Preparation and Use of 7-Azaindenoisoquinoline

Abstract

This specification describes novel 8,9-dialkoxy-7-azaindenoisoquinoline compounds (I), processes for their preparation, and their use in the treatment of diseases responsive to inhibition of topoisomerase I and / or binding to the c-MYC G-quadruplex. 【Chemical 1】 TIFF2025516331000079.tif41159

Description

Technical Field

[0001] Government Rights This invention was made with government support under grants U01CA089566, P30CACA023168, U01CA240346, and R01CA177585 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 337,248, filed May 2, 2022, and U.S. Provisional Application No. 63 / 351,550, filed Jun. 13, 2022, which are hereby incorporated by reference herein under 35 U.S.C. § 119(e).

[0003] The present disclosure relates to novel 7 - azaindenoisoquinoline compounds, processes for their preparation, and their use for various therapeutic applications, particularly for the treatment of cancer. The present disclosure relates to novel inhibitors of topoisomerase 1 (Top1) and compounds that block the activity of the MYC cancer gene by binding to, for example, the MYC promoter G - quadruplex. In some embodiments, the present disclosure relates to methods for treating patients having cancer by targeting the MYC cancer gene or otherwise blocking the activity of the MYC cancer gene. In some embodiments, the present disclosure relates to methods for treating patients having cancer by targeting human topoisomerase I. In some other embodiments, the present disclosure relates to methods for treating patients having cancer by a dual - mechanism of action that targets both the MYC cancer gene and human topoisomerase I.

Background Art

[0004] Cancer is a group of diverse diseases involving abnormal cell growth. Currently, over 100 types of cancer that affect humans and animals have been identified. In 2018 alone in the United States, an estimated 1,708,921 new human cancer cases were diagnosed, and 599,265 people died from cancer (Cancer Statistics 2018 - Centers for Disease Control and Prevention). The need for new treatments for cancer remains unmet and is increasing.

[0005] DNA is the target of many important anticancer agents, including human topoisomerase I inhibitors. Recently, there have been significant advances in the development of molecular targeted therapies. Therapeutic advantages can be obtained from DNA - targeting drugs with cancer - specific molecular targeting properties. Indenoisoquinolines are human topoisomerase I inhibitors with improved physicochemical and biological properties compared to the conventional camptothecin topoisomerase I inhibitors that are clinically used in the treatment of various solid tumors. Three indenoisoquinolines, indotecan (LMP400), indimitecan (LMP776), and LMP744, entered Phase I clinical trials in adults with recurrent solid tumors and lymphomas. However, some indenoisoquinolines with potent anticancer activity were surprisingly found not to show strong topoisomerase I inhibition. A particular indenoisoquinoline was also found to target the MYC G - quadruplex (MYC - G4).

[0006] MYC is one of the most important cancer genes and is overexpressed in more than 80% of all cancer types. The transcription factor MYC protein is involved in cell proliferation, differentiation, and apoptosis and plays a central role in tumor initiation, progression, and drug resistance. MYC is known to be a general transcription "amplifier" in cancer cells. Even short inhibition of MYC expression has been observed to permanently arrest tumor growth and induce tumor regression in vivo due to the "cancer gene dependence" of tumor cells. Therefore, MYC is a potential therapeutic target. However, the MYC protein is not an easy drug target because of its short half-life and lack of obvious small molecule binding pockets.

[0007] G4 detected in immortalized pre-cancer cells is detected at levels 10-fold higher than those in normal human cells, and G4 sites are found to be particularly enriched in regulatory transcriptionally active regions of chromatin, particularly in the MYC promoter region.

[0008] The nuclease hypersensitive element (NHE) III1 of the MYC promoter, which controls 85 - 90% of MYC transcriptional activity, forms a DNA G-quadruplex (G4) under negative supercoiling associated with transcription and functions as a transcriptional silencer. Compounds of formula (I) have been found to be useful for the treatment of cancer, particularly MYC-positive cancers. Compounds of formula (I) may target MYC quadruplex (MYC G4) and / or topoisomerase I and are thought to be effective against many cancers, including MYC-positive cancers containing MYC G4. SUMMARY OF THE INVENTION

[0009] According to some embodiments, the present disclosure provides a compound of formula (I), or a salt, hydrate, or solvate thereof:

Chemical formula

[0010] In some embodiments, A is (CH 2 )n, where n is selected from the group consisting of 1, 2, and 3. In some embodiments, A is (CH 2 ) 3 .

[0011] According to some embodiments, R 3 is hydrogen.

[0012] According to some embodiments, R 1 and R 2 are independently selected from the group consisting of hydrogen and CH 3 . In other embodiments, R 1 and R 2 form a ring together with the atoms to which they are attached.

[0013] In some embodiments, R 4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, where each of heteroaryl, heterocyclyl, and heterocyclylamino is optionally substituted.

[0014] In some embodiments, R 1 and R 2 together form -CH 2 ]-, R 3 is hydrogen, and R 5represents 2-MeO and 3-MeO, A is (CH 2 ) 3 and R 4 is selected from the group consisting of heteroaryl, heteroaryloxy, heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, trialkylammonium, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein each of heteroaryl, heteroaryloxy, and heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl, and heterocyclylamino is optionally substituted.

[0015] According to some embodiments, the compound of formula (I) is

Chemical formula

[0016] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound of formula (I), or a salt, hydrate, or solvate thereof, wherein formula (I) is as described herein.

[0017] According to some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound of formula (I), or a salt, hydrate, or solvate thereof:

Chemical formula

[0018] In some embodiments, the pharmaceutical composition further comprises at least one additional component selected from the group consisting of diluents, excipients, and combinations thereof.

[0019] According to an embodiment, the pharmaceutical composition is for treating cancer.

[0020] According to some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a compound of formula (I), or a salt, hydrate, or solvate thereof.

[0021] According to embodiments of the present disclosure, there is provided a method for treating a disease responsive to topoisomerase I inhibition or binding to MYC quadruplex in a host animal. In some embodiments, the method comprises administering to the host animal a composition comprising a therapeutically effective amount of one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a pharmaceutical composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, Formula (I) is [Chemical formula] wherein R 1 and R 2 are selected from the group consisting of hydrogen, (C 1 -C 6 )alkyl, (C 2 -C 6 )alkenyl, (C 2 -C 6 )alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R 1 and R 2 together with the atoms to which they are attached form a 5- or 6-membered ring, R3 is hydrogen, halo, nitro, cyano, CF 3 , (C 1 ~C 6 )alkyl, (C 1 ~C 6 )alkylthio, or (C 1 -C 6 )alkoxy, and A is alkylene, R 4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein each of heteroaryl, heterocyclyl, and heterocyclylamino is optionally substituted, R 5 is amino, (C 1 ~C 6 )alkylamino, di(C 1 ~C 6 )alkylamino, hydroxy(C 1 ~C 6 )alkyl, (C 1 ~C 6 )alkenyl, (C 1 ~C 6 )alkynyl, (C 1 ~C 6 )heteroalkyl, (C 3 ~C 8 )cycloalkyl, (C 3 ~C 8 )cycloheteroalkyl, (C 1 ~C 6 )alkoxy, (C 1 ~C 6 )alkyl(CO)O-, (C 1 ~C 6 )alkyl-O(CO)O- and (C 1 ~C 6 )alkylthio, representing one to two substituents independently selected from the group consisting of, or R 5 represents two adjacent substituents that together with the attached carbon form an optionally substituted ring or heterocycle, The pharmaceutical composition optionally further comprises one or more carriers, diluents, or excipients, or combinations thereof.

[0022] In some embodiments, the host animal is a human.

[0023] According to embodiments of the present disclosure, as described herein, a process for preparing a compound of formula (I) is provided. In some embodiments, the process comprises brominating a compound of formula II to obtain compound III, wherein R 1 , R 2 , and R 3 are as defined in claim 1:

Chemical formula

[0024] In some embodiments, R 3 is hydrogen.

[0025] In some embodiments, wherein R 1 and R 2 together form -CH 2 -.

[0026] In some embodiments, the bromination step comprises treating an acetic acid solution containing the compound of formula (II) with N-bromosuccinimide.

Brief Description of the Drawings

[0027]

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Mode for Carrying Out the Invention

[0028] Definition In each of the embodiments, unless otherwise indicated, the transitional phrase “consisting essentially of” is understood to mean that the corresponding composition, unit dose, method or use encompasses those that do not materially affect the basic and novel features of the invention described herein, including the specific compounds or recited steps. For example, a method described herein that consists essentially of a single compound, or a genus of compounds, is understood to represent monotherapy for the recited disease. Monotherapy may include co-administration of one or more carriers, vehicles, diluents, adjuvants, excipients, etc., and / or combinations thereof, and / or co-administration of one or more additional active pharmaceutical ingredients, where the latter additional active pharmaceutical ingredients are for treating diseases and / or symptoms different from the underlying disease described herein, such as cancer itself.

[0029] In each of the embodiments, unless otherwise indicated, the formula is understood to include not only all pharmaceutically acceptable salts of the compound, but also any and all hydrates and / or solvates of the formula of the compound. It will be understood that certain functional groups, such as hydroxy groups, amino groups, etc., form complexes and / or coordination compounds with water and / or various solvents in various physical forms of the compound. Thus, the formula should be understood as a description of such hydrates and / or solvates, including pharmaceutically acceptable solvates.

[0030] As used herein, the term "solvate" refers to a compound that forms a complex with solvent molecules. The compound can form such a complex with the solvent by simply mixing the compound with the solvent or dissolving the compound in the solvent. When the compound is used as a medicine, such a solvent is a pharmaceutically acceptable solvent. When the compound is used as a medicine, the relative amount of the solvent forming the solvate should be less than established guidelines for such pharmaceutical uses, such as less than the International Conference on Harmonization (ICH) guidelines. The solvate can be isolated from the excess solvent by evaporation, precipitation, and / or crystallization. In some embodiments, the solvate is amorphous, and in other embodiments, the solvate is crystalline.

[0031] In each of the embodiments, unless otherwise indicated, the formula includes any crystalline form, partially crystalline form, as well as amorphous and / or non-crystalline form of the compound, including the partially arranged form, disordered form, liquid crystal form, and mesophase described above. It should also be understood that it represents.

[0032] In each of the embodiments, unless otherwise indicated, the formula also includes any possible isomers, such as stereoisomers and geometric isomers, both individually and in any possible mixture. It should also be understood that it represents.

[0033] The pharmaceutical composition includes any compound of the embodiments described herein and optionally includes one or more carriers, diluents, excipients, etc., and combinations thereof.

[0034] Exemplary derivatives include both compounds that can be synthetically prepared from the described compounds and compounds that can be prepared by a method similar to the described method but with a different selection of starting materials, but are not limited thereto. For example, R 3 and R 5Compounds of formula (I) containing various functional groups on an aromatic ring such as are described. Derivatives of those compounds also include compounds having functional groups on their aromatic rings that are different from, for example, those explicitly stated in the definition of formula (I). Further, derivatives of those compounds also include compounds having the same or different of these functional groups at different positions on the aromatic ring. Similarly, derivatives include parallel variations of other functional groups on the compounds described herein, such as R 4 and the like.

[0035] Such derivatives may include compounds containing one or more protecting or protecting groups and prodrugs of the compounds, including compounds used in the preparation of other compounds described.

[0036] Exemplary analogs include, but are not limited to, compounds that share a functional and in some cases a structural similarity with the compounds described herein. For example, compounds of formula (I) containing a 7-aza-indenoisoquinoline ring system are described. Exemplary analogs include, but are not limited to, the corresponding ring-expanded compounds. Other exemplary analogs include, but are not limited to, the corresponding ring systems containing additional heteroatoms.

[0037] It should be understood that each of the embodiments may be combined in chemically relevant ways to produce subsets of the embodiments. Thus, it will be further understood that all such subsets are also exemplary embodiments.

[0038] The compounds described herein may contain one or more chiral centers or may otherwise exist as multiple stereoisomers. In one embodiment, the present disclosure is not limited to any particular stereochemical requirement, and the compounds, as well as compositions, methods, uses, and agents comprising them, may be optically pure or any of a variety of stereoisomeric mixtures, including racemates and other mixtures of enantiomers, other mixtures of diastereomers, and the like. Such mixtures of stereoisomers may contain a single stereochemical configuration at one or more chiral centers, but may contain a mixture of stereochemical configurations at one or more other chiral centers.

[0039] Similarly, the compounds may include geometric centers such as cis, trans, E, and Z double bonds on the ring, or spatial configurations such as cis, trans, syn, and anti. In another embodiment, the present disclosure is not limited to any particular geometric isomer requirement, and the compounds, as well as compositions, methods, uses, and agents comprising them, may be pure or any of various geometric isomeric mixtures. Such mixtures of geometric isomers may include a single configuration at one or more double bonds, but may include a mixture of geometric configurations at one or more other double bonds.

[0040] As used herein, the term "alkyl" includes a chain of carbon atoms that is optionally branched. As used herein, the terms "alkenyl" and "alkynyl" each include a chain of carbon atoms that is optionally branched and each include at least one double or triple bond. Alkynyl may also include one or more double bonds. In certain embodiments, alkyl is advantageously selected from C 1 ~C 24 , C 1 ~C 12 , C 1 ~C 8 , C 1 ~C 6 , and C 1 ~C 4 , and C 2 ~C 24 , C 2 ~C 12 , C2 ~C 8 、C 2 ~C 6 、and C 2 ~C 4 including ~C, etc., is of limited length. Exemplarily, C 1 ~C 8 、C 1 ~C 6 、and C 1 ~C 4 、as well as C 2 ~C 8 、C 2 ~C 6 、and C 2 ~C 4 such alkyl groups of such particularly limited length including ~C, etc. may be referred to as lower alkyl. In certain embodiments, alkenyl and / or alkynyl are each, advantageously, C 2 ~C 24 、C 2 ~C 12 、C 2 ~C 8 、C 2 ~C 6 、and C 2 ~C 4 、as well as C 3 ~C 24 、C 3 ~C 12 、C 3 ~C 8 、C 3 ~C 6 、and C 3 ~C 4 including ~C, etc., can be of limited length. Exemplarily, C 2 ~C 8 、C 2 ~C 6 、and C 2 ~C 4 、as well as C 3 ~C 8 、C 3 ~C 6 、and C 3 ~C 4Alkenyl and / or alkynyl groups of such particularly limited lengths, including etc., may be referred to as lower alkenyl and / or lower alkynyl. The shorter the alkyl, alkenyl, and / or alkynyl groups, the lower the lipophilicity added to the compound may be, and for this reason, they may have different pharmacokinetic behaviors. In certain embodiments, the recitation of alkyl refers to alkyl and optionally lower alkyl. In certain embodiments, the recitation of alkenyl refers to alkenyl and optionally lower alkenyl. In certain embodiments, the recitation of alkynyl refers to alkynyl and optionally lower alkynyl. Exemplary alkyl, alkenyl, and alkynyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl, hexyl, heptyl, octyl, etc., and the corresponding groups containing one or more double bonds and / or triple bonds, or combinations thereof.

[0041] The term "alkylene" includes a divalent chain of carbon atoms that is optionally branched. The terms "alkenylene" and "alkynylene" include a divalent chain of carbon atoms that is optionally branched and contain at least one double bond or triple bond, respectively. Alkynylene may also contain one or more double bonds. In certain embodiments, alkylene is advantageously C 1 ~C 24 、C 1 ~C 12 、C 1 ~C 8 、C 1 ~C 6 、and C 1 ~C 4 、as well as C 2 ~C 24 、C 2 ~C 12 、C 2 ~C 8 、C 2 ~C 6 、and C 2 ~C 4is of limited length and includes, for example, C 1 ~C 8 、C 1 ~C 6 、and C 1 ~C 4 、and C 2 ~C 8 、C 2 ~C 6 、and C 2 ~C 4 Such alkylene groups of such particularly limited length, including those mentioned above, may be referred to as lower alkylene. In certain embodiments, alkenylene and / or alkynylene are advantageously C 2 ~C 24 、C 2 ~C 12 、C 2 ~C 8 、C 2 ~C 6 、and C 2 ~C 4 、as well as C 3 ~C 24 、C 3 ~C 12 、C 3 ~C 8 、C 3 ~C 6 、and C 3 ~C 4 can be of limited length and includes, for example, C 2 ~C 8 、C 2 ~C 6 、and C 2 ~C 4 、as well as C 3 ~C 8 、C 3 ~C 6 、and C 3 ~C 4Such alkenylene and / or alkynylene groups of such particularly limited lengths, including etc., may be referred to as lower alkenylene and / or lower alkynylene. The shorter the alkylene, alkenylene, and / or alkynylene groups, the lower the lipophilicity added to the compound may be, and for this reason, different pharmacokinetic behaviors may result. In certain embodiments, the listing of alkylene, alkenylene, and alkynylene refers to alkylene, alkenylene, and alkynylene, as well as optionally lower alkylene, lower alkenylene, and lower alkynylene. Exemplary alkyl groups include, but are not limited to, methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, pentylene, 1,2-pentylene, 1,3-pentylene, hexylene, heptylene, octylene, etc.

[0042] As used herein, the term "cycloalkyl" includes a chain of carbon atoms that is optionally branched, at least a portion of which chain is cyclic. Cycloalkylalkyl is a subset of cycloalkyl. Cycloalkyl may be polycyclic. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, 2-methylcyclopropyl, cyclopentylta-2-yl, adamantyl, etc. As used herein, the term "cycloalkenyl" includes a chain of carbon atoms that is optionally branched, includes at least one double bond, and at least a portion of which chain is cyclic. One or more of its double bonds may be in the cyclic portion and / or the acyclic portion of the cycloalkenyl. Cycloalkenylalkyl and cycloalkylalkenyl are each subsets of cycloalkenyl. Cycloalkyl may be polycyclic. Exemplary cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclohexylethene-2-yl, cycloheptenylpropenyl, etc. Chain-forming cycloalkyl and / or cycloalkenyl are advantageously C 3 ~C 24 、C 3 ~C12 , C 3 ~C 8 , C 3 ~C 6 , and C 5 ~C 6 is of limited length and includes. The shorter the alkyl chain and / or alkenyl chain that forms a cycloalkyl and / or cycloalkenyl, respectively, the less lipophilicity is added to the compound, and thus different pharmacokinetic behaviors may result.

[0043] The term "heteroalkyl" includes a chain of atoms that contains both carbon and at least one heteroatom and is optionally branched. Exemplary heteroatoms include nitrogen, oxygen, and sulfur. In certain alternative forms, exemplary heteroatoms also include phosphorus and selenium. The term "cycloheteroalkyl", which includes heterocyclyl and heterocycles, includes a chain of atoms that contains both carbon and at least one heteroatom such as heteroalkyl, is optionally branched, and at least a portion of the chain is cyclic. Exemplary heteroatoms include nitrogen, oxygen, and sulfur. In certain alternative forms, exemplary heteroatoms also include phosphorus and selenium. Exemplary cycloheteroalkyls include, but are not limited to, tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl, and the like.

[0044] The term "aryl" includes monocyclic and polycyclic aromatic carbocyclic groups, each of which may optionally be substituted. Exemplary aromatic carbocyclic groups described herein include, but are not limited to, phenyl, naphthyl, and the like. The term "heteroaryl" includes aromatic heterocyclic groups, each of which may optionally be substituted. Exemplary aromatic heterocyclic groups include, but are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzoisoxazolyl, benzoisothiazolyl, and the like.

[0045] The term "amino" includes the group NH 2 , alkylamino, and dialkylamino, and the two alkyl groups in the dialkylamino may be the same or different, i.e., it may be alkylalkylamino. Exemplarily, amino includes methylamino, ethylamino, dimethylamino, methylethylamino, and the like. Further, when amino modifies another term, such as aminoalkyl or acylamino, or is modified by another term, the above variations of the term amino are included. Exemplarily, aminoalkyl includes H 2 N-alkyl, methylaminoalkyl, ethylaminoalkyl, dimethylaminoalkyl, methylethylaminoalkyl, and the like. Exemplarily, acylamino includes acylmethylamino, acylethylamino, and the like.

[0046] The term "amino and its derivatives" includes amino as described herein, and alkylamino, alkenylamino, alkynylamino, heteroalkylamino, heteroalkenylamino, heteroalkynylamino, cycloalkylamino, cycloalkenylamino, cycloheteroalkylamino, cycloheteroalkenylamino, arylamino, arylalkylamino, arylalkenylamino, arylalkynylamino, heteroarylamino, heteroarylalkylamino, heteroarylalkenylamino, heteroarylalkynylamino, acylamino, etc., each of which is optionally substituted. The term "amino and its derivatives" also includes alkylalkylamino, alkylalkenylamino, alkylalkynylamino, alkylheteroalkylamino, alkylheteroalkynylamino, alkylheteroalkynylamino, alkylcycloalkylamino, alkylcycloalkenylamino, alkylcycloheteroalkylamino, alkylcycloheteroalkenylamino, arylamino, arylalkylamino, arylalkenylamino, arylalkynylamino, heteroarylamino, heteroarylalkylamino, heteroarylalkenylamino, heteroarylalkynylamino, alkylacylamino, etc., each of which is optionally substituted. The term "amino derivative" also includes urea, carbamate, etc.

[0047] The term "hydroxy and its derivatives" includes OH, and alkyloxy, alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy, heteroalkynyloxy, cycloalkyloxy, cycloalkenyloxy, cycloheteroalkyloxy, cycloheteroalkenyloxy, aryloxy, arylalkyloxy, arylalkenyloxy, arylalkynyloxy, heteroaryloxy, heteroarylalkyloxy, heteroarolalkenyloxy, heteroarylalkynyloxy, acyloxy, etc., each of which is optionally substituted. The term "hydroxy derivative" also includes carbamate, etc.

[0048] The term "thio and its derivatives" includes SH, and alkylthio, alkenylthio, alkynylthio, heteroalkylthio, heteroalkenylthio, heteroalkynylthio, cycloalkylthio, cycloalkenylthio, cycloheteroalkylthio, cycloheteroalkenylthio, arylthio, arylalkylthio, arylalkenylthio, arylalkynylthio, heteroarylthio, heteroarylalkylthio, heteroarylalkenylthio, heteroarylalkynylthio, acylthio, etc., each of which is optionally substituted. The term "thio derivative" also includes thiocarbamate, etc.

[0049] The term "acyl" includes formyl, and alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, heteroalkylcarbonyl, heteroalkenylcarbonyl, heteroalkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl, cycloheteroalkylcarbonyl, cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl, arylalkenylcarbonyl, arylalkynylcarbonyl, heteroarylcarbonyl, heteroarylalkylcarbonyl, heteroarylalkenylcarbonyl, heteroarylalkynylcarbonyl, acylcarbonyl, etc., each of which is optionally substituted.

[0050] The term "carbonyl and its derivatives" includes the groups C(O), C(S), C(NH), and their substituted amino derivatives.

[0051] The term "carboxylic acid and its derivatives" includes the group CO 2 H and its salts, as well as its esters and amides, and CN.

[0052] The term "sulfinic acid or its derivatives" includes SO 2 H and its salts, as well as its esters and amides.

[0053] The term "sulfonic acid and its derivatives" includes SO 3 H and its salts, as well as its esters and amides.

[0054] The term "sulfonyl" includes alkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, heteroalkylsulfonyl, heteroalkenylsulfonyl, heteroalkynylsulfonyl, cycloalkylsulfonyl, cycloalkenylsulfonyl, cycloheteroalkylsulfonyl, cycloheteroalkenylsulfonyl, arylsulfonyl, arylalkylsulfonyl, arylalkenylsulfonyl, arylalkynylsulfonyl, heteroarylsulfonyl, heteroarylalkylsulfonyl, heteroarylalkenylsulfonyl, heteroarylalkynylsulfonyl, acylsulfonyl, etc., each of which is optionally substituted.

[0055] The term "hydroxylamino and its derivatives" includes NHOH, and alkyloxyl NH, alkenyloxyl NH, alkynyloxyl NH, heteroalkyloxyl NH, heteroalkenyloxyl NH, heteroalkynyloxyl NH, cycloalkyloxyl NH, cycloalkenyloxyl NH, cycloheteroalkyloxyl NH, cycloheteroalkenyloxyl NH, aryloxyl NH, arylalkyloxyl NH, arylalkenyloxyl NH, arylalkynyloxyl NH, heteroaryloxyl NH, heteroarylalkyloxyl NH, heteroarylalkenyloxyl NH, heteroarylalkynyloxyl NH, acyloxyl NH, etc., each of which is optionally substituted.

[0056] The term "hydrazino and its derivatives" includes alkyl NHNH, alkenyl NHNH, alkynyl NHNH, heteroalkyl NHNH, heteroalkenyl NHNH, heteroalkynyl NHNH, cycloalkyl NHNH, cycloalkenyl NHNH, cycloheteroalkyl NHNH, cycloheteroalkenyl NHNH, aryl NHNH, arylalkyl NHNH, arylalkenyl NHNH, arylalkynyl NHNH, heteroaryl NHNH, heteroarylalkyl NHNH, heteroarylalkenyl NHNH, heteroarylalkynyl NHNH, acyl NHNH, etc., each of which is optionally substituted.

[0057] The term "optionally substituted" includes substitution of a hydrogen atom by another functional group on the optionally substituted radical. Exemplary such other functional groups include, but are not limited to, amino, hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acid and its derivatives, carboxylic acid and its derivatives, etc. Exemplarily, any of amino, hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, and / or sulfonic acid is optionally substituted.

[0058] The terms "optionally substituted aryl" and "optionally substituted heteroaryl" include substitution of a hydrogen atom on the optionally substituted aryl or heteroaryl by other functional groups. Such other functional groups, which are also referred to herein as aryl substituents or heteroaryl substituents respectively, include, by way of example, amino, hydroxyl, halo, thio, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acid and its derivatives, carboxylic acid and its derivatives, and the like, but are not limited thereto. By way of example, any one of amino, hydroxyl, thio, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, and / or sulfonic acid is optionally substituted.

[0059] Exemplary substituents include the radical -(CH 2 ) x Z X which is also not limited thereto, wherein x is an integer from 0 to 6, and Z X is halogen, hydroxy, C 1 ~C 6 alkanoyloxy including alkanoyloxy, optionally substituted aroyloxy, C 1 ~C 6 alkyl including alkyl, C 1 ~C 6 alkoxy including alkoxy, C 3 ~C 8 cycloalkyl including cycloalkyl, C 3 ~C 8 cycloalkoxy including cycloalkoxy, C 2 ~C 6 alkenyl including alkenyl, C 2 ~C 6 alkynyl including alkynyl, C 1 ~C 6 haloalkyl including haloalkyl, C 1 ~C 6Haloalkoxy, C containing haloalkoxy 3 ~C 8 Halocycloalkyl, C containing halocycloalkyl 3 ~C 8 Halocycloalkoxy, amino, C containing halocycloalkoxy 1 ~C 6 Alkylamino, (C 1 ~C 6 alkyl)(C 1 ~C 6 alkyl)amino, alkylcarbonylamino, N-(C 1 ~C 6 alkyl)alkylcarbonylamino, aminoalkyl, C 1 ~C 6 alkylaminoalkyl, (C 1 ~C 6 alkyl)(C 1 ~C 6 alkyl)aminoalkyl, alkylcarbonylaminoalkyl, N-(C 1 ~C 6 alkyl)alkylcarbonylaminoalkyl, cyano, and nitro, or Z X is -CO 2 R 4 and -CONR 5 R 6 selected from, where R 4 , R 5 and R 6 are each independently hydrogen, C 1 ~C 6 alkyl, aryl-C 1 ~C 6 alkyl, and heteroaryl-C 1 ~C 6 alkyl, each selected with its respective frequency of occurrence.

[0060] As used herein, the term "leaving group" refers to a reactive functional group that generates an electrophilic site on the atom to which it is attached, such that a nucleophile can be added to the electrophilic site on the atom. Exemplary leaving groups include, but are not limited to, halogen, optionally substituted phenol, acyloxy group, sulfonium group, sulfonoxy group, etc. Such leaving groups can be present on alkyl, acyl, etc. Such leaving groups may also be referred to herein as activating groups, such as when the leaving group is present on an acyl. Further, without limitation, conventional peptide, amide, and ester coupling agents such as PyBop, BOP-Cl, BOP, pentafluorophenol, isobutyl chloroformate, etc. form various intermediates containing leaving groups as defined herein on a carbonyl group.

[0061] The recitation of an integer range for any variable describes the recited range, all individual elements within that range, and all possible sub-ranges for that variable. For example, the recitation that n is an integer from 0 to 8 describes the range, the individual and selectable values 0, 1, 2, 3, 4, 5, 6, 7, and 8, such that, for example, n is 0, or n is 1, or n is 2, etc. Further, the recitation that n is an integer from 0 to 8 also describes any sub-ranges, each of which can form the basis for further embodiments, for example, n is an integer in the ranges 1 to 8, 1 to 7, 1 to 6, 2 to 8, 2 to 7, 1 to 3, 2 to 4, etc.

[0062] The terms "processing," "contacting," and "reacting," when referring to a chemical reaction, generally mean adding or mixing two or more reagents under appropriate conditions that allow a chemical transformation or chemical reaction to occur, and / or producing the indicated product and / or the desired product. The reaction that produces the indicated product and / or the desired product does not necessarily result directly from the combination of the two initially added reagents. In other words, one or more intermediates may be present in the mixture, which ultimately leads to the formation of the indicated product and / or the desired product.

[0063] The term "protecting group" generally refers to any radical that is reversibly bonded to a functional group and is used to block or partially block the reactivity of that functional group against a given set of conditions such as reaction conditions. Exemplarily, a nitrogen protecting group is reversibly bonded to an amine and blocks or partially blocks the reactivity of the amine under a given set of conditions. Exemplary nitrogen protecting groups include, but are not limited to, carbamates such as t-Boc and Fmoc.

[0064] The term "composition" generally refers to any product containing specific components, as well as any product directly or indirectly obtained from a combination of specific components.

[0065] Certain functional groups such as hydroxy groups and amino groups form complexes and / or coordination compounds with water and / or various solvents in various physical forms of the compound. The compositions described herein can be prepared from isolated compounds or from salts, solutions, hydrates, solvates, and other forms of the compounds. Compositions can be prepared from compounds in various amorphous, non-amorphous, partially crystalline, crystalline, and / or other morphological forms. Compositions can be prepared from various hydrates and / or solvates of the compounds. Furthermore, compositions can be prepared from various co-crystals of the compounds. Thus, it will be understood that such pharmaceutical compositions listing the compounds include each of the various morphological forms and / or solvate or hydrate forms of the compounds, or any combination thereof.

[0066] Exemplarily, the composition can include one or more carriers, diluents, and / or excipients. The compounds, or compositions containing them, can be formulated in a therapeutically effective amount in any conventional dosage form appropriate for the method. The compounds, or compositions containing them (including such formulations), can be administered in a variety of dosage forms by a variety of conventional routes for the method and using known procedures (generally, Remington: The Science and Practice of Pharmacy, (23rd See (ed., 2020).

[0067] This compound can be used for both human clinical medicine and veterinary applications. Thus, a host animal having cancer and being treated with the compound can be a human, or in the case of veterinary applications, can be an experimental animal, an agricultural animal, a breeding animal, or a wild animal. The present disclosure can be applied to host animals including, but not limited to, humans, rodents (e.g., mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees, etc. as experimental animals, dogs, cats, and rabbits as breeding animals, cows, horses, pigs, sheep, goats, etc. as agricultural animals, and bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, and whales, etc. as captive wild animals.

[0068] This method can be utilized to treat cancers such as carcinomas, sarcomas, lymphomas, Hodgkin's disease, melanomas, mesotheliomas, Burkitt's lymphomas, nasopharyngeal carcinomas, leukemias, and myelomas. Examples of cancer cell populations include, but are not limited to, oral cancer, thyroid cancer, endocrine cancer, skin cancer, gastric cancer, esophageal cancer, laryngeal cancer, pancreatic cancer, colon cancer, bladder cancer, bone cancer, ovarian cancer, cervical cancer, uterine cancer, breast cancer, testicular cancer, prostate cancer, rectal cancer, kidney cancer, liver cancer, and lung cancer.

[0069] The term "therapeutically effective amount" refers to the amount of an active compound or pharmaceutical that elicits a biological or pharmacological response in a tissue system, animal, or human, including the alleviation of the symptoms of the disease or disorder being treated, which a researcher, veterinarian, physician, or other clinician is seeking. In one aspect, a therapeutically effective amount is an amount that can treat or alleviate a disease or the symptoms of a disease with a reasonable benefit / risk ratio applicable to any medical treatment. However, the total daily usage of the compounds and compositions can be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective amount level for any particular patient depends on a variety of factors, such factors including the disorder being treated and the severity of the disorder; the activity of the specific compound being used; the specific composition being used; the age, weight, general health, gender, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound being used; the duration of the treatment; drugs used in combination with or concurrently with the specific compound being used; and similar factors well known to researchers, veterinarians, physicians, and other clinicians with ordinary skill.

[0070] A therapeutically effective amount is advantageously selected taking into account any toxicity or other undesirable side effects that may occur during the administration of one or more of the compounds described herein, whether for monotherapy or combination therapy. Further, combination therapy may permit the administration of lower dosages of compounds that exhibit such toxicity, or other undesirable side effects, where such lower dosages are below the threshold of toxicity or are lower than the amounts that would be administered without combination therapy within the therapeutic concentration range.

[0071] The effective amount of any one or a mixture of the compounds described herein can be readily determined by the attending diagnostician or physician using known techniques and / or by observing the results obtained in similar circumstances. In determining the effective amount or dosage, factors including, but not limited to, the mammalian species, including humans, its size, age, and general health, the particular disease or disorder involved, the degree or involvement or severity of the disease or disorder, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dosage regimen selected, the use of concomitant medications, and other related circumstances, are considered by the attending diagnostician or physician.

[0072] The dosage of each compound of the claimed combination depends on several factors including the mode of administration, the condition being treated, the severity of the condition, whether the condition is being treated or prevented, and the age, weight, and health of the person being treated. Additionally, pharmacogenomic information (the effect of genotype on the pharmacokinetic, pharmacodynamic, or efficacy profile of a therapeutic agent) regarding a particular patient can affect the dosage used.

[0073] In the method, the individual components, or combinations, of the co-administration can be administered contemporaneously, simultaneously, sequentially, separately, or in a single pharmaceutical formulation by any suitable means. When the compounds or compositions being co-administered are administered in separate dosage forms, the number of administrations per day for each compound may be the same or different. The compounds or compositions may be administered via the same or different routes of administration. The compounds or compositions may be co-administered simultaneously in divided or single form at the same or different times during the course of therapy according to the same or alternating regimens.

[0074] Furthermore, in embodiments focusing on combination therapies that include administration of a compound of formula (I) and another anti-cancer agent known in the art, "therapeutically effective amount" refers to the combined amount of the agents administered together such that the combined effect elicits the desired biological or pharmaceutical response. For example, a therapeutically effective amount of a compound of formula (I) and imatinib is the amount of the compound of formula (I) and the amount of imatinib, etc., which, when administered together or sequentially, have a combined effect that is therapeutically effective. Further, in some embodiments of such methods that include co-administration, the amount of the compound of formula (I) or imatinib, etc., of that co-administration dose may or may not be therapeutically effective when administered individually.

[0075] The term "administering" includes all means of introducing the compounds and compositions into a host animal, including, but not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal, intraocular, sublingual, vaginal, rectal, etc. The compounds and compositions may be administered in unit dosage forms and / or formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and / or vehicles.

[0076] Exemplary forms for oral administration include tablets, capsules, elixirs, syrups, etc.

[0077] Exemplary routes for parenteral administration include intravenous, intraarterial, intraperitoneal, epidural, intraurethral, intrasternal, intramuscular, and subcutaneous, as well as any other parenteral administration routes recognized in the art.

[0078] Exemplary means of parenteral administration include needle (including microneedle) injection, needleless injection, and infusion techniques, as well as any other parenteral administration means recognized in the art. Parenteral formulations are typically aqueous solutions that may contain excipients such as salts, carbohydrates, and buffering agents (preferably at a pH in the range of about 3 to about 9), but in some applications may be more preferably formulated as a sterile non-aqueous solution or in a dry form for use in combination with a suitable vehicle such as sterile pyrogen-free water. Preparation of parenteral formulations under aseptic conditions, for example by lyophilization, can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art. Parenteral administration of the compounds is exemplified by administration in the form of physiological saline or by incorporating the compounds into liposomes. If the compound is not sufficiently soluble to dissolve, solubilizing agents such as ethanol may be applied.

[0079] Exemplarily, administration includes topical use as when administered locally to the site of the disease or to a particular organ or tissue system. Exemplary topical administration can be performed during laparotomy or during other procedures that provide access to the site of the disease. Alternatively, topical administration may be performed using parenteral delivery, in which case the compound or composition is deposited locally at the site without generally distributing to multiple other non-target sites in the host animal being treated. Similar variations regarding topical delivery to specific tissue types, such as organs, are also described. Exemplarily, the compounds can be administered directly to the nervous system, including but not limited to administration routes such as intracerebral, intraventricular, intrathecal, intracisternal, intramedullary, and / or perispinal, with or without a pump device, via intracranial or intraspinal needles and / or catheters.

[0080] When manufacturing a pharmaceutical composition of a compound, a therapeutically effective amount of one or more compounds in any of the various forms described herein is mixed with one or more excipients, diluted with one or more excipients, or encapsulated in a carrier which can be in the form of a capsule, sachet, paper, or other container. The excipient can function as a diluent and can be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredient. Thus, the composition can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. The composition can contain from about 0.1% to about 99.9% of the active ingredient, depending on the selected dosage and dosage form.

[0081] Specific examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The formulation can further include lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweetening agents; and flavoring agents. The composition can be formulated to provide rapid, sustained, or delayed release of the active ingredient after administration to a host animal by using procedures known in the art. One or more carriers, one or more diluents, one or more excipients, and combinations thereof can be used in making the pharmaceutical composition. It will be understood that the carriers, diluents, and excipients used in preparing the compositions described herein are advantageously GRAS (Generally Recognized as Safe) compounds. It will also be understood that the acids and bases used to make the salts as described herein and / or the solvents used to make the solvates as described herein are also advantageously GRAS compounds.

[0082] Specific examples of emulsifiers include naturally occurring gums (such as gum arabic or tragacanth gum) and naturally occurring phosphatides (such as soy lecithin and sorbitan monooleate derivatives). Examples of antioxidants are butylated hydroxyanisole (BHA), ascorbic acid and its derivatives, tocopherol and its derivatives, butylated hydroxyanisole, and cysteine. Examples of preservatives are, for example, methyl or propyl p-hydroxybenzoate, and parabens such as benzalkonium chloride. Examples of wetting agents are glycerin, propylene glycol, sorbitol, and urea. Examples of penetration enhancers are propylene glycol, DMSO, triethanolamine, N,N-dimethylacetamide, N,N-dimethylformamide, 2-pyrrolidone and its derivatives, tetrahydrofurfuryl alcohol, and AZONE. Examples of chelating agents are sodium EDTA, citric acid, and phosphoric acid. Examples of gelling agents are CARBOPOL, cellulose derivatives, bentonite, alginate, gelatin, and polyvinylpyrrolidone. Examples of ointment bases are beeswax, paraffin, cetyl palmitate, vegetable oil, sorbitan esters of fatty acids (Span), polyethylene glycol, and condensation products of sorbitan esters of fatty acids and ethylene oxide (such as polyoxyethylene sorbitan monooleate (TWEEN)).

[0083] The therapeutically effective dose administered in an animal model can be used to calculate the corresponding therapeutically effective dose for administration to other host animals, including humans. Exemplary corresponding doses can be calculated using the "Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" published by the FDA, which can be found at https: / / www.fda.gov / media / 72309 / download and is hereby incorporated by reference in its entirety.

[0084] The compound of formula (I) In one embodiment, the present disclosure provides a compound of formula (I), or a salt, hydrate or solvate thereof:

Chemical formula

[0085] In an exemplary embodiment, compounds of formula (I), or salts, hydrates or solvates thereof are described: [Chemical formula] wherein A is (CH 2 )n, where n is 1, 2, or 3, R 1 and R 2 are each independently selected from the group consisting of hydrogen, (C 1 ~C 6 ) alkyl, (C 2 ~C 6 ) alkenyl, (C 2 ~C 6 ) alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R 1 and R 2 together with the atoms to which they are attached form a 5- or 6-membered ring, R 3 is hydrogen, halo, nitro, cyano, CF 3 , (C 1 ~C6 ) alkyl, (C 1 ~C 6 ) alkylthio, or (C 1 ~C 6 ) alkoxy, and R 4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein each of heteroaryl, heterocyclyl, and heterocyclylamino is optionally substituted, R 5 is amino, (C 1 ~C 6 ) alkylamino, di(C 1 ~C 6 ) alkylamino, hydroxy(C 1 ~C 6 ) alkyl, (C 1 ~C 6 ) alkenyl, (C 1 ~C 6 ) alkynyl, (C 1 ~C 6 ) heteroalkyl, (C 3 ~C 8 ) cycloalkyl, (C 3 ~C 8 ) cycloheteroalkyl, (C 1 ~C 6 ) alkoxy, (C 1 ~C 6 ) alkyl(CO)O-, (C 1 ~C 6 ) alkyl-O(CO)O- and (C 1 ~C 6 ) alkylthio, representing one to two substituents independently selected in each case from the group consisting of, or R 5 represents two adjacent substituents that together with the attached carbon form an optionally substituted ring or heterocycle.

[0086] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate, or solvate thereof is described, wherein n is an integer from 1 to 3, or n is an integer from 2 to 3, or n is 3.

[0087] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate or solvate thereof is described, wherein A is (CH 2 ) 3 is.

[0088] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate or solvate thereof is described, wherein R 3 is hydrogen.

[0089] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate, or solvate thereof is described, wherein R 1 and R 2 are, independently, hydrogen or C 1 ~C 6 alkyl.

[0090] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate or solvate thereof is described, wherein R 1 and R 2 are, independently, hydrogen or CH 3 is.

[0091] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate or solvate thereof is described, wherein R 1 and R 2 is CH 3 is.

[0092] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate, or solvate thereof is described, wherein R 1 and R 2 together with the atoms to which they are attached form a ring.

[0093] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate or solvate thereof, is described, wherein R 1 and R 2 together form -CH 2 -.

[0094] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate or solvate thereof, is described, wherein R 3 is hydrogen, (C 1 ~C 6 )alkyl, (C 1 ~C 6 )alkylthio, or (C 1 ~C 6 )alkoxy.

[0095] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate or solvate thereof, is described, wherein R 3 is hydrogen.

[0096] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate, or solvate thereof, is described, wherein R 4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein each of heteroaryl, heterocyclyl, and heterocyclylamino is optionally substituted.

[0097] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate, or solvate thereof, is described, wherein R 4 is selected from the group consisting of:

Chemical formula

[0098] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate, or solvate thereof, is described, wherein R 4 is selected from the group consisting of:

Chemical formula

[0099] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate, or solvate thereof, is described, wherein R 1 and R 2 together form -CH 2 -, R 3 is hydrogen, R 5 represents 2-MeO and 3-MeO, A is (CH 2 ) 3 and R 4 is selected from the group consisting of heteroaryl, heteroaryloxy, heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, trialkylammonium, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein heteroaryl, heteroaryloxy, and heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl, and heterocyclylamino are optionally substituted.

[0100] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate, or solvate thereof, is described, wherein R 1 and R 2 together form -CH 2 -, R 3is hydrogen, and R 5 represents 2-MeO and 3-MeO, and A is (CH 2 ) 3 and R 4 is selected from the group consisting of:

Chemical formula

[0101] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate, or solvate thereof, is described, wherein R 1 and R 2 together form -CH 2 -, R 3 is hydrogen, R 5 represents 2-MeO and 3-MeO, A is (CH 2 ) 3 and R 4 is selected from the group consisting of:

Chemical formula

[0102] In another exemplary embodiment, a compound of the following formula, or a salt, hydrate, or solvate thereof, is described:

Chemical formula

[0103] In another exemplary embodiment, a compound of the following formula, or a salt, hydrate, or solvate thereof, is described:

Chemical formula

[0104] In another exemplary embodiment, a compound of the following formula, or a salt, hydrate, or solvate thereof, is described.

[0105]

Chemical formula

[0106] In another exemplary embodiment, compounds of the following formula, or salts, hydrates, or solvates thereof, are described: [ka]

[0107] In another exemplary embodiment, compounds of the following formula, or salts, hydrates, or solvates thereof, are described: [ka]

[0108] In another exemplary embodiment, compounds of the following formula, or salts, hydrates, or solvates thereof, are described: [ka]

[0109] In another exemplary embodiment, compounds of the following formula, or salts, hydrates, or solvates thereof, are described: [ka]

[0110] In another exemplary embodiment, compounds of the following formula, or salts, hydrates, or solvates thereof, are described: [ka]

[0111] A and R listed 1 , R 2 , R 3 , R 4 , R 5 , R O , R S , R N1 , and R N2It should be understood that all possible combinations of each of the various genera and subgenera represent additional exemplary embodiments of the compounds. It will be further understood that each of those additional exemplary embodiments of the compounds can be used in any of the described compositions, methods, unit dosages, kits, and / or uses.

[0112] In one embodiment, the present disclosure provides a pharmaceutical composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof. In one embodiment, the pharmaceutical composition comprises one or more of the compounds in a therapeutically effective amount for treating a host animal having cancer. The composition may include other therapeutically active compounds and / or one or more carriers, vehicles, diluents, adjuvants, excipients, etc., and other components and / or ingredients, including but not limited to combinations thereof.

[0113] In another embodiment, unit dosages of one or more compounds of formula (I) and pharmaceutical compositions containing one or more of the compounds of formula (I) are also described herein. The unit dosage comprises one or more of the compounds of formula (I) in a therapeutically effective amount for treating a host animal having cancer. The unit dosage may be in single or divided form and may be adjusted to a shorter periodic amount, including multiple daily dosages, or to a longer periodic amount, including weekly or monthly dosages, corresponding to a daily dosage. The composition may include other therapeutically active compounds and / or one or more carriers, vehicles, diluents, adjuvants, excipients, etc., and other components and / or ingredients, including but not limited to combinations thereof.

[0114] In another embodiment, a method for treating a host animal having cancer is also described, the method comprising administering to the host animal having cancer one or more of the compounds of formula (I), or a salt, hydrate, or solvate thereof, and / or one or more of the compositions. In another embodiment, the method comprises administering a therapeutically effective amount of one or more of the compounds of formula (I) and / or the composition for treating a host animal having cancer.

[0115] In another exemplary embodiment, a method for treating a host animal having cancer is described, the method comprising administering to the host animal one or more compounds of formula (I), or a salt, hydrate or solvate thereof, [Chemical formula] wherein R 1 and R 2 are independently selected from the group consisting of hydrogen, (C 1 -C 6 ) alkyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R 1 and R 2 together with the atoms to which they are attached form a 5- or 6-membered ring, R 3 is hydrogen, halo, nitro, cyano, CF 3 , (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkylthio, or (C 1 -C 6 ) alkoxy, A is alkylene, R 4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, each of heteroaryl, heterocyclyl, and heterocyclylamino being optionally substituted, R 5 is amino, (C 1 -C 6 ) alkylamino, di(C 1 -C 6 ) alkylamino, hydroxy(C 1 -C 6 ) alkyl, (C 1 -C6 ) alkenyl, (C 1 ~C 6 ) alkynyl, (C 1 ~C 6 ) heteroalkyl, (C 3 ~C 8 ) cycloalkyl, (C 3 ~C 8 ) cycloheteroalkyl, (C 1 ~C 6 ) alkoxy, (C 1 ~C 6 ) alkyl(CO)O-, (C 1 ~C 6 ) alkyl-O(CO)O- and (C 1 ~C 6 ) alkylthio, representing one to two substituents independently selected in each case from the group consisting of, or R 5 represents two adjacent substituents which, together with the carbon to which they are attached, form an optionally substituted ring or heterocycle.

[0116] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal a compound of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising a compound of formula (I), or a salt, hydrate, or solvate thereof, wherein A is (CH 2 )n, where n is 1, 2, or 3, R 1 and R 2 are hydrogen, (C 1 ~C 6 ) alkyl, (C 2 ~C 6 ) alkenyl, (C 2 ~C 6 ) alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, independently selected in each case from the group consisting of, or R 1 and R 2 form a 5- or 6-membered ring together with the atoms to which they are attached, R 3 is hydrogen, halo, nitro, cyano, CF3 , (C 1 ~C 6 ) alkyl, (C 1 -C 6 ) alkylthio, or (C 1 ~C 6 ) alkoxy, and R 4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein each of heteroaryl, heterocyclyl, and heterocyclylamino is optionally substituted, R 5 is amino, (C 1 ~C 6 ) alkylamino, di(C 1 ~C 6 ) alkylamino, hydroxy(C 1 ~C 6 ) alkyl, (C 1 ~C 6 ) alkenyl, (C 1 ~C 6 ) alkynyl, (C 1 ~C 6 ) heteroalkyl, (C 3 ~C 8 ) cycloalkyl, (C 3 ~C 8 ) cycloheteroalkyl, (C 1 ~C 6 ) alkoxy, (C 1 ~C 6 ) alkyl(CO)O-, (C 1 ~C 6 ) alkyl-O(CO)O- and (C 1 ~C 6 ) alkylthio, representing one to two substituents independently selected in each case from the group consisting of, or R 5 represents two adjacent substituents that together with the attached carbon form an optionally substituted ring or heterocycle.

[0117] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein A is (CH 2 )n, where n is an integer from 1 to 3, or n is an integer from 2 to 3, or n is 3.

[0118] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein R 1 and R 2 are independently selected from hydrogen or C 1 -C 6 alkyl.

[0119] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein R 1 and R 2 are independently hydrogen or CH 3 .

[0120] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein R 1 and R 2 are CH 3 .

[0121] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein R 1 and R 2 together with the atoms to which they are attached form a ring.

[0122] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein R 1 and R 2 together form -CH 2 -.

[0123] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein R 3 is hydrogen, (C 1 ~C 6 )alkyl, (C 1 ~C 6 )alkylthio, or (C 1 ~C 6 )alkoxy.

[0124] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein R 3 is hydrogen.

[0125] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein R 4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein each of heteroaryl, heterocyclyl, and heterocyclylamino is optionally substituted.

[0126] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein R 4 is selected from the group consisting of:

Chemical formula

[0127] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein R 4 is selected from the group consisting of: [Chem.]

[0128] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein R 1 and R 2 together form -CH 2 -, R 3 is hydrogen, R 5 represents 2-MeO and 3-MeO, A is (CH 2 ) 3 , and R 4 is selected from the group consisting of heteroaryl, heteroaryloxy, heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, trialkylammonium, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein each of heteroaryl, heteroaryloxy, and heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl, and heterocyclylamino is optionally substituted.

[0129] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein R 1 and R 2 together form -CH 2 -, R 3 is hydrogen, R 5 represents 2-MeO and 3-MeO, A is (CH 2 ) 3 , and R4 is selected from the group consisting of:

Chem.

[0130] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein R 1 and R 2 together form -CH 2 -, R 3 is hydrogen, R 5 represents 2-MeO and 3-MeO, A is (CH 2 ) 3 and R 4 is selected from the group consisting of:

Chem.

[0131] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G101)

Chem.

[0132] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G102)

Chem.

[0133] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G103) [Chemical formula] or a salt, hydrate, or solvate thereof, or a composition comprising the compound, or a salt, hydrate, or solvate thereof.

[0134] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G104) [Chemical formula] or a salt, hydrate, or solvate thereof, or a composition comprising the compound, or a salt, hydrate, or solvate thereof.

[0135] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G105) [Chemical formula] or a salt, hydrate, or solvate thereof, or a composition comprising the compound, or a salt, hydrate, or solvate thereof.

[0136] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G108) [Chemical formula] or a salt, hydrate, or solvate thereof, or a composition comprising the compound, or a salt, hydrate, or solvate thereof.

[0137] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G106) [Chemical formula] or administering a composition comprising the compound, or a salt, hydrate, or solvate thereof, or a compound, or a salt, hydrate, or solvate thereof.

[0138] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G107) [Chemical formula] or administering a composition comprising the compound, or a salt, hydrate, or solvate thereof, or a compound, or a salt, hydrate, or solvate thereof.

[0139] In another embodiment, the use of the compound and composition in the manufacture of a medicament for treating a host animal having cancer is also described. In another embodiment, the medicament comprises a therapeutically effective amount of one or more compounds, or salts, hydrates, or solvates thereof, and / or a composition comprising the same for treating a host animal having cancer.

[0140] The present compounds, compositions, unit dosages, and methods may be used alone or in combination with other compounds useful for the treatment of cancer, including compounds that may be therapeutically effective by the same or different modes of action. Further, the present compounds may be used in combination with other compounds administered for treating other symptoms of cancer, such as compounds administered to cancer.

[0141] Topoisomerase I inhibitors are classified as either inhibitors that inhibit the DNA cleavage reaction catalyzed by the enzyme or poisons that inhibit the DNA religation reaction catalyzed by the enzyme after cleavage has occurred (Scheme 1). Since the discovery that indenoisoquinoline NSC314622 (1) 1 acts as a topoisomerase I poison by inserting between base pairs at the cleavage site, 2 topoisomerase II, 3~7 retinoid X receptor (RXR), 8~10 PARP-1, 11Estrogen receptor, 12 tyrosyl-DNA phosphodiesterase 1 (TDP1), 13~17 and tyrosyl-DNA phosphodiesterase 2 (TDP2) 13 Several additional biological macromolecules have been found to be targeted by variously substituted indenoisoquinolines that include estrogen receptor, 12 , tyrosyl-DNA phosphodiesterase 1 (TDP1), 13~17 , and tyrosyl-DNA phosphodiesterase 2 (TDP2) 13 . More recently, an array of indenoisoquinolines has been reported to downregulate MYC protein expression via a mechanism that stabilizes the G-quadruplex of the MYC promoter and involves terminal stacking of the indenoisoquinoline with the outer quartet of the G-quadruplex. 18 Three indenoisoquinoline topoisomerase 1 toxicants, LMP400 (indotecan, 2), 19 LMP776 (indimitecan, 3), 19 and LMP744 (4) 20 have successfully completed, or are about to complete, phase I clinical trials, 21 and all three drugs, in particular, have also been found to be MYC promoter G-quadruplex stabilizers that downregulate MYC protein expression. 18 Furthermore, indenoisoquinolines can stabilize G-quadruplexes by two mechanisms: (1) inhibition of topoisomerase I to maintain the negative DNA supercoiling associated with G-quadruplex formation, and (2) terminal stacking with two outer G-quadruplex quartets. 18 Indeed, the two activities (topoisomerase I inhibition and G-quadruplex stabilization) have been found to act synergistically with respect to the cytotoxicity of cancer cells. 18 This specification describes the results of tests aimed at optimizing indenoisoquinolines for both targets in an attempt to maximize the anticancer activity of indenoisoquinolines.

[0142]

Chemical formula

[0143] Chemical properties In one embodiment, a process for preparing a compound of formula (I) is described.

[0144] In known routes to the analogs 2,3-methoxy and 8,9-methylenedioxy-indenoisoquinoline, it was found that the target compounds could not be provided. For example, the following compounds could not be cyclized under known conditions:

Chemical formula

[0145] The route used to prepare 9-MeO-7-aza-indenoisoquinoline requires the preparation of the following compounds (WO2018 / 118852A1):

Chemical formula

[0146] The route to this intermediate based on known chemistry failed. The unexpected bromination of the ring shown below was an important step in preparing the required intermediate:

Chemical formula

[0147] The process described herein for preparing the compound of formula (I) overcomes the obstacles caused by the known synthetic routes to 7-azaindenoisoquinoline. Exemplary processes include the following:

Chemical formula

[0148] In another embodiment, a compound prepared by one or more of the processes described herein is provided.

[0149] Depending on whether the 3-bromopropyl chain of the relevant intermediate is attached to the basic part of the side chain (16, Scheme 2) or to 7-azaindenoisoquinoline (20, Scheme 3), two different routes were used to synthesize the target compound. As outlined in Scheme 2, treating furfuraldehyde (5) with aqueous bromine solution under acidic conditions and subsequently adding sulfamic acid (6) to the reaction mixture gave 5-bromopyridine-2,3-diol (7), which was converted to intermediate 8 in low yield (12%) using dibromomethane in the presence of potassium carbonate and cupric oxide. 27、28Interestingly, the details of the experiment for synthesizing 7 from 8 were not reported, but the 12% yield claimed for the conversion was reproduced as described herein. 28 The bromide of 8 was replaced with a methyl group using Suzuki-Miyaura cross-coupling to obtain the desired methylated product 9. 29 Surprisingly, when 5-methylpyridine 9 on the aromatic ring was regioselectively brominated with NBS in AcOH / CH 3 CH at room temperature, the bromopyridine derivative 10 was obtained, 30~32 and then it was found that nucleophilic aromatic substitution occurred with CuCN in DMF under reflux to give the intermediate 11. The desired primary bromide 12 was produced by free radical bromination of the methyl group of 11 with NBS in the presence of perbenzoic acid during reflux in carbon tetrachloride. 12 and 6,7-dimethoxyhomophthalic anhydride were condensed in refluxing acetonitrile in the presence of triethylamine to obtain 7-azaindenoisoquinoline 14, 33 which was oxidized with selenium dioxide to produce the penultimate intermediate 15. 23 15 was deprotonated with sodium hydride, and then the resulting anion was alkylated with the appropriate bromide 16 to obtain the target compounds 17, 18, and 19.

[0150] Scheme 2. Synthesis of 7-aza-8,9-methylenedioxyindenoisoquinoline derivatives a of

Chemical Structure

[0151] The first step of the alternative synthesis outlined in Scheme 3 involves deprotonating intermediate 15 with sodium hydride, followed by reacting it with 1,3-dibromopropane in the presence of DMF containing potassium iodide, resulting in the formation of intermediate 20 with a 3-bromopropyl side chain. 22、24 The target compounds shown in Scheme 3 were prepared by reacting 20 with the appropriate amine using DIPEA as the base in DMF at various times and temperatures as shown in the general example. 19

[0152] Scheme 3. 7-Aza-8,9-methylenedioxyindenoisoquinoline derivatives a Synthesis

Chemical Structure

[0153] Biological results and discussion 1. In-vitro binding to the c-Myc promoter G-quadruplex (MycG4) MycG4 stabilization measured by FRET melting. To determine whether 7-aza-8,9-methylenedioxyindenoisoquinoline derivatives can bind to and stabilize the c-Myc promoter G-quadruplex (MycG4, Figure 1), a Förster resonance energy transfer (FRET) melting assay was performed. MycG4 DNA was labeled with a FRET donor (6-FAM) at the 5'-end and a FRET acceptor (TAMRA) at the 3'-end. Due to the proximity of the donor fluorophore and the acceptor fluorophore in the G-quadruplex secondary structure, the fluorescence of 6-FAM is quenched by FRET transfer to TAMRA. When the secondary structure melts into single-stranded DNA, the proximity of the FRET pair decreases and the 6-FAM-based fluorescence emission recovers. Monitoring the emission of 6-FAM during the thermal denaturation of the G-quadruplex provides a melting curve from which the melting temperature (T m )(the temperature at which folded DNA and unfolded DNA are equally present) is derived. The T m value of MycG4 was determined by FRET melting experiments in 10 mM K +Measured in the presence of the compound. All 8 tested 7-aza-8,9-methylenedioxyindenoisoquinolines showed a distinct thermal stabilization (ΔT m ) of MycG4 (Table 1 and Figure 2), with the melting temperature increasing by more than 10 °C. Among these, G103, G104, G105, and G108 showed the maximum thermal stabilization (ΔT m ) of MycG4. The data of the reference LMP compound are provided in Figure 3, and the structure of the reference compound 26 (LMP517) is shown below.

[0154]

Table 1

[0155] Binding affinity to Myc G-quadruplex. The binding affinity of eight 7-aza-8,9-methylenedioxyindenoisoquinolines to MycG4 was determined using a fluorescence binding assay with 3'-TAMRA-labeled MycG4 DNA. The binding isotherm was obtained by monitoring the TAMRA emission during drug titration, from which the apparent binding affinity K d,app was derived. Six of the 7-aza-8,9-methylenedioxyindenoisoquinolines (G-103 to G-108) showed strong binding with an apparent binding affinity K d,app value of less than 100 nM (Table 2). Indenoisoquinoline shows very little fluorescence whether in the free or bound state. The data of the reference LMP compound are shown in Table 3.

[0156]

Table 2

[0157]

Table 3

[0158] Binding interaction with MycG4 by NMR. The binding interaction between 7-aza-8,9-methylenedioxyindenoisoquinoline and MycG4 was determined by K +1D in solution was examined using 1 1 H NMR titration. The unbound Myc G-quadruplex exhibits 12 guanine imino protons, four from each of the three tetramers (Figure 4). Changes in the linewidth and chemical shift of the imino proton resonances upon addition of the compound indicate binding to the G-quadruplex. With the exception of G101, all 7-aza compounds are seen to bind to MycG4 DNA at both ends, as indicated by the clearly shifted imino proton peaks corresponding to the 3'- and 5'-tetramers upon drug addition. The binding was seen to be in the intermediate to fast exchange regime on the NMR timescale, as indicated by the broadening of the DNA proton peaks upon drug addition.

[0159] 2. In vivo Myc inhibition Western blot. The G-quadruplex formed at the MYC promoter functions as a transcriptional silencer. To identify the effect of 7-aza-8,9-methylenedioxyindenoisoquinoline on the MYC protein level, Western blotting experiments were performed using MCF-7 breast cancer cells. Western blotting experiments were conducted to examine the downregulatory effect of 7-aza-8,9-methylenedioxyindenoisoquinoline on the Myc protein level (Figure 5). MCF-7 breast cancer cells were incubated with 0, 0.5, 1.0, and 2.0 μM of G-101 to 108 for 24 hours. G101 to G105 showed a strong Myc inhibitory effect. For G106 to G108, only G107 was seen to exhibit moderate Myc inhibition, while G106 and G108 showed weak Myc downregulation.

[0160] qRT-PCR. To confirm the effects of the selected 7-aza-8,9-methylenedioxyindenoisoquinolines, G103 and G105, on the transcription of the MYC gene in cancer cells, the MYC mRNA levels in MCF-7 breast cancer cells were measured by qRT-PCR (Figure 6). MCF-7 cells were incubated with 0 and 1.0 μM of G103 and G105 for 6 hours. Consistent with the Western blotting data, G103 and G105 significantly decreased the MYC mRNA levels at 6 hours after treatment with 1.0 μM of the drug. The data of the reference LMP compounds are shown in Table 7.

[0161] 3. Results of in vivo cytotoxicity in cancer cell lines Single-dose cell survival assay in CA-46 cells and Raji cells. Both CA-46 and Raji are Myc-dependent and Myc-overexpressing non-Hodgkin lymphoma cell lines. Both cell lines overexpress Myc due to a translocation that inserts the IgH enhancer upstream of the Myc gene. The translocation in CA-46 eliminates the G-quadruplex-forming region in one allele, while the translocation in Raji preserves the G-quadruplex-forming regions in both alleles. Thus, Raji is more sensitive to G-quadruplex-mediated Myc inhibition than CA-46, which can be reflected in the lower IC 50 value. To evaluate the selectivity of the cell lines, an MTS single-dosing assay was performed to compare the antiproliferative effects of 7-aza-8,9-methylenedioxyindenoisoquinoline on CA-46 cells and Raji cells at a drug concentration of 250 nM (Figure 8). The values of the average survival rate % of CA-46 cells and Raji cells for each drug were shown side by side in a bar plot. Among G101 - G108 at 250 nM, G101 and G106 - G108 showed no growth inhibition in either cell line. In contrast, G102 - G105 showed obvious antiproliferative effects on both cell lines and showed a statistically significantly higher survival rate % of CA46 than Raji. The data of the reference LMP compounds are shown in Figure 9.

[0162] Results of cytotoxicity in Raji non-Hodgkin lymphoma cells. Next, to test the anti-proliferative effect of 7-aza-8,9-methylenedioxyindenoisoquinoline in Raji cells, an MTS assay was performed. Cells were incubated with each drug at 2 - 2000 nM for 72 hours, and cell viability was quantified using the MTS assay. The measured IC 50 values of all compounds are shown in Table 4. Among all the compounds, G102 - G105 are clearly more potent than G106 - G108. This trend in potency generally correlates with the results of Western blot, in which G106 and G108 did not show weak Myc downregulation. Data for the reference LMP compounds are shown in Table 5.

[0163]

Table 4

Table 5

[0164] Results of cytotoxicity in Myc-dependent cancer cell lines. Due to the high potency of the G101 - G105 compounds shown in the Raji MTS IC 50 assay, these compounds were further tested in a selected panel of Myc-dependent / overexpressing cancer cell lines. Similar to the CA-46 and Raji MTS IC 50 assay, each cell line was treated with each compound at 2 - 2000 nM for 72 hours.

[0165] Among 7-aza-8,9-methylenedioxyindenoisoquinolines (Table 6), G103 - G105 were generally more potent than G101 - G102 across all cell lines. Among all the cell lines, MOLT-4, RS4;11, and CHLA-99, as well as RD-ES, were highly sensitive to G103 - G105, showing GI 50 values close to nM. Data for the reference LMP compounds are shown in Table 7.

[0166]

Table 6

Table 7

[0167] 5. Inhibition of Topoisomerase I The inhibitory effect of 7-aza-8,9-methylenedioxyindenoisoquinoline compounds on Topo1-mediated DNA relaxation was examined (Figure 10A), and Topo1 toxicity was examined using a Topo1-mediated DNA cleavage assay (Figure 10B).

[0168] Topo1-mediated DNA relaxation assay. Using a Topogen human topoisomerase I assay kit (TG1015-1A), the inhibitory effect of the compound on Topo1-mediated DNA relaxation was examined. The assay was performed according to the manufacturer's instructions. A typical assay sample had a total volume of 20 μl containing 100 ng of pHOT1 DNA, 4 U of human Topo1, and 10 μM of the test compound. The assay sample was incubated at 37 °C for 30 minutes, followed by termination of the reaction with 10% SDS. Subsequently, digestion with proteinase K was performed at 37 °C for 15 minutes to degrade Topo1, and 1:1 volume chloroform:isoamyl alcohol DNA extraction was performed. Next, the sample was electrophoresed at 46 v for 4 hours on a 1% agarose gel in TBE buffer. Before imaging, the gel was stained with a 0.5 μg / ml ethidium bromide solution for 15 minutes and washed three times with Milli-Q water at 5-minute intervals. The results are shown in Figure 10A.

[0169] Topo1-mediated DNA cleavage assay. The Topo1 toxicity of the compounds was examined using the Topogen human topoisomerase I assay kit (TG1015-1A). The assay was performed according to the manufacturer's instructions. 10 μM CPT was used as a positive control. A typical assay sample had a total volume of 20 μl containing 100 ng of pHOT1 DNA, 4 U of human Topo1, and 10 μM of the test compound. The assay samples were incubated at 37 °C for 30 min, followed by termination of the reaction with 10% SDS. Subsequently, digestion with proteinase K was performed at 37 °C for 15 min to degrade Topo1, and 1:1 volume chloroform:isoamyl alcohol DNA extraction was carried out. The samples were electrophoresed on a 1% agarose gel in TBE buffer at 46 v for 2 h. Both the agarose gel and the buffer contained 0.5 μg / ml ethidium bromide. The results are shown in Figure 10B.

[0170] As shown by the data in Figure 10A, none of the compounds inhibited the DNA cleavage reaction catalyzed by Top1. In contrast, compounds G103 - G107 inhibited the DNA drop reaction, while G101, G102, and G108 had only a very slight effect on DNA drop, as shown in Figure 10B. In other words, G103 - G107 act as Top1 toxins and do not act as Top1 inhibitors, and G105 - G107 are the most potent. This is consistent with the Top1 inhibitory activity of indenoisoquinolines having 7 carbon atoms.

[0171] 6. Results of Pharmacokinetics Pharmacokinetics. The pharmacokinetic results obtained after IV and PO administration of Compounds 19, 21, and 22 to CD1 mice are listed in Tables 8 and 9, respectively. The half-lives of the drugs after IV administration were 3.29 hours for 19, 2.73 hours for 21, and 1.84 hours for 22. The half-lives after oral administration were 6.88 hours for 19, 6.91 hours for 21, and 5.20 hours for 22. There was a significant variation in the percentage of the dose absorbed after oral administration, with 41% of the dose of 19 being absorbed, 14.2% of the dose of 21 being absorbed, and 1.19% of the dose of 22 being absorbed. The absorption percentages generally corresponded to the CLogP values of 1.67 for 19, 1.36 for 21, and 0.363 for 22, indicating that more absorption from the GI tract is expected for more lipophilic compounds.

[0172]

Table 8

Table 9

[0173] Plasma protein binding and stability analysis. The percentage of protein binding and stability of Compounds 19, 21, and 22 were determined in both human and mouse plasma. The results are listed in Table 10. The order of protein binding was in the order of lipophilicity of the compounds, with Compound 19 being the most highly protein-bound and Compound 22 being the least protein-bound in both mouse plasma and human plasma. The free drug (unbound) concentrations of all three compounds were sufficient to drive pharmacological efficacy, and all three compounds were relatively stable in plasma.

[0174]

Table 10

[0175] Stability analysis in human and mouse liver microsomes. The stabilities of Compounds 19, 21, and 22 were determined in the presence of human and mouse liver microsomes, and the results are listed in Table 11. All three compounds showed robust microsomal stability that was consistent with their in vivo stability in male CD1 mice (Table 11). The half-lives for human microsomes ranged from 227 to 372 minutes, and the order of stability was 19 > 21 > 22. Interestingly, both Compounds 19 and 21 were more stable towards human microsomes than mouse microsomes, but for Compound 22, the order was reversed, and it was more stable towards mouse microsomes than human microsomes. Despite observing a longer half-life and lower in vitro CL for Compound 22 in mouse microsomes compared to human microsomes, the calculated scaled-up CL was higher in mice due to the much larger scaling factor for mice compared to humans (Table 20a, Experimental section). int Despite observing a longer half-life and lower in vitro CL for Compound 22 in mouse microsomes compared to human microsomes, the calculated scaled-up CL Int was higher in mice due to the much larger scaling factor for mice compared to humans (Table 20a, Experimental section).

[0176]

Table 11

[0177] Caco-2 cell permeability studies. Caco-2 permeability tests were performed on Compounds 19, 21, and 22, and the results are listed in Table 12. All three compounds showed high efflux ratios in Caco-2 cells, indicating that all three compounds are likely substrates of efflux transporter(s) (P-gp, BCRP, and / or MRP2). All three compounds showed moderate permeability in the apical-to-basolateral direction, which is the direction of intestinal absorption.

Table 12

[0178] In summary, Compound 19 (G103) has the longest IV half-life, the maximum absorption %PO, the maximum stability against human liver microsomes, and the lowest efflux ratio in Caco-2 cells. Compound 21 (G104) has the highest cytotoxicity against CA46 and Raji cells, as well as excellent selectivity against Raji cells comparable to that of 22 (G105).

[0179] Determination of the maximum tolerated dose. The maximum tolerated doses (MTDs) of G103, G104, and G105 were determined in NOD SCID mice, and the results are shown in Table 13 and Figures 11 and 12. The MTD of G103 was 50 mg / kg, and the MTDs of G104 and G105 were 30 mg / kg and 20 mg / kg, respectively. Experiments containing all three drugs at a dose of 100 mg / kg were terminated after 1 day due to toxicity, while the test containing G105 at a dose of 50 mg / kg was also stopped after 1 day (Figure 11). Similarly, the test using G105 at a dose of 40 mg / kg was interrupted after 2 days (Figure 12). The large difference in MTD between G103 and G104 (50 vs. 30 mg / kg) is unexpected because the chemical structure changes are small (one methyl group for the hydrogen of the side chain).

[0180]

Table 13

[0181] Xenograft studies. The efficacy of G103, G104, and G105 was investigated in female NOD SCID mice after inoculation with MOLT-4 human leukemia cells, RD-ES human sarcoma cells, and RS4;11 human leukemia cells. JQ1 was used as a positive control. Ten days after inoculation, the tumors were approximately 100 - 150 mm 3Treatment was initiated when the size reached [the specified size]. The drug was administered IV on days 1 - 5 and 11 - 16 after the start of treatment, and body weight and tumor values were monitored on days 10, 14, 17, 21, and 24 after inoculation. As shown in Figure 13, the drug generally had a relatively good tolerance as judged by the decrease in body weight, and the largest decrease was observed after administration of G105 at a dose of 20 mg / kg (the average body weight of the animals was 80.9 ± 1.1% of the initial body weight on day 24 after tumor inoculation).

[0182] Figure 14 shows a plot of tumor size over time after inoculation of MOLT-4 human leukemia cells into female NOD SCID mice, and after IV treatment with various doses of G103, G104, and G105, and after IP administration of the positive control JQ1. Each of the "G compounds" was administered at higher and lower doses as detailed in Figure 13. The data reveals the relative effectiveness of the various treatments: G105 (20 mg / kg) > G103 (50 mg / kg) > G103 (25 mg / kg) > G105 (10 mg / kg) > G104 (30 mg / kg) > G104 (15 mg / kg) > JQ1 (50 mg / kg). Each of the drugs decreased the rate of increase in tumor size compared to the control.

[0183] A similar test was conducted after inoculating female NOD SCID mice with RD-ES human sarcoma tumor cells (Figures 15 and 16). The tumors grew much more rapidly, treatment was initiated on day 8 after tumor inoculation, and measurements of body weight and tumor size were taken on days 8, 12, 15, 19, and 22 after tumor inoculation. The most potent treatment was the same as for MOLT-4, and G105 at a dose of 20 mg was the most potent. However, the overall order of efficacy was not the same as above: G105 (20 mg / kg) > G103 (50 mg / kg) > G104 (30 mg / kg) > G103 (25 mg / kg) > G105 (10 mg / kg) > JQ-1 (50 mg / kg) > G104 (15 mg / kg). The average RD-ES tumor size was much larger than that observed in MOLT-4 (2327 ± 285 vs 546 ± 59 mm 3 )

[0184] The compounds were also evaluated in the RS4;11 human leukemia xenograft model (Figures 17 and 18). Treatment was initiated 12 days after tumor inoculation, and the drugs were administered on days 1-5 and 11-15 after the start of treatment. Animal weight and tumor volume were determined on days 12, 15, 19, 22, and 25 after tumor inoculation. As in other cases, treatment with G105 at a dose level of 20 mg / kg was found to be the most toxic, as estimated from the decrease in animal body weight (Figure 16), and was the most potent in reducing tumor volume (Figure 17). The relative efficacy of the various treatments against RS4;11 was different from that observed in the other two cases: G105 (20 mg / kg) > G103 (50 mg / kg) > G103 (25 mg / kg) > G105 (10 mg / kg) > JQ-1 (50 mg / kg) > G104 (15 mg / kg) > G104 (30 mg / kg). Overall, the various treatments had low to moderate effects in the RS4;11 xenograft model, and the tumor volumes after treatment with G104 at both dose levels (15 and 30 mg / kg) were close to those observed in untreated animals at day 25.

[0185] Brain permeability test. One possible use of indenoisoquinolines is the treatment of malignant brain tumors. Previous studies have demonstrated CNS penetration by LMP400 (2), suggesting that G103, G104, and G105 may also be able to penetrate the blood-brain barrier. 40Therefore, after intravenous administration to CD1 mice, the concentrations of these compounds in plasma and brain, as well as LMP744(4), were monitored by LC-MS / MS, and the brain / plasma concentration was calculated (Table 14a). Four compounds were tested by intravenous administration at a dose of 20 mg / kg to four groups of 24 male CD1 mice, and three animals were used at each time point. A total of 96 animals were utilized to generate the data in Table 14a, and plasma and brain tissue samples were collected from each mouse. Concentrations were determined by LC-MS / MS that directly measures plasma samples. Brain tissue was homogenized with water using a 3:1 ratio of brain (g) to water (mL), then the concentration (ng / mL) in the brain homogenate was determined, and then the homogenate value was multiplied by 4 to obtain the brain concentration (ng / g). Next, the ng / g concentration value in the brain was converted to nM concentration in the brain using a conversion factor of 1.03 based on the fact that the weight of 1 cc of brain is 1.03 g. 41 The initial brain concentration was proportional to the lipophilicity of the side chain, while the initial plasma concentration was inversely proportional to the lipophilicity of the side chain, which reflects that compounds with more lipophilic side chains partitioned more rapidly from plasma into the brain. The highest initial brain concentration was achieved with G103, and the average brain / plasma ratio observed 15 minutes after administration was 7.97, and the relatively high brain / plasma ratio of G103 was maintained throughout the 24-hour observation period. The second highest initial brain concentration and average brain / plasma ratio were detected with G104, resulting in a ratio of 1.88 at the 15-minute time point. With G105 and LMP744, lower initial brain concentrations and ratios were observed. Low concentrations of the compounds were measured in both plasma and brain at the 24-hour time point, but high brain / plasma ratios were calculated, and higher numbers were observed for the compounds with the lowest initial ratios. This reflects the fact that the brain concentrations of G105 and LMP744 were relatively low but surprisingly stable over the 24-hour period, while G103 and G104 were associated with "normal" brain pharmacokinetics, and the concentrations of G103 and G104 decreased over time as expected. On the other hand, the plasma concentrations of all compounds decreased as expected as a function of time.

[0186] Similar analyses were performed with LMP744 and LMP400 in combination with Temodar and Lynparza. Table 14b lists the brain concentrations, plasma concentrations, and brain / plasma ratios of LMP744 and LMP400 when administered in combination with 100 mg / kg PO of Lynparza or 125 mg / kg of Temodar. Clearly, LMP400 is able to penetrate the blood-brain barrier more effectively than LMP744, as evidenced by the much higher brain concentrations and brain / plasma ratios of LMP400 compared to LMP744. The low permeability of the compound with the hydroxyethylaminopropyl side chain (i.e., LMP744) described in Table 14b is consistent with the results shown in Table 14a, which explains the lower levels of LMP744 and G105 compared to G103 and G104. It is interesting that the brain concentrations of the LMPs are consistently high after Lynparza administration at all time points, suggesting that Lynparza may facilitate the penetration of LMP400 into the brain compared to Temodar. Finally, the "flat" concentration levels of LMP744 as a function of time shown in Table 14b are also consistent with the results for both LMP744 and G105 shown in Table 14a, indicating that the hydroxyethylaminopropyl side chain appears to maintain these drugs in the brain at relatively low levels over a long binding time (5 minutes to 24 hours after administration). This is in contrast to LMP400, which shows a "normal" decrease in concentration as a function of time.

[0187] The concentrations of Temodar and Lynparza during these experiments were also followed as shown in Table 14c. Temodar can enter the brain after oral administration, but the partitioning of Lynparza into the brain after oral administration is very inefficient. Based on these results, the IV use of LMP400 in combination with orally administered Temodar may be effective, but the combination with Lynparza does not appear promising for the treatment of CNS cancers.

[0188] The pharmacokinetics observed during plasma and brain monitoring are detailed in Tables 15a and 16a. G103 and G104 were rapidly expressed in brain tissue after IV administration (T max 5 minutes, Table 16a), and the plasma half-life of G104 was 7.06 hours (Table 14a). %Extrap Because the _obs was greater than 20%, it was not possible to calculate the effective plasma half-life of G103. The plasma half-lives of G104 and G105 observed after a 20 mg / kg dose (Table 15a) were longer than those seen after a 1 mg / kg dose (Table 8), but they were in the same order (G104 T 1 / 2 >G105 T 1 / 2 ) AUC_ of G103, G104, G105, and LMP744 in the brain %Extrap Since all of the _obs values ​​were well above 20%, the effective brain T 1 / 2 , AUC Inf and MRT Inf It was not possible to calculate the _obs parameter (Table 16b). The initial brain concentrations of G103 were significantly higher than those of G104 (C max 3809 vs. 1613 ng / g, Table 16a), reflecting the greater lipophilicity of G103 compared to G104 (cLog P values ​​= 1.674 vs. 1.365, respectively).

[0189] The plasma and brain pharmacokinetic parameters of LMP400 and LMP744 when administered in combination with Lynparza and Temodar are presented in Tables 15b and 16b, respectively. LMP400 had a T of 5 minutes max As shown by the T (0.0830 h, Table 16b), LMP744 was rapidly expressed in the brain. max The time to cerebral C in the presence of LMP400 was 1 hour in the presence of Lynparza and 2 hours in the presence of Temodar (Table 16b), both of which were delayed compared to the 0.25 hours observed when LMP744 was administered alone (Table 16a). As expected, the time to cerebral C in the presence of LMP400 was 1 hour in the presence of Lynparza and 2 hours in the presence of Temodar (Table 16b), both of which were delayed compared to the 0.25 hours observed when LMP744 was administered alone (Table 16a). max The values ​​were significantly higher than LMP744, regardless of coadministration of Temodar or Lymparza, and both Temodar and Lymparza significantly increased the Cmax It has no impact (compare the values in Table 16b with those in 16a).

[0190] The plasma C of LMP744 0 AUC last values and AUC Inf Note that the values of AUC and the AUC values are different when co - administered with Lynparza and when co - administered with Temodar (Table 15b). LMP744 has a much larger volume of distribution at steady state than LMP400. The half - life and volume of distribution at steady state are not significantly affected by being co - administered with Lynparza compared to being co - administered with Temodar.

[0191]

Table 14

Table 15

Table 16

[0192]

Table 17

[0193]

Table 18

[0194]

Table 19

[0195]

Table 20

[0196] Tumor permeability test. In addition to the brain / plasma ratio, the tumor / plasma ratios of JQ1, G103, G104, and G105 were determined in mice bearing MOLT-4, RS4;11, and RD-ES tumor xenografts (Table 16). The ratios of MOLT-4 and RD4;11 tumors were determined at 3 and 9 hours, and the ratio of RD-ES was measured at 3 and 6 hours. All of the "G compounds" were administered IV at the maximum tolerated dose level and half of the maximum tolerated dose level, except in the experiment with JQ1, where a standard IP administration of 50 mg / kg was performed. From the results reported in Table 16, it is clear that all of the "G compounds" were highly concentrated in tumor tissue, while the tumor-plasma ratio of JQ1 was significantly lower. These results are consistent with the previously reported results for the related indenoisoquinoline LMP744 in dogs, which explain the extensive distribution in tumor tissue and that the average tumor concentration is at least 100-fold higher than the plasma concentration at the same time point. 42 In a related study of LMP400 in BALB / c female mice bearing CT26 colon tumors, the concentration in tumor tissue compared to plasma was also shown, but the ratio was lower (5 - 14.6 after IV administration). 40 Except for G104 (30 mg / kg) and G105 (10 mg / kg) for RE-ES, the ratios determined at earlier time points were larger than the ratios determined at earlier time points.

[0197] [Table 21]

[0198] The expression levels of c-Myc and γ-H2AX were determined by Western blotting in RS4;11, MOLT-4, and RD-ES xenografts after administration of JQ1, G103, G104, and G105 to female NOD SCID mice, and the results are listed in Tables 17 - 19. Except for JQ1, a maximal decrease in c-Myc expression was observed at the 9-hour time point after administration of G103 to mice bearing RS4;11 xenografts, and the effect was greater at higher dose levels (Table 17). G103 also decreased c-Myc relatively substantially in MOLT-4 xenografts, but the effect was not as great as that observed in RS4;11 cells (Table 18). Administration of G103 actually resulted in an unexpected increase in the level of c-Myc protein in RD-ES xenografts, and the effect was greater at higher dose levels (Table 19). Some moderate increases in c-Myc levels were observed in RS4;11 and MOLT-4 xenografts, but they were not as large or as common as those seen in RD-ES tumors, where a large increase was observed 3 hours after normal drug administration, followed by a decrease at the normal 6-hour time point (Table 19) (the exception being G103 at the 25 mg / kg dose level). For G104 and G105 against RD-ES xenografts, c-Myc levels were below control values at the 6-hour time point, but not so for G103 (Table 19). In some cases, c-Myc levels decreased more substantially at lower dose levels. See, for example, the 9-hour data for G104 in Tables 17 and 18 and the 6-hour data in Table 19. It should be noted that at the 3-hour time point in RD-ES cells, c-Myc expression was consistently higher at higher doses than at lower doses, and the levels at higher doses were significantly higher than the control. The maximal decrease in c-Myc was seen with JQ1 at the 3-hour time point in MOLT-4 cells (Table 18), but in RD-ES xenografts, it resulted in a substantial increase in c-Myc at the 3-hour time point (Table 19). Overall, the data in Tables 17 - 19 have many unexpected and unexplained aspects, which are highly variable and also highly dependent on the cell line. In particular, the drug-induced increase in c-Myc expression compared to the control and the greater changes in c-Myc expression seen at lower doses compared to higher doses are counterintuitive and unexplained.

[0199] 7-Azaindenoisoquinoline is known to induce phosphorylation of histone H2AX at Ser139 (γ-H2AX) by causing DNA double-strand breaks. 25 As shown in Tables 17 to 19, G103, G104, and G105 consistently increased γ-H2AX production compared to the control at earlier time points, with the exception of G104 in RD-ES cells. The degree of DNA damage generally became higher at earlier time points. The exceptions were the low dose of G104 and both doses of G105 in RD-ES cells (Table 19), as well as both doses of G103 in RS4;11 xenografts (Table 17). There were many examples where γ-H2AX decreased below the control level, which generally occurred at the 3-hour time point. As an exception, the levels decreased at both time points in JQ1 of RD-ES xenografts. (Table 19).

[0200] Among the three compounds (G103 - G105) tested in more detail, G103 has the longest IV half-life, the greatest oral absorption, the greatest stability in human liver microsomes, the lowest efflux ratio in Caco-2 cells, the largest and most tolerable dose in NOD SCID mice, and the greatest brain permeability. However, as an anti-cancer agent in vivo, G105 seems to be the most effective among the three compounds.

[0201]

Table 22

[0202]

Table 23

[0203]

Table 24

[0204] Experimental section 5-Bromopyridine-2,3-diol (7). A cold aqueous solution was prepared by adding formaldehyde (5, 20.10 g, 17.3 mL, 209.2 mmol, 1 equiv) to ice (200 g) from a newly opened container. The solution was stirred vigorously and bromine (33.43 g, 10.78 mL, 209.2 mmol, 1 equiv) was added dropwise at a rate such that the reaction temperature was maintained at 0 °C (using an external ice bath). The reaction mixture was stirred for 30 min. Concentrated HCl (10 mL) was added all at once and the reaction mixture was stirred at 0 °C for 30 min. While maintaining the reaction temperature at -5 to 0 °C, additional bromine (33.43 g, 10.78 mL, 209.2 mmol, 1 equiv) was added dropwise over 1 h. The reaction mixture was then filtered and sulfamic acid (6) (20.31 g, 209.2 mmol, 1 equiv) was added to the filtrate and the mixture was stirred vigorously at 50 °C for 3 h. The reaction mixture was cooled to 0 °C and the resulting solid precipitate was collected by filtration. The solid was dried at 25 °C for 22 h to give 5-bromopyridine-2,3-diol (7, 19.8 g, 104 mmol, 49.8%) as a brown solid. 1 H NMR (300 MHz, DMSO-d 6 ) δ 11.87 (s, 1H), 9.69 (s, 1H), 7.09 (d, J = 2.5 Hz, 1H), 6.79 (d, J = 2.5 Hz, 1H).

[0205] 6-Bromo-[1,3]dioxolo[4,5-b]pyridine (8). Dibromomethane (14.6 g, 5.88 mL, 84.2 mmol, 2.0 equiv), potassium carbonate (11.6 g, 84.2 mmol, 2.0 equiv) and copper(II) oxide (1.0 g, 12.6 mmol, 0.30 equiv) were added to a solution of 5-bromopyridine-2,3-diol (3, 8.0 g, 42.1 mmol, 1 equiv) in DMF (80 mL). The reaction mixture was stirred and heated to 90 °C for 14 h. The reaction mixture was cooled to room temperature and filtered through a Celite bed, which was washed with ethyl acetate (2 x 100 mL). The filtrate was extracted with ethyl acetate (2 × 200 mL). The combined ethyl acetate extracts were washed with water (100 mL) and then brine (100 mL) and dried over anhydrous MgSO 4It was dried. The solvent was evaporated to dryness to prepare a crude product, which was purified by flash column chromatography [silica gel (40 g), eluted with hexane containing 0 - 100% ethyl acetate] to obtain 6-bromo-[1,3]dioxolo[4,5-b]pyridine (8, 1.0 g, 4.95 mmol, 12%) as a white solid. 1 H NMR (300 MHz, DMSO-d 6 ) δ 7.71 (d, J = 2.0 Hz, 1H), 7.55 (dd, J = 2.0, 0.9 Hz, 1H), 6.20 (s, 2H).

[0206] 6-Methyl-[1,3]dioxolo[4,5-b]pyridine (9). 6-Bromo-[1,3]dioxolo[4,5-b]pyridine (8, 10.0 g, 49.5 mmol, 1 equivalent), methylboronic acid (5.93 g, 99.0 mmol, 2 equivalents), sodium carbonate (21.0 g, 198 mmol, 4.0 equivalents), 2-dicyclohexylphosphino-2’,6’-diisopropoxybiphenyl (RuPHOS) (2.31 g, 4.95 mmol, 0.1 equivalent), and Pd 2 dba 3 (2.27 g, 2.48 mmol, 0.050 equivalent) in a mixture of toluene (200 mL) and water (20 mL) was degassed and then filled with nitrogen. The reaction mixture was heated at 110 °C for 12 hours and then cooled to room temperature, and diluted with ethyl acetate (100 mL) and water (50 mL). The reaction mixture was stirred and then allowed to stand to separate the layers. The aqueous layer was back-extracted with ethyl acetate (100 mL). The combined organic extracts were dried over anhydrous MgSO 4 and filtered, and evaporated to dryness. The remaining residue was purified twice by flash column chromatography [silica gel (2 × 120 g), eluted with hexane containing 0 - 100% ethyl acetate] to obtain 6-methyl-[1,3]dioxolo[4,5-b]pyridine (9, 5.6 g, 41 mmol, 82%) as a viscous yellow syrup. 1 H NMR (300 MHz, DMSO-d 6 ) δ 7.45 - 7.34 (m, 1H), 7.10 (d, J = 1.8 Hz, 1H), 6.07 (s, 2H), 2.18 (s, 3H).

[0207] 5-Bromo-6-methyl-[1,3]dioxolo[4,5-b]pyridine (10). N-Bromosuccinimide (10.9 g, 61.0 mmol, 1.1 eq) was added to a stirred solution of 6-methyl-[1,3]dioxolo[4,5-b]pyridine (9) (7.60 g, 55.4 mmol, 1 eq) and acetic acid (1.14 mL) in acetonitrile (150 mL) at room temperature. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with water (300 mL), and the product was extracted with ethyl acetate (2 × 300 mL). The combined ethyl acetate extracts were dried over anhydrous MgSO 4 and filtered, and the solvent was evaporated to dryness. The remaining residue was purified twice by flash column chromatography [silica gel (2 × 120 g), eluting with hexane solution containing 0 - 100% ethyl acetate] to afford 5-bromo-6-methyl-[1,3]dioxolo[4,5-b]pyridine (10; 6.5 g, 30 mmol, 54%) as a pale red solid. 1 H NMR (300 MHz, DMSO-d 6 ) δ 7.32 (s, 1H), 6.17 (s, 2H), 2.22 (s, 3H).

[0208] 6-Methyl-[1,3]dioxolo[4,5-b]pyridine-5-carbonitrile (11). CuCN (5.4 g, 60 mmol, 2.0 eq) was added to a stirred solution of 5-bromo-6-methyl-[1,3]dioxolo[4,5-b]pyridine (10, 6.5 g, 30 mmol, 1 eq) in dehydrated DMF (40 mL) and toluene (80 mL). The reaction mixture was stirred for 18 h while heating at 110 °C. After completion of the reaction, brine (300 mL) was added, and the product was extracted with ethyl acetate (2 × 500 mL). The combined extracts were dried over anhydrous MgSO 4 and filtered, and the solvent was evaporated. The remaining residue was purified by flash column chromatography [silica gel (120 g), eluting with hexane containing 0 - 100% 9:1 mixture of ethyl acetate / methanol] to afford 6-methyl-[1,3]dioxolo[4,5-b]pyridine-5-carbonitrile (11, 2.3 g, 19 mmol, 47%) as a white solid. 11H NMR (300 MHz, DMSO-d 6 ) δ 7.35 (s, 1H), 6.26 (s, 2H), 2.38 (s, 3H).

[0209] 6-(Bromomethyl)-[1,3]dioxolo[4,5-b]pyridine-5-carbonitrile (12). N-Bromosuccinimide (1.0 g, 5.61 mmol, 1.30 equiv) and benzoyl peroxide (83.7 mg, 75 wt%, 259 μmol, 0.060 equiv) were added to a stirred solution of 6-methyl-[1,3]dioxolo[4,5-b]pyridine-5-carbonitrile (11, 0.70 g, 4.32 mmol, 1.0 equiv) in carbon tetrachloride (35 mL) at room temperature. The mixture was heated to reflux for 16 h. The reaction mixture was concentrated and the residue was purified by flash column chromatography [silica gel (12 g), eluting with hexane containing 0–100% ethyl acetate] to afford 6-(bromomethyl)-[1,3]dioxolo[4,5-b]pyridine-5-carbonitrile (12, 0.488 g, 2.02 mmol, 46.9%) as a white solid. 1 1H NMR (300 MHz, DMSO-d 6 ) δ 7.54 (s, 1H), 6.31 (s, 2H), 4.71 (s, 2H).

[0210] 2,3-Dimethoxy-6,12-dihydro-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinolin-5-one (14). 6,7-Dimethoxyisochroman-1,3-dione (13, 409 mg, 1.84 mmol, 1.2 eq) was added to acetonitrile (22 mL, 60 volume eq) containing 6-(bromomethyl)[1,3]dioxolo[4,5-b]pyridine-5-carbonitrile (12, 0.370 g, 1.54 mmol, 1 eq). Triethylamine (388 mg, 535 μL, 3.84 mmol, 2.50 eq) was then added and the mixture became a clear solution. The reaction mixture was heated to reflux for 15 h, then cooled to room temperature and the precipitated solid was collected by filtration, washed with acetonitrile to give 2,3-dimethoxy-6,12-dihydro-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinolin-5-one (14, 0.310 g, 916 μmol, 59.7%) as a pale yellow solid. 1 H NMR (300 MHz, DMSO-d 6 ) δ 11.84 (s, 1H), 7.61 (s, 1H), 7.54 (s, 1H), 7.13 (s, 1H), 6.21 (s, 2H), 3.95 (s, 3H), 3.87 (s, 3H), 3.76 (s, 2H).

[0211] 2,3-Dimethoxy-5H-[1,3]dioxolo[4″,5″:5′,6′]pyrido[3′,2′:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (15). A mixture of 2,3-dimethoxy-6,12-dihydro-5H-[1,3]dioxolo[4″,5″:5′,6′]pyrido[3′,2′:4,5]cyclopenta[1,2-c]isoquinolin-5-one (14, 50 mg, 0.15 mmol, 1 equiv) and selenium dioxide (66 mg, 0.59 mmol, 4.0 equiv) in dioxane (7 mL, 140 volume equiv) and acetic acid (8.9 mg, 8.5 μL, 0.15 mmol, 1 equiv) was heated to reflux for 11 h. The reaction mixture was filtered and the resulting solid was washed with dioxane to afford 2,3-dimethoxy-5H-[1,3]dioxolo[4″,5″:5′,6′]pyrido[3′,2′:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (15, 0.041 g, 0.12 mmol, 79%) as a brown solid. 1 H NMR (300 MHz, DMSO-d 6 ) δ 7.67 (s, 1H), 7.50 (s, 1H), 7.35 (s, 1H), 6.28 (s, 2H), 3.90 (s, 3H), 3.86 (s, 3H).

[0212] 2,3-Dimethoxy-6-(3-morpholinopropyl)-5H-[1,3]dioxolo[4″,5″:5′,6′]pyrido[3′,2′:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (17). NaH (12 mg, 60 wt%, 0.30 mmol, 3.0 equiv) and KI (1.6 mg, 0.1 equiv) were added to a suspension of 2,3-dimethoxy-5H-[1,3]dioxolo[4″,5″:5′,6′]pyrido[3′,2′:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (15, 0.035 g, 1 equiv) in anhydrous DMF (3 mL) at 5 °C. The reaction mixture was warmed to room temperature and stirred for 1 h to give a dark brown solution. The solution was cooled to 0 °C and 4-(3-bromopropyl)morpholine hydrobromide (16, 37 mg, 0.13 mmol, 1.3 equiv) was added and the solution was stirred at room temperature for 12 h. The reaction was quenched with water (100 mL) and the product was extracted with CH2 Cl 2 Extracted with (2 × 100 mL), CH 2 Cl 2 The extract was washed with brine (100 mL) and dried over anhydrous MgSO 4 and the solvent was evaporated to dryness. The remaining residue was purified by flash column chromatography [silica gel (24 g), CH containing 0 - 50% methanol 2 Cl 2 eluted] to afford 2,3 - dimethoxy - 6 - (3 - morpholinopropyl) - 5H - [1,3] dioxolo[4”,5”:5’,6’] pyrido[3’,2’:4,5] cyclopenta[1,2 - c] isoquinoline - 5,12(6H) - dione (17, 0.018 g, 38 μmol, 38%) as a dark brown solid. 1 1H NMR (300 MHz, CDCl 3 ) δ 7.94 (s, 1H), 7.68 (s, 1H), 7.17 (s, 1H), 6.18 (s, 2H), 4.88 (t, J = 7.6 Hz, 2H), 4.05 (s, 3H), 3.99 (s, 3H), 3.70 - 3.55 (m, 4H), 2.56 (t, J = 6.6 Hz, 2H), 2.46 (s, 4H), 2.01 (s, 2H); MS (ES+): 480.4.

[0213] Alternative synthesis of 17. DIPEA (0.15 g, 0.20 mL, 1.2 mmol, 10 equiv) and morpholine (0.20 g, 0.20 mL, 2.3 mmol, 20 equiv) were added to a stirred solution of 6-(3-bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (20, 55 mg, 0.12 mmol, 1 equiv) in DMF (11 mL). The reaction mixture was stirred at room temperature for 12 h and then heated to 50 °C over 12 h. The solvent was evaporated and the residue was triturated with water to a solid. The solid was filtered, washed with water, and 2,3-dimethoxy-6-(3-morpholinopropyl)-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (17, 24 mg, 50 μmol, 43%) was obtained as a purple solid, which was treated with water / acetonitrile (0.8 mL / 0.2 mL), followed by 5 equiv of 4N HCl, then soaked for several minutes and lyophilized to dryness to give the HCl salt of 17 (26 mg, 42%). 1 H NMR (300 MHz, CDCl 3 ) δ 7.94 (s, 1H), 7.68 (s, 1H), 7.16 (s, 1H), 6.18 (s, 2H), 5.02 - 4.80 (m, 2H), 4.05 (s, 3H), 4.00 (s, 3H), 3.66 (s, 4H), 2.64 2.53 (m, 2H), 2.46 (s, 4H), 2.11 - 1.93 (m, 2H); MS (ES+): 480.4 (M+1). 6-(3-(1H-Imidazol-1-yl)propyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (18). NaH (28 mg, 60 wt%, 0.71 mmol, 5.0 equiv) was added to a suspension of 2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (15, 50 mg, 0.14 mmol, 1 equiv) and 1-(3-bromopropyl)-1H-imidazole (95 mg, 0.35 mmol, 2.5 equiv) in dehydrated DMF (10 mL) at room temperature. The reaction mixture was stirred for 3 h. Then, the reaction mixture was quenched with ice-cold water and extracted with CH 2 Cl 2 to form a turbid solution, which was filtered using Whatman filter paper to obtain a gummy solid. LCMS analysis indicated the presence of the desired substance in this crude product. The gummy solid was redissolved in methanol / CH 2 Cl 2 and mixed with silica gel (2 g), and the solvent was evaporated to form a slurry. The silica gel slurry was placed on top of a column containing silica gel (24 g), and the product was eluted with CH 2 Cl 2 containing 0 - 10% of DMA80 (80% v / v DCM, 18% v / v methanol, and 2% v / v ammonia) to afford 6-(3-(1H-imidazol-1-yl)propyl)-2,3-dimethoxy-5H[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (18, 8 mg, 0.02 mmol, 10%) as a light brown solid. 1 1H NMR (300 MHz, CDCl 3)δ 7.94(s,1H),7.68(s,1H),7.57(s,1H),7.18(s,1H),7.07(s,1H),7.02(s,1H),6.21(s,2H),4.84(t,J=7.3Hz,2H),4.16(t,J=7.5Hz,2H),4.05(s,3H),4.01(s,3H),0.95-0.77(m,2H);MS(ES+):461。

[0214] Alternative synthesis of 18. 6-(3-Bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4″,5″:5′,6′]pyrido[3′,2′:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (20, 55 mg, 0.12 mmol, 1 equiv), DIPEA (0.15 g, 0.20 mL, 10 equiv, 1.2 mmol), and 1H-imidazole (0.16 g, 2.3 mmol, 20.0 equiv) were dissolved in DMF (11 mL). The reaction mixture was stirred at room temperature for 16 h. Then, the reaction mixture was heated to 70 °C for 18 h. The solvent was evaporated to dryness, and the residue was treated with CH 2 Cl 2 (200 mL) and brine (100 mL). The mixture was filtered, and the layers were separated. The aqueous layer was back-extracted with CH 2 Cl 2 (100 mL), and the combined organic extracts were dried over anhydrous MgSO 4 . The solution was filtered, and the solvent was evaporated to dryness. The residue was purified by flash column chromatography [silica gel (24 g), eluting with CH 2 Cl 2 containing 0–10% MeOH] to afford 6-(3-(1H-imidazol-1-yl)propyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4″,5″:5′,6′]pyrido[3′,2′:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (18, 7 mg, 0.02 mmol, 10%) as a purple solid. The compound was treated with water / acetonitrile (8:2 ratio) containing 7 equiv of 4N HCl and then lyophilized to dryness to convert it to the HCl salt, giving 18 (9.1 mg) of the HCl salt. 1 H NMR(300MHz,DMSO-d 6)δ 9.14(s,1H),7.84 - 7.81(m,1H),7.80(s,1H),7.71 - 7.66(m,1H),7.52(s,1H),7.43(s,1H),6.30(s,2H),4.69(t,J = 6.3Hz,2H),4.32(t,J = 6.8Hz,2H),3.92(s,3H),3.88(s,3H),2.40 - 2.25(m,2H); MS(ES+): 461.4(M + 1).

[0215] 6-(3-(Isopropylamino)propyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (19). NaH (28 mg, 60 wt%, 0.71 mmol, 5.0 equiv) was added to a suspension of 2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (15, 50 mg, 0.14 mmol, 1 equiv) in DMF (3 mL). Subsequently, dehydrated DMF (10 mL) containing 3-bromo-N-isopropylpropan-1-amine hydrobromide (92 mg, 0.35 mmol, 2.5 equiv) was added at room temperature, and the reaction mixture was stirred for 3 h. Then, the reaction mixture was quenched with ice-cold water and extracted with CH 2 Cl 2 The CH 2 Cl 2 layer was dried over anhydrous MgSO 4 and filtered, and washed with CH 2 Cl 2 (50 mL). The solvent was evaporated to dryness, and the resulting residue was subjected to flash column chromatography [silica gel (24 g), CH containing 0 - 10% DMA80 (80 vol% DCM, 18 vol% methanol, and 2 vol% ammonia) 2 Cl 2Purified by extraction with [extraction method], 6-(3-(isopropylamino)propyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (19, 3 mg, 6.6 μmol, 4.7%) was obtained as a brown solid. 1 H NMR (300 MHz, CDCl 3 ) δ 7.94 (s, 1H), 7.61 (s, 1H), 7.16 (s, 1H), 6.20 (s, 2H), 5.03 - 4.86 (m, 2H), 4.07 (s, 3H), 4.01 (s, 3H), 3.45 - 3.28 (m, 1H), 2.93 (s, 2H), 2.61 (s, 2H), 1.49 (d, J = 6.5 Hz, 6H); MS (ES+): 452.52.

[0216] Alternative synthesis of 19. 6-(3-Bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (20, 52 mg, 0.11 mmol, 1 equiv), DIPEA (0.14 g, 0.19 mL, 1.1 mmol, 10 equiv) and isopropylamine (0.13 g, 0.19 mL, 2.2 mmol, 20 equiv) were dissolved in DMF (11 mL). The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was treated with brine (100 mL) and CH 2 Cl 2 (100 mL), and stirred for 10 min. The layers were separated, and the aqueous layer was back-extracted with CH 2 Cl 2 (100 mL). The combined organic extracts were dried over anhydrous MgSO 4 , filtered, and the solvent was evaporated to dryness. The residue was purified by flash column chromatography [silica gel (24 g), 0 - 20% methanol-containing CH 2 Cl 2Purified by elution to give the product (10 mg) as a purple solid in the form of the free base. The solid was treated with water (0.8 mL) and acetonitrile (0.2 mL), followed by 5 equivalents of 4N aqueous HCl. The components were mixed for several minutes and then lyophilized to dryness to give 6-(3-(isopropylamino)propyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione·2HCl (19, 5.8 mg, 13 μmol, 12%). 1 H NMR(300MHz,DMSO-d 6 )δ 8.36(s,2H),7.85(s,1H),7.55(s,1H),7.50(s,1H),6.33(s,2H),4.84-4.63(m,2H),3.93(s,3H),3.89(s,3H),3.01(s,2H),2.21-2.03(m,2H),1.21(d,J=6.5Hz,6H).MS(ES+):452.5(M+1).

[0217] (3-Bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4″,5″:5′,6′]pyrido[3′,2′:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (20). NaH (54.5 mg, 60 wt%, 1.36 mmol, 1.2 equiv) was added to a suspension of 2,3-dimethoxy-5H-[1,3]dioxolo[4″,5″:5′,6′]pyrido[3′,2′:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (15, 400 mg, 1.14 mmol, 1 equiv) and KI (37.7 mg, 227 μmol, 0.2 equiv) in DMF (36 mL) at 0 °C. The reaction mixture was warmed to room temperature, stirred for 2 h, then cooled to 0 °C. 1,3-Dibromopropane (275 mg, 139 μL, 1.36 mmol, 1.2 equiv) was added and the solution was warmed to room temperature and stirred for 20 h. The reaction mixture was diluted with water (120 mL), and the resulting solid was collected by filtration, washed with water (2 × 30 mL), and dried at room temperature to afford (3-bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4″,5″:5′,6′]pyrido[3′,2′:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (20, 0.256 g, 541 μmol, 47.6%) as a purple solid. 1 H NMR (300 MHz, CDCl 3 ) δ 7.94 (s, 1H), 7.69 (s, 1H), 7.17 (s, 1H), 6.19 (s, 2H), 4.94 (t, J = 7.1 Hz, 2H), 4.05 (s, 3H), 4.00 (s, 3H), 3.56 (t, J = 7.0 Hz, 2H), 2.43 (p, J = 7.3 Hz, 2H).

[0218] 6-(3-(Ethylamino)propyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (21). 6-(3-Bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (20, 61 mg, 0.13 mmol, 1 equiv), DIPEA (0.17 g, 0.22 mL, 1.3 mmol, 10 equiv) and ethylamine (0.12 g, 1.3 mL, 2.6 mmol, 20 equiv) were dissolved in DMF (12 mL). The reaction mixture was stirred at room temperature for 12 h. More DMF (11 mL), DIPEA (0.17 g, 0.22 mL, 1.3 mmol, 10 equiv) and ethylamine (0.12 g, 1.3 mL, 2.6 mmol, 20 equiv) were added and the reaction mixture was stirred at room temperature for 12 h. The solvent was evaporated to dryness and the residue was treated with brine (100 mL) and CH 2 Cl 2 (100 mL) and then stirred for 10 min. The layers were separated and the aqueous layer was back-extracted with CH 2 Cl 2 (100 mL). The combined organic extracts were dried over anhydrous MgSO 4 and filtered and evaporated to dryness. The residue was purified by flash column chromatography [silica gel (24 g), eluting with CH 2 Cl 2 containing 0 - 20% methanol] to afford 6-(3-(ethylamino)propyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (21, 11 mg, 25 μmol, 20%) as a purple solid. The compound was treated with water (0.8 mL) and acetonitrile (0.2 mL) containing 5 equiv of 4N HCl and then lyophilized to give the HCl salt 21 (11 mg, 18%). 1 H NMR (300 MHz, DMSO-d 6)δ 8.56(s,2H),7.79(s,1H),7.52(s,1H),7.44(s,1H),6.33(s,2H),4.71(t,J=6.9Hz,2H),3.92(s,3H),3.88(s,3H),3.04-2.87(m,4H),2.19-2.02(m,2H),1.16(t,J=7.2Hz,3H);MS(ES+):438.4(M+1).

[0219] 6-(3-((2-Hydroxyethyl)amino)propyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (22). DIPEA (628 mg, 847 μL, 4.86 mmol, 20 eq) was added to a stirred solution of 6-(3-bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (20, 115 mg, 243 μmol, 1 eq) and ethanolamine (148 mg, 147 μL, 2.43 mmol, 10 eq) in DMF (22 mL). The resulting reaction mixture was stirred for 6 h. More ethanolamine (148 mg, 147 μL, 2.43 mmol, 10 eq) was added and the reaction mixture was stirred overnight. The solution was diluted with brine (100 mL) and extracted with CH 2 Cl 2 (2 x 100 mL). The combined extracts were dried over anhydrous MgSO 4 and filtered, and the solvent was evaporated to dryness. The residue was purified by flash column chromatography [silica gel (24 g), 0 - 100% methanol in CH 2 Cl 2Purified by elution with [eluent details] to afford 6-(3-((2-hydroxyethyl)amino)propyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (22, 11 mg, 24 μmol, 10%) as a purple solid. The product was treated with water (0.8 mL) containing 5 equivalents of 4N HCl and acetonitrile (0.2 mL), followed by lyophilization to afford 11 mg of the HCl salt 22. 1 H NMR(300MHz,DMSO-d 6 ) δ 8.58(s,2H),7.82(s,1H),7.54(s,1H),7.46(s,1H),6.33(s,2H),4.72(t,J = 6.5Hz,2H),3.92(s,3H),3.88(s,3H),3.67 - 3.57(m,2H),3.09 - 2.90(m,4H),2.22 - 2.06(m,2H); MS(ES+): 454.4(M + 1).

[0220] 2,3-Dimethoxy-6-(3-(pyrrolidin-1-yl)propyl)-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (23). DIPEA (819 mg, 1.10 mL, 6.34 mmol, 10 equivalents) and pyrrolidine (902 mg, 1.04 mL, 12.7 mmol, 20 equivalents) were added to a stirred solution of 6-(3-bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (20, 300 mg, 634 μmol, 1 equivalent) in DMF (30 mL). The reaction mixture was stirred at room temperature for 16 h. The solvent was evaporated to dryness and the residue was treated with brine (100 mL) and CH 2 Cl 2 (100 mL), then stirred for 10 min. The organic layer was separated and the aqueous layer was back-extracted with CH 2 Cl 2 (100 mL). The combined organic extracts were dried over anhydrous MgSO 4It was dried above, filtered, and the solvent was evaporated to dryness. The residue was purified by flash column chromatography [silica gel (24 g), elution with CH containing 0 - 20% methanol 2 Cl 2 to give 2,3 - dimethoxy - 6-(3-(pyrrolidin - 1 - yl)propyl)-5H - [1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2 - c]isoquinoline - 5,12(6H)-dione (23, 10 mg, 3.4%) as a purple solid. The product was mixed with water (0.8 mL) and acetonitrile (0.2 mL), followed by addition of 5 equivalents of 4N aqueous HCl, and the mixture was lyophilized to dryness to obtain the HCl salt of 23 (11 mg). 1 1H NMR (300 MHz, DMSO - d 6 ) δ 7.83 (s, 1H), 7.55 (s, 1H), 7.46 (s, 1H), 6.32 (s, 2H), 4.73 (s, 2H), 3.93 (s, 3H), 3.88 (s, 3H), 3.63 - 3.43 (m, 1H), 3.14 - 2.71 (m, 2H), 2.34 - 2.10 (m, 2H), 2.05 - 1.75 (m, 7H); MS (ES+): 464.3 (M + 1).

[0221] (S)-1-(3-(2,3 - dimethoxy - 5,12 - dioxo - 5,12 - dihydro - 6H - [1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2 - c]isoquinolin - 6 - yl)propyl)pyrrolidine - 2 - carboxamide (24). DIPEA (819 mg, 1.10 mL, 6.34 mmol, 10 equivalents) and (S)-pyrrolidine - 2 - carboxamide (1.45 g, 12.7 mmol, 20 equivalents) were added to a stirred solution of DMF (30 mL) containing 6-(3 - bromopropyl)-2,3 - dimethoxy - 5H - [1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2 - c]isoquinoline - 5,12(6H)-dione (20, 300 mg, 634 μmol, 1 equivalent). The reaction mixture was stirred at room temperature for 12 h. The solvent was evaporated to dryness, and the residue was partitioned between brine (100 mL) and CH 2 Cl 2(100 mL) was used for treatment and stirred for 10 minutes. The organic layer was separated, and the aqueous layer was back-extracted with CH 2 Cl 2 (100 mL). The combined organic extracts were dried over anhydrous MgSO 4 and filtered, and the solvent was evaporated to dryness. The residue was purified by flash column chromatography [silica gel (12 g), elution with CH 2 Cl 2 containing 0 - 20% methanol] to afford (S)-1-(3-(2,3-dimethoxy-5,12-dioxo-5,12-dihydro-6H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinolin-6-yl)propyl)pyrrolidine-2-carboxamide (24, 10 mg, 20 μmol, 3.1%) as a purple solid. The solid product was mixed with water (0.8 mL) and acetonitrile (0.2 mL), then treated with 5 equivalents of 4N aqueous HCl solution and lyophilized to dryness to obtain the HCl salt (24). 1 1H NMR (300 MHz, DMSO-d 6 ) δ 9.42 (s, 1H), 8.09 (s, 1H), 7.82 (s, 2H), 7.54 (s, 1H), 7.45 (s, 1H), 6.32 (s, 1H), 4.80 - 4.58 (m, 2H), 4.08 (dd, J = 14.3, 7.1 Hz, 1H), 3.92 (s, 3H), 3.88 (s, 3H), 3.68 - 3.53 (m, 1H), 3.25 - 3.09 (m, 1H), 2.22 - 1.79 (m, 7H); MS (ES+): 507.3 (M + 1).

[0222] (S)-6-(3-(2-(Hydroxymethyl)pyrrolidin-1-yl)propyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (25). DIPEA (819 mg, 1.10 mL, 6.34 mmol, 10 equiv) and (2S)-2-pyrrolidinemethanol (1.28 g, 1.24 mL, 12.7 mmol, 20 equiv) were added to a stirred solution of DMF (30 mL) containing 6-(3-bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (20, 300 mg, 634 μmol, 1 equiv). The reaction mixture was stirred at room temperature for 12 h. The solvent was evaporated to dryness and the residue was treated with brine (100 mL) and CH 2 Cl 2 (100 mL), and stirred for 10 min. The organic layer was separated and the aqueous layer was back-extracted with CH 2 Cl 2 (100 mL). The combined organic extracts were dried over anhydrous MgSO 4 and filtered, and the solvent was evaporated to dryness. The residue was purified by flash column chromatography [silica gel (24 g), eluting with CH 2 Cl 2 containing 0 - 20% methanol] to afford (S)-6-(3-(2-(Hydroxymethyl)pyrrolidin-1-yl)propyl)-2,3-dimethoxy-5H-[1,3]dioxolo[4”,5”:5’,6’]pyrido[3’,2’:4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (25, 8 mg, 0.02 mmol, 3%) as a purple solid. The solid was mixed with water (0.8 mL) and acetonitrile (0.2 mL), treated with 5 equiv of 4N HCl, and the mixture was lyophilized to dryness to afford the HCl salt of 25 (8 mg). 1H NMR (300 MHz, CDCl 3)δ 7.92(s,1H),7.63(s,1H),7.16(d,J=2.3Hz,1H),6.20(s,2H),4.94-4.78(m,2H),4.06(s,3H),4.00(s,4H),3.83-3.39(m,2H),2.7-2.26(m,1H),2.12-2.02(m,3H),1.93-1.42(m,3H),0.97-0.81(m,3H);MS(ES+):494.3(M+1).

[0223] Examples of the method NCI-60 cancer cell line drug screening The antiproliferative activity of indenoisoquinoline compounds is determined in the NCI-60 cancer cell lines of the National Cancer Institute Developmental Therapeutics Program (NCI-DTP). Compounds that showed sufficient cytotoxicity during prescreening were subjected to a 5-dose assay to determine the 50% growth inhibition (GI 50 ) value. Cancer cells are incubated with the test compound for 48 hours at five concentrations in the range of 100 μM to 10 nM. After staining the treated cancer cells with sulforhodamine B dye, the percentage of proliferation is plotted as a function of the common logarithm of the test compound concentration. GI 50 values are determined by interpolation between points located above and below 50% cell growth. GI -4 ~10 -8 M) values above and below the test range are used as the maximum (10-4 M) and minimum (10 50 M) drug concentrations used in the screening test, respectively. The approximate average of the GI -8 values across the entire panel of NCI-60 cancer cell lines for each compound is recorded as the MGM value. 50

[0224] The cytotoxicity days of G101 - G105 are shown in Tables 20a and 20b below.

[0225]

Table 25

Table 26

Table 27

[0226] Test method: DNA melting experiment. The FRET-based DNA melting experiment was carried out on a QuantStudio 6 Flex Real-Time PCR system. The 5'-Fluorescein (6-FAM), 3'-Rhodamine (TAMRA) double-labeled MycG4 DNA sequence (Sigma-Aldrich) was diluted to 0.25 μM using 7.5 mM KCl / 2.5 mM potassium phosphate buffer, pH 7.0. The diluted DNA was annealed by heating at 95 °C for 2 minutes. The labeled MycG4 and the aqueous solution of the drug were added to each well of a 96-well plate, and the final volume was made to 20 μL at a concentration of 0.2 μM DNA and 2.0 μM drug in 10 mM K + buffer, pH 7.0. The drug-DNA mixture was incubated in the dark at 25 °C for 1 hour, and then heated to 95 °C at a rate of 0.9 °C / min while measuring the fluorescence of 6-FAM at 520 nm (for example, 470 nm). The G-quadruplex melting temperature, T m was determined from the maximum value of the first derivative of the melting curve. ΔT m was calculated as the difference between T m,DNA+薬物 and T m,DNA and T.

[0227] Fluorescence-based binding assay. The fluorescence-based binding assay was performed using a Jasco FP-8300 spectrofluorometer equipped with a temperature controller at 20 °C in a quartz cuvette with a path length of 10 mm. 3’-TAMRA-labeled MycG4 DNA (Sigma-Aldrich) was diluted to 20 nM using 75 mM KCl / 25 mM potassium phosphate buffer, pH 7.0. The emission spectra of the free DNA and the emission spectra after each titration step were recorded from 570 to 600 nm using an excitation wavelength of 555 nm, a bandwidth of 5 nm, a scan speed of 200 nm / min, and a response time of 1 s. The fluorescence intensity at 580 nm, which is the maximum value of the free DNA, was plotted against the drug:DNA ratio. For the binding of each compound, the apparent binding dissociation constant K d,app was derived. The change in fluorescence intensity was fitted to a one-site binding model using OriginPro software (OriginLab Corp., United States). [Number] where B 0 is the initial fluorescence intensity, B is the maximum change in fluorescence intensity, R is the DNA concentration, and L is the drug:DNA ratio.

[0228] NMR titration experiment. The NMR titration experiment was performed at 25 °C using a Bruker AV-500 spectrometer equipped with a Prodigy cryoprobe. MycG4 DNA was diluted to 150 μM in 75 mM KCl / 25 mM potassium phosphate buffer (pH 7.0), 10%D 2 O / H 2 O. The diluted DNA was annealed by heating to 95 °C for 2 min and slowly cooled to room temperature. 1D 1 H NMR was measured for the unbound DNA and after subsequent titration with the compound (in DMSO- d 6 6). The measurements were performed when the drug:DNA equivalent was 0.5, 1.0, 2.0, 3.0, 4.0. After adding the compound, the sample was incubated for 5 min.

[0229] Western blotting. MCF-7 cells were grown and treated in 6-well tissue culture plates. After collection, the cells were suspended in 140 μL of lysis buffer containing 1X RIPA buffer, 1X protease inhibitor cocktail (A32965, Thermo Scientific), 1X NuPAGE LDS sample buffer (NP0007, Invitrogen), and 1X sample reducing agent (NP0009, Invitrogen). The cell lysates were heated at 75 °C for 10 minutes. After sonication, 8 μL of each sample was loaded onto a 4–15% Mini-PROTEAN TGX gel (4561086, Bio-Rad). The gel was transferred to a nitrocellulose membrane (IB23002, Invitrogen) using an iBlot 2 transfer device (Invitrogen). Myc and GAPDH were detected with 1:1000-diluted monoclonal anti-Myc antibody (5605S, Cell Signaling Technology) and 1:2000-diluted monoclonal anti-GAPDH antibody (5174S, Cell Signaling Technology), respectively. The primary antibody was detected with an anti-rabbit IgG HRP-linked antibody (7074S, Cell Signaling Technology). Blotting was developed with an ECL substrate (1863096, Thermo Scientific) and imaged with a Bio-Rad ChemiDoc Touch imaging system.

[0230] Quantitative reverse transcription polymerase chain reaction (qRT-PCR). MCF-7 cells were grown and processed in 12-well tissue culture plates until harvest. Total RNA was extracted using TRIzol reagent (15596018, Life Technologies). The purified RNA was dissolved in DEPC water and quantified. Using qScript cDNA SuperMix (84034, Quantabio), 1 μg of purified RNA per sample was converted to cDNA according to the manufacturer's instructions. Real-time PCR reagents were prepared for each sample by mixing 1 μL of synthesized cDNA, 3 μL of PCR-grade water, 5 μL of SYBR Green reagent (A25742, Applied Biosystem), and 0.25 μL of each primer for Myc and GAPDH. The reaction was set up in triplicate for each target of each sample. Real-time PCR was performed in a 96-well PCR plate using a QuantStudio 6 Flex real-time PCR system (Life Technologies). The cycling conditions were 5 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C, 15 seconds at 60°C, and 15 seconds at 72°C. The relative Myc expression level was calculated using the 2 -ΔΔCt method using GAPDH as an endogenous control.

[0231] MTS cell viability IC 50 assay. The chemical solutions for the MTS IC 50 assay were prepared from DMSO stocks by serial dilution in 1X PBS (10010-031, Gibco). To set up the treatments, the chemical solutions were plated in triplicate in 96-well tissue culture plates, and cell-containing medium was added thereto to make a total volume of 100 μL / well. The control with 100% viability was untreated cells. 2000 cells / well were plated for all floating cell lines except 3000 cells / well of KMS-27 and 10000 cells / well of CHLA-32. The blank was cell-free medium. The drug treatment was carried out in 5% CO 2It was maintained at 37 °C for 72 hours using [the relevant method]. To quantify the cells, CellTiter 96 AQueous MTS reagent powder (G1111, Promega) was dissolved according to the manufacturer's instructions and mixed with phenazine methosulfate (PMS, Sigma-Aldrich P9625). MTS / PMS mix (20 μL / well) was added to the treated wells, control wells with 100% viability, and blank wells. 2 Color development was carried out at 37 °C for 4 hours using [the relevant method], and the absorbance at 490 nm was measured by a BioTek Synergy 4 system. To obtain the percentage of viability, the A 490 value was corrected by blank subtraction, normalized to the 100% viability control, and averaged. The average percentage of viability was plotted against the drug dosage to obtain a dose-response curve. Using Prism software (GraphPad Prism version 9.1.0 for macOS, www.graphpad.com), the equation "log (inhibitor) vs. normalized response - variable slope" implemented in the program: Y = 100 / (1 + 10 ((LogIC50-X)*HillSlope) ) was used to IC50 calculate. For adherent cell lines, except for cell plating and drug treatment, the MTS IC 50 assay was performed in the same way as for suspension cell lines. Regarding cell plating, 50 μL of 2000 cells were plated in triplicate in a 96-well tissue culture plate. Sufficient time was given for the cells to become adherent. Serial dilution of the drug solution was prepared in the same way as for the MTS IC 50 assay. To set up the treatment, the drug solution was mixed with the medium until it reached twice the final concentration, and then 50 μL of such a drug-medium mix was added to 50 μL of the plated cells.

[0232] MTS single-dose cell viability assay. The drug solution for the MTS single-dose assay was prepared from the DMSO stock by dilution with 1X PBS. To set up the treatment, the drug solution was plated in triplicate in a 96-well tissue culture plate, and the medium containing CA46 or Raji 2000 cells / well was added. The control and blank with 100% viability were IC 50It was set up in the same way as the assay. Cells were 50 quantified in the same way as the assay. To obtain the survival rate %, the A 490 value was subtracted from the blank, normalized to the 100% survival rate control, and averaged. The results were visualized as bar plots using Prism software.

[0233] NCI-60 cytotoxicity assay. Compounds were submitted to the National Cancer Institute Developmental Therapeutic Program to determine their potential for growth inhibition in the NCI-60 cell lines. All compounds that met the pre-screening advanced to the 5-dose cell survival assay, and the cells were treated with 5 doses in the range of 100 μM to 10 nM for 48 hours. After treatment, the percentage of growth was plotted against Log 10 (drug dose) to extrapolate the growth inhibition at 50% (IC 50 ). If the GI 50 exceeded 100 μM or was less than 10 nM, the GI 50 was reported as 100 μM or 10 nM, respectively.

[0234] Pharmacokinetics. The pharmacokinetic profiles of Compounds 19 (G103), 21 (G104), and 22 (G105) were determined by IV (dose 1 mg / kg) and PO (dose 10 mg / kg) administration to male CD1 mice. The mice were obtained from Si Bei Fu Laboratory Animal Technology Co., Ltd. The animals were 6 - 8 weeks old and their body weights at the time of drug administration were 20 - 30 g. Plasma samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours for both the IV and PO experiments. Three animals were used for each experiment, and the plasma samples were pooled and analyzed by LC / MS / MS. The pharmacokinetic study was conducted at Pharmaron Beijing Co., Ltd. after appropriate ethical considerations and review by Pharmaron's IACUC (Institutional Animal Care and Use Committee).

[0235] Analysis of plasma protein binding and stability in human and mouse plasma by equilibrium dialysis. Frozen plasma (stored at -80 °C) was thawed immediately in a 37 °C water bath. Working solutions of the test compound and the control compound were prepared at a concentration of 200 μM in DMSO, and the working solutions were added to the plasma. The final concentration of the compound was 1 μM. The final concentration of DMSO was 0.5%. Ketoconazole was used as a positive control for the assay. The dialysis membrane was immersed in ultrapure water for 60 minutes to separate the strip, then immersed in 20% ethanol for 20 minutes, and finally immersed in dialysis buffer for 20 minutes. In equilibrium dialysis, plasma samples (150 μL) were placed in each cell and dialyzed against an equal volume of dialysis buffer (PBS). Each assay was repeated. The dialysis plate was sealed and incubated at 5% CO 2 2, 100 rpm, 37 °C for 6 hours. At the end of the incubation, samples (50 μL) from both the buffer chamber and the plasma chamber were transferred to wells of a 96-well plate. Plasma (50 μL) was added to each buffer sample, and an equal volume of PBS was added to the plasma samples collected. Cold acetonitrile (400 μL) containing internal standards (IS, 100 nM alprazolam, 200 nM labetalol, 200 nM imipramine, and 2 μM ketoprofen) was added to precipitate the protein and release the compound. The samples were vortexed for 2 minutes and centrifuged at 3,220 g for 30 minutes. An aliquot (100 μL) of the supernatant was diluted with ultrapure water H 2 2O (100 μL), and the mixture was used for LC-MS / MS analysis. To measure the stability in plasma, spiked plasma samples (50 μL) were transferred to a new plate and incubated at 37 °C, 5% CO 2 2 for 0 hours and 6 hours. At the designated time points, PBS (50 μL) was added and mixed well, followed by addition of room temperature quenching solution [400 μL of acetonitrile containing internal standards (IS, 100 nM alprazolam, 200 nM labetalol, 200 nM imipramine and 2 μM ketoprofen)] to precipitate the protein and release the compound. The samples were vortexed for 2 minutes and centrifuged at 3,220 g for 30 minutes. An aliquot (400 μL) of the supernatant was diluted with ultrapure water H 2It was diluted with O and the mixture was used for LC-MS / MS analysis. The concentrations of the test compound in the buffer and the plasma chamber were determined from the peak area ratio.

[0236] Stability analysis against human and mouse liver microsomes. The microsome solution (0.5 mg / mL) was prepared in phosphate buffer (100 mM) containing MgCl 2 (5 mM). NADPH solution (40 μL, 10 mM) was added to each well. The final concentration of NADPH was 1 mM. The mixture was preheated at 37 °C for 5 minutes. The negative control sample was prepared by replacing the 40 μL of NADPH solution with 40 μL of ultrapure water H 2 O. Samples containing NADPH were prepared in duplicate and the negative control was prepared as a single line. The reaction was initiated by adding the addition control compound (2 μL of a 200 μM solution) or the test compound solution. Verapamil was used as a positive control. The final concentration of the test compound or the control compound was 1 μM. Aliquots (50 μL) were taken from the reaction solution at 0, 15, 30, 45, and 60 minutes. The reaction was stopped by adding four volumes of cold acetonitrile containing alprazolam (100 nM), imipramine (200 nM), labetalol (200 nM), and ketoprofen (2 μM). The samples were centrifuged at 3,220 g for 40 minutes. An aliquot (90 μL) of the supernatant was mixed with ultrapure water H 2 O (90 μL) and then used for LC-MS / MS analysis. The peak area was determined from the extracted ion chromatogram. The slope value k was determined by linear regression of the natural logarithm of the percentage of the parent drug remaining against the incubation time curve. The in vitro half-life (in vitro t 1 / 2 ) was determined from the slope value: in vitro t 1 / 2 =-(0.693 / k). The conversion of in vitro t 1 / 2 (minutes) to in vitro intrinsic clearance (in vitro CL Int , μL / min / mg protein) was performed using the following equation (average of two measurements):

Equation

Equation

[0237] The scaling factors used in the calculation are shown in Table 21.

Table 28

[0238] Caco-2 permeability study. The Caco-2 plates were taken out of the incubator, washed twice with pre-warmed HBSS (10 mM HEPES, pH 7.4), and then incubated at 37 °C for 30 minutes. The stock solution of the control compound was diluted with DMSO to obtain a 1 mM solution, and then diluted with HBSS (10 mM HEPES, pH 7.4) to obtain a 5 μM working solution. The stock solution of the test compound was diluted with DMSO to obtain a 1 mM solution, and then diluted with HBSS (10 mM HEPES, pH 7.4) to obtain a 5 μM working solution. The final concentration of DMSO in the incubation system was 0.5%. To determine the drug transport rate in the apical-to-basolateral direction, 75 μL of the 5 μM working solution of the test compound was added to the Transwell insert (apical compartment), and the well of the receiver plate (basolateral compartment) was filled with 235 μL of HBSS (10 mM HEPES, pH 7.4). To determine the drug transport rate in the basolateral-to-apical direction, 235 μL of the 5 μM working solution of the test compound was added to the well of the receiver plate (basolateral compartment), and then the Transwell insert (apical compartment) was filled with 75 μL of HBSS (10 mM HEPES, pH 7.4). 50 μL of the 5 μM working solution was transferred to the well of a 96-deep well plate, and then 200 μL of cold methanol containing the appropriate internal standard (IS) was added to prepare the sample at time point 0. The plate was incubated at 37 °C for 2 hours. At the end of the incubation, 50 μL of the sample was transferred from the donor side (apical compartment for Ap→Bl flux and basolateral compartment for Bl→Ap) and the receiver side (basolateral compartment for Ap→Bl flux and apical compartment for Bl→Ap) to the well of a new 96-well plate, and then 4 volumes of cold acetonitrile or methanol containing the appropriate internal standard (IS) were added. After vortexing the sample for 5 minutes, it was centrifuged at 3,220 g for 40 minutes. Before LC-MS / MS analysis, 100 μL of an aliquot of the supernatant was mixed with an appropriate amount of ultrapure water.To measure the leakage of lucifer yellow after a 2-hour transport period, a stock solution of lucifer yellow was prepared in ultrapure water, diluted with HBSS (10 mM HEPES, pH 7.4) to a final concentration of 100 μM, and then 100 μL of the lucifer yellow solution was added to each Transwell insert (apical compartment). Subsequently, the wells of the receiver plate (basolateral compartment) were filled with 300 μL of HBSS (10 mM HEPES, pH 7.4). The plate was incubated at 37 °C for 30 minutes. Samples (80 μL) were taken directly from the apical well and the basolateral well (using the basolateral access holes) and transferred to the wells of a new 96-well plate. The lucifer yellow fluorescence signal (to monitor the integrity of the monolayer) was measured with a fluorescence plate reader at an excitation of 485 nM and an emission of 530 nM.

[0239] Embodiment E1. A compound of formula (I), or a salt, hydrate, or solvate thereof, wherein

Chemical formula

[0240] E2. Use of the compound of E1, or a salt, hydrate, or solvate thereof, for treating a patient in need of reducing cancer.

[0241] A pharmaceutical composition comprising one or more compounds of E3.E1, or a salt, hydrate, or solvate thereof, and one or more carriers, diluents, or excipients, or a combination thereof.

[0242] E4. A compound of E1, or a salt, hydrate, or solvate thereof, for treating cancer.

[0243] E5. A pharmaceutical composition for treating cancer, comprising one or more compounds of E1, or a salt, hydrate, or solvate thereof.

[0244] E6. A pharmaceutical composition for treating cancer, said composition comprising a therapeutically effective amount of one or more compounds of E1, or a salt, hydrate, or solvate thereof.

[0245] E7. A method for treating a disease responsive to topoisomerase I inhibition or binding to MYC quadruplex in a host animal, said method comprising administering to said host animal a composition comprising a therapeutically effective amount of one or more compounds of E1, or a salt, hydrate, or solvate thereof, or a pharmaceutical composition comprising one or more compounds of claim 1, or a salt, hydrate, or solvate thereof, said pharmaceutical composition optionally comprising one or more carriers, diluents, or excipients, or a combination thereof.

[0246] E8. The method of E7, wherein said host animal is a human.

[0247] E9. A method for treating cancer in a host animal, said method comprising administering to said host animal a composition comprising a therapeutically effective amount of one or more compounds of claim 1, or a salt, hydrate, or solvate thereof.

[0248] E10. The method of E9, wherein said composition further comprises one or more carriers, diluents, or excipients, or a combination thereof.

[0249] A process for preparing a compound of E11.E1, which comprises the step of brominating a compound of formula II to obtain compound III, where R 1 , R 2 , and R 3 are as defined in E1, said process.

Chemical formula

[0250] E12. The process of E11, where R 3 is hydrogen.

[0251] E13. The process of E11 or E12, where R 1 and R 2 together form -CH 2 -.

[0252] E14. The process of any one of E11 - E13, where said bromination step comprises treating an acetic acid solution containing the compound of formula (II) with N-bromosuccinimide.

[0253] References Each of the following publications, and additional publications cited herein, are hereby incorporated by reference into this specification. (1) Cushman, M.; Cheng, L. Stereoselective Oxidation by Thionyl Chloride Leading to the Indeno[1,2 - c]isoquinoline System. J. Org. Chem. 1978, 43, 3781 - 3783. (2) Kohlhagen, G.; Paull, K.; Cushman, M.; Nagafuji, P.; Pommier, Y. Protein - Linked DNA Strand Breaks Induced by NSC 314622, a Novel Noncamptothecin Topoisomerase I Poison. Mol. Pharmacol. 1998, 54, 50 - 58. (3) Ryckebusch, A.; Garcin, D.; Lansiaux, A.; Goossens, J.F.; Baldeyrou, B.; Houssin, R.; Bailly, C.; Henichart, J.P. Synthesis, Cytotoxicity, DNA Interaction, and Topoisomerase II Inhibition Properties of Novel Indeno[2,1-c]quinolin-7-one and indeno[1,2-c]isoquinolin-5,11-dione Derivatives. J. Med. Chem. 2008, 51, 3617-3629. (4) Ahn, G.; Lansiaux, A.; Goossens, J.F.; Bailly, C.; Baldeyrou, B.; Schifano-Faux, N.; Grandclaudon, P.; Couture, A.; Ryckebusch, A. Indeno[1,2-c]Isoquinolin-5,11-diones Conjugated to Amino Acids: Synthesis, Cytotoxicity, DNA Interaction, and Topoisomerase II Inhibition Properties. Bioorg. Med. Chem. 2010, 18, 8119-8133. (5) Ahn, G.; Schifano-Faux, N.; Goossens, J.F.; Baldeyrou, B.; Couture, A.; Grandclaudon, P.; Lansiaux, A.; Ryckebusch, A. Synthesis, Cytotoxicity and Topoisomerase Inhibition Properties of Multifarious Aminoalkylated Indeno[1,2-c]isoquinolin-5,11-diones. Bioorg. Med. Chem. Lett. 2011, 21, 2259-2263. (6)Wambang, N.; Schifano-Faux, N.; Aillerie, A.; Baldeyrou, B.; Jacquet, C.; Bal-Mahieu, C.; Bousquet, T.; Pellegrini, S.; Ndifon, P.T.; Meignan, S.; Goossens, J.F.; Lansiaux, A.; Pelinski, L. Synthesis and Biological Activity of Ferrocenyl Indeno[1,2-c]isoquinolines as Topoisomerase II Inhibitors. Bioorg. Med. Chem. 2016, 24, 651 - 660. (7)Antony, S.; Agama, K.K.; Miao, Z.H.; Hollingshead, M.; Holbeck, S.L.; Wright, M.H.; Varticovski, L.; Nagarajan, M.; Morrell, A.; Cushman, M.; Pommier, Y. Bisindenoisoquinoline Bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propylamino}propane bis(trifluoroacetate)(NSC 727357), a DNA Intercalator and Topoisomerase Inhibitor with Antitumor Activity. Mol. Pharmacol. 2006, 70, 1109 - 1120. (8)Park, E.J.; Kondratyuk, T.P.; Morrell, A.; Kiselev, E.; Conda-Sheridan, M.; Cushman, M.; Ahn, S.; Choi, Y.; White, J.J.; van Breemen, R.B.; Pezzuto, J.M. Induction of Retinoid X Receptor Activity and Consequent Upregulation of p21 (WAF1 / CIP1)by Indenoisoquinolines in MCF7 Cells.Cancer Prev. Res.2011,4,592-607. (9)Chen,L.;Conda-Sheridan,M.;Reddy,P.V.N.;Morrell,A.;Park,E.J.;Kondratyuk,T.P.;Pezzuto,J.M.;van Breemen,R.;Cushman,M.Identification,Synthesis,and Biological Evaluation of the Metabolites of 3-Amino-6-(3′-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11-(6H)dione(AM6-36),a Promising Rexinoid Lead Compound for the Development of Cancer Chemotherapeutic and Chemopreventive Agents.J.Med.Chem.2012,55,5965-5981. (10)Conda-Sheridan,M.;Park,E.J.;Beck,D.E.;Reddy,P.V.N.;Nguyen,T.X.;Hu,B.;Chen,L.;White,J.L.;Van Breemen,R.B.;Pezzuto,J.M.;Cushman,M.Design,Synthesis,and Biological Evaluation of Indenoisoquinoline Rexinoids with Chemopreventive Potential.J.Med.Chem.2013,56,2581-2605. (11) Jagtap, P.G.; Baloglu, E.; Southan, G.J.; Mabley, J.G.; Li, H.S.; Zhou, J.; van Duzer, J.; Salzman, A.L.; Szabo, C. Discovery of Potent Poly(ADP-ribose) Polymerase-1 Inhibitors from the Modification of Indeno[1,2-c]isoquinolinone. J. Med. Chem. 2005, 48, 5100 - 5103. (12) Tang, Z.C.; Wu, C.Z.; Wang, T.L.; Lao, K.J.; Wang, Y.J.; Liu, L.Y.; Muyaba, M.; Xu, P.; He, C.H.; Luo, G.S.; Qian, Z.Y.; Niu, S.X.; Wang, L.J.; Wang, Y.; Xiao, H.; You, Q.D.; Xiang, H. Design, Synthesis and Evaluation of 6-Aryl-indenoisoquinolone Derivatives Dual Targeting ER Alpha and VEGFR-2 as Anti-breast Cancer Agents. Eur. J. Med. Chem. 2016, 118, 328 - 339. (13) Wang, P.; Elsayed, M.S.A.; Plescia, C.B.; Ravji, A.; Redon, C.E.; Kiselev, E.; Marchand, C.; Zeleznik, O.; Agama, K.; Pommier, Y.; Cushman, M. Synthesis and Biological Evaluation of the First Triple Inhibitors of Human Topoisomerase 1, Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), and Tyrosyl-DNA Phosphodiesterase 2 (Tdp2). J. Med. Chem. 2017, 60, 3275 - 3288. (14) Nguyen, T. X.; Morrell, A.; Conda-Sheridan, M.; Marchand, C.; Agama, K.; Bermingam, A.; Stephen, A. G.; Chergui, A.; Naumova, A.; Fisher, R.; O’Keefe, B. R.; Pommier, Y.; Cushman, M. Synthesis and Biological Evaluation of the First Dual Tyrosyl-DNA Phosphodiesterase I (Tdp1)-Topoisomerase I (Top1) Inhibitors. J. Med. Chem. 2012, 55, 4457 - 4478. (15) Conda-Sheridan, M.; Reddy, P. V. N.; Morrell, A.; Cobb, B. T.; Marchand, C.; Agama, K.; Chergui, A.; Renaud, A.; Stephen, A. G.; Bindu, L. K.; Pommier, Y.; Cushman, M. Synthesis and Biological Evaluation of Indenoisoquinolines That Inhibit Both Tyrosyl-DNA Phosphodiesterase I (Tdp1) and Topoisomerase I (Top1). J. Med. Chem. 2013, 56, 182 - 200. (16) Lv, P. C.; Agama, K.; Marchand, C.; Pommier, Y.; Cushman, M. Design, Synthesis, and Biological Evaluation of O-2-Modified Indenoisoquinolines as Dual Topoisomerase I-Tyrosyl-DNA Phosphodiesterase I Inhibitors. J. Med. Chem. 2014, 57, 4324 - 4336. (17) Nguyen, T. X.; Abdelmalak, M.; Marchand, C.; Agama, K.; Pommier, Y.; Cushman, M. Synthesis and Biological Evaluation of Nitrated 7-, 8-, 9-, and 10-Hydroxyindenoisoquinolines as Potential Dual Topoisomerase I (Top1)-Tyrosyl-DNA Phosphodiesterase I (TDP1) Inhibitors. J. Med. Chem. 2015, 58, 3188 - 3208. (18) Wang, K. B.; Elsayed, M. S. A.; Wu, G. H.; Deng, N. J.; Cushman, M.; Yang, D. Z. Indenoisoquinoline Topoisomerase Inhibitors Strongly Bind and Stabilize the MYC Promoter G-Quadruplex and Downregulate MYC. J. Am. Chem. Soc. 2019, 141, 11059 - 11070. (19) Nagarajan, M.; Morrell, A.; Antony, S.; Kohlhagen, G.; Agama, K.; Pommier, Y.; Ragazzon, P. A.; Garbett, N. C.; Chaires, J. B.; Hollingshead, M.; Cushman, M. Synthesis and Biological Evaluation of Bisindenoisoquinolines as Topoisomerase I Inhibitors. J. Med. Chem. 2006, 49, 5129 - 5140. (20)Cushman,M.;Jayaraman,M.;Vroman,J.A.;Fukunaga,A.K.;Fox,B.M.;Kohlhagen,G.;Strumberg,D.;Pommier,Y.Synthesis of New Indeno[1,2-c]isoquinolines:Cytotoxic Non-Camptothecin Topoisomerase I Inhibitors.J.Med.Chem.2000,43,3688-3698. (21)Pommier,Y.;Cushman,M.;Doroshow,J.H.Novel Clinical Indenoisoquinoline Topoisomerase I Inhibitors:a Twist Around the Camptothecins.Oncotarget 2018,9,37286-37288. (22)Kiselev,E.;DeGuire,S.;Morrell,A.;Agama,K.;Dexheimer,T.S.;Pommier,Y.;Cushman,M.7-Azaindenoisoquinolines as Topoisomerase I Inhibitors and Potential Anticancer Agents.J.Med.Chem.2011,54,6106-6116. (23)Kiselev,E.;Agama,K.;Pommier,Y.;Cushman,M.Azaindenoisoquinolines as Topoisomerase I Inhibitors and Potential Anticancer Agents:A Systematic Study of Structure-Activity Relationships.J.Med.Chem.2012,55,1682-1697. (24) Kiselev, E.; Sooryakumar, D.; Agama, K.; Cushman, M.; Pommier, Y. Optimization of the Lactam Side Chain of 7-Azaindenoisoquinoline Topoisomerase I Inhibitors and Mechanism of Action Studies in Cancer Cells. J. Med. Chem. 2014, 57, 1289-1298. (25) Elsayed, M. S. A.; Su, Y.; Wang, P.; Sethi, T.; Agama, K.; Ravji, A.; Redon, C. E.; Kiselev, E.; Horzmann, K. A.; Freeman, J. L.; Pommier, Y.; Cushman, M. Design and Synthesis of Chlorinated and Fluorinated 7-Azaindenoisoquinolines as Potent Cytotoxic Anticancer Agents That Inhibit Topoisomerase I. J. Med. Chem. 2017, 60, 5364-5376. (26) Nagarajan, M.; Morrell, A.; Fort, B. C.; Meckley, M. R.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. Synthesis and Anticancer Activity of Simplified Indenoisoquinoline Topoisomerase I Inhibitors Lacking Substituents on the Aromatic Rings. J. Med. Chem. 2004, 47, 5651-5661. (27) Dallacker, F.; Fechter, P.; Mues, V. 1,3-Dioxolohetarenes. 3. Preparation and Reactions of Pyrido[2.3-d]- and Pyrido[3.4-d][1.3]dioxoles. Z. Naturforsch. B Chem. Sci. 1979, 34, 1729 - 1736. (28) Vshyvenko, S.; W’Giorgis, Z.; Weber, A.; Neverova, N.; Hedberg, B.; Hudlicky, T. Synthesis and Biological Activity of 10-Aza-narciclasine. Adv. Syn. Catal. 2015, 357, 83 - 87. (29) Falb, E.; Ulanenko, K.; Tor, A.; Gottesfeld, R.; Weitman, M.; Afri, M.; Gottlieb, H.; Hassner, A. A Highly Efficient Suzuki-Miyaura Methylation of Pyridines Leading to the Drug Pirfenidone and Its CD 3 Version(SD-560). Green Chem. 2017, 19, 5046 - 5053. (30) Li, H.J.; Wu, Y.C.; Dai, J.H.; Song, Y.; Cheng, R.J.; Qiao, Y.Y. Regioselective Electrophilic Aromatic Bromination: Theoretical Analysis and Experimental Verification. Molecules 2014, 19, 3401 - 3416. (31) Fukuda, T.; Iwao, M. Synthesis of Both Enantiomers of Protoberberines via Laterally Lithiated (S)-4-Isopropyl-2-(o-tolyl)oxazolines. Heterocycles 2007, 74, 701 - 720. (32) Wang, B. J.; Qin, L. L.; Neumann, K. D.; Uppaluri, S.; Cerny, R. L.; DiMagno, S. G. Improved Arene Fluorination Methodology for I(III) Salts. Organic Lett. 2010, 12, 3352 - 3355. (33) Jagtap, P. G.; Baloglu, E.; Southan, G.; Williams, W.; Roy, A.; Nivorozhkin, A.; Landrau, N.; Desisto, K.; Salzman, A. L.; Szabo, C. Facile and Convenient Syntheses of 6,11 - Dihydro - 5H - indeno[1,2 - c]isoquinolin - 5 - ones and 6,11 - Dihydro - 5H - indolo[3,2 - c]isoquinolin - 5 - one. Org. Lett. 2005, 7, 1753 - 1756. (34) Beck, D. E.; Lv, W.; Abdelmalak, M.; Plescia, C. B.; Agama, K.; Marchand, C.; Pommier, Y.; Cushman, M. Synthesis and Biological Evaluation of New Fluorinated and Chlorinated Indenoisoquinoline Topoisomerase I Poisons. Bioorg. Med. Chem. 2016, 24, 1469 - 1479. (35) Marzi, L.; Agama, K.; Murari, J.; Difilippantonio, S.; James, A.; Peer, C. J.; Figg, W. D.; Beck, D. E.; Elsayed M. S. A.; Cushman, M.; et al. Novel Fluoroindenoisoquinoline Non - Camptothecin Topoisomerase I Inhibitors. Mol. Cancer Ther. 2018, 17, 1694 - 1704. (36)Marzi,L.;Sun,Y.L.;Huang,S.Y.N.;James,A.;Difilippantonio,S.;Pommier,Y.The Indenoisoquinoline LMP517:A Novel Antitumor Agent Targeting both TOP1 and TOP2.Mol.Cancer Ther.2020,19,1589-1597. (37)Shoemaker,R.H.The NCI60 Human Tumour Cell Line Anticancer Drug Screen.Nat.Rev.Cancer 2006,6,813-823. (38)Strumberg,D.;Pommier,Y.;Paull,K.;Jayaraman,M.;Nagafuji,P.;Cushman,M.Synthesis of Cytotoxic Indenoisoquinoline Topoisomerase I Poisons.J.Med.Chem.1999,42,446-457. (39)Stacker,B.L.;Feese,M.D.;Cushman,M.;Pommier,Y.;Zembower,D.;Stewart,L.;Burgin,A.B.Structures of Three Classes of Anticancer Agents Bound to the Human Topoisomerase I-DNA Covalent Complex.J.Med.Chem.2005,48,2336-2345. (40)Gao, J.; Holleran, J.; Schmitz, J.C.; Czambel, K.; Beumer, J.H.; Eiseman, J.L. Abstract 4513: Pharmacokineetic and Pharmacodynamics of Indenoisoquinoline LMP400 (Indotecan) in BALB / s Female Mice Bearing CT-26 Colon Tumors. In Proceedings of the 106th Annual Meeting of the American Association for Cancer Research, 2015. (41)Weight of Brain. https: / / www.aqua-calc.com / calculate / volume-to-weight / substance / brain (accessed March 22, 2023). (42)Eisman, J.L.; Holeran, J.; McCormick, D.L.; Muzzio, M.; Covey, J.M.; Khanna, C.; Mazcko, C.; Pommier, Y.; Paolini, M.; Leblanc, A.; et al. Abstract 4517: Plasma and Tumor Pharmacokinetics of IV LMP744, a Novel Indenoisoquinoline Topoisomerase I Inhibitor, in a Canine Phase I Study. Cancer Res. 2015, 75(15_Supplement), 4517 - 5417. (43)Davies, B.; Morris, T. Physiological Parameters in Laboratory Animals and Humans. Pharm. Res. 1993, 10, 1093 - 1095. (44)Iwatsubo, T.; Suzuki, H.; Sugiyama, Y. Prediction of Species Differences (Rats, Dogs, Humans) in the In Vivo Metabolic Clearance of YM796 by the Liver from In Vitro Data. J. Pharm. Exp. Ther. 1997, 283, 462 - 469. (45)Barter, Z. E.; Bayliss, M. K.; Beaune, P. H.; Boobis, A. R.; Carlile, D. J.; Edwards, R. J.; Houston, J. B.; Lake, B. G.; Lipscomb, J. C.; Pelkonen, O. R.; Tucker, G. T.; Rostami-Hodjegan, A. Scaling Factors for the Extrapolation of In Vivo Metabolic Drug Clearance from In Vitro Data: Reaching a Consensus on Values of Human Microsomal Protein and Hepatocellularity Per Gram of Liver. Curr. Drug Metab. 2007, 8, 33 - 45.

Claims

1. A compound of formula (I), or a salt, hydrate, or solvate thereof, wherein 【Chemical 1】 wherein R １ and R ２ are independently selected from the group consisting of hydrogen, (C １ -C ６ )alkyl, (C ２ -C ６ )alkenyl, (C ２ -C ６ )alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R １ and R ２ together with the atom to which they are attached form a 5- or 6-membered ring, R ３ is hydrogen, halo, nitro, cyano, CF ３ , (C １ -C ６ )alkyl, (C １ -C ６ )alkylthio, or (C １ -C ６ )alkoxy, and A is alkylene, R ４ is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein each of heteroaryl, heterocyclyl, and heterocyclylamino is optionally substituted, R ５ represents one or two substituents independently selected from the group consisting of amino, (C １ -C ６ alkyl)amino, di(C １ -C ６ alkyl)amino, hydroxy(C １ -C ６ alkyl), (C １ -C ６ alkenyl), (C １ -C ６ alkynyl), (C １ -C ６ heteroalkyl), (C ３ -C ８ cycloalkyl), (C ３ -C ８ cycloheteroalkyl), (C １ -C ６ alkoxy), (C １ -C ６ alkyl)(CO)O-, (C １ -C ６ alkyl)-O(CO)O- and (C １ -C ６ alkylthio), or R ５ represents two adjacent substituents which together with the attached carbon form an optionally substituted ring or heterocycle, said compound, or a salt, hydrate, or solvate thereof.

2. A is (CH ２ ), where n is selected from the group consisting of 1, 2, and 3, the compound according to claim 1.

3. A is (CH ２ ) ３ The compound according to claim 2, wherein

4. R ３ The compound according to claim 1, wherein R is hydrogen.

5. R １ and R ２ are independently selected from the group consisting of hydrogen and CH ３ The compound according to claim 1, wherein

6. R １ and R ２ The compound according to claim 1, wherein together with the atoms to which they are attached, they form a 5- or 6-membered ring.

7. R ４ is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein each of heteroaryl, heterocyclyl, and heterocyclylamino is optionally substituted, the compound according to claim 1.

8. R １ and R ２ together form -CH ２ -, R ３ is hydrogen, R ５ represents 2-MeO and 3-MeO, A is (CH ２ ) ３ and R ４ is selected from the group consisting of heteroaryl, heteroaryloxy, heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, trialkylammonium, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein each of heteroaryl, heteroaryloxy, and heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl, and heterocyclylamino is optionally substituted, the compound according to claim 1.

9. wherein the compound is 【Chemical 2】 The compound according to claim 1, selected from the group consisting of

10. A pharmaceutical composition comprising the compound according to claim 1, or a salt, hydrate, or solvate thereof

11. A pharmaceutical composition comprising a compound of formula (I), or a salt, hydrate, or solvate thereof, wherein [Chemical Formula 3] wherein R １ and R ２ are independently selected from the group consisting of hydrogen, (C １ ~C ６ )alkyl, (C ２ ~C ６ )alkenyl, (C ２ ~C ６ )alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R １ and R ２ together with the atoms to which they are attached form a 5- or 6-membered ring. R ３ is hydrogen, halo, nitro, cyano, CF ３ , (C １ ~C ６ )alkyl, (C １ -C ６ )alkylthio, or (C １ -C ６ )alkoxy, and A is (CH ３ ) ｎ , where n is from 2 to 3, R ４ is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein each of heteroaryl, heterocyclyl, and heterocyclylamino is optionally substituted, R ５ represents one or two substituents independently selected from the group consisting of amino, (C １ ~C ６ alkyl)amino, di(C １ ~C ６ alkyl)amino, hydroxy(C １ ~C ６ alkyl, (C １ ~C ６ alkenyl, (C １ ~C ６ alkynyl, (C １ ~C ６ heteroalkyl, (C ３ ~C ８ cycloalkyl, (C ３ ~C ８ cycloheteroalkyl, (C １ ~C ６ alkoxy, (C １ ~C ６ alkyl)(CO)O-, (C １ ~C ６ alkyl-O(CO)O- and (C １ ~C ６ alkylthio, or R ５ represents two adjacent substituents which, together with the carbon to which they are attached, optionally form a substituted ring or heterocycle, said pharmaceutical composition.

12. The pharmaceutical composition according to claim 11, further comprising at least one additional component selected from the group consisting of diluents, additives, and combinations thereof

13. The pharmaceutical composition according to claim 11 for treating cancer

14. The pharmaceutical composition according to claim 11, comprising a therapeutically effective amount of the compound of formula (I), or a salt, hydrate, or solvate thereof

15. A method for treating a disease responsive to topoisomerase I inhibition or binding to MYC quadruplex in a host animal, comprising administering to the host animal a composition comprising a therapeutically effective amount of one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, or a pharmaceutical composition comprising one or more compounds of formula (I), or a salt, hydrate, or solvate thereof, wherein Formula (I) is 【Chemical Formula 4】 wherein, R １ and R ２ are independently selected from the group consisting of hydrogen, (C １ -C ６ )alkyl, (C ２ -C ６ )alkenyl, (C ２ -C ６ )alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R １ and R ２ together with the atom to which they are attached form a 5- or 6-membered ring, R ３ is hydrogen, halo, nitro, cyano, CF ３ , (C １ ~C ６ )alkyl, (C １ -C ６ )alkylthio, or (C １ -C ６ )alkoxy, and A is alkylene, R ４ is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halo, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein each of heteroaryl, heterocyclyl, and heterocyclylamino is optionally substituted, R ５ represents one or two substituents independently selected from the group consisting of amino, (C １ ~C ６ alkylamino, di(C １ ~C ６ alkylamino, hydroxy(C １ ~C ６ alkyl, (C １ ~C ６ alkenyl, (C １ ~C ６ alkynyl, (C １ ~C ６ heteroalkyl, (C ３ ~C ８ cycloalkyl, (C ３ ~C ８ cycloheteroalkyl, (C １ ~C ６ alkoxy, (C １ ~C ６ alkyl(CO)O-, (C １ ~C ６ alkyl-O(CO)O- and (C １ ~C ６ alkylthio, or R ５ represents two adjacent substituents which together with the attached carbon form an optionally substituted ring or heterocycle, The method, wherein the pharmaceutical composition optionally further comprises one or more carriers, diluents, or excipients, or combinations thereof

16. The method according to claim 15, wherein the host animal is a human

17. A process for preparing the compound according to claim 1, comprising the step of brominating the compound of formula II to obtain compound III, wherein R １ , R ２ , and R ３ are as defined in claim 1, said process. 【Chemical Formula 5】

18. R ３ The process according to claim 17, wherein R is hydrogen.

19. R １ and R ２ together form -CH ２ - to form the process according to claim 18.

20. The process according to claim 19, wherein the brominating step comprises treating an acetic acid solution comprising the compound of formula (II) with N-bromosuccinimide

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