Preparation and application of 7-azaindeno isoquinoline

Abstract

Described herein are novel 8, 9-dioxy-7-azaindeno isoquinoline compounds (I), processes for their preparation, and methods of their use in the treatment of diseases responsive to inhibition of topoisomerase I and / or binding to c-MYC G-quadruplex. # imgabs0 #

Description

[0001] Government Rights

[0002] 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.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 337,248, filed on May 2, 2022, and U.S. Provisional Application No. 63 / 351,550, filed on June 13, 2022, under 35 U.S.C. §119(e), which are incorporated herein by reference. Technical Field

[0005] The present disclosure relates to novel 7-azaindenoisoquinoline compounds, their preparation processes and their use for various therapeutic purposes, especially for the treatment of cancer. The present disclosure relates to novel inhibitors of topoisomerase 1 (Top1) and compounds that block the activity of MYC oncogenes (such as by binding to the MYC promoter G-quadruplex). In some embodiments, the present disclosure relates to methods for treating patients with cancer by targeting the MYC oncogene or otherwise blocking the activity of the MYC oncogene. In some embodiments, the present disclosure relates to methods for treating cancer patients by targeting human topoisomerase I. In some other embodiments, the present disclosure relates to methods for treating patients with cancer by targeting the dual mechanism of action of the MYC oncogene and human topoisomerase I.

[0006] background

[0007] Cancer is a group of diverse diseases involving abnormal cell growth. There are currently more than 100 recognized types of cancer affecting humans as well as animals. In 2018, there were an estimated 1,708,921 new human cancer cases diagnosed in the United States alone, and 599,265 cancer deaths (2018 Cancer Statistics - Centers for Disease Control and Prevention). There is an increasing unmet need for new therapies for cancer treatment.

[0008] DNA is the target of many important anticancer agents, including human topoisomerase I inhibitors. Recently, significant progress has been made in the development of molecular targeted therapies. DNA-targeted drugs with cancer-specific molecular targeting properties may gain therapeutic advantages. Indenoisoquinolines are human topoisomerase I inhibitors with improved physicochemical and biological properties compared to traditional camptothecin topoisomerase I inhibitors used clinically for the treatment of various solid tumors. Three indenoisoquinolines, indotecan (LMP400), indimitecan (LMP776) and LMP744, have entered Phase I clinical trials for recurrent solid tumors and lymphomas in adults. However, it has been found that some indenoisoquinolines with potent anticancer activity surprisingly do not exhibit strong topoisomerase I inhibition. It has been found that certain indenoisoquinolines also target MYC G-quadruplex (MYC-G4).

[0009] MYC is one of the most important oncogenes and is overexpressed in more than 80% of all types of cancer. The transcription factor MYC protein is involved in cell proliferation, differentiation and apoptosis, and plays a key role in tumorigenesis and progression, as well as drug resistance. MYC was found to be a universal transcriptional "amplifier" in cancer cells. It has been observed that even transient inhibition of MYC expression can permanently stop tumor growth and induce tumor regression in vivo due to the "oncogene addiction" 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.

[0010] The G4 levels detected in immortalized precancerous cells were 10-fold higher than those in normal human cells, and G4 sites were found to be specifically enriched in regulatory, transcriptionally active regions of chromatin, particularly the MYC promoter region.

[0011] The nuclease hypersensitive element (NHE) III1 in the MYC promoter, which controls 85-90% of the MYC transcriptional activity, forms a DNA G-quadruplex (G4) under transcription-related negative supercoiling and functions as a transcriptional silencer. It has been found that the compounds of formula (I) can be used to treat cancer, particularly MYC-positive cancers. It is believed that the compounds of formula (I) can target MYC quadruplexes (MYCG4) and / or topoisomerase I and are effective against many cancers including MYC-positive cancers containing MYC G4. Summary of the invention

[0012] According to some embodiments, the present disclosure provides a compound of formula (I) or a salt, hydrate or solvate thereof,

[0013]

[0014] wherein R1 and R2 are independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R1 and R2 together with the atoms to which they are attached form a 5- or 6-membered ring;

[0015] R3 is hydrogen, halogen, nitro, cyano, CF3, (C1-C6) alkyl, (C1-C6) alkylthio or (C1-C6) alkoxy;

[0016] A is an alkylene group;

[0017] R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxy, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl, and heterocyclylamino are each optionally substituted;

[0018] R5 represents 1 to 2 substituents independently selected from the group consisting of amino, (C1-C6)alkylamino, di(C1-C6)alkylamino, hydroxy(C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, (C1-C6)heteroalkyl, (C3-C8)cycloalkyl, (C3-C8)cycloheteroalkyl, (C1-C6)alkoxy, (C1-C6)alkyl(CO)O-, (C1-C6)alkyl-O(CO)O- and (C1-C6)alkylthio; or R5 represents 2 adjacent substituents which, together with the attached carbon, form an optionally substituted ring or heterocycle.

[0019] In some embodiments, A is (CH2)n, wherein n is selected from the group consisting of 1, 2, and 3. In some embodiments, A is (CH2)3.

[0020] According to some embodiments, R3 is hydrogen.

[0021] According to some embodiments, R1 and R2 are independently selected from the group consisting of hydrogen and CH3. In other embodiments, R1 and R2 together with the atoms to which they are attached form a ring.

[0022] In some embodiments, R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl, and heterocyclylamino are each optionally substituted.

[0023] In some embodiments, R1 and R2 are taken together to form -CH2-, R3 is hydrogen, R5 represents 2-MeO and 3-MeO, A is (CH2)3, and R4 is selected from the group consisting of heteroaryl, heteroaryloxy, heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl, heterocyclylamino, amino, hydroxyl, halogen, cyano, alkylamino, dialkylamino, trialkylammonium, hydroxyalkylamino, bis(hydroxyalkyl)amino and hydroxyalkylaminoalkylamino, wherein heteroaryl, heteroaryloxy and heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl and heterocyclylamino are each optionally substituted.

[0024] According to some embodiments, the compound of formula (I) is selected from the group consisting of:

[0025]

[0026] 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.

[0027] According to some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound of formula (I) or a salt, hydrate or solvate thereof.

[0028]

[0029] in

[0030] R1 and R2 are independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R1 and R2 together with the atoms to which they are attached form a 5- or 6-membered ring;

[0031] R3 is hydrogen, halogen, nitro, cyano, CF3, (C1-C6) alkyl, (C1-C6) alkylthio or (C1-C6) alkoxy;

[0032] A is an alkylene group;

[0033] R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxy, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl, and heterocyclylamino are each optionally substituted;

[0034] R5 represents 1 to 2 substituents independently selected from the group consisting of amino, (C1-C6)alkylamino, di(C1-C6)alkylamino, hydroxy(C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, (C1-C6)heteroalkyl, (C3-C8)cycloalkyl, (C3-C8)cycloheteroalkyl, (C1-C6)alkoxy, (C1-C6)alkyl(CO)O-, (C1-C6)alkyl-O(CO)O- and (C1-C6)alkylthio; or R5 represents 2 adjacent substituents which, together with the attached carbon, form an optionally substituted ring or heterocycle.

[0035] In some embodiments, the pharmaceutical composition further comprises at least one additional component selected from the group consisting of a diluent, an excipient, and a combination thereof.

[0036] According to some embodiments, the pharmaceutical composition is for use in treating cancer.

[0037] 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.

[0038] According to some embodiments of the present disclosure, methods for treating diseases in a host animal that respond to topoisomerase I inhibition or binding to a MYC quadruplex are provided. 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,

[0039] Wherein formula (I) is

[0040]

[0041] wherein R1 and R2 are independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R1 and R2 together with the atoms to which they are attached form a 5- or 6-membered ring;

[0042] R3 is hydrogen, halogen, nitro, cyano, CF3, (C1-C6) alkyl, (C1-C6) alkylthio or (C1-C6) alkoxy;

[0043] A is an alkylene group;

[0044] R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxy, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl, and heterocyclylamino are each optionally substituted;

[0045] R5 represents 1 to 2 substituents independently selected from the group consisting of amino, (C1-C6)alkylamino, di(C1-C6)alkylamino, hydroxy(C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, (C1-C6)heteroalkyl, (C3-C8)cycloalkyl, (C3-C8)cycloheteroalkyl, (C1-C6)alkoxy, (C1-C6)alkyl(CO)O-, (C1-C6)alkyl-O(CO)O-, and (C1-C6)alkylthio; or R5 represents 2 adjacent substituents which, together with the attached carbon, form an optionally substituted ring or heterocycle, and

[0046] The pharmaceutical composition optionally further comprises one or more carriers, diluents, excipients or a combination thereof.

[0047] In some embodiments, the host animal is a human.

[0048] According to an embodiment of the present disclosure, a process for preparing a compound of formula (I) as described herein is provided. In some embodiments, the process comprises the step of brominating a compound of formula II to produce compound III, wherein R1, R2 and R3 are as defined in claim 1.

[0049]

[0050] In some embodiments, R3 is hydrogen.

[0051] In some embodiments, R1 and R2 are taken together to form -CH2-.

[0052] In some embodiments, the bromination step comprises treating a solution of the compound of formula (II) in acetic acid with N-bromosuccinimide. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 is a schematic diagram of MycG4;

[0055] Figure 2 is a graph using a FRET-based melting experiment showing the melting temperature (ΔT m ) or MycG4 G-quadruplexes, under the following conditions: 10 mM K + 0.2 μM 5′-g-FAM / 3′-TAMRA-MycG4 DNA in buffer (pH 7.0), 2.0 μM (10 equivalents) ligand;

[0056] Figure 3 To display the Figure 2 Graphs of the results for the reference compounds in the data;

[0057] Figure 4 1D shows the 1D expression of 7-aza-8,9-methylenedioxindole to MycG4 DNA 1 H NMR titration of the imino proton region at 25 °C, 150 μM DNA, 100 mM K + Buffer, pH 7.0;

[0058] Figure 5 is a Western blot showing the Myc protein level in MCF-7 cells treated with or without 0.5, 1.0, and 2.0 μM 7-aza-8,9-methylenedioxindoisoquinoline for 24 h, with GAPDH as a loading control;

[0059] Figure 6 qRT-PCR results are shown, which show Myc mRNA levels in MCF-7 cells with or without 1.0 μM drug treatment for 6 h, wherein the relative Myc mRNA levels were calculated by normalization to the endogenous control GAPDH, and all data points of five biological replicates were plotted, wherein each data point contained duplicate repeats, and the statistical significance between each treatment group and the control was assessed by one-way ANOVA with Dunnett (**** indicates P<0.0001);

[0060] Figure 7 Shown for Figure 6 Results for reference compounds in the data;

[0061] Figure 8 Shown are the results of MTS single dose assays showing the % survival values ​​(calculated by normalizing to the control minus the blank value for the same cell line) of CA-46 and Raji cells treated with and without 250 nM 7-aza-8,9-methylenedioxindenoisoquinoline, wherein each bar graph represents the mean of three biological replicates, each biological replicate containing four technical replicates, and the treatment groups with statistically significant differences in % survival values ​​between CA-46 and Raji cells are marked, with p value <0.005 assessed by Welch t-test;

[0062] Fig. 9 Shown for Figure 8 Reference compound results for data with and without 250 nM indenoisoquinoline treatment;

[0063] Fig.10Shown are the effects of 7-aza-8,9-methylenedioxindenoisoquinoline compounds on Topol inhibition, which were measured by (A) Topo1-mediated relaxation assay using native agarose gel stained with ethidium bromide to measure Topo1 relaxation activity, and (B) Topo1-mediated DNA cleavage assay using agarose gel containing ethidium bromide to optimize the visualization of Topo1 / DNA cleavage complex formation for each drug compound, wherein lane 1 is a DNA relaxation marker, lane 2 is supercoiled DNA, lane 3 is supercoiled DNA+Topol (CON), and lanes 4-11 are supercoiled DNA+Topol+10 μM of each compound;

[0064] Fig.11 is a graph showing the percentage of growth as a function of time following administration of G103, G104 and G105 to female NOD SCID mice at low and high doses;

[0065] Fig.12 is a graph showing the percentage of growth as a function of time following administration of G103, G104 and G105 to female NOD SCID mice over a mid-dose range;

[0066] Fig.13 is a graph of body weight as a function of time following administration of G103, G104, G105 and JQ-1 to female NOD SCID mice inoculated with MOLT-4 tumor cells;

[0067] Fig.14 is a graph showing the change in tumor size over time after administration of G103, G104, G105 and JQ-1 to female NOD SCID mice inoculated with MOLT-4 tumor cells;

[0068] Fig.15 is a graph of body weight as a function of time following administration of G103, G104, G105, and JQ-1 to female NOD SCID mice following inoculation with RD-ES tumor cells;

[0069] Fig.16 is a graph of tumor size as a function of time following administration of G103, G104, G105, and JQ-1 to female NOD SCID mice following inoculation with RD-ES tumor cells;

[0070] Fig.17 is a graph of body weight as a function of time following administration of G103, G104, G105, and JQ-1 to female NOD SCID mice following inoculation with RS4;11 tumor cells; and

[0071] Fig.18Graph of tumor size as a function of time following administration of G103, G104, G105, and JQ-1 to female NOD SCID mice following inoculation with RS4;11 tumor cells.

[0072] Detailed Description

[0073] definition

[0074] In each embodiment, unless otherwise indicated, the transitional phrase "consisting essentially of" should be understood to mean that the scope of the corresponding composition, unit dosage, method, or use is understood to cover the specific compound or steps recited, as well as those that do not materially affect the basic and novel characteristics of the invention described herein. For example, the methods described herein consisting essentially of a single compound or class of compounds are understood to represent a monotherapy for the recited disease. Although a monotherapy may include co-administration of one or more carriers, vehicles, diluents, adjuvants, excipients, etc. and combinations thereof, and / or include co-administration of one or more additional active pharmaceutical ingredients, those subsequent additional active pharmaceutical ingredients are understood to be used to treat diseases and / or symptoms different from the underlying conditions described herein (such as cancer itself).

[0075] In each embodiment, unless otherwise indicated, it is understood that the formula not only includes and represents all pharmaceutically acceptable salts of the compound, but also includes any and all hydrates and / or solvates of the compound formula. It should be understood that certain functional groups in various physical forms of the compound, such as hydroxyl, amino and other groups, form complexes and / or coordination compounds with water and / or various solvents. Therefore, the formula is understood as a description of such hydrates and / or solvates, including pharmaceutically acceptable solvates.

[0076] As used herein, the term "solvate" refers to a compound complexed with a solvent molecule. The compound can form such a complex with a solvent by simply mixing the compound with the solvent or dissolving the compound in the solvent. When the compound is used as a drug, such a solvate is a pharmaceutically acceptable solvent. When the compound is used as a drug, the relative amount of the solvent forming the solvate should be less than the established guidelines for the use of such drugs, such as less than the International Conference on Harmonization (ICH) guidelines. The solvate can be separated 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.

[0077] In each embodiment, unless otherwise indicated, the formula is understood to include and represent any and all crystalline forms, partially crystalline forms, and non-crystalline and / or amorphous forms of the compound, including partially ordered forms, disordered forms, liquid crystal forms, and meso phases of any of the foregoing.

[0078] In each embodiment, unless otherwise indicated, it is understood that the formula includes and represents every possible isomer, such as stereoisomers and geometric isomers, both individually and in any and all possible mixtures.

[0079] A pharmaceutical composition comprising a compound of any embodiment described herein, and optionally comprising one or more carriers, diluents, excipients, etc. and combinations thereof.

[0080] Exemplary derivatives include, but are not limited to, compounds that can be synthesized from the described compounds, as well as compounds that can be prepared in a manner similar to those described but with different starting material selections. For example, compounds of formula (I) are described that include various functional groups on the aromatic ring, such as R3 and R5. Derivatives of those compounds also include, for example, compounds having functional groups different from those explicitly listed in the definition of formula (I) on those aromatic rings. In addition, derivatives of those compounds also include compounds having those same or different functional groups at different positions on the aromatic rings. Similarly, derivatives include parallel variants of other functional groups of the compounds described herein, such as R4.

[0081] Such derivatives may include prodrugs of the compounds and compounds containing one or more protecting groups or protective groups, including compounds used in the preparation of other compounds described.

[0082] Exemplary analogs include, but are not limited to, those compounds that share functional and, in some cases, structural similarities with those compounds described herein. For example, compounds of formula (I) that include a 7-aza-indenoisoquinoline ring system are described. Exemplary analogs include, but are not limited to, corresponding ring-expanded compounds. Other exemplary analogs include, but are not limited to, corresponding ring systems that include additional heteroatoms.

[0083] It should be understood that each embodiment can be combined in a chemically related manner to generate a subset of embodiments. Therefore, it should be further understood that all such subsets are also illustrative embodiments.

[0084] 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 requirements, and the compounds and compositions, methods, uses, and medicaments comprising them may be optically pure, or may be any of a variety of stereoisomer mixtures, including racemates and other enantiomeric mixtures, other diastereomeric mixtures, and the like. Such mixtures of stereoisomers may include a single stereochemical configuration at one or more chiral centers, while including a mixture of stereochemical configurations at one or more other chiral centers.

[0085] Similarly, the compound may include a geometric center, such as cis, trans, E and Z double bonds, or a spatial arrangement on the ring, such as cis, trans, syn and anti on the ring. In another embodiment, the disclosure is not limited to any specific geometric isomer requirements, and the compound and the compositions, methods, uses and medicines including them may be pure, or may be any of various geometric isomer mixtures. Such a mixture of geometric isomers may include a single configuration at one or more double bonds, while including a geometric mixture at one or more other double bonds.

[0086] As used herein, the term "alkyl" includes a chain of carbon atoms, which is optionally branched. As used herein, the terms "alkenyl" and "alkynyl" each include a chain of carbon atoms, which is optionally branched, and each include at least one double bond or triple bond. Alkynyl groups may also include one or more double bonds. In certain embodiments, alkyl groups advantageously have a limited length, including C1-C 24 、C1-C 12 , C1-C8, C1-C6, and C1-C4 and C2-C 24 、C2-C 12 , C2-C8, C2-C6 and C2-C4, etc. Exemplarily, such specific alkyl groups of limited length including C1-C8, C1-C6 and C1-C4, and C2-C8, C2-C6 and C2-C4, etc. may be referred to as low alkyl groups. In certain embodiments, alkenyl and / or alkynyl groups may each advantageously have limited length, including C2-C 24 、C2-C 12 , C2-C8, C2-C6 and C2-C4, and C3-C 24 、C3-C 12 In some embodiments, the narration of alkyl group refers to alkyl and optional low alkyl group. In some embodiments, the narration of alkyl group refers to alkyl and optional low alkyl group. In some embodiments, the narration of alkyl group refers to alkyl and optional low alkyl group. In some embodiments, the narration of alkynyl refers to alkynyl and optional low alkynyl group. 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, and the like, as well as corresponding groups or combinations thereof containing one or more double and / or triple bonds.

[0087] The term "alkylene" includes a divalent carbon atom chain, which is optionally branched. The terms "alkenylene" and "alkynylene" include a divalent carbon atom chain, which is optionally branched, and includes at least one double bond or triple bond, respectively. Alkynylenes may also include one or more double bonds. In certain embodiments, alkylenes advantageously have a limited length, including C1-C 24 、C1-C 12 , C1-C8, C1-C6 and C1-C4, and C2-C 24 、C2-C 12 , C2-C8, C2-C6 and C2-C4, etc. Exemplarily, such specific limited length alkylene groups including C1-C8, C1-C6 and C1-C4, and C2-C8, C2-C6 and C2-C4, etc. may be referred to as lower alkylene. In certain embodiments, alkenylene and / or alkynylene may each advantageously have a limited length, including C2-C 24 、C2-C 12 , C2-C8, C2-C6 and C2-C4, and C3-C 24 、C3-C 12 , C3-C8, C3-C6 and C3-C4, etc. Exemplarily, such specific limited length alkenylene and / or alkynylene groups including C2-C8, C2-C6 and C2-C4, and C3-C8, C3-C6 and C3-C4, etc., may also be referred to as low alkenylene and / or alkynylene. Shorter alkylene, alkenylene and / or alkynylene groups can make the lipophilicity of the compound lower, and therefore will have different pharmacokinetic behaviors. In certain embodiments, the description of alkylene, alkenylene and alkynylene relates to alkylene, alkenylene and alkynylene, and optionally low alkylene, alkenylene and alkynylene. Exemplary alkyl groups include, but are not limited to, methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, secondary butylene, pentylene, 1,2-pentylene, 1,3-pentylene, hexylene, heptylene, octylene, etc.

[0088] As used herein, the term "cycloalkyl" includes a chain of carbon atoms, which is optionally branched, wherein at least a portion of the chain is cyclic. Cycloalkylalkyl is a subset of cycloalkyl. Cycloalkyl can be polycyclic. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, 2-methylcyclopropyl, cyclopentyleth-2-yl, adamantyl, and the like. As used herein, the term "cycloalkenyl" includes a chain of carbon atoms, which is optionally branched, and includes at least one double bond, wherein at least a portion of the chain is cyclic. One or more double bonds may be located in the cyclic portion of the cycloalkenyl and / or the non-cyclic portion of the cycloalkenyl. Cycloalkenylalkyl and cycloalkylalkenyl are each a subset of cycloalkenyl. Cycloalkyl can be polycyclic. Exemplary cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclohexylethen-2-yl, cycloheptenylpropenyl, and the like. The chain-formed cycloalkyl and / or cycloalkenyl groups advantageously have limited lengths, including C3-C 24 、C3-C 12 , C3-C8, C3-C6 and C5-C6. Shorter alkyl and / or alkenyl chains forming cycloalkyl and / or cycloalkenyl groups, respectively, can make the compound less lipophilic and thus will have different pharmacokinetic behavior.

[0089] The term "heteroalkyl" includes an atom chain comprising carbon and at least one heteroatom, and is optionally a branched chain. Exemplary heteroatoms include nitrogen, oxygen and sulfur. In some variants, exemplary heteroatoms also include phosphorus and selenium. The term "cycloheteroalkyl" including heterocyclic radicals and heterocycles includes an atom chain comprising carbon and at least one heteroatom, such as heteroalkyl, and is optionally a branched chain, wherein at least a portion of the chain is cyclic. Exemplary heteroatoms include nitrogen, oxygen and sulfur. In some variants, exemplary heteroatoms also include phosphorus and selenium. Exemplary cycloheteroalkyl includes, but is not limited to, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl, etc.

[0090] The term "aryl" includes monocyclic and polycyclic aromatic carbocyclic groups, each of which can be optionally substituted. Exemplary aromatic carbocyclic groups described herein include, but are not limited to, phenyl, naphthyl, etc. The term "heteroaryl" includes aromatic heterocyclic groups, each of which can be optionally substituted. Exemplary aromatic heterocyclic groups include, but are not limited to, pyridyl, pyrimidyl, pyrazinyl, triazinyl, tetrazinyl, quinolyl, quinazolinyl, quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzisoxazolyl, benzisothiazolyl, etc.

[0091] The term "amino" includes groups NH2, alkylamino and dialkylamino, wherein the two alkyl groups in the dialkylamino can be the same or different, i.e., alkylalkylamino. Exemplarily, amino includes methylamino, ethylamino, dimethylamino, methylethylamino, etc. In addition, when amino is modified or modified by other terms such as aminoalkyl or acylamino, the above-mentioned variants of the term amino are included. Exemplarily, aminoalkyl includes H2N-alkyl, methylaminoalkyl, ethylaminoalkyl, dimethylaminoalkyl, methylethylaminoalkyl, etc. Exemplarily, acylamino includes acylmethylamino, acylethylamino, etc.

[0092] The term "amino and derivatives thereof" includes amino groups described herein, as well as alkylamino, alkenylamino, alkynylamino, heteroalkylamino, heteroalkenylamino, heteroalkynylamino, cycloalkylamino, cycloalkenylamino, cycloheteroalkylamino, cycloheteroalkenylamino, arylamino, arylalkylamino, arylalkenylamino, arylalkynylamino, heteroarylamino, heteroarylalkylamino, heteroarylalkenylamino, heteroarylalkynylamino, acylamino, and the like, each of which is optionally substituted. The term "amino and derivatives thereof" also includes alkylalkylamino, alkylalkenylamino, alkylalkynylamino, alkylheteroalkylamino, alkylheteroalkenylamino, alkylheteroalkynylamino, alkylcycloalkylamino, alkylcycloalkenylamino, alkylcycloheteroalkylamino, alkylcycloheteroalkenylamino, arylamino, arylalkylamino, arylalkenylamino, arylalkynylamino, heteroarylamino, heteroarylalkylamino, heteroarylalkenylamino, heteroarylalkynylamino, alkylacylamino, and the like, each of which is optionally substituted. The term "amino derivative" also includes urea, carbamate and the like.

[0093] The term "hydroxyl and its derivatives" includes OH, as well as alkoxy, alkenyloxy, alkynyloxy, heteroalkoxy, heteroalkenyloxy, heteroalkynyloxy, cycloalkoxy, cycloalkenyloxy, cycloheteroalkoxy, cycloheteroalkenyloxy, aryloxy, aralkyloxy, aralkenyloxy, aralkynyloxy, heteroaryloxy, heteroarylalkoxy, heteroarylalkenyloxy, heteroarylalkynyloxy, acyloxy, and the like, each of which is optionally substituted. The term "hydroxyl derivatives" also includes carbamates and the like.

[0094] The term "sulfenyl and its derivatives" includes SH, and alkylthio, alkenylthio, alkynylthio, heteroalkylthio, heteroalkenylthio, heteroalkynylthio, cycloalkylthio, cycloalkenylthio, cycloheteroalkylthio, cycloheteroalkenylthio, arylthio, arylalkylthio, aralkenylthio, aralkynylthio, heteroarylthio, heteroarylalkylthio, heteroarylalkenylthio, heteroarylalkynylthio, acylthio and the like, each of which is optionally substituted. The term "sulfenyl derivatives" also includes sulfenylcarbamates and the like.

[0095] The term "acyl" includes formyl, as well as alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, heteroalkylcarbonyl, heteroalkenylcarbonyl, heteroalkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl, cycloheteroalkylcarbonyl, cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl, arylalkenylcarbonyl, arylalkynylcarbonyl, heteroarylcarbonyl, heteroarylalkylcarbonyl, heteroarylalkenylcarbonyl, heteroarylalkynylcarbonyl, acylcarbonyl and the like, each of which is optionally substituted.

[0096] The term "carbonyl and its derivatives" includes the groups C(O), C(S), C(NH) and their substituted amino derivatives.

[0097] The term "carboxylic acid and its derivatives" includes the group CO2H and its salts, and its esters and amides, and CN.

[0098] The term "sulfinic acid or its derivatives" includes SO2H and its salts, and its esters and amides.

[0099] The term "sulfonic acid or its derivatives" includes SO3H and its salts, and its esters and amides.

[0100] The term "sulfonyl" includes alkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, heteroalkylsulfonyl, heteroalkenylsulfonyl, heteroalkynylsulfonyl, cycloalkylsulfonyl, cycloalkenylsulfonyl, cycloheteroalkylsulfonyl, cycloheteroalkenylsulfonyl, arylsulfonyl, arylalkylsulfonyl, arylalkenylsulfonyl, arylalkynylsulfonyl, heteroarylsulfonyl, heteroarylalkylsulfonyl, heteroarylalkenylsulfonyl, heteroarylalkynylsulfonyl, acylsulfonyl and the like, each of which is optionally substituted.

[0101] The term "hydroxyamino and derivatives thereof" includes NHOH, as well as alkoxy NH, alkenyloxy NH, alkynyloxy NH, heteroalkoxy NH, heteroalkenyloxy NH, heteroalkynyloxy NH, cycloalkoxy NH, cycloalkenyloxy NH, cycloheteroalkoxy NH, cycloheteroalkenyloxy NH, aryloxy NH, arylalkoxy NH, arylalkenyloxy NH, arylalkynyloxy NH, heteroaryloxy NH, heteroarylalkoxy NH, heteroarylalkenyloxy NH, heteroarylalkynyloxy NH, acyloxy NH and the like, each of which is optionally substituted.

[0102] The term “hydrazino and derivatives thereof” 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, heteroaryl alkyl NHNH, heteroarylalkenyl NHNH, heteroarylalkynyl NHNH, acyl NHNH and the like, each of which is optionally substituted.

[0103] The term "optionally substituted" includes replacing hydrogen atoms with other functional groups on the optionally substituted free radical. Such other functional groups illustratively include, but are not limited to, amino, hydroxyl, halogen, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acid and derivatives thereof, carboxylic acid and derivatives thereof, etc. Exemplarily, any one of amino, hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl and / or sulfonic acid is optionally substituted.

[0104] The terms "optionally substituted aryl" and "optionally substituted heteroaryl" include replacing hydrogen atoms with other functional groups on the optionally substituted aryl or heteroaryl. Such other functional groups are also referred to herein as aryl substituents or heteroaryl substituents, respectively, and illustratively include but are not limited to amino, hydroxyl, halogen, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acid and derivatives thereof, carboxylic acid and derivatives thereof, etc. Exemplarily, any of amino, hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl and / or sulfonic acid is optionally substituted.

[0105] Exemplary substituents also include, but are not limited to, the radical -(CH2) x Z X , where x is an integer from 0 to 6, and Z X is selected from the group consisting of halogen, hydroxy, alkanoyloxy (including C1-C6 alkanoyloxy), optionally substituted aroyloxy, alkyl (including C1-C6 alkyl), alkoxy (including C1-C6 alkoxy), cycloalkyl (including C3-C8 cycloalkyl), cycloalkoxy (including C3-C8 cycloalkoxy), alkenyl (including C2-C6 alkenyl), alkynyl (including C2-C6 alkynyl), haloalkyl (including C1-C6 haloalkyl), haloalkoxy (including C1-C6 haloalkoxy), halogen or Z is substituted with alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, alkyl or alkyl groups, XSelected from -CO2R4 and -CONR5R6, wherein R4, R5 and R6 at each occurrence are independently selected from hydrogen, C1-C6 alkyl, aryl-C1-C6 alkyl and heteroaryl-C1-C6 alkyl.

[0106] As used herein, the term "leaving group" refers to a reactive functional group that produces an electrophilic site on the atom to which it is attached, so that a nucleophilic substance can be added to the electrophilic site on the atom. Exemplary leaving groups include, but are not limited to, halogen elements, optionally substituted phenols, acyloxy groups, sulfonium groups, sulfonoxy groups, etc. Such leaving groups may be located on alkyl, acyl, etc. Such leaving groups may also be referred to as activating groups herein, such as when the leaving group is present on an acyl group. In addition, conventional peptides, amides, and ester coupling agents, such as, but not limited to, PyBop, BOP-Cl, BOP, pentafluorophenol, isobutyl chloroformate, etc., form various intermediates including leaving groups defined herein on a carbonyl group.

[0107] The recitation of an integer range for any variable describes the recited range, each individual member of the range, and each possible subrange of the variable. For example, the recitation of n as an integer from 0 to 8 describes the range, with individual and alternative values ​​of 0, 1, 2, 3, 4, 5, 6, 7, and 8, such as n is 0, or n is 1, or n is 2, etc. In addition, the recitation of n as an integer from 0 to 8 also describes each subrange, each of which can serve as the basis for further embodiments, such as n as an integer from 1 to 8, 1 to 7, 1 to 6, 2 to 8, 2 to 7, 1 to 3, 2 to 4, etc.

[0108] When referring to a chemical reaction, the terms "treating", "contacting" and "reacting" generally refer to the addition or mixing of two or more reagents under appropriate conditions so that a chemical transformation or chemical reaction occurs and / or an indicated and / or desired product is produced. The reaction that produces the indicated and / or desired product may not necessarily result directly from the combination of the two reagents initially added. In other words, there may be one or more intermediates produced in the mixture that ultimately lead to the formation of the indicated and / or desired product.

[0109] The term "protecting group" generally refers to any free radical that reversibly binds to a functional group and serves to block or partially block the reactivity of the functional group under a predetermined set of conditions (such as reaction conditions). Exemplarily, a nitrogen protecting group reversibly binds to an amine to block or partially block the reactivity of the amine under a predetermined set of conditions. Exemplary nitrogen protecting groups include, but are not limited to, carbamates such as t-Boc, Fmoc, and the like.

[0110] The term "composition" generally refers to any product comprising the specified ingredients, as well as any product which results, directly or indirectly, from combination of the specified ingredients.

[0111] Certain functional groups in various physical forms of compounds, such as hydroxyl, amino and other groups, form complexes and / or coordination compounds with water and / or various solvents. The compositions described herein can be prepared from isolated compounds or salts, solutions, hydrates, solvates and other forms of the compounds. The compositions can be prepared from various amorphous, non-amorphous, partially crystalline, crystalline and / or other forms of the compounds. The compositions can be prepared from various hydrates and / or solvates of the compounds. In addition, the compositions can be prepared from various co-crystals of the compounds. Therefore, it should be understood that such pharmaceutical compositions describing compounds include each or any combination of various morphological forms and / or solvates or hydrate forms of the compounds.

[0112] Exemplarily, the composition may include one or more carriers, diluents and / or excipients. The compound or the composition comprising them can be formulated in a therapeutically effective amount as any conventional dosage form suitable for the method. The compound or the composition comprising them (including such preparations) can be applied by a variety of conventional routes for the method, and in various dosage forms (generally referring to Remington: The Science and Practice of Pharmacy, (23rd edition, 2020)) using known procedures.

[0113] Compound can be used for human clinical medicine and veterinary application.Therefore, the host animal suffering from cancer and treated with the compound can be a human, or in the case of veterinary application, can be a laboratory animal, agricultural animal, domestic animal or wild animal.The disclosure can be applied to host animals, which include but are not limited to humans, laboratory animals such as rodents (such as mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees, domestic animals such as dogs, cats and rabbits, agricultural animals such as cattle, horses, pigs, sheep, goats, and captive wild animals such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins and whales.

[0114] The method can be used to treat such cancers, such as: carcinoma, sarcoma, lymphoma, Hodgkin's disease, melanoma, mesothelioma, Burkitt's lymphoma, nasopharyngeal carcinoma, leukemia and myeloma. Cancer cell populations can include, but are not limited to, oral cancer, thyroid cancer, endocrine cancer, skin cancer, stomach 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.

[0115] The term "therapeutically effective amount" refers to the amount of active compound or agent that causes a biological or drug response in a tissue system, animal or human that is sought by a researcher, veterinarian, doctor or other clinician, including relieving the symptoms of the disease or condition being treated. In one aspect, a therapeutically effective amount is an amount that can treat or alleviate the symptoms of a disease or disease at a reasonable benefit / risk ratio applicable to any medical treatment. However, the total daily usage of the compound and composition can be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors, including the severity of the disease and condition being treated; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health, sex and diet; the administration time, route of administration and excretion rate of the specific compound used; the duration of treatment; drugs used in combination or simultaneously with the specific compound used; and similar factors well known to researchers, veterinarians, doctors or other clinicians with ordinary skills.

[0116] The therapeutically effective amount, whether referring to monotherapy or combination therapy, is advantageously selected with reference to any toxicity or other undesirable side effects that may occur during administration of one or more compounds described herein. Further, co-therapy may allow for administration of low doses of the compounds that exhibit such toxicity or other undesirable side effects, where those low doses are below the toxicity threshold or therapeutic window is below the dose in the absence of co-therapy.

[0117] The attending diagnostician or physician readily determines the effective amount of any one of the compounds or mixtures of compounds described herein by using known techniques and / or by observing the results obtained under analogous circumstances. In determining the effective amount or dosage, the attending diagnostician or physician considers a variety of factors, including, but not limited to, the species of mammal (including humans), its size, age and general health, the specific disease or condition involved, the extent of the disease or condition, the involvement or severity of the disease or condition, the response of the individual patient, the specific compound being administered, the mode of administration, the bioavailability characteristics of the administered formulation, the dosage regimen selected, the use of concurrent medications, and other relevant circumstances.

[0118] The dosage of each compound in the claimed combination depends on a variety of factors, including: the method of administration, the condition being treated, the severity of the condition, whether the condition is therapeutic or preventive, and the age, weight, and health of the person being treated. In addition, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic, or efficacy characteristics of a therapeutic agent) information about a particular patient may affect the dosage used.

[0119] In the method, the single components of co-administration or combination can be administered in any suitable manner, contemporaneously, simultaneously, sequentially, individually or in the form of a single pharmaceutical preparation. Wherein the co-administered compound or composition is administered in a separate dosage form, and the dosage number of each compound administered per day can be the same or different. The compound or composition can be administered via the same or different routes of administration. The compound or composition can be administered in parallel (concurrently) in a separate or single form at the same or different times during the course of the therapy according to a simultaneous or alternating scheme.

[0120] In addition, in those embodiments involving combination therapy comprising administration of a compound of formula (I) and another anticancer drug known in the art, "therapeutically effective amount" refers to the amount of the combination of agents together such that the combined effect causes the desired biological or drug response. For example, a therapeutically effective amount of a compound of formula (I) and imatinib, etc., is an amount of a compound of formula (I) and an amount of imatinib, etc., that have a therapeutically effective combined effect when administered together or sequentially. Further, in some embodiments of such methods involving co-administration, the co-administered amount of a compound of formula (I) or imatinib, etc., may or may not be therapeutically effective when administered alone.

[0121] The term "administering" includes all ways of introducing 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 can be administered in unit dosage forms and / or formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and / or vehicles.

[0122] Exemplary oral administration forms include tablets, capsules, elixirs, syrups and the like.

[0123] Exemplary routes of parenteral administration include intravenous, intraarterial, intraperitoneal, epidural, intraurethral, ​​intrasternal, intramuscular, and subcutaneous, as well as any other art-recognized route of parenteral administration.

[0124] Exemplary parenteral administration methods include needle (including microneedle) injectors, needleless injectors and infusion techniques, and any other parenteral administration means recognized in the art. Parenteral preparations are generally aqueous solutions, which may include excipients such as salts, carbohydrates and buffers (preferably pH values ​​in the range of about 3 to about 9), but for some applications, they may be more suitable for being formulated as sterile non-aqueous solutions or dry forms, to be used in combination with suitable vehicles (such as sterile, pyrogen-free water). The preparation of parenteral preparations under sterile conditions (e.g., by lyophilization) can be easily achieved using standard pharmaceutical techniques well known to those skilled in the art. The parenteral administration of the compound is exemplarily carried out in the form of saline solution or in the form of the compound being incorporated into liposomes. In the case where the compound is not sufficiently soluble to be dissolved, a solubilizing agent such as ethanol may be applied.

[0125] Exemplarily, administration includes topical use, such as when topically applied to the site of the disease or to a specific organ or tissue system. Exemplary topical administration can be performed during open surgery, or during other operations accessible to the site of the disease. Alternatively, topical administration can be performed using parenteral delivery, wherein the compound or composition is locally deposited to the site, and is not generally distributed in a plurality of other non-target sites of the host animal to be treated. Similar changes regarding topical delivery to specific tissue types (such as organs, etc.) are also described. Exemplarily, the compound can be directly administered to the nervous system, including but not limited to intracerebral, intraventricular (intraventricular), intraventricular (intracerebroventricular), intrathecal, intracisternal, intraspinal and / or perispinal administration routes, by delivering via intracranial or intravertebral needles and / or catheters (with or without pump devices).

[0126] In preparing a pharmaceutical composition of a compound, a therapeutically effective amount of one or more compounds described herein in any of a variety of forms may be mixed with one or more excipients, diluted with one or more excipients, or encapsulated in such a carrier, which may be in the form of a capsule, sachet, paper or other container. An excipient may act as a diluent and may be a solid, semisolid or liquid material that acts as a vehicle, carrier or medium for the active ingredient. Thus, the formulation composition may be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), ointments, soft and hard gelatin capsules, suppositories, sterile injection solutions and sterile packaged powders. Depending on the dosage and dosage form selected, the composition may contain from about 0.1% to about 99.9% active ingredient.

[0127] Illustrative examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup and methylcellulose. The formulation may additionally include lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweeteners; and flavoring agents. The composition can be formulated by methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to the host animal. When preparing a pharmaceutical composition, one or more carriers, one or more diluents, one or more excipients and combinations of the foregoing may be used. It should be understood that the carriers, diluents and excipients used to prepare the compositions described herein are advantageously GRAS (generally recognized as safe) compounds. It should also be understood that the acids and bases used to prepare the salts described herein, and / or the solvents used to prepare the solvates described herein are also advantageously GRAS compounds.

[0128] Examples of exemplary emulsifiers include naturally occurring gums (e.g., gum arabic or tragacanth) and naturally occurring phospholipids (e.g., soy lecithin and sorbitol 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 parabens (such as methyl or propyl parabens) and benzalkonium chloride. Examples of humectants are glycerol, 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 ethylenediaminetetraacetate, citric acid, and phosphoric acid. Examples of gel forming agents are CARBOPOL, cellulose derivatives, bentonite, alginate, gelatin, and polyvinylpyrrolidone. Examples of ointment bases are beeswax, paraffin, cetyl palmitate, vegetable oils, sorbitan fatty acid esters (Span), polyethylene glycols and condensation products of sorbitan fatty acid esters with ethylene oxide (eg polyoxyethylene sorbitan monooleate (TWEEN)).

[0129] The therapeutically effective dose administered in the animal model can be used to calculate the corresponding therapeutically effective dose for administration to other host animals (including humans). An exemplary corresponding dose 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 incorporated herein by reference in its entirety.

[0130] Compound of formula (I)

[0131] In an embodiment, the present disclosure provides a compound of formula (I) or a salt, hydrate or solvate thereof:

[0132] in:

[0133] R1 and R2 are independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R1 and R2 together with the atoms to which they are attached form a 5- or 6-membered ring;

[0134] R3 is hydrogen, halogen, nitro, cyano, CF3, (C1-C6) alkyl, (C1-C6) alkylthio or (C1-C6) alkoxy;

[0135] A is an alkylene group;

[0136] R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxy, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl, and heterocyclylamino are each optionally substituted;

[0137] R5 represents 1 to 2 substituents independently selected from the group consisting of amino, (C1-C6)alkylamino, di(C1-C6)alkylamino, hydroxy(C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, (C1-C6)heteroalkyl, (C3-C8)cycloalkyl, (C3-C8)cycloheteroalkyl, (C1-C6)alkoxy, (C1-C6)alkyl(CO)O-, (C1-C6)alkyl-O(CO)O- and (C1-C6)alkylthio; or R5 represents 2 adjacent substituents which, together with the attached carbon, form an optionally substituted ring or heterocycle.

[0138] In an exemplary embodiment, a compound of formula (I) or a salt, hydrate or solvate thereof is described:

[0139]

[0140] wherein A is (CH2)n, wherein n is 1, 2 or 3;

[0141] In each instance, R1 and R2 are independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R1 and R2 together with the atoms to which they are attached form a 5- or 6-membered ring;

[0142] R3 is hydrogen, halogen, nitro, cyano, CF3, (C1-C6) alkyl, (C1-C6) alkylthio or (C1-C6) alkoxy;

[0143] R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxy, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl, and heterocyclylamino are each optionally substituted;

[0144] In each instance, R5 represents 1 to 2 substituents independently selected from the group consisting of amino, (C1-C6)alkylamino, di(C1-C6)alkylamino, hydroxy(C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, (C1-C6)heteroalkyl, (C3-C8)cycloalkyl, (C3-C8)cycloheteroalkyl, (C1-C6)alkoxy, (C1-C6)alkyl(CO)O-, (C1-C6)alkyl-O(CO)O- and (C1-C6)alkylthio; or R5 represents 2 adjacent substituents which, together with the attached carbon, form an optionally substituted ring or heterocycle.

[0145] 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.

[0146] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate or solvate thereof, is described, wherein A is (CH2)3.

[0147] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate or solvate thereof, is described, wherein R3 is hydrogen.

[0148] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate, or solvate thereof, is described, wherein R1 and R2 are independently hydrogen or C1-C6 alkyl.

[0149] In another exemplary embodiment, a compound of formula (I) or a salt, hydrate or solvate thereof is described, wherein R1 and R2 are independently hydrogen or CH3.

[0150] In another exemplary embodiment, a compound of formula (I) or a salt, hydrate or solvate thereof is described, wherein R1 and R2 are CH3.

[0151] In another exemplary embodiment, a compound of Formula (I), or a salt, hydrate, or solvate thereof, is described, wherein R1 and R2 together with the atoms to which they are attached form a ring.

[0152] In another exemplary embodiment, a compound of formula (I) or a salt, hydrate or solvate thereof is described, wherein R1 and R2 together form -CH2-.

[0153] In another exemplary embodiment, a compound of formula (I) or a salt, hydrate or solvate thereof is described, wherein R3 is hydrogen, (C1-C6)alkyl, (C1-C6)alkylthio or (C1-C6)alkoxy.

[0154] In another exemplary embodiment, a compound of formula (I), or a salt, hydrate or solvate thereof, is described, wherein R3 is hydrogen.

[0155] In another exemplary embodiment, a compound of formula (I) or a salt, hydrate or solvate thereof is described, wherein R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl and heterocyclylamino are each optionally substituted.

[0156] In another exemplary embodiment, a compound of formula (I) or a salt, hydrate or solvate thereof is described, wherein R4 is selected from the group consisting of:

[0157]

[0158] Where R O , R S , R N1 and R N2 is independently selected at each instance from hydrogen and optionally substituted (C1-C6)alkyl, or R N1 and R N2 Together with the attached nitrogen, an optionally substituted heterocyclic ring is formed.

[0159] In another exemplary embodiment, a compound of formula (I) or a salt, hydrate or solvate thereof is described, wherein R4 is selected from the group consisting of:

[0160]

[0161] In another exemplary embodiment, a compound of formula (I) or a salt, hydrate or solvate thereof is described, wherein R1 and R2 together form -CH2-, R3 is hydrogen, R5 represents 2-MeO and 3-MeO, A is (CH2)3, and R4 is selected from the group consisting of heteroaryl, heteroaryloxy, heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl, heterocyclylamino, amino, hydroxyl, halogen, cyano, alkylamino, dialkylamino, trialkylammonium, hydroxyalkylamino, bis(hydroxyalkyl)amino and hydroxyalkylaminoalkylamino, wherein heteroaryl, heteroaryloxy and heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl and heterocyclylamino are each optionally substituted.

[0162] In another exemplary embodiment, a compound of formula (I) or a salt, hydrate or solvate thereof is described, wherein R1 and R2 together form -CH2-, R3 is hydrogen, R5 represents 2-MeO and 3-MeO, A is (CH2)3, and R4 is selected from the group consisting of:

[0163]

[0164] In another exemplary embodiment, a compound of formula (I) or a salt, hydrate or solvate thereof is described, wherein R1 and R2 together form -CH2-, R3 is hydrogen, R5 represents 2-MeO and 3-MeO, A is (CH2)3, and R4 is selected from the group consisting of:

[0165]

[0166] In another exemplary embodiment, a compound of the following formula or a salt, hydrate or solvate thereof is described:

[0167]

[0168] In another exemplary embodiment, a compound of the following formula or a salt, hydrate or solvate thereof is described:

[0169]

[0170] In another exemplary embodiment, a compound of the following formula or a salt, hydrate or solvate thereof is described:

[0171]

[0172] In another exemplary embodiment, a compound of the following formula or a salt, hydrate or solvate thereof is described:

[0173]

[0174] In another exemplary embodiment, a compound of the following formula or a salt, hydrate or solvate thereof is described:

[0175]

[0176] In another exemplary embodiment, a compound of the following formula or a salt, hydrate or solvate thereof is described:

[0177]

[0178] In another exemplary embodiment, a compound of the following formula or a salt, hydrate or solvate thereof is described:

[0179]

[0180] In another exemplary embodiment, a compound of the following formula or a salt, hydrate or solvate thereof is described:

[0181]

[0182] It should be understood that the A, R1, R2, R3, R4, R5, R O , R S , R N1 and R N2 All possible combinations of different genera and subgenera of each represent additional exemplary embodiments of the compound. It is further understood that each of those additional exemplary embodiments of the compound can be used in any of the described compositions, methods, unit doses, kits and / or uses.

[0183] In an embodiment, the present disclosure provides a pharmaceutical composition comprising one or more compounds of formula (I) or salts, hydrates or solvates thereof. In an embodiment, the pharmaceutical composition comprises a therapeutically effective amount of one or more compounds for treating a host animal suffering from cancer. It should be understood that the composition may include other components and / or ingredients, including but not limited to other therapeutically active compounds, and / or one or more carriers, vehicles, diluents, adjuvants, excipients, etc. and combinations thereof.

[0184] In another embodiment, the unit dose of one or more compounds of formula (I) and pharmaceutical compositions comprising one or more compounds of formula (I) are also described herein. The unit dose includes a therapeutically effective amount of one or more compounds of formula (I) for treating a host animal suffering from cancer. The unit dose is in a single or separate form and may correspond to a daily dosage, or be adjusted to a shorter cycle amount (including multiple daily doses), or a longer cycle amount (including weekly or monthly doses). The composition may include other components and / or ingredients, including but not limited to other therapeutically active compounds and / or one or more carriers, vehicles, diluents, adjuvants, excipients, etc. and combinations thereof.

[0185] In another embodiment, a method for treating a host animal with cancer is also described, wherein the method comprises administering one or more compounds of formula (I) or salts, hydrates or solvates thereof, and / or compositions to the host animal with cancer. In another embodiment, the method comprises administering a therapeutically effective amount of one or more compounds of formula (I) and / or compositions to treat a host animal with cancer.

[0186] In another exemplary embodiment, a method for treating a host animal having cancer is described, wherein the method comprises administering to the host animal one or more compounds of formula (I) or a salt, hydrate or solvate thereof,

[0187]

[0188] wherein R1 and R2 are independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R1 and R2 together with the atoms to which they are attached form a 5- or 6-membered ring;

[0189] R3 is hydrogen, halogen, nitro, cyano, CF3, (C1-C6) alkyl, (C1-C6) alkylthio or (C1-C6) alkoxy;

[0190] A is an alkylene group;

[0191] R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxy, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl, and heterocyclylamino are each optionally substituted;

[0192] In each instance, R5 represents 1 to 2 substituents independently selected from the group consisting of amino, (C1-C6)alkylamino, di(C1-C6)alkylamino, hydroxy(C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, (C1-C6)heteroalkyl, (C3-C8)cycloalkyl, (C3-C8)cycloheteroalkyl, (C1-C6)alkoxy, (C1-C6)alkyl(CO)O-, (C1-C6)alkyl-O(CO)O- and (C1-C6)alkylthio; or R5 represents 2 adjacent substituents which, together with the attached carbon, form an optionally substituted ring or heterocycle.

[0193] 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 salts, hydrates or solvates thereof, or a composition comprising one or more compounds of formula (I) or salts, hydrates or solvates thereof, wherein

[0194] A is (CH2)n, wherein n is 1, 2 or 3;

[0195] R1 and R2 in each instance are each independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R1 and R2 together with the atoms to which they are attached form a 5- or 6-membered ring;

[0196] R3 is hydrogen, halogen, nitro, cyano, CF3, (C1-C6) alkyl, (C1-C6) alkylthio or (C1-C6) alkoxy;

[0197] R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxy, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl, and heterocyclylamino are each optionally substituted;

[0198] In each instance, R5 represents 1 to 2 substituents independently selected from the group consisting of amino, (C1-C6)alkylamino, di(C1-C6)alkylamino, hydroxy(C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, (C1-C6)heteroalkyl, (C3-C8)cycloalkyl, (C3-C8)cycloheteroalkyl, (C1-C6)alkoxy, (C1-C6)alkyl(CO)O-, (C1-C6)alkyl-O(CO)O- and (C1-C6)alkylthio; or R5 represents 2 adjacent substituents which, together with the attached carbon, form an optionally substituted ring or heterocycle.

[0199] 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 (CH2)n, wherein n is an integer from 1 to 3, or n is an integer from 2 to 3, or n is 3.

[0200] 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 R1 and R2 are independently hydrogen or C1-C6 alkyl.

[0201] 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 R1 and R2 are independently hydrogen or CH3.

[0202] 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 salts, hydrates or solvates thereof, or a composition comprising one or more compounds of formula (I) or salts, hydrates or solvates thereof, wherein R1 and R2 are CH3.

[0203] 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 R1 and R2 together with the atoms to which they are attached form a ring.

[0204] 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 R1 and R2 together form -CH2-.

[0205] 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 R3 is hydrogen, (C1-C6) alkyl, (C1-C6) alkylthio or (C1-C6) alkoxy.

[0206] 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 R3 is hydrogen.

[0207] 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 salts, hydrates or solvates thereof, or a composition comprising one or more compounds of formula (I) or salts, hydrates or solvates thereof, wherein R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl and heterocyclylamino are each optionally substituted.

[0208] 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 R4 is selected from the group consisting of:

[0209]

[0210] In each instance, R O , R S , R N1 and R N2 are independently selected from hydrogen and optionally substituted (C1-C6)alkyl, or R N1 and R N2 Together with the attached nitrogen, an optionally substituted heterocyclic ring is formed.

[0211] 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 R4 is selected from the group consisting of:

[0212]

[0213] In another exemplary embodiment, a method for treating a host animal suffering from cancer comprises administering to the host animal one or more compounds of formula (I) or salts, hydrates or solvates thereof, or a composition comprising one or more compounds of formula (I) or salts, hydrates or solvates thereof, wherein R1 and R2 together form -CH2-, R3 is hydrogen, R5 represents 2-MeO and 3-MeO, A is (CH2)3, and R4 is selected from the group consisting of heteroaryl, heteroaryloxy, heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl, heterocyclylamino, amino, hydroxyl, halogen, cyano, alkylamino, dialkylamino, trialkylammonium, hydroxyalkylamino, bis(hydroxyalkyl)amino and hydroxyalkylaminoalkylamino, wherein heteroaryl, heteroaryloxy and heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl and heterocyclylamino are each optionally substituted.

[0214] 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 R1 and R2 together form -CH2-, R3 is hydrogen, R5 represents 2-MeO and 3-MeO, A is (CH2)3, and R4 is selected from the group consisting of:

[0215]

[0216] 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 R1 and R2 together form -CH2-, R3 is hydrogen, R5 represents 2-MeO and 3-MeO, A is (CH2)3, and R4 is selected from the group consisting of:

[0217]

[0218] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G101):

[0219]

[0220] or a salt, hydrate or solvate thereof, or a composition comprising the compound or a salt, hydrate or solvate thereof.

[0221] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G102):

[0222]

[0223] or a salt, hydrate or solvate thereof, or a composition comprising the compound or a salt, hydrate or solvate thereof.

[0224] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G103):

[0225]

[0226] or a salt, hydrate or solvate thereof, or a composition comprising the compound or a salt, hydrate or solvate thereof.

[0227] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G104):

[0228]

[0229] or a salt, hydrate or solvate thereof, or a composition comprising the compound or a salt, hydrate or solvate thereof.

[0230] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G105):

[0231]

[0232] or a salt, hydrate or solvate thereof, or a composition comprising the compound or a salt, hydrate or solvate thereof.

[0233] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G108):

[0234]

[0235] or a salt, hydrate or solvate thereof, or a composition comprising the compound or a salt, hydrate or solvate thereof.

[0236] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G106):

[0237]

[0238] or a salt, hydrate or solvate thereof, or a composition comprising the compound or a salt, hydrate or solvate thereof.

[0239] In another exemplary embodiment, a method for treating a host animal having cancer comprises administering to the host animal the following compound (G107):

[0240]

[0241] or a salt, hydrate or solvate thereof, or a composition comprising the compound or a salt, hydrate or solvate thereof.

[0242] In another embodiment, the use of the compounds and compositions in the preparation of a medicament for treating a host animal with 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 compositions comprising the same, for treating a host animal with cancer.

[0243] The compounds, compositions, unit doses and methods can be used alone or in combination with other compounds for treating cancer, including those compounds that may be therapeutically effective through the same or different modes of action. In addition, the compounds can be used in combination with other compounds that are administered to treat other symptoms of cancer (such as compounds administered to cancer, etc.).

[0244] Topoisomerase I inhibitors are classified as either inhibitors (which inhibit the enzyme-catalyzed DNA cleavage reaction) or poisons (which inhibit the enzyme-catalyzed DNA religation reaction (Scheme 1) after cleavage has occurred). Since the discovery of indenoisoquinoline NSC314622 (1) 1 Acts as a topoisomerase I poison by inserting between base pairs at the cleavage site 2 Since then, several additional biomacromolecules have been found to be targeted by various substituted indenoisoquinolines, including topoisomerase II 3-7 , Retinoid X receptor (RXR) 8-10 、PARP-1 11 , estrogen receptor 12 , tyrosyl-DNA phosphodiesterase 1 (TDP1) 13-17 and tyrosyl-DNA phosphodiesterase 2 (TDP2) 13 Recently, a series of indenoisoquinolines were reported to stabilize the G-quadruplex in the MYC promoter and downregulate MYC protein expression through a mechanism involving stacking of indenoisoquinolines with the outer quadruplex ends of the G-quadruplex. 18 Three indenoisoquinoline topoisomerase 1 poisons, LMP400 (indotecan, 2) 19 、LMP776 (indoline, 3) 19 and LMP744(4) 20Phase 1 clinical trial has been successfully completed or is about to be completed 21 , and notably, all three drugs were also found to be MYC promoter G-quadruplex stabilizers and downregulate MYC protein expression 18 In addition, indenoisoquinolines can stabilize G-quadruplexes through two mechanisms: (1) inhibition of topoisomerase I to maintain the negative DNA supercoil associated with the G-quadruplex, and (2) stacking with the ends of the two outer G-quadruplex quadruplexes. 18 In fact, the two activities (topoisomerase I inhibition and G-quadruplex stabilization) have been found to act synergistically in cancer cell cytotoxicity 18 This article describes the results of studies aimed at optimizing indenoisoquinolines against two targets in an attempt to maximize their anticancer activity.

[0245] Solution 1

[0246]

[0247]

[0248] Previous attempts to optimize the anticancer activity of indenoisoquinolines involved implanting single nitrogen atoms at all possible positions of the two aromatic rings. More specifically, the carbons at positions 1-4 and 7-10 were each replaced individually with nitrogen atoms, one at a time. 13,22-24 The principle is that the electronegative nitrogen promotes π–π stacking of the azaindenoisoquinoline with adjacent base pairs in the ternary drug-DNA-topoisomerase I cleavage complex by promoting the formation of a charge transfer complex involving a base pair as an electron donor and an azaindenoisoquinoline as an electron acceptor. These studies showed that the 7-aza analogues were the most cytotoxic in cancer cell cultures, especially when combined with a methoxy substituent at position 9 and a nitro, fluorine, or chlorine substituent at position 3. 13,23-25 Quantum mechanical calculations of the π–π stacking model of 7-, 8-, 9-, and 10-azaindenoisoquinolines with adjacent DNA base pairs appear to confirm that electronegative nitrogen atoms do in fact increase charge transfer complex formation. However, this effect appears to be counteracted in 8-, 9-, and 10-azaindenoisoquinolines (but not 7-azaindenoisoquinoline) because the contribution of the distribution (electron correlations) to the π–π stacking is reduced. 23 Furthermore, results from previous studies of indenoisoquinolines lacking substituents on the aromatic ring suggest that the 2,3-methoxy and 8,9-methylenedioxy substituents are not absolutely necessary for biological activity, but they do appear to contribute consistently to anticancer activity. 26 .

[0249] Chemical

[0250] In the embodiments, processes for preparing compounds of formula (I) are described.

[0251] It was found that the known routes for similar 2,3-methoxy and 8,9-methylenedioxy-indenoisoquinolines were unsuccessful in providing the target compounds. For example, the following compound could not be cyclized under the known conditions.

[0252]

[0253] The route for preparing 9-MeO-7-aza-indenoisoquinoline requires the preparation of the following compound (WO2018 / 118852A1):

[0254]

[0255] The route to this intermediate based on known chemical reactions failed. The unexpected ring bromination shown below was the key step in the preparation of the desired intermediate:

[0256]

[0257] The processes described herein for preparing compounds of formula (I) overcome the obstacles presented by previously known synthetic routes to 7-azaindenoisoquinolines.

[0258] Exemplary processes include the following:

[0259]

[0260] In another embodiment, provided are compounds prepared by one or more of the processes described herein.

[0261] The target compounds were synthesized using two different routes, depending on whether the 3-bromopropyl chain in the relevant intermediate was attached to a basic moiety in the side chain (16, Scheme 2) or to a 7-azaindenoisoquinoline (20, Scheme 3). As outlined in Scheme 2, treatment of furfural (5) with aqueous bromine under acidic conditions followed by addition of sulfonic acid (6) to the reaction mixture afforded 5-bromopyridine-2,3-diol (7), which was converted to intermediate 8 in low (12%) yield using dibromomethane in the presence of potassium carbonate and copper oxide. 27,28 Interestingly, the experimental details of the synthesis of 8 from 7 have never been reported, although the claimed transformation was repeated with a 12% yield as described in the paper. 28 The bromide of 8 was replaced by a methyl group using Suzuki-Miyaura cross-coupling to give the desired methylated species 9. 29 Surprisingly, it was found that regioselective bromination of 5-methylpyridine 9 on the aromatic ring with NBS in AcOH / CH3CH at room temperature afforded bromopyridine derivative 10 30-32, which then underwent nucleophilic aromatic substitution with CuCN in refluxing DMF to produce intermediate 11. Free radical bromination of the methyl group of 11 with NBS in the presence of perbenzoic acid in refluxing carbon tetrachloride produced the desired primary bromide 12. Condensation of 12 with 6,7-dimethoxyhomophthalic anhydride in the presence of triethylamine in refluxing acetonitrile gave 7-azaindenoisoquinoline 14. 33 , which was oxidized with selenium dioxide to generate the penultimate intermediate 15 23 Deprotonation of 15 with sodium hydride followed by alkylation of the resulting anion with the appropriate bromide 16 afforded the target compounds 17, 18, and 19.

[0262] Scheme 2. Synthesis of 7-aza-8,9-methylenedioxindenoisoquinoline derivatives a

[0263]

[0264] a Reagents and conditions: (a) (1) Br2, ice, 0°C, 30 min; (2) HCl, 0°C, 30 min; (3) Br2, -5 to 0°C, 1 h; (4) 6, 50°C, 3 h. (b) CH2Br2, K2CO3, CuO, DMF, 90°C, 14 h. (c) (HO)2BCH3, Na2CO3, RuPHOS, Pd2dba3, toluene, H2O, 110°C, 12 h. (d) NBS, acetic acid, H3CCN, room temperature, 3 h. (e) CuCN, DMF, toluene, 110°C, 18 h. (f) NBS, PhCOOOH, CCl4, reflux, 16 h. (g) 13, H3CCN, Et3N, reflux, 15 h. (h) SeO2, dioxane, acetic acid, reflux, 11 h. (i)(1) For 17: (1) NaH, KI, DMF, 5°C, 1 h; (2) 4-(3-bromopropyl)morpholine hydrobromide, room temperature, 12 h; For 18: (1) 1-(3-bromopropyl)-1H-imidazole, NaH, DMF, room temperature, 3 h; For 19: (1) NaH, KI, (2) 3-bromo-N-isopropylpropan-1-amine hydrobromide, DMF, room temperature, 3 h.

[0265] The first step of the alternative synthesis outlined in Scheme 3 involves deprotonation of intermediate 15 with sodium hydride followed by reaction with 1,3-dibromopropane in the presence of potassium iodide in DMF, leading to 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 appropriate amines in DMF at various times and temperatures using DIPEA as a base. 19 , as indicated in the legend.

[0266] Scheme 3. Synthesis of novel 7-aza-8,9-methylenedioxindenoisoquinoline derivatives a

[0267]

[0268] a Reagents and conditions: (a) NaH, KI, DMF, room temperature, 2h; (3) 1,3-dibromopropane, room temperature, 20h. (b) For 17: DIPEA, morpholine, DMF, room temperature, 12h, then 50°C, 12h; For 18: DIPEA, 1H-imidazole, DMF, room temperature, 16h, then 70°C for 18h; For 19: DIPEA, isopropylamine, DMF, room temperature, 12h; For 21: DIPEA, ethylamine, DMF, room temperature, 24h; For 22: DIPEA, ethanolamine, DMF, room temperature, overnight; For 23: DIPEA, pyrrolidine, DMF, room temperature, 16h; For 24: DIPEA, (S)-pyrrolidine-2-carboxamide, DMF, room temperature, 12h; For 25: DIPEA, (2S)-2-pyrrolidinemethanol, DMF, room temperature, 12h.

[0269] Biological Results and Discussion.

[0270] 1. In vitro binding to c-Myc promoter G-quadruplex (MycG4).

[0271] MycG4 stability was measured by FRET melting. Resonance resonance energy transfer (FRET)-melting assay to determine whether 7-aza-8,9-methylenedioxindenoisoquinoline derivatives can bind to and stabilize the c-Myc promoter G-quadruplex (MycG4, Figure 1 ). MycG4 DNA is labeled with a FRET donor (6-FAM) at the 5'-end and a FRET acceptor (TAMRA) at the 3'-end. The close proximity of the donor and acceptor fluorophores in the G-quadruplex secondary structure quenches the 6-FAM fluorescence due to FRET transfer to TAMRA. Melting of the secondary structure into single-stranded DNA reduces the proximity of the FRET pair, thereby restoring 6-FAM-based fluorescence emission. Monitoring the 6-FAM emission during thermal denaturation of the G-quadruplex provides a melting curve, from which the melting temperature (T m ) (temperature at which folded and unfolded DNA are equally populated). By FRET-melting assay, the + Measuring the T of MycG4 in the presence of the compounds mAll eight 7-aza-8,9-methylenedioxindoleisoquinolines tested showed significant MycG4 thermal stability (ΔT m )(Table 1 and Figure 2 ), where the melting temperature increased by more than 10°C. Among them, G103, G104, G105, and G108 showed the highest MycG4 thermal stability (ΔT m ). Figure 3 Data for reference LMP compounds are provided, and the structure of reference compound 26 (LMP517) is shown below.

[0272] Table 1. Increase in melting temperature ΔT of MycG4 G-quadruplex in the presence of 7-aza-8,9-methylenedioxindenoisoquinoline determined by FRET-based melting experiments m .

[0273]

[0274] a Conditions: 10 mM K + 0.2 μM 5'-6-FAM / 3'-TAMRA-MycG4 DNA, 2.0 μM (10 equivalents) ligand in buffer (pH 7.0).

[0275] Binding affinity to Myc G-quadruplex. The binding affinities of eight 7-aza-8,9-methylenedioxindole isoquinolines to MycG4 were determined using a fluorescence binding assay using 3'-TAMRA-labeled MycG4 DNA. The binding isotherms were monitored after drug titration to derive the apparent binding affinity, K d,app Six 7-aza-8,9-methylenedioxindenoisoquinolines (G-103 to G-108) showed strong binding, with apparent binding affinities K d,app The values ​​were less than 100 nM (Table 2). Indenoisoquinoline showed negligible fluorescence in both free and bound states. Table 3 provides the data for reference LMP compounds.

[0276] Table 2. Apparent binding affinity of 7-aza-8,9-methylenedioxindole to 3'-TAMRA-labeled MycG4 DNA, K d .

[0277]

[0278] a Conditions: 20°C, 100 mM K + Buffer, pH 7.0.

[0279] Table 3. Data of reference compounds. Results are shown in Table 3. Apparent binding affinity of LMP-compounds to 3'-TAMRA-labeled MycG4 DNA, K d .

[0280]

[0281]

[0282] a Conditions: 20°C, 100 mM K + Buffer, pH 7.0.

[0283] Binding interaction with MycG4 by NMR. 1 H NMR titration in K-containing + The binding interaction between 7-aza-8,9-methylenedioxindanoisoquinoline and MycG4 was examined in solution. Unbound Myc G-quadruplexes showed twelve guanine imino protons, with four protons in each of the three quadruplexes ( Figure 4 ). Changes in line width and chemical shift of the imino proton resonances upon compound addition indicate binding to the G-quadruplex. All 7-aza compounds except G101 appear to bind to MycG4 DNA at both ends, as indicated by the distinct shifted imino proton peaks corresponding to the 3'- and 5'-quadruplexes upon drug addition. Binding appears to be at a moderate to fast exchange rate on the NMR time scale, as indicated by the broadening of the DNA proton peaks upon drug addition.

[0284] 2. In vivo Myc inhibition

[0285] Western blot. The G-quadruplex formed in the MYC promoter functions as a transcriptional silencer. To determine the effect of 7-aza-8,9-methylenedioxindoisoquinoline on MYC protein levels, Western blot experiments were performed using MCF-7 breast cancer cells. Western blot experiments were performed to examine the downregulatory effect of 7-aza-8,9-methylenedioxindoisoquinoline on Myc protein levels ( Figure 5 ). MCF-7 breast cancer cells were incubated in 0, 0.5, 1.0 and 2.0 μM of G-101-108 for 24 h. G101-G105 showed strong Myc inhibition effect. For G106-G108, only G107 seemed to show moderate Myc inhibition, while G106 and G108 showed weak Myc downregulation.

[0286] qRT-PCR. To confirm the effects of the selected 7-aza-8,9-methylenedioxindole isoquinolines G103 and G105 on the transcription of the MYC gene in cancer cells, the MYC mRNA level in MCF-7 breast cancer cells was measured by qRT-PCR ( Figure 6 MCF-7 cells were incubated with 0 and 1.0 μM of G103 and G105 for 6 h. Consistent with the Western blot data, G103 and G105 significantly reduced MYC mRNA levels 6 h after treatment with 1.0 μM of the drugs. Figure 7 Data for reference LMP compounds are provided.

[0287] 3. In vivo cytotoxicity results in cancer cell lines

[0288] Single-dose cell viability assay in CA-46 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. While the CA-46 translocation eliminates the G-quadruplex-forming region in one allele, the Raji translocation preserves the G-quadruplex-forming region in both alleles. Therefore, Raji is more sensitive to G-quadruplex-mediated Myc inhibition than CA-46, which is reflected in the IC of Raji. 50 To evaluate cell line selectivity, an MTS single-dose assay was performed to compare the antiproliferative effects of 7-aza-8,9-methylenedioxindole on CA-46 and Raji cells at a drug concentration of 250 nM ( Figure 8 ). The mean % survival values ​​for CA-46 and Raji cells for each drug are presented side by side in a bar graph. At 250 nM of G101–G108, G101 and G106–G108 showed no growth inhibition in either cell line. In contrast, G102-G105 showed significant antiproliferative effects on both cell lines, and the % survival of CA46 was statistically significantly higher than that of Raji. Fig. 9 Data for reference LMP compounds are provided.

[0289] Cytotoxicity results in Raji non-Hodgkin lymphoma cells. MTS experiments were then performed to test the antiproliferative effects of 7-aza-8,9-methylenedioxindanoisoquinolines in Raji cells. Cells were incubated in 2-2000 nM of each drug for 72 h, and cell viability was quantified using the MTS assay. Table 4 shows the measured IC values ​​for all compounds. 50 Values. Among all compounds, G102-G105 were significantly more potent than G106-G108. This potency trend generally correlates with the western blot results, where G106 and G108 did not show weak Myc downregulation. Table 5 provides data for reference LMP compounds.

[0290] Table 4. IC of 7-aza-8,9-methylenedioxindole isoquinoline in RAJI lymphoma cells 50 (nM) value.

[0291]

[0292] Table 5. Reference compound results for the data in Table 4. IC of non-7-aza-8,9-methylenedioxindenoisoquinolines in RAJI lymphoma cell line 50 (nM) value.

[0293]

[0294] Cytotoxicity results in Myc-dependent cancer cell lines. 50 Given the high potency of the G101-G105 compounds demonstrated in the assay, these compounds were further tested in a selected panel of Myc-dependent / overexpressing cancer cell lines, such as CA-46 and Raji MTS IC 50 In the assay, each cell line received 2-2000 nM of each compound for 72 h.

[0295] Among the 7-aza-8,9-methylenedioxindoleisoquinolines (Table 6), G103-G105 was generally more potent than G101-G102 across all cell lines. Among all cell lines, MOLT-4, RS4;11, and CHLA-99, as well as RD-ES, were very sensitive to G103-G105, with GI values ​​approaching nM. 50 Table 7 provides data for reference LMP compounds.

[0296] Table 6. GI of 7-aza-8,9-methylenedioxindoisoquinolines in a selected panel of Myc-dependent / overexpressing cancer cell lines 50 (nM) value.

[0297]

[0298]

[0299] Table 7. Reference compound results for the data in Table 6. LMP and Camptothecin GI in a Selected Panel of Myc-Dependent / Overexpressing Cancer Cell Lines 50 (nM) value.

[0300]

[0301]

[0302] 5. Inhibition of topoisomerase I

[0303] To examine the inhibitory effects of 7-aza-8,9-methylenedioxindoleisoquinoline compounds on Topo1-mediated DNA relaxation ( Fig.10A), and the toxic effect of Topo1 on DNA cleavage using Topo1 ( Fig.10 B).

[0304] Topo1-mediated DNA relaxation assay. The inhibitory effect of compounds on Topo1-mediated DNA relaxation was examined using the human topoisomerase I assay kit (TG1015-1A) from Topogen. The assay was performed according to the manufacturer's instructions. The typical assay sample total volume was 20 μl, including 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 10% SDS termination of the reaction. This was followed by proteinase K digestion at 37°C for 15 min to degrade Topo1, and 1:1 volume chloroform:isoamyl alcohol DNA extraction. The samples were then electrophoresed on a 1% agarose gel in TBE buffer at 46 v for 4 h. Before imaging, the gel was stained with 0.5 μg / ml ethidium bromide solution for 15 min and washed 3 times with Milli-Q water with an interval of 5 min. The results are shown in Fig.10 As shown in A.

[0305] Topo1-mediated DNA cleavage assay. The toxic effects of compounds on Topo1 were examined using the human topoisomerase I assay kit (TG1015-1A) from Topogen. The assay was performed according to the manufacturer's instructions. 10 μM CPT was used as a positive control. The typical assay sample total volume was 20 μl, including 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 10% SDS termination of the reaction. This was followed by proteinase K digestion at 37°C for 15 min to degrade Topo1, and 1:1 volume chloroform:isoamyl alcohol DNA extraction. 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 of ethidium bromide. The results are shown in Fig.10 As shown in B.

[0306] like Fig.10 As shown in the data in A, no compound inhibited the DNA cleavage reaction catalyzed by Top1. In contrast, compounds G103-G107 inhibited the DNA degradation reaction, while G101, G102 and G108 had the least effect on DNA degradation, as shown in Fig.10 B. In other words, G103-G107 act as Top1 poisons rather than Top1 inhibitors, with G105-G107 being the most effective. This is consistent with the Top1 inhibitory activity of indenoisoquinolines with 7 carbon atoms.

[0307] 6. Pharmacokinetic results

[0308] 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. After IV administration, the half-life of the drug was 3.29h for 19, 2.73h for 21, and 1.84h for 22. After oral administration, the half-life was 6.88h for 19, 6.91h for 21, and 5.20h for 22. There were significant differences in the dose fraction absorbed after oral administration, with 41% of the dose of 19 absorbed, 14.2% of the dose of 21 absorbed, and 1.19% of the dose of 22 absorbed. The absorption percentage was roughly consistent with the CLogP value, which was 1.67 for 19, 1.36 for 21, and 0.363 for 22, indicating that higher absorption of more lipophilic compounds is expected from the gastrointestinal tract.

[0309] Table 8. IV pharmacokinetic parameters of compounds 19 (G103), 21 (G104), and 22 (G105) in male CD1 mice (dose = 1 mg / kg).

[0310]

[0311] Table 9. PO pharmacokinetic parameters of compounds 19 (G103), 21 (G104) and 22 (G105) in male CD1 mice (dose = 10 mg / kg).

[0312]

[0313] Plasma protein binding and stability analysis. The protein binding percentage and stability of compounds 19, 21 and 22 were determined in both human and mouse plasma. The results are listed in Table 10. The protein binding was ranked according to the lipophilicity of the compound, with compound 19 having the highest protein binding and compound 22 having the lowest protein binding in both human and mouse plasma. The free drug (unbound) concentrations of all three compounds were sufficient to exert pharmacological effects, and all three compounds were also relatively stable in plasma.

[0314] Table 10. Protein binding results of compounds 19, 21, 22 and control compounds in human and mouse plasma.

[0315]

[0316]

[0317] Stability analysis in human and mouse liver microsomes. The stability of compounds 19, 21 and 22 was determined in the presence of human and mouse liver microsomes, and the results are listed in Table 11. All three compounds showed strong microsomal stability, consistent with their stability in male CD1 mice (Table 11). The half-life for human microsomes ranged from 227-372 min, with the order of stability being 19>21>22. Interestingly, both compounds 19 and 21 were more stable for human microsomes than mouse microsomes, but for compound 22, the order was reversed, with mouse microsomes having higher stability than human microsomes. Although 22 was observed to have a longer half-life in mouse microsomes than in human microsomes and a longer in vitro CL int smaller, but because the scaling factor for mice is much larger than that for humans, the magnified CL calculated for mice is int larger (Table 20a, Experimental part).

[0318] Table 11. Metabolic stability of test compounds in pooled human and male mouse liver microsomes.

[0319]

[0320] Caco-2 cell permeability study. Caco-2 permeability studies were performed on compounds 19, 21 and 22, and the results are listed in Table 12. All three compounds showed high efflux rates in Caco-2 cells, indicating that all three compounds should be substrates for efflux transporters (P-gp, BCRP and / or MRP2). All three compounds showed moderate permeability in the apical to basolateral direction (i.e., the direction of intestinal absorption).

[0321] Table 12. Permeability results of test compounds in Caco-2 cells

[0322]

[0323]

[0324] In summary, compound 19 (G103) had the longest IV half-life, the highest % absorbed PO, the highest stability to human liver microsomes, and the lowest efflux rate in Caco-2 cells. Compound 21 (G104) had the best cytotoxicity to CA46 and Raji cells, and had excellent selectivity for Raji cells, which was comparable to 22 (G105).

[0325] Maximum tolerated dose determination. The maximum tolerated dose (MTD) of G103, G104 and G105 was determined in NOD SCID mice, and the results are shown in Tables 13 and Fig.11 and 12The MTD of G103 was 50 mg / kg, while that of G104 and G105 was 30 mg / kg and 20 mg / kg, respectively. Experiments involving 100 mg / kg doses of all three drugs were terminated after one day due to toxicity, and studies involving 50 mg / kg doses of G105 were also stopped after one day ( Fig.11 Likewise, the investigational study involving a 40 mg / kg dose of G105 was discontinued after two days ( Fig.12 The large difference in MTD between G103 and G104 (50 vs. 30 mg / kg) was unexpected because of the small change in chemical structure (one methyl vs. hydrogen in the side chain).

[0326] Table 13. Determination of Maximum Tolerated Dose (MTD)

[0327]

[0328]

[0329] a IV, QD x 5. b Based on 4 mice per group, the mice were observed 7 days after the first administration.

[0330] The average percentage of initial body weight observed. c The maximum tolerated dose,

[0331] The tolerated dose was defined by ≥80% of body weight.

[0332] Xenograft studies. Female NOD SCID mice were inoculated with MOLT-4 human leukemia cells, RD-ES human sarcoma cells, and RS4;11 human leukemia cells to investigate the efficacy of G103, G014, and G105. JQ1 was used as a positive control. Tumors reached approximately 100-150 mm at 10 days post-inoculation. 3 Treatment was started after the size of the mice reached 1.5. The drug was administered by 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. Fig.13 As shown, the drug was generally well tolerated as judged by body weight loss, with the greatest loss observed after administration of a 20 mg / kg dose of G105 (mean animal weight was 80.9±1.1% relative to initial body weight 24 days after tumor inoculation).

[0333] Fig.14A graph showing the change in tumor size over time in female NOD SCID mice inoculated with MOLT-4 human leukemia cells and treated IV with various doses of G103, G104, and G105, as well as IP administration of the positive control JQ1. Each "G compound" was administered at higher and lower doses, as Fig.13 The data revealed the relative efficacy 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 drug reduced the rate of increase in tumor size relative to the control.

[0334] Similar studies were performed in female NOD SCID mice inoculated with RD-ES human sarcoma tumor cells ( Fig.15 and 16 ). Tumors grew much faster and treatment was initiated on day 8 after tumor inoculation, with body weight and tumor size measured on days 8, 12, 15, 19, and 22 after tumor inoculation. The most effective treatments were the same as MOLT-4, with G105 at a dose of 20 mg being the most effective. However, the overall order of efficacy was different from that described 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 size of RD-ES tumors was much larger than that observed with MOLT-4 (2327 ± 285 vs. 546 ± 59 mm 3 ).

[0335] The compounds were also evaluated in the RS4;11 human leukemia xenograft model ( Fig.17 and 18 Treatment was initiated 12 days after tumor inoculation, with drug administration on days 1-5 and 11-15 after treatment initiation. Animal body weights and tumor volumes were determined on days 12, 15, 19, 22, and 25 after tumor inoculation. As in the other cases, treatment with G105 at the 20 mg / kg dose level demonstrated the greatest toxicity, as assessed by animal weight loss ( Fig.16 ), and it was also most effective in reducing tumor volume ( Fig.17). The relative efficacy of the various treatments for RS4;11 was significantly 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). In summary, the various treatments had low to moderate efficacy in the RS4;11 xenograft model, with tumor volumes following G104 treatment at both dose levels (15 and 30 mg / kg) approaching those of untreated animals observed at the 25-day time point.

[0336] Brain penetration studies. One possible application of indenoisoquinolines is the treatment of malignant brain tumors. Previous studies have demonstrated CNS penetration via LMP400 (2), suggesting that G103, G104, and G105 may also be able to penetrate the blood-brain barrier. 40 . Therefore, after IV administration to CD1 mice, the concentrations of these compounds and LMP744 (4) in plasma and brain were monitored by LC-MS / MS, and brain / plasma concentrations were calculated (Table 14a). These four compounds were tested by IV administration of 20 mg / kg doses to four groups of twenty-four male CD1 mice, three animals were used at each time point, and a total of ninety-six animals were used to generate the data in Table 14a, and plasma and brain tissue samples were collected from each mouse. The concentration was determined by directly measuring the plasma samples by LC-MS / MS. The brain tissue was homogenized with water using a 3:1 ratio of water (mL) and brain (g), and the concentration in the brain homogenate (ng / mL) was then determined, and the homogenate value was multiplied by a factor of 4 to obtain the brain concentration (ng / g). Then, based on the fact that 1cc of brain weighs 1.03g, the ng / g concentration value in the brain was converted to nM concentration in the brain using a conversion factor of 1.03. 41. The initial brain concentration is proportional to the lipophilicity of the side chain, while the initial plasma concentration is inversely proportional to the lipophilicity of the side chain, reflecting the faster initial partitioning of compounds with more lipophilic side chains from plasma to the brain. The highest initial brain concentration was achieved with G103, with an average brain / plasma ratio of 7.97 observed 15 min after administration, and a 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, which produced a ratio of 1.88 at the 15 min time point. Lower initial brain concentrations and ratios were observed with G105 and LMP744. Although low concentrations of compounds were measured in plasma and brain at the 24-hour time point, high brain / plasma ratios were calculated, with higher values ​​observed for compounds with the lowest initial ratios. This reflects the fact that brain concentrations of G105 and LMP744 were relatively low but surprisingly stable over the entire 24-hour period, whereas G103 and G104 were associated with "normal" brain pharmacokinetics, with concentrations of G103 and G104 decreasing as expected over time. On the other hand, plasma concentrations of all compounds decreased as a function of time as expected.

[0337] A similar analysis was performed with the combination of LMP744 and LMP400 with Temodar and Olaparib. In Table 14b, brain concentration, plasma concentration and brain / plasma ratio are listed when LMP744 and LMP400 are administered in combination with 100mg / kg PO Olaparib or 125mg / kg Temodar. Obviously, LMP400 can penetrate the blood-brain barrier more effectively than LMP744, as demonstrated by the much higher brain concentration and brain / plasma ratio of LMP400 relative to LMP744. The lower permeability of the compound (i.e., LMP744) with a hydroxyethylaminopropyl side chain recorded in Table 14b is consistent with the results provided in Table 14a (which records the lower levels of LMP744 and G105 relative to G103 and G104). It is worth noting that Olaparib seems to promote LMP400 to penetrate into the brain relative to Temodar, because after applying Olaparib at all time points, the brain concentration of LMP is consistently higher. Finally, the "flat" concentration levels of LMP744 shown in Table 14b as a function of time are also consistent with the results for LMP744 and G105 shown in Table 14a, indicating that the hydroxyethylaminopropyl side chain appears to maintain relatively low levels of these drugs in the brain over long periods of time (5 min to 24 h after administration). This is in contrast to LMP400, which showed a "normal" decline in concentration as a function of time.

[0338] The concentrations of tamodar and olaparib were also tracked during these experiments, as shown in Table 14c. Tamodar is able to enter the brain after oral administration, while olaparib is very inefficiently distributed into the brain after oral administration. Based on these results, IV use of LMP400 in combination with oral administration of tamodar may be effective, but combination with olaparib does not seem promising for the treatment of CNS cancers.

[0339] The pharmacokinetics observed during the plasma and brain monitoring are detailed in Tables 15a and 16a. G103 and G104 rapidly appeared in brain tissue after IV administration (T max The effective plasma half-life of G103 could not be calculated because the AUC _％Extrap_ The plasma half-lives of G104 and G105 observed after the 20 mg / kg dose (Table 15a) were longer than those seen after the 1 mg / kg dose (Table 8), although they were of the same order (G104 T 1 / 2 >G105 T 1 / 2 ). It is impossible to calculate effective brain T 1 / 2 , AUC Inf and MRT Inf_ obs parameter, because the AUC of G103, G104, G105 and LMP744 in the brain _％Extrap_ The obs values ​​were all far above 20% (Table 16b). The initial brain concentration of G103 was significantly higher than that of G104 (C max 3809 vs. 1613 ng / g, respectively, Table 16a), which reflects the greater lipophilicity of G103 relative to G104 (cLog P value = 1.674 vs. 1.365, respectively).

[0340] The plasma and brain pharmacokinetic parameters for LMP400 and LMP744 after combined administration with olaparib and termodar are shown in Tables 15b and 16b, respectively. LMP400 appears rapidly in the brain, as shown by a T of 5 min. max On the other hand, the T max In the presence of olaparib, it was 1 h, and in the presence of temoda, it was 2 h (Table 16b), both of which were delayed relative to the 0.25 h observed when LMP744 was administered alone (Table 16a). As expected, brain C of LMP400 was significantly increased regardless of co-administration of temoda or olaparib. max The values ​​of both groups were significantly higher than those of LMP744, and neither tamoda nor olaparib affected the C max (Compare the values ​​in Tables 16b and 16a).

[0341] It should be noted that when LMP744 was co-administered with olaparib relative to temoda, the plasma C0 AUC for LMP744 was 最后 and AUC Inf The values ​​were different (Table 15b). LMP744 has a much larger volume of distribution at steady state than LMP400. There was no significant effect on the half-life and volume of distribution at steady state by administration of olaparib relative to tamoda.

[0342]

[0343]

[0344] Table 15a. Plasma pharmacokinetic parameters observed after IV administration of 20 mg / kg of G103, G104, G105, and LMP744 to male CD1 mice a

[0345]

[0346] a PK parameters were estimated by non-compartmental modeling using WinNonlin software. b It is impossible to calculate meaningful Cl_obs and T for G103 1 / 2、 AUC Inf , MRT Inf_ obs and V ss_ obs parameter, because AUC _％Extrap_ obs is greater than 20%.

[0347] Table 15b. Plasma pharmacokinetic parameters of LMP400 and LMP744 observed after IV administration of 10 mg / kg of LMP400 and 10 mg / kg or 20 mg / kg of LMP744 in combination with PO administration of 100 mg / lg of olaparib or 125 mg / kg of temoda to mice a

[0348]

[0349] a PK parameters were estimated by non-compartmental modeling using WinNonlin software. b LMP400 10 mg / kg was administered IV plus olaparib 100 mg / kg was administered PO. c LMP400 at 10 mg / kg was administered IV plus Temmodar at 125 mg / kg was administered PO. d LMP744 was administered IV at 10 mg / kg plus olaparib was administered PO at 100 mg / kg. e20 mg / kg of LMP744 plus 125 mg / kg of Temoda were administered IV.

[0350] Table 16a. Brain pharmacokinetic parameters observed after IV administration of 20 mg / kg of G103, G104, G105, and LMP744 to male CD1 mice a

[0351]

[0352] a PK parameters were estimated by noncompartmental modeling using WinNonlin software. It was not possible to calculate a meaningful T 1 / 2 , AUC Inf and MRT Inf_ obs parameter, because AUC _％Extrap_ The obs value exceeds 20%.

[0353] Table 16b. Brain pharmacokinetic parameters of LMP400 and LMP744 observed after IV administration of 10 mg / kg of LMP400 and 10 mg / kg or 20 mg / kg of LMP744 in combination with PO administration of 100 mg / kg of olaparib or 125 mg / kg of temoda to mice a

[0354]

[0355] a PK parameters were estimated by non-compartmental modeling using WinNonlin software.

[0356] b LMP400 was administered IV at 10 mg / kg plus olaparib was administered PO at 100 mg / kg.

[0357] c LMP400 at 10 mg / kg was administered IV plus Temmodar at 125 mg / kg was administered PO.

[0358] d LMP744 was administered IV at 10 mg / kg plus olaparib was administered PO at 100 mg / kg.

[0359] e 20 mg / kg of LMP744 plus 125 mg / kg of Temoda were administered IV.

[0360] Tumor Penetration Studies. In addition to the brain / plasma ratios, 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 in MOLT-4 and RD4;11 tumors were determined at the 3 and 9 hour time points, while the ratios in RD-ES were measured at the 3 and 6 hour time points. The "G compounds" were all administered IV at the maximum tolerated dose level and at half the maximum tolerated dose level, while the experiments with JQ1 involved standard IP administration of 50 mg / kg. The results reported in Table 16 indicate that all "G compounds" were highly concentrated in tumor tissue, while the tumor-plasma ratio of JQ1 was significantly lower. These results are consistent with previously reported results for the related indenoisoquinoline LMP744 in dogs, which documented that the drug was widely distributed in tumor tissue, with average tumor concentrations at least 100 times higher than plasma concentrations at the same time points. 42 Related studies of LMP400 in BALB / c female mice bearing CT26 colon tumors also demonstrated concentrations in tumor tissue relative to plasma, albeit at a lower ratio (5 to 14.6 after IV administration). 40 With the exception of G104 (30 mg / kg) and G105 (10 mg / kg) relative to RE-ES, the ratios determined at the longer time points were greater than those determined at the shorter time points.

[0361]

[0362] After administration of JQ1, G103, G104 and G105 to female NOD SCID mice, c-Myc and γ-H2AX expression levels in RS4;11, MOLT-4 and RD-ES xenografts were determined by Western blot, and the results are listed in Tables 17-19. In addition to JQ1, the greatest reduction 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 significantly reduced c-Myc in MOLT-4 xenografts, although 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 c-Myc protein levels in RD-ES xenografts, and the effect was greater at higher dose levels (Table 19). Several moderate increases in c-Myc levels were observed in RS4;11 and MOLT-4 xenografts, but they were not as large or widespread as seen (Table 19), with large increases usually occurring 3 hours after drug administration in the case of RD-ES tumors, usually followed by a decline at the 6 hour time point (the exception being G103 at the 25 mg / kg dose level). For RD-ES xenografts, c-Myc levels were suppressed below control values ​​at the 6 hour time point with G104 and G105, but they were not with G103 (Table 19). In several cases, c-Myc levels were significantly suppressed at lower dose levels. For example, see the 9 hour data for G104 in Tables 17 and 18 and the 6 hour data in Table 19. It is noteworthy that at the 3 hour time point in RD-ES cells, c-Myc expression at high doses was consistently more than that at low doses, and the levels at high doses were significantly higher than those at controls. The greatest reduction in c-Myc was seen with JQ1 at the 3 hour time point in MOLT-4 cells (Table 18), although it produced a large increase in c-Myc at the 3 hour time point in RD-ES xenografts (Table 19). Overall, the data in Tables 17-19 have many unexpected and unexplained aspects that vary widely and are also highly dependent on cell type. Specifically, drug-induced c-Myc expression increases relative to controls, and the greater changes in c-Myc expression seen at lower doses relative to higher doses are counterintuitive and unexplained.

[0363] 7-Azaindenoisoquinoline is known to induce phosphorylation of histone H2AX at Ser139 (γ-H2AX) by causing DNA double-strand breaks 25As shown in Tables 17-19, G103, G104, and G105 consistently increased γ-H2AX production relative to controls at longer time points, with one exception being G104 in RD-ES cells. The extent of DNA damage was generally higher at longer time points, with the exceptions being the lower dose of G104 and both doses of G105 in RD-ES cells (Table 19), and both doses of G103 in RS4;11 xenografts (Table 17). There were many instances of γ-H2AX being suppressed below control levels, and this generally occurred at the 3 hour time point, with the exception of JQ1 in RD-ES xenografts, which was suppressed at both time point levels (Table 19).

[0364] Of the three compounds tested in more detail (G103-G105), G103 had the longest IV half-life, greatest oral absorption, highest stability in human liver microsomes, lowest efflux rate in Caco-2 cells, highest maximum tolerated dose in NODS CID mice, and greatest brain penetration. However, G105 appears to be the most potent of the three compounds as an in vivo anticancer agent.

[0365]

[0366]

[0367] a time after administration of vehicle or drug. b This value is obtained after excluding the outlier value 4115.66.

[0368]

[0369] a time after administration of vehicle or drug.

[0370]

[0371]

[0372] a time after administration of vehicle or drug.

[0373] Experimental Section

[0374] 5-Bromopyridine-2,3-diol (7). A cold aqueous solution was prepared by adding furfural (5, 20.10 g, 17.3 mL, 209.2 mmol, 1 equivalent) from a freshly opened container to ice (200 g). The solution was stirred vigorously, and bromine (33.43 g, 10.78 mL, 209.2 mmol, 1 equivalent) was added dropwise at a rate to keep the reaction temperature at 0 ° C (using an external ice bath). The reaction mixture was stirred for 30 min. A portion of concentrated HCl (10 mL) was added, and the reaction mixture was stirred at 0 ° C for 30 min. Additional bromine (33.43 g, 10.78 mL, 209.2 mmol, 1 equivalent) was added dropwise over a period of 1 h, while keeping the reaction temperature at -5 to 0 ° C. The reaction mixture was then filtered, and sulfonic acid (6) (20.31 g, 209.2 mmol, 1 equivalent) 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 afford 5-bromopyridine-2,3-diol (7, 19.8 g, 104 mmol, 49.8%) as a brown solid. 1 HNMR (300MHz, DMSO-d6) δ11.87(s,1H),9.69(s,1H),7.09(d,J=2.5Hz,1H),6.79(d,J=2.5Hz,1H).

[0375] 6-Bromo-[1,3]dioxo[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), and the reaction mixture was stirred and heated to 90 ° C for 14 h. The reaction mixture was cooled to room temperature, filtered through a celite bed, and the bed was washed with ethyl acetate (2x100 mL). The filtrate was extracted with ethyl acetate (2×200 mL). The combined ethyl acetate extracts were washed with water (100 mL), and then with brine (100 mL), and dried over anhydrous MgSO4. The solvent was evaporated to dryness to provide the crude product, which was purified by flash column chromatography [silica gel (40 g), eluting with 0-100% ethyl acetate in hexanes] to afford 6-bromo-[1,3]dioxo[4,5-b]pyridine (8, 1.0 g, 4.95 mmol, 12%) as a white solid. 1H NMR (300MHz, DMSO-d6) δ7.71 (d, J = 2.0 Hz, 1H), 7.55 (dd, J = 2.0, 0.9 Hz, 1H), 6.20 (s, 2H).

[0376] 6-Methyl-[1,3]dioxo[4,5-b]pyridine (9). A mixture of 6-bromo-[1,3]dioxo[4,5-b]pyridine (8, 10.0 g, 49.5 mmol, 1 eq), methylboronic acid (5.93 g, 99.0 mmol, 2 eq), sodium carbonate (21.0 g, 198 mmol, 4.0 eq), 2-dicyclohexylphosphino-2',6'-diisopropoxybiphenyl (RuPHOS) (2.31 g, 4.95 mmol, 0.1 eq) and Pd2dba3 (2.27 g, 2.48 mmol, 0.050 eq) in toluene (200 mL) and water (20 mL) was degassed and then filled with nitrogen. The reaction mixture was heated to 110° C. for 12 h, 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 extracted with ethyl acetate (100 mL). The combined organic extracts were dried over anhydrous MgSO4, filtered and evaporated to dryness. The remaining residue was purified twice by rapid column chromatography [silica gel (2 x 120 g), eluted with 0-100% ethyl acetate in hexane] to obtain 6-methyl-[1,3]dioxo[4,5-b]pyridine (9, 5.6 g, 41 mmol, 82%) as a thick yellow syrup. 1 H NMR (300MHz, DMSO-d6) δ7.45-7.34 (m, 1H), 7.10 (d, J = 1.8Hz, 1H), 6.07 (s, 2H), 2.18 (s, 3H).

[0377] 5-Bromo-6-methyl-[1,3]dioxo[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]dioxo[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 x 300 mL). The combined ethyl acetate extracts were dried over anhydrous MgSO4, filtered, and the solvent evaporated to dryness. The remaining residue was purified twice by flash column chromatography [silica gel (2 x 120 g), eluting with 0-100% ethyl acetate in hexanes] to provide 5-bromo-6-methyl-[1,3]dioxo[4,5-b]pyridine (10; 6.5 g, 30 mmol, 54%) as a light red solid. 1 H NMR (300MHz, DMSO-d6) δ7.32(s,1H),6.17(s,2H),2.22(s,3H).

[0378] 6-Methyl-[1,3]dioxo[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]dioxo[4,5-b]pyridine (10, 6.5 g, 30 mmol, 1 eq) in dry DMF (40 mL) and toluene (80 mL). The reaction mixture was stirred and heated at 110 °C for 18 h. After the reaction was complete, brine (300 mL) was added and the product was extracted with ethyl acetate (2×500 mL). The combined extracts were dried over anhydrous MgSO4, filtered and the solvent evaporated. The remaining residue was purified by flash column chromatography [silica gel (120 g), eluting with a 9:1 ethyl acetate / methanol mixture in hexanes] to provide 6-methyl-[1,3]dioxo[4,5-b]pyridine-5-carbonitrile (11, 2.3 g, 19 mmol, 47%) as a white solid. 1 H NMR (300MHz, DMSO-d6) δ7.35(s,1H),6.26(s,2H),2.38(s,3H).

[0379] 6-(Bromomethyl)-[1,3]dioxo[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]dioxo[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 0-100% ethyl acetate in hexanes] to provide 6-(bromomethyl)-[1,3]dioxo[4,5-b]pyridine-5-carbonitrile (12, 0.488 g, 2.02 mmol, 46.9%) as a white solid. 1 H NMR (300MHz, DMSO-d6) δ7.54(s,1H),6.31(s,2H),4.71(s,2H).

[0380] 2,3-Dimethoxy-6,12-dihydro-5H-[1,3]dioxo[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 6-(bromomethyl)[1,3]dioxo[4,5-b]pyridine-5-carbonitrile (12, 0.370 g, 1.54 mmol, 1 eq) in acetonitrile (22 mL, 60 vol 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 and then cooled to room temperature, and the precipitated solid was collected by filtration and rinsed with acetonitrile to provide 2,3-dimethoxy-6,12-dihydro-5H-[1,3]dioxo[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 light yellow solid. 1 H NMR (300MHz, DMSO-d6) δ11.84(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).

[0381] 2,3-Dimethoxy-5H-[1,3]dioxo[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]dioxo[4",5":5',6']pyrido[3',2':4,5]cyclopenta[1,2-c]isoquinolin-5-one (14,50 mg, 0.15 mmol, 1 eq) and selenium dioxide (66 mg, 0.59 mmol, 4.0 eq) in dioxane (7 mL, 140 vol eq) and acetic acid (8.9 mg, 8.5 μL, 0.15 mmol, 1 eq) was heated at reflux for 11 h. The reaction mixture was filtered and the obtained solid was rinsed with dioxane to provide 2,3-dimethoxy-5H-[1,3]dioxo[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 (300MHz, DMSO-d6) δ7.67(s,1H),7.50(s,1H),7.35(s,1H),6.28(s,2H),3.90(s,3H),3.86(s,3H).

[0382] 2,3-Dimethoxy-6-(3-morpholinopropyl)-5H-[1,3]dioxo[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 eq) and KI (1.6 mg, 0.1 eq) were added to a suspension of 2,3-dimethoxy-5H-[1,3]dioxo[4",5":5',6']pyrido[3',2':4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (15, 0.035 g, 1 eq) in dry DMF (3 mL) at 5 °C. The reaction mixture was warmed to room temperature and stirred for 1 h to form a dark red solution. The solution was cooled to 0 ° C, and 4- (3-bromopropyl) morpholine hydrobromide (16,37 mg, 0.13 mmol, 1.3 equivalents) 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 CH2Cl2 (2 × 100 mL) and the CH2Cl2 extract was washed with brine (100 mL), dried over anhydrous MgSO4, and the solvent was evaporated to dryness. The remaining residue was purified by flash column chromatography [silica gel (24 g), eluted with 0-50% methanol in CH2Cl2] to provide 2,3-dimethoxy-6-(3-morpholinopropyl)-5H-[1,3]dioxo[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 H NMR (300MHz, CDCl3) δ7.94(s,1H),7.68(s,1H),7.17(s,1H),6.18(s,2H),4.88(t,J=7.6Hz,2H),4.05(s ,3H),3.99(s,3H),3.70–3.55(m,4H),2.56(t,J=6.6Hz,2H),2.46(s,4H),2.01(s,2H); MS(ES+):480.4.

[0383]

[0136] 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]dioxo[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 for 12 h. The solvent was evaporated and the residue was triturated with water to form a solid. The solid was filtered and rinsed with water to provide 2,3-dimethoxy-6-(3-morpholinopropyl)-5H-[1,3]dioxo[4",5":5',6']pyrido[3',2':4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (17, 24 mg, 50 μmol, 43%) as a purple solid, which was treated with water / acetonitrile (0.8 mL / 0.2 mL) followed by 5 equivalents of 4N HCl and then soaked for a few minutes and lyophilized to dryness to provide the hydrochloride salt of 17 (26 mg, 42%). 1 H NMR (300MHz, CDCl3) δ7.94(s,1H),7.68(s,1H),7.16(s,1H),6.18(s,2H),5.02-4.80(m,2H),4.05(s,3 H),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).

[0384] 6-(3-(1H-imidazol-1-yl)propyl)-2,3-dimethoxy-5H-[1,3]dioxo[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]dioxo[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 dry DMF (10 mL) at room temperature. The reaction mixture was stirred for 3 h. The reaction mixture was quenched with ice water and extracted with CHCl. ​​A turbid solution was formed and filtered through Whatman filter paper to obtain a sticky solid. LCMS analysis indicated the presence of the desired material in the crude product. The sticky solid was redissolved in methanol / CH2Cl2 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 0-10% DMA80 (80% DCM, 18% methanol and 2% ammonia by volume) in CH2Cl2 to provide 6-(3-(1H-imidazol-1-yl)propyl)-2,3-dimethoxy-5H[1,3]dioxo[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 H NMR (300MHz, CDCl3) δ7.94(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.

[0385]

[00136] Alternative synthesis of 18. 6-(3-Bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxo[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 eq), DIPEA (0.15 g, 0.20 mL, 10 eq, 1.2 mmol) and 1H-imidazole (0.16 g, 2.3 mmol, 20.0 eq) were dissolved in DMF (11 mL). The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was then heated to 70 °C for 18 h. The solvent was evaporated to dryness and the residue was treated with CH2Cl2 (200 mL) and brine (100 mL). The mixture was filtered and the layers were separated. The aqueous layer was back extracted with CH2Cl2 (100 mL) and the combined organic extracts were dried over anhydrous MgSO4. The solution was filtered and the solvent was evaporated to dryness. The residue was purified by flash column chromatography [silica gel (24 g), eluted with 0-10% MeOH in CH2Cl2] to provide 6-(3-(1H-imidazol-1-yl)propyl)-2,3-dimethoxy-5H-[1,3]dioxo[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 converted to the hydrochloride salt by treatment with 7 equivalents of 4N HCl in water / acetonitrile (8:2 ratio) and then lyophilized to dryness to give the hydrochloride salt of 18 (9.1 mg). 1 H NMR(300MHz,DMSO-d6)δ9.14(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).

[0386] 6-(3-(Isopropylamino)propyl)-2,3-dimethoxy-5H-[1,3]dioxo[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]dioxo[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). Then at room temperature add 3-bromo-N-isopropylpropan-1-amine hydrobromide (92mg, 0.35mmol, 2.5 equivalents) dry DMF (10mL), and the reaction mixture is stirred for 3h. Use ice water to quench the reaction mixture, and use CH2Cl2 extraction. With CH2Cl2 layer through anhydrous MgSO4 drying, filter and use CH2Cl2 (50mL) rinse. The solvent was evaporated to dryness and the obtained residue was purified by flash column chromatography [silica gel (24 g), eluted with 0-10% DMA80 (80% DCM, 18% methanol and 2% ammonia by volume) in CH2Cl2] to afford 6-(3-(isopropylamino)propyl)-2,3-dimethoxy-5H-[1,3]dioxo[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%) as a brown solid. 1 H NMR (300MHz, CDCl3) δ7.94(s,1H),7.61(s,1H),7.16(s,1H),6.20(s,2H),5.03-4.86(m,2H),4.07(s,3 H), 4.01 (s, 3H), 3.45-3.28 (m, 1H), 2.93 (s, 2H), 2.61 (s, 2H), 1.49 (d, J = 6.5Hz, 6H); MS (ES+): 452.52.

[0387]

[00136] Alternative synthesis of 19. 6-(3-Bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxo[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 eq), DIPEA (0.14 g, 0.19 mL, 1.1 mmol, 10 eq) and isopropylamine (0.13 g, 0.19 mL, 2.2 mmol, 20 eq) 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 CHCl (100 mL) and stirred for 10 min. The layers were separated and the aqueous layer was back extracted with CHCl (100 mL). The combined organic extracts were dried over anhydrous MgSO4, filtered, and the solvent was evaporated to dryness. The residue was purified by flash column chromatography [silica gel (24 g), eluted with 0-20% methanol in CH2Cl2] to provide the product as a purple solid in the free base form (10 mg). The solid was treated with water (0.8 mL) and acetonitrile (0.2 mL), followed by 5 equivalents of 4N HCl aqueous solution. The components were mixed for a few minutes and then lyophilized to dryness to provide 6-(3-(isopropylamino)propyl)-2,3-dimethoxy-5H-[1,3]dioxo[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-d6)δ8.36(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).

[0388] Bromopropyl-2,3-dimethoxy-5H-[1,3]dioxo[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 eq) was added to a suspension of 2,3-dimethoxy-5H-[1,3]dioxo[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 eq) and KI (37.7 mg, 227 μmol, 0.2 eq) in DMF (36 mL) at 0° C., and the reaction mixture was warmed to room temperature, stirred for 2 h, and then cooled to 0° C. 1,3-Dibromopropane (275 mg, 139 μL, 1.36 mmol, 1.2 eq) 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, rinsed with water (2 x 30 mL) and dried at room temperature to provide 6-(3-bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxo[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 (300MHz, CDCl3) δ7.94(s,1H),7.69(s,1H),7.17(s,1H),6.19(s,2H),4.94(t,J =7.1Hz,2H),4.05(s,3H),4.00(s,3H),3.56(t,J=7.0Hz,2H),2.43(p,J=7.3Hz,2H).

[0389] 6-(3-(Ethylamino)propyl)-2,3-dimethoxy-5H-[1,3]dioxo[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]dioxo[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 eq), DIPEA (0.17 g, 0.22 mL, 1.3 mmol, 10 eq) and ethylamine (0.12 g, 1.3 mL, 2.6 mmol, 20 eq) 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 eq) and ethylamine (0.12 g, 1.3 mL, 2.6 mmol, 20 eq) 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 CH2Cl2 (100 mL), and then stirred for 10 min. The layers were separated, and the aqueous layer was back extracted with CH2Cl2 (100 mL). The combined organic extracts were dried over anhydrous MgSO4, filtered, and evaporated to dryness. The residue was purified by flash column chromatography [silica gel (24 g), eluted with 0-20% methanol in CH2Cl2] to provide 6-(3-(ethylamino)propyl)-2,3-dimethoxy-5H-[1,3]dioxo[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 5 equivalents of 4N aqueous HCl (0.8 mL) and acetonitrile (0.2 mL) followed by lyophilization to give the hydrochloride salt 21 (11 mg, 18%). 1 H NMR (300MHz, DMSO-d6) δ8.56(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).

[0390] 6-(3-((2-Hydroxyethyl)amino)propyl)-2,3-dimethoxy-5H-[1,3]dioxo[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 equiv) was added to a stirred solution of 6-(3-bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxo[4",5":5',6']pyrido[3',2':4,5]cyclopenta[1,2-c]isoquinoline-5,12(6H)-dione (20, 115 mg, 243 μmol, 1 equiv) and ethanolamine (148 mg, 147 μL, 2.43 mmol, 10 equiv) in DMF (22 mL). The resulting reaction mixture was stirred for 6 h. More ethanolamine (148 mg, 147 μL, 2.43 mmol, 10 equiv) was added and the reaction mixture was stirred overnight. The solution was diluted with brine (100 mL) and extracted with CH2Cl2 (2 x 100 mL). The combined extracts were dried over anhydrous MgSO4, filtered and the solvent was evaporated to dryness. The residue was purified by flash column chromatography [silica gel (24 g), eluted with 0-100% methanol in CH2Cl2] to provide 6-(3-((2-hydroxyethyl)amino)propyl)-2,3-dimethoxy-5H-[1,3]dioxo[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 5 equivalents of 4N HCl in water (0.8 mL) and acetonitrile (0.2 mL) followed by lyophilization to provide 11 mg of the hydrochloride salt 22. 1 H NMR (300MHz, DMSO-d6) δ8.58(s,2H),7.82(s,1H),7.54(s,1H),7.46(s,1H),6.33(s,2H),4.72(t,J=6.5Hz,2 H),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).

[0391] 2,3-Dimethoxy-6-(3-(pyrrolidin-1-yl)propyl)-5H-[1,3]dioxo[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 equiv) and pyrrolidine (902 mg, 1.04 mL, 12.7 mmol, 20 equiv) were added to a stirred solution of 6-(3-bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxo[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) 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 CHCl (100 mL) and then stirred for 10 min. The organic layer was separated, and the aqueous layer was back extracted with CHCl (100 mL). The combined organic extracts were dried over anhydrous MgSO4, filtered and the solvent was evaporated to dryness. The residue was purified by flash column chromatography [silica gel (24 g), eluted with 0-20% methanol in CH2Cl2] to provide 2,3-dimethoxy-6-(3-(pyrrolidin-1-yl)propyl)-5H-[1,3]dioxo[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), then 5 equivalents of 4N aqueous HCl were added, and the mixture was lyophilized to dryness to provide the hydrochloride salt of 23 (11 mg). 1 H NMR(300MHz,DMSO-d6)δ7.83(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).

[0392] (S)-1-(3-(2,3-Dimethoxy-5,12-dioxo-5,12-dihydro-6H-[1,3]dioxo[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 equiv) and (S)-pyrrolidine-2-carboxamide (1.45 g, 12.7 mmol, 20 equiv) were added to a stirred solution of 6-(3-bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxo[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) in DMF (30 mL). 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 CHCl (100 mL) and stirred for 10 min. The organic layer was separated and the aqueous layer was back extracted with CHCl (100 mL). The combined organic extracts were dried over anhydrous MgSO4, filtered and the solvent was evaporated to dryness. The residue was purified by flash column chromatography [silica gel (12 g), eluted with 0-20% methanol in CH2Cl2] to provide (S)-1-(3-(2,3-dimethoxy-5,12-dioxo-5,12-dihydro-6H-[1,3]dioxo[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), and then treated with 5 equivalents of 4N aqueous HCl and lyophilized to dryness to provide the hydrochloride salt (24). 1 H NMR(300MHz,DMSO-d6)δ9.42(s,1H),8.09(s,1H),7.82(s,2H),7.54(s,1H),7.45(s,1H),6.32(s,2H),4.80-4.58(m,2H),4.08(d d, J=14.3, 7.1Hz, 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).

[0393] (S)-6-(3-(2-(Hydroxymethyl)pyrrolidin-1-yl)propyl)-2,3-dimethoxy-5H-[1,3]dioxo[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 6-(3-bromopropyl)-2,3-dimethoxy-5H-[1,3]dioxo[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) in DMF (30 mL). 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 CHCl (100 mL) and stirred for 10 min. The organic layer was separated and the aqueous layer was back extracted with CHCl (100 mL). The combined organic extracts were dried over anhydrous MgSO4, filtered, and the solvent was evaporated to dryness. The residue was purified by flash column chromatography [silica gel (24 g), eluted with 0-20% methanol in CH2Cl2] to provide (S)-6-(3-(2-(hydroxymethyl)pyrrolidin-1-yl)propyl)-2,3-dimethoxy-5H-[1,3]dioxo[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) and treated with 5 equivalents of 4N HCl, and the mixture was lyophilized to dryness to give the hydrochloride salt of 25 (8 mg). 1 H NMR (300MHz, CDCl3) δ7.92(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).

[0394] Method Examples

[0395] Drug screening in NCI-60 cancer cell lines

[0396] The antiproliferative activity of indenoisoquinoline compounds was determined in the NCI-60 cancer cell line of the National Cancer Institute Developmental Therapeutics Program (NCI-DTP). Compounds that showed adequate cytotoxicity during prescreening were tested at five doses to determine the 50% growth inhibition (GI 50 ) values. Cancer cells were incubated with five concentrations of the test compound ranging from 100 μM to 10 nM for 48 h. After staining the treated cancer cells with sulforhodamine B dye, the growth percentage was plotted as a function of the common logarithm of the test compound concentration. GI 50 Values ​​were determined by interpolation between points above and below 50% cell growth. -4 Up to 10 -8 M) GI above and below 50 The values ​​were used as the maximum (10 -4 M) and minimum (10 -8 M) Drug concentration. GI of each compound across the entire panel of NCI-60 cancer cell lines 50 The approximate mean of the values ​​is recorded as the MGM value.

[0397] The cytotoxicity data for G101-G105 are shown in Tables 20a and 20b below.

[0398]

[0399]

[0400]

[0401] Test Method

[0402] DNA melting assay. FRET-based DNA melting assays were performed on the QuantStudio 6Flex Real-Time PCR System. 5'-Fluorescein (6-FAM), 3'-Rhodamine (TAMRA) dual-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 to 95 °C for 2 min. The labeled MycG4 and drug aqueous solutions were added to each well of a 96-well plate in a final volume of 20 μL, with a concentration of 0.2 μM DNA and 2.0 μM drug in 10 mM KCl. +Buffer (pH 7.0). The drug-DNA mixture was incubated at 25°C for 1 h in the dark and then heated to 95°C at a rate of 0.9°C / min while measuring the fluorescence of 6-FAM at 520 nm (ex. 470 nm). G-quadruplex melting temperature T m Determined by the maximum of the first derivative of the melting curve. ΔT m Calculated as T m,DNA+药物 With T m,DNA The difference between .

[0403] Fluorescence-based binding assays. Fluorescence-based binding assays were performed in a quartz cuvette with a path length of 10 mm at 20°C on a Jasco FP-8300 spectrofluorometer equipped with a temperature controller. 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 spectrum of free DNA after each titration step was recorded from 570 to 600 nm using an excitation wavelength of 555 nm, a 5 nm bandwidth, a 200 nm / min scan speed, and a 1 s response time. The fluorescence intensity at 580 nm (which is the largest for free DNA) was plotted against the drug: DNA ratio. The apparent binding dissociation constant K for the binding of each compound was derived. d,app The changes in fluorescence intensity were fitted to the single-site binding equation using OriginPro software (OriginLab Corp., USA).

[0404]

[0405] where B0 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.

[0406] NMR titration experiments. NMR titration experiments were performed on a Bruker AV-500 spectrometer equipped with a Prodigy cryoprobe at 25°C. MycG4 DNA was diluted to 150 μM in 75 mM KCl / 25 mM potassium phosphate buffer (pH 7.0) in D2O / H2O. The diluted DNA was annealed by heating to 95°C for 2 min and slowly cooled to room temperature. The 1D of unbound DNA was measured. 1 H NMR and subsequent titration of the compound (in DMSO-d6). Measurements were made at drug:DNA equivalents of 0.5, 1.0, 2.0, 3.0, 4.0. After addition of the compound, the sample was incubated for 5 min.

[0407] Western blot. MCF-7 cells were grown and treated in 6-well tissue culture plates. After collection, cells were suspended in 140 μL 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). Cell lysates were heated to 75°C for 10 min. After ultrasound, 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 monoclonal anti-Myc antibody (5605S, Cell Signaling Technology) diluted 1:1000 and monoclonal anti-GAPDH antibody (5174S, Cell Signaling Technology) diluted 1:2000, respectively. The primary antibody was detected by anti-rabbit IgG HRP-linked antibody (7074S, Cell Signaling Technology). The blot was developed by ECL substrate (1863096, ThermoScientific) and imaged by Bio-Rad ChemiDoc Touch imaging system.

[0408] Quantitative reverse transcriptase PCR (qRT-PCR). MCF-7 cells were grown and processed in 12-well tissue culture plates until collection. Total RNA was extracted using TRIzol reagent (15596018, Life Technologies). Purified RNA was dissolved in DEPC water and quantified. According to the manufacturer's instructions, 1 μg of purified RNA per sample was converted into cDNA using qScript cDNA SuperMix (84034, Quantabio). Real-time PCR reagents were prepared for each sample by mixing 1 μL synthetic cDNA, 3 μL PCR grade water, 5 μL SYBR Green reagent (A25742, Applied Biosystem) and 0.25 μL of each primer for Myc and GAPDH. Reactions were set up in triplicate for each target of each sample. Real-time PCR was performed in 96-well PCR plates using QuantStudio 6Flex real-time PCR system (Life Technologies). Cycling conditions were 95°C 5min, followed by 40 cycles (95°C 15s, 60°C 15s, and 72°C 15s). The results were normalized to GAPDH as an endogenous control using 2 -ΔΔCt The relative Myc expression was calculated by the method.

[0409] MTS cell viability IC 50 Assay. MTS IC of suspension cell lines 50 The drug solution measured was prepared by serial dilution of DMSO stock solution in 1XPBS (10010-031, Gibco). In order to set up the treatment, the drug solution was plated in triplicate to a 96-well tissue culture plate, to which the culture medium containing the cells was added to a total volume of 100 μL / well. The 100% viability control was untreated cells. Except for KMS-27, which was 3000 cells / well and CHLA-32, which was 10,000 cells / well, 2000 cells / well were plated for all suspension cell lines. The blank was a cell-free culture medium. Drug treatment was maintained for 72 hr at 37°C with 5% CO2. In order to quantify the cells, CellTiter 96AQueous MTS reagent powder (G1111, Promega) was dissolved and mixed with phenazine methyl sulfate (PMS, Sigma-Aldrich P9625) according to the manufacturer's instructions. MTS / PMS mixture (20 μL / well) was added to the treated wells, 100% viability control wells, and blank wells. Color was developed for 4 h at 37°C with 5% CO2, and the absorbance at 490 nm was measured using a BioTek Synergy 4 system. To obtain % viability, A was corrected by subtracting the blank. 490The values ​​were normalized to 100% survival control and averaged. The mean % survival was plotted against drug dose to obtain dose response curves. IC was calculated using Prism software (GraphPad Prism version 9.1.0 for macOS, www.graphpad.com). 50 , using the equation "log(inhibitor) vs. normalized response-variable slope" implemented by the program: Y = 100 / (1+10 ((LogIC50-X)*HillSlope) ). For adherent cell lines, in addition to cell plating and drug treatment, MTS IC 50 The assay was performed in the same manner as for suspension cell lines. For cell plating, 2000 cells in 50 μL were plated in triplicate into 96-well tissue culture plates. The cells were allowed sufficient time to adhere. 50 Serial dilutions of drug solutions were prepared in the same manner as the assay. To set up treatments, drug solutions were mixed with culture medium to twice the final concentration, and then 50 μL of this drug-culture medium mixture was added to 50 μL of plated cells.

[0410] MTS Cell Viability Single-Dose Assay. Drug solutions for the MTS single-dose assay were prepared by dilution from DMSO stocks in 1X PBS. To set up treatments, drug solutions were plated in triplicate into 96-well tissue culture plates and medium containing CA46 or Raji 2000 cells / well was added. 100% viability controls and blanks were set up in the same manner as IC 50 The assay is the same as that of IC 50 To obtain % survival, use A 490 Values ​​were blank subtracted, normalized to 100% viability control, and averaged. Results were visualized as bar graphs using Prism software.

[0411] NCI-60 Cytotoxicity Assay. Compounds were submitted to the National Cancer Institute Developmental Therapeutics Program to determine their growth inhibitory potential in the NCI-60 cell line. All compounds that met the prescreening criteria entered a 5-dose cell viability assay, in which cells were treated with 5 doses ranging from 100 μM to 10 nM for 48 h. After treatment, the percentage growth was compared to Log 10 (drug dosage) to infer the 50% growth inhibition (IC 50 ). If GI 50 If the concentration is above 100 μM or below 10 nM, GI is reported respectively. 50 100 μM or 10 nM.

[0412] Pharmacokinetics. The pharmacokinetic characteristics of compounds 19 (G103), 21 (G104) and 22 (G105) were determined by administration to male CD1 mice via IV (dose 1 mg / kg) and PO (dose 10 mg / kg). Mice were obtained from Si Bei Fu Laboratory Animal Technology Co. Ltd. The animals were 6-8 weeks old and weighed 20-30 g when the drug was administered. Plasma samples for IV and PO experiments were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 h. Three animals were used for each experiment, and plasma samples were pooled together and analyzed by LC / MS / MS. Pharmacokinetic studies were conducted at Pharmaron Beijing Co. Ltd after appropriate ethical considerations and by review of Pharmaron's IACUC (Institutional Animal Care and Use Committee).

[0413] Plasma protein binding and stability in human and mouse plasma were analyzed by equilibrium dialysis. Frozen plasma (stored at -80 ° C) was immediately thawed in a 37 ° C water bath. Working solutions of test compounds and control compounds were prepared at 200 μM concentration in DMSO, and the working solutions were spiked into 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 in the assay. The dialysis membrane was soaked in ultrapure water for 60 min to separate the bands, then soaked in 20% ethanol for 20 min, and then finally soaked in dialysis buffer for 20 min. For equilibrium dialysis, each unit contained a plasma sample (150 μL) and was dialyzed for an equal volume of dialysis buffer (PBS). Determined in duplicate. The dialysis plate was sealed and incubated for 6 h at 37 ° C with 5% CO2 at 100 rpm. At the end of the incubation, the samples (50 μL) in both the buffer and plasma chambers were transferred to the 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 collected plasma samples. 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 protein and release compound. The sample was vortexed for 2 min and centrifuged at 3,220 g for 30 min. The sample (100 μL) of the supernatant was diluted with ultrapure water (100 μL), and the mixture was used for LC-MS / MS analysis. In order to determine the stability in plasma, the spiked plasma sample (50 μL) was transferred to a new plate and incubated at 37 ° C, 5% CO2 for 0 and 6 h. At designated time points, PBS (50 μL) was added and mixed thoroughly, followed by the 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 proteins and release compounds. The samples were vortexed for 2 min and centrifuged at 3,220 g for 30 min. A sample (400 μL) of the supernatant was diluted with ultrapure water, and the mixture was used for LC-MS / MS analysis. The concentration of the test compound in the buffer and plasma compartments was determined by the peak area ratio.

[0414] Stability analysis of human and mouse liver microsomes. Microsome solution (0.5 mg / mL) was prepared in phosphate buffer (100 mM) containing MgCl2 (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 min. Negative control samples were prepared by replacing NADPH solution with 40 μL of ultrapure H2O. Samples with NADPH were prepared in duplicate, and negative controls were prepared in duplicate. The reaction was started by adding control compounds (2 μL of 200 μM solution) or test compound solutions. Verapamil was used as a positive control. The final concentration of the test compound or control compound was 1 μM. Samples (50 μL) were taken out from the reaction solution at 0, 15, 30, 45 and 60 min. The reaction was terminated by adding 4 volumes of cold acetonitrile containing alprazolam (100 nM), imipramine (200 nM), labetalol (200 nM) and 2 μM ketoprofen (2 μM). The sample was centrifuged at 3,220 g for 40 min. A sample of the supernatant (90 μL) was mixed with ultrapure H2O (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 residual percentage of the prototype drug relative to the incubation time curve. The in vitro half-life (in vitro t 1 / 2 ) is determined by the slope value: in vitro t 1 / 2 =–(0.693 / k). The in vitro t 1 / 2 (min) converted to in vitro intrinsic clearance (in vitro CL int , in μL / min / mg protein).

[0415]

[0416] The in vitro t 1 / 2 (min) converted to amplified unbound intrinsic clearance (amplified CL int , in mL / min / kg):

[0417]

[0418] The scaling factors used in the calculations are listed in Table 21.

[0419] Table 21. Scaling factors for intrinsic clearance prediction in liver microsomes.

[0420]

[0421] a From references 43 .b From references 44,45 .

[0422] Caco-2 permeability study. The Caco-2 plate was taken out from the incubator and washed twice with preheated HBSS (10mM HEPES, pH 7.4), and then incubated at 37°C for 30min. The stock solution of the control compound was diluted in DMSO to obtain a 1mM solution, and then diluted with HBSS (10mM HEPES, pH 7.4) to obtain a 5μM working solution. The stock solution of the test compound was diluted in DMSO to obtain a 1mM solution, and then diluted with HBSS (10mM HEPES, pH7.4) to obtain a 5μM working solution. The final concentration of DMSO in the incubation system was 0.5%. To determine the drug transport rate from the top to the basolateral direction, 75μL of a working solution of 5μM test compound was added to a Transwell insert (insert) (top compartment), and 235μL of HBSS (10mM HEPES, pH7.4) was filled in the hole of the receiving plate (basolateral compartment). To determine the drug transport rate from the basolateral to the apical direction, 235 μL of a 5 μM working solution of the test compound was added to the receiving plate well (basolateral compartment), and then filled with 75 μL of HBSS (10 mM HEPES, pH 7.4) in the Transwell insert (apical compartment). Time 0 samples were prepared by transferring 50 μL of the 5 μM working solution to the wells of a 96-deep well plate, followed by the addition of 200 μL of cold methanol containing an appropriate internal standard (IS). The plate was incubated at 37° C. for 2 h. At the end of the incubation, 50 μL samples from the donor side (apical compartment for Ap→Bl flux and basolateral compartment for Bl→Ap) and the acceptor side (basolateral compartment for Ap→Bl flux and apical compartment for Bl→Ap) were transferred to the wells of a new 96-well plate, followed by the addition of 4 volumes of cold acetonitrile or methanol containing an appropriate internal standard (IS). The sample was vortexed for 5 min and then centrifuged at 3,220 g for 40 min. Before LC-MS / MS analysis, 100 μL of supernatant samples were mixed with appropriate volumes of ultrapure water. In order to determine the leakage of fluorescein after the 2 h transport period, a stock solution of fluorescein was prepared in ultrapure water and diluted with HBSS (10 mM HEPES, pH 7.4) to reach a final concentration of 100 μM, and then 100 μL of fluorescein solution was added to each Transwell insert (top compartment), and then the holes in the receiving plate (basal side compartment) were filled with 300 μL of HBSS (10 mM HEPES, pH 7.4). The plate was incubated at 37 ° C for 30 min. Samples (80 μL) were taken out directly from the top and basal side holes (using the basal side interface holes) and transferred to the holes of a new 96-well plate. Measure the fluorescein fluorescence (to monitor the integrity of the monolayer) signal at 485 nM excitation and 530 nM emission in a fluorescence microplate reader.

[0423] Implementation

[0424] E1. A compound of formula (I) or a salt, hydrate or solvate thereof,

[0425]

[0426] in

[0427] R1 and R2 are independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R1 and R2 together with the atoms to which they are attached form a 5- or 6-membered ring;

[0428] R3 is hydrogen, halogen, nitro, cyano, CF3, (C1-C6) alkyl, (C1-C6) alkylthio or (C1-C6) alkoxy;

[0429] A is an alkylene group;

[0430] R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxy, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl, and heterocyclylamino are each optionally substituted;

[0431] R5 represents 1 to 2 substituents independently selected from the group consisting of amino, (C1-C6) alkylamino, di(C1-C6) alkylamino, hydroxy(C1-C6) alkyl, (C1-C6) alkenyl, (C1-C6) alkynyl, (C1-C6) heteroalkyl, (C3-C8) cycloalkyl, (C3-C8) cycloheteroalkyl, (C1-C6) alkoxy, (C1-C6) alkyl(CO)O-, (C1-C6) alkyl-O(CO)O- and (C1-C6) alkylthio; or R5 represents 2 adjacent substituents that together with the attached carbon form an optionally substituted ring or heterocycle. E2. Use of a compound described in E1 or a salt, hydrate or solvate thereof for treating a patient in need of relief of cancer. E3. A pharmaceutical composition comprising one or more compounds of E1 or a salt, hydrate or solvate thereof, and one or more carriers, diluents or excipients or a combination thereof.

[0432] E4. The compound of E1 or its salt, hydrate or solvate, for use in treating cancer.

[0433] E5. A pharmaceutical composition comprising one or more compounds of E1 or salts, hydrates or solvates thereof, for use in treating cancer.

[0434] E6. A pharmaceutical composition for treating cancer, comprising a therapeutically effective amount of one or more compounds of E1 or salts, hydrates or solvates thereof.

[0435] E7. A method for treating a disease in a host animal that responds to topoisomerase I inhibition or binding to a MYC quadruplex, the method comprising administering to the host animal a composition comprising a therapeutically effective amount of one or more E1 compounds or salts, hydrates or solvates thereof, or a pharmaceutical composition comprising one or more compounds of claim 1 or salts, hydrates or solvates thereof, wherein the pharmaceutical composition optionally further comprises one or more carriers, diluents or excipients or a combination thereof.

[0436] E8. The method described in E7, wherein the host animal is a human.

[0437] E9. A method for treating cancer in a host animal, the method comprising the step of administering to the host animal a composition comprising a therapeutically effective amount of one or more compounds of claim 1 or a salt, hydrate or solvate thereof.

[0438] E10. The method described in E9, wherein the composition further comprises one or more carriers, diluents or excipients or a combination thereof.

[0439] E11. A process for preparing a compound of E1, said process comprising the step of brominating a compound of formula II to produce compound III, wherein R1, R2 and R3 are as defined in E1.

[0440]

[0441] E12. A process according to E11, wherein R3 is hydrogen.

[0442] E13. A process according to E11, wherein R1 and R2 together form -CH2-.

[0443] E14. A process as described in any one of E11-13, wherein the bromination step comprises treating a solution of the compound of formula (II) in acetic acid with N-bromosuccinimide.

[0444] References

[0445] The following publications, and every other publication cited herein, are hereby incorporated by reference:

[0446] (1)Cushman,M.;Cheng,L.Stereoselective Oxidation by Thionyl ChlorideLeading to the Indeno[1,2-c]isoquinoline System.J.Org.Chem.1978,43,3781-3783.

[0447] (2)Kohlhagen,G.;Paull,K.;Cushman,M.;Nagafuji,P.;Pommier,Y.Protein-Linked DNA Strand Breaks Induced by NSC 314622,a Novel NoncamptothecinTopoisomerase IPoison.Mol.Pharmacol.1998,54,50-58.

[0448] (3)Ryckebusch,A.;Garcin,D.;Lansiaux,A.;Goossens,J.F.;Baldeyrou,B.;Houssin,R.;Bailly,C.;Henichart,J.P.Synthesis,Cytotoxicity,DNA Interaction,andTopoisomerase II Inhibition Properties of Novel Indeno[2,1-c]quinolin-7-oneand indeno[1,2-c]isoquinolin-5,11-dione Derivatives.J.Med.Chem.2008,51,3617-3629.

[0449] (4)Ahn,G.Lansiaux,A.Goossens,JFBailly,C.Baldeyrou,B.Schifano-Faux,N.Grandclaudon,P.Couture,A.Ryckebusch,A.Indeno[1,2-c]isoquinoline-5,11-diones Conjugated to Amino Acids:Synthesis,Cytotoxicity,DNA Interaction,and Topoisomerase II Inhibition Properties.Bioorg.Med.Chem.2010,18,8119-8133.

[0450] (5)Ahn,G.Schifano-Faux,N.Goossens,JF;Baldeyrou,B.Couture,A.Grandclaudon,P.Lansiaux,A.Ryckebusch,A.Synthesis,Cytotoxicity andTopoisomerase Inhibition Properties of Multifarious Aminoalkylated Indeno[1,2-c]isoquinoline-5,11-diones.Bioorg.Med.Chem.Lett.2011,21,2259-2263.

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Claims

1. A compound of formula (I) or a salt, hydrate or solvate thereof, in R1 and R2 are independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R1 and R2 together with the atoms to which they are attached form a 5- or 6-membered ring; R3 is hydrogen, halogen, nitro, cyano, CF3, (C1-C6) alkyl, (C1-C6) alkylthio or (C1-C6) alkoxy; A is an alkylene group; R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxy, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl, and heterocyclylamino are each optionally substituted; R5 represents 1 to 2 substituents independently selected from the group consisting of amino, (C1-C6)alkylamino, di(C1-C6)alkylamino, hydroxy(C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, (C1-C6)heteroalkyl, (C3-C8)cycloalkyl, (C3-C8)cycloheteroalkyl, (C1-C6)alkoxy, (C1-C6)alkyl(CO)O-, (C1-C6)alkyl-O(CO)O- and (C1-C6)alkylthio; or R5 represents 2 adjacent substituents which, together with the attached carbon, form an optionally substituted ring or heterocycle.

2. The compound according to claim 1, wherein A is (CH2)n, wherein n is selected from the group consisting of 1, 2 and 3.

3. The compound according to claim 2, wherein A is (CH2)3.

4. The compound according to claim 1, wherein R3 is hydrogen.

5. The compound of claim 1, wherein R1 and R2 are independently selected from the group consisting of hydrogen and CH3.

6. The compound of claim 1, wherein R1 and R2 together with the atoms to which they are attached form a 5- or 6-membered ring.

7. The compound of claim 1, wherein R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxyl, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl, and heterocyclylamino are each optionally substituted.

8. The compound according to claim 1, wherein R1 and R2 together form -CH2-, R3 is hydrogen, R5 represents 2-MeO and 3-MeO, A is (CH2)3, and R4 is selected from the group consisting of heteroaryl, heteroaryloxy, heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl, heterocyclylamino, amino, hydroxyl, halogen, cyano, alkylamino, dialkylamino, trialkylammonium, hydroxyalkylamino, bis(hydroxyalkyl)amino and hydroxyalkylaminoalkylamino, wherein heteroaryl, heteroaryloxy and heteroarylamino, heteroarylalkylaminoalkylamino, heterocyclyl and heterocyclylamino are each optionally substituted.

9. The compound according to claim 1, wherein the compound is 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 in R1 and R2 are independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R1 and R2 together with the atoms to which they are attached form a 5- or 6-membered ring; R3 is hydrogen, halogen, nitro, cyano, CF3, (C1-C6) alkyl, (C1-C6) alkylthio or (C1-C6) alkoxy; A is (CH3) n , wherein n is 2 to 3; R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxy, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl, and heterocyclylamino are each optionally substituted; R5 represents 1 to 2 substituents independently selected from the group consisting of amino, (C1-C6)alkylamino, di(C1-C6)alkylamino, hydroxy(C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, (C1-C6)heteroalkyl, (C3-C8)cycloalkyl, (C3-C8)cycloheteroalkyl, (C1-C6)alkoxy, (C1-C6)alkyl(CO)O-, (C1-C6)alkyl-O(CO)O- and (C1-C6)alkylthio; or R5 represents 2 adjacent substituents which, together with the attached carbon, form an optionally substituted ring or heterocycle.

12. The pharmaceutical composition according to claim 11, further comprising at least one additional component selected from the group consisting of a diluent, an excipient, and a combination thereof.

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

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

15. A method for treating a disease in a host animal that responds to topoisomerase I inhibition or binding to a MYC quadruplex, the method comprising administering to the host animal a composition comprising a therapeutically effective amount of one or more compounds of formula (I) or salts, hydrates or solvates thereof, or a pharmaceutical composition comprising one or more compounds of formula (I) or salts, hydrates or solvates thereof, Wherein formula (I) is wherein R1 and R2 are independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, optionally substituted heteroalkyl, and optionally substituted acyl, or R1 and R2 together with the atoms to which they are attached form a 5- or 6-membered ring; R3 is hydrogen, halogen, nitro, cyano, CF3, (C1-C6) alkyl, (C1-C6) alkylthio or (C1-C6) alkoxy; A is alkylene; R4 is selected from the group consisting of heteroaryl, heterocyclyl, heterocyclylamino, amino, hydroxy, halogen, cyano, alkylamino, dialkylamino, hydroxyalkylamino, bis(hydroxyalkyl)amino, and hydroxyalkylaminoalkylamino, wherein heteroaryl, heterocyclyl, and heterocyclylamino are each optionally substituted; R5 represents 1 to 2 substituents independently selected from the group consisting of amino, (C1-C6)alkylamino, di(C1-C6)alkylamino, hydroxy(C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, (C1-C6)heteroalkyl, (C3-C8)cycloalkyl, (C3-C8)cycloheteroalkyl, (C1-C6)alkoxy, (C1-C6)alkyl(CO)O-, (C1-C6)alkyl-O(CO)O-, and (C1-C6)alkylthio; or R5 represents 2 adjacent substituents which, together with the attached carbon, form an optionally substituted ring or heterocycle, and The pharmaceutical composition optionally further comprises one or more carriers, diluents or excipients or a combination thereof.

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

17. A process for preparing the compound of claim 1, said process comprising the step of brominating a compound of formula II to produce compound III, wherein R1, R2 and R3 are as defined in claim 1.

18. A process according to claim 17, wherein R3 is hydrogen.

19. A process according to claim 18, wherein R1 and R2 together form -CH2-.

20. A process according to claim 19, wherein the bromination step comprises treating a solution of the compound of formula (II) in acetic acid with N-bromosuccinimide.

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