Formula I  and Formula II

OR

PNQ 103

STRUCTURE COMING…………

for the potential treatment of COPD & sickle cell disease (SCD)

Adenosine A2b receptor antagonist

Advinus Therapeutics Ltd

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PNQ-103 is a proprietary A2B Adenosine receptor (A2BAdoR antagonist), currently in the pre-clinical development stage for the potential treatment of COPD & sickle cell disease (SCD). Advinus is looking for partnering/co-development opportunities.

COPD

Chronic Obstructive Pulmonary Disease (COPD) is a disease that damages lung tissue or restricts airflow through the bronchioles and bronchi, and commonly leads to chronic bronchitis and emphysema. COPD, along with asthma, forms the third leading cause of death in both developed and developing countries and an annual direct and indirect cost of healthcare of more than $50 billion in the US alone. Current therapies suffer from lack of long term efficacy, patient compliance and a narrow therapeutic index.

Adenosine is a powerful bronchoconstrictor and pro-inflammatory agent in COPD and asthma. Adenosine regulates tissue function by activating its receptors: A₁AdoR and A_2AAdoR are high affinity receptors and A_2BAdoR and A₃AdoR are low affinity receptors. During pathological conditions in lung, local adenosine concentrations rise to high levels and activate A_2BAdoR. A_2BAdoR agonized by adenosine induces both bronchoconstriction and pro-inflammatory effects in lung by acting on multiple cell types that lead to airway hyperreactivity and chronic inflammation. Therefore, A_2BAdoR antagonists are expected to be beneficial in COPD and asthma.

PNQ-103 is a proprietary A_2BAdoR antagonist, currently in the pre-clinical development stage for the potential treatment of COPD.  It is a potent, selective, orally bio-available agent with low clearance and small volume of distribution. PNQ-103 is efficacious in standard rodent asthma and lung fibrosis models. PNQ-103 was found to be safe in exploratory safety studies including a Drug Matrix Screen, mini-AMES test, and a test for cardiovascular liability in dog telemetry as well as a 30- day repeat dose study in rats.

SCD

Sickle Cell Disease (SCD) affects millions of people worldwide. It is caused by an autosomal mutation in the hemoglobin gene (substitution of amino-acid valine [Hb A] for glutamic acid [Hb S]. Hb S in low O2 condition polymerizes, leading to distortion of the cell membrane of red blood cells (RBC) into an elongated sickle shape. Sickled RBCs accumulate in capillaries causing occlusions, impair circulation and cause tissue damage and severe disabilities. Unfortunately, there is no targeted therapy for SCD.

Adenosine levels are elevated in SCD patients. Activation of the A_2BAdoR by adenosine increases 2,3-DPG levels in RBCs, which reduces Hb S affinity to O2 and promotes its polymerization leading to RBC sickling. A recent study published in Nature Medicine (2011; 17:79-86) demonstrated potential utility of an A_2BAdoR antagonist for the treatment of SCD, through selective inhibition of 2,3-DPG production in RBCs.  Therefore, PNQ-103, a selective A_2BAdoR antagonist, is expected to be useful for the treatment of SCD.  In support, ex vivo PoC (selective inhibition of 2,3-DPG production) has been established for PNQ-103 in RBCs from normal and SCD patients.

EXAMPLES………

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2012035548

Example 1: Phosphoric acid mono-{2-cyano-6-oxo-l-propyl-8-[l-(3-trifluoromethyl-benzyl)-lH-pyraz -4-yl]-l,6-dihydr»-purin-7-ylmethyl} ester

Step I: Synthesis of l-(3-Trifiuoroirethyl-ben:ijl)-lH-pyrazole-4-carboxylic acid (6-amino-2,4-dioxo-3-propyl-l,2,3₅4-tetrahydro-pyrimidin-5-yl)-amide

A mixture of 5,6-diamino-3-propyl-l H-pyrimidine-2,4-dione (4.25 g, 0.023 mol), l-(3-Trifluoromethyl-benzyl)-lH-pyrazole-4-carboxylic acid (6.23 g, 0.023 mol), prepared by conventional methods starting from pyrazole-4-carboxylic ester, in methanol (50 ml) were cooled to 0 °C and added EDCI.HC1 (8.82 g, 0.046 mol). The reaction mixture was stirred at 25 °C for 6 h and the organic volatiles were evaporated. To this residue water (50 ml) was added and the precipitate was filtered off, and washed with cold water (50 ml) to obtain l-(3-Trifluoromethyl-benzyl)- 1 H-pyrazole-4-carboxylic acid (6-amino-2,4-dioxo-3-propyl-l,2,3,4-tetrahydro-pyrimidin-5-yl)-amide (7.2 g, 72 %) as a pale yellow solid.

‘HNMR(400MHz, DMSO d6): δ 0.82 (t, J=7.6Hz, 3H); 1.46-1.51 (m, 2H); 3.64 (t, J=7.2Hz, 2H); 5.49 (s, 2H); 6.01 (s, 2H); 7.55-7.63 (m, 2H); 7.68-7.72 (m, 2H); 7.99 (s, 1H); 8.37 (s, 1H); 8.55 (s, 1H); 10.42 (s, 1H).

Step II: Preparation of l-Propyl-8-[l-(3-trifluoromethyl-benzyl)-lH-pyrazoI-4-yl]-3,7-dihydro-purine-2,6-dione

A mixture of l-(3-Trifluoromethyl-benzyl)-lH-pyrazole-4-carboxylic acid (6-amino-2,4-dioxo-3-propyl-l,2,3,4-tetrahydro-pyrimidin-5-yl)-amide (30 g, 0.068 mol), P₂0₅(34.0g, 0.240.8 mol) and DMF (300ml) were heated at 100 °C for 30 minutes. The reaction mixture was cooled to 20-25 °C. The reaction mixture was slowly poured into water (1.5 L) with vigorous stirring. Solid material separated was filtered off, and washed with water (200ml) to obtain 1 -Propyl-8-[l -(3-trifluoromethyl-benzyl)-l H-pyrazol-4-yl]-3,7-dihydro-purine-2,6-dione (25 g, 88 %) as a pale yellow solid.

‘HNMR(400MHz, DMSO d6): δ 0.87 (t, J=7.2Hz, 3H); 1.53-1.60 (m, 2H); 3.98 (t, J=7.2Hz, 2H); 5.53 (s, 2H); 7.57-7.64 (m, 2H); 7.69-7.71 (m, 2H); 8.08 (s, 1H); 8.47 (s, 1H); 1 1.83 (s, 1H); 13.39 (s, 1H)

Step III: Preparation of 2-ChIoro-l-propyI-8-[l-(3-trifluoromethyI-benzyl)-lH-pyrazol-4-yl]-l,7-dihydro-purin-6-one

A mixture of l-Propyl-8-[l-(3-trifluoromethyl-benzyl)-lH-pyrazol-4-yl]-3,7-dihydro-purine-2,6-dione (7.2 g, 0.017 mol), NH₄C1 (4.54 g, 0.085 mol) and POCl₃ (220 ml) were heated at 120-125 °C for 72 h. Reaction mixture was cooled to 20-25 °C. It was then concentrated under vacuum and quenched with cold water slowly and solid material was separated. It was filtered off and washed with water. The solid material was dried under vacuum. The crude product was purified by column chromatography using silica gel (230-400 mesh) and 0.5 to 4 % methanol in chloroform as an eluent to obtain 2-Chloro-l-propyl-8-[l-(3-trifluoromethyl-benzyl)- lH-pyrazol-4-yl]-l,7-dihydro-purin-6-one (4.2 g, 58 %) as a pale yellow solid.

‘HNMR(400MHz, CD₃OD): 6 1.02 (t, J=7.2Hz, 3H); 1.78-1.84 (m, 2H); 4.29 (t, J=7.6Hz,

2H); 5.52 (s, 2H); 7.56-7.57 (m, 2H); 7.63 (m, 2H); 8.12 (s, 1H); 8.35 (s, 1 H)

Step IV: Preparation of 6-Oxo-l-propyl-8-[l-(3-trifluoromethyl-benzyl)-lH-pyrazol-4-yl]-6,7-dihydro-lH-purine-2-carbonitrile

A mixture of 2-Chloro-l-propyl-8-[l-(3-trifluoromethyl-benzyl)-l H-pyrazol-4-yl]-l ,7-dihydro-purin-6-one (O. lg, 0.23 mmol), NaCN (0.016 g, 0.35 mmol), Nal (0.069g, 0.46 mmol) and DMF (2 ml) were stirred for 48 h at 65-70 °C. Reaction mixture was cooled to 20-25 °C and water was added. Solid material was separated. It was filtered off and washed with water. The product was dried under vacuum to obtain 6-Oxo-l-propyl-8-[l-(3-

trifluoromethyl-benzyl)-lH-pyrazol-4-yl]-6,7-dihydro-lH-puriiAe-2-carbonitrile (0.075 g, 77 %) as an off white solid.

‘HNMR(400MHz, DMSO d6): δ 0.97 (t, J=7.6Hz, 3H); 1.71-1.77 (m, 2H); 4.12 (t, J=7.6Hz, 2H); 5.51 (s, 2H); 7.57-7.67 (m, 4H); 8.14 (s, 1H); 8.55 (s, 1H); 14.01 (bs, 1H)

Preparation of hosphoric acid di-tert-butyl ester chloromethyl ester:

Step I: Phosphoric acid di-tert-butyl ester

A mixture of di-tert-butylphosphite (5 g, 0.026 mol), NaHC0₃ (3.71 g, 0.044 mol) and water (50 ml) were taken and cooled to 0-(-5 , °C. KMn0₄ (6.18 g, 0.039 mol) was added to the reaction mixture in portion wise over ¾ period of 30 minutes at that temperature. The reaction mixture was allowed to warm to 20-25 °C ana stirred for 1.5 hours at that temperature. To this reaction mixture activated charcoal (25 g) was added and stirred at 55-60 °C for 1 hour. The reaction mixture was cooled to room temperature and filtered off and washed with water (200 ml). The filtrate was concentrated to half of its volume and cooled to 0 °C. It was then acidified with con. HC1 (pH~l-2) to obtain solid. The solid material was filtered off, washed with ice cold water and dried under vacuum to obtain Phosphoric acid di-tert-butyl ester as white solid (3.44 g, 63 %).

Step II. Phosphoric acid di-tert-butyl ester chloromethyl ester

A mixture of Phosphoric acid di-tert-butyl ester (1 g, 0.0048 mol), NaHC0₃ (0.806 g, 0.0096 mol), tetra butyl ammonium hydrogen sulphate (0.163 g, 0.00048 mol), water (40 ml) and DCM (25 ml) were taken. The mixture was cooled to 0 °C and stirred at that temperature for 20 minutes. Chloromethyl chlorosulphatc (0.943g, 0.0057 mol) in DCM (15 ml) was added to it at 0 °C. The reaction mixture allc ed to warm to room temperature and stirred for 18 hours. The organic layer was separated and aqueous layer was extracted with DCM (30 ml). The organic layer was washed with brine (60 ml) solution and dried over Na₂SC>4. The organic layer was evaporated to obtain Phosphoric acid di-tert-butyl ester chloromethyl ester as colorless oil (0.79 g, 64%).

Step I: Phosphoric acid di-tert-butyl ester 2-cyano-6-oxo-l-propyl-8-[l-(3-trifluoromethyl-benzyl)-lH-pyrazol-4-yl]-l,6-dihydro-purin-7-ylmethyl ester

A mixture of 6-Oxo-l-propyl-8-[l-(3-trifluoromethyl-benzyl)-lH-pyrazol-4-yl]-6,7-dihydro-lH-purine-2-carbonitrile (0.5 g, 0.0012mol), K₂C0₃ (0.485 g, 0.0036 mol ) and acetone ( 10 ml) were taken and stirred for 20 minutes at room temperature. Nal (0.702 g, 0.0047 mol) was added and then Phosphoric acid di-ten-butyl ester chloromethyl ester (0.619 g, 0.0024 mol in 2 ml acetone) was added to the reaction mixture drop wise. The reaction mixture was heated at 45 °C for 16 h. The reaction mixture was filtered through celite and washed with acetone. The organic layer was concentrated and the residue was taken in ethyl acetate (30 ml) and saturated NaHC0₃ solution (20 ml). The organic layer was separated and washed with saturated sodium thiosulphate solution (20 ml). The organic layer was washed with 0.5 N HC1 solution (20 ml) and brine solution (20 ml). The organic layer was dried over sodium sulphate and evaporated to obtain brown colored mass. The crude product, which is a mixture of N7 and N9 isomers was purified by column chromatography (230-400 mesh silica gel and it was first treated with 5% triethyl amine in hexane) using 5-20 % acetone in hexane (with 0.5 to 1% triethyl amine) as an eluent to obtain N7 isomer (0.34g, 45 % ) and N9 isomer ( 0.1 lg, 14 % )

Phosphoric acid di-tert-butyl ester 2-cyano-6-oxo-l-propyl-8-[l-(3-trifluoromethyl-benzyl)-lH-pyrazol-4-yl]-l,6-dihydro-purin-7-ylmethyl ester (N7-isomer).

Ή NMR (400MHz, DMSO d6):6 0.95 (t J=8Hz, 3H); 125 (s, 18 H); 1.75-1.80 (m, 2H); 4.18 (t, J=7.2Hz, 2H); 5.58 (s, 2H); 6.34 (d,
2H); 7.61-7.63 (m, 2H); 7.70-7.73 (m, 2H); 8.19 (s, 1H); 8.75 (s, 1H)

Phosphoric acid di-tert-butyl ester 2-cyano-8-[l-(3-trifluoromethyI-benzyl)-lH-pyrazol-4-yl]-6-oxo-l-propyl-l,6-dihydro-purin-9-ylmethyl ester (N9-isomer)

Ή NMR (400MHz, DMSO d6): δ 0.94 (t, J=8Hz, 3H); 125 (s, 18 H); 1.74-1.78 (m, 2H); 4.21 (t, J=7.2Hz, 2H); 5.59 (s, 2H); 6.05 (d, J=10.8Hz, 2H); 7.62-7.63 (m, 2H); 7.69-7.71 (m, 2H); 8.16 (s, 1H); 8.71 (s, 1H)

Step II: Phosphoric acid mono-{2-cyano-6-oxo-l-propyl-8-[l-(3-trifluoromethyl-benzyl)-lH-pyrazol-4-yl]-l,6-dihydro-purin-7-ylmethyl} ester (N7-isomer).

The above product, N7 isomer (0.34 g, 0.52 mmol) was dissolved in DCM (20 ml) and TFA (0.29 ml, 4.2 mmol) was added to it. The reaction mixture was stirred at room temperature for 7 hours. The organic volatiles were evaporated and the residue was stirred with pentane: diethyl ether (3:1, 10 ml) and the solid material obtained was filtered off and washed with 10 % diethyl ether in pentane (10 ml) to obtain Phosphoric acid mono- {2-cyano-6-oxo-l -propyls’ [ 1 -(3 -trifluoromethyl-benzyl)- 1 H-pyrazol-4-yl]- 1 ,6-dihydro-purin-7-ylmethyl } ester (0.239g, 85 %) as an off white solid.

(400MHz, DMSO d6): δ 0.96 (t, J=7.6Hz, 3H); 1.75-1.81 (m, 2H); 4.16 (t, J=7.2Hz, 2H); 5.58 (s, 2H); 6.23 (d, J=6Hz, 2H); 7.61-7.63 (m, 2H); 7.69-7.75 (m, 2H); 8.22 (s, 1 H); 8.80 (s, 1H); (M+1): 538.2

Phosphoric acid mono-{2-cyano-6-oxo-l-propyl-8-[l-(3-trifluoromethyl-benzyl)-lH-pyrazol-4-yl]-l,6-dihydro-purin-9-ylmethyl} ester (N9-isomer, 28%)

(400MHz, DMSO d6): δ 0.93 (t, J=7.6Hz, 3H); 1.72-1.80 (m, 2H); 4.16 (t, J=7.2Hz, 2H); 5.54 (s, 2H); 5.95 (d, J=6Hz, 2H); 7.59-7.60 (m, 2H); 7.67-7.73 (m, 2H); 8.17 (s, 1H); 8.72 (s, 1H).

Step III: Phosphoric acid mon -{2-cyano-6-oxo-l-propyl-8-[l-(3-trifluoromethyl-benzyI)-lH-pyrazol-4-yl]-l,6-dihydro-purin-7-yimethyl} ester di sodium salt

The above product (0.239g, 0.44 mmol) and water (25 ml) were taken. To the suspension formed, NaHC0₃ solution (0.1 12g, 1.3 mmol in 20 ml water) was added. The reaction mixture was stirred at room temperature for 1.5 h and the solid material obtained was filtered off. The clear solution was passed through reverse phase column chromatography (LCMS). The fraction obtained was evaporated. It was lyophilized to obtain pure Phosphoric acid mono-{2-cyano-6-oxo- 1 -propyl-8-[ 1 -(3 -trifluoromethyl-benzyl)- 1 H-pyrazol-4-yl]- 1 ,6-dihydro-purin-7-ylmethyl} ester di sodium salt (0.208g; 80%) as an off white solid.

Ή NMR: (400MHz, D₂0): δ 0.97 (t, J=7.6Hz, 3H); 1.80-1.86 (m, 2H); 4.28 (t, J=7.6Hz, 2H); 5.53 (s, 2H); 6.04 (d, J=3.2Hz, 2H); 7.52-7.53 (m, 2H); 7.62-7.64 (m, 2H); 8.22 (s, 1H); 8.74 (s, 1H)

³¹P NMR: (400MHz, D20): δ 0.447

EXAMPLES…………..

Patent

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2009118759

Example Al: 1, 3-Dipropyl-8-[l-(3-p-tolyl-prop-2ynyl)-lH-pyrazol-4-yI]-3, 7-dihydro-purine-2, 6-dione

Step I: l-(3-p-ToIyl-prop-2-ynyl)-lH-pyrazole-4-carboxylic acid ethyl ester

A mixture of l-prop-2-ynyl-lH-pyrazole-4-carboxylic acid ethyl ester obtained as given in example Bl (0.20Og, l.lmmol), 4-iodo toluene (0.254g, 1.1 mol), copper iodide (0.021g, O.l lmmol), dichlorobis (triphenylphosphine)-palladium (II) (39mg, O.Oόmmol), triethylamine (2ml), DMF (2ml) was degassed for lOmin. and stirred for 20hrs at 25-25 ⁰C. Reaction mixture was diluted with water (10ml) and extracted with

• ethyl acetate. Organic layer was washed with brine solution and dried over Na₂SO₄.

The solvent was evaporated and crude product was purified by column chromatography

(Ethyl acetate: hexane-12:78) to obtain pure l-(3-p-tolyl-prop-2-ynyl)-lH-pyrazole-4- carboxylic acid ethyl ester compound (0.226g, 75%). ¹HNMR^OOMHZ, CDCl₃): δ 1.35 (t, J=6.8Hz, 3H); 2.37 (s, 3H); 4.31 (q, J=6.8Hz, 2H); 5.18 (s, 2H); 7.16 (d, J=7.6Hz, 2H); 7.38 (d, J=8Hz, 2H); 7.95 (s, IH); 8.21 (s, IH)

Step II: l-(3-p-Tolyl-prop-2-ynyl)-lH-pyrazole-4-carboxy!ic acid l-(3-p-Tolyl-prop-2-ynyl)-lH-pyrazole-4-carboxylic acid ethyl ester (0.226g, 0.84 mmol) was dissolved in a mixture of solvents THF: methanol: water (3:1:1, 10ml) and LiOH (0.07 Ig, 1.7mol) was added to the reaction mixture with stirring. The reaction mixture was then stirred at 20-25 ⁰C for 2 hours. Solvents were evaporated and the residue was diluted with water (0.5 ml) and acidified with dil. HCl, filtered and dried to obtain off white precipitate, l-(3-p-Tolyl-prop-2-ynyl)-lH-pyrazole-4-carboxylic acid (0.182g, 90%).

¹HNMR^OOMHZ, CDCl₃): δ 2.37 (s, 3H); 5.2 (s, 2H); 7.16 (d, J=7.6Hz, 2H); 7.38 (d, J=8Hz, 2H); 8.01 (s, IH); 8.29 (s, IH) Step III: 1, 3-Dipropyl-8-[l-(3-p-tolyl-prop-2ynyl)-lH-pyrazol-4-yI]-3, 7-dihydro-_‘ purine-2, 6-dione

A mixture of 5,6-diamino-l,3-dipropyl-lH-pyrimidine-2,4-dione (0.075g, 0.33 mmol), l-(3-p-tolyl-prop-2-ynyl)-lH-pyrazole-4-carboxylic acid (0.080gm, 0.33mmol), methanol (5ml), EDCI (0.089g, 0.46mmol) were taken and stirred for 12 hours at 20-25 ⁰C. The reaction mixture was concentrated to obtain intermediate l-(3-p-tolyl-prop-2-ynyl)-lH-pyrazole-4-carboxylic acid (6-amino-2, 4-dioxo-l, 3-dipropyl)-l, 2, 3, 4-tetrahydro-pyrimidine-5yl) amide (50mg, 34%) which was dissolved in hexamethyldisilazane (HMDS). To this reaction mixture ammonium sulphate (0.01 Og) was added. The reaction mixture was refluxed at 140 ⁰C for 18hrs. The organic volatiles were evaporated and the residue was treated with crushed ice, the precipitate formed was filtered off. The product was then purified by column chromatography (l%MeOH in CHCl₃) to obtain 1, 3-dipropyl-8~[l-(3-p-tolyl-prop-2ynyl)-lH-pyrazol-4-yl]-3, 7-dihydro-purine-2, 6-dione (0.035g, 92%). ‘HNMR(400MHz, DMSO d6): δ 0.76-0.87 (m, 6H); 1.51-1.57 (m, 2H); 1.68-1.74 (m, 2H); 2.29 (s, 3H); 3.82 (t, J=7.2Hz, 2H); 3.95 (t, J=7.2Hz, 2H); 5.36 (s, 2H); 7.18 (d, J=8Hz, 2H); 7.35 (d. J=8Hz, 2H); 8.08 (s, IH); 8.49 (s, IH); 13.9 (bs,lH)

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