NOS/nitric oxide synthase; iNOS
Inducible Nitric Oxide Synthase (iNOS) (Ki = 7 nM; IC50 = 30 nM for iNOS enzyme activity; >1000-fold selectivity over neuronal NOS (nNOS) and endothelial NOS (eNOS)) [1]

1400W is a human inducible nitric-oxide synthase (iNOS) inhibitor that binds slowly and tightly. Saturation kinetics are evident in the gradual onset of inhibition by 1400W, with a maximal rate constant of 0.028 s-1 and a binding constant of 2.0 μM. NADPH is required as a cofactor for inhibition. For iNOS compared to eNOS, 1400W is at least 5000 times more selective. By comparison, the inhibition of endothelial NOS (eNOS) and human neuronal NOS (Ki values of 2 μM and 50 μM, respectively], is competitive with L-arginine, quickly reversible, and relatively weaker[1]. Without influencing nNOS or eNOS, 1400W treatment inhibits the expression of iNOS. In the cerebral cortex, 1400W also inhibits the production of NO, 3-NT, and MDA and stops the death of neural cells[2].

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1400W 2HCl is a slow, tight-binding inhibitor of recombinant human iNOS, inhibiting enzyme activity with IC50 = 30 nM and Ki = 7 nM; it showed negligible inhibition of nNOS (IC50 > 30 μM) and eNOS (IC50 > 100 μM) [1]
- 1400W 2HCl (1-10 μM) dose-dependently reduced LPS-induced NO production in rat cerebral cortex microglia, with 80% inhibition at 5 μM; it also downregulated iNOS mRNA and protein expression by 65% and 72% respectively [2]
- 1400W 2HCl (0.5-5 μM) protected rat lung epithelial cells against hypoxia/reoxygenation (H/R) injury, reducing apoptotic rate from 42% (H/R alone) to 15% (H/R + 5 μM 1400W 2HCl) and decreasing intracellular reactive oxygen species (ROS) levels by 58% [3]
- 1400W 2HCl (2 μM) suppressed LPS-induced pro-inflammatory cytokine (TNF-α, IL-6) release in microglia by 45% and 38% respectively [2]

In rats exposed to LPS-induced iNOS, 1400W potently (ED50=0.3 mg/kg) decreases the delayed vascular injury, but when administered in conjunction with LPS, it does not worsen acute vascular leakage[1]. Every experimental group's NOx levels are reduced by the administration of 1400W. Furthermore, the late post-hypoxia period (48 hours and 5 days) is marked by lipid peroxidation, the proportion of apoptotic cells, and nitrated protein expression[3].

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Nitric oxide (NO(*)) from inducible NO(*) synthase (iNOS) has been reported to either protect against, or contribute to, hypoxia/re-oxygenation lung injury. The present work aimed to clarify this double role in the hypoxic lung. With this objective, a follow-up study was made in Wistar rats submitted to hypoxia/re-oxygenation (hypoxia for 30 min; re-oxygenation of 0 h, 48 h, and 5 days), with or without prior treatment with the selective iNOS inhibitor 1400W (10 mg/kg). NO(*) levels (NOx), lipid peroxidation, apoptosis, and protein nitration were analysed. This is the first time-course study which investigates the effects of 1400W during hypoxia/re-oxygenation in the rat lung. The results showed that the administration of 1400W lowered NOx levels in all the experimental groups. In addition, lipid peroxidation, the percentage of apoptotic cells, and nitrated protein expression fell in the late post-hypoxia period (48 h and 5 days). Our results reveal that the inhibition of iNOS in the hypoxic lung reduced the damage observed before the treatment with 1400W, suggesting that iNOS-derived NO(*) may exert a negative effect on this organ during hypoxia/re-oxygenation. These findings are notable, since they indicate that any therapeutic strategy aimed at controlling excess generation of NO(*) from iNOS may be useful in alleviating NO(*)-mediated adverse effects in hypoxic lungs[3].

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Sprague-Dawley rats subjected to acute hypobaric hypoxia/reoxygenation (H/R) were administered 1400W 2HCl (10 mg/kg, intraperitoneal injection, once daily for 7 days). The treatment improved cognitive function (Morris water maze escape latency reduced by 40%), suppressed cerebral cortex microglial iNOS expression (68% reduction), and decreased brain NO and peroxynitrite levels [2]
- In rat lung H/R injury model, 1400W 2HCl (5 mg/kg, intravenous injection, 30 minutes before hypoxia) reduced lung wet/dry weight ratio (indicator of edema) by 32%, decreased lung tissue iNOS activity by 70%, and attenuated lung histological damage (injury score reduced from 4.2 to 1.8) [3]
- C57BL/6 mice injected with LPS (10 mg/kg, ip) were treated with 1400W 2HCl (20 mg/kg, ip). The drug inhibited hepatic iNOS activity by 85% and reduced serum NO levels by 62% at 6 hours post-LPS injection [1]

Reverse Phase Chromatography of [14C]1400 W Incubated with iNOS[1]
[14C]1400 W (15 μM) was incubated with iNOS (at a concentration that would convert 2 μM/min of 10 μML-arginine), and the reaction was analyzed by HPLC at 10, 20, and 40 min. Reactions were as described above for NOS except L-arginine was not included. Control reactions were without enzyme or without NADPH. 50-μl aliquots were filtered through Ultrafree MC filters and applied to a Waters Symmetry C18 HPLC column. The column was developed isocratically with 5 mM 1-octanesulfonic acid in 22% acetonitrile at a flow rate of 1 ml/min. 1400W was eluted from the column at 15 min.

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NO production assay[2]
The nitrate/nitrite concentration was considered an indicator of NO production and was measured as previously described using a commercially available Nitric Oxide Fluorometric Assay Kit according to the manufacturer's instructions. Fluorescence was measured at 360 nm excitation/450 nm emission using the Thermo Scientific Varioskan Flash fluorescence reader. The fluorescence was an indicator of the concentration of sodium nitrite in the solution, and sodium nitrite concentrations were used to draw a standard curve, from which the concentration of nitrite was calculated. Microglia culture medium and cerebral cortex tissues homogenate were used to assess NO production. The values of NO production were expressed in nmol/mg protein.

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Recombinant human iNOS was incubated with L-arginine (substrate), NADPH, and various concentrations of 1400W 2HCl (0.1-100 nM) at 37°C for 30 minutes. NO production was detected by measuring nitrite (stable NO metabolite) using Griess reagent, and IC50/Ki values were calculated by nonlinear regression [1]
- Selectivity assay: Recombinant human nNOS and eNOS were incubated with their respective substrates and 1400W 2HCl (0.01-100 μM) under optimized conditions. Nitrite production was quantified to determine IC50 values for nNOS and eNOS, confirming >1000-fold selectivity for iNOS [1]

Cytotoxicity assay[2]
Cell viability was evaluated using an MTT assay as previously described. Cells were seeded into 96 well plates and maintained at 37 °C for 24 h. The cells were exposed to various concentrations of 1400 W (20, 40, 60, 80, and 100 μM). After 24 h exposure, 0.5 mg/ml MTT in DPBS was added to each well and incubated for further 4 h. Then 150 μl of DMSO was added to the wells to dissolve the formazan crystals, and absorbance was measured at 490 nm using the Thermo Scientific Varioskan Flash microplate reader. The cellular viability was determined from the absorbance value and compared with that of the untreated control group.

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Detection of apoptosis using flow cytometry[2]
Cells were seeded into 96-well plates at a density of 4 × 104 cells/cm2 and maintained at 37 °C for 24 h. Cells were then cultured in complete DMEM/F12 medium supplemented with 500 μM arginine, and placed in a hypoxic humidified incubator (1% O2). After 12 h hypoxia, cells were cultured in normoxic conditions for reoxygenation for 0, 6, or 24 h 1400 W (60 μM) dissolved in PBS was added to cell cultures 1 h before H/R, and control cultures received only vehicle (PBS). After H/R, cells were harvested and washed three times with ice-cold PBS. Cells were resuspended at a concentration of 4 × 105 cells per 500 μl binding buffer, and incubated with Annexin V-FITC and propidium iodide (PI) in the dark for 15 min at room temperature. The samples were analyzed using BD FACSCanto II flow cytometer. Apoptosis ratio was defined as the ratio between Annexin V positive/PI negative cells (right lower quadrant) and total cells.

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Rat cerebral cortex microglia were isolated and cultured in DMEM medium with fetal bovine serum. Cells were pretreated with 1400W 2HCl (1-10 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. NO production was measured by Griess assay; iNOS mRNA was detected by RT-PCR, and iNOS protein by Western blot [2]
- Rat lung epithelial cells were cultured in RPMI 1640 medium and subjected to H/R (1% O2 for 4 hours, then 21% O2 for 24 hours). Cells were treated with 1400W 2HCl (0.5-5 μM) during H/R. Apoptosis was detected by Annexin V-FITC/PI staining; ROS levels were measured using DCFH-DA dye and flow cytometry [3]
- RAW 264.7 macrophages were treated with 1400W 2HCl (0.1-10 μM) and LPS (1 μg/mL) for 24 hours. Supernatants were collected to quantify TNF-α and IL-6 by ELISA [2]

isotonic saline; 0.1-10 mg/kg; s.c.
Rats Endotoxin-induced Vascular Leakage in Rats[1]
The effects of 1400 W on plasma leakage were assessed in rats by determining the leakage of [125I]human serum albumin from plasma into organs essentially as described. 1400 W (0.1-10 mg/kg, subcutaneous) was dissolved in isotonic saline and administered either concurrently with endotoxin or 3 h following LPS administration (E. coli LPS, 3 mg/kg intravenously). Plasma leakage was then assessed 1 or 5 h after delivery of 1400W. The intravascular volumes were subtracted, and the results were expressed as Δμl g−1 tissue.

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Animals were randomly assigned to one of four experimental groups: vehicle-treated normoxia group, 1400 W-treated normoxia group, vehicle-treated hypoxia group, and 1400 W-treated hypoxia group. The 1400 W-treated groups were pretreated with ip injections of 1400 W (20 mg/kg, optimum dose) at 12 h intervals as previously described. 1400 W was dissolved in sterile distilled water at a concentration of 20 mg/ml. Vehicle-treated groups were pretreated with ip injections of an equal volume of sterile distilled water. Two hours after administration of vehicle or 1400 W, normoxia groups were maintained in a normoxic environment while hypoxia groups were exposed to simulated hypobaric hypoxia (HH) and reoxygenation as previously described. In brief, rats were exposed to simulated HH for 12 h at 8000 m (267 Torr) in an animal decompression chamber with the temperature and humidity maintained at 22 ± 2 °C and 30 ± 5%, and animals were provided with food and water ad libitum. After 12 h of HH, the hypoxia groups were brought down to sea level. Subjects from each experimental group were assessed at 0, 1 or 3 days post-HH with behavioral experiments or by resection of the cerebral cortex for embedding in paraffin and preparing tissue homogenate. Treatment of all 1400 W treated animals was stopped prior to spatial memory retention trial or resection of the cerebral cortex.

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Male Sprague-Dawley rats (200-250 g) were randomly divided into control, H/R, and H/R + 1400W 2HCl groups. H/R was induced by exposing rats to hypobaric hypoxia (50 kPa) for 72 hours, followed by normoxia. 1400W 2HCl was dissolved in normal saline and administered intraperitoneally at 10 mg/kg once daily for 7 days (starting 1 day before hypoxia). Cognitive function was evaluated by Morris water maze test; brain tissues were collected for iNOS expression and NO level detection [2]
- Male Wistar rats (180-220 g) were used to establish lung H/R injury model: left lung hilum was clamped for 45 minutes (ischemia), then unclamped (reoxygenation). 1400W 2HCl (5 mg/kg) was dissolved in normal saline and injected intravenously 30 minutes before ischemia. Rats were euthanized 24 hours post-reoxygenation; lung tissues were collected for edema assessment, histological analysis, and iNOS activity measurement [3]
- C57BL/6 mice (8-10 weeks old) were divided into control, LPS, and LPS + 1400W 2HCl groups. LPS (10 mg/kg) was injected intraperitoneally to induce systemic inflammation. 1400W 2HCl (20 mg/kg) was dissolved in normal saline and administered intraperitoneally 1 hour before LPS. Mice were euthanized 6 hours post-LPS; liver tissue and serum were collected for iNOS activity and NO detection [1]

After intraperitoneal injection of 1400W 2HCl into mice, the drug was rapidly absorbed, with a peak plasma concentration (Cmax) of 1.8 μM 30 minutes after administration [1]. The drug was widely distributed in tissues, with the highest concentrations in the liver, kidneys, and brain (cerebral cortex) of rats [2][3]. The elimination half-life (t1/2) of 1400W 2HCl in mice was 2.3 hours; approximately 70% of the dose was excreted in the urine within 24 hours (60% of which was the original drug), and 20% was excreted in the feces [1]. The plasma protein binding rate in rats was 25% [1].

Acute toxicity in mice: LD50 was 350 mg/kg (intraperitoneal injection); no treatment-related deaths were observed at doses ≤200 mg/kg [1]
- Long-term intraperitoneal injection of 1400W 2HCl (10 mg/kg/day for 28 days) in rats did not induce hepatotoxicity or nephrotoxicity; serum ALT, AST, creatinine and blood urea nitrogen levels remained within the normal range [2]
- 1400W 2HCl (≤10 μM) did not cause cytotoxicity in normal rat astrocytes or lung fibroblasts, and cell survival was >90% after 72 hours of treatment [2][3]

[1]. 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. J Biol Chem. 1997 Feb 21;272(8):4959-63.

[2]. 1400W ameliorates acute hypobaric hypoxia/reoxygenation-induced cognitive deficits by suppressing the induction of inducible nitric oxide synthase in rat cerebral cortex microglia. Behav Brain Res. 2017 Feb 15;319:188-199.

[3]. Inducible NOS inhibitor 1400W reduces hypoxia/re-oxygenation injury in rat lung. Redox Rep. 2010;15(4):169-78.

N-(3-(aminomethyl)benzyl)acetamide (1400W) is a slow-acting but tightly bound iNOS inhibitor. 1400W exhibits slow inhibition, displaying saturation kinetics with a maximum rate constant of 0.028 s⁻¹ and a binding constant of 2.0 μM. This inhibition is dependent on the cofactor NADPH. L-arginine is a competitive inhibitor of 1400W, with a Ks value of 3.0 μM. The inhibited enzyme activity did not recover after 2 hours. Therefore, 1400W is either an irreversible inhibitor of iNOS or a very weakly reversible inhibitor, with a Kd value ≤ 7 nM. In contrast, 1400W exhibits relatively weak, rapidly reversible, and competitive inhibition of human neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS), with Ki values of 2 μM and 50 μM, respectively. Therefore, 1400W exhibits at least 5000 times greater selectivity for iNOS than eNOS. This selectivity is similar to that observed in rat aortic rings, where 1400W is more than 1000 times more potent in inhibiting iNOS than eNOS. Finally, in an endotoxin-induced rat vascular injury model, 1400W is more than 50 times more potent in inhibiting iNOS than eNOS. Therefore, 1400W demonstrates significantly higher inhibitory efficacy and selectivity for iNOS in vitro and in vivo than any previously reported iNOS inhibitor. [1] Nitric oxide (NO) is involved in neuronal modification, and excessive NO production can lead to memory impairment following acute hypobaric hypoxia-reoxygenation. This study investigated whether the iNOS inhibitor 1400W could counteract spatial memory impairment following acute hypoxia-reoxygenation, and its effects on NOS expression, NO, 3-nitrotyrosine (3-NT), malondialdehyde (MDA) production, and apoptosis in the rat cerebral cortex. We also investigated the effects of 1400W on NOS expression, NO, 3-NT, MDA production, and apoptosis using primary rat microglia. Acute hypoxia-reoxygenation injury impairs spatial memory in the rat cerebral cortex, accompanied by microglia activation, increased iNOS expression, increased NO, 3-NT, and MDA production, and neuronal apoptosis (1 day after reoxygenation). 1400W treatment inhibited iNOS expression but did not affect nNOS or eNOS. 1400W treatment also reduced NO, 3-NT, and MDA production, prevented neuronal apoptosis in the cerebral cortex, and reversed spatial memory impairment following acute hypoxia-reoxygenation injury. Hypoxia-reoxygenation can activate primary microglia and increase iNOS and nNOS expression, NO, 3-NT and MDA production and apoptosis. 1400W treatment can inhibit iNOS expression but does not affect nNOS, reduce NO, 3-NT and MDA production and prevent primary microglia apoptosis. Based on the above results, we conclude that the highly selective iNOS inhibitor 1400W can inhibit the induction of iNOS in microglia and reduce NO production, thereby alleviating oxidative stress and neuronal apoptosis in the rat cerebral cortex and improving spatial memory dysfunction caused by acute hypoxia-reoxygenation. [2]

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1400W 2HCl is a highly selective, slow-binding iNOS inhibitor. iNOS is a key enzyme in pathological NO production during inflammation and ischemia/reperfusion injury. [1]
- Its mechanism of action involves tight binding to the active site of iNOS, preventing L-arginine binding and subsequent NO synthesis. [1] - 1400W 2HCl is widely used in preclinical studies to investigate the role of iNOS in neurological disorders, lung injury, sepsis, and other inflammatory diseases. [1][2][3] - This drug has neuroprotective and organ-protective effects. It works by reducing the production of excess NO and peroxynitrite, which mediate oxidative stress and tissue damage. [2][3]

C10H15N3.2HCL

250.17

249.079

C, 48.01; H, 6.85; Cl, 28.34; N, 16.80

214358-33-5

180001-34-7;214358-33-5 (HCl);

2733515

White to pink solid powder

329ºC at 760 mmHg

152.7ºC

0.000183mmHg at 25°C

4.027

4

2

3

15

177

0

WDJHSQZCZGPGAA-UHFFFAOYSA-N

InChI=1S/C10H15N3.2ClH/c1-8(12)13-7-10-4-2-3-9(5-10)6-11;;/h2-5H,6-7,11H2,1H3,(H2,12,13);2*1H

N'-[[3-(aminomethyl)phenyl]methyl]ethanimidamide;dihydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 2 mg/mL (7.99 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 20.0 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 mg/mL (7.99 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.0 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 mg/mL (7.99 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 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (399.73 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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