SCH 58261

X I Meeting Heterocyclic Struttures in Medicinal Chemistry Palermo, May 23-26, 2004

New Heterocyclic Ligands for the Adenosine Receptors P1 and for the ATP Receptors P2 P.G.Baraldi, F.Fruttarolo, M.Aghazadeh Tabrizi, Hussein ElKashef, R.Romagnoli, M.G.Pavani, A.Bovero, D. Preti, K.Varani, P.A.Borea

Ferrara University, Italy

RECEPTOR CLASSIFICATION

P1

Adenosine

P2

Nucleotides

Purinergic Receptors ATP, ADP, UTR,UDP

A1, A2A A2B, A3

High affinity receptors Low affinity receptoers

A1 A2A A2B A3 P2X P2Y

SIGNAL TRANSDUCTION A1

A3 A2A A2B

INHIBITION ADENYLYL CICLASE

AMPc

ACTIVATION ADENYLYL CICLASE

AMPc

Regulation of Adenylyl Cyclase by Adenosine Receptor Stimulation

Main effects of Adenosine to A1 receptors Through interaction with A1 receptors, adenosine is involved in: • • • • •

 heart rate  atrioventricular nodal conduction  lipolysis  synaptic activity in the CNS  neutrophil chemotaxis

Main effects of Adenosine to A2A receptors Through interaction with A2A receptors, adenosine is involved in:

• dilating the coronary vessels of the heart • preventing ischemic arrythmias • tratment of CNS disorders (Parkinson’s disease) • antiinflammatory effects • inhibiting platelet aggregation (antithrombotic effect)

Main effects of Adenosine to A2B receptors Through interaction with A2B receptors, adenosine is involved in:

• Bronchoconstriction

Main effects of Adenosine to A3 receptors DISTRIBUTION

EFFECTS

AGONIST

ANTAGONIST

SNC

Cytoprotective or cytotoxic

Neuroprotective (nM) Apoptotic (M)

Neuroprotective

Heart

Cytoprotective or Cardioprotective (nM) cytotoxic Protective in ischaemic preconditioning Apoptotic (M)  degranulation

Mast cells

Macrophages

 TNF- release

Anti-inflammatory

Eosinophils

 degranulation

Anti-inflammatory Anti-asthmatic

Lymphocytes

Immunosuppres.

Antiinflammatory Anti-asthmatic

Immunotherapy of cancer

To fully evaluate the great variety of therapeutic implications in the adenosine receptor area, subtype-selective agonists and antagonists with high affinity are required

Strategies to activate A1 adenosine receptors  Agonists (CPA, CHA, CCPA)  Inhibitors of adenosine uptake (dipiridamole)  Kinases inhibitors  Allosteric modulators (Enhancers)

What is an allosteric modulator (enhancer) An enhancer is a molecule which binds a receptor binding site different from the one of the endogenous ligand. It is able to amplify the activation of the receptor by an agonist, stabilizing the complex between the ligand and the high affinity receptor state. This effect is carried out by the retardation of the agonist receptor dissociation process.

Why use the enhancers?  Adenosine release is a local process which happens in tissues

during hypoxic and ischemic injuries.  Endogenous adenosine release exerts a protective action in a tissue affected by a degenerative process.  Adenosine decreases the neuronal activity and the oxygen use ,which are mediated by A1 adenosine receptors. Allosteric modulators could be useful when is necessary a local increase of adenosine action on A1 adenosine receptors.

Identification of PD 81,723, as a model for the development of A1 adenosine allosteric enancers

CF3

O S NH2 PD 81,723

Bruns R. F. et al. J.. Mol. Pharmacol. 1990, 38, 950-958

General procedure for the synthesis of 2-amino3-aroyl thiophenes (GEWALD REACTION) O Ar

CN

+ R1

R2

O

S8, morpholine EtOH O R2 R1

Ar S

NH2 Ar= aryl or heteroaryl R1=R2=CH3 R1,R2=-(CH2)3-, -(CH2)4-

Structure-Activity-Relationship of 2-amino-3benzoyl-thiophenes

R

R

O

n=0, 1 e 2 n(H2C)

O N

S

NH2

S

NH2

Lipophylicity of the R substituent is preferred in para and meta positions

Baraldi et al. Exp. Op. on Therapeutic Patent 2004, 71-79

Effects of the substitution of he benzoyl at the 3 position with a naftoyl moiety

O

(CH2)n

O

(CH2)n

S

NH2

n=0, E=10 n=1, E=29 n=2, E=44

S

NH2

n=0, E=45 n=1, E=60 n=2, E=52

Baraldi et al. Exp. Op. on Therapeutic Patent 2004, 71-79

2-Amino-3-naftoyl thiophenes: effects of the substituents at the 4 and 5 positions

O

O S

NH2

S

NH2

E=60

E=45

O

O S

NH2

E=52

O

O N

S E=37

NH2

S E=21

NH2

S

NH2

attività antagonista a 10 M E=21 a 10 nM E=7 a 100 nM E=25 a 1 M

Conclusions a) In the examined series of 2-amino-3-aroyl-thiophenes, the derivatives substituted at the 3-position with a naphtoyl group resulted to be more active then the corresponding 3-benzoyl derivatives. b) The substitution of the naftoyl nucleous with an halogen determined an encrease of affinity.

c) The biological activity is affected by the hydrophobic effects of the examined substituents more then by the electronic aspects. d)The presence of lipophylic substituents at the positions 4 and 5 of the thiophene ring is essential for the biological activity.

STYRYL-XANTHINE A2A ANTAGONISTS O CH3 O

N

O

CH3 N

Cl

C3H7

N N CH3

O

CSC A1 28200 nM; A2A 54 nM A1/A2A 522

O

N

N N C3H7

OCH3 OCH3

KF 17837 A1 62 nM; A2A 1 nM A1/A2A 62 O

C3H7

N

CH3 N

CH3 N

N N C3H7

S

1,3-dipropyl-7-methyl-8-[2(3-thienyl)ethenyl] xanthine A1 561 nM; A2A 19 nM A1/A2A 30

Baraldi, P.G. et al. Curr. Med. Chem. 1995, 2, 707

Summary A2A adenosine receptor antagonists •SCH 58261 story

•FSPTP, the first A2A irreversible adenosine antagonist •[ 3H]-SCH 58261 •[11C]SCH442416 the first non-xanthine ligand for the in vivo imaging of A2A adenosine receptors using PET • Water-soluble A2A adenosine antagonists

A2A adenosine receptor antagonists: the starting point NH2 N

N

N

N

Cl

CGS15943

O

rA1 6.4 nM; rA2A1.2 nM A1/A2A 5.3

NH2 NH N

N

F

N

2

N N

N

N

N

O N

N

O

N F

N

8FB PTP rA1 3.3 nM; rA2A 1.2 nM A1/A2A 2.8

7FB PTP rA1 189 nM; rA2A 12 nM A1/A2A 15.8

Gatta et al. Eur.J. Med. Chem. 1993, 28, 569

New A 2A Adenosine Antagonists NH2

NH2 N

N N

N

N

N O

N N N

N

O

N

N

SCH 63390

SCH 58261

rA1 504 nM; rA2A 2.4 nM A1/A2A 210

rA1 121 nM; rA2A 2.3 nM A1/A2A 52.6 NH2 N

N

N N

N

O

N

rA1 2705 nM; rA2A 21 nM A1/A2A 129

Baraldi, P.G. et al. J. Med. Chem. 1996, 39, 1164

Synthesis of Pyrazolo[4,3-e] 1,2,4 Triazolo [1,5-c] Pyrimidine Derivatives CN

CN

K2CO3, RX N N

2

NH2

2

N

NH2

N

reflux

N N

OEt

N

1

R

1

R

H

CN

H(COEt)3

R= alkyl, aryl, aralkyl 1) 2-furoic hydrazide 2) Ph2O, 260 °C

N N N

8

R

N 7

N

N

NH2CN

N 2

pTsOH

N NH2

O

O

O

HCl, reflux

N 1

NH2

N 8

N H

N R

N N

N R

N 7

N

Baraldi, P.G. et al. Bioorg. Med. Chem. Lett. 1994, 4, 2539

Design of Tritiated SCH 58261

NH2

NH2 N

N

N

N

3

H

N

N

SCH 58261

O

3

H

3

H

N N

N N N

N

[3H]SCH 58261

O

Synthesis of [ 3H]-SCH 58261 CH2-CH2-NH-NH2

CH2-CH2-Cl

CH2-CH2-Cl

Br

Br

Br2, Fe

NH2-NH2

Br

Br

Br

Br

O

O N

N

N

N N

N

N

3

H2, C/Pd

N

N

N N

NH2

NH2

N 3

H

Br 3

Br

H 3

Br

H

Baraldi, P.G. et al. J. Label. Compd. and Radiopharm. 1996, 8 , 725

II Generation of A2A Antagonists NH2

NH2 N

N

N N

N

N O

SCH 63390 rA1 504 nM; A2A r2.4 nM A1/A2A 210 NH2

NH2

N

O

N

SCH 58261 rA1 121 nM; rA2A 2.3 nM A1/A2A 52.6

N

N N

N

N

HO

N

N

N N

N O

N

rA1 444 nM; rA2A 1.7 nM A1/A2A 261

N

N

N N

O

N HO

rA1 725 nM; rA2A 0.85 nM A1/A2A 853

Baraldi, P.G. et al. J. Med. Chem. 1998, 41, 2126

Comparison Between II Generation Derivatives and ZM 241385 NH2

NH2 HO

N

N

N N

N

N O

N

N N N

O

N

N

HO

rA1 444 nM; rA2A 1.7 nM A1/A2A 261

rA1 725 nM; rA2A 0.85 nM A1/A2A 853 NH2

HO N N H

N N

N N

O

ZM241385 rA1 810 nM; rA2A 1.5 nM A1/A2A 520

Structural Correlation Between II Generation Derivative and ZM 241385 NH2

NH2 HO

N N

N

HO N N

N O

N

N N

N N

O

N

ZM241385 A1 444 nM; A2A 1.7 nM A1/A2A 261

A1 810 nM; A2A 1.5 nM A1/A2A 520

Replacement of pyrazole ring with different heterocycles NH2

N

N

N

O

N

R1

N N

NH2

N

NH2 N

N

N N

S

N

O

N

N

O

N R1

Het NH2

N

R1

N

N

N

N N

O

FSPTP, the first A2A irreversible adenosine antagonist NH2

N

N

N

N

O

N N FO2S

FSPTP FSPTP, the fluorosulfonyl derivative of SCH 63390, showed an irreversible antagonism for A2A receptors and it has been useful to demonstrate the presence of a large receptor reserve (spare receptors) for the A2A subtype. This large receptor reserve can explain the greater coronary vasodilation in the guinea pig heart observed for fixed agonist concentrations

L.Belardinelli et al. Circulation, 1998, 98, 711

Biological and Physico-Chemical Characterization NH2 N R

N

N N

N

O

N

Binding studies hA1, hA2A, hA3

Thermodynamic studies

Rm values

Concluding remarks on A2A adenosine antagonist studies All

the new derivatives are endowed with good affinity and selectivity for A2A adenosine receptor subtype The best lenght of the chain at 7 position is three methylene groups The oxygenated moieties on the phenyl ring increase the affinity and selectivity The water solubility is slightly increased

Preparation of Key Intermediate 7H-compound NC NHNH2

CN

CN

+

N OEt

H(COEt)3

NH2

N

CN N

reflux

OEt

N

N 1

1) 2-fu roic hydrazide 2) Ph2O, 260°C

O

N N N

8

N N

7

H

N

HCO2 H

N N

8

N 7

N

N N

8

NH2CN NH2

N

HCl

N N

NH2

O

O

N N

N 7

Direct Alkylation of 7H-key intermediate

O

O

N

N N N

8

N

RX

N

N N

N

7 H

N

NH2

NaH

N

R

N

+ N8 - isomer NH2

Chlorosulphonation of SCH 58261 NH2 N

NH2

N N

N N

SCH 58261

O

N

N

N N

Cl SO3H, O°C, 1h

Cl

S

O O

N N N

O

Sulphonamide derivatives of SCH 58261 NH2 N N

N

HO

S

N

N N

HCl 10%

O

glycine LiOH

O O

A1 Ki = 4300 nM A2A Ki = 140 nM

Cl

NH3 (g)

S

HO2C

O O

diethanol amine

S

O N

O

A1 Ki = 4200 nM A2A Ki = 1.31 nM

S

O O

N-methyl A1 Ki = 2500 nM piperazine A2A K i = 2.2 nM

A1 Ki = 523 nM A2AKi = 3.8 nM

HO H2N

NH

S

O

N

O

S

N HO

A1 Ki = 3400 nM A2A Ki = 0.8 nM

Baraldi, P.G. et al., J.Med.Chem, 2002, 45, 115 - 126

O O

New synthetic methodology to prepare the key 7H-tricyclic intermediate NH H2N

NH2

NH2

NH2

N

N

N

NH2

N

N

N

+

EtO

OEt O

HO

Cl

OH

Cl CHO

O

N

NH2

NH2 N

N

N N

HN N

Cl

HN

O

N HN N

N N H

O

H N O

Dimroth-Type Rearrangement NH2 N HN N

NH2

N N H

O

H N O

N

BSA HMDS DMF 230°C

N N N

HN N

This step is crucial for yield optimization, reagents ratio and product isolation

O

Direct alkylation of 7H – tricyclic intermediate NH2 N HN N

NH2

N N

O

N

R-Cl K2CO3, DMF 100°C, 12h

N 7N

R

N7 R

N N

O

N

N 8

N7 R

NO2

CN

NHCOCH3

COOEt

N(CH2CH2OH)2

OCH2COOEt

N(CH2CH2OH)2

Baraldi, P.G. et al., J.Med.Chem, 2002, 115

Synthetic elaborations of the cathecolic system NH2 N

N N

O

N

N N R

R = OH, R1 = OH R1

Br2C(CO2Et)2

R=R1 =

O CO2Et O CO2Et

, NaCl

KOH

NaBH4 O R=R1 O

CO2Et

R=R1

O CH2OH O CH2OH

R=R1

NaOH

10% NaOH O R=R1 O

CO2H

O CO2H O CO2H

R=R1

O CO2Na O CO2Na

New A2A water-soluble adenosine antagonists NH2 N

N

N

O

N

N N

O HO

O HO

hA1 = 432 nM hA2A = 0,19 nM hA2B >10000 hA3>10000 hA1/hA2A = 2273

New water-soluble A2A adenosine antagonists NH2 N

N

N

N

N N

Cl

H3N

hA1 = 2160 nM hA2A = 0,22 nM hA2B >10000 hA3>10000 hA1/hA2A = 9818

O

New A2A water-soluble adenosine antagonists NH2 N

N

N

O

N

N N

N HO

OH

hA1 = 123 nM hA2A = 0,12 nM hA2B >10000 hA3>10000 hA1/hA2A = 1025

Influence of phenyl substitutions on adenosine receptor affinity NH2

N

N

7

N

O

N

N N 8 R R = SO3H hA1 = 139 nM hA2A = 140 nM hA2B = > 10,000 nM hA3 = > 10,000 nM

R = CO2H hA1 = 4,965 nM hA2A = 4.4 nM hA2B = > 10,000 nM hA3 = > 10,000 nM

R =NH2 hA1 = 2,160 nM hA2A = 0.22 nM hA2B = > 10,000 nM hA3 = > 10,000 nM

Antagonists for A2B Adenosine Receptor NH2

N 7

N

N

N

R

N

O

N 8

SCH 58261 R = N7 - CH2CH2-Ph hA1 = 549 nM hA2A = 1.1 nM hA2B = > 10,000 nM hA3 = > 10,000 nM

R = N8 - CH2CH2-Ph hA1 = 1 nM hA2A = 0.31 nM hA2B = 5 nM hA3 = 2,030 nM

O

HN

N

R

N

N

N

7N N 8

8

O

R =CH2NH3+Cl-

R =CH2CH2NH3+Cl-

hA1 = 4.5 nM

hA1 = 2.5 nM

hA2A = 182 nM

hA2A = 50 nM

hA2B = 70 nM

hA2B = 37.6 nM

hA3 = 163 nM

hA3 = 80 nM

Baraldi, P.G. et al., DDR, 2001,53, 225

Summary • A3 adenosine receptor antagonists • MRE3008FE20 story • [3H] MRE 3008FE20

• Irreversible A3 adenosine antagonists • Modelling studies on A3 adenosine receptor antagonists •

Water-soluble A3 adenosine antagonists

A3 adenosine agonists: previous results R = 3-Cl, R1 = H A1 = 45 nM A2A = 420 nM A3 = 4.4 nM O R HN N

EtHN

N

O O OH

N N

N

H R1

R = 4-OMe, R1 = H A1 = 33 nM A2A = 3363 nM A3 = 6.6 nM R = 4-SO2NH2, R1 = H A1 = 453 nM A2A = 1180 nM A3 = 9.7 nM

OH

R = 4-OMe, R1 = Cl A1 = 22 nM A2A = 59 nM A3 = 17 nM

Baraldi, P.G. et al, J.Med.Chem.1996, 802 and 1998,3174

A2A Adenosine Antagonists: Previous Results NH2 N

N

N

NH2

NH2

5

5

N

N N

N

O

N

R

N

N N

N O

O

N A1= 121 nM

A1= 4,7 nM A1/A2A=3,4 A2A=1,4 nM

N N

N

7

N8

N

SCH 58261

A1/A2A=52,6 A2A=2,3 nM

Baraldi, P.G. et al, J.Med.Chem.1996, 39

A3 Adenosine Antagonists: Recent Results NH2 N N

N

N

N

O

O

R

N R

N H

A2A antagonist

NH N

R O HN N

EtHN

O O OH

N H

N

R = 3-Cl, 4-OMe OH

A3 Agonist

N N

N N

N

N

N

R

A3 antagonist

O

A3 adenosine antagonists: affinity and selectivity R = Et, R1 = 4-MeO A1 = 1026 nM A2A = 1045 nM A3 = 0.28 nM

R1

R = Et, R1 = 3-Cl A1 = 249 nM A2A = 185 nM A3 = 2.09 nM

O N H

NH

5 N

N N

N N R

N

8

O

R = Propyl, R1 = 4-MeO A1 = 1197 nM A2A = 141 nM A3 = 0.29 nM R = Phenethyl, R1 = 4-MeO A1 = 201nM A2A = 124 nM A3 = 1.47 nM R = Phenylpropyl, R1 = 4-MeO A1 = 251 nM A2A = 1015 nM A3 = 19.8 nM

Baraldi, P.G. et al., J.Med.Chem, 1999, 4474

Synthetic scheme for the preparation of MRE3000FE20 series CN

CN

CN a

N

NH2

N

c

b

2 N

2 N

NH2

N1

N1

OEt

R

R

H

N

NH2 N

N

N

N

N

NH2 O

d

R

N N

N

N

HN

5

N

O

e

7

R

N

N

N

N

N

N R

8

a: NaH, RX; b: HC(OEt)3; c: furoic hydrazide, PhOPh; d: HCl 10%; e: NH2CN

O

Regiospecific synthesis of a key intermediate to prepare the basic structure MRE3000F20 CN EtO

CN

reflux

+

CN

N

Me

N

CN N Ph

NH Me

reflux, HCL

NC NH2

N Me

N

Preparation of the MRE3000 series R1 NH2 O N

N

N

N

7

N N

H

XPhN=C=O O

N

N

R

N R

NH2 N

N

N

N

N

7

8

N H

N

8

MRE3000F20 series

N XPhN=C=O

7

R

N

N

O

N 8

SCH like compounds

O

NO REACTION

Structural requirements to obtain potent and selective A3 adenosine receptor antagonists OCH3 O

HN

5 6N

N H

4

3

O

N

N

2 N

7

1

N N

8

H3C

9

MRE 3002F20

Baraldi, P.G. et al., Med.Res.Rew., 2000, 20, 103

Modelling studies on some A3 adenosine receptor antagonists

Concluding Remarks on SAR Studies • By utilizing the synthetic procedure here described, we have synthesized more than 100 compounds showing A3 adenosine receptor antagonistic properties • Some of these compounds belonging to the MRE3000F20 chemical series show to be potent (< 1nM) and selective (> 100) at A3 adenosine receptors

[3H] MRE 3008FE20: the first potent and selective radiolabeled A3 adenosine antagonist OCH3 O H

N

N

N 3

H

H

A1 = 1100 nM A2A = 140 nM A2B= 2100 nM A3 = 0.29 nM

N N

N

3

N H

N

O

A1/A3= 1294 A2A/A3= 165 A2B/A3= 2471

Inhibition curves of MRE 3008F20 to human cloned A3, A2A, A1 and A2B adenosine receptors, respectively 100

100

80

80

60

60

Specific binding (%) 40

40

20

20

0 1E-12

1E-11

1E-10

1E-9

1E-8

1E-7

[MRE 3008F20] inhibitor (M)

1E-6

1E-5

0 1E-4

Design of C9 substituted A3 adenosine antagonists R1

O N H

NH

5 N

N N

N N H3C

N

O

9 8

R

R = S - alkyl, NH – alkyl R1 = 4-OMe, 3-Cl

Synthetic Scheme CN

CN

R

CS2 RX K2CO3 DMF

H3CS

CN

H3CS

CN

R'

N

CN CN CH3NHNH2

H3CS

R' NH R

O

R R R'

N

CN

1. HC(OEt)3

R'

N

N

N H3C

N

2. Furoic acid H3C hydrazide 3. Diphenyl ether

NH2

N

N

N N

N

O R, R' = CH2CH3, H;

R R'

OCH3 , H;

N

N

N

1. HCl 10% 2. H2NCN

H3C

N

N N

N

NH2

N

N CH3

Concluding remarks on the C9 substitution • The substitution at C9 position with thioeters, primary, secondary and aromatic amines is detrimental both for affinity and selectivity

• The preparation of Mannich bases on the furane ring produces water-soluble compounds with concomitant loss of affinity Baraldi, P.G. et al., J.Med.Chem, 2003, 48, 1229-1241

Irreversible A3 Adenosine Antagonists FO2S

O HN

NH

5 N

N

N N

N N

8

O

To the concentration of 100 nM the irreversible ligand blocks the A3 human receptor of 79%

Baraldi, P.G. et al., J.Med.Chem, 2001, 44, 2735

Irreversible A3 adenosine antagonists : molecular modeling studies

Design of Water-Soluble A3 Adenosine Antagonists • The A3 adenosine antagonists we have developed showed high affinity ( less than 1 nM) and great selectivity ( more than 100) for the human A3 receptor subtype, but they were only slightly water-soluble compounds. • The next step of our investigation has been of designing new water-soluble A3 adenosine antagonists retaining the same potency and selectivity

Water-Soluble A3 Adenosine Antagonists O R NH

N H

5 N

N N

N N H3C

N

O

8

R = PhSO3H, PhCOOH, PhN(CH3)2, 2, 3, 4- PYRIDINE

The most potent and selective A3 adenosine antagonists water-soluble Cl

O

HN N

NH

H

5 N

N H3C

N

N

NH

H

5 N

N N

N

O

N

O

hA1 = 350 nM hA2A = 100 nM hA2B = 250 nM hA3 = 0.01 nM

H3C

N N

N N

8

N

8

hA1 = 250 nM hA2A = 60 nM hA2B = 200 nM hA3 = 0.04 nM

Baraldi, P.G. et al., J.Med.Chem, 2002, 45, 3579

O

Affinities, expressed as KD values, and selectivity of agonists and antagonists radioligands to human ARs A1 [3H]R-PIA [3H]DPCPX A2A [ H]CGS 21680 [3H]SCH 58261 [3H]ZM241385 3

KD (nM) 2.0 3.9

A2A/A1 429 33

A2B/A1 1915 8.0

A3/A1 37 308

27 0.6 0.8

A1/A2A 11 478 319

A2B/A2A 0.6 >16667 >40

A3/A2A 36 >16667 >12500

A1/A2B

A2A/A2B

A3/A2B

30 32 1.97

0.13 8 204

4.3 0.02 255

40 >312 289

0.8 0.8 2.3

A1/A3 11 1294 190

A2A/A3 589 165 >900

A2B/A3 1100 2471 >900

A2B [3H]DPCPX [3H]ZM241385 [3H]MRS - 1754 A3 [ I]ABMECA [3H]MRE3008F20 [3H]PSB - 11 125

SAR Profile of Pyrazolo-Triazolo-Pyrimidines Free is essential for A2A affinity Arylcarbamoyl moieties give high hA3 affinity Its role for A2B and A1 affinities is not still clear Cyclopentyl and cyclohexyl substituents increase affinity and selectivity versus A1 adenosine subtype Aralkyl chains confer good affinity and selectivity for A2A subtype. Any substitutions resulted to be uneffective in terms of affinity for all the other receptor subtypes

Indispensable for affinity at all four adenosine receptor subtypes NH2

N 7

N

N

O

N N N 8

Both pyrazole nitrogens are strongly involved in selectivity

Branched or aralkyl chains confer A1 and A2B affinity but low selectivity vs A2A, while small substituents (H, Me, Et) confer good affinity and selectivity for hA3 adenosine subtype

Baraldi, P.G., Borea, P.A., Trends in Pharmacol.Sc, 2000, 21, 456

XANTHINES AND PYRAZOLO[4,3-e] 1,2,4 TRIAZOLO [1,5-c]PYRIMIDINES O R1

R5

R7 N

N

N

N

N

R8 O

N R3

N

N

N

R8/R7

N

COMMON TEMPLATES FOR ALL ADENOSINE RECEPTORS: A1, A2A, A2B, A3

O

8-Heterocyclic Xanthine Derivatives: Synthesis and Biological Evaluation of New Potent A2B Selective Adenosine Receptor Antagonists.

selection of several classes of non-xanthine and xanthine adenosine antagonists that have been found to be potent and slightly selective versus A2B receptor. O N O

N

H N

NH2

O O

NH2

N H

N

NN N

N

Cl

CGS15943

XAC NH2 HO N H

O

N

N

O

N

ALLOXAZINE

N

N

ENPROFYLLINE O

N N H

H N

N

N

O N

O N

O

ZM241385 H

H

O

N O

N

H N

O O

N MRS1754

N H

CN

GENERAL STRUCTURE OF THE NEW A2B ADENOSINE RECEPTOR ANTAGONISTS O R

N

H N

H N

Y

N N R R = methyl, n-propyl O

X

R1

O

R2 R3

Y = N1-methylpyrazolo, N2-methylpyrazolo, phenyl X = CH2, NH

O R O

N N R

H N

Y

R1

O

N

O

R = n-propyl, iso-butyl, allyl. Y = N1-methylpyrazolo, N2-methylpyrazolo, isoxazole, pyridine, pyridazine. R1 = substituted aniline, aminopyridine, piperazine rings

O R1 O

H

N

N

N N

N

N

R2

H

N

Binding Data of 8-phenylacetamidopyrazole-xanthine Derivatives

R3 O R4

CH3

R1

Ki(nM) hA1a

Compd

R1

R2

R3

R4

15

n-C3H7

H

H

H 900 (811-996)

16

n-C3H7

OCH3

H

H

> 10000

17

n-C3H7

H

CH2CH(CH3)2 H

> 10000

18

n-C3H7

H

NO2

H

> 10000

19

n-C3H7

H

OCH2C6H5

H

> 10000

20

n-C3H7

H

OH

H

> 10000

21

n-C3H7

H

F

H

200

24

n-C3H7

F

H

H

3227

hA2Ab

hA2Bc

hA3d

> 10000

35 (27-45)

> 10000

> 10000

96 (80-114)

> 10000

> 10000

> 10000

> 10000

> 10000

78 (63-96)

> 10000

> 10000

56 (42-77)

> 10000

> 10000

103 (79-136)

> 10000

> 10000

88 (84-92)

> 10000

> 10000

50 (41-60)

> 10000

O R1 O

H

N

N

N N

N

R2

H

N

N

R3 O R4

CH3

R1

Binding Data of phenylacetamido-pyrazolexanthine Derivatives Ki(nM)

Compd

R1

R2

R3

R4

hA1a

hA2Ab

hA2Bc

hA3d

25

n-C3H7

H

N(CH3)2

H

> 10000

> 10000

1628 (1374-1930)

> 10000

26

n-C3H7

H

Cl

H

520 (484-558)

> 10000

28 (23-33)

> 10000

27

n-C3H7

OCH3

OCH3

H

> 10000

> 10000

38 (33-43)

> 10000

28

n-C3H7

H

OCH2-o-CF3-C6H5

H

56 (47-67)

> 10000

13 (11-16)

> 10000

34

n-C3H7

H

566 (516-621)

1249 (856-1822)

18 (12-27)

> 10000

35

n-C3H7

OCH3

OH

H

> 10000

175 (1343-2292)

342 (274-426)

> 10000

36

CH3

H

H

H

> 10000

> 10000

569 (506-640)

> 10000

OCH2O

O N

N O

H N

H

N

N

N

N

R1

H N

R2

O

H 3C

Binding Data of Phenylureidic-pyrazolexanthine Derivatives

Ki(nM)

Compd.

R1

R2

hA1a

hA2Ab

hA2Bc

hA3d

40

H

N(CH3)2

2410 (1760-3301)

> 10000

59 (44-81)

> 10000

41

Cl

H

448 (365-550)

> 10000

39 (33-46)

> 10000

42

OCH3

H

1993 (1658-2397)

> 10000

90 (73-110)

> 10000

43

H

OCH3

1440 (1250-2211)

> 10000

81 (70-110)

> 10000

SYNTHESIS H N HOOC a

O

O N N CH3

2

O RN O

NH2 N R

a

n-C3H7

N

O

H N HOOC

O

NH2 N N

N N n-C3H7

CH3

6

O O

NN

H N

O 3

CH3

R

N

O

NH2

N R

H N N

NH2 N N H3C 7a, b

1a, b a: R = CH3 b: R = n-C3H7 HOOC a

H N

O

O O

4

n-C3H7

N

a

O n-C3H7 O

NHR2

N n-C3H7 8 : R1 = CH3, R2 = H 9 : R1 = H, R2 = H

HOOC COOC2H5 N CH3 5

R1

N

O

N

N

N

H N

N N n-C3H7

COOH N N

CH3

10 a : i) methanol, DCI, 4-5 hrs; ii) methanol, NaOH 2.5 N, 70 °C, 12 hrs.

Synthesis O n-C3H7

N

O

N N

O

N

N R

N N H3C

O

N

N

N

O

R NH2 O

R1

HO O

N R

O

R1

N N n-C3H7

(i):

i

R

R2 , SOCl2, TEA, CH2Cl2 R3

N N R

H N

H N

N N n-C3H7

O

CH3

NH2

O n-C3H7

N

O

H N

N

n-C3H7

NH2

N 6N n-C3H7

O R

O

H N

N N

H N N

R1

H N

H N N

O CH3

N N H3C

R2 O

H N

R3

R1 O

R2 R3

Binding and Functional Data of Selected A2B Adenosine Compounds. [3H]-DPCPX binding in HEK 293

cAMP assay in CHO cells

membranes expressing human A2B

expressing human A2B adenosine

adenosine receptors Ki (nM)

receptors IC50 (nM)

15

35 (27-45)

103 (92-115)

19

56 (42-77)

185 (154-198)

24

50 (41-60)

160 (146-188)

26

28 (23-33)

128 (114-144)

27

38 (33-43)

120 (103-140)

28

13 (11-16)

93 (84-102)

34

18 (12-27)

95 (90-101)

39

34 (26-46)

152 (136-170)

Compd

Conclusions of our preliminary studies: 1) introduction of a N1 or N2 methyl-pyrazol-3-yl ring in place of a phenyl ring, gave compounds showing a moderate A2B adenosine receptor affinity. 2) Amine function (positive charge) on pyrazole ring has importance rather than a negatively charged group. Trying to enhance this interaction by acylation permits to decrease affinity for both A1 and A2B and decreasing selectivity for A2B receptors 3) substitution (o-CF3, m-CF3, p-NO2) of the 4-benzyloxy group at para position of phenyl ring produced increasa of affinity and selettivity vs A2B adenosine receptor. 4) Introduction of bulky groups at 3, 4 position in the phenylacetic acid chain, such as 3,4-dimethoxy (compound 27) led to potent and selective A2B AR antagonist, whereas 3,4,5-trimethoxy (compound 33) showed no affinity.

OUR PROJECTS IN THE SECOND PART The purpose of our investigation was to synthesize a new series of 8-substituted xanthine derivatives in which the xanthine nucleus was linked to a differently substituted oxyacetamide chain by an heterocylic spacer (pyrazole, isoxazole, pyridine, pyridazine), and to determinate the effects of such substitutions on the affinity and selectivity of compounds obtained as A2B adenosine receptor antagonist. Baraldi, P.G. et al., J.Med.Chem, 2004, 47, 1434-1437

O R1 O

N N R1

Binding Data of Oxymethylene-anilidepyrazole-xanthine Derivatives

O H N N

O N

N

N H

R2

Ki nM Compd

R1

R2

hA1a

hA2Ab

hA2Bc

hA3d

11

n-C3H7

4-F-phenyl

65 (48-86)

> 10000

12 (7-21)

> 10000

12

n-C3H7

4-Br-phenyl

150 (132-170)

> 10000

20 (16-25)

> 10000

13

iso-C4H9

4-F-phenyl

467 (400-546)

> 10000

303 (260-352)

> 10000

14

iso-C4H9

4-Br-phenyl

2427 (2067-2850) > 10000

132 (98-178)

> 10000

15

n-C3H7

3,4-methylendioxy-phenyl

200 (180-226)

> 10000

5.5 (4.6-6.5)

> 10000

19

n-C3H7

4-methyl-phenyl

79 (72-86)

> 10000

19 (12-29)

> 10000

20

n-C3H7

4-N-morpholin-phenyl

> 10000

> 10000

86 (78-93)

> 10000

O R1

N

O

N R1

H N N

Adenosine Receptor Affinities of N2-methylpyrazole-Xanthine Derivatives

O O N N

N H

R2

Ki(nM) Compd

R1

R2

hA1a

hA2Ab

hA2Bc

hA3d

21

n-C3H7 4-ethoxy-carbonyl-phenyl

> 10000

> 10000

32 (22-45)

> 10000

22

n-C3H7

4-carboxy-phenyl

> 10000

> 10000

36 (27-47)

> 10000

23

n-C3H7

3,4-dimethyl-phenyl

700 (650-760)

> 10000

10 (8-13)

> 10000

24

n-C3H7

3,4-dichloro-phenyl

300 (240-380)

> 10000

16 (12-20)

> 10000

25

n-C3H7

3,4-dimethoxy-phenyl

> 10000

> 10000

12 (8-17)

> 10000

26

n-C3H7

pyridin-4-yl

955 (896-1017) > 10000

41 (35-48)

> 10000

O R1

N

O

O

H N N

N R1

O N N

N H

R2

Adenosine Receptor Affinities of N1-methyl-pyrazoleXanthine Derivatives

Ki(nM) Compd

R1

R2

hA1a

hA2Ab

hA2Bc

hA3d

27

n-C3H7

4-Br-phenyl

168 (140-201)

>10000

93 (82-105)

>10000

28

n-C3H7

4-F-phenyl

181 (127-258)

> 10000

185 (163-210)

> 10000

29

iso-C4H9

4-Br-phenyl

49 (34-72)

> 10000

66 (38-116)

> 10000

30

iso-C4H9

4-F-phenyl

72 (45-114)

> 10000

207 (162-265)

> 10000

O

O

H N

R1 N O

OH N

N

R1 N

ii

O

O

NH2

iii

O R1 N O

N R1

O

i:

HO N

OC2H5

, DCI, CH3OH, r.t;

N

3

O

ii: Amine derivative, DCI, HOBt, DMF, rt, 2-6 h O O

iii: HO N O

N

4

N

NH2

R1 = n-propyl: 2a, isobutyl: 2b, allyl: 2c

O

R'

11-26, 31-34, 37

R1 = n-propyl: 44a isobutyl: 44b allyl: 44c

i

N R1

N

R1

O N

O

N

N

R1

R1

H N

O

N

N

O

O

OC2H5

, DCI,CH3OH, r.t;

O H N N

O N

O OH

N

R1 = n-propyl: 45a isobutyl: 45b

ii

R1 N O

N R1

O H N N

O N

N

27-30

R'

O

O R1

N

O

O

H N

N N

N R1

N

N

N

Compd

R1

R

31

n-C3H7

32

R

Adenosine A2B Receptor Affinities of Piperazinyl Xanthine Derivatives Ki(nM)

phenyl

hA1a 250 (181-348)

hA2Ab > 10000

hA2Bc 15 (10-21)

hA3d > 10000

n-C3H7

4-F-phenyl

> 10000

> 10000

55 (46-65)

> 10000

33

n-C3H7

CH3

> 10000

> 10000

122 (108-136)

> 10000

34

n-C3H7

benzyl

810 (763-859)

> 10000

85 (66-95)

> 10000

35

n-C3H7

phenyl

260 (232-287)

> 10000

12 (7-19)

> 10000

36

n-C3H7

CH3

> 10000

> 10000

76 (67-86)

> 10000

37

CH2CH=CH2

phenyl

> 10000

> 10000

24 (18-32)

> 10000

Hydrochloride Derivatives: Compoundes 35 and 36

R1 O N O

N

R2

O H N

O

N H

O N

N

Binding Data of Isoxazole-xanthine Derivatives at Adenosine Receptors Ki(nM)

Compd 38

R1

R2

OCH2O

hA1a

hA2Ab

hA2Bc

hA3d

> 10000

> 10000

47 (43-52)

> 10000

39

OCH3

OCH3

> 10000

> 10000

51 (44-58)

> 10000

40

H

F

> 10000

> 10000

70 (61-80)

> 10000

41

H

OCH3

> 10000

> 10000

53 (40-69)

> 10000

Synthesis of Isoxazole Xanthine Derivatives O

O NH2

N O

N

i

N O

NH2

N

O

H N

O O

N

OH

N

46 ii R1 O N O

N

N

O O

N

38-41 O O

i : HO O

N

OC2H5

, DCI, CH3OH, r.t.

5

O R1

ii: H2N

R2

O

H N

R2 , DCI, HOBt, DMF, r.t.

N H

O

H N

N O

H N

O N

N

X N

O

I

Binding Data of Pyridine/Pyridazinexanthine Derivatives

Ki(nM) Compd

X hA1a

hA2Ab

hA2Bc

42

CH

> 10000

> 10000

108 (75-155)

hA3d > 10000

43

N

> 10000

> 10000

> 10000

> 10000

Preparation of radiolabeled A2B adenosine antagonist O O N O

N

H N

O N N

N

hA1 200 hA2A >10000 hA2B 5 nM hA3 > 10000

O

O N H

MRE2029F20

O O

O N O

N

N

O

O

H N

O N N

N H

hA1 200 hA2A >10000 hA2B 9 nM hA3 > 10000

H3

H3

O N

O

N

N

O

O

H N

O N N

N H

H3 H3

[3H] MRE2029F20

Baraldi et al. Bioorg. Med. Chem. Lett. In press.

Conclusions: 1) The presence of pyrazole ring at 8 position of xanthine moiety confirmed to be an important structural requirement for A2B adenosine receptor binding. . Among them, derivatives bearing the N1-methyl-pyrazole isomer, exhibited lower affinity than the corresponding N2-methyl analogues for A2B receptor. In addition, it was observed that N1-methyl derivatives demonstrated a global increase of affinity toward A1 receptor with consequent loss of selectivity. 2) Compounds bearing the isoxazole nucleus at 8 position showed lower affinity at A2B receptor than 8-pyrazole derivatives, anyway replacing the pyrazole ring with the isoxazole one, enhanced selectivity versus A1 adenosine receptor

3) Another result that supported the fundamental importance of the pyrazole is the complete loss of affinity produced by the substitution of pyrazole with pyridine or pyridazine rings. Although pyridine and pyridazine showed to be detrimental in terms of affinity, the pyridine derivative seemed to interact with A2B better than the pyridazine derivative, this suggested that the electronic and lipophilic characters play a fundamental role to influence A2B binding parameters, in addition to steric factors. 4) Further important information was furnished by the introduction of different moiety on the side chain of the heterocyclic spacer differed for electronic, steric and lipophilic features. Small hydrophobic residue in 3 and/or 4 position of the phenyl ring seemed to increase affinity toward A2B receptor. In particular the presence of electron donating groups, such as -OCH3 or 3,4-methylendioxy, increased affinity .

5) Introduction of substituents containing a carbonylic function (17, 21, 22), determined a light loss of affinity but an improvement of selectivity versus other adenosine receptors subtypes, in particular versus A1 adenosine receptors. The most interesting compound in terms of affinity was 15 (hA2B = 5.5 nM) presenting 3,4-methylendioxy function on the phenyl ring, while the 3,4dimethoxy derivative 25 (hA2B = 12 nM) exhibited a good retention of affinity with improved selectivity for A2B 6) In order to obtain water-soluble compounds, we replaced the phenyl ring in the side chain with a 4-substituted-piperazine, introducing different kind of substituents in N4 position of piperazine. The choice of 4-F-phenyl ring appeared to be the most interesting modulation in terms of both affinity and selectivity. 1,3di-n-propyl-8-{5-[2-oxo-2-(4-phenyl-piperazin-1-yl)-ethoxy]-2methyl-2H-pyrazole-3-yl}-xanthine (31) and its hydrochloride salt (35) showed high affinity and good selectivity at A2B receptor (respectively hA2B = 15 nM; hA2B = 12 nM).

7) In our SAR study we have also investigated the importance of the alkyl substitution at 1,3-positions of xanthine structure. Introduction of isobutyl chains produced a marked decrease of affinity toward A2B adenosine receptors. Another interesting finding was the substitution of 1,3-dipropyl pattern with 1,3-diallyl one, which produced an increase of selectivity.

Synthesis and Biological Activity of P2X7 (P2Z) Receptor Antagonists Structurally Related to KN62

A1 A2A P1

A2B A3

Purinergic receptors

P2Y1 P2Y2 P2Y3

P2

P2Y

P2Y4 P2Y5

P2X

P2Y6 P2Y7

P2X1 P2X2 P2X3 P2X4 P2X5 P2X6

P2X7 o P2Z

P2X7 Receptor: -P2X7 is a ligand-activated ion channels (ionotropic receptor), permeable to hydrophilic molecules with molecular weight up to 900 Da. -This receptor is mainly, if not exclusively, expressed in cells of hematopoietic origin (mast cells, macrophages, lymphocytes), where it mediates cytotoxic responses, cytochine release and cell fusion - Activation of the P2X7 receptor in macrophages and microglial cells causes a large a rapid release of mature interleukin-1b (IL-1b). IL-1b is of prime importance in the induction of the immune responses, including facilitating responses to antigens, synthesis of prostaglandins, proliferation of fibroblasts, blood neutrophils, and inducing the synthesis of other cytokines.

…P2X7 Receptor:

-P2X7 receptor is considered a promising new target for antiinflammatory drug development. - Due to its likely involvement in immunomodulation, it would be of the most importance to develop selective P2X7 antagonists. - Actually there is only one specific antagonist of the P2X7 receptor active in the nanomolar range.

Structure of selected P2Z antagonists NaO3S

N NH2

SO3Na

N

N

O

N N H

N

N

PPPO CHOCHO

Brilliant Blue G (noncompetitive) IC50=400 nM

oxidized-ATP (irreversible)

N

N

O S O O

O S O O N

N

H N N

N

S O O

KN04 (noncompetitive) IC50=85 nM

N

N

CH3 N S O O O

KN62 (noncompetitive) IC50=51 nM (measured by ATP-stimulated calcium influx into human macrophages)

Conformationally constrained analogues of KN62 N O S O O N N O S O O

N N

CH3

N

N S O O O

Conformationally constrained analogue of KN62 completely inactive

N

N

N

S O O O

N

KN62 IC50=51 nM

O S O O

N

N

N S O O O

IC50=316 nM 6-fold less active than KN62

Which modifications could improve the activity of the reference compound? N O S O O

O

N

N

CH3 N S O O N O KN62

B

S O O B

W N n(H2C) A

N

Z

X Y

Y X Z

S O O O

R1 X,Y,Z=CH or N W=H or CH3 A=N or CH B=H or halogen n= from 0 to 4 R1 =electron-releasing or electron-withdrawing groups

Synthetic metodology OH

OH

A=CH or N n=0,1,2 R=NO2, CH3, halogen, ,CN, acetyl, methoxy X,Y=N when R=H

a O CO2H Boc

N

N

Boc

N

A (CH2)n X

Y R

b

Reaction. a: substituted aryl or heteroaryl piperazine, HOBt (1.1 equiv.), EDCI (1.1 equiv.), DMF, r.t., 24 hours; b: TFA, DCM; c: TEA, isoquinoline sulphonyle chloride, DMF.

N

O O S O

OH c O

O

N

N N O S O

A (CH2)n X N

Y R

HN

A (CH2)n R

X

Y

Modification of the phenyl moiety: effect of the introduction of a substituent on the para position Substituent (X)

Activity (IC50)

H (KN62)

51

F

1.33

Cl

105

I

176

CH3

13.5

OCH3

132

NO2

5.76

NH2

101

CN

98

CH3CO

71

N O S O O N

X

N

N

CH3 N S O O O

Baraldi, P.G. et al., J.Med.Chem., 2003, 46, 1318-1329

Modification of the linker between phenyl and piperazine N O S O O N

N

N

CH3 N S OO O

KN62 IC50 = 51 nM N

N O S O O

O S O O

N

N

N

N

CH3 N S OO O

Benzyl derivative IC50 = 21 nM

N

N

CH3 N S OO O

Phenylethyl derivative IC50 = 600 nM

Modification on the phenyl ring linked to the piperazine moiety N O S O O N

N

N

CH3 N S OO O

KN62 IC50 = 51 nM

N

N O S O O

O S O O

N

N

N N

N

CH3 N S OO O

Pyridine derivative IC50 = 170 nM

N N

N

CH3 N S OO O

N Pyrimidine derivative IC50 = 80 nM

Modification of the isoquinoline moiety: effect of the substitution with a pyridine N

N

N

O S O O

O S O O

O S O O

N

N

N

N

CH3 N S OO O

N

KN62 IC50=51 nM

N

CH3 N S OO O

N

N

IC50=955 nM

N

IC50 = 15 nM

N

N

N

O S O O

O

O

S O O

S O O

CH3 N S OO O

N

N

N

CH3 N S OO O

IC50 = 9120 nM

N F

N

N

CH3 N S OO O

IC50 = 1.33 nM

F

N

N

CH3 N S OO O

IC50 = 1148 nM

N

Structure-activity relationship (SAR) in the series of synthesized compounds:

-the 5-isoquinoline moieties are essential for the activity - the heteroaryl piperazine derivatives do not improve the activity - the unsubtituted benzyl piperazine is more active than the phenyl piperazine counterpart - about the effect on the biological activity of substituents on the phenyl ring linked to the piperazine, for the same substituent the ortho and meta was favorited than the para position. - the N-methyl seems not basic for the biological activity

Acknowledgements • • • • • • • • • • •

Dott.Romeo Romagnoli Dott.ssa Mojgan Tabrizi Prof.Hussein El-Kashef Dott.ssa Francesca Fruttarolo Dott.ssa Delia Preti Dott.Andrea Bovero Prof.Pier Andrea Borea Dott.ssa Katia Varani Dott.Ennio Ongini (Schering-Plough) Dott.Eddie Leung (Medco/Kings) Dott.Allan Moorman (Medco/Kings)