Patent Application
20240391928
G protein-coupled receptors (GPCRs) regulate various physiological functions and are frequently targeted in the treatment of disease. The GPCR ghrelin receptor (GHSR1a) is a critical regulator of food intake, energy homeostasis, and reward-seeking behaviors. Therefore, pharmacological agents targeting GHSR1a may have utility in the treatment of several mental health disorders, including eating disorders (e.g., obesity, anorexia) and drug addiction. Ghrelin, the endogenous ligand of GHSR1a, is a brain-gut peptide composed of 28 amino acids. The ghrelin-GHSR1a system can regulate the release of growth hormone (GH), affect learning and memory, and play an anti-inflammatory and anti-apoptotic role when GHSR1a is activated by ghrelin. Numerous peripheral actions of ghrelin can include, but are not limited to, regulation of glucose metabolism, lipogenesis, suppression of brown fat thermogenesis and improvement of cardiovascular and/or neurologic functions. Accordingly, pharmacological intervention of ghrelin-GHSR1a system could benefit a myriad of disorders and diseases. Despite the tremendous therapeutic potential for targeting GHSR1a, there are no GHSR1a agonists or GHSR1a antagonists currently approved for clinical use. Accordingly, there is a need in the field for the development of safe and effective pharmacologic agents that modulate GHSR1a activity.
The present disclosure provides, in part, novel compounds, compositions, and methods for treating an imbalance in brain dopamine homeostasis in a subject.
In one aspect, disclosed herein, are compounds of Formula (I), analogs, isomers, pharmaceutically acceptable salts, and prodrugs thereof or a pharmaceutically acceptable salt thereof:
In another aspect, as disclosed herein, are compounds of Formula (II), analogs, isomers, prodrugs, or a pharmaceutically acceptable salt thereof:
In another aspect, as disclosed herein, provides a pharmaceutical composition comprising, consisting of, or consisting essentially of a compound of Formula (I) and/or Formula (II) and a pharmaceutically acceptable carrier and/or excipient.
In yet another aspect, as disclosed herein, provides a method of modulating GHSR1a activity in a cell and/or subject comprising, consisting of, or consisting essentially of administering to the cell and/or subject an effective amount of a compound of Formula (I) and/or Formula (II) such that the GISR1a activity is modulated in the cell and/or subject.
In still another aspect, as disclosed herein, provides a method of modulating β-arrestin (β-arr) activity in a cell and/or subject comprising, consisting of, or consisting essentially of administering to the cell and/or subject an effective amount of a compound of Formula (I) or Formula (II) such that the β-arrestin (β-arr) activity is modulated in the cell and/or subject.
In yet another aspect, as disclosed herein, provides a method of modulating G protein activity in a cell and/or subject comprising, consisting of, or consisting essentially of administering to the cell and/or subject an effective amount of a compound of Formula (I) or Formula (II) such that the G protein activity is modulated in the cell and/or subject.
In some embodiments, the compound activates the G protein, f-arrestin, and/or GHSR1a activity.
In another aspect, as disclosed herein, provides a method of treating and/or preventing a GHSR1a-associated condition in a subject, the method comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a compound of Formula (I) and/or Formula (II) such that GISR1a-associated condition is treated and/or prevented in the subject.
In some embodiments, the GHSR1a-associated condition is selected from the group consisting of eating disorders, addiction, and combinations thereof. In one embodiment, the GISR1a-associated condition comprises an eating disorder. In some embodiments, the eating disorder is selected from the group consisting of bulimia nervosa, anorexia nervosa, binge-eating disorder, and combinations thereof. In another embodiment, the GHSR1a-associated condition comprises addiction. In some embodiments, the addition comprises a chemical addiction.
Another aspect of the present disclosure provides all that is described and illustrated herein.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein.
GPCRs, including GHSR1a, are known to signal via distinct pathways: pathways mediated by either G proteins or beta-arrestins (β-arr). GHSR1a activation results in pleiotropic physiological outcomes through distinct and pharmacologically separable C protein- and β-arr-dependent signaling pathways. Thus, as provided in the present disclosure, pathway-selective modulation can enable improved pharmacotherapeutics that may promote therapeutic efficacy while mitigating one or more side effects that have thus far prevented clinical use of pharmacologic agents that modulate GHSR1a activity.
The present disclosure is based, in part, on the discovery of a novel GHSR1a-agonist (NCG00536164-01) and its oxidized version (NCGC00538279-01). NCGC00538279 is also referred to in the present disclosure as “N8279” and “NCATS-SM8864” interchangeably. In certain embodiments, GHSR1a-agonists disclosed herein may target one or more pathophysiological changes in CNS dopamine homeostasis. In certain embodiments, GHSR1a-agonists disclosed herein may normalize dysfunctional dopamine signaling in the brain. In certain embodiments, GHSR1a-agonists disclosed herein may be used to treat and/or prevent one or more brain disorders of mood, cognition, or movement, including addiction, Alzheimer's Disease (AD), Parkinson's Disease (PD), and the like. Accordingly, disclosed herein are GHSR1a-agonists (e.g., NCG00536164-01 and NCGC00538279-01) and methods of making such, pharmaceutical compositions comprising such GHSR1a-agonists, and methods for administering GHSR1a-agonists for treating a target disease, such as a disease originating from the brain.
For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.
As used in the specification, articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.
“About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. The term “about” in association with a numerical value means that the numerical value can vary plus or minus by 5% or less of the numerical value.
Throughout this specification, unless the context requires otherwise, the word “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other integer or step or group of integers or steps.
As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).
As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”
Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise-Indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.
As used herein, “treatment,” “therapy” and/or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder, or condition.
As used herein, “prevent” or “prevention” refers to eliminating or delaying the onset of a particular disease, disorder, or physiological condition, or to the reduction of the degree of severity of a particular disease, disorder or physiological condition, relative to the time and/or degree of onset or severity in the absence of intervention.
The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. In some embodiments, the subject comprises a human. In other embodiments, the subject comprises a human in need of bone repair or bone formation.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
The present disclosure is based, in part, on the discovery novel GHSR1a-agonists. As used herein, a GHSR1a-agonist refers to a small molecule (e.g., a chemical) that activates a GHSR1a receptor to produce a biological response. Examples of biological responses stimulated by GHSR1a-agonists disclosed herein may include, but are not limited to, activation and accumulation of phospholipase C γ (PLCγ), activation of β-arrestin, accumulation of inositol phosphatase 3 kinase (IP3K), production of diacylglycerol (DAG), release of Ca+ from the endoplasmic reticulum, facilitating calcium/calmodulin kinase (CaCMK)-catalyzed phosphorylation of adenosine monophosphate-activated protein kinase (pAMPK), increased phosphorylation of ERK via the β-arrestin pathway, increased AKT phosphorylation via increased levels of PI3K, and the like. Other examples of biological responses stimulated by GHSR1a-agonists disclosed herein may include, but are not limited to, food intake stimulation, lower addiction to drugs (e.g., alcohol, cocaine), promote growth hormone secretion, and the like.
In some embodiments, the present disclosure provides a GHSR1a-agonist compound comprising, consisting of, or consisting essentially of the general Formula (I), analogs, isomers, pharmaceutically acceptable salts, and prodrugs thereof or a pharmaceutically acceptable salt thereof.
In general, R1, R2, R3, and R4 are independently selected from a group consisting of H, NO2, CN, CHO, F, Cl, Br, I, CF3, unsubstituted C1-C6 alkyl, substituted alkyl, COR12, CO2H, CO2R13, CONH2, CONHR14, and CONR15R16 wherein R12, R13, R14, R15, and R16 are independently selected from a group consisting of H, unsubstituted C1-C6 alkyl, substituted C1-C6 alkyl, unsubstituted phenyl, substituted phenyl, heterocyclic, R15 and R16 may be taken together to form an unsubstituted C1-C6 alkyl or a substituted C1-C6 alkyl. In some embodiments, R1, R2, R3, and R4 are independently selected from a group consisting of H, NO2, CN, CHO, F, Cl, Br, I, CF3, unsubstituted C1-C6 alkyl, substituted alkyl, COR1, CO2H, CO2R13, CONH2, CONHR14, and CONR15R16 wherein R12, R13, R14, R15, and R16 are independently selected from a group consisting of H, C1-C4 alkyl, unsubstituted phenyl, substituted phenyl, heterocycle. In certain embodiments, R1, R2, R3, and R4 are independently selected from a group consisting of H, F, Cl, Br, I, CF3, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, and C(CH3)3. In specific embodiments, R1, R3, and R4 are H; R2 is Cl.
Generally, R5 is selected from a group consisting of OH, OR17, NH2, NHR18, NR19R20, NHCOR21, NHCO2R22, and heterocyclic wherein R17, R18, R19, R20, R21, and R22 are independently selected from a group consisting of H, unsubstituted C1-C6 alkyl, substituted C1-C6 alkyl, unsubstituted phenyl, substituted phenyl, heterocyclic, R15 and R16 may be taken together to form an unsubstituted C1-C6 alkyl or a substituted C1-C6 alkyl, and R19 and R20 may be taken together to form an unsubstituted C1-C6 alkyl or a substituted C1-C6 alkyl. In some embodiments, R5 is selected from a group consisting of OH, OR17, NH2, NHR18, NR19R20, NHCOR21, and NHCO2R22 wherein R17, R18, R19, R20, R21, and R22 are independently selected from a group consisting of H, C1-C4 alkyl, unsubstituted phenyl, substituted phenyl, heterocycle, and R19 and R20 may be taken together to form an unsubstituted C1-C6 alkyl or a substituted C1-C6 alkyl. In certain embodiments, R5 is selected from a group consisting of NHCH3, N(CH3)2, NHCH2CH3, N(CH2CH3)2, NCH3CH2CH3, NHCH2CH2CH3, N(CH2CH2CH3)2, NHCH(CH3)2, N(CH(CH3)2)2, NCH3Ph, NCH2CH3Ph, NCH3CH(CH3)2, NH(C(CH3)3, pyridine, pyrazole, pyridazine, and pyrimidine. In specific embodiments, R5 is N(CH3)2.
In general, R6 is H or an unsubstituted C1-C6 alkyl. In some embodiments, R6 is H or an unsubstituted C1-C6 alkyl. In certain embodiments, R6 is H or CH3. In specific embodiments, R6 is H.
In general, Ar is
In some embodiments, Ar is
In certain embodiments, Ar is
In specific embodiments, Ar is
Generally, R7, R8, R9, R10, and R11 are independently H, F, Cl, Br, I, CF3, CN, OH, OR23, NH2, NHR24, NR25R26, NHCOR27, NHCO2R28, unsubstituted C1-C6 alkyl, substituted C1-C6 alkyl, unsubstituted C2-C6 cycloalkyl, substituted C2-C6 cycloalkyl, unsubstituted C1-C6 alkenyl, substituted C1-C6 alkenyl, unsubstituted C1-C6 alkynyl, substituted C1-C6 alkynyl, unsubstituted phenyl, substituted phenyl, R7 and R8 may be taken together to form an unsubstituted C1-C6 alkyl, or a substituted C1-C6 alkyl, R8 and R9 may be taken together to form an unsubstituted C1-C6 alkyl, or a substituted C1-C6 alkyl, R9 and R10 may be taken together to form an unsubstituted C1-C6 alkyl, or a substituted C1-C6 alkyl, R10 and R11 may be taken together to form an unsubstituted C1-C6 alkyl, or a substituted C1-C6 alkyl wherein R23, R24, R25, R26, R27, and R28 are independently selected from a group consisting of H, unsubstituted C1-C6 alkyl, substituted C1-C6 alkyl, unsubstituted phenyl, substituted phenyl, heterocyclic, R15 and R16 may be taken together to form an unsubstituted C1-C6 alkyl or a substituted C1-C6 alkyl, and R19 and R20 may be taken together to form an unsubstituted C1-C6 alkyl or a substituted C1-C6 alkyl. In some embodiments, R7, R8, R9, R10, and R11 are independently H, F, Cl, Br, I, CF3, CN, OH, OR23, unsubstituted C1-C4 alkyl, substituted C1-C4 alkyl, unsubstituted phenyl, or substituted phenyl, R7 and R8 may be taken together to form an unsubstituted C1-C4 alkyl, or a substituted C1-C4 alkyl, R8 and R9 may be taken together to form an unsubstituted C1-C4 alkyl, or a substituted C1-C4 alkyl, R9 and R10 may be taken together to form an unsubstituted C1-C4 alkyl, or a substituted C1-C4 alkyl, R10 and R11 may be taken together to form an unsubstituted C1-C4 alkyl, or a substituted C1-C4 alkyl wherein R23 are independently selected from a group consisting of H, C1-C4 alkyl, unsubstituted phenyl, substituted phenyl, and heterocycle. In certain embodiments, R7, R8, R9, R10, and R11 are independently selected from a group consisting of H, F, Cl, Br, I, CF3, CN, OH, OCH3, OCH2CH3, OPh, CH3, CHR7, R8, and R11 are H; R8 and R9 are OCH3.
In general, n is an integer from 1 to 6. In some embodiments, n is an integer from 1 to 4. In certain embodiments, n is an integer from 1 to 4. In specific embodiments, n is 3.
In one preferred embodiment, R1, R3, R4, and R6 are H; R2 is Cl; R5 is N(CH3)2; Ar is
R7, R8, and R11 are H; R8 and R9 are OCH3 and n=3 as shown in the compound comprising Formula (111):
also referred to as NCGC00538279.
In another embodiment, the present disclosure provides a GHSR1a-agonist compound comprising, consisting of, or consisting essentially of the general Formula (II), analogs, isomers, pharmaceutically acceptable salts, and prodrugs thereof or a pharmaceutically acceptable salt thereof:
In general, R1, R2, R3, and R4 are independently selected from a group consisting of H, NO2, CN, CHO, F, Cl, Br, I, CF3, unsubstituted C1-C6 alkyl, substituted alkyl, COR12, CO2H, CO2R1, CONH2, CONHR14, and CONR15R16 wherein R1, R13, R14, R15, and R16 are independently selected from a group consisting of H, unsubstituted C1-C6 alkyl, substituted C1-C6 alkyl, unsubstituted phenyl, substituted phenyl, heterocyclic, R15 and R16 may be taken together to form an unsubstituted C1-C6 alkyl or a substituted C1-C6 alkyl. In some embodiments, R1, R2, R3, and R4 are independently selected from a group consisting of H, NO2, CN, CHO, F, Cl, Br, I, CF3, unsubstituted C1-C6 alkyl, substituted alkyl, COR2, CO2H, CO2R13, CONH2, CONHR14, and CONR15R16 wherein R12, R13, R14, R15, and R16 are independently selected from a group consisting of H, C1-C4 alkyl, unsubstituted phenyl, substituted phenyl, heterocycle. In certain embodiments, R1, R2, R3, and R4 are independently selected from a group consisting of H, F, Cl, Br, I, CF3, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, and C(CH3)3. In specific embodiments, R1, R3, and R4 are H; R2 is C1.
Generally, R5 is selected from a group consisting of OH, OR1, NH2, NHR18, NR19R20, NHCOR21, NHCO2R22, and heterocyclic wherein R17, R18, R19, R20, R21, and R22 are independently selected from a group consisting of H, unsubstituted C1-C6 alkyl, substituted C1-C6 alkyl, unsubstituted phenyl, substituted phenyl, heterocyclic, R15 and R16 may be taken together to form an unsubstituted C1-C6 alkyl or a substituted C1-C6 alkyl, and R19 and R20 may be taken together to form an unsubstituted C1-C6 alkyl or a substituted C1-C6 alkyl. In some embodiments, R5 is selected from a group consisting of OH, OR12, NH2, NHR18, NR19R20, NHCOR21, and NHCO2R22 wherein R17, R18, R19, R20, R21, and R22 are independently selected from a group consisting of H, C1-C4 alkyl, unsubstituted phenyl, substituted phenyl, heterocycle, and R19 and R20 may be taken together to form an unsubstituted C1-C6 alkyl or a substituted C1-C6 alkyl. In certain embodiments, R5 is selected from a group consisting of NHCH3, N(CH3)2, NHCH2CH3, N(CH2CH3)2, NCH3CH2CH3, NHCH2CH2CH3, N(CH2CH2CH3)2, NHCH(CH3)2, N(CH(CH3)2)2, NCH3Ph, NCH2CH3Ph, NCH3CH(CH3)2, NH(C(CH3)3, pyridine, pyrazole, pyridazine, and pyrimidine. In specific embodiments, R5 is N(CH3)2.
In general, Ar is
In some embodiments, Ar is
In certain embodiments, Ar is
In specific embodiments, Ar is
Generally, R7, R8, R9, R10, and R11 are independently H, F, Cl, Br, I, CF3, CN, OH, OR3, NH2, NHR24, NR25R26, NHCOR27, NHCO2R28, unsubstituted C1-C6 alkyl, substituted C1-C6 alkyl, unsubstituted C2-C6 cycloalkyl, substituted C2-C6 cycloalkyl, unsubstituted C1-C6 alkenyl, substituted C1-C6 alkenyl, unsubstituted C1-C6 alkynyl, substituted C1-C6 alkynyl, unsubstituted phenyl, substituted phenyl, R7 and R8 may be taken together to form an unsubstituted C1-C6 alkyl, or a substituted C1-C6 alkyl, R8 and R9 may be taken together to form an unsubstituted C1-C6 alkyl, or a substituted C1-C6 alkyl, R9 and R10 may be taken together to form an unsubstituted C1-C6 alkyl, or a substituted C1-C6 alkyl, R10 and R11 may be taken together to form an unsubstituted C1-C6 alkyl, or a substituted C1-C6 alkyl wherein R2, R24, R25, R26, R27, and R28 are independently selected from a group consisting of H, unsubstituted C1-C6 alkyl, substituted C1-C6 alkyl, unsubstituted phenyl, substituted phenyl, heterocyclic, R11 and R16 may be taken together to form an unsubstituted C1-C6 alkyl or a substituted C1-C6 alkyl, and R19 and R20 may be taken together to form an unsubstituted C1-C6 alkyl or a substituted C1-C6 alkyl. In some embodiments, R7, R8, R9, R10 and R11 are independently H, F, Cl, Br, I, CF3, CN, 01-1, OR23, unsubstituted C1-C4 alkyl, substituted C1-C4 alkyl, unsubstituted phenyl, or substituted phenyl, R7 and R8 may be taken together to form an unsubstituted C1-C4 alkyl, or a substituted C1-C4 alkyl, R8 and R9 may be taken together to form an unsubstituted C1-C4 alkyl, or a substituted C1-C4 alkyl, R9 and R10 may be taken together to form an unsubstituted C1-C4 alkyl, or a substituted C1-C4 alkyl, R10 and R11 may be taken together to form an unsubstituted C1-C4 alkyl, or a substituted C1-C4 alkyl wherein R23 are independently selected from a group consisting of H, C1-C4 alkyl, unsubstituted phenyl, substituted phenyl, and heterocycle. In certain embodiments, R7, R8, R9, R10, and R11 are independently selected from a group consisting of H, F, Cl, Br, I, CF3, CN, OH, OCH3, OCH2CH3, OPh, CH3, CHR7, R8, and R11 are H; R8 and R9 are OCH3.
In general, n is an integer from 1 to 6. In some embodiments, n is an integer from 1 to 4. In certain embodiments, n is an integer from 1 to 4. In specific embodiments, n is 3.
In one preferred embodiment, R1, R3, and R4 are H; R2 is Cl; R3 is N(CH3)2; Ar is
R7, R8, and R11 are H; R8 and R9 are OCH13 and n=3 as shown in the compound comprising Formula (IV):
also referred to as NCG00536164-01.
Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.
The term “alkyl” refers to a straight or branched saturated hydrocarbon chain. Alkyl groups may include a specified number of carbon atoms. For example, C1-C12 alkyl indicates that the alkyl group may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. An alkyl group may be, e.g., a C1 1-C12 alkyl group, a C1-C10 alkyl group, a C1-C6 alkyl group, a C1-C6 alkyl group or a C1-C4 alkyl group. For example, exemplary C1-C4 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl groups. An alkyl group may be optionally substituted with one or more substituents.
The term “alkylenyl” refers to a divalent alkyl group, examples of which include but are not limited to —C2—, —CH2CH2—, —CH2CH2CH2— and —CH2CH(CH3)CH2—. An alkylenyl group may be optionally substituted with one or more substituents.
The term “alkenyl” refers to a straight or branched hydrocarbon chain having one or more double bonds. Alkenyl groups may include a specified number of carbon atoms. For example, C2-C12 alkenyl indicates that the alkenyl group may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. An alkenyl group may be, e.g., a C2-C12 alkenyl group, a C2-C10 alkenyl group, a C2-C8 alkenyl group, a C2-C6 alkenyl group or a C2-C4 alkenyl group. Examples of alkenyl groups include but are not limited to allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. One of the double bond carbons may optionally be the point of attachment of the alkenyl substituent. An alkenyl group may be optionally substituted with one or more substituents.
The term “alkenylenyl” refers to a divalent alkenyl group, examples of which include but are not limited to —CH═CH—, —CH═CH—CH—=2, —CH═CH—CH2— and —CH2—CH═CH—CH2—. An alkenylenyl group may be optionally substituted with one or more substituents.
The term “alkynyl” refers to a straight or branched hydrocarbon chain having one or more triple bonds. Alkynyl groups may include a specified number of carbon atoms. For example, C2-C12 alkynyl indicates that the alkynyl group may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. An alkynyl group may be, e.g., a C2-C12 alkynyl group, a C2-C1m alkynyl group, a C2-C12 alkynyl group, a C2-C6 alkynyl group or a C2-C4 alkynyl group. Examples of alkynyl groups include but are not limited to ethynyl, propargyl, and 3-hexynyl. One of the triple bond carbons may optionally be the point of attachment of the alkynyl substituent. An alkynyl group may be optionally substituted with one or more substituents.
The term “alkynylenyl” refers to a divalent alkynyl group, examples of which include but are not limited to —CC—, —CC—CH2—, —C—CH2—CH2— and —CH2—CC—CH2—. An alkynylenyl group may be optionally substituted with one or more substituents.
The term “amino” as used herein refers to —NRN1RN2 wherein RN1 and RN2 independently may be H, alkyl, aryl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heteroaryl, or heterocyclyl.
The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom capable of substitution can be substituted (e.g., with one or more substituents). Examples of aryl moieties include but are not limited to phenyl, naphthyl, and anthracenyl. Aryl groups may be optionally substituted with one or more substituents.
The term “arylalkyl” refers to an alkyl moiety in which at least one alkyl hydrogen atom is replaced with an aryl group. Arylalkyl includes groups in which more than one hydrogen atom has been replaced with an aryl group. Examples of arylalkyl groups include but are not limited to benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups. Arylalkyl groups may be optionally substituted with one or more substituents, on either the aryl moiety or the alkyl moiety.
The term “carbonyl” as used herein refers to a —C(O)R group, wherein R is alkyl, aryl, alkenyl, alkynyl, alkoxy, heteroalkyl, cycloalkyl, heteroaryl, heterocyclyl, or amino. The term “C1-4 carbonyl” refers to a group that may be preceded by an alkyl group of up to 3 carbon atoms. It may also be called an “alkylcarbonyl”. Examples of C1-4 carbonyl include —C(O)R, —CH2C(O)R, —CH2CH2C(O)R, and —CH2CH2CH2C(O)R.
The term “carboxyl” as used herein refers to a —OC(O)R group, wherein R is alkyl, aryl, alkenyl, alkynyl, alkoxy, heteroalkyl, cycloalkyl, heteroaryl, heterocyclyl, or amino. The term “C1-4 carboxyl” refers to a group that may be preceded by an alkyl group of up to 3 carbon atoms. It may also be called an “alkylcarboxyl”. Examples of C.sub.1-4 carbonyl include —OC(O)R, —CH2OC(O)R, —CH2CH2OC(O)R, and —CH2CH2CH2OC(O)R.
The term “cycloalkyl” as used herein refers to non-aromatic, saturated or partially unsaturated cyclic, bicyclic, tricyclic or polycyclic hydrocarbon groups having 3 to 12 carbons. Any ring atom can be substituted (e.g., with one or more substituents). Cycloalkyl groups can contain fused rings. Fused rings are rings that share one or more common carbon atoms. Examples of cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, methylcyclohexyl, adamantyl, norbornyl, norbornenyl, tetrahydronaphthalenyl and dihydroindenyl. Cycloalkyl groups may be optionally substituted with one or more substituents.
The term “cycloalkylalkyl”, as used herein, refers to an alkyl group in which at least one hydrogen atom is replaced with a cycloalkyl group. Cycloalkylalkyl groups include those in which more than one hydrogen atom of the alkyl group is replaced with a cycloalkyl group. Examples of cycloalkylalkyl groups include but are not limited to cyclohexylmethyl, cyclopentylmethyl, cyclobutylmethyl and cyclopropylmethyl. Cycloalkylalkyl groups can be optionally substituted with one or more substituents, on either the cycloalkyl moiety or the alkyl moiety.
The term “halo” or “halogen” as used herein refers to any radical of fluorine, chlorine, bromine or iodine.
“Heteroalkyl” refers to an alkyl, alkenyl or alkynyl group as defined herein, wherein at least one carbon atom of the alkyl group is replaced with a heteroatom. Heteroalkyl groups may contain from 1 to 18 non-hydrogen atoms (carbon and heteroatoms) in the chain, or 1 to 12 atoms, or 1 to 6 atoms, or 1 to 4 atoms. Heteroalkyl groups may be straight or branched, and saturated or unsaturated. Unsaturated heteroalkyl groups have one or more double bonds and/or one or more triple bonds. Heteroalkyl groups may be unsubstituted or substituted. Exemplary heteroalkyl groups include but are not limited to alkoxyalkyl (e.g., methoxymethyl), and aminoalkyl (e.g., alkylaminoalkyl and dialkylaminoalkyl). Heteroalkyl groups may be optionally substituted with one or more substituents.
The term “heteralkylenyl” refers to a divalent heteroalkyl group, examples of which include but are not limited to —CH2OCH2—, —CH2NHCH2—, polyethyleneglycol groups (e.g., —(CH2CH2O)n—), polyethyleneimine groups (e.g., —(CH2CH2NH)n—), and the like. A heteroalkylenyl group may be optionally substituted with one or more substituents.
The term “heteroaryl” as used herein refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 13 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms independently selected from O, N, S, P and Si (e.g., carbon atoms and 13, 1-6, or 1-9 heteroatoms independently selected from O, N, S, P and Si if monocyclic, bicyclic, or tricyclic, respectively). Any ring atom can be substituted (e.g., with one or more substituents). Heteroaryl groups can contain fused rings, which are rings that share one or more common atoms. Examples of heteroaryl groups include but are not limited to radicals of pyridine, pyrimidine, pyrazine, pyridazine, pyrrole, imidazole, pyrazole, oxazole, isoxazole, furan, thiazole, isothiazole, thiophene, quinoline, isoquinoline, quinoxaline, quinazoline, cinnoline, indole, isoindole, indolizine, indazole, benzimidazole, phthalazine, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, phenazine, naphthyridines and purines. Heteroaryl groups may be optionally substituted with one or more substituents.
The term “heteroarylalkyl” refers to an alkyl moiety in which at least one alkyl hydrogen atom is replaced with a heteroaryl group. Heteroarylalkyl includes groups in which more than one hydrogen atom has been replaced with a heteroaryl group. Examples of heteroarylalkyl groups include but are not limited to imidazolylmethyl (e.g., 1H-imidazol-2-ylmethyl and 1H-imidazol-4-ylmethyl), pyridinylmethyl (e.g., pyridin-3-ylmethyl and pyridin-4-ylmethyl), pyrimidinylmethyl (e.g., pyrimidin-5-ylmethyl), furylmethyl (e.g., fur-2-ylmethyl and fur-3-ylmethyl), and thienylmethyl (e.g., thien-2-ylmethyl and thien-3-ylmethyl) groups. Heteroarylalkyl groups may be optionally substituted with one or more substituents, on either the heteroaryl moiety or the alkyl moiety.
The term “heteroatom”, as used herein, refers to a non-carbon or hydrogen atom such as a nitrogen, sulfur, oxygen, silicon, or phosphorus atom. Groups containing more than one heteroatom may contain different heteroatoms.
The term “heterocyclyl” or “heterocyclic”, as used herein, refers to a nonaromatic, saturated or partially unsaturated 3-10 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, S, Si and P (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, S, Si and P if monocyclic, bicyclic, or tricyclic, respectively). Any ring atom can be substituted (e.g., with one or more substituents). Heterocyclyl groups can contain fused rings, which are rings that share one or more common atoms. Examples of heterocyclyl groups include but are not limited to radicals of tetrahydrofuran, tetrahydrothiophene, tetrahydropyran, oxetane, piperidine, piperazine, morpholine, pyrroline, pyrimidine, pyrrolidine, indoline, tetrahydropyridine, dihydropyran, thianthrene, pyran, benzopyran, xanthene, phenoxathiin, phenothiazine, furazan, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. Heterocyclyl groups may be optionally substituted with one or more substituents.
The term “heterocyclylalkyl” refers to an alkyl moiety in which at least one alkyl hydrogen atom is replaced with a heterocyclyl group. Heterocyclylalkyl includes groups in which more than one hydrogen atom has been replaced with a heterocyclyl group. Examples of heterocyclylalkyl groups include but are not limited to oxetanylmethyl, morpholinomethyl, and pyrrolidinylmethyl groups, and the like. Heterocyclylalkyl groups may be optionally substituted with one or more substituents, on either the heterocyclyl moiety or the alkyl moiety.
The term “hydroxy” refers to an —OH radical. The term “alkoxy” refers to an —O— alkyl radical. The term “aryloxy” refers to an —O-aryl radical.
The term “oxo” refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur (i.e. ═O).
The term “substituents” refers to a group “substituted” on an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl group at any atom of that group. Any atom can be substituted. Suitable substituents include, without limitation: acyl, acylamido, acyloxy, alkoxy, alkyl, alkenyl, alkynyl, amido, amino, carboxy, cyano, ester, halo, hydroxy, imino, nitro, oxo (e.g., C═O), phosphonate, sulfinyl, sulfonyl, sulfonate, sulfonamino, sulfonamido, thioamido, thiol, thioxo (e.g., C═S), and ureido. In embodiments, substituents on a group are independently any one single, or any combination of the aforementioned substituents. In embodiments, a substituent may itself be substituted with any one of the above substituents.
The above substituents may be abbreviated herein. For example, the abbreviations Me, Et, Ph and Bn represent methyl, ethyl, phenyl and benzyl, respectively. A more comprehensive list of standard abbreviations used by organic chemists appears in a table entitled Standard List of Abbreviations of the Journal of Organic Chemistry. The abbreviations contained in said list are hereby incorporated by reference.
For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, and such that the selections and substitutions result in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
Where substituent groups are specified by their conventional chemical formulae, written from left to right, they optionally encompass substituents resulting from writing the structure from right to left, e.g., —CH2O— optionally also recites —OCH2—.
In some embodiments, GHSR1a-agonist compounds described herein, where applicable, can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. In certain embodiments, GHSR1a-agonist compounds described herein may exist in one or more particular geometric, optical, enantiomeric, diastereomeric, epimeric, atropic, stereoisomer, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; .alpha.- and .beta.-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and half chair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”). Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981). The disclosure additionally encompasses GHSR1a-agonist compounds disclosed herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers. In some embodiments, GHSR1a-agonist compounds disclosed herein may be (R)-isomers. Alternatively, GHSR1a-agonist compounds disclosed herein may be (S)-isomers. In some embodiments, GHSR1a-agonist compounds disclosed herein may be a mixture of (R) and (S) isomers.
In some embodiments, a GHSR1a-agonist compound disclosed herein may be an enantiomerically enriched isomer of a stereoisomer described herein. For example, a GHSR1a-agonist compound herein may have an enantiomeric excess of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Enantiomer, when used herein, refers to either of a pair of chemical compounds whose molecular structures have a mirror-image relationship to each other.
In some embodiments, preparation of a GHSR1a-agonist compound disclosed herein may be enriched for an isomer of the compound having a selected stereochemistry, e.g., R or S, corresponding to a selected stereocenter. For example, a GHSR1a-agonist compound herein may have a purity corresponding to a compound having a selected stereochemistry of a selected stereocenter of at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
In some embodiments, a GHSR1a-agonist compound disclosed herein may include a preparation of a compound disclosed herein that is enriched for a structure or structures having a selected stereochemistry, e.g., R or S, at a selected stereocenter. Exemplary R/S configurations can be those provided in an example described herein. An “enriched preparation,” as used herein, is enriched for a selected stereoconfiguration of one, two, three or more selected stereocenters within the subject compound. Exemplary selected stereocenters and exemplary stereoconfigurations thereof can be selected from those provided herein, e.g., in an example described herein. By enriched is meant at least 60%, e.g., of the molecules of compound in the preparation have a selected stereochemistry of a selected stereocenter. In an embodiment it is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Enriched refers to the level of a subject molecule(s) and does not connote a process limitation unless specified.
In some embodiments, GHSR1a-agonist compounds disclosed herein can be in the form of an ester prodrug. The term “ester” herein can refer a compound which is produced by modifying a functional group (e.g. hydroxyl, carboxyl, amino or the like group). Examples of the “ester” include “esters formed with a hydroxyl group” and “esters formed with a carboxyl group.” The term “ester” can mean an ester whose ester residue is a “conventional protecting group” or a “protecting group removable in vivo by a biological method such as hydrolysis”. In some embodiments, the term “conventional protecting group” can mean a protecting group removable by a chemical method such as hydrogenolysis, hydrolysis, electrolysis, or photolysis. In some embodiments, the term “protecting group removable in vivo by a biological method such as hydrolysis” can mean a protecting group removable in vivo by a biological method such as hydrolysis to produce a free acid or its salt.
In some embodiments, GHSR1a-agonist compounds disclosed herein can be in the form of a pharmaceutically acceptable salt. By “salt” or “pharmaceutically acceptable salt”, it is meant those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit to risk ratio, and effective for their intended use. A “pharmacologically acceptable salt” can refer to a salt, which can be formed when a GHSR1a-agonist compound has an acidic group such as carboxyl or a basic group such as amino or imino. In some embodiments, GHSR1a-agonist salt formed with an acidic group herein can include alkali metal salts such as a sodium salt, potassium salt or lithium salt, alkaline earth metal salts such as a calcium salt or magnesium salt, metal salts such as an aluminum salt or iron salt; amine salts, e.g., inorganic salts such as an ammonium salt and organic salts such as a t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N′-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzylphenethylamine salt, piperazine salt, tetramethylammonium salt or tris(hydroxymethyl)aminomethane salt; and amino acid salts such as a glycine salt, lysine salt, arginine salt, ornithine salt, glutamate or aspartate. In some embodiments, a GHSR1a-agonist salt formed with a basic group herein can include hydro-halides such as a hydrofluoride, hydrochloride, hydrobromide or hydroiodide, inorganic acid salts such as a nitrate, perchlorate, sulfate or phosphate; lower alkanesulfonates such as a methanesulfonate, trifluoromethanesulfonate or ethanesulfonate, arylsulfonates such as a benzenesulfonate or p-toluenesulfonate, organic acid salts such as an acetate, malate, fumarate, succinate, citrate, ascorbate, tartrate, oxalate or maleate; and amino acid salts such as a glycine salt, lysine salt, arginine salt, omithine salt, glutamate or aspartate. In certain embodiments, when a pharmacologically acceptable salt of a GHSR1a-agonist remains in the atmosphere or is recrystallized, it can absorb water to form a hydrate of use in formulations disclosed herein.
In some embodiments, GHSR1a-agonist compounds disclosed herein can be in the form of another aminoglycoside derivative. In some embodiments, the term “other derivative” can mean a derivative of the GHSR1a-agonist compound other than the above-described “ester” or the above-described “pharmacologically acceptable salt” which can be formed, if it has an amino and/or carboxyl group or other conjugate form or other active derivative thereof.
In some embodiments, GHSR1a-agonist compounds disclosed herein may be in a chemically protected form. The term “chemically protected form” is used herein in the conventional chemical sense and pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions under specified conditions (e.g., pH, temperature, radiation, solvent, and the like). In practice, well known chemical methods are employed to reversibly render unreactive a functional group, which otherwise would be reactive, under specified conditions. In a chemically protected form, one or more reactive functional groups are in the form of a protected or protecting group (also known as a masked or masking group or a blocked or blocking group). By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting group may be removed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example, Protective Groups in Organic Synthesis (T. Green and P. Wuts; 3rd Edition; John Wiley and Sons, 1999). Unless otherwise specified, a reference to a particular compound also includes chemically protected forms thereof.
A wide variety of such “protecting,” “blocking,” or “masking” methods are widely used and well known in organic synthesis. For example, a compound which has two nonequivalent reactive functional groups, both of which would be reactive under specified conditions, may be derivatized to render one of the functional groups “protected,” and therefore unreactive, under the specified conditions; so protected, the compound may be used as a reactant which has effectively only one reactive functional group. After the desired reaction (involving the other functional group) is complete, the protected group may be “deprotected” to return it to its original functionality. A hydroxy group may be protected as an ether (—OR) or an ester (—OC(O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl (diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester (—OC(O)CH3, —OAc). An aldehyde or ketone group may be protected as an acetal (RCH(OR)2) or ketal (R2C(OR)2), respectively, in which the carbonyl group (R2C═O) is converted to a diether (R2C(OR)2), by reaction with, for example, a primary alcohol. The aldehyde or ketone group is readily regenerated by hydrolysis using a large excess of water in the presence of acid. An amine group may be protected, for example, as an amide (—NRC(O)R) or a urethane (—NRC(O)OR), for example, as: a methyl amide (—NHC(O)CH3); a benzyloxy amide (—NHC(O)OCH2C6H5, —NH-Cbz); as a t-butoxy amide (—NHC(O)OC(CH)3, —NH-Boc); a 2-biphenyl-2-propoxy amide (—NHCO(O)C(CH3)2C6H4C6H5, —NH-Bpoc), as a 9-fluorenylmethoxy amide (—N1-Fmoc), as a 6-nitroveratryloxy amide (—NH—Nvoc), as a 2-trimethylsilylethyloxy amide (—NH-Teoc), as a 2,2,2-trichloroethyloxy amide (—NH-Troc), as an allyloxy amide (—NH-Alloc), as a 2(-phenylsulphonyl)ethyloxy amide (—NH—Psec); or, in suitable cases (e.g., cyclic amines), as a nitroxide radical (>N-0<<). A carboxylic acid group may be protected as an ester, for example, as: an alkyl ester (e.g., a methyl ester; a t-butyl ester); a haloalkyl ester (e.g., a haloalkyl ester); a trialkylsilylalkyl ester; or an arylalkyl ester (e.g., a benzyl ester; a nitrobenzyl ester); or as an amide, for example, as a methyl amide. A thiol group may be protected as a thioether (—SR), for example, as: a benzyl thioether; an acetamidomethyl ether (—S—CH2NHC(O)CH3).
In addition to salt forms, the present disclosure may also provide compounds that are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds described herein. Prodrugs can be converted to the compounds of the present disclosure by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present disclosure when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.
Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. Examples of isotopes suitable for inclusion in the compounds of the invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to 2H, 3H, 13C, 14C, 15N, 18O, 17O, 31P, 32P, 35S, 18F, and 36Cl, respectively. Substitution with heavier isotopes such as deuterium, i.e. 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. The compound may incorporate positron-emitting isotopes for medical imaging and positron-emitting tomography (PET) studies for determining the distribution of receptors. Suitable positron-emitting isotopes that can be incorporated in compounds of formula (I) are 11C, 13N, 15O, and 18F. Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using appropriate isotopically-labeled reagent in place of non-isotopically-labeled reagent. In some embodiments, in compounds of Formula (I) and Formula (III), any hydrogen atom may be deuterium.
In some embodiments, GHSR1a-agonist compounds disclosed herein may be modified to enhance selective biological properties. Such modifications are known in the art and may include those that increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism, and/or alter rate of excretion. Examples of these modifications include, but are not limited to, esterification with polyethylene glycols, derivatization with pivolates or fatty acid substituents, conversion to carbamates, hydroxylation of aromatic rings, and heteroatom substitution in aromatic rings. In some embodiments, GHSR1a-agonist compounds disclosed herein may be modified to enhance blood brain barrier permeability.
In certain embodiments, GHSR1a-agonist compounds herein (e.g., a compound of Formula I, II, III and/or IV) can have kinetic solubility. Higher kinetic solubility can be important in bioavailability of formulations, such as oral and/or peritoneal formulations. In some embodiments, compounds herein can have a kinetic solubility of at least about 0.1 μM. In some embodiments, compounds herein can have a kinetic solubility ranging from about 0.1 μM to about 110 μM (e.g., about 0.1 μM, about 0.5 μM, about 1 μM, about 5 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 110 μM). In some embodiments, compounds herein can maintain a kinetic solubility for about 1 hours to about 48 hours (e.g., about 1 hour, about 3 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, 48 hours). In some embodiments, compounds herein can maintain a kinetic solubility ranging from about 0.1 μM to about 110 μM (e.g., about 0.1 μM, about 0.5 μM, about 1 μM, about 5 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 110 μM) for about 1 hours to about 48 hours (e.g., about 1 hour, about 3 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, 48 hours). In some embodiments, compounds herein can maintain a kinetic solubility at temperatures ranging from about 4° C. to about 80° C. (e.g., about 4° C., about 6° C., about 8° C., about 10° C., about 20° C., about 30° C., about 40° C., about 50° C., about 60° C., about 70° C., about 80° C.). In some embodiments, compounds herein can maintain a kinetic solubility ranging from about 0.1 μM to about 110 μM (e.g., about 0.1 μM, about 0.5 μM, about 1 μM, about 5 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 110 μM) at temperatures ranging from about 4° C. to about 80° C. (e.g., about 4° C., about 6° C., about 8° C., about 10° C., about 20° C., about 30° C., about 40° C., about 50° C., about 60° C., about 70° C., about 80° C.). In some embodiments, compounds herein can maintain a kinetic solubility ranging from about 0.1 μM to about 110 μM (e.g., about 0.1 μM, about 0.5 μM, about 1 μM, about 5 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 110 μM) at room temperature (i.e., 25° C.±3° C.). In some embodiments, compounds herein can maintain a kinetic solubility ranging from about 0.1 μM to about 110 μM (e.g., about 0.1 μM, about 0.5 μM, about 1 μM, about 5 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 110 μM) at room temperature (i.e., 25° C.±3° C.) for about 1 hours to about 48 hours (e.g., about 1 hour, about 3 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours, 48 hours or more depending on the compound).
In certain embodiments, GHSR1a-agonist compounds disclosed herein (e.g., a compound of Formula I, II, III and/or IV) can have a central nervous system multiparameter optimization (CNS MPO) score. The CNS MPO score is determined by an algorithm that uses a weighted scoring function to assess six fundamental physicochemical properties [(a) lipophilicity, calculated partition coefficient (C log P); (b) calculated distribution coefficient at pH 7.4 (C log D); (c) molecular weight (MW); (d) topological polar surface area (TPSA); (e) number of hydrogen-bond donors (HBDs); and (f) most basic center (pKa)]. The algorithm assigns a compound a collective score ranging from 0 to 6, wherein higher CNS MPO scores are indicative of the compound's capability of crossing the blood-brain-barrier (BBB). In some embodiments, compounds herein can have a CNS MPO score indicative of BBB permeability. In some embodiments, compounds disclosed herein can have a CNS MPO score greater than or equal to 4.0.
In certain embodiments, GHSR1a-agonist compounds disclosed herein (e.g., a compound of Formula I, II, III and/or IV) may maintain sustained concentrations in the brain following administration. In some embodiments, compounds disclosed herein may maintain sustained concentrations in the brain following systemic administration (e.g., by oral administration, intraperitoneal administration, intravenous administration). In some embodiments, compounds disclosed herein may maintain sustained concentrations in the brain for about 5 minutes to about 24 hours following administration. In some embodiments, compounds disclosed herein may maintain sustained concentrations in the brain for about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, or about 24 hours following administration. In some embodiments, compounds disclosed herein (e.g., a compound of Formula I, II, III and/or IV) may maintain a brain:plasma ratio of about 0.2:1 to about 1:1 following administration. In some embodiments, compounds disclosed herein may maintain a brain:plasma ratio of about 0.2:1, about 0.3:1, about 0.4:1, about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, or about 1:1 following administration. In some embodiments, compounds disclosed herein may maintain a brain:plasma ratio of about 0.2:1 to about 1:1 for about 5 minutes to about 24 hours following administration.
GHSR1a-agonist compounds disclosed herein may modulate heterotrimeric G protein activity, β-arrestin activity, or a combination thereof. In certain embodiments, GHSR1a-agonist compounds disclosed herein may be biased agonists. Ligand bias, or “true” biased agonism, refers to differences in signaling due to the molecular variation that governs the interaction between the ligand (e.g., GHSR1a-agonist compounds disclosed herein) and the transduction proteins at the receptor (e.g., GHSR1a). For GPCRs, such as GHSR1a, the easiest bias to observe is that between selective activation of heterotrimeric 0 proteins (G protein-bias) and β-arrestin (β-arrestin-bias) adapter proteins because G proteins and f-arrestins typically activate distinct signaling pathways, with G proteins typically activating second messengers and β-arrestins regulating receptor desensitization, internalization and activation of MAP kinases. In this regard, “agonists,” as used herein, may also be “biased agonists” with the ability to selectively stimulate a subset of GHSR1a's signaling activities, for example, the selective activation of G-protein or β-arrestin function. In some embodiments, GHSR1a-agonist compounds disclosed herein may be biased agonists that select for G-protein coupled signaling over arrestin coupled signaling. In some embodiments, GHSR1a-agonist compounds disclosed herein may be biased agonists that selectively activate one or more heterotrimeric G proteins (i.e., “G protein-bias”). In some embodiments, GHSR1a-agonist compounds disclosed herein may be biased agonists that do not selectively activate β-arrestin (i.e., “β-arrestin-bias”) adapter proteins.
In some embodiments, GHSR1a-agonist compounds disclosed herein may be biased agonists that selectively activate one or more of the G protein subunits. In accordance with these embodiments, biased GHSR1a-agonist disclosed herein may selectively activate one or more of the G protein subunits comprising Gα, Gβ, and/or Gγ. In some embodiments, GHSR1a-agonist compounds disclosed herein may be biased agonists that selectively activate one or more Gα subunits. In some embodiments, GHSR1a-agonist compounds disclosed herein may be biased agonists that selectively activate Gαs, Gαolf, Gαi1, Gαi2, Gαi3, GαoA, GαoB, Gαz, Gαq, Gα11, Gα14, Gα15, Gα12, Gα13, or any combination thereof. In some embodiments, GHSR1a-agonist compounds disclosed herein may be biased agonists that selectively activate Gαq. In some embodiments, GHSR1a-agonist compounds disclosed herein may be biased agonists that selectively activate Gαq/11.
In some embodiments, GHSR1a-agonist compounds disclosed herein may not compete with ghrelin peptide binding to the GHSR1a. In some embodiments, a GHSR1a-agonist compound disclosed herein and a ghrelin peptide may bind to the GHSR1a simultaneously.
In certain embodiments, compositions disclosed herein are pharmaceutical compositions. In some embodiments, pharmaceutical compositions can include at least one compound disclosed herein and at least one pharmaceutically acceptable carrier. In some embodiments, pharmaceutical compositions can include pharmaceutically acceptable carriers, excipients, and/or stabilizers are nontoxic to recipients at dosages and/or concentrations used to practice the methods disclosed herein. In some embodiments, pharmaceutically acceptable carriers, excipients, and/or stabilizers can include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
In certain embodiments, pharmaceutical compositions disclosed herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral, or rectal administration, or administration by inhalation or insufflation. In some embodiments, for preparing solid compositions such as tablets, the principal active ingredient (e.g., a compound disclosed herein) can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid pre-formulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these pre-formulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. In some embodiments, solid pre-formulation compositions herein can then subdivided into unit dosage forms of the type described above containing from 0.1 mg to about 500 mg (e.g., about 0.1 mg, about 0.5 mg, about 1.0 mg, about 5.0 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg) of a compound disclosed herein.
In some embodiments, tablets and/or pills disclosed herein can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. In some embodiments, a tablet and/or pill herein can have an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. In accordance with embodiments herein, the two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. In some embodiments, tablets and/or pills disclosed herein can include one or more materials that can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate. Non-limiting surface-active agents (surfactants) suitable for use herein can include non-ionic agents, such as polyoxyethylenesorbitans (e.g., Tween 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span 20, 40, 60, 80 or 85). In some embodiments, compositions herein with a surface-active agent can have between about 0.05 and about 5% surface-active agent. In some embodiments, other ingredients can be added to pharmaceutical compositions herein, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.
In some embodiments, pharmaceutical compositions herein can be tablets. In accordance with some embodiments herein, tablets contemplated herein can contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. In accordance with some embodiments herein, tablets contemplated herein can contain lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc. In some embodiments, pharmaceutical compositions herein can be solid compositions employed as fillers in gelatin capsules. In accordance with some embodiments herein, excipients included in gelatin capsules contemplated herein can include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols.
In some embodiments, pharmaceutical compositions herein can be emulsions. In some embodiments, emulsions herein can be prepared using commercially available fat emulsions, such as Intralipid, Liposyn, Infonutrol, Lipofundin, and Lipiphysan. The active ingredient (e.g., the one or more aminopeptidase inhibitors and/or one or more platinum-based chemotherapeutics) can be either dissolved in a pre-mixed emulsion composition or alternatively it can be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g., egg phospholipids, soybean phospholipids or soybean lecithin) and water. In some embodiments, other ingredients can be added to the compositions herein, for example glycerol or glucose, to adjust the tonicity of the emulsion. In other embodiments, emulsions can contain up to about 20% oil, for example, between about 5% and about 20%. In some embodiments, emulsions can have fat droplets between about 0.1 μm and about 1.0 μm in diameter and/or have a pH in the range of about 5.5 to about 8.0.
In certain embodiments, pharmaceutical compositions herein can be formulated for parenteral administration, such as intravenous, intracerebroventricular injection, intra-cisterna magna injection, intra-parenchymal injection, or a combination thereof. In some embodiments, pharmaceutical compositions herein formulated for parenteral administration can include one or more sterile liquids as pharmaceutically acceptable carriers. Non-limiting examples of sterile liquids suitable for use as pharmaceutically acceptable carriers herein can be water and oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and the like. Saline solutions and aqueous dextrose, polyethylene glycol (PEG) and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions disclosed herein may further comprise additional ingredients, for example preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, viscosity-increasing agents, and the like. In some embodiments, pharmaceutical compositions disclosed herein can be packaged in single unit dosages or in multi-dosage forms.
In some embodiments, pharmaceutical compositions herein suitable for parenteral administration can include aqueous and non-aqueous sterile injection solutions which can further contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Aqueous solutions may be suitably buffered (preferably to a pH of from about 3 to about 9). The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
In some embodiments, pharmaceutical compositions described herein can further include an anti-microbial agent. In accordance with these embodiments, the anti-microbial agent can, in an example, be an anti-viral, bactericidal agent, anti-fungal, or anti-bacterial agent. For example, the anti-microbial agent can be an anti-bacterial agent (antibiotic) such as doxycycline or other antibiotics such as a general antibiotic.
Any of the GHSR1a-agonist compounds (e.g., a compound of Formula I, II, III and/or IV) disclosed herein may be used for preventing, alleviating and/or treating pathological disruptions of brain dopamine (DA) homeostasis. DA, or 4-(2-aminoethyl)-1,2-benzenediol, is an organic chemical of the catecholamine and phenethylamine families and constitutes about 80% of the catecholamine content in the brain. The brain includes several distinct dopamine pathways, some of which play a major role in the motivational component of reward-motivated behavior. Both imbalances in brain dopamine homeostasis and alterations of brain circuits where dopamine is a key factor are involved in a variety of neurological and neuropsychiatric diseases. Non-limiting examples of disorders and/or diseases that arise from disruptions of brain dopamine homeostasis can include, but are not limited to, Parkinson's disease, attention deficit hyperactivity disorder, Tourette syndrome, schizophrenia, bipolar disorder, Alzheimer's Disease, eating disorders, and addiction (e.g., food/alcohol/drug).
In certain embodiments, the present disclosure provides methods for modulating dopaminergic neurotransmission in a subject in need thereof. In some embodiments, methods for modulating dopaminergic neurotransmission in a subject in need thereof may include administering a subject in need thereof an effective amount of any of the GHSR1a-agonist compounds (e.g., a compound of Formula I, II, III and/or IV) and/or pharmaceutical compositions disclosed herein. In some embodiments, a subject in need of modulation dopaminergic neurotransmission may have or be suspected of having one or more disruptions of brain dopamine (DA) homeostasis. In some embodiments, a subject in need of modulation dopaminergic neurotransmission may have or be suspected of having one or more of (but not limited to) the following: Parkinson's disease, attention deficit hyperactivity disorder, Tourette syndrome, one or more eating disorders, schizophrenia, bipolar disorder, Alzheimer's Disease, narcolepsy, depression, obesity, addiction (e.g., food/alcohol/drug), or any combination thereof.
To perform the methods disclosed herein, an effective amount of the compounds or compositions disclosed herein may be administered to a subject who needs treatment via a suitable route (e.g., orally, intravenous, intracerebroventricular injection, intra-cisterna magna injection, or intra-parenchymal injection) at a suitable amount as disclosed herein.
As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who is in need of the treatment, for example, having a target disease or disorder, a symptom of the disease/disorder, or a predisposition toward the disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder.
Alleviating a target disease/disorder includes delaying the development or progression of the disease or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, “delaying” the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.
“Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and/or recurrence.
In certain embodiments, methods disclosed herein may be used for preventing, alleviating and/or treating a neurological disorder associated with involuntary motor movements. In some embodiments, methods disclosed herein may be used for preventing, alleviating and/or treating Parkinson's disease. Thus, in some aspects, the present disclosure provides methods for alleviating one or more symptoms and/or for treating Parkinson's disease in a subject in need thereof by administration of any of the compounds disclosed herein, as well as a pharmaceutical composition comprising such. In some embodiments, methods herein may reduce hyperlocomotion in a subject treated with a compound or composition disclosed herein compared to an untreated subject with identical disease condition and predicted outcome. In some embodiments, methods herein may reduce hyperlocomotion by about 2% to about 99% (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%) in a subject treated with a compound or composition disclosed herein compared to an untreated subject with identical disease condition and predicted outcome.
In certain embodiments, methods disclosed herein may be used for preventing, alleviating and/or treating at least one addictive behavior in a subject. In some embodiments, methods disclosed herein may be used for preventing, alleviating and/or treating a drug addition, an alcohol addition, an addiction to food, or a combination thereof. The “drugs” according to the present disclosure can be, but are not limited to, narcotics, medicaments, tobacco, and/or alcohol. In some aspects, the drugs that are the source of the addition to be treated herein may be cocaine, crack, cannabis, morphine, opioids, heroin, ecstasy, LSD, amphetamines, ketamine, tobacco, alcohol, or any combination thereof. As will be clear to those skilled in the art, tobacco addiction is generally a nicotine addiction and alcohol addiction according is generally an ethanol addiction. In some embodiments, methods disclosed herein may be used for preventing relapse into addiction. In some embodiments, methods disclosed herein may be useful in reducing the addictive effect of re-exposure or continuous exposure to at least one agent, behavior, and/or stimulus which induces the addictive behavior in an addicted subject or in a subject having a risk of developing an addiction. In some embodiments, the addiction is not induced by re-exposure or continuous exposure to at least one agent or behavior in a subject after administration of any one of the compounds and/or compositions disclosed herein.
In certain embodiments, methods disclosed herein may be used for preventing, alleviating and/or treating at least one eating disorder in a subject. Eating disorders referred to herein may be characterized by abnormal compulsions to avoid eating or uncontrollable impulses to consume abnormally large amounts of food. In some embodiments, methods disclosed herein may be used for preventing, alleviating and/or treating bulimia nervosa, anorexia nervosa, binge-eating disorder, a food addition, or any combinations thereof. In some embodiments, methods disclosed herein may be used for preventing, alleviating and/or treating binge eating disorders, obesity resulting from binge eating behavior, and/or depression. In some embodiments, methods disclosed herein may treat an underlying mechanism of an eating disorders, including but not limited to hunger and satiety; palatability and aversion; hedonism; reward/addiction behavior; mood; anxiety; depression, taste, smell, and the like. In some embodiments, methods disclosed herein may be used for preventing, alleviating and/or treating an eating disorder that is an undesired side-effect of a therapeutic (e.g., chemotherapy, pharmacological treatments for mood disorders).
Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer any of the compounds or compositions disclosed herein to a subject in need thereof. In some embodiments, a compound or composition disclosed herein can be administered parenterally, e.g., by intravenous injection, intracerebroventricular injection, intra-cisterna magna injection, intra-parenchymal injection, or a combination thereof. In some embodiments, a compound or composition disclosed herein can be administered orally.
In certain embodiments, a compound or composition disclosed herein can be administered in combination with at least one additional therapeutic agent. In some embodiments, a compound or composition disclosed herein can be administered in combination with at least one additional therapeutic agent for treatment and/or prevention of any one of the diseases and/or disorders disclosed herein. In some embodiments, a compound or composition disclosed herein can be administered in combination with at least one additional therapeutic agent for treatment and/or prevention of one or more GHSR1a-associated conditions. In accordance with these embodiments, therapeutic agents suitable for administration to a subject in combination with any of the compounds or compositions disclosed herein may include, but are not limited to, methadone, naltrexone, acamprosate, disulfiram, bupropion, varenicline, nicotine replacement therapies, and any combination thereof. In some embodiments, a compound or composition disclosed herein can be administered before at least one additional therapeutic agent is administered to a subject. In some embodiments, a compound or composition disclosed herein can be administered after at least one additional therapeutic agent is administered to a subject. In some embodiments, a compound or composition disclosed herein can be administered during the course of treatment with at least one additional therapeutic agent administered to a subject.
In certain embodiments, a compound disclosed herein may be administered to a subject orally at a concentration ranging from about 0.5 mg/kg to about 50 mg/kg. In some embodiments, a compound disclosed herein may be administered to a subject orally at a concentration of about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, or about 50 mg/kg. In some embodiments, a compound disclosed herein may be administered to a subject orally at least once a day, at least twice a day, at least three times a day or more.
In certain embodiments, a compound disclosed herein may be administered to a subject intraperitoneally (IP) at a concentration ranging from about 0.5 mg/kg to about 50 mg/kg. In some embodiments, a compound disclosed herein may be administered to a subject intraperitoneally at a concentration of about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, or about 50 mg/kg. In some embodiments, a compound disclosed herein may be administered to a subject intraperitoneally at least once a day, at least twice a day, at least three times a day or more.
In certain embodiments, a compound disclosed herein may be administered to a subject intravenously at a concentration ranging from about 0.05 mg/kg to about 20 mg/kg. In some embodiments, a compound disclosed herein may be administered to a subject intravenously at a concentration of about 0.05 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, or about 20 mg/kg. In some embodiments, a compound disclosed herein may be administered to a subject intravenously at least once a day, at least twice a day, at least three times a day or more.
In certain embodiments, methods disclosed herein may be used to administer any of the compounds or compositions disclosed herein to a subject with high bioavailability. In some embodiments, a compound disclosed herein may have an oral bioavailability of about 1% to about 15%. As used herein “oral bioavailability” and “bioavailability upon oral administration” refer to the systemic availability (i.e., blood/plasma levels) of a given amount of a compound administered orally to a subject according to the methods herein. In some embodiments, a compound disclosed herein may have an oral bioavailability of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%.
In some embodiments, a compound disclosed herein may have an IP bioavailability of about 10% to about 35%. As used herein “IP bioavailability” and “bioavailability upon IP administration” refer to the systemic availability (i.e., blood/plasma levels) of a given amount of a compound administered intraperitoneally (IP) to a subject according to the methods herein. In some embodiments, a compound disclosed herein may have an IP bioavailability of about 10%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, or about 35%.
In certain embodiments, kits are provided herein for use in treating or alleviating a targeted disease (e.g., Parkinson's disease, addiction, eating disorder) or condition treatable by use of a compound disclosed herein. In some embodiments, kits herein can include instructions for use in accordance with any of the methods described herein. In other embodiments, instructions can include a description of administering a compound and/or pharmaceutical composition disclosed herein to a subject at risk of the target disease. In certain embodiments, kits disclosed herein can include instructions for using the components of the kit, for example relating to the use of a compound and/or pharmaceutical composition disclosed herein. In accordance with embodiments herein, kits can include instructions that provide information as to dosage, dosing schedule, and route of administration for the intended treatment.
In some embodiments, kits disclosed herein can include at least one container. In accordance with embodiments herein, containers can be any container such as tubes, vials, bottles, syringe, such as unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention can be written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. The label or package insert indicates that the composition is used for treating, delaying the onset and/or alleviating the disease (e.g., Parkinson's disease, an addiction). Instructions can be provided for practicing any of the methods described herein.
Kits disclosed herein can include suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated herein are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container can also have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition can be a compound disclosed herein.
Kits can optionally provide additional components such as buffers and interpretive information. Normally, the kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture including contents of the kits described above.
While the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the disclosure.
To discover novel, biased GHSR1a ligands, a cell-based, human GHSR1a/beta-arrestin1 (β-arr1) chemiluminescent assay (DiscoverX, PathHunter) was used to screen 47K compounds from the Sytravon library and the NCATS Pharmacological collection (NPC) (
From these experiments, NCGC141956 (N1956) (
To determine target selectivity, N8279 activity was evaluated across the GPCRome (at 320 receptors) using the PRESTO-Tango platform by high-throughput screening with a β-arr2 recruitment assay similar to the method described in Kroeze et al., Nat Struct Mol Biol. 2015; 22(5):362-9, the disclosure of which is incorporated herein in its entirety. Hits were defined as >3-fold activation above baseline. N8279 stimulated 6-fold activation at the GHSR1a and did not exceed >3-fold activation at any other GPCR (
The following analytical methods were used to evaluate the GHSR1a-selective compounds, in the following examples. Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Method 1: A 7-min gradient of 4% to 100% acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8-min run time at a flow rate of 1.0 mL/min.
Method 2: A 3-min gradient of 4% to 100% acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5-min run time at a flow rate of 1.0 mL/min. A Phenomenex Luna Cis column (3-micron, 3×75 nm) was used at a temperature of 50° C. Purity determination was performed using an Agilent diode array detector for both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode.
1H NMR data was collected on a Bruker 400 MHz NMR.
Solvents from Sigma Aldrich were purchased commercially and used without further purification.
The preparation of 6-chloro-3-(3,4-dimethoxybenzoyl)-N-(3-(dimethylamino)propyl)-4-oxo-4H-chromene-2-carboxamide (5) was conducted according to the Chemical Scheme 1 shown below.
1-(5-Chloro-2-hydroxyphenyl) ethan-1-one (5 g, 29.3 mmol) was dissolved in DMF (100 ml), then the solution was placed under an ice bath. Potassium 2-methylpropane-2-olate (64.5 ml, 64.5 mmol) was slowly added to the solution. After 5 min, diethyl oxalate (5.97 ml, 44.0 mmol) was added to the reaction mixture with one portion. A yellow solid appeared. The reaction mixture was kept at room temperature overnight. The reaction mixture was diluted with ethyl acetate. Then, 1N HCl was added to the reaction mixture to extract the organic layer. The organic layer containing insoluble yellow solid was removed by filtration. The organic layer was dried with sodium sulfate. The crude product was obtained by evaporation of the solvent, which was purified by silica column (10 to 30% EA in Hex). Yield (92%). 1H NMR (400 MHz, DMSO-d6) δ 8.33 (d, J=1.9 Hz, 1H), 7.71-7.60 (m, 2H), 7.15 (d, J=8.7 Hz, 1H), 4.22 (qd, J=7.1, 1.0 Hz, 2H), 3.32 (d, J=1.7 Hz, 1H), 2.91 (dd, J=16.5, 1.1 Hz, 1H), 1.22 (td, J=7.1, 1.1 Hz, 3H).
3,4-Dimethoxybenzaldehyde (100 mg, 0.602 mmol) was placed in the microwave reaction vessel and dissolved in MeOH (5 ml). To the solution was added N1,N1-dimethylpropane-1,3-diamine (76 pal, 0.602 mmol). The microwave-assisted reaction was run at 110° C. for 30 min. After cooling the reaction vessel to room temperature, ethyl 6-chloro-2-hydroxy-4-oxochromane-2-carboxylate (200 mg, 0.739 mmol) was added to the reaction vessel and the microwave-assisted reaction was run at 70° C. for 30 min. The reaction mixture was rotovaped and the crude product was fractionally filtered through silica gel (Methanol/DCM) to afford the desired crude product. This crude product was taken directly to the next step.
7-Chloro-1-(3,4-dimethoxyphenyl)-2-(3-(dimethylamino)propyl)-3a-hydroxy-1,2,3a,9a-tetrahydrochromeno[2,3-c]pyrrole-3,9-dione (100 mg, 0.211 mmol) was placed in the microwave reaction vessel and dissolved in acetic acid (5 ml). The microwave-assisted reaction was run at 70° C. for one hour. The reaction mixture was rotovaped and then dissolved in EtOH. After heating, the product came out upon cooling to room temperature. The product was filtered and dried (86 mg, 90%). 1H NMR (400 MHz, CDCl3) δ 8.12 (d, J=2.3 Hz, 1H), 7.71-7.61 (m, 2H), 6.92-6.81 (m, 2H), 6.65 (d, J=1.9 Hz, 1H), 5.48 (s, 1H), 3.81-3.90 (m, 7H), 2.99 (ddd, J=14.0, 8.2, 5.9 Hz, 1H), 2.29-2.19 (m, 2H), 2.15 (s, 6H), 1.85-1.63 (m, 2H); LCMS RT (Method 1)=4.035 min, m/z 457.2 [M+H+]; HRMS (ESI) m/z calcd for C24H26ClN2O5+ [M+H+] 457.1530, found 457.1537.
The title compound was prepared according to Vydzhak, R. N.; Panchishin, S. Y. Synthesis of 2-Alkyl-1-aryl-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione Derivatives. Russ. J. Gen. Chem. 2008, 78, 2391-2397.
A solution of 7-chloro-1-(3,4-dimethoxyphenyl)-2-(3-(dimethylamino)propyl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione (200 mg, 0.438 mmol) was dissolved in DMSO (10 mL). To this solution was bubbled air until complete conversion was observed by LCMS. The reaction mixture was blown dry and purified by column chromatography (Methanol/DCM) to afford the desired product (2:1 ratio 5a:5b) as a light brown solid (86.7 mg, 42%).
5a: 1H NMR (400 MHz, CD2Cl2) δ 9.48 (s, 1H), 8.12 (d, J=2.5 Hz, 1H), 7.73-7.62 (m, 2H), 7.08 (d, J=2.2 Hz, 1H), 6.98 (dd, J=8.4, 2.2 Hz, 1H), 6.84 (d, J=8.4 Hz, 1H), 3.83 (s, 3H), 3.82 (s, 3H), 3.69 (ddd, J=14.7, 5.5, 3.9 Hz, 2H), 2.88 (ddd, J=14.1, 10.4, 3.2 Hz, 2H), 2.33 (m, 2H), 2.22 (s, 6H); 5b: 1H NMR (400 MHz, CD2Cl2) δ 8.15 (d, J=2.6 Hz, 1H), 7.77 (dd, J=8.9, 2.6 Hz, 1H), 7.73-7.64 (m, 1H), 7.58 (d, J=2.0 Hz, 1H), 7.30 (dd, J=8.4, 2.0 Hz, 1H), 6.84 (d, J=8.4 Hz, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.51-3.43 (m, 1H), 2.74 (m, 1H), 2.60-2.52 (m, 1H), 2.53 (s, 6H), 2.33 (m, 1H), 1.87 (m, 1H), 1.71 (m, 1H); LCMS RT (Method 1)=3.921 min, m/z 473.2 [M+H+]; HRMS (ESI) m/z calcd for C24H26ClN2O6+ [M+H+] 473.1479, found 473.1467.
The preparation of 6-chloro-9a-(3,4-dimethoxyphenyl)-1-(2-(dimethylamino)ethyl)-3a,9a-dihydrochromeno[2,3-b]pyrrole-2,3,4(1H)-trione was conducted according to the Chemical Scheme 2 shown below.
To a suspension of 1-(5-chloro-2-hydroxyphenyl)ethan-1-one (2 g, 11.72 mmol) and 3,4-dimethoxybenzoyl chloride (2.58 g, 12.86 mmol) in pyridine (30 mL) was added a catalytic amount of 4-DMAP and heated to 50° C. under N2. Pyridine was rotovaped off and the resulting crude product was dissolved in DCM and washed with 1N HCl. The organic layer was dried with MgSO4, filtered, and rotovaped. The crude product was purified by column chromatography (hexanes/EtOAc) to yield the desired product as a white solid (3.6 g, 92%). 1H NMR (400 MHz, DMSO-d6) δ 7.96 (d, J=2.6 Hz, 1H), 7.75 (ddd, J=13.4, 8.5, 2.3 Hz, 2H), 7.56 (d, J=2.1 Hz, 1H), 7.42 (d, J=8.7 Hz, 1H), 7.16 (d, J=8.6 Hz, 1H), 3.88 (s, 3H), 3.84 (s, 3H); 2.51 (s, 3H).
A suspension of 2-acetyl-4-chlorophenyl 3,4-dimethoxybenzoate (2 g, 5.97 mmol) and potassium hydroxide (503 mg, 8.97 mmol) in pyridine (20 ml) was flushed with N2, sealed, and heated to 50° C. Pyridine was rotovaped off and the resulting crude product was dissolved in DCM and washed with 1N HCL. The organic layer was dried With MgSO4, filtered and rotovaped. The crude product was purified by column chromatography (hexanes/EtOAc) to yield the desired product (3:1 conformational isomers) as a yellow solid (1.55 g, 78%). 1H NMR (400 MHz, DMSO-d6) δ 16.73 (s, 1H), 11.34 (s, 1H), 7.97 (d, J=2.7 Hz, 1H), 7.71 (dd, J=8.5, 2.1 Hz, 1H), 7.54 (d, J=2.1 Hz, H), 7.51-7.45 (m, 1H), 7.37 (s, 1H), 7.14 (d, J=8.5 Hz, 1H), 7.03 (d, J=8.8 Hz, 1H), 3.87 (s, 3H), 3.86 (s, 3H).
A solution of 1-(5-chloro-2-hydroxyphenyl)-3-(3,4-dimethoxyphenyl)propane-1,3-dione (1.55 g, 4.63 mmol) in pyridine (40 mL) was cooled to 0° C. Then, ethyl 2-chloro-2-oxoacetate (1.03 mL, 9.22 mmol) was added dropwise. The reaction was stirred under N2 and slowly warmed to room temperature. Pyridine was rotovaped off and the resulting crude product was dissolved in DCM and washed with 1N HCl. The organic layer was dried with MgSO4, filtered, and rotovaped. The crude product was purified by column chromatography (hexanes/EtOAc) to yield the desired products as a yellow solid. product A (520 mg, 26.9%) and product B (750 mg, 38.9%). Product A: 1H NMR (400 MHz, CD2Cl2) a 8.15 (d, J=2.6 Hz, 1H), 7.79 (dd, J=9.0, 2.6 Hz, 1H), 7.66 (d, J=9.0 Hz, 1H), 7.58 (d, J=2.0 Hz, 1H), 7.33 (dd, J=8.4, 2.0 Hz, 1H), 6.87 (d, J=8.4 Hz, 1H), 4.27 (q, J=7.1 Hz, 2H), 3.92 (d, J=4.5 Hz, 6H), 1.17 (t, J=7.1 Hz, 3H). LCMS RT (Method 1)=5.411 min, m/z 417.0 [M+H+]; HRMS (EST) in z calcd for C21H18ClO7+ [M+H+] 417.0741, found 417.0752. Product B: 1H NMR (400 MHz, CD2C2) δ 8.17 (d, J=2.6 Hz, 1H), 7.74 (dd, J=8.9, 2.6 Hz, 1H), 7.59 (d, J=8.9 Hz, 1H), 7.25 (dd, J=8.4, 2.2 Hz, 1H), 7.19 (d, J=2.1 Hz, 1H), 6.98 (d, J=8.4 Hz, 1H), 4.23 (q, J=7.1 Hz, 2H), 3.93 (s, 3H), 3.90 (s, 3H), 1.27 (t, J=7.1 Hz, 3H). LCMS RT (Method 2)=3.426 min, m/z 417.1 [M+H+].
To a solution of ethyl 2-(6-chloro-2-(3,4-dimethoxyphenyl)-4-oxo-4H-chromen-3-yl)-2-oxoacetate (30 mg, 0.072 mmol) in acetonitrile (3 mL) was added N1,N1-dimethylethane-1,2-diamine (10.5 mL, 0.096 mmol). The reaction was stirred at room temperature under N2. The reaction mixture was rotovaped off and purified by column chromatography (hexanes/EtOAc) to yield the desired product as a white solid (30 mg, 91%).
1H NMR (400 MHz, DMSO-d6) δ 9.64 (bs, 1H), 7.48 (d, J=2.7 Hz, 1H), 7.37 (dd, J=8.7, 2.7 Hz, 1H), 7.24 (d, J=8.7 Hz, 1H), 7.12 (s, 1H), 6.99 (dd, J=8.5, 2.1 Hz, 1H), 6.79 (d, J=8.5 Hz, 1H), 3.76-3.62 (m, 7H), 3.25-2.05 (m, 2H), 2.95-2.80 (m, 1H), 2.81 (s, 61H); LCMS RT (Method 1)=4.268 min, m/z 458.8 [M+H+]; HRMS (ESI) nm/z calcd for C23H24ClN2O6+ [M+H+] 459.1323, found 459.1218.
The preparation of 6-chloro-9a-(3,4-dimethoxyphenyl)-1-(3-(dimethylamino)propyl)-3a,9a-dihydrochromeno[2,3-b]pyrrole-2,3,4(1H)-trione followed the above Chemical Scheme 2 denoted above. 1H NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1H), 7.48 (d, J=2.7 Hz, 1H), 7.36 (dd, J=8.7, 2.8 Hz, 1H), 7.18 (d, J=8.7 Hz, 1H), 6.91 (dd, J 8.4, 2.2 Hz, 1H), 6.85-6.79 (m, 2H), 3.67 (s, 3H), 3.64 (s, 3H), 3.44-3.37 (m, 1H), 3.01-2.85 (m, 3H), 2.74 (s, 6H), 1.85-1.74 (m, 2H); L CMS RT (Method 1)=3.911 min, m/z 473.1 [M+H+]; HRMS (EST) m/z calcd for C24H26ClN2O6+ [M+H+] 473.1479, found 473.1246.
The preparation of 3-(3,4-dimethoxybenzoyl)-N-(3-(dimethylamino)propyl)-6-iodo-4-oxo-4H-chromene-2-carboxamide was conducted according to Chemical Scheme 3 shown below.
1-(5-Iodo-2-hydroxyphenyl) ethan-1-one (200 mg, 0.763 mmol) was dissolved in THF (10 ml), then the solution was placed under an ice bath. 1M Potassium 2-methylpropane-2-olate in THF (1.7 mL, 2.227 mmol) was slowly added to the solution. After 5 min, diethyl oxalate (103.5 mL, 0.763 mmol) was added to the reaction mixture in one portion. A yellow solid appeared. The reaction mixture was kept at room temperature overnight. The reaction mixture was diluted with ethyl acetate. Then, 1N HCl was added to the reaction mixture to extract the organic layer. The organic layer containing the insoluble yellow solid was removed by filtration. The organic layer was dried over sodium sulfate. The crude product was obtained by evaporation of the solvent, which was purified by silica gel chromatography (10 to 30% EA in Hex). Yield (227 mg, 92%). 1H NMR (400 MHz, DMSO-d6) δ 8.31 (d, J=1.9 Hz, 1H), 7.94 (d, J=2.2 Hz, 1H), 7.87 (dd, J=8.7, 2.3 Hz, 1H), 6.93 (d, J=8.6 Hz, 1H), 4.20 (q, J=7.1 Hz, 2H), 3.30 (dd, J=16.4, 1.9 Hz, 1H), 2.88 (d, J=16.5 Hz, 1H), 1.21 (t, J=7.1 Hz, 3H).
3,4-Dimethoxybenzaldehyde (52.8 mg, 0.318 mmol) was placed in the microwave reaction vessel and dissolved in MeOH (5 ml). To the solution was added N1,N1-dimethylpropane-1,3-diamine (40 μl, 0.318 mmol). The microwave-assisted reaction was run at 110° C. for 30 min. After cooling the reaction vessel to room temperature, ethyl 6-chloro-2-hydroxy-4-oxochromane-2-carboxylate (115 mg, 0.318 mmol) was added to the reaction vessel and the microwave-assisted reaction was conducted at 70° C. for 30 min. The reaction mixture was rotovaped and the crude product was fractionally filtered through silica gel (Methanol/DCM) to afford the desired crude product. This was taken directly to the next step. Crude 1-(3,4-dimethoxyphenyl)-2-(3-(dimethylamino)propyl)-3a-hydroxy-7-iodo-1,2,3a,9a-tetrahydrochromeno [2,3-c]pyrrole-3,9-dione was placed in the microwave reaction vessel and dissolved in acetic acid (5 ml). The microwave-assisted reaction was run at 70° C. for one hour. The reaction mixture was rotovaped and then dissolved in EtOH. The EtOH solution was warmed and then cooled to room temperature. Upon cooling to room temperature, the product precipitated. The product was filtered and dried (90 mg, 52%). 1H NMR (400 MHz, DMSO-d6) δ 8.26 (d, J=2.2 Hz, 1H), 8.16 (dd, J=8.8, 2.2 Hz, 1H), 7.70 (d, J=8.8 Hz, 1H), 6.97-6.81 (m, 3H), 5.61 (s, 1H), 3.73 (s, 3H), 3.66 (s, 3H), 3.56 (dt, J=14.4, 7.8 Hz, 1H), 2.88 (ddd, J=14.2, 8.6, 6.0 Hz, 1H), 2.21-2.08 (m, 2H), 2.03 (s, 6H), 1.62 (dq, J=13.6, 7.0 Hz, 1H), 1.45 (dq, J=14.1, 7.1 Hz, 1H); LCMS RT (Method 2)=2.864 min, m/z 549.0 [M+H+].
The title compound was prepared according to Vydzhak, R. N.; Panchishin, S. Y. Synthesis of 2-Alkyl-1-aryl-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione Derivatives. Russ. J. Gen. Chem. 2008, 78, 2391-2397.
The compound was prepared as described above. 1H NMR (400 MHz, CD2Cl2) δ 8.48 (d, J=2.2 Hz, 1H), 8.01 (dd, J=8.8, 2.2 Hz, 1H), 7.45 (d, J=8.8 Hz, 1H), 7.08 (d, J=2.1 Hz, 1H), 6.98 (dd, J=8.4, 2.2 Hz, 1H), 6.84 (d, J=8.4 Hz, 1H), 3.83 (s, 3H), 3.82 (s, 3H), 3.68 (ddd, J=14.6, 5.3, 3.8 Hz, 1H), 2.87 (ddd, J=14.2, 10.5, 3.2 Hz, 1H), 2.60-2.47 (m, 2H), 2.39-2.26 (m, 1H), 2.20 (s, 6H), 1.75-1.65 (m, 1H). 2:1 ratio. Major to minor. Only major NMR shown; LCMS RT (Method 1)=3.975 min, m/z 565.1 [M+H+]; HRMS (ESI) in m/z calcd for C24H26IN2O6+ [M+H+] 565.0836, found 565.0718.
A solution of 4-hydroxy-3-methoxybenzaldehyde (28.2 mg, 0.185 mmol) and N1,N1-dimethylpropane-1,3-diamine (23.3 mL, 0.185 mmol) in MeOH (5 mL) was heated to 70° C. for 30 minutes, cooled, then ethyl 6-chloro-2-hydroxy-4-oxochromane-2-carboxylate (50 mg, 0.185 mmol) was added. The reaction mixture was heated to 50° C. and stirred for 2 hrs. Reaction mixture was filtered and the resulting solid was collected and taken to the next step. The crude product was dissolved in acetic acid (5 mL) and heated to 50° C. for 3 hrs. The reaction mixture was rotovaped, triturated in EtOH, and then the desired product was filtered to yield a white solid (35 mg, 43%). 1H NMR (400 MHz, CDCl3) δ 8.13 (d, J=2.3 Hz, 1H), 7.72-7.62 (m, 2H), 6.91 (d, J=8.1 Hz, 1H), 6.81 (dd, J=8.1, 2.0 Hz, 1H), 6.67 (s, 1H), 5.66 (s, 1H), 5.48 (s, 1H), 3.85 (s, 3H), 3.00 (dt, J=14.0, 6.9 Hz, 1H), 2.30-2.21 (m, 21H), 2.16 (s, 61H), 1.85-1.63 (m, 211), 1.26-1.23 (m, 1H); LCMS RT (Method 2)=2.019 min, m/z 443.1 [M+H+].
1H NMR (400 MHz, CD2Cl2) δ 8.43 (s, 1H), 8.12 (d, J=2.5 Hz, 1H), 7.73-7.63 (m, 2H), 7.28-7.16 (m, 2H), 6.84 (s, 1H), 3.92 (s, 3H), 3.68 (ddd, J=14.6, 5.8, 3.8 Hz, 1H), 2.89 (ddd, J=14.1, 10.1, 3.2 Hz, 1H), 2.61-2.55 (m, 2H), 2.35-2.22 (m, 1H), 2.25 (s, 6H), 1.78-1.72 (m, 1H). 3:2 ratio. Major to minor. Only major NMR shown; LCMS RT (Method 1)=3.639 min, nm/z 459.1 [M+H+]; HRMS (ESI) m/z calcd for C23H24ClN2O6+[M+H+] 459.1323, found 459.1105.
1-(2-Chloro-6-hydroxyphenyl) ethan-1-one (300 mg, 1.759 mmol) was dissolved in 20 mL of THF. The reaction was cooled in an ice-bath and potassium t-butoxide (3.9 mL, 3.9 mmol) was added. After 5 minutes with stirring, to the reaction mixture was added diethyl oxalate (0.240 mL, 1.767 mmol). The reaction mixture was heated to 50° C. and kept for 1 hour. Then the reaction was cooled down to room temperature, followed by quenching it with 0.1N HCl solution. The ethyl acetate was added to the reaction solution. The organic phase was separated and dried with anhydrous Na2SO4. The organic phase was evaporation to obtain the crude product. The product was purified by silica column (20-80% ethyl acetate in hexane). Yield=92%. 1H NMR (400 MHz, CDCl3) δ 7.42-7.30 (m, 1H), 7.10 (dt, J=7.9, 1.0 Hz, 1H), 6.92 (dq, J=8.4, 1.1 Hz, 1H), 4.58-4.48 (m, 1H), 4.42-4.30 (i, 2H), 3.40 (ddd, J=16.0, 2.6, 1.5 Hz, 1H), 2.96 (dt, J=16.0, 0.9 Hz, 1H), 1.35 (ddd, J 8.1, 6.7, 1.0 Hz, 3H).
3,4-Dimethoxybenzaldehyde (123 mg, 0.739 mmol) was placed in the microwave reaction vessel and dissolved in MeOH (5 ml). To the solution was added N1,N1-dimethylpropane-1,3-diamine (93 μl, 0.739 mmol). The microwave-assisted reaction was run at 110° C. for 30 min. After cooling the reaction vessel to room temperature, ethyl 5-chloro-2-hydroxy-4-oxochromane-2-carboxylate (200 mg, 0.739 mmol) was added to the reaction vessel and the microwave-assisted reaction was conducted at 70° C. for 30 min. The yellow solid was precipitated, filtered, and washed with MeOH three times. The product was used for the next step without further purification.
8-Chloro-1-(3,4-dimethoxyphenyl)-2-(3-(dimethylamino)propyl)-3a-hydroxy-1,2,3a,9a-tetrahydrochromeno[2,3-c]pyrrole-3,9-dione (328.9 mg, 0.693 mmol) was dissolved in AcOH (10 ml). The solution was heated up to 70° C. and stirred for 1 hour. Yield 91%. 1H NMR (400 MHz, CDCl3) δ 7.64-7.54 (m, 2H), 7.43 (dd, J=6.3, 2.8 Hz, 1H), 6.92-6.81 (m, 2H), 6.66 (d, J=2.0 Hz, 1H), 5.53 (s, 1H), 3.84 (d, J=11.7 Hz, 6H), 3.82-3.77 (m, 1H), 3.00 (dt, J=14.3, 5.9 Hz, 1H), 2.84 (td, J=11.7, 5.2 Hz, 1H), 2.68 (ddd, J=12.0, 10.5, 5.4 Hz, 1H), 2.53 (s, 6H), 1.98-1.86 (m, 2H). LCMS RT (Method 1)=2.598 min, m/z=457.1 [M+H+].
3-Hydroxy-4-methoxybenzaldehyde (225 mg, 1.478 mmol) and N1,N1-dimethylpropane-1,3-diamine (186 μl, 1.478 mmol) was dissolved in MeOH (5 ml). The reaction mixture was microwave-assisted heated up to 110° C. for 30 min. The ethyl 6-chloro-2-hydroxy-4-oxochromane-2-carboxylate (400 mg, 1.478 mmol) was added to the imine solution of MeOH. The mixture was heated to 70° C. for one hour. Yellow solid was precipitated. The solid was filtered and washed with methanol. The product was used for the next step without further purification. 7-Chloro-2-(3-(dimethylamino)propyl)-3a-hydroxy-1-(3-hydroxy-4-methoxyphenyl)-1,2,3a,9a-tetrahydrochromeno[2,3-c]pyrrole-3,9-dione (550 mg, 1.193 mmol) was placed in the microwave reaction vessel and dissolved in acetic acid (10 ml). The microwave-assisted reaction was run at 70° C. for one hour. The acetic acid in the vessel was dried and dissolved in ethanol. The ethanol was evaporated and dried under vacuum. Overall yield=80%. 1H NMR (400 MHz, CDCl3) δ 8.16-8.06 (m, 1H), 7.67 (dd, J=3.9, 1.4 Hz, 2H), 6.85 (d, J=3.4 Hz, 2H), 6.69 (d, J=1.6 Hz, 1H), 5.45 (s, 1H), 3.95-3.72 (m, 4H), 2.99 (ddd, J=14.0, 7.9, 6.3 Hz, 1H), 2.24 (q, J=7.0 Hz, 2H), 2.16 (s, 6H), 1.82-1.65 (m, 2H). LCMS RT (Method 1)=2.715 min, m/z=443.1 [M+H+].
5-Formyl-2-methoxybenzonitrile (125 mg, 0.739 mmol) was placed in the microwave reaction vessel and dissolved in MeOH (5 ml). To the solution was added N1,N1-dimethylpropane-1,3-diamine (93 μl, 0.739 mmol). The microwave-assisted reaction was run at 110° C. for 30 min. After cooling the reaction vessel to room temperature, ethyl 6-chloro-2-hydroxy-4-oxochromane-2-carboxylate (200 mg, 0.739 mmol) was added to the reaction vessel and the microwave-assisted reaction was run at 70° C. for 30 min. A yellow solid precipitated and removed by filtration. The solid was washed with MeOH three times. The product was used for the next step without further purification.
The preparation of 5-(7-chloro-2-(3-(dimethylamino)propyl)-3,9-dioxo-1,2,3,9-tetrahydrochromeno[2,3-c]pyrrol-1-yl)-2-methoxybenzonitrile was conducted according to the chemical reaction scheme as shown above. 1H NMR (400 MHz, CDCl3) δ 8.12 (d, J=2.4 Hz, 1H), 7.71 (dd, J=9.0, 2.5 Hz, 1H), 7.66 (d, J=9.0 Hz, 1H), 7.55 (dd, J=8.8, 2.4 Hz, 1H), 7.43 (d, J=2.4 Hz, 1H), 7.00 (d, J=8.8 Hz, 1H), 5.64 (s, 1H), 3.93 (s, 3H), 3.92-3.84 (m, 1H), 2.96 (ddd, J=14.3, 6.7, 5.1 Hz, 1H), 2.83-2.74 (m, 1H), 2.68-2.59 (m, 1H), 2.49 (s, 6H), 2.00-1.89 (m, 2H). LCMS RT (Method 1) 2.889 min, m/z=468.1 [M+H+].
The preparation of 6-chloro-N-(3-(dimethylamino)propyl)-4-oxo-3-(3,4,5-trimethoxybenzoyl)-4H-chromene-2-carboxamide was conducted according to the chemical reaction scheme as shown above. [00174] 4H NMR (400 MHz, CD2Cl2) δ 8.14-8.13 (m, 1H), 7.73-7.65 (m, 2H), 6.72 (s, 2H), 3.82 (s, 6H), 3.77 (s, 3H), 3.70 (dt, J=14.6, 4.6 Hz, 1H), 2.90 (td, J=11.2, 5.5 Hz, 1H), 2.57-2.60 (m, 2H), 2.37-2.25 (m, 1H), 2.25 (s, 6H), 1.80-1.72 (m, 1H). 4:1 ratio. Major to minor. Only major NMR shown; LCMS RT (Method 1)=3.991 min, m/z 503.2 [M+H+]; HRMS (ESI) m/z calcd for C25H28ClN2O7+[M+H+]503.1585, found 503.1568.
The preparation of 6-chloro-3-(3,4-dimethoxybenzoyl)-N-(3-morpholinopropyl)-4-oxo-4H-chromene-2-carboxamide was conducted according to the chemical reaction scheme as shown above.
1H NMR (400 MHz, CD2Cl2) δ 8.13 (d, J=2.5 Hz, 1H), 7.74-7.62 (m, 2H), 7.07-7.02 (m, 2H), 6.86 (d, J=7.9 Hz, 1H), 3.83-3.82 (m, 6H), 3.70-3.58 (m, 5H), 2.90 (ddd, J=14.5, 10.8, 3.4 Hz, 1H), 2.70-2.33 (m, 7H), 1.70-1.78 (m, 1H). 2:1 ratio. Major to minor. Only major NMR shown; LCMS RT (Method 1)=3.902 min, m/z 515.2 [M+H+]; HRMS (ESI) m/z calcd for C26H28ClN2O7+ [M+H+] 515.1585, found 515.1451.
The preparation of 6-chloro-N-(3-(dimethylamino)propyl)-3-(3-methoxybenzoyl)-4-oxo-4H-chromene-2-carboxamide was conducted according to the chemical reaction scheme as shown above. 1H NMR (400 MHz, CD2Cl2) δ 8.12 (d, J=2.4 Hz, 1H), 7.71-7.65 (m, 2H), 7.27 (t, J=8.0 Hz, 1H), 7.15 (d, J=2.6 Hz, 1H), 6.99 (d, J=7.7 Hz, 1H), 6.87 (dd, J=8.2, 2.6 Hz, 1H), 3.81 (s, 3H), 3.68 (dt, J=14.7, 4.7 Hz, 1H), 2.87 (ddd, J=14.1, 10.0, 3.2 Hz, 1H), 2.61 (m, 2H), 2.35-2.25 (3, 7H), 1.70-1.78 (m, 1H). 10:1 ratio. Major to minor. Only major NMR shown; LCMS RT (Method 1)=4.031 min, m/z 443.1 [M+H+]; HRMS (ESI) m/z calcd for C23H24ClN2O5+[M+H+] 443.1374, found 443.1203.
The preparation of 6-chloro-3-(4-chlorobenzoyl)-N-(3-(dimethylamino)propyl)-4-oxo-4H-chromene-2-carboxamide was conducted according to the chemical reaction scheme as shown above. 1H NMR (400 MHz, CD2Cl2) δ 8.11 (m, 1H), 7.72-7.64 (m, 2H), 7.49-7.44 (m, 2H), 7.39-7.32 (m, 2H), 3.70 (dt, J=14.7, 4.7 Hz, 1H), 2.85 (ddd, J=14.1, 10.0, 3.1 Hz, 1H), 2.65-2.55 (m, 2H), 2.35-2.22 (m, 7H), 1.72-1.78 (m, 1H). 10:1 ratio. Major to minor. Only major NMR shown; LCMS RT (Method 1)=4.347 min, m/z 447.1 [M+H+]; HRMS (ESI) m/z calcd for C22H21Cl2N2O4+ [M+H+] 447.0878, found 447.0700.
The preparation of 6-chloro-N-(3-(dimethylamino)propyl)-3-(4-isopropylbenzoyl)-4-oxo-4H-chromene-2-carboxamide was conducted according to the chemical reaction scheme as shown above. 1H NMR (400 MHz, CD2Cl2) δ 8.11 (d, J=2.4 Hz, 1H), 7.80-7.62 (m, 21H), 7.41 (d, J=7.9 Hz, 211), 7.23 (d, J=8.0 Hz, 2H), 3.67 (dt, J=14.5, 4.5 Hz, 1H), 2.99-2.79 (m, 2H), 2.60-2.45 (m, 2H), 2.36-2.22 (m, 1H), 2.22 (s, 6H), 1.78-1.65 (0, 1H), 1.24 (d, J=6.9 Hz, 6H); LCMS RT (Method 1)=4.688 min, m/z 455.2 [M+H+]; HRMS (ESI) m/z calcd for C25H28ClN2O4+[M+H+] 455.1738, found 455.1575.
The preparation of 6-chloro-N-(3-(dimethylamino)propyl)-3-(2-fluorobenzoyl)-4-oxo-4H-chromene-2-carboxamide was conducted according to the chemical reaction scheme as shown above. 1H NMR (400 MHz, CD2Cl2) δ 8.19 (td, J=8.1, 1.9 Hz, 1H), 8.10 (d, J=2.4 Hz, 1H), 7.74-7.63 (m, 2H), 7.34 (dtd, J=16.5, 7.5, 1.7 Hz, 2H), 6.97-6.88 (m, 1H), 3.74-3.62 (m, 1H), 2.80 (ddd, J=14.2, 10.3, 3.1 Hz, 1H), 2.63-2.54 (m, 2H), 2.35-2.22 (m, 7H), 1.78-1.65 (m, 1H); LCMS RT (Method 1)=4.108 min, n z 431.1 [M+H+]; HRMS (ESI) m/z calcd for C22H21ClFN2O4+ [M+H+] 431.1174, found 431.0986.
The preparation of 6-chloro-N-(3-(diethylamino)propyl)-3-(3,4-dimethoxybenzoyl)-4-oxo-4H-chromene-2-carboxamide was conducted according to the chemical reaction scheme as shown above. 1H NMR (400 MHz, CD2Cl2) δ 8.12 (d, J=2.5 Hz, 1H), 7.74-7.62 (m, 2H), 7.09 (s, 1H), 6.98 (d, J=8.9 Hz, 1H), 6.84 (d, J=8.4 Hz, 1H), 3.82 (d, J=2.9 Hz, 6H), 3.77-3.65 (m, 1H), 2.89-2.83 (m, 1H), 2.63-2.55 (m, 6H), 2.25 (bs, 1H), 1.81 (bs, 1H), 1.00-0.99 (m, 6H). 1:1 ratio of 2 conformational isomers; LCMS RT (Method 1)=4.092 min, m/z 501.2 [M+H+]; HRMS (ESI) m/z calcd for C26H30ClN2O6+ [M+H+] 501.1792, found 501.1613.
The preparation of 6-chloro-3-(4-chlorobenzoyl)-N-(3-(dimethylamino)propyl)-4-oxo-4H-chromene-2-carboxamide was conducted according to the chemical reaction scheme shown above. 1H NMR (400 MHz, CD2Cl2) δ 8.11 (m, 1H), 7.72-7.64 (m, 2H), 7.49-7.44 (m, 2H), 7.39-7.32 (m, 2H), 3.70 (dt, J=14.7, 4.7 Hz, 1H), 2.85 (ddd, J=14.1, 10.0, 3.1 Hz, 1H), 2.65-2.55 (m, 2H), 2.35-2.22 (m, 7H), 1.72-1.78 (m, 1H). 10:1 ratio. Major to minor. Only major NMR shown; LCMS RT (Method 1)=4.347 min, n z 447.1 [M+H+]; HRMS (ESI) m/z calcd for C22H21Cl2N2O4+ [M+H+] 447.0878, found 447.0700.
The preparation of 6-chloro-N-(3-(dimethylamino)propyl)-3-(4-fluorobenzoyl)-4-oxo-4H-chromene-2-carboxamide. was conducted according to the chemical reaction scheme as shown above. 1H NMR (400 MHz, CD2Cl2) δ 8.11 (d, J=2.4 Hz, 1H), 7.73-7.63 (m, 2H), 7.51 (dd, J=8.6, 5.3 Hz, 2H), 7.06 (t, J=8.6 Hz, 2H), 3.70 (dt, J=14.6, 4.6 Hz, 1H), 2.85 (ddd, J=14.2, 10.2, 3.2 Hz, 1H), 2.67-2.46 (m, 2H), 2.38-2.29 (m, 1H), 2.24 (s, 6H), 1.78-1.70 (m, 1H). 8:1 ratio. Major to minor. Only major NMR shown; LCMS RT (Method 1)=4.133 min, m/z 431.1 [M+H+]; HRMS (ESI) in z calcd for C22H21ClFN2O4+[M+H+] 431.1174, found 431.1045.
The preparation of 3-(4-(2-amino-2-oxoethoxy)benzoyl)-6-chloro-N-(3-(dimethylamino)propyl)-4-oxo-4H-chromene-2-carboxamide was conducted according to the chemical reaction scheme as shown above. 1H NMR (400 MHz, CD2Cl2) δ 8.11 (d, J=2.4 Hz, 1H), 7.73-7.62 (m, 2H), 7.47 (d, J=8.4 Hz, 2H), 6.94 (d, J=8.5 Hz, 2H), 6.49 (s, 1H), 5.58 (s, 1H), 4.48 (s, 2H), 3.68 (dt, J=14.6, 4.7 Hz, 1H), 2.95-2.73 (m, 1H), 2.62-2.55 (m, 2H), 2.39-2.27 (m, 1H), 2.24 (s, 6H), 1.75-1.73 (n, 1H). 3:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=3.463 min, m/z 486.2 [M+H+]; HRMS (ESI) m/z calcd for C24H25ClN3O6+ [M+H+] 486.1432, found 486.1247.
The preparation of 3-benzoyl-6-chloro-N-(3-(dimethylamino)propyl)-4-oxo-4H-chromene-2-carboxamide was conducted according to the chemical reaction scheme as shown above. 1H NMR (400 MHz, CD2Cl2) δ 8.11 (d, J=2.4 Hz, 1H), 7.73-7.64 (m, 2H), 7.54-7.48 (m, 2H), 7.42-7.31 (m, 3H), 3.68 (dt, J=14.5, 4.6 Hz, 1H), 2.84 (ddd, J=14.2, 10.4, 3.1 Hz, 1H), 2.60-2.43 (m, 2H), 2.38-2.25 (m, 1H), 2.22 (s, 6H), 1.75-1.62 (m, 1H). 10:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=3.976 min, m/z 413.1 [M+H+]; HRMS (ESI) m/z calcd for C22H22ClN2O4+ [M+H+] 413.1268, found 413.1115.
The preparation of 6-chloro-3-(3,4-dichlorobenzoyl)-N-(3-(dimethylamino)propyl)-4-oxo-4H-chromene-2-carboxamide was conducted according to the chemical reaction scheme as shown above. 1H NMR (400 MHz, CD2Cl2) δ 8.10 (d, J=2.5 Hz, 1H), 7.75-7.62 (m, 31H), 7.45 (d, J=8.5 Hz, 1H), 7.34 (dd, J=8.5, 2.2 Hz, 1H), 3.72 (ddd, J=14.6, 5.7, 3.5 Hz, 1H), 2.84 (ddd, J=14.0, 10.1, 3.0 Hz, 1H), 2.75-2.52 (m, 21H), 2.38-2.25 (m, 1H), 2.27 (s, 6H), 1.82-1.72 (m, 1H). 20:1 ratio. Major to minor. Only major NMR shown; LCMS RT (Method 1)=4.601 min, m/z 481.1 [M+H+]; HRMS (ESI) n z calcd for C22H20Cl3N2O4+ [M+H+] 481.0489, found 481.0309.
The preparation of 6-chloro-N-(3-(dimethylamino)propyl)-3-(4-ethoxy-3-methoxybenzoyl)-4-oxo-4H-chromene-2-carboxamide was conducted according to the chemical reaction scheme as shown above. 1H NMR (400 MHz, CD2Cl2) δ 8.12 (d, J=2.5 Hz, 1H), 7.73-7.63 (m, 2H), 7.08 (s, 1H), 6.96 (dd, J=7.8, 1.9 Hz, 1H), 6.83 (d, J=8.4 Hz, 1H), 4.04 (q, J=7.0 Hz, 2H), 3.84 (s, 3H), 3.68 (dt, J=14.4, 4.5 Hz, 1H), 2.88 (ddd, J=14.2, 10.4, 3.2 Hz, 1H), 2.60-2.48 (m, 2H), 2.37-2.25 (m, 1H), 2.21 (s, 6H), 1.75-1.65 (m, 1H), 1.41 (t, J=7.0 Hz, 3H). 2:1 ratio. Major to minor. Only major NMR shown; LCMS RT (Method 1)=4.159 min, m/z 487.1 [M+H+]; HRMS (ESI) m/z calcd for C25H28ClN2O6+ [M+H+] 487.1636, found 487.1545.
The preparation of 6-chloro-3-(3,4-dimethoxybenzoyl)-N-(3-(dimethylamino)propyl)-N-methyl-4-oxo-4H-chromene-2-carboxamide was conducted according to the chemical reaction scheme as shown above.
To a solution of ethyl 6-chloro-3-(3,4-dimethoxybenzoyl)-4-oxo-4H-chromene-2-carboxylate (20 mg, 0.048 mmol) in MeOH (3 mL) was added 1N NaOH (245 mL, 0.245 mmol). The reaction mixture was stirred for 1 hr at room temperature under N2. The reaction as quenched with 1N HCl (aq) and extracted with EtOAc. The combined organic layers were dried with MgSO4, filtered and rotovaped. The crude product was purified by column chromatography (hexanes/EtOAc) to yield the desired product as a white solid (7.5 mg, 40%). 1H NMR (400 MI-z, DMSO-d6) δ 8.00-7.93 (m, 21H), 7.88 (d, J=9.7 Hz, 1H), 7.52 (dd, J=8.3, 2.0 Hz, 1H), 7.47 (d, J=2.0 Hz, 1H), 6.97 (d, J=8.4 Hz, 1H), 3.83 (s, 3H), 3.81 (s, 3H). LCMS RT (Method 2)=2.780 min, m/z 389.0 [M+H+].
To a solution of 6-chloro-3-(3,4-dimethoxybenzoyl)-4-oxo-4H-chromene-2-carboxylic acid (16 mg, 0.041 mmol) in DCM (3 mL) was added 1 drop of DMF then oxalyl chloride (10.8 mL, 0.123 mmol). Reaction mixture was stirred at room temperature under N2. After 1 hr, the reaction mixture was rotovaped and dried under high vacuum. To this residue was added DCM (3 mL) then N1,N1,N3-trimethylpropane-1,3-diamine (12.2 mL, 0.083 mmol). Reaction was stirred at room temperature overnight under N2. Reaction was rotovaped and crude product was purified by column chromatography (DCM/MeOH) to yield the desired product as a brown oil (5.2 mg, 21%). 1H NMR (400 m/z, CD2Cl2) δ 8.17-8.16 (m, 1H), 7.78-7.75 (m, 1H), 7.63-7.59 (m, 1H), 7.47-7.40 (m, 2H), 6.92-6.83 (m, 1H), 3.97-3.87 (m, 6H), 3.59-3.51 (m, 2H), 3.13 (s, 3H), 2.99-2.96 (m, 2H), 2.84 (s, 1H, minor), 2.80 (s, 5H, major), 2.11-2.05 (m, 2H); LCMS RT (Method 1)=4.364 min, n z 487.1 [M+H+]; FIRMS (ESI) i/z calcd for C25H28ClN2O6+[M+H+] 487.1636, found 487.1571.
7-Chloro-2-(3-(dimethylamino) propyl)-1-(4-methoxyphenyl)-1,2-dihydrochromeno[2,3-c] pyrrole-3,9-dione (101.4 ng, 0.238 mmol) was dissolved in DMSO (10 ml) with heating. The clear DMSO solution was stirred with air bubbling at 70° C. for 12 hours. The DMSO was evaporated. The crude product was purified by HPLC (H2O/MeCN). Yield=20.82%. 1H NMR (400 MHz, CD2Cl2) δ 8.11 (d, J=2.4 Hz, 1H), 7.71-7.62 (m, 21H), 7.44-7.38 (m, 2H), 6.91-6.85 (m, 2H), 3.80 (d, J=0.9 Hz, 3H), 3.67 (dt, J=14.7, 4.7 Hz, 1H), 2.90-2.83 (m, 1H), 2.57 (s, 2H), 2.35-2.29 (m, 1H), 2.25 (s, 6H), 1.76 (s, 1H). 2:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=4.055 min, m/z=443.1 [M+H+]; HRMS (ESI) m/z calcd for C23H24ClN2O5+ [M+H+] 443.1374, found 443.1242.
7-Chloro-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-(3-(dimethylamino)propyl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione (180.7 mg, 0.397 mmol) was dissolved in DMSO. The DMSO solution was stirred with air-bubbling for 12 hr. The crude product was purified by HPLC (H2O/MeCN). Yield 18.33%. 1H NMR (400 MHz, CD2Cl2) δ 8.12 (d, J=2.5 Hz, 1H), 7.72-7.62 (m, 2H), 7.03 (d, J=2.2 Hz, 1H), 6.95-6.89 (m, 1H), 6.82 (d, J=8.5 Hz, 1H), 4.24 (s, 4H), 3.71-3.60 (m, 1H), 2.91-2.84 (m, 1H), 2.60 (m, 2H), 2.29 (s, 1H), 2.25 (s, 6H), 1.76 (d, J=10.0 Hz, 1H). 3:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=3.970 min, m/z=471.1 [M+H+]; HRMS (ESI) m/z calcd for C24H24ClN2O6+[M+H+] 471.1323, found 471.1169.
7-Chloro-1-(3,5-dimethoxyphenyl)-2-(3-(dimethylamino) propyl)-1,2-dihydrochromeno[2,3-c] pyrrole-3,9-dione (330 ng, 0.722 mmol) was dissolved in DMSO (10 ml). The solution was stirred overnight with air bubbling. The crude product was purified by HPLC (H2O/MeCN). Yield=6.70%. 1H NMR (400 MHz, CD2Cl2) δ 8.13 (d, J=2.5 Hz, 1H), 7.74-7.63 (m, 21H), 6.65 (d, J=2.3 Hz, 2H), 6.42 (t, J=2.3 Hz, 1H), 3.77 (s, 6H), 3.68 (ddd, J=14.7, 5.7, 3.7 Hz, 1H), 2.88 (ddd, J=14.0, 10.3, 3.2 Hz, 1H), 2.57 (dd, J=9.0, 3.9 Hz, 2H), 2.38-2.26 (m, 1H), 2.23 (s, 6H), 1.72 (d, J=15.4 Hz, 1H). LCMS RT (Method 1)=4.058 min, m/z=473.14 [M+H+]; HRMS (ESI) m/z calcd for C24H26ClN2O6+ 473.1479, found 473.1407.
7-Chloro-1-(3,4-difluorophenyl)-2-(3-(dimethylamino) propyl)-1,2-dihydrochromeno[2,3-c] pyrrole-3,9-dione (190 mg, 0.439 mmol) was dissolved in 10 mL DMSO with heating. The DMSO solution was stirred with air-bubbling until the starting compound was consumed completely. The crude product was purified by HPLC (H2O/MeCN). Yield=29.9%. 1H NMR (400 MHz, CD2Cl2) δ 8.12 (d, J=2.5 Hz, 1H), 7.74-7.63 (m, 2H), 7.47 (ddd, J=11.6, 7.6, 2.1 Hz, 1H), 7.22-7.10 (m, 2H), 3.71 (ddd, J=14.6, 5.6, 3.6 Hz, 1H), 2.83 (ddd, J=14.2, 10.5, 3.1 Hz, 1H), 2.65-2.46 (m, 2H), 2.35 (dddd, J=15.8, 9.4, 6.7, 3.7 Hz, 1H), 2.22 (s, 6H), 1.77-1.66 (m, 1H). LCMS RT (Method 1)=4.172 min, m/z=449.10 [M+H+]; HRMS (ESI) m/z calcd for C22H20ClF2NO4+[M+H+] 449.1080, found 449.0957.
1-(3-Bromo-4-methoxyphenyl)-7-chloro-2-(3-(dimethylamino)propyl)-1,2-dihydrochromeno[2,3-c] pyrrole-3,9-dione (106.1 mg, 0.210 mmol) was dissolved in DMSO (10 ml) with heating. The solution was stirred at 70° C. for 12 hr with air bubbling. The crude product was purified by HPLC (H2O/MeCN). Yield=16.90%. 1H NMR (400 MHz, CD2Cl2) δ 8.12 (d, J=2.5 Hz, 1H), 7.73-7.64 (m, 31), 7.46 (dd, J=8.7, 2.4 Hz, 1H), 6.92 (d, J=8.1 Hz, 1H), 3.88 (d, J=0.8 Hz, 3H), 3.70 (dt, J=14.0, 4.5 Hz, 1H), 2.86 (ddd, J=13.7, 10.5, 2.9 Hz, 1H), 2.63-2.55 (m, 2H), 2.38-2.29 (m, 1H), 2.23 (s, 6H), 1.72 (d, J=15.4 Hz, 1H). 5:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=4.248 min, m z=521.10 [M+H+]; HRMS (ESI) m/z calcd for C23H23BrClN2O5+ [M+H+] 523.0458, found 523.0346.
7-Chloro-2-(3-(dimethylamino)propyl)-1-(3-fluoro-4,5-dimethoxyphenyl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione (308 mg, 0.649 mmol) was dissolved in DMSO (10 ml). The DMSO solution was stirred overnight at 70° C. with air-bubbling. The crude product was purified by HPLC (H2O/MeCN). Yield=15.11%. 1H NMR (400 Hz, CD2Cl2) δ 8.13 (dd, J=2.5, 0.5 Hz, 1H), 7.74-7.64 (m, 2H), 6.94 (t, J=1.9 Hz, 1H), 6.82 (dd, J=11.4, 2.1 Hz, 1H), 3.89-3.85 (m, 6H), 3.70 (ddd, J=14.4, 5.4, 3.6 Hz, 1H), 2.86 (ddd, J=14.6, 10.5, 3.2 Hz, 1H), 2.63-2.50 (m, 21H), 2.39-2.27 (m, 1H), 2.22 (s, 61H), 1.77-1.68 (m, 1H). LCMS RT (Method 1)=4.060 min, m/z=491.1 [1M+H+]; HRMS (ESI) m/z calcd for C24H25ClFN2O6+ [M+H+] 491.1380, found 491.1256.
7-Chloro-2-(3-(dimethylamino)propyl)-1-(3-hydroxy-4-methoxyphenyl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione (36 mg, 0.081 mmol) was dissolved in DMSO (10 ml). The solution was stirred at room temperature with air bubbling for 2 days. The crude product was purified by HPLC (H2O/MeCN). Yield=19.57%. 1H NMR (400 Hz, CD2Cl2) δ 8.11 (d, J=2.5 Hz, 1H), 7.72-7.62 (m, 2H), 7.08 (dd, J=8.4, 2.3 Hz, 1H), 6.94 (d, J=2.3 Hz, 1H), 6.87 (d, J=8.4 Hz, 1H), 3.88 (s, 3H), 3.65 (dt, J=14.5, 4.6 Hz, 1H), 2.85 (ddd, J=14.3, 10.6, 3.3 Hz, 1H), 2.57-2.46 (m, 2H), 2.36-2.27 (m, 1H), 2.20 (s, 6H), 1.69 (ddd, J=12.3, 6.1, 3.1 Hz, 1H). 3:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=3.433 min, m/z=459.1 [M+H+]; HRMS (ESI) m/z calcd for C23H24ClN2O6+ [M+H+] 459.1323, found 459.1169.
5-Chloro-1-(3,4-dimethoxyphenyl)-2-(3-(dimethylamino)propyl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione (201.3 mg, 0.441 mmol) was dissolved in DMSO (10 ml). The DMSO solution was stirred for 12 hours at 70° C. with air bubbling. The crude product was purified by HPLC (H2O/MeCN). Yield=11.33%. 1H NMR (400 MHz, CD2Cl2) δ 8.07 (dt, J=8.1, 1.6 Hz, 1H), 7.82 (dt, J=7.8, 1.6 Hz, 1H), 7.40 (td, J=7.9, 1.4 Hz, 1H), 7.08 (d, J=1.9 Hz, 1H), 7.03-6.98 (m, 1H), 6.85 (dt, J=8.5, 1.6 Hz, 1H), 3.83 (dd, J=4.2, 1.4 Hz, 6H), 3.75-3.68 (m, 1H), 2.93 (t, J=11.5 Hz, 1H), 2.68 (s, 2H), 2.36 (m, 1H), 2.33 (s, 6H), 1.87 (s, 1H). 3:2 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=3.728 min, m/z=473.2 [M+H+]; HRMS (ESI) m/z calcd for C24H25ClN2O6+ [M+H+] 473.1479, found 473.1379.
1-(Benzo[d][1,3]dioxol-5-yl)-7-chloro-2-(3-(dimethylamino)propyl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione (259 mg, 0.587 mmol) was dissolved in DMSO (10 ml). The DMSO solution was stirred overnight at 70° C. The crude product was purified by HPLC (H2O/MeCN). Yield=8.16%. 1H NMR (400 MHz, CD2Cl2) δ 8.12 (d, J=2.5 Hz, 1H), 7.72-7.64 (m, 2H), 7.39 (d, J=6.7 Hz, 1H), 6.98 (d, J=1.9 Hz, 1H), 6.79 (d, J=8.1 Hz, 1H), 5.98-5.95 (m, 2H), 3.67 (ddd, J=14.3, 5.5, 3.6 Hz, 1H), 2.91-2.82 (m, 1H), 2.61-2.51 (m, 2H), 2.36-2.26 (m, 1H), 2.22 (s, 6H), 1.78-1.68 (m, 1H). 3:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=3.910 min, m/z=457.2 [M+H+]; HRMS (ESI) m/z calcd for C23H22ClN2O6+ [M+H+] 457.1166, found 457.1037.
1-(3,4-Dimethoxyphenyl)-2-(3-(dimethylamino) propyl)-7-fluoro-1,2-dihydrochromeno[2,3-c] pyrrole-3,9-dione (299 mg, 0.679 mmol) was dissolved in DMSO (10 ml). The DMSO solution was stirred overnight at 70° C. with air bubbling. The crude product was purified by HPLC (H2O/MeCN). Yield=3.68%. 1H NMR (400 MHz, CD2Cl2) δ 7.80 (dd, J=8.4, 3.3 Hz, 1H), 771 (dd, J=9.3, 4.2 Hz, 1H), 7.48 (ddd, J=8.9, 7.3, 3.0 Hz, 1H), 7.10 (d, J=2.2 Hz, 1H), 6.99 (dd, J=8.6, 2.1 Hz, 1H), 6.84 (d, J=8.4 Hz, 1H), 3.83 (d, J=5.7 Hz, 6H), 3.69 (dt, J=14.3, 4.6 Hz, 1H), 2.93 (d, J=12.1 Hz, 1H), 2.64 (m, 2H), 2.32 (m, 1H), 2.29 (s, 6H), 1.79 (s, 1H). 1:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=3.621 min, m/z=457.10 [M+H+]; HRMS (ESI) m/z calcd for C24H26FN2O6+[M+H+] 457.1775, found 457.1688.
7-(tert-Butyl)-1-(3,4-dimethoxyphenyl)-2-(3-(dimethylamino) propyl)-1,2-dihydrochromeno[2,3-c] pyrrole-3,9-dione (175.8 mg, 0.367 mmol) was dissolved in DMSO (10 ml). The solution was stirred at RT for 12 hours with air-bubbling. The crude product was purified by HPLC (H2O/MeCN). Yield=10.79%. 1H NMR (400 MHz, CD2Cl2) δ 8.17-8.13 (m, 1H), 7.62-7.54 (m, 2H), 7.13-7.08 (m, 1H), 6.98 (dt, J=8.4, 1.9 Hz, 1H), 6.83 (dt, J=8.5, 1.6 Hz, 1H), 3.83 (dd, J=8.1, 1.6 Hz, 6H), 3.69 (dt, J=14.9, 4.5 Hz, 1H), 2.90 (t, J=11.2 Hz, 1H), 2.53 (m, 21H), 2.33 (s, 1H), 2.23 (s, 61H), 1.71 (s, 1H), 1.37 (d, J=1.7 Hz, 9H). 1:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=4.332 min, m/z=495.2 [M+H+]; HRMS (ESI) m/z calcd for C28H35N2O6+ [M+H+] 495.2495, found 495.2406.
7-Bromo-1-(3,4-dimethoxyphenyl)-2-(3-(dimethylamino)propyl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione (265.4 mg, 0.529 mmol) was dissolved in DMSO (10 ml). The solution was stirred at 50° C. overnight with air-bubbling. The crude product was purified by HPLC (H2O/MeCN). Yield=2.85%. 1H NMR (400 Mz, CD2Cl2) δ 8.27 (d, J=2.5 Hz, 1H), 7.83 (dd, J=8.9, 2.5 Hz, 1H), 7.08 (d, J=2.3 Hz, 1H), 6.98 (dd, J=8.4, 2.2 Hz, 1H), 6.84 (d, J=8.3 Hz, 2H), 3.83 (d, J=4.7 Hz, 6H), 3.68 (dt, J=14.7, 4.2 Hz, 1H), 2.94-2.83 (m, 1H), 2.58 (s, 2H), 2.32 (s, 1H), 2.25 (s, 6H), 1.74 (s, 1H). 3:2 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)==3.933 min, m/z=517.1 [M+H+]; HRMS (ESI) m/z calcd for C24H26BrN2O6+[M+H+] 517.0974, found 519.0835.
1-(3,4-Dimethoxyphenyl)-2-(3-(dimethylamino) propyl)-1,2-dihydrochromeno[2,3-c] pyrrole-3,9-dione (305 mg, 0.722 mmol) was dissolved in DMSO (10 ml) with heating. Then the solution was stirred for 16 hr with air-bubbling at 70° C. The crude product was purified by HPLC (H2O/MeCN). Yield=15.86%. 1H NMR (400 MHz, CD2C2) δ 8.16 (dd, J=8.0, 1.7 Hz, 1H), 7.75 (ddd, J=8.6, 7.0, 1.8 Hz, TH), 7.68 (d, J=7.5 Hz, 1H), 7.49-7.42 (m, 1H), 7.10 (d, J=2.2 Hz, 1H), 6.99 (dd, J=8.4, 2.2 Hz, 1H), 6.84 (d, J=8.4 Hz, 1H), 3.83 (d, J=5.7 Hz, 6H), 3.73-3.63 (m, 1H), 2.89 (ddd, J=13.8, 10.1, 3.1 Hz, 1H), 2.58 (dd, J=8.8, 4.3 Hz, 2H), 2.37-2.28 (m, 1H), 2.24 (s, 6H), 1.73 (d, J=15.1 Hz, 1H). 3:2 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1) 3.406 min, m/z=439.2 [M+H+]; HRMS (ESI) m/z calcd for C24H27N2O6+ [M+H+] 439.1869, found 439.1769.
1-(3,4-dimethoxyphenyl)-2-(3-(dimethylamino) propyl)-7-ethyl-1,2-dihydrochromeno[2,3-c] pyrrole-3,9-dione (125 mg, 0.277 mmol) was dissolved in DMSO (10 ml) and stirred with air bubbling for 12 hours. The product was purified by HPLC (H2O/MeCN) Yield=18.16%. 1H NMR (400 MHz, CD2Cl2) δ 7.98-7.95 (m, 1H), 7.61-7.57 (m, 2H), 7.10 (d, J=2.2 Hz, 1H), 6.98 (dd, J=8.4, 2.2 Hz, 1H), 6.83 (d, J=8.4 Hz, 1H), 3.82 (d, J=6.2 Hz, 6H), 3.72-3.63 (m, 1H), 2.89 (ddd, J=13.5, 10.2, 3.1 Hz, 1H), 2.78 (dd, J=15.6, 7.8 Hz, 2H), 2.60-2.52 (m, 2H), 2.36-2.27 (m, 1H), 2.22 (s, 6H), 1.72 (d, J=14.6 Hz, 1H), 1.28 (dt, J=16.1, 7.6 Hz, 3H). 3:2 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=3.980 mi, m/z=467.2 [M+H+]; HRMS (ESI) m/z calcd for C26H31N2O6+ [M+H+] 467.2182, found 467.2078.
8-Chloro-1-(3,4-dimethoxyphenyl)-2-(3-(dimethylamino)propyl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione (308 mg, 0.674 mmol) was dissolved in DMSO (10 ml). The solution was stirred overnight with air-bubbling. The crude product was purified by HPLC (H2O/MeCN). Yield=1.456%. 1H NMR (400 MHz, CD2Cl2) δ 7.66 (d, J=6.7 Hz, 1H), 7.62-7.56 (m, 2H), 7.06 (d, J=2.3 Hz, 1H), 7.04-6.98 (m, 1H), 6.85 (d, J=8.5 Hz, 2H), 3.83 (d, J=3.3 Hz, 6H), 3.66 (dt, J=14.4, 4.7 Hz, 1H), 2.92-2.82 (m, 1H), 2.55 (s, 2H), 2.35-2.27 (m, 1H), 2.24 (s, 6H), 1.71 (s, 1H). 1:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=3.683 min, m/z=473.1 [M+H+]; HRMS (ESI) m/z calcd for C24H26ClN2O6+ [M+H+] 473.1479, found 473.1398.
1-(3,4-Dimethoxyphenyl)-2-(3-(dimethylamino) propyl)-7-(trifluoromethyl)-1,2-dihydrochromeno[2,3-c] pyrrole-3,9-dione (219 mg, 0.447 mmol) was dissolved in DMSO (10 ml) with air bubbling for 12 hours. The crude product was purified by HPLC (H2O/MeCN). Yield=11.36%. 1H NMR (400 MHz, CD2Cl2) δ 8.46 (d, J=2.3 Hz, 1H), 7.97 (dd, J=8.8, 2.4 Hz, 1H), 7.83 (d, J=8.8 Hz, 1H), 7.10 (d, J=2.2 Hz, 1H), 6.99 (dd, J=8.4, 2.2 Hz, 1H), 6.84 (d, J=8.4 Hz, 1H), 3.83 (d, J=7.1 Hz, 6H), 3.71 (ddd, J=14.5, 7.5, 3.7 Hz, 1H), 2.88 (ddd, J=14.3, 10.7, 3.3 Hz, 1H), 2.56 (td, J=9.1, 4.5 Hz, 2H), 2.38-2.31 (m, 1H), 2.20 (s, 611), 1.72-1.67 (m, 1H). 3:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=4.039 min, m/z=507.1 [M+H+]; HRMS (ESI) m/z calcd for C25H26F3N2O6+ [M+H+] 507.1743, found 507.1604.
7-Chloro-2-(3-(dimethylamino) propyl)-1-(5-m-ethoxypyridin-3-yl)-1,2-dihydrochromeno[2,3-c] pyrrole-3,9-dione (338 mg, 0.790 mmol) was dissolved in DMSO (10 ml). The DMSO solution was stirred overnight at 70° C. The crude product was purified by HPLC (1120/MeCN). Yield=5.36%. 1H NMR (400 MHz, CD2Cl2) δ 8.23 (d, J=3.0 Hz, 1H), 8.18 (d, J=1.7 Hz, 1H), 8.11 (d, J=2.6 Hz, 1H), 7.73-7.62 (m, 2H), 7.55-7.51 (m, 1H), 3.89 (s, 3H), 3.76 (dt, J=14.6, 4.5 Hz, 1H), 2.90 (ddd, J=14.3, 10.2, 3.3 Hz, 1H), 2.68-2.50 (m, 2H), 2.34 (td, J=9.6, 5.2 Hz, 1H), 2.25 (s, 6H), 1.80-1.71 (m, 1H). LCMS RT (Method 1) 3.266 min, m/z=444.1 [M+H+]; HRMS (ESI) m/z calcd for C22H23ClN3O6+ [M+H+] 444.1326, found 444.1193.
7-Chloro-1-(5,6-dimethoxypyridin-3-yl)-2-(3-(dimethylamino)propyl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione (306 mg, 0.668 mmol) was dissolved in DMSO (10 ml). The DMSO solution was stirred overnight at 70° C. with air bubbling. The crude product was purified by HPLC (H2O/MeCN). Yield=11.34%. 1H NMR (400 MHz, CD2Cl2) δ 8.99 (s, 1H), 8.12 (d, J=2.5 Hz, 1H), 8.10 (1, J=2.2 Hz, 1H), 8.04 (d, J=2.0 Hz, 1H), 7.76 (dd, J=8.8, 2.4 Hz, 2H), 4.00 (s, 3H), 3.84 (s, 3H), 3.51-3.46 (m, 2H), 3.08 (t, J=6.8 Hz, 2H), 2.77 (s, 6H), 2.09 (q, J=6.2 Hz, 2H). 1:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=5.292 min, m/z=474.1 [M+H+]; HRMS (ESI) n/z calcd for C23H25ClN3O6+ [M+H+] 474.1432, found 474.1314.
5-(7-Chloro-2-(3-(dimethylamino) propyl)-3,9-dioxo-1,2,3,9-tetrahydrochromeno[2,3-c] pyrrol-1-yl)-2-methoxybenzonitrile (280 mg, 0.620 mmol)] was dissolved in DMSO (10 ml). The DMSO solution was stirred for 16 hours with air-bubbling. Yield=1.483%. 1H NMR (400 MHz, CD2Cl2) δ 8.10 (d, J=2.5 Hz, 1H), 7.76-7.60 (m, 4H), 7.00 (d, J=8.9 Hz, 1H), 3.94 (s, 3H), 3.71 (ddd, J=14.5, 5.7, 3.8 Hz, 1H), 2.82 (ddd, J=14.3, 10.3, 3.3 Hz, 1H), 2.57-2.47 (m, 2H), 2.38-2.28 (m, 1H), 2.24 (s, 6H), 1.73 (d, J=15.4 Hz, 1H). LCMS RT (Method 1)=4.026 min, m/z=468.1 [M+H+]; HRMS (ESI) m/z calcd for C24H23ClN3O5+ [M+H+] 468.1326, found 468.1072.
6-Chloro-1-(3,4-dimethoxyphenyl)-2-(3-(dimethylamino) propyl)-1,2-dihydrochromeno[2,3-c] pyrrole-3,9-dione (220 mg, 0.481 mmol) was dissolved in DMSO with heating. Then the solution was stirred for 16 hours with air-bubbling at 70° C. The crude product was purified by HPLC (H2O/MeCN). Yield=7.38%. 1H NMR (400 MHz, CD2Cl2) a 8.10 (d, J=8.6 Hz, 1H), 7.70 (d, J=1.7 Hz, 1H), 7.43 (dd, J=8.6, 1.8 Hz, 1H), 7.08 (d, J=1.9 Hz, 1H), 6.99 (dd, J=8.4, 2.0 Hz, 1H), 6.84 (d, J=8.4 Hz, 1H), 3.83 (d, J=5.5 Hz, 6H), 3.68 (dt, J=14.7, 4.7 Hz, 1H), 2.87 (ddd, J=14.5, 10.5, 3.6 Hz, 1H), 2.60-2.54 (m, 2H), 2.31 (tt, J=7.6, 4.2 Hz, 1H), 2.19 (s, 6H), 1.71-1.64 (m, 1H). 2:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=3.751 min, m/z=473.1 [M+H+]; HRMS (ESI) m/z calcd for C24H26ClN2O6+ [M+H+] 473.1479, found 473.1078.
6,7-Dichloro-1-(3,4-dimethoxyphenyl)-2-(3-(dimethylamino) propyl)-1,2-dihydrochromeno[2,3-c] pyrrole-3,9-dione (172.5 mg, 0.351 mmol) was dissolved in DMSO with heating. Then the solution was stirred for 16 hr with air-bubbling at 70° C. The crude product was purified by HPLC (H2O/MeCN). Yield=23.47%. 1H NMR (400 MHz, CD2Cl2) δ 8.23 (s, 1H), 7.84 (s, 1H), 7.08 (d, J=1.9 Hz, 1H), 6.98 (dd, J=8.5, 2.0 Hz, 1H), 6.84 (d, J=8.4 Hz, 1H), 3.83 (d, J=4.9 Hz, 6H), 3.68 (dt, J=14.3, 4.6 Hz, 1H), 2.93-2.82 (m, 1H), 2.56 (q, J=5.9 Hz, 2H), 2.30 (d, J=10.0 Hz, 1H), 2.23 (s, 6H), 1.73 (d, J=15.6 Hz, 1H). 2:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=4.159 min, m/z=507.1 [M+H+]; HRMS (ESI) m/z calcd for C24H25Cl2N2O6+ [M+H+] 507.1090, found 507.0685.
7-Chloro-1-(3,4-dihydroxyphenyl)-2-(3-(dimethylamino)propyl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione (141 mg. 0.329 mmol) was dissolved in DMSO (10 ml). The solution was stirred for 12 hours with air bubbling. The crude product was purified by normal phase column (20˜50% MeOH in EA). Yield=6.91%. 1H NMR (400 MHz, Acetic acid-d4) δ 8.16 (t, J=2.4 Hz, 1H), 7.82 (ddd, J=11.8, 8.9, 2.7 Hz, 2H), 7.50 (d, J=2.2 Hz, 1H), 7.38 (dd, J=8.4, 2.1 Hz, 1H), 6.91 (dd, J=8.3, 2.3 Hz, 1H), 3.48 (q, J=6.1 Hz, 2H), 3.12 (q, J=6.7 Hz, 2H), 2.81 (s, 6H), 2.75 (d, J=9.5 Hz, 2H). 2:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=3.406 min, m/z=445.1 [M+H+]; HRMS (ESI) m/z calcd for C22H22ClN2O6+[M+H+] 445.1166, found 445.0875.
To the solution of 6-chloro-3-(3,4-dimethoxybenzoyl)-4-oxo-4H-chromene-2-carboxylic acid (10 mg, 0.026 mmol) and N1,N1-dimethylpropane-1,3-diamine (4.21 μl, 0.033 mmol) in acetonitrile (2 ml) was added 1-methyl-1H-imidazole (7.18 μl, 0.090 mmol), followed by N-(chloro(dimethylamino)methylene)-N-methylmethananinium hexafluorophosphate(V) (8.30 mg, 0.030 mmol). The reaction mixture was stirred at room temperature overnight. The acetonitrile solution was dried up and the residue was dissolved in ethylacetate, followed by addition of 3 mL of water. The separated organic phase was dried with sodium sulfate to obtain the crude product.
To the solution of pyrimidin-5-ylmethanamine (10 mg, 0.092 mmol) and 6-chloro-3-(3,4-dimethoxybenzol)-4-oxo-4H-chromene-2-carboxylic acid (27.4 mg, 0.070 mmol) in acetonitrile was added 1-methyl-1H-imidazole (19.67 μl, 0.247 mmol), followed by N-(chloro(dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V) (22.74 mg, 0.081 mmol). The reaction mixture was stirred overnight at room temperature. The crude product was purified by silica column (50-100% EA in Hex). Yield=16.20%. 1H NMR (400 MHz, Acetic acid-d4) δ 9.11 (s, 1H), 8.77 (s, 2H), 7.71 (d, J=2.4 Hz, 1H), 7.51 (dd, J=8.8, 2.5 Hz, 1H), 7.23 (d, J=9.0 Hz, 1H), 7.02 (d, J=8.7 Hz, 1H), 6.87 (s, 1H), 6.75 (d, J=8.1 Hz, 1H), 4.86-4.50 (m, 2H), 3.81-3.62 (m, 6H). LCMS RT (Method 1)=4.238 min, m/z=480.1 [M+H+]; HRMS (ESI) m/z calcd for C24H19ClN3O6[M+H+] 480.0962, found 480.0645.
4-(Aminomethyl)pyridine (3.41 μl, 0.033 mmol) and 6-chloro-3-(3,4-dimethoxybenzoyl)-4-oxo-4H-chromene-2-carboxylic acid (10 mg, 0.026 mmol) were dissolved in acetonitrile (3 ml). To the solution was added 1-methyl-1H-imidazole (7.18 μl, 0.090 mmol), followed by the addition of N-(chloro(dimethylamino)methylene)-N-methylmethananinium hexafluorophosphate(V) (8.30 mg, 0.030 mmol). The reaction mixture was stirred overnight at room temperature. The crude product was purified by normal phase column (50-100% EA in Hex). Yield=44.6%. 1H NMR (400 MHz, CD2Cl2) δ 8.52 (d, J=5.6 Hz, 2H), 8.14 (d, J=2.7 Hz, 1H), 7.77 (ddd, J=9.1, 2.7, 1.3 Hz, 11), 7.60-7.54 (m, 2H), 7.31 (dt, J=8.4, 1.7 Hz, 1H), 7.22 (d, J=5.8 Hz, 2H), 6.83 (t, =8.6 Hz, 1H), 4.57 (d, J=6.2 Hz, 2H), 3.93-3.88 (m, 6H). 3:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=3.895 min, m/z=479.1 [M+H+]; HRMS (ESI) m/z calcd for C25H20ClN2O6+ [M+H+] 479.1010, found 479.0705.
To the solution of N1-methyl-N1-phenylpropane-1,3-diamine (5.46 μl, 0.033 mmol) and 6-chloro-3-(3,4-dimethoxybenzoyl)-4-oxo-6,7-dihydro-4H-chromene-2-carboxylic acid (10 mg, 0.026 mmol) in MeCN(3 mL) was added 1-methyl-1H-imidazole (7.14 μl, 0.090 mmol) and N-(chloro(dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V) (8.26 mg, 0.029 mmol) sequentially. The reaction mixture was stirred overnight at room temperature. The crude product was purified by normal phase column (20-50% EA in Hex). Yield=40.7%. 1H NMR (400 MHz, CD2Cl2) δ 8.12 (t, J=2.6 Hz, 1H), 7.77-7.65 (m, 2H), 7.57 (t, J=2.1 Hz, 1H), 7.30-7.21 (m, 3H), 6.87-6.76 (m, 4H), 3.90 (dd, J=9.4, 2.3 Hz, 6H), 3.51-3.44 (m, 2H), 3.41 (td, J=6.7, 2.2 Hz, 2H), 2.91 (d, J=2.3 Hz, 3H), 1.91 (td, J=6.8, 2.3 Hz, 2H). 5:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=4.934 and 5.134 min, m/z=535.1 [M+H+]; HRMS (ESI) m/z calcd for C29H28ClN2O6+ [M+H+] 535.1636, found 535.1654.
To the solution of 6-chloro-3-(1-(3,4-dimethoxyphenyl)ethyl)-4-hydroxy-4H-chromene-2-carboxylic acid (10 mg, 0.026 mmol) in MeCN (3 ml) was added pyridin-3-ylmethanamine (3.39 μl, 0.033 mmol), 1-methyl-1H-imidazole (7.14 μl, 0.090 mmol), and N-(chloro(dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V) (8.26 mg, 0.029 mmol) sequentially. The reaction mixture was stirred overnight at room temperature. The crude product was purified by normal phase column (50-100% EA in Hex). Yield=55.5%. 1H NMR (400 MHz, CD2Cl2) δ 8.55-8.51 (m, 1H), 8.49 (dd, J=5.3, 1.3 Hz, 1H), 8.13 (d, J=2.5 Hz, 1H), 7.75 (dd, J=9.0, 2.6 Hz, 1H), 7.68-7.64 (m, 1H), 7.58 (d, J=2.1 Hz, 1H), 7.55 (d, J=9.0 Hz, 1H), 7.31 (dd, J=8.3, 2.1 Hz, 1H), 7.28 (dd, J=7.9, 4.7 Hz, 1H), 6.85 (dd, J=8.5, 1.1 Hz, 1H), 4.56 (d, J=6.1 Hz, 2H), 3.91 (d, J=4.6 Hz, 6H). 2:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=2.756 min, m/z=479.1 [M+H+]; HRMS (ESI) m/z calcd for C25H20ClN2O6+[M+H+] 479.1010, found 479.1004.
To the solution of 6-chloro-3-(3,4-dimethoxybenzoyl)-4-oxo-4H-chromene-2-carboxylic acid (10 mg, 0.026 mmol) in MeCN (3 ml) was added (1-methyl-1H-imidazol-5-yl)methanamine (3.10 μl, 0.033 mmol), 1-methyl-1H-imidazole (7.18 μl, 0.090 mmol), and N-(chloro(dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V) (8.30 mg, 0.030 mmol) sequentially. The reaction mixture was stirred overnight at room temperature. The crude product was purified by normal phase column (50 to 100% EA in Hex and 0 to 30% MeOH in EA). Yield=20.98%. 1H NMR (400 MHz, CD2Cl2) a 8.14 (d, J=3.8 Hz, 1H), 7.74 (dd, J=9.2, 3.5 Hz, 1H), 7.63-7.53 (m, 3H), 7.31 (dt, J=8.3, 1.6 Hz, 1H), 6.92-6.82 (m, 2H), 4.54 (d, J=5.7 Hz, 2H), 3.91 (dd, J=6.5, 1.7 Hz, 6H), 3.51 (s, 3H). 2:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=3.898 min, m/z=482.1 [M+H+]; HRMS (ESI) m/z calcd for C24H21ClN3O6+ [M+H+] 482.1119, found 482.1127.
To the solution of 6-chloro-3-(3,4-dimethoxybenzoyl)-4-oxo-4H-chromene-2-carboxylic acid (10 mg, 0.026 mmol) in MeCN (3 ml) was added (1-methyl-1H-imidazol-4-yl)methanamine (3.20 μl, 0.033 mmol), 1-methyl-TH-imidazole (7.18 μl, 0.090 mmol), and N-(chloro(dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V) (8.30 mg, 0.030 mmol) sequentially. The reaction mixture was stirred overnight at room temperature. The crude product was purified by normal phase column (50 to 100% EA in Hex and 0 to 20% MeOH in EA). Yield=23.40%. 1H NMR (400 MHz, CD2Cl2) δ 8.13 (dd, J=2.6, 1.4 Hz, 1H), 7.79-7.66 (m, 2H), 7.64 (dd, J=8.9, 1.2 Hz, 1H), 7.48 (s, 1H), 7.40 (s, 1H), 6.94-6.88 (m, 2H), 4.42 (d, J=6.1 Hz, 2H), 3.85-3.79 (m, 6H), 3.69 (d, J=1.3 Hz, 3H). 1:1 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=3.863 min, m/z=482.1 [M+H+]; HRMS (ESI) n/z calcd for C24H21ClN3O6+[M+H+] 482.1119, found 482.1122.
To the solution of 6-chloro-3-(3,4-dimethoxybenzoyl)-4-oxo-4H-chromene-2-carboxylic acid (20 mg, 0.051 mmol) in DCM (4 mL) under ice-bath was added oxalyl dichloride (9.79 mg, 0.077 mmol) and two drops of DMF with stirring for one hour, then continued stirring for three hours at room temperature. The DCM in the reaction mixture was removed by flushing with nitrogen gas. The residue was dissolved in DCM (2 ml). To the solution was added triethylamine (35.9 μl, 0.257 mmol), pyridazin-4-ylmethanamine (7.30 mg, 0.067 mmol) in DMF (2.0 ml). The reaction mixture was stirred overnight. The crude product was purified by normal phase column (50 to 100% EA in Hex and 0 to 20% MeOH in EA). Yield=8.1%. 1H NMR (400 MHz, CD2Cl2) δ 9.09 (d, J=5.4 Hz, 2H), 8.18-8.14 (i, 1H), 7.78 (dd, J=8.9, 2.6 Hz, 1H), 7.72 (dd, J=3.7, 1.6 Hz, 1H), 7.59 (s, 1H), 7.40-7.32 (m, 2H), 6.87 (d, J=9.2 Hz, 1H), 4.60 (d, J=6.4 Hz, 2H), 3.91 (dd, J=3.6, 1.4 Hz, 6H). 3:2 ratio. Major to minor. Only major NMR shown. LCMS RT (Method 1)=2.604 min, m/z=480.1 [M+H+]; HRMIS (ESI) m/z calcd for C24H19ClN3O6+[M+H+ ] 480.0962, found 480.0972.
Initial SAR screening suggested that the N8279 precursors N1965 and N6164 may exhibit Gαq/11 bias (
The iCa2+ evoked by EC50 N8279 (
The effect of N8279 on ghrelin-induced iCa2+ signaling was next evaluated to test for ago-allosteric activity. Parenthetically, ago-allosteric agonists interacted with topographically-distinct receptor sites (allosteric) from the endogenous ligand (orthosteric), elicit agonist behavior on their own, and cooperatively act as a positive (PAM), negative (NAM), or silent (SAM) modulator of orthosteric ligand affinity, potency, and/or efficacy. N8279 displayed intrinsic GHSR1a agonism on its own (
To model how N8279 could co-occupy the monomeric GHSR1a with ghrelin, molecular docking was employed with an NMR-based homology model of the ghrelin-bound GHSR1a. Concomitant N8279 docking to ghrelin (1-17)-bound GHSR1a suggested that the propylamine moiety of N8279 could form a strong, ionic bond with a negatively charged ECL2 (Asp191) (
To evaluate the effect of N8279 on Gα proximal to the GHSR1a, parallel NanoBiT- and bioluminescence resonance energy transfer (BRET, TRUPATH)-based heterotrimeric G protein dissociation approaches were employed. In both assays, N8279 was a full agonist for Gαq activation with a potency comparable to that of iCa2+ (
In an independent set of experiments, N8279 signaling was evaluated through other G proteins to compare to Gαq. Gα subunits that are both expressed highly in midbrain DAergic neurons and reported to exhibit GHSR1a coupling, including GαsS (Gαs), Gαi1, Gαi2, GαoA, Gα12, and Gα13 were selectively tested. Relative to Gαq, ghrelin potency was statistically equivalent for each Gαi/o and was reduced moderately for Gα12 and Gα13 (
Together, the data in
Having established that N8279 was a potent activator Gαq signaling, its effect on GHSR1a-mediated β-arr recruitment was next assessed using a NanoBiT-based approach. In brief, cells expressing a fixed ratio of GHSR1aLgBiT and smBiTβ-arr2 were treated with ghrelin, L585, or N8279. Here, N8279 was ˜20-fold less potent than ghrelin and it approached full agonism (
Next, cells expressing a variable ratio of GHSR1aLgBiT and SmBiTβ-arr2 were treated with ghrelin (100 nM) or ˜EC80 N8279 for Gα signaling (100-200 nM,
Next, it was assessed if N8279 could behave functionally as a β-arr2 antagonist in the presence ghrelin. Cells expressing GHSR1aLgBiT and smBiTβ-arr2 were pretreated with increasing concentrations of the antagonists YIL781 or JMV2959, or N8279, followed by EC80 ghrelin. N8279 inhibited ghrelin-induced β-arr2 recruitment significantly, but incompletely, in a concentration-dependent manner and was 1.7 and 2.9-fold less potent than JMV2959 and YIL781 (
While the GHSR1aL149G reduced Emax similarly for both ghrelin and N8279 relative to GHSR1aWT (˜35%), N8279-induced β-arr2 recruitment potency to the GHSR1aL149G was reduced by ˜5-fold, whereas ghrelin potency was decreased by merely 2-fold (
To further characterize N8279 modulation of β-arr2 recruitment to the ghrelin-bound GHSR1a, cells expressing GHSR1aLgBiT and SmBiTβ-arr2 were pretreated with increasing concentrations of YIL781, JMV2959, or N8279 followed by EC80 ghrelin. N8279 inhibited ghrelin-induced β-arr2 recruitment to the GHSR1a in a concentration-dependent manner, albeit incompletely and with a potency 1.7-2.9-fold less than JMV2959 and YIL781, respectively (
Qualitative microscopy of U2OS cells expressing the GHSR1a and β-arr2GFP showed minimal response to 100 nM N8279 and displayed diffusely distributed cytosolic β-arr2GFP Similar to vehicle-treated cells (
β-arr2 is required for GHSR1a-mediated RhoA GTPase/ROCK signaling, leading to transcriptional activation and cytoskeletal rearrangement by induction of actin polymerization. To test if N8279 affected these processes, the RhoA-dependent transcriptional reporter, SRF-RE, was utilized. Here, N8279 was a full agonist with mildly increased maximal efficacy relative to ghrelin (
To quantitatively assess N8279 bias between the Gαq and β-arr2 (
Collectively, the data in
Next it was evaluated whether determinants outside the orthosteric binding pocket are required for N8279 signaling by first using a naturally-occurring variant, GHSR1aA204E (
GHSR1aA204E showed no basal iCa2+ activity (
Next, the NMR-based homology model of the ghrelin-bound GHSR1a (
To test the model, point mutations were made to predict N8279-GHSR1a interaction sites or residue clusters (
Relative to the WT receptor, the surface expression of the GHSR1aE197A was reduced moderately, the GHSR1aR199A was comparable, and the GHSR1aP200A was increased mildly (
For comparison, ghrelin-stimulated Gαq dissociation and iCa2+ was evaluated for each GHSR1aECD mutant. Ghrelin signaling efficacy was reduced markedly in both GU& dissociation and iCa2+ assays respectively (
To assess if N8279 signaling required receptor sites within and/or conformational states are conserved from the mouse receptor to the human receptor, the amino acid sequences for variations within the ECL2 were first determined (
Collectively, the data in
Pharmacokinetic studies with oral gavage (PO; 5 mg/kg), intraperitoneal (IP; 5 mg/kg), and intravenous (IV; 1 mg/kg) administration in C57BL/6 mice revealed a PO bioavailability of 7% and IP bioavailability of 27% (
To evaluate the effect of N8279 on DAergic-modulated behavior in vivo, DAT knockout (KO) mice, which have constitutively elevated extracellular DA levels and consequently, spontaneous hyperactivity in a novel, open-field were used. After a 30 minute acclimation period, male and female DAT KO mice were administered vehicle or pharmacologically-relevant, brain penetrant doses of N8279 (
Next, cocaine-induced behavioral sensitization was assessed in male and female C57BL/6J mice following subchronic (8-day) administration of the vehicle or N8279 (5 mg/kg, IP) in the home-cage. Subsequently, mice were given the same treatments as described above, followed with an injection (IP) of vehicle or cocaine (20 mg/kg) in the open-field once per day for 5 days (
The following exemplary methods were performed in Examples 1 and 69-74 as described herein:
Chemicals and Compounds. Full-length, human acyl-ghrelin (Cat. no. 1463), L-692,585 (Cat. no. 2261), YIL781 hydrochloride (Cat. no. 3959), and MK-0677 (Cat No. 5272) were purchased from Tocris Biosciences (Bristol, United Kingdom). JMV2959 was purchased from Sigma Aldrich (Cat no. 345888). [125I]ghrelin was purchased from Perkin-Elmer (Waltham, MA; Cat no. NEX3880101UC). Ghrelin peptide was maintained as a 1 mM stock in 50% glycerol, whereas all other small molecule ligands (including N8279) were maintained as 10 mM stocks in dimethyl sulfoxide (DMSO).
Plasmids. The 3×HA-hGHSR1aWT plasmid was originally purchased from the cDNA Resource Center and consisted of an N-terminally 3×HA-tagged human GHSR1aWT coding sequence cloned into a pcDNA3.1+ backbone. This construct was used to generate 3×HA-hGHSR1aA204E by QuikChange site-directed mutagenesis (Agilent Technologies, Santa Clara, CA). The expression vector for the bioluminescent, mitochondria-targeted apo-aequorin Ca2+ sensor (miAeq 1) was a gift from Dr. Stanley Thayer (University of Minnesota). To generate a C-terminally-tagged 3×HA-hGHSR1aWT plasmid with LgBiT, the coding sequence for 3×HA-hGHSR1aWT was inserted 5′ into a vector containing the coding sequence for the LgBiT NanoLuc fragment (pBiT1.2-C). To generate a complementary N-terminally-tagged β-arrestin2WT plasmid, β-arrestin2WT was inserted 3′ of the coding sequence for the SmBiT NanoLuc fragment (pBiT2.2-N). Both LgBiT and SmBiT vector backbones were purchased from Promega. GαqLgBiT, smBiT-J31, untagged Gα2, and RIC8A were generous gifts from Dr. Asuka Inoue. The hGHSR1aWT-RLuclI and 2×-FYVEmVenus plasmids used for bBRET analyses were made by the Caron lab. The RhoA-dependent serum response element plasmid SRF-RE-Luciferase, cloned into the pGL4.34[luc2P/SRF-RE/Hygro] vector backbone, was purchased from Promega (Cat. no. E1350). pcDNA3.1+ was used as empty vector in all experiments and all constructs were validated by sequencing.
Cell Culture & Transfections. U2OS, HEK293/T, and HEK293/N cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) of Fetal Bovine Serum (FBS) and 1× antibiotic-antimycotic solution (100 IU-1 penicillin, 100 μg/mL streptomycin and 250 ng/mL amphotericin B; MilliporeSigma). HEK293/S Gαq/11 KO and its parental WT line were a generous gift from Dr. Asuka Inoue (Tohoku University, Miyagi, Japan). U2OS cells stably expressing 3×HA-hGHSR1aWT and green fluorescent protein (GFP) tagged-β-arrestin2 and HEK293/N cells stably expressing 3×HA-hGHSR1aWT and the luminescent Ca2+ sensor mitochondrial-Aequorin (miAeq) were made by the Caron lab. All cell lines were grown in a humidified incubator at 37° C. (5% CO2). All transient transfections were performed using a standard calcium phosphate method.
High-Throughput and Directed Library Compound Screening. Quantitative high-throughput screening (qHTS). qHTS was performed at the National Center for Advancing Translational Sciences (NCATS) against 47,000 compounds from the NCATS Sytravon library and Pharmacological collection (NPC) using the PathHunter U2OS GHSR1a β-arr1 cells and the β-arr Assay kit (DiscoverX, Fremont, CA), which measures recruitment of β-arr to the GHSR1a. Prior to the screening, the assay was miniaturized to a 1536-well format and optimized in terms of signal-to-background window (S/B), Z factor, and potency of ghrelin control. The initial assay validation was performed with Library of Pharmacologically Active Compounds (LOPAC1280, Sigma-Aldrich) to confirm plate-to-plate reproducibility of parameters, hit rate identification, etc. For qHTS, two doses of the compounds—11 μM and 57 μM—were used to measure GHSR1a activation using a fully automated robotic screening system (Kalypsys, San Diego, CA). Briefly, 1.2×103 cells were seeded with MultiDrop Combi dispenser (Thermo Scientific, Logan, UT) into white solid-bottom tissue culture-treated 1536-well plates (Aurora Microplates, Whitefish, MT) in 3 μL of AssayComplete Cell Plating 5 Reagent (DiscoverX) and cultured overnight. Next, 1 μl/well of 1 μM Ghrelin diluted in HBSS+10 mM HEPES (HH buffer) was added to one column of the plate, while all other wells were dispensed with 1 μL/well of HH buffer for matching the assay conditions. Subsequently, the libraries' compounds in DMSO solution were transferred from the source plates to the assay plates at 23 mL/well. The plates were incubated for 90 minutes followed by addition of 1.5 μL/well PathHunter Detection Reagent prepared according to the manufacturer's instructions. After 60 minutes of incubation at ambient temperature, the luminescent signal was measured on ViewLux uHTS Microplate Imager (Perkin Elmer, Waltham, MA) with 20 seconds exposure. Quality of the screening was evaluated based on the median characteristics of Z factor and S/B which were 0.53 and ˜4.5-fold, respectively. Hit detection window parameters were calculated based on results obtained from the vehicle control (ECO) and ghrelin as the positive control (EC100=250 nM final concentration) conditions. A cut-off of >60% activation by either the lower or higher compound's dose was used to select primary hits. A follow-up PathHunter U2OS GHSR1a β-arr1 assay was performed on 145 selected primary compounds. They were re-tested at 7 doses in the range [57 μM-3.7 nM] applying the same protocol as for qHTS. Thirty-six hits were selected based on curve response class (CRC) 1,2,3 for further validation.
X-Ray Diffraction. Single crystal X-ray diffraction studies were conducted on a Bruker Kappa Photon 11 CPAD diffractometer equipped with Cu Ka radiation (λ=1.54178 Å). Crystals of the subject compound were grown by dissolving approximately 1 mg of sample in 350 μL of 90/10 Dichloroethane/Methanol solution, which was then vapor diffused with Pentane over several days. A 0.267×0.243×0.228 mm piece of a colorless block was mounted on a Cryoloop with Paratone oil. Data were collected in a nitrogen gas stream at 285K using Φ and ω scans. The crystal-to-detector distance was 40 mm using variable exposure time (20 s-90 s) depending on Θ with a scan width of 2.0°. Data collection was 99.5% complete to 59.009° in Θ (0.90 Å). A total of 58959 reflections were collected covering the indices, −17<=h<=17, −16<=k<=16, −24<=1<=24. 6947 reflections were found to be symmetry independent, with a Rint of 0.0596. Indexing and unit cell refinement indicated a primitive, monoclinic lattice. The space group was found to be P21/c. The data were integrated using the Bruker SAINT software program and scaled using the SADABS software program. Solution by direct methods (SHELXT) produced a complete phasing model for refinement. All nonhydrogen atoms were refined anisotropically by full-matrix least-squares (SHELXL-2014). All carbon bonded hydrogen atoms were placed using a riding model. Their positions were constrained relative to their parent atom using the appropriate HFIX command in SHELXL-2014. All other hydrogen atoms (H-bonding) were located in the difference map. Their relative positions were restrained using DFIX commands and their thermals freely refined. Crystallographic data are summarized in Table 2.
GHSR1a Radioligand Binding Assays. Saturation: [I125]ghrelin (Perkin-Elmer, Waltham, MA) saturation binding experiments were performed as described. Briefly, hGHSR1aWT (1 μg) was transiently transfected into HEK293/T cells cultured on 1 cm dishes. After 48 hours, cells were collected, resuspended in hypotonic membrane lysis buffer (50 mM Tris-HCl, pH 7.4), then centrifuged at 21,000×G for 20 minutes to obtain crude membrane pellets. Membranes were resuspended in assay buffer (50 mM Tris-HCl, 5 mM EDTA, 5 mM MgCl2, 1% BSA), protein content was measured by BCA protein assay, and diluted to 2 μg/mL. Ten μL of GHSR1a-expressing membrane (20 μg total protein) was then incubated with increasing concentrations of cold ghrelin (0.8-10 nM) and [I125]ghrelin with its specific activity diluted 1:100. Non-specific binding was defined in parallel reactions containing 10 μM YIL781. Binding reactions proceeded for 1 hour on ice and were terminated by rapid filtration over 0.3% PEI-soaked GF/B filters with a 96-well Brandel harvester, followed by four washes with ice-cold wash buffer (Tris HCl, pH 7.4).
Competition: Equilibrium [I125]ghrelin competition binding assays were performed by incubating 2-20 μg of hGHSR1aWT (1 μg transfected, 10 cm dish) or hGHSR1aL149G (0.5 μg transfected, 10 cm dish)-expressing HEK293 cell membranes with a fixed concentration of [I125]ghrelin (3.2 nM) and increasing concentrations of cold acyl-ghrelin (1-28), N8279, N6164, or N1956. hGHSR1aL149G was transfected at half the total amount of cDNA relative to hGHSR1aWT because it exhibited enhanced cell surface expression. The data for each ligand was normalized to its respective vehicle condition, representing 100% binding. Equilibrium binding reactions proceeded for 1 hour and were terminated by rapid filtration over 0.3% PEI-soaked GF/B filters using a 96-well Brandel harvester, followed by four washes with ice-cold wash buffer.
Gαq-Dependent Intracellular Ca2+ Mobilization. HEK293/N cells stably expressing 3×HA-hGHSR1aWT and the mitochondrial-Aequorin (miAeq) Ca2+ sensor were grown in standard DMEM supplemented with 10% FBS and antibiotic-antimycotic, as well as selection media (G418 and Puromycin). Cells were split into and seeded into 10 cm dishes without antibiotic selection and grown to confluency overnight. The next morning, cells were serum starved in 10 mL of clear opti-MEM (supplemented with L-glutamine and HEPES; Gibco, Waltham, MA) for 3-4 hours in a 37° C. incubator with 5% CO2. Then, 25 μL of coelenterazine H (NanoLight Technology, Pinetop, AZ) was added to each 10 mL dish and incubated for another 1-2 hours. For antagonist assays, test compounds were either added directly to the media in 10 cm dishes, or cells were collected by gentle washing/trituration and plated onto white, clear-bottomed 96-well plates for 30 minutes—1 hour. Once the coelenterazine H (or antagonist) incubation was complete, cells were harvested from the 10 cm dish by gentle washing/trituration and collected in a 15 mL conical tube. Cell suspensions were then immediately subjected to assay by injecting cells onto a white, clear-bottomed 96-well plate containing 2× test compounds. Luminescence was read without delay and for 10 seconds per well. To control for cell number variability, cells were lysed in 2× lysis buffer (100 mM CaCl2+0.2% Triton-X) immediately following the assay. The max ligand-induced response (Ca2+ mobilization: net-miAeq) over 10 seconds was normalized by dividing the ligand-induced luminescence counts (L-miAeq) by the ligand-induced luminescence counts (L-MiAeq) plus the luminescence measured upon cell lysis (Lysis-miAeq). Relative, lysis-normalized data were then normalized to the appropriate control/reference condition.
For experiments with WT and Gt KO HEK293/S cells, 2.5 pig of hGHSR1aWT, 10 μg of miAEQ, and 2.5 μg of pcDNA3.1 was transiently transfected into 10 cm dishes and incubated overnight. For experiments evaluating hGHSR1aA204E-mediated Ca2+ mobilization, HEK293/T (10 cm dishes) cells were transiently transfected with hGHSR1aWT (2.5 μg) or hGHSR1aA204E (5 μg), 10 μg of miAEQ, and 2.5 or 0 μg of pcDNA3.1, then incubated overnight. hGHSR1aA204E was expressed at 2×hGHSR1aWT because it exhibited ˜50% the cell surface expression of the WT receptor (see
β-arr2GFP Translocation. Compound Screening: β-arr2 translocation was assessed using U2OS cells stably expressing the human hGHSR1a-vasopressin receptor 2 tail chimera (hCHSR1aV2T) and green fluorescent protein (GFP)-tagged β-arr2. Replacement of the C terminal sequence of GHSR1a to the C terminal tail of a class B GPCR, vasopressin receptor 2 leads to the formation of stable GHSR1a/β-arrs complexes in endocytic vesicles. On day 1, stable cells were split into MGB101-1-2-LG glass-bottom 384-well plates (MatriCal, Spokane, WA) using a Multidrop 384 liquid dispenser (Thermo Scientific, Hudson, Nil). Each well contained 30 μL aliquots of 8,000 cells in Minimum Eagle's medium (MEM) containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin (Life Technologies, Grand Island, NY). The plates were incubated overnight at 37° C. in 5% CO2, and on the following day, media was changed to 30 LL clear MEM without serum. Compounds at 50 μM in 5% DMSO from the Molecular Libraries Small Molecule Repository (MLSMR) collection were added to each well using a MicroLab StarLET liquid handler (Hamilton Robotics, Reno, NV) and diluted 10-fold to 5 μM final concentration. The plates were returned to the incubator for 40 min, and the cells were fixed by adding 30 μL of 2% paraformaldehyde-phosphate buffered saline (PBS) to each well. Plates were stored at 4° C. until analysis on an ImageXpress Ultra (Molecular Devices, Sunnyvale, CA) at 488 nm. Images were analyzed using a wavelet algorithm to measure formation of fluorescence aggregates. Image results were also confirmed visually.
Evaluation of Ghrelin- and N8279-Induced β-arr2GFP Translocation. U2OS cells stably expressing the human GHSR1aWT and β-arr2GFP were seeded onto 35 mm MatTek (Ashland, MA, USA) glass coverslip dishes in opti-MEM supplemented with 2% FBS and antibiotic-antimycotic and grown overnight in a humified incubator (5% CO2, 37° C.). The next morning, media was aspirated and replaced with fresh, serum-free opti-MEM (supplemented with antibiotic-antimycotic). Four hours later, cells were treated with vehicle, ghrelin (100 nM), or N8279 (100 nM), placed back in a humified incubator (5% CO2, 37° C.) and incubated for 45 minutes. Next, treatment was terminated by aspiration of media, a PBS wash, and fixation with 4% paraformaldehyde (20 minutes, room temperature (RT)). Fixed cells were either stored at 4° C. or immediately imaged for β-arr2GFP translocation with a Zeiss LSM 510 meta confocal microscopy.
NanoBiT-based Gaq Dissociation & β-arr2 Recruitment Assays. Gαq dissociation/activation: GαqLgBiT and smBiT-β1 were transiently co-transfected with human 3×HA-GHSR1aWT, untagged Gγ2, and RIC8A into monolayers of HEK293S-Gαq knockout cells. 3×HA-GHSR1aWT and GαqLgBiT cDNA was transfected at a 1:1 ratio. The next morning, media (DMEM supplemented with 10% FBS and antibiotic/antimycotic) was exchanged and after 4-6 hours, cells were plated on white 96-well, clear-bottomed assay plates at a density of 45,000 cells/well in opti-MEM supplemented with 2% FBS and antibiotic/antimycotic. The next morning, opti-MEM was removed and cells were incubated with 80 μL HBSS supplemented with 20 mM HEPES (pH 7.4) for 4 hours. Cells were then treated with 10 μL of the luminescent substrate, coelenterazine H (5 μM final), for 15 minutes at room temperature prior to the addition of 10 μL of test compound. Two minutes ligand application, a stable luminescence signal was established and measured by a Mithras 940 plate reader over 20 minutes. The average response was utilized for generating C/R curves in
β-arr2 recruitment: In agonist and antagonist assays of NanoBiT-based β-arr2 recruitment, hGHSR1aWT-LgBiT and SmBiT-βWT were transiently co-transfected at a 1:1 ratio (250 μg each) into monolayers of HEK293/T cells on 6-well plates along with 2 μg of pcDNA3.1. For assays employing the Ala204Glu variant, hGHSR1aA204E-LgBiT was transfected at 2×hGHSR1aWT-LgBiT, as performed for iCa2+ assays with hGHSR1aA204E described above. For GHSR1a-β-arr2 saturation assays, a fixed amount of hGHSR1aWT-LgBiT cDNA (250 μg) was transfected with 0, 62.5 (1:0.25), 125 (1:0.5), 250 (1:1), 500 (1:2), or 1000 (1:4) μg of SmBiTβ-arrWT. The morning after transfection, media was exchanged and after 4-6 hours, cells were then plated on white 96-well, clear-bottomed assay plates at a density of 45,000 cells/well in opti-MEM supplemented with 2% FBS and antibiotic/antimycotic. The following morning, opti-MEM was removed and cells were incubated with 70-80 μL HBSS supplemented with 20 mM HEPES (pH 7.4) for 4 hours. Cells were then treated with 10 μL of coelenterazine H (5 μM final) for 15-20 minutes at room temperature. Cells were then stimulated with 10 μL of test compounds and 2 minutes later, luminescence was measured over 5 minutes, which encompassed the peak β-arr2 recruitment signal. For antagonist assays, cells were pretreated with the indicated ligand for 5 minutes prior to stimulation with EC80 ghrelin (40 nM).
BRET-based Gαq Dissociation & β-arr2 Recruitment Assays. TRUPATH Gα dissociation: Gαq/i1/i2/OA/12/13/sSRLuc8 and Gγ8GFP2 or Gγ9GFP2 were transiently co-transfected with human 3×HA-GHSR1aWT and untagged Gβ3 at a 1:1:1:1 ratio (100 μg each) into monolayers of HEK293/T cells along with 1.6 μg pcDNA3.1. The next morning, media (DMEM supplemented with 10% FBS and antibiotic/antimycotic) was exchanged and after 4-6 hours, cells were plated on white 96-well, clear-bottomed assay plates at a density of 40,000-50,000 cells/well in opti-MEM supplemented with 2% FBS and antibiotic/antimycotic. The next morning, opti-MEM was removed and cells were incubated with 80 μL HBSS supplemented with 20 mM HEPES (pH 7.4) for 4 hours. Cells were then treated with 10 μL of the luminescent substrate, coelenterazine 400a (5 μM final), for 5 minutes at room temperature prior to the addition of 10 μL of test compound. Five minutes after ligand application, luminescence was measured by a Mithras 940 plate reader over 30 minutes and the maximal response was utilized for generating all C/R curves.
β-arr2 recruitment: 3×HA-hGHSR1WT-RLucII and mVenus-βarr2WT were transiently co-transfected at a 1:15 ratio (100 ng:1.5 μg) into monolayers of HEK293/T cells on 6-well plates along with 0.9 μg of pcDNA3.1. Due to increased surface expression of GHSR1aL149G, 50 ng of 3×HA-hGHSR1aL149G-RLucII was transfected with 750 ng of mVenus-β-arr2WT (1:15 ratio). The morning after transfection, media was exchanged and after 4-6 hours, cells were then plated on white 96-well, clear-bottomed assay plates at a density of 45,000 cells/well in opti-MEM supplemented with 2% FBS and antibiotic/antimycotic. The following morning, opti-MEM was removed and cells were incubated with 70-80 μL HBSS supplemented with 20 mM HEPES (pH 7.4) for 4 hours. Cells were then treated with 10 μL of coelenterazine H (5 μM final) for 5-10 minutes at room temperature. Cells were then stimulated with 10 μL of test compounds and 2 minutes later, luminescence was measured over 60 minutes and the maximal response was utilized for generating C/R curves in
Chemiluminescent Fixed-Cell ELISA. 3×HA-hGHSR1aWT or pcDNA3.1 was transiently transfected by the calcium phosphate method into monolayers of HEK293/T cells on 6-well plates. The next morning, media (DMEM supplemented with 10% FBS and antibiotic/antimycotic) was exchanged. Later that afternoon, cells were plated on white 96-well, clear-bottomed assay plates at a density of 40,000 cells/well in opti-MEM (supplemented with 2% FBS and antibiotic/antimycotic). The next morning, media was removed and cells were serum starved with 90 μL of opti-MEM (containing no serum) for 2-3 hours at 37° C. Cells were then stimulated with 10× test compounds (diluted in serum-free opti-MEM) for 45 minutes at 37° C. in a humidified incubator. After 45 minutes, cells were fixed with 4% paraformaldehyde for 15 minutes (RT). Fixed cells were then washed three times with PBS (pH 7.4, Gibco) and blocked with fish gelatin in TBS (Rockland, Gilbertsville, PA) for 45 minutes. Cells were then incubated with a rabbit, horseradish peroxidase (HRP)-conjugated anti-HA antibody (1:2, 500; Novus Biologicals, Littleton, CO) for 1 hour (RT). After three washes with PBS, cells were treated with 50 μL of SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Waltham, MA) and luminescence was measured by a Mithras 940 plate reader over ˜12 minutes. The average luminescence was used and the data for each ligand were normalized its corresponding vehicle treatment conditions.
Bystander BRET (bBRET). hGHSR1aWT-RLucII and plasma membrane marker, MyrPalmmVenus, or the early endosomal marker, 2×-FYVEmVenus, was transiently co-transfected in HEK293/T cells at a ratio of 1:5 or 1:12.5, respectively. The next morning, media (DMEM supplemented with 10% FBS and antibiotic/antimycotic) was exchanged. Later that afternoon, cells were plated on white 96-well, clear-bottomed assay plates at a density of 45,000 cells/well in opti-MEM (supplemented with 2% FBS and antibiotic/antimycotic). The next morning, opti-MEM was removed, and cells were incubated with 70-80 μL HBSS+20 mM HEPES for 4 hours at 37° C. Cells were treated with 10 μL of coelenterazine H (5 μM final) for 5-15 minutes at 37° C., then stimulated with 10 μL of 10× test compound for 5 minutes at 37° C. Plates were then read by a Mithras 940 plate reader every 5 minutes over 1 hour (MyrPalmmVenus) or 2 hours (2×FYVEmVenus) to determine a BRET ratios. To generate CR curves, the average net BRET over 60 minutes post-treatment was used.
SRF-RE Transcriptional Activity. hGHSR1aWT and SRF-RE (Promega) were transiently co-transfected at a 1:40 ratio by the calcium phosphate method into monolayers of HEK293/T cells on 6-well plates. The next morning, standard media was exchanged and later that afternoon, cells were plated on white 96-well, clear-bottomed assay plates at a density of 45,000 cells/well in opti-MEM supplemented with 2% FBS and antibiotic/antimycotic. The next morning, media was removed and cells were serum starved with 90 μL of opti-MEM (containing no serum) for 2 hours at 37° C. Cells were then stimulated with 10× test compounds (diluted in serum-free opti-MEM) for 5 hours at 37° C. in a humidified incubator. Cells were then lysed in 20 μL of 1× passive lysis buffer (Promega) for 10 minutes on a shaker (RT). To measure luminescence, 40 μL of luciferin (in HBSS supplemented with 20 mM HEPES) was injected into in each well and immediately read by a Mithras 940 plate reader for 10 seconds.
Molecular Docking. Molecular docking studies were performed using the Glide and Maestro user interface (Release 2019-4, Schrodinger LLC, New York, NY). The model structure of ghrelin-bound GHSR1a and the X-ray crystal structure of antagonist-bound GHSR1a were used to represent the active and inactive state of the GHSR1a, respectively. The Protein Preparation Wizard function was used to assign bond orders, add hydrogen atoms, and remove water molecules that did not participate in interactions. The GHSR1a models were subjected to energy minimization using the OPLS3 force field. A receptor grid box of 30×30×30 Å3 with a default inner box (10×10×10 Å3) was centered on the ligand binding pocket. The ligand structures were generated and prepared using the LigPrep function with the OPLS3 force field. Flexible ligand docking was performed using the “standard precision” Glide algorithm, and after the post-docking minimization the pose with the best docking score was evaluated.
Pharmacokinetic Analysis. Male C57BL/6 mice were used for pharmacokinetic studies and were purchased from Charles River Laboratories. DAT KO mice were backcrossed for >10 generations onto a C57BL/6J (Jackson Laboratory, Bar Harbor, ME) genetic background. Mice were bred and maintained on a standard 12:12 hour light:dark cycle, socially housed, and supplied with standard laboratory chow and water ad libitum, except during testing. Male C57BL/6 mice (n=3/time-point) were administered N8279 at 1 mg/kg IV, 5 mg/kg PO and 5 mg/kg IP. Plasma, brain and liver samples were collected over 24 hours. N8279 concentrations in plasma, brain and liver homogenates were determined by LC-MS/MS. The mean concentration from 3 animals at each time-point was used in the pharmacokinetic (PK) analysis. PK parameters were calculated with Phoenix WinNonlin Software (Ver. 8.0, Certara).
Novelty-induced locomotor activity in DAT KO and inbred C57BL/6J mice. Open-field locomotor activity in mice was performed similar to that described in Barak et al., ACS Chem Biol. (2016)11(7):1880-90, the disclosure of which is incorporated herein in its entirety. In brief, Age- and sex-matched, littermate DAT KO and inbred C57BL/6J mice between 2-6 months of age were used to measure locomotor activity in an Omnitech Digiscan activity monitor (20×20 cm2; Accuscan Instruments, Columbus, OH). Locomotor activity was measured at 5 minutes intervals. To evaluate the effects of compounds on locomotor behavior, the mice were placed in an activity monitor for a 20-minute habituation period, and afterwards injected with drug or vehicle, returned to the monitor, and locomotor activity was recorded over a period of 90 minutes.
Cocaine Sensitization. Male and female C57BL6/J mice (Jackson Labs, Bar Harbor, ME) were administered (IP) vehicle or N8279 (5 mg/kg) subchronically for 8 consecutive days (once/day) (
Statistics. All data are presented as the mean±SEM derived from multiple independent experiments or animals. For binding and signaling assays, >2 technical replicates were included in each experiment. These data were plotted and analyzed in GraphPad Prism version 9.0 with a statistical significance threshold defined as p<0.05. Non-linear regression parameters and best-fit models for all C/R data were determined statistically by an extra sum-of-squares F-test. The behavioral data were analyzed by repeated measures ANOVA followed by the appropriate post-hoc multiple comparisons test.
One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present disclosure described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present disclosure. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the present disclosure as defined by the scope of the claims.
No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise.
The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.
This application claims priority to U.S. Provisional Patent Application Ser. No. 63/246,934, filed Sep. 22, 2021, the contents of which is hereby incorporated by reference in its entirety.
This invention was made with Government support under Federal Grant Nos. U18DA052417 and P30DA029925 awarded by the National Institutes of Health. The Federal Government has certain rights to this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/016801 | 2/17/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63246934 | Sep 2021 | US |
