GMX1778 (CHS828) is a potent and selective inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD⁺ salvage biosynthesis pathway. It exhibits an IC50 of 9 nM against recombinant human NAMPT in an enzyme activity assay. It does not inhibit nicotinic acid phosphoribosyltransferase 1 (NAPRT1), another NAD⁺ biosynthetic enzyme, at concentrations up to 10 μM [1]

CHS-828 (GMX1778) showed a phosphoribosyltransferase activity inhibition threshold of less than 25 nM for recombinant NAMPT, but not for recombinant NMNAT1's adenyltransferase activity. Using a fluorescent tag (CHS-828 (GMX1778)-Alexa Fluor), the recombinant NAMPT for GMX1778 had a Kd of 120 nM. In response to a 3 nM GMX1778 challenge, overexpression of wild-type NAMPT was able to sustain a certain level of NAD+; however, this effect was eliminated when cells were subjected to 300 nM CHS-828 (GMX1778). Through a rise in superoxide and a decrease in intracellular NAD(+), CHS-828 (GMX1778) raises intracellular ROS in cancer cells. Interestingly, ROS are not produced in normal cells by CHS-828 (GMX1778) treatment. In a way that is dependent on NA phosphoribosyltransferase 1 (NAPRT1), nicotinic acid (NA) can reduce ROS produced by CHS-828 (GMX1778)[2].

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Antiproliferative activity in tumor cells: GMX1778 (0.1-1000 nM) showed dose-dependent antiproliferative effects on various human tumor cell lines, with significantly lower IC50 values in NAPRT1-deficient cells: IC50 = 12 nM for A2780 (ovarian cancer, NAPRT1⁻), vs. IC50 = 280 nM for SKOV3 (ovarian cancer, NAPRT1⁺); IC50 = 15 nM for HCT116 p53⁺/⁺ (colorectal cancer, NAPRT1⁻), vs. IC50 = 320 nM for HCT116 p53⁻/⁻ (NAPRT1⁺) (SRB assay, 72-hour treatment) [1]
- NAD⁺ and ATP depletion: In A2780 cells, GMX1778 (20 nM) treatment for 24 hours reduced intracellular NAD⁺ levels by 85% (cyclic enzyme assay) and ATP levels by 70% (luciferase-based ATP assay) compared to the vehicle control. No significant NAD⁺ depletion was observed in SKOV3 cells treated with 200 nM GMX1778 [1]
- Apoptosis induction: GMX1778 (20 nM) induced apoptosis in A2780 cells, with the apoptotic rate increasing from 3% (vehicle) to 45% (Annexin V-FITC/PI staining) after 48 hours. Western blot showed a 3.5-fold increase in cleaved caspase-3 and a 2.8-fold increase in cleaved PARP [1]
- ROS-mediated cytotoxicity (p53- and NAPRT1-dependent): In HCT116 p53⁺/⁻ cells, GMX1778 (50 nM) increased intracellular ROS levels by 2.3-fold (DCFH-DA staining) in p53⁺/⁺ cells, but only by 0.5-fold in p53⁻/⁻ cells. Pretreatment with N-acetylcysteine (NAC, a ROS scavenger) reversed GMX1778-induced cytotoxicity in p53⁺/⁺ cells (viability from 35% to 82%), but not in p53⁻/⁻ cells [2]
- NAPRT1-dependent resistance: Transfection of NAPRT1 cDNA into A2780 cells (NAPRT1⁻) increased the IC50 of GMX1778 from 12 nM to 250 nM, confirming NAPRT1 expression confers resistance [2]

In the NAPRT1-deficient xenograft trials, a 24-hour intravenous infusion of CHS-828 (GMX1778) at a dose of 150 mg/kg or 650 mg/kg did not negatively impact the antitumor efficacy of a 4-hour intravenous infusion of NA (120 mg/kg of body weight). At 650 mg/kg, CHS-828 (GMX1778) exceeds the highest dosage that can be tolerated. Following treatment with 750 mg/kg CHS-828 (GMX1778), the administration of NA as a 4-hour intravenous infusion decreased the mortality linked to hazardous levels of GMX1777[2].

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Nude mouse ovarian cancer xenograft model (A2780, NAPRT1⁻): Female BALB/c nu/nu mice (6-8 weeks old) were subcutaneously injected with 5×10⁶ A2780 cells. When tumors reached 100 mm³, mice were randomized into 3 groups (n=6/group):
1. Vehicle: Intraperitoneal injection of 5% DMSO + 5% Tween 80 + saline (10 mL/kg/day);
2. GMX1778 10 mg/kg: Intraperitoneal injection of 10 mg/kg/day GMX1778 (dissolved in vehicle);
3. GMX1778 20 mg/kg: Intraperitoneal injection of 20 mg/kg/day GMX1778 (dissolved in vehicle).
After 21 days, GMX1778 reduced tumor volume by 58% (10 mg/kg) and 75% (20 mg/kg) compared to the vehicle group. Tumor tissue analysis showed NAD⁺ levels reduced by 65% (20 mg/kg group) and ATP levels reduced by 55% [1]
- Nude mouse ovarian cancer xenograft model (SKOV3, NAPRT1⁺): Mice bearing SKOV3 tumors treated with GMX1778 (20 mg/kg/day, intraperitoneal) for 21 days showed no significant tumor inhibition (tumor volume reduction <15%), confirming NAPRT1 expression limits GMX1778 efficacy [1]

Recombinant Human NAMPT Activity Inhibition Assay: The 50 μL reaction system contained 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 2 mM nicotinamide, 1 mM 5-phosphoribosyl-1-pyrophosphate (PRPP), 5 μg recombinant human NAMPT, and GMX1778 (0.1-100 nM). The reaction was initiated at 37°C and incubated for 60 minutes, then terminated by adding 50 μL 10% trichloroacetic acid (TCA). The product (nicotinamide mononucleotide, NMN) was detected via a cyclic enzyme assay (coupled with NMN adenylyltransferase and luciferase), and luminescence intensity was measured with a luminometer. Inhibition rates were calculated relative to the vehicle control, and IC50 was derived via nonlinear regression (four-parameter logistic model) [1]
- NAPRT1 Activity Assay: The protocol was similar to the NAMPT assay, except recombinant human NAPRT1 (5 μg) and nicotinic acid (2 mM) were used instead of NAMPT and nicotinamide. GMX1778 concentrations up to 10 μM showed <5% inhibition of NAPRT1 activity, confirming selectivity [1]

Tumor Cell Proliferation Assay (SRB Method): A2780, SKOV3, HCT116 p53⁺/⁺, and HCT116 p53⁻/⁻ cells were seeded in 96-well plates at 3×10³ cells/well and cultured in RPMI 1640 with 10% FBS for 24 hours. GMX1778 (0.1-1000 nM) was added, and cells were incubated for 72 hours. Cells were fixed with 10% TCA, stained with 0.4% sulforhodamine B (SRB) in 1% acetic acid, and excess dye was washed with 1% acetic acid. Bound dye was dissolved in 10 mM Tris base, and absorbance at 515 nm was measured to calculate cell viability and IC50 [1,2]
- Intracellular NAD⁺ Detection: A2780 cells (2×10⁵ cells/well in 6-well plates) were treated with GMX1778 (20 nM) for 6, 12, 24 hours. Cells were lysed with 0.5 M perchloric acid, and lysates were neutralized with 2 M KOH. NAD⁺ levels were measured using a cyclic enzyme assay (as in Enzyme Assay) and normalized to protein concentration (BCA assay) [1]
- Apoptosis Detection (Annexin V-FITC/PI Staining): A2780 cells (1×10⁵ cells/well) were treated with GMX1778 (20 nM) for 48 hours. Cells were harvested, washed with cold PBS, stained with Annexin V-FITC and propidium iodide (PI) for 15 minutes at room temperature, and analyzed via flow cytometry. The apoptotic rate was calculated as the percentage of Annexin V-positive cells [1]
- ROS Detection (DCFH-DA Staining): HCT116 p53⁺/⁺ and p53⁻/⁻ cells (2×10⁵ cells/well) were treated with GMX1778 (50 nM) for 24 hours. Cells were loaded with 10 μM DCFH-DA (a ROS probe) for 30 minutes at 37°C, washed with PBS, and fluorescence intensity was measured via flow cytometry (excitation 488 nm, emission 525 nm) [2]
- Western Blot for Apoptotic Proteins: HCT116 p53⁺/⁺ cells (1×10⁶ cells/10-cm dish) were treated with GMX1778 (50 nM) for 48 hours. Cells were lysed with RIPA buffer containing protease inhibitors, 30 μg protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with anti-cleaved caspase-3, anti-cleaved PARP, and anti-β-actin antibodies. HRP-conjugated secondary antibodies and ECL reagent were used for detection [2]

Dissolved in 2% carboxymethyl cellulose in 0.9% saline; 250 mg/kg; oral administration
Nude mice bearing midgut carcinoid (GOT1), pancreatic carcinoid (BON) and medullary thyroid cancer (GOT2) tumors

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Nude Mouse A2780 Xenograft Model: Female BALB/c nu/nu mice (6-8 weeks old, 18-22 g) were housed under SPF conditions (22±2°C, 12-hour light/dark cycle, free access to food/water). Mice were subcutaneously injected with 5×10⁶ A2780 cells (suspended in 100 μL PBS + 50 μL Matrigel) into the right flank. When tumors reached 100 mm³, mice were randomized into 3 groups (n=6/group):
1. Vehicle group: Intraperitoneal injection of 5% DMSO + 5% Tween 80 + sterile saline (10 mL/kg) once daily for 21 days;
2. 10 mg/kg GMX1778 group: Intraperitoneal injection of GMX1778 (10 mg/kg, dissolved in vehicle) once daily for 21 days;
3. 20 mg/kg GMX1778 group: Intraperitoneal injection of GMX1778 (20 mg/kg, dissolved in vehicle) once daily for 21 days.
Tumor volume was measured every 3 days (volume = length × width² / 2). On day 21, mice were euthanized with CO₂, tumors were harvested for NAD⁺ and ATP detection, and major organs (liver, kidney) were collected for histopathology [1]
- Nude Mouse SKOV3 Xenograft Model: The protocol was identical to the A2780 model, except 5×10⁶ SKOV3 cells were used, and mice were treated with GMX1778 (20 mg/kg/day, intraperitoneal) for 21 days. Tumor volume and weight were measured, and no significant inhibition was observed [1]

In vitro acute toxicity: No significant cytotoxicity was observed in normal human fibroblasts (NHF) after treatment with GMX1778 (0.1-100 nM) for 72 hours—cell viability remained above 90% (SRB method), indicating its selectivity for tumor cells [1]. In vivo acute toxicity: No abnormal behavior (e.g., lethargy, diarrhea), weight loss (less than 5% of baseline), or changes in serum ALT/AST/BUN/creatinine levels were observed after treatment of nude mice with GMX1778 (10-20 mg/kg/day, intraperitoneal injection) for 21 days. Histopathological examination of the liver, kidneys, spleen, and lungs revealed no tissue damage [1].

[1]. The small molecule GMX1778 is a potent inhibitor of NAD+ biosynthesis: strategy for enhanced therapy in nicotinic acid phosphoribosyltransferase 1-deficient tumors. Mol Cell Biol. 2009 Nov;29(21):5872-88.

[2]. Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) activity by small molecule GMX1778 regulates reactive oxygen species (ROS)-mediated cytotoxicity in a p53- and nicotinic acid phosphoribosyltransferase1 (NAPRT1)-dependent manner. J Biol Chem. 2012 Jun 22;287(26):22408-17.

2-[6-(4-chlorophenoxy)hexyl]-1-cyano-3-pyridin-4-ylguanidine is an aromatic ether. CHS-828 has been used in clinical trials investigating the treatment of undefined adult solid tumors under specific regimens. Pyridylcyanoguanidine CHS 828 is a pyridylcyanoguanidine whose antitumor activity mechanism is not yet clear. (NCI) Mechanism of action: GMX1778 (CHS828) inhibits NAMPT, blocking the NAD⁺ salvage pathway, leading to intracellular NAD⁺ depletion. Decreased NAD⁺ levels impair ATP production (through mitochondrial respiration) and activate apoptosis pathways (through PARP activation and caspase cleavage). Its efficacy depends on NAPRT1 deficiency (because NAPRT1 positive cells can bypass NAMPT inhibition by using the de novo synthesized NAD⁺ pathway)[1]
- p53-dependent ROS amplification: Reference [2] adds that GMX1778-induced NAD⁺ depletion increases ROS levels, and p53 enhances this effect by upregulating pro-oxidative genes (e.g., NOX2) and downregulating antioxidant genes (e.g., SOD2). This ROS amplification further promotes cytotoxicity in p53 wild-type, NAPRT1-deficient tumors[2]
- Therapeutic potential: GMX1778 is a preclinical candidate for NAPRT1-deficient tumors (e.g., ovarian cancer, colorectal cancer) because these tumors depend on the NAMPT-mediated NAD⁺ rescue pathway. This drug has not yet undergone clinical trials and has not been approved by the FDA [1,2]
- Resistance Mechanism: NAPRT1 expression is the main resistance mechanism to GMX1778 because it enables cells to synthesize NAD⁺ from nicotinic acid via a de novo synthesis pathway. Combining GMX1778 with a NAPRT1 inhibitor may overcome this resistance [1,2]

C19H22CLN5O

371.86

371.151

200484-11-3

1160589-73-0 (nicotinate);200484-11-3 (free);

148198

White to off-white solid powder

1.2±0.1 g/cm3

526.9±58.0 °C at 760 mmHg

272.4±32.3 °C

0.0±1.4 mmHg at 25°C

1.587

4.95

2

4

11

26

450

0

BOIPLTNGIAPDBY-UHFFFAOYSA-N

InChI=1S/C19H22ClN5O/c20-16-5-7-18(8-6-16)26-14-4-2-1-3-11-23-19(24-15-21)25-17-9-12-22-13-10-17/h5-10,12-13H,1-4,11,14H2,(H2,22,23,24,25)

2-[6-(4-chlorophenoxy)hexyl]-1-cyano-3-pyridin-4-ylguanidine

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.72 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.)