PDE1 INHIBITOR

Abstract

The present invention provides a compound of Formula I:

##STR00001##

useful for treating chronic kidney disease and diabetic kidney disease.

Claims

1. A compound of the formula: ##STR00022##

2. A method of treating chronic kidney disease in a patient, comprising administering to a patient in need thereof an effective amount of a compound of claim 1.

3. A method of treating diabetic kidney disease in a patient, comprising administering to a patient in need thereof an effective amount of a compound of claim 1.

4. A pharmaceutical composition, comprising a compound according to claim 1, with one or more pharmaceutically acceptable carriers, diluents, or excipients.

5. A process for preparing a pharmaceutical composition, comprising admixing a compound according to claim 1, with one or more pharmaceutically acceptable carriers, diluents, or excipients.

Description

EXAMPLE 1

1-(1-cyclopropylcyclopropyl)-5-(cyclopropylmethyl)-6,7,8,9-tetrahydro-[1,2,4]triazolo[4,3-a]quinoxalin-4-one

[0067] ##STR00020##

[0068] Scheme 1, step I: Add LHMDS (1.6 g, 1.8 mmol) to a solution of 1-(1-cyclopropylcyclopropyl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]quinoxalin-4-one (164.2 mg, 607.4 μmol) in DMF (5 mL). After stirring the mixture for 1 hour at ambient temperature, add KI (10 mg, 60.7 μmol) and bromomethylcyclopropane (246.0 mg, 1.8 mmol) and stir the mixture at ambient temperature for 2 days. Dilute the mixture with EtOAc and wash with saturated aqueous NaCl. Dry over Na.sub.2SO.sub.4, filter, and concentrate under reduced pressure to give an oil. Purify by flash chromatography on silica, eluting with EtOAc (100%), to give the title compound (108.2 mg, 55%) as a tan solid. LC-ES/MS (m/z): 325.2 (M+H).

Alternative Procedure for Example 1

1-(1-cyclopropylcyclopropyl)-5-(cyclopropylmethyl)-6,7,8,9-tetrahydro-[1,2,4]triazolo[4,3-a]quinoxalin-4-one

[0069] ##STR00021##

[0070] Scheme 2, step H: Combine 1-cyclopropyl-N′-[4-(cyclopropylmethyl)-3-oxo-5,6,7,8-tetrahydroquinoxalin-2-yl]cyclopropanecarbohydrazide (35.7 g, 0.1 mol), HMDS (357 mL), and DBU (2.9 g, 0.02 mol) and heat the mixture at 125° C. for 16 hr. Cool the mixture to ambient temperature and pour into H.sub.2O (260 mL). Collect the precipitate by vacuum filtration and dissolve the solid in DCM (200 mL). Wash the organic solution with saturated aqueous NaCl, separate the layers, dry the organic layer over Na.sub.2SO.sub.4, and concentrate under reduced pressure to give a solid. Triturate the solid with ACN (2 mL/g) and collect by vacuum filtration. Dissolve the collected solid in EtOAc and concentrate under reduced pressure to give the title compound (20 g, 59%) as a pale yellow solid. LC-ES/MS (m/z): 325.2 (M+H).

Generation of PDE Proteins

[0071] The nucleotide sequences encoding full-length human PDE1A (NP_001003683.1), PDE1C (NP_005011.1), PDE5A (NP_001074.2), PDE7B (NP_061818.1) and PDE9A (NP_002597.1) are inserted into pFastBac1 (Invitrogen) vector with an N-terminal HIS tag. The nucleotide sequences encoding full-length human PDE4D (NP_006194.2) and catalytic domain (residue 641-1141) of PDE3A (NP_000912.3) are inserted into pFastBac1 (Invitrogen) vector with a C-terminal HIS tag. The nucleotide sequences encoding full-length human PDE8A (NP_002596.1) and PDE11A (AAI12394.1) are inserted into pFastBac1 (Invitrogen) vector with an N-terminal Flag tag. The nucleotide sequences encoding full-length human PDE10A (AAD32595.1) are inserted into pFastBac1 (Invitrogen) vector with a C-terminal Flag-His tag. The nucleotide sequences encoding full-length human PDE6A (NP_000431.2) and PDE6B (AAH00249.1) are inserted into pFastBacDua1 (Invitrogen) vector with an N-terminal HIS tag and N-terminal Flag tag, respectively, for production of PDE6A/6B dimer. Baculovirus generation and protein expression in Sf9 cells are carried out according to the protocol of Bac-to-Bac Baculovirus Expression system (Invitrogen). The nucleotide sequences encoding full-length human PDE1B (NP_000915.1) and PDE2A (NP_002590.1) are inserted into pIEX4 (Novagen) with a C-terminal HIS tag, and both protein productions in Sf9 cells are carried out according to the vendor's protocol (Novagen). The His tagged PDE proteins are purified using Ni-NTA agarose (Qiagen) followed by size exclusion chromatography on a SUPERDEX® 200 column (GE Healthcare) in storage buffer (20 mM Tris-HCl, pH7.5, 150 mM NaCl, 10% Glycerol). The Flag tagged PDE proteins including PDE6A/6B are purified using anti-Flag M2-agarose (Sigma), after purification through NiNTA column chromatography and eluted in storage buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 10% Glycerol, 0.1 mg/ml Flag peptide). All purified proteins are stored at −80° C. in small aliquots.

Phosphodiesterase Enzyme Assays

[0072] All 3′, 5′ cyclic nucleotide phosphodiesterase (PDE) enzyme activities are measured with a radiometric enzyme assay based on SPA detection system (scintillation proximity assay). Compounds to be tested are diluted in pure dimethyl sulfoxide (DMSO) using ten point concentration response curves. Maximal compound concentration in the reaction mixture is either 10 or 100 μM. Compounds at the appropriate concentration are pre-incubated with either of the PDE enzymes for 30 minutes before the reaction is started by the addition of substrate. Reactions are allowed to proceed for 60 minutes at room temperature. Next, reactions are stopped by addition of SPA beads. Samples are read 12 hours later in a MICROBETA™ TRILUX® Counter. “IC.sub.50” refers to the concentration of the compound that produces 50% of the maximal inhibitory response possible for that compound. IC.sub.50 values are calculated by plotting the normalized data vs. log [compound] and fitting the data using a four parameter logistic equation.

Ca.SUP.2+.-Calmodulin Dependent PDE Enzyme Assays

[0073] PDE1B, PDE1A, and PDE1C are cloned and purified in house following standard protein generation procedures. The assay buffer is prepared to give a final concentration in the assay of 50 mM Tris-HCl, 50 mM MgCl.sub.2, 4 mM CaCl.sub.2, 0.1% Bovine serum albumin and 6 U/ml Calmodulin in water, at pH 7.5. The final enzyme concentration is 0.25, 0.074 and 0.0012 nM, for PDE1A, PDE1B and PDE1C respectively. The reactions are started by addition of the substrate, [.sup.3H]cAMP, to give a final concentration of 47 nM.

TABLE-US-00001 TABLE 1 In vitro potency of Example 1 against PDE1A, PDE1B, and PDE1C. PDE enzymes IC.sub.50 (nM) of Example 1 PDE 1A 3.41 PDE 1B 4.91 PDE 1C 3.05

[0074] The data in Table 1 demonstrate that the compound of Example 1 inhibits PDE1A, PDE1B, and PDE1C enzyme activity in vitro.

PDE Enzyme Assays Using [.SUP.3.H]cAMP as Substrate

[0075] The following phosphodiesterase activities are measured using [.sup.3H]cAMP as reaction substrate: PDE3A (catalytic domain), PDE4D, PDE7B and PDE8A. All these enzymes are cloned and purified in house following standard procedures. The assay buffer is prepared to give a final concentration in the assay of 50 mM Tris-HCl, 8.3 mM MgCl.sub.2, 1.7 mM ethylenediaminetetraacetic acid (EDTA) and 0.1% Bovine serum albumin at pH 7.5. Final enzyme concentrations are 0.008, 0.021, 0.5 and 0.06 nM for PDE3A, PDE4D, PDE7B and PDE5A respectively. Reactions are also started by addition of the substrate, [.sup.3H]cAMP, to give a final concentration of 47 nM.

TABLE-US-00002 TABLE 2 In vitro potency of Example 1 against PDE3A (catalytic domain), PDE4D, PDE7B and PDE8A. PDE enzymes IC.sub.50 (nM) of Example 1 PDE3A >100000 PDE4D 7060 PDE7B 2180 PDE8A >10000

PDE Enzyme Assays Using [.SUP.3.H]cGMP as Substrate

[0076] The following phosphodiesterase activities are measured using [.sup.3H]cGMP as reaction substrate: PDE2A, PDE5A, PDE6A/6B, PDE9A, PDE10A and PDE11A. The catalytic active form of PDE6 is a dimer composed of a α (PDE6A) and β subunits (PDE6B). The dimer of PDE6A/6B is produced by the expression and purification strategy, using two purification steps, i.e., NiNTA and anti-FLAG Sepharose chromatography. The rest of the enzymes are cloned and purified in house following standard procedures. The assay buffer is prepared to give a final concentration in the assay of 50 mM Tris-HCl, 8.3 mM MgCl.sub.2, 1.7 mM EDTA and 0.1% Bovine serum albumin at pH 7.5. Final enzyme concentrations are 0.2, 0.002, 5, 1, 0.03 and 0.03 nM for PDE2A, PDE5A, PDE6AB, PDE9A, PDE10A and PDE11A, respectively. The reactions are started by addition of the substrate, [.sup.3H]cGMP, to give a final concentration of 80 nM in the case of PDE2A, PDE10A, PDE5A, PDE6AB and PDE11A assays, whereas for PDE9A 20 nM of [.sup.3H]cGMP is used.

TABLE-US-00003 TABLE 3 In vitro potency of Example 1 against PDE2A, PDE5A, PDE6AB, PDE9A, PDE10A and PDE11A. PDE enzymes IC.sub.50 (nM) of Example 1 PDE2A >10000 PDE5A >10000 PDE 6AB >10000 PDE9A >10000 PDE10A >10000 PDE11A 2970

[0077] The data in Tables 2 and 3 demonstrate that the compound of Example 1 is a selective inhibitor of PDE1A, PDE1B, and PDE1C relative to PDE2A, PDE3A, PDE4D, PDE5A, PDE6AB, PDE7B, PDE8A, PDE9A, PDE10A, and PDE11A in vitro.