Flavor composition, food product comprising the same, and method for increasing umami taste intensity of food product

Abstract

The present invention provides a flavor composition comprising at least one nucleotide derivative and / or at least one transmembrane compound that modulates, increases and / or enhances the activity of umami receptors for enhancing the umami taste and / or palatability of a food product. In certain non-limiting embodiments, the flavor composition comprises at least one nucleotide derivative and / or at least one transmembrane compound, optionally at least one nucleotide, optionally at least one first amino acid, optionally at least one second amino acid. The present invention also relates to a food product comprising the above flavor composition, and a method of using the flavor composition to increase the umami taste intensity of a food product. The flavor composition of the present invention, the food product comprising the flavor composition, and the method are capable of achieving a significantly increased umami taste.

Description

[0001] This application is a divisional application of the application filed on December 10, 2015, with international application number PCT / US2015 / 065046, national application number 201580075915.1, and entitled "Flavor Composition and Pet Food Containing the Composition".

[0002] Cross-reference related applications

[0003] This application claims priority to U.S. Provisional Application Serial No. 62 / 090,138, filed December 10, 2014, the contents of which are incorporated herein by reference in their entirety, and claims priority thereto. Technical Field

[0004] This document discloses compounds that modulate the activity of umami taste receptors, flavor compositions comprising at least one such compound, and methods for identifying such compounds. Flavor compositions can be used to enhance or modulate the palatability, taste, and / or flavor of pet food. Flavor compositions may comprise combinations of compounds and can be added to pet food in various delivery system forms. Compounds that modulate the activity of umami taste receptors may include one or more transmembrane compounds, nucleotide derivatives, nucleotides, a first amino acid, a second amino acid, or combinations thereof. Background Technology

[0005] The taste profile of edible compositions includes basic tastes such as sweet, salty, bitter, sour, umami, and kokumi. Taste profiles have also been described as including tastes derived from free fatty acids. The chemical compounds that elicit these tastes are commonly referred to as taste enhancers. It is presumed that taste enhancers are sensed by taste receptors in the mouth and throat, which transmit signals to the brain, where taste enhancers are recorded and taste profiles are generated. Taste receptors include T1R-type taste receptors such as T1R1, T1R2, and T1R3, which interact as heterodimers to function as taste receptors. For example, T1R2 / T1R3 responds to sweet stimuli, and the T1R1 / T1R3 heterodimer recognizes umami tastes. Cats and other members of the Felidae family have failed to express functional T1R2 monomers, suggesting that the primary functional T1R-type taste receptors in cats are the umami receptors T1R1 / T1R3. Furthermore, cats have been shown to prefer food compositions with umami flavors.

[0006] Several significant differences exist between human T1R1 / T1R3 and cat T1R1 / T1R3 receptors. For example, human T1R1 / T1R3 responds to the amino acid glutamate as an agonist and nucleotides, particularly IMP and GMP, as orthoallosteric regulators. Human T1R1 / T1R3 also responds to compounds that bind to the transmembrane domain of the receptor. For example, N-(hept-4-yl)benzo[d][1,3]dioxanepentene-5-carboxamide, an orthoallosteric regulator of the receptor, has been shown to bind to the transmembrane domain of human T1R1 by domain exchange experiments (Zhang et al., Proc Natl Acad Sci USA, 105(52):20930-4, 2008). Allosteric regulators that bind to the transmembrane domain are also known to regulate the activity of several members of class III GPCRs, including the mGluR and CaSR receptors. However, both human and cat T1R1 / T1R3 respond synergistically to combinations of nucleotides and amino acids.

[0007] Pet food manufacturers have long sought to provide pet food with high nutritional value. Furthermore, with particular consideration for cat and dog food, manufacturers aim for high palatability so that pets can obtain the full nutritional benefits from their food. Domestic animals, especially cats, are notoriously fickle in their food preferences, often refusing to eat pet food they've accepted for some time or even refusing only the minimum amount. As a result, pet owners frequently change the type and brand of pet food to keep their pets healthy and satisfied.

[0008] Despite recent advancements in palatability and flavor technologies, there remains a need for compounds that enhance or alter the palatability of pet foods by strengthening or modifying their taste profile, texture profile, and / or flavor profile. This enhancement or modification can involve increasing the intensity of a desired attribute, replacing a desired attribute that is absent or somehow lost in the pet food, or reducing the intensity of an undesirable attribute. In particular, increasing the intensity of flavor enhancers in pet foods is desirable. Therefore, there remains a need in the art for compositions that enhance the palatability and / or umami flavor of pet foods. Summary of the Invention

[0009] The subject matter disclosed herein relates to flavor compositions and methods for preparing and improving such compositions across a variety of pet foods. Specifically, the present invention relates to compositions comprising one or more transmembrane compounds and / or nucleotide derivatives that enhance, increase, and / or modulate the activity of umami receptors T1R1 / T1R3.

[0010] In some embodiments of the invention, the flavor composition comprises a nucleotide derivative of formula Nt-1:

[0011]

[0012] Where X, R1, R2, R4, R5, R 14 and R 15 Described below. The present invention provides salts and stereoisomers of compounds of formula Nt-1.

[0013] In some embodiments of the invention, the flavor composition comprises one or more nucleotide derivatives of formula Nt-2 to Nt-55 as described herein.

[0014] In some embodiments of the invention, the flavor composition further comprises one or more transmembrane compounds of formulas Tm-1 to Tm-104 as described herein.

[0015] In some embodiments, the flavor composition further comprises one or more first amino acids and / or one or more second amino acids. In some embodiments, the flavor composition further comprises at least one, two, three, four, five, or more first amino acids and / or at least one, two, three, four, five, or more second amino acids. In some embodiments, the flavor composition further comprises at least one first amino acid and / or at least one second amino acid. In some embodiments, the flavor composition comprises at least two first amino acids and / or at least one second amino acid. In some embodiments, the flavor composition further comprises at least one first amino acid and / or at least two second amino acids. In some embodiments, the flavor composition further comprises at least two first amino acids and / or at least two second amino acids.

[0016] Non-limiting examples of the first amino acid include tryptophan, phenylalanine, histidine, glycine, cysteine, alanine, tyrosine, serine, methionine, asparagine, leucine, and combinations thereof.

[0017] Non-limiting examples of the second amino acid include asparagine, threonine, isoleucine, proline, glutamic acid, aspartic acid, hydroxyproline, arginine, cystine, glutamine, lysine, valine, ornithine, monosodium glutamate, taurine, and combinations thereof.

[0018] In some embodiments, the flavor composition also contains alanine.

[0019] In some embodiments, the flavor composition also contains glycine.

[0020] In some embodiments, the flavor composition also contains histidine.

[0021] In some embodiments, the flavor composition may also comprise at least one nucleotide or nucleotide derivative. Non-limiting examples of nucleotides include guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), inosine monophosphate (IMP), inosine diphosphate (IDP), inosine triphosphate (ITP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), xanthoside monophosphate (XMP), xanthoside diphosphate (XDP), xanthoside triphosphate (XTP), and combinations thereof.

[0022] In one embodiment, the flavor composition may further comprise nucleotides selected from the group consisting of guanosine monophosphate (GMP), inosine monophosphate (IMP), and combinations thereof.

[0023] In other embodiments, the flavor composition also includes alanine and guanosine monophosphate (GMP).

[0024] In other embodiments, the flavor composition also includes glycine and guanosine monophosphate (GMP).

[0025] In other embodiments, the flavor composition also includes histidine and guanosine monophosphate (GMP).

[0026] In other embodiments, the flavor composition also includes alanine and inosine monophosphate (IMP).

[0027] In other embodiments, the flavor composition also includes glycine and inosine monophosphate (IMP).

[0028] In other embodiments, the flavor composition also includes histidine and inosine monophosphate (IMP).

[0029] In other embodiments, the flavor composition further comprises proline and an amino acid selected from the group consisting of histidine, alanine, glycine, phenylalanine, tryptophan, and tyrosine.

[0030] In other embodiments, the flavor composition further comprises threonine and amino acids selected from the group consisting of histidine, alanine, glycine, phenylalanine, tryptophan, and tyrosine.

[0031] In some embodiments, the flavor composition further comprises glutamic acid and amino acids selected from the group consisting of histidine, alanine, glycine, phenylalanine, tryptophan, and tyrosine.

[0032] In some embodiments, the present invention provides a food comprising a flavor composition as described herein, wherein the flavor composition is present at a concentration of about 0.0001% by weight to about 10% by weight of the food. In other embodiments, the flavor composition is present at a concentration of about 0.001 ppm to about 1000 ppm of the food. In still other embodiments, the flavor composition is present at a concentration of about 1 pM to about 1 M, or about 1 μM to about 1 M of the food.

[0033] In some embodiments, the flavor composition is present in an effective amount to increase the palatability of the food, as determined by a panel of taste testers.

[0034] In other embodiments, the present invention provides a method for increasing the umami flavor intensity of food, comprising mixing the food with a flavor composition described herein, wherein the flavor composition is present at a concentration of about 0.0001 wt% to about 10 wt% of the mixture. In other embodiments, the flavor composition is present at a concentration of about 0.001 ppm to about 1000 ppm of the mixture. In still other embodiments, the flavor composition is present at a concentration of about 1 pM to about 1 M, or about 1 μM to about 1 M of the mixture.

[0035] In other embodiments, the present invention provides a method for increasing the umami flavor intensity of a food, comprising mixing the food with a flavor composition described herein, wherein the flavor composition is present in an effective amount to increase the palatability of the food, as determined by a panel of taste testers. In some embodiments, the increase in umami flavor intensity includes an increase in umami aftertaste.

[0036] The present invention also provides a method for preparing a food comprising the flavor composition described herein, wherein the method includes heat-treating a food precursor, wherein the flavor composition is produced during heat treatment. Examples of heat treatment include, for example, sterilization, retorting, extrusion, injection molding, or combinations thereof.

[0037] In some embodiments, the food described herein includes pet food, such as feline pet food, including wet and / or dry feline pet food. In other instances, pet food includes canine pet food, including wet and / or dry canine pet food.

[0038] In other embodiments, the food described herein includes food for humans.

[0039] The foregoing has provided a fairly broad overview of the features and technical advantages of this application in order to better understand the detailed description that follows. Other features and advantages of this application, which constitute the subject matter of the claims, will be described below. Those skilled in the art will understand that the disclosed concepts and specific embodiments can be readily used as a basis for modifying or designing other structures to perform the same purpose of this application. Those skilled in the art will also recognize that such equivalent structures do not depart from the spirit and scope of this application as set forth in the appended claims. The following description will provide a better understanding of the new features, further objectives, and advantages that are considered characteristic of this application in terms of its organization and operation. Attached Figure Description

[0040] Figure 1 The dose-response curves of guanosine monophosphate (GMP) as the test compound for in vitro activation of feline T1R1 / T1R3 umami receptors are shown. In this figure (and in... Figure 2-12 In Figures 14 and 38-45, four curves are shown: the test compound in buffer, the test compound in buffer with 20 mM alanine, the test compound in buffer with 0.2 mM IMP, and the test compound in 20 mM alanine with 0.2 mM IMP. The horizontal axis represents the concentration of the test compound in mM. The vertical axis represents the receptor response as ΔF / F as measured by fluorescence assay or as Lum as measured by luminescence assay.

[0041] Figure 2 The dose-response curves for 2'-deoxyadenosine-3',5'-O-bisphosphate are shown.

[0042] Figure 3 The dose-response curves for sodium inosine 5'-bisphosphate (IDP) are shown.

[0043] Figure 4 The dose-response curves for 2'- / 3'-O-(2-aminoethylcarbamoyl)adenosine-5'O-monophosphate are shown.

[0044] Figure 5 The dose-response curves of 2'- / 3'-O-(2-aminoethylcarbamoyl)guanosine-5'-O-monophosphate are shown.

[0045] Figure 6 The dose-response curve for inosine triphosphate (ITP) trisodium salt is shown.

[0046] Figure 7 The dose-response curve for N6-benzoyladenosine-5'-O-monophosphate is shown.

[0047] Figure 8 The dose-response curves for adenosine 5'-O-thiophosphate dilithium salt are shown.

[0048] Figure 9 The dose-response curves for adenosine 3',5' diphosphate sodium salt and alanine (1:1000) are shown.

[0049] Figure 10 The dose-response curves for adenosine 3',5' diphosphate sodium salt and alanine (1:100) are shown.

[0050] Figure 11 The dose-response curves for adenosine 3',5' diphosphate sodium salt and alanine (1:10) are shown.

[0051] Figure 12 The dose-response curves for sodium adenosine 3',5'-bisphosphate are shown.

[0052] Figure 13 The dose-response curves of inosine monophosphate (IMP) for activating cat T1R1 / T1R3 are shown. IMP is used as a derivative of... Figure 1-13 A control of the experiments described in 38-45.

[0053] Figure 14 The overall structural model of the VFT domain of cat T1R1 / T1R3, which binds IMP and L-alanine, is shown. The active sites for L-alanine and IMP binding are shown between the upper and lower lobes of the VFT domain.

[0054] Figure 15 A model of the cat T1R1 VFT bound to L-alanine is shown. The hinge region of the L-alanine-bound VFT domain is shown. The putative hydrogen bonds, salt bridges, and Pi-cation interactions between L-alanine and the following T1R1 amino acids are shown: Thr149, Ser172, Tyr220, Thr148, Glu170, and Asp302. These interactions are shown as dashed lines. Glu170 and Asp302 coordinate the zwitterionic nitrogen of the amino acids, while electrostatically not supporting the binding of L-glutamate and L-aspartic acid (which are natural ligands for human umami receptors).

[0055] Figure 16 A computer model of cat T1R1 VFT binding to GMP is shown. The putative hydrogen bond and salt bridge interactions between the phosphate group of GMP and His47, His71, Arg277, and Asn69, between the sugar group of GMP and Asp302 and Ser306, and between the base group of GMP and Ser384, His308, and Ala380 are shown as dashed lines.

[0056] Figure 17The computer model of the cat T1R1 VFT is shown, indicating that the Asp302 of T1R1 can simultaneously coordinate the binding of the zwitterionic backbone nitrogen of the amino acid (L-alanine, left figure) and the bound nucleotide (GMP, right figure) sugar molecule.

[0057] Figure 18 A computer model of the cat T1R1 VFT is shown, illustrating the putative interaction between T1R1 and bound 3',5' diphosphate.

[0058] Figure 19 A computer model of the cat T1R1 VFT is shown, illustrating the putative interaction between T1R1 and the bound XMP.

[0059] Figure 20 A computer model of the cat T1R1 VFT is shown, illustrating the putative interaction between T1R1 and the bound N-acetyl-5'-GMP.

[0060] Figure 21 A computer model of the cat T1R1 VFT is shown, illustrating the putative interaction between T1R1 and the combined 2'-3'AEC-5'-AMP.

[0061] Figure 22 A computer model of the cat T1R1 VFT is shown, illustrating the putative interaction between T1R1 and the bound 5'd-GMPS.

[0062] Figure 23 A computer model of the cat T1R1 VFT is shown, illustrating the putative interaction between T1R1 and bound 5'-O-2-thiodiphosphoric acid.

[0063] Figure 24 A computer model of the cat T1R1 VFT is shown, illustrating the putative interaction between T1R1 and the bound 6-thioguanosine-5'-O-monophosphate.

[0064] Figure 25 A computer model of the cat T1R1 VFT is shown, illustrating the putative interaction between T1R1 and the bound CMP.

[0065] Figure 26 A computer model of the cat T1R1 VFT of adenosine 3'5'-bisphosphate is shown. The putative hydrogen bond and salt bridge interactions between the phosphate group of adenosine 3'5'-bisphosphate and His71, His47, Asn69, Arg281, Arg277, His308, and Ile309, between the sugar group of adenosine 3'5'-bisphosphate and Asp302 and Ser306, and between the adenosine 3'5'-bisphosphate base and Ser384 are shown as dashed lines.

[0066] Figure 27 A computer model of the transmembrane domain of cat T1R1 is shown. The transmembrane compound N-benzyl-L-phenylalanine methyl ester is shown docked into the transmembrane region of T1R1.

[0067] Figure 28 A computer model is shown of N-benzyl-L-phenylalanine methyl ester docking into the transmembrane region of T1R1.

[0068] Figure 29 A computer model is shown of 2-amino-N-phenylethylbenzamide docking into the transmembrane region of T1R1.

[0069] Figure 30 A computer model is shown of N-(2-(1H-indol-3-yl)ethyl)nicotinamide docking into the transmembrane region of T1R1.

[0070] Figure 31 A computer model is shown of the docking of 1-benzyl-3-(2-(5-chlorothiophen-2-yl)-2-oxoethyl)imidazoline-2,4,5-trione into the transmembrane region of T1R1.

[0071] Figure 32 A computer model is shown of (2,2-diphenylacetyl)carbamate docking into the transmembrane region of T1R1.

[0072] Figure 33 The computer model shown is of 2-((3,5-dichlorophenyl)carbamoyl)cyclohexanecarboxylic acid docked into the transmembrane region of T1R1.

[0073] Figure 34 A computer model is shown showing the docking of N-(hept-4-yl)benzo[d][1,3]dioxanepentene-5-carboxamide into the transmembrane region of T1R1.

[0074] Figure 35 Computer modeling is shown of the docking of 1-benzyl-3-(2-oxo-2-phenylethyl)imidazoline-2,4,5-trione into the transmembrane region of T1R1.

[0075] Figure 36 Computer modeling of 1H-imidazo[4,5-c]pyridine-2(3H)-one derivative compounds docking into the transmembrane region of T1R1 is shown.

[0076] Figure 37 The dose-response curves for 1-benzyl-3-(2-oxo-2-phenylethyl)imidazoline-2,4,5-trione are shown.

[0077] Figure 38The dose-response curve of 1-(2-bromophenyl)-3-((1R,2S)-2-hydroxy-2,3-dihydro-1H-indene-1-yl)urea is shown.

[0078] Figure 39 The dose-response curves for N-(benzo[d][1,3]dioxacyclopenten-5-yl)-2-propylpentanamide are shown.

[0079] Figure 40 The dose-response curves for N-(hept-4-yl)benzo[d][1,3]dioxane-5-carboxamide are shown.

[0080] Figure 41 The dose-response curves for N-(2-amino-2-oxo-1-phenylethyl)-3-chloro-4,5-dimethoxybenzamide are shown.

[0081] Figure 42 The dose-response curves for (E)-3-(4-methoxyphenyl)-N-(pent-3-yl)acrylamide are shown.

[0082] Figure 43 The dose-response curves for 2-((5-(4-(methylthio)phenyl)-2H-tetrazole-2-yl)methyl)pyridine are shown.

[0083] Figure 44 The dose-response curves for N-(hept-4-yl)benzo[d][1,3]dioxane-5-carboxamide are shown in the presence of GMP and phenylalanine.

[0084] Figure 45 The graph shows the ΔF / F0 values ​​of N-(hept-4-yl)benzo[d][1,3]dioxane-5-carboxamide in the presence of GMP and phenylalanine.

[0085] Figure 46 Showing the use of Figure 1-14 Dose-response curves for positive and negative controls of activated cat T1R1 / T1R3 in the experiments described in 38-45. Dose-response curves for amino acids were determined in the presence of 0.2 mM IMP. Dose-response curves for nucleotides were determined in the presence of 20 mM alanine.

[0086] Figure 47 Computer modeling of a transmembrane compound docking into the transmembrane region of T1R1, according to an exemplary embodiment of the disclosed subject matter.

[0087] Figure 48 Computer modeling of a transmembrane compound docking into the transmembrane region of T1R1, according to an exemplary embodiment of the disclosed subject matter.

[0088] Figure 49 Computer modeling of a transmembrane compound docking into the transmembrane region of T1R1, according to an exemplary embodiment of the disclosed subject matter.

[0089] Figure 50 Computer modeling of a transmembrane compound docking into the transmembrane region of T1R1, according to an exemplary embodiment of the disclosed subject matter.

[0090] Figure 51 Computer modeling of a transmembrane compound docking into the transmembrane region of T1R1, according to an exemplary embodiment of the disclosed subject matter.

[0091] Figure 52 Computer modeling of a transmembrane compound docking into the transmembrane region of T1R1, according to an exemplary embodiment of the disclosed subject matter.

[0092] Figure 53 Computer modeling of a transmembrane compound docking into the transmembrane region of T1R1, according to an exemplary embodiment of the disclosed subject matter.

[0093] Figure 54 Computer modeling of a transmembrane compound docking into the transmembrane region of T1R1, according to an exemplary embodiment of the disclosed subject matter.

[0094] Figure 55 Computer modeling of a transmembrane compound docking into the transmembrane region of T1R1, according to an exemplary embodiment of the disclosed subject matter.

[0095] Figures 56A-B show the dose-response curves for (A) activation of T1R1 / T1R3 in the presence of 1,3-dibenzylpyrimidine-2,4,6(1H,3H,5H)-trione in buffer, or in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) activation of T1R1 / T1R3 in the presence of 1,3-dibenzylpyrimidine-2,4,6(1H,3H,5H)-trione alone in buffer, or in the presence of 20 mM L-alanine and 0.2 mM IMP, or both.

[0096] Figures 57A-B show the dose-response curves for (A) activation of T1R1 / T1R3 in the presence of 4-benzyl-3-butyl-1-(2-oxo-2-(pyrrolidone-1-yl)ethyl)-1H-1,2,4-triazol-5(4H)-one in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) activation of T1R1 / T1R3 in the presence of 4-benzyl-3-butyl-1-(2-oxo-2-(pyrrolidone-1-yl)ethyl)-1H-1,2,4-triazol-5(4H)-one alone in buffer or in the presence of both 20 mM L-alanine and 0.2 mM IMP.

[0097] Figures 58A-B show the dose-response curves for (A) activation of T1R1 / T1R3 in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) activation of T1R1 / T1R3 in buffer alone or in the presence of 20 mM L-alanine and 0.2 mM IMP or both.

[0098] Figures 59A-B show the dose-response curves for (A) activation of T1R1 / T1R3 by 4-acetamido-N-(1-(2-hydroxyethyl)-3-phenyl-1H-pyrazol-5-yl)benzamide in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) activation of T1R1 / T1R3 by 4-acetamido-N-(1-(2-hydroxyethyl)-3-phenyl-1H-pyrazol-5-yl)benzamide alone in buffer or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

[0099] Figures 60A-B show the dose-response curves for (A) (diphenylacetyl)-carbamate ethyl ester activating T1R1 / T1R3 in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) (diphenylacetyl)-carbamate ethyl ester activating T1R1 / T1R3 alone in buffer or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

[0100] Figures 61A-B show the dose-response curves for (A) activation of T1R1 / T1R3 by N,N'-(butane-1,4-diyl)nicotinamide in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) activation of T1R1 / T1R3 by N,N'-(butane-1,4-diyl)nicotinamide alone in buffer or in the presence of both 20 mM L-alanine and 0.2 mM IMP or both 20 mM L-alanine and 0.2 mM IMP.

[0101] Figures 62A-B show the dose-response curves for (A) N-phenylethyl nicotinamide activating T1R1 / T1R3 in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) N-phenylethyl nicotinamide alone in buffer or in the presence of both 20 mM L-alanine and 0.2 mM IMP or 20 mM L-alanine and 0.2 mM IMP.

[0102] Figures 63A-B show the dose-response curves for (A) activation of T1R1 / T1R3 by 2-amino-N-phenylethylbenzamide in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) activation of T1R1 / T1R3 by 2-amino-N-phenylethylbenzamide alone in buffer or in the presence of 20 mM L-alanine, 0.2 mM IMP or both 20 mM L-alanine and 0.2 mM IMP.

[0103] Figures 64A-B show the dose-response curves for (A) N-phenylethylbenzo[d][1,3]dioxacyclopentene-5-carboxamide activating T1R1 / T1R3 in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) N-phenylethylbenzo[d][1,3]dioxacyclopentene-5-carboxamide alone in buffer or in the presence of 20 mM L-alanine and 0.2 mM IMP or both 20 mM L-alanine and 0.2 mM IMP.

[0104] Figures 65A-B show the dose-response curves for (A) N-phenylethylbenzamide activating T1R1 / T1R3 in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) N-phenylethylbenzamide alone in buffer or in the presence of 20 mM L-alanine, 0.2 mM IMP or both 20 mM L-alanine and 0.2 mM IMP.

[0105] Figures 66A-B show the dose-response curves for (A) N-benzoyl-DL-leucine amide activating T1R1 / T1R3 in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) N-benzoyl-DL-leucine amide alone in buffer or in the presence of 20 mM L-alanine, 0.2 mM IMP or both 20 mM L-alanine and 0.2 mM IMP.

[0106] Figures 67A-B show the dose-response curves for (A) N-(2-(1H-indol-3-yl)ethyl)nicotinamide activating T1R1 / T1R3 in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) N-(2-(1H-indol-3-yl)ethyl)nicotinamide alone in buffer or in the presence of 20 mM L-alanine, 0.2 mM IMP or both 20 mM L-alanine and 0.2 mM IMP.

[0107] Figures 68A-B show the dose-response curves for (A) N-benzyl-L-phenylalanine methyl ester hydrochloride activating T1R1 / T1R3 in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) N-benzyl-L-phenylalanine methyl ester hydrochloride activating T1R1 / T1R3 alone in buffer or in the presence of 20 mM L-alanine, 0.2 mM IMP or both 20 mM L-alanine and 0.2 mM IMP.

[0108] Figures 69A-B show the dose-response curves for (A) activation of T1R1 / T1R3 by 6-thioguanine nucleoside-5'-O-bisphosphate (6-T-GDP) in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) activation of T1R1 / T1R3 by 6-thioguanine nucleoside-5'-O-bisphosphate (6-T-GDP) alone in buffer or in the presence of 20 mM L-alanine and 0.2 mM IMP or both 20 mM L-alanine and 0.2 mM IMP.

[0109] Figures 70A-B show the dose-response curves for (A) activation of T1R1 / T1R3 by 6-chloropurine nucleoside-5'-O-triphosphate (6-Cl-PuTP) in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) activation of T1R1 / T1R3 by 6-chloropurine nucleoside-5'-O-triphosphate (6-Cl-PuTP) alone in buffer or in the presence of 20 mM L-alanine and 0.2 mM IMP or both 20 mM L-alanine and 0.2 mM IMP.

[0110] Figure 71The response curves for positive and negative controls of activated cat T1R1 / T1R3, as described in Figures 57-75, are shown. The dose-response curves for amino acids were determined in the presence of 0.2 mM IMP. The dose-response curves for nucleotides were determined in the presence of 20 mM alanine.

[0111] Figures 72A-D illustrate the activation of T1R1 / T1R3: (A) by the transmembrane compound 1-benzyl-3-(2-(3,4-dihydro-2H-benzo[b][1,4]dioxane-7-yl)-2-oxoethyl)imidazoline-2,4,5-trione alone (agonist characterization assessment), and (B) and (C) in the presence of GMP and alanine (PAM characterization assessment). (D) shows a graphical representation of the data from (A) and (B). The transmembrane compound exhibits PAM activity.

[0112] Figures 73A-F illustrate T1R1 / T1R3 activation: (A) by the transmembrane compound 1-benzyl-3-(2-(5-chlorothiophene-2-yl)-2-oxoethyl)imidazoline-2,4,5-trione alone (agonist characterization), (B) and (C) in the presence of GMP and alanine (PAM characterization). (D) shows a graphical representation of the data from (A) and (B). (E) and (F) show the dose-response curves of the transmembrane compound when GMP concentration is kept constant and Ala concentration varies, and the dose-response curves of the transmembrane compound when Ala concentration is kept constant and GMP concentration varies in cells expressing T1R1 / T1R3 (induced) and in mock control cells not expressing T1R1 (uninduced). The transmembrane compound exhibits PAM activity.

[0113] Figures 74A-E illustrate T1R1 / T1R3 activation: (A) by the transmembrane compound N-(hept-4-yl)benzo[d][1,3]dioxane-5-carboxamide alone (agonist characterization), and (B) in the presence of GMP and alanine (PAM characterization). (C) shows a graphical representation of the data from (A) and (B). (D) and (E) show the dose-response curves of the transmembrane compound when the GMP concentration is kept constant and the Ala concentration varies, and the dose-response curves of the transmembrane compound when the Ala concentration is kept constant and the GMP concentration varies in cells expressing T1R1 / T1R3 (induced, (D)) and blank control cells not expressing T1R1 (uninduced, (E)). The transmembrane compound exhibits both agonist and PAM activities.

[0114] Figures 75A-E illustrate T1R1 / T1R3 activation: (A) by the transmembrane compound N-benzyl-L-phenylalanine methyl ester hydrochloride alone (agonist characterization), and (B) in the presence of GMP and alanine (PAM characterization). (C) shows a graphical representation of the data from (A) and (B). (D) and (E) show the dose-response curves of the transmembrane compound when GMP concentration is kept constant and Ala concentration varies, and the dose-response curves of the transmembrane compound when Ala concentration is kept constant and GMP concentration varies in cells expressing T1R1 / T1R3 (induced, (D)) and blank control cells not expressing T1R1 (uninduced, (E)). The transmembrane compound exhibits both agonist and PAM activities.

[0115] Figures 76A-E illustrate T1R1 / T1R3 activation: (A) by the transmembrane compound N-benzyl-D-phenylalanine methyl ester hydrochloride alone (agonist characterization), and (B) in the presence of GMP and alanine (PAM characterization). (C) shows a graphical representation of the data from (A) and (B). (D) and (E) show the dose-response curves of the transmembrane compound when GMP concentration is kept constant and Ala concentration varies, and the dose-response curves of the transmembrane compound when Ala concentration is kept constant and GMP concentration varies in cells expressing T1R1 / T1R3 (induced, (D)) and blank control cells not expressing T1R1 (uninduced, (E)). The transmembrane compound exhibits both agonist and PAM activities.

[0116] Figures 77A-E illustrate T1R1 / T1R3 activation: (A) by the transmembrane compound benzyl-L-leucine methyl ester hydrochloride alone (agonist characterization), and (B) in the presence of GMP and alanine (PAM characterization). (C) shows a graphical representation of the data from (A) and (B). (C) and (E) show the dose-response curves of the transmembrane compound when GMP concentration is kept constant and Ala concentration varies, and the dose-response curves of the transmembrane compound when Ala concentration is kept constant and GMP concentration varies in cells expressing T1R1 / T1R3 (induced, (D)) and blank control cells not expressing T1R1 (uninduced, (E)). The transmembrane compound exhibits both agonist and PAM activities.

[0117] Figures 78A-E illustrate T1R1 / T1R3 activation: (A) by the transmembrane compound methyl 2-benzylamino-2-phenylacetate alone (agonist characterization), and (B) in the presence of GMP and alanine (PAM characterization). (C) shows a graphical representation of the data from (A) and (B). (D) and (E) show the dose-response curves of the transmembrane compound when GMP concentration is kept constant and Ala concentration varies, and the dose-response curves of the transmembrane compound when Ala concentration is kept constant and GMP concentration varies in cells expressing T1R1 / T1R3 (induced, (D)) and blank control cells not expressing T1R1 (uninduced, (E)). The transmembrane compound exhibits both agonist and PAM activities.

[0118] Figures 79A-E illustrate T1R1 / T1R3 activation: (A) by the transmembrane compound L-phenylalanine benzyl ester hydrochloride alone (agonist characterization), and (B) in the presence of GMP and alanine (PAM characterization). (C) shows a graphical representation of the data from (A) and (B). (D) and (E) show the dose-response curves of the transmembrane compound when GMP concentration is kept constant and Ala concentration varies, and the dose-response curves of the transmembrane compound when Ala concentration is kept constant and GMP concentration varies in cells expressing T1R1 / T1R3 (induced, (D)) and blank control cells not expressing T1R1 (uninduced, (E)). The transmembrane compound exhibits both agonist and PAM activities.

[0119] Figures 80A-B illustrate the activation of T1R1 / T1R3: (A) by the transmembrane compound 1,3-dibenzylpyrimidine-2,4,6(1H,3H,5H)-trione (agonist characterization assessment), and (B) in the presence of GMP and alanine (PAM characterization assessment). The transmembrane compound exhibits agonist activity.

[0120] Figure 81 The nucleic acid sequence of the feline T1R1 receptor is shown (SEQ ID NO: 1).

[0121] Figure 82 The amino acid sequence of the feline T1R1 receptor is shown (SEQ ID NO: 2).

[0122] Figure 83 The nucleic acid sequence of the feline T1R3 receptor is shown (SEQ ID NO: 3).

[0123] Figure 84 The amino acid sequence of the feline T1R3 receptor is shown (SEQ ID NO: 4). Detailed Implementation

[0124] To date, there remains a need for a flavor enhancer that can provide a desired level of umami flavor to increase and / or enhance the palatability of various cat pet foods. This application relates to a flavor composition comprising at least one nucleotide derivative and / or a transmembrane compound. This flavor composition can be used to increase palatability and / or enhance or improve the taste of various pet foods, such as nutritionally complete pet foods. In some embodiments, the flavor composition can be used to enhance the umami flavor of pet foods. The flavor composition may also comprise a combination of compounds including nucleotides and / or amino acids, and can be added to pet foods in various delivery system forms.

[0125] 1. Definition

[0126] The terms used in this specification generally have their usual meaning in the context of the invention and in the specific context in which each term is used. Some terms will be discussed below or elsewhere in the specification to provide further guidance to those skilled in the art in describing the compositions and methods of the invention and how they are made and used.

[0127] As used herein, the words “a” or “an” used in conjunction with the term “comprising” in the claims and / or specification may mean “an”, but they are also consistent with the meanings of “one or more,” “at least one,” and “one or more.” Furthermore, the terms “having,” “comprising,” “containing,” and “including” are interchangeable, and those skilled in the art will recognize that these terms are open-ended.

[0128] The terms “about” or “approximately” are intended to indicate an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about” can be within three or more standard deviations. Alternatively, “about” can indicate a range of up to 20%, preferably up to 10%, more preferably up to 5%, and still more preferably up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term can indicate a range within an order of magnitude of a value, preferably within five times, more preferably within two times.

[0129] As used herein, “taste” refers to the sensation caused by the activation or inhibition of receptor cells in an individual’s oral cavity. In some embodiments, taste may be selected from the group consisting of sweet, sour, salty, bitter, kokumi, and umami. In some embodiments, “taste” may include the taste of free fatty acids. See, for example, Cartoni et al., J. of Neuroscience, 30(25):8376-8382 (2010), the contents of which are incorporated herein by reference. In some embodiments, taste is caused by a “taste enhancer” in the individual. In some embodiments, the taste enhancer is a synthetic taste enhancer. In some embodiments, the taste enhancer is prepared from a natural source.

[0130] As used herein, "flavor characteristic" refers to a combination of flavors, such as one or more of sweet, sour, salty, bitter, umami, full-bodied, and free fatty acid flavors. In some embodiments, the flavor characteristic is generated by one or more flavor enhancers present in the composition at the same or different concentrations. In some embodiments, the flavor characteristic refers to the intensity of a flavor or combination of flavors, such as sweet, sour, salty, bitter, umami, full-bodied, and free fatty acid flavors as detected by an individual or by any assay known in the art. In some embodiments, modifying, altering, or changing the combination of flavor enhancers in the flavor characteristic can change an individual's sensory experience.

[0131] In some implementations, "aftertaste" refers to the intensity of the taste of food perceived after it has been removed from the mouth or oral cavity.

[0132] As used herein, “flavor” refers to one or more sensory stimuli, such as taste (guest), odor (olfaction), touch (touch), and temperature (heat). In some non-limiting embodiments, an individual’s sensory experience of exposure to a flavor can be categorized as a particular flavor-specific experience. For example, a flavor can be identified by an individual as, but is not limited to, the flavors of flowers, citrus, berries, nuts, caramel, chocolate, pepper, smoke, cheese, meat, etc. As used herein, flavor compositions can be selected from liquids, solutions, dry powders, sprays, pastes, suspensions, and any combination thereof. Flavoring agents can be natural compositions, artificial compositions, natural equivalents, or any combination thereof.

[0133] As used interchangeably in this article, "aromatic" and "odor" refer to the olfactory response to a stimulus. For example, rather than in a restrictive way, aroma can be produced by aromatic substances perceived by odor receptors in the olfactory system.

[0134] As used herein, "flavor characteristic" refers to sensory stimuli such as tastes, including sweet, sour, bitter, salty, umami, full-bodied, and free fatty acid tastes, and / or combinations of olfactory, tactile, and / or thermal stimuli. In some embodiments, flavor characteristics include one or more flavors that contribute to an individual's sensory experience. In some embodiments, modifying, altering, or changing the combination of flavor characteristic stimuli can change an individual's sensory experience.

[0135] As used herein, “mixing,” for example, “mixing the flavor composition or a combination thereof with a food product,” refers to a process in which the flavor composition or its components are mixed or added to a finished product or mixed with some or all of the components of a product during product formation or in some combination of these steps. When used in the context of mixing, the term “product” refers to a product or any of its components. This mixing step may include processes selected from the following steps: adding the flavor composition to a product, spraying the flavor composition onto the product, coating the product with the flavor composition, suspending the product in the flavor composition, applying the flavor composition to the product, adhering the flavor composition to the product, encapsulating the product with the flavor composition, mixing the flavor composition with the product and any combination thereof. The flavor composition may be a solution, liquid, dry powder, spray, paste, suspension, and any combination thereof.

[0136] In some embodiments, nucleotide derivatives and / or transmembrane compounds of the flavor composition can be generated from precursor compounds present in the pet food during heat treatment, such as sterilization, dry distillation, injection molding, and / or extrusion. In some embodiments, nucleotide derivatives and / or transmembrane compounds of the flavor composition can be generated during the processing of the pet food and additional components of the flavor composition, such as nucleotides and / or amino acids, which can be added to the pet food by mixing.

[0137] As used herein, “ppm” refers to parts per million, which is a weight-relative parameter. Parts per million is the number of micrograms per gram, such that a component present at 10 ppm is 10 micrograms of a specific component per gram of aggregate mixture.

[0138] As used herein, “palliativeness” can refer to the overall willingness of a human or non-human animal, such as a companion animal, to consume a particular food. Increasing the “palliativeness” of a food can lead to an increase in the enjoyment and acceptance of the food by a human or non-human animal, ensuring that the human or non-human animal consumes a “healthy amount” of food. The term “healthy amount” of food, as used herein, refers to the amount of micronutrients, macronutrients, and calories that enables a human or non-human animal to maintain or achieve an intake that contributes to its overall health, as described in, for example, the Mars Petcare Essential Nutrient Standards. In some embodiments, “palliativeness” can mean a relative preference of a human or non-human animal for one food over another. For example, when a human or non-human animal shows a preference for one of two or more foods, the preferred food is more “palatable” and has “enhanced palliativeness.” In some embodiments, a relative preference for one food over one or more other foods can be determined, for example, by a free choice comparison, such as by relative consumption of the food or other appropriate measures of preference indicative of palliativeness. Palatability can be determined through standard testing protocols in which animals have equitable access to two foods, such as the so-called "two-bowl test" or "relative test." This preference can be induced by any of the animal's senses, but can be related to taste, aftertaste, aroma, mouthfeel, and / or texture, in particular.

[0139] The term "pet food" or "pet supplement" refers to a product or composition intended for consumption by companion animals such as cats, dogs, guinea pigs, rabbits, birds, and horses. For example, rather than by limitation, a companion animal can be a "domestic" cat such as the domestic cat (Felis domesticus). In some embodiments, a companion animal can be a "domestic" dog, such as the domestic dog (Canis lupus familiaris). "Pet food" or "pet supplement" includes any food, feed, snacks, food supplements, liquids, beverages, treats, toys (chewable and / or edible toys), meal substitutes, or dietary alternatives.

[0140] The term "human food" or "human food" refers to a product or composition intended for human consumption. "Human food" or "human food" includes any food, animal feed, snacks, food supplements, liquids, beverages, treats, toys (chewable and / or edible toys), meal substitutes, or dietary alternatives.

[0141] In some implementations, "food" includes human and / or pet food.

[0142] As used in this article, "nutritionalally complete" refers to foods that contain all known essential nutrients for the intended recipient of the food, such as pet food, in appropriate amounts and proportions based on those recognized or recommended by regulatory authorities in the field of companion animal nutrition. Such foods can therefore serve as the sole source of dietary intake necessary to sustain life, without the need for supplemental nutrients.

[0143] As used herein, a "flavor composition" means at least one compound or a biologically acceptable salt thereof, the modulation of which includes enhancing, increasing, strengthening, reducing, inhibiting, or inducing the taste, odor, flavor, and / or texture of natural or synthetic flavor enhancers, flavoring agents, taste characteristics, flavor features, and / or texture characteristics in animals or humans. In some embodiments, the flavor composition comprises a combination of compounds or biologically acceptable salts thereof. In some embodiments, the flavor composition comprises one or more excipients.

[0144] As used herein, “agonist” means at least one compound or its biologically acceptable salt that modulates the activity of a receptor by enhancing, increasing, strengthening, or inducing its binding or otherwise interacting activity. In some embodiments, the term is used to describe compounds that act alone to activate receptors or to describe compounds that are “positive allosteric modulators” (also known as “PAMs”) that actively enhance the effects of other agonists.

[0145] As used herein, “synergistic,” “synergistically,” or “synergistically effective” refers to an effect produced by two or more individual components, where the overall effect is produced by these components and the combined effect is greater than the sum of the effects of the individual components acting alone. As used herein, the term “synergistically effective” refers to any combination of nucleotide derivatives and / or transmembrane compounds and other compounds (e.g., amino acids, nucleotides, or compounds binding to the transmembrane domains of T1R1 or T1R3) that exhibit synergistic activation of the T1R1 / T1R3 receptor or increased palatability of pet food.

[0146] The term "alkyl" refers to a straight-chain or branched C1-C chain consisting only of carbon and hydrogen atoms. 20 (Preferably C1-C6) hydrocarbon group (without unsaturated bonds) that is connected to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl).

[0147] The term "alkenyl" refers to a C2-C group containing at least one carbon-carbon double bond, which can be straight-chain or branched. 20 (Preferred C2-C)12 Aliphatic hydrocarbon groups, such as vinyl, 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl.

[0148] The term "alkynyl" refers to a C2-C group containing at least one carbon-carbon triple bond, which can be straight-chain or branched. 20 (Preferred C2-C) 12 Aliphatic hydrocarbon groups, such as ethynyl, 1-propynyl, and 2-propynyl.

[0149] The term "cycloalkyl" refers to unsaturated non-aromatic monocyclic or polycyclic hydrocarbon ring systems (including, for example, C3-C6), such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Polycyclic cycloalkyl (including, for example, C6-C6) 15 Examples include bridged cycloalkanes or spirobycycloalkanes such as perhydronaphthyl, adamantyl, and norbornyl, for example, spiro(4,4)non-2-yl.

[0150] The term "cycloalkylalkyl" refers to a cycloalkyl group as defined above that is directly attached to an alkyl group as defined above, resulting in a stable structure such as cyclopropylmethyl, cyclobutylethyl, or cyclopentylethyl.

[0151] The term "ether" refers to an alkyl or cycloalkyl group incorporated into an alkyl chain as defined above, having at least one oxygen atom, such as methyl ethyl ether, diethyl ether, or tetrahydrofuran. These groups may also be described as alkoxyalkyl or alkoxycycloalkyl.

[0152] The term "aminoalkyl" refers to an alkyl or cycloalkyl group having at least one nitrogen atom as defined above, such as n-butylamine and tetrahydrobenzene. Azine.

[0153] The term "aryl" refers to an aromatic group having about 6 to about 14 carbon atoms, such as phenyl, naphthyl, tetrahydronaphthyl, indenyl, and biphenyl.

[0154] The term "aralkyl" refers to an aryl group as defined above directly bonded to an alkyl group as defined above, such as -CH2C6H5 and -C2H4C6H5.

[0155] The term "heterocyclic" refers to a stable 3- to 15-membered ring group consisting of a carbon atom and one or more heteroatoms, for example, one to five heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. For the purposes of this application, the heterocyclic group can be a monocyclic or bicyclic ring system, which may include fused or bridged ring systems, and the nitrogen, carbon, oxygen, or sulfur atom in the heterocyclic group may optionally be oxidized to various oxidation states. Furthermore, the nitrogen atom may optionally be quaternized; the ring group may be partially or fully saturated, or the ring group may be completely unsaturated (i.e., aromatic heterocycles or heteroaryl aromatics). The heterocyclic group may be attached to the host structure at any heteroatom or carbon atom that results in a stable structure.

[0156] The term "heteroaryl" refers to a heterocyclic ring in which the aromatic ring is a heterocyclic ring.

[0157] The term "heteroarylalkyl" refers to a heteroaryl group as defined above that is directly bonded to an alkyl group. Heteroarylalkyl groups can be attached to the main structure at any carbon atom of the alkyl group that results in a stable structure.

[0158] The term "heterocyclic group" refers to a heterocyclic cyclic group as defined above. A heterocyclic cyclic group can be attached to the main structure at any heteroatom or carbon atom that results in a stable structure.

[0159] In some implementations, the term "umami receptor" refers to a G protein-coupled receptor (GPCR), such as the T1R1 / T1R3 GPCR. Umami receptors can be, for example, umami receptors of cats, dogs, humans, or non-human mammals.

[0160] In some embodiments, cat T1R1 is a protein comprising an amino acid sequence as shown in SEQ ID NO: 2 or a sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homologous to it (homology, as used herein, can be measured using standard software such as BLAST or FASTA), and is encoded by a nucleic acid comprising a sequence as shown in SEQ ID NO: 1 or a sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homologous to it (homology, as used herein, can be measured using standard software such as BLAST or FASTA).

[0161] In some embodiments, cat T1R3 is a protein comprising an amino acid sequence as shown in SEQ ID NO: 4 or a sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homologous to it (homology, as used herein, can be measured using standard software such as BLAST or FASTA), and is encoded by a nucleic acid comprising a sequence as shown in SEQ ID NO: 3 or a sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% homologous to it (homology, as used herein, can be measured using standard software such as BLAST or FASTA).

[0162] 2. Nucleotide derivatives

[0163] This invention relates to flavor compositions comprising at least one nucleotide derivative. In some embodiments, the nucleotide derivative is a umami-enhancing compound. The nucleotide derivatives disclosed herein are identified by computer modeling of the binding pocket of the feline T1R1 / T1R3 receptor (“umami receptor”). Flavor compositions can be used to enhance or improve the palatability, taste, or flavor of pet foods. Flavor compositions may include combinations of compounds, such as one or more nucleotide derivatives and / or one or more amino acids and / or one or more nucleotides and / or one or more transmembrane compounds, as described herein, and may be added to pet food compositions in various delivery system forms.

[0164] In some embodiments, the nucleotide derivative may be a compound listed in Tables 2 and 5-13 below.

[0165] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-1:

[0166] R1 is selected from the group consisting of substituted or unsubstituted purines or substituted or unsubstituted pyrimidines;

[0167] Wherein R2 is selected from monophosphate, diphosphate, triphosphate, OP(W)(OH)2, -OP(W)(OH)OP(W)(OH)2, -OP(W)(OH)OP(W)(OH)OP(W)(OH)2, -OS(O)2 aryl(H), -OS(O)2 aryl(CH3), -P(W)(OH)2, -OP(W)(OH)OS(O)2(OH), -OP(W)(OH)Z, -P(W)(OH)OP(W)(OH)2, -O(CH2) 1-4OP(W)(OH)2, -OS(W)(OH)2, -OP(W)(OH)CH2OP(W)(OH)2, -OP(W)(OH)OP(W)O(CH2) 1-4 R 18 -(CH2) 0-4 COOH, -(CH2) 0-4 S(O)(OH)2-(CH2) 0-4 C(O)NHOH and -(CH2) 0-4 B(OH)2;

[0168] Where X is selected from O, S, N(R3) and CH2;

[0169] Where W is selected from O and S;

[0170] R3 is selected from H and CH3;

[0171] Among them, R4, R5, R 14 R 15 R 18 Independently selected from H, OH, SH, CH2, CH3, OR6, SR6, CH2CH3, lower alkyl branches and unbranched (C1-C6), XC(O) lower alkyl, -XC(O)CH2Ph, -P(W)(OH)2, -XC(O)PhR 11 -OP(O)(OH)O, OCH3, N(R) 11 ,R 17 ), -O(C)n R 11 R 17 O-, N (H or independent lower alkyl group) 2-3 and COOR 11 -OC(W)NH(CH2) 1- 6NH2、-OC(W)NH(CH2) 1-6 R 44 ;

[0172] Where R 44 It includes H, OH, SH, CH2, CH3, OR6, SR6, CH2CH3, lower alkyl branches and unbranched chains (C1-C6), XC(O) lower alkyl, -XC(O)CH2Ph, -P(W)(OH)2, -XC(O)PhR 11 -OP(O)(OH)O, OCH3, N(R) 11 ,R 17 ), -O(C)n R 11 R 17 O-, N (H or independent lower alkyl group) 2-3 or COOR 11-OC(W)NH(CH2) 1-6 NH2;

[0173] Z is selected from piperidine, morpholine, piperazine, N-methylpiperazine, and N(R). 16 (R) 17 );and

[0174] Among them, R6, R 11 R 16 and R 17 Independently selected from H, OH, SH, CH2, CH3, OCH3, COOR, N(R) 12 (R) 13 CH2CH3, lower alkyl branched and unbranched (C1-C6), XC(O) lower alkyl, -XC(O)CH2Ph, -P(W)(OH)2, -XC(O)PhR 12 -OP(O)(OH)O, OCH3,

[0175]

[0176] N(R 12 ,R 13 ), -O(C)n R 12 R 13 O-, N (H or independent lower alkyl group) 2-3 and COOR 12 ;and

[0177] Where R 12 and R 13 It is independently selected from H, OH, SH, CH2, CH3, OCH3 and CH2CH3.

[0178] In some implementations, R1 in equation Nt-1 is selected from...

[0179]

[0180] In some embodiments, R1 in formula Nt-1 is selected from the compounds listed in Table 1.

[0181] Table 1. R1 groups of nucleotide derivatives of formula Nt-1

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188] Substituents in the substituent groups described herein, such as "substituted alkyl", "substituted aryl", "substituted furan", "substituted thiophene", "substituted alkyl", "substituted phenyl", "substituted pyrimidine", or "substituted naphthalene", may be the same as or different from one or more of the following groups selected from those described in this application: hydrogen, halogen, methyl, amide, acetyl, nitro (-NO2), hydroxyl (-OH), oxo (=O), thio (=S), OCH3, methylenedioxy, CN, NO2, COOH, SO3H, S(O) 1- 2CH3、S(O) 1-2 Aryl, SCH3, OH, N(R) 1-2 The group may be substituted with one or more substituents, such as COOCH3, OC(O)CH3, SH, sulfonyl, sulfinylamino, sulfate, cyano, azide, trifluoromethyl (-CF3), methoxy (-OCH3), tert-butyl carbamate (-Boc), or a group optionally substituted from the following: alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aryloxy, aralkyl, ether, carboxyl, hydroxyl, heteroaryl, heteroarylalkyl, sulfonyl, and heterocyclic. The "substituted" function may have one or more substituents.

[0189] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-2:

[0190]

[0191] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-3:

[0192]

[0193] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-4:

[0194]

[0195] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-5:

[0196]

[0197] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-6:

[0198]

[0199] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-7:

[0200]

[0201] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-8:

[0202]

[0203] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-9:

[0204]

[0205] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-10:

[0206]

[0207] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-11:

[0208]

[0209] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-12:

[0210]

[0211] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-13:

[0212]

[0213] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-14:

[0214]

[0215] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-15:

[0216]

[0217] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-16:

[0218]

[0219] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-17:

[0220]

[0221] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-18:

[0222]

[0223] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-19:

[0224]

[0225] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-20:

[0226]

[0227] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-21:

[0228]

[0229] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-22:

[0230]

[0231] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-23:

[0232]

[0233] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-24:

[0234]

[0235] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-25:

[0236]

[0237] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-26:

[0238]

[0239] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-27:

[0240]

[0241] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-28:

[0242]

[0243] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-29:

[0244]

[0245] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-30:

[0246]

[0247] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-31:

[0248]

[0249] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-32:

[0250]

[0251] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-33:

[0252]

[0253] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-34:

[0254]

[0255] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-35:

[0256]

[0257] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-36:

[0258]

[0259] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-37:

[0260]

[0261] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-38:

[0262]

[0263] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-39:

[0264]

[0265] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-40:

[0266]

[0267] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-41:

[0268]

[0269] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-42:

[0270]

[0271] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-43:

[0272]

[0273] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-44:

[0274]

[0275] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-45:

[0276]

[0277] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-46:

[0278]

[0279] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-47:

[0280]

[0281] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-48:

[0282]

[0283] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-49:

[0284]

[0285] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-50:

[0286]

[0287] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-51:

[0288]

[0289] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-52:

[0290]

[0291] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-53:

[0292]

[0293] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-54:

[0294]

[0295] In some embodiments, the nucleotide derivative comprises a compound having the structure Nt-55:

[0296]

[0297] In some embodiments, the nucleotide derivative may be a salt, stereoisomer, or edible form of a compound of formula NT-1 to Nt-55 and / or a compound listed in Tables 2 and 6-14.

[0298] Table 2: Nucleotide Derivatives

[0299]

[0300]

[0301]

[0302] In some embodiments, the nucleotide derivatives of the present invention comprise salts of nucleotide derivatives, such as, but not limited to, acetates or formates. In some embodiments, the salts of said nucleotide derivatives comprise salts containing cations (+) via ionic bonds (e.g., but not limited to, Al). 3+ Ca 2+ Na + K + Cu 2+ H + Fe 3+ Mg 2+ NH4 + and H3O + Bonded anions (-) (e.g., but not limited to, Cl-) - O 2- CO3 2- HCO3 - OH - NO3 - PO4 3- SO4 2- CH3COO - HCOO - and C2O4 2- In other embodiments, the salt of the nucleotide derivative includes a cation (+) bonded to an anion (-) via an ionic bond.

[0303] In some embodiments, the nucleotide derivatives of this application can be identified by computer modeling of the feline T1R1 / T1R3 receptor (“umami receptor”), wherein the nucleotide derivatives of this application comprise structures assembled into the binding site of the feline T1R1 / T1R3 receptor.

[0304] In some embodiments, the nucleotide derivatives of this application are identified by an in vitro assay, wherein the nucleotide derivative activates the feline T1R1 / T1R3 receptor expressed in vitro by cells. In some embodiments, the nucleotide derivative activates the receptor alone, or in combination with other T1R1 / T1R3 binding agents such as the nucleotides, amino acids, and transmembrane compounds described herein, and / or in combination with amino acids described herein that bind one or more other receptors. In some embodiments, the in vitro assay includes the in vitro assays described in the Examples section of this application.

[0305] 2.1 T1R1 nucleotide binding site

[0306] This application also provides compositions for modulating umami receptors, such as T1R1 / T1R3 receptors, wherein the composition interacts with one or more amino acids in the Venus Flytrap domain of the umami receptor. In some embodiments, the Venus Flytrap (VFT) domain is present in T1R1. In some embodiments, the amino acids interacting with the composition include one or more of Thr449, Ser172, Glu170, Glu301, His71, His47, Arg277, His308, Asn69, Asn302, Ser306, Ser384, Asp302, Ser306, and Ala380.

[0307] In one non-limiting embodiment, the composition comprises nucleotides and / or nucleotide derivatives, wherein the nucleotides and / or nucleotide derivatives interact with one, two, three, four, five, six, seven, eight or more of His71, Arg277, His308, Ser306, Ser384, Ala380, His47, Asn69 and Asp302 of T1R1.

[0308] In another non-limiting embodiment, the composition comprises nucleotides and / or nucleotide derivatives, wherein the nucleotides and / or nucleotide derivatives interact with one, two, three, four or more of Met383, Ser385, Ile309, Ser107 and Asp49 of T1R1.

[0309] When bound to nucleotides and / or nucleotide derivatives, the amino acid residues of the VFT may coordinate the phosphate, diphosphate, triphosphate, bisphosphate, phosphate analogs (e.g., acidic functional groups such as COOH, SO3H, NHOH, etc.) of the nucleotides and / or nucleotide derivatives, and include one or more of His71, His47, Arg277, His308, Ile309, Asn69, Ser107 and Asp49 of T1R1.

[0310] In some embodiments, at least one phosphate group of the nucleotide and / or nucleotide derivative interacts with one, two, three, four, or more of His71, His47, Arg277, His308, and Asn69 of T1R1. In a non-limiting example, the binding of the nucleotide and / or nucleotide derivative to the VFT involves an interaction between a negatively charged group of an amino acid in the phosphate-binding region of the VFT and the phosphate group of the nucleotide and / or nucleotide derivative.

[0311] The amino acid residues of the VFT may coordinate the sugar atoms of nucleotides and / or nucleotide derivatives (or, for example, modified sugars or sugar substitutions), and may include Asp302 and / or Ser306 of T1R1.

[0312] In some embodiments, at least one sugar molecule of the nucleotide and / or nucleotide derivative interacts with the amino acids Asp302 and / or Ser306 of T1R1.

[0313] The amino acid residues of the VFT may coordinate nitrogenous bases of nucleotides and / or nucleotide derivatives, and may include one or more of T1R1's Ser384, Ser385, Ala380, Met383, Glu170 and Asp302.

[0314] In some embodiments, the nitrogenous bases of nucleotides and / or nucleotide derivatives interact with one, two or more of Ser384, His308 and Ala380 of T1R1.

[0315] In other non-limiting embodiments, the sugar molecules of nucleotides and / or nucleotide derivatives interact with the Asp302 of the VFT, wherein the Asp302 is tuned to simultaneously coordinate the binding of the zwitterionic backbone nitrogen of the amino acid and the sugar of the nucleotide or nucleotide derivative.

[0316] In some embodiments, the composition interacts with the umami Venus flytrap domain (VFT) according to any combination of interactions described herein, such as one, two, three or more interactions. The interaction between the nucleotide and / or nucleotide derivative and the VFT may also include additional hydrophobic interactions added to the interaction energy between the nucleotide and / or nucleotide derivative and the VFT.

[0317] In some embodiments, the interaction between the composition and one or more amino acids includes one or more hydrogen bonds, covalent bonds, non-covalent bonds, salt bridges, physical interactions, and combinations thereof. The interaction can also be any ligand-receptor interaction known in the art. Such interactions can be determined by, for example, site-directed mutagenesis, X-ray crystallography, X-ray or other spectroscopic methods, nuclear magnetic resonance (NMR), crosslink assessment, mass spectrometry or electrophoresis, shift determination based on known agonists, structure determination, and combinations thereof. In some embodiments, the interaction is determined by computer, for example by theoretical methods such as using molecular docking, molecular modeling, molecular simulation, or other means known to those skilled in the art to dock the compound into the VFT domain.

[0318] This application also provides a method for identifying compounds that regulate the activity of umami receptors, such as T1R1, wherein the compounds are identified based on their ability to interact with one or more amino acids present in the VFT domain of T1R1 as described herein.

[0319] In some embodiments, the method includes contacting a test agent with a feline T1R1 umami receptor, detecting an interaction between the test agent and another amino acid at a VFT interaction site of the feline T1R1 umami receptor, and selecting a test agent that interacts with one or more amino acids as the compound.

[0320] 3. Transmembrane compounds

[0321] This invention relates to flavor compositions comprising at least one transmembrane compound. In some embodiments, the transmembrane compound is an umami flavor-enhancing compound. The transmembrane compounds disclosed herein are identified by computer modeling of a putative agonist within the transmembrane region of the T1R1 of the feline T1R1 / T1R3 receptor (“umami receptor”). Thus, in some embodiments, the transmembrane compound is a composition that interacts (e.g., binds) to a region of T1R1 comprising a transmembrane domain of T1R1. In some embodiments, this interaction with the transmembrane domain of T1R1 activates T1R1 / T1R3 or the umami receptor. In other embodiments, the transmembrane compound synergizes with other T1R1 agonists or modulators to modulate the activity of T1R1 / T1R3 or the umami receptor.

[0322] Flavor compositions can be used to enhance or improve the palatability, taste, or flavor of pet food. Flavor compositions may include combinations of compounds, such as transmembrane compounds and / or nucleotides and / or nucleotide derivatives and / or amino acids, and may be added to pet food compositions in various delivery system forms.

[0323] 3.1 Amine Derivatives I

[0324] In some embodiments, the transmembrane compound includes a compound of formula Tm-1 having the following structure:

[0325]

[0326] X1 is selected from the group consisting of O and S;

[0327] Where n1 is 1-3;

[0328] Where n2 is 0-4 (where when n is 0, there is a chemical bond);

[0329] Where n4 is 0-3;

[0330] R1, R2 and R3 are independently selected from the group consisting of H, =O, =S, branched or unbranched and substituted or unsubstituted lower alkyl groups (C1-C8) and R5;

[0331] R4 is selected from the group consisting of: H, branched or unbranched lower alkyl groups (C1-C8), and (CH2). n2 Aryl;

[0332] R5 is selected from the group consisting of: H, CH3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3, CH2CH2SCH3, CH2SH, CH2SeH, CH2OH, CH(OH)CH3, CH2C(O)NH2, CH2CH2C(O)NH2, CH2CH2CH2NHC(NH)NH2, CH2(1H-imidazolyl))(CH2(1H-imadazole-yl)), CH2(CH2)2CH2NH2, CH2COOH, CH2CH2COOH, CH2C6H5, CH2(4-hydroxyphenyl), CH2[3-yl-(1H-indole)], CH2(cyclopentyl), CH2(cyclohexyl), CH2(indanyl), independent branched or unbranched lower alkyl groups (C1-C2). 10 (CH2) 0-4 PH, c-C3H5, c-C4H7, c-C5H9, c-C6H 10 , phenyl, biaryl, (CH2) n2 Aryl, pyridine, thiophine, CH2Ph, CH2pyridine, and CH2thiophine;

[0333] The aryl and alkyl (branched and unbranched) groups may optionally be substituted with the following: methyl, OH, SH, OCH3, SCH3, COOH, COOR. 13 S(O)n4R1, C(O)R 11 C(O)NR11 R 12 CN, NR 11 R 12 NR 11 C(O)R 12 , aryl, methylenedioxy, alkyl (C1–C5), CH2SSCH2CH(COOH)(NH2), halogen (F, Cl, Br, I), NO2, NHC(=NH)NH2, CHO, CF3, P(=X1)(OR1)2, OP(=X1)(OR1)2;

[0334] R5 and R6 can be connected to form cyclic rings, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl (i.e., spirocyclic);

[0335] R6 is selected from the group consisting of H and branched or unbranched lower alkyl groups (C1-C4);

[0336] R7 is selected from the group consisting of: H, AA, OH, O, branched or unbranched lower alkyl groups (C1-C6), and O (CH2). n1 Aryl, NR 11 R 12 、N(R 14 OH, C(R8)(R9), aryl and heteroaryl;

[0337] R8 and R9 are independently selected from the following group: H, branched or unbranched lower alkyl groups (C1-C6), aryl, alkylaryl and alkylheteroaryl;

[0338] Where R 11 and R 12 Independently selected from the group consisting of: H, CH3, branched or unbranched lower alkyl groups (C1-C6), phenyl, aryl, and (CH2). n1 Aryl;

[0339] Where R 13 Choose the group consisting of H, CH3, CH2CH3, CH2 aryl, and tert-butyl;

[0340] Where R 14 Choose the group consisting of H and CH3; and

[0341] AA is selected from the group consisting of naturally occurring α-amino acids or (R) or (S)-configurations (i.e., protein amino acids).

[0342] In one non-limiting embodiment, all asymmetric configurations are considered for use in the formulations disclosed in this invention.

[0343] In some embodiments, the aryl group has its standard chemical meaning and may include, but is not limited to, the following: benzene (Ph), pyridine, thiophene, furan, naphthyl, indole, benzothiophene, benzofuran, quinolone, isoquinoline, pyrrole, N-(methyl)pyrrole, imidazole, thiazole, pyrimidine, isoquinoline, pyrrole, N-(methyl)pyrrole, imidazole, thiazole, pyrimidine, and so on. azole, Oxazole, isoindole, indazine, purine, pyrazine, and pyrieazine.

[0344] In some implementations, the aryl group may be linked at different carbon centers, as will be understood by those skilled in the art.

[0345] In other non-limiting embodiments, when the compound comprises two aromatic rings (e.g., phenyl), one or both rings may be substituted by a biaryl ring system. Such biaryl ring systems include, for example, phenyl-phenyl, phenyl-pyridyl, phenyl-thiophene, thiophene-thiophene, and phenyl-furan.

[0346] In some embodiments, the transmembrane compound includes a compound of formula Tm-2 having the following structure:

[0347]

[0348] R4, R5, R6, R7, X1, and n1 are defined as in equation Tm-1.

[0349] In some embodiments, the transmembrane compound comprises a compound having the structure Tm-20:

[0350]

[0351] In some embodiments, the transmembrane compound includes a compound of formula Tm-21 having the following structure:

[0352]

[0353] In some embodiments, the transmembrane compound includes a compound having the structure Tm-22:

[0354]

[0355] In some embodiments, the transmembrane compound includes a compound of formula Tm-23 having the following structure:

[0356]

[0357] In some embodiments, the transmembrane compound includes a compound having the structure Tm-24:

[0358]

[0359] In some embodiments, the transmembrane compound includes a compound of formula Tm-25 having the following structure:

[0360]

[0361] In some embodiments, the transmembrane compound includes a compound having the structure Tm-26:

[0362]

[0363] In some embodiments, the transmembrane compound includes a compound of formula Tm-27 having the following structure:

[0364]

[0365] In some embodiments, the transmembrane compound includes a compound of formula Tm-28 having the following structure:

[0366]

[0367] In some embodiments, the transmembrane compound includes a compound of formula Tm-29 having the following structure:

[0368]

[0369] In some embodiments, the transmembrane compound includes a compound having the structure Tm-30:

[0370]

[0371] In some embodiments, the transmembrane compound includes a compound of formula Tm-31 having the following structure:

[0372]

[0373] In some embodiments, the transmembrane compound includes a compound having the structure Tm-32:

[0374]

[0375] In some embodiments, the transmembrane compound includes a compound having the structure Tm-33:

[0376]

[0377] In some embodiments, the transmembrane compound includes a compound having the structure Tm-34:

[0378]

[0379] In some embodiments, the transmembrane compound includes a compound having the structure Tm-35:

[0380]

[0381] In some embodiments, the transmembrane compound includes a compound of formula Tm-36 having the following structure:

[0382]

[0383] In some embodiments, the transmembrane compound includes a compound having the structure Tm-37:

[0384]

[0385] In some embodiments, the transmembrane compound includes a compound having the structure Tm-38:

[0386]

[0387] In some embodiments, the transmembrane compound includes a compound of formula Tm-39 having the following structure:

[0388]

[0389] In some embodiments, the transmembrane compound includes a compound having the structure Tm-40:

[0390]

[0391] 3.2 Amine Derivatives II

[0392] In some embodiments, the transmembrane compound includes a compound of formula Tm-3 having the following structure:

[0393]

[0394] X1 is selected from the group consisting of O and S;

[0395] Where n1 and n2 are 0-4 (where chemical bonds exist when n1 and / or n2 are 0);

[0396] R1 is selected from the group consisting of: branched or unbranched lower alkyl groups (C1-C1). 10 (CH2) n2 Ph, c-C3H5, c-C4H7, c-C5H9, c-C6H 10 , phenyl, (CH2) n2Aryl, Ph, pyridine, thiophine, CH2Ph, CH2pyridine, CH2thiophine, O-aryl, Ph, pyridine, thiophene, furan, naphthyl, indole, benzothiophene, benzofuran, quinolone, isoquinoline, pyrrole, N-(methyl)pyrrole, imidazole, thiazole, pyrimidine, iso azole, Zyrazoles, isoindole, indazines, purines, pyrazines, pyrieazines, O-alkyl (C1-C6), biaryl groups, and OR1 (e.g., carbamates).

[0397] The aryl and alkyl (branched and unbranched) groups may optionally be substituted with the following: methyl, OH, SH, OCH3, SCH3, COOH, COOR. 13 S(O) n4 R1、C(O)R 11 C(O)NR 11 R 12 CN, NR 11 R 12 NR 11 C(O)R 12 , aryl, methylenedioxy, alkyl (C1–C5), CH2SSCH2CH(COOH)(NH2), halogen (F, Cl, Br, I), NO2, NHC(=NH)NH2, CHO, CF3, P(=X1)(OR1)2 or OP(=X1)(OR1)2;

[0398] R2 is selected from the group consisting of H, CH3, (CH2) and aryl groups;

[0399] R3, R4, and R5 are independently selected from the group consisting of: H, branched or unbranched lower alkyl groups (C1-C8), (CH2). n2 Aryl and R1;

[0400] Where R 11 and R 12 It is independently selected from H, CH3, branched or unbranched lower alkyl groups (C1-C6), phenyl, aryl, and (CH2). n1 Groups composed of aryl groups;

[0401] Where R 13 Choose the group consisting of H, CH3, CH2CH3, CH2 aryl and tert-butyl.

[0402] In some embodiments, the aryl group has its standard chemical meaning and may include, but is not limited to, the following: benzene (Ph), pyridine, thiophene, furan, naphthyl, indole, benzothiophene, benzofuran, quinolone, isoquinoline, pyrrole, N-(methyl)pyrrole, imidazole, thiazole, pyrimidine, isoquinoline, pyrrole, N-(methyl)pyrrole, imidazole, thiazole, pyrimidine, and so on. azole, Azole, isoindole, indazine, purine, pyrazine, and pyrieazine.

[0403] In some implementations, the aryl group may be linked at different carbon centers, as will be understood by those skilled in the art.

[0404] In some implementations, the asymmetry center may be of (R) or (S) configuration, as will be understood by those skilled in the art.

[0405] In one non-limiting embodiment, all asymmetric configurations are considered for use in the formulations disclosed in this invention.

[0406] In other non-limiting embodiments, when the compound comprises two aromatic rings (e.g., phenyl), one or both rings may be substituted by a biaryl ring system. Such biaryl ring systems include, for example, phenyl-phenyl, phenyl-pyridyl, phenyl-thiophene, thiophene-thiophene, and phenyl-furan.

[0407] In some embodiments, the transmembrane compound includes a compound of formula Tm-4 having the following structure:

[0408]

[0409] R1 and R2 are independently selected from the group consisting of: aryl, cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl), and heteroaryl (e.g., but not limited to Ph, pyridine, thiophene, furan, naphthyl, indole, benzothiophene, benzofuran, quinolone, isoquinoline, pyrrole, N-(methyl)pyrrole, imidazole, thiazole, pyrimidine, iso... azole, Azole, isoindole, indazine, purine, pyrazine, and pyrieazine;

[0410] The aryl or cycloalkyl group may optionally be substituted with the following: methyl, OH, SH, OCH3, SCH3, COOH, COOR. 13 S(O) n4 R1、C(O)R 11 C(O)NR 11 R 12 CN, NR 11 C(O)R 12, aryl, methylenedioxy, alkyl (C1–C5), CH2SSCH2CH(COOH)(NH2), halogen (F, Cl, Br, I), NO2, NHC(=NH)NH2, CHO, CF3, P(=X1)(OR1)2 or OP(=X1)(OR1)2;

[0411] The cycloalkyl group may optionally include heteroatoms (e.g., O, N and / or S) in the ring, such as piperidine, piperazine, tetrahydrothiophene, pyran, pyrrolidine or tetrahydrofuran;

[0412] Where n1 = 0 - 4;

[0413] Where R 11 R 12 R 13 And X1 as defined in this paper; and

[0414] Where n4 = 0 - 4.

[0415] In some embodiments, the transmembrane compound includes a compound of formula Tm-41 having the following structure:

[0416]

[0417] In some embodiments, the transmembrane compound includes a compound having the structure Tm-42:

[0418]

[0419] In some embodiments, the transmembrane compound includes a compound having the structure Tm-43:

[0420]

[0421] In some embodiments, the transmembrane compound includes a compound of formula Tm-44 having the following structure:

[0422]

[0423] In some embodiments, the transmembrane compound includes a compound having the structure Tm-45:

[0424]

[0425] In some embodiments, the transmembrane compound includes a compound having the structure Tm-46:

[0426]

[0427] In some embodiments, the transmembrane compound includes a compound having the structure Tm-47:

[0428]

[0429] In some embodiments, the transmembrane compound includes a compound of formula Tm-48 having the following structure:

[0430]

[0431] In some embodiments, the transmembrane compound includes a compound of formula Tm-49 having the following structure:

[0432]

[0433] In some embodiments, the transmembrane compound includes a compound having the structure Tm-50:

[0434]

[0435] In some embodiments, the transmembrane compound includes a compound of formula Tm-51 having the following structure:

[0436]

[0437] In some embodiments, the transmembrane compound includes a compound having the structure Tm-52:

[0438]

[0439] In some embodiments, the transmembrane compound includes a compound having the structure Tm-53:

[0440]

[0441] In some embodiments, the transmembrane compound includes a compound of formula Tm-54 having the following structure:

[0442]

[0443] In some embodiments, the transmembrane compound comprises a compound having the structural formula Tm-55:

[0444]

[0445] 3.3 Pericarpicolone derivatives

[0446] In some embodiments, the transmembrane compound includes a compound of formula Tm-6 having the following structure:

[0447]

[0448] Where R1 or R 2 Independently selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, hydroxyl, hydrogen, substituted or unsubstituted ether, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted naphthyl, substituted or unsubstituted thiophenyl, substituted or unsubstituted benzothiaphenyl, substituted or unsubstituted indolyl, substituted or unsubstituted isoquinolinyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted indole, or substituted or unsubstituted indoleyl.

[0449] In some implementations, R 1 or R 2 It can contain the following structures:

[0450]

[0451] Where R 3 R 4 R 5 R 6 and R 7 Independently selected from the group consisting of: hydrogen, halogen, cyano, azide, hydroxyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfinamide, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted amide, substituted or unsubstituted heterocycle, substituted or unsubstituted alkoxy, substituted or Unsubstituted aryloxy groups, substituted or unsubstituted ethers, substituted or unsubstituted carboxyl groups, substituted or unsubstituted acyl groups, substituted or unsubstituted benzothiazolyl groups, substituted or unsubstituted pyridyl groups, substituted or unsubstituted naphthyl groups, substituted or unsubstituted thiophene groups, substituted or unsubstituted benzothiaphene groups, substituted or unsubstituted indolyl groups, substituted or unsubstituted isoquinolinyl groups, substituted or unsubstituted quinolinyl groups, or substituted or unsubstituted heteroarenyl groups, or substituted or unsubstituted indene groups, or substituted or unsubstituted indene groups. In some embodiments, R 3 R 4 R 5 R 6 and R 7Two or more of them can form a ring together, which can be a heterocycle (i.e., containing one or more heteroatoms) or it can be a complete carbon ring and can be saturated or unsaturated independently.

[0452] Substituents in the substituent groups described herein include, for example, "substituted ether", "substituted carboxyl", "substituted acyl", "substituted sulfonyl", "substituted alkyl", "substituted alkenyl", "substituted cycloalkyl", "substituted cycloalkylalkyl", "substituted aralkyl", "substituted aryl", "substituted heterocyclic", "substituted heteroarylalkyl", "substituted heteroaryl", "substituted naphthyl", "substituted phenyl", "substituted thiophene", "substituted benzothiophene", "substituted pyridinyl", "substituted indolyl", "substituted isoquinolinyl", "substituted quinolinyl", "substituted benzothiazolyl", and "substituted..." "Heteroaryl", "substituted indene", or "substituted indene" may be the same as or different from one or more of the following groups selected from those described in this application: hydrogen, halogen, amide, acetyl, nitro (-NO2), hydroxyl (-OH), oxo (=O), thio (=S), sulfonyl, sulfinylamino, sulfate, mercapto, cyano, azide, trifluoromethyl (-CF3), methoxy (-OCH3), tert-butyl carbamate (-Boc), or selected from the following optionally substituted groups: alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryl, aryloxy, arylalkyl, ether, carboxyl, hydroxyl, heteroaryl, sulfonyl, and heterocyclic. The "substituted" function may have one or more substituents.

[0453] In one non-limiting implementation, R 1 and R 2 It is an unsubstituted phenyl group.

[0454] In one non-limiting implementation, R 1 It is an unsubstituted phenyl and R 2 It is a substituted phenyl group.

[0455] In some embodiments, the transmembrane compound includes a compound of formula Tm-7 having the following structure:

[0456]

[0457] R1 and R2 are defined as above for equation Tm-6.

[0458] In some embodiments, the transmembrane compound includes a compound of formula Tm-5 having the following structure:

[0459]

[0460] X1 is selected from the group consisting of O, N(R3), and S;

[0461] X2 and X3 are independently selected from the groups composed of O and S;

[0462] R3 is selected from the group consisting of: H and branched or unbranched lower alkyl groups (C1-C4); and

[0463] R1 and R2 are as defined above for equation Tm-6, and can also be (CH2). n C(=X1)R1,(CH2) n C(=X1)R2, where X1 is O or S, and n is 0-4.

[0464] In some embodiments, the transmembrane compound includes a compound of formula Tm-9 having the following structure:

[0465]

[0466] Where X 1-5 Choose independently the group consisting of O and S; and where R1 and R2 are as defined above for equation Tm-6.

[0467] In some embodiments, the transmembrane compound includes a compound of formula Tm-10 having the following structure:

[0468]

[0469] Where X 1-5 Choose independently the group consisting of O and S; and where R1 and R2 are as defined above for equation Tm-6.

[0470] In some embodiments, the transmembrane compound comprises a compound having the structure Tm-11:

[0471]

[0472] Where X 1-3 Choose independently the group consisting of O and S; and where R1 and R2 are as defined above for equation Tm-6.

[0473] In some embodiments, the transmembrane compound includes a compound having the structure Tm-56:

[0474]

[0475] In some embodiments, the transmembrane compound includes a compound having the structure Tm-57:

[0476]

[0477] In some embodiments, the transmembrane compound includes a compound of formula Tm-58 having the following structure:

[0478]

[0479] In some embodiments, the transmembrane compound includes a compound of formula Tm-59 having the following structure:

[0480]

[0481] In some embodiments, the transmembrane compound includes a compound having the structure Tm-60:

[0482]

[0483] In some embodiments, the transmembrane compound includes a compound having the structure Tm-61:

[0484]

[0485] In some embodiments, the transmembrane compound includes a compound having the structure Tm-62:

[0486]

[0487] In some embodiments, the transmembrane compound includes a compound having the structure Tm-63:

[0488]

[0489] In some embodiments, the transmembrane compound includes a compound having the structure Tm-64:

[0490]

[0491] In some embodiments, the transmembrane compound includes a compound having the structure Tm-65:

[0492]

[0493] In some embodiments, the transmembrane compound includes a compound having the structure Tm-66:

[0494]

[0495] In some embodiments, the transmembrane compound includes a compound having the structure Tm-67:

[0496]

[0497] In some embodiments, the transmembrane compound includes a compound having the structure Tm-68:

[0498]

[0499] In some embodiments, the transmembrane compound includes a compound having the structure Tm-69:

[0500]

[0501] In some embodiments, the transmembrane compound includes a compound having the structure Tm-70:

[0502]

[0503] In some embodiments, the transmembrane compound includes a compound having the structure Tm-71:

[0504]

[0505] In some embodiments, the transmembrane compound includes a compound having the structure Tm-72:

[0506]

[0507] In some embodiments, the transmembrane compound includes a compound having the structure Tm-73:

[0508]

[0509] In some embodiments, the transmembrane compound includes a compound having the structure Tm-74:

[0510]

[0511] 3.4 Imidazolidinedione derivatives

[0512] In some embodiments, the transmembrane compound includes a compound of formula Tm-19 having the following structure:

[0513]

[0514] Where R 1 or R 2Independently selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, hydroxyl, hydrogen, substituted or unsubstituted ether, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted naphthyl, substituted or unsubstituted thiophenyl, substituted or unsubstituted benzothiaphenyl, substituted or unsubstituted indolyl, substituted or unsubstituted isoquinolinyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted indole, or substituted or unsubstituted indoleyl.

[0515] In some implementations, R 1 or R 2 It can contain the following structures:

[0516]

[0517] Where R 3 R 4 R 5 R 6 and R 7 Independently selected from the group consisting of: hydrogen, halogen, cyano, azide, hydroxyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfinamide, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted amide, substituted or unsubstituted heterocycle, substituted or unsubstituted alkoxy, substituted or Unsubstituted aryloxy groups, substituted or unsubstituted ethers, substituted or unsubstituted carboxyl groups, substituted or unsubstituted acyl groups, substituted or unsubstituted benzothiazolyl groups, substituted or unsubstituted pyridyl groups, substituted or unsubstituted naphthyl groups, substituted or unsubstituted thiophene groups, substituted or unsubstituted benzothiaphene groups, substituted or unsubstituted indolyl groups, substituted or unsubstituted isoquinolinyl groups, substituted or unsubstituted quinolinyl groups, or substituted or unsubstituted heteroarenyl groups, or substituted or unsubstituted indene groups, or substituted or unsubstituted indene groups. In some embodiments, R 3 R 4 R 5 R 6 and R 7 Two or more of them can form a ring together, which can be a heterocycle (i.e., containing one or more heteroatoms) or it can be a complete carbon ring and can be saturated or unsaturated independently.

[0518] Substituents in the substituent groups described herein include, for example, "substituted ether", "substituted carboxyl", "substituted acyl", "substituted sulfonyl", "substituted alkyl", "substituted alkenyl", "substituted cycloalkyl", "substituted cycloalkylalkyl", "substituted aralkyl", "substituted aryl", "substituted heterocyclic", "substituted heteroarylalkyl", "substituted heteroaryl", "substituted naphthyl", "substituted phenyl", "substituted thiophene", "substituted benzothiophene", "substituted pyridinyl", "substituted indolyl", "substituted isoquinolinyl", "substituted quinolinyl", "substituted benzothiazolyl", and "substituted..." "Heteroaryl", "substituted indene", or "substituted indene" may be the same as or different from one or more of the following groups selected from those described in this application: hydrogen, halogen, amide, acetyl, nitro (-NO2), hydroxyl (-OH), oxo (=O), thio (=S), sulfonyl, sulfinylamino, sulfate, mercapto, cyano, azide, trifluoromethyl (-CF3), methoxy (-OCH3), tert-butyl carbamate (-Boc), or selected from the following optionally substituted groups: alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryl, aryloxy, arylalkyl, ether, carboxyl, hydroxyl, heteroaryl, sulfonyl, and heterocyclic. The "substituted" function may have one or more substituents.

[0519] In one non-limiting embodiment, R1 and R2 are unsubstituted phenyl groups.

[0520] In one non-limiting embodiment, R1 is an unsubstituted phenyl and R2 is a substituted phenyl.

[0521] In some embodiments, the transmembrane compound includes a compound having the structure Tm-12:

[0522]

[0523] Y is selected from the group consisting of O, S, and N (R4);

[0524] Where X 1-3 It is a group that is independently selected from C, O, N, and S;

[0525] R3 is a substituted or unsubstituted aromatic substituent, such as H, OR4, or S(O). nR4, N(R4)(R5), CN, COOH, COOR, C(O)N(R4)(R5), SO2N(R4)(R5), halogens (e.g., Cl, Br, F, I), branched or unbranched substituted or unsubstituted lower alkyl groups (C1-C8), aryl, biaryl, P(O)(OH)2, NHOH, B(OH)2, C(=NH)NH2, NHC(=NH)NH2, NO2, CF3, -OCH2O- (i.e., methylenedioxy), branched or unbranched substituted or unsubstituted lower alkynes (C2-C6), branched or unbranched substituted or unsubstituted lower alkenes (C2-C6), (CH2) n Aryl;

[0526] R4 and R5 are independently H, branched or unbranched substituted or unsubstituted lower alkyl groups (C1-C8), branched or unbranched substituted or unsubstituted lower alkynes (C2-C6), branched or unbranched substituted or unsubstituted lower alkenes (C2-C6), aryl groups, and (CH2). n Aryl;

[0527] Where n is 0-4; and

[0528] Where R1 and R2 are described for equation Tm-19.

[0529] In some embodiments, the transmembrane compound includes a compound of formula Tm-13 having the following structure:

[0530]

[0531] Y is selected from the group consisting of O, S, and N (R4);

[0532] Where X 1-4 It is a group that is independently selected from C, O, N, and S;

[0533] R3 is a substituted or unsubstituted aromatic substituent, such as H, OR4, or S(O). n R4, N(R4)(R5), CN, COOH, COOR, C(O)N(R4)(R5), SO2N(R4)(R5), halogens (e.g., Cl, Br, Fl, I), H, branched or unbranched substituted or unsubstituted lower alkyl groups (C1-C8), aryl, biaryl, P(O)(OH)2, NHOH, B(OH)2, C(=NH)NH2, NHC(=NH)NH2, NO2, CF3, -OCH2O- (i.e., methylenedioxy), branched or unbranched substituted or unsubstituted lower alkynes (C2-C6), branched or unbranched substituted or unsubstituted lower alkenes (C2-C6), (CH2) n Aryl;

[0534] R4 and R5 are independently H, branched or unbranched substituted or unsubstituted lower alkyl groups (C1-C8), branched or unbranched substituted or unsubstituted lower alkynes (C2-C6), branched or unbranched substituted or unsubstituted lower alkenes (C2-C6), aryl groups, and (CH2). n Aryl;

[0535] Where n is 0-4; and

[0536] Where R1 and R2 are described for equation Tm-19.

[0537] In some embodiments, the transmembrane compound includes a compound of formula Tm-14 having the following structure:

[0538]

[0539] X is selected from the group consisting of C, O, N, and S; and

[0540] Where R 1-3 As described above for equations Tm-19, Tm-12, and Tm-13.

[0541] In some embodiments, the transmembrane compound comprises a compound having the following structure: Tm-15

[0542]

[0543] Where R 1-2 As described above for equations Tm-19, Tm-12, and Tm-13.

[0544] In some embodiments, the transmembrane compound includes a compound of formula Tm-16 having the following structure:

[0545]

[0546] X is selected from the group consisting of C, O, N, and S; and

[0547] Where R 1-2 As described above for equations Tm-19, Tm-12, and Tm-13.

[0548] In some embodiments, the transmembrane compound includes a compound having the structure Tm-17:

[0549]

[0550] Where R 1-2 As described above for equations Tm-19, Tm-12, and Tm-13.

[0551] In some embodiments, the transmembrane compound includes a compound of formula Tm-18 having the following structure:

[0552]

[0553] Where R2 is as described above for equations Tm-19, Tm-12, and Tm-13.

[0554] In some embodiments, the transmembrane compound includes a compound having the structure Tm-75:

[0555]

[0556] 3,5-Pyrimidine-2,4,6-trione derivatives

[0557] In some embodiments, the transmembrane compound comprises a compound having the following structure: Tm-8

[0558]

[0559] X1 is selected freely from O and N(R). 12 A group consisting of ) and S;

[0560] X2 and X3 are independently selected from the groups composed of O and S;

[0561] X4 is selected from the group consisting of NH, N(R7) and C(R8,R9);

[0562] Where n1 is 0-1, and when n1 is 0, there is a chemical bond between the two carbons with X2 and X3;

[0563] R7, R8, and R9 are independently selected from the group consisting of: H, substituted or unsubstituted branched or unbranched lower alkyl groups (C1-C1). 20 ), aryl, heteroaryl, cycloalkyl (C3-C7), and substituted, unsubstituted, branched or unbranched C(CH2). n2 Aryl,

[0564] Substitutions on alkyl and aryl groups include functional groups suitable for those skilled in the art, such as OH, NH2, halogen, SH, nitro, aryl, olefin, COOH, COOR, C(O)N(R4)(R5), SO2N(R4)(R5), NO2, P(O)(OH)2, NHOH, B(OH)2, C(=NH)NH2, NHC(=NH)NH2, NO2, and CF3;

[0565] R4 and R5 are independently: H, branched or unbranched substituted or unsubstituted lower alkyl groups (C1-C8), branched or unbranched substituted or unsubstituted lower alkynes (C2-C6), branched or unbranched substituted or unsubstituted lower alkenes (C2-C6), aryl, (CH2). n Aryl;

[0566] Where n2 is 0-10;

[0567] Where R 12 Choose from the group consisting of: H, branched or unbranched lower alkyl groups (C1-C4); and

[0568] R1 and R2 are independently selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, hydroxyl, hydrogen, substituted or unsubstituted ether, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted naphthyl, substituted or unsubstituted thiophenyl, substituted or unsubstituted benzothiaphenyl, substituted or unsubstituted indolyl, substituted or unsubstituted isoquinolinyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted indenyl, or substituted or unsubstituted indenyl.

[0569] In some implementations, R1 and / or R2 may include the following structures:

[0570]

[0571] Where R 3 R 4 R 5 R 6 and R 7Independently selected from the group consisting of: hydrogen, halogen, cyano, azide, hydroxyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfinamide, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted amide, substituted or unsubstituted heterocycle, substituted or unsubstituted alkoxy, substituted or Unsubstituted aryloxy groups, substituted or unsubstituted ethers, substituted or unsubstituted carboxyl groups, substituted or unsubstituted acyl groups, substituted or unsubstituted benzothiazolyl groups, substituted or unsubstituted pyridyl groups, substituted or unsubstituted naphthyl groups, substituted or unsubstituted thiophene groups, substituted or unsubstituted benzothiaphene groups, substituted or unsubstituted indolyl groups, substituted or unsubstituted isoquinolinyl groups, substituted or unsubstituted quinolinyl groups, or substituted or unsubstituted heteroarenyl groups, or substituted or unsubstituted indene groups, or substituted or unsubstituted indene groups. In some embodiments, R 3 R 4 R 5 R 6 and R 7 Two or more of them can form a ring together, which can be a heterocycle (i.e., containing one or more heteroatoms) or it can be a complete carbon ring and can be saturated or unsaturated independently.

[0572] In one non-limiting embodiment, R1 and R2 are unsubstituted phenyl groups.

[0573] In one non-limiting embodiment, R1 is an unsubstituted phenyl and R2 is a substituted phenyl.

[0574] In a non-limiting embodiment, R1 and R2 are as defined above for formulas Tm-6, Tm-19, Tm-12, and Tm-13, and may also be (CH2). n C(=X1)R1、(CH2) n C(=X1)R2, where X1 is O or S, and n is 0-4.

[0575] In some embodiments, the transmembrane compound includes a compound having the structure Tm-76:

[0576]

[0577] 3.6 Other transmembrane compounds

[0578] In some embodiments, the transmembrane compound includes a compound having the structure Tm-77:

[0579]

[0580] In some embodiments, the transmembrane compound includes a compound having the structure Tm-78:

[0581]

[0582] In some embodiments, the transmembrane compound includes a compound having the following structural formula: Tm-79

[0583]

[0584] In some embodiments, the transmembrane compound includes a compound having the structure Tm-80:

[0585]

[0586] In some embodiments, the transmembrane compound includes a compound of formula Tm-81 having the following structure:

[0587]

[0588] In some embodiments, the transmembrane compound includes a compound having the structure Tm-82:

[0589]

[0590] In some embodiments, the transmembrane compound includes a compound having the structure Tm-83:

[0591]

[0592] In some embodiments, the transmembrane compound includes a compound of formula Tm-84 having the following structure:

[0593]

[0594] In some embodiments, the transmembrane compound includes a compound having the structure Tm-85:

[0595]

[0596] In some embodiments, the transmembrane compound includes a compound having the structure Tm-86:

[0597]

[0598] In some embodiments, the transmembrane compound includes a compound of formula Tm-87 having the following structure:

[0599]

[0600] In some embodiments, the transmembrane compound includes a compound having the structure Tm-88:

[0601]

[0602] In some embodiments, the transmembrane compound includes a compound having the structure Tm-89:

[0603]

[0604] In some embodiments, the transmembrane compound includes a compound having the structure Tm-90:

[0605]

[0606] In some embodiments, the transmembrane compound includes a compound of formula Tm-91 having the following structure:

[0607]

[0608] In some embodiments, the transmembrane compound includes a compound of formula Tm-92 having the following structure:

[0609]

[0610] In some embodiments, the transmembrane compound includes a compound having the structure Tm-93:

[0611]

[0612] In some embodiments, the transmembrane compound includes a compound having the structure Tm-94:

[0613]

[0614] In some embodiments, the transmembrane compound includes a compound having the structure Tm-95:

[0615]

[0616] In some embodiments, the transmembrane compound includes a compound having the structure Tm-96:

[0617]

[0618] In some embodiments, the transmembrane compound includes a compound having the structure Tm-97:

[0619]

[0620] In some embodiments, the transmembrane compound includes a compound of formula Tm-98 having the following structure:

[0621]

[0622] In some embodiments, the transmembrane compound includes a compound having the structural formula Tm-99:

[0623]

[0624] In some embodiments, the transmembrane compound includes a compound having the structure Tm-100:

[0625]

[0626] In some embodiments, the transmembrane compound includes a compound of formula Tm-101 having the following structure:

[0627]

[0628] In some embodiments, the transmembrane compound includes a compound having the structure Tm-102:

[0629]

[0630] In some embodiments, the transmembrane compound includes a compound having the structure Tm-103:

[0631]

[0632] In some embodiments, the transmembrane compound includes a compound of formula Tm-104 having the following structure:

[0633]

[0634] In some embodiments of the present invention, the transmembrane compound includes the compounds described in Table 3 below.

[0635] Table 3. Transmembrane compounds

[0636]

[0637]

[0638] 3.7 Transmembrane compound salts

[0639] In some embodiments, the transmembrane compound of the present invention comprises a salt of a transmembrane compound, such as, but not limited to, acetate, trifluoroacetate, or formate. In some embodiments, the transmembrane compound salt comprises a salt containing a cation (+) via an ionic bond (e.g., but not limited to Al). 3+Ca 2+ Na + K + Cu 2+ H + Fe 3+ Mg 2+ Ag + NH4 + H3O + Hg2 2+ Bonded anions (-) (e.g., but not limited to Cl) - F - ,Br - O 2- CO3 2- HCO3 - OH - NO3 - PO4 3- SO4 2- CH3COO - HCOO - C2O4 2- and CN - In other embodiments, the transmembrane compound salt comprises a cation (+) bonded to an anion (-) via an ionic bond.

[0640] In some embodiments, the transmembrane compound may be a salt, stereoisomer, or edible form of the transmembrane compound described herein, such as compounds of formulas Tm-1 to Tm-104.

[0641] 3.8T1R1 transmembrane compound binding sites

[0642] This application provides compositions for modulating the activity of umami receptors, such as T1R1 / T1R3 receptors, wherein the composition interacts with one or more amino acids, such as the seven transmembrane domains (7TM) of T1R1, in the transmembrane domains of the umami receptor. In some embodiments, the amino acids interacting with the composition include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more of Ala795, Ala796, Asn792, Trp773, Phe776, Ala731, Phe728, Leu730, Phe732, Asn735, Ala689, Ser686, Gln690, Ile693, Cys694, Leu695, Arg634, Gln635, Phe642, Ala639, Ala643, and Leu638.

[0643] In one specific non-limiting embodiment, the amino acids that interact with the composition include Asn735 and / or Ser686.

[0644] In other non-limiting embodiments, the amino acids that interact with the composition include 1, 2, 3, 4, 5, 6, 7 or more of Trp773, Phe776, Phe732, Phe728, Leu730, Leu695, Leu638 and Phe642.

[0645] In several other non-limiting embodiments, the amino acids interacting with the composition include one, two, three, four, or more of Trp773, Phe776, Phe732, Phe728, and Phe642. In one non-limiting example, the amino acids interacting with the composition undergo cyclic stacking interactions with the bound composition.

[0646] In some embodiments, the composition interacts with the umami 7TM domain according to any combination of interactions described herein, such as one, two, three or more interactions. The interaction between the composition and 7TM may also include additional hydrophobic interactions added to the interaction energy between the composition and 7TM.

[0647] In some embodiments, the interaction between the composition and one or more amino acids in the T1R1 7TM domain includes one or more hydrogen bonds, covalent bonds, non-covalent bonds, salt bridges, physical interactions, and combinations thereof. The interaction can also be any ligand-receptor interaction known in the art. Such interactions can be determined, for example, by site-directed mutagenesis, X-ray crystallography, X-ray or other spectroscopic methods, nuclear magnetic resonance (NMR), crosslink assessment, mass spectrometry or electrophoresis, cryo-microscopy, shift determination based on known agonists, structure determination, and combinations thereof. In some embodiments, the interaction is determined by computer, for example by theoretical methods such as using molecular docking, molecular modeling, molecular simulation, or other means known to those skilled in the art to dock the compound into the T1R1 7TM domain.

[0648] This application also provides a method for identifying compounds that regulate the activity of umami receptors, such as T1R1, wherein the compounds are identified based on their ability to interact with one or more amino acids present in the 7TM domain of T1R1 as described herein.

[0649] In some embodiments, the method includes contacting a test agent with a feline T1R1 umami receptor, detecting an interaction between the test agent and another amino acid at the 7TM interaction site of the feline T1R1 umami receptor, and selecting a test agent that interacts with one or more amino acids as the compound.

[0650] 4. Flavor composition

[0651] In some embodiments, the flavor compositions of the present invention can be used to enhance the umami flavor of pet foods such as cat foods and / or increase their palatability. The flavor compositions may include combinations of compounds and can be added to pet foods using various delivery systems.

[0652] In some embodiments, the present invention relates to a method for adjusting the umami flavor of pet food, comprising: a) providing at least one pet food or a precursor thereof, and b) combining said pet food or the precursor thereof with at least one flavor composition in an amount of at least umami flavor adjustment to form an enhanced pet food, the flavor composition comprising, for example, one or more nucleotide derivatives and / or one or more transmembrane compounds or food-acceptable salts thereof.

[0653] In some embodiments, the flavor compositions of the present invention can enhance the umami flavor and / or palatability of pet foods, for example, pet foods including wet pet foods, dry pet foods, slightly moist pet foods, pet beverage products, and / or snack pet foods.

[0654] In some embodiments, one or more flavor compositions of the present invention may be added to pet food in an effective amount to adjust, enhance, or otherwise alter the taste or flavor characteristics of the pet food. Adjustment may include, for example, increasing or enhancing the umami flavor of the pet food, as determined by animals such as cats and / or dogs, or, in the case of formulation testing, as determined by a panel of taste testers of animals such as cats and / or dogs, using procedures known in the art.

[0655] In some embodiments of the invention, pet food comprising a sufficient amount of at least one flavor composition described herein, such as a nucleotide derivative, such as a compound of formula Nt-1, can be prepared to produce pet food having a desired flavor, such as umami.

[0656] In some embodiments of the invention, pet food comprising a sufficient amount of at least one flavor composition described herein, such as a transmembrane compound, such as compounds of formulas Tm-1 to Tm-19, can be prepared to produce pet food having a desired flavor, such as umami.

[0657] In some embodiments of the invention, pet food comprising a sufficient amount of a flavor composition comprising at least one, two, three, four, five, six or more nucleotide derivatives can be prepared.

[0658] In some embodiments of the invention, pet food comprising a sufficient amount of a flavor composition comprising at least one, two, three, four, five, six or more transmembrane compounds can be prepared.

[0659] In some embodiments, an adjusted amount of one or more flavor compositions of the present invention may be added to pet food, thereby giving the pet food enhanced palatability compared to pet food prepared without the flavor composition, as determined by animals such as cats and / or dogs, or in the case of formulation testing, as determined by a panel of taste testers of animals such as cats and / or dogs, using procedures known in the art.

[0660] In some embodiments of the invention, flavor compositions are added to pet food in an effective amount to increase, enhance, and / or improve the palatability of the pet food.

[0661] The concentration of a flavor composition that is mixed with pet food to adjust and / or improve the palatability and / or umami flavor of the pet food can vary depending on variables such as, for example, the specific type of pet food, the type and concentration of umami compounds present in the pet food, and the enhancing effect of the specific flavor composition on such umami compounds.

[0662] Flavor compositions with a wide range of concentrations can be used to provide such umami flavor and / or palatability improvements. In some embodiments of this application, the flavor composition is mixed with pet food, wherein the amount of the flavor composition present is from about 0.001 ppm to about 1000 ppm. For example, but not in a limiting manner, the flavor composition may be present in amounts of about 0.001 ppm to about 750 ppm, about 0.001 ppm to about 500 ppm, about 0.001 ppm to about 250 ppm, about 0.001 ppm to about 150 ppm, about 0.001 ppm to about 100 ppm, about 0.001 ppm to about 75 ppm, about 0.001 ppm to about 50 ppm, about 0.001 ppm to about 25 ppm, about 0.001 ppm to about 15 ppm, about 0.001 ppm to about 10 ppm, about 0.001 ppm to about 5 ppm, about 0.001 ppm to about 4 ppm, about 0.001 ppm to about 3 ppm, about 0.001 ppm to about 2 ppm, about 0.001 ppm to about 1 ppm, about 0.01 pp m to about 1000 ppm, about 0.1 ppm to 1000 ppm, about 1 ppm to 1000 ppm, about 2 ppm to about 1000 ppm, about 3 ppm to about 1000 ppm, about 4 ppm to about 1000 ppm, about 5 ppm to about 1000 ppm, about 10 ppm to about 1000 ppm, about 15 ppm to about 1000 ppm, about 25 ppm to about 1000 ppm, about 50 ppm to about 1000 ppm, about 75 ppm to about 1000 ppm, about 100 ppm to about 1000 ppm, about 150 ppm to about 1000 ppm, about 250 ppm to about 1000 ppm, about 250 ppm to about 1000 ppm, about 500 ppm to about 1000 ppm or about 750 ppm to about 1000 ppm, and values ​​in between.

[0663] In some embodiments, the flavor composition is present in the pet food in amounts greater than about 0.001 ppm, greater than about 0.01 ppm, greater than about 0.1 ppm, greater than about 1 ppm, greater than about 2 ppm, greater than about 3 ppm, greater than about 4 ppm, greater than about 5 ppm, greater than about 10 ppm, greater than about 25 ppm, greater than about 50 ppm, greater than about 75 ppm, greater than about 100 ppm, greater than about 250 ppm, greater than about 500 ppm, greater than about 750 ppm, or greater than about 1000 ppm, and values ​​between therewith.

[0664] In some embodiments, the nucleotide derivatives of the present invention are present in food in an amount sufficient to modulate, activate, and / or enhance umami receptors such as T1R1 / T1R3 receptors. For example, but not by way of limitation, the nucleotide derivatives may be present in food in amounts of about 1 pM to about 1 M, about 1 nM to about 1 M, about 1 μM to about 1 M, about 1 mM to about 1 M, about 10 mM to about 1 M, about 100 mM to about 1 M, about 250 mM to about 1 M, about 500 mM to about 1 M, about 750 mM to about 1 M, about 0.001 μM to about 1 M, and about 0.001 μM to about 750 mM. M, about 0.001 μM to about 500 mM, about 0.001 μM to about 250 mM, about 0.001 μM to about 100 mM, about 0.001 μM to about 50 mM, about 0.001 μM to about 25 mM, about 0.001 μM to about 10 mM, about 0.001 μM to about 1 mM, about 0.001 μM to about 100 μM or about 0.001 μM to about 10 μM, and values ​​in between.

[0665] In some embodiments, the transmembrane compounds of the present invention are present in food in amounts sufficient to modulate, activate, and / or enhance umami receptors such as feline T1R1 / T1R3 receptors. For example, but not in a restrictive manner, transmembrane compounds may be present in food in amounts of about 1 pM to about 10 M, about 1 pM to about 1 M, about 1 nM to about 1 M, about 1 μM to about 1 M, about 1 mM to about 1 M, about 10 mM to about 1 M, about 100 mM to about 1 M, about 250 mM to about 1 M, about 500 mM to about 1 M, about 750 mM to about 1 M, about 1 μM to about 1 M, about 1 μM to about 750 mM, about 1 μM to about 500 mM, about 1 μM to about 250 mM, about 1 μM to about 100 mM, about 1 μM to about 50 mM, about 1 μM to about 25 mM, about 1 μM to about 10 mM, about 1 μM to about 100 μM, or about 1 μM to about 10 μM, and values ​​between them.

[0666] In some embodiments of this application, the flavor composition is mixed with a food product, wherein the amount of the flavor composition present is between about 0.0001% and about 10% by weight (w / w) of the food product. For example, but not in a limiting manner, the amount of the flavor composition may be about 0.0001% to about 10%, about 0.0001% to about 1%, about 0.0001% to about 0.1%, about 0.0001% to about 0.01%, about 0.0001% to about 0.001%, about 0.001% to about 10%, about 0.001% to about 1%, about 0.01% to about 1%, or about 0.1% to about 1%, and values ​​between thereto.

[0667] In some embodiments, the nucleotide derivatives and / or transmembrane compounds of this application are mixed together in various proportions or with other compounds such as nucleotides and / or amino acids and / or furanones to form various flavor compositions. Non-limiting examples of nucleotides, amino acids, and furanones are disclosed in patent applications PCT / EP2013 / 072788, PCT / EP2013 / 072789, PCT / EP2013 / 072790, and PCT / EP2013 / 072794, which are incorporated herein by reference in their entirety. In some embodiments, the nucleotide derivatives and / or transmembrane compounds mixed with other compounds are one or more compounds of formulas Nt-1 to Nt-55 and / or one or more compounds of formulas Tm-1 to Tm-104.

[0668] In some embodiments, the nucleotide derivatives mixed with other compounds include nucleotide derivatives of formulas Nt-1 to Nt-55 and those listed in Tables 2 and 6-14.

[0669] In some embodiments, the transmembrane compound mixed with other compounds includes transmembrane compounds of formulas Tm-1 to Tm-104.

[0670] 4.1 Nucleotides

[0671] In some embodiments of the invention, the flavor composition comprises at least one nucleotide derivative and / or at least one transmembrane compound, as well as at least one nucleotide described herein.

[0672] In certain embodiments of the invention, the flavor composition comprises at least two, three, four, five, or more nucleotides as described herein. Non-limiting examples of nucleotides include guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), inosine monophosphate (IMP), inosine diphosphate (IDP), inosine triphosphate (ITP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), and xanthoside monophosphate (XMP), xanthoside diphosphate (XDP), and xanthoside triphosphate (XTP), or any nucleotide derivative as described in Formula Nt-1.

[0673] In some embodiments, the flavor composition may include nucleotides present in food, which may be present in amounts of about 1 pM to about 1 M, about 1 nM to about 1 M, about 1 μM to about 1 M, about 1 mM to about 1 M, about 10 mM to about 1 M, about 100 mM to about 1 M, about 250 mM to about 1 M, about 500 mM to about 1 M, about 750 mM to about 1 M, about 1 μM to about 1 M, about 1 μM to about 750 mM, about 1 μM to about 500 mM, about 1 μM to about 250 mM, about 1 μM to about 100 mM, about 1 μM to about 50 mM, about 1 μM to about 25 mM, about 1 μM to about 10 mM, about 1 μM to about 10 mM, about 1 μM to about 100 μM, or about 1 μM to about 10 μM, and values ​​between thereto.

[0674] In some embodiments, the nucleotide may be present in amounts greater than about 1 mM or about 2.5 mM of the pet food. In some non-limiting embodiments, the nucleotide and / or nucleotide derivative may be present in amounts less than about 100 mM, less than about 50 mM, less than about 20 mM, or less than about 10 mM of the pet food. In some non-limiting embodiments, the nucleotide is present in an amount of about 5 mM of the pet food.

[0675] In some embodiments, the flavor composition comprises at least one transmembrane compound and at least one nucleotide and / or nucleotide derivative, which may be IMP, GMP, or a mixture thereof. In some embodiments, at least one nucleotide may be a combination of GMP and IMP, comprising about 1% to about 99% GMP and about 1% to about 99% IMP, or about 20% to about 80% GMP and about 20% to about 80% IMP, or about 50% GMP and about 50% IMP, or about 10% GMP and about 90% IMP, or about 20% GMP and about 80% IMP, or about 30% GMP and about 70% IMP, or about 40% GMP and about 60% IMP, or about 60% GMP and about 40% IMP, or about 70% GMP and about 30% IMP, or about 80% GMP and about 20% IMP, or about 10% GMP and about 90% IMP.

[0676] In some embodiments of the invention, the flavor composition further comprises at least one amino acid described herein.

[0677] 4.2 amino acids

[0678] In some embodiments of the invention, the flavor composition comprises at least one nucleotide derivative and / or at least one transmembrane compound, and at least one amino acid as described herein. In some embodiments, the flavor composition comprises at least two, three, four, five or more amino acids as described herein.

[0679] In some embodiments, the flavor composition comprises at least one, two, three, four, five or more first amino acids and / or at least one, two, three, four, five or more second amino acids.

[0680] In some embodiments of the present invention, the flavor composition comprises at least one first amino acid and at least one second amino acid.

[0681] In some embodiments of the present invention, the flavor composition comprises at least two first amino acids and at least one second amino acid.

[0682] In some embodiments of the invention, the flavor composition comprises at least one first amino acid and at least two second amino acids.

[0683] In some embodiments of the present invention, the flavor composition comprises at least two first amino acids and at least two second amino acids.

[0684] In some embodiments of the invention, the flavor composition further comprises at least one nucleotide as described herein.

[0685] Non-limiting examples of the first amino acid include tryptophan, phenylalanine, histidine, glycine, cysteine, alanine, tyrosine, serine, methionine, asparagine, leucine, and combinations thereof.

[0686] Non-limiting examples of the second amino acid include asparagine, threonine, isoleucine, proline, glutamic acid, aspartic acid, hydroxyproline, arginine, cystine, glutamine, lysine, valine, ornithine, taurine, monosodium glutamate (MSG), and combinations thereof.

[0687] In some embodiments, at least one first amino acid and / or a second amino acid, alone or in combination, may be present in amounts ranging from about 1 mM to about 1 M in the pet food, or from about 250 mM to about 1 M, or from about 5 mM to about 500 mM, or from about 10 mM to about 100 mM, or from about 15 mM to about 50 mM, or from about 20 mM to about 40 mM. In some embodiments, one or more amino acids may be present in amounts less than about 1 M in the pet food, less than about 200 mM, less than about 100 mM, less than about 50 mM, less than about 20 mM, or less than about 10 mM. In some embodiments, the first amino acid and / or a second amino acid, alone or in combination, may be present in amounts of about 25 mM in the pet food.

[0688] 4.2.1 T1R1 amino acid binding site

[0689] The amino acids in the compositions described herein that regulate umami receptors, such as T1R1 / T1R3 receptors, can interact with one or more amino acids in the Venus flytrap domain of the umami receptor. In some embodiments, the Venus flytrap domain (VFT) is present in T1R1. In some embodiments, the VFT amino acids that interact with the composition include one or more of Thr149, Tyr220, Thr148, Thr449, Ser172, Glu170, Glu301, His71, His47, Arg277, His308, Asn69, Asn302, Ser306, Ser384, Asp302, Ser306, and Ala380.

[0690] In one non-limiting embodiment, the composition comprises an amino acid, wherein the amino acid interacts with one, two, three, four, five, six or more of Ser172, Thr149, Thr148, Glu301, Tyr220, Glu170 and Asp302 of T1R1.

[0691] In other non-limiting embodiments, the composition interacts with one, two, three, four, five or more of Thr149, Ser172, Tyr220, Thr148, Glu170 and / or Asp302, wherein said interaction may include, for example, hydrogen bonding, salt bridging and / or Pi-cation interaction.

[0692] In one non-limiting example, the composition interacts with Glu170 and / or Asp302 of the VFT domain, wherein the composition does not contain L-glutamic acid or L-aspartic acid. In one embodiment, Glu170 and Asp302 help coordinate the zwitterionic nitrogen of the amino acid ligands of the composition suitable for the active site of T1R1, while establishing an electrostatic environment unsuitable for binding L-glutamic acid and L-aspartic acid.

[0693] In some embodiments, the composition interacts with the VFT according to any combination of interactions described herein, such as one, two, three, or more interactions. The interaction between the amino acid and the VFT may also include additional hydrophobic interactions added to the interaction energy between the amino acid and the VFT.

[0694] In some embodiments, the interaction between the composition and one or more VFT amino acids includes one or more hydrogen bonds, covalent bonds, non-covalent bonds, salt bridges, physical interactions, and combinations thereof. The interaction can also be any ligand-receptor interaction known in the art. Such interactions can be determined, for example, by site-directed mutagenesis, X-ray crystallography, X-ray or other spectroscopic methods, nuclear magnetic resonance (NMR), crosslink assessment, mass spectrometry or electrophoresis, shift determination based on known agonists, structure determination, and combinations thereof. In some embodiments, the interaction is determined by computer, for example by theoretical methods such as using molecular docking, molecular modeling, molecular simulation, or other means known to those skilled in the art to dock the compound into the VFT domain.

[0695] This application also provides a method for identifying compounds that regulate the activity of umami receptors, such as T1R1, wherein the compounds are identified based on their ability to interact with one or more amino acids present in the VFT domain of T1R1 as described herein.

[0696] In some embodiments, the method includes contacting a test agent with a feline T1R1 umami receptor, detecting an interaction between the test agent and another amino acid at a VFT interaction site of the feline T1R1 umami receptor, and selecting a test agent that interacts with one or more amino acids as the compound.

[0697] 5. Delivery system

[0698] In some embodiments, the flavor composition of this application can be incorporated into a delivery system for pet food. The delivery system can be liquid or solid, aqueous or non-aqueous. The delivery system is typically adapted to meet the needs of the flavor composition and / or the pet food in which the flavor composition will be incorporated.

[0699] The flavor composition can be used in liquid, dry, and / or solid form. When used in a dry form, a suitable drying method such as spray drying can be used. Optionally, the flavor composition can be encapsulated or absorbed into a water-soluble material, including but not limited to materials such as cellulose, starch, sugar, maltodextrin, gum arabic, etc. Practical techniques for preparing such a dry form are well known in the art and are applicable to the subject matter disclosed herein.

[0700] The flavor compositions disclosed in this invention can be used in many unique physical forms well known in the art to provide an initial burst of taste, flavor, and / or texture; and / or a sustained sensation of taste, flavor, and / or texture. However, this is not an limitation; such physical forms include free forms, such as spray-dried, powdered, and beaded forms, as well as encapsulated forms and mixtures thereof.

[0701] In some embodiments, nucleotide derivatives and / or transmembrane compounds of the flavor composition may be generated during the processing of pet food. For example, rather than in a restrictive manner, nucleotide derivatives and / or transmembrane compounds may be generated from precursor compounds during the heat treatment of pet food, such as dry distillation, extrusion, and / or sterilization.

[0702] In some embodiments, as described above, encapsulation techniques can be used to modify flavor systems. In some embodiments, flavor compounds, flavor components, or the entire flavor composition can be encapsulated, either completely or partially. The encapsulation materials and / or techniques can be selected to determine the type of flavor system modification.

[0703] In some embodiments, encapsulation materials and / or techniques are selected to improve the stability of flavor compounds, flavor components, or flavor compositions; while in other embodiments, encapsulation materials and / or techniques are selected to improve the release profile of the flavor composition.

[0704] Suitable encapsulation materials may include, but are not limited to: hydrocolloids, such as alginate, pectin, agar, guar gum, cellulose, etc.; proteins; polyvinyl acetate, polyethylene, cross-linked polyvinylpyrrolidone, polymethyl methacrylate, polylactic acid, polyhydroxyalkanoates, ethyl cellulose, polyvinyl acetate phthalate, polyethylene glycol, methacrylate-methyl methacrylate copolymer, ethylene-vinyl acetate (EVA) copolymer, etc., and combinations thereof. Suitable encapsulation techniques may include, but are not limited to: spraying, spray drying, spray cooling, absorption, adsorption, inclusion complexation (e.g., to generate a flavor / cyclodextrin complex), coagulation, fluidized bed coating, or other methods that can be used to encapsulate components with encapsulation materials.

[0705] Delivery systems for encapsulating flavorings or sweeteners may contain a hydrophobic matrix of fat or wax surrounding a sweetener or flavoring core. The fat may be selected from any number of conventional materials, such as fatty acids, glycerides or polyglycerides, sorbitol esters, and mixtures thereof. Examples of fatty acids include, but are not limited to, hydrogenated and partially hydrogenated vegetable oils, such as palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, soybean oil, cottonseed oil, sunflower oil, safflower oil, and combinations thereof. Examples of glycerides include, but are not limited to, monoglycerides, diglycerides, and triglycerides.

[0706] Waxes can be selected from the group consisting of natural waxes and synthetic waxes, and mixtures thereof. Non-limiting examples include paraffin wax, petrolatum, carbon wax, microcrystalline wax, beeswax, carnauba wax, candelilla wax, lanolin, myrica wax, sugarcane wax, cetacean wax, rice bran wax, and mixtures thereof.

[0707] The fats and waxes can be used alone or in combination, in an amount of about 10% to about 70% of the weight of the encapsulation system, optionally about 30% to about 60%. When used in combination, the fats and waxes can be present in a ratio of about 70:10 to 85:15, respectively.

[0708] Typical encapsulated flavor compositions, flavoring agents, or sweetener delivery systems are disclosed in U.S. Patent Nos. 4,597,970 and 4,722,845, which are incorporated herein by reference in their entirety.

[0709] Liquid delivery systems may include, but are not limited to, systems containing dispersions of the flavor compositions of this application, such as in carbohydrate syrups and / or emulsions. Liquid delivery systems may also include extracts in which nucleotide derivatives, transmembrane compounds, and / or flavor compositions are dissolved in a solvent. Solid delivery systems may be created using spray drying, spraying, spray cooling, fluidized bed drying, absorption, adsorption, coagulation, complexation, or any other standard techniques. In some embodiments, the delivery system may be selected to be compatible with or function within an edible composition. In some embodiments, the delivery system will include an oily material, such as fat or oil. In some embodiments, the delivery system will include sugary fats, such as cocoa butter, cocoa butter substitutes, cocoa butter alternatives, or cocoa butter equivalents.

[0710] When used in a dry form, a suitable drying method such as spray drying can be used. Alternatively, the flavor composition may be adsorbed or absorbed onto a substrate, such as water-soluble substances like cellulose, starch, sugar, maltodextrin, gum arabic, etc., or may be encapsulated. Practical techniques for preparing this dry form are well known in the art.

[0711] 6. Pet food

[0712] The flavor compositions of the subject matter of this invention can be used in a wide variety of pet foods. Non-limiting examples of suitable pet foods include wet foods, dry foods, slightly moist foods, pet food supplements (e.g., vitamins), pet beverage products, snacks and treats, and the pet food categories described herein.

[0713] When needed, one or more flavor compositions of the subject matter of this invention, combined with pet food and optional ingredients, provide a flavoring agent with an unexpected taste and imparts a sensory experience such as umami and / or saltiness. The flavor compositions disclosed herein may be added before, during, or after the formulation or packaging of pet food, and the components of the flavor composition may be added sequentially or simultaneously. In some embodiments, one or more components of the flavor compositions disclosed herein may be generated from precursor compounds during the production of pet food, such as during heat treatment. For example, rather than by limitation, nucleotide derivatives and / or transmembrane compounds of the flavor composition may be generated during the production of pet food and additional components of the flavor composition, and may be added before, during, or after the formulation or packaging of pet food.

[0714] In some implementations, pet food is a nutritionally complete dry food. Nutritionally complete dry or low-moisture pet food may contain less than about 15% moisture and about 10% to about 60% fat, about 10% to about 70% protein, and about 30% to about 80% carbohydrates, such as dietary fiber and ash.

[0715] In some embodiments, the pet food is a nutritionally complete wet food. A nutritionally complete wet or high-moisture pet food may contain more than about 50% water. In some embodiments, the wet pet food contains about 40% fat, about 50% protein, and about 10% carbohydrates, such as dietary fiber and ash.

[0716] In some implementations, pet food is a nutritionally complete, slightly moist food. Slightly moist (e.g., semi-moist, semi-dry, dry-soft, slightly moist-soft, or containing intermediate or moderate moisture) nutritionally complete pet food contains approximately 15% to approximately 50% moisture.

[0717] In some implementations, pet food is a pet food snack product. Non-limiting examples of pet food snack products include treat bars, pet chews, crunchy treats, compressed dry food, snacks, biscuits, and desserts.

[0718] In some embodiments, the protein source may be derived from plant sources, such as lupin protein, wheat protein, soy protein, and combinations thereof. Optionally or additionally, the protein source may be derived from a variety of animal sources. Non-limiting examples of animal protein include beef, pork, poultry, lamb, or fish, including, for example, muscle meat, meat by-products, meat meal, or fish meal.

[0719] 7. Methods for measuring taste attributes

[0720] In some embodiments of the invention, the taste, flavor, and / or palatability properties of pet food may be modified by mixing a flavor composition with the food, or produced under food preparation conditions, as described herein. In some embodiments, one or more properties may be enhanced or weakened by increasing or decreasing the concentration of the flavor composition mixed with or produced with the food. In some embodiments, the modified taste properties of the food may be evaluated as described herein, and the concentration of the flavor composition mixed with or produced with the food may be increased or decreased based on the evaluation results.

[0721] In some embodiments of the invention, taste and / or palatability properties can be determined using an in vitro assay, wherein the ability of a compound to activate feline umami receptors expressed by cells is measured in vitro at different concentrations. In some embodiments, increased receptor activation is associated with enhanced taste and / or palatability properties of the compound. In some embodiments, the composition is measured alone or in combination with other compounds. In some embodiments, the in vitro assay includes the in vitro assay described in the Examples section of this application. In some embodiments, the in vitro assay includes recombinant cells expressing umami receptors encoded by nucleic acids (e.g., exogenous nucleic acids) introduced into the cells. In other non-limiting embodiments, the in vitro assay includes cells expressing umami receptors derived from cells. Examples of such cells expressing natural umami receptors include, for example, but not limited to, cat and / or canine taste cells. In some embodiments, cat and / or canine taste cells expressing umami receptors are isolated from cats and / or dogs and cultured in vitro.

[0722] In some embodiments of the invention, a panel of taste testers may be used to determine taste and / or palatability properties. For example, instead of being restrictive, the panel may include members of a cat panel. In some embodiments, the panel may include members of a dog panel. In some embodiments, the palatability of pet food can be determined by consuming pet food containing only the flavor composition (e.g., single-bowl test, monadicranking). In some embodiments, the palatability of pet food can be determined by preferentially consuming pet food containing the flavor composition of the invention relative to pet food without the flavor composition or another flavor composition (e.g., two-bowl test, difference and / or choice test for testing preferences).

[0723] In some embodiments, the palatability and / or umami flavor of the flavor composition can be determined by preferentially consuming the pet food containing the flavor composition of the present invention relative to pet food without the flavor composition or containing another flavor composition (e.g., a two-bowl test). For example, solution groups can be used to compare the palatability of compounds at a certain concentration range under single exposure. In some embodiments, the solution may contain a palatability enhancer, such as L-histidine, as an ingestion / positive flavor enhancer to increase baseline solution intake, thus enabling the identification of potential negative effects of the test compound.

[0724] The intake ratio of each pet food or aqueous solution can be determined by dividing the measured amount consumed by the total amount consumed. The consumption ratio (CR) can then be calculated to compare the consumption of one amount relative to another, thereby determining the preferred consumption of one food or aqueous solution relative to the other. Optionally or additionally, the difference in intake (g) can be used to assess the average difference measured at a selected significance level, such as 5%, in a two-bowl test for two solutions or two pet foods, to determine the average difference in intake with a confidence interval of 95%. However, any significance level can be used, such as 1%, 2%, 3%, 4%, 5%, 10%, 15%, 2%, 25%, or 50%. In some embodiments, a percentage preference score can also be calculated; for example, the animal's percentage preference for a solution or food is the percentage of solution or food ingested during the test relative to the total liquid or food intake.

[0725] 8. Generation Method

[0726] In some embodiments, standard chemical synthesis methods can be used to produce the nucleotide derivatives and / or transmembrane compounds of the present invention. In some embodiments, the chemical synthesis methods provide nucleotide derivatives and / or transmembrane compounds having a purity of at least 99.999%, or at least 99%, or at least 95%, or at least 90%, or at least 85%, or at least 80%. In some embodiments, standard hydrolysis methods, such as those using acids, enzymes, or combinations of acids and enzymes, can be used to prepare the nucleotide derivatives and / or transmembrane compounds.

[0727] The nucleotide derivatives and / or transmembrane compounds of the present invention can also be produced under food preparation conditions, such as in the production of pet food. For example, rather than in a restrictive manner, the nucleotide derivatives and / or transmembrane compounds of the present invention can be produced from precursor compounds present in pet food during hot food processing processes such as sterilization, dry distillation, and / or extrusion. In some embodiments, liquid and / or powdered flavoring agents may also be added to improve the flavor of pet food, for example, by adding them to dry pet food to increase palatability. The flavoring agent may be a digestive product of meat (e.g., liver) and / or a digestive product of vegetables, and may optionally include other flavoring agents known in the art. In some embodiments, the nucleotide derivatives and / or transmembrane compounds may be mixed with or generated in liquid and / or powdered flavoring agents prior to addition to the pet food. Optionally or additionally, the nucleotide derivatives and / or transmembrane compounds may be mixed with or generated in liquid and / or powdered flavoring agents after addition to the pet food.

[0728] In some embodiments, the flavor compositions of the present invention comprise one or more transmembrane compounds of formulas Tm-1 to Tm-104. In some embodiments, such compounds can be synthesized, but are not limited to, by any method known in the art. In some embodiments, secganic acid derivatives and transmembrane compounds can be synthesized according to the following synthetic schemes:

[0729]

[0730] 9. Non-limiting examples of flavor compositions of the present invention

[0731] As described herein, at least three distinct binding sites exist on the feline T1R1 / T1R3 receptor (i.e., umami receptor), allowing the binding of small molecules and / or compounds. One of the binding sites on the feline T1R1 / T1R3 receptor can bind the nucleotides and / or nucleotide derivatives described herein. A second binding site on the feline T1R1 / T1R3 receptor can bind the first group of amino acids described herein, and a third binding site on the feline T1R1 / T1R3 receptor (i.e., the T1R1 7TM domain) can bind transmembrane compounds described herein. Without being bound by any particular theory, the binding of the first group of amino acids disclosed herein can alter the conformation of the feline T1R1 / T1R3 receptor to allow greater contact with the bound nucleotides and / or nucleotide derivatives, resulting in co-activation of the umami receptor. The second group of amino acids disclosed herein can interact with one or more other receptors and does not compete with the first amino acid for binding to the umami receptor. The addition of the second group of amino acids to a flavor composition can enhance the flavor sensation of the composition. As described in this article, the binding of transmembrane compounds to receptors further activates the receptors, thereby enhancing or improving the palatability of foods containing such compounds.

[0732] In some embodiments, the present invention provides a flavor composition comprising at least one nucleotide and / or nucleotide derivative bound to a first binding site on an umami receptor, and / or at least one first group of amino acids bound to a second binding site on an umami receptor, and / or at least one transmembrane compound bound to a third binding site on an umami receptor (e.g., within the 7TM domain of the umami receptor), and / or at least one second group of amino acids bound to a different receptor.

[0733] The subject of this invention provides flavor compositions comprising at least one, two, three, four, five or more nucleotide derivatives and / or at least one, two, three, four, five or more nucleotides and / or at least one, two, three, four, five or more transmembrane compounds and / or at least one, two, three, four, five or more Group 1 amino acids and / or at least one, two, three, four, five or more Group 2 amino acids.

[0734] In some embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative and at least one, two, three, four, five or more first group amino acids and / or at least one, two, three, four, five or more second group amino acids selected from Table 4.

[0735] In some embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative and at least one group 1 amino acid selected from Table 4.

[0736] In some embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative and at least one second group amino acid selected from Table 4.

[0737] In some embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative and at least one first group amino acid and at least one second group amino acid selected from Table 4.

[0738] In some embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative and at least two first group amino acids and at least one second group amino acid selected from Table 4.

[0739] In some embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative and at least one first group amino acid selected from Table 4 and at least two second group amino acids.

[0740] In some embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative and at least two first group amino acids and at least two second group amino acids selected from Table 4.

[0741] In some embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, at least one nucleotide, and at least one group I amino acid selected from Table 4.

[0742] In some embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, at least one nucleotide, and at least one amino acid selected from the second group of amino acids in Table 4.

[0743] In some embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, at least one nucleotide, and at least one, two, three, four, five, or more amino acids selected from Group 1 amino acids and / or at least one, two, three, four, five, or more amino acids from Group 2 amino acids selected from Table 4.

[0744] In some embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, at least one nucleotide, and at least one first group amino acid and at least one second group amino acid selected from Table 4.

[0745] Table 4. Amino Acids

[0746]

[0747]

[0748] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, histidine, and proline.

[0749] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, alanine, and proline.

[0750] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, glycine, and proline.

[0751] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, phenylalanine, and proline.

[0752] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, tryptophan, and proline.

[0753] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, tyrosine, and proline.

[0754] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, histidine, and threonine.

[0755] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, alanine, and threonine.

[0756] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, glycine, and threonine.

[0757] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, phenylalanine, and threonine.

[0758] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, tryptophan, and threonine.

[0759] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, tyrosine, and threonine.

[0760] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, histidine, and glutamic acid.

[0761] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, alanine, and glutamic acid.

[0762] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, glycine, and glutamic acid.

[0763] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, phenylalanine, and glutamic acid.

[0764] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, tryptophan, and glutamic acid.

[0765] In some non-limiting embodiments, the present invention provides a flavor composition comprising at least one nucleotide derivative, tyrosine, and glutamic acid.

[0766] In some embodiments, any of the flavor compositions disclosed above may also contain at least one nucleotide and / or at least one transmembrane compound as described herein.

[0767] In some non-limiting embodiments, the present invention provides a flavor composition comprising adenosine 3',5'-bisphosphate and alanine.

[0768] In some non-limiting embodiments, the present invention provides a flavor composition comprising 6-thioguanosine-5'-O-monophosphate and alanine.

[0769] In some non-limiting embodiments, the present invention provides a flavor composition comprising 2'-,3'-O-(N'-methyl-o-aminobenzoyl)guanosine 5'-O-monophosphate and alanine.

[0770] In some non-limiting embodiments, the present invention provides a flavor composition comprising 2-amino-6-chloropurine riboside-5'-O-monophosphate and alanine.

[0771] In some non-limiting embodiments, the present invention provides a flavor composition comprising 6-chloropurine riboside-5'-O-monophosphate and alanine.

[0772] In some non-limiting embodiments, the present invention provides a flavor composition comprising inosine triphosphate (ITP) and alanine.

[0773] In some non-limiting embodiments, the present invention provides a flavor composition comprising inosine triphosphate (ITP), alanine, and IMP.

[0774] In some non-limiting embodiments, the present invention provides flavor compositions comprising 1-(2-bromophenyl)-3-((1R,2S)-2-hydroxy-2,3-dihydro-1H-indene-1-yl)urea, alanine, and IMP.

[0775] In some non-limiting embodiments, the present invention provides a flavor composition comprising N-(benzo[d][1,3]dioxacyclopenten-5-yl)-2-propylpentanamide, alanine, and IMP.

[0776] In some non-limiting embodiments, the present invention provides a flavor composition comprising N-(hept-4-yl)benzo[d][1,3]dioxane-5-carboxamide, alanine, and IMP.

[0777] In some non-limiting embodiments, the present invention provides a flavor composition comprising N-(2-amino-2-oxo-1-phenylethyl)-3-chloro-4,5-dimethoxybenzamide, alanine, and IMP.

[0778] In some non-limiting embodiments, the present invention provides a flavor composition comprising (E)-3-(4-methoxyphenyl)-N-(pent-3-yl)acrylamide, alanine, and IMP.

[0779] In some non-limiting embodiments, the present invention provides a flavor composition comprising 1-benzyl-3-(2-oxo-2-phenylethyl)imidazoline-2,4,5-trione, alanine, and IMP.

[0780] In some non-limiting embodiments, the present invention provides flavor compositions comprising 1H-imidazo[4,5-c]pyridine-2(3H)-one, alanine, and IMP.

[0781] In some non-limiting embodiments, the present invention provides a flavor composition comprising N-(hept-4-yl)benzo[d][1,3]dioxane-5-carboxamide, phenylalanine, and GMP.

[0782] In some non-limiting embodiments, the present invention provides a flavor composition comprising a combination of N-(heptyl-4-yl)benzo[d][1,3]dioxane-5-carboxamide with a first amino acid (Historic acid A), a second amino acid (Historic acid B), and one or more nucleotides, as described below.

[0783] Table 5. Flavor compositions containing transmembrane compounds, first amino acids (Group A), second amino acids (Group B), and nucleotides.

[0784]

[0785]

[0786]

[0787] In some non-limiting embodiments, the present invention provides a pet food comprising the flavor composition described herein, wherein the flavor composition is present in an amount of about 0.001 ppm to about 1000 ppm.

[0788] In some non-limiting embodiments, the present invention provides a pet food comprising the flavor composition described herein, wherein the flavor composition is present at a concentration of about 0.0001% by weight to about 10% by weight of the pet food.

[0789] In some non-limiting embodiments, the present invention provides a pet food comprising the flavor composition described herein, wherein the flavor composition is present in an amount greater than about 1 ppm.

[0790] In some non-limiting embodiments, the present invention provides a pet food comprising the flavor composition described herein, wherein the flavor composition is present in an amount greater than about 10 ppm.

[0791] In some non-limiting embodiments, the present invention provides a pet food comprising the flavor composition described herein, wherein the flavor composition is present in an amount greater than about 100 ppm.

[0792] In some non-limiting embodiments, the present invention provides a pet food comprising the nucleotide derivative described herein, wherein the nucleotide derivative is present in an amount of about 1 pM to about 1 M.

[0793] 10. Non-limiting examples of the method of the present invention

[0794] In some non-limiting embodiments, the present invention provides a method for improving the palatability of pet food, comprising mixing the pet food with a flavor composition comprising nucleotide derivatives and / or transmembrane compounds described herein, wherein the concentration of the nucleotide derivatives and / or transmembrane compounds present in the mixture is from about 1 pM to about 10 M, or from about 1 pM to about 1 M.

[0795] In some non-limiting embodiments, the present invention provides a method for improving the palatability of pet food, comprising mixing the pet food with a flavor composition comprising a nucleotide derivative and / or transmembrane compound described herein, wherein the concentration of the nucleotide derivative and / or transmembrane compound is present in the product from about 1 pM to about 10 M, or from about 1 pM to about 1 M.

[0796] In some non-limiting embodiments, the present invention provides a method for enhancing the umami flavor of pet food, comprising mixing the pet food with a flavor composition comprising nucleotide derivatives and / or transmembrane compounds described herein, wherein the nucleotide derivatives are present in the mixture at a concentration of 0.001 ppm to 1000 ppm.

[0797] In some non-limiting embodiments, the present invention provides a method for improving the palatability of pet food, comprising mixing the pet food with a flavor composition comprising nucleotide derivatives and / or transmembrane compounds described herein, wherein the flavor composition is present in the mixture at a concentration of about 0.001 ppm to 1000 ppm.

[0798] In some non-limiting embodiments, the present invention provides a method for enhancing the umami flavor of pet food, comprising mixing the pet food with a flavor composition comprising nucleotide derivatives and / or transmembrane compounds as described herein, wherein the flavor composition is present in the mixture at a concentration of about 0.0001% by weight to about 10% by weight.

[0799] 11. Example

[0800] The subject matter of this disclosure will be better understood by referring to the following embodiments, which are provided as examples of the invention and not by way of limitation.

[0801] Example 1 - Activation of T1R1 / T1R3 receptors via nucleotide derivatives

[0802] This embodiment describes the in vitro activation of the feline T1R1 / T1R3 receptor via a nucleotide derivative.

[0803] Nucleotide derivatives that could function as T1R1 / T1R3 activators were identified using computer modeling of the cat umami receptors T1R1 / T1R3 and selected for further in vitro testing. In vitro functional characterization of the selected nucleotide derivatives was used to evaluate their effectiveness in activating the T1R1 / T1R3 receptor alone and in combination with amino acids and / or nucleotides.

[0804] Methods: HEK293 cells stably expressing T1R3 and inducibly expressing T1R1 were exposed to separate nucleotide derivatives to activate umami receptors. Activation of the T1R1 / T1R3 receptors was detected by changes in intracellular calcium levels using a calcium-sensitive fluorescent dye. Cells expressing T1R3 but not T1 served as controls. Tetra or 3 is used for data capture.

[0805] For each nucleotide derivative, dose-response curves were generated and the following properties were determined: EC50 of each nucleotide derivative individually. 50 ; EC50 of nucleotide derivatives with 20mM alanine 50 Nucleotide derivatives and 0.2 mM IMP EC 50 ; Nucleotide derivatives with 20 mM alanine and 0.2 mM IMP EC 50 .

[0806] The term "half-maximum effective concentration" (EC50) is used to describe the concentration of substances at half maximum effective concentration. 50 The concentration of a compound is the concentration that induces half the response between the baseline and the maximum after a specified exposure time. In each experiment, serial dilutions of up to 0.1 mM, 1 mM, or 10 mM of the nucleotide derivative were added to T1R1 / T1R3 expressing cells.

[0807] Results: Treatment of HEK293 cells expressing the T1R1 / T1R3 receptor alone (e.g., in buffer) or in combination with 20 mM alanine resulted in activation of the T1R1 / T1R3 receptor, as indicated by changes in intracellular calcium levels (ΔF / F0). In the presence of alanine or in buffer, 2'-deoxyadenosine-3',5'-O-bisphosphate resulted in EC50 at an observed value of 0.02 mM. 50 (Table 6 and Figure 2 These results indicate that 2'-deoxyadenosine-3',5'-O-bisphosphate is a positive activator of the T1R1 / T1R3 receptor.

[0808] It was observed that the nucleotide derivative adenosine 5'-O-thiophosphate dilithium salt was used alone as an activator for T1R1 / T1R3. Figure 8 (and Table 6). In the presence of 20 mM alanine, it alone has an EC50 of 13.6 mM. 50 Value, EC in the form of adenosine 5'-O-thiobisphosphate dilithium salt 50 The value decreased from greater than 1 mM to 0.06 mM, while ΔF / F0 expanded significantly higher. Figure 8 (and Table 6). These results indicate that alanine and nucleotide derivatives, such as adenosine 5'-O-thiophosphate, work synergistically to activate T1R1 / T1R3.

[0809] Unbound by any particular theory, these results suggest that nucleotide derivatives, alone or in combination with amino acids such as alanine, can act as positive regulators of the T1R1 / T1R3 receptor.

[0810]

[0811]

[0812]

[0813] Example 2 - Activation of T1R1 / T1R3 receptors through combination of nucleotide derivative compounds and amino acids

[0814] This embodiment describes the in vitro activation of the cat T1R1 / T1R3 receptor by a combination of nucleotide derivative compounds and amino acids.

[0815] Evaluate nucleotide derivatives to determine the effectiveness of nucleotide derivatives in activating T1R1 / T1R3 receptors in combination with one or more nucleotides and / or one or more amino acids.

[0816] Methods: HEK293 cells stably expressing T1R3 and inducibly expressing T1R1 were exposed to a single nucleotide derivative or a combination thereof with one or more amino acids and / or one or more nucleotides to activate umami receptors. Activation of the T1R1 / T1R3 receptors was detected by changes in intracellular calcium levels using a calcium-sensitive fluorescent dye and / or a luminescent reporter system. Cells expressing T1R3 but not T1 served as controls. Tetra or 3 is used for data capture.

[0817] For each nucleotide derivative, dose-response curves were generated and the following properties were determined in the presence of 20 mM alanine: EC50 of the nucleotide derivative. 50 The maximum receptor response in the presence of nucleotide derivatives, relative to the receptor response in the presence of IMP, is the threshold amount of nucleotide derivatives leading to T1R1 / T1R3 receptor activation. EC2000 was used as a positive and negative control. 50 The values ​​are summarized in Table 16.

[0818] Results: Table 15 shows the effects of all tested nucleotide derivatives on T1R1 / T1R3 activation. Treatment of HEK293 cells expressing the T1R1 / T1R3 receptor with a combination of nucleotide derivatives, sodium adenosine 3',5'-bisphosphate (ADP), and 20 mM alanine resulted in T1R1 / T1R3 receptor activation, as evidenced by the maximum change in intracellular calcium levels (ΔF / F0) and the observed EC50 at 0.001 mM. 50 The value indicated. Conversely, in the presence of 20 mM alanine, adenosine monophosphate (AMP) resulted in an observed EC50 of 0.011 mM. 50Values ​​(Table 7). These results indicate that adenosine-based nucleotide derivatives exhibit improved activity in activating the T1R1 / T1R3 receptor compared to the standard nucleotides from which they are derived. Similar results were observed for 2'- / 3'-O-(N'-o-aminobenzoyl)adenosine-5'-O-monophosphate sodium salt (Table 7). Furthermore, adenosine-based nucleotide derivatives also exhibited a lower threshold for activating the receptor compared to AMP (Table 7).

[0819] Guanosine-based nucleotide derivatives, including 6-thioguanosine-5'-O-monophosphate, 2'-deoxyguanosine-5'-O-monophosphate (sodium salt), 2'-,3'-O-(N'-methyl-o-aminobenzoyl)guanosine-5'-O-monophosphate, guanosine-5'-monophosphate (sodium salt), 2'-deoxy-3'-O-(N'-methyl-o-aminobenzoyl)guanosine-5'-O-monophosphate, guanosine-5'-O-(2-thiodiphosphate), 2'-deoxyguanosine-3',5'-O-diphosphate, and 2'-deoxyguanosine-5'-O-monophosphate, acted as activators of T1R1 / T1R3 in the presence of alanine (Table 8). As shown in Table 8, these guanosine-based nucleotide derivatives exhibited improved activity compared to the standard nucleotide guanosine monophosphate (GMP). For example, 6-thioguanosine-5'-O-monophosphate showed an EC50 of 0.0009 mM in the presence of alanine. 50 The value and the threshold of 0.0002 mM; while GMP shows an EC of 0.02 mM. 50 Values ​​and a threshold of 0.008 mM (Table 8 and Figure 1 ).

[0820] Purine-based nucleotide derivatives, 2-amino-6-chloropurine riboside-5'-O-monophosphate (2-NH2-6-Cl-5'-PuMP) and 6-chloropurine riboside-5'-O-monophosphate, were also observed as activators of the T1R1 / T1R3 receptor (Table 9). For example, 2-amino-6-chloropurine riboside-5'-O-monophosphate showed an EC50 of 0.0005 mM in the presence of alanine. 50 With a threshold of 0.00013 mM, 6-chloropurine riboside-5'-O-monophosphate showed an EC50 of 0.002 mM in the presence of alanine. 50 The values ​​and threshold of 0.0005 mM are shown in Table 9. In contrast, purine riboside-5'-O-monophosphate (5'-PuMP) showed an EC50 of 0.02 mM in the presence of alanine. 50 The values ​​and the threshold of 0.005 mM indicate that purine-based nucleotides activate cat umami receptors at lower concentrations compared to the standard nucleotide 5'-PuMP (Table 9).

[0821] Inosine-based nucleotide derivatives were observed to activate the receptor in the presence of alanine (Table 11). 6-Thioinosine phosphate showed an EC50 of 0.02 mM in the presence of alanine. 50 The EC50 value for inosine triphosphate (ITP) in the presence of alanine was 0.08 mM. 50 Values ​​(Table 11 and Figure 6 Standard nucleotide inosine monophosphate (IMP) showed an EC50 of 0.07 mM. 50 The value indicates that 6-thioinosine phosphate exhibits increased activity compared to IMP. Similar results were observed for uridine-based nucleotide derivatives, which showed improved activity compared to uridine monophosphate (UMP). For example, the uridine-based nucleotide derivative, uridine 5'-monophosphate morpholinate 4-morpholino-N,N'-dicyclohexylformamidin, showed improved activity compared to UMP (EC). 50 Values ​​greater than 30mM) indicate EC values ​​greater than 3mM. 50 Values ​​(Table 12).

[0822] Unbound by any particular theory, these results indicate that the nucleotide derivatives function as positive regulators of the T1R1 / T1R3 receptor and exhibit improved activity compared to the standard nucleotide.

[0823]

[0824]

[0825]

[0826]

[0827]

[0828]

[0829]

[0830]

[0831]

[0832]

[0833]

[0834]

[0835]

[0836]

[0837]

[0838]

[0839]

[0840]

[0841]

[0842]

[0843]

[0844]

[0845] Table 16. Positive and negative controls: EC50 of amino acids determined in the presence of 0.2 mM IMP 50 .

[0846]

[0847] Example 3 - Identification of the T1R1 nucleotide and amino acid interaction domains

[0848] This embodiment describes the identification of amino acids within T1R1 that interact with nucleotides and amino acids that bind to T1R1 on a computer chip.

[0849] Methods: Feline T1R1 is a group C G protein-coupled receptor (GPCR) that complexes with T1R3 to form a heterodimer of the umami taste receptor. A model of the Venus flytrap domain (VFT domain) of feline T1R1 was constructed using the crystal structure of another group C GPCR, the metabolite glutamate receptor 1EWT (Kunishima et al., Nature 407:971-977 (2000)), which was available from a protein database (Berman et al., Nucleic Acids Research, 28:235-242 (2000)). The crystal structures of the VFTs of the group C GPCRs, including the metabolite glutamate receptors mGluR1, mGluR3, mGluR5, and mGluR7, showed very similar patterns of ligands binding to the active site cleft of the VFT. These ligand binding patterns were used to manually compare the feline T1R1 VFT sequence with the sequence distribution of the metabolite glutamate receptor. Subsequently, the comparison was performed using homology modeling with the Modeller software package (Eswar et al., Curr Protoc Bioinformatics, John Wiley & Sons, Supplement 15, 5.6.1-5.6.30 (2006)).

[0850] Modeling amino acids to the active site of T1R1 After placing the zwitterionic framework of glutamate into the mGluR crystal structure, alanine (L-alanine) was first located at the active site of the cat T1R1 VFT model. The resulting complex was refined using molecular dynamics and energy minimization. The binding energy was estimated using a continuum model for water by calculating the difference between the calculated energy of the complex and the calculated energy of the isolated ligands and apolipoproteins. Other amino acids were constructed into the bound alanine scaffold using Discovery Studio (Dassault Systemes, BIOVIA Corp, San Diego, CA, USA) and refined using molecular dynamics and energy minimization (Brooks et al., J Comput Chem. 30(10):545-614(2009)). The final model was selected for those amino acids whose calculated binding energies were comparable to those of alanine and which also preserved the conserved interactions with the hinges observed in the mGluR crystal structure.

[0851] Nucleotides are modeled to the active site of T1R1.After modeling previously published IMPs into human T1R1, IMPs and GMPs were first localized to the VFT active sites of cat T1R1, as described by Zhang et al. (Proc Natl Acad Sci US A. 105(52):20930-4(2008)). The resulting models were refined by altering nucleotide twists and using molecular dynamics and energy minimization. The final models were selected that had binding energies comparable to GMPs and also exhibited nucleotide interactions with conserved residues established as important for IMP binding to human T1R1 via site-directed mutagenesis (Zhang et al. Proc Natl Acad Sci US A. 105(52):20930-4(2008)). Other nucleotides and nucleotide analogues were first overlaid onto the IMP and GMP models, followed by the same refinement, evaluation, and selection procedures as those used for the IMP and GMP descriptions.

[0852] Result: As Figure 14 As shown, the cat T1R1 VFT domain consists of two lobes. The upper lobe and lower lobe are connected by a three-stranded protein called the hinge. Figure 14 In the diagram, the upper leaf is at the top; the lower leaf is at the bottom. The hinge points to the left. Upon agonist binding, the Venus flytrap domain changes from an open to a closed conformation. Active amino acids and nucleotides bind to the VFT domain between the two leaves (see [link to diagram]). Figure 14 Amino acids bind to the region near the hinge (see...). Figure 14 Alanine is shown in CPK space-filled form, rendered to the left of the figure. Nucleotides bind to regions further from the hinge but still located between the leaves of the Venus flytrap (see [reference]). Figure 14 : IMP indicates CPK space filling, which is rendered to the right side of the graph.

[0853] amino acid binding :like Figure 14 As shown, amino acid ligands bind to the hinge region of the VFT and are well coordinated through interactions near the hinge. Figure 15 Exemplary binding modes of L-alanine are shown, illustrating possible hydrogen bonds, salt bridge interactions, and Pi-cation interactions with Thr149, Ser172, Tyr220, Thr148, Glu170, and Asp302. These interactions are shown as dashed lines.

[0854] Site-directed mutagenesis identified Thr149, Ser172, and Tyr220 residues as important for binding to L-glutamate in human umami receptors (Zhang et al., Proceedings of the National Academy of Sciences 105(52):20930-4(2008)). Mouse Asn149, which corresponds to cat Thr148, was shown to be important for binding to amino acids in mice (Toda et al., Journal of Biochemistry 288:36863-36877(2013)). Glu170 and Asp302 are present in cat and mouse T1R1 but not in humans. In humans, the amino acid at these positions is alanine. Human T1R1 / T1R3 is highly selective for L-glutamate and L-aspartate. In contrast, cat and mouse T1R1 / T1R3 respond to a wide range of amino acids. In the model described in this embodiment, Glu170 and Asp302 help coordinate the zwitterionic nitrogen of amino acid ligands suitable for the active site of T1R1, while establishing an electrostatic environment unsuitable for binding L-glutamate and L-aspartate. Using site-directed mutagenesis, Toda et al. showed that Glu170 and Asp302 are responsible for the observed differences in amino acid ligand preferences between cats and humans (Toda et al., Journal of Biochemistry 288:36863-36877 (2013)).

[0855] Nucleotide binding Nucleotide binding ratios specify the position of amino acids further from the hinge, such as... Figure 14 As shown. Figure 16 Exemplary binding modes of GMP are shown, including possible hydrogen bonds and salt bridge interactions with GMP phosphate and T1R1 His47, His71, Arg277 and Asn69, GMP sugar and T1R1 Asn302 and Ser306, and GMP bases and T1R1 Ser384, His308 and Ala380. These interactions are shown as dashed lines.

[0856] His308 shows coordination of the GMP bases, but can also wiggle to coordinate the GMP phosphate. Additional hydrophobic interactions also exist that add to the interaction energy of the ligand with the VFT, and the flexibility of different binding sites can vary (data not shown). Different nucleotides can show different interactions with T1R1, but can overlap with the GMP interactions described herein. Structure-activity relationship (SAR) and T1R1 modeling suggest that the presence of negatively charged groups in the phosphate-binding region of T1R1 is important for the binding of nucleotides to T1R1. SAR and modeling suggest that the presence of nucleotide bases, extended bases, substituted bases, or other bioelectron isostatic substitutions of nucleotide bases can form interactions in the nucleotide base-binding region of T1R1, which are equally important for binding (for examples of bases, see Limbach et al., Nucleic Acids Research 22(12):2183-2196(1994)).

[0857] Similarly, SAR and modeling indicate that interactions between nucleotide sugars (or sugar substitutes) are important for the successful binding of nucleotides to T1R1. The sugar can orient appropriately negatively charged groups to similarly facilitate the establishment of interactions between T1R1 and both the phosphate region and the base region of the nucleotide.

[0858] Although different nucleotides may exhibit different interactions with T1R1, this interaction will likely largely conform to this set of possible interactions.

[0859] Bridging interactions between amino acid and nucleotide binding Asp302 is present as a residue in the VFT of T1R1 in various species, including cats and mice. However, in humans, the amino acid at this position is alanine. Asp302 has a flexible side chain that can be oriented to coordinate with the zwitterionic nitrogen or side chain of the amino acid ligand. Figure 15 Or alternatively, sugars can be directed to coordinate the binding of nucleotides. Figure 16 Furthermore, Asp302 can also be directed to simultaneously coordinate the binding of the zwitterionic backbone nitrogen of amino acids and the sugar of nucleotides. Figure 17 This bridging interaction can enhance the synergistic effect between the bound amino acid and the bound nucleotide. Because the amino acid at this position is alanine in humans, such a bridging interaction is impossible in humans. Alternating conformations of the nucleotide bases can establish further bridging interactions between Glu170 and the selected nucleotide bases (data not shown).

[0860] Example 4 - Computer Identification of Transmembrane Compounds

[0861] This embodiment describes computational modeling of the T1R1 / T1R3 receptor to identify putative transmembrane compounds.

[0862] Computational methods were used to analyze the three-dimensional structure of T1R1 to identify transmembrane regions that could be utilized to selectively activate the T1R1 / T1R3 receptor. Although the crystal structure of cat T1R1 is not yet determined, structural models of the transmembrane region of T1R1 were generated based on the crystal structures of human GPCR metabotropic glutamate receptor 1 (mGluR1) (Wu et al., Science, 2014, Vol. 344, pp. 58–64) and human GPCR metabotropic glutamate receptor 5 (mGluR5) (Dore et al., Nature, July 31, 2014; 511(7511):557–62; Epub, July 6, 2014). Computer modeling was then used to identify small chemical compounds that could potentially interact with the transmembrane domains of the T1R1 monomer of the T1R1 / T1R3 receptor.

[0863] Figure 28 The interaction between N-benzyl-L-phenylalanine methyl ester and T1R1 is shown. The asparagine (ASN)735 of the T1R1 transmembrane domain interacts with the ester of the ligand. The aryl residue cluster is located at the active site, which can coordinate with the ligand by forming a cyclic stacking interaction with the phenyl group of the ligand on the right side of the figure. Figure 35 Modeling of 1-benzyl-3-(2-oxo-2-phenylethyl)imidazoline-2,4,5-trione within the transmembrane region of T1R1 is shown. Phe642 of helix 6 and Phe776 of helix 2 interact with the benzene ring of 1-benzyl-3-(2-oxo-2-phenylethyl)imidazoline-2,4,5-trione, and Asn792 of helix 6 interacts with the carbonyl group via hydrogen bonding. Figure 35 Modeling of 1H-imidazo[4,5-c]pyridin-2(3H)-one in the T1R1 transmembrane region reveals hydrogen bonds between Asn735 in the T1R1 transmembrane region and 1H-imidazo[4,5-c]pyridin-2(3H)-one. Figure 36 ).

[0864] Example 5 - Activation of T1R1 / T1R3 receptors via transmembrane compounds

[0865] This embodiment describes the in vitro activation of T1R1 / T1R3 receptors by transmembrane compounds.

[0866] Based on the computer modeling described in Example 4, presumed T1R1 / T1R3 transmembrane compounds were identified and selected for further in vitro testing. Functional characterization of the selected compounds in vitro was used to evaluate the effectiveness of the presumed transmembrane compounds in activating the T1R1 / T1R3 receptor alone or in combination with one or more nucleotides and / or one or more amino acids.

[0867] Methods: HEK293 cells stably expressing T1R3 and inducibly expressing T1R1 were exposed to transmembrane compounds alone or in combination with one or more amino acids and / or one or more nucleotides to activate umami receptors. Activation of the T1R1 / T1R3 receptors was detected by changes in intracellular calcium levels using a calcium-sensitive fluorescent dye. Cells expressing T1R3 but not T1 served as controls. Tetra or 3 is used for data capture.

[0868] For each transmembrane compound, dose-response curves were generated and the following properties were determined: EC50 of the transmembrane compound alone. 50 ; EC of transmembrane compounds with 20 mM alanine 50 Transmembrane compounds with 0.2 mM IMP EC 50 Transmembrane compounds with 20 mM alanine and 0.2 mM IMP EC 50 The term "half-maximum effective concentration" (EC50) refers to the concentration at half maximum effective concentration. 50 The concentration of the compound that induces half the response between the baseline and the maximum after a specified exposure time is defined as 10 mM. In each experiment, serial dilutions of the transmembrane compound, up to 10 mM, are added to T1R1 / T1R3 expressing cells.

[0869] Results: Treatment of HEK293 cells expressing the T1R1 / T1R3 receptor with 1-benzyl-3-(2-oxo-2-phenylethyl)imidazoline-2,4,5-trione (e.g., in buffer) alone or in combination with 20 mM alanine resulted in activation of the T1R1 / T1R3 receptor, as indicated by changes in intracellular calcium levels (ΔF / F0), and led to observed EC50 greater than 1 mM. 50 Value. Conversely, in the presence of 0.2 mM IMP or 20 mM alanine and 0.2 mM IMP, 1-benzyl-3-(2-oxo-2-phenylethyl)imidazoline-2,4,5-trione leads to EC. 50 The values ​​decreased to 0.32±0.05 mM and 0.33±0.04 mM, respectively. Figure 37 (and Table 17). These results indicate that IMP is a positive allosteric modulator of the transmembrane compound 1-benzyl-3-(2-oxo-2-phenylethyl)imidazoline-2,4,5-trione, meaning that the compound has a synergistic effect on T1R1 / T1R3.

[0870] The compound N-(benzo[d][1,3]dioxacyclopenten-5-yl)-2-propylpentanamide was observed to act as a separate agonist for T1R1 / T1R3. Figure 39 (and Table 17). In the presence of IMP or IMP and alanine, the EC of N-(benzo[d][1,3]dioxacyclopenten-5-yl)-2-propylpentanamide... 50 The decrease leads to a significantly higher expansion of ΔF / F0. Figure 39 (and Table 17). These results indicate that alanine and IMP synergistically activate T1R1 / T1R3 with N-(benzo[d][1,3]dioxacyclopenten-5-yl)-2-propylpentanamide. Similar results were observed for the transmembrane compound N-(hept-4-yl)benzo[d][1,3]dioxacyclopenten-5-carboxamide. Figure 40 (and Table 17). IMP acts as a positive allosteric modifier for N-(hept-4-yl)benzo[d][1,3]dioxanepentene-5-carboxamide.

[0871] The proposed transmembrane compound N-(2-amino-2-oxo-1-phenylethyl)-3-chloro-4,5-dimethoxybenzamide failed to activate T1R1 / T1R3 alone; however, in the presence of IMP or IMP and alanine, N-(2-amino-2-oxo-1-phenylethyl)-3-chloro-4,5-dimethoxybenzamide activated T1R1 / T1R3, as by increasing ΔF / F0 and EC at lower concentrations. 50 The decrease indicated by ( Figure 41 (and Table 17). Similar results were observed with 2-((5-(4-(methylthio)phenyl)-2H-tetrazole-2-yl)methyl)pyridine. Figure 43 (and Table 17).

[0872] Compound (E)-3-(4-methoxyphenyl)-N-(pent-3-yl)acrylamide functions as a transmembrane compound in the T1R1 / T1R3 configuration, EC 50 The value was 0.45 ± 0.01. In the presence of IMP or IMP and alanine, the activity of (E)-3-(4-methoxyphenyl)-N-(pent-3-yl)acrylamide was greatly enhanced, leading to EC... 50 The values ​​were 0.15±0.02 and 0.08±0.01, respectively, indicating that IMP and alanine act as allosteric modifiers for (E)-3-(4-methoxyphenyl)-N-(pent-3-yl)acrylamide. Figure 42 (and Table 17).

[0873] Further analysis was conducted on the transmembrane compound N-(hept-4-yl)benzo[d][1,3]dioxane-5-carboxamide. Figure 44 The dose-response curves of N-(hept-4-yl)benzo[d][1,3]dioxacyclopenten-5-carboxamide are shown in the presence of GMP and phenylalanine; GMP, phenylalanine and alanine; GMP, phenylalanine and IMP; or GMP, phenylalanine, alanine and IMP. EC50 values ​​are compared with those of N-(hept-4-yl)benzo[d][1,3]dioxacyclopenten-5-carboxamide. 50 The EC50 values ​​of N-(hept-4-yl)benzo[d][1,3]dioxane-5-carboxamide were significantly reduced in the presence of phenylalanine and GMP or in the presence of IMP alone. 50 Values ​​(Table 19). As shown above, IMP acts as an allosteric modulator of the agonist activity of N-(hept-4-yl)benzo[d][1,3]dioxane-5-carboxamide; however, these results indicate that the combination of IMP and alanine leads to a lower EC value compared to IMP alone. 50 The value also shows a higher shift in ΔF / F0, indicating that the combination exhibits a synergistic effect with N-(hept-4-yl)benzo[d][1,3]dioxane-5-carboxamide activating T1R1 / T1R3. Figure 44 (and Table 19). Furthermore, compared to the combination of phenylalanine and GMP in the absence of the agonist, ΔF / F0 is approximately 10X higher in the presence of a ternary mixture of N-(hept-4-yl)benzo[d][1,3]dioxane-5-carboxamide, phenylalanine, and GMP. Figure 45 ).

[0874] Figure 46 The dose-response curves for the positive and negative controls used are shown, and the results for the active compounds are summarized in Table 17. Table 18 shows the results for all compounds tested. For the positive and negative controls, dose-response curves for amino acids were determined in the presence of 0.2 mM IMP. The dose-response curves for nucleotides were determined in the presence of 20 mM alanine.

[0875] Unbound by any particular theory, these results suggest that individual nucleotides, such as IMP, or combinations of nucleotides and amino acids, such as IMP and alanine, act as positive regulators of the disclosed transmembrane compounds, thereby leading to a reduction in the amount of agonist necessary for full activation of the T1R1 / T1R3 receptor.

[0876]

[0877]

[0878]

[0879]

[0880]

[0881]

[0882]

[0883]

[0884]

[0885] Table 19. Activation of T1R1 / T1R3 via nucleotides, amino acids, and transmembrane compounds

[0886]

[0887] Table 20. Positive and negative controls determined using 0.2 mM IMP.

[0888]

[0889] Example 6 - Activation of T1R1 / T1R3 receptors via transmembrane compounds

[0890] This embodiment describes the in vitro activation of T1R1 / T1R3 receptors by transmembrane compounds.

[0891] Based on the computer modeling described in Example 4, proposed T1R1 / T1R3 transmembrane compounds were identified and selected for further in vitro testing. The in vitro functional characterization of the selected compounds was used to evaluate the effectiveness of the proposed transmembrane compounds in activating the T1R1 / T1R3 receptor alone or in combination with one or more nucleotides and / or one or more amino acids.

[0892] Method: As described in Example 5, the selected compounds were characterized in vitro.

[0893] Results: As shown in Table 21, treatment of HEK293 cells expressing the T1R1 / T1R3 receptor alone with N-benzyl-L-phenylalanine methyl ester HCl (e.g., in buffer) led to activation of the T1R1 / T1R3 receptor and EC 50 The concentration was 0.03 ± 0.002 mM. The combination of this compound with 20 mM L-alanine increased the EC50. 50 The concentration decreased to 0.05 ± 0.001 mM. The combination of this compound with 0.2 mM IMP reduced EC50. 50 The concentration decreased to 0.02 ± 0.001 mM, and the combination of this compound with 20 mM L-alanine and 0.2 mM IMP reduced EC. 50 Up to 0.02±0.021mM.

[0894] Table 21. EC52 of N-benzyl-L-phenylalanine methyl ester HCl activated T1R1 / T1R3 50

[0895]

[0896] As shown in Table 22, treatment of HEK293 cells expressing the T1R1 / T1R3 receptor alone with N-(2-(1H-indol-3-yl)ethyl)nicotinamide (e.g., in buffer) resulted in activation of the T1R1 / T1R3 receptor, EC 50 The concentration was 0.15 ± 0.03 mM. The combination of this compound with 20 mM L-alanine increased the EC50. 50 Up to greater than 0.1 mM. The combination of this compound with 0.2 mM IMP reduces EC50. 50 The concentration decreased to 0.05 ± 0.01 mM, and the combination of this compound with 20 mM L-alanine and 0.2 mM IMP reduced EC. 50 Up to 0.04±0.01mM.

[0897] Table 22. EC50 of N-(2-(1H-indol-3-yl)ethyl)nicotinamide activating T1R1 / T1R3 50

[0898]

[0899] As shown in Table 23, treatment of HEK293 cells expressing the T1R1 / T1R3 receptor alone with 2-amino-N-phenylethylbenzamide (e.g., in buffer) resulted in activation of the T1R1 / T1R3 receptor, EC 50 The concentration was 0.42 ± 0.01 mM. The combination of this compound with 20 mM L-alanine increased the EC50. 50 The concentration decreased to 0.48 ± 0.01 mM. The combination of this compound with 0.2 mM IMP reduced EC50. 50 The concentration decreased to 0.14 ± 0.03 mM, and the combination of this compound with 20 mM L-alanine and 0.2 mM IMP reduced EC. 50 Up to 0.09±0.01mM.

[0900] Table 23. EC50 of 2-amino-N-phenylethylbenzamide activating T1R1 / T1R3 50

[0901]

[0902] Unbound by any particular theory, these results suggest that individual nucleotides, such as IMP, or combinations of nucleotides and amino acids, such as IMP and alanine, act as positive regulators of the disclosed transmembrane compounds, thereby leading to a reduction in the amount of agonist necessary for full activation of the T1R1 / T1R3 receptor.

[0903] Example 7 - Activation of T1R1 / T1R3 receptors via transmembrane compounds and nucleotide derivatives

[0904] This embodiment describes the in vitro activation of the T1R1 / T1R3 receptor using transmembrane compounds and nucleotide derivatives.

[0905] Based on the computer modeling of Example 4, proposed T1R1 / T1R3 transmembrane compounds were identified and selected for further in vitro testing. In vitro functional characterization of the selected compounds was used to evaluate the effectiveness of the proposed transmembrane compounds in activating the T1R1 / T1R3 receptor alone or in combination with one or more nucleotides and / or one or more amino acids. The selected nucleotide derivatives were tested alone or in combination with one or more nucleotides and / or one or more amino acids.

[0906] Methods: The selected compounds were characterized in vitro as described in Examples 1, 2 and 5.

[0907] Results: Table 26 shows the effects of all tested transmembrane compounds and nucleotide derivatives on T1R1 / T1R3 activation. Treatment of HEK293 cells expressing the T1R1 / T1R3 receptor alone with several transmembrane compounds in buffer resulted in T1R1 / T1R3 activation, as indicated by changes in intracellular calcium levels (ΔF / F0), as shown in Tables 24A-O. When these compounds were combined with 0.2 mM IMP or with a mixture of 0.2 mM IMP and 20 mM alanine, the transmembrane compounds were more effective in activating T1R1 / T1R3, as indicated by EC50 of the transmembrane compounds. 50 The concentration reduction is evidenced by (Tables 24A-O and Figures 58-75). Tables 24A-O show the EC50 values ​​for each active transmembrane compound alone (in buffer) and in the presence of L-alanine, IMP, and L-alanine + IMP. 50 Table 25 and Figure 71 EC50 of the control compound (20 mM amino acids in the presence of 0.2 mM IMP) is shown. 50 And dose-response curves. Unbound by any particular theory, these results suggest that individual nucleotides, such as IMP, or combinations of nucleotides and amino acids, such as IMP and alanine, act as positive regulators of the disclosed transmembrane compounds, resulting in a reduction in the amount of transmembrane compounds necessary for full activation of the T1R1 / T1R3 receptor.

[0908] Table 24A-O. EC5 activation of T1R1 / T1R3 by transmembrane compounds 50

[0909] Table 24A

[0910] 1,3-Dibenzylpyrimidine-2,4,6(1H,3H,5H)-trione <![CDATA[T1R1 / T1R3 EC 50 ]]> Alone in buffer 0.26±0.01mM +20mM L-alanine 0.31±0.05mM +0.2mM IMP 0.52±0.19mM +20mM L-alanine +0.2mM IMP >0.3mM

[0911] Table 24B

[0912]

[0913] Table 24C

[0914]

[0915] Table 24D

[0916]

[0917] Table 24E

[0918] (diphenylacetyl)-ethyl carbamate <![CDATA[T1R1 / T1R3 EC 50 ]]> Alone in buffer 0.25±0.01mM +20mM L-alanine 0.38±0.01mM +0.2mM IMP 0.12±0.02mM +20mM L-alanine +0.2mM IMP 0.10±0.01mM

[0919] Table 24F

[0920] N,N'-(butane-1,4-diyl)nicotinamide <![CDATA[T1R1 / T1R3 EC 50 ]]> Alone in buffer N / A +20mM L-alanine N / A +0.2mM IMP 0.60±0.09mM +20mM L-alanine +0.2mM IMP 0.61±0.36mM

[0921] Table 24G

[0922] N-Phenylacetamide <![CDATA[T1R1 / T1R3 EC 50 ]]> Alone in buffer 0.37±0.02mM +20mM L-alanine >0.3mM +0.2mM IMP 0.35±0.1mM +20mM L-alanine +0.2mM IMP 0.22±0.02mM

[0923] Table 24H

[0924] 2-Amino-N-phenylethylbenzamide <![CDATA[T1R1 / T1R3 EC 50 ]]> Alone in buffer 0.42±0.01mM +20mM L-alanine 0.48±0.01mM +0.2mM IMP 0.14±0.02mM +20mM L-alanine +0.2mM IMP 0.09±0.01mM

[0925] Table 24I

[0926] N-Phenethylbenzo[d][1,3]dioxacyclopentene-5-carboxamide <![CDATA[T1R1 / T1R3 EC 50 ]]> Alone in buffer 0.12±0.07mM +20mM L-alanine >1.0mM +0.2mM IMP 0.04±0.01mM +20mM L-alanine +0.2mM IMP 0.03±0.01mM

[0927] Table 24J

[0928] N-Phenethylbenzamide <![CDATA[T1R1 / T1R3 EC 50 ]]> Alone in buffer 0.33±0.01mM +20mM L-alanine >1.0mM +0.2mM IMP 0.12±0.03mM +20mM L-alanine +0.2mM IMP 0.09±0.02mM

[0929] Table 24K

[0930] N-benzoyl-DL-leucine amide <![CDATA[T1R1 / T1R3 EC 50 ]]> Alone in buffer >0.6mM +20mM L-alanine >1.0mM +0.2mM IMP 0.47±0.07mM +20mM L-alanine +0.2mM IMP 0.91±0.23mM

[0931] Table 24L

[0932] N-(2-(1H-indol-3-yl)ethyl)nicotinamide <![CDATA[T1R1 / T1R3 EC 50 ]]> Alone in buffer 0.15±0.03mM +20mM L-alanine >0.1mM +0.2mM IMP 0.05±0.01mM +20mM L-alanine +0.2mM IMP 0.04±0.10mM

[0933] Table 24M

[0934] N-Benzyl-L-Phenylalanine Methyl Ester Hydrochloride <![CDATA[T1R1 / T1R3 EC 50 ]]> Alone in buffer 0.03±0.002mM +20mM L-alanine 0.05±0.001mM +0.2mM IMP 0.02±0.001mM +20mM L-alanine +0.2mM IMP 0.02±0.002mM

[0935] Table 24N

[0936] 6-Thioguanosine-5'-O-bisphosphate (6-T-GDP) <![CDATA[T1R1 / T1R3 EC 50 ]]> Alone in buffer N / A +20mM L-alanine 0.18±0.02mM +0.2mM IMP N / A +20mM L-alanine +0.2mM IMP 0.28±0.05mM

[0937] Table 24O

[0938] 6-Chloropurinoriboside-5'-O-triphosphate (6-Cl-PuTP) <![CDATA[T1R1 / T1R3 EC 50 ]]> Alone in buffer >0.03mM +20mM L-alanine >0.03mM +0.2mM IMP >0.03mM +20mM L-alanine +0.2mM IMP >0.03mM

[0939] Table 25. Positive and negative controls for transmembrane compound activation of T1R1 / T1R3

[0940]

[0941]

[0942]

[0943]

[0944]

[0945]

[0946]

[0947]

[0948]

[0949]

[0950]

[0951]

[0952]

[0953] Example 8 - Activation of T1R1 / T1R3 receptors via transmembrane compounds

[0954] This embodiment describes the in vitro activation of the T1R1 / T1R3 receptor by a transmembrane compound, wherein the transmembrane compound is used as a receptor agonist and / or a positive allosteric modulator (PAM). When used as a PAM, the transmembrane compound increases the effect of nucleotides and amino acids on receptor activity.

[0955] Based on the computer modeling described in Example 4, proposed T1R1 / T1R3 transmembrane compounds were identified and selected for further testing as in vitro receptor agonists and / or PAMs. In vitro functional characterization of the selected compounds was used to evaluate the effectiveness of the proposed transmembrane compounds as agonists and / or PAMs in activating the T1R1 / T1R3 receptor.

[0956] Agonist screening method: HEK293 cells stably expressing T1R3 and inducibly expressing T1R1 were exposed to transmembrane compounds. Activation of the T1R1 / T1R3 receptor was detected by changes in intracellular calcium levels using calcium-sensitive fluorescent dyes and / or luminescent reporter systems. Cells not expressing the T1R1 / T1R3 receptor served as controls. Tetra or 3 is used for data capture.

[0957] Each transmembrane compound was tested at concentrations of 0.01 mM, 0.1 mM, and 1 mM. Dose-response profiles were generated for each transmembrane compound that activated T1R1 / T1R3. To generate dose-response profiles, transmembrane compounds were tested at concentrations ranging from 0.0001 mM to 1.0 mM in the presence of GMP and Ala. Dose-response profiles were created where Ala remained constant at 20 mM, and GMP was increased from 0.001 mM to 1 mM (i.e., specifically 0.001 mM, 0.003 mM, 0.01 mM, 0.03 mM, 0.1 mM, 0.3 mM, 0.6 mM, and 1 mM). Similarly, GMP remained constant at 1 mM, while Ala concentrations varied between 0.1 mM and 100 mM (specifically 0.1 mM, 0.3 mM, 1 mM, 3 mM, 10 mM, 30 mM, 60 mM, and 100 mM).

[0958] PAM screening method: HEK293 cells stably expressing T1R3 and inducibly expressing T1R1 were exposed to transmembrane compounds alone or in combination with Ala and GMP to activate umami receptors. Activation of the T1R1 / T1R3 receptors was detected by changes in intracellular calcium levels using calcium-sensitive fluorescent dyes and / or luminescent reporter systems. Cells not expressing T1R1 / T1R3 receptors served as controls. Tetra or 3 is used for data capture.

[0959] Dose-response curves were generated for each transmembrane compound at concentrations of 0 (buffer only), 0.01 mM, 0.1 mM, and 1 mM, with combinations of the three concentrations of transmembrane compound tested with 0.03 mM GMP + 100 mM Ala (T1), 0.6 mM GMP + 10 mM Ala (T2), 0.1 mM GMP + 60 mM Ala (T3), and 0.3 mM GMP + 60 mM Ala (T4). Mixtures were also tested in a 2-fold dilution series (T4(×) concentration). In these experiments, the first test concentration was 2× (2-fold) of the T4 concentration. Subsequent test concentrations were 2-fold dilutions of it (1×, 0.5×, etc.). This dilution series was tested with and without the addition of a constant concentration of the test compound at 0.3 mM.

[0960] Furthermore, it was determined that umami receptor activation occurs in the presence of 1 mM GMP + 100 mM Ala to produce the “maximum” umami receptor activation level. Compounds are classified as PAM if the response to any combination of compound + Ala + GMP exceeds the sum of the responses to either the compound alone or to GMP alone + alanine.

[0961] Results: As shown in Table 27, transmembrane tests were conducted on 24 different compounds, nine of which were identified as T1R1 / T1R3 agonists, PAM, or both.

[0962] Table 27. Transmembrane compounds as T1R1 / T1R3 agonists, PAM, or both

[0963]

[0964]

[0965] As described in Table 28, the nine compounds tested were active as T1R1 / T1R3 agonists and / or PAMs. Figures 72-80 show dose-response curves for the agonist and PAM characteristics of each compound identified as a T1R1 / T1R3 agonist and / or PAM.

[0966] Unbound by any particular theory, these results suggest that transmembrane compounds can act as agonists to activate T1R1 / T1R3 alone, and also as GMP and Ala activation modulators of T1R1 / T1R3, thereby reducing the amount of agonist required to fully activate the T1R1 / T1R3 receptor.

[0967] Example 9 - Identification of T1R1 transmembrane compound interacting residues

[0968] This embodiment describes the computer-aided identification of amino acids that interact with transmembrane compounds bound to T1R1 within T1R1.

[0969] Methods: Feline T1R1 is a group C G protein-coupled receptor (GPCR), such as T1R2, T1R3, CaSR, GabaB, and mGlu. Group C GPCRs contain (1) a large outer domain called the Venus flytrap domain (VFT), (2) a 7-transmembrane domain (7TM), and (3) a cysteine-rich domain connecting the VFT and 7TM. Homology models of the feline T1R1 7TM domain were constructed based on crystal structures 4OR2 and 4OO9 from a protein database (Berman et al., Nucleic Acids Research, 28:235-242 (2000)). 4OR2 and 4OO9 are partial crystal structures of two metabolites of glutamate receptors from group C GPCRs. 4OR2 is the crystal structure of mGluR1 with a transmembrane domain containing a bound negative allosteric modulator (NAM) (Wu et al., Science, April 4, 2014, 344(6179):58-64; Epub March 6, 2014). 4OO9 is the crystal structure of mGluR5 with a transmembrane domain containing a bound NAM (Dore et al., Nature, July 31, 2014; 511(7511):557-62; Epub July 6, 2014). The model was constructed using the I-TASSER program suite (Yang et al., Nature Methods, 12:7-8 (2015)) and the Modeller software package (Eswar et al., Curr Protoc Bioinformatics, John Wiley & Sons, Supplement 15, 5.6.1-5.6.30 (2006)) (which is part of a program suite from Dassault Systemes, BIOVIA Corp., San Diego, CA, USA). There was approximately 25% sequence identity between the mGluR1 and cat T1R1 7TM domains. N-benzyl-L-phenylalanine methyl ester was docked into the allosteric site of cat T1R1 7TM in the T1R1 model using the docking program BioDock (Ollard, NJ, USA) from BioPredict. For docking amino acids and nucleotides with VFT domains, a similar scheme as described for modeling nucleotides docked with VFT domains is used.

[0970] Results: In the docking model, N-benzyl-L-phenylalanine methyl ester interacted with the following amino acids at the allosteric 7TM binding site of cat T1R1: Ala795, Ala796, and Asn792, which are on helix 7 of 7TM; Trp773 and Phe776, which are on helix 6 of 7TM; Ala731, Phe728, Leu730, Phe732, and Asn735, which are on helix 5 of 7TM; Ala689, Ser686, Gln690, Ile693, Cys694, and Leu695, which are on helix 4 of 7TM; and Arg634, Gln635, Phe642, Ala639, Ala643, and Leu638, which are on helix 3 of 7TM. Figure 28 The ester group of N-benzyl-L-phenylalanine methyl ester forms a hydrogen bond with Asn735. Models of other transmembrane compounds illustrate that the ligand can form hydrogen bond interactions with Asn735, Ser686, or both.

[0971] Most of the hydrophobic interactions between N-benzyl-L-phenylalanine methyl ester and the 7TM domain of T1R1 occur between the ligand and Trp773, Phe776, Phe732, Phe728, Leu730, Leu695, Leu638, and Phe642. These amino acids also provide for hydrophobic interactions between other T1R1 transmembrane ligands and the T1R1 transmembrane domain.

[0972] A significant characteristic of the T1R1 active site is the number of residues capable of ring-stacking interactions with the bound ligand. The model of N-benzyl-L-phenylalanine methyl ester binding to T1R1 7TM illustrates a ring-stacking interaction from the benzyl group to the transmembrane domain. This characteristic is common to models of other active transmembrane compounds binding to T1R1 7TM. These may contribute to the binding of both and the stabilization of the T1R1 / T1R3 active conformation. T1R1 7TM amino acids capable of forming this interaction include Trp773, Phe776, Phe732, Phe728, and Phe642.

[0973] ***

[0974] Although the subject matter and advantages disclosed herein have been described in detail, it should be understood that various changes, substitutions, and modifications can be made without departing from the spirit and scope of the invention as defined by the appended claims. Furthermore, the scope of this application is not intended to be limited to specific embodiments of the treatment, processing, manufacture, and compositions, apparatuses, methods, and steps of the substances described herein. As will be readily apparent to those skilled in the art from the content of this disclosure, based on the subject matter disclosed herein, treatment, processing, manufacture, and compositions, apparatuses, methods, or steps of substances that perform substantially the same function or achieve substantially the same results as the corresponding embodiments described herein can be utilized using existing or future embodiments. Therefore, the appended claims are intended to be included within the scope of such treatment, processing, manufacture, compositions, apparatuses, methods, or steps of substances.

[0975] This application incorporates patents, patent applications, publications, product descriptions, and agreements throughout its contents, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

Claims

1. Use of a flavor composition in the preparation of an agonist of the feline umami receptor T1R1 / T1R3, wherein the flavor composition comprises a compound or combination of compounds selected from the group consisting of: (diphenylacetyl)-ethyl carbamate, or A combination of (diphenylacetyl)-carbamate and IMP, or A combination of (diphenylacetyl)-carbamate ethyl ester with alanine and IMP.

2. A feline pet food comprising the flavor composition of claim 1, wherein the flavor composition is present at a concentration of 0.0001% by weight to 10% by weight of the feline pet food.

3. A feline pet food comprising the flavor composition of claim 1, wherein the flavor composition is present at a concentration of 0.001 ppm to 1000 ppm in the feline pet food.

4. A feline pet food comprising the flavor composition of claim 1, wherein the flavor composition is present at a concentration of 1 μM to 1 M in the feline pet food.

5. A feline pet food comprising the flavor composition of claim 1, wherein the flavor composition is present in an effective amount to increase the palatability of the feline pet food, as determined by a panel of taste testers.

6. The feline pet food according to claim 5, wherein the feline pet food is a wet feline pet food.

7. The feline pet food according to claim 5, wherein the feline pet food is dry feline pet food.

8. A method for increasing the umami flavor intensity of feline pet food, the method comprising mixing the feline pet food with the flavor composition of claim 1, wherein the flavor composition is present at a concentration of 0.0001% by weight to 10% by weight of the mixture.

9. A method for increasing the umami flavor intensity of feline pet food, the method comprising mixing the feline pet food with the flavor composition of claim 1, wherein the flavor composition is present at a concentration of 0.001 ppm to 1000 ppm of the mixture.

10. A method for increasing the umami flavor intensity of feline pet food, the method comprising mixing the feline pet food with the flavor composition of claim 1, wherein the flavor composition is present at a concentration of 1 μM to 1 M in the mixture.

11. The method according to any one of claims 8-10, wherein the increase in umami flavor intensity includes an increase in umami aftertaste.

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