WO2021050547A1

WO2021050547A1 - Mitochondrial complex i inhibitors and methods of use

Info

Publication number: WO2021050547A1
Application number: PCT/US2020/049944
Authority: WO
Inventors: Dieter A. Wolf; Arkadii Vaisburg
Original assignee: Sanford Burnham Prebys Medical Discovery Institute
Priority date: 2019-09-10
Filing date: 2020-09-09
Publication date: 2021-03-18

Abstract

Disclosed herein are methods of using mitochondrial complex I inhibitors in the treatment of a disease or condition that would benefit from the inhibition of mitochondrial respiration. Also described herein are methods of treating a disease or condition such as prostate cancer and breast cancer by administering a mitochondrial complex I inhibitor in combination with a second therapeutic agent.

Description

MITOCHONDRIAL COMPLEX I INHIBITORS AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/898,287 filed on September 10, 2019. Priority is claimed pursuant to 35 U.S.C. § 119. The above noted patent application is incorporated by reference as if set forth fully herein.

GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with government support under R01 GM105802, R21 CA190588 and R01 GM121834 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] Described herein are inhibitors of NADH:ubiquinone oxidoreductase for cancer therapy.

BACKGROUND

[0004] Prostate cancer is the most common male malignancy in the Western World and the second most common cause of cancer-related death in men. There are currently about 2.6 Million prostate cancer patients living in the U.S., a figure expected to rise to about 3.9 million over the next ten years. Approximately 30,000 men are dying of prostate cancer each year. Standard -of-care therapy for advanced prostate cancer is androgen deprivation therapy combined with chemotherapy. Most advanced prostate cancers initially shrink in response to chemo -hormonal therapy but progression to castration resistant prostate cancer (CRPC) occurs within 24-48 months. Next generation androgen receptor (AR) blockers such as enzalutamide are facing limitations due to rapid development of resistance through AR point mutations, some of which dangerously convert AR antagonists into agonists. There is thus a strong unmet need for alternative therapies.

SUMMARY

[0005] The present invention relates to methods of treating a disease or condition that would benefit from the inhibition of mitochondrial respiration such as an androgen-resistant cancer. Particularly, the present invention relates to methods of treating a disease or condition by administering to a subject in need thereof a small molecule compound that inhibits mitochondrial respiration through an inhibition of mitochondrial complex I. The present invention further relates to methods of using the mitochondrial complex I inhibitors as described herein. [0006] In one aspect, described herein is a method of treating a disease or condition that would benefit from inhibition of mitochondrial respiration by inhibiting mitochondrial NADH: ubiquinone reductase (Complex I), comprising administering to a subject in need thereof a therapeutically effective amount of a mitochondrial Complex I inhibitor, wherein the inhibitor is a compound that has the structure of Formula I, or a pharmaceutically acceptable salt, or solvate thereof:

wherein:

X is CH orN;

Ri is hydrogen, Ci-Cx alkyl, halogen, C₁-C₆ alkenyl, C₁-C₆ alkynyl, -OR₅, Ci-Cr, alkyl -O-R_5. or C0-C6 alkyl-N(R5)2;

R₃ is hydrogen, halogen, C₁-C₆ alkyl, -OR₅, or C₁-C₆ alkyl -O-R₅; each of R₂ and R₄ is independently hydrogen, C₁-C₆ alkyl, or -L-R₅, wherein L is -0-, - NC(=0)R₅-, or -NR5-;

A is hydrogen, C1-C6 alkyl -O-R5, C1-C6 alkyl -N(Rs)2, -OR5, or Ci-Cx alkyl; and each R₅ is independently hydrogen or C₁-C₆ alkyl.

[0007] In some embodiments, the disease or condition is a cancer.

[0008] In one aspect, described herein is a method of treating cancer in a mammal comprising administering an inhibitor of the mitochondrial NADFfubiquinone reductase (Complex I) to the mammal in need thereof.

[0009] In another aspect, described herein is a method of inhibiting the activity of the mitochondrial NADFfubiquinone reductase (Complex I) in a mammal with cancer comprising administering to the mammal with cancer an inhibitor of the mitochondrial NADFfubiquinone reductase (Complex I). [0010] In some embodiments, the inhibitor of the mitochondrial NADFfubiquinone reductase (Complex I) interacts with the catalytic subunit NDUFS2 of the mitochondrial NADFfubiquinone reductase (Complex I). In some embodiments, the inhibitor is a small molecule. In some embodiments, the inhibitor is a compound that has the structure of Formula (I), or a pharmaceutically acceptable salt, or solvate thereof:

Formula I wherein:

X is CH orN;

Ri is hydrogen, Ci-Cx alkyl, halogen, C1-C6 alkenyl, C1-C6 alkynyl, -OR₅, Ci-Cr, alkyl -O-R_5. or C0-C6 alkyl-N(R5)2;

R3 is hydrogen, halogen, C1-C6 alkyl, -OR5, or C1-C6 alkyl -O-R5; each of R2 and R4 is independently hydrogen, C1-C6 alkyl, or -L-R5, wherein L is -0-, - NC(=0)R₅-, or -NR5-;

A is hydrogen, C1-C6 alkyl -O-R5_, C1-C6 alkyl -N(Rs)2, -OR5, or Ci-Cx alkyl; and each R5 is independently hydrogen or C1-C6 alkyl.

[0011] In some embodiments, the inhibitor is a compound that is selected from the group consisting of: 4-Butyl -2 -methylaniline, N-(4-butyl-2-methylphenyl)-N-methylacetamide, 4-butyl -2- ethylphenylamine, (4-butyl-2-methylphenyl)methylamine, 2-methyl-4-pentylphenylamine, 4-butyl- 2,5-dimethylphenylamine, 4-(2-methoxyethyl)-2-methylphenylamine, 4-(3-aminopropyl)-2- methylphenylamine, 2-methyl-4-propoxyaniline, 4-butyl-2-chlorophenylamine, (4-butyl-2- methylphenyl)dimethyl amine, 2-methyl-4-propylphenylamine, 4-butyl-2-methoxyphenylamine, 3-(4- amino-3-methylphenyl)propan-l-ol, 4-butyl -2, 6-dimethylphenylamine, 5-butyl-2-methylaniline, 6- butyl-4-methylpyri din-3 -amine, 6-butyl -2 -methylpyri din-3 -amine, 4-butyl -2 -methyl phenol, and 4- butyl-2,3-dimethylphenylamine. In some embodiments, the inhibitor is a compound in Table 1.

[0012] In some embodiments, the inhibitor is a compound that has the structure of Formula (II), or a pharmaceutically acceptable salt, or solvate thereof:

Formula (II) wherein:

Z is hydrogen, Ci-Cs alkyl, C1-C6 alkenyl, C1-C6 alkynyl, -ORx, Ci-Cealkyl-O-Rs, or -N(Rx)2; R₆ is hydrogen or C₁-C₆ alkyl;

R₇ is hydrogen or C₁-C₆ alkyl; each R₈ is independently hydrogen or C₁-C₆ alkyl; each of R₉, Rio and Rn is independently selected from hydrogen and C₁-C₆ alkyl, or R₉ and Rio are taken together with the carbon atoms to which they are attached to form a double bond; and each of Y₂, Y₃, and Y₄ is independently CH or N.

[0013] In one aspect, described herein is a method of treating a disease or condition that would benefit from inhibition of mitochondrial respiration by inhibiting mitochondrial NADH: ubiquinone reductase (Complex I), comprising administering to a subject in need thereof a therapeutically effective amount of a mitochondrial Complex I inhibitor, wherein the inhibitor is a compound that has the structure Formula II, or a pharmaceutically acceptable salt, or solvate thereof:

wherein:

Z is hydrogen, Ci-Cs alkyl, C1-C6 alkenyl, C1-C6 alkynyl, -ORx, Ci-Cealkyl-O-Rs, or -N(Rx)2; R_X5 is hydrogen or C₁-C₆ alkyl;

[0014] In some embodiments, the disease or condition is a cancer. In some embodiments, the inhibitor is a compound that is selected from the group consisting of: 2-tert-Butyl-lH-indol-5-amine, 5 -butyl - lH-indole, and 5-butyl-2,3-dihydro-lH-indole.

[0015] In some embodiments, the cancer is prostate cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, colon cancer, colorectal cancer, brain cancer, head and neck cancer, melanoma, kidney cancer, or heart cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is a hormone dependent cancer. In some embodiments, the cancer is a hormone refractory cancer. In some embodiments, the cancer is resistant to treatment with an antiandrogen agent or the cancer is refractory to treatment with an antiandrogen agent. In some embodiments, the cancer is prostate cancer. In some embodiments, the prostate cancer is castration- resistant prostate cancer. In some embodiments, the prostate cancer is acinar adenocarcinoma, ductal adenocarcinoma, transitional cell (or urothelial) cancer, squamous cell cancer, small cell prostate cancer, or soft tissue sarcoma. In some embodiments, the prostate cancer is a grade 1 prostate cancer, grade 2 prostate cancer, grade 3 prostate cancer, grade 4 prostate cancer, or grade 5 prostate cancer. In some embodiments, the antiandrogen agent is a steroidal antiandrogen or a nonsteroidal antiandrogen. In some embodiments, the nonsteroidal antiandrogen is a first-generation nonsteroidal antiandrogen. In some embodiments, the first-generation nonsteroidal antiandrogen is flutamide, nilutamide, bicalutamide, or topilutamide. In some embodiments, the nonsteroidal antiandrogen is a second- generation nonsteroidal antiandrogen. In some embodiments, the second-generation nonsteroidal antiandrogen is apalutamide or enzalutamide. In some embodiments, the inhibitor is administered in combination with an androgen suppression therapy.

[0016] In one aspect, described herein is a method of treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a mitochondrial Complex I inhibitor and a second therapeutic agent, wherein the inhibitor is a compound that has the structure of Formula (I), or a pharmaceutically acceptable salt, or solvate thereof:

wherein:

X is CH orN;

A is hydrogen, C1-C6 alkyl -O-R5, C1-C6 alkyl -N(Rs)2, -OR5, or Ci-Cx alkyl; and each R₅ is independently hydrogen or C₁-C₆ alkyl. [0017] In another aspect, described herein is a method of treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a mitochondrial Complex I inhibitor and a second therapeutic agent, wherein the inhibitor is a compound that has the structure of Formula (II), or a pharmaceutically acceptable salt, or solvate thereof:

wherein:

Z is hydrogen, Ci-Cs alkyl, C1-C6 alkenyl, C1-C6 alkynyl, -ORx, Ci-Cealkyl-O-Rs, or -N(Rs)2; R6 is hydrogen or C1-C6 alkyl;

R₇ is hydrogen or C1-C6 alkyl; each R₈ is independently hydrogen or C1-C6 alkyl; each of R₉, Rio and Rn is independently selected from hydrogen and C1-C6 alkyl, or R₉ and Rio are taken together with the carbon atoms to which they are attached to form a double bond; and each of Y2, Y3, and Y4 is independently CH or N.

[0018] In some embodiments, the disease or condition is prostate cancer. In some embodiments, the second therapeutic agent is an antiandrogen. In some embodiments, the antiandrogen is a steroidal antiandrogen or a nonsteroidal antiandrogen. In some embodiments, the nonsteroidal antiandrogen is a first-generation nonsteroidal antiandrogen. In some embodiments, the first-generation nonsteroidal antiandrogen is flutamide, nilutamide, bicalutamide, or topilutamide. In some embodiments, the nonsteroidal antiandrogen is a second-generation nonsteroidal antiandrogen. In some embodiments, the second-generation nonsteroidal antiandrogen is apalutamide or enzalutamide. In some embodiments, the disease or condition is breast cancer. In some embodiments, the breast cancer is a triple negative breast cancer. In some embodiments, the second therapeutic agent is a BRAF inhibitor. In some embodiments, the BRAF inhibitor is Vemurafenib, dabrafenib, or encorafenib. In some embodiments, the second therapeutic agent is a CDK4/6 inhibitor. In some embodiments, the CDK4/6 inhibitor is palbociclib, ribociclib, or abemaciclib.

[0019] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0021] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0022] FIG.1 A-FIG.1 J illustrate the effect of SMIP004-7 on cells with sternness features.

[0023] FIG.2A-FIG.2D illustrate the effect of SMIP004-7 on mitochondrial respiration complexes. [0024] FIG.3A-FIG.3M illustrate that SMIP004-7 mediated cytotoxicity is rescued by overexpression of NDUFS2.

[0025] FIG.4A-FIG.4B illustrate the effect of SMIP004-7 on NADH oxidation by Cl in vitro.

[0026] FIG.5 A-FIG.5D illustrate cellular thermal shift assays (CETSA) showing stabilization of NDUFS2 by SMIP004-7.

[0027] FIG.6A-FIG.6H illustrate the effect of an indole analog of SMIP004-7 on cell viability and NDUFS2 stability.

[0028] FIG.7 A-FIG.7F illustrate the effect of SMIP004-7 on 4T1 tumor growth in immunocompetent and immunodeficient hosts

DETAILED DESCRIPTION

[0029] The present invention relates to the methods of using mitochondrial complex I inhibitors in the treatment of a disease or condition that would benefit from the inhibition of mitochondrial respiration. The present invention also relates to the methods of treating a disease or condition such as prostate cancer by administering a mitochondrial complex I inhibitor.

[0030] The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this present disclosure, which are encompassed within its scope. [0031] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment.

[0032] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0033] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

Definitions

[0034] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0035] In this application, the use of “or” means “and/or” unless stated otherwise. The terms “and/or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0036] The term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0037] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0038] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0039] The term “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

[0040] A “pharmaceutically acceptable excipient, carrier or diluent” refers to an excipient, carrier or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

[0041] A “pharmaceutically acceptable salt” suitable for the present disclosure may be an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication. Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2 -hydroxy ethyl sulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC-(CH2)n-COOH where n is 0-4, and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. Those of ordinary skill in the art will recognize from this disclosure and the knowledge in the art that further pharmaceutically acceptable salts include those listed by Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 ( 1985). In general, a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in an appropriate solvent. [0042] As used herein, the terms “prevent,” “preventing,” “prevention,” and the like, refer to reducing the probability of developing a disease or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease or condition.

[0043] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,

22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0044] The term “subject” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, bovine, equine, canine, ovine, or feline.

[0045] The terms “treat,” “treated,” “treating,” “treatment,” and the like are meant to refer to reducing or ameliorating a disorder and/or symptoms associated therewith (e.g., cancer). “Treating” may refer to administration of the inhibitors to a subj ect after the onset, or suspected onset, of a cancer. “Treating” includes the concepts of “alleviating”, which refers to lessening the frequency of occurrence or recurrence, or the severity, of any symptoms or other ill effects related to a cancer and/or the side effects associated with cancer therapy. The term “treating” also encompasses the concept of “managing” which refers to reducing the severity of a particular disease or disorder in a patient or delaying its recurrence, e.g., lengthening the period of remission in a patient who had suffered from the disease. The term “treating” further encompasses the concept of “prevent,” “preventing,” and “prevention,” as previously stated. It is appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

[0046] The term “therapeutic effect” refers to some extent of relief of one or more of the symptoms of a disorder (e.g., cancer) or its associated pathology. “Therapeutically effective amount” as used herein refers to an amount of an agent which is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survivability of the patient with such a disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying, and the like beyond that expected in the absence of such treatment. “Therapeutically effective amount” is intended to qualify the amount required to achieve a therapeutic effect. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the “therapeutically effective amount” (e.g., ED50) of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in a pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0047] The term “alkyl” refers to a straight or branched hydrocarbon chain radical, having from one to twenty carbon atoms, and which is attached to the rest of the molecule by a single bond. An alkyl comprising up to 10 carbon atoms is referred to as a Ci-Cio alkyl, likewise, for example, an alkyl comprising up to 6 carbon atoms is a C1-C6 alkyl. Alkyls (and other moieties defined herein) comprising other numbers of carbon atoms are represented similarly. Alkyl groups include, but are not limited to, Ci-Cio alkyl, C1-C9 alkyl, Ci-C₈ alkyl, C1-C7 alkyl, Ci-C₆ alkyl, C1-C5 alkyl, C1-C4 alkyl, C₁-C₃ alkyl, C₁-C₂ alkyl, C₂-C₈ alkyl, C₃-C₈ alkyl and C₄-C₈ alkyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, «-propyl, 1 -methyl ethyl (/-propyl), «-butyl, /-butyl, 5-butyl, «- pentyl, 1,1 -dimethyl ethyl (/-butyl), 3-methylhexyl, 2-methylhexyl, 1 -ethyl -propyl, and the like. In some embodiments, the alkyl is methyl or ethyl. In some embodiments, the alkyl is -CH(CH3)2 or - C(CH3)3. Unless stated otherwise specifically in the specification, an alkyl group may be optionally substituted as described below. “Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group. In some embodiments, the alkylene is -CH₂-, -CH₂CH₂-, or -CH₂CH₂CH₂-. In some embodiments, the alkylene is -CH₂-. In some embodiments, the alkylene is -CH₂CH₂-. In some embodiments, the alkylene is -CH₂CH₂CH₂-.

[0048] The term “alkenyl” refers to a type of alkyl group in which at least one carbon-carbon double bond is present. In one embodiment, an alkenyl group has the formula -C(R)=CR2, wherein R refers to the remaining portions of the alkenyl group, which may be the same or different. In some embodiments, R is H or an alkyl. In some embodiments, an alkenyl is selected from ethenyl (i.e., vinyl), propenyl (i.e., allyl), butenyl, pentenyl, pentadienyl, and the like. Non-limiting examples of an alkenyl group include -CH=CH₂, -C(CH )=CH₂, -CH=CHCH , -C(CH )=CHCH , and -CH₂CH=CH₂. [0049] The term “alkynyl” refers to a type of alkyl group in which at least one carbon-carbon triple bond is present. In one embodiment, an alkenyl group has the formula -CºC-R, wherein R refers to the remaining portions of the alkynyl group. In some embodiments, R is H or an alkyl. In some embodiments, an alkynyl is selected from ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Non-limiting examples of an alkynyl group include -CºCH, -CºCCH3 -CºCCH2CH3, -CH2CºCH. Technical Description

Characteristics of the Compounds

[0050] Mitochondrial Complex I (NADH: ubiquinone oxidoreductase, NADH Coenzyme Q reductase, or NADH dehydrogenase) is an enzyme that catalyzes the first step of the mitochondrial electron transport chain. It transfers electrons from NADH to a soluble lipid carrier, ubiquinone, and translocates protons across the membrane, such providing electrons for respiration and driving ATP synthesis. A minimum active form of Complex I found in bacteria contains 14 subunits. A mammalian Complex I contains 44 subunits, including the 14 “core” subunits and an additional 30 “supernumerary” subunits. See Vinothkumar et al., Nature 515, 80-84 (Nov 06, 2014). The 14 cores subunits include 7 hydrophilic domains (NDUFSl, NDUFVl, NDUFS2, NDUFS3, NDUFV2, NDUFS7, and NDUFS8) and 7 membrane domains (DN1, DN2, ND3, ND4, ND5, ND6, and ND4L). NADH dehydrogenase (ubiquinone) iron-sulfur protein 2 (NDUFS2) is one of the core Complex I subunits, and mutations in the NDUFS2 gene, as well as mutations in NDUFSl, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFVl, and NDUFV2 gene, have been identified as a cause for Complex I deficiency. See Kirby, J Clin Invest. 2004;114(6):837-845.

[0051] The present disclosure relates to a novel approach for targeting respiratory cancer metabolism. The disclosed compounds exhibit low micromolar cancer cell selective cytotoxic activity and act as effective Complex I inhibitors with unique ubiquinone-noncompetitive mechanism of inhibition involving catalytic subunit NDUFS2. The induction of mitochondrial reactive oxygen species (ROS) triggers apoptosis downstream of the unfolded protein response (UPR), which is rescued by overexpression of the mitochondrial target. The disclosed compounds exhibit in vivo anti -cancer activity in rodent xenograft models, e.g., active against enzalutamide-resistant prostate cancer cells, and shows synergism with androgen deprivation therapy. In the rodent models, the administration of the compounds does not lead to any weight loss, organ pathology, or blood chemistry changes. An overexpression of target (NDUFS2 and other Complex I subunits) and LKB1 mutations in prostate and other cancers may be used as potential biomarkers.

[0052] In one aspect, the inhibitors described herein such as SMIP004-7 are inhibitors of NADH:ubiquinone oxidoreductase. In some embodiments, the N-terminal domain of NDUFS2 that is identified as the likely binding site of the described inhibitors such as SMIP004-7 is ~70A distal to the quinone binding cleft targeted by most other Cl inhibitors. In certain embodiments, this is consistent with enzyme kinetic studies that revealed that SMIP004-7 reduces the Km for quinones ~10-fold, suggesting that the compound can uncompetitively interfere with the conformational change required for the release of reduced ubiquinone (ubiquinol) and the coupling of electron transfer with proton transport. In some embodiments, this coupling can occur through the formation of high energy quinol derivatives that drive a conformational change involving subunits ND1, NDUFS3, and NDUFS2. This change can then be transferred to the membrane arm, resulting in realignment of discontinuous transmembrane helices of the transporters to enable proton translocation. The realignment can be mediated by horizontal movements of the transverse helix of proton channel ND5, which also embraces proton channels ND2 and ND4. In some embodiments, the coupling between the conformational change in NDUFS2 caused by ubiquinone reduction and the movement of the transverse helix of ND5 may occur through the N-terminal domain of NDUFS2, which harbors the SMTP004-7 binding site. In some embodiments, the traverse helix of ND5 is the only element of Cl close to Trp 61 of NDUFS2 In some embodiments, the SMIP004-7 binding in this region blocks the propagation of the conformational change in NDUFS2 to ND5 and the other proton channels. In some embodiments, the inhibition of Cl by SMIP004-7 leads to NADH accumulation and increased superoxide formation. In some embodiments, high ROS levels rather than depletion of ATP and/or NAD+ is the main trigger of cell death.

Advantages of the Compounds

[0053] A common target in prostate cancer is the androgen receptor (AR). Standard-of-care therapy for advanced prostate cancer is therefore androgen deprivation therapy combined with chemotherapy. On the basis that castration resistant prostate cancer (CRPC) continues to depend on androgen receptor (AR) function, new anti-androgens and androgen synthesis blockers were introduced to the clinic with substantial therapeutic success. However, rapid development of resistance indicates a strong unmet need for alternative therapies.

[0054] CRPC arising after prolonged AR blockade originates from drug-resistant prostate cancer stem cells (CSCs). Regimens to eliminate CSCs would therefore appear a promising novel approach to combating CRPC and other drug-resistant cancers. Targets to specifically attack CSCs have just recently emerged. Inhibition of mitochondrial respiration (OXPHOS) is an approach in drug-resistant cancers. Accumulating evidence has suggested that CSCs are in a unique metabolic state characterized by a PGCla-driven transcriptional program that upregulates OXPHOS. Unlike the bulk proliferative tumor mass which can utilize glycolysis and respiration, CSCs display a strong dependence on mitochondrial respiration for ATP and NAD production. Tumors inoculated from cells deficient in mitochondria grow very slowly and are under selective pressure to acquire mitochondria from their surrounding microenvironment. Likewise, circulating tumor cells have increased OXPHOS and this increase is necessary for breast cancer metastasis.

[0055] Laboratory use inhibitors of mitochondrial respiration (e.g. oligomycin, CCCP) are effective in driving the death of CSCs but are not used clinically due to off-target effects. In addition, mitochondrial inhibition synergizes with classical cancer therapeutics (cisplatin, adriamycin, 5-FU, B- RAF inhibitors). The approved anti-diabetic metformin is an inhibitor of mitochondrial electron transport chain (ETC) complex I (Cl, NADFfubiquinone reductase) and selectively targets CSCs in rodent xenograft models. Based on these findings, metformin is in clinical testing as an anti -cancer agent and is already recommended as a first-line therapy for diabetic men with prostate cancer. However, metformin has drawbacks: Its IC50 for inhibition of Cl is 19.4 mM and low mM concentrations are needed to detect anti-tumor activity in tissue culture models. In addition, the pharmacokinetics of metformin is complex and there is uncertainty whether therapeutic concentrations can be reached in tumors at anti-diabetic doses. In conclusion, whereas metformin provides proof-of- concept for the use of mitochondrial inhibitors in cancer therapy, there is considerable potential for new compounds with improved characteristics. Compounds described herein rapidly inhibit mitochondrial respiration at a dose over 1000-fold lower than metformin thus giving them a considerable advantage over existing clinical compounds.

Complex I Inhibitors

[0056] Disclosed herein are inhibitors of mitochondrial Complex I. In embodiments, the inhibitor is an inhibitor of mitochondrial respiration. In some embodiments, the inhibitors are small molecules. A small molecule, as used herein, may refer to organic compounds having a molecular weight less than 1000, 900, 800, 700, 600, 500, 400, 300, or 200 Da. In embodiments, small molecules refer to organic compounds having a molecular weight less than 900, 700, or 500 Da. In some embodiments, the inhibitor is a compound that has the structure of Formula (I), or a pharmaceutically acceptable salt, or solvate thereof:

wherein:

X is CH orN;

Ri is hydrogen, Ci-Cx alkyl, halogen, C1-C6 alkenyl, C1-C6 alkynyl, -OR5, C1-C6 alkyl -O-R5_, or Co-C₆ alkyl-N(R₅)2;

R3 is hydrogen, halogen, C1-C6 alkyl, -OR5, or C1-C6 alkyl -O-R5; each of R₂ and R₄ is independently hydrogen, C₁-C₆ alkyl, or -L-R₅, wherein L is -0-, - NC(=0)R₅-, or -NR5-;

A is hydrogen, C1-C6 alkyl -O-R5, C1-C6 alkyl -N(Rs)2, -OR5, or Ci-Cx alkyl; and each R5 is independently hydrogen or C1-C6 alkyl.

[0057] In some embodiments, the inhibitor is a compound that is selected from the group consisting of: 4-Butyl -2 -methylaniline, N-(4-butyl-2-methylphenyl)-N-methylacetamide, 4-butyl -2- ethylphenylamine, (4-butyl-2-methylphenyl)methylamine, 2-methyl-4-pentylphenylamine, 4-butyl- 2,5-dimethylphenylamine, 4-(2-methoxyethyl)-2-methylphenylamine, 4-(3-aminopropyl)-2- methylphenylamine, 2-methyl-4-propoxyaniline, 4-butyl-2-chlorophenylamine, (4-butyl-2- methylphenyl)dimethyl amine, 2 -methyl -4-propylphenyl amine, 4-butyl-2-methylphenylamine, 4- butyl-2-methoxyphenylamine, 3-(4-amino-3-methylphenyl) propan-l-ol, 4-butyl-2,6- dimethylphenylamine, 5-butyl-2-methylaniline, 6-butyl-4-methylpyridin-3-amine, 6-butyl-2- methylpyri din-3 -amine, 4-butyl -2 -methylphenol, and 4-butyl-2,3-dimethylphenylamine.

[0058] In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, X is CH. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, X is N.

[0059] In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, Ri is hydrogen, C1-C6 alkyl, halogen, -OR5, C1-C3 alkyl -O-R5_. or C0-C3 alkyl-N(R5)2_. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, Ri is hydrogen, C1-C3 alkyl, halogen, -OR5, or -N(¾)2_. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, Ri is H. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, Ri is CFF. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, Ri is CH2CH3. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, Ri is halogen. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, Ri is Br, Cl, or F. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, Ri is Cl. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, Ri is -OR5_. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, Ri is -O-C1-C3 alkyl. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, Ri is -OCH3. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, Ri is N(Rs)2_. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, Ri is NH_2.

[0060] In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R2 is C1-C₆ alkyl, or -L-R5, wherein L is -0-, -NC(=0)Rs-, or -NR5-. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, II_I is N(¾)2. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R2 is NH2. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R^isMIRs. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R2 is NHCFF. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R2 is N(0¾)_2. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R2 is CFF. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R2 is N(C=0)R_5. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R2IS N(C=0)CH_3. [0061] In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R₃ is hydrogen, halogen, C₁-C₃ alkyl, -OR5, or C₁-C₃ alkyl-O-Rs. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R₃ is hydrogen or C₁-C₃ alkyl. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R₃ is hydrogen. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R3IS CFF_.

[0062] In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R₄ is hydrogen, C₁-C₆ alkyl, or -L-R₅, wherein L is -0-, -NC(=0)Rs-, or -NR₅-. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R₄ is hydrogen or C₁-C₆ alkyl. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R₄ is hydrogen or C₁-C₃ alkyl. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R₄ is hydrogen. In some embodiments of Formula (I), R₄ is CH_3.

[0063] In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, A is C1-C₆ alkyl -O-R5_, C1-C₆ alkyl-N(Rs)2, -OR5, or C1-C₆ alkyl. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, A is C₁-C₃ alkyl -O-R5, C1-C3 alkyl -N(Rs)2, -OR5, or C1-C₆ alkyl. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, A is C₁-C₃ alkyl-O-Rs_. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, A is CH₂CH₂OCH₃ or CH₂CH₂CH₂OCH_3. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, A is CH₂CH₂OH or CH₂CH₂CH₂OH. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, A is C₁-C₃ alkyl -N(¾)_2. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, A is C₁-C₃ alkyl-NFF. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, A is CH₂CH₂NH₂ or CH₂CH₂CH₂NH₂. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, A is C₁-C₅ alkyl. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, A is CFF, CH₂CH₃, n-C₃FF, n-C₄Hc>, or n- C₅H₁₁.

[0064] In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R5 is hydrogen. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R5 is C1-C6 alkyl. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R₅ is C₁-C₃ alkyl. In some embodiments of a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, R₅ is methyl, ethyl, «-propyl, or 1 -methyl ethyl (/-propyl).

[0065] In some embodiments, the inhibitor has a structure that is

[0066] In some embodiments, the inhibitor is a compound in Table 1. Table 1. List of Compounds

[0067] In some embodiments, the inhibitor is a compound that has the structure of Formula (II), or a pharmaceutically acceptable salt, or solvate thereof:

Formula (II) wherein:

R₇ is hydrogen or C₁-C₆ alkyl; each R₈ is independently hydrogen or C1-C6 alkyl; each of R₉, Rio and Rn is independently selected from hydrogen and C1-C6 alkyl, or R₉ and Rio are taken together with the carbon atoms to which they are attached to form a double bond; and each of Y2, Y3, and Y4 is independently CH or N.

[0068] In some embodiments of a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, each of Y₂, Y₃, and Y₄ is CH. In some embodiments of a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, one of Y₂, Y₃, and Y₄ is N.

[0069] In some embodiments of a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, 5 is C1-C4 alkyl. In some embodiments of a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, 5 i_s methyl, ethyl, «-propyl, /-propyl, «-butyl, /- butyl, 5-butyl, or /-butyl. In some embodiments of a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, R₆ is hydrogen or -C(C¾)3.

[0070] In some embodiments of a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, R₉ and Rio are taken together with the carbon atoms to which they are attached to form a double bond.

[0071] In some embodiments of a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, R₇ is hydrogen.

[0072] In some embodiments of a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, Z is Ci-Cs alkyl or N(Rs)_2. In some embodiments of a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, Z is C1-C6 alkyl. In some embodiments of a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, Z is C₁-C₄ alkyl. In some embodiments of a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, Z is n-C4H9. In some embodiments of a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, Z is N(Rs)2. In some embodiments of a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, Z is NFL. In some embodiments of a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof, Z is N(CH3)2.

[0073] In some embodiments, the inhibitor is a compound in Table 2.

Table 2. List of Compounds

Compound Compound Structure Number Names

Methods of Treatment

[0074] Described herein are methods of treating a disease or condition that would benefit from the inhibition of mitochondrial respiration. For example, the methods can be used to treat, prevent or inhibit progression of cancer cells in a subject in need thereof. Disclosed herein are methods of inhibiting mitochondrial Complex I through a ubiquinone-noncompetitive mechanism. In some embodiments, the method comprises administering an inhibitor as described herein. In some embodiments, the methods of inhibiting mitochondrial Complex I through a ubiquinone- noncompetitive mechanism comprise administering to a subject in need thereof a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the methods of inhibiting mitochondrial Complex I through a ubiquinone-noncompetitive mechanism comprise administering to a subject in need thereof a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the ubiquinone-noncompetitive mechanism involves subunit NDUFS2. According, the methods may be used to treat a disease or condition that would benefit from the inhibition of NDUFS2. The methods further relate to treating a disease or condition by administering a mitochondrial Complex I inhibitor to a subject in need thereof.

[0075] In one aspect, described herein is the use of an inhibitor of the mitochondrial Complex I in the treatment or prevention of a disease or condition in a mammal that would benefit from the inhibition or reduction of mitochondrial Complex I activity. In some embodiments, the inhibitor is a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the inhibitor is a compound of Formula (II), or a pharmaceutically acceptable salt or solvate thereof. Subjects

[0076] A suitable subject for the treatment may be a mammal such as human, dog, cat, horse, or any animal in which the inhibition of mitochondrial Complex I may potentially be desirable. In embodiments, the subject is a human.

[0077] In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is a solid tumor. Exemplary solid tumors include, but are not limited to, breast, ovarian, prostate, lung, kidney, gastric, colon, testicular, head and neck, pancreas, brain, melanoma, and other tumors of tissue organs and hematological tumors, such as lymphomas and leukemias, including acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia, and B cell lymphomas.

[0078] In embodiments, the subject has or is diagnosed with a prostate cancer. The prostate cancer may be non-invasive, invasive, and/or metastatic prostate cancer. In embodiments, the prostate cancer is a grade 1, grade 2, grade 3, grade 4, or grade 5 prostate cancer. In embodiments, the prostate acinar is adenocarcinoma, ductal adenocarcinoma, transitional cell (or urothelial) cancer, squamous cell cancer, small cell prostate cancer, or soft tissue sarcoma.

[0079] In some embodiments, the cancer is an antiandrogen-resistant cancer, i.e., a cancer that is resistant to antiandrogen therapy. Antiandrogen therapy prevents androgen from binding to androgen receptors in prostate cancer cells and inhibits the growth of cancer cells. Antiandrogen therapy may comprise the administration of an androgen receptor antagonist, an androgen synthesis inhibitor, an antigonadotropin, or other modulators that affect the production and/or activity of androgen such as estrogens, anticorticotropins, ovandrotone albumin, and androstenedione albumin. In some embodiments, the cancer is resistant to an androgen synthesis inhibitor. In some embodiments, the cancer is resistant to an androgen receptor antagonist.

[0080] Based on the structure of the androgen receptor antagonist, they are generally classified into two classes: steroidal and nonsteroidal. Steroidal AR antagonists are those structurally similar to steroids, and nonsteroidal AR antagonists are those not steroids and not structurally similar to steroids. In some embodiments, the cancer is resistant to steroidal antiandrogen (SAA), i.e., an SAA-resistant cancer. In some embodiments, the cancer is resistant to nonsteroidal antiandrogen (NSAA), i.e., an NSAA-resistant cancer. In further embodiments, the cancer is resistant to a first generation NSAA such as flutamide, nilutamide, bicalutamide, and topilutamide. In some embodiments, the cancer is resistant to a second generation NSAA such as apalutamide and enzalutamide. In some embodiments, the cancer is resistant to an NSAA that is not a first generation or a second generation NSAA, for example, cimetidine.

[0081] In some particular embodiments, the cancer is resistant to flutamide, nilutamide, bicalutamide, topilutamide, apalutamide, enzalutamide, or any combination thereof. In further embodiments, the cancer is resistant to enzalutamide.

Combination Therapy

[0082] In one aspect, the inhibitor described herein is administered with a second therapeutic agent, i.e., a combination therapy. The second therapeutic agent can be an androgen suppression therapy, a BRAF inhibitor, or a CDK4/6 inhibitor. In some embodiments, the inhibitor described herein is administered in combination with an androgen suppression therapy. Accordingly, disclosed herein are combination therapies comprising the administration of a herein described mitochondrial inhibitor and an androgen suppression therapy. In embodiments, the inhibitor is an inhibitor of mitochondrial Complex I.

[0083] Due to varying degrees of tumor metabolic flexibility, OXPHOS inhibitors such as the compounds of Table 1 can be efficacious in combination with other drugs. Based on metabolic reprogramming toward OXPHOS induced by mutant BRAF and CDK4/6 inhibitors, both drugs can be suitable for combination with OXPHOS inhibitors. BRAF mutations could serve as biomarker. In addition, wildtype RB can be a biomarker for sensitivity to OXPHOS inhibitors in combination with CDK4/6 inhibitors. Likewise, mutations in the tumor suppressor LKB1 could serve as biomarker of the sensitivity of KRAS mutant NSCLC to OXPHOS inhibition.

[0084] In some embodiments, the inhibitor described herein is administered in combination with one or more BRAF inhibitors. Exemplary BRAF inhibitors include, but are not limited to, vemurafenib, dabrafenib, encorafenib (Braftovi), BMS-908662, LGX818, PLX3603, RAF265, R05185426, or GSK2118436. In some embodiments, the second therapeutic agent is a BRAF inhibitor that is selected from vemurafenib, dabrafenib, and encorafenib.

[0085] In some embodiments, the inhibitor described herein is administered in combination with one or more CDK4/6 inhibitors. In some embodiments, the CDK4/6 inhibitor is Abemaciclib (Verzenio), Palbociclib (Ibrance), or Ribociclib (Kisqali). In some embodiments, the second therapeutic agent is a CDK4/6 inhibitor that is selected from Abemaciclib (Verzenio), Palbociclib (Ibrance), and Ribociclib (Kisqali).

[0086] In some embodiments, OXPHOS inhibition can synergize with androgen deprivation therapy in prostate cancer, potentially by targeting the androgen-dependence of the proliferative bulk compartment simultaneously with the CSC compartment. The significant single agent activity of SMIP004-7 toward LNCaP cells (Figure 1) may be due to its suppression of the cyclinDl/RB/E2F axis, resulting in the transcriptional downregulation of the androgen receptor thus effectively targeting both cellular compartments. Through the same transcriptional pathway, the inhibitors described herein, such as the compounds of Table 1, can also downregulate SKP2. SMIP004 was recently shown to enhance radiotherapy of breast cancer xenografts through SKP2 downregulation, suggesting that high SKP2 levels may indicate sensitivity to SMIP004-7. Finally, due to frequent overexpression in major human cancers, including breast and prostate cancer, NDUFS2, the molecular target of the inhibitors described herein such as SMIP004-7, as well as other Cl subunits can serve as biomarkers of the sensitivity of the inhibitors.

[0087] In some embodiments, the androgen suppression therapy used in combination is a surgery based therapy, such as orchiectomy. In some embodiments, the androgen suppression therapy is a drug based therapy that affects the production of androgen or its binding to the androgen receptor. Accordingly, in some specific embodiments, the inhibitor is administered in combination with an orchiectomy; for example, the administration of the inhibitor may be initiated before or after the orchiectomy. In other embodiments, the inhibitor is administered in combination with a drug that affects the production or binding of androgen. For example, in embodiments, the inhibitor is administered in combination with a compound that lowers the production of androgen, which includes, but is not limited to, leuprorelin, goserelin, triptorelin, histrelin, buserelin, and degarelix. In some embodiments, the inhibitor is administered in combination with a compound that interferes the binding between androgen and androgen receptors, e.g., antiandrogens. In some embodiments, the inhibitor is administered in combination with cyproterone acetate, flutamide, nilutamide, bicalutamide, enzalutamide, apalutamide, and darolutamide. In some embodiments, the second therapeutic agent is an antiandrogen that is selected from cyproterone acetate, flutamide, nilutamide, bicalutamide, enzalutamide, apalutamide, and darolutamide. In specific embodiments, the inhibitor is administered in combination with enzalutamide.

[0088] As used herein, the term “in combination” or “combination therapy” are intended to embrace administration of these compounds (or therapies) in a sequential manner, that is, wherein each compound or therapy is administered at a different time, as well as administration of these compounds (or therapies) in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a dose having a fixed ratio of each compound or in multiple, single dose for each of the compound. As used herein, the term “simultaneously” is meant to refer to administration of one or more compounds at the same time. For example, in certain embodiments, an inhibitor and enzalutamide are administered simultaneously. The term “simultaneously” includes administration contemporaneously, that is during the same period of time. In certain embodiments, the one or more compounds (or therapies) are administered simultaneously in the same hour, or simultaneously in the same day. Sequential or substantially simultaneous administration of each compound can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, sub-cutaneous routes, intramuscular routes, direct absorption through mucous membrane tissues (e.g., nasal, mouth, vaginal, and rectal), and ocular routes (e.g., intravitreal, intraocular, etc.). The therapeutic agents can be administered by the same route or by different routes. For example, one component of a particular combination may be administered by intravenous injection while the other component(s) of the combination may be administered orally. The components may be administered in any therapeutically effective sequence. The phrase “combination” also embraces groups of compounds or non-drug therapies useful as part of a combination therapy.

[0089] The present invention is directed to methods of treating a disease or condition by administering an effective amount of the inhibitors, optionally in combination with a second therapeutic agent such as an androgen suppression therapy, a BRAF inhibitor or a CDK4/6 inhibitor. The inhibitors may be administered once daily, twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every two weeks, once every three weeks, once every four weeks, once every two months, once every six months, or once per year. The dosing interval can be adjusted according to the needs of individual patients. For longer intervals of administration, extended release or depot formulations can be used. In certain embodiments, the inhibitors of the disclosure are administered for time periods exceeding two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, two years, three years, four years, or five years. In some cases, it may be advantageous for the compounds to be administered for the remainder of the subj ecf s life. In some embodiments, the subj ect is monitored to check the progression of the disease or disorder, and the dose is adjusted accordingly. [0090] When administered as a combination, the described inhibitor and the second therapeutic agent can be formulated as separate compositions that are given at the same time or different times, or they can be given as a single composition. The combination therapy may be administered once daily, twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every two weeks, once every three weeks, once every four weeks, once every two months, once every six months, or once per year.

[0091] In certain embodiments, the inhibitors and/or the combination therapies of the disclosure are administered for time periods exceeding two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, two years, three years, four years, or five years, ten years, or fifteen years; or for example, any time period range in days, months or years in which the low end of the range is any time period between 14 days and 15 years and the upper end of the range is between 15 days and 20 years (e.g., 4 weeks and 15 years, 6 months and 20 years). In some cases, it may be advantageous for the inhibitors and/or the combination therapies to be administered for the remainder of the patient’s life. In some embodiments, the patient is monitored to check the progression of the disease or disorder, and the dose is adjusted accordingly. In some embodiments, treatment according to the disclosure is effective for at least two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, two years, three years, four years, or five years, ten years, fifteen years, twenty years, or for the remainder of the subject’s life.

[0092] The administration of the inhibitors and/or the combination therapies may begin at the diagnosis, detection, or surgical removal of tumors. Surgical resection uses surgery to remove abnormal tissue in cancer, such as mediastinal, neurogenic, or germ cell tumors, or thymoma. In certain embodiments, administration of the inhibitor and/or the combination therapy is initiated following tumor resection. In some embodiments, administration of the inhibitor and/or combination therapy is initiated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more weeks after tumor resection. In some embodiments, administration of the inhibitor and/or the combination therapy is initiated 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks after tumor resection. In certain embodiments, administration of the inhibitor and/or the combination therapy is initiated no more than 10 weeks, 20 weeks, 30 weeks, 1 year, 2 years, 5 years, 10 years, or 20 years after tumor resection.

[0093] In certain embodiments, the initiations of administration for the inhibitor and the second therapeutic agent are performed within a period of time, such as 1 hour, 12 hours, 24 hours, or on the same day. In certain embodiments, the inhibitor and the second therapeutic agent is administered simultaneously. Simultaneous administrations may be performed by the same or different routes of administration. For example, the inhibitor and the second therapeutic agent may be administered simultaneously by oral administration and subcutaneous injection, respectively. As another example, a simultaneous administration of the inhibitor and the second therapeutic agent may be performed by subcutaneous injection and intravenous injection, respectively. As a further example, the inhibitor and the second therapeutic agent may be administered simultaneously, both by subcutaneous injections. For simultaneous administrations, the inhibitor and the second therapeutic agent may be combined before administration. For example, the inhibitor and the second therapeutic agent may be combined prior to or at the administration site, or a pre-combined package of the inhibitor and the second therapeutic agent may be used.

Pharmaceutical Composition, Method of Delivery, and Dose

[0094] The herein described methods of treatment comprise administering pharmaceutical compositions comprising the inhibitors and optionally an androgen suppression therapy. [0095] The compounds of the current disclosure may be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and, if desired for local treatment, intralesional administration. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intrasternal, intraperitoneal, and infusion techniques. In some embodiments, the inhibitor is administered orally.

[0096] The compounds of the present disclosure may be administered in any convenient administrative form, e.g., tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain components conventional in pharmaceutical preparations, e.g., diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents. In some embodiments, the inhibitors are administered in solid form such as tablets and capsules. In some embodiments, the inhibitor is formulated in a liquid form and administered by injection.

[0097] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient, carrier or diluent. Suitable carriers, excipients, and dilutes are well known to those skilled in the art and are described in detail in, e.g., Ansel, Howard C, et ah, Ansel’s Pharmaceutical Dosage Forms and DrugDelivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. The compositions may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, and other known additives.

[0098] In embodiments, for solid dosage forms used in oral administration (e.g., capsules, tablets, pills, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, excipients, or diluents, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents, in the case of capsules, tablets, and pills, the pharmaceutical compositions can also comprise buffering agents. Solid compositions of a similar type can also be prepared using fillers in soft and hard-filled gelatin capsules, and excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0099] Actual dosage levels and time course of administration of the compounds in the pharmaceutical compositions of the invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0100] The amounts and dosage regimens administered to a subject can depend on a number of factors, such as the mode of administration, the nature of the condition being treated, the body weight of the subject being treated and the judgment of the prescribing physician; all such factors being within the ambit of the skilled artisan from this disclosure and the knowledge in the art. The amount of compound included within therapeutically active formulations according to the present invention is an effective amount for treating the disease or condition.

[0101] In general, a therapeutically effective amount of a compound in dosage form can range from slightly less than about 0.025 mg/kg/day to about 2.5 g/kg/day, about 0.1 mg/kg/day to about 100 mg/kg/day of the patient or considerably more, depending upon the compound used, the condition or disease treated and the route of administration, although exceptions to this dosage range may be contemplated by the present invention.

EXAMPLES

A. Chemical Synthesis

[0102] The compounds of Table 1 and Table 2 have been synthesized according to organic synthesis techniques known to those skilled in this art, starting from commercially available chemicals and/or from compounds described in the chemical literature.

Example Al- 2-Methyl-4-pentylphenylamine (HC1 salt)

[0103] Compound 2-Methyl-4-pentylphenylamine was synthesized according to scheme 1 and the following steps. Scheme 1

NM* NH ec

[0104] Step 1: (4-iodo-2 -methyl -phenyl)-carbamic acid tert-butyl ester

[0105] To a mixture of 4-iodo-2-methyl-phenylamine (1.29 g, 5.5 mmol) in toluene (40 mL) was added B0C2O (1.81 g, 8.3 mmol). The resulting mixture was then heated at 80°C for 16 h. The solvent was removed in vacuum and the residue was purified by silica gel column chromatography (PE to PE/EA = 20/1) to give (4-iodo-2-methyl-phenyl)-carbamic acid tert- butyl ester (1.8 g, 99% yield) as white solid. ¾NMR (300 MHz, CDCh): d = 7.64 (d, J= 9.7 Hz, 1H), 7.55 - 7.41 (m, 2H), 6.24 (brs, 1H), 2.21 (s, 3H), 1.53 (s, 9H). MS: m/z 334.0 (M⁺H⁺ ).

[0106] Step 2: (2-methyl-4-pent-l-ynyl-phenyl)-carbamic acid tert-butyl ester [0107] A vial charged with (4-iodo-2-methyl-phenyl)-carbamic acid tert-butyl ester (350 mg, 1.05 mmol), trimethyl -pent-l-ynyl-silane (290 mg, 2.1 mmol), Ag2CC>3 (290 mg, 1.05 mmol), (n-Bu)4N⁺CT (580 mg, 2.1 mmol) and Pd(PPh₃)2Cl₂ (70 mg, 0.1 mmol) in THF (12 mL) was bubbled with Ar for about 3 min and quickly sealed. The sealed vial was then heated at 60°C for 20 h. The reaction mixture was filtered, and the filter cake was washed with EA (10 mL x 3). The filtrate and washings were combined and concentrated. The residue was purified by silica gel column chromatography (PE to PE/EA = 20/1) to give (2 -methyl -4-pent-l-ynyl-phenyl)-carbamic acid tert-butyl ester (200 mg, 70% yield) as a yellow solid. ¾NMR (300 MHz, CDCh): d = 7.82 (d, J= 8.8 Hz, 1H), 7.27-7.11 (m, 2H), 6.29 (brs, 1H), 2.38 (t, J= 6.9 Hz, 2H), 2.21 (s, 3H), 1.72-1.58 (m, 2H), 1.53 (s, 9H), 1.05 (t, J= 7.2 Hz, 3H). MS: m/z 174.1 (M-100⁺H⁺ ).

[0108] Step 3: 2 -Methyl -4-pentyl -phenylamine

[0109] A flask charged with (2-methyl-4-pent-l -ynyl-phenyl)-carbamic acid tert-butyl ester (120 mg, 0.44 mmol) and PtCE (23 mg, O.lmml) in MeOH (10 mL) and EA (10 mL) was degassed and filled with hydrogen using a balloon. The resulting mixture was hydrogenated at room temperature for 4 h then filtered. The filtrate was concentrated under reduced pressure and the residue was purified by prep-TLC (PE/EA = 20/1) to give (2-methyl-4-pentyl-phenyl)-carbamic acid tert-butyl ester as a yellow oil. The material was treated with HC1 (cone., 5 mL) and MeOH (5 mL), stirred for 15 min at room temperature and concentrated to dryness. The dry product was triturated with MeCN (5 mL) and collected by filtration to give pure 2-methyl-4-pentyl-phenylamine (45 mg, HC1 salt, 48% yield) as a white solid. ¾ NMR (400 MHz, DMSO-r/d): d =10.02 (brs, 3H), 7.31 (d, J = 8.0 Hz, 1H), 7.16 (s, 1H), 7.11 (dd, J= 8.0, 1.5 Hz, 1H), 2.59-2.5 (m, 2H), 2.32 (s, 3H), 1.62-1.50 (m, 2H), 1.38-1.19 (m, 4H), 0.86 (t, J= 7.0 Hz, 3H). MS: m/z 178.1 (M⁺H⁺ ).

Example A2- 2-Methyl-4-propylphenylamine (HC1 salt)

[0110] The title compound was prepared by following the procedures similar to the ones used in the synthesis of 2-methyl -4-pentylphenylamine. ¹HNMR(400 MHz, DMSO-r/d/· 5 =10.03 (brs, 3H), 7.30 (d, J= 8.0 Hz, 1H), 7.14 (s, 1H), 7.10 (d, J= 8.0 Hz, 1H), 2.54-2.50 (m, 2H), 2.31 (s, 3H), 1.58-1.52 (m, 2H), 0.87 (t, J= 7.2 Hz, 3H). MS: m/z 150.1 (M⁺H⁺).

Example A3- 4-Butyl-2-methoxyphenylamine (HC1 salt)

[0111] The title compound was prepared by following the procedures similar to the ones used in the synthesis of 2 -methyl -4-pentylphenylamine. ¾ NMR (400 MHz, DMSO-r/d/· 5 = 9.98 (brs, 3H), 7.34 (d, J= 8.0 Hz, 1H), 7.04 (d, J= 1.2 Hz, 1H), 6.84 (d, J= 8.0 Hz, 1.2H), 3.87 (s, 3H), 2.59 (t, J= 8.0 Hz, 2H), 1.59-1.54 (m, 2H), 1.34-1.28 (m, 2H), 0.90 (t, J= 7.2 Hz, 3H). MS: m/z 180.1 (M⁺H⁺).

Example A4- 4-Butyl-2-chloro-phenylamine (HC1 salt)

[0112] The title compound was prepared by following the procedures similar to the ones used in the synthesis of 2 -methyl -4-pentylphenylamine. ¾ NMR (400 MHz, DMSO-r/d/· 5 = 7.34 (brs, 3H), 7.20 (s, 1H), 7.08-7.01 (m, 2H), 2.49-2.45 (m, 2H), 1.52-1.44 (m, 2H), 1.31-1.23 (m, 2H), 0.86 (t, J= 7.2 Hz, 3H). MS: m/z 184.0 (M⁺H⁺).

Example A5- 4-(2-Methoxy-ethyl)-2-methyl-phenylamine (HC1 salt)

[0113] The title compound was synthesized according to scheme 2 and the following steps. Scheme 2

[0114] Step 1: 2-Methyl-l-nitro-4-vinyl -benzene

[0115] A flask charged with 4-iodo-2-methyl-l-nitro-benzene (1 g, 4.6 mmol), potassium vinyltrifluoroborate (930 mg, 6.9 mmol), Pd(dppf)Ch (335 mg, 0.46 mmol) and K2CC>3 (1.26 g, 9.2 mml) in dioxane (70 mL) and water (10 mL) was degassed and backfilled with N2. The mixture was heated at 90° C for 16 h. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (PE to PE/EA = 10/1) to give 2-methyl-l-nitro-4-vinyl-benzene (550 mg, 73% yield) as a yellow oil. ¾ NMR (400 MHz, CD₃OD -d4): d = 7.93 (d, J= 9.0 Hz, 1H), 7.54-7.32 (m, 2H), 6.77 (dd, J= 17.6, 11.0 Hz, 1H), 5.96 (d, J= 17.6 Hz, 1H), 5.44 (d, J= 11.0 Hz, 1H), 2.56 (s, 3H).

[0116] Step 2: 2-(3-Methyl-4-nitro-phenyl)-ethanol

[0117] To a mixture of 2-methyl-l-nitro-4-vinyl-benzene (300 mg, 1.84 mmol) in THF (10 mL) at 0°C under N² was added dropwise B¾ x SMe2 (10 M in THF, 1.8 mL). After addition, the resulting mixture was stirred for 4 h at room temperature and slowly quenched with MeOH (2 mL). Then aq. NaOH (4N, 9 mL) and H2O2 (35%, 5 mL) were added and the mixture was stirred for an additional 14 h at room temperature. The reaction mixture was then diluted with water (30 mL) and extracted with EA (25 mL x3). The extracts were dried over INfeSCri and concentrated to dryness. The residue was purified by prep-TLC (PE/EA = 2:1) to give the desired 2-(3-methyl-4-nitro-phenyl)-ethanol (165 mg, 49% yield) as brown oil. ¾NMR (300 MHz, CDC13): d = 7.97 (d, J= 9.0 Hz, 1H), 7.26-7.18 (m, 2H), 3.92 (t, J= 6.0 Hz, 2H), 2.93 (t, J= 6.6 Hz, 2H), 2.62 (s, 3H). MS: m/z 182.2 (M⁺H⁺).

[0118] Step 3: 4-(2-Methoxy-ethyl)-2-methyl-l-nitro-benzene

[0119] To a mixture of 2-(3-methyl-4-nitro-phenyl)-ethanol (165 mg, 0.91 mmol) in THF at 0°C was added NaH (-60% in mineral oil, 55 mg, 1.37 mmol) under N2, and the mixture was stirred for 20 min at room temperature. Mel (0.5 mL, 9.1 mmol) was added in one portion. The resulting mixture was stirred for 16 h at room temperature, quenched with MeOH (1 mL) and concentrated. The residue was purified by prep-TLC (PE/EA = 5/1) to give 4-(2 -methoxy-ethyl)-2 -methyl-1 -nitro-benzene (125 mg, 70% yield) as a yellow solid. MS: m/z 196.1 (M⁺H⁺). [0120] Step 4: 4-(2-Methoxy-ethyl)-2-methyl-phenylamine

[0121] A flask charged with 4-(2 -methoxy-ethyl)-2 -methyl-1 -nitro-benzene (125 mg, 0.64 mmol) and Pd/C (~50 mg, 40% wt.) in MeOH (10 mL) was degassed and backfilled with hydrogen using a balloon. The resulting mixture was then hydrogenated at room temperature for 4 h and filtered. To the filtrate was added HC1 (cone., 0.2 mL) and the solution was concentrated to dryness. The residue was triturated with MeCN (3 mL x2) and the solid was collected by filtration to give 4 -(2-m ethoxy -ethyl)- 2-methyl-phenylamine (HC1 salt, 42 mg, 33% yield) as a white solid.

[0122] ¾ NMR (400 MHz, DMSO-i/d): d = 10.31 (brs, 3H), 7.36 (d, J= 8.0 Hz, 1H), 7.18 (s, 1H), 7.14 (d, J= 8.0 Hz, 1H), 3.51 (t, J= 6.7 Hz, 2H), 3.22 (s, 3H), 2.78 (t, J= 6.7 Hz, 2H), 2.34 (s, 3H). MS: m/z 166.1 (M⁺H⁺).

Example A6- 4-Butyl-2-ethyl-phenylamine (HC1 salt)

[0123] The title compound was synthesized according to scheme 3 and the following steps.

Scheme 3

[0124] Step 1: 2-Ethyl -4-iodo-phenylamine

[0125] To a mixture of 2-ethyl-phenylamine (1.00 g, 8.3 mmol) in DCM/MeOH (20 mL/10 mL) was added aqueous NaHCCh (1.39 g in 10 mL of water, 16.5 mmol) at 0°C. After addition, a solution of benzyltriethylammonium dichloroiodate (3.86 g, 9.9 mmol) in DCM (10 mL) added dropwise over a 10 min period. The reaction mixture was allowed to reached room temperature and stirred for an additional 30 min, quenched with water (10 mL) and extracted with DCM (50 mL x 3). The combined organic extracts were washed with brine (50 mL) and water (50 mL) then evaporated to dryness. The crude product was purified by silica gel chromatography (PE to PE/EA = 50/1) to give 2-ethyl -4-iodo- phenylamine (1.76 g, 87 % yield) as brown solid. ¾ NMR (400 MHz, DMSO-<¾. d = 7.16-7.14 (m, 2H), 6.44 (d, 7 = 8.0 Hz, 1H), 5.08 (s, 2H), 2.38 (q, 7 = 7.6 Hz, 2H), 1.08 (t, 7 = 7.6 Hz, 3H).

[0126] Step 2: (2-Ethyl-4-iodo-phenyl)-carbamic acid tert- butyl ester

[0127] To a mixture of 2-ethyl -4-iodo-phenylamine (1.76 g, 7.0 mmol) in THF (30 mL) was added (BOC)20 (3.06 g, 14.0 mmol). The resulting mixture was heated at 70°C overnight. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (PE to PE/EA = 50/1) to give (2-ethyl-4-iodo-phenyl)-carbamic acid tert- butyl ester (2.43 g, 100% yield) as brown solid, which was used in the next step with no purification. ¾ NMR (300 MHz, DMSO-£¾. d = 8.58 (S, lH), 7.51-7.46 (m, 2H), 7.11 (d, 7= 8.1 Hz, 1H), 2.55 (q, 7= 7.5 Hz, 1H), 1.44 (s, 9H), 1.07 (t, 7 = 7.5 Hz, 3H).

[0128] Step 3: (4-But-l-ynyl-2-ethyl-phenyl) -carbamic acid tert- butyl ester

[0129] A vial charged with (2-ethyl-4-iodo-phenyl)-carbamic acid tert- butyl ester (100 mg, 0.29 mmol), but-l-ynyl-trimethyl-silane (73 mg, 0.58 mmol), Ag2CC>3 (80 mg, 0.29 mmol), tetrabutyl ammonium chloride (160 mg, 0.58 mmol) and Pd(PPli3)2Cl2 (20 mg, 0.03 mmol) in THF (10 mL) was bubbled with Ar for about 3 min and was quickly sealed. The sealed vial was then heated at 60oC overnight. The reaction mixture was filtered, and the filter cake was washed with EA (10 mL x3). The filtrate and washings were combined, concentrated and the residue was purified by perp-TLC (PE/EA = 20/1) to give (4-but-l-ynyl -2-ethyl-phenyl) -carbamic acid tert- butyl ester (70 mg, 89 % yield) as yellow solid. ¾NMR (300 MHz, DMSO^ d = 8.57 (s, 1H), 7.28 (d, 7= 8.4 Hz, 1H), 7.18- 7.13 (m, 2H), 2.55 (q, 7= 7.5 Hz, 2H), 2.39 (q, 7= 7.5 Hz, 2H), 1.44 (s, 9H), 1.14 (t, 7= 7.5 Hz, 3H), 1.08 (t, 7= 7.5 Hz, 3H).

[0130] Step 4: (4 -Butyl -2 -ethyl- phenyl)-carbamic acid tert- butyl ester

[0131] A flask charged with (4-but-l-ynyl-2-ethyl-phenyl)-carbamic acid tert- butyl ester (200 mg, 0.73 mmol) and PtCh (23 mg, O.lmml) inMeOH(10 mL) and EA(10 mL) was degassed and backfilled with hydrogen using a balloon. The resulting mixture was then hydrogenated at room temperature for 4 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give (4-butyl-2 -ethyl- phenyl)-carbamic acid tert- butyl ester (200 mg, 99 % yield) as yellow oil. 'H NMR (300 MHz, DMSO-76 d = 8.41 (brs, 1H), 7.09 (d, 7= 8.4 Hz, 1H), 6.98 (s, 1H), 6.94 (d, 7= 8.4 Hz, 1H), 2.54 (q, 7= 7.5 Hz, 2H), 1.56-1.22 (m, 15H), 1.08 (t, 7= 7.5 Hz, 3H), 0.88 (t, 7= 7.4 Hz, 3H). [0132] Step 5: 4-Butyl -2-ethyl -phenylamine

[0133] To a mxiture of (4-butyl-2-ethyl-phenyl)-carbamic acid tert- butyl ester (200 mg, 0.72 mmol) in MeOH (5 mL) was added HC1 (cone., 2 mL) at room temperature. The resulting mixture was concentrated under reduced pressure. The crude product was triturated with MeCN (5 mL x 2) and collected by filtration to give 4-butyl-2-ethyl-phenylamine (HC1 salt, 94 mg, 61% yield) as white solid. ¾NMR (400 MHz, DMSO-i¾ d = 9.93 (brs, 3H), 7.28 (d, J= 8.4 Hz, 1H), 7.16 (s, 1H), 7.11 (d, J = 8.0 Hz, 1H), 2.65 (q, J= 7.6 Hz, 2H), 2.57 (t, J= 7.8 Hz, 2H), 1.57-1.49 (m, 2H), 1.34-1.25 (m, 2H), 1.19 (t, J= 7.6 Hz, 3H), 0.89 (t, J= 7.2 Hz, 3H). MS: m/z 178.1 (M⁺H⁺ ).

Example A7- 4-Butyl-2,6-dimethylphenylamine (HC1 salt)

[0134] The title compound was prepared by following the procedures similar to the ones used in the synthesis of 2-ethyl-4-pentylphenylamine. 'H NMR (400 MHz, DMSO-r/d/· d =9.68 (brs, 3H), 6.95 (s, 2H), 2.50 (t, J= 7.2 Hz, 2H), 2.33 (s, 6H), 1.55-1.47 (m, 2H), 1.32-1.23 (m, 2H), 0.88 (t, J= 7.6 Hz, 3H). MS: m/z 178.1 (M⁺H⁺).

Example A8- 4-Butyl-2,5-dimethylphenylamine (HC1 salt)

[0135] The title compound was prepared by following the procedures similar to the ones used in the synthesis of 2-ethyl-4-pentylphenylamine. 'H NMR (400 MHz, DMSO-r/d/· d = 10.02 (brs, 3H), 7.14 (s, 1H), 7.07 (s, 1H), 2.52 (t, J= 8.0 Hz, 2H), 2.27 (s, 3H), 2.23 (s, 3H), 1.50-1.43 (m, 2H), 1.38-1.29 (m, 2H), 0.91 (t, J= 7.2 Hz, 3H). MS: m/z 178.1 (M⁺H⁺).

Example A9- (4-Butyl-2-methyl-phenyl)-dimethyl-amine (HC1 salt)

[0136] To a mixture of commercially available 4-butyl-2-methylaniline (SMIP004-7, 80 mg, 0.49 mmol) and K2CO3 (140 mg, 1 mmol) in DMF (5 mL) was added CH3I (282 mg, 2 mmol) in one portion at room temperature. The resulting mixture was then stirred for 4 h at room temperature diluted with water (20 mL) and extracted with EA (20 mL x 3). The extracts were combined and concentrated. The residue was purified by prep-TLC (PE/EA = 2:1) and treated with several drops of cone. HC1 to give (4-butyl-2-methyl-phenyl)-dimethyl-amine (HC1 salt, 20 mg, 21% yield) as yellow oil after freeze drying. ¾ NMR (400 MHz, DMSO-i/dj: d = 7.72 (d, J= 8.4 Hz, 1H), 7.25-7.20 (m, 2H), 3.15 (s, 6H), 2.58 (s, 3H), 2.56-2.53 (m, 2H), 1.59-1.50 (m, 2H), 1.32-1.24 (m, 2H), 0.89 (t, J= 7.4 Hz, 3H). MS: m/z 192.1 (M⁺H⁺).

Example A10- N-(4-Butyl-2-methylphenyl)-N-methylacetamide

[0137] The title compound was synthesized according to scheme 4 and the following steps. Scheme 4

[0138] Step 1: /V-(4-Butyl -2 -methyl -phenyl)-acetamide

[0139] A mixture of 4-butyl -2 -methyl -phenyl amine (100 mg, 0.61 mmol) and AC2O (122 mg, 1.2 mmol) in EtOH (5 mL) was stirred for 16 h at room temperature. Solvent was removed to give N-( 4- butyl-2-methyl-phenyl)-acetamide (120 mg, crude) as a yellow oil, which was used in the next step without further purification. MS: m/z 192.1 (M⁺H⁺).

[0140] Step 2. L-64-Butyl -2- ethyl -phenyl )-A-m ethyl -acetami de

[0141] To a mixture of A-/4-butyl -2-methyl -phenyl )-acetamide (120 mg, 0.61 mmol) in DMF (3 mL) at 0°C was added NaH (-60% in mineral oil, 48 mg, 1.2 mmol) in portions under N2. The resulting mixture was stirred for 20 min at 0°C, and then CH3I (130 mg, 0.92 mmol) was added in one portion. The reaction mixture was stirred for another 2 h at room temperature, quenched with aq. NH4CI (20 mL) and the aqueous phase was extracted with EA (20 mL x 3). The extracts were concentrated and the residue was purified by prep-TLC (PE/EA = 4:1) to give A-/4-butyl -2-methyl -phenyl )-A-methyl - acetamide (35 mg, 26% yield over two steps) as a yellow oil. ¾ NMR (400 MHz, CDC13): d = 7.09 (s, 1H), 7.06-6.99 (m, 2H), 3.16 (s, 3H), 2.59 (t, J= 8.0 Hz, 2H), 2.20 (s, 3H), 1.77 (s, 3H), 1.63-1.56 (m, 2H), 1.39-1.33 (m, 2H), 0.94 (t, J= 7.2 Hz, 3H). MS: m/z 220.1 (M⁺H⁺).

Example All- 3-(4-Amino-3-methyl-phenyl)-propan-l-ol (HC1 salt)

[0142] The title compound was synthesized according to scheme 5 and the following steps.

Scheme 5

[0143] Step 1: (4-Iodo-2 -methyl -phenyl)-carbamic acid /er/-butyl ester

[0144] To a mixture of 4-iodo-2-methyl-phenylamine (1.29 g, 5.5 mmol) in toluene (40 mL) was added B0C2O (1.81 g, 8.3 mmol). The resulting mixture was heated at 80°C for 16 h. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (PE to PE/EA = 20/1) to give (4-iodo-2-methyl-phenyl)-carbamic acid /er/-butyl ester (1.8 g, 99% yield) as white solid. 1H NMR (300 MHz, CDC13): d = 7.64 (d, J= 9.7 Hz, 1H), 7.55-7.41 (m, 2H), 6.24 (brs, 1H), 2.21 (s, 3H), 1.53 (s, 9H). MS: m/z 334.0 (M+H+ ).

[0145] Step 2: /tvV-Butyl (4-(3-hydroxyprop-l-yn-l-yl)-2-methylphenyl)-carbamate [0146] A flask charged with (4-iodo-2-methyl-phenyl)-carbamic acid tert- butyl ester (2.5 g, 7.5 mmol), prop-2 -yn-l-ol (2.1 g, 37.5 mmol), Cul (142 mg, 0.75 mmol), Pd(PPh3)2Cl2 (1.1 g, 1.5 mmol) and TEA (2.2 g, 21.5 mmol) in THF (25 mL) was degassed and backfilled with N2. The resulting mixture was then heated at 40oC for 14 h. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (PE to PE/EA = 10/1) to give /cvV-butyl (4- (3-hydroxyprop-l-yn-l-yl)-2-methylphenyl)-carbamate (970 mg, 56% yield) as yellow oil. 1H NMR (300 MHz, CDC13): d = 7.87 (d, J= 9.0 Hz, 1H), 7.30-7.22 (m, 2H), 6.36 (brs, 1H), 4.47 (s, 2H), 2.22 (s, 3H), 1.53 (s, 9H). MS: m/z 262.1 (M+H+).

[0147] Step 3: [4-(3-Hydroxy-propyl)-2 -methyl -phenyl]-carbamic acid tert- butyl ester [0148] A flask charged with give /cvV-butyl (4-(3-hydroxyprop-l-yn-l-yl)-2- methylphenyl)carbamate (970 mg, 3.7 mmol) and Pd/C (-100 mg, wet, 10% wt.) in MeOH (20 mL) was degassed and backfilled with hydrogen using a balloon. The resulting mixture was hydrogenated at room temperature for 5 h, filtered. The filtrate was evaporated under reduced pressure and the residue was purified by prep-TLC (PE/EA = 5:1) to give [4-(3 -hy droxy-propyl)-2 -methyl -phenyl] - carbamic acid /er/-butyl ester (950 mg, 96% yield) as a yellow oil. MS: m/z 266.1 (M+H+).

[0149] Step 4: 3 -(4-Amino-3 -methyl -phenyl)-propan-l-ol

[0150] A mixture of [4-(3-hydroxy-propyl)-2-methyl-phenyl]-carbamic acid tert-butyl ester (100 mg, 0.38 mmol) in dioxane (5 mL) and HCI (cone., 3 mL) was stirred for 30 min at room temperature. The solvent was removed under reduced pressure and the residue was purified by prep-HPLC (0.1% HCI) to give 3-(4-amino-3-methyl-phenyl)-propan-l-ol (HCI salt, 22 mg, 40 % yield) as a white solid. 1H NMR (400 MHz, DMSO^d): d = 10.09 (brs, 3H), 7.32 (d, J= 8 Hz, 1H), 7.15 (s, 1H), 7.13-7.09 (m, 1H), 3.39 (t, J = 6.4 Hz, 2H), 2.60-2.55 (m, 2H), 2.31 (s, 3H), 1.72-1.65 (m, 2H). MS: m/z 166.1 (M+H+ ).

Example A12- 4-(3-Aminopropyl)-2-methylphenylamine (HCI salt)

[0151] The title compound was synthesized according to scheme 6 and the following steps.

Scheme 6

[0152] Step 1: (4-Iodo-2 -methyl -phenyl)-carbamic acid tert-butyl ester

[0153] To a mixture of 4-iodo-2-methyl-phenylamine (1.29 g, 5.5 mmol) in toluene (40 mL) was added B0C2O (1.81 g, 8.3 mmol). The resulting mixture was then heated at 80°C for 16 h. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (PE to PE/EA = 20/1) to give (4-iodo-2 -methyl -phenyl)-carbamic acid tert- butyl ester (1.8 g, 99% yield) as white solid. 1H NMR (300 MHz, CDC13): d = 7.64 (d, J = 9.7 Hz, 1H), 7.55- 7.41 (m, 2H), 6.24 (brs, 1H), 2.21 (s, 3H), 1.53 (s, 9H). MS: m/z 334.0 (M+H+ ).

[0154] Step 2: 3 -(4-Amino-3 -methyl -phenyl)-propan-l-ol

[0155] A flask charged with (4-iodo-2-methyl-phenyl)-carbamic acid tert-butyl ester (2.5 g, 7.5 mmol), prop-2 -yn-l-ol (2.1 g, 37.5 mmol), Cul (142 mg, 0.75 mmol), Pd(PPh3)2Cl2 (1.1 g, 1.5 mmol) and TEA (2.2 g, 21.5 mmol) in THF (25 mL) was degassed and backfilled with N2. The resulting mixture was then heated at 40°C for 14 h. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (PE to PE/EA = 10/1) to give 3-(4-amino- 3-methyl-phenyl)-propan-l-ol (970 mg, 56% yield) as yellow oil. 1H NMR (300 MHz, CDCb): d = 7.87 (d, J= 9.0 Hz, 1H), 7.30-7.22 (m, 2H), 6.36 (brs, 1H), 4.47 (s, 2H), 2.22 (s, 3H), 1.53 (s, 9H). MS: m/z 262.1 (M+H+).

[0156] Step 3: [4-(3-Hydroxy-propyl)-2 -methyl -phenylj-carbamic acid tert- butyl ester [0157] Aflask charged with 3 -(4-amino-3 -methyl -phenyl)-propan-l-ol (970 mg, 3.7 mmol) andPd/C (-100 mg, wet, 10% wt.) in MeOH (20 mL) was degassed and backfilled with hydrogen using a balloon. The resulting mixture was then hydrogenated at room temperature for 5 h. The reaction mixture was filtered and the filtrate was purified by prep-TLC (PE/EA = 5:1) to give [4-(3-hydroxy-propyl)-2- methyl-phenyl]-carbamic acid tert- butyl ester (950 mg, 96% yiled) as a yellow oil. MS: m/z 266.1 (M+H+).

[0158] Step 4: Methanesulfonic acid 3 -(4-/<3/7-butoxycarbonyl ami no-3 -methyl -phenyl )-propyl _ester [0159] To a mixture of [4-(3-hydroxy-propyl)-2-methyl-phenyl]-carbamic acid tert-butyl ester (100 mg, 0.61 mmol) and TEA(183mg, 1.82 mmol)inDCM(10 mL) was added MsCl (103 mg, 0.91 mmol) at 0 °C under N2. The resulting mixture was stirred for 2 h at room temperature. The reaction mixture was quenched with aq. NaHCCh (10 mL) and the aqueous phase was extracted with DCM (15 mL x3). The extracts were concentrated. The residue was purified by prep-TLC (PE/EA =1/1) to give methanesulfonic acid 3-(4-tert-butoxycarbonylamino-3-methyl-phenyl)-propyl ester (170 mg, 82% yield) as a yellow oil. MS: m/z 344.1 (M+H+).

[0160] Step 5: [4-(3-Azido-propyl)-2-methyl-phenyl]-carbamic acid tert- butyl ester [0161] A mixture of methanesulfonic acid 3 -(4-tert-butoxycarbonylamino-3 -methyl -phenyl)-propyl ester (170 mg, 0.49 mmol) and NaN3 (48 mg, 0.74 mmol) in DMF (5 mL) was heated at 100°C for 16 h. The reaction mixture was cooled, diluted with water (20 mL), and extracted with EA (15 mL x3). The extracts were dried over anhydrous NaiSCE, filtered and concentrated to give [4-(3-azido-propyl)- 2-methyl-phenyl]-carbamic acid tert- butyl ester (100 mg, crude) as a yellow oil, which was used in the next step with no purification.

[0162] Step 6: [4-(3-Amino-propyl)-2-methyl-phenyl]-carbamic acid tert- butyl ester [0163] A mixture of [4-(3-azido-propyl)-2-methyl-phenyl]-carbamic acid tert-butyl ester (100 mg, 0.35 mmol) and Pd/C (-20 mg, wet, 20% wt.) in MeOH (10 mL) was hydrogenated at room temperature for 3 h (balloon). The reaction mixture was filtered through a celite pad, and the filtrate was concentrated under reduced pressure to give [4-(3-amino-propyl)-2-methyl-phenyl]-carbamic acid tert- butyl ester (80 mg, crude) as a yellow oil, which was used in the next step with no purification. MS: m/z 265.2 (M+H+).

[0164] Step 7: 4-(3-Aminopropyl)-2-methylphenylamine

[0165] A mixture of [4-(3-amino-propyl)-2 -methyl -phenyl]-carbamic acid /er/-butyl ester (80 mg, 0.30 mmol) in dioxane (3 mL) and HC1 (cone., 2 mL) was stirred for 30 min at room temperature. The solvent was removed under reduced pressure and the residue was purified by prep-HPLC (0.1% HC1) to give 4-(3-aminopropyl)-2-methylphenylamine (2 HC1 salt, 8 mg, 13% yield (over 3 steps)) as a white solid. 1HNMR (400 MHz, CD OD): d = 7.30-7.27 (m, 2H), 7.23-7.21 (m, 1H), 2.95 (t, J= 7.6 Hz, 2H), 2.73 (t, J= 8.0 Hz, 2H), 2.39 (s, 3H), 1.99-1.93 (m, 2H). MS: m/z 165.1 (M+H+ ).

Example A13- (4-Butyl-2-methylphenyl)-methylamine (HC1 salt)

[0166] The title compound was synthesized according to scheme 7 and the following steps.

Scheme 7

[0167] Step 1: (4-Butyl -2 -methyl -phenyl)-carbamic acid tert- butyl ester

[0168] To a solution of 4-butyl -2 -methyl -phenylamine (150 mg, 0.92 mmol) in toluene (5 mL) was added (Boc)₂0 (300mg, 1.38 mmol). The resulting mixture was then heated at 90°C overnight, the solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (PE to PE/EA = 3/1) to give (4-butyl-2-methyl-phenyl)-carbamic acid tert- butyl ester (220 mg, 91% yield) as white solid. MS: m/z 264.2 (M+H+).

[0169] Step 2: (4-Butyl-2-methyl-phenyl)-methyl-carbamic acid tert- butyl ester [0170] To a mixture of 4-butyl-2 -methyl -phenyl)-carbamic acid tert- butyl ester (220 mg, 0.84 mmol) in THF (5 mL) was added NaH (-60%, 67 mg, 1.67 mmol) in portions at 0°C under N2. The resulting mixture was stirred for 20 min at 0°C, and then CH3I (176 mg, 1.2 mmol) was added in one portion. The reaction mixture was stirred for 16 h at room temperature. The reaction mixture was quenched with aq. NH4CI (20 mL) and extracted with EA (20 mL x3). The extracts were concentrated. The residue was purified by silica gel column chromatography (PE to PE/EA =5/1) to give (4-butyl-2- methyl-phenyl)-methyl-carbamic acid tert- butyl ester (110 mg, 48% yield) as white solid. MS: m/z 278.2 (M+H+). [0171] Step 3: (4-Butyl -2 -methyl -phenyl)-methylamine

[0172] A mixture of (4-butyl-2-methyl-phenyl)-methyl-carbamic acid tert- butyl ester (110 mg, 0.39 mmol) in dioxane (3 mL) and HC1 (cone., 2 mL) was stirred at room temperature for 30 min. The solvent was removed under reduced pressure and the residue was purified by prep-HPLC (0.1% HC1) to give (4-butyl-2-methyl-phenyl)-methylamine (HC1 salt, 54 mg, 77% yield) as a white solid. 1H NMR (400 MHz, DMSO-i/d): d = 10.9 (brs, 2H), 7.38 (d, J= 7.6 Hz, 1H), 7.18-7.15 (m, 2H), 2.86 (s, 3H), 2.58-2.51 (m, 2H), 2.37 (s,3H), 1.57-1.49 (m, 2H), 1.33-1.24 (m, 2H), 0.91-0.84 (m, 3H). MS: m/z 178.1 (M+H+ ).

Example A14- 5-Butyl-lH-indole

[0173] The title compound was synthesized according to scheme 8 and the following steps.

Scheme 8

[0174] Step 1: 5-But-l-enyl-lH-indole

[0175] To a mixture of lH-indole-5-carbaldehyde (290 mg, 2 mmol) in dry THF (10 mL) at -20 °C was added dropwise NaHMDS (2M, 1.5 mL) via syringe. The resulting mixture was stirred for 1 hr at -20 °C, and was allowed to warm to 0 °C. The reaction mixture was then cooled to -78 °C and a solution of bromotriphenyl(propyl)phosphorane (1.07 g, 3 mmol) in THF (5 mL) was added dropwise over 20 min. The mixture was stirred for 2 hrs at -78 °C, then allowed to warm to room temperature and stirred for another 14 hrs. The mixture was quenched with aq. NH4C1 (about 10 mL) dropwise at 0 °C and the layers were separated. The aqueous phase was extracted with EA (10 mL x3), and the organic phases were combined and concentrated. The residue was purified by prep-TLC (PE/EA = 5:1) to give 5-but- 1-enyl-lH-indole (100 mg, 29% yield) as a brown oil. 1HNMR (400 MHz, CDC13): d = 8.10 (br, 1H), 7.57 (s, 1H), 7.36-7.32 (m, 1H), 7.20-7.18 (m, 1H), 7.17-7.12 (m, 1H), 6.56-6.50 (m, 2H), 5.64-5.56 (m, 1H), 2.41-2.34 (m, 2H), 1.53-4.45 (m, 3H).

[0176] Step 2: 5-Butyl-lH-indole

[0177] A mixture of 5-but-l-enyl-lH-indole (100 mg, 0.58 mmol) and Pd/C (~20 mg, 20% wt) in EtOH (8 mL) was hydrogenated under H2 using a balloon for 16 hrs at room temperature. The mixture was filtered and the residue was purified by prep-TLC (PE/EA =5 : 1) to give desired 5-butyl-lH-indole (30 mg, 29% yield) as a yellow oil. 1H NMR (400 MHz, DMSO-i/d) d = 10.91 (brs, 1H), 7.40-7.15 (m, 3H), 7.00-6.79 (m, 1H), 6.32 (s, 1H), 2.64-2.57 (m, 2H), 1.70 - 1.49 (m, 2H), 1.37 - 1.23 (m, 2H), 0.86 (t, J= 7.0 Hz, 3H).

Example A15- 4-Butyl-2,3-dimethyl-phenylamine (HC1 salt)

[0178] The title compound was synthesized according to scheme 9 and the following steps.

[0179] Step 1: 4-Iodo-2, 3-dimethyl -phenylamine

[0180] To a solution of 2, 3-dimethyl -phenylamine (1.00 g, 8.3 mmol) in DCM/MeOH (20 mL/10 mL) was added aqueous NaHC03 (1.39 g, 16.5 mmol) at 0 °C followed by a solution of benzyltriethylammonium dichloroiodate (3.86 g, 9.9 mmol) in DCM (5 mL), added dropwise over a period of 5 min. The resulting mixture was stirred for 16 hrs, over which time it was allowed to warm to room temperature. The mixture was then diluted with DCM (50 mL) and water (40 mL) and the phases were separated. The organic phase was washed with brine (40 mL) and concentrated. The residue was purified by flash chromatography (PE/EA = 87/13) to give 4-iodo-2,3-dimethyl- phenylamine (1.0 g, 49% yield) as a brown solid. 1HNMR (300 MHz, DMSO-<¾. d = 7.48 (d, J= 8.4 Hz, 1H), 6.35 (d, J= 8.4 Hz, 1H), 3.65 (brs, 2H), 2.48 (s, 3H), 2.24 (s,3H). MS: m/z 247.9 (M+H+ ). [0181] Step 2: (4-Iodo-2,3-dimethyl-phenyl)-carbamic acid tert-butyl ester

[0182] To a mixture of 4-iodo-2, 3-dimethyl -phenylamine (1.0 g, 4 mmol) in toluene (40 mL) was added B0C2O (1.32 g, 6 mmol). The resulting mixture was then heated at 80 °C for 16 h. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (PE/EA = 93/7) to give (4-iodo-2,3-dimethyl-phenyl)-carbamic acid tert-butyl ester (1.2 g, 86% yield) as a yellow solid. MS: m/z 291.9 (M-56+H+).

[0183] Step 3: (4-But-l-ynyl-2, 3-dimethyl -phenyl)-carbamic acid tert- butyl ester [0184] A vial charged with (4-iodo-2, 3 -dimethyl -phenyl)-carbamic acid tert- butyl ester (200 mg, 0.58 mmol), trimethyl -pent- 1-ynyl -silane (145 mg, 1.15 mmol), Ag2CC>3 (159 mg, 0.58 mmol), (nBu)4N⁺CT (319 mg, 1.15 mmol) and Pd(PPh3)2Cl₂ (41 mg, 0.058 mmol) in THF (10 mL) was bubbled with Ar for about 3 min and was quickly sealed. The sealed vial was then heated at 65 °C for 16 hrs, filtered and the filter cake was washed with EA (10 mL x3). The filtrate and washings were combined, concentrated and the residue was purified by prep-TLC (PE/EA = 15/1) to give (4-but-l- ynyl-2, 3-dimethyl -phenyl)-carbamic acid tert- butyl ester (150 mg, 95% yield) as a brown oil. MS: m/z 174.0 (M-100+H+).

[0185] Step 4: 4-Butyl-2,3-dimethyl-phenylamine

[0186] A flask charged with (4-but-l-ynyl-2,3-dimethyl-phenyl)-carbamic acid tert- butyl ester (150 mg, 0.55 mmol) and PtCh (30 mg, 0.13 mml) in MeOH (8 mL) and EA (4 mL) was degassed and filled with hydrogen using a balloon. The resulting mixture was then hydrogenated for 2 hrs at room temperature. The mixture was filtered, and the residue was purified by prep-HPLC (NH4HC03 system) to give (4-butyl -2, 3 -dimethyl -phenyl)-carbamic acid tert- butyl ester as a yellow solid, which was treated with HC1 (cone., 5 mL) and MeOH (5 mL), stirred for 15 min at room temperature, and concentrated. The residue was triturated with MeCN (3 mL x2) and collected by filtration to give 4- butyl -2, 3 -dimethyl -phenylamine (21 mg, HC1 salt, 41% yield over 2 steps) as a white solid. 1HNMR (400 MHz, DMSO-i/d) d = 9.58 (brs, 3H), 7.14-6.98 (m, 2H), 2.64-2.55 (m, 2H), 2.20 (s, 3H), 2.17 (s, 3H), 1.50-1.38 (m, 2H), 1.37-1.29 (m, 2H), 0.90 (t, J =1 Hz, 3H). MS: m/z 178.1 (M+H+ ).

B. Biological Experiments

Example Bl- Effect of SMIP004-7 on cancer cell viability

[0187] The effects of SMIP004-7 on cancer cell viability under conditions that mimic the CSC microenvironment were tested. Various prostate (LNCaP, 22RV1, LAPC4), colon cancer (HCT116), and mouse triple negative breast cancer (4T1) cell lines were grown either as 2D monolayers or as 3D sphere cultures to enrich for CSCs. Cells were treated with SMIP004-7 (10 mM) for 7 days after which single cell suspensions were assayed for their ability to form colonies. SMIP004-7 suppressed colony formation substantially more efficiently when cells were treated as 3D rather than 2D cultures (see, FIG.1 A). We also assessed the effect of SMIP004-7 by exposing 2D monolayer and 3D sphere cultures to increasing doses of the compound followed by determination of cell viability. In both LNCaP and LNCaP-SKP2 cells, which stably overexpress the SKP2 oncogene, the dose-response curve shifted to lower IC50 values when cells were grown as 3D spheres compared to 2D monolayers (see, FIG. IB and FIG.1C).

[0188] We tested whether glucose limitation affects the sensitivity to SMIP004-7. Switching cells to low glucose media or to galactose media, where cells are rendered dependent on OXPHOS, sensitized them to SMIP004-7-mediated cytotoxicity (see, FIG. ID and FIG. IE).

[0189] We also tested the effect of SMIP004-7 on LNCaP cells rendered castration resistant by long term culture in the presence of the androgen receptor blocker enzalutamide. Enzalutamide-resistant LNCaP cells retained the same sensitivity to SMIP004-7 as seen for parental, androgen-dependent LNCaP cells (see, FIG. IF and FIG.1G). At the same time, androgen withdrawal from LNCaP and LNCaP-SKP2 cells strongly synergized with SMIP004-7, suggesting that the compound may be effectively combined with androgen deprivation therapy.

[0190] Lastly, we assessed the effect of SMIP004-7 on well characterized human patient derived breast cancer stem cells (BCSCs). These CD24-/CD44+ and EpCAM+/CD49f+ cells were derived from triple negative primary breast cancers after extensive chemotherapy (Metzger et al., 2017). Despite their multi -drug resistance, the growth of these BCSCs was effectively inhibited by SMIP004- 7 but not by the inactive analog SMIP004-4 (see, FIG.IH-FIG.J). FIG.1A-FIG.1J illustrate the effect of SMIP004-7 on cells with sternness features. As shown in FIG.1A, the indicated cancer cell lines were grown as 2D monolayers or as 3D tumor spheres and exposed to 10 mM SMIP004-7 for 72 h. Single cell suspensions were plated on culture dishes and colony forming ability was detected after 21 days by staining with crystal violet. The graph indicates the percentage of inhibition of colony formation.

[0191] In FIG. IB and FIG.1C, LNCaP and LNCaP-SKP2 cells were grown in 2D monolayers or as 3D spheres for 7 days. Single cell suspensions were plated in 2D format for 1 day, followed by exposure to SMIP004-7 for 96 h and viability measurement by CCK8 assay. As shown in FIG. ID and FIG. IE, LNCaP and LNCaP-SKP2 cells were maintained in media containing the indicated carbon sources and sensitivity to SMIP004-7 was measured by CCK8 assay after 96 h. FIG. IF and FIG.1G illustrates that parental LNCaP cells and LNCaPEr cells rendered resistant to enzalutamide were exposed to increasing doses of enzalutamide or SMIP004-7, followed by determination of cell viability with the MTT assay after 96 h. FIG. 1H-FIG.J illustrates that three BCSC lines were exposed to the indicated concentrations of SMIP004-7 or the inactive analog SMIP004-4 for increasing periods. Cell density was monitored in real time in the Incucyte device.

Example B2- SMIP004-7 Targets NADHrUbiquinone Oxidoreductase

[0192] It is believed that the suppression of mitochondrial respiration by SMIP004-7 occurs through direct inhibition of one of the five complexes of the respiration chain. To obtain an indication of which complex may be targeted, we compared quantitative proteomic profiles of cells treated with SMIP004- 7 or with known inhibitors of each of the five respiration chain complexes. Hierarchical clustering revealed that the proteomic signature of cells treated with SMIP004-7 most closely resembled the signature imposed by the Complex I (Cl) inhibitor rotenone (see, FIG.2A), suggesting that NADH:ubiquinone oxidoreductase may be targeted by SMIP004-7. [0193] This conjecture was confirmed when we observed inhibition of Cl - but not CII, III, or IV - activity by SMIP004-7 assayed in submitochondrial particles (SMPs) in vitro (see, FIG.2B). Cl was inhibited by SMIP004-7 and its active analog SMIP004, but not by the inactive analog SMIP004-4 (see, FIG.2B). The ~50-fold difference in the potencies of SMIP004-7 in cell killing (IC50 ~ 2 mM) versus inhibition of NADH oxidation in vitro (IC50 ~ 100 mM) strongly suggests active enrichment of the compound in the mitochondrial matrix of cells.

[0194] In order to determine whether Cl is the relevant target mediating the cytotoxic activity of SMIP004-7, cell viability was rescued by stable overexpression of yeast Ndil, a structurally distinct NADH dehydrogenase that can functionally replace human Cl. Whereas cells overexpressing Ndil were ~5 times less sensitive to SMIP004-7 than empty vector controls (see, FIG.2C and FIG.2D), no such difference was observed in sensitivity toward other cytotoxic agents, including doxorubicin and bortezomib. The rescue was also observed upon transient overexpression of Ndil. These suggested that inhibition of Cl underlies cancer cell selective cytotoxicity mediated by SMIP004-7.

[0195] FIG.2A-FIG.2D illustrate the effect of SMIP004-7 on mitochondrial respiration complexes. As shown in FIG. 2A, quantitative proteomic profiles were obtained by TMT labeling and LC -MS/MS analysis of LNCaP cells treated for 6 h with the indicated mitochondrial inhibitors at their respective IC50 for cytotoxicity. Hierarchical clustering revealed that the protein profile of cells treated with SMIP004-7 most closely resembles that of cells treated with Cl inhibitor rotenone. In FIG.2B, the effect of the indicated SMIP004-7 analogs on the activity of NADH:ubiquinone oxidoreductase in vitro activity was measured in submitochondrial particles isolated from DAI -3b leukemia cells. In FIG.2C and FIG.2D, parental LNCaP cells or LNCaP cells stably expressing yeast Ndi 1 were assessed for their sensitivity to SMIP004-7 by CCK8 assay after 96 h.

Example B3- SMIP004-7 Mediated Cytotoxicity

[0196] Experiments have been carried out to determine which of the 45 subunits of Cl is targeted by SMIP004-7. It is believed that the compound interfered with one of the 7 catalytic subunits of the matrix arm, and thus cell lines stably overexpressing each of these subunits were created. Overexpression of a single subunit, NDUFS2, conferred resistance to SMIP004-7 similar to what was observed with Ndil (see, FIG.3 A-FIG.3C). The same rescue was observed by transient overexpression of NDUFS2 but not NDUFS3. NDUFS2 overexpression did not rescue cytotoxicity induced by metformin, another inhibitor of Cl.

[0197] Cl in vitro activity was ~2-fold increased in SMPs isolated from cells overexpressing NDUFS2 but ~2-fold decreased upon knockdown of NDUFS2 (FIG.3D-FIG.3F). Even high concentrations of SMIP004-7 did not inhibited Cl in SMPs from NDUFS2 overexpressing cells below the level obtained with SMPs from vector controls, suggesting that the rescue of cell death is due to increased catalytic activity of Cl.

[0198] Rescue by NDUFS2 also depended on the mitochondrial localization of the overexpressed protein as it was abolished by the removal of the N-terminal 33 residues comprising the mitochondrial targeting signal (see, FIG.3G-FIG.3I). Also at the N-terminus, NDUFS2 has a helical extension stretching from the matrix arm far into the membrane arm, thereby potentially coupling NADH oxidation with proton transport (FIG.3A-FIG.3C). Removal of this extension (residues 34 - 78) completely abolished the ability of NDUFS2 to rescue SMIP004 -7 -induced cytotoxicity despite efficient mitochondrial localization of the truncated protein (FIG.3G-FIG.3I). Further mapping by successive deletion of three helical segments narrowed the region required for rescue to helix 2 (FIG.3J-FIG.3L). Alanine scanning along helix 2 pinpointed two consecutive residues of NDUFS2, His 60 and Trp 61, as critical for conferring resistance to SMIP004-7 (FIG.3M). Taken together, these data strongly suggest that SMIP004-7 inhibits Cl through interaction with His 60 and Trp 61 of subunit NDUFS2.

[0199] FIGs. 3 A-3E illustrate that SMIP004-7 mediated cytotoxicity is rescued by the overexpression of NDUFS2. As shown in FIG.3A, LNCaP cells stably expressing one of the 7 catalytic subunits of the matrix arm (highlighted in space fill) were assessed for sensitivity to SMIP004-7 after 96 h. Ectopic expression of subunit NDUFS2 (indicated in blue) rescues SMIP004-7 mediated cytotoxicity. Cl structure is from PBD 5xtd (Guo et al., 2017). In FIG.3D-FIG.3F, the effect of SMIP004-7 on the in vitro activity of Cl was assessed in SMPs isolated from cells overexpressing NDUFS2 or from cells in which NDUFS2 was knocked down by siRNA. In FIG.3G-FIG.3I, NDUFS2 missing either the mitochondrial targeting sequence (residues 2 - 33) or the N-terminal extension (residues 34 - 78) was ectopically expressed in LNCaP cells for 48 h, and the sensitivity of these cells to SMIP004-7 mediated cytotoxicity was determined by CCK8 assay after 96 h. The subcellular localization of the ectopically expressed NDUFS2 variants was determined by immunofluorescence staining. In FIG.3J-FIG.3L, successive N-terminal truncation mutants of NDUFS2 were ectopically expressed in LNCaP cells for 48 h to determine their efficacy in rescuing SMIP004-7 mediated cytotoxicity 96 h after exposure to the compound. FIG.3M shows that NDUFS2 proteins with single amino acid mutations to alanine along N-terminal helix 2 were ectopically expressed in LNCaP cells for 48 h and tested for their ability to rescue SMIP004-7 mediated cytotoxicity after 96 h. Residues His 60 and Trp 61 which are required for rescue are highlighted in the structure in red. Example B4- Enzyme Kinetics

[0200] To determine whether SMIP004-7 targets the ubiquinone- 10 binding site, steady-state enzyme kinetic parameters of Cl in the presence of piericidin A and SMIP004-7 were compared using SMPs. When assayed with decylubiquinone or ubiquinone- 1 (Ql), SMIP004-7, despite some SMP batch- dependent variability, robustly reduced both Vmax and Km, indicating that SMIP004-7 is an uncompetitive inhibitor with regards to the quinones (FIG.4A). In contrast, inhibition by piericidin A was mostly a Km effect (FIG.4B), a finding consistent with the established partial quinone competitive mechanism of this inhibitor. Despite limitations in interpreting Michaelis-Menten type kinetics for the complex catalytic mechanism of Cl, the different kinetics obtained with piericidin A and SMIP004-7 indicate that SMIP004-7 and its analogs interact with a site distinct from the quinone pocket and thus define a new class of uncompetitive Cl inhibitors.

[0201] FIGs. 4A-4B illustrate the effect of SMIP004-7 on NADH oxidation by Cl in vitro. As shown in FIG. 4A, Cl mediated NADH oxidation was assayed in SMPs in the presence of different concentrations of SMIP004-7 and ubiquinone analogs (ubiquinone Ql or decylubiquinone). The enzymatic data were fitted to Michaelis-Menten kinetics and Vmax and Km for ubiquinones were calculated. FIG. 4B shows the same experiment as in FIG. 4 A, but in the presence of SMIP004-7 (100 mM) or the ubiquinone-competitive Cl inhibitor piericidin A (100 nM).

Example B5- Target Engagement of SMIP004-7 In Cells and In Vivo

[0202] The above experiments suggest that SMIP004-7 inhibits Cl through interaction with the N- terminal extension of NDUFS2. To assess target engagement in cells, cellular thermal shift assays (CETSA) (Molina et ak, 2013) were performed. LNCaP cells were treated with 20 pM SMIP004-7 for 2 hours after which cell aliquots were incubated at increasing temperatures to denature cellular proteins. Following lysate preparation and separation of the soluble and insoluble fractions, the thermal stability of NDUFS2 was assessed by immunoblotting of the soluble protein. The melting temperature of NDUFS2 in cells treated with the inactive analog SMIP004-4 was ~58 °C (FIG.5A). SMIP004-7 shifted the melting temperature to > 64 °C, indicating considerable stabilization of NDUFS2 by the compound (FIG.5A). The stability of NDUFSl, another subunit of the matrix arm, was not affected by SMIP004 compounds.

[0203] Isothermal CETSA carried out at 61 °C revealed dose-dependent stabilization of NDUFS2 by SMIP004-7 with an EC50 of 20.38 pM (FIG.5B). Similar selective stabilization of NDUFS2 by SMIP004-7 was also observed in HCT116 cells and in the triple negative mouse breast cancer cell line 4T1. In addition, NDUFS2 but not NDUFSl was stabilized in 4T1 mouse xenograft tumors 2 hours after injection of 100 mg/kg SMIP004-7 (FIG.5C). These results strongly suggest that SMIP004-7 engages its target NDUFS2 in cancer cells in vitro and in tumors in vivo.

[0204] FIG.5A-FIG.5D illustrate cellular thermal shift assays (CETSA) showing stabilization of NDUFS2 by SMIP004-7. As shown in FIG. 5A, LNCaP cells were incubated in the presence of 20 mM SMIP004-7 or 2 hours and equal aliquots of cells were exposed to the indicated temperatures for 4 minutes. Cell lysate was prepared, the insoluble fraction was pelleted by centrifugation and the soluble fraction was analyzed by immunoblotting with the indicated antibodies detecting NDUFS2, NDUFS1, and GAPDH. FIG. 5B shows that Isothermal CETSA performed at 61 °C after adding increasing concentrations of SMIP004-7 to LNCaP cells. The stabilized NDUFS2 band was quantified and the EC50 was calculated. In FIG.5C, CETSA assay, as shown in FIG. 5A, was carried out with the indicated cell lines. As shown in FIG. 5D, 5xl0⁴4T1 cancer cells were injected into the mammary fat pad of Balb/C mice and allowed to form a tumor. When tumor size reached -500 mm³, SMIP004- 7 was injected (100 mg/kg, i.p.) and the tumor was harvested after 2 hours. The tumor was dissociated into a single cell suspension which was used for CETSA to determine the stability of NDUFS2 and NDUFS 1.

Example B6- Structure-Activity Relationship

[0205] As shown in Table 3 and Table 4, initial structure-activity relationships were obtained by performing an exploratory round of chemical synthesis and assays.

Table 3. Structure-Activity Relationship

Table 4. Structure-Activity Relationship

Compound ID Structure Name IC50 in vitro LNCaP

[0206] In particular, the indole analog SBI-0802936 (see, Table 4) inhibited Cl in vitro activity 4 - 6 times more potently than SMIP004-7, acting in the same uncompetitive fashion as SMIP004-7 (FIG.6 A). SBI-0802936 also stabilized NDUFS2 in cells (FIG.6B), killed LNCaP and 4T1 cells at low or sub-micromolar IC50 (FIG.6C and FIG.6D) in a manner that was rescued by overexpression of NDUFS2 depending on the N-terminal extension, inhibited the growth of human BCSCs (FIG.6E), and inhibited orthotopic 4T1 xenografts in immunocompetent mice without causing weight loss (FIG.6F and FIG.6G). The indole analog eliminated the aniline group while maintaining full on target anti-cancer activity. The exploratory experiments suggested that further modifications of SMIP004-7 toward higher potency and improved pharmacology are feasible.

[0207] FIG.6A-FIG.6H illustrate the effect of an indole analog of SMIP004-7 on cell viability and NDUFS2 stability. FIG.6A shows the dose-dependent inhibition of Cl in vitro activity by SMIP004-7 and indole analog SBI-0802936. In FIG.6B, CETSA with SMIP004-7 and indole analog SBI-0802936 (each at 20 mM, 2 hours) were added to LNCaP cells. FIG.6C-FIG.6D shows that the cytotoxicity of SMTP004-7 and indole analog SBI-0802936 was assessed in LNCaP and 4T1 cells by CCK8 assay after 96 h. In FIG.6E, BCSC1 breast cancer stem cells were exposed to the indicated concentrations of SMIP004-7 or indole analog SBI-0802936 for increasing periods. Cell density was monitored in real time in the Incucyte device. As shown in FIG.6F and FIG.6G, 5xl0⁴ 4T1 cells were injected into the mammary fat pad of Balb/C mice. When tumors reached -100 mm³, mice were administered 100 mg/kg SBI-0802936 (i.p., every other day for 3 weeks). Tumor volumes and body weights were measured.

Example B7-Effect of SMIP004-7 on 4T1 tumor growth in immunocompetent and immunodeficient hosts

[0208] In FIG.7A-FIG.7D, 5 x 10⁴4T1 cells were injected into the mammary fat pad of BALB/c or SCID mice. When tumors reached -50 mm³, mice were administered 100 mg/kg SMIP004-7 (i.p., 5 days on, 2 days off). Tumor volumes and body weights were measured. In FIG.7E-FIG.7F, 5 x 10⁴ 4T1 cells were injected into the mammary fat pad of BALB/c mice. When tumors reached -50 mm³, mice were administered 100 mg/kg SMIP004-7 (i.p., 5 days on, 2 days off) and/or anti-PD-1 antibody (5 mg/kg twice per week). Tumor volumes and body weights were measured.

[0209] The activity of SMIP004-7 observed against 4T1 xenografts in immunocompetent BALB/c hosts (FIG.7A and FIG.7C) was pronounced. We therefore compared the activity of SMIP004-7 observed against 4T1 xenografts in immunocompetent BALB/c mice with the activity in immunodeficient SCID mice under an identical dosing scheme (5 days on, 2 days off). Remarkably, unlike in BALB/c hosts, SMIP004-7 had minimal activity against 4T1 tumors in SCID mice (FIG.7B and FIG.7D), indicating that SMIP004-7 activity against 4T1 tumors is largely dependent on a functional host immune system. [0210] We thus asked whether SMIP004-7 treatment modulates the sensitivity of 4T1 xenografts grown in BALB/c mice to immune checkpoint inhibition by anti-PDl antibodies. As shown in FIG.7E and FIG.7F, anti-PD-1 treatment showed little activity against orthotopic 4T1 tumors, whereas SMIP004-7 showed consistent single agent tumoristatic activity. Combination of anti-PD-1 with SMIP004-7 showed increased anti -turn or activity, leading to complete eradication of 4T1 tumors in 2 out of 6 mice in the cohort (FIG.7E and FIG.7F). These data suggest that inhibition of Cl by SMTP004- 7 promotes anti -cancer immune surveillance and the efficacy of immune checkpoint blockade.

[0211] The examples and embodiments described herein are for illustrative purposes only and various modifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of treating a disease or condition that would benefit from inhibition of mitochondrial respiration by inhibiting mitochondrial NADH: ubiquinone reductase (Complex I), comprising administering to a subject in need thereof a therapeutically effective amount of a mitochondrial Complex I inhibitor, wherein the inhibitor is a compound that has the structure of Formula I, or a pharmaceutically acceptable salt, or solvate thereof:

wherein:

X is CH orN;

Ri is hydrogen, Ci-Cs alkyl, halogen, C₁-C₆ alkenyl, C₁-C₆ alkynyl, -OR₅, Ci-Cr, alkyl -O-R_5. or C0-C₆ alkyl-N(R5)2;

R3 is hydrogen, halogen, C1-C₆ alkyl, -OR5, or C1-C₆ alkyl -O-R5; each of R₂ and R₄ is independently hydrogen, C₁-C₆ alkyl, or -L-R₅, wherein L is -0-, - NC(=0)R₅-, or -NR5-;

A is hydrogen, C1-C₆ alkyl -O-R5, C1-C₆ alkyl -N(Rs)2, -OR5, or Ci-Cx alkyl; and each R₅ is independently hydrogen or C₁-C₆ alkyl.

2. The method of claim 1, wherein the disease or condition is a cancer.

3. A method of treating cancer in a mammal comprising administering an inhibitor of the mitochondrial NADFfubiquinone reductase (Complex I) to the mammal in need thereof.

4. A method of inhibiting the activity of the mitochondrial NADFfubiquinone reductase (Complex I) in a mammal with cancer comprising administering to the mammal with cancer an inhibitor of the mitochondrial NADFfubiquinone reductase (Complex I).

5. The method of claim 3 or claim 4, wherein the inhibitor of the mitochondrial NADFfubiquinone reductase (Complex I) interacts with the catalytic subunit NDUFS2 of the mitochondrial NADFfubiquinone reductase (Complex I).

6. The method of any one of claims 3-5, wherein the inhibitor is a small molecule.

7. The method of claim 6, wherein the inhibitor is a compound that has the structure of Formula (I), or a pharmaceutically acceptable salt, or solvate thereof:

wherein:

X is CH orN;

Ri is hydrogen, Ci-Cx alkyl, halogen, C1-C6 alkenyl, C1-C6 alkynyl, -OR₅, Ci-Cr, alkyl -O-R_5. or Co-C₆ alkyl-N(R₅)2;

A is hydrogen, C1-C6 alkyl -O-R5_, C1-C6 alkyl -N(Rs)2, -OR5, or Ci-Cx alkyl; and each R₅ is independently hydrogen or C₁-C₆ alkyl.

8. The method of any one of claims 1-7, wherein:

X is CH.

9. The method of any one of claims 1-7, wherein:

X is N.

10. The method of any one of claims 1-9, wherein:

Ri is hydrogen, C1-C6 alkyl, halogen, -OR5, C1-C3 alkyl-O-Rs, or C0-C3 alkyl-N(Rs)2.

11. The method of any one of claims 1-10, wherein:

Ri is hydrogen, C₁-C₃ alkyl, halogen, -OR₅, or -N(R₅)_2..

12. The method of any one of claims 1-11, wherein:

Ri is H, -CH3, CH2CH3, Cl, -OCH3, or NH_2.

13. The method of any one of claims 1-12, wherein:

R₂ is C₁-C₆ alkyl or -L-R₅, wherein L is -0-, -NC(=0)Rs-, or -NR₅-.

14. The method of any one of claims 1-13, wherein:

R2 is -L-R5, wherein L is -0-, -NC(=0)Rs-, or -NR5-.

15. The method of any one of claims 1-14, wherein:

R₂ is OH, NH₂, NHCH3, N(CH )₂, orN(C=0)CH_3.

16. The method of any one of claims 1-13, wherein:

R₂ is CH _.

17. The method of any one of claims 1-16, wherein:

R.₃ is hydrogen, halogen, C1-C3 alkyl, -OR₅, or C1-C3 alkyl-O-Rs

18. The method of any one of claims 1-17, wherein:

R₃ is hydrogen or C₁-C₃ alkyl.

19. The method of any one of claims 1-18, wherein:

R₂ is hydrogen or CH₃.

20. The method of any one of claims 1-19, wherein:

R4 is hydrogen, C1-C3 alkyl, or -L-R5, wherein L is -0-, -NC(=0)Rs-, or -NR5-.

21. The method of any one of claims 1-20, wherein:

R₄ is hydrogen or C₁-C₃ alkyl.

22. The method of any one of claims 1-21, wherein:

R₄ is hydrogen or CH₃.

23. The method of any one of claims 1-22, wherein:

A is C1-C6 alkyl-O-Rs, C1-C6 alkyl-N(Rs)2, -OR5, or C1-C6 alkyl.

24. The method of any one of claims 1-23, wherein:

A is C1-C3 alkyl-O-Rs, C1-C3 alkyl -NH2, -OR5, or C1-C6 alkyl.

25. The method of any one of claims 1-24, wherein:

A is CH₂CH₂OH, CH₂CH₂CH₂OH, CH₂CH₂OCH₃, CH₂CH₂CH₂OCH₃, CH₂CH₂NH₂, CH2CH2CH2NH2, CH3, CH2CH3, n-C₃H₇, n-C₄H₉, or n-CsHn.

26. The method of any one of claims 1-25, wherein:

R₅ is hydrogen.

27. The method of any one of claims 1-25, wherein:

R5 is C1-C3 alkyl.

28. The method of any one of claims 1-25, wherein:

R5 is methyl, ethyl, «-propyl, or 1 -methyl ethyl (/-propyl).

29. The method of any one of claims 1-7, wherein the inhibitor is a compound that is selected from the group consisting of:

4-Butyl -2 -methylaniline,

N-(4-butyl-2-methylphenyl)-N-methylacetamide,

4-butyl -2-ethylphenylamine,

(4-butyl -2 -methylphenyl)methylamine,

2-methyl -4-pentylphenylamine, 4-butyl-2,5-dimethylphenylamine,

4-(2-methoxyethyl)-2-methylphenylamine,

4-(3-aminopropyl)-2-methylphenylamine,

2-methyl -4-propoxyaniline,

4-butyl -2-chlorophenylamine,

(4-butyl -2 -methylphenyl)dimethylamine,

2-methyl -4-propylphenylamine,

4-butyl -2 -methoxyphenylamine,

3 -(4-amino-3 -methylphenyl)propan- 1 -ol,

4-butyl -2, 6-dimethylphenylamine,

5-butyl -2 -methylaniline,

6-butyl -4-methylpyri din-3 -amine,

6-butyl -2 -methylpyri din-3 -amine,

4-butyl-2-methylphenol, and 4-butyl -2, 3-dimethylphenylamine.

30. The method of any one of claims 1-7, wherein the inhibitor is a compound in Table 1.

31. The method of any one of claims 3-6, wherein the inhibitor is a compound that has the structure of Formula (II), or a pharmaceutically acceptable salt, or solvate thereof:

Formula (II) wherein:

Z is hydrogen, Ci-Cs alkyl, C1-C6 alkenyl, C1-C6 alkynyl, -ORx, Ci-Cealkyl-O-Rs, or -N(Rx)2; R₆ is hydrogen or C1-C6 alkyl;

R7 is hydrogen or C1-C6 alkyl; each R₈ is independently hydrogen or C1-C6 alkyl; each of R₉, Rio and Rn is independently selected from hydrogen and C1-C6 alkyl, or R₉ and Rio are taken together with the carbon atoms to which they are attached to form a double bond; and each of Y2, Y3, and Y4 is independently CH or N.

32. A method of treating a disease or condition that would benefit from inhibition of mitochondrial respiration by inhibiting mitochondrial NADH: ubiquinone reductase (Complex I), comprising administering to a subject in need thereof a therapeutically effective amount of a mitochondrial Complex I inhibitor, wherein the inhibitor is a compound that has the structure Formula II, or a pharmaceutically acceptable salt, or solvate thereof:

wherein:

Z is hydrogen, Ci-Cs alkyl, C1-C6 alkenyl, C1-C6 alkynyl, -ORx, Ci-Cealkyl-O-Rs, or -N(Rs)2; R₆ is hydrogen or C1-C6 alkyl;

R₇ is hydrogen or C1-C6 alkyl; each R₈ is independently hydrogen or C1-C6 alkyl; each of R₉, Rio and Rn is independently selected from hydrogen and C1-C6 alkyl, or R9 and Rio are taken together with the carbon atoms to which they are attached to form a double bond; and each of Y2, Y3, and Y4 is independently CH or N.

33. The method of claim 32, wherein the disease or condition is a cancer.

34. The method of any one of claims 31 to 33, wherein: each of Y2, Y3, and Y4 is CH.

35. The method of any one of claims 31 to 33, wherein: one of Y2, Y3, and Y4 is N.

36. The method of any one of claims 31 to 35, wherein: i is C1-C4 alkyl.

37. The method of any one of claims 31 to 36, wherein: i is methyl, ethyl, «-propyl, /-propyl, «-butyl, /-butyl, 5-butyl, or /-butyl.

38. The method of any one of claims 31 to 37, wherein:

R₉ and Rio are taken together with the carbon atoms to which they are attached to form a double bond.

39. The method of any one of claims 31 to 38, wherein:

R₇ is hydrogen.

40. The method of any one of claims 31 to 39, wherein:

Z is Ci-Ce alkyl orN(Rs)2.

41. The method of any one of claims 31 to 40, wherein:

Z is Ci-C₆ alkyl orN(Rs)2.

42. The method of any one of claims 31 to 41, wherein:

Z is n-C₄H₉, N(CH )₂ or NH_2.

43. The method of any one of claims 31 to 33, wherein the inhibitor is a compound that is selected from the group consisting of:

2-tert-Butyl - 1 H-indol -5 -amine,

5-butyl-lH-indole, and 5 -butyl -2,3 -dihydro- 1 H-indole.

44. The method of any one of claims 2-31 or 33-43, wherein the cancer is prostate cancer, breast cancer, lung cancer, liver cancer, pancreatic cancer, colon cancer, colorectal cancer, brain cancer, head and neck cancer, melanoma, kidney cancer, or heart cancer.

45. The method of any one of claims 2-31 or 33-44, wherein the cancer is breast cancer.

46. The method of any one of claims 2-31 or 33-44, wherein the cancer is a hormone dependent cancer.

47. The method of any one of claims 2-31 or 33-44, wherein the cancer is a hormone refractory cancer.

48. The method of any one of claims 2-31 or 33-44, wherein the cancer is resistant to treatment with an antiandrogen agent or the cancer is refractory to treatment with an antiandrogen agent.

49. The method of any one of claims 2-31 or 33-44, wherein the cancer is prostate cancer.

50. The method of claim 49, wherein the prostate cancer is castration-resistant prostate cancer.

51. The method of claim 49, wherein the prostate cancer is acinar adenocarcinoma, ductal adenocarcinoma, transitional cell (or urothelial) cancer, squamous cell cancer, small cell prostate cancer, or soft tissue sarcoma.

52. The method of any one of claims 49-51, wherein the prostate cancer is a grade 1 prostate cancer, grade 2 prostate cancer, grade 3 prostate cancer, grade 4 prostate cancer, or grade 5 prostate cancer.

53. The method of claim 48, wherein the antiandrogen agent is a steroidal antiandrogen or a nonsteroidal antiandrogen.

54. The method of claim 48, wherein the nonsteroidal antiandrogen is a first-generation nonsteroidal antiandrogen.

55. The method of claim 54, wherein the first-generation nonsteroidal antiandrogen is flutamide, nilutamide, bicalutamide, or topilutamide.

56. The method of claim 48, wherein the nonsteroidal antiandrogen is a second-generation nonsteroidal antiandrogen.

57. The method of claim 56, wherein the second-generation nonsteroidal antiandrogen is apalutamide or enzalutamide.

58. The method of any one of the claims 1-57, wherein the inhibitor is administered in combination with an androgen suppression therapy.

59. A method of treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a mitochondrial Complex I inhibitor and a second therapeutic agent, wherein the inhibitor is a compound that has the structure of Formula (I), or a pharmaceutically acceptable salt, or solvate thereof:

Formula I wherein:

X is CH orN;

Ri is hydrogen, Ci-Cx alkyl, halogen, C1-C6 alkenyl, C1-C6 alkynyl, -OR₅, Ci-Cr, alkyl -O-R_5. or C0-C₆ alkyl-N(R5)2;

60. A method of treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a mitochondrial Complex I inhibitor and a second therapeutic agent, wherein the inhibitor is a compound that has the structure of Formula (II), or a pharmaceutically acceptable salt, or solvate thereof:

wherein:

61. The method of claim 59 or 60, wherein the disease or condition is prostate cancer.

62. The method of any one of claims 59-61, wherein the second therapeutic agent is an anti androgen.

63. The method of claim 62, wherein the antiandrogen is a steroidal antiandrogen or a nonsteroidal antiandrogen.

64. The method of claim 63, wherein the nonsteroidal antiandrogen is a first-generation nonsteroidal antiandrogen.

65. The method of claim 64, wherein the first-generation nonsteroidal antiandrogen is flutamide, nilutamide, bicalutamide, or topilutamide.

66. The method of claim 63, wherein the nonsteroidal antiandrogen is a second-generation nonsteroidal antiandrogen.

67. The method of claim 66, wherein the second-generation nonsteroidal antiandrogen is apalutamide or enzalutamide.

68. The method of claim 59 or 60, wherein the disease or condition is breast cancer.

69. The method of claim 68, wherein the breast cancer is a triple negative breast cancer.

70. The method of any one of claims 59-60, 68 or 69, wherein the second therapeutic agent is a BRAF inhibitor.

71. The method of claim 70, wherein the BRAF inhibitor is Vemurafenib, dabrafenib, or encorafenib.

72. The method of any one of claims 59-60, 68 or 69, wherein the second therapeutic agent is a CDK4/6 inhibitor.

73. The method of claim 72, wherein the CDK4/6 inhibitor is palbociclib, ribociclib, or abemaciclib.