Syntheses and pharmacological evaluations of novel N-substituted bicyclo-heptane-2-amines at N-methyl-d-aspartate receptors

Article abstract of DOI:10.1111/j.1747-0285.2011.01124.x

Several novel norcamphor (bicycloheptane)-based compounds were designed and synthesized as non-competitive N-methyl-d-aspartate receptor antagonists at the phencyclidine binding sites. The heterocyclic ring was also varied to examine piperidine, pyrrolidi

Full text of DOI:10.1111/j.1747-0285.2011.01124.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01124.x
Chem Biol Drug Des 2011; 78: 25–32
Research Article
Syntheses and Pharmacological Evaluations of
Novel N-Substituted Bicyclo-Heptane-2-amines at
N-Methyl-D-Aspartate Receptors
Zeynep Ates-Alagoz1,2, Shengguo Sun1,
and D-serine. In addition to these sites, allosteric modulatory sites
for Mg2+, phenylcyclidine (PCP), polyamines, and Zn2+are known (7).
While glutamate, glycine, and polyamine sites are found outside
the ion channel; the sites for Mg2+and PCP are located within the
channel itself (8). The NMDA receptor has been implicated in the
pathophysiology of a variety of neurological and neuropsychiatric
diseases including Alzheimer's disease (9), epilepsy, chronic pain
syndrome, schizophrenia, Parkinson's disease, Huntington's disease
(10,11), major depression, addiction, and anxiety (12). Excessive glu-
tamate and subsequent over-stimulation of NMDA receptors leading
to excessive Ca2+influx has been implicated in the pathophysiology
of these disease states (13,14). Several preclinical paradigms have
found that non-competitive NMDA antagonism can effectively
reduce NMDAR-mediated neurotoxicity (15).
Jason Wallach1 and Adeboye Adejare1,*
1Department of Pharmaceutical Sciences, Philadelphia College of
Pharmacy, University of the Sciences in Philadelphia, Philadelphia,
PA 19104, USA
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Ankara University, 06100 Tandogan, Ankara, Turkey
*Corresponding author: Adeboye Adejare, a.adejar@usp.edu
Several novel norcamphor (bicycloheptane)-based
compounds were designed and synthesized as
non-competitive N-methyl-D-aspartate receptor
antagonists at the phencyclidine binding sites.
The heterocyclic ring was also varied to examine
piperidine, pyrrolidine, and morpholine groups. We
examined pharmacological activities of these com-
pounds in vitro (binding studies) and in vivo (maxi-
A major limitation for therapeutically available NMDA antagonists
is the essential role of NMDAR in neuro-physiology. While blockade
of excessive NMDAR activity is desirable, it must be achieved with-
out complete amelioration of normal glutamate function. As a result
of this dichotomy, many competitive antagonists have failed in clini-
cal trials (16). Utilization of non-competitive antagonists working
through open-channel blockade has been proposed as an attractive
alternative, as this mechanism requires initial activation of the
channel for inhibition to occur, possibly leading to a higher likeli-
hood of channel blockade in the presence of excessive levels of
glutamate and a lower likelihood of antagonism with normal physio-
logical levels of glutamate (16).
mal
electroshock
test).
Pharmacological
evaluations revealed one of the compounds, 5a, to
be a good lead, exhibiting moderate binding at N-
methyl-D-aspartate
receptors
(IC50 = 7.86 lM;
Ki = 5.28 lM), maximal electroshock neuroprotec-
tion activity at 100 mg ⁄ kg and acceptable toxicity
profile.
Key words: anticonvulsant activity, bicycloheptane derivatives, max-
imal electroshock test, N-methyl-D-aspartate receptor antagonist, phen-
cyclidine binding site
Received 22 September 2010, revised 28 February 2011 and accepted
for publication 1 April 2011
Phenylcyclidine is a non-competitive open-channel antagonist of
NMDAR. PCP and it`s derivatives have attracted medicinal chemists
for years, as these drugs have exhibited a variety of therapeutically
desirable effects including anti-convulsant (9), anxiolytic (11), and
neuroprotective effects against neural damage resulting from ische-
mia, anoxia, hypoglycemia, and endogenous neurotoxins (12–14).
Unfortunately, in addition to this array of therapeutically desirable
effects, most of these ligands exhibit an undesirable side effect
profile, most noteworthy of which are the psychotomimetic effects.
Glutamate is one of the principal excitatory neurotransmitters in the
mammalian central nervous system (CNS). A major function of glu-
tamate is control of ion flow at excitatory synapses. Glutamate
receptors are subdivided into two, namely metabotropic and iono-
tropic. Three ionotropic receptor types have been identified based
on ligand selectivity; AMPA, N-methyl-D-aspartate (NMDA), and kai-
nate. In addition to ionotropic receptors, three classes of metabo-
tropic receptors are acknowledged (mGluRs) (1). The ionotropic
NMDA receptor (NMDAR) is noteworthy in that it requires binding
by agonist glutamate and co-agonist D-serine or glycine for it to be
activated (open state). NMDAR is also distinct in that it exhibits
slow kinetics, is permeable to Na+, K+, and Ca2+(2–4); and is both
ligand and voltage gated (5,6). It is a complex made up of distinct
binding sites including sites for amino acids L-glutamate, glycine,
Some authors have speculated that high affinity at the NMDA
receptor may contribute to undesirable effects. There has thus been
some interest in obtaining low to moderate affinity non-competitive
NMDA antagonists (17–20). This hypothesis is supported by the fact
that several therapeutically used moderate affinity NMDA antago-
nists, such as ketamine, DXM, memantine, and adamantine are
generally well tolerated (18,20–22).
25

Ates-Alagoz et al.
Our group is involved in the design and syntheses of NMDA recep-
tor inhibitors as possible therapies for neurodegenerative diseases.
Several novel norcamphor (bicyloheptane) derivatives were designed
and synthesized as non-competitive antagonists of the NMDA
receptor. Syntheses of these compounds are displayed in Scheme 1.
Pharmacological activities of these compounds were evaluated in vi-
tro (binding studies) and in vivo (MES test).
give Grignard reagent and was added to norcamphor (5.75 g,
52.2 mmol) according to modification of the method of Geneste
et al. (23–25) to give a crude alcohol as a red oil (2a, 9.2 g, 93%
yield). Treatment of this alcohol (9.0 g, 47.8 mmol) with TFA (32 mL,
430 mmol) in the presence of sodium azide (9.3 g, 143 mmol)
resulted in the tertiary azide as a red oil (3a, 9.3 g, 91% yield).
This azide (9.3 g, 40 mmol) was reduced to the corresponding
amine with LiAlH4 (2.5 g, 67 mmol) to give 2-phenylbicy-
clo[2.2.1]heptan-2-amine (4a) as a clear oil (7.8 g, 90% yield). This
oil (0.2 g) was purified by preparative TLC developed using a mix-
ture of chloroform and diethyl ether (9:1, v ⁄ v) as mobile phase. A
pale yellow oil was obtained which solidified at 0 ꢀC. 1H NMR
(CDCl3): d ppm 7.3–7.4 (m, 5H), 1.0–2.6 (b, 10 H, 4CH2 and 2CH).
13C NMR (CDCl3): d 148.3, 128.6, 127.0, 126.4, 64.1, 48.5, 45.2,
37.1, 36.9, 28.8, 24.8. MS (ESI+) m ⁄ z: 188 (10%), [M+H], 171
(100%), [M-16]. The amine hydrochloride salt was obtained by bub-
bling hydrogen chloride gas through the ethyl ether solution. The
solvent used for crystallization was the mixture of methanol and
ethyl ether. Anal. Calcd. for compound 4a hydrochloride, C13H18ClN-
0.1H2O: C, 69.23; H, 8.13; N, 6.21. Found: C, 69.22; H, 8.11; N,
6.10. m.p. 243–244 ꢀC (26).
Materials and Methods
Melting points were determined with a Mel-Temp electrothermal
1
apparatus and are uncorrected. The H, 13C, and 19F NMR spectra
were recorded with a 400-MHz Bruker NMR spectrophotometer
with TMS as internal standard and CDCl3 as solvent. The mass
spectra were recorded with a Varian 1200 Triple Quadrupole instru-
ment using electrospray ionization (ESI) technique. NMR and MS
were obtained on the free base of each amine. Column chromatog-
raphy was conducted using Merck silica gel, grade 9385, 230–400
mesh, 60 ꢀ. Compound purity was determined by elemental analy-
sis conducted by Galbraith Laboratories, (Knoxville, TN, USA). The
chemical reagents used were purchased from Aldrich (St. Louis,
MO, USA), Acros (Pittsburgh, PA, USA), and Alfa Aesar (Ward Hill,
MA, USA).
Synthesis of intermediate 4b
Crude alcohol 2b (9.4 g, 99% yield) was obtained from p-bromoflu-
orobenzene (5.00 mL, 45.5 mmol), magnesium turnings (3.32 g,
137 mmol), and norcamphor (5.00 g, 45.5 mmol) as described for
Synthesis of intermediate 4a
A mixture of bromobenzene (5.5 mL, 52.2 mmol), magnesium turn-
ings (3.81 g, 157 mmol), and a few iodine crystals was stirred to
1
synthesis of compound 2a. H NMR (CDCl3): d ppm 7.63–7.43 (m,
Br
X
X
b
a
+
O
1
N3
OH
X
3a X = H
3b X = F
2a X = H
2b X = F
X = -H, -F
X
X
d
c
Cl
Y
N
HN
.HCl
NH2
4a X = H
4b X = F
N
Y
d
5a X = H, Y = CH2
5b X = F, Y = CH2
5c X = H, Y = O
5d X = F, Y = O
Cl
N
.HCl
X
HN
N
Scheme 1: Syntheses of tar-
get compounds 5a–5f (a) Mg, I2,
THF; (b) NaN3, TFA, CHCl3; (c)
LiAlH4, THF; (d) TEA, Et-OH.
5e X = H,
5f X = F,
26
Chem Biol Drug Des 2011; 78: 25–32

Syntheses and Pharmacological Evaluations of NMDA Receptor Antagonists
2H), 7.15–6.96 (m, 2H), 2.58 (s, 1H), 2.44–2.11 (m, 3H), 1.81–1.26
the general procedure and was purified by column chromatography
(EtOAc ⁄ i-prOH ⁄ TEA 8:2:0.1) to give 5b (0.43 g, 25% yield) as color-
less crystals: m.p 66–68 ꢀC. 1H NMR (400 MHz, CDCl3) d ppm
7.03–6.96 (m, 2H), 7.34–7.28 (m, 2H), 2.51 (d, J = 4.06 Hz, 1H), 2.36
(s, 1H), 2.30–2.02 (m, 9H), 1.88–1.67 (m, 3H), 1.55–1.26 (m, 9H),
1.12–0.98 (m, 2H); 13C (400 MHz, CDCl3) d ppm 162.24, 140.59,
129.56, 129.48, 114.40, 114.19, 67.55, 58.42, 54.28, 46.03, 42.08,
38.81, 37.01, 36.86, 29.15, 26.09, 24.52, 24.40; 19F (400 MHz,
CDCl3) d ppm -117.75; MS (ESI+) m ⁄ z: 317 (100%), [M+H]. Anal.
Calcd. for C20H29FN2: C, 75.91; H, 9.24; N, 8.85. Found: C, 75.80; H,
8.86; N, 8.65. HCl salt was prepared, colorless crystals, m.p. 185–
187 ꢀC.
(m, 7H); 13C NMR (CDCl3): d ppm 162.8, 144.89, 127.7, 127.6, 114.9,
114.7, 80.45, 47.6, 46.9, 38.8, 37.6, 29.1, 22.28. Treatment of the alco-
hol (9.4 g, 46 mmol) with TFA (30.5 mL, 410 mmol) in the presence of
sodium azide (8.89 g, 137 mmol) resulted in the tertiary azide as a
red oil (3b, 10.5 g, 93.6% yield). This azide (10.5 g, 45.4 mmol) was
then reduced to the corresponding amine with LiAlH4 (2.60 g,
68 mmol) to give 2-(4-fluorophenyl)bicyclo[2.2.1]heptan-2-amine (4b)
as a clear oil (6.6 g, 70.8% yield). This oil (0.4 g) was purified by pre-
parative TLC developed using a mixture of chloroform and diethyl
ether (9:1, v ⁄ v) as mobile phase. A pale yellow oil was obtained
1
which solidified at 0 ꢀC. H NMR (CDCl3): d ppm 7.2 (m, 2H), 6.8 (m,
2H), 1.0–2.6 (b, 10 H, 4CH2 and 2CH); 13C NMR (CDCl3): d 163.0,
160.4, 128.6, 128.5, 115.3, 115.0, 63.6, 48.7, 45.6, 37.1, 36.8, 28.7,
24.7; 19F NMR (CDCl3): d -117.6 ppm. MS (ESI+) m ⁄ z: 206 (10%),
[M+H], 189 (100%), [M-16]. The amine hydrochloride salt was
obtained as described for compound 4a. Anal. Calcd. for compound
4b hydrochloride, C13H17ClFN: C, 64.59; H, 7.09; F, 7.86; N, 5.79.
Found: C, 64.30; H, 7.29; F, 7.56; N, 5.62. m.p. 214–216 ꢀC (26).
N-(2-morpholinoethyl)-2-phenylbicyclo [2.2.1]
heptan-2-amine (5c)
The compound was prepared from 4c (0.84 g, 4.5 mmol) and 4-(2-
chloroethyl) morpholine hydrochloride (0.91 g, 4.91 mmol) according
to the general procedure and was purified by column chromatogra-
phy (EtOAc ⁄ i-prOH ⁄ TEA 7:3:0.1) to give 5c (0.22 g, 16% yield) as
1
colorless crystals: m.p. 29–31 ꢀC. H NMR (400 MHz, CDCl3) d ppm
General procedure for syntheses of compounds
5a–5f
7.31 (dq, J = 8.20, 7.90 Hz, 4H), 7.22–7.15 (m, 1H), 3.59 (td,
J = 20.60, 4.55 Hz, 4H), 2.56 (d, J = 3.27 Hz, 1H), 2.36 (s, 1H),
2.33–2.17 (m, 4H), 2.15–2.02 (m, 5H), 1.92–1.74 (m, 3H), 1.53–1.34
(m, 2H), 1.30 (d, J = 9.53 Hz, 1H), 1.18–0.98 (m, 2H); 13C (400 MHz,
CDCl3) d ppm 144.55, 128.00, 127.69, 125.89, 68.07, 67.02, 57.90,
53.13, 45.45, 41.71, 38.07, 36.90, 36.83, 29.29, 24.43; MS (ESI+)
m ⁄ z: 301 (80%), [M+H]. Anal. Calcd. for C19H28N2O: C, 75.96; H,
9.39; N, 9.32. Found: C, 75.71; H, 9.29; N, 9.33. HCl salt was pre-
pared, colorless crystals, m.p. 197–199 ꢀC.
The crude base of compound 4, 2-phenylbicyclo[2.2.1] heptan-2-
amine or 2-(4-fluoro-phenyl)-bicyclo[2.2.1]hept-2-ylamine (5.4 mmol)
and appropriate alkyl halides derivatives (5.6 mmol) dissolved in
ethanol (30 mL) and triethylamine (1 mL, 7.1 mmol) was stirred and
the mixture heated at 50 ꢀC for 20–48 h. After cooling, the solvent
was removed under reduced pressure and the residue was treated
with water. The mixture was extracted with ethyl acetate
(3 · 20 mL). Organic phase extracts were combined, dried over
sodium sulfate, and purified by column chromatography. Hydrochlo-
ride salt was obtained as described for compound 4a. Fumarate
salt was obtained using equal molar amounts of fumaric acid and
compound in methanol. The solvent used for crystallization was the
mixture of methanol and ethyl ether.
2-(4-Fluorophenyl)-N-(2-morpholinoethyl)bicyclo
[2.2.1]heptan-2-amine (5d)
The compound was prepared from 4d (0.75 g, 3.66 mmol) and 4-(2-
chloroethyl) morpholine hydrochloride (0.75 g, 4.0 mmol) according
to the general procedure and was purified by column chromatogra-
phy (EtOAc ⁄ i-prOH ⁄ TEA 7:3:0.2) to give 5d (0.16 g, 14% yield) as
1
2-Phenyl-N-(2-(piperidin-1-yl)
colorless crystals: m.p. 47–49 ꢀC. H NMR (400 MHz, CDCl3) d ppm
ethyl)bicyclo[2.2.1]heptan-2-amine (5a)
7.35–7.26 (m, 2H), 7.09–6.95 (m, 2H), 3.70–3.55 (m, 4H), 2.51 (d,
J = 3.60 Hz, 1H), 2.40–2.02 (m, 10H), 1.90–1.61 (m, 3H), 1.55–1.27
(m, 3H), 1.15–0.94 (m, 2H); 13C (400 MHz, CDCl3) d ppm 162.31,
140.56, 129.51, 129.43, 114.46, 114.25, 67.56, 67.00, 58.01, 53.26,
45.81, 42.06, 38.12, 36.94, 36.85, 29.19, 24.38; 19F (400 MHz,
CDCl3) d ppm -117.45; MS (ESI+) m ⁄ z: 319 (80%), [M+H]. Anal.
Calcd. for C19H27FN2O: C, 71.66; F, 5.96; H, 8.53; N, 8.79. Found: C,
71.54; F, 5.81; H, 8.13; N, 8.76. Hydrogen fumarate salt was pre-
pared, white crystal, m.p. 152–154 ꢀC.
The compound was prepared from 4a (1.25 g, 6.7 mmol) and 1-(2-
chloroethyl)piperidine hydrochloride (1.2 g, 8.1 mmol) according to
the general procedure and was purified by column chromatography
(EtOAc ⁄ i-prOH ⁄ TEA 8:2:0.2) to give 5a (0.36 g, 18% yield) as color-
less crystals: m.p. 32–34 ꢀC. 1H NMR (400 MHz, CDCl3) d ppm
7.37–7.27 (m, 4H), 7.22–7.16 (m, 1H), 2.54 (d, J = 3.82 Hz, 1H), 2.36
(s, 1H), 2.31–2.02 (m, 9H), 1.80 (m, 3H), 1.53–1.27 (m, 9H), 1.15–
1.00 (m, 2H); 13C (400 MHz, CDCl3) d ppm 144.75, 128.06, 127.63,
125.70, 68.00, 58.48, 54.24, 45.80, 41.78, 38.84, 36.95, 36.84, 29.23,
26.09, 24.55, 24.45. MS (ESI+) m ⁄ z: 299 (100%), [M+H]. Anal. Calcd.
for C20H30N2: C, 80.48; H, 10.13; N, 9.39. Found: C, 80.70; H, 10.20;
N, 9.15. HCl salt was prepared, colorless crystals, m.p. 202–204 ꢀC.
2-Phenyl-N-(2-(pyrrolidin-1-yl) ethyl)
bicyclo[2.2.1]heptan-2-amine (5e)
The compound was prepared from 4e (1.83 g, 9.82 mmol) and 1-(2-
chloroethyl) pyrrolidine hydrochloride (1.70 g, 10 mmol) according to
the general procedure and was purified by column chromatography
(CHCl3 ⁄ i-prOH ⁄ TEA 7:3:0.5) to give 5e (0.64 g, 23% yield) as color-
less oil. H NMR (400 MHz, CDCl3) d ppm 7.43–7.27 (m, 4H), 7.27–
7.14 (m, 1H), 2.55 (s, 1H), 2.49–2.18 (m, 9H), 2.10 (dd, J = 9.44 Hz,
2-(4-Fluorophenyl-N-(2-(piperidin-1-yl)
ethyl)bicyclo[2.2.1]heptan-2-amine (5b)
The compound was prepared from 4b (1.11 g, 5.4 mmol) and 1-(2-
chloroethyl)piperidine hydrochloride (1.10 g, 5.6 mmol) according to
1
Chem Biol Drug Des 2011; 78: 25–32
27

Ates-Alagoz et al.
1H), 1.94–1.63 (m, 7H), 1.53–1.25 (m, 3H), 1.17–0.99 (m, 2H); MS
(ESI+) m ⁄ z: 285 (100%), [M+H]. Anal. calcd. for C19H28N2-0.1CHCl3:
C, 77.40; H, 9.55; N, 9.45. Found: C, 77.43; H, 9.33; N, 9.82. HCl
salt was prepared, colorless crystal, m.p. 180–182 ꢀC.
National Institutes of Health (NIH). Compounds evaluated by this
program are typically subjected to a 'primary assay' designed to
identify receptors, transporters, and ion channels for which the
compounds display affinity. Compounds found active in the primary
screening (>10 000 nM) are subjected to a secondary screen where
affinity (Ki) is calculated. Experimental details and procedure can be
found through Assay Protocol Book, National Institute of Mental
Health Psychoactive Drug Screening Program, and University of
North Carolina at Chapel Hilla.
2-(4-Fluorophenyl-N-(2-(pyrrolidin-1-yl) ethyl)
bicyclo[2.2.1]heptan-2-amine (5f)
The compound was prepared from 4f (1.80 g, 8.78 mmol) and 1-(2-
chloroethyl) pyrrolidine hydrochloride (1.63 g, 9.58 mmol) according
to the general procedure and was purified by column chromatogra-
phy (CHCl3 ⁄ i-prOH ⁄ TEA 6.5:3:0.5) to give 5f (0.28 g, 11% yield) as
In vivo biological studies
1
colorless oil. H NMR (400 MHz, CDCl3) d ppm 7.37–7.26 (m, 2H),
7.08–6.95 (m, 2H), 2.51 (d, J = 3.83 Hz, 1H), 2.47–2.16 (m, 8H), 2.06
(dd, J = 6.07 Hz, 1H), 1.89–1.78 (m, 2H), 1.76–1.60 (m, 5H), 1.53–
1.26 (m, 4H), 1.05 (td, J = 12.13, 6.58 Hz, 2H); MS (ESI+) m ⁄ z: 303
(100%), [M+H]. Hydrogen fumarate salt was prepared, white crys-
tals, m.p. 118–120 ꢀC. Anal. calcd. for fumarate salt; C23H31FN2O4-
H2O: C, 63.28; F, 4.35; H, 7.61; N, 6.41. Found: C, 62.92; F, 4.29; H,
7.40; N, 6.36.
Maximal electroshock (MES) test
The MES test is a model for generalized tonic–clonic seizures and
provides an indication of a compound's ability to prevent seizure
spread when all neuronal circuits in the brain are maximally active.
These seizures are highly reproducible and are electrophysiologically
consistent with human seizures. For this test, 60 Hz of alternating
current (50 mA in mice, 150 mA in rats) is delivered for 0.2 seconds
by corneal electrodes which have been primed with an electrolyte
solution containing an anesthetic agent (0.5% tetracaine HCl). In
Test 1, mice are evaluated at various intervals following doses of
30, 100, and 300 mg ⁄ kg of test compound given by i.p. injection of
a volume of 0.01 mL ⁄ g. In Test 2, rats are tested after a dose of
30 mg ⁄ kg (p.o.) in a volume of 0.04 mL ⁄ 10 g. Final test uses vary-
ing doses administered via i.p. injection, again in a volume of
0.04 mL ⁄ 10 g. An animal is considered 'protected' from MES-
induced seizures upon abolition of the hindlimb tonic extensor com-
ponent of the seizure (30–32).
In vitro biological studies
Receptor binding studies
To evaluate in vitro affinities of the compounds at the PCP site of
the NMDA receptor complex, radioligand binding studies were con-
ducted in accordance with published protocol by Reynolds and Shar-
ma (27). Thoroughly washed rat forebrain homogenate was used as
receptor source [whole brain obtained from Pel-Freez Biologicals,
forebrain tissue preparations prepared as in Reynolds and Sharma
(27)].
Subcutaneous Metrazol seizure threshold test
(scMET)
Suspensions of 10 mM HEPES (pH 7.4 at room temperature) contain-
ing 30 lg ⁄ mL protein, 1 nM (+)-[3H]MK-801, 100 lM glutamate,
30 lM glycine, and various concentrations of unlabeled competitor
or 30 lM (+)-MK-801 for non-specific binding, were incubated at
room temperature for 2 h. Termination of reaction was performed
via vacuum filtration using a 24-well cell harvester (Brandel, Gai-
thersburg, MD, USA) over GF ⁄ B glass fiber filters (Brandel). Filters
were washed with three 5-mL aliquots of assay buffer. Radioactivity
trapped on filters was measured via liquid scintillation counting,
using a Beckman LS 6500 multipurpose scintillation counter (Beck-
man Coulter, Brea, CA, USA) at 64% efficiency.
Subcutaneous injection of the convulsant Metrazol produces clonic
seizures in laboratory animals. The scMET test detects the ability
of a test compound to raise the seizure threshold of an animal and
thus protect it from exhibiting clonic seizure. Animals are pretreated
with various doses of the test compound (in a similar manner to
MES test, although a dose of 50 mg ⁄ kg (p.o.) is the standard for
Test 2 scMET). At the previously determined therapeutic plasma
exchange (TPE) of the test compound, the dose of Metrazol which
will induce convulsions in 97% of animals (CD97: 85 mg ⁄ kg mice;
70 mg ⁄ kg rats) is injected into a loose fold of skin in the midline
of the neck. The animals are placed in isolation cages to minimize
stress (33) and observed for the next 30 min for the presence or
absence of seizure. An episode of clonic spasms, approximately 3–
5 seconds, of the fore and ⁄ or hindlimbs, jaws or vibrissae is taken
as the end-point. Animals that do not meet this criterion are con-
sidered protected.
IC50 values were determined with GRAPHPAD (GraphPad Software, La
Jolla, CA, USA) using log-concentration plotted against percent spe-
cific binding. Percent specific binding for [3H]-MK-801 in control
experiment was 70% of total. Ki values were calculated using the
equation of Cheng and Prusoff (28). The Kd for (+)-MK-801 binding
under the saturation conditions was 1.747 nM and this is consistent
with that reported in literature (27). The Kd of (+)-MK-801 was
determined via homologous binding assay as described by Reynolds
and Sharma. The protein concentration was determined by the
method of Bradford (29) using coomassie protein assay reagent.
Acute toxicity-minimal motor impairment (MMI)
To assess a compound's undesirable side effects (toxicity), ani-
mals are monitored for overt signs of impaired neurological or
muscular function. In mice, the rotorod (34) procedure is used to
evaluate such impairment. When a mouse is placed on a rod
that rotates at a speed of 6 rpm, the animal can maintain its
The compounds were evaluated through the National Institute of
Mental Health (NIMH) Psychoactive Drug Screening Program (PDSP),
28
Chem Biol Drug Des 2011; 78: 25–32

Syntheses and Pharmacological Evaluations of NMDA Receptor Antagonists
equilibrium for long periods of time. The compound is considered
presence of sodium azide resulted in the tertiary azide, which was
then reduced to the corresponding amine with lithium aluminum
hydride (LiAlH4) to give intermediates 4. Treatment of these amines
with alkyl halides in the presence of ethanol and triethylamine com-
pleted syntheses of the target compounds (Scheme 1).
toxic if the animal falls off this rotating rod three times during
a 1-min period. In rats, minimal motor deficit is indicated by
ataxia, which is manifested by an abnormal, uncoordinated gait.
Rats used for evaluating toxicity are examined before the test
drug is administered, as individual animals may have peculiarities
in gait, equilibrium, placing response, among others which might
be attributed erroneously to the test substance. In addition to
MMI, animals may exhibit a circular or zigzag gait, abnormal
body posture and spread the legs, tremors, hyperactivity, lack of
exploratory behavior, somnolence, stupor, catalepsy, loss of plac-
ing response and changes in muscle tone.
Radioligand binding studies were utilized to evaluate in vitro affini-
ties of the target compounds at the PCP site of the NMDA receptor
complex. A representative plot from which the IC50 values were
extracted is depicted in Figure 1. All 8 novel amines (4a, 4b, 5a–
f) at 10 000 nM were screened for % inhibition of 1 nM [3H] (+)-
MK-801. As compounds with moderate NMDAR activity were
desired, only those possessing greater than 90% inhibition at
10 lM were subjected to further analysis. The IC50 and Ki values
were calculated for the 4 compounds (4a, 5a, 5e, and 5f) with
greater than 90% inhibition. As depicted in Table 1, the binding
affinities of the compounds are comparable, though lower than that
of (+)-MK-801.
Results and Discussion
Our studies examined design, syntheses, and pharmacological evalu-
ations of novel, non-competitive, NMDAR antagonists as potential
therapies for neurodegenerative diseases (35,36). The syntheses of
N-substitued bicyclo-heptan-2-amines (5a–5f) were carried out
starting from commercially available norcamphor and a phenyl Grig-
nard reagent. Phenyl magnesium bromides were added to norcam-
phor 1 to give alcohols 2a and 2b. This addition has been
reported to give exo-phenyl tertiary alcohols (37). Treatment of the
resulting tertiary alcohol with trifluoroacetic acid (TFA) in the
Each compound was evaluated for affinity at human or rat recep-
tors through the NIMH-PDSP program. In vitro binding affinities of
these novel NMDAR antagonists at Serotonin (5-HT2A), Dopamine
2A
1 (D1), Dopamine 2 (D2), the dopamine transporter (DAT), kappa opi-
oid receptor (KOR), l-opioid receptors (MOR), Norepinephrine trans-
porter (NET), Serotonin transporter (SERT), Sigma-1, and Sigma-2
receptors are shown in Table 2. The receptors evaluated, radio-
labeled ligand, and receptor source are depicted in Table 3. The
compounds lacked significant affinity for the majority of receptors
screened. However, many of the compounds possessed low affinity
at KOR and the DAT. In almost all cases these affinities were
110
100
90
80
70
60
50
40
30
20
10
0
Table 1: The IC50 and Ki for the four compounds subjected to
further analysis and (+)-MK-801 run under experimental condition as
reference
N-methyl-D-aspartate
Compound
IC50
affinity Ki
–10
(+)-MK-801
2.02 € 0 .797 nM
13.27 € 0.63 lM
7.86 € 1.64 lM
8.48 € 3.106 lM
8.657 € 2.564 lM
1.357 nM
8.45 lM
5.28 lM
5.67 lM
5.82 lM
–10
–9
–8
–7
–6
–5
–4
–3
4a
5a
5e
5f
Log (Conc)
Figure 1: Concentration % specific binding curve for compound
5a.
Table 2: In vitro binding affinities of novel N-methyl-D-aspartate receptor antagonists for 5-HT2A, D1, D2, DAT, KOR, MOR, NET, SERT,
Sigma-1, and Sigma-2 receptors
Compound
5HT2A (nM)
D1 (nM)
D2 (nM)
DAT (nM)
KOR (nM)
MOR (nM)
NET (nM)
SERT (nM)
Sigma 1 (nM)
Sigma 2 (nM)
4a
4b
5a
5b
5c
5d
5e
5f
>10 000
>10 000
2643
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
1231
2570
6482
>10 000
>10 000
876 nM
3546
988
>10 000
4507
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
4607
2201
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
995
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
8822
>10 000
>10 000
>10 000
>10 000
3624
226
2111
Chem Biol Drug Des 2011; 78: 25–32
29

Ates-Alagoz et al.
Table 3: Novel N-methyl-D-aspartate receptor antagonists were
screened for the following receptors, and sources
mediate their NMDA effects through active metabolites. A slightly
similar case is known with the antitussive agent dextromethorphan
(DXM) which itself has low activity at NMDA, whereas one of its
metabolites, dextrophan (DX), has lM affinity (38,39). It has been
speculated that some of the therapeutic effects of DXM are mediated
by DX (40). DXM has been found to have neuroprotective activity in
the MES test.
Receptor
Source
Hot ligand
Serotonin2A (5-HT2A
Dopamine 1 (D1)
Dopamine 1(D2)
Dopamine transporter (DAT)
j-opioid receptors (KOR)
l-opioid receptors (MOR)
Norepinephrine transporter (NET)
Serotonin transporter (SERT)
Sigma1
)
Human cloned
Human cloned
Human cloned
Human cloned
Rat cloned
Human cloned
Human cloned
Human cloned
Rat brain
Ketanserin
SCH23390
N-methylspiperone
WIN35428
U69593 (2007-07-27)
DAMGO (2007-07-27)
Nisoxetine
Citalopram
Pentazocine(+)
DTG
Toxicity was observed for every compound at ‡100 mg ⁄ kg. Severe
mortality (either 3 ⁄ 4 or 4 ⁄ 4) was also noted for all these molecules
in the TOX evaluation at the higher dose of 300 mg ⁄ kg. Other com-
ments on these novel NMDAR antagonists are displayed on
Table 5.
Sigma2
Rat C12
Conclusions and Future Directions
moderate at best. A notable exception is compound 5f, which
showed significant affinity (226 nM) at DAT (Table 2).
All target compounds were synthesized in hundred milligram quanti-
ties showing feasibility of the synthetic scheme. They were all pro-
tective in the MES test at the dose of 100 mg ⁄ kg except for
compound 5e. None of the compounds showed protection in the
scMET model. When tested in rats, 5b and 5d did not display any
protection or toxicity at the dose of 30 mg ⁄ kg. None of the target
compounds exhibited significant toxicity at 30 mg ⁄ kg based on the
rotorod TOX assessment. At 100 mg ⁄ kg, several test compounds
exhibited toxicity and at 300 mg ⁄ kg all compounds exhibited toxicity
(Table 4).
The novel compounds also were accepted into the Anticonvulsant
Screening Program (ASP), National Institute of Neurological Disorders
and stroke (NINDS). Their anticonvulsant activities were evaluated by
MES test and scMET, and their neurotoxicities were measured by the
rotorod test. These tests showcase the ability of the compound to
enter the CNS and infer NMDA receptor antagonism. Compound 5a
exhibited the highest affinity for NMDA and greatest degree of pro-
tection from MES-induced neural damage and death. However, com-
pounds 5b, 5c, and 5d which did not exhibit significant affinity also
displayed MES neuroprotective activity (Tables 4 and 5). This could be
explained by several factors one of which may be the high doses
tested in vivo may have made up for the low affinity. Other possibili-
ties are differences between in vitro versus in vivo characteristics
and ⁄ or bioavailabilities of the compounds in the brain. Yet another
possibility is that the compounds displaying minimal NMDA affinity
and significant MES protection may in fact be prodrugs and thus
Non-competitive low-affinity NMDA antagonists have received
attention as a means of reducing the intolerable side effects of
higher-affinity NMDA antagonists while still retaining the therapeu-
tic profile (17,41,42). Thus, compound 5a may be worthy of further
investigation as well as serve as a good lead for the discovery of
more suitable compounds.
Table 4: The MES, scMET and rotorod TOX assessments of novel N-methyl-D-aspartate receptor antagonists
MES MES MES 6 Hz scMET scMET scMET TOX TOX
TOX
TOX
Compound Time (h) (30) N ⁄ F (100) N ⁄ F (300) N ⁄ F (50) (30) N ⁄ F (100) N ⁄ F (300) N ⁄ F (30) N ⁄ F (65) N ⁄ F (100) N ⁄ F (300) N ⁄ F
4a
4b
5a
5b
5c
5d
5e
5f
0.5
4.0
0.5
4.0
0.5
4.0
0.5
4.0
0.5
4.0
0.5
4.0
0.5
4.0
0.5
4.0
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 0
1 ⁄ 1
3 ⁄ 3
0 ⁄ 3
3 ⁄ 3
1 ⁄ 3
2 ⁄ 3
0 ⁄ 3
2 ⁄ 3
0 ⁄ 3
2 ⁄ 3
0 ⁄ 3
0 ⁄ 3
0 ⁄ 3
1 ⁄ 3
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 1
0 ⁄ 4
0 ⁄ 2
0 ⁄ 4
0 ⁄ 2
0 ⁄ 4
0 ⁄ 2
0 ⁄ 4
0 ⁄ 2
0 ⁄ 4
0 ⁄ 2
0 ⁄ 4
0 ⁄ 2
0 ⁄ 4
0 ⁄ 2
0 ⁄ 4
0 ⁄ 2
8 ⁄ 8
0 ⁄ 2
8 ⁄ 8
0 ⁄ 4
8 ⁄ 8
0 ⁄ 4
3 ⁄ 8
0 ⁄ 4
6 ⁄ 8
0 ⁄ 4
1 ⁄ 8
0 ⁄ 4
0 ⁄ 8
0 ⁄ 4
5 ⁄ 8
0 ⁄ 4
4 ⁄ 4
⁄
4 ⁄ 4
⁄
4 ⁄ 4
⁄
4 ⁄ 4
⁄
4 ⁄ 4
1 ⁄ 1
4 ⁄ 4
⁄
4 ⁄ 4
⁄
0 ⁄ 4
2 ⁄ 4
2 ⁄ 4
1 ⁄ 4
0 ⁄ 1
0 ⁄ 0
1 ⁄ 1
0 ⁄ 0
1 ⁄ 4
1 ⁄ 4
0 ⁄ 4
0 ⁄ 4
0 ⁄ 1
0 ⁄ 0
4 ⁄ 4
⁄
Doses are in () and are mg ⁄ kg.
N ⁄ F, number of the animals active or toxic over the number tested.
30
Chem Biol Drug Des 2011; 78: 25–32

Syntheses and Pharmacological Evaluations of NMDA Receptor Antagonists
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0.5
Severe tremors,
death following clonic seizure
Death
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Clonic seizures
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Death
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TOX 100
TOX 300
TOX 100
TOX 300
TOX 50
TOX 100
TOX 300
TOX 100
TOX 300
TOX 300
TOX 300
TOX 65
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