WO2008132211A1

WO2008132211A1 - Amino-quinazolinone derivatives for use as radiotracers and imaging agents

Info

Publication number: WO2008132211A1
Application number: PCT/EP2008/055244
Authority: WO
Inventors: William R. Antonios-Mccrea; Yves Auberson; Mats Bergstroem; Emmanuelle Briard; Kristin Brinner; Barry Haskell Levine; Timothy D. Machajewski; Daniel Poon
Original assignee: Novartis Ag
Priority date: 2007-05-01
Filing date: 2008-04-29
Publication date: 2008-11-06

Abstract

The present invention relates to compounds of formula (I) which includes a radiolabeled atom is used to probe Hsp90 in vivo using molecular imaging modalities. A method of making compounds of formula (I), and uses in monitoring disorders associated with disfunction of heat shock protein 90 are also included within the invention.

Description

AMINO-QUINAZOLINONE DERIVATIVES FOR USE AS RADIOTRACERS AND

IMAGING AGENTS

FIELD OF THE INVENTION The present invention relates to novel quinazolinone derivatives, their preparation, their use as radiotracer s/markers and compositions containing them.

BACKGROUND OF THE INVENTION

The Hsp90 family of chaperones is comprised of four known members: Hsp90α and Hsp90β both in the cytosol, grp94 in the endoplasmic reticulum and trap-1 in the mitochondria. Hsp90 is an abundant cellular chaperone required for the ATP-dependent refolding of denatured or "unfolded" proteins and for the conformational maturation of a variety of key proteins involved in the growth response of the cell to extracellular factors. These proteins, which are called client proteins, include the steroid receptors as well as various protein kinases. Hsp90 is essential for eukaryotic cell survival and is overexpressed in many tumors. Cancer cells seem to be sensitive to transient inhibition of Hsp90 ATPase activity suggesting that Hsp90 inhibitors could have a potential as new anticancer drugs. Each Hsp90 family member possesses a conserved ATP -binding site at its N-terminal domain, which is found in few other ATP-binding proteins. The weak ATPase activity of Hsp90 is stimulated upon its interaction with various co-chaperone proteins. Several natural compounds such as geldanamycin or radicicol bind at the ATP-binding site of Hsp90 inhibiting its ATPase activity. In cellular systems and in vivo, these drugs upon binding to Hsp90 prevent the folding of the client proteins, which are then degraded in the proteasome. 17-allylamino-17-demethoxygeldanarnycin (17- AA G), a geldanamycin derivative, is currently in Phase I clinical trial at several institutions. Initial clinical experiences with 17- AAG have offered preliminary evidence that concentrations of the drug associated with activity in pre-clinical systems can be achieved in humans with tolerable toxicity, and provided early evidence of target modulation in at least certain surrogate and tumor compartments. The dose limiting toxicity of 17-AAG is hepatic. 17-AAG poor solubility makes it difficult to formulate/administer and its synthesis is difficult (it is generally obtained by fermentation). Therefore alternative compounds with better physicochemical properties and maybe of higher specificity (17- A AG inhibits all of the four Hsp90 paralogs) are needed. Noninvasive, nuclear imaging techniques can be used to obtain basic and diagnostic information about the physiology and biochemistry of living subjects, including experimental animals, patients and volunteers. These techniques rely on the use of imaging instruments that can detect radiation emitted from radiotracers administered to living subjects. The information obtained can be reconstructed to provide planar and tomographic images which reveal the distribution and/or concentration of the radiotracer as a function of time.

Positron emission tomography (PET) is a noninvasive imaging technique that offers the highest spatial and temporal resolution of all nuclear medicine imaging modalities and has the added advantage that it can allow for true quantitation of tracer concentrations in tissues. The technique involves the use of radiotracers, labelled with positron emitting radionuclides, that are designed to have in vivo properties which permit measurement of parameters regarding the physiology or biochemistry of a variety of processes in living tissue.

Compounds can be labelled with positron or gamma emitting radionuclides. The most commonly used positron emitting radionuclides are ^$0, ^N, HC and ^F, which are accelerator produced and have half lifes of 2, 10, 20 and 110 minutes respectively. The most widely used gamma emmitting radionuclides are 99m-p_C; 201-j/i ^d 123j

For in vitro labelling of HSP90 binding sites and for competition assays compounds that incorporate H, I or Br would be preferred.

Summary of the Invention

We have now found that amino-quinazolinone derivatives can be also be used to probe Hsp90 in vivo using molecular imaging modalities.

Accordingly the present invention provides a compound of formula I

or a stereoisomer, tautomer, or salt thereof, wherein

Y is H or lower alkyl; W is CH₂, NH, orN-alkyl;

X is hydrogen or halo;

Ra is het which may be unsubstituted or substituted with one or more substituents selected from halo, alkoxy, alkyl, nitro, amino, alkylamino, haloalkyl, and haloalkoxy, wherein Ra bears a radio-labeled carbon or fluoro atom. In an embodiment, the compound of formula I includes compounds where Y is H or

CH₃, and Ra is selected from the group of pyridyl, pyrazynil, and thiazolyl. In another embodiment of the compound of formula I, Ra is selected from the group of: 6- [^πC]methoxy-pyridin-2-yl, 6-[^πC]methoxy-pyrazin-2-yl, 2-[¹¹C]methoxy-thiazol-4-yl, 6- (2-[¹⁸F]fluoroethoxy)- pyridin-2-yl, 6-(2-[¹⁸F]fluoroethoxy)- pyrazin-2-yl, 2-(2- [¹⁸F]fluoroethoxy)- thiazol-4-yl, 6-(2-[¹⁸F]fluoromethoxy)- pyridin-2-yl, 6-(2- [¹⁸F]fluoromethoxy)- pyrazin-2-yl, 2-(2-[¹⁸F]fluoromethoxy)- thiazol-4-yl, 5-[¹⁸F]fluoro-6- methoxy- pyridin-2-yl or 5-[ F]fluoro-6-methoxy- pyrazin-2-yl

In another embodiment, the compound of formula I may be in free base or acid addition salt form together with at least one pharmaceutically acceptable carrier or excipient.

In an embodiment of the present invention, the use of a compound of formula I

(I) or a stereoisomer, tautomer, or salt thereof for the manufacture in a composition for the diagnosis of a disorder(s) mediated by HSP90 is included, wherein

Y is H or methyl; W is CH₂, NH, or N-alkyl;

X is hydrogen or halo;

Ra is het which may be unsubstituted or substituted with one or more substituents selected from halo, alkoxy, alkyl, nitro, amino, alkylamino, haloalkyl, and haloalkoxy; and wherein Ra bears a radio-labeled carbon or fluoro atom.

In a further embodiment, the present invention includes a method of making a compound of the formula I

or a stereoisomer, tautomer, or salt thereof, wherein

Y is H or methyl; W is CH₂, NH, or N-alkyl; X is hydrogen or halo;

Ra is het which may be unsubstituted or substituted with one or more substituents selected from halo, alkoxy, alkyl, nitro, amino, alkylamino, haloalkyl, and haloalkoxy, and has a radio-labeled carbon or fluoro atom; said method comprising providing a precursor compound of Ra and reacting said precursor compound of Ra with a compound having a radio-labeled atom.

In an embodiment, Ra may be selected from the group of 6-f¹ C]methoxy-pyridin-2- yl, 6-[^πC]methoxy-pyrazin-2-yl, 2-[ⁿC]methoxy-thiazol-4-yl, 6-(2- [¹⁸F] fluoroethoxy)- pyridin-2-yl, 6-(2-[¹⁸F]fluoroethoxy)- pyrazin-2-yl, 2-(2-[¹⁸F]fluoroethoxy)- thiazol-4-yl, 6-

(2-[ rl^l8^βF]fluoromethoxy)- pyridin-2-yl, 6-(2-[ 1^l8^δτF]fluoromethoxy)- pyrazin-2-yl, or 2-(2- [' Fjfluoromethoxy)- thiazol-4-yl. The precursor compound of Ra may be selected from the group of 6-hydroxy-pyridin-2-yl, 6-hydroxy-pyrazin-2-yl or 2-hydroxy-thiazol-4-yl. The compound having a radio-labeled atom may be selected from the group of [ⁿC]alkyliodide, [ Hjalkyliodide, [ Fjfluoromethylbromide, [ FJfluoroethylbromide, or [¹⁸F]fluoroethyltosylate.

In another embodiment, Ra may be selected from the group of 6-[ Fjfluoro-pyridin-

2-yl, 6-[¹⁸F]fluoro-pyrazin-2-yl, 2-[¹⁸F]fluoro-thiazol-4-yl, 5-[¹⁸F]fluoro-6-methoxy- pyrazin-2-yl. The Ra precursor may be selected from the group of 6-halo- pyridin-2-yl, 6- halo- pyrazin-2-yl, 2-halo- thiazol-4-yl, and the compound having a radio-labeled atom is [¹⁸F]F^".

In another embodiment, the Ra precursor compound is selected from the group of 6- nitro- pyridin-2-yl, 6-nitro- pyrazin-2-yl, 2-nitro- thiazol-4-yl, 5-nitro-6-methoxy-pyrazin-2- yl, and the compound having a radio-labeled atom is [¹⁸F]F^".

In yet another embodiment, a method of monitoring HSP90 activity is included within the present invention. The method includes providing an amino-quinazolinone derivative of formula (I), administering said derivative to a mammal, and observing said derivative within said mammal.

In yet another embodiment, the present invention includes a method of labeling tumors, brain and other tissues exhibiting overexpression, activation, or dysregulation of Hsp90 in vitro or in vivo. The method includes contacting tumor, brain tissues and other tissues with a compound of formula I in free base or salt form.

Detailed Description of the Invention

Ra is selected from the group consisting of pyridyl, pyrazynil and triazolyl. Groups are substituted with one or two sustituents selected from the group consisting of O¹¹CH₃, OC(³H)₃, (CH₂)IiHaI, 0(CH₂)IiHaI, (O(CH₂)n)nHal, Hal where Hal is ¹²³1, ⁷⁶Br, ¹⁸F and n is 1, 2, 3 or 4.

The general terms used hereinbefore and hereinafter preferably have within the context of this disclosure the following meanings, unless otherwise indicated

"Alkyl" includes lower alkyl preferably alkyl with up to 10 carbon atoms, preferably from 1 to and including 5, and is linear or branched; preferably, lower alkyl is methyl, ethyl, propyl, such as n-propyl or isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, straight or branched pentyl, straight or branched hexyl, straight or branched heptyl, straight or branched nonyl or straight or branched decyl. Preferably alkyl is C₁ to C₄-alkyl especially methyl, ethyl, propyl, 2-methyl propyl and t-butyl. Most preferably the alkyl group is a lower alkyl of 1-4 carbon atoms, preferably methyl, ethyl, propyl, butyl, isobutyl, terfbutyl, and isopropyl.

"Alkoxy" denotes a radical R'-O-, wherein R' represents alkyl and alkyl has the meaning as specified above. The term "lower alkyl" as used herein when used alone or in combination refers to alkyl containing 1-6 carbon atoms. The alkyl group may be branched or straight-chained, and is as defined hereinabove.

"Het" as used herein, refers to heteroaryl and heterocyclic compounds containing at least one S, O or N ring heteroatom. More specifically, "Het" is a 5-7 membered heterocyclic ring containing 1- 4 heteroatoms selected from N, O and S, or an 8-12 membered fused ring system including at least one 5-7 membered heterocyclic ring containing 1, 2 or 3 heteroatoms selected from N, O, and S. Examples of het, as used herein, include but are not limited to unsubstituted and substituted pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuryl, piperidyl, piperazyl, purinyl, tetrahydropyranyl, morpholino, 1,3- diazapanyl, 1 ,4-diazapanyl, 1 ,4-oxazepanyl, 1 ,4-oxathiapanyl, furyl, thienyl, pyrryl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, oxadiazolyl, imidazolyl, pyrrolidyl, pyrrolidinyl, thiazolyl, oxazolyl, pyridyl, pyrazolyl, pyrazinyl, pyrimidinyl, isoxazolyl, pyrazinyl, quinolyl, isoquinolyl, pyridopyrazinyl, pyrrolopyridyl, furopyridyl, indolyl, benzofuryl, benzothiofuryl, benzoindolyl, benzothienyl, pyrazolyl, piperidyl, piperazinyl, indolinyl, morpholinyl, benzoxazolyl, pyrroloquinolyl, pyrrolo[2,3-b]pyridinyl, benzotriazolyl, oxobenzo-oxazolyl, benzo[l,3]dioxolyl, benzoimidazolyl, quinolinyl, indanyl and the like. Heteroaryls are within the scope of the definition of het. Examples of heteroaryls are pyridyl, pyrimidinyl, quinolyl, thiazolyl and benzothiazolyl. The most preferred het are pyridyl, pyrimidinyl and thiazolyl. The het may be unsubstituted or substituted as described herein. It is preferred that it is unsubstituted or if substituted it is substituted on a carbon atom by halogen, especially fluorine or chlorine, hydroxy, C₁-C₄ alkyl, such as methyl and ethyl, Cj-C₄ alkoxy, especially methoxy and ethoxy, nitro, -O- C(O)-C _rC₄alkyl or -C(O)-O-C i-C₄alkyl, SCN or nitro or on a nitrogen atom by Ci-C₄ alkyl, especially methyl or ethyl, -O-C(O)-C_rC₄alkyl or -C(O)-O-C_rC₄alkyl, such as carbomethoxy or carboethoxy.

"Halo" as used herein means fluoro, chloro, bromo or iodo, preferably fluoro. Natural occurring carbon consists of the isotopes ¹²C (98.90%), ¹³C (1.10 %) and traces of ¹⁴C. The term "¹¹C" denotes that a higher ratio of the respective carbon atoms in a radical or moiety correspond to ¹¹C isotopes as compared to natural occurring carbon, especially a radical or moiety wherein at least 25 %, preferably at least 90 %, more preferably at least 95 %, of the carbon atoms are ¹¹C isotopes. The ¹¹C isotope can be prepared by methods known as such. Since the half-life period is short (about 20 min), the isotope needs to be freshly prepared shortly before usage in reagents by employment of a cyclotron. The methylation reaction using ^πC-methyl iodide is the most used reaction in ¹¹C labeling synthesis. A suitable method is based on the reduction of ⁿC-carbon dioxide with hydrogen/Ni followed by a free radical iodination in a circulating phase while the [ C]methyl iodide formed is continuously trapped on a solid phase to prevent further iodination.

Natural occurring fluorine consists of the isotope ¹⁹F (100 %). The term "¹⁸F" denotes that a higher ratio of the respective fluorine atoms in a radical or moiety correspond to ¹⁸F isotope as compared to natural occurring fluorine, especially a radical or moiety wherein at least 25 %, preferably at least 90 %, more preferably at least 95 %, of the fluorine atoms are F isotopes.

The F isotope can be prepared by methods known as such. Since the half-life period is short (about 110 min), the isotope needs to be freshly prepared shortly before usage in reagents by employment of a cyclotron. F can be obtained, e.g., by irradiating a target containing ' 0-H₂O with a proton beam, in a cyclotron. In this process, ¹⁸F is obtained from the ¹⁸O(p,n) ¹⁸F nuclear reaction and used immediately to generate final radiomarkers as described, e.g. in "Fundamentals of Positron Emission tomography and Applications in Preclinical Drug Development", Simon R Cherry, J Clin Pharmacol 2001; 41 :482-491, and the references cited therein, which publication is included in the present patent filing by reference.

Salts are especially the pharmaceutically acceptable salts of compounds of formula I. For isolation or purification purposes it is also possible to use pharmaceutically unacceptable salts, for example picrates or perchlorates.

The agents of the invention are useful, for instance, for determining the levels of HSP90 inhibition of a drug acting on HSP90, or diagnostic purposes for diseases resulting from an imbalance or dysfunction of HSP90, and for monitoring the effectiveness of pharmacotherapies of such diseases. Properly isotope-labeled agents of the invention (i.e. compounds of formula (I)) exhibit valuable properties as histopathological labeling agents, imaging agents and/or biomarkers, hereinafter "markers", for the selective labeling of HSP90. More particularly the agents of the invention are useful as markers for labeling HSP90 in vitro or in vivo. Radio-labeled analogues of compound (I) may be used in clinical studies to evaluate the role of HSP90 ligands in a variety of disease areas where HSP90 ligands are believed to be involved.

The present invention also provides a composition, especially for labeling histopathological structures in vitro or in vivo, which comprises a compound of formula (I) and a pharmaceutically acceptable carrier or excipient.

More particularly the agents of the invention are useful as markers for labeling Hsp90 in vitro or in vivo.

The agents of the invention are therefore useful, for instance, for determining the levels of receptor occupancy of a drug acting at Hsp90, or diagnostic purposes for disorders mediated by Hsp90, and for monitoring the effectiveness of pharmacotherapies of such diseases.

The expression "disorders mediated by Hsp90" as used herein denotes disorders, such as tumor diseases, which result from overexpression, activation or dysregulation of Hsp90 or wherein such overexpression, activation or dysregulation play a prominent role. In accordance with the above, the present invention provides an agent of the invention for use as a marker for cancer imaging or neuroimaging.

In a further aspect, the present invention provides a composition for labeling tumors, brain and other tissues involving overexpression activation, or dysregulation of Hsp90 in vivo and in vitro comprising an agent of the invention. In still a further aspect, the present invention provides a method for labeling tumors, brain and other tissues exhibiting overexpression, activation, or dysregulation of Hsp90 in vitro or in vivo, which comprises contacting tumor, brain tissues and other tissues with an agent of the invention.

An agent of the invention may be prepared by processes that, though not applied hitherto for the new compounds of the present invention, are known per se, especially a process characterized in that: a) for the synthesis of a compound of the formula I wherein Ra represents 6- [^πC]methoxy-pyridin-2-yl, 6-[ⁿC]methoxy-pyrazin-2-yl, 2-[¹¹C]rnethoxy-thiazol-4-yl, 6- (2-[¹⁸F]fluoroethoxy)- pyridin-2-yl, 6-(2-[¹⁸F]fluoroethoxy)- pyrazin-2-yl, 2-(2- [¹⁸F]fluoroethoxy)- thiazol-4-yl, 6-(2-[¹⁸F]fluoromethoxy)- pyridin-2-yl, 6-(2- [¹⁸F]fluoromethoxy)- pyrazin-2-yl or 2-(2-[¹⁸F]fluoromethoxy)- thiazol-4-yl, and the other radicals have the meaning as provided above for a compound of formula I, an Ra precursor of 6-hydroxy-pyridin-2-yl, 6-hydroxy-pyrazin-2-yl or 2-hydroxy-thiazol-4-yl is reacted with a compound containing a radiolabeled atom selected from the group of [ⁿC]alkyliodide, [³H]alkyliodide, [¹⁸F]fluoromethylbromide, [¹⁸F]fluoroethylbromide, or [¹⁸F]fluoroethyltosylate. b) for the synthesis of a compound of the formula I wherein Ra represents 6- [¹⁸F]fluoro-pyridin-2-yl, 6-[¹⁸F]fluoro-pyrazin-2-yl, 2-[¹⁸F]fluoro-thiazol-4-yl, 5- [¹⁸F]fluoro-6-methoxy-pyrazin-2-yl and the other radicals have the meaning as provided above for a compound of formula I, an Ra precursor selected from the group of 6-halo- pyridin-2-yl, 6-halo- pyrazin-2-yl, 2-halo- thiazol-4-yl with halo being chloro, bromo or iodo, preferably bromo, or 6-nitro- pyridin-2-yl, 6-nitro- pyrazin-2-yl, 2-nitro- thiazol-4-yl, 5-nitro-6-methoxy-pyrazin-2-yl, 5-halo-6-methoxy-pyrazin-2-yl with halo being chloro, bromo or iodo is reacted with a compound containing a radiolabeled atom, such as [ F]F^".

All process steps described here can be carried out under known reaction conditions, preferably under those specifically mentioned, in the absence of or usually in the presence of solvents or diluents, preferably such as are inert to the reagents used and able to dissolve these, in the absence or presence of catalysts, condensing agents or neutralising agents, for example ion exchangers, typically cation exchangers, for example in the H⁺ form, depending on the type of reaction and/or reactants at reduced, normal, or elevated temperature, for example in the range from -100°C to about 190°C, preferably from about - 80⁰C to about 15O⁰C, for example at -80 to -6O⁰C, at room temperature, at - 20 to 4O⁰C or at the boiling point of the solvent used, under atmospheric pressure or in a closed vessel or in a HPLC loop, where appropriate under pressure, and/or in an inert atmosphere, for example under argon or nitrogen.

The solvents from which those can be selected which are suitable for the reaction in question include for example water, esters, typically lower alkyl-lower alkanoates, e.g diethyl acetate, ethers, typically aliphatic ethers, e.g. diethylether, or cyclic ethers, e.g. tetrahydrofuran, liquid aromatic hydrocarbons, typically benzene or toluene, alcohols, typically methanol, ethanol or 1- or 2-propanol, nitriles, typically acetonitrile, halogenated hydrocarbons, typically dichloromethane, acid amides, typically dimethylformamide, bases, typically heterocyclic nitrogen bases, e.g. pyridine, carboxylic acids, typically lower alkanecarboxylic acids, e.g. acetic acid, carboxylic acid anhydrides, typically lower alkane acid anhydrides, e.g. acetic anhydride, cyclic, linear, or branched hydrocarbons, typically cyclohexane, hexane, or isopentane, or mixtures of these solvents, e.g. aqueous solutions, unless otherwise stated in the description of the process. Such solvent mixtures may also be used in processing, for example through chromatography or distribution. Salts may be present in all starting compounds and transients, if these contain salt- forming groups. Salts may also be present during the reaction of such compounds, provided the reaction is not thereby disturbed.

At all reaction stages, isomeric mixtures that occur can be separated into their individual isomers, e.g. diastereomers or enantiomers, or into any mixtures of isomers, e.g. racemates or diastereomeric mixtures.

Working up the reaction mixtures and purification of the compounds thus obtained may be carried out in accordance to known procedures.

During any of the described synthetic sequences it may be necessary and /or desirable to protect sensitive or reactive groups on any molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed J.F.W. McOmie, Plenum Press, 1973; and T. W. Greene amd P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991. The protecting groups may be removed at a convenient stage using methods known from the art. Where hydroxyl groups require protection, this may be achieved by forming, esters, trialkylsilyl, tetrahydropyran, benzyl or alkyl ethers. Such derivatives may be deprotected by standard procedures thus, for example, a methoxymethyl ether derivative may be deprotected using hydrochloric acid in methanol. New starting materials and/or intermediates, as well as processes for the preparation thereof, are likewise the subject of this invention. In the preferred embodiment, such starting materials are used and reaction conditions so selected as to enable the preferred compounds to be obtained.

EXAMPLES

The following examples are intended to illustrate, but not further limit, the invention. The following compounds are prepared by methods analog to those described herein utilizing analogous starting materials. Temperatures are measured in degrees Celsius (⁰C). Unless otherwise indicated, the reactions take place at room temperature.

Radiolabeled compounds are analyzed by HPLC equipped with a reverse phase column (C 18, 10m particle size 250 x 4,60 mm od) and are identified by retention time and co-injection of the corresponding reference time.

Abbreviations cone. concentrated

DMF dimethyl formamide

Et ethyl

HPLC high performance liquid chromatography

Rt retention time iv intravenous

Me methyl

During any of the described synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any molecules concerned.

Example 1: (R)-2-amino-7-(4-fluoro-2-(6-[¹¹C]methoxy-pyridin-2yl)-phenyl)-4- methyl-7,8-dihydro-6H-quinazolin-5-one

A nitrogen-14 gas target containing ca. 0.5% oxygen is bombarded with a proton beam to generate [¹¹C]CO₂. The [¹¹C]CO₂ is converted to [¹¹C]MeI using a GE Medical Systems PETtrace MeI Microlab instrument. The produced [¹¹C]MeI is delivered to a reaction mixture composed of 0.3 mg of (R)-2-amino-7-(4-fluoro-2-(6-hydroxy-pyridin- 2yl)-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one (prepared according to the procedures disclosed in WO2006113498 and applications US Serial No. 60/722,796 and No. 60/836,886) and sodium hydride in DMF (0.3mg). When the amount of radioactivity in the reaction vessel has reached its peak, the mixture is heated at 130⁰C for 30 min and then diluted with CH₃CN-H₂O for direct injection onto a semi-preparative reverse phase HPLC column for purification. Following collection of the peak corresponding to (R)-2-amino-7- (4-fluoro-2-(6-[ⁿC] methoxy-pyridin-2yl)-phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5- one, the HPLC mobile phase is removed in vacuo and the title compound reformulated in saline for iv injection.

Example 2: [¹¹C] labeled quinazolinones

The compounds of formula I as specified below can be prepared using the same method as described in Example 1, i.e. O-alkylation with [¹¹C]MeI followed by removing the protection group when needed. The starting materials can be prepared according to the procedures disclosed in WO2006113498 and applications US Serial No. 60/722,796 and

No. 60/836,886.

Example 3: [ H] -labeled quinazolinones

The compounds of formula I as specified below can be prepared using the same method as described in Example 1, i.e. O-alkylation with [³H]MeI followed by removing the protection group. The starting materials can be prepared according to the procedures disclosed in WO2006113498 and applications US Serial No. 60/722,796 and No. 60/836,886.

Example 4: (R)-2-amino-7-(4-fluoro-2-(6-(2- r[l8 ^β,F]fluoroethoxy)- pyridin-2yl)- phenyl)-4-methyl-7,8-dihydro-6H-quinazolin-5-one

[^I8F]fluoride prepared by proton bombardment of [¹⁸O]H₂O is concentrated onto an ion exchange resin and recovered by elution using a aqueous potassium carbonate solution.

The aqueous fluoride is added to a micro-vial containing 1 niL of a solution of 10 mg

Kryptofix in CH₃CN. The solution is heated at 116⁰C for 3.5 min after which three additional portions of 1 niL CH₃CN are added and evaporated to dry the fluoride which is then used to react with ethylene glycol bis-tosylate (2.0 mg) in CH₃CN (250 μL) according to the procedures described in J. L. Musachio et al J Label Compd Radiopharm, 2005, 48,

1 S

735-747 to generate [ F]2-fluoroethyl tosylate. The above solution is transferred to a micro-vial containing (R)-2-amino-7-(4-fluoro-2-(6-hydroxy-pyridin-2yl)-phenyl)-4- methyl-7,8-dihydro-6H-quinazolin-5-one. This residue is heated at 13O⁰C for 30 min and then diluted with CH₃CN-H₂O for direct injection onto a semi-preparative reverse phase HPLC column for purification. Following collection of the peak corresponding to (R)-2- amino-7-(4-fluoro-2-(6-(2-[¹⁸F]fluoroethoxy)- pyridin-2yl)-phenyl)-4-methyl-7,8-dihydro- 6H-quinazolin-5-one, the HPLC mobile phase was removed in vacuo and the title compound reformulated in saline for iv injection.

Example 5: _r [18 FJalkoxy labeled quinazolinones The compounds of formula I specified below can be prepared using the same method as described in Example 4, i.e. aklylation using f¹ F]2-fluoroethyl tosylate or [¹⁸F]fluoromethyl bromide. The starting materials can be prepared according to the procedures disclosed in WO2006113498 and applications US Serial No. 60/722,796 and No.

Example 6: (R)-2-amino-7-(4-fluoro-2-(6-(2-[¹⁸F]fluoro-pyridin-2yl)-phenyl)-4-methyl- 7,8-dihydro-6H-quinazolin-5-one

[¹⁸F]fluoride prepared by proton bombardment of [¹⁸O]H₂O is concentrated onto an ion exchange resin and recovered by elution using a aqueous potassium carbonate solution. The aqueous fluoride is added to a micro-vial containing 1 mL of a solution of 10 mg Kryptofix in CH₃CN. The solution is heated at 116⁰C for 3.5 min after which three additional portions of 1 mL CH₃CN are added and evaporated to dry the fluoride, which is then used to react with (R)-2-amino-7-(4-fluoro-2-(6-(2-nitro-pyridin-2yl)-phenyl)-4- methyl-7,8-dihydro-6H-quinazolin-5-one according to the procedures described in M. R. Kilbourn, Fluorine- 18 labeling of Radiopharmaceuticals, National Academy Press, Washington, D.C. 1990; or E.F.J. DeVries et al J Nucl Med, 2003, 44, 1700-1706.

Following that step the reaction mixture is injected onto a semi -preparative reverse phase HPLC column for purification. Following collection of the peak corresponding to (R)-2-amino-7-(4-fluoro-2-(6-(2-[¹⁸F]fluoro-pyridin-2yl)-phenyl)-4-methyl-7,8-dihydro-6H- quinazolin-5-one, the HPLC mobile phase was removed in vacuo and the title compound reformulated in saline for iv injection.

1 fi

Example 7: [ F] labeled quinazolinones The compounds of formula I specified below can be prepared using the same method as described in Example 6, i.e. nitro-to-fluorine exchange. The starting materials can be prepared according to the procedures disclosed in WO2006113498 and applications US Serial No. 60/722,796 and No. 60/836,886.

Example 8: KΪ/IC50 determination (binding assay)

The inhibition of Hsp90 is measured using the procedure described in Schilb et al.

Development and Implementation of a Highly Miniaturized Confocal 2D-FIDA-Based

Analysis-Based High-Throughput Screening Assay to Search for Active Site Modulators of the Human Heat Shock Protein 90β, J of Biomolecular Screeninfi, 2003. The procedure is repeated for different concentrations of test compound selected to cover the range of 0% to

100% inhibition and the concentration at which 50% inhibition of Hsp90 occurs (IC₅₀) for each compound is determined from concentration-inhibition curves in a conventional manner. The compounds of the Examples 1 to 7 have IC₅₀ values of the order of 50-100OnM or less in the above mentioned FIDA assay, specifically <10OnM.

Example 9; Pharmacokinetics and organ distribution

Tissue distribution of radioactivity is determined in normal and tumor-bearing male Fischer rats (200-250 g) after intravenous injection of the radiolabelled compound (1-100 MBq). The animals are allowed food and water ad libitum before the experiment. Following anesthesia radiopharmaceutical is injected into the rats via a tail vein catheter. Groups of four rats are sacrificed at various time points after injection of the dose. The animals are dissected, and selected tissues are weighed and counted along with dose standards in a Gamma Counter. The raw counts are decay-corrected, and the counts normalized as the percent of total injected dose per gram of tissue (% ID/g). The tissue distribution of radioactivity is also determined in tumor-bearing rats following intravenous injection. The same tissues are assayed as in normal rats with the addition of the tumor tissue, and the corresponding region of brain contralateral to the tumor is excised and used for comparison.

Example 10: Ex vivo autoradiography

The regional distribution and uptake of the radiopharmaceuticals can be investigated using a quantitative autoradiographic technique. Radiopharmaceuticals are injected into the tail vein of the animal. Tissues are immediately removed and frozen in isopentane, which is cooled to -70⁰C. The frozen samples are cut into 20 μm sagittal sections using a cryostat, mounted on glass microscope slides and without any washing placed on a phosphor imager screen for 2 h. The imaging plate data is analysed with a BAS800 II system (Fuji Film).

The above preferred embodiments are given to illustrate the scope and spirit of the present invention. The descriptions provided herein will make apparent to those skilled in the art other embodiments and examples. These other embodiments and examples are within the contemplation of the present invention. Therefore, the present invention should be limited only by the appended claims.

Example 11: EC50 determination using multicellular tumor speroid model

The inhibition of Hsp90 is measured using the procedure described in Monazzam et al. Application of the multicellular tumor spheroid model to screen PET tracers for analysis of early response of chemotherapy in breast cancer, Breast Cancer Research, 2007. A selection of compounds from Examples 1 to 7 demonstrate EC50 values below 10 mM.

Claims

What is claimed is:

1. A radio-labeled quinazolinone compound of formula I

or a stereoisomer, tautomer, or salt thereof, wherein

Y is H or lower alkyl;

W is CH₂, NH, orN-alkyl;

X is hydrogen or halo;

Ra is het which may be unsubstituted or substituted with one or more substituents selected from halo, alkoxy, alkyl, nitro, amino, alkylamino, haloalkyl, and haloalkoxy, wherein Ra bears a radio-labeled carbon or fluoro atom.

2. The compound of claim 1 wherein Y is H or CH₃, and Ra is selected from the group of pyridyl, pyrazynil, and thiazolyl.

3. The compound of claim 2 wherein Ra is selected from the group of: 6- [^πC]methoxy-pyridin-2-yl, 6-[ⁿC]methoxy-pyrazin-2-yl, 2-[^uC]methoxy-thiazol-4-yl, 6-

(2-[^lsF]fluoroethoxy)- pyridin-2-yl, 6-(2- [ 1¹8⁸τF] fluoroethoxy)- pyrazin-2-yl, 2-(2- [ rl¹8⁸F]fluoroethoxy)- thiazol-4-yl, 6-(2-[ 1¹8Tτ ]fluoromethoxy)- pyridin-2-yl, 6-(2-

[ _r18τ F]fluoromethoxy)- pyrazin-2-yl, 2-(2-[ rl¹8⁸F]fluoromethoxy)- thiazol-4-yl, 5-[ 1¹8⁸τF]fluoro-6- methoxy- pyridin-2-yl or 5-[ rl8 F]fluoro-6-methoxy- pyrazin-2-yl.

4. The composition according to formula I of claim 1 in free base or acid addition salt form together with at least one pharmaceutically acceptable carrier or excipient.

5. Use of a compound of formula I

or a stereoisomer, tautomer, or salt thereof, wherein

Y is H or methyl;

W is CH₂, NH, orN-alkyl;

X is hydrogen or halo;

Ra is het which may be unsubstituted or substituted with one or more substituents selected from halo, alkoxy, alkyl, nitro, amino, alkylamino, haloalkyl, and haloalkoxy; and wherein Ra bears a radio-labeled carbon or fluoro atom for the manufacture of a composition for the diagnosis of a disorders mediated by HSP90.

6. The use of a compound of formula I according to claim 5 wherein Ra is selected from the group of pyridyl, pyrazynil, and thiazolyl.

7. The use of a compound of formula I according to claim 6 wherein Ra is selected from the group of: 6-[ⁿC]methoxy-pyridin-2-yl, 6-[ⁿC]methoxy-pyrazin-2-yl, 2- [ⁿC]methoxy-thiazol-4-yl, 6-(2-[¹⁸F]fluoroethoxy)- pyridin-2-yl, 6-(2-[¹⁸F]fluoroethoxy)- pyrazin-2-yl, 2-(2-[¹⁸F]fluoroethoxy)- thiazol-4-yl, 6-(2-[¹⁸F]fluoromethoxy)- pyridin-2-yl, 6-(2-[¹⁸F]fluoromethoxy)- pyrazin-2-yl, 2-(2-[¹⁸F]fluoromethoxy)- thiazol-4-yl, 5- [¹⁸F]fluoro-6-methoxy- pyridin-2-yl or 5-[¹⁸F]fluoro-6-methoxy- pyrazin-2-yl.

8. A method of making a compound of the formula I

or a stereoisomer, tautomer, or salt thereof, wherein

Y is H or methyl;

W is CH₂, NH, orN-alkyl;

X is hydrogen or halo;

Ra is het which may be unsubstituted or substituted with one or more substituents selected from halo, alkoxy, alkyl, nitro, amino, alkylamino, haloalkyl, and haloalkoxy, and has a radio-labeled carbon or fluoro atom; said method comprising

Providing a precursor compound of Ra and reacting said precursor compound of Ra with a compound having a radio-labeled atom.

9. The method of claim 8 wherein Ra is selected from the group of 6-[ 1 1/ CJmethoxy- pyridin-2-yl, 6-[ C]methoxy-pyrazin-2-yl, 2-[ C]methoxy-thiazol-4-yl, 6-(2- [¹⁸F]fluoroethoxy)- pyridin-2-yl, 6-(2-[¹⁸F]fluoroethoxy)- pyrazin-2-yl, 2-(2- [¹⁸F]fluoroethoxy)- thiazol-4-yl, 6-(2-[¹⁸F]fluoromethoxy)- pyridin-2-yl, 6-(2-

[ FJfluoromethoxy)- pyrazin-2-yl, or 2-(2-[ 18τ F]fluoromethoxy)- thiazol-4-yl, and said precursor compound of Ra is selected from the group of 6-hydroxy-pyridin-2-yl, 6-hydroxy- pyrazin-2-yl or 2-hydroxy-thiazol-4-yl; and said compound having a radio-labeled atom is selected from the group of [ⁿC]alkyliodide, [³H]alkyliodide, [¹⁸F]fluoromethylbromide,

[ rl8 FJfluoroethylbromide, or [ 18 Fτ Jfluoroethyltosylate.

10. The method of claim 8 wherein Ra is selected from the group of 6-[ 18τ F]fluoro- pyridin-2-yl, 6-[ rl¹8⁸F]fluoro-pyrazin-2-yl, 2-[ 1¹8⁸τF]fluoro-thiazol-4-yl, 5-[ rl¹8^!Tτ ]fluoro-6- methoxy-pyrazin-2-yl, and wherein said Ra precursor is selected from the group of 6-halo- pyridin-2-yl, 6-halo- pyrazin-2-yl, 2-halo- thiazol-4-yl, 5-halo-6-methoxy-pyrazin-2-yl and wherein said compound having a radio-labeled atom is [ F]F^".

11. The method according to claim 8 wherein said Ra precursor compound is selected from the group of 6-nitro- pyridin-2-yl, 6-nitro- pyrazin-2-yl, 2-nitro- thiazol-4-yl, 5-nitro- 6-methoxy-pyrazin-2-yl, and wherein said compound having a radio-labeled atom is [ F]F^".

12. A method of monitoring HSP90 activity comprising:

Providing an amino-quinazolinone derivative of formula (I) according to claim 1; Administering said derivative to a mammal; and Observing said derivative within said mammal.

13. A method for labeling tumors, brain and other tissues exhibiting overexpression, activation, or dysregulation of Hsp90 in vitro or in vivo, which comprises contacting tumor, brain tissues and other tissues with a compound of formula I according to any one of claims 1 to 4, in free base or salt form.