PET imaging for soluble epoxide hydrolase (sEH) 18 F-FNDP

Abstract

The present application relates to PET imaging of soluble epoxide hydrolase (sEH) 18 F-FNDP. Disclosed herein are radiofluorinated FNDPs for PET imaging of soluble epoxide hydrolase (sEH) and methods of use thereof, in particular, compounds of Formula (I) of the present application and uses thereof are disclosed.

Description

[0001] This application is filed on May 4, 2017, with application number 201780041727.6, and is titled “PET imaging of soluble epoxide hydrolase (sEH)”. 18 F-FNDP” application.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 331,691, filed May 4, 2016, which is incorporated herein by reference in its entirety.

[0004] Federally funded research or development

[0005] This invention was made with government support under Grants NS089437, NS038684, NS060703 awarded by the National Institute of Health (NIH), and DoD Grant GW130098. The government has certain rights in this invention. background

[0006] Epoxyeicosatrienoic acids (EETs) are important signaling molecules in vasodilation of cerebral blood vessels associated with neuronal activity. They also regulate the activity of many molecular targets and signaling pathways (Spector and Norris, Am. J. Physiol. Cell Physiol 2009). Soluble epoxide hydrolases (sEHs) (Sura et al., J. Histochem. Cytochem, 2008; Marowski et al., Neuroscience, 2009) distributed throughout mammals catalyze the hydrolysis of EETs into molecules with lower biological activity (Newman et al., Prog. Lipid. Res., 2005). Over the past decade, sEH has become a drug target, and many small molecule sEH inhibitors have been developed. Those sEH inhibitors increase the level of EETs, which can then benefit various conditions, including hypertension, atherosclerosis, inflammation, diabetes, pain, and lung disease and others (Shen and Hammock, J. Med. Chem., 2012).

[0007] The regulation of sEH is altered in many conditions, including vascular cognitive impairment (VCI), and stroke, among others. The contribution of cerebrovascular pathology to Alzheimer's disease (AD) and dementia is becoming increasingly understood. Postmortem studies have shown that one-third of patients with dementia have comorbid cerebrovascular pathology (White et al., Ann. NY Acad. Sci., 2002; Knopman et al., Arch. Neurol., 2003). Recent reports have found that sEH activity in subjects with VCI is 50% greater than in age-matched controls (Neslon et al., Prostaglandins Other Lipid. Mediat., 2014). The most common type of VCI is associated with white matter hyperintensities, which are early predictors of conversion to mild cognitive impairment (Neslon et al., Prostaglandins Other Lipid. Mediat., 2014), which in turn represent an increased risk of developing AD.

[0008] As mentioned above, the change of sEH expression changes the biological effect of EET. The effect of EET observed consistently is their ability to prevent apoptosis after ischemic damage and other forms of damage (Iliff and Alkayed, Future neurology, 2009). Various studies have shown that EET protects the brain during stroke and the inhibition of sEH enhances this effect (Ingraham et al., Curr. Med. Chem, 2011). Patients with aneurysmal subarachnoid hemorrhage are at high risk of delayed cerebral ischemia and stroke (Martini et al., J. Neurosurg., 2014). Patients with the common K55R polymorphism in the sEH gene (Ephx2) demonstrated 30% lower levels of EETs due to increased sEH activity (Lee et al., Hum. Mol. Genet., 2006), and they presented a 28.6% mortality rate after stroke, compared to a 5.3% mortality rate in control subjects (Martini et al., J. Neurosurg., 2014). Other studies have demonstrated highly increased sEH expression in animal models of epilepsy (Hung et al., Brain Behav. Immun., 2015) and Parkinson's disease (Qin et al., Mol. Neurob., 2015).

[0009] In addition to promoting drug development (Shen and Hammock, J. Med. Chem., 2012), the importance of PET imaging agents targeting sEH lies in obtaining a better understanding of stroke and dementia, i.e., the vascular aspects of the latter, non-invasively, repeatedly, and with high resolution. Clinically, stroke is mainly evaluated by anatomical and functional magnetic resonance imaging, with molecular methods being limited by the lack of viable radiotracers for this indication, except for methods for measuring perfusion with single-photon emission computed tomography (Heiss, Neurosci. Bull., 2014). PET agents for sEH may enable the in vivo differentiation of AD and VCI, rather than having to rely on postmortem observation of Aβ plaques and neurofibrillary tangles (Morris et al., Eur. J. Nucl. Med. Mol, Imaging, 2015; Couto and Millis, International Journal of Alzheimer's disease, 2015).

[0010] Overview

[0011] In some aspects, the presently disclosed subject matter provides compounds of formula (I):

[0012]

[0013] wherein X is selected from the group consisting of F, Br, and I and radioactive isotopes thereof; Y is -NR-C(=O)- or -C(=O)-NR-; m is an integer selected from the group consisting of 1, 2, 3, and 4; n is an integer selected from the group consisting of 1, 2, 3, 4, and 5; p is an integer selected from the group consisting of 1, 2, and 3; and R is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted aryl alkyl, substituted or unsubstituted alkylheteroaryl, substituted or unsubstituted heteroalkylaryl, and substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl; each R1 may independently be the same or different and is selected from the group consisting of hydrogen, halogen, alkoxy, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted naphthyl, and substituted or unsubstituted biphenyl;

[0014] and stereoisomers or pharmaceutically acceptable salts thereof.

[0015] In other aspects, the compound of formula (I) is a compound of formula (II):

[0016]

[0017] In certain aspects, the compounds of Formula (I) further comprise a radioisotope suitable for imaging.

[0018] In specific aspects, the radioisotope suitable for imaging is 18 F. 76 Br, 123 I. 124 I. 125 I and 131 I.

[0019] In a still more specific aspect, the compound of formula (I) is

[0020]

[0021] In other aspects, the presently disclosed subject matter provides methods for imaging soluble epoxide hydrolase (sEH), comprising contacting the sEH with an effective amount of a compound of formula (I) and producing an image.

[0022] In some other aspects, the presently disclosed subject matter provides methods for inhibiting soluble epoxide hydrolase (sEH) in the treatment of sEH-mediated diseases, the methods comprising administering to a subject a therapeutically effective amount of a compound of formula (I), thereby inhibiting sEH.

[0023] In yet other aspects, the presently disclosed subject matter provides kits comprising a compound of Formula (I).

[0024] This application provides the following:

[0025] 1. A compound of formula (I):

[0026]

[0027] in

[0028] X is selected from the group consisting of F, Br, and I, and radioactive isotopes thereof;

[0029] Y is -NR-C(=O)- or -C(=O)-NR-;

[0030] m is an integer selected from the group consisting of 1, 2, 3, and 4;

[0031] n is an integer selected from the group consisting of 1, 2, 3, 4 and 5;

[0032] p is an integer selected from the group consisting of 1, 2, and 3;

[0033] R is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted alkylheteroaryl, substituted or unsubstituted heteroalkylaryl, and substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl;

[0034] each R1 may independently be the same or different and is selected from the group consisting of hydrogen, halogen, alkoxy, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted naphthyl, and substituted or unsubstituted biphenyl;

[0035] and stereoisomers or pharmaceutically acceptable salts thereof.

[0036] 2. The compound according to item 1, wherein the compound of formula (I) is a compound of formula (II):

[0037]

[0038] 3. The compound according to item 1, wherein the compound of formula (I) contains a radioactive isotope suitable for imaging.

[0039] 4. The compound of claim 3, wherein the radioisotope suitable for imaging is selected from the group consisting of: 18 F. 76 Br, 123 I. 124 I. 125 I and 131 I.

[0040] 5. A compound as described in claim 4, wherein the radioisotope suitable for imaging is 18 F.

[0041] 6. The compound according to item 1, wherein the compound of formula (I) is:

[0042]

[0043] 7. A method for imaging soluble epoxide hydrolase (sEH), the method comprising contacting sEH with an effective amount of a compound of formula (I) and producing an image, the compound of formula (I) comprising:

[0044]

[0045] in:

[0046] X is selected from the group consisting of: 18 F. 76 Br, 123 I. 124 I. 125 I and 131 I;

[0047] Y is -NR-C(=O)- or -C(=O)-NR-;

[0048] m is an integer selected from the group consisting of 1, 2, 3 and 4;

[0049] n is an integer selected from the group consisting of 1, 2, 3, 4 and 5;

[0050] p is an integer selected from the group consisting of 1, 2, and 3;

[0051] R is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted alkylheteroaryl, substituted or unsubstituted heteroalkylaryl, and substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl;

[0052] each R1 may independently be the same or different and is selected from the group consisting of hydrogen, halogen, alkoxy, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted naphthyl, and substituted or unsubstituted biphenyl;

[0053] and stereoisomers or pharmaceutically acceptable salts thereof.

[0054] 8. The method according to item 7, wherein the compound of formula (I) is a compound of formula (II):

[0055]

[0056] 9. The method according to item 7, wherein X is 18 F.

[0057] 10. The method according to claim 7, wherein the compound of formula (I) is:

[0058]

[0059] 11. The method of item 7, wherein the image is obtained by using positron emission tomography.

[0060] 12. The method of claim 7, wherein the compound of formula (I) is highly specific for sEH.

[0061] 13. The method of claim 12, wherein the specificity is up to about 95%.

[0062] 14. The method of claim 7, wherein sEH is in vitro, in vivo or ex vivo.

[0063] 15. The method of claim 7, wherein sEH is present in the subject.

[0064] 16. The method of claim 15, wherein the subject is a human.

[0065] 17. The method of claim 15, wherein the compound of formula (I) is capable of crossing the blood-brain barrier, and wherein sEH is present in the brain of the subject.

[0066] 18. The method of claim 15, wherein the method is non-invasive.

[0067] 19. The method of claim 15, wherein the compound of formula (I) readily enters the brain of the subject.

[0068] 20. The method of claim 15, wherein the compound of formula (I) is cleared from the brain of the subject.

[0069] 21. A method for inhibiting soluble epoxide hydrolase (sEH) in the treatment of a sEH-mediated disease, the method comprising administering to a subject a therapeutically effective amount of a compound of formula (I), thereby inhibiting sEH.

[0070] 22. The method of claim 21, wherein the soluble epoxide hydrolase-mediated disease is selected from the group consisting of hypertension, atherosclerosis, inflammation, diabetes-related diseases, pain, lung disease, Alzheimer's disease, vascular cognitive impairment (VCI), and stroke.

[0071] 23. A pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier, diluent or excipient.

[0072] 24. A kit comprising a packaged pharmaceutical composition comprising a compound according to any one of items 1 to 6.

[0073] Having set forth above certain aspects of the presently disclosed subject matter, which are fully or partially addressed by the presently disclosed subject matter, other aspects will become apparent as the description proceeds when used in conjunction with the accompanying examples and drawings as best described herein below. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale, and in which:

[0075] Figure 1A and Figure 1B (A) N-(3,3-diphenylpropyl)-6-fluoronicotinamide (FNDP) and the radiolabeled [ 18 Synthesis of the precursor of FNDP-FNDP; Reagents and conditions: (a) 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIPEA), dimethylformamide (DMF), room temperature; (B) Radiotracer for PET imaging of sEH 18 Radiosynthesis of F-FNDP;

[0076] Figure 2 Representative sEH inhibitors known in the art, 1-cyclohexyl-3-dodecyl-urea (CDU) and 12-(3-(adamantan-1-yl)ureido)dodecanoic acid (AUDA) are shown (Shen, Expert. Opin. Ther. Pat., 2010); these compounds are considered unsuitable leads for PET radiotracer development due to the large hydrophobic domains in their structures;

[0077] Figure 3 A comparative study of the relative fluorescence of the compounds FNDP, des-fluoro-FNDP (N-(3,3-diphenylpropyl)-nicotinamide), and AUDA is shown; FNDP demonstrated low nanomolar inhibitory activity against sEH, comparable to the nanomolar inhibitory activity of AUDA and des-fluoro-FNDP;

[0078] Figure 4 Shown is the effect of the sEH inhibitor des-fluoro-FNDP (subcutaneous) on the brain of CD-1 mice 60 min after radiotracer injection. 18Dose-dependent blockade of F-FNDP (0.1 mCi) uptake; data are mean % ID / g tissue ± SD (n = 3); Abbreviations: Str, striatum; Ctx, cortex; Hip, hippocampus; CB, cerebellum; blockade curves demonstrate 18 F-FNDP specifically labels sEH binding sites in all brain regions studied; residual binding at the highest dose of blocker corresponds to nonspecific binding;

[0079] Figure 5 Shown are the changes in sEH-KO and control C57BL / 6 mice 60 min after radiotracer injection. 18 Baseline and blockade of F-FNDP (0.1 mCi) uptake; data are mean % ID / g ± SD (n = 5); blockade was performed with the sEH inhibitor des-fluoro-FNDP (1 mg / kg, subcutaneous);

[0080] Figure 6 Representative plasma time-activity curves (TACs) obtained from a baseline PET study in baboons are shown; a total of 13 brain regions were analyzed, 6 of which are shown above for clarity;

[0081] Figure 7 Shown in the same baboon at baseline 18 Comparison of regional time-uptake curves after blocking with des-FNDP (two upper curves) and after blocking with des-FNDP (2 mg / kg) (two lower curves), showing a significant reduction in radioactivity in the blocking scan; two representative regions, putamen (squares) and cerebellum (triangles), are shown;

[0082] Figure 8 Figure 3 shows the difference between baseline and blocked scans for 13 brain regions in the baboon brain. 18 F-FNDP PET regional distribution volume (V T ) comparison; data = average V T ±SD (baseline n = 3, blockade n = 1);

[0083] Figure 9 Showing the baboon brain 18 PET baseline (average, 3 scans, top row) and blockade (single scan, bottom row) parameters V for F-FNDP T Image; PET image shown in pseudocolor overlaid with baboon brain MR image (grayscale);

[0084] Figure 10 Representative radioactive metabolite HPLC of baboon plasma is shown, 60 min time point (maternal 18F-FNDP - 9.5 min, two radioactive metabolites - 0.7 min and 6.8 min); and

[0085] Figure 11 shows maternal 18 HPLC radiometabolite analysis of F-FNDP, time-% curve; data = mean % parent ± SD, n = 3.

[0086] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. Detailed description

[0087] The presently disclosed subject matter will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the invention are shown. Like numerals refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that the present disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will occur to those skilled in the art to which the presently disclosed subject matter relates, having the benefit of the teachings presented in the foregoing description and the associated drawings. Therefore, it should be understood that the presently disclosed subject matter is not limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0088] I. PET Imaging of Soluble Epoxide Hydrolases 18 F-FNDP

[0089] Soluble epoxide hydrolase (sEH) is a bifunctional enzyme located in the cytosol and peroxisomes that converts epoxides into corresponding diols and hydrolyzes phosphate monoesters. It is used to inactivate epoxyeicosatrienoic acids (EETs) that have vasoactive and anti-inflammatory properties. Inhibitors of sEH are sought as agents to reduce neuronal damage after stroke. However, to date, many sEH inhibitors have large hydrophobic domains that make them unlikely to be viable radiotracers due to potentially high nonspecific binding ( Figure 2 In the presently disclosed subject matter, N-(3,3-diphenylpropyl)-6- 18 F-Fluoronicotinamide ( 18 Synthesis, biodistribution, and baboon brain PET imaging of F-FNDP). 18F-FNDP is a radiotracer for sEH that is structurally similar to the potent sEH inhibitor N-(3,3-diphenylpropyl)-nicotinamide, also known as des-fluoro-FNDP (Eldrup et al., J. Med. Chem., 2009). 18 F-FNDP readily enters the brains of mice and baboons and selectively labels sEH with remarkable specificity.

[0090] at present, 18 F-FNDP is the first and only radiotracer with properties suitable for PET imaging of sEH in animal brain.

[0091] A. Compounds of formula (I)

[0092] Thus, in some embodiments, the presently disclosed subject matter provides compounds of formula (I):

[0093]

[0094] wherein X is selected from the group consisting of F, Br, and I, and radioactive isotopes thereof; Y is -NR-C(=O)- or -C(=O)-NR-; m is an integer selected from the group consisting of 1, 2, 3, and 4; n is an integer selected from the group consisting of 1, 2, 3, 4, and 5; p is an integer selected from the group consisting of 1, 2, and 3; and R is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted aromatic R1 is selected from the group consisting of: alkyl, substituted or unsubstituted alkylheteroaryl, substituted or unsubstituted heteroalkylaryl, and substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl; each R1 may independently be the same or different and is selected from the group consisting of: hydrogen, halogen, alkoxy, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted naphthyl, and substituted or unsubstituted biphenyl;

[0095] and stereoisomers or pharmaceutically acceptable salts thereof.

[0096] In other embodiments, the compound of formula (I) is a compound of formula (II):

[0097]

[0098] In a specific embodiment, the compound of formula (I) comprises a radioisotope suitable for imaging. In a more specific embodiment, the radioisotope suitable for imaging is selected from the group consisting of: 18 F. 76 Br, 123 I. 124 I. 125 I and 131 1. In yet more specific embodiments, the radioisotope suitable for imaging is 18 F.

[0099] In yet more specific embodiments, the compound of formula (I) is

[0100]

[0101] B. Methods of using compounds of formula (I) for imaging soluble epoxide hydrolases (sEH)

[0102] In some embodiments, the presently disclosed subject matter provides a method for imaging a soluble epoxide hydrolase (sEH), the method comprising contacting the sEH with an effective amount of a compound of formula (I) comprising:

[0103]

[0104] in:

[0105] X chooses freedom 18 F. 76 Br, 123 I. 124 I. 125 I and 131I; Y is -NR-C(=O)- or -C(=O)-NR-; m is an integer selected from the group consisting of 1, 2, 3 and 4; n is an integer selected from the group consisting of 1, 2, 3, 4 and 5; R is selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted substituted alkylheteroaryl, substituted or unsubstituted heteroalkylaryl, and substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl; R1 is selected from the group consisting of hydrogen, halogen, alkoxy, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted naphthyl, and substituted or unsubstituted biphenyl;

[0106] and stereoisomers or pharmaceutically acceptable salts thereof.

[0107] In some embodiments, the compound of formula (I) is a compound of formula (II):

[0108]

[0109] In specific embodiments, X is 18 F.

[0110] In a more specific embodiment, the compound of formula (I) is a compound of formula (II):

[0111]

[0112] "Contacting" means any action that causes at least one compound of the presently disclosed subject matter comprising an imaging agent to physically contact sEH. Contacting can include exposing sEH to a compound in an amount sufficient to cause at least one compound to contact sEH. The method can be practiced in vitro or ex vivo by introducing, and preferably mixing, the compound and sEH in a controlled environment such as a culture dish or tube. The method can be practiced in vivo, in which case contacting means exposing sEH in a subject to at least one compound of the presently disclosed subject matter, such as by administering the compound to the subject via any suitable route. According to the presently disclosed subject matter, contacting can include introducing, exposing, etc., a compound at a site remote from the sEH to be contacted, and allowing the subject's body functions or natural (e.g., diffusion) or artificially induced (e.g., swirling) fluid movement to cause the compound and sEH to contact.

[0113] By "imaging" is meant using positron emission tomography (PET) to form images of cells, tissues, tumors, parts of the body, and the like.

[0114] In other embodiments, the compounds of Formula (I) are highly specific for sEH. In some embodiments, the specificity is as high as about 95%.

[0115] In other embodiments, the sEH is in vitro, in vivo, or ex vivo. In yet other embodiments, the sEH is present in a subject.

[0116] The "subject" treated by the presently disclosed methods, in many embodiments thereof, is desirably a human subject, however it is understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included within the term "subject." Thus, a "subject" can include a human subject used for medical purposes, such as for treatment of an existing condition or disease or for prophylactic treatment to prevent the onset of a condition or disease, or an animal subject used for medical, veterinary, or developmental purposes. Suitable animal subjects include mammals, including but not limited to primates, such as humans, monkeys, apes, etc.; bovines, such as cattle, oxen, etc.; ovines, such as sheep, etc.; caprines, such as goats, etc.; porcines, such as pigs, hogs, etc.; equines, such as horses, donkeys, zebras, etc.; felines, including wild cats and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, etc.; and rodents, including mice, rats, etc. The animal can be a transgenic animal. In some embodiments, the subject is a human, including but not limited to fetuses, newborns, infants, adolescents, and adult subjects. In addition, a "subject" may include a patient suffering from or suspected of suffering from a condition or disease. Thus, the terms "subject" and "patient" are used interchangeably herein.The term "subject" also refers to an organism, tissue, cell, or collection of cells from a subject.

[0117] In some embodiments, a detectably effective amount of the imaging agent of the presently disclosed methods is administered to a subject. According to the presently disclosed subject matter, a "detectably effective amount" of an imaging agent is defined as an amount sufficient to produce an acceptable image using a device that can be used clinically. A detectably effective amount of an imaging agent can be administered in more than one injection. The detectably effective amount of an imaging agent can vary according to factors such as the individual's degree of susceptibility, the individual's age, sex, and weight, the individual's idiosyncrasy response, dosimetry, and instrument and film-related factors. Optimization of such factors is well within the skill level of the art.

[0118] In specific embodiments, the compound of formula (I) is able to cross the blood-brain barrier, ie, sEH is present in the brain of the subject. In other embodiments, the method is non-invasive.

[0119] The term "non-invasive" as used herein refers to a method in which no instruments are introduced into the body.

[0120] It is preferred that the compound comprising the imaging agent localize to the sEH rapidly after administration in order to minimize any side effects to the subject.Thus, in some embodiments, the compound of formula (I) readily enters the brain of the subject.

[0121] In some embodiments, the presently disclosed methods utilize compounds that are stable in vivo such that substantially all, e.g., greater than about 50%, 60%, 70%, 80%, or more preferably 90%, of the injected compound is not metabolized by the body prior to excretion. In other embodiments, the compound comprising the imaging agent is stable in vivo.

[0122] It is also preferred that the compounds of the presently disclosed subject matter are rapidly excreted from the body's tissues to prevent prolonged exposure to radiation from the radiolabeled compound administered to the patient. Typically, the compounds of the presently disclosed subject matter are cleared from the body in less than about 24 hours. More preferably, the compounds of the presently disclosed subject matter are cleared from the body in less than about 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, or 2 hours.

[0123] C. Methods of using compounds of formula (I) for inhibiting soluble epoxide hydrolase (sEH) in the treatment of sEH-mediated diseases

[0124] In other embodiments, the presently disclosed subject matter provides methods for inhibiting soluble epoxide hydrolase (sEH) in the treatment of sEH-mediated diseases, the methods comprising administering to a subject a therapeutically effective amount of a compound of formula (I), thereby inhibiting sEH.

[0125] As used herein, the term "inhibit" means to reduce or decrease the excess soluble epoxide hydrolase activity found in a subject. The term "inhibit" can also mean to reduce, suppress, weaken, reduce, prevent or stabilize the development or progression of a disease, disorder or condition. Inhibition can occur, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or even 100% compared to an untreated control subject or a subject without the disease or disorder.

[0126] As used herein, generally, an "effective amount" of an active agent refers to an amount sufficient to produce a desired effect, e.g., to deliver an amount of the active agent that can be detected in the brain or used for imaging, diagnosis, and / or treatment of the brain. A "therapeutically effective amount" of a therapeutic agent refers to the amount of the agent required to elicit a desired biological response. As will be understood by one of ordinary skill in the art, the effective amount of an agent may vary depending on factors such as the desired biological endpoint, the agent to be delivered, the composition of the pharmaceutical composition, the target tissue or cell, etc. In some embodiments, the term "effective amount" refers to an amount sufficient to reduce or ameliorate the severity, duration, progression, or onset of a disease, disorder, or condition, or one or more symptoms thereof; sufficient to prevent the progression of a disease, disorder, or condition; sufficient to cause regression of a disease, disorder, or condition; sufficient to prevent the recurrence, development, onset, or progression of symptoms associated with a disease, disorder, or condition; or sufficient to enhance or improve the prophylactic or therapeutic effect of another therapy.

[0127] As used herein, active agents can be combined and administered in a single dosage form, can be administered as a separate dosage form simultaneously, or can be administered as a separate dosage form administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, active agents are combined and administered in a single dosage form. In another embodiment, active agents are administered in a separate dosage form (e.g., wherein the amount of one dosage form is changed without changing the amount of another dosage form is desirable). A single dosage form may include another active agent for treating a disease state.

[0128] In specific embodiments, the soluble epoxide hydrolase mediated disease is selected from the group consisting of hypertension, atherosclerosis, inflammation, diabetes-related diseases, pain, lung disease, Alzheimer's disease, vascular cognitive impairment (VCI), and stroke.

[0129] D. Pharmaceutical Compositions and Administration

[0130] In some embodiments, the present disclosure provides pharmaceutical compositions comprising a compound of formula (I) alone or in combination with one or more additional therapeutic agents in admixture with a pharmaceutically acceptable excipient. Thus, in some embodiments, the presently disclosed subject matter provides pharmaceutical compositions comprising a compound of formula (I) and a pharmaceutically acceptable carrier, diluent, or excipient. Those skilled in the art will recognize that pharmaceutical compositions comprise pharmaceutically acceptable salts of the compounds described above.

[0131] Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the art and include salts of the active compounds prepared with relatively nontoxic acids or bases, depending on the specific substituent moieties found on the compounds described herein. When the compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent, or by ion exchange, whereby one base counterion (base) in an ionic complex is replaced by another. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts.

[0132] When the compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent, or by ion exchange, whereby one acidic counterion (acid) in the ionic complex is replaced by another. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, and the like, as well as salts derived from relatively nontoxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginine salts, and salts of organic acids such as glucuronic or galacturonic acid (see, e.g., Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into base or acid addition salts.

[0133] Thus, pharmaceutically acceptable salts suitable for use with the presently disclosed subject matter include, but are not limited to, for example, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, edetate, carnsylate, carbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, glucoheptonate, gluconate, glutamate, glycolyl arsenate, hexylresorcinate, Pharmaceutically acceptable salts include, but are not limited to, hydroxybenzoate, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, methanesulfonate, mucate, naphthenate, nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Other pharmaceutically acceptable salts can be found, for example, in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000).

[0134] In a specific embodiment, the salt is a tri(alkyl)ammonium salt or a tetra(alkyl)ammonium salt. In yet more specific embodiments, the salt is selected from the group consisting of tri(C1-C8-alkyl)ammonium salts, tetra(C1-C8-alkyl)ammonium salts, triphenylammonium salts, tri(hydroxy-C1-C8-alkyl)ammonium salts, and tetra(hydroxy-C1-C8-alkyl)ammonium salts. In an even more specific embodiment, the salt is selected from the group consisting of trimethylammonium salts, triethylammonium salts, tri(hydroxyethyl)ammonium salts, tripropylammonium salts, and tri(hydroxypropyl)ammonium salts.

[0135] In therapeutic and / or diagnostic applications, the compounds of the present disclosure can be formulated for a variety of modes of administration, including oral (sublingual, buccal) administration, oral administration, sublingual administration, systemic administration, and topical or localized administration. Techniques and formulations can generally be found in Remington: The Science and Practice of Pharmacy (20th edition) Lippincott, Williams & Wilkins (2000).

[0136] Depending on the specific circumstances treated, such an agent can be formulated as a liquid dosage form (for example, a solution, a suspension or an emulsion) or a solid dosage form (capsule or tablet), and is administered systemically or topically. Agent can, for example, be delivered in the form of time-, controlled or sustained-slow release as known to those skilled in the art. The technology for preparation and application can be found in Remington: The Science and Practice of Pharmacy (20th edition) Lippincott, Williams & Wilkins (2000). Suitable approaches can include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injection and intrathecal, direct intraventricular, intravenous, intraarticular, intrasternal, intrasynovial, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal or intraocular injection or other delivery modes. In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered intramuscularly. In some embodiments, the pharmaceutical composition is administered intrathecally. In some embodiments, the pharmaceutical composition is administered subcutaneously.

[0137] For injection, the agent of the present disclosure can be prepared and diluted in an aqueous solution, for example, in a physiologically compatible buffer such as Hank's solution, Ringer's solution or normal saline buffer. For such transmucosal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art.

[0138] It is within the scope of the present disclosure to formulate the compounds disclosed herein for the practice of the present disclosure into dosage forms suitable for systemic administration using pharmaceutically acceptable inert carriers. With appropriate selection of carriers and suitable manufacturing practices, the compositions of the present disclosure, particularly those formulated as solutions, can be administered parenterally, for example, by intravenous injection. The compounds can be easily formulated into dosages suitable for oral administration using pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the present disclosure to be formulated into tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral intake by subjects to be treated (e.g., patients).

[0139] For nasal or inhaled delivery, the agents of the present disclosure may also be formulated by methods known to those skilled in the art and may include, but are not limited to, examples of solubilizing, diluting, or dispersing substances, such as saline; preservatives, such as benzyl alcohol; absorption enhancers; and fluorocarbons.

[0140] The pharmaceutical composition that is suitable for using in the present disclosure includes the composition that wherein active ingredient is contained in the effective amount that realizes its intended purpose.The determination of effective amount is fully within the ability of those skilled in the art, especially according to the detailed disclosure provided herein.Usually, according to the compound of the present disclosure, it is effective in a wide dosage range.For example, in the treatment of adults, the dosage of from 0.01mg to 1000mg, from 0.5mg to 100mg, from 1mg to 50mg and from 5mg to 40mg every day is the example of the dosage that can be used.Non-limiting dosage is 10mg to 30mg every day.Exact dosage will depend on the form of administration route, compound being applied, subject to be treated, subject to be treated's body weight, bioavailability of compound, absorption of compound, distribution, metabolism and excretion (ADME) toxicity and attending physician's preference and experience.

[0141] In addition to the active ingredient, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers containing excipients and adjuvants which facilitate processing of the active compound into preparations which can be used pharmaceutically. Preparations formulated for oral administration may be in the form of tablets, dragees, capsules or solutions.

[0142] Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding the resulting mixture, and processing the granule mixture as desired after adding suitable auxiliary agents to obtain tablets or dragee cores. Suitable excipients are in particular fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, for example corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose (CMC), and / or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate can be added.

[0143] Dragee cores are provided with a suitable coating. For this purpose, concentrated sugar solutions can be used, which can optionally comprise gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol (PEG) and / or titanium dioxide, lacquer solution and a suitable organic solvent or solvent mixture. Dye or pigment can be added to tablet or dragee coating for identification or characterization of the different combinations of active compound dosage.

[0144] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin and soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient in admixture with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active compound can be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or liquid polyethylene glycol (PEG). In addition, a stabilizer can be added.

[0145] E. Kit

[0146] In yet other embodiments, the presently disclosed subject matter provides kits comprising compounds of formula (I). In certain embodiments, the kit provides packaged pharmaceutical compositions comprising a pharmaceutically acceptable carrier, diluent, or excipient, and a presently disclosed compound. In certain embodiments, the packaged pharmaceutical composition will contain the reactive precursors necessary to produce the compounds of the invention upon combination with a radiolabeled precursor. Other packaged pharmaceutical compositions provided by the present invention also include a label comprising at least one of the following: instructions for preparing a compound according to the invention from the supplied precursors, instructions for using the composition to image cells or tissues expressing PSMA, or instructions for using the composition to image glutamatergic neurotransmission in patients with stress-related disorders, or instructions for using the composition to image prostate cancer.

[0147] II. Definitions

[0148] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently described subject matter belongs.

[0149] Although the following terms with respect to compounds of formula (I) are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to aid in explaining the presently disclosed subject matter. These definitions are intended to supplement and illustrate, but not to exclude, definitions that would be obvious to those of ordinary skill in the art after reading this disclosure.

[0150] As used herein, the term substituted, whether or not preceded by the term "optionally", and substituents, refers to the ability to change one functional group on a molecule to another functional group, as understood by those skilled in the art, provided that the valence of all atoms is maintained. When more than one position in any given structure can be substituted by more than one substituent selected from a specified group, the substituents can be the same or different at each position. Substituents can also be further substituted (e.g., an aryl group substituent can have another substituent away from it, such as another aryl group, which is further substituted at one or more positions).

[0151] Where substituent groups or linking groups are specified by their conventional chemical formulae written from left to right, they likewise encompass chemically equivalent substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to -OCH2-; -C(=O)O- is equivalent to -OC(=O)-; -OC(=O)NR- is equivalent to -NRC(=O)O-, and the like.

[0152] When the term "independently selected" is used, the substituents referred to (e.g., R groups, such as groups R1, R2, etc., or variables such as "m" and "n") can be the same or different. For example, both R1 and R2 can be substituted alkyl, or R1 can be hydrogen and R2 can be substituted alkyl, etc.

[0153] When used in reference to a group of substituents herein, the term "a," "an," or "a(n)" means at least one. For example, where a compound is substituted with "an" alkyl or aryl group, the compound is optionally substituted with at least one alkyl group and / or at least one aryl group. Additionally, where a moiety is substituted with R substituents, the group may be referred to as "R-substituted." Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different.

[0154] A named "R" or group will generally have a structure recognized in the art as corresponding to the group having that name, unless otherwise indicated herein. For illustrative purposes, certain representative "R" groups as set forth above are defined below.

[0155] The description of the compounds of the present disclosure is subject to the principles of chemical bonding known to those skilled in the art. Thus, where a group may be substituted by one or more of a number of substituents, such substituents are selected so as to adhere to the principles of chemical bonding and provide compounds that are not inherently unstable and / or would be considered by one of ordinary skill in the art to be potentially unstable under environmental conditions, such as aqueous, neutral, and several known physiological conditions. For example, a heterocycloalkyl or heteroaryl group is attached to the remainder of the molecule via a ring heteroatom in accordance with the principles of chemical bonding known to those skilled in the art, thereby avoiding inherently unstable compounds.

[0156] Unless expressly defined otherwise, a "substituent group" as used herein includes a functional group selected from one or more of the following moieties as defined herein:

[0157] As used herein, the term hydrocarbon refers to any chemical group comprising hydrogen and carbon. Hydrocarbon can be substituted or unsubstituted. As will be known to those skilled in the art, all valences must be satisfied when any replacement is carried out. Hydrocarbon can be unsaturated, saturated, branched, unbranched, cyclic, polycyclic or heterocyclic. Illustrative hydrocarbons are also defined herein below and include, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl etc.

[0158] Unless otherwise stated, the term "alkyl" by itself or as part of another substituent means a straight chain (i.e., unbranched) or branched, acyclic or cyclic hydrocarbon group, or combinations thereof, which may be fully saturated, monounsaturated, or polyunsaturated and may include groups having the specified number of carbon atoms (i.e., C1-C 10 means divalent and multivalent groups of one to ten carbons, including 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 carbons. In a specific embodiment, the term "alkyl" refers to a C 1-20 linear (i.e., "straight-chain"), branched, or cyclic, saturated or at least partially unsaturated and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon radicals, inclusive (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 carbons), derived from a hydrocarbon moiety containing between 1 and 20 carbon atoms by the removal of a single hydrogen atom.

[0159] Representative saturated hydrocarbon groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers thereof.

[0160] "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. "Lower alkyl" refers to an alkyl group having from 1 to about 8 carbon atoms, for example, 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms (i.e., C 1-8 "Higher alkyl" refers to an alkyl group having from about 10 to about 20 carbon atoms, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, "alkyl" specifically refers to a C 1-8 In other embodiments, "alkyl" refers specifically to a C 1-8 Branched chain alkyl.

[0161] An alkyl group may be optionally substituted with one or more alkyl group substituents ("substituted alkyl") which may be the same or different. The term "alkyl group substituent" includes, but is not limited to, alkyl, substituted alkyl, halogen, arylamino, acyl, hydroxy, aryloxy, alkoxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms may be optionally inserted along the alkyl chain, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as "alkylaminoalkyl"), or aryl.

[0162] Thus, as used herein, the term "substituted alkyl" includes alkyl groups as defined herein wherein one or more atoms or functional groups of the alkyl group are replaced by another atom or functional group, including, for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxy, hydroxy, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

[0163] Unless otherwise stated, the term "heteroalkyl," by itself or in combination with another term, means a stable linear or branched, or cyclic hydrocarbon group, or combinations thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen, phosphorus, and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The one or more heteroatoms O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0164] As described above, heteroalkyl groups as used herein include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)NR', ​​-NR'R", -OR', -SR, -S(O)R, and / or -S(O2)R'. Where "heteroalkyl" is recited followed by a specific heteroalkyl group (such as -NR'R, etc.), it will be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl group is recited to increase clarity. Therefore, the term "heteroalkyl" should not be interpreted herein to exclude specific heteroalkyl groups such as -NR'R", etc.

[0165] "Cyclic" and "cycloalkyl" refer to non-aromatic monocyclic or polycyclic ring systems of about 3 to about 10 carbon atoms, for example, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Cycloalkyl groups may optionally be partially unsaturated. Cycloalkyl groups may also be optionally substituted with alkyl group substituents, oxo and / or alkylene groups as defined herein. One or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms may be optionally inserted along the cyclic alkyl chain, wherein the nitrogen substituent is hydrogen, unsubstituted alkyl, substituted alkyl, aryl or substituted aryl, thereby providing a heterocyclic group. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl and cycloheptyl. Polycyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decahydronaphthalene, camphor, camphane and n-adamantyl, as well as fused ring systems such as dihydronaphthalene and tetrahydronaphthalene.

[0166] The term "cycloalkylalkyl" as used herein refers to a cycloalkyl group, as defined above, attached to the parent molecular moiety through an alkyl group, also as defined above. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

[0167] The term "cycloheteroalkyl" or "heterocycloalkyl" refers to a non-aromatic ring system, an unsaturated or partially unsaturated ring system, such as a 3- to 10-membered substituted or unsubstituted cycloalkyl ring system, containing one or more heteroatoms which may be the same or different and are selected from the group consisting of nitrogen (N), oxygen (O), sulfur (S), phosphorus (P) and silicon (Si), and optionally may contain one or more double bonds.

[0168] Cycloheteroalkyl rings may optionally be fused or otherwise attached to other cycloheteroalkyl rings and / or non-aromatic hydrocarbon rings. Heterocyclic rings include those having from 1 to 3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. In certain embodiments, the term heterocycle refers to a non-aromatic 5-, 6-, or 7-membered ring or polycyclic group in which at least one ring atom is a heteroatom selected from O, S, and N (wherein the nitrogen and sulfur heteroatoms may optionally be oxidized), including but not limited to bicyclic or tricyclic groups, including fused six-membered rings having between one and three heteroatoms independently selected from oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds, and each 7-membered ring has 0 to 3 double bonds, (ii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl ring. Representative cycloheteroalkyl ring systems include, but are not limited to, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidinyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like.

[0169] Unless otherwise stated, the terms "cycloalkyl" and "heterocycloalkyl" by themselves or in combination with other terms represent the cyclic forms of "alkyl" and "heteroalkyl", respectively. In addition, for heterocycloalkyl, heteroatoms can occupy the position where the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, etc. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidyl, 2-piperidyl, 3-piperidyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophene-2-yl, tetrahydrothiophene-3-yl, 1-piperazinyl, 2-piperazinyl, etc. The terms "cycloalkylene" and "heterocycloalkylene" refer to divalent derivatives of cycloalkyl and heterocycloalkyl, respectively.

[0170] Unsaturated alkyl groups are alkyl groups with one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl and higher homologues and isomers. The alkyl group that is limited to hydrocarbon groups is referred to as "homoalkyl".

[0171] More specifically, the term "alkenyl" as used herein refers to a C alkylene radical having at least one carbon-carbon double bond derived from a C alkylene radical by removing a single hydrogen molecule. 1-20 Alkenyl groups include, for example, ethenyl (i.e., vinyl), propenyl, butenyl, 1-methyl-2-butene-1-yl, pentenyl, hexenyl, octenyl, allenyl, and butadienyl.

[0172] As used herein, the term "cycloalkenyl" refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, 1,3-cyclohexadienyl, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.

[0173] As used herein, the term "alkynyl" refers to a linear or branched C-alkyl group containing at least one carbon-carbon triple bond and derived from the specified number of carbon atoms. 1-20 Examples of "alkynyl" include an ethynyl group, a 2-propynyl (propargyl) group, a 1-propynyl group, a pentynyl group, a hexynyl group, a heptynyl group, and the like.

[0174] The term "alkylene" by itself or as part of another substituent refers to a straight or branched divalent aliphatic hydrocarbon radical derived from an alkyl group having from 1 to about 20 carbon atoms, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group can also be optionally unsaturated and / or substituted with one or more "alkyl group substituents". One or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as "alkylaminoalkyl") can be optionally inserted along the alkylene group, wherein the nitrogen substituent is an alkyl group as previously described. Exemplary alkylene groups include methylene (-CH2-); ethylene (-CH2-CH2-); propylene (-(CH2)3-); cyclohexylene (-C6H 10 -); -CH=CH-CH=CH-; -CH=CH-CH2-;-CH2CH2CH2CH2-, -CH2CH=CHCH2-, -CH2CsCCH2-, -CH2CH2CH(CH2CH2CH3)CH2-, -(CH2) q -N(R)-(CH2) r -, wherein each of q and r is independently an integer from 0 to about 20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, and R is hydrogen or lower alkyl; methylenedioxy (-O-CH2-O-); and ethylenedioxy (-O(CH2)2-O-). Alkylene groups can have from about 2 to about 3 carbon atoms and can also have 6-20 carbons. Typically, alkyl (or alkylene) groups will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being some embodiments of the present disclosure. "Lower alkyl" or "lower alkylene" are shorter chain alkyl or alkylene groups, typically having 8 or fewer carbon atoms.

[0175] The term "heteroalkylene" itself or as a part of another substituent means a divalent group derived from heteroalkyl, such as exemplified, but not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms can also occupy any one or two (e.g., alkylene oxo, alkylenedioxo, alkyleneamino, alkylenediamino etc.) of the chain ends. Additionally, for alkylene and heteroalkylene linking groups, the orientation of the linking group is not implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)OR'- represents both -C(O)OR'- and -R'OC(O)-.

[0176] Unless otherwise stated, the term "aryl" means an aromatic hydrocarbon substituent which may be a single ring or multiple rings (e.g., from 1 to 3 rings) fused together or linked covalently. The term "heteroaryl" refers to an aryl group (or ring) containing from 1 to 4 heteroatoms selected from N, O, and S (in each individual ring in the case of multiple rings), wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. The heteroaryl group may be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl , 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl and 6-quinolyl. Substituents for each of the above-mentioned aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. The terms "arylene" and "heteroarylene" refer to the divalent forms of aryl and heteroaryl groups, respectively.

[0177] For the sake of brevity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the terms "arylalkyl" and "heteroarylalkyl" are meant to include those groups in which an aryl group or heteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, furylmethyl, etc.), including those in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.). However, the term "haloaryl" as used herein is meant to include only aryl groups substituted by one or more halogens.

[0178] Where a heteroalkyl, heterocycloalkyl, or heteroaryl group includes a specific number of members (eg, "3- to 7-membered"), the term "member" refers to a carbon or heteroatom.

[0179] Additionally, as used herein, structures generally represented by the formula:

[0180]

[0181] Refers to a ring structure comprising a substituent R group, such as, but not limited to, a 3-carbon, 4-carbon, 5-carbon, 6-carbon, 7-carbon, etc., aliphatic and / or aromatic cyclic compounds, including saturated ring structures, partially saturated ring structures and unsaturated ring structures, wherein the R group may be present or absent, and when present, one or more R groups may each be substituted on one or more available carbon atoms of the ring structure. The presence or absence of the R group and the number of the R group are determined by the value of the variable "n", which is an integer generally having a value in the range of from 0 to the number of carbon atoms available for substitution on the ring. If more than one, each R group is substituted on an available carbon of the ring structure rather than on another R group. For example, wherein n is 0 to 2 above structures will include groups of compounds, including but not limited to:

[0182]

[0183] wait.

[0184] A dotted line representing a bond in a cyclic ring structure indicates that the bond may or may not be present in the ring. That is, a dotted line representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure.

[0185] symbol Indicates the point of attachment of the moiety to the rest of the molecule.

[0186] When a named atom of an aromatic or heterocyclic aromatic ring is defined as "absent," the named atom is replaced by a direct bond.

[0187] Each of the above terms (e.g., "alkyl," "heteroalkyl," "cycloalkyl," and "heterocycloalkyl," "aryl," "heteroaryl," "phosphonate," and "sulfonate," and divalent derivatives thereof) is intended to include both substituted and unsubstituted forms of the indicated group. Optional substituents for each type of group are provided below.

[0188] Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl monovalent and divalent derivative groups (including those groups commonly referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, -OR', =O, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R'" in a number ranging from zero to (2m'+1). , -OC(O)R', -C(O)R', -C02R', -C(O)NR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R'", -NR"C(O)OR', -NR-C(NR'R")=NR'", -S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -CN and -NO2, where m' is the total number of carbon atoms in such a group. R', R", R"' and R"" can each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl groups, alkoxy groups or thioalkoxy groups or arylalkyl groups. As used herein, an "alkoxy" group is an alkyl group attached to the remainder of the molecule through a divalent oxygen. When a compound of the present disclosure contains more than one R group, for example, each of the R groups is independently selected, such as being an R', R", R"' and R"" group when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 4-membered, 5-membered, 6-membered or 7-membered ring. For example, -NR'R" means, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" is meant to include groups containing carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl groups (e.g., -CF3 and -CH2CF3) and acyl groups (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0189] Similar to the substituents described for the alkyl groups above, exemplary substituents for aryl and heteroaryl groups (and their divalent derivatives) vary and are selected from, for example, halogen, -OR', -NR'R", -SR', SiR'R"R'", -OC(O)R', -C(O)R', -C02R', -C(O)NR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R'", - 2-C4)alkyl, said substituents being in a number ranging from zero to the total number of open valences on the aromatic ring system; and wherein R', R", R'"' and R"" can be independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When a compound of the disclosure contains more than one R group, for example, each of the R groups is independently selected, such as each of the R', R", R'" and R"" groups when more than one of these groups is present.

[0190] Two of the substituents on adjacent atoms of an aryl or heteroaryl ring may optionally form a group of the formula -TC(O)-(CRR') q -U-, wherein T and U are independently -NR-, -O-, -CRR'- or a single bond, and q is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally replaced by a -A-(CH2) r -B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer from 1 to 4.

[0191] One of the single bonds of the new ring thus formed may optionally be replaced by a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by a double bond of the formula -(CRR') s -X'-(C"R'") d-, wherein s and d are independently integers from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2- or -S(O)2NR'-. The substituents R, R', R" and R'" may be independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0192] As used herein, the term "acyl" refers to an organic acid group in which the -OH of the carboxyl group has been replaced by another substituent and has the general formula RC(=O)-, wherein R is an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a carbocyclic group, a heterocyclic group, or an aromatic heterocyclic group as defined above. Thus, the term "acyl" specifically includes aryl acyl groups such as 2-(furan-2-yl)acetyl)- and 2-phenylacetyl groups. Specific examples of acyl groups include acetyl and benzoyl. Acyl groups are also intended to include amides-RC(=O)NR', ​​esters-RC(=O)OR', ketones-RC(=O)R', and aldehydes-RC(=O)H.

[0193] The terms "alkoxyl" or "alkoxy" are used interchangeably herein and refer to a saturated (i.e., alkyl-O-) or unsaturated (i.e., alkenyl-O- and alkynyl-O-) group attached to the parent molecular moiety through an oxygen atom, wherein the terms "alkyl," "alkenyl," and "alkynyl" are as previously described and may include C 1-20 The term "inclusive" refers to a linear, branched, or cyclic saturated or unsaturated oxyhydrocarbon chain, including, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy, and the like.

[0194] The term "alkoxyalkyl" as used herein refers to an alkyl-O-alkyl ether, for example, a methoxyethyl group or an ethoxymethyl group.

[0195] "Aryloxy" refers to an aryl-O- group, wherein the aryl group is as previously described, including substituted aryl groups. As used herein, the term "aryloxy" may refer to phenyloxy or hexyloxy, and phenyloxy or hexyloxy substituted with alkyl, substituted alkyl, halogen, or alkoxy.

[0196] "Aralkyl" refers to an aryl-alkyl- group in which the aryl and alkyl are as previously described, and include substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.

[0197] "Aralkyloxy" refers to an aralkyl-O- group, wherein the aralkyl group is as previously described. An exemplary aralkyloxy group is benzyloxy, i.e., C6H5-CH2-O-. The aralkyloxy group may be optionally substituted.

[0198] "Alkoxycarbonyl" refers to an alkyl-OC(=O)- group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butoxycarbonyl, and tert-butoxycarbonyl.

[0199] "Aryloxycarbonyl" refers to an aryl-OC(=O)- group. Exemplary aryloxycarbonyl groups include phenoxy-carbonyl and naphthoxy-carbonyl.

[0200] "Aralkyloxycarbonyl" refers to an aralkyl-OC(=O)- group. An exemplary aralkyloxycarbonyl group is benzyloxycarbonyl.

[0201] "Carbamoyl" refers to an amide group of the formula -C(=O)NH. "Alkylcarbamoyl" refers to an R'RN-C(=O)- group, wherein one of R and R' is hydrogen and the other of R and R' is alkyl and / or substituted alkyl as previously described. "Dialkylcarbamoyl" refers to an R'RN-C(=O)- group, wherein each of R and R' is independently alkyl and / or substituted alkyl as previously described.

[0202] The term carbonyldioxy as used herein refers to a carbonate group of formula -OC(=O)-OR.

[0203] "Acyloxy" means an acyl-O- group in which the acyl group is as previously described.

[0204] The term "amino" refers to an -NH2 group and also refers to nitrogen-containing groups as known in the art derived from ammonia by replacing one or more hydrogen radicals with an organic group. For example, the terms "acylamino" and "alkylamino" refer to specific N-substituted organic groups having acyl and alkyl substituent groups, respectively.

[0205] As used herein, "aminoalkyl" refers to an amino group covalently bonded to an alkylene linker. More specifically, the terms alkylamino, dialkylamino, and trialkylamino, as used herein, refer to one, two, or three alkyl groups, as previously defined, respectively, attached to the parent molecular moiety through a nitrogen atom. The term alkylamino refers to a group having the structure -NHR', wherein R' is an alkyl group as previously defined; while the term dialkylamino refers to a group having the structure -NR'R", wherein R' and R" are each independently selected from the group consisting of alkyl groups. The term trialkylamino refers to a group having the structure -NR'R"R"', wherein R', R" and R'" are each independently selected from the group consisting of alkyl groups. In addition, R', R" and / or R'" together may optionally be -(CH2) k -, wherein k is an integer from 2 to 6. Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, isopropylamino, piperidinyl, trimethylamino, and propylamino.

[0206] An amino group is -NR'R", wherein R' and R" are typically selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0207] The terms alkylthioether and thioalkoxy refer to saturated (i.e., alkyl-S-) or unsaturated (i.e., alkenyl-S- and alkynyl-S-) groups attached to the parent molecular moiety through a sulfur atom. Examples of thioalkoxy moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

[0208] "Acylamino" means an acyl-NH- group in which the acyl group is as previously described. "Aroylamino" means an aroyl-NH- group in which the aroyl group is as previously described.

[0209] The term "carbonyl" refers to a -C(=O)- group and may include an aldehyde group represented by the general formula RC(=O)H.

[0210] The term "carboxyl" refers to a -COOH group. Such groups are also referred to herein as "carboxylic acid" moieties.

[0211] As used herein, the term "halo", "halide" or "halogen" refers to a fluoro group, a chloro group, a bromo group and an iodo group. In addition, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo (C1-C4) alkyl" is intended to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc.

[0212] The term "hydroxy" refers to an -OH group.

[0213] The term "hydroxyalkyl" refers to an alkyl group substituted with an -OH group.

[0214] The term "mercapto" refers to a -SH group.

[0215] The term "oxo" as used herein means an oxygen atom double-bonded to a carbon atom or another element.

[0216] The term "nitro" refers to a -NO2 group.

[0217] The term "thio" refers to compounds previously described herein wherein a carbon or oxygen atom is replaced by a sulfur atom.

[0218] The term "sulfate" refers to a -SO4 group.

[0219] As used herein, the term thiol or thiol refers to a group of formula -SH.

[0220] More specifically, the term "sulfide" refers to a compound having a group of formula -SR.

[0221] The term "sulfone" refers to a compound having a sulfonyl group -S(O2)R.

[0222] The term "sulfoxide" refers to a compound having a sulfinyl group -S(O)R.

[0223] The term urea refers to a urea group of the formula -NH-CO-NH2.

[0224] Throughout the specification and claims, a given chemical formula or name shall encompass all tautomers, congeners, optical isomers, and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist.

[0225] Certain compounds of the present disclosure may have asymmetric carbon atoms (optical or chiral centers) or double bonds; enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms, and individual isomers that can be defined in terms of absolute stereochemistry as (R)- or (S)-, or as (D)- or (L)- for amino acids, are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include those compounds known in the art that are too unstable to be synthesized and / or isolated. The present disclosure is intended to include compounds in racemic, non-racemic (scalemic) and optically pure forms. Optically active (R)- and (S)-isomers or D- and L-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless otherwise indicated, it is intended that the compounds include both E and Z geometric isomers.

[0226] Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; that is, the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the present disclosure.

[0227] It will be apparent to those skilled in the art that certain compounds of the present disclosure may exist in tautomeric forms, and all such tautomeric forms of the compounds are within the scope of the present disclosure. As used herein, the term "tautomer" refers to one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another.

[0228] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, in the replacement of hydrogen with deuterium or tritium, or in the replacement of 13 C- or 14 Compounds having the present structure where a C-rich carbon replaces the carbon are within the scope of the present disclosure.

[0229] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more atoms that constitute such compounds. For example, the compounds may be treated with radioactive isotopes, such as, for example, tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C) Radiolabeling. All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.

[0230] The compounds of the present disclosure may exist as salts. The present disclosure includes such salts. Examples of applicable salt forms include hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof, including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid. These salts can be prepared by methods known to those skilled in the art. Also included are base addition salts such as sodium salts, potassium salts, calcium salts, ammonium salts, organic amino salts, or magnesium salts, or similar salts. When the compounds of the present disclosure contain relatively basic functionality, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either pure or in a suitable inert solvent, or by ion exchange. Examples of acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and the like, and organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginine salts, and salts of organic acids such as glucuronic acid or galacturonic acid. Certain specific compounds of the present disclosure contain both basic and acidic functionalities, allowing the compounds to be converted into either base or acid addition salts.

[0231] The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0232] Some compounds of the present disclosure may exist in unsolvated forms as well as solvated forms (including hydrated forms). Generally, solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Some compounds of the present disclosure may exist in multiple crystalline forms or amorphous forms. Generally, all physical forms are equivalent and intended to be within the scope of the present disclosure for the intended purposes of the present disclosure.

[0233] In addition to salt forms, the present disclosure provides compounds in the form of prodrugs. The prodrugs of the compounds described herein are those compounds that easily undergo chemical changes under physiological conditions to provide the compounds of the present disclosure. In addition, prodrugs can be converted into compounds of the present disclosure by chemical or biochemical methods in an isolated environment. For example, prodrugs can be slowly converted into compounds of the present disclosure when placed in a transdermal patch reservoir (transdermal patch reservoir) with a suitable enzyme or chemical reagent.

[0234] In accordance with long-standing patent law practice, the terms "a," "an," and "the" when used in this application (including the claims) mean "one or more." Thus, for example, reference to "a subject" includes more than one subject unless the context clearly dictates otherwise (e.g., more than one subject), etc.

[0235] Throughout this specification and claims, the terms "comprise," "comprises," and "comprising" are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term "include" and its grammatical variations are intended to be non-limiting, such that the recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

[0236] For the purposes of this specification and the appended claims, unless otherwise indicated, all numbers used in the specification and claims to express amounts, sizes, dimensions, ratios, shapes, formulations, parameters, percentages, quantities, features, and other numerical values ​​should be understood to be modified by the term "about" in all cases, even if the term "about" may not explicitly appear with the value, amount, or range. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are not exact and need not be exact, but may be close and / or larger or smaller as desired, reflecting tolerances, conversion factors, rounding, measurement errors, etc., as well as other factors known to those skilled in the art, depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, when the term "about" refers to a value, it can be meant to encompass differences of ±100%, ±50%, ±20%, ±10%, ±5%, ±1%, ±0.5%, and ±0.1% from the specific amount, as such differences are appropriate for performing the disclosed methods or employing the disclosed compositions.

[0237] In addition, when used in connection with one or more numerical values ​​or numerical ranges, the term "about" should be understood to refer to all such values, including all values ​​within the range and to modify the range by extending the boundaries to above and below the values ​​listed. Numerical ranges recited by endpoints include all values ​​within the range, such as all integers, including fractions thereof (e.g., a recitation of 1 to 5 includes 1, 2, 3, 4, and 5, and fractions thereof, such as 1.5, 2.25, 3.75, 4.1, etc.) and any range within the range. Example

[0238] The following examples have been included to provide guidance to those of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. Based on the present disclosure and the general level of skill in the art, it will be understood that the following examples are intended to be illustrative only and that many variations, modifications, and alterations may be employed without departing from the scope of the presently disclosed subject matter. The following descriptions of the synthesis and specific examples are intended for illustrative purposes only and are not to be construed as limiting in any way the preparation of the compounds of the present disclosure by other methods.

[0239] Example 1

[0240] Overview

[0241] Soluble epoxide hydrolase (sEH) is a bifunctional enzyme located in the cytosol and peroxisomes that converts epoxides to the corresponding diols and hydrolyzes phosphate monoesters. It is used to inactivate epoxyeicosatrienoic acids (EETs) that have vasoactive and anti-inflammatory properties. Inhibitors of sEH are sought as agents to reduce neuronal damage after stroke. The presently disclosed subject matter provides N-(3,3-diphenylpropyl)-6- 18 F-Fluoronicotinamide ( 18 F-FNDP) and its analogs and derivatives, which have been shown to be highly specific for imaging sEH in the brain of mice and non-human primates using PET.

[0242] 18 F-FNDP is synthesized from the corresponding bromine precursor. 18 The sEH inhibitory activity of F-FNDP was measured using a sEH inhibitor screening assay kit (Cayman Chemical, MI). Biodistribution was performed in CD-1 mice. Binding specificity was determined in CD-1 and sEH knockout mice and Papio anubis (baboons) by pretreatment with sEH inhibitors to block sEH binding. Dynamic PET imaging of arterial blood sampling was performed in three baboons, using the volume of distribution (V T ) Quantitative regional tracer binding in baboons. 18The metabolism of F-FNDP was assessed using high performance liquid chromatography (HPLC).

[0243] 18 F-FNDP(K i =1.73nM) was prepared in one step using an automated radiosynthesis module with a radiochemical yield of 14% ± 7%, specific radioactivity in the range of 888GBq / μmol-3,774GBq / μmol, and radiochemical purity of >99%. Preparation time was approximately 75 min. In CD-1 mice, regional uptake followed a pattern of striatum > cortex > hippocampus > cerebellum, consistent with the known brain distribution of sEH, with 5.2% of the injected dose per gram of tissue at peak uptake. Blockade of 80%-90% was demonstrated in all brain regions. Minimal radiotracer uptake was present in sEH-KO mice. PET baboon brain distribution was paralleled to that seen in mice, with blockade of annotated markers (95%) in all regions indicating sEH-mediated 18 Uptake of F-FNDP. Two hydrophilic metabolites were identified in baboon plasma 90 min after injection, along with 20% of the parent compound present.

[0244] 18 F-FNDP can be synthesized with suitable radiochemical yield, high specific radioactivity and purity. In vivo imaging experiments have shown that 18 F-FNDP specifically targets sEH in the brains of mice and non-human primates. 18 F-FNDP is a promising PET radiotracer that may be useful in understanding the role of sEH in various conditions affecting the central nervous system.

[0245] Example 2

[0246] Materials and methods

[0247] All reagents were used as purchased from Sigma-Aldrich (St. Louis, MO). Des-Fluoro-FNDP was prepared as previously described (Edrup et al. J. Med. Chem. 2009). Flash column chromatography was performed using E. Merck silica gel 60F (230-400 mesh) (Sigma-Aldrich). 1 H NMR spectra were recorded in CDCl3 on a Bruker-500 MHz NMR spectrometer (Billerica, Massachusetts) (referenced to internal Me4Si at δ H0 ppm). The high-performance liquid chromatography (HPLC) system consisted of two Varian ProStar pumps (Palo Alto, California), a single Rheodyne Model 7725i manual injector, a ProStar 325 UV-Vis variable wavelength detector, and a BioScan Flow-Count radioactivity detector (Poway, California). Analytical and semi-preparative chromatography was performed using Phenomenex Luna C-18 10 μm columns (4.6 mm × 250 mm and 10 mm × 250 mm, respectively) (Torrance, California). The experimental animal protocol was approved by the Animal Care and Use Committee of the Johns Hopkins Medical Institutions.

[0248] synthesis.

[0249] N-(3,3-diphenylpropyl)-6-fluoronicotinamide (FNDP). 3,3-Diphenylpropane-1-amine (105.5 mg, 0.5 mmol) was added to a solution of 6-fluoronicotinic acid (70.5 mg, 0.5 mmol) in 3 mL of N,N-dimethylformamide, and then 1-hydroxybenzotriazole (135 mg, 1 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (191 mg, 1 mmol), and diisopropylethylamine (195.5 mg, 1.5 mmol) were added. The reaction was stirred at room temperature for 48 h, the solvent was evaporated under vacuum, and the residue was separated by flash LC (silica gel, hexane-ethyl acetate 5:1→2:1) to give the desired product, FNDP (121 mg, 72%). 1 H NMR (CHCl3-d3, 500 MHz) δ 8.32 (d, J = 2.5 Hz, 1H), 8.10-8.06 (m, 1H), 7.27-7.24 (m, 8H), 7.25 (m, 2H), 7.01-6.98 (m, 1H), 5.92 (broad singlet, 1H), 4.07 (t, J = 8 Hz, 1H), 3.57 (m, 2H), 2.48 (m, 2H).

[0250] 6-Bromo-N-(3,3-diphenylpropyl)nicotinamide (precursor-FNDP). Precursor-FNDP was prepared similarly to FNDP using 6-bromonicotinic acid as the starting material. Yield: 59%. 1H NMR (CHCl3-d3, 500 MHz) δ 8.45 (d, J = 2.5 Hz, 1H), 7.79 (m, 1H), 7.56 (d, J = 8 Hz, 1H), 7.37-7.32 (m, 8H), 7.27-7.24 (m, 2H), 5.92 (broad singlet, 1H), 4.07 (t, J = 9 Hz, 1H), 3.56 (m, 2H), 2.49 (m, 2H).

[0251] Radiosynthesis.

[0252] N-(3,3-diphenylpropyl)-6- 18 F-Fluoronicotinamide ( 18 F-FNDP). will be produced from proton bombardment in the General Electric PETtrace cyclotron. 18 O-water obtained 18 A solution of F-fluoride, 2 mg of K2CO3 in 0.4 mL of water and 15 mg-20 mg of The reaction vessel was added to a GE MicroLab module (Cincinnati, OH). After adding 2 mL of CH 3 CN, the mixture was evaporated at 140 ° C under an argon stream. A solution of precursor-FNDP (2 mg) in DMSO (0.8 mL) was added to the reaction vessel and the mixture was heated at 160 ° C for 12 min. The reaction mixture was cooled, diluted with 0.7 mL of water, and injected onto a reverse phase semi-preparative high performance liquid chromatography (HPLC) column. The radioactive product peak was collected in 50 mL of HPLC grade water. The aqueous solution was transferred through an activated Waters C-18 Sep-Pak optical cassette (Milford, MA). After washing the cassette with 10 mL of saline, the product was eluted with 1 mL of ethanol through a 0.2 μM sterile filter into a sterile, pyrogen-free vial, and 10 mL of 0.9% saline was added through the same filter. The final product was then 18F-FNDP was analyzed by analytical HPLC to determine radiochemical purity and specific activity. The total preparation time, including quality control, was 75 min. Semi-preparative HPLC conditions: Luna C18, 10 micron, 10 mm x 250 mm; mobile phase: 45:55 (acetonitrile: 0.1 M aqueous ammonium formate); flow rate: 10 mL / min; UV-254 nm; retention times: 13 min and 21 min (FNDP and precursor-FNDP, respectively). Analytical HPLC conditions: Luna C18, 10 micron, 4.6 mm x 250 mm; mobile phase: 55:45 (acetonitrile: 0.1 M aqueous ammonium formate); flow rate: 3 mL / min; UV-254 nm; retention times: 3.9 min and 6.5 min (FNDP and precursor-FNDP, respectively).

[0253] In vitro studies.

[0254] In vitro inhibition of sEH by FNDP. The inhibitory activity of FNDP and des-fluoro-FNDP, analogs of FNDP, and known sEH inhibitors (Eldrup et al., J. Med. Chem., 2009) was measured using the sEH inhibitor assay kit (Cayman Chemical, MI). Briefly, the IC 50 The value was determined by measuring the inhibition of hydrolysis of (3-phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl)-methyl ester by sEH. AUDA (Cayman Chemical, MI) (Imig et al., Hypertension, 2005), a known inhibitor of sEH, was used as a positive control. All reactions were performed in triplicate and the data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA) and the inhibition constant (K) was generated. i value).

[0255] In mice 18 Biodistribution studies of F-FNDP.

[0256] Baseline study in CD1 mice. Male CD-1 mice weighing 25 g to 27 g were obtained from Charles River (Wilmington, MA). 3.7 MBq (0.1 mCi) of 18After F-FNDP (specific radioactivity = 814GBq / μmol (22,000mCi / μmol)) was injected into the lateral tail vein (n=3), animals were sacrificed by cervical dislocation at 5min, 15min, 30min, 60min and 90min. The brain was removed and dissected on ice. The rest of the striatum, cortex, hippocampus, hypothalamus, cerebellum and brain were weighed and their radioactivity content was determined in a γ counter LKB / Wallac 1283 CompuGamma CS (Perkin Elmer, Bridgeport, CT). Aliquots of the injection were prepared as standards, and their radioactivity content was determined together with the tissue samples. The percentage of injected dose per gram of tissue (%ID / g tissue) was calculated.

[0257] Blockade in CD1 mice 18 F-FNDP binding. Different doses (0 mg / kg, 0.03 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg) of des-FNDP were administered subcutaneously, followed by an IV injection of 3.7 MBq (0.1 mCi) 15 minutes later. 18 F-FNDP was used for in vivo binding specificity (blocking) studies (n=3). 90 minutes after administration of the radiotracer, animals were sacrificed by cervical dislocation, brain tissues were harvested, and their radioactivity content was determined.

[0258] 18 Baseline and blocking studies of F-FNDP in sEH knockout (SEH-KO) mice and C57BL / 6 control mice. Similar studies were performed as described above using the same batch of 18 Baseline and blocking studies of F-FNDP (des-Fluoro-FNDP, 1 mg / kg, subcutaneous) were performed in sEH-KO mice (Ephx2 gene deletion; Jackson Labs) and mice on a C57Bl / 6 background (n=5). All animals were sacrificed 60 min after radiotracer injection.

[0259] In baseline and blocking studies, the sample sizes for CD-1 and sEH-KO mice were 3 and 5, respectively, corresponding to statistical power values ​​> 0.9 as calculated by G*Power, v. 3.1.9.2 free software.

[0260] Baboon PET and radioactive metabolite studies

[0261] Dynamic baseline and blockade PET experiments (90 min) were performed on three male baboons (Papio papio) weighing 23.9 kg, 25.0 kg, and 28.2 kg using high-resolution research tomography (HRRT, CPS Innovations, Inc., Knoxville, TN). Briefly, dynamic PET acquisition was performed after intravenous injection of 248 MBq (6.7 mCi) 18 F-FNDP (specific radioactivity 2634 GBq / μmol (71,177 mCi / μmol), carrier mass = 0.0011 μg / kg) was used. For the blocking scan in the same baboon, 307 MBq (8.3 mCi) was injected intravenously with a bolus. 18 F-FNDP (specific radioactivity 1420 GBq / μmol (38,386 mCi / μmol), carrier mass = 0.0025 μg / kg) and des-fluoro-FNDP (2 mg / kg) were administered subcutaneously 1 h before the start of the scan. Analysis of radioactive metabolites in baboon arterial blood was performed under the general conditions previously published (Hilton et al., Nucl. Med. Biol., 2000).

[0262] Baboon PET study. PET experiments were performed on male baboons (Papio papio) weighing 28.2 kg using high-resolution research tomography (HRRT, CPS Innovations, Inc., Knoxville, TN). The animals underwent a baseline PET scan and a blockade scan three weeks later. Prior to each PET study, the animals were fasted for 12 h. Anesthesia was induced with intramuscular ketamine (7.5 mg / kg-10 mg / kg) and maintained with a continuous intravenous infusion of propofol at 0.3 mg / kg / min-0.4 mg / kg / min throughout the PET experiment. A venous catheter was inserted for radioligand injection, and an arterial catheter was inserted to obtain arterial blood samples. During the 90-min dynamic PET scan, arterial plasma input function was measured by collecting 43 blood samples. The baboons were also intubated to assist breathing, and circulatory volume was maintained by continuous infusion of isotonic saline. Physiological vital signs including heart rate, blood pressure, electrocardiogram, and oxygen saturation were continuously monitored throughout the study.

[0263] The animal was placed in the PET scanner with the head fixed with a thermoplastic hood. For attenuation correction, a rotating [ 137 A 6-min transmission scan was acquired using a cesium [Cs] source. Then, a 90-min dynamic PET acquisition was started in 3D list mode with a simultaneous intravenous bolus injection of 248 MBq (6.7 mCi) 18F-FNDP (specific radioactivity 2634 GBq / μmol (71,177 mCi / μmol)). For blocking scans, 307 MBq (8.3 mCi) was injected intravenously as a bolus. 18 F-FNDP (specific radioactivity value 1420 GBq / μmol (38,386 mCi / μmol)) and des-fluoro-FNDP (2 mg / kg) were administered subcutaneously 1 h before the start of 90 min dynamic PET imaging.

[0264] PET image reconstruction. The 90-min PET list-mode data were divided into 22 frames (3 20-s, 2 30-s, 2 1-min, 3 2-min, 8 5-min, and 4 10-min frames). The data were then reconstructed using the iterative ordered subset expectation maximization (OS-EM) algorithm (with 6 iterations and 16 subsets) to correct for radioactive decay, dead time, attenuation, scatter, and randomness. Before the emission scan, the data were reconstructed using [ 137 The attenuation map was generated by a 6-minute transmission scan using a cesium [Cs] point source. The reconstructed image space consists of cubic voxels, each with a size of 1.22 mm. 3 , and has a span of 31 cm x 31 cm (lateral) and 25 cm (axial).

[0265] Brain volumes of interest (VOIs) and regional time-activity curves (TACs): The software package PMOD (v3.3, PMOD Technologies Ltd, Zurich, Switzerland) was used for the following image processing and subsequent kinetic analysis steps. Previously acquired T1-weighted images of baboon brain MRI were co-registered with the reconstructed dynamic PET images collected in this study. Thirteen representative baboon brain VOIs were defined by manually matching the co-registered MRIs with the INIA19 Template and NeuroMaps Atlas for primate brain image segmentation and spatial normalization (Rohlfing et al., Frontiers in neuroinformatics, 2012), including the frontal and temporal gyri, thalamus, hippocampus, caudate nucleus, putamen, amygdala, globus pallidus, insula, hypothalamus, cerebellum, corpus callosum, and white matter. These VOIs were then used to generate brain regional TACs for both baseline and blocked PET scans.

[0266] PET dynamic analysis: calculation of brain regional distribution volume (V T ): Based on the regional TAC obtained above, metabolite-corrected arterial plasma input function was used to quantitatively characterize 18Binding of F-FNDP to sEH. Following the consensus nomenclature for in vivo imaging of reversibly binding radioligands (Innis et al., J. Cereb. Blood Flow Metab., 2007), the primary outcome measure was the regional distribution volume (V T ), is defined as the ratio of the concentration of radioligand in regional brain tissue to the concentration of radioligand in plasma at equilibrium. T Proportional to the receptor density within the defined VOI. For each VOI, V is calculated using the Logan plot method. T (Logan et al., J. Cereb. Blood Flow Metab., 1990).

[0267] HPLC analysis of radioactive metabolites: Throughout the PET study, baboon arterial blood samples were collected initially at very short intervals (<5 s) and gradually at extended intervals for determination of plasma radioactivity. Selected samples collected at 0, 5, 10, 20, 30, 60, and 90 min were analyzed by HPLC using a general method described previously (Hilton et al., Nucl. Med. Biol., 2000). 18 The presence of F-FNDP and its radioactive metabolites ( Figure 9 Briefly, 3 mL of plasma in 8 M urea was passed through a capture column (19 mm × 4.6 mm Strata-X, Phenomenex, Torrance, CA), followed by washing of plasma proteins from the column with 1% acetonitrile in water. The effluent from the capture column, containing only highly polar components, flowed through a dual BGO detector (Bioscan, Washington, DC). The solvent was then switched to a mixture of 60% acetonitrile / 40% 0.1 M ammonium formate aqueous solution, pH = 2.7 (2 mL / min) to elute the radiolabeled components bound to the capture column onto an analytical column (Gemini C18, 4.6 x 254 mm, Phenomenex, Torrance, CA).

[0268] Example 3

[0269] Chemical

[0270] N-(3,3-diphenylpropyl)-6-fluoronicotinamide (FNDP) and N-(3,3-diphenylpropyl)-6-bromonicotinamide (precursor-FNDP) were synthesized in high yields (59%-72%). Figure 1A The molecular structures of FNDP and precursor FNDP were confirmed by NMR analysis.

[0271] prepared by nucleophilic radiofluorination of the bromine precursor FNDP. 18 F-FNDP, with a radiochemical yield of 14% ± 7% (n = 6) (not decay corrected) at the end of the synthesis, a specific radioactivity in the range of 888 GBq / mmol-3,774 GBq / mmol (24,000 mCi / μmol-102,000 mCi / μmol), and a radiochemical purity greater than 99% ( Figure 1B ). The final product 18 F-FNDP is formulated as a sterile, pyrogen-free solution in 7% ethanolic saline with a pH of 5.5-6.5.

[0272] FNDP exhibits an IC comparable to that of the potent sEH inhibitor AUDA 50 and K i IC values ​​of FNDP, des-fluoro-FNDP, and AUDA 50 The values ​​were 8.66nM±0.06nM, 18.53nM±0.04nM and 6.48nM±0.05nM ( Figure 3 ). The corresponding K values ​​of FNDP, des-fluoro-FNDP and AUDA i The values ​​were 1.73 nM, 3.71 nM and 1.30 nM, respectively.

[0273] Example 4

[0274] Regional brain distribution studies in CD-1 mice.

[0275] Baseline study. 18 The regional distribution of F-FNDP is shown in Table 1 .

[0276]

[0277] In CD-1 mice 18 The sEH inhibitor des-fluoro-FNDP blocked SEH binding specifically in all studied brain regions (striatum, hippocampus, cortex, and cerebellum) in a dose-dependent manner 60 min after injection. 18 F-FNDP binding ( Figure 4 At the highest blocker dose of 3 mg / kg, radioactivity uptake was reduced to approximately 90% in the striatum, hippocampus, and cortex, and to approximately 75% in the cerebellum.

[0278] SEH-KO and C57BL6 control mice 18 Baseline and blockade studies of F-FNDP. In baseline experiments, 60 min after injection, the striatum, hippocampus, and cortex of C57BL6 control mice were 18The regional uptake of F-FNDP was approximately 1% of the injected dose per gram of tissue (%ID / g) and was 18 The regional uptake of F-FNDP was 0.5% ID / g ( Figure 5 In blocking experiments in C57BL6 mice, in all studied regions 18 F-FNDP brain uptake was reduced to 0.2% ID / g tissue in sEH-KO mice at 60 min. 18 The regional brain uptake of F-FNDP was almost the same in baseline (0.11% ID / g-0.12% ID / g) and blocking experiments (0.10% ID / g-0.11% ID / g) ( Figure 5 ).

[0279] Example 5

[0280] PET imaging in East African baboons.

[0281] In bolus injection 18 During baseline scans following F-FNDP, elevated and heterogeneous uptake of radioactivity was observed in the baboon brain, as evident by representative regional TACs ( Figure 6 ). TAC peaked in all regions approximately 5 minutes after injection, with peak SUV ranging from 2.5 g / mL to 4.0 g / mL. The peak SUV for the entire brain was 3.2 and gradually decreased to 1.8 by the end of the 90-minute dynamic scan. The highest accumulation of radioactivity occurred in regions such as the putamen, insula, frontal cortex, and amygdala, while lower uptake was seen in the white matter and cerebellum. Notably, the time-activity curve for the cerebellum decreased more rapidly than that for the other regions studied ( Figure 6 ).

[0282] When using V T When quantified, among the 13 brain volumes of interest studied, the highest radioligand binding occurred in the insula, putamen, caudate putamen, and amygdala (V T >10.0), with moderate uptake in the frontal / temporal gyri, hippocampus, and globus pallidus (V T >8.3), followed by the corpus callosum, white matter, hypothalamus, and thalamus (V T >7.2). The lowest binding was in the cerebellum, V T It is 6.97.

[0283] Comparison of TAC between baseline and blocking studies Figure 7 During the blocking study, regional TAC peaked earlier, approximately 1 min after injection, with a mean peak SUV of 2.3 g / mL throughout the brain, and rapidly declined to a mean SUV of only 0.18 g / mL (10%) of baseline values ​​by the end of the 90-min scan.T For quantification, all regions in the blocked scan show V T <0.8, and when compared with the baseline V T The percentage reductions were comparable between high- and low-binding regions identified during the baseline study, with, for example, the insula and amygdala showing a 95% reduction, while the cerebellum demonstrated a 93% reduction ( Figure 8 ). Generate parameters V for both baseline and block scans T Images for comparison ( Figure 9 ).

[0284] In the blocking and baseline baboon studies 18 The specific activity of the F-FNDP dose ranged from 1420 GBq / μmol to 2634 GBq / μmol. Because the specific activity was so high, the corresponding FNDP carrier mass was only 0.0011 μg / kg-0.0025 μg / kg, which is 6 orders of magnitude lower than the de-fluoro-FNDP blocker dose (2 mg / kg). Without wishing to be bound by any particular theory, it is assumed that the specific activity variability would not affect the results of this study.

[0285] Analysis of radioactive metabolites in blood samples from baboons using reversed-phase high performance liquid chromatography (RP-HPLC) showed that the parent compound 18 F-FNDP is metabolized into two hydrophilic species ( Figure 10 ). At 90 min after injection, the incorporated radioactive metabolite in the plasma of the baboons reached a value of 80% ( Figure 10 and Figure 11 ).

[0286] Example 6

[0287] Summary and Discussion

[0288] Due to a lack of available radiotracers, only about 39 of the hundreds of known binding sites (receptors and enzymes) in the human brain have been imaged by PET (Table of CNS Radiotracers). Until now, sEH is one of the many binding sites that lacks a specific PET radiotracer. With the development of numerous sEH inhibitors by researchers in the pharmaceutical industry and academia, the opportunities for the development of sEH PET radiotracers are vast. However, many potent sEH inhibitors have large hydrophobic domains, making them unlikely to be viable radiotracers due to the potential for high nonspecific binding ( Figure 2 ).

[0289] In the presently disclosed subject matter, N-(3,3-diphenylpropyl)-6-fluoronicotinamide (FNDP, Figure 1A), a potent sEH inhibitor with optimal molecular properties for brain PET radiotracers (log P of 2.9 and a molecular weight of 334 Da) (Horti et al., Springer, 2014). FNDP is structurally similar to the sEH inhibitor N-(3,3-diphenylpropyl)-nicotinamide (de-fluoro-FNDP), which was identified by Boehringer Ingelheim as a sEH inhibitor with improved "drug-like" properties (human IC 50 7 nM) (Eldrup et al., J. Med. Chem., 2009) and was used herein as a lead compound for the development of FNDP and as a blocker in animal experiments. In vitro assays demonstrated that FNDP is a sEH inhibitor with greater potency than the lead compound des-fluoro-FNDP and comparable to the common sEH inhibitor AUDA ( Figure 3 ).

[0290] FNDP contains a fluorine atom at position 2 of the pyridine ring that is activated for nucleophilic substitution and can be used in previously developed 18 F-pyridine exchanged Br-pyridine was readily radiofluorinated via the corresponding bromine precursor under general conditions (Gao et al., J. Med. Chem., 2008). 18 F-Fluoronicotinamide ( 18 The radiosynthesis of F-FNDP) was performed in a conventional FDG-radiochemistry module by nucleophilic radiofluorination of the precursor-FNDP ( Figure 1B ), followed by semi-preparative HPLC separation and formulation of the final radiolabeled product as a sterile, pyrogen-free solution in saline. The radiotracer was prepared with very high specific radioactivity and radiochemical purity. The precursor-FNDP was easily separated by preparative HPLC and in the final product 18 F-FNDP was not detectable by analytical HPLC.

[0291] Mouse studies. In CD-1 mice, 18 F-FNDP showed a heterogeneous pattern of brain uptake, comparable to the expected regional expression of sEH in the mouse brain (Marowsky et al., Neuroscience, 2009). At 5 min after injection, the peak uptake value was 5.2% ID / g, which then declined rapidly. Brain uptake is considered moderately high because an uptake of 1% ID / g has traditionally been used as the minimum standard for selecting central nervous system radiotracers for research in our PET center. Among the brain regions studied, the highest accumulation of F-FNDP was found in the striatum, cortex, hippocampus, and the rest of the brain. 18F-FNDP radioactivity was significantly higher in the mouse brain than in the control group (Marowsky et al., Neuroscience, 2009).

[0292] To prove 18 To investigate the specificity of F-FNDP binding, two types of studies were performed: dose-escalation blockade in CD-1 mice and biodistribution in sEH knockout (sEH-KO) mice. 18 Regional brain uptake of F-FNDP is highly sensitive to increasing doses of the sEH inhibitor des-fluoro-FNDP ( Figure 4 Blocked binding in CD-1 mouse brain was considered specific (90% in striatum, hippocampus, and cortex), while residual binding at high doses of blocker was considered nonspecific (10%). These findings indicate that 18 F-FNDP uptake is highly specific and mediated by sEH. This study did not reveal regions with low sEH binding. The 75% blockade of radioactivity in the cerebellum is consistent with sEH expression in this region (Marowsky et al., Neuroscience, 2009) and suggests that the cerebellum cannot be used as a reference in the mouse brain.

[0293] As a further test of binding specificity, sEH-KO mice and control animals with the same genetic background (C57BL / 6) (Sinal et al., J. Biol. Chem., 2000) were used. Because sEH-KO mouse brains do not contain sEH (Qin et al., Mol. Neurobiol., 2015), it is expected that the expression of sEH in these mice will be 18 F-FNDP binding will be nonspecific, and the difference between sEH-KO and control brain uptake will represent specific sEH binding. 60 min after radiotracer injection, sEH-KO and control animals (C57BL / 6) were compared in a baseline experiment. 18 F-FNDP uptake ( Figure 5 ). Compared with the control, sEH-KO mice had 18 A significant reduction in F-FNDP uptake (approximately 90%) was observed in all tested regions (10.3-striatum, 9.4-cortex, 9.2-hippocampus, 4.8-cerebellum). Furthermore, in blocking studies using the sEH inhibitor des-FNDP, the control / sEH-KO mice showed a significant decrease in F-FNDP uptake (approximately 90%). 18 The decrease in F-FNDP uptake was negligible ( Figure 4 ). This negligible effect indicates that 18Any nonspecific binding of F-FNDP to other proteins such as the product of the Ephx1 gene (microsomal epoxide hydrolase) (Marowsky et al., Neuroscience, 2009) was negligible in sEH-KO brains. In C57BL / 6 controls, the blocking effect (approximately 80%) ( Figure 5 ) demonstrated a very similar blocking effect to that in CD-1 mice ( Figure 4 ).

[0294] Studies in mice have shown that 18 F-FNDP readily entered the brain (at a peak of 5% ID / g) and labeled brain sEH with high specificity (80%-90%) in both strains of control mice (CD-1 and C57BL / 6). Consistent with the low expression of sEH in KO animals, sEH-KO mice 18 Brain uptake of F-FNDP was 10-fold lower than that in controls and was essentially nonspecific.

[0295] Baboon PET imaging. Three baseline 18 During F-FNDP PET scanning, a high and rapid heterogeneous uptake of radioactivity was observed in the baboon brain ( Figure 9 ). 18 The regional distribution of F-FNDP is consistent with semiquantitative assessments of sEH expression in human brain (Sura et al., J. Histochem. Cytochem, 2008) and mouse brain (Marowsky et al., Neuroscience, 2009). Regional TACs confirmed the properties of optimal reversible PET radioligand binding. Notably, the regional distribution of F-FNDP in baboon brain was consistent with semiquantitative assessments of sEH expression in human brain (Sura et al., J. Histochem. Cytochem, 2008) and mouse brain (Marowsky et al., Neuroscience, 2009). 18 The washout rate of F-FNDP ( Figure 6 ) was not as rapid as the washout rate in mouse brain (Table 1) and was robust to the mathematical model (see below).

[0296] Blockade PET studies have shown that 18 F-FNDP labels sEH in baboon brain with very high specificity ( Figure 7 、 Figure 8 and Figure 9 Blockade was observed in all baboon brain regions studied, including the cerebellum.

[0297] Soluble epoxide hydrolase inhibitors can increase peripheral vasodilation and lower blood pressure, which in turn can increase cerebral blood flow and affect radiotracer delivery. To examine this possibility, radiotracer kinetics were modeled using a classic two-tissue three-compartment model from which the rate constant (K1) for transfer from arterial plasma to tissue can be reliably estimated. The mean K1 values ​​at baseline and occlusion were found to be 0.18 ml / cm2, respectively. 3 / min and 0.14ml / cm 3 / min, a difference of 23%. This observation proves that the change in K1 cannot be attributed to V T The reason for the significant decrease (>90%) in the values ​​from baseline to blockade was confirmed by 18 F-FNDP labeled sEH with very high specificity in baboon brain. The currently disclosed subject matter does not reveal an irreplaceable reference region in baboon brain that does not contain sEH binding, which is consistent with the widespread abundance of sEH in mammalian brain (Sura et al., J. Histochem. Cytochem, 2008; Marowsky et al., Neuroscience, 2009).

[0298] Analysis of radioactive metabolites in baboon plasma demonstrated that 18 F-FNDP is metabolized into two hydrophilic radioactive metabolites. By the end of the 90-min PET scan, the remaining parent 18 F-FNDP represents ∼20% of the radioactivity in plasma, which is comparable to many other PET radiotracers. Because the radioactive metabolites are hydrophilic, they are unlikely to enter the brain to an appreciable extent, suggesting that kinetic modeling of radioactive metabolites may not be necessary for quantification of sEH.

[0299] The first specific PET radiotracer for sEH imaging has been developed 18 F-FNDP. 18 F-FNDP, a potent sEH inhibitor, readily enters the brains of mice (5% ID / g tissue) and baboons (SUV=4) and radiolabels sEH with very high specificity (up to 95%) in both animal species while exhibiting reversible brain kinetics suitable for quantitative analysis. 18 F-FNDP holds promise for further preclinical studies and human PET imaging to evaluate the role of sEH in various conditions and disorders, including VCI, mild cognitive impairment, and stroke.

[0300] References

[0301] All publications, patent applications, patents and other references mentioned in the specification represent the level of those skilled in the art of the subject matter disclosed at present.All publications, patent applications, patents and other references are incorporated by reference at this, and their degree is specifically and individually indicated as being incorporated by reference to the same degree as each individual publication, patent application, patent and other references. It will be understood that although some patent applications, patents and other references are mentioned in this article, such references do not constitute the admission that any of these files form the part of common general knowledge in this area. If there is a conflict between this specification and any reference incorporated into, it should be based on this specification (including any modification thereof, which may be based on the reference incorporated into). The standard field acceptance meaning of the term is used herein, unless otherwise indicated. The standard abbreviations of various terms are used herein.

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[0334] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

1. A compound of formula (II): Where X is selected from 18 F. 76 Br, 123 I. 124 I. 125 I and 131 Radioisotopes of group I; or a stereoisomer or a pharmaceutically acceptable salt thereof.

2. Use of a compound of formula (II) or a stereoisomer or pharmaceutically acceptable salt thereof in the preparation of an agent for imaging soluble epoxide hydrolase (sEH): Where X is selected from 18 F. 76 Br, 123 I. 124 I. 125 I and 131 I is composed of radioactive isotopes.

3. The use according to claim 2, wherein the image is obtained by using positron emission tomography. The use according to claim 2 , wherein the compound of formula (II) is highly specific for sEH.

5. Use according to claim 4, wherein the specificity is up to 95%.

6. The use according to claim 2, wherein sEH is in vitro, in vivo or ex vivo.

7. The use according to claim 2, wherein sEH is present in the subject.

8. The use according to claim 7, wherein the subject is a human.

9. The use according to claim 7, wherein the compound of formula (II) is capable of crossing the blood-brain barrier, and wherein sEH is present in the brain of the subject.

10. The use according to claim 8, wherein the imaging is non-invasive.

11. The use according to claim 8, wherein the compound of formula (II) readily enters the brain of the subject.

12. The use according to claim 7, wherein the compound of formula (II) is cleared from the brain of the subject.

13. Use of a compound of formula (II) according to claim 1 in the preparation of a pharmaceutical composition for inhibiting soluble epoxide hydrolase (sEH) in the treatment of sEH-mediated diseases, wherein when the pharmaceutical composition is used, a therapeutically effective amount of the compound of formula (II) is administered to a subject, thereby inhibiting sEH.

14. The use according to claim 13, wherein the soluble epoxide hydrolase mediated disease is selected from the group consisting of hypertension, atherosclerosis, inflammation, diabetes-related diseases, pain, lung disease, Alzheimer's disease, vascular cognitive impairment (VCI) and stroke.

15. A pharmaceutical composition comprising the compound of formula (II) according to claim 1, and a pharmaceutically acceptable carrier, diluent or excipient.

16. A kit comprising a packaged pharmaceutical composition comprising a compound of formula (II) according to claim 1.

Images