Novel trpv1 agonists and uses thereof

Abstract

Novel TRPV1 agonists and uses thereof are provided, including modified resiniferatoxin (RTX) to desensitize TRPV1 -expressing neurons. The modified RTX disclosed herein may comprise at least one substitution on the distal 6-membered ring of RTX, a replacement of the 6-membered distal ring of RTX, or at least one alteration of resiniferonol 9, 13, 14-ortho-phenylacetate, which can further comprise at least one substitution on the distal 6-membered ring of RTX and / or a replacement of the 6-membered distal ring of RTX

Description

NOVEL TRPV1 AGONISTS AND USES THEREOFBACKGROUND

[0001] Transient receptor potential vanilloid (TRPV) channels are a family of nonselective cation channels that are present on the cell membranes of various cells throughout the body. Of this family, transient receptor potential vanilloid subtype 1 (TRPV1) channels located on sensory neurons and are responsible for the transduction of noxious stimuli such as heat and pain. Exogenous agonists of TRPV1 such as capsaicin and resiniferatoxin (RTX) depolarize TRPV1- expressing neurons and can be specifically used to desensitize this population of neurons. Thus, the use of exogenous TRPV1 agonists is emerging as a method to provide analgesia (e.g., inflammatory pain, neuropathic pain, cancer pain) as well as to treat a range of clinical conditions such as cardiovascular diseases (e.g., heart failure, arrhythmias), overactive bladder, gastrointestinal disease (e.g., irritable bowel syndrome), cough, and asthma.

[0002] Accordingly, there is a need for novel TRPV1 agonists that can desensitize neurons.SUMMARY

[0003] The present disclosure is directed to a family of compounds comprising a modified RTX. In an embodiment, the modified RTX comprises at least one substitution on the distal 6-membered ring of RTX. In another embodiment, the modified RTX comprises a replacement of the 6- membered distal ring of RTX. In another embodiment, the modified RTX comprises at least one alteration of resiniferonol 9,13,14-ortho-phenylacetate, which can further comprise at least one substitution on the distal 6-membered ring of RTX and / or a replacement of the 6-membered distal ring of RTX.

[0004] The present disclosure is also directed to a compound comprising certain chemical structures disclosed herein and / or a composition comprising the chemical structures disclosed herein and further comprising an excipient. In an embodiment, the excipient is selected from at least one of the group consisting of ethanol, methanol, polyethylene glycol, dimethyl sulfoxide, sodium chloride, or a cyclodextran. In another embodiment, the composition further comprises a solubility enhancer. In an embodiment, the solubility enhancer comprises a protein, including but not limited to albumin or a derivative thereof.[0051 The present disclosure is also directed to a method of administering the compounds or compositions disclosed herein to a subject in need thereof. In an embodiment, the method comprises administration by at least one of topical, subcutaneous, epicardial, epidural, intrathecal, peri or intra-ganglionic, vascular, intraarticular, interarticular, pericardial, intrapericardial, or intravesical administration.DRAWINGS

[0006] FIG. 1 depicts the synthesis schematic of COMPOUND 3.

[0007] FIG. 2 depicts the synthesis schematic of COMPOUND 5.

[0008] FIG. 3 depicts the synthesis schematic of COMPOUND 7.

[0009] FIG. 4 depicts the synthesis schematic of COMPOUND 9.

[0010] FIG. 5 depicts the synthesis schematic of COMPOUND 11.

[0011] FIG. 6 depicts the synthesis schematic of COMPOUND 13.

[0012] FIG. 7 depicts the synthesis schematic of COMPOUND 15.

[0013] FIG. 8 depicts the synthesis schematic of COMPOUND 17.

[0014] FIG. 9 depicts the synthesis schematic of COMPOUND 19.

[0015] FIG. 10 depicts the synthesis schematic of COMPOUND 21.

[0016] FIG. 11 depicts the synthesis schematic of COMPOUND 23.

[0017] FIG. 12 depicts the TRPV1 agonist assay data for COMPOUNDS 3, 5, 7, 9, and 11, RTX, and Capsaicin.

[0018] FIG. 13A depicts the TRPV1 agonist assay data for COMPOUNDS 13, 15, 17, 19, and 21.

[0019] FIG. 13B depicts the TRPV1 agonist assay data for COMPOUNDS 23, 9, and 11, RTX, and Capsaicin.

[0020] FIG. 14 depicts the estimated EC50 (nM) of COMPOUNDS 13, 15, 17, 19, 21, 23, 9, and 11, and RTX.

[0021] FIG. 15 depicts the TRPV1 agonist assay data for RTX and capsaicin.

[0022] FIG. 16 depicts desensitization studies of COMPOUNDS 13 and 21, RTX, and Capsaicin.

[0023] FIG. 17A presents the data for a TRPV1 agonist assay performed on COMPOUND 21.

[0024] FIG. 17B presents the data for a TRPV1 agonist assay performed on RTX, capsaicin, and capsazepine.

[0025] FIG. 18A graphically depicts the data from the TRPV1 agonist assay performed on COMPOUND 21.

[0026] FIG. 18B graphically depicts the data from the TRPV1 agonist assay performed on RTX.

[0027] FIG. 18C graphically depicts the data from the TRPV1 agonist assay performed on capsaicin.

[0028] FIG. 18D graphically depicts the data from the TRPV1 agonist assay performed on capsazepine.

[0029] FIG. 19 depicts a study protocol for evaluation of COMPOUND 21 and COMPOUND 9 in a pig model.

[0030] FIG. 20A depicts the heart rate change during the study protocol depicted in FIG. 19 for COMPOUND 21.

[0031] FIG. 20B depicts the blood pressure change during the study protocol depicted in FIG. 19 for COMPOUND 21.

[0032] FIG. 20C depicts the heart rate change during the study protocol depicted in FIG. 19 for COMPOUND 9.

[0033] FIG. 20D depicts the blood pressure change during the study protocol depicted in FIG. 19 for COMPOUND 9.DETAILED DESCRIPTION

[0034] The present disclosure provides compounds and compositions of novel TRPV1 agonists and methods of treating a subject in need comprising administering the compositions disclosed herein.

[0035] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. The use of the term “or” in the claims and the present disclosure is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0036] As used herein, abbreviations for chemical compounds and elements are consistent with their customary use in the art unless otherwise noted. For example, “N” refers to Nitrogen, “C” refers to Carbon, “F” refers to Fluorine, “H” refers to Hydrogen, “S” refers to Sulfur, “Me” and / or “CH3” refers to a Methyl group, and “CH2” refers to Methylene.

[0037] The present disclosure provides compounds that are novel agonists of the TRPV1 receptor.

[0038] In an embodiment, agonists of the TRPV1 receptor are equivalent to or no less than 1000 fold less potent than RTX.

[0039] In an embodiment, agonists of the TRPV1 receptor can stimulate and desensitize the TRPV1 receptor. One method to test desensitization is as follows: a series of compound dilutions are prepared and applied to cells that express the TRPV1 receptor to test the amount of calcium (flux) into the cell with application of the agonist. As the concentration of the agonist increases, the amount of calcium (flux) increases until it hits a maximal plateau. The EC50 is the concentration that causes 50% of the maximal stimulation. Following this, the agonist is washed out and the cells are allowed to rest for a few hours. The cells are then stimulated with capsaicin, a known agonist of the TRPV1 receptor, and amount of calcium (flux) is again observed. Use of capsaicin as the agonist in both rounds of stimulation will produce essentially the same EC50 curve, i.e., capsaicin does not desensitize the TRPV1 receptor. Desensitization is observed where a second round of stimulation with capsaicin does not result in any stimulation of the calcium (flux). RTX is a compound known to produce this desensitization effect — RTX applied the first round of stimulation produces calcium (flux), and the following round of stimulation with capsaicin will not result in stimulation. One of skill in the art will understand that variations of the above-described desensitization test can also aid in identifying TRPV1 agonists that desensitize the TRPV 1 receptor.

[0040] In some embodiments, the compounds comprise the base structure of RTX (reproduced below) with modifications.

[0041] In some embodiments, the compounds comprise substitutions on the distal 6-membered ring of RTX (circled in dotted line below).By way of example but not limitation, such compounds comprising substitutions on the distal 6- membered ring of RTX comprise COMPOUNDS 3, 5, 7, 9, 11, 15, OR 17 depicted below:

[0042] In some embodiments, the compounds comprise a replacement of the 6-membered distal ring of RTX (circled in dotted line above). By way of example but not limitation, such compounds comprising a replacement of the 6-membered distal ring of RTX comprise COMPOUNDS 13, 19, 21, or 23 depicted below:

[0043] In some embodiments, the compounds comprise alterations of the RTX structures circled in dotted line below.

[0044] By way of example, but not limitation, such alterations of the RTX structured circled above (resiniferonol 9,13,14-ortho-phenylacetate (ROPA)) may comprise replacement of the circled structure as disclosed in Kim et al., Bioorg Med Chem Lett, 2014 January 01; 24(1): 382- 385. doi: 10.1016 / j.bmcl.2013.10.064.

[0045] In some embodiments, the compound comprises:

[0046] By way of example but not limitation, in the above disclosed compound, X=CH2 and n=l- 3; R1 is selected from H, Me, or alkyl, haloalkyl, cycloalkyl and R2= H. In another configuration, by way of example but not limitation, X=CH2 and n=l or n=2-3; R2 is selected from H, Me, or alkyl, haloalkyl, cycloalkyl and Rl= H.

[0047] In some embodiments the compound comprises:

[0048] By way of example but not limitation, in the above disclosed compound, X= CH2, ni=l- 3; Y= CH2, and n2=l-3.

[0049] In some embodiments the compound comprises:

[0050] By way of example but not limitation, in the above disclosed compound X=CH2, m=l-3;Y= CH2, n2=l-3; A and B=N, or A=C and B=N; or A=N and B=C.

[0051] In some embodiments the compound comprises:

[0052] By way of example but not limitation, in the above disclosed compound X= CH2, m=l- 3; Y= CH2, n2=l-3, and A is selected from S, N-Me, or NH.

[0053] In some embodiments the compound comprises:

[0054] By way of example but not limitation, in the above disclosed compound X=CH2, m=l-3;Y= CH2, n2=l-3, and B is selected from S, N-Me, or NH.

[0055] In some embodiments the compound comprises:

[0056] By way of example but not limitation, in the above disclosed compound R3=CH3 H, X=N, Y=C, and R4 is selected from Me, alkyl, haloalkyl, cycloalkyl or a branched alkyl. In another configuration, by way of example but not limitation, R3=CH3 H, Y=N, X=C, and R4 is selected from Me, alkyl, haloalkyl, cycloalkyl or a branched alkyl.

[0057] In some embodiments the compound comprises:

[0058] By way of example but not limitation, in the above disclosed compound R3=CH3 H, X=N, Y=C, R4 is selected from Me, alkyl, haloalkyl, cycloalkyl or a branched alkyl, and R5= F. In another configuration, by way of example but not limitation, R3=CH3 H, Y=N, X=C, R4 is selected from Me, alkyl, or a branched alkyl, and R5=F.

[0059] The present disclosure also provides compositions comprising the compounds described above.

[0060] In some embodiments, the composition further comprises one or more excipients. Such excipients include those known in the art, by way of example but not limitation, said excipients include bulking agents, fillers, or diluents. By way of example but not limitation, said excipients include ethanol, methanol, polyethylene glycol, tween, dimethyl sulfoxide (“DMSO”), sodium chloride, and cyclodextrans, or any other bulking agent, filler, or diluent.

[0061] In some embodiments, the composition further comprises one or more solubility enhancers that increase solubility of a TRPV1 modulator. Solubility can be measured by various means known by those of skill in the art, including but not limited to measuring the amount of a compound of interest in the supernatant by HPLC / MS. Solubility enhancers may include, but are not limited to proteins such as albumin and derivatives thereof.

[0062] The present disclosure also provides a method of administering the compounds or compositions disclosed herein to a subject in need thereof.

[0063] Administration of the compounds or compositions disclosed herein can be performed by methods known in the art, including but not limited to topical, subcutaneous, epicardial, epidural, intrathecal, peri or intra-ganglionic, vascular, intraarticular, interarticular, pericardial, intraperi cardial, and intravesical routes of administration.

[0064] In some embodiments, the methods disclosed herein are used to treat acute or chronic cardiovascular disease, acute or chronic pain, acute or chronic lung disease such as chronic asthma or obstructive pulmonary disease, acute or chronic arthritis, acute or chronic radiculopathy, hypertension, myocardial infarction, arrhythmias, heart failure, or other conditions for which inflammation and / or the TRPV1 -neural pathway is an important pathophysiologic factor and neural desensitization is desired.EXAMPLES

[0065] The following examples are provided to better illustrate the methods of the present disclosure and the resultant effects on a chronic disease (specifically myocardial infarction). These examples are not intended to be limited or to otherwise alter the scope of the compounds, compositions, or methods disclosed in the present disclosure.Example 1

[0066] Novel compounds of the present disclosure were synthesized as follows.

[0067] COMPOUND 3. FIG. 1 depicts the synthesis schematic of COMPOUND 3 (chemical name ((3aR,6aR,9aR,9bR,10R,l laR)-2-benzyl-6a-hydroxy-8,10-dimethyl-7-oxo-l la-(prop-l- en-2-yl)-3a,3b,6,6a,9a,10,l 1,1 la-octahydro-7H-2,9b-epoxyazuleno[5',4':3,4]benzo[l,2- d][l,3]dioxol-5-yl)methyl 2-(benzo[d][l,3]dioxol-5-yl)acetate). COMPOUND 3 was synthesized as follows: Ten milligrams of resiniferonol-9,13,14-orthophenyl acetate (also referred to as daphnetoxin, 6,7-deepoxy-6,7-didehydro-5-deoxy- 2 l-dephenyl-21 -(phenylmethyl)) (compound 1) was dissolved in 0.5 mL of dimethylformamide (also referred to as N,N-dimethylmethanamide) (DMF). Eight milligrams or 2 equivalents of COMPOUND 2 (chemical name 2-(l,3-benzodioxol- 5-yl)acetic acid) was added followed by 3 equivalents of diisopropylethylamine (also referred to as N,N-diisopropylethylamine) (DIEA) and 2 equivalents of hexafluorophosphate benzotriazole tetramethyl uranium (also referred to as 3-[Bis(dimethylamino)methyliumyl]-3H-benzotriazol-l- oxide hexafluorophosphate) (HBTU). The mixture was stirred at room temperature overnight. The reaction mixture was loaded directly in a 4 g silica gel column and 0-100% ethyl acetate / hexane gradient was used to produce 6.5 mg of COMPOUND 3. Note LCMS ESI (M+Na)+649.4; HPLC retention time: 4.25 min.

[0068] COMPOUND 5. FIG. 2 depicts the synthesis schematic of COMPOUND 5 (chemical name ((3aR,6aR,9aR,9bR,10R,l laR)-2-benzyl-6a-hydroxy-8,10-dimethyl-7-oxo-l la-(prop-l- en-2-yl)-3 a, 3b, 6, 6a, 9a, 10,11,11 a-octahydro-7H-2,9b-epoxyazuleno[5',4' : 3 ,4]benzo[ 1 ,2- d][l,3]dioxol-5-yl)methyl 2-(2,3-dihydrobenzo[b][l,4]dioxin-6-yl)acetate). COMPOUND 5 was synthesized as follows: ten milligrams of resiniferonol-9,13,14-orthophenyl acetate (compound 1) was dissolved in 0.5 mL of dimethylformamide (DMF). Nine milligrams or 2 equivalents of COMPOUND 4 (chemical name 2-(2,3-dihydro-l,4-benzodioxin-6-yl)acetic acid) was added followed by 3 equivalents of diisopropylethylamine (DIEA) and 2 equivalents of hexafluorophosphate benzotriazole tetramethyl uranium (HBTU). The mixture was stirred at room temperature overnight. The reaction mixture was loaded directly in a 4 g silica gel column and 0- 20% ethyl acetate / hexane gradient was used to produce 7 mg of COMPOUND 5. Note LCMS ESI (M+Na)+663.2 HPLC retention time: 4.20 min.

[0069] COMPOUND 7. FIG. 3 depicts the synthesis schematic of COMPOUND 7 (chemical name ((3aR,6aR,9aR,9bR,10R,l laR)-2-benzyl-6a-hydroxy-8,10-dimethyl-7-oxo-l la-(prop-l- en-2-yl)-3a,3b,6,6a,9a,10,l 1,1 la-octahydro-7H-2,9b-epoxyazuleno[5',4':3,4]benzo[l,2- d][l,3]dioxol-5-yl)methyl 2-(3,4-dihydro-2H-benzo[b][l,4]dioxepin-7-yl)acetate). COMPOUND7 was synthesized as follows: Ten milligrams of resiniferonol-9,13, 14-orthophenyl acetate (compound 1) was dissolved in 0.5 mL of dimethylformamide (DMF). 13.6 mg or 3 equivalents of COMPOUND 6 (chemical name 2-(3,4-dihydro-2H-l,5-benzodioxepin-7-yl)acetic acid) was added followed by 3 equivalents of diisopropylethylamine (DIEA) and 2 equivalents of hexafluorophosphate benzotriazole tetramethyl uranium (HBTU). The mixture was stirred at room temperature overnight. The reaction mixture was loaded directly in a 4 g silica gel column and 0- 40% ethyl acetate / hexane gradient was used to produce 10 mg of COMPOUND 7. Note, LCMS ESI (M+Na)+677.6; HPLC retention time: 4.21 min.

[0070] COMPOUND 9. FIG. 4 depicts the synthesis schematic of COMPOUND 9 (chemical name ((3aR,6aR,9aR,9bR,10R,l laR)-2-benzyl-6a-hydroxy-8,10-dimethyl-7-oxo-l la-(prop-l- en-2-yl)-3a,3b,6,6a,9a,10,l 1,1 la-octahydro-7H-2,9b-epoxyazuleno[5',4':3,4]benzo[l,2- d][l,3]dioxol-5-yl)methyl 2-(3,4-dimethoxyphenyl)acetate). COMPOUND 9 was synthesized as follows: Ten milligrams of resiniferonol-9,13,14-orthophenyl acetate (compound 1) was dissolved in 0.5 mL of dimethylformamide (DMF). Nine milligrams or 2 equivalents of COMPOUND 8 (chemical name 2-(3,4-dimethoxyphenyl)acetic acid) was added followed by 3 equivalents of diisopropylethylamine (DIEA) and 2 equivalents of hexafluorophosphate benzotriazole tetramethyl uranium (HBTU). The mixture was stirred at room temperature overnight. The reaction mixture was loaded directly in a 4 g silica gel column and 0-35% ethyl acetate / hexane gradient was used to produce 10 mg of COMPOUND 9. Note, LCMS ESI (M+Na)+665; HPLC retention time: 4.13 min.

[0071] COMPOUND 11. FIG. 5 depicts the synthesis schematic of COMPOUND 11 (chemical name [((3aR,6aR,9aR,9bR,10R,l laR)-2-benzyl-6a-hydroxy-8,10-dimethyl-7-oxo-l la-(prop-l- en-2-yl)-3 a, 3b, 6, 6a, 9a, 10,11,11 a-octahydro-7H-2,9b-epoxyazuleno[5',4' : 3 ,4]benzo[ 1 ,2- d][l,3]dioxol-5-yl)methyl-2-(3,4-dimethoxyphenyl)propanoate]). COMPOUND 11 was synthesized as follows: Ten milligrams of resiniferonol-9,13,14-orthophenyl acetate (compound 1) was dissolved in 0.5 mL of dimethylformamide (DMF). Ten milligrams or 2 equivalents of COMPOUND 10 (chemical name 2-(3,4-dimethoxyphenyl)propanoic acid) was added followed by 3 equivalents of diisopropylethylamine (DIEA) and 2 equivalents of hexafluorophosphate benzotriazole tetramethyl uranium (HBTU). The mixture was stirred at room temperature overnight. The reaction mixture was loaded directly in a 4 g silica gel column and 0-35% ethylacetate / hexane gradient was used to produce 10 mg of COMPOUND 11. Note, LCMS ESI (M+H)+657.8; (M+Na)+679.7; HPLC retention time: 4.13 min.

[0072] COMPOUND 13. FIG. 6 depicts the synthesis schematic of COMPOUND 13 (chemical name ((3aR,6aR,9aR,9bR,10R, l laR)-2-benzyl-6a-hydroxy-8,10-dimethyl-7-oxo-l la-(prop-l- en-2-yl)-3 a, 3b, 6, 6a, 9a, 10,11,11 a-octahydro-7H-2,9b-epoxyazuleno[5',4' : 3 ,4]benzo[ 1 ,2- d][l,3]dioxol-5-yl)methyl 2-(2-methoxypyridin-4-yl)acetate). COMPOUND 13 was synthesized as follows: Ten milligrams of resiniferonol-9,13,14-orthophenyl acetate (compound 1) was dissolved in 0.5 m of dimethylformamide (DMF). Seven milligrams or 2 equivalents of COMPOUND 12 (chemical name 2-(2-methoxy-4-pyridyl)acetic acid) was added followed by 3 equivalents of diisopropylethylamine (DIEA) and 2 equivalents of hexafluorophosphate benzotriazole tetramethyl uranium (HBTU). The mixture was stirred at room temperature overnight. The reaction mixture was loaded directly in a 4 g silica gel column and 0-30% ethyl acetate / hexane gradient was used to produce 4.5 mg of COMPOUND 13. Note, LCMS ESI (M+H)+614.4; HPLC retention time: 4.16 min.

[0073] COMPOUND 15. FIG. 7 depicts the synthesis schematic of COMPOUND 15 (RTX) (chemical name ((3aR,6aR,9aR,9bR,10R,l laR)-2-benzyl-6a-hydroxy-8,10-dimethyl-7-oxo-l la- (prop- 1 -en-2-yl)-3 a, 3b, 6, 6a, 9a, 10, 11 , 11 a-octahydro-7H-2,9b-epoxyazuleno[5',4' : 3,4]benzo[ 1 ,2- d][l,3]dioxol-5-yl)methyl 2-(3-methoxy-4-methylphenyl)acetate). COMPOUND 15 was synthesized as follows: Ten milligrams of resiniferonol-9,13,14-orthophenyl acetate (compound 1) was dissolved in 0.5 mL of dimethylformamide (DMF). Eight milligrams or 2 equivalents of COMPOUND 14 (chemical name 2-(3-methoxy-4-methyl-phenyl)acetic acid) was added followed by 3 equivalents of diisopropylethylamine (DIEA) and 2 equivalents of hexafluorophosphate benzotriazole tetramethyl uranium (HBTU). The mixture was stirred at room temperature overnight. The reaction mixture was loaded directly in a 4 g silica gel column and 0-20% ethyl acetate / hexane gradient was used to produce 4.5 mg of COMPOUND 15. Note, LCMS ESI (M+H)+627.6; HPLC retention time: 4.08 min.

[0074] COMPOUND 17. FIG. 8 depicts the synthesis schematic of COMPOUND 17 (chemical name ((3aR,6aR,9aR,9bR,10R,l laR)-2-benzyl-6a-hydroxy-8,10-dimethyl-7-oxo-l la-(prop-l- en-2-yl)-3 a, 3b, 6, 6a, 9a, 10,11,11 a-octahydro-7H-2,9b-epoxyazuleno[5',4' : 3 ,4]benzo[ 1 ,2- d][l,3]dioxol-5-yl)methyl 2-(4-fluoro-3-methoxyphenyl)acetate). COMPOUND 17 was synthesized as follows: Ten milligrams of resiniferonol-9, 13,14-orthophenyl acetate (compound1) was dissolved in 0.5 mL of dimethylformamide (DMF). Eight milligrams or 2 equivalents of COMPOUND 16 (chemical name 2-(4-fluoro-3-methoxy-phenyl)acetic acid) was added followed by 3 equivalents of diisopropylethylamine (DIEA) and 2 equivalents of hexafluorophosphate benzotriazole tetramethyl uranium (HBTU). The mixture was stirred at room temperature overnight. The reaction mixture was loaded directly in a 4 g silica gel column and 0-30% ethyl acetate / hexane gradient was used to produce 10 mg of COMPOUND 17. Note, LCMS ESI (M+H)+631.1; HPLC retention time: 4.13 min.

[0075] COMPOUND 19. FIG. 9 depicts the synthesis schematic of COMPOUND 19 (chemical name ((3aR,6aR,9aR,9bR,10R,l laR)-2-benzyl-6a-hydroxy-8,10-dimethyl-7-oxo-l la-(prop-l- en-2-yl)-3 a, 3b, 6, 6a, 9a, 10,11,11 a-octahydro-7H-2,9b-epoxyazuleno[5',4' : 3 ,4]benzo[ 1 ,2- d][l,3]dioxol-5-yl)methyl (2R)-2-(3,4-dimethoxyphenyl)propanoate). COMPOUND 19 was synthesized as follows: Ten milligrams of resiniferonol-9, 13,14-orthophenyl acetate (compound 1) was dissolved in 0.5 mL of dimethylformamide (DMF). Eight milligrams or 2 equivalents of COMPOUND 18 (chemical name (2S)-2-(3,4-dimethoxyphenyl)propanoic acid) was added followed by 3 equivalents of diisopropylethylamine (DIEA) and 2 equivalents of hexafluorophosphate benzotriazole tetramethyl uranium (HBTU). The mixture was stirred at room temperature overnight. The reaction mixture was loaded directly in a 4 g silica gel column and 0- 40% ethyl acetate / hexane gradient was used to produce 6 mg of COMPOUND 19. Note, LCMS ESI (M+H) 657.1; HPLC retention time: 4.12 min.

[0076] COMPOUND 21. FIG. 10 depicts the synthesis schematic of COMPOUND 21 (chemical name ((3aR,6aR,9aR,9bR,10R,l laR)-2-benzyl-6a-hydroxy-8,10-dimethyl-7-oxo-l la-(prop-l- en-2-yl)-3a,3b,6,6a,9a,10,l 1,1 la-octahydro-7H-2,9b-epoxyazuleno[5',4':3,4]benzo[l,2- d][l,3]dioxol-5-yl)methyl 3,4-dimethoxybicyclo[4.2.0]octa-l(6),2,4-triene-7-carboxylate). COMPOUND 21 was synthesized as follows: Ten milligrams of resiniferonol-9, 13,14- orthophenyl acetate (compound 1) was dissolved in 0.5 mL of dimethylformamide (DMF). Nine milligrams or 2 equivalents of COMPOUND 20 (chemical name 3,4- dimethoxybicyclo[4.2.0]octa-l,3,5-triene-7-carboxylic acid) was added followed by 3 equivalents of diisopropylethylamine (DIEA) and 2 equivalents of hexafluorophosphate benzotriazole tetramethyl uranium (HBTU). The mixture was stirred at room temperature overnight. The reaction mixture was loaded directly in a 4 g silica gel column and 0-30% ethyl acetate / hexanegradient was used to produce 12 mg of COMPOUND 21. Note, LCMS ESI (M+H)+655.4; HPLC retention time: 4.12 min.

[0077] COMPOUND 23. FIG. 11 depicts the synthesis schematic of COMPOUND 23 (chemical name ((3aR,6aR,9aR,9bR,10R, l laR)-2-benzyl-6a-hydroxy-8,10-dimethyl-7-oxo-l la-(prop-l- en-2-yl)-3 a, 3b, 6, 6a, 9a, 10,11,11 a-octahydro-7H-2,9b-epoxyazuleno[5',4' : 3 ,4]benzo[ 1 ,2- d][l,3]dioxol-5-yl)methyl (2S)-2-(3,4-dimethoxyphenyl)propanoate). COMPOUND 23 was synthesized as follows: Ten milligrams of resiniferonol-9, 13,14-orthophenyl acetate (compound 1) was dissolved in 0.5 mL of dimethylformamide (DMF). Ten milligrams or 2 equivalents of COMPOUND 22 (chemical name (2R)-2-(3,4-dimethoxyphenyl)propanoic acid) was added followed by 3 equivalents of diisopropylethylamine (DIEA) and 2 equivalents of hexafluorophosphate benzotriazole tetramethyl uranium (HBTU). The mixture was stirred at room temperature overnight. The reaction mixture was loaded directly in a 4 g silica gel column and 0- 25% ethyl acetate / hexane gradient was used to produce 6 mg of COMPOUND 23. Note, LCMS ESI (M+H)+657.1; HPLC retention time: 4.15 min.Example 2

[0078] Novel compounds disclosed herein were tested to determine their agonist effect on TRPV1.

[0079] Materials and Methods. COMPOUNDS 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 (described above in Example 1), RTX, and capsaicin were prepared in assay buffer to the final dilutions of 0.0001 to 0.3 pM. The compound wells, reference agonist, and background vehicle controls were prepared in DMSO at 0.3%. All wells were prepared using Eurofins’ FLIPR Assay Buffer. Reference agonist for each Ion Channel assayed was prepared in a similar manner to serve as assay control. The reference agonist for each Ion Channel was included at Emax (the concentration where the reference agonist elicited a maximal response). The agonist assay was conducted on a FLIPRTETRAinstrument where the test compounds, vehicle controls, and reference agonist were added to the assay plate after a fluorescence baseline was established. The agonist assay was a total of 180 seconds and was used to assess each compound’s ability to activate each Ion Channel assayed.

[0080] Data Calculation and Analysis. All plates were subjected to appropriate baseline corrections. Once baseline corrections were processed, maximum fluorescence / luminescence values were exported and data manipulated to calculate percentage activation and percentageinhibition. Data manipulation calculation is as followed: ((Max RLU)-(Baseline Avg.)) / ((Positive Avg.)-(Baseline Avg.)). All data were first exported to an Excel compatible data file and then analyzed using Graph Pad Prism software. EC50 values were determined by a non-linear, least squares regression analysis. Reference standards were run as an integral part of each assay to ensure the validity of the results obtained.

[0081] Results. The results of the TRPV1 agonist assay described above are depicted in FIGs. 12-14. FIGs. 12-13 depict the data collected at each concentration and FIG. 14 depicts the EC50 values that could be estimated from the data. As depicted in FIG. 14, several of the novel compounds appear to be potent agonists of TRPV1 — for example, COMPOUND 9 has EC50 of 2.3nM, COMPOUND 13 has EC50 of 3. InM, COMPOUND 17 has EC50 of 2.6nM— though none of these appear as potent as RTX — EC50 of 0.559nM. We expect that the agonistic effect of other compounds disclosed herein can be tested in the same way described above to determine if other novel compounds are TRPV1 agonists.Example 3

[0082] We profiled COMPOUNDS 13 and 21 for their ability to desensitize the TRPV1 ion channel.

[0083] Materials and Methods. The compounds (COMPOUND 13, COMPOUND 21, RTX, and Capsaicin) were prepared in cell culture media to the final dilutions of 0.3 pM. The compound wells and background vehicle controls were prepared in DMSO at 0.3%. Compounds were preincubated in cell culture media for 15 minutes. Compounds were removed and Eurofins’ FLIPR Dye Buffer was added to the cells for 90 minutes. The assay was conducted on a FLIPRTETRAinstrument where challenge concentrations of the reference agonist were added to the plate. The assay was a total of 180 seconds and was used to assess each compound’s ability to inhibit or desensitize each Ion Channel assayed.

[0084] Results. FIG. 16 depicts the results of the above-described desensitization experiments. As expected, capsaicin percentage activation remains high upon the second activation (FIG. 16, far right 3 columns labelled “% response of Capsaicin Emax”)). Also as expected, desensitization observed with use of RTX in the first stimulation round followed by capsaicin in the second round (FIG. 16). We observed the desensitization effect in COMPOUND 13 and COMPOUND 21 (FIG. 16).

[0085] We expect that other TRPV1 agonists disclosed herein, such as COMPOUNDS 1, 9, and 17, as well as additional compounds disclosed herein that have not yet been tested for TRPV1 agonist activity, may have such a desensitization effect, and intend to run the same desensitization test to identify other TRPV1 agonists that desensitize stimulation of calcium (flux) upon exposure to capsaicin.

[0086] Moreover, we expect that the TRPV1 agonists disclosed herein will exhibit desensitization using other assays, such as a cube assay that measures electrical potential across the membrane. We intend to test at least COMPOUNDS 2 and 21 disclosed herein using a Qube assay, which can record activation or inhibition of outward TRPV1 current from a population of cells. Such Qube assay can be performed as follows: Cells such as recombinant HEK293 expressing human TRPV1 (accession number AF 196175) are harvested by Accutase and maintained in Serum Free Medium at room temperature before the assay. The cells are pipetted into each well of a 384-well plate in external solution. Compound stock solution is prepared in DMSO at 300x the final assay concentration(s), and stored at -20°C until the day of assay. On the day of the assay, an aliquot of the stock solution is thawed and diluted into external solution to make final test concentrations. A final concentration of 0.33% DMSO is maintained for each concentration of the assay compounds and reference controls. The assay is conducted at room temperature. The intracellular solution (mM) comprises 120 KF, 20 KC1, 10 HEPES, 10 EGTA (pH 7.2 by KOH) and the extracellular solution (mM) comprises 45 NaCl, 4 KC1, 1 MgC12, CaC12, 10 HEPES, 10 Glucose (pH 7.4 by NaOH). Agonist and antagonist assays are run independently.

[0087] The agonist assay proceeds as follows: After whole cell configuration is achieved, the cell is held at -80mV for 100ms. Then the cell is ramped to +60mV over 400ms then stepped back to -80mV for 100ms. This paradigm is delivered once every 45s to monitor the current amplitude. The assay is conducted at room temperature. The Extracellular Solution (vehicle control) is applied first and the cell is stabilized in the solution for 3 min. Then Capsaicin Emax is added to evoke TRPV1 currents for 3 min. One concentration of the test compound is then applied for 3 min. Capsaicin and / or Capsazepine, along with a negative vehicle control(0.33% DMSO) are used as the are tested concurrently with the test compounds. The maximum outward current elicited on stepping to +60 mV for 500msec from -80mV is measured. Compound interaction is assessed by normalizing the Post current amplitude (at the end of each three (3) minute compound application)from peak Capsacin control response, these values are then plotted and estimated EC50 curve fits calculated if applicable.

[0088] The antagonist assay proceeds as follows: After whole cell configuration is achieved, the cell is held at -80mV for 100ms. Then the cell is ramped to +60mV over 400ms then stepped back to -80mV for 100ms. This paradigm is delivered once every 45s to monitor the current amplitude. The assay is conducted at room temperature. The Extracellular Solution (vehicle control) is applied first and the cell is stabilized in the solution for 3 min. Then Capsaicin EC80 is added to evoke TRPV1 currents for 3 min. One concentration of the test compound is then applied for 3 min in the presence of Capsaicin EC80 conentration. Finally, a maximal dose of Capsazepine is added in the presence of Capsaicin EC80 to block the channel. Capsaicin and / or Capsazepine, along with a negative vehicle control(0.33% DMSO) are used as the are tested concurrently with the test compounds. The maximum outward current elicited on stepping to +60 mV for 500msec from - 80mV is measured. Compound interaction is assessed by subtracting Post current amplitude (at the end of each three (3) minute compound application) from baseline Capsacin EC80 response and dividing by the vehicle control amplitude. These values are then plotted and estimated IC50 curve fits calculated.Example 4

[0089] We demonstrate that RTX dissolved in bovine serum albumin (BSA) is fully soluble.

[0090] Methods. Make a stock of 0.1 mg / ml RTX in 50% Saline / DMSO. Pre-make BSA 0.5% solution in 13.33 ml saline, vortex for 10 min and let it sit overnight (16h)- Solution is very clear no precipitation. This was centrifuged and supernatant transferred to another tube just to insure no particulates. Add 0.623 mb of the RTX stock to the 5% BSA solution (4.433 mL) and vortex for 10 min.- 12.5 ug / ml solution. Centrifuge and then take supernatant. Take 50 uL of the supernatant and then add 150uL of Methanol containing IS (warfarin). Centrifuge and inject (40uL) supernatant into HPLC / MS.

[0091] Results. As shown in the table below, RTX-BSA and RTX-Control (RTX dissolved in methanol) have essentially the same AUC. Therefore, RTX dissolved in BSA is fully soluble.

[0092] We expect that other TRPV1 agonists, including the novel TRPV1 agonists disclosed herein, will similarly exhibit increased solubility with solubility enhancers comprising albumin or derivatives thereof.Example 5

[0093] We demonstrate that COMPOUND 21 TRPV1 agonist activity desensitizes the TRPV1 response to capsaicin in a Qube assay.

[0094] We evaluated the TRPV1 agonist activity of COMPOUND 21 in an electrophysiological assay by Eurofins. COMPOUND 21, RTX, capsaicin, and capsazepine were prepared in assay buffer to the final dilutions indicated in the Concentration columns of 0.1 to 33 pM (FIGs. 17A- 17B). The compound wells, reference, agonist and background vehicle controls were prepared in DMSO at 0.33%. Reference agonist for each Ion Channel assayed was prepared in a similar manner to serve as assay control. The reference agonist for each Ion Channel was included at Emax (the concentration where the reference agonist elicited a maximal response). The agonist assay was conducted using a Qube electrophysiological platform. In short, all compound concentrations were applied to a well for 10 minutes followed by a 5-minute wash-off and recovery period. After the 5-minute recovery period, EC80 concentration (IpM) of capsaicin was applied to the well to assess the level of ion channel desensitization. The results of the agonist assay are presented in FIGs. 17A-17B and FIGs. 18A-18D.

[0095] Specifically, all compounds were tested in the presence of 0.1% Pluronic F-68 Non-Ionic Surfactant and at approximately room temperature. Eurofins Panlabs performed a custom assay on the Qube automated patch clamp platform using a ramp protocol and the following methodology. Cells were clamped and maintained at a holding potential of -80mV. The cells were subjected to a voltage ramp protocol from -80mV to +60mV before returning to the holding potential. The current was be measured at the peak of the ramp (+60mV) in the absence of compound to establish a baseline and indicator of any endogenous or leak current. Individual compound concentrations or vehicle control were applied to the cells. Currents were measured every 60 seconds for a period of ten (10) minutes. The peak current amplitudes for agonist activity at +60mV are depicted in FIGs. 18A-18D as (•). These are labelled as “Addition 1 : Compound Pre-Incubation”. Immediately following the ten (10) minute recording in the presence of compound, cells were washed with external recording solution and left to recover for five (5) minutes at the -80mV holding potential. Current amplitudes at -80mV are depicted in FIGs. 18A-18D as (■). These are labelled as “Wash-Off period / Intermediate Holding (-80m V)”. Immediately following the five (5) minute recovery period, an EC80 concentration (IpM) Capsaicin was applied to all wells. Currents were measured every 60 seconds for a period of five (5) minutes. The peak current amplitudes are depicted in FIGs. 18A-18D as (A). These are labelled as “Addition 2: EC80 Capsaicin”. The dotted horizontal line depicted in each of FIGs. 18A-18D represents the mean amplitude of vehicle control wells and the solid horizontal line depicted in each of FIGs. 18A-18D represents the zero current level (the average EC80 capsaicin response for wells that did not any compound in the agonist portion of the assay).

[0096] COMPOUND 21 agonist activity desensitized the subsequent TRPV1 response to EC80 Capsaicin at 30pM and lOpM only, with the four lower concentrations response to Capsaicin falling in line with the vehicle control (FIG. 18A; see also FIGs. 17A, 17B). RTX agonist activity appeared to desensitize the subsequent TRPV1 response to EC80 Capsaicin at all concentrations tested with the response to EC80 Capsaicin falling well below that of vehicle control in a concentration dependent manner (FIG. 18B; see also FIG. 17B). Reference compound Capsaicin agonist activity appeared to desensitize the subsequent TRPV1 response to EC80 Capsaicin at 30pM, lOpM, 3pM, and IpM only, with the two lower concentrations response to EC80 Capsaicin falling in line with the vehicle control (FIG. 18C; see also FIG. 17B). Reference compound Capsazepine did not exhibit any agonist activity on TRPV 1 ion channels and inhibited the response to EC80 Capsaicin at 30pM, lOpM, 3pM, and IpM only, with the two lower concentrations response to EC80 Capsaicin falling in line with the vehicle control (FIG. 18D; see also FIG. 17B).Example 6

[0097] We evaluated the activity of COMPOUND 21 and COMPOUND 9 in a pig model using a study protocol depicted in FIG. 19. Pigs were sedated, mechanically ventilated, and instrumented with an electrocardiogram and an intraarterial pressure catheter to measure heart rate and blood pressure, respectively. Percutaneous access into the pericardial space would be obtained under fluoroscopic guidance and a sheath would be placed. In each animal, one of the test compounds was administered into the pericardial space and completely aspirated out minutes later. The hemodynamic response to the test compound was measured. After hemodynamic parameters returned to baseline, about 30 minutes following application of the test compound, an acute challenge was performed by administering 80 pg / ml capsaicin, a TRPV1 agonist, into the pericardial space to determine if the test compound acutely desensitized the TRPV1 receptor. Theanimal was then recovered. Two weeks after the acute experiments, the animal was again sedated, mechanically ventilated, and instrumented with an electrocardiogram and an intraarterial pressure catheter to measure heart rate and blood pressure, respectively. The pericardial space was accessed. The animal was rechallenged with 40 pg / ml bradykinin, a TRVP1 agonist, 80 pg / ml bradykinin, and 80 pg / ml capsaicin and the hemodynamic response was measured to determine if the test compound chronically desensitize the TRPV 1 receptor.

[0098] The results of the above-described experiments are show in in FIGs. 20A-20D. Data demonstrate that COMPOUND 21 and COMPOUND 9 acutely and chronically desensitize the TRPV1 receptor as seen by the diminished response to the TRPV1 agonist capsaicin and bradykinin as indicated by heart rate (FIGs. 20A, 20C) and blood pressure (FIGs. 20B, 20D).

Claims

CLAIMS:What is claimed is:

1. A compound comprising a modified RTX.

2. The compound of claim 1, wherein the modified RTX comprises at least one substitution on the distal 6-membered ring of RTX.

3. The compound of claim 1, wherein the modified RTX comprises a replacement of the 6- membered distal ring of RTX.

4. The compound of claim 1, wherein the modified RTX comprises at least one alteration of resiniferonol 9, 13,14-ortho-phenylacetate.

5. The compound of claim 4, wherein the modified RTX further comprises at least one substitution on the distal 6-membered ring of RTX.

6. The compound of claim 4, wherein the modified RTX further comprises a replacement of the 6-membered distal ring of RTX.

7. A compound comprising:

8. A compound comprising:

9. A compound comprising:

10. A compound comprising:

11. The compound of claim 10, wherein X comprises CH2.

12. The compound of claim 10, wherein n comprises 1-3.

13. The compound of claim 10, wherein R1 is selected from the group consisting of H, Me, haloalkyl, cycloalkyl, and alkyl and R2 comprises H.

14. The compound of claim 10, wherein R2 is selected from the group consisting of H, Me, haloalkyl, cycloalkyl, and alkyl and R1 comprises H.

15. A compound comprising:

16. The compound of claim 15, wherein X comprises CH2.

17. The compound of claim 15, wherein m comprises 2-3.

18. The compound of claim 15, wherein Y comprises CH2.

19. The compound of claim 15, wherein comprises 1-3.

20. A compound comprising:

21. The compound of claim 20, wherein X comprises CH2.

22. The compound of claim 20, wherein m comprises 2-3.

23. The compound of claim 20, wherein Y comprises CH2.

24. The compound of claim 20, wherein comprises 1-3.

25. The compound of claim 20, wherein A and B comprise N.

26. The compound of claim 20, wherein A comprises C, and B comprises N.

27. The compound of claim 20, wherein A comprises N and B comprises C.

28. A compound comprising:

29. The compound of claim 28, wherein X comprises CH2.

30. The compound of claim 28, wherein m comprises 2-3.

31. The compound of claim 28, wherein Y comprises CH2.

32. The compound of claim 28, wherein comprises 1-3.

33. The compound of claim 28, wherein A is selected from the group consisting of S, N-Me, and NH.

34. A compound comprising:

35. The compound of claim 34, wherein X comprises CH2.

36. The compound of claim 34, wherein m comprises 2-3.

37. The compound of claim 34, wherein Y comprises CH2.

38. The compound of claim 34, wherein n2 comprises 1-3.

39. The compound of claim 34, wherein B is selected from the group consisting of S, N-Me, and NH.

40. A compound comprising:

41. The compound of claim 40, wherein R3 comprises CH3 H.

42. The compound of claim 40, wherein X comprises N and Y comprises C.

43. The compound of claim 40, wherein X comprises C and Y comprises N.

44. The compound of claim 40, wherein R4 is selected from the group consisting of Me, alkyl, haloalkyl, cycloalkyl, and branched alkyl.

45. A compound comprising:

46. The compound of claim 45, wherein R3 comprises CH3 H.

47. The compound of claim 45, wherein X comprises N and Y comprises C.

48. The compound of claim 45, wherein X comprises C and Y comprises N.

49. The compound of claim 45, wherein R4 is selected from the group consisting of Me, alkyl, haloalkyl, cycloalkyl, and branched alkyl.

50. The compound of claim 45, wherein R5 comprises F.

51. A composition comprising any of the compounds of claims 1-50.

52. The composition of claim 51, wherein the composition further comprises an excipient.

53. The composition of claim 52, wherein the excipient is selected from at least one of the group consisting of ethanol, methanol, polyethylene glycol, dimethyl sulfoxide, sodium chloride, or a cyclodextran.

54. The composition of any of claims 51-53, wherein the composition further comprises a solubility enhancer.

55. The composition of claim 54, wherein the solubility enhancer comprises albumin or a derivative thereof.

56. A method of administering the compound of any of claims 1-50 or the composition of any of claims 51-55 to a subject in need thereof.

57. The method of claim 56, wherein administering the compound or administering the composition comprises administration by at least one of topical, subcutaneous, epicardial, epidural, intrathecal, peri or intra-ganglionic, vascular, intraarticular, interarticular, pericardial, intrapericardial, or intravesical administration.

58. The method of claim 56, wherein administering the compound or administering the composition stimulates the TRPV1 receptor in the subject.

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