IMPDH/inosine 5'-monophosphate dehydrogenase

Although the phenyl series provided many highly potent analogues, substituted pyridyl acridones demonstrated optimal in vivo efficacy with improved pharmacokinetic and in vitro profiling properties. Based on this analysis, analogue 4m/BMS-566419 was selected as our lead candidate. Steady-state enzyme kinetic studies determined that 4m/BMS-566419 was a reversible and uncompetitive inhibitor of IMPDH II with a Ki of 25 ± 3 nM with respect to IMP and 20 ± 4 nM with respect to NAD. A partial list of profiling data is presented in Table 4. [1]

Acridone 4m/BMS-566419 was evaluated in the fully preventative Lewis rat adjuvant arthritis (AA rat) model, a widely utilized preclinical model of human rheumatoid arthritis,to establish the effect of compound treatment on paw swelling post-inoculation with complete Freund's adjuvant. The rat was chosen because of its sensitivity to the immunosuppressive effects of IMPDH inhibitors and its predisposition to the analogous mechanism-based toxicities such as anemia and GI toxicity observed clinically with MMF.
Arthritis was induced by the subcutaneous injection of Freund's complete adjuvant into the base of the tail of male Lewis rats. Acridone 4m/BMS-566419 was administered at doses of 50 and 25 mg/kg PO/QD in the first experiment, along with MMF at 15 mg/kg (Figure 3A), and doses of 10 and 5 mg/kg PO/QD in a second experiment (Figure 3B) daily for 21 days starting at day 0. Baseline measurements of hind paw volume were obtained by plethysmometry. Additional paw volume measurements were performed over the next three weeks, and the increases in paw volume above baseline were calculated. Significant inhibition of paw swelling was seen at doses of 10, 25, and 50 mg/kg PO/QD (p < 0.05 vs water vehicle, Student's t test, Figures 3). Additionally, the rats showed no signs of GI toxicity at any dose by either gross examination or histopathology. MMF demonstrated significant inhibition at 15 mg/kg (Figure 3A) and only modest (∼30%) but not statistically significant inhibition of paw swelling at a dose of 10 mg/kg PO/QD (data not shown), also showing no signs of GI toxicity at these doses. This study clearly demonstrated the efficacy of this novel, orally active acridone inhibitor (4m)/BMS-566419 in the rat adjuvant arthritis model.[1]

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Chronic allograft nephropathy (CAN) is a major cause of late allograft loss. One morphological characteristic of CAN is renal interstitial fibrosis. Mycophenolate mofetil (MMF), the inosine 5'-monophosphate dehydrogenase (IMPDH) inhibitor, has been reported to attenuate the progression of renal interstitial fibrosis. However, the question of whether the newly synthesized IMPDH inhibitors with structures different from MMF have an antifibrotic effect remains unanswered. We evaluated the antifibrotic effects of BMS-566419, a chemically synthesized IMPDH inhibitor, using an experimental rat model, unilateral ureteral obstruction (UUO), in comparison with those of MMF. Expression levels of monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-beta1 (TGF-β1), which play important roles in UUO-induced renal fibrosis, were also investigated to determine the mechanism by which BMS-566419 affects the progression of renal fibrosis. After 14 days of UUO, interstitial fibrosis was frequently observed in the renal cortex of rats administered vehicle control. BMS-566419 by oral administration showed a significant and dose-dependent suppressive effect on UUO-induced renal fibrosis in histopathological experiments. BMS-566419 treatment also decreased collagen content, as indicated by hydroxyproline concentration, and the expression of collagen type 1 mRNA. BMS-566419 also decreased the expression of mRNA for both MCP-1 and TGF-β1. The antifibrotic effects of treatment with BMS-566419 at 60 mg/kg seemed comparable to those with MMF at 40 mg/kg. These results suggest that BMS-566419 and other chemically synthesized IMPDH inhibitors have beneficial pharmacological effects similar to those of MMF, and are potential pharmaceutical candidates in the treatment of fibrotic renal disease, including CAN [2].

IMPDH Type I and II Enzymatic Activity. [1]
The enzymatic activity of human IMPDH type I or II was measured using a procedure similar to that previously reported.17 The conversion of NAD+ to NADH was followed spectrophotometrically at 340 nm. A reaction mixture containing 0.1 M Tris, 0.1 M KCl, 3 mM EDTA, pH 8.0, 400 μM IMP, 2 mM DTT, and 40 nM of either IMPDH I or II was added to the wells of flat bottom UV-transparent 96-well plates. To test inhibitors, 4m/BMS-566419 dissolved in DMSO was diluted in the reaction to give a final DMSO concentration of 2.5%. IMPDH I and II used in these assays was purified from E. coli expressing the gene for the human Type I or Type II enzyme, respectively. The reaction was initiated by addition of NAD+ to a final concentration of 400 μM. After a 2-hour incubation at 25 °C, readings were taken at 340 nM. The concentrations of compound required to inhibit NADH accumulation by 50% (IC50) were calculated using a four-parameter logistic plot.

Human T-Lymphoblast (CEM) Proliferation Inhibition Assay. [1]
The human T-lymphoblast CEM cell line (ATCC) was cultured in RPMI 1640 containing 10% heat inactivated FBS and 100 units/mL of penicillin and streptomycin. Cells were seeded in 96-well Costar flat bottom tissue culture plates at a concentration of 3000 cells/well in the presence of 0.5% DMSO. Test compounds were added in triplicate at a final concentration of 10 μM with 3-fold serial dilutions. Cell cultures were maintained in a 5% CO2 humidified atmosphere for 72 h. Cell viability was measured after a final 5 h incubation with 10% (v/v) Alamar Blue dye. The fluorometric conversion of Alamar Blue was read on a Cytoflour II multi-well plate reader with excitation/emission settings of 530/590 nm, respectively. The IC50 values were calculated using a four-parameter logistic plot.

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Human Peripheral Blood Mononuclear Cells Proliferation Inhibition Assay. [1]
Human PBMCs were isolated from heparinized whole blood by density gradient centrifugation according to ICN/Cappel LSM specifications. PBMCs were maintained in RPMI 1640 medium containing 10% heat inactivated FBS and 100 units/mL of penicillin and streptomycin. Directly following whole blood isolation, 5 × 104 cells/well were plated into 96-well tissue culture plates and stimulated with anti-CD3 mAb OKT3 at 200 ng/mL and 1 μg/mL soluble anti-CD28 antibody. Test compounds were added in triplicate at a final starting concentration of 3.3 μM with 3-fold serial dilutions. All culture wells included 0.5% DMSO. Cell cultures were maintained in a 5% CO2 humidified atmosphere for 72 h followed by the addition of 1 μCi 3H-thymidine/well. Radiolabeled thymidine [3H] incorporation was determined after a 5 h incubation. The concentrations of compound required to inhibit PBMC proliferation by 50% (IC50) were calculated using a four-parameter logistic plot.

UUO model [2]
7-week-old male Sprague Dawley rats were intraperitoneally anesthetized with phenobarbital (40 mg/kg). The left ureter was ligated with 5-0 silk at two points and cut between the ligatures. Animals whose ureter was exposed but not ligated were regarded as the sham control. MMF and BMS-566419 were used. MMF (20 or 40 mg/kg) suspended in 0.5% methylcellulose water or BMS-566419 (30 or 60 mg/kg) dissolved in 0.1 N HCl was administered orally once daily for 14 consecutive days beginning on the day of surgery. On 14 day after surgery the rats were exsanguinated and killed under pentobarbital anesthesia (40 mg/kg), and the left kidneys were harvested for the following studies. The effect of drugs on renal fibrosis was evaluated on day 14 after UUO treatment, on the basis that histopathological and biochemical changes induced by UUO were small at least on day 7 and significantly increased thereafter.

In pharmacokinetic (PK) studies (Table 5), 4m/BMS-566419 showed a bioavailability of 43% and a half-life of 3.6 hours in rats, and a bioavailability of 88% and a half-life of 4.0 hours in cynomolgus monkeys. These studies suggest that 4m/BMS-566419 is metabolically stable and, due to the absence of phenolic residues, is not expected to undergo enterohepatic circulation like MMF. [1]

In a Lewis rat AA model, the gastrointestinal tolerability of acridinium BMS-566419/4m and MMF was evaluated. To determine the maximum tolerated dose for gastrointestinal toxicity, rats were administered escalating doses of MMF or 4m for up to 14 days. Diarrhea and weight loss were then monitored in the rats. Rats receiving 50 mg/kg MMF or 125 mg/kg BMS-566419/4m daily survived the two-week study with only mild, transient weight loss observed, and occasional soft stools in some rats. Higher doses of MMF and 4m/BMS-566419 (e.g., 75 mg/kg and 150 mg/kg, respectively) caused moderate to severe diarrhea, confirmed histopathologically (crypt cell necrosis), leading to morbidity. Based on these findings, MMF 50 mg/kg and 4m 125 mg/kg were selected as the maximum tolerated doses for gastrointestinal toxicity. Based on the exposure values (AUC) in these rat studies, the gastrointestinal toxicity therapeutic index (TI) for 4m and MMF was determined. Based on the AUC at the lowest effective dose and the highest tolerated dose, the gastrointestinal toxicity TI of 4m in the AA rat model was determined to be 19-fold. In contrast, the TI of MMF was 6.9-fold. [1]

[1]. Acridone-based inhibitors of inosine 5'-monophosphate dehydrogenase: discovery and SAR leading to the identification of N-(2-(6-(4-ethylpiperazin-1-yl)pyridin-3-yl)propan-2-yl)-2- fluoro-9-oxo-9,10-dihydroacridine-3-carboxamide (BMS-566419). J Med Chem. 2007;50(15):3730-3742.
[2]. Effect of the inosine 5'-monophosphate dehydrogenase inhibitor BMS-566419 on renal fibrosis in unilateral ureteral obstruction in rats. Int Immunopharmacol. 2010 Nov;10(11):1434-9.

Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme in the de novo synthesis of guanosine nucleotides, catalyzing the irreversible oxidation of inosine-5'-monophosphate to xanthine nucleotide-5'-monophosphate, a process dependent on nicotinamide adenine dinucleotide (NAD+). Mycophenolate mofetil (MMF) is a prodrug of mycophenolic acid, and its mechanism of action is based on the inhibition of IMPDH, thus it is clinically used to treat transplant rejection. The overall clinical efficacy of MMF is limited by its generally accepted dose-limiting gastrointestinal (GI) toxicity, which is associated with its specific pharmacokinetic characteristics. Therefore, developing an IMPDH inhibitor with a novel structure and different pharmacokinetic characteristics may reduce the incidence of gastrointestinal toxicity and improve its efficacy. This article will detail the discovery and structure-activity relationship study of the novel, highly effective acridinium IMPDH inhibitor BMS-566419/4m, and explore its efficacy and gastrointestinal tolerability after oral administration in an adjuvant-induced arthritis rat model. [1]

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In summary, we have identified a series of novel acridinone IMPDH II inhibitors. Structure-activity relationship studies yielded several highly effective analogs that not only significantly inhibited IMPDH activity but also suppressed the proliferation of CEM lymphoblasts and human peripheral blood mononuclear cells (PBMCs). Based on their in vitro, in vivo, pharmacokinetic, and safety profiles, we selected 4m/BMS-566419 as a lead candidate. Compound 4m/BMS-566419 demonstrated efficacy at a dose of 10 mg/kg in a rat adjuvanted arthritis model (a preclinical model of human rheumatoid arthritis). The in vitro and in vivo pharmacokinetic and metabolic profiles of this compound differ from those of MMF; therefore, 4m/BMS-566419 was evaluated in rats to determine the relationship between its efficacy and toxicity in vivo. The results showed that, compared with MMF, 4m/BMS-566419 had a therapeutic index that was about 3 times higher in terms of gastrointestinal toxicity. Overall, preclinical data suggest that acridinone 4m may have better safety in humans in terms of gastrointestinal adverse reactions compared with mycophenolate mofetil (MMF). [1]

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In summary, this study showed that BMS-566419 had the same antifibrotic effect on UUO-treated kidneys as MMF, with a decrease in MCP-1 and TGF-β1 expression. These findings suggest that newly synthesized IMPDH inhibitors (such as BMS-566419) are potent and potentially effective new drugs for the treatment of acute rejection of CAN and renal fibrosis in the transplant field. [2]

C28H30FN5O2

487.57

487.238

C, 68.97; H, 6.20; F, 3.90; N, 14.36; O, 6.56

566161-24-8

9913339

Light yellow to yellow solid powder

4.6

2

7

5

36

801

0

FC1=CC2C(C3C=CC=CC=3NC=2C=C1C(NC(C)(C)C1=CN=C(C=C1)N1CCN(CC)CC1)=O)=O

XEVJUIZOZCFECP-UHFFFAOYSA-N

InChI=1S/C28H30FN5O2/c1-4-33-11-13-34(14-12-33)25-10-9-18(17-30-25)28(2,3)32-27(36)20-16-24-21(15-22(20)29)26(35)19-7-5-6-8-23(19)31-24/h5-10,15-17H,4,11-14H2,1-3H3,(H,31,35)(H,32,36)

N-[2-[6-(4-ethylpiperazin-1-yl)pyridin-3-yl]propan-2-yl]-2-fluoro-9-oxo-10H-acridine-3-carboxamide

BMS566419; BMS-566419; BMS-566,419; 566161-24-8; BMS 566,419; UNII-9688E11ZQ0; 9688E11ZQ0; N-(1-(6-(4-Ethyl-1-piperazinyl)-3-pyridinyl)-1-methylethyl)-2-fluoro-9,10-dihydro-9-oxo-3-acridinecarboxamide; 3-Acridinecarboxamide, N-(1-(6-(4-ethyl-1-piperazinyl)-3-pyridinyl)-1-methylethyl)-2-fluoro-9,10-dihydro-9-oxo-; N-(2-(6-(4-ethylpiperazin-1-yl)pyridin-3-yl)propan-2-yl)-2-fluoro-9-oxo-9,10-dihydroacridine-3-carboxamide; BMS 566419

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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