NOVEL COMPOUNDS AND THERAPEUTIC USES THEREOF
20230322815 · 2023-10-12
Inventors
- Simon John Allison (Huddersfield, GB)
- Roger Mortimer Phillips (Huddersfield, GB)
- Craig Raymond Rice (Huddersfield, GB)
Cpc classification
A61K31/444
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
C07F13/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to compounds of Formula I:
##STR00001##
wherein R.sup.1, L.sup.1, A, X.sup.a, L.sup.2, B and X.sup.b are each as defined herein. The present invention also relates to compounds of Formula II and III defined herein, formed by self-assembly of the compounds of Formula I with a metal M and an anion Q. Compounds of Formula II and III are useful in the treatment of proliferative disorders, such as cancer. The present invention also relates to pharmaceutical compositions comprising compounds of Formula I, II or III, and to the use of these compounds and compositions in the treatment of proliferative disorders, such as cancer.
Claims
1. A compound having a structure according to Formula I shown below, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use as a medicament: ##STR00089## wherein R.sup.1 is selected from the group consisting of N, CR.sup.2, aryl, heteroaryl, carbocyclyl and heterocyclyl, where any aryl, heteroaryl, carbocyclyl or heterocyclyl in R.sup.1 is optionally substituted with one or more R.sup.3; each R.sup.3 is independently selected from the group consisting of hydroxy, cyano, halogen, (1-4C)alkyl, (1-4C)haloalkyl, (2-4C)alkenyl, (2-4C)alkynyl, aryl, aryl(1-3C)alkyl, heteroaryl, heteroaryl(1-3C)alkyl, carbocyclyl, carbocyclyl(1-3C)alkyl, heterocyclyl, heterocyclyl(1-3C)alkyl, —OR.sup.3a, —NR.sup.3aR.sup.3b, —C(O)—R.sup.3a, —C(O)—OR.sup.3a, —O—C(O)—R.sup.3a, —C(O)—NR.sup.3aR.sup.3b, —N(R.sup.3a)C(O)—R.sup.3b and —S(O).sub.0-2R.sup.3a, where any (1-4C)alkyl, (1-4C)haloalkyl, (2-4C)alkenyl, (2-4C)alkynyl, aryl, aryl(1-3C)alkyl, heteroaryl, heteroaryl(1-3C)alkyl, carbocyclyl, carbocyclyl(1-3C)alkyl, heterocyclyl or heterocyclyl(1-3C)alkyl in R.sup.3 is optionally substituted with one or more R.sup.3c; R.sup.3a and R.sup.3b are each independently selected from the group consisting of hydrogen, (1-3C)alkyl and (1-3C)haloalkyl; each R.sup.3c is independently selected from the group consisting of hydroxy, halogen, cyano, amino, (1-3C)alkyl, (1-3C)alkoxy and (1-3C)haloalkyl; R.sup.2 is selected from the group consisting of hydrogen, hydroxy, cyano, halogen, (1-4C)alkyl, (1-4C)haloalkyl, (2-4C)alkenyl, (2-4C)alkynyl, aryl, aryl(1-3C)alkyl, heteroaryl, heteroaryl(1-3C)alkyl, carbocyclyl, carbocyclyl(1-3C)alkyl, heterocyclyl, heterocyclyl(1-3C)alkyl, —OR.sup.2a, —NR.sup.2aR.sup.2b, —C(O)—R.sup.2a, —C(O)—OR.sup.2a, —O—C(O)—R.sup.2a, —C(O)—NR.sup.2aR.sup.2b, —N(R.sup.2a)C(O)—R.sup.2b and —S(O).sub.0-2R.sup.9a, where any (1-4C)alkyl, (1-4C)haloalkyl, (2-4C)alkenyl, (2-4C)alkynyl, aryl, aryl(1-3C)alkyl, heteroaryl, heteroaryl(1-3C)alkyl, carbocyclyl, carbocyclyl(1-3C)alkyl, heterocyclyl or heterocyclyl(1-3C)alkyl in R.sup.2 is optionally substituted with one or more R.sup.2c; R.sup.2a and R.sup.2b are each independently selected from the group consisting of hydrogen, (1-3C)alkyl and (1-3C)haloalkyl; each R.sup.2c is independently selected from the group consisting of hydroxy, halogen, cyano, amino, (1-3C)alkyl, (1-3C)alkoxy and (1-3C)haloalkyl; each L.sup.1 is a group:
—(W).sub.n—(X).sub.m—(Y).sub.o—(Z).sub.p— in which n and o are each independently 0, 1 or 2, and m and p are each independently 0 or 1, with the provisos that when m and p are both 1, o is not 0; each W is selected from the group consisting of (1-3C)alkylene, (2-3C)alkenylene, (2-3C)alkynylene, arylene, heteroarylene, carbocyclylene and heterocyclylene, where any (1-3C)alkylene, (2-3C)alkenylene, (2-3C)alkynylene, arylene, heteroarylene, carbocyclylene or heterocyclylene in W is optionally substituted with one or more W.sup.a, where each W.sup.a is independently selected from the group consisting of hydroxy, cyano, halogen, amino, (1-2)alkoxy and (1-2C)haloalkyl; X is selected from the group consisting of —O—, —C(O)—, —C(O)—O—, —O—C(O)—, —S(O).sub.0-2—, —C(O)—N(R.sup.x)—, —N(R.sup.x)—C(O)—, —NR.sup.x—, —N(R.sup.x)—C(O)—NR.sup.x—, —SO.sub.2N(R.sup.x)—, and —N(R.sup.x)SO.sub.2, where each R.sup.x is independently selected from the group consisting of hydrogen, hydroxy, cyano, (1-4C)alkyl, (2-4C)alkenyl and (2-4C)alkynyl; each Y is selected from the group consisting of (1-3C)alkylene, (2-3C)alkenylene, (2-3C)alkynylene, arylene, heteroarylene, carbocyclylene and heterocyclylene, where any (1-3C)alkylene, (2-3C)alkenylene, (2-3C)alkynylene, arylene, heteroarylene, carbocyclylene or heterocyclylene in Y is optionally substituted with one or more Y.sup.a, where each Y.sup.a is independently selected from the group consisting of hydroxy, cyano, halogen, amino, (1-2)alkoxy and (1-2C)haloalkyl; Z is selected from the group consisting of —O—, —C(O)—, —C(O)—O—, —O—C(O)—, —S(O).sub.0-2—, —C(O)—N(R.sup.z)—, —N(R.sup.z)—C(O)—, —NR.sup.z—, —N(R.sup.z)—C(O)—NR.sup.z—, —SO.sub.2N(R.sup.z)—, and —N(R.sup.z)SO.sub.2, where each R.sup.z is independently selected from the group consisting of hydrogen, hydroxy, cyano, (1-4C)alkyl, (2-4C)alkenyl and (2-4C)alkynyl; X.sup.a is a ring heteroatom located within ring A and is selected from N and O; each ring A is a monocyclic heteroaryl, bicyclic heteroaryl, monocyclic heterocycle or bicyclic heterocycle, any one of which is optionally substituted with one or more R.sup.4, where each R.sup.4 is independently selected from the group consisting of hydroxy, cyano, halogen, (1-6C)alkyl, (1-6C)haloalkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, aryl(1-3C)alkyl, heteroaryl, heteroaryl(1-3C)alkyl, carbocyclyl, carbocyclyl(1-3C)alkyl, heterocyclyl, heterocyclyl(1-3C)alkyl, —R.sup.4a—OR.sup.4b, —R.sup.4a—NR.sup.4bR.sup.4c, —R.sup.4a—C(O)—R.sup.4b, —R.sup.4a—C(O)—OR.sup.4b, —R.sup.4a—O—C(O)—R.sup.4b, —R.sup.4a—C(O)—NR.sup.4bR.sup.4c, —R.sup.4a—N(R.sup.4b)C(O)—R.sup.4c and —R.sup.4a—S(O).sub.0-2R.sup.4b, where any (1-6C)alkyl, (1-6C)haloalkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, aryl(1-3C)alkyl, heteroaryl, heteroaryl(1-3C)alkyl, carbocyclyl, carbocyclyl(1-3C)alkyl, heterocyclyl or heterocyclyl(1-3C)alkyl in R.sup.4 is optionally substituted with one or more R.sup.4d; R.sup.4a is absent or is (1-3C)alkylene that is optionally substituted with one or substituents selected from group consisting of hydroxy, halo and amino; R.sup.4b and R.sup.4c are each independently selected from the group consisting of hydrogen, (1-3C)alkyl and (1-3C)haloalkyl; each R.sup.4d is independently selected from the group consisting of hydroxy, halogen, cyano, amino, (1-3C)alkyl, (1-3C)alkoxy and (1-3C)haloalkyl; X.sup.b is a ring heteroatom located within ring B and is selected from N and O each ring B is a monocyclic heteroaryl, bicyclic heteroaryl, monocyclic heterocycle or bicyclic heterocycle, any one of which is optionally substituted with one or more R.sup.5, where each R.sup.5 is independently selected from the group consisting of hydroxy, cyano, halogen, (1-6C)alkyl, (1-6C)haloalkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, aryl(1-3C)alkyl, heteroaryl, heteroaryl(1-3C)alkyl, carbocyclyl, carbocyclyl(1-3C)alkyl, heterocyclyl, heterocyclyl(1-3C)alkyl, —R.sup.5a—OR.sup.5b, —R.sup.5a—NR.sup.5bR.sup.5c, —R.sup.5a—C(O)—R.sup.5b, —R.sup.5a—C(O)—OR.sup.5b, —R.sup.5a—O—C(O)—R.sup.5b, —R.sup.5a—C(O)—NR.sup.5bR.sup.5c, —R.sup.5a—N(R.sup.5b)C(O)—R.sup.5c and —R.sup.5a—S(O).sub.0-2R.sup.5b, where any (1-6C)alkyl, (1-6C)haloalkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, aryl(1-3C)alkyl, heteroaryl, heteroaryl(1-3C)alkyl, carbocyclyl, carbocyclyl(1-3C)alkyl, heterocyclyl or heterocyclyl(1-3C)alkyl in R.sup.5 is optionally substituted with one or more R.sup.5d; R.sup.5a is absent or is (1-3C)alkylene that is optionally substituted with one or substituents selected from group consisting of hydroxy, halo and amino; R.sup.5b and R.sup.5c are each independently selected from the group consisting of hydrogen, (1-5C)alkyl (e.g. (1-3C)alkyl) and (1-3C)haloalkyl; each R.sup.5d is independently selected from the group consisting of hydroxy, halogen, cyano, amino, (1-3C)alkyl, (1-3C)alkoxy and (1-3C)haloalkyl; and each L.sup.2 is selected from the group consisting of absent (in which case ring A is bonded directly to ring B), (1-2C)alkylene, ethenylene and ethynylene, where any (1-2C)alkylene, ethenylene and ethynylene in L.sup.2 is optionally substituted with one or more substituents selected form the group consisting of hydroxy, halogen, cyano, amino, (1-3C)alkyl, (1-3C)alkoxy and (1-3C)haloalkyl; and wherein the compound of Formula I, or the pharmaceutically acceptable salt, hydrate or solvate thereof, is for use in combination with a source of M, wherein M is selected from the group consisting of Zn.sup.2+, Mn.sup.2+, Cu.sup.2+, Fe.sup.2+, CO.sup.2+ and Ni.sup.2+.
2. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to claim 1, wherein each ring A is a 5-6 membered monocyclic heteroaryl containing 1, 2 or 3 ring heteroatoms in total independently selected from N, O and S, or a 5-6 membered monocyclic heterocycle containing 1, 2 or 3 ring heteroatoms in total independently selected from N, O and S, wherein each ring A is optionally substituted with one or more R.sup.4, and X.sup.a is located immediately adjacent the carbon atom bonded to L.sup.1;
3. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to claim 1 or 2, wherein each ring B is: i) a 5-6 membered monocyclic heterocycle containing 1, 2 or 3 ring heteroatoms in total that are independently selected from N, O and S; ii) a 5-6 membered monocyclic heteroaryl containing 1, 2 or 3 ring heteroatoms in total that are independently selected from N, O and S; iii) a 9-10 membered bicyclic heterocycle containing 1, 2 or 3 ring heteroatoms in total that are independently selected from N, O and S; or iv) a 9-10 membered bicyclic heteroaryl containing 1, 2 or 3 ring heteroatoms in total that are independently selected from N, O and S, wherein any ring in B is optionally substituted with one or more R.sup.5, and X.sup.b is located immediately adjacent the carbon atom bonded to L.sup.2.
4. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to claim 1, 2 or 3, wherein each ring A is group: ##STR00090## wherein a is 0 or 1.
5. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to any preceding claim, wherein each ring B is any of the following: ##STR00091## wherein: b.sup.1 is 0, 1 or 2, and b.sup.2 is 0, 1, 2 or 3.
6. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to any preceding claim, wherein each W is selected from the group consisting of (1-3C)alkylene or phenylene, where any (1-3C)alkylene or phenylene in W is optionally substituted with one or more W.sup.a; X is selected from the group consisting of —C(O)—N(R.sup.x)—, —N(R.sup.x)—C(O)— and —NR.sup.x—; each Y is selected from the group consisting of (1-3C)alkylene or phenylene, where any (1-3C)alkylene or phenylene in Y is optionally substituted with one or more Y.sup.a; and Z is selected from the group consisting of —C(O)—N(R.sup.z)—, —N(R.sup.z)—C(O)— and —NR.sup.z—.
7. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to any preceding claim, wherein: n is 0 or 1 and W is selected from the group consisting of (1-3C)alkylene or phenylene, where any (1-3C)alkylene or phenylene in W is optionally substituted with one or more W.sup.a, where each W.sup.a is independently selected from the group consisting of hydroxy, halogen, (1-2)alkoxy and (1-2C)haloalkyl; m is 0; o is 0 or 1 and Y is selected from the group consisting of (1-3C)alkylene or phenylene, where any (1-3C)alkylene or phenylene in Y is optionally substituted with one or more Y.sup.a, where each Y.sup.a is independently selected from the group consisting of hydroxy, halogen, (1-2)alkoxy and (1-2C)haloalkyl; and p is 1 and Z is —NR.sup.z—, where R.sup.z is hydrogen.
8. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to any preceding claim, wherein: n is 0 or 1 and W is selected from the group consisting of (1-3C)alkylene or phenylene, where any (1-3C)alkylene or phenylene in W is optionally substituted with one or more W.sup.a, where each W.sup.a is independently selected from the group consisting of hydroxy, halogen, (1-2)alkoxy and (1-2C)haloalkyl; m is 0; o is 0 or 1 and Y is selected from the group consisting of (1-3C)alkylene or phenylene, where any (1-3C)alkylene or phenylene in Y is optionally substituted with one or more Y.sup.a, where each Y.sup.a is independently selected from the group consisting of hydroxy, halogen, (1-2)alkoxy and (1-2C)haloalkyl; and p is 1 and Z is —NR.sup.z—, where R.sup.z is hydrogen.
9. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to any preceding claim, wherein L.sup.1 has a structure according to any one of the following: ##STR00092##
10. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to any preceding claim, wherein L.sup.2 is selected from the group consisting of absent and (1-2C)alkylene, where any (1-2C)alkylene in L.sup.2 is optionally substituted with one or more substituents selected form the group consisting of hydroxy, halogen and (1-2C)haloalkyl.
11. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to any preceding claim, wherein R.sup.1 is selected from the group consisting of N, CR.sup.2, phenyl and cyclohexyl, where any phenyl or cyclohexyl in R.sup.1 is optionally substituted with one or more R.sup.3; each R.sup.3 is independently selected from the group consisting of hydroxy, halogen, (1-4C)alkyl, (1-4C)haloalkyl and —OR.sup.3a, where any (1-4C)alkyl or (1-4C)haloalkyl in R.sup.3 is optionally substituted with one or more R.sup.3c; R.sup.2 is selected from the group consisting of hydrogen, and (1-3C)alkyl, where any (1-4C)alkyl in R.sup.2 is optionally substituted with one or more R.sup.2c.
12. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to any preceding claim, wherein R.sup.1 has a structure according to any one of the following: ##STR00093##
13. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to any preceding claim, wherein R.sup.5b and R.sup.5c are each independently selected from the group consisting of hydrogen, (1-3C)alkyl and (1-3C)haloalkyl.
14. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to any preceding claim, wherein M is selected from the group consisting of Zn.sup.2+, Cu.sup.2+, Mn.sup.2+ and Fe.sup.2+.
15. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to any preceding claim, wherein the compound of Formula I, or a pharmaceutically acceptable salt, hydrate or solvate thereof, is in further combination with a source of Q, wherein Q is an anion selected from the group consisting of spherical monoanionic anions, trigonal planar anions, dianionic tetrahedral anions, trianionic tetrahedral anions, dianionic octahedral anions and trianionic octahedral anions.
16. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to claim 15, wherein Q is an anion selected from the group consisting of dianionic tetrahedral oxoanions and trianionic tetrahedral oxoanions.
17. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to claim 15 or 16, wherein Q is sulfate (SO.sub.4.sup.2−), phosphate (PO.sub.4.sup.3−) or organophosphate (such as monophenylphosphate).
18. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to any preceding claim, wherein the compound of Formula I has a structure according to any one of the following: ##STR00094## ##STR00095## ##STR00096## ##STR00097## or a pharmaceutically-acceptable salt, hydrate and/or solvate thereof.
19. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to any preceding claim, wherein the compound of Formula I has the structure: ##STR00098## or a pharmaceutically-acceptable salt, hydrate and/or solvate thereof.
20. A compound having a structure according to Formula II shown below, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use as a medicament: ##STR00099## wherein M, R.sup.1, L.sup.1, A, X.sup.a, L.sup.2, B, X.sup.b and any associated subgroups are as defined in any preceding claim.
21. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to claim 20, wherein the compound of Formula II, or a pharmaceutically acceptable salt, hydrate or solvate thereof, is in further combination with a source of Q as defined in any one of claims 15, 16 and 17.
22. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to claim 20 or 21, wherein the compound of Formula II has a structure according to any one of the following: ##STR00100## or a pharmaceutically-acceptable salt, hydrate and/or solvate thereof.
23. A compound having a structure according to Formula III shown below, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use as a medicament: ##STR00101## wherein M, Q, R.sup.1, L.sup.1, A, X.sup.a, L.sup.2, B, X.sup.b and any associated subgroups are as defined in any one of claims 1-19.
24. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to claim 23, wherein the compound of Formula III has a structure according to any one of the following: ##STR00102## or a pharmaceutically-acceptable salt, hydrate and/or solvate thereof.
25. The compound, pharmaceutically acceptable salt, hydrate or solvate for use according to any preceding claim, wherein the medicament is for the treatment of a proliferative disorder (e.g. cancer).
Description
EXAMPLES
[0599] One or more examples of the invention will now be described, for the purpose of illustration only, with reference to the accompanying figures:
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PART A
Synthesis and Characterisation
[0625] Unless otherwise stated, all solvents and materials were purchased from either Sigma Aldrich, Fisher Scientific or Fluorochem and were used without further purification. .sup.1H, .sup.13C, DEPT-135 and DEPT-90 NMR data was recorded on either a Bruker Fourier 300 MHz or Bruker Avance III (AVIII) 400 MHz spectrometer or a Bruker Avance Neo 600 MHz NMR spectrometer. Mass spectra were obtained on an Agilent 6210 TOF MS with electrospray ionisation operating in positive ion mode and mass spectra of metal complexes were obtained on a Bruker Micro TOF-q LC mass spectrometer with electrospray ionisation operating in positive ion mode. Phosphorylated ‘SAMS’ peptide HMRSAMS*GLHLVKRR (phosphorylated on the serine residue) was obtained from PeptideSynthetics, Peptide Protein Research Ltd (>95% purity).
[0626] Single crystal X-ray diffraction data was collected at 150(2) K on a Bruker D8 Venture diffractometer equipped with a graphite monochromated Mo(Kα) radiation source and a cold stream of N2 gas. Solutions were generated by conventional heavy atom Patterson or direct methods and refined by full-matrix least squares on all F.sup.2 data, using SHELXS-97 and SHELXL software respectively..sup.26 Absorption corrections were applied based on multiple and symmetry-equivalent measurements using SADABS..sup.27 Almost all the structures contained some form of disordered ether with solvent molecules and/or counter anions (generally substitutional or rotation disorder). In these cases, the atoms were modelled using the PART instruction in the least squares refinement and refined over two positions. The anisotropic displacement parameters were treated with S/MU, DELU and in some cases ISOR where needed. Due to the diffuse nature of the electron density map the hydrogen atoms were not added to disordered solvent molecules. The structure [L.sub.2Zn.sub.3SO.sub.4](BF.sub.4).sub.3.5 contained extensively disordered tetrafluoroborate counter anions, one of which refined poorly and was modelled with 50% occupancy.
Ligand Synthesis
Synthesis of L
[0627] The ligand L was prepared as described previously..sup.28 However, procedures for the synthesis of the benzoylated thioamide (1) and the thioamide (2) were slightly modified.
##STR00087##
[0628] Synthesis of (1): To a solution of tris(2-aminoethyl)amine (1.0 g, 6.84 mmol) in acetone (50 mL) under an atmosphere of dinitrogen was added benzoyl isothiocyanate (3.7 g, 22.6 mmol) at such a rate to cause the reaction to gently reflux. After addition the reaction was stirred overnight during which time a colourless precipitate formed. The precipitate was isolated by filtration and washed with acetone (3×5 mL) giving (1) as a white solid. Yield=2.05 g (47%). .sup.1H NMR (400 MHz, DMSO-d.sup.6) δ (ppm) 11.20 (s, 3H, —NH), 11.0 (t, 3H, J=4.8, —CH.sub.2Nh), 7.81 (d, 6H, J=7.2, Ph), 7.56 (t, 3H, J=7.4, Ph), 7.38 (t, 6H, J=7.6, Ph), 3.75 (q, 6H, J=5.7, —CH.sub.2CH.sub.2NH), 2.89 (t, 6H, J=6.0 Hz, —CH.sub.2CH.sub.2NH). .sup.13C NMR [100 MHz, DMSO-d.sup.6]: δ.sub.C=180.5 (C═S), 168.2 (C═O), 133.1 (CH), 132.7 (Q), 128.9 (CH), 128.6 (CH), 51.9 (CH.sub.2), 42.6 (CH.sub.2). ESI-MS m/z 636 (M+H.sup.+), HR ESI-MS found 636.1882 C.sub.30H.sub.33N.sub.7S.sub.3O.sub.3 requires 636.1880 (error 0.46 ppm).
[0629] Synthesis of (2): The benzoylated urea derivative (1) (1.3 g, 2.05 mmol) was suspended in water (20 mL) and NaOH (820 mg, 20.5 mmol) added. The reaction was then heated to 60° C. and after 48 hrs the temperature was incrementally decreased allowing the solution to slowly cool to room temperature, avoiding formation on an oil and resulting in the formation of a colourless precipitate. Isolation by filtration and washing with ice cold water (2×1 mL) gave the tri-thiourea (2) as a colourless solid. Yield=503 mg (76%). .sup.1H NMR (400 MHz, DMSO-d.sup.6) δ (ppm) 7.55 (brs, 3H, —NH), 7.08 (brs, 6H, —NH.sub.2), 3.44 (brs, 6H, —CH.sub.2CH.sub.2NH), 2.58 (brs, 6H, —CH.sub.2CH.sub.2NH). .sup.13C NMR [100 MHz, DMSO-d.sup.6]: δ.sub.C=183.5 (C═S), 52.9 (CH.sub.2), 42.3 (CH.sub.2). ESI-MS m/z 324 (M+H.sup.+), HR ESI-MS found 324.1089 C.sub.9H.sub.21N.sub.7S.sub.3 requires 324.1093 (error 1.03 ppm).
Synthesis of L.SUP.1
[0630] Ligand L.sup.1 (an analogue of ligand L) shown below was prepared according to the following protocol.
[0631] Synthesis of α-bromo-2-acetylisoquinoline: To a solution of 2-acetylisoquinoline (1.0 g, 6.28 mmol) in carbon tetrachloride (20 mL) at 80° C. was added liquid bromine (0.39 mL, 7.55 mmol) in carbon tetrachloride (0.6 mL) slowly and dropwise until TLC (SiO.sub.2, 1% MeOH in DCM) showed the absence of the starting material. The reaction was cooled, diluted in dichloromethane (100 mL) and washed strenuously with saturated sodium carbonate solution (2×50 mL). The organic layer was removed, dried over anhydrous magnesium sulfate and solvents removed by reduced pressure to leave the crude product as a yellow oil. This was purified by column chromatography (1% MeOH in DCM, SiO.sub.2) and solvents removed to leave the pure product as a slightly yellow oil (1.08 g, 72%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ (ppm) 9.06-9.00 (m, 1H), 8.62 (d, J=5.5 Hz, 1H), 7.90-7.95 (m, 2H), 7.81-7.73 (m, 2H), 5.04 (s, 2H). .sup.13C NMR [100 MHz, CDCl.sub.3]: δ.sub.C=194.1 (Q), 150.1 (Q), 141.1 (CH), 137.1 (Q), 130.7 (CH), 129.7 (CH), 127.1 (CH), 126.53 (Q), 126.49 (CH), 125.6 (CH), 34.7 (CH.sub.2). ESI-MS m/z 249.9864 (M+H.sup.+), observed neutral mass 248.9788, C.sub.11H.sub.8NOBr requires 248.9789 (error 0.37 ppm).
[0632] Synthesis of L.sup.1: To a solution of α-bromo-2-acetylisoquinoline (500 mg, 2.10 mmol) in ethanol (20 mL), 1,1′, 1″-(nitrilotris(ethane-2,1-diyl))tris(thiourea) (226 mg, 0.70 mmol) was added. The reaction was then heated to 80° C. for 12 h, during which time all the solid dissolved and the solution turned yellow. The solution was then allowed to cool and the solvent removed under reduced pressure. The resultant oil was diluted in dichloromethane (50 mL) and washed with saturated aqueous sodium bicarbonate solution (2×25 mL). The combined organic layers were dried over anhydrous magnesium sulfate and solvent removed under reduced pressure to give the crude product as a brown/yellow oil. This was purified by column chromatography (SiO.sub.2, 10% methanol in dichloromethane) to afford L.sup.1 as an orange solid (277 mg, 51%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ (ppm) 8.88 (d, J=8.5, 3H), 8.56 (d, J=5.6, 3H), 7.81 (d, J=8.2, 3H), 7.64-7.57 (m, 6H), 7.48 (ddd, J=8.3, 7.1, 1.0, 3H), 7.09 (s, 3H), 6.39 (br s, 3H), 3.50 (q, J=5.3, 6H), 2.90 (t, J=5.56 Hz, 6H). .sup.13C NMR [100 MHz, CDCl.sub.3]: δ.sub.C=169.2 (Q), 153.7 (Q), 150.9 (Q), 142.0 (CH), 137.1 (Q), 129.9 (CH), 127.9 (CH), 127.1 (CH), 126.72 (Q), 126.70 (CH), 120.5 (CH), 108.8 (CH), 53.3 (CH.sub.2), 43.4 (CH.sub.2). ESI-MS m/z 777.2363 (M+H.sup.+), observed neutral mass 776.2291, C.sub.42H.sub.36N.sub.10S.sub.3 requires 776.2287 (error 0.63 ppm).
##STR00088##
Synthesis of Complexes
Mononuclear Complexes
[0633] Synthesis of [LZn](ClO.sub.4).sub.2. To a solution of Zn(ClO.sub.4).sub.2.Math.6H.sub.2O (10 mg. 0.027 mmol) in MeCN (1 ml) was added a suspension of ligand L (12 mg, 0.019 mmol) in MeCN and the reaction sonicated until a clear solution had formed. To this was added water (˜1 ml) and the solution slowly allowed to evaporate, during which time pale yellow crystals were formed which were isolated by filtration and dried (yield=9 mg. 53%*). The [LMn](ClO.sub.4).sub.2 complex was prepared in an analogous fashion using Mn(ClO.sub.4).sub.2.Math.6H.sub.2O giving yellow crystals (yield=10 mg, 60%*). *percentage yield based on the moles of ligand used.
Trinuclear Complexes
[0634] Synthesis of [L.sub.2Zn.sub.3(SO.sub.4)](ClO.sub.4).sub.4. To a solution of Zn(ClO.sub.4).sub.2.Math.6H.sub.2O (10 mg. 0.027 mmol) in MeCN (1 ml) was added a suspension of ligand L (12 mg, 0.019 mmol) in MeCN and the reaction sonicated until a clear solution had formed. To this was added water (˜1 ml) containing Bu.sub.4NHSO.sub.4 (3.1 mg, 0.009 mmol) and the solution slowly allowed to evaporate during which time colourless crystals were formed which were isolated by filtration and dried (yield=11 mg, 60%). The [L.sub.2Mn.sub.3(SO.sub.4)](ClO.sub.4).sub.4 complex was prepared in an analogous fashion using Mn(ClO.sub.4).sub.2.Math.6H.sub.2O giving yellow crystals (yield=9 mg, 50%).
[0635] Synthesis of [L.sub.2Cu.sub.3(O.sub.3POPh)](ClO.sub.4).sub.4. To a solution of Cu(ClO.sub.4).sub.2.Math.6H.sub.2O (10 mg. 0.027 mmol) in MeCN (1 ml) was added a suspension of ligand L (12 mg, 0.019 mmol) in MeCN and the reaction sonicated until a clear solution had formed. To this was added water (˜1 ml) containing Na.sub.2O.sub.3POPh (2.3 mg, 0.009 mmol) and the solution slowly allowed to evaporate giving green crystals which were isolated by filtration and dried (yield=9 mg, 47%).
[0636] Synthesis of [L.sub.2Zn.sub.3(PO.sub.4)](ClO.sub.4).sub.3. To a solution of Zn(ClO.sub.4).sub.2.Math.6H.sub.2O (10 mg. 0.027 mmol) in MeCN (1 ml) was added a suspension of ligand L (12 mg, 0.019 mmol) in MeCN and the reaction sonicated until a clear solution had formed. To this was added water (˜1 ml) containing Na.sub.2O.sub.3POPh (2.3 mg, 0.009 mmol) and the solution slowly allowed to evaporate giving yellow crystals which were isolated by filtration and dried (yield=10 mg, 54%). The [L.sub.2Mn.sub.3(PO.sub.4)](ClO.sub.4).sub.3 complex was prepared in an analogous fashion using Mn(ClO.sub.4).sub.2.Math.6H.sub.2O giving yellow crystals (yield=9 mg, 50%).
DISCUSSION
[0637] Reaction of 1.5 equivalents of L with either Zn(ClO.sub.4).sub.2 or Mn(ClO.sub.4).sub.2 results in a mononuclear complex (e.g. [LM].sup.2+) as demonstrated by X-ray crystallography and ESI-MS. In the solid-state the zinc complex contains a 6-coordinate Zn.sup.2+ cation coordinated by six nitrogen atoms from three pyridyl-thiazole bidentate units from the same ligand. In the Mn.sup.2+ analogue the metal ion is again 6-coordinate, but this arises from coordination by four N-donor atoms from two bidentate pyridyl-thiazole units and two water O-donor atoms (
[0638] Reaction of L with either Mn.sup.2+ or Zn.sup.2+ with (Bu.sub.4N)HSO.sub.4 (in the correct stoichiometric proportions) results in the formation of the capsule in which sulfate anions are encapsulated (e.g. [L.sub.2M.sub.3(SO.sub.4)].sup.4+) (
[0639] Ions in the ESI-MS at m/z 1844 and 1812 corresponding to {[L.sub.2Zn.sub.3(SO.sub.4)(ClO.sub.4).sub.3}.sup.+ and {[L.sub.2Mn.sub.3(SO.sub.4)(ClO.sub.4).sub.3}.sup.+ coupled with doubly charged ions indicate that these species are also observed in the gas phase.
[0640] Addition of disodium phenylphosphate to a solution of [L.sub.2Cu.sub.3].sup.6+ in MeCN/H.sub.2O results in a colour change from light blue to green. Crystals were then deposited after several days and analysis by X-ray crystallography shows that the trimetallic capsule is still formed but held inside the host is a PhOPO.sub.3.sup.2− anion. In a very similar fashion to the other oxoanions, PhOPO.sub.3.sup.2− is coordinated to the three Cu.sup.2+ metal ions supplemented by a series of —NH— anion interactions. However, due to the phenyl substituent the ligands adopt a slightly different conformation allowing the phenyl unit to occupy a cleft formed by two pyridyl-thiazole units (
[0641] Reaction of two equivalents of L, three equivalents of either M(ClO.sub.4).sub.2(where M=Zn.sup.2+ or Mn.sup.2+) and PhOPO.sub.3Na.sub.2 results in a very different species. In the solid-state both structures contain a central PO.sub.4.sup.3− anion held within the molecule by a series of interactions between the metal ions and amine hydrogen atoms (
[0642] This metal-dependent reactivity is also observed in the ESI-MS. Reaction of [L.sub.2Cu.sub.3](ClO.sub.4).sub.6 with PhOPO.sub.3Na.sub.2 in water and MeCN gives ions at m/z 1914, 1024, 788 and 463 corresponding to {[L.sub.2Cu.sub.3(PhOPO.sub.3)](ClO.sub.4).sub.3}+, {[LCu.sub.2(PhOPO.sub.3)](ClO.sub.4)}+, {[LCu](ClO.sub.4)}+ and {[LCu.sub.2(PhOPO.sub.3)]}.sup.2+. Heating this sample at 80° C. shows no change in the ESI-MS spectrum indicating that the phenylphosphate dianion remains intact. A similar reaction of PhOPO.sub.3Na.sub.2 with Zn(ClO.sub.4).sub.2 and L gave an ESI-MS with ions at m/z 1920 and 910 corresponding to {[L.sub.2Zn.sub.3(PhOPO.sub.3)](ClO.sub.4).sub.3}.sup.+ and {[L.sub.2Zn.sub.3(PhOPO.sub.3)](ClO.sub.4).sub.2}.sup.2+ respectively. Lower molecular weight ions at m/z 1029, 791 and 464 corresponding to {[LZn.sub.2(PhOPO.sub.3)](ClO.sub.4)}.sup.+, {[LZn](ClO.sub.4)}.sup.+ and {[LZn.sub.2(PhOPO.sub.3)]}.sup.2+ were also observed. However, heating this sample at 80° C. results in a dramatic change in the ESI-MS with the spectrum now much simplified with ions at m/z 1743 and 822 corresponding to {[L.sub.2Zn.sub.3(PO.sub.4)](ClO.sub.4).sub.2}.sup.+ and {[L.sub.2Zn.sub.3(PO.sub.4)](ClO.sub.4)}.sup.2+. This demonstrates that initially the Zn.sup.2+ containing complex reacts with phenyl phosphate dianion and in a similar fashion to the Cu.sup.2+ analogue and forms the trinuclear complex incorporating this anion (e.g. [L.sub.2Zn.sub.3(PhOPO.sub.3)].sup.4+). However, after either a few days at room temperature or heating at 80° C. for 1 hr the anion is hydrolysed and phosphate is encapsulated within the cryptand (see
[0643] The Zn.sup.2+ complex shows substrate specific differences in the rates of hydrolysis and its phosphatase activity. Analysis of the hydrolysis of phenyl phosphate dianion by the Zn.sup.2+ complex (in a 25%:75% mixture of DMSO and buffered H.sub.2O solution (HEPES pH 7.5)) by .sup.31P NMR shows a signal at −1.5 ppm corresponding to unhydrolysed PhOPO.sub.3.sup.2− at t=0 (
[0644] Given the importance of protein phosphorylation in inter- and intra-cellular signalling and to cell function, and its common dysregulation in cancers,.sup.30, 31 it was next analysed whether the Zn.sup.2+ complex could dephosphorylate the phosphorylated amino acids serine, threonine and tyrosine. Indeed, the Zn.sup.2+ complex resulted in dephosphorylation of serine-PO.sub.3.sup.2− and tyrosine-OPO.sub.3.sup.2− at similar hydrolysis rates to PhOPO.sub.3.sup.2− with substantial hydrolysis occurring over 24 hrs and completion after 48 hrs (
[0645] The rate of substrate hydrolysis is also dependent upon the metal used in the self-assembly process. The solid-state and ESI-MS data suggest that [L.sub.2Cu.sub.3].sup.6+ does not hydrolyse phenyl phosphate but incorporates this anion within the assembly e.g. [L.sub.2Cu.sub.3(PhOPO.sub.3)].sup.4+. Comparison of the reactivity of the Zn.sup.2+ species verses the Mn.sup.2+ by monitoring the hydrolysis of 4-nitrophenyl phosphate by UV-Vis spectroscopy shows after 24 hrs the Mn.sup.2+ has hydrolysed with three times more phosphate, indicating that the Mn.sup.2+ is more active than the Zn.sup.2+ complex.
Biological Studies
Cell Lines and Culture
[0646] All cell lines used were maintained at low passage in antibiotic-free media and were obtained from ATCC (LGC Standards, Middlesex, UK) unless otherwise stated (Methods). HT29, DLD-1, HCT116 p53.sup.+/+ and HCT116 p53.sup.−/− are all colorectal adenocarcinoma cell lines derived from different individuals and harbor different combinations of oncogenic lesions (except for the HCT116 isogenic cancer cell clones that are genetically identical except for p53 status). PSN-1, BxPC-3 and MiaPaCa2 are pancreatic carcinoma cell lines and A549 and H460 are lung carcinoma cell lines. HT29, DLD-1, PSN-1, BxPC-3, A549 and H460 cell lines were cultured in RPMI-1640 growth media (Sigma) containing 2 mM L-glutamine, 1 mM sodium pyruvate and 10% fetal bovine serum (FBS). HCT116 (p53.sup.+/+ and p53.sup.−/−) and MiaPaCa2 cell lines were cultured in Dulbecco's Modified Eagle's Medium (Sigma), 2 mM L-glutamine and 10% FBS. The ARPE-19 retinal epithelial non-cancer cell line (Dunn et al., 1996) was cultured in DMEM/F12 media (Gibco), 2 mM L-glutamine, 1 mM sodium pyruvate and 10% FBS. The MCF10A non-cancerous human breast epithelial cell line was cultured in Minimal Essential Media Eagle (Sigma), 2 mM L-glutamine, 1 mM sodium pyruvate, 10% FBS and 1×non-essential amino acids (NEAA). NP1 and GBM1 cells were cultured on plasticware coated with poly-L-ornithine (5 μg/ml) and laminin (2.4 μg/ml) (Polson et al., 2018; Da Silva et al., 2019). NP1 cells were grown in DMEM/F12 media (Gibco) supplemented with 5% FBS, 20 ng/ml hFGF, 20 ng/ml rhEGF, 0.5×B-27 supplement (Gibco), 0.5×N-2 supplement (Gibco) and 1×GLUTAMAX (Gibco). GBM1 cells were cultured in Neurobasal media (Gibco) supplemented with 40 ng/ml hFGF, 40 ng/ml rhEGF, 0.5×B-27 supplement (Gibco) and 0.5×N-2 supplement (Gibco).
Chemosensitivity Studies
[0647] [L.sub.2M.sub.3].sup.6+ and all the other self-assembling complexes were freshly formed by adding DMSO to individual components mixing together by pipetting. These were then further diluted in cell culture media such that the final DMSO concentration that cells were exposed to was 0.2% (vehicle control). Cisplatin, oxaliplatin and carboplatin were dissolved in phosphate buffered saline. Cell lines were seeded into 96 well plates at 2×10.sup.3 cells per well and incubated overnight at 37° C. GBM1 cancer stem-like cells were seeded at 3×10.sup.3 cells per well and NP1 neural progenitors were seeded at 1.5×10.sup.3 cells per well. The following day, media was removed and replaced with fresh media containing test compounds at a range of concentrations. Cells were incubated with test compounds for a further 96 hours after which the media was removed and replaced with fresh media (200 μl/well). MTT was added (20 μl at 5 mg/ml) and cells were incubated for a further 4 hours. Media and MTT were removed and formazan crystals were dissolved in 150 μl of DMSO and the absorbance of the resulting solution determined at 540 nm. Dose response curves were constructed, and the concentration required to reduce cell growth by 50% (IC.sub.50) determined. The selectivity index was defined as the IC.sub.50 for non-cancer cells divided by the IC.sub.50 for cancer cell lines with values >1 representing selectivity for cancer cells as opposed to non-cancer cells.
Immunoblotting
[0648] Protein lysates and recombinant proteins were resolved on 15% SDS polyacrylamide gels. Proteins were electroblotted onto nitrocellulose membrane by wet transfer in 1×Tris Glycine buffer (Biorad) at 35 mA overnight at room temperature (Allison et al., 2014). After blocking of membranes, these were incubated with primary antibody overnight at 4° C. before addition of rabbit or mouse secondary antibody (HRP-conjugated) for 1 h at room temperature and development of blots by enhanced chemiluminescence. Primary antibodies were: anti-Src (total) (Cell Signalling Technology #2123 1:1000), anti-phosphorylated Src (Y527) (Cell Signalling Technology #21055 1:1000), anti-phosphorylated Src (Y416) (Cell Signalling Technology #69435 1:1000), anti-AMPKα (total) (Cell Signalling Technology #2532 1:1000), anti-phosphorylated AMPKα (T172) (Cell Signalling Technology #2532 1:1000), anti-phospho-Tyr (pan) (Cell Signalling Technology #8954 1:2000), anti-p53 (Santa Cruz, DO-1 clone, 1:1000), anti-β-actin (Merck MAB1501, 1:40,000). Quantification of signals was performed by densitometry using Image J software.
Results and Discussion
[0649] [L.sub.2Cu.sub.3].sup.6+ and [L.sub.2Zn.sub.3].sup.6+ possess both potent and selective activity against most of the cancer cell lines tested compared to three non-cancer cell models utilised (
[0650] The magnitude of selectivity towards cancer cells was marked and for both [L.sub.2Cu.sub.3].sup.6+ and [L.sub.2Zn.sub.3].sup.6+ was over 10-fold for most of the cancer cell lines compared to all three non-cancer cell models (
[0651] Whilst the potency of both the Zn.sup.2+ and Cu.sup.2+ complexes (
[0652] The encapsulation of different specific anions (e.g. PO.sub.4.sup.3−, SO.sub.4.sup.2− or PhOPO.sub.3.sup.2−) into the [L.sub.2Zn.sub.3].sup.6+ and [L.sub.2Cu.sub.3].sup.6+ complexes at the point of self-assembly prior to any cell exposure impacts on both activity and selectivity. The effect is both anion and cell line dependent (see
Mechanistic Studies
[0653] Given the ability of the Zn.sup.2+ complex to dephosphorylate amino acids serine, tyrosine and threonine (
[0654] There are several potential mechanisms which could lead the observed kinase inhibition ‘readout’ of this screen including, i) direct ATP hydrolysis by the complex(es) and differing Km of the kinases for ATP, and, ii) dephosphorylation of the peptide substrate by the phosphatase activity of the Zn.sup.2+ complex resulting in cycles of peptide phosphorylation by active recombinant enzyme and dephosphorylation by the complex. However, mass spectroscopy showed little or no hydrolysis of ATP when incubated with Zn.sup.2+ or Cu.sup.2+ complex alone (see Further results section). Similarly, neither complex resulted in dephosphorylation of a purified phosphorylated AMPK peptide (see Further results section) although AMPK is one of the most potently inhibited kinases by both complexes (
[0655] An alternative explanation of the observed effects which can be reconciled with both selective kinase inhibition and activation is that that the Zn.sup.2+ and Cu.sup.2+ complexes are modulating key regulatory phosphor-sites on the kinases themselves leading to enhanced or repressed kinase activity. In the case of the Zn.sup.2+ complex, dephosphorylation of specific phosphorylated regulatory amino acids through its phosphatase activity (
[0656] To investigate these hypotheses further, regulatory phospho-amino acids of two key kinases identified by the kinase screen, AMPK and Src, were analysed following incubation of the recombinant kinases with Zn.sup.2+ or Cu.sup.2+ complexes. Phosphorylation of AMPKα at threonine 172 (p-T172) stimulates AMPK activity.sup.37 and treatment of AMPK with either [L.sub.2Zn.sub.3].sup.6+ or [L.sub.2Cu.sub.3].sup.6+ significantly reduced p-T172 levels detectable by immunoblotting at a molecular weight of ˜63 kDa (
[0657] These results are consistent with the inhibition of AMPK by both [L.sub.2Zn.sub.3].sup.6+ and [L.sub.2Cu.sub.3].sup.6+ observed in the kinase screen (
[0658] For Src, effects of the complexes on two key regulatory phospho-amino acids were examined, Y527 and Y416 (
Selective Induction of Autophagy and Cancer Cell ATP Depletion
[0659] Given the kinase screen results indicating that the [L.sub.2Cu.sub.3].sup.6+ and [L.sub.2Zn.sub.3].sup.6+ complexes can inhibit multiple kinases as well as activating several others (
[0660] Autophagy is a catabolic process that is induced in response to metabolic stresses including low ATP levels and starvation..sup.37 It was hypothesised that the induction of autophagy by Zn.sup.2+ and Mn.sup.2+ complexes could be due to cellular ATP depletion resulting from their protein phosphatase activity and repeated futile cycles between protein phosphorylation by constitutively active oncogenic kinases.sup.39 and dephosphorylation by the complexes. In support of this hypothesis, Zn.sup.2+ and Mn.sup.2+ complexes both caused a dose-dependent decrease in ATP levels (
[0661] Immunoblot analyses suggest that the autophagy is a compensatory catabolic response to sustain ATP levels and prevent bioenergetic failure and death with [L.sub.2Zn.sub.3].sup.6+ inducing activation of the ‘low ATP’ sensing kinase AMPK.sup.37 in HCT116 cancer cells but not in ARPE19 non-cancer cells (
[0662] Thus, cellular levels of p-T172 of AMPKα were increased relative to total AMPKα levels specifically in the HCT116 cancer cells by [L.sub.2Zn.sub.3].sup.6+ but not by [L.sub.2Cu.sub.3].sup.6+ (
[0663] Following [L.sub.2Zn.sub.3].sup.6+ treatment of HCT116 cancer cells, a small decrease (˜10%) in total levels of Tyr-phosphorylated proteins was detected by immunoblotting using a pan phospho-tyrosine antibody although there was also evidence of increased p-Y of some proteins (*) (
Further Results
[0664] Chemosensitivity studies results: The effect of anions on the potency and selectivity of all test compounds evaluated is presented in
[0665] Inhibition of kinase activity: The effect of [L.sub.2Cu.sub.3].sup.6+ and [L.sub.2Zn.sub.3].sup.6+ on the activity of 140 human recombinant kinases is presented in
[0666] Autophagy studies: The effects of 3.125 μM [L.sub.2Cu.sub.3].sup.6+, [L.sub.2Zn.sub.3].sup.6+ and [L.sub.2Mn.sub.3].sup.6+ treatments (40 h) on the induction of cellular vacuoles and autophagy in the HCT116 p53.sup.−/−, HCT116 p53.sup.+/+ and ARPE19 cells are presented in
[0667] Cellular ATP studies: The effects of 20 h treatment with a range of different concentrations of [L.sub.2Cu.sub.3].sup.6+, [L.sub.2Zn.sub.3].sup.6+ and [L.sub.2Mn.sub.3].sup.6+ on total cellular ATP levels in HCT116 p53.sup.+/+ and ARPE19 cells are presented in
SUMMARY
[0668] In summary, in this study three different metal-containing, self-assembled anion binding complexes are characterised and shown to have distinctive chemical and biological properties. Depending on the metal, the reactivity of the complexes towards different anionic species varied with the Zn.sup.2+ and Mn.sup.2+ complexes both showing significant phosphatase activity but with different rates of hydrolysis (Mn.sup.2+>>Zn.sup.2+) whereas the Cu.sup.2+ complex bound to, rather than hydrolysed, phospho-containing species. Remarkable selective activity towards particular cancer cells compared to non-cancer cells was shown by the Zn.sup.2+ and Cu.sup.2+ complexes by different mechanisms with the modulation of multiple kinases via either binding (Cu.sup.2+) or by de-phosphorylation (Zn.sup.2+) of regulatory sites on kinases. Zn.sup.2+ and Mn.sup.2+ complexes both induced cancer cell autophagy consistent with cellular ATP deficiency and bioenergetic failure as a result of their phosphatase activity and futile cycles of re-phosphorylation by oncogenic kinases. Further modulation of activity and selectivity profile by incorporation of different anions (eg. PO.sub.4.sup.3−, SO.sub.4.sup.2− or PhOPO.sub.3.sup.2−) pre-cell exposure indicates the ease of generating numeromodular' combinations of metal/anion binding self-assembling complexes that can differ in potency, selectivity and mechanism(s) of action towards disease.
PART B
Overview
[0669] [L.sub.2Cu.sub.3].sup.6+ and [L.sub.2Zn.sub.3].sup.6+ were evaluated at three different doses for in ovo anti-cancer activity, toxicity and anti-metastatic activity against tumors initiated from three different human cancer cell line models (H460, HT29, HCT116), in the chick embryo chorioallantoic membrane (CAM) assay.
Methodology
[0670] Each compound was provided to the contract research organisation (CRO) Inovotion (La Tronche, France) conducting the in ovo study, as a powder pre-weighed in single vials (2 equivalents of ligand, 3 equivalents of metal; and stored at room temperature). Before use, this was then dissolved in DMSO to 50 mM to generate a stock solution and further freshly diluted in RPM11640 or McCoys aqueous cell culture media for administration.
[0671] A standard of care (SoC) drug for the particular tumour type was tested concurrently as a comparative control for in vivo efficacy and toxicity evaluation. A solvent control served as a negative control for both SoC and experimental compounds.
[0672] 10.sup.6 tumor cells (H460, HT29 or HCT116) were grafted onto the upper chorioallantoic membrane (CAM) of the developing chick embryo on embryonic day 9 (E9). Access to the upper CAM was through a small hole drilled through the eggshell of fertilized White Leghorn eggs (into the air sac) on E9 (15 eggs per experimental group).
[0673] 100 μl of freshly prepared experimental compound, vehicle control or standard-of-care (SoC) drug at their working concentrations were pipetted onto the tumor graft at E11, E13, E15 and E17. On day E18, the upper CAM (with tumor) was removed, washed by PBS buffer and then directly transferred in PFA (fixation for 48 hrs). After that, tumors were carefully cut away from normal CAM tissue and weighed.
[0674] Embryonic viability was checked daily. The number of dead embryos was also counted on E18, to evaluate treatment-induced embryo toxicity.
[0675] On day E18, a 1 cm.sup.2 portion of the lower CAM was collected (n=8 per group), stored at −20° C. and later processed to extract DNA. Samples were analysed for human genomic DNA (qPCR for Alu sequences) as a quantitative marker of tumor metastasis to the lower CAM.
Results
[0676] 1. In Vivo Efficacy Against Tumors Initiated from Human Lung Cancer Cell Line H460
Reduction of H460 Tumor Growth
[0677] Statistically significant regression of tumor growth (as determined by mean tumor weight at the end of the study, E18) was observed for [L.sub.2Zn.sub.3].sup.6+ at all three tested doses and for [L.sub.2Cu.sub.3].sup.6+ at the two highest tested doses (
Analysis of Anti-Metastatic Activity
[0678] Statistically significant decreases in H460 metastatic load were observed for all three doses of [L.sub.2Zn.sub.3].sup.6+ with reduction by the highest dose comparable to that observed with SoC (
Analysis of Embryonic Toxicity
[0679] In term of toxicity, [L.sub.2Cu.sub.3].sup.6+ and [L.sub.2Zn.sub.3].sup.6+ were well tolerated at all doses tested with no statistically significant difference observed compared to the solvent (negative) control group (Table 1).
TABLE-US-00001 TABLE 1 Percentage of alive and dead embryos per experimental group at the end of the H460 in ovo study. Group Gr. # Description Total Alive Dead % Alive % Dead 1 Neg. Ctrl. 14 12 2 85.71 14.29 2 SoC 14 12 2 85.71 14.29 3 L2Zn3 [1] 14 13 1 92.86 7.14 4 L2Zn3 [2] 13 10 3 76.92 23.08 5 L2Zn3 [3] 11 10 1 90.91 9.09 6 L2Cu3 [1] 14 12 2 85.71 14.29 7 L2Cu3 [2] 14 13 1 92.86 7.14 8 L2Cu3 [3] 14 12 2 85.71 14.29
Summary (H460 Tumours)
[0680] Overall, these results show that both [L.sub.2Cu.sub.3].sup.6+ and [L.sub.2Zn.sub.3].sup.6+ are well tolerated at all tested doses with [L.sub.2Zn.sub.3].sup.6+ shown to have comparable anti-cancer efficacy to the SoC in ovo, both in decreasing primary H460 tumor weight and metastatic burden. For [L.sub.2Cu.sub.3].sup.6+, at the highest dose tested, comparable anti-cancer activity to the SoC was also observed in decreasing H460 tumor weight, however, no statistically significant metastatic regression was observed.
[0681] This provides the first evidence of in vivo efficacy of these compounds (at non-optimised doses) validating the anti-cancer activity and selectivity observed in vitro. The lack of embryonic toxicity observed suggests testing of higher doses is warranted (for potentially improved in vivo efficacy compared to the SoC).
2. In Vivo Efficacy Against Tumors Initiated from Human Colon Cancer Cell Line HT29
Reduction of HT29 Tumor Growth
[0682] Statistically significant regression of tumor growth (as determined by mean tumor weight at the end of the study, E18) was observed for both [L.sub.2Zn.sub.3].sup.6+ and [L.sub.2Cu.sub.3].sup.6+ at their highest tested dose that was comparable that obtained with the SoC (
Analysis of Anti-Metastatic Activity
[0683] There was no reduction in HT29 tumor cell metastasis to the lower CAM either with the SoC or any of the tested doses of [L2Cu.sub.3].sup.6+ and [L.sub.2Zn.sub.3].sup.6+ (data not shown).
Analysis of Embryonic Toxicity
[0684] [L.sub.2Cu.sub.3].sup.6+ and [L.sub.2Zn.sub.3].sup.6+ were well tolerated in the chick embryos grafted with HT29 colon cancer cells with increasing dose escalation showing no increased embryonic toxicity (Table 2).
TABLE-US-00002 TABLE 2 Percentage of alive and dead embryos per experimental group at the end of the HT29 in ovo study. Group Gr. # Description Total Alive Dead % Alive % Dead 1 Neg. Ctrl. 15 14 1 93.33 6.67 2 SoC 14 14 0 100 0 3 L2Zn3 [1] 14 12 2 85.71 14.29 4 L2Zn3 [2] 14 13 1 92.86 7.14 5 L2Zn3 [3] 14 13 1 92.86 7.14 6 L2Cu3 [1] 14 10 4 71.43 28.57 7 L2Cu3 [2] 14 12 2 85.71 14.29 8 L2Cu3 [3] 13 12 1 92.31 7.69
Summary (HT29 Tumours)
[0685] Overall, these results show that both [L.sub.2Cu.sub.3].sup.6+ and [L.sub.2Zn.sub.3].sup.6+ are well tolerated at all tested doses with both [L.sub.2Cu.sub.3].sup.6+ and [L.sub.2Zn.sub.3].sup.6+ at the highest dose tested shown to decrease tumor weight to a similar level to that seen with the SoC. This provides further evidence of in vivo efficacy of these compounds (at non-optimised doses) against a second tumour type (colorectal cancer) and further validates the anti-cancer activity and selectivity observed in vitro. The lack of embryonic toxicity observed suggests testing of higher doses is warranted (for potentially improved in vivo efficacy compared to the SoC and anti-metastatic activity).
3. In Vivo Efficacy Against Tumors Initiated from Human Colon Cancer Cell Line HCT116
Reduction of HCT116 Tumor Growth
[0686] Statistically significant regression of HCT116 tumor growth (as determined by mean tumor weight at the end of the study, E18) was observed for [L.sub.2Zn.sub.3].sup.6+ at the highest two doses tested and for [L.sub.2Cu.sub.3].sup.6+ at all three tested doses, although effects were not as pronounced as obtained with the SoC (
Analysis of Anti-Metastatic Activity
[0687] Decreases in HCT116 metastatic load were observed for all three doses of [L.sub.2Zn.sub.3].sup.6+ comparable to that observed with SoC (
Analysis of Embryonic Toxicity
[0688] In term of embryo toxicity, L2Zn3 and L2Cu3 were well tolerated at all doses tested with no statistically significant difference observed compared to the solvent (negative) control group (Table 3).
TABLE-US-00003 TABLE 3 Percentage of alive and dead embryos per experimental group at the end of the HCT116 in ovo study. Group Gr. # Description Total Alive Dead % Alive % Dead 1 Neg. Ctrl. 17 15 2 88.24 11.76 2 SoC 15 14 1 93.33 6.67 3 L2Zn3 [1] 15 15 0 100 0 4 L2Zn3 [2] 15 13 2 86.67 13.33 5 L2Zn3 [3] 15 12 3 80.00 20.00 6 L2Cu3 [1] 14 14 0 100 0 7 L2Cu3 [2] 15 13 2 86.67 13.33 8 L2Cu3 [3] 15 13 2 86.67 13.33
Summary (HCT116 Tumours)
[0689] These results show that both [L.sub.2Cu.sub.3].sup.6+ and [L.sub.2Zn.sub.3].sup.6+ are well tolerated at all tested doses with [L.sub.2Zn.sub.3].sup.6+ at the highest two tested doses and for [L.sub.2Cu.sub.3].sup.6+ at all three doses resulting in statistically significant regression of tumor growth. In term of metastatic invasion, none of the conditions induce a significant regression of the metastatic load. However a strong regression tendency was observed for [L.sub.2Zn.sub.3].sup.6+ (all doses) similar to that obtained with the SoC. This provides further evidence of in vivo efficacy of these compounds (at non-optimised doses). The lack of embryonic toxicity observed suggests testing of higher doses is warranted (for potentially improved in vivo efficacy and anti-metastatic activity compared to the SoC).
CONCLUSIONS
[0690] [L.sub.2Cu.sub.3].sup.6+ and [L.sub.2Zn.sub.3].sup.6+ were both tested at three non-optimised doses (based on their in vitro IC.sub.50 values) for in vivo anti-cancer activity against tumors initiated from three different human cancer cell line models (H460, HT29, HCT116) in the chick embryo chorioallantoic membrane (CAM) assay. Against each tumour model, for both [L.sub.2Cu.sub.3].sup.6+ and [L.sub.2Zn.sub.3].sup.6+ statistically significant regression of tumour growth was observed, with effects most pronounced against the H460 lung cancer model (
[0691] While specific embodiments of the invention have been described herein for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims.
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