Substituted 1,2-dihydrophthalazines: Potent, selective, and noncompetitive inhibitors of the AMPA receptor

Full text of DOI:10.1021/jm950740w

© Copyright 1996 by the American Chemical Society
Volume 39, Number 2
J anuary 19, 1996
Communications to the Editor
Ch a r t 1
Su bstitu ted 1,2-Dih yd r op h th a la zin es:
P oten t, Selective, a n d Non com p etitive
In h ibitor s of th e AMP A Recep tor
J effrey C. Pelletier,* David P. Hesson,
Kenneth A. J ones, and Anna-Maria Costa
Symphony Pharmaceuticals, 76 Great Valley Parkway,
Malvern, Pennsylvania 19355
Received October 6, 1995
The amino acid L-glutamate 1 (Chart 1) is the major
excitatory neurotransmitter of the central nervous
system (CNS) and plays a key role in normal function
of the CNS.1 However, under certain pathological
conditions, when extracellular concentrations of glu-
tamate are excessive, neuronal damage and cell death
can occur. This phenomenon has been termed excito-
toxicity and there is increasing evidence that it plays a
role in neurodegeneration associated with both acute
(stroke, trauma) and chronic (Alzheimers disease, epi-
lepsy) neurological disorders.2 It is now recognized that
agents which selectively antagonize certain ionotropic
glutamate receptor subtypes reduce injury in animal
models of stroke and epilepsy.3 However, severe side
effects, lack of solubility, and poor biodistribution
profiles have hampered the clinical development of these
agents.2a,4 Therefore, valuable therapeutic potential
exists for a new generation of centrally acting ionotropic
glutamate receptor modulators.
Studies based on biochemistry and molecular biology
have clearly shown that several subtypes of ionotropic
glutamate receptors exist in the mammalian CNS.5 The
most widely studied receptor subtype is the N-methyl-
D-aspartate (NMDA) receptor.6 Two non-NMDA recep-
tor subtypes, the kainate (KA) and R-amino-3-hydroxy-
4-methylisoxazolepropionic acid (AMPA) receptors, have
also been identified. The three receptor subtypes have
been pharmacologically classified according to the ligands
that selectively activate them (i.e. NMDA (2),6 KA (3),6
and AMPA (4)7). While numerous NMDA antagonists
have been discovered, only a small number of selective
AMPA antagonists have been reported.8
Modulation of the AMPA receptor can occur through
several modes of action. Selective ligands such as
AMPA (4) (agonist)7 and NBQX (5) (antagonist)8b are
competitive binders. As seen in whole cell electrophysi-
ology assays the diuretic cyclothiazide (6), a noncom-
petitive agonist, potentiates AMPA receptor-mediated
currents.9 The 2,3-benzodiazepines, GYKI 52466 (7)
and GYKI 53655 (8) (racemic mixture), noncompeti-
tively inhibit AMPA-receptor currents.10 The 2,3-ben-
zodiazepines have also been shown to inhibit seizure
activity10b as well as both focal and global ischemic
damage in vivo.11 The desirable effects of these latter
agents prompted us to investigate other heterocycles
with similar pharmacological properties. We now report
preliminary studies on the preparation, electrophysiol-
ogy, ancillary binding, and anticonvulsant activity of
structurally novel 1,2-dihydrophthalazines, a new series
0022-2623/96/1839-0343$12.00/0 © 1996 American Chemical Society

344 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Communications to the Editor
Sch em e 1a
Sch em e 2a
a
(a) n-BuLi, THF, -78 °C; (b) H2NNH2‚HCl, MeOH, H2O.
of compounds possessing selective, noncompetitive in-
hibitory properties at the AMPA receptor.
Ch em istr y. The fully aromatic phthalazine 12 was
synthesized as shown in Scheme 1. Bromo acetal 912
underwent rapid lithium halogen exchange upon treat-
ment with n-butyllithium at -78 °C for 2 min. Subse-
quent treatment of this lithio species with the protected
aminophenyl carboxamide 1013 provided the expected
benzophenone derivative 11 (64%) which, when further
reacted with hydrazine monohydrochloride (2 equiv),13
resulted in a 94% yield of the fully deprotected (ami-
nophenyl)phthalazine 12.
a
(a) Ac2O; (b) MeLi (4 equiv), TMEDA, THF; (c) R′NCO, CH2Cl2;
(d) 1 N NaOH, MeOH, reflux; (e) n-PrNCS, toluene reflux.
Compound 12 also served as an intermediate to the
dihydrophthalazines 16a -g and 18 (Scheme 2). Hence,
the amino group of 12 was reprotected as an acetanilide
(acetic anhydride, 20 °C, 3 h, 85%) and the product 13
was treated with methyllithium which resulted in a 45-
50% yield of the key intermediate 14.14 The final
products 16a -g were obtained in good yields via treat-
ment of methyldihydrophthalazine 14 with various
isocyanates (isocyanate, dichloromethane, 20 °C) fol-
lowed by selective hydrolysis of the acetate. Refluxing
14 in toluene with n-propyl isothiocyanate with subse-
quent deprotection of the product 17 as above gave 18.
Biologica l Resu lts a n d Discu ssion . Since no reli-
able ligand-binding assay has been reported for non-
competitive agonists and antagonists affecting the
AMPA receptor, attention was focused on an electro-
physiological assay to serve as the primary screen.
Voltage-clamped cortical neurons, which exhibit robust
AMPA receptor-mediated currents, have previously
been used to assess the potency of substituted 2,3-
benzodiazepines. AMPA receptor responses were stimu-
lated by brief applications of kainic acid, an agonist that
induces large, non-desensitizing (steady-state) currents
in these cells.10a,d Using this assay, compounds 5, 7,
12, 16a -g, and 18 were screened for their ability to
inhibit AMPA receptor currents using initial concentra-
tions of 100 or 10 µM. Concentrations providing 50%
inhibition (IC50’s) were determined from current traces
F igu r e 1. Inhibition of current in a voltage-clamped neuron
isolated from rat cerebral cortex by increasing concentrations
of 16d . Application of 50 µM kainic acid (hollow bar) to a cell
elicits a current date to stimulation of AMPA receptors.10a,d
Subsequent addition of 16d (filled bars) at concentrations
ranging from 0.3 to 30 mM causes a stepwise progressive
inhibition of the current. After a 30 s wash in control saline,
the kainic acid-stimulated current recovers completely (right-
hand trace).
similar to that shown for dihydrophthalazine 16d
(Figure 1). The resulting data are shown in Table 1.
The 2,3-benzodiazepine 7 displayed an IC50 value simi-
lar to that found in the literature (10 µM)10b while
(aminophenyl)phthalazine 12 provided minimal activity

Communications to the Editor
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 345
Ta ble 1. Inhibition of AMPA Currents in Rat Cortical Cells
Stimulated with 50 µM Kainic Acid
reported to be active in in vivo electrical seizure
models.19 1,2-Dihydrophthalazine 16d was tested in the
maximum electroshock (MES) test in mice. The ED50
was determined to be 30 mg/kg (ip dose). The results
clearly indicate that 16d penetrates the blood-brain
barrier and inhibits the onset of electrically stimulated
seizure activity.20
In summary, novel heterocycles consisting of a 1,2-
dihydrophthalazine core have been shown to selectively
and noncompetitively inhibit currents associated with
activation of the AMPA subtype of the glutamate
receptor. The activity of the most potent compound, 16e
(SYM 2207), was similar to the literature value reported
for GYKI 53655 (2,3-benzodiazepine, 8) in the same
electrophysiology assay. Unlike 8, the 1,2-dihydro-
phthalazine 16d was shown to be inactive at the central
benzodiazepine binding site. Furthermore, 16d was
active in an in vivo anticonvulsant assay. The potent
and selective activity of the 1,2-dihydrophthalazines
coupled with their relative ease of synthesis and im-
proved solubility properties should make them valuable
tools for the pharmacological study of glutamate recep-
tors. Further structure activity and in vivo studies are
in progress.
% inhibn at
10 µM (100 µM)
no.a
R′
IC50b((SE)
12
(38)
13(88)
55
45
77
89
70
79
16a
16b
16c
16d
16e
16f
18
CH3
C2H5
i-C3H7
n-C3H7
n-C4H9
t-C4H9
23(5.9)
7.2(0.7)
2.8(0.4)
1.8(0.2)
5.4(1.5)
16g
7
5
C6H5
(35)
51
10(0.8)
0.14
98c
a
b
Compounds 16a -g and 18 are racemic mixtures. IC50 (µM)
values represent an average of at least four cells. c Percent
inhibition at 3 µM.
at 100 µM (<40% inhibition). When 12 was converted
to the 1,2-dihydrophthalazines 16a -f (racemic mix-
tures), a dramatic increase in AMPA receptor inhibition
was observed (Table 1). Antagonism increased with the
length of the pendant alkyl chain. The trend is appar-
ent for dihydrophthalazines with pendant groups bear-
ing normal alkyl functionality (IC50’s of 23, 7.2, 2.8, and
1.8 µM for R ) methyl, ethyl, n-propyl, and n-butyl,
respectively), a pattern that contrasts with the data
reported for the 2,3-benzodiazepine analogues.15 Par-
ticularly notable is dihydrophthalazine 16e (SYM 2207)
which had an IC50 value of 1.8 µM and was similar to
the literature value of the most potent noncompetitive
AMPA antagonist GYKI 53655 (8, IC50 ) 1.0 µM).10b
Size limitations of the side chain are evident with the
phenyl derivative 16g (35% inhibition at 100 µM).
Interestingly, the thio derivative 18 showed approxi-
mately the same level of activity as its oxygenated
counterpart 16d (79% inhibition at 10 µM for 18 vs 77%
inhibition at 10 µM for 16d ).
Numerous ligands for the AMPA receptor show activ-
ity at the kainate receptor subtype as well.16 In order
to define a selectivity profile for the dihydrophthalazines
(AMPA vs kainate receptor activity), selected com-
pounds were tested in the voltage-clamp assay for
modulation of kainate receptor currents. Human em-
bryonic kidney (HEK) cells were used to express the
GluR6 receptor.17 This homomeric ligand gated ion
channel is known to have properties similar to the
native kainate receptor.17 When 16b and 16d were
tested for inhibition of GluR6 currents, neither com-
pound showed greater than 10% inhibition at a concen-
tration of 100 µM (data not shown).
Further selectivity data was obtained by screening
compounds 16d and 8 for their ability to displace
tritiated ligands in a variety of binding assays.18 Di-
hydrophthalazine 16d showed very weak binding in a
nonselective adenosine assay (32% inhibition at 10 µM)
while displaying no binding in all others tested (<10%
inhibition of tritiated ligand binding at 10 µM). The
2,3-benzodiazepine 8, however, showed approximately
62% inhibition of binding (at 10 µM) to the central
benzodiazepine receptor while remaining relatively
inactive at the other receptors assayed (data not shown).
None of the compounds bound competitively to glutamate
receptors (NMDA, AMPA, kainate, and strychnine-
insensitive glycine assays).
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails with spectral data (6 pages). Ordering information is
available on any current masthead page.
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