Syntheses and Pharmacological Evaluations of Novel N-substituted Bicyclo-heptan-2-amines at NMDA Receptors

Abstract

Several novel norcamphor (bicycloheptane) based compounds were designed and synthesized as noncompetitive NMDA receptor antagonists at the Phencyclidine (PCP) binding sites. The heterocyclic ring was also varied to examine piperidine, pyrrolidine and morpholine groups. We examined pharmacological activities of these compounds in vitro (binding studies) and in vivo (MES test). Pharmacological evaluations revealed one of the compounds, 5a, to be a good lead, exhibiting moderate binding at NMDA receptors (IC₅₀ = 7.86 μM; K_i = 5.28 μM), MES neuroprotection activity at 100 mg/kg and acceptable toxicity profile.

Introduction

Glutamate is one of the principal excitatory neurotransmitters in the mammalian central nervous system (CNS). A major function of glutamate is control of ion flow at excitatory synapses. Glutamate receptors are subdivided into two, namely metabotropic and ionotropic. Three ionotropic receptor types have been identified based on ligand selectivity; AMPA, N-methyl-D-aspartate (NMDA), and kainate. In addition to ionotropic receptors, three classes of metabotropic 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 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 glutamate 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).

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 without complete amelioration of normal glutamate function. As a result of this dichotomy, many competitive antagonists have failed in clinical 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 likelihood of channel blockade in the presence of excessive levels of glutamate and a lower likelihood of antagonism with normal physiological levels of glutamate (16).

PCP 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 ischaemia, anoxia, hypoglycaemia, 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.

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 antagonists, such as ketamine, DXM, memantine and adamantine are generally well tolerated (18, 20-22).

Our group is involved in the design and syntheses of NMDA receptor inhibitors as possible therapies for neurodegenerative diseases. Several novel norcamphor (bicyloheptane) derivatives were designed and synthesized as noncompetitive antagonists of the NMDA receptor. Syntheses of these compounds are displayed in Scheme 1. Pharmacological activities of these compounds were evaluated in vitro (binding studies) and in vivo (MES test).

Scheme 1.

Syntheses of target compounds 5a-5f a) Mg, I₂, THF b) NaN₃, TFA, CHCl₃ c) LiAlH₄, THF d) TEA, Et-OH

Material and Methods

Melting points were determined with a Mel-Temp electrothermal apparatus and are uncorrected. The ¹H, 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 instrument using electrospray ionization (ESI) technique. NMR and MS were obtained on the free base of each amine. Column chromatography was conducted using Merck silica gel, grade 9385, 230-400 mesh, 60 Å. Compound purity was determined by elemental analysis conducted by Galbraith Laboratories, Knoxville, TN. The chemical reagents used were purchased from Aldrich, Acros and Alfa Aesar.

Synthesis of intermediate 4a

A mixture of bromobenzene (5.5 ml, 52.2 mmol), magnesium turnings (3.81 g, 157 mmol), and a few iodine crystals was stirred to 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-phenylbicyclo[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 mixture 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): δ ppm 7.3-7.4 (m, 5H), 1.0-2.6 (b, 10 H, 4CH₂ and 2CH). ¹³C NMR (CDCl₃): δ 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 bubbling 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).

Synthesis of intermediate 4b

Crude alcohol 2b (9.4 g, 99% yield) was obtained from p-bromofluorobenzene (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 compound 2a. ¹H NMR (CDCl₃): δ ppm 7.63-7.43 (m, 2H), 7.15-6.96 (m, 2H), 2.58 (s, 1H), 2.44-2.11 (m, 3H), 1.81-1.26 (m, 7H); ¹³C NMR (CDCl₃): δ 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 alcohol (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 preparative TLC developed using a mixture 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): δ ppm 7.2 (m, 2H), 6.8 (m, 2H), 1.0-2.6 (b, 10 H, 4CH₂ and 2CH); ¹³C NMR (CDCl₃): δ 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): δ −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).

General Procedure for Syntheses of Compounds 5a-5f

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. Hydrochloride 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-Phenyl-N-(2-(piperidin-1-yl) ethyl)bicyclo[2.2.1]heptan-2-amine (5a)

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 colorless crystals: m.p. 32-34 °C. ¹H NMR (400 MHz, CDCl₃) δ 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) δ 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-(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 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 colorless crystals: m.p 66-68 °C. ¹H NMR (400 MHz, CDCl₃) δ 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) δ 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) δ 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.

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 chromatography (EtOAc/i-prOH/TEA 7:3:0.1) to give 5c (0.22 g, 16% yield) as colorless crystals: m.p. 29-31 °C. ¹H NMR (400 MHz, CDCl₃) δ ppm 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) δ 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 prepared, colorless crystals, m.p. 197-199 °C.

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 chromatography (EtOAc/i-prOH/TEA 7:3:0.2) to give 5d (0.16 g, 14% yield) as colorless crystals: m.p. 47-49 °C. ¹H NMR (400 MHz, CDCl₃) δ ppm 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) δ 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) δ 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 prepared, white crystal, m.p. 152-154 °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 colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 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, 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.

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 chromatography (CHCl₃/i-prOH/TEA 6.5:3:0.5) to give 5f (0.28 g, 11% yield) as colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 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 crystals, 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.

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 conducted in accordance with published protocol by Reynolds and Sharma (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 1999).

Suspensions of 10 mM HEPES (pH 7.4 at room temperature) containing 30 ug/mL protein, 1 nM (+)-[³H]MK-801, 100 uM glutamate, 30 uM glycine, and various concentrations of unlabeled competitor or 30 uM (+)-MK-801 for nonspecific binding, were incubated at room temperature for 2 hours. Termination of reaction was performed via vacuum filtration using a 24 well cell harvester (Brandel, Gaithersburg, MD) over GF/B glass fiber filters (Brandel, Gaithersburg, MD). 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 (Beckman Coulter, USA) at 64% efficiency.

IC₅₀ values were determined with graphpad using log-concentration plotted against percent specific binding. Percent specific binding for [³H]-MK-801 in control experiment was 70% of total. K_i values were calculated using the equation of Cheng and Prusoff (28). The K_d for (+)-MK-801 binding under the saturation conditions was 1.747 nM and this is consistent with that reported in literature (27). The K_d 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 coomasssie protein assay reagent.

The compounds were evaluated through the National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program (PDSP), 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 (K_i) 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 Hill (30).

In vivo Biological Studies

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 s 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 varying 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 component of the seizure (31-33).

Subcutaneous Metrazol Seizure Threshold Test (scMET)

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 (34) and observed for the next 30 minutes 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 endpoint. Animals which do not meet this criterion are considered protected.

Acute Toxicity-Minimal Motor Impairment (MMI)

To assess a compound’s undesirable side effects (toxicity) animals are monitored for overt signs of impaired neurological or muscular function. In mice, the rotorod (35) 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 equilibrium for long periods of time. The compound is considered 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, since individual animals may have peculiarities in gait, equilibrium, placing response, amongst 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 placing response and changes in muscle tone.

Results and Discussion

Our studies examined design, syntheses and pharmacological evaluations of novel, non-competitive, NMDAR antagonists as potential therapies for neurodegenerative diseases (36, 37). The syntheses of N-substitued bicyclo-heptan-2-amines (5a-5f) were carried out starting from commercially available norcamphor and a phenyl Grignard reagent. Phenyl magnesium bromides were added to norcamphor 1 to give alcohols 2a and 2b. This addition has been reported to give exo-phenyl tertiary alcohols (38). Treatment of the resulting tertiary alcohol with trifluoroacetic acid (TFA) in the 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 completed syntheses of the target compounds (Scheme 1).

Radioligand binding studies were utilized to evaluate in vitro affinities 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 μM were subjected to further analysis. The IC₅₀ and K_i 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.

Figure 1.

Concentration % specific binding curve for Compound 5a

Table 1.

The IC₅₀ and K_i for the 4 compounds subjected to further analysis and (+)-MK-801 run under experimental condition as reference

Compound	IC50	NMDA affinity   Ki
(+)-MK-801	2.02 ± 0 .797 nM	1.357 nM
4a	13.27 ± 0.63 μM	8.45 μM
5a	7.86 ± 1.64 μM	5.28 μM
5e	8.48 ± 3.106 μM	5.67 μM
5f	8.657 ± 2.564 μM	5.82 μM

Each compound was evaluated for affinity at human or rat receptors through the NIMH-PDSP program. In vitro binding affinities of these novel NMDAR antagonists at Serotonin _2A (5-HT_2A), Dopamine 1 (D₁), Dopamine 2 (D₂), the dopamine transporter (DAT), kappa opioid receptor (KOR), μ- opioid receptors (MOR), Norepinephrine transporter (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 moderate at best. A notable exception is compound 5f which showed significant affinity (226 nM) at DAT (Table 2).

Table 2.

In vitro binding affinities of novel NMDAR antagonists for 5-HT_2A, D₁, D₂, DAT, KOR, MOR, NET, SERT, sigma-1, and sigma-2 receptors

Comp.	5HT2A (nM)	D1 (nM)	D2 (nM)	DAT (nM)	KOR (nM)	MOR (nM)	NET (nM)	SERT (nM)	Sigma 1 (nM)	Sigma 2 (nM)
4a	>10,000	>10,000	>10,000	>10,000	>10,000	>10,000	>10,000	>10,000	2,201	995
4b	>10,000	>10,000	>10,000	>10,000	>10,000	>10,000	>10,000	>10,000	>10,000	>10,000
5a	2,643	>10,000	>10,000	>10,000	876 nM	>10,000	>10,000	>10,000	>10,000	>10,000
5b	8,822	>10,000	>10,000	1,231	3,546	>10,000	>10,000	>10,000	>10,000	>10,000
5c	>10,000	>10,000	>10,000	2,570	988	>10,000	>10,000	>10,000	>10,000	>10,000
5d	>10,000	>10,000	>10,000	6,482	>10,000	>10,000	>10,000	>10,000	>10,000	>10,000
5e	>10,000	>10,000	>10,000	3,624	4,507	>10,000	>10,000	>10,000	>10,000	>10,000
5f	>10,000	>10,000	>10,000	226	2,111	>10,000	>10,000	4,607	>10,000	>10,000

Table 3.

Novel NMDAR antagonists were screened for the following receptors, and sources

Receptor	Source	Hot ligand
Serotonin 2A (5-HT2A)	Human Cloned	Ketanserin
Dopamine 1 (D1)	Human Cloned	SCH23390
Dopamine 1(D2)	Human Cloned	N-Methylspiperone
Dopamine Transporter (DAT)	Human Cloned	WIN35428
κ- opioid receptors (KOR)	Rat Cloned	U69593 (2007-07-27)
μ- opioid receptors (MOR)	Human Cloned	DAMGO (2007-07-27)
Norepinephrine Transporter (NET)	Human Cloned	Nisoxetine
Serotonin Transporter (SERT)	Human Cloned	Citalopram
Sigma1	Rat Brain	Pentazocine(+)
Sigma2	Rat C12	DTG

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 maximal electroshock (MES) test and Subcutaneous Metrazol Seizure Threshold test (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 protection from MES induced neural damage and death. However, compounds 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 possibilities 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 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 it’s metabolites, dextrophan (DX) has μM affinity (39, 40). It has been speculated that some of the therapeutic effects of DXM are mediated by DX (41). DXM has been found to have neuroprotective activity in the MES test.

Table 4.

The MES, scMET and rotorod TOX assessments of novel NMDAR antagonists

Comp.	Time (h)	MES (30) N/F	MES (100) N/F	MES (300) N/F	6HZ (50)	scMET (30) N/F	scMET (100) N/F	scMET (300) N/F	TOX (30) N/F	TOX (65) N/F	TOX (100) N/F	TOX (300) N/F
4a	0.5 4.0	0/1 0/1	0/0 1/1			0/1 0/1	0/1 0/1		0/4 0/2		8/8 0/2	4/4 /
4b	0.5 4.0	0/1 0/1	3/3 0/3			0/1 0/1	0/1 0/1		0/4 0/2		8/8 0/4	4/4 /
5a	0.5 4.0	0/1 0/1	3/3 1/3			0/1 0/1	0/1 0/1		0/4 0/2		8/8 0/4	4/4 /
5b	0.5 4.0	0/1 0/1	2/3 0/3		0/4 2/4	0/1 0/1	0/1 0/1		0/4 0/2	2/4 1/4	3/8 0/4	4/4 /
5c	0.5 4.0	0/1 0/1	2/3 0/3			0/1 0/1	0/1 0/1	0/1 0/0	0/4 0/2		6/8 0/4	4/4 1/1
5d	0.5 4.0	0/1 0/1	2/3 0/3	1/1 0/0	1/4 1/4	0/1 0/1	0/1 0/1		0/4 0/2	0/4 0/4	1/8 0/4	4/4 /
5e	0.5 4.0	0/1 0/1	0/3 0/3			0/1 0/1	0/1 0/1	0/1 0/0	0/4 0/2		0/8 0/4	4/4 /
5f	0.5 4.0	0/1 0/1	1/3 0/1			0/1 0/1	0/1 0/1		0/4 0/2		5/8 0/4	4/4 /

N/F= number of the animals active or toxic over the number tested.

Doses are in ( ) and are mg/kg.

Table 5.

Other comments on the novel NMDAR antagonists

Comp.	Test	Dose (mg/kg)	Time (h)	Comments
4a	TOX TOX	100 300	0.5 0.5	Severe Tremors, Death following clonic seizure Death
4b	TOX TOX	100 300	0.5 0.5	Severe Tremors, Clonic seizures Death following clonic seizure
5a	TOX TOX	100 300	0.5 0.5	Clonic seizures Death
5b	TOX TOX TOX	50 100 300	0.25 0.5 0.5	Clonic seizures Clonic seizures Death
5c	TOX TOX TOX TOX	100 300 300 300	0.5 0.5 0.5 4	Clonic seizures Death following clonic seizure Clonic seizures Death
5d	TOX TOX TOX TOX	65 100 300 300	0.25 0.5 0.5 0.5	Clonic seizures Clonic seizures Loss of righting reflex Death
5e	TOX TOX TOX	300 300 300	0.5 0.5 0.5	Diarrhea Unable to grasp rotorod Death
5f	TOX TOX	100 300	0.5 0.5	Clonic seizures Death

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 comments on these novel NMDAR antagonists are displayed on Table 5.

Conclusions and Future Directions

All target compounds were synthesized in hundred milligram quantities showing feasibility of the synthetic scheme. They were all protective 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).

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 therapeutic profile (17, 42, 43). Thus, compound 5a may be worthy of further investigation as well as serve as a good lead for the discovery of more suitable compounds.