1, cis-isomer
21: n = 2, trans-isomer
22: n = 2, cis-isomer
Scheme 1.

R. L. Johnson, K. S. S. P. Rao / Bioorg. Med. Chem. Lett. 15 (2005) 57–60
59
the NH peak in 20 (9.04ppm) was downfield of that in
19 (8.7ppm), while the phosphorous resonance in 20
(29.2ppm) was upfield of that in 19 (30.4ppm). These
chemical shift differences were analogous to those seen
between the cis-cyclopentyl derivative 22 (dNH =
9.8ppm, dP = 31.6ppm) and the trans-cyclopentyl deriv-
ative 21 (dNH = 9.2ppm, dP = 32.3ppm). In addition,
the chemical shift of the methylene hydrogens next to
the phosphorous atom in 5 and 6 showed a distinct pat-
tern that was analogous to that seen for the correspond-
ing methylene hydrogens in 7 and 8. In the case of 6,
these methylene protons appear as two sets of multiplets
at 2.30–2.56 and 2.82–2.93ppm, whereas in the case of 5,
the methylene protons appear as a single multiplet at
2.28–2.38ppm. This pattern is analogous to that seen
in the cyclopentyl cis-isomer 8 (2.20–2.25 and 2.25–
2.50ppm) and the cyclopentyl trans-isomer 7 (2.15–
2.25ppm). Molecular modeling (Insight, version 2.0)
suggested a possible explanation for the different chem-
ical shift patterns seen between the trans- and cis-iso-
mers in the cyclobutyl and cyclopentyl series. The
energy-minimized structures of both sets of isomers
reveal that the CH2P hydrogens are diastereotopic
with respect to the protonated amino group. The
distance between the nitrogen atom and the two methyl-
for the mGluR4 receptor. This implies that the area
on the mGluR4 receptor that interacts with the a- and
b-carbon atoms of AP4 is quite restricted as anything
larger than a methylene bridge is not allowed. Also,
the fact that the cyclopropyl AP4 analogues with the
methylene bridge retain agonist activity, while breaking
the methylene bridge to yield the a-methyl analogue of
AP4 results in an antagonist further indicates that this
region of the receptor is very sensitive to where the steric
bulk is projected. The results obtained in this study
should prove useful in the design of future ligands for
the mGluR4 receptor and they should be of value in
the refinement of the molecular models of the mGluR4
receptor that have been developed.21,22
Acknowledgements
This work was supported in part from a grant from NIH
(NS35608) and a Development Grant in Drug Design
from the Department of Medicinal Chemistry, Univer-
sity of Minnesota. The crystallographic analysis carried
out by Neil R Brooks and Victor G. Young, Jr. of the
University of Minnesota X-ray Crystallographic Labo-
ratory is gratefully acknowledged. The authors thank
Dr. James Monn and Renee Emkey of Eli Lilly and
Company for arranging and conducting, respectively,
the biological testing.
˚
ene hydrogens was 4.23 and 4.57A in trans-isomer 5,
whereas the distance between the nitrogen atom
˚
and methylene hydrogens was 2.92 and 4.15A in cis-iso-
mer 6. An analogous pattern was observed in cyclopen-
˚
tane trans-isomer 7 (4.16 and 4.42A) and cis-isomer 8
˚
(2.89 and 4.11A).
References and notes
Racemic compounds 5–8 were evaluated in a fluoromet-
ric imaging plate reader (FLIPR) assay utilizing
mGluR1–5, 8 that were stably expressed in AV12 cells
co-expressing the rat glutamate transporter.20 Cells
expressing mGluR2–4, 8 also expressed the promiscuous
G-protein Ga15. The four AP4 analogues, 5–8, did not
show significant activity as agonists at the mGluR4
receptor when tested up to a concentration of 200lM.
Neither did they show significant antagonist activity at
mGluR4 receptors when tested up to a concentration
of 100lM. Similar results also were seen at another
Group III receptor, mGluR8. Not surprisingly, 5–8
did not show significant affinity toward either the Group
I metabotropic receptors mGluR1 and 5 or the Group II
metabotropic receptors mGluR2 and 3.
1. Conn, P. J.; Pin, J.-P. Annu. Rev. Pharmacol. Toxicol.
1997, 37, 205.
2. Tanabe, Y.; Nomura, A.; Masu, M.; Shigemoto, R.;
Mizuno, M.; Nakanishi, S. J. Neurosci. 1993, 13, 1372.
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4. Okamoto, N.; Hori, S.; Akazawa, C.; Hayashi, Y.;
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1994, 269, 1231.
5. Duvoisin, R. M.; Zhang, C.; Rammonell, K. A. J.
Neurosci. 1995, 15, 3075.
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cology 1999, 38, 1431.
7. Johansen, P. A.; Robinson, M. B. Eur. J. Pharmacol.
1995, 290, R1.
8. Crooks, S. L.; Freund, R. K.; Halsrud, D. A.; Koerner, J.
F.; Johnson, R. L. Brain Res. 1985, 329, 346.
9. Crooks, S. L.; Robinson, M. B.; Koerner, J. F.; Johnson,
R. L. J. Med. Chem. 1986, 29, 1988.
10. Kroona, H. B.; Peterson, N. L.; Koerner, J. F.; Johnson,
R. L. J. Med. Chem. 1991, 34, 1692.
11. Peterson, N. L.; Kroona, H. B.; Johnson, R. L.; Koerner,
J. F. Brain Res. 1992, 571, 162.
12. Gasparini, F.; Inderbitzin, W.; Francotte, E.; Lecis, G.;
Richert, P.; Dragic, Z.; Kuhn, R.; Flor, P. J. Bioorg. Med.
Chem. Lett. 2000, 10, 1241.
13. Peterson, N. L.; Thoreson, W. B.; Johnson, R. L.;
Koerner, J. F.; Miller, R. F. Brain Res. 1991, 568, 15.
14. Johansen, P. A.; Chase, L. A.; Sinor, A. D.; Koerner, J.
F.; Johnson, R. L.; Robinson, M. B. Mol. Pharmacol.
1995, 48, 140.
In contrast to the inactivity of 5–8, the racemic cyclo-
propyl AP4 analogues 3 and 4 were found to have
EC50 values of 7.9 and 0.58lM, respectively, at the
mGluR4 receptor.14 The possible juxtaposition in space
of the amino, carboxyl, and phosphonic acid moieties is
similar for the corresponding isomers in the cyclopropyl,
cyclobutyl, and cyclopentyl AP4 analogues. The differ-
ence in these analogues lies in the nature of the bridging
unit between the a- and b-carbon atoms of the AP4
backbone. Clearly, these results show that a methylene
bridge allows for retention of affinity toward the
mGluR4 receptor. Increasing the bridging unit to the
ethylene group as in the cyclobutyl isomers or to the tri-
methylene group as in the cyclopentyl isomers intro-
duces sufficient steric hindrance to eliminate affinity
15. Frank, J.; Pierle, M. J. Am. Chem. Soc. 1951, 73, 724.

60
R. L. Johnson, K. S. S. P. Rao / Bioorg. Med. Chem. Lett. 15 (2005) 57–60
16. Spectral properties of ( )-19 and ( )-20. ( )-19: 1H NMR
(CDCl3) d 8.70 (s, 1H, NH), 7.85–7.88 (d, 2H, Ph),
7.40–7.50 (m, 3H, Ph), 4.10–4.18 (m, 4H, POCH2), 2.88–
2.95 (m, 2H, PCH2CH), 2.33–2.45 (m, 2H), 1.98–2.23
(m, 3H), 1.31–1.38 (apparent t, 6H, OCH2CH3); 13C NMR
18. Spectral properties for 5–8. ( )-5: 1H NMR (D2O) d 1.58–
1.74 (m, 2H), 1.78–1.92 (m, 2H), 2.00–2.10 (m, 1H,
PCH2CH), 2.28–2.38 (m, 2H, PCH2); 31P NMR (D2O) d
26.4; FABMS m/z 210.0 (M+H)+. ( )-6: 1H NMR (D2O) d
1.68–1.95 (m, 3H), 2.00–2.25 (m, 2H), 2.30–2.56 (m, 1H,
PCH2), 2.82–2.93 (m, 1H, PCH2); 31P NMR (D2O) d 27.0;
FABMS m/z 210.0 (M+H)+. ( )-7: 1H NMR (D2O) d
1.40–1.80 (m, 6H), 1.80–2.00 (m, 1H, PCH2CH), 2.15–2.25
(m, 2H, PCH2); 31P NMR (D2O) d 24.4; FABMS m/z
224.0 (M+H)+. ( )-8: 1H NMR (D2O) d 1.40–1.80 (m,
6H), 2.00–2.10 (m, 1H, PCH2CH), 2.20–2.25 (m, 1H,
PCH2), 2.25–2.50 (m, 1H, PCH2); 31P NMR (D2O) d 27.0;
FABMS m/z 224.0 (M+H)+.
3
(CDCl3, DEPT assignment) d 16.4 (d, JC–P = 6.3Hz,
POCH2CH3), 16.5 (d, JC–P = 5.6Hz, POCH2CH3),
3
3
24.1 (d, JC–P = 16.4Hz, PCH2CHCH2), 28.2 (d, JC–P
1
=
=
2
135.8Hz, PCH2), 32.9 (CH2C(CN)), 39.6 (d, JC–P
3
4.7Hz, PCH2CH), 54.9 (d, JC–P = 6.8Hz, NC(CN)),
=
2
62.1 (d, JC–P = 6.8Hz, POCH2CH3), 62.7 (d, JC–P
3
6.3Hz, POCH2CH3), 119.3 (CN), 127.4, 128.7, 132.1,
132.9 (Ph), 167.6 (C@O); 31P NMR (CDCl3) d 30.4; CIMS
1
m/z 351.2 (M+H)+. ( )-20: H NMR (CDCl3) d 9.04 (s,
19. Crystallographic data (excluding structure factors) for the
structure in this paper has been deposited with the
Cambridge Crystallographic Data Centre as the supple-
mentary publication number CCDC 248532. Copies of the
data can be obtained, free of charge, on application to
CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK [fax:
+44 (0)1223 336033 or e-mail: deposit@ccdc.cam.ac.uk].
20. Cells were seeded at 50,000 per well in poly D-lysine coated
96-well black walled clear bottom plates one day prior to
the assay. The day of the FLIPR assay cells were dye
loaded with 8lM Fluo-3AM (Molecular Probes, F-1241)
in FLIPR Buffer (HBSS Biowhittaker, 10-527F) supple-
mented with 20mM HEPES (Biowhittaker, 17-737E) at
25°C for 1.5h (mGluR1, 2, 5) or 2h (mGluR3, 4, 8). The
dye was removed from the plate and replaced with 50lL
per well of FLIPR Buffer, which was pre-warmed to 37°C.
The stage of the FLIPR was set to 34.5°C and the
compounds and glutamate (Sigma, G-1626) used in the
assay was also pre-warmed to 37°C. A two addition
FLIPR assay was performed by addition of compound
(50lL of 50lM compound in 2.5% DMSO for a final
concentration of 25lM in 1.25% DMSO) in the first
addition. The second addition was either an EC10
concentration of glutamate (for potentiator assays) or an
EC90 concentration of glutamate (for antagonist assays).
In both cases the volume of the second addition was
100lL resulting in a compound concentration of 12.5lM
(0.625% DMSO) in the second read. Data was collected
every sec for the first 30s and then every 3s until the
second addition was made at 2min. Following the second
addition data was collected every second for 30s and then
every 3s until the end of the run at total time of 3.25min.
21. Bertrand, H.-O.; Bessis, A. S.; Pin, J.-P.; Acher, F. C.
J. Med. Chem. 2002, 45, 3171.
1H, NH), 7.96–8.15 (m, 2H, Ph), 7.45–7.58 (m, 3H, Ph),
4.05–4.18 (m, 4H, POCH2CH3), 3.05–3.18 (m, 1H,
PCH2CH), 2.81–2.95 (m, 1H, PCH2CH), 2.05–2.67 (m,
4H), 1.85–2.00 (m, 1H), 1.20–1.35 (m, 6H, POCH2CH3);
13C NMR (CDCl3) d 16.8 (d, 3JC–P = 5.6Hz, POCH2CH3),
21.6 (PCH2CHCH2), 29.8 (PCH2), 32.8 (CH2C(CN)),
2
41.06 (d, JP–C = 4.6Hz, PCH2CH), 56.6 (NC(CN)), 62.2
(d, 2JP–C = 6.6Hz, POCH2CH3), 117.0 (CN), 128.4, 128.7,
128.8, 130.1, 134.2 (Ph), 164.6 (C@O); 31P NMR (CDCl3)
d 29.2; CIMS m/z 351.2 (M+H)+.
17. Spectral properties of ( )-21 and ( )-22. ( )-21: 1H NMR
(CDCl3) d 9.20 (s, 1H, NH), 7.95–7.99 (dd, 2H, Ph),
7.35–7.46 (m, 3H, Ph), 3.95–4.15 (m, 4H, POCH2CH3),
3.05–3.20 (m, 1H, PCH2CH), 2.43–2.60 (m, 1H), 1.60–
2.40 (m, 6H) 1.21 (t, J = 7.5Hz, 3H, POCH2CH3),
1.30 (t, J = 7.5Hz, 3H, POCH2CH3); 13C NMR (CDCl3,
DEPT assignment) d 16.5 (d, 3JP–C = 6.3Hz, POCH2CH3),
20.5 (CH2), 26.8 (d, 1JP–C = 138Hz, PCH2), 31.1 (d, 3JP–C
=
14.3Hz, PCH2CHCH2), 39.0 (CH2C(CN)), 43.2 (d,
2JP–C = 3.4Hz, PCH2CH), 61.2 (d, 3JP–C = 4.6Hz,
2
NC(CN)), 62.7 (d, JP–C = 6.3Hz, POCH2CH3), 119.2
(CN), 127.8, 128.6, 132.0, 133.2 (Ph), 167.6 (C@O); 31P
NMR (CDCl3) d 32.3; CIMS m/z 365.2 (M+H)+. ( )-22:
1H NMR (CDCl3) d 9.80 (s, 1H, NH), 7.95–7.99 (dd, 2H,
Ph), 7.35–7.46 (m, 3H, Ph), 3.95–4.15 (m, 4H,
POCH2CH3), 2.75–2.85 (m, 2H), 2.50–2.60 (m, 2H),
2.30–2.40 (m, 2H), 2.00–2.20 (m, 1H), 1.9–2.00 (m, 2H),
1.20–1.35 (m, 6H); 13C NMR (CDCl3, DEPT assignment)
3
d 16.6 (d, JP–C = 6.3Hz, POCH2CH3), 21.0 (CH2), 24.3
(d, JP–C = 139.7Hz, PCH2), 29.8 (d, JP–C = 6.9Hz,
1
3
2
PCH2CHCH2), 36.9 (CH2C(CN)), 43.7 (d, JP–C
=
3.4Hz, PCH2CH), 59.1 (d, JP–C = 4.6Hz, NC(CN)),
3
2
62.7 (d, JP–C = 6.9Hz, POCH2CH3), 121.2 (CN), 127.9,
128.7, 132.2, 133.8 (Ph), 167.8 (C@O); 31P NMR (CDCl3)
d 31.6; CIMS m/z 365.2 (M+H)+.
22. Rosemond, E.; Peltekova, V.; Naples, M.; Thogersen, H.;
Hampson, D. R. J. Biol. Chem. 2002, 277, 7333.