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Title:
SARTAN ANALOGUE
Document Type and Number:
WIPO Patent Application WO/2019/134765
Kind Code:
A1
Abstract:
The invention concerns a sartan analogue on basis of a sartan which sartan comprises an alkyl group or an alkoxy group, wherein the sartan analogue only differs from the sartan by a replacement of the alkyl group or the alkoxy group or replacement of a methyl residue or a hydrogen residue of the alkyl group or of the alkoxy group by a fluorine atom.

Inventors:
CHEN, Xinyu (Straubmühlweg 2, Haus A10 Zimmer 213, Würzburg, 97078, DE)
DECKER, Michael (Institut für Pharmazie und LebensmittelchemieAm Hubland, Würzburg, 97074, DE)
HIGUCHI, Takahiro (Universitätsklinikum WürzburgOberdürrbacher Str. 6, Würzburg, 97080, DE)
HOFFMANN, Matthias (Fichtenstr. 1, Rottendorf, 97228, DE)
Application Number:
EP2018/077897
Publication Date:
July 11, 2019
Filing Date:
October 12, 2018
Export Citation:
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Assignee:
JULIUS-MAXIMILIANS-UNIVERSITAET WUERZBURG (Sanderring 2, Würzburg, 97070, DE)
International Classes:
C07D257/04; A61K31/41; A61P9/12
Domestic Patent References:
WO2004062568A22004-07-29
WO2004062568A22004-07-29
Other References:
M. HACHEM ET AL: "Characterization of 18F-FPyKYNE-Losartan for Imaging AT1 Receptors", THE JOURNAL OF NUCLEAR MEDICINE, vol. 57, no. 10, 19 May 2016 (2016-05-19), US, pages 1612 - 1617, XP055483242, ISSN: 0161-5505, DOI: 10.2967/jnumed.115.170951
DION VAN DER BORN ET AL: "Fluorine-18 labelled building blocks for PET tracer synthesis", CHEMICAL SOCIETY REVIEWS, vol. 46, no. 15, 1 January 2017 (2017-01-01), pages 4709 - 4773, XP055484032, ISSN: 0306-0012, DOI: 10.1039/C6CS00492J
A. M. AMIN ET AL: "Radioiodination and biological evaluation of valsartan as a tracer for cardiovascular disorder detection", NATURAL SCIENCE, vol. 05, no. 04, 1 January 2013 (2013-01-01), pages 526 - 531, XP055484080, ISSN: 2150-4091, DOI: 10.4236/ns.2013.54066
HACHEM M; TIBERI M; ISMAIL B; HUNTER CR; ARKSEY N; HADIZAD T; BEANLANDS RS; DEKEMP RA; DASILVA JN: "Characterization of 18F-FPyKYNE-losartan for imaging AT receptors", J. NUCL. MED., vol. 57, no. 10, 2016, pages 1612 - 7, XP055483242, DOI: doi:10.2967/jnumed.115.170951
ARKSEY N; HADIZAD T; ISMAIL B; HACHEM M; VALDIVIA AC; BEANLANDS RS; DEKEMP RA; DASILVA JN: "Synthesis and evaluation of the novel 2-[18F]fluoro-3-propoxy-triazole-pyridine-substituted losartan for imaging AT receptors", BIOORG. MED. CHEM., vol. 22, no. 15, 2014, pages 3931 - 7, XP029009845, DOI: doi:10.1016/j.bmc.2014.06.011
MATHEWS WB; BURNS D; DANNALS RF; RAVERT HAT; NAYLOR EM: "Carbon-11 labeling of a potent, nonpeptide, ATi-selective angiotensin-II receptor antagonist: MK-996", JOURNAL OF LABELLED COMPOUNDS AND RADIO-PHARMACEUTICALS, vol. 36, no. 8, 1995, pages 729 - 737
MATHEWS WB; YOO SE; LEE SH; SCHEFFEL U; RAUSEO PA; ZOBER TG; GOCCO G; SANDBERG K; RAVERT HT; DANNALS RF: "A novel radioligand for imaging the AT angiotensin receptor with PET", NUCL. MED. BIOL., vol. 31, no. 5, 2004, pages 571 - 574, XP004517603, DOI: doi:10.1016/j.nucmedbio.2003.10.014
ZOBER TG; MATHEWS WB; SECKIN E; YOO SE; HILTON J; XIA J; SANDBERG K; RAVERT HT; DANNALS RF; SZABO Z: "PET imaging of the AT receptor with [''C]KR31173", NUCL. MED. BIO., vol. 33, no. 1, 2006, pages 5 - 13
HIGUCHI T; FUKUSHIMA K; XIA J; MATHEWS WB; LAUTAMAKI R; BRAVO PE; JAVADI MS; DANNALS RF; SZABO Z; BENGEL FM: "Radionuclide imaging of angiotensin II type 1 receptor upregulation after myocardial ischemia-reperfusion injury", J. NUCL. MED., vol. 51, no. 12, 2010, pages 1956 - 1961
Attorney, Agent or Firm:
DR. GASSNER & PARTNER MBB (Wetterkreuz 3, Erlangen, 91052, DE)
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Claims:
Claims

1. Sartan analogue on basis of a sartan which sartan comprises an alkyl group or an alkoxy group, wherein the sartan analogue only differs from the sartan by a replacement of the alkyl group or the alkoxy group or replacement of a methyl resi- due or a hydrogen residue of the alkyl group or of the alkoxy group by a fluorine atom.

2. Sartan analogue according to claim 1 , wherein the sartan is tasosartan or wherein the alkyl group or the alkoxy group comprises at least two consecutive ter- minal carbon atoms.

3. Sartan analogue according to claim 1 or 2, wherein the sartan is saprisartan or eprosartan or includes i. a biphenyl group, ii. an imidazole group or a benzimidazole group and/or iii. a tetrazole residue, an oxadiazolone residue, or a carboxy residue.

4. Sartan analogue according to any of the preceding claims, wherein the sar- tan is candesartan or azilsartan or the alkyl group is linear and comprises more than two carbon atoms.

5. Sartan analogue according to any of the preceding claims, wherein the sar- tan is losartan, irbesartan, olmesartan, candesartan, valsartan, telmisartan, mil- fasartan, pomisartan, pratosartan, ripisartan, or fimasartan, in particular losartan, irbesartan, candesartan, or valsartan.

6. Sartan analogue according to any of the preceding claims, wherein the fluo- rine atom is a radioactive isotope of fluorine, in particular 18F.

7. Sartan analogue according to any of the preceding claims, wherein the sar- tan analogue is F-valsartan ((S)-2-(N-((2'-(1 H-tetrazol-5-yl)-[1 ,1 '-biphenyl]-4-yl)me- thyl)-5-fluoropentanamido)-3-methylbutanoic acid), in particular 18F-valsartan.

8. Sartan analogue according to any of the preceding claims for use as a me- dicament.

9. Sartan analogue according to claim 6 or 7 for use in a diagnostic method for detecting and localizing angiotensin II receptors for diagnosing disorders in a hu- man or animal body by an imaging technique in vivo, wherein the sartan analogue is administered to the human or animal body and subsequently an image of at least a part of said body is generated on basis of the radioactive emission of the radioactive isotope.

10. Sartan analogue according to claim 9 for use in a diagnostic method in vivo according to claim 8, wherein the imaging technique is or comprises positron emis- sion tomography (PET).

11. Method for synthesizing a sartan analogue according to any of claims 1 to 6 comprising the following steps: a) Providing a precursor 10 of the sartan analogue and in case said pre- cursor 10 comprises a/the carboxy residue, protecting the carboxy res- idue by coupling a protection group to the C-atom of the carboxy group via an ester binding and in case said precursor 10 comprises a/the te- trazole residue or an/the oxadiazolone residue, protecting the acidic NFI-group in said residue by coupling a protection group to the N-atom of said NFI-group, b) replacing the alkyl group, the alkoxy group, the methyl residue or the hydrogen residue to be replaced by the fluorine atom by a leaving group coupled via an ester binding to the rest of the precursor 10 or by iodine or bromide, c) incubating the molecule resulting from step b) together with a fluoride and a fluoride complexing cryptand or crown ether in a polar aprotic solvent at a temperature above 90 °C and d) in case the carboxy residue, the tetrazole residue or the oxadiazolone residue has been protected, deprotecting said residue by incubating the molecule resulting from step c) with an acid or base for achieving hydrolysis of the ester binding or with an acid for achieving deprotec- tion of the tetrazole residue or the oxadiazolone residue.

12. Method according to claim 11 , wherein the protection group is benzyl or te/f-butyl, the cryptand is 4,7,13,16,21 ,24-hexaoxa-1 ,10-diazabicyclo[8.8.8]hexa- cosane, the leaving group is a tosyl group, iodine, or bromine, the crown ether is 18-crown-6, the polar aprotic solvent is dimethylformamide (DMF) or acetonitrile, and/or the acid is hydrochloric acid, hydrobromic acid, trifluoroacetic acid, trichlo- roacetic acid, or formic acid.

13. Method according to claim 11 or 12, wherein the sartan analogue is fluorine valsartan and the method comprises the following steps: a) Providing 5-chlorovaleric acid, b) protecting the carboxyl group by esterification with a benzyl group for obtaining a compound 12, c) replacing chlorine in compound 12 by iodine through Finkelstein re- action for obtaining a compound 13, d) replacing the iodine in compound 13 with tosylate using silver tosyl- ate for obtaining a compound 14, e) removing the benzyl ester in compound 14 selectively under hydro- genation condition without affecting the tosylate for obtaining an acid 16, f) reacting said acid 16 with oxalyl chloride in dichloromethane to form an acid chloride 17, g) reacting said acid chloride 17 with (S)-terf-butyl 3-methyl-2-(((2'-(1 - trityl-1 H-tetrazol-5-yl)-[1 ,1 '-biphenyl]-4-yl)methyl)amino)butanoate 7 thus forming the precursor 10, h) incubating a mixture of a fluoride, K2CO3, 4,7,13,16,21 ,24-hexaoxa-

1 ,10-diazabicyclo[8.8.8]hexacosane and the precursor 10 in acetoni- trile at a temperature between 90 and 150 °C, and i) hydrolyzing terf-butyl ester and removing trityl protection group by addition of HCI and heating to a temperature between 90 and 150 °C.

14. Method according to claim 11 or 12, wherein the sartan analogue is fluorine valsartan and the method comprises the following steps: a) Providing 5-chlorovaleric acid, b) protecting the carboxyl group by esterification with a benzyl group for obtaining a compound 12, c) replacing chlorine in compound 12 by iodine through Finkelstein re- action for obtaining a compound 13, d) replacing the iodine in compound 13 with tosylate using silver tosyl- ate for obtaining a compound 14, e) Incubating compound 14, a fluoride and 18-crown-6 in dimethylfor- mamide at a temperature between 90 and 150 °C for obtaining a compound 15, f) removing the benzyl ester in compound 15 selectively under hydro- genation condition without affecting the tosylate for obtaining an acid 18, g) reacting said acid 18 with oxalyl chloride in dichloromethane to form an acid chloride 19, h) reacting said acid chloride 19 with (S)-benzyl 3-methyl-2-(((2'-(1 -trityl- 1 H-tetrazol-5-yl)-[1 ,1 '-biphenyl]-4-yl)methyl)amino)butanoate 20 thus forming a compound 21 , i) adding palladium/charcoal in ethanol to compound 21 and stirring or agitating the resulting mixture under hydrogen atmosphere, j) filtering the mixture and concentrating the resulting filtrate under vac- uum, and k) dissolving the resulting residue in dichloromethane, adding formic acid and stirring or agitating it.

15. Method according to any of claims 11 to 14, wherein the fluoride is 18F-fluo- ride.

Description:
Sartan analogue

The invention concerns a sartan analogue comprising a fluorine atom, in particular a radioisotope of fluorine, in particular 18 F.

Sartan analogues comprising a fluorine atom are known in the art.

Sartans are angiotensin II subtype 1 receptor antagonists also called ATi receptor antagonists.

The renin angiotensin aldosterone system (RAS) is a hormonal cascade that gen- erates angiotensin peptides and is the main regulator of blood pressure as well as fluid and electrolyte balance. The key factor is octapeptide angiotensin II, which is generated in a well described cascade and stimulates mainly angiotensin II type 1 receptor (ATi), a G-protein coupled receptor (GPCR), and thereby initiates further downstream effects. ATi receptors are mainly located in the heart, blood vessels and kidney. They are considered responsible for cardiovascular reactions, such as hypertension and heart failure (HF), the treatment of which is the major therapeutic target of ATi antagonists.

In addition to the central role of RAS in regulation of cardiovascular system men- tioned above, it is also involved in much broader functions in the body, including important actions on growth factors, mitosis and cancer pathogenesis.

In contrast to anatomical techniques, such as computed tomography (CT), ultra- sound or radiography, molecular imaging using radionuclide techniques focuses on small-scale molecular events. It provides a non-invasive method for the moni- toring of functional changes in individual organs, with higher sensitivity, specificity and the possibility of quantifying these alterations. Molecular imaging using radio- nucleotides can be performed, e. g., by means of positron emission tomography (PET) or single-photon emission computed tomography (SPECT). WO 2004/062568 A2 discloses a contrast agent of formula V - L - Z, wherein V is a non-peptidic vector having affinity for the angiotensin II receptor, L is a bond, a spacer or a linker moiety and Z represents a moiety detectable in an in vivo imag- ing procedure of a human or animal body. V may be losartan, valsartan, candesar- tan, eprosartan or derivatives thereof. Z may comprise an imaging moiety compris- ing radionucleotides such as 90 Y, 99m Tc, 111 In, 47 Sc, 67 Ga, 51 Cr, 177m Sn, 67 Cu,

167 Tm, 97 Ru, 188 Re, 177 Lu, 199 Au, 203 Pb, 141 Ce or 18 F. The compound may comprise a moiety Z carrying one or more imaging moieties M useful in the PET imaging modality. M may then be a radioemitter with positron-emitting properties, such as 18 F. The document also discloses preparation of a contrast medium comprising a radioactive tracer for use in a method of diagnosis involving administering that contrast medium to a human or animal body and generating an image of at least part of that body.

Chen, X., 2016, http://gepris.dfg.de/gepris/projekt/326751428 discloses develop- ment of new 18 F-PET-radiotracers on basis of drug molecules, such as valsartan and losartan, for the imaging of cardial angiotensin II type 1 receptor.

The thesis "Characterization of [ 18 F]FPyKYNE-Losartan as a Novel PET Tracer for Imaging ATi Receptors" submitted by Maryam Flachem to the Faculty of Graduate and Postdoctoral Studies as a partial fulfillment of the M.Sc. program in Cellular and Molecular Medicine at the University of Ottawa and the publication Flachem M, Tiberi M, Ismail B, Flunter CR, Arksey N, Fladizad T, Beanlands RS, deKemp RA, DaSilva JN, "Characterization of 18F-FPyKYNE-losartan for imaging ATi re- ceptors", J. Nucl. Med., 2016, 57(10): 1612-7 disclose a specific 18 F-losartan de- rived PET-tracer for the angiotensin II type 1 receptor. Synthesis of this tracer has been described in Arksey N, Fladizad T, Ismail B, Flachem M, Valdivia AC, Bean- lands RS, deKemp RA, DaSilva JN, "Synthesis and evaluation of the novel 2- [18F]fluoro-3-propoxy-triazole-pyridine-substituted losartan for imaging ATi recep- tors", Bioorg. Med. Chem., 2014, 22(15): 3931 -7 and in the thesis "Synthesis and Preliminary Evaluation of an F-18 Labeled Fluoropyridine Losartan Analog as a Novel PET Tracer for Imaging ATi Receptors" submitted by Natasha Arksey to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the require- ments for the Master of Science (M.Sc.) in the Department of Cellular and Molecu- lar Medicine at the University of Ottawa.

A 11 C-labelled ATi-selective angiotensin-ll receptor antagonist is known from Mathews WB, Burns D, Dannals RF, Ravert FIAT, Naylor EM, "Carbon-11 labeling of a potent, nonpeptide, ATi-selective angiotensin-ll receptor antagonist: MK-996", Journal of Labelled Compounds and Radio-pharmaceuticals, 1995, 36(8): 729- 737.

The methoxyl analog [ 11 C]KR31173 of non-peptide ATi-selective antagonist SK1080 is the most intensively developed and investigated RAS tracer (Mathews WB, Yoo SE, Lee SH, Scheffel U, Rauseo PA, Zober TG, Gocco G, Sandberg K, Ravert HT, Dannals RF, Szabo Z, "A novel radioligand for imaging the ATi angio- tensin receptor with PET", Nucl. Med. Biol. 2004, 31 (5): 571 -574; Zober TG, Mathews WB, Seckin E, Yoo SE, Hilton J, Xia J, Sandberg K, Ravert HT, Dannals RF, Szabo Z, "PET imaging of the ATi receptor with [ 11 C]KR31173", Nucl. Med. Bio. 2006, 33(1 ): 5-13; Higuchi T, Fukushima K, Xia J, Mathews WB, Lautamaki R, Bravo PE, Javadi MS, Dannals RF, Szabo Z, Bengel FM, "Radionuclide imaging of angiotensin II type 1 receptor upregulation after myocardial ischemia-reperfusion injury" J. Nucl. Med. 2010, 51 (12): 1956-1961 ).

The problem to be solved by the present invention is to provide an alternative an- giotensin II receptor antagonist and in particular an alternative angiotensin II re- ceptor antagonist detectable by PET. A further problem to be solved by the pre- sent invention is to provide such an angiotensin II receptor antagonist for specific uses and a method for synthesizing such an angiotensin II receptor antagonist.

The problems are solved by the features of claims 1 , 8, 9 and 11. Embodiments are subject-matter of claims 2 to 7, 10 and 12 to 15. According to the invention a sartan analogue on basis of a sartan which sartan comprises an alkyl group or an alkoxy group is provided. The sartan analogue only differs from the sartan by a replacement of the alkyl group or the alkoxy group or replacement of a methyl residue or a hydrogen residue of the alkyl group or of the alkoxy group by a fluorine atom. The alkyl group may be methyl, e. g. in case of tasosartan. In case of valsartan, losartan and irbesartan the alkyl group is butyl. The advantage of the sartan analogue according to the invention is that the struc- ture activity relationship (SAR) of the sartan analogue differs not or only very little from the SAR of the sartan on which a sartan analogue is based. Fluorine is of smaller size than hydrogen and has neutral properties as well. The modification maintains the affinity of the basic sartan to the ATi receptor or influences this affin ity only little. The modification of the basic sartan for introducing the fluorine atom is sterically smaller than the modifications known in the state of the art described above.

Furthermore, in case of valsartan fluorination at the end of the alkyl group blocks the formation of its potential non-active metabolite 4-hydroxyvalsartan. Therefore, the pharmacodynamics and pharmacokinetics of the sartan analogue according to the invention can be improved with respect to its basic sartan, for instance, by de- creased liver uptake and/or decreased metabolism.

Though the fluorine atom may be a radioactive isotope of fluorine and in particular 18 F allowing to use the sartan analogue as a PET tracer the sartan analogue corn- prising a non-radioactive fluorine atom may be advantageous with respect to its pharmacodynamics and pharmacokinetics and therefore may be used as a medic- ament. The medicament may be a medicament for the treatment of cardiovascular diseases such as hypertension or heart failure.

Furthermore, the sartan analogue comprising the non-radioactive fluorine atom can be used to explore different ways of syntheses that can then be used for syn- thesis of a sartan analogue comprising a radioactive isotope of fluorine. The sartan analogue comprising a non-radioactive fluorine atom may also be used for exploring pharmacokinetics, pharmacodynamics and receptor binding of the sartan analogue.

In one embodiment of the invention the sartan is tasosartan or the alkyl group or the alkoxy group comprises at least two consecutive terminal carbon atoms. The fluorine atom in the sartan analogue may then be located at the terminal carbon atom.

In a further embodiment of the invention the sartan is saprisartan or eprosartan. Alternatively, the sartan analogue may include i. a biphenyl group, ii. an imidazole group or a benzimidazole group and/or iii. a tetrazole residue, an oxadiazolone residue, or a carboxy residue.

The sartan may be candesartan or azilsartan. Alternatively, the alkyl group may be linear and comprise more than two carbon atoms.

The sartan may be losartan, irbesartan, olmesartan, candesartan, valsartan, telmisartan, milfasartan, pomisartan, pratosartan, ripisartan, or fimasartan, in par- ticular losartan, irbesartan, candesartan, or valsartan. In particular the sartan ana- logue may be F-valsartan ((S)-2-(N-((2'-(1 H-tetrazol-5-yl)-[1 ,T-biphenyl]-4-yl)me- thyl)-5-fluoropentanamido)-3-methylbutanoic acid), in particular 18 F-valsartan.

If the fluorine atom is a radioactive isotope of fluorine the sartan analogue accord- ing to the invention may be used in a diagnostic method for detecting and localiz ing angiotensin II receptors for diagnosing disorders in a human or animal body by an imaging technique in vivo. For this purpose, the sartan analogue is adminis- tered to the human or animal body and subsequently an image of at least a part of that body is generated on basis of a radioactive emission of the radioactive isotope. The imaging technique may be or may comprise positron emission tomog- raphy (PET).

The invention further concerns a method for synthesizing a sartan analogue ac- cording to the invention comprising the following steps: a) Providing a precursor of the sartan analogue and in case said precursor comprises a/the carboxy residue, protecting the carboxy residue by coupling a pro- tection group to the C-atom of the carboxy group via an ester binding and in case said precursor comprises a/the tetrazole residue or an/the oxadiazolone residue, protecting the acidic NH-group in said residue by coupling a protection group to the N-atom of said NH-group, b) replacing the alkyl group, the alkoxy group, the methyl residue or the hydro- gen residue to be replaced by the fluorine atom by a leaving group coupled via an ester binding to the rest of the precursor or by iodine or bromide, c) incubating the molecule resulting from step b) together with a fluoride and a fluoride complexing cryptand or crown ether in a polar aprotic solvent at a temper- ature above 90 °C and d) in case the carboxy residue, the tetrazole residue or the oxadiazolone resi- due has been protected, deprotecting said residue by incubating the molecule re- sulting from step c) with an acid or a base for achieving hydrolysis of the ester binding or with an acid for achieving deprotection of the tetrazole residue or the oxadiazolone residue.

From the method it is clear that the precursor may be a molecule that differs from the sartan analogue according to the invention only at the position that will be oc cupied by the fluorine atom in the sartan analogue. However, it may also be that the precursor is a molecule comprising the alkyl group or the alkoxy group, wherein the molecule is a compound used in synthesis of the sartan analogue.

The protection group may be benzyl or terf-butyl. The cryptand may be

4,7,13,16,21 ,24-hexaoxa-1 ,10-diazabicyclo[8.8.8]hexacosane. The leaving group may be a tosyl group, iodine, or bromine. The crown ether may be 18-crown-6, the polar aprotic solvent may be dimethylformamide (DMF) or acetonitrile. The acid may be hydrochloric acid, hydrobromic acid, trifluoroacetic acid, trichloroacetic acid, or formic acid.

The sartan analogue may be fluorine valsartan. In this case the method may corn- prise the following steps: a) Providing 5-chlorovaleric acid, b) protecting the carboxyl group by esterification with a benzyl group for ob- taining a compound 12, c) replacing chlorine in compound 12 by iodine through Finkelstein reaction for obtaining a compound 13, d) replacing the iodine in compound 13 with tosylate using silver tosylate for obtaining a compound 14, e) removing the benzyl ester in compound 14 selectively under mild hydro- genation condition without affecting the tosylate for obtaining an acid, f) reacting said acid with oxalyl chloride in dichloromethane to form an acid chloride, g) reacting said acid chloride with (S)-terf-butyl 3-methyl-2-(((2'-(1 -trityl-1 H-te- trazol-5-yl)-[1 ,T-biphenyl]-4-yl)methyl)amino)butanoate thus forming the precursor, h) incubating a mixture of a fluoride, K2CO3, 4,7,13,16,21 ,24-hexaoxa-1 ,10-di- azabicyclo[8.8.8]hexacosane and the precursor in acetonitrile at a temperature be- tween 90 and 150 °C, and i) hydrolyzing terf-butyl ester and removing trityl protection group by addition of HCI and heating to a temperature between 90 and 150 °C.

Alternatively, the method may comprise the following steps when the sartan ana- logue is fluorine valsartan: a) Providing 5-chlorovaleric acid, b) protecting the carboxyl group by esterification with a benzyl group for ob- taining a compound 12, c) replacing chlorine in compound 12 by iodine through Finkelstein reaction for obtaining a compound 13, d) replacing the iodine in compound 13 with tosylate using silver tosylate for obtaining a compound 14, e) Incubating compound 14, a fluoride and 18-crown-6 in dimethylformamide at a temperature between 90 and 150 °C for obtaining a compound 15, f) removing the benzyl ester in compound 15 selectively under mild hydro- genation condition without affecting the tosylate for obtaining an acid, g) reacting said acid with oxalyl chloride in dichloromethane to form an acid chloride, h) reacting said acid chloride with (S)-benzyl 3-methyl-2-(((2'-(1 -trityl-1 H-te- trazol-5-yl)-[1 ,1 '-biphenyl]-4-yl)methyl)annino)butanoate thus forming a compound 21 i) adding palladium/charcoal in ethanol to compound 21 and stirring or agitat- ing the resulting mixture under hydrogen atmosphere, j) filtering the mixture and concentrating the resulting filtrate under vacuum, and k) dissolving the resulting residue in dichloromethane, adding formic acid and stirring or agitating it.

In all of the methods described above the fluoride may be 18 F-fluoride. Embodiments of the invention:

Fig. 1 shows the chemical structures of valsartan and 18 F-valsartan,

Fig. 2 shows a reaction scheme for two different syntheses of fluorine valsar- tan,

Fig. 3 shows PET images of rat kidneys 10 minutes after i. v. injection of 18 F- valsartan,

Fig. 4 shows dynamic coronal PET images of rat kidneys at different times af- ter i. v. injection of 18 F-valsartan,

Fig. 5 shows a reaction scheme for the synthesis of w-F-irbesartan (MD149),

Fig. 6 shows a reaction scheme for synthesis of a-F-irbesartan (MD147), Fig. 7 shows a reaction scheme for the preparation of precursor compound 39 and radiolabeling of this compound to achieve [ 18 F]MD147,

Fig. 8 shows the results of radioligand competition binding studies on human

ATi receptors,

Fig. 9 shows dynamic coronal and sagittal PET images of pig kidneys at differ ent times after i. v. injection of [ 18 F]MD147,

Fig. 10 A shows PET images of rat kidneys after i. v. injection of 18 F-irbesartan,

Fig. 10 B shows results of kidney tracer uptake in rats after i. v. injection of 18 F- irbesartan and

Fig. 10 C shows results of tracer uptake in several rat organs after i. v. injection of

1 8 F-irbesartan.

1 . Structure of sartan analogue F-valsartan (FV45)

The chemical structures of valsartan and the 18 F-labelled radiotracer [ 18 F] F-valsar- tan (FV45) are shown in Fig. 1 . Valsartan is shown on the left side of Fig. 1 and [ 18 F]FV45 on the right side of Fig. 1 .

2. Syntheses of sartan analogues

Common reagents and solvents were obtained from commercial suppliers and were used without any further purification. Tetrahydrofuran (TFIF) was distilled from sodium/benzophenone under an argon atmosphere. Reaction progress was monitored by using analytical thin-layer chromatography (TLC) on precoated silica gel GF254 plates (Macherey-Nagel GmbFI & Co. KG, Ddren, Germany), and spots were detected under UV light (l = 254 nm) or by staining with iodine. Nuclear mag- netic resonance spectra were performed with a Bruker AV-400 NMR instrument (Bruker, Karlsruhe, Germany) in [D6]DMS0 or CDCI3. Chemical shifts are ex- pressed in ppm relative to CHCI3/DMSO (d = 7.26/2.50 and 77.16/39.52 ppm for 1 H and 13 C NMR, respectively). For purity and reaction analyzes, analytical HPLC analysis was performed with a system from Shimadzu equipped with a DGU- 20A3R controller, LC20AB liquid chromatograph, and a SPD-20A UVA/is detector. Stationary phase was a Synergi 4 pm fusion-RP (150 c 4.6 mm) column (Phenom- enex, Aschaffenburg, Germany). As mobile phase, H2O (phase A) and methanol (phase B) were used with 1 mL/min (cone. B: 5®90% from 0 to 8 min; 90% from 8 to 13 min; 90®5% from 13 to 15 min; 5% from 15 to 18 min). ESI mass spectral data were acquired with a Shimadzu LCMS-2020. a) Syntheses of 18 F-valsartan ([ 18 F]FV45) and F-valsartan (FV45)

Syntheses of the radioactive [ 18 F]FV45 and the non-radioactive FV45 is schemati- cally shown in Fig. 2. Flowever, it is clear that it is possible to synthesize [ 18 F]FV45 in the same way as shown for FV45 and also that it is possible to synthesize FV45 in the same way as shown for [ 18 F]FV45 in Fig. 2.

In detail, the single steps shown in Fig. 2 were performed as follows:

5-Chlorovaleric acid 5 was first esterified to protect the carboxyl group. "Bn" means benzyl in the figures. Chlorine in compound 12 was replaced by iodine through Finkelstein reaction. This gave the possibility of replacing the iodine in compound 13 with tosylate using silver tosylate, which was obtained by mixing equal molar quantities of silver oxide with 4-toluenesulfonic acid in acetonitrile in darkness. The fluorine could therefore be introduced to the "tail" of the acyl chain for subsequent synthesis of non-radioactive "cold" reference. By this means fluori- nation conditions could also be tested as a reference for the radiolabeling proce- dure.

After removal of the benzyl ester selectively under mild hydrogenation condition without affecting the tosylate, the acid 16 was reacted with oxalyl chloride in dry dichloromethane to form the acid chloride 17, which was then reacted with diphe- nyl valinate moiety 7, and formed the precursor 10 available for labeling after puri fication and characterization. The "cold" reference FV45 was synthesized analo- gously as the precursor, with the only difference of using benzyl valinate 20 in- stead of terf-butyl ester. Benzyl ester is more stable during the preparation, whereas terf-butyl ester in the precursor will be conveniently removed after the la- beling together with trityl group in acidic condition.

Substitution of the good leaving group tosyl with radioactive K[ 18 F]F was per- formed in high purity as well as reasonable yield. [ 18 F]F produced via proton bom- bardment of Fte 18 0 was isolated by trapping on Sep-Pak Light QMA cartridge, fol lowed by washing with 3 mL water. Fluoride was eluted with a mixture of a solution of K2CO3 in 0.3 mL of water (50.6 mM) and a solution of Kryptofix 2.2.2. (14 mg) in 0.7 mL of acetonitrile into a sealed glass vial. The solution was dried with azeo- tropic condition under argon flow at 120 °C. The solution of 5 mg precursor 10 in 0.3 mL dry acetonitrile was added to the residue followed by heating at 110 °C for 10 min under argon atmosphere. Subsequent hydrolysis of terf-butyl ester as well as the removal of trityl protection group were performed in the same vessel by the addition of 0.3 mL 1 N HCI and continued heating at 110 °C for 10 min. The mix- ture was cooled, diluted with 1 mL of mixture solution of water and acetonitrile (1 : 1 ), and applied to the semi-preparative HPLC column (ZORBAX Eclipse XDB- C18, 5 pm, 9.4 x 250 mm, linear gradient of 50-95% methanol with 0.1 % formic acid, 3 mL/min). The average overall radiochemical yield was 21.8 ± 8.5% (decay- corrected based on starting activity, calculated from 5 times of labeling records) and > 99% radiochemical purity. After purification, 5 mL water was added to the collected solution containing radioactive tracer. The solution was then passing through a Sep-Pak plus cartridge (C18), washed with 4 mL of water and eluted with 3 mL of diethyl ether. The organic solution was concentrated at 50 °C and di- luted with saline to appropriate concentration for the imaging studies. (S)-tert-butyl 3-methyl-2-(((2'-( 1-trityl-1H-tetrazol-5-yl)-[ 1, 1 '-biphenyl]-4-yl)me- thyl)amino)butanoate (7)

2-tetrazol(2-trityl)-4'-methylbromide-biphenyl 4 (2.00 g, 3.59 mmol), tert-butyl (2S)- 2-amino-3-methylbutanoate hydrochloride 6 (753 mg, 3.59 mmol) and DIPEA (1.25 ml, 7.18 mmol) in toluene were stirred over night at 65 °C under argon at- mosphere. The solvent was removed under vacuum and the crude product was purified via column chromatography (petroleum ether : ethyl acetate 10 : 1 ). The coupling compound was obtained as light yellow oil (671 mg, 53%).

ESI-MS: 650.2 m/z [M+H] +

1 H-NMR (CDCIs) d: 7.94-7.91 (m, 1 H), 7.49-7.39 (m, 3H), 7.35-7.24 (m, 9H), 7.14- 7.07 (m, 4H), 6.94-6.91 (m, 6H), 3.60 (dd, 2H, J = 3.63 Hz), 2.89 (d, 1 H, J = 2.89 Hz), 1.93-1.88 (m, 1 H), 1.51 (s, 9H), 0.96 (dd, 6H, J = 0.96 Hz) ppm;

13 C-NMR (CDCIs) d: 174.86, 164.40, 142.51 , 141.58, 140.01 , 139.30, 131.01 , 130.57, 130.54, 130.15, 129.43, 128.48, 127.95, 127.91 , 127.63, 126.74, 83.16, 67.85, 52.59, 32.03, 28.54, 19.67, 18.96 ppm.

(S)-benzyl 3-methyl-2-(((2'-( 1-trityl-1H-tetrazol-5-yl)-[ 1, 1 '-biphenyl]-4-yl)me- thyl)amino)butanoate (20)

The corresponding benzyl ester was synthesized analogously to terf-butyl ester as described above.

ESI-MS: 684.3 m/z [M+H] +

1 H-NMR (CDCIs) d: 7.85-7.83 (m, 1 H), 7.41 -7.36 (m, 2H), 7.30-7.20 (m, 9H), 7.18- 7.14 (m, 7H), 7.01 -6.96 (m, 4H), 6.83-6.81 (m, 5H), 5.11 (s, 2H), 3.50 (dd, 2H, J = 3.52 Hz), 3.00 (d, 1 H, J = 2.99 Hz), 1.89-1.84 (m, 1 H), 0.84 (dd, 6H, J = 0.85 Hz) ppm;

13 C-NMR (CDCIs) d: 175.02, 164.09, 142.16, 141.26, 139.85, 138.52, 135.91 , 130.70, 130.27, 130.23, 130.20, 129.86, 129.15, 128.61 , 128.43, 128.37, 128.30, 128.19, 127.70, 127.65, 127.61 , 127.37, 126.42, 82.87, 66.96, 66.31 , 52.27,

40.88, 31.70, 19.39, 18.61 ppm. Benzyl 5-chlorovalerate (12)

5-Chlorovaleric acid 5 (1 g, 7.3 mmol) and benzylbromide (867 mI_, 7.3 mmol) were dissolved in acetonitrile. Sodium carbonate was added to the above solution. The mixture was heated to reflux under argon for 15 h. The reaction solution was cooled and concentrated under vacuum. The residue was diluted with diethyl ether (30 ml_) and washed with water (10 ml_) and then brine (10 ml_). The organic phase was dried over sodium sulfate and concentrated under vacuum to afford the product as colorless oil (1 .65 g, 100%), which was used in the next step.

Benzyl 5-iodovalerate (13)

Benzyl 5-chlorovalerate 12 (3.17 g, 13.98 mmol) was dissolved in acetone. So- dium iodide (2.60 g, 17.35 mmol) was added to the solution. This mixture was heated to reflux under argon for 5 h. The formed white solid was filtered off and the filtrate was removed under vacuum. The residue was diluted with diethyl ether (30 ml_) and washed with water (10 ml_) and then brine (10 ml_). The organic phase was dried over sodium sulfate and concentrated under vacuum to afford the product as colorless oil (3.51 g, 79%).

1 H-NMR (CDC ) d: 7.38-7.34 (m, 5H), 5.12 (s, 2H), 3.18 (t, 2H, J = 3.18 Hz), 2.39 (t, 2H, J = 2.39 Hz), 1 .88-1 .74 (m, 4H) ppm;

13 C-NMR (CDCIs) d: 173.04, 136.07, 128.73, 128.41 , 128.37, 66.43, 33.23, 32.84, 25.92, 5.85 ppm.

Benzyl 5-(tosyloxy)pentanoate (14)

To the cooled solution of benzyl 5-iodovalerate 13 (3.6 g, 1 1 .3 mmol) in acetoni- trile (10 mL) was added silver tosylate (3.46 mg, 12.4 mmol). The resulting solu- tion was protected from light (aluminium foil) and was stirred at room temperature overnight. The solid was filtered off. The solvent was removed under vacuum. The residue was diluted in ethyl acetate and water. The organic phase was separated and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with brine and dried over anhydrous sodium sulfate. The solvent was removed and the residue was purified via column chromatography (petroleum ether : ethyl acetate 3 : 1 ). The compound was obtained as colorless oil (2.03 g, 49.5%).

1 H-NMR (CDCb) d: 7.79 - 7.32 (m, 9H), 5.09 (s, 2H), 4.04 - 4.01 (t, 2H), 2.44 (s, 3H), 2.34 - 2.31 (t, 2H), 1 .70 - 1 .67 (m, 4H) ppm;

13 C-NMR (CDC ) d: 172.78, 69.88, 66.28, 33.37, 28.20, 21 .63, 20.89 ppm.

5-(Tosyloxy)pentanoic acid (16)

To the solution of benzyl 5-(tosyloxy)pentanoate 14 (500 mg, 1 .38 mmol) in etha- nol (10 ml_) was added palladium on charcoal (50 mg) under argon atmosphere. The flask was equipped with a hydrogen balloon. The gas in the flask was ex- changed. After stirring vigorously for 3 h at room temperature, the catalyst was fil- tered off through celite and the filtrate was concentrated. The compound was ob- tained as colorless oil (360 mg, 96%).

1 H-NMR (CDCb) d: 8.56 (br. s, 1 H), 7.79 - 7.77 and 7.35 - 7.33 (m, 2H), 4.05 - 4.02 (m, 2H), 2.44 (s, 3H), 2.33 - 2.30 (m, 2H), 1 -71 - 1 .65 (m, 4H) ppm;

13 C-NMR (CDCb) d: 178.82, 69.83, 33.04, 28.1 1 , 21 .62, 20.61 ppm.

(S)-tert-Butyl 3-methyl-2-(5-(tosyloxy)-N-((2'-( 1 -trityl-1 H-tetrazol-5-yl)-[ 1, 1 '-bi- phenyl]-4-yl)methyl)pentanamido)butanoate (10)

The solution of 5-(tosyloxy)pentanoic acid 16 (100.5 mg, 0.37 mmol) in dry THF (5 ml_) was cooled with an ice/water bath under argon atmosphere. Oxylyl chloride (32 mI_, 0.37 mmol) was added the cooled solution followed with catalytic amount of DMF (4.75 mg, 5 mI_, 0.065 mmol). The solution was stirred for 10 min in ice/wa- ter bath and then in room temperature for 3 h. The solution was concentrated un- der vacuum. After adding dry TFIF (5 ml_) and DIPEA (80.4 mI_, 0.31 mmol), the so- lution was cooled again with ice/water bath. A solution of compound 7 (200 mg, 0.31 mmol) in dry TFIF (5 ml_) was added dropwise to the above solution. The re- action was allowed to rise to room temperature and stirred overnight. The reaction solution was diluted with ethyl acetate and water. The organic phase was sepa- rated and the aqueous phase was extracted with ethyl acetate. The combined or- ganic phases were washed with brine and dried over anhydrous sodium sulfate. The compound was obtained after concentration and purification via column chro- matography (petroleum ether : ethyl acetate 3 : 1 ) as yellow oil (100 mg, 36%). ESI-MS: 926.4 m/z [M+Na] +

1 H-NMR (CDCb) d: 7.80-7.62 (m, 3H), 7.44-7.36 (m, 3H), 7.22-7.19 (m, 14H), 7.05-6.86 (m, 7H), 4.74-4.30 (m, 3H), 3.99 & 3.82 (dt, 2H, J = 3.82 Hz), 2.47-1.92 (m, 3H), 2.34 (s, 3H), 1.68-1.67 (m, 2H), 1.44-1.41 (m, 2H), 1.22-1.20 (d, 9H), 0.91 -0.75 (m, 6H) ppm;

13 C-NMR (CDCb) d: 171.16, 164.15, 146.90, 141.32, 133.11 , 130.79, 130.21 , 129.81 , 129.53, 129.27, 128.29, 128.22, 128.08, 127.94, 127.90, 127.67, 127.26, 127.23, 125.73, 82.88, 81.59, 70.29, 60.40, 32.79, 27.82, 27.71 , 23.85, 21.61 ,

21.04, 14.20 ppm.

Benzyl 5-fluorovalerate (15)

The mixture of benzyl 5-(tosyloxy)pentanoate 14 (500 mg, 1.38 mmol), potassium fluoride (80 mg, 1.38 mmol) and 18-crown-6 (364 mg, 1.38 mmol) in dry DMF (10 ml_) was stirred overnight at 115 °C. The solvent was removed under vacuum. The residue was diluted with water and extracted with ethyl acetate. The combined organic phases were washed with brine, dried over sodium sulfate and concen- trated. The crude product was purified via column chromatography (petroleum ether : ethyl acetate 10 : 1 ). The fluoride compound was obtained as colorless oil (40 mg, 14%).

1 H-NMR (CDCb) d: 7.37-7.34 (m, 5H), 5.12 (s, 2H), 4.52^1.37 (dt, 2H), 2.44-2.40 (t, 2H), 1.81-1.70 (m, 4H) ppm;

13 C-NMR (CDCb) d: 173.21 , 136.13, 128.71 , 128.38, 128.34, 84.52, 82.88, 66.37, 33.86, 29.98, 29.79, 21.00, 20.95 ppm. 5-Fluorovaleric acid (18)

The hydrogenation was performed with benzyl 5-fluorovalerate 15 (62 mg,

0.29 mmol) in the presence of palladium on charcoal (6 mg) in ethanol. After three times of degasification, the mixture was stirred overnight. The catalyst was filtered off and the filtrate concentrated (13 mg, 37% colorless volatile oil).

1 H-NMR (CDCb) d: 10.85 (br. s, 1 H), 4.54-4.40 (dt, 2H), 2.45-2.42 (t, 2H), 1 .81- 1 .76 (4H) ppm;

13 C-NMR (CDC ) d: 179.49, 84.35, 82.71 , 33.42, 29.75, 29.55, 20.60, 20.55 ppm.

(S)-Benzyl 2-(5-fluoro-N-((2'-( 1-trityl-1H-tetrazol-5-yl)-[ 1, 1 '-biphenyl]-4-yl)me- thyl)pentanamido)-3-methylbutanoate (21 )

To the solution of 5-fluorovaleric acid 18 (60 mg, 0.5 mmol) in dry dichloromethane (5 ml_) was added oxalyl chloride (51 mI_, 0.6 mmol). The solution was cooled us- ing ice/water bath under argon atmosphere. Dry DMF (5 mI_) was added to the above solution and was thereafter stirred at room temperature for 2 h. This solu- tion was transferred with a syringe and added slowly to a precooled solution of compound 20 (324, 0.5 mmol) and DIPEA (130 mI_, 0.75 mmol) in dry dichloro- methane (5 ml_). Ice/water bath was removed after the addition, and the resulting solution was stirred at room temperature overnight. Water was added to the reac- tion solution. The organic phase was separated, dried over dry sodium sulfate and concentrated under vacuum. The residue was purified via column chromatography (petroleum ether : ethyl acetate 5 : 1 ). The compound was obtained as yellow oil (125 mg, 32 %).

ESI-MS: 786.4 m/z [M+H] +

1 H-NMR (CDCb) d: 7.80-7.75 (m, 1 4H), 7.40-7.35 (m, 3H), 7.27-7.16 (m, 23H), 7.10-7.08 (m, 1 H), 6.97-6.95 (m, 4H), 6.89-6.87 (m, 11 H), 4.83-4.69 (m, 3H), 4.59- 4.49 (m, 2H), 4.37-4.26 (m, 3H), 4.17-4.14 (m 1 .5H), 3.95-3.92 (m, 0.5H), 2.30- 2.24 (m, 1 H), 2.16-2.06 (dt, 2H), 1 .61 -1 .42 (m, 4H), 0.89 (d, 3H), 0.80 (d, 3H) ppm; 13 C-NMR (CDCb) d: 173.71 , 170.31 , 164.16, 141 .57, 141 .33, 140.27, 135.64, 135.49, 130.71 , 130.45, 130.22, 129.97, 129.57, 129.06, 128.49, 128.27, 127.88, 127.67, 125.70, 84.64, 83.01 , 82.91 , 66.68, 63.1 1 , 60.40, 49.35, 32.66, 31 .90, 28.24, 22.53, 21 .05, 20.17, 19.1 1 , 14.21 ppm.

(S)-2-(N-((2'-( 1 H-tetrazol-5-yl)-[ 1, 1 '-biphenyl]-4-yl)methyl)-5-fluoropentanamido)-3- methylbutanoic acid (FV45)

To the solution of compound 21 in methanol was added palladium/charcoal. The resulting mixture was stirred vigorously under hydrogen atmosphere at room tem- perature overnight. The catalyst was filtered and the filtrate was concentrated un- der vacuum. The residue was dissolved in dry dichloromethane following with for- mic acid. The solution was stirred at room temperature for 2 h. The reaction solu- tion was concentrated under vacuum. The residue was purified via column chro- matography using petroleum ether : ethyl acetate : formic acid 1 : 1 : 0.05 as the eluent system. The target cold compound was obtained as colorless oil.

ESI-MS: 454.3 m/z [M+H] +

1 H-NMR (CDCb) d: 7.98-7.96 (m, 1 H), 7.60-7.44 (m, 3H), 7.17-7.1 1 (m, 3H), 6.97- 6.95 (m, 1 H), 4.90 & 4.31 (dd, 2H), 4.53 & 4.41 (dt, 2H), 4.02 & 3.68 (dd, 1 H), 2.63-2.39 (m, 3H), 1 .87-1 .73 (m, 4H), 0.98 (d, 3H), 0.94 (d, 3H) ppm;

13 C-NMR (CDCb) d: 176.33, 172.74, 154.60, 140.31 , 139.25, 135.19, 131 .31 , 131 .09, 130.31 , 130.23, 129.91 , 128.85, 128.42, 128.15, 127.67, 122.99, 84.67, 83.04, 77.22, 53.47, 33.81 , 29.82, 29.63, 26.83, 21 .33, 21 .29, 19.83, 19.49, 19.15, 18.53 ppm. b) Syntheses of w-F-irbesartan (MD149), a-F-irbesartan (MD147) and [ 18 F]MD147

Synthesis of MD149 is schematically shown in Fig. 5, synthesis of MD147 is sche- matically shown in Fig. 6 and synthesis of [ 18 F]MD147 is schematically shown in Fig. 7.

In detail, the single steps of syntheses were performed as follows: 1-(5-Fluoropentanamido)cyclopentane-1 -carboxamide (23)

1 -Fluoropentanoic acid (22, 350 mg, 2.91 mmol) were dissolved in dry dimethylfor- mamide (= DMF) (5 ml_) and triethylamine (= Et3N) (486 mI_, 353 mg, 3.49 mmol) was added. Then 1 -aminocyclopentanecaroxamide (373 mg, 2.91 mmol) and (2- (1 FI-benzotriazol-1 -yl)-1 ,1 ,3,3-tetramethyluronium hexafluorophosphate) (= FIBTU) (1324 mg, 3.49 mmol) were added. The mixture was stirred for 24 h at room tem- perature (= r.t.). Then water was added and the mixture was extracted wit ethyl ac- etate (3x). The combined organic layer was washed with water, saturated NaFIC03 solution and brine (2x), dried over Na2S04, filtered and evaporated to dryness in vacuo. The residue was purified by flash column chromatography (dichloro- methane/methanol = 98/2; Rf = 0.2) to yield 23 (668 mg, 99%) as colorless solid.

1 H NMR (400 MHz, CDCIs): d = 4.76 (t, J = 5.7 Hz, 1 H), 4.61 (t, J = 5.7 Hz, 1 H), 2.43 - 2.34 (m, 2H), 2.30 - 2.22 (m, 2H), 2.07 - 1 .99 (m, 2H), 1 .94 - 1 .73 (m, 8H) ppm.

13 C NMR (101 MHz, CDCIs): d = 175.57, 170.07, 82.94, 69.19, 34.94, 34.64,

29.77, 29.62, 23.00, 21 .95 ppm.

MS: 231 .05 [M+H] +

2-(4-Fluorobutyl)-1,3-diazaspiro[4.4]non-1-en-4-one (24)

Compound 23 (300 mg, 1 .30 mmol) was dissolved in methanol (10 ml_) and a 10 M solution of KOH (10 ml_) was added. The mixture was heated to 60 °C for 1 h. After cooling to reach room temperature, it was neutralized with 1 M HCI. The aqueous layer was extracted with ethyl acetate (3x) and the combined organic lay ers were washed with water, saturated NaHCOs solution and brine, dried over Na2S04, filtered and evaporated to dryness in vacuo. The residue was purified by flash column chromatography (dichloromethane/methanol = 98/2; Rf = 0.3) to yield 24 (180 mg, 65%) as yellow oil.

1 H NMR (400 MHz, CDCIs): d = 5.76 (s, 1 H), 4.55 (t, J = 5.7 Hz, 1 H), 4.41 (t, J = 5.7 Hz, 1 H), 2.46 - 2.35 (m, 2H), 2.32 - 2.23 (m, 2H), 2.10 - 2.00 (m, 2H), 1 .94 - 1 .75 (m, 8H) ppm.

13 C NMR (101 MHz, CDCIs): d = 172.46, 120.77, 84.96, 83.28, 55.19, 39.16,

35.64, 29.82, 29.62, 23.10, 21 .59 ppm. MS: 213.05 [M+H] +

2-(4-Fluorobutyl)-3-((2'-(1 -trityl-1 H-tetrazol-5-yl)-[1 , 1 '-biphenyl]-4-yl)methyl)-1 ,3-di- azaspiro[4.4]non-1-en-4-one (25)

Compound 24 (160 mg, 0.75 mmol) was dissolved in dry DMF and K2CO3

(207 mg, 1 .50 mmol) and compound 4 (836 mg, 1 .50 mmol) were added. The re- action mixture was stirred for 24 h at room temperature. Afterwards, the reaction mixture was diluted with water and it was extracted with ethyl acetate (3x). The combined organic layers were washed with water, saturated NaHC03 solution and brine, dried over Na2S04, filtered and evaporated to dryness in vacuo. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate = 6/1 ; Rf = 0.2) to yield 25 as white solid.

1 H NMR (400 MHz, DMSO-cfe): d = 7.82 (dd, J = 7.6, 1 .2 Hz, 1 H), 7.67 (td, J = 7.5, 1 .5 Hz, 1 H), 7.59 (td, J = 7.5, 1 .4 Hz, 1 H), 7.46 (dd, J = 7.6, 1 .1 Hz, 1 H), 7.44 - 7.30 (m, 9H), 7.09 (d, J = 8.3 Hz, 2H), 7.04 -6.98 (m, 2H), 6.91 - 6.83 (m, 6H), 4.88 (d, J = 16.4 Hz, 1 H), 4.77 (d, J = 16.4 Hz, 1 H), 4.73 (t, 1 H), 4.61 (t, 1 H), 3.32

- 3.27 (m, 2H), 2.29 - 2.18 (m, 2H), 1 .99 - 1 .93 (m, 6H), 1 .75 - 1 .68 (m, 2H), 1 .55

- 1 .46 (m, 2H), 1 .39 - 1 .27 (m, 2H) ppm.

13 C NMR (101 MHz, DMSO-cfe): d = 186.17, 163.99, 162.98, 141 .55, 130.00, 129.65, 129.63, 128.76, 128.31 , 126.56, 123.02, 1 18.10, 1 17.03, 76.77, 60.19, 58.69, 40.63, 39.16, 35.64, 29.82, 29.62, 23.10, 21 .59 ppm.

MS: 689.20 [M+H] +

3-((2'-(1 H-Tetrazol-5-yl)-[ 1, 1 '-biphenyl]-4-yl)methyl)-2-(4-fluorobutyl)-1, 3-dia- zaspiro[4.4]non-1-en-4-one (MD149)

Compound 25 (380 mg, 0.55 mmol) was dissolved in methanol (10 ml_) and HCI (1 .25 M solution in methanol, 2 ml_, 2.5 mmol) was added. The solution was stirred for 30 min at room temperature. The solvent was evaporated and the residue was purified by flash column chromatography (dichloromethane/methanol = 98/2, Rf = 0.2) to yield MD149 (241 mg, 98%) as white solid.

1 H NMR (400 MHz, DMSO-cfe): d = 7.77 - 7.64 (m, 2H), 7.49 - 7.33 (m, 2H), 7.09 -6.99 (m, 4H), 4.88 (d, J = 16.4 Hz, 1 H), 4.82 (d, J = 16.4 Hz, 1 H), 4.77 (t, 1 H), 4.67 (t, 1 H), 3.33 - 3.27 (m, 2H), 2.32 - 2.26 (m, 2H), 1 .99 - 1 .90 (m, 6H), 1 .70 - 1 .64 (m, 2H), 1 .55 - 1 .46 (m, 2H), 1 .39 - 1 .27 (m, 2H) ppm.

13 C NMR (101 MHz, DMSO-cfe): d = 185.97, 189.85, 141 .57, 138.00, 137.65, 131 .33, 131 .06, 130.75, 128.31 , 127.89, 126.21 , 84.96, 83.28, 76.76, 58.69,

42.73, 39.66, 29.86, 29.68, 23.19, 21 .59 ppm.

MS: 447.15 [M+H] +

Ethyl 2-(benzyloxy)pentanoate (27)

Ethyl 2-bromopentanoate (26, 1 .01 g, 4.83 mmol) was dissolved in dry DMF and benzyl alcohol (= BnOH) (0.502 ml_, 522 mg, 4.83 mmol) was added. The resulting solution was cooled down to 0 °C before NaH (60% suspension in paraffin oil,

232 mg, 5.8 mmol) was added portionwise. After complete addition the reaction mixture was stirred for 1 h at 0 °C, then for 23 h at room temperature. After careful addition of water, the mixture was extracted with ethyl acetate (3x). The combined organic layers were washed with water, saturated NaHC03 solution (2x) and brine, dried over Na2S04, filtered and evaporated to dryness in vacuo to yield 27 (1 .14 g, quantitative) as colorless oil.

1 H NMR (400 MHz, CDCIs): d = 4.95 (t, J = 6.0 Hz, 1 H), 4.85 - 4.80 (m, 1 H), 4.25 (q, J = 7.1 Hz, 2H), 1 .93 - 1 .78 (m, 1 H), 1 .56 - 1 .44 (m, 1 H), 1 .31 (t, J = 7.1 Hz, 2H), 0.97 (t, J = 7.4 Hz, 2H) ppm.

13 C NMR (101 MHz, CDCIs): d = 175.88, 138.07, 128.77, 128.74, 128.65, 128.43, 127.99, 77.43, 71 .73, 61 .80, 37.76, 18.08, 14.45, 13.90 ppm.

MS: 237.05 [M+H] +

Ethyl 2-hydroxypentanoate (28)

Compound 27 (1 .1 g, 4.65 mmol) was dissolved in ethanol (20 ml_) and palladium on carbon (= Pd/C) (1 10 mg, 10%) was added. The atmosphere was saturated with H2 and the mixture was stirred for 1 h at room temperature, then 2 h to reflux. Afterwards, the catalyst was filtered off and the solvent was removed under re- duced pressure. The residue was purified by flash column chromatography (petro- leum ether/ethyl acetate 9/1 , Rf = 0.3) to give 28 (250 mg, 37%) as colorless, vola- tile oil. 1 H NMR (400 MHz, CDCIs): d = 4.28 - 4.20 (m, 2H), 4.17 (dt, J = 7.5, 3.7 Hz, 1 H),

1.81 - 1.70 (m, 1 H), 1.67 - 1.55 (m, 1 H), 1.52 - 1.39 (m, 2H), 1.30 (t, J = 7.1 Hz, 3H), 0.94 (t, J = 7.3 Hz, 3H) ppm.

13 C NMR (101 MHz, CDCIs): d = 175.61 , 70.41 , 61.74, 36.66, 18.19, 14.34, 13.91 ppm.

Ethyl 2-fluoropentanoate (29)

Compound 28 (1 g, 6.84 mmol) was dissolved in dry dichloromethane and the so- lution was cooled to -78 °C. Then diethylaminosulfur trifluoride (= DAST) (1 ml_,

1.22 g, 7.57 mmol) was added portionwise and the reaction mixture was stirred at -78 °C for 2 h. Then the reaction mixture was allowed to reach room temperature and it was stirred for 48 h. Then ice water was added carefully and the mixture was extracted with ethyl acetate (3x). The combined organic layers were washed with water, saturated NaHCOs solution (2x) and brine, dried over Na2S04, filtered and evaporated to dryness in vacuo. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate 98/2, Rf = 0.35) to yield 29

(380 mg, 37%) as colorless oil.

1 H NMR (400 MHz, CDCIs): d = 4.95 (t, J = 6.0 Hz, 1 H), 4.85 - 4.80 (m, 1 H), 4.25 (q, J = 7.1 Hz, 2H), 1.93 - 1.78 (m, 1 H), 1.56 - 1.44 (m, 1 H), 1.31 (t, J = 7.1 Hz, 2H), 0.97 (t, J = 7.4 Hz, 2H) ppm.

13 C NMR (101 MHz, CDCIs): d = 172.3, 170.5, 90.1 , 88.9, 61.4, 34.7, 34.3, 18.1 , 17.8, 14.2, 13.6 ppm.

2-Fluoropentanoic acid (30)

Compound 29 (350 mg, 2.3 mmol) was dissolved in methanol (5 ml_) and 1 M NaOH solution in water (5 ml_) was added. The mixture was stirred for 5 h at room temperature. Then HCI (1 M) was added until acidic reaction and the mixture was extracted with ethyl acetate (3x). The combined organic layers were washed with water and brine, dried over Na2S04, filtered and evaporated to dryness in vacuo to give 30 (280 mg, 99%) as colorless, volatile oil, which was used directly in the next step without further purification. 1-(2-Fluoropentanamido)cyclopentane-1 -carboxamide (31 )

Compound 30 (280 mg, 2.33 mmol) were dissolved in dry DMF (5 ml_) and triethyl- amine (64 pl_, 46 mg, 4.66 mmol) was added. Then compound 22 (299 mg,

2.33 mmol) and HBTU (972 mg, 2.56 mmol) were added. The mixture was stirred for 24 h at room temperature. Then water was added and the mixture was ex- tracted wit ethyl acetate (3x). The combined organic layer was washed with water, saturated NaHC03 solution and brine (2x), dried over Na2S04, filtered and evapo- rated to dryness in vacuo. The residue was purified by flash column chromatog- raphy (dichloromethane/methanol = 98/2; Rf = 0.2) to yield 31 (320 mg, 60%) as white solid.

1 H NMR (CDCIs): d = 4.92 (dd, J = 7.4, 3.7 Hz, 0.5H), 4.80 (dd, J = 7.4, 3.8 Hz, 0.5H), 2.08 - 1.96 (m, 2H), 1.83 - 1.69 (m, 8H), 1.51 - 1.40 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H) ppm.

13 C NMR (CDCIs): d = 175.71 , 171.08, 92.90, 91.05, 67.46, 38.73, 36.65, 34.48, 23.99, 17.70, 13.71 ppm.

2-(1-Fluorobutyl)-1,3-diazaspiro[4.4]non-1-en-4-one (32)

Compound 31 (300 mg, 1.3 mmol) was dissolved in methanol (10 ml_) and KOH (10 M solution in water, 10 ml_, 100 mmol) was added. The mixture was stirred for 1 h at 60 °C. Then the mixture was allowed to reach room temperature and it was neutralized with 1 M HCI. The aqueous layer was extracted with ethyl acetate (3x) and the combined organic layers were washed with water, saturated NaHCOs so- lution and brine, dried over Na2S04, filtered and evaporated to dryness in vacuo. The residue was purified by flash column chromatography (dichloromethane/meth- anol = 98/2; Rf = 0.28) to yield 32 (184 mg, 67%) as yellow oil.

1 H NMR (400 MHz, CDCIs): d = 5.31 (dd, 0.5H), 5.19 (dd, 0.5H), 2.05 - 1.86 (m, 8H), 1.86 - 1.74 (m, 2H), 1.62 - 1.43 (m, 2H), 0.98 (t, J = 7.4 Hz, 3H) ppm.

13 C NMR (101 MHz, CDCIs): d = 175.71 , 171.08, 92.90, 91.05, 67.46, 38.73,

36.65, 34.48, 23.99, 17.70, 13.71 ppm.

MS: 213.05 [M+H] + 2-(1 -Fluorobutyl)-3-((2'-(1 -trityl-1 H-tetrazol-5-yl)-[1 , 1 '-biphenyl]-4-yl)methyl)-1 ,3-di- azaspiro[4.4]non-1-en-4-one (33)

Compound 32 (170 mg, 0.8 mmol) was dissolved in dry DMF (5 ml_) and K2CO3 (122 mg, 0.88 mmol) was added. Then compound 4 (491 mg, 0,88 mmol) was added and the mixture was stirred for 18 h at room temperature. After the reaction was finished, the reaction mixture was diluted with water and was extracted with ethyl acetate (3x). The combined organic layers were washed with water and brine, dried over Na2S04 and evaporated to dryness in vacuo. The residue was purified with flash column chromatography (petroleum ether/ethyl acetate = 6/1 , Rf = 0.3) to give 33 (351 mg, 64%) as white solid.

1 H NMR (400 MHz, DMSO-cfe): d = 7.77 (dd, J = 7.6, 1 .2 Hz, 1 H), 7.61 (td, J = 7.5, 1 .5 Hz, 1 H), 7.53 (td, J = 7.5, 1 .4 Hz, 1 H), 7.43 (dd, J = 7.6, 1 .1 Hz, 1 H), 7.40 - 7.30 (m, 10H), 7.09 - 7.01 (m, 3H), 6.92 - 6.85 (m, 6H), 5.31 (dd, J = 8.1 , 5.1 Hz, 0.5H), 5.20 (dd, J = 8.3, 4.8 Hz, 0.5H), 4.77 (d, J = 16.4 Hz, 1 H), 4.66 (d, J = 16.4 Hz, 1 H), 1 .92 - 1 .63 (m, 10H), 1 .38 - 1 .18 (m, 2H), 0.78 (t, J = 7.4 Hz, 3H) ppm. 13 C NMR (101 MHz, DMSO-cfe): d = 185.59, 163.51 , 158.53, 158.32, 141 .12, 140.81 , 139.29, 135.61 , 130.55, 130.50, 130.36, 129.55, 129.19, 128.28, 127.84, 126.28, 125.75, 87.96, 86.28, 82.29, 76.30, 59.75, 43.08, 36.77, 33.04, 32.84, 25.46, 20.74, 17.36, 17.32, 13.41 ppm.

MS: 689.20 [M+H] +

3-((2'-(1 H-Tetrazol-5-yl)-[ 1, 1 '-biphenyl]-4-yl)methyl)-2-( 1 -fluorobutyl)-1 , 3-dia- zaspiro[4.4]non-1-en-4-one (MD147)

Compound 33 (310 mg, 0.45 mmol) was dissolved in methanol (10 ml_) and HCI (1 .25 M solution in methanol, 2 ml_, 2.5 mmol) was added. The solution was stirred for 30 min at room temperature. The solvent was evaporated and the residue was purified by flash column chromatography (dichloromethane/methanol = 98/2, Rf = 0.2) to yield MD147 (198 mg, 99%) as white solid.

1 H NMR (400 MHz, DMSO-cfe): d = 7.72 - 7.64 (m, 2H), 7.61 - 7.50 (m, 2H), 7.14 - 7.04 (m, 4H), 5.76 (s, 1 H), 5.39 (dd, J = 7.9, 5.2 Hz, 0.5H), 5.28 (dd, J = 8.2, 4.9 Hz, 0.5H), 4.76 (dd, J = 42.5, 16.6 Hz, 2H), 1 .94 - 1 .67 (m, 10H), 1 .46 - 1 .25 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H) ppm. 13 C NMR (101 MHz, DMSO-cfe): d = 185.60, 158.56, 158.36, 141.01 , 138.33, 136.05, 131.06, 130.59, 129.09, 127.80, 126.27, 87.96, 86.30, 76.29, 54.89,

42.98, 36.75, 33.08, 32.87, 25.44, 17.38, 17.33, 13.44 ppm.

MS: 447.25 [M+H] +

1-(2-(Benzyloxy)pentanamido)cyclopentane-1 -carboxamide (35)

2-(Benzyloxy)pentanoic acid (34, 850 mg, 4.08 mmol) were dissolved in dry DMF (10 ml_) and triethylamine (690 pl_) was added. Then compound 22 (471 mg,

3.67 mmol) and HBTU (1547 mg, 4.08 mmol) were added. The mixture was stirred for 24 h at room temperature. Then water was added and the mixture was ex- tracted wit ethyl acetate (3x). The combined organic layer was washed with water, saturated NaHC03 solution and brine (2x), dried over Na2S04, filtered and evapo- rated to dryness in vacuo. Compound 35 was obtained as colorless solid

(1162 mg, 99%) and was directly used in the next step without further purification. MS: 319.10 [M+H] +

2-(1-(Benzyloxy)butyl)-1,3-diazaspiro[4.4]non-1-en-4-one (36)

Compound 35 (500 mg, 1.57 mmol) was dissolved in methanol (10 ml_) and KOH (10 M solution in water, 10 ml_, 100 mmol) was added. The mixture was stirred for 1 h at 60 °C. Then the mixture was allowed to reach room temperature and it was neutralized with 1 M HCI. The aqueous layer was extracted with ethyl acetate (3x) and the combined organic layers were washed with water, saturated NaHC03 so- lution and brine, dried over Na2S04, filtered and evaporated to dryness in vacuo. The residue was purified by flash column chromatography (dichloromethane/meth- anol = 98/2; Rf = 0.25) to yield 36 (180 mg, 38%) as yellow oil.

1 H NMR (400 MHz, CDCIs): d = 7.31 - 7.19 (m, 5H), 4.46 (dd, J = 11.6, 9.9 Hz, 2H), 4.19 (dd, J = 7.7, 5.9 Hz, 1 H), 1.99 - 1.82 (m, 6H), 1.78 - 1.67 (m, 4H), 1.32 - 1.14 (m, 2H), 0.85 (t, J = 7.4 Hz, 3H) ppm.

13 C NMR (101 MHz, CDCIs): d = 188.12, 176.63, 163.73, 137.28, 128.68, 128.33, 78.00, 76.45, 72.49, 37.69, 37.63, 35.72, 26.17, 18.53, 13.91 ppm.

MS: 301.05 [M+H] + 2-(1 -Hydroxybutyl)-1 , 3-diazaspiro[4.4]non-1 -en-4-one (37)

Compound 36 (550 mg, 1.83 mmol) was dissolved in dry methanol (20 ml_) and Pd/C (70 mg; 13%) was added. Then the atmosphere was evaporated three times and was replaced with hydrogen. The reaction mixture was heated to 50 °C for 24 h. Afterwards the catalyst was filtered off and the solvent was evaporated in vacuo. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate = 1/1 , Rf = 0.15) to yield 37 (73 mg, 13%) as white solid.

1 H NMR (400 MHz, CDCIs): d = 4.51 (dd, J = 7.7, 5.1 Hz, 1 H), 2.02 - 1.86 (m, 6H), 1.84 - 1.69 (m, 4H), 1.57 - 1.38 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H) ppm.

13 C NMR (101 MHz, CDCIs): d = 190.90, 171.85, 76.34, 68.64, 37.58, 37.48, 37.18, 26.15, 18.46, 13.99 ppm.

MS: 211.05 [M+H] +

2-(1 -Hydroxybutyl)-3-((2'-(1 -trityl-1 H-tetrazol-5-yl)-[1 , 1 '-biphenyl]-4-yl)methyl)-1 ,3- diazaspiro[4.4]non-1-en-4-one (38)

Compound 37 (240 mg, 1.14 mmol) was dissolved in dry DMF (5 ml_) and K2CO3 (189 mg, 1.37 mmol) was added. Then compound 4 (613 mg, 1.1 mmol) was added and the mixture was stirred for 48 h at room temperature. After the reaction was finished, the reaction mixture was diluted with water and was extracted with ethyl acetate (3x). The combined organic layers were washed with water and brine, dried over Na2S04 and evaporated to dryness in vacuo. The residue was purified with flash column chromatography (petroleum ether/ethyl acetate = 1/1 , Rf = 0.2) to give 38 (543 mg, 72%) as white solid.

1 H NMR (400 MHz, aceton e-cfe): d = 7.88 - 7.82 (m, 1 H), 7.58 (td, J = 7.5, 1.5 Hz,

1 H), 7.52 (td, J = 7.5, 1.5 Hz, 1 H), 7.45 - 7.32 (m, 10H), 7.10 (s, 4H), 7.01 - 6.95 (m, 6H), 4.85 (q, J = 16.0 Hz, 2H), 4.29 (dt, J = 12.5, 6.2 Hz, 1 H), 1.96 - 1.87 (m, 4H), 1.78 - 1.57 (m, 2H), 1.43 - 1.22 (m, 4H), 0.92 - 0.81 (m, 2H), 0.77 (t, J = 7.4 Hz, 3H) ppm.

13 C NMR (101 MHz, aceton e-cfe): d = 186.90, 164.73, 163.49, 142.54, 142.27, 140.85, 137.22, 131.47, 131.10, 130.92, 130.84, 130.16, 129.01 , 128.51 , 128.35, 127.36, 127.27, 83.51 , 76.90, 68.15, 43.98, 37.88, 37.74, 36.88, 26.42, 26.41 , 19.02, 13.98 ppm. MS: 867.15 [M+H] +

1-(4-Oxo-3-((2'-(1-trityl-1H-tetrazol-5-yl)-[1, 1'-biphenyl]-4-yl)methyl)-1,3-dia- zaspiro[4.4]non-1-en-2-yl)butyl 4-methylbenzenesulfonate (39)

Compound 38 (530 mg; 0.77 mmol) was dissolved in dry dichloromethane (10 ml_) and the solution was cooled to 0 °C. Then triethylamine (128 mI_, 0.92 mmol) and p-tosylchloride (= TsCI) (176 mg, 0.92 mmol) were added. The solution was stirred for 1 h at 0 °C, then 72 h at room temperature. Afterwards the solvent was evapo- rated and the residue was purified by flash column chromatography (petroleum ether/ethyl acetate = 3/1 , Rf = 0.26) to yield 39 (180 mg, 28%) as yellow solid.

1 H NMR (400 MHz, DMSO-cfe): d = 7.78 (dd, J = 7.7, 1.3 Hz, 1 H), 7.66 - 7.52 (m, 4H), 7.42 - 7.33 (m, 12H), 7.10 (d, J = 8.3 Hz, 2H), 6.99 (d, J = 8.2 Hz, 2H), 6.92 - 6.86 (m, 6H), 4.99 (dd, J = 8.5, 5.2 Hz, 1 H), 4.65 (s, 2H), 2.01 - 1.85 (m, 4H), 1.87 - 1.76 (m, 2H), 1.30 - 1.18 (m, 4H), 0.89 - 0.76 (m, 2H), 0.54 (t, J = 7.4 Hz, 3H) ppm.

13 C NMR (101 MHz, DMSO-cfe): d = 185.55, 164.18, 163.47, 158.34, 158.05, 145.93, 141.28, 138.00, 135.70, 132.87, 130.58, 130.50, 130.02, 129.80, 128.78, 128.38, 128.34, 128.15, 126.93, 126.89, 126.28, 126.20, 82.82, 76.70, 37.16, 34.51 , 25.88, 21.54, 17.89, 17.81 , 15.66, 14.40, 13.14, 12.85 ppm.

MS: 841.20 [M+H] +

Radiolabeling

Radiolabeling of compound 39 to [ 18 F]MD147 was performed analogously to that of compound 10 to [ 18 F]FV45. [ 18 F]F _ produced via proton bombardment of H2 18 0 was isolated by trapping on Sep-Pak Light QMA cartridge, followed by washing with 3 mL water. Fluoride was eluted with a mixture of a solution of K2CO3 in 0.3 mL of water (50.6 mM) and a solution of Kryptofix 2.2.2. (14 mg) in 0.7 mL of acetonitrile into a sealed glass vial. The solution was dried with azeotropic condi- tion under argon flow at 120 °C. The solution of 5 mg precursor 39 in 0.3 mL dry acetonitrile was added to the residue followed by heating at 110 °C for 10 min un- der argon atmosphere. Subsequent removal of trityl protection group were per- formed in the same vessel by the addition of 0.3 mL 1 N HCI and continued to stir at room temperature for 10 min. The mixture was diluted with 1 ml_ of mixture solu- tion of water and acetonitrile (1 : 1 ), and applied to the semi-preparative HPLC col- umn (ZORBAX Eclipse XDB-C-is, 5 pm, 9.4x250 mm, linear gradient of 50-95% methanol with 0.1 % formic acid, 3 mL/min). The radiolabeled [ 18 F]MD147 was ob- tained at retention time 14 min. Overall, the total synthesis time of labeling is ~120 min. The average overall radiochemical yield was 25.6 ± 7.1 % (decay-cor- rected based on starting activity, calculated from 5 times of labeling records) and > 99% radiochemical purity. After purification, 5 ml_ water was added to the collected solution containing radioactive tracer. The solution was then passed through a Sep-Pak plus cartridge (C18), washed with 4 ml_ of water and eluted with 3 ml_ of diethyl ether. The organic solution was concentrated at 50 °C and could be diluted with saline to appropriate concentration for the imaging studies.

3. Renal imaging studies in rats using 18 F-valsartan

[ 18 F]FV45 was obtained as described above. Rats were maintained under anes- thesia by 2% isoflurane during the whole experiment. [ 18 F]FV45 (20-25MBq) was administered via the tail vain. A 60 min list-mode PET acquisition was started shortly after injection. The data obtained in rats demonstrated distinct [ 18 F]FV45 accumulation in the kidneys: tissue activity, mainly renal cortex, was fast and high in the control animal after 10 min of injection (Fig. 3 left and Fig. 4 above). Cold (non-labeled) valsartan, which was administered 3 hours (oral, 30 mg) and 10 min (i. v., 30 mg/kg in saline), respectively, before the injection of [ 18 F]FV45, visibly in- hibited kidney uptake of [ 18 F]FV45 (Fig. 3 right and Fig. 4 below). Despite the unfa- vorable uptake into the liver compared to kidney, the uptake of [ 18 F]FV45 into renal cortex could be clearly observed (shown as control in Fig. 3 and Fig. 4). Further- more, this uptake could be blocked by cold valsartan (shown as co-injection in Fig. 3 and Fig. 4), which indicates that the tracer uptake is specific to the ATi receptor. 4. Radioligand binding studies on human ATi receptors

Radioligand binding assays were performed by using cell membrane preparations expressing human ATi receptors (Membrane Target Systems™; PerkinElmer, Waltham, MA). Human ATi receptors (0.6 pg of membrane protein/well) were in- cubated with assay buffer (50 mM Tris*HCI, 5 mM MgC , pH 7.4) containing vari- ous concentrations of test compounds angiotensin II, MD147, MD149 and FV45 and tracer 125 l-Sar 1 -lle 8 -Angiotensin II (= All) (final concentration 0.3 nM) in 200 pl_ total volume in 96 well plate at room temperature. After 1 hour, the plate was washed 9 times with 250 mI/well of wash buffer (50 mM Tris*HCI, pH 7.4) to re- move unbound tracer. Membrane-bound radioactivity was counted using a gamma-counter (FH 412; Frieseke & Hopfner, Erlangen, Germany). The results are shown in Fig. 8. Nonspecific binding of 125 l-Sar 1 -lle 8 -AII was estimated in the presence of 10 M unlabeled All. Specific binding was defined as total binding mi- nus nonspecific binding. All data were generated in triplicates. Results are ex- pressed as percent of control 125 l-Angiotensin II binding. In Fig. 8 MD147 (ICso = 2.44 nM) is a-F-irbesartan, MD149 (ICso = 7.46 nM) is w-F-irbesartan, FV45 (ICso = 17.4 nM) is w-F-valsartan. ICso of angiotensin II is 1 .96 nM. The results show clear binding of all tested compounds with ICso values similar to that of All.

5. Renal imaging studies in pigs using 18 F- a-F-irbesartan Healthy female Gottingen pigs (weight 22 kg for control and 26 kg for blocking experiment) were used. The pigs were kept anaesthetized by isoflurane during the whole experi- ments. [ 18 F]MD147 (33 MBq) was injected via the ear vein followed by PET meas- urements (30-45min after tracer injection) using a clinical PET scanner (PCA- 2000A, Toshiba Medical Systems Corporation, Otawara, Japan). For blocking ex- periments, 2 mg/kg irbesartan was injected 10 min prior to injection of the tracer ([ 18 F]MD147 16MBq).

Fig. 9 shows dynamic kidney uptake of [ 18 F]MD147 as control (top) and after blocking by selective ATi receptor blocker irbesartan (bottom) with time frames of 30 to 45 min after tracer administration. "SUV" means "standardized uptake value". Fig. 9, top shows a high tracer uptake as there is a high density of ATi re- ceptors present. This effect was reduced by the injection of 2 mg/kg of irbesartan 10 min before injection of tracer proving specific binding (Fig. 9, bottom).

6. Studies in rats using 18 F-irbesartan

After preparation of [ 18 F]MD147 as described above and radioactive purity control, [ 18 F]MD147 (10 MBq) was injected intravenously in anaesthetized rats (Wister, anaesthetized with 2% isoflurane). PET-data were acquired and reconstructed into images of 5 min time frames. Fig. 10 A shows dynamic kidney uptake of

[ 18 F]MD147 as control (left) and after blocking by selective ATi receptor blocker irbesartan (right) 60 min after tracer injection with 5 min timeframes. The good up- take of tracer into kidney tissue proves the suitability of [ 18 F]MD147 for renal imag- ing. Selective binding of tracer was confirmed by the pretreatment with non-la- belled irbesartan 5 min before tracer injection. The pretreatment significantly re- duced renal retention with no clear delineation of kidneys 10 min after tracer ad- ministration.

Tracer time-activity curves for kidney uptake in rats are presented as SUVs from 0 to 50 min as control (·) and after blocking with irbesartan (5 mg/kg;■) in Fig. 10 B.

Fig. 10 C shows the results of biodistribution studies in rats 15 min after tracer in- jection as control (hatched columns, n = 3) and after blocking by selective ATi re- ceptor blocker irbesartan (black columns, n = 3) in rats. Uptake of [ 18 F]MD147 is expressed as SUV. The data reveal that tracer [ 18 F]MD147 is taken up into all rele- vant tissues expressing ATi receptors such as heart, kidney and lung, while tracer uptake can be reduced significantly by blocking with non-radioactive drug irbesar- tan 5 min before tracer injection. Almost no uptake into other organs such as mus- cle could be detected. Metabolic degradation might be the cause of bone accumu- lation of 18 F-fluoride, but there was minimal bone uptake at current imaging stud- ies, indicating high metabolic stability of the tracer during the timeframe of the PET-study.




 
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