Positive allosteric modulator (PAM) of the homomeric α7 nicotinic acetylcholine receptor (α7 nAChR). It does not bind to the orthosteric (agonist) site and potentiates acetylcholine (ACh)-evoked currents. The potency for modulating human α7 nAChR expressed in Xenopus oocytes was EC50 = 3.4 μM (Emax = 322%, nH = 2.0). The potency for modulating human α7 nAChR expressed in GH4C1 cells (patch-clamp) was EC50 = 1.6 μM (Emax = 1170%, nH = 3.5).
It also shows inhibitory effects on heteromeric nAChRs at high concentrations: IC50 = 27 μM for α3β4 nAChR and IC50 = 84 μM for α4β2 nAChR (determined in Xenopus oocytes). It does not affect neuromuscular-type (α1β1γδ) nAChRs in TE671 cells at 10 μM. [1]

Acetylcholine (ACh)-evoked currents have a peak amplitude that NS 1738 rises at all doses, enhancing the maximum effectiveness of ACh. The preincubation NS 1738 concentration logarithm was plotted against the peak current amplitude, and the resulting concentration-response relationship was sigmoidal and well-fitted to the Hill equation (EC50=3.4 μM). Comparable efficacy and potency were demonstrated by NS 1738 against rat α7 nAChR (EC50=3.9 μM) in similar experimental circumstances [1].

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In Xenopus oocytes expressing human α7 nAChR, preincubation with NS1738 (EC50 = 3.4 μM) followed by ACh (100 μM) application led to an approximately 2- to 3-fold increase in peak current amplitude. In the presence of 10 μM NS1738, the ACh concentration-response curve showed a leftward shift (ACh EC50 changed from 139 μM to 15 μM) and an increase in maximal efficacy (Emax from 109% to 184%). [1]
In whole-cell patch-clamp recordings from GH4C1 cells transiently transfected with human α7 nAChR, application of 10 μM NS1738 alone elicited no current, while co-application with a saturating ACh concentration (1 mM) resulted in an approximately 10-fold increase in peak current amplitude compared to ACh alone. The concentration-response relationship for NS1738-mediated potentiation gave an EC50 of 1.6 μM and Emax of 1170%. In the presence of 10 μM NS1738, the ACh concentration-response curve showed a marked increase in maximal efficacy (Emax from 100% to 600%) but no significant change in ACh potency (EC50 ~97 μM vs. 119 μM). [1]
In cultured rat hippocampal neurons (a native system), NS1738 (10 μM) also potentiated ACh (1 mM)-evoked currents, confirming its activity on native α7 nAChRs. [1]
NS1738 had only marginal effects on the desensitization kinetics of α7 nAChRs. In transfected GH4C1 cells, the desensitization time constant (τ) increased modestly from 30 ± 4 ms to 53 ± 7 ms in the presence of 10 μM NS1738. In hippocampal neurons, τ increased from 14 ± 3 ms to 21 ± 2 ms. [1]
NS1738 showed selectivity for α7 nAChR over other subtypes. At 10 μM, it slightly inhibited currents mediated by α3β4 and α4β2 nAChRs expressed in oocytes. It had no effect on ACh-evoked currents in TE671 cells expressing neuromuscular nAChRs (α1β1γδ). [1]

Rats were given an intraperitoneal injection of 10 mg/kg of NS 1738 in order to evaluate its ability to cross the blood-brain barrier. At this dose, peak brain concentrations were measured about half an hour after injection, reaching about 80 ng/mL (approximately 200 nM). The ratio of the amount of compound entering the brain to the amount in the plasma is AUCbrain/AUCplasma=0.50, and the half-life in plasma is estimated to be 42 minutes. In vitro incubation of NS 1738 with isolated liver microsomes revealed that in mice and rats, respectively, approximately 60% and 75% of NS 1738 were metabolized by the cytochrome P450 system within an hour. Adult rats were injected with NS 1738 ip at 10 and 30 mg/kg immediately following the first exposure to juvenile rats (T1) and two hours later

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In the rat Morris water maze spatial learning paradigm, administration of NS1738 (30 mg/kg, i.p.) significantly reversed the acquisition learning deficits induced by (-)-scopolamine (0.1 mg/kg, s.c.), bringing latencies to find the submerged platform to near control levels over days 2-4 of training. A dose of 10 mg/kg showed an intermediate but non-significant effect. [1]
In the rat social recognition test (a model of short-term memory), administration of NS1738 (10 and 30 mg/kg, i.p.) immediately after the initial exposure (T1) significantly decreased the investigative duration during a second exposure (T2) 2 hours later. The T2/T1 ratios were 0.69 ± 0.13 and 0.61 ± 0.07 for 10 and 30 mg/kg, respectively, compared to ~1.0 for vehicle controls, indicating improved short-term recognition memory. The effect was comparable to that of (-)-nicotine (0.1 mg/kg, i.p.). [1]
In mice, NS1738 at 10 and 30 mg/kg (i.p.) did not affect novelty-induced exploratory locomotor activity, while a slight but statistically significant increase was noted at 100 mg/kg. [1]

A saturation binding assay was performed to assess the interaction of NS1738 with the orthosteric site of α7 nAChR. Rat cortical and hippocampal membrane homogenates were incubated with varying concentrations of [³H]α-bungarotoxin ([³H]α-BgTx, 0.05 to 10 nM) in the absence or presence of 10 μM NS1738 for 2 hours at 37°C. The reaction was terminated by adding ice-cold HEPES buffer with 0.05% polyethyleneimine, and the mixture was filtered through glass fiber filters presoaked in 0.1% polyethyleneimine. Filters were washed and radioactivity was measured by liquid scintillation counting. NS1738 at 10 μM did not affect the Kd or Bmax values for [³H]α-BgTx binding, indicating it does not compete with the orthosteric ligand. [1]
Additionally, displacement binding assays showed that NS1738 (up to 100 μM) was unable to displace [³H]α-BgTx, [³H]methyllycaconitine, [³H]cytisine, or [³H]epibatidine from rat brain membranes or membranes from TE671/RD cells, confirming lack of binding to orthosteric sites of various nAChR subtypes. [1]

Cell Culture and Transfection for Electrophysiology: Rat pituitary carcinoma GH4C1 cells were cultured in Ham's F-10 medium supplemented with serum. Cells were plated on poly-D-lysine-coated coverslips and transiently transfected with a mixture of cDNAs encoding human α7 nAChR subunit and green fluorescent protein using a lipid-based transfection method. After transfection, cells were incubated in growth medium supplemented with 50 mM KCl to facilitate α7 nAChR expression. Cells were used for patch-clamp recording 1-2 days post-transfection. [1]
Primary Hippocampal Neuron Culture: Hippocampi from neonatal rats were dissected, dissociated by mild trypsinization, and plated on poly-L-lysine-coated plates at a density of 0.5 × 10⁶ cells/ml. Neurons were maintained in culture for 14 days with an antimitotic agent added from day 3-4 to limit glial growth. These neurons were used for patch-clamp recordings to study native α7 nAChRs. [1]
Electrophysiological Recording (Patch-Clamp): Transfected GH4C1 cells or cultured hippocampal neurons grown on coverslips were placed in a recording chamber on an inverted microscope and superfused with extracellular buffer. Whole-cell voltage-clamp recordings were performed using borosilicate micropipettes filled with intracellular buffer. The holding potential was -60 mV. Agonists and drugs were applied via an ultrafast solution exchange system using a θ-tube controlled by a piezo-ceramic device, allowing solution exchange within 200-400 μs. For α7 nAChR stimulation, agonist pulses lasted 200 ms, delivered at a frequency of 1 pulse per 30 seconds. Cells were preincubated with NS1738 for at least 60 seconds before ACh application in its presence. Peak current amplitudes were measured and normalized as described. [1]

Morris Water Maze (Rat): Male Wistar rats were used. (-)-Scopolamine HBr was dissolved in 0.9% saline and administered subcutaneously at 1 ml/kg, 30 minutes before the first acquisition trial each day. NS1738 was dissolved in 10% Tween 80 and administered intraperitoneally at 1 ml/kg, 15 minutes before the first trial. The water maze consisted of a black pool filled with water at 23°C. A submerged escape platform was placed in a fixed quadrant. Acquisition training consisted of four trials per day for four consecutive days. The latency to find the platform was recorded. On day 5, a probe trial (platform removed) and a reversal trial (platform moved to a new location) were conducted, but rats were drug-free on this day. [1]
Social Recognition Test (Rat): Adult Sprague-Dawley rats were acclimated to the test room. After habituation in a test cage, an adult rat was allowed to interact with a juvenile rat for 5 minutes (T1). Immediately after T1, the adult rat was administered NS1738 [dissolved in 5% ethanol/95% hydroxypropyl-β-cyclodextrin (34% solution)] intraperitoneally at 2.0 ml/kg or vehicle, and returned to its home cage. A second 5-minute interactive trial (T2) with the same juvenile was conducted 120 minutes later in the same test cage. The investigative behavior duration was recorded for both trials. The recognition ratio (T2/T1) was calculated. [1]
Pharmacokinetics Study (Rat): Rats were administered NS1738 intraperitoneally at 10 mg/kg. Plasma and brain samples were collected at various time points for concentration measurement. [1]
Locomotor Activity (Mouse): Mice were injected intraperitoneally with vehicle or NS1738 (10, 30, 100 mg/kg). Novelty-induced exploratory locomotor activity was measured at various time points (0-3 hours) after injection. [1]

After intraperitoneal injection of NS1738 (10 mg/kg) into rats, the peak plasma concentration (Tmax) was approximately 30 minutes. The plasma half-life (t1/2) was estimated to be 42 minutes. The maximum plasma concentration (Cmax) was 3042 ng/ml, and the area under the concentration-time curve (AUC) was 172,133 ng·min/ml. [1] NS1738 has some brain permeability. The peak concentration in brain tissue was approximately 80 ng/ml (~200 nM) 30 minutes after injection. The AUC ratio of brain tissue to plasma (AUCbrain/AUCplasma) was 0.50. [1] In vitro incubation experiments with isolated liver microsomes showed that approximately 60% (mice) and 75% (rats) of NS1738 were metabolized by the cytochrome P450 system within 1 hour. Preliminary analysis indicated that its main metabolic pathway was sulfation or glucuronidation of hydroxyl groups. [1]

Side effects in behavioral studies were limited to assessments of motor activity. High doses (100 mg/kg, intraperitoneal) of NS1738 resulted in a slight but statistically significant increase in motor activity in mice, but no sedation or significant toxicity was observed at lower effective doses (10 and 30 mg/kg). [1]

[1]. An allosteric modulator of the alpha7 nicotinic acetylcholine receptor possessing cognition-enhancing properties in vivo. J Pharmacol Exp Ther. 2007 Oct;323(1):294-307.

NS1738 (1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethylphenyl)urea) is a novel small-molecule positive allosteric modulator of the α7 nicotinic acetylcholine receptor. It was discovered by screening for compounds that enhance acetylcholine-induced currents in Xenopus laevis oocytes expressing human α7 nAChR. [1] Its mechanism of action is through allosteric-dependent regulation. It does not activate the receptor itself, but rather enhances the response to the endogenous agonist acetylcholine, primarily by increasing maximal potency (peak current amplitude), and in some experimental systems (oocytes), it also enhances the potency of acetylcholine. It has little effect on the desensitization kinetics of the receptor. [1]
NS1738 is selective for α7 nAChR, superior to other nAChR subtypes (α3β4, α4β2, neuromuscular) and 5-HT3A receptors. [1]
In rodent models (Morris water maze and social recognition test), the cognitive-enhancing effect of NS1738 supports the potential of α7 nAChR positive allosteric modulators as a novel treatment for disease-related cognitive impairments such as Alzheimer's disease and schizophrenia. [1]
The pharmacological properties of NS1738 are drastically different from another α7 PAM PNU-120596, which significantly inhibits desensitization and has shown neurotoxicity in vitro. NS1738 and similar compounds (“Compound 6”) represent a class of modulators that enhance peak current without significantly altering desensitization, thereby avoiding excessive calcium ion influx and potentially offering safer properties. [1]

C14H9CL2F3N2O2

365.1347

363.999

501684-93-1

310378

White to off-white solid powder

5.507

3

5

2

23

425

0

OUDXRNQPVSMGDW-UHFFFAOYSA-N

InChI=1S/C14H9Cl2F3N2O2/c15-8-2-4-12(22)11(6-8)21-13(23)20-10-5-7(14(17,18)19)1-3-9(10)16/h1-6,22H,(H2,20,21,23)

3-(5-chloro-2-hydroxyphenyl)-1-[2-chloro-5-(trifluoromethyl)phenyl]urea

NSC 213859; NSC213859; NSC-213859; NS1738; NS 1738; NS-1738.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 100 mg/mL (~273.88 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.85 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.85 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.85 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)