AU2018101501A4 - Method of treating cancer - Google Patents

Abstract

The present invention relates to the administration of a compound of formula (1) advantageous efficacious as PDE6 inhibitor and its effects on subjects with cancer. More specifically, the present invention is directed to a method for administering a compound having favorable geometric properties for interacting with the PDE6 prenyl-binding pocket for treating a subject suffering from cancer, in particular lung cancer. The structural components contribute an advantageous interaction with PDE6, in particular with amino acids in the binding pocket. The present invention further provides a method of inhibiting the growth or proliferation of cancer cells as well as pharmaceutical compositions comprising said compound. IL -L CY) KA r b) U-

Description

The present invention relates to the administration of a compound of formula (I) advantageous efficacious as PDE6 inhibitor and its effects on subjects with cancer. More specifically, the present invention is directed to a method for administering a compound having favorable geometric properties for interacting with the PDE6 prenyl-binding pocket for treating a subject suffering from cancer, in particular lung cancer. The structural components contribute an advantageous interaction with PDE6, in particular with amino acids in the binding pocket. The present invention further provides a method of inhibiting the growth or proliferation of cancer cells as well as pharmaceutical compositions comprising said compound.

2018101501 10 Oct 2018

2018101501 10 Oct 2018

METHOD OF TREATING CANCER

TECHNICAL FIELD

The present invention relates to the administration of a compound efficacious as PDE5 inhibitor and its effects on subjects with cancer. More specifically, the present invention is directed to a method of administering a compound having certain structural components for treating a subject suffering from a disease such as cancer, in particular lung cancer. The present invention further provides a method of inhibiting the growth or proliferation of cancer cells harboring a RAS gene mutation as well as pharmaceutical compositions comprising said compound.

BACKGROUND OF THE INVENTION

RAS proteins belong to a family of membrane-associated 21-kDa guanosine triphosphate (GTP)-binding proteins by cycling between Off’ and On’ conformations that are conferred by the binding of guanosine diphosphate (GDP) and GTP, respectively. Namely, they cycle between inactive GDP-bound and active GTP-bound forms, wherein interconversion between both forms is catalyzed, for example, by GTPase activating proteins (GAP).

RAS proteins are central mediators involved in a variety of intracellular signaling pathways critical for cell proliferation, survival, and differentiation of cells. Three different mammalian RAS proteins and encoding genes have been identified, namely K-RAS (with two splice variants K-RAS4A and K-RAS4B with K-RAS4B being the more abundant isoform), H-RAS, and N-RAS. All RAS isoforms are reported to share 82% to 90% overall sequence identity as well as sequence identity in all of the regions responsible for GDP/GTP-binding, but they exhibit different C-terminal variable regions (prenylated cysteine) that target them to different cellular compartments and are responsible for membrane association and cellular localization (Spiegel, J. et al., Nat Chem Biol., 2014, 10:613-622, Cox, A.D. et al., Nat Rev Drug Discov., 2014, 13:828-851). They all are farnesylated and H-RAS, N-RAS and KRAS4A are additionally S-palmitoylated in their variable regions.

RAS proteins interact with and can activate several downstream effectors in particular including rat protein kinases and phosphoinositide 3-kinases (PI3K) involved in cell survival and proliferation. Downstream signaling pathways activated by RAS are, for example, the PI3K-AKT-mTOR pathway and the raf-MEK-ERK pathway (Wang, Y. et al., J Med Chem., 2013, 56:5219-5230, Acquaviva, J. et al., Mol Cancer Ther., 2012, 11:2633-2643). Said RAS signaling strongly depends on the correct intracellular localization of the RAS proteins.

RAS proteins have been reported to be involved in the pathogenesis of several cancers. In particular, several mutations within the RAS protein encoding genes are reported to result in permanently activated RAS signaling pathways. It is generally assumed that about 30% of all human cancers harbor activating RAS mutations while being often not responsive to established therapies, making such RAS mutations, thus, to one of the most common known genetic causes of cancer. In this context, K-RAS is considered for being the most frequent mutated isomer in various cancers such as colon cancer, lung cancer, pancreatic cancer,

2018101501 10 Oct 2018 and hematologic malignancies (Wang, Y. et al., J Med Chem., 2013, 56:5219-5230, Spiegel, J. et al., Nat Chem Biol., 2014, 10:613-622, Cox, A.D. et al., Nat Rev Drug Discov., 2014, 13:828-851). N-RAS and/or H-RAS mutations are frequently reported in colorectal cancer, bladder cancer, kidney cancer, thyroid carcinomas, melanoma, hepatocellular carcinoma, and hematologic malignancies (Cox, A.D. et al., Nat Rev Drug Discov., 2014, 13:828-851). Namely, Prior et al. found K-RAS as most frequent mutated isoform in analyzed tumors, namely in 22% of all tumors analyzed compared to about 8% for N-RAS and 3% for H-RAS (Prior, I. A. et al., Cancer Res., 2012, 72:2457-2467). In the majority of cases, these mutations are point mutations which introduce an amino acid substitution at position 12, 13, or 61 (Wang, Y. et al., J Med Chem., 2013, 56:5219-5230, Spiegel, J. et al., Nat Chem Biol., 2014, 10:613-622). Presence of said point mutations impairs GTPase activity, in particular renders RAS insensitive to GAP action with a resulting constitutive activation of RAS signaling pathways (Zimmermann, G. et al., J Med Chem., 2014, 57:5435-5448). Prior et al., for example, found that 80% of K-RAS mutations occur at codon 12, whereas very few mutations were observed at codon 61 or 13 (Prior, I. A. et al., Cancer Res., 2012, 72:24572467).

K-RAS mutations are reported to be present in more than 25% of non-small cell lung cancers (NSCLC) usually associated with unfavorable clinical outcomes, and they have been reported to occur frequently in patients with lung adenocarcinoma (20-30%). K-RAS mutations are comparable uncommon in lung squamous cell carcinoma (Cox, A.D. et al., Nat Rev Drug Discov., 2014, 13:828-851). Constitutive activation of K-RAS leads to persistent stimulation of signaling pathways that promote tumorigenesis, including the raf/MEK/ERK and PI3K/AKT/mTOR signaling cascades that are downstream to K-RAS.

In the absence of such activating RAS mutations, an increased RAS activity such as by overexpression or increased activation of growth signaling pathways has been reported in tumors, too (Wang, Y. et al., J Med Chem., 2013, 56:5219-5230).

Different approaches have been described for inhibiting RAS protein signaling pathways including strategies to influence the distribution of RAS in the cell such as inhibition of farnesylation of RAS or inhibition of RAS membrane interactions as well as to specifically address the signaling pathways or to inhibit RAS protein directly (e.g. Spiegel, J. et al., Nat Chem Biol., 2014, 10:613-622). Although RAS makes up the most frequently mutated oncogene family in human cancer and more than three decades of intensive effort has been spent in the past decade to provide RAS inhibitors, no effective pharmacological inhibitor of the RAS protein has reached the clinic, which has garnered the view of RAS being an “undruggable” protein.

Recently, Zimmermann et al. described a specific approach aimed at disrupting K-RAS membrane association by inhibiting cGMP phosphodiesterase delta subunit (“PDE5”), a protein that can assist in RAS protein intracellular trafficking, in particular bind to farnesyl moieties and regulate the trafficking of RAS proteins to plasma membranes, i.e. facilitate the intracellular RAS diffusion and enhance its trapping at the right compartment. They identified and characterized a small-molecule PDE5 inhibitor, named deltarasin that inhibited the K3

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RAS-PDE5 interaction and impaired K-RAS signaling. In addition, deltarasin also strongly suppressed the proliferation of human pancreatic ductal adenocarcinoma cells in vitro and in vivo (Zimmermann, G. et al., Nature, 2013, 497:638-642, Zimmermann, G. et al., J Med Chem, 2014, 57:5435-5448).

In view of the limited clinical applicability of the majority of the approaches described so far and in view of frequent resistance mechanisms, there remains a strong need for compounds suitable for treating cancer, in particular for those being suitable to sufficiently and specifically inhibit RAS signaling.

SUMMARY OF THE INVENTION

The present invention relates in a first aspect to a method of treating cancer, such as lung cancer like non-small cell lung cancer (NSCLC), in a subject, in particular a mammal having at least one RAS gene mutation such as a K-RAS gene mutation.

Said method comprises administering an effective amount of a compound having Formula (1), or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof to the subject:

Formula (I)

The compound of the present invention proved to comprise favorable geometric properties allowing for unexpected exceptional interaction with the PDE5 binding site and inhibition of PDE5, in particular the compound was found to advantageously bind to, and interact with, amino acids in the hydrophobic pocket of PDE5. It has been unexpectedly found that the presence of the structural components in the compound for formula (I) allow for exceptional inhibition of the interaction of PDE6 with RAS and respective RAS signaling pathways in cells, thereby impairing oncogenic RAS signaling and leading to an advantageous inhibition of cancer cell proliferation and induction of apoptosis

According to the invention is also the compound of Formula (I), or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, for use as a medicament, preferably for use in the treatment of cancer such as lung cancer like NSCLC, including for example NSCLC adenocarcinoma or NSCLC bronchioalveolar carcinoma.

In another aspect, the present invention provides a method of inhibiting the growth or proliferation of cancer cells harboring at least one RAS gene mutation comprising the step of

2018101501 10 Oct 2018 contacting said cancer cells with a compound having Formula (I), or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof.

In particular, the method for inhibiting the growth or proliferation of cancer cells provided comprises the step of contacting cancer cells, including cancer cells harboring at least one RAS gene mutation, particularly a K-RAS gene mutation with an effective amount of the compound of Formula (I), or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof; wherein PDE6 is inhibited and the proliferation or growth of the cells harboring a RAS, particular a K-RAS, gene mutation is selectively inhibited.

In still another aspect, the present invention provides a pharmaceutical composition comprising a compound of Formula (I), or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof as active ingredient. The pharmaceutical composition further comprises a physiologically tolerable excipient and may additionally contain further active ingredient, in particular a therapeutic compound, such as a anticancer agent, for treating cancer such as lung cancer, like NSCLC adenocarcinoma or NSCLC bronchioalveolar carcinoma. The present invention also refers to the use of said pharmaceutical composition for inhibiting PDE6, such as for inhibiting the signaling pathways downstream to RAS, in particular to K-RAS.

Other features and aspects of the invention will become apparent by consideration of the following detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A shows a 3D schematic representation of the structure of the compound of Formula (I), 1-benzyl-2-phenyl-1H-benzimidazole and the interaction mode of the 1-benzyl-2-phenyl1H-benzimidazole with the binding pocket on PDE6 of the K-RAS protein. Hydrogen bonds formed by the 1-benzyl-2-phenyl-1 H-benzimidazole are indicated.

Fig. 1B shows a 3D schematic representation of the binding mode and binding interactions between the compound of Formula (I) and the binding pocket of K-RAS protein. Hydrogen bonds formed by the compound of Formula (I) are indicated.

Fig. 2A shows the cell viability relative to untreated controls of A549 cells after treatment with the compound of Formula (I) with a concentration of 0-20 μΜ for 72 h.

Fig. 2B shows the cell viability relative to untreated controls of H358 cells after treatment with the compound of Formula (I) with a concentration of 0-20 pM for 72 h.

Fig. 2C shows the cell viability relative to untreated controls of H2122 cells after treatment with the compound of Formula (I) with a concentration of 0-20 pM for 72 h.

Fig. 2D shows the cell viability relative to untreated controls of CCD19-Lu normal lung cells after treatment with the compound of Formula (I) with a concentration of 0-40 pM for 72 h.

Fig. 3A shows the formation of A549 cell colonies after treatment with 5 pM deltarasin (positive control group).

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Fig. 3B shows the formation of A549 cell colonies after treatment with a control group.

Fig. 3C shows the formation of A549 cell colonies after treatment with 2.5 μΜ of the compound of Formula (I).

Fig. 3D shows the formation of A549 cell colonies after treatment with 5 μΜ of the compound of Formula (I).

Fig. 3E shows the formation of A549 cell colonies after treatment with 10 μΜ of the compound of Formula (I).

Fig. 3F shows the formation of A549 cell colonies after treatment with 20 μΜ of the compound of Formula (I).

Fig. 3G is a bar chart illustrating the average number of colonies formed in the colony formation assay as shown in Fig. 3A-3F, i.e. with 2.5 μΜ, 5 μΜ, 10 μΜ and 20 μΜ of the compound of Formula (I) (referenced as “G642-7049”) compared with 5 μΜ deltarasin and control group.

Fig. 4A is a flow cytometry pattern of A549 cells treated with 5 μΜ deltarasin.

Fig. 4B is a flow cytometry pattern of A549 cells treated with a control group.

Fig. 4C is a flow cytometry pattern of A549 cells treated with 5 μΜ of the compound of Formula (I).

Fig. 4D is a flow cytometry pattern of A549 cells treated with 10 μΜ of the compound of Formula (I).

Fig. 4E is a flow cytometry pattern of A549 cells treated with 20 μΜ of the compound of Formula (I).

Fig. 4F is a bar chart showing the percentage of apoptotic A549 cells after treatment with 5 μΜ, 10 μΜ, 20 μΜ of the compound of Formula (I) (referenced as “G642-7049”) compared to 5 μΜ deltarasin and a control group.

Fig. 5 is a western blot and shows the expression of p-C-raf, C-raf, p-AKT, AKT, p-ERK, ERK and GAPDH of A549 cells treated with 5 μΜ deltarasin or 5 μΜ, 10 μΜ and 20 μΜ of the compound of Formula (I) (referenced as “G642-7049”) compared to a control group.

Fig. 6 is a western blot and shows the expression of KRAS-GTP and total KRAS in A549 cells treated with 5 μΜ deltarasin or 20 μΜ of the compound of Formula (I) (referenced as “G642-7049”) compared to a control group.

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DESCRIPTION OF THE EMBODIMENTS

The following embodiments and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representing preferred embodiments thereof. The technical terms used in the present patent application have the meaning as commonly understood by a respective skilled person unless specifically defined otherwise.

The present invention refers in a first aspect to a method of treating cancer in a subject. Said method comprises administering an effective amount of a compound having Formula (I) to the subject:

o

Formula (I)

Formula (I) is also referenced as “G642-7049” herein and may include a derivative, or a pharmaceutically acceptable salt or solvate thereof.

Said compound having Formula (I) proved to comprise favorable geometric properties for interacting with the phosphodiesterase δ (PDE6) prenyl-binding pocket, in particular structural components including a core cyclic backbone that includes highly electronegative atoms, namely N and/or O atoms. The inventors found that the presence of such structural components in the compound having Formula (I) provides the compound having Formula (I) with a high affinity for, and advantageous interaction with, the PDE6 prenyl-binding pocket. In particular, said compound of Formula (I) advantageously binds deep in the PDE6 hydrophobic pocket formed by Leu22, Leu54, Val59, Gln78, Trp90, Pro113, Ile129 and Tyr149, particularly interacting with Gln78 and Trp90.

Further moieties which may be attached to the backbone or side chain according to Formula (I) do not impede the interaction of compound of Formula (I) with the prenyl-binding pocket of PDE6 and preferably allow for additional interactions including van der Waals forces and hydrogen bonds or hydrophobic interactions with the prenyl-binding pocket of PDE5 and, thus, further contribute to the exceptional interaction with PDE6.

As contemplated by the present invention are any derivatives of the compound of Formula (I) which may further add to the advantageous interaction with PDE6 or contribute to the facilitation of said compound having Formula (I) as a pharmaceutical formulation. A derivative includes any compound produced from said compound having Formula (I) by

2018101501 10 Oct 2018 modification or partial substitution of the original Formula (I) compound core, or by replacement of one or more atoms or a group of atoms from the compound having Formula (I), for example modification or substitution of one or more side chains of the compound having Formula (I) with other moieties.

A derivative may also refer to a pro-drug of the compound having Formula (I), i.e. a compound that is converted (e.g. hydrolyzed, metabolized, etc.) in vivo to the compound having Formula (I). A pro-drug includes any compound which releases the active compound of Formula (I) in vivo when the pro-drug is administered to a subject. Pro-drugs are prepared by modifying functional groups present in such a way that the modifications may be cleaved in vivo to release the parent compound. Pro-drugs include, for example, compounds wherein a hydroxy, amino, or sulfhydryl group is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, or sulfhydryl group, respectively.

Said compound may also be a pharmaceutically acceptable salt or solvate of the derivative of the compound of Formula (I).

Also contemplated by the present invention are any pharmaceutically acceptable salts, hydrates, solvates, as well as enantiomers and their mixtures, stereoisomeric forms, racemates, diastereomers and their mixtures of the compound having Formula (I).

As used herein, the term solvate refers to a complex of variable stoichiometry formed by a solute, i.e. compound of Formula (I), and a solvent. If the solvent is water, the solvate formed is a hydrate.

Suitable pharmaceutically acceptable salts are those which are suitable to be administered to subjects, in particular mammals such as humans and can be prepared with sufficient purity and used to prepare a pharmaceutical formulation. The terms stereoisomers, diastereomers, enantiomers and racemates are known to the skilled person.

The effective amount of the compound having Formula (I) may depend on the species, body weight, age and individual conditions of the subject and can be determined by standard procedures such as with cell cultures or experimental animals.

The subject of the treatment is preferably a mammal, in particular a human. In particular, the method of the present invention refers to the treatment of the subject suffering from cancer.

The term “cancer” refers to or describes a physiological condition in subjects in which a population of cells are characterized by unregulated cell growth.

The cancer is selected from pancreatic cancer, lung cancer, colorectal cancer, bladder cancer, kidney cancer, thyroid carcinomas, melanoma, hepatocellular carcinoma, and hematologic malignancies, preferably pancreatic cancer, lung cancer or colorectal cancer, further preferably lung cancer, more preferably NSCLC, still more preferably NSCLC of the adenocarcinoma type, i.e. NSCLC adenocarcinoma, or NSCLC of the bronchioalveolar carcinoma type, i.e. NSCLC bronchioalveolar carcinoma.

Said compound having Formula (I) is particularly effective for treating cancer associated with a RAS gene mutation. RAS gene mutation is in particular accompanied by an aberrant

2018101501 10 Oct 2018 function of the expressed RAS mutant protein favoring GTP binding and producing constitutive activation of RAS mutant protein with a resulting upregulation of signaling pathways thereby stimulating cell proliferation and inhibiting apoptosis and leading to uncontrolled cell growth. Preferably, the RAS gene mutation concerns codons 12, 13 and/or 61 of the RAS encoding genes. Preferably, the RAS gene mutation is a K-RAS gene mutation that concerns codon 12 of the K-RAS encoding gene and includes G12C, G12A, G12D, G12S and/or G12V.

In a preferred embodiment, the subject is preferably a mammal having at least one K-RAS gene mutation, in particular G12C and/or G12S.

In a most preferred embodiment of the present invention, the disease is a K-RAS dependent NSCLC adenocarcinoma. In a further preferred embodiment, the disease is a K-RAS dependent NSCLC bronchioalveolar carcinoma.

“RAS-dependent”, in particular “K-RAS-dependent” as used herein refers to a cancer or cancer cells having an enhanced expression or activity of a RAS protein such as K-RAS protein. This can be assessed by the activation of one or more downstream pathways to RAS such as to K-RAS. “Enhanced expression” or “Enhanced activity” preferably means an increase in RAS protein expression or RAS protein activity by at least 5% compared to a reference control, i.e. normal (healthy) cells, i.e. non-cancerous cells. In particular, the RAS protein is a RAS mutant protein, in particular a K-RAS mutant protein. The skilled person is able to determine the level of the expression of RAS such as K-RAS protein and/or the RAS such as K-RAS protein activity with common methods, for example, with well-known immunological assays that utilize antibody methods, Northern blotting, in-situ hybridization or similar techniques or qRT-PCR, RAS Activation Kits for determining the active form of RAS or by measuring the level of downstream effectors of the signaling pathway downstream to RAS.

In particular, said enhanced RAS expression or enhanced activity is essentially required for viability of the cells, i.e. the RAS protein expression or activity is highly correlated with the growth of the cancer cells and its inhibition results in a further enhanced growth suppression and cell death, i.e. the enhanced RAS such as K-RAS protein expression or activity is preferably the decisive factor essentially required for the survival of the cancer cells in RASdependent such as K-RAS-dependent cancers.

Whether a subject has such RAS gene mutation can be detected with methods known to the skilled person such as DNA sequencing or commercially available test systems, DNA-DNA hybridization and the like.

The method of the present invention may further include steps carried out before administering the compound of Formula (I) to the subject comprising:

• Obtaining a sample, in particular cancer cells from the subject;

• Testing said sample for the RAS expression levels, in particular the K-RAS expression levels, or identifying at least one RAS gene mutation, in particular K-RAS

2018101501 10 Oct 2018 gene mutation such as selected from G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, Q61K, Q61L, Q61R and/or Q61H;

• Optionally correlating the level of RAS expression, in particular K-RAS expression, with outcome and if conditions are met, administering the compound of Formula (I) to said subject.

“RAS” as used in the present invention comprises N-RAS, H-RAS and K-RAS isoforms.

According to the invention is also the compound of Formula (I) for use as a medicament, preferably for use in the treatment of cancer such as lung cancer, especially NSCLC such as NSCLC adenocarcinoma and/or NSCLC bronchioalveolar carcinoma, in particular RASdependent such as K-RAS-dependent NSCLC adenocarcinoma and/or K-RAS-dependent NSCLC bronchioalveolar carcinoma. The compound of Formula (I) can be used in an effective amount for treating a human. Another aspect of the invention refers to the use of the compound of Formula (I) for preparing a medicament for treatment of cancer, especially lung cancer, in particular NSCLC including NSCLC adenocarcinoma and/or NSCLC bronchioalveolar carcinoma, especially RAS-dependent such as K-RAS-dependent NSCLC adenocarcinoma and/or NSCLC bronchioalveolar carcinoma.

The compound having Formula (I) was found to inhibit PDE6 and disrupted interaction between PDE6 and RAS proteins, thereby deviating intracellular localization of RAS proteins and thus advantageously impairing the oncogenic RAS signaling pathway, particularly the KRAS signaling pathway. Said compound disrupts the interaction between PDE6 and RAS proteins by directly binding to PDE6 and preventing the binding between PDE6 and RAS proteins, particularly K-RAS, thereby interfering with intracellular localization of K-RAS. Said compound also suppresses K-RAS binding to GTP and K-RAS downstream cell growth signaling pathways, for example, RAF, PI3K and RAL pathways.

Thus, the compound of the present invention can be advantageously used for inhibiting, reducing or preventing the growth or proliferation of cancer cells.

The present invention provides in a further aspect a method of inhibiting the growth or proliferation of cancer cells, comprising the step of contacting the cancer cells with a compound having Formula (I) or a pharmaceutically acceptable salt or solvate thereof:

o

Formula (I).

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The compound includes derivatives of the compound having Formula (I) or pharmaceutically acceptable salts or solvates of the derivative of the compound having Formula (I).

The compound having Formula (I) is preferably used in a concentration of at least 1.25 μΜ, more preferably at least 2.5 μΜ, more preferably at least 5 μΜ and more preferably 10 μΜ or 20 μΜ. In a preferred embodiment, compound having Formula (I) is used in a concentration of at least 2.5 μΜ and preferably in a concentration of 10 μΜ. In particular, contacting said cells with the compound having Formula (I) leads to an inhibition, reduction or prevention of the proliferation of the cancer cells or induction of apoptosis of the cancer cells. The cancer cells are preferably contacted with the compound having Formula (I) for at least 10 hours, more preferably for at least 12 hours. In a further example embodiment, the cancer cells are treated with the compound having Formula (I) for 48 hours.

The term ‘proliferation’ as described is a process that results in an increase of the number of cells as a result of cell growth and cell division. Proliferation is generally increased and/or abnormal in cancer cells.

The cancer cells are preferably from a lung cancer, further preferably from a NSCLC including for example a NSCLC adenocarcinoma and/or a NSCLC bronchioalveolar carcinoma. Preferably the cancer cells are from a RAS-dependent cancer, particularly a KRAS dependent cancer wherein the cancer cells harbor at least one RAS gene mutation. Preferably, the RAS gene mutation is selected from a mutation in the RAS, in particular in the K-RAS, protein encoding genes at codons 12, 13 and/or 61, more preferably at codon 12. More preferably, the RAS gene mutation is a K-RAS gene mutation selected from G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, Q61K, Q61L, Q61R and/or Q61H. More preferably, the K-RAS gene mutation is selected from one or more of G12C, G12A, G12D, G12S and G12V. Most preferably, the K-RAS gene mutation is selected from one or more of G12S and/or G12C.

In a preferred embodiment, contacting the cancer cells with the compound having Formula (I) inhibits PDE5 and selectively inhibits the proliferation of the cancer cells harboring a K-RAS gene mutation, particularly a G12S and/or a G12C gene mutation. In yet another preferred embodiment, contacting the cancer cells with the compound having Formula (I) inhibits PDE5 and induces apoptosis of cancer cells harboring a K-RAS gene mutation, particularly a G12S and/or a G12C gene mutation.

In still another aspect, the present invention provides a pharmaceutical composition comprising a compound of Formula (I):

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Formula (I), or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, as active ingredient and further comprising physiologically active excipients.

The term ‘pharmaceutically acceptable salt’ refers to salts which retain the biological effectiveness and properties of compounds which are not biologically or otherwise undesirable. Pharmaceutically acceptable salts refer to pharmaceutically-acceptable salts of the compounds, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

Said pharmaceutical composition further comprises a physiologically tolerable excipient. The skilled person is able to select suitable excipients depending on the form of the pharmaceutical composition and is aware of methods for manufacturing pharmaceutical compositions as well as able to select a suitable method for preparing the pharmaceutical composition depending on the kind of excipients and the form of the pharmaceutical composition. The excipients may, for example, improve the solubility and stability of said pharmaceutical composition whilst being non-toxic and non-reactive with other substances.

Said pharmaceutical composition may further be a pharmaceutically acceptable salt or solvate of the derivative of the compound of Formula (I). In an alternative embodiment, the pharmaceutical composition may be a pro-drug of the compound of Formula (I).

The pharmaceutical composition may include a pharmaceutically acceptable carrier. As described herein, this is a carrier that is useful in preparing a pharmaceutical composition or formulation that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes a carrier that is acceptable for veterinary use as well as human pharmaceutical use.

The skilled person is able to prepare the compound of Formula (I) with suitable purity and/or respective compounds are commercially available with sufficient purity.

The present invention also refers to the use of the pharmaceutical formulation of the present invention for inhibiting PDE6, especially for inhibiting the signaling pathways downstream to RAS mutant protein, in particular K-RAS mutant protein, and deviating the intracellular localization of RAS proteins, particular K-RAS, and impairing the oncogenic RAS signaling pathway.

The pharmaceutical composition according to the invention can be present in solid, semisolid or liquid form to be administered by an oral, rectal, topical, parenteral or transdermal or inhalative route to a subject, preferably a human.

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The pharmaceutical composition may comprise further active ingredients, such as therapeutic compounds used for treating cancer, in particular lung cancer such as NSCLC, in particular NSCLC adenocarcinoma and/or NSCLC bronchioalveolar carcinoma. For example, anticancer agents including but not limited to alkylating agents such as cisplatin and procarbazine; antimetabolites such as methotrexate; anti-microtubule agents such as paclitaxel; topoisomerase inhibitors such as doxorubicin; and cytotoxic agents.

In a further example embodiment, the pharmaceutical composition including the compound of Formula (I) may be applied in the treatment of multidrug resistant (MDR) cancer.

EXAMPLES

A549 (K-RAS^G12S), H358 (K-RAS^G12C) , H2122 (K-RAS^G12C) and CCD19-Lu cells were obtained from the American Type Culture Collection and cultured in a humidified environment of 5% CO₂ at 37°C RPMI-1640 medium containing 10 % fetal bovine serum (FBS; Gibco), 100 U/mL penicillin and 100 pg/mL streptomycin.

Compound of Formula (I), i.e.G642-7049, was purchased from ChemDiv. Deltarasin was purchased from Selleck Chemicals, dissolved in DMSO and stored in small aliquots at -20°C Antibodies to GAPDH, ERK, p-ERK (Thr202/Thy204), C-Raf, p-C-Raf p-AKT (Ser473), were purchased from Cell signaling Technology. Anti-AKT and K-RAS antibodies were acquired from Santa Cruz Biotechnology.

Descriptive analytical data are presented as means ± standard deviation (SD). Statistical analysis was conducted using Graph Prism 5.0. One-way analysis of variance (ANOVA) was used to assess significant differences between datasets. Values of P<0.05 were considered statistically significant.

EXAMPLE 1

Firstly, the binding mode between the compound of Formula (I) and K-RAS has been determined.

In this context, molecular docking calculation has been performed to study the interaction between the compound of Formula (I) and PDE5 by Induced Fit Docking module in Schrodinger software (Schrodinger, Inc., New York, NY, 2015). The studied compound of Formula (I) is prepared and optimized in the LigPrep module. The 3D structure of PDE5 in complex with a benzimidazole compound is derived from the PDB database (PDB ID: 4JV6) and prepared using the Protein Preparation Wizard. During the induced fit docking, centroid of the co-crystalized inhibitor was used to define the active site. The poses of the studied compound are evaluated by extra precision (XP) docking score and the conformation with the highest score is selected for binding mode analysis.

The binding affinity of compound of Formula (I) to PDE5 was evaluated by the XP docking score. The docking score of compound of Formula (I) is -13.415 Kcal/mol. The conformation of compound of Formula (I) has been superimposed with the co-crystal ized benzimidazole compound to compare their binding modes. As shown in Fig. 1A, the scaffold of compound

2018101501 10 Oct 2018 of Formula (I) overlapped well with the location of two molecules of benzimidazole compound which was in the location of the two residues, Tyr149 and Arg61. As shown in Fig 1B, the compound of Formula (I) was buried in a hydrophobic pocket formed by Leu22, Leu54, Val59, Gln78, Trp90, Pro113, Ile129, Tyr149. Among these residues, Gln78 and Trp90 formed hydrogen bonds with the compound of Formula (I).

EXAMPLE 2

In order to prove that the compound of Formula (I) is highly cytotoxic and selective to cancer cells, the cytotoxic effect of the compound of Formula (I) on lung cancer cell lines that have K-RAS gene mutation and normal lung epithelial cells (CCD19-Lu) has been determined.

3000 cells were seeded on 96-well plates and treated with the compound of Formula (I),

i.e.G642-7049, for 72 hours. 10μΙ MTT reagent was added to each well and the crystals were solubilized in 100 μΙ of the resolved solution (10% SDS and 0.1 mM HCL).The absorbance at 570 nm was measured using a microplate reader. The cell viability was calculated relative to untreated control cells, with results based on at least three independent experiments.

MTT assay showed that the compound of Formula (I) inhibited proliferation and viability in A549, H358 and H2122 cell lines with IC50 of 18.35 ±4.23μΜ, 10.66±3.97μΜ and 5.63±1.08μΜ, respectively (Fig. 2A to 2C), and showed lower cytotoxicity in normal lung epithelial cells (CCD19-Lu). The IC50 in CCD19-Lu is more than 40 μΜ (Table 1).

Table 1: IC50 of the compound of Formula (I) in different cell lines

Cell lines	IC50 (μΜ)
A549	18.35 ±4.23
H358	10.66 ±3.97
H2122	5.63 ± 1.08
CCD-19 Lu	>40

EXAMPLE 3

The inhibitory effect of the compound of Formula (I) on the colony formation in A549 cells has been analyzed to provide further evidence that the compound of Formula (I) inhibits the formation of colonies of cancerous cells.

A549 cells were cultured overnight on a 6-well plate at a density of 500 cells per well. The cells were exposed to various concentrations of the compound of formula (I) (2.5 μΜ, 5 μΜ, 10 μΜ or 20 μΜ) or 5 μΜ deltarasin. After 10-14 days, the colonies were fixed with 4% ice14

2018101501 10 Oct 2018 cold paraformaldehyde for 15 minutes at 4°C and stained with a 0.1% crystal violet solution for 15 minutes at room temperature. The colonies were then photomicrographed and counted under a microscope.

The analysis of the effect of the compound of Formula (I) on colony formation activity revealed that the compound of Formula (I) significantly inhibited the colony formation capacity of A549 (Fig. 3C to Fig. 3G). Notably, when the concentration of the compound of Formula (I) reached 10 μΜ, A549 cells formed no visible colonies (Fig. 3E and Fig. 3G).

EXAMPLE 4

Further, to provide additional evidence that the compound of Formula (I) is highly effective in inducing apoptosis in cancer cells, the induced apoptosis in A549 cells has been analyzed.

Apoptosis was measured using Annexin V-FITC/PI flow cytometry. Briefly, A549 cells (1.0><105 cells/well) were allowed to attach in a 6-well plate overnight, cells were treated with the compound of Formula (I) (5 pM, 10 pM or 20 pM) or 5 pM deltarasin for 48 h. Subsequently, cells were trypsinized, washed twice with PBS and stained with 2 pi AnnexinV FITC and 2 pi propidine iodide (PI) and incubated in the dark at room temperature for 15 min. The stained cells were analyzed quantitatively using a Flow Cytometer (BD Biosciences, San Jose, California, USA). Data were analyzed by Flow Jo software.

Flow cytometry analysis showed that the compound of Formula (I) exhibited anti-cancer ability through induction of apoptosis of A549 cells in a concentration-dependent manner. Compared with the control group, treatment on A549 cells with the compound of Formula (I) induced significant cell apoptosis as shown in Fig. 4A to 4F.

EXAMPLE 5

Mutant K-RAS oncogenic function is exerted through constitutive activation of RAF/MEK/ERK and PI3K/AKT signaling cascades. Thus, the suppression of the downstream signaling pathways to RAS by the compound of Formula (I) has been tested.

A549 cells were exposed to different concentrations of the compound of Formula (I), namely 5 pM, 10 pM and 20 pM or 5 pM deltarasin. Total protein was collected with cold lysis buffer containing protease and phosphatase inhibitors. Protein concentration of the cell lysates was determined using a Bio-Rad protein Assay kit, 5χ laemmli buffer was added and boiled at 100 °C for 5 min. 30 pg protein was loaded onto each lane and subjected to a 10% SDSPAGE gel, then the separated proteins were transferred to a Nitrocellulose (NC) membrane, The membranes were blocked at room temperature with 5% non-fat dry milk in TBS containing 0.1% Tween 20 (0.1% TBST) for 1 hour with constant agitation, subsequently incubated overnight at 4° C with primary antibody(1:1000). After washing, the membrane was incubated with secondary rabbit or mouse fluorescent antibodies (1:10000) and the signal intensity of the membranes was detected by a LI-COR Odessy scanner.

Treatment of A549 cells with the compound of Formula (I) decreased the levels of p-C-raf, pErk and pAkt when compared with the untreated cells (Fig. 5), which proves that the

2018101501 10 Oct 2018 compound of Formula (I) is exceptionally suitable to suppress signaling pathways downstream to K-RAS.

EXAMPLE 6

Further, a GTP pull-down assay was carried out. A549 cells were treated with the compound of Formula (I) at 20 μΜ for 48 h. Cells were lysed in lysis buffer and the volume of each sample was adjusted to 1 mL with 1X Assay Lysis Buffer. The protein extracts were incubated while rotating at 4°C for 1 h with an equal volume of Rhotekin-RBD bound to glutathione-agarose beads and the beads were washed three times with 1X lysis buffer. Proteins were eluted with 2 xSDS-loading buffer and boiled at 100°C for 5 min. Sepharose beads were removed by centrifugation and the proteins were analyzed by SDS-PAGE followed by Western Blot with a monoclonal antibody against K-RAS.

Treatment of A549 cells with the compound of Formula (I) at 20 μΜ prior to probing with desthiobiotin-GTP caused a decreased amount of K-RAS being pulled down with streptavidin as compared to the untreated control (Figure 6). Deltarasin was used as positive control to demonstrate suppression of GTP binding with K-RAS. The compound of Formula (I) advantageous decreased K-RAS-GTP levels in A549 cells (Fig.6).

2018101501 10 Oct 2018

Claims (22)

1. A method of treating cancer in a subject comprising administering an effective amount of a compound having Formula (I), or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, to the subject:

Formula (I).

2. The method of claim 1, wherein the compound is a pharmaceutically acceptable salt or solvate of the derivative of the compound having Formula (I).

3. The method of claim 1, wherein the cancer is lung cancer.

4. The method of claim 1, wherein the cancer is non-small cell lung cancer (NSCLC).

5. The method of claim 1, wherein the cancer is K-RAS-dependent NSCLC adenocarcinoma.

6. The method of claim 1, wherein the subject is a mammal having at least one RAS gene mutation and wherein the mutation concerns codons 12, 13 and/or 61 of the RAS encoding genes.

7. The method of claim 6, wherein the subject is a human having at least one KRAS gene mutation and wherein the mutation concerns codon 12 of the K-RAS encoding gene and is selected from G12C, G12A, G12D, G12S and/or G12V.

8. The method of claim 1, wherein the compound inhibits phosphodiesterase δ (PDE5).

9. The method of any one of claims 5 to 8, wherein the compound treats the cancer by inhibiting the interaction of K-RAS and PDE5.

2018101501 10 Oct 2018

10. A method of inhibiting the growth or proliferation of cancer cells comprising contacting the cancer cells with a compound having Formula (I), or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof:

Formula (I).

11. The method of claim 10, wherein the compound is a derivative of the compound having Formula (I) or a pharmaceutically acceptable salt or solvate thereof.

12. The method of claim 10, wherein apoptosis of the cancer cells is induced.

13. The method of claim 10, wherein the cancer cells are from a lung cancer.

14. The method of claim 10, wherein the cancer cells are from a NSCLC.

15. The method of claim 10, wherein the compound of Formula (I) is used in a concentration of at least 2.5 μΜ.

16. The method of claim 10, wherein the cancer cells harbor at least one RAS gene mutation at codon 12 of the RAS protein encoding genes.

17. The method of claim 10, wherein the cancer cells harbor at least one K-RAS gene mutation at codon 12 of the K-RAS protein encoding gene selected from G12C, G12A, G12D, G12S and/or G12V.

18. A pharmaceutical composition comprising a compound of Formula (I):

2018101501 10 Oct 2018

Formula (I) or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, as active ingredient and further comprising a physiologically tolerable excipient.

19. The pharmaceutical composition of claim 18, wherein the compound is a pharmaceutically acceptable salt or solvate of the derivative of the compound having Formula (I).

20. The pharmaceutical composition of claim 18, further comprising a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition is formulated for oral administration or injection.

22. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition further comprises an anticancer agent.

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