AZOARYLS AS REVERSIBLY MODULATABLE TUBULIN INHIBITORS

Abstract

The invention concerns a new class of tubulin polymerisation inhibitors and their applications in research and medicine, notably in chemotherapy. The invention proposes new azoaryl derivatives of formula (I):

##STR00001##

as defined in Claim 1, which may be fully reversibly interconverted between non-tubulin-binding trans and tubulin-binding as isomeric forms, either by irradiation or spontaneously. The invention also concerns compounds with a azoaryl structure for use in studying the cytoskeleton and/or its associated processes, or in the treatment of a disease for which a tubulin polymerisation inhibition activity has a beneficial effect, wherein the compound is administered to the cell, organism or patient in need of such treatment in the trans form of the diazenyl bond, and where this trans form is inactive as regards a tubulin polymerisation inhibition effect, and where after photoisomerisation in vitro, in cellulo or in vivo to an azoaryl compound in its cis isomeric form of the diazenyl bond by the application of light, optionally with modification in vitro, in cellulo or in vivo of one or more substituents, the resultant cis form is active as regards a tubulin polymerisation inhibition effect.

Claims

1-55. (canceled)

56. Compounds corresponding to one of the following formulae: ##STR00069## wherein: the aryl ring bearing R.sub.3 is denoted the south ring, dotted lines indicate sites where a fused ring may be present; R.sub.2 is chosen among OCH.sub.3, OCF.sub.3, F, CH.sub.3, CF.sub.3, CH.sub.2CH.sub.3, OCH.sub.2CH.sub.3, SCH.sub.3, SCF.sub.3, NHCH.sub.3, N(CH.sub.3).sub.2 and CN; and R.sub.1, R.sub.6 and R.sub.7 are defined as follows: R.sub.1 is chosen among hydrogen, Y.sub.1R.sub.a, S.sub.2R.sub.b, NHR.sub.d, OR.sub.e, OPO.sub.3H.sub.2, NO.sub.2, B(OH).sub.2, B(OR.sub.b).sub.2, B(OR.sub.bO), N.sub.3, F, Cl, Br, I, CHO, CO.sub.2H, CONH.sub.2, CN, NC, SO.sub.3H, CO.sub.2R.sub.b, SO.sub.2NH.sub.2 and R.sub.b; R.sub.7, and when R.sub.6 is not linked to R.sub.5, R.sub.6 also, identical or different, are chosen among hydrogen, Y.sub.2R.sub.f, S.sub.2R.sub.g, NHR.sub.i, OR.sub.j, OPO.sub.3H.sub.2, NO.sub.2, B(OH).sub.2, B(OR.sub.g).sub.2, B(OR.sub.gO), N.sub.3, F, Cl, Br, I, CHO, CO.sub.2H, CONH.sub.2, CN, NC, SO.sub.3H, CO.sub.2R.sub.g, SO.sub.2NH.sub.2, R.sub.g, CO.sub.2NHR.sub.g, CO.sub.2NR.sub.gR.sub.h, N-piperazinyl, N-morpholinyl, N-pyrrolidinyl, N-piperidinyl, and -linker-reporter units; and when R.sub.6 is linked to R.sub.5 then they are linked together forming a fused phenyl, 2-pyridinyl or 3-pyridinyl ring, the said phenyl, 2-pyridinyl or 3-pyridinyl being unsubstituted or substituted with one or several groups R.sub.n, identical or different, such that the south ring is respectively a naphthalene, quinoline or isoquinoline; considering that at least one of the substituents R.sub.6, R.sub.7 and R.sub.1 is different from hydrogen; and R.sub.3 is chosen among OCH.sub.3, OCF.sub.3, F, CH.sub.3, CF.sub.3, CH.sub.2CH.sub.3, OCH.sub.2CH.sub.3, SCH.sub.3, SCF.sub.3, NHCH.sub.3, N(CH.sub.3).sub.2 and CN; R.sub.4 and, when R.sub.6 is not linked to R.sub.5, R.sub.5 also, identical or different, are chosen among OCH.sub.3, OCF.sub.3, F, CH.sub.3, CF.sub.3, CH.sub.2CH.sub.3, OCH.sub.2CH.sub.3, SCH.sub.3, SCF.sub.3, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2 and CN; Y.sub.1O, S, NH or NR.sub.k; Y.sub.2O, S, NH or NR.sub.1; R.sub.a is chosen among hydrogen, R.sub.b, COR.sub.b, CO.sub.2R.sub.b, CONH.sub.2, CONR.sub.bR.sub.c, CONHR.sub.b, CH.sub.2OC(O)R.sub.b, and cleavable groups which after cleavage, for instance in vivo, lead either to R.sub.1OH when Y.sub.1O, or to R.sub.1NH.sub.2 when Y.sub.1NH, or to R.sub.1NHR.sub.k when Y.sub.1NR.sub.k, or to R.sub.1SH when Y.sub.1S; R.sub.b, R.sub.c, R.sub.g, R.sub.h, R.sub.k and R.sub.l, identical or different, are chosen among (C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkyl-OH, (C.sub.1-C.sub.6)alkenyl, (C.sub.1-C.sub.6)alkynyl, (C.sub.3-C.sub.7)cycloalkyl, aryl, heteroaryl, heterocycle, (C.sub.1-C.sub.6)alkyl(C.sub.3-C.sub.7)cycloalkyl, (C.sub.1-C.sub.6)alkylaryl, (C.sub.1-C.sub.6)alkylheteroaryl, and (C.sub.1-C.sub.6)alkylheterocycle; R.sub.d and R.sub.i are identical or different, and are a peptidic group attached via its carboxyl terminus; R.sub.e and R.sub.j are identical or different, and are a glycosidyl group; R.sub.f is chosen among hydrogen, R.sub.g, COR.sub.g, CO.sub.2R.sub.g, CONH.sub.2, CONR.sub.gR.sub.h, CONHR.sub.g, OCH.sub.2OC(O)R.sub.g, and cleavable groups which after, for instance in vivo, lead either to R.sub.6 or R.sub.7OH when Y.sub.2O, to R.sub.6 or R.sub.7NH.sub.2 when Y.sub.2NH, or to R.sub.6 or R.sub.7NHR.sub.1 when Y.sub.2NR.sub.1, or to R.sub.6 or R.sub.7SH when Y.sub.2S; R.sub.n is chosen among CH.sub.3, OH, NH.sub.2, NHCOCH.sub.3, SO.sub.3H, CO.sub.2H, CONH.sub.2, CO.sub.2CH.sub.3, PO.sub.3H.sub.2, NO.sub.2, B(OH).sub.2, N.sub.3, CN, CCH, and SO.sub.2NH.sub.2; and their hydrates, pharmaceutically acceptable salts and solvates, as a mixture of isomers in any proportions and also as pure isomer.

57. Compounds according to claim 56, wherein R.sub.1Y.sub.1R.sub.a, with Y.sub.1O, NH or S and R.sub.a being as defined in claim 56.

58. Compounds according to claim 56, wherein R.sub.1 is chosen among OH, NH.sub.2 or SH.

59. Compounds according to claim 56, wherein R.sub.7Y.sub.2R.sub.f, Y.sub.2O, NH or S and R.sub.f is as defined in claim 1 and/or R.sub.6Y.sub.2R.sub.f, Y.sub.2O, NH or S, and R.sub.f is as defined in claim 56.

60. Compounds according to claim 56, wherein R.sub.1NH-peptidic group and/or R.sub.7NH-peptidic group and/or R.sub.6 is a NH-peptidic group.

61. Compounds according to claim 56, wherein R.sub.6 is a linker-reporter unit -Link1-Rep1 and/or R.sub.7 is a linker-reporter unit -Link2-Rep2 where: the reporter Rep1 and Rep2, identical or different, are chosen among fluorophores, chromophores, antennas and tag moieties, and especially among fluorescein, rhodamine, coumarin, phenoxazine, acridine, boron-dipyrromethene, dansyl, propidium, nitrobenzofurazan, resorufin, cyanine, Cascade Yellow, Nile Red, carborhodamine, silarhodamine, DABCYL, black hole quencher moieties, (E)-4,4-bis(diethylamino)stilbene, biotin, and tag protein substrates, especially O.sup.6-benzylguanine, O.sup.2-benzylcytosine or ((CH.sub.2).sub.2O).sub.2(CH.sub.2).sub.6Cl, and their derivatives, and the linker Link1 and Link2, identical or different, are chosen among bivalent (C.sub.1-C.sub.12)alkyl; bivalent (C.sub.1-C.sub.12)alkenyl; (CH.sub.2).sub.m1(C.sub.3-C.sub.7)cycloalkyl(CH.sub.2).sub.m2, (CH.sub.2).sub.m1aryl(CH.sub.2).sub.m2; moieties including between 1 to 10 carbon atoms and 1 to 6 heteroatoms chosen from among oxygen, nitrogen and sulfur, such as (CH.sub.2).sub.m1heteroaryl(CH.sub.2).sub.m2 especially when heteroaryl is a triazole, tetrazole or pyridazine, (CH.sub.2).sub.m1heterocycle(CH.sub.2).sub.m2, oligo(ethyleneglycol), (CH.sub.2).sub.m1C(O)O(CH.sub.2).sub.m2, (CH.sub.2).sub.m1C(O)NH(CH.sub.2).sub.m2, C(O), (CH.sub.2).sub.m1SS(CH.sub.2).sub.m2, (CH.sub.2).sub.m1N-succinimide-3-S(CH.sub.2).sub.m2, C(O)-(4-cyclohexyl)-CH.sub.2N-succinimide-3-S(CH.sub.2).sub.m2 and (CH.sub.2).sub.m1SCH.sub.2C(O)(CH.sub.2).sub.m2 with m1 and m2, identical or different, being integers chosen in the range 0 to 6.

62. Compounds according to claim 56, wherein R.sub.6 and R.sub.7 are chosen among H, F, Cl, NO.sub.2, NHCOCH.sub.3, N(CH.sub.3).sub.2, and OCH.sub.3.

63. Compounds according to claim 56, wherein R.sub.6H.

64. Compounds according to claim 56, wherein R.sub.2 and R.sub.3 are chosen separately among OCH.sub.3, OCF.sub.3, F, CH.sub.3, CF.sub.3, CH.sub.2CH.sub.3 and OCH.sub.2CH.sub.3.

65. Compounds according to claim 56, wherein R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are chosen separately among OCH.sub.3, OCF.sub.3, F, CH.sub.3, CF.sub.3, CH.sub.2CH.sub.3 and OCH.sub.2CH.sub.3.

66. Compounds according to claim 56, wherein R.sub.2R.sub.3R.sub.4R.sub.5OCH.sub.3.

67. Compounds according to claim 56, wherein R.sub.2R.sub.3R.sub.4R.sub.5OCH.sub.3; R.sub.6 and R.sub.7 are chosen among H, F, Cl, NO.sub.2, NHCOCH.sub.3, N(CH.sub.3).sub.2, and OCH.sub.3.

68. Compounds according to claim 56 chosen among: 2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenol (I.1): ##STR00070## 2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)aniline (I.2): ##STR00071## 1-(3-fluoro-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.3): ##STR00072## 1-(2-fluoro-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.4): ##STR00073## 1-(2,3-difluoro-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.5): ##STR00074## 1-(2-fluoro-4-methoxy-3-nitrophenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.7): ##STR00075## 5-methoxy-2-((3,4,5-trimethoxyphenyl)diazenyl)phenol (I.8): ##STR00076## (2-methoxy 5-((3,4,5-trimethoxyphenyl)diazenyl)phenyl) 2-((L-leucinamido)methyl)piperidine 1-carboxylate 2,2,2-trifluoroacetate salt (I.10): ##STR00077## and its free form, 1-(3,4-dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.11): ##STR00078## 1-(2,4-dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.12): ##STR00079## 5-methoxy-2-((3,4,5-trimethoxyphenyl)diazenyl)aniline (I.16): ##STR00080## 3-methoxy-2-methyl-6-((3,4,5-trimethoxyphenyl)diazenyl)phenol (I.17): ##STR00081## 2-(3-methoxy-2-methyl-6-((3,4,5-trimethoxyphenyl)diazenyl)phenoxy)ethan-1-ol (I.18): ##STR00082## 2-(5-methoxy-2-((3,4,5-trimethoxyphenyl)diazenyl)phenoxy)ethan-1-ol (I.19): ##STR00083## (2-methoxy 5-((3,4,5-trimethoxyphenyl)diazenyl)phenyl)) 1-L-serinamide 2,2,2-trifluoroacetate salt (I.20): ##STR00084## and its free form, 3-(3-methoxy-2-methyl-6-((3,4,5-trimethoxyphenyl)diazenyl)phenoxy)propan-1-ol (I.21): ##STR00085## 2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenyl phosphate disodium salt (I.24): ##STR00086## and its free form; N-(6-(diethylamino)-9-(2-(4-(3-(3-methoxy-2-methyl-6-((3,4,5-trimethoxyphenyl)diazenyl)phenoxy)propyl)piperazine-1-carbonyl)phenyl)-3H-xanthen-3-ylidene)-N-ethylethanaminium bis(formate) salt (I.25): ##STR00087## N-(5-methoxy-2-((3,4,5-trimethoxyphenyl)diazenyl)phenyl)acetamide (I.26): ##STR00088## 1-(3-bromo-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.27): ##STR00089## 2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)benzaldehyde (I.28): ##STR00090## and 2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)benzoic acid (I.29): ##STR00091## as a mixture of cis and trans isomers in any proportions and also as a pure isomer either cis or trans, and their hydrates, pharmaceutically acceptable salts and solvates.

69. Compounds according to claim 56 chosen among: 5-((3,5-dimethoxy-4-(trifluoromethoxy)phenyl)diazenyl)-2-(trifluoromethoxy)phenol; 2-(trifluoromethoxy)-5-((3,4,5-trimethoxyphenyl)diazenyl)phenol; 5-((3,5-dimethoxy-4-(trifluoromethoxy)phenyl)diazenyl)-2-methoxyphenol; 2-fluoro-6-methoxy-3-((3,4,5-trimethoxyphenyl)diazenyl)aniline; N-(2-hydroxy-3-methoxy-6-((3,4,5-trimethoxyphenyl)diazenyl)phenyl)acetamide; 5-((3,5-dimethoxy-4-(trifluoromethoxy)phenyl)diazenyl)-2-(trifluoromethoxy)phenyl dihydrogen phosphate; 2-(trifluoromethoxy)-5-((3,4,5-trimethoxyphenyl)diazenyl)phenyl dihydrogen phosphate and 5-((3,5-dimethoxy-4-(trifluoromethoxy)phenyl)diazenyl)-2-methoxyphenyl dihydrogen phosphate; as a mixture of cis and trans isomers in any proportions and also as a pure isomer either cis or trans, and their hydrates, pharmaceutically acceptable salts and solvates.

70. Compounds according to claim 56 for their use as a medicament, and in particular as an anti-mitotic, anti-angiogenic, antitumoral or chemotherapeutic agent.

71. Compounds according to claim 56 for their use in the treatment of a disease for which the administration of a compound with antitubulin activity has a beneficial effect.

72. Compounds according to claim 56 for their use in the treatment of a cancer, such as melanoma, adenocarcinoma of the lung, neuroblastoma, small cell carcinoma of the lung, breast carcinoma, colon carcinoma, ovarian carcinoma, or bladder carcinoma, or of a disease characterized by abnormal vascularisation such as diabetic retinopathy, psoriasis or endometriosis, or of rheumatoid arthritis or atherosclerosis.

73. A pharmaceutical composition comprising a compound according to claim 56 with at least one pharmaceutically acceptable excipient.

74. A compound with an azoaryl structure for use in the treatment of a disease for which a tubulin polymerisation inhibitor activity has a beneficial effect, wherein the compound is administered to the patient in need of such treatment, at least partially in its trans isomeric form of the diazenyl bond, and where this trans form is inactive as regards a tubulin polymerisation inhibitory effect, and where the compound is photoisomerised in vivo to an azoaryl compound in its cis isomeric form of the diazenyl bond by the application of light, with optional modification in vivo of one or more substituents either before or after this photoisomerisation, and where this resultant cis form is active as regards a tubulin polymerisation inhibitory effect.

75. A compound for use according to claim 74, wherein the azoaryl compound is administered: in its trans isomeric form of the diazenyl bond, and where its cis form is active as regards a tubulin polymerisation inhibitory effect; or in a mixture of cis and trans isomeric forms of the diazenyl bond, and where its cis form is active as regards a tubulin polymerisation inhibitory effect.

76. A compound for use according to claim 74, wherein the application of light is localized.

77. A compound for use according to claim 74, wherein the isomerisation in vivo of the diazenyl bond from trans to cis form is followed by a conversion of cis to trans form by spontaneous thermal reversion or by application of light.

78. A compound for use according to claim 77, wherein the isomerisation in vivo of the diazenyl bond from trans to cis form leads to an inactive form as regards a tubulin polymerisation inhibitory effect.

79. A compound for use according to claim 74, wherein the disease is a cancer, such as melanoma, adenocarcinoma of the lung, neuroblastoma, small cell carcinoma of the lung, breast carcinoma, colon carcinoma, ovarian carcinoma, or bladder carcinoma, or a disease characterized by abnormal vascularisation such as diabetic retinopathy, psoriasis or endometriosis, or rheumatoid arthritis or atherosclerosis.

80. A compound for use according to claim 74, wherein the compound is selected among the compounds: ##STR00092## wherein: the aryl ring bearing R.sub.3 is denoted the south ring, dotted lines indicate sites where a fused ring may be present; R.sub.2 is chosen among OCH.sub.3, OCF.sub.3, F, CH.sub.3, CF.sub.3, CH.sub.2CH.sub.3, OCH.sub.2CH.sub.3, SCH.sub.3, SCF.sub.3, NHCH.sub.3, N(CH.sub.3).sub.2 and CN; and R.sub.1, R.sub.6 and R.sub.7 are defined as follows: R.sub.1 is chosen among hydrogen, Y.sub.1R.sub.a, S.sub.2R.sub.b, NHR.sub.d, OR.sub.e, OPO.sub.3H.sub.2, NO.sub.2, B(OH).sub.2, B(OR.sub.b).sub.2, B(OR.sub.bO), N.sub.3, F, Cl, Br, I, CHO, CO.sub.2H, CONH.sub.2, CN, NC, SO.sub.3H, CO.sub.2R.sub.b, SO.sub.2NH.sub.2 and R.sub.b; R.sub.7, and when R.sub.6 is not linked to R.sub.5, R.sub.6 also, identical or different, are chosen among hydrogen, Y.sub.2R.sub.f, S.sub.2R.sub.g, NHR.sub.1, OR.sub.j, OPO.sub.3H.sub.2, NO.sub.2, B(OH).sub.2, B(OR.sub.g).sub.2, B(OR.sub.gO), N.sub.3, F, Cl, Br, I, CHO, CO.sub.2H, CONH.sub.2, CN, NC, SO.sub.3H, CO.sub.2R.sub.g, SO.sub.2NH.sub.2, R.sub.g, CO.sub.2NHR.sub.g, CO.sub.2NR.sub.gR.sub.h, N-piperazinyl, N-morpholinyl, N-pyrrolidinyl, N-piperidinyl, and -linker-reporter units; and when R.sub.6 is linked to R.sub.5 then they are linked together forming a fused phenyl, 2-pyridinyl or 3-pyridinyl ring, the said phenyl, 2-pyridinyl or 3-pyridinyl being unsubstituted or substituted with one or several groups R.sub.n, identical or different, such that the south ring is respectively a naphthalene, quinoline or isoquinoline; considering that at least one of the substituents R.sub.6, R.sub.7 and R.sub.1 is different from hydrogen; and R.sub.3 is chosen among OCH.sub.3, OCF.sub.3, F, CH.sub.3, CF.sub.3, CH.sub.2CH.sub.3, OCH.sub.2CH.sub.3, SCH.sub.3, SCF.sub.3, NHCH.sub.3, N(CH.sub.3).sub.2 and CN; R.sub.4 and, when R.sub.6 is not linked to R.sub.5, R.sub.5 also, identical or different, are chosen among OCH.sub.3, OCF.sub.3, F, CH.sub.3, CF.sub.3, CH.sub.2CH.sub.3, OCH.sub.2CH.sub.3, SCH.sub.3, SCF.sub.3, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2 and CN; Y.sub.1O, S, NH or NR.sub.k; Y.sub.2O, S, NH or NR.sub.l; R.sub.a is chosen among hydrogen, R.sub.b, COR.sub.b, CO.sub.2R.sub.b, CONH.sub.2, CONR.sub.bR.sub.c, CONHR.sub.b, CH.sub.2OC(O)R.sub.b, and cleavable groups which after cleavage, for instance in vivo, lead either to R.sub.1OH when Y.sub.1O, or to R.sub.1NH.sub.2 when Y.sub.1NH, or to R.sub.1NHR.sub.k when Y.sub.1NR.sub.k, or to R.sub.1SH when Y.sub.1S; R.sub.b, R.sub.c, R.sub.g, R.sub.h, R.sub.k and R.sub.l, identical or different, are chosen among (C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkyl-OH, (C.sub.1-C.sub.6)alkenyl, (C.sub.1-C.sub.6)alkynyl, (C.sub.3-C.sub.7)cycloalkyl, aryl, heteroaryl, heterocycle, (C.sub.1-C.sub.6)alkyl(C.sub.3-C.sub.7)cycloalkyl, (C.sub.1-C.sub.6)alkylaryl, (C.sub.1-C.sub.6)alkylheteroaryl, and (C.sub.1-C.sub.6)alkylheterocycle; R.sub.d and R.sub.i are identical or different, and are a peptidic group attached via its carboxyl terminus; R.sub.e and R.sub.j are identical or different, and are a glycosidyl group; R.sub.f is chosen among hydrogen, R.sub.g, COR.sub.g, CO.sub.2R.sub.g, CONH.sub.2, CONR.sub.gR.sub.h, CONHR.sub.g, OCH.sub.2OC(O)R.sub.g, and cleavable groups which after, for instance in vivo, lead either to R.sub.6 or R.sub.7OH when Y.sub.2O, to R.sub.6 or R.sub.7=NH.sub.2 when Y.sub.2NH, or to R.sub.6 or R.sub.7=NHR.sub.1 when Y.sub.2NR.sub.1, or to R.sub.6 or R.sub.7=SH when Y.sub.2S; R.sub.n is chosen among CH.sub.3, OH, NH.sub.2, NHCOCH.sub.3, SO.sub.3H, CO.sub.2H, CONH.sub.2, CO.sub.2CH.sub.3, PO.sub.3H.sub.2, NO.sub.2, B(OH).sub.2, N.sub.3, CN, CCH, and SO.sub.2NH.sub.2; and their hydrates, pharmaceutically acceptable salts and solvates, as a mixture of isomers in any proportions and also as pure isomer; wherein the compounds are in the trans form of the diazenyl bond, or in a mixture of cis and trans forms of the diazenyl bond.

81. A compound for use according to claim 74, wherein the azoaryl compound is an azobenzene compound.

82. A method of studying the cytoskeleton and/or its associated processes wherein cells or a sample are treated with an azoaryl compound, at least partially in its trans isomeric form of the diazenyl bond, which is inactive as regards a tubulin polymerisation inhibitory effect, and where the compound is photoisomerised in vitro to an azoaryl compound in its cis isomeric form of the diazenyl bond by the application of light, with optional modification in vitro of one or more substituents either before or after this photoisomerisation, and where this cis form is active as regards a tubulin polymerisation inhibitory effect.

83. A method according to claim 82, wherein: either, the azoaryl compound in its pure trans isomeric form of the diazenyl bond is the form of the compound used for treating the cells or the sample and where its cis form is active as regards a tubulin polymerisation inhibitory effect; or, the azoaryl compound in a mixture of its cis and trans isomeric forms of the diazenyl bond is the form of the compound used for treating the cells or the sample and where its cis form is active as regards a tubulin polymerisation inhibitory effect.

84. A method according to claim 82, wherein the application of light is localized.

85. A method according to claim 82, wherein the conversion from the trans to the cis form of the diazenyl bond is followed by its conversion from the cis to the trans form by spontaneous thermal reversion or by application of light with a wavelength able to isomerise the compound from its cis form to its trans form of the diazenyl bond.

86. A method according to claim 85, wherein the isomerisation of the diazenyl bond from the cis to the trans form leads to an inactive form as regards a tubulin polymerisation inhibitory effect.

87. A method according to claim 82, wherein the compound for treating the cells or sample is selected among the compounds: ##STR00093## wherein: the aryl ring bearing R.sub.3 is denoted the south ring, dotted lines indicate sites where a fused ring may be present; R.sub.2 is chosen among OCH.sub.3, OCF.sub.3, F, CH.sub.3, CF.sub.3, CH.sub.2CH.sub.3, OCH.sub.2CH.sub.3, SCH.sub.3, SCF.sub.3, NHCH.sub.3, N(CH.sub.3).sub.2 and CN; and R.sub.1, R.sub.6 and R.sub.7 are defined as follows: R.sub.1 is chosen among hydrogen, Y.sub.1R.sub.a, S.sub.2R.sub.b, NHR.sub.d, OR.sub.e, OPO.sub.3H.sub.2, NO.sub.2, B(OH).sub.2, B(OR.sub.b).sub.2, B(OR.sub.bO), N.sub.3, F, Cl, Br, I, CHO, CO.sub.2H, CONH.sub.2, CN, NC, SO.sub.3H, CO.sub.2R.sub.b, SO.sub.2NH.sub.2 and R.sub.b; R.sub.7, and when R.sub.6 is not linked to R.sub.5, R.sub.6 also, identical or different, are chosen among hydrogen, Y.sub.2R.sub.f, S.sub.2R.sub.g, NHR.sub.i, OR.sub.j, OPO.sub.3H.sub.2, NO.sub.2, B(OH).sub.2, B(OR.sub.g).sub.2, B(OR.sub.gO), N.sub.3, F, Cl, Br, I, CHO, CO.sub.2H, CONH.sub.2, CN, NC, SO.sub.3H, CO.sub.2R.sub.g, SO.sub.2NH.sub.2, R.sub.g, CO.sub.2NHR.sub.g, CO.sub.2NR.sub.gR.sub.h, N-piperazinyl, N-morpholinyl, N-pyrrolidinyl, N piperidinyl, and -linker-reporter units; and when R.sub.6 is linked to R.sub.5 then they are linked together forming a fused phenyl, 2-pyridinyl or 3-pyridinyl ring, the said phenyl, 2-pyridinyl or 3-pyridinyl being unsubstituted or substituted with one or several groups R.sub.n, identical or different, such that the south ring is respectively a naphthalene, quinoline or isoquinoline; considering that at least one of the substituents R.sub.6, R.sub.7 and R.sub.1 is different from hydrogen; and R.sub.3 is chosen among OCH.sub.3, OCF.sub.3, F, CH.sub.3, CF.sub.3, CH.sub.2CH.sub.3, OCH.sub.2CH.sub.3, SCH.sub.3, SCF.sub.3, NHCH.sub.3, N(CH.sub.3).sub.2 and CN; R.sub.4 and, when R.sub.6 is not linked to R.sub.5, R.sub.5 also, identical or different, are chosen among OCH.sub.3, OCF.sub.3, F, CH.sub.3, CF.sub.3, CH.sub.2CH.sub.3, OCH.sub.2CH.sub.3, SCH.sub.3, SCF.sub.3, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2 and CN; Y.sub.1O, S, NH or NR.sub.k; Y.sub.2O, S, NH or NR.sub.l; R.sub.a is chosen among hydrogen, R.sub.b, COR.sub.b, CO.sub.2R.sub.b, CONH.sub.2, CONR.sub.bR.sub.c, CONHR.sub.b, CH.sub.2OC(O)R.sub.b, and cleavable groups which after cleavage, for instance in vivo, lead either to R.sub.1OH when Y.sub.1O, or to R.sub.1NH.sub.2 when Y.sub.1NH, or to R.sub.1NHR.sub.k when Y.sub.1NR.sub.k, or to R.sub.1SH when Y.sub.1S; R.sub.b, R.sub.c, R.sub.g, R.sub.h, R.sub.k and R.sub.l, identical or different, are chosen among (C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkyl-OH, (C.sub.1-C.sub.6)alkenyl, (C.sub.1-C.sub.6)alkynyl, (C.sub.3-C.sub.7)cycloalkyl, aryl, heteroaryl, heterocycle, (C.sub.1-C.sub.6)alkyl(C.sub.3-C.sub.7)cycloalkyl, (C.sub.1-C.sub.6)alkylaryl, (C.sub.1-C.sub.6)alkylheteroaryl, and (C.sub.1-C.sub.6)alkylheterocycle; R.sub.d and R.sub.i are identical or different, and are a peptidic group attached via its carboxyl terminus; R.sub.e and R.sub.j are identical or different, and are a glycosidyl group; R.sub.f is chosen among hydrogen, R.sub.g, COR.sub.g, CO.sub.2R.sub.g, CONH.sub.2, CONR.sub.gR.sub.h, CONHR.sub.g, OCH.sub.2OC(O)R.sub.g, and cleavable groups which after, for instance in vivo, lead either to R.sub.6 or R.sub.7OH when Y.sub.2O, to R.sub.6 or R.sub.7NH.sub.2 when Y.sub.2NH, or to R.sub.6 or R.sub.7NHR.sub.1 when Y.sub.2NR.sub.1, or to R.sub.6 or R.sub.7SH when Y.sub.2S; R.sub.n is chosen among CH.sub.3, OH, NH.sub.2, NHCOCH.sub.3, SO.sub.3H, CO.sub.2H, CONH.sub.2, CO.sub.2CH.sub.3, PO.sub.3H.sub.2, NO.sub.2, B(OH).sub.2, N.sub.3, CN, CCH, and SO.sub.2NH.sub.2; and their hydrates, pharmaceutically acceptable salts and solvates, as a mixture of isomers in any proportions and also as pure isomer; wherein the compounds are in the trans form of the diazenyl bond, or in a mixture of cis and trans forms of the diazenyl bond.

88. A method according to claim 82, wherein the azoaryl compound is an azobenzene compound.

89. A method according to claim 82, wherein the compound is 1-(4-methoxynaphthalen-1-yl)-2-(3,4,5-trimethoxyphenyl)diazene or 1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)diazene in its trans form or in a mixture of its trans and cis forms.

Description

EXAMPLES

[0247] The syntheses and descriptions of biological tests hereinafter, with reference to the appended schemes and figures, illustrate the invention without, however, limiting it.

[0248] FIG. 1: Typical absorption spectra of trans (solid lines) and as (dotted lines) isomers of selected example compounds.

[0249] FIG. 2: typical () (dotted lines) and E() (solid lines) of selected example compounds.

[0250] FIG. 3: Raw absorbance values A(.sub.strong=378 nm) of a sample of I.1 (18 M in non-degassed PBS left open to the atmosphere, containing 10% MeCN, at 37 C.) measured over time, while being reversibly photoisomerised between majority-cis and majority-trans forms by alternating the irradiating wavelength between .sub.1=388 nm (50 s; bulk trans->cis) and .sub.2=508 nm (180 s, bulk cis->trans). Higher absorbance corresponds to a greater amount of trans isomer.

[0251] FIG. 4: Raw data from UV-Vis measurements of the absorbance of a sample of compound I.25 at 33 M in PBS containing 20% MeCN at 37 C. Top panel: absorbance spectra of the same sample photoisomerised to contain either 100%-trans (spectrum E-I.25, generated by irradiation at 554 nm) or a majority of as (spectrum Z-I.25, >3:1 ratio of cis:trans isomers, generated by irradiation at 384 nm). Bottom panel: A(.sub.strong=375 nm) measured while the irradiating wavelength was held alternately at .sub.1=384 nm (140 s; bulk trans->cis, decrease of absorption) then .sub.2=554 nm (40 s, quantitative cis->trans; return of absorption to 0.51). Note that at 6 minutes, the sample is in the dark-adapted (all-trans) state, as irradiations have not yet commenced.

[0252] FIG. 5: Fluorescence spectrophotometry measurements of a sample of compound I.25 at 10 M in 60:32:8 EtOH:PBS:MeCN at 25 C. Top panel: emission spectra showing that I.25 can be excited at either 380 nm (dotted line) or 554 nm (solid line) to produce fluorescence with an emission maximum at 590 nm (note: vertical scale is in arbitrary units not comparable between measurements). Bottom panel: excitation spectrum showing that I.25 can be excited over a range of wavelengths to produce fluorescence at 590 nm.

[0253] FIG. 6: Schematic presentation of a computer-controlled LED-based automatic lighting system for cell culture experiments. The system was designed to evaluate potential in vivo medicinal uses of the compounds of the invention in an in vitro cell culture model. For example, both toxic regimes (eg. 390 nm irradiation for 250 ms pulsed every 5 min) and strong rescue regimes (eg. 410 nm irradiation for 250 ms then 525 nm irradiation for 600 ms synchronously pulsed every 5 min) could easily and cheaply be applied in parallel to many standard multiwell cell culture plates, during incubations maintained over several days.

[0254] FIG. 7: Immunofluorescence microscopy staining images showing in cellulo light-controlled effects of compound I.1 on the structure of microtubules. MDA-MB-231 cells were treated for 20 h with the indicated concentrations of compound I.1 and kept in the dark, or exposed to the 390 nm protocol (200 ms every 2 min), or exposed to the double irradiation rescue protocol (200 ms of 390 nm, then immediately 600 ms of 505 nm, every 2 min). Representative confocal microscope images are shown. White scale bars in the lower right of each panel correspond to a scale of 20 m.

EXAMPLES PART A

Chemical Synthesis

Reagents and Procedures:

[0255] Unless stated otherwise, (1) all reactions and characterisations were performed with unpurified, undried, non-degassed solvents and reagents, used as obtained, under closed air atmosphere without special precautions; (2) hexane used for chromatography was distilled from commercial crude isohexane fraction on rotavap; (3) column and chromatography refer to flash column chromatography, which was performed on Merck silica gel Si-60 (40-63 m); (4) procedures and yields are unoptimized; (5) yields refer to isolated chromatographically and spectroscopically pure materials, corrected for residual solvent content; (6) all eluent and solvent mixtures are given as volume ratios unless otherwise specified, thus 1:1 Cy:EA indicates a 1:1 mixture of cyclohexane and ethyl acetate by volume.

[0256] Thin-layer chromatography (TLC) was run on 0.25 mm Merck silica gel plates (60, F-254). UV light (254 nm) was used as a visualising agent, and standard TLC dips based on p-anisaldehyde (Anis), Hanessian's cerium ammonium molybdate formulation (Han), 0.6% methanolic FeCl.sub.3 (FeCl.sub.3), basic KMnO.sub.4 (KMnO.sub.4), phosphomolybdic acid (PMA), Dragendorff's reagent (Drag), vanillin (Van) and ninhydrin (Nin) followed by heating where necessary were used as developing agents. R.sub.f values were usually determined in hexane:ethyl acetate (Hx:EA) or cyclohexane:ethyl acetate (Cy:EA) eluents, reported as volume ratio compositions (v:v). TLC characterisations are thus abbreviated as per (R.sub.f=0.09 on 6:1 Hx:EA, Anis).

NMR:

[0257] Standard NMR characterisation was by .sup.1H and .sup.13C 1D-NMR spectra. Known compounds were checked against literature data and their spectral analysis is not detailed unless necessary. Spectrometers used were Bruker DPX 200 (200 MHz & 50 MHz for .sup.1H and .sup.13C respectively), Bruker Ascend 300 (300 MHz, 75 MHz and 282 MHz for .sup.1H, .sup.13C and .sup.19F respectively), Bruker Ascend 400 (400 MHz & 100 MHz for .sup.1H and .sup.13C respectively), Bruker AVANCE 500 (500 MHz & 125 MHz for .sup.1H and .sup.13C respectively), as indicated, at 300K. Where not indicated otherwise, the NMR solvent was CDCl.sub.3. Chemical shifts () are reported in ppm calibrated to residual non-perdeuterated solvent as an internal reference..sup.[42] The following peak descriptions are used: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broad (br), quintet (quin), sextet (sext); apparent multiplicities (resolved by 2D experiments or determined by complete spectral assignment) are denoted by a tilde, eg. doublet of doublets, appears as a triplet with apparent coupling constant J=3 Hz is denoted (t, 3 Hz).

Mass Spectra:

[0258] Unit mass measurements were performed on AGILENT 1100 SL and AGILENT 1200 SL coupled LC-MS systems with ESI mode ionisation, with binary eluent mixtures of water-acetonitrile, with the water containing sodium/ammonium formate or formic acid. Both direct injection of the sample (abbreviated DIMS) and LCMS were performed as specified. For LCMS, unless stated otherwise, an Eclipse Plus 3.5 m/4.6100 mm C18 column was used at 25 C. with a 2 mL/min flow rate such that the sample solvent front eluted at t.sub.ret=0.76 min. A linear gradient of eluent composition from 90:10->10:90 water:acetonitrile was applied over the first 4.5 min, then 10:90 maintained until all peaks of interest had been observed (typically a further 3 min). Ion peaks from (positive/negative mode) are reported as (+/) with units Th (m/z), thus DIMS(+): 328 @ 100, 227 @ 80 indicates ESI with direct injection giving two positive ion peaks at m/z=328 and 227 Th, with the peak at 227 Th being 80% of the height of the peak at 328 Th (isotopic peak patterns were sometimes useful to confirm molecular identity on DIMS spectra). Unless stated otherwise, all reported peaks in the positive mode were [MH].sup.+ peaks, and all observed peaks in the negative mode were [M-H].sup. peaks. HRMS was carried out by the Service Central d'Analyse du CNRS, Solaize, France, and by the Zentrale Analytik of the LMU, Munich using ESI or EI ionisation as specified.

Other Information:

[0259] The following abbreviations are used: Hxdistilled isohexanes, Cycyclohexane, EAethyl acetate, pethpetroleum ether 40-60 fraction, DCMdichloromethane, TFA2,2,2-trifluoroacetic acid, iPenONOisopentyl nitrite, PBSphosphate buffer saline, HOBt1-hydroxybenzotriazole, DCCdicyclohexylcarbodiimide, DMFdimethylformamide, brsmbased on recovered starting material, Ts or tosylpara-toluenesulfonyl, Boctert-butoxycarbonyl, SerL-serinyl, LeuL-leucyl, TBStert-butyldimethylsilyl, Etethyl, Acacetyl, Memethyl, MeCNacetonitrile, iPrisopropyl, iPeniso-pentyl, Bubutyl, DMAP4-(dimethylamino)pyridine, DBU1,8-diazabicycloundec-7-ene, DMAP4-dimethylaminopyridine, RBpipN-(6-(diethylamino)-9-(2-(piperazine-1-carbonyl)phenyl)-3H-xanthen-3-ylidene)-N-ethylethanaminium, wt % percentage by weight. Where Standard Procedures were used in synthesis, the amounts of reactants, reagents and solvents employed were implicitly adjusted to maintain the same molar ratios as in the given Procedure, and no other alterations from the Standard Procedure (eg reaction time, choice of extraction solvent, temperature) were made, unless stated otherwise. In these synthetic descriptions in Part A, azobenzenes are drawn by default in their cis-isomeric form to enable easier comparison with the SAR literature of their isosteric antitubulin stilbenes and stilbenoid compounds such as the combretastatins. However, this should be understood to imply either or both of the trans & as forms constituting a given sample depending on light exposure, therefore they are also named without E/Z-designations.

Standard Procedure A: Diazo Coupling Using Isopentyl Nitrite

[0260] To the aniline (1 mmol) were added MeOH (5 mL) and conc. HCl (0.25 mL), and the mixture cooled in an icebath. A solution of iso-pentyl nitrite (1.02 mmol) in methanol (0.6 mL) was added dropwise and the reaction stirred for 30 min in the cold. A cold solution of the phenol (1.05 mmol) in methanol (2 mL) and NaOH (2.0 M, 1.8 mL) was prepared, and to it was added the solution of the diazonium, dropwise over 1 minute. After typically 30 minutes stirring in the cold, the pH was adjusted to 7 with phosphate buffer, chloroform (10 mL) was added, and the aqueous phase was extracted with CHCl.sub.3 (210 mL). The combined organic layers were washed with water (15 mL) and brine (10 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated. Chromatography with a Hx:EA gradient was used to separate the para-phenolic azobenzene product which typically ran as a single isomer during chromatography.

Standard Procedure B: Phenol Methylation in Acetone

[0261] To the phenol (1 mmol) were added K.sub.2CO.sub.3 (3 mmol), technical grade acetone (10 mL), and MeI (2 mmol), and the mixture stirred at RT for 2-12 h, until TLC showed satisfactory conversion of the starting material. Note that TLC often separated the trans and cis azobenzene isomers, with the major spot apparently being the faster-running trans isomer; the as isomer typically appeared at near-identical R.sub.f to that of the starting phenol. The volatiles were evaporated on the rotavap, then the crude mixture was separated by chromatography with a Hx:EA gradient. Since the para-O-methylated trans and as product isomers typically were separable by chromatography, the crude product could optionally be kept in the dark overnight and protected from light during loading and chromatography (eg wrapping the column with aluminium foil) to ensure cleaner separation of the desired product (as the trans isomer) from other impurities, though this was typically not necessary.

Standard Procedure C: Phenol Methylation Using MeI and Ag.sub.2CO.sub.3 in Toluene

[0262] To the phenol (1 mmol) in a screw-cap pressure tube were added toluene (6 mL), Ag.sub.2CO.sub.3 (1 mmol, supported on Celite or not), and MeI (1.5 mmol). The tube was sealed, protected from light, and the reaction heated to 110 C. overnight with stirring. After cooling, the crude reaction mixture was filtered, the residue washed with chloroform (2 mL), and the combined filtrates concentrated and separated on column as for Standard Procedure B.

[0263] Azocombretastatin A-4 (I.1) and Methylated Derivative (I.11)

[0264] The synthesis of I.1 and I.11 is presented on Scheme 4 hereafter.

##STR00046##

2-((tert-butyldimethylsilyl)oxy)phenol (III.1)

[0265] Catechol (580 mg, 5.27 mmol) was added to a stirred solution of TBSCl (658 mg, 4.4 mmol) and imidazole (850 mg, 11.6 mmol) in DMF (15 mL), then NEt.sub.3 (1 mL, 7.5 mmol) was added and a precipitate formed. The reaction mixture was stirred overnight, concentrated on the rotavap, and partitioned between water (75 mL) and ethyl acetate (25 mL). The aqueous phase was extracted twice with ethyl acetate (225 mL), then the combined organic extracts were washed with water (225 mL), brine (10 mL), dried on Na.sub.2SO.sub.4, filtered and evaporated to yield a pale yellow crude (980 mg) of which 817 mg was purified by chromatography on 100:0->20:1->10:1 Hx:EA giving III.1 as colourless oil (567 mg, 75%; R.sub.f=0.56 on 9:1 Hx:EA, Han). .sup.1H-NMR matched literature data.sup.[43].

2-((tert-butyldimethylsilyl)oxy)-4-((3,4,5-trimethoxyphenyl)diazenyl)phenol (IV.1)

[0266] By Standard Procedure A, commercial 3,4,5-trimethoxyaniline (II.1; 236 mg, 1.29 mmol) was reacted with III.1 (250 mg, 1.12 mmol) to yield a deep red crude oil. Chromatography on 5:1->1:1 Hx:EA returned IV.1 as a yellow oil (102 mg, 0.244 mmol, 22%; R.sub.f=0.24 on 5:1 Hx:EA). NMR of the product as isolated ex organic solvent revealed a roughly 55:44 proportion of [presumably trans and cis] isomers when analysed in CDCl.sub.3 without precautions to block ambient light. Their spectra could for some peaks be separated (denoted .sub.E or .sub.Z): .sup.1H-NMR (400 MHz): =7.57 (td, Hz, 1H), 7.47-7.45 (m, 1H), 7.23 (s, 2H.sub.Z) & 7.21 (s, 2H.sub.E), 7.07 (d, 8.5 Hz, 1H.sub.E) & 6.97 (d, 8.5 Hz, 1H.sub.Z), 5.88 (s br, 1H.sub.E) & 5.66 (s br, 1H.sub.Z), 3.98 (s, 6H.sub.E) & 3.97 (5, 6 Hz), 3.94 (s, 3H), 1.06 (s, 9H.sub.E) & 1.05 (s, 9H.sub.Z), 0.36 (s, 6H.sub.E) & 0.34 (s, 6H.sub.Z) ppm. .sup.13C-NMR (100 MHz): =153.5 (2, E & Z), 150.3 & 148.6 (E & Z), 148.5 & 142.9 (E & Z), 147.9 (E & Z), 146.5 & 145.4 (E & Z), 140.2 & 140.1 (E & Z), 119.6 & 118.1 (E & Z), 117.5 & 114.6 (Z & E), 110.7 & 106.9 (E & Z), 100.2 & 100.1 (2, E & Z), 61.0 (E & Z), 56.2 & 56.2 (2, E & Z), 25.8 & 25.7 (3, E & Z), 18.3 & 18.2 (E & Z), 4.2 & 4.3 (3, E & Z) ppm. LCMS(+): t.sub.ret=5.6 & 5.8 min, each 419 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak showed a secondary absorption band between 450-510 nm (cis), while the second peak was generally more intense especially between 320-380 nm (trans) but without this secondary band. HRMS (ESI+) calcd for [C.sub.21H.sub.33N.sub.2O.sub.6Si].sup.+[M.H.sub.3O.sup.+]: m/z 437.210. found 437.236.

2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenol, Azocombretastatin A-4 (I.1) and 1-(3,4-dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.11): procedure 1

[0267] To IV.1 (73 mg, 0.175 mmol) were added K.sub.2CO.sub.3 (44 mg, 0.32 mmol), dry DMF (2 mL), and MeI solution (1.37 g of a 4.3 wt % solution in DMF, 0.43 mmol), and the mixture stirred at RT for 2 h until TLC (5:1 Hx:EA) showed complete conversion of the starting material to a faster-running product. The volatiles were evaporated at 60 C. and 5 mbar, then THF (8 mL) and an aqueous solution of KF (1 M, 5 mL) were added to the residue and the mixture stirred at RT for 3 h 30 min. The bulk of the THF was removed on the rotavap, then water (15 mL), brine (2 mL), and KH.sub.2PO.sub.4/K.sub.2HPO.sub.4 buffer (2 M, pH=6.8, 4 mL) were added and the aqueous phase extracted with dichloromethane (315 mL). The combined organic layers were washed with water (15 mL) and brine (10 mL) and dried on Na.sub.2SO.sub.4, filtered and concentrated to a crude oil. Flash chromatography with a very gentle gradient covering 5:1->2.4:1 Hx:EA in the dark (aluminium foil wrapped around the column) separated the crude components cleanly without problems due to the different R.sub.f values of their trans and cis isomers, returning I.11 (8.0 mg, 0.024 mmol, 14% over 2 steps) then I.1 (19.7 mg, 0.062 mmol, 35% over 2 steps).

I.1:

[0268] R.sub.f (trans/cis)=0.36 and 0.18 on 1.7:1 Hx:EA (Anis); orange solid. NMR of the product as isolated with precautions to block ambient light revealed a single geometric isomer. .sup.1H-NMR (400 MHz): =7.59-7.53 (m, 1H), 7.24 (s, 2H), 7.00 (d, 8.4 Hz, 1H), 5.84-5.66 (s br, 1H), 4.01 (s, 3H), 3.99 (s, 6H), 3.95 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =153.5 (2), 149.2, 148.5, 147.3, 146.2, 140.2, 119.0, 110.1, 106.0, 100.2 (2), 61.0, 56.2 (2), 56.2 ppm. LCMS(+): t.sub.ret=3.12 & 3.89 min, each 319 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the first peak had a secondary absorption band at 445 nm. HRMS (EI+) calcd for [C.sub.16H.sub.13N.sub.2O.sub.5].sup.+[M.sup.+]: m/z 318.1288. found 318.1287.

I.11:

[0269] R.sub.f (trans/cis)=0.34 and 0.14 on 2.4:1 Hx:EA; R.sub.f (trans/cis)=0.53 and 0.28 on 1.7:1 Hx:EA (Anis); yellow solid. .sup.1H-NMR (400 MHz, CD.sub.3CN): =7.60 (dd, 8.5&2.2 Hz, 1H), 7.52 (d, 2.2 Hz, 1H), 7.26 (s, 2H), 7.12 (d, 8.5 Hz, 1H), 3.93 (s, 6H), 3.93 (s, 3H), 3.91 (s, 3H), 3.83 (s, 3H) ppm. .sup.13C-NMR (100 MHz, CD.sub.3CN): =154.3 (2), 152.8, 150.4, 149.1, 147.1, 140.7, 120.9, 111.6, 102.4, 100.5 (2), 60.6, 56.3 (2), 56.2, 55.9 ppm. LCMS(+): t.sub.ret=3.45 & 4.35 min, each 333.1 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the first peak had a secondary absorption band at 440 nm. HRMS (ESI+) calcd for [C.sub.17H.sub.21N.sub.2O.sub.5].sup.+[MH.sup.+]: m/z 333.1445. found 333.1443.

(I.1) and (I.11): Procedure 2:

[0270] Alternatively, by Standard Procedure B, IV.1 (100 mg, 0.23 mmol) was methylated in acetone (5 mL) using MeI (80 mg, 0.56 mmol) and K.sub.2CO.sub.3 (420 mg, 3 mmol). After evaporation of the volatiles, the residue was partitioned between CHCl.sub.3 (10 mL) and aqueous phosphate buffer (pH=3), extracted with CHCl.sub.3 (210 mL), and pad filtered on silica using 2.4:1 Hx:EA eluent, yielding 94 mg crude red oil. To this under nitrogen atmosphere were added MeCN (6 mL) and HF (70% in pyridine, 165 mg), and the reaction stirred for 15 minutes. CaCO.sub.3 (1.0 g), CaCl.sub.2 (0.5 g) and water (10 mL) were added to quench excess HF, the pH adjusted to 3 with KH.sub.2PO.sub.4, then the mixture was extracted with CHCl.sub.3 (310 mL). The combined organic layers were washed with brine (10 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated to a black crude powder (70 mg). Flash chromatography with 5:1->2.4:1 Hx:EA in the dark (aluminium foil wrapped around the column) separated the crude components cleanly without problems due to the different R.sub.f values of their trans and as isomers, giving I.11 (5 mg, 0.015 mmol, 6%) and I.1 (14 mg, 0.044 mmol, 19% over 2 steps), both spectroscopically identical to the products of procedure 1.

Azocombretastatin A-4 (I.1), Alternative Procedure

[0271] An alternative synthesis giving I.1 without generating I.11, using tosyl instead of TBS as a protecting group, is presented on Scheme 5 hereafter.

##STR00047##

2-hydroxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenyl 4-methylbenzenesulfonate (IV.30a)

[0272] Commercial 3,4,5-trimethoxyaniline (II.1; 1.045 g, 5.71 mmol) was reacted with known 2-hydroxyphenyl 4-para-toluenesulfonate.sup.[44] (1.508 g, 5.71 mmol) by Standard Procedure A except that stirring of the mixture of phenolate and diazonium was continued for 5 h at 0 C. to allow for more complete conversion. Following workup, the deep red crude oil was chromatographed on 5:1->1:1 Hx:EA returning IV.30a as a yellow oil (1.130 g, 2.47 mmol, 43%; R.sub.f=0.43 on 1:1 Hx:EA). .sup.1H-NMR (400 MHz): =7.76 (d, 8.4 Hz, 2H), 7.72 (dd, 8.7 & 2.3 Hz, 1H), 7.51 (d, 2.3 Hz, 1H), 7.29 (dd, 8.5 & 0.8 Hz, 2H), 7.11 (s, 2H), 7.04 (d, 8.7 Hz, 1H), 3.89 (s, 6H), 3.86 (s, 3H), 2.39 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =153.5 (2), 150.7, 148.1, 146.4, 146.4, 140.7, 137.3, 131.2, 130.1 (2), 128.7 (2), 124.0, 118.1, 117.4, 100.4 (2), 61.1, 56.2 (2), 21.8 ppm. LCMS(+): t.sub.ret=4.60 min, 459 Th[MH].sup.+. HRMS (ESI+) calcd for [C.sub.22H.sub.23N.sub.2O.sub.7S].sup.+[MH.sup.+]: m/z 459.12205. found 459.12168.

2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenyl 4-methylbenzenesulfonate (IV.30b)

[0273] By Standard Procedure B, IV.30a (700 mg, 1.53 mmol) was methylated overnight. Chromatography of the red crude solid on 5:1->1:1 Hx:EA returned IV.30b (712 mg, 1.51 mmol, 99%; R.sub.f=0.62 and 0.46 on 1:1 Hx:EA, FeCl.sub.3) as a red oil. .sup.1H-NMR (400 MHz, DMSO): =7.93 (dd, 8.8 & 2.4 Hz, 1H), 7.75 (d, 8.4 Hz, 2H), 7.63 (d, 2.4 Hz, 1H), 7.49 (d, 8.5 Hz, 2H), 7.28 (d, 8.9 Hz, 1H), 7.23 (s, 2H), 3.91 (s, 6H), 3.77 (s, 3H), 3.61 (s, 3H), 2.44 (s, 3H) ppm. .sup.13C-NMR (100 MHz, DMSO): =154.2, 153.8 (2), 148.0, 146.3, 145.6, 140.7, 138.5, 132.3, 130.4 (2), 128.8 (2), 126.2, 115.6, 113.9, 100.7 (2), 60.7, 56.6, 56.5 (2), 21.6 ppm. LCMS(+): t.sub.ret=4.48 & 5.17 min, 473 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (cis) featured a shoulder centred around 450 nm. HRMS (ESI+) calcd for [C.sub.23H.sub.25N.sub.2O.sub.7S].sup.+[MH.sup.+]: m/z 473.13770. found 473.13730.

2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenol, Azocombretastatin A-4 (I.1)

[0274] To IV.30b (525 mg, 1.10 mmol) were added KOH (1.25 g) and MeOH (25 mL) and the solution heated to 80 C. for 1 hour. After evaporation of the volatiles, the residue was partitioned between EtOAc (20 mL) and aqueous KH.sub.2PO.sub.4 solution (10%, 30 mL), then the aqueous layer was extracted with EtOAc (210 mL). The combined organic layers were washed with water (20 mL), brine (10 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated. The crude oil was chromatographed on 5:1->1:1 Hx:EA, giving I.1 (320 mg, 1.01 mmol, 92%) as an orange solid, identical by NMR and LCMS to that synthesised previously from IV.1b (shown above).

North Ring Meta-Amino Derivative (I.2)

[0275] The synthesis of I.2 is presented on Scheme 6 hereafter.

##STR00048##

tert-butyl (2-hydroxyphenyl)carbamate (III.2)

[0276] 2-aminophenol (3.93 g, 36 mmol) was stirred with tert-butoxycarbonyl dicarbonate (8.32 g, 38 mmol) in dry pyridine (30 mL) with triethylamine (4 mL) warming from 0 C. to 25 C. over 12 h. The volatiles were evaporated and the residue partitioned between diethyl ether and phosphate buffer (pH=10); the ether layer was washed with phosphate buffer then brine, dried on Na.sub.2SO.sub.4, filtered and evaporated to yield 8.1 g of dark crude product which could be purified by column chromatography (20:1->5:1 Hex:EA), or by fractional crystallisations from acetone-hexane followed by hot hexane trituration. NMR spectra matched literature data.sup.[45]: .sup.1H-NMR (400 MHz): =8.15 (s br, 1H), 7.08-7.00 (m, 2H), 6.99 (d, 7.9 Hz, 1H), 6.88 (t, 7.5 Hz, 1H), 6.69 (s, 1H), 1.56 (s, 9H) ppm. .sup.13C-NMR (100 MHz): =155.1, 147.6, 125.7, 125.5, 121.5, 120.7, 119.1, 82.2, 28.3 (3) ppm. DIMS(+): 210 Th=[MH].sup.+.

tert-butyl (2-hydroxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenyl)carbamate (IV.2)

[0277] By Standard Procedure A, II.1 (368 mg, 2.01 mmol) was reacted with III.2 (406 mg, 1.94 mmol). Chromatography on 5:1->2.4:1 Hx:EA returned IV.2 (642 mg, 1.59 mmol, 82%; R.sub.f=0.22 on 2.4:1 Hx:EA, FeCl.sub.3) as a brown viscous oil. .sup.1H-NMR (400 MHz, CD.sub.3CN): =8.36 (s, 1H), 8.32 (d, 2.3 Hz, 1H), 7.54 (dd, 8.5 & 2.4 Hz, 1H), 7.35 (s, 1H), 7.19 (s, 2H), 7.02 (d, 8.5 Hz, 1H), 3.89 (s, 6H), 3.80 (s, 3H) ppm. .sup.13C-NMR (100 MHz, CD.sub.3CN): =153.7 (2), 153.5, 149.0, 148.5, 146.1, 140.1, 127.5, 120.6, 115.4, 112.3, 100.0 (2), 80.6, 60.0, 55.8 (2), 27.52 (3) ppm. LCMS(+): t.sub.ret=4.65 min, 404 Th[MH].sup.+. HRMS (ESI+) calcd for [C.sub.20H.sub.26N.sub.3O.sub.6].sup.+[MH.sup.+]: m/z 404.1816. found 404.1817.

tert-butyl (2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenyl)carbamate (I.41)

[0278] By Standard Procedure B, IV.2 (637 mg, 1.58 mmol) was methylated overnight with MeI (448 mg, 3.13 mmol) and K.sub.2CO.sub.3 (873 mg, 6.32 mmol). Chromatography of the black crude solid on 5:1->2.4:1 Hx:EA returned I.41 (593 mg, 1.42 mmol, 90%; R.sub.f=0.41 on 2.4:1 Hx:EA, FeCl.sub.3) as a red oil. .sup.1H-NMR (400 MHz): =8.64 (s br, 1H), 7.58 (dd, 8.7, 2.4 Hz, 1H), 7.20 (s, 2H), 7.09 (s, 1H), 6.90 (d, 8.7 Hz, 1H), 3.90 (s, 9H), 3.86 (s, 3H), 1.49 (s, 9H) ppm. .sup.13C-NMR (100 MHz): =153.4 (2), 152.6, 149.8, 148.4, 146.7, 140.2, 128.8, 119.1, 111.2, 109.6, 100.4 (2), 80.7, 61.0, 56.2 (2), 56.0, 28.4 (3) ppm. LCMS(+): t.sub.ret=4.57 & 5.42 min, 418 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (cis) featured a shoulder centred around 440 nm. HRMS (ESI+) calcd for [C.sub.21H.sub.28N.sub.3O.sub.6].sup.+[MH.sup.+]: m/z 418.19726. found 418.19718.

2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)aniline (I.2)

[0279] To I.41 (590 mg, 1.41 mmol) were added CH.sub.2Cl.sub.2 (6 mL) and CF.sub.3COOH (5 mL) and the purple solution stirred overnight at room temperature. The volatiles were removed under high vacuum, the residual TFA neutralised with addition of CHCl.sub.3 (10 mL) and K.sub.2CO.sub.3 (618 mg) and the residue chromatographed on 1:1:0->1:1:1 Hx:EA:MeOH, giving I.2 as a green-black powder (394 mg, 1.24 mmol, 88%; R.sub.f=0.56 on 1:1 Hx:EA (Van)). NMR when analysed in CDCl.sub.3 without precautions to block ambient light showed two isomers in approximately 2:1 ratio [presumably trans and cis forms, attributed by HSQC, denoted .sub.E and .sub.Z]. .sup.1H-NMR (400 MHz): =8.84 (s br, 1H, NH.sub.2), 7.72 (dd, 8.5 & 1.9 Hz, 1H.sub.E), 7.58 (d, 2.2 Hz, 1H.sub.Z), 7.56 (d, 1.9 Hz, 1H.sub.E), 7.53 (dd, 8.6 & 2.4 Hz, 1H.sub.Z), 7.26 (d, 8.5 Hz, 1H.sub.E), 7.19 (s, 2H.sub.E), 7.16 (s, 2H.sub.Z), 6.90 (d, 8.6 Hz, 1H.sub.Z), 3.92-3.84 (m, 12H.sub.E & 12H.sub.Z) ppm. .sup.13C-NMR (100 MHz): =153.6 & 153.5 (2, E&Z), 151.6 & 149.4 (1, E&Z), 148.5 & 148.1 (1, E&Z), 146.8 & 145.7 (1, E&Z), 140.8 & 140.2 (1, E&Z), 130.0 (1, E&Z), 121.7 & 120.7 (1, E&Z), 110.4 & 110.3 (1, E&Z), 110.1 & 101.3 (1, E&Z), 100.5 & 100.2 (2, E&Z), 61.1 & 61.0 (1, E&Z), 56.2 & 56.2 (2, E&Z), 56.1 (1, E&Z) ppm. LCMS(+): t.sub.ret=3.04 & 3.94 min, each 318 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the first peak featured an absorption shoulder at 450 nm which was absent in the second peak (trans). HRMS (ESI+) calcd for [C.sub.16H.sub.20N.sub.3O.sub.4].sup.+[MH.sup.+]: m/z 318.1448. found 318.1449.

North Ring Ortho-Oxy, Meta-Hydro Derivatives (I.8) and (I.12)

[0280] The syntheses of I.8 and I.12 are presented on Scheme 7 hereafter.

##STR00049##

4-((3,4,5-trimethoxyphenyl)diazenyl)benzene-1,3-diol (IV.8)

[0281] A mono-protected resorcinol could be chosen to reduce byproduct formation during the diazo coupling. By Standard Procedure A, II.1 (590 mg, 3.22 mmol) was reacted with commercial resorcinol monobenzoate (III.8, 724 mg, 3.38 mmol), where the phenol was dissolved in NaOH only one minute prior to diazonium addition to reduce ester hydrolysis prior to reaction, and where the coupling was run for only 15 minutes before neutralisation and extraction. Chromatography of the red crude oil on 5:1->1:1 Hx:EA returned the major product IV.8 (693 mg, 2.28 mmol, 71%; R.sub.f=0.37 on 1.7:1 Hx:EA, FeCl.sub.3) as a deep red powder. .sup.1H-NMR (400 MHz, DMSO): =12.15 (s, 1H), 10.50 (s, 1H), 7.68 (d, 8.8 Hz, 1H), 7.26 (s, 2H), 6.49 (dd, 8.8 & 2.5 Hz, 1H), 6.36 (d, 2.5 Hz, 1H), 3.88 (s, 6H), 3.74 (s, 3H) ppm. .sup.13C-NMR (100 MHz, DMSO): =163.1, 156.6, 153.9 (2), 147.2, 139.7, 132.5, 129.5, 109.4, 103.5, 99.9 (2), 60.7, 56.5 (2) ppm. HRMS (ESI+) calcd for [C.sub.15H.sub.17N.sub.2O.sub.5].sup.+[MH].sup.+: m/z 305.11320. found 305.11322.

5-methoxy-2-((3,4,5-trimethoxyphenyl)diazenyl)phenol (I.8)

[0282] By Standard Procedure B, IV.8 (670 mg, 2.20 mmol) was methylated overnight with MeI (618 mg, 4.35 mmol, 1.98 eq). Chromatography on a gentle gradient of 10:1->1:1 Hx:EA returned para-monomethylated 1.8 (255 mg, 0.80 mmol, 36%; R.sub.f=0.52 on 2.4:1 Hx:EA, FeCl.sub.3) as a red solid, then bismethylated byproduct 1.12 (100 mg, 0.30 mmol, 14%; R.sub.f=0.19 on 2.4:1 Hx:EA, FeCl.sub.3) as a viscous red oil. The identity of 1.8 as the para-methoxy isomer was confirmed by comparison to independently synthesised ortho-methoxy isomer IV.12 (detailed below). I.8: .sup.1H-NMR (400 MHz): =7.80 (d, 8.9 Hz, 1H), 7.11 (s, 2H), 6.63 (dd, 8.9 & 2.7 Hz, 1H), 6.50 (d, 2.7 Hz, 1H), 3.97 (s, 6H), 3.94 (s, 3H), 3.89 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =163.8, 156.2, 153.7 (2), 146.0, 140.0, 134.5, 132.7, 108.3, 101.4, 99.0 (2), 61.1, 56.3 (2), 55.7 ppm. LCMS(+): t.sub.ret=4.83 min, 319 Th[MH].sup.+: this peak was assigned to the trans isomer since the UV absorption profile did not feature any shoulder band in the visible; ion-sim mode indicated a possible second peak with 319 Th at t.sub.ret=3.37 min however no clear UV-Vis spectrum was seen for that peak. HRMS (ESI) calcd for [C.sub.16H.sub.17N.sub.2O.sub.5].sup.[M-H].sup.: m/z 317.11430. found 317.11410.

3-methoxy-4-((3,4,5-trimethoxyphenyl)diazenyl)phenol (IV.12)

[0283] By Standard Procedure A, II.1 (366 mg, 2.00 mmol) was reacted with commercial 3-methoxyphenol (III.12, 240 mg, 2.05 mmol). Chromatography of the red crude oil on 5:1->1:1 Hx:EA returned IV.12 (564 mg, 1.77 mmol, 89%; R.sub.f=0.11 on 2.4:1 Hx:EA, FeCl.sub.3) as a deep red powder. .sup.1H-NMR (400 MHz, DMSO): =10.32 (s br, 1H), 7.55 (d, 8.8 Hz, 1H), 7.12 (s, 2H), 6.60 (d, 2.4 Hz, 1H), 6.45 (dd, 8.8 & 2.4 Hz, 1H), 3.91 (s, 3H), 3.87 (s, 6H), 3.74 (s, 3H) ppm. .sup.13C-NMR (100 MHz, DMSO): =163.0, 159.3, 153.7 (2), 149.0, 139.6, 135.2, 118.0, 108.4, 100.4, 100.1 (2), 60.7, 56.4 (2), 56.3 ppm. LCMS(+): t.sub.ret=3.57 min, 319 Th[MH].sup.+. HRMS (ESI+) calcd for [C.sub.16H.sub.19N.sub.2O.sub.5].sup.+[MH].sup.+: m/z 319.12157. found 319.12856.

1-(2,4-di methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.12)

[0284] By Standard Procedure B, IV.12 (550 mg, 1.73 mmol) was methylated for 6 hours. Chromatography on a gradient of 5:1->1:1 Hx:EA returned I.12 (460 mg, 1.38 mmol, 78%; R.sub.f=0.19 on 2.4:1 Hx:EA, FeCl.sub.3) as an orange powder. .sup.1H-NMR (400 MHz): =7.60 (d, 8.9 Hz, 1H), 7.06 (s, 2H), 6.49-6.39 (m, 2H), 3.88 (s, 3H), 3.83 (s, 6H), 3.78 (s, 3H), 3.76 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =163.6, 158.5, 153.5 (2), 149.2, 140.0, 136.7, 118.2, 105.6, 100.1 (2), 99.0, 61.0, 56.3, 56.2 (2), 55.6 ppm. LCMS(+): t.sub.ret=3.72 and 4.42 min, 333 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (cis) featured a shoulder at around 440 nm which was absent in the second peak. HRMS (ESI+) calcd for [C.sub.17H.sub.21N.sub.2O.sub.5].sup.+[MH].sup.+: m/z 333.14450. found 333.14430.

North Ring Ortho-Amino, Meta-Hydro Derivative (I.16)

[0285] The synthesis of I.16 is presented on Scheme 8 hereafter.

##STR00050##

tert-butyl (5-hydroxy-2-((3,4,5-trimethoxyphenyl)diazenyl)phenyl)carbamate (IV.16)

[0286] By Standard Procedure A, II.1 (921 mg, 5.01 mmol) was reacted with III.16 (1.04 g, 4.97 mmol). Chromatography on 5:1->2.4:1 Hx:EA returned IV.16 (1.846 g, 4.57 mmol, 92%; R.sub.f=0.44 on 2.4:1 Hx:EA, FeCl.sub.3) as an orange foam. .sup.1H-NMR (400 MHz, DMSO): =10.44 (s br, 1H), 9.84 (s, 1H), 7.70-7.65 (m, 2H), 7.17 (s, 2H), 6.57 (dd, 8.9 & 2.6 Hz, 1H), 3.88 (s, 6H), 3.75 (s, 3H), 1.50 (s, 9H) ppm. .sup.13C-NMR (100 MHz, DMSO): =162.3, 153.8 (2), 152.5, 148.4, 139.9, 138.6, 133.1, 123.1, 111.1, 105.6, 100.2 (2), 80.5, 60.7, 56.3 (2), 28.3 (3) ppm. LCMS(+): t.sub.ret=4.86 and 5.14 min, each 404 Th[MH].sup.+. HRMS (ESI+) calcd for [C.sub.20H.sub.26N.sub.3O.sub.6].sup.+[MH.sup.+]: m/z 404.18161. found 404.18188.

tert-butyl (5-methoxy-2-((3,4,5-trimethoxyphenyl)diazenyl)phenyl)carbamate (I.42)

[0287] By Standard Procedure B, IV.16 (1830 mg, 4.54 mmol) was methylated for 6 hours with MeI (1.13 g, 8.0 mmol) and K.sub.2CO.sub.3 (2.2 g, 16 mmol). Chromatography of the black crude oil on 5:1->2.4:1 Hx:EA returned I.42 (1787 mg, 4.28 mmol, 94%; R.sub.f=0.42 and 0.56 on 2.4:1 Hx:EA, trans and cis isomers; FeCl.sub.3) as an orange solid. .sup.1H-NMR (400 MHz): =9.77 (s br, 1H), 7.91 (d, 2.7 Hz, 1H), 7.78 (d, 9.0 Hz, 1H), 7.09 (s, 2H), 6.58 (dd, 9.0 & 2.7 Hz, 1H), 3.89 (s, 6H), 3.86 (s, 3H), 3.85 (s, 3H), 1.48 (s, 9H) ppm. .sup.13C-NMR (100 MHz): =163.8, 153.6 (2), 152.4, 147.9, 140.2, 138.6, 132.8, 124.2, 110.0, 101.8, 99.7 (2), 80.7, 61.1, 56.1, 55.8, 28.3 (3) Ppm. LCMS(+): t.sub.ret=4.72 & 5.81 min, 418 Th[MH].sup.+. HRMS (ESI+) calcd for [C.sub.21H.sub.27N.sub.3O.sub.6Na].sup.+[MNa.sup.+]: m/z 440.17921. found 440.17938.

5-methoxy-2-((3,4,5-trimethoxyphenyl)diazenyl)aniline (I.16)

[0288] To I.42 (1.20 g, 2.87 mmol) were added CH.sub.2Cl.sub.2 (10 mL) and CF.sub.3COOH (12 mL) and the purple solution stirred overnight at room temperature. The volatiles were removed under high vacuum, the purple residue was partitioned between CH.sub.2Cl.sub.2 (30 mL) and K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 buffer (pH=6.8, 30 mL), the aqueous layer extracted with DCM (15 mL), then the combined organic layers were washed with brine (20 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated to give a red crude oil. Chromatography on 5:1->1:1 Hx:EA returned I.16 as a red oil (870 mg, 2.74 mmol, 95%; R.sub.f=0.55 on 1:1 Hx:EA (FeCl.sub.3)). .sup.1H-NMR (400 MHz, CD.sub.3CN): =7.67 (d, 8.9 Hz, 1H), 7.18 (s, 2H), 6.48 (s, 2H), 6.36 (dd, 8.9 & 2.7 Hz, 1H), 6.32 (d, 2.6 Hz, 1H), 3.90 (s, 6H), 3.81 (s, 3H), 3.78 (s, 3H) ppm. .sup.13C-NMR (100 MHz, CD.sub.3CN): =163.1, 153.7 (2), 149.0, 145.8, 139.1, 131.5, 129.4, 105.2, 99.2 (2), 99.0, 60.0, 55.7 (2), 55.1 ppm. LCMS(+): t.sub.ret=4.35 min, 318 Th[MH]+. HRMS (ESI+) calcd for [C.sub.16H.sub.20N.sub.3O.sub.4].sup.+[MH.sup.+]: m/z 318.1448. found 318.14454.

North Ring Ortho-Oxy, Meta-Methyl Derivative (I.17)

[0289] The synthesis of I.17 is presented on Scheme 9 hereafter.

##STR00051##

2-methyl-4-((3,4,5-trimethoxyphenyl)diazenyl)benzene-1,3-diol (IV.17)

[0290] By Standard Procedure A, II.1 (916 mg, 5.01 mmol) was reacted with commercial 2-methylresorcinol (III.17, 618 mg, 4.98 mmol). Chromatography of the red crude oil on 5:1->1:1 Hx:EA returned IV.17 (1079 mg, 3.39 mmol, 68%; R.sub.f=0.63 on 1:1 Hx:EA, FeCl.sub.3) as a red solid. .sup.1H-NMR (400 MHz, DMSO): =12.98 (s, 1H), 10.48 (s br, 1H), 7.56 (d, 8.8 Hz, 1H), 7.26 (s, 2H), 6.60 (d, 8.9 Hz, 1H), 3.88 (s, 6H), 3.74 (s, 3H), 2.04 (s, 3H) ppm. .sup.13C-NMR (100 MHz, DMSO): =160.9, 154.3, 153.9 (2), 146.7, 139.6, 132.0, 128.7, 111.2, 108.6, 99.7, 60.7, 56.5 (2), 8.3 ppm. LCMS(+): t.sub.ret=4.26 min, 319 Th[MH].sup.+. HRMS (ESI+) calcd for [C.sub.16H.sub.19N.sub.2O.sub.5].sup.+[MH].sup.+: m/z 319.12157. found 319.12859.

3-methoxy-2-methyl-6-((3,4,5-trimethoxyphenyl)diazenyl)phenol (I.17)

[0291] By Standard Procedure B, IV.17 (1.05 g, 3.30 mmol) was methylated for four hours with MeI (1.02 eq). Chromatography on a gradient of 5:1->1:1 Hx:EA returned I.17 (520 mg, 1.56 mmol, 47%; R.sub.f=0.51 on 2.4:1 Hx:EA, FeCl.sub.3) as a red-orange solid which could be crystallised as fine red needles from Hx/EtOAc. .sup.1H-NMR (400 MHz): =7.67 (d, 8.9 Hz, 1H), 7.04 (s, 2H), 6.57 (d, 8.9 Hz, 1H), 3.88 (s, 6H), 3.86 (s, 3H), 3.85 (s, 3H), 2.08 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =161.4, 153.7 (2), 152.9, 146.3, 139.9, 132.9, 132.0, 113.6, 102.8, 99.1 (2), 61.1, 56.2 (2), 55.8, 7.57 ppm. LCMS(+): t.sub.ret=5.24 min, 333 Th[MH].sup.+; this peak was attributed as the trans-isomer due to the single band structure with absorption maximum at 390 nm; no peak for the cis-isomer could be observed even after pre-irradiation of the sample with 390 nm before injection. HRMS (ESI+) calcd for [C.sub.17H.sub.21N.sub.2O.sub.5].sup.+[MH].sup.+: m/z 333.14450. found 333.14421.

North Ring Ortho- and/or Meta-Fluoro Derivatives (I.3), (I.4) and (I.5)

[0292] The syntheses of I.3, I.4 and I.5 are presented on Scheme 10 hereafter.

##STR00052##

3-fluoro-4-((3,4,5-trimethoxyphenyl)diazenyl)phenol (IV.3)

[0293] By Standard Procedure A, II.1 (196 mg, 1.07 mmol) was reacted with commercial 3-fluorophenol (III.3; 120 mg, 1.07 mmol), and the product was extracted with ethyl acetate. Chromatography on 5:1->2.5:1 Hx:EA returned IV.3 (161 mg, 0.53 mmol, 49%; R.sub.f=0.20 on 2.4:1 Hx:EA, KMnO.sub.4) as a yellow oil only sparingly soluble in CH.sub.2Cl.sub.2 or CHCl.sub.3. .sup.1H-NMR (400 MHz): =7.68-7.60 (m, 2H), 7.14 (s, 2H), 7.07 (t, 8.8 Hz, 1H), 5.47 (d br, 4.2 Hz, 1H), 3.89 (s, 6H), 3.86 (s, 3H) ppm. .sup.13C-NMR (100 MHz) showed the expected CF couplings, as did the spectra of the other fluorinated compounds: 5=153.5 (2), 151.3 (d, 240.1 Hz), 148.2, 146.7 (d, 5.2 Hz), 146.1 (d, 15.3 Hz), 140.5, 122.6 (d, 2.9 Hz), 117.0 (d, 2.2 Hz), 107.8 (d, 19.3 Hz), 100.3 (2), 61.1, 56.2 (2) ppm. HRMS (ESI) calcd for [C.sub.15H.sub.14N.sub.2O.sub.4F].sup.[M-H].sup.: m/z 305.09431. found 305.09427.

1-(2-fluoro-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.3)

[0294] By Standard Procedure B, IV.3 (159 mg, 0.52 mmol) was methylated overnight in a mixture of acetone (15 mL), EA (0.6 mL), CHCl.sub.3 (0.7 mL) and DMSO (0.7 mL). Chromatography on 10:1->4:1 Hx:EA cleanly returned I.3 (158 mg, 0.49 mmol, 94%; R.sub.f=0.44 and 0.20 on 2.4:1 Hx:EA, FeCl.sub.3: trans and cis isomers) as a red oil. .sup.1H-NMR (400 MHz, DMSO): =7.83 (ddd, 8.7 & 2.4 & 1.2 Hz, 1H), 7.69 (dd, 12.4 & 2.3 Hz, 1H), 7.39 (t, 8.9 Hz, 1H), 7.24 (s, 2H), 3.96 (s, 3H), 3.89 (s, 6H), 3.77 (s, 3H) ppm. .sup.13C-NMR (100 MHz, DMSO): =153.85 (2), 152.3 (d, 247.1 Hz), 150.3 (d, 11.1 Hz), 148.0, 146.0 (d, 5.1 Hz), 140.6, 123.3 (d, 2.9 Hz), 114.1 (d, 2.2 Hz), 107.4 (d, 19.1 Hz), 100.6 (2), 60.7, 56.8, 56.5 (2) ppm. .sup.19F-NMR (282 MHz, DMSO): =133.45 (ddd, 12.2 & 10.2 & 1.3 Hz) ppm. LCMS(+): t.sub.ret=3.86 & 4.80 min, each 321 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (cis) featured a shoulder centred at 440 nm which was absent in the second peak. HRMS (EI+) calcd for [C.sub.16H.sub.17N.sub.2O.sub.4F].sup.+[M].sup.+: m/z 320.1172. found 320.1170.

2-fluoro-4-((3,4,5-trimethoxyphenyl)diazenyl)phenol (IV.4)

[0295] By Standard Procedure A, II.1 (183 mg, 1.00 mmol) was reacted with commercial 2-fluorophenol (III.4; 116 mg, 1.04 mmol). Chromatography on 5:1->2.5:1 Hx:EA returned IV.4 (198 mg, 0.65 mmol, 65%; R.sub.f=0.25 on 2.4:1 Hx:EA, KMnO.sub.4) as a yellow oil. .sup.1H-NMR (400 MHz, DMSO): =10.77 (s, 1H), 7.68 (t, 8.9 Hz, 1H), 7.18 (s, 2H), 6.80 (dd, 12.7 & 2.5 Hz, 1H), 6.74 (dd, 8.9 & 2.5 Hz, 1H), 3.88 (s, 6H), 3.76 (s, 3H) ppm. .sup.13C-NMR (100 MHz, DMSO): =162.8 (d, 12.1 Hz), 161.2 (d, 255.7 Hz), 153.8 (2), 148.6, 140.3, 133.4 (d, 6.9 Hz), 119.0 (d, 2.0 Hz), 112.9 (d, 2.4 Hz), 103.8 (d, 21.8 Hz), 100.4 (2), 60.7, 56.4 (2) ppm. HRMS (ESI) calcd for [C.sub.15H.sub.14N.sub.2O.sub.4F].sup.[M-H].sup.: m/z 305.09431. found 305.09433.

1-(3-fluoro-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.4)

[0296] By Standard Procedure B, IV.4 (190 mg, 0.62 mmol) was methylated overnight. Chromatography on 10:1->4:1 Hx:EA cleanly returned I.4 (173 mg, 0.54 mmol, 91%; R.sub.f=0.42 and 0.25 on 2.4:1 Hx:EA, FeCl.sub.3: trans and as isomers) as fine orange crystals. .sup.1H-NMR (400 MHz, DMSO): =7.75 (t, 9.0 Hz, 1H), 7.22 (s, 2H), 7.12 (dd, 13.0 & 2.6 Hz, 1H), 6.92 (ddd, 9.2 & 2.7 & 0.9 Hz, 1H), 3.89 (s, 6H), 3.89 (s, 3H), 3.77 (s, 3H) ppm. .sup.13C-NMR (100 MHz, DMSO): =163.8 (d, 11.2 Hz), 161.1 (d, 255.9 Hz), 153.8 (2), 148.5, 140.6, 134.3 (d, 7.2 Hz), 118.8 (d, 2.1 Hz), 112.0 (d, 2.6 Hz), 102.7 (d, 23.4 Hz), 100.6 (2), 60.7, 56.7, 56.4 (2) ppm. .sup.19F-NMR (282 MHz, DMSO): =121.31 (ddd, 13.2 & 8.8 & 1.1 Hz) ppm. LCMS(+): t.sub.ret=3.96 & 4.82 min, each 321 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (cis) featured a shoulder centred at 445 nm which was absent in the second peak. HRMS (EI+) calcd for [C.sub.16H.sub.17N.sub.2O.sub.4F].sup.+[M].sup.+: m/z 320.1172. found 320.1167.

2,3-difluoro-4-((3,4,5-trimethoxyphenyl)diazenyl)phenol (IV.5)

[0297] By Standard Procedure A, II.1 (186 mg, 1.02 mmol) was reacted with commercial 2,3-difluorophenol (III.5; 140 mg, 1.08 mmol). Chromatography on 5:1->2.5:1 Hx:EA returned IV.5 (91 mg, 0.28 mmol, 28%; R.sub.f=0.26 on 1.7:1 Hx:EA, Van) as a yellow oil. .sup.1H-NMR (400 MHz): =7.46 (ddd, 9.2 & 7.5 & 2.3 Hz, 1H), 7.17 (s, 2H), 6.78 (ddd, 9.3 & 8.0 & 2.1 Hz, 1H), 5.79 (s br, 1H), 3.89 (s, 6H), 3.87 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =153.5 (2), 149.1 (dd, 260.6 & 11.3 Hz), 148.6, 147.3 (d, 11.4 Hz), 140.4 (dd, 240.2 & 13.4 Hz), 140.9, 135.4 (d, 4.7 Hz), 112.7 (d, 3.7 Hz), 111.9 (d, 3.6 Hz), 100.6 (2), 61.1, 56.24 (2) ppm. HRMS (ESI) calcd for [C.sub.15H.sub.13N.sub.2O.sub.4F.sub.2].sup.[M-H].sup.: m/z 323.08489. found 323.08495.

1-(2,3-difluoro-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.5)

[0298] By Standard Procedure B, IV.5 (89 mg, 0.27 mmol) was methylated overnight. Chromatography on 10:1->2:1 Hx:EA cleanly returned I.5 (79 mg, 0.23 mmol, 83%; R.sub.f=0.34 and 0.17 on 2.4:1 Hx:EA, FeCl.sub.3: trans and as isomers) as fine orange crystals. .sup.1H-NMR (400 MHz, DMSO): =7.59 (ddd, 9.3 & 8.0 & 2.3 Hz, 1H), 7.23 (s, 2H), 7.19 (ddd, 9.7 & 8.0 & 1.9 Hz, 1H), 3.98 (s, 3H), 3.89 (s, 6H), 3.78 (s, 3H) ppm. .sup.13C-NMR (100 MHz, DMSO): =153.8 (2), 151.3 (dd, 7.8 & 3.1 Hz), 148.7 (dd, 255.9 & 10.8 Hz), 148.3, 141.1, 140.9 (dd, 246.4 & 12.9 Hz), 134.9 (d, 4.8 Hz), 112.8 (d, 3.9 Hz), 109.2 (d, 3.1 Hz), 100.8 (2), 60.7, 57.4, 56.5 (2) ppm. .sup.19F-NMR (282 MHz, DMSO): =148.54 (ddd, 20.0 & 8.1 & 2.1 Hz), 159.65 (ddd, 20.1 & 8.1 & 2.5 Hz) ppm. LCMS(+): t.sub.ret=4.09 & 4.84 min, each 338 Th=[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (cis) had a shoulder centred at 440 nm which was absent in the second peak. HRMS (EI+) calcd for [C.sub.16H.sub.16N.sub.2O.sub.4F.sub.2].sup.+[M].sup.+: m/z 338.1078. found 338.1074.

South Ring Bis(Meta-Desoxyfluoro) Derivative (I.9)

[0299] The synthesis of I.9 is presented on Scheme 11 hereafter.

##STR00053##

2,6-difluoro-4-((4-methoxyphenyl)diazenyl)phenol (IV.9)

[0300] By Standard Procedure A, commercial para-anisidine (V.9; 185 mg, 1.50 mmol) was reacted with commercial 2,6-difluorophenol (VI.9; 199 mg, 1.53 mmol), with stirring at 0 C. continued for 2 hours to allow for greater reaction completion. Chromatography of the dark red crude oil on 5:1->2.5:1 Hx:EA returned IV.9 (80 mg, 0.30 mmol, 20%; R.sub.f=0.49 on 2.4:1 Hx:EA, Van) as a red solid. .sup.1H-NMR (400 MHz): =7.81 (d, 9.0 Hz, 2H), 7.47 (d, 8.9 Hz, 2H), 6.94 (d, 9.1 Hz, 2H), 3.83 (s, 3H) ppm. .sup.13C-NMR (100 MHz): 5=162.4, 151.7 (dd, 243.7 & 6.1 Hz; 2), 146.4, 145.1 (t, 7.2 Hz), 134.7 (t, 16.8 Hz), 124.9 (2), 114.3 (2), 106.6-106.30 (m, 2), 55.63 ppm. .sup.19F-NMR (282 MHz): =134.75 (d, 8.8 Hz) ppm. HRMS (ESI+) calcd for [C.sub.13H.sub.11N.sub.2O.sub.2F.sub.2].sup.+[MH].sup.+: m/z 265.07831. found 265.07832. LCMS(+): t.sub.ret=4.46 min, 265 Th[MH].sup.+.

1-(3,5-difluoro-4-methoxyphenyl)-2-(4-methoxyphenyl)diazene (I.9)

[0301] By Standard Procedure B, IV.9 (75 mg, 0.28 mmol) was methylated overnight. Chromatography of the orange crude oil on 7.5:1->5:1 Hx:EA returned I.9 (58 mg, 0.21 mmol, 74%; R.sub.f=0.78 & 0.68 on 2.4:1 Hx:EA, trans & cis isomers, FeCl.sub.3) as an orange oil. .sup.1H-NMR (400 MHz): =7.89 (d, 9.0 Hz, 2H), 7.63 (d, 9.5 Hz, 2H), 7.15 (d, 9.1 Hz, 2H), 4.03 (t, 1.3 Hz, 3H), 3.88 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =163.0, 155.5 (dd, 248.6 & 6.7 Hz; 2), 147.0 (t, 8.0 Hz), 146.1, 138.1 (t, 14.5 Hz), 125.4 (2), 115.2 (2), 107.3-106.9 (m; 2), 62.3, 56.2 ppm. .sup.19F-NMR (282 MHz, DMSO): =127.35 (dd, 9.6 & 1.3 Hz) ppm. HRMS (EI+) calcd for [C.sub.14H.sub.13N.sub.2O.sub.2F.sub.2].sup.+[M].sup.+: m/z 278.0867. found 278.0873. LCMS(+): t.sub.ret=4.36 & 5.44 min, 279 Th[MH].sup.+, cis and trans isomers respectively (cis isomer has a shoulder at 440 nm).

North Ring Ortho-Fluoro, Meta-Nitro Derivative (I.7)

[0302] The synthesis of I.7 is presented on Scheme 12 hereafter.

##STR00054##

3-fluoro-2-nitro-4-((3,4,5-trimethoxyphenyl)diazenyl)phenol (IV.7)

[0303] By Standard Procedure A, II.1 (185 mg, 1.01 mmol) was reacted with 3-fluoro-2-nitrophenol (III.7; 162 mg, 1.03 mmol), where the reaction between the phenolate and the diazonium was stirred for 5 h in the dark in the cold to allow for better conversion of the slow-reacting materials. Chromatography on 1:1:0->5:1:0->5:1:0.5 Hx:EA:MeOH returned IV.7 (134 mg, 0.38 mmol, 38%; R.sub.f=0.16 on 1:5 Hx:EA, Han) as a red solid. .sup.1H-NMR (400 MHz): =10.68 (s, 1H), 8.02 (dd, 9.4 & 7.6 Hz, 1H), 7.26 (s, 2H), 6.98 (dd, 9.4 & 1.9 Hz, 1H), 3.90 (s, 6H), 3.88 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =157.4 (s), 155.8 (d, 279 Hz), 153.6 (2), 148.3, 141.5, 134.4 (d, 6.3 Hz), 125.8 (d, 7.4 Hz), 125.0 (d, 3.3 Hz), 114.7 (d, 4.3 Hz), 100.9 (2), 61.10, 56.3 (2) ppm. LCMS(+): t.sub.ret=4.25, 352 Th[MH].sup.+. HRMS (ESI) calcd for [C.sub.15H.sub.13N.sub.3O.sub.6F].sup.[M-H].sup.: m/z 350.0794. found 350.0796.

1-(2-fluoro-4-methoxy-3-nitrophenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.7)

[0304] By Standard Procedure C, IV.7 (130 mg, 0.37 mmol) was methylated using MeI (69 mg, 0.48 mmol) and Ag.sub.2CO.sub.3 (50% on Celite, 210 mg, 0.38 mmol) in toluene (6 mL). The crude was separated with 3:1->2:1 Hx:EA to afford 1.7 (30 mg, 0.08 mmol, 22%; R.sub.f=0.19 on 2.4:1 Hx:EA, FeCl.sub.3) as an orange solid. .sup.1H-NMR (400 MHz): =7.85 (dd, 9.3 & 8.1 Hz, 1H), 7.17 (s, 2H), 6.84 (dd, 9.4 & 1.8 Hz, 1H), 3.94 (s, 3H), 3.89 (s, 6H), 3.87 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =153.8 (d, 2.6 Hz), 153.6 (2), 152.2 (d, 270 Hz), 148.4, 141.4, 134.5 (d, 6.1 Hz), 131.4 (d, 14.6 Hz), 120.0 (d, 2.1 Hz), 107.7 (d, 3.6 Hz), 100.8 (2), 61.1, 57.2, 56.2 (2) ppm. .sup.19F-NMR (282 MHz): =132.69 (dd, 8.1 & 1.9 Hz) ppm. LCMS(+): t.sub.ret=4.18 & 4.83 min, each 366 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the first peak showed a shoulder at 445 nm whereas the second peak had a single-band structure. HRMS (ESI+) calcd for [C.sub.16H.sub.17N.sub.3O.sub.6F].sup.+[MH.sup.+]: m/z 366.1096. found 366.10944.

North Ring Heteroarylic, Meta-Pyridinyl, Derivative (I.13)

[0305] The synthesis of I.13 is presented on Scheme 13 hereafter.

##STR00055##

2,6-dimethoxy-4-((6-methoxypyridin-3-yl)diazenyl)phenol (IV.13)

[0306] By Standard Procedure A, 6-methoxypyridin-3-amine (V.13; 260 mg, 2.10 mmol) was reacted with 2,6-dimethoxyphenol (VI.13; 316 mg, 2.05 mmol). Chromatography of the red crude oil on 5:1->2.5:1 Hx:EA returned IV.13 (195 mg, 0.67 mmol, 33%; R.sub.f=0.23 on 2.4:1 Hx:EA, FeCl.sub.3) as an orange solid. .sup.1H-NMR (400 MHz, DMSO): =9.31 (s br, 1H), 8.75 (dd, 2.6 & 0.6 Hz, 1H), 8.11 (dd, 9.0 & 2.6 Hz, 1H), 7.24 (s, 2H), 6.98 (dd, 8.9 & 0.6 Hz, 1H), 3.96 (s, 3H), 3.87 (s, 6H) ppm. .sup.13C-NMR (100 MHz, DMSO): =165.2, 148.6 (2), 146.6, 144.5, 143.9, 139.9, 129.1, 112.1, 101.1 (2), 56.5 (2), 54.3 ppm. HRMS (ESI+) calcd for [C.sub.14H.sub.16N.sub.3O.sub.4].sup.+[MH].sup.+: m/z 290.11353. found 290.11351.

2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)pyridine (I.13)

[0307] By Standard Procedure B, IV.13 (191 mg, 0.66 mmol) was methylated overnight. Chromatography on 3:1->2.4:1 Hx:EA returned I.13 (61 mg, 0.20 mmol, 31% or 45% brsm; R.sub.f=0.54 on 2.4:1 Hx:EA, FeCl.sub.3) as an orange solid, followed by unreacted IV.13 (64 mg, 0.22 mmol). .sup.1H-NMR (400 MHz): =8.73 (dd, 2.6 & 0.6 Hz, 1H), 8.04 (dd, 8.9 & 2.6 Hz, 1H), 7.16 (s, 2H), 6.77 (dd, 8.9 & 0.6 Hz, 1H), 3.97 (s, 3H), 3.90 (s, 6H), 3.87 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =165.6, 153.5 (2), 148.5, 147.6, 143.8, 140.6, 128.5, 111.8, 100.3 (2), 61.1, 56.2 (2), 54.1 ppm. LCMS(+): t.sub.ret=3.57 & 4.65 min, each 304 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (ds) featured a shoulder centred around 440 nm which was absent in the second peak. HRMS (ESI+) calcd for [C.sub.15H.sub.18N.sub.3O.sub.4].sup.+[MH].sup.+: m/z 304.12918. found 304.12919.

North Ring Heteroarylic, Quinolinyl, Derivative (I.14)

[0308] The synthesis of I.14 is presented on Scheme 14 hereafter.

##STR00056##

5-((3,4,5-trimethoxyphenyl)diazenyl)quinolin-8-ol (IV.14)

[0309] By Standard Procedure A, II.1 (366 mg, 2.00 mmol) was reacted with commercial 8-hydroxyquinoline (III.14; 300 mg, 2.07 mmol). Chromatography of the red crude solid on 1:1:0->1:5:0->1:5:0.3 Hx:EA:MeOH returned IV.14 (514 mg, 1.52 mmol, 76%; R.sub.f=0.07 on 1:5 Hx:EA, FeCl.sub.3) as a deep orange solid. .sup.1H-NMR (400 MHz): =9.20 (dd, 8.5 & 1.4 Hz, 1H), 8.81 (dd, 4.2 & 1.6 Hz, 1H), 7.95 (d, 8.3 Hz, 1H), 7.56 (dd, 8.6 & 4.1 Hz, 1H), 7.26 (s, 2H), 7.20 (d overlapped, 7 Hz, 1H), 3.93 (s, 6H), 3.88 (s, 3H) ppm. .sup.13C-NMR (100 MHz) showed 2 isomers in >4:1 ratio, only the major isomer's peaks are reported: 5=155.2, 153.6 (2), 149.0, 148.5, 140.5, 139.9, 137.7, 132.9, 127.0, 122.8, 115.6, 110.1, 100.3 (2), 61.1, 56.3 (2) ppm. HRMS (ESI+) calcd for [C.sub.18H.sub.18N.sub.3O.sub.4].sup.+[MH].sup.+: m/z 340.12918. found 340.12898. LCMS(+): t.sub.ret=4.02 min, 340 Th[MH].sup.+.

8-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)quinoline (I.14)

[0310] By Standard Procedure B, IV.14 (505 mg, 1.49 mmol) was methylated overnight. Chromatography of the red crude on 1:5:0->1:5:1 Hx:EA:MeOH returned I.14 (422 mg, 1.20 mmol, 80%; R.sub.f=0.32 on 1:5:0.1 Hx:EA:MeOH, FeCl.sub.3) as a brown solid. .sup.1H-NMR (400 MHz): =9.25 (d, 8.5 Hz, 1H), 9.03-8.97 (m, 1H), 7.92 (d, 8.6 Hz, 1H), 7.59 (dd, 8.6 & 3.8 Hz, 1H), 7.25 (s, 2H), 7.12 (d, 8.6 Hz, 1H), 4.13 (s, 3H), 3.94 (s, 6H), 3.89 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =157.5, 153.6 (2), 149.4, 149.4, 149.0, 140.7, 140.7, 133.1, 127.8, 122.5, 114.0, 107.7, 100.5 (2), 61.1, 56.5, 56.3 (2) ppm. LCMS(+): t.sub.ret=2.69 & 3.55 min, each 354 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (cis) featured a very broad shoulder centred around 400 nm and extending to 480 nm at half-maximum, which was absent in the second peak. HRMS (ESI+) calcd for [C.sub.19H.sub.20N.sub.3O.sub.4].sup.+[MH].sup.+: m/z 354.14483. found 354.14462.

North Ring Heteroarylic, Quinolinyl, Derivative (I.15)

[0311] The synthesis of I.15 is presented on Scheme 15 hereafter.

##STR00057##

methyl 8-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)quinoline-2-carboxylate (I.15)

[0312] By Standard Procedure A, II.1 (187 mg, 1.02 mmol) was reacted with commercial 8-hydroxyquinoline-2-carboxylic acid (III.15; 195 mg, 1.03 mmol), using EtOAc as the extraction solvent while maintaining the aqueous phase at pH=3. Pad filtration of the black crude on 1:0->1:1 CHCl.sub.3:MeOH removed both fast-running and immobile crude components, and the resultant black crude IV.15 containing residual III.15 and other contaminants was carried over to the next step directly (247 mg; LCMS(+): t.sub.ret=4.02 min, 384 Th[MH].sup.+). Crude IV.15 (120 mg) was methylated by Standard Procedure C using MeI (88 mg, >2 equivalents). Chromatography on 5:1:0->1:1:0->1:1:1 Hx:EA:MeOH returned I.15 (19 mg, 0.046 mmol; 9% over 2 steps; R.sub.f=0.27 on 1:5 Hx:EA, UV) as an orange oil. .sup.1H-NMR (400 MHz) without precautions to block ambient light revealed a 3:1 proportion of [presumably trans:cis] isomers: =9.37 (d, 8.9 Hz, 1H.sub.Z), 9.34 (d, 8.8 Hz, 1H.sub.E), 9.04 (d, 8.7 Hz, 1H.sub.Z), 8.33 (d, 8.8 Hz, 1H.sub.E), 8.29 (d, 8.8 Hz, 1H.sub.Z), 7.99 (d, 8.6 Hz, 1H.sub.E), 7.58 (s, 2H.sub.Z), 7.25 (s, 2H.sub.E), 7.13 (d, 8.6 Hz, 1H.sub.E), 7.10 (d, 8.9 Hz, 1H.sub.Z), 4.12 (s, 3H.sub.E), 4.11 (s, 3H.sub.Z), 4.02 (s, 3H.sub.E), 4.01 (s, 3H.sub.Z), 3.94 (s, 6H.sub.E), 3.94 (s, 6H.sub.Z), 3.89 (s, 3H.sub.E), 3.88 (s, 3H.sub.Z) ppm. .sup.13C-NMR (100 MHz; only major isomer peaks are reported): =165.9, 158.4, 153.6 (2), 148.9, 147.2, 140.8, 140.5, 139.1, 133.6, 128.9, 122.3, 116.0, 107.8, 100.5 (2), 61.1, 56.6, 56.3 (2), 53.2 ppm. HRMS (ESI+) calcd for [C.sub.21H.sub.22N.sub.3O.sub.6].sup.+[MH].sup.+: m/z 412.15031. found 412.15036. LCMS(+): t.sub.ret=3.61 & 4.47 min, 412 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (cis) featured a shoulder centred around 455 nm.

(I.10)a Water-Soluble, Peptidase-Activatable Prodrug of (I.1)

[0313] The synthesis of I.10 is presented on Scheme 16 hereafter.

##STR00058##

[0314] Caution: phosgene, liberated by amine-mediated decomposition of triphosgene, has boiling point 8 C., is highly toxic and corrosive and can react violently with water or other nucleophiles especially if the reaction is in homogenous media. Reactions were kept cold to avoid boil-off of phosgene. Excess phosgene was caught apparently quantitatively during evaporation in a primary liquid nitrogen trap (a backup trap was employed but always found empty); it was destroyed when still cold by its dropwise addition to a vigorously stirred, cold mixture of 2-aminoethanol or piperidine (1 mL) and ethanol (5 mL) in dichloromethane (20 mL) in a well-ventilated hood.

(2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenyl) 2-((L-leucinamido)methyl)piperidine-1-carboxylate 2,2,2-trifluoroacetate salt (I.10)

[0315] To a solution of I.1 (13 mg, 0.041 mmol) in CH.sub.2Cl.sub.2 (3 mL) under nitrogen atmosphere in an ice bath, were added a solution of triphosgene (60 mg, 0.20 mmol) in CH.sub.2Cl.sub.2 (1 mL), then, dropwise, triethylamine (0.10 mL). The solution was stirred in the cold for 30 min then the volatiles were evaporated at high vacuum. To the residue under nitrogen were added a solution of NEt.sub.3 (0.15 mL) and N-tert-butoxycarbonyl-L-leucyl-(piperidin-2-ylmethyl)amide (S1, 16 mg, 0.045 mmol; prepared according to known procedure.sup.[22]) in CH.sub.2Cl.sub.2 (3 mL), and the mixture stirred for 2 h at room temperature. The volatiles were evaporated, and a solution of TFA (2 mL) in DCM (2 mL) was added. The purple solution was stirred at room temperature for 30 min. The volatiles were removed at 0.4 mbar until the purple residue had become yellow-brown, indicating removal of excess TFA. Chromatography on 5:1:0->1:1:0->1:1:1 Hx: EA: MeOH returned I.10 2,2,2-trifluoroacetate salt (9.5 mg, 0.014 mmol, 34%; R.sub.f=0.54 on 1:1:1 Hx:EA:MeOH, FeCl.sub.3) as a brown viscous oil. .sup.1H-NMR (400 MHz, DMSO): =7.87 (dd, 8.7 & 2.4 Hz, 1H), 7.63 (d, 2.5 Hz, 1H), 7.32 (d, 8.9 Hz, 1H), 7.23 (s, 2H), 6.21 (s, 1H), 3.90 (s, 3H), 3.90 (s, 3H), 3.89 (s, 6H), 4.51-4.23 (m, 2H), 3.86-3.02 (m, 4H overlapped), 1.82-1.61 (m, 1H), 1.58-1.34+1.12-1.06 (m+m, 8H), 0.85-0.80 (m, 6H) ppm. LCMS(+): t.sub.ret=2.92 & 3.41 min, each 572 Th[MH].sup.+; the first peak was assigned as the as isomer due to its absorbance shoulder centred at 450 nm. HRMS (ESI+) calcd for [C.sub.29H.sub.42N.sub.5O.sub.7].sup.+[MH].sup.+: m/z 572.30788. found 572.30867.

Azoombrabulin (I.20)a Water-Soluble Prodrug of (I.2)

[0316] The synthesis of I.20 is presented on Scheme 17 hereafter.

##STR00059##

(2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenyl))-1-(N-tert-butoxycarbonyl)L-serinamide (I.22)

[0317] To commercial N-tert-butoxycarbonyl-L-serine (65 mg, 0.32 mmol) in an icebath were added DCM (15 mL), HOBt (50 mg, 0.37 mmol) and DCC (68 mg, 0.33 mmol), and the mixture stirred for 5 min. I.2 (100 mg, 0.31 mmol) was added, and stirring continued overnight while warming slowly to room temperature. The organic phase was washed with saturated aqueous sodium carbonate solution (20 mL), pH=10 phosphate buffer (20 mL), and brine (20 mL), then dried on Na.sub.2SO.sub.4, filtered, concentrated and chromatographed on 5:1->1:5 Hx:EA, returning I.22 (70 mg, 0.14 mmol, 43%; R.sub.f=0.24 & 0.12 on 1:1 Hx:EA, trans & cis isomers, FeCl.sub.3) as a brown solid. .sup.1H-NMR (400 MHz): =8.87 (d, 2.4 Hz, 1H), 7.65 (dd, 8.6 & 2.4 Hz, 1H), 7.18 (s, 2H), 6.94 (d, 8.8 Hz, 1H), 5.61 (s br, 1H), 4.38-4.12 (m, 3H), 3.90 (s, 3H), 3.89 (s, 6H), 3.86 (s, 3H), 1.44 (s, 9H) ppm. .sup.13C-NMR (100 MHz): =169.7, 157.4, 153.5 (2), 150.5, 148.5, 146.7, 140.2, 127.7, 121.2, 112.9, 109.9, 100.2 (2), 80.8, 62.7, 61.0, 56.2 (2), 56.1, 50.1, 28.3 (3) ppm. HRMS (ESI+) calcd for [C.sub.24H.sub.33N.sub.4O.sub.8].sup.+[MH].sup.+: m/z 505.22929. found 505.22945. LCMS(+): t.sub.ret=3.52 & 4.22 min, each 505 Th=[MH].sup.+, cis and trans isomers respectively.

(2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenyl))-1-L-serinamide 2,2,2-trifluoroacetate salt (I.20)

[0318] To I.22 (55 mg, 0.11 mmol) were added DCM (1 mL) and TFA (1 mL) and the purple solution stirred at room temperature for 1 hr. The volatiles were removed at 1 mbar until a brown crude oil was obtained. Chromatography on 1:1:0->1:1:0.3 Hx:EA:MeOH returned unreacted I.22 (18 mg, 0.036 mmol) then I.20 2,2,2-trifluoroacetate salt (21 mg, 0.041 mmol, 37% or 55% brsm; R.sub.f=0.35 on 1:1:0.3 Hx:EA:MeOH, FeCl.sub.3) as a brown viscous oil. .sup.1H-NMR (400 MHz, DMSO): =8.63 (d, 2.5 Hz, 1H), 8.38 (s br, 3H), 7.80 (dd, 8.7 & 2.5 Hz, 1H), 7.32 (d, 8.9 Hz, 1H), 7.23 (s, 2H), 5.70 (s br, 1H), 4.70 (dd, 9.1 & 4.2 Hz, 1H), 4.54 (t, 8.9 Hz, 1H), 4.34 (dd, 8.8 & 4.1 Hz, 1H), 3.99 (s, 3H), 3.91 (s, 6H), 3.77 (s, 3H) ppm. .sup.13C-NMR (100 MHz, DMSO): =172.0, 158.0 (q, 31.6 Hz), 153.4 (2), 152.1, 147.8, 145.4, 139.9, 127.2, 122.5, 117.1 (q, 294 Hz), 113.7, 111.4, 99.9 (2), 60.5, 60.2, 56.4, 56.0 (2), 54.5 ppm. HRMS (ESI+) calcd for [C.sub.19H.sub.25N.sub.4O.sub.6].sup.+[MH].sup.+: m/z 405.17686. found 405.17695. LCMS(+): t.sub.ret=2.30-2.65 min (broad), 405 Th[MH].sup.+.

North Ring Ortho-Oxy-Linker-Bearing Derivatives (I.18) and (I.19)

[0319] The syntheses of I.18 and I.19 are presented on Scheme 18 hereafter.

##STR00060##

2-(3-methoxy-2-methyl-6-((3,4,5-trimethoxyphenyl)diazenyl)phenoxy)ethan-1-ol (I.18)

[0320] To I.8 (72 mg, 0.23 mmol) were added 2-bromoethanol (360 mg, 2.9 mmol), K.sub.2CO.sub.3 (400 mg, 2.8 mmol), and DMF (6 mL) and the mixture stirred overnight. Water (60 mL) was added and the aqueous phase extracted with EtOAc (215 mL); the combined organic layers were washed with water (10 mL), aqueous LiCl (10%, 5 mL), and brine (15 mL), then dried on Na.sub.2SO.sub.4, filtered and concentrated. After column chromatography on 5:1->1:1 Hx:EA, I.18 (44 mg, 0.12 mmol, 54%; R.sub.f=0.18 on 1:1 Hx:EA, Han) was returned as a red oil. .sup.1H-NMR (400 MHz, CD.sub.3CN): =7.67 (d, 9.0 Hz, 1H), 7.20 (s, 2H), 6.75 (d, 2.6 Hz, 1H), 6.64 (dd, 9.0 & 2.6 Hz, 1H), 4.27 (dd, 5.3 & 4.3 Hz, 2H), 3.92 (s, 6H), 3.91-3.87 (m overlapped, 2H), 3.88 (s, 3H), 3.82 (s, 3H) ppm. .sup.13C-NMR (100 MHz, CD.sub.3CN): =164.3, 158.9, 154.2 (2), 149.6, 140.5, 137.3, 118.3, 107.8, 101.9, 100.5 (2), 72.5, 61.0, 60.6, 56.3 (2), 56.1 ppm. HRMS (ESI+) calcd for [C.sub.18H.sub.23N.sub.2O.sub.6].sup.+[MH].sup.+: m/z 363.38945. found 363.15459. LCMS(+): t.sub.ret=3.12 & 3.85 min, each 363 Th[MH].sup.+; the first peak was assigned as the as isomer due to its absorbance shoulder centred at 445 nm.

2-(5-methoxy-2-((3,4,5-trimethoxyphenyl)diazenyl)phenoxy)ethan-1-ol (I.19)

[0321] To I.17 (150 mg, 0.45 mmol) were added 2-bromoethanol (220 mg, 1.8 mmol), K.sub.2CO.sub.3 (250 mg, 1.8 mmol), and DMF (5 mL) and the mixture stirred overnight. Water (20 mL) and aqueous KH.sub.2PO.sub.4 solution (10%, 5 mL) were added and the aqueous phase extracted with Et.sub.2O (320 mL); the combined organic layers were washed with water (10 mL), aqueous LiCl (10%, 10 mL), and brine (10 mL), then dried on Na.sub.2SO.sub.4, filtered and concentrated. After column chromatography on 5:1->1:1 Hx:EA, 1.19 (114 mg, 0.30 mmol, 67%; R.sub.f=0.72 on 1:5 Hx:EA, Van) was returned as a red oil. .sup.1H-NMR (400 MHz): =7.58 (dd, 9.1 Hz, 1H), 7.14 (s, 2H), 6.66 (d, 9.1 Hz, 1H), 4.22-4.16 (m, 2H), 3.89 (s, 6H), 3.85 (s, 3H), 3.84 (s, 4H), 3.84-3.80 (m, 2H), 2.18 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =161.7, 156.3, 153.6 (2), 148.5, 140.4, 140.0, 120.7, 115.4, 106.5, 100.2 (2), 61.7, 61.1, 56.3 (2), 56.0, 9.3 ppm. HRMS (ESI+) calcd for [C.sub.19H.sub.25N.sub.2O.sub.6].sup.+[MH].sup.+: m/z 377.17071. found 377.17016. LCMS(+): t.sub.ret=3.48 & 4.37 min, 377 Th=[MH].sup.+; the first peak was assigned as the cis isomer due to its absorbance shoulder centred at 450 nm.

(I.24)a Phosphatase-Activated Prodrug of (I.1)

[0322] The syntheses of I.24 and its precursor IV.24 were carried out similarly to a described procedure.sup.[19], as is presented on Scheme 19 hereafter.

##STR00061##

dibenzyl (2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenyl) phosphate (IV.24)

[0323] Similarly to the described procedure.sup.[19], I.1 (100 mg, 0.31 mmol) was dissolved in dry acetonitrile (4 mL) under nitrogen, then the solution cooled to 30 C. CCl.sub.4 (242 mg, 1.57 mmol) was added, then NEt.sub.3 which had been stood on KOH (71 mg), and 4-(N,N-dimethylamino)pyridine (DMAP; 5 mg). Dibenzyl phosphite (122 mg, 0.46 mmol) was then added dropwise. The reaction was stirred for 3 h at 30 C., then as LCMS showed incomplete conversion of the starting material, additional dibenzyl phosphite (180 mg, 0.68 mmol) and CCl.sub.4 (300 mg, 1.94 mmol) were added. The mixture was stirred warming to room temperature overnight. Aqueous KH.sub.2PO.sub.4 solution (10%, 10 mL) was added and the aqueous phase extracted with EtOAc (410 mL). The combined organic layers were washed with water (10 mL), brine (10 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated. The crude oil thus obtained was chromatographed on 5:1->1:1 Hx:EA, giving IV.24 (105 mg, 0.18 mmol, 59%; R.sub.f=0.38 and 0.22 on 1:1 Hx:EA (trans and cis isomers), Han) as a yellow oil. .sup.1H-NMR (400 MHz): =7.82 (dd, 2.3 & 1.5 Hz, 1H), 7.73 (ddd, 8.7 & 2.4 & 1.0 Hz, 1H), 7.33-7.21 (m, 10H), 7.14 (s, 2H), 6.96 (dd, 8.8 & 1.0 Hz, 1H), 5.14 (d, 7.9 Hz, 4H), 3.90 (s, 6H), 3.87 (s, 3H), 3.80 (s, 3H) ppm. .sup.13C-NMR (100 MHz) showed some peaks split, perhaps for diastereotopicity around the phosphoester: =153.5 (2), 153.1 & 153.0 (1), 148.3, 146.3 & 146.3 (1), 140.4, 140.2 & 140.1 (1), 135.6 & 135.6 (2), 128.6 (4), 128.5 (2), 127.9 (4), 123.2 & 123.2 (1), 114.2 & 114.2 (1), 112.0, 100.3 (2), 70.0 & 70.0 (2), 61.1, 56.2 (2), 56.2 ppm. HRMS (ESI+) calcd for [C.sub.30H.sub.32N.sub.2O.sub.8P].sup.+[MH].sup.+: m/z 579.18908. found 579.18938. LCMS(+): t.sub.ret=4.69 & 5.27 min, 579 Th[MH].sup.+; the first peak was assigned as the cis isomer due to its absorbance shoulder centred at 450 nm.

2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenyl phosphate disodium salt (I.24)

[0324] Similarly to a described, analogous procedure.sup.[19], to IV.24 (100 mg, 0.165 mmol) were added under nitrogen, NaI (49 mg, 0.33 mmol), dry acetonitrile (2.5 mL), and TMSCl (37 mg, 0.34 mmol). The mixture was stirred for 4 h at room temperature. Water (1.5 mL) and aqueous Na.sub.2S.sub.2O.sub.3 solution (10%, 0.05 mL) were added and the aqueous phase extracted with EtOAc (310 mL). The combined organic layers were dried on Na.sub.2SO.sub.4, filtered and concentrated to a red crude oil. To the crude oil were added under nitrogen, dry MeOH (3 mL) and NaOMe (18.5 mg) and the reaction was stirred overnight. The volatiles were evaporated, and the yellow oily residue (principally containing 1.24 disodium salt together with its monobenzyl ester, which was identified by LCMS as the peak with t.sub.ret=3.08 min, 489 Th) was repeatedly triturated with cyclohexane (33 mL), 1:3 cyclohexane:ethyl acetate (52 mL), ethyl acetate (22 mL), and lastly acetone (22 mL), leaving 1.24 disodium salt as a yellow-brown powder (25 mg, 0.056 mmol, 34%) which was fully soluble in PBS to at least 25 mM. .sup.1H-NMR (400 MHz, D.sub.2O): =8.33 (s, 1H), 7.63 (s, 1H), 7.39 (d, 8.7 Hz, 1H), 6.98 (d, 9.5 Hz, 1H), 6.97 (s, 2H), 3.80 (s, 3H), 3.77 (s, 6H), 3.70 (s, 3H) ppm. .sup.13C-NMR (100 MHz, D.sub.2O): =171.0, 153.4 & 153.3 (1), 152.7, 148.2, 145.5, 142.5 & 142.4 (1), 138.8, 128.5 & 127.6 (1), 121.5, 112.1 (2), 100.0 (2), 60.9, 55.9 (2), 55.9 ppm. HRMS (ESI) calcd for [C.sub.16H.sub.18N.sub.2O.sub.8P].sup.[M-H].sup.: m/z 397.08008. found 397.08029. LCMS(+): t.sub.ret=2.04 & 2.44 min, 399 Th=[MH].sup.+; the first peak was assigned as the as isomer due to its absorbance shoulder centred at 450 nm.

North Ring Ortho-Linked Fluorophore-Bearing Derivative (I.25)

[0325] The synthesis of I.25 is presented on Scheme 20 hereafter.

##STR00062## ##STR00063##

3-(3-methoxy-2-methyl-6-((3,4,5-trimethoxyphenyl)diazenyl)phenoxy)propan-1-ol (I.21)

[0326] To I.17 (165 mg, 0.50 mmol) were added NaI (15 mg, 0.1 mmol), 3-bromopropanol (210 mg, 1.53 mmol), K.sub.2CO.sub.3 (207 mg, 1.46 mmol), and DMF (6 mL) and the mixture stirred overnight at room temperature. Water (10 mL), aqueous LiCl (10%, 10 mL) and aqueous KH.sub.2PO.sub.4 solution (10%, 10 mL) were added and the aqueous phase extracted with CHCl.sub.3 (10 mL) then Et.sub.2O (215 mL); the combined organic layers were washed with water (10 mL), aqueous LiCl (10%, 10 mL), and brine (10 mL), then dried on Na.sub.2SO.sub.4, filtered and concentrated. After column chromatography on 5:1->1:1 Hx:EA, 1.21 (158 mg, 0.405 mmol, 81%; R.sub.f=0.35 and 0.12 on 1:1 Hx:EA (trans and cis isomers), Van) was returned as a yellow oil. .sup.1H-NMR (400 MHz): =7.61 (d, 9.0 Hz, 1H), 7.22 (s, 2H), 6.66 (d, 9.1 Hz, 1H), 4.15 (t, 5.6 Hz, 2H), 3.91 (s, 6H), 3.91-3.77 (m, 2H), 3.86 (s, 3H), 3.84 (s, 3H), 2.18 (s, 3H), 2.03 1.98 (m, 2H) ppm. .sup.13C-NMR (100 MHz): =161.5, 156.6, 153.6 (2), 148.6, 140.3, 139.9, 120.6, 115.2, 106.4, 100.3 (2), 75.0, 61.6, 61.1, 56.2 (2), 55.9, 32.3, 8.96 ppm. HRMS (ESI+) calcd for [C.sub.20H.sub.27N.sub.2O.sub.6].sup.+[MH].sup.+: m/z 391.18636. found 391.18604. LCMS(+): t.sub.ret=3.52 & 4.48 min, 391 Th=[MH].sup.+; the first peak was assigned as the as isomer due to its absorbance shoulder centred at 450 nm.

N-(6-(diethylamino)-9-(2-(4-(3-(3-methoxy-2-methyl-6-((3,4,5-trimethoxyphenyl)diazenyl)phenoxy)propyl)piperazine-1-carbonyl)phenyl)-3H-xanthen-3-ylidene)-N-ethylethanaminium bis(formate) salt (I.25)

[0327] Known compound N-(6-(diethylamino)-9-(2-(piperazine-1-carbonyl)phenyl)-3H-xanthen-3-ylidene)-N-ethylethanaminium chloride (abbreviated RBpip chloride) was made by the reported method and confirmed by NMR.sup.[46].

[0328] To I.21 (140 mg, 0.36 mmol) were added CH.sub.2Cl.sub.2 (5 mL), Et.sub.3N (89 mg, 0.88 mmol), 4-dimethylaminopyridine (10 mg), and TsCl (69 mg, 0.36 mmol), and the mixture stirred at room temperature for 4 hours until TLC indicated complete conversion of the starting material, presumably to the tosylate IV.21. After evaporation of the volatiles, to the residue were added EtOH (15 mL), NaI (20 mg), RBpip chloride (160 mg, 0.29 mmol) and NEt.sub.3 (81 mg), and the mixture stirred at 80 C. for 2 days under closed air atmosphere. After cooling and evaporation of the volatiles, the crude residue was chromatographed on a small volume of silica gel to separate the bulk of the impurities, using a gradient of 1:1:0:0->1:1:1:0->1:1:0:0->1:1:0:1->0:0:0:1->0:0:1:1 Hx:EA:MeOH:CH.sub.2Cl.sub.2. The third red fraction to elute contained the pink-fluorescent product cation of 1.25 (R.sub.f=0.0 on 1:1:0.2 Hx:EA:MeOH, 0.25<R.sub.f<0.4 on 9:1 DCM:MeOH) as well as substantial impurities. This fraction was then concentrated (dry weight: 45 mg) then separated by semi-preparative HPLC on reverse-phase column with a 10:90->60:40 MeCN:water eluent gradient (water component contains 0.1% formic acid), and pure I.25 as the bis(formate) salt (2.0 mg, 2.0 mol, 1%) was recovered from the pure fractions as a dark purple solid. .sup.1H-NMR (400 MHz, CD.sub.3CN) showed overlapping peaks from two components at roughly 5:2 ratio (possibly conformers about the piperazine moiety, as suggested by the .sup.13C-NMR spectrum): =8.37 (s, 2H), 7.74-7.70 (m, 2H), 7.63-7.58 (m, 2H), 7.47-7.42 (m, 1H), 7.23 (s, 2H), 7.19 (s, 1H), 7.00-6.94 (m, 2H), 6.87-6.82 (m, 4H), 4.16 (t, 6.3 Hz, 2H), 3.91 (s, 3H), 3.90 (s, 6H), 3.82 (s, 3H), 3.66-3.58 (m, 11H), 3.36-3.24 (m, 5H), 2.47-2.40 (m, 2H), 2.20 (s, 3H), 2.01-1.96 (m, 2H), 1.25 (t, 7.1 Hz, 12H) ppm. .sup.13C-NMR (100 MHz): =167.2, 165.0 (2), 161.8, 158.3 (2), 157.5, 156.7, 156.2 (2), 154.3 (2), 149.6, 140.7, 140.2, 136.6, 132.7 (2), 131.2, 130.5, 130.4, 130.0, 128.0, 120.5, 114.8 (2), 114.6 (2), 114.2, 106.8, 100.6 (2), 96.4 (2), 74.6, 60.6, 56.4 (2), 56.3, 55.2, 53.3, 52.9, 48.8, 47.7, 46.2 (4), 27.9, 12.4 (4), 8.9 ppm. LCMS(+): t.sub.ret=3.25 & 3.45 min, each 883 Th[MH].sup.+: peaks assigned as cis & trans isomers respectively since the second peak has substantially greater absorbance at 390 nm. HRMS (ESI+) calcd for [C.sub.52H.sub.63N.sub.6O.sub.7].sup.+[M].sup.+: m/z 883.47582. found 883.47453.

Meta-Hydro, Ortho-Anilide Derivative (I.26)

[0329] The synthesis of I.26 is presented on Scheme 21 hereafter.

##STR00064##

N-(5-methoxy-2-((3,4,5-trimethoxyphenyl)diazenyl)phenyl)acetamide (I.26)

[0330] To I.16 (51 mg, 0.16 mmol) were added pyridine (5 mL) and acetic anhydride (0.5 mL) and the mixture stirred overnight. After evaporation of the volatiles at 2 mbar and 30 C., the residue was partitioned between aqueous HCl (1 M, 5 mL) and EtOAc (5 mL), then the aqueous layer was extracted with EtOAc (210 mL); the combined organic layers were washed with aqueous HCl (1 M, 5 mL) and brine (5 mL), then dried on Na.sub.2SO.sub.4, filtered and concentrated to an olive powder which was spectroscopically pure I.26 (56 mg, 0.16 mmol, 97%; R.sub.f=0.15 on 2.4:1 Hx:EA (trans and as isomers overlapped), FeCl.sub.3). .sup.1H-NMR (400 MHz): =10.53 (s, 1H), 8.24 (d, 2.7 Hz, 1H), 7.76 (d, 9.0 Hz, 1H), 7.04 (s, 2H), 6.65 (dd, 9.0 & 2.8 Hz, 1H), 3.89 (s, 6H), 3.87 (s, 3H), 3.84 (s, 3H), 2.20 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =168.8, 163.3, 153.7 (2), 148.2, 140.4, 137.1, 133.1, 124.9, 110.8, 103.7, 99.7 (2), 61.2, 56.2 (2), 55.8, 25.6 ppm. HRMS (ESI) calcd for [C.sub.18H.sub.20N.sub.3O.sub.5].sup.[M-H].sup.: m/z 358.14030. found 358.14059. LCMS(+): t.sub.ret=3.12 & 4.41 min, each 360 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (cis) featured a shoulder centred around 450 nm which was absent in the second peak.

Meta-Bromo Derivative (I.27)

[0331] The synthesis of I.27 is presented on Scheme 22 hereafter.

##STR00065##

2-bromo-4-((3,4,5-trimethoxyphenyl)diazenyl)phenol (IV.27)

[0332] By Standard Procedure A, II.1 (3.67 g, 20.0 mmol) was reacted with commercial 2-bromophenol (III.27; 3.46 g, 20.0 mmol). Chromatography of the orange crude oil on 5:1->1:1 Hx:EA returned IV.27 (3.52 g, 9.62 mmol, 48%; R.sub.f=0.66 on 1:1 Hx:EA, Han) as a yellow solid. .sup.1H-NMR (400 MHz): =8.03 (d, 2.3 Hz, 1H), 7.80 (dd, 8.7 & 2.3 Hz, 1H), 7.15 (s, 2H), 7.09 (d, 8.7 Hz, 1H), 3.89 (s, 6H), 3.87 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =154.4, 153.5 (2), 148.2, 147.1, 140.6, 125.5, 125.5, 116.1, 111.1, 100.3 (2), 61.1, 56.2 (2) ppm. HRMS (ESI+) calcd for [C.sub.15H.sub.16N.sub.2O.sub.4Br].sup.+[MH].sup.+: m/z 367.02880. found 367.02857. LCMS(+): t.sub.ret=4.38 min, 367 and 369 Th=[MH].sup.+.

1-(3-bromo-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.27)

[0333] By Standard Procedure B, IV.27 (2.40 g, 6.54 mmol) was methylated overnight. Chromatography on 5:1->1:1 Hx:EA returned I.27 (2.37 g, 6.21 mmol, 95%; R.sub.f=0.69 and 0.51 on 1:1 Hx:EA, trans and as isomers, FeCl.sub.3) as orange crystals. .sup.1H-NMR (400 MHz): =8.19 (d, 2.3 Hz, 1H), 7.94 (dd, 8.7 & 2.4 Hz, 1H), 7.24 (s, 2H), 7.05 (d, 8.8 Hz, 1H), 4.01 (s, 3H), 3.99 (s, 6H), 3.96 (s, 3H ppm. .sup.13C-NMR (100 MHz): =157.8, 153.5 (2), 148.3, 146.9, 140.5, 126.0, 125.6, 112.7, 111.4, 100.3 (2), 61.1, 56.6, 56.2 (2) ppm. LCMS(+): t.sub.ret=4.20 & 5.28 min, each 381 and 383 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (cis) featured a shoulder centred around 445 nm which was absent in the second peak. HRMS (ESI+) calcd for [C.sub.16H.sub.18H.sub.2O.sub.4Br].sup.+[MH].sup.+: m/z 381.04445. found 381.04419.

Meta-Electrophile Derivatives (I.28) and (I.29)

[0334] Compound (I.27) could be used as a convenient starting point for divergent synthesis of a variety of meta-substituted polar derivatives, via lithium-halogen exchange followed by a range of electrophilic quenches. The syntheses of I.28 and I.29 are presented on Scheme 23 hereafter.

##STR00066##

(2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)phenyl)lithium stock solution ALi

[0335] To I.27 (1.20 g, 3.15 mmol) under nitrogen at 80 C. were added dry THF (9 mL) and, dropwise, n-butyllithium (2.5 M in hexanes, 1.32 mL, 3.30 mmol). The solution darkened significantly over the course of the addition. This stock solution ALi of the azoaryllithium intermediate (approximately 0.30 M) was aged at 80 C. for 1 hour, then used without further delay.

2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)benzaldehyde (I.28)

[0336] To a solution of DMF (0.2 mL) in dry THF (3 mL) at 80 C. under nitrogen was added dropwise the stock solution ALi (1.0 mL, approx. 0.30 mmol). The solution was warmed to room temperature and stirred for 30 min, then quenched by its dropwise addition into a rapidly-stirred mixture of aqueous KH.sub.2PO.sub.4 solution (10%, 15 mL) and Et.sub.2O (10 mL). The aqueous layer was extracted with Et.sub.2O (210 mL), then the combined organic layers were washed with water (10 mL), brine (10 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated. The brown crude oil was separated by chromatography on 5:1->1:1 Hx:EA gradient yielding 1.28 (67 mg, 0.21 mmol, 67%; R.sub.f=0.50 and 0.29 on 1:1 Hx:EA, trans and cis isomers, Han) as a yellow oil. .sup.1H-NMR (400 MHz): =10.45 (s, 1H), 8.34 (d, 2.6 Hz, 1H), 8.08 (dd, 8.9 & 2.6 Hz, 1H), 7.17 (s, 2H), 7.06 (d, 8.9 Hz, 1H), 3.96 (s, 3H), 3.90 (s, 6H), 3.86 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =189.4, 163.3, 153.5 (2), 148.3, 146.3, 140.6, 130.0, 125.0, 123.4, 112.1, 100.3 (2), 61.1, 56.2 (3) ppm. HRMS (ESI+) calcd for [C.sub.17H.sub.19N.sub.2O.sub.5].sup.+[MH].sup.+: m/z 331.12885. found 331.12855. LCMS(+): t.sub.ret=3.61 & 4.59 min, each 331 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (cis) featured a shoulder centred around 440 nm which was absent in the second peak.

2-methoxy-5-((3,4,5-trimethoxyphenyl)diazenyl)benzoic acid (I.29)

[0337] To a mixture of solid CO.sub.2 (1 g) in dry THF (5 mL) at 80 C. under nitrogen was added dropwise the stock solution ALi (1.0 mL, approx. 0.30 mmol). The drops of ALi lightened in colour immediately upon leaving the needle. The resultant yellow solution was warmed to room temperature and stirred for 5 min, then poured into a rapidly-stirred mixture of aqueous KH.sub.2PO.sub.4 solution (10%, 15 mL) and Et.sub.2O (10 mL), rinsing the flask once with Et.sub.2O (5 mL). The aqueous layer was extracted with Et.sub.2O (210 mL), then the combined organic layers were washed with water (10 mL), brine (10 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated. The brown crude oil was separated by chromatography on 5:1:0->1:1:0->1:1:0.5 Hx:EA:MeOH gradient yielding 1.29 (48 mg, 0.14 mmol, 46%; R.sub.f=0.13 on 1:1:0.5 Hx:EA:MeOH, trans and as isomers overlap, Han) as a brown solid. .sup.1H-NMR (400 MHz, CD.sub.3CN): =8.46 (d, 2.6 Hz, 1H), 8.13 (dd, 8.9 & 2.6 Hz, 1H), 7.35 (d, 8.9 Hz, 1H), 7.30 (s, 2H), 4.09 (s, 3H), 3.93 (s, 6H), 3.83 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =165.6, 159.7, 153.6 (2), 148.3, 147.0, 140.9, 129.0, 128.6, 118.4, 112.3, 100.5 (2), 61.2, 57.2, 56.3 (2) ppm. HRMS (ESI+) calcd for [C.sub.17H.sub.19N.sub.2O.sub.6].sup.+[MH].sup.+: m/z 347.12376. found 347.12343. LCMS(+): t.sub.ret=2.95 & 3.69 min, each 347 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (cis) featured a shoulder centred around 440 nm which was absent in the second peak.

Para-Trifluoromethoxy Derivative (I.30) Formed by Mills Reaction

[0338] The synthesis of I.30 is presented on Scheme 24 hereafter.

##STR00067##

1-(4-(trifluoromethoxy)phenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.30)

[0339] To commercial 4-(trifluoromethoxy)aniline V.30 (250 mg, 1.41 mmol) were added DCM (10 mL) and water (10 mL). Oxone (867 mg, 2.83 mmol) was added and the mixture was stirred vigorously at room temperature for 16 hours. The organic layer was separated, washed with aqueous HCl (1M, 210 mL) and brine (10 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated quickly to 40 mbar at 40 C. on the rotavap. Glacial acetic acid (6 mL) and II.1 (256 mg, 1.40 mmol) were added and the mixture stirred at 60 C. for 6 hours. The reaction was neutralised by pouring into a saturated solution of NaHCO.sub.3 and K.sub.2CO.sub.3 (50 mL), then extracted with EtOAc (220 mL). The combined organic layers were washed with brine (10 mL), dried on Na.sub.2SO.sub.4, filtered and concentrated. The crude residue was separated by chromatography with 10:1->2.4:1 Hx:EA gradient, giving I.30 (25 mg, 0.070 mmol, 5%; R.sub.f=0.75 and 0.58 on 2.4:1 Hx:EA, Han) as a red solid. .sup.1H-NMR (400 MHz): =7.87 (d, 8.9 Hz, 2H), 7.28 (d, 8.0 Hz, 1H), 7.18 (s, 2H), 3.90 (s, 6H), 3.87 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =153.6 (2), 150.7, 150.7, 148.3, 141.0, 124.2 (2), 121.3 (2), 120.4 (q, 258 Hz), 100.5 (2), 61.1, 56.2 (2) ppm. HRMS (ESI+) calcd for [C.sub.16H.sub.16N.sub.2O.sub.4F.sub.3].sup.+[MH].sup.+: m/z 357.10567. found 357.1054. LCMS(+): t.sub.ret=4.53 and 5.41 min, 357 Th=[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (cis) featured a shoulder centred around 435 nm which was absent in the second peak.

Prior Art Hydro-Derivative (I.6)

[0340] Compound I.6.sup.[13,14] was also synthesized, and its synthesis (presented on Scheme 25 hereafter) and properties are therefore reported.

##STR00068##

4-((3,4,5-trimethoxyphenyl)diazenyl)phenol (IV.6).SUP.[13]

[0341] By Standard Procedure A, II.1 (174 mg, 0.95 mmol) was reacted with commercial phenol (III.6; 102 mg, 1.08 mmol). Chromatography of the red crude oil on 5:1->2.4:1 Hx:EA returned IV.6 (227 mg, 0.78 mmol, 82%; R.sub.f=0.64 on 1:1 Hx:EA, FeCl.sub.3) as a red oil. .sup.1H-NMR (400 MHz): =7.81 (d, 8.8 Hz, 2H), 7.19 (s, 2H), 6.89 (d, 8.9 Hz, 2H), 3.89 (s, 6H), 3.86 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =159.1, 153.5 (2), 148.2, 146.6, 140.2, 125.1 (2), 116.0 (2), 100.1 (2), 61.1, 56.2 (2) ppm. HRMS (ESI+) calcd for [C.sub.15H.sub.17N.sub.2O.sub.4].sup.+[MH].sup.+: m/z 289.11828. found 289.11813.

1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)diazene (I.6).SUP.[13]

[0342] By Standard Procedure B, IV.6 (226 mg, 0.77 mmol) was methylated overnight. Chromatography on 5:1->3:1 Hx:EA returned I.6 (231 mg, 0.76 mmol, 97%; R.sub.f=0.76 and 0.60 on 1:1 Hx:EA, trans and cis isomers, FeCl.sub.3) as a red oil. .sup.1H-NMR (400 MHz): =7.85 (d, 8.9 Hz, 2H), 7.16 (s, 2H), 6.95 (d, 9.0 Hz, 2H), 3.90 (s, 6H), 3.86 (s, 3H), 3.83 (s, 3H) ppm. .sup.13C-NMR (100 MHz): =162.0, 153.5 (2), 148.5, 146.7, 140.2, 124.7 (2), 114.3 (2), 100.1 (2), 61.1, 56.2 (2), 55.6 ppm. LCMS(+): t.sub.ret=3.80 & 4.78 min, each 303 Th[MH].sup.+: these peaks were assigned to the cis & trans isomers respectively since the UV absorption profile of the first peak (cis) featured a shoulder centred around 440 nm which was absent in the second peak. HRMS (ESI+) calcd for [C.sub.16H.sub.19N.sub.2O.sub.4].sup.+[MH].sup.+: m/z 303.13393. found 303.13371.

EXAMPLES PART B

Photocharacterisation In Vitro

Rationale for In Vitro Studies:

[0343] The purpose of these in vitro photocharacterisations was to analyse and compare qualitatively the behaviour of the synthesized azoaryls according to the invention, as regards important factors for their photoisomerisation, under conditions which could be easily measured, and which could most easily and generally be translated into qualitatively correct behaviour in cellulo and hopefully in vivo. Therefore favoured measurement conditions were taken at 37 C. and in aqueous solution at pH7 containing a minimum of cosolvent. As discussed in the literature.sup.[7], three key isomerisation parameters for sophisticated biological applications were: (1) PSS(), the fraction of as isomer established in a sample at the photostationary state (when the trans<->cis photoisomerisations are in equilibrium under saturating photon flux) as a function of wavelength. PSS() depends strongly on the relative ratio between .sub.E() and .sub.Z() (the absorption coefficients of the trans and cis forms at wavelength ), among other factors; (2) the relative efficiency E() of approaching the PSS() as a function of the applied wavelength . E() reflects the magnitude of the photon flux one would need to apply to photoisomerise a mixture of trans and cis forms from a starting cis/trans ratio by a given percentage towards the cis/trans ratio at PSS(). E() depends strongly on the absolute magnitudes of both .sub.E() and .sub.Z(), among other factors; (3) the thermal reversion halflife for the spontaneous cis->trans isomerisation.

[0344] PSS(), E() and inform the design of lighting conditions for realistic long-term biological experiments where samples should not be irradiated at high intensities or with high net flux.sup.[10,47]. To illustrate: a typical biological study examining light-dependent inhibition of tubulin polymerisation (See Part D for details) incubated cells with a known concentration of azoaryl according to the invention, maintaining a negative control group in the dark, while irradiating active groups using light at wavelengths (), each with relatively narrow bandwidth such as 15 nm. Irradiation could be applied in pulses, with the interval t.sub.pause between pulses chosen to be significantly less than . Then, knowing I() (the relative intensity of the light source as a function of wavelength), 1/[I()E()] could be used as a scaling factor to determine the relative pulse durations to apply as a function of the chosen wavelengths, thus ensuring that the PSS was approximately reached at each wavelength applied. If the PSS() were known, an estimation of [Z]* (the time-average concentration of cis-azoaryl present during the assay) could then be possible for each wavelength applied. The local net antitubulin or cytotoxic effect generated in the assay protocol could then be understood simplistically as the product of [Z]* and a factor which would describe the cis-azoaryl's strength of tubulin polymerisation inhibition or cytotoxicity. Therefore it was considered important to determine at least some estimates for PSS(), E() and which could preferably be intercomparable between compounds, in order to design and analyse biological experimental data later.

Sufficiently determining .sub.E() and .sub.Z(), PSS(), E() and :

[0345] A(.sub.meas, .sub.irrad) is used to indicate the measured UV-Vis absorption spectrum in the range 340 nm<.sub.meas<650 nm, as a function of the irradiating wavelength .sub.irrad once PSS(.sub.irrad) has been established. UV-Vis measurement of A(.sub.meas, .sub.irrad) was performed. It was considered that the PSS had been established when the absorbance profile ceased to alter under continued irradiation. An isosbestic point (wavelength .sub.iso) was found for each compound by examining where the absorption A(.sub.meas) was invariant with respect to changing .sub.irrad. A(.sub.meas, dark) was also acquired with samples that had been kept in the dark for at least several hours, and it was assumed that no significant spectral contribution from the cis isomer was present in these spectra. Standard cell culture purpose phosphate buffer saline at pH7.4 (PBS) with a fixed percentage of cosolvent, at 37 C., under air atmosphere, was used throughout.

[0346] HPLC (high-performance liquid chromatography) was then used as described in Part A to measure *.sub.E() and *.sub.Z() (the separated absorption profiles of trans and cis isomers respectively, in arbitrary units which are not intercomparable in an absolute sense). It was approximated that these spectra would be identical in shape if measured under the UV-Vis conditions, so once *.sub.E() and *.sub.Z() were scaled appropriately relative to each other, these scaled spectra could be used to deconvolute A(.sub.meas, .sub.irrad). This approximation was considered useful especially in the regions of greatest spectral interest, ie. regions of relatively strong absorption (since only rather well-absorbed wavelengths will allow significant photoisomerisation under finite photon flux). To place the arbitrary-unit absorption spectra on an absolute scale, the maximal absorption wavelength .sub.strong of the trans-isomer was first selected by examining A(.sub.meas, dark): typically, .sub.strong380 nm. with Expressing A(.sub.strong, dark) in units M.sup.1cm.sup.1, *.sub.E(.sub.strong) was then scaled so that .sub.E(.sub.strong)=A(.sub.strong, dark), giving:

.sub.E()=*.sub.E()[A(.sub.strong,dark)/*.sub.E(.sub.strong)]

and the isosbestic point was used to scale the cis-isomer spectrum as per:

.sub.Z()=*.sub.Z()[.sub.E(.sub.iso)/*.sub.Z(.sub.iso)]

[0347] A(.sub.iso) could also be used to scale the spectra, with the advantage that finding A(.sub.iso) does not require samples to be kept in the dark prior to UV-Vis measurement. However, using A(.sub.strong, dark) was preferred, since A(.sub.iso) was typically so much smaller that the standard deviation in the scaled absorptivities which it generated was far greater. It should be noted that certain compounds displayed a rather strong dependency of absorption spectrum upon the pH, possibly connected to their protonation state; and some compounds were markedly solvatochromic; therefore .sub.E() and .sub.Z() are considered only as reasonable approximations to the absorptivities that could be expected under diverse biological conditions.

[0348] In a simple analysis of the PSS() and E(), it was approximated that .sub.EZ and .sub.ZE (the quantum efficiencies of the photoisomerisations trans->cis and cis->trans, respectively) are equal and independent of wavelength, and also that the kinetics of spontaneous reversion could be ignored (eg. high-intensity light source). A function () was then calculated as per:

()=.sub.E()/[.sub.Z()+.sub.E()]

If .sub.EZ and .sub.ZE are equal and independent of wavelength, () gives the true PSS(). The wavelength independence of .sub.EZ and .sub.ZE may be an acceptable approximation in regions of relatively strong absorption within the visible spectrum. Their equality may also be an acceptable assumption: since if .sub.EZ and .sub.ZE are unequal but only depend weakly on wavelength in regions of relatively strong absorption, then () is a transform of the true PSS(); this transform preserves the features of PSS() which were most desired for evaluation in this study as long as .sub.EZ and .sub.ZE are not too different (eg less than a factor of ten difference). Indeed, the calculated () were later confirmed by in cellulo experiments to give reliable indications of favourable wavelengths for bulk trans->cis and bulk cis->trans photoisomerisation, which is the benchmark for a suitable analysis of the PSS(). The advantages of calculating () rather than exhaustively measuring the PSS() experimentally are that (a) the throughput is much faster, (b) the true PSS() may be difficult to achieve at weakly-absorbing wavelengths or with fast-relaxing compounds, (c) the true PSS() may be difficult to measure accurately at wavelengths where one isomer strongly dominates the sample even if both are relatively strongly absorbing, and (d) the variation of PSS() with changes in the local biological microenvironment (polarity, pH etc) may anyway be larger than the error incurred by the assumption ()PSS().

[0349] Also following the assumption that .sub.EZ and .sub.ZE are equal and independent of wavelength, a photoisomerisation efficiency E() was calculated as per:

E()=[.sub.E()+.sub.Z()]/[.sub.E(390)+.sub.Z(390)]

[0350] Larger values of E() therefore denote higher efficiency photoisomerisation (less photon flux is required to approach the PSS()). E() was parametrised to the absorption coefficients at 390 nm, since typically that wavelength gave satisfactorily strong absorption and efficient photoisomerisation. It should be noted that E() are not intercomparable between different compounds. This reflects the realistic scenario that the .sub.EZ (or the .sub.ZE) of two arbitrarily chosen compounds may differ by a significant amount, even if the approximation that .sub.EZ and .sub.ZE are equal and independent of wavelength applies independently to each compound.

[0351] Finally, trends in were ascertained by establishing the PSS at .sub.strong, then turning off the light source at time t.sub.0, and measuring A(.sub.strong, t) over time. Data were acquired for half an hour, or else the first three halflives (whichever was the shorter period), then fitted to the equation:

A(.sub.strong,t)=A(.sub.strong,dark)[A(.sub.strong,dark)A(.sub.strong,t.sub.0)]e.sup.kt

[0352] This fit was used to return an in vitro halflife * for the cis->trans spontaneous thermal reversion isomerisation process, defined as =ln 2/k. It should be noted however, that azoaryl compounds' thermal reversion rates are known to depend strongly upon their microenvironment, such as pH and solvent/solvation effects.sup.[7,38]. This was shown for compound (I.1), where a change of only 1.4 pH units gave a 25% change in *; note that different compounds are expected to respond very differently to changes in pH so no unifying trends are expected. Therefore the * acquired in vitro were considered appropriate only for qualitative intercomparison between samples (trend of * vs structure), rather than quantitative prediction of the values which might be experienced in cellulo or in vivo. Indeed, these in vitro * values appeared at least three times longer than what would have been expected based on in cellulo experiments where the pause time between activating pulses was varied.

Spectrophotometry Equipment:

[0353] Absorption spectra in cuvette (UV-Vis) were acquired on a Varian CaryScan 50 (1 cm, 100 L or 1 mL volume) with Peltier cell temperature control unit maintained at 37 C., in PBS at pH7.4 containing a low percentage of cosolvent if needed (typically 2% MeCN or 5% DMSO). A ThorLabs Polychrome V monochromator with a fibre optic cable output directed into the cuvette was used to perform photoisomerisation studies by UV-Vis spectrophotometry although single LEDs of approximately 10-20 mW output, 15-20 cone angle, and 10-15 nm bandwidth FWHM, obtained commercially from Roithner Lasertechnik GmbH, were equally successful in providing repeatable monochromatic photoisomerisation. Separated spectra of trans and as forms were acquired from the inline Diode Array Detector on the AGILENT 1260 SL coupled LC-MS system after HPLC separation. Fluorescence spectrophotometry (excitation and emission spectra) were acquired on a Varian CaryEclipse fluorescence spectrophotometer.

Photocharacterisation Results 1: Trans- and Cis-Absorption Spectra

[0354] By the above procedure, the trans- and cis-isomers of each species were separated by HPLC as described in Part A, and *.sub.E() and *.sub.Z() were measured on the inline ultraviolet/visible Diode Array Detector; these were then scaled as outlined above. The parameters .sub.strong, .sub.E(.sub.strong) and .sub.iso are tabulated for selected compounds below (Table 1).

TABLE-US-00001 TABLE 1 .sub.strong, .sub.E(.sub.strong) and .sub.iso for selected compounds, measured in PBS, at pH = 7.4 unless indicated otherwise, containing 20% MeCN to ensure solubility. nd indicates not determined, ie. no clear isosbestic point could be established due to insufficient measurable photoisomerisation. Compound I.25 displayed a bimodal absorbance spectrum, mainly attributable to the azoaryl moiety in the spectral range 360-450 nm, and mainly attributable to the fluorophore moiety above 510 nm, hence values are given for each range; 510 ff indicates that A( > 510) was invariant upon trans<->cis photoswitching. .sub.strong .sub.E(.sub.strong) Compound (nm) (M.sup.1cm.sup.1) .sub.iso (nm) I.1 375 17700 470 I.1, pH = 6 380 17100 465 I.2 365 11400 470 I.3 365 13300 445 I.4 365 19804 455 I.5 365 19578 450 I.6 365 16300 445 I.7 365 13600 455 I.8 390 14900 nd I.9 355 11400 430 I.11 375 18163 460 I.12 380 12400 485 I.13 355 14200 440 I.14 390 18000 485 I.16 410 14430 nd I.17 385 10872 nd I.25 375 15300 460 (565) (66600) (510 ff) I.26 380 16300 475 I.27 365 19540 445 I.28 360 17813 452 I.29 365 20102 447

[0355] The complete absorbance spectra of selected compounds are presented in FIG. 1.

[0356] Table 1 and FIG. 1 illustrate the large single-photon absorption coefficients within the in vivo compatible wavelength range which are typical for the compounds of the invention, and which especially distinguish them from stilbenes. Such strong absorption coefficients, coupled with the high quantum efficiencies known to be typical of azobenzene photoisomerisation.sup.[7,32], enable efficient single-photon photoisomerisation of the compounds of the invention in both directions cis->trans and trans->cis, with low power irradiation as is in vivo compatible, cheap and practical.

Photocharacterisation Results 2: Calculated () and E()

[0357] () and E() were calculated as described above and typical examples are presented in FIG. 2.

[0358] FIG. 2 indicates that structural modifications within the scope of the compounds of the invention may substantially alter both the proportion of the as form in the photostationary state at different wavelengths, and the relative efficiency of approaching those photostationary states. This illustrates the possibility of structural modifications within the scope of the compounds of the invention being used to give spectral tuning, both for better biological light penetration (red-shifting), and so that both trans->cis and (especially) cis->trans photoisomerisations may be conducted more efficiently and more completely.

[0359] Photocharacterisation Results 3: Trans<->Cis Photoisomerisations are Fully Reversible Over Thousands of Cycles Under Biologically Relevant Conditions

[0360] A(.sub.strong) was measured over time in the UV-Vis cuvette in non-degassed PBS left open to the atmosphere, containing 10% MeCN, at 37 C., while the monochromator was used to apply .sub.irrad alternating between two values .sub.1 and .sub.2, chosen to induce net trans->cis and net cis->trans isomerisation respectively. Typical results are presented for compound I.1 (FIG. 3). The absorbance of I.1 (18 M) was measured at .sub.strong=378 nm, while the irradiating wavelength was held alternately at .sub.1=388 nm (50 s; bulk trans->cis) then .sub.2=508 nm (180 s, bulk cis->trans). Higher absorbance corresponds to a greater amount of trans isomer: an absorbance of 0.27 here corresponds to an approximately 5:1 ratio of trans:cis isomer in the sample, and an absorbance of 0.12 here corresponds to an approximately 1:2 ratio of trans:cis isomer in the sample. Note that this bulk trans->cis isomerisation at 388 nm is significantly faster than the bulk cis->trans isomerisation at 508 nm, although the monochromator equipment used delivers light at 508 nm with approximately 1.5 times the intensity of the light delivered at 388 nm. This is consistent with the expectation that photoisomerisation toward the respective photostationary states should be significantly more efficient at 388 nm than at 508 nm for this compound, since E(388)/E(508)=0.04. Typically, compounds of the invention could be bulk-photoisomerised hundreds of times over a timescale of hours (50 representative cycles are shown in FIG. 3 for I.1), such that the majority species in a sample was photocontrollably alternated between the trans-isomer and the cis-isomer.

[0361] However, the photoisomerisation behaviour of compound I.25, which features a reporter moiety (a rhodamine B fluorophore derivative) attached to the azoaryl moiety via a linker as explained in the Description, was qualitatively different in a very important fashion. As noted in Table 1 and shown in FIG. 4, the absorbance spectra of the trans and cis forms of I.25 are bimodal, with the region <450 nm dominated by the azoaryl moiety and the region >510 nm dominated by the rhodamine moiety. This particular rhodamine was chosen due to a literature report that its fluorescence emission occurs with high brightness (the product of the excitation wavelength absorption coefficient and the fluorescence quantum yield), and only above 525 nm (peak emission reported at 561 nm)..sup.[46] Above 525 nm is a spectral region where the absorbance of only the as (but not the trans) isomers of prototypical azoaryl moieties of compounds of the invention (such as I.1) is non-negligible. Therefore it was considered possible that irradiation of a mixture of cis and trans isomers of I.25 in the spectral region dominated by the strongly-absorbing rhodamine moiety might result in favourable resonant energy transfer from the rhodamine moiety to the azoaryl moiety only when the azoaryl is in the as form, and that this energy transfer could result in isomerisation of the diazenyl bond, thus that such irradiation could rapidly decrease the subpopulation in the as form, perhaps even without substantial photoisomerisation of the subpopulation in the trans form. Firstly, irradiation at 554 nm of a sample of I.25 which had been kept in the dark resulted in visible fluorescence, but without the absorbance spectrum of the sample in the region 360-450 nm being changed. This is consistent with the hypothesis that irradiation at this wavelength does not substantially decrease the population of the trans form. As was observed previously with similar azoaryl moieties, such as for compound I.1, irradiation of this sample of I.25 at 384 nm induced a rapid decrease of the absorbance around 375 nm, consistent with the interpretation that irradiation at this wavelength effects bulk trans->cis photoisomerisation. Subsequent irradiation of this sample at 554 nm then resulted in the very rapid return of the absorbance spectrum of the sample entirely to its dark-adapted state (all-trans). This is consistent with the hypothesis that irradiation at this wavelength effects quantitative cis->trans photoisomerisation of I.25, presumably by highly selective resonant energy transfer to the cis isomer. For illustrative purposes, the A(.sub.strong=375 nm) of I.25 was monitored while .sub.irrad was alternated between .sub.1=384 nm and .sub.2=554 nm, and the results are also shown in FIG. 4.

[0362] The unusual rapidity and the quantitative nature of the cis->trans photoisomerisation of I.25 should especially be noted, and may be compared with the slower and non-quantitative nature of the cis->trans photoisomerisation of I.1 which was depicted in FIG. 3.

[0363] The cyclical photoisomerisations of all compounds tested proceeded without any detectable photobleaching, or decreases in photoswitching speed, or photoswitching efficiency or limiting isomeric percentage obtained, despite the fact that measurements were conducted in non-degassed aqueous solution under open air atmosphere at 37 C. This highlights the extreme robustness of the azoaryl compounds of the invention towards photochemical reaction or damage in a biologically relevant setting. This robustness is a key advantage of the compounds of the invention when compared to the prior art relying on stilbene photoisomerisation, which is a process known to give extensive and rapid degradation (principally to phenanthrenes) when biologically relevant conditions (eg. presence of dissolved oxygen) are used.sup.[11]. These results thus demonstrate that photocontrolled trans<->cis isomerisation of the compounds of the invention is fully reversible, in a highly robust fashion which it is practical to implement under biologically relevant conditions.

Photocharacterisation Results 4: Thermal Reversion Times

[0364] Thermal reversion times were measured as described above. Those for selected compounds are presented below (Table 2).

TABLE-US-00002 TABLE 2 reversion times for selected compounds, measured in PBS at pH = 7.4 unless indicated otherwise, containing 20% MeCN to ensure solubility. Too Fast indicates that no clear isosbestic point could be established due to insufficient measurable photoisomerisation, which was attributed to very fast thermal reversion. Compound (min) I.1 6.2 I.1, pH = 6 8.4 I.2 0.75 I.3 61 I.4 119 I.5 134 I.6 83 I.7 50 I.8 TOO FAST I.9 116 I.11 40 I.12 28 I.13 270 I.14 3.3 I.16 TOO FAST I.17 TOO FAST I.25 16.2 I.26 8.4 I.27 129 I.28 248 I.29 104

[0365] Table 2 shows that structural modifications within the scope of the compounds of the invention can greatly alter the timescale of spontaneous cis->trans reversion. Therefore different compounds of the invention may be appropriate for different types of biological experiments, especially when these are carried out over significantly different timescales (eg. seconds to minutes for experimental biology applications, or hours to days for biomedical applications), or if weak (ie. non-saturating) light intensities are to be used. Note that compounds I.8, I.16 and I.17 showed no measurable change in absorbance spectrum upon irradiation at 390 nm. This is interpreted as being due to very fast kinetics of spontaneous reversion which deplete the cis population before it can contribute to the absorbance; thus they are indicated to possess too fast a reversion time to be measured by this method. Proton transfer to the diazenyl bond from the hydroxyl or amino group in ortho to this diazenyl bond, is the assumed mechanism underlying these fast kinetics.

Photocharacterisation Results 5: Fluorescence of I.25

[0366] Fluorescence imaging is commonly used to sensitively, conveniently and non-invasively determine the local concentration of fluorescent species in biological and medical settings. Considering I.25 as an example of a compound of the invention bearing a fluorescent reporter, it was considered desirable that its rhodamine moiety would allow fluorescence detection of I.25. Fluorescence excitation and emission spectra of I.25 were therefore acquired and are shown in FIG. 5.

[0367] The fluorescence emission spectra in the top panel of FIG. 5 show that I.25 can give a relatively broad fluorescence signal with an emission maximum at 590 nm when excited either at wavelengths which are also appropriate for effecting bulk trans->cis isomerisation (eg. 380 nm, dotted line), or else at wavelengths which are also appropriate for effecting near-quantitative cis->trans isomerisation (eg. 554 nm, solid line; note that the vertical scale is in arbitrary units not comparable between measurements).

[0368] The excitation spectrum of I.25 in the bottom panel of FIG. 5 shows the relative intensity of fluorescent emission at 590 nm, depending on the excitation wavelength used. While excitation at 570 nm gives the maximum emission intensity, many other wavelengths provide satisfactory fluorescent readout, eg. the spectral range between 350 nm and 440 nm, and that between 470 nm and 580 nm. It should also be noted that irradiation between 455-465 nm does not result in significant fluorescence output, which may be useful if azoaryl trans<->cis photoisomerisation is desired without risking fluorescent output.

[0369] Therefore, I.25 provides an example of a compound of the invention bearing a reporter chosen such that the fluorescence output of I.25 is well-defined, and can be produced by a broad range of excitation wavelengths covering much of the wavelength range commonly used for fluorescence imaging in biological and medical settings, and can be produced either by excitation wavelengths favouring the generation of the as isomer (eg. 384 nm) or favouring the generation of the trans isomer (eg. 554 nm), which factors should allow sophisticated biological applications eg. in fluorescent tracking, as well as benefiting from the advantage of resonant energy transfer allowing near-quantitative cis->trans photoisomerisation as described above.

Part C: Biochemistry In Vitro

[0370] Turbidimetric tubulin polymerisation assays were performed as described in the literature.sup.[29], following the increase in absorbance at 340 nm, but with the addition of irradiation supplied by the monochromator setup described in Part B. Two wavelengths were chosen for each compound, .sub.E->Z (chosen to effect bulk trans->cis isomerisation, typically 390 nm), and .sub.Z->E (chosen to effect bulk cis->trans isomerisation, typically 505 nm). The raw absorbance A(t) could be more clearly discussed by first correcting for any change in azoaryl absorbance over time by subtracting the absorbance of a control run performed without tubulin, yielding the corrected absorbance A*(t), and then taking its derivative R=dA*(t)/dt. Experiments were performed under six sets of conditions to illustrate the light control of tubulin polymerisation inhibition effected by the compounds of the invention: (a) in the dark (typical result: large R at the start decreasing rapidly over time), or (b) with constant irradiation at .sub.E->Z (typical result: small R throughout the experiment), or (c) with constant irradiation at .sub.Z->E (typical result: moderate R at the start decreasing slowly over time), or (d) with irradiation at .sub.E->Z for an initial period then darkness (typical result: initial period with small R, then R increases after a while), or (e) with irradiation at .sub.E->Z for a given period (typically 30 min) then irradiation at .sub.Z->E (typical result: 30 minutes with small R, then R increases quickly to a moderate value which decreases slowly over time), or (f) with irradiation at .sub.E->Z for a given period (usually 30 min) then a short irradiation at .sub.Z->E (usually 30 seconds) then darkness (typical result: 30 minutes with small R, then R increases quickly to a moderate-to-large value which decreases slowly over time); the last three conditions required correction of A(t) to A*(t) to reveal the changes in absorption which were due only to tubulin polymerisation. It would be costly to determine light-dependent in vitro tubulin polymerisation IC.sub.50 values for each compound, eg. under protocols (a) and (b) (and/or (c)). This was not performed here since it was considered that such parameters have little predictive value in their intended complex biological settings, especially in light of the importance of biodistribution, tubulin-binding kinetics and lability (rather than thermodynamic binding strength) for tubulin disruption in biological settings.

[0371] Table 3 below illustrates typical results from experiments of type (a) (DARK) and (b) (390 nm) as described above, with compound (I.1) at concentrations well above the IC.sub.50 for the toxic regime, compared to a PBS-only control CTRL (no I.1 present).

TABLE-US-00003 TABLE 3 A turbidimetric tubulin polymerization assay showing the absorbance A(t) as defined above, comparing the behaviour of a control run CTRL (no azoaryl added) vs runs using compound I.1 at 50 and 25 M, with constant 390 nm illumination or else in the dark. A( = 340 nm) time CTRL I.1, 50 M I.1, 25 M (min) DARK DARK 390 nm DARK 390 nm 0.0 0.0000 0.0000 0.0000 0.0000 0.0000 0.5 0.0011 0.0010 0.0014 0.0001 0.0026 1.0 0.0015 0.0014 0.0020 0.0016 0.0022 1.5 0.0061 0.0034 0.0033 0.0018 0.0022 2.0 0.0220 0.0128 0.0027 0.0119 0.0010 2.5 0.0486 0.0305 0.0052 0.0274 0.0001 3.0 0.0754 0.0552 0.0066 0.0492 0.0004 3.5 0.1013 0.0734 0.0074 0.0689 0.0029 4.0 0.1195 0.0926 0.0100 0.0851 0.0027 4.5 0.1324 0.1046 0.0084 0.0984 0.0034 5.0 0.1398 0.1139 0.0122 0.1073 0.0100 5.5 0.1456 0.1209 0.0166 0.1133 0.0094 6.0 0.1474 0.1269 0.0163 0.1150 0.0132 6.5 0.1491 0.1284 0.0172 0.1212 0.0174 7.0 0.1537 0.1262 0.0213 0.1249 0.0171 7.5 0.1545 0.1379 0.0209 0.1228 0.0222 8.0 0.1553 0.1338 0.0221 0.1250 0.0241 8.5 0.1544 0.1359 0.0268 0.1259 0.0259 9.0 0.1559 0.1367 0.0268 0.1240 0.0288 9.5 0.1562 0.1374 0.0293 0.1276 0.0313 10.0 0.1552 0.1397 0.0320 0.1276 0.0341 10.5 0.1545 0.1370 0.0318 0.1255 0.0358 11.0 0.1576 0.1405 0.0327 0.1290 0.0402 11.5 0.1556 0.1417 0.0333 0.1299 0.0413 12.0 0.1584 0.1402 0.0359 0.1333 0.0443 12.5 0.1567 0.1444 0.0391 0.1330 0.0457 13.0 0.1588 0.1440 0.0385 0.1317 0.0473 13.5 0.1569 0.1483 0.0381 0.1311 0.0519 14.0 0.1577 0.1453 0.0420 0.1355 0.0539 14.5 0.1567 0.1478 0.0436 0.1345 0.0531 15.0 0.1592 0.1515 0.0448 0.1403 0.0576

[0372] Table 3 indicates that tubulin polymerisation is very strongly inhibited by (I.1) in a dose-dependent fashion when it is exposed to 390 nm irradiation, but is not inhibited at these concentrations in the dark. This can be understood as a strong tubulin polymerisation inhibition effected only by (I.1)-cis, since if darkness is maintained, (I.1)-trans is the isomeric form present in the sample, and tubulin polymerisation is seen here to proceed identically to the control case.

[0373] These results indicate that the compounds of the invention can act in vitro as inhibitors of tubulin polymerisation whose inhibitory activity may be controlled (activated or deactivated) by the choice of illumination conditions over time.

Part D: Cell Biology

From Photopharmacology Principles to Biology Assay Design:

[0374] For clarity of discussion, [Z] is defined as the instantaneous local concentration of the cis-azoaryl isomer; [Z]* is defined as the time-average [Z] experienced during a significant phase of an experiment (eg. the first phase of a two-phase experiment; see below); and t.sub.pause t is defined as the interval between light pulses in a pulsed experiment (if the experiment is a dual-wavelength experiment, each pulse is defined as containing both .sub.ACT and .sub.DEACT). Recall that the target is defined as the spatiotemporal region where it is desired that the biological effects of the azoaryl compound be most strongly applied, while it is considered beneficial to avoid generating biological effects in off-target zones, whether far from or near to the target.

[0375] Two strong light-dependent steady-state biomedically relevant protocols are evident: (1) a toxic regime designed to maximise the pharmacological toxicity of the azoaryl compound, by applying activating irradiation at wavelength .sub.ACT so as to generate a significant [Z]* in the target; continuous irradiation could be used, or else pulsed irradiation if t.sub.pause were significantly shorter than ; an example pulsed toxic regime for compound I.1 ( estimated as 6 minutes) could thus be 390 nm applied in pulses of 200 ms with t.sub.pause=30 s; and (2) a strong rescue regime, designed to deliver a rigorous test of the degree of photocontrol which can be exerted over the azoaryl compound's toxicity, by applying .sub.ACT as for the toxic regime, but competitively applying deactivating irradiation at wavelength .sub.DEACT in order to reduce [Z]* relative to the value experienced in the toxic regime; like the toxic regime, this regime could be pulsed or continuous; an example pulsed strong rescue regime for compound I.1 could thus be 390 nm applied for 200 ms then 505 nm applied for 600 ms, with t.sub.pause=30 s.

[0376] One likely design for localised therapeutic applications of the compounds of the invention is by spatially separated application of a toxic regime on a target synchronously with the application of a deactivating regime (featuring only the .sub.DEACT component of an optimised strong rescue regime) in a thin protection zone surrounding this target (in order to reduce the exposure of the rest of the organism or sample to any cis-azoaryl isomer escaping the target). Especially with strongly nonlinear dose-response relationships, as the present compounds feature, this may maximise the biological effects in a target while keeping the off-target [Z]* below the minimal response concentration, thereby avoiding side effects. In the context of biomedical applications therefore, the toxic regime thus gives an estimate of the maximum strength of the biological effects that can be exerted in a target zone by a given concentration of the azoaryl compound; and assuming that a deactivating regime can be applied in those off-target zones which are the very closest neighbours to this target zone, then the strong rescue regime estimates the maximum strength of the (undesirable) biological effects that could be experienced in the very nearest off-target zones, eg. due to the diffusion of cis isomer out of the target zone or due to a degree of scattering of .sub.ACT; weaker biological effects are to be anticipated in off-target zones still further from the target.

[0377] Weak light-dependent protocols are defined as those where spontaneous reversion plays a significant role in reducing [Z]*, and these may also be very important in medical and especially fundamental research applications. Examples include (3) a dark rescue regime, where a toxic regime would be applied for the first phase of an experiment, then all light switched off and darkness maintained throughout a second phase of the experiment thus allowing [Z] to reach zero; and (4) a weak pulsed rescue regime similar to the strong pulsed rescue regime but where t.sub.pause t is instead significantly longer than , such that the component of .sub.DEACT in each pulse primes the sample to decay more rapidly to [Z]0 than would be possible by spontaneous reversion alone. Note that a weak pulsed rescue regime will always display lesser biological effects than the corresponding strong rescue regime which has a shorter t.sub.pause. Note also that dynamic protocols exploiting the spatiotemporal control of appropriate irradiation could find even more sophisticated applications than these steady-state protocols.

[0378] In any chosen regime, light could be applied either continuously (allowing very low intensities to be used), or in pulses (allowing for fixed source intensity and probably permitting higher-performance implementation of rescue protocols). Example experiments showed that pulsed protocols were well-adapted to illustrating the relationship between observed photopharmacological effects and the underlying qualitative trends of PSS(), E() and described previously, so pulsed protocols were used throughout biological studies. Results from the strong light-dependent steady-state protocols are given here, as these provide a more demanding proof of the principle of fully reversibly light-controllable biological effects than do the weak protocols described above. The predictable and successful outcome of experiments under these protocols serves as a generalised indication that weak protocols can also be applied with at least as much, if not more, success. Lastly, note that .sub.E->Z and .sub.Z->E as defined above also give initial estimates for biologically good values of .sub.ACT and .sub.DEACT by balancing the need to restrict the light flux applied, while still favouring one or the other isomer's formation as much as possible. The values of .sub.ACT and .sub.DEACT used in optimised experiments could be refined empirically from these initial guesses, however this was typically not necessary.

Use of Photoswitchable Compounds in Cell Culture:

[0379] Irradiated cell culture was performed using a self-built computer-controlled system of arrays of LEDs, where each array irradiated a standard 24-well or 96-well cell culture plate, and these were contained in separate light-proof gas-permeable boxes in a cell culture incubator; the system is illustrated schematically in FIG. 6. Either one or two arrays were conveniently used per wellplate (typically, an array at an activating wavelength illuminating from the bottom, with an optional second array at a deactivating wavelength illuminating from the top down on a different timing sequence if desired), thus enabling pulsed or continuous implementation of eg. toxic regime, strong rescue regime, weak rescue regime, or dark rescue regime protocols, in a straightforward manner.

Results 1: Photoactuated Toxicity of a Range of Compounds:

[0380] Seven of the compounds according to the invention were selected for biological tests. Their cytotoxicity was assessed using crystal violet staining as adapted from standard procedure.sup.[31]. Briefly, HeLa cells were seeded on 96-well plates, treated with the given concentrations of the selected compound, and exposed or not to the pulse protocol of illumination with light at 390 nm (pulses of 75 ms every 15 s). After 40 h cells were stained with crystal violet solution (0.5% crystal violet in 20% methanol) for 10 min. Unbound crystal violet was removed by rinsing with distilled water and cells were air-dried. Crystal violet was subsequently eluted from cells with 0.1 M sodium citrate in 50% ethanol. The absorbance of crystal violet is proportional to the cell number and was determined at 590 nm with a FLUOstar Omega microplate reader (BMG Labtech).

[0381] Each compound was tested at 6 doses: 100 nM, 500 nM, 1 M, 2 M, 5 M and 10 M. In order to ensure their solubility in the cell culture media, DMSO was used as a co-solvent, and to provide comparability between all samples the volume of DMSO was adjusted to obtain its final concentration as 1% in the cell culture media for all the compounds at all concentrations tested.

[0382] Results presented in Table 4 below are expressed as a fold growth relative to the control growth of the cells treated only with a co-solvent (1% DMSO), and are represented as a mean value from triplicates coming from a representative experiment.

TABLE-US-00004 TABLE 4 Cytotoxicity assessed by crystal violet staining, followed 40 h of treatment with indicated concentrations of the panel of indicated compounds in the dark or upon application of a pulsed toxic regime protocol (390 nm for 75 ms every 15 s). Dark 390 nm mean mean I.1 fold fold No. concentration growth StDev growth StDev 1 0 nM 1.00 0.05 1.00 0.006 2 100 nM 0.95 0.04 0.95 0.04 3 500 nM 0.89 0.008 0.85 0.03 4 1 M 0.87 0.03 0.43 0.01 5 2 M 0.89 0.02 0.35 0.004 6 5 M 0.84 0.009 0.27 0.04 7 10 M 0.75 0.02 0.35 0.04 Dark 390 nm mean mean I.2 fold fold No. concentration growth StDev growth StDev 1 0 nM 1.00 0.03 1.00 0.04 2 100 nM 0.97 0.04 1.10 0.16 3 500 nM 0.90 0.02 0.83 0.07 4 1 M 0.89 0.04 0.50 0.07 5 2 M 0.81 0.03 0.47 0.05 6 5 M 0.86 0.008 0.32 0.08 7 10 M 0.70 0.05 0.34 0.06 Dark 390 nm mean mean I.3 fold fold No. concentration growth StDev growth StDev 1 0 nM 1.00 0.08 1.00 0.08 2 100 nM 1.03 0.04 0.93 0.08 3 500 nM 1.00 0.01 0.88 0.06 4 1 M 0.95 0.03 0.82 0.06 5 2 M 0.90 0.02 0.62 0.06 6 5 M 0.96 0.02 0.39 0.02 7 10 M 0.99 0.04 0.47 0.07 Dark 390 nm mean mean I.4 fold fold No. concentration growth StDev growth StDev 1 0 nM 1.00 0.09 1.00 0.08 2 100 nM 0.93 0.04 0.98 0.11 3 500 nM 0.93 0.08 0.78 0.02 4 1 M 0.89 0.07 0.53 0.08 5 2 M 0.94 0.07 0.48 0.04 6 5 M 0.94 0.07 0.44 0.06 7 10 M 0.78 0.07 0.36 0.09 Dark 390 nm mean mean I.6 fold fold No. concentration growth StDev growth StDev 1 0 nM 1.00 0.03 1.00 0.02 2 100 nM 1.00 0.03 0.99 0.02 3 500 nM 1.07 0.02 1.09 0.04 4 1 M 0.96 0.01 0.79 0.11 5 2 M 0.96 0.01 0.47 0.07 6 5 M 0.93 0.05 0.68 0.06 7 10 M 0.88 0.14 0.37 0.12 Dark 390 nm mean mean I.20 fold fold No. concentration growth StDev growth StDev 1 0 nM 1.00 0.04 1.00 0.01 2 100 nM 0.99 0.06 1.09 0.11 3 500 nM 1.11 0.05 1.10 0.19 4 1 M 1.12 0.12 1.00 0.14 5 2 M 1.10 0.04 0.96 0.04 6 5 M 1.03 0.04 0.68 0.02 7 10 M 1.01 0.03 0.51 0.06 Dark 390 nm mean mean I.10 fold fold No. concentration growth StDev growth StDev 1 0 nM 1.00 0.008 1.000 0.05 2 100 nM 0.95 0.08 0.986 0.08 3 500 nM 0.99 0.01 0.945 0.03 4 1 M 0.92 0.07 0.387 0.02 5 2 M 0.90 0.009 0.357 0.03 6 5 M 0.99 0.18 0.217 0.03 7 10 M 0.78 0.08 0.293 0.009

[0383] For all the compounds tested, strong dose-dependent toxicity was observed with activating irradiation (390 nm regimen), while in the dark no such significant cytotoxic effect was observed. The results of this experiment therefore support the conclusion that the compounds of the invention act as light-inducible cytotoxins. Based on the strong performance of compound I.1 and the similarity of its behavior to the other compounds tested, further tests in detail were pursued focusing on compound I.1 exclusively.

Results 2: Photocontrolled Cytoxicity of I.1

[0384] The cytotoxic properties of I.1 were subsequently confirmed with another method, using the quantification of mitochondrial dehydrogenase activity of cells as determined by the level of 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT) reduction to its purple formazan, according to standard protocol.sup.[30]. Briefly, HeLa cells were seeded on 96-well plates and treated with compound I.1 at concentrations ranging from 10 nM-2 M in PBS containing 0.5% MeCN to ensure the compound's solubility. Cells were kept in the dark, or exposed to a pulsed toxic regime of 390 nm only, or exposed to a strong rescue protocol with pulses of 390 nm then 505 nm light. Pulses of light were applied every 30 s; 390 nm light pulses lasted 150 ms every 30 s, while the 505 nm light pulses were applied for 500 ms; in the strong rescue regime, the 505 nm pulse was synchronised so that it began immediately after the 390 nm pulse ended.

[0385] Cellular viability was measured 48 h later, namely, cells were incubated with MTT for three hours and after dissolution of the formazan crystals with DMSO, absorbance at 550 nm was measured using a FLUOstar Omega microplate reader. Results are shown in Table 5 below and are expressed as a fold growth relative to the control growth of the cells treated only with a co-solvent (0.5% MeCN) and represent mean and standard deviation of quadruplicates of the optical density values, which are proportional to the cell number.

TABLE-US-00005 TABLE 5 Values of formazan absorbance measured by MTT assay were used to derive fold values correlated to cell count number, which were used to quantify the cytotoxicity of compound I.1. Light pulses were applied every 30 s; 390 nm light pulses lasted 150 ms, 505 nm light pulses lasted 500 ms. rescue: 390 nm then Dark 390 nm 505 nm mean mean mean fold fold fold No. compound growth StDev growth StDev growth StDev 1 I.1 0 nM 1.00 0.08 1.00 0.05 2 I.1 10 nM 0.93 0.08 0.95 0.03 3 I.1 20 nM 1.02 0.04 0.96 0.03 4 I.1 50 nM 1.01 0.03 0.96 0.04 5 I.1 100 nM 1.01 0.05 0.93 0.02 6 I.1 400 nM 1.00 0.05 0.28 0.02 0.46 0.04 7 I.1 600 nM 1.05 0.06 0.21 0.01 8 I.1 800 nM 0.98 0.07 0.17 0.02 9 I.1 1 M 0.93 0.02 0.18 0.02 10 I.1 2 M 0.98 0.03 0.21 0.01 11 CA4P 50 nM 0.29 0.05 0.38 0.10

[0386] In the dark no toxicity of compound I.1 was observed within the concentration range tested, while upon exposure to the light, compound I.1 showed an induction of severe cytotoxicity at a concentration 400 nM and the cytotoxic effect was comparable to that of the 50 nM CA4P positive control (Table 5). In the strong rescue protocol, tested here only with compound I.1 at 400 nM, approximately 5700 pulses were applied over the 48 hours of the experiment, each time cycling between bulk trans->cis then bulk cis->trans photoisomerisation. It should be noted that the rescue protocol cells were under irradiation for significantly longer than the toxic protocol cells due to the double irradiation, however this rescue protocol reduced the cytotoxic effect of compound I.1 compared to the 390 nm-only protocol, which is strong evidence of the possibility of fully reversible light-control of the anti-proliferative properties of compound I.1.

Results 3: Cell Cycle Arrest

[0387] The effect of compound I.1 on cell cycle progression was assessed by flow cytometry. Briefly, following the application of compound I.1 and exposure to the indicated light regime, cells were harvested on ice and incubated in a hypotonic buffer [0.1% sodium citrate, 0.1% Triton X-100 and 50 g/mL propidium iodide (PI)] for 30 min at 4 C. Following the PI staining cells were analysed by flow cytometry using FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany) and Cell Quest Pro Software (Becton Dickinson). Subsequently the cell cycle analysis was performed using the FlowJo software (Tree Star Inc., Ashland, Oreg., USA).

[0388] As is typical for tubulin-binding agents, compound I.1 induced cell cycle arrest in G.sub.2/M phase, but for compound I.1 this was dependent on the light regimen. Table 6 below shows the repartition of cells between different phases of the cell cycle. This shows dose-dependent arrest in G.sub.2/M phase in MDA-MB-231 cells treated with compound I.1 and exposed to a 390 nm toxic regime. This effect is not cell type specific: it was observed in all the tested cell types MDA-MB-231, HEK-293, HeLa and HepG2 (Tables 7-8); a significantly higher response threshold was only observed for the HepG2 cells. Therefore the invention shows a light-activated mode of action, and one which is generalizable across cell types (as appropriate to their generalisable mechanism of cytotoxicity).

[0389] Controls supported these experimental conclusions as to the light-dependency of the cytotoxicity of compound I.1. Analysis of control cells treated with CA4P showed the same level of cell cycle arrest regardless of illumination conditions; notably though, when compound I.1 was not subjected to light, no cell cycle arrest was evident within the whole concentration range tested (Table 7 and Table 8 below).

[0390] Again, compound I.1 was shown to be able to induce toxicity in a more sophisticated light-dependent manner when different wavelengths were considered. Table 9 below presents results again illustrating that the biological effects induced under the toxic regime can be reduced by applying a strong rescue regime. This strong rescue protocol succeeds in reducing the toxicity to levels approximately the same as are seen when only the deactivating pulse component is applied, although applying a total dose of light approximately four times higher than in the toxic regime (either in terms of illumination time and/or applied energy), which supports the idea that the cis-form of compound I.1 was the main determinant of toxicity. Again, the rescue protocol thus isomerised the compounds of the invention, to a proportion clearly significant for determining toxicity, back and forth between cis and trans forms more than 5000 times over the experiment, thereby supporting the claim of full and reversible light control of the toxicity of the compounds of the invention, demonstrable in a robust and practical setting.

[0391] Result values in Tables 6-9 below are presented as mean+/standard deviation calculated for triplicates from one representative experiment out of three independent trials.

TABLE-US-00006 TABLE 6 Repartition of cells between the phases of the cell cycle in MDA-MB-231 cells treated with compound I.1 and exposed for 48 h to 390 nm regimen (150 ms pulses every 30 s). Concen- 390 nm tration % G1 % S % G2 No of I.1 mean StDev mean StDev mean StDev 1 0 77.4 7.1 16.9 1.5 2.7 0.5 2 10 nM 74.6 6.0 17.1 0.8 1.9 0.3 3 20 nM 79.5 8.7 18.0 1.9 2.5 0.6 4 50 nM 77.1 4.8 18.0 1.9 1.8 0.2 5 100 nM 76.6 4.8 18.5 1.9 2.0 0.3 6 400 nM 53.1 3.6 27.6 0.7 15.1 1.4 7 600 nM 18.0 1.6 31.0 0.4 42.6 0.8 8 800 nM 9.5 4.0 27.5 2.1 54.2 10.9 9 1 M 5.5 0.7 27.6 3.4 52.9 3.9 10 2 M 4.8 0.2 26.4 4.0 60.1 1.3 11 5 M 5.0 0.7 23.9 2.1 70.2 9.1

TABLE-US-00007 TABLE 7 G2/M phase arrest in the panel of cell lines: HEK-293, HeLa and MDA-MB-231. Cells were exposed to compound I.1 at indicated concentrations and exposed to a 390 nm regimen (1 s pulses every 15 min), or not (dark). The cell cycle analysis was performed 30 h post-treatment. HEK HeLa MDA mean % mean % mean % light G2/M G2/M G2/M No. type Compound phase StDev phase StDev phase StDev 1 dark 19.8 0.4 22.7 0.4 14.1 0.9 2 dark I.1 200 nM 20.2 0.4 23.5 0.9 14.3 0.9 3 dark I.1 600 nM 21.2 0.3 24.8 0.3 12.8 1.5 4 dark I.1 1.5 M 21.1 0.1 27.1 0.5 13.1 0.6 5 dark I.1 5 M 24.6 0.07 28.4 0.8 13.8 1.4 6 dark CA4P 15 nM 72.0 0.5 69.0 1.2 51.3 2.3 7 390 nm 18.5 0.5 24.4 0.4 10.5 3.3 8 390 nm I.1 200 nM 19.0 0.4 26.2 0.6 8.2 2.0 9 390 nm I.1 600 nM 19.4 1.6 33.7 1.8 16.3 0.07 10 390 nm I.1 1.5 M 65.3 3.0 59.9 2.1 40.6 2.6 11 390 nm I.1 5 M 71.5 2.6 68.7 0.9 49.4 1.9 12 390 nm CA4P 15 nM 70.1 5.5 69.5 1.1 50.9 1.6

TABLE-US-00008 TABLE 8 Cell cycle analysis of HepG2 cells following 48 h treatment with compound I.1 and exposure to a 390 nm light regimen (250 ms pulses every 15 s) or not (dark). HepG2 mean % lighting G2/M No type Compound phase StDev 1 dark 9.9 0.5 2 dark 1.1 100 nM 10.0 1.0 3 dark 1.1 600 nM 8.6 0.9 4 dark 1.1 1 M 10.1 0.5 5 dark 1.1 3 M 11.2 2.1 6 dark 1.1 10 M 11.7 1.3 7 390 nm 8.7 1.2 8 390 nm I.1 100 nM 7.8 0.6 9 390 nm I.1 600 nM 9.4 0.7 10 390 nm I.1 1 M 10.7 1.5 11 390 nm I.1 3 M 9.3 1.5 12 390 nm I.1 10 M 42.3 2.8

TABLE-US-00009 TABLE 9 Cell cycle analysis of MDA-MB-231 cells treated with compound I.1 and exposed for 48 h to a 390 nm regimen (a 150 ms pulse at 390 nm every 30 s), or a 515 nm regimen (a 500 ms pulse at 515 nm every 30 s), or a rescue regimen (a 150 ms pulse at 390 nm then immediately a 500 ms pulse at 515 nm, one such pulse pair every 30 s). 390 nm + 515 nm 390 nm 515 nm (rescue) mean % mean % mean % G2/M G2/M G2/M No Compound phase StDev phase StDev phase StDev 1 2.7 0.5 1.6 1.5 1.2 0.6 2 I.1 10 nM 1.9 0.3 1.1 1.1 0.8 0.5 3 I.1 100 nM 2.0 0.3 1.2 1.2 0.9 0.5 4 I.1 400 nM 15.1 1.4 8.2 9.7 6.4 4.5 5 I.1 600 nM 42.6 0.8 21.7 29.5 17.3 14.8 6 I.1 800 nM 54.2 10.9 32.5 30.7 24.7 12.0 7 I.1 1 M 52.9 3.9 28.4 34.7 22.3 16.3

Results 4: Light-Dependent Cell Proliferation Studied in Depth:

[0392] MTT assays on HEK-293T cells were performed with the MTT-assay procedure described in Results 2 above, but examining the light-dependency of the antiproliferative effect of compound I.1 in more depth. Cells were incubated in the dark, or with pulses of light at single wavelengths ranging from 525 nm to 390 nm, or under strong rescue regimes. The results are presented alongside the appropriate E() values from the in vitro modelling above (Table 10 below).

TABLE-US-00010 TABLE 10 HEK 293T cells were treated for 72 h with compound I.1 while being exposed to different irradiation patterns, each of which was applied every 2 min (pulse durations and wavelengths are as indicated in column headings). 390 nm, 0.35s 410 nm, then 0.35 s 525 505 490 475 410 390 490 nm, then 505 nm, nm, nm, nm, nm, nm, 3 s nm, 3 s HEK Dark 3 s 3 s 3 s 3 s 0.35 s 0.35 s (rescue) (rescue) PBS only 0.99 1.18 1.12 1.21 1.03 1.06 1.18 1.27 1.16 Cosolvent 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 CA4P 5 nM 0.82 0.86 1.07 1.43 1.12 1.19 1.29 1.28 1.20 CA4P 15 nM 0.79 0.81 0.75 0.97 0.70 0.80 0.88 0.82 0.81 I.1 100 nM 1.29 1.12 1.22 1.10 1.11 1.19 1.31 1.26 1.29 I.1 400 nM 0.87 0.82 0.87 0.69 0.55 0.70 0.67 0.69 0.86 I.1 800 nM 0.95 1.00 0.92 0.43 0.37 0.51 0.38 0.33 0.67 I.1 1.5 M 0.71 0.66 0.42 0.19 0.19 0.25 0.22 0.18 0.25 I.1 3 M 0.66 0.64 0.27 0.20 0.15 0.18 0.17 0.14 0.22 I.1 6 M 0.80 0.39 0.27 0.34 0.21 0.23 0.29 0.18 0.30 I.1 15 M 0.87 0.29 0.20 0.31 0.17 0.23 0.28 0.21 0.24 E() 0.02 0.04 0.08 0.14 0.59 1.00

[0393] It was observed that under the given experimental conditions, to achieve high cytotoxicity, either a high-efficiency wavelength could be applied in short pulses (eg. 390 nm or 410 nm), or else relatively long pulses of less efficient wavelengths could be applied (compare results for 3 s pulses of 475 nm with those for 0.35 s pulses of 410 nm), or else high doses of the compound of the invention could be applied even with a low-efficiency wavelength (results for 525 nm irradiation with 6 M of compound I.1 are similar to those for 390 nm with 800 nM). It was also observed that the rescue protocol using 490 nm was not successful in reducing toxicity, while the rescue using 505 nm was successful (each considered relative to the toxicity given by their activating wavelength components, 390 nm and 410 nm respectively; see eg. the effects at 400-800 nM where the toxicity difference is clearest). The difference in rescue success can be understood by reference to the relative difference in the calculated values (490)=0.30 and (505)=0.23 (see Photocharacterisation Results). 505 nm light is thereby understood as producing a more complete overall isomerisation of active cis isomer towards the inactive trans isomer than 490 nm, despite its comparatively lower overall efficiency of producing photoisomerisation at all. These data are therefore consistent with the hypothesis that a certain threshold amount of cis isomer, formed by overall light-induced trans->cis isomerism, is needed to achieve a cytotoxic effect, and that this threshold may be reached by modulating the combination of (a) the total dose of the compound applied, (b) the wavelength(s) applied (with regards to their E() and PSS()), and (c) the time-average duration of each applied wavelength(s). This highlights the principle of rationally-understood light control of the compounds of the invention, as well as their reversible light control. It also shows that lighting and dose conditions to be applied can be chosen and rationally tuned to respond to experimental constraints while achieving the desired biochemical outcome, and that such tuning can be understood to depend on the key isomerisation parameters which can be influenced by chemical design.sup.[38,48] and then measured experimentally as performed above in vitro.

Results 5: Membrane Permeability

[0394] Membrane integrity was assessed as a marker of cellular viability, via examining the uptake of propidium iodide (PI) in nonpermeabilized cells according to a standard protocol. Namely, cells were harvested and incubated with 5 g/mL PI in PBS containing 2% FCS (foetal calf serum), and immediately analysed by flow cytometry using a FACSCalibur flow cytometer.

[0395] The effect of compound I.1 was analysed in three cell lines: HeLa and MDA-MB-231 (Table 11 below) and Jurkat cells (Table 12 below). Treatment with compound I.1 combined with exposure to a 390 nm regimen led to increased membrane permeability in all cell lines tested. Similarly to the other experiments presented in this section, no dose-dependent toxicity of compound I.1 was observed in the dark. Therefore the cytotoxic effect of compound I.1 was light dependent. This is in notable contrast to the prior art of always-active compounds, as represented here by CA4P, which in both lighting conditions showed the same level of toxicity.

[0396] Data presented in Table 11 and Table 12 below are expressed as means and standard deviations of one representative experiment out of three independent trials performed in triplicate.

TABLE-US-00011 TABLE 11 The effects of compound I.1 on cell membrane permeability are presented. HeLa and MDA-MB-231 cells were treated for 70 h with compound I.1 while being exposed to irradiation at 390 nm (1 s every 15 min), or not (dark). The percentage of PI positive cells in the total amount of cells is shown. HeLa MDA-MB-231 light mean % mean % No conditions Compound cells PI+ StDev cells PI+ StDev 1 dark 4.2 0.1 5.1 0.7 2 dark I.1 200 nM 4.5 0.02 5.0 0.9 3 dark I.1 600 nM 4.3 0.3 4.6 0.2 4 dark I.1 1.5 M 4.2 0.5 4.3 0.4 5 dark I.1 5 M 5.1 0.1 5.0 0.5 6 dark CA4P 15 nM 62.5 0.8 45.1 0.9 7 390 nm 4.1 0.6 5.0 1.6 8 390 nm I.1 200 nM 4.5 0.1 4.4 0.8 9 390 nm I.1 600 nM 8.2 0.1 8.2 0.5 10 390 nm I.1 1.5 M 46.1 0.7 24.1 1.3 11 390 nm I.1 5 M 73.0 0.07 47.2 1.7 12 390 nm CA4P 15 nM 63.9 1.3 43.2 1.8

TABLE-US-00012 TABLE 12 The effects of compound I.1 on cell membrane permeability in Jurkat cells treated for 48 h with compound I.1 and exposed to irradiation at 390 nm (350 ms every 5 min) or not (dark). The percentage of PI positive cells in the total amount of cells is shown. Jurkat light mean % No conditions Compound cells PI+ StDev 1 dark 1.4 0.5 2 dark I.1 50 nM 1.6 0.02 3 dark I.1 100 nM 2.2 0.3 4 dark I.1 200 nM 2.3 0.5 5 dark I.1 400 nM 2.3 0.3 6 dark I.1 600 nM 2.8 0.4 7 dark CA4P 15 nM 8.0 1.6 8 390 nm 1.1 0.08 9 390 nm I.1 50 nM 1.3 0.3 10 390 nm I.1 100 nM 2.3 0.2 11 390 nm I.1 200 nM 3.4 0.3 12 390 nm I.1 400 nM 6.3 0.3 13 390 nm I.1 600 nM 8.1 1.4 14 390 nm CA4P 15 nM 5.7 0.9

Results 6: Nuclear Fragmentation

[0397] In order to determine whether cells treated with compound I.1 exhibit functional parameters of apoptosis, a quantification of nuclear fragmentation was performed according to the protocol established by Nicoletti.sup.[49]. Different cell types, including MDA-MB-231, Jurkat, HeLa and HepG2 cells, were treated with indicated concentrations of compound I.1 and exposed to a 390 nm toxic regime, a rescue regime with both 390 nm and 515 nm light, or kept in the dark. Following the treatment, cells were stained with PI and the percentage of cells containing a hypodiploid amount of DNA (subG1 phase of cell cycle), was determined. Briefly, prior to analysis, cells were harvested on ice and incubated in a hypotonic buffer [0.1% sodium citrate, 0.1% Triton X-100 and 50 g/mL propidium iodide (PI)] for 30 min at 4 C. Following the PI staining cells were analysed by flow cytometry using FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany) and Cell Quest Pro Software (Becton Dickinson). Subdiploid cells containing an amount of DNA inferior to that of the cells in the G1 phase were considered as apoptotic.

[0398] Results shown in Tables 13-17 represent means and standard deviations calculated for triplicates from one representative experiment out of three independent trials.

[0399] Compound I.1 showed similar behavior in a range of cell lines tested. Jurkat cells (Table 15) were most sensitive to compound I.1, while HepG2 cells showed higher resistance to this compound than HeLa cells, even when irradiated under more favourable conditions (Table 16), however all responded in the same qualitative way. This indicates that the invention's compounds have a generalizable mode of action as claimed for its mechanism of cytotoxicity.

[0400] EC.sub.50 values (concentration required for a 50% increase of the subG1 population percentage from control conditions towards the plateau maximum value) were calculated for MDA-MB-231 cells treated with compound I.1 in two different lighting conditions. Compound I.1 exposed to the 390 nm regimen showed an EC.sub.50 of approximately 1 M, while in the dark it showed an EC.sub.50 of approximately 120 M (Tables 13 and 14). Moreover a reduction of the cytotoxic effect of compound I.1 (by comparison to the 390 nm regimen) was observed upon application of the rescue protocol (Table 17), similar to results reported in the context of cell cycle arrest. The results for control compound CA4P (Table 15) showed the same response regardless of the illumination protocol. Therefore taken together, the results demonstrate that the ability of compound I.1 to induce this key indicator of apoptosis can be fully reversibly controlled by light.

TABLE-US-00013 TABLE 13 Effect of compound I.1 on the induction of apoptosis in MDA-MB-231 cells exposed to irradiation at 390 nm (350 ms pulses every 5 min), or not (dark), for 48 h. The mean percentage of cells in sub-G1 phase is shown. MDA-MB-231 DARK 390 nm mean % mean % subG1 subG1 No Compound phase StDev phase StDev 1 6.3 5.6 5.7 3.6 2 I.1 10 nM 6.7 3.6 3.6 1.2 3 I.1 100 nM 6.0 2.6 3.9 1.6 4 I.1 400 nM 3.3 0.2 4.2 0.8 5 I.1 600 nM 3.9 0.6 16.8 8.4 6 I.1 800 nM 3.0 0.9 32.1 3.2 7 I.1 1 M 3.1 1.2 55.7 5.5 8 I.1 1.2 M 3.6 0.3 56.6 11.0 9 I.1 1.5 M 3.3 1.2 76.1 5.6 10 I.1 3 M 6.0 1.1 79.0 6.7 11 I.1 6 M 3.9 0.2 83.5 0.06 12 I.1 15 M 5.4 1.8 83.7 4.8

TABLE-US-00014 TABLE 14 Effect of high concentrations of compound I.1 on the induction of apoptosis in MDA-MB-231 cells kept in the dark for 48 hours. The mean percentage of cells in sub-G1 phase is shown. MDA-MB-231 DARK mean % subG1 standard No Compound phase deviation 1 1.6 0.2 2 I.1 100 nM 2.1 0.6 3 I.1 1 M 2.0 0.6 4 I.1 10 M 2.0 0.2 5 I.1 50 M 4.5 1.1 6 I.1 75 M 7.9 1.2 7 I.1 85 M 12.7 3.5 8 I.1 100 M 26.8 6.1 9 I.1 125 M 51.1 25.5 10 I.1 150 M 92.2 4.9 11 I.1 175 M 95.7 0.5 12 I.1 200 M 95.5 0.2

TABLE-US-00015 TABLE 15 Effect of compound I.1 on the induction of apoptosis in Jurkat cells exposed to irradiation at 390 nm (350 ms pulses every 5 min) or not (dark) for 48 h. The mean percentage of cells in sub-G1 phase is shown. Jurkat DARK 390 nm mean % mean % subG1 subG1 No Compound phase StDev phase StDev 1 3.0 0.3 3.7 0.8 2 I.1 5 nM 3.0 0.5 2.7 0.4 3 I.1 50 nM 3.4 0.3 4.9 0.7 4 I.1 100 nM 3.5 0.6 9.1 0.06 5 I.1 150 nM 3.3 0.7 18.5 2.0 6 I.1 200 nM 3.0 0.5 49.7 5.7 7 I.1 400 nM 4.4 0.6 88.5 0.9 8 I.1 800 nM 5.5 0.9 89.5 0.6 9 I.1 1.5 M 7.9 1.2 89.6 0.6 10 I.1 5 M 12.0 1.3 91.0 0.6 11 CA4P 5 nM 37.5 2.2 40.4 1.0 12 CA4P 15 nM 89.7 1.2 91.5 1.4

TABLE-US-00016 TABLE 16 Effect of compound I.1 on the induction of apoptosis in HeLa and HepG2 cells. HeLa cells were kept in dark conditions or exposed to 390 nm light (1 s pulses every 15 min) for 30 h, while HepG2 cells were kept in dark conditions or exposed to 390 nm light (250 ms pulses every 15 s) for 48 h. HeLa mean % light subG1 No regime Compound phase StDev 1 dark 1.8 0.1 2 dark I.1 200 nM 1.8 0.2 3 dark I.1 600 nM 2.3 0.1 4 dark I.1. 1.5 M 2.9 0.01 5 dark I.1 5 M 3.2 0.1 6 dark CA4P 15 nM 11.5 0.6 7 390 nm 1.9 0.04 8 390 nm I.1 200 nM 2.4 0.2 9 390 nm I.1 600 nM 6.2 0.2 10 390 nm I.1 1.5 M 16.1 0.9 11 390 nm I.1 5 M 12.8 0.8 12 390 nm CA4P 15 nM 12.3 0.07 HepG2 mean % light subG1 No regime Compound phase StDev 1 dark 1.8 0.2 2 dark I.1 100 nM 2.5 0.3 3 dark I.1 600 nM 3.1 0.5 4 dark I.1 1 M 2.3 0.2 5 dark I.1 3 M 1.9 0.2 6 dark I.1 10 M 2.8 0.1 7 390 nm 4.7 0.6 8 390 nm I.1 100 nM 5.4 0.9 9 390 nm I.1 600 nM 5.2 0.1 10 390 nm I.1 1 M 4.6 1.2 11 390 nm I.1 3 M 6.5 0.5 12 390 nm I.1 10 M 14 2.1

TABLE-US-00017 TABLE 17 The rescue protocol of double illumination reduced the induction of apoptosis in MDA-MB-231 cells relative to a 390 nm regimen. Cells were illuminated for 48 h either with 390 nm light only (pulses of 150 ms pulse every 30 s) or in a rescue protocol where each such 390 nm pulse was immediately followed by a pulse at 515 nm (500 ms). 390 nm + 390 nm 515 nm mean mean % % subG1 subG1 No Compound phase StDev phase StDev 1 0.3 0.1 0.5 0.2 2 I.1 10 nM 0.3 0.05 0.3 0.06 3 I.1 100 nM 0.3 0.1 0.4 0.1 4 I.1 400 nM 14.0 1.6 1.7 0.4 5 I.1 600 nM 22.9 0.4 5.4 1.1 6 I.1 800 nM 19.4 0.3 11.7 1.0 7 I.1 1 M 22.5 0.6 15.3 2.0 8 I.1 2 M 35.4 2.6 25.0 1.3

Results 7: In Cellulo Tubulin Imaging

[0401] In order to investigate the influence of compound I.1 on microtubule structure in cellulo, immunostaining of the microtubule cytoskeleton in MDA-MB-231 cells was performed. Cells were treated with compound I.1 and then kept in dark conditions, or exposed to 390 nm light (200 ms every 2 min), or exposed to a rescue protocol with sequential pulses of 390 nm light (200 ms every 2 min) then immediately 505 nm (600 ms every 2 min). After 20 h of treatment cells were fixed, stained and subjected to confocal microscopy.

[0402] Briefly, prior to analysis, cells were washed with pre-warmed PBS then cell extraction buffer (80 mM piperazine-N,N-bis(2-ethanesulfonic acid) [abbreviated PIPES], 1 mM MgCl.sub.2, 5 mM EGTA-K and 0.5% Triton X-100, at pH 6.8) was added to remove monomeric and dimeric tubulin subunits. After 30 s of extraction, cells were fixed for 10 min with 0.5% glutaraldehyde. Following glutaraldehyde quenching with 0.1% NaBH.sub.4 in PBS for 7 min, coverslips were blocked with PBS containing 0.2% BSA (bovine serum albumin). Immunostaining was performed using anti--tubulin antibody (ab18251) and the AlexaFluor 488 secondary antibody (A 11008), purchased from Abcam (Cambridge, UK) and Invitrogen (Darmstadt, Germany), respectively. Hoechst 33342 (bisbenzimide), purchased from Sigma-Aldrich (catalogue number B2261; Taufkirchen, Germany), was used at a concentration of 1 ug/ml for nuclear staining. Cells were mounted with PermaFluor mounting medium (Beckman Coulter) and analyzed with a Zeiss LSM 510 Meta confocal microscope (Jena, Germany). Acquired images were processed using the ImageJ program (National Institutes of Health) and representative images are collected in FIG. 7.

[0403] Non-treated cells showed intact, long and polarized microtubules. In the 390 nm regimen, treatment with 1.5 M of compound I.1 led to degradation of the microtubule cytoskeleton, and 4 M of compound I.1 led to complete microtubule breakdown plus fragmentation of the nuclei (typical for apoptotic cells). Such nuclear fragmentation and microtubule destruction were not observed in control cells treated with compound I.1 but kept in the dark, indicating the light-controlled toxic effect of compound I.1. The rescue protocol also led to a dose-dependent reduction of the signs of cytotoxic effects of compound I.1, indicating the reversible photo-control of the tubulin polymerisation inhibitor properties of compound I.1 in cellulo.

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