3-pyrazolone analogues of the 3-isoxazolol metabotropic excitatory amino acid receptor agonist homo-AMPA. Synthesis and pharmacological testing

Article abstract of DOI:10.1016/S0223-5234(99)00122-1

We have previously shown that the higher homologue of (S)-glutamic acid [(S)-Glu], (S)-α-aminoadipic acid [(S)-α-AA] is selectively recognized by the mGlu₂ and mGlu₆ subtypes of the family of metabotropic glutamic acid (mGlu) receptors. Furthermore, a number of analogues of (S)-α-AA, in which the terminal carboxyl group has been replaced by various bioisosteric groups, such as phosphonic acid or 3-isoxazolol groups, have been shown to interact selectively with different subtypes of mGlu receptors. In this paper we report the synthesis of the 3-pyrazolone bioisosteres of α-AA, compounds (RS)-2-amino-4-(1,2-dihydro-5-methyl-3-oxo-3H-pyrazol-4-yl)butyric acid (1) and (RS)-2-amino-4-(1,2-dihydro-1,5-dimethyl-3-oxo-3H-pyrazol-4-yl)butyric acid (2). At a number of steps in the reaction sequences used, the reactions took unexpected courses and provided products which could not be transformed into the target compounds, and attempts to synthesize the 2,5-dimethyl isomer of 2, compound 3, failed. An X-ray crystallographic analysis of the intermediate 1,2-dihydro-4-(2-hydroxyethyl)-2,5-dimethyl-3H-pyrazol-3-one (5b) confirmed the expected regioselectivity of the reaction between methylhydrazine and α-acetylbutyrolactone (4). Neither 1 nor 2 showed significant effects at the different types of ionotropic glutamic acid receptors or at mGlu(1a) (group I), mGlu₂ (group II), and mGlu(4a) and mGlu₆ (group III) receptors, representing the three indicated groups of mGlu receptors.

Full text of DOI:10.1016/S0223-5234(99)00122-1

Eur. J. Med. Chem. 34 (1999) 967−976
© 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
967
Original article
3-Pyrazolone analogues of the 3-isoxazolol metabotropic excitatory amino acid
receptor agonist homo-AMPA. Synthesis and pharmacological testing
Diane Zimmermann, Yves Louis Janin, Lotte Brehm, Hans Bräuner-Osborne, Bjarke Ebert,
Tommy Nørskov Johansen, Ulf Madsen, Povl Krogsgaard-Larsen*
Neuroscience PharmaBiotec Research Center, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, 2
Universitetsparken, DK-2100 Copenhagen, Denmark
(Received 31 May 1999; accepted 10 June 1999)
Abstract – We have previously shown that the higher homologue of (S)-glutamic acid [(S)-Glu], (S)-α-aminoadipic acid [(S)-α-AA] is
selectively recognized by the mGlu2 and mGlu6 subtypes of the family of metabotropic glutamic acid (mGlu) receptors. Furthermore, a
number of analogues of (S)-α-AA, in which the terminal carboxyl group has been replaced by various bioisosteric groups, such as phosphonic
acid or 3-isoxazolol groups, have been shown to interact selectively with different subtypes of mGlu receptors. In this paper we report the
synthesis of the 3-pyrazolone bioisosteres of α-AA, compounds (RS)-2-amino-4-(1,2-dihydro-5-methyl-3-oxo-3H-pyrazol-4-yl)butyric acid
(1) and (RS)-2-amino-4-(1,2-dihydro-1,5-dimethyl-3-oxo-3H-pyrazol-4-yl)butyric acid (2). At a number of steps in the reaction sequences
used, the reactions took unexpected courses and provided products which could not be transformed into the target compounds, and attempts
to synthesize the 2,5-dimethyl isomer of 2, compound 3, failed. An X-ray crystallographic analysis of the intermediate 1,2-dihydro-4-(2-
hydroxyethyl)-2,5-dimethyl-3H-pyrazol-3-one (5b) confirmed the expected regioselectivity of the reaction between methylhydrazine and
α-acetylbutyrolactone (4). Neither 1 nor 2 showed significant effects at the different types of ionotropic glutamic acid receptors or at mGlu1a
(group I), mGlu2 (group II), and mGlu4a and mGlu6 (group III) receptors, representing the three indicated groups of mGlu receptors. © 1999
Éditions scientifiques et médicales Elsevier SAS
excitatory amino acids / metabotropic glutamic acid receptors / homologues of glutamic acid / heterocyclic bioisosteres / 3-pyrazolone
bioisosteres
1. Introduction
(NMDA), (RS)-2-amino-3-(3-hydroxy-5-methyl-4-iso-
xazolyl)propionic acid (AMPA), and kainic acid recep-
tors [1–3], a heterogeneous class of metabotropic EAA
receptors (mGlu receptors) has been shown to have
important functions in the central excitatory neurotrans-
mission processes [4]. It is now generally agreed that
iGlu as well as mGlu receptors play important roles in the
healthy as well as the diseased CNS, and that all subtypes
of these receptors are potential targets for therapeutic
intervention in a number of diseases [5, 6].
(S)-Glutamic acid [(S)-Glu], which is the main excita-
tory neurotransmitter in the central nervous system
(CNS), and other excitatory amino acids (EAAs) operate
through four different classes of receptors. In addition to
the three heterogeneous classes of ionotropic EAA recep-
tors (iGlu receptors), named N-methyl-D-aspartic acid
*Correspondence and reprints
Abbreviations: EAA: excitatory amino acid; iGlu receptor: iono-
tropic glutamic acid receptor; mGlu receptor: metabotropic gluta-
mic acid receptor; (S)-Glu: (S)-glutamic acid; NMDA: N-methyl-
D-aspartic acid; AMPA: (RS)-2-amino-3-(3-hydroxy-5-methyl-4-
isoxazolyl)propionic acid; homo-AMPA: (RS)-2-amino-4-(3-
hydroxy-5-methyl-4-isoxazolyl)butyric acid; HIBO: (RS)-2-
amino-3-(3-hydroxy-5-isoxazolyl)propionic acid; (S)-α-AA: (S)-
α-aminoadipic acid; (S)-AP5: (S)-2-amino-5-phosphonovaleric
acid.
mGlu receptors belong to the family of G-protein
coupled receptors which regulate the intracellular content
of second messengers [6–8]. Based on molecular cloning,
eight different subtypes of mGlu receptors have been
identified, and on the basis of pharmacological character-
ization, sequence homology, and second messenger path-
ways, these receptors have been subdivided into three

968
groups [6–12]: mGlu1 and mGlu5, which are coupled to
phosphoinositide hydrolysis (group I); mGlu2 and mGlu3
form group II and are coupled to inhibition of adenylate
cyclase; mGlu4 and mGlu6–8, which also are coupled to
inhibition of adenylate cyclase form group III.
We have recently shown that the homologue of (S)-
Glu, (S)-α-aminoadipic acid [(S)-α-AA], activates the
mGlu2 and mGlu6 receptor subtypes without interacting
detectably with other mGlu receptors [13]. Subsequently,
it was shown that substitution of various heterocyclic
bioisosteric groups for the terminal carboxyl group of
(S)-α-AA provided a series of heterocyclic (S)-Glu ho-
mologues showing different mGlu receptor pharmacol-
ogy. Thus, (S)-homoquisqualic acid is an antagonist at
mGlu1a but an agonist at mGlu2 and mGlu5a, whereas
(S)-2-amino-5-phosphonovaleric acid [(S)-AP5] (fig-
ure 1) selectively blocks (S)-Glu activation of
mGlu2 [14]. Within this class of mGlu receptor ligands,
even minor structural modifications can have quite pro-
found pharmacological consequences. Thus, whereas
(S)-2-amino-4-(3-hydroxy-5-methyl-4-isoxazolyl)butyric
acid [(S)-homo-AMPA] is a specific mGlu6 receptor
agonist [15],
(S)-2-amino-3-(3-hydroxy-5-isoxazolyl)
propionic acid [(S)-HIBO] as well as (S)-4-methyl-HIBO
(figure 1) are antagonists at the mGlu1a and mGlu5a
receptors, the latter compound being the more potent
antagonist at both receptors [16].
We have now synthesized and pharmacologically
tested the 3-pyrazolone amino acids 1 and 2, and we also
report our attempts to synthesize compound 3 (figure 1).
Figure 1. Structures of (S)-glutamic acid [(S)-Glu], its higher
homologue (S)-α-aminoadipic acid [(S)-α-AA], a number of
bioisosteres of (S)-α-AA, and compounds 1–3. The synthesis of
1 and 2, and the unsuccessful attempts to synthesize 3 are
described in this paper.
2. Chemistry
2.1. Synthesis
Our first synthetic approach was initiated by conden-
sations of a-acetylbutyrolactone (4) with hydrazine to
give 5a, and with methylhydrazine to give 5b and very
small amounts of the N-1 methyl isomer (6) (figure 2).
The regioselectivity of the latter reaction, which is in
agreement with previous observations for reactions be-
tween methylhydrazine and â-oxo ester [17–19], was
confirmed by an X-ray crystallographic analysis of 5b
(see subsequent section). Attempts to convert 7a and b,
prepared from 5a and b, by heating with hydrobromic
acid (62%), into the respective acetamidomalonates 10a
and b were unsuccessful. However, treatment of 7a and b
with acetamidomalonate anion gave the spiro compounds
9a and b as the only products. Accordingly, addition of
potassium carbonate to the evaporated reaction products
from the treatment of 5a and b with hydrobromic acid
(62%) exclusively gave 9a and b. Prolonged heating
(80 °C) of 9a and b with acetamidomalonate anion in
DMF failed to give detectable amounts of 10a and b by
cyclopropyl ring opening reactions (figure 2). The NMR
signals recorded for 9a and b were compatible with the
1
assigned spiro structures (second order H-NMR signals
centered at 1.4 and 1.7 ppm for both 9a and 9b,
corresponding to only one methylenic 13C-NMR signal at
17.2 and 17.6 ppm for 9a and 9b, respectively).
Alkylation of 5a and b with isopropyl bromide in the
presence of potassium carbonate gave the O-alkylated
derivatives 8a and b as the main products. Compounds 8a
and b were converted into the corresponding bromides
11a and b by treatment with phosphorus tribromide, and
prior to alkylation experiments, 11a was further con-
verted into 13. Attempts to synthesize 14a and b by

969
Figure 3. (a) NaH; (b) Cl(CH2)2Br; (c) CH3COCHNaCOOEt;
(d) NaH.
1-bromo-2-chloroethane did, however, fail to give 19, but
led to extensive decomposition. In order to elucidate the
underlying reaction(s), 16 was subjected to the same
basic reaction conditions, i.e. sodium hydride in DMF,
but in the absence of ethyl acetoacetate, and under these
conditions, compound 18 was formed and assigned the
structure shown in figure 3 on the basis of NMR spectro-
scopic data. From the crude product of 16, obtained by
alkylation of 15, compound 17 was isolated in low yields
(4%). The mechanism(s) for the formation of 17, which
could be detected in all reaction mixtures formed after
prolonged treatment of acetamidomalonate anion with
different alkylating agents, are unclear, but may be
analogous to a previously reported oxidative dimerization
of b-ketoesters [20].
In the third approach to the synthesis of the target
3-pyrazolone amino acids, compound 20 [21] was used as
starting material (figure 4). Treatment of 20 with hydra-
zine gave 21 in good yields, and 21 was subsequently
deprotected to give 1. The reaction between 20 and
methylhydrazine was less straightforward. From the re-
action product only compound 22 (17%) could be iso-
lated, whereas the isomeric condensation product 23
could not be detected in or isolated from the crude
product. The position of the N-1 methyl group in 22 was
established by a positive NOE effect with the methyl
substituent at C-5 of the 3-pyrazolone ring. The formation
of 22 rather than 23 in this reaction probably indicates
that the initial attack is of the primary amino group of
methylhydrazine at the lactam carbonyl group of 20 with
subsequent ring closure. This may reflect a pronounced
activation of the N-acylated lactam bond [22, 23]. A
perhaps less likely [19] explanation of the formation of
Figure 2. (a) NH2NH2 or NH2NHCH3; (b) 62% HBr; (c)
iPrBr/K2CO3; (d) (EtOOC)2CNaNHAc; (e) PBr3; (f) Boc2O.
treatment of 11a and b with acetamidomalonate anion
were, however, unsuccessful, and attempts to alkylate
acetamidomalonate anion by 13 also failed to provide the
desired alkylated product. In analogy with 7a and b,
compound 11a also cyclized to cyclopropyl 12 under
these conditions (figure 2). However, NMR monitoring of
the reactions between 11b or 13 and acetamidomalonate
anion indicated that the starting material was the major
component and that some elimination of hydrogen bro-
mide from 11b and 13 occurred, but no alkylated deriva-
tives could be observed in the mixture.
Our second, and equally unsuccessful, approach to the
synthesis of the acetamidomalonate precursors 10a and b
for the target compounds 1 and 3, respectively, is outlined
in figure 3. The idea was to synthesize 10a and b by
condensation of the key intermediate 19 with hydrazine
or methylhydrazine, respectively. Treatment of ethyl
acetoacetate with 16, prepared by alkylation of 15 with

970
Figure 5. Perspective drawing [24] of 1,2-dihydro-4-(2-hydro-
xyethyl)-2,5-dimethyl-3H-pyrazol-3-one (5b) monohydrate
with atomic labelling of the non-hydrogen atoms. Displacement
ellipsoids of the non-hydrogen atoms are shown at the 50%
probability level. Hydrogen atoms are represented by spheres of
arbitrary size. Hydrogen bond is indicated by a thin line.
Figure 4. (a) NH2NH2; (b) NH2NHCH3; (c) CF3COOH.
22 rather than 23 by treatment of 20 with methylhydra-
zine may be that the reaction is initiated by the formation
of an unreactive enamine from the secondary amino
group and the keto group of 20 prior to cyclization.
Boc-deprotection of 20 and treatment of the resulting
lactam with methylhydrazine gave a very complex reac-
tion mixture, which was not worked up. Deprotection of
22 gave the 3-pyrazolone amino acid 2.
show an appreciable degree of π-delocalization over the
whole of the heterocyclic system. Thus, resonance forms
of the molecule include charged species. The partial
negative charge on the carbonylic oxygen atom O1 makes
it a very strong hydrogen bond acceptor. The O1 atom is
engaged in two hydrogen bonds, with the N1-H fragment
of a c glideplane-related dihydropyrazolone molecule and
with the water molecule. In accordance with literature
data [26], the appreciable delocalization of the π-
conjugated system with a lengthening of the carbonyl
bond [C3-O1 = 1.276 (1)] is coupled with a very strong
intermolecular N1-H...O1=C3 hydrogen bond (resonance
assisted hydrogen bonding) [26–28] (table I).
2.2. X-ray analysis of 1,2-dihydro-4-(2-hydroxyethyl)-
2,5-dimethyl-3H-pyrazol-3-one (5b) monohydrate
A perspective drawing [24] of the molecular structure
of compound 5b monohydrate as determined by an X-ray
analysis is shown with atomic labelling in figure 5. Of the
possible tautomers, which may exist for the molecule, the
1,2-dihydropyrazol-3-one is found in the solid state. A
search within the Cambridge Structural Database [25]
reveals that the 1,2-dihydropyrazol-3-one fragment dis-
plays in crystals a variety of geometries, which depend on
the nature and the number of substituents as well as the
crystal packing. In the present structure the bond lengths
In the crystal structure (figures 5 and 6) the N1-
H...O1=C3 hydrogen bonded c glideplane-related mol-
ecules form infinite zigzag chains along the c axis. The
packing of the zigzag chains is attained via the water
molecules. Each water molecule is hydrogen bonded, two
Table I. Hydrogen bond geometries (Å, degrees) for 1,2-dihydro-4-(2-hydroxyethyl-2,5-dimethyl-3H-pyrazol-3-one (5b) monohydratea,b
.
D-H...A
D-H
H...A
D...A
< DHA
N1-H1...O1ii
0.93 (2)
0.86 (2)
0.72 (2)
0.82 (2)
0.92 (2)
1.72 (2)
1.83 (2)
2.02 (2)
1.91 (2)
2.54 (2)
2.650 (1)
2.687 (2)
2.738 (1)
2.733 (2)
3.349 (2)
170 (2)
172 (2)
171 (2)
172 (2)
146 (2)
O2-H2...OWi
OW-HWa...O1iii
OW-HWb...O2iv
C6-H6c...OWv
aEstimated standard deviations are given in parentheses.
bSymmetry code: (i) x, y, z; (ii) x, 1 – y, –½ + z; (iii) ½ + x, ½ – y, –½ + z; (iv) 1 + x, y, z; (v) –½ + x, ½ + y, z.

971
Although the distribution of mGlu receptors in the CNS
and their synaptic localization are far from being fully
mapped out, it has been shown that members of this
heterogeneous family of receptors are functioning as
postsynaptic receptors as well as autoreceptors [36].
Thus, at least in principle, both agonists and antagonists
at a number of mGlu receptors have therapeutic interest.
The first step in the rational development of drugs
targeted at distinct subtypes of mGlu receptors is the
design of ligands interacting specifically with the recep-
tors in question. So far, the design of such compounds has
primarily been achieved by conformational immobiliza-
tion of various parts of the molecule of (S)-Glu [37–42].
We have recently shown that bioisosteric replacements of
the terminal carboxyl group of the (S)-Glu homologue,
(S)-α-AA, by different acidic functionalities have pro-
vided a series of compounds (figure 1) showing different
kinds and degrees of selectivety at mGlu receptor sub-
types [13–16].
Figure 6. Molecular packing [24] of 1,2-dihydro-4-(2-hydroxy-
ethyl)-2,5-dimethyl-3H-pyrazol-3-one (5b) monohydrate. For
clarity only hydrogen atoms bonded to oxygen and nitrogen
atoms are shown, except for the (x, y, z) molecule, where all
hydrogen atoms are shown. Displacement ellipsoids are shown
at the 50% probability level for the non-hydrogen atoms.
Hydrogen atoms are represented by spheres of arbitrary size.
Hydrogen bonds [O(N)-H O] are indicated by thin lines.
In this paper is described the synthesis of two
3-pyrazolone analogues of α-AA, compounds 1 and 2.
The synthesis of a third target compound 3 (figure 1)
failed in spite of quite extensive synthetic efforts (fig-
ures 2–4). Neither 1 nor 2 showed detectable affinity for
representative members of the mGlu receptor family, or
for any of the iGlu receptors.
as donors (OW-H...O1=C3 and OW-H...O2-H) and two as
acceptors (O2-H...OW and C6-H...OW) [29]. Hydrogen
bond geometries are given in table I.
We have previously shown that among a number of
heterocyclic analogues of the inhibitory amino acid
neurotransmitter, γ-aminobutyric acid (GABA), various
3-pyrazolone analogues stood out as the only compounds
devoid of affinity for GABAA receptors [18, 43]. The
inactivity of 1 and 2 at mGlu or iGlu receptors and the
lack of affinity of 3-pyrazolone GABA analogues for
GABAA receptors may mechanistically be an unrelated
phenomena, but it is tempting to speculate that certain
structural and/or physico-chemical characteristics of the
pyrazolone unit impede these receptor interactions. 4,5-
Di- or 1,4,5-trisubstituted 3-pyrazolones incorporated in
GABA analogues have pKa values in the range of
6–7 [18], and 4,5-disubstituted 3-isoxazolols incorpo-
rated in Glu analogues have pKa values in the range of
4–6 [44], indicating that these heterocyclic units are
predominantly ionized at physiological pH values. The
pKa values of compound 1 have been determined by
titration to be 2.9, 8.2 and 9.8. Thus, the degree of
ionization of the 3-pyrazolone moiety of compound 1
(pKa 8.2) is substantially lower than for the above
mentioned analogues. This may, to some extent, explain
the lack of receptor affinity of these 3-pyrazolones.
Alternatively, the degree of hydration of 3-pyrazolones
may be markedly different from those of the carboxyl
group or the bioisosteric groups illustrated in figure 1, and
3. In vitro pharmacological testing
Compounds 1 and 2 were tested for affinity at the
NMDA, AMPA, and kainic acid classes of iGlu receptors,
using the radioligands [3H]-(RS)-3-(2-carboxy-4-
piperazinyl)propyl-1-phosphonic acid ([3H]CPP) [30],
[3H]AMPA [31], and [3H]kainic acid [32], respectively.
No significant affinity (IC50 > 100 µM) was observed in
any of these binding assays. Using the rat cortical slice
preparation [33] in a modified version [34] as an in vitro
electrophysiological model, compounds 1 and 2 were
shown not to act as agonists or antagonists (1 mM
concentrations) at any of the iGlu receptors. Using
established second messenger assay systems [13], com-
pounds 1 and 2 were shown not to exbibit agonist or
antagonist effects (1 mM concentrations) at the mGlu1a,
mGlu2, mGlu4a, or mGlu6 subtypes of mGlu receptors.
4. Discussion
There is a rapidly growing pharmacological and thera-
peutic interest in mGlu receptor ligands [4, 6, 35, 36].

972
may obstruct interaction of such groups with appropriate
sites at the receptors.
give 5b (6.29 g; 78%): M.p. 74–76 °C. Anal.
C7H12N2O2 H2O (C, H, N). 1H-NMR (MeOH-d4): δ 2.18
(3H, s), 2.51 (2H, t, J = 6.7 Hz), 3.43 (3H, s), 3.65 (2H,
t, J = 6.7 Hz). 13C-NMR (MeOH-d4): δ 8.8, 24.7, 28.8,
60.6, 100.7, 143.9, 162.9. Crude 6 was recrystallized
(EtOAc) to give 6 (210 mg; 3%): M.p. 149–150 °C. Anal.
C7H12N2O2 (C, H, N). 1H-NMR (DMSO-d6): δ 2.04 (3H,
s), 2.33 (2H, t, J = 7 Hz), 3.36 (2H, t, J = 7 Hz), 3.44 (3H,
s). 13C-NMR (DMSO-d6): δ 9.5, 26.1, 35.2, 61.4, 98.6,
136.8, 158.3.
5. Experimental protocols
5.1. Chemistry
Column chromatography (CC) was performed on silica
gel C60-H (230–400 mesh, Rhône-Poulenc) and flash
chromatography was performed as described by Still et
al. [45]. Solvents were dried using 3 Å or 4 Å molecular
sieves. Compounds containing the 3-pyrazolone unit
5.2.3. 1,2-Dihydro-4-(2-bromoethyl)-5-methyl-3H-pyr-
azol-3-one hydrobromide 7a
were visualized on TLC plates (Merck silica gel 60 F254
)
A solution of 5a (2.00 g; 14.1 mmol) in hydrobromic
acid (62%; 30 mL) was heated at 80 °C for 16 h in a
sealed flask. The reaction mixture was evaporated to give
crude crystalline 7a, which was very prone to cyclize to
9a and was therefore not subjected to further purification.
1H-NMR (DMSO-d6): δ 2.25 (3H, s), 2.88 (2H, t, J = 6.6
Hz), 3.54 (2H, t, J = 6.6 Hz). 13C-NMR (DMSO-d6): δ
10.4, 24.3, 33.3, 100.4, 145.0, 155.2.
using UV light and a FeCl3 spray reagent. Compounds
containing amino groups were visualized using a ninhy-
drin spray reagent. Melting points were determined in
1
capillary tubes and are uncorrected. H-NMR and 13C-
NMR spectra were recorded on a Bruker AC-200 MHz
spectrometer and NOESY experiments were performed
on a Bruker AM 400 MHz spectrometer. Chemical shifts
are in parts per million (ppm) with respect to TMS unless
otherwise stated. Mass spectra were recorded on an LKB
2091 spectrometer and elemental analyses were per-
formed by the Analytical Department, H. Lundbeck A/S
Denmark, and were within ± 0.4% of the calculated
values unless otherwise stated. The pKa values were
determined by titration with 0.1 M NaOH using a
published procedure [44] and the program Grafit 4.0 for
curvefitting.
5.2.4. 1,2-Dihydro-4-(2-bromoethyl)-2,5-dimethyl-3H-
pyrazol-3-one hydrobromide 7b
Compound 7b was prepared as described above for 7a.
Crude 7b was recrystallized (H2O) to give 7b (2.41 g;
57%): M.p. 91.1–93.6 °C. Anal. C71H11N2OBr HBr (H, N,
Br) C: calcd. 27.93, found 27.50. H-NMR (DMSO-d6):
δ 2.25 (3H, s), 2.91 (2H, t, J = 7.4 Hz), 3.52 (2H, t, J =
7.4 Hz), 3.62 (3H, s). 13C-NMR (DMSO-d6): δ 10.1,
24.4, 32.2, 33.0, 100.2, 143.5, 153.9.
5.2. Synthesis of new compounds
5.2.5. 7-Methyl-5,6-diazaspiro[2,4]hept-6-ene-4-one 9a
The conversion of 7a into 9a was accomplished in an
NMR tube. To a solution of 7a (30 mg; 0.10 mmol) in
DMSO-d6 (0.7 mL) was added potassium carbonate
(30 mg; 0.22 mmol) at 22 °C. NMR recordings indicated
that the conversion of 7a into 9a was completed after 2 h.
No attempts were made to isolate and purify 9a. 1H-NMR
(DMSO-d6): δ 1.32 (2H, m), 1.69 (2H, m), 1.78 (3H, s).
13C-NMR (DMSO-d6): δ 12.5, 17.2, 31.6, 159.6, 176.2.
5.2.1. 1,2-Dihydro-4-(2-hydroxyethyl)-5-methyl-3H-pyr-
azol-3-one 5a
A solution of 4 (5.95 g; 46.4 mmol) and hydrazine
hydrate (2.32 g; 46.4 mmol) in H2O (20 mL) was re-
fluxed for 2 h. The evaporated reaction product was
subjected to CC (CH2Cl2/MeOH, 9:1), and crude 5a was
recrystallized (2-propanol) to give 5a (4.31 g; 65%): M.p.
180–181 °C. Anal. C6H10N2O2 (C, H, N). 1H-NMR
(MeOH-d4): δ 2.17 (3H, s), 2.53 (2H, t, J = 6.7 Hz), 3.66
(2H, t, J = 6.7 Hz). 13C-NMR (MeOH-d4): δ 10.3, 26.3,
62.9, 100.9, 143.3, 163.9.
5.2.6. 5,7-Dimethyl-5,6-diazaspiro[2,4]hept-6-ene-4-one
9b
Compound 7b was converted into 9b as described
above for 9a. 1H-NMR (DMSO-d6): δ 1.40 (2H, m), 1.74
(2H, m), 1.83 (3H, s) 3.22 (3H, s). 13C-NMR (DMSO-
d6): δ 11.3, 17.6, 32.1, 33.2, 164.0, 178.0.
5.2.2. 1,2-Dihydro-4-(2-hydroxyethyl)-2,5-dimethyl-3H-
pyrazol-3-one monohydrate 5b and 1,2-dihydro-4-(2-
hydroxyethyl)-1,5-dimethyl-3H-pyrazol-3-one 6
A solution of 4 (5.95 g; 46.4 mmol) and methylhydra-
zine (2.13 g; 46.4 mmol) in acetonitrile (20 mL) was
refluxed for 2 h. The evaporated reaction product was
subjected to CC (CH2Cl2/MeOH, 19:1) to give two
products. Crude 5b was recrystallized (acetonitrile) to
5.2.7. 4-(2-Hydroxyethyl)-3-isopropyloxy-5-methyl-2H-
pyrazole 8a
A solution of 5a (2.27 g; 16 mmol), isopropyl bromide
(1.65 mL; 17.6 mmol), and potassium carbonate (2.43 g;

973
17.6 mmol) in dry DMF was heated at 60 °C for 48 h.
The reaction mixture was evaporated and upon addition
of H2O (20 mL), the mixture was extracted with EtOAc
(3 × 20 mL). The combined organic phases were dried
(MgSO4) and evaporated, and the residue was subjected
to CC (CH2Cl2/MeOH, 9:1) to give crude 8a (2.36 g; ca.
80%) as a crystalline compound, which was converted
into 11a without further purification. Spectroscopic data
for 8a: 1H-NMR (DMSO-d6): δ 1.33 (6H, d, J = 6.1 Hz),
2.17 (3H, s), 2.56 (2H, t, J = 6.1 Hz), 3.72 (2H, t, J = 6.1
Hz), 4.86 (1H, septet, J = 6.1 Hz). 13C-NMR (DMSO-d6):
δ 10.1, 22.3, 25.6, 62.5, 71.5, 100.0, 138.2, 159.9.
5.2.11. 4-Isopropyl-7-methyl-5,6-diazaspiro[2,4]hept-6-
ene 12
Compound 11a was converted into 12 as described
1
above for 9a. H-NMR (DMSO-d6): δ 1.27 (6H, d, J =
6.2 Hz), 1.55 (2H, m) 1.80 (2H, m), 1.80 (3H, s), 3.22
(3H, s), 4.97 (1H, septet, J = 6.2 Hz). 13C-NMR (DMSO-
d6): δ 11.6, 16.5, 21.4, 33.8, 72.0, 168.3, 176.5.
5.2.12. Tert-Butyl 4-(2-bromoethyl)-3-isopropyloxy-5-
methyl-1H-pyrazole-1-carboxylate 13
A solution of 11a (500 mg; 2.02 mmol), 4-di-
methylaminopyridine (254 mg; 2.02 mmol), triethy-
lamine (0.28 mL; 2.02 mmol), and di-tert-butyl dicarbon-
ate (661 mg; 3.03 mmol) in dry CH2Cl2 (50 mL) was
stirred at 25 °C for 2 h and then evaporated. Flash
chromatography (hexane/EtOAc, 9:1) of the residue gave
5.2.8. 4-(2-Hydroxyethyl)-3-isopropyloxy-2,5-dimethyl-
2H-pyrazole 8b
1
13 (645 mg; 92%) as an oil. H-NMR (CDCl3): δ 1.31
Compound 8b was synthesized from 5b as described
above for 8a. CC (CH2Cl2/MeOH, 24:1) gave 8b (2.38 g;
ca. 75%) as an oil. 1H-NMR (DMSO-d6): δ 1.24 (6H, d,
J = 6.1 Hz), 2.00 (3H, s), 2.39 (2H, t, J = 8.1 Hz), 3.38
(2H, m), 3.46 (3H, s), 4.30 (1H, septet, J = 6.1 Hz), 4.65
(1H, t, J = 5 Hz). 13C-NMR (DMSO-d6): δ 12.8, 22.3,
26.6, 36.7, 60.9, 77.0, 100.3, 144.7, 149.7.
(6H, d, J = 6.1 Hz), 1.59 (9H, s), 2.36 (3H, s) 2.82 (2H,
t, J = 7.3 Hz), 3.44 (2H, t, J = 7.3 Hz), 5.08 (1H, septet,
J = 6.1 Hz). 13C-NMR (CDCl3): δ 13.2, 22.1, 26.0, 28.0,
31.4, 71.4, 83.8, 108.2, 141.4, 148.8, 162.0.
5.2.13. Ethyl 2-acetamido-4-chloro-2-(ethyloxycarbonyl)
butyrate 16 and ethyl 2-acetamido-3,3-(bisethoxy-
carbonyl)acrylate 17
To a solution of 15 (10.0 g; 46.0 mmol) in dry DMF
(150 mL) was added sodium hydride (65% in oil disper-
sion; 1.86 g; 46.5 mmol). The suspension was stirred at
25 °C for 1 h, and after cooling of the reaction mixture to
0 °C, 1-bromo-2-chloroethane (4.2 mL; 50 mmol) was
added. The mixture was stirred at 25 °C for 4 d and then
evaporated. To the residue was added diethylether
(60 mL) and H2O (60 mL). The organic phase was dried
(MgSO4) and evaporated. CC (light petroleum/EtOAc,
3:2) of the residue gave, in addition to starting material 15
(36%), two reaction products. Compound 17 (540 mg;
4%): pale yellow oil, which solidified upon standing. MS:
5.2.9.
pyrazole 11a
4-(2-Bromoethyl)-3-isopropyloxy-5-methyl-2H-
To a solution of 8a (920 mg; 5 mmol) in dry toluene
(50 mL) and pyridine (0.12 mL) was slowly added a
solution of phosphorus tribromide (0.18 mL; 0.35 mmol)
in dry toluene (10 mL), and the solution was heated at
65 °C for 16 h. The solution was poured on ice, washed
with an aqueous solution of sodium hydrogen carbonate
(5%), dried (MgSO4), and evaporated. CC (CH2Cl2/
MeOH, 30:1) of the residue gave 11a (940 mg; 76%) as
an oil, which solidified upon standing: M.p. 79–81 °C.
MS: m/z (IE): 247 (M[79Br]), 249 (M[81Br]). H-NMR
1
1
m/z (IE): 301 (M). H-NMR (CDCl3): δ 1.28 (9H, m),
(CDCl3): δ 1.33 (6H, d, J = 6.1 Hz), 2.18 (3H, s), 2.84
(2H, t, J = 7.5 Hz), 3.47 (2H, t, J = 7.5 Hz), 4.83 (1H,
septet, J = 6.1 Hz). 13C-NMR (CDCl3): δ 10.3, 22.3,
26.1, 32.5, 71.2, 101.0, 138.1, 161.7.
2.18 (3H, s), 4.24 (6H, s), 11.0 (1H, s). 13C-NMR
(CDCl3): δ 13.7, 13.9, 14.0, 23.9, 61.8, 61.9, 62.6, 105.5,
146.3, 162.3, 164.0, 166.7, 167.8. Compound 16 (2.91 g;
23%) colourless crystals: M.p. 86.6–87.4 °C (light petro-
leum). Anal. C11H18NO5Cl (C, H, N). MS: m/z (IE): 279
1
(M). H-NMR (CDCl3): δ 1.25 (6H, t, J = 7.4 Hz), 2.05
5.2.10. 4-(2-Bromoethyl)-3-isopropyloxy-2,5-dimethyl-
2H-pyrazole 11b
(3H, s), 2.85 (2H, t, J = 6.6 Hz), 3.49 (2H, t, J = 6.6 Hz),
4.24 (4H, q, J = 7.4 Hz), 6.92 (1H, s). 13C-NMR (CDCl3):
δ 13.9, 28.3, 35.2, 39.7, 62.9, 64.9, 167.7, 169.6.
Compound 11b was prepared from 8b as described
above for 11a. CC (light petroleum/EtOAc, 3:2) of the
evaporated reaction product gave 11b (835 mg; 64%) as
an oil. 1H-NMR (CDCl3): δ 1.33 (6H, d, J = 6.1 Hz), 2.16
(3H, s) 2.87 (2H, t, J = 7.7 Hz), 3.39 (2H, t, J = 7.7 Hz),
3.59 (3H, s) 4.31 (1H, q, J = 6.1 Hz). 13C-NMR (CDCl3):
δ 12.7, 22.4, 27.1, 31.1, 33.8, 77.3, 101.4, 145.7, 150.1.
5.2.14. Ethyl 5,6-dihydro-4-ethoxycarbonyl-2-methyl-
1,3-oxazine-4-carboxylate 18
To a solution of 16 (200 mg; 0.7 mmol) in dry DMF
(5 mL) was added sodium hydride (65% in oil suspen-
sion; 30 mg; 0.7 mmol), and the suspension was stirred at

974
80 °C for 16 h. To the reaction mixture was then added
diethylether (20 mL) and H2O (10 mL). The organic
phase was dried (MgSO4) and evaporated to give 18 as
the only detectable product. Attempts to further purify 18
by CC led to decomposition. Spectroscopic data for 18:
1H-NMR (CDCl3): δ 1.26 (6H, t, J = 7 Hz), 2.00 (3H, s),
2.27 (2H, t, J = 5.9 Hz), 4.17 (2H, t, J = 5.9 Hz), 4.26
(4H, m). 13C-NMR (CDCl3): δ 14.1, 22.2, 26.2, 61.8,
62.2, 64.4, 160.6, 169.5.
63%) after recrystallization (H2O): M.p. 268–269 °C
(decomp). pKa 2.9, 8.2 and 9.8. Anal. C8H13N3O3 (C, H,
N). 1H-NMR [D2O, tert-BuOH (1.26 ppm)]: δ 1.95–2.07
(2H, m), 2.19 (3H, s), 2.41 (2H, t, J = 7.7 Hz), 3.70 (1H,
t, J = 6.4 Hz). 13C-NMR [D2O, tert-BuOH (31.6 ppm)]:
δ 11.6, 18.5, 32.3, 56.1, 103.3, 147.7, 165.8, 176.7.
5.2.18. (RS)-2-Amino-4-(1,2-dihydro-1,5-dimethyl-3-oxo-
3H-pyrazol-4-yl)butyric acid 2
Compound 2 was prepared from 22 (270 mg;
0.79 mmol) as described above for 1. Yield of 2: 45 mg
(27%) after recrystallization (H2O): M.p. 237.5–239.0 °C
5.2.15. Ethyl (RS)-2-(tert-butyloxycarbonylamino)-4-
(1,2-dihydro-5-methyl-3-oxo-3H-pyrazol-4-yl)butyrate 21
A solution of 20 [21] (2.00 g; 6.4 mmol) and hydrazine
(64% aqueous solution; 0.73 mL; 9.6 mmol) in acetoni-
trile was refluxed for 3 h under inert atmosphere. The
solution was evaporated, and the residue subjected to
flash chromatography on silica gel (CH2Cl2/MeOH, 12:1)
to give 21 (1.41 g; 67%) as a wax. MS: m/z (IE): 272
(13), 255 (16), 227 (M-100, 46), 154 (37), 125 (M-202,
100), 111 (M-216, 94), 57 (tert-butyl, 57). 1H-NMR
(CDCl3): δ 1.22 (3H, t, J = 7.1 Hz), 1.42 (9H, s), 1.93
(2H, m), 2.11 (3H, s), 2.37 (2H, m) 4.10 (2H, q, J = 7.1
Hz), 4.22 (1H, m) 5.81 (1H, broad d). 13C-NMR
(CDCl3): δ 10.4, 14.2, 17.8, 28.4, 32.1, 53.3, 61.3, 80.1,
101.4, 140.5, 155.8, 162.3, 173.0.
1
(decomp). Anal. C9H15N3O3 (C, H, N). H-NMR [D2O,
tert-BuOH (1.26 ppm)]: δ 2.00 (2H, m), 2.17 (3H, s),
2.42 (2H, t, J = 7.6 Hz), 3.51 (3H, s), 3.69 (1H, t, J = 6.5
Hz). 13C-NMR [D2O, tert-BuOH (31.6 ppm)]: δ 10.8,
18.9, 32.6, 35.5, 56.1, 102.9, 146.8, 163.5, 176.6. Positive
NOE effect between the two ring methyl groups was
observed.
5.3. X-ray analysis of 1,2-dihydro-4-(2-hydroxyethyl)-
2,5-dimethyl-3H-pyrazol-3-one (5b) monohydrate
Crystal data: C7H12N2O2. H2O, Mr = 174.20, colour-
less elongated prisms recrystallized from acetonitrile;
monoclinic, space group Cc (no. 9); a = 4.717(4) Å, b =
19.170(4) Å, c = 10.056(4) Å, â = 102.92(4)°, V =
886.3(9) Å3, Z = 4, Dc = 1.306 Mg m–3, F(000) = 376,
µ(Mo Kα) = 0.102 mm–1, T = 122 K. Crystal dimensions
0.14 × 0.28 × 0.32 mm.
Diffraction data were collected on an Enraf-Nonius
CAD-4 diffractometer [46] using graphite monochro-
mated Mo Kα radiation (λ = 0.71073 Å) at 122 ± 0.5 K;
ω/2θ scan mode. Unit cell dimensions were determined
by least-squares refinement of the setting angles of 21
reflections (θ range: 18.48–22.43°). A total of 6 239
(space group allowed) reflections (θ = 2–35°: h: –7→3, k:
–30→30, l: –16→16) were averaged according to the
point group symmetry m (Rint = 0.024 on F2) resulting in
3 195 unique reflections. Data were reduced using the
programs of Blessing (DREADD) [47, 48]. Four standard
reflections were monitored every 104 s, and appropriate
scaling was performed using the program SCALE3 [47,
48]. No absorption correction was made.
5.2.16. Ethyl (RS)-2-(tert-butyloxycarbonylamino)-4-
(1,2-dihydro-1,5-dimethyl-3-oxo-3H-pyrazol-4-yl)buty-
rate 22
Compound 22 was prepared as described above for 21,
using 20 (6.4 mmol) and methylhydrazine (7.7 mmol).
Yield of 22: 360 mg (17%) as an amorphous solid. MS:
m/z (IE): 284 (M-57, 5), 267 (M-74, 11), 241 (M-100, 5),
139 (M-CH(COOEt)NHBoc, 51), 125 (M-CH2-
CH(COOEt)NHBoc, 100), 59 (18). 1H-NMR (CDCl3): δ
1.24 (3H, t, J = 7.1 Hz), 1.44 (9H, s), 1.82–2.05 (2H, m),
2.08 (3H, s), 2.37 (2H, t, J = 7.6 Hz), 3.55 (3H, s), 4.12
(2H, q, J = 7.1 Hz), 4.22 (1H, m), 5.22 (1H, broad d).
13C-NMR (CDCl3): δ 9.5, 14.2, 18.1, 28.4, 32.3, 34.9,
53.2, 61.2, 79.9, 100.7, 137.5, 155.7, 159.6, 172.9.
Positive NOE effect between the two ring methyl groups
was observed.
5.2.17.
(RS)-2-Amino-4-(1,2-dihydro-5-methyl-3-oxo-
3H-pyrazol-4-yl)butyric acid 1
The structure was solved by direct methods using the
program SHELXS-97 [49, 50]. Full-matrix least-squares
refinement (SHELXL-97) [51] was performed on F2 for
all unique reflections, minimizing Σw(Fo – Fc ) , with
anisotropic displacement parameters for the nonhydrogen
atoms. The positions of the hydrogen atoms were located
on intermediate difference electron density maps and
refined with isotropic displacement parameters, except
for the methyl hydrogen atoms of C9, which were
Asolution of 21 (1.34 g; 4.1 mmol) in aqueous trifluoro-
acetic acid (80 mL; 1 M) was refluxed for 18 h, and was
then evaporated to dryness. To the residue was added
H2O (10 mL) and the solution was re-evaporated. This
procedure was repeated four times. A solution of the
residue in H2O (10 mL) was subjected to ion-exchange
on a Dowex 50 WX4 column (H2O/pyridine, 19:1).
Appropriate fractions were evaporated to give 1 (514 mg;
2
2 2

975
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included in idealized positions (riding model) with Uiso
set to 1.5 Uiso of the parent atom, in order to keep
reasonable bond lengths and bond angles. The refinement
(154 parameters, 3 195 reflections) converged at R(F) =
0.0330, wR(F2) = 0.0869 for 3 033 reflections with Fo >
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2
2
4σ(Fo); w–1 = σ2(Fo ) + (0.0606 P)2 where P = (Fo
+
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2
2Fc )/3; S = 1.072. The residual electron density varied
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Supplementary material is available for 1,2-dihydro-4-
(2-hydroxyethyl)-2,5-dimethyl-3H-pyrazol-3-one (5b)
monohydrate. Tables listing final atomic coordinates,
equivalent isotropic (non-H atoms) or isotropic (H atoms)
displacement parameters and anisotropic displacement
parameters of the non-H atoms, and selected bond
lengths, bond angles and torsion angles (3 pages); a list of
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Acknowledgements
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Tetrahedron 49 (1993) 3801–3808.
This work was supported by grants from the Lundbeck
Foundation and H. Lundbeck A/S, EEC (BIO2-CT93-
0243), the Danish State Biotechnology programme
(1991–1999), and the Danish Natural Science Research
Council. The assistance of Mr Flemming Hansen with the
X-ray data collection and of Dr Jean-Daniel Ehrhardt
with mass spectra recordings and the secretarial assis-
tance of Mrs Anne-Mette Nielsen are gratefully acknowl-
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