Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives

Definitions

• the present invention relates to pyrazolo pyrimidinone based compounds as PDE5 inhibitors for the treatment of impotence.
• the pyrazolopyrimidinone based compounds have been designed, synthesized and the PDE5 inhibitory potential is provided in this invention. These designer compounds have shown nanomolar potency when screened for PDE5 inhibitory activity. These compounds are having better efficacy and P profile than the existing PDE5 inhibitors.
• the use of a cGMP PDE inhibitors and their method of treating a male mammal, including human, to cure or prevent erectile dysfunction comprising effective amount of compound of formula 1 or pharmaceutically acceptable salts thereof or a pharmaceutical composition in provided in this invention.
• the present invention also relates to the process for the preparation of pyrazolopyrimidinone based compounds.
• Pyrazolopyrimidinones are very well reported for their PDE5 (Phosphodiesterase) inhibitory activity.
• the well known drug used for erectile dysfunction is 'Sildenafil', it contains 5-(2-ethoxyphenyl)- 1 -methyl-3-propyl- 1 H-pyrazolo[4,3-d]pyrimidin-7(6H)- one as an active ingredient in Viagra.
• Phosphodiesterase is an ubiquitous enzyme which selectively catalyzes the hydrolysis of the 3 ' -cyclic phosphate bonds of adenosine and/or guanosine 3 ',5'- cyclicmonophosphate into their respective 5 '-nucleosidemonophosphates.
• cAMP (3 ',5'- cyclic adenosine monophosphate)
• cGMP (3 ',5 '-cyclic guanosine monophosphate) are second messengers within cells. These signaling molecules can be generated in two ways. They can carry signals generated by extra-cellular signaling molecules that are incapable of entering the cells.
• extracellular signaling molecules such as hormones or neurotransmitters, bind to membrane bound proteins that in turn activate the particulate forms of adenylate or guanylate cyclase.
• This activation results in the generation of cAMP or cGMP from adenosine triphosphate (ATP) and guanosine triphosphate (GTP), respectively.
• Soluble (cytosolic) forms of adenylate and guanylate cyclase also exist and can be activated by messengers within the cell. Soluble adenylate cyclase is activated by calcium signaling to generate cAMP, and soluble guanylate cyclase is activated by nitric oxide (NO) which in turn generates cGMP.
• NO nitric oxide
• Temporal and spatial regulation of cAMP and cGMP falls to the phosphodiesterases. This is done through the hydrolysis of cAMP and cGMP into their non signaling forms AMP and GMP, respectively (Chappie T. A., Helal C. J., Hou X. J. Med. Chem.im, 55, 7299-7331).
• PDE-1 Vinpocetine
• PDE-2 EHNA (erythro-9-(2-hydroxy-3- nonyl)adenine
• PDE-3 Amrinone, Anagrelide, Cilostazol
• PDE-4 Piclamilast, Tibenelast, Benafentrine and Zardaverine
• PDE-5 Sildenafil, Tadalafin, Vardenafil and Avanafil
• PDE5 inhibitors Phosphodiesterase (V) inhibitors (PDE5 inhibitors) are an important class of pharmaceutical compounds.
• sildenafil as an efficacious, orally active agent for the treatment of male erectile dysfunction (MED), (Terrett N. K., Bell A. S., Brown D., Ellis P.; Bioorg. Med. Chem. Lett. 1996, 6, 1819-1824) has created significant interest in the discovery of additional phosphodiesterase type 5 (PDE5) inhibitors.
• PDE5 is the primary cGMP-hydrolyzing enzyme present in the corpus cavernosum, the smooth muscle in the penis which helps control vascular tone. When a man is sexually stimulated, nitric oxide is released from the cavernosal nerve.
• the main object of the present invention is to provide compounds of pyrazolo pyrimidinone as PDE5 inhibitors.
• Another object of the present invention is to provide a process for the preparation of pyrazolo pyrimidinone compounds.
• Still another object of the present invention is to evaluate biological activity of pyrazolo pyrimidinone compounds as PDE5 inhibitors.
• Yet another object of the present invention is to identify isoform selectivity of these compounds for different PDE enzymes to find enzyme specificity.
• Still another object of the present invention is to evaluate in vivo biological activity in animals (rabbit or rat models) of pyrazolo pyrimidinone compounds as PDE5 inhibitors. Yet another object of the present invention is that the compounds of the present invention are useful for the treatment of the male erectile dysfunction and for the treatment of impotence.
• the present invention relates to the novel compounds of pyrazolo pyrimidinone based scaffold as PDE5 inhibitors.
• Several compounds in the series have been designed and synthesized based on pyrazolo pyrimidinone scaffold. These molecules were screened for PDE5 activity and they are found to be potential PDE5 inhibitors. Some of the compounds have shown IC 50 value in nano molar range.
• a and B represents -N, -S, -CH, -CR, -NH, -NR; wherein, R is BocHN, substituted aryl, heteroaryl, alkyl, heterocycloalkane with substitution selected from the group consisting of ketone, aryl, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl optionally having hydroxyl, amino, halo group at the terminal position of the carbon chain optionally having unsaturation on carbon chain at any position with different substitutions, wherein, Ri to R 5 are either independently selected from H, alkyl, aryl, halo, oxy, hydroxy, alkoxy, alkyl halide, alkyne ether, allyl ether, substituted alkene, amino, formyl, nitro with substitutions optionally having heteroaryl substitutions, wherein, R in general represents an independently
• Formula 1A represents general structure for cyclic ring containing compounds wherein, the cyclic ring is directly bonded to -S0 2 placed on arylring at 4 th position of -OEt substitution, wherein, the cyclic ring is selected from the group consisting of five membered, six membered and seven membered, wherein,the cyclic ring contains substitutions, wherein, A and B represents -N, -S, - CH, -CR, -NH, -NR; wherein, R is BocHN, substituted aryl, heteroaryl, alkyl, heterocycloalkane with substitution selected from the group consisting of ketone, aryl, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl optionally having hydroxyl, amino, halo group at the terminal position of the carbon chain optionally
• Ri to R 5 are either independently selected from H, alkyl, aryl, halo, oxy, hydroxy, alkoxy, alkyl halide, alkyne ether, allyl ether, substituted alkene, amino, formyl, nitro with substitutions optionally having heteroaryl substitutions, wherein, R in general represents an independently selected groups with substitutions on aryl ring selected from the group consisting of halo, alkoxy, nitro, amino, oxy, thio, carboxylic, formyl, hydroxyl, prenyl and isoprenyl,
• heteroaryl group is selected from the group consisting of pyridyl, furyl, thiphenyl, thiobenzyl, indolyl, thioindolyl, quinolyl, quinazolinyl, isoquinolyl, benzopyranyl, benzothiozolyl, benzooxazolyl, oxazolyl, triazolyl and tetrazolyl.
• the representative compounds of formula 1 A are selected from the group consisting of
• the present invention also provides a process for the preparation of the compound of claim 1 , wherein the steps comprising-
• step (iii) treating compound 2 of step (ii) with NaOH solution to obtain l-methyl-3- propyl-lH-pyrazole-5-carboxylic acid (compound 3);
• step (iv) reacting compound 3 of step (iii) with cone. H 2 S0 4 and nitric acid to obtain l-methyl-4-nitro-3-propyl-lH-pyrazole-5-carboxylic acid (compound 4);
• step (vi) treating compound 5 of step (v) with EtOH:H 2 0, Fe powder and NH CI to obtain 4-amino- 1 -methyl-3-propyl- 1 H-pyrazole-5-carboxamide (compound 6);
• step (vii) reacting compound 6 of step (vi) with a compound selected from the group consisting of substituted aryl and heterocyclic aldehydes in the presence of a solvent and CuCl 2 at a temperature ranging between 40 to 80 °C for a period ranging between 2 to 5 hr under oxygen atmosphere or under ordinary conditions to obtain compound of formula IB;
• step (ix) treating compound 7 of step (viii) with chlorosulphonic acid to obtain 4- ethoxy-3-(6,7-dihydro-l-methyl-7-oxo-3 -propyl- lH-pyrazolo [4,3- d]pyrimidin-5-yl)benzene-l-sulfonyl chloride (compound 8);
• step (x) reacting compound 8 of step (ix) with an amino compound selected from the group consisting of cyclic, acyclic, aliphatic, aromatic amino in the presence of a base in a solvent at a temperature ranging between 10 to 35 °C for a period ranging between 45 min to 4 hrs, adding cold water to quench the reaction and obtaining the compound of formula 1 A
• Formula 1A In an embodiment of the present invention there is provided a process for the preparation of pyrazolo-pyrimidinone compound, wherein the solvent is selected from the group consisting of DMF, DCM, CHC1 3 , DCE, dioxane, acetonitrile, acetone and ethanol.
• the solvent is selected from the group consisting of DMF, DCM, CHC1 3 , DCE, dioxane, acetonitrile, acetone and ethanol.
• a pharmaceutical composition comprising an effective amount of the compound of formula 1 , optionally along with the pharmaceutically acceptable salt, excipient, diluent, and carrier.
• composition wherein pharmaceutically acceptable carrier containing aqueous solution is selected from the group consisting of water, buffered saline, glycol, glycerol, olive oil and liposome.
• a pharmaceutical composition wherein the dose of compound of formula 1 is ranging between 0.1 mg/kg to 100 mg/kg.
• a method of treating a subject in a need to cure or prevent erectile dysfunction comprising, administering the effective amount of compound of formula 1 or pharmaceutically acceptable salts thereof or a pharmaceutical composition containing either entity.
• a method of inhibiting PDE5 enzyme wherein the IC 50 value for compound of claim 1 ranges from 0.3 nM to 100 ⁇ , wherein the in vivo activity of said compound is with the AUC range from 100 to 1000 at the dose of 3 mg/kg and it may vary with the dose pattern.
• Fig. 1 Results of structural binding (in silico) studies of a representative compound viz. A) Sildenafil; (B) Compound Aa with PDE5 enzyme.
• Fig. 2 Graph showing IC 50 value of (a) Sildenafil and (b) most potent PDE5 inhibitor i.e. 'Compound Aa' of the present invention.
• Fig. 3 In vivo study result for a representative compound Aa and comparative graph with reference molecule Sildenafil (Length of uncovered penile mucosa in rabbits was measured after intravenous administration of compound Aa (3 mg/kg) and Sildenafil (3mg/kg) in a group of 5 animals each followed 5mins later by an injection of Sodium nitroprusside (0.2mg/kg). SNP alone was also used as a control and the values obtained were subtracted from both the molecules).
• Fig. 4 Scheme for synthesis of compound Aa.
• Fig. 5 Scheme for synthesis of compound of formula 1 A.
• Fig. 6 Scheme for synthesis of compound of formula IB.
• the present invention relates to the pyrazolopyrimidinone scaffold that are well reported for their PDE5 (Phosphodiesterase) inhibitory activity.
• Sildenafil is the well known drug used for erectile dysfunction and it contains 5-(2-ethoxyphenyl)-l-methyl- 3-propyl- 1 H-pyrazolo[4,3-d]pyrimidin-7(6H)-one as a active ingredient in Viagra.
• This invention is based on the design, synthesis and biological evaluation of pyrazolopyrimidinone compounds and their pharmaceutical salts as PDE5 inhibitors and their use in the treatment of male erectile dysfunction and for the treatment of impotence.
• ⁇ ' is selected from
• A, B, and R ⁇ to R 5 are independently selected from the groups described below; wherein, A and B represents -N, -S, -CH, -CR, -NH, -NR; wherein, R is BocHN, substituted aryl, heteroaryl, alkyl, heterocycloalkane with substitution selected from the group consisting of ketone, aryl, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl optionally having hydroxyl, amino, halo group at the terminal position of the carbon chain optionally having unsaturation on carbon chain at any position with different substitutions, wherein, R ⁇ to R 5 are either independently selected from H, alkyl, aryl, halo, oxy, hydroxy, alkoxy, alkyl halide, alkyne ether, allyl ether, substituted alkene, amino, formyl
• Formula 1 comprises of two representative structures that independently represents different compounds based on pyrazolopyrimidinone scaffold, wherein, A and B represents -N, -S, -CH, -CR, -NH, - NR; wherein, R is BocHN, substituted aryl, heteroaryl, alkyl, heterocycloalkane with substitution selected from the group consisting of ketone, aryl, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl optionally having hydroxyl, amino, halo group at the terminal position of the carbon chain optionally having unsaturation on carbon chain at any position with different substitutions,
• R ⁇ to R 5 are either independently selected from H, alkyl, aryl, halo, oxy, hydroxy, alkoxy, alkyl halide, alkyne ether, allyl ether, substituted alkene, amino, formyl, nitro with substitutions optionally having heteroaryl substitutions,
• R in general represents an independently selected groups with substitutions on aryl ring selected from the group consisting of halo, alkoxy, nitro, amino, oxy, thio, carboxylic, formyl, hydroxyl, prenyl and isoprenyl,
• heteroaryl group is selected from the group consisting of pyridyl, furyl, thiphenyl, thiobenzyl, indolyl, thioindolyl, quinolyl, quinazolinyl, isoquinolyl, benzopyranyl, benzothiozolyl, benzooxazolyl, oxazolyl, triazolyl and tetrazolyl.
• all formulas (1A) in formula 1 represents 5-(2- ethoxyphenyl)- 1 -methyl-3-propyl- 1 H-pyrazolo[4,3 -d]pyrimidin-7(6H)-one as common core in the structure and formula IB represents aryl and heteroaryl pyrazolo pyrimidinone structures.
• the formula IB, having R on aryl, heteroaryl ring represents different substitutions selected from the group consisting of halo, alkyl, amino, substituted amino, nitro, carboxylates, ethers and arylethers.
• the method used for the in silico bioinformatics study of pyrazolo pyrimidinone based compounds is peformed wherein all the computational studies were carried out in the Schrodinger suite 2010 molecular modeling software. The 2D structures of all the molecules were built in the maestro window (Maestro, version 9.2, Schrodinger, LLC, New York, NY, 2011).
• Bond length and bond order correction was also carried out for preparing the protein for docking studies.
• the active site grid was generated based on the already co-crystalised ligand of the receptor using receptor grid generation module.
• the ligands were docked on to the receptor through this grid using Glide module and flexible docking was carried out for all the conformers in order to find out the binding mode of these ligands.
• the extra precision (XP) scoring function of Glide was used for carrying out these studies. (Glide, version 5.6, Schrodinger, Inc., New York, NY, 2010.; Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D.
• step (ii) reacting compound 1 of step (i) with dimethyl sulfate to obtain ethyl 1- methyl-3-propyl-lH-pyrazole-5-carboxylate (compound 2);
• step (iii) treating compound 2 of step (ii) with NaOH solution to obtain l-methyl-3- propyl-lH-pyrazole-5-carboxylic acid (compound 3);
• step (iv) reacting compound 3 of step (iii) with cone. H2SO4 and nitric acid to obtain l-methyl-4-nitro-3 -propyl- lH-pyrazole-5-carboxylic acid (compound 4);
• step (vi) treating compound 5 of step (v) with EtOH:H 2 0, Fe powder and NH 4 CI to obtain 4-amino-l-methyl-3-propyl-lH-pyrazole-5-carboxamide (compound 6);
• step (vii) reacting compound 6 of step (vi) with a compound selected from the group consisting of substituted aryl and heterocyclic aldehydes in the presence of a solvent and CuCl 2 at a temperature ranging between 40 to 80 °C for a period ranging between 2 to 5 hr under oxygen atmosphere or under ordinary conditions to obtain compound of formula IB;
• step (viii) alternatively, reacting compound 6 of step (vi) with 2-ethoxybenz- aldehyde to obtain 5-(2-ethoxyphenyl)-l-methyl-3-propyl-lH-pyrazolo[4,3- d]pyrimidin-7(6H)-one (compound 7);
• step (x) reacting compound 8 of step (ix) with an amino compound selected from the group consisting of cyclic, acyclic, aliphatic, aromatic amino in the presence of a base in a solvent at a temperature ranging between 10 to 35 °C for a period ranging between 45 min to 4 hrs, adding cold water to quench the reaction and obtaining the compound of formula 1 A
• the representative compounds of Formula 1 A are selected from the group consisting of:5-(2-ethoxy-5-((4-oxopiperidin-l-yl)sulfonyl)phenyl)-l- methyl-3-propyl-lH-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-(2-ethoxy-5-((4- hydroxypiperidin- 1 -yl)sulfonyl)phenyl)- 1 -methyl-3 -propyl- 1 H-pyrazolo[4,3 - d]pyrimidin-7(6H)-one, 5-(2-ethoxy-5-((3-oxopiperazin-l-yl)sulfonyl)phenyl)-l- methyl-3-propyl-lH-pyrazolo[4,3-d]pyrimidin-7(6H)-one, 5-(2-ethoxy-5-((4- methoxypiperidin- 1
• the representative compounds of Formula IB are selected from the group consisting of: 5-(3 -bromo-4-methoxyphenyl)- 1 -methyl-3 -propyl- 1 H-pyra zolo[4,3 -djpyrimidin- 7(6H)-one, l-methyl-5-(3-nitrophenyl)-3-propyl-lH-pyrazolo[4,3-d] pyrimidin-7(6H)- one, 5-(3-bromo-4-fluorophenyl)- 1 -methyl-3 -propyl- 1 H-pyrazolo [4,3-d]pyrimidin- 7(6H)-one, l-methyl-5-(5-nitrofuran-2-yl)-3-propyl-lH-pyrazolo[4,3-d] pyrimidin- 7(6H)-one, 5-(4-hydroxyphenyl)-l -methyl-3 -propyl- 1 H-pyrazolo[4,3-d]
• Step 1 Synthesis of ethyl 3-propyl-lH-pyrazole-5-carboxylate:- Diethyl oxalate (20 gm 13.7 m.ml) and 2-pentanone (11.8 gm 13.7 m.ml) are mixed and then the reaction mix is cooled to 2 °C and sodium methoxide (8.12 gm 15.0 m. ml) are added in four portions of each according to the following procedure.
• the first lot of sodium methoxide (4.03 gm) is added at 0 °C to the mixture maintained over an ice bath at 5-0 °C, then the temperature is allowed to reach 40 °C and when stabilized the ice bath is removed and the reaction mixture is stirred for 30 minutes and cooled again to 5 °C over an ice bath.
• the second lot of sodium methoxide (4.03 gm) is added at 5 °C to the mixture kept over an ice bath at 5 °C for 10 minutes, the temperature is allowed to reach 40 °C and when stabilized the ice bath is removed and the reaction mixture is stirred for 30 minutes and cooled again to 5 °C over an ice bath.
• the third lot of sodium methoxide (4.03 gm) is added at 5 °C to the mixture kept over an ice bath at 5 °C for 10 minutes, the temperature is allowed to reach 40 °C and when stabilized the ice bath is removed, the reaction mixture is stirred for 30 minutes and cooled again to 5 °C over an ice bath.
• the fourth lot of sodium methoxide (4.03 gm) is added at 5 °C to the mixture kept over an ice bath at 5 °C for 10 minutes, the temperature is allowed to reach 55 °C stirred for 2 hours at 55 °C and the mixture is cooled at 10 °C and 300 ml ethyl acetate is added, stirred for 20 minutes and added water to the reaction mixture.
• Step 2 Synthesis of ethyl l-methyl-3-propyl-lH-pyrazole-5-carboxylate:- Take ethyl 3- propyl- 1 H-pyrazole-5-carboxylate (20 gm 10.9 m.ml) in round bottom flask cooled to 5 °C and then added dimetyl sulfate (10.93 ml 11.5 m.ml) over a period of lh. The temperature is allowed to reach 25 °C and it is kept under stirring for 8 hours. Then it is cooled to 10 °C by adding 200 ml DCM, 100 ml water. The pH is adjusted to 7-7.3 by adding 30% NaOH solution while the temperature is kept below 15 °C.
• Step 3 Synthesis of l-methyl-3 -propyl- lH-pyrazole-5-carboxylic acid:-Ethyl 1-methyl- 3-propyl-lH-pyrazole-5-carboxylate (19 gm 9.6 mml) was suspended in 6N NaOH solution (48 ml 20.0 m.ml). The mixture was heated to 75 °C for 3 hours then cooled to 20 °C, diluted with 50 ml water and acidified with cone. HC1. Filtration gave the carboxylic acid as pale brown compound; Yield 90%.
• Step 4 Synthesis of l-methyl-4-nitro-3-propyl-lH-pyrazole-5-carboxylic acid:- Dissolve l-methyl-3 -propyl- lH-pyrazole-5-carboxylic acid (13.0 gm 7.7 m.ml) in 20 ml of conc.H 2 S0 4 and heated at 50 °C. After that nitric mixture (9 ml) was added slowly over an 1 hour after the addition mixture was stirred at 50 °C for 7 hours and then cooled to room temperature before being poured onto ice. Filteration of the precipitate gave the nitropyrazole as a white solid; Yield 88%.
• Step 5 Synthesis of l-methyl-4-nitro-3-propyl-lH-pyrazole-5-carboxamide:- 1-methyl-
• Step 6 Synthesis of 4-amino-l-methyl-3-propyl-lH-pyrazole-5-carboxamide:- 1- methyl-4-nitro-3-propyl-lH-pyrazole-5-carboxamide (11.0 gm 5.1 m.ml) was dissolved in 40 ml EtOH/H 2 0 (3:1), added Fe powder (8.7 gm 15.5 m.ml) and NH4CI (2.75gm 5.1 m.ml). The mixture was heated up to 80 °C for 4.5 hours. The reaction mixture was cooled to 25 °C and filtered through celite and filterate was concentrated under vacuum. Trituration of the residue with either gave the 4-amino- l-methyl-3 -propyl- lH-pyrazole-
• Step 7 Synthesis of 5-(2-ethoxyphenyl)-l-methyl-3-propyl-lH-pyrazolo[4,3- d]pyrimidin-7(6H)-one:- 4-amino- 1 -methyl-3-propyl- 1 H-pyrazole-5-carboxamide (7.0 gm 3.84 m.ml) and 2-ethoxybenz- aldehyde (6.0 gm 4.0 m.ml) were suspended in ethanol and the mixture was heated at 70 °C for 1.5 hours after conformation of forming of imine by TLC. Added CuCl 2 (15.4 gm 11.5 m ml) and the reaction mixture heated at 70 °C under 0 2 for 1.5 hours.
• Step 8 Synthesis of 4-ethoxy-3-(6, 7-dihydro-l-methyl-7-oxo-3 -propyl- lH-pyrazolo [4, 3-d] pyrimidin-5-yl) benzene- 1-sulfonyl chloride:- To the chlorosulphonic acid (10 eq) was added 5-(2-ethoxyphenyl)-l-methyl-3-propyl-lH-pyrazolo[4,3-d]pyrimidin-7(6H)- one (1 eq) while maintaining the temperature 0 °C. Then reaction was allowed to proceed at 5 °C until TLC analysis indicated the absence of starting material.
• Piperidin-4-one (1 eq) was dissolved in dry DCM and added DIPEA (3 eq), stirred the reaction mixture for 10 minutes at 15 °C, added 4-ethoxy-3-(l-methyl-7-oxo-3-propyl- 6,7-dihydro-lH-pyrazolo[4,3-d]pyrimidin-5-yl)benzene-l-sulfonyl chloride (1 eq) and stirred the reaction for 6 hours at 25 °C. After completion of the reaction, added 30 ml DCM, 30 ml water organic layer was separated. Water layer re-extracted with 20 ml DCM and the combined organic layer are washed with brine solution, concentrated under vacuum Yield 90%.
• step 1 All the compounds of formula 1A mentioned below are prepared by employing the similar method for step 1 to step 8 containing different substitutions at R and Rl to R5 positions, as described for the preparation of compound Aa.
• the intermediates from 1 to 8 are prepared as per the literature reported methods (Peter J. Dunn, Org. Process Res. Dev. 2005, 9, 88-97). Furthermore, the procedure for step 9 is provided for some representative examples, the similar conditions are used for all the examples.
• Example 2 Compound Ab (5-(2-ethoxy-5-((4-hydroxypiperidin-l- yl)suIfonyl)phenyl)-l-methyl-3-propyl-lH-pyrazolo[4,3-d]pyrimidin-7(6H)-one):
• Example 3 Compound Ac (5-(2-ethoxy-5-((3-oxopiperazin-l-yl)sulfonyl)phenyl)-l- methyl-3-propyl- 1 H-py razolo [4,3-d] pyrimidin-7(6H)-one) :
• Example 4 Compound Ad (5-(2-ethoxy-5-((4-methoxypiperidin-l- yl)sulfonyl)phenyl)-l-methyl-3-propyl-lH-pyrazolo[4,3-dlpyrimidin-7(6H)-one): Synthesis of Ad; Step 9: Dissolve 4-methoxypiperidine (1 eq) dry acetone and added K 2 C0 3 (3 eq) stirred the reaction mixture for 10 minutes at 20 °C, added 4-ethoxy-3-(l- methyl-7-oxo-3-propyl-6,7-dihydro-lH-pyrazolo[4,3-d]pyrimidin-5-yl)benzene-l- sulfonyl chloride (1 eq) and stirred the reaction for 5 hours at 35 °C.
• Example 5 Compound Ae (5-(2-ethoxy-5-((4-(hydroxymethyl)piperidin-l- yl)sulfonyl) phenyl)- l-methyl-3-propyl- 1 H-py r azolo [4,3-d] pyrimidin-7(6H)-one) :
• Example 6 Compound Af (5-(2-ethoxy-5-((4-(2-hydroxyethyl)piperidin-l- yl)sulfonyl) phenyl)-l-methyl-3-propyl-lH-pyrazolo[4,3-d]pyrimidin-7(6H)-one):
• Example 7 Compound Ag (5-(2-ethoxy-5-((4-(2-hydroxyphenyl)piperazin-l- yl)sulfonyl) phenyl)-l-methyl-3-propyl-lH-pyrazolo[4,3-d]pyrimidm-7(6H)-one):
• Step 9 Dissolve 2-(l-piperazinyl)phenol (1 eq) dry DCM and added Pyridine (2 eq) stirred the reaction mixture for 10 minutes at 25 °C, added 4-ethoxy-3- (l-methyl-7-oxo-3-propyl-6,7-dihydro-lH-pyrazolo[4,3-d]pyrimidin-5-yl)benzene-l- sulfonyl chloride (1 eq) and stirred the reaction for 5.5 hours at 25 °C. After completion of the reaction, added 30 ml DCM, 30 ml water organic layer was separated.
• Example 8 Compound Ah (5-(5-((4-(4-acetylphenyl)piperazm-l-yl)sulfonyl)-2- ethoxy phenyl)-l-methyl-3-propyl-lH-pyrazolo[4,3-d]pyrimidin-7(6H)-one):
• Example 10 Compound Aj (5-(2-ethoxy-5-((4-(pyridin-2-yl)piperazin-l- yl)sulfonyl) phenyl)- l-methyI-3-propyl-l H-pyr azolo [4,3-d] pyrimidin-7(6H)-one) : Synthesis of Aj; Step 9: Dissolve l-(2-pyridinyl)piperazine (1 eq) dry DMF and added CsC0 3 (3 eq), stirred the reaction mixture for 10 minutes at 15 °C, added 4-ethoxy-3- ( 1 -methyl-7-oxo-3-propyl-6,7-dihydro- 1 H-pyrazolo[4,3-d]pyrimidin-5-yl)benzene- 1 - sulfonyl chloride (1 eq) and stirred the reaction for 4 hours at 35 °C.
• Example 11 Compound Ak (5-(2-ethoxy-5-((4-(pyrimidin-2-yl)piperazin-l- yl)sulfonyl) phenyl)-l-methyl-3-propyl-lH-pyrazolo[4,3-d)pyrimidin-7(6H)-one): Synthesis of Ak; Step 9: Dissolve 2-(l-piperazinyl)pyrimidine (1 eq) dry 1,4 dioxane and added TMA (3 eq) stirred the reaction mixture for 10 minutes at 15 °C, added 4- ethoxy-3-(l -methyl-7-oxo-3-propyl-6,7-dihydro- 1 H-pyrazolo[4,3-d]pyrimidin-5- yl)benzene-l-sulfonyl chloride (1 eq) and stirred the reaction for 6 hours at 27 °C.
• Example 12 Compound Al (5-(5-([l,4 » -bipiperidin]-l'-ylsulfonyl)-2-ethoxyphenyl)- l-methyl-3-propyl-lH-pyrazolo[4,3-d]pyrimidin-7(6H)-one):
• Example 13 Compound Am (5-(5-((4-benzylpiperidin-l-yl)sulfonyl)-2- ethoxyphenyl)- 1 -methyl-3-propyl- 1 H-py razolo [4,3-d] py rimidin-7(6H)-one) :
• Step 9 Dissolve 4-methylpiperidine (1 eq) dry DCE and added DIPEA (3 eq) stirred the reaction mixture for 10 minutes at 20 °C, added 4-ethoxy-3-(l-methyl- 7-oxo-3-propyl-6,7-dihydro-lH-pyrazolo[4,3-d]pyrimidin-5-yl)benzene-l-sulfonyl chloride (1 eq) and stirred the reaction for 6 hours at 30 °C. After completion of the reaction, added 30 ml DCM, 30 ml water organic layer was separated. Water layer was re-extracted with 20 ml DCM and the combined organic layer are washed with brine solution, concentrated under vacuum. The residue was purified by column chromatography on silica the desired product An as a white solid; yield 90%.
• Example 15 Compound Ao (tert-butyl (l-((4-ethoxy-3-(l-methyl-7-oxo-3-propyl- 6,7-dihydro- 1 H-pyr azolo [4,3-d] pyrimidin-5-yl)phenyl)sulf onyl)piperidin-4- yl)carbamate):
• Example 16 Compound Ap (5-(5-((4-aminopiperidin-l-yl)sulfonyl)-2- ethoxyphenyl)-l-me thyl-3-propyl-lH-pyrazolo[4,3-d]pyrimidin-7(6H)-one): Synthesis of Ap; Step 9: Dissolve tert-butyl (l-((4-ethoxy-3-(l-methyl-7-oxo-3-propyl- 6,7-dihydro-lH-pyrazolo[4,3-d]pyrimidin-5-yl)phenyl)sulfonyl)piperidin-4- yl)carbamate (1 eq) dry DCM and added 20%TFA in DCM stirred the reaction mixture at 30 °C for 6 hours.
• Example 17 Compound Ba (5-(3-bromo-4-methoxyphenyl)-l-methyl-3-propyl-lH- pyr a zolo [4,3-d] pyrimidin-7(6H)-one) : Synthesis ofBa; Step 7: 4-amino-l-methyl-3-propyl-lH-pyrazole-5-carboxamide (1 eq) and 3-Bromo-4-methoxybenzaldehyde (1.1 eq) were suspended in ethanol 5 ml and the mixture heated at 70 °C for 2 hours after conformation of forming of imine by TLC. Added CuC12 (3 eq) and the reaction mixture heated at 75 °C under 0 2 for 3 hours.
• Example 18 Compound Bb (l-methyl-5-(3-nitrophenyl)-3-propyl-lH- pyrazolo [4,3-d] pyrimidin-7(6H)-one) :
• Example 19 Compound Be (5-(3-bromo-4-fluorophenyl)-l-methyl-3-propyI-lH- pyrazolo [4,3-d] pyrimidin-7(6H)-one) :
• Step 7 4-amino-l-methyl-3-propyl-lH-pyrazole-5-carboxamide (1 eq) and 3-bromo-4-fluorobenzaldehyde (1.1 eq) were suspended in isopropanol 5 ml and the mixture heated at 75 °C for 2 hours after conformation of forming of imine by TLC. Added CuC12 (3 eq) and the reaction mixture heated at 75 °C under 0 2 for 3 hours. After completion of the reaction, the isopropanol was removed under vacuum. Then workup with ethyl acetate and water. Separate the organic layer and Water layer re- extracted with 2x25 ml ethyl acetate.
• Ci 5 Hi 4 BrFN 4 0 Ci 5 Hi 4 BrFN 4 0; C, 49.33; H, 3.86; Br, 21.88;F,5.20; N, 15.34; O, 4.38; found C, 49.30; H, 3.87; Br, 21.85; F, 5.22; N, 15.34; O, 4.41.
• Example 20 Compound Bd (l-methyl-5-(5-nitrofuran-2-yl)-3-propyl-lH- py r azolo [4,3-d] pyrimidin-7(6H)-one) : Synthesis ofBd; Step 7: 4-amino-l-methyl-3-propyl-lH-pyrazole-5-carboxamide (1 eq) and 5-Nitro-2-furaldehyde (1.1 eq) were suspended in ethanol 5 ml and the mixture heated at 60 °C for 0.5 hours after conformation of forming of imine by TLC. Added CuC12 (3 eq) and the reaction mixture heated at 65 °C under 0 2 for 0.5 hours.
• MASS ESI [M + H] + : 304.10 ; Elemental anal, calcd. for Ci 3 Hi 3 N 5 0 4 ; C, 51.48; H, 4.32; N, 23.09; O, 21.10; found C, 51.39; H, 4.34; N, 23.12; O, 21.14.
• Example 21 Compound Be (5-(4-hydroxyphenyl)-l-methyl-3-propyl-lH- pyrazolo [4,3-d] pyrimidin-7(6H)-one) :
• Step 7 4-amino-l-methyl-3-propyl-lH-pyrazole-5-carboxamide (1 eq) and 4-hydroxybenzaldehyde (1.1 eq) were suspended in ethanol 5 ml and the mixture heated at 60 °C for 1.5 hours after conformation of forming of imine by TLC. Added CuC12 (3 eq) and the reaction mixture heated at 70 °C under 0 2 for 2 hours. After completion of the reaction, the ethanol was removed under vacuum. Then workup with ethyl acetate and water. Separate the organic layer and Water layer re-extracted with 2x25 ml ethyl acetate. The combined organic layers are washed with brine solution, concentrated under vacuum.
• Example 22 Compound Bf (5-(4-methoxy-3-nitrophenyl)-l-methyl-3-propyl-lH- pyrazolo [4,3-d]pyrimidin-7(6H)-one):
• Example 23 Compound Bg (5-(4-methoxyphenyl)-l-methyl-3-propyl-lH- pyrazolo [4,3-d] pyrimidin-7(6H)-one):
• Example 24 Compound Bh (5-(4-chlorophenyl)-l-methyl-3-propyl-lH- pyrazolo [4,3-d] pyrimidin-7(6H)-one) :
• Example 25 Compound Bi (l-methyI-3-propyl-5-(2,4,5-trimethoxyphenyl)-lH- pyr azolo [4,3-d] pyrimidin-7(6H)-one) : Synthesis ofBi; Step 7: 4-amino-l-methyl-3-propyl-lH-pyrazole-5-carboxamide (1 eq) and 2,4,5-trimethoxybenzaldehyde (1 eq) were suspended in ethanol 5ml and the mixture heated at 60 °C for 0.5 hours after conformation of forming of imine by TLC. Added CuC12 (3 eq) and the reaction mixture heated at 75 °C under 0 2 for 0.5 hours.
• Example 26 Compound Bj (5-(3-fluoro-4-methoxyphenyl)-l-methyl-3-propyl-l Ilpyrazolo [4,3-d]pyrimidin-7(6H)-one):
• Example 27 Compound Bk (5-(3,5-dimethoxyphenyl)-l-methyl-3-propyl-lH- py razolo [4,3-d] pyrimidin-7(6H)-one) : Synthesis ofBk; Step 7: 4-amino-l-methyl-3-propyl-lH-pyrazole-5-carboxamide (1 eq) and 3,5-dimethoxybenzaldehyde (1.1 eq) were suspended in ethanol 5 ml and the mixture heated at 60 °C for 1 hours after conformation of forming of imine by TLC. Added CuC12 (3 eq) and the reaction mixture heated at 70 °C under 0 2 for 0.5 hours.
• MASS ESI [M + H] + : 329.16 ; Elemental anal, calcd. for Ci 7 H 20 N 4 O 3 ; C, 62.18; H, 6.14; N, 17.06; O, 14.62; found C, 62.21; H, 6.11 ; N, 17.12; 0, 14.56.
• Example 28 Compound Bl (5-(3-chlorophenyl)-l-methyl-3-propyl-lH- pyrazolo [4,3-d] pyrimidin-7(6H)-one) : Synthesis ofBl; Step 7: 4-amino-l-methyl-3-propyl-lH-pyrazole-5-carboxamide (1 eq) and 3-chlorobenzaldehyde (1.1 eq) were suspended in ethanol 5 ml and the mixture heated at 75 °C for 1.5 hours after conformation of forming of imine by TLC. Added CuC12 (3 eq) and the reaction mixture heated at 75 °C under 0 2 for 1.5 hours. After completion of the reaction, the ethanol was removed under vacuum.
• MASS ESI [M + H] + : 287.12; Elemental anal, calcd. for Ci 5 Hi 5 FN 4 0 ; C, 62.93; H, 5.28; F, 6.64; N, 19.57; O, 5.59; found C, 62.88; H, 5.31 ; F, 6.64; N, 19.57; 0, 5.61.
• Example 30 Compound Bn (5-(3-fluorophenyl)-l-methyl-3-propyl-lH- pyrazolo [4,3-d] pyrimidin-7(6H)-one) :
• Example 31 Compound Bo (5-(4-hydroxy-3-nitrophenyl)-l-methyl-3-propyl-lH- py r azolo [4,3-d] pyrimidin-7(6H)-one) : Synthesis of Bo; Step 7: 4-amino-l-methyl-3-propyl-lH-pyrazole-5-carboxamide (1 eq) and 4-hydroxy-3-nitrobenzaldehyde (1.1 eq ) were suspended in ethanol 5 ml and the mixture heated at 70 °C for 1.5 hours after conformation of forming of imine by TLC. Added CuC12 (3 eq) and the reaction mixture heated at 75 °C under 0 2 for 1 hours.
• Example 32 Compound Bp (5-(3,4-dimethoxyphenyl)-l-methyl-3-propyl-lH- pyrazolo [4,3-d] pyrimidin-7(6H)-one) : Synthesis of Bp; Step 7: 4-amino-l-methyl-3-propyl-lH-pyrazole-5-carboxamide (1 eq) and 3,4-dimethoxybenzaldehyde (1.1 eq) were suspended in ethanol 5 ml and the mixture heated at 70 °C for 1.5 hours after conformation of forming of imine by TLC. Added CuC12 (3 eq) and the reaction mixture heated at 70 °C under 0 2 for 1.5 hours.
• Example 33 Compound Bq (5-(benzo[d][l,3]dioxol-5-yl)-l-methyl-3-propyl-lH- pyrazolo [4,3-d] pyrimidin-7(6H)-one) :
• the PDE5 inhibitory activity (IC50) of the invented compound was checked by using commercially available purified human PDE5A active (Signalchem, Canada: Cat No. P93-31G), expressed by baculovirus in sf9 insect cells and PDE Glo Phosphodiesterase Assay kit from Promega (Cat No.V1361). The assays were performed by following the manufacturer recommended protocol.
• the IC 5 o value of reference molecule i.e. sildenafil was (5.6nM), which was almost similar with literature reported value.
• the screening for PDE5 inhibitory activity of all the pyrazolopyrimidinone based compounds was determined at 750nM and 1.5 ⁇ concentrations. The results showed these molecules were highly active against the PDE5A. Some of the compounds have shown better IC 50 values compared to reference compound.
• IC 5 0 determination In order to determine the IC50 of the active molecules a nine point titration in duplicate against the PDE5A was performed and found some of the invented compounds were more active than that of reference. Some of the representative molecules showed an IC50 viz. compound Aa: 0.8nM ( Figure 2), compound Ab 4.8nM. Data analysis was performed with GraphPad Prism®, version 5.00, for Windows using a sigmoidal dose- response (variable slope) equation. Each point represents an average of two replicates per concentration.
• compound Aa was dissolved in Transcutol and diluted with 20% Cremophor- EL in distilled water at a ratio of 3:7.
• This solution was injected into the ear vein of a group of five healthy male rabbit (3 -3.5kg weight) in a volume of 0.5ml/kg. The event was followed 5 minutes later by another equal volume saline injection containing Sodium nitroprusside (0.2mg/kg), which acts as a donor of Nitric oxide (NO). Length of uncovered penile mucosa was measured with a sliding caliper at different time points for an hour after administration of the test compounds. The area under the curve (AUC) was calculated by Graph Pad Software. Controls experiments were performed with the SNP alone injections.
• the present invention provides the novel structural entities as PDE5 inhibitors for the treatment of impotence.
• the present invention provides new generation pyrazolopymidinone compounds with better PK parameters as PDE5 inhibitors for the treatment of impotence.
• the present invention covers diversity in the claimed structures as per formula 1, representing 2 classes of compounds 1A and IB.
• the process offered is scalable.
• the present invention also provides isoform selectivity of these compounds for different PDE enzymes to find enzyme specificity.
• AMP adenosine monophosphate
• cAMP cyclic adenosine monophosphate
• DIPEA diisopropyl ethyl amine
• GMP guanosine monophosphate
• GTP guanosine triphosphate
• PKA protein kinase A

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