Neurochem Res DOI 10.1007/s11064-014-1332-0
ORIGINAL PAPER
Analogues of 3-Hydroxyisoxazole-Containing Glutamate Receptor Ligands Based on the 3-Hydroxypyrazole-Moiety: Design, Synthesis and Pharmacological Characterization Lars Jørgensen • Birgitte Nielsen • Darryl S. Pickering Anders S. Kristensen • Karla Frydenvang • Ulf Madsen • Rasmus P. Clausen
•
Received: 10 April 2014 / Revised: 9 May 2014 / Accepted: 10 May 2014 Ó Springer Science+Business Media New York 2014
Abstract A series of analogues of the glutamate receptor ligands (S)-2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propionic acid (AMPA) and AMOA were synthesized in which the 3-hydroxyisoxazole moiety was exchanged for a 3-hydroxypyrazole moiety. This exchange enables further substitution at the additional nitrogen atom in the heterocyclic core. Several of the analogues have activity at AMPA receptors equipotent to the antagonist ATPO, demonstrating that additional substitution can be accommodated in the antagonist binding site. Modelling studies offer an explanation for the pharmacological pattern observed for the compounds and suggest that this scaffold may be developed further to obtain subtype selective antagonists. Keywords Glutamate receptors 3-hydroxypyrazole AMPA Kainic acid Antagonists
Ulf Madsen and Rasmus P. Clausen have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s11064-014-1332-0) contains supplementary material, which is available to authorized users. L. Jørgensen B. Nielsen D. S. Pickering A. S. Kristensen K. Frydenvang U. Madsen (&) R. P. Clausen (&) Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, Copenhagen DK-2100, Denmark e-mail: [email protected] R. P. Clausen e-mail: [email protected]
Introduction (S)-Glutamic acid (1, Fig. 1) is the major excitatory neurotransmitter in the central nervous system, and is able to activate the highly heterogeneous group of glutamate receptors comprising both G-protein coupled metabotropic receptors (mGluRs) and ligand gated ionotropic receptors (iGluRs) [1]. These receptors are crucial in the normal function of the brain, but are also implicated in a range of disorders in the brain [2, 3]. Therefore, compounds that can affect these receptors are important pharmacological tools and may potentially lead to new drugs. The iGluRs have been divided into three major groups based on the selective activation by (S)-2-amino-3-(3-hydroxy-5methyl-isoxazol-4-yl)propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartic acid (NMDA) [1, 3] and this division is also reflected in the sequence homology within each pharmacological group. Each group is comprised of several cloned subunits and the functional ion channel consists of four of these subunits. Since both homo- and heteromeric combinations are functional, the heterogeneity of physiological iGluRs and thereby their pharmacological and biophysical properties are quite extensive. So far, 16 different iGluR subunits have been cloned and AMPA receptors are made up of GluA1-4 subunits, KA receptors of GluK1-5 subunits and NMDA receptors of GluN1, GluN2A-D, GluN3A-B. The recent X-ray crystal structure of a full-length tetrameric glutamate receptor [4] has confirmed that iGluRs consist of three domains: a transmembrane domain (TMD), an agonist binding domain (ABD) and an amino terminal domain (ATD) [5]. A large number of X-ray crystallographic studies of the ABD with various ligands have yielded important information on ligand recognition and understanding of the mechanisms governing receptor activation and desensitization. These studies have disclosed
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Neurochem Res R2
HO OH
N O
O
OH NH2
O
HO N O
R1 OH
R
NH2
AMPA (3a, R=Me) ATPA (3b, R=tBu)
OH
NH2 Ibotenic acid (2)
(S)-glutamic acid (1)
O
O N O
OH R2
N N R1
R2
NH2
NH2 R
AMOA (4a, R1=CO2H, R2=Me) ATOA (4b, R1=CO2H, R2=tBu) AMPO (5a, R=PO3H2, R2=Me) ATPO (5b, R=PO3H2, R2=tBu)
that the ABD has a clamshell-like structure and led to the hypothesis that ABD closes around agonists leading to opening of the ion-channel whereas antagonists stabilize an open form of the ABD maintaining a closed ion-channel. Ibotenic acid (2) is a natural product from the fly-agaric mushroom Amanita Muscaria, and is an agonist at both iGluRs and mGluRs [6]. The redesign of ibotenic acid led to the selective agonist AMPA (3a) [7] that is recognized by all 4 cloned AMPA (3a) receptor subunits (GluA1-4) [8]. Later ATPA (3b) was identified as a selective agonist of the KA receptor subunit GluK1. Addition of a methylene carboxylic acid to the 3-hydroxyisoxazole groups of AMPA and ATPA led to AMOA (4a) and ATOA (4b), respectively, and similar addition of a methylene phosphonic acid gave AMPO (5a) and ATPO (5b). [9] These four compounds are all AMPA receptor antagonists and (S)-ATPO has been shown to antagonize GluK1 and marginally discriminate AMPA receptor subtypes [10, 11]. X-ray crystallographic studies of ATPO in GluA2 and GluK1 have been reported [12, 13] and show that ATPO stabilizes an open conformation of the agonist binding domain of the receptor that is believed to correspond to an inactive state of the whole receptor. In the current study we explore the possibility of using 3-hydroxypyrazole as a bioisoster for the carboxylic acid instead of the 3-hydroxyisoxazole moiety from ibotenic acid. The 3-hydroxypyrazole has an increased pKa compared to the 3-hydroxyisoxazole [14, 15], but also an added possibility for substitution on the extra ring nitrogen. This is interesting since it enables insertion of substituents that may reach non-conserved regions of the receptor and thereby the possibility to design subtype selective ligands. We here present the synthesis and pharmacological characterization of 3-hydroxypyrazole analogues of the agonist AMPA and the antagonists AMOA and AMPO. The synthetic route allowed us to explore other isomeric analogues as well.
O N N
O
O
OH
N N R1
OH
6 (R1=Me, R2=Me) 7 (R1=Et, R2=Me) 8 (R1=Bn, R2=Me) (RS)-9 (R1=Me, R2=CF3)
O
NH2
10a (R1=H, R2=CO2H) 10b (R1=H, R2=PO3H2) 11a (R1=Me, R2=CO2H) 11b (R1=Me, R2=PO3H2) 12a (R1=Et, R2=CO2H) 12b (R1=Et, R2=PO3H2) 13a (R1=Bn, R2=CO2H) 13b (R1=Bn, R2=PO3H2)
OH
O
O
NH2
14a (R=CO2H) 14b (R=PO3H2)
Fig. 1 Structures of agonists (1, 2, 3a and 3b) and antagonists (4a, 4b, 5a, and 5b) at ionotropic glutamate receptors
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O
HO
O
O
OH
N N HO2C
NH2 15
Fig. 2 Synthesized 3-hydroxypyrazole analogues of AMPA (6, 7, 8 and (RS)-9) and of AMOA and AMPO (10a-b, 11a-b, 12a-b, 13a-b, 14a-b and 15)
Design We decided to synthesize AMPA analogues 6–8 (Fig. 2) where the 3-hydroxypyrazole is substituted at N1 with methyl, ethyl and benzyl to explore whether these substitutions can be accommodated in the ABD of the iGluRs. For the methyl substituted analogues we also synthesized a trifluoromethyl-AMPA analogue (RS)-9. Furthermore, 3-hydroxypyrazole analogues of AMOA and AMPO were synthesized, both N1-unsubstituted (10a and 10b) as well as N1-methyl (11a, 11b), N1-ethyl (12a, 12b) and N1benzyl (13a, 13b) substituted analogues to explore the binding pocket in the antagonized state of the iGluRs. Furthermore the synthetic route enabled us to prepare the analogues 14a, 14b and 15. Collectively, these pyrazole analogues allow us to investigate if substitution is allowed in the positions corresponding to the ring oxygen of AMPA and the ring oxygen and nitrogen of AMOA and AMPO.
Chemistry The synthetic route for most of the compounds started with tert-butyl N-Boc-pyroglutamate (16) (Scheme 1) that was deprotonated with LDA and acetylated with N-acetylimidazole to give 17. Reacting 17 with hydrazine gave 3-hydroxypyrazole 18 that was alkylated with ethyl bromoacetate or diethyl [(p-tosyloxy)methyl]phosphonate to give 19a and 19b, respectively. The ester 19a was hydrolyzed with NaOH followed by treatment with HCl yielding 10a. The phosphonoester 19b was deprotected with TMS-Br followed by
Neurochem Res Scheme 1 Synthetic routes to the new analogues. (a) LDA, Nacetylimidazole, THF, -78 to -20 °C. (b) NH2NH2, MeCN, reflux. (c) Ethyl bromoacetate, K2CO3, DMF, r.t. (d) Diethyl [(ptosyloxy)methyl]phosphonate, K2CO3, DMF, r.t. (e) TBS-Cl, Et3N, CH2Cl2, r.t. (f) (1) 4 M NaOH (aq), EtOH, r.t. (2) TFA, CH2Cl2, r.t. (g) (1) TMS-Br, CH3CN, r.t. 2) 1 M HCl (aq), reflux. (h) MeNHNH2, MeCN, reflux. (i) NaH, methyl iodide, DMF, -50 °C to r.t.. (j) NaH, ethyl bromide, DMF, -50 °C to r.t. (k) NaH, benzyl bromide, DMF, -50 °C to r.t.. (l) 1 M HCl (aq) or 1 M TFA (aq), reflux. (m) KF, DMF, r.t. (n) Iodomonochloride, AcOH, H2O, 85 °C. (o) n-BuLi, tert-butyl 2-[bis(tert-butoxycarbonyl) amino]acrylate, THF, -78 to -40 °C
O O-tBu
O O-tBu O
N Boc
a O
O
N Boc
b
16
HO
h
O-tBu
NHBoc N
O
N 20
17 O
c or d
O-tBu HO
O
NHBoc N
18 c, d, or e
O
O-tBu NHBoc
N N
O-tBu
R
O
O N H
NHBoc
+
N
EtO2C
O O-tBu
N
22a (R=CO2Et) 22b (R=PO3Et2)
32
RO f
NHBoc N
N H
15
f
14a, 14b
10a 10b
19a (R=CH2CO2Et) 19b (R=CH2PO3Et2) g
21 (R=TBS) i, j, or k
f or g
O
O
O
R2
O-tBu
O-tBu HO
TBSO NHBoc N
NHBoc
m N
N R
N
N R1
29a (R1=Me, R2=CO2Et) 29b (R1=Me, R2=PO3Et2) 30a (R1=Et, R2=CO2Et) 30b (R1=Et, R2=PO3Et2) 31a (R1=Bn, R2=CO2Et) 31b (R1=Bn, R2=PO3Et2)
f or g
l 11a, 11b, 12a, 12b, 13a, 13b
6, 7, 8
NHBoc
c or d
N R
26 (R=Me) 27 (R=Et) 28 (R=Bn)
23 (R=Me) 24 (R=Et) 25 (R=Bn)
O-tBu O
O
O O-tBu
R
HO
I
TBSO e
N
n
N
CF3
33 (R=H) 34 (R=I)
HCl treatment giving 10b. When 17 was reacted with methyl hydrazine compound 20 was obtained as the only isomer, which was alkylated with ethyl bromoacetate or diethyl [(ptosyloxy)methyl]phosphonate to give 22a and 22b, respectively. These were deprotected with the same conditions as 19a and 19b to give 14a and 14b, respectively. TBS protection of the 3-hydroxy group in 18 gave compound 21, which was selectively alkylated at N1 in the pyrazole ring with methyl iodide, ethyl and benzyl bromide to give 23, 24 and 25. These were deprotected in aqueous HCl to yield 6, 7
HO NBoc2
o N
N 35
OH
TBSO CF3
N
N
CF3
(RS)-36
NH2
l N
N
CF3
(RS)-9
and 8, respectively. Selective removal of the TBS group of 23–25 yielded 26–28 that could be alkylated with ethyl bromoacetate or diethyl [(p-tosyloxy)methyl]phosphonate to give 29a, 30a, 31a or 29b, 30b, 31b, respectively. These were deprotected under the same conditions as 19a and 19b to give 11a, 11b, 12a, 12b, 13a, and 13b. The isomeric assignment based on 1H NMR was confirmed by an X-ray crystal structure of 11b (Fig. 3). Alkylation of 20 with ethyl bromoacetate also yielded 32 as a by-product that could be deprotected under the same conditions as 19a to give 15.
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Fig. 3 Perspective drawing of compound 11b. Displacement ellipsoids of the non-hydrogen atoms are shown at the 50 % probability level. Hydrogen atoms are represented by spheres of arbitrary size
were 14a, 14b and 15. Furthermore, all compounds were probed for activity towards cloned homomeric GluK1 and GluK3 subunits expressed in Sf9 insect cells at a concentration of 100 lM, since these activities are not revealed in the KA binding assay on rat cortical synaptosomes. In this binding assay only compound (RS)-9 could reduce the radioligand binding towards GluK1 to \50 %. At GluK3 only compound 11a reduced radioligand binding below 50 %. Thus, the GluK1 activity is abolished in these analogues compared to ATPO, but for 11a a modest GluK3 affinity is observed. The functional activity of the active analogues were assessed in two-electrode voltage clamp experiments at GluA2 expressing Xenopus oocytes (Fig. 4). Analogues 6 and (RS)-9 showed agonist activity whereas 11a, 11b, 12a and 12b showed antagonist activity at glutamate induced agonist responses.
Docking The synthesis of (RS)-9 followed a different route starting with 1-methyl-5-(trifluoromethyl)-pyrazol-3-ol (33) that was iodinated with ICl to give 34. This compound was TBS protected with TBS-Cl to give 35 followed by treatment with n-BuLi and tert-butyl 2-[bis(tert-butoxycarbonyl)amino]acrylate giving 36. This was followed by treatment with HCl giving (RS)-9.
Pharmacology The compounds were characterized pharmacologically by investigation of their binding affinity towards the major iGluR groups, AMPA, KA and NMDA receptors in radioligand binding assays using rat cortical synaptosomes and [3H]AMPA, [3H]KA and [3H]CGP39653 as ligands, respectively (Table 1). The AMPA analogue 6 with an Nmethyl substituent on the heterocycle showed weak activity towards AMPA binding sites but no activity towards KA and NMDA binding sites. Compounds 7 and 8 were both inactive in all binding assays. Compound (RS)-9 showed slightly increased activity at AMPA binding sites compared to 6, but no activity at KA and NMDA sites. The AMOA and AMPO analogues were also probed for binding affinity towards the major iGluR groups. The analogues 10a and 10b were inactive at all sites. In contrast, N-methyl substituted 11a and 11b as well as ethyl substituted 12a and 12b showed affinity for AMPA binding sites but not for KA and NMDA binding sites. Thus, the carboxylates were more potent than phosphonates and ethyl substituted slightly more active than methyl substituted while benzyl substituted 13a and 13b were inactive as
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To get an impression of the binding modes of the pyrazole compounds we docked 10a, 11a and 12a in the X-ray crystal structures of ATPO in GluA2 and GluK1. These suggested that the compounds displayed binding modes resembling ATPO as exemplified in Fig. 5 showing 11a docked in GluA2. Thus, the amino acid moiety and heterocycle of ATPO and the new analogues are overlapping. The order of the docking scores reflected the order of activity in the binding assay (10a \ 11a < 12a) in GluA2. Furthermore, it seemed that the higher activity could be ascribed to positive Van Der Waals interactions of the Nmethyl group in 11a with M708 and of the N-ethyl group (not shown) in 12a with M708 and T686 (not shown). These interactions are predicted in a grid calculation of the GluA2 ATPO structure [12]. These residues are conserved among AMPA receptor subtypes, but correspond to S741 and S721 and GluK1, and T743 and N723 in GluK3. Thus, the changed pharmacological profile can be explained by interactions with non-conserved residues in this region. In the binding mode of 12a the methyl (Me) group on the heterocycle point into the conserved hydrophobic region that is occupied by the tert-butyl group of ATPO, whereas the N-ethyl group (Et) points towards a region containing residues that are non-conserved (indicated with red colors) not only among the major iGluR groups but also among the otherwise highly homologous AMPA receptor subtypes. This suggests that extended substitution in this direction could lead to subtype selective AMPA receptor antagonists, although the lack of activity in compounds 13a and 13b suggest that there is not enough room for accommodating very large groups like the benzyl group.
Neurochem Res Table 1 Receptor binding affinities of compounds at the three major groups of iGluRs in rat cortical synaptosome assays and at cloned rat kainate receptors GluK1 and GluK3. The numbers in brackets [min; max] indicate mean ± SEM according to a logarithmic distribution
CGP39653 is a competitive NMDA receptor antagonist and AMPA and KA are agonists at AMPA and KA receptors, respectively. Ki values are calculated using GraphPad Prism a
Vogensen et al. [8]
b
Madsen et al. [9]
c
3
[ H]CPP binding
d
Møller et al. [11]
nd not determined
[3H]AMPA
[3H]KA
[3H]CGP39653
% Binding (100 lM) or Ki
IC50 (lM)
IC50 (lM)
Ki (lM)
GluK1
GluK3
AMPA
0.021a
24a
[100a
1.15 ± 0.51 lM (Ki)
43 ± 13 lM (Ki)
(RS)AMOA
90b
[100b
[100b,c
84 ± 18 lM (Ki)
[100 lM (Ki)
(RS)ATOA
33b
[100d
[100b,c
4.1 ± 0.2 lM (Ki) [100 lM (Ki)
(RS)AMPO
31b
[100b
[100b,c
19 ± 3 lM (Ki)
(RS)ATPO
35b (16 (S-)d)
[100b
[100b,c
2.6 ± 0.7 lM (Ki) [100 lM (Ki)
6
8.9 [8.3;9.5] [5.05 ± 0.03]
[100
[100
91
91
7
[100
[100
[100
75
93
8
[100
[100
[100
96
111
(RS)-9
[100
[100
44
81
10a
3.6 [3.5;3.7] [5.45 ± 0.01] [100
[100
[100
93
74
10b
[100
[100
[100
65
86
11a
23 [22;24] [4.64 ± 0.02]
[100
[100
76
33
11b
77 [75;79] [4.11 ± 0.01]
[100
[100
65
68
12a
31 [30;32] [4.51 ± 0.01]
[100
[100
70
68
12b
87 [83;91] [4.06 ± 0.02]
[100
[100
51
95
13a
[100
[100
[100
91
91
Compound
[100 lM (Ki)
13b
[100
[100
[100
nd
nd
14a
[100
[100
[100
105
109
14b
[100
[100
[100
106
111
15
[100
[100
[100
110
104
Conclusion We have synthesized a series of 3-hydroxypyrazole analogues of the agonist AMPA and the antagonists AMPO and AMOA. The synthesis offers an efficient stereoselective route for making such analogues with differing substituents on the 3-hydroxypyrazole moiety. N1 alkylated pyrazole analogues of AMPA showed very weak or no activity at AMPA receptors, whereas some of the pyrazole analogues of AMOA and AMPO showed activities comparable to the parent compounds. Interestingly the pyrazole analogues showed the reverse order of activity for the carboxy versus the phosphonate analogues compared to AMOA and AMPO. Thus, the carboxy analogues in the pyrazole series (11a, 12a) showed higher potency than the phophonate analogues (11b, 12b), whereas for the isoxazoles the phosphonate analogue (AMPO) shows higher activity than the carboxy analogue AMOA. Docking results
explain the difference in activity between unsubstituted 10a and 11a/12a, with hydrophobic VDW interactions with M708. This amino acid is not conserved in GluK1 and GluK3 and thus offers an explanation for the change in selectivity profile when substituents protrude into this region. The pyrazole analogues offer, in contrast to isoxazoles, the possibility for substitution at the N1 position, and the results observed indicate that further elaboration of the substituent pattern on such pyrazole analogues may lead to subtype selective compounds.
Experimental Section Chemistry General procedures. All reactions involving dry solvents or sensitive reagents were performed under a nitrogen or
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Neurochem Res
Fig. 4 Representative current responses obtained from a two-electrode voltage-clamp recording on Xenopus oocytes expressing homomeric GluA2(Q)i receptors. Currents were evoked by compounds alone (top) or co-application of Glu and the compounds at 100 lM (bottom)
Fig. 5 Left X-ray crystal structure GluA2 ABD (PDB:1N0T) showing the binding mode of (S)-ATPO (grey carbons) together with the binding mode of compound N-methyl substituted 11a proposed from docking into the X-ray structure. Right Proposed docking mode of N-
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argon atmosphere and glassware was flame dried prior to use under vacuum. Solvents were dried according to standard procedures, and reactions were monitored by analytical thin-layer chromatography (TLC) analysis. TLC was carried out using Merck silica gel 60 F254 aluminum sheets. The compounds were detected as single spots on TLC plates (using n-BuOH/water/EtOAc/AcOH 1/1/1/1) and visualized using UV light (254 nm) and staining reagents (ex. Ninhydrin). Flash chromatography was carried out using Merck silica gel 60A (35–70 lm). 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recorded on a 300 MHz Varian Mercury 300BB. Chemical shifts (d) are given in parts per million (ppm), and coupling constants (J) are given in Hertz (Hz). Optical rotations were measured on a Perkin-Elmer 241 polarimeter. Elemental analysis was performed by Mr. J. Theiner at the University of Vienna. Melting point was determined in an open capillary tube and is uncorrected. All purchased chemicals were used without further purification. Pyrazole regioisomers were established by both 13C-NMR signal assignment and 1H-NMR NOESY experiments showing correlation between the N-alkyl group and other substituents in the ring. In case of 11b this was further confirmed with an X-ray crystal structure determination. (S)-2-amino-3-(3-hydroxy-1,5-dimethyl-1H-pyrazol4-yl)propanoic acid (6) A solution of 23 (100 mg, 0.213 mmol) in 1 M HCl (15 ml) was refluxed for 45 min. The solvent was evaporated in vacuo, and the residue was re-evaporated twice from water. The crude
ethyl substituted 12a docked in PDB:1N0T. The red regions represent regions containing residues that are not fully conserved among AMPA receptor subunits GluA1–GluA4
Neurochem Res
product was purified by HPLC (X-Terra Ò Prep MS C 18 10 lm, 10 9 300 mm; 15 mM AcOH (aq); flow: 10 ml/min). The product was re-crystallized from water to give colorless crystals of 6 (29 mg, 68 %). R f = 0.24. Mp. [ 240 °C. 1H NMR (D2O): d 2.13 (s, 3 H), 2.86 (d, J = 5 Hz, 2 H), 3.50 (s, 3 H), 3.88 (t, J = 5 Hz, 1 H). 13C NMR (D2O): d 9.5, 24.4, 34.3, 55.9, 97.8, 145.8, 162.5, 174.3. EA: calcd for C 8 H 13 N 3 O 3 , C: 48.23, H: 6.58, N: 21.09; found: C: 48.01, H: 6.49, N: 20.89. [a]365 = -198 (c = 0.18, 0.04 M HCl). (S)-2-amino-3-(1-ethyl-3-hydroxy-5-methyl-1H-pyrazol-4-yl)propanoic acid (7) Compound 24 (960 mg, 1.98 mmol) was dissolved in 1 M TFA (aq) (30 ml) and the mixture was refluxed for 2 h. The solvent was evaporated and the residue was re-evaporated twice from water. The residue was dissolved in water (2 ml) and ethanol (10 ml), and the pH was adjusted to 3–4 by addition of triethylamine. No crystallization was observed. Therefore the mixture was evaporated and the residue was subjected to reverse-phase chromatography (RP-18 silicagel, AcOH/ H2O 1/99 to AcOH/MeCN/H2O 1/4/95). Recrystallization using water and 2-propanol gave colorless crystals of 7 (190 mg, 0.79 mmol, 40 %).1H NMR (D2O): d 1.24 (t, J = 7.2 Hz, 3 H), 2.16 (s, 3 H), 2.86 (d, J = 5.4 Hz, 2 H), 3.89 (t, J = 5.3 Hz, 1 H), 3.89 (q, J = 7.2 Hz, 2 H).13C NMR (D2O): d 9.4, 14.2, 24.4, 43.0, 55.9, 98.4, 145.3, 162.9, 174.2. Mp. = 210 °C (decomp.). EA: calcd for C9H15N3O31 H2O, C: 44.99, H: 7.55, N: 17.49; found: C: 45.34, H: 7.71, N: 17.44. (S)-2-amino-3-(1-benzyl-3-hydroxy-5-methyl-1H-pyrazol-4-yl)propanoic acid (8) Compound 25 (350 mg, 0.641 mmol) was stirred at 70 °C in 1 M HCl (15 ml) for 4 h. The solvent was evaporated and re-evaporated twice from water. The residue was dissolved in water (0.5 ml) and ethanol (2.5 ml). The pH was adjusted to 3–4 by addition of triethylamine. No crystallization was observed. Therefore the mixture was evaporated and subjected to reverse-phase chromatography (RP-18 silicagel, AcOH/ H2O 1/99 to AcOH/MeCN/H2O 1/9/90). Recrystallization using water/2-propanol gave colorless crystals of 8 (105 mg, 59 %). 1H NMR (300 MHz, D2O): d 2.19 (s, 3 H), 2.90 (d, J = 5.5 Hz, 2 H), 3.90 (t, J = 5.5 Hz, 1 H); 5.10 (s, 2 H), 7.11–7.44 (m, 5 H). 13C NMR (75 MHz, D2O): d 9.8, 24.4, 51.2, 55.8, 99.0, 127.1, 128.6, 129.5, 136.3, 145.6, 162.7, 174.2. Mp. [ 230 °C. EA: calcd for C14H17N3O3, C: 61.08, H: 6.22, N: 15.26; found: C: 60.98, H: 6.03, N: 15.13. (RS)-2-amino-3-(3-hydroxy-1-methyl-5-(trifluoromethyl)-1H-pyrazol-4-yl)propanoic acid ((RS)-9) Aqueous 1 M HCl (15 ml) was stirred and heated to reflux temperature. A solution of 36 (0.44 g, 0.71 mmol) in 1,4dioxane (5 ml) was added dropwise. The solution was
allowed to reflux for 3 h. The solvent was evaporated and the residue was re-evaporated twice from water. The crude product was subjected to reverse-phase chromatography (RP-18 silicagel, AcOH/H2O 1/99). Recrystallization with water gave (RS)-9 (126 mg, 71 %) as colorless crystals.1H NMR (300 MHz, D2O): d 3.01 (dd, J = 7.7, 15.6 Hz, 1 H), 3.10 (dd, J = 5.8, 15.7 Hz, 1 H), 3.77 (s, 3 H), 3.86 (dd, J = 7.6, 5.6 Hz, 1 H). 13C NMR (75 MHz, D2O with NaOD): d 29.0, 37.0, 57.4, 104.8, 121.2 (q, J = 269.3 Hz), 130.0 (q, J = 36.1 Hz), 168.1, 183.1. Mp. [ 220 °C. EA: calcd for C8H10F3N3O3 H2O, C: 36.03, H: 4.35, N: 15.76; found: C: 35.89, H: 4.55, N: 15.54. (S)-2-amino-3-(3-(carboxymethoxy)-1,5-dimethyl1H-pyrazol-4-yl)propanoic acid (10a) Compound 19a (300 mg, 0.70 mmol) was dissolved in EtOH (20 ml) and 4 M NaOH (3 ml) was added. The mixture was stirred for 2 h. The volume was reduced in vacuo to 3 ml. Water (50 ml) and EtOAc (50 ml) were added. The pH of the aqueous phase was adjusted to 3–4 with 1 M HCl and extracted with EtOAc (3 9 50 ml). The combined extracts were dried over MgSO4 and the solvent was removed in vacuo. The residue was dissolved in CH2Cl2 (10 ml) and TFA (10 ml) and stirred at r.t. for 3 h. The solvent was evaporated. Re-evaporation twice from water and twice from 1 M HCl gave crystals, which were dissolved in as little water as possible. Propylenoxide (few drops) were added, giving precipitation of crystals. Recrystallization twice from water gave colorless crystals of 10a (40 mg, 22 %). Rf = 0.36. 1H NMR (300 MHz, D2O): d 2.18 (s, 3 H), 3.00 (dd, J = 15.7, 6.1 Hz, 1 H), 3.02 (dd, J = 15.7, 4.9 Hz, 1 H),3.96–4.07 (m, 1 H), 4.70 (d, J = 16.2 Hz, 1 H), 4.77 (d, J = 16.3 Hz, 1 H). 13C NMR (75 MHz, D2O): d 9.7, 23.4, 55.2, 66.5, 95.7, 142.0, 161.2, 173.6, 175.2. Mp. = 197–199 °C. EA: calcd for C9H13N3O5H2O, C: 41.38, H: 5.79, N: 16.09; found: C: 41.50, H: 5.96, N: 16.10. [a]365 = -9.68 (c = 0.15, 0.04 M HCl). (S)-2-amino-3-(3-(phosphonomethoxy)-1,5-dimethyl1H-pyrazol-4-yl)propanoic acid (10b) Compound 19b (400 mg, 0.814 mmol) was dissolved in CH3CN (20 ml). TMSBr (0.50 ml, 3.86 mmol) was added and the mixture was stirred for 5 h. The solvent was evaporated and the residue was re-dissolved in 1 M HCl (15 ml). The mixture was refluxed for 24 h. The solvent was removed in vacuo. The residue was re-evaporated twice from water. The product was crystallized by dissolving it in water and adding propyleneoxide (few drops). Recrystallization from water gave colorless crystals of 10b (130 mg, 54 %). Rf = 0.16. 1H NMR (300 MHz, D2O): d 2.18 (s, 3 H), 3.01 (dd, J = 15.8, 6.1 Hz, 1 H), 3.04 (dd, J = 15.7, 5.0 Hz, 1 H), 4.07 (dd, J = 6.0, 5.3 Hz, 1 H), 4.24 (dd, J = 17.0, 9.1 Hz, 1 H), 4.28 (dd, J = 17.0, 9.0 Hz, 1 H). 13 C NMR (75 MHz, D2O with NaOD): d 10.1, 27.9, 56.8, 67.0 (d, J = 151 Hz), 98.5, 141.9, 163.9 (d, J = 12.5 Hz),
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183.3. Mp. [ 230 °C. EA: calcd for C8H14N3O6PH2O, C: 32.33, H: 5.43, N: 14.14; found: C: 32.02, H: 5.65, N: 13.83. [a]365 = ?6.98 (c = 0.23, 0.5 M HCl). (S)-2-amino-3-(3-(carboxymethoxy)-1,5-dimethyl-1Hpyrazol-4-yl)propanoic acid (11a) To a solution of 29a (220 mg, 0.499 mmol) in EtOH (10 ml) was added 4 M NaOH (10 ml). The mixture was stirred for 3 h. Water (10 ml) was added and the solvent was reduced to 10 ml. 1 M KHSO4 (80 ml) was added and the mixture was extracted with EtOAc (5 9 100 ml). The combined extracts were washed with brine (50 ml), dried over MgSO4, and the solvent was evaporated. The residue was dissolved in CH2Cl2 (5 ml) and TFA (5 ml) and stirred for 1 h. The solvent was evaporated. The crude product was purified by HPLC (X-TerraÒ Prep MS C18 10 lm, 10 9 300 mm; 15 mM AcOH (aq); flow: 10 ml/min). Evaporation three times with water followed by recrystallization from water yielding colorless crystals of 11a (78 mg, 60 %). Rf = 0.30. 1H NMR (300 MHz, D2O): d 2.15 (s, 3 H), 2.98 (dd, J = 6.0, 15.7 Hz, 1 H), 3.01 (dd, J = 4.9, 15.7 Hz, 1 H), 3.57 (s, 3 H), 4.01 (dd, J = 4.9, 5.9 Hz, 1 H), 4.68 (d, J = 16.2 Hz, 1 H), 4.75 (d, J = 16.2 Hz, 1 H). 13C NMR (75 MHz, D2O): d 9.7, 23.7, 35.6, 55.3, 66.4, 96.0, 142.0, 159.7, 173.5, 175.1. Mp. [ 240 °C. EA: calcd for C10H15N3O5H2O, C: 45.89, H: 5.97, N: 16.05; found: C: 45.74, H: 5.71, N: 15.94. [a]365 = -228 (c = 0.25, 0.04 M HCl). (S)-2-amino-3-(3-(phosphonomethoxy)-1,5-dimethyl1H-pyrazol-4-yl)propanoic acid (11b) To a solution of 29b (300 mg, 0.594 mmol) in CH3CN (30 ml) was added TMSBr (1.5 ml, 11.6 mmol) and stirred for 24 h. The solvent was evaporated. Water was added and evaporated twice. The crude product was purified by HPLC (X-TerraÒ Prep MS C18 10 lm, 10 9 300 mm; 15 mM AcOH (aq); flow: 10 ml/min). Evaporation three times with water followed by recrystallization from water yielding colorless crystals of 11b (50 mg, 30 %). Rf = 0.13. 1H NMR (300 MHz, D2O): d 2.15 (s, 3 H), 3.02 (dd, J = 6.1, 15.7 Hz, 1 H), 3.05 (dd, J = 5.4, 15.7 Hz, 1 H), 3.60 (s, 3 H), 4.16 (t, J = 5.5 Hz, 1 H), 4.19 (dd, J = 9.2, 12.8 Hz, 1 H), 4.25 (dd, J = 9.1, 12.9 Hz, 1 H). 13C NMR (75 MHz, D2O): d 9.6, 23.5, 35.6, 54.2, 65.4 (d, J = 157.5 Hz), 95.4, 142.2, 160.8 (d, J = 12.2 Hz), 172.4. [a]365 = -238 (c = 0.25, 0.04 M HCl). (S)-2-amino-3-(3-(carboxymethoxy)-1-ethyl-5-methyl1H-pyrazol-4-yl)propanoic acid (12a) Compound 30a (1.00 g, 2.19 mmol) was dissolved in a mixture of EtOH (20 ml) and 4 M NaOH (10 ml) and stirred at r.t. for 4 h. Water (20 ml) was added and the mixture was evaporated to 15 ml. 1 M KHSO4 (15 ml) was added and the mixture was extracted with EtOAc (5 9 100 ml). The combined extracts were washed with brine, dried over MgSO4, and the solvent was evaporated. The residue was dissolved in CH2Cl2 (10 ml) and TFA (10 ml) and then stirred at r.t. for 1 h, followed by
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evaporation of solvent and re-evaporation twice from water. Recrystallization of the crude product using water and 2-propanol gave 12a (400 mg, 67 %) as colorless crystals. 1H NMR (300 MHz, D2O): d 1.24 (t, J = 7.2 Hz, 3 H), 2.17 (s, 3 H), 2.92–3.07 (m, 2 H), 3.93 (q, J = 7.2 Hz, 2 H), 4.03 (br t, J = 5.4 Hz, 1 H), 4.71 (d, J = 16.3 Hz, 1 H), 4.78 (d, 1 H). 13 C NMR (75 MHz, D2O with NaOD): d 9.6, 15.2, 28.2, 43.7, 57.0, 67.9, 99.1, 140.4, 160.4, 177.1, 183.0. Mp. [ 230 °C. EA: calcd for C11H17N3O5, C: 48.70, H: 6.32, N: 15.49; found: C: 48.73, H: 6.21, N: 15.33. (S)-2-amino-3-(3-(phosphonomethoxy)-1-ethyl-5methyl-1H-pyrazol-4-yl)propanoic acid (12b) Compound 30b (1.00 g, 1.92 mmol) was dissolved in MeCN (60 ml). TMSBr (3.0 ml, 23.2 mmol) was added. The mixture was stirred at r.t. for 24 h and then the solvent was evaporated. The residue was re-dissolved in CH2Cl2 (20 ml) and TFA (20 ml), and then stirred for 3 h at r.t. The solvent was evaporated followed by re-evaporation twice from water. Recrystallization using water and 2-propanol gave colorless crystals of 12b (485 mg, 82 %). 1 H NMR (300 MHz, D2O): d 1.27 (t, J = 7.2 Hz, 3 H), 2.17 (s, 3 H), 3.04 (m, 2 H), 3.96 (q, J = 7.2 Hz, 2 H), 4.18 (t, J = 5.6 Hz, 1 H), 4.18–4.33 (m, 2 H). 13C NMR (75 MHz, D2O with NaOD): d 9.4, 15.1, 23.5, 44.0, 54.1, 65.5 (d, J = 158 Hz), 95.6, 141.2, 160.9 (d, J = 12.4 Hz), 172.3. Mp. [ 230 °C. EA: calcd for C10H18N3O6P, C: 39.09, H: 5.91, N: 13.68; found: C: 38.96, H: 5.63, N: 13.42. (S)-2-amino-3-(3-(carboxymethoxy)-1-benzyl-5methyl-1H-pyrazol-4-yl)propanoic acid (13a) Compound 31a (450 mg, 0.869 mmol) was dissolved in EtOH (15 ml) and 2 M NaOH (15 ml). The mixture was stirred at r.t. for 3 h. Water (15 ml) was added and the mixture was evaporated to 15 ml. 1 M NaHSO4 was added and extracted with EtOAc (3 9 100 ml). The combined extracts were washed with brine, dried over MgSO4, and the solvent was evaporated. The residue was dissolved in CH2Cl2 (15 ml) and TFA (15 ml) and stirred at r.t. for 1 h. After evaporation of the solvent and re-evaporation twice from water, recrystallization from water gave colorless crystals of 13a (224 mg, 76 %). 1H NMR (300 MHz, D2O): d 2.14 (s, 3 H), 2.95–3.10 (m, 2 H), 3.98 (t, J = 5.3 Hz, 1 H), 4.65 (d, J = 16.0 Hz, 1 H), 4.73 (d, J = 16.0 Hz, 1 H), 5.17 (s, 2 H), 7.00–7.40 (m, 5 H). 13C NMR (75 MHz, D2O with NaOD): d 9.9, 28.25, 51.9, 56.9, 67.9, 100.1, 126.9, 128.1, 129.4, 138.0, 141.5, 160.9, 177.0, 183.0. Mp. [ 230 °C. EA: calcd for C16H19N3O5 H2O, C: 56.88, H: 5.82, N: 12.44; found: C: 56.69, H: 6.01, N: 12.30. (S)-2-amino-3-(3-(phosphonomethoxy)-1-benzyl-5methyl-1H-pyrazol-4-yl)propanoic acid (13b) Compound 31b was dissolved in CH3CN (30 ml) and TMSBr (1.5 ml) was added. The solution was allowed to stir for
Neurochem Res
22 h at r.t. and the solvent was evaporated. The residue was dissolved in CH2Cl2 (5 ml) and TFA (5 ml) and stirred for 1 h. The solvent was evaporated. The crude product was dissolved in aqueous 1 M TFA (30 ml) and the solution was washed with EtOAc (4 9 30 ml). By evaporation of the aqueous solution colorless crystals were formed. Recrystallization using water gave colorless crystals of 13b (96 mg, 19 % for 2 steps). 1H NMR (300 MHz, D2O): d 2,15 (s, 3H); 2,94–3,10(2 9 dd, 2H, J = 5,0 and 6,1 and 15,8 Hz); 4,0 (t, IH, J = 5,5 Hz); 4,16–4,30 (2 x dd, 2H, J = 9,0 and 9,2 and 15,8 Hz); 5,19 (s, 2H); 7,05–7,45 (m, 5H). 13C NMR (75 MHz, D2O with NaOD): d 9,9; 28,2; 51,9; 56,9; 67,2 (d, J = 151 Hz,); 100,1; 126,9; 128,l; 129,4; 138,0; 141,6; 162,9; 183,1. Mp. [ 230 °C. EA: calcd for C15H20N3O6P, C: 48.78, H: 5.46, N: 11.38; found: C: 48.54, H: 5.37, N: 11.25. (S)-2-amino-3-(5-(carboxymethoxy)-1,3-dimethyl-1Hpyrazol-4-yl)propanoic acid (14a) A solution of 22a (250 mg, 0.566 mmol) in EtOH (20 ml) was added 4 M NaOH (3 ml) and stirred for 2 h. Water (10 ml) was added and the volume was reduced to 10 ml. 1 M KHSO4 (30 ml) was added and the mixture was extracted with EtOAc (9 9 40 ml). The combined extracts were washed with brine (30 ml), dried over MgSO4, and the solvent was evaporated in vacuo. The residue was dissolved in CH2Cl2 (5 ml) and TFA (5 ml) and stirred for 2 h. The solvent was evaporated. The residue was evaporated twice from 1 M HCl. The residue was purified on a strong basic ionexchange column [Amberlite IRA-400(Cl)] eluting with 1 M AcOH. The product was evaporated twice from water, triturated with EtOAc. The crude product was purified by HPLC [X-TerraÒ Prep MS C18 10 lm, 10 9 300 mm; 15 mM AcOH (aq); flow: 10 ml/min]. The product was reevaporated twice from water to give 14a (73 mg, 48 %) as colorless crystals. Rf = 0.36. 1H NMR (300 MHz, D2O): d 2.18 (s, 3 H), 3.01 (dd, J = 6.6, 15.6 Hz, 1 H), 3.06 (dd, J = 6.1, 15.6 Hz, 1 H), 3.69 (s, 3 H), 3.97 (t, J = 6.3 Hz, 1 H), 4.62 (d, J = 15.5 Hz, 1 H), 4.68 (d, J = 15.6 Hz, 1 H). 13 C NMR (75 MHz, D2O): d 11.6, 24.0, 34.1, 54.5, 72.3, 99.5, 147.4, 153.2, 173.2, 174.6. Mp. = 155 °C (decomp.). EA: calcd for C10H15N3O5H2O, C: 44.36, H: 6.14, N: 15.52; found: C: 44.25, H: 5.79, N: 15.33. [a]365 = ?468 (c = 0.24, 0.04 M HCl). (S)-2-amino-3-(5-(phosphonomethoxy)-1,3-dimethyl1H-pyrazol-4-yl)propanoic acid (14b) A solution of 22b (400 mg, 0.791 mmol) in CH3CN (30 ml) was added TMSBr (1.60 ml, 12.3 mmol) and stirred for 4 days. The solvent was evaporated. The residue was dissolved in CH2Cl2 (10 ml) and TFA (10 ml) and stirred for 1 h. The solvent was evaporated. Water was added and evaporated, twice. The residue was triturated with EtOAc. The crude product was purified by HPLC (X-TerraÒ Prep MS C18 10 lm, 10 9 300 mm; 15 mM AcOH (aq); flow: 10 ml/
min). The product was re-evaporated twice from water to give 14b (183 mg, 75 %) as colorless crystals. Rf = 0.30. 1 H NMR (300 MHz, D2O): d 2.19 (s, 3 H), 3.07 (dd, J = 6.5, 15.6 Hz, 1 H), 3.11 (dd, J = 6.2, 15.6 Hz, 1 H), 3.72, (s, 3 H), 4.00 (t, J = 6.3 Hz, 1 H), 4.25 (dd, J = 8.9, 12.9 Hz, 1 H), 4.30 (d, J = 8.7, 12.8 Hz, 1 H). 13C NMR (75 MHz, D2O): d 11.3, 23.7, 34.0, 54.1, 71.6 (d, J = 154 Hz), 100.1, 147.0, 154.4 (d, J = 11.0 Hz), 172.7. Mp. = 195 °C (decomp.). EA: calcd for C9H16N3O6 PH2O, C: 35.24, H: 5.75, N: 13.70; found: C: 35.38, H: 5.77, N: 13.68. [a]365 = ?358 (c = 0.19, 0.04 M HCl). (S)-2-amino-3-(1-(carboxymethyl)-2,5-dimethyl-3oxo-2,3-dihydro-1H-pyrazol-4-yl)propanoic acid (15) To a solution of 32 (200 mg, 0.452 mmol) in EtOH (10 ml) was added 4 M NaOH (10 ml). The mixture was stirred for 2 h. Water (10 ml) was added and the solvent was reduced to 6 ml. 1 M KHSO4 (70 ml) was added and the mixture was extracted with EtOAc (10 9 80 ml). The combined extracts were washed with brine (50 ml), dried over MgSO4, and the solvent was evaporated. The residue was dissolved in CH2Cl2 (5 ml) and TFA (5 ml) and stirred for 2 h. The solvent was evaporated. The crude product was purified by HPLC (X-TerraÒ Prep MS C18 10 lm, 10 9 300 mm; 15 mM AcOH (aq); flow: 10 ml/min). Evaporation three times with water followed by recrystallization from water yielding colorless crystals of 15 (89 mg, 0.328 mmol, 73 %). Rf = 0.23. 1H NMR (300 MHz, D2O): d 2.17 (s, 3 H), 2.94 (dd, J = 6.4, 15.9 Hz, 1 H), 2.97 (dd, J = 5.1, 15.7 Hz, 1 H), 3.40 (s, 3 H), 4.11 (bs, 1 H), 4.69 (s, 2H). 13C NMR (75 MHz, D2O): d 9.9, 24.3, 29.1, 47.7, 54.5, 99.2, 147.3, 163.3, 172.9. Mp. 176 °C (decomp). EA: calcd for C10H15N3O53/4H2O, C: 44.36, H: 6.14, N: 15.52; found: C: 44.68, H: 5.87, N: 15.60.
Pharmacology Binding Affinity at Native Receptors All native receptor binding experiments were performed using rat brain synaptic membranes of cortex and the central hemisphere from male SPRD rats with tissue preparation as described in the literature [16]. Affinity for AMPA, KA, and NMDA receptors were determined using 5 nM [3H]AMPA [17], 5 nM [3H]KA [18], and 2 nM [3H]CGP 39653 [19], respectively, with modifications previously described [20]. Binding Affinity at Cloned Receptors Cloned rat GluK1 and GluK3 receptors were expressed in sf9 insect cells and ligand binding evaluated as previously
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described using either [3H]-(2S,4R)-4-methylglutamic acid (47.9 Ci/mmol; ARC Inc., St. Louis, MO) or [3H]Vinylidene kainic acid (44.0 Ci/mmol; PerkinElmer, DK) as the radiolabel [21]. [3H]Vinylidene kainic acid Kd values are: 67 nM (GluK1) and 8.0 nM (GluK3). Electrophysiology Cloned rat GluA1 receptors were expressed in Xenopus laevis oocytes and used for evaluation of agonist or antagonist activity using two-electrode voltage-clamp electrophysiological recordings as previously described [22]. Computational Docking Studies The Schro¨dinger 2013 suite was used for modelling and docking studies (Schro¨dinger, Portland, OR). The crystal structure of the soluble GluA2-ABD construct in complex with (S)-ATPO (PDB code 5N0T) was employed in the protein preparation wizard in Maestro (Schro¨dinger, Portland, OR) with standard parameters for assigning charges and protons and performing a minimization. This model was then used to generate Van der Waals and electrostatic grids with the docking module Glide using default parameters (Schro¨dinger, Portland, OR). These grids were then used for ligand docking. Compounds 11a and 12b were submitted to minimization in Macromodel (Schro¨dinger, Portland, OR) in triionized forms using default parameters. The best scoring poses according to G-score were used. Figures were created using Pymol (ver. 0.99, Delano Scientific). X-ray Crystallographic Analysis of Compound 11b Trihydrate Crystal data: single crystals suitable for X-ray diffraction studies were grown from a solution in water by slow cooling. C9H16N3O6P, 3H2O, Mr 347.27, orthorhombic, space group P212121 (No 19), a = 9.2206(9), b = 9.7995(7), c = ˚ , V = 1560.3(3) A ˚ 3, Z = 4, Dc = 1.478 Mg/m3, 17.269(2) A F(000) = 736, l(MoKa) = 0.225 mm-1, crystal size: 0.13 90.21 9 0.56 mm. CCDC 994918 contains the supplementary crystallographic data for this paper (compound 11b). These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_ request/cif. Acknowledgments The Lundbeck Foundation, the Hørslev Foundation and the University of Copenhagen Programme of Excellence GluTarget are gratefully acknowledged for financial support. The technical assistance of Flemming Hansen, Department of Chemistry,
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University of Copenhagen, with collecting X-ray data is gratefully acknowledged.
References 1. Bra¨uner-Osborne H, Egebjerg J, Nielsen EØ et al (2000) Ligands for glutamate receptors: design and therapeutic prospects. J Med Chem 43:2609–2645. doi:10.1021/jm000007r 2. Javitt DC (2004) Glutamate as a therapeutic target in psychiatric disorders. Mol Psychiatry 9:984–997. doi:10.1038/sj.mp.4001551 3. Traynelis SF, Wollmuth LP, McBain CJ et al (2010) Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev 62:405–496. doi:10.1124/pr.109.002451 4. Sobolevsky AI, Rosconi MP, Gouaux E (2009) X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor. Nature 462:745–756. doi:10.1038/nature08624 5. Hansen KB, Yuan H, Traynelis SF (2007) Structural aspects of AMPA receptor activation, desensitization and deactivation. Curr Opin Neurobiol 17:281–288 6. Hermit MB, Greenwood JR, Nielsen B et al (2004) Ibotenic acid and thioibotenic acid: a remarkable difference in activity at group III metabotropic glutamate receptors. Eur J Pharmacol 486:241–250. doi:10.1016/j.ejphar.2003.12.033 7. Krogsgaard-Larsen P, Honore´ T, Hansen JJ et al (1980) New class of glutamate agonist structurally related to ibotenic acid. Nature 284:64–66. doi:10.1038/284064a0 8. Vogensen SB, Greenwood JR, Varming AR et al (2004) A stereochemical anomaly: the cyclised (R)-AMPA analogue (R)-3hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-5-carboxylic acid [(R)-5-HPCA] resembles (S)-AMPA at glutamate receptors. Org Biomol Chem 2:206–213. doi:10.1039/b310450h 9. Madsen U, Bang-Andersen B, Brehm L et al (1996) Synthesis and pharmacology of highly selective carboxy and phosphono isoxazole amino acid AMPA receptor antagonists. J Med Chem 39:1682–1691 10. Wahl P, Anker C, Traynelis SF et al (1998) Antagonist properties of a phosphono isoxazole amino acid at glutamate R1–4 (R, S)-2amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid receptor subtypes. Mol Pharmacol 53:590–596 11. Møller EH, Egebjerg J, Brehm L et al (1999) Resolution, absolute stereochemistry, and enantiopharmacology of the GluR1–4 and GluR5 antagonist 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl] propionic acid. Chirality 11:752–759 12. Hogner A, Greenwood JR, Liljefors T et al (2003) Competitive antagonism of AMPA receptors by ligands of different classes: crystal structure of ATPO bound to the GluR2 ligand-binding core, in comparison with DNQX. J Med Chem 46:214–221. doi:10.1021/jm020989v 13. Hald H, Naur P, Pickering DS et al (2007) Partial agonism and antagonism of the ionotropic glutamate receptor iGLuR5: structures of the ligand-binding core in complex with domoic acid and 2-AMINO-3-[5-tert-Butyl-3-(Phosphonomethoxy)-4-isoxazolyl]Propionic acid. J Biol Chem 282:25726–25736. doi:10.1074/ jbc.M700137200 14. Honore T, Hjeds H (1979) Synthesis and GABA-receptor bindingactivity of 2 fluoroaminophenols. Eur J Med Chem 14:285–287 15. Hjeds H, Krogsgaard-Larsen P (1976) Synthesis of some 4-Aminoalkyl-5-methyl-3-isoxazolols structurally related to muscimol and gamma-Aminobutyric Acid (GABA). Acta Chem Scand 30b:567–573. doi:10.3891/acta.chem.scand.30-0567 16. Ransom RW, Stec NL (1988) Cooperative modulation of [3H]MK801 binding to the N-Methyl-D-aspartate receptor-ion channel complex by L-glutamate, glycine, and polyamines. J Neurochem 51:830–836. doi:10.1111/j.1471-4159.1988.tb01818.x
Neurochem Res 17. Honore´ T, Nielsen M (1985) Complex structure of quisqualatesensitive glutamate receptors in rat cortex. Neurosci Lett 54:27–32. doi:10.1016/S0304-3940(85)80113-0 18. Braitman DJ, Coyle JT (1987) Inhibition of [3H]kainic acid receptor binding by divalent cations correlates with ion affinity for the calcium channel. Neuropharmacology 26:1247–1251. doi:10.1016/0028-3908(87)90083-9 19. Sills MA, Fagg G, Pozza M et al (1991) [3H]CGP 39653: a new N-methyl-D-aspartate antagonist radioligand with low nanomolar affinity in rat brain. Eur J Pharmacol 192:19–24 20. Assaf Z, Larsen AP, Venskutonyt_e R et al (2013) Chemoenzymatic synthesis of new 2,4-syn-functionalized (S)-glutamate
analogues and structure-activity relationship studies at ionotropic glutamate receptors and excitatory amino acid transporters. J Med Chem 56:1614–1628. doi:10.1021/jm301433m 21. Sagot E, Pickering DS, Pu X et al (2008) Chemo-enzymatic synthesis of a series of 2,4-Syn-functionalized (S)-glutamate analogues: new insight into the structure–activity relation of ionotropic glutamate receptor subtypes 5, 6, and 7. J Med Chem 51:4093–4103. doi:10.1021/jm800092x 22. Poulsen MH, Lucas S, Strømgaard K, Kristensen AS (2014) Evaluation of PhTX-74 as subtype-selective inhibitor of GluA2containing AMPA receptors. Mol Pharmacol 85:261–268. doi:10. 1124/mol.113.089961
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ORIGINAL PAPER
Analogues of 3-Hydroxyisoxazole-Containing Glutamate Receptor Ligands Based on the 3-Hydroxypyrazole-Moiety: Design, Synthesis and Pharmacological Characterization Lars Jørgensen • Birgitte Nielsen • Darryl S. Pickering Anders S. Kristensen • Karla Frydenvang • Ulf Madsen • Rasmus P. Clausen
•
Received: 10 April 2014 / Revised: 9 May 2014 / Accepted: 10 May 2014 Ó Springer Science+Business Media New York 2014
Abstract A series of analogues of the glutamate receptor ligands (S)-2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propionic acid (AMPA) and AMOA were synthesized in which the 3-hydroxyisoxazole moiety was exchanged for a 3-hydroxypyrazole moiety. This exchange enables further substitution at the additional nitrogen atom in the heterocyclic core. Several of the analogues have activity at AMPA receptors equipotent to the antagonist ATPO, demonstrating that additional substitution can be accommodated in the antagonist binding site. Modelling studies offer an explanation for the pharmacological pattern observed for the compounds and suggest that this scaffold may be developed further to obtain subtype selective antagonists. Keywords Glutamate receptors 3-hydroxypyrazole AMPA Kainic acid Antagonists
Ulf Madsen and Rasmus P. Clausen have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s11064-014-1332-0) contains supplementary material, which is available to authorized users. L. Jørgensen B. Nielsen D. S. Pickering A. S. Kristensen K. Frydenvang U. Madsen (&) R. P. Clausen (&) Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, Copenhagen DK-2100, Denmark e-mail: [email protected] R. P. Clausen e-mail: [email protected]
Introduction (S)-Glutamic acid (1, Fig. 1) is the major excitatory neurotransmitter in the central nervous system, and is able to activate the highly heterogeneous group of glutamate receptors comprising both G-protein coupled metabotropic receptors (mGluRs) and ligand gated ionotropic receptors (iGluRs) [1]. These receptors are crucial in the normal function of the brain, but are also implicated in a range of disorders in the brain [2, 3]. Therefore, compounds that can affect these receptors are important pharmacological tools and may potentially lead to new drugs. The iGluRs have been divided into three major groups based on the selective activation by (S)-2-amino-3-(3-hydroxy-5methyl-isoxazol-4-yl)propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartic acid (NMDA) [1, 3] and this division is also reflected in the sequence homology within each pharmacological group. Each group is comprised of several cloned subunits and the functional ion channel consists of four of these subunits. Since both homo- and heteromeric combinations are functional, the heterogeneity of physiological iGluRs and thereby their pharmacological and biophysical properties are quite extensive. So far, 16 different iGluR subunits have been cloned and AMPA receptors are made up of GluA1-4 subunits, KA receptors of GluK1-5 subunits and NMDA receptors of GluN1, GluN2A-D, GluN3A-B. The recent X-ray crystal structure of a full-length tetrameric glutamate receptor [4] has confirmed that iGluRs consist of three domains: a transmembrane domain (TMD), an agonist binding domain (ABD) and an amino terminal domain (ATD) [5]. A large number of X-ray crystallographic studies of the ABD with various ligands have yielded important information on ligand recognition and understanding of the mechanisms governing receptor activation and desensitization. These studies have disclosed
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Neurochem Res R2
HO OH
N O
O
OH NH2
O
HO N O
R1 OH
R
NH2
AMPA (3a, R=Me) ATPA (3b, R=tBu)
OH
NH2 Ibotenic acid (2)
(S)-glutamic acid (1)
O
O N O
OH R2
N N R1
R2
NH2
NH2 R
AMOA (4a, R1=CO2H, R2=Me) ATOA (4b, R1=CO2H, R2=tBu) AMPO (5a, R=PO3H2, R2=Me) ATPO (5b, R=PO3H2, R2=tBu)
that the ABD has a clamshell-like structure and led to the hypothesis that ABD closes around agonists leading to opening of the ion-channel whereas antagonists stabilize an open form of the ABD maintaining a closed ion-channel. Ibotenic acid (2) is a natural product from the fly-agaric mushroom Amanita Muscaria, and is an agonist at both iGluRs and mGluRs [6]. The redesign of ibotenic acid led to the selective agonist AMPA (3a) [7] that is recognized by all 4 cloned AMPA (3a) receptor subunits (GluA1-4) [8]. Later ATPA (3b) was identified as a selective agonist of the KA receptor subunit GluK1. Addition of a methylene carboxylic acid to the 3-hydroxyisoxazole groups of AMPA and ATPA led to AMOA (4a) and ATOA (4b), respectively, and similar addition of a methylene phosphonic acid gave AMPO (5a) and ATPO (5b). [9] These four compounds are all AMPA receptor antagonists and (S)-ATPO has been shown to antagonize GluK1 and marginally discriminate AMPA receptor subtypes [10, 11]. X-ray crystallographic studies of ATPO in GluA2 and GluK1 have been reported [12, 13] and show that ATPO stabilizes an open conformation of the agonist binding domain of the receptor that is believed to correspond to an inactive state of the whole receptor. In the current study we explore the possibility of using 3-hydroxypyrazole as a bioisoster for the carboxylic acid instead of the 3-hydroxyisoxazole moiety from ibotenic acid. The 3-hydroxypyrazole has an increased pKa compared to the 3-hydroxyisoxazole [14, 15], but also an added possibility for substitution on the extra ring nitrogen. This is interesting since it enables insertion of substituents that may reach non-conserved regions of the receptor and thereby the possibility to design subtype selective ligands. We here present the synthesis and pharmacological characterization of 3-hydroxypyrazole analogues of the agonist AMPA and the antagonists AMOA and AMPO. The synthetic route allowed us to explore other isomeric analogues as well.
O N N
O
O
OH
N N R1
OH
6 (R1=Me, R2=Me) 7 (R1=Et, R2=Me) 8 (R1=Bn, R2=Me) (RS)-9 (R1=Me, R2=CF3)
O
NH2
10a (R1=H, R2=CO2H) 10b (R1=H, R2=PO3H2) 11a (R1=Me, R2=CO2H) 11b (R1=Me, R2=PO3H2) 12a (R1=Et, R2=CO2H) 12b (R1=Et, R2=PO3H2) 13a (R1=Bn, R2=CO2H) 13b (R1=Bn, R2=PO3H2)
OH
O
O
NH2
14a (R=CO2H) 14b (R=PO3H2)
Fig. 1 Structures of agonists (1, 2, 3a and 3b) and antagonists (4a, 4b, 5a, and 5b) at ionotropic glutamate receptors
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O
HO
O
O
OH
N N HO2C
NH2 15
Fig. 2 Synthesized 3-hydroxypyrazole analogues of AMPA (6, 7, 8 and (RS)-9) and of AMOA and AMPO (10a-b, 11a-b, 12a-b, 13a-b, 14a-b and 15)
Design We decided to synthesize AMPA analogues 6–8 (Fig. 2) where the 3-hydroxypyrazole is substituted at N1 with methyl, ethyl and benzyl to explore whether these substitutions can be accommodated in the ABD of the iGluRs. For the methyl substituted analogues we also synthesized a trifluoromethyl-AMPA analogue (RS)-9. Furthermore, 3-hydroxypyrazole analogues of AMOA and AMPO were synthesized, both N1-unsubstituted (10a and 10b) as well as N1-methyl (11a, 11b), N1-ethyl (12a, 12b) and N1benzyl (13a, 13b) substituted analogues to explore the binding pocket in the antagonized state of the iGluRs. Furthermore the synthetic route enabled us to prepare the analogues 14a, 14b and 15. Collectively, these pyrazole analogues allow us to investigate if substitution is allowed in the positions corresponding to the ring oxygen of AMPA and the ring oxygen and nitrogen of AMOA and AMPO.
Chemistry The synthetic route for most of the compounds started with tert-butyl N-Boc-pyroglutamate (16) (Scheme 1) that was deprotonated with LDA and acetylated with N-acetylimidazole to give 17. Reacting 17 with hydrazine gave 3-hydroxypyrazole 18 that was alkylated with ethyl bromoacetate or diethyl [(p-tosyloxy)methyl]phosphonate to give 19a and 19b, respectively. The ester 19a was hydrolyzed with NaOH followed by treatment with HCl yielding 10a. The phosphonoester 19b was deprotected with TMS-Br followed by
Neurochem Res Scheme 1 Synthetic routes to the new analogues. (a) LDA, Nacetylimidazole, THF, -78 to -20 °C. (b) NH2NH2, MeCN, reflux. (c) Ethyl bromoacetate, K2CO3, DMF, r.t. (d) Diethyl [(ptosyloxy)methyl]phosphonate, K2CO3, DMF, r.t. (e) TBS-Cl, Et3N, CH2Cl2, r.t. (f) (1) 4 M NaOH (aq), EtOH, r.t. (2) TFA, CH2Cl2, r.t. (g) (1) TMS-Br, CH3CN, r.t. 2) 1 M HCl (aq), reflux. (h) MeNHNH2, MeCN, reflux. (i) NaH, methyl iodide, DMF, -50 °C to r.t.. (j) NaH, ethyl bromide, DMF, -50 °C to r.t. (k) NaH, benzyl bromide, DMF, -50 °C to r.t.. (l) 1 M HCl (aq) or 1 M TFA (aq), reflux. (m) KF, DMF, r.t. (n) Iodomonochloride, AcOH, H2O, 85 °C. (o) n-BuLi, tert-butyl 2-[bis(tert-butoxycarbonyl) amino]acrylate, THF, -78 to -40 °C
O O-tBu
O O-tBu O
N Boc
a O
O
N Boc
b
16
HO
h
O-tBu
NHBoc N
O
N 20
17 O
c or d
O-tBu HO
O
NHBoc N
18 c, d, or e
O
O-tBu NHBoc
N N
O-tBu
R
O
O N H
NHBoc
+
N
EtO2C
O O-tBu
N
22a (R=CO2Et) 22b (R=PO3Et2)
32
RO f
NHBoc N
N H
15
f
14a, 14b
10a 10b
19a (R=CH2CO2Et) 19b (R=CH2PO3Et2) g
21 (R=TBS) i, j, or k
f or g
O
O
O
R2
O-tBu
O-tBu HO
TBSO NHBoc N
NHBoc
m N
N R
N
N R1
29a (R1=Me, R2=CO2Et) 29b (R1=Me, R2=PO3Et2) 30a (R1=Et, R2=CO2Et) 30b (R1=Et, R2=PO3Et2) 31a (R1=Bn, R2=CO2Et) 31b (R1=Bn, R2=PO3Et2)
f or g
l 11a, 11b, 12a, 12b, 13a, 13b
6, 7, 8
NHBoc
c or d
N R
26 (R=Me) 27 (R=Et) 28 (R=Bn)
23 (R=Me) 24 (R=Et) 25 (R=Bn)
O-tBu O
O
O O-tBu
R
HO
I
TBSO e
N
n
N
CF3
33 (R=H) 34 (R=I)
HCl treatment giving 10b. When 17 was reacted with methyl hydrazine compound 20 was obtained as the only isomer, which was alkylated with ethyl bromoacetate or diethyl [(ptosyloxy)methyl]phosphonate to give 22a and 22b, respectively. These were deprotected with the same conditions as 19a and 19b to give 14a and 14b, respectively. TBS protection of the 3-hydroxy group in 18 gave compound 21, which was selectively alkylated at N1 in the pyrazole ring with methyl iodide, ethyl and benzyl bromide to give 23, 24 and 25. These were deprotected in aqueous HCl to yield 6, 7
HO NBoc2
o N
N 35
OH
TBSO CF3
N
N
CF3
(RS)-36
NH2
l N
N
CF3
(RS)-9
and 8, respectively. Selective removal of the TBS group of 23–25 yielded 26–28 that could be alkylated with ethyl bromoacetate or diethyl [(p-tosyloxy)methyl]phosphonate to give 29a, 30a, 31a or 29b, 30b, 31b, respectively. These were deprotected under the same conditions as 19a and 19b to give 11a, 11b, 12a, 12b, 13a, and 13b. The isomeric assignment based on 1H NMR was confirmed by an X-ray crystal structure of 11b (Fig. 3). Alkylation of 20 with ethyl bromoacetate also yielded 32 as a by-product that could be deprotected under the same conditions as 19a to give 15.
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Neurochem Res
Fig. 3 Perspective drawing of compound 11b. Displacement ellipsoids of the non-hydrogen atoms are shown at the 50 % probability level. Hydrogen atoms are represented by spheres of arbitrary size
were 14a, 14b and 15. Furthermore, all compounds were probed for activity towards cloned homomeric GluK1 and GluK3 subunits expressed in Sf9 insect cells at a concentration of 100 lM, since these activities are not revealed in the KA binding assay on rat cortical synaptosomes. In this binding assay only compound (RS)-9 could reduce the radioligand binding towards GluK1 to \50 %. At GluK3 only compound 11a reduced radioligand binding below 50 %. Thus, the GluK1 activity is abolished in these analogues compared to ATPO, but for 11a a modest GluK3 affinity is observed. The functional activity of the active analogues were assessed in two-electrode voltage clamp experiments at GluA2 expressing Xenopus oocytes (Fig. 4). Analogues 6 and (RS)-9 showed agonist activity whereas 11a, 11b, 12a and 12b showed antagonist activity at glutamate induced agonist responses.
Docking The synthesis of (RS)-9 followed a different route starting with 1-methyl-5-(trifluoromethyl)-pyrazol-3-ol (33) that was iodinated with ICl to give 34. This compound was TBS protected with TBS-Cl to give 35 followed by treatment with n-BuLi and tert-butyl 2-[bis(tert-butoxycarbonyl)amino]acrylate giving 36. This was followed by treatment with HCl giving (RS)-9.
Pharmacology The compounds were characterized pharmacologically by investigation of their binding affinity towards the major iGluR groups, AMPA, KA and NMDA receptors in radioligand binding assays using rat cortical synaptosomes and [3H]AMPA, [3H]KA and [3H]CGP39653 as ligands, respectively (Table 1). The AMPA analogue 6 with an Nmethyl substituent on the heterocycle showed weak activity towards AMPA binding sites but no activity towards KA and NMDA binding sites. Compounds 7 and 8 were both inactive in all binding assays. Compound (RS)-9 showed slightly increased activity at AMPA binding sites compared to 6, but no activity at KA and NMDA sites. The AMOA and AMPO analogues were also probed for binding affinity towards the major iGluR groups. The analogues 10a and 10b were inactive at all sites. In contrast, N-methyl substituted 11a and 11b as well as ethyl substituted 12a and 12b showed affinity for AMPA binding sites but not for KA and NMDA binding sites. Thus, the carboxylates were more potent than phosphonates and ethyl substituted slightly more active than methyl substituted while benzyl substituted 13a and 13b were inactive as
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To get an impression of the binding modes of the pyrazole compounds we docked 10a, 11a and 12a in the X-ray crystal structures of ATPO in GluA2 and GluK1. These suggested that the compounds displayed binding modes resembling ATPO as exemplified in Fig. 5 showing 11a docked in GluA2. Thus, the amino acid moiety and heterocycle of ATPO and the new analogues are overlapping. The order of the docking scores reflected the order of activity in the binding assay (10a \ 11a < 12a) in GluA2. Furthermore, it seemed that the higher activity could be ascribed to positive Van Der Waals interactions of the Nmethyl group in 11a with M708 and of the N-ethyl group (not shown) in 12a with M708 and T686 (not shown). These interactions are predicted in a grid calculation of the GluA2 ATPO structure [12]. These residues are conserved among AMPA receptor subtypes, but correspond to S741 and S721 and GluK1, and T743 and N723 in GluK3. Thus, the changed pharmacological profile can be explained by interactions with non-conserved residues in this region. In the binding mode of 12a the methyl (Me) group on the heterocycle point into the conserved hydrophobic region that is occupied by the tert-butyl group of ATPO, whereas the N-ethyl group (Et) points towards a region containing residues that are non-conserved (indicated with red colors) not only among the major iGluR groups but also among the otherwise highly homologous AMPA receptor subtypes. This suggests that extended substitution in this direction could lead to subtype selective AMPA receptor antagonists, although the lack of activity in compounds 13a and 13b suggest that there is not enough room for accommodating very large groups like the benzyl group.
Neurochem Res Table 1 Receptor binding affinities of compounds at the three major groups of iGluRs in rat cortical synaptosome assays and at cloned rat kainate receptors GluK1 and GluK3. The numbers in brackets [min; max] indicate mean ± SEM according to a logarithmic distribution
CGP39653 is a competitive NMDA receptor antagonist and AMPA and KA are agonists at AMPA and KA receptors, respectively. Ki values are calculated using GraphPad Prism a
Vogensen et al. [8]
b
Madsen et al. [9]
c
3
[ H]CPP binding
d
Møller et al. [11]
nd not determined
[3H]AMPA
[3H]KA
[3H]CGP39653
% Binding (100 lM) or Ki
IC50 (lM)
IC50 (lM)
Ki (lM)
GluK1
GluK3
AMPA
0.021a
24a
[100a
1.15 ± 0.51 lM (Ki)
43 ± 13 lM (Ki)
(RS)AMOA
90b
[100b
[100b,c
84 ± 18 lM (Ki)
[100 lM (Ki)
(RS)ATOA
33b
[100d
[100b,c
4.1 ± 0.2 lM (Ki) [100 lM (Ki)
(RS)AMPO
31b
[100b
[100b,c
19 ± 3 lM (Ki)
(RS)ATPO
35b (16 (S-)d)
[100b
[100b,c
2.6 ± 0.7 lM (Ki) [100 lM (Ki)
6
8.9 [8.3;9.5] [5.05 ± 0.03]
[100
[100
91
91
7
[100
[100
[100
75
93
8
[100
[100
[100
96
111
(RS)-9
[100
[100
44
81
10a
3.6 [3.5;3.7] [5.45 ± 0.01] [100
[100
[100
93
74
10b
[100
[100
[100
65
86
11a
23 [22;24] [4.64 ± 0.02]
[100
[100
76
33
11b
77 [75;79] [4.11 ± 0.01]
[100
[100
65
68
12a
31 [30;32] [4.51 ± 0.01]
[100
[100
70
68
12b
87 [83;91] [4.06 ± 0.02]
[100
[100
51
95
13a
[100
[100
[100
91
91
Compound
[100 lM (Ki)
13b
[100
[100
[100
nd
nd
14a
[100
[100
[100
105
109
14b
[100
[100
[100
106
111
15
[100
[100
[100
110
104
Conclusion We have synthesized a series of 3-hydroxypyrazole analogues of the agonist AMPA and the antagonists AMPO and AMOA. The synthesis offers an efficient stereoselective route for making such analogues with differing substituents on the 3-hydroxypyrazole moiety. N1 alkylated pyrazole analogues of AMPA showed very weak or no activity at AMPA receptors, whereas some of the pyrazole analogues of AMOA and AMPO showed activities comparable to the parent compounds. Interestingly the pyrazole analogues showed the reverse order of activity for the carboxy versus the phosphonate analogues compared to AMOA and AMPO. Thus, the carboxy analogues in the pyrazole series (11a, 12a) showed higher potency than the phophonate analogues (11b, 12b), whereas for the isoxazoles the phosphonate analogue (AMPO) shows higher activity than the carboxy analogue AMOA. Docking results
explain the difference in activity between unsubstituted 10a and 11a/12a, with hydrophobic VDW interactions with M708. This amino acid is not conserved in GluK1 and GluK3 and thus offers an explanation for the change in selectivity profile when substituents protrude into this region. The pyrazole analogues offer, in contrast to isoxazoles, the possibility for substitution at the N1 position, and the results observed indicate that further elaboration of the substituent pattern on such pyrazole analogues may lead to subtype selective compounds.
Experimental Section Chemistry General procedures. All reactions involving dry solvents or sensitive reagents were performed under a nitrogen or
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Neurochem Res
Fig. 4 Representative current responses obtained from a two-electrode voltage-clamp recording on Xenopus oocytes expressing homomeric GluA2(Q)i receptors. Currents were evoked by compounds alone (top) or co-application of Glu and the compounds at 100 lM (bottom)
Fig. 5 Left X-ray crystal structure GluA2 ABD (PDB:1N0T) showing the binding mode of (S)-ATPO (grey carbons) together with the binding mode of compound N-methyl substituted 11a proposed from docking into the X-ray structure. Right Proposed docking mode of N-
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argon atmosphere and glassware was flame dried prior to use under vacuum. Solvents were dried according to standard procedures, and reactions were monitored by analytical thin-layer chromatography (TLC) analysis. TLC was carried out using Merck silica gel 60 F254 aluminum sheets. The compounds were detected as single spots on TLC plates (using n-BuOH/water/EtOAc/AcOH 1/1/1/1) and visualized using UV light (254 nm) and staining reagents (ex. Ninhydrin). Flash chromatography was carried out using Merck silica gel 60A (35–70 lm). 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recorded on a 300 MHz Varian Mercury 300BB. Chemical shifts (d) are given in parts per million (ppm), and coupling constants (J) are given in Hertz (Hz). Optical rotations were measured on a Perkin-Elmer 241 polarimeter. Elemental analysis was performed by Mr. J. Theiner at the University of Vienna. Melting point was determined in an open capillary tube and is uncorrected. All purchased chemicals were used without further purification. Pyrazole regioisomers were established by both 13C-NMR signal assignment and 1H-NMR NOESY experiments showing correlation between the N-alkyl group and other substituents in the ring. In case of 11b this was further confirmed with an X-ray crystal structure determination. (S)-2-amino-3-(3-hydroxy-1,5-dimethyl-1H-pyrazol4-yl)propanoic acid (6) A solution of 23 (100 mg, 0.213 mmol) in 1 M HCl (15 ml) was refluxed for 45 min. The solvent was evaporated in vacuo, and the residue was re-evaporated twice from water. The crude
ethyl substituted 12a docked in PDB:1N0T. The red regions represent regions containing residues that are not fully conserved among AMPA receptor subunits GluA1–GluA4
Neurochem Res
product was purified by HPLC (X-Terra Ò Prep MS C 18 10 lm, 10 9 300 mm; 15 mM AcOH (aq); flow: 10 ml/min). The product was re-crystallized from water to give colorless crystals of 6 (29 mg, 68 %). R f = 0.24. Mp. [ 240 °C. 1H NMR (D2O): d 2.13 (s, 3 H), 2.86 (d, J = 5 Hz, 2 H), 3.50 (s, 3 H), 3.88 (t, J = 5 Hz, 1 H). 13C NMR (D2O): d 9.5, 24.4, 34.3, 55.9, 97.8, 145.8, 162.5, 174.3. EA: calcd for C 8 H 13 N 3 O 3 , C: 48.23, H: 6.58, N: 21.09; found: C: 48.01, H: 6.49, N: 20.89. [a]365 = -198 (c = 0.18, 0.04 M HCl). (S)-2-amino-3-(1-ethyl-3-hydroxy-5-methyl-1H-pyrazol-4-yl)propanoic acid (7) Compound 24 (960 mg, 1.98 mmol) was dissolved in 1 M TFA (aq) (30 ml) and the mixture was refluxed for 2 h. The solvent was evaporated and the residue was re-evaporated twice from water. The residue was dissolved in water (2 ml) and ethanol (10 ml), and the pH was adjusted to 3–4 by addition of triethylamine. No crystallization was observed. Therefore the mixture was evaporated and the residue was subjected to reverse-phase chromatography (RP-18 silicagel, AcOH/ H2O 1/99 to AcOH/MeCN/H2O 1/4/95). Recrystallization using water and 2-propanol gave colorless crystals of 7 (190 mg, 0.79 mmol, 40 %).1H NMR (D2O): d 1.24 (t, J = 7.2 Hz, 3 H), 2.16 (s, 3 H), 2.86 (d, J = 5.4 Hz, 2 H), 3.89 (t, J = 5.3 Hz, 1 H), 3.89 (q, J = 7.2 Hz, 2 H).13C NMR (D2O): d 9.4, 14.2, 24.4, 43.0, 55.9, 98.4, 145.3, 162.9, 174.2. Mp. = 210 °C (decomp.). EA: calcd for C9H15N3O31 H2O, C: 44.99, H: 7.55, N: 17.49; found: C: 45.34, H: 7.71, N: 17.44. (S)-2-amino-3-(1-benzyl-3-hydroxy-5-methyl-1H-pyrazol-4-yl)propanoic acid (8) Compound 25 (350 mg, 0.641 mmol) was stirred at 70 °C in 1 M HCl (15 ml) for 4 h. The solvent was evaporated and re-evaporated twice from water. The residue was dissolved in water (0.5 ml) and ethanol (2.5 ml). The pH was adjusted to 3–4 by addition of triethylamine. No crystallization was observed. Therefore the mixture was evaporated and subjected to reverse-phase chromatography (RP-18 silicagel, AcOH/ H2O 1/99 to AcOH/MeCN/H2O 1/9/90). Recrystallization using water/2-propanol gave colorless crystals of 8 (105 mg, 59 %). 1H NMR (300 MHz, D2O): d 2.19 (s, 3 H), 2.90 (d, J = 5.5 Hz, 2 H), 3.90 (t, J = 5.5 Hz, 1 H); 5.10 (s, 2 H), 7.11–7.44 (m, 5 H). 13C NMR (75 MHz, D2O): d 9.8, 24.4, 51.2, 55.8, 99.0, 127.1, 128.6, 129.5, 136.3, 145.6, 162.7, 174.2. Mp. [ 230 °C. EA: calcd for C14H17N3O3, C: 61.08, H: 6.22, N: 15.26; found: C: 60.98, H: 6.03, N: 15.13. (RS)-2-amino-3-(3-hydroxy-1-methyl-5-(trifluoromethyl)-1H-pyrazol-4-yl)propanoic acid ((RS)-9) Aqueous 1 M HCl (15 ml) was stirred and heated to reflux temperature. A solution of 36 (0.44 g, 0.71 mmol) in 1,4dioxane (5 ml) was added dropwise. The solution was
allowed to reflux for 3 h. The solvent was evaporated and the residue was re-evaporated twice from water. The crude product was subjected to reverse-phase chromatography (RP-18 silicagel, AcOH/H2O 1/99). Recrystallization with water gave (RS)-9 (126 mg, 71 %) as colorless crystals.1H NMR (300 MHz, D2O): d 3.01 (dd, J = 7.7, 15.6 Hz, 1 H), 3.10 (dd, J = 5.8, 15.7 Hz, 1 H), 3.77 (s, 3 H), 3.86 (dd, J = 7.6, 5.6 Hz, 1 H). 13C NMR (75 MHz, D2O with NaOD): d 29.0, 37.0, 57.4, 104.8, 121.2 (q, J = 269.3 Hz), 130.0 (q, J = 36.1 Hz), 168.1, 183.1. Mp. [ 220 °C. EA: calcd for C8H10F3N3O3 H2O, C: 36.03, H: 4.35, N: 15.76; found: C: 35.89, H: 4.55, N: 15.54. (S)-2-amino-3-(3-(carboxymethoxy)-1,5-dimethyl1H-pyrazol-4-yl)propanoic acid (10a) Compound 19a (300 mg, 0.70 mmol) was dissolved in EtOH (20 ml) and 4 M NaOH (3 ml) was added. The mixture was stirred for 2 h. The volume was reduced in vacuo to 3 ml. Water (50 ml) and EtOAc (50 ml) were added. The pH of the aqueous phase was adjusted to 3–4 with 1 M HCl and extracted with EtOAc (3 9 50 ml). The combined extracts were dried over MgSO4 and the solvent was removed in vacuo. The residue was dissolved in CH2Cl2 (10 ml) and TFA (10 ml) and stirred at r.t. for 3 h. The solvent was evaporated. Re-evaporation twice from water and twice from 1 M HCl gave crystals, which were dissolved in as little water as possible. Propylenoxide (few drops) were added, giving precipitation of crystals. Recrystallization twice from water gave colorless crystals of 10a (40 mg, 22 %). Rf = 0.36. 1H NMR (300 MHz, D2O): d 2.18 (s, 3 H), 3.00 (dd, J = 15.7, 6.1 Hz, 1 H), 3.02 (dd, J = 15.7, 4.9 Hz, 1 H),3.96–4.07 (m, 1 H), 4.70 (d, J = 16.2 Hz, 1 H), 4.77 (d, J = 16.3 Hz, 1 H). 13C NMR (75 MHz, D2O): d 9.7, 23.4, 55.2, 66.5, 95.7, 142.0, 161.2, 173.6, 175.2. Mp. = 197–199 °C. EA: calcd for C9H13N3O5H2O, C: 41.38, H: 5.79, N: 16.09; found: C: 41.50, H: 5.96, N: 16.10. [a]365 = -9.68 (c = 0.15, 0.04 M HCl). (S)-2-amino-3-(3-(phosphonomethoxy)-1,5-dimethyl1H-pyrazol-4-yl)propanoic acid (10b) Compound 19b (400 mg, 0.814 mmol) was dissolved in CH3CN (20 ml). TMSBr (0.50 ml, 3.86 mmol) was added and the mixture was stirred for 5 h. The solvent was evaporated and the residue was re-dissolved in 1 M HCl (15 ml). The mixture was refluxed for 24 h. The solvent was removed in vacuo. The residue was re-evaporated twice from water. The product was crystallized by dissolving it in water and adding propyleneoxide (few drops). Recrystallization from water gave colorless crystals of 10b (130 mg, 54 %). Rf = 0.16. 1H NMR (300 MHz, D2O): d 2.18 (s, 3 H), 3.01 (dd, J = 15.8, 6.1 Hz, 1 H), 3.04 (dd, J = 15.7, 5.0 Hz, 1 H), 4.07 (dd, J = 6.0, 5.3 Hz, 1 H), 4.24 (dd, J = 17.0, 9.1 Hz, 1 H), 4.28 (dd, J = 17.0, 9.0 Hz, 1 H). 13 C NMR (75 MHz, D2O with NaOD): d 10.1, 27.9, 56.8, 67.0 (d, J = 151 Hz), 98.5, 141.9, 163.9 (d, J = 12.5 Hz),
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183.3. Mp. [ 230 °C. EA: calcd for C8H14N3O6PH2O, C: 32.33, H: 5.43, N: 14.14; found: C: 32.02, H: 5.65, N: 13.83. [a]365 = ?6.98 (c = 0.23, 0.5 M HCl). (S)-2-amino-3-(3-(carboxymethoxy)-1,5-dimethyl-1Hpyrazol-4-yl)propanoic acid (11a) To a solution of 29a (220 mg, 0.499 mmol) in EtOH (10 ml) was added 4 M NaOH (10 ml). The mixture was stirred for 3 h. Water (10 ml) was added and the solvent was reduced to 10 ml. 1 M KHSO4 (80 ml) was added and the mixture was extracted with EtOAc (5 9 100 ml). The combined extracts were washed with brine (50 ml), dried over MgSO4, and the solvent was evaporated. The residue was dissolved in CH2Cl2 (5 ml) and TFA (5 ml) and stirred for 1 h. The solvent was evaporated. The crude product was purified by HPLC (X-TerraÒ Prep MS C18 10 lm, 10 9 300 mm; 15 mM AcOH (aq); flow: 10 ml/min). Evaporation three times with water followed by recrystallization from water yielding colorless crystals of 11a (78 mg, 60 %). Rf = 0.30. 1H NMR (300 MHz, D2O): d 2.15 (s, 3 H), 2.98 (dd, J = 6.0, 15.7 Hz, 1 H), 3.01 (dd, J = 4.9, 15.7 Hz, 1 H), 3.57 (s, 3 H), 4.01 (dd, J = 4.9, 5.9 Hz, 1 H), 4.68 (d, J = 16.2 Hz, 1 H), 4.75 (d, J = 16.2 Hz, 1 H). 13C NMR (75 MHz, D2O): d 9.7, 23.7, 35.6, 55.3, 66.4, 96.0, 142.0, 159.7, 173.5, 175.1. Mp. [ 240 °C. EA: calcd for C10H15N3O5H2O, C: 45.89, H: 5.97, N: 16.05; found: C: 45.74, H: 5.71, N: 15.94. [a]365 = -228 (c = 0.25, 0.04 M HCl). (S)-2-amino-3-(3-(phosphonomethoxy)-1,5-dimethyl1H-pyrazol-4-yl)propanoic acid (11b) To a solution of 29b (300 mg, 0.594 mmol) in CH3CN (30 ml) was added TMSBr (1.5 ml, 11.6 mmol) and stirred for 24 h. The solvent was evaporated. Water was added and evaporated twice. The crude product was purified by HPLC (X-TerraÒ Prep MS C18 10 lm, 10 9 300 mm; 15 mM AcOH (aq); flow: 10 ml/min). Evaporation three times with water followed by recrystallization from water yielding colorless crystals of 11b (50 mg, 30 %). Rf = 0.13. 1H NMR (300 MHz, D2O): d 2.15 (s, 3 H), 3.02 (dd, J = 6.1, 15.7 Hz, 1 H), 3.05 (dd, J = 5.4, 15.7 Hz, 1 H), 3.60 (s, 3 H), 4.16 (t, J = 5.5 Hz, 1 H), 4.19 (dd, J = 9.2, 12.8 Hz, 1 H), 4.25 (dd, J = 9.1, 12.9 Hz, 1 H). 13C NMR (75 MHz, D2O): d 9.6, 23.5, 35.6, 54.2, 65.4 (d, J = 157.5 Hz), 95.4, 142.2, 160.8 (d, J = 12.2 Hz), 172.4. [a]365 = -238 (c = 0.25, 0.04 M HCl). (S)-2-amino-3-(3-(carboxymethoxy)-1-ethyl-5-methyl1H-pyrazol-4-yl)propanoic acid (12a) Compound 30a (1.00 g, 2.19 mmol) was dissolved in a mixture of EtOH (20 ml) and 4 M NaOH (10 ml) and stirred at r.t. for 4 h. Water (20 ml) was added and the mixture was evaporated to 15 ml. 1 M KHSO4 (15 ml) was added and the mixture was extracted with EtOAc (5 9 100 ml). The combined extracts were washed with brine, dried over MgSO4, and the solvent was evaporated. The residue was dissolved in CH2Cl2 (10 ml) and TFA (10 ml) and then stirred at r.t. for 1 h, followed by
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evaporation of solvent and re-evaporation twice from water. Recrystallization of the crude product using water and 2-propanol gave 12a (400 mg, 67 %) as colorless crystals. 1H NMR (300 MHz, D2O): d 1.24 (t, J = 7.2 Hz, 3 H), 2.17 (s, 3 H), 2.92–3.07 (m, 2 H), 3.93 (q, J = 7.2 Hz, 2 H), 4.03 (br t, J = 5.4 Hz, 1 H), 4.71 (d, J = 16.3 Hz, 1 H), 4.78 (d, 1 H). 13 C NMR (75 MHz, D2O with NaOD): d 9.6, 15.2, 28.2, 43.7, 57.0, 67.9, 99.1, 140.4, 160.4, 177.1, 183.0. Mp. [ 230 °C. EA: calcd for C11H17N3O5, C: 48.70, H: 6.32, N: 15.49; found: C: 48.73, H: 6.21, N: 15.33. (S)-2-amino-3-(3-(phosphonomethoxy)-1-ethyl-5methyl-1H-pyrazol-4-yl)propanoic acid (12b) Compound 30b (1.00 g, 1.92 mmol) was dissolved in MeCN (60 ml). TMSBr (3.0 ml, 23.2 mmol) was added. The mixture was stirred at r.t. for 24 h and then the solvent was evaporated. The residue was re-dissolved in CH2Cl2 (20 ml) and TFA (20 ml), and then stirred for 3 h at r.t. The solvent was evaporated followed by re-evaporation twice from water. Recrystallization using water and 2-propanol gave colorless crystals of 12b (485 mg, 82 %). 1 H NMR (300 MHz, D2O): d 1.27 (t, J = 7.2 Hz, 3 H), 2.17 (s, 3 H), 3.04 (m, 2 H), 3.96 (q, J = 7.2 Hz, 2 H), 4.18 (t, J = 5.6 Hz, 1 H), 4.18–4.33 (m, 2 H). 13C NMR (75 MHz, D2O with NaOD): d 9.4, 15.1, 23.5, 44.0, 54.1, 65.5 (d, J = 158 Hz), 95.6, 141.2, 160.9 (d, J = 12.4 Hz), 172.3. Mp. [ 230 °C. EA: calcd for C10H18N3O6P, C: 39.09, H: 5.91, N: 13.68; found: C: 38.96, H: 5.63, N: 13.42. (S)-2-amino-3-(3-(carboxymethoxy)-1-benzyl-5methyl-1H-pyrazol-4-yl)propanoic acid (13a) Compound 31a (450 mg, 0.869 mmol) was dissolved in EtOH (15 ml) and 2 M NaOH (15 ml). The mixture was stirred at r.t. for 3 h. Water (15 ml) was added and the mixture was evaporated to 15 ml. 1 M NaHSO4 was added and extracted with EtOAc (3 9 100 ml). The combined extracts were washed with brine, dried over MgSO4, and the solvent was evaporated. The residue was dissolved in CH2Cl2 (15 ml) and TFA (15 ml) and stirred at r.t. for 1 h. After evaporation of the solvent and re-evaporation twice from water, recrystallization from water gave colorless crystals of 13a (224 mg, 76 %). 1H NMR (300 MHz, D2O): d 2.14 (s, 3 H), 2.95–3.10 (m, 2 H), 3.98 (t, J = 5.3 Hz, 1 H), 4.65 (d, J = 16.0 Hz, 1 H), 4.73 (d, J = 16.0 Hz, 1 H), 5.17 (s, 2 H), 7.00–7.40 (m, 5 H). 13C NMR (75 MHz, D2O with NaOD): d 9.9, 28.25, 51.9, 56.9, 67.9, 100.1, 126.9, 128.1, 129.4, 138.0, 141.5, 160.9, 177.0, 183.0. Mp. [ 230 °C. EA: calcd for C16H19N3O5 H2O, C: 56.88, H: 5.82, N: 12.44; found: C: 56.69, H: 6.01, N: 12.30. (S)-2-amino-3-(3-(phosphonomethoxy)-1-benzyl-5methyl-1H-pyrazol-4-yl)propanoic acid (13b) Compound 31b was dissolved in CH3CN (30 ml) and TMSBr (1.5 ml) was added. The solution was allowed to stir for
Neurochem Res
22 h at r.t. and the solvent was evaporated. The residue was dissolved in CH2Cl2 (5 ml) and TFA (5 ml) and stirred for 1 h. The solvent was evaporated. The crude product was dissolved in aqueous 1 M TFA (30 ml) and the solution was washed with EtOAc (4 9 30 ml). By evaporation of the aqueous solution colorless crystals were formed. Recrystallization using water gave colorless crystals of 13b (96 mg, 19 % for 2 steps). 1H NMR (300 MHz, D2O): d 2,15 (s, 3H); 2,94–3,10(2 9 dd, 2H, J = 5,0 and 6,1 and 15,8 Hz); 4,0 (t, IH, J = 5,5 Hz); 4,16–4,30 (2 x dd, 2H, J = 9,0 and 9,2 and 15,8 Hz); 5,19 (s, 2H); 7,05–7,45 (m, 5H). 13C NMR (75 MHz, D2O with NaOD): d 9,9; 28,2; 51,9; 56,9; 67,2 (d, J = 151 Hz,); 100,1; 126,9; 128,l; 129,4; 138,0; 141,6; 162,9; 183,1. Mp. [ 230 °C. EA: calcd for C15H20N3O6P, C: 48.78, H: 5.46, N: 11.38; found: C: 48.54, H: 5.37, N: 11.25. (S)-2-amino-3-(5-(carboxymethoxy)-1,3-dimethyl-1Hpyrazol-4-yl)propanoic acid (14a) A solution of 22a (250 mg, 0.566 mmol) in EtOH (20 ml) was added 4 M NaOH (3 ml) and stirred for 2 h. Water (10 ml) was added and the volume was reduced to 10 ml. 1 M KHSO4 (30 ml) was added and the mixture was extracted with EtOAc (9 9 40 ml). The combined extracts were washed with brine (30 ml), dried over MgSO4, and the solvent was evaporated in vacuo. The residue was dissolved in CH2Cl2 (5 ml) and TFA (5 ml) and stirred for 2 h. The solvent was evaporated. The residue was evaporated twice from 1 M HCl. The residue was purified on a strong basic ionexchange column [Amberlite IRA-400(Cl)] eluting with 1 M AcOH. The product was evaporated twice from water, triturated with EtOAc. The crude product was purified by HPLC [X-TerraÒ Prep MS C18 10 lm, 10 9 300 mm; 15 mM AcOH (aq); flow: 10 ml/min]. The product was reevaporated twice from water to give 14a (73 mg, 48 %) as colorless crystals. Rf = 0.36. 1H NMR (300 MHz, D2O): d 2.18 (s, 3 H), 3.01 (dd, J = 6.6, 15.6 Hz, 1 H), 3.06 (dd, J = 6.1, 15.6 Hz, 1 H), 3.69 (s, 3 H), 3.97 (t, J = 6.3 Hz, 1 H), 4.62 (d, J = 15.5 Hz, 1 H), 4.68 (d, J = 15.6 Hz, 1 H). 13 C NMR (75 MHz, D2O): d 11.6, 24.0, 34.1, 54.5, 72.3, 99.5, 147.4, 153.2, 173.2, 174.6. Mp. = 155 °C (decomp.). EA: calcd for C10H15N3O5H2O, C: 44.36, H: 6.14, N: 15.52; found: C: 44.25, H: 5.79, N: 15.33. [a]365 = ?468 (c = 0.24, 0.04 M HCl). (S)-2-amino-3-(5-(phosphonomethoxy)-1,3-dimethyl1H-pyrazol-4-yl)propanoic acid (14b) A solution of 22b (400 mg, 0.791 mmol) in CH3CN (30 ml) was added TMSBr (1.60 ml, 12.3 mmol) and stirred for 4 days. The solvent was evaporated. The residue was dissolved in CH2Cl2 (10 ml) and TFA (10 ml) and stirred for 1 h. The solvent was evaporated. Water was added and evaporated, twice. The residue was triturated with EtOAc. The crude product was purified by HPLC (X-TerraÒ Prep MS C18 10 lm, 10 9 300 mm; 15 mM AcOH (aq); flow: 10 ml/
min). The product was re-evaporated twice from water to give 14b (183 mg, 75 %) as colorless crystals. Rf = 0.30. 1 H NMR (300 MHz, D2O): d 2.19 (s, 3 H), 3.07 (dd, J = 6.5, 15.6 Hz, 1 H), 3.11 (dd, J = 6.2, 15.6 Hz, 1 H), 3.72, (s, 3 H), 4.00 (t, J = 6.3 Hz, 1 H), 4.25 (dd, J = 8.9, 12.9 Hz, 1 H), 4.30 (d, J = 8.7, 12.8 Hz, 1 H). 13C NMR (75 MHz, D2O): d 11.3, 23.7, 34.0, 54.1, 71.6 (d, J = 154 Hz), 100.1, 147.0, 154.4 (d, J = 11.0 Hz), 172.7. Mp. = 195 °C (decomp.). EA: calcd for C9H16N3O6 PH2O, C: 35.24, H: 5.75, N: 13.70; found: C: 35.38, H: 5.77, N: 13.68. [a]365 = ?358 (c = 0.19, 0.04 M HCl). (S)-2-amino-3-(1-(carboxymethyl)-2,5-dimethyl-3oxo-2,3-dihydro-1H-pyrazol-4-yl)propanoic acid (15) To a solution of 32 (200 mg, 0.452 mmol) in EtOH (10 ml) was added 4 M NaOH (10 ml). The mixture was stirred for 2 h. Water (10 ml) was added and the solvent was reduced to 6 ml. 1 M KHSO4 (70 ml) was added and the mixture was extracted with EtOAc (10 9 80 ml). The combined extracts were washed with brine (50 ml), dried over MgSO4, and the solvent was evaporated. The residue was dissolved in CH2Cl2 (5 ml) and TFA (5 ml) and stirred for 2 h. The solvent was evaporated. The crude product was purified by HPLC (X-TerraÒ Prep MS C18 10 lm, 10 9 300 mm; 15 mM AcOH (aq); flow: 10 ml/min). Evaporation three times with water followed by recrystallization from water yielding colorless crystals of 15 (89 mg, 0.328 mmol, 73 %). Rf = 0.23. 1H NMR (300 MHz, D2O): d 2.17 (s, 3 H), 2.94 (dd, J = 6.4, 15.9 Hz, 1 H), 2.97 (dd, J = 5.1, 15.7 Hz, 1 H), 3.40 (s, 3 H), 4.11 (bs, 1 H), 4.69 (s, 2H). 13C NMR (75 MHz, D2O): d 9.9, 24.3, 29.1, 47.7, 54.5, 99.2, 147.3, 163.3, 172.9. Mp. 176 °C (decomp). EA: calcd for C10H15N3O53/4H2O, C: 44.36, H: 6.14, N: 15.52; found: C: 44.68, H: 5.87, N: 15.60.
Pharmacology Binding Affinity at Native Receptors All native receptor binding experiments were performed using rat brain synaptic membranes of cortex and the central hemisphere from male SPRD rats with tissue preparation as described in the literature [16]. Affinity for AMPA, KA, and NMDA receptors were determined using 5 nM [3H]AMPA [17], 5 nM [3H]KA [18], and 2 nM [3H]CGP 39653 [19], respectively, with modifications previously described [20]. Binding Affinity at Cloned Receptors Cloned rat GluK1 and GluK3 receptors were expressed in sf9 insect cells and ligand binding evaluated as previously
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described using either [3H]-(2S,4R)-4-methylglutamic acid (47.9 Ci/mmol; ARC Inc., St. Louis, MO) or [3H]Vinylidene kainic acid (44.0 Ci/mmol; PerkinElmer, DK) as the radiolabel [21]. [3H]Vinylidene kainic acid Kd values are: 67 nM (GluK1) and 8.0 nM (GluK3). Electrophysiology Cloned rat GluA1 receptors were expressed in Xenopus laevis oocytes and used for evaluation of agonist or antagonist activity using two-electrode voltage-clamp electrophysiological recordings as previously described [22]. Computational Docking Studies The Schro¨dinger 2013 suite was used for modelling and docking studies (Schro¨dinger, Portland, OR). The crystal structure of the soluble GluA2-ABD construct in complex with (S)-ATPO (PDB code 5N0T) was employed in the protein preparation wizard in Maestro (Schro¨dinger, Portland, OR) with standard parameters for assigning charges and protons and performing a minimization. This model was then used to generate Van der Waals and electrostatic grids with the docking module Glide using default parameters (Schro¨dinger, Portland, OR). These grids were then used for ligand docking. Compounds 11a and 12b were submitted to minimization in Macromodel (Schro¨dinger, Portland, OR) in triionized forms using default parameters. The best scoring poses according to G-score were used. Figures were created using Pymol (ver. 0.99, Delano Scientific). X-ray Crystallographic Analysis of Compound 11b Trihydrate Crystal data: single crystals suitable for X-ray diffraction studies were grown from a solution in water by slow cooling. C9H16N3O6P, 3H2O, Mr 347.27, orthorhombic, space group P212121 (No 19), a = 9.2206(9), b = 9.7995(7), c = ˚ , V = 1560.3(3) A ˚ 3, Z = 4, Dc = 1.478 Mg/m3, 17.269(2) A F(000) = 736, l(MoKa) = 0.225 mm-1, crystal size: 0.13 90.21 9 0.56 mm. CCDC 994918 contains the supplementary crystallographic data for this paper (compound 11b). These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_ request/cif. Acknowledgments The Lundbeck Foundation, the Hørslev Foundation and the University of Copenhagen Programme of Excellence GluTarget are gratefully acknowledged for financial support. The technical assistance of Flemming Hansen, Department of Chemistry,
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University of Copenhagen, with collecting X-ray data is gratefully acknowledged.
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