BR112017019983B1 - ANTI-CANCER THERAPEUTIC AGENTS - Google Patents

Abstract

ANTI-CANCER THERAPEUTIC AGENTS. The present invention relates to anticancer therapeutic agents that exhibit preferential cytotoxicity to malignant cells expressing a cancer-specific isoform of proliferating cell nuclear antigen (caPCNA) as compared to cytotoxicity to comparable non-malignant cells, to pharmaceutical compositions comprising the agents and its use in cancer therapy.

Description

GOVERNMENT RIGHTS

[0001] This invention was made with government support in part under grant no. W81XWH-07-1-0707 awarded by the Congressionally Directed Medical Research Programs (CDMRP) Breast Cancer Research Program of the Department of Defense; grant no. ROl CA 121289, granted by the National Institutes of Health/National Cancer Institute; and a generous donation from the A.N.N.A Foundation.

[0002] The US government has certain rights in the invention.

FIELD OF TECHNIQUE

[0003] The invention described herein relates to anticancer therapeutic agents that exhibit preferential cytotoxicity to malignant cells expressing a cancer-specific isoform of proliferating cell nuclear antigen (caPCNA) compared to cytotoxicity to comparable non-malignant cells, pharmaceutical compositions that understand the agents and their use in cancer therapy.

BACKGROUND

[0004] Proliferating cell nuclear antigen (PCNA) plays an important role in the process of DNA replication, repair, chromosomal recombination, cell cycle checkpoint control and other cellular proliferative activities. In conjunction with an adapter protein, replication factor C (RFC), PCNA forms a movable claw that is the attachment point for delta and epsilon DNA polymerases. Different isoforms of proliferating cell nuclear antigen (PCNA) that exhibit acidic and basic isoelectric points (pI) have been demonstrated. Analysis of PCNA by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) of malignant and non-malignant breast tissues and cells (termed non-malignant PCNA or nmPCNA) revealed the presence of an acidic form of PCNA only in malignant cells (termed specific PCNA for cancer or csPCNA or caPCNA, herein, caPCNA). This difference in isoelectric point between these two forms of PCNA appears to result from a change in the ability of the malignant cells to post-translationally modify the PCNA polypeptide and is not due to a genetic change within the PCNA gene.

[0005] It has been shown that antibodies or peptides that bind only to the caPCNA isoform and not to the nmPCNA isoform interfere with intracellular protein-protein interactions, thus causing a reduction in the proliferative potential of cancer. See, for example, documents numbers WO 2006/1 16631 and WO 2007 098/415.

[0006] Also, PCNA is known to interact with other factors such as FEN-1, DNA ligase and DNA methyl transferase. Additionally, PCNA has also been shown to be an essential player in multiple DNA repair trajectories. Interactions with proteins such as the mismatch recognition protein, MSH2, and the nucleotide excision repair endonuclease, XPG, have implicated PCNA in processes distinct from DNA synthesis. Interactions with multiple partners often rely on mechanisms that allow PCNA to interact selectively in an energetically favored and ordered manner.

SUMMARY OF THE INVENTION

[0007] The present invention arises from the disclosure of small molecule therapeutic agents that exhibit preferential cytotoxicity to malignant cells expressing the cancer-specific isoform of proliferating cell nuclear antigen (caPCNA) compared to cytotoxicity to comparable non-malignant cells, see, for example, Publication number U.S. 2013/034523 (incorporated herein by reference in its entirety). Without being bound by theory and as described below, these small molecule therapeutic agents are believed to exert their action through specific binding modes that inhibit protein-protein interactions involving caPCNA. Once attached with caPCNA, these molecules either reduce or prevent caPCNA from interacting with its natural set of binding partners. This disruption in the binding partner interaction results in the inhibition of specific cellular functions that both caPCNA and its binding partner exhibit (e.g., DNA replication and DNA repair). See, for example, Figure 1.

[0008] Thus, small molecules bound to the protein-protein interaction domain of caPCNA or its binding partners (including, but not limited to, DNA polymerase δ, Xeroderma Pigmentosum G protein (XPG) or Flap-endonuclease (FEN- 1)) would, in turn, reduce/eliminate the ability of a cancer cell to properly replicate and/or repair its DNA; leading to the extermination of the cancer cell. Also, small molecule inhibitors of caPCNA-mediated function may have better therapeutic efficacy than the caPCNA-derived octapeptides described above, because of the intrinsic stability properties of these small molecules within the bloodstream and tissues, relative to the stability of peptides, and the problem of selectively targeting sufficient amounts of the peptide to cancer cells without having the bulk of the peptide being taken up by cells in the bloodstream or surrounding tissues.

[0009] In an illustrative embodiment of the invention, a method for reducing cellular proliferation of malignant cells expressing a cancer-specific isoform of proliferating cell nuclear antigen (caPCNA) in a patient in need thereof, comprises administering a therapeutically effective amount of a compound from the Formula (hereinafter referred to as AOH39), or a substituted derivative thereof, or a pharmaceutically acceptable salt thereof, is described herein.

[0010] In another embodiment, there is described the use of a compound as described above or a substituted derivative thereof, or a pharmaceutically acceptable salt thereof, to reduce cellular proliferation of malignant cells expressing a cancer-specific isoform of the nuclear antigen of proliferating cell (caPCNA).

[0011] In another embodiment, a pharmaceutical composition is described which comprises a compound as described above or a substituted derivative thereof, or a pharmaceutically acceptable salt thereof, and which additionally comprises one or more carriers, diluents or excipients or a combination thereof. same.

[0012] It is understood herein that the compounds described herein can be used alone or in combination with other compounds useful to treat cancer, including those compounds that can be therapeutically effective through the same or different modes of action.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 shows a proposed Scheme for the action of caPCNA.

[0014] Figure 2 shows the viability of breast cancer and Neuroblastoma cell lines incubated for 72 hours with the indicated concentrations of AOH39 or a phosphate-buffered saline (PBS)/drug solution without DMSO control.

[0015] Figure 3 shows the inhibition of SV40 origin-dependent in vitro DNA replication of MCF7 Cell by AOH39.

[0016] Figure 4 shows the correlation between AOH39-mediated cytotoxicity in MCF7 cells and in vitro DNA replication activity mediated by the DNA syntesome isolated from these cells.

[0017] Figure 5 shows the result of an Energy Minimization of the interaction of AOH39 with the PIP Box Domain of caPCNA. Figure 6 shows the effect of AOH39 on the proliferation of cancerous and non-cancerous cell lines.

[0018] Figure 7 shows the effect of AOH39 on the proliferation of pancreatic cancer cell lines.

[0019] Figure 8 shows the effect of AOH39 on cell viability in a variety of malignant and non-malignant breast cell lines.

[0020] Figure 9 shows the effect of AOH39 on cell viability in neuroblastoma cell lines and peripheral blood monocytes.

DETAILED DESCRIPTION

[0021] According to the invention, small molecule therapeutic agents exert their action through specific binding modes that inhibit protein-protein interactions involving caPCNA. Once attached with caPCNA, these molecules either reduce or prevent caPCNA from interacting with its natural set of binding partners. This disruption in the binding partner interaction results in the inhibition of specific cellular functions that both caPCNA and its binding partner exhibit (e.g., DNA replication and DNA repair).

[0022] Figure 1 shows a proposed Scheme for action of caPCNA in accordance with a theory behind the invention. Panel A represents how doxorubicin (DOX)-induced DNA damage is normally repaired in cancer cells. caPCNA would interact with DNA repair proteins to facilitate the repair of damaged DNA. Panel B represents the conditions when the small molecule (SM) therapeutic agent is present in a cell that has DOX-induced DNA damage. In this case, the small molecule (SM) therapeutic agent that binds to caPCNA or its binding partner competes with the full-length caPCNA protein that binds to its DNA repair protein partners, thereby preventing repair. of damaged DNA.

[0023] Embodiments of the invention are further described by the following enumerated clauses:

[0024] 1. A method for reducing cellular proliferation of malignant cells expressing a cancer-specific isoform of proliferating cell nuclear antigen (caPCNA) in a patient in need thereof, comprising administering a therapeutically effective amount of a compound of formula or a substituted derivative thereof or a pharmaceutically acceptable salt thereof.

[0025] 2. Use of a compound as described in clause 1 or a substituted derivative thereof, or a pharmaceutically acceptable salt thereof, to reduce cellular proliferation of malignant cells expressing a cancer-specific isoform of proliferating cell nuclear antigen (caPCNA).

[0026] 3. A pharmaceutical composition comprising a compound as described in clause 1 or a substituted derivative thereof, or a pharmaceutically acceptable salt thereof, and which additionally comprises one or more carriers, diluents or excipients or a combination thereof.

[0027] 4. The method, use or composition of any one of clauses 1 to 3 or a substituted derivative thereof or a pharmaceutically acceptable salt thereof.

[0028] 5. The method, use or composition of any of clauses 1 to 4 in which the cancer is breast cancer.

[0029] 6. The method, use or composition of any of clauses 1 to 4 wherein the cancer is pancreatic cancer.

[0030] 7. The method, use or composition of any of clauses 1 to 4 in which the cancer is neuroblastoma.

[0031] 8. The method or use of any of clauses 1 to 7 in which the use is to extend another chemotherapy method.

[0032] 9. A pharmaceutical composition comprising a compound as described in clause 3 and an additional chemotherapeutic agent.

[0033] As used herein, a substituted derivative of an illustrated compound includes one in which one or more hydrogens have been replaced, for example, by a halo group, hydroxy and derivatives thereof, amino and derivatives thereof, thio and derivatives thereof. of the same, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heteroalkyl, cycloheteroalkyl, heteroaryl, heteroarylalkyl, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl, heteroarylsulfinyl or heteroarylsulfonyl, each of which may have one or more substituents, as well as a derivative in which, for example, one or more halo, hydroxy or alkyl groups have been replaced by a hydrogen.

[0034] In each of the preceding and following embodiments, it should be understood that the formulas include and represent not only all pharmaceutically acceptable salts of the compounds, but also include any and all hydrates and/or solvates of the compound formulas. It is understood that certain functional groups, such as hydroxy, amino groups and the like, form complexes and/or coordination compounds with water and/or various solvents, in the various physical forms of the compounds. Accordingly, the above formulas should be understood as including and representing those various hydrates and/or solvates. In each of the foregoing and following embodiments, it should also be understood that the formulas include and represent each possible isomer, such as stereoisomers and geometric isomers, both individually and in any and all possible mixtures. In each of the foregoing and following embodiments, it should also be understood that the formulas include and represent any and all crystalline forms, partially crystalline forms, and non-crystalline and/or amorphous forms of the compounds.

[0035] Illustrative derivatives include, however, without limitation, both those compounds that can be synthetically prepared from the compounds described herein and those compounds that can be prepared in a manner similar to those described herein, but differing in selection of starting materials. For example, compounds including aromatic rings are described herein. It should be understood that derivatives of these compounds also include compounds that have, for example, substituents on these aromatic rings other than those explicitly presented in the definition above. Additionally, it should be understood that derivatives of these compounds also include compounds that have the same or different functional groups at different positions on the aromatic ring. Similarly, derivatives include variations of other substituents in the compounds described herein, such as an alkyl group or an amino group and the like.

[0036] It should be understood that such derivatives may include prodrugs of the compounds described herein, compounds described herein that include one or more protecting or protecting groups, including compounds that are used in the preparation of other compounds described in this document,

[0037] It should be understood that such derivatives can be mixed with other compounds that improve the stability and biological distribution of the compounds described in this document.

[0038] Illustrative derivatives include, however, without limitation, those compounds that share functional and, in some cases, structural similarity with those compounds described herein. For example, compounds that include a ring system are described herein. Illustrative substituted derivatives include, but are not limited to, corresponding ring-expanded compounds and corresponding ring systems that include one or more heteroatoms, such as by replacing a methylene group with an optionally substituted oxa, thia, or amino group, or by replacing an aromatic C-H group with an N.

[0039] The compounds described herein may contain one or more chiral centers or may otherwise have the ability to exist as multiple stereoisomers. It is to be understood that, in one embodiment, the invention described herein is not limited to any particular stereochemical requirement and that the compounds and compositions, methods, uses and medicaments comprising the same may be optically pure or may be any of a variety of stereoisomeric mixtures, including racemic mixtures and other mixtures of enantiomers, other mixtures of diastereomers and the like. It should also be understood that such mixtures of stereoisomers may include a single stereochemical configuration at one or more chiral centers, while including mixtures of stereochemical configurations at one or more other chiral centers.

[0040] Similarly, the compounds described herein may include geometric centers, such as cis, trans, E and Z double bonds. It should be understood that, in another embodiment, the invention described herein is not limited to any requirement of particular geometric isomer and that the compounds and compositions, methods uses and medicaments comprising the same may be pure or may be any of a variety of mixtures of geometric isomers. It should also be understood that such mixtures of geometric isomers may include a single configuration in one or more double bonds, while including mixtures of geometry in one or more other double bonds.

[0041] As used herein, the term "alkyl" includes a chain of carbon atoms that is optionally branched. As used herein, the terms "alkenyl" and "alkynyl" include a chain of carbon atoms, which is optionally branched, and includes at least one double bond or triple bond, respectively. It should be understood that alkynyl may also include one or more double bonds. It should be further understood that, in certain embodiments, alkyl is advantageously of limited length, including C1-C24, C1-C12, C1-Cs, C1-C6 and C1-C4. It should be further understood that, in certain embodiments, alkenyl and/or alkynyl may each advantageously be of limited length, including C2-C24, C2-C12, C2-C8, C2-C6 and C2-C4. It is understood herein that shorter alkyl, alkenyl and/or alkynyl groups may add less lipophilicity to the compound and, consequently, will have different pharmacokinetic behavior. Illustrative alkyl groups are, however, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl, hexyl, heptyl octyl and the like.

[0042] As used herein, the term "cycloalkyl" includes a chain of carbon atoms, which is optionally branched, wherein at least a portion of the chain is cyclic. It should be understood that cycloalkylalkyl is a subset of cycloalkyl. It should be understood that cycloalkyl can be polycyclic. Illustrative cycloalkyls include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, 2-methylcyclopropyl, cyclopentylet-2-yl, adamantyl and the like. As used herein, the term "cycloalkenyl" includes a chain of carbon atoms, which is optionally branched, and includes at least one double bond, wherein at least a portion of the chain is cyclic. It should be understood that the one or more double bonds may be in the cyclic portion of the cycloalkenyl and/or in the non-cyclic portion of the cycloalkenyl. It should be understood that cycloalkenylalkyl and cycloalkylalkenyl are each subsets of cycloalkenyl. It should be understood that cycloalkyl can be polycyclic. Illustrative cycloalkenyls include, but are not limited to, cyclopentenyl, cyclohexylethen-2-yl, cycloheptenylpropenyl and the like. It should further be understood that the chain forming cycloalkyl and/or cycloalkenyl is advantageously of limited length, including C3-C24, C3-C12, C3-C8, C3-6 and C5-C6. It is understood herein that shorter alkyl and/or alkenyl chains that form cycloalkyl and/or cycloalkenyl, respectively, may add less lipophilicity to the compound and will consequently have different pharmacokinetic behavior.

[0043] As used herein, the term "heteroalkyl" includes a chain of atoms that includes carbon and at least one heteroatom and is optionally branched. Illustrative heteroatoms include nitrogen, oxygen, and sulfur. In certain variations, illustrative heteroatoms also include phosphorus and selenium. As used herein, the term "cycloheteroalkyl" including heterocyclyl and heterocycle includes a chain of atoms that includes carbon and at least one heteroatom, such as heteroalkyl, and is optionally branched, wherein at least a portion of the chain is cyclic. . Illustrative heteroatoms include nitrogen, oxygen, and sulfur. In certain variations, illustrative heteroatoms also include phosphorus and selenium. Illustrative cycloheteroalkyls include, but are not limited to, tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl and the like.

[0044] As used herein, the term "aryl" includes monocyclic and polycyclic aromatic carbocyclic groups, each of which may be optionally substituted. Illustrative aromatic carbocyclic groups described herein include, but are not limited to, phenyl, naphthyl and the like. As used herein, the term "heteroaryl" includes aromatic heterocyclic groups, each of which may be optionally substituted. Illustrative aromatic heterocyclic groups include, but are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzthiazolyl, benzisoxazolyl, benzisothiazolyl and the like.

[0045] As used herein, the term "amino" includes the group N34, alkylamino and dialkylamino, where the two alkyl groups in dialkylamino can be the same or different, that is, alkylalkylamino. Illustratively, amino includes methylamino, ethylamino, dimethylamino, methylethylamino and the like. Additionally, it should be understood that when amino modifies or is modified by another term, such as aminoalkyl or acylamino, the above variations of the term amino are included therein. Illustratively, aminoalkyl includes H2N-alkyl, methylaminoalkyl, ethylaminoalkyl, dimethylaminoalkyl, methylethylaminoalkyl and the like. Illustratively, acylamino includes acylmethylamino, acylethylamino and the like.

[0046] As used herein, the term "amino and derivatives thereof" includes amino as described herein and alkylamino, alkenylamino, alkynylamino, heteroalkylamino, heteroalkenylamino, heteroalkynylamino, cycloalkylamino, cycloalkenylamino, cycloheteroalkylamino, cycloheteroalkenylamino, arylamino, arylalkylamino, arylalkynylamino, arylalkynylamino, heteroarylamino, heteroarylalkylamino, heteroarylalkynylamino, heteroarylalkynylamino, acylamino and the like, each of which is optionally substituted. The term "amino derivative" also includes urea, carbamate and the like.

[0047] As used herein, the term "hydroxy and derivatives thereof" includes OH and alkyloxy, alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy, heteroalkynyloxy, cycloalkyloxy, cycloalkenyloxy, cycloheteroalkyloxy, cycloheteroalkenyloxy, aryloxy, arylalkyloxy, arylalkenyloxy , aryl alkynyloxy, heteroaryloxy, heteroarylalkyloxy, heteroarylalkynyloxy, heteroarylalkynyloxy, acyloxy and the like, each of which is optionally substituted. The term "hydroxy derivative" also includes carbamate and the like.

[0048] As used herein, the term "thio and derivatives thereof" includes SH, and alkylthio, alkenylthio, alkynylthio, heteroalkylthio, heteroalkenylthio, heteroalkynylthio, cycloalkylthio, cycloalkenylthio, cycloheteroalkylthio, cycloheteroalkenylthio, arylthio, arylalkylthio, arylalkynylthio, aryl alkynylthio, heteroarylthio, heteroarylalkylthio, heteroarylalkynylthio, heteroarylalkynylthio, acylthio and the like, each of which is optionally substituted. The term "thio derivative" also includes thiocarbamate and the like.

[0049] As used herein, the term "acyl" includes formyl and alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, heteroalkylcarbonyl, heteroalkenylcarbonyl, heteroalkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl, cycloheteroalkylcarbonyl, cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl, arylalkynylcarbonyl, , heteroarylcarbonyl , heteroarylalkylcarbonyl, heteroarylalkynylcarbonyl, heteroarylalkynylcarbonyl, acylcarbonyl and the like, each of which is optionally substituted.

[0050] As used herein, the term "carbonyl and derivatives thereof" includes the group C(O), C(S), C(NH) and substituted amino derivatives thereof.

[0051] As used herein, the term "carboxylate and derivatives thereof" includes the group CO2H and salts thereof and esters and amides thereof and CN.

[0052] The term "optionally substituted" as used herein includes the replacement of hydrogen atoms with other functional groups in the radical that is optionally substituted. Such other functional groups illustratively include, but are not limited to, amino, hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroaryl-heteroalkyl, nitro, sulfonic acids and derivatives thereof. , carboxylic acids and derivatives thereof and the like. Illustratively, any of amino, hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl and/or sulfonic acid is optionally substituted.

[0053] As used herein, the terms "optionally substituted aryl" and "optionally substituted heteroaryl" include the replacement of hydrogen atoms with other functional groups in the aryl or heteroaryl that is optionally substituted. Such other functional groups illustratively include, but are not limited to, amino, hydroxy, halo, thio, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids and derivatives thereof. , carboxylic acids and derivatives thereof and the like. Illustratively, any of amino, hydroxy, thio, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl and/or sulfonic acid is optionally substituted.

[0054] The term "prodrug" as used herein generally refers to any compound that, when administered to a biological system, generates a biologically active compound as a result of one or more spontaneous chemical reactions, chemical reactions catalyzed by enzyme and/or metabolic chemical reactions or a combination thereof. In vivo, the prodrug is typically acted upon by an enzyme (such as esterases, amidases, phosphatases and the like), simple biological chemistry or other in vivo process to release or regenerate the more pharmacologically active drug. This activation can occur through the action of an endogenous host enzyme or a non-endogenous enzyme that is administered to the host before, after or during administration of the prodrug. Further details of the use of prodrug are described in U.S. Patent No. 5,627,165; and Pathalk et al., Enzymic group protecting techniques in organic synthesis, Stereosel. Biocatalyst. 775 to 797 (2000) (incorporated into this document by reference in its entirety). It is understood that the prodrug is advantageously converted into the original drug once the objective, such as targeted delivery, safety, stability and the like, is achieved, followed by the subsequent rapid elimination of the released remnants of the group forming the prodrug.

[0055] Prodrugs can be prepared from the compounds described in this document by linking the groups that ultimately cleave in vivo to one or more functional groups present in the compound, such as -OH-, -SH, -CO2H, -NR2. Illustrative prodrugs include, but are not limited to, carboxylate esters in which the group is alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, acyloxyalkyl, alkoxycarbonyloxyalkyl as well as hydroxyl, thiol and amine esters in which the attached group is a acyl, an alkoxycarbonyl, aminocarbonyl, phosphate or sulfate. It is understood that prodrugs themselves may not possess significant biological activity, but instead undergo one or more spontaneous chemical reactions, enzyme-catalyzed chemical reactions, and/or metabolic chemical reactions, or a combination thereof following administration. in vivo to produce the compound described herein that is biologically active or is a precursor of the biologically active compound. However, it is understood that in some cases the prodrug is biologically active. It is also understood that prodrugs can often serve to improve the efficacy or safety of the drug through improved oral bioavailability, improved pharmacodynamic half-life, and the like. Prodrugs also refer to derivatives of the compounds described herein that include groups that simply mask undesirable properties of the drug or enhance drug delivery. For example, one or more compounds described herein that may exhibit an undesirable property that is advantageously blocked or minimized may become pharmacological, pharmaceutical or pharmacokinetic barriers in clinical drug application, such as poor oral drug absorption, lack of drug specificity. site, chemical instability, toxicity and poor patient acceptance (bad taste, odor, pain at the injection site and similar) and others. It is understood herein that a prodrug, or other strategy that uses reversible derivatives, may be useful in optimizing the clinical application of a drug.

[0056] The term "patient" includes both human and non-human patients, such as mammals, including pets and other captive animals, such as zoo animals.

[0057] The term "therapeutically effective amount", as used herein, refers to that amount of the active compound or pharmaceutical agent that produces the biological or medicinal response in a tissue system, animal or human being sought for a researcher, veterinarian, doctor, or other clinician, which includes relieving the symptoms of the disease or disorder being treated. In one aspect, the therapeutically effective amount is that which can treat or alleviate the disease or symptoms of the disease at a reasonable risk/benefit ratio applicable to any medical treatment. However, it should be understood that the total daily use of the compounds and compositions described herein can be decided by the responsible physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; activity of the specific compound used; the specific composition used; the age, body weight, general health, gender and diet of the patient: the time of administration, route of administration and the excretion rate of the specific compound used; the duration of treatment; the drugs used in combination or coincidentally with the specific compound employed and similar factors well known to the researcher, veterinarian, physician or other clinician of ordinary skill.

[0058] Additionally, in those embodiments described herein that deal with combination therapy comprising the administration of a chemotherapeutic agent and a small molecule therapeutic agent of the present invention, "therapeutically effective amount" refers to this amount of the combination of agents taken together so that the combined effect produces the desired biological or medicinal response. For example, the therapeutically effective amount of doxorubicin and a small molecule therapeutic agent of the present invention would be those amounts that, when taken together or sequentially, have a combined effect that is therapeutically effective. Furthermore, it is understood that, in some embodiments, of such methods that include coadministration, this coadministration amount of the chemotherapeutic agent or small molecule therapeutic agent of the present invention, when taken individually, may be therapeutically effective or not.

[0059] It is also appreciated that the therapeutically effective amount, whether referring to monotherapy or combination therapy, is advantageously selected with reference to any toxicity, or other undesirable side effect, that may occur during administration of one or more of the compounds. described in this document. Furthermore, it is understood that the cotherapies described herein may permit the administration of lower doses of compounds that show such toxicity, or other undesirable side effect, wherein such lower doses are below the toxicity thresholds or lower in the therapeutic window. than would otherwise be administered in the absence of cotherapy.

[0060] As used herein, the term "composition" generally refers to any product that comprises the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combinations of the specified ingredients in the specified amounts. It should be understood that the compositions described herein can be prepared from the isolated compounds described herein or from salts, solutions, hydrates, solvates and other forms of the compounds described herein. It should also be understood that the compositions can be prepared from various amorphous, non-amorphous, partially crystalline, crystalline and/or other morphological forms of the compounds described herein. It should also be understood that compositions can be prepared from various hydrates and/or solvates of the compounds described herein. Consequently, such pharmaceutical compositions citing compounds described herein should be understood as including each, or any combination, of the various morphological forms and/or solvate or hydrate forms of the compounds described herein. Illustratively, the compositions may include one or more carriers, diluents and/or excipients. The compounds described herein, or compositions containing the same, can be formulated in a therapeutically effective amount in any conventional dosage forms appropriate for the methods described herein. The compounds described herein, or compositions containing the same, including such formulations, can be administered by a wide variety of routes conventional to the methods described herein and in a wide variety of dosage formats using known procedures (see , generally, Remington: The Science and Practice of Pharmacy, (21st edition, 2005)).

[0061] The term "administer" as used herein includes all means of introducing the compounds and compositions described herein to the patient, including, but not limited to, oral (po), intravenous (iv), intramuscular (im ), subcutaneous (sc), transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal and the like. The compounds and compositions described herein can be administered in unit dosage forms and/or formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.

[0062] It should be understood that, in the methods described herein, the individual components of a co-administration or combination can be administered by any suitable means, contemporaneously, simultaneously, sequentially, separately or in a single pharmaceutical formulation. When co-administered compounds or compositions are administered in separate dosage forms, the number of dosages administered per day for each compound may be the same or different. The compounds or compositions may be administered via the same or different routes of administration. The compounds or compositions can be administered according to simultaneous or alternating regimens, at the same time or at different times during the course of therapy, concomitantly in divided or single forms.

[0063] Illustrative oral administration routes include tablets, capsules, elixirs, syrups and the like.

[0064] Illustrative routes of parenteral administration include intravenous, intra-arterial, intraperitoneal, epidural, intraurethral, intrasternal, intramuscular and subcutaneous, as well as any other route of parenteral administration recognized in the art.

[0065] Illustrative parenteral administration means include needle injectors (including microneedle), needleless injectors and infusion techniques, as well as any other parenteral administration means recognized in the art. Parenteral formulations are typically aqueous solutions that may contain excipients such as salts, carbohydrates, and buffering agents (preferably at a pH in the range of about 3 to about 9), but for some applications they may be most suitably formulated as a sterile non-aqueous solution or as a dry form to be used in conjunction with a suitable vehicle such as sterile pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for example, by lyophilization, can be readily carried out using standard pharmaceutical techniques well known to those skilled in the art. Parenteral administration of a compound is illustratively carried out in the form of saline solutions or with the compound incorporated in liposomes. In cases where the compound itself is not soluble enough to be dissolved, a solubilizer such as ethanol, DMSO, Cremophor, etc., can be applied.

[0066] The dosage of each compound of the claimed combinations depends on several factors, including: the method of administration, the condition to be treated, the severity of the condition, whether the condition should be treated or prevented and the age, weight and health of the patient. person to be treated. Additionally, pharmacogenomic information (the effect of genotype on the pharmacokinetic, pharmacodynamic, or efficacy profile of a therapeutic product) about a particular patient may affect the dosage used.

[0067] Depending on the disease, as described herein, and the route of administration, a wide range of permissible dosages is contemplated herein, including doses that fall in the range of about 1 μg/kg to about 1 g /kg. Dosages may be single or divided and may be administered according to a wide variety of protocols, including q.d., b.i.d., t.i.d. or even every two days, once a week, once a month, once a quarter and the like. In each of these cases, the total daily, weekly, monthly or quarterly dose is understood to correspond to the therapeutically effective amounts described herein. When given locally, such as by injection at or near the site of disease, illustrative doses include those in the range of about 1 μg/kg to about 10 mg/kg or about 0.01 mg/kg to about 10 mg /kg or about 0.01 mg/kg to about 1 mg/kg or about 0.1 mg/kg to about 10 mg/kg or about 0.1 mg/kg to about 1 mg/kg . When given locally, such as by injection at or near the site of disease, or locally into tissues surrounding the site of disease, illustrative doses include those in the range of about 0.01 mg/kg to about 10 mg/kg or about 0.01 mg/kg to about 1 mg/kg or about 0.1 mg/kg to about 10 mg/kg or about 0.1 mg/kg to about 1 mg/kg. When given systematically, such as parenterally, illustrative doses include those in the range of about 0.01 mg/kg to about 100 mg/kg or about 0.01 mg/kg to about 10 mg/kg or about 0.1 mg/kg to about 100 mg/kg or about 0.1 mg/kg to about 10 mg/kg. When given systematically, such as orally, illustrative doses include those in the range of about 0.1 mg/kg to about 1,000 mg/kg or about 0.1 mg/kg to about 100 mg/kg or about 0.1 mg/kg to about 10 mg/kg or about 1 mg/kg to about 1,000 mg/kg or about 1 mg/kg to about 100 mg/kg or about 1 mg/kg to about of 10 mg/kg.

[0068] In another illustrative embodiment, such as when treating a disease described herein, the compound is administered parenterally and locally q.d. at a dose of about 0.01 mg/kg or about 0.05 mg/kg or about 0.1 mg/kg or about 0.5 mg/kg or about 1 mg/kg or about 5 mg/kg of the patient's body weight.

[0069] In another illustrative embodiment, such as when treating a systemic condition, the compound is administered parenterally and systemically q.d. at a dose of about 0.1 mg/kg or about 0.5 mg/kg or about 1 mg/kg or about 5 mg/kg or about 10 mg/kg or about 50 mg/kg of patient's body weight.

[0070] In addition to the foregoing illustrative dosages and dosage protocols, it should be understood that an effective amount of any one or a mixture of the compounds described herein can be readily determined by the diagnostician or physician responsible using known techniques and/or observing the results obtained under similar circumstances. In determining the effective amount or dose, several factors are considered by the diagnostician or treating physician, including, but not limited to, the species of mammal, including human, its size, age and general health, the specific disease or disorder involved, the degree or involvement or severity of the disease or disorder, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication and other relevant circumstances .

[0071] In producing pharmaceutical compositions of the compounds described herein, a therapeutically effective amount of one or more compounds in any of the various forms described herein may be mixed with one or more excipients, diluted by one or more excipients or enclosed within such a carrier which may be in the form of a capsule, sachet, paper or container. Excipients can serve as a diluent and can be solid, semisolid, or liquid materials that act as a vehicle, carrier, or medium for the active ingredient. Thus, formulation compositions may be in the form of tablets, pills, powders, tablets, sachets, wafers, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, gelatin capsules. soft and hard, suppositories, sterile injectable solutions and sterile packaged powders. The compositions may contain from about 0.1% to about 99.9% active ingredients, depending on the dose and dosage form selected.

[0072] Parenteral Compositions. The pharmaceutical composition may also be administered parenterally by injection, infusion or implantation (intravenous, intramuscular, subcutaneous or the like) in dosage forms, formulations or by means of suitable delivery devices or implants containing conventional non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

[0073] Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules) or in vials containing several doses and to which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device or an implantation delivery device, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. . In addition to the active drug (or drugs), the composition may include parenterally acceptable carriers and/or excipients. The active drug (or drugs) can be incorporated into microspheres, microcapsules, nanoparticles, liposomes or similar for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting and/or dispersant agents.

[0074] As indicated above, the pharmaceutical compositions described herein may be in form suitable for sterile injection. To prepare such a composition, the suitable active drug (or drugs) is dissolved or suspended in a parenterally acceptable liquid vehicle. Among the acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by the addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butaneediol, Ringer's solution and chloride solution. isotonic sodium. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only moderately or slightly soluble in water, a solubilizing agent or dissolution enhancer may be added, or the solvent may include 10 to 60% w/w propylene glycol or the like.

[0075] The following examples illustrate specific embodiments of the invention; however, the following illustrative examples should not be construed in any way to limit the invention.

EXAMPLES

[0076] The compounds exemplified herein, as denoted by "AOH" numbers, were obtained or derived from compounds contained in chemical screening libraries used by AMRI (formerly Albany Medical Research, Inc.), Albany, New York to identify AOH 39.

[0077] Other small molecule inhibitors of caPCNA-mediated function of the invention can be prepared by conventional synthetic routes.

EFFECT OF AOH39 ON CELL VIABILITY:

[0078] The effect of AOH39 on cell viability was studied by examining the viability of breast cancer and Neuroblastoma cell lines incubated for 72 hours with the indicated concentrations of AOH39 or a phosphate-buffered saline (PBS)/drug-free solution. DMSO control. See Figure 2; HCC1937- is a BRCA1-defective breast cancer cell line; MCF7 - is an ER+ Breast Cancer cell line; MCF10A is a non-malignant but immortalized breast cell line; the SK-N-AS, SK-N-BE2c and SK-N-DZ lines are neuroblastoma cell lines; the X axis lists the various cell lines tested. The Y axis shows proliferation as a percentage of proliferation in cultures grown in the presence of PBS/DMSO without added AOH39. Proliferation was monitored using the CellTiter Glo Assay (Promega Inc., Madison, WI) (https://www.promega.com/products/cell- health-and-metabolism/cell-viability- assays/celltiter-glo -luminescent-cell-viability-assay/). The histogram was generated using Prism 6.

INHIBITION OF IN VITRO DNA REPLICATION OF SV40 BY AOH39

[0079] In vitro DNA replication activity was determined using the standard T antigen and origin-dependent in vitro DNA replication reaction (L. Malkas et al., Biochemistry 29: 6,362 to 6,374 (1990), a percent inhibition was determined relative to a reaction containing Tris-HCl/DMSO in place of AOH39). The reaction was initiated by adding the T antigen to the reaction mixture. The results are shown in Figure 3, with AOH39 inhibiting replication by approximately 50% at concentrations of 5 μM and by more than 75% at 10 μM.

CORRELATION BETWEEN MCF7 CELL CYTOTOXICITY MEDIATED BY AOH39 AND INHIBITION OF IN VITRO DNA REPLICATION MEDIATED BY MCF7 CELL EXTRACT.

[0080] There is an accurate correlation between the ability of AOH39 to inhibit the SV40 origin-dependent in vitro DNA replication process and MCF7 cell viability, Figure 4. Cell viability was determined colorimetrically using the assay MTT after a 72-hour incubation with 10 μM AOH39. In vitro DNA replication activity was determined using the SV40 origin- and T antigen-dependent in vitro DNA replication reaction (L. Malkas et al., Biochemistry, 29, 6362 to 6374 (1990)), and percent inhibition was determined relative to a reaction containing Tris-HC-1/DMSO in place of AOH39 for the activity assay and PBS/DMSO for the viability assay. AOH39 significantly inhibited DNA replication relative to a control replication reaction containing PBS/DMSO in place of the compound and the inhibition precisely correlated with a significant decrease in MCF7 cell viability.

PREDICTED INTERACTION OF AOH 39 WITH THE PIP BOX INTERACTION DOMAIN OF cPCNA.

[0081] Figure 5 shows the result of an Energy Minimization of the interaction of AOH39 with the PIP Box Domain of caPCNA. The binding modes of AOH39 were initially constructed using All-Around Docking (AAD) (Nam S, Wen W, Schroeder A, Herrmann A, Yu H, Cheng X, Merz KH, Eisenbrand G, Li H, Yuan YC , Jove R, "Dual inhibition of Janus and Src family kinases by novel indirubin derivative blocks constitutively-activated Stat3 signaling associated with apoptosis of human pancreatic cancer cells", Mol. Oncol Jun 2013;7(3):369 - 378. doi: 10.1016/j.molonc.2012.10.013 PMID: 23206899) and refined by Metadynamics (Laio, A.; Parrinello, M. (2002). "Escaping free-energy minimum". Proceedings of the National Academy of Sciences of the United States of America 99 (20): 12,562 to 12,566.) to a 50 ns simulation. The binding affinity of AOH39 to caPCNA is predicted to be -4.62 kcal/mol using Schrodinger Glide Docking software at XP accuracy.

ANALYSIS OF AOH 39 GROWTH INHIBITION IN CANCEROUS CELLS.

[0082] The effect of AOH39 on the proliferation of cancer and non-cancer cell lines was studied, with the results summarized in Figure 6. Viability of Breast Cancer and Neuroblastoma cell lines incubated with AOH39 or a drug without DMSO control . Proliferation was monitored using the CellTiter Glo Assay (Promega Inc., Madison, WI) (https://www.promega.com/products/cell-health-and-metabolism/cell-viability-assays/celltiter-glo -luminescent-cell-viability-assay/). The geometric axis X is the log value of the AOH39 concentration in uM, where 0 corresponds to 1 uM and 1 corresponds to 10 uM. Graphs were generated using Prism 6. MDA-MB231 and MDA-MB468 are ER breast cancer cell lines; the SK-N-AS, SK-N-BE2c, SK-N-DZ lines are neuroblastoma cell lines; LAN5 cells - neuroblastoma bone metastasis; HCC1937 is a BRCA1-defective breast cancer cell line; MCF7 is an ER+ breast cancer cell line; MCF10A is a non-malignant but immortalized breast cell line; PBMC - peripheral blood monocytes normal proliferating cells). The X axis is plotted as the log of AOH39 concentration, where 1.0 corresponds to 10 uM. The Y axis shows cell proliferation as a percentage of proliferation in PBS/DMSO without added AOH39. EFFECT OF AOH39 ON THE VIABILITY OF PANCREATIC CANCER CELL LINES The effect of AOH39 on the proliferation of pancreatic cancer cell lines is summarized in Figure 7. The percent viability of Panc 1 and Panc 2 cell lines incubated with AOH39 at the concentrations indicated in The ratio of PBS/DMSO (without drug) is plotted on the Y axis, and the X axis shows the concentration of AOH39 in uM. Proliferation was monitored using the CellTiter Glo Assay (Promega Inc., Madison, WI). (https://www.promega.com/products/cell-health-and-metabolism/cell-viability-assays/celltiter-glo-luminescent-cell-viability-assay/). The graph was generated using Prism 6.

EFFECT OF AOH39 ON CELL VIABILITY IN A VARIETY OF MALIGNANT AND NON-MALIGNANT CELL LINES.

[0083] The effect of AOH39 on cell viability in a variety of malignant and non-malignant cell lines is shown in Table 1 and Figures 8 and 9. TABLE 1

[0084] The IC50's listed in Table 1 and Figures 8 and 9 were determined by monitoring cell proliferation in the absence or presence of increasing concentrations of AOH39 using the Cell-Titer Glo Assay (Promega Inc., Madison, WI) which monitors the energy status of the entire culture and the viability of the cell population, (https://www.promega.com/products/cell-health- and-metabolism/cell-viability- assays/celltiter_glo-luminescent-cell- viability-assay/). As the population doubles, the quantum yield of the assay also doubles. HCC1937 is a BRCA1-defective breast cancer cell line; MCF7 is an ER+ breast cancer cell line; MDA-MB231 and MDA-MB468 are ER aggressive breast cancer cell lines. SK-N-AS, SK-N-BE2c, SK-N-DZ are neuroblastoma cell lines, and LAN5 cells are a cell line derived from a bone metastasis of neuroblastoma; MCF10A is a non-malignant but immortalized breast cell line. PBMC- peripheral blood monocytes (normal proliferating cells), and MCF

Claims (2)

1. Pharmaceutical composition CHARACTERIZED by the fact that it comprises the compound of the formula or a pharmaceutically acceptable salt thereof, and which additionally comprises one or more carriers, diluents or excipients. 2. Pharmaceutical composition, according to claim 1, CHARACTERIZED by the fact that it further comprises a chemotherapeutic agent.