NO-RELEASING NONOATE(OXYGEN-BOUND)CHROMENE CONJUGATES

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of U.S. Provisional Application No. 61/927,352, filed on 14 January 2014. The entire disclosure of the above application is incorporated herein by reference.

FIELD

[002] The present invention generally relates to NO-releasing chromene conjugate compounds, pharmaceutical compositions comprising the compounds, methods useful for treating a subject by administering a therapeutically effective amount of the compounds, and methods for making the compounds. More specifically, the present invention relates to a class of NO-releasing NONOate(oxygen-bound)chromene gastro-protective compounds, pharmaceutical compositions thereof, and methods useful for healing wounds, preventing and treating cancer, and treating actinic keratosis, cystic fibrosis, and acne.

BACKGROUND

[003] Despite decades of effort, cancer remains an especially difficult disease for development of therapeutics. According to the Cancer Prevention Coalition (University of Illinois), cancer rates have increased 24% in the past thirty years even after adjusting for aging of the population. Remarkably, despite significant progress during this period, the overall five-year survival rates have remained virtually static (approximately 50% depending on the cancer). Thus, new drugs are required to develop more effective life-saving cancer therapies.

[004] Celecoxib, a selective COX-2 inhibitor, is one of the world's most successful drugs, alleviating pain and inflammation for millions of patients. In addition, COX-2 over-expression has been found in several types of human cancers, such as colon, breast, lung, prostate, and pancreas, and appears to control many cellular processes. COX-2 plays a role in carcinogenesis, apoptosis, and angiogenesis and, therefore, represents an excellent drug target for the development of novel medicines for prevention and/or treatment of human cancers. Currently, celecoxib is approved for limited use in the reduction of polyps in familial adenomatous polyposis (FAP). [005] The Adenoma Prevention with Celecoxib (APC) trial demonstrated human efficacy of celecoxib in the prevention of sporadic colorectal adenoma. However, this trial also showed that the elevated dose of celecoxib required for anti-cancer efficacy was accompanied by concomitant increase in adverse cardiovascular (CV) events {Cancer P rev. Res. 2, 310-321(2009)).

[006] Development of more potent or selective COX-2 inhibitors does not improve CV safety; this liability is thought to be a mechanism-based effect. This was demonstrated in the VIGOR trial by Vioxx®, an extremely potent and highly selective COX-2 inhibitor withdrawn from the market in 2004 due to CV concerns about increased risk of heart attack and stroke with long term, high dose use. These facts have undermined the development of novel COX-2 inhibitors and slowed research to expand their utility to other disease indications, such as cancer.

[007] Chromene coxibs represent a class of coxibs that could fulfill an unmet medical need in inflammation and cancer. Chromene coxibs have a carboxylate moiety and, uniquely among the coxib class of molecules, do not bind in the hydrophobic binding pocket of the COX-2 active site. Selected chromene derivatives have comparable potency, efficacy, and selectivity to the older diaryl heterocyclic coxibs (e.g., celecoxib, valdecoxib, rofecoxib, and etoricoxib) in the standard rat models of inflammation and pain (Bioorg. Med. Chem. Lett. 20(23):7155-7158 (2010); Bioorg.Med. Chem. Lett. 20(23):7159-7163 (2010); Bioorg. Med. Chem. Lett. 20(23) :7164-716 (2010)). One benzopyran derivative was demonstrated to be effective in mitigating acute dental pain (Clin.Pharmacol.Ther. 83(6):857-866 (2008)).

[008] Nitric oxide (NO) is an important endogenous signaling molecule and vasodilator. NO is synthesized from L-arginine by the enzyme NO synthase (NOS), which exists in three distinct isoforms, namely, the constitutively expressed endothelial (eNOS) and neuronal (nNOS) forms, and the mainly inducible form (iNOS). Arginine administration has been shown to reduce blood pressure and renal vascular resistance in essential hypertensive patients with normal or insufficient renal function (Am. J. Hypertens. 12, 8-15 (1999)). It has also been shown that NO deficiency promotes vascular side-effects of celecoxib and other COX inhibitors (Blood 108, 4059-4062 (2006)).

[009] The role of NO in cancer is complex; however, pharmacological evidence using NO- releasing compounds of NSAIDs has shown increased anti-tumor efficacy in cell culture and animal cancer models. The different molecular mechanisms of NO are expected to simultaneously enhance anti-cancer efficacy of celecoxib, and improve CV safety by preventing an increase in blood pressure associated with COX-2 inhibition, while maintaining gastric- sparing properties superior to NSAIDs.

[010] Diverse molecular mechanisms of NO delivery are well known. For example, it is reported that nitric oxide-donating NSAIDs (NO-sulindac, NO-ibuprofen, NO-indomethacin, and NO-aspirin) inhibit the growth of various cultured human cancer cells, providing evidence of a tissue type-independent effect (J. Pharmacol. Exp. Ther. 303, 1273-1282 (2002)).

[011] In another example, it is reported that nitric oxide-donating aspirin prevented pancreatic cancer in a hamster tumor model {Cancer Res. 66, 4503-4511 (2006)).

[012] Two isoforms of cyclooxygenase (COX) are known to exist, a constitutive form (COX-1) present in nearly all tissues and an inducible form (COX-2) upregulated in response to inflammatory stimuli. The discovery of COX-2 led to the development of selective COX-2 inhibitors as anti-inflammatory drugs (coxibs), which were shown to be largely devoid of the antiplatelet activity and gastrointestinal ulcerogenicity believed to be associated with inhibition of COX-1.

[013] NSAIDs are among the most widely used treatments for pain, fever, and inflammation, and have long been known to reduce the risk of cancer in multiple organ sites. The use of aspirin in treatment and prevention of cancer has wide- spread support in the medical community; however, the risks of regular aspirin use are also well established and the risk-benefit profile is not sufficient to recommend aspirin treatment for cancer prevention. With the advent of coxibs, research has focused on COX-2 as a target for the treatment and prevention of certain cancers. Compelling data from the APC trial, described above, demonstrated that celecoxib was useful in preventing sporadic colorectal adenoma in patients at high risk for colorectal cancer.

[014] Lung cancer is the leading cause of cancer-related deaths in the US and is responsible for more deaths than breast, prostate, and colon cancers combined. Current research suggests that COX-2 and epidermal growth factor receptor (EGFR) are important mediators in non-small cell lung cancer (NSCLC). One study demonstrates a strong cooperative effect on slowing tumor progression by blocking both the EGFR and COX-2 pathways using gefitinib and celecoxib (Zhang, X, Clin. Cancer Res. i l, 6261-6269 (2005)).

[015] In human NSCLC patients, a combination of erlotinib (a tyrosine kinase inhibitor) and celecoxib showed high response rates, and demonstrable clinical benefit (Reckamp, K. L, Clin. Cancer Res. 12, 3381-3388 (2006)). NSCLC currently represents one of the preferred indications for COX-2 inhibition cancer therapy (Brown, J. R., Clin. Cancer Res. 10, 4266s-4269s (2004); and Gadgeel, S. M., Cancer 110, 2775-2784 (2007)).

[016] A key feature of COX-2 biology is its ability alone to cause cancer formation in a number of transgenic mouse models. COX-2 derived PGE2 plays a prominent role in tumor growth and is the most abundant prostanoid in many human malignancies. Metabolism of arachidonic acid by COX-2 leads to the formation of several prostaglandins (PGs) that bind to tumor suppressor p53, preventing p53-mediated apoptosis. COX-2-derived PGE2 promotes epithelial-to- mesenchymal transition and, thus, increases resistance to EGFR tyrosine kinase inhibitors in lung cancer (Krysan, K., J. Thorac. Oncol. 3, 107-110 (2008)).

[017] Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths in the US. Colorectal cancer progression and metastasis occurs through aberrant signaling through the prostaglandin-endoperoxide synthase 2 (PTGS2) and epithelial growth factor (EGF) signaling pathways (Wang, D., Cancers 3, 3894-3908 (2011)). COX-2 over-expression contributes to PTGS2 signaling and therefore COX-2 inhibitors may provide a successful treatment modality for colorectal neoplasia (Eberhart, C.E., Gastroenterology 107, 1183-1188 (1994)).

[018] Nitric oxide exhibits a number of important pharmacological actions including vascular relaxation (vasodilatation) and inhibition of platelet aggregation and adhesion. Inhibition of NO synthesis leads to an increase in systemic blood pressure. NO also prevents atherogenesis by inhibiting vascular smooth muscle cell proliferation, and preventing low-density lipoprotein oxidation and macrophage activation. Vascular NO generation is important in controlling blood pressure, and a growing body of evidence indicates that NO signaling is a key factor in counteracting the onset and development of several CV diseases including hypertension, myocardial infarction, and stroke. NO can be used to counteract CV liabilities associated with COX-2 inhibition.

[019] NO-releasing COX inhibitors were originally created to improve gastrointestinal (GI) tolerability (Inflammopharmacology 11(4), 415-22 (2003)). Naproxcinod is a NO-releasing prodrug of the NSAID naproxen. Naproxcinod showed significantly improved GI tolerability compared to naproxen alone in a chronic rat study (Life Sciences 62, 235-240 (1998)). In another example, L-arginine, coadministered with the NSAID ibuprofen, showed a protective effect on gastric mucosa against ibuprofen-induced mucosal lesions (Free Radic. Res. 38(9), 903-11 (2004)). [020] NO modulates the activity of transcription factor NF-κΒ, which represents a potential mechanism for inflammation control, but also regulation of apoptotic mechanisms. NO promotes apoptosis and can reverse tumor cell resistance to chemotherapeutic agents. Studies with NO- releasing NSAIDs have shown that NO contributes to anti-cancer activity in cell culture and enhanced in vivo efficacy in rodent cancer models. For example, it is reported that nitric oxide- naproxen is an effective agent against carcinogenesis in rodent models of colon and urinary bladder cancers (Cancer Prev. Res. 2, 951-956 (2009)). Nitric oxide-releasing prodrugs useful in the treatment of inflammation and the reduction of adverse cardiovascular and/or ulcerogenic events associated with chronic use of COX-2 inhibitors are reported by Velazquez et al. WO2007/127725. Also disclosed is a method of preventing or treating cancer or treating inflammation or an inflammation-related condition. Compounds proposed therein include proline-based acyloxymethylene diazen-l-ium-l,2-diolate prodrugs of NSAIDs and celecoxib compound.

[021] Nitric oxide-releasing prodrugs useful in the treatment of inflammation and the reduction of adverse cardiovascular and/or ulcerogenic events associated with chronic use of COX-2 inhibitors are reported by Abdellatif et al, Bioorg. Med. Chem. (20) 4544-4549 (2010). The compounds described therein include celecoxib substituted with an acetyloxymethylenediazeniumdiolate radical attached to non- sulfonamide substituted phenyl, yielding the structure:

[022] Nitric oxide-releasing prodrugs useful in the treatment of inflammation and the reduction of adverse cardiovascular and/or ulcerogenic events associated with chronic use of COX-2 inhibitors are reported by Abdellatif et al, Bioorg. Med. Chem. (16) 9694-98 (2008). The compounds described therein include celecoxib substituted with an acetyloxymethylenediazeniumdiolatepyrrolidinylcarbonyloxymet hylene radical attached to non- sulfonamide substituted phenyl, yielding the structure:

[023] Nitric oxide-releasing prodrugs useful in the treatment of inflammation and the reduction of adverse cardiovascular and/or ulcerogenic events associated with chronic use of COX-2 inhibitors are reported by Abdellatif et al, Bioorg. Med. Chem. (14) 5182-88 (2009). The compounds described therein include celecoxib substituted with a acetyloxymethylenediazeniumdiolatepyrrolidinylmethyleneoxyca rbonylmethylene or a acetyloxymethylenediazeniumdiolateaminoethyleneoxycarbonylme thylene radical attached to

[024] Nitric oxide-releasing prodrugs useful in the treatment of inflammation and the reduction of adverse cardiovascular and/or ulcerogenic events associated with chronic use of COX-2 inhibitors are reported by Abdellatif et al, Bioorg. Med. Chem. (16) 6528-34 (2008). The compounds described therein include methylsulfonylcelecoxib substituted with a acetyloxymethylenediazeniumdiolateaminoethyleneoxycarbonyl radical attached to pyrazolyl, yielding the structure:

[025] Chromenes useful in the treatment of dermatological disorders, including acne and inflammation, have been reported in US 2005/0014729. The compounds described therein for the aforementioned use include a chromene of the structure:

[026] Nitric oxide-releasing chromene prodrugs useful in the treatment of inflammation and the reduction of adverse cardiovascular and/or ulcerogenic events associated with chronic use of COX-2 inhibitors have been reported in WO 2001/045703, including chromenes substituted with an nitrooxyalkyl of the structure:

[027] Nitric oxide-releasing agents non-covalently combined with chromenes useful in the treatment of inflammation and the reduction of adverse cardiovascular and/or ulcerogenic events associated with chronic use of COX-2 inhibitors are reported in US 2005/0113409, including (S)- 6-chloro-7-(l,l-dimethylethyl)-2-(trifluoromethyl)-2H-l-benz opyran-3-carboxylic acid of the structure:

[028] Nitric oxide-releasing chromene prodrugs useful in the treatment of inflammation, cancer, and the reduction of adverse cardiovascular and/or ulcerogenic events associated with chronic use of COX-2 inhibitors are reported in WO 2006/040676, including chromenes substituted with an nitrooxyalkyl of the following structures:

[029] Chromene-based coxib drugs possess a number of advantages over existing medicines for the treatment of inflammation, pain, and cancer. The molecules of the present invention have the potential to be renal-sparing, safer on the gastrointestinal tract, and will not show coxib-induced hypertension due to their intrinsic and distinct structural, pharmacological and physiochemical properties.

[030] Herein described is a family of NO-releasing chromene conjugates which provides a therapeutic benefit to a subject with a disease indication, such as cancer, actinic keratosis, cystic fibrosis, acne, or provides a wound healing benefit to a subject. Such NO-releasing chromene conjugates can reduce gastric erosion of cancer therapy, improve CV safety, permit higher dose of cancer-treating compound, enhance cancer-treating efficacy, and/or maintain gastric-sparing properties superior to NSAIDs.

[031] In one embodiment, there is provided a compound of Formula (I):

and pharmaceutically acceptable salts thereof, wherein Z, R 1, R2, R 3, R 4, R 5, R 6, and R V are as defined in the detailed description:

[032] Compounds of the present invention can exist in tautomeric, geometric or stereoisomeric forms. Ester, metabolite, oxime, prodrug, onium, hydrate, solvate, and N-oxide forms of a compound of Formula (I) are also embraced by the invention. The present invention considers all such compounds, including, but not limited to, cis- and trans-geometric isomers (Z- and E- geometric isomers), R- and S-enantiomers, diastereomers, d-isomers, 1-isomers, atropisomers, epimers, conformers, rotamers, mixtures of isomers, and racemates thereof, as falling within the scope of the invention.

DETAILED DESCRIPTION

A. Compounds

[033] One embodiment of the invention is a compound, or a pharmaceutically acceptable salt, or solvate of a compound or salt of Formula (I):

wherein Z is selected from the group consisting of CF ₃ , OCF ₃ , and 1 2 3

C ₂ F ₅ ; each of R , R , R , and R ⁴ is independently selected from a group consisting of H, alkyl, aralkyl, cycloalkyl, cycloalkenyl, halo, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, pentafluorosulfanyl, hydroxyalkyl, trialkylsilyl, alkynyl, and alkenyl; R ⁵ is selected from the group consisting of H, alkyl, cycloalkyl, cycloalkenyl, aryl, aralkyl, and heterocyclyl; and each of R ⁶ and R ⁷ is independently selected from the group consisting of H, C C ₄ alkylenyl, cycloalkyl, cycloalkenyl, aryl, aralkyl, and heteroaryl, optionally substituted from the group consisting of H, alkoxycarbonyl, alkoxycarbonylalkylenyl, alkylcarbonylamino, alkylcarbonylalkylenylamino, alkylcarbonyloxy, carboxy, carboxyalkylenyl, carboxyamino, carboxyalkylenylamino, hydroxy, hydroxyalkylenyl, halo, amino, aryl amino, dialkyl amino, aralkyl amino, alkyl ether, aryl ether, heteroaryl ether, amido, and alkyl amido; or taken together, R ⁶ and R ⁷ may form a 4, 5, 6 or 7- membered heterocycle wherein said heterocycle may be optionally substituted from the group consisting of H, alkyl, alkoxycarbonyl, alkoxycarbonylalkylenyl, alkylcarbonylamino, alkylcarbonylalkylenylamino, alkylcarbonyloxy, carboxy, carboxyalkylenyl, carboxyamino, carboxyaminoalkylenyl, hydroxy, hydroxyalkylenyl, halo, amino, amino alkylenyl, aryl amino, dialkyl amino, aralkyl amino, alkyl ether, aryl ether, heteroaryl ether, amido, and alkyl amido.

[034] In another family of the compounds of Formula (I), R ¹ is selected from a group consisting of H, alkyl, and halo; R is selected from a group consisting of alkyl, halo, haloalkyl, alkoxy, haloalkoxy, alkylthio, haloalkylthio, and pentafluorosulfanyl; R is selected from a group consisting of H, alkyl, cycloalkyl, cycloalkenyl, halo, haloalkyl, hydroxyalkyl, and trialkylsilyl; R ⁴ is selected from a group consisting of H, alkyl, halo, alkynyl, and alkenyl; R ⁵ is H or alkyl; R ⁶ is selected from the group consisting of alkyl, CrC ₆ hydroxyalkylenyl, and CrC ₆ carboxyalkylenyl; and R is selected from the group consisting of C -C hydroxyalkylenyl and C -C carboxyalkylenyl, optionally substituted from the group consisting of alkyl, aryl, aralkyl, and heteroaryl; or taken together, R ⁶ and R ⁷ may form a 4, 5, or 6-membered heterocycle wherein said heterocycle may be optionally substituted from the group consisting of alkyl, amido, amido C C ₄ alkylenyl, amino, amino C C ₄ alkylenyl, aryl, aralkyl, heteroaryl, hydroxy, hydroxy C C ₄ alkylenyl, carboxy, carboxy C C ₄ alkylenyl, carboxyamino, carboxyamino C C ₄ alkylenyl, and CrC ₄ alkylcarbonyloxy.

[035] In another family of the compounds of Formula (I), R ¹ is selected from the group consisting of H, methyl, CI, and F; R is selected from the group consisting of CI, Br, methyl, trifluoromethoxy, pentafluorosulfanyl, OCH ₃ , OCH ₂ CH ₃ , OCF ₂ H, SCH ₃ , SCH ₂ CH ₃ , SCF ₃ , SCF ₂ H, CF ₃ , and CF ₂ CF ₃ ; R is selected from the group consisting of H, methyl, tert-butyl, ethyl, w-propyl, isopropyl, w-butyl, CH(CH ₃ )CH ₃ CH ₂ , CH ₂ CH(CH ₃ ) ₂ , C(CH ₃ ) ₂ CH ₂ OH, CI, F, Br, CF ₃ , and Si(CH ₃ ) ₃ ; R ⁴ is selected from the group consisting of H, CI, methyl, ethyl, C≡CH, CH=CH ₂ , and Br; R ⁶ is selected from the group consisting of methyl, ethyl, isopropyl, tert-butyl, C C ₆ hydroxyalkylenyl, and CrC ₆ carboxyalkylenyl; and R is selected from the group consisting of CrC ₆ hydroxyalkylenyl and CrC ₆ carboxyalkylenyl, optionally substituted from the group consisting of alkyl, aryl, aralkyl, and heteroaryl; or taken together, R ⁶ and R ⁷ may form a 4 or 5-membered heterocycle wherein said heterocycle may be optionally substituted from the group consisting of alkyl, aryl, aralkyl, heteroaryl, hydroxy, hydroxyl CrC ₄ alkylenyl, carboxy, and carboxy C C ₄ alkylenyl.

[036] The present invention is also directed to a subclass of compounds, including pharmaceutically acceptable salts of compounds, wherein compounds have the structure of Formula (II):

1 2

wherein R is H or methyl; R is selected from the group consisting of CI, Br, methyl, trifluoromethoxy, and pentafluorosulfanyl; R is selected from the group consisting of H, methyl, and ieri-butyl; R ⁴ is selected from the group consisting of H, CI, methyl, and ethyl; R ⁵ is H or methyl; and R is CH ₂ CH ₂ OH or CH ₂ CO ₂ H. Non-limiting examples include:

3-methyltriaz-l-ene 2-oxide

[037] The present invention is also directed to a subclass of compounds, including pharmaceutically acceptable salts of compounds, wherein compounds have the structure of Formula (III):

wherein R is Η or methyl; R is selected from the group consisting of CI, Br, methyl, trifluoromethoxy, and pentafluorosulfanyl; R is selected from the group consisting of Η, methyl, and ieri-butyl; R ⁴ is selected from the group consisting of H, CI, methyl, and ethyl; R ⁵ is H or methyl; and R is OH or H. Non-limiting examples include:

yl)diazene oxide (S,Z)-2-(((8-ethyl-6-(trifluoromethoxy)-2- (trifluoromethyl) -2H-chromene- 3 -

169

carbonyl)oxy)methoxy)- l-(azetidin- 1-

Et yl)diazene oxide

(5,Z)-2-(((7-(ieri-butyl)-6-chloro-2- (trifluoromethyl) -2H-chromene- 3 -

172

carbonyl)oxy)methoxy)- l-(azetidin- 1- yl)diazene oxide

[038] The present invention is also directed to a subclass of compounds, including pharmaceutically acceptable salts of compounds, wherein compounds have the structure of Formula (IV):

1 2

wherein R is Η or methyl; R is selected from the group consisting of CI, Br, methyl, trifluoromethoxy, and pentafluorosulfanyl; R is selected from the group consisting of Η, methyl, and ieri-butyl; R ⁴ is selected from the group consisting of Η, CI, methyl, and ethyl; R ⁵ is Η or methyl; and R ⁹ is CH ₂ OH or C0 ₂ H. Non-limiting examples include:

(Z)-2-((((5)-6-bromo-8-methyl-2- (trifluoromethyl) -2H-chromene- 3 - carbonyl)oxy)methoxy)- l-(( _l S')-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide

(Z)-2-((((5)-8-ethyl-6-(trifluoromethoxy)- 2-(trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- l-((S)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide

(Z)-2-((((5)-7-(ieri-butyl)-6-chloro-2- (trifluoromethyl) -2H-chromene- 3 - carbonyl)oxy)methoxy)- l-(( _l S')-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide

(Z)-l-((5 -2-carboxypyrrolidin- l-yl)-2- ((((5)-6-chloro-8-methyl-2- (trifluoromethyl) -2H-chromene- 3 - carbonyl)oxy)methoxy)diazene oxide

(Z)-2-((((5 6-bromo-8-methyl-2- (trifluoromethyl) -2H-chromene- 3 - carbonyl)oxy)methoxy)- l-((5j-2- carboxypyrrolidin- l-yl)diazene oxide

(Z)-l-(f ')-2-carboxypyrrolidin- l-yl)-2- ((((S)-8-ethyl-6-(trifluoromethoxy)-2- (trifluoromethyl) -2H-chromene- 3 - carbonyl)oxy)methoxy)diazene oxide

(Z)-2-((((5)-7-(ieri-butyl)-6-chloro-2- (trifluoromethyl) -2H-chromene- 3 - carbonyl)oxy)methoxy)- l-(( _l S')-2-

carboxypyrrolidin- l-yl)diazene oxide B. Other Embodiments

[039] In another embodiment, there is provided a pharmaceutical composition comprising a compound of the structural formulae herein, and a pharmaceutically-acceptable carrier.

[040] In another embodiment, the pharmaceutical composition further comprises one or more additional pharmaceutically active compounds.

[041] In another embodiment, there is provided a method for treating or preventing a disease condition comprising administering to a subject a therapeutically effective amount of a compound of the structural formulae herein, wherein the condition to be treated or prevented includes, for example, cancer. Further non-limiting examples include non-small cell lung cancer, skin cancer, liver cancer, colorectal cancer (including metastatic colorectal cancer, and FAP), glioblastoma (and other CNS related cancers), squamous cell cancer, bladder cancer, breast cancer, biliary tract cancer, cervical cancer, prostate cancer, small cell lung cancer, ovarian cancer, pancreatic cancer, gastrointestinal cancer, and CNS cancer.

[042] In another embodiment, there is provided a method for healing wounds, comprising administering to a subject a therapeutically effective amount of a compound of the structural formulae herein.

[043] In another embodiment, there is provided a method for treating a condition, comprising administering to a subject a therapeutically effective amount of a compound of the structural formulae herein, wherein the condition to be treated includes, for example, actinic keratosis, cystic fibrosis, and/or acne.

[044] In another embodiment, there is provided a method for treating a condition comprising administering to a subject a therapeutically effective amount of a compound of the structural formulae herein, wherein the condition to be treated includes, for example, autoimmune disorder, inflammatory disorder, and/or auto-inflammatory disorder.

[045] In another embodiment, there is provided a method that comprises administering a combination of a compound of the structural formulae herein, and at least one additional pharmaceutically active compound.

[046] In another embodiment, there is provided a use of a compound of the structural formulae herein for manufacture of a medicament for treatment of a disease condition in a subject. [047] In another embodiment, there is provided a method for preparing a compound of the structural formulae herein.

[048] In another embodiment, there is provided an intermediate useful in making a compound of the structural formulae herein.

[049] In another embodiment, there is provided a method of enhancing cancer-treating efficacy by activating both NO and COX-2-inihibitor anti-tumor mechanisms in a subject, by

administering a therapeutically effective amount of a compound of the structural formulae herein.

[050] In another embodiment, there is provided a method of treating a subject suffering from a disease condition caused by COX-2 over-expression, including but not limited to cancer, an autoimmune disorder such as rheumatoid arthritis, and other disorders characterized by pain and/or inflammation, by administering a therapeutically effective amount of a compound of the structural formulae herein.

[051] In another embodiment, there is provided a method of improving CV safety in a subject, by administering a therapeutically effective amount of a compound of the structural formulae herein.

[052] In another embodiment, there is provided a method of treating a subject suffering from a disease condition, including but not limited to cancer, by administering a high dose of a compound of the structural formulae herein.

[053] In another embodiment, there is provided a method of gastro-protection in a subject, comprising administering a therapeutically effective amount of a compound of the structural formulae herein.

[054] In another embodiment, there is provided a method of releasing NO in a subject, comprising administering a therapeutically effective amount of a compound of the structural formulae herein.

[055] In another embodiment, there is provided a method of gastro-protection in a subject, comprising administering a therapeutically effective amount of a compound of the structural formulae herein, which releases NO in the subject, preferably by sustained release.

[056] In another embodiment, there is provided a method of gastro-protection in a subject, comprising administering a therapeutically effective amount of a compound of the structural formulae herein, which releases NO in the subject, preferably by sustained release, wherein the NO release is likely caused by an enzymatic mechanism acting on the NONOate moiety of a compound of the structural formulae herein.

[057] In another embodiment, there is provided a method of gastro-protection in a subject, comprising administering a therapeutically effective amount of a compound of the structural formulae herein, which releases NO in the subject, preferably by sustained release, wherein the NO release is likely caused by a non-enzymatic mechanism acting on the NONOate moiety of a compound of the structural formulae herein.

[058] In another embodiment, there is provided a method of gastro-protection in a subject, comprising administering a therapeutically effective amount of a compound of the structural formulae herein, which releases NO in the subject, preferably by sustained release, wherein the NO release is likely caused by both enzymatic and non-enzymatic mechanisms acting on the NONOate moiety of a compound of the structural formulae herein. Non-enzymatic mechanisms can include chemical mechanisms.

[059] In another embodiment, there is provided a method of treating a subject suffering from a disease condition, including but not limited to cancer, comprising administering a therapeutically effective amount of a compound of the structural formulae herein, without causing substantial adverse, cardiovascular events.

[060] In another embodiment, there is provided a method of treating a subject suffering from a disease condition, including but not limited to cancer, comprising administering a therapeutically effective amount of a compound of the structural formulae herein, without causing substantial changes in blood pressure, while maintaining gastric- sparing properties.

[061] It will be recognized that compounds of this invention can exist in radiolabeled form, i.e., compounds may contain one or more atoms containing an atomic mass or mass number different from the atomic mass or mass number usually found in nature (e.g., an isotope). Alternatively, a plurality of molecules of a single structure may include at least one atom that occurs in an isotopic ratio that is different from the isotopic ratio found in nature. Radioisotopes of hydrogen, carbon, phosphorous, fluorine, chlorine and iodine include 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 35 S, 18 F,

36 Cl, 125 I, 124 I and 131 I respectively. Compounds that contain those radioisotopes and/or other radioisotopes of other atoms are within the scope of this invention. Tritiated, i.e. H, and carbon- 14, i.e., ¹⁴ C, radioisotopes are particularly preferred for their ease in preparation and

detectability. Compounds that contain isotopes ^U C, ¹³ N, ¹⁵ 0, ¹²⁴ I and ¹⁸ F are well suited for positron emission tomography. Radiolabeled compounds of the structural formulae herein and prodrugs thereof can generally be prepared by methods well known to those skilled in the art. Conveniently, such radiolabeled compounds can be prepared by carrying out the procedures disclosed in the Examples and Schemes by substituting a readily available radiolabeled reagent for a non-radiolabeled reagent.

C. Definitions

[062] The terms "substituent", "radical", "group", "moiety", and "fragment" may be used interchangeably.

[063] If a substituent is described as being "optionally substituted," the substituent may be either (1) not substituted or (2) substituted on a substitutable position. If a substitutable position is not substituted, the default substituent is H.

[064] Singular forms "a", and "an" may include plural reference unless the context clearly dictates otherwise.

[065] The number of carbon atoms in a substituent can be indicated by the prefix "C _A - _B " where A is the minimum and B is the maximum number of carbon atoms in the substituent.

[066] The term "hydrido" denotes a single -H atom (H) and may be used interchangeably with the symbol "H". Hydrido may be attached, for example, to an oxygen atom to form a "hydroxy" radical (i.e., -OH) or two hydrido radicals may be attached to a carbon atom to form a "methylene" (-CH ₂ -) radical.

[067] The terms "hydroxyl" and "hydroxy" may be used interchangeably.

[068] The term "ester" denotes

[069] The term "O-linked ester" denotes an ester that is linked to the parent scaffold through the covalent O in the ester.

[070] The term "halo" refers to fluoro (-F), chloro (-C1), bromo (-Br), or iodo (-1).

[071] The term "alkyl" denotes a linear or branched acyclic alkyl radical containing from 1 to about 15 carbon atoms and less than or about equal to the natural abundance of deuterium (i.e. (i.e. -0.0156%). In some embodiments, alkyl is a Crioalkyl, C _h alky!, C ₆ alkyl or Crsalkyl radical. Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentan-3-yl (i.e., ) and the like.

[072] The term "alkylcarbonyl" denotes an alkyl radical attached to carbonyl.

[073] The term "hydroxyalkyl" embraces a radical wherein any one or more of an alkyl carbon is substituted with a hydroxyl radical as defined above, for example, monohydroxyalkyl, dihydroxyalkyl, and trihydroxyalkyl. More specific examples of hydroxyalkyl include hydroxymethyl, hydroxyethyl, and hydroxypropyl.

[074] Hydroxyalkyl may be substituted with, for example, alkyl, hydroxyalkyl, hydroxyalkoxy, hydroxyalkoxyalkyl, amino, aminoalkyl, aryl, aralkyl, and heterocyclyl. Further non-limiting examples include hydroxyalkyl substituted with methyl, isobutyl, benzyl, isopropyl, benzyl, and sec-butyl.

[075] The term "hydroxyalkoxy" denotes a hydroxy radical attached to an alkoxy radical (e.g., hydroxyl-C-O-scaffold).

[076] The term "hydroxyalkoxyalkyl" denotes a hydroxyalkoxy radical attached to an alkyl radical. Non-limiting examples include hydroxyethyl-O-ethyl and hydroxylmethyl-O-ethyl.

[077] Hydroxyalkoxyalkyl may, for example, be substituted with alkyl, hydroxyalkyl, hydroxyalkoxy, hydroxyalkoxyalkyl, amino, aminoalkyl, aryl, aralkyl, and heterocyclyl. Further non-limiting examples include hydroxyalkoxyalkyl substituted with methyl, isobutyl, benzyl, isopropyl, and sec-butyl. More specific non-limiting examples of substituted hydroxyalkoxyalkyl include hydroxyethyl-O-ethyl substituted with methyl, isobutyl, benzyl, isopropyl, and sec-butyl.

[078] The term "haloalkyl" embraces an alkyl radical wherein any one or more of the alkyl carbon atoms is substituted with halo as defined above. For example, monohaloalkyl, dihaloalkyl, and trihaloalkyl. A monohaloalkyl radical, for one example, may have either a bromo, chloro or a fluoro atom within the radical. A dihalo radical may have two of the same halo radicals or a combination of different halo radicals. A trihaloalkyl radical may have three of the same halo radicals or a combination of different halo radicals. Non-limiting examples of haloalkyl include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, trifluoroethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, dichloropropyl, iodomethyl, diiodomethyl, and triiodomethyl.

[079] The term "alkylenyl" denotes a divalent linear or branched saturated carbon chain containing from 2 to about 15 carbon atoms. The terms "alkylene", and "alkylenyl" may be used interchangeably. Non-limiting examples of alkylenyl radicals include methylene, ethylenyl (

^^^^), propylenyl ^^), butylenyl ^), and pentylenyl ^ ^^)-

[080] One or more substitutable carbons in an alkylenyl radical may be replaced with, for

example,-CH(Z ⁶ )-, -CH(Z ⁶ )-0-, -C(Z ⁶ ) ₂ -,

. _where z ⁶ , z ⁷ , and Z may be, for example, independently selected from the group consisting of H, alkyl, hydroxy, aminoalkyl, acylamino, amido, carboxy, carboxyalkylenyl, hydroxyalkyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, (e.g., heteroaryl, more specifically phthalimido) aralkyl, alkyl-

0-, alkyl-S-, and alkyl-NH-, or Z 7 may be taken together with Z 8 to form a cyclic ring; Z 7 may be, for example, H, alkyl, hydroxyalkyl, aryl, heterocyclyl, alkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, carboxyalkylcarbonyl, alkyloxycarbonylalkylcarbonyl, alkylsulfonyl, arylsulfonyl, and heteroarylsulfonyl.

[081] Examples of substituted alkylenyl include, ethyleneoxypropylene ( ^^ ^^^^ ), r ), ethyleneoxymethylene ( ^ ^"0 ^) ethyleneoxypropylene (V^^ ^^^^), ethylenecarbonyl (

[082] One bstitutable carbons in an alkylenyl radical may be replaced with a

\ or

[083] When one or more substitutable carbons in alkylenyl are substituted, and the resulting radical has multiple orientations (e.g., ¾ ^N vs \ ), both orientations are embraced by the display of a single orientation.

[084] The term "alkoxy" is RO- where R is alkyl as defined above. Non-limiting examples of alkoxy radicals include methoxy, ethoxy and propoxy. The terms "alkyloxy" and "alkoxy" and "alkyl-O-" may be used interchangeably.

[085] The term "haloalkoxy" is RO- where R is halo-substituted alkyl. Non-limiting examples of haloalkoxy radicals include trifluoromethoxy and tribromomethoxy.

[086] The term "alkoxyalkyl" refers to an alkoxy moiety substituted with an alkyl radical. Examples of alkoxyalkyl radicals include methoxymethyl, methoxyethyl, methoxypropyl and ethoxyethyl.

[087] The term "alkoxycarbonyl" refers to an alkoxy radical substituted with carbonyl. Non- limiting examples include methoxycarbonyl and ethoxycarbonyl.

[088] The term "alkoxycarbonylalkylenyl" refers to an alkoxycarbonyl radical attached to an alkylenyl radical.

[089] The term "alkyloxycarbonylalkylcarbonyl" refers to an alkoxycarbonylalkyl radical

substituted with carbonyl

[090] The term "alkylcarbonylalkylenylamino" refers to an alkylcarbonylalkylenyl radical attached to an amino radical.

[091] The term "alkylcarbonylamino" refers to an alkylcarbonyl radical attached to an amino radical.

[092] The term "alkylcarbonxyloxy" refers to an alkylcarbonylradical attached to the parent

scaffold through an oxy radical (e.g.,

[093] The term "alkenyl" refers to an unsaturated, acyclic hydrocarbon radical with at least one double bond. Such alkenyl radicals contain from 2 to about 15 carbon atoms.

[094] The term "alkynyl" refers to an unsaturated, acyclic hydrocarbon radical with at least one triple bond. Such alkynyl radicals containing from 2 to about 15 carbon atoms. A non-limiting example is propargyl.

[095] The term "cyano" denotes a carbon radical having three of four covalent bonds shared by a single nitrogen atom. [096] The term "silyl" denotes a radical.

[097] The term "alkylsilyl" denotes an alkyl substituted silyl radical.

[098] The term "carbonyl" denotes a carbon radical having two of four covalent bonds shared with a single oxygen atom.

[099] The term "alkylcarbonyl" denotes an alkyl radical attached to a carbonyl radical.

[0100] The term "haloalkylcarbonyl" denotes a haloalkyl radical ttached to a carbonyl radical.

[0101] The term "carbonylalkylenyl" denotes a carbonyl radical attached to an alkylenyl radical.

[0102] The term "alkylcarbonylalkylenyl" denotes an alkyl radical attached to a carbonyl alkylenyl radical.

[0103] The term "carbonylalkylenylcarbonyl" denotes a carbonylalkylenyl radical attached to a carbonyl radical.

[0104] The term "carbonyloxy" denotes an oxygen radical having one of two covalent bonds shared with a carbonyl radical.

[0105] The term "alkylcarbonylamino" embraces an alkylcarbonyl radical attached to an amino

[0106] The term "alkylcarbonylalkylenylamino" embraces an alkylcarbonylalkylenyl radical

attached to an amino radical (e.g.

[0107] The term "alkylcarbonyloxy" denotes an alkyl radical attached to a carbonyloxy radical.

[0108] The term "alkylcarbonyloxyalkyenel" denotes an alkylcarbonyloxy radical attached to an alkylenyl radical.

[0109] The term "alkylcarbonyloxyalkylenylcarbonyl" denotes an alkylcarbonyloxyalkylenyl radical attached to an carbonyl radical.

[0110] The term "thiocarbonyl" denotes a carbon radical having two of four covalent bonds shared with a single sulfur atom, i.e., o

[0111] The term "ureido" denotes H H and may be used interchangeably with carbamido.

[0112] The term "azetidinyl" denotes a fully saturated 4-membered heterocyclic moiety containing one nitrogen atom and three carbon atoms, connected to the scaffold through the

nitrogen or one of the ring carbons. A non-limiting example is:

[0113] The term "pyrrolidinyl" denotes a fully saturated 5-membered heterocyclic moiety containing one nitrogen atom and 4 carbon atoms, scaffold through the nitrogen

or one of the ring carbons. A non-limiting example

[0114] The term "piperidinyl" denotes a fully saturated 6-membered heterocyclic moiety containing one nitrogen atom and 5 carbon atoms, connected to the scaffold through the nitrogen

or one of the ring carbons. A non-limiting example is

[0115] The term "acyl", is R may be, for example, H, alkyl, nitrooxyalkylenyl, aryl and aralkyl. More specific examples of acyl include formyl, acetyl, benzoyl, nitrooxymethylcarbonyl and nitrooxyethylcarbonyl.

O

[0116] The term "acylamino" is R , where R may be, for example, H, alkyl, nitrooxyalkylenyl, aryl and aralkyl. A more specific example of acylamino is acetylamino.

[0117] The term "carboxy" embraces a hydroxy radical attached to one of two unshared bonds in a carbonyl radical.

[0118] The term "carboxyamino" embraces a carboxy radical attached to an amino radical (e.g.,

[0119] The term "carboxyalkylenylamino" embraces a carboxyamino radical attached to an alkylenyl radical (e.g.,

[0120] The term "carboxyaminoalkylenyl" embraces a carboxyamino radical attached to an alkyenel radical.

[0121] The term "carboxyalkylenyl" embraces a carboxy radical attached to an alkylenyl radical

(e.g., HO alkylene Non-limiting examples of carboxyalkyene include carboxymethylene and carboxyethylenyl. The terms "carboxyalkylenyl" and "hydroxycarbonylalkylenyl" may be used interchangeably.

[0122] The term "carboxyalkylcarbonyl" denotes a carboxyalkyl radical attached to a carbonyl radical.

[0123] The term "thiocarboxy" embraces a hydroxyl radical, as defined above, attached to one of two unshared bonds in a thiocarbonyl radical.

[0124] The term "thiocarboxyalkylenyl" embraces a thiocarboxy radical, as defined above, attached to an alkylenyl radical. Non-limiting examples include thiocarboxymethylene and thiocarboxyethylenyl.

[0125] The term "amido" embraces an amino radical attached to a parent molecular scaffold

through carbonyl (e.g., Z ² , where Z and Z ² may be, H, alkyl, or aralkyl, or Z may be taken together with Z to form heterocyclyl, wherein at least one heteroatom is an amido nitrogen). The terms "amido" and "carboxamido" may be used interchangeably. Examples of amido radicals include monoalkylaminocarbonyl, dialkylaminocarbonyl. More specific examples of amido radicals include N-methylamino carbonyl and N,N-dimethylaminocarbonyl.

O

I

[0126] The term "carbamate" is R , where R may be, for example, H, alkyl or acyl.

[0127] The term "cyclic ring" embraces any aromatic or non-aromatic cyclized carbon radical (e.g., aryl and cycloalkyl respectively) which may contain one or more ring heteroatoms (e.g., heteroaryl and heterocyclyl).

[0128] The term "cycloalkyl" embraces any monocyclic, bicyclic or tricyclic cyclized carbon radical of 3 to about 15 carbon atoms that is fully saturated. Cycloalkyl may be attached to an aryl, cycloalkyl, cycloalkenyl, or a heterocyclyl radical in a fused or pendant manner.

[0129] Cycloalkyl may be substituted with alkyl, alkoxy, carboxyalkylenyl, hydroxyalkyl, amino, acylamino, amido, alkylamino, nitrooxyalkylenyl, nitrooxy, carbonyl, acyl, aralkyl, aryl, heterocyclyl, cycloalkenyl, or cycloalkyl.

[0130] The term "cycloalkenyl" embraces any monocyclic, bicyclic, or tricyclic cyclized carbon radical, fused or pendant, of 3 to about 15 carbon atoms that is partially saturated, containing one or more double bonds, but is not aromatic.

[0131] Cycloalkenyl may be substituted with alkyl, alkoxy, carboxyalkylenyl, hydroxyalkyl, amino, acylamino, amido, alkylamino, nitrooxyalkylenyl, nitrooxy, carbonyl, acyl, aralkyl, aryl, heterocyclyl, cycloalkenyl, or cycloalkyl.

[0132] The term "aryl" refers to any monocyclic, bicyclic or tricyclic cyclized carbon radical, wherein at least one ring is aromatic. An aromatic radical may be attached to a non-aromatic cycloalkyl, cycloalkenyl, or heterocyclyl radical in a fused or pendant manner. Examples of aryl radicals include, but are not limited to, phenyl and naphthyl.

[0133] The term "arylcarbonyl" denotes an aryl radical attached to a carbonyl radical. The terms "aroyl" and "arylcarbonyl" may be used interchangeably. Examples of arylcarbonyl include benzoyl and toluoyl.

[0134] The term "haloarylcarbonyl" denotes a halo radical attached to a carbonyl radical.

[0135] The term "aralkyl" embraces aryl attached to an alkyl radical and may be used interchangeably with arylalkyl. Examples of aralkyl include benzyl, diphenylmethyl, triphenylmethyl, phenylethyl and diphenylethyl. The terms "benzyl" and "phenylmethyl" may be used interchangeably.

[0136] The term "heterocyclyl" refers to any monocyclic, bicyclic or tricyclic ring system having from 5 to about 15 ring members selected from carbon, nitrogen, sulfur and oxygen, wherein at least one ring member is a heteroatom. Heterocyclyl embraces a fully saturated, partially saturated and fully unsaturated radical (e.g., heteroaryl). Heterocyclyl may be fused or attached in a pendant manner to another heterocyclyl, aryl, cycloalkenyl, or cycloalkyl radical.

[0137] Heterocyclyl embraces combinations of different heteroatoms within the same cyclized ring system. When nitrogen is a ring member, heterocyclyl may be attached to the parent molecular scaffold through a ring nitrogen. Non-limiting examples of fully saturated five and six-membered heterocyclyl include: pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl, tetrahydrofuranyl, morpholinyl and thiazolidinyl. Examples of partially saturated heterocyclyl dihydropyranyl, dihydrofuranyl and dihydrothiazolyl.

[0138] Heterocyclyl may be substituted, for example, with alkyl, alkoxy, carboxyalkylenyl, hydroxyalkyl, amino, acylamino, amido, alkylamino, nitrooxyalkylenyl, nitrooxy, carbonyl, acyl, aralkyl, aryl, heterocyclyl, cycloalkenyl, or cycloalkyl. Non-limiting examples include, five- membered heterocyclyl substituted with hydroxyalkyl, alkoxyalkyl, acyl, carbonyl or alkylaminocarbonyl. More specifically, pyrrolidinyl may be substituted with hydroxyalkyl, alkoxyalkyl, acyl, carbonyl or alkylaminocarbonyl. Substituted and un-substituted 5-membered heterocyclyl may be fused or attached in a pendant manner to an additional heterocyclyl, aryl, cycloalkenyl, or cycloalkyl radical. For example, pyrrolidinyl-2,5-dione may be fused to phenyl giving isoindolinyl,l,3-dione (also termed "phthalimido").

[0139] The term "heterocycloalkyl" embraces a heterocyclyl radical attached to the parent molecular scaffold through an alkyl radical (e.g., heterocyclyl-alkyl-scaffold).

[0140] The term "alkylheterocyclylcarbonyl" embraces an alkyl substituted heterocylyl radical attached to the parent molecular scaffold through a carbonyl radical (e.g., alkyl-heterocyclyl- carbonyl-scaffold).

[0141] Six-membered heterocyclyl may be substituted with, for example, hydroxyalkyl, alkoxyalkyl, acyl, carbonyl or alkylaminocarbonyl. More specifically, piperidinyl, piperazinyl and morpholinyl may be substituted with hydroxyalkyl, alkoxyalkyl, acyl, carbonyl or alkylaminocarbonyl. Substituted and un-substituted 6-membered heterocyclyl may be fused or attached in a pendant manner to an additional heterocyclyl, aryl, cycloalkenyl, or cycloalkyl radical.

[0142] The term "heteroaryl" refers to an aromatic heterocyclyl radical. Heteroaryl may be fused or attached in a pendant manner to another heterocyclyl, aryl, cycloalkenyl, or cycloalkyl radical. Heteroaryl embraces combinations of different heteroatoms within the same cyclized radical. When nitrogen is a ring member, heteroaryl may be attached to the parent molecular scaffold through a ring nitrogen. Non-limiting examples of heteroaryl include pyridyl, thienyl, furanyl, pyrimidyl, imidazolyl, pyrazolyl, thiazolyl, thiadiazolyl, isothiazolyl, oxazolyl, isoxazoyl, pyrrolyl, pyridazinyl, pyrazinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothienyl, indolyl,

and thianaphthenyl. The term "heteroaryl" is also understood to include the N-oxide derivative of any nitrogen containing heteroaryl.

[0143] The term "heteroaryloxy" embraces a heteroaryl radical attached through an oxygen atom to the parent molecular scaffold (e.g., heteroaryl-O-scaffold).

[0144] The term "heteroarylcarbonyl" embraces a heteroaryl radical attached to a molecular scaffold through a carbonyl radical (e.g., heteroaryl-carbonyl-scaffold).

[0145] The term "haloheteroarylcarbonyl" embraces a halo-substituted heteroaryl radical attached to a molecular scaffold through a carbonyl radical (e.g., haloheteroaryl-carbonyl- scaffold).

[0146] The term "alkylamino" embraces an alkyl radical attached to a molecular scaffold through an amino radical (e.g., alkyl-NH-scaffold). Specific non-limiting examples of alkylamino include N,N-dimethylamino-scaffold and N-methylamino- scaffold.

[0147] The term "aminoalkyl" embraces an amino radical attached to a molecular scaffold through an alkyl radical (e.g., NH ₂ -alkyl-scaffold).

[0148] The term "aminoaryl" embraces an amino substituted aryl radical.

[0149] The term "aminoarylcarbonyl" embraces an aminoaryl radical attached to a molecular scaffold through a carbonyl radical (e.g., NH ₂ -aryl-carbonyl-scaffold).

[0150] The term "aminocarbonyl" embraces an amino radical attached to a carbonyl radical.

[0151] The term "arylaminocarbonyl" embraces an aryl radical attached to a molecular scaffold through an aminocarbonyl radical.

[0152] The term "aralkoxy" embraces an arylalkyl radical attached through an oxygen atom to the parent molecular scaffold. The terms "arylalkoxy" and "aralkoxy" may be used interchangeably.

[0153] The term "aralkoxycarbonyl" embraces an aralkoxy radical attached to a carbonyl radical.

[0154] The term "heteroaralkoxycarbonyl" embraces a heteroaralkoxy radical attached to a molecular scaffold through a carbonyl radical.

[0155] The term "heteroaralkylcarbonyl" embraces a heteroaralkyl radical attached to a molecular scaffold through a carbonyl radical. [0156] The term "aryloxy" is RO-, where R is aryl.

[0157] The term "arylthio" is RS-, where R is aryl.

[0158] The term "alkylthio" is RS-, where R is alkyl (e.g., alkyl-S-scaffold).

[0159] The term "haloalkylthio" is RS-, where R is halo-substituted alkyl (e.g., haloalkyl-S- scaffold).

[0160] The term "thiolalkyl" is HSR-, where R is alkyl (e.g., HS-alkyl-scaffold).

[0161] The term "aryloxyalkyl" embraces an aryloxy radical attached to an alkyl radical.

[0162] The term "sulfonyl" is -S0 ₂ -.

[0163] The term "alkylsulfonyl" embraces an alkyl radical attached to a sulfonyl radical, where alkyl is defined as above.

[0164] The term "arylsulfonyl" embraces an aryl radical attached to a sulfonyl radical.

[0165] The term "heteroarylsulfonyl" embraces a heteroaryl radical attached to a sulfonyl radical.

[0166] The term "alkylsulfonylalkyl", embraces an alkylsulfonyl radical attached to an alkyl radical, where alkyl is defined as above.

[0167] The term "haloalkylsulfonyl" embraces a haloalkyl radical attached to a sulfonyl radical, where haloalkyl is defined as above.

[0168] The term "pentafluorosulfanyl" denotes a sulfur moiety substituted with five fluoro radicals (i.e., -SF ₅ ).

[0169] The term "sulfonamide" denotes sulfonyl attached to an amino radical. For example: NH ₂ S0 ₂ - and -NHS0 ₂ -. Sulfonamide may be used interchangeably with sulfamyl, sulfonamido and amino sulfonyl.

[0170] The term "guanidino" denotes and may be used interchangeably with guanido.

[0171] The term "diazeniumdiolate" denotes an anion containing N=N linkage ("diazen") which has a formal positive charge ("ium") and which bridges two negatively charged oxygen atoms

("diolate") (e.g., ). Diazeniumdiolate can act as a linker by forming a bond through N+

N=N-0 to giveV ^{' 0} . This bonding configuration is denoted as "diazen-l-ium-l,2-diolate."

[0172] When diazeniumdiolate is attached to nitrogen at position "1", the resulting radical is

termed "NONOate" (e.g., ~L ). Species-compounds containing NONOate chemical formula, are denoted as "NONOates."

term "nitrooxy" denotes

"nitrooxyalkylenyl" embraces a nitrooxy radical attached to an alkylenyl radical

is

equivalent to ; and ~J~ is equivalent to ~1~

The term "oxygen-bound" denotes NONOate bound to a parent molecular scaffold through

oxygen. For example; _; wherein R and R are independently selected from the group consisting of H, C C ₄ alkylenyl, cycloalkyl, cycloalkenyl, aryl, aralkyl, and heteroaryl, optionally substituted from the group consisting of H, hydroxy, hydroxyalkyl, halo, amino, aryl amino, dialkyl amino, aralkyl amino, alkyl ether, aryl ether, heteroaryl ether, and alkyl amido; or taken together, R ⁶ and R ⁷ may form a 4, 5, 6 or 7-membered heterocycle wherein said

heterocycle may be optionally substituted from the group consisting of H, hydroxy,

hydroxyalkyl, halo, amino, aryl amino, dialkyl amino, aralkyl amino, alkyl ether, aryl ether, heteroaryl ether, and alkyl amido. More specific examples include R ⁶ taken together with R ⁶ to form five or six-membered heterocyclyl. Non-limiting examples of five or six-membered

heterocyclyl formed when R 6 is taken together with R 7 include,

[0176] The term "alkylenyloxyalkylenyl" is ^alkylene ^/ ^alkylene^ _where aikylenyl is defined as above.

[0177] The term "alkylenyloxycarbonyl" is ₅ where aikylenyl is defined as above.

[0178] The term "succinyl" denotes .

[0179] The term "imine" denotes a compound containing the structure >C=N-.

Represents point of attachment to molecular

[0180] Scaffold

[0181] The term "coxib" characterizes any member of a class of nonsteroidal anti-inflammatory drugs that causes fewer gastrointestinal side effects by selective inhibition of prostaglandin formation. The terms "coxib" and "selective COX-2 inhibitor" may be used interchangeably.

[0182] The term "pharmaceutically-acceptable salt" refers to a salt which may enhance desired pharmacological activity or may enhance stability of a compound. Examples of pharmaceutically-acceptable salts include acid addition salts formed with inorganic or organic acids, metal salts, and amine salts. Examples of acid addition salts formed with inorganic acids include salts with hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, nitric acid, and phosphoric acid. Examples of acid addition salts formed with organic acids include acetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, citric acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, o-(4-hydroxy-benzoyl)-benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2- hydroxyethane-sulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, 2- naphthalenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methyl- bicyclo[2.2.2]oct-2-enel-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis(3-hydroxy-2- naphthoic) acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acids, salicylic acid, stearic acid, and muconic acid. Examples of metal salts include salts with sodium, potassium, calcium, magnesium, aluminum, iron, barium, bismuth, lithium, and zinc ions. Examples of amine salts include salts with ammonia, arecoline, arginine, benethamine, benzathamine, betaine, chloroprocaine, choline, clemizole, cytosine, deanol, diethanolamine, diethylamine, diethylamine, diethylaminoethanol, epolamine, ethanolamine, ethylenediamine, guanine, imidazole, lysine, meglumine, morpholineethanol, niacinamide, piperazine, procaine, pyridoxine, ie/ -butlamine (erbumine), thiamine, thymine, trolamine, tromethamine, and uracil.

[0183] The term "therapeutically-effective amount" refers to an amount of a compound that, when administered to a subject for treating a disease, is sufficient to effect treatment for the disease. "Therapeutically effective amount" can vary depending on the compound, the disease and its severity, the age, the weight, etc. of the subject to be treated.

[0184] The term "solvate" denotes a molecular or ionic complex of molecules or ions of solvent with those of a compound of the present invention. The term "solvate" embraces the term "hydrate".

[0185] The term "hydrate" denotes a compound of the present invention containing water combined in the molecular form.

[0186] Some of the compounds described contain one or more stereocenters and are meant to include R, S and mixtures of R and S forms for each stereocenter present.

[0187] The term "NO-releasing" means releasing, liberating or generating nitric oxide (NO).

[0188] The term "patient" refers to both humans and non-human animals afflicted with the conditions described herein. Non-human animals could be companion animals such as, but not limited to, canine and feline species.

[0189] The terms "patient" and "subject" are meant to be interchangeable. [0190] The term "subject" refers to suitable subjects for the methods described herein, which include mammalian subjects. Mammals according to the present invention include, but are not limited to, human, canine, feline, bovine, caprine, equine, ovine, porcine, rodents, lagomorphs, primates, and the like and encompass mammals in utero. Subjects may be of either gender and at any stage of development.

[0191] The term "chromene" refers to a compound with a 6-carbon aromatic ring fused to a six- membered heterocyclic pyran ring of the structure:

[0192] The term "chromene" is intended to embrace compounds with substitution by any substituent at any point on the structure above (denoted by "R" groups). The term "chromene" can also refer to a compound which contains a radical of the chromene structure above. The term "benzopyran" is intended to be interchangeable.

[0193] The term "conjugate" refers to a compound formed by the covalent joining of two or more chemical moieties or entities.

[0194] List of Suitable Protecting Groups and Abbreviations:

Acetyl (Ac)

Acylals

Benzoyl (Bz)

Benzyl (Bn, Bnl)

Benzyl esters

Carbamate

Carbobenzyloxy (Cbz)

Dimethoxytrityl, [bi5 ^, -(4-methoxyphenyl)phenylmethyl] (DMT)

Dithianes

Ethoxyethyl ethers (EE)

Methoxymethyl ether (MOM)

Methoxytrityl [(4-methoxyphenyl)diphenylmethyl], (MMT) Methyl Ethers

Methyl (Me)

Methyl esters

Methylthiomethyl ether

Orthoesters

Oxazoline

Pivaloyl (Piv)

Phthalimido

/7-Methoxybenzyl carbonyl (Moz or MeOZ)

/7-Methoxybenzyl (PMB)

Propargyl alcohols

Silyl groups (e.g., trimethylsilyl (TMS), ieri-butyldimethylsilyl (TBDMS), propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS))

Silyl esters

ieri-Butyl esters

ieri-Butyloxycarbonyl (BOC or tBOC)

Tetrahydropyranyl (THP)

Tosyl (Ts or Tos)

Trimethylsilylethoxymethyl (SEM)

Trityl (triphenylmethyl, Tr)

β-Methoxyethoxymethyl ether (MEM)

(4-nitrophenyl)sulfonyl or (4-nitrophenyl)(dioxido)-lambda(6)-sulfanyl) (Nosyl) 2-cyanoethyl

2-nitrophenylsulfenyl (Nps)

3,4-Dimethoxybenzyl (DMPM)

9-Fluorenylmethyloxycarbonyl (FMOC)

[0195] List of abbreviations:

ACN acetonitrile

DCC dicyclohexylcarbodiimide

DCI dicyclohexylcarbodiimide

DCM dichloromethane or methylenechloride diisopropylethylamine

4-dimethylaminopyridine or N,N-dimethylaminopyridine

DME 1 ,2-dimethoxyethane

DMF N,N-dimethylformamide

DMSO dimethylsulfoxide

eq. equivalents

EtOAC ethyl acetate

EtOH ethanol

Fmoc fluorenylmethyloxycarbonyl chloride

HPLC high performance liquid chromatography

h hour

K ₂ C0 ₃ potassium carbonate

LC/MS liquid chromatography mass spectrometry

LC/MS/MS liquid chromatography tandem mass spectrometry

MeOH methanol

MgS0 ₄ magnesium sulfate

min. minute(s)

mL milliliter

mmol millimole

Na ₂ S ₂ 0 ₃ sodium thiosulfate

Na ₂ S0 ₄ sodium sulfate

NaH sodium hydride

NaHC0 ₃ sodium bicarbonate

Nal sodium iodide

NaI0 ₄ sodium periodate

NaOCH ₃ sodium methoxide

NBS N-bromo succinimide

NIS N-iodo succinimide

NMR nuclear magnetic resonance

NO nitric oxide

psi pounds per square inch PyBOP benzotriazole-l-yl-oxy-tris-(dimethylamino)-phosphonium

hexafhioropho sphate

RuCl ₃ ruthenium trichloride hydrate

TEA triethylamine

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

TSA /7-toluenesulfonic acid

D. General Synthetic Schemes

[0196] Compounds of the present invention can be prepared using methods illustrated in general synthetic schemes and experimental procedures detailed below. These general synthetic schemes and experimental procedures are presented for purposes of illustration and are not intended to be limiting. Starting materials used to prepare compounds of the present invention are commercially available or can be prepared using routine methods known in the art.

[0197] Haloalkyl Chromene Esters: In step 1, chromene acids are made by reaction of salicylic aldehydes (made from corresponding phenols) with ethyl 4,4,4-trifluorocrotonate (Scheme 1) according to procedures described in literature (i.e., U.S. Pat. 6,034,256) for when Z = CF ₃ , or with ethyl 4,4,5,5,5-pentafluorobut-2-enoate (CAS# [37759-78-7]) for when Z = CF ₂ CF ₃ . Alternatively, chiral chromene acids, where Z = CF ₃ , are made by reaction of salicylic aldehydes with 4,4,4-trifluorocrotonaldehyde and chiral catalyst followed by oxidation (Scheme 2) according to procedures described in ACS Med. Chem. Lett. 2014, 5, 1162-1166. Chiral chromene acids where Z = CF ₂ CF ₃ are made by an analogous approach using 4,4,5,5,5- pentafluoropent-2-enal, which is made from 4,4,5,5, 5-pentafluoropent-2-en-l-ol using the same procedure to make 4,4,4-trifluorocrotonaldehyde (INT-07) outlined below. Chromene acids are converted to haloalkyl esters (Scheme 2) by standard methods as described in Syn. Comm., 1984, 14(9), 857-864 using reagents such as chloromethyl chlorosulfate and chloroethyl chlorosulfate in inert solvent (e.g., dichloromethane, chloroform, etc.). Scheme 1

Scheme 2 - Step 1

Scheme 2 - Step 2

Scheme 3

Z is CF ₃ or CF ₂ CF ₃ ;

R ¹ = H or methyl;

R = CI or Br or methyl or trifmoromethoxy or pentafluorosulfanyl;

R = H or methyl or tert-butyl;

R ⁴ = H or CI or methyl or ethyl; and

R ⁵ = H or methyl.

[0198] Conversion of N-Boc-protected amino alcohols to N-alkylamino alcohols: (S)-2-Boc- alaninol, (^-l-Boc-leucinol, (S)-2-(Boc-amino)-3-phenyl-l-propanol, (^- -Boc-S-methyl-l- butanol and ( _l S')-2-Boc-isoleucinol are converted to ( _l S')-2-N-methyl-alaninol, ( _l S')-2-N-methyl- leucinol, ( _l S')-2-(N-methylamino)-3-phenyl-l-propanol, ( _l S')-2-N-methyl-3-methyl-l-butanol and (5 ^, )-2-N-methyl-isoleucinol, respectively, using established procedures (See also Reddy, G.V., Tetrahedron Lett. 2000, 41, 949-951). A N-protected amino alcohol (1.0 eq.), paraformaldehyde (3 eq.) and p-toluenesulfonic acid (0.1 eq.) are dissolved in toluene and refluxed for 30 min using a Dean-Stark trap. The solvent is evaporated under reduced pressure to give a crude residue that is purified by silica gel column chromatography to afford pure N-protected oxazolines. The N- protected oxazoline (1 eq.) is dissolved in dry acetonitrile under nitrogen atmosphere and treated with sodium cyanoborohydride (1 eq.) followed by trimethylsilyl chloride (1 eq.). The reaction mixture is stirred at room temperature for 30 min and the solvent is evaporated under reduced pressure. The residue is suspended in ethyl acetate and washed with a solution of 0.5 N sodium hydroxide. The organic layer is separated and the aqueous layer is extracted again with ethyl acetate. The combined organic layers are washed with brine, dried over sodium sulfate, filtered and evaporated under reduced pressure. The residue is purified by silica gel column chromatography to afford pure Boc-N-methyl amino alcohols. Boc-Deprotection is accomplished using standard conditions stirring at room temperature in 5: l-methylene chloride (methylene chloride)/trifluoroacetic acid (TFA)(v/v). After 4h the reaction is evaporated, the residue is dissolved in ethyl acetate and washed with saturated sodium bicarbonate solution. The organic layer is separated and the aqueous layer is extracted again with ethyl acetate. The organic layers are pooled, dried over sodium sulfate, filtered to remove solids and evaporated to dryness resulting in N-methylamino alcohols. In addition to paraformaldehyde, numerous aldehydes, such as paraldehyde, benzaldehyde, isobutyraldehyde, trimethylacetaldehyde, 2- methylbutyraldehyde, 2-ethylbutyraldehyde, etc., are commercially available and are used in this reaction to generate starting materials (See Table 3).

[0199] Sodium NONOate (SNO) Salts (See also, Velazquez, C.A., J. Med. Chem. 2008, 51, 1954-1961): N-Alkylamino alcohols (See Table 3) (10 g, 0.13 mol) are added to a solution of sodium methoxide (7.2 g, 0.13 mol, 30.5 mL of a 25% w/v solution in methanol) and ether (150 mL) with stirring at 25 °C. The flask is evacuated and then charged with nitric oxide (NO) (40 psi internal pressure) with stirring at 25 °C for 72 h. The product is isolated by filtration and then suspended in ether (100 mL) with stirring for 15 min. The suspension is filtered, collected and dried under reduced pressure until a constant weight is achieved. Starting materials and corresponding sodium NONOate salts are listed in Table 3.

[0200] Chloroalkyl NONOates (See also, Tang, X., Tett. Lett, 2001, 42, 2625-2629): In step 1, sodium NONOate salts and an inorganic base (e.g., sodium carbonate, potassium carbonate, etc.) are suspended in solvent (e.g., tetrahydrofuran, acetonitrile, N,N-dimethylformamide, etc.). The reaction is cooled to 0 °C and chloroalkyl methylsulfide is added drop-wise, the ice bath is removed, a catalytic amount of potassium iodide is added and the reaction is stirred overnight to afford methylthio alkyl ethers (Scheme 4). In step 2, ethers are treated with sulfuryl chloride at room temperature in inert solvent (e.g., dichloromethane, chloroform, etc.).

Scheme 4 - Step 1

Scheme 4 - Step 2

R H or methyl;

R ⁶ is selected from the group consisting of methyl, ethyl, isopropyl, tert-butyl, CrC ₆

hydroxyalkylenyl, and CrC ₆ carboxyalkylenyl; and

R is selected from the group consisting of C -C hydroxyalkylenyl and C -C carboxyalkylenyl optionally substituted from the group consisting of alkyl, aryl, and aralkyl; or

taken together, R ⁶ and R ⁷ may form an azetidinyl moiety or a pyrrolidinyl moiety or a piperidinyl moiety, wherein said azetidinyl moiety or pyrrolidinyl moiety or piperidinyl moiety may be optionally substituted from the group consisting of alkyl, aryl, aralkyl, hydroxy, hydroxyl C C ₄ alkylenyl, carboxy, and carboxy C C ₄ alkylenyl.

[0201] O-Linked Chromene NONOates (See also, WO 2006/125016; WO 2007/127725; WO 2011/116336; WO 2007;044963): Procedure A (Scheme 5) - Haloalkyl chromene esters are reacted directly with sodium NONOate salts in polar solvents (e.g., acetonitrile, DMSO, DMF, NMP, etc.) at room temperature overnight. Procedure B (Scheme 6) - Chromene acids are reacted directly with chloroalkyl NONOates in polar solvents (e.g., acetonitrile, DMSO, DMF, NMP, etc.). Postassium iodide may be added to improve the reaction.

heme 5

heme 6

R ⁶ is selected from the group consisting of methyl, ethyl, isopropyl, tert-butyl, CrC ₆

hydroxyalkylenyl, and C -C carboxyalkylenyl; and

R is selected from the group consisting of Ci-Ce hydroxyalkylenyl and Ci-Ce carboxyalkylenyl optionally substituted from the group consisting of alkyl, aryl, and aralkyl; or

taken together, R ⁶ and R ⁷ may form an azetidinyl moiety or a pyrrolidinyl moiety or a piperidinyl moiety, wherein said azetidinyl moiety or pyrrolidinyl moiety or piperidinyl moiety may be optionally substituted from the group consisting of alkyl, aryl, aralkyl, hydroxy, hydroxyl CrC ₄ alkylenyl, carboxy, and carboxy CrC ₄ alkylenyl.

[0202] O-Linked Chromene NONOate Carboxylates (See also, Chakrapani, H., Org. Lett. 2008, 10, 5155-5158): O-Linked chromene NONOates are treated with sodium metaperiodate and ruthenium (III) chloride (catalytic) in acetonitrile, ethyl acetate and water to afford carboxylic acids that can be made into ammonium or inorganic salts (Scheme 7).

Scheme 7

Z = CF ₃ or CF ₂ CF ₃ ;

R ¹ = H or methyl;

R = CI or Br or methyl or trifhioromethoxy or pentafhiorosulfanyl;

R = H or methyl or tert-butyl;

R ⁴ = H or CI or methyl or ethyl;

R ⁵ = H or methyl; and R ⁶ is selected from the group consisting of methyl, ethyl, isopropyl, ieri-butyl, C]_-C hydroxyalkylenyl, and CrC ₆ carboxyalkylenyl.

CHROMENE ACID

[0203] 6,8-Dichloro-2-(trifluoromethyl)-2H-chromene-3-carboxylic acid (CA-Ola)

[0204] Stepl : 3,5-Dichloro-2-hydroxybenzaldehyde (INT-01)

[0205] Chromene acids are made starting from phenols via salicylic aldehydes. 2,4- Dichlorophenol (10.0 g, 61.3 mmol) and hexamethylenetetramine (17.2 g, 122.6 mmol) were dissolved in 80 mL methanesulfonic acid and heated at 100 C for 1.5 h. The reaction was diluted with ethyl acetate and the organic layer was washed with water, followed by saturated sodium bicarbonate, dried over magnesium sulfate, and evaporated. The product 3,5-dichloro-2- hydroxybenzaldehyde (INT-01) was purified by chromatography using ethyl acetate/hexane gradient to give a yellow oil (7.5 g, 64% yield). 1H NMR (400 MHz, CDC1 ₃ ) δ 11.39 (s, 1H), 7.64 (s, 1H), 7.52 (s, 1H). LC t _r = 3.95 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.7 mL/min with detection 254 nm, at 23 C).

[0206] Step 2: Ethyl 6,8-dichloro-2-(trifluoromethyl)-2H-chromene-3-carboxylate (INT-02)

[0207] 3,5-Dichloro-2-hydroxybenzaldehyde (INT-01) (7.5 g, 39.3 mmol) was dissolved in 15 mL of dimethylsulf oxide. Ethyl 4,4,4-trifluorocrotonate (9.38 mL, 62.8 mmol) and triethylamine (11.0 mL, 78.6 mmol) were added, and heated to 85 C for 3 days. The reaction was cooled, diluted with water, and extracted with ethyl acetate. The organic layer was washed with 3N hydrochloric acid solution, saturated sodium bicarbonate solution and brine, dried over magnesium sulfate, and evaporated to give a tan solid INT-02 (12.5 g, 93% yield). 1H NMR (400 MHz, CDC1 ₃ ) δ 7.66 (s, 1H), 7.40 (d, J=2.4 Hz, 1H), 7.16 (d, J= 2.4 Hz, 1H), 5.84 (q, J=6.6 Hz, 1H), 4.43-4.40 (m, 2H), 1.38 (t, J=7.1 Hz, 3H). LC t _r = 5.20 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.7 mL/min with detection 254 nm, at 23 C). ES(pos)MS m/z 341 (M+H calcd for CnHgCliFaCh requires 341).

[0208] Step 3: 6,8-Dichloro-2-(trifluoromethyl)-2H-chromene-3-carboxylic acid (CA-Ola)

[0209] INT-02 (12.5 g, 36.6 mmol) was dissolved in 15 mL methanol and 1.5 mL water. Solid sodium hydroxide (4.47 g, 111.8 mmol) was added, and the reaction was stirred at room temperature overnight. The reaction was acidified with IN hydrochloric acid solution and the resulting precipitate was filtered, washed with water and hexane, and dried to a tan solid CA-Ola (10.2 g, 89% yield). 1H NMR (400 MHz, CDC1 ₃ ) δ 7.66 (s, 1H), 7.40 (d, J=2.4 Hz, 1H), 7.16 (d, J= 2.4 Hz, 1H), 5.84 (q, J=6.6 Hz, 1H), 4.43-4.40 (m, 2H), 1.38 (t, J=7.1 Hz, 3H). LC t _r = 4.33 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.7 mL/min with detection 254 nm, at 23 ^° C). ES(pos)MS m/z 313 (M+H calcd for C11H5CI2F3O3 requires 313).

[0210] ( _l S ^, )-7-(ieri-Butyl)-6-chloro-2-(trifluoromethyl)-2H-chrom ene-3-carboxylic acid (CA-11)

[0211] Step 1: 3-ieri-Butyl-4-chlorophe -03)

[0212] 3-ie/ -Butyl phenol (50.0 g, 333 mmol) and ferric chloride (162.6 mg, 1.0 mmol) were heated to 60 C in dichloromethane. Sulfuryl chloride (35.0 mL, 433 mmol) was added drop- wise and the mixture was heated at 60 C overnight. The reaction was evaporated and dissolved in ethyl acetate. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated. The crude product INT-03 (68.4 g, 111% yield) was taken directly into the next step. 1H NMR (400 MHz, CDC1 ₃ ) δ 7.21 (d, J=8.5Hz, 1H), 6.93 (d, J=3.0Hz, 1H), 6.63 (dd, J=8.5, 3.0Hz, 1H), 4.79 (s, 1H), 1.48 (s, 9H). LC t _r = 4.18 min (C- 18 column, 5 to 95% acetonitrile/water over 6 min at 1.3 mL/min with detection 254 nm, at 23 C).

[0213] Step 2: 4-i<?/ -Butyl-5-chloro-2-hydroxybenzaldehyde (INT-04)

[0214] INT-03 (68.4 g, 370.4 mmol) was dissolved in methanesulfonic acid (200 mL) and the reaction was cooled to 0 C. Hexamethylene tetraamine (103.9 g, 740.8 mmol) was added, followed by methane sulfonic acid (200 mL) added portion-wise, keeping the exothermic reaction below 100 C. The reaction was then stirred at 100 C overnight then cooled to room temperature and poured into cold water (3 L). The product was extracted with ethyl acetate and the organic layer was washed with water, saturated sodium bicarbonate and brine, dried over magnesium sulfate and evaporated to afford INT-04 as a dark oil (58.8 g, 75% yield). 1H NMR (400 MHz, CDCI ₃ ) δ 9.84 (s, 1H), 7.53 (s, 1H), 7.12 (s, 1H), 1.51 (s, 9H). LC t _r = 4.66 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.3 mL/min with detection 254 nm, at 23 C). ES(pos)MS m/z 213 (M+H calcd for CnH ₁₃ C10 ₂ requires 213).

[0215] Step 3: Ethyl 7-(ieri-butyl)-6-chloro-2-(trifluoromethyl)-2H-chromene-3-ca rboxylate (INT-05)

[0216] INT-04 (58.8g, 276 mmol), ethyl 4,4,4-trifluorocrotonate (45.4 mL, 304 mmol) and potassium carbonate (49.6 g, 359 mmol) were heated in DMSO (175 mL) to 85 C. After 3 h, the reaction was diluted with water and extracted with ethyl acetate. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated to afford INT-05 as a dark oil (62.8 g, 63% yield). 1H NMR (400 MHz, CDC1 ₃ ) δ 7.65 (s, 1H), 7.22 (s, 1H), 7.06 (s, 1H), 5.71 (q, J=6.8Hz, 1H), 4.39-4.30 (m, 2H), 1.49 (s, 9H), 1.38 (t, J=7.1Hz, 3H). LC t _r = 5.76 min (C- 18 column, 5 to 95% acetonitrile/water over 6 min at 1.3 mL/min with detection 254 nm, at 23 ^° C). ES(pos)MS m/z 363 (M+H calcd for C ₁₇ H ₁₈ C1F ₃ 0 ₃ requires 363). [0217] Step 4: 7-(ieri-Butyl)-6-chloro-2-(trifluoromethyl)-2H-chromene-3-ca rboxylic acid (CA- 11a).

[0218] INT-05 (62.8 g, 173 mmol) was dissolved in methanol (1.25 L) and sodium hydroxide (22.7 g, 568 mmol) in water (125 mL) was added. The reaction was stirred at room temperature overnight. The methanol was evaporated and the resulting aqueous layer was washed with diethyl ether, acidified with 3N aqueous hydrochloric acid, then extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and evaporated to a dark orange, oily solid. The solid was broken up and washed with a minimal amount of methylene chloride to remove the colored impurities to afford CA-lla as an off-white solid (34.5 g, 59% yield). 1H NMR (400 MHz, CDC1 ₃ ) δ 7.85 (s, 1H), 7.60 (s, 1H), 7.05 (s, 1H), 5.95 (q, J=7.3Hz, 1H), 1.43 (s, 9H). LC t _r = 4.87 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.3 mL/min with detection 254 nm, at 23 ^° C). ES(pos)MS m/z 335 (M+H calcd for C ₁₅ Hi ₄ ClF ₃ 0 ₃ requires 335).

[0219] Step 5: ( _l S')-7-(ieri-Butyl)-6-chloro-2-(trifluoromethyl)-2H-chr omene-3-carboxylic acid (CA-11).

[0220] Preparative enantiomer separation was conducted on a Thar 350 preparative SFC using a ChiralPak AD column (10μ, 300x50mmI.D.; Mobile phase A: C0 ₂ and Mobile phase B: Isopropanol; Gradient: B 25% ; Flow rate: 200 mL/min; Back pressure: lOObar; Column temperature: 38 C; Wavelength: 220nm). Samples were dissolved in methanol at ~45mg/mL and injected in 3-mL portions. Compound CA-lla (20.0 g) underwent chiral chromatography to yield each enantiomer. The initial peak off the column is the (R)-isomer (8.44 g; Chiral HPLC:

AD (n-hexane/i-PrOH 9: 1, λ = 254nm), t _R = 4.28min, 93.8%ee), and the second peak off the column is the (S)-isomer CA-11 (8.16g; Chiral HPLC: AD (n-hexane/i-PrOH 9: 1, λ = 254 nm), t _r = 5.87 min, 90.4%ee). [0221] (^-G-Pentafluorosulfanyl-l-itrifluoromethy^-lH-chromene-S-ca rboxylic acid (CA-08)

[0222] Step 1. 2-Hydroxy-5-(pentafluorosulfanyl)benzaldehyde (INT-06)

OH

[0223] Pentafluorosulfanyl phenol (10.0 mmol) and hexamethylenetetramine (20.0 mmol) are dissolved in methanesulfonic acid (15 mL) and heated to 100 C for 1.5 h. The reaction is diluted with ethyl acetate and the organic layer is washed with water, saturated sodium bicarbonate, dried over magnesium sulfate, and evaporated. The product is purified by silica gel column chromatography using ethyl acetate/hexane gradient to give INT-06.

[0224] Step 2: 4,4,4-Trifluorobut-2-en -07)

[0225] 4,4,4-Trifluorobut-2-en-l-ol (3.28 g, 26.0 mmol) was dissolved in dichloromethane (80 mL). Pyridinium chlorochromate (5.61 g, 26.0 mmol) was added and the reaction was stirred at room temperature overnight. The dark red reaction mixture was filtered through Celite and decolorizing carbon. The dark brown/green filtrate containing about 25 mmol of INT-07 in -80 mL of dichloromethane (0.31 M) was used directly in the next step.

[0226] Step 3: ( _l S ^, )-6-(Pentafluorosulfanyl)-2-(trifluoromethyl)-2H-chrom ene-3-carbaldehyde (INT-08)

[0227] To a solution of 4,4,4-trifluorobut-2-enal (INT-07; trifluorocrotonaldehyde) in dichloromethane (-0.31 M, -20 mmol, 66 mL) is added INT-06 (10.0 mmol), (5 (-)-α,α- diphenyl-2-pyrrolidine methanol trimethylsilyl ether (2.0 mmol) and 2-nitrobenzoic acid (2.0 mmol). The reaction is stirred at room temperature overnight, concentrated, and purified by silica gel column chromatography (0-15% ethyl acetate -hexane gradient) to afford (INT-08).

[0228] Step 4: ( _l S ^, )-6-pentafluorosulfanyl-2-(trifluoromethyl)-2H-chromen e-3-carboxylic acid (CA-08)

[0229] INT-08 (5.0 mmol) is dissolved in DMF (20 mL) and oxone (monopersulfate) (7.10 mmol) is added. The reaction is stirred at room temperature for 48 h, diluted with water, and extracted with ethyl acetate. The ethyl acetate layer is dried over anhydrous magnesium sulfate and concentrated in vacuo. The crude product is purified by silica gel column chromatography to yield (CA-08).

[0230] ( _l S ^, )-6-Pentafluorosulfanyl-8-methyl-2-(trifluoromethyl)-2 H-chromene-3-carboxylic acid (CA-09)

[0232] INT-06 (20 mmol) is dissolved in tetrahydrofuran (40 mL) and cooled to 0 °C. To the mixture is added water (16 mL) followed by sodium borohydride (20 mmol) and the reaction is warmed to room temperature and stirred for 48 h. The reaction is diluted with IN aqueous hydrochloric acid to adjust the pH to 6 and extracted with diethyl ether. The organic layer is washed with brine, dried over magnesium sulfate and evaporated. The crude product is purified by silica gel column chromatography (ethyl acetate/hexane gradient) to obtain INT-09. [0233] Step 2: 2-Hydroxy-3-methyl-5-(pentafluorosulfanyl)benzaldehyde (INT-10)

[0234] INT-09 (10.0 mmol) and hexamethylenetetramine (20.0 mmol) are dissolved in methanesulfonic acid (15 mL) and heated to 100 C for 1.5 h. The reaction is diluted with ethyl acetate and the organic layer is washed with water, saturated sodium bicarbonate, dried over magnesium sulfate, and evaporated. The product is purified by silica gel column chromatography using ethyl acetate/hexane gradient to give INT-10.

[0235] Steps 4 & 5: ( _l S ^, )-6-Pentafluorosulfanyl-8-methyl-2-(trifluoromethyl)-2 H-chromene-3- carboxylic acid (CA-09)

[0236] Using a similar procedure to make CA-08, INT-10 is reacted with INT-07 to give the corresponding chromene aldehyde and subsequent oxidation with oxone provides CA-09.

[0237] ( _l S')-6-Pentafluorosulfanyl-8-ethyl-2-(trifluoromethyl)- 2H-chromene-3-carboxylic acid (CA-10)

[0238] Step 1 : 2-(l-Hydroxyethyl)- enol (INT-11)

[0239] INT-06 (20 mmol) is dissolved in tetrahydrofuran (40 mL) and the mixture is cooled to 0 °C. Methylmagnesium bromide (3.0 M; 20 mmol) in diethyl ether is added drop-wise to the cold solution. The reaction is monitored by TLC and upon completion; the mixture is poured into water and extracted with ethyl acetate. The organic layer is washed with saturated sodium bicarbonate, dried over magnesium sulfate, and evaporated. The product is purified by silica gel column chromatography using ethyl acetate/hexane gradient to give INT-11. [0240] Step 2: 2-Ethyl-4-(pentafluorosulfanyl)phenol (INT-12)

[0241] INT-11 (15 mmol) is dissolved in tetrahydrofuran (30 mL) and cooled to 0 °C. To the mixture is added water (12 mL) followed by sodium borohydride (15 mmol) and the reaction is warmed to room temperature and stirred for 48 h. The reaction is diluted with IN aqueous hydrochloric acid to adjust the pH to 6 and extracted with diethyl ether. The organic layer is washed with brine, dried over magnesium sulfate and evaporated. The crude product is purified by silica gel column chromatography (ethyl acetate/hexane gradient) to obtain INT-12.

[0242] Step 3: 3-Ethyl-2-hydroxy-5-(pentafluorosulfanyl)benzaldehyde (INT-13)

[0243] INT-12 (10.0 mmol) and hexamethylenetetramine (20.0 mmol) are dissolved in methanesulfonic acid (15 mL) and heated to 100 C for 1.5 h. The reaction is diluted with ethyl acetate and the organic layer is washed with water, saturated sodium bicarbonate, dried over magnesium sulfate, and evaporated. The product is purified by silica gel column chromatography using ethyl acetate/hexane gradient to give INT-13.

[0244] Steps 4 & 5: (^-e-Pentafluorosulfanyl-S-ethyl^-itrifluoromethy^^H-chromen e-S- carboxylic acid (CA-10)

[0245] Using a similar procedure to make CA-08, INT-13 is reacted with INT-07 to give the corresponding chromene aldehyde and subsequent oxidation with oxone provides CA-10.

[0246] Using a similar procedure to make CA-Ola and CA-11 additional chromene acids (Table 1) are made using procedures described in U.S. Pat. 6,034,256. [0247] Table 1. Chromene acids.

CA-11 chromene-3-carboxylic acid

HALOALKYL CHROMENE ESTERS

[0248] Iodomethyl 6,8-dichloro-2-(trifluoromethyl)-2H-chromene-3-carboxylate (C-Ola)

[0249] Step 1: Chloromethyl 6,8-dichloro-2-(trifluoromethyl)-2H-chromene-3-carboxylate (C- 02a)

[0250] Chromene acids are converted to chloromethyl esters as described in Syn. Comm., 1984, 14(9), 857-864. Compound CA-Ola (200 mg, 0.64 mmol), tetrabutylammonium hydrogensulfate (22 mg, 0.06 mmol) and sodium bicarbonate (204 mg, 2.43 mmol) were dissolved in 1: 1 dichloromethane- water mixture (1.2 mL). Chloromethyl chlorosulfate (75 μΐ _^ , 0.74 mmol) in 150 μL· of dichloromethane was added drop-wise and stirred at room temperature overnight. The reaction was diluted with ethyl acetate and the organic layer was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate, and evaporated to give C-02a (197 mg, 85% yield). 1H NMR (400 MHz, CDC1 ₃ ) δ 7.77 (s, 1H), 7.44 (d, J=2.5 Hz, 1H), 7.19 (dd, J= 2.5, 0.3 Hz, 1H), 5.91 (abq, J=36.4, 6.2 Hz, 2H), 5.84 (q, J=6.6 Hz, 1H). LC t _r = 5.04 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.7 mL/min with detection 254 nm, at 23 C). [0251] Step 5: Iodomethyl 6,8-dichloro-2-(trifluoromethyl)-2H-chromene-3-carboxylate (C-Ola)

[0252] Compound C-02a (530 mg, 1.69 mmol) and sodium iodide (381 mg, 2.54 mmol) were heated to 60 C in 1.5 mL of acetonitrile for 5h, then stirred at room temperature for 48 h. The reaction was diluted with ethyl acetate. The organic layer was washed with 0.2 M sodium thiosulfate solution and brine, dried over magnesium sulfate, and evaporated to give C-Ola as a light yellow oil (505 mg, 66% yield). 1H NMR (400 MHz, CDC1 ₃ ) δ 7.72 (s, 1H), 7.44 (d, J=2.4 Hz, 1H), 7.19 (d, J= 2.4 Hz, 1H), 6.11 (abq, J=32.7, 5.0 Hz, 2H), 5.82 (q, J=6.6 Hz, 1H). LC t _r =5.21 min (C- 18 column, 5 to 95% acetonitrile/water over 6 min at 1.7 mL/min with detection 254 nm, at 23 ^° C).

[0253] 1-Chloroethyl 6,8-dichloro-2-(trifluoromethyl)-2H-chromene-3-carboxylate (C-03a)

[0254] Compound CA-Ola (0.50 mmol), tetrabutylammonium hydrogensulfate (0.05 mmol) and sodium bicarbonate (2.0 mmol) are dissolved in 1: 1 dichloromethane-water mixture (1.0 mL). 1-

Chloroethyl chlorosulfate (0.60 mmol, prepared as described in U.S. Pat. 2,860,123) in 150 μΐ _^ of dichloromethane is added drop-wise and stirred at room temperature overnight. The reaction is diluted with ethyl acetate and the organic layer is washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate, and evaporated to afford C-03a.

[0255] Using a similar procedure to make C-Ola to C-03a, additional haloalkyl chromene esters

(Table 2) are made by replacing CA-Ola with other suitable chromene acids from Table 1 and known in the literature.

[0256] Table 2. Haloalkyl chromene esters.

Chromene Structure Product Name

C-30 (trifluoromethyl)-2H-chromene-3-carboxylate

SODIUM NONOATES

[0257] (Z)-l-Hydroxy-3-(2-hydroxyethyl)-3-methyltriaz-l-ene 2-oxide sodium salt (N-01)

HO J

I

o-

[0258] N-methylethanolamine (2.0 g, 26.63 mmol) was added to a solution of sodium methoxide (1.44 g, 26.63 mmol, 6.0 mL of a 25% w/v solution in methanol) and ether (30 mL) with stirring at 25 °C. The flask was evacuated and then charged with nitric oxide (NO) (40 psi internal pressure) and stirred at 25 °C for 24 h. The product was isolated by filtration and then suspended in ether (30 mL) and stirred for 15 min. The suspension was filtered, collected and dried at 25 °C under reduced pressure to give (N-01) as a white fine powder (2.72 g, 65% yield). 1H NMR (400 MHz, D ₂ O) δ 3.42-3.40 (m, 2H), 2.95-2.92 (m, 2H), 2.63 (s, 3H).

[0259] ( _l S',Z)-2-Hydroxy-l-(2-(hydroxymethyl)pyrrolidin-l-yl)di azene oxide sodium salt (N-02)

[0260] (S)-(+)-2-Pyrrolidinemethanol (5.0 g, 49.4 mmol) was added to a solution of sodium methoxide (11.3 mL of a 25% w/v solution in MeOH, 49.4 mmol) and ether (50 mL) with stirring at 25 °C. The flask was evacuated and then charged with nitric oxide (NO) (40 psi internal pressure) with stirring at 25 °C for 24 h. The product was isolated by filtration and then suspended in ether (30 mL) with stirring for 15 min. The suspension was filtered, collected, and dried at 25 °C under reduced pressure to give (N-02) as a white fine powder (4.36 g, 48% yield). 1H NMR (400 MHz, d ₆ -DMSO) δ 3.37-3.19 (br m, 4H), 2.97-2.90 (br m, 2H), 1.87-1.50 (m, 4H). LC t _r = 0.90 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.7 mL/min with detection 254 nm, at 23 C).

[0261] (Z)-2-Hydroxy-l-(3-hydroxyazetidin-l-yl)diazene oxide sodium salt (N-03)

[0262] 3-Hydroxyazetidine hydrochloride (5.0 g, 46.1 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of N-01: (8.98 g, >100% yield(contaminated with -25% NaCl)). 1H NMR (400 MHz, D ₂ 0) δ 4.43-4.38 (m, 1H), 4.04- 4.00 (m, 2H), 3.88-3.84 (m, 2H).

[0263] (S,Z)-l-Hydroxy-3-(l-hydroxypropan-2-yl)-3-methyltriaz-l-ene 2-oxide sodium salt (N- 04)

HO N >. .ONa

N N

[0264] A solution of iodine (4.92 g, 19.39 mmol) in tetrahydrofuran (10 mL) was slowly added dropwise to a suspension of /V-methyl-L-alanine (2.00 g, 10.39 mmol) and sodium borohydride (1.83 g, 48.49 mmol) in tetrahydrofuran (50 mL), pre-cooled in an ice-bath. Once the addition was complete the reaction mixture was heated to reflux. After 18 h the reaction was allowed to cool to ambient temperature and then methanol (5 mL) was slowly added. The mixture was stirred at ambient temperature for 30 min and then concentrated under reduced pressure. The residue was dissolved in 20% potassium hydroxide (w/w) (30 mL) and then stirred at ambient temperature for 4 h. The aqueous reaction mixture was extracted with dichloromethane. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to provide a clear oil (2.07 g, 100% yield).

[0265] Crude N-methyl-L-alaninol (19.39 mmol) was added to a solution of sodium methoxide (1.05 g, 19.39 mmol, 4.5 mL of a 25% w/v solution in methanol) and ether (25 mL) with stirring at 25 °C. The flask was evacuated and then charged with nitric oxide (NO) (40 psi internal pressure) with stirring at 25 °C for 2 days. The product was isolated by filtration and the filtrate was concentrated under reduced pressure. The residue was treated with acetonitrile and the precipitate that developed was collected by filtration. This process was repeated a second time and the solids collect were combined with the original product to give an off-white solid N-04 (0.935 g, 28% yield).

[0266] (Z)-3-Ethyl-l-hydroxy-3-(2-hydroxyethyl)triaz-l-ene 2-oxide sodium salt (N-05)

[0267] 2-(Ethylamino) ethanol (5.0 g, 56.1 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of N-01: (9.58 g, 50% yield). 1H NMR (400 MHz, D ₂ 0) δ 3.41 (t, 5.6 Hz, 2H), 2.94 (t, J=5.7 Hz, 2H), 2.84 (q, J=7.1 Hz, 2H), 0.83 (t, 7.1 Hz, 3H).

[0268] (Z)-l-Hydroxy-3-(2-hydroxyethyl)-3-isopropyltriaz-l-ene 2-oxide sodium salt (N-06)

[0269] 2-(Isopropylamino) ethanol (13.0 g, 126 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of N-01: (4.02 g, 17% yield). 1H NMR (400 MHz, D ₂ 0) δ 3.44 (t, 5.8 Hz, 2H), 3.21-3.15 (m, 1H), 3.08 (t, J=5.9 Hz, 2H), 1.00 (d, J=6.3 Hz, 6H).

[0270] (Z)-3-(ieri-Butyl)-l-hydroxy-3-(2-hydroxyethyl)triaz-l-ene 2-oxide sodium salt (N-07) HO „ONa

[0271] 2-(ieri-Butylamino) ethanol (10.0 g, 85.6 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of N-01: (3.07 g, 18% yield). 1H NMR (400 MHz, D ₂ 0) δ 8.38 (s, 1H), 3.41 (t, 5.9 Hz, 2H), 3.12 (t, J=5.9 Hz, 2H), 1.11 (s, 9H).

[0272] (Z)-l-Hydroxy-3-(3-hydroxypropyl)-3-methyltriaz-l-ene 2-oxide sodium salt (N-08)

[0273] N-Methylamino-l-propanol (4.75 g, 53.29 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of N-01: (2.80 g, 31% yield).

[0274] (Z)-l-Hydroxy-3-(6-hydroxyhexyl)-3-methyltriaz-l-ene 2-oxide sodium salt (N-09)

[0275] 6-(Methylamino)hexan-l-ol (1.0 g, 7.62 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of N-01: (1.67 g as colorless oil, 100% yield. 1H NMR (400 MHz, D ₂ 0) δ 3.54-3.48 (m, 2H), 2.84-2.79 (m, 2H), 2.63 (s, 3H), 1.51-1.42 (m, 4H), 1.31-1.21 (m, 4H).

[0276] (Z)-l-(Azetidin-l-yl)-2-hydroxydiazene oxide sodium salt (NS-26)

[0277] Azetidine hydrochloride (25.0 g, 267.2 mmol) was added to a solution of sodium methoxide (61 mL of a 25% w/v solution in methanol, 267.2 mmol) and diethyl ether (75 mL) with stirring at 25 °C. After 5 min, the resulting sodium chloride precipitate was filtered and the filtrate was placed in a pressure vessel. Another aliquot of sodium methoxide (61 mL of a 25% w/v solution in methanol, 267.2 mmol) was added and the flask was evacuated with vacuum and then charged with nitric oxide (NO) (40 psi internal pressure) with stirring at 25 °C for 2 days. Diethyl ether (100 mL) was added to the resulting suspension to fully precipitate the product. The precipitate was isolated by filtration to give NS-26 as a white solid (13.05 g, 35% yield). 1H NMR (400 MHz, D ₂ 0) δ 3.87 (t, J=7.5Hz, 4H), 2.07 (pent, J=7.5Hz, 2H).

[0278] (Z)-2 -Hydroxy- 1 -morpholinodiazene oxide sodium salt (NS-27)

[0279] Morpholine (11.6 g, 133.1 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of N-01: (9.28 g, 44% yield). 1H NMR (400 MHz, D20) 3.83 (br m, 4H), 3.09 (br m, 4H).

[0280] Using a similar procedure to make N-01 to N-09, N-26 & N-27 additional NONOate salts can be made by replacing the N-methylethanolamine in the synthesis of N-01 with other suitable amines listed in Table 3.

[0281] Table 3. Sodium NONOates.

^H °- o- yl)diazene oxide sodium salt

\ f Vl-ONa salt

CHLOROALKYL NONOATES

[0282] The conversion of NONOates to chloroalkyl NONOates can be done based on procedures described in Tett. Lett, 2001, 42, 2625-2629.

[0283] (Z)-l-(Chloromethoxy)-3-(2-hydroxyethyl)-3-methyltriaz-l-ene 2-oxide (CN-01) cr OH

I

o-

[0284] Step 1: (Z)-3-(2-Hydroxyethyl)-3-methyl-l-((methylthio)methoxy)triaz -l-ene 2-oxide (INT-14)

[0285] NONOate sodium salt N-01 (500 mg, 3.18 mmol) and sodium carbonate (337 mg, 3.18 mmol) were suspended in 6 mL tetrahydrofuran and cooled to 0 °C. Chloromethyl methylsulfide (354 μί, 4.14 mmol) was added drop-wise, followed by the addition of DMF (2.5 mL). After 5 min, the ice bath was removed and a catalytic amount of potassium iodide was added. The reaction was allowed to stir at room temperature overnight. The pink reaction was quenched with water and extracted five times with n-butanol. The combined organic layer was washed with water and evaporated under reduced pressure, which also removed the pink color, to afford INT- 14 as a yellow oil (145 mg, 23% yield). 1H NMR (400 MHz, CDC1 ₃ ) δ 5.29 (s, 2H), 3.80 (t, J= 5.1 Hz, 2H), 3.47 (t, J= 5.1 Hz, 2H), 3.16 (s, 1H), 3.09 (s, 3H), 2.30 (s, 3H). LC t _r = 1.65 min (C- 18 column, 5 to 95% acetonitrile/water over 6 min at 1.7 mL/min with detection 254 nm, at 23 ^° C). ES(pos)MS m/z 218 (M+Na calcd for C ₅ H ₁₃ N ₃ 0 ₃ S requires 218).

[0286] Step 2: (Z)-l-(Chloromethoxy)-3-(2-hydroxyethyl)-3-methyltriaz-l-ene 2-oxide (CN-01)

[0287] Compound INT- 14 (140 mg, 0.72 mmol) was dissolved in 5 mL dichloro methane and cooled to 0 °C. Sulfuryl chloride (58 μί, 0.72 mmol) was added drop-wise and the reaction was allowed to stir while warming to room temperature. After lh, the reaction was evaporated and CN-01 was used immediately in the next step (160 mg, quant). LC t _r = 1.54 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.7 mL/min with detection 254 nm, at 23 C).

[0288] (Z)-l-(l-Chloroethoxy)-3- -hydroxyethyl)-3-methyltriaz-l-ene 2-oxide (CN-02)

[0289] Step 1: (Z)-l-(l-(Ethylthio)ethoxy)-3-(2-hydroxyethyl)-3-methyltriaz -l-ene 2-oxide

(INT-15)

[0290] Compound N-01 (3.0 mmol) and sodium carbonate (3.0 mmol) are suspended in 6 mL tetrahydrofuran and cooled to 0 °C. Freshly prepared 1-chloroethyl ethylsulfide (4.0 mmol) is added drop-wise, followed by the addition of DMF (2.5 mL). After 5 min, the ice bath is removed and a catalytic amount of potassium iodide is added. The reaction is stirred at room temperature overnight. The reaction is quenched with water and extracted with n-butanol. The combined organic layers are washed with water and evaporated under reduced pressure to afford

INT-15.

[0291] Step 2: (Z)-l-(l-Chloroethoxy)-3-(2-hydroxyethyl)-3-methyltriaz-l-en e 2-oxide (CN-02) [0292] Compound INT-15 (0.50 mmol) is dissolved in 5 mL dichloromethane and cooled to 0 °C. Sulfuryl chloride (0.50 mmol) is added drop-wise and the reaction is allowed to stir while warming to room temperature. After 1 h, the reaction is evaporated and CN-02 is used immediately in the next step.

[0293] (Z)-2-(Chloromethoxy)-l-( -hydroxyazetidin-l-yl)diazene oxide (CN-05)

[0294] Step 1: (Z)-l-(3-Hydroxy methoxy)diazene oxide (INT-16)

[0295] Using a procedure similar to that described for the synthesis of CN-01, N-03 (500 mg, 3.79 mmol) was converted to INT-16 (100 mg, 14% yield). 1H NMR (400 MHz, CDC1 ₃ ) δ 5.26 (s, 2H), 4.39-4.30 (m, 3H), 4.10-4.06 (m, 2H), 2.29 (s, 3H). LC t _r = 1.69 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.7 mL/min with detection 254 nm, at 23 C). ES(pos)MS m/z 216 (M+Na calcd for C ₅ HnN ₃ 0 ₃ S requires 216).

[0296] Step 2: (Z)-2-(Chloromethoxy)-l-(3-hydroxyazetidin-l-yl)diazene oxide (CN-05)

[0297] Using a procedure similar to that described for the synthesis of CN-01, INT-16 (95 mg, 0.49 mmol) was converted to CN-05. The product was used immediately in the next step. LC t _r = 1.58 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.7 mL/min with detection 254 nm, at 23 C).

[0298] (Z)-l-(Azetidin-l-yl)-2-(chloromethoxy)diazene oxide (CN-51)

[0299] Step 1: (Z)-l-(Azetidin-l-yl)-2-((methylthio)methoxy)diazene oxide (INT-17) O

[0300] Compound N-26 (2.25 g, 16.2 mmol) was dissolved in DMF (15 mL), followed by the addition of chloromethyl methyl sulfide (4.15 mL, 48.6 mmol) and 15-crown-5 (481 μί, 2.43 mmol). A catalytic amount of potassium iodide was added and the reaction was stirred at room temperature overnight. The pink reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate, filtered, and evaporated. The resulting oil was dried under high vacuum using mild heat to afford INT- 17 as a crude oil (920 mg, 32% yield), which was used directly in the next step. 1H NMR (400 MHz, CDC1 ₃ ) δ 5.24 (s, 2H), 4.12 (t, J=7.4Hz, 4H), 2.28 (s, 3H), 2.23-2.18 (m, 2H). LC t _r = 2.39 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.3 mL/min with detection 254 nm, at 23 ^° C). ES(pos)MS m/z 200 (M+Na calcd for C ₅ HnN ₃ 0 ₂ S requires 200).

[0301] Step 2: (Z)-l-(Azetidin-l-yl)-2-(chloromethoxy)diazene oxide (CN-51)

[0302] INT-17 (900 mg, 5.08 mmol) was dissolved in dichloromethane (10 mL) and cooled to 0 C. Sulfuryl chloride (410 μί, 5.08 mmol) was added drop-wise while stirring at 0 C. After 1 min, the reaction solvent was evaporated with a stream of nitrogen gas. The residue was placed under high vacuum for 10 min to afford CN-51 as an oil (298 mg, 35% yield), which was used directly in the next step.

[0303] (Z)-2-(Chloromethoxy)-l-morpholinodiazene oxide oxide (CN-53)

[0304] Step 1: (Z)-2-((Methylthio)methoxy)-l-morpholinodiazene oxide (INT-18)

[0305] Compound N-27 (2.0 g, 11.8 mmol) was dissolved in DMF (20 mL), followed by the addition of chloromethyl methyl sulfide (3.03 mL, 35.5 mmol) and 15-crown-5 (351 μί, 1.77 mmol). A catalytic amount of potassium iodide was added to the reaction and the mixture was stirred at room temperature overnight. The pink reaction was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate, filtered, and evaporated. The residue was placed under high vacuum for 10 min to afford INT-18 as a crude oil (1.2 g, 49% yield), which was used directly in the next step. 1H NMR (400 MHz, CDC1 ₃ ) δ 5.28 (s, 2H), 3.88-3.85 (m, 4H), 3.50-3.48 (m, 4H) 2.29 (s, 3H). LC t _r = 2.29 min (C- 18 column, 5 to 95% acetonitrile/water over 6 min at 1.3 mL/min with detection 254 nm, at 23 ^° C). ES(pos)MS m/z 230 (M+Na calcd for C ₆ H ₁₃ N ₃ 0 ₃ S requires 230).

[0306] Step 2: (Z)-2-(Chloromethoxy)-l-morpholinodiazene oxide (CN-53)

[0307] INT-18 (1.2 g, 5.79 mmol) was dissolved in dichloromethane (15 mL) and cooled to 0 C. Sulfuryl chloride (468 μί, 5.79 mmol) was added drop-wise while stirring at 0 C. After 1 min, the reaction solvent was evaporated with a stream of nitrogen gas. The residue was placed under high vacuum for 10 min to afford CN-53 as an oil (5.79 mmol), which was used directly in the next step.

[0308] Using similar procedures to make CN-01, CN-02, CN-05, CN-51 & CN-53 additional chloroalkyl NONOates (Table 4) are made by replacing NONOate sodium salt N-01 in the synthesis of CN-01 with other suitable NONOate sodium salts shown in Table 3.

[0309] Table 4. Chloroalkyl NONOates.

oxide 0 ^" methyltriaz-l-ene 2-oxide 1

l-yl)diazene oxide o-

l-yl)diazene oxide ^HO sA CI methyltriaz-l-ene 2-oxide 1 I

l-yl)diazene oxide

-l-yl)diazene oxide r-C ^N Y CI

O-LINKED CHROMENE NONOATES

[0310] Example la: (Z)-2-(((6,8-Dichloro-2-(trifluoromethyl)-2H-chromene-3-carb onyl)oxy) methoxy)- 1 -(3-hydroxy

[0311] Procedure A: Compound CA-Ola (164 mg, 0.52 mmol), CN-05 (95 mg, 0.52 mmol) and cesium carbonate (204 mg, 0.62 mmol, 1.2 eq.) were stirred in DMF (1.5 mL) at room temperature overnight. The reaction was diluted with ethyl acetate and washed with sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated. The residue was purified by chromatography using an ethyl acetate/hexane gradient to afford Example la (7.1 mg, 3% yield). 1H NMR (400 MHz, CDC1 ₃ ) δ 7.76 (s, 1H), 7.43 (d, J=2.4 Hz, 1H), 7.19 (d, J= 2.4 Hz, 1H), 5.96 (abq, J=19.2, 7.1 Hz, 1H), 5.83 (q, J=6.6 Hz, 1H), 4.62-4.56 (m, 1H), 4.40- 4.35 (m, 2H), 4.11-4.06 (m, 2H). LC t _r =4.32 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.7 mL/min with detection 254 nm, at 23 ^° C). ES(pos)MS m/z 480 (M+Na calcd for C15H12CI2F3N3O6 requires 480).

[0312] Example 9a: (Z)-2-(((7-(ieri-Butyl)-6-chloro-2-(trifluoromethyl)-2H-chro mene-3- carbonyl)oxy)methoxy)- l-(3-hydroxyazetidin-l-yl)diazene oxide

[0313] Procedure A: Compound CA-lla (301 mg, 0.90 mmol) was dissolved in DMF (3 mL). Crude CN-05 (0.9 mmol) and cesium carbonate (352 mg, 1.08 mmol) were added and stirred at room temperature overnight. The reaction was diluted with ethyl acetate and washed with saturated sodium bicarbonate, water and brine, then dried over magnesium sulfate and evaporated. The resulting oil was purified by chromatography (CH ₂ Cl ₂ /methanol/0.1% triethylamine gradient) to obtain Example 9a as a white solid (67 mg, 15% yield). 1H NMR (400 MHz, CDCI ₃ ) δ 7.75 (s, 1H), 7.25 (s, 1H), 7.08 (s, 1H), 5.96 (abq, J=24.2, 7.1Hz, 2H), 5.72 (q, J=6.9Hz, 1H), 4.62-4.57 (m, 1H), 4.40-4.36 (m, 2H), 4.11-4.05 (m, 2H), 1.48 (s, 9H). LC t _r = 5.52 min (C- 18 column, 5 to 95% acetonitrile/water over 6 min at 1.3 mL/min with detection 254 nm, at 23 ^° C). ES(pos)MS m/z 480 (M+H calcd for C ₁₉ H ₂₁ C1F ₃ N ₃ 0 ₆ requires 480).

[0314] Example 25a: (Z)-l-(((6,8-Dichloro-2-(trifluoromethyl)-2H-chromene-3-carb onyl)oxy) methoxy)-3-(2-hydroxyethyl)-3-methyltriaz- 1-ene 2-oxide

[0315] Procedure A: Compound CA-Ola (273 mg, 0.87 mmol), CN-01 (160 mg, 0.87 mmol) and cesium carbonate (340 mg, 1.04 mmol) were stirred in DMF (2 mL) at room temperature overnight. The reaction was diluted with ethyl acetate and washed with sodium bicarbonate solution and brine, dried over magnesium sulfate, and evaporated. The residue was purified by chromatography using an ethyl acetate/hexane gradient to afford Example 25a (32.8 mg, 8% yield). 1H NMR (400 MHz, CDC1 ₃ ) δ 7.76 (s, 1H), 7.42 (d, J=2.4 Hz, 1H), 7.18 (d, J= 2.4 Hz, 1H), 6.00 (abq, J=21.2, 7.2 Hz, 1H), 5.83 (q, J=6.6 Hz, 1H), 3.82 (t, J=5.1 Hz, 2H), 3.56 (t, J=5.1 Hz, 2H), 3.14 (s, 3H). LC t _r =4.32 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.7 mL/min with detection 254 nm, at 23 ^° C). ES(pos)MS m/z 482 (M+Na calcd for C15H14CI2F3N3O6 requires 482).

[0316] Example 33a: (Z)-l-(((7-(ieri-Butyl)-6-chloro-2-(trifluoromethyl)-2H-chro mene-3- carbonyl)oxy)methoxy)-3-( -hydroxyethyl)-3-methyltriaz- l-ene 2-oxide

[0317] Procedure A: Compound CA-lla (2.19 g, 6.54 mmol) was dissolved in DMF (45 mL). Crude CN-01 (13.3 mmol) and cesium carbonate (5.2 g, 16.0 mmol) were added and stirred at room temperature overnight. The reaction was quenched with water and extracted with ethyl acetate. The organic layer was washed with saturated sodium bicarbonate, water and brine, dried over magnesium sulfate and evaporated. The product was purified by chromatography (ethyl acetate/hexane gradient) to obtain Example 33a as a white foam (39 mg, 0.6% yield). 1H NMR (400 MHz, CDCI ₃ ) δ 7.75 (s, 1H), 7.24 (s, 1H), 7.08 (s, 1H), 5.99 (abq, J=25.8, 7.1Hz, 2H), 5.71 (q, J=6.8Hz, 1H), 3.81 (t, J=5.2Hz, 2H), 3.55 (t, J=5.3Hz, 2H), 3.14 (s, 3H), 1.48 (s, 9H). LC t _r = 4.83 min (C- 18 column, 5 to 95% acetonitrile/water over 6 min at 1.3 mL/min with detection 254 nm, at 23 ^° C). ES(pos)MS m/z 482 (M+H calcd for CigHasClFaNsOe requires 482).

[0318] Example 49a: (Z)-2-(((6,8-Dichloro-2-(trifluoromethyl)-2H-chromene-3-carb onyl)oxy) methoxy)- l-(( _l S')-2-(hydroxymethyl)pyrrolidin-l-yl)diazene oxide

[0319] Procedure B: Compound C-02a (190 mg, 0.53 mmol), NONOate sodium salt N-02 (106 mg, 0.58 mmol) and sodium iodide (357 mg, 2.39 mmol) were dissolved in 2 mL acetonitrile and stirred at room temperature overnight. The reaction was evaporated and dissolved in ethyl acetate. The organic layer was washed with 0.2 N sodium thiosulfate solution and brine, dried over magnesium sulfate, and evaporated. The residue was purified by chromatography using an ethyl acetate/hexane gradient to afford Example 49a (6.6 mg, 2.6% yield). 1H NMR (400 MHz, CDCI ₃ ) δ 7.76 (s, 1H), 7.43 (d, J=2.4 Hz, 1H), 7.18 (d, J= 2.4 Hz, 1H), 5.98 (abq, J=13.0, 7.2 Hz, 2H), 5.84 (q, J=6.6 Hz, 1H), 4.20-4.12 (m, 1H), 3.83-3.79 (m, 1H), 3.73-3.57 (m, 3H), 2.17-2.08 (m, 1H), 2.03- 1.96 (m, 2H), 1.90-1.84 (m, 1H). LC t _r =4.53 min (C- 18 column, 5 to 95% acetonitrile/water over 6 min at 1.7 mL/min with detection 254 nm, at 23 C). ES(pos)MS m/z 508 (M+Na calcd for C ₁₇ Hi ₆ Cl ₂ F ₃ N ₃ 0 ₆ requires 508).

[0320] Example 57a: (Z)-2-(((7-(ieri-Butyl)-6-chloro-2-(trifluoromethyl)-2H-chro mene-3- carbonyl)oxy)methoxy)- l-(( _l S' -2-(hydroxymethyl)pyrrolidin-l-yl)diazene oxide

[0321] Procedure A: Compound CA-lla (3.31 g, 9.90 mmol) was dissolved in DMF (40 mL). Crude CN-03 (2.20 g, 9.90 mmol) and cesium carbonate (3.87 g, 11.9 mmol) were added and stirred at room temperature overnight. The reaction was quenched with water and extracted with ethyl acetate. The organic layer was washed with saturated sodium bicarbonate, water and brine, dried over magnesium sulfate and evaporated. The product was purified by chromatography (ethyl acetate/hexane gradient) to obtain Example 57a as a white solid (100 mg, 53% yield based on recovered starting material). 1H NMR (400 MHz, CDC1 ₃ ) δ 7.75 (s, 1H), 7.24 (s, 1H), 7.08 (s, 1H), 5.97 (abq, J=24.2, 7.2Hz, 2H), 5.72 (q, J=6.8Hz, 1H), 4.19-4.13 (m, 1H), 3.81 (dd, J=11.2, 6.0Hz, 1H), 3.73-3.57 (m, 2H), 2.15-2.07 (m, 1H), 2.01-1.95 (m, 2H), 1.90-1.83 (m, 1H), 1.48 (s, 9H). LC t _r = 5.02 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.3 mL/min with detection 254 nm, at 23 ^° C). ES(pos)MS m/z 508 (M+H calcd for C ₂ iH ₂₅ ClF ₃ N ₃ 0 ₆ requires 508).

[0322] Example 164a: (Z)-2-(((6,8-Dichloro-2-(trifluoromethyl)-2H-chromene-3-carb onyl)oxy) methoxy)- 1 -(azetidin- 1 -yl)diazene oxide

[0323] Procedure A: Compound CA-Ola (188 mg, 0.60 mmol) was dissolved in DMF (2 mL). Crude CN-51 (298 mg, 1.8 mmol) and cesium carbonate (684 mg, 2.1 mmol) were added and stirred at room temperature overnight. The reaction was quenched with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate and evaporated. The resulting oil was purified by chromatography (ethyl acetate/hexane gradient) to obtain Example 164a as a white foam (17.5 mg, 7% yield). 1H NMR (400 MHz, CDC1 ₃ ) δ 7.75 (s, 1H), 7.42 (d, J=2.4Hz, 1H), 7.18 (dd, J=2.3, 0.2Hz, 1H), 5.96 (abq, J=16.8, 7.1Hz, 2H), 5.83 (q, J=6.6Hz, 1H), 4.14 (dt, J=7.5, 0.7Hz, 4H), 2.22 (pent, J=7.3Hz, 2H). LC t _r = 4.76 min (C- 18 column, 5 to 95% acetonitrile/water over 6 min at 1.3 mL/min with detection 254 nm, at 23 C). ES(pos)MS m/z 466 (M+Na calcd for C ₁₅ H ₁₂ Cl ₂ F ₃ N ₃ 0 ₅ requires 466).

[0324] Example 172: ( _l S ^, ,Z)-2-(((7-(ieri-Butyl)-6-chloro-2-(trifluoromethyl)-2 H-chromene-3- carbonyl)oxy)methoxy)- l-(3-hydroxyazetidin-l-yl)diazene oxide

[0325] Procedure A: Compound CA-11 (200 mg, 0.60 mmol) was dissolved in DMF (2 mL). Crude CN-51 (298 mg, 1.8 mmol) and cesium carbonate (684 mg, 2.1 mmol) were added and stirred at room temperature overnight. The reaction was quenched with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate and evaporated. The resulting oil was purified by chromatography (ethyl acetate/hexane gradient) to obtain Example 172 (98.5 mg, 35% yield). 1H NMR (400 MHz, CDC1 ₃ ) δ 7.74 (s, 1H), 7.24 (s, 1H), 7.08 (s, 1H), 5.95 (abq, J=22.4, 7.2Hz, 2H), 5.71 (q, J=6.8Hz, 1H), 4.14 (dt, J=7.5, 0.9Hz, 4H), 2.22 (pent, J=7.4Hz, 2H), 1.48 (s, 9H). ¹⁹ F NMR (400 MHz, CDC1 ₃ ) δ -78.4 (d, J=6.8Hz, 3F). ¹³ C NMR (400 MHz, CDC1 ₃ ) δ 162.2, 152.8, 151.7, 137.3, 131.9, 127.0, 117.5, 1 15.7, 87.9, CF ₃ (71.1, 70.8, 70.4, 70.1), 56.5, 36.7, 29.3, 14.6. LC t _r = 5.24 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.3 mL/min with detection 254 nm, at 23 C). ES(pos)MS m/z 464, 481 (M+H calcd for C ₁₉ H ₂₁ C1F ₃ N ₃ 0 ₅ requires 464), (M+H ₂ 0 calcd for C ₁₉ H ₂ iClF ₃ N ₃ 0 ₅ requires 481). Chiral HPLC: AD (n-hexane/i-PrOH 9: 1, λ = 254 nm), t _r = 5.89 min, 96.8%ee.

[0326] Example 197a: (Z)-2-(((6,8-Dichloro-2-(trifluoromethyl)-2H-chromene-3-carb onyl)oxy) methoxy)- 1-morpholinodiazene oxide

[0327] Procedure A: Compound CA-Ola (906 mg, 2.9 mmol) was dissolved in DMF (20 mL). Crude CN-53 (5.79 mmol) and cesium carbonate (2.36 g, 7.25 mmol) were added and stirred at room temperature overnight. The reaction was quenched with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate and evaporated. The resulting oil was purified by chromatography (ethyl acetate/hexane gradient) to obtain Example 197a (850 mg, 31 % yield). 1H NMR (400 MHz, CDC1 ₃ ) δ 7.75 (s, 1H), 7.43 (d, J=2.4Hz, 1H), 7.18 (d, J=2.4Hz, 1H), 6.00 (abq, J=22.4, 7.2Hz, 2H), 5.83 (q, J=6.6Hz, 1H), 3.88- 3.84 (m, 4H), 3.51-3.48 (m, 4H). LC t _r = 4.66 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.3 mL/min with detection 254 nm, at 23 ^° C). ES(pos)MS m/z 494 (M+Na calcd for C ₁₆ Hi ₄ Cl ₂ F ₃ N ₃ 0 ₆ requires 494).

[0328] Using Procedure A, additional O-linked chromene NONOates are made by replacing CN- 01 or CN-05 with other chloroalkyl NONOates from Table 4 and an appropriate chromene acid, including but not limited to examples in Table 1. Alternatively, additional examples are made using Procedure B by replacing C-02a with other haloalkyl chromenes from Table 2 and N-02 with other NONOate sodium salts from Table 3. Table 5 lists non-limiting examples of additional O-linked chromene NONOates.

[0329] Table 5. O-linked Chromene NONOates.

(S,Z)-2-(((6-bromo-8-methyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- 1-(3- hydroxyazetidin- l-yl)diazene oxide

(5,Z)-l-(3-hydroxyazetidin- l-yl)-2-(((6-

(trifluoromethoxy)-2-(trifluoromethyl)-2H- chromene-3- carbonyl)oxy)methoxy)diazene oxide

(5,Z)-l-(3-hydroxyazetidin- l-yl)-2-(((8- methyl- 6- (trifluoromethoxy) -2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)diazene oxide

(S,¾-2-(((8-ethyl-6-(trifluoromethoxy)-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- 1-(3- hydroxyazetidin- l-yl)diazene oxide

(S,Z)-2-(((6,8-dimethyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- 1-(3- hydroxyazetidin- l-yl)diazene oxide

(5,Z)-2-(((6-chloro-5,7-dimethyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- 1-(3- hydroxyazetidin- l-yl)diazene oxide (Z)-2-(((7-(ieri-butyl)-6-chloro-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- 1-(3- hydroxyazetidin- l-yl)diazene oxide

(S,Z)-2-(((7-(ieri-butyl)-6-chloro-2- (trifluoromethyl)-2H-chromene-3- 8 ^/ ~Τ ^0Η carbonyl)oxy)methoxy)- 1-(3- hydroxyazetidin- l-yl)diazene oxide

(S,Z)-2-(( (6- (pentafluoro sulf anyl) -2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- 1-(3- hydroxyazetidin- l-yl)diazene oxide

(S,Z)-2-(( (6- (pentafluoro sulf anyl) - 8 - methyl-2-(trifluoromethyl)-2H-chromene- 3-carbonyl)oxy)methoxy)- l-(3- hydroxyazetidin- l-yl)diazene oxide

(S,Z)-2-(( (6- (pentafluoro sulf anyl) - 8 - ethyl- 2- (trifluoromethyl) -2H-chromene- 3 - carbonyl)oxy)methoxy)- 1-(3- hydroxyazetidin- l-yl)diazene oxide

(Z)-2-(l-(((5)-6,8-dichloro-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)- l-(3- hydroxyazetidin- l-yl)diazene oxide CI F ^F

hydroxyazetidin- l-yl)diazene oxide (Z)-2-(l-(((S)-6-chloro-5,7-dimethyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)- l-(3- hydroxyazetidin- l-yl)diazene oxide

(Z)-2-(l-(((5)-7-(ierf-butyl)-6-chloro-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)- l-(3- hydroxyazetidin- l-yl)diazene oxide

(Z)-2-(l-(((5)-6-(pentafluorosulfanyl)-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)- l-(3- hydroxyazetidin- l-yl)diazene oxide

(Z)-2-(l-(((5)-6-(pentafluorosulfanyl)-8- methyl-2-(trifluoromethyl)-2H-chromene- 3-carbonyl)oxy)ethoxy)- l-(3- hydroxyazetidin- l-yl)diazene oxide

(Z)-2-(l-(((5)-6-(pentafluorosulfanyl)-8- ethyl-2-(trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)- l-(3- hydroxyazetidin- l-yl)diazene oxide

(Z)-l-(((6,8-dichloro-2-(trifluoromethyl)- 2H-chromene-3-carbonyl)oxy)methoxy)-3- (2-hydroxyethyl)-3-methyltriaz- 1-ene 2- oxide (S,Z)-l-(((6,8-dichloro-2-(trifluoromethyl)- 2H-chromene-3-carbonyl)oxy)methoxy)-3- (2-hydroxyethyl)-3-methyltriaz- 1-ene 2- oxide

(S,Z)-l-(((6-chloro-8-methyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)-3-(2- hydroxyethyl)-3-methyltriaz- 1-ene 2-oxide

(S,Z)-l-(((6-bromo-8-methyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)-3-(2- hydroxyethyl)-3-methyltriaz- 1-ene 2-oxide

(S,Z)-3-(2-hydroxyethyl)-l-(((6- (trifluoromethoxy)-2-(trifluoromethyl)-2H- chromene-3-carbonyl)oxy)methoxy)triaz- 1-ene 2-oxide

(S,Z)-3-(2-hydroxyethyl)-3-methyl-l-(((8- methyl- 6- (trifluoromethoxy) -2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)triaz- 1-ene 2-oxide

(S,Z)-l-(((8-ethyl-6-(trifluoromethoxy)-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)-3-(2- hydroxyethyl)-3-methyltriaz- 1-ene 2-oxide (S,Z)-l-(((6,8-dimethyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)-3-(2- hydroxyethyl)-3-methyltriaz- 1-ene 2-oxide

(S,Z)-l-(((6-chloro-5,7-dimethyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)-3-(2- hydroxyethyl)-3-methyltriaz- 1-ene 2-oxide

(Z)-l-(((7-(ie/ -butyl)-6-chloro-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)-3-(2- hydroxyethyl)-3-methyltriaz- 1-ene 2-oxide

(5,Z)-l-(((7-(ieri-butyl)-6-chloro-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)-3-(2- hydroxyethyl)-3-methyltriaz- 1-ene 2-oxide

(S,Z)-l-(( (6- (pentafluoro sulf anyl) -2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)-3-(2- hydroxyethyl)-3-methyltriaz- 1-ene 2-oxide

(S,Z)-l-(((6-(pentafluorosulfanyl)-8- methyl-2-(trifluoromethyl)-2H-chromene- 3-carbonyl)oxy)methoxy)-3-(2- hydroxyethyl)-3-methyltriaz- 1-ene 2-oxide (S,Z)-l-(( (6- (pentafluoro sulf anyl) - 8 - ethyl- 2- (trifluoromethyl) -2H-chromene- 3 - carbonyl)oxy)methoxy)-3-(2- hydroxyethyl)-3-methyltriaz- 1-ene 2-oxide

(Z)-l-(l-(((5)-6,8-dichloro-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)-3-(2-hydroxyethyl)- 3-methyltriaz- 1-ene 2-oxide

(Z)-l-(l-(((S)-6-chloro-8-methyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)-3-(2-hydroxyethyl)- 3-methyltriaz- 1-ene 2-oxide

(Z)-l-(l-(((S)-6-bromo-8-methyl-2- (trifluoromethyl)-2H-chromene-3- 8 1 ¹ carbonyl)oxy)ethoxy)-3-(2-hydroxyethyl)- L _o i ,F 0 ^" 3-methyltriaz- 1-ene 2-oxide

(Z)-3-(2-hydroxyethyl)-3-methyl- 1 -( 1 - (((S)-6-(trifluoromethoxy)-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)triaz- 1-ene 2-oxide

(Z)-3-(2-hydroxyethyl)-3-methyl- 1 -( 1 - ( ((S) - 8 -methyl- 6- (trifluoromethoxy) -2- 8 I ¹ (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)triaz- 1-ene 2-oxide (Z)-l-(l-(((S)-8-ethyl-6- (trifluoromethoxy)-2-(trifluoromethyl)-2H- chromene-3-carbonyl)oxy)ethoxy)-3-(2- hydroxyethyl)-3-methyltriaz- 1-ene 2-oxide

(Z)-l-(l-(((S)-6,8-dimethyl-2- (trifluoromethyl)-2H-chromene-3- s i ¹ carbonyl)oxy)ethoxy)-3-(2-hydroxyethyl)- 3-methyltriaz- 1-ene 2-oxide

(Z)-l-(l-(((S)-6-chloro-5,7-dimethyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)-3-(2-hydroxyethyl)- 3-methyltriaz- 1-ene 2-oxide

(Z)-l-(l-(((5)-7-(ierf-butyl)-6-chloro-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)-3-(2-hydroxyethyl)- 3-methyltriaz- 1-ene 2-oxide

(Z)-l-(l-(((5)-6-(pentafluorosulfanyl)-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)-3-(2-hydroxyethyl)- 3-methyltriaz- 1-ene 2-oxide

(Z)-l-(l-(((5)-6-(pentafluorosulfanyl)-8- methyl-2-(trifluoromethyl)-2H-chromene- 3-carbonyl)oxy)ethoxy)-3-(2- hydroxyethyl)-3-methyltriaz- 1-ene 2-oxide (Z)- l-(l-(((5)-6-(pentafluorosulfanyl)-8- ethyl-2-(trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)-3-(2-hydroxyethyl)- 3-methyltriaz-l-ene 2-oxide

(Z)-2-(((6,8-dichloro-2-(trifluoromethyl)- 2H-chromene-3-carbonyl)oxy)methoxy)- l- ( (S) -2- (hydroxymethyl)pyrrolidin- 1 - yl)diazene oxide

(Z)-2-((((5)-6,8-dichloro-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- l-((S)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene

oxide CI F ^F

(Z)-2-((((S)-6-chloro-8-methyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- l-((S)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene

oxide

(Z)-2-((((S)-6-bromo-8-methyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- l-((S)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene

oxide (Z)- 1 - ((S)-2- (hydroxymethyl)pyrrolidin- 1 - yl)-2-((((5)-6-(trifluoromethoxy)-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)diazene oxide

(Z)- 1 - ((S)-2- (hydroxymethyl)pyrrolidin- 1 - yl)-2-((((5')-8-methyl-6-(trifluoromethoxy)- 2- (trifluoromethyl) -2H-chromene- 3 - carbonyl)oxy)methoxy)diazene oxide

(Z)-2-((((5)-8-ethyl-6-(trifluoromethoxy)- 2- (trifluoromethyl) -2H-chromene- 3 - carbonyl)oxy)methoxy)- l-((S)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene

oxide

(Z)-2-((((S)-6,8-dimethyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- l-((S)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene l¾Jl , Jv .F 0- oxide

(Z)-2-((((S)-6-cmoro-5,7-dimethyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- l-((S)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene

oxide (Z)-2-(((7-(ieri-butyl)-6-chloro-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- l-((S)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide

(Z)-2-((((5)-7-(ierf-butyl)-6-chloro-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- l-((S)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide

(Z)-2-((((5)-6-(pentafluorosulfanyl)-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- l-((S)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide

(Z)-2-((((5)-6-(pentafluorosulfanyl)-8- methyl-2-(trifluoromethyl)-2H-chromene- 3-carbonyl)oxy)methoxy)- l-(( _l S')-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide

(Z)-2-((((5)-6-(pentafluorosulfanyl)-8- ethyl-2-(trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- l-((S)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide (Z)-2-(l-(((S)-6,8-dichloro-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)- l-(( _l S')-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide

(Z)-2-(l-(((S)-6-chloro-8-methyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)- l-(( _l S')-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide

(Z)-2-(l-(((S)-6-bromo-8-methyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)- l-(( _l S')-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide

(Z)- 1 - ((S)-2- (hydroxymethyl)pyrrolidin- 1 - yl)-2-(l-(((5)-6-(trifluoromethoxy)-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)diazene oxide

(Z)- 1 - ((S)-2- (hydroxymethyl)pyrrolidin- 1 - yl)-2-(l-(((5)-8-methyl-6-

(trifluoromethoxy)-2-(trifluoromethyl)-2H- chromene-3-carbonyl)oxy)ethoxy)diazene oxide (Z)-2-(l-(((S)-8-ethyl-6- (trifluoromethoxy)-2-(trifluoromethyl)-2H- chromene-3-carbonyl)oxy)ethoxy)-l-(( _l S')- 2-(hydroxymethyl)pyrrolidin- 1 -yl)diazene

oxide

(Z)-2-(l-(((S)-6,8-dimethyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)- l-(( _l S')-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene

oxide

(Z)-2-(l-(((S)-6-chloro-5,7-dimethyl-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)- l-(( _l S')-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene

oxide F ^F

(Z)-2-(l-(((5)-7-(ieri-butyl)-6-chloro-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)- l-(( _l S')-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene

oxide

(Z)-2-(l-(((S)-6-(pentafluorosulfanyl)-2- (trifluoromethyl)-2H-chromene-3- carbonyl)oxy)ethoxy)- l-(( _l S')-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene

oxide F ^F

oxide

oxide

hydroxypiperidin- 1 -yl)diazene oxide

yl)diazene oxide

O-LINKED CHROMENE NONOATE CARBOXYLATES

[0330] Example 95a: (Z)-3-(Carboxymethyl)-l-(((6,8-dichloro-2-(trifluoromethyl)- 2H- chromene-3-carbonyl)oxy)methoxy)-3-methyltriaz- 1-ene 2-oxide

[0331] Example 25a (26 mg, 0.06 mmol) was dissolved in acetonitrile (0.5 mL), ethyl acetate (0.5 mL) and water (0.75 mL). Sodium metaperiodate (51 mg, 0.24 mmol) was added, followed by catalytic ruthenium (III) chloride hydrate and the reagents were stirred at room temperature overnight. The reaction was filtered and the filtrate was quenched with 1 N hydrochloric acid solution, then extracted three times with ethyl acetate. The combined organic layers were washed with brine and dried over magnesium sulfate, then filtered, and evaporated to yield 21.8 mg (0.046 mmol) of desired acid Example 95a as a tan oil.

[0332] Example 163: Sodium (Z)-2-(3-(((6,8-dichloro-2-(trifluoromethyl)-2H-chromene-3- carbonyl)oxy)methoxy)- l-methyl-2-oxidotriaz-2-en- l-yl)acetate

[0333] Example 95a was dissolved in acetonitrile (0.2 mL) and treated with 0.4935 M NaOH solution (93.2 μί, 0.046 mmol) and the solvent was evaporated. The resulting beige amorphous solid was washed twice with diethyl ether and dried to provide Example 163 (10.7 mg, 36% yield). 1H NMR (400 MHz, CD ₃ OD) δ 7.87 (d, J=2.5 Hz, 1H), 7.81 (d, J=2.5 Hz, 1H), 7.76 (d, J= 2.7 Hz, 1H), 5.77 (abq, J=47.8, 7.2 Hz, 2H), 5.28 (q, J=6.3 Hz, 1H), 4.07 (s, 2H), 3.17 (s, 3H). LC t _r =4.32 min (C-18 column, 5 to 95% acetonitrile/water over 6 min at 1.7 mL/min with detection 254 nm, at 23 C).

[0334] Using a similar procedure to make Example 95a, additional O-linked chromene NONOate carboxylates are made by replacing Example 25a with other O-linked chromene NONOates from Table 5 having a primary alcohol for oxidation to the carboxylic acid. In addition to carboxylic acids, each compound in Table 6 is converted to the corresponding lithium, sodium, potassium, calcium, etc. salts using standard methods as shown for Example 163.

[0335] Table 6. O-linked Chromene NONOate Carboxylates.

(carboxymethyl)-3-methyltriaz- 1 -ene F ^F

carbonyl)oxy)ethoxy)triaz-l-ene 2- F ^F

2-oxide (Z)- l-((S)-2-carboxypyrrolidin- l-yl)-2- ((((S)-6,8-dimethyl-2-(trifluoromethyl)- 2H-chromene-3- carbonyl)oxy)methoxy)diazene oxide

(Z)-l-((5')-2-carboxypyrrolidin- l-yl)-2- ((((S)-6-chloro-5,7-dimethyl-2- (trifluoromethyl) -2H-chromene- 3 - carbonyl)oxy)methoxy)diazene oxide

(Z)-2-((((5)-7-(ieri-butyl)-6-chloro-2- (trifluoromethyl) -2H-chromene- 3 - carbonyl)oxy)methoxy)- l-(( _l S')-2- carboxypyrrolidin- l-yl)diazene oxide

(Z)-2-((((5)-6-(pentafluorosulfanyl)-2- (trifluoromethyl) -2H-chromene- 3 - carbonyl)oxy)methoxy)- l-(( _l S')-2 carboxypyrrolidin- l-yl)diazene oxide

2- oxide

methyltriaz- 1-ene 2-oxide

hydroxyethyl)triaz-l-ene 2-oxide

carboxypiperidin- 1 -yl)diazene oxide

carboxypyrrolidin- l-yl)diazene oxide

E. Method of Treatment

[0336] A compound of the structural formulae herein is meant to include a pharmaceutically acceptable salt, or solvate of a compound or salt, of the structural formulae herein.

[0337] The present invention further provides methods for treating a disease condition in a subject having or susceptible to having such a disease condition, by administering to the subject a therapeutically-effective amount of one or more compounds as described by the structural formulae herein. In one embodiment, the treatment is preventative treatment. In another embodiment, the treatment is palliative treatment. In another embodiment, the treatment is restorative treatment, for example, treatments for wound healing, acne, and inflammation. In another embodiment, the subject is a mammal. In yet another embodiment, the subject is a human.

1. Conditions

[0338] The conditions that can be treated in accordance with the present invention include, but are not limited to, autoimmune disorders, chronic inflammatory disorders, acute inflammatory disorders, auto-inflammatory disorders, pain, cancer, neoplasia, lung cancer, colorectal cancer, and the like.

[0339] In one embodiment, methods described herein are used to treat, prevent, or ameliorate a disease condition comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the structural formulae herein, wherein the condition is selected from the group consisting of cancer pain, Barrett's esophagus, Lynch syndrome, non- small cell lung cancer, head and neck cancer, skin cancer, liver cancer, metastatic colorectal cancer (and FAP), renal cell cancer, glioblastoma, squamous cell cancer, bladder cancer, breast cancer, biliary tract cancer, cervical cancer, prostate cancer, small cell lung cancer, ovarian cancer, pancreatic cancer, gastrointestinal cancer, and CNS cancer.

[0340] In another embodiment, methods described herein are used to treat, prevent, or ameliorate a disease condition comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the structural formulae herein, wherein the condition is selected from the group consisting of cancer, actinic keratosis, cystic fibrosis, and acne.

[0341] In yet another embodiment, methods described herein are used for healing wounds by administering to a subject in need thereof a therapeutically effective amount of a compound of the structural formulae herein.

[0342] In one embodiment, methods described herein are used to treat, prevent, or ameliorate a disease condition comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the structural formulae herein, wherein the condition is selected from a group consisting of colorectal cancer, non- small cell lung cancer, and head and neck cancer.

[0343] In some embodiments the methods described herein are used for administering to a patient in need thereof, a therapeutically effective amount of a compound of the structural formulae herein, to treat, prevent, or ameliorate a disease condition or disorder arising from dysregulated enzymes, and/or dysregulated inflammatory mediator production, stability, secretion, and posttranslational processing. Examples of inflammatory mediators that may be dysregulated include nitric oxide, prostaglandins, and leukotrienes. Examples of enzymes which may be dysregulated include cyclooxygenase and nitric oxide synthase.

[0344] In some embodiments, the methods described herein are used for administering to a patient in need thereof a therapeutically effective amount of a compound of the structural formulae herein, to treat, prevent, or ameliorate a disease condition or disorder that is, arises from, or is related to an autoimmune disorder, chronic, and/or acute inflammatory disorder, and/or auto-inflammatory disorder. Examples of disorders include, but are not limited to arthritis, rheumatoid arthritis, osteoarthritis, juvenile arthritis, psoriatic arthritis.

[0345] In some embodiments, the methods described herein can be used for administering to a patient in need thereof a therapeutically effective amount of a compound of the structural formulae herein, to treat, prevent, or ameliorate neoplasia and the symptoms thereof. Examples of these conditions include but are not limited to the following:

carcinosarcoma large cell carcinoma serous carcinoma

skin cancer

cavernous leiomyosarcoma

cervical cancer

lentigo maligna melanomas small cell carcinoma cholangiocarcinoma liver cancer small cell lung cancer chondosarcoma malignant melanoma soft tissue carcinomas choroid plexus malignant mesothelial tumors somatostatin-secreting tumor papilloma/carcinoma clear cell carcinoma medulloblastoma squamous carcinoma

CNS cancer

medulloepithelioma squamous cell carcinoma cystadenoma melanoma submesothelial endodermal sinus tumor meningeal superficial spreading

melanoma

undifferentiatied carcinoma endometrial hyperplasia mesothelial endometrial stromal sarcoma metastatic carcinoma uveal melanoma endometrioid adenocarcinoma metastatic colorectal cancer verrucous carcinoma ependymal mucoepidermoid carcinoma vipoma epitheloid neuroblastoma well differentiated carcinoma

Ewing's sarcoma neuroepithelial Wilm's tumor

adenocarcinoma nodular

melanoma familial adenomatous

polyposis (FAP) [0346] In one embodiment, the methods described herein can be used for administering to a patient in need thereof a therapeutically effective amount of a compound of the structural formulae herein, to treat, prevent, or ameliorate metastatic colorectal cancer.

[0347] In an additional embodiment, the methods described herein can be used for administering to a patient in need thereof a therapeutically effective amount of a compound of the structural formulae herein, to treat, prevent, or ameliorate a disease condition characterized by or related to COX-2 over-expression, including but not limited to cancer, an autoimmune disorder such as rheumatoid arthritis, and other disorders characterized by pain and/or inflammation.

[0348] COX-2 over-expression is found in a variety of medical conditions. Examples of conditions characterized by COX-2 over-expression given herein are not intended to be limiting and are solely for illustrative purposes. The journal article Transgenic mouse for conditional, tissue-specific Cox-2 over expression (Kamei et al. Genesis. 2006 Apr; 44(4): 177-82.) states that COX-2 over-expression is found in, for example, cardiovascular conditions, acute and chronic inflammatory responses, neurodegenerative diseases, and cancer. Exemplary and non-limiting cardiovascular conditions include septicemia (Cuenca et al., Infiltration of Inflammatory Cells Plays an Important Role in Matrix Metalloproteinase Expression and Activation in the Heart during Sepsis. 2006; Am J Pathol. 169(5): 1567-1576.), aortic aneurysms (King et al., Selective Cyclooxygenase-2 Inhibition with Celecoxib Decreases Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Mice. Nov 2006; Arterioscler Thromb Vase Biol. 26: 1137-1143.), and mycardial infarction (LaPointe et al., Inhibition of cyclooxygenase-2 improves cardiac function after myocardial infarction in the mouse. 2004; Am J Physiol Heart Circ Physiol. 286: H1416-H1424,) Exemplary and non-limiting acute and chronic inflammatory responses include injury-related inflammation and Rhematoid Arthritis respectively. Exemplary and non-limiting neurodegenerative diseases include Parkinson's disease (Teismann, Peter. COX-2 in the neurodegenerative process of Parkinson's disease. Nov 2012; 38(6): 395-397.) and Alzheimer's disease (Rogers, Joseph. Neuroinflammatory Mechanisms in Alzheimer's Disease: Basic and Clinical Research. Springer Science and Business Media, Jan 2001., 203- 204). Exemplary and non-limiting cancers include non-small cell lung cancer and colorectal cancer.

[0349] In another embodiment, patients with high baseline COX-2 activity are more likely to improve upon administration of a therapeutically effective amount of a compound of the structural formulae herein. Baseline levels of COX-2 activity can be determined by urinary PGE- M content.

[0350] The term patient refers to both humans and non-human animals with the abovementioned conditions. Non-human animals could be companion animals such as, but not limited to, canine and feline species. The terms "patient" and "subject" are meant to be interchangeable.

2. Subjects

[0351] Suitable subjects for the methods described herein include mammalian subjects. Mammals according to the present invention include, but are not limited to, human, canine, feline, bovine, caprine, equine, ovine, porcine, rodents, lagomorphs, primates, and the like and encompass mammals in utero. Subjects may be of either gender and at any stage of development.

3. Administration and Dosing

[0352] A compound of the present invention may be administered in the form of a prodrug in a therapeutically effective amount.

[0353] A compound of the present invention can be administered by any suitable route in the form of a pharmaceutical composition adapted to such a route and in a dose effective for the treatment intended. Therapeutically effective doses of a compound of the present invention required to prevent or arrest the progress of, to treat or ameliorate the medical condition, or to alleviate symptoms thereof, such as pain or inflammation, are readily ascertained by one of ordinary skill in the art using preclinical and clinical approaches familiar to the medicinal arts.

[0354] For convenience a compound of the present invention can be administered in a unit dosage form. If desired, multiple doses per day of the unit dosage form can be used to increase the total daily dose. The unit dosage form, for example, may be a tablet or capsule containing about 0.01, about 0.05, about 0.1, about 0.5, about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 125, about 150, about 175, about 200, about 250, or about 500 mg of a compound of the present invention. In one embodiment, the unit dosage form contains from about 0.01 mg to about 500 mg of a compound of the present invention. In another embodiment, the unit dosage form contains from about 0.02 to about 400 mg of a compound of the present invention. In another embodiment, the unit dosage form contains from about 0.05 mg to about 250 mg of a compound of the present invention. In another embodiment, the unit dosage form contains from about 0.1 mg to about 200 mg of a compound of the present invention. In another embodiment, the unit dosage form contains from about 0.5 mg to about 150 mg of a compound of the present invention. In another embodiment, the unit dosage form contains from about 1.0 mg to about 100 mg of a compound of the present invention.

[0355] The dosage regimen required for therapeutic effect for compounds of the present invention and/or compositions containing compounds of the present invention is based on a variety of factors, including the type, age, weight, sex, and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the particular compound employed. Thus, the dosage regimen may vary based on patient to patient variability of individual factors, including but not limited to those listed here. Dosage levels from about 0.001 mg to about 100 mg of a compound of the present invention per kilogram of body weight per day are useful in the treatment of the above-indicated conditions. In one embodiment, the total daily dose of a compound of the present invention (administered in single or divided doses) is typically from about 0.001 mg/kg to about 20 mg/kg (i.e., mg compound/kg body weight). In another embodiment, the total daily dose of a compound of the present invention is from about 0.005 mg/kg to about 10 mg/kg. In another embodiment, the total daily dose is from about 0.005 mg/kg to about 5 mg/kg. In another embodiment, the total daily dose is from about 0.01 mg/kg to about 1 mg/kg. In another embodiment, the total daily dose is from about 0.8 mg/kg to about 15 mg/kg. In another embodiment, the total daily dose is from about 0.2 mg/kg to about 4 mg/kg. These dosages are based on an average human subject having a weight of about 65 kg to about 75 kg. A physician will readily be able to determine doses for subjects whose weight falls outside of this range, such as infants or children. The administration of a compound of the present invention can be repeated a plurality of times in a day (typically no greater than 4 times) to achieve the desired daily dose.

[0356] The present invention further comprises use of a compound of the present invention as a medicament (such as a unit dosage tablet or unit dosage capsule).

[0357] In another embodiment, the present invention comprises the use of a compound of the present invention for the manufacture of a medicament (such as a unit dosage tablet or unit dosage capsule) to treat one or more of the conditions previously identified in the above sections discussing methods of treatment. In one embodiment, the condition is cancer. In another embodiment the condition is an inflammatory condition. F. Pharmaceutical Compositions

[0358] For treatment of the conditions referred to above, a compound or several compounds described herein can be administered as follows, these representations are not meant to be limiting:

1. Oral Administration

[0359] A compound or several compounds of the present invention may be administered orally, including by swallowing, so that the compound enters the gastrointestinal tract, or absorbed into the blood stream directly from the mouth (e.g., buccal or sublingual administration).

[0360] Suitable compositions for oral administration include, but are not limited to, solid formulations such as tablets, lozenges, pills, cachets, and hard and soft capsules, which can contain liquids, gels, or powders.

[0361] Compositions for oral administration may be formulated as immediate or modified release, including delayed or sustained release, optionally with enteric coating.

[0362] Liquid formulations can include, but are not limited to, solutions, syrups, and suspensions, which can be used in soft or hard capsules. Such formulations may include a pharmaceutically acceptable carrier, for example, water, ethanol, polyethylene glycol, cellulose, or an oil or oils. The formulation may also include one or more emulsifying agents and/or suspending agents.

[0363] In a tablet dosage form the amount of drug present may be from about 0.05% to about 95% by weight, more typically from about 2% to about 50% by weight of the dosage form. In addition, tablets may contain a disintegrant, comprising from about 0.5% to about 35% by weight, more typically from about 2% to about 25% of the dosage form. Examples of disintegrants include, but are not limited to, methyl cellulose, sodium or calcium carboxymethyl cellulose, croscarmellose sodium, polyvinylpyrrolidone, hydroxypropyl cellulose, starch, and the like.

[0364] Suitable lubricants, for use in a tablet, may be present in amounts from about 0.1% to about 5% by weight and include, but are not limited to, calcium, zinc or magnesium stearate, sodium stearyl fumarate, and the like.

[0365] Suitable binders, for use in a tablet, include, but are not limited to, gelatin, polyethylene glycol, sugars, gums, starch, hydroxypropyl cellulose, and the like. Suitable diluents, for use in a tablet, include, but are not limited to, mannitol, xylitol, lactose, dextrose, sucrose, sorbitol, and starch.

[0366] Suitable surface active agents and glidants, for use in a tablet, may be present in amounts from about 0.1% to about 3% by weight and include, but are not limited to, polysorbate 80, sodium dodecyl sulfate, talc, and silicon dioxide.

[0367] In another embodiment, a pharmaceutical composition comprises a therapeutically effective amount of a compound of the structural formulae herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

2. Parenteral Administration

[0368] Compounds of the present invention may be administered directly into the blood stream, muscle, or internal organs. Suitable means for parenteral administration include, but are not limited to, intravenous, intra-muscular, subcutaneous intraarterial, intraperitoneal, intrathecal, intracranial, and the like. Suitable devices for parenteral administration include, but are not limited to, injectors (including needle and needle-free injectors) and infusion methods.

[0369] Compositions for parenteral administration may be formulated as immediate or modified release, including delayed or sustained release.

[0370] Most parenteral formulations are aqueous solutions containing excipients, including, but not limited to, salts, buffering agents, and carbohydrates.

[0371] Parenteral formulations may also be prepared in a dehydrated form (e.g., by lyophilization) or as sterile non-aqueous solutions. These formulations can be used with a suitable vehicle, such as sterile water. Solubility-enhancing agents may also be used in preparation of parenteral solutions.

3. Topical Administration

[0372] Compounds of the present invention may be administered topically to the skin or transdermally. Formulations for this topical administration can include, but are not limited to, lotions, solutions, creams, gels, hydrogels, ointments, foams, implants, patches, and the like. Pharmaceutically acceptable carriers for topical administration formulations can include, but are not limited to, water, alcohol, mineral oil, glycerin, polyethylene glycol, and the like. Topical administration can also be performed by electroporation, iontophoresis, phonophoresis, and the like. [0373] Compositions for topical administration may be formulated as immediate or modified release, including delayed or sustained release.

4. Rectal Administration

[0374] Suppositories for rectal administration of a compound of the present invention can be prepared by mixing the active agent with a suitable non-irritating excipient including, but not limited to, cocoa butter, synthetic mono-, di-, or triglycerides, fatty acids, or polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature, and which will therefore melt in the rectum and release the drug.

[0375] Other carrier materials and modes of administration known in the pharmaceutical art may also be used. Pharmaceutical compositions of the invention may be prepared by any of the well- known techniques of pharmacy, such as effective formulation and administration procedures. The above considerations in regard to effective formulations and administration procedures are well known in the art, and are described in standard textbooks. Formulation of drugs is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1975; Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New

York, N.Y., 1980; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients (3 rd Ed.), American Pharmaceutical Association, Washington, 1999.

G. Combinations and Combination Therapy

[0376] A compound of the present invention can be used, alone or in combination with other pharmaceutically active compounds, to treat conditions such as those previously described above. A compound or several compounds of the present invention and other pharmaceutically active compound(s) can be administered simultaneously (either in the same dosage form or in separate dosage forms) or sequentially. Accordingly, in one embodiment, the present invention comprises methods for treating a condition by administering to the subject a therapeutically- effective amount of one or more compounds of the present invention, and one or more additional pharmaceutically active compounds.

[0377] In another embodiment, there is provided a pharmaceutical composition comprising one or more compounds of the present invention, one or more additional pharmaceutically active compounds, and a pharmaceutically acceptable carrier.

[0378] In another embodiment, the one or more additional pharmaceutically active compounds is selected from the group consisting of anti-inflammatory drugs, cytostatic drugs, cytotoxic drugs, anti-proliferative agents, and angiogenesis inhibitors.

[0379] In another embodiment, the one or more additional pharmaceutically active compounds is selected from the group consisting of anti-cancer drugs and anti-inflammatory drugs.

[0380] NO-releasing chromene conjugate described herein are also optionally used in combination with other therapeutic reagents that are selected for their therapeutic value for the condition to be treated. In general, the compounds described herein, and in embodiments where combinational therapy is employed, other agents, do not have to be administered in the same pharmaceutical composition and, because of different physical and chemical characteristics, are optionally administered by different routes. The initial administration is generally made according to established protocols and then, based upon the observed effects, the dosage, modes of administration, and times of administration subsequently modified. In certain instances, it is appropriate to administer an NO-releasing chromene conjugate, as described herein, in combination with another therapeutic agent or NO-releasing chromene conjugate. By way of example only, the therapeutic effectiveness of an NO-releasing chromene conjugate is enhanced by administration of another therapeutic agent (which also includes a therapeutic regimen) that also has therapeutic benefit. Regardless of the disease, disorder or condition being treated, the overall benefit experienced by the patient is either simply additive of the two therapeutic agents or the patient experiences an enhanced benefit.

[0381] Therapeutically effective dosages vary when the drugs are used in treatment combinations. Methods for experimentally determining therapeutically effective dosages of drugs and other agents for use in combination treatment regimens are documented methodologies. Combination treatment further includes periodic treatments that start and stop at various times to assist with the clinical management of the patient. In any case, the multiple therapeutic agents (one of which is an NO-releasing chromene conjugate as described herein) are administered in any order, or even simultaneously. If simultaneously, the multiple therapeutic agents are optionally provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills).

[0382] In some embodiments, one of the therapeutic agents is given in multiple doses, or both are given as multiple doses. If not simultaneous, the timing between the multiple doses optionally varies from more than zero weeks to less than twelve weeks. [0383] In addition, the combination methods, compositions, and formulations are not to be limited to the use of only two agents, the use of multiple therapeutic combinations are also envisioned. It is understood that the dosage regimen to treat, prevent, or ameliorate the condition(s) for which relief is sought, is optionally modified in accordance with a variety of factors. These factors include, but are not limited to, the disorder from which the subject suffers, as well as the age, weight, sex, diet, and medical condition of the subject. Thus, the dosage regimen actually employed varies widely, in some embodiments, and therefore deviates from the dosage regimens set forth herein.

[0384] The pharmaceutical agents which make up the combination therapy disclosed herein are optionally a combined dosage form or in separate dosage forms intended for substantially simultaneous administration. The pharmaceutical agents that make up the combination therapy are optionally also administered sequentially, with either agent being administered by a regimen calling for two-step administration. The two-step administration regimen optionally calls for sequential administration of the active agents or spaced-apart administration of the separate active agents. The time period between the multiple administration steps ranges from a few minutes to several hours, depending upon the properties of each pharmaceutical agent, including, but not limited to, potency, solubility, bioavailability, plasma half-life, and kinetic profile of the pharmaceutical agent.

[0385] In another embodiment, an NO-releasing chromene conjugate is optionally used in combination with procedures that provide additional benefit to the patient. An NO-releasing chromene conjugate and any additional therapies are optionally administered before, during or after the occurrence of a disease or condition, and the timing of administering the composition containing an NO-releasing chromene prodrug varies in some embodiments. Thus, for example, an NO-releasing chromene conjugate is used as a prophylactic, and is administered continuously to subjects with a propensity to develop conditions or diseases in order to prevent the occurrence of the disease or condition. An NO-releasing chromene conjugate is optionally administered to a subject during or as soon as possible after the onset of the symptoms. While embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that in some embodiments of the invention various alternatives to the embodiments described herein are employed in practicing the invention.

[0386] A NO-releasing chromene conjugate can be used in combination with anti-cancer drugs, including but not limited to the following classes: alkylating agents, anti-metabolites, plant alkaloids and terpenoids, topoisomerase inhibitors, cytotoxic antibiotics, angiogenesis inhibitors, and tyrosine kinase inhibitors.

[0387] For use in cancer and neoplastic diseases, an NO-releasing chromene conjugate may be optimally used together with one or more of the following non-limiting examples of anti-cancer agents. As a first example, alkylating agents include but are not limited to cisplatin (PLATIN), carboplatin (PARAPLATIN), streptozocin (ZANOSAR), busulfan (MYLERAN), and cyclophosphamide (ENDOXAN). As a second example, anti-metabolites include but are not limited to mercaptopurine (PURINETHOL), thioguanine, pentostatin (NIPENT), cytosine arabinoside (ARA-C), and methotrexate (RHEUMATREX). As a third example, plant alkaloids and terpenoids include but are not limited to vincristine (ONCOVIN), vinblastine, and paclitaxel (TAXOL). As a fourth example, topoisomerase inhibitors include but are not limited to irinotecan (CAMPTOSAR), topotecan (HYCAMTIN), and etoposide (EPOSIN). As a fifth example, cytotoxic antibiotics include but are not limited to actinomycin D (COSMEGEN), doxorubicin ( ADRIAM YCIN) , bleomycin (BLENOXANE), and mitomycin (MITOSOL). As a sixth example, angiogenesis inhibitors include but are not limited to sunitinib (SUTENT) and bevacizumab (AVASTIN). As a seventh example, tyrosine kinase inhibitors include but are not limited to imatinib (GLEEVEC), erlotinib (TARCEVA), lapatininb (TYKERB), and axitinib (INLYTA). As an eigth example, EGFR inhibitors include but are not limited to the monoclonal antibody cetuximab (ERBITUX). As a nineth example, agents that target HER2 include but are not limited to the monoclonal antibodies pertuzumab (PERJETA) and trastuzumab (HERCEPTIN) which have strong co-expression links to COX-2 in prostrate and breast cancer.

[0388] Where a subject is suffering from or at risk of suffering from an inflammatory condition, an NO-releasing chromene conjugate described herein is optionally used together with one or more agents or methods for treating an inflammatory condition in any combination. Therapeutic agents/treatments for treating an autoimmune and/or inflammatory condition include, but are not limited to any of the following examples. As a first example, corticosteroids include but are not limited to cortisone, dexamethasone, and methylprednisolone. As a second example, nonsteroidal anti-inflammatory drugs (NSAIDs) include but are not limited to ibuprofen, naproxen, acetaminophen, aspirin, fenoprofen (NALFON), flurbiprofen (ANSAID), ketoprofen, oxaprozin (DAYPRO), diclofenac sodium (VOLTAREN), diclofenac potassium (CATAFLAM), etodolac (LODINE), indomethacin (INDOCIN), ketorolac (TORADOL), sulindac (CLINORIL), tolmetin (TOLECTIN), meclofenamate (MECLOMEN), mefenamic acid (PONSTEL), nabumetone (RELAFEN), and piroxicam (FELDENE). As a third example, immunosuppressants include but are not limited to methotrexate (RHEUMATREX), leflunomide (ARAVA), azathioprine (IMURAN), cyclosporine (NEORAL, SANDIMMUNE), tacrolimus, and cyclophosphamide (CYTOXAN). As a fourth example, CD20 blockers include but are not limited to rituximab (RITUXAN). As a fifth example, Tumor Necrosis Factor (TNF) blockers include but are not limited to etanercept (ENBREL), infliximab (REMICADE), and adalimumab (HUMIRA). As a sixth example, interleukin-1 receptor antagonists include but are not limited to anakinra (KINERET). As a seventh example, interleukin-6 inhibitors include but are not limited to tocilizumab (ACTEMRA). As an eighth example, interleukin-17 inhibitors include but are not limited to AIN457. As a ninth example, Janus kinase inhibitors include but are not limited to tasocitinib. As a tenth example, syk inhibitors include but are not limited to fostamatinib.

H. Kits

[0389] The present invention further comprises kits that are suitable for use in performing the methods of treatment or prevention described above. In one embodiment, the kit contains a first dosage form comprising a compound or several compounds of the present invention, and a container for the dosage, in quantities sufficient to carry out the methods of the present invention.

I. Biological Assays

Compound Metabolism in Plasma & Microsomal Stability

[0390] The present invention includes compounds that are enzymatically activated in vivo to produce chromenes. Compounds are analyzed, after incubation in plasma or serum, for the rate of disappearance of the compound species and appearance of chromene and/or intermediate compounds.

[0391] Prodrugs are dissolved into DMSO to make 3 mM stocks and stored at -20 deg C. Solutions of 5% rat serum (Sigma Cat. # S7648) or 15% human serum (Bioreclaimation, Cat. # HMSRM) are made in PBS (% v/v). In a 96-well plate, 3 μΐ. of DMSO in 127 μΐ. PBS are placed in blank wells and 3 μΐ _^ of DMSO in 127 μϊ _^ of diluted serum are placed in wells for reference standards. Test wells are charged with 127 μL· of diluted serum followed by 3 μΐ _^ of 3 mM compound DMSO stocks tested in quadruplicate. Plates are incubated at 37 °C for 90 min, removed from the incubator and placed on ice. Reference wells are diluted with 150 μΐ _^ of sodium nitrite stock solutions in PBS. Remaining wells are diluted with 150 μΐ _^ of PBS and then every well received 20 μΐ _^ of Griess Reagent (Promega Cat. # G2930). Wells are mixed, the plates are incubated for 30 min at room temperature and then absorbance is measured at 562 nm using a microplate reader. Reference standards final concentrations are 100, 33.3, 11.1, 3.7, 1.23 and 0.41 μΜ nitrite and final test concentrations for all compounds is 30 μΜ. The average DMSO blank readings are subtracted from test readings and a standard curve is generated from the reference standard wells. Nitrite levels are determined and percent release of nitric oxide is calculated, relative to theoretical maximum (60 μΜ), for each compound.

[0392] Compounds are dissolved in DMSO to make 30 mM stocks. Compounds (2 μί) are incubated with pure rat or human serum (98 μΐ _^ ; 600 μΜ) at 37 °C for various time points (T = 3h, 6h, and 24h). Reactions are stopped with 1.0 N HC1 (400 μί) and extracted with ethyl acetate (500 μΐ,). Each tube is vortexed for three 5-second intervals and the tubes are spun down at maximum speed (13,000 rpm) for 10 min to pellet protein. The ethyl acetate layer is separated (top 300 μΐ,), concentrated, diluted with 30 μΐ _^ methanol, and analyzed using HPLC by UV (254 nm) for the presence of starting compound, chromene, and intermediate compounds.

[0393] Table 7. In vitro release of chromene after 6 h or NO release after 90 min upon incubation in rat or human serum.

172 90% 10% <1% 0% 100% 64%

164a 47% 53% 5% 0% 100% 63%

[0394] Compounds (1 μΜ) are incubated, in triplicate or quadruplicate, in plasma or S9 liver microsomes fractions (rat or human) at 37 °C, reactions are quenched by acetonitrile, and samples are analyzed by LC/MS/MS (T = 0, 10, 20, 30, 45, and 60 min). Standard reverse phase HPLC and API 4000 triple quadrupole mass spectrometry are used for analysis.

[0395] Table 8. Maximal release of chromene acid after 60 min of incubation in rat or human S9 liver microsome fractions or fresh plasma.

Measurement of Nitric Oxide Release In Vivo

[0396] The compounds of the present invention contain NO-releasing moieties, which release NO in vivo. Pharmacokinetics (PK) of nitric oxide release is measured by administering a single oral (PO) gavage dose to Sprague Dawley rats. For each test compound, 2-6 Sprague Dawley (CD® IGS) male rats are used. Animals are fasted before the study and fed only after the 8-hour blood draw. Animals are weighed and dosed individually by body weight on the day of treatment. Compounds are administered orally (PO) in 2% DMSO/0.5% methylcellulose/0.1% Tween 20 in water or 2% DMSO/25% ΗΡ-β-CD (hydroxypropyl-beta-cyclodextrin) in water at 30-100 mg/kg using 10 mL/kg volume per animal. Compounds are formulated by making a 150 mg/mL DMSO compound stock and adding to warm vehicle at 35-40°C to make a clear solution or fine suspension. Animals found in severe distress or a moribund condition are euthanized. Peripheral blood collections are done primarily through venipuncture of the tail or saphenous veins or by jugular catheter at various times (e.g. pre-dose, 15min, 30min, lh, 2h, 4h, 8h, and 24h). Whole blood samples are collected in K ₂ EDTA microtainer (Fisher # 02-669-38), processed to plasma by centrifugation, and the plasma is frozen at -80°C.

[0397] Thawed plasma samples (30 are dilute with into PB along with control rat plasma. Samples are spun at 2000x g for 10 min and then 80 PBS-diluted plasma samples are transferred into the appropriate well of a 96-well plate. Sodium nitrate is used in standard curve wells at 100, 33.3, 11.1, 3.7, 1.23, and 0.41 μΜ. To each well is added ΙΟμΙ _^ of the nitrate reductase solution and 10 μΐ _^ of the enzyme co-factors solution to convert nitrate to nitrite (Cayman Chemical # 780001 Nitrate/Nitrite Colorimetric Assay Kit). The plate is incubated at room temperature for 2 h and then 50μί of Griess Reagent A is added to each well, and mixed. After 5 min., 50 μΐ _^ of Griess Reagent B is added to each well, and mixed. The plate is incubated for 10 min at room temperature, and the absorbance is measured at 540nm with a microplate reader. A standard curve is generated from the reference standard wells and nitrate/nitrite (NO _x ) levels are determined (μΜ) and standard deviation (+/-S.D.) for each blood draw and plotted against time of blood draw.

[0398] Table 9. Nitric oxide release in vivo after oral administration in rats.

* Average of two rats ** Average of three rats

Rat Air-Pouch Model for Acute Inflammation

[0399] A study of chromene release is assessed by measurement of PGE2 levels, which are indicative of inflammatory response. Compounds of the present invention lower the inflammatory response by selective COX-2 inhibition, decreasing PGE-2 levels in patients. [0400] Animals: Sprague-Dawley rats (Charles River Laboratories, R #3234, PO #738990, male, 160-180g) are received, individually examined, and housed in cages of five rats each. The rats are ear notched for identification purposes.

[0401] Compounds and dosing solutions: The vehicle is prepared by dissolving 40 g (2- hydroxypropyl)-P-cyclodextrin (ΗΡ-β-CD, Sigma, Cat. 332593, lot MKBJ5858V) in 160 mL sterile saline for injection, USP (Hospira, lot 26-801-FW) making a 25% solution which is filter sterilized (0.2 μιη, Nalgene, Cat. 151-4020, lot 1095610). A 1% carrageenan solution is prepared by dissolving 0.6 g λ-carrageenan (Fluka, Cat. 22049, lot 1318338) in hot 60 mL sterile saline for injection, USP. This solution is stored at 4-8 C. Test compounds are dissolved in DMSO (Fisher Scientific, Cat. D 128-500, lot 874999) to make 75 mM stocks. 0.25mL of compound DMSO stocks are mixed with 12.5 mL of ΗΡ-β-CD solution at 50 ^° C (maximum DMSO concentration is 2% of the final volume of vehicle,). Final concentration of all test compounds is 1.5 mM and compounds are dosed within 2 h of preparation at 0.01 mmol/kg (12 nmol of test compound per rat).

[0402] Day 0 - Air pouch initiation: The rats are anesthetized in a biological cabinet, the nape of the neck is cleansed with 70% isopropanol (Butler Schein Animal Health, Cat. 002498, lot 29EMS07104547) followed by 1% povidone-iodine solution (Ricca Chemical Co., Cat. 3955-16, lot 2205469). Twenty mL of sterile (0.22 μπι, Millipore, Cat. SLGP033RS, lot R2KA55925, exp 08/2015) air is injected subcutaneously (SC) using a 23G x 1½ inch needle fixed to a 20 mL syringe. The rats are returned to routine housing.

[0403] Day 3 - Air pouch maintenance: The rats are anesthetized in a biological cabinet, the nape of the neck is cleansed with 70% isopropanol followed by 1% povidone-iodine solution. Ten mL of sterile air is injected SC using a 23G x 1½ inch needle fixed to a 20 mL syringe. The rats are returned to routine housing in clean cages.

[0404] Day 6 - Compound administration and carrageenan insult: At commencement of the study, each rat is weighed and sorted into treatment groups of 5 rats/group based upon average weight. Each rat is dosed orally via gavage at 6.809 mL/kg (1.6 mL/235 g) with their respective test material/vehicle. Two hours after test material/vehicle administration, the rats are injected with 1.0 mL of the room temperature 1% carrageenan saline solution into the air pouch. Four hours after carrageenan injection, the rats are anesthetized, and 5 mL of the exudate buffer is injected into the air pouch. The pouch is gently massaged, the exudate immediately removed, and exudate volume recorded. The exudate is collected in a serum separator tube on an ice bath. The exudates are centrifuged (refrigerated) and an aliquot of the supernatant is stored in a labeled Eppendorf tube at -80 C.

[0405] Termination of Study: Animals are euthanized via C0 ₂ asphyxiation at the completion of the in-life portion of this study and carcasses are disposed of according to standard protocols.

[0406] Data analysis: The exudate samples are thawed to room temperature and assayed by ELISA for PGE2 (R&D Systems, Cat. KGE004B, lot 307711). Statistical significance of treatments on mean exudate volumes are determined by comparison of means for treatment groups with vehicle group. Mean cytokine concentrations and standard deviations are determined for each group. Statistical significance of treatments on cytokine concentrations are determined for each compound group compared to vehicle group. Statistical significance (p-value) is calculated vs control groups by Student's t-Test. Percent PGE2 produced relative to control is calculated using the following equations:

% PGE2 Production = (100 / Mean Vehicle Control)* (Mean Test)

S.D. % PGE2 Production = (100 / Mean Vehicle Control Value)*(S.D.)

[0407] Table 10. In vivo percent inhibition of PGE2 production relative to control.

Evaluation of COX- 1 & COX-2 Activity In Vitro

[0408] The present invention includes compounds that are chromene conjugates, therefore they are evaluated for selective COX-1 or COX-2 inhibition. Assays for COX-1 and COX-2 activity in vitro are described in U.S. Pat. 5,760,068.

[0409] Preparation of recombinant COX-1 and COX-2:

(1) A fragment containing the coding region of either human or murine COX-1 or COX-2 is cloned into a BamHl site of a baculovirus transfer vector to generate transfer vectors for COX-I and COX-II.

(2) Recombinant baculoviruses are isolated by transfecting baculovirus transfer vector DNA into SF9 insect cells.

(3) Recombinant viruses are purified and high titer stocks of virus are prepared.

(4) SF9 insect cells are infected with the recombinant baculovirus stock. After 72 hours the cells are centrifuged and the cell pellet homogenized. The homogenate is centrifuged and the supernatant is assayed for COX activity.

[0410] Assay for COX-1 and COX-2 activity:

(1) COX activity is assayed as PGE2 formed^g protein/time using an ELISA to detect the prostaglandin formed.

(2) Insect cell membranes containing the appropriate COX enzyme are incubated in buffer containing arachidonic acid.

(3) Compounds are pre-incubated with the enzyme for 10-20 min prior to the addition of arachidonic acid.

(4) Reaction between the arachidonic acid and the enzyme is stopped after ten min and the PGE2 formed is measured by standard ELISA technology.

Assessment of Anti-Proliferative Activity

[0411] Compounds of the present invention are intended to inhibit growth of tumors in patients. Anti-tumor growth potential of test compounds are evaluated in vitro using various human tumor cells, available from the American Type Culture Collection (ATCC), such as A549 lung tumor cells, DU145 prostate tumor cells, HT29 colon cancer cells, MIA PaCa-2 pancreatic cancer cells, MCF-7 (ER ⁺ ) breast tumor cells, and BEAS-2B cells (immortalized normal lung epithelial cells) as control (Clin. Cancer Res. 6, 2006-2011 (2000)). Test compound effect on cell proliferation is determined using the MTT based cell proliferation assay. MTT based cell proliferation assays are described in U.S. Pat. 8,143,237.

[0412] MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] incorporation based cell proliferation assay is performed using the MTT cell proliferation assay kit (Roche Applied Sciences, Germany). The assay is carried out according to the instruction provided by the vendor. Briefly, equal numbers of cells are plated in 96-well flat-bottomed plates and are incubated with test compounds at various concentrations for a period of three days. Vehicle control culture wells receive an equal volume of vehicle solution. Thereafter, 0.5 mg/mL of MTT reagent is added to each well and the microplate is incubated further for 4 h at 37 °C in presence of 5% C0 ₂ . Cells are then solubilized by adding solubilizing solution and allowed to incubate at 37°C overnight. After complete solubilization of the formazan crystals, the absorbance is read at 540 nm in a microplate reader (BioRad, USA). The results (mean optical density (OD) + standard dethroughtion (SD)) obtained from quadruplicate wells are used to calculate the inhibition of cell proliferation (50% of inhibitory concentration, IC ₅₀ ) of the test compounds.

Suppression of Lung Cancer Cell Migration

[0413] Compounds of the present invention are intended to have anti-lung cancer effects. Efficacy testing is done to evaluate test compound suppression of lung cancer cell migration, a model of metastasis. Methods to evaluate lung cancer cell migration are described in Mol. Med. Reports 3, 1007-1013 (2010).

[0414] Cell Culture: Human lung cancer cells A549 are obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells are incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (GibcoBRL, Grand Island, NY, USA).

Monolayer Wound Healing Assay

[0415] Compounds of the present invention are intended to have wound-healing properties. Cell proliferation in confluent A549 monolayers is blocked by a 30 min pre-incubation in the presence of mitomycin C (3 μg/mL). Test compounds, in cell culture buffer, are added to confluent monolayers 30 min before wound induction. A549 monolayers are subsequently scratched with a pipette tip. Wound areas are evaluated with phase contrast microscopy on an inverted microscope. Images of the same areas are obtained at intervals from zero to 96 h. Cell migration rate through wound healing is evaluated from the images using Paint.Net v.3.10 software. Cell migration is expressed as the fold change in the migration area, relative to untreated control cells at the same time period.

Compound Formulations for Intravenous (IV), Oral Gavage (PO) or Intraperitoneal (IP)

Administration

[0416] Compounds are formulated for administration using 25% hydroxypropyl-P-cyclodextrin- PBS buffer (ΗΡ-β-CD -PBS) at 1 mg/mL. HBCD-PBS is the preferred formulation media for compound administration. Additional formulation vehicles may also be used, including 2% Tween 80 in saline and 20% polyethylene glycol (PEG-300) in 0.9% sodium chloride in water.

Determination of Maximum Tolerated Dose (MTD) of Test Compounds in Rats

[0417] In order to estimate the doses of test compounds for use in efficacy testing in animal models of cancer, the dosage at which adverse events occur is determined. Methods to determine MTD in rats are described in Mol. Cancer Ther. 5, 1530-1538 (2006).

[0418] In order to determine doses for efficacy studies, the maximum tolerated dose (MTD) is determined. Male F344 rats are fed various concentrations of test compounds for six weeks. MTD is determined based on the highest dose that causes a 10% loss in body weight without mortality or signs of toxicity. Body weights are recorded twice weekly. Animals are examined daily for signs of toxicity. At termination, animals are euthanized, and organs dissected and examined.

Compound Metabolism (PK) in Rats

[0419] The pharmacokinetics (PK) of compounds is tested by single dose IV administration to Sprague Dawley rats.

[0420] For each test compound, three (3) Sprague Dawley (CD® IGS) male rats are used. Animals are weighed and dosed individually by body weight on the day of treatment. Compounds are administered intravenously (IV), through surgically placed jugular catheters, at 10 mg/kg using 10 mL/kg volume per animal. Animals found in severe distress or a moribund condition are euthanized. Peripheral blood collections are done primarily through venipuncture of the tail or saphenous veins at various times (T = 15min, 30min, lh, 2h, 4h, 8h, and 24h). Whole blood samples are collected in an EDTA microtainer, processed to plasma by centrifugation and the plasma frozen at -80°C. Bioanalysis is done using LC/MS/MS methods using standard reverse phase HPLC and API 4000 triple quadrupole mass spectrometry. The amount of compound present is used to calculate PK parameters C _max , T _max , and AUC.

Compound Effects on Blood Pressure

[0421] COX-2 inhibitors have been shown to have adverse effects on blood pressure in vivo, the effect of the present compounds is evaluated for blood pressure effects in spontaneously hypertensive rats (SHR). [0422] Thirty-two male, spontaneously hypertensive rats (SHR), 12-weeks old (four groups of eight) are used in this study. Initially, mean arterial blood pressure (MAP) is measured through tail-cuff daily, throughout the study. Animals undergo 2 days of blood pressure training and 1 day of baseline blood pressure measurements. Animals are weighed and dosed individually by body weight on the day of treatment. Compounds are administered orally (PO) or by intraperitoneal (IP) injection once on Day 1 at 10 mg/kg using 10 mL/kg volume per animal. Blood pressures are monitored for 6 days post-dose. A total of 7 time points are measured: Day 0 for baseline and Days 1, 2, 3, 4, 5, and 6 of the study. Animals found in severe distress or in a moribund condition are euthanized. Celecoxib is the positive control tested in these studies.

[0423] Anti-inflammatory Efficacy: Rat Carrageenan Foot Pad Edema: The compounds of the present invention are conjugates of chromenes, therefore they are evaluated for efficacy in vivo in a model of inflammation. Methods to determine efficacy in rat carrageenan foot pad edema are described in U.S. Pat. 5,760,068.

[0424] Male Sprague Dawley rats are selected for equal average body weight per group. After fasting, with free access to water sixteen hours prior to test, animals are dosed orally (1 mL) with test compounds in a vehicle containing 0.5% methylcellulose and 0.025% surfactant. The control group is dosed with vehicle alone.

[0425] One hour after dosing, a subplantar injection of 0.1 mL of 1% solution of carrageenan/sterile 0.9% saline is administered in one foot, to all animals. The volume of the injected foot is measured using a displacement plethysmometer. Foot volume is measured again three hours after carrageenan injection. The three hour foot volume measurement is compared between treated and control groups; the percent inhibition of edema is calculated.

Anti-inflammatory Efficacy - Rat Carrageenan-Induced Analgesia Test

[0426] The compounds of the present invention are conjugates of chromenes, therefore they are evaluated for efficacy in vivo in a model of inflammatory analgesia. Methods to determine efficacy in rat carrageenan-induced analgesia test are described in U.S. Pat. 5,760,068.

[0427] Male Sprague Dawley rats are selected for equal average body weight per group. After fasting, with free access to water sixteen hours prior to test, animals are dosed orally (1 mL) with test compounds in vehicle containing 0.5% methylcellulose and 0.025% surfactant. Control groups are dosed with vehicle alone. [0428] One hour after dosing, a subplantar injection of 0.1 mL of 1% solution of carrageenan/sterile 0.9% saline is administered in one foot, to all animals. Three hours after carrageenan injection, rats are placed in a plexiglass container with a high intensity lamp under the floor. After 20 min, thermal stimulation is begun on either the injected or the uninjected foot. Foot withdrawal is determined by a photoelectric cell. The time until foot withdrawal is measured and compared between treated and control groups. The percent inhibition of the hyperalgesic foot withdrawal is calculated.

Tumor Growth Inhibition in Xenograft Mouse Model of Colon Cancer

[0429] Compounds of the present invention are intended to have anti-colon cancer effects in patients. Efficacy testing is done in animal models of cancer tumors. Methods to determine tumor growth inhibition in xenograft mouse models of colon cancer are described in J. Drug Delivery 2011, 1-9 (Article ID 869027) and Invest. New Drugs 2014, 32(6), 1105-12.

[0430] HT-29 cells are trypsinized, resuspended in sterile PBS, and pelleted by brief centrifugation at 200 x g. The cell pellet is resuspended in sterile PBS and counted using a hemocytometer. Cells are resuspended in PBS to a final concentration of 5 x 10 cells /mL. Female HRLN nu/nu mice are injected subcutaneously into the high axilla region with 5 x 10 ⁶ HT-29 cells in 0.1 mL of PBS. Mice are triaged into treatment groups (10 mice/group) when mean tumor burden is 100-200mg (target 150mg, -10 days of logarithmic growth), at which point treatment is initiated. Mice are distributed into treatment groups such that the mean tumor burden in each group is within 10% of the overall mean. Body weights and tumor measures are recorded 3x/week, and clinical signs are recorded daily. Tumor volume is determined using digital calipers and calculated according to the equation V = (L x W )/2, where V is the volume, L is the length, and W is the width. Mice are dosed individually by body weight once daily (QD) or twice daily (bid or Q12Hx2). Testing compounds are formulated in 1% methylcellulose/0.1% Tween-80/2% DMSO or 2% DMSO/25% hydroxypropyl-P-cyclodextrin (ΗΡ-β-CD) in water and administered via oral dosing (p.o.) at 1, 3, or 10 mpk. 5-FU (i.p. dosing; Q7Dx3; 100 mpk) and celecoxib (p.o. dosing; Q12Hx2; 30 mpk) are administered as positive controls and vehicle alone as the negative control. Animals with tumor burdens greater than 2g or found in a moribund condition are euthanized, otherwise animals are euthanized, and tumors are harvested and measured after 28 days of treatment. Gross necropsy is performed on every animal leaving the study and abnormal findings are recorded. Drug efficacy is measured based on animal survival and tumor growth inhibition relative to negative control.

Tumor Growth Inhibition in Xenograft Mouse Model of NSCLC

[0431] Compounds of the present invention are intended to inhibit the growth of cancerous tumors in patients. Efficacy testing is done in animal models of cancer tumors. Methods to determine tumor growth inhibition in xenograft mouse models of NSCLC are described in Clin. Cancer Res. 7, 724-733 (2001) and are similar to the detail method described above for Colon Cancer.

[0432] Female HRLN nu/nu mice are injected subcutaneously with 1 x 10 ⁷ MV-522 cells in 0.1 mL of phosphate-buffered saline. Treatment is initiated when tumors measure 5x5 mm. Mice are weighed and tumors measured by calipers twice weekly. Animals are euthanized, and tumors are harvested and measured after 67 days or when animal dies. Drug efficacy is measured based on animal survival and tumor growth.

Growth Inhibition of Gallbladder Adenocarcinoma in Transgenic Mice

[0433] Compounds of the present invention are intended to inhibit the growth of cancerous tumors in patients. Efficacy testing is done in animal models of cancer tumors. Gallbladder adenocarcinoma in transgenic mice is described in Mol. Cancer Ther. 6, 1709-1717 (2007).

[0434] Homozygous BK5.ErbB-2 transgenic mice, that overexpress rat ErbB-2 and nontransgenic littermates receive a control AIN76A diet or an experimental diet containing the test compound for one month. The transgenic mice develop adenocarcinoma of the gallbladder with a 90% incidence. Ultrasound image analysis and histologic evaluation are used to determine compound effects on gall bladder tumor reversion to a milder phenotype and inhibition of tumor progression.

Inhibition of Colon Cancer in Azomethane-Treated Rats

[0435] Compounds of the present invention are intended to have anti-colon cancer effects in patients. Efficacy testing is done in animal models of cancer tumors. Colon cancer in azomethane-treated rats is described in Mol. Cancer Ther. 5, 1530-1538 (2006).

[0436] Male F344 rats (Charles River Breeding Laboratories) are given test compounds blended into the diet. Efficacy of test compounds are determined following initiation of azoxymethane- induced colon cancer. Rats are randomly distributed by weight into various groups and housed in cages. Azomethane treated animals are injected subcutaneous (s.c), twice weekly, at 15 mg/kg body weight. Vehicle-treated groups are injected with normal saline. Rats are placed on control diet or diets containing test compounds, two weeks after the second injection of azomethane or saline. Body weights are measured every two weeks until termination, 52 weeks after the last azoxymethane treatment. Organs are dissected and examined using a dissecting microscope.

[0437] Colon tumors with a diameter of >0.4 cm are fixed in 10% neutral buffered formalin for histopathologic evaluation. Test compounds are evaluated for effect on colonocyte proliferation. Proliferating cell nuclear antigen (PCNA) expression is determined by immunohistochemistry. Paraffin-embedded colons are sectioned and mounted on slides. PCNA antibody (PharMingen, San Diego, CA), at a 1:200 dilution, is added for 1 hour. Sections are washed, then incubated with secondary anti-rabbit IgG (30 min). Following washing, avidin biotin-complex reagent

(Vector Laboratories, Burlingame, CA) is added. Sections are washed, 3,3"-diaminobenzidine is added, and sections are counterstained with hematoxylin. Proliferation index is calculated based on the number of positive cells (brown nucleus) per crypt.

Patient Selection Using Urinary PGE-M

[0438] Compounds of the present invention inhibit COX-2 activity. Urinary PGE-M can serve as a diagnostic marker of aberrant COX-2 over-expression in patients with COX-2 dependent cancers. Accordingly, urinary PGE-M levels can be useful in patient selection, as patients with elevated PGE-M levels can be targeted for therapy with compounds of the present invention. Urinary PGE-M level is typically measured using a liquid chromatography/tandem mass spectrometric method as described in Murphey, L. J. et al.: "Quantification of major urinary metabolite of PGE2 by a liquid chromato graphic/mass spectrometric assay: Determination of cyclooxygenase specific PGE2 synthesis in healthy humans and those with lung cancer" Anal. Biochem. 2004, 334, 266-75 and US Patent Application 2012/0016002 Al, the entire contents of which are hereby incorporated by reference. Alternatively, urinary PGE-M level is also measured using commercially available ELISA kits from vendors such as Cayman Chemical (Item Number 514531) following protocols outlined in accompanying technical documents, the entire contents of which are hereby incorporated by reference. [0439] Urinary PGE-M LCMS Protocol: Briefly, 0.75 mL urine is acidified to pH 3 with dilute aqueous hydrochloric acid and endogenous PGE-M is then converted to O-methyloxime derivative by treatment with methyloxime hydrochloride. The methoximated PGE-M is extracted with ethyl acetate, applied to a C-18 Sep-Pak, and eluted with ethyl acetate. An [ FLJ-O- methyloxime PGE-M internal standard is then added. Liquid chromatography is performed on a Zorbax Eclipse XDB-C18 column attached to a Thermo Finnigan Surveyor MS Pump (Thermo Finnigan, San Jose, Calif.). For endogenous PGE-M, the predominant product ion m/z 336 representing [M-OCH ₃ +H ₂ 0] ^" and the analogous ion, m/z 339 (M-OC[ ² H] ₃ +H ₂ 0)] ^" , for the deuterated internal standard, are monitored in the selected reaction monitoring (SRM) mode. Quantification of endogenous PGE-M utilizes the ratio of the mass chromatogram peak areas of the m/z 336 and m/z 339 ions. Urinary creatinine levels are measured using a test kit from SIGMA Company (St. Louis, Mo.). Urine samples for each case-control pair are analyzed in the same batch and adjacently to eliminate between-assay variability. Individuals having elevated PGE-M levels relative to control urine are identified and administered therapy as described herein.

[0440] All mentioned documents are incorporated by reference as if herein written. When introducing elements of the present invention or the exemplary embodiment(s) thereof, the articles "a," "an," "the", and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including", and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. Although this invention has been described with respect to specific embodiments, the details of these embodiments are not to be construed as limitations.