Patent Application
20200276181
This disclosure relates to thienoisoquinoline compounds and their derivatives, and more particularly to methods for targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM using thienoisoquinoline compounds.
Cancer is now the leading cause of death in Canada. According to statistics from the Canadian Cancer Society, 1 out of 4 Canadians will die from the disease, and 2 out of 5 Canadians will develop cancers over their lifetime. Since the incidence of cancer is higher in people aged 50 and older, these numbers are expected to soar as the number of senior citizens in Canada increases. While the mortality rates from some cancers have decreased due to success in the clinic or through prevention (e.g. breast and prostate cancers), many aggressive, hard-to-treat cancers persist (e.g. lung, pancreatic and brain cancers). One of the main methods used to treat cancers is via the use of chemotherapies, which often has severe side-effects for patients, because most chemotherapeutic drugs also target healthy cells. Further, patients often develop resistance to drugs through various mechanisms. Combinatorial therapies are now being used to reduce side-effects and resistance, where two or more drugs are administered simultaneously or concurrently at lower doses. In addition, personalized medicine, where genetic profiling of individual tumours is used to tailor treatments more specifically to each patient, is another, more recent, treatment option. Thus, it is important to expand the repertoire of drugs to increase the number of cancers that can be treated effectively. However, current methods that are being used to search for novel anti-cancer compounds are often not successful[1]. They may be too restrictive, because they search for compounds with a specific molecular target that may not be optimal, or their drug-like qualities and ease of synthesis are not considered. For example, poor quality compounds may have solubility issues, they may aggregate and require high concentrations to be effective in vivo[2-6].
A subset of successful anti-cancer drugs used to treat a wide spectrum of cancers target mitosis, which is important for cell division[12,13]. One of the hallmarks shared by cancer cells is that they divide rapidly in an uncontrolled manner. The mitotic spindle is a structure that forms to align and segregate chromosomes, and to ensure that each daughter cell inherits the appropriate genetic content during division[14]. If the mitotic spindle fails to attach to the chromosomes properly, then the spindle assembly checkpoint (SAC) is not satisfied and the cell will arrest and undergo apoptosis[14-17]. Alternatively, chromosomes can be mis-segregated, leading to aneuploidy and mitotic catastrophe in subsequent divisions[18]. As healthy somatic cells enter mitosis, two centrosomes move apart and nucleate microtubules to form a bipolar spindle that then captures the chromosomes[19]. Many metastatic cancer cells have aberrant centrosomes, which are structurally or functionally defective[20-22]. Since centrosomes are the main sites for nucleating microtubules, cells with aberrant centrosomes often have defective mitotic spindles, such as multipolar spindles, with defective chromosome attachment[23]. Therefore, cancer cells rely on mechanisms to cluster fragmented or amplified centrosomes to form two poles[16,17,20-22]. The mechanisms that cancer cells use to cluster aberrant centrosomes are not well-understood, but are attractive to target via chemotherapies because their requirement is selective to cancer cells. In support of this, several publications have described searching for compounds that specifically target centrosome clustering[24-29] However, the compounds described in these papers do not achieve high efficacy and are not ideal for clinical phase trials.
Thienoisoquinoline-phenyl sulfonamide compounds have been described in U.S. Pat. No. 7,696,221 (herein incorporated by reference in its entirety) for use as ER-NF kappa B inhibitors. Synthesis of thienoisoquinolines by a 5-step linear synthesis employing a palladium-catalyzed decarboxylative cross-coupling and functionalization sequence has been reported by Chen et al.[11], herein incorporate by reference in its entirety.
In accordance to a first aspect disclosed herein, there is provided a method for targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM expressed in a cancer cell and selectively inhibiting growth therein, comprising exposing said cancer cell to a compound of Formula I:
In accordance to another aspect disclosed herein, there is provided a method for targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM expressed in a cancer cell and selectively inhibiting growth therein, comprising exposing said cancer cell to a compound of Formula
According to another aspect, there is provided herein a method for targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM expressed in a cancer cell and selectively inhibiting growth therein, comprising exposing said cancer cell to a combination of a compound of Formula I and an anti-cancer agent and/or an anti-mitotic agent.
Another aspect herein disclosed relates to a method for targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM expressed in a cancer cell and inhibiting growth therein, comprising exposing said cancer cell to a synergistic combination of a compound of Formula I and an anti-cancer agent and/or an anti-mitotic agent, wherein said combination more than additively inhibits growth of said cancer cell.
In yet another aspect there is provided a use of a compound of Formula I for targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM expressed in a cancer cell and selectively inhibiting growth therein.
According to another aspect, there is provided a use of a combination of a compound of Formula I and an anti-cancer agent and/or an anti-mitotic agent for targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM expressed in a cancer cell and selectively inhibiting growth therein
According to an aspect, there is provided herein a method for selectively inhibiting growth in a cancer cell, comprising exposing the cancer cell to a compound of Formula I:
According to an aspect, there is provided herein a method for selectively inhibiting growth in a cancer cell, comprising exposing the cancer cell to a compound of Formula I:
According to another aspect, there is provided herein a method for disrupting centrosome integrity, preventing and/or reducing centrosome clustering, declustering centrosomes, regulating centrosome clustering and/or altering microtubule dynamics including microtubule depolymerization in a cancer cell, comprising exposing the cancer cell to a compound of Formula I.
According to yet another aspect, there is provided herein a method for selectively inhibiting growth in a cancer cell, comprising exposing the cancer cell to a combination of a compound of Formula I and an anti-cancer agent and/or an anti-mitotic agent.
According to another aspect, there is provided herein a method for inhibiting growth in a cancer cell, comprising exposing the cancer cell to a synergistic combination of a compound of Formula I and an anti-cancer agent and/or an anti-mitotic agent, wherein the combination more than additively inhibits growth of the cancer cell.
According to another aspect, there is provided herein a method for increasing selectivity of an anti-cancer agent and/or an anti-mitotic agent to a cancer cell, comprising exposing the cancer cell with a compound of Formula I and the anti-cancer agent and/or the anti-mitotic agent.
According to another aspect, there is provided herein a method of treating a cancer in a subject, comprising administering to the subject an effective amount of a compound of Formula I.
According to another aspect, there is provided herein a method of treating a cancer in a subject, comprising administering to the subject an effective amount of a combination of a compound of Formula I and an anti-cancer agent and/or an anti-mitotic agent.
According to a further aspect, there is provided herein a use of a compound of Formula I for selectively inhibiting growth in a cancer cell.
According to another aspect, there is provided herein a use of a compound of Formula I for disrupting centrosome integrity, preventing and/or reducing centrosome clustering, declustering centrosomes, regulating centrosome clustering and/or altering microtubule dynamics including microtubule depolymerization in a cancer cell.
According to yet another aspect, there is provided herein a use of a combination of a compound of Formula I and an anti-cancer agent and/or an anti-mitotic agent for selectively inhibiting growth in a cancer cell.
According to another aspect, there is provided herein a use of a compound of Formula I for the treatment of cancer in a subject.
According to another aspect, there is provided herein a use of a combination of a Formula I and an anti-cancer agent and/or an anti-mitotic agent for the treatment of cancer in a subject.
According to another aspect, there is provided herein a use of a compound of Formula I in combination with an anti-cancer agent and/or an anti-mitotic agent for increasing selectivity of the anti-cancer agent and/or the anti-mitotic agent to a cancer cell.
According to a further aspect, there is provided herein a compound of Formula I:
According to a further aspect, there is provided herein a compound of Formula IA:
According to a further aspect, there is provided herein a compound chosen from:
According to a further aspect, there is provided herein a compound of Formula IB:
According to a further aspect, there is provided herein a compound of Formula IC:
According to a further aspect, there is provided herein a compound of Formula ID:
Further features and advantages of the disclosure will become more readily apparent from the following description of specific embodiments as illustrated by way of examples in the appended figures wherein:
2L and 2M show that C75 selectively causes mitotic arrest in cancer cells and targets centrosomes.
Accordingly, a class of anti-cancer compounds with potential for clinical use has been identified. By synthesizing compounds with a quinoline scaffold that already has been proven to have successful drug-like properties[7-11], it has been made possible to overcome limitations related to quality. These compounds appear to have a unique mechanism of action in comparison to known anti-cancer drugs, by targeting a process that occurs uniquely in cancer cells, making the compounds selective for cancer cells. In addition, these compounds enhance the selectivity of other anti-cancer drugs, making them suitable for use in combinatorial therapies.
As used herein, the term “ch-TOG” (colonic and hepatic tumor overexpressed gene protein), or “CKAP5” (cytoskeleton associated protein 5) refers to a microtubule polymerase that plays a role in bipolar mitotic spindle assembly and that is overexpressed in certain cancer cells such as for example colorectal adenocarcinoma cells. ch-TOG includes, without limitation, all known ch-TOG molecules, including human, naturally occurring variants as well as Uni-ProtKB ID of Q14008, herein incorporated by reference in its entirety.
As used herein “Aurora A kinase” means an enzyme that regulates the function of multiple proteins that control mitotic spindle assembly. Aurora A kinase is differentially expressed in certain cancers including breast, colorectal and lung cancer cells. Aurora A kinase includes without limitation, all known Aurora A kinase molecules, including human, naturally occurring variants as well as Uni-ProtKB ID of 014965, herein incorporated by reference in its entirety.
The term “TPX2” as used herein means a protein that mediates microtubule nucleation from the centrosomes and recruits Aurora A kinase. TPX2 is differentially expressed in certain cancers such as gastric cancer. TPX2 includes without limitation, all known TPX2 molecules, including human, naturally occurring variants as well as Uni-ProtKB ID of Q9ULW0, herein incorporated by reference in its entirety.
The term “tubulin” as used herein means alpha or beta protein that forms heterodimers to make microtubules. Tubulin also includes gamma tubulin, which forms the gamma-tubulin ring complex that nucleates microtubules. Tubulin includes without limitation, all known tubulin molecules, including human, naturally occurring variants as well as Uni-ProtKB ID of Q71U36, P07437 or P23258, herein incorporated by reference in its entirety.
The term “Cdk5rap2” as used herein means CDK5 Regulatory Subunit Associated Protein 2. Cdk5rap2 includes without limitation, all known Cdk5rap2 molecules, including human, naturally occurring variants as well as Uni-ProtKB ID of Q96SN8, herein incorporated by reference in its entirety.
The term “ASPM” as used herein means Abnormal spindle-like microcephaly-associated protein. ASPM includes without limitation, all known ASPM molecules, including human, naturally occurring variants as well as Uni-ProtKB ID of Q81ZT6, herein incorporated by reference in its entirety. ASPM is an ortholog of the Drosophila melanogaster abnormal spindle (asp) gene.
As used herein, the phrase “targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM” means that a compound of the present disclosure that binds, for example selectively, one or more of tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM expressed in a cancer cell, and causes dysregulation of mitotic spindle assembly leading to growth inhibition of the cancer cell.
The expression “compound(s) of the present disclosure” as used in the present document refers to compounds of formulae I and IA presented in the present disclosure, isomers thereof, such as stereoisomers (for example, enantiomers, diastereoisomers, including racemic mixtures) or tautomers, or to pharmaceutically acceptable salts, solvates, hydrates and/or prodrugs of these compounds, isomers of these latter compounds, or racemic mixtures of these latter compounds. The expression “compound(s) of the present disclosure” also refers to mixtures of the various compounds or variants mentioned in the present paragraph.
It is to be understood that the present disclosure includes isomers, racemic mixtures, pharmaceutically acceptable salts, solvates, hydrates and prodrugs of compounds described therein and mixtures comprising two or more of such compounds.
The compounds of the disclosure may have at least one asymmetric centre. Where the compounds disclosed herein possess more than one asymmetric centre, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present disclosure. It is to be understood that while the stereochemistry of the compounds of the present disclosure may be as provided for in any given compound listed herein, such compounds of the disclosure may also contain certain amounts (for example less than 30%, less than 20%, less than 10%, or less than 5%) of compounds of the present disclosure having alternate stereochemistry.
The term “alkyl” as used herein means straight and/or branched chain, saturated alkyl groups containing from one to n carbon atoms and includes (depending on the identity of n) methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, 2,2-dimethylbutyl, n-pentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, n-hexyl and the like, wherein n is the maximum number of carbon atoms in the group.
The term “aryl” as used herein refers to a cyclic or polycyclic aromatic ring. For example, the aryl group can be phenyl or napthyl.
The expression “aromatic heterocycle” as used herein refers to an aromatic cyclic or fused polycyclic ring system having at least one heteroatom selected from the group consisting of N, O, S and P. Non-limitative examples include heteroaryl groups are furyl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, triazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl, pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl, naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl, and so on.
The expression “non-aromatic heterocycle” includes non-aromatic rings or ring systems that contain at least one ring having at least having at least one heteroatom selected from the group consisting of N, O, S and P. This term includes, in a non-limitative manner all of the fully saturated and partially unsaturated derivatives of the above mentioned aromatic heterocycles groups. Examples of non-aromatic heterocycle groups include, in a non-limitative manner, pyrrolidinyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, thiazolidinyl, isothiazolidinyl, and imidazolidinyl.
The term “suitable”, as in for example, “suitable counter anion” or “suitable reaction conditions” means that the selection of the particular group or conditions would depend on the specific synthetic manipulation to be performed and the identity of the molecule but the selection would be well within the skill of a person trained in the art. All process steps described herein are to be conducted under conditions suitable to provide the product shown. A person skilled in the art would understand that all reaction conditions, including, for example, reaction solvent, reaction time, reaction temperature, reaction pressure, reactant ratio and whether or not the reaction should be performed under an anhydrous or inert atmosphere, can be varied to optimize the yield of the desired product and it is within their skill to do so.
The expression “pharmaceutically acceptable” means compatible with the treatment of subjects such as animals or humans.
The expression “pharmaceutically acceptable salt” means an acid addition salt or basic addition salt which is suitable for or compatible with the treatment of subjects such as animals or humans.
The expression “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any compound of the present disclosure, or any of its intermediates. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluenesulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of the compounds of the present disclosure are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g. oxalates, may be used, for example, in the isolation of the compounds of the present disclosure, for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
The term “pharmaceutically acceptable basic addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acid compound of the disclosure, or any of its intermediates. Acidic compounds of the disclosure that may form a basic addition salt include, for example, where CO2H is a functional group. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art. Other non-pharmaceutically acceptable basic addition salts, may be used, for example, in the isolation of the compounds of the disclosure, for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
The formation of a desired compound salt is achieved using standard techniques. For example, the neutral compound is treated with an acid or base in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method.
The term “solvate” as used herein means a compound or its pharmaceutically acceptable salt, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a “hydrate”. The formation of solvates will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.
Compounds of the present disclosure include prodrugs. In general, such prodrugs will be functional derivatives of these compounds which are readily convertible in vivo into the compound from which it is notionally derived. Prodrugs of the compounds of the present disclosure may be conventional esters formed with available hydroxy, or amino group. For example, an available OH or nitrogen in a compound of the present disclosure may be acylated using an activated acid in the presence of a base, and optionally, in inert solvent (e.g. an acid chloride in pyridine). Some common esters which have been utilized as prodrugs are phenyl esters, aliphatic (C8-C24) esters, acyloxymethyl esters, carbamates and amino acid esters. In certain instances, the prodrugs of the compounds of the present disclosure are those in which one or more of the hydroxy groups in the compounds is masked as groups which can be converted to hydroxy groups in vivo. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs” ed. H. Bundgaard, Elsevier, 1985.
The term “cancer” as used herein means a primary or a secondary cancer and includes a non-metastatic cancer and/or a metastatic cancer. Reference to cancer includes reference to cancer cells. For example, the cancer is cervical cancer, breast cancer, ovarian cancer, brain cancer, melanoma, colorectal cancer, glioblastoma, liver cancer, lung cancer, prostate cancer, head cancer, gastric cancer, kidney cancer, endometrial cancer, testis cancer, urothelial cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Hodgkin lymphoma, neuroblastoma, non-Hodgkin lymphoma, soft tissue cancer, bone sarcoma, thyroid cancer, transitional cell bladder cancer, Wilm's tumour, glioma, pancreatic cancer or spleen cancer. For example, the cancer includes any cancer with centrosome aberrations in the cancer cell.
The term “cancer cell” as used herein refers to in vitro cancer cells but also to in vivo cancer cells, e.g. cancer cells present in a subject such as a mammal or a human. For example, in vitro cancer cells may include (human breast cancer) cells, (e.g. BT-549 and MCF-7 cells) mouse neuroblastoma cells (e.g. N1E-115 cells), human non-small cell lung cancer cells (e.g. A549 and H1299 cells), colorectal cancer cells (e.g. HCT116 cells) or human cervical cancer cells (e.g. HeLa).
The term “anti-cancer agent” as used herein means an agent capable of producing a therapeutic effect by inhibiting, suppressing or reducing a cancer (e.g., as determined by clinical symptoms or the amount of cancerous cells) in a subject as compared to a control. Examples of anti-cancer agents include for example non-tubulin-targeting drugs such as doxorubicin, and tubulin-targeting drugs such as taxanes (e.g. paclitaxel), vinca alkaloids (e.g. vinblastine).
The term “anti-mitotic agent” as used herein means an agent that can be used for blocking cancer cell proliferation. For example, the anti-mitotic agent can be an agent that causes microtubule depolymerization such as for example nocodazole or colchicine.
The term “mixture” as used herein, means a composition comprising two or more compounds. In an embodiment a mixture is a mixture of two or more distinct compounds, for example a mixture comprising a compound herein disclosed and an anti-cancer agent such as a taxane for example. In a further embodiment, when a compound is referred to as a “mixture”, it may comprise two or more “forms” of the compounds, such as, salts, solvates, prodrugs or, where applicable, stereoisomers of the compound in any ratio. A person of skill in the art would understand that a compound in a mixture can also exist as a mixture of forms. For example, a compound may exist as a hydrate of a salt or as a hydrate of a salt of a prodrug of the compound. All forms of the compounds disclosed herein are within the scope of the present application.
The term “subject” as used herein includes all members of the animal kingdom including a mammal. In an embodiment, the mammal is a mouse. In another embodiment, the mammal is a human.
The terms “suitable” and “appropriate” refer to the selection of particular groups or conditions that would depend for example on the specific synthetic manipulation to be performed and the identity of the compound, however the selection remains well within the skill of a person trained in the art. All method steps described herein are to be conducted under conditions suitable to provide the product described. A person skilled in the art would understand that all reaction conditions, including, for example, reaction solvent, reaction time, reaction temperature, reaction pressure, reactant ratio and whether or not the reaction should be performed under an anhydrous or inert atmosphere, can be varied to optimize the yield of the desired product and it is within their skill to do so.
The expression an “effective amount” of a compound or composition of the present disclosure is a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of treating cancer, for example, it is an amount of the compound or composition, alone or in combination with an anti-cancer agent and/or an anti-mitotic agent, sufficient to achieve treatment of the cancer as compared to a response in the absence of administration of the compound or composition, alone or in combination with an anti-cancer agent and/or an anti-mitotic agent. The amount of a given compound or composition of the present disclosure that corresponds to an effective amount will vary depending upon various factors, such as for example the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. Also, as used herein, an “effective amount” of a compound of the present disclosure is an amount which inhibits, suppresses or reduces a cancer (e.g., as determined by clinical symptoms or the amount of cancerous cells) in a subject as compared to a control. As further used herein, the “effective amount” is an amount that is sufficient to induce mitotic arrest, disrupt centrosome integrity, prevent and/or reduce centrosome clustering, decluster centrosomes and/or alter microtubule dynamics including microtubule depolymerization in a cancer cell.
As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” or “treating” can also mean prolonging survival as compared to expected survival if not receiving treatment. “Palliating” a disease or disorder, means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder.
The term “administered” as used herein means administration of a therapeutically effective dose of a composition of the application to a cell either in cell culture or in a patient.
In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. Finally, terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±10% of the modified term if this deviation would not negate the meaning of the word it modifies.
As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus for example, a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
In compositions comprising an “additional” or “second” component, the second component as used herein is chemically different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.
The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.
In accordance with a first aspect, there is provided a method for targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM expressed in a cancer cell and selectively inhibiting growth therein, comprising exposing said cancer cell to a compound of Formula I:
For example, targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM induces mitotic arrest in said cancer cell.
For example, targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM is effective for disrupting centrosome integrity, preventing and/or reducing centrosome clustering, declustering centrosomes, regulating centrosome clustering and/or altering microtubule dynamics including microtubule depolymerization in a cancer cell.
According to an aspect, there is provided herein a method for targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM expressed in a cancer cell and selectively inhibiting growth therein, comprising exposing said cancer cell to a combination of a compound of Formula I and an anti-cancer agent and/or an anti-mitotic agent.
According to an aspect, there is provided herein a method for targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM expressed in a cancer cell and inhibiting growth therein, comprising exposing said cancer cell to a synergistic combination of a compound of Formula I and an anti-cancer agent and/or an anti-mitotic agent, wherein said combination more than additively inhibits growth of said cancer cell.
For example, the cancer cell expresses ch-TOG.
For example, the cancer cell expresses Aurora A kinase.
For example, the cancer cell expresses TPX2.
For example, the cancer cell expresses tubulin.
For example, the cancer cell expresses Cdk5rap2.
For example, the cancer cell expresses ASPM.
For example, targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM is effective for treating a cancer in a subject.
According to an aspect, there is provided herein a method for selectively inhibiting growth in a cancer cell, comprising exposing the cancer cell to a compound of Formula I:
For example, inhibiting growth comprises inducing mitotic arrest in the cancer cell.
According to another aspect, there is provided herein a method for disrupting centrosome integrity, preventing and/or reducing centrosome clustering, declustering centrosomes, regulating centrosome clustering and/or altering microtubule dynamics including microtubule depolymerization in a cancer cell, comprising exposing the cancer cell to a compound of Formula I.
According to yet another aspect, there is provided herein a method for selectively inhibiting growth in a cancer cell, comprising exposing the cancer cell to a combination of a compound of Formula I and an anti-cancer agent and/or an anti-mitotic agent.
According to another aspect, there is provided herein a method for inhibiting growth in a cancer cell, comprising exposing the cancer cell to a synergistic combination of a compound of Formula I and an anti-cancer agent and/or an anti-mitotic agent, wherein the combination more than additively inhibits growth of the cancer cell.
According to another aspect, there is provided herein a method for increasing selectivity of an anti-cancer agent and/or an anti-mitotic agent to a cancer cell, comprising exposing the cancer cell with a compound of Formula I and the anti-cancer agent and/or the anti-mitotic agent.
For example, the cancer cells are contacted with a thienoisoquinoline compound herein disclosed at a concentration in the nanomolar range. For example, the method comprises exposing the cancer cell to the compound having a concentration of about 1 nM, about 5 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 550 nM, about 600 nM, about 1000 nM, about 5000 nM or about 10000 nM.
For example, the method comprises exposing the cancer cell to the compound for at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 60 hours, at least 72 hours or at least 80 hours. For example, the cancer cells can also be contacted with a thienoisoquinoline compound herein disclosed for a population doubling time. The term “population doubling time” is to be understood as the period of time required to double the cell population.
For example, the cancer cell is a cancer cell with aberrant centrosomes.
For example, the cancer cell is a breast cancer cell, a cervical cancer cell, a lung cancer cell, a pancreatic cancer cell, a colorectal cancer cell, a neuroblastoma cancer cell or an ovarian cancer cell.
For example, the cancer cell is a mammal cancer cell.
For example, the mammal cancer cell is a human cancer cell.
For example, the method is carried out in vitro.
For example, the method is carried out in vivo.
For example, the cancer cell is present in a subject.
For example, the subject is a mammal.
For example, the mammal is a human.
According to another aspect, there is provided herein a method of treating a cancer in a subject, comprising administering to the subject an effective amount of a compound of Formula I.
According to another aspect, there is provided herein a method of treating a cancer in a subject, comprising administering to the subject an effective amount of a combination of a compound of Formula I and an anti-cancer agent and/or an anti-mitotic agent.
For example, the cancer is a cancer with aberrant centrosomes.
For example, the cancer is breast cancer, cervical cancer, lung cancer, pancreatic cancer, colorectal cancer, neuroblastoma cancer or ovarian cancer.
For example, the subject is a mammal.
For example, the mammal is a human.
For example, the compound for Formula I and anti-cancer agent and/or anti-mitotic agent are administered sequentially or concomitantly.
For example, the anti-cancer agent is a taxane, a vinca alkaloid or a colchicine-site binder.
For example, the anti-cancer agent is a non-mitotic anti-cancer agent.
For example, the anti-mitotic agent is nocodazole.
For example, the cancer cells are further contacted with nocodazole at a concentration of about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 33 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 66 nM, about 70 nM, about 75 nM, about 100 nM, about 125 nM or about 135 nM.
For example, the taxane is paclitaxel, cabazitaxel, or docetaxel.
For example, the cancer cells are further contacted with a taxane at a concentration of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, about 19 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM or about 100 nM.
For example, the vinca alkaloid is vinblastine, vincristine, vindesine, and vinorelbine.
For example, the colchicine-site binder is colchicine, a combrestatin or podophyllotoxin.
For example, the non-mitotic anti-cancer agent is doxorubicin, an anthracycline, an alkylating drug or an antimetabolite.
For example, the compound of Formula I is a compound of Formula IA:
For example, the compound of Formula I is a compound of Formula IA:
For example, the compound of Formula I is a compound of Formula IA:
For example,
For example,
For example, R1, RA, RB said C6-C12 aryl and said three- to seven-membered aromatic heterocycle can be each independently unsubstituted or substituted with 2 or 3 or 4 substituents chosen from C1-C6 alkyl, C1-C6 alkoxy, C6-C12 aryl, three- to seven-membered aromatic heterocycle, C1-C6 hydroxyalkyl, C1-C6 alkythio, C1-C6 thioalkyl, C1-C6 sulfonylakyl, C1-C6 aminoalkyl, C1-C6 alkylamino, CN, NO2, 4,5-dioxoyl, NH2, CF3, CF2H, CFH2, F, Cl, Br and I, OH, CHO, COOH and COORC, wherein RC is a C1-C6 alkyl.
For example, the compound is chosen from
For example, the compound is
For example, the compound is
For example, the compound is
For example, the compound is
According to a further aspect, there is provided herein a use of a compound of Formula I for targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM expressed in a cancer cell and selectively inhibiting growth therein.
For example, targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM is effective for disrupting centrosome integrity, preventing and/or reducing centrosome clustering, declustering centrosomes, regulating centrosome clustering and/or altering microtubule dynamics including microtubule depolymerization in a cancer cell.
Yet another aspect provided herein relates to a use of a combination of a compound of Formula I and an anti-cancer agent and/or an anti-mitotic agent for targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM expressed in a cancer cell and selectively inhibiting growth therein.
For example, targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM induces mitotic arrest in said cancer cell.
For example, targeting tubulin, ch-TOG, Aurora A kinase, TPX2, Cdk5rap2 and/or ASPM is effective for treating a cancer in a subject.
For example, the compound of Formula I is used in combination with an anti-cancer agent and/or an anti-mitotic agent.
For example, the use is effective for increasing selectivity of said anti-cancer agent and/or said anti-mitotic agent to a cancer cell.
For example, the cancer cell expresses ch-TOG.
For example, the cancer cell expresses Aurora A kinase.
For example, the cancer cell expresses TPX2.
For example, the cancer cell expresses tubulin.
For example, the cancer cell expresses Cdk5rap2.
For example, the cancer cell expresses ASPM.
According to a further aspect, there is provided herein a use of a compound of Formula I for selectively inhibiting growth in a cancer cell.
According to another aspect, there is provided herein a use of a compound of Formula I for disrupting centrosome integrity, preventing and/or reducing centrosome clustering, declustering centrosomes, regulating centrosome clustering and/or altering microtubule dynamics including microtubule depolymerization in a cancer cell.
According to yet another aspect, there is provided herein a use of a combination of a compound of Formula I and an anti-cancer agent and/or an anti-mitotic agent in a cancer cell.
For example, inhibiting growth in the cancer cell is selective.
For example, inhibiting growth comprises inducing mitotic arrest in the cancer cell.
According to another aspect, there is provided herein a use of a compound of Formula I for the treatment of cancer in a subject.
According to another aspect, there is provided herein a use of a combination of a Formula I and an anti-cancer agent and/or an anti-mitotic agent for the treatment of cancer in a subject.
According to another aspect, there is provided herein a use of a compound of Formula I in combination with an anti-cancer agent and/or an anti-mitotic agent for increasing selectivity of the anti-cancer agent and/or the anti-mitotic agent to a cancer cell.
For example, the compound herein disclosed and/or the combination comprising the compound and an anti-cancer agent and/or an anti-mitotic agent herein disclosed are comprised in a composition that comprises an injectable dosage form. For example, the composition is administered by intratumoral injection.
For example, the compound herein disclosed and/or the combination comprising the compound and an anti-cancer agent and/or an anti-mitotic agent herein disclosed are comprised in a composition administered by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol or oral administration.
According to a further aspect, there is provided herein a compound of Formula IA:
For example,
For example,
For example,
For example, the compound is chosen from
For example, the compound is
According to a further aspect, there is provided herein a compound chosen from:
These examples are not to be construed as limiting the scope of the present disclosure in any way.
A family of high-quality compounds with drug-like properties and potential for medicinal use were identified. A novel synthesis was designed to improve the yield of these compounds in a cost-effective manner and their ability to treat cancers was evaluated. More specifically, a novel synthesis for a high quality, small molecular weight thienoisoquinoline scaffold was designed[10,11].
To date, 30 variants with different structural modifications have been made. Table 3 below shows IC50 values for a subset of thienoisoquinoline compounds in HeLa (cervical adenocarcinoma), BT549 (breast ductal carcinoma), A549 (lung carcinoma) and MCF-7 (mammary gland carcinoma) cells and HFF1 (fibroblast) and MCF-10A (mammary gland fibrocystic disease) non-cancer cells. Several thienoisoquinoline derivatives, for example C75, C91 and C207, show higher efficacy, i.e. more strongly affect viability, in cancer cells vs. non-cancer cells. In particular, it is shown that C75 causes death in multiple cancer cells with IC50 values in the 100-400 nM range, including breast cancers (MCF7, BT549), lung cancer (A549), colorectal cancer (HCT116) in addition to HeLa cells (cervical cancer).
C75 was tested using high-throughput automated analysis, which is more robust compared to prior testing and found to selectively cause toxicity in HeLa, A549 and HCT116 cancer cells compared to HFF-1 non-cancer cells (
Table 4 shows the calculated IC50 values for C75 and C87 for HFF-1, HeLa, A549 and HCT 116 cell lines.
Another active derivative, C140, has higher efficacy in A549 cells in comparison to C75 (Table 5), and causes spindle phenotypes similar to C75 (
C75 was also tested for efficacy in different cancer cell lines. In HCT116 (colorectal cancer), HeLa (cervical cancer) cells, A549 (lung cancer) cells, C75 caused an increase in the proportion of G2/M cells after treatment with 300 nM or 400 nM for 8 hours (
The thienoisoquinoline compounds may also have potential for use in combinatorial therapies. Several known anti-cancer drugs cause mitotic arrest by disrupting microtubule dynamics, and have been used to combat a spectrum of cancers, including paclitaxel (Taxol™) and vinblastine[12,13]. Taxanes (e.g. paclitaxel), vinca alkaloids (e.g. vinblastine) and other drugs such as colchicine and nocodazole bind to β-tubulin or to the α-β-tubulin lattice. Evidence that C75 can be used in combination with other anti-cancer drugs and enhances the effects of tubulin-targeting drugs is in
Typically, α-tubulin forms a dimer with β-tubulin that then assembles into the polymers that make microtubules[30]. At low concentrations, tubulin-targeting drugs stabilize microtubules without changing their polymer mass, effectively ‘freezing’ the mitotic spindle. C75 does not appear to do this and enhances the efficacy and selectivity of paclitaxel in HeLa cells vs. HFF1 cells (e.g.
An in-depth analysis of HeLa cells treated with both C75 and nocodazole have higher efficacy and selectivity for cancer cells, and have more extreme phenotypes in HeLa cells when treated together vs. on their own (
HeLa, A549 and HCT116 cells treated with subthreshold, non-toxic doses of colchicine in combination with C75 lowers its IC50 by ˜2-fold, and adding a low dose of colchicine enhances the mitotic spindle phenotypes caused by C75 in HeLa and HCT116 cells (
In addition, the thienoisoquinoline compounds disclosed herein may have the potential for use in combinatorial therapies with non-mitotic anti-cancer drugs (non-tubulin-targeting drugs) such as doxorubicin, an anthracycline, an alkylating drug, or an antimetabolite. Specifically, by not targeting tubulin or by targeting tubulin differently, and still disrupting mitosis, the thienoisoquinoline compounds may be used as an anti-mitotic agent in cells that are resistant to tubulin-targeting drugs such as taxanes (e.g. due to the upregulation of alternate beta-tubulin isoforms). To evaluate this, cancer cell lines resistant to tubulin-targeting drugs or having higher resistance to tubulin-targeting drugs may be treated with a thienoisoquinoline compound in combination with a non-mitotic anti-cancer drug, for example doxorubicin. Efficacy, for example in terms of mitotic arrest, may be evaluated using the methods described in Example 1.
The anti-mitotic activity of a thienoisoquinoline compound can be evaluated in vivo using subcutaneous xenografts in rodents. Human cancer cells, e.g. obtained from patients and/or from cancer cell lines, are injected into each of the lower legs (one as a control) of nude rats or SCID mice. Other types of grafting in animal models to test the compounds, e.g. colorectal cancers with different metastases, are also contemplated. Different regiments of treatment will be tested by injection before tumors form, or after ˜2 weeks when tumors are palpable (e.g. 50 mm3). The treatment may also be administered orally, in particular to test for safety and bioavailability. The rodents are treated daily, after every 2-3 days, or weekly with an effective amount of a thienoisoquinoline compound or saline (control) under suitable conditions for a determined duration. Tumors will be monitored daily for change in growth (% treated vs. control). At the end of the study, tumors will be collected and fixed for more in depth analyses of tumor morphology. Toxicity will be monitored by weight loss and/or death[31-33].
The anti-mitotic effect as well as synergistic and protective effects of a thienoisoquinoline compound in combination with an anti-cancer agent such as for example a taxane compound or vinca alkaloid, can also be assessed in vivo using similar methods as described in Example 3.
To show that C75 has a different effect on the mitotic spindle compared to microtubule polymerizing agents, toxicity, mitotic arrest and spindle phenotypes were compared in HCT116 cells after treatment with C75+/− paclitaxel (
To show that C75 has a different effect on the mitotic spindle compared to microtubule depolymerizing agents, the spindle phenotypes were compared in HeLa cells after treatment with C75+/− nocodazole or colchicine, both of which target tubulin and induce microtubule depolymerization. As shown in
C75 was further tested in multicellular tumour spheroids, which are more representative of in vivo tumours in comparison to growing cells as monolayers. These spheroids were produced according to the methods of Friedrich et al. 2009 [34]. Briefly, 96-well plates coated with 1.5% agarose were seeded with 500-1000 HeLa or HCT116 cells as outlined for the liquid-overlay technique. They were left to aggregate with gravity in optimal growth conditions, and individual spheroids were transferred to 24-well dishes for further growth and treatment. A549 spheroids were initiated using the hanging-drop method outlined by Froehlich et al. 2016[35], and transferred to 24-well dishes for further growth and treatment. HeLa, HCT116 and A549 spheroids were treated with C75 alone, or C75-loaded into biodegradable polymeric nanoparticles. Nanoparticles can be produced for example according to the methods reviewed in Zhang et al. 2012[36] and Hong et al. 2018[37]. Both C75 alone and C75-loaded nanoparticles were shown to disrupt HeLa spheroids (
Another novel synthesis for thienoisoquinoline scaffold derivatives was designed.
General Procedure for Bromination of Nitrotoluene
2-bromo-1-methyl-3-nitrobenzene (1 equiv.) and NBS (1.1 equiv) are mixed in 0.8M of anhydrous CCl4 in an oven-dried vessel. The mixture is purged with Argon gas, and heated under reflux for 7 hours. The mixture is cooled to room temperature and diluted with DCM, then follow by washing with distilled water. The Aqueqous layer is extract with DCM. The combined organic layers are washed three times with distilled water, and dried over Na2SO4. The compound is purified with column chromatography. The product is white solid. Isolated yield 72%.
General Procedure for Benzylation
Methyl 3-((4-methoxyphenyl)sulfonamido)thiophene-2-carboxylate (1 equiv.), 2-bromo-1-(bromomethyl)-3-nitrobenzene (1.2 equiv.), and K2CO3 (3 equiv.) are mixed in 0.3M of DMF. Solution is heated to 50° C. for 18 hours. Reaction mixture is cooled to room temperature after 18 hours. The mixture is diluted with EtOAc and washed with distilled water, and aqueous layer is extracted with EtOAc. The combined organic layers are washed with distill water three times, saturated salt solution three times, and dried over Na2SO4 The solvent is evaporated under reduced pressure, and solid residue is recrystallized with EtOAc and hexane. The final product is pale yellow crystal. Isolated yield: 92%
General Procedure for Saponification
Methyl 3-((N-(2-bromo-3-nitrobenzyl)-4-methoxyphenyl)sulfonamido)thiophene-2-carboxylate (1 equiv.) and NaOH powder (5 equiv.) was mixed and dissolved with THF, water, and MeOH (2:1:1 respectively, 0.1M overall). The mixture is heated under reflux, then cooled to room temperature after 1.5 hours for completion, TLC is used to monitor the reaction. The mixture is treated with 1M HCl solution until pH reaches 1. The mixture is diluted with distilled water and extracted with DCM two times. The combined organic layers are washed with water three time, saturated salt solution two times, and dried over Na2SO4. The solvent is evaporated under reduced pressure, and residue is recrystallized with EtOAc and hexane. The final product is yellow solid. Isolated yield: 95%
General Procedure for Decarboxylative Cross-Coupling
3-((N-(2-bromo-3-nitrobenzyl)-4-methoxyphenyl)sulfonamido)thiophene-2-carboxylic acid (1 equiv.) in DMA (0.1M) was added to an oven-dried microwave vial contain PdCl2 (0.1 equiv.), P(tBu)3.HBF4 (0.2 equiv.), Cs2CO3 (3 equiv.). The mixture is heated under microwave radiation at 170° C. for 8 min, then cooled to room temperature, and diluted with EtOAc. The mixture is washed with distilled water, and aqueous layer is extracted with EtOAc. The combined layers are washed with saturated NaHCO3, distilled water, saturated NaCl solution, and dried over Na2SO4. The solvent is evaporated under reduced pressure. The product is purified by column chromatography and recrystallization from CHCl3 and MeOH. The final product is yellow crystal. Isolated yield: 41%.
General Procedure for Reduction
4-((4-methoxyphenyl)sulfonyl)-9-nitro-4,5-dihydrothieno[3,2-c]isoquinoline (1 equiv.) and Pd/C (0.1 equiv. 10 mol %) is mixed with THF and MeOH (1:1 ratio, 0.2M overall). Hydrazine hydrate (10 equiv.) is added slowly to the mixture. The mixture is heated under reflux for 20 mins for completion. TLC is used to monitor the reaction. Mixture is filtered from a pad of celite and washed with EtOAc. The mixture is washed with distilled water, and aqueous layer is extracted with EtOAc, The combined organic layer is washed with distilled water one time, saturated salt solution one time, and dried over Na2SO4. The solvent is evaporated under reduced pressure. The residue is recrystallized with CHCl3 and MeOH. Isolated yield: 70%.
General Procedure for Amine Protection
4-((4-methoxyphenyl)sulfonyl)-4,5-dihydrothieno[3,2-c]isoquinolin-9-amine (1 equiv.) and Di-tert-butyl dicarbonate (2 equiv.) are dissolved in Et3N (0.4 M), DMAP (1 equiv.) is added into mixture. The mixture is placed under room temperature for 48 hours. The mixture is diluted with EtOAc and washed with distilled water. The aqueous layer is extracted with EtOAc. The combined organic layers are washed with 1M HCl one time, distilled water three times, saturated salt solution one time, and dried over Na2SO4. The solvent is evaporated under reduced pressure. The product is purified by column chromatography. The product is colorless solid. Isolated yield: 60%
General Procedure for Bromination
tert-butyl (4-((4-methoxyphenyl)sulfonyl)-4,5-dihydrothieno[3,2-c]isoquinolin-9-yl)carbamate (1 equiv.) and NBS (1.1 equiv.) are mixed with CHCl3 (0.1M) in an ember vial, and mixture is placed in ice bath follow by 2 v/v % AcOH. The reaction is slowly return to room temperature and run for 18 hours. The mixture is diluted with EtOAc then washed with distilled water, and aqueous layer is extracted with EtOAc. The combined organic layer is washed with distilled water one time, saturated salt solution one time, and dried over Na2SO4. The solvent is evaporated under reduced pressure. The product is purified by column chromatography. The product is light brown solid. Isolated yield: 30%
General Procedure for Boc Group Deprotection
tert-butyl (2-bromo-4-((4-methoxyphenyl)sulfonyl)-4,5-dihydrothieno[3,2-c]isoquinolin-9-yl)carbamate (1 equiv.) is dissolved on DCM (0.1M) and placed in ice bath. 50 v/v % TFA is added slowly to mixture. The reaction is monitored by TLC, and stopped after 5 hours. The mixture is diluted with EtOAc then washed with distilled water, and aqueous layer is extracted with EtOAc. The combined organic layer is washed with saturated NaHCO3 one time, distilled water one time, saturated NaCl solution one time, and dried over NasSO4. The solvent is evaporated under reduced pressure. The product is purified by column chromatography. The product is brown solid. Isolated yield: 25%
General Procedure for Sulfonylation
Methyl 3-amino-4-methylthiophene-2-carboxylate (1 equiv.) and 4-methoxybenzenesulfonyl chloride (1.5 equiv.) are mixed in 0.8M of Pyridine. Solution is heated to 50° C. for 1.5 hour, and is cooled to room temperature. The mixture is diluted with EtOAc and washed with distilled water, and aqueous layer is extracted with EtOAc. The combined organic layers are washed with distill water three times and Saturated salt solution three times. The solvent is evaporated under reduced pressure, and solid residue is recrystallized with EtOAc and hexane. Product is colorless crystal. Isolated yield: 74%
General Procedure for Benzylation
methyl 3-((4-methoxyphenyl)sulfonamido)thiophene-2-carboxylate (1 equiv.), 1-bromo-2-(bromomethyl)benzene (1.2 equiv.), and K2CO3 (3 equiv.) are mixed in 0.3M of DMF. Solution is heated to 50° C. for 16 hours. Reaction mixture is cooled to room temperature after 18 hours. The mixture is diluted with EtOAc and washed with distilled water, and aqueous layer is extracted with EtOAc. The combined organic layers are washed with distill water three times, saturated salt solution three times, and dried over Na2SO4 The solvent is evaporated under reduced pressure, and solid residue is recrystallized with EtOAc and hexane. The final product is colorless crystal. Isolated yield: 73%
General Procedure for Saponification
methyl 3-((N-(2-bromobenzyl)-4-methoxyphenyl)sulfonamido)-4-methylthiophene-2-carboxylate (1 equiv.) and NaOH powder (5 equiv.) was mixed and dissolved with THF, water, and MeOH (2:1:1 respectively, 0.1M overall). The mixture is heated under reflux, then cooled to room temperature after 2 hours for completion, TLC is used to monitor the reaction. The mixture is treated with 1M HCl solution until pH reaches 1. The mixture is diluted with distilled water and extracted with DCM two times. The combined organic layers are washed with water three time, saturated salt solution two times, and dried over Na2SO4. The solvent is evaporated under reduced pressure, and residue is recrystallized with EtOAc and hexane. The final product is colorless solid. Isolated yield: 84%
General Procedure for Decarboxylative Cross-Coupling
3-((N-(2-bromobenzyl)-4-methoxyphenyl)sulfonamido)-4-methylthiophene-2-carboxylic acid (1 equiv.) in DMA (0.1M) was added to an oven-dried microwave vial contain PdCl2 (0.1 equiv.), P(tBu)3.HBF4 (0.2 equiv.), nBu4NBr (0.15 equiv.), and Cs2CO3 (3 equiv.). The mixture is heated under microwave radiation at 170° C. for 8 min, then cooled to room temperature, and diluted with EtOAc. The mixture is washed with distilled water, and aqueous layer is extracted with EtOAc. The combined layers are washed with saturated NaHCO3, distilled water, saturated NaCl solution, and dried over Na2SO4. The solvent is evaporated under reduced pressure. The product is purified by column chromatography and recrystallization from CHCl3 and MeOH. The final product is colorless crystal. Isolated yield: 76%.
General Procedure for Bromination
4-((4-methoxyphenyl)sulfonyl)-3-methyl-4,5-dihydrothieno[3,2-c]isoquinoline (1 equiv.) and NBS (1.1 equiv.) are mixed with CHCl3 (0.1M) in an ember vial, and mixture is placed in ice bath follow by 1 v/v % AcOH. The reaction is slowly return to room temperature and run for 18 hours. The mixture is diluted with EtOAc then washed with distilled water, and aqueous layer is extracted with EtOAc. The combined organic layer is washed with distilled water one time, saturated salt solution one time, and dried over Na2SO4. The solvent is evaporated under reduced pressure. The product is purified by column chromatography. The product is colorless solid. Isolated yield: 71%
C75 targets a protein, a structural component or enzyme that regulates the centrosomes responsible for assembly and organization of the mitotic spindle. The target is likely differentially expressed and/or functionally required in cancer cells in comparison to non-cancer cells.
As shown in
ch-TOG contains multiple TOG domains that bind to tubulin dimers, and a microtubule-binding domain that binds to the microtubule lattice. If C75 were to bind and inhibit one or more of the TOG domains, this would shift its affinity onto microtubules, while inhibiting the microtubule domain would cause ch-TOG to become cytosolic. As shown in
The phenotypes of C75-treated cells are reminiscent of phenotypes caused by ch-TOG (CKAP5), a protein known to regulate microtubule polymerization and mitotic spindle assembly. As shown in
As shown in
Prior studies showed that the fragmented/multipolar spindle phenotypes caused by ch-TOG RNAi can be suppressed by MCAK RNAi. As shown in
As shown in
It was found that the molecular target of C75 is conserved in Drosophila. Using S2 cells stably expressing GFP-tagged tubulin, adding 300 nM C75 to live cells caused spindle phenotypes similar to what was observed in mammalian cells. Varying concentrations of C75 in Drosophila S2 cells vs. colchicine were added to determine if the molecular target is conserved. It was found that the cells respond similar to HeLa cells, supporting that the target is conserved. This will permit using Drosophila proteins for binding studies in vitro, and genetics-based tools for further study of the target.
Aurora A kinase regulates the function of multiple proteins that control mitotic spindle assembly, including TPX2, and a complex that comprises ch-TOG, TACC3 and clathrin. The mitotic phenotypes caused by loss of Aurora A kinase function via RNAi or inhibition share similarities to what we observe for C75, as shown in
Aurora A kinase inhibition+C75 vs. colchicine in HeLa and HCT116 cells with images and graphs showing the proportion of spindle phenotypes will be assessed. As above, we will compare phenotypes, and determine if C75 enhances/synergizes Aurora A kinase phenotypes, which would rule it out as a target. We will use the Aurora A kinase inhibitor Alisertib, which has been shown to have selectivity for Aurora A kinase, with affinity in the low nanomolar range (˜1 nM in vitro). We will use colchicine as a control, since it should show enhancement.
TPX2 RNAi+C75 vs. DMSO and vs. colchicine in HeLa and HCT116 cells with images and graphs showing the proportion of spindle phenotypes. As above, we will compare phenotypes, and determine if C75 enhances/synergizes TPX2 phenotypes, which would rule it out as a target. As above, we are using colchicine as a control, since it should show enhancement.
C75 and its potential target will be further characterized by performing cold treatments of HeLa cells to collapse microtubules followed by recovery by temperature upshift in C75 vs. colchicine or other tubulin-targeting drugs. If C75 targets tubulin, the effect on microtubules should be similar to other tubulin-targeting drugs.
To further verify the target, we will perform in vitro binding studies. We have constructs for recombinant expression of the ch-TOG domains from Drosophila and human ch-TOG and have begun expressing and purifying these proteins. We will determine if there are spectral shifts after adding C75 to these purified protein fragments. We also will try DARTs to determine if C75 can protect the various protein fragments from degradation via proteases, which would support binding. We will do similar experiments with other recombinant proteins (e.g. TPX2, tubulin).
We will use a modified compound, functionalized at a site that does not perturb binding affinity, to couple to a solid matrix for pulldown experiments using cell lysates. This will permit to identify potential binding partners based on affinity, which will be determined via mass spectrometry.
HCT116 cells, colorectal adenocarcinoma, were particularly sensitive to C75. ch-TOG/CKAP5, a microtubule polymerase, is highly overexpressed in these cells, and counteracts the function of MCAK, a microtubule depolymerase, to regulate spindle length during mitosis. Indeed, C75 phenocopies ch-TOG knockdown, and similar to ch-TOG knockdown, the spindle phenotypes caused by C75 can be suppressed by MCAK RNAi. Changes in endogenous ch-TOG localization in the presence of C75 using ch-TOG antibodies were also observed. Despite causing spindle phenotypes, ch-TOG becomes more highly enriched on the centrosomes and microtubules after C75 treatment. This suggests that C75 could bind to at least one of the TOG domains required for binding to free tubulin dimers, causing an increase in ch-TOG affinity to the polymerized microtubules. It is expected, without being bound to this theory that ch-TOG is the molecular target of C75, and it will be determined if other cancer cells that are more sensitive to C75 also have high levels of ch-TOG, making it a suitable biomarker for a subset of highly progressive cancers. Similar logic applies to the other candidates, such as Aurora A kinase or TPX2.
As shown in
C75 could also target ASPM (abnormal spindle-like microcephaly-associated) protein. Asp (abnormal spindle protein), which is conserved and functions in human and Drosophila cells to focus the poles of the mitotic spindle[38-42]. It localizes to the minus ends of microtubules at the poles as well as centrosomes, and its depletion causes displacement of gamma tubulin ring complexes and disorganized spindles, a phenotype that is consistent with what is observed with C75[38-41]. In addition, Asp can nucleate microtubules in vitro and has a microtubule cross-linking domain[38,41]. It is required during prometaphase-metaphase, the time at which C75 causes cell cycle arrest[38,41].
A recent study found that Asp functions redundantly with Cdk5rap2 (also called CEP215), another potential target of C75 that is conserved in Drosophila[42]. Cdk5rap2/CEP215 localizes to the pericentriolar material (PCM) of centrosomes where it tethers gamma-tubulin ring complexes, and its depletion causes their displacement giving rise to disorganized mitotic spindles, which is similar to what is observed after treatment with C75[43-45. Cancer cells with amplified/fragmented centrosomes may be sensitive to loss of Cdk5rap2 function, because it forms a complex with HSET, a protein that functions to cluster centrosomes[45].
The embodiments of paragraphs [0036] to [00232] of the present disclosure are presented in such a manner in the present disclosure so as to demonstrate that every combination of embodiments, when applicable can be made. These embodiments have thus been presented in the description in a manner equivalent to making dependent claims for all the embodiments that depend upon any of the preceding claims (covering the previously presented embodiments), thereby demonstrating that they can be combined together in all possible manners. For example, all the possible combination, when applicable, between the embodiments of paragraphs [0036] to [00232] and the methods, processes, uses, compounds and compositions of paragraphs [0006] to [0035] are hereby covered by the present disclosure.
This is a Patent Cooperation Treaty Application claims priority to U.S. Provisional Patent Application No. 62/586,553, filed Nov. 15, 2017; U.S. Provisional Patent Application No. 62/681,093, filed Jun. 6, 2018; and International Patent Application No. PCT/CA2017/051473 filed Dec. 6, 2017. These documents are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2018/051454 | 11/15/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62586553 | Nov 2017 | US | |
| 62681093 | Jun 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CA2017/051473 | Dec 2017 | US |
| Child | 16764370 | US |
