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Title:
P-GP RADIOTRACERS FOR IMAGING AS BIOMARKER INVOLVED IN ONSET OF NEURODEGENERATIVE DISEASES
Document Type and Number:
WIPO Patent Application WO/2016/174496
Kind Code:
A1
Abstract:
The invention includes a new class of radiolabeled compounds of formula I able to bind P-glycoprotein with high affinity and selectivity. The protein prevents accumulation in the brain of a wide range of drugs and, changes in P-gp expression and function, are also believed to occur in several neurological disorder, such as Alzheimer's disease (AD). Indeed, it is widely reported that decreased expression of P- gp and/or activity at the BBB level could cause accumulation of I3- amyloid plaques, the main hallmark of AD onset. 13-amyloid is a P-gp substrate, and these results support the importance of P-gp in the clearance of 13-amyloid peptide from brain parenchyma. For this reason, P-gp can be considered a useful biomarker for the early diagnosis of neurodegeneration. There is considerable interest in the development of P-gp PET radiotracers for imaging expression and function of P-gp. Formula I wherein the compound is labeled with an isotope selected from H, 11C 14C, 13N, 150 18F 19F, 75Br, 76Br, 79Br.

Inventors:
GHAFIR EL IDRISSI IMANE (IT)
CANTORE MARIANGELA (IT)
BERARDI FRANCESCO (IT)
LEOPOLDO MARCELLO (IT)
PERRONE ROBERTO (IT)
COLABUFO NICOLA ANTONIO (IT)
Application Number:
PCT/IB2015/053046
Publication Date:
November 03, 2016
Filing Date:
April 27, 2015
Export Citation:
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Assignee:
BIOFORDRUG S R L (IT)
International Classes:
C07D217/04; A61K31/472; A61P25/28; C07D217/06
Domestic Patent References:
WO2012159666A12012-11-29
Foreign References:
US20120171119A12012-07-05
Other References:
SEVERIN MAIRINGER ET AL: "Synthesis and preclinical evaluation of the radiolabeled P-glycoprotein inhibitor [11C]MC113", NUCLEAR MEDICINE AND BIOLOGY, vol. 39, no. 8, 1 November 2012 (2012-11-01), pages 1219 - 1225, XP055210338, ISSN: 0969-8051, DOI: 10.1016/j.nucmedbio.2012.08.005
ELENA CAPPARELLI ET AL: "SAR Studies on Tetrahydroisoquinoline Derivatives: The Role of Flexibility and Bioisosterism To Raise Potency and Selectivity toward P-glycoprotein", JOURNAL OF MEDICINAL CHEMISTRY, vol. 57, no. 23, 11 December 2014 (2014-12-11), pages 9983 - 9994, XP055196172, ISSN: 0022-2623, DOI: 10.1021/jm501640e
BARTELS, A. L.: "Blood-Brain Barrier P-glycoprotein function in neurodegenerative disease", CURR. PHARM. DES., vol. 17, 2011, pages 2771 - 2777
PALMEIRA, A.; SOUSA, E.; VASCONCELOS, M. H.; PINTO, M. M.: "Three decades of P-gp inhibitors: skimming through several generations and scaffolds", CURR. MED. CHEM., vol. 19, 2012, pages 1946 - 2025
ZINZI, L.; CAPPARELLI, E.; CANTORE, M.; CONTINO, M.; LEOPOLDO, M.; COLABUFO, N. A.: "Small and innovative molecules as new strategy to revert MDR Front", ONCOL., vol. 4, 2014, pages 2
COLABUFO, N. A.; BERARDI, F.; PERRONE, M. G.; CAPPARELLI, E.; CANTORE, M.; INGLESE, C.; PERRONE, R: "Substrates, inhibitors and activators of P-glycoprotein: candidates for radiolabeling and imaging perspectives", CURR. TOP. MED. CHEM., vol. 10, 2010, pages 1703 - 1714
DORNER, B. ET AL., J. MED. CHEM, vol. 52, 2009, pages 6073 - 6082
LUURTSEMA, G. ET AL., NUCL. MED. BIOL., vol. 36, 2009, pages 6073 - 6082
BANKSTAHL, J. P. ET AL., J. NUCL. MED. CHEM, vol. 49, 2008, pages 1328 - 1335
COLABUFO, N. A. ET AL.: "Small P-gp modulating molecules: SAR studies on tetrahydroisoquinoline derivatives", BIOORG. MED. CHEM., vol. 16, 2008, pages 362 - 373
VAN WAARDE, A. ET AL.: "Synthesis and preclinical evaluation of novel PET probes for P-glycoprotein function and expression", J. MED. CHEM., vol. 52, 2009, pages 4524 - 4532
COLABUFO, N. A. ET AL.: "4-Biphenyl and 2-naphthyl substituted 6,7-dimethoxytetrahydroisoquinoine derivatives as potent P-gp modulators", BIOORG. MED. CHEM., vol. 16, 2008, pages 3732 - 3743
AZZARITI, A. ET AL.: "MC70 potentiates doxorubicin efficacy in colon and breast cancer in vitro treatment", EUR. J. PHARMACOL., vol. 670, 2011, pages 74 - 84
MAIRINGER, S. ET AL.: "Synthesis and preclinical evaluation of the radiolabeled P-glycoprotein inhibitor [11C]MC113", NUCL. MED. BIOL., vol. 39, 2012, pages 1219 - 1225
CHOPRA, A.: "0-[11 C]Methyl Derivative of 6,7-Dimethoxy-2-(4-methoxybiphenyl-4-ylmethyl)-1,2,3,4-tetrahydro-isoquinoline", IMAGING AND CONTRAST AGENT DATABASE (MICAD), 2013
WANEK, T.; MAIRINQER, S.; LANQER, O.: "Radioligands targeting P-glycoprotein and other drug efflux proteins at the blood-brain barrier", J. LABELLED COMPD. RADIOPHARM., vol. 56, 2013, pages 68 - 77
BAUER, S.; OCHOA-PUENTES, C.; SUN, Q.; BAUSE, M.; BERNHARDT, G.; KÖNIG, B.; BUSCHAUER, A.: "Quinolinecarboxamide type ABCG2 modulators: indole and quinoline moieties as anilide replacements", CHEM.MED. CHEM., vol. 8, 2013, pages 1773 - 1778
MAIRINQER, S.; ERKER, T.; MULLER, M.; LANQER, O.: "PET and SPECT radiotracers to assess function and expression of ABC transporters in vivo", CURR. DRUG METAB., vol. 12, 2011, pages 774 - 792
FENG ET AL.: "In vitro P-glycoprotein assays to predict the in vivo interactions of P-glycoprotein with drugs in the central nervous system", DRUG METAB. DISPOS., vol. 36, 2008, pages 268 - 275
RAUTIO, J. ET AL.: "In vitro P-glycoprotein inhibition assays for assessment of clinical drug interaction potential of new drug candidates: a recommendation for probe substrates", DRUG METAB. DISPOS., vol. 34, 2006, pages 786 - 792
POLLI, J. W. ET AL.: "Rational use of in vitro P-glycoprotein assays in drug discovery", J. PHARMACOL. EXP. THER., vol. 299, 2001, pages 620 - 628
Attorney, Agent or Firm:
GERMINARIO, Claudio et al. (Piazza di Pietra 39, Rome, IT)
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Claims:
CLAIMS

1. A radiolabelled compound having formula I:

wherein:

R is selected from: OCH3, OH, F, N02, NH2, Br, COCH3, NHCH3, N(CH3)2 or N(C2H5)2.

X is selected from: CH2, CH2CO, NH, O, S, NH(CH3), NH(C2H5).

n is an integer number selected from: 0 and 5.

Ar is a phenyl or an heterocyclic group selected from the following groups:

2. The radiolabelled compound according to claim 1 having formula II:

where R, Ar, X and n have the same meaning as in claim 1.

3. The radiolabelled compound according to claim 1 having formula III:

where Ar and R have the same meaning indicated in claim 1.

4. The compound according to any one of claims 1 or 2 wherein Ar is a phenyl group.

5. The compound according to any one of claims 1 to 4 wherein and R is 4'-methoxy group. 6. The compound according to anyone of claims 1 to 4 wherein R is a 3'- methoxy group.

7. The compound according to anyone of claims 1 to 4 wherein R is a 4'- or 3'- F group.

8. The compound according to any one of claims 1 , 2, 4 to 7, wherein X is oxygen.

9. The compound according to any one of claims 1 , 2, 4 to 7, wherein X is a -CH2- group.

10. The compound according to any one of claims 1 , 2, 4 to 7, wherein X is a -CH2CO- group.

11. The compound according to any one of claims 1 , 2, 4 to 10, wherein n is 0, 1 , 2, 3 or 4.

12. The compound according to anyone of claims 1 to 1 1 wherein the labeling isotope selected from 11C, 18F.

13. A compound selected from the group comprising :

1 -(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)-2-(4'-methoxy-[1 , 1 '- biphenyl]-4-yl)ethanone.

6,7-Dimethoxy-2-(2-(4'-methoxy-[1 , 1 '-biphenyl]-4-yl)ethyl)-1 ,2,3,4- tetrahydroisoquinoline.

2-(2-(4'-Fluoro-[1 , 1 '-biphenyl]-4-yl)ethyl)-6,7-dimethoxy-1 ,2,3,4- tetrahydroisoquinoline.

2-((4'-Fluoro-[1 , 1 '-biphenyl]-4-yl)methyl)-6,7-dimethoxy-1 ,2,3,4- tetrahydroisoquinoline.

6,7-Dimethoxy-2-(2-((4'-methoxy-[1 , 1 '-biphenyl]-4-yl)oxy)-ethyl)-1 ,2,3,4- tetrahydroisoquinoline.

6,7-Dimethoxy-2-(3-((4'-methoxy-[1 , 1 '-biphenyl]-4-yl)oxy)-propyl)-1 ,2,3,4- tetrahydroisoquinoline.

6,7-Dimethoxy-2-(4-((4'-methoxy-[1 , 1 '-biphenyl]-4-yl)oxy)-butyl)-1 ,2,3,4- tetrahydroisoquinoline.

/V-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)-propyl)-4'-methoxy- [1 , 1 '-biphenyl]-4-amine.

4'-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)-butoxy)-[1 , 1 -biphenyl]- 4-ol. 2'-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)-ethyl)-[1 , 1 '-biphenyl]-3- ol.

6,7-Dimethoxy-2-(2-(3'-methoxy-[1 , 1 '-biphenyl]-2-yl)ethyl)-1 ,2,3,4- tetrahydroisoquinoline.

14. The compound according any one of claims 1 to 13 for use in an in vivo diagnostic method of a neurodegenerative disease involving P- glycoprotein. 15. The compound according to claim 14 wherein the neurodegenerative disease is the Alzheimer's or Parkinson's disease.

16. The compound according to claims 14 or 15 wherein the diagnosis is performed by PET o SPECT analysis.

17. A diagnostic imaging composition comprising as imaging agent a compound according to anyone of claims 1 to 13 and a carrier.

18. The diagnostic imaging composition according to claim 17 for use in vivo diagnosis of a neurodegenerative disease involving P-glycoprotein.

19. The diagnostic imaging composition according to claim 18 wherein the neurodegenerative disease is the Alzheimer's or Parkinson's disease. 20. The diagnostic imaging composition according to any one of the claims 17 to 19 in the form of an injectable solution wherein the imaging agent is from 4 mg/ml_ to 7.5 mg/ml_.

21. A compound according to any one of the claims 1 to 13 for use as medicament.

22. A compound according to claim 21 for use as inhibitor, inducer or modulator of the P-glycoprotein function in the prevention or treatment of neurodegenerative pathologies involving P-glycoprotein.

23. A compound according to claim 22 wherein said pathology is Alzheimer's or Parkinson's disease.

24. A pharmaceutical composition comprising a compound according to any one of the claims 1 to 13 and a pharmacologically acceptable excipient and/or diluent.

25. A pharmaceutical kit formed by a first element containing a precursor of a compound according to any one of the claims 1 to 13 and a second element containing radionuclide isotope suitable for preparing the radiolabeled compound according to claims 1 to 13.

26. A method for preparing the compounds according to claims 1 or 2 comprising the following steps illustrated in Scheme 1 :

• preparing the carboxylic acid C by coupling reaction of precursor A and B;

• condensing carboxylic acid C with 6,7-dimethoxytetrahydoisoquinoline to obtain amide D;

• reducing amide D, with LiAIH4,

• isolating and optionally purifying amines I,

wherein at least one passage comprises introducing in the compound a radiolabelling isotope.

and wherein R' is selected from: -COOH, -CH2COOH, -OCH3, OH, and all other substituents are as defined above.

27. A method of preparing the compounds according to claims 1 or 3 comprising the following steps illustrated in Scheme 3:

• preparing the carboxylic acid G by coupling reaction of precursor E and F;

• condensing carboxylic acid G with 6,7-dimethoxy-1 ,2,3,4- tetrahydoisoquinoline to obtain amide H;

• reducing amide H with LiAIH4,

• reduction to final amines III;

• isolating and optionally purifying amine III,

wherein at least one passage comprises introducing in the compound a radiolabelling isotope.

and wherein all substituents are as defined above.

Description:
P-GP RADIOTRACERS FOR IMAGING AS BIOMARKER INVOLVED IN ONSET OF NEURODEGENERATIVE DISEASES

DESCRIPTION TECHNICAL FIELD OF THE INVENTION

The invention relates to a new class of radiolabelled compounds with high affinity and selectivity towards P-glycoprotein. The invention also relates to the utilization of such compounds for the in vivo diagnosis of neurodegenerative diseases. Several compounds are useful as medicaments for the prevention and treatment of neurodegenerative diseases involving P- glycoprotein.

BACKGROUND OF THE INVENTION

P-glycoprotein (P-gp) is an ATPase protein, belonging to the ATP- Binding Cassette (ABC) transporters family, that uses the energy of ATP hydrolysis to efflux drugs and xenobiotics out of cells and therefore plays a key role in absorption and detoxification processes. P-gp is encoded by MDR1 and MDR2 genes in humans.

P-gp is a transmembrane protein localized at the apical side of cell membranes involved in the adsorption and elimination of xenobiotics such as liver, kidney, and gut, and in some barriers such as the Blood-Brain Barrier (BBB), Blood-Testis Barrier (BTB), and Blood-CerebroSpinal Fluid Barrier (BCSFB).

Moreover, changes in P-gp expression and function are involved in several neurological disorders, such as Alzheimer's disease or Parkinson's disease. (Bartels, A. L. "Blood-Brain Barrier P-glycoprotein function in neurodegenerative disease" Curr. Pharm. Des. 201 1 , 17, 2771-2777).

Therefore, P-gp is important not only as a target for the prevention and treatment of the neurodegenerative disorders, but also for the development of radiotracers useful in imaging method such as PET and SPECT analyses to detect the activity of this protein, and therefore the onset of neurodegenerative disease.

In recent years, many different classes of ligands have been tested for their ability to modulate P-gp activity (Palmeira, A.; Sousa, E.; Vasconcelos, M. H.; Pinto, M. M. "Three decades of P-gp inhibitors: skimming through several generations and scaffolds" Curr. Med. Chem. 2012, 19, 1946-2025. Zinzi, L; Capparelli, E.; Cantore, M.; Contino, M.; Leopoldo, M.; Colabufo, N. A "Small and innovative molecules as new strategy to revert MDR Front". Oncol. 2014, 4, 2).

Several P-gp ligands have also been used, as radiolabeling probe with 11 C or 18 F, for imaging P-gp function and expression in vivo by Positron Emission Tomography (PET). (Colabufo, N. A.; Berardi, F.; Perrone, M. G.; Capparelli, E.; Cantore, M.; Inglese, C; Perrone, R. Substrates, inhibitors and activators of P-glycoprotein: candidates for radiolabeling and imaging perspectives Curr. Top. Med. Chem. 2010, 10, 1703-1714). However their diagnostic use was limited by their low non-specific uptake, the production of radiometabolites falsifying the analysis and their poor selectivity towards other ABC transporters.

P-gp inhibitors such as [ 11 C]elacridar, [ 11 C]laniquidar and [ 11 C]tariquidar were in vivo tested for the detection of P-gp expression (Dorner, B. et al. 2009, J. Med. Chem, 52, 6073-6082, Luurtsema, G. et al., 2009, Nucl. Med. Biol., 36, 6073-6082; Bankstahl, J. P. et al., 2008, J. Nucl. Med. Chem., 49, 1328-1335), but the absence of selectivity towards other ABC transporters, such as BCRP and MRP1 , the low brain uptake and a different dose-dependent behavior has limited their diagnostic use.

Starting from two small molecules namely [ 11 C]MC18 and [ 11 C]MC266, having the formula showed below, that displayed potent inhibitory and substrate profiles respectively, (see Colabufo, N. A.; et al., "Small P-gp modulating molecules: SAR studies on tetrahydroisoquinoline derivatives", Bioorg. Med. Chem. 2008, 16, 362-373; or Van Waarde, A. et al. "Synthesis and preclinical evaluation of novel PET probes for P-glycoprotein function and expression" J. Med. Chem. 2009, 52, 4524^1532), new molecules have been designed, wherein the spacer has been constrained into a biphenyl fragment, obtaining several P-gp ligands such as MC70 and MC113, having formula showed below, wherein the biphenyl fragment is linked to 6-7 dimethoxytetrahydoisoquinoline and displays P-gp activity in the submicromolar range (see Colabufo, N. A. et al. "4-Biphenyl and 2-naphthyl substituted 6,7-dimethoxytetrahydroisoquinoline derivatives as potent P-gp modulators" Bioorg. Med. Chem. 2008, 16, 3732-3743 or Azzariti, A. et al.

"MC70 potentiates doxorubicin efficacy in colon and breast cancer in vitro treatment" Eur. J. Pharmacol. 201 1 , 670, 74-84.

Moreover, the reference molecule [ 11 C]MC18 has been studied in vivo for its ability to target P-gp in specific mouse models for neurodegenerative disease. The radioligand demonstrated a low P-gp specific binding signal in vivo, probably due to its poor selectivity (see Mairinger, S.; et al. "Synthesis and preclinical evaluation of the radiolabeled P-glycoprotein inhibitor [1 1 C]MC1 13" Nucl. Med. Biol. 2012, 39, 1219-1225 or Chopra, A. "0- [1 1 C]Methyl Derivative of 6,7-Dimethoxy-2-(4-methoxybiphenyl-4-ylmethyl)- 1 ,2,3,4-tetrahydro-isoquinoline" Imaging and Contrast Agent Database (MICAD); NCBI: Bethesda, MD, 2013.

We have also observed that the choice of the most suitable radiotracer elements and the method for radiolabelling the selected molecules may influence the stability, selectivity, uptake of the ligands.

Therefore scope of the present invention is to provide novel compounds useful as radiotracers in vivo diagnosis of neurodegenerative diseases involving P-glycoprotein activity without the disadvantages of prior art's compounds.

SUMMARY OF THE INVENTION

The present invention is based on the radiolabelling of a novel family of compounds able to interfere with P-gp activity as inhibitors or as modulators thereof, but with poor or no activity towards the other efflux pumps Multidrug Resistance associated Protein (MRP1 ) and Breast Cancer Resistence Protein (BCRP), another transporter belonging to the ABC superfamily.

The compounds developed by the inventors present different points of radiolabelling. This property together with the properties described above makes such compounds high specific radiotracers useful for in vivo diagnosis of neurodegenerative diseases involving P-gp activity.

Moreover the pharmacological activity of these compounds is highly advantageous in terms of new therapeutic approach to neurodegenerative diseases involving P-gp activity. Indeed, P-gp inducers should be employed in neurodegenerative diseases to clean CNS areas from beta amyloid plaques.

Hence, aim of the present invention is a family of radiolabeled compounds having the general formula as indicated in claim 1 .

A second aim of the invention is a method for preparing said radiolabeled compounds.

A third object are compounds selected from the above-indicated family for use in a method of imaging and in vivo diagnosis of a neurodegenerative disease involving P-gp activity.

A fourth object of the invention are compounds selected from the above-indicated family for use as medicaments, in particular as inhibitor or as modulator or inducers of the P-glycoprotein function in the prevention or treatment of pathologies involving P-glycoprotein, in particular wherein said pathology is Alzheimer's or Parkinson's disease. A fifth object of the invention is a diagnostic imaging composition comprising as imaging agent a compound selected from the above-indicated family isotopically radio labeled and a carrier.

A sixth object of the invention is a pharmaceutical composition comprising the compounds selected from the above-indicated family and a pharmacologically acceptable excipient and/or diluent.

A further object of the invention is a pharmaceutical kit formed by a first element containing a compound selected from the above-indicated family and a second element containing radionuclide suitable for imaging.

Other objects will be made evident in the light of the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION.

Compounds of the Invention

The invention relates to a family of novel radiolabeled compounds having the following general formula I:

wherein:

- R is selected from: OCH 3 , OH, F, N0 2 , NH 2 , Br, COCH 3 , NHCH 3 , N(CH 3 ) 2 or N(C 2 H 5 ) 2 .

- X is selected from: CH 2 , CH 2 CO, NH, O, S, NH(CH 3 ), NH(C 2 H 5 ).

n is an integer number selected from: 0 and 5.

- Ar is a phenyl or an heterocyclic group, preferably selected from the following groups:

wherein the indicated bonds represent the respective position for the phenyl- Ar linkage and for the Ar-R linkage.

Specific embodiments of formula I are the compounds of formula II, having the Ar- group in position para as regard to the linking chain,

where R, Ar, X and n have the same meaning indicated above in relation to Formula I; or compounds of formula III, having the Ar-group in position ortho as regard to the linking chain.

where Ar and R have the same meaning indicated above in relation to Formula I.

In one specific embodiment of the invention Ar is a phenyl group, and the so obtained compound is a 1 , 1'-diphenyl derivative.

In further specific embodiments of Formula I, II or III, R is a methoxy group, an hydroxyl-group, a fluorine-group. When Ar is a phenyl group, R is preferably in position 4' of the phenyl group.

Preferred compounds are:

1-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-2-(4'-meth oxy-[1 , 1 '-biphenyl]-4-yl) ethanone.

6,7-Dimethoxy-2-(2-(4'-methoxy-[1 , 1 '-biphenyl]-4-yl)ethyl)-1 , 2,3,4- tetrahydroisoquinoline. 2-(2-(4'-Fluoro-[1 , 1 '-biphenyl]-4-yl)ethyl)-6,7-dimethoxy-1 ,2,3,4- tetrahydroisoquinoline.

2-((4'-Fluoro-[1,1'-biphenyl]-4-yl)methyl)-6,7-dimethoxy-1,2 ,3,4- tetrahydroisoquinoline.

6,7-Dimethoxy-2-(2-((4'-methoxy-[1 , 1 '-biphenyl]-4-yl)oxy)-ethyl)-1 ,2,3,4- tetrahydroisoquinoline.

6,7-Dimethoxy-2-(3-((4'-methoxy-[1 , 1 '-biphenyl]-4-yl)oxy)-propyl)-1 ,2,3,4- tetrahydroisoquinoline.

6,7-Dimethoxy-2-(4-((4'-methoxy-[1 , 1 '-biphenyl]-4-yl)oxy)-butyl)-1 ,2,3,4- tetrahydroisoquinoline.

N-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)-propyl)-4'-methoxy-[1,1'- biphenyl]-4-amine.

4'-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)-butoxy)-[1 , 1 '-biphenyl]-4-ol. 2'-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)-ethyl)-[1 , 1 '-biphenyl]-3-ol. 6,7-Dimethoxy-2-(2-(3'-methoxy-[1,1'-biphenyl]-2-yl)ethyl)-1 ,2,3,4- tetrahydroisoquinoline.

Synthesis process

The compounds of general formula I or II were synthetized as illustrated in Scheme 1. The synthesis process started from the preparation of carboxylic acid C by Suzuki-Miyaura' 11 coupling reaction followed by their condensation with 6,7- dimethoxytetrahydoisoquinoline to obtain amide D, which underwent LiAIH 4 reduction to afford final amines I. The reagents used in Schemes 1 and 2 are: Pd(PPh 3 ) 4 , Cs 2 CO 3 , toluene; CDI, 6,7-dimethoxy-1,2,3,4-tetrahydoisoquinoline, THF; LiAIH 4 , THF.

Scheme 1

wherein R' is selected from: -COOH, -CH 2 COOH, -OCH 3 , OH, and all other substituents are as defined above. Scheme 2, illustrates the synthesis of a specific compound of general Formiula II.

The compounds of general formula I or III were synthetized as illustrated in Scheme 3 in which carboxylic acids G were prepared by Suzuki-Miyaura coupling reaction, and they were condensed with 6,7-dimethoxy-1 ,2,3,4-tetrahydoisoqui to afford amide H, which underwent LiAIH 4 reduction to final amines III.

The reagents used are: K 2 CO 3 , Pd(OAc) 2 , DMGE/H 2 0 (3: 1); CDI, dimethoxy, 1 ,2,3,4-tetrahydroisoquinoline, THF; LiAIH 4 , THF.

Scheme 3

where all substituents are as defined above.

Radiolabeled compounds

The compounds selected from the above-indicated fam ily may be radiolabelling for use in vivo diagnosis of a neurodegenerative disease involving P-glycoprotein function.

All the radioisotopes and methods known in the art, suitable for labelling the compounds of the invention, may be used. For example isotopes such as 3 H, 11 C, 14 C, 13 N, 15 0, 18 F 19 F, 75 Br, 76 Br, 79 Br, may be used. Preferred isotopes are 3 H, 18 F and 11 C.

Advantageously, the compounds may be labeled in the last synthetic steps. In this way the radiosynthesis will be done directly in nuclear medicine centers where the radioligand will be injected for in vivo evaluation. Radiolabelling in the last step is also advantageous for the short decay time of radioisotopes (for example 2 h for 18 F and 20 minutes for 11 C).

Imaging composition and methods

A further object of the invention is a diagnostic imaging composition comprising as imaging agent the compounds selected from the above- indicated families isotopically radiolabeled and a carrier. In accordance with the invention, the radiolabeled compounds according to Formulas I, II or III may be formulated in a single unit injectable dose. Any of the common carriers known to those with skill in the art, such as sterile saline solution or plasma, can be utilized after radiolabelling for preparing the injectable solution to diagnostically imaging in accordance with the invention. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or diluent(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with the liquid carrier.

Generally, the unit dosage to be administered for a diagnostic agent has a radioactivity of about 10 MBq to about 15 MBq. The solution to be injected at unit dose is from about 4 mg/ml_ to about 7.5 mg/ml_. For diagnostic purposes after intravenous administration, imaging of the organ in vivo can take place in a matter of a few minutes. However, imaging takes place, if desired, in hours or even longer, after injecting into patients. In most instances, a sufficient amount of the administered dose will accumulate in the area to be imaged for example within about 1 hour to permit the taking of diagnostic images. Any conventional method of imaging for diagnostic purposes can be utilized in accordance with this invention as Positron Emission Tomography (PET) or Single Photon Pmission Computed Tomography (SPECT).

The diagnostic imaging compositions of the invention are useful for use in vivo diagnosis of a neurodegenerative disease involving P-gp activity such as Alzheimer's or Parkinson's disease.

Kit

The pharmaceutical kit of the present invention is formed by a first element containing a compound of the family described above and a second element containing radionuclide suitable for labelling the first compound.

The kit will allow user to obtain compound of Formula I, II or III ready to use for imaging.

Method for in vivo diagnosis

It is a further object of the invention a method for diagnosis of neurodegenerative disease involving P-gp activity comprising:

delivering to a mammal, preferably a human, an effective amount of a isotopically radio labelled compound of the family described above;

imaging the in vivo P-gp.

The imaging technique may be for example Positron Emission

Tomography (PET) or Single Positron Emission Computerized Tomography (SPECT).

In the method of in vivo diagnosis the effective amount administered of the isotopically labelled compounds of the present invention will depend on the particular condition to be diagnosed, the age, weight and the overall physical condition of the particular patient as it is well known to the experts in the field.

Pharmaceutical compositions

A further object of the invention is a pharmaceutical composition comprising the compounds selected from the above-indicated family and a pharmacologically acceptable excipient and/or diluent. Preferably these compositions are in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, auto-injector devices or suppositories; for oral, parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dose of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Typical unit dose contain from 1 to 50 mg, of the active ingredient. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, acetyl alcohol and cellulose acetate.

The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavoured syrups, aqueous or oil suspensions, and flavoured emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or gelatin. Thus, based on the above, a variety of pharmaceutically acceptable doses are provided.

Also, it is noted that the term "pharmaceutically acceptable salt(s)" refers to salts derived from treating a compound of formula 1 with an organic or inorganic acid such as, for example, acetic, lactic, citric, cinnamic, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, oxalic, propionic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, glycolic, pyruvic, methanesulfonic, ethanesulfonic, toluenesulfonic, salicylic, benzoic, or similar.

Compositions comprising compounds of Formula I and a pharmaceutical acceptable carrier or diluent may advantageously be used in the treatment of pathologies involving P-glycoprotein, in particular pathologies such as Alzheimer's and Parkinson's disease.

Unless otherwise specified, when referring, to the compounds of formula I per se, to any pharmaceutical composition thereof, the present invention includes all of the salts, hydrates, solvates, complexes, and prodrugs of the compounds of the invention. Prodrugs are any covalently bonded compounds, which releases the active parent pharmaceutical according to formula I.

Therapeutic application

It is a further object of the invention a method for treating of a neurodegenerative disease by modulating P-glycoprotein activity, comprising administering to a patient an effective amount of a compound of the families described above. In particular, the Alzheimer's or Parkinson's disease.

In the method of treatment the effective amount administered and frequency of administration of the compounds of the present invention will depend on the particular condition to be treated, the severity of the condition, age, weight and the overall physical condition of the particular patient as well as on other medicaments the patient is taking, as it is well known to the experts in the field. Effective dosages that can be administered are from 2 mg/Kg to 10 mg/Kg body weight.

Biological assays

In this section are reported biological data of compounds that are listed as examples. In order to establish the ability of the final compounds to modulate the activity of P-gp and MRP1 as well as their interacting mechanisms, each prepared ligand was studied in vitro using the following assays: (a) Calcein-AM assay to determine P-gp potency and selectivity and (b) the Apparent Permeability assay to investigate the interacting mechanism of the studied compound.

In particular, the effect of the tested molecules on the activities of P-gp and MRP1 were determined by evaluating Calcein accumulation in MDCK-MDR1 and MDCK-MRP1 cells overexpressing the P-gp and MRP1 transporters, respectively. Calcein-AM is a lipophilic pro-fluorescent probe that is a P-gp and MRP1 substrate. In the presence of a P-gp/MRP1 inhibitor, Calcein-AM is able to permeate the cell membrane, and it is hydrolyzed by cytosolic esterases to fluorescent Calcein. Because Calcein is hydrophilic and is not a P-gp/MRP1 substrate, it cannot cross the cell membrane, and a rapid increase in fluorescence can be measured. In Table 1 , EC 50 values are reported for each tested compound. EC 50 values were determined by fitting the percent fluorescence increase versus log[dose] (Wanek,

T.; Mairinger, S.; Langer, 0. "Radioligands targeting P-glycoprotein and other drug efflux proteins at the blood-brain barrier", J. Labelled Compd.

Radiopharm. 2013, 56, 68-77.

Moreover, the activity of all synthesized ligands toward BCRP were studied in a Hoechst 33342 experiment [3] in cells overexpressing BCRP

(MDCK-BCRP) (Bauer, S.; Ochoa-Puentes, C; Sun. Q.; Bause, M.;

Bernhardt, G.; Konig, B.; Buschauer, A. "Quinolinecarboxamide type ABCG2 modulators: indole and quinoline moieties as anilide replacements", Chem. Med. Chem. 2013, 8, 1773-1778.)

In this case, the inhibition of the pump was measured by the accumulation of the fluorescent dye Hoescht 33342 (a BCRP substrate) in the cells.

The Apparent Permeability assay allows two fluxes through the cell monolayer to be studied, from the basolateral to the apical (B→A) and from the apical to the basolateral (A→B) compartments, thus predicting the interacting mechanism of the tested compound with the pump. In particular, if a studied compound is a P-gp inhibitor, then it displayed BA/AB (P app ,

Apparent Permeability) < 2, whereas a P-gp substrate showed BA/AB > 2.

(Mairinger, S.; Erker, T; Muller, M.; Langer, O. "PET and SPECT radiotracers to assess function and expression of ABC transporters in vivo",

Curr. Drug Metab. 201 1 , 12, 774-792.

Biological data on several compounds of the present invention are reported in Table 1 .

EXPERIMENTAL WORK

The invention is detailed hereinafter via the following experimental examples of preparation and through the following biological testing.

General Procedures. Column chromatography was performed with 1 :30 Merck silica gel 60 A (63-200 μm) as the stationary phase. Melting points were determined in open capillaries on a Gallenkamp electrothermal apparatus. 1 H NMR spectra were recorded in CDCI 3 or DMSO-d 6 at 300 MHz on a Varian Mercury-VX spectrometer. All spectra were recorded on the free bases. All chemical shift values are reported in ppm (δ). Recording of mass spectra was done on an HP6890-5973MSD gas chromatograph/mass spectrometer; only significant m/z peaks, with their percentage of relative intensity given in parentheses, are reported. All spectra were in accordance with the assigned structures. ESI-MS analyses were performed on an Agilent 1 100LC/MSD trap system VL. Purity of final compounds was established by combustion analysis of the corresponding hydrochloride salts, confirming a purity > 95%.

For some biphenyl derivatives of formula II and formula III of present invention are reported the chemistry and biology. The invention is detailed hereinafter via the following examples:

Examplel . 1 -(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)-2-(4'-methoxy- [1 , 1 '-biphenyl]-4-yl) ethanone.

The compound was synthetized starting from a mixture of the appropriate carboxylic acid (1 mmol) and 1 , 1 '-carbonyldiimidazole (1 .1 mmol) in dry THF. The mixture was stirred at room temperature overnight. A solution of 6,7- dimethoxy-1 ,2,3,4-tetrahydroisoquinoline (1 mmol) in dry THF was dropped into the reaction complex. The mixture was stirred at room temperature for 4 h. 15 mL of H 2 0 was added to the reaction mixture, the aqueous phase was re-extracted with AcOEt (3 x 50 mL), and the combined solution was dried over Na 2 S0 4 and evaporated. The residue was purified by silica gel column chromatography for to obtain a yellow solid. Eluted with CHCI 3 . Yield 36%. 1 H NMR (CDCI 3 ) δ 2.63 (m, 1 H), 2.83 (m, 1 H), 3.64 (m, 2H), 3.91 -4.22 (m, 1 1 H), 4.53 (s, 1 H), 4.73 (s, 1 H), 6.55 (s, 1 H), 6.62 (s, 1 H), 6.95 (d, 2H, J = 8 Hz), 7.25-7.30 (m, 2H), 7.48-7.53 (m, 4H). ESI+/MS m/z: 418 [M + H] + ; ESr/MS/MS m/z: 194 (100), 165 (54). Anal. C, H, N [C 26 H 27 NO 4 mp 144-146 °C.

Example 2. 6,7-Dimethoxy-2-(2-(4'-methoxy-[1 , 1 '-biphenyl]-4-yl)ethyl)-1 ,2,3, 4-tetrahydroisoquinoline.

Starting from a solution of the amide Example 1 , 1 -(6,7-dimethoxy-3,4- dihydroisoquinolin-2(1 H)-yl)-2-(4'-methoxy-[1 , 1 '-biphenyl]-4-yl)ethanone, (1 mmol) was added to a suspension of LiAIH 4 (2 mmol) in dry THF and refluxed for 2 h. Water was added to the cooled reaction until effervescence ceased. The aqueous phase was extracted with Et 2 0 (3 x 50 mL), and the combined solution was dried over Na 2 S0 4 and evaporated. The residue was purified by silica gel column chromatography and I was obteining a white solid. Eluted with CHCI 3 . Yield 26%. 1 H NMR (CDCI 3 ) δ 2.67-3.10 (m, 8H), 3.67 (s, 2H), 3.85 (s, 9H), 6.56 (s, 1 H), 6.68 (s, 1 H), 7.01 (d, 2H, J = 8 Hz) 7.26-7.30 (m, 2H), 7.47-7.55 (m, 4H). ESI + /MS m/z: 404 [M + H] + ; ESr/MS/MS m/z: 240 (36), 21 1 (100), 179 (78). Anal. C, H, N [C 2 6H 2 9N03-2H 2 O 2 - HCI]. mp 214-216 °C. Example 3. 2-(2-(4'-Fluoro-[1 , r-biphenyl]-4-yl)ethyl)-6,7-dimethoxy-1 ,2,3,4 - tetrahydroisoquinoline.

The compound was prepared as described for Example 2 starting from amide 1 -(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)-2-(4'-fluoro-[1 , r- biphenyl]-4-yl)ethanone to obtain a white solid. Eluted with CHCI 3 . Yield 58%. 1 H NMR (CDCI 3 ) δ 2.77-2.98 (m, 8H) 3.64 (s, 2H), 3.85 (s, 6H), 6.54 (s, 1 H), 6.60 (s, 1 H), 7.09-7.12 (m, 2H) 7.29-7.32 (d, 2H, J = 8 Hz), 7.46-7.59 (m, 4H). ESr/MS m/z: 414 [M + Na] + ; ESI + /MS/MS m/z: 240 (36), 21 1 (100), 179 (78). Anal. C, H, N [C 26 H 29 NO 2 F HCI]. mp 202-203 °C.

Example 4. 2-((4'-Fluoro-[1 , 1 '-biphenyl]-4-yl)methyl)-6,7-dimethoxy-1 ,2,3, 4- tetrahydroisoquinoline.

The compound was prepared as described for Example 2 starting from amide (6,7-dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)(4'-fluoro-[1 , 1 '- biphenyl]-4-yl)methanone to obtain a white solid. Eluted with CHCIs/MeOH (9: 1 ). Yield 40%. 1 H NMR (CDCI 3 ) δ 2.77-2.98 (m, 4H) 3.57 (s, 2H), 3.73 (s, 2H), 3.77 (s, 3H), 3.84 (s, 3H), 6.49 (s, 1 H), 6.60 (s, 1 H), 7.09-7.12 (m, 2H) 7.40-7.58 (m, 6H). Anal. C, H, N [C 24 H 24 N0 2 F-2H 2 O HCI]. mp 198-201 °C. Example 5. 6,7-Dimethoxy-2-(2-((4'-methoxy-[1 , 1 -biphenyl]-4-yl)oxy)-ethyl)- 1 ,2,3,4-tetrahydroisoquinoline.

This compound was prepared as described for Example 2 starting from amide 1 -(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)-2-((4'-methoxy-[1 , 1 '- biphenyl]-4-yl)oxy)ethanone to obtain a yellow solid. Eluted with CHCIs/AcOEt 1 : 1 . Yield 76%. 1 H NMR (CDCI 3 ) δ 2.86 (m, 4H), 2.95-3.00 (m, 2H), 3.76 (s, 2H), 3.83 (s, 6H), 3.86 (s, 3H), 4.25 (m, 2H), 6.52 (s, 1 H), 6.59 (s, 1 H), 6.94-6.99 (m, 4H), 7.35 (d, 2H, J = 8 Hz), 7.55 (d, 2H, J = 8 Hz). ESr/MS m/z: 420 [M +H] + ; ESI + /MS/MS m/z: 218 (100), 179 (61 ).

Example 6. 6,7-Dimethoxy-2-(3-((4'-methoxy-[1 , 1 -biphenyl]-4-yl)oxy)-ropyl)- 1 ,2,3,4- tetrahydroisoquinoline.

4'-Hydroxy-4-methoxy-biphenyl (1 mmol) dissolved in dry DMF (1 mL) was dropped into a suspension of NaH (2 mmol) in dry DMF (4 mL). The mixture was stirred at room temperature for 1 h. A solution of compound 10 (2 mmol) in dry DMF (3 mL) was added to the mixture reaction, which was then heated to reflux overnight. Water was added until effervescence ceased. The solvent was evaporated, and the residue was partitioned between H 2 0 (20 mL) and CHCI 3 (20 mL). The organic phase was separated, the aqueous phase was extracted with CHCI 3 (3 x 50 mL), and the collected organic fractions were dried over Na 2 S0 4 and evaporated. Eluted with CHCI 3 . The product was transformed in the hydrochloric salt and recrystallized from MeOH. Yield 90%. 1 H NMR (CDCI3) δ 1 .82-1 .84 (m, 2H), 2.45-2.47 (m, 2H), 2.74-2.93 (m, 4H), 3.68 (s, 2H), 3.84 (s, 9H), 4.12-4.14 (m, 2H), 6.81 (s, 1 H), 6.83 (s, 1 H), 7.10-7.15 (m, 4H), 7.65-7.67 (m, 4H). ESr/MS m/z: 434 [M +H] + ; ESI + /MS/MS m/z: 208 (45), 179 (100).

Example 7. 6,7-Dimethoxy-2-(4-((4'-methoxy-[1 , 1 -biphenyl]-4-yl)oxy)-butyl)- 1 ,2,3,4-tetrahydroisoquinoline.

Amine was synthesized by alkylation of 6, 7-dimethoxy-1 ,2,3,4- tetrahydroisoquinoline (1 .2 mmol) by 4-(4-chlorobutoxy)-4'-methoxy-1 , 1 '- biphenyl (1 mmol) in DMF (20 mL) using Na 2 CO 3 as base (2 mmol). The mixture was refluxed overnight. The DMF was evaporated, and the residue was partitioned between H 2 0 (20 mL) and CHCI3 (20 mL). The organic phase was separated, the aqueous phase was extracted with CHCI3 (3 x 50 mL), and the collected solutions were dried over Na 2 S0 4 and evaporated. The residue was purified by silica gel column chromatography.

White solid. Eluted with CH 2 CI 2 /MeOH (98:2). Yield 29%. 1 HNMR(CDCI 3 ) δ 1 .79-1 .90 (m, 4H), 2.55-2.60 (m, 2H), 2.70-2.80 (m, 2H), 2.82-2.84 (m, 2H), 3.57 (s, 2H), 3.83 (s, 9H,), 4.01 -4.05 (m, 2H), 6.52(s, 1 H), 6.59 (s, 1 H), 6.93-6.96 (m, 4 H), 7.44-7.48 (m, 4H). ESI + /MS m/z: 448 [M + H] + ; ESr/MS/MS m/z: 284 (47), 255 (61 ), 231 (89), 179 (100). Anal. [C 28 H 3 3N0 4 HCI H 2 0] C, H, N. mp 222-223 °C.

Example 8. /V-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)-propyl)-4'- methoxy-[1 , 1 '-biphenyl]-4-amine.

This amine was synthetized as described for Example 7 in this case alkylation of 6, 7-dimethoxy-1 ,2,3,4-tetrahydroisoquinoline was did by Λ/-(3- chloropropyl)-4'-methoxybiphenyl-4-amine for to obtain a yellow solid. Eluted with CHCIs/MeOH (97:3). Quantitative yield. 1 H NMR (CDCI 3 ) δ 1 .67-1.69 (m, 2H), 2.45-2.47 (m, 2H), 2.78-2.82 (m, 4H), 3.65-3.67 (m, 2H), 3.72 (s, 2H), 3.84 (s, 9H), 4.21 (br s, 1 H, NH exchangeable with D 2 0), 6.51 -6.52 (m, 2H), 6.85 (s, 1 H), 6.87 (s, 1 H), 7.10 (d, 2H, J = 8 Hz), 7.56 (d, 2H, J = 8 Hz), 7.68 (d, 2H, J = 8 Hz). ESI + /MS m/z: 455 [M+ Na] + , ESI + /MS/MS m/z: 451 (100), 425 (45), 258 (17), 192 (23). Anal. [C 27 H 32 N 2 O 3 HCI] C, H, N.

Example 9. 4'-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)-butoxy)- [1 , 1 '-biphenyl]-4-ol.

This amine was synthetized as described for Example 7 the alkylation of 6,7- dimethoxy-1 ,2,3,4-tetrahydroisoquinoline was did 4'-(4- chlorobutoxy)biphenyl-4-ol for to obtain a white solid. Eluted with CH 2 CI 2 /MeOH (9: 1 ). Yield 16%. 1 H NMR (CDCI 3 ) δ 1 .85 (s, 4H), 2.62-2.65 (m, 2H), 2.78-2.85 (m, 4H), 3.62 (s, 2H), 3.83 (s, 6H), 4.01 -4.10 (m, 2H), 5.35 (br s, 1 H, OH exchangeable with D 2 O), 6.52 (s, 1 H), 6.59 (s, 1 H) 6.83-6.88 (m, 4H), 7.39-7.42 (m, 4H). ESI + /MS m/z: 456 [M + Na] + ; ESr/MS/MS m/z: 248 (100), 179 (12). Anal. [C 27 H 31 NO 4 HCI] C, H, N. mp 178-180 °C.

Example 10. 2'-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)-ethyl)- [1 , 1 '-biphenyl]-3-ol.

A solution of amide 1 -(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)-2-(3'- hydroxy-[1 , 1 '-biphenyl]-2-yl)ethanone (1 mmol) was added to a suspension of LiAIH 4 (2 mmol) in dry THF and refluxed for 2 h. Water was added to the cooled reaction until effervescence ceased. The aqueous phase was extracted with Et 2 O (3 χ 50 mL), and the combined solution was dried over Na 2 SO 4 and evaporated. The residue was purified by silica gel column chromatography.

White solid. Eluted with CHCI 3 . Yield 26%. 1 H NMR (CDCI 3 ) δ 2.66-2.69 (m, 4H), 2.79-2.95 (m, 4H), 3.49 (s, 2H), 3.75 (s, 3H), 3.79 (s, 3H), 5.40 (br s, 1 H, exchangeable with D 2 O), 6.38 (s, 1 H), 6.51 (s, 1 H), 6.53-6.60 (m, 1 H), 6.70-6.80 (m, 1 H), 7.12-7.29 (m, 2H), 7.33-7.35 (m, 3H), 7.8 (m, 1 H). ESI7MS m/z: 388 [M - H] " ; ESI7MS/MS m/z: 373 (100). Anal. C, H, N [C 25 H 27 NO 3 HCI]. mp > 250 °C.

Example 11. 6,7-Dimethoxy-2-(2-(3'-methoxy-[1 , 1 '-biphenyl]-2-yl)ethyl)-1 ,2,3, 4-tetrahydroisoquinoline.

This compound was synthetized as described for Example 10 starting from a solution of another amide, 1 -(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl)- 2-(3'-methoxy-[1 , 1 '-biphenyl]-2-yl)ethanone to obtain a white solid. Eluted with CHCI 3 . Yield 64%. 1 H NMR (CDCI 3 ) δ 2.66-2.68 (m, 4H), 2.71 -2.79 (m, 2H), 2.81 -2.92 (m, 2H), 3.42 (s, 2H), 3.82 (s, 9H), 6.43 (s, 1 H), 6.54 (s, 1 H), 6.86-6.92 (m, 2H), 7.21 -7.35 (m, 6H). ESI7MS m/z: 426 [M + Na] + ;

ESI7MS/MS m/z: 365 (100). Anal. C, H, N [C 26 H 29 NO 3 HCI]. mp 183-186.

The compounds may be radiolabeled according to following methods. Procedure for 18 F radiosynthesis

In the synthesis of [ 18 F]Compound (4) 15 μί of 2-bromoethyl tosylate (1 ) in 1 mL of 1 ,2-dichlorobenzene was added to the dried fluoride complex. Distillation of the formed [ 18 F]bromoethyl fluoride (2) at 90 °C was quickly started with a helium gas flow to the second vial in room temperature containing 2 mg of compound precursor (3) and 3 mg of NaH in 0.5 mL of DMF. After 15 min, vial 2 was reacted for 10 min at 80 °C in the synthesis of [ 18 F] Compound (4). After reaction, 0.5 mL of HPLC eluent was added (0.1 M NaOAc/MeCN 5.5:4.5 (v/v)) and the product was purified by RP-HPLC (column Symmetryshield RPS 5 μm 7.8 x 300 mm, flow 3 mL/min, 254 nm, Rt ([ 18 F]Compound (4) = 10 min). Radiopharmaceutical tracer was collected into a bottle containing 80 mL of sterile water. After mixing with helium, the solution was transferred to an Oasis HLB 1 cc (30 mg) extraction cartridge. The product was trapped to the cartridge which was washed twice with 8 mL of water and the product was eluted with 1 mL of ethanol through a Millipore Millex LG filter (0.2 μm). A volume of 4 mL of 0.9 % NaCI was also added to reduce the ethanol concentration to 20 %. Alltech Alltima C18 5 urn 4.6 χ 250 mm was used as a column in the QC with either the same eluent as in preparation. HPLC or MeCN/water 1 : 1 + 0.1 % TFA (1 mL/min).

Procedure for 11 C radiosynthesis

[ 11 C]Methyl iodide was trapped in a solution (0.5 mg in 0.5 mL dry DMSO) of Compound-OH. Shortly, before the arrival of [ 11 C]methyl iodide, 7 μί of NaOH (5 M) was added to the precursor solution. The reaction mixture was heated for 4 min at 80 °C. After addition of water (2 mL), [ 11 C]-Compound was purified on a reversed-phase column (Phenomenex C18, 300 mm 8 mm) with 100 mM NaH 2 PO 4 :EtOH 1 : 1 (v/v) as mobile phase (flow rate 5 mL/min, UV detection at 254 nm). The retention time of both compounds was approximately 12 min. The eluted fraction containing [ 11 C]-Compound was diluted with at least 4 volumes of saline (0.9% NaCI) before injection into experimental animals. Quality control for the radiolabeled products was based on the data from the preparative HPLC (specific activity).

Procedure for [ 3 H]tracer

Radiosynthesis for obtaining [ 3 H]t

Precursor was suspended in potassium hydroxide solution (0.1 M), and methylene chloride. A toluene solution of Aliquat 336 and of [ 3 H]methyl iodide in toluene was added to the reaction to obtaine [ 3 H]tracer. .

BIOLOGICAL EXPERIMENTATION

Calcein-AM Experiment.

These experiments were carried out as described by Feng et al (In vitro P- glycoprotein assays to predict the in vivo interactions of P-glycoprotein with drugs in the central nervous system Drug Metab. Dispos. 2008, 36, 268-275) with minor modifications. Each cell line (50 000 cells per well) was seeded into black 96-well plates with 100 μL of medium and allowed to become confluent overnight. Test compounds were solubilized in 100 μL of culture medium and were added to the cell monolayers. The plates were then incubated at 37 °C for 30 min. Calcein-AM was added in 100 μL of phosphate-buffered saline (PBS) to yield a final concentration of 2.5 μΜ, and the plate was incubated for another 30 min. Each well was washed three times with ice-cold PBS. Saline buffer was added to each well, and the plates were read with a Victor3 fluorimeter (PerkinElmer) at excitation and emission wavelengths of 485 and 535 nm, respectively. Under these experimental conditions, Calcein cell accumulation in the absence and presence of tested compounds was evaluated, and basal-level fluorescence was estimated from the fluorescence of untreated cells. In treated wells, the increase in fluorescence was measured relative to that of the basal level. EC 50 values were determined by fitting the percent fluorescence increase versus log[dose] (Rautio, J.et al. In vitro P-glycoprotein inhibition assays for assessment of clinical drug interaction potential of new drug candidates: a recommendation for probe substrates Drug Metab. Dispos. 2006, 34, 786-792).

Permeability Experiment.

Preparation of Caco-2 Monolayer: Caco-2 cells were harvested with trypsin-EDTA and seeded onto a Multiscreen Caco-2 assay system at a density of 10000 cells/well. The culture medium was replaced every 48 h for the first 6 days and every 24 h thereafter, and after 21 days in culture, the Caco-2 monolayer was utilized for the permeability experiments. The transepithelial electrical resistance (TEER) of the monolayers was measured daily before and after the experiment using an epithelial voltohmmeter (Millicell-ERS; Millipore, Billerica, MA). Generally, TEER values obtained are greater than 1000 Ω for a 21 day culture (Polli, J. W. et al. Rational use of in vitro P-glycoprotein assays in drug discovery J. Pharmacol. Exp. Ther. 2001, 299, 620-628).

Drug Transport Experiment: Apical to Basolateral (P app A→B) and Basolateral to Apical (P app B→A) permeability of drugs was measured at 120 min at the concentration of 100 μΜ. Drugs were dissolved in Hank's balanced salt solution (HBSS, pH 7.4) and sterile filtered. After 21 days of cell growth, the medium was removed from filter wells and from the receiver plate. The filter wells were filled with 75 μL of fresh HBSS buffer, and the receiver plate, with 250 μL per well of the same buffer. This procedure was repeated twice, and the plates were incubated at 37 °C for 30 min. After incubation, the HBSS buffer was removed, and, in some wells, drug solutions were added to the filter well (75 μL); HBSS without drug was added to the corresponding receiver plate (250 μL). For other wells, the drug solutions were added to the Basolateral side (250 μL), and HBSS without drug was added to the corresponding filter wells. The plates were incubated at 37 °C for 120 min. After incubation, samples were removed both from the apical (filter well) and Basolateral (receiver plate) sides of the monolayer and then were stored in a freezer (-20 °C) pending analysis. The concentration of compounds was analyzed using UV-Vis spectroscopy. The Apparent Permeability (P apP ) , in units of nm/s, was calculated using the following equation

where VA is the volume (in ml_) in the acceptor well, area is the surface area of the membrane (0.11 cm2 of the well), time is the total transport time in seconds (7200 s), [drug]acceptor is the concentration of the drug measured by UV spectroscopy, and [drug] in itiai is the initial drug concentration (1 χ 10 -4 M) in the apical or basolateral well (Polli, J. W. et al. Rational use of in vitro P-glycoprotein assays in drug discovery J. Pharmacol. Exp. Ther. 2001, 299, 620-628).

Results

The results obtained in the above-described assays with some representative compounds are reported in the Table 1 below.

The reported data are the mean of three-independent determinations of duplicate samples and show that all compounds are inactive toward BCRP pump.

Only for the compound of Example 1 CH 2 CO is present as a spacer and it displays moderate activity (EC 50 = 0.8 μΜ) and low selectivity toward P-gp (MRP1 EC 50 = 10 μΜ). Since P app was 3.9, its profile was superimposed to MC113 (P app = 4.4). Also the compound of Example 2 displayed same activity and permeability values obtained for the corresponding amide derivative of Example 1. The amide of Example 1 resulted in a loss of selectivity, but no relevant changes in the activity and interacting mechanism were observed with respect amine of Example 2. Another modification was the insertion of a fluorine substituent in place of the methoxy group, with two different spacers (n = 0, 1 as in Example 3 and Example 4, respectively). The compounds of Example 3 and Example 4 displayed less activity toward P-gp (EC 50 = 2 and 4.9 μΜ, respectively) with respect to MC113 (EC 50 = 0.60 μΜ) and Example 2 (EC 50 = 0.57 μΜ). A severe loss of activity was also observed in fluorine derivatives with respect to their corresponding methoxy- substituted (Ex. 2) . Example 5, Example 6, and Example 7 were designed by inserting an O-alkyl spacer, which different (from 2 to 4) methylene units. In this series the highest activity has been obtained with the compound of Example 7 (EC 50 = 0.004 μΜ) whereas the other compounds displayed only moderate activity in micromolar range. All compounds bearing an O-alkyl spacer were inactive toward MRP1 , and they displayed P app values greater than 2, and so are considered P-gp substrates. Specifically, the increase in P-gp potency could be explained with a higher degree of flexibility although all compounds bound the substrate sites of the protein.

In this series was also investigated the effect of the presence of a hydroxyl group as a substituent on the biphenyl moiety. Example 9 derivative was designed by inserting an O-alkyl spacer, which has four methylene units, and a hydroxyl group on the 4-biphenyl fragment. This compound was inactive toward the target protein, whereas its P app value, less than 2, suggests an interaction with other efflux transporters.

In addition, the presence of a NH group was evaluated in order to determine how the nature of the spacer influences activity and selectivity toward P-gp. The NH group was inserted in the alkyl spacer of compound of Example 8, which has three methylene units. The replacement of O with NH did not lead to changes in potency or selectivity (see Ex 6 vs Ex8).

Another effort was to investigate compounds representative for the cisoid

configuration of MC18in order to establish if this configuration is more useful to interact with the pump with respect the lead compound. Both compounds of

Example 10 and Example 11 displayed good potency toward P-gp (EC 50 = 0.23 and 0.069 μΜ, respectively), but Example 11 displayed low selectivity although it is the most potent in this series . Finally P app was greater than 2 for both 2-biphenyl derivatives, so they can be considered P-gp substrates.