The present invention relates to a novel compound 3-(3-fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine, and the 18F labelled version 3-(3-[18F]-fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine, and to intermediates for preparation of these compounds, and to methods of using these compounds for tau imaging, and to compositions and formulations of these compounds for diagnostic imaging, and to methods of imaging using these compounds, compositions, and formulations.

Alzheimer’s disease (AD), a leading cause of dementia, develops in one percent of the population between the ages 65 and 69, and increases to 40-50% in those 95 years and older. AD patients exhibit telltale clinical symptoms that include cognitive impairment and deficits in memory function. In these patients, the presence of AD is confirmed by heavy senile plaque burden and neurofibrillary tangles (NFT) found in the cerebral cortex upon post mortem histopathological examination. The mature senile plaques consist of extracellular ȕ-amyloid peptides derived from enzymatic processing of amyloid precursor protein and intracellular neurofibrillary tangles (NFT), which are derived from filaments of hyperphosphorylated tau proteins. Aggregates of hyperphosphorylated tau, such as neurofibrillary tangles, are linked to the degree of cognitive impairment in Alzheimer's disease. In AD and various other tauopathies, tau aggregates appear in particular brain regions and patterns that are linked to disease risk, onset, and or progression, and these regions and patterns are known to skilled artisans. In AD patients, tau-containing tangles first appear in brain regions that are very closely linked to memory, and pathologic studies show that tangles may correlate even more strongly with cognition than plaques. Signals arising from a tau imaging agent in these regions and patterns can be used by skilled artisans to better monitor and diagnose the risk, onset and progression of the particular disease state. (See Correlation of Alzheimer disease neuropathologic changes with cognitive status: a review of the literature. Nelson PT, et al., J Neuropathol Exp Neurol.2012 May; 71(5):362-81.) Thus, simple noninvasive methods, for detecting and/or quantitation of tau deposits in patients are eagerly sought. (See M. Maruyama et al.,“Imaging of tau pathology in a tauopathy mouse model and in Alzheimer patients compared to normal controls”, Neuron, 79: 1094-1108, 2013, C. Mathis and W. Klunk,

“Imaging Tau Deposits In Vivo: Progress in Viewing More of The Proteopathy Picture”, Neuron, 79: 1035-10-37, 2013).

Existing agents for PET imaging of tau are known in the art, for example such agents are recited in WO2009/102498, and a compound recently in clinical evaluation, [18F]T807 (also known as AV-1451), is recited in WO 2013/176698. (See also

[(18)F]T807, a novel tau positron emission tomography imaging agent for Alzheimer's disease. Xia CF, et al., Alzheimer’s Dement.2013 Nov; 9(6):666-76.)

However, existing tau imaging
compounds have technical attributes that could be improved by the design of innovative agents which may provide enhanced tau images, with improved tau signaling and minimal non-tau signaling. Thus, improved methods for detecting and/or quantitation of tau in patients are eagerly sought.

There are several potential benefits of imaging tau in the brain with improved imaging agents. Enhanced tau imaging will improve diagnosis by identifying potential patients, those having high levels of tau in the brain, who may have increased chance of developing AD. Imaging with an improved PET agent will also be useful to monitor tau accumulation and localization, and or progression of AD and or other tauopathies, and when anti-tau drug treatments become available, tau imaging may provide an essential tool for monitoring treatment.

The present invention provides novel compounds, compositions, formulations and methods for tau imaging. Improved technology advancing the capacity to image tau in patients is thus also needed to expand the clinical benefits and impact of diagnostic tau imaging. An improved imaging agent will provide enhanced tau images, as compared with known agents, producing images with better clarity due to strong tau signals and decreased non-tau signals.

The present invention provides the compound 3-(3-[18F]-fluoroazetidin-1-yl)-8- methylbenzo[4,5]imidazo[1,2-a]pyridine, also referred to herein as“Compound 8”, which can be structurally represented as the compound of formula I:

The present invention also provides the compound 3-(3-fluoroazetidin-1-yl)-8- methylbenzo[4,5]imidazo[1,2-a]pyridine, also referred to herein as“Compound 4”, which can be structurally represented as the compound of formula II:

The present invention further provides the use of the compound of formula I and/or the compound of formula II, and/or mixtures thereof, for the preparation of tau imaging agents, and tau PET imaging.

The present invention further provides intermediates for preparation of the compound of formula I or the compound of formula II. The present invention provides a compound of formula II (indicated as Compound 8 below) prepared from a compound of formula 7:

The invention further provides a compound of formula I prepared from a compound of formula Ia or formula Ib. A preferred species of the present invention is a compound of formula Ia.

Another preferre
d species of the present invention is a compound of formula Ib:

The present invention provides the use of compounds of formula I, Ia, Ib or II, for the manufacture of a radiopharmaceutical agent for imaging tau in humans. In another aspect the invention provides methods of preparing compounds of formula I, Ia, Ib or II. In another aspect the invention provides methods of preparing Compound 8 from compounds of formula Ia, or Ib. Particularly preferred is the method of preparing Compound 8, or pharmaceutically acceptable salt thereof, from the compound of formula Ia. In another aspect the invention provides a pharmaceutical composition comprising Compound 8, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent or carrier. In another aspect the invention provides a pharmaceutical composition comprising Compound 8, or pharmaceutically acceptable salt thereof, and Compound 4, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent or carrier. In another aspect the invention provides a pharmaceutical composition comprising Compound 8, or pharmaceutically acceptable salt thereof, which is formulated in 10% EtOH (v/v), 0.45% (w/v) sodium ascorbate in 0.9% sodium chloride, preferably for use in humans. In another aspect the invention provides a pharmaceutical composition comprising Compound 8, or pharmaceutically acceptable salt thereof, prepared from a compound of formula Ia or Ib, which is formulated in 10% EtOH (v/v), 0.45% (w/v) sodium ascorbate in 0.9% sodium chloride, preferably for use in humans. The present invention also provides methods of imaging tau comprising introducing into a patient a detectable quantity of Compound 8, or pharmaceutically acceptable salt thereof, or a composition thereof, preferably prepared from a compound of a compound of formula Ia or Ib.

The present invention provides a process of making a compound of the formula I:

comprising reacting 1-(8-methy
lbenzo[4,5]imidazo[1,2-a]pyridin-3-yl)azetidin-3-yl 4-methylbenzenesulfonate, represented by the formula:

with a source of [18F]fluoride.

The present invention provides a process of making a compound of the formula I:

comprising reacting a compound of formula Ib:

with a source of [18F]fluoride.

The present invention provides a method of imaging tau comprising: introducing into a mammal a detectable quantity of the compound:

,

allowing sufficient time for said compound to become associated with tau, and detecting said compound.

The following Schemes, Preparations, and Examples are provided to better elucidate the practice of the present invention. Suitable reaction conditions for the steps of these Schemes, Preparations, and Examples are well known in the art and appropriate modification of reaction conditions, including substitution of solvents and co-reagents are within the ability of the skilled artisan.

Furthermore, the skilled artisan will appreciate that in some circumstances, the order in which moieties are introduced is not critical. The particular order of steps required to produce the compounds of Formula I or Formula II is dependent upon the particular compound being synthesized, the starting compound, and the relative lability of the substituted moieties, as is well appreciated by the skilled chemist. The skilled artisan will appreciate that not all substituents are compatible with all reaction conditions. These compounds may be protected or modified at a convenient point in the synthesis by methods well known in the art. The intermediates and final products of the present invention may be further purified, if desired by common techniques such as

recrystallization or chromatography over solid supports such as silica gel or alumina.

The compounds of the present invention are preferably formulated as

radiopharmaceutical compositions administered by a variety of routes. Preferably, such compositions are for intravenous use, preferably in humans. Such pharmaceutical compositions and processes for preparing same are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy (P.P. Gerbino, 21st ed., Lippincott Williams & Wilkins, 2006). Methods of using tau imaging agents for PET imaging of tau are known to those of skill in the art. See e.g. [(18)F]T807, a novel tau positron emission tomography imaging agent for Alzheimer's disease. Xia CF, et al., Alzheimer’s Dement. 2013 Nov; 9(6):666-76.). [(18)F]T807 is also known as [18F]AV-1451.

Preferred formulations of the present invention are preparations of Compound 8 prepared from a compound of formula Ia. Particularly preferred is Compound 8 prepared from the compound of formula Ia according to the procedures described herein according to Scheme 2. Particularly preferred is Compound 8 prepared from the compound of formula Ia according to the procedures described herein according Example 2. A preferred formulation of Compound 8 is formulated in 10% EtOH (v/v), 0.45% (w/v) sodium ascorbate in 0.9% sodium chloride, preferably for use in humans. Another embodiment of the invention is a formulation of Compound 8 prepared from the compound of formula Ia and formulated in 10% EtOH (v/v), 0.45% (w/v) sodium ascorbate in 0.9% sodium chloride. Particularly preferred is Compound 4 prepared according to the procedures described herein according to Scheme 1. Particularly preferred is Compound 8 prepared from the compound of formula Ia according to the procedures described herein according to Example 2 and formulated in 10% EtOH (v/v), 0.45% (w/v) sodium ascorbate in 0.9% sodium chloride. The present invention provides a method of imaging tau comprising introducing into a mammal a detectable quantity of a diagnostic composition as described according to the embodiments herein, and allowing sufficient time for said diagnostic composition to become associated with tau; and detecting the diagnostic composition. Particularly preferred is a method of imaging tau comprising introducing into a mammal a detectable quantity of a diagnostic composition of Compound 8, prepared from the compound of formula Ia according to the procedures described herein according to Example 2, and formulated in 10% EtOH (v/v), 0.45% (w/v) sodium ascorbate in 0.9% sodium chloride.

Novel compounds of Formula I and II have been discovered to be surprisingly and unexpectedly advantageous for tau imaging, preferably including human clinical imaging. A preferred compound, the compound of formula I, also referred to herein as Compound 8, possesses a combination of particularly useful properties for tau imaging, including high affinity for tau, selectivity, uptake, washout, and metabolic profile. In vivo

Compound 8 demonstrates advantageous tissue distribution, pharmacokinetics, and metabolic stability. Ex vivo and/or in vitro, Compound 8 demonstrates high affinity binding to tau, and labels tau containing tissue samples from AD brain with high selectivity with respect to Aȕ and/or non-tau binding. Compound 8 demonstrates high affinity and selectivity for tau, exhibiting radiographic signals which are disease state, tissue, and cellular location specific. The radiographic signals generated by Compound 8 reflect improved detection of tau as compared to undesired non-tau signals, and an in vivo tissue distribution and metabolic profile which are useful for a clinical

radiopharmaceutical imaging agent. Compound 8, having this combination of particularly useful properties, provides for enhanced tau images, as compared with known agents, producing images with improved clarity due to robust tau signals and decreased non-tau signals. This surprisingly advantageous combination of properties provides an effective clinical tau imaging agent which facilitates imaging of patients for tau. The use of Compound 8 in clinical tau PET imaging would have important positive impact on assessment and or diagnosis of AD, and would advance the detection, treatment, monitoring, and evaluation of tau and diagnosis of diseases involving tau.

Description of the Figures:

Figure 1. Representative Semi-Preparative HPLC Chromatogram of 3-(3-[18F]-Fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine (Compound 8)

Radiosynthesis. The upper panel illustrates an HPLC chromatograph with gamma detection. The lower panel illustrates an HPLC chromatograph with UV detection. The segment indicated as“Cut Peak” indicates the corresponding fractions collected to obtain the product 3-(3-[18F]-Fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine (Compound 8).

Figure 2. Representative Analytical HPLC (QC) Chromatogram of 3-(3-[18F]-Fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine (Compound 8). The upper panel labelled (HPLC Gamma Detector) illustrates a radio-chromatogram of 3-(3-[18F]-Fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine (Compound 8). The lower panel labelled (HPLC UV Detector) illustrates an ultraviolet chromatogram of 3-(3-[18F]-Fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine (Compound 8). The peak retention time of 6.589 minutes is indicated on the main peak in the upper panel, and peak retention time of 6.607 minutes is indicated on the main peak in the lower panel.

Figure 3.3-(3-[18F]-Fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine (Compound 8) autoradiography on AD brain sections for Kd determination. See Assay Example 4 for an explanation of the experimental setup and analysis.

Figure 4: 3-(3-[18F]-Fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine autoradiography on AD brain sections for selectivity determination. See Assay Example 5 for an explanation of the experimental setup and analysis.

Figure 5: 3-(4-(2-[18F]-Fluoroethyl)piperidin-1-yl)-8-methoxybenzo[4,5]imidazo[1,2-a]pyridine autoradiography on AD brain sections for selectivity determination See Assay Example 5 for an explanation of the experimental setup and analysis.

Figure 6: 3-(4-[18F]-Fluoropiperidin-1-yl)-8-methoxybenzo[4,5]imidazo[1,2-a]pyridineautoradiography on AD brain sections for selectivity determination See Assay Example 5 for an explanation of the experimental setup and analysis.

Figure 7: 3-(3-[18F]-Fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine, Compound 8 autoradiography on normal versus AD brain sections. See Assay Example 6 for an explanation of the experimental setup and analysis.

Figure 8: 3-(3-[18F]-Fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine (Compound 8) and [18F]AV-1451 (also known as [18F]T807), autoradiography on normal versus AD brain sections using alcohol free washes. See Assay Example 6 for an explanation of the experimental setup and analysis.

Figure 9: Mouse PET/CT time activity curves of 3-(4-(2-[18F]-Fluoroethyl)piperidin-1-yl)-8-methoxybenzo[4,5]imidazo[1,2-a]pyridine (AKA T821) versus 3-(3-[18F]-Fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine (Compound 8).

Figure 10: Mouse PET/CT time activity curves of 3-(4-[18F]-Fluoropiperidin-1-yl)-8-methoxybenzo[4,5]imidazo[1,2-a]pyridine (AKA T798) versus 3-(3-[18F]-Fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine (Compound 8).

Examples and Preparations

General Methods

All reactions are run under a nitrogen atmosphere unless otherwise noted.

Products are purified using an automated Teledyne Isco Flash® Chromatography System. 1H, 19F, and 13C NMR spectra are recorded on a Bruker® HD Avance III 400 spectrometer in CDCl3 (Cambridge Isotope Laboratories, Cat. No. DLM-7-100) or DMSO-d6, (Cambridge Isotope Laboratories, Cat. No. DLM-10-25). HRMS data are obtained on a Waters® QTof mass spectrometer using an electrospray ionization positive scan mode. Elemental analysis is performed at Galbraith Laboratories using GLI Procedure ME-14 (Galbraith Inc., 2323 Sycamore Drive, Knoxville, TN 37921).

Reagents, solvents, and supplies are known to the skilled chemist. The names for the compounds of the present invention can be generated for example using Symyx Version 3.2.NET with the IUPAC naming functionality.

Abbreviations represent the common and ordinary usage known to one of skill in the art and particular abbreviations used herein have the following meanings:

Abbreviations:

BPV bulk product vial

bs Broad singlet

CDCl3 deuterated chloroform

CH2Cl2 methylene chloride

d doublet

DAD diode array detector

dd doublet of doublets

dt doublet of triplets

DMPAO [(2,6-dimethylphenyl)amino](oxo)acetic acid

DMSO dimethyl sulfoxide

DMSO-d6 hexadeuterodimethyl sulfoxide

EtOH ethanol

HPLC high performance liquid chromatography

HRMS high resolution mass spectrometry

IHC immunohistochemistry

K2CO3 potassium carbonate

LCMS liquid chromatography mass spectrometry

N normal

NMR nuclear magnetic resonance

PHF paired helical filaments

ppm parts per million

QTof quaternary time of flight

s singlet

SUV standardized uptake value

SUVr standardized uptake value ratio

t triplet

UPLC ultra-high performance liquid chromatography

PBS phosphate buffered saline

WFI water for injection

Schemes

Scheme 1 provides the synthesis of 3-(3-fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine. The synthesis commences with the formation of the benzo[4,5]imidazo[1,2-a]pyridine core via a copper catalyzed coupling of

commercially available 2-bromo-4-methylaniline and 2,4-dibromopyridine followed by intramolecular cyclization. The desired product, 3-(3-fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine, is obtained via a second copper catalyzed coupling of 3-bromo-8-methylbenzo[4,5]imidazo[1,2-a]pyridine (3) and 3-fluoroazetidine hydrogen chloride. After column chromatography on silica gel, metal scavenging with Quadrasil MP resin, and trituration, 3-(3-fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine (Compound 4) is obtained as a yellow solid (4.44 g, 21% overall yield).

Scheme 1: Synthesis of 3-(3-Fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2- a]pyridine.

Br CuI

Scheme 2 provides the synthesis of 1-(8-methylbenzo[4,5]imidazo[1,2-a]pyridin- 3-yl)azetidin-3-yl 4-methylbenzenesulfonate, which is the precursor for 3-(3-[18F]- fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine. Copper catalyzed coupling of 3-bromo-8-methylbenzo[4,5]imidazo[1,2-a]pyridine (3) and 3- hydroxyazetidine affords hydroxyl intermediate 1-(8-methylbenzo[4,5]imidazo[1,2- a]pyridin-3-yl)azetidin-3-ol (5), which is also purified by silica gel column

chromatography. The clean intermediate is then reacted with tosyl anhydride and triethylamine, followed by silica gel column chromatography to give the desired product, 1-(8-methylbenzo[4,5]imidazo[1,2-a]pyridin-3-yl)azetidin-3-yl 4-methylbenzenesulfonate (6), as a beige solid (3.21 g, 31% overall yield).

Scheme 2: Synthesis of 1-(8-Methylbenzo[4,5]imidazo[1,2-a]pyridin-3-yl)azetidin-3-yl 4-methylbenzenesulfonate (6).

,

methylbenzo[4,5]imidazo[1,2-a]pyridine, Compound 8, is prepared from a compound of Formula 7 where R is a suitable leaving group. More specifically, a compound of Formula 7 where R is a leaving group such as methanesulfonyl (mesyl) or 4- methylbenzenesulfonyl (tosyl), can be reacted with a suitable source of 18F fluoride ([18F]F-) in the presence of a suitable base such as potassium carbonate. Sources of 18F fluoride ([18F]F-) include [18F]F K222. Suitable solvents include dimethylsulfoxide.

Scheme 3: Synthesis of 3-(3-[18F]-Fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2- a]pyridine (8).

Example 1

Synthesis of 3-(3-Fluoroazetidin-1-yl)-8-methylbenzo[4,5]imidazo[1,2-a]pyridine (Compound 4).

Step 1: Synthesis of 3-Bromo-8-methylbenzo[4,5]imidazo[1,2-a]pyridine (Compound 3).

In a 1 L round-bottom flask are combined 2,4-dibromopyridine (20.0 g, 84.6 mmol), copper iodide (3.22 g, 16.9 mmol), 1,10-phenanthroline (6.10 g, 33.8 mmol), cesium carbonate (110 g, 338 mmol), Celite® (16 g) and p-xylene (170 mL). To the resulting slurry is added 2-bromo-4-methylaniline (10.6 mL, 84.6 mmol) and nitrogen is bubbled through the vigorously stirred mixture for 10 minutes. The flask is fitted with a reflux condenser and the system is heated at 135°C for 24 hours. The reaction mixture is cooled to room temperature and filtered. The filter cake is rinsed with methylene chloride and ethyl acetate, and the combined organic filtrates are concentrated under reduced pressure over silica gel. The crude reaction product is purified by chromatography on silica gel using a gradient of 0 to 10% ethyl acetate in methylene chloride. The resulting brown solid is slurried in methylene chloride and triturated using hexanes, then isolated by filtration to provide the title compound (6.52 g, 25.0 mmol, 30% yield) as a shiny yellow solid: 1H NMR (400.13 MHz, DMSO-d6 with TFA-d) į ppm: 9.40 (dd, J=0.9, 7.2Hz, 1H), 8.45 (dd, J=0.7, 1.8Hz, 1H), 8.42 (bs, 1H), 7.86 (dd, J=2.1, 7.3Hz, 1H), 7.83 (d, J=8.4Hz, 1H), 7.64 (dd, J=0.9, 8.4Hz, 1H), 2.57 (s, 3H); 13C NMR (100.62 MHz, DMSO-d6 with TFA-d) į ppm 142.6, 134.6, 131.9, 130.8, 129.9, 129.4, 127.0, 119.7, 115.0, 113.8, 113.4, 21.1; HRMS (m/z): found: 261.0013 (M+H), calcd for C12H10N2Br: 261.0027, Err=–5.4 ppm.

We Claim:

1. A compound of the formula:

, or pharmaceutically acceptable salt thereof.

2. A compound of the formula:

, or pharmaceutically acceptable salt thereof.

3. A compound of the formula:

. 4. A compound of the formula:

. 5. A process of making a compound of the formula: ,

comprising reacting a compound of the formula:

,

with a source of [18F]fluoride.

6. A process of making a compound of the formula:

,

comprising reacting a compound of the formula:

,

with a source of [18F]fluoride.

7. A composition comprising

,

or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.

8. A composition comprising

,

or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.

9. A composition comprising

,

and 10% EtOH (v/v), 0.45% (w/v) sodium ascorbate in 0.9% sodium chloride.

10. A composition comprising

made by the process of claim 5 or 6; and 10% EtOH (v/v), 0.45% (w/v) sodium ascorbate in 0.9% sodium chloride.

11. A method of imaging aggregated tau comprising:

a.introducing into a mammal a detectable quantity of the compound:

, or a pharmaceutically acceptable salt thereof,

b. allowing sufficient time for said compound to become associated with tau; and

c. detecting said compound.

12. A method of imaging aggregated tau comprising:

a. introducing into a mammal a detectable quantity of a composition of claim 7, 9 or 10;

b. allowing sufficient time for said composition to become associated with tau; and

c. detecting the composition.

13. A method of claim 11 wherein the mammal is a human suspected of having

Alzheimer’s Disease.

14. A method of claim 12 wherein the mammal is a human suspected of having

Alzheimer’s Disease.

15. A method of claim 11 wherein the mammal is a human suspected of having

Chronic Traumatic Encephalopathy (CTE).

16. A method of claim 12 wherein the mammal is a human suspected of having

Chronic Traumatic Encephalopathy (CTE).

17. An intermediate for preparing a compound of claim 2 wherein the intermediate is 3-bromo-8-methylbenzo[4,5]imidazo[1,2-a]pyridine.

18. An intermediate for preparing a compound of claim 3 wherein the intermediate is 1-(8-methylbenzo[4,5]imidazo[1,2-a]pyridin-3-yl)azetidin-3-ol.