BENZODIAZEPINE ANALOGS AND METHODS OF USE IN TREATING CANCER
20250313537 ยท 2025-10-09
Assignee
Inventors
- Soma Sengupta (Cincinnati, OH, US)
- Daniel Pomeranz Krummel (Cincinnati, OH, US)
- Donatien Kamdem Toukam (Cincinnati, OH, US)
- James Cook (Whitefish Bay, WI)
- Taukir Ahmed (Milwaukee, WI, US)
- Sepideh Rezvanian (Milwaukee, WI, US)
- Laura Kallay (Cincinnati, OH, US)
Cpc classification
A61K31/5517
HUMAN NECESSITIES
A61K31/5513
HUMAN NECESSITIES
International classification
A61K31/5517
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
Provided herein are benzodiazepine analogs that are modified at the 7-position on the benzodiazepine ring to include a moiety selected from C.sub.2-C.sub.4 alkynyl, C.sub.3-C.sub.6 cycloalkyl, d(5)-cyclopropyl, methyl alkynyl, alkynyl-CD.sub.3, and alkynyl-CF.sub.3. Also provided are methods of use of the compounds in treating cancer, sensitizing a tumor to radiation, sensitizing a tumor to immunotherapy or chemotherapy, and treating neurological conditions associated with Type-A GABA neurotransmitter receptor function. Pharmaceutical compositions including the compounds are also provided.
Claims
1. A compound according to Formula I, or a pharmaceutically acceptable salt, racemate, or enantiomer thereof: ##STR00050## wherein: R.sub.1 is selected from the group consisting of C.sub.2-C.sub.4 alkynyl, C.sub.3-C.sub.6 cycloalkyl, d(5)-cyclopropyl, methyl alkynyl, alkynyl-CD.sub.3, and alkynyl-CF.sub.3; R.sub.2 is selected from the group consisting of hydrogen, methyl, and trideuteromethyl; R.sub.3 and R.sub.4 are independently selected from the group consisting of hydrogen, deuterium, and methyl; and R.sub.5 is selected from the group consisting of hydrogen, methyl, trideuteromethyl, tritritiomethyl, halogen, and trifluoromethyl; wherein when R.sub.2 is trideuteromethyl, R.sub.1 is not ethynyl; and wherein when R.sub.1 is ethynyl, R.sub.5 is not hydrogen.
2. The compound according to claim 1, wherein R.sub.3 and R.sub.4 are each hydrogen or are each deuterium.
3. (canceled)
4. The compound according to claim 1, wherein R.sub.3 is hydrogen and R.sub.4 is methyl.
5. The compound according to claim 1, wherein R.sub.5 is Cl, F, or Br.
6. The compound according to claim 1, wherein R.sub.1 is cyclopropyl.
7. The compound according to claim 6, wherein the compound is selected from the group consisting of: ##STR00051## ##STR00052##
8. The compound according to claim 1, wherein R.sub.1 is ethynyl.
9. The compound according to claim 8, wherein the compound is selected from the group consisting of: ##STR00053##
10. The compound according to claim 1, wherein R.sub.1 is cyclopropyl or ethynyl; R.sub.2 is hydrogen or methyl; R.sub.3 and R.sub.4 are each deuterium; and R.sub.5 is selected from the group consisting of hydrogen, methyl, trideuteromethyl, tritritiomethyl, trifluoromethyl, fluorine, and chlorine.
11. A pharmaceutical composition comprising: an effective amount of the compound according to claim 1; and a pharmaceutically acceptable carrier.
12. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound according to Formula I, or a pharmaceutically acceptable salt, racemate, or enantiomer thereof: wherein: R.sub.1 is selected from the group consisting of C.sub.2-C.sub.4 alkynyl, C.sub.3-C.sub.6 cycloalkyl, d(5)-cyclopropyl, methyl alkynyl, alkynyl-CD.sub.3, and alkynyl-CF.sub.3; R.sub.2 is selected from the group consisting of hydrogen, methyl, and trideuteromethyl; R.sub.3 and R.sub.4 are independently selected from the group consisting of hydrogen, deuterium, and methyl; and R.sub.5 is selected from the group consisting of hydrogen, methyl, trideuteromethyl, tritritiomethyl, halogen, and trifluoromethyl; wherein when R.sub.2 is trideuteromethyl, R.sub.1 is cyclopropyl; and wherein when R.sub.1 is ethynyl, R.sub.5 is not hydrogen.
13. The method according to claim 12, wherein R.sub.3 and R.sub.4 are each hydrogen or are each deuterium.
14. (canceled)
15. The method according to claim 12, wherein R.sub.3 is hydrogen and R.sub.4 is methyl.
16. The method according to claim 12, wherein R.sub.5 is Cl, F, or Br.
17. The method according to claim 12, wherein R.sub.1 is cyclopropyl.
18. The method according to claim 17, wherein the compound is selected from the group consisting of: ##STR00054##
19. The method according to claim 12, wherein R.sub.1 is ethynyl.
20. The method according to claim 19, wherein the compound is selected from the group consisting of: ##STR00055##
21. A method of sensitizing a tumor to radiation in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound according to claim 1.
22. A method of sensitizing a tumor to immunotherapy or chemotherapy in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound according to claim 1.
23. A method of treating a neurological condition associated with Type-A GABA neurotransmitter receptor function in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound according to claim 1.
24. The method according to claim 23, wherein the neurological condition is selected from the group consisting of sleep disorder, generalized anxiety disorder, social anxiety disorder, seizure disorder, panic disorder, tic disorder, bipolar disorder, and alcohol withdrawal.
25. The method according to claim 24, wherein the sleep disorder is insomnia.
26. The method according to claim 24, wherein the seizure disorder is epilepsy.
27. The compound according to claim 8, wherein when R.sub.3 and R.sub.4 are each hydrogen, R.sub.2 is not methyl.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The details of embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided herein.
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DETAILED DESCRIPTION
[0029] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document.
[0030] While the following terms are believed to be well understood in the art, definitions are set forth to facilitate explanation of the presently disclosed subject matter. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently-disclosed subject matter belongs.
[0031] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term about. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
[0032] As used herein, the term about, when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments 20%, in some embodiments 10%, in some embodiments 5%, in some embodiments 1%, in some embodiments 0.5%, and in some embodiments 0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
[0033] For the purposes of defining the present technology, the transitional phrase consisting of may be introduced in the claims as a closed preamble term limiting the scope of the claims to the recited components or steps and any naturally occurring impurities. For the purposes of defining the present technology, the transitional phrase consisting essentially of may be introduced in the claims to limit the scope of one or more claims to the recited elements, components, materials, or method steps as well as any non-recited elements, components, materials, or method steps that do not materially affect the novel characteristics of the claimed subject matter. The transitional phrases consisting of and consisting essentially of may be interpreted to be subsets of the open-ended transitional phrases, such as comprising and including, such that any use of an open ended phrase to introduce a recitation of a series of elements, components, materials, or steps should be interpreted to also disclose recitation of the series of elements, components, materials, or steps using the closed terms consisting of and consisting essentially of. For example, the recitation of a composition comprising components A, B, and C should be interpreted as also disclosing a composition consisting of components A, B, and C as well as a composition consisting essentially of components A, B, and C. Any quantitative value expressed in the present application may be considered to include open-ended embodiments consistent with the transitional phrases comprising or including as well as closed or partially closed embodiments consistent with the transitional phrases consisting of and consisting essentially of.
[0034] When the term independently selected is used, the substituents being referred to (e.g., R groups, such as groups R.sub.3 and R.sub.4), can be identical or different. For example, both R.sub.3 and R.sub.4 can be the same substituent, or R.sub.3 and R.sub.4 can each be different substituents selected from a specified group.
[0035] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
[0036] As used in this specification and the appended claims, the singular forms a, an, and the include plural references unless the content clearly dictates otherwise.
[0037] A pharmaceutically acceptable salt is a cationic salt formed at any acidic (e.g., hydroxamic or carboxylic acid) group, or an anionic salt formed at any basic (e.g., amino) group. Many such salts are known in the art, as described in WO 1987/005297, by Johnston et al., published Sep. 11, 1987. Specific cationic salts include the alkali metal salts (such as sodium and potassium), and alkaline earth metal salts (such as magnesium and calcium) and organic salts. Specific anionic salts include halide (such as chloride, bromide, or fluoride salts), sulfate, and maleate. In embodiments, suitable pharmaceutically acceptable salts include, but are not limited to, halide, sodium, sulfate, acetate, phosphate, diphosphate, potassium, maleate, calcium, citrate, mesylate, nitrate, tartrate, aluminum, gluconate, carboxylate, and the like.
[0038] Such salts are well understood by the skilled artisan and the skilled artisan is able to prepare any number of salts given the knowledge in the art. Furthermore, it is recognized that the skilled artisan may select one salt over another for reasons of solubility, stability, formulation ease and the like. Determination and optimization of such salts is within the purview of the skilled artisan's practice.
[0039] The terms enantiomer and racemate have the standard art recognized meanings (see, e.g., Hawley's Condensed Chemical Dictionary, 16th ed. (2016)). The illustration of specific protected forms and other derivatives of the compounds of the instant invention is not intended to be limiting. The application of other useful protecting groups, salt forms, esters, and the like is within the purview of the skilled artisan.
[0040] The terms halo or halogen, as used herein, refer to fluoro (F), chloro (Cl), bromo (Br), and iodo (I) groups.
[0041] Alkynyl, as used herein, refers to a univalent hydrocarbon radical containing a triple bond. In embodiments, an alkynyl bond is represented as CC. In embodiments, an alkynyl substituent is a C.sub.2-C.sub.4 alkynyl. In a specific embodiment, the alkynyl group is an ethynyl (also called ethinyl) group. Optionally, an alkynyl group may be substituted. Such substituted alkynyls include, but are not limited to, methyl alkynyl, alkynyl-D.sub.3, alkynyl-CF.sub.3, and the like.
[0042] Cycloalkyl, as used herein, refers to a C.sub.3-C.sub.6 inclusive hydrocarbon ring. Exemplary cycloalkyls include, but are not limited to, cyclopropyl, d(5)-cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl moieties. Optionally, a cycloalkyl group may be substituted with one or more short alkyls (C.sub.1-C.sub.6alkyl), deuterium, tritium, halogen, and the like.
[0043] D(5)-cyclopropyl refers to a cyclopropyl group wherein the hydrogen atoms are replaced with deuterium:
##STR00002##
[0044] Deuterium (D), also known as heavy hydrogen or hydrogen-2, refers to an isotope of hydrogen that has one proton and one neutron in its nucleus and has twice the mass of hydrogen. A deuterated compound is a compound to which a deuterium atom has been introduced to replace hydrogen. Trideuteromethyl, or CD.sub.3, is a methyl group wherein the hydrogen atoms have been replaced with deuterium.
[0045] Tritium (T), also known as hydrogen-3, refers to a radioactive isotope of hydrogen that has one proton and two neutrons. A tritiated compound is a compound to which a tritium atom has been introduced to replace hydrogen. Tritritiomethyl, or CT.sub.3, is a methyl group wherein the hydrogen atoms have been replaced with tritium.
[0046] As used herein, the terms treatment or treating of a condition and/or a disease in an individual, including a human or lower mammal, means: [0047] (i) preventing the condition or disease, that is, avoiding any clinical symptoms of the disease, particularly in individuals at risk for developing the condition or disease; [0048] (ii) inhibiting the condition or disease, that is, arresting the development or progression of clinical symptoms; and/or [0049] (iii) relieving the condition or disease, that is, causing the regression of clinical symptoms.
[0050] The terms effective amount or therapeutically effective amount as defined herein in relation to the treatment of cancer or neurological disorders, refer to an amount that will decrease, reduce, inhibit, or otherwise abrogate the growth of a cancer cell or tumor or arrest the development or progression of clinical symptoms of the neurological disorder. The specific therapeutically effective amount will vary with such factors as the particular disease being treated, the physical condition of the individual being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed.
[0051] As used herein, the terms administer or administration may comprise administration routes such as enteral (e.g., oral, sublingual, buccal, or rectal), parenteral (e.g., intravenous, intramuscular, subcutaneous, intraarterial, intratumoral), intranasal, inhaled, vaginal, transdermal, etc., so long as the route of administration results in an anti-cancer effect or treats the neurological disorder in the subject. In specific embodiments, the administration route is oral, intravenous, or intratumoral.
[0052] As used herein, the term subject generally refers to a living being (e.g., animal or human) capable of suffering from cancer or a neurological disorder. In a specific embodiment, the subject is a mammal. In a more specific embodiment, the subject is a human subject.
Compounds
[0053] Provided herein are benzodiazepine analogs that enhance chloride anion efflux in cancer cells, thereby initiating a cascade of events that impairs cancer cell viability. The disclosed benzodiazepine analogs have further application in the treatment of neurological disorders associated with GABA.sub.A receptor function.
[0054] The compounds disclosed herein are analogs of benzodiazepine compounds such as diazepam and QH-II-066, which compounds have the following structures:
##STR00003##
[0055] In one embodiment, a compound according to Formula I is provided, or a pharmaceutically acceptable salt, racemate, or enantiomer thereof:
##STR00004##
wherein R.sub.1 is selected from the group consisting of C.sub.2-C.sub.4 alkynyl, C.sub.3-C.sub.6 cycloalkyl, d(5)-cyclopropyl, methyl alkynyl, alkynyl-CD.sub.3, and alkynyl-CF.sub.3; R.sub.2 is selected from the group consisting of hydrogen, methyl, and trideuteromethyl; R.sub.3 and R.sub.4 are independently selected from the group consisting of hydrogen, deuterium, and methyl; and R.sub.5 is selected from the group consisting of hydrogen, methyl, trideuteromethyl, tritritiomethyl, halogen, and trifluoromethyl. In certain embodiments, when R.sub.2 is trideuteromethyl, R.sub.1 is not ethynyl. In certain embodiments, when R.sub.1 is ethynyl, R.sub.5 is not hydrogen.
[0056] In some embodiments, R.sub.3 and R.sub.4 are each deuterium. In other embodiments, R.sub.3 and R.sub.4 are each hydrogen. In other embodiments, one or both of R.sub.3 or R.sub.4 is methyl.
[0057] In some embodiments, R.sub.5 is a halogen selected from the group consisting of Cl, F, and Br.
[0058] In some embodiments, R.sub.1 is cyclopropyl. In other embodiments, R.sub.1 is ethynyl.
[0059] In a specific embodiment, R.sub.1 is cyclopropyl or ethynyl; R.sub.2 is hydrogen or methyl; R.sub.3 and R.sub.4 are each deuterium; and R.sub.5 is selected from the group consisting of hydrogen, methyl, trideuteromethyl, tritritiomethyl, trifluoromethyl, fluorine, and chlorine.
[0060] In some embodiments, the compound is selected from the compounds set forth in Table 1:
TABLE-US-00001 TABLE 1 Exemplary Compounds Formula/Molecular Name Chemical Name Compound Weight TA-IV-08 7-bromo-1-methyl-5-(o- tolyl)-1,3-dihydro-2H- benzo[e][1,4]diazepin-2- one
Exemplary Synthetic Routes
Scheme 1: Preparation of TA-II-73
[0061] In embodiments, benzodiazepine analogs of the present disclosure differ from diazepam by a 2 methyl and an ethynyl moiety in place of Cl.
##STR00023##
Scheme 2: Preparation of N-H TA-II-59
##STR00024##
Scheme 3: Preparation of SRE-III-35 and SRE-III-43
##STR00025##
2-bromo-N-(4-bromo-2-(2-methylbenzoyl) phenyl) acetamide (3)
##STR00026##
[0062] To a mixture of (2-amino-5-bromophenyl) (o-tolyl) methanone 1 (10 g, 34.46 mmol), sodium bicarbonate (5.79 g, 68.92 mmol), and dichloromethane (100 mL), bromoacetyl bromide (2) (3.60 mL, 41.35 mmol) was added dropwise. The temperature was kept between 10 C.-0 C. with continuous stirring. The white colored reaction mixture, which resulted, was then allowed to stir for longer than 3 h at room temperature (rt). The completion of the reaction was verified by analysis by TLC (silica gel) and 50% ethyl acetate/hexanes. The reaction mixture was then slowly diluted over 30 min with water (100 mL) as carbon dioxide bubbles occurred. The biphasic mixture, which resulted, was allowed to stand for 15 min and the layers were separated. The aq layer was extracted with dichloromethane (100 mL) and the combined organic layers were washed with 5% aq sodium bicarbonate solution (100 mL) and then 10% aq sodium chloride solution (300 mL). The organic layer was dried (Na.sub.2SO.sub.4). The solvents were removed under reduced pressure and the residue was slurried with ethanol (100 mL) at 50-55 C. for 30 min. Upon cooling to rt and after holding the temperature for 1 h, the solid, which formed, was filtered, and washed with ethanol (60 mL3). The solid was dried under vacuum at 40 C. to afford the product 2-bromo-N-(4-bromo-2-(2-methylbenzoyl) phenyl) acetamide 2 as an off-white solid (3) (13.56 g, 95.5%).
[0063] .sup.1H NMR (300 MHz, CDCl.sub.3) 12.01 (s, 1H), 8.66 (d, J=9.0 Hz, 1H), 7.71 (d, J=9.0 Hz, 1H), 7.55 (s, 1H), 7.44 (s, 1H), 7.35 (s, 2H), 4.07 (s, 2H), 2.35 (s, 3H). .sup.13C NMR (300 MHz, CDCl.sub.3) 190.64 (s), 172.73 (s), 139.27 (s), 138.47 (s), 137.96 (s), 131.86 (s), 130.16 (s), 129.35 (s), 128.66 (s), 127.56 (s), 125.65 (s). 124.35 (s), 122.76 (s), 36.02 (s), 19.82 (s); HRMS (ESI/IT-TOF) m/z: [M+H]+ Calcd for C.sub.16H.sub.13BrNO.sub.2 411.0869. found 411.0851.
7-bromo-5-(o-tolyl)-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (4)
##STR00027##
[0064] A mixture of 2-bromo-N-(4-bromo-2-(2-methylbenzoyl) phenyl) acetamide, 3 (10 g, 24.3 mmol), hexamethylenetetramine (HMTM, 7.50 g, 53.5 mmol), ammonium acetate (4.12 g, 53.5 mmol), and isopropanol (100 mL) was heated to reflux (82 C.). The reaction mixture was held at reflux for 6 h, at which point the reaction progress was deemed complete on analysis by TLC (silica gel and 1:1, ethyl acetate/hexanes). The reaction mixture was then cooled to 0-5 C. using an ice bath. The solid, which resulted, was filtered, and washed with cold isopropanol (100 mL2) and then water (100 mL4). The solid was dried under vacuum at 40 C. to afford 3.2 g of the benzodiazepine 72 as an off-white solid. The IPA was removed from the mother liquor under reduced pressure. The solid was then extracted with ethyl acetate. The ethyl acetate was removed under reduced pressure and the residue was purified by column chromatography using 1:4 ethyl acetate/hexanes to afford 2.01 grams more of 7-bromo-5-(o-tolyl)-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (4) (5.21 g, 65%).
[0065] .sup.1H NMR (300 MHz, CDCl.sub.3) 11.95 (s, 1H), 8.68 (d, J=9.0 Hz, 1H), 7.65 (d, J=9.0 Hz, 1H), 7.45 (d, J=22.1 Hz, 2H), 7.27-7.17 (m, 2H), 7.14 (d, J=7.6 Hz, 1H), 3.65 (s, 2H), 2.23 (s, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3) 200.64, 170.52, 139.35, 138.24, 137.51, 136.52, 136.16, 131.20, 130.77, 128.31, 125.50, 122.95, 115.09, 53.65, 19.80. HRMS (ESI/IT-TOF) m/z: [M+H]+ Calcd for C.sub.17H.sub.15BrN.sub.2O, 329.1892. found 329.1922.
7-bromo-1-methyl-5-(o-tolyl)-1,3-dihydro-2H-benzo[e] [1,4] diazepin-2-one (5)
##STR00028##
[0066] The 7-bromo-5-(o-tolyl)-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one 4, (5.0 g, 15.2 mmol) was dissolved in THF, (30 mL) and the solution was cooled to 0 C. using an ice bath. Then potassium tert butoxide (1.87 g, 16.7 mmol) which was dissolved in 30 mL of THF was added dropwise by using an addition funnel. Then methyl iodide (1.14 mL, 18.2 mmoL) was added dropwise to the reaction mixture over a 1 min period, while maintaining the temperature at 0 C. Upon completion of the addition, the reaction mixture was allowed to warm to rt and stir for 60 min, at which point the reaction was deemed complete on analysis by TLC (silica gel). The reaction mixture was then diluted with ethyl acetate (20 mL) and a solution of 10% aq sodium chloride (200 mL) was added. The biphasic mixture, which resulted, was allowed to stand for 15 min and the layers were separated. The aq layer was then extracted with ethyl acetate (50 mL) and the combined organic layers were washed with 10% aq sodium chloride solution (50 mL). The organic layer was dried (Na.sub.2SO.sub.4). The solvent was removed under reduced pressure. The brown solid which was obtained was purified by crystallization using 15:85 (ethyl acetate/hexanes), to give 7-bromo-1-methyl-5-(o-tolyl)-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (5) (3.91 g, 75%) as a brownish white solid.
[0067] .sup.1H NMR (300 MHz, CDCl.sub.3) 7.42 (d, J=6.4 Hz, 4H), 7.28 (d, J=4.1 Hz, 1H), 7.17 (d, J=8.1 Hz, 1H), 7.01 (d, J=2.2 Hz, 1H), 4.33 (s, 2H), 3.87 (s, 3H), 2.10 (s, 3H). .sup.13C NMR (126 MHz, CDCl3) 169.93, 168.86, 143.10, 138.19, 134.37, 132.89, 130.73, 129.50, 128.45, 122.79, 116.82, 56.94, 34.85, 19.98. HRMS (ESI/IT-TOF) m/z: [M+H]+ Calcd for C.sub.17H.sub.15BrN.sub.2O, 343.2169. found 343.2152.
1-methyl-5-(o-tolyl)-7-((triisopropylsilyl)ethynyl)-1,3-dihydro-2H-benzo[e] [1,4] diazepin-2-one (6)
##STR00029##
[0068] In a 500 mL round bottom flask, Pd(OAc).sub.2 (112 mg, 0.5 mmol) and P(o-tolyl)3 (304.37 mg, 1.0 mmol) was added to 50 mL of acetonitrile. The mixture was stirred until a slurry appeared, which took about 20 min. Then 7-bromo-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one 5, (3.50 g, 10.2 mmol), triethylamine (4.26 mL, 30.6 mmol), (triisopropylsilyl)acetylene (3.43 mL, 15.3 mmol) and additional acetonitrile (50 mL) was added. The reaction mixture was then heated to reflux (75 C.) and held for 6 h, at which point the reaction was deemed complete on analysis by TLC (Silica gel). Upon completion of the reaction progress, the mixture was cooled to rt and filtered through celite. After washing with acetonitrile (100 mL2), the solvents were removed under reduced pressure and the residue was dissolved in dichloromethane (400 mL) and 5% aq sodium bicarbonate (400 mL) was added. The biphasic mixture, which resulted, was allowed to stand for 15 min and the layers were separated. The aq layer was then extracted with dichloromethane (300 mL) and the combined organic layers were washed with 5% aq sodium bicarbonate solution (300 mL) and then 10% aq sodium chloride solution (300 mL3). The organic layer was dried (Na.sub.2SO.sub.4) and it was purified by flash chromatography on silica gel. This process gave a dark orange liquid that gets solidified eventually on standing to give 1-methyl-5-phenyl-7-((tripropan-2-ylsilyl) ethynyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one (6) (3.4, 71% crude yield).
[0069] .sup.1H NMR (500 MHz, CDCl.sub.3) 7.24-7.52 (m, 7H), 3.48 (s, 2H), 3.33 (s, 3H), 2.21 (s, 3H), 1.27 (m, 3H), 1.0 (d, 18H); .sup.13C NMR (500 MHz, CDCl3) 177.23 (s), 168.21 (s), 137.86 (s), 132.36 (s), 129.35 (s), 128.97 (s), 128.56 (s), 128.37 (s), 125.65 (s), 122.47 (s), 92.37 (s), 91.53 (s), 43.84 (s), 33.82 (s), 19.82 (s), 15.53 (s, 3C), 9.43 (s, 6C).
7-ethynyl-1-methyl-5-(o-tolyl)-1,3-dihydro-2H-benzo[e] [1,4] diazepin-2-one (7)
##STR00030##
[0070] 1-Methyl-5-phenyl-7-((tripropan-2-ylsilyl) ethynyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one 6, (3.40 g, 7.6 mmol), water (0.5 mL) and tetrahydrofuran (30 mL) were cooled to 20 C. using a dry ice/IPA bath. Then tetrabutylammonium fluoride hydrate, [1 M in THF (10.9 mL, 10.9 mmoL)] was added dropwise to the reaction mixture over a 30 min period, while maintaining the temperature at 20 to 15 C. Upon completion of the addition, the reaction mixture was allowed to warm to rt and stir for an additional 60 min at which point the reaction progress was deemed complete on analysis by TLC (silica gel). The reaction mixture was then diluted with ethyl acetate (50 mL) and 10% aq sodium chloride (50 mL). The biphasic mixture, which resulted, was allowed to stand for 15 min and the layers were separated. The aq layer was then extracted with ethyl acetate (50 mL3) and the combined organic layers were washed with 10% aq sodium chloride solution (150 mL). The organic layer was dried (Na.sub.2SO.sub.4). The solvents were removed under reduced pressure. Then the mixture was dissolved in 50 mL of ethyl acetate and then stirred with 20 g of silica gel for 2 hours and filtered. The amount of solvent was reduced to about 40 mL under reduced pressure. Then 20 mL of hexanes was added dropwise to the mixture, and it was allowed to stir overnight. The solid, which formed, was filtered and the grey solid was recrystallized from 1:4 (ethyl acetate/hexanes) to obtain cream white colored TA-II-73 (7). (1.98 g, 90%).
[0071] .sup.1H NMR (500 MHz, CDCl.sub.3) 7.63-7.60 (m, 1H), 7.38-7.27 (m, 4H), 7.24-7.18 (m, 2H), 4.86 (t, J=11.4 Hz, 1H), 3.81 (dd, J=18.3, 10.8 Hz, 1H), 3.46 (s, 3H), 3.05 (s, 1H), 1.99 (d, J=8.0 Hz, 3H). .sup.13C NMR (126 MHz, CDCl.sub.3) 171.32, 169.86, 143.10, 138.91, 136.26, 134.67, 133.12, 130.84, 130.49, 129.67, 128.39, 125.97, 121.12, 118.19, 81.95, 78.27, 56.79, 34.72, 19.94. HRMS (ESI/IT-TOF) m/z: [M+H]+ Calcd for C.sub.19H.sub.16N.sub.2O, 288.3420. found 288.3406.
7-cyclopropyl-5-phenyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (39)
##STR00031##
[0072] To a solution of palladium (II) acetate (0.099 g, 0.44 mmol) and tri-o-tolyl phosphine (0.27 g, 0.88 mmol) in toluene, the 7-bromo-5-phenyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (2.0 g, 6.3 mmol) was added. Then in sequence, cyclopropyl boronic acid (2.45 g, 28.52 mmol), and bis(triphenylphosphine) (5.38 g, 25.37 mmol) and water (0.5 mL) were added under argon. A reflux condenser was attached, and the reaction mixture was heated to reflux. The mixture was stirred and heated to 100 C. After 12 h the reaction was completed on analysis by mass spectroscopy, and it was then cooled to rt and silica gel (3 g) was added. After stirring for 30 minutes open to the air, the spent catalyst on silica gel was removed by filtration through a pad of celite and washed with EtOAc. Then the filtrate was concentrated under reduced pressure. The residue which resulted was purified by a wash column (silica gel, EtOAc) to afford the desired product as a white solid (39) (1.29 g, 74%).
[0073] .sup.1H NMR (500 MHz, CDCl.sub.3) 9.67 (s, 1H), 7.65 (d, J=7.5 Hz, 1H), 7.55 (dd, J=14.8, 7.3 Hz, 2H), 7.47 (t, J=7.5 Hz, 2H), 7.25 (d, J=7.6 Hz, 1H), 7.21 (d, J=8.0 Hz, 1H), 7.02 (d, J=1.7 Hz, 1H), 4.29 (s, 2H), 1.89-1.82 (m, 1H), 0.98-0.94 (m, 2H), 0.64-0.57 (m, 2H). .sup.13C NMR (126 MHz, CDCl.sub.3) 170.3, 166.00, 139.66, 138.35, 137.24, 132.71, 131.34, 130.98, 130.68, 129.95, 129.57, 129.22, 121.85, 121.69, 47.10, 14.83, 9.32, 9.18.
7-cyclopropyl-5-(2-fluorophenyl)-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (40)
##STR00032##
[0074] To a solution of palladium (II) acetate (0.07 g, 0.31 mmol) and tri-o-tolyl phosphine (0.19 g, 0.63 mmol) in toluene, the 7-bromo-5-(2-fluorophenyl)-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (1.5 g, 4.5 mmol) was added. Then in sequence, cyclopropyl boronic acid (1.74 g, 20.25 mmol), and bis(triphenylphosphine) (3.81 g, 18 mmol) and water (0.36 mL) were added under argon. A reflux condenser was attached, and the reaction mixture was heated to reflux. The mixture was stirred and heated to 100 C. After 12 h the reaction was completed on analysis by mass spectroscopy, and it was then cooled to rt and silica gel (2.5 g) was added. After stirring for 30 minutes open to the air, the spent catalyst on silica gel was removed by filtration through a pad of celite and washed with EtOAc. Then the filtrate was concentrated under reduced pressure. The residue which resulted was purified by a wash column (silica gel, EtOAc) to afford the desired product as a white solid (40) (0.99 g, 75%).
[0075] .sup.1H NMR (500 MHz, CDCl.sub.3) 9.66 (s, 1H), 7.62 (t, J=7.2 Hz, 1H), 7.57 (t, J=7.4 Hz, 1H), 7.31 (d, J=8.2 Hz, 1H), 7.29 (s, 1H), 7.26 (d, J=7.1 Hz, 1H), 7.24 (s, 1H), 6.95 (s, 1H), 4.36 (s, 2H), 1.86-1.80 (m, 1H), 0.94 (dt, J=6.3, 3.1 Hz, 2H), 0.59 (dt, J=10.0, 4.9 Hz, 2H). .sup.13C NMR (126 MHz, CDCl.sub.3) 170.2, 165.27, 161.59, 159.57, 140.13, 139.0, 134.6, 132.61, 132.14, 124.40, 123.95, 121.72, 116.46, 116.40, 116.23, 14.80, 9.25, 9.05.
Synthesis of N-CD.SUB.3 .Analogs of QH-II-066 and KRM-II-08
##STR00033##
[0076] Two N-CD.sub.3 compounds were synthesized designated TA-I-16 and MYM-III-85. TA-I-16 can be synthesized from NOR KRM-II-08 by treating the amide with deuterated methyl iodide in the presence of potassium tert-butoxide in THF at 0 C. to room temperature. MYM-III-85 can be synthesized from nor QH-II-066 via a similar route. The final products can be purified by column chromatography with ethyl acetate-hexanes (20:80). KRM-II-08 has the following structure:
##STR00034##
Synthesis of D.SUB.2.-QH-II-066
[0077] To synthesize D.sub.2-QH-II-066, one must o exchange the hydrogen atoms at the C-3 position of QH-II-066. To do this, a sufficiently strong base and a deuterated solvent are needed. 50 mg of QH-II-066 are dissolved in in 1 mL of D4-methanol. 1 equivalent of base is added and the mixture is stirred for 1 h at room temperature. The precipitate is then filtered using a PTFE filter. The D4-methanol is evaporated on a rotary evaporator under reduced pressure. Exchange of H for D is confirmed via .sup.1H NMR. The solid is dissolved in regular methanol and the solvent is evaporated 5-6 times to regenerate the acetylene hydrogen atom.
[0078] Potassium tert-butoxide is a suitable strong base. Applying heat after the workup increases the percent of deuterium exchange. Without heat, one cannot regenerate back the acetylene hydrogen. 95+% deuterium is preferred for use in in vivo assays.
##STR00035##
Synthesis of D.SUB.5.-QH-II-066
##STR00036##
[0079] D.sub.3-MYM-III-85 was used as a starting material. This compound contains an N-CD.sub.3 moiety. A similar procedure was followed as was done for D.sub.2-QH-II-066 for the deuteration of MYM-III-85, but the results were the same as observed in the case of D.sub.2-QH-II-066. Potassium tert-butoxide was used as the base and D4-methanol was used as the solvent.
[0080] MYM-III-85 was dissolved in D4-methanol and 1.1 equivalents of potassium tert-butoxide was added. The mixture was sonicated at 50 C. for one hour. Then, 4 mL of DI water were added to the mixture at room temperature. This solution was extracted with ethyl acetate and the solvent was removed under an argon flow. After this, a 96% deuterium exchange at the C-3 position of MYM-III-85 was observed, providing 95%+D.sub.5-QH-II-066, which was assigned code number of TA-III-72.
7-ethynyl-1-(methyl-d3)-5-phenyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one. (22)
##STR00037##
[0081] The 5-phenyl-7-((ethynyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one (7), (4.0 g, 15.34 mmol) was dissolved in THF (15 mL) and the solution was cooled to 0 C. using an ice bath. Then potassium tert butoxide (1.90 g, 16.92 mmol), which was dissolved in 20 mL of THF, was added dropwise by using an addition funnel. Then trideutero methyl iodide (1.24 mL, 19.94 mmoL) was added dropwise to the reaction mixture over a 5 min period, while maintaining the temperature at 0 C. Upon completion of the addition, the reaction mixture was allowed to warm to rt and stirred for 60 min, at which point the reaction was deemed complete on analysis by TLC (silica gel). The reaction mixture was then diluted with ethyl acetate (20 mL) and a solution of 10% aq sodium chloride solution (20 mL) was added. The biphasic mixture, which resulted, was allowed to stand for 15 min and the layers were separated. The aq layer was then extracted with ethyl acetate (20 mL) and the combined organic layers were washed with 10% aq sodium chloride solution (20 mL). The organic layer was dried (Na.sub.2SO.sub.4). The solvent was removed under reduced pressure. The brown solid, which was obtained, was purified by crystallization using 15:85 (ethyl acetate/hexanes), to give 7-ethynyl-1-(methyl-d3)-5-phenyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (22)(3.7 g, 86%) as a white solid.
[0082] .sup.1H NMR (500 MHz, CDCl.sub.3): 7.82 (d, 1H) 7.66-7.64 (m, 2H, 2ArH), 7.60-7.57 (m, 2H, 2ArH), 7.52-7.49 (m, 2H, 2ArH), 7.38 (dd, 1H, J=8.5, 2.0 Hz, H-4), 4.6 (D, 1H), 3.6 (D, 1H), 3.1 (s, 1H). .sup.13C NMR (500 MHz, CDCl.sub.3): 171.2, 168.21, 149.4, 139.5, 134.4, 133.4, 131.7, 129.3, 128.5, 120.3, 119.2, 118.8, 82.3, 79.2, 56.2, 34.2. Rf=0.5 (silica gel, ethyl acetate/hexanes 1:2) HRMS (ESI/IT-TOF): m/z [M+H]+ calcd for C.sub.18H.sub.11D.sub.3N.sub.2O: 277.3350. found 277.3329.
7-Ethynyl-1-(methyl-d3)-5-phenyl-1,3-dihydro-2H-benzo[e] [1,4] diazepin-2-one-3,3-d2 (24)
##STR00038##
[0083] The 7-ethynyl-1-(methyl-d3)-5-phenyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (22), (0.5 g, 1.8 mmol) was dissolved in D4-methanol (1 mL). Then potassium tert butoxide (0.22 g, 1.9 mmol) was added to the solution. The mixture was sonicated for 45 min starting from rt to 50 C. After the reaction was completed (checked by NMR) 5 ml of de-ionized water was added and the mixture was extracted with (5 mL2) ethyl acetate. It was dried by first with blowing argon over it and then placing it in high vacuum. This gave 1-CD3-methyl-5-phenyl-7-ethynyl-1,3-dihydro-2d-1,4-benzodiazepin-2-one with 97% deuteration at the C-3 position via integration by NMR spectroscopy, to give 7-ethynyl-1-(methyl-d3)-5-phenyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one-3,3-d2 (D5-QH-II-066) (24) (0.49 g, 97%) as a white solid.
[0084] .sup.1H NMR (500 MHz, CDCl.sub.3): 7.82 (d, 1H) 7.66-7.64 (m, 2H, 2ArH), 7.60-7.57 (m, 2H, 2ArH), 7.52-7.49 (m, 2H, 2ArH), 7.38 (dd, 1H, J=8.5, 2.0 Hz, H-4), 3.1 (s, 1H). .sup.13C NMR (500 MHz, CDCl.sub.3): 171.2, 168.21, 149.4, 139.5, 134.4, 133.4, 131.7, 129.3, 128.5, 120.3, 119.2, 118.8, 82.3, 79.2, 56.2, 34.2. Rf=0.5 (silica gel, ethyl acetate/hexanes 1:2), HRMS (ESI/IT-TOF): m/z [M+H]+ calcd for C.sub.18H.sub.9D.sub.5N.sub.2O: 279.3473. found 279.3422.
Synthesis of Acetylene Analogs
##STR00039##
Synthesis of d(5)-Cyclopropyl Analogs
##STR00040##
Synthesis of Substituted Cycloalkyl Analogs
##STR00041##
Pharmaceutical Compositions
[0085] In another embodiment, a pharmaceutical composition is provided, the composition comprising a compound according to Formula I, or a pharmaceutically acceptable salt, racemate, enantiomer, or derivative thereof; and at least one pharmaceutically acceptable carrier. In embodiments, the pharmaceutical compositions disclosed herein are formulated for the treatment of cancer. In embodiments, the pharmaceutical compositions disclosed herein are formulated for the treatment of a neurological disorder associated with GABA.sub.A receptor function.
[0086] The pharmaceutically acceptable excipient, or carrier, must be acceptable in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof. The disclosure further includes a pharmaceutical composition, in combination with packaging material suitable for the pharmaceutical composition, including instructions for the use of the composition in the treatment of subjects in need thereof.
[0087] Pharmaceutical compositions include those suitable for enteral (e.g., oral, sublingual, buccal, or rectal), parenteral (e.g., intravenous, intramuscular, subcutaneous, intraarterial, intratumoral), intranasal, inhaled, vaginal, or transdermal administration. In a specific embodiment, the pharmaceutical compositions are formulated for intravenous administration, e.g., by injection or infusion. In another specific embodiment, the pharmaceutical compositions are formulated for oral administration.
[0088] The pharmaceutical compositions may be prepared by any methods well known in the art of pharmacy, for example, using methods such as those described in Remington: The Science and Practice of Pharmacy (21st ed., Lippincott Williams and Wilkins, 2005, see Part 5: Pharmaceutical Manufacturing). Suitable pharmaceutical carriers are well-known in the art. See, for example, Handbook of Pharmaceutical Excipients, Sixth Edition, edited by Raymond C. Rowe (2009). The skilled artisan will appreciate that certain carriers may be more desirable or suitable for certain modes of administration of an active ingredient. It is within the purview of the skilled artisan to select the appropriate carriers for a given composition.
[0089] For parenteral administration, suitable compositions include aqueous and non-aqueous sterile suspensions for intravenous administration. The compositions may be presented in unit dose or multi-dose containers, for example, sealed vials and ampoules.
[0090] For oral administration, suitable compositions include liquids, capsules, tablets, chewable tablets, soluble films, powders, and the like.
[0091] As will be understood by those of skill in this art, the specific dose level for any particular subject will depend on a variety of factors, including the activity of the agent employed; the age, body weight, general health, and sex of the individual being treated; the particular disease to be treated; the time and route of administration; the rate of excretion; and the like.
[0092] In embodiments, an effective dose of a Formula I compound according to the present disclosure may range from about 0.01 mg/kg/day to about 100 mg/kg/day, or from about 0.01 mg/kg/day to about 10 mg/kg/day, or from about 0.1 mg/kg/day to about 100 mg/kg/day, or from about 0.1 mg/kg/day to about 10 mg/kg/day, or from about 1 mg/kg/day to about 10 mg/kg/day. In embodiments, the dose of a Formula 1 compound is at least about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg/kg/day, or any selected range of values there between.
Methods of Use
[0093] In another embodiment, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a compound according to Formula I as disclosed herein, or a pharmaceutically acceptable salt, racemate, or enantiomer thereof.
[0094] In embodiments, the subject is a mammal. In a more specific embodiment, the subject is a human.
[0095] In embodiments, the cancer is any primary or metastatic solid tumor, including pediatric and adult tumors. In specific embodiments, the cancer is selected from the group consisting of melanoma, glioblastoma, medulloblastoma, neuroblastoma, and lung cancer. In a more specific embodiment, the lung cancer is non-small cell lung cancer (NSCLC).
[0096] In embodiments, administering comprises enteral or parenteral administration. In more specific embodiments, enteral administration comprises oral, sublingual, or buccal administration. In other specific embodiments, parenteral administration comprises intravenous, intramuscular, subcutaneous, intraarterial, or intratumoral administration. Compositions comprising Formula I compounds can be formulated for administration by any suitable enteral or parenteral administration.
[0097] In embodiments, the compound is administered at a dose of from about 0.1 mg/kg/day to about 100 mg/kg/day. In a more specific embodiment, the compound is administered at a dose of from about 1 mg/kg/day to about 30 mg/kg/day.
[0098] In embodiments, the methods disclosed herein further comprise administering to the subject one or more additional active agents. Illustratively, the one or more additional active agents are selected from the group consisting of an anti-inflammatory agent, an immunosuppressive agent, a corticosteroid, and a chemotherapeutic agent selected from the group consisting of an alkylating agent, a platinum drug, an antimetabolite, an anti-tumor antibiotic, a topoisomerase inhibitor, a mitotic inhibitor, a differentiating agent, an immune checkpoint inhibitor, and a hormone therapy.
[0099] In other embodiments, the methods disclosed herein further comprise administering radiation therapy to the subject. In embodiments, the methods disclosed herein further comprise administration of an immune checkpoint inhibitor, including but not limited to PD-1 inhibitors (e.g., pembrolizumab, nivolumab, cemiplimab, etc.); PD-L1 inhibitors (e.g., atezolizumab, avelumab, durvalumab, etc.); CTLA-4 inhibitors (e.g., ipilimumab, tremelimumab, etc.); and LAG-3 inhibitors (e.g., relatlimab, opdualag, etc.); and combinations thereof. In a specific embodiment, the checkpoint inhibitor is a PD-L1 inhibitor.
[0100] In another embodiment, a method of sensitizing a tumor to radiation in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a compound according to any of the embodiments of Formula I disclosed herein, or a pharmaceutically acceptable salt, racemate, or enantiomer thereof.
[0101] In another embodiment, a method of sensitizing a tumor to immunotherapy or chemotherapy in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a compound according to any of the embodiments of Formula I disclosed herein, or a pharmaceutically acceptable salt, racemate, or enantiomer thereof.
[0102] It is well known in the field that benzodiazepine drugs have utility in treating various neurological conditions. Thus, in another embodiment, a method of treating a neurological condition associated with Type-A GABA neurotransmitter receptor function in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a compound according to any of the embodiments of Formula I disclosed herein, or a pharmaceutically acceptable salt, racemate, or enantiomer thereof.
[0103] In a specific embodiment, the neurological condition is selected from the group consisting of sleep disorder, generalized anxiety disorder, social anxiety disorder, seizure disorder, panic disorder, tic disorder, bipolar disorder, and alcohol withdrawal. In a more specific embodiment, the sleep disorder is insomnia. In another more specific embodiment, the seizure disorder is epilepsy.
EXAMPLES
[0104] The following examples are given by way of illustration are not intended to limit the scope of the disclosure.
Example 1. Materials and Methods
Cell Lines and Culture Conditions
[0105] Cell lines tested were purchased from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Corning) or Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco). Media for lines was supplemented with 10% (v/v) Fetal Bovine Serum (FBS) (Corning) and 100 U/mL penicillin/streptomycin (Sigma). Lines were grown at 37 C. with 5% (v/v) CO.sub.2. Cells were passaged twice a week at 1:10 ratio and reached 80 to 90% confluency between 72 and 96 hours.
Drug Preparation
[0106] For in vitro studies drug was solubilized in dimethyl sulfoxide (DMSO, 0.125%) prior to use. For mouse studies drug was solubilized in a co-solvent formulation: propylene glycol (40%); ethanol (10%); benzyl alcohol (2%); benzoic acid (2%); sodium benzoate (2%).
Electrophysiology
[0107] Functional characterization employed a Port-a-Patch single cell automated patch-clamp electrophysiology instrument (Nanion Technologies, Germany). Recording solutions were purchased from Nanion Technologies. The composition of external solution consisted of: 140 mM NaCl; 4 mM KCl; 1 mM MgCl.sub.2; 2 mM CaCl.sub.2; 10 mM HEPES; 5 mM D-Glucose. High Ca.sup.2+ seal enhancer solution was composed of: 130 mM NaCl; 4 mM KCl; 1 mM MgCl.sub.2; 10 mM CaCl.sub.2); 10 mM HEPES; 5 mM D-Glucose. The internal solution composition was: 110 mM KF; 10 mM NaCl; 10 mM KCl; 10 mM EGTA; 10 mM HEPES, pH 7.2 adjusted using KOH.
[0108] To increase the current amplitude in single cell recordings, GABA and QH-II-066 were dissolved in high sodium containing external solution composed of: 161 mM NaCl; 3 mM KCl; 1 mM MgCl.sub.2; 1.5 mM CaCl.sub.2); 10 mM HEPES; 6 mM D-Glucose. Whole-cell recordings were performed on cells (held at 80 mV) using a gap-free protocol under continuous perfusion of external solutions and drug applications. GABA (1 M) and QH-II-066 (4 M) were applied briefly for 5 sec to record the current potentiation. Data acquisition was obtained using HEKA Elektronik software (Dr. Schulze GmBH, Germany). Data were low-pass filtered at 1 kHz and digitalized at 100 kHz. Data analysis was performed by computing the maximum current amplitude using Nest-O-Patch Software (Open Source).
Mitochondrial Depolarization
[0109] Drug was diluted to 4 M in RPMI-1640 culture media. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Sigma) was diluted to 20 M in culture medium. Tetramethylrhodamine ethyl ester perchlorate (TMRE) (Sigma) was diluted to 400 nM in culture medium. H1792 cells were grown in culture to 75-90% confluency. Cells (5105 cells/mL) were harvested and resuspended in media. Cell suspension (200 L) was dispensed and drug or FCCP (200 L) added to final concentrations of 2 M and 10 M, respectively. Solution was briefly vortexed and incubated for 10 min. TMRE (40 L of 400 nM stock) was added (final concentration 10 nM), vortexed, and sample reading acquired using a BD LSR Fortessa (Beckton Dickinson, San Diego). Data was analyzed using Flowjo v10 software (Flowjo, LLC).
Immunoblotting
[0110] DNA was sheared by sonication. Lysates were kept on ice for 30 min, centrifuged 10 min (13,500g, 4 C.), and protein concentration of supernatant determined by a Bradford assay (Protein Assay Dye Reagent, Bio-Rad). Lysates were mixed 1:1 with 2 Laemmli sample buffer containing -mercaptoethanol and heated 5 min at 95 C. Protein was resolved by SDS-PAGE using 4-20% gradient polyacrylamide gels (Bio-Rad), then transferred to nitrocellulose membranes (Bio-Rad) for 2 hr at 100 V in tris-glycine transfer buffer containing 20% methanol. Membranes were blocked at room-temperature in 5% 1TBST blocking buffer (TBS with 0.1% Tween-20 and 5% non-fat dry milk) for 1 hr with gentle agitation, followed by overnight incubation with primary Ab and gentle shaking at 4 C. The primary Abs were diluted as follows: GAPDH (1:1000, Cell Signaling Technology); GABARAP (1:1000, Cell Signaling Technology); NIX (1:1000, Cell Signaling Technology). Immunodetection was performed with anti-rabbit horseradish-peroxidase-conjugated secondary antibody (1:10000, Cell Signaling Technology). Post-primary Ab incubation, membranes were washed (3 for 10 min in TBST at room-temperature), probed with rabbit HRP tagged secondary antibody (1:3000, Cell Signaling Technology), and processed for chemiluminescence detection using ECL kit (Thermo Scientific). Chemiluminescence images were acquired using ChemiDoc Touch Imaging System (Bio-Rad).
Immunofluorescence Microscopy
[0111] Cells were seeded on sterile glass cover slips and grown overnight at 37 C. Cells were then rinsed with cold PBS and fixed in 4% paraformaldehyde (30 min, room temperature). Fixed cells were rinsed in PBS (35 min at room-temperature), and permeabilized (15 min in PBS containing 0.1% Triton X-100). Cells were washed in PBS (3 for 5 min at room-temperature) and incubated with blocking buffer (PBS containing 0.3% Triton X-100 and 3% BSA) for 1 hr at room-temperature with gentle shaking. Blocking solution was aspirated, cells washed with ice-cold PBS, and incubated overnight with primary Ab (LC3B or NIX) diluted 1:200 in sterile 0.5% BSA in PBS at 4 C. on the cover slip placed on a glass slide kept inside a humidified 10 cm dish with gentle shaking 60. Cells were washed (3 for 5 min in PBS) and incubated with fluorophore conjugated secondary Ab goat anti-rabbit Alexa Fluor 594 (Abcam) in 2% normal donkey serum at room-temperature for 1 hr in dark. Cells were washed (3 for 5 min in PBS) under low light and mounted on a glass slide in VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories). The following Abs were used (1:200 dilution): rabbit anti-human LC3B (Cell Signaling Technology) and rabbit NIX (Cell Signaling Technology). Slides were imaged on a Zeiss LSM 710 laser scanning confocal microscope and analyzed using NIH Fiji ImageJ2 (version 1.53c).
In Vitro Viability Assays
[0112] For cell proliferation viability studies, cells were assayed using the Cell Titer 96 Aqueous One Solution Assay (Promega). IC.sub.50 values were determined using the [Inhibitor] versus normalized response nonlinear regression function and the log of inhibitor concentration in Prism 8 software (version 8.3.0 for MacOS).
In Vitro Cell Survival Assay with Inhibitory Peptide
[0113] H1792 cells (2500 cells per well in 100 L media) were plated in 96 well plates with colorless phenol-red free RPMI-1640 media and allowed to grow overnight. On the next day, the media was removed and inhibitory peptide pen-3-ortho (gift of J. Kritzer, Tufts University) diluted in fresh 100 L of phenol-red free RPMI media was added in two sets of wells (6 wells in each set) at two different concentrations, 15 M and 25 M.
[0114] Cells in another set of 6 wells were treated with 3 M QH-II-066 in 100 L phenol-red free media in each well. Two other treatment groups (each group having 6 wells) were treated with a combination of QH-II-066 (3 M) and pen-3-ortho inhibitory peptide in two different concentrations, 15 M and 25 M. One set of 6 wells with cells were kept as control with no treatment and one set of 6 cells was kept as media only control with no cells plated in them. After adding the drug and DMSO (in control) all groups of cells including the controls, treated and media-only groups in the culture plate were incubated at 37 C., 5% CO.sub.2 in a humidified environment in an incubator for 48 hours. Following the 48-hr incubation, 20 L of diluted MTS reagent was added to each control (DMSO) and treated group of wells and in the wells containing only media. The cells were incubated at 37 C. for 1 hr and the absorbance were measured at 490 nm using a microplate reader (Molecular Devices).
[0115] The mean of the absorbance of media only samples was calculated, and the value was subtracted from each control and test samples. The percentage of inhibition in each group was calculated by the formula (CT)/C100% where C is the mean absorbance reading for Control group, T is the mean absorbance reading for each treated group. The percentage of survival was calculated by subtracting percentage of inhibition of each group from 100%. Percentage survival data is expressed as meanSEM, analyzed with GraphPad Prism 8.0.1 software (San Diego, CA, USA). Student's t test (paired) for two groups were used for statistical comparison. p<0.05 was significant.
Mouse Experiments
[0116] For subcutaneous xenograft tumor growth delay experiments, black 6 mice (C57Bl6) were purchased (Charles River Laboratories) and housed at University of Cincinnati LAMS. Purchased mice were allowed to accommodate for a week prior to experiment. Mice were housed in pathogen-free rooms and clinical health evaluated weekly by veterinary staff of the University of Cincinnati LAMS. All animal studies were conducted in accordance with approval of a University of Cincinnati IACUC.
[0117] LLC1 cells (a million) grown in RPMI medium were washed in cold PBS and mixed with Matrigel 25%, then injected subcutaneously into left and right flanks above the hind limbs of 6 to 8-week-old mice. When subcutaneous tumors were palpable (100 mm.sup.3 in size) the following treatments groups were initiated: (1) vehicle (2) drug. In mice receiving vehicle alone, the vehicle was injected i.p for 7 days. In mice receiving drug, drug (2.5 mg/kg body weight) was injected i.p. for 7 days. Following end of treatment, tumor volume were taken for growth delay studies. Mouse tumors were measured by Vernier calipers. Tumor volume was calculated using the formula: 4/3(lh2), where: l and h are radii of the tumor taken perpendicular to each other. Tumor size was measured three times a week using a caliper.
Example 2. GABA.SUB.A .Receptors are Functional in Cancer Cells
[0118] Patch clamp electrophysiology of single primary patient-derived cancer cells from lung adenocarcinoma was employed to demonstrate intrinsic, functional GABA.sub.A receptors. A current signal was observed in response to GABA, as illustrated for lung adenocarcinoma cell line H1792 (
Example 3. Enhanced GABA.SUB.A .Receptor Activity is Depolarizing to Cancer Cells
[0119] QH-II-066 mediates enhanced GABA.sub.A receptor activation and leads to an efflux of chloride anions across the extracellular plasma membrane, which contributes to a depolarization of the mitochondrial transmembrane. QH-II-066 creates a shift in electric charge distribution in different cancer cell types. Newly synthesized benzodiazepine analogs TA-II-59; SRE-III-35; SRE-III-43 (
Example 4. Enhanced GABA.SUB.A .Receptor Activity Triggers Cell Death
[0120] Depolarization can trigger cell death via activation of the intrinsic (mitochondrial) apoptotic pathway and we have reported this phenomenon in cell lines of the pediatric brain cancer medulloblastoma and melanoma using QH-II-066. We report here an examination of eighteen novel benzodiazepine analogs for their ability to impair the viability of cancer cells. These benzodiazepine analogs can be grouped into three classes based on the R1 moiety: (1) bromine; (2) ethynyl; (3) cyclopropyl (
Example 5. New Benzodiazepine Analog are Potent In Vivo
[0121] Mouse efficacy studies of three of the novel benzodiazepine variants were conducted: the ethynyl variant TA-II-59; and cyclopropyl variants SRE-III-35 and TA-IV-74. Xenograft tumors were generated in left and right flanks of black 6 mice using LLC1 cells. The treatment protocol involved administering a single i.p. dose of drug (2.5 mg drug/kg body weight) for seven consecutive days, once the tumor was palpable (
Example 6. GABA.SUB.A .Receptor Activation Enhances Autophagy
[0122] The contribution of molecular events to the observed cytotoxic effect of this new class of benzodiazepine analogs has been examined, i.e., those containing an ethynyl. In considering how this class of benzodiazepines may mediate cell death and tumor control, several observations suggested that autophagy may be a contributing factor. First, these compounds depolarize the mitochondrial transmembrane. It has been reported that depolarization of mitochondria serves to trigger autophagy. Second, a central protein to autophagy induction is GABARAP, which prior to its role in autophagy was shown to interact with GABA.sub.A receptors. GABARAP is also a NIX interacting factor, which has a significant role in autophagosome formation.
[0123] Key proteins to the assembly of autophagy granules or puncta, a hallmark of autophagy induction, are the ATG8 sub-family associated protein LC3B and NIX. Importantly, NIX mediates an association between the mitochondria and GABARAP, which is associated with the GABA.sub.A receptor at the extracellular plasma membrane. Subcellular distribution of autophagosomal proteins by immunofluorescence (IF) (e.g., LC3 puncta formation) is a well-established assay to monitor autophagy. Confocal IF was employed to observe assembly of puncta in lung adenocarcinoma cells (H1792) treated with QH-II-066. There is a background level of both LC3B- and NIX-positive puncta in H1792 cells in the control (DMSO treated) group (
[0124] Having observed enhanced assembly of LC3B and NIX-positive puncta, assays focused on the protein GABARAP, since NIX binds to GABARAP. In a time-course experiment it was found that lung adenocarcinoma cells (H1792) treated with QH-II-066 show a pronounced accumulation of both GABARAP and NIX at 72 hours post-treatment. Importantly, GABARAP and NIX also dimerize at 72 hours (
Example 7. GABARAP Structural Perturbation Inhibits GABA.SUB.A .Receptor Mediated Cytotoxicity Commensurate with NIX Destabilization
[0125] To establish that the autophagic response that was observed contributed to the cytotoxicity of QH-II-066, a peptide designed to target the protein GABARAP was employed and reported to inhibit autophagy in ovarian cancer cells. This peptide, pen-3-ortho (gift of J. Kritzer, Tufts University), has several advantages: (1) it is highly specific for GABARAP, binding with a low nanomolar affinity; and (2) a crystal structure has been determined of pen-3-ortho in complex with GABARAP, thus its mode of action delineated. Mechanistically, pen-3-ortho is a competitive inhibitor of NIX for binding to GABARAP. Pen-3-ortho alone is not cytotoxic to H1792 cells or nominally at exceedingly high concentrations (
Example 8. Model of Mechanism of Action
[0126] It has previously been reported that, in cancer cells, GABA.sub.A receptors are functional and that their activation using a member of our benzodiazepine analogs enhances the effect of its natural agonist, GABA (
[0127] As well as enhancing expression of GABRARAP and Nix, the investigators found that GABA.sub.A receptor activation contributes to their multimerization, thus augmenting the autophagic response. While not desiring to be bound by theory, the data suggests a model wherein multimerization of GABARAP occurs commensurate with multimerization of GABA.sub.A receptors, and that this macromolecular assembly assumes a cytotoxic-state as it drives a significant efflux of chloride anion locally. This model is consistent with experimental analysis and theoretical modeling, which shows that multimerization of GABA.sub.A receptors, and in turn GABARAP, generates an enhanced localized charge differential. This would also occur commensurate with a stabilization and multimerization of NIX, as observed. In this way, perturbation of ion homeostasis sensed and regulated by GABA.sub.A receptors would lead to assembly of an autophagosome and potential enclosure and subsequent recycling of mitochondria.
[0128] Aspects of the present disclosure can be described with reference to the following numbered clauses, with preferred features laid out in dependent clauses.
[0129] 1. A compound according to Formula I, or a pharmaceutically acceptable salt, racemate, or enantiomer thereof:
##STR00042##
wherein: R.sub.1 is selected from the group consisting of C.sub.2-C.sub.4 alkynyl, C.sub.3-C.sub.6 cycloalkyl, d(5)-cyclopropyl, methyl alkynyl, alkynyl-CD.sub.3, and alkynyl-CF.sub.3; R.sub.2 is selected from the group consisting of hydrogen, methyl, and trideuteromethyl; R.sub.3 and R.sub.4 are independently selected from the group consisting of hydrogen, deuterium, and methyl; and R.sub.5 is selected from the group consisting of hydrogen, methyl, trideuteromethyl, tritritiomethyl, halogen, and trifluoromethyl; optionally, wherein when R.sub.2 is trideuteromethyl, R.sub.1 is not ethynyl; and optionally, wherein when R.sub.1 is ethynyl, R.sub.5 is not hydrogen.
[0130] 2. The compound according to clause 1, wherein R.sub.3 and R.sub.4 are each hydrogen.
[0131] 3. The compound according to clause 1, wherein R.sub.3 and R.sub.4 are each deuterium.
[0132] 4. The compound according to clause 1, wherein R.sub.3 is hydrogen and R.sub.4 is methyl.
[0133] 5. The compound according to any of the preceding clauses, wherein R.sub.5 is Cl, F, or Br.
[0134] 6. The compound according to any of the preceding clauses, wherein R.sub.1 is cyclopropyl.
[0135] 7. The compound according to clause 6, wherein the compound is selected from the group consisting of:
##STR00043## ##STR00044## ##STR00045##
[0136] 8. The compound according to any of clauses 1-7, wherein the compound is selected from Table 1.
[0137] 9. The compound according to any of clauses 1-7, wherein the compound is selected from the group consisting of:
##STR00046## ##STR00047##
[0138] 10. The compound according to any of clauses 1-5, wherein R.sub.1 is ethynyl.
[0139] 11. The compound according to clause 10, wherein the compound is selected from the group consisting of:
##STR00048##
[0140] 12. The compound according to clause 10, wherein the compound is selected from the group consisting of:
##STR00049##
[0141] 13. The compound according to any of the preceding clauses, wherein R.sub.1 is cyclopropyl or ethynyl; R.sub.2 is hydrogen or methyl; R.sub.3 and R.sub.4 are each deuterium; and R.sub.5 is selected from the group consisting of hydrogen, methyl, trideuteromethyl, tritritiomethyl, trifluoromethyl, fluorine, and chlorine.
[0142] 14. A pharmaceutical composition comprising: [0143] an effective amount of the compound according to any of clauses 1-13; and [0144] a pharmaceutically acceptable carrier.
[0145] 15. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound according to any of clauses 1-13 or the pharmaceutical composition of clause 14.
[0146] 16. A method of sensitizing a tumor to radiation in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound according to any of clauses 1-13 or the pharmaceutical composition of clause 14.
[0147] 17. A method of sensitizing a tumor to immunotherapy or chemotherapy in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound according to any of clauses 1-13 or the pharmaceutical composition of clause 14.
[0148] 18. A method of treating a neurological condition associated with Type-A GABA neurotransmitter receptor function in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound according to any of clauses 1-13 or the pharmaceutical composition of clause 14.
[0149] 19. The method according to clause 18, wherein the neurological condition is selected from the group consisting of sleep disorder, generalized anxiety disorder, social anxiety disorder, seizure disorder, panic disorder, tic disorder, bipolar disorder, and alcohol withdrawal.
[0150] 20. The method according to clause 19, wherein the sleep disorder is insomnia.
[0151] 21. The method according to clause 19, wherein the seizure disorder is epilepsy.
[0152] All documents cited are incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.
[0153] It is to be further understood that where descriptions of various embodiments use the term comprising, and/or including those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language consisting essentially of or consisting of.
[0154] The foregoing description is illustrative of particular embodiments of the invention but is not meant to be a limitation upon the practice thereof. While particular embodiments have been illustrated and described, it would be obvious to one skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.