All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
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This invention is directed to anticancer compounds, pharmaceutical compositions comprising the same, methods of making anticancer compounds, and methods of treating cancer with compounds and pharmaceutical compositions.
Glial tumors account for nearly 50% of all adult primary intracranial neoplasms, among which GBM is the most aggressive and practically incurable. Glioblastoma multiforme (GBM) is a highly lethal brain tumor for which therapeutic options are limited. Rapidly growing and highly invasive GBM cells rely on both glycolysis and mitochondrial respiration to generate sufficient amounts of ATP and intermediate metabolites (anaplerosis). A large variety of different genetic and epigenetic modifications have been found in GBMs, among which p53 mutations, EGF receptor amplification, and PTEN mutations are most common. However, gene therapy, molecular and immunological approaches targeting these molecules and their pathways, as well as recently tested antibodies against immune checkpoint inhibitors have yet to produce improvements in patient outcomes.
The present invention provides anticancer compounds, pharmaceutical compositions comprising anticancer compounds, methods of synthesizing anticancer compounds, and methods of using anticancer compounds to treat disease.
Aspects of the invention are directed towards an anticancer compound comprising the following formula:
In an embodiment of Formula (I), R13 can comprise substituted cycloalkyl, substituted aryl, substituted heterocycle, or substituted aromatic heterocycle; R14 can comprise hydrogen or double bounded oxygen; R15 comprises hydrogen, substituted alkyl, or substituted aryl; R16 can comprise hydrogen, substituted alkyl, or substituted aryl; R17 can comprise nitrogen or carbon; R19 can comprise hydrogen, hydroxy, amino, substituted amino, alkyl, hydroxyalkyl, cycloalkyl, heterocycle, aryl, hydroxyaryl, aromatic heterocycle, or hydroxyaryl; R20 can comprise hydrogen, hydroxy, amino, substituted amino, alkyl, hydroxyalkyl, cycloalkyl, heterocycle, aryl, hydroxyaryl, aromatic heterocycle, or hydroxyaryl; or any combination thereof.
In embodiments, an anticancer compound comprises the following formula:
In an embodiment of Formula (II), R1 can comprise hydrogen, halogen, alkyl, oxyalkyl, aryl, oxyaryl, halogen, cyano, nitro, carboxyl, or carboxyalkyl; R2 can comprise hydrogen, halogen, alkyl, oxyalkyl, aryl, oxyaryl, halogen, cyano, nitro, carboxyl, or carboxyalkyl; R3 can comprise hydrogen, halogen, alkyl, or aryl; R4 can comprise hydrogen, substituted alkyl, or substituted aryl; R5 can comprise hydrogen, substituted alkyl, or substituted aryl; R6 can comprise hydrogen, hydroxy, amino, substituted amino, alkyl, hydroxyalkyl, cycloalkyl, heterocycle, aryl, hydroxyaryl, aromatic heterocycle, or hydroxyaryl; R7 can comprise hydrogen, hydroxy, amino, substituted amino, alkyl, hydroxyalkyl, cycloalkyl, heterocycle, aryl, hydroxyaryl, aromatic heterocycle, or hydroxyaryl; or any combination thereof. In embodiments, R6-R7 can be part of cycle, such as but not limited to pyrrolidine, piperidine, morpholine, pyrrole, and their alkyl substituents. In embodiments, Z can comprise 2H, O, NNH2, and NNHR, where R can be alkyl, aryl, acyl, aryloyl or any combination thereof.
In embodiments, the anticancer compound comprises one of the following structures:
In embodiments, R1, R2, R3 can comprise H, alkyl, O-alkyl, nitro, cyano, or halogen; R4, R5, R6, R9 can comprise H or alkyl; R7 can comprise alkyl, substituted alkyl, hydroxy alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, or amino; R5 can comprise aryl, substituted aryl, heteroaryl, substituted heteroaryl, CO-aryl, CO-heteroaryl, or CO-linker; R10 can comprise alkyl, hydroxyalkyl, substituted aryl, or substituted heteroaryl; R1, R12 can comprise H, alkyl, aryl, heteroaryl, alkyl with linker, or aryl with linker; X can comprise CO, CH2, O, CC, or CH═CH; Z can comprise O, or H2; any combination thereof
For example, the anticancer compound comprises any one of the structures of Table 2.
In embodiments, the anticancer compound comprises any of the following structures:
a pharmaceutically acceptable salt thereof, or a derivative thereof.
In embodiments, the anticancer compound comprises the following formula:
In embodiments, R1 can be H, alkyl, hydroxyalkyl. In embodiments, R2 can be H, alkyl, hydroxyalkyl, polyhydroxyalkyl, aminoalkyl, dialkylaminoalkyl. In embodiments, R1-R2 can be (CH2)n, (CH2)nO(CH2)m, (CH2)nCHOH(CH2)m, (CH2)nNCH3(CH2)m, where n and m are 1, 2, 3, 4, or 5.
For example, the anticancer compound comprises any one of the structures of Table 3.
In embodiments, the anticancer compound comprises any one of the structures of Table 3. For example, the anticancer compound comprises any of the following structures:
a pharmaceutically acceptable salt thereof, or a derivative thereof.
In embodiments, the anti cancer compound comprises the following formula:
In embodiments, R1 can be H or CH3; R2 can be OH, OCH3, or any combination thereof; R3 can be OH, OCH3, or any combination thereof; R4 can be OH, OCH3, or any combination thereof; R5 can be OH, OCH3, or any combination thereof,
For example, the anti cancer compound can comprise any of the following structures:
pharmaceutically acceptable salt thereof, or a derivative thereof.
In embodiments, the anti cancer compound comprises the following formula:
In embodiments, R1 can be H, CH3, or (CH2)5; R2 can be H, CH3, (CH2)5.
For example, the anti cancer compound comprises any of the following structures:
pharmaceutically acceptable salt thereof, or a derivative thereof.
In embodiments, the anti cancer compound can be the following formula:
In embodiments, R1 can be H or CH3, R2 can be OH and/or OCH3, R3 can be OH and/or OCH3, R4 can be OH and/or OCH3, and/or R5 can be OH and/or OCH3.
For example, the anti cancer compound comprises any of the following structures:
pharmaceutically acceptable salt thereof, or a derivative thereof.
In embodiments, the anti cancer compound can be the following formula:
In embodiments, n can be 0, 1 or 2; and m can be 1 or 2.
For example, the anti cancer compound comprises any of the following structures:
pharmaceutically acceptable salt thereof, or a derivative thereof.
In embodiments, the anti cancer compound can be the following formula:
In embodiments, R1 can be H, OH, Cl, Br, NO2, and R2 can be H, OH, Cl, Br, NO2.
For example, the anti cancer compound can be any of the following structures:
pharmaceutically acceptable salt thereof, or a derivative thereof.
In embodiments, the anti cancer compound comprises the following formula:
In embodiments, R can be H, Cl, or Br; and n can be 0, 1, 2, or 3.
For example, the anti cancer compound can be any of the following structures:
a pharmaceutically acceptable salt thereof, or a derivative thereof.
In embodiments, the anti cancer compound comprises the following formula:
In embodiments, R can be H, Cl, or Br; n can be 0 or 1; m can be 1, 2, or 3.
For example, the anti cancer compound comprises any of the following structures:
pharmaceutically acceptable salt thereof, or a derivative thereof.
Further, aspects of the invention are directed towards a pharmaceutical composition comprising an anticancer compound described herein and a pharmaceutically acceptable carrier and/or pharmaceutically acceptable excipient. For example, the pharmaceutically acceptable carrier is albumin. For example, the pharmaceutical compositions can comprise an anticancer compound of Table 2, Table 3, or Example 4.
Still further, aspects of the invention are directed towards a method of synthesizing an anticancer compound as described herein. For example, the method comprises the schematic as indicated in
Embodiments are directed towards a method of synthesizing 2-[4-(4-chlorobenzoyl)phenoxy]-N-(2-hydroxyethyl)-N,2-dimethylpropanamide (PP1), the method comprising: Dichloromethane (20 ml) suspension of fenofibric acid (318.75 mg; 1 mmol) and oxalyl chloride (0.25 mL; 380.1 mg; 3 mmol), and one drop od N,N-dimethylformamide was stirred at room temperature for 5 hours. Solvent was evaporated under reduced pressure. White solid residue was resolved in in dichloromethane (10 ml) and again evaporated to the solid residue. This solid residue was dissolved in dichloromethane (20 ml) and at room temperature with stirring dichloromethane (10 ml) solution of 2-(methylamino)ethanol (0.24 ml; 225 mg; 3 mmol) was gradually added. Reaction mixture was stirred at room temperature. Dichloromethane reaction mixture was washed with water (3×15 ml), 5% hydrochloric acid (3×15 ml), water (3×15 ml), 10% sodium carbonate (3×15 ml), and finally with water (3×15 ml) and dried over anhydrous sodium carbonate. Solvent was evaporated under reduced pressure to result in viscous pale-yellow liquid (390 mg). Product was crystalized from dichloromethane/hexane (30 ml; 1:4) at room temperature by slow solvent evaporation to ⅕ original volume and formed crystals were washed with ice cold hexane. Isolated yield 340 mg (90%).
Embodiments are also directed towards a method of synthesizing 2-[4-(4-chlorobenzoyl)phenoxy]-N-(2-hydroxyethyl)-N,2-dimethylpropanamide (PP1), the method comprising: Dichloromethane (100 ml) solution of fenofibric acid (637 mg; 2 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride (EDC, 576; 3 mmol), and 2-(methylamino)ethanol (600 mg; 8 mmol) was stirred at room temperature overnight. Dichloromethane solution was washed with 5% hydrochloric acid (5×20 ml), water (5×20 ml), 10% sodium carbonate (5×20 ml), water (3×20 ml) and dried over anhydrous sodium carbonate. After solvent evaporation, oily residue was crystalized from dichloromethane/hexane (1:4) to give pure product (640 mg; 85% yield).
Embodiments are also directed towards a method of synthesizing 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl-1-(4-methylpiperazin-1-yl)propan-1-one (PP2), the method comprising: Dichloromethane (30 ml) of fenofibric chloride (1 mmol; prepared as described above for PP1 preparation) was slowly added in stirring water (5 ml) solution of sodium carbonate (216 mg; 2 mmol) with tetrahydrofuran (10 ml) and of 1-methylpiperazine (0.13 ml; 120 mg; 1.2 mmol). Resulting reaction mixture was stirred at room temperature for one hour. Additional water (30 ml) was added and organic layer was separated, washed with water (3×10 ml), 5% hydrochloric acid (3×10 ml), 10% sodium carbonate (3×10 ml), water and dried over anhydrous sodium carbonate. After evaporation oily residue was crystalized from dichloromethane hexane to give 350 mg (88%) of pure product.
Further, embodiments are directed towards a method of synthesizing 2-[4-(4-chlorobenzoyl)phenoxy]-N,2-dimethyl-N-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]propenamide (PP3), the method comprising: Fenofibric acid chloride prepared from fenofibric acid (318.75 mg; 1 mmol) and oxalyl chloride (0.25 mL; 380.1 mg; 3 mmol) as described for preparation of PP1 was dissolved in dichloromethane (15 ml) and mixed with acetonitrile (20 ml) and water (10 ml) solution of N-methyl-D-glucamine (196 mg; 1 mmol) and sodium carbonate (212 mg; 2 mmol). Resulting mixture was stirred at room temperature for five minutes and solvent was evaporated under reduce pressure at room temperature. Resulting solid residue was mixed with dichloromethane (50 ml) and water (20 ml). Dichloromethane layer was separated, washed with 10% sodium carbonate (3×10 ml), water (3×10 ml) and dried over anhydrous sodium carbonate. After solvent was evaporated solid residue was washed with hexane (3×5 ml) and dried in vacuum under reduce pressure to give 350 mg (71%) of pure product.
Still further, embodiments are directed towards a method of synthesizing 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl-1-[4-(morpholin-4-yl)piperidin-1-yl]propan-1-one (PP4), the method comprising: Fenofibric acid chloride prepared from fenofibric acid (160 mg; 0.5 mmol) and oxalyl chloride (0.25 mL; 380.1 mg; 3 mmol) as described for preparation of PP1 was dissolved in dichloromethane (30 ml) and mixed with tetrahydrofuran (10 ml) solution of 4-morpholinopiperidine (100 mg; 0.6 mol), and water (10 ml) solution of sodium carbonate (106 mg; 1 mmol). Resulting mixture was stirred at room temperature for one hour. Water (20 ml) was added and organic layer was separated and extensively washed with 5% hydrochloric acid (5×20 ml), water (3×10 ml), 10% sodium carbonate (5×20 ml) and again with water (3×10 ml). After drying over anhydrous sodium carbonate solvent was evaporated to give pure product (200 mg; 85%) as oil that crystallized by standing at room temperature overnight. If necessary, the product can be further purified by crystallization from hexane or by silica gel chromatography with ethyl acetate-ethanol (5:1).
Also, embodiments are directed towards a method of synthesizing 4-(1-{2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoyl}piperidin-4-yl)morpholin-4-iumchloride (PP4HCl)—A mixture of concentrated hydrochloric acid (2 ml) and PP4 (94 mg; 0.2 mmol) was sonicated at room temperature for half an hour. Clear solution was evaporated to dryness at room temperature under reduced pressure. Residue was dissolved in dry ether (20 ml) and clear solution was left at room temperature for solvent to slowly evaporate. For white crystalline material was separated by filtration and washed with ice-cold hexane to give 80 mg (79%) of pure product.
Aspects of the invention are also directed towards a method of treating a subject afflicted with a disease, such as cancer, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein, wherein the pharmaceutical composition comprises an anticancer compound described herein. Non-limiting examples of the cancer comprise a solid tumor or a liquid cancer; a brain cancer, such as glioma; a glioma, such as an astrocytoma, an ependymoma, or an oligodendrogliuma; glioblastoma; a blood cancer, such as leukemia, lymphoma, or hemangiosarcoma.
Further, aspects of the invention are directed towards a method of attenuating abnormal cell proliferation comprising administering to the subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 12, wherein the composition attenuates abnormal cell proliferation. For example, the cell can comprise a cancer cell, such as a primary cancer cell, a brain cancer cell, or a blood cancer cell.
Still further, aspects of the invention are directed towards a method of inhibiting or delaying metastatic invasion of a cancer cell comprising administering to the subject in need thereof a therapeutically effective amount of the pharmaceutical composition described herein, wherein the composition inhibits or delays metastatic invasion of a cancer cell.
In embodiments, the pharmaceutical composition is administered orally to the subject.
Other objects and advantages of this invention will become readily apparent from the ensuing description.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
TABLE 1 shows computed properties for piper derivatives.
Anticancer effects of the lipid-lowering drug, fenofibrate (FF) have been described in the literature for a quite some time, however, FF has not been used as a direct anticancer therapy. FF in its unprocessed form accumulates in mitochondria, inhibits mitochondrial respiration, triggering a severe energy deficit and extensive glioblastoma cell death. However, FF does not cross blood brain barrier, and is quickly processed by blood and tissue esterases to form PPARα agonist, fenofibric acid (FA). In comparison to unprocessed FF, FA is much less effective in triggering cancer cell death.
The present invention provides anticancer compounds comprising chemical modifications to improve stability, water solubility, tissue penetration, and ultimately, anti-glioblastoma efficacy relative to FF. As described herein, data shows that the embodiments of the invention have improved cytotoxicity against glioblastoma cells in vitro in comparison to FF, and block mitochondrial respiration similarly to FF. In addition, embodiments of the invention are significantly more stable than FF when exposed to human blood, and have much better solubility in water when compared to FF. Mice orally administered embodiments of the invention demonstrated accumulation of the compound at therapeutically relevant concentrations in several tissues, including intracranial glioblastoma tumors, and survived the treatment without any major signs of distress. Importantly, treatment of mice bearing large intracranial glioblastoma tumors with embodiments of the invention resulted in extensive areas of necrosis within the tumor mass, thus demonstrating anti-glioblastoma efficacy of such novel metabolically active compounds. The anticancer effect of embodiments described herein is attributed to targeting cancer cell energy metabolism, which is very different in comparison to normal cells (see, for example, the Warburg effect).
Detailed descriptions of one or more preferred embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.
The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.
The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.
The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.
As used herein the term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).
Compounds of the Invention
Aspects of the invention are directed towards anticancer compounds comprising chemical compounds with improved stability, water solubility, tissue penetration, and ultimately, anti-glioblastoma efficacy relative to fenofibrate (FF).
An “anticancer compound” can refer to a compound effective in the treatment of cancer. This includes compounds, which kill the tumor cells and/or reduce the size of the tumor and/or reduce the growth and/or spreading or migration of the tumor or cancer cells. The term also encompasses traditional chemotherapeutic drugs and cytotoxic drugs. Other exemplary anti-cancer compounds include, e.g., neomycin, podophyl-lotoxin(s), TNF-alpha, calcium ionophores, calcium-flux inducing compounds, anti-tubulin drugs, colchicine, taxol, vinblastine, vincristine, vindescine, and combretastatin.
In one aspect of the invention, the anticancer compound is a compound of Formula I.
In an embodiment of Formula (I), R13 can comprise substituted cycloalkyl, substituted aryl, substituted heterocycle, or substituted aromatic heterocycle; R14 can comprise hydrogen or double bounded oxygen; R15 can comprise hydrogen, substituted alkyl, or substituted aryl; R16 comprises hydrogen, substituted alkyl, or substituted aryl; R17 can comprise nitrogen or carbon; R19 comprises hydrogen, hydroxy, amino, substituted amino, alkyl, hydroxyalkyl, cycloalkyl, heterocycle, aryl, hydroxyaryl, aromatic heterocycle, or hydroxyaryl; R20 can comprise hydrogen, hydroxy, amino, substituted amino, alkyl, hydroxyalkyl, cycloalkyl, heterocycle, aryl, hydroxyaryl, aromatic heterocycle, or hydroxyaryl; or any combination thereof.
In another aspect of the invention, the anticancer compound is a compound of Formula II.
In an embodiment of Formula (II), R1 can comprise hydrogen, halogen, alkyl, oxyalkyl, aryl, oxyaryl, halogen, cyano, nitro, carboxyl, or carboxyalkyl; R2 can comprise hydrogen, halogen, alkyl, oxyalkyl, aryl, oxyaryl, halogen, cyano, nitro, carboxyl, or carboxyalkyl; R3 can comprise hydrogen, halogen, alkyl, or aryl; R4 can comprise hydrogen, substituted alkyl, or substituted aryl; R5 can comprise hydrogen, substituted alkyl, or substituted aryl; R6 can comprise hydrogen, hydroxy, amino, substituted amino, alkyl, hydroxyalkyl, cycloalkyl, heterocycle, aryl, hydroxyaryl, aromatic heterocycle, or hydroxyaryl; R7 can comprise hydrogen, hydroxy, amino, substituted amino, alkyl, hydroxyalkyl, cycloalkyl, heterocycle, aryl, hydroxyaryl, aromatic heterocycle, or hydroxyaryl; or any combination thereof. In embodiments, R6-R7 can be part of cycle, such as but not limited to pyrrolidine, piperidine, morpholine, pyrrole, and their alkyl substituents. In embodiments, Z can comprise 2H, O, NNH2, and NNHR, where R can be alkyl, aryl, acyl, aryloyl or any combination thereof.
In still another aspect of the invention, the anticancer compound is a compound of Structure (I):
In another aspect of the invention, the anticancer compound is a compound of Structure (II):
In another aspect of the invention, the anticancer compound is a compound of Structure (III):
In another aspect of the invention, the anticancer compound is a compound of Structure (IV):
In another aspect of the invention, the anticancer compound is a compound of Structure (V):
In another aspect of the invention, the anticancer compound is a compound of Structure (VI):
In embodiments described herein R1, R2, R3 can be H, alkyl, O-alkyl, nitro, cyano, halogen; R4, R5, R6, R9 can be H or alkyl; R7 can be alkyl, substituted alkyl, hydroxy alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, hydroxy, amino; R5 can be aryl, substituted aryl, heteroaryl, substituted heteroaryl, CO-aryl, CO-heteroaryl, and CO-linker; R10 can be alkyl, hydroxyalkyl, substituted aryl, substituted heteroaryl; R1, R12 can be H, alkyl, aryl, heteroaryl, alkyl with linker, aryl with linker; X can be CH2, CHCH, CC, CO, or O; Z can be O, H2, or any combination thereof.
Non-limiting examples of compounds of the invention comprise those compounds of Table 2.
Table 2 refers to compounds of the invention.
Aspects of the invention are also directed towards an anticancer compound comprising the following formula:
For example, R1 can be H, alkyl, hydroxyalkyl. In embodiments, R2 can be H, alkyl, hydroxyalkyl, polyhydroxyalkyl, aminoalkyl, dialkylaminoalkyl. In embodiments, R1-R2 can be (CH2)n, (CH2)nO(CH2)m, (CH2)nCHOH(CH2)m, (CH2)nNCH3(CH2)m, where n and m are 1, 2, 3, 4, or 5.
Non-limiting examples of the anticancer compound of Formula (III) comprise the compounds in Table 3.
For example, the anticancer compound of Formula (III) can be:
a pharmaceutically acceptable salt thereof, or a derivative thereof.
Non-limiting examples of compounds of the invention comprise those compounds of Table 3.
Table 3 refers to compounds of the invention:
See also, for example, U.S. Pat. No. 4,235,896 (1980); U.S. Pat. No. 4,378,373 (1983); United State Patent Application US 20090054450A1; Yu, et al. European Journal of Medicinal Chemistry 2015, 106, 50-59; U.S. Pat. No. 4,146,385 (1979); Hogberg, T., et al. Acta Pharmaceutica Suecica 1976, 13, 427-438; Schimler, S. D., et al. Journal of the American Chemical Society, 2017, 137, 1452-1455; Giampietro, Letizia, et al. Medicinal Chemistry, 2014, 10, 59-65, each of which are incorporated by reference herein in their entireties.
Aspects of the invention are also directed towards an anti cancer compound comprising the following formula:
For example, R1 can be H or CH3; R2 can be OH, OCH3, or any combination thereof; R3 can be OH, OCH3, or any combination thereof; R4 can be OH, OCH3, or any combination thereof; R5 can be OH, OCH3, or any combination thereof.
Non-limiting examples of anti cancer compounds of Formula (IV) can be found in Example 4. For example, the anti cancer compound can be any of the following structures:
a pharmaceutically acceptable salt thereof, or a derivative thereof.
Aspects of the invention are also directed towards an anti cancer compound comprising the following formula:
For example, R1 can be H, CH3, or (CH2)5; R2 can be H, CH3, (CH2)5.
Non-liming examples of anti cancer compounds of Formula (V) can be found in Example 4. For example, the anti cancer compound can be any of the following structures:
pharmaceutically acceptable salt thereof, or a derivative thereof.
Aspects of the invention are also directed towards an anti cancer compound comprising the following formula:
For example, R1 can be H or CH3, R2 can be OH and/or OCH3, R3 can be OH and/or OCH3, R4 can be OH and/or OCH3, and/or R5 can be OH and/or OCH3.
Non-liming examples of anti cancer compounds of Formula (VI) can be found in Example 4. For example, the anti cancer compound can be any of the following structures:
a pharmaceutically acceptable salt thereof, or a derivative thereof.
Aspects of the invention are also directed towards an anti cancer compound comprising the following formula:
For example, n can be 0, 1 or 2; and wherein m can be 1 or 2.
Non-liming examples of anti cancer compounds of Formula (VII) can be found in Example 4. For example, the anti cancer compound can be any of the following structures:
a pharmaceutically acceptable salt thereof, or a derivative thereof.
Aspects of the invention are also directed towards an anti cancer compound comprising the following formula:
For example, R1 is H, OH, Cl, Br, NO2, and wherein R2 is H, OH, Cl, Br, NO2.
Non-liming examples of anti cancer compounds of Formula (VIII) can be found in Example 4. For example, the anti cancer compound can be any of the following structures:
a pharmaceutically acceptable salt thereof, or a derivative thereof.
Aspects of the invention are also directed towards an anti cancer compound comprising the following formula:
For example, R can be H, Cl, or Br; and wherein n can be 0, 1, 2, or 3.
Non-liming examples of anti cancer compounds of Formula (IX) can be found in Example 4. For example, the anti cancer compound can be any of the following structures:
Aspects of the invention are also directed towards an anti cancer compound comprising the following formula:
a pharmaceutically acceptable salt thereof, or a derivative thereof.
For example, R can be H, Cl, or Br; wherein n can be 0 or 1; wherein m can be 1, 2, or 3.
Non-liming examples of anti cancer compounds of Formula (X) can be found in Example 4. For example, the anti cancer compound can be any of the following structures:
a pharmaceutically acceptable salt thereof, or a derivative thereof.
Aspects of the invention are further directed towards pharmaceutical compositions comprising an anticancer compound as described herein. A “pharmaceutical composition” can refer to preparation of a compound as described herein with other chemical components such as physiologically suitable carriers and/or excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. For example, a pharmaceutical composition can comprise a compound of formula (I) and a pharmaceutically acceptable carrier. For example, a pharmaceutical composition can comprise a compound of formula (II) and a pharmaceutically acceptable carrier. For example, a pharmaceutical composition can comprise a compound of formula (III) and a pharmaceutically acceptable carrier. For example, a pharmaceutical composition can comprise a compound of formula (IV) and a pharmaceutically acceptable carrier. For example, a pharmaceutical composition can comprise a compound of formula (V) and a pharmaceutically acceptable carrier. For example, a pharmaceutical composition can comprise a compound of formula (VI) and a pharmaceutically acceptable carrier. For example, a pharmaceutical composition can comprise a compound of formula (VII) and a pharmaceutically acceptable carrier. For example, a pharmaceutical composition can comprise a compound of formula (VIII) and a pharmaceutically acceptable carrier. For example, a pharmaceutical composition can comprise a compound of formula (IX) and a pharmaceutically acceptable carrier. For example, a pharmaceutical composition can comprise a compound of formula (X) and a pharmaceutically acceptable carrier.
The terms “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used can refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered chelator.
The term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
According to the invention, a pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions.
Non-limiting examples of pharmaceutically acceptable carriers comprise solid or liquid fillers, diluents, and encapsulating substances. Including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl benzoate, propyl benzoate, talc, magnesium stearate, and mineral oil.
A pharmaceutical composition of the invention can be formulated to be compatible with its intended route of administration. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference in its entirety. Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes
Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
Embodiments of the invention can be provided as a pharmaceutically acceptable salt. The term “pharmaceutically acceptable” can refer to salts or chelating agents are acceptable from a toxicity viewpoint. The term “pharmaceutically acceptable salt” can refer to refer to ammonium salts, alkali metal salts such as potassium and sodium (including mono, di- and tri-sodium) salts (which are preferred), alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth.
Methods of Synthesis
Embodiments of the invention comprise synthetic schemes and methods to produce, make, or manufacture anticancer compounds described herein.
Exemplary synthetic schemes can be found in, for example,
For example, embodiments are directed towards a method of synthesizing 2-[4-(4-chlorobenzoyl)phenoxy]-N-(2-hydroxyethyl)-N,2-dimethylpropanamide (PP1), the method comprising: Dichloromethane (20 ml) suspension of fenofibric acid (318.75 mg; 1 mmol) and oxalyl chloride (0.25 mL; 380.1 mg; 3 mmol), and one drop od N,N-dimethylformamide was stirred at room temperature for 5 hours. Solvent was evaporated under reduced pressure. White solid residue was resolved in in dichloromethane (10 ml) and again evaporated to the solid residue. This solid residue was dissolved in dichloromethane (20 ml) and at room temperature with stirring dichloromethane (10 ml) solution of 2-(methylamino)ethanol (0.24 ml; 225 mg; 3 mmol) was gradually added. Reaction mixture was stirred at room temperature. Dichloromethane reaction mixture was washed with water (3×15 ml), 5% hydrochloric acid (3×15 ml), water (3×15 ml), 10% sodium carbonate (3×15 ml), and finally with water (3×15 ml) and dried over anhydrous sodium carbonate. Solvent was evaporated under reduced pressure to result in viscous pale-yellow liquid (390 mg). Product was crystalized from dichloromethane/hexane (30 ml; 1:4) at room temperature by slow solvent evaporation to ⅕ original volume and formed crystals were washed with ice cold hexane. Isolated yield 340 mg (90%).
As another example, embodiments are directed towards a method of synthesizing 2-[4-(4-chlorobenzoyl)phenoxy]-N-(2-hydroxyethyl)-N,2-dimethylpropanamide (PP1), the method comprising: Dichloromethane (100 ml) solution of fenofibric acid (637 mg; 2 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride (EDC, 576; 3 mmol), and 2-(methylamino)ethanol (600 mg; 8 mmol) was stirred at room temperature overnight. Dichloromethane solution was washed with 5% hydrochloric acid (5×20 ml), water (5×20 ml), 10% sodium carbonate (5×20 ml), water (3×20 ml) and dried over anhydrous sodium carbonate. After solvent evaporation, oily residue was crystalized from dichloromethane/hexane (1:4) to give pure product (640 mg; 85% yield).
As yet another example, embodiments are directed towards a method of synthesizing 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl-1-(4-methylpiperazin-1-yl)propan-1-one (PP2), the method comprising: Dichloromethane (30 ml) of fenofibric chloride (1 mmol; prepared as described above for PP1 preparation) was slowly added in stirring water (5 ml) solution of sodium carbonate (216 mg; 2 mmol) with tetrahydrofuran (10 ml) and of 1-methylpiperazine (0.13 ml; 120 mg; 1.2 mmol). Resulting reaction mixture was stirred at room temperature for one hour. Additional water (30 ml) was added and organic layer was separated, washed with water (3×10 ml), 5% hydrochloric acid (3×10 ml), 10% sodium carbonate (3×10 ml), water and dried over anhydrous sodium carbonate. After evaporation oily residue was crystalized from dichloromethane hexane to give 350 mg (88%) of pure product.
As still another example, embodiments are directed towards a method of synthesizing 2-[4-(4-chlorobenzoyl)phenoxy]-N,2-dimethyl-N-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]propenamide (PP3), the method comprising: Fenofibric acid chloride prepared from fenofibric acid (318.75 mg; 1 mmol) and oxalyl chloride (0.25 mL; 380.1 mg; 3 mmol) as described for preparation of PP1 was dissolved in dichloromethane (15 ml) and mixed with acetonitrile (20 ml) and water (10 ml) solution of N-methyl-D-glucamine (196 mg; 1 mmol) and sodium carbonate (212 mg; 2 mmol). Resulting mixture was stirred at room temperature for five minutes and solvent was evaporated under reduce pressure at room temperature. Resulting solid residue was mixed with dichloromethane (50 ml) and water (20 ml). Dichloromethane layer was separated, washed with 10% sodium carbonate (3×10 ml), water (3×10 ml) and dried over anhydrous sodium carbonate. After solvent was evaporated solid residue was washed with hexane (3×5 ml) and dried in vacuum under reduce pressure to give 350 mg (71%) of pure product.
As an example, embodiments are directed towards a method of synthesizing 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl-1-[4-(morpholin-4-yl)piperidin-1-yl]propan-1-one (PP4), the method comprising: Fenofibric acid chloride prepared from fenofibric acid (160 mg; 0.5 mmol) and oxalyl chloride (0.25 mL; 380.1 mg; 3 mmol) as described for preparation of PP1 was dissolved in dichloromethane (30 ml) and mixed with tetrahydrofuran (10 ml) solution of 4-morpholinopiperidine (100 mg; 0.6 mol), and water (10 ml) solution of sodium carbonate (106 mg; 1 mmol). Resulting mixture was stirred at room temperature for one hour. Water (20 ml) was added and organic layer was separated and extensively washed with 5% hydrochloric acid (5×20 ml), water (3×10 ml), 10% sodium carbonate (5×20 ml) and again with water (3×10 ml). After drying over anhydrous sodium carbonate solvent was evaporated to give pure product (200 mg; 85%) as oil that crystallized by standing at room temperature overnight. If necessary, the product can be further purified by crystallization from hexane or by silica gel chromatography with ethyl acetate-ethanol (5:1).
As an example, embodiments are directed towards a method of synthesizing 4-(1-{2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoyl}piperidin-4-yl)morpholin-4-iumchloride (PP4HCl)—A mixture of concentrated hydrochloric acid (2 ml) and PP4 (94 mg; 0.2 mmol) was sonicated at room temperature for half an hour. Clear solution was evaporated to dryness at room temperature under reduced pressure. Residue was dissolved in dry ether (20 ml) and clear solution was left at room temperature for solvent to slowly evaporate. For white crystalline material was separated by filtration and washed with ice-cold hexane to give 80 mg (79%) of pure product.
The skilled artisan will recognize the synthetic schemes and mechanisms can be adapted to provide different embodiments of the invention, derivative compounds, and/or pharmaceutically acceptable salts, such as those described herein.
Methods of Treatment
Aspects of the invention are directed towards methods of treating a subject with a cancer. As used herein, the terms “tumor” and “cancer” can be used interchangeably, and generally refer to a physiological condition characterized by the abnormal and/or unregulated growth, proliferation or multiplication of cells.
The terms “treat,” “treating” or “treatment” can refer to the lessening of severity of a tumor or cancer, delay in onset of a tumor or cancer, slowing the growth of a tumor or cancer, slowing metastasis of cells of a tumor or cancer, shortening of duration of a tumor or cancer, arresting the development of a tumor or cancer, causing regression of a tumor or cancer, relieving a condition caused by a tumor or cancer, or stopping symptoms which result from a tumor or cancer. The terms “treat,” “treating” or “treatment”, can include, but are not limited to, prophylactic and/or therapeutic treatments. For example, the invention is directed towards methods of reducing cell viability and/or promoting apoptosis of cancer cells by administering to a subject in need thereof a therapeutically effective amount of an anticancer compound or composition. The invention is also directed towards methods of attenuating abnormal cell proliferation, and methods of delaying or inhibiting metastatic invasion of a cancer cell.
The approach as described herein (i.e., administration of an anticancer compound or pharmaceutical composition to a subject in need thereof) will provide clinical benefit, defined broadly as any of the following: inhibiting an increase in cell volume, slowing or inhibiting worsening or progression of cancer cell proliferation, reducing primary tumor size, reducing occurrence or size of metastasis, reducing or stopping tumor growth, inhibiting tumor cell division, killing a tumor cell, sensitizing a tumor cell to a drug, radiation, or chemical, inducing apoptosis in a tumor cell, reducing or eliminating tumor recurrence.
In embodiments, the method comprises administering to the subject a therapeutically effective amount of an anticancer compound or pharmaceutical composition described herein. The term “administer” or “administration” can refer to introducing an anticancer compound or pharmaceutical composition into a subject. In general, any route of administration can be utilized. Non-limiting examples of routes of administration comprise parenteral (e.g., intravenous), intraperitoneal, oral, topical, subcutaneous, peritoneal, intraarterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into body compartments. In some embodiments, administration is intraperitoneal. Additionally, or alternatively, in some embodiments, administration is parenteral. In some embodiments, administration is intravenous. In other embodiments, administration is orally.
An anticancer compound or pharmaceutical composition can be administered to a subject in need thereof one time (e.g., as a single injection or deposition). Alternatively, administration can be once or twice daily to a subject in need thereof for a period of from about 2 to about 28 days, or from about 7 to about 10 days, or from about 7 to about 15 days. It can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof. It can also be administered once or twice daily to a subject for a period of years or until the death of the subject, such as can be the case for a subject suffering from pancreatic cancer.
An anticancer compound or pharmaceutical composition can be incorporated into a delivery system for administration to a subject. The term “delivery system” can refer to any form of a composition, such as a solid, semi-solid, or liquid, having an anticancer compound or such pharmaceutical composition incorporated therein which can deliver the anticancer compound to/into a cell, such as a cancer cell. The delivery system can be a biodegradable delivery system. The pharmaceutical composition can be designed to have a desired release rate of the anticancer compound incorporated therein. The delivery system comprising the anticancer compound can be administered to a subject as described herein.
In embodiments, the delivery system comprises a nanoparticle. The term “nanoparticle” can refer to a carrier structure which is biocompatible with and sufficiently resistant to chemical and/or physical destruction by the environment of use such that a sufficient amount of the nanoparticles remain substantially intact after injection into the blood stream, or given intraperitoneally or orally, so as to be able to reach a cancer cell. Nanoparticles can be solid colloidal particles ranging in size from 1 to 1000 nm. Drugs, such as an anticancer compound described herein, or other relevant materials (e.g., those used for diagnostic purposes in nuclear medicine or in radiation therapy) can be dissolved within the nanoparticles, entrapped, encapsulated and/or adsorbed or attached.
Nanoparticles can be made from a broad number of materials including acrylates, methacrylates, methylmethacrylates, cyanoacrylates, acrylamides, polyacetates, polyglycolates, polyanhydrides, polyorthoesters, gelatin, polysaccharides, albumin, polystyrenes, polyvinyls, polyacroleines, polyglutataldehydes, and derivatives, copolymers, and derivatives thereof. Monomer materials particularly suitable to fabricate biodegradable nanoparticles by emulsion polymerization in a continuous aqueous phase include methylmethacrylates, polyalkycyanoacrylates, hydroxyethylmethacrylates, methacrylate acid, ethylene glycol dimethacrylate, acrylamide, N,N′-bismethyleneacrylamide and 2-dimethylaminoethyl methacrylate. Other nanoparticles are made by different techniques from N, N-L-lysinediylterephthalate, alkylcyanoacrylate, polylactic acid, polylactic acid-polyglycolic acid-copolymer, polyanhydrates, polyorthoesters, gelatin, albumin, and desolvated macromolecules or carbohydrates. Further, non-biodegradable materials can be used such as polystyrene, poly (vinylpyridine), polyacroleine and polyglutaraldehyde. A summary of materials and fabrication methods for making nanoparticles has previously been published. See Kreuter, J. (1991) “Nanoparticles-preparation and applications.” In: M. Donbrow (Ed.) “Microcapsules and nanoparticles in medicine and pharmacy.” CRC Press, Boca Raton, Fla., pp. 125-148.
Nanoparticles can be produced by conventional methods, including emulsion polymerization in a continuous aqueous phase, emulsion polymerization in continuous organic phase, interfacial polymerization, solvent deposition, solvent evaporation, dissolvation of an organic polymer solution, cross-linking of water-soluble polymers in emulsion, dissolvation of macromolecules, and carbohydrate cross-linking. These fabrication methods can be performed with a wide range of polymer materials mentioned above.
A “therapeutically effective amount” of an anticancer composition or compound can refer to an amount of an anticancer compound or composition sufficient to provide a benefit in the treatment of cancer, to delay or minimize symptoms associated with cancer, or to cure or ameliorate cancer. In particular, a therapeutically effective amount means an amount of an anticancer compound sufficient to provide a therapeutic benefit in vivo. The term preferably encompasses a non-toxic amount of an anticancer compound that improves overall therapy, reduces or avoids symptoms or causes of disease, or enhances the therapeutic efficacy of or synergies with another therapeutic agent.
A therapeutically effective dose of an anticancer compound or composition can depend upon a number of factors known to those of ordinary skill in the art. The dose(s) can vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires. It is understood that a medical professional will typically determine the dosage regimen in accordance with a variety of factors. These factors include the cancer and/or tumor from which the subject suffers, the degree of metastasis, as well as the age, weight, sex, diet, and medical condition of the subject.
In some embodiments, the therapeutically effective amount is at least about 0.1 mg/kg body weight, at least about 0.25 mg/kg body weight, at least about 0.5 mg/kg body weight, at least about 0.75 mg/kg body weight, at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, at least about 100 mg/kg body weight, at least about 200 mg/kg body weight, at least about 250 mg/kg body weight, at least about 300 mg/kg body weight, at least about 3500 mg/kg body weight, at least about 400 mg/kg body weight, at least about 450 mg/kg body weight, at least about 500 mg/kg body weight, at least about 550 mg/kg body weight, at least about 600 mg/kg body weight, at least about 650 mg/kg body weight, at least about 700 mg/kg body weight, at least about 750 mg/kg body weight, at least about 800 mg/kg body weight, at least about 900 mg/kg body weight, or at least about 1000 mg/kg body weight.
Referring to the Examples, anticancer effects and minimal toxicity of compound PP1 were observed in mice bearing intracranial glioblastomas at a doses of 25-75 mg/kg body weight. The exact dosage will be determined by the practitioner, in light of factors related to the patient who requires treatment. Dosage and administration are adjusted to provide sufficient levels of the anticancer compound or to maintain the desired effect. Factors that can be taken into account include the type of subject (i.e., human, dog, or otherwise), severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
Described herein are methods of treating a subject afflicted with cancer comprising administering to a subject a therapeutically effective amount of an anticancer compound or composition. The terms “individual”, “patient” and “subject” can be used interchangeably. They refer to a mammal (e.g., a human) which is the object of treatment, or observation. Typical subjects to which the anticancer compound or composition can be administered will be mammals, particularly primates, especially humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like.
Aspects of the invention are directed towards methods of inhibiting or delaying metastasis of a cancer cell, and/or inhibiting or delaying invasion of a cancer cell. A cancer cell is a cell(s) that divide uncontrollably, forming solid tumors or flooding the blood with abnormal cells. The term “metastasis” refers to the ability of tumor cells to invade host tissues and metastasize to distant, often specific organ sites. As is known, this is the salient feature of lethal tumor growths. Metastasis formation occurs via a complex series of unique interactions between tumor cells and normal host tissues and cells. “Metastasis” is distinguished from invasion, which can refer to the direct migration and penetration by cancer cells into neighboring tissues. In other words, invasion refers to the direct extension and penetration by cancer cells into neighboring tissues. The proliferation of transformed cells and the progressive increase in tumor size eventually leads to a breach in the barriers between tissues, leading to tumor extension into adjacent tissue. Local invasion is also the first stage in the process that leads to the development of secondary tumors or metastases.
Aspects of the invention are also directed towards methods of treating a subject afflicted with a disease, such as cancer or a tumor.
Cancers are classified by the type of cell that the tumor cells resemble and is therefore presumed to be the origin of the tumor. Cancer types include carcinoma (cancers derived from epithelial cells), sarcomas (cancers arising from connective tissue), lymphoma and leukemia (cancers arising from hematopoietic cells), germ cell tumors (cancers derived from pluripotent cells), and blastoma (cancer derived from immature “precursor” cells or embryonic tissue).
Carcinomas refer to malignancies of epithelial or endocrine tissue, and include respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from the cervix, lung, prostate, breast, head and neck, pancreas, colon, liver and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. Adenocarcinoma includes a carcinoma of a glandular tissue, or in which the tumor forms a gland like structure.
Sarcomas comprise cancers arising from connective tissue (i.e. bone, cartilage, fat, nerve), each of which develops from cells originating in mesenchymal cells outside the bone marrow. Exemplary sarcomas include for example, lymphosarcoma, liposarcoma, osteosarcoma, and fibrosarcoma.
Lymphoma and leukemia arise from hematopoietic (blood-forming) cells that leave the marrow and tend to mature in the lymph nodes and blood, respectively. Non-limiting examples include acute leukemia, erythroblastic leukemia and acute megakaryoblastic leukemia, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML); lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional malignant lymphomas include, but are not limited to, non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.
Germ cell tumors are cancers derived from pluripotent cells, most often presenting in the testicle or the ovary (seminoma and dysgerminoma, respectively).
Blastomas are cancers derived from immature “precursor” cells or embryonic tissue.
The term “solid tumor” refers to hyperplasias, neoplasias or metastases that typically aggregate together and form a mass. Non-limiting examples include visceral tumors such as gastric or colon cancer, hepatomas, venal carcinomas, lung and brain tumors/cancers.
A “liquid tumor” generally refers to neoplasias of the haematopoetic system, such as lymphomas, myelomas and leukemias, or neoplasias that are diffuse in nature, as they do not typically form a solid mass. Non-limiting examples of leukemias include acute and chronic lymphoblastic, myeloblastic and multiple myeloma.
Neoplasms or cancers can affect virtually any cell or tissue type, e.g., carcinoma, sarcoma, melanoma, metastatic disorders or haematopoietic neoplastic disorders. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to breast, lung, thyroid, head and neck, brain, lymphoid, gastrointestinal (mouth, esophagus, stomach, small intestine, colon, rectum), genito-urinary tract (uterus, ovary, cervix, bladder, testicle, penis, prostate), kidney, pancreas, liver, bone, muscle, skin, etc.
“Glioma” refers to a tumor that arises from glial cells or their precursors of the brain or spinal cord. Gliomas are histologically defined based on whether they exhibit primarily astrocytic or oligodendroglial morphology, and are graded by cellularity, nuclear atypia, necrosis, mitotic figures, and microvascular proliferation-all features associated with biologically aggressive behavior. Astrocytomas are of two main types-high-grade and low-grade. High-grade tumors grow rapidly, are well-vascularized, and can easily spread through the brain. Low-grade astrocytomas are usually localized and grow slowly over a long period of time. High-grade tumors are much more aggressive, require very intensive therapy, and are associated with shorter survival lengths of time than low grade tumors. The majority of astrocytic tumors in children are low-grade, whereas the majority in adults are high-grade. These tumors can occur anywhere in the brain and spinal cord. Some of the more common low-grade astrocytomas are: Juvenile Pilocytic Astrocytoma (JPA), Fibrillary Astrocytoma Pleomorphic Xantroastrocytoma (PXA) and Desembryoplastic Neuroepithelial Tumor (DNET). The two most common high-grade astrocytomas are Anaplastic Astrocytoma (AA) and Glioblastoma Multiforme (GBM).
Embodiments herein can be used to treat ependymomas, for example, or oligodendrogliomas. Ependymomas arise from ependymal cells that line the ventricles of the brain and the center of the spinal cord. Oligodendrogliomas are a type of glioma that are believed to originate from the oligodendrocytes of the brain or from a glial precursor cell.
Kits
The anticancer compounds and pharmaceutical compositions described herein can also be provided in a kit for treating cancer, such as gliomas. In one embodiment, the kit includes (a) a container that contains the anticancer compound or pharmaceutical composition, and optionally (b) informational material for treating a specific type of cancer, such as a glioma or leukemia. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the compound for therapeutic benefit. In an embodiment, the kit includes also includes a second agent for treating a cancer. For example, the kit includes a first container that contains an anticancer compound or pharmaceutical composition, and a second container that includes the second agent.
The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods of administering the compound, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein), to treat a subject who has cancer. The information can be provided in a variety of formats, include printed text, computer readable material, video recording, or audio recording, or any information that provides a link or address to substantive material.
Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
Chemically Modified Variants of Fenofibric Acid (PP Compounds) with High Anticancer Efficacy
PP compounds are unique chemical modifications of Fenofibric Acid. When compared to unprocessed fenofibrate, PP compounds show significantly higher anticancer efficacy in vitro, are much more stable in blood and tissues, and penetrate blood-brain tumor barrier. As described herein, administration of PP compounds, such as oral administration of PP compounds, can attenuate primary tumor growth and/or metastatic invasion.
According to our previous work, a common lipid lowering drug, fenofibrate, targets energy metabolism of tumor cells, including glioblastoma, triggering severe metabolic deficit, which is followed extensive tumor cell death. Importantly, fenofibrate is practically harmless to normal differentiated cells including cells from the Central Nervous System (CNS). However, fenofibrate does not cross blood brain tumor barrier, and is highly unstable in blood and tissue environment. Therefore, its anticancer efficacy in vivo is limited.
Described herein are new chemical modifications of fenofibric acid generated a series of compounds (can be referred to as PP compounds), which in comparison to unprocessed fenofibrate demonstrate a superior anticancer efficacy in vitro. The compounds are significantly more stable, reaching therapeutically relevant concentrations of the drug in different tissues, and importantly penetrate blood brain tumor barrier, which is critical for developing new PP-based anti-glioblastoma therapy.
Additional evidence supporting overall anti-cancer efficacy of the PP compounds will be demonstrated by more experimental data in tumor animal models. Without wishing to be bound by theory, for example, such experiments will demonstrate that PP compounds are highly effective against brain tumors, including glioblastoma, and different solid and blood cancers.
Chemically Modified Variants of Fenofibrate with High Anti-Glioblastoma Efficacy
Abbreviations: 13C-NMR, carbon-13 nuclear magnetic resonance; 1H-NMR, proton nuclear magnetic resonance; ATP, adenosine triphosphate; BBTB, blood brain tumor barrier; CDCl3, deuterated chloroform; C Log P, Calculated Partitioning; DAD, Diode Array Detector; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; ECAR, extracellular acidification rate; EDC, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide; EDTA, ethylenediaminetetraacetic acid; ETC, Electron Transport Chain; FA, fenofibric acid; FBS, fetal bovine serum; FC, fenofibric chloride; FCCP, carbonylcyanide-p-trifluoromethoxyphenylhydrazone; FF, fenofibrate; GBM, glioblastoma multiforme; HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; HPLC, high performance liquid chromatography; MP, Molecular Polarizability; MSA, Molecular Surface Area; MW, molecular weight; NAD, nicotinamide adenine dinucleotide; OCR, oxygen consumption rate; PBS, phosphate buffered saline; PPARa, peroxisome proliferator activated receptor alpha; PPRE, PPAR responsive element; PSA, Polar Surface Area; siRNA, small interfering RNA.
Abstract
Anticancer effects of a common lipid-lowering drug, fenofibrate (FF) have been described in the literature for a quite some time, however, FF has not been used as a direct anticancer therapy. We have previously reported that FF in its unprocessed form (ester) accumulates in mitochondria, inhibits mitochondrial respiration, triggering a severe energy deficit and extensive glioblastoma cell death. However, FF does not cross blood brain barrier, and is quickly processed by blood and tissue esterases to form PPARα agonist, fenofibric acid (FA). In comparison to unprocessed FF, FA is much less effective in triggering cancer cell death.
To address these issues, we have made several chemical modifications in FF structure to increase its stability, water solubility, tissue penetration, and ultimately, anticancer potential. As exemplary embodiments, our data show that four new compounds designated as PP1, PP2, PP3 and PP4 (see Table 1) have improved anticancer activity when compared to FF. Like FF, they block mitochondrial respiration and trigger massive glioblastoma cell death in vitro. In addition, PP1 has improved water solubility, and is much more stable when exposed to human blood in comparison to FF. In comparison to controls, mice bearing large intracranial tumors demonstrated extensive necrotic areas within the tumor mass following two weeks of daily oral administration of PP1. We also demonstrated that the treated mice accumulated PP1 in different tissues, including intracranial tumors, and survived the treatment without major signs of distress.
Introduction
Glioblastoma multiforme (GBM) is a highly lethal brain tumor for which therapeutic options are limited. Rapidly growing and highly invasive GBM cells rely on both glycolysis and mitochondrial respiration to generate sufficient amounts of ATP and intermediate metabolites (anaplerosis). Interfering with these pathways may be a promising therapeutic strategy to induce “metabolic catastrophe” in these practically incurable brain neoplasms. We have demonstrated that tumor cells of neuroectodermal origin, including melanoma, medulloblastoma and glioblastoma, are highly sensitive to the metabolic drug, fenofibrate (FF) (1-11). FF is routinely used as a lipid-lowering drug through the ability of its metabolite, fenofibric acid (FA), to activate peroxisome proliferator activated receptor alpha (PPARa) (12). Although activation of PPARa may explain some of the observed anticancer effects, glioblastoma cells treated with PPARa siRNA retain sensitivity to FF, indicating a PPARa-independent mechanism of its anticancer action (11). Indeed, our previously published data demonstrate that unprocessed FF (ester) accumulates in mitochondrial membranes, with evidence that mitochondrial FF triggers a severe and immediate inhibition of mitochondrial respiration. This leads to a severe decline in intracellular ATP, and apoptotic tumor cell death (11). We also reported, that FF is promptly processed to FA by blood and tissue esterases, and FA is much less effective in triggering tumor cell death, and that both FF and FA do not cross the blood brain tumor barrier (BBTB) (4). To address these issues, which are hampering development of more effective FF-based anti-tumoral therapies, we have made several chemical modifications to improve FF stability, water solubility, tissue penetration, and ultimately, anti-glioblastoma efficacy. Our data show that in comparison to FF, four compounds, designated as PP1, PP2, PP3, and PP4 (
Materials and Methods
Chemical procedures to prepare PP compounds—All starting materials were reagent grade purchased from Sigma-Aldrich or Ark Pharm. 1H-NMR spectra were recorded on Varian Mercury Plus 400 MHz instrument in CDCl3 or DMSO-d6, with the solvent chemical shifts as an internal standard. All computed molecular descriptors were generated by Chemaxon MarvinSketch version 18.8.0.
Preparation of 2-[4-(4-chlorobenzoyl)phenoxy]-N-(2-hydroxyethyl)-N,2-dimethylpropanamide (PP1)—
Method A: Dichloromethane (20 ml) suspension of fenofibric acid (318.75 mg; 1 mmol) and oxalyl chloride (0.25 mL; 380.1 mg; 3 mmol), and one drop od N,N-dimethylformamide was stirred at room temperature for 5 hours. Solvent was evaporated under reduced pressure. White solid residue was resolved in dichloromethane (10 ml) and again evaporated to the solid residue. This solid residue was dissolved in dichloromethane (20 ml) and at room temperature with stirring dichloromethane (10 ml) solution of 2-(methylamino)ethanol (0.24 ml; 225 mg; 3 mmol) was gradually added. Reaction mixture was stirred at room temperature. Dichloromethane reaction mixture was washed with water (3×15 ml), 5% hydrochloric acid (3×15 ml), water (3×15 ml), 10% sodium carbonate (3×15 ml), and finally with water (3×15 ml) and dried over anhydrous sodium carbonate. Solvent was evaporated under reduced pressure to result in viscous pale-yellow liquid (390 mg). Product was crystalized from dichloromethane/hexane (30 ml; 1:4) at room temperature by slow solvent evaporation to ⅕ original volume and formed crystals were washed with ice cold hexane. Isolated yield 340 mg (90%).
Method B: Dichloromethane (100 ml) solution of fenofibric acid (637 mg; 2 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride (EDC, 576; 3 mmol), and 2-(methylamino)ethanol (600 mg; 8 mmol) was stirred at room temperature overnight. Dichloromethane solution was washed with 5% hydrochloric acid (5×20 ml), water (5×20 ml), 10% sodium carbonate (5×20 ml), water (3×20 ml) and dried over anhydrous sodium carbonate. After solvent evaporation, oily residue was crystalized from dichloromethane/hexane (1:4) to give pure product (640 mg; 85% yield). 1H-NMR (CDCl3) δ 7.74 (2H, 6, J=8.8 Hz), 7.71 (2H, d, J=8.8 Hz), 7.45 (2H, d, J=8.8 Hz), 6.92 (2H, d, J=8.8 Hz), 3.78 (2H, t, J=4.8 Hz), 3.53 (2H, t, J=4.8 Hz), 3.6 (1H, broad s), 3.17 (3H, s), and 1.71 (6H, s) ppm. 13C-NMR (CDCl3) δ 194.1, 173.5, 159.3, 138.3, 136.1, 132.2, 131.1, 130.3, 128.5, 116.5, 81.4, 60.3, 52.8, 36.7, and 25.7 ppm.
Preparation of 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl-1-(4-methylpiperazin-1-yl)propan-1-one (PP2)—Dichloromethane (30 ml) of fenofibric chloride (1 mmol; prepared as described above for PP1 preparation) was slowly added in stirring water (5 ml) solution of sodium carbonate (216 mg; 2 mmol) with tetrahydrofuran (10 ml) and of 1-methylpiperazine (0.13 ml; 120 mg; 1.2 mmol). Resulting reaction mixture was stirred at room temperature for one hour. Additional water (30 ml) was added and organic layer was separated, washed with water (3×10 ml), 5% hydrochloric acid (3×10 ml), 10% sodium carbonate (3×10 ml), water and dried over anhydrous sodium carbonate. After evaporation oily residue was crystalized from dichloromethane hexane to give 350 mg (88%) of pure product. 1H-NMR (DMSO-d6) δ 7.71 (2H, d, J=8.8 Hz), 7.67 (2H, d, J=8.8 Hz), 7.58 (2H, d, J=8.4 Hz), 6.90 (2H, d, J=8.4 Hz), 3.64 (2H, broad s), 3.46 (2H, broad s), 2.12 (2H, broad s), 1.98 (3H, s), and 1.59 (6H, s) ppm. 13C-NMR (DMSO-d6) δ 193.6, 169.0, 159.5, 137.6, 136.6, 132.5, 131.6, 130.1, 129.1, 116.8, 81.7, 54.7, 45.9, 42.9 and 26.1 ppm.
Preparation of 2-[4-(4-chlorobenzoyl)phenoxy]-N,2-dimethyl-N-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]propenamide (PP3)—Fenofibric acid chloride prepared from fenofibric acid (318.75 mg; 1 mmol) and oxalyl chloride (0.25 mL; 380.1 mg; 3 mmol) as described for preparation of PP1 was dissolved in dichloromethane (15 ml) and mixed with acetonitrile (20 ml) and water (10 ml) solution of N-methyl-D-glucamine (196 mg; 1 mmol) and sodium carbonate (212 mg; 2 mmol). Resulting mixture was stirred at room temperature for five minutes and solvent was evaporated under reduce pressure at room temperature. Resulting solid residue was mixed with dichloromethane (50 ml) and water (20 ml). Dichloromethane layer was separated, washed with 10% sodium carbonate (3×10 ml), water (3×10 ml) and dried over anhydrous sodium carbonate. After solvent was evaporated solid residue was washed with hexane (3×5 ml) and dried in vacuum under reduce pressure to give 350 mg (71%) of pure product. Selected signals for 1H-NMR (CDCl3) δ 7.71 (2H, d, J=8.8 Hz), 7.67 (2H, d, J=8.8 Hz), 3.17 (3H, s), and 1.66 (6H, s) ppm.
Preparation of 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl-1-[4-(morpholin-4-yl)piperidin-1-yl]propan-1-one (PP4)—Fenofibric acid chloride prepared from fenofibric acid (160 mg; 0.5 mmol) and oxalyl chloride (0.25 mL; 380.1 mg; 3 mmol) as described for preparation of PP1 was dissolved in dichloromethane (30 ml) and mixed with tetrahydrofuran (10 ml) solution of 4-morpholinopiperidine (100 mg; 0.6 mol), and water (10 ml) solution of sodium carbonate (106 mg; 1 mmol). Resulting mixture was stirred at room temperature for one hour. Water (20 ml) was added and organic layer was separated and extensively washed with 5% hydrochloric acid (5×20 ml), water (3×10 ml), 10% sodium carbonate (5×20 ml) and again with water (3×10 ml). After drying over anhydrous sodium carbonate solvent was evaporated to give pure product (200 mg; 85%) as oil that crystallized by standing at room temperature overnight. If necessary, the product can be further purified by crystallization from hexane or by silica gel chromatography with ethyl acetate-ethanol (5:1). 1H-NMR (CDCl3) δ 7.71 (2H, d, J=7.6 Hz), 7.68 (2H, d, J=7.6 Hz), 7.47 (2H, d, J=7.6 Hz), 6.92 (2H, d, J=7.6 Hz), 4.66 (1H, d, J=13.2 Hz), 4.60 (1H, d, J=13.2 Hz), 3.64 (4H, t, J=4.8 Hz), 2.90 (1H, t, J=12.8 Hz), 2.57 (1H, t, J=13.2 Hz), 4.38 (4H, m), 2.28 (1H, t of t, J1=11.2 Hz, J2=4 Hz), 1.83 (1H, d, J=13.2 Hz), 1.71 (6H, s), 1.64 (1H, d, J=13.2 Hz), 1.30 (1H, m), and 0.95 (1H, m) ppm.
Preparation of 4-(1-{2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoyl}piperidin-4-yl)morpholin-4-iumchloride (PP4HCl)—A mixture of concentrated hydrochloric acid (2 ml) and PP4 (94 mg; 0.2 mmol) was sonicated at room temperature for half an hour. Clear solution was evaporated to dryness at room temperature under reduced pressure. Residue was dissolved in dry ether (20 ml) and clear solution was left at room temperature for solvent to slowly evaporate. For white crystalline material was separated by filtration and washed with ice-cold hexane to give 80 mg (79%) of pure product. 1H-NMR (CDCl3) δ 12.97 (1H, s), 7.71 (4H, d, J=8.4 Hz), 7.47 (2H, d, J=8.4 Hz), 6.93 (2H, d, J=8.4 Hz), 4.93 (1H, d, J=12.0 Hz), 4.74 (1H, d, J=12.0 Hz), 4.31 (1H, t), 4.22 (1H, t), 3.92 (2H, d, J=11.6 Hz), 2.4-3.2 (7H, m), 2.13 (2H, m), 1.60 (6H, s), 1.10 (1H, m), and 0.88 (1H, m) ppm.
Detection of PP compounds by high performance liquid chromatography (HPLC) —All HPLC data were obtained from the Agilent 1100 apparatus equipped with a line degasser, binary pump (high pressure mixer), autosampler, column thermostat and Diode Array Detector (DAD) (Agilent Technologies, Santa Clara, Calif.). The analytical column 3 μm, 4.6×150 mm (Octyl Silane C8; YMC America, Inc.), solvent A—50 mM acetic acid in water, and solvent B—acetonitrile, with isocratic flow were used to detect and quantify PP compounds in culture media, in cells, tissues and body fluids. The flow rate was set to 1 ml/min, column temperature was 20° C., and the sample volume was 5 μl. DAD wavelength was set to 285 nm. Sample preparation. Blood, cell culture media, cellular and tissue lysates, were deproteinized by adding 150 μl of acetonitrile to 150 μl of sample, mixed well and centrifuged (15 000 g, 5 min). The lysates were sonicated on ice and centrifuged (15 000 g, 5 min). Finally, 150 μl of the supernatant was mixed with the equal volume of acetonitrile, filtered through 0.22 μm centrifuge filter (Sigma) and analyzed by High Performance Liquid Chromatography (HPLC).
Cell Culture—We have used two human glioblastoma cell lines LN-229 (ATCC #CRL-2611) and U-87MG (ATCC #HTB14); GBM12, which are patient-derived human glioblastoma cells (13, 14); and mouse glioblastoma cell line Bioware Brite GL261-Red-FLuc (PerkinElmer #BW134246). All cell lines were maintained as semi-confluent monolayer cultures in DMEM (1 g/L glucose; with sodium pyruvate and L-glutamine) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS) at 37° C. in a 5% CO2 atmosphere. The cells were treated with the PP compounds at different doses ranging from 5 to 50 μM. In addition, the cells were treated with fenofibrate (FF; Sigma Aldrich, St. Louis), at concentrations ranging from 10 to 50 μM. Control cultures were treated with the corresponding volumes of DMSO (vehicle control; final concentration 0.1%). GBM12 cells were routinely propagated in the subcutaneous tissue of nude mice and isolated from the tumor tissue for short-term cultures as previously described (11), and according to IACUC protocol #3444 5 (LSUHSC, New Orleans).
Evaluation of metabolic parameters—Metabolic responses of human glioblastoma cells were evaluated with Extracellular Flux Analyzer XFe24 (Agilent Technologies). Day prior to each assay the cells were plated at 4×104 cells/well in Agilent Seahorse 24-well XF cell culture microplates in growth supporting media and incubated overnight. At the time of measurement, growth media were replaced with serum-free XF assay medium (Seahorse XF Base Medium supplemented with 1 mM sodium pyruvate, 2 mM glutamine, and 5.5 mM glucose) and cartridges equipped with oxygen-sensitive and pH-sensitive fluorescent probes (Seahorse) were placed above the cells. The oxygen consumption rate (OCR; indicative of mitochondrial respiration) and acidification rate (ECAR; indicative of glycolysis) were evaluated after injecting the PP compounds (all used at 25 μM), FF (50 μM) or DMSO (0.1%; vehicle control) followed by injections of metabolic toxins: oligomycin (inhibitor of ATP synthase; complex V of the Electron Transport Chain, ETC; 0.5 μM), carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; uncoupling factor; 0.5 μM), rotenone (inhibitor of mitochondrial complex I of ETC; 0.3 μM), and antimycin A (inhibitor of mitochondrial complex III of ETC; 0.3 μM).
PPAR Luciferase Assay—The PPAR transcriptional activity was determined by utilizing the JsTkpGL3 reporter plasmid, which contains a firefly luciferase gene driven by the PPAR responsive element (PPRE), which consists of three copies of the J site from the apo-AII gene promoter. Together with JsTkpGL3 plasmid HepG2 cells were transfected with pSV40-GLuc (New England Biolabs, Ipswich, Mass.) control plasmid expressing Gaussia luciferase under the control of the constitutive SV40 early promoter to normalize for efficiency of transfection. Twenty-four hours after transfection the cells were incubated with ciglitazone (30 μM), fenofibrate, PP1, PP2, PP3 and PP4 (all 25 μM) for an additional 24 hrs. The luciferase activity was detected with Dual-Luciferase reporter assay system (Promega, Madison, Wis.), and the resulting luminescence measured with Synergy 2 microplate reader (BioTek, Winooski, Vt.).
Cell death assays—Cell death was evaluated by assays based on cell membrane integrity. We used either the trypan blue exclusion test (15) or GUAVA easyCyte 8HT flow cytometer with ViaCount reagent (Millipore) and Guava/ViaCount software for data analysis. Briefly, the cells were plated at 1×104 cells/cm2 in 24-well plates in growth medium. After 24 hours, the medium was replaced by fresh growth medium containing PP compounds, FF or 0.1% DMSO (vehicle control) and further incubated for the amount of time specified for each experiment. The cells were then harvested with 0.05% trypsin/EDTA, centrifuged, re-suspended in PBS and counted in a hemocytometer with trypan blue (0.4%, 1:1) or incubated with the ViaCount reagent (1:10; 5 minutes at room temperature) before cell viability was assessed by Guava/ViaCount according to the manufacturer's recommendations.
Animal studies—All described procedures involving experimental animals were performed in accordance to the IACUC protocol #3444 at LSUHSC, New Orleans. C57BL/6NHsd mice, 11-12 weeks of age (Envigo), were anesthetized with 4% isoflurane and secured in a stereotaxic head frame (Harvard Apparatus, Holliston Mass.). GL-261-luc cells (1×105) (PerkinElmer Inc.) were suspended in PBS and 2 μl aliquots were injected into the brain parenchyma (coordinates: 3 mm anterior to Bregma; 1.5 mm lateral to Sagital suture; 3 mm down from surface) through a burr hole in the skull using a 10 μl Hamilton syringe. Biophotonic images of the skull were captured using a Xenogen IVIS 200 imaging system (Palo Alto, Calif.) two weeks after initial cell implantation (
Pathological evaluations—After harvesting the brain and tumors, liver, spleen, kidneys, heart and lungs, the tissues were placed in 10% buffered formalin for 24 hours, processed and embedded into paraffin blocks. Sections were cut at 4 microns in thickness, placed in electromagnetically charged slides, deparaffinized, rehydrated and stained with Hematoxylin and Eosin for routine histopathological examination.
Statistical analysis—The data were analyzed with Student's t-test corrected for multiple comparisons using Bonferroni-Dunn method. The difference between control and experimental groups were considered significant and marked with an asterisk (*) for P values lower or equal 0.05.
Results
Synthesis of new FF-based compounds for glioblastoma therapy—We have modified certain specific physiochemical properties of FF to improve its resistance to blood and tissue esterases, water solubility, tissue uptake and ultimately anticancer activity. As a result, we have initially generated 23 new compounds, which have been designated here as PP compounds. They all contain a structural motif outlined in
Computed physicochemical properties of the four preselected PP compounds are presented in Table 1. Using computational methods for the estimation of physicochemical properties of potential new lead compounds is well established in medicinal chemistry (24, 25). If we compare our computed descriptors to one obtained from lead compounds with anticancer activity (25), our amides PP1, PP2, and PP4 are well in the desired range (Table 1). Molecular weight should be around 380, C log P around 3.7, PSA around 80, and HBA around 5. On the other hand, PP3 was designed to substantially increase water solubility and it is a saccharide derivative that was perfectly reflected on its estimated physicochemical properties. It is well hydrated (5 hydrogen bond donors and 16 hydrogen bond acceptors) in water media, it is hydrophilic (low C log P), however, it has a large polar surface area that might decrease its cell membrane permeability (26).
In vitro anti-cancer effects of PP compounds compared to fenofibrate (FF)—Since fenofibric acid (FA), was used as a primary substrate for the proposed chemical modifications (
On the bases of these initial findings, we have selected PP1 for additional experiments and confirmed its high in vitro cytotoxicity in another human glioblastoma cell line, U87MG (
Physiochemical and Metabolic effects of PP compounds compared to FF—The main purpose for the described chemical modifications was to generate new compounds, which in comparison to FF, are more stable, resistant to blood and tissue esterases, better soluble in water, and possibly more effective in penetrating blood brain tumor barrier (BBTB). Our data show that in comparison to FF, PP1 was significantly more resistant to blood esterases (
We have also demonstrated that PP1, PP2 and PP4 attenuate PPAR responsive elements (PPRE) (
Since FF anticancer effects are mediated mainly by the inhibition of mitochondrial respiration (11), we used an Extracellular Flux Analyzer (XF24, Seahorse Biosciences) to measure real-time oxygen consumption rate (OCR; indicative of mitochondrial respiration) and extracellular acidification rate (ECAR; indicative of glycolytic activity) in LN229 human glioblastoma cells treated with the PP1 at 25 μM concentration. The cells treated with DMSO (vehicle) or with 25 μM FF were used as a background control and positive control, respectively. These metabolic parameters were measured in monolayer cultures after sequential injections of the following metabolic toxins: oligomycin [inhibitor of complex V (ATP synthase)], FCCP (mitochondrial uncoupling factor); rotenone [inhibitor of mitochondrial complex I (NADH dehydrogenase)], and antimycin A (inhibitor of mitochondrial complex III). In a typical “mitochondrial stress” experiment (
PP1 tissue distribution and toxicity in intracranial glioblastoma—We used a syngeneic mouse glioblastoma model in which GL-261-luc cells (PerkinElmer Inc.) were injected into the brain parenchyma of C57BL/6 mice. Following 2 weeks of a continuous tumor growth, mice with large intracranial tumors were selected by using biophotonic imaging (Xenogen IVIS 200) (
Results in
Discussion
Glial tumors account for nearly 50% of all adult primary intracranial neoplasms, among which GBM is the most aggressive and practically incurable (28, 29). A large variety of different genetic and epigenetic modifications have been found in GBMs, among which p53 mutations, EGF receptor amplification, and PTEN mutations are most common (30). However, gene therapy, molecular and immunological approaches targeting these molecules and their pathways, as well as recently tested antibodies against immune checkpoint inhibitors (31) have yet to produce improvements in patient outcomes. In addition to the introduction of personalized medicine approach to target these specific pathways in glioblastoma patients (32-34), metabolic methods including calorie restriction and ketogenic diet, are surprisingly effective as supplemental therapies for glioblastoma patients (35-37). In addition, interesting anticancer effects of lipid lowering drugs, fibrates and statins have been also reported (3, 8, 38-42), among which a ten-year all-cause mortality study involving 7,722 patients treated with different fibrates revealed that the use of these metabolic compounds was associated with a significantly lower total mortality and reduced probability of death from cancer (43). In cell culture and in animal studies, various members of the fibrate family demonstrated a broad range of anticancer activities (1-3, 10, 41, 42, 44-47). These multiple reports encouraged recent clinical trials in which chronic administration of low doses of FF was tested along with chemotherapeutic agents, minimizing their toxicity and acute side effects in patients with recurrent brain tumors and leukemia (48, 49). Although these beneficial anticancer effects of FF are still suspected to rely on PPAR-dependent mechanism/s of action, we have recently demonstrated that brain tumor cells retain sensitivity to FF in the presence of PPARα antagonists or PPARα siRNA (1, 11), which strongly suggests PPARa-independent mechanism. Other labs also demonstrated that FF could have PPAR-independent cellular effects including: PPAR-independent activation of GDF15 (50); effects of FF on cell membrane fluidity (51); and the FF-induced inhibition of mitochondrial respiration in isolated cardiac and liver mitochondria (52, 53). Therefore, a growing line of evidence supports the interaction of unprocessed FF (ester) with biological membranes, which could be a reason for the observed strong anticancer activity of this lipid-lowering drug. In this respect, we have demonstrated accumulation of FF in the mitochondrial membrane fraction of different glioblastoma cells (11). As a consequence, the affected cells underwent immediate impairment of mitochondrial respiration, followed by a compensatory attempt of increasing glycolysis, which prolonged cell survival for nearly 48 hours. However, prolonged exposure to FF depleted intracellular ATP, and activated AMPK-mTOR-dependent autophagy pathway. Although autophagy gave the affected tumor cells additional 24 hours of survival, it was followed by a massive tumor cell death between 72 and 96 hours of the treatment (11). We have also demonstrated that this metabolic approach (single intratumoral injection of FF) was effective in suppressing growth of small intracranial glioblastoma tumors in mice (11).
In spite of these promising results, FF and FA do not cross blood brain tumor barrier (BBB) (4), and FF is quickly converted to FA in blood and tissues (4) and
The fact that orally administered PP1 can be found in both intact mouse brain and in brain tumor tissues is extremely important. Brain tumors are particularly difficult to treat due to distinct anatomical and physiological traits of neural tissue and vasculature. The blood-brain tumor barrier (BBTB), although more permeable than blood-brain barrier (BBB), still represents the major obstacles preventing chemotherapeutic agents from reaching therapeutically relevant concentrations. The only FDA-approved chemotherapy drug against GBM, which can cross BBTB is temozolomide (TMZ), however glioblastoma cells quickly develop TMZ-resistance and recurrent tumors are practically incurable (13).
Several strategies have been proposed to circumvent BBB and BBTB, however, their efficacy for glioblastoma treatment are still very low. For instance, carotid artery infusion with hyperosmotic mannitol was shown to temporarily open BBB by inducing shrinkage of endothelial cells and disrupting tight junctions. However, the opening lasts about 30 minutes, leaving a very narrow window for potential drug deliver (54, 55). Another strategy involves the use of vasomodulators including bradykinin or nitric oxide donors, which are also able to transiently increase capillary permeability (56, 57). However, the main caveat associated with the use of bradykinin is its ability to promote glioblastoma cell migration, invasiveness, and tumor angiogenesis (58, 59). Intracranial drug deliveries have been also tested, including intratumoral and intreventricular drug injections, and post-surgical implantation of biodegradable wafers (Glaidel). The major limitation of these intracranial interventions is a slow and diffusion of the drug which is not effective in large tumors (60, 61).
The ability of PP1 to cross BBTB is a highly desirable feature, however, it raises another relevant question: why PP1 triggers extensive cell death in the intracranial tumor tissues (
Conclusions
We have evaluated the anti-glioblastoma effects of four new compounds, which are modifications of a common lipid lowering drug, fenofibrate (FF). These 4 chemical modifications were developed specifically to increase the compound/s resistance to blood and tissue esterases, to improve their water solubility, tissue penetration, and ultimately anticancer potential. Our data show that the PP compounds, for example PPT, is highly effective in blocking mitochondrial respiration, and in eliminating glioblastoma cells in vitro. In comparison to the original compound, PP1 is also more stable when exposed to human blood, and is also better soluble in water. Our animal data show that PP1 accumulates in the intracranial glioblastomas following oral administration, and causes extensive necrotic damage in the intracranial tumors. Importantly, PP1-treated mice did not lose weight during the treatment, and no major pathological changes were observed in the adjacent brain tissue or in the other tested organs, with the exception of enlarged spleen, and minor necrosis and inflammation in the liver. Without wishing to be bound by theory, these results indicate the use of this new metabolic compound as a part of anti-glioblastoma therapy.
10.4161/cc.21015. PubMed PMID: 22732497; PMCID: 3409008.
52. Nadanaciva S, Dykens J A, Bernal A, Capaldi R A, Will Y. Mitochondrial impairment by PPAR agonists and statins identified via immunocaptured OXPHOS complex activities and respiration. Toxicol Appl Pharmacol. 2007; 223(3):277-87. Epub 2007/07/31.
Exploring Anticancer Activity of Structurally Modified Benzylphenoxyacetamide (BPA). I: Synthesis Strategies and Computational Analyses of Substituted BPA Variants with High Anti-Glioblastoma Potential
Abstract
Structural variations of the benzylphenoxy acetamide (BPA) molecular skeleton were explored as a viable starting point for designing new anti-glioblastoma drug candidates. Hand-to-hand computational evaluation, chemical modifications, and cell viability testing were performed to explore the importance of some of the structural properties in order to generate, retain, and improve desired anti-glioboastoma characteristics. It was demonstrated that several structural features are required to retain the anti-glioblastoma activity, including a carbonyl group of the benzophenone moiety, as well as 4′-chloro and 2,2-dimethy substituents. In addition, the structure of the amide moiety can be modified in such a way that desirable anti-glioblastoma and physical properties can be improved. Via these structural modifications, more than 50 compounds were prepared and tested for anti-glioblastoma activity. Some of the compounds, for example HR28, HR32, HR37, HR40(PP1), and HR46, have been determined to have desirable physical and biological properties with potential to become anti-glioblastoma drugs.
Introduction
Glioblastoma is the most aggressive and prevalent malignancy of the central nervous system (CNS), with a median patient survival rate of about 15 months1-4. Surprisingly, the most effective way to increase survival of glioblastoma patients is still extensive surgical resection. However in many instances, this approach is not feasible due to the tumor's location and its infiltration of highly specialized brain areas5. The current standard of care therapies include maximal surgical resection, followed by radiotherapy plus concomitant and maintenance temozolomide (TMZ), which is one of the few anticancer drugs capable of crossing the blood brain barrier (BBB)6. Unfortunately, TMZ-treated tumors develop TMZ-resistance, and recurrent glioblastomas are practically incurable7-9. Moreover, numerous clinical trials targeting a variety of glioblastoma-specific pathways, as well as, those testing immune checkpoint inhibitors, have been implemented, but have failed to produce a positive outcome in glioblastoma patients2,10,11.
Therapeutic strategies that include TMZ in combination with other drugs have been also explored. For instance, glioblastomas are characterized by exaggerated lipogenesis, enhanced LDL and cholesterol uptake, and extensive phagocytosis and exosome formation. All of these processes require high cholesterol metabolism and uptake for a continuous biogenesis of cellular membranes. Therefore, a combination of cholesterol lowering drugs with TMZ might be a good approach for glioblastoma treatment12. One lipid-lowering drug that has attracted attention as a candidate for an anticancer regimen is fenofibrate (FF) 13-18 In recent studies, it was demonstrated that 50 μM FF has a strong anticancer activity with low systemic toxicity13,16,19 We have previously reported that fenofibrate, in its unprocessed form (ester), blocks mitochondrial respiration, resulting in a severe energy deficit, followed by extensive glioblastoma cell death19. However, fenofibrate does not cross the BBB, and is quickly processed by the blood and tissue esterases to form fenofibric acid (FFA). This acid functions as a potent peroxisome proliferator activated receptor a (PPARa) agonist, however, it is no longer effective in triggering tumor cell death19,20.
We have made several chemical modifications to the FF structure in order to improve the prospective anticancer drug stability, water solubility, tissue penetration, and ultimately, the anti-glioblastoma efficacy. One of the compounds, PP1 (
Design of the Molecular Target
The basic molecular skeleton of FF contains a benzylphenoxy acetate structural arrangement (
Preparation Methods
All preparations (
The starting point for the preparation of each BPA modification in this study is the benzylphenoxyacetic acid derivative. For amides, although there is a plethora of synthetic methods for transformation of acids into amides, many of these methods cannot be successfully applied for preparation of all the amides included in this study. For instance, employing a well-developed amide preparation that utilizes DCC and EDC based activation agents23 for direct carboxylic acid conversion to amide cannot be used in the case of 2,2-dimethylbenzylphenoxyacetic acid. This is due to a combination of steric hindrance and lower reactivity of the activated carboxylic acid. However, when the corresponding carboxylic acid chloride is used, almost quantitative conversion of carboxylic acid into amide is achieved (
Reduced forms of the studied amides were prepared by selective reduction (
To further increase the water solubility of basic BPA, ammonium salts were prepared. Hydrochloric salts were prepared by simple mixing of the corresponding BPA with concentrated hydrochloric acid followed by water evaporation. Then, alkylation of basic BPA was performed with acetone as the solvent and methyl iodide as the alkylating reagent. The product (HR36) crystalized directly from the reaction mixture (
Results and Discussion
As mentioned above, it was demonstrated that FF possesses anti-glioblastoma activity. However, there are several FF properties that make it impractical for anticancer treatment: low water solubility, fast hydrolysis into FFA, and a required relatively high therapeutic concentration19,20 In our previous study, we reported that some amide derivatives of FF, including PP1, were more potent in eliminating glioblastoma cells than FF21. These amides belong to the large family of BPA24.
One fundamental challenge for the design of CNS penetrant drugs is the need to cross the blood-brain barrier (BBB). But, BBB-permeable compounds form a very small subset of oral drugs currently in existence, and experimental models for testing BBB penetration are quite complex. Therefore, an independent indicator of the BBB penetration is needed for the initial screening and selection of large number of compounds (BPA variants) to evaluate their potential for reaching intracranial tumor site at therapeutically relevant concentrations. Therefore, prior to the preparation of all BPA variant compounds in this study, we performed extensive molecular modeling to describe their physicochemical properties. Cell viability (CV) assay was then performed using LN229 human glioblastoma cell line, and the cells were treated with BPA variants at 25 M for 72 hours. The results of these computational and cell viability testing are outlined in
One currently accepted way to define physicochemical properties is by using a weighted scoring approach, known as the Central Nervous System—Multiparameter Optimization (CNS-MPO)25,26 The CNS-MPO algorithm uses a weighted scoring function that assesses 6 key physicochemical properties (c log P, c log D, MW, TPSA, HBD, and pKa) that indicate relative BBB penetration. The CNS-MPO scale is between 0 and 6.0, with scores ≥4.0 widely used as a cut-off to select compounds for hit CNS therapeutic drug discovery programs26. The validation of this approach utilized a library of 616 compounds to evaluate the experimental distribution of the computed parameters incorporated into CNS-MPO scores25,26. It was found that CNS-MPO scores of 1-2 (0%), 2-3 (11.6%), 3-4 (40.8%), 4-5 (53.8%) and 5-6 (81.1%) increase the probability of drugs to be found in the brain27.
In addition, parameters that are routinely used for Quantitative Structure Activity Relationship (QSAR) study are molecular polarizability (MP), minimal molecular projection area (MPA), and water solubility (Log S). These parameters are not incorporated into the CNS-MPO score, however they are also expected as possible contributing factors in BBB penetration. Therefore, in the associated figures herein, these additional parameters were defined and calculated for each BPA variant compound. MP of a molecule characterizes the capability of its electronic system to be distorted by the external field, and it plays an important role in modeling many molecular properties and biological activities28. Without wishing to be bound by theory, an MP between 30-40 is optimal for a molecule to bind to a biotarget29. Minimal projected area (MPA) is also very important for drug transport and ultimately for drug activity. For instance, in recent studies by Cha, Müller, and Pos, a distinct phenotypical pattern of drug recognition and transport for the G616N variant was reported, indicating that drug substrates with MPA of >70 Å2 are less well transported than other substrates30. Finally, Log S of −4.5 and greater are indicators of acceptable water solubility31.
The BBB permeation propensity of all studied compounds is indicated by the decimal logarithm of brain to-plasma concentration ratio (log BB) value, which is derived from the modified Clark's equation: log BB=0.152 C log P −0.0148PSA+0.139 30. This parameter was also calculated and is listed in the tables. It has been shown that chemical compounds with log BB>0.3 readily cross the BBB, while those with log BB<−1.0 are poorly distributed to the brain32. Finally, the rate of passive diffusion is inversely proportionate to the square root of molecular size (Graham's law33), which is also included in our compound analysis.
Minimal projected area (MPA,
Our previously reported drug candidate PP1 (HR40) has an excellent activity against glioblastoma tumor cells21, therefore, we are using this drug candidate as a standard to evaluate the structural modifications made on the BPA skeleton. Therefore, we investigated first the importance of the second aromatic ring in BPA, the conjugation of the carbonyl group, and the presence of a chlorine atom. All computed parameters (
Replacing the 4-chlorobenzoyl moiety of PP1 with a cyclohexylmethyl generates a new drug candidate HR1. However, this modification results in a decrease of anti-glioblastoma activity (
In addition, it is also important to assess the significance of the halogen atoms on the BPA skeleton. Substituting hydrogen by fluorine substantially changes molecular polarizability (MP) and lipophilicity, and increases binding affinity to targeted proteins36. Also, halogen bonding is stronger between chloro-aryls and carbonyl compounds, than between corresponding fluoro-aryls [33, 34]. Therefore, the computed data, as well as, the cell viability data of modified variants of PP1 in which the chlorine atom was replaced with fluorine (HR8-HR11) were collected (
Fluoro-PP1 (HR9) has lower potency than PP1, and therefore, halogen bonding appears to be very important for anti-glioblastoma activity in BPAs (
The BPA structure was further modified in order to explore the importance of two methyl groups in the alpha position (
The computed physical properties for monomethylated amides (shown in
Comparison of electrostatic potential for di-, mono-, and non-methylated compounds (HR13, HR18, HR21) suggests that a large positive area in the molecule is lost by removing the methyl groups (
After establishing the importance of the 2-(4-chlorobenzoyl)phenoxy-2,2-dimethylacetamide (AA) structural skeleton for anti-glioblastoma activity, it is essential to assess the nature of the amide moiety (
Next, amides with a basic amide moiety are presented in
In the case of the alkylated drug candidates, such as methylated HR34, the activity is substantially diminished because it cannot be deprotonated. If one examines the computed parameters for HR36, it is apparent that the compound is very hydrophilic (C log P=−0.31; log S=0.88) and has a very low estimated BBB (log BB=−0.6). Surprisingly the CNS-MPO of HR36 is 4.0, which would suggest that this compound should be capable of accumulating in CNS, however it is completely inactive at 25 μM (CV=98%) (
Water solubility is a major obstacle in the proper administration of drug candidates 37. One approach to increasing water solubility is to introduce hydroxy groups in the non-essential structural area of the compound. The amide moiety of new compounds listed in
Introducing rigidity to stabilize a desired drug conformation could result in increased drug potency 38 in many instances. One of the ways to introduce molecular rigidity is by replacing a linear carbon skeleton with a cyclic carbon skeleton. This modification to PP1 (
Four drug candidates containing two hydroxy groups (HR42, HR43, HR44 and HR46) are presented in
In conclusion, we have identified four drug candidates, similar to PP1 (HR40), that have strong in vitro anti-glioblastoma activity but have physical properties that may contribute to the improved brain tumor penetration (
Experimental Strategies.
All starting materials were reagent grade and purchased from Sigma-Aldrich, ArkPharm, TCI America, and AbaChemScene. 1H-NMR spectra were recorded on Varian Mercury 300 and Varian Mercury 400 Plus instruments in CDCl3, DMSO-d6, using the solvent chemical shifts as an internal standard. Electrospray Mass Spectroscopy (EMS) was recorded on Waters LCT Premier XE (that's a Tof MS) with an ESI source, scanning 100-2000 m/z with direct injections of 5 μl sample, using a 0.2 ml/min flow of acetonitrile. All molecular physical properties were calculated using Marvin Sketch software. All computed molecular descriptors were generated by ChemAxon MarvinSketch version 19.4. Electrostatic potential maps were calculated with PM3 semi-empirical method as implemented in Spartan '18 v 1.1.0.
Cell culture and viability assays. Human glioblastoma LN-229 cells were maintained as semi-confluent monolayer culture in DMEM with 1 g/L glucose, sodium pyruvate, L-glutamine (Corning) supplemented with 10% heat inactivated FBS (Gibco) and P/S (50 units/mL of penicillin and 50 μg/mL of streptomycin) at 37° C. in a 5% CO2 atmosphere. Prior to treatment, cells were plated well in 96-well plates (BD Falcon) at initial density of 2×103 cells/cm2. Stock solutions of the compounds were prepared in DMSO, diluted in cell culture medium and added to the cells in triplicate for every experimental condition 24 h after plating (final concentration 25 μM). For the vehicle control, DMSO was used at 0.5%. MTT assay40 (measuring cell metabolic activity) was performed after a 72 h incubation in the presence of the compounds as previously described. Following 1 h incubation with MTT, formazan crystals were dissolved in 5 mM HCl in isopropanol and absorbance read at 540 nm. Data represent mean values expressed as percentage of vehicle control±SD. Phase contrast images of treated cells were taken 48 hours following the treatment with a BZ-X800 fluorescence microscope (Keyence) equipped with a 20× objective.
Method A. Preparation of isopropyl 2-(4-(4-chlorobenzoyl)phenoxy)acetate (3n). Water (10 ml) solution of sodium hydroxide (410 mg; 10.25 mmol), (4-chlorophenyl)(4-hydroxyphenyl)methanone (2.3 g; 0.1 mol) and benzene (100 ml) was refluxed for 10 minutes and water was azeotropically removed by using Deen-Stark distillation apparatus followed by removal of benzene under reduced pressure. White powdery sodium phenoxide was mixed with dry isopropanol (100 ml) and isopropyl bromoacetate (1.9 g; 10.5 mmol). Resulting mixture was stirred with sonication for 1 hour and refluxed for 4 hours. Solvent was evaporated to solid residue and mixed with dichloromethane (100 ml) and water (100 ml). Water layer was discarded, and organic layer was washed with 5% sodium carbonate (3×50 ml) and dried over anhydrous sodium carbonate. After solvent evaporation white residue was dried under vacuum to give white solid product in 93% (3.1 g) isolated yield. 1H-NMR (400, DMSO-d6) δ 7.79 (2H, d, J=9.2 Hz), 7.71 (2H, d, J=8.8 Hz), 6.97 (2H, d, J=8.8 Hz), 5.16 (1H, septet, J=7.2 Hz), 4.67 (2H, s), and 1.28 (6H, d, J=7.2 Hz) ppm.
Method B. Isopropyl 2-(3-benzoylphenoxy)-2-methylpropanoate (4f). Isopropanol (300 ml) suspension of sodium carbonate (21.2 g; 200 mmol), (3-hydroxyphenyl)(phenyl)methanone (4 g; 20 mmol) and isopropyl 2-bromo-2-isobutirate (4.2 g; 20 mmol) was refluxed with stirring for 3 days. After cooling to room temperature white solid was separated by filtration washed with isopropanol (3×20 ml). Combined filtrates were evaporated to an oily residue. This residue was mixed with water/chloroform (100 ml/200 ml). Water layer was discarded, and chloroform layers was washed with 5% sodium hydroxide (3×50 ml), water (3×50 ml), and dried over anhydrous sodium carbonate. After filtration chloroform was evaporated to oily residue that standing at 5° C. overnight give white solid in 92% (6 g) yield. 1H-NMR (300 MHz, CDCl3) δ 7.77 (2H, d, J=6.9 Hz), 7.68 (1H, t, J=6.9 Hz), 7.48 (2H, d, J=7.5 Hz), 7.5-7.3 (3H, m), 7.27 (1H, s), 7.07 (1H, d, J=6.9 Hz), 5.06 (1H, septet, J=6.3 Hz), 1.60 (6H, s), and 1.18 (6H, d, J=6.9 Hz). 13C-NMR (200 MHz, CDCl3) δ 173.2, 155.4, 128.6, 137.5, 129.9, 129.0, 128.2, 127.5, 123.8, 123.1, 120.3, 79.4, 69.1, 25.3 and 21.5 ppm
Method C. Acid preparation. Preparation of 2-methyl-2-(4-phenoxyphenoxy)propanoic acid (le). Water (10 ml) solution of sodium hydroxide (0.42 mg; 10.5 mmol), benzene (100 ml) and 4-phenoxyphenol (1.86 g; 10 mmol) was stirred at room temperature for one hour. Water was evaporated away by Dean-Stark distillation. Remaining benzene was removed under reduced pressure. White powdery residue was mixed with isopropanol (200 ml) and isopropyl 2-bromo-2-methylpropanoate (2.1 g; 10 ml). Resulting mixture was stirred with sonication at 60° C. for two hours followed by refluxing overnight. After cooling to room temperature 5% sodium hydroxide (100 ml) was added and resulted mixture was refluxed for 2 hours. Solvent was evaporated to solid residue mixed with water (150 ml) and acidified with concentrated hydrochloric acid to pH ˜3. Resulting white suspension was mixed with chloroform (100 ml) and chloroform layer was separated, washed with water (3×50 ml) and then with 10% sodium carbonate (100 ml). Sodium carbonate layer was acidified with concentrated hydrochloric acid to pH˜ 3. Formed white precipitate was separated by filtration, washed with water and dried at room temperature under vacuum to give pure product in 92% yield (2.5 g). 1H-NMR (400 MHz, CDCl3) δ 7.31 (2H, t, J=7.6 Hz), 7.08 (1H, t, J=7.6 Hz), 6.96 (2H, d, J=7.6 Hz), 6.93 (4H, s), and 1.58 (6H, s) ppm.
Method D (nitrogen unsubstituted amides). Preparation of 2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropanamide (AA). Dichloromethane (10 ml) suspension of fenofibric acid (FFA, 318 mg; 1 mmol) and two drops of DMF was stirred at room temperature for 3 hours. Clear dichloromethane solution was evaporated at 30° C. under reduced pressure. Remaining white solid residue was dissolved in tetrahydrofuran (20 ml) and mixed with aqueous ammonia (10 ml; 3.08 g; 0.17 mol) and stirred at room temperature for one hour. Resulting mixture was mixed with dichloromethane (50 ml) and water (50 ml). Organic solvent was separated, washed with water (3×50 ml), 5% sodium carbonate (50 ml), and dried over anhydrous sodium carbonate. Solvent was evaporated to give white solid product that was recrystallized from dichloromethane-cyclohexane to give pure product in 93% (295 mg). 1H-NMR (400, DMSO-d6) δ 7.69 (4H, d+d, J1=J2=8.4 Hz), 7.58 (2H, d, J=8.4 Hz), 7.57 (1H, s), 7.32 (1H, s), 6.97 (2H, d, J=8.4 Hz), and 1.50 (6H, s) ppm. 13C-NMR (400, DMSO-d6) δ 193.7, 175.4, 159.8, 137.5, 136.7, 132.1, 131.6, 130.0, 129.0, 118.5, 80.9, and 25.4 ppm.
Method E (N-methyl amides). Preparation of 2-(4-(4-chlorobenzoyl)phenoxy)-N,2-dimethylpropanamide (MA). Aqueous (20 ml) solution of methylamine hydrochloride (675 mg; 10 mmol) and sodium carbonate (530 mg; 5 mmol) were mixed with tetrahydrofuran (20 ml) solution of acid chloride prepared as explain above from fenofibric acid (FFA, 318 mg; 1 mmol) and stirred at room temperature for two hours. Dichloromethane was added (50 ml) and organic layer washed with water (3×50 ml), 5% sodium carbonate (50 ml) and dried over anhydrous sodium carbonate. After evaporation of the oily residue was mixed with cyclohexane (5 ml) and left at room temperature overnight. Formed white needles were separated by filtration and dried at 60° C. under vacuum to give pure product in 90% (300 mg). 1H-NMR (400, DMSO-d6) δ 8.09 (1H, q, J=4.8 Hz), 7.67 (2H, d, J=8.8 Hz), 7.66 (2H, d, J=8.8 Hz), 7.56 (2H, d, J=8.8 Hz), 6.93 (2H, d, J=8.8 Hz), 2.58 (3H, d, J=4.8 Hz), and 1.47 (6H, s) ppm. 13C-NMR (400, DMSO-d6) δ 194.0, 174.0, 159.6, 137.6, 136.5, 132.2, 131.6, 130.2, 129.1, 118.9, 81.2, 30.7, and 25.4 ppm.
Method F (N,N-dimethyl amides). Preparation 2-(4-(4-chlorobenzoyl)phenoxy)-N,N,2-trimethylpropanamide (DMA). Mixture of 40% dimethylamine in water (10 ml; 88 mmol) and the acid chloride of fenofibric acid (FFA, 1 mmol) was stirred at room temperature for two hours. Reaction mixture was worked up as described above. Product was purified by crystallization from cyclohexane. Isolated yield 89% (310 mg). 1H-NMR (400, DMSO-d6) δ 7.71 (2H, d, J=8.8 Hz), 7.69 (2H, d, J=8.8 Hz), 6.89 (2H, d, J=8.8 Hz), 3.02 (3H, s), 2.82 (3H, s), and 1.59 ppm. 13C-NMR (400, DMSO-d6) δ 193.9, 171.5, 159.6, 137.5, 136.7, 132.6, 131.6, 130.1, 129.1, 116.8, 81.6, 37.3, and 25.9 ppm.
Method G (pH neutral nitrogen substituted phenoxyacetamides). Preparation of 2-(4-(4-chlorophenoxy)phenoxy)-N,N-bis(2-hydroxyethyl)-2-methylpropanamide (HR5). Dry dichloromethane (20 ml) solution of acid 1k (306 mg; 1 mmol), oxalyl chloride (0.260 ml; 380 mg; 3 mmol) and two drops of DMF was stirred at room temperature for 4 hours. Solvent was evaporated, and residue was dissolved in dichloromethane and mixed with mixture of tetrahydrofuran (10 ml) with diethanolamine (160 mg; 1.5 mmol) and water (10 ml) with sodium carbonate (212 mg; 2 mmol). Resulting mixture was stirred at room temperature 2 hours and solvent was evaporated under reduced pressure. Solid residue was mixed with dichloromethane (100 ml) and water (100 ml). Water layer was discarded, and organic layer was washed with 5% hydrochloric acid (3×50 ml), 5% sodium carbonate (3×50 ml), water (3×50 ml) and dried over anhydrous sodium carbonate. Solvent was evaporated under reduced pressure to give pure product in 84% (330 mg) yield. 1H-NMR (300 MHz, CDCl3) δ 6.92 (6H, m), 6.82 (2H, d, J=9.3 Hz), 3.92 (4H, m), 3.66 (2H, t, J=5.8 Hz), 3.60 (2H, t, J=5.8 Hz), and 1.65 (6H) ppm.
Method H (basic nitrogen substituted aryloxyacetamides). Preparation of 2-(4-(4-chlorobenzoyl)phenoxy)-N-(2-(diethylamino)ethyl)-2-methylpropanamide (HR32). Tetrahydrofuran (20 ml) mixture of FFA (318.75; 1 mmol), oxalyl chloride (0.26 ml; 3 mmol), and two drops of DMF was stirred at room temperature for 2 hours. Solvent was evaporated, and solid residue was mixed with dry tetrahydrofuran (20 ml) and cooled down to 5° C. This solution was mixed at 5° C. with ice cold water solution (20 ml) of sodium carbonate (212 mg; 2 mol) and N,N-diethylethylenediamine (127 mg; 1.1 mmol). Resulting mixture was stirred at room temperature for two hours and evaporated to an oily residue. This residue was mixed with dichloromethane (100 ml) and 5% sodium carbonate (50 ml). Water layer was discarded, and organic layer was extensively washed with water (10×50 ml) and dried over anhydrous sodium carbonate. After solvent evaporation product was further purified by silica gel filtration with 5% triethylamine in ethyl acetate as solvent. Isolated yield 75% (317 mg). 1H-NMR (400 MHz, CDCl3) δ 7.73 (2H, d, J=8.8 Hz), 7.69 (2H, d, J=8.4 Hz), 7.45 (2H, d, J=8.4 Hz), 7.730 (1H, broad s), 6.95 (2H, d, J=8.8 Hz), 3.31 (2H, q, J=6.0 Hz), 2.49 (2H, t, J=6.0 Hz), 2.41 (4H, q, J=7.2 Hz), 1.61 (6H, s), and 0.87 (6H, t, J=7.2 Hz) ppm. 13C-NMR (300, CDCl3) δ 194.1, 173.8, 158.9, 138.5, 136.2, 131.8, 131.1, 128.5, 119.4, 119.0, 81.7, 51.3, 46.6, 36.8, 25.1, and 11.6 ppm.
Method I (catalytic hydrogenation of benzoylphenoxyacetamides) Preparation of 2-(4-(cyclohexylmethyl)phenoxy)-N-(2-hydroxyethyl)-N,2-dimethylpropanamide (HR1). Ethanol (50 ml) suspension of amide HR40 (190 mg; 0.5 mmol) and 3% Pd/C (150 mg) was stirred at room temperature overnight under atmospheric hydrogen pressure. The catalyst was removed by filtration, solvent was evaporated under reduce pressure to give pure product in 96% (320 mg) isolated yield. 1H-NMR (400, CDCl3) δ 6.99 (2H, d, J=8.4 Hz), 6.74 (2H, d, J=8.4 Hz, 3.79 (2H, t, J=5.2 Hz), 3.54 (2H, t, J=5.2 Hz), 3.24 (3H, s), 3.21 (1H, m), 2.39 (2H, d, J=7.6 Hz), 1.64 (4H, m), 1.62 (6H, s), 1.45 (2H, m), 1.17 (2H, m), and 0.98 (2H, m) ppm
Method J (sodium borohydride—trifluoracetic acid reduction of benzolphenoxyacetamide carbonyl group). Preparation of 2-(4-(4-chlorobenzyl)phenoxy)-N-(2-hydroxyethyl)-N,2-dimethylpropanamide (HR2). Dichloromethane (30 ml) suspension of HR40 (190 mg; 0.5 mmol) and fine grinded sodium borohydride (110 mg; 3 mmol) was kept at −5° C. for one hour. Into this stirring suspension at −5° C. slowly dropwise trifluoracetic acid (15 ml) was added in period of half an hour. Resulting suspension was then stirred at 0° C. for one hour and at room temperature for additional two hours. The suspension was filtered, solid was discarded and dichloromethane filtrate was washed with 5% sodium carbonate (3×30 ml), dried over anhydrous sodium carbonate and evaporated to oily residue to give crude product. The product was purified by silica gel column chromatography with ethyl acetate-dichloromethane (7:3). Isolated yield 72%. 1H-NMR (400, CDCl3) δ 7.23 (2H, d, J=8.4 Hz), 7.08 (2H, d, J=8.4 Hz), 7.01 (2H, d, J=8.4 Hz), 6.76 (2H, d, J=8.4 Hz), 3.85 (2H, s), 3.78 (2H, q, J=4.8 Hz), 3.53 (2H, t, J=4.8 Hz), 3.21 (3H, s), 2.73 (1H, t, J=5.2 Hz), and 1.62 (6H, s) pppm.
Method K (hydrochloric salts of basic phenoxyacetamide). Preparation of 4-{2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoyl}6-8,13-20,24-26,28,34,38-1-methylpiperazin-1-ium chloride (HR35). Mixture of concentrated hydrochloric (3 ml) and HR34 (200 mg; 0.5 mmol) was sonicated at room temperature for five minutes. Clear water solution was left under stream of nitrogen in hood for several hours to result in white powder. White powder was dissolved in dichloromethane (10 ml) and dried over 4 Å molecular sieve overnight. The solvent was evaporated, and white residue was dried in vacuum to give 208 mg (95%). 1H-NMR (CDCl3) δ 13.20 (1H, broad s), 7.75 (2H, d. J=8.8 Hz), 7.71 (2H, d, J=8.0 Hz), 7.56 (2H, d, J=8.0 Hz), 6.90 (2H, d, J=8.8 Hz), 4.78 (2H, d, J=13.2 Hz), 3.93 (1H, t, J=12.8 Hz), 3.52 (1H, t, J=13.2 Hz), 3.45 (1H, d, J=12.4 Hz), 3.27 (1H, d, J=10.0 Hz), 2.63 (3H, d, J=4 Hz), 2.53 (1H, q, J=10.0 Hz), 1.98 (1H, q, J=9.6 Hz) and 1.71 (6H, s) ppm.
Method L (ammonium salts of basic phenoxyacetamides. Preparation of 4-{2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoyl}-1,1-dimethylpiperazin-1-ium iodide (HR36). Acetone (20 ml) solution of HR34 (100 mg; 0.25 mmol) and methyl Iodide (141 mg; 0.06 ml; 1 mmol) was left in dark at room temperature for three days (˜70 hours). During the reaction white solid precipitate forms. Solid product was separated by filtration. Washed with acetone (3×3 ml) and dried at 110° C. for two hours to give pure product in 96% (130 mg). 1H-NMR (DMSO-d6) δ 7.73 (2H, d, J=8.8 Hz), 7.71 (2H, d, J=8.8 Hz), 7.60 (2H, d, J=7.6 Hz), 6.93 (2H, d, J=8.4). 4.06 (2H, broad singlet), 3.86 (2H, broad singlet), 3.30 (2H, broad singlet), 3.25 (2H, broad singlet), 3.12 (6H, s) and 1.60 (3H, s) ppm. 13C-NMR (DMSO-d6) δ 193.7, 170.7, 159.1, 137.6, 136.5, 132.7, 131.7, 130.6, 129.1, 117.7, 81.8, 60.7, 51.2, and 26.2 ppm.
Non-limiting examples of anti-cancer compounds comprises those described in Example 4.
Exploring Anticancer Activity of structurally Modified Benzoylphenoxyacetamide (BPA) II: Synthesis Strategies and Computational Analyses of Phenolic BPAs with High Anti-Glioblastoma Potential.
Extensive computational studies of phenol and naphthol modifying benzoylphenoxyacetamide (BPA) were performed with target to generated physicochemical properties that can be used as to select several molecular skeletons for preparation and study with target to develop amt-glioblastoma drug candidate. Overall extensive computational studies of 71 structural variants of BPA were performed and their physicochemical properties such as solubility (log S), brain-blood partitioning (log BB) and probability to penetrate central nervous system (MPO-CNS), polar surface area, molecular polarizability, electrostatic surface maps, and frontier orbitals were evaluated. From these set of generated data statistical cut off values were used to select eighteen BPA based candidates with phenol and naphthol moieties be prepared and experimentally evaluated their anti-glioblastoma activity. Nine of these compounds show acceptable anti-glioblastoma activity at 25 μM concentration on LN229 cell line. However only four (HR49, HR50, HR51, HR59) are selected as anti-glioblastoma drug candidates considering that for these compounds acceptable water solubility and brain penetration were calculated. In addition, their IC50 values are below 10 μM. These compounds will be further evaluated as anti-glioblastoma drug candidates.
Introduction
Glioblastoma (GBM) is aggressive malignant tumor in adult brain and one of the most challenging malignancies for treatment in the oncology [Holland, E, C. “Glioblastoma multiforme: The terminator” PNAS 2000, 97, 6242-6244]. Standard therapeutic approach to treat GMB for many years has been surgical resection and postoperative radiotherapy. This standard treatment has resulted in a poor median survival of about 12 months [Weller M, van den Bent M, Tonn J C, Stupp R, Preusser M, Cohen-Jonathan-Moyal E, Henriksson R, Le Rhun E, Balana C, Chinot O, Bendszus M, Reijneveld J C, Dhermain F, French P, Marosi C, Watts C, Oberg I, Pilkington G, Baumert B G, Taphoorn M J B, Hegi M, Westphal M, Reifenberger G, Soffietti R, Wick W; “European Association for Neuro-Oncology (EANO) Task Force on Gliomas” Lancet Oncol. 2017, 18, e315-e329]. Addition temozolomide (TMZ) to surgery and radiotherapy has become the standard first-line treatment for GBM, but with an increase of the median survival for only about 2.5 months [Jaoude, D. A.; Moore, J. A.; Moore, M. B.; Twumasi-Ankrah, P.; Ablah, E.; Moore, D. F. “Glioblastoma and Increased Survival with Longer Chemotherapy Duration” Kans. J. Med. 2019, 12, 65-69.]. Considering that there are many FDA-approved drugs [As of May 2019 the FDA approved drugs for brain cancer are Afinitor (Everolimus), Afinitor Disperz (Everolimus), Avastin (Bevacizumab), Bevacizumab), NICNU (Carmustine), Carmustine Implant, Everolimus, Gliadel Wafer (Carmustine Implant), Lamustine, Mvasi (Bebvacizumab), Temodar (Temozolomide), Temozolomide] with one drug combination (PCV combination include Procarbazzine Hydrochloride, Lomustine, Vincristine Sulfate) for cancer treatment and that there is also noticeable progress made in the molecular and cellular profiling of GBM the increase of the survival moderate [Paolollo, M.; Boselli, C.; Schinelli, S. “Glioblastoma under Siege: An Overview of Current Therapeutic Strategies” Brain Sci. 2018, 8(1): 15.]
An efficient treatment of GBM is difficult to develop for a series of reasons. First, GBM is characterized by many dysregulated pathways that can hardly be all blocked and repaired at the same time with a single therapy [Alifieris, C.; Trafalis, D. T. “Glioblastoma multiforme: pathogenesis and treatment” Pharmacol. Ther. 2015, 152, 63-82.]. Second, GBM partly consists of infiltrating cells that cannot easily be all removed by surgery. Third, GBM early diagnosis is not carried out routinely. Sensitive imaging techniques, such as MRI are still too expensive to be carried out on a regular basis over the whole population. Fourth, the optimization of a clinical protocol for GBM treatment requires the use of an accurate and representative preclinical GBM model. Currently used mouse and rat models are not appropriate because their tumors are typically ˜103-104 smaller than human GBM [Biasibetti, E.; Valazza, A.; Capucchio, M. T.; Annovazzi, L.; Battaglia. L.; Chirio, D.; Gallarate, M.; Mellai, M.; Muntoni, E.; Peira, E.; Riganti, C.; Schiffer, D.; Panciani, P.; Lanotte, M. “Comparison of Allogeneic and Syngeneic Rat Glioma Models by Using MRI and Histopathologic Evaluation” Comp. Med. 2017, 67, 147-156.]. Fifth, the blood brain barrier (BBB) often prevents drugs from efficiently reaching glioblastoma cells, and methods to enable drugs to efficiently cross the BBB should therefore be developed [Harder, B. G.; Blomquest, M. R.; Wang, J.; Kim, A. J.; Woodworth, G. F.; Winkles, J. A.; Loftus, J. C.; Tran, N. L. “Developments in Blood-Brain Barrier Penetrance and Drug Repurposing for Improved Treatment of Glioblastoma” Front. Oncol. 2018, 8, 462]. In fact, there are actually 3 structural variation that are commonly used as anti-glioblastoma drugs: Temozolomine (Temodar, TMZ), Lomustine and Carmustine as chloroethylnitrosoureas, Avastin (Bevacizumab, Mvasi) as monoclonal antibody. TMZ is rapidly bsorbed and eliminated [Agarwala, S. S.; Kirkwood, J. M. “Temozolomide, a Novel Alkylating Agent with Activity in the Central Nervous System, May Improve the Treatment of Advanced Metastatic Melanoma” The Oncologist 2000, 5, 144-151.]. Upon oral administration maximum plasma concentration was reach less the one our and the elimination half-life is approximately 1.8 hours. Penetration of TMZ into CNS has been studied in 35 patients with newly diagnosed or recurrent malignant gliomas showing that the drug concentration of the drug in brain and cerebrospinal fluid is approximately 20% of the plasma concentration [Ostermann, S.; Csajka, C.; Buclin, T.; Leyvraz, S.; Lejeune, F.; Descosterd, L. A.; Stupp, R. “Plasma and Cerebrospinal Fluid Population Pharmacokinetics of Temozolomidine in Malignant Glioma Patients” Clin. Cancer Res. 2004, 100, 3728-36]. This makes experimentally estimated log BB (Brain-Blood Distribution) to be around −0.7.
It is quite clear that none of these therapeutic strategies are producing actable response. There are more and more studies that include most widely used anti-glioblastoma drug, TMZ in combination with other drugs [Gao, L.; Huang, S.; Zhang, H.; Hua, W.; Xin, S.; Cheng, L.; Guan, W.; Yu, Y.; Mao, Y.; Pei, G. “Suppression of glioblastoma by a drug cocktail reprogramming tumor cells into neuron like cells” Nature Scientific Reports 2019, 9, 3462]. One of the approaches is combination of TMZ with lipid lowering drugs [Vasilev, A.; Sofi, R.; Tong, L.; Teschemascher, A. G.; Kasparov, S. “In search of a Breakthrough Therapy for Glioblastoma Multiforme” Neuroglioma 2018, 1, 292-310.] One lipid-lowering drug that has attracted attention as a candidate for an anticancer regimen is fenofibrate (FF) [Majeed, Y.; Upadhayay, R.; Alhousseiny, S.; Taha, T.; Musthak, A. Shaheen, Y.; Jameel, M.; Triggle, C. R.; Ding, H. “Potent and PPARalfa-independent anti-proliferative action of the hypolipidemic drug fenofibrate in VEGF-dependent angiosarcomas in vitro” Nature Scientific Reports 2019, 9, 6316.] Recently we have demonstrated that 50 μM FF has a strong anticancer activity with low systemic toxicity [Grabacka, M; Waligorski, P.; Zapata, A.; Blake, D. A.; Wyczechowska, D.; Wilk, A.; Rutkowska, M.; Vishistha, H.; Ayyala, R.; Ponnusamy, T.; John, V. T.; Culicchia, F.; Wisniewska-Becker, A.; Reiss, K. “Fenofibrate Subcellular Distribution as a Rational for the Intercranial Delivery Through Biodegradable Carrier” J. Physiol. Pharmacol. 2015, 66, 233-247.]. However, FF does not cross the BBB, and is quickly processed by the blood and tissue esterases to form fenofibric acid (FFA) Wilk, A.; Wyczechowska, D.; Zapata, A.; Dean, M.; Mullinax, J.; Marrero, L.; Parsons, C.; Peruzzi, F.; Culicchia, F.; Ochoa, A.; Grabacka, M.; Reiss, K. “Molecular Mechanism of Fenofibrate-Induced Metabolic Catastrophe and Glioblastoma Cell Death” Mol. Cell. Biol. 2015, 35, 182-198.]. This acid is no longer effective in triggering tumor cell death.
To eliminate both problem with hydrolysis as well as to increase solubility, tissue penetration and ultimately anti-glioblastoma activity we have made several chemical modifications to the FF molecular skeleton. One of these modifications has resulted in a new compounds, PP1 (
Results and Discussion
Overall Chemical Design of Therapeutic Compounds: In our previous studies we have explore importance of basic BPA skeleton and we have demonstrated that this is the pharmacophore [Gao, Q.; Yang, L.; Zhu, Y. “Pharmacophore based drug design approach as a practical process in drug discovery” Curr. Comput Aided Drug Des. 2010, 6, 37-49.] necessary to retain anti-glioblastoma activity. Amide part of the BPA skeleton can be modified in such a way to obtain desirable biological activity in combination with acceptable physicochemical molecular properties. We have selected phenol and naphthol BPA variants because phenol derivatives, besides many other health benefits, can have anti-cancer activity {Dzialo, M.; Mierziak, J.; Korzun, U.; Preisner, M.; Szopa, J.; Kulma, A. “The Potential of Plant Phenolics in Prevention and Therapy of Skin Disorders” Int. J. Mol. Sci. 2016, 17, 160; Bhuyan, J.; Basu, A. “Phenolic Compounds Potential Health Benefits and Toxicity” in Utilization of Bioactive Compounds from Agricultural and Food Waste. Chapter 2, Editor: Voug, Q. V. CRC Press, 2017]. The basic molecular skeleton for all prepared and tested compounds in this paper therefore contains phenolic BPA skeleton (
The starting point for the preparation of each and all phenolic BPA starts with readily available fenofibric acid (FFA) and corresponding aminophenol or aminonaphthol (
Biological, Chemical and Computational Testing of Therapeutic Compounds: There are many challenges in the development stage of drugs for central nervous system (CNS) [Palmer, A. M; Stephenson, F. A. “CNS drug discovery: challenges and solutions” Drug News Perspect. 2005, 18, 51-57.’ Danon, J. J.; Reekie, T. A.; Kassiou, M. “Challenges and Opportunities in Central Nervous System Drug Discovery” Trends in Chemistry 2019, 1, 612-624.]. Major question is how to find appropriate lead molecular structural skeleton that will result in acceptable drug candidate and ultimately desired drug. In our case there were plethora reported research that some lipid lowering drugs alone or in combination with other anticancer compounds noticeably decrease cancer prevalence [Papanagnou, P.; Stivarou, T.; Papageorgiou, I.; Papadopoulso, G. E.; Pappas, A. “Marketed drugs used for the management of hypercholesterolemia as cancer armament” Onco. Targets Ther. 2017, 10, 4393-4411.; Jang, H. J.; Kim, H. S.; Kim, J. H.; Lee, J. “The Effect od Statin Added to Systematic Anticancer Therapy: A Meta-Analysis of Randomized Controlled Trials” Journal of Clinical Medicine 2018, 7, 325]. On of lipid lowering drug that was used as lead molecule in two of our previous studies is fenofibrate (FF) [Lian, X.; Wang, G.; Zhou, H.; Zheng, Z.; Fu, Y.; Cai, L. “Anticancer Properties of Fenofibrate: Repurposing Use” Journal of Cancer, 2018, 9, 1527-1537.; Majeed, Y.; Upadhayay, R.; Alhousseiny, S.; Taha, T.; Musthak, A. Shaheen, Y.; Jameel, M.; Triggle, C. R.; Ding, H. “Potent and PPARalfa-independent anti-proliferative action of the hypolipidemic drug fenofibrate in VEGF-dependent angiosarcomas in vitro” Nature Scientific Reports 2019, 9, 6316.] from which one we have derived BPA molecular skeleton as an important pharmacophore as anti-glioblastoma drug. Here we evaluate the suitability of phenolic and naphtholic BPA as viable anti-glioblastoma compound. Before any of these compounds were to be prepared intensive computational studies were performed regarding expected physicochemical properties of simple phenoxy BPAs. First group of 3 computed parameters for elimination of drug candidates include log S, Log BB, and MPO-CNS. It was applied on unsubstituted phenolic BPAs (
Certainly, replacing phenyl group of HR48 with phenol group will substantially change physico-chemical properties of new compounds. There are 3 possible isomers HR49, HR50, and HR51 (
As mentioned before frontier molecular orbitals are routinely used in molecular modeling for drug discovery [Aminpour, M.; Nontemagno, C.; Tuszynski, J. “An Overview of Nolecular Modeling for Drug Discovery with Specific Illustrative Examples of Applications” Molecules 2019, 24, 1693]. The atomic orbital contribution to LUMO is entirely from the BPA molecular part and therefore it is not surprising that their estimated energies are almost identical (from −6.66 to −6.72 eV;
One can consider that HR49 is the most potent compounds of three phenolic BPAs. We have demonstrated that with changing electrostatic potential surface one can alter ant-glioblastoma activity of these compounds. We place on the phenol ring moderate electron donating (methyl), slightly electron withdrawing (chloro) and strong mesomeric electron withdrawing group (carboxylic). Computational studies all of 30 isomers were performed and four candidates were selected to be prepared and their anti-glioblastoma activity was evaluated (
Almost all computed indicators for carboxylic acid derivatives of phenolic BPAs indicate that these compounds are not good anti-glioblastoma drug candidates. To demonstrate this finding only one typical example of these group of compounds were presented. Compound HR55 has only one desirable physicochemical property that are better than for good glioblastoma drug candidate phenolic BPA HR49. Its water solubility (log S for HR55 is −3.20 in comparison with −6.61 for HR49) may be better. To demonstrate large deviation for desirable physicochemical properties when screen for molecular targets we will focus on basic principles of computational studies [Pajouhesh, H.; Lenz, G. R. “Medicinal Chemical Properties of Successful Central Nervous System Drugs” NeuroRx 2005, 2, 541-553]. Introducing carboxylic acid group in HR55 compounds is an acid with computed pKa of 4.23. Therefore, at philological pH this compound is deprotonated, and it is more important to use log D (partition dependence regarding pH) that is 2.13 [Kokate, A.; Li, Z.; Jasti, B. “Effect of drug Lipophilicity and Ionization on Permeability Across the Buccal Mucosa: A Technical Note” AAPS Pharm Sci Tech 2008, 9, 501-504; Lindsley C. W. “Lipophilicity. In: Stolerman I., Price L.; Price L. H (eds) Encyclopedia of Psychopharmacology” 2014 Springer, Berlin, Heidelberg.]. In comparison estimated log D for HR49 is 5.1. In addition, electrostaic potential map indication noticeable change in the phenolic part of the molecule. Considering that this is electron rich expected HVOMO energy should be substantially altered (−8.65 vs −9.32 respectively) while LUMO energy should be basically unchanged (−0.66 vs −0.69 eV respectively). That was perfectly demonstrated in
One can argue that one contribution to the phenolic BPA activity (
Introducing hydroxy group will substantially increase electron density on the phenolic moiety as well as solubility in water due to one more hydrogen donor group. One and two hydroxy derivatives of phenolic BPA (di and triphenols) was subject of our computational studies. From 11 compounds we have selected HR58 and HR59 candidates with best computed physicochemical descriptors for preparation and their anti-glioblastoma activity evaluation. These two compounds, HR58 and HR59, can be considered as monohydroxy derivatives of HR49 (Panel A). Both computed lipophilicity and water solubility are at an acceptable level. The predicted lipophilicity is slightly higher and predicted water solubility is slightly lower than what would be optimal values. There is not substantial difference in pKa1 values and ˜8.6 these molecules are unionized form at physiological pH. Predicted PSA are in an acceptable range. Major difference between these two compounds is in minimal projected area and HOMO energies. It is predicted that HR59 will penetrated slight better (MPA is 47.89 vs 49.54 Å2 for HR58, Panel A) and it is predicted to bind slightly stringer (HOMO is −8.54 vs −8.44 eV for HR58). Therefore, without wishing to be bound by theory, HR59 is more potent than any of the four studied drugs candied in this series of phenolic BPA. In fact, it is experimentally confirmed that this is the most potent compound and it should be considered viable for further studies as anti-glioblastoma drug.
Both computational studies and experimental data confirm that phenolic BPAs are an excellent anti-glioblastoma drug candidate. Beside lipophilicity, water solubility, blood-brain distribution, molecular size, frontier orbital energy (particularly HOMO), and electrostatic potential maps seems to be additional selecting physicochemical parameters to determine molecular suitability as anti-glioblastoma drug candidate. Computational studies of 1- and 2-naphylyl derivatives of BPA and 14 corresponding naphthols of BPA are performed. From this study we have selected six compounds for synthesis and further studies. Results are presented in
As mentioned above it is very difficult for drug to penetrated Central Nervous System. It is absolute necessity to develop reliable models that can accurately predict drugs blood-brain-barrier penetration ability [Gribkoff, V. K.; Kaczmarek, L. K. “The Need for New Approaches in CNS Srug Discovery: Why Drugs Have Failed, and What Can be Done to Improve Outcomes” Neuropharmacology 2017, 120, 11-19.; Banks, W. A.; Greig, N. H. “Small molecules as central nervous system therapeutics: old challenges, new directions, and a philosophic divide” Future Med. Chem. 2019, 11, 489-493.; Miao, R.; Xia, L.-Y.; Chen, H. H.; Huang, H.-H.; Liang, Y. “Improved Classification of Blood-Brain-Barrier Drugs Using Deep Learning” Scientific Reports 2019, 9, 8802.; Plisson, F.; Piggott, A. M. “Predicting Blood-Brain Barrier Permeability of Marine-Derived Kinase Inhibitors Using Ensemble Classifiers Reveals Potential Hits for Neurodegenerative Disorders” Mar. Drugs 2019, 17, 81.]. We used several models to calculate log BB values and compare them to experimental values to select best one that is suitable to our studies. We have applied these models to evaluate brain penetration ability of our compounds presented in this paper as well as for our earlier BPA compound PP1 that penetrates the brain (5 μM) with its estimated log BB to be zero [Stalinska, J.; Zimolag, E.; Pianovich, N. A.; Zapata, A.; Lassak, A.; Rak, M.; Dean, M.; Ucar-Bilyeu, D.; Culicchia, F.; Marrero, L.; Del Valle, L.; Sarkaria, J.; Peruzzi, F.; Jursic, B. S.; Reiss, K. “Chemically Modified Variants of Fenofibrate with Antiglioblastoma Potential” Trans. Oncol. 2019, 12, 895-907.]. Calculated values are presented in
In conclusion, although there nine compounds from eighteen selected by from computational studies that have acceptable anti-glioblastoma activity on LN229 cell line at 25 M concentration only four (HR49, HR50, HR51, and HR59) are selected as drug candidates. This is in addition to recently reported BPA based compounds (HR28, HR32, HR37, and HR46) [Stalinska, J H.; Houser, L.; Rak, M.; Colley, S. B.; Reiss, K.; Jursic, B. S. “Exploring anticancer activity of structurally modified benzylphenoxyacetamide (BPA); I: Synthesis strategies and computational analyses of substituted BPA variants with high anti-glioblastoma potential” Scientific Reports 2019, 9, 17021.]. All of these compounds have strong in vitro anti-glioblastoma activity in a combination of physical properties that may contribute to the improved brain tumor penetration (
Methods
All starting materials were reagent grade and purchased from Sigma-Aldrich, ArkPharm, and TCI America. 1H-NMR spectra were recorded on Varian Mercury 300 and Varian Mercury 400 Plus instruments in CDCl3 and DMSO-d6, using the solvent chemical shifts as an internal standard. All computed molecular descriptors were generated by ChemAxon MarvinSketch version 19.20. Electrostatic potential maps were calculated with PM3 semi-empirical method as implemented in Spartan '18 v 1.1.0. 1H-NMR and 13C-NMR spectra for all HR compounds generated in this study are included in Supplementary Materials.
Cell culture and viability assays. Human glioblastoma LN-229 cells were maintained as semi-confluent monolayer culture in DMEM with 1 g/L glucose, sodium pyruvate, L-glutamine (Corning) supplemented with 10% heat inactivated FBS (Gibco) and P/S (50 units/mL of penicillin and 50 μg/mL of streptomycin) at 37° C. in a 5% CO2 atmosphere. Prior to treatment, cells were plated well in 96-well plates (BD Falcon) at initial density of 2×103 cells/cm2. Stock solutions of the compounds were prepared in DMSO, diluted in cell culture medium and added to the cells in triplicate for every experimental condition 24 h after plating (final concentration 25 μM). For the vehicle control, DMSO was used at 0.5%. MTT assay40 (measuring cell metabolic activity) was performed after a 72 h incubation in the presence of the compounds as previously described. Following 1 h incubation with MTT, formazan crystals were dissolved in 5 mM HCl in isopropanol and absorbance read at 540 nm. Data represent mean values expressed as percentage of vehicle control±SD. Phase contrast images of treated cells were taken 48 hours following the treatment with a BZ-X800 fluorescence microscope (Keyence) equipped with a 20× objective.
Method A (Larger scale preparation without extraction or crystallization). 2-(4-(4-chlorobenzoyl)phenoxy)-N-(2-hydroxyphenyl)-2-methylpropanamide (HR49). Freshly prepared fenofibric chloride (FFC) from FFA (9.6 g; 0.03 mol) and oxalyl chloride (5.2 ml; 7.6 g; 0.09 mol) in dichloromethane (50 ml) by stirring at room temperature overnight. After solvent evaporation and solid residue drying under argon FFC was dissolved in dichloromethane (50 ml) and mixed with pyridine (50 ml) solution of 2-aminophenol (2.6 g; 0.025 mol). Resulting solution was stirred at room temperature for 3 hours and then refluxed for additional 3 hours. Solvent was evaporated under reduced pressure and solid residue mixed with ethanol (100 ml) and refluxed with stirring until all solid material was dissolved. This clear alcohol solution was mixed with hot (70° C.) 3% sodium carbonate solution (400 ml) and refluxed for half an hour. Volume of the mixture was reduced to ˜½ by destiling off solvent at room temperature. Resulting mixture was left at room temperature for several hours. Resulting solid product was separated by filtration, extensively washed with water (10×100 ml) and dried at 110° C. for 1 hour. Isolated yield 97% (4.77 g). 1H-NMR (DMSO-d6, 400 MHz) δ 9.95 (1H, s, OH), 9.09 (1H, s, NH), 7.93 (1H, d, J=8.0 Hz), 7.73 (2H, d, J=8.0 Hz), 7.70 (2H, d, J=8.4 Hz), 7.59 (2H, d, J=8.4 Hz), 7.11 (2H, d, J=8.4 Hz), 6.23 (1H, t, J=7.6 Hz), 6.83 (1H, d, J=8.0 Hz), 6.78 (1H, t, J=8.0 Hz), and 1.60 (6H, s) ppm. 13C-NMR (DMSO-d6) δ 193.8, 171.7, 158.9, 147.7, 137.6, 136.5, 132.2, 131.7, 131.3, 129.1, 126.1, 125.1, 121.2, 119.9, 119.6, 115.5, 82.3 and 25.3 ppm.
Method B (small scale preparation). Preparation of 2-(4-(4-chlorobenzoyl)phenoxy)-N-(2-hydroxy-5-methylphenyl)-2-methylpropanamide (HR52). Dichloromethane (10 ml) suspension of fenofibric acid (FFA) (191 mg; 0.6 mmol) and oxalyl chloride (0.2 ml; 384 mg; 2 mmol) was stirred at room temperature for five minutes. Few drops of N,N-dimethylformamide (DMF) was added and immediately bubbles start to form resulting for reaction mixture to become clear solution in approximately 30 minutes. This solution was stirred at 60° C. with slow solvent evaporation. Residue of solvent and oxalyl chloride was removed by drying under argon flow at room temperature. Resulting yellow solid was dissolved in dichloromethane (10 ml) and mixed with 2-amino-4-methylphenol (62 mg; 0.5 mmol) in THF (10 ml) and Na2CO3 (1.06 g; 10 mmol) in water (10 ml). Resulting mixture was stirred at room temperature for five hours. Solvent was evaporated under reduced pressure and solid residue was mixed with dichloromethane (50 ml) and water (50 ml). Resulting mixture was stirred with sonication at room temperature until all solid was dissolved. Water layer was discarded, and dichloromethane layer was washed with 5% Na2CO3 (3×50 ml), water (50 ml), 5% HCl (3×50 ml), water (50 ml) and dried over anhydrous Na2CO3. After solvent evaporation product was purified by crystallization from dichloromethane (˜3 ml) and hexane (20 ml). Isolated yield 90% (190 mg). 1H-NMR (DMSO-d6, 400 MHz) δ 9.74 (1H, broad s, OH), 9.06 (1H, s, NH), 7.81 (1H, s), 7.73 (2H, d, J=8.8 Hz), 7.70 (2H, d, J=8.8 Hz), 7.58 (2H, d, J=8.8 Hz), 7.11 (2H, d, J=8.4 Hz), 6.73 (2H, s), 2.18 (3H, s), and 1.60 (6H, s) ppm. 13C-NMR (DMSO-d6) δ 193.8, 171.6, 158.8, 145.4, 137.7, 136.5, 132.2, 131.7, 131.3, 129.1, 128.1, 125.9, 125.3, 121.5, 119.9, 115.2, 82.2, 25.3, and 20.9 ppm.
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.
This application claims priority from U.S. Provisional Application No. 62/791,252, filed Jan. 11, 2019, and U.S. Provisional Application No. 62/884,529, filed Aug. 8, 2019, the entire contents of each which are incorporated herein by reference.
This invention was made with government support under Grant No. P20-GM121288 awarded by the National Institutes of Health. The government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
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PCT/US20/13317 | 1/13/2020 | WO |
Number | Date | Country | |
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62791252 | Jan 2019 | US | |
62884529 | Aug 2019 | US |