CHALCONE-UREA DERIVATIVES AS INSULIN SENSORS

Abstract

The invention relates to compounds of formula (I) or derivatives, tautomeric forms, stereoisomers, polymorphs, hydrates, solvates, pharmaceutically acceptable salts, pharmaceutical compositions, metabolites or prodrugs thereof, in which the substituents are as defined in the specification. These compounds may be useful as insulin sensors; in insulin secretion studies and as probes for insulin detection.

Claims

1. A compound of formula (I) ##STR00133## wherein the configuration around the double bond is E or Z; wherein R.sub.1 is selected from the group consisting of: hydrogen, cyclic aliphatic, acyclic aliphatic, branched aliphatic, unbranched aliphatic, substituted aliphatic, unsubstituted aliphatic, cyclic heteroaliphatic, acyclic heteroaliphatic, branched heteroaliphatic, unbranched heteroaliphatic, substituted heteroaliphatic, unsubstituted heteroaliphatic, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, cyclic acyl, acyclic acyl, substituted acyl and unsubstituted acyl; wherein when the group R.sub.1 is substituted, the one or more substituents are selected from the group consisting of halogen, hydroxyl, nitro, cyano, oxo (O), thioxo (S), azido, nitroso, amino, hydrazine, formyl, alkyl, alkene, alkyne, -alkyalkoxy, aryl, haloalkyl, haloalkoxy, -arylalkoxy, arylalkoxy-, cycloalkyl, O-cycloalkyl, heterocyclyl, heteroaryl, alkylamino-, -alkyleneamine, alkoxyamino-, OCH.sub.2-cycloalkyl; COOR.sup.a; C(O)R.sup.b; C(S)R.sup.a; C(O)NR.sup.aR.sup.b; NR.sup.aC(O)NR.sup.bR.sup.c; N(R.sup.a)SOR.sup.b; N(R.sup.a) SO.sub.2R.sup.b; NR.sup.aC(O)OR.sup.b; NR.sup.aR.sup.b; NR.sup.aC(O)R.sup.b; NR.sup.aC(S)R.sup.b; SONR.sup.aR.sup.b; SO.sub.2NR.sup.aR.sup.b; OR.sup.a; OR.sup.aC(O)OR.sup.b; OC(O)NR.sup.aR.sup.b; OC(O)R.sup.a; R.sup.aNR.sup.bR.sup.c; R.sup.aOR.sup.b; SR.sup.a; SOR.sup.a and SO.sub.2R.sup.a; each of R.sup.a, R.sup.b and R.sup.c are independently a hydrogen atom or an optionally substituted alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heteroaryl or heteroarylalkyl; wherein the substituents are further optionally substituted by one or more halogen, hydroxy, nitro, cycloalkyl, cyano, azido, nitroso, amino, hydrazino, formyl, alkyl, alkoxy, oxo, aryl, alkoxyamino- or haloalkyl; wherein when the group R.sub.1 is cyclic, R.sub.1 is a residue formed by removing a hydrogen atom from a saturated or unsaturated monocyclic carbocyclic ring or a saturated or unsaturated monocyclic heterocyclic ring having one or two or three heteroatoms selected from a nitrogen atom, an oxygen atom and a sulfur atom in the ring, and a saturated or unsaturated monocyclic 3, 4, 5, 6, 7 or 8 membered heterocyclic ring having 3 to 5 carbon atoms, or a bicyclic or tricyclic condensed polycyclic heterocyclic ring having one or two or three heteroatoms in the ring, and a bicyclic or tricyclic condensed polycyclic heterocyclic ring having 7 to 13 carbon atoms, wherein the rings are optionally substituted with at least one substituent; wherein R.sub.2 is hydrogen or alkyl; wherein R.sub.3 is NR.sup.dR.sup.e; each of R.sup.d and R.sup.e are independently selected from the group consisting of hydrogen or optionally substituted alkyl, alkenyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heteroaryl or heteroarylalkyl; or R.sup.d and R.sup.e together with the N, form an optionally substituted heterocyclic or heteroaryl ring; wherein the substituents are further optionally substituted by one or more halogen, hydroxy, nitro, cycloalkyl, cyano, azido, nitroso, amino, hydrazino, formyl, alkyl, aryl, heterocyclyl, alkoxy, mercapto, alkylthio, alkylcarbonyl, carboxy, alkxoycarbonyl, alkoxycarbonyloxy, oxo, or haloalkyl; wherein these further substituents are in turn further optionally substituted by one or more halogen, hydroxy, nitro, cycloalkyl, cyano, azido, nitroso, amino, hydrazino, formyl, alkyl, or haloalkyl; their derivatives, analogs, tautomeric forms, stereoisomers, polymorphs, hydrates, solvates, intermediates, pharmaceutically acceptable salts, pharmaceutical compositions, metabolites and prodrugs thereof.

2. The compound of claim 1, wherein R.sub.1 represents hydrogen, optionally hydroxy-, amino-, carboxy-, C.sub.1-C.sub.6-alkyl-C.sub.1-C.sub.6 alkanoate-substituted C.sub.1-C.sub.20alkyl, C.sub.2-C.sub.20alkenyl, C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl, or poly-C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl, or represents mono-, bi-, or tricyclic C.sub.3-C.sub.13 cycloalkyl in which optionally one or two not directly adjacent methylene groups are replaced by oxygen and/or nitrogen, wherein cycloalkyl is optionally substituted with halogen, hydroxyl, amino, oxo, phenyl or C.sub.1-C.sub.8-alkoxy, or represents phenyl-C.sub.1-C.sub.6-alkyl-, wherein phenyl is optionally substituted with one or more halogen, hydroxyl, amino, C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-alkoxy, or represents optionally halogen- or C.sub.1-C.sub.6-alkyl-substituted 5- or 6-membered heteroaryl having one or two heteroatoms selected from the group consisting of oxygen and nitrogen, or represents formula (IIa), (IIb), (IIc), (IId) or (IIe): ##STR00134## and wherein R.sub.2 represents hydrogen or C.sub.1-C.sub.6 alkyl.

3. The compound of claim 1, wherein R.sub.1R.sub.2N is selected from the groups consisting of: ##STR00135## ##STR00136## ##STR00137##

4. (canceled)

5. The compound according to claim 1, wherein R.sub.3 represents the formula NR.sup.dR.sup.e, wherein each of R.sup.d and R.sup.e is independently selected from the group consisting of a hydrogen atom, or an optionally substituted group selected from the group consisting of: an alkyl group having 1 to 20 carbon atoms which may have one or two or three substituents selected from the group consisting of: a halogen atom, a cycloalkyl group having 3 to 10 carbon atoms, an aryl group, a residue formed by removing a hydrogen atom from a saturated or unsaturated monocyclic heterocyclic ring having one or two or three heteroatoms selected from the group consisting of a nitrogen atom, an oxygen atom and a sulfur atom in the ring, a saturated or unsaturated monocyclic 5 or 6 membered heterocyclic ring having 3 to 5 carbon atoms, a bicyclic or tricyclic condensed polycyclic heterocyclic ring having one or two or three heteroatoms in the ring, a bicyclic or tricyclic condensed polycyclic heterocyclic ring having 7 to 13 carbon atoms group, and an alkoxy group having an alkyl moiety having 1 to 8 carbon atoms; an alkenyl group having 2 to 8 carbon atoms; a cycloalkyl group having 3 to 10 carbon atoms; an aryl group having one or two or three substituents selected from the group consisting of: a halogen atom, an alkyl group having 1 to 8 carbon atoms, a hydroxy group, an alkoxy group having an alkyl moiety having 1 to 8 carbon atoms, a mercapto group, an alkylthio group having an alkyl moiety having 1 to 8 carbon atoms, a formyl group, an alkylcarbonyl group having an alkyl moiety having 1 to 8 carbon atoms, a carboxy group, an alkoxycarbonyl group having an alkoxy group having an alkyl moiety having 1 to 8 carbon atoms, an alkylcarbonyloxy group having an alkylcarbonyl group having an alkyl moiety having 1 to 8 carbon atoms, a nitro group, a cyano group, a residue formed by removing a hydrogen atom from a saturated or unsaturated monocyclic heterocyclic ring having one or two or three heteroatoms selected from a nitrogen atom, an oxygen atom and a sulfur atom in the ring, and a saturated or unsaturated monocyclic 5 or 6 membered heterocyclic ring having 3 to 5 carbon atoms, or a bicyclic or tricyclic condensed polycyclic heterocyclic ring having one or two or three heteroatoms in the ring, and a bicyclic or tricyclic condensed polycyclic heterocyclic ring having 7 to 13 carbon atoms; an optionally cyano-, hydroxy substituted C.sub.1-C.sub.20 alkyl; an optionally halogen-, hydroxy-, amino-, oxo-, C.sub.1-C.sub.6-alkoxy substituted C.sub.3-C.sub.10 cycloalkyl, or C.sub.1-C.sub.6-alkoxy substituted C.sub.3-C.sub.10 heterocyclyl, wherein the heterocyclyl has one or two heteroatoms selected from the group consisting of nitrogen and/or oxygen; a C.sub.1-C.sub.6-alkyl-C.sub.1-C.sub.6-alkanoate; wherein the substituents in turn are further optionally substituted by one or more halogen, hydroxy, nitro, cycloalkyl, cyano, azido, nitroso, amino, hydrazino, formyl, alkyl, haloalkyl; or wherein R.sub.3 is represented by the formula ##STR00138## or their derivatives, analogs, tautomeric forms, stereoisomers, polymorphs, hydrates, solvates, intermediates, pharmaceutically acceptable salts, pharmaceutical compositions, metabolites and prodrugs thereof.

6. (canceled)

7. (canceled)

8. The compound of claim 1 having the formula selected from the group consisting of: ##STR00139## ##STR00140## their derivatives, analogs, tautomeric forms, stereoisomers, polymorphs, hydrates, solvates, intermediates, pharmaceutically acceptable salts, pharmaceutical compositions, metabolites and prodrugs thereof.

9. A method of detecting insulin, wherein the method comprises the use of the compound of claim 1.

10. (canceled)

11. (canceled)

12. The method according to claim 9, wherein insulin is to be detected in a sample selected from the group consisting of a cell culture, an isolated sample from a subject, a solution and an isolated cell from a tissue, preferably wherein detection comprises evaluating insulin secretion in the sample.

13. (canceled)

14. The method according to claim 9, wherein insulin is selected from the group consisting of human, bovine, murine and porcine insulin; or wherein insulin comprises the amino acid sequence selected from the group consisting of SEQ ID NO. 1 (GIVEQCCTSICSLYQLENYCN), SEQ ID NO. 2 (FVNQHLCGSHLVEALYLVCGERGFFYTPKT), SEQ ID NO. 3 (GIVEQCCASVCSLYQLENYCN), SEQ ID NO. 4 (FVNQHLCGSHLVEALYLVCGERGFFYTPKA), SEQ ID NO. 5 (GIVEQCCTSICSLYQLENYCN), SEQ ID NO. 6 (FVNQHLCGSHLVEALYLVCGERGFFYTPKA), SEQ ID NO. 7 (GIVDQCCTSICSLYQLENYCN) and SEQ ID NO. 8 (FVKQHLCGPHLVEALYLVCGERGFFYTPKS), wherein SEQ ID NOs. 1 to 8 is the amino acid sequence of the A and B chains of human, bovine, porcine and murine insulin, respectively; or wherein the insulin is encoded by a nucleic acid having the sequence selected from the group consisting of SEQ ID NO: 9 to 16: TABLE-US-00007 SEQ IDNO. NUCLEICACIDSEQUENCE 9 ggcattgtggaacagtgctgcaccagcatttgcagcct gtatcagctggaaaactattgcaac 10 tttgtgaaccagcatctgtgcggcagccatctggtgga agcgctgtatctggtgtgcggcgaacgcggcttttttt ataccccgaaaacc 11 ggcattgtggaacagtgctgcgcgagcgtgtgcagcct gtatcagctggaaaactattgcaac 12 tttgtgaaccagcatctgtgcggcagccatctggtgga agcgctgtatctggtgtgcggcgaacgcggcttttttt ataccccgaaagcg 13 ggcattgtggaacagtgctgcaccagcatttgcagcct gtatcagctggaaaactattgcaac 14 tttgtgaaccagcatctgtgcggcagccatctggtgga agcgctgtatctggtgtgcggcgaacgcggcttttttt ataccccgaaagcg 15 ggcattgtggatcagtgctgcaccagcatttgcagcct gtatcagctggaaaactattgcaac 16 tttgtgaaacagcatctgtgcggcccgcatctggtgga agcgctgtatctggtgtgcggcgaacgcggcttttttt ataccccgaaaagc.

15. (canceled)

16. (canceled)

17. The method of claim 14, wherein the nucleic acid is comprised in a vector.

18. The method according to claim 9, wherein detecting insulin in a test sample comprises the steps of: (a) contacting the test sample to be evaluated with a compound of formula (I): ##STR00141## wherein the configuration around the double bond is E or Z; wherein R.sub.1 is selected from the group consisting of: hydrogen, cyclic aliphatic, acyclic aliphatic, branched aliphatic, unbranched aliphatic, substituted aliphatic, unsubstituted aliphatic, cyclic heteroaliphatic, acyclic heteroaliphatic, branched heteroaliphatic, unbranched heteroaliphatic, substituted heteroaliphatic, unsubstituted heteroaliphatic, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, cyclic acyl, acyclic acyl, substituted acyl and unsubstituted acyl; wherein when the group R.sub.1 is substituted, the one or more substituents are selected from the group consisting of halogen, hydroxyl, nitro, cyano, oxo (O), thioxo (S), azido, nitroso, amino, hydrazine, formyl, alkyl, alkene, alkyne, -alkyalkoxy, aryl, haloalkyl, haloalkoxy, -arylalkoxy, arylalkoxy-, cycloalkyl, O-cycloalkyl, heterocyclyl, heteroaryl, alkylamino-, -alkyleneamine, alkoxyamino-, OCH.sub.2-cycloalkyl; COOR.sup.a; C(O)R.sup.b; C(S)R.sup.a; C(O)NR.sup.aR.sup.b; NR.sup.aC(O)NR.sup.bR.sup.c; N(R.sup.a)SOR.sup.b; N(R.sup.a)SO.sub.2R.sup.b; NR.sup.aC(O)OR.sup.b; NR.sup.aR.sup.b; NR.sup.aC(O)R.sup.b; NR.sup.aC(S)R.sup.b; SONR.sup.aR.sup.b; SO.sub.2NR.sup.aR.sup.b; OR.sup.a; OR.sup.aC(O)OR.sup.b; OC(O); OC(O)R.sup.a; R.sup.aNR.sup.bR.sup.c; R.sup.aOR.sup.b; SR.sup.a; SOR.sup.a and SO.sub.2R.sup.a; each of R.sup.a, R.sup.b and R.sup.c are independently a hydrogen atom or an optionally substituted alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heteroaryl or heteroarylalkyl; wherein the substituents are further optionally substituted by one or more halogen, hydroxy, nitro, cycloalkyl, cyano, azido, nitroso, amino, hydrazino, formyl, alkyl, alkoxy, oxo, aryl, alkoxyamino- or haloalkyl; wherein when the group R.sub.1 is cyclic, R.sub.1 is a residue formed by removing a hydrogen atom from a saturated or unsaturated monocyclic carbocyclic ring or a saturated or unsaturated monocyclic heterocyclic ring having one or two or three heteroatoms selected from a nitrogen atom, an oxygen atom and a sulfur atom in the ring, and a saturated or unsaturated monocyclic 3, 4, 5, 6, 7 or 8 membered heterocyclic ring having 3 to 5 carbon atoms, or a bicyclic or tricyclic condensed polycyclic heterocyclic ring having one or two or three heteroatoms in the ring, and a bicyclic or tricyclic condensed polycyclic heterocyclic ring having 7 to 13 carbon atoms, wherein the rings are optionally substituted with at least one substituent; wherein R.sub.2 is hydrogen or alkyl; wherein R.sub.3 is NR.sup.dR.sup.e; each of R.sup.d and R.sup.e are independently selected from the group consisting of hydrogen or optionally substituted alkyl, alkenyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heteroaryl or heteroarylalkyl; or R.sup.d and R.sup.e together with the N, form an optionally substituted heterocyclic or heteroaryl ring; wherein the substituents are further optionally substituted by one or more halogen, hydroxy, nitro, cycloalkyl, cyano, azido, nitroso, amino, hydrazino, formyl, alkyl, aryl, heterocyclyl, alkoxy, mercapto, alkylthio, alkylcarbonyl, carboxy, alkxoycarbonyl, alkoxycarbonyloxy, oxo, or haloalkyl; wherein these further substituents are in turn further optionally substituted by one or more halogen, hydroxy, nitro, cycloalkyl, cyano, azido, nitroso, amino, hydrazino, formyl, alkyl, or haloalkyl; their derivatives, analogs, tautomeric forms, stereoisomers, polymorphs, hydrates, solvates, intermediates, pharmaceutically acceptable salts, pharmaceutical compositions, metabolites and prodrugs thereof; (b) exciting the sample with a radiation source at a wavelength of about 430 nm, suitable to detect the compound; (c) observing the sample in conjunction with means for detecting fluorescence intensity of the compound at an emission wavelength of about 560 nm; (d) comparing the fluorescence in the test sample with the fluorescence in a control sample, wherein the control sample does not contain insulin; wherein an increase in fluorescence intensity above a basal level of fluorescence in the control sample denotes the presence of insulin in the test sample.

19. The method of claim 18, wherein the compound is represented by the formula ##STR00142##

20. The method according to claim 18, wherein detection comprises evaluating secretion of insulin by living cells, wherein said step of contacting the sample with the compound of formula (I) comprises incubating said cells in a medium containing said compound for sufficient time, such that said insulin is secreted in the medium where said insulin can be detected.

21. The method according to claim 20, wherein evaluating secretion comprises means for detecting fluorescence of the compound of formula (I) in real time, thereby providing real time monitoring of secretion of insulin by living cells.

22. A method for screening a compound that is suspected to affect secretion of insulin comprised in a test sample, comprising the steps of: (a) contacting the compound to be screened with the test sample; (b) contacting a compound of formula (I) with the test sample, wherein contacting is simultaneous, sequential or separate: ##STR00143## wherein the configuration around the double bond is E or Z; wherein R.sub.1 is selected from the group consisting of: hydrogen, cyclic aliphatic, acyclic aliphatic, branched aliphatic, unbranched aliphatic, substituted aliphatic, unsubstituted aliphatic, cyclic heteroaliphatic, acyclic heteroaliphatic, branched heteroaliphatic, unbranched heteroaliphatic, substituted heteroaliphatic, unsubstituted heteroaliphatic, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, cyclic acyl, acyclic acyl, substituted acyl and unsubstituted acyl; wherein when the group R.sub.1 is substituted, the one or more substituents are selected from the group consisting of halogen, hydroxyl, nitro, cyano, oxo (O), thioxo (S), azido, nitroso, amino, hydrazine, formyl, alkyl, alkene, alkyne, -alkyalkoxy, aryl, haloalkyl, haloalkoxy, -arylalkoxy, arylalkoxy-, cycloalkyl, O-cycloalkyl, heterocyclyl, heteroaryl, alkylamino-, -alkyleneamine, alkoxyamino-, OCH.sub.2-cycloalkyl; COOR.sup.a; C(O)R.sup.b; C(S)R.sup.a; C(O)NR.sup.aR.sup.b; NR.sup.aC(O)NR.sup.bR.sup.c; N(R.sup.a)SOR.sup.b; N(R.sup.a)SO.sub.2R.sup.b; NR.sup.aC(O)OR.sup.b; NR.sup.aR.sup.b; NR.sup.aC(O)R.sup.b; NR.sup.aC(S)R.sup.b; SONR.sup.aR.sup.b; SO.sub.2NR.sup.aR.sup.b; OR.sup.a; OR.sup.aC(O)OR.sup.b; OC(O); OC(O)R.sup.a; R.sup.aNR.sup.bR.sup.c; R.sup.aOR.sup.b; SR.sup.a; SOR.sup.a and SO.sub.2R.sup.a; each of R.sup.a, R.sup.b and R.sup.c are independently a hydrogen atom or an optionally substituted alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heteroaryl or heteroarylalkyl; wherein the substituents are further optionally substituted by one or more halogen, hydroxy, nitro, cycloalkyl, cyano, azido, nitroso, amino, hydrazino, formyl, alkyl, alkoxy, oxo, aryl, alkoxyamino- or haloalkyl; wherein when the group R.sub.1 is cyclic, R.sub.1 is a residue formed by removing a hydrogen atom from a saturated or unsaturated monocyclic carbocyclic ring or a saturated or unsaturated monocyclic heterocyclic ring having one or two or three heteroatoms selected from a nitrogen atom, an oxygen atom and a sulfur atom in the ring, and a saturated or unsaturated monocyclic 3, 4, 5, 6, 7 or 8 membered heterocyclic ring having 3 to 5 carbon atoms, or a bicyclic or tricyclic condensed polycyclic heterocyclic ring having one or two or three heteroatoms in the ring, and a bicyclic or tricyclic condensed polycyclic heterocyclic ring having 7 to 13 carbon atoms, wherein the rings are optionally substituted with at least one substituent; wherein R.sub.2 is hydrogen or alkyl; wherein R.sub.3 is NR.sup.dR.sup.e; each of R.sup.d and R.sup.e are independently selected from the group consisting of hydrogen or optionally substituted alkyl, alkenyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heteroaryl or heteroarylalkyl; or R.sup.d and R.sup.e together with the N, form an optionally substituted heterocyclic or heteroaryl ring; wherein the substituents are further optionally substituted by one or more halogen, hydroxy, nitro, cycloalkyl, cyano, azido, nitroso, amino, hydrazino, formyl, alkyl, aryl, heterocyclyl, alkoxy, mercapto, alkylthio, alkylcarbonyl, carboxy, alkxoycarbonyl, alkoxycarbonyloxy, oxo, or haloalkyl; wherein these further substituents are in turn further optionally substituted by one or more halogen, hydroxy, nitro, cycloalkyl, cyano, azido, nitroso, amino, hydrazino, formyl, alkyl, or haloalkyl; their derivatives, analogs, tautomeric forms, stereoisomers, polymorphs, hydrates, solvates, intermediates, pharmaceutically acceptable salts, pharmaceutical compositions, metabolites and prodrugs thereof; (c) detecting a signal from the compound of formula (I), which is indicative of the presence of insulin in the test sample to obtain a first value; and (d) comparing the first value with a control value, wherein the control value corresponds to a signal in a control sample, and the control value is determined from the control sample which is the same as provided in (a), except that the control sample is not contacted with the compound to be screened; wherein if the test value is greater or smaller than the control value, then the compound affects secretion of insulin in the sample.

23. The method of claim 22, wherein the test sample is selected from the group consisting of a cell culture, an isolated sample from a subject and an isolated islet; or wherein the compound to be screened is selected from the group consisting of KCl, glucose, Forskolin ((3R,4aR,5S, 6S, 6aS, 10S, 10aR, 10bS)-6, 10, 10b-trihydroxy-3, 4a, 7, 7, 10a-pentamethyl-1-oxo-3-vinyldodecahydro-1H-benzo[f]chromen-5-yl acetate), 3-isobutyl-1-methylxanthine (IBMX; 1-methyl-3-(2-methylpropyl)-7H-purine-2,6-dione), Glibenclamide (5-chloro-N-(4-[N-(cyclohexylcarbamoyl)sulfamoyl]phenethyl)-2-methoxybenzamide), amiodarone ((2-{4-[(2-butyl-1-benzofuran-3-yl)carbonyl]-2,6-diiodophenoxy}ethyl)diethylamine), nifedipine (3,5-dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate), Leucine, Tolbutamide (N-[(butylamino)carbonyl]-4-methylbenzenesulfonamide), Ouabain (1,3, 5, 11, 14, 19-Hexahydroxycard-20 (22)-enolide 3-(6-deoxy--L-mannopyranoside), 4-[(1R,3S,5S, 8R, 9S, 10R, 11R, 13R, 14S, 17R)-1,5,11,14-tetrahydroxy-10-(hydroxymethyl)-13-methyl-3-((2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yloxy)hexadecahydro-1H-cyclopenta[a]phenanthren-17-yl]furan-2 (5H)-one) and adrenalin.

24. The method of claim 22, wherein the contacting as provided in step (b) is done simultaneously, separately, or sequentially; or wherein detecting as provided in step (c) is real time detection.

25.-28. (canceled)

29. A method of assessing whether a condition or a stimulus affects secretion of insulin in cultured cells, comprising: (a) culturing cells expressing insulin under a condition or stimulus to be assessed for its effect on secretion of insulin, wherein the cells are cultured in the presence of a compound of formula (I), wherein the cells are referred to as test cells: ##STR00144## wherein the configuration around the double bond is E or Z; wherein R.sub.1 is selected from the group consisting of: hydrogen, cyclic aliphatic, acyclic aliphatic, branched aliphatic, unbranched aliphatic, substituted aliphatic, unsubstituted aliphatic, cyclic heteroaliphatic, acyclic heteroaliphatic, branched heteroaliphatic, unbranched heteroaliphatic, substituted heteroaliphatic, unsubstituted heteroaliphatic, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, cyclic acyl, acyclic acyl, substituted acyl and unsubstituted acyl; wherein when the group R.sub.1 is substituted, the one or more substituents are selected from the group consisting of halogen, hydroxyl, nitro, cyano, oxo (O), thioxo (S), azido, nitroso, amino, hydrazine, formyl, alkyl, alkene, alkyne, -alkyalkoxy, aryl, haloalkyl, haloalkoxy, -arylalkoxy, arylalkoxy-, cycloalkyl, O-cycloalkyl, heterocyclyl, heteroaryl, alkylamino-, -alkyleneamine, alkoxyamino-, OCH.sub.2-cycloalkyl; COOR.sup.a; C(O)R.sup.b; C(S)R.sup.a; C(O)NR.sup.aR.sup.b; NR.sup.aC(O)NR.sup.bR.sup.c; N(R.sup.a)SOR.sup.b; N(R.sup.a)SO.sub.2R.sup.b; NR.sup.aC(O)OR.sup.b; NR.sup.aR.sup.b; NR.sup.aC(O)R.sup.b; NR.sup.aC(S)R.sup.b; SONR.sup.aR.sup.b; SO.sub.2NR.sup.aR.sup.b; OR.sup.a; OR.sup.aC(O)OR.sup.b; OC(O); OC(O)R.sup.a; R.sup.aNR.sup.bR.sup.c; R.sup.aOR.sup.b; SR.sup.a; SOR.sup.a and SO.sub.2R.sup.a; each of R.sup.a, R.sup.b and R.sup.c are independently a hydrogen atom or an optionally substituted alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heteroaryl or heteroarylalkyl; wherein the substituents are further optionally substituted by one or more halogen, hydroxy, nitro, cycloalkyl, cyano, azido, nitroso, amino, hydrazino, formyl, alkyl, alkoxy, oxo, aryl, alkoxyamino- or haloalkyl; wherein when the group R.sub.1 is cyclic, R.sub.1 is a residue formed by removing a hydrogen atom from a saturated or unsaturated monocyclic carbocyclic ring or a saturated or unsaturated monocyclic heterocyclic ring having one or two or three heteroatoms selected from a nitrogen atom, an oxygen atom and a sulfur atom in the ring, and a saturated or unsaturated monocyclic 3, 4, 5, 6, 7 or 8 membered heterocyclic ring having 3 to 5 carbon atoms, or a bicyclic or tricyclic condensed polycyclic heterocyclic ring having one or two or three heteroatoms in the ring, and a bicyclic or tricyclic condensed polycyclic heterocyclic ring having 7 to 13 carbon atoms, wherein the rings are optionally substituted with at least one substituent; wherein R.sub.2 is hydrogen or alkyl; wherein R.sub.3 is NR.sup.dR.sup.e; each of R.sup.d and R.sup.e are independently selected from the group consisting of hydrogen or optionally substituted alkyl, alkenyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heteroaryl or heteroarylalkyl; or R.sup.d and R.sup.e together with the N, form an optionally substituted heterocyclic or heteroaryl ring; wherein the substituents are further optionally substituted by one or more halogen, hydroxy, nitro, cycloalkyl, cyano, azido, nitroso, amino, hydrazino, formyl, alkyl, aryl, heterocyclyl, alkoxy, mercapto, alkylthio, alkylcarbonyl, carboxy, alkxoycarbonyl, alkoxycarbonyloxy, oxo, or haloalkyl; wherein these further substituents are in turn further optionally substituted by one or more halogen, hydroxy, nitro, cycloalkyl, cyano, azido, nitroso, amino, hydrazino, formyl, alkyl, or haloalkyl; their derivatives, analogs, tautomeric forms, stereoisomers, polymorphs, hydrates, solvates, intermediates, pharmaceutically acceptable salts, pharmaceutical compositions, metabolites and prodrugs thereof; (b) detecting a signal from the compound, which is indicative of the presence of insulin in an extracellular environment of the test cells to obtain a first value; (c) comparing the first value with a control value, wherein the control value corresponds to a signal in the extracellular environment of the control cells, and the control value is determined from control cells which are the same cells as cultured in (a), and which are cultured under the same conditions as in (a), except that the control cells are not cultured under the condition or stimulus to be assessed, wherein if the test value is smaller or greater than the control value, then the condition or stimulus affects secretion of insulin in the cells.

30. The method of claim 29, wherein the condition or stimulus to be assessed is selected from the group consisting of one or more of pH, temperature, pressure, medium composition comprising serum, salts, vitamins, hormones, proteins, carbohydrates, lipids and chemical compounds, age and origin of the culture, atmosphere composition comprising CO.sub.2 and O.sub.2 concentration, cell culture support comprising two-dimensional culture, three-dimensional scaffold, and suspension culture.

31. The method of claim 29, wherein detecting as provided in step (c) is real time detection.

32. The method of claim 29, wherein the compound is represented by a formula ##STR00145##

Description

BRIEF DESCRIPTION OF DRAWINGS

[0164] The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

[0165] FIG. 1a is a graph showing the fluorescence response of Insulin Green to different concentrations of insulin.

[0166] FIG. 1b is a graph showing the interactions between Insulin Green and insulin.

[0167] FIG. 1c is a graph showing the selectivity of Insulin Green to insulin over bovine serum albumin (BSA), human serum albumin (HSA) and HSA lipid free.

[0168] FIG. 2 is a graph showing the quantification of insulin concentration released from cultured beta cells.

[0169] FIG. 3a is a graph showing the fluorescence response of Insulin Green with cultured beta cells challenged with four different conditions: Series 1: Glucose concentration 2.8 mM was used as control; Series 2: 16.7 mM glucose and 1 M of forskoline to induce insulin release; Series 3: Insulin release was further stimulated with 16.7 mM glucose, 5 M forskoline and 100 M IBMX; Series 4: Adrenaline was pre-incubated with the cells for 5 min to inhibit insulin secretion upon the treatment of series 3.

[0170] FIG. 3b is a graph showing the fluorescence response of Insulin Green with isolated islets challenged with four different conditions: Series 1: Glucose concentration 2.8 mM was used as control; Series 2: 16.7 mM glucose and 1 M of forskoline to induce insulin release; Series 3: Insulin release was further stimulated with 16.7 mM glucose, 5 M forskoline and 100 M IBMX; Series 4: Adrenaline was pre-incubated with the cells for 5 min to inhibit insulin secretion upon the treatment of series 3.

[0171] FIG. 4 shows the structure of Formula (I).

EXAMPLES

[0172] Non-limiting examples of the invention will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

1. Materials and Methods

[0173] All the reagents and solvents were purchased from Aldrich, Alfa and Acros organics and used without further purification. All reactions were performed in oven-dried glassware under a positive pressure of nitrogen. Analytical TLC was carried out on Merck 60 F254 silica gel plate (0.25 mm layer thickness) and visualization was done with UV light. Column chromatography was performed on Merck 60 silica gel (230-400 mesh). NMR spectra were recorded on a Bruker Avance 300 NMR spectrometer. Chemical shifts are reported as 6 in units of parts per million (ppm) and coupling constants are reported as a J value in Hertz (Hz). Mass of all the compounds was determined by LC-MS of Agilent Technologies with an electrospray ionization source. Spectroscopic measurements were performed on a fluorometer and UV/VIS instrument, Synergy 4 of bioteck company and Gemini XS fluorescence plate reader. The slit width was 1 nm for both excitation and emission.

1a. Cell Culture

[0174] Beta TC6 cell was cultured in high-glucose (4500 mg/L) contained Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 g/ml streptomycin, and incubated at 37 C. with 5% CO2. Cells were plated on plates 24-36 hours prior to experiment.

1b. Isolation of Islets

[0175] Extracted pancreas is immediately transferred into a vial containing cold Krebs-Ringer-Hepes (KRH) medium (containing, mM: 130 NaCl, 4.7 KCl, 1.2 KH.sub.2PO.sub.4, 1.2 MgSO.sub.4, and 2.56 CaCl.sub.2, 1 mg/mL BSA, 20 mM HEPES, pH 7.4) and cut into smaller pieces. 0.5 mg/mL of Collagenase-P (Roche-applied-science) was added and placed into a 37 C. water bath shaker (180-200 rpm/min) for 15-20 min. After digestion, cold KRH buffer with 0.1% BSA was added and inverted several times. The suspension was washed away after allowing islets to settle to the bottom of the vial for 10 mins and this wash process was repeated twice more.

Example 1: Calcone-Urea (CLU) Library

[0176] The present invention includes the synthesis of fluorescent chalcone-urea library (CLU), which was prepared on solid support. The CLU library was screened against four different concentrations of insulin (0.2, 0.1, 0.05 and 0.025 mg/mL) in HEPES buffer (20 mM, pH=7.4). Based on fluorescent intensity, 5 hits with more than 50-fold increase were selected (Table 2). CLU-381 was further derivatized due to the synthetic convenience to render 8 derivatives (Table 3). These 8 compounds were then tested against insulin to identify CLU-381-L257 (Insulin Green) as the most responsive compound.

##STR00016##

TABLE-US-00003 TABLE 2 CLU compound R.sub.1 CLU-C5 [00017] CLU-C10 [00018] CLU-D5 [00019] CLU-D11 [00020] CLU-F2 [00021]

1.1: Synthesis of Compound 1

[0177] ##STR00022##

[0178] To a solution of 4-Nitroacetophenone (100 mg, 0.6 mmole) and N,N2-4-((2-hydroxyethyl)(methyl)amino)benzaldehyde (100 mg, 0.55 mmole) in EtOH, pyrrolidine 0.1 mL was added and heat up with commercial Microwave Reactor (150 MW) for 3 min. Resulted. solution was cooled down and kept at the room temperature for 2 hrs to precipitate dark red solid. Solid was filtered and washed with ethylacetate and hexane (1:1) solution and dried as a dark red solid (100 mg, 60%). Obtained solid was used for further reaction without any purification. 1H-NMR (DMSO-d6) 8.34 (m, 4H), 7.71 (m, 4H), 6.75 (d, J=9, 2H), 4.75 (bt, 1H), 3.57 (bt, 2H), 3.49 (bt, 2H), 3.03 (s, 3H); 13C-NMR (CDCl.sub.3) 187.4, 151.6, 149.4, 146.9, 143.5, 131.4, 129.5, 128.3, 125.4, 123.7, 121.3, 115.3, 111.5, 58.2, 53.9, ESI-MS m/z (M+H) calc'd: 323.1, found 323.0.

1.2: Synthesis of Compound 2

[0179] ##STR00023##

[0180] 2-ChloroTrityl Resin (200 mg, 1 mmole/g) was pre-swelled in DCM. To the Resin solution compound 1 (100 mg) and 3 eq. of pyridine were added and kept for overnight. The reaction mixture was filtered and washed with DMF, MeOH, DCM after 1 hr MeOH capping procedure for extra resin chloride deactivation. Washed resin was dried with high vacuum dessicator and used for further reaction.

1.3: Synthesis of Compound 5

[0181] ##STR00024##

[0182] Resin loaded compound 2 was treated with SnCl.sub.2 hydrate eq. in DMF solution. Reaction mixture was shaken overnight and washed with DMF, MeOH, DCM. DCM swelled resin was placed in 50 mL tube and trichloroehtyl chlorocarbonate (3 eq) was added with DIEA (3 eq) at room temperature. After shaking for 2 hours the reaction mixture was washed with DMF, MeOH, DCM.

[0183] After high vacuum drying, the resin was divided by 60 mg each in the 5 mL syringe. The reaction syringes were washed with DMF and amines (3 eq) in DMF 3 mL was loaded in syringe and shake at 60 C. for 3 hrs. After reaction the reaction syringes were washed out with DCM, MeOH, DCM 3 times each.

1.4: Cleavage Protocol

[0184] 2% TFA in DCM solution was loaded for cleavage. For 30 min, incubated with cleavage solution, aliquot was squeezed out and collected in 20 mL vial. 2.5% ammonia water in ACN was added for TFA neutralization and filter with silica end filled Tip. Dark brown solid was obtained after removing solvent and purity was analyzed by LC/MS instrument. LC/MS gradient condition was 5% ACN to 100% ACN in water with 4.350 mm C18 column. All HPLC solvent contains 0.1% formic acid for the LC/MS analysis.

Example 2: CLU-381 Derivatives

[0185] ##STR00025##

Scheme 2: General Synthetic Scheme for CLU-381 Derivatives

[0186] To an EtOH soln. (40 ml) of the acetophenone (1 mmol each) and a mixture of the aldehyde (1.5 mmol) was added a solution of aq. 4 N NaOH (0.5 mL). The mixture was exposed to microwave irradiation until EtOH is dried. The mass obtained was cooled and neutralized with cold dil. aq. HCl. The chalcones were successively extracted with AcOEt (50 mL) and purified by column chromatography in the solvent system of MeOH (0 to 5%) in DCM.

2.1: Synthesis of Compound 6

[0187] ##STR00026##

[0188] In the 50 mL of round bottomed flask, 4-aminoacetophenon was dissolved in DCM with DIEA, at 0 C. the trichloroethoxychloroformate was added slowly. After reaction completion reaction mixture was concentrated and redissolved with DMF, p-methoxybenzyl amine (3 eq) addition was followed and heat to 70 C. for 3 hrs. Reaction mixture was concentrated with vacuum and purified with column chromatography on silica. Reaction yield was 90%. 1H-NMR (CDCl.sub.3+CD3OD) 7.80 (d, J=8.4, 2H), 7.42 (d, J=8.4, 2H), 7.18 (d, J=8.1, 2H), 6.80 d, J=8.1, 2H), 4.29 (s, 2H), 3.73 (s, 3H), 2.49 (s, 3H); 13C-NMR (CDCl3+CD3OD) 187.4, 151.6, 149.4, 146.9, 143.5, 131.4, 129.5, 128.3, 125.4, 123.7, 121.3, 115.3, 111.5, 58.2, 53.9.

TABLE-US-00004 TABLE 3 CLU381 Compound derivative R.sub.2 structure CLU-F2-L2 [00027] [00028] CLU-F2-L3 [00029] [00030] CLU-F2-L107 [00031] [00032] CLU-F2-L114 [00033] [00034] CLU-F2-L257 [00035] [00036] CLU-F2-L499 [00037] [00038] CLU-F2-L501 [00039] [00040] CLU-F2-Ac [00041] [00042]

TABLE-US-00005 TABLE 4 CLU381 Compound derivative structure CLU-F2-L2 [00043] CLU-F2-L3 [00044] CLU-F2-L107 [00045] CLU-F2-L114 [00046] CLU-F2-L257 [00047] CLU-F2-L499 [00048] CLU-F2-L501 [00049] CLU-F2-Ac [00050]

Example 3: R.SUB.1.-Amine Building Blocks Used in the Synthesis of CLU Library

[0189]

TABLE-US-00006 [00051] Code Structure Amine-6 [00052] Amine-11 [00053] Amine-25 [00054] Amine-28 [00055] Amine-33 [00056] Amine-48 [00057] Amine-77 [00058] Amine-78 [00059] Amine-89 [00060] Amine-92 [00061] Amine-95 [00062] Amine-100 [00063] Amine-101 [00064] Amine-102 [00065] Amine-107 [00066] Amine-111 [00067] Amine-124 [00068] Amine-131 [00069] Amine-164 [00070] Amine-165 [00071] Amine-167 [00072] Amine-177 [00073] Amine-181 [00074] Amine-184 [00075] Amine-193 [00076] Amine-201 [00077] Amine-216 [00078] Amine-218 [00079] Amine-220 [00080] Amine-221 [00081] Amine-222 [00082] Amine-225 [00083] Amine-262 [00084] Amine-266 [00085] Amine-267 [00086] Amine-272 [00087] Amine-273 [00088] Amine-274 [00089] Amine-282 [00090] Amine-319 [00091] Amine-335 [00092] Amine-346 [00093] Amine-347 [00094] Amine-351 [00095] Amine-356 [00096] Amine-364 [00097] Amine-370 [00098] Amine-373 [00099] Amine-374 [00100] Amine-376 [00101] Amine-381 [00102] Amine-382 [00103] Amine-384 [00104] Amine-395 [00105] Amine-396 [00106] Amine-397 [00107] Amine-404 [00108] Amine-405 [00109] Amine-414 [00110] Amine-417 [00111] Amine-419 [00112] Amine-420 [00113] Amine-427 [00114] Amine-434 [00115] Amine-442 [00116] Amine-446 [00117] Amine-454 [00118] Amine-477 [00119] Amine-478 [00120] Amine-480 [00121] Amine-487 [00122] Amine-526 [00123] Amine-574 [00124] Amine-599 [00125] Amine-615 [00126] Amine-616 [00127] Amine-618 [00128] Amine-677 [00129] Amine-N154 [00130] Amine-N154A [00131]

Example 4: Insulin Green

[0190] ##STR00132##

[0191] 1H-NMR (CDCl3+CD3OD) 7.84 (d, J=8.7, 2H), 7.63 (d, J=15.4, 1H), 7.42 (m, 4H), 7.25 (d, J=15.4, 1H), 7.14 (d, J=8.7, 2H), 6.76 (d, J=8.7, 2H), 6.62 (d, J=8.8, 2H), 4.25 (s, 2H), 3.72 (t, J=5.2, 4H), 3.68 (s, 3H), 3.52 (t, J=5.1, 4H), 13C-NMR (CDCl.sub.3+CD.sub.3OD) 189.8, 149.8, 145.2, 144.0, 130.3, 129.6, 128.5, 117.2, 113.7, 111.8, 59.6, 55.0, 54.5, 42.9, 29.4.

[0192] Insulin Green showed a 94-fold fluorescent increase to 200 g/mL insulin (FIG. 1a). The job plot reveals that a maximum fluorescence increase was observed for 1:1 insulin: Insulin Green ratio (FIG. 1b). Furthermore, Insulin Green also showed good selectivity over other proteins, such as the most abundant protein in blood (i.e., serum albumin) (FIG. 1c).

[0193] The good sensitivity and selectivity of Insulin Green displays its potential in a wide variety of applications.

[0194] Insulin quantification in cell culture medium was explored using Insulin Green. KCl is known to induce the insulin release from both beta cell and isolated islet. After beta cell was plated on 6-well plates, the cells were pre-incubated for 30 min in 2.8 mM glucose and then challenged with 50 mM KCl. The media was collected at 2 min intervals and measured with both Insulin Green and enzyme-linked immunosorbent assay (ELISA). The results correlate well with each other, indicating that Insulin Green can provide a fast way for insulin quantification (FIG. 2). However, the real time dynamics are still beyond observation due to time averaging.

[0195] Therefore, Insulin Green was applied to the real-time measurement of insulin secretion from both cultured cells and isolated islets. Forskolin and 3-isobutyl-1-methylxanthine (IBMX) are known to be able to stimulate insulin release, while adrenaline can inhibits insulin secretion. After beta cells were plated on 96-well plates, cell were pre-incubated for 30 min in 2.8 mM glucose and treated with 10 M Insulin Green. To test whether Insulin Green can respond the dynamic insulin concentration change in real time, the cells were further challenged with four different conditions. With reference to FIGS. 3a and 3b, the glucose concentration remaining as 2.8 mM was used as control (series 1). The cells were challenged with 16.7 mM glucose and 1 M of forskoline to induce insulin release (series 2). The insulin release was further stimulated with 16.7 mM glucose, 5 M forskoline and 100 M IBMX (series 3). Adrenaline was pre-incubated to the cells for min to inhibit insulin secretion upon the treatment of series 3 (series 4). After the cells were challenged with four different conditions, the 96-well plates were immediately applied to a fluorescent plate reader to monitor the fluorescence change of Insulin Green at 20 seconds intervals. The fluorescence changes of Insulin Green showed clear differences with different conditions and correlated well with reported values. Similar experiments were also performed on isolated islets. Insulin Green showed similar response to the four different conditions. These results indicate that Insulin Green can measure the insulin secretion from both cultured cells and isolated islets in real time.

Applications

[0196] The disclosed compounds may be useful as insulin sensors.

[0197] The disclosed compounds may display good sensitivity and selectivity for insulin.

[0198] The disclosed compounds may be able to detect insulin rapidly and directly.

[0199] The disclosed compounds may display sensitive fluorescent intensity enhancement to insulin.

[0200] The disclosed methods may allow for fast detection of insulin and may display low detection limits.

[0201] The disclosed methods may provide real time monitoring of insulin secretion in both cultured cells and isolated islets.

[0202] The disclosed methods may not require labelling insulin with expensive antibodies, fluorescent tags or radioisotopes. Therefore, the disclosed methods may be cost-effective.

[0203] The disclosed methods may be simple to perform and may not require time consuming preparation steps.

[0204] The disclosed methods may provide for fast insulin detection and insulin secretion study in both cultured cells and isolated islets.

[0205] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.