WO2021113605A1 - Methods, compounds and compositions for the treatment of cancer by inhibiting notch-1 maturation - Google Patents

Abstract

Sarcoendoplasmic reticulum calcium ATPase (SERCA) is a critical intracellular regulator of Ca⁺² influx into the endoplasmic reticulum to promote proper protein folding. Perturbation of intracellular Ca⁺² can enhance protein misfolding and is found in cancers. To accommodate protein misfolding, cancer cells upregulate unfolded protein response (UPR). However, when UPR exceeds a tolerable threshold, this can trigger pro-apoptotic mechanisms. We have developed a novel anti-cancer compound, C1, an orally bioavailable specific inhibitor of SERCA2 that is able to selectively inhibit triple negative breast cancer (TNBC) in vitro and in vivo using a xenograft model. C1 also served as a platform for the optimization and development of other effective compounds disclosed herein.

Description

METHODS, COMPOUNDS AND COMPOSITIONS FOR THE TREATMENT OF CANCER BY INHIBITING NOTCH-1 MATURATION

RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62/944,273 filed December 5, 2019, which is incorporated herein by reference.

FIELD OF THE INVENTION This invention relates to methods, compounds, and compositions for the treatment of cancer by inducing Notchl maturation inhibition.

BACKGROUND OF THE INVENTION

Protein synthesis is an essential component for cancer survival, migration, proliferation and metastasis. Cancer adaptive mechanisms include upregulation of endoplasmic reticulum (ER) activities to accommodate increases in protein misfolding. Cancerous growth is a combination of malignant cells infiltrating in a tumor microenvironment that include non-malignant and immune cells. This malignant growth is an interaction between normal and malignant cells, where the end result is proliferation, invasion and metastasis of the original cancerous growth.

Metastatic spread of the primary cancer growth requires malignant cells to invade and breakthrough the basement membrane and entering into the blood or lymphatic circulation. These malignant cells gaining access into vascular system can circulate and establish a new cancerous site. This requires a number of events to take place. All of these steps require that the malignant cell continues to be 1 ) viable while in the circulation, 2) capable of establishing a new site, 3) extravasate into the new tissue, and 4) establish a new site for malignant propagation.

Cancer cells with stem cell like characteristics have been implicated as an important determinant in metastatic disease. The presence of circulating cancer stem cells has shown to increase the risk for metastatic potential.

Numerous studies have shown that the presence of metastatic disease significantly reduces the chance for complete remission and disease-free survival.

Thus, the ability to not only reduce the primary tumor size but to also reduce the number of circulating cancer stem cells in the circulation may significantly improve overall clinical outcomes. Therefore, additional new therapeutic agents are needed for the effective treatment of cancer.

SUMMARY OF THE INVENTION This invention discloses compounds, compositions, and methods of use that are able to inhibit tumor growth and to also reduce the formation of circulating cancer stem cells. This invention is based on the ability of the provided methods, compounds and compositions to inhibit tumor growth and to also inhibit the Notch-1 formation.

In order to have an effective anticancer activity, a compound must first be safe at the pharmacological effect concentration. Although an anticancer drug can be delivered systemically either through parenteral or oral route, the ability to develop an orally bioavailable drug is far more advantageous. This mode of delivery allows daily dosing without the need of inserting a vascular catheter, which can be a source for systemic infections. Sarcoendoplasmic Reticulum Calcium ATPase (SERCA) are important regulators of calcium influx found in the endoplasmic reticulum, where inhibition can lead to the reduction of calcium which is necessary for proper folding of the proteins. Slight inhibition of SERCA can lead to protein misfolding triggering cellular apoptosis.

Breast cancer cells, in particular triple negative breast cancers (TNBCs), express higher level of SERCA2 and thus are more susceptible to SERCA2 inhibition. This invention discloses a new class of compounds capable of inhibiting the expression of sarcoendoplasmic reticulum calcium ATPase isoform 2 (SERCA2). This is correlated with the reduction of Notch 1 maturation by inhibiting enzymatic cleavage of the pro protein precursor to the active form of Notchl . This class of compounds is able to target cancer cells since they have preexisting misfolded protein levels, where only a small addition can trigger tumor selective apoptosis. Moreover, these compounds are orally bioavailable, and are active alone or in combination with standard chemotherapeutic agents that enhance antitumor properties.

Inhibition of SERCA-2 isoform not only inhibits the production of Notch 1 maturation but can also enhance protein misfolding and thus can induce misfolded protein pathways. The ability to inhibit Notchl may potentiate its antitumor properties by inhibiting circulating cancer stem cells from entering the circulation and reestablishing in another tissue cell type to form metastatic satellites. Cancer survival is dependent on the ability to increase protein synthesis required for cell migration, proliferation and metastasis. Cancer adaptive mechanisms include upregulation of endoplasmic reticulum (ER) activities to accommodate increases in misfolded proteins. SERCAs are important regulators of calcium found in the endoplasmic reticulum, where inhibition can lead to protein misfolding. Since breast cancer cells express higher level of SERCA2, its overexpression maybe a target for drug development.

We have screened several new analogs to obtain Compound 1 (C1 ). Compound 1 lacks COX-2 inhibitory activity but targets SERCA 2 to induce ER stress. We have demonstrated that targeting SERCA 2 leads to the activation of unfolded protein response pathway and cell death is effective in an in-vitro model.

Accordingly, this disclosure provides A compound represented by Formula I:

or a pharmaceutically acceptable salt thereof; wherein

R¹ is -(Ci-C4)alkyl substituted with one or more halo;

R² and R³ are each independently — (Ci-C6)alkyl;

R⁴ and R⁵ are each independently H ,-(Ci-C6)alkyl, or -C(=0)(Ci-Cs)alkyl wherein the (Ci-Cs)alkyl moiety is substituted with 1-6 heteroatoms, interrupted with 1-6 heteroatoms, forms a heterocycle, or a combination thereof; and

R⁶ is H or -(Ci-Ce)alkyl.

Also, this disclosure provides composition comprising the compound described above and a pharmaceutically acceptable carrier.

Additionally, this disclosure provides a method for the treatment of cancer in a subject in need of cancer therapy comprising administering an effective amount of the compound described above or a composition of the compound described above, thereby treating the cancer in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention described herein. Figure 1 : Concentration dependent inhibition of SERCA 2 using Compound-1 from this new class of agents.

Figure 2: Time-dependent exposure of 20 mM Compound 1 leads to reduction of SERCA2 expression, where mature Notchl after 6 hours is reduced. This coincides with Multiple Resistant Protein-1 (MRP1) expression inhibition, suggesting that SERCA2 inhibition can suppress the formation of circulating cancer stem cells, where MRP1 is one of the hallmarks of chemoresistance.

Figure 3: Compound-1 activity pathway: Inhibition of SERCA2 expression causes a depletion of ER calcium level leading to the upregulation of GRP78 (78-kDa Glucose-Regulated Protein) and the Unfolded Protein Response (UPR). Prolonged activation of UPR leads to cell death by apoptosis. Prolonged upregulation of GRP78 activates the Unfolded Protein Response (UPR) involving Activating Transcription Factors 3/4 (ATF3/4) to elevate the expression of transcription factor CHOP. Increased expression of CHOP leads to the activation of apoptosis pathway and the cleavage of Caspase-3. Figure 4: Antitumor activity of Compound 1 in MDA-MB-231 in a dosage escalation study design.

Figure 5: Combination antitumor activity of Compound 1 with Paclitaxel (PTX) or Gemcitabine (GEM) in 4T1 xenograft mice model.

Figure 6: (A) Compound 1 and related lead analogues were studied for their accumulation in MDA-MB-231 cell line. Accumulation in cells increases with analogs compared to Compound 1. C11 accumulation profile follows similar pattern to

Compound 1 while C8 and C9 accumulates exponentially and linearly, respectively.

No data point was reported for C9 at 60 mM because the cells were not viable with treatment. (B) Compound 1 lead analogs molecular activity was evaluated in MDA- MB-231 cell line. After 1 hour of treatment, new analogues reduced SERCA2 and increased expression of GRP78 indicating the activation of UPR pathway.

Figure 7: The molecular activity of Compound 1 was evaluated in MDA-MB-231 in comparison to Dimethyl Celecoxib and Celecoxib after 12 hours of treatment. Compound 1 inhibits SERCA2 expression at lower dose showing subsequent activation of UPR through the increased expression of GRP78. In response, the cell death pathway is activated through ATF-3, DR5 and the eventual cleavage of caspase-3. After 12 hours of treatment, immature Notch 1 was accumulated and PARP expression was decreased while apoptotic marker DR5 and ATF-3 were induced after prolonged UPR activity.

Figure 8: The antitumor activity of Compound 1 was evaluated in MDA-MB-31 xenografted into nu/nu mice treated with orally administered 25-50mg/kg C1 given either once or twice daily showed a >85% tumor reduction (Upper Panel). Relative RNA expression of SERCA2 (A), CHOP (B), GRP78 (C), Notchl (D), Vegfa (E) and DR5 (F), and), were significantly reduced at dosages >25 mg/kg/d.

Figure 9: Tumor from in vivo studies using MDA-MB-231 xenograft was evaluated for microvessel density after 28 days of Compound 1 treatment using anti- CD31 antibody. Compound 1 treatment was able to decrease microvessel as determined by anti-CD31 staining.

DETAILED DESCRIPTION

The compound disclosed herein (Compound 1) has significantly improved potency against various breast cancer cell lines as compared to Di-methyl Celecoxib (DMC) and Celecoxib (CBX). The activity could further be explained through the improved inhibition of SERCA2 with Compound 1 treatment, in comparison to DMC and CBX. The depletion also led to the increase of GRP78 expression and unfolded protein response (UPR) pathway with Compound 1 showing a dosage dependent effect leading to tumor apoptosis with cleaved caspase-3 cleavage and D5R up regulation for the activation of necrosis. Based on time course, Compound 1 decrease of SERCA2 expression is shown after 12 hours of treatment followed by the activation of GRP78 expression. In this process, protein misfolding accumulates in the same time frame with the increased detection of immature Notchl and the degradation of MRP1 . Prolonged treatment of 24 and 48 hours later shows cells exhibiting apoptosis and necrosis. In dosage escalation in vivo studies using Compound 1 dosages up to 100 mg/kg/day, no detectable biochemical and histological toxicities were detected. Using 25 mg/kg/day of Compound 1 treatment resulted to the increased the mRNA expression of DR5 and the decrease of Notchl in tumor tissues. When the dosage of Compound 1 was increased to 50 mg/kg/day, the tumor expression for Vegfa was decrease suggesting that Compound 1 also has anti-angiogenesis properties. Further exploration of angiogenesis with Compound 1 treatment shows decrease of microvessel density in tumor tissue with increasing dosages of Compound 1 treatment in mice. Based on these properties, Compound 1 will be an effective component in the treatment of Triple Negative Breast Cancer (TNBC), where new and effective therapies are much needed.

Definitions

The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley’s Condensed Chemical Dictionary 14^th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described. The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds

X. It is further noted that the claims may be drafted to exclude any optional element.

As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and/or the recitation of claim elements or use of "negative" limitations.

The term "and/or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is disubstituted.

As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect.

The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than and/or less than a recited integer at each end of the range. Unless indicated otherwise herein, the terms "about" and "approximately" are intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The terms "about" and "approximately" can also modify the end-points of a recited range as discussed above in this paragraph.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units are also disclosed. For example, if 10 to 15 is disclosed, then 11 , 12, 13, and 14 are also disclosed, individually, and as part of a range. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

This disclosure provides ranges, limits, and deviations to variables such as volume, mass, percentages, ratios, etc. It is understood by an ordinary person skilled in the art that a range, such as “numberl ” to “number2”, implies a continuous range of numbers that includes the whole numbers and fractional numbers. For example, 1 to

10 means 1 , 2, 3, 4, 5, ... 9, 10. It also means 1.0, 1.1 , 1.2. 1.3, ..., 9.8, 9.9, 10.0, and also means 1.01 , 1.02, 1.03, and so on. If the variable disclosed is a number less than “numberl 0”, it implies a continuous range that includes whole numbers and fractional numbers less than numberl 0, as discussed above. Similarly, if the variable disclosed is a number greater than “numberl 0”, it implies a continuous range that includes whole numbers and fractional numbers greater than numberl 0. These ranges can be modified by the term “about”, whose meaning has been described above. One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo. An "effective amount" refers to an amount effective to treat a disease, disorder, and/or condition, or to bring about a recited effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect.

Alternatively, The terms "effective amount" or "therapeutically effective amount," as used herein, refer to a sufficient amount of an agent or a composition or combination of compositions being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic uses is the amount of the composition comprising a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study. The dose could be administered in one or more administrations. Flowever, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of the disease, route of administration of the compositions, the type or extent of supplemental therapy used, ongoing disease process and type of treatment desired (e.g., aggressive vs. conventional treatment). The terms "treating", "treat" and "treatment" include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving the disease, pathologic or medical condition; and/or (iv) diminishing symptoms associated with the disease, pathologic or medical condition. Thus, the terms "treat", "treatment", and "treating" can extend to prophylaxis and can include prevent, prevention, preventing, lowering, stopping or reversing the progression or severity of the condition or symptoms being treated. As such, the term "treatment" can include medical, therapeutic, and/or prophylactic administration, as appropriate. As used herein, "subject" or “patient” means an individual having symptoms of, or at risk for, a disease or other malignancy. A patient may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles {e.g., children). Moreover, patient may mean any living organism, preferably a mammal {e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. In one embodiment of the methods provided herein, the mammal is a human.

As used herein, the terms “providing”, “administering,” “introducing,” are used interchangeably herein and refer to the placement of a compound of the disclosure into a subject by a method or route that results in at least partial localization of the compound to a desired site. The compound can be administered by any appropriate route that results in delivery to a desired location in the subject.

The compound and compositions described herein may be administered with additional compositions to prolong stability and activity of the compositions, or in combination with other therapeutic drugs.

The terms "inhibit", "inhibiting", and "inhibition" refer to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells.

The inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting.

The term “substantially” as used herein, is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified. For example, the term could refer to a numerical value that may not be 100% the full numerical value. The full numerical value may be less by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.

Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of” or “consisting essentially of” are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

This disclosure provides methods of making the compounds and compositions of the invention. The compounds and compositions can be prepared by any of the applicable techniques described herein, optionally in combination with standard techniques of organic synthesis. Many techniques such as etherification and esterification are well known in the art. However, many of these techniques are elaborated in Compendium of Organic Synthetic Methods (John Wiley & Sons, New York), Vol. 1 , Ian T. Harrison and Shuyen Harrison, 1971 ; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade, Jr., 1980; Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 6; as well as standard organic reference texts such as March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Ed., by M. B. Smith and J. March (John Wiley & Sons, New York, 2001); Comprehensive Organic Synthesis. Selectivity, Strategy & Efficiency in Modern Organic Chemistry. In 9 Volumes, Barry M. Trost, Editor-in-Chief

(Pergamon Press, New York, 1993 printing); Advanced Organic Chemistry, Part B: Reactions and Synthesis, Second Edition, Cary and Sundberg (1983); for heterocyclic synthesis see Hermanson, Greg T, Bioconjugate Techniques, Third Edition, Academic Press, 2013.

The formulas and compounds described herein can be modified using protecting groups. Suitable amino and carboxy protecting groups are known to those skilled in the art (see for example, Protecting Groups in Organic Synthesis, Second Edition, Greene, T. W., and Wutz, P. G. M., John Wiley & Sons, New York, and references cited therein; Philip J. Kocienski; Protecting Groups (Georg Thieme Verlag Stuttgart, New York, 1994), and references cited therein); and Comprehensive Organic Transformations, Larock, R. C., Second Edition, John Wiley & Sons, New York (1999), and referenced cited therein.

The term "halo" or "halide" refers to fluoro, chloro, bromo, or iodo. Similarly, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

The term "alkyl" refers to a branched or unbranched hydrocarbon having, for example, from 1 -20 carbon atoms, and often 1-12, 1 -10, 1 -8, 1 -6, or 1 -4 carbon atoms; or for example, a range between 1-20 carbon atoms, such as 2-6, 3-6, 2-8, or 3-8 carbon atoms. As used herein, the term “alkyl” also encompasses a “cycloalkyl”, defined below. Examples include, but are not limited to, methyl, ethyl, 1 -propyl, 2- propyl (/so- propyl), 1 -butyl, 2-methyl-1 -propyl ( isobutyl ), 2-butyl (sec-butyl), 2-methyl- 2-propyl (f-butyl), 1 -pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3- methyl-1 -butyl, 2-methyl-1 -butyl, 1 -hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3- methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3- dimethyl-2-butyl, 3,3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like. The alkyl can be unsubstituted or substituted, for example, with a substituent described below or otherwise described herein. The alkyl can also be optionally partially or fully unsaturated. As such, the recitation of an alkyl group can include an alkenyl group or an alkynyl group. The alkyl can be a monovalent hydrocarbon radical, as described and exemplified above, or it can be a divalent hydrocarbon radical (i.e., an alkylene).

An alkylene is an alkyl group having two free valences at a carbon atom or two different carbon atoms of a carbon chain. Similarly, alkenylene and alkynylene are respectively an alkene and an alkyne having two free valences at two different carbon atoms.

The term "cycloalkyl" refers to cyclic alkyl groups of, for example, from 3 to 10 carbon atoms having a single cyclic ring or multiple condensed rings. Cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantyl, and the like. The cycloalkyl can be unsubstituted or substituted.

The term "heterocycloalkyl" or “heterocyclyl” refers to a saturated or partially saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3 to 10 membered, more preferably 4 to 7 membered. Examples of suitable heterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, morpholino, 1 ,3-diazapane, 1 ,4-diazapane, 1 ,4-oxazepane, and 1 ,4-oxathiapane. The group may be a terminal group or a bridging group.

As used herein, the term "substituted" or “substituent” is intended to indicate that one or more (for example, in various embodiments, 1 -10; in other embodiments, 1 -6; in some embodiments 1 , 2, 3, 4, or 5; in certain embodiments, 1 , 2, or 3; and in other embodiments, 1 or 2) hydrogens on the group indicated in the expression using “substituted” (or “substituent”) is replaced with a selection from the indicated group(s), or with a suitable group known to those of skill in the art, provided that the indicated atom’s normal valency is not exceeded, and that the substitution results in a stable compound. Suitable indicated groups include, e.g., alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, carboxyalkyl, alkylthio, alkylsulfinyl, and alkylsulfonyl. Substituents of the indicated groups can be those recited in a specific list of substituents described herein, or as one of skill in the art would recognize, can be one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, and cyano.

Stereochemical definitions and conventions used herein generally follow S.P.

Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book

Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic

Compounds”, John Wiley & Sons, Inc., New York, 1994. The compounds of the invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof, such as racemic mixtures, which form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S. are used to denote the absolute configuration of the molecule about its chiral center(s).

The term “EC50” is generally defined as the concentration required to kill 50% of the cells in 24 hours.

Embodiments of the Invention

This disclosure provides a compound represented by Formula I:

or a pharmaceutically acceptable salt thereof; wherein

R¹ is -(Ci-C4)alkyl substituted with one or more halo;

R² and R³ are each independently — (Ci-C6)alkyl;

R⁴ and R⁵ are each independently H, CFte, -(Ci-C6)alkyl, or-C(=0)(Ci-C6)alkyl wherein the (Ci-C6)alkyl moiety is substituted with at least one heteroatom, or a combination thereof; and

R⁶ is H or -(Ci-Ce)alkyl.

In various embodiments, R¹ is CF3, CFIF2, CF2CF3, or -(Ci-C4)alkyl substituted with one or more fluorine atoms. In various embodiments, R² and R³ are each independently methyl or ethyl. In various embodiments, R⁴ and R⁵ are H.

In various embodiments, R⁴ is FI and R⁵ is -C(=0)CFl2NFl2, -C(=0)CFl2CFl2NFl2, -C(=0)CH(NH2)CH 0H, -C(=0)CH(NH2)CH2C(=0)0H, -C(=0)CH(NH2)CH2-

(imidazolyl), -C(=0)(pyrrolidinyl), -CH2CH2NH2, -CH2CH2NHCH3. In some embodiments R⁵ is -C(=0)CH(NH2)CH 0H, -C(=0)CH(NH2)CH2C(=0)0H,

-C(=0)CFI(NFl2)CFl2-(imidazolyl), or — C(=0)(pyrrolidinyl). In some embodiment, stereoisomers of Formula I exist in (R) or (S) chirality. In various embodiments, R⁶ is H.

In various embodiments, the compound is any one of compounds 1 to 9:

or a pharmaceutically acceptable salt thereof.

In various embodiments, the compound is N-(3-aminophenyl)-4-(5-(2,5- dimethylphenyl)-3-(trifluoromethyl)-1 H-pyrazol-1-yl)benzenesulfonamide (1) or a pharmaceutically acceptable salt thereof. In various embodiments, the methine moiety of one of compounds 4, 5, 6 or 7 has the (S)-configuration. In various embodiments, the methine moiety of one of compounds 4, 5, 6 or 7 has the (^-configuration.

In some embodiments, the compound is any one of compounds 4a to 7a: (S)-2- amino-N-(3-((4-(5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1 H-pyrazol-1 - yl)phenyl)sulfonamido)phenyl)-3-hydroxypropanamide (4a); (S)-2-amino-N-(3-((4-(5- (2,5-dimethylphenyl)-3-(trifluoromethyl)-1 H-pyrazol-1 -yl)phenyl)sulfonamido)phenyl)- 3-(1 H-imidazol-4-yl)propenamide (5a); (R)-N-(3-((4-(5-(2,5-dimethylphenyl)-3-

(trifluoromethyl)-l H-pyrazol-1 -yl)phenyl)sulfonamido)phenyl)pyrrolidine-2- carboxamide (6a); (S)-3-amino-4-((3-((4-(5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-

1 H-pyrazol-1 -yl)phenyl)sulfonamido)phenyl)amino)-4-oxobutanoic acid (7a); or an enantiomer thereof and their pharmaceutically acceptable salts.

In various embodiments, the compound can selectively inhibit Sarcoendoplasmic Reticulum Calcium ATPase isoform 2 (SERCA2) at an ECso at least two-fold more effectively compared to the compound’s ECso for inhibition of cyclooxygenase-2 (COX- 2). In various embodiments, the compound is at least three-fold more selective at inhibiting SERCA2 compared to COX-2. In various embodiments, the selectivity is at least five-fold, at least 10-fold, at least 25-fold, at least 100-fold, or at least 1000-fold better.

Also, this disclosure provides a composition comprising a compound disclosed herein and a pharmaceutically acceptable carrier.

Additionally, this disclosure provides a method for the treatment of cancer in a subject in need of cancer therapy comprising administering an effective amount of a compound disclosed herein or a composition of a compound disclosed herein, thereby treating the cancer in the subject. In various embodiments, the cancer is breast cancer. In various embodiments, the breast cancer is triple negative breast cancer. In various embodiments, the cancer is treated by inhibiting the production of Notch-1 maturation via inhibition of SERCA2 wherein apoptosis is selectively induced in the cancer.

In various embodiments, the effective amount is about 10 mg/kg/day to about 200 mg/kg/day. In various embodiments, the effective amount is about 25 mg/kg/day to about 50 mg/kg/day. In various embodiments, the effective amount is about 1 mg/kg or greater. In various embodiments, the effective amount is about 2000 mg/kg or less. In various embodiments, the effective amount is up to the maximum tolerated dose in the subject. In various embodiments, administration is for 1 day, 7 days, 28 days, 90 days, at least 1 day, or at least 5 days.

In various embodiments, the compound is N-(3-aminophenyl)-4-(5-(2,5- dimethylphenyl)-3-(trifluoromethyl)-1 H-pyrazol-1-yl)benzenesulfonamide (1) or a pharmaceutically acceptable salt thereof. In various embodiments, the method further comprises administering a second agent, such as an anticancer agent, sequentially or simultaneously. In various embodiments, the second anticancer agent is Paclitaxel or Gemcitabine.

BRIEF DESCRIPTION OF THE METHODS AND RESULTS

Experimental Methods: Using an in vitro screening method to guide our development, we have developed a pharmacophore and made structural changes to enhance anti-cancer activity while reducing of COX-2 inhibitory activity. In this effort, we have identified compound 1 , a coxib analog with improved antitumor activities and minimal COX-2 inhibitory properties. We have further tested compound 1 in nu/nu athymic mice xenograft with human triple negative breast cancer cell line (MDA MB 231 ). T umor volume and animal weights were measured three times a week with for 28 days. At the end of the study, tumor volume was correlated with molecular markers using RT-PCR and IHC staining.

Experimental Results: Our screening identified compound 1 as a promising anti-cancer compound with in vitro and affirmed in xenograft triple negative breast cancer (TNBC) animal model. Compound 1 has EC50 <20 mM which was more potent than Di-methyl Celecoxib (DMC) and Celecoxib (CBX) with EC50 of ~30 mM and ~40 mM, respectively. Molecular dissection using western blot showed that compound 1 inhibited SERCA2 in both a time- and exposure-dependent manner. SERCA2 inhibition correlated with reduction of Notch 1 maturation. In vivo studies using oral administration of compound 1 shown optimal antitumor activity between 25-50 mg/kg/day, which resulted in >85% tumor reduction when compared to vehicle after 28 days of treatment. The safety of compound 1 was assessed for 48 days at 100 mg/kg/day, where no detectable biochemical, hematologic or histological toxicities were detected. RT-PCR analysis of residual tumor correlated with cell death indicated through the activation of DR5 marker. Finally, compound 1 treatment decreased angiogenesis markers VEGF-oc and IL-8, corresponding to reduction in microvessel formation (Table 1). Table 1 : C1 EC50 was evaluated several breast cancer cell lines against DMC and CBX using cell viability assay. Across all cell lines, potency of compounds follows a specific pattern, C1 > DMC > CBX as the average EC50 for C1 , DMC and CBX are 16.76 , 28.0, and 41.1 mM, respectively.

Using C1 as our prototype, we have identified three promising anti-cancer compounds: C8, C9, and C11. MDA-MB-231 cellular viability of C8, C9 and C11 were evaluated, where the corresponding EC50 of 12.37, 10.15 and 8.60 mM, respectively. The antitumor activity of these compounds corresponded to intracellular accumulation when MDA-MB-231 were with 10 mM of C1 analogues when using a quantitative LCMS assay. Our current finding showed C11 was the most potent as compare to other analogues (Table 2).

Molecular characterization of C1 analogues showed that these compounds continue to target SERCA2, where the reduction of SERCA2 corresponded with increased GRP78 expression of GRP78 after 1 hour of treatment. This verify that our new analogues inhibition of SERCA2, can activate UPR in MDA-MB-231

Table 2: C1 analogues were synthesized and tested in MDA-MB-231 cell line for efficacy using MTT assay. Screening results show three lead compounds, C7, C8 and C9, that have improved EC50 compared to C1.

Cell viability to determine EC50 was evaluated through calorimetric (3-(4, 5- dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide OR MTT assay using. To study C1 molecular activity, we treated MDA-MB-231 with C1 and measured protein regulation through western blot. Cell lysate was collected on ice using M-PER solution (Thermofisher, cat: 78501) with protease inhibitor cocktail. Samples were separated using gradient PAGE 4-12%. Protein is transferred onto PVDF through semi-dry transfer. PDVF is then blocked for 1-hour in 5% milk before adding primary antibody and incubating at 4°C overnight. Protein detection using chemiluminescence was analyzed using Chemidoc. We have further tested C1 in nu/nu athymic mice xenograft with human breast cancer cell line (MDA MB 231 ). Tumor volume and animal weights were measured three times a week for 28 days. At the end of the study, tumor volume was correlated with molecular markers using RT-PCR and IHC staining.

Compound 1 is a bioavailable coxib analogue with minimal COX-2 inhibition while possessing broad spectrum antitumor activity, including TNBC. Treatment with compound 1 leads to dosage dependent antitumor properties as a consequence of specific SERCA2 inhibition. In dosage escalation studies up to 100 mg/kg/day, no detectable biochemical and histological toxicities were detected. With these properties, compound 1 will be an effective component in the treatment of TNBCs, where new and effective therapies are much needed.

C1 has a significantly improved antitumor potency against various breast cancer lines as compared to Di-Methyl Celecoxib (DMC) and Celecoxib (CBX). The activity could further be explained through the improved inhibition of SERCA2 with C1 treatment compared to DMC and CXB. Reduced expression of SERCA2 correlated with increase of GRP78 expression, an important component in the unfolded protein response (UPR) pathway, where C1 treatment increased caspase-3 cleavage and DR5 up-regulation for the activation of necrosis. C1 was able to decrease SERCA2 expression after 12 hours of treatment followed by the activation of GRP78 expression. In this process, protein misfolding accumulates in the same time frame with the increased detection of immature Notchl and the degradation of MRP1. Prolonged treatment of 24 and 48 hours later show cells exhibiting apoptosis and necrosis.

In dosage escalation tumor xenograft studies up to 100 mg/kg/day, no detectable biochemical and histological toxicities were detected. Treatment with 25mg/kg/day given orally resulted to the increased mRNA expression of DR5 and the decrease of Notchl . Treatment with 50mg/kg of C1 was more potent in reducing RNA expression of the targeted gene when compared to vehicle.

Vegfa expression was also seen to decrease with compound 1 treatment, suggesting that C1 also has anti-angiogenesis properties. Further exploration of angiogenesis with C1 treatment shows decrease of microvessel density with increase of C1 treatment in mice. Based on these properties, compound 1 may be an effective component for the treatment of triple-negative-breast-cancer (TNBCs), where effective treatments are needed. Key Findings: (a) C1 has increased inhibition of SERCA2, which leads to the greater activation of apoptosis and necrosis, seen with cleaved caspase-3 and DR5 respectively.

(b) C1 activation leads to the inhibition of SERCA2 expression, followed by increased expression of GRP78, immature Notchl , and degradation of MRP1. (c) Prolonged treatment with compound 1 after 24 and 48 hours leads to the activation of apoptosis and necrosis.

(d) An in-vivo study has shown significant reduction of tumor growth after 28 days of treatment and reverse transcription-polymerase chain reaction (RT-PCR) of ER stress markers in tumors, were reduced with increasing compound 1 dosing. (e) An increasing dose of compound 1 also reduces angiogenesis biomarkers in tumor models with reduction of gene expression and tumor micro-vessel density wit anti-CD33 staining.

Conclusions: Compound 1 is a bioavailable compound with minimal COX-2 inhibition while retaining antitumor activity in a variety of tumor types, including colon, lung, hematologic (e.g. leukemia and lymphomas) and breast cancers. Treatment with Compound 1 leads to dosage-dependent antitumor properties as a consequence of specific SERCA2 inhibition. In dosage escalation tumor xenograft studies up to 100 mg/kg/day, no detectable biochemical and histological toxicities were detected. With these properties, Compound 1 will be an effective therapeutic for the treatment of TNBCs, where new and effective therapies are much needed.

Pharmaceutical Formulations: The compounds described herein can be used to prepare therapeutic pharmaceutical compositions, for example, by combining the compounds with a pharmaceutically acceptable diluent, excipient, or carrier. The compounds may be added to a carrier in the form of a salt or solvate. For example, in cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate.

Examples of pharmaceutically acceptable salts are organic acid addition salts, formed with acids that form a physiologically acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, a-ketoglutarate, and b- glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable ionic compound. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be prepared by analogous methods.

The compounds of the formulas described herein can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms. The forms can be specifically adapted to a chosen route of administration, e.g., oral or parenteral administration, by intravenous, intramuscular, topical or subcutaneous routes.

The compounds described herein may be systemically administered in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. For oral administration, compounds can be enclosed in hard- or soft-shell gelatin capsules, compressed into tablets, or incorporated directly into the food of a patient's diet. Compounds may also be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations typically contain at least 0.1% of active compound.

The percentage of the compositions and preparations can vary and may conveniently be from about 0.5% to about 60%, about 1% to about 25%, or about 2% to about 10%, of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions can be such that an effective dosage level can be obtained.

The tablets, troches, pills, capsules, and the like may also contain one or more of the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; and a lubricant such as magnesium stearate. A sweetening agent such as sucrose, fructose, lactose or aspartame; or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring, may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and flavoring such as cherry or orange flavor. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices. The active compound may be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can be prepared in glycerol, liquid polyethylene glycols, triacetin, or mixtures thereof, or in a pharmaceutically acceptable oil. Under ordinary conditions of storage and use, preparations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions, dispersions, or sterile powders comprising the active ingredient adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and/or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by agents delaying absorption, for example, aluminum monostearate and/or gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, optionally followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation can include vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the solution.

Useful dosages of the compounds described herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Patent No. 4,938,949 (Borch et al.). The amount of a compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular compound or salt selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will be ultimately at the discretion of an attendant physician or clinician. The compound can be conveniently administered in a unit dosage form, for example, containing 5 to 1000 mg/m², conveniently 10 to 750 mg/m2, most conveniently, 50 to 500 mg/m² of active ingredient per unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

The invention provides therapeutic methods of treating cancer in a mammal, which involve administering to a mammal having cancer an effective amount of a compound or composition described herein. A mammal includes a primate, human, rodent, canine, feline, bovine, ovine, equine, swine, caprine, bovine and the like. Cancer refers to any various type of malignant neoplasm, for example, colon cancer, breast cancer, melanoma and leukemia, and in general is characterized by an undesirable cellular proliferation, e.g., unregulated growth, lack of differentiation, local tissue invasion, and metastasis.

The ability of a compound of the invention to treat cancer may be determined by using assays well known to the art. For example, the design of treatment protocols, toxicity evaluation, data analysis, quantification of tumor cell kill, and the biological significance of the use of transplantable tumor screens are known The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

EXAMPLES

Example 1. Pharmaceutical Dosage Forms: The following formulations illustrate representative pharmaceutical dosage forms that may be used for the therapeutic or prophylactic administration of a compound of a formula described herein, a compound specifically disclosed herein, or a pharmaceutically acceptable salt or solvate thereof (hereinafter referred to as 'Compound X'): (i) Tablet 1 mq/tablet

'Compound X' 100.0

Lactose 77.5

Povidone 15.0

Croscarmellose sodium 12.0

Microcrystalline cellulose 92.5

Magnesium stearate 3.0

300.0

(ii) Tablet 2 mq/tablet

'Compound X' 20.0

Microcrystalline cellulose 410.0

Starch 50.0

Sodium starch glycolate 15.0

Magnesium stearate 5.0

500.0

(iii) Capsule mq/capsule

'Compound X' 10.0

Colloidal silicon dioxide 1.5

Lactose 465.5

Pregelatinized starch 120.0

Magnesium stearate 3.0

600.0

(iv) Injection 1 (1 mq/mU mq/mL

'Compound X' (free acid form) 1.0

Dibasic sodium phosphate 12.0

Monobasic sodium phosphate 0.7

Sodium chloride 4.5

1.0 N Sodium hydroxide q.s. solution

(pH adjustment to 7.0-7.5)

Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mq/mU mq/mL

'Compound X' (free acid form) 10.0

Monobasic sodium phosphate 0.3

Dibasic sodium phosphate 1.1

Polyethylene glycol 400 200.0

0.1 N Sodium hydroxide q.s. solution

(pH adjustment to 7.0-7.5) Water for injection q.s. ad 1 mL

(vi) Aerosol mq/can

'Compound X' 20

Oleic acid 10

Trichloromonofluoromethane 5,000

Dichlorodifluoromethane 10,000

Dichlorotetrafluoroethane 5,000

(vii) Topical Gel 1 wt.%

'Compound X' 5% Carbomer 934 1.25% Triethanolamine q.s. (pH adjustment to 5-7) Methyl paraben 0.2% Purified water q.s. to 100g

(viii) Topical Gel 2 wt.%

'Compound X' 5% Methylcellulose 2% Methyl paraben 0.2% Propyl paraben 0.02% Purified water q.s. to 100g Topical Ointment wt.% ompound X' 5%

Propylene glycol 1%

Anhydrous ointment base 40%

Polysorbate 80 2%

Methyl paraben 0.2%

Purified water q.s. to 100g

(x) Topical Cream 1 wt.%

'Compound X' 5%

White bees wax 10%

Liquid paraffin 30%

Benzyl alcohol 5%

Purified water q.s. to 100g wt.%

(xi) Topical Cream 2

'Compound X' 5%

Stearic acid 10%

Glyceryl monostearate 3%

Polyoxyethylene stearyl ether 3%

Sorbitol 5% Isopropyl palmitate 2 % Methyl Paraben 0.2% Purified water q.s. to 100g

These formulations may be prepared by conventional procedures well known in the pharmaceutical art. It will be appreciated that the above pharmaceutical compositions may be varied according to well-known pharmaceutical techniques to accommodate differing amounts and types of active ingredient 'Compound X'. Aerosol formulation (vi) may be used in conjunction with a standard, metered dose aerosol dispenser. Additionally, the specific ingredients and proportions are for illustrative purposes. Ingredients may be exchanged for suitable equivalents and proportions may be varied, according to the desired properties of the dosage form of interest.

While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

What is claimed is:

1. A compound represented by Formula I:

or a pharmaceutically acceptable salt thereof; wherein

R¹ is CF₃, CHF , or CF2CF3;

R² and R³ are each independently -(Ci-C6)alkyl;

R⁴ and R⁵ are each independently H,— (Ci -Cejalkyl, or -C(=0)(Ci-Cs)alkyl wherein the (Ci-Cs)alkyl moiety is substituted with 1-6 heteroatoms, interrupted with 1-6 heteroatoms, forms a heterocycle, or a combination thereof; and R⁶ is H or -(Ci-Ce)alkyl.

2. The compound of claim 1 wherein R¹ is CF3.

3. The compound of claim 1 wherein R² and R³ are each independently methyl or ethyl.

4. The compound of claim 1 wherein R⁴ and R⁵ are H.

5. The compound of claim 1 wherein R⁴ is FI and R⁵ is -C(=0)CFl2NFl2,

-C(=0)CH2CH NH2, -C(=0)CH(NH2)CH 0H, -C(=0)CH(NH2)CH2C(=0)0H,

-C(=0)CH(NH2)CH2-(imidazolyl), -C(=0)(pyrrolidinyl), -CH2CH2NH2, or -CH2CH2NHCH3.

6. The compound of claim 1 wherein R⁶ is FI.

7. The compound of claim 1 wherein the compound is N-(3-aminophenyl)-4-(5- (2,5-dimethylphenyl)-3-(trifluoromethyl)-1 H-pyrazol-1-yl)benzenesulfonamide (1) or a pharmaceutically acceptable salt thereof.

8. The compound of claim 1 wherein the compound is any one of compounds 1 to 9:

or a pharmaceutically acceptable salt thereof.

9. The compound of claim 8 wherein the methine moiety of one of compounds 4, 5, 6 or 7 has the (S)-configuration.

10. The compound of claim 8 wherein the methine moiety of one of compounds 4, 5, 6 or 7 has the (^-configuration.

11. The compound of any one of claims 1-10 wherein the compound selectively inhibits Sarcoendoplasmic Reticulum Calcium ATPase isoform 2 (SERCA2) at an EC50 at least two-fold greater compared to the compound’s EC50 for inhibition of cyclooxygenase-2 (COX-2).

12. A composition comprising the compound of any one of claims 1-11 and a pharmaceutically acceptable carrier.

13. A method for the treatment of cancer in a subject in need of cancer therapy comprising administering an effective amount of the compound of any one of claims 1-11 or the composition of claim 12, thereby treating the cancer in the subject.

14. The method of claim 13 wherein the cancer is breast cancer.

15. The method of claim 14 wherein the breast cancer is triple negative breast cancer.

16. The method of claim 13 wherein the cancer is treated by inhibiting the production of Notch-1 maturation via inhibition of SERCA2 wherein apoptosis is selectively induced in the cancer.

17. The method of claim 13 wherein the effective amount is about 10 mg/kg/day to about 200 mg/kg/day.

18. The method of claim 17 wherein the effective amount is about 25 mg/kg/day to about 50 mg/kg/day for at least 5 days.

19. The method of claim 13 wherein the compound is N-(3-aminophenyl)-4-(5-(2,5- dimethylphenyl)-3-(trifluoromethyl)-1 H-pyrazol-1-yl)benzenesulfonamide (1) or a pharmaceutically acceptable salt thereof.

20. The method of claim 13 further comprising administering a second anticancer agent sequentially or simultaneously.

21. The method of claim 20 wherein the second anticancer agent is Paclitaxel or Gemcitabine.