Design, synthesis, and biological activity of a potent inhibitor of the neuropeptidase N-acetylated alpha-linked acidic dipeptidase.

Article abstract of DOI:10.1021/jm950801q

A series of substituted phosphonate derivatives were designed and synthesized in order to study the ability of these compounds to inhibit the neuropeptidase N-acetylated alpha-linked acidic dipeptidase (NAALADase). The molecules were shown to act as inhib

Full text of DOI:10.1021/jm950801q

J . Med. Chem. 1996, 39, 619-622
619
Design , Syn th esis, a n d Biologica l Activity of a P oten t In h ibitor of th e
Neu r op ep tid a se N-Acetyla ted r-Lin k ed Acid ic Dip ep tid a se
Paul F. J ackson,*,†,§ Derek C. Cole,† Barbara S. Slusher,‡,§ Susan L. Stetz,‡ Laurie E. Ross,‡
Bruce A. Donzanti,‡ and Diane Amy Trainor†
Departments of Medicinal Chemistry and Pharmacology, ZENECA Pharmaceuticals, Wilmington, Delaware 19897
Received October 17, 1995X
A series of substituted phosphonate derivatives were designed and synthesized in order to
study the ability of these compounds to inhibit the neuropeptidase N-acetylated R-linked acidic
dipeptidase (NAALADase). The molecules were shown to act as inhibitors of the enzyme, with
the most potent (compound 3) having a Ki of 0.275 nM. The potency of this compound is more
than 1000 times greater than that of previously reported inhibitors of the enzyme. NAALADase
is responsible for the catabolism of the abundant neuropeptide N-acetyl-L-aspartylglutamate
(NAAG) into N-acetylaspartate and glutamate. NAAG has been proposed to be a neurotrans-
mitter at a subpopulation of glutamate receptors; alternatively, NAAG has been suggested to
act as a storage form of synaptic glutamate. As a result, inhibition of NAALADase may show
utility as a therapeutic intervention in diseases in which altered levels of glutamate are thought
to be involved.
In tr od u ction
The dipeptide N-acetyl-L-aspartate-L-glutamate (NA-
AG) is a major peptidic component of the brain, with
levels comparable to that of the major inhibitory neu-
rotransmitter γ-aminobutyric acid (GABA). Although
NAAG was first isolated in 1964, there was little activity
F igu r e 1. Catabolism of NAAG by the peptidase NAALA-
toward elucidating its role in the central nervous system
(CNS) until the deleterious nature of excess glutamate
in a variety of disease states became apparent. Due to
its structural similarity to glutamate, NAAG has been
suggested to have a variety of roles similar to those of
glutamate itself, including functioning as a neurotrans-
mitter or a cotransmitter in the CNS.1 In 1985, NAAG
was reported to cause excitation of neurons of the lateral
olfactory tract; however, it was subsequently demon-
strated that these findings were due to an artifact of
the preparation.2 More recently, NAAG was shown to
elicit excitatory responses both in vitro and in vivo.3
However, under both scenarios, NAAG was less potent
than glutamate. In 1988, a brain enzyme was identified
which hydrolyzes NAAG to N-acetylaspartate (NAA)
and glutamate (Figure 1).
Dase.
reported in animal models of epilepsy and amyotrophic
lateral sclerosis (ALS)5,7 and in postmortem brain tissue
of patients with ALS, schizophrenia, and Alzhei-
mer’s disease.6,8,9 The further investigation of NAALA-
Dase activity, however, has been limited by the avail-
ability of potent and selective inhibitors of this enzyme.
Although there have been several inhibitors of NAALA-
Dase reported, they have only weak affinity and speci-
ficity for the enzyme.10,11 As a result, there is a need
for the development of potent inhibitors of NAALADase
in order to ascertain its role in the CNS. In this paper,
we describe the synthesis and activity of such an
inhibitor.
This enzyme was found to be specific for the cleavage
of N-acetylated R-linked acidic dipeptides and as a result
was named NAALADase.4 The enzyme is a membrane-
bound peptidase, with an apparent weight of 94 kDa.
NAALADase exhibits a requirement for divalent cations
and is inhibited by known metal-chelating agents,
suggesting that it belongs to the class of metallopepti-
dases. The identification and subsequent purification
of NAALADase led to the proposal of another role for
NAAG: specifically that the dipeptide may serve as a
storage form of synaptic glutamate.
Ch em istr y
Since there was substantial evidence that NAALA-
Dase was a metallopeptidase, we initially focused our
attention on small molecules with functional groups that
are known to inhibit metallopeptidases, such as hy-
droxyphosphinyl derivatives, thio derivatives, and hy-
droxamic acids.12 On the basis of an initial screening
of compounds from our corporate compound collection
which contained these functional groups, we began to
examine a variety of hydroxyphosphinyl derivatives. In
addition, on the basis of literature precedent, we made
the further assumption that the glutamate moiety of
NAAG was important for recognition by the enzyme and
that the aspartate region played a less critical role.13
As a result, we focused our attention on a series of
glutamate-derived hydroxyphosphinyl derivatives whose
synthesis is outlined in Schemes 1 and 2.
In order to further ascertain the exact role of NAAL-
ADase and NAAG in the CNS, a number of in vitro and
in vivo studies have been conducted. For example,
altered levels of NAAG and NAALADase have been
† Department of Medicinal Chemistry.
‡ Department of Pharmacology.
§ Current address: Guilford Pharmaceuticals, 6611 Tributary St.,
Baltimore, MD 21224.
Treatment of commercially available benzyl acrylate
with hexamethylphosphoramide (HMPA) afforded the
X Abstract published in Advance ACS Abstracts, December 15, 1995.
0022-2623/96/1839-0619$12.00/0 © 1996 American Chemical Society

620 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Notes
Ta ble 2. Effects of Compound 3 on Basal Glutamate Efflux
and KCl-Evoked Glutamate Release in Vitroa
Sch em e 1
K+-evoked release
(% of base line)
concentrationb (nM)
basal efflux (%)
control
0.1
1.0
100
285 ( 16
316 ( 27
368 ( 46
437 ( 99c
151 ( 33
137 ( 8
167 ( 24
10.0
a
Sch em e 2
For all studies, Sprague-Dawley rats were used; n ) 3-6.
b
See ref 20 for protocol. Compound dissolved in artificial cere-
brospinal fluid.c p < 0.05 vs control.
Microdialysis probes were then lowered into the stria-
tum, a brain region which contains NAAG, NAALA-
Dase, and glutamatergic nerve terminals. Ten minute
dialysate samples were collected and analyzed for
glutamate content using HPLC with electrochemical
detection as previously described.20 Our findings dem-
onstrate that compound 3, when perfused directly
through the dialysis probe, did not decrease the basal
or potassium-evoked levels of synaptic glutamate (Table
2). Thus, these results tentatively argue against NAAG
serving as a storage form of synaptically available
glutamate. It is possible, however, that in our experi-
ments NAAG could have multiple activities including
acting as an agonist at presynaptic glutamate receptors
or interacting with the glutamate transporters.21 Fur-
ther studies will be necessary to explore these possibili-
ties and will be reported in due course.
Ta ble 1. In Vitro Activity of NAALADAse Inhibitors
compd
Ki (nM)
3
4
7
0.275 ( 0.08
700 ( 67.3
1.89 ( 0.19
R-methylene dibenzyl glutamate 1a 14 in 60% yield
(Scheme 1). A trimethylaluminum-promoted conjugate
addition of dibenzyl phosphite then gave the fully
protected compound 2a 15 (50% yield) which was depro-
tected to afford the (phosphonomethyl)pentanedioic acid
3 in 90% yield. Compound 4 was also prepared by this
route starting with the dibenzyl itaconate 1b16 (40%
overall yield).
The disubstituted phosphinic acid 7 was prepared as
shown in Scheme 2. Conjugate addition of the in situ
generated bis(trimethylsilyl)phosphonite to benzyl acryl-
ate afforded the protected phosphinate 5. Repetition
of the in situ phosphonite generation followed by
conjugate addition to R-methylene dibenzyl glutarate 1a
provided the protected triester 6, which was then
deprotected to afford compound 7 (Scheme 2).17,18
In conclusion, we have synthesized a potent inhibitor
of the neuropeptidase NAALADase. This compound is
greater than 1000 times more potent than previously
described inhibitors and should serve as a useful
pharmacological tool in order to ascertain the function
of NAAG in the CNS.
Exp er im en ta l Section
Ch em istr y. Proton NMR spectra were obtained using a
Bruker AM-300 spectrometer. Chemical shifts are reported
in parts per million relative to tetramethylsilane as internal
standard. Mass spectra (MS) were recorded on a Kratos MS-
80 instrument operating in the chemical ionization (DCI)
mode. Elemental analyses for carbon and hydrogen were
determined by the ZENECA Pharmaceuticals Analytical De-
partment on a Perkin-Elmer 241 elemental analyzer and are
within (0.4% of theory for the formula given. Analytical thin-
layer chromatography (TLC) was conducted on prelayered
silica gel GHLF plates (Analtech, Newark, DE). Visualization
of the plates was accomplished by using UV light, phospho-
molybdic acid-ethanol, and/or iodoplatinate charring. Flash
chromatography was conducted on Kieselgel 60, 230-400
mesh (E. Merck, Darmstadt, West Germany). Solvents were
either reagent or HPLC grade. Reactions were run at ambient
temperature and under a nitrogen atmosphere unless other-
wise noted. Solutions were evaporated under reduced pressure
on a Buchi rotary evaporator. HPLC purification was carried
out on a Dynamax 300 Å C8 21.4 × 250 mm reversed phase
column with a Rainin HPXL solvent delivery system. Frac-
tions were analyzed on a Zorbax Rx-C8 4.6 × 250 mm reversed
phase column with a Hewlett Packard HP 1090 liquid chro-
matograph and a Hewlett Packard HP 1047A RI detector.
Resu lts a n d Discu ssion
Compounds 3, 4, and 7 were tested for inhibition of
NAALADase activity. Of the compounds tested, the
phosphonate 3 showed the best activity, with a Ki of
0.275 nM (Table 1). The activity of this compound is
>1000 times more potent than that of previously de-
scribed inhibitors.
The aspartate analog of compound 3 was also pre-
pared. This molecule (4) showed a large decrease in
efficacy in inhibiting the activity of NAALADase (Table
1), suggesting that a glutamate analog attached to the
phosphonic acid is required for potent inhibition of the
enzyme. In addition, compound 7, which contains an
additional carboxylic acid side chain similar to the
aspartate residue found in NAAG, did not lead to an
increase in potency.
The effect of NAALADase inhibition by compound 3
was subsequently examined in vivo using intracranial
microdialysis, a technique that allows the assessment
of endogenous glutamate levels and release in the
extracellular space of brain tissue.19 Male Sprague-
Dawley rats (300-350 g) were anesthetized with ure-
thane (1.5 g/kg, ip) and placed in a stereotaxic frame.
Diben zyl 2-Meth ylen ep en ta n ed ioa te (1a ). HMPA (2.06
g, 11.5 mmol) was added to benzyl acrylate (19.33 g, 119
mmol), and the mixture stirred at room temperature for 16 h.
The crude material was then applied directly to a silica gel
flash chromatography column (4 × 18 cm) and subsequently
eluted with a hexanes-EtOAc (19:1 f 9:1) gradient. This
afforded compound 1a as a clear oil (11.5 g, 59.5%): TLC Rf
0.23 (4:1 hexanes-EtOAc); 1H NMR (300 MHz, CDCl3) δ 2.55-
2.60 (m, 2 H), 2.65-2.71 (m, 2 H), 5.10 (s, 2 H), 5.19 (s, 2 H),

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 621
5.59 (s, 1 H), 6.22 (s, 1 H), 7.29-7.36 (m, 10 H); MS (methane,
DCI) m/z 325 (M+ + 1).
mL/min, 20 × 10 mL fractions. HPLC analysis, Zorbax C8
column, isocratic water-methanol (4:1), showed the product
(tR ) 1.6 min) was contained in fractions 7-10. These fractions
were combined and lyophilized to afford 3 (0.44 g, 38%): 1H
NMR (300 MHz, D2O) δ 1.91 (ddd, J ) 17.4, 15.5, 7.7 Hz, 1
H), 2.13 (dd, J ) 18.0, 15.5, 6.1 Hz, 1 H), 2.71-2.92 (m, 2 H)
3.01-3.15 (m, 1 H); MS (methane, DCI-) m/z 211 (M+ - 1).
Anal. Calcd for C5H9O7P‚2H2O: C, 24.2; H. 5.28. Found: C,
24.2; H, 5.34.
Diben zyl 2-[[Bis(ben zyloxy)p h osp h or yl]m eth yl]p en t-
a n ed ioa te (2a ). To a solution of dibenzyl phosphite (0.53 g,
2.02 mmol) in CH2Cl2 (20 mL) at 0 °C was added trimethyl-
aluminum (1.01 mL, 2.0 M solution in hexanes, 2.02 mmol).
After 20 min a solution of 1a (0.66 g, 2.02 mmol) in CH2Cl2 (5
mL) was added. The cooling bath was removed, and stirring
at room-temperature continued for 16 h. The reaction was
quenched by the slow addition of 1 N hydrochloric acid (20
mL). After stirring for 10 min the phases were separated, and
the aqueous phase was extracted with CH2Cl2 (2 × 10 mL).
The organic phases were combined, dried (MgSO4), and
concentrated. Flash chromatography over silica gel (3 × 18
cm) with a hexanes-EtOAc (4:1 f 1:1) gradient gave 2a as a
Ben zyl 3-(Hyd r oxyp h osp h in yl)p r op ion a te (5). Hypo-
phosphorous acid (1.82 g, 13.8 mmol, 50% aqueous solution)
and triethylamine (0.97 g, 13.8 mmol) were mixed and dried
by azeotropic removal of toluene (3 × 20 mL) in vacuo at 50
°C. The residue was dissolved in dry CH2Cl2 (50 mL) and
cooled to 0 °C. Triethylamine (2.4 g, 24.2 mmol) and chloro-
trimethylsilane (2.56 g, 23.6 mmol) were added followed after
5 min by benzyl acrylate (0.32 g, 1.98 mmol) in CH2Cl2 (5 mL).
The mixture was stirred for 24 h at room temperature and
then filtered. The filtrate was washed with 1 N hydrochloric
acid (10 mL) and water (10 mL), dried (MgSO4), and concen-
1
clear oil (0.58 g, 48%): TLC Rf 0.50 (1:1 hexanes-EtOAc); H
NMR (300 MHz, CDCl3) δ 1.80-1.99 (m, 3 H), 2.25-2.35 (m,
3 H), 2.78-2.90 (m, 1 H), 4.88-5.01 (m, 6 H), 5.05 (s, 2 H),
7.23-7.35 (m, 20 H); MS (methane, DCI) m/z 587 (M+ + 1).
Anal. Calcd for C34H35O7P: C, 69.61; H, 6.01. Found: C, 69.21;
H, 6.00.
1
trated to afford 5 in quantitative yield: H NMR (300 MHz,
CDCl3) δ 2.05 (dquintet, J ) 1.8, 7.7 Hz, 2 H), 2.66 (quintet,
J ) 7.7 Hz, 2 H), 5.12 (s, 2 H), 7.25-7.38 (m, 5 H), 10.89 (brs,
1 H).
2-(P h osp h on om eth yl)p en ta n ed ioic Acid (3). To a solu-
tion of 2a (2.89 g, 4.93 mmol) in methanol (10 mL) was added
10% palladium on carbon (0.26 g, 5 mol %). After shaking at
40 psi for 24 h, the mixture was filtered through Celite and
the filter cake washed with methanol. The combined filtrate
was concentrated in vacuo to afford 3 in quantitative yield.
Purification of 3 was carried out by HPLC: semipreparative
C8 column, linear gradient of water and methanol (0% f 20%)
over 20 min, 10 mL/min, 20 × 10 mL fractions. HPLC
analysis, Zorbax C8 column, isocratic water-methanol (4:1),
showed the product (tR ) 1.6 min) was contained in fractions
7-10. These fractions were combined and lyophilized to afford
Dib en zyl 2-[[[2-(Ben zylca r b oxy)et h yl]h yd r oxyp h os-
p h in oyl]m eth yl]p en ta n ed ioa te (6). Compound 5 (0.82 g,
3.63 mmol) was dried by azeotropic removal of toluene (3 ×
20 mL) in vacuo at 50 °C. The residue was dissolved in dry
CH2Cl2 (50 mL) and cooled to 0 °C. Triethylamine (1.5 g, 14.6
mmol) and chlorotrimethylsilane (1.34 mL, 12.4 mmol) were
added followed after 5 min by compound 1a (1.28 g, 3.94 mmol)
in CH2Cl2 (5 mL). The mixture was stirred for 24 h at room
temperature and then washed with 1 N hydrochloric acid (10
mL) and water (10 mL), dried (MgSO4), and concentrated.
Flash chromatography over silica gel (3 × 18 cm) with a
hexane-EtOAc (4:1 f 1:1) gradient gave 6 as a clear oil (0.74
g, 37%): TLC Rf 0.1 (1:1 hexanes-EtOAc); 1H NMR (300 MHz,
CDCl3) δ 1.70-1.84 (m, 1 H), 1.84-2.05 (m, 4 H), 2.12-2.35
(m, 3 H), 2.51-2.66 (m, 2 H), 2.76-2.94 (m, 1 H), 5.00-5.17
(m, 6 H), 7.20-7.40 (m, 15 H); MS (methane, DCI) m/z 553
(M+ + 1).
1
3 (0.62 g, 55%): H NMR (300 MHz, D2O) δ 1.75-2.21 (m, 4
H), 2.39-2.49 (m, 2 H), 2.66-2.83 (m, 1 H); MS (methane,
DCI-) m/z 225 (M+ - 1). Anal. Calcd for C6H11O7P‚1.25H2O:
C, 28.98; H, 5.47. Found: C, 28.98; H, 5.45.
Diben zyl Ita con a te (1b). A solution of itaconic acid (1.23
g, 9.45 mmol), triethylamine (18.9 mmol, 1.92 g), and benzyl
bromide (23.62 mmol, 4.04 g) in toluene (30 mL) was heated
at 80 °C for 16 h. After cooling to room temperature, the
solution was diluted with ether (100 mL), washed with 1 N
hydrochloric acid (20 mL) and water (20 mL), dried (MgSO4),
and concentrated. Flash chromatography over silica gel (3 ×
18 cm) with a hexane-EtOAc (20:1 f 10:1) gradient gave 2b
as a clear oil (1.84 g, 63%): TLC Rf 0.28 (9:1 hexanes-EtOAc);
1H NMR (300 MHz, CDCl3) δ 3.41 (s, 2 H), 5.09 (s, 2 H), 5.17
(s, 2 H), 5.73 (s, 1 H), 6.39 (s, 1 H), 7.23-7.36 (m, 10 H); MS
(methane, DCI) m/z 311 (M+ + 1).
2-[[(2-Ca r b oxye t h yl)h yd r oxyp h osp h in oyl]m e t h yl]-
p en ta n ed ioic Acid (7). The procedure used for the prepara-
tion of 3 was followed using 6 (0.58 g, 1.06 mmol) in methanol
(10 mL) with 10% palladium on carbon (0.11 g, 10 mol %) to
afford 0.31 g of compound 7. Purification of 7 was carried out
by HPLC: semipreparative C8 column, linear gradient of water
and methanol (0% f 20%) over 20 min, 10 mL/min, 20 × 10
mL fractions. HPLC analysis, Zorbax C8 column, isocratic
water-methanol (4:1), showed the product (tR ) 1.9 min) was
contained in fractions 12-15. These fractions were combined
Dib en zyl 2-[[Bis(b en zyloxy)p h osp h or yl]m et h yl]su c-
cin a te (2b). To a solution of dibenzyl phosphite (3.04 g, 11.6
mmol) in THF (20 mL) at 0 °C was added n-butyllithium (11.6
mmol, 4.6 mL, 2.5 M solution in hexanes) followed after 10
min by trimethylaluminum (11.6 mmol, 5.8 mL, 2.0 M solution
in hexanes). After 20 min a solution of 1b (3.0 g, 9.7 mmol)
in THF (5 mL) was added. The cooling bath was removed,
and the resulting solution stirred at room temperature for 16
h. The reaction was quenched by the slow addition of 1 N
hydrochloric acid (20 mL). After stirring for 10 min the phases
were separated, and the aqueous phase was extracted with
ethyl acetate (2 × 10 mL). The organic phases were combined,
dried (MgSO4), and concentrated. Flash chromatography over
silica gel (3 × 18 cm) with a hexane-EtOAc (4:1 f 1:1)
gradient gave 2b as a clear oil (2.6 g, 55%): TLC Rf 0.39 (1:1
1
and lyophilized to afford 7 (0.17 g, 56%): H NMR (300 MHz,
MeOD) δ 1.82-2.08 (m, 5 H), 2.14-2.27 (m, 1 H), 2.31-2.43
(m, 2 H), 2.51-2.63 (m, 2 H), 2.74-2.91 (m, 1 H); MS (FAB)
m/z 283 (M+).
NAALADa se Assa y. NAALADase activity was assayed as
was previously described.22 In brief, the assay measured the
amount of [3H]Glu liberated from [3H]NAAG in 50 mM Tris-
Cl buffer in 15 min at 37 °C using 30-50 µg of synaptosomal
protein; substrate and product were resolved by anion-
exchange liquid chromatography. Duplicate assays were
always performed so that no more than 20% of the NAAG was
digested, representing the linear range of peptidase activity.
Quis (100 µM) was included in parallel assay tubes to confirm
the specificity of measurements.
1
hexanes-EtOAc); H NMR (300 MHz, CDCl3) δ 2.08 (ddd, J
) 17.6, 15.5, 8.2 Hz, 1 H), 2.35 (ddd, J ) 19.2, 15.5, 5.5 Hz, 1
H), 2.83 (d, J ) 6.5 Hz, 2 H), 3.10-3.27 (m, 1 H), 4.82-5.06
(m, 8 H), 7.22-7.37 (m, 20 H); MS (methane, DCI) m/z 573
(M+ + 1). Anal. Calcd for C33H33O7P: C, 69.22; H, 5.81.
Found: C, 69.28; H, 5.86.
Ack n ow led gm en t. The authors would like to thank
Dr. Andrew Shaw for helpful discussions during the
course of this work.
2-(P h osp h on om eth yl)su ccin ic Acid (4). The procedure
used for the preparation of 3 was followed using 2b (1.4 g,
2.44 mmol) in methanol (10 mL) with 10% palladium on carbon
(0.25 g, 5 mol %) to afford 4 (0.55 g). Purification of 4 was
carried out by HPLC: semipreparative C8 column, linear
gradient of water and methanol (0% f 20%) over 20 min, 10
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