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 pharmocokenetic (PK) studies (Table 5), 4m/BMS-566419 had a bioavailability of 43% in rats with a half-life of 3.6 h and a bioavailability of 88% in cynologous monkey with a half-life of 4.0 h. Through these studies, 4m/BMS-566419 was found to be metabolically stable, and in the absence of a phenolic residue, 4m/BMS-566419 is not expected to exhibit the corresponding enterohepatic recirculation associated with MMF.[1]

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GI tolerability was assessed for both acridone BMS-566419/4m and MMF in Lewis rats in the context of the rat AA model. To determine the maximum tolerated dose with respect to GI toxicity, the rats were given escalating doses of either MMF or 4m for up to 14 days. The rats were then monitored for the development of diarrhea and weight loss. Rats receiving either a daily dose of 50 mg/kg of MMF or 125 mg/kg of BMS-566419/4m survived the two week study with only mild transient weight loss and occasional soft stools observed for some rats. Higher doses of MMF and 4m/BMS-566419 (e.g., 75 mg/kg and 150 mg/kg, respectively) resulted in moderate to severe diarrhea, confirmed by histopathology (crypt cell necrosis), leading to morbidity. As a result of these findings, doses of 50 mg/kg for MMF and 125 mg/kg for 4m were selected as the maximum tolerated doses for GI toxicity. The therapeutic index (TI) of 4m and MMF for GI toxicity was established based on exposure (AUC) in these rat studies. Based on the AUCs for 4m at the lowest efficacious and maximally tolerated doses, the TI for GI toxicity in the rat AA model was determined to be 19-fold. Comparatively, the TI for MMF was determined to be 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), a key enzyme in the de novo synthesis of guanosine nucleotides, catalyzes the irreversible nicotinamide-adenine dinucleotide dependent oxidation of inosine-5'-monophosphate to xanthosine-5'-monophosphate. Mycophenolate Mofetil (MMF), a prodrug of mycophenolic acid, has clinical utility for the treatment of transplant rejection based on its inhibition of IMPDH. The overall clinical benefit of MMF is limited by what is generally believed to be compound-based, dose-limiting gastrointestinal (GI) toxicity that is related to its specific pharmacokinetic characteristics. Thus, development of an IMPDH inhibitor with a novel structure and a different pharmacokinetic profile may reduce the likelihood of GI toxicity and allow for increased efficacy. This article will detail the discovery and SAR leading to a novel and potent acridone-based IMPDH inhibitor BMS-566419/4m and its efficacy and GI tolerability when administered orally in a rat adjuvant arthritis model. [1]

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In summary, a novel series of acridone-based inhibitors of IMPDH II has been identified. Structure−activity relationship studies led to multiple highly potent analogues exhibiting significant IMPDH inhibition as well as the inhibition of the proliferation of CEM lymphoblastoid cells and human PBMCs. Based on its in vitro potency, in vivo activity, and pharmacokinetic and safety profiles, 4m/BMS-566419 was selected as our lead drug candidate. Compound 4m/BMS-566419 was efficacious at 10 mg/kg in the rat adjuvant arthritis model, a preclinical model of human rheumatoid arthritis. This compound has a different in vitro and in vivo pharmacokinetic and metabolic profile than MMF and, as a result, 4m/BMS-566419 was evaluated in rat to establish in vivo the relationship between efficacy and toxicity and was found to have an approximately 3-fold improvement over MMF in its therapeutic index with respect to GI toxicity. Overall, preclinical data suggest that acridone 4m may have an improved safety window in humans over MMF with respect to GI adverse events.[1]

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In conclusion, the present study demonstrates that the antifibrotic effects of BMS-566419 on UUO-treated kidney are identical to those of MMF, and are accompanied by decreased expression of MCP-1 and TGF-β1. These findings suggest that newly synthesized IMPDH inhibitors such as BMS-566419 are potent and possibly beneficial new drugs for the treatment of both acute rejection and renal fibrosis in CAN in the transplantation 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.)