Patent Application
20190290656
The invention concerns novel anticancer agents combining a platinum(IV) moiety with therapeutically active ligands for use as prodrugs for e.g., cancer treatment.
Platinum anticancer agents are among the most widely used chemotherapeutic drugs and are administered in about 50% of all chemotherapeutic regimens. Two of the major drawbacks involved in their administration are the need for intravenous administration, which requires hospitalization, incurring significant costs, and the ability of tumors to develop resistance to these drugs.
One approach clinicians use for the treatment of unresponsive cancer patients is to use drug cocktails that act by different mechanisms.
Histone deacetylase inhibitors (HDACs) are emerging as a new class of anticancer drugs that can alter gene transcription and exert antitumor effects such as growth arrest, differentiation, apoptosis, and inhibition of tumour angiogenesis. In 2006, the FDA approved the first HDAC inhibitor—suberoylanilide hydroxamic acid (SAHA, Vorinostat), to treat the rare cutaneous T-cell lymphoma (CTCL). There are several clinical trials where HDAC inhibitors have been utilized in combination with platinum anticancer drugs. An ongoing trial (phase 2) entitled “Valproic Acid and Platinum-based Chemoradiation in Locally Advanced Head and Neck Squamous Cell Carcinoma” aimed to evaluate if the addition of valproic acid to standard platinum-based chemo-radiation as definitive treatment of locally advanced head and neck squamous cell carcinoma could improve treatment outcomes, such as response rate.
A Phase I Clinical Trial of Vorinostat in combination with Gemcitabine plus Platinum in patients with advanced Non-Small Cell Lung Cancer was completed in 2011.
Another approach to treating unresponsive cancer patients is to improve the therapeutic profiles of the anticancer drug.
Yang et al. described the synthesis of a complex of Pt(IV) prodrug with valproic acid, a specific histone deacetylase inhibitor, and its experiments in treatment of human carcinoma cell lines W.
The inventors of the present invention have developed a family of complex (conjugate) molecules comprising an anticancer platinum drug associated with one or more another therapeutically active anticancer drug, for the treatment of a variety of cancers.
The presence of more than one active moiety and the ability to permit simultaneous release, in cancer cells, of the active moieties, each acting via a different anticancer pathway permits eradication of cancer cells by acting on, or activating, more than one different cellular targets, increasing the chances of effective cancer cells eradication, including those resistant to anticancer drugs, and patient survivability.
Thus, the invention provides platinum based multifunctional compounds, each containing one or more platinum center and one or more therapeutically active anticancer moiety.
In one aspect the invention provides a compound comprising at least one platinum atom associated to one or more phenylbutyrate ligand, and optionally one or more therapeutically active anticancer moiety. The one or more therapeutically active anticancer moiety may be associated, bonded or coordinated to the platinum atom or to any one atom, group or moiety present in the compound.
In some embodiments, the compound of the invention is in the form of a prodrug, capable of releasing the phenylbutyrate ligand or any of the one or more optionally present therapeutically active anticancer moieties, thereby affecting anticancer activity.
In another aspect, there is provided a Pt-anticancer agent comprising one or more phenylbutyrate.
In some embodiments, the Pt-anticancer agent comprises at least two phenylbutyrate ligands; in other embodiments, the Pt-anticancer agent comprises at least three phenylbutyrate ligands, and yet in other embodiments, the Pt-anticancer agent comprises at least four phenylbutyrate ligands, such that in each of the aforesaid embodiments, the one or more optionally present therapeutically active anticancer moiety is not phenylbutyrate or a derivative thereof.
In some embodiments, the Pt-anticancer agent is associated to 2 or 3 or 5 or 6 phenylbutyrate ligands. Some embodiments relating to compounds of the invention are depicted below:
Typically, the phenylbutyrate ligand associated with the Pt atom is a monodentate ligand. Where the complex of the invention includes a single phenylbutyrate ligand, the complex may comprise in addition one or more polydentate ligand or one or more additional monodentate ligand, being different from phenylbutyrate. The “polydentate ligand”, being a ‘donor group’, is a ligand having more than one atom that can associate (or link, coordinate) directly to the Pt atom in a complex according to the invention; wherein a “monodentate” ligand forms a single bond with the metal atom.
In some embodiments, the complex has at least one monodentate ligand.
In some embodiments, the complex has at least one polydentate ligand. In some embodiments, the at least one polydentate ligand is a bidentate ligand. In some embodiments, the at least one polydentate ligand is a tridentate ligand. In some embodiments, the at least one polydentate ligand is a tetradentate ligand.
In some embodiments, the complex of the invention comprises two monodentate phenylbutyrate ligands.
The complex of the invention may be in any structural isomerization or stereoisomerization or optical isomers. In some embodiments, the complex is a cis isomer. In some embodiments, the complex is a trans isomer. In some embodiments, the complex is a mer-isomer. In some embodiments, the complex is a fac-isomer.
In some embodiments, the complex is in an octahedral geometry, wherein at least two of the ligands are in the axial positions of the octahedral complex. In some embodiments, the complex is in an octahedral geometry, wherein two ligands are phenyl butyrate ligands positioned in the axial positions of the octahedral complex. In some embodiments, the complex is in an octahedral geometry, wherein one of the ligands positioned in an axial position of the octahedral complex is phenyl butyrate.
In some embodiments, the complex of the invention comprises one or more phenylbutyrate ligand and at least one additional ligand selected from ligands designated herein L1 through L47:
L14, wherein the ligand associates to the Pt via the amine moiety;
L21, wherein the ligand associates to the Pt via the amine moiety;
In each of the above ligands, the various substituting groups or radicals are selected as defined herein.
In some embodiments, at least one of the ligands is halide (Cl, Br, I or F). In some embodiments, at least one of the ligands is Cl.
In some embodiments, at least one of the ligands is an amine selected from ammonia, a primary amine, a secondary amine, a non-planar heterocyclic aliphatic amine or a heterocyclic aromatic amine. In some embodiments, at least one of the ligands is —NH3. In some embodiments, at least one of the ligands is —NH2.
In some embodiments, at least one of the ligands is a primary amine Non-limiting examples of a primary amine are methylamine, ethylamine, n-propylamine, isopropylamine, n-butylamine, n-hexylamine, n-heptylamine or n-nonylamine.
In some embodiments, at least one of the ligands is a secondary amine. Non limiting examples of a secondary amine are dimethylamine, diethylamine, dipropylamine, dibutylamine.
In some embodiments, at least one of the ligands is a non-planar heterocyclic aliphatic amine Non-limiting examples of a non-planar heterocyclic aliphatic amine are piperazine, 2-methylpiperazine, piperadine, 2-, 3- or 4-hydroxypiperidine, 4-piperidino-piperidine, pyrrolidine, 4-(2-hydroxyethyl)piperazine or 3-aminopyrrolidine.
In some embodiments, at least one of the ligands is a heterocyclic aromatic amine Non-limiting examples of a heterocyclic aromatic amine are pyridine, 2-, 3-, or 4-aminopyridine, 2-, 3-, or 4-picoline, quinoline, 3-, or 4-aminoquinoline, thiazole, imidazole, 3-pyrroline, pyrazine, 2-methylpyrazine, 4-aminooquinaldine, pyridazine, 1,10-phenanthroline and 5,6-dimethyl-1,10-phenanthroline.
In some embodiments, at least one of the ligands is selected from ligands herein designated L48 through L63:
In some embodiments, in compounds of the invention, each of the ligands is different from the other.
In some embodiments, in compounds of the invention, at least two of the ligands are identical.
The platinum atom may be selected from platinum(IV).
As stated herein, compounds of the invention may comprise one or more phenylbutyrate ligand, and optionally one or more moiety or ligand that is a therapeutically active anticancer moiety or ligand. The therapeutically active anticancer moiety or ligand is one that affects, modulates, inhibits or enhances at least one cancer-related pathway. The therapeutically active anticancer moiety or ligand is thus selected amongst:
Pyruvate dehydrogenase kinase (PDK) inhibitors;
Histone deacetylase (HDAC) inhibitors;
DNA methylation enhancers;
COX inhibitors;
PARP inhibitors;
DNA repair inhibitors; and
Cancer targeting moieties.
As known in the art, “pyruvate dehydrogenase kinase (PDK) inhibitors” are ligands or moieties which may be directly associated to the metal center in compounds of the invention or to any one atom, group or moiety, that are known to inactivate enzyme pyruvate dehydrogenase by phosphorylating it using ATP.
“Histone deacetylase (HDAC) inhibitors” are ligands or moieties which may be directly associated to the metal center in compounds of the invention or to any one atom, group or moiety, that are known to inhibit histone deacetylase.
“DNA methylation enhancers” are ligands or moieties which may be directly associated to the metal center in compounds of the invention or to any one atom, group or moiety, that are known to directly or indirectly cause methylation of a DNA molecule, thereby changing or modifying or affecting DNA activity without substantially changing the DNA sequence.
As known, “COX inhibitors” are ligands or moieties which may be directly associated to the metal center in compounds of the invention or to any one atom, group or moiety, that are known to directly or indirectly inhibit activity of Cyclooxygenase (COX), or prostaglandin-endoperoxide synthase (PTGS).
“PARP inhibitors” are ligands or moieties which may be directly associated to the metal center in compounds of the invention or to any one atom, group or moiety, that are known to inhibit function of poly ADP ribose polymerase (PARP).
“DNA repair inhibitors” are ligands or moieties which may be directly associated to the metal center in compounds of the invention or to any one atom, group or moiety, that are known to inhibit or interrupt one or more DNA repair mechanisms.
Cancer targeting moieties are typically tumor-cell-specific small-molecule ligand or moieties capable of targeting a therapeutic agent, e.g., a compound of the invention, to tumor cells.
In some embodiments, the PDK inhibitors may be dichloro acetate (DCA), pyrazols described in U.S. Patent Application No. 2003/236294, hereby incorporated by reference in its entirety, pyrimadine derivatives described in U.S. Application No. 2004/186118, hereby incorporated by reference in its entirety, pyrrolo[3,4-c]pyrazoles described in U.S. Application No. 2005/101594, hereby incorporated by reference in its entirety, and other inhibitors as disclosed in U.S. Application No. 2014/0005127, hereby incorporated by reference in its entirety.
In some embodiments, the PARP inhibitors may be those described in U.S. Pat. Nos. 8,623,872, 5,177,075, European Patent No. 103,6073 and U.S. Pat. No. 6,635,642 each being incorporated by reference in its entirety.
In some embodiments, the targeting moieties are selected amongst acids such as folic acid (folate), and materials such as those disclosed in U.S. Application No. US 20090061010, herein incorporated by reference.
In some embodiments, the DNA repair inhibitors are selected amongst nitrogen mustards and derivatives thereof, reported in Future Med. Chem. 2012; 4(9): 1093-1111 and in Front Pharmacol. 2013; 4: 5; and others.
In some embodiments, the COX inhibitors may be selected amongst NSAIDs such as ibuprofen, sulindac, celecoxib and aspirin.
In some embodiments, the DNA methylation enhancers may be selected from octanoate and octanoate derivatives, folate and others.
In some embodiments, the HDAC inhibitors may be phenyl butyrate, valproic acid and derivatives thereof, romidepsin, belinostat, panobinostat and vorinostat.
In some embodiments, the therapeutically active moieties or ligands are selected amongst ligands herein designated L48 (in some embodiments, n=4, 5, 6), L53, L54, L59, L60, L61, L62, L63.
In some embodiments, the therapeutically active moieties or ligands, one or more, may be associated or bonded to the metal atom, or to any one atom in a compound of the invention, directly through a native atom or bond present on the therapeutically active moiety or ligand, or through a linker moiety such as —O—C(═O)—, wherein one end of the linker moiety is bonded to the therapeutically active moiety or ligand and another is bonded to the compound (directly to the metal atom or to an atom of the compound).
As stated above, in a compound according to the invention, the platinum atom is associated to one or more phenylbutyrate ligand and at least one additional ligand, e.g., therapeutically active anticancer moiety or ligand. The term “associate”, or any lingual variation thereof, refers to any chemical or physical bond (linkage), such as covalent, ionic, Van der Walls or coordinative which holds the Pt atom and at least one of the ligand atoms together. Typically, the platinum atom is associated to the ligand(s) via coordinative bond(s).
In further embodiments, at least one ligand is bound to the platinum atom via at least one heteroatom selected from nitrogen, oxygen and sulfur. In some embodiments, some of the bonds between the metal atom and the heteroatoms are covalent and some of the bonds are coordinative bonds.
In some embodiments, the covalent bonds to the metal atom are via oxygen or sulfur atoms. In some embodiments, the coordinative bonds to the metal atoms are via nitrogen or sulfur atoms.
In another aspect the invention provides a compound of Formula (I):
wherein
L is a ligand moiety selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted cycloalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, halogen, substituted or unsubstituted —NR1R2, substituted or unsubstituted —OR3, substituted or unsubstituted —SR4, substituted or unsubstituted —S(O)R5, substituted or unsubstituted alkylene-COOH, substituted or unsubstituted ester, —OH, —SH, and —NH;
wherein each of said R1, R2, R3, R4 and R5, independently of the other, is selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted cycloalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl;
and wherein
n is the number of ligand moieties, being 1, 2, 3, 4, or 5.
As used herein, “alkyl”, “alkenyl” and “alkynyl” carbon chains, if not specified, contain from 1 to 20 carbons, or 1 or 2 to 16 carbons, and are straight or branched. In some embodiments, the carbon chain contains 1 to 10 carbon atoms. In some embodiments, the carbon chain contains 1 to 6 carbon atoms. In some embodiments, the carbon chain contains 2 to 6 carbon atoms. Alkenyl carbon chains of from 2 to 20 carbons, in certain embodiments, contain 1 to 8 double bonds and alkenyl carbon chains of 2 to 16 carbons, in certain embodiments, contain 1 to 5 double bonds. Alkynyl carbon chains of from 2 to 20 carbons, in certain embodiments, contain 1 to 8 triple bonds, in yet other embodiments, contain 1 to 5.
Exemplary alkyl, alkenyl and alkynyl groups herein include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isohexyl, allyl (propenyl) and propargyl (propynyl).
As used herein, “cycloalkyl” refers to a saturated mono- or multi-cyclic ring system, in certain embodiments of 3 to 10 carbon atoms, in other embodiments of 3 to 6 carbon atoms; cycloalkenyl and cycloalkynyl refer to mono- or multicyclic ring systems that respectively include at least one double bond and at least one triple bond. Cycloalkenyl and cycloalkynyl groups may, in certain embodiments, contain 3 to 10 carbon atoms, with cycloalkenyl groups, in further embodiments, containing 4 to 7 carbon atoms and cycloalkynyl groups, in further embodiments, containing 8 to 10 carbon atoms. The ring systems of the cycloalkyl, cycloalkenyl and cycloalkynyl groups may be composed of one ring or two or more rings which may be joined together in a fused, bridged or spiro-connected fashion.
As used herein, “aryl” refers to aromatic monocyclic or multicyclic groups containing from 6 to 10 carbon atoms. Aryl groups include, but are not limited to groups such as unsubstituted or substituted fluorenyl, unsubstituted or substituted phenyl, and unsubstituted or substituted naphthyl.
As used herein, “heteroaryl” refers to a monocyclic or multicyclic aromatic ring system, in certain embodiments, of about 5 to about 15 members where one or more, in one embodiment 1 to 3, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur. The heteroaryl group may be optionally fused to a benzene ring. Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, quinolinyl and isoquinolinyl,
As used herein, “heterocyclyl” refers to a saturated mono- or multi-cyclic ring system, in one embodiment of 3 to 10 members, in another embodiment of 4 to 7 members, in a further embodiment of 5 to 6 members, where one or more, in certain embodiments, 1 to 3, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur. In embodiments where the heteroatom(s) is nitrogen, the nitrogen is optionally substituted with alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, acyl, guanidine, or the nitrogen may be quaternized to form an ammonium group where the substituents are selected as above.
As used herein, “halogen” or “halide” refers to F, Cl, Br or I.
As used herein, “—NR1R2” refers to an amine group wherein R1 and R2 are independently selected from hydrogen, alkyl, alkenyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, halogen, —C(O)NR6R7, sulfinyl, ester, carbonyl.
In some embodiments, amine group is selected from a primary amine (wherein each of R1 and R2 is —H), a secondary amine (wherein one of R1 and R2 is a —C1-C6alkyl) or a tertiary amine (wherein each of R1 and R2 is a —C1-C6alkyl, R1 and R2 need not be the same). In some embodiments, the —NR1R2 may represent a quaternary amine, wherein the N atom is further protonated or alkylated to a charged state, forming a salt with, e.g., at least one pharmaceutically acceptable counter-ion.
In some embodiments, R1 and R2 in —NR1R2 form a cyclic structure with the N atom they are bonded to; the cyclic amine having between 3 and 6 atoms in the hetero-ring structure. In some embodiments, the hetero-ring comprises, apart from the N atom, one or more additional heteroatoms selected from N, O and S. In further embodiments, the hetero-ring comprises a single heteroatom (the N atom of the —NR1R2 group) with the remaining atoms being carbon atoms.
As used herein, —C(O)NR6R7 wherein R3 and R4 are independently selected from hydrogen, alkyl, alkenyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, halogen, —NR1R2, sulfinyl, ester, carbonyl, —OH, —SH and —NH.
As used herein, “—OR3” refers to an hydroxyl group wherein R3 is selected from hydrogen, alkyl, alkenyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, halogen, sulfinyl, ester, carbonyl.
As used herein, “—SR4” refers to an thiol group wherein R4 is selected from hydrogen, alkyl, alkenyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, halogen, sulfinyl, ester, carbonyl.
As used herein, “—S(O)R5” refers to an sulfinyl group wherein R5 is selected from hydrogen, alkyl, alkenyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, halogen, sulfinyl, ester, carbonyl. As used herein, “ester” refers —C(O)OR8 in which R8 is selected from hydrogen, alkyl, alkenyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, halogen, —NR1R2, sulfinyl, carbonyl, —OR3, SR4, —S(O)R5—OH, —SH and —NH.
The term “substituted”, a ligand as defined herein above having (further substituted) one or more substituent, wherein the substituent is a ligand as defined herein above. In some embodiments the substituent is selected as specifically indicated and/or from a substituent selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, halogen, —NR1R2, —OR3, —SR4, —S(O)R5, alkylene-COOH, ester, —OH, —SH, and —NH. In some embodiments, the number of substituent (on certain ligand) is 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 20 substituents.
In some embodiments, n is 1.
In some embodiments, n is 2.
In some embodiments, n is 3.
In some embodiments, n is 4.
In some embodiments, n is 5.
In some embodiments, in a compound of formula (I), ligand L is selected amongst ligands herein designated L1 through L63.
In some embodiments, in a compound according to formula (I), L is L1 or L2 or L3 or L4 or L5 or L6 or L7 or L8 or L9 or L10 or L11 or L12 or L13 or L14 or L15 or L16 or L17 or L18 or L19 or L20 or L21 or L22 or L23 or L24 or L25 or L26 or L27 or L28 or L29 or L30 or L31 or L32 or L33 or L34 or L35 or L36 or L37 or L38 or L39 or L40 or L41 or L42 or L43 or L44 or L45 or L46 or L47 or L48 or L49 or L50 or L51 or L52 or L53 or L54 or L55 or L56 or L57 or L58 or L59 or L60 or L61 or L62 or L63.
In some embodiments, L is selected from L1 or L2 or L3 or L4 or L5 or L6 or L7 or L8 or L9 or L10 or L11 or L12. In some embodiments, L is selected from L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12 L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L25, L26, L27, L28, L29, L30, L31, L32, L33, L34, L35, L36, L37, L38, L39, L40, L41, L42, L43, L44, L45, L46, L47, L48, L49, L50, L51, L52, L53, L54, L55, L56, L57, L58, L59, L60, L61, L62 and L63.
In some embodiments, in a compound of formula (I), wherein n is larger than 1, each L is independently selected from L1 through L63.
In some embodiments, in a compound of formula (I), wherein n is larger than 1, two ligands are identical and the rest of the ligands are independently L1 or L2 or L3 or L4 or L5 or L6 or L7 or L8 or L9 or L10 or L11 or L12 or L13 or L14 or L15 or L16 or L17 or L18 or L19 or L20 or L21 or L22 or L23 or L24 or L25 or L26 or L27 or L28 or L29 or L30 or L31 or L32 or L33 or L34 or L35 or L36 or L37 or L38 or L39 or L40 or L41 or L42 or L43 or L44 or L45 or L46 or L47 or L48 or L49 or L50 or L51 or L52 or L53 or L54 or L55 or L56 or L57 or L58 or L59 or L60 or L61 or L62 or L63.
In some embodiments, in a compound of formula (I), wherein n is larger than 1, two ligands are independently selected from L48 through L63, and the rest of the ligands are selected independently from L1 through L63.
In some embodiments, in a compound of formula (I), wherein n is larger than 1, two ligands are independently selected from L48 through L63, wherein said ligands are positioned in the axial positions of an octahedral complex, and the rest of the ligands are selected independently from L1 through L63.
In some embodiments, in a compound of formula (I), wherein n is larger than 1, two ligands are identical and selected from L48 through L63, and the rest of the ligands are selected independently from L1 through L63.
In some embodiments, in a compound of formula (I), wherein n is larger than 1, two ligands are identical and selected from L48 through L63, wherein said ligands are positioned in the axial positions of an octahedral complex, and the rest of the ligands are selected independently from L1 through L63.
In some embodiments, in a compound of formula (I), wherein n is larger than 1, one pairs of ligands are identical and selected from L48 through L63, and the rest of the ligands are selected independently from L1 through L63.
In some embodiments, in a compound of formula (I), wherein n is larger than 2, two pairs of ligands are identical and the rest of the ligands are L1 or L2 or L3 or L4 or L5 or L6 or L7 or L8 or L9 or L10 or L11 or L12 or L13 or L14 or L15 or L16 or L17 or L18 or L19 or L20 or L21 or L22 or L23 or L24 or L25 or L26 or L27 or L28 or L29 or L30 or L31 or L32 or L33 or L34 or L35 or L36 or L37 or L38 or L39 or L40 or L41 or L42 or L43 or L44 or L45 or L46 or L47 or L48 or L49 or L50 or L51 or L52 or L53 or L54 or L55 or L56 or L57 or L58 or L59 or L60 or L61 or L62 or L63.
In some embodiments, in a compound of formula (I), wherein n is larger than 2, two pairs of ligands are identical, one pair of ligands is selected from L1 through L47, the other pair is selected from L48 through L63, and the rest of the ligands are selected independently from L1 through L63.
In some embodiments, the compound of the invention comprises one or more phenylbutyrate ligand. In some embodiments, the compound of the invention is a compound of Formula (Ia):
In some embodiments, the compound of the invention is a compound of Formula (II):
wherein L is as defined hereinabove; and p is the number of ligand moieties, being 1, 2, 3, or 4.
In some embodiments, p is 1.
In some embodiments, p is 2.
In some embodiments, p is 3.
In some embodiments, p is 4.
In some embodiments, the compound of the invention is a compound of Formula (IIa):
wherein L and p are as defined above.
In some embodiments, the compound of the invention is a compound of Formula (IIb):
wherein L and p are as defined above.
In some embodiments, the compound of the invention is a compound of Formula (IIc):
In some embodiments, the compound of the invention is a compound of Formula (IId):
wherein L and p are as defined above.
In some embodiments, the compound of the invention is a compound of Formula (IIe):
In some embodiments, the compound of the invention is a compound of Formula (IIf):
In some embodiments, the compound of the invention is a compound of Formula (IIg):
wherein L and p are as defined above.
In some embodiments, the compound of the invention is a compound of Formula (IIh):
wherein L and p are as defined above.
In some embodiments, the compound of the invention is a compound of Formula
In some embodiments, the compound of the invention is a compound of Formula (IIj):
wherein L and p are as defined above.
In some embodiments, the compound of the invention is a compound of Formula (IIk):
wherein L and p are as defined above.
In some embodiments, the compound of the invention is a compound of Formula (IIl):
In some embodiments, the compound of the invention is a compound of Formula (IIm):
wherein L and p are as defined above.
In some embodiments, the compound of the invention is a compound of Formula (IIn):
wherein L and p are as defined above.
In some embodiments, the compound of the invention is a compound of Formula (IIo):
In some embodiments, the compound of the invention is a compound of Formula (IIp):
wherein L and p are as defined above.
In some embodiments, the compound of the invention is a compound of Formula (IIq):
In some embodiments, the compound of the invention is a compound of Formula (IIr):
wherein L and p are as defined above.
In some embodiments, the compound of the invention is a compound of Formula (IIs):
In some embodiments, the compound of the invention comprises one or more phenylbutyrate ligand and one or more therapeutically active anticancer moiety, as defined herein. In some embodiments, the one or more therapeutically active anticancer moiety is selected from pyruvate dehydrogenase kinase (PDK) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methylation enhancers, COX inhibitors, PARP inhibitors, inhibitors of DNA repair and cancer targeting moieties.
In some embodiments, the compound of the invention is a compound of Formula (I):
wherein L is a therapeutically active anticancer moiety and n is an integer as defined hereinabove. In some embodiments, L is selected from pyruvate dehydrogenase kinase (PDK) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methylation enhancers, COX inhibitors, PARP inhibitors, inhibitors of DNA repair and cancer targeting moieties.
In some embodiments, the compound of the invention comprises two or more Pt atoms (centers), each Pt atom being linked to the other via a ligand group which may be selected amongst therapeutically active anticancer moieties, as defined, or via a linker moiety or group.
In some embodiments, the compound is of the Formula (III):
wherein L is a ligand different than phenylbutyrate; optionally being selected from therapeutically active anticancer moieties, as defined hereinabove.
In some embodiments, L is selected from pyruvate dehydrogenase kinase (PDK) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methylation enhancers, COX inhibitors, PARP inhibitors, inhibitors of DNA repair and cancer targeting moieties.
In some embodiments, in a compound of Formula (III), L is selected from ligands herein designated L48 through L63. In some embodiments, in a compound of Formula (III), L is L48 or L49 or L50 or L51 or L52 or L53 or L54 or L55 or L56 or L57 or L58 or L59 or L60 or L61 or L62 or L63.
In some embodiments, the compound is a compound selected from:
In some embodiments, the compound is of the Formula (IV):
wherein L is a ligand different than phenylbutyrate; optionally being selected from therapeutically active anticancer moieties, as defined hereinabove.
In some embodiments, L is selected from pyruvate dehydrogenase kinase (PDK) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methylation enhancers, COX inhibitors, PARP inhibitors, DNA repair inhibitors and cancer targeting moieties.
In some embodiments, in a compound of Formula (IV), L is selected from ligands herein designated L48 through L63. In some embodiments, in a compound of Formula (IV), L is L48 or L49 or L50 or L51 or L52 or L53 or L54 or L55 or L56 or L57 or L58 or L59 or L60 or L61 or L62 or L63.
In some embodiments, the compound is a compound selected from:
In some embodiments, the compound is of the Formula (V):
wherein L is a ligand different than phenylbutyrate; optionally being selected from therapeutically active anticancer moieties, as defined hereinabove.
In some embodiments, L is selected from pyruvate dehydrogenase kinase (PDK) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methylation enhancers, COX inhibitors, PARP inhibitors, DNA repair inhibitors and cancer targeting moieties.
In some embodiments, in a compound of Formula (V), L is selected from ligands herein designated L48 through L63. In some embodiments, in a compound of Formula (V), L is L48 or L49 or L50 or L51 or L52 or L53 or L54 or L55 or L56 or L57 or L58 or L59 or L60 or L61 or L62 or L63.
In some embodiments, the compound is a compound selected from:
In some embodiments, the compound is of the Formula (VI):
wherein L is a ligand selected as defined herein; n is the number of ligands, selected as herein. The compound of Formula (VI) comprises two or more Pt atoms, each may be labeled Pt1, Pt2, Pt3 . . . , and may be independently substituted with or associated to the same or different ligands or groups or therapeutically active anticancer moieties. In such compounds of the invention, each Pt atom is connected to another Pt atom via a linker group or a ligand designated in Formula (VI) by a curved line, and each Pt atom being connected to one or more ligand groups as disclosed herein. As shown in the compound of Formula (VI), in some embodiments, at least one ligand is phenylbutyrate.
In some embodiments, each of the Pt atoms is bonded to at least one ligand herein designated L1 through L63.
In some embodiments, a Pt atom in a compound of Formula (VI) may be associated to at least one therapeutically active anticancer moieties, as defined hereinabove, optionally selected from pyruvate dehydrogenase kinase (PDK) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methylation enhancers, COX inhibitors, PARP inhibitors, DNA repair inhibitors and cancer targeting moieties.
In some embodiments, in a compound of Formula (VI), one or more of the Pt atoms is associated to a ligand herein designated L48 through L63.
In some embodiments, the linker group linking the two or more Pt atoms may be any carbon chain comprising between 6 and 25 carbon atoms.
In some embodiments, the linker group linking the two or more Pt atoms may be a ligand group selected from:
and
In some embodiments, the linker group is a ligand selected from: L49, wherein each of n and m is between 1 and 3; L50, wherein each of n and m is between 1 and 3; L51, wherein each of n and m is between 0 and 3; and L52, wherein each of n and m is between 1 and 3.
In some embodiments, the linker group is:
In some embodiments, the compound is a compound of the Formula (VII):
wherein L and n are each as defined herein.
In some embodiments, the linker group linking the two or more Pt atoms may be any carbon chain comprising between 6 and 25 carbon atoms.
In some embodiments, the linker group linking the two or more Pt atoms may be a ligand group selected from:
and
In some embodiments, the linker group is a ligand selected from: L49, wherein each of n and m is between 1 and 3; L50, wherein each of n and m is between 1 and 3; L51, wherein each of n and m is between 0 and 3; and L52, wherein each of n and m is between 1 and 3.
In some embodiments, the linker group is:
In some embodiments, the compound is a compound of the Formula (VIII):
wherein L and n are each as defined herein.
In some embodiments, the linker group linking the two or more Pt atoms may be any carbon chain comprising between 6 and 25 carbon atoms.
In some embodiments, the linker group linking the two or more Pt atoms may be a ligand group selected from:
and
In some embodiments, the linker group is a ligand selected from: L49, wherein each of n and m is between 1 and 3; L50, wherein each of n and m is between 1 and 3; L51, wherein each of n and m is between 0 and 3; and L52, wherein each of n and m is between 1 and 3.
In some embodiments, the linker group is:
In some embodiments, the compound is a compound of the Formula (IX):
wherein L and n are each as defined herein.
In some embodiments, the linker group linking the two or more Pt atoms may be any carbon chain comprising between 6 and 25 carbon atoms.
In some embodiments, the linker group linking the two or more Pt atoms may be a ligand group selected from:
and
In some embodiments, the linker group is a ligand selected from: L49, wherein each of n and m is between 1 and 3; L50, wherein each of n and m is between 1 and 3; L51, wherein each of n and m is between 0 and 3; and L52, wherein each of n and m is between 1 and 3.
In some embodiments, the linker group is:
In some embodiments, compounds of Formulae (VI) through (IX), are selected from Formulae (X), (XI) and (XII):
wherein L and n are each as defined herein.
In some embodiments, in a compound of any one of Formulae (X), (XI) and (XII), each of L may be selected from L48 through L63.
In some embodiments, compounds of the invention are of Formulae (XIII) through (XV):
wherein each of L and n are as defined herein.
In some embodiments, the compound is a compound of the Formula (XVI):
wherein L and n are each as defined herein.
In some embodiments, the linker group linking the two or more Pt atoms may be any carbon chain comprising between 6 and 25 carbon atoms.
In some embodiments, the linker group linking the two or more Pt atoms may be a ligand group selected from:
and
In some embodiments, the linker group is a ligand selected from: L49, wherein each of n and m is between 1 and 3; L50, wherein each of n and m is between 1 and 3; L51, wherein each of n and m is between 0 and 3; and L52, wherein each of n and m is between 1 and 3.
In some embodiments, the linker group is:
In some embodiments, the compound is a compound of the Formula (XVII):
wherein L and n are each as defined herein.
In some embodiments, the linker group linking the two or more Pt atoms may be a ligand group selected from:
and
In some embodiments, the linker group is a ligand selected from: L49, wherein each of n and m is between 1 and 3; L50, wherein each of n and m is between 1 and 3; L51, wherein each of n and m is between 0 and 3; and L52, wherein each of n and m is between 1 and 3.
In some embodiments, the linker group is:
In some embodiments, the compound is a compound of the Formula (XVIII):
wherein L and n are each as defined herein.
In some embodiments, the linker group linking the two or more Pt atoms may be a ligand group selected from:
and
In some embodiments, the linker group is a ligand selected from: L49, wherein each of n and m is between 1 and 3; L50, wherein each of n and m is between 1 and 3; L51, wherein each of n and m is between 0 and 3; and L52, wherein each of n and m is between 1 and 3.
In some embodiments, the linker group is:
In some embodiments, compounds of the invention are of Formulae (XIX), (XX) and (XXI):
In some embodiments, in a compound of any one of Formulae (XIX), (XX) and (XXI), each of L may be selected from L48 through L63.
In some embodiments, compounds of the invention are of Formulae (XXII) through (XXIV):
wherein each of L and n is as defined herein.
In some embodiments, compounds of the invention are of Formulae (XXV) and (XXVI):
wherein each of L and n is as defined herein.
In some embodiments, compounds of the invention are of Formulae (XXVII) and (XXVIII):
wherein each of L and n is as defined herein.
In some embodiments, compounds of the invention are of the Formula (XXIX):
wherein L and n are each as defined herein.
In some embodiments, the linker group linking the two or more Pt atoms may be a ligand group selected from:
and
In some embodiments, the linker group is a ligand selected from: L49, wherein each of n and m is between 1 and 3; L50, wherein each of n and m is between 1 and 3; L51, wherein each of n and m is between 0 and 3; and L52, wherein each of n and m is between 1 and 3.
In some embodiments, the linker group is:
In some embodiments, the compound is a compound of the Formula (XXX):
wherein L and n are each as defined herein.
In some embodiments, the linker group linking the two or more Pt atoms may be a ligand group selected from:
and
In some embodiments, the linker group is a ligand selected from: L49, wherein each of n and m is between 1 and 3; L50, wherein each of n and m is between 1 and 3; L51, wherein each of n and m is between 0 and 3; and L52, wherein each of n and m is between 1 and 3.
In some embodiments, the linker group is:
In some embodiments, compounds of the invention are of Formulae (XXXI):
In some embodiments, in a compound of Formula (XXXI), L may be selected from L48 through L63.
In another aspect, the invention further provides a compound selected from Compounds herein designated:
wherein L is a therapeutically active anticancer moiety as disclosed herein.
wherein L is a therapeutically active anticancer moiety as disclosed herein,
wherein L is a therapeutically active anticancer moiety as disclosed herein,
wherein L is a therapeutically active anticancer moiety as disclosed herein,
In another aspect, the invention provides a compound selected from:
In some embodiments of compounds of the invention, the compounds are not of formula (M):
wherein
Pt is a platinum atom;
A is a C8-C22 fatty acid associated with the Pt atom via an oxygen atom of the fatty acid;
B is a C2-C22 fatty acid associated with the Pt atom via an oxygen atom of the fatty acid;
provided that each of A and B is not C6-C9 branched alkyl fatty acid;
L is a ligand atom or group of atoms selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted cycloalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, halide atom, substituted or unsubstituted amine —NR1R2, substituted or unsubstituted —OR3, substituted or unsubstituted —SR4, substituted or unsubstituted —S(O)R5, substituted or unsubstituted alkylene-COOH, —OH, —SH, —NH, or any one of ligands L1 to L5 as designated herein; and
n is the number of ligand moieties, being 1, 2, 3, or 4;
R1 and R2 are each independently selected from hydrogen, alkyl, alkenyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, halide, —C(O)NR6R7, sulfinyl, ester, and carbonyl; or wherein R1 and R2 in form a cyclic structure with the N atom they are bonded to;
each of R3, R4, and 123 is independently selected from hydrogen, alkyl, alkenyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, halide, sulfinyl, ester, and carbonyl; and
R6 and R7 are each independently selected from hydrogen, alkyl, alkenyl, alkenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, halide, sulfinyl, ester, carbonyl, —OH, —SH and —NH.
In some embodiments, excluded is Oxaliplatin palmitate acetate.
In some embodiments, excluded are Compounds of formula (M):
wherein L is as defined in claim 1 and p is the number of ligand moieties, being 0, 1 or 2.
wherein L is independently as defined in claim 1 and p in Formula (M4) is 0, 1 or 2.
wherein L is as defined in claim 1 and p is the number of ligand moieties, being 0, 1 or 2.
wherein L is as defined in claim 1 and p is the number of ligand moieties, being 0, 1 or 2.
wherein p is independently the number of ligand moieties, being selected from 0, 1 and 2.
In some embodiments, compounds of the invention are not compounds disclosed in International Patent Application No. PCT/IL2015/050448 (WO 2015/166498).
In some embodiments, compounds of disclosed in International Patent Application No. PCT/IL2015/050448 (WO 2015/166498) are hereby excluded.
In another aspect, the invention provides a method of preparation of compounds of the invention, the method comprising:
to thereby obtain the complex of the invention.
In another aspect, the invention provides a method of preparation of compounds of the invention, the method comprising:
The term “Pt complex being in its active form” refers to Pt complex having a therapeutic effect. In some embodiments Pt being in its active form refers to Pt(II) complex.
Typically, oxidation of an active Pt complex is carried out in the presence of an oxidizing agent. In some embodiments, the oxidizing agent is selected from peroxide agents. In some embodiments, the oxidizing agent is an inorganic peroxide or an organic peroxide.
In some embodiments the oxidizing agent is an inorganic peroxide selected from H2O2, Na2O2, K2O2, K2CO3.H2O2, CaO2.2H2O2, peroxymonosulfuric acid (HOSO2OOH), peroxydisulfuric acid (HOSO2OOSO2OH). In some embodiments, the oxidizing agent is H2O2.
In some embodiments the oxidizing agent is an organic peroxide. Non limiting examples of organic peroxide is ROOR or RCOOOCOR or ROOH or RCOOOH or R3SiOOLi or R2BOOR or CF3OOOCF3, wherein R is an organic moiety, typically selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl. In some embodiments, the inorganic peroxide is tert-butyl hydrogen peroxide.
In some embodiments the oxidizing agent (in particular peroxides) are presence in a solvent. The solvent may be aqueous solvent or organic solvent. In some embodiments the solvent is water. In some embodiments the solvent is organic solvent. Non-limiting examples of organic solvents are acetonitrile, tetrahydrofuran, dimethyl formamide, ethanol, chloroform, hexane or any combination thereof.
In some embodiments, the oxidizing agent is inorganic peroxide in water. In some embodiments, the oxidizing agent is H2O2 in water.
In some embodiments, the oxidizing agent is inorganic peroxide or an organic peroxide in an organic solvent. In some embodiments, the oxidizing agent is H2O2 in an organic solvent.
In some embodiments (e.g., when the oxidizing agent is H2O2 in water), the oxidizing step results in a Pt complex having dihydroxido species. In some embodiments, the dihydroxido species having axial hydroxides on the Pt complex.
In some embodiments, the axial hydroxides (dihydroxide) form an axially symmetric complex, i.e., a pair of two identical ligands on the axial axis.
In some embodiments (e.g., when the oxidizing agent is organic peroxide in organic solvent or inorganic peroxide in organic solvent), the oxidizing step results in a Pt complex having a single hydroxide (monohydroxido species) or dihydroxide (dihydroxido species).
In some embodiments, the dihydroxido species having axial hydroxides on the Pt complex.
In some embodiments, the monohydroxide or the dihydroxide form a non-symmetric axial complex, i.e., non-identical ligands on the axial axis. The hydroxides on the Pt complex may be reacted with 4-phenylbutirate or reactive 4-phenylbutirate.
The term “reactive 4-phenylbutyrate” or “phenylbutyrate in a reactive form” refers to any form of 4-phenylbutyrate precursor which enables formation of the complex of the invention, when reacted with the Pt complex. In other words, the phenylbutyrate in a reactive form is a phenylbutyrate in the form of a reactant or precursor in the reaction of the inactive Pt complex to form the complex of the invention. In some embodiments, the reactive 4-phenylbutyrate is an anhydride of 4-phenylbutyrate. In some embodiments, the reactive 4-phenylbutyrate is a chloric acid of 4-phenylbutyrate. In some embodiments, the reactive 4-phenylbutyrate is acyl chloride of 4-phenylbutyrate. In some embodiments, the reactive 4-phenylbutyrate is an activated esters of 4-phenylbutyrate. In some embodiments, the reactive 4-phenylbutyrate is an isocynates of 4-phenylbutyrate. In some embodiments, the reactive form of 4-phenylbutyrate is 4-phenylbutyrate.
The reactive 4-phenylbutyrate may be obtained by direct synthesis or from a commercial source. For example, anhydride of 4-phenylbutyrate may be obtained by reacting 4-phenyl butyric acid with N,N′-dicyclohexylcarbodiimide).
In some embodiments (e.g. when obtaining a non-symmetric axial complex of the invention), the step of reacting said inactive complex with said reactive 4-phenylbutyrate refers to reacting a single hydroxide (e.g., carboxylation) and having another ligand (which may be hydroxide, carboxyl group, carbonyl group or any other ligand) subjected to a further/another chemical reaction.
In another aspect, the invention provides a compound of the invention as a prodrug. As used herein the term “prodrug” refers to an agent which is converted into the parent drug (active agent) in vivo by some physiological chemical process (e.g., a prodrug converted to the desired drug form under physiological conditions). The prodrugs of the invention are useful as they may be easier to administer than the parent drug, they are less toxic and present improved bioavailability.
After administration, the prodrug is enzymatically or chemically cleaved to deliver the active drug and the ligand moieties as free molecules in the blood or tissue.
In some embodiments, the prodrug may release at least one active agent. In other embodiments, the prodrug may release one or two or three active agents.
In some embodiments, the prodrug of the invention releases a Pt(II) complex as an active agent. In some embodiments, the prodrug of the invention releases phenyl butyrate or phenyl butyrate derivative (an anion or a salt) as an active agent. In some embodiments, the prodrug of the invention releases Pt(II) complex and phenyl butyrate or phenyl butyrate derivative as active agents.
In some embodiments, the prodrug releases the active agent (activated) in a physiological pH (7.4). In some embodiments, the prodrug is activated at a pH lower than the physiological pH. In some embodiments, the prodrug is activated at a pH of about 6.
A person of skill in the art would realize that a complex of the invention generally releases two (or more) active agent drugs, which may induce, depending on the nature of the ligands, two different mechanisms of action, typically on two different cellular targets. Without wishing to be bound to theory the complex releases Pt(II) active complex which targets DNA molecule. The complex of the invention also releases 4-phenyl butyrate which targets (modify, inhibit) histone deacetylase.
Thus, in another aspect, the invention provides a complex according to the above for targeting DNA and inhibiting histone deacetylase.
In another aspect, the invention provides a pharmaceutical composition comprising at least one compound of any one of the above.
As used herein, “pharmaceutical composition” comprises a therapeutically effective amount of a compound of the present invention, together with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers. Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g.; Tris-HCL, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils).
In another aspect, the invention provides at least one compound of any one of the above for use in treatment of proliferation disorders.
In a further aspect, the invention provides use of at least one compound of any of the above Formulae for the preparation of a pharmaceutical composition for use in treatment of proliferation disorders.
In yet a further aspect, the invention provides a method for the treatment of a proliferative disorder, the method comprising administering an effective amount of the compound of the invention to a subject suffering from said disorder.
The term “proliferative disorders” encompass diseases or disorders that effect a cellular growth, differentiation or proliferation processes. In some embodiments, the proliferation disorder is cancer. The term “cancer” as used herein encompasses any neoplastic disease which is characterized by abnormal and uncontrolled cell division causing malignant growth or tumor. Cancer as used herein may refer to either a solid tumor or tumor metastasis.
Non-limiting examples of cancer are ovary cancer, and pancreatic cancer, squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer. Solid cancers appear in many forms, for example, breast cancer, prostate cancer, sarcomas, and skin cancer. One form of skin cancer is melanoma.
The term “treatment” as used herein refers to the administering of a therapeutic amount of the composition of the present invention which is effective to ameliorate undesired symptoms associated with a disease, to prevent the manifestation of such symptoms before they occur, to slow down the progression of the disease, slow down the deterioration of symptoms, to enhance the onset of remission period, slow down the irreversible damage caused in the progressive chronic stage of the disease, to delay the onset of said progressive stage, to lessen the severity or cure the disease, to improve survival rate or more rapid recovery, or to prevent the disease from occurring or a combination of two or more of the above.
The term “effective amount” as used herein is determined by such considerations as may be known in the art. The amount must be effective to achieve the desired therapeutic effect as described above, depending, inter alia, on the type and severity of the disease to be treated and the treatment regime. The effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount. As generally known, an effective amount depends on a variety of factors including the affinity of the ligand to the receptor, its distribution profile within the body, a variety of pharmacological parameters such as half-life in the body, on undesired side effects, if any, on factors such as age and gender, etc.
In some embodiments, the effective amount of the compound is administrated by one or more of the following routes: oral, rectal, transmucosal, transnasal, intestinal, parenteral, intramuscular, subcutaneous, intramedullary injections, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
The compounds utilized in accordance with the present invention may be used in their free base or free acid form or as “pharmaceutically acceptable salt(s)”, namely as salts that are safe and effective for pharmaceutical use in mammals (e.g., humans) and that possess the desired biological activity.
In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
Synthesis of the Compounds of the Invention:
Cisplatin and oxaliplatin are commercially available and can be easily synthesized in the lab. 4-phenybutyrate is also commercially available.
Cisplatin or oxaliplatin were oxidized with H2O2 to yield the Pt(IV) complexes, as shown in Scheme 1:
The anhydride of 4-phenybutyrate was prepared by standard methods, as shown in Scheme 2: 4-phenyl butyric acid (1 eq.) and N,N′-Dicyclohexyl carbodiimide (DCC, 0.5 eq) were dissolved in Chloroform (15 ml) and stirred at room temperature overnight. The dicyclohexylurea byproduct was removed by celite filtration. The crude mixture was re-dissolved in DCM (30 mL), concentrated and filtered, and was repeated until no urea was observed.
The final compound were obtained by reacting the Pt(IV) dihydroxido complexes with the anhydride, as shown in Scheme 3:
The final compounds were purified by HPLC and characterized by NMR spectroscopy and elemental analysis.
NMR Spectroscopy:
1H NMR and 1H NMR data were recorded on Varian Unity-500 MHz and Varian Unity-300 MHz spectrometers respectively and Data were processed using MestreNova software.
The purities of tested compound are >95% (HPLC). HPLC analyses were performed in a Varian ProStar HPLC system equipped with a UV detector, set at 220 nm, using a RP-C18 column (Phenomenex, Luna, 250×4.6 mm, 5 μm, 100 Å).
Synthesis of compounds (see
Ctc-Pt(NH3)2(C8H15O2)2Cl2] (1)
Starting material [Pt(NH3)2(OH)2Cl2] was obtained by H2O2 oxidation of cisplatin.
[Pt(NH3)2(OH)2Cl2] (50 mg, 1.49×10−4 mol) was suspended in N,N-dimethylformamide (1 mL) and valproic anhydride was added (101.18 mg, 3.74×10−4 mol, 2.5 eq.). The reaction mixture was stirred for 5 h (complete dissolution) at 40° C. Solvent was evaporated at the end of the reaction and diethyl ether was added to precipitate the desired compound as a light yellow powder. The precipitate was separated by centrifugation and washed twice with ethyl ether and dried in vacuo.
Yield: 71 mg, 77.2%, 195Pt-NMR (DMF): 1174 ppm.
1H-NMR (DMSO-d6): δ ppm 1.19 (t, 12H), 1.49-1.89 (m, 16H), 2.6 (m, 2H), 6.51-6.98 (b, 6H).
Pt(NH3)2Cl2(C2H3O2)(C8H15O2)] (II)
Starting material [Pt(NH3)2(OH) (C2H3O2) Cl2] was prepared by oxidizing cisplatin in acetic acid.
To a solution of [Pt(NH3)2(OH) (C2H3O2) Cl2] (50 mg, 1.32×10−4 mol) in N,N-dimethylformamide (2 mL), valproic anhydride (53.92 mg, 1.96×10−4 mol, 1.5 eq.) was added. After stirring for 2 h at rt, solvent was evaporated at reduced pressure. Acetone was added to the residue and the precipitated compound was collected by filtration, washed twice with acetone, and dried in vacuo to afford the desired compound as a light yellow powder.
Yield: 59 mg, 88%. 195Pt-NMR (DMF):1177 ppm.
1H-NMR (DMSO): δ ppm 0.89 (t, 6H), 1.13-1.49 (m, 8H), 1.89 (s, 3H), 6.51-6.90 (b, 6H).
[Pt(DACH)(C8H15O2)2(ox)] (III)
The starting material, [Pt(DACH)(OH)2(ox)] was prepared according to Hall et al. (J Biol Inorg Chem. 2003 September; 8(7):726-32.)
To a suspension of [Pt(DACH) (OH)2 (ox)] (50 mg, 1.16×10−4 mol) in N,N-dimethylformamide (1 mL), valproic anhydride (78.37 mg, 2.88×10−4 mol, 2.5 eq) was added. The mixture was stirred at rt for 12 h. Solvent was evaporated at the end of the reaction and the residue was redissolved in 5 mL dichloromethane. Diethyl ether was added to precipitate the desired compound as a white powder. The precipitate was separated by centrifugation, washed twice with diethyl ether and dried under vacuum.
Yield: 68 mg, 86%
195Pt-NMR (DMF):1569 ppm. 1H-NMR (DMSO): δ ppm 1.10 (t, 12H), 1.30-1.90 (m, 24H), 2.4-2.8 (m, 4H), 8.74-9.09 (b, 4H).
[Pt(DACH)(C8H15O2)(OH)(ox)] (IV)
Oxaliplatin diOH (10 mg, 2.3×10-5 mol) and Valproic anhydride (70 uL, 2.3×10-4 mol) were dissolved in DMF (2 mL) and stirred at RT overnight.
DMF was evaporated. The residue was dissolved in DCM and ethyl ether was added to induce precipitation. The white precipitated was collected by centrifuge and washed with ether. Remaining ether was evaporated under a nitrogen stream. Yield: 81%, 10.4 mg, 1.86×10-5 mol.
ctc-[Pt(NH3)2(C10H11O2)2Cl2] (VI)
[Pt(NH3)2(OH)2Cl2] (100 mg, 2.99×10−4 mol) was suspended in N,N-dimethylformamide (1 mL) and 4phenylbutiric anhydride was added (232.28 mg, 7.47×10−4 mol, 2.5 eq). The reaction mixture was stirred for 1 h (complete dissolution) at 40° C. The solvent was evaporated at reduced pressure. The residue was redissolved in acetone and diethyl ether was added to precipitate the compound. The precipitate was collected by filtration, washed twice with diethyl ether and dried in vacuo.
Yield: 138 mg, 74%
195Pt-NMR (DMF):1174 ppm. 1H-NMR (DMSO): δ ppm 1.72 (m, 4H), 2.20 (t, 4H), 2.57 (t, 4H), 6.27-6.80 (b, 6H), 7.24 (m, 10H).
Ctc-[Pt(NH3)2(C2H3O2)(C10H11O2)Cl2] (VII)
To a solution of [Pt(NH3)2(OH) (C2H3O2) Cl2] (50 mg, 1.32×10−4 mol) in N,N-dimethylformamide (2 mL), 4phenylbutirc anhydride (61.89 mg, 1.99×10−4 mol, 1.5 eq) was added. After stirring for 1 h at rt, solvent was evaporated at reduced pressure. Acetone was added to the residue and the precipitated compound was collected by filtration, washed twice with acetone, and dried in vacuo to afford the desired compound as a light yellow powder.
Yield: 60 mg, 87%
195Pt-NMR(DMF):1176 PPM, 1H-NMR (DMSO): δ ppm 1.704 (m, 2H), 1.87 (s, 3H), 2.18 (t, 2H), 2.55 (t, 2H), 6.5 (b, 6H), 7.18 (m, 5H).
[Pt(DACH)(C10H11O2)2(ox)] (VIII)
To a suspension of [Pt(DACH) (OH)2 (ox)] (100 mg, 2.32×10−4 mol) in N,N-dimethylformamide (1 mL), 4phenylbutyric anhydride (179.91 mg, 5.795×10−4 mol, 2.5 eq) was added. The mixture was stirred at RT for 2.5 h. Solvent was evaporated at the end of the reaction and dichloromethane was added to precipitate the desired compound as a white powder. The compound was separated by centrifugation, washed twice with dichloromethane and dried under vacuo.
Yield: 121 mg, 72%
195Pt-NMR(DMF):1588 PPM. 1H-NMR (DMSO): δ ppm 1.0-1.59 (m, 8H), 1.69 (m, 4H), 2.02-2.34 (m, 8H), 7.2 (m, 10H), 8.32 (b, 4H).
Experiments with Cultured Human Cells.
Platinum(IV) complexes were dissolved in DMSO to stock solutions of 1 mg/mL just before the experiment, and a calculated amount of drug solution was added to the cell growth medium to a final solvent concentration of 0.5%, which had no discernible effect on cell killing. Cisplatin (CDDP) and oxaliplatin (OXP) were dissolved just before the experiment in a 0.9% NaCl solution.
Cisplatin, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 4-phenylbutyric acid, staurosporin, Z-VAD-fmk and Hoechst 33258 (2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole trihydrochloride hydrate) were obtained from Sigma Chemical Co, St. Louis, USA.
Cell Cultures.
Human lung (A549), breast (MCF-7), pancreatic (BxPC3), kidney (A498) and colon (HCT-15) carcinoma cell lines along with melanoma (A375) were obtained from American Type Culture Collection (ATCC, Rockville, Md.). Human prostate adenocarcinoma (PC3) cells were obtained from European Collection of Cell Cultures (ECACC, Salisbury, UK). Human ovarian cancer cell lines A2780 and its cisplatin resistant variant, A2780cisR, were kindly provided by Prof. G. Marverti (Dept. of Biomedical Science of Modena University, Italy. Cell lines were maintained in the logarithmic phase at 37° C. in a 5% carbon dioxide atmosphere using the following culture media containing 10% fetal calf serum (Euroclone, Milan, Italy), antibiotics (50 units·mL−1 penicillin and 50 μg·mL−1 streptomycin) and 2 mM 1-glutamine: i) RPMI-1640 medium (Euroclone) for MCF-7, HCT-15, A431, BxPC3, A2780 and A2780 cisR cells; ii) F-12 HAM'S (Sigma Chemical Co.) for A549 and PC3 cells; iii) D-MEM medium (Euroclone) for A375 cells; iv) EMEM for A498 cells.
Cytotoxicity MTT Assay.
The growth inhibitory effect towards human cell lines was evaluated by means of MTT (tetrazolium salt reduction) assay [Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 1988, 48, 589-601]. Briefly, 3-8.103 cells/well, dependent upon the growth characteristics of the cell line, were seeded in 96-well microplates in growth medium (100 μL) and then incubated at 37° C. in a 5% carbon dioxide atmosphere. After 24 h, the medium was removed and replaced with a fresh one containing the compound to be studied at the appropriate concentration. Triplicate cultures were established for each treatment. After 72 h, each well was treated with 10 μL of a 5 mg·mL−1 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) saline solution, and after 5 h additional incubation, 100 μL of a sodium dodecylsulfate (SDS) solution in HCl 0.01 M were added. After overnight incubation, the inhibition of cell growth induced by the tested complexes was detected by measuring the absorbance of each well at 570 nm using a Bio-Rad 680 microplate reader (Bio-Rad, Hercules, Calif.). Mean absorbance for each drug dose was expressed as a percentage of the control untreated well absorbance and plotted vs drug concentration. IC50 values represent the drug concentrations that reduced the mean absorbance at 570 nm to 50% of those in the untreated control wells.
Cellular Accumulation.
MCF-7 cells (2.5×106) were seeded in 75 cm2 flasks in growth medium (20 ml). After overnight incubation, the medium was replaced and the cells were treated with tested compounds for 24 h. Cell monolayers were washed twice with cold PBS, harvested and counted. Cell nuclei were isolated by means of the nuclei isolation kit Nuclei EZ Prep (Sigma Co.). Samples were than subjected to three freezing/thawing cycles at −80° C., and then vigorously vortexed. The samples were treated with highly pure nitric acid (Pt: ≤0.01 μg kg−1, TraceSELECT® Ultra, Sigma Chemical Co.) and transferred into a microwave teflon vessel. Subsequently, samples were submitted to standard procedures using a speed wave MWS-3 Berghof instrument (Eningen, Germany) After cooling, each mineralized sample was analyzed for platinum by using a Varian AA Duo graphite furnace atomic absorption spectrometer (Varian, Palo Alto, Calif.; USA) at the wavelength of 324.7 nm. The calibration curve was obtained using known concentrations of standard solutions purchased from Sigma Chemical Co. purchased from Sigma Chemical Co.
DNA platination. MCF-7 cells (3×106) were seeded in 10 cm Petri dishes in 10 mL of culture medium. Subsequently, cells were treated with tested complexes for 24 h. DNA was extracted and purified by a commercial spin column quantification kit (Qiagen DNeasy Blood and Tissue Kit). Only highly purified samples (A260/A230≅1.8 and A280/A260≅2.0) were included for analysis to avoid any artifacts. The samples were completely dried and re-dissolved in 200 μL of Milli-Q water (18.2 MΩ) for at least 20 min at 65° C. in a shaking thermo-mixer, mineralized and analyzed for total Pt content by GF-AAS as described above.
Histone Deacetylase Assay.
Histone deacetylase activity was determined using Fluor-de-Lys® HDAC fluorometric activity assay kit (Enzo Life Sciences International, Inc., Plymount Meeting, Pa., U.S.A.). MCF-7 cells (5.104 seeded in 96-well microplates) were treated for 24 h with equi-toxic concentrations of tested complexes, corresponding to the IC50 values, and then processed as reported by the manufacturer's instructions. Fluorescence was measured using a Fluoroskan Ascent FL (Labsystem, Finland) plate reader, with excitation at 360 nm and emission at 460 nm. For comparison purpose, the HDAC activity was also measured in nuclear extracts of MCF-7-treated cells by means of the same fluorometric activity assay kit. In this latter case, MCF-7 nuclear extracts were obtained as follow: cell pellets were re-suspended in lysis buffer [Tris.HCl (10 mM, pH 8.0), KCl (60 mM), EDTA (1.2 mM), DTT (1 mM), PMSF (0.05 mM), NP-40 (0.05%)] and kept on ice for 10 min. Subsequently, samples were centrifuged at 1000 g and re-suspended in the nuclear extraction buffer [Tris.HCl (20 mM, (pH 8.0), NaCl (420 mM), MgCl2 (0.7 mM), EDTA (0.25 mM), glycerol (25%)] for 30 min at 4° C., and then centrifuged at 15000 g for 15 min at 4° C.
Caspase-3/-7 Activation.
Caspase-3/-7 activity was detected by using the Apo-One® 3/7 Homogeneous Caspase-3/7 Assay (Promega, Madison, Wis., USA) according to the manufacturer's recommended procedures. MCF-7 cells were seeded in 96-well microplates in growth medium (100 μL) and then incubated at 37° C. in a 5% carbon dioxide atmosphere. After overnight incubation, cells were subjected to a 24 h treatment with tested compounds (at concentrations corresponding to IC50 values). Subsequently, each well was treated with 120 μl of the Apo-ONE® Caspase-3/7 Assay Reagent containing the specific substrate (rhodamine 110 bis-(N-CBZ-1-aspartyl-1-glutamyl-1-valyl-aspartic acid amide), Z-DEVD-R110). The fluorescence was determined after one hour with a PerkinElmer 550 spectrofluorometer (excitation 499 nm, emission 521 nm).
Hoechst 33258 Staining.
MCF-7 cells were seeded into 8-well tissue-culture slides (BD Falcon, Bedford, Mass., USA) at 5.104 cells/well (0.8 cm2). After 24 h, cells were washed twice with PBS and following 72 h of treatment with IC50 concentration of tested complexes, cells were stained for 5 min with 1 mg/mL Hoechst 33258 (Sigma-Aldrich) in PBS before being examined by fluorescence microscopy (Olympus BX41, Cell F software, Olympus, Munster, Germany)
A group of the compounds synthesized for this study are depicted in
The non-symmetric compound ctc-[Pt(NH3)2(OH)(OAc)Cl2] was prepared by oxidizing cisplatin in acetic acid. It was then reacted with 1.5 equivalents of the appropriate anhydride in DMF to yield ctc-[Pt(NH3)2(L)(OAc)Cl2] where L=VPA, PhB.
All compounds were characterized by 1H NMR spectroscopy and when possible by mass spectrometry. They were purified by preparative HPLC and their purity was ascertained by elemental analysis.
Compounds I-VIII, the corresponding uncoordinated axial ligands (VPA and PhB) as well as cisplatin and oxaliplatin were screened against seven different cancer cell lines representative of lung (A549), breast (MCF-7), pancreatic (BxPC3), kidney (A498) and colon (HCT-15) carcinoma, along with melanoma (A375). Furthermore, Pt(IV) complexes were also tested for their in vitro cytotoxicity on a pair of human ovarian adenocarcinoma cell lines which have been selected for sensitivity/resistance to cisplatin (cancer cells A2780/A2780cisR). The cytotoxicity parameters, in terms of IC50 (the median growth inhibitory concentration calculated from dose-survival curves) obtained after 72 h exposure, are reported in Table 1 below.
Uncoordinated HDAC inhibitor ligands VPA and PhB displayed very low cytotoxic activities, with IC50 values in the millimolar range, as already reported in the literature. Mono- and bis-VPA Pt(IV) derivatives of cisplatin showed cytotoxic activities significantly higher than those elicited by cisplatin against all tested cell lines, having average IC50 values (μM), for the nine cancer cell lines, of 1.15 for ctc-[Pt(NH3)2(VPA)2Cl2], 4.48 for ctc-[Pt(NH3)2(VPA)(OAc)Cl2] and 9.18 for cisplatin. These results, in accordance with the results reported in the literature, clearly indicate that Pt(IV) pro-drugs with bioactive VPA ligands are significantly more potent than the parent cisplatin. In particular, ctc-[Pt(NH3)2(VPA)2Cl2] was about 19, 14 and 10 times more effective than cisplatin against breast (MCF-7), colon (HCT-15) and pancreatic (BxPC3) cancer cells, respectively. In the sensitive and resistant ovarian cancer cells A2780 and A2780cisR, ctc-[Pt(NH3)2(VPA)2Cl2] is 11 and 46 fold more potent than cisplatin, with the resistance factor (being the ratio between IC50 values calculated for the resistant cells and those obtained with the sensitive ones) of 1.2, indicating its ability to circumvent acquired resistance to cisplatin.
Interestingly, when one VPA ligand of ctc-[Pt(NH3)2(VPA)2Cl2] is replaced with an acetato to give ctc-[Pt(NH3)2(VPA)(OAc)Cl2], there is a 1.7-7.9 fold reduction in the potency compared with ctc-[Pt(NH3)2(VPA)2Cl2] and on the average of the nine cell lines, the bis-VPA derivative of cisplatin was 4.6 fold more potent than the mono-VPA monoacetato analog. Yet, with the exception of the PC3 cells, ctc-[Pt(NH3)2(VPA)(OAc)Cl2] was as or more potent than cisplatin in all tested cells lines and showed the ability to overcome acquired cisplatin resistance in A2780cisR cells (resistance factor of 1.6).
Conversely, the behavior of the Pt(IV) oxaliplatin derivatives with VPA axial ligands was very different than that observed for the Pt(IV) cisplatin derivatives with VPA ligands. The antiproliferative potencies of compounds III, [Pt(DACH)(VPA)2(ox)] and IV, [Pt(DACH)(VPA)(OH)(ox)], were significantly lower than those of the parent Pt(II) complex oxaliplatin, having average IC50 values (μM) over the nine cell lines of 4.9, 9.62 and 37.6 for oxaliplatin, III and IV, respectively. Moreover, both complexes showed cross resistance with cisplatin in the A2780cisR cells (resistance factor of 6.8 and 3.0 for III and IV, respectively).
The transplatinum derivative (IV), trans-[Pt(n-butylamine)(VPA)(OH) (piperidine) Cl2], which possesses one VPA ligand in axial position, elicited an average IC50 value over the nine tested cell lines that is very similar to that of cisplatin (9.27 μM). However, it retained a better in vitro antitumor potency with respect to cisplatin against five out of the nine cell lines tested (A2780, A2780cisR, MCF-7, A549, A498 and BxPC3 cells). Notably it was roughly 11 times more effective than cisplatin against the ovarian cancer cell lines A2780cisR, eliciting a resistance factor of 1.1, thus attesting the absence of cross-resistance with cisplatin.
Compounds VI and VII, the Pt(IV) derivatives of cisplatin with either two or one 4-phenylbutyrate (PhB) ligands in the axial positions, showed a significant antiproliferative activity against all tested cell lines. Like with mono- and bis-VPA cisplatin derivatives, the bis-PhB cisplatin derivatives VI and VII are significantly more potent than cisplatin, with average IC50 values (μM) of 0.23 (0.12-0.43) and 4.8 (0.65-10.14) respectively. Remarkably, the bis-PhB derivative, ctc-[Pt(NH3)2(PhB)2Cl2] (compound VI) was nearly 100-, 60- and 50-fold more potent than cisplatin against MCF-7, BxPC3 and A549 cells, respectively. Furthermore, it showed similar cytotoxicity against the cisplatin sensitive (0.14 μM) and resistant ovarian cancer cells (0.12 μM), denoting its ability to overcome cisplatin resistance.
As in the case of ctc-[Pt(NH3)2(VPA)2Cl2] and ctc-[Pt(NH3)2(VPA)(OAc)Cl2], where the symmetric bis-VPA complex was significantly more potent than the non-symmetric mono-VPA complex, the symmetric ctc-[Pt(NH3)2(PhB)2Cl2] was significantly more effective than ctc-[Pt(NH3)2(PhB)(OAc)Cl2]. The bis-PhB is 4.6-53 fold more potent than the non-symmetric mono-PhB monoacetate complex and on the average about 24 fold more potent than cisplatin. Ctc-[Pt(NH3)2(PhB)(OAc)Cl2] was more potent than cisplatin in five out of the nine cells lines by factors of 2.1-9. Replacing a PhB in ctc-[Pt(NH3)2(PhB)(OAc)Cl2] with an acetato ligand significantly diminished the potency of the Pt(IV) complex.
By comparing the cytotoxicity results of I and VI, it is evident that replacing both VPA ligands with PhB in the Pt(IV) derivatives of cisplatin increases the potency of the complex (on the average there is nearly a 5 fold improvement in potency) whereas when the VPA in ctc-[Pt(NH3)2(VPA)(OAc)Cl2] was replaced with PhB, ctc-[Pt(NH3)2(PhB)(OAc)Cl2] and ctc-[Pt(NH3)2(VPA)(OAc)Cl2], elicited a similar cytotoxicity profile.
The Pt(IV) derivative of oxaliplatin, with two PhB, [Pt(DACH)(PhB)2(ox)] was significantly (4-40 fold) less potent than its cisplatin analog (compound VI) and had similar potency to its VPA analog (compound III).
Cellular Accumulation and Binding to Nuclear DNA
With the aim of identifying a possible correlation between cytotoxic activity and cellular uptake, the cellular Pt content was measured in MCF-7 cells treated for 24 h with equi-Pt concentrations (5 μM) of the Pt(IV) compounds. The intracellular platinum levels were quantified by means of GF-AAS analysis, and the results expressed as pg Pt/106 cells, are shown in
Interestingly, the levels of cell associated platinum depend strictly on the structures of the Pt(IV) complexes. Compound VI accumulated most efficiently whereas compound IV was not internalized nearly as efficiently by MCF-7 cells.
The internalization pattern was VI>I>III≈VI>II≈VII>V>IV (see
Although accumulation of the platinum based drugs in the cancer cell is an essential step in the cascade of events leading to the death of the cancer cells, it does not necessarily correlate with cytotoxicity. It is believed that for platinum drugs the binding to nuclear DNA is the critical step and hence it is important to quantify the levels of nuclear DNA and DNA-bounded platinum. As with whole cell uptake experiments, the highest nuclear Pt accumulation and DNA platination levels were reached with compound VI whereas the lowest values were detected for complex IV. Notably, for all tested complexes the intranuclear accumulation and DNA platination profiles were comparable suggesting that nuclear internalization correlates with DNA-platination.
In addition the amount of platinum was also measured in the cytoplasm. These experiments were performed in two cell lines: MCF-7 (breast cancer) and A375 (melanoma).
The cells were incubated for 24 h either with equal concentrations of the complexes (
The log P of compounds VI and VIII was measured to see if a correlation existed between lipophilicity and cell association. To quantify the hydrophobicity of the platinum complexes, partition coefficients (log P) values for octanol/water partition were measured using the shake-flask method. The log P values for cisplatin and several compounds I, II, III, VI and VIII are shown in the Table 2 below:
Interestingly, there was no correlation between lipophilicity and cellular association. The highest accumulation was observed for ctc-[Pt(NH3)2(PhB)2Cl2] followed by ctc-[Pt(NH3)2(VPA)2Cl2]. Although ctc-[Pt(NH3)2(VPA)2Cl2] and [Pt(DACH)(PhB)2(ox)] have the same log P values the former has much higher accumulation than the latter and [Pt(DACH)(VPA)2(ox)] with the highest log P (0.37) does not accumulate as well as ctc-[Pt(NH3)2(PhB)2Cl2] and ctc-[Pt(NH3)2(VPA)2Cl2].
A very different picture unfolds when the cells (MCF-7 and A375) are incubated for 24 h with the IC50 concentrations for each complex (
HDAC Inhibition
The complexes described here were specifically designed to release either VPA of PhB inside the cells in the hope that they will inhibit HDAC activity paving the way for more efficient DNA platination. Thus, in addition to measuring the levels of DNA platination, we also measured the ability of these pro-drugs to inhibit HDAC activity in cells. MCF-7 cells were incubated for 24 h with IC50 concentrations of compounds I-VIII and their HDAC activity was determined in cells (
MCF-7 cells were incubated for 24 h with IC25 or IC50 concentrations of tested complexes or TSA (trichostatin A, HDAC inhibitor). The HDAC activity was determined by the Fluor de Lys® Fluorescent Cellular Activity Assay (Enzo Life Sciences).
In both experiments, the pattern of HDAC inhibition was very similar; resembling those obtained from cytotoxicity and platination assays. As expected, cisplatin, oxaliplatin as well as Pt(IV) complexes IX-XII lacking the HDAC inhibitors in the axial position were not effective in modifying HDAC activity. Compounds I and VI are significantly more potent HDAC inhibitors than the other derivatives. More importantly, their IC50 values (μM) calculated in term of cellular HDCA inhibitory activity were three orders of magnitude lower than those reported for free VPA or PhB, respectively. As a general consideration, the bis-HDACi compounds were more efficient than the corresponding mono-HDACi. However, all the complexes were significantly less potent HDAC inhibitors compared with Trichostatin A (TSA).
Also, MCF-7 cells were incubated for 24 h with increasing concentrations of compounds I-VIII and their HDAC activity was determined. The IC50 values for the inhibition of HDAC activity in the cells are depicted in
The IC50 values for cellular inhibition of HDAC activity for all the compounds that release either VPA (I-V) or PhB (VI-VIII) in the cell are in the low μM range, similar to the cytotoxicity IC50 values, while those for valproate (˜8 mM) and PhB are significantly higher.
To gain more insights into the mode of action of newly synthesized Pt(IV) derivatives, compounds 1 and 6 were chosen for further experiments. In particular, their capacity to induce apoptosis against human breast MCF-7 cells treated with equitoxic concentrations (IC50 values) of these drugs was evaluated and compared. Nuclear DNA fragmentation and apoptosome complex formation are critical steps in the apoptotic process. DNA fragmentation tests performed on MCF-7 cells treated for 12 or 24 h with IC50 concentrations showed the ability of both Pt(IV) derivatives to increase mono- and oligo-nucleosome formation to a very similar extent in a time-dependent manner (
To assess the mechanism of the triggered apoptotic process, the activity of the two initiator caspases (-8 and -9), and the downstream effectors (-3/-7 and -6) was determined in MCF-7 treated cultures. As shown in
Interestingly, treatment with zVAD, a cell-permeable pan-caspase inhibitor, strongly decrease caspase activation, thus confirming the role played by these proteases in cell death determined by the novel Pt(IV) complexes. Since caspases 3/-7 and 9 are mainly involved in intrinsic apoptosis pathway, whereas caspase 6 and 8 are extrinsic caspase executors, these data attest for 1 and 6 a predominant induction of apoptotic signaling via the mitochondrial pathway. Based on these findings, we thought it would be interesting to evaluate the effect induced by Pt(IV) derivatives on mitochondria membrane potential (ΔΨ, MMP). MMP depletion generally precedes mitochondrial-driven apoptotic cell death. By treating MCF-7 cells with IC50 concentrations of 1 and 6 for 12 and 24 h, JC-10 fluorescence intensity decrease by increasing exposure times (
Satraplatin, ctc-[Pt(NH3)(c-hexylamine)(OAc)2Cl2], and other Pt(VI) derivatives have been recently shown to induce cell death in human cancer cells via p53 and p21 induction. Accordingly, 24 h-treatment of MCF-7 cells with 1 and 6 resulted in an increase in p53 activation. However, by taking into account cytotoxicity profiles of 1 and 6, rather similar cytotoxicities were observed against either p53 wild-type MCF-7 cells and p53 null PC3 cell line. These data indicate that the p53 status barely influenced the sensitivity of the cancer cells to the studied compounds, thus indicating that these complexes can activate apoptotic cell death pathways by mechanisms that are both dependent and independent of p53, and can probably recruit more than one pathway to trigger cell death.
In Vivo Studies on Compound VI ctc-[Pt(NH3)2(PhB)2Cl2]
A preliminary in vivo efficacy study was carried out on a mouse model (see Table 3 below):
a vehicle (0.5% (v/v) of DMSO, 99.5% (v/v) of 0.9% NaCl).
Lewis lung carcinoma (LLC) was implanted i.m. (2·106 cells inoculum) into the right hind leg of 8-week old inbred C57BL mice (24±3 g body weight). Chemotherapy was delayed until the tumor became visible (day 7). Day 7-14: animal received daily 20 mg/kg of I or IV orally as well as daily 1.5 and 3 mg/kg of cisplatin i.p. At day 15 animals were sacrificed, legs amputated at the proximal end of the femur, and the inhibition of tumor growth was determined as the difference in weight of the tumor-bearing leg and the healthy leg expressed as % referred to the control animals.
This preliminary results show that compound VI has curative potential by oral administration and is nearly as effective in this model as cisplatin that was administered ip.
The hypothesis underlying this work was that by combining agents that can increase the accessibility of nuclear DNA with DNA damaging agents that lead to the death of the cancer cells we might be able to increase the efficiency of killing cancer cells. Towards this end we designed Pt(IV) derivatives of cisplatin and oxaliplatin that have one or two HDACis as axial ligands in the hope that the cellular accumulation of these complexes will be high due to the lipophilicity of the HDAC is and once inside the cell these pro-drugs will be activated by reduction simultaneously releasing the Pt drug and the HDACi. To check this hypothesis we synthesized eight such complexes and compared their cytotoxicity to the parent Pt drugs (cisplatin or oxaliplatin) and to the HDACis (VPA of PhB).
The compounds were screened against nine different cancer cells and looking at the average IC50 values of all cell lines we see that oxaliplatin is about two fold more potent than cisplatin (4.9 vs 9.9 μM). Therefore, it is worth examining whether the Pt(IV) derivatives of oxaliplatin with the same axial ligands are be more potent than their cisplatin analogs. This is clearly not the case as we see that ctc-[Pt(NH3)2(VPA)2Cl2] is significantly more potent than [Pt(DACH)(VPA)2(ox)] with average IC50s of 1.15 and 4.48 μM respectively. Ctc-[Pt(NH3)2(PhB)2Cl2] and [Pt(DACH)(PhB)2(ox)] behave in a similar manner where their IC50s are 0.23 and 6.81 μM respectively. Interestingly, attaching the two bioactive VPA ligands to the axial positions of the Pt(IV) derivative of cisplatin increased its average potency by 8 fold compared with cisplatin while doing the same for the oxaliplatin derivatives resulted in a twofold reduction in potency relative to oxaliplatin. Ctc-[Pt(NH3)2(PhB)2Cl2] is 50 times more potent than cisplatin while [Pt(DACH)(PhB)2(ox)] is slightly less potent that oxaliplatin. Clearly, conjugation of either two VPA or two PhB to the axial positions of cisplatin significantly enhance their potency relative to cisplatin and especially when compared with free VPA or PhB whose IC50 values are in the mM range.
An essential step in the cascade of events leading to the death of the cancer cells by these compounds is the accumulation in the cells. Often higher cellular accumulation correlates with improved cytotoxicty. Comparing the cellular association of the complexes following 24 h incubation to equi-Pt concentration (5 μM) showed a correlation between the cell-associated Pt levels and the IC50 values. The most potent compounds, I and VI, also displayed the highest levels of cellular accumulation while IV, the least potent complex, had the lowest levels of cellular associated Pt.
The same general correlation patterns were observed for Pt accumulation in the nucleus and platination of nuclear DNA and potency. The most potent compounds (I and VI) were also the most abundant in the nucleus and displayed the highest levels of DNA platination. It seems like there is a correlation between the Pt levels associated with whole cells and their levels in the nucleus and bound to DNA. Interestingly, when the cells were exposed for 24 h to IC50 values of each of the compounds the Pt levels associated with the cells for all the compounds (with the exception of IV) were very similar. This result seems to suggest that regardless of the structure of the Pt complex, in order to kill 50% of the cells, a certain number of molecules have to accumulate in the cell. In the case of MCF-7 cells approximately 350 pg Pt/106 cells are required (1×106 molec/cell) while for A375 cells approximately 500 pg Pt/106 cells are required (1.5×106 molec/cell). However, the extracellular Pt concentrations necessary to attain these conditions are different.
To check the validity of the working hypotheses that these complexes will inhibit HDAC activity in the cells, the ability of each of the complexes to inhibit HDAC activity in MCF-7 cells was evaluated. Cells were exposed to increasing concentration of the compounds and the HDAC activity was measured and the concentrations of the complexes that caused 50% inhibition of HDAC activity are depicted in
One of the interesting questions that arises is whether the high potency of compounds I and VI is due primarily to a synergistic effect between the HDAC inhibitors and the Pt complexes or is it merely due to the enhances cellular accumulation. It is generally accepted that lipophilic ligands enhance the cellular uptake of Pt complexes and that higher accumulation usually results in enhanced cytotoxicity. Thus attaching the lipophilic VPA or PhB ligands to the Pt pro-drug is expected to enhance its potency by virtue of higher cellular accumulation. The situation is quite different for the HDACis used in this study. Both valproic acid and 4-phenylbutyruc acid are ionized at neutral pH and are monoanionic and therefore will not efficiently accumulate in the cancer cells. The results show that valproate does not accumulate in the cells very efficiently. Attempts to increase the cellular accumulation of valproate include neutralization of the charge by esterifying the carboxylate and forming for instance valproyl ester-valpramide of acyclovir. Upon ligating the VPA or PhB to the axial positions of the Pt(IV) complex a metalloester is formed and the charges of the VPA and PhB are neutralized facilitating the cellular uptake. Thus, conjugation of VPA or PhB to the Pt(IV) yields complexes that facilitate the cellular uptake of both its components.
Higher intracellular concentrations of both VPA/PhB and the cytotoxic Pt(II) drugs are likely to be more effective at inducing apoptosis. Although VPA/PhB were selected because they have the ability to inhibit HDAC activity, but the activity of HDAC enzymes is not limited to histones and they can modify other proteins such as chaperones, transcription regulators, transcription factors, signal transduction mediators and others. VPA can deplete GSH levels in rat cells.
The growth inhibitory effect of PBA on gastric cancer cells is associated with alteration of the cell cycle. In another publication, phenyl butyric acid is reported to rescue endoplasmic reticulum stress-induced suppression of APP proteolysis and prevents apoptosis in neuronal cells. These examples demonstrate that efficient importation of VPA or PhB into cell by the Pt(IV) pro-drugs can lead to many different cellular outcomes, including HDAC inhibition and suppression of expressions of HDAC enzymes, that could in conjunction with cisplatin enhance the efficiency of killing the cancer cells. It is not clear which of the cellular events, or combination of event triggered by the HDAC is contributes to the enhances potency in conjunction with the Pt moiety.
As indicated herein, the present invention also contemplates quadruple action Pt(IV) dinuclear compounds that incorporate in one molecular entity cisplatin, [Pt(DACH)(56Phen)], phenylbutyrate and dichloroacetate. This compound, after intracellular reduction, can aim DNA, histones and mitochondria as cellular targets.
Commercial reagents and solvents were used as received, without further purification.
c,t,c-[Pt(DACH)(OH)2(56Phen)]Cl2, (PhBu)2O were synthesized according procedures previously reported in the literature.
Reaction mixtures and purified products were analyzed on a Thermo Scientific UltimaMate 3000 station, equipped with a reverse-phase C18 column (Phenomenex Kinetex 250×4.6 mm, 5 μm, 100 Å). UV detection was set at 220 and 260 nm. The samples were eluted with a 0-90% linear gradient of acetonitrile in water with 0.1% trifluoroacetic acid (TFA) over 30 min.
Reaction mixtures were separated on a Thermo Scientific UltimaMate 3000 station, equipped with a reverse-phase C18 column (Phenomenex Luna 250 X 21.2 mm, 10 μm, 100 Å). UV detection was set at 220 nm. The samples were eluted with a 0-90% linear gradient of acetonitrile in water with 0.1% trifluoroacetic acid (TFA) over 30 min.
c,c,t-[Pt(NH3)2Cl2(DCA)(GABA)](TFA) (1a, Scheme 4).
c,c,t-[Pt(NH3)2Cl2(OH)2] (96.2 mg, 0.29 mmol) was suspended in 10 mL of DMSO. GABA-BOC anhydride (123.0 mg, 0.32 mmol) was added and it was left to react overnight at RT. The mixture turned to a yellow solution and was centrifuged. The mother liquor was treated with ether, obtaining a two phases system that was centrifuged in order to remove the ether phase. This process was repeated several times until a sticky yellow solid was obtained. It was dissolved in methanol and precipitated with ether, affording a pale yellow solid. It was recovered by centrifugation, dried and dissolved in 3 mL of DMF. DCA anhydride (265 μL, 1.74 mmol) was added to the mixture. After 4 h at RT, the mixture had turned to a bright yellow solution. It was filtered and then evaporated to dryness, affording a yellow sticky solid. It was dissolved in methanol and precipitated with ether. The solid was recovered by centrifugation and dried. It was stirred at room temperature in a mix of DCM and TFA (1:1) for 45 min. The solvents were removed with a stream of compressed air. A sticky yellow solid was obtained. It was dissolved in methanol and added I a dropwise fashion to ice-cold ether, inducing the formation of a white precipitate. The solid was collected by centrifugation and dried under a stream of compressed air. 52.0 mg were recovered (28% yield). This complex was used for the synthesis of 3a (Scheme 4) without further purification. It was purified on preparative HPLC (retention time=11.8 min) and lyophilized, when used for biological studies. 1H NMR δ (D2O): 6.24 (s, 1H), 2.96 (t, 2H), 2.48 (t, 2H), 1.82 (m, 2H). 1H NMR δ (D2O): 1076 ppm. Retention time on analytical HPLC: 8.4 min ESI-MS: calculated for [M+H]+ (M=C6H15 Cl4N3O4Pt): m/z=531.10; experimental: m/z=531.92.
c,c,t-[Pt(NH3)2Cl2(OAc)(GABA)](TFA) (1b, Scheme 4).
c,c,t-[Pt(NH3)2Cl2(OAc)(OH)] (113.3 mg, 0.30 mmol) was suspended in 5 mL of DMF. GABA-BOC anhydride (234.0 mg, 0.60 mmol) was added and then the mixture was stirred overnight at 40° C., affording a yellow solution that was filtered and then reduced to dryness under reduce pressure. The sticky yellow solid was then dissolved in methanol and precipitated with ether. The precipitate was collected by centrifugation and dried. It was then stirred at room temperature in a mix of DCM and TFA (1:1) for 45 min. The solvents were removed with a stream of compressed air. The sticky solid was dissolved in methanol and added dropwise to ice-cold ether, inducing the formation of a white precipitate. The solid was collected by centrifugation and dried under a stream of compressed air. 59.2 mg were recovered (34% yield). This complex was used for the synthesis of 3b (Scheme 4) without further purification. 1H NMR δ (D2O): 2.94 (t, 2H), 2.44 (t, 2H), 2.00 (s, 3H), 1.81 (q, 2H). 1H NMR δ (D2O): 1077 ppm. Retention time on analytical HPLC: 3.4 min ESI-MS: calculated for [M+H]+ (M=C6H17Cl2N3O4Pt): m/z=462.21; experimental: m/z=462.14.
c,c,t-[Pt(DACH)(56Phen)(PhBu)(Succ)]Cl2 (2a, Scheme 4).
c,c,t-[Pt(DACH)(56Phen)(OH)2]Cl2 (111.5 mg, 0.18 mmol) was stirred in 4 mL of DMSO together with phenylbutyric anhydride (67.0 mg, 0.22 mmol) at RT for 2 h. Afterwards, succinic anhydride (72.0 mg, 0.72 mmol) was added to the mixture. After one night, the reaction mixture was filtered. The filtrate was diluted with EtOAc, and then precipitated with ether. The brown sticky precipitate was recovered by centrifugation, dried and then dissolved in methanol and precipitated with ether, affording a beige precipitate that was recovered by centrifugation and then dried. 92.0 mg were recovered (60% yield). This complex was used for the synthesis of 3a (Scheme 4) without further purification. It was purified on preparative HPLC (retention time=15.6 min), obtaining the TFA salt c,t,c-[Pt(DACH)(PhBu)(Succ)(56Phen)](TFA), and lyophilized, when used for biological studies. 1H NMR δ (D2O): 9.38 (d, 2H), 9.18 (m, 2H), 8.29 (m, 2H), 7.16 (m, 3H), 6.47 (m, 2H), 3.30 (d, 1H), 3.20 (d, 1H), 2.71 (s, 6H), 2.46 (m, 2H), 2.32 (t, 2H), 2.14 (t, 2H), 2.08 (M, 2H), 1.80 (m, 2H), 1.74 (M, 2h), 1.1 (M, 4H), 1.18 (t, 2H). 105Pt NMR δ (D2O): 700 ppm. Retention time on analytical HPLC: 14.6 min. ESI-MS: calculated for [M−H]+ (M=C34H42N4O6Pt): m/z=796.27; experimental: m/z=795.94.
c,c,t-[Pt(DACH)(56Phen)(OAc)(Succ)]Cl2 (2b, Scheme 4).
c,c,t-[Pt(DACH)(56Phen)(OH)2]Cl2 (337.8 mg, 0.54 mmol) was suspended in 12 mL of DMSO. Succinic anhydride (54.3 mg, 0.54 mmol) was added and the mixture was stirred overnight at room temperature in the dark. The next day, an excess of acetic anhydride (6 mL) was added. After 4 h, the mixture was filtered, diluted with EtOAc and precipitated with ether. The brown sticky precipitate was collected by centrifugation, dried and then dissolved in methanol and reprecipitated with ether. The beige precipitate was collected by centrifugation and dried. 614.8 mg were recovered (80% yield). This complex was used for the following reactions without further purification. This complex was used for the synthesis of 3b (Scheme 4) without further purification. 1H NMR δ (D2O): 9.14 (d, 2H), 9.07 (d, 2H), 8.09 (t, 2H), 3.07 (d, 2H), 2.73 (s, 6H). 2.29 (d, 2H), 2.10 (m, 2H), 1.99 (m, 2H), 1.61 (b, 2H), 1.60 (s, 3H), 1.56 (b, 2H), 1.20 (b, 2H). 1H NMR δ (D2O): 702 ppm. Retention time on analytical HPLC: 9.5 min ESI-MS: calculated for [M−H]+ (M=C26H34N4O6Pt): m/z=692.20; experimental: m/z=692.18.
c,c,t,t,c,c-[(DACH)(56Phen)(PhBu)Pt(μ-OOC—(CH2)2—CONH(CH2)3COO)—Pt(DCA)(NH3)2 (Cl2)](TFA)2 (3a, Scheme 1).
2a (86.8 mg, 0.10 mmol) was stirred in 1.0 mL of DMSO. EDC (38.3 mg, 0.20 mmol) and NHS (23.0 mg, 0.20 μmol) were added to the mixture, and it was stirred at room temperature in the dark for 45 minutes. 1a (64.9 mg, 0.10 mmol) was dissolved in 1.0 mL of DMSO in the presence of TEA (28.0 μl, 0.20 mmol) and added to the mixture. The mixture was kept under magnetic stirring at RT for 4 h. Afterwards, the mixture was diluted with EtOAc and precipitated with ether, affording a sticky dark brown precipitate. It was recovered by centrifugation, dried, dissolved in methanol and precipitated with ether. The resulting beige precipitate was collected by centrifugation and dried. Afterwards, it was dissolved in a mixture of water and ACN (1:1), purified on preparative HPLC (retention time=17.6 min), and finally lyophilized. 30.0 mg were recovered (20% yield). 1H NMR δ (D2O): 9.41 (d, 2H), 9.22 (m, 2H), 8.34 (m, 2H), 7.07 (d, 2H), 7.06 (m, 1H), 6.36 (m, 2H), 6.25 (s, 1H), 3.31 (m, 1H), 3.23 (m, 1H), 2.73 (s, 3H), 2.71 (t, 2H), 2.48 (m, 2H), 2.44 (m, 2H), 2.25 (m, 2H), 2.13 (m, 2H), 2.09 (m, 2H), 1.77 (m, 2H), 1.47 (m, 2H), 1.38 (m, 4H), 1.32 (m, 2H), 1.17 ppm (m, 2H). 1H NMR δ (D2O): 1082 and 701 ppm. Retention time on analytical HPLC: 16.6 min ESI-MS: calculated for [M−H]+ (M=C40H55 Cl4N7O9Pt2): m/z=1308.54; experimental: m/z=1308.87.
c,c,t,t,c,c-[(DACH)(56Phen)(OAc)Pt(μ-OOC—(CH2)2—CONH(CH2)3COO)-Pt(OAc)(NH3)2(Cl2)](TFA)2 (3b, Scheme 4).
2b (104.3 mg, 0.14 mmol) was stirred in 1.2 mL of DMSO. EDC (53.7 mg, 0.28 mmol) and NHS (32.2 mg, 0.28 mmol) were added and the mixture was stirred at RT for 45 min. Afterwards, 1b (78.5 mg, 0.14 mmol), previously dissolved in 1.2 mL of DMSO in the presence of TEA (39.0 μl, 0.28 mmol), was added to the mixture. The mixture was kept under magnetic stirring at RT for 4 h. Afterwards, the mixture was diluted with EtOAc and precipitated with ether, affording a sticky dark brown precipitate. It was recovered by centrifugation, dried, dissolved in methanol and precipitated with ether. The resulting beige precipitate was collected by centrifugation and dried. Afterwards, it was dissolved in a mixture of water and ACN (1:1), purified on preparative HPLC (retention time=11.6 min), and finally lyophilized. 20.0 mg were recovered (10% yield). 1H NMR δ (D2O): 9.21 (d, 2H), 9.14 (d, 2H), 8.17 (t, 2H), 3.12 (d, 2H), 2.79 (s, 6H, CH3), 2.55 (t, 2H), 2.35 (d, 2H), 2.22 (m, 2H), 2.14 (t, 2H), 2.01 (s, 3H), 1.96 (m, 2H), 1.66 (b, 2H), 1.64 (s, 3H), 1.63 (b, 2H), 1.35 (q, 2H), 1.26 (b, 2H) ppm. 1H NMR δ (D2O): 1081 and 707 ppm. Retention time on analytical HPLC: 10.0 min. ESI-MS: calculated for [M−H]+ (M=C32H49Cl2N7O9Pt2): m/z=1134.22; experimental: m/z=1134.86.
Study of Stability in Buffer.
A solution 2.0 mM of 3a (Scheme 4) in sodium phosphate buffer (50 mM, pH=7) was kept at 37° C. for 3 days. NMR and HPLC measurements were performed every day.
Study of Stability in Cell Culture Medium.
A solution 2.0 mM of 3a (Scheme 4) in DMEM was kept at 37° C. for 3 days. NMR measurements were performed every day.
Study of Reduction in Buffer in the Presence of Ascorbic Acid.
A solution 2.0 mM of 3a (Scheme 4) in buffer was treated with 1 Eq ascorbic acid. NMR measurements were performed over a 2 hour time frame.
Cytotoxicity
MTT assay. The growth inhibitory effect towards human cell lines was evaluated by means of MTT (tetrazolium salt reduction) assay. Briefly, 3-8.103 cells/well, dependent upon the growth characteristics of the cell line, were seeded in 96-well microplates in growth medium (100 μL) and then incubated at 37° C. in a 5% carbon dioxide atmosphere. After 24 h, the medium was removed and replaced with a fresh one containing the compound to be studied at the appropriate concentration. Triplicate cultures were established for each treatment. After 72 h, each well was treated with 10 μL of a 5 mg·mL−1 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) saline solution, and after 5 h additional incubation, 100 μL of a sodium dodecylsulfate (SDS) solution in HCl 0.01 M were added. After overnight incubation, the inhibition of cell growth induced by the tested complexes was detected by measuring the absorbance of each well at 570 nm using a Bio-Rad 680 microplate reader (Bio-Rad, Hercules, Calif.). Mean absorbance for each drug dose was expressed as a percentage of the control untreated well absorbance and plotted vs drug concentration. IC50 values represent the drug concentrations that reduced the mean absorbance at 570 nm to 50% of those in the untreated control wells.
Acid phosphatase (APH) assay. A modified APH assay, which is based on quantification of cytosolic acid phosphatase activity, was used for determining cell viability in spheroids. Briefly, the pre-seeded spheroids were treated with fresh medium containing the compound to be studied at the appropriate concentration. Triplicate cultures were established for each treatment. After 72 h, each well was treated with 100 μL of the assay buffer (0.1 M sodium acetate, 0.1% Triton-X-100, supplemented with ImmunoPure p-nitrophenyl phosphate; Sigma Chemical Co.) and, following 3 h of incubation, 10 μL of 1 M NaOH solution were added. The inhibition of the cell growth induced by the tested complexes was detected by measuring the absorbance of each well at 405 nm, using a Bio-Rad 680 microplate reader. Mean absorbance for each drug dose was expressed as a percentage of the control untreated well absorbance (T/C) and plotted vs drug concentration. IC50 values, the drug concentrations that reduce the mean absorbance at 405 nm 50% of those in the untreated control wells, were calculated by four parameter logistic (4-PL) model. Evaluation was based on means from at least four independent experiments.
Cellular accumulation and distribution. 2008 and PSN1 cells (2.5.106) were seeded in 75 cm2 flasks in growth medium (20 mL). After overnight incubation, the medium was replaced and the cells were treated with tested compounds for 24 h. Cell monolayers were washed twice with cold PBS, harvested and counted. Cell nuclei were isolated by means of the nuclei isolation kit Nuclei EZ Prep (Sigma Co.) as well as cellular mitochondrial fractions were isolated by Mitochondria Isolation Kit (Sigma Co.). Samples were than subjected to three freezing/thawing cycles at −80° C., and then vigorously vortexed. The samples were treated with highly pure nitric acid (Pt: ≤0.01 μg·kg−1, TraceSELECT® Ultra, Sigma Chemical Co.) and transferred into a microwave teflon vessel. Subsequently, samples were submitted to standard procedures using a speed wave MWS-3 Berghof instrument (Eningen, Germany) After cooling, each mineralized sample was analyzed for platinum by using a Varian AA Duo graphite furnace atomic absorption spectrometer (Varian, Palo Alto, Calif.; USA) at the wavelength of 324.7 nm. The calibration curve was obtained using known concentrations of standard solutions purchased from Sigma Chemical Co. purchased from Sigma Chemical Co.
DNA platination. 2008 and PSN1 cells (3.106) were seeded in 10 cm Petri dishes in 10 mL of culture medium. Subsequently, cells were treated with tested complexes for 24 h. DNA was extracted and purified by a commercial spin column quantification kit (Qiagen DNeasy Blood and Tissue Kit). Only highly purified samples (A260/A230 □ 1.8 and A280/A260 □ 2.0) were included for analysis to avoid any artifacts. The samples were completely dried and re-dissolved in 200 μL of Milli-Q water (18.2 MΩ) for at least 20 min at 65° C. in a shaking thermo-mixer, mineralized and analyzed for total Pt content by GF-AAS as described above.
Histone deacetylase assay. Histone deacetylase activity was determined using Fluor-de-Lys® HDAC fluorometric activity assay kit (Enzo Life Sciences International, Inc., Plymount Meeting, Pa., U.S.A.). 2008 and PSN1 cells (5.104 seeded in 96-well microplates) were treated for 24 h with tested complexes, and then processed as reported by the manufacturer's instructions. Fluorescence was measured using a Fluoroskan Ascent FL (Labsystem, Finland) plate reader, with excitation at 360 nm and emission at 460 nm.
Mitochondrial membrane potential (ΔΨ). ΔΨ was assayed using the Mito-ID Membrane Potential Kit according to the manufacturer's instructions (Enzo Life Sciences, Farmingdale, N.Y.). Briefly, 2008 and PSN1 cells were seeded onto 96-well microplates at 5.104 cells/well. After 24 h, cells were treated with tested compounds for 6, 12 and 24 h. The mitochondrial depolarizing agent, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was employed as positive control. An equal volume of cationic dye loading solution was added to each well and cell plates were incubated for additional 30 min at 37° C. Plates were read at excitation/emission wavelengths 490 and 590 nm using a fluorescence microplate reader (Fluoroskan Ascent FL, Labsystem, Finland).
Glycolysis. The cellular glycolytic activity was assessed using the Mito-ID® O2 Extracellular Sensor Kit according to the manufacturer's instructions (Enzo Life Sciences, Farmingdale, N.Y.). Briefly, PSN-1 cells (15·103 per well) were seeded in 96-well plates. After 24 h, cells were treated with Mito-ID® O2 Sensor Probe Solution containing the tested compounds at the appropriate concentration. Fluorescence was estimated using a plate reader (Fluoroskan Ascent FL, Labsystem, Finland) at 350 nm (excitation) and 610 nm (emission).
Transmission electron microscopy analyses. About 106 PSN-1 cells were seeded in 24-well plates and, after 24 h incubation, were treated with the tested compounds and incubated for additional 24 h. Cells were then washed with cold PBS, harvested and directly fixed with 1.5% glutaraldehyde buffer with 0.2 M sodium cacodylate, pH 7.4. After washing with buffer and postfixation with 1% OsO4 in 0.2 M cacodylate buffer, specimens were dehydrated and embedded in epoxy resin (Epon Araldite). Sagittal serial sections (1 μm) were counterstained with toluidine blue; thin sections (90 nm) were given contrast by staining with uranyl acetate and lead citrate. Micrographs were taken with a Hitachi H-600 electron microscope (Hitachi, Tokyo, Japan) operating at 75 kV. All photos were typeset in Corel Draw 11.
Hoechst 33258 staining. PSN-1 cells were seeded into 8-well tissue-culture slides (BD Falcon, Bedford, Mass., USA) at 5.104 cells/well (0.8 cm2). After 24 h, cells were washed twice with PBS and following 48 and 72 h of treatment with IC50 doses of the tested compound, cells were stained for 5 min with 10 μg/mL of Hoechst 33258 (2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole 3HCl hydrate, Sigma-Aldrich) in PBS before being examined by fluorescence microscopy (Olympus).
Synthesis and Characterization
In order to obtain the quadruple action Pt(IV) dinuclear compound 3a (Scheme 4), we synthesized two separate Pt(IV) units, 1a and 2a (Scheme 4), each of them with both one bioactive and one bridging moiety in the axial positions, and finally we condensed the two units, obtaining the dinuclear complex.
In the case of the unit 1a (Scheme 4), we started from oxoplatin, ctc-[Pt(NH3)2(OH)2Cl2], the axial dihydroxido Pt(IV) derivative of cisplatin. We performed the first carboxylation reaction in DMSO using a small excess of the anhydride (1.1 Eq), under dilute conditions (ca. 10 mg of Pt complex/mL). Initially, a GABA-BOC moiety was conjugated to one of the axial positions. The initial suspension turned to a solution after one day. The monocarboxy derivative, that was identified by 1H NMR=1050 ppm), was isolated by precipitation with ether dissolved in DMF and was then reacted with DCA anhydride. The reaction was monitored by 1H NMR spectroscopy: the Pt resonance shifted from 1050 to 1170 ppm upon carboxylation. We chose to begin the carboxylation with the least reactive anhydride (GABA-BOC anhydride) in order to minimize biscarboxylation andoptimize the yield for the monocarboxylation. The second carboxylation proceeded smoothly with the more reactive and less hindered DCA. Finally, the protecting BOC group was removed in a mixture of DCM and TFA, leaving a free aminic function suitable for further reactions.
Unit 2a (Scheme 4) was prepared by initial reaction of Pt56MeSS(OH)2 with a small excess (1.2 Eq) of phenylbutyric anhydride. The reaction was monitored by 195Pt NMR spectroscopy: upon carboxylation the Pt resonance shifted from 349 to 462 ppm. Once the reaction was complete, an excess of the second anhydride was added to the mixture, without isolating the monocaroxylic derivative. Pt resonance shifted to 699 ppm after the reaction was complete. This second unit had a free carboxylic function on the succinic anhydride, suitable for further reactions.
Finally, units 1a and 2a (Scheme 4) were used for the formation of a peptide bond, to afford 3a. DMSO turned out to be a good solvent for this reaction, although DMF also afforded good results. 1H NMR assignments for 1a, 2a, and 3a (Scheme 4) are summarized in Table 4. Cylic voltammetric measurements (CV) were performed on complex 3a and 3b finding Ep values of −0.462 and −0.058 V. The first reduction peak was assigned to the reduction of the cisplatin-derivative portion of the dinuclear complex, because of similarity with the values reported for similar complexes. The less negative reduction peak was assigned to the Pt-Phenantroline metal center, because of similarity to the values reported for the bis axial phenylbutyrato Pt(IV) complex Pt56MeSS(PhB)2.
The synthesis compound 3b (Scheme 4), a control compound that has acetates instead of bioactive moieties in the axial positions, but acetates, was achieved via a similar pathway. Unit 1b (Scheme 4) was synthesized starting from the mono acetato Pt(IV) derivative of cisplatin, obtained by oxidation of cisplatin in acetic acid, which then reacted with GABA-BOC anhydride, to afford the biscarboxylato species, that underwent a deprotection process to free the aminic end of GABA. For the synthesis of the unit 2b (Scheme 4), the first carboxylation step was carried out with succinic anhydride, and the second with acetic anhydride. Finally, 1b and 2b (Scheme 4) were condensed to afford 3b (Scheme 4). 1H NMR assignments for 1b, 2b, and 3b (Scheme 4) are summarized in Table 4.
1H and 195Pt NMR chemical shift in D2O (ppm).
195Pt NMR
1H NMR chemical shift (ppm)
Stability in Buffer
A 2.0 mM solution of 3a in phosphate buffer at pH=7 was kept at 37° C. in order to assay its stability by mean of NMR spectroscopy (
After one day, a new series of low intensity peaks was observed in the 1H NMR. These new signals at 9.06, 8.88 and 8.02 ppm were assigned to the aromatic protons of the Pt(II) complex [Pt(DACH)(56Phen)]2+, indicating that some reduction of the Pt(IV) that is ligated to the phenanthroline moiety occurred. In the 7.45-7.28 ppm range, a series of new signals overlapping with the signal of the amidic proton of 3a appeared. These new signals belong to the free phenylbutyric acid, resulting from the reductive elimination. From the relative integrals of the phenanthroline protons it can be estimated that after one day approximately 6.5% of the Pt(IV)-phen was reduced, after 2 days about 9% and after 3 days 10%.
The new signal at 6.06 ppm was assigned to free DCA. The free DCA could result from hydrolysis or from reduction of the Pt(IV)-cisPt moiety. To sort this out, 1H NMR spectra were measured at time zero and every day. Initially two peaks with nearly equal intensity were observed in the Pt(IV) region: one peak at 1101 ppm assigned to Pt(IV)-cisPt and the other at 703 ppm assigned to Pt(IV)-phen. After a day the signal at 1101 was drastically reduced and was only 15% of the signal at 703 ppm. After the second day only the signal of the Pt(IV)-phen could be observed suggesting that all the Pt(IV)-cisPt was reduced or completely hydrolysed. We looked for the potential reduction products (cisplatin and its hydrolysis products) in the Pt(II) region but did not see any signals at all.
These observations, together with the presence of a solid in the mixture, led us to hypothesize that the DCA ligand bound to the cisplatin-like metal center was rapidly hydrolyzed and was replaced by an OH releasing free DCA into solution. Then the linker chain was also hydrolyzed releasing oxoplatin, c,c,t-[Pt(NH3)2Cl2(OH)2], as a precipitate. The instability of Pt(IV) derivatives of cisplatin with DCA was previously demonstrated by us. The poorly soluble complex c,c,t-[Pt(NH3)2Cl2(OH)2], that precipitated cannot be observed in the spectrum.
This led to the formation of a new species:
c,c,t-[Pt(DACH)(phen)(PhB)(O2CCH2CH2CONHCH2CH2CH2CO2H)]2+ (4). (Scheme 5). The HPLC of the reaction mixture at this stage showed a peak at 14.6 min. This peak was isolated and analyzed by 1H NMR and ESI-MS experiments, confirming the hypothesized structure of 4.
Stability in Cell Culture Medium
A phosphate buffer solution is an oversimplified model for medium of biological relevance in order to test the stability of a drug candidate. On one hand its simplicity allows to study degradation reactions easily, since the number of potentially interfering species is reduced to the minimum; on the other hand it does not provide a very reliable overview of what the fate of the drug candidate could be once administered. Thus, as following step, we evaluated the stability of 3a in cell culture medium DMEM, where the biomolecules present can affect the overall degradation process. A 2.0 mM solution of 3a in DMEM monitored by 1H NMR spectroscopy for 3 days at 37° C. (
Immediately after the preparation of the NMR sample, it was possible to find the signals of H4, H2 and H3 of the phenanthroline moiety of 3a, at 9.41, 9.21 and 8.34 ppm, respectively. The signal at 7.40 ppm was assigned to the amidic proton. The signals at 7.18 and 6.48 belonged to the aromatic ring of the coordinated phenylbutyrate. Finally, the peak at 6.37 belongs to the coordinated DCA. After 1 day, the signal of the reduction product [Pt(DACH)(56Phen)]2+ appeared at 9.00, 8.88 and 8.03 ppm. These signal were broad, while the signals of 3a stayed sharp, suggesting that the Pt(II) species interacts with the high MW proteins in the cell culture medium (probably BSA), while the starting Pt(IV) dimer does not. Integration of the phenanthroline peaks of Pt(IV)-phen and Pt56MeSS indicated that after 1 day about 22% of the Pt(IV)-phen moiety was reduced and after 2 days 39% and after 3 days 58%. It is interesting to note that compound 3a is more stable in buffer than in cell culture medium since the monomeric compounds Pt56MeSS(OAc)2 and Pt56MeSS(PhB)2 were unstable in buffer but were stable in human serum. In the range 7.43-7.25 ppm the signals of free phenylbutyrate were detected, overlapping with the amidic signal of 3a and 4. Finally, a signal belonging to free DCA was detected at 6.06 ppm. The following day, the signals belonging to [Pt(DACH)(56Phen)]2+ and free PhBu, increased in intensity, suggesting that the reduction of the Pt metal center of 3a bond to the phenanthroline was continuing. On the other hand, almost no more coordinated DCA was detected, while free DCA grew in intensity, suggesting that the hydrolysis of the cisplatin-like part of 3a was almost complete. The same trend was observed on the third day.
Compared to the pervious experiment, in which the stability of 3a was tested in phosphate buffer, the experiment carried out in cell culture medium showed the same degradation pathway, in which the Pt metal center bond to the phenanthroline ligand undergoes reduction, while the Pt metal center that has DCA as axial ligand undergoes hydrolysis. The process seems to occur faster in cell culture medium.
Study of Reduction in Buffer in the Presence of Ascorbic Acid.
In order to assay which of the two Pt metal centers of 3a is more prone to be reduced, we added 1 Eq of ascorbic acid to a solution of 3a in phosphate buffer. We added 1 Eq of ascorbic instead of 2 Eq that would be needed for the reduction of both Pt cores, to assay if one of the two is preferably reduced over the other. Finally, we carried out the reaction at RT and not at 37° C. to avoid a fast reduction process that would have made more complicated its study.
The reaction was monitored by 1H NMR spectroscopy (
This behavior is in agreement with the CV measurements, that showed a reduction potential more negative for the cisplatin-derived Pt metal center of 3a, that is, so, less prone to undergo reduction.
The two dimers, 3a and 3b, as well as compounds 1a and 2a were screened against six human cancer cell lines representative of lung (H157), colon (HCT-15), cervical (A431), ovarian (2008) and pancreatic (PSN1) cancers, along with melanoma (A375). Cytotoxicity was evaluated by means of the MTT test after 72 h of treatment and the in vitro antitumor potential of newly synthesized derivatives was compared to that of the reference metallo-drugs CDDP and OXP. The results, expressed as IC50 values calculated from dose-survival curves, are reported in Table 5. Compound 1a showed a pattern of cytotoxicity similar to that of CDDP against colon cancer cells and melanoma cells whereas it was more effective than the reference metallodrug against pancreatic cancer cells PSN1. Compound 2a proved to be more effective than cisplatin against colon and pancreatic cancer cells as well as melanoma cells whereas it retains an in vitro antitumor potential lower than that of cisplatin against cervical cancer cells A431. On the other hand, the “quadrupole action” complex (3a) was more potent than CDDP and OXP in all tested cancer cell lines and its cytotoxicity resembles that of the parental compound [Pt(DACH)(56Phen)]. Interestingly, compound 3a was significantly (on average about 12 times) more potent than the “dual action” compound 3b. This clearly attests to the importance of tethering bioactive ligands such as DCA and PhB to the axial position in order to attain superior in vitro antitumor potential. It is interesting to note that 3a was up to 200 and 92 times more effective than CDDP and OXP, respectively, against PSN1 cells, that a notorious highly aggressive p53 and KRAS mutated pancreatic cancer cells.
In Vitro Antitumor Activity on 3D Human Cancer Cell Cultures
Cytotoxicity results obtained by MTT test showed that compounds 3a and 3b elicited a significant antiproliferative activity against cancer cells derived from solid tumors. However, in two-dimensional (2D) monolayer cell cultures cellular activities are often altered and their typical in vivo functions resulted to be lost. As a result, the conventional 2D cell culture provides limited predictive capacity for drug testing.
As an attractive alternative, 3D cell cultures have proven to be a physiologic mimic of the in vivo tissue because they produce a similar cellular microenvironment. Actually, on a three-dimensional architectural organization, tumor cells are not uniformly exposed to nutrients or oxygen in vitro, thus closely mimicking the organization of human tumors. Thus, significant differences were described in terms of sensitivity to drugs among 2D and 3D cultures due to fundamental differences in terms of growth, migration, morphology and gene expression.
In order to improve the predictive capacity of the in vitro assays, we tested the activity in three three-dimensional (3D) cell cultures; human colon cancer cells, HCT-15, and human pancreatic cancer cells, KRAS wild type BxPC3 cells KRAS mutated PSN-1 cells. The cancer spheroids were treated with 3a and 3b for 72 h and the cell viability was assessed by means of the APH assay (Table 6). For comparison purposes, the cytotoxicities of CDDP and OXP were evaluated under the same experimental conditions.
In all human cancer spheroid models, derivatives 3a and 3b yielded IC50 values lower than that showed by CDDP. In the HCT-15 3D model, 3a was about 2 times more effective than CDDP and its antitumor potential was almost comparable to that of OXP. Notably, against PSN-1 pancreatic cancer cells the activity of 3a exceeded that of the reference drugs by a factor of about 6. These results, besides clearly indicating a significant antitumor potential for compound 3a, suggest its preferential activity toward highly aggressive KRAS mutated pancreatic cancer cells PSN-1.
Cytotoxicity Against Non-Cancer Cells
The cytotoxic activity of derivatives 3a and 3b was also evaluated against non-tumor cells, namely human embryonic kidney HEK293 cells (Table 7). Both compounds 3a and 3b were more cytotoxic than CDDP. However, the selectivity index (SI=ratio between average IC50 of non-tumor cells and IC50 of malignant cells) calculated for complex 3b was lower than that elicited by CDDP whereas the SI for compound 3a was about 2 times better than that of cisplatin, thus attesting the preferential cytotoxicity of 3a towards neoplastic cells.
Cell Accumulation and Distribution
With the aim of identifying a possible correlation between cytotoxic activity and cellular accumulation, the cellular Pt content and distribution were measured in human ovarian 2008 and pancreatic PSN-1 cancer cells treated for 24 h with equimolar concentrations (0.1 μM) of the platinum compounds. The cellular Pt levels in cellular sub-fractions were quantified by means of GF-AAS analysis, and the results expressed as pg Pt per 105 cells, are summarized in
Although the IC50 values of 3a and Pt56MeSS are practically identical in the ovarian cancer cell line 2008, there is nearly a fourfold difference in their cellular association and a twofold difference in their accumulation in their nuclie and mitochondria, in favor of 3a. Also, cisplatin is nearly 3 fold more potent than 3b against 2008 cells despite it much lower levels of cell association and nuclear and mitochondrial accumulation. The cell association, nuclear and mitochondrial accumulation in the PSN1 cells seem to correlate qualitatively with cytotoxicity.
In both cell lines, the lowest level of platinum associated with the cells was observed after exposure to cisplatin and, remarkably, the replacement of axial acetato groups in 3a by PhB and DCA (3b) resulted in a very significant enhancement of the level of platinum associated with the cells.
Concerning cellular distribution of Pt, 3a and 3b as well as the reference [Pt(DACH)(56Phen)] were able to highly accumulate into the mitochondrial fraction. This is probably due to the reduction of 3a and 3b in the cells that releases that cationic and lipophilic Pt56MeSS that accumulates in the mitochondria. Maybe another way to look at this is the % in the nucleus compared to the whole cell association. It seems to me that in 2008 there is more cisplatin in the nucleus (compared to mitochondria) and the other way around in PSN1.
HDAC Inhibition and DNA Platination
The newly prepared 3a complex described here was designed as quadruple action prodrug able to intracellularly release four bioactive compounds with different cellular targets and mechanisms of action. In particular, the release of PhB moiety inside the cells should grant the inhibit HDAC activity, thus allowing for a more efficient DNA platination.
Thus, we measured both the levels of DNA platination and the ability of these pro-drugs to inhibit HDAC activity in cancer cells. 2008 and PSN-1 cells were incubated for 24 h with cytotoxic IC50 concentrations of 3a, 3b, CDDP or [Pt(DACH)(56Phen)], and their DNA-platination ability as well as the HDAC inhibitory activity was determined (
DNA-platination studies (
Concerning HDAC inhibitory activity, as expected CDDP and the Pt(IV) complex 3b were not effective in modifying HDAC activity (
DCA Activity (Similar to Lippard Paper)
It has been previously demonstrated that DCA inhibits the activity of pyruvate dehydrogenase kinase (PDK), thereby shifting cellular metabolism from glycolysis to glucose oxidation, altering mitochondrial homeostasis and ultimately leading to cell death through apoptosis.
Based on the above findings, we investigated the effects determined by derivatives 3a and 3b on 2008 and PSN-1 cancer cell aerobic glycolysis and mitochondria function.
Treatment of 2008 cells with IC50 concentrations of both 3a and 3b for 24 h resulted in a reduction (about 25 and 14% for 3a and 3b, respectively) of glycolysis processes compared with untreated control cells (
In addition, it is interesting to note that treatment of 2008 and PSN-1 cells with 3a and 3b determined a significant time-dependent increase in the number of cells with depolarized mitochondria (data not shown). In particular, after 12 h of incubation, 3a elicited an increase of about 39% of PSN-1 cells with depolarized mitochondrial potential
Morphological analyses by TEM of PSN-1 cells treated for 28 h with both Pt(IV) derivatives showed disrupted cristae and significant increase in volume (swelling) with respect to control cells (
Cytochrome c Release and Apoptosis Induction
It has been previously shown that DCA facilitates translocation of proapoptotic mediators, like cytochrome c, thus stimulating apoptosis. In addition, DNA-damaging agents as well as HDAC inhibitors are known to drive cancer cells to commit suicide by apoptosis. Therefore, in order to highlight the nature of the cell death determined by 3a, cytochrome c release and classical apoptosis markers were monitored.
By monitoring cytochrome c release in PSN-1 cells after 12 and 24 h of treatment with IC50 of compound 3a, we observed that the compound caused a substantial time-dependent release of cytochrome c from the mitochondria to the cytosol (
In addition, Hoechst staining of PSN-1 cells after 72 h incubation with 3a clearly indicated the occurrence of nuclear condensation (pyknosis) and blebbing (
Pancreatic cancer is one of the most aggressive tumors with a dramatically poor prognosis and an incidence rate equaling mortality rate. Current evidence has reveal that pancreatic cancer cells possess an altered metabolic/energetic redox regulation primarily due to the oncogenic Kras mutation, which enhances glucose and glutamine metabolisms by upregulating the expression of a variety of key enzymes involved in this peculiar pathways. These peculiar characteristic make this cells particularly sensitive to cellular redox alterations, highlighting the therapeutic potential of targeting pancreatic cells by compounds able to shift the redox metabolism toward an oxidative one.
This application is a National Stage of International Application No. PCT/IL2016/051398, filed Dec. 29, 2016 which claims priority to U.S. Patent Application No. 62/272,741, filed Dec. 30, 2015, both of which are hereby incorporated by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2016/051398 | 12/29/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62272741 | Dec 2015 | US |
