# Patent

## Title
Substituted quinobenzoxazine analogs

## Abstract

The present invention relates to methods for ameliorating a cell proliferative disorder using quinobenzoxazines analogs having the general formula:




    
    
        and pharmaceutically acceptable salts, esters and prodrugs thereof;
        wherein A, U, V, W, X and Z are substituents.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 10/821,243, filed Apr. 7, 2004, now U.S. Pat. No. 7,141,565 which claims the benefit of U.S. provisional application 60/461,271, filed Apr. 7, 2003; U.S. provisional application 60/463,171, filed Apr. 15, 2003; U.S. provisional application 60/519,535, filed Nov. 12, 2003; and U.S. provisional application 60/532,727, filed Dec. 23, 2003. The contents of these documents are incorporated herein by reference.


FIELD OF THE INVENTION
The invention relates to substituted quinobenzoxazines analogs, and methods of using such compounds.
BACKGROUND
Quadruplexes can form in certain purine-rich strands of nucleic acids. In duplex nucleic acids, certain purine rich strands are capable of engaging in a slow equilibrium between a typical duplex helix structure and in unwound and non-B-form regions. These unwound and non-B forms can be referred to as “paranemic structures.” Some forms are associated with sensitivity to S1 nuclease digestion, which can be referred to as “nuclease hypersensitivity elements” or “NHEs.” A quadruplex is one type of paranemic structure and certain NHEs can adopt a quadruplex structure. Considerable circumstantial evidence suggests that quadruplex structures can exist in vivo in specific regions of the genome, including the telomeric ends of chromosomes and oncogene regulatory regions. (Han, et al., Trends Pharm. Sci. (2000) 21:136-142). Thus, quadruplex forming regions of DNA may be used as molecular targets for anticancer agents.
SUMMARY OF THE INVENTION
Compounds described herein interact with regions of DNA that can form quadruplexes and act as tumor suppression agents with reduced side effects. Such compounds reduce expression of highly proliferate genes and are utilized for treating cancers. Furthermore, the compounds may also exhibit antibacterial or antiviral activity, and may be used for treating bacterial and viral infections.
Various embodiments of the present invention are described below. The present invention encompasses other compounds having formula 1, with substituents independently selected from compounds in Tables 1-3. Thus, the present invention is not limited to the specific combination of substituents described in various embodiments below.
The compounds have the general formula:




and pharmaceutically acceptable salts, esters and prodrugs thereof;

    
    
        wherein V is H, halo, or NR1R2;
        A is H, fluoro, or NR12;
        Z is O, S, NR1 or CH2;
        U is OR2 or NR1R2;
        X is OR2, NR1R2, halo, azido, or SR2;
        n is 1-3;
        wherein in NR1R2, R1 and R2 may form a double bound or a ring, each of which is optionally substituted;
        R1 is H or a C1-6 alkyl;
        R2 is H or a C1-10 alkyl or C2-10 alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O, and S, and optionally substituted with a carbocyclic or heterocyclic ring; or R2 is an optionally substituted heterocyclic ring, aryl or heteroaryl;
        W is selected from the group consisting of
    
    












    
    
        
        
            wherein Q, Q1, Q2, and Q3 are independently CH or N;
            Y is independently O, CH, ═O or NR1;
            and R5 is a substituent at any position on the fused ring; and is H, OR2, C1-6 alkyl, C2-6 alkenyl, each optionally substituted by halo, ═O or one or more heteroatoms; or R5 is an inorganic substituent; or two adjacent R5 is linked to obtain a 5-6 membered substituted or unsubstituted carbocyclic or heterocyclic ring, optionally fused to an additional substituted or unsubstituted carbocyclic or heterocyclic ring;
        
        
        provided that U is not OR1 when X is pyrrolidinyl; A is F; Z is O; and W is naphthalenyl or phenylene;
        U is not morpholinyl or 2,4-difluoroaniline when X is F or pyrrolidinyl; A is F; Z is O; and W is phenylene; and
        further provided that if U is OH, then W represents multiple fused aromatic rings and X is not halo; and X is NH2, or a moiety that does not contain N, or contains more than 6 carbons.
    
    


In the above formula 1, A and X may independently be halo. In one example, A and X may independently be fluoro.
In the above formula, V may be H. Alternatively, V may be NH2 or a compound having the formula NR1—(CR12)n—NR3R4;

    
    
        wherein R1 and R3 are independently H or C1-6 alkyl;
        n is 1-6; and
        R4 is H, C1-6 alkyl optionally substituted with a carbocyclic or heterocyclic ring, or aryl; and wherein in NR3R4, R3 and R4 may form an optionally substituted ring.
    
    


In the above formula 1, U and X may independently be NR1R2. In one example, R1 is H and R2 is a C1-10 alkyl optionally containing one or more heteroatoms, and optionally substituted with a C3-6 cycloalkyl, aryl or a 5-14 membered heterocyclic ring containing one or more N, O or S. In another example, R1 is H and R2 is an aryl or a 5-14 membered heterocyclic ring containing one or more N, O or S, each optionally substituted with an amino or another heterocyclic ring. In yet another example, R1 and R2 in NR1R2 form an optionally substituted 5-14 membered ring containing one or more N, O or S. In particular examples, NR1R2 is morpholine, thiomorpholine, piperazine, piperidine or diazepine.
In the above formula 1, U and X may independently have the formula

NR1—(CR12)n—NR3R4  (2)

    
    
        wherein R1 and R3 are independently H or C1-6 alkyl;
        n is 1-6; and
        R4 is H or a C1-10 alkyl or C2-10 alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O and S, and optionally substituted with a carbocyclic or heterocyclic ring; and
        wherein in NR3R4, R3 and R4 may form an optionally substituted ring.
    
    


In the above formula 2, n may be 2-3. In one example, NR3R4 is an acyclic amine, or guanidinyl or a tautomer thereof; or R3 and R4 optionally form a substituted ring containing one or more N, O or S. In particular examples, NR3R4 is morpholine, thiomorpholine, imidazole, pyrrolidine, piperazine, pyridine or piperidine.
In the above formula 1, X may be NR1R2; and U has the formula

NR1—(CR12)n—NR3R4  (2)

    
    
        wherein R1 and R2 are as defined in claim 1;
        R3is H or C1-6 alkyl;
        n is 1-6; and
        R4is H or a C1-10 alkyl or C2-10 alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O and S, and optionally substituted with a carbocyclic or heterocyclic ring; and
        wherein in NR1R2 and NR3R4, R1 and R2, and R3 and R4 each independently may form a substituted ring.
    
    


In the above formula, where X is NR1R2 and U has the formula NR1—(CR12)n—NR3R4 (2), R1 and R2 in NR1R2, and R3 and R4 in NR3R4 each may independently form a substituted ring containing one or more N, O or S. For example, X is optionally substituted with amino, carbamate, a C1-10 alkyl containing one or more non-adjacent N, O or S, and optionally substituted with a heterocyclic ring; aryl or a saturated or unsaturated heterocyclic ring, each of which is optionally substituted. In one example, X and NR3R4 are independently morpholine, thiomorpholine, imidazole, pyrrolidine, piperazine, pyridine or piperidine. In one example, X and NR3R4 are independently pyrrolidine. In another example, X is pyrrolidine substituted with pyrazine. In this example, V is H; A is fluoro; and W is naphthalenyl.
Examples of 5-6 membered heterocyclic rings include but are not limited to tetrahydrofuran, 1,3-dioxolane, 2,3-dihydrofuran, tetrahydropyran, benzofuran, isobenzofuran, 1,3-dihydro-isobenzofuran, isoxazole, 4,5-dihydroisoxazole, piperidine, pyrrolidine, pyrrolidin-2-one, pyrrole, pyridine, pyrimidine, octahydro-pyrrolo[3,4-b]pyridine, piperazine, pyrazine, morpholine, thiomorpholine, imidazole, imidazolidine-2,4-dione, benzimidazole, 1,3-dihydrobenzimidazol-2-one, indole, thiazole, benzothiazole, thiadiazole, thiophene, tetrahydro-thiophene 1,1-dioxide, diazepine, triazole, guanidine, diazabicyclo[2.2.1]heptane, 2,5-diazabicyclo[2.2.1]heptane, and 2,3,4,4a,9,9a-hexahydro-1H-β-carboline.
In the above formula 1, W may be benzene, pyridine, biphenyl, naphthalene, phenanthrene, quinoline, isoquinoline, quinazoline, cinnoline, phthalazine, quinoxaline, indole, benzimidazole, benzoxazole, benzthiazole, benzofuran, anthrone, xanthone, acridone, fluorenone, carbazolyl, pyrimido[4,3-b]furan, pyrido[4,3-b]indole, pyrido[2,3-b]indole, dibenzofuran, acridine or acridizine.
In the above formula 1, U may be OR2 and R2 is a C1-6 alkyl optionally substituted with a carbocyclic or heterocyclic ring.
In the above formula 1, each optionally substituted moiety is substituted with one or more halo, OR2, NR1R2, carbamate, C1-10 alkyl, C2-10 alkenyl, each optionally substituted by halo, ═O, aryl or one or more heteroatoms; inorganic substituents, aryl, carbocyclic or a heterocyclic ring.
The compounds of the present invention may be chiral. As used herein, a chiral compound is a compound that is different from its mirror image, and has an enantiomer. Methods of synthesizing chiral compounds and resolving a racemic mixture of enantiomers are well known to those skilled in the art. See, e.g., March, “Advanced Organic Chemistry,” John Wiley and Sons, Inc., New York, (1985), which is incorporated herein by reference.
The present invention also provides pharmaceutical compositions comprising compounds having formula 1, and a pharmaceutically acceptable excipient.
Furthermore, the present invention provides methods for ameliorating a cell proliferative disorder, comprising administering to a subject in need thereof an effective amount of a compound having formula 1 or a pharmaceutical composition thereof, thereby ameliorating said cell-proliferative disorder. In one example, the cell proliferative disorder is cancer. In another example, cell proliferation is reduced, or cell death is induced. The subject may be human or animal.
The present invention also provides methods for reducing cell proliferation or inducing cell death, comprising contacting a system with an effective amount of a compound having formula I or a pharmaceutical composition thereof, thereby reducing cell proliferation or inducing cell death in said system. The system may be a cell or tissue.
The present invention further provides methods for reducing microbial titers, comprising contacting a system with an effective amount of a compound having formula 1 or a pharmaceutical composition thereof, thereby reducing microbial titers. The system may be a cell or tissue. In one example, the microbial titers are viral, bacterial or fungal titers.
Further, the present invention provides methods for ameliorating a microbial infection, comprising administering to a subject in need thereof an effective amount of a compound having formula 1 or a pharmaceutical composition thereof, thereby ameliorating said microbial infection. The subject may be human or animal. In one example, the microbial infection is viral, bacterial or fungal.



DESCRIPTION OF THE FIGS.
FIG. 1 shows antitumor activity of a compound having formula 1 tested in a Ramos xenograft model.
FIG. 2 shows antitumor activity of a compound having formula 1 tested in HCT 116.



DESCRIPTION OF THE INVENTION
The present invention relates to quinoline derivatives having formula 1, and pharmaceutically acceptable salts, esters, and prodrugs thereof. In particular embodiments, the compounds interact with regions of DNA that can form quadruplexes. The present invention also relates to methods for treating cancer, bacterial and viral infections using such compounds.
Because regions of DNA that can form quadruplexes are regulators of biological processes such as oncogene transcription, modulators of quadruplex biological activity can be utilized as cancer therapeutics. Molecules that interact with regions of DNA that can form quadruplexes can exert a therapeutic effect on certain cell proliferative disorders and related conditions. Particularly, abnormally increased oncogene expression can cause cell proliferative disorders and quadruplex structures typically down-regulate oncogene expression. Examples of oncogenes include but are not limited to MYC, HIF, VEGF, ABL, TGF, PDGFA, MYB, SPARC, HUMTEL, HER, VAV, RET, H-RAS, EGF, SRC, BCL1, BCL2, and other oncogenes known to one of skill in the art.
Molecules that bind to regions of DNA that can form quadruplexes can exert a biological effect according to different mechanisms, which include for example, stabilizing a native quadruplex structure, inhibiting conversion of a native quadruplex to duplex DNA by blocking strand cleavage, and stabilizing a native quadruplex structure having a quadruplex-destabilizing nucleotide substitution and other sequence specific interactions. Thus, compounds that bind to regions of DNA that can form quadruplexes described herein may be administered to cells, tissues, or organisms for the purpose of down-regulating oncogene transcription and thereby treating cell proliferative disorders. The terms “treatment” and “therapeutic effect” as used herein refer to reducing or stopping a cell proliferation rate (e.g., slowing or halting tumor growth) or reducing the number of proliferating cancer cells (e.g., removing part or all of a tumor).
Determining whether the biological activity of native DNA that can form quadruplexes is modulated in a cell, tissue, or organism can be accomplished by monitoring quadruplex biological activity. Quadruplex forming regions of DNA biological activity may be monitored in cells, tissues, or organisms, for example, by detecting a decrease or increase of gene transcription in response to contacting the quadruplex forming DNA with a molecule. Transcription can be detected by directly observing RNA transcripts or observing polypeptides translated by transcripts, which are methods well known in the art.
Molecules that interact with quadruplex forming DNA and quadruplex forming nucleic acids can be utilized to treat many cell proliferative disorders. Cell proliferative disorders include, for example, colorectal cancers and hematopoietic neoplastic disorders (i.e., diseases involving hyperplastic/neoplastic cells of hematopoietic origin such as those arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof). The diseases can arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (Vaickus, Crit. Rev. in Oncol./Hemotol. 11:267-297 (1991)). Lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease. Cell proliferative disorders also include cancers of the colorectum, breast, lung, liver, pancreas, lymph node, colon, prostate, brain, head and neck, skin, liver, kidney, and heart. Compounds that interact with regions of DNA that can form quadruplexes also can be utilized to target cancer related processes and conditions, such as increased angiogenesis, by inhibiting angiogenesis in a subject.
The present invention provides a method for reducing cell proliferation or for treating or alleviating cell proliferative disorders, comprising contacting a system having a DNA capable of forming a quadruplex with a compound having formula 1. The system may be a group of cells or one or more tissues. In one embodiment, the system is a subject in need of a treatment of a cell proliferative disorder (e.g., a mammal such as a mouse, rat, monkey, or human). The present invention also provides a method for treating colorectal cancer by administering a compound that interacts with a c-MYC quadruplex forming region to a subject in need thereof, thereby reducing the colorectal cancer cell proliferation. Furthermore, the present invention provides a method for inhibiting angiogenesis and optionally treating a cancer associated with angiogenesis, comprising administering a compound that interacts with a vascular endothelial growth factor (VEGF) quadruplex forming region to a subject in need thereof, thereby reducing angiogenesis and optionally treating a cancer associated with angiogenesis.
As used herein, the term “alkyl” refers to a carbon-containing compound, and encompasses compounds containing one or more heteroatoms. The term “alkyl” also encompasses compounds substituted with one or more non-interfering substituents. Examples of non-interfering substituents include but are not limited to OR1, amino, amido, halo, ═O, aryl, heterocyclic groups, or inorganic substituents, and other substituents that do not interfere with the activity of the compound.
As used herein, the term “carbocycle” refers to a cyclic compound containing only carbon atoms in the ring.
As used herein, the term “heterocycle” refers to a cyclic compound comprising a heteroatom, including monocyclic or bicyclic heterocycles. As used herein, the term “heteroatom” refers to any atom that is not carbon or hydrogen, such as nitrogen, oxygen or sulfur. Examples of heterocycles include but are not limited to oxirane, oxetane, pyran, tetrahydropyran, dioxane, lactones, aziridine, azetidine, pyrrolidine, piperidine, morpholine, lactams, and tetrahydrofuran.
As used herein, the term “bicyclic compound” refers to a compound having two rings which share a pair of bridgehead carbon atoms. Examples of bicyclic compounds include but are not limited to decalin, norbornane, camphor, and diazabicyclo[2.2.1]heptane.
As used herein, the terms “heteroaryl” or “heteroaromatic” refer to an aromatic hetercycle. Examples of heteroaryls include but are not limited to furan, pyrrole, pyridine, pyrimidine, imidazole, or triazole.
The terms “treat,” “treatment” and “therapeutic effect” as used herein refer to reducing or stopping a cell proliferation rate (e.g., slowing or halting tumor growth) or reducing the number of proliferating cancer cells (e.g., removing part or all of a tumor). These terms also are applicable to reducing a titre of a microorganism in a system (i.e., cell, tissue, or subject) infected with a microorganism, reducing the rate of microbial propagation, reducing the number of symptoms or an effect of a symptom associated with the microbial infection, and/or removing detectable amounts of the microbe from the system. Examples of microorganism include but are not limited to virus, bacterium and fungus.
Compounds that interact with quadruplex forming regions of DNA can also be used to reduce a microbial infection, such as a viral infection. Retroviruses offer a wealth of potential targets for G-quadruplex targeted therapeutics. G-quadruplex structures have been implicated as functional elements in at least two secondary structures formed by either viral RNA or DNA in HIV, the dimer linker structure (DLS) and the central DNA flap (CDF). Additionally, DNA aptamers which are able to adopt either inter- or intramolecular quadruplex structures are able to inhibit viral replication. In one example, DNA aptamers are able to inhibit viral replication by targeting the envelope glycoprotein (putatively). In another example, DNA aptamers inhibit viral replication by targeting the HIV-integrase respectively, suggesting the involvement of native quadruplex structures in interaction with the integrase enzyme.
Dimer linker structures, which are common to all retroviruses, serve to bind two copies of the viral genome together by a non-covalent interaction between the two 5′ ends of the two viral RNA sequences. The genomic dimer is stably associated with the gag protein in the mature virus particle. In the case of HIV, the origin of this non-covalent binding may be traced to a 98 base-pair sequence containing several runs of at least two consecutive guanines (e.g., the 3′ for the formation of RNA dimers in vitro). An observed cation (potassium) dependence for the formation and stability of the dimer in vitro, in addition to the failure of an antisense sequence to effectively dimerize, has revealed the most likely binding structure to be an intermolecular G-quadruplex.
Prior to integration into the host genome, reverse transcribed viral DNA forms a pre-integration complex (PIC) with at least two major viral proteins, integrase and reverse transcriptase, which is subsequently transported into the nucleus. The Central DNA Flap (CDF) refers to 99-base length single-stranded tail of the +strand, occurring near the center of the viral duplex DNA, which is known to a play a role in the nuclear import of the PIC. Oligonucleotide mimics of the CDF have been shown to form intermolecular G-quadruplex structures in cell-free systems.
Thus, compounds that recognize quadruplex forming regions can be used to stabilize the dimer linker structure and thus prevent de-coupling of the two RNA strands. Also, by binding to the quadruplex structure formed by the CDF, protein recognition and/or binding events for nuclear transport of the PIC may be disrupted. In either case, a substantial advantage can exist over other anti-viral therapeutics. Current Highly Active Anti-Retroviral Therapeutic (HAART) regimes rely on the use of combinations of drugs targeted towards the HIV protease and HIV integrase. The requirement for multi-drug regimes is to minimize the emergence of resistance, which will usually develop rapidly when agents are used in isolation. The source of such rapid resistance is the infidelity of the reverse transcriptase enzyme which makes a mutation approximately once in every 10,000 base pairs. An advantage of targeting viral quadruplex structures over protein targets, is that the development of resistance is slow or is impossible. A point mutation of the target quadruplex can compromise the integrity of the quadruplex structure and lead to a non-functional copy of the virus. A single therapeutic agent based on this concept may replace the multiple drug regimes currently employed, with the concomitant benefits of reduced costs and the elimination of harmful drug/drug interactions.
The present invention provides a method for reducing a microbial titer in a system, comprising contacting a system having a native DNA quadruplex forming region with a compound having formula 1. The system may be one or more cells or tissues. Examples of microbial titers include but are not limited to viral, bacterial or fungal titers. In a particular embodiment, the system is a subject in need of a treatment for a viral infection (e.g., a mammal such as a mouse, rat, monkey, or human). Examples of viral infections include infections by a hepatitis virus (e.g., hepatitis B or C), human immunodeficiency virus (HIV), rhinovirus, herpes-zoster virus (VZV), herpes simplex virus (e.g., HSV-1 or HSV-2), cytomegalovirus (CMV), vaccinia virus, influenza virus, encephalitis virus, hantavirus, arbovirus, West Nile virus, human papilloma virus (HPV), Epstein-Barr virus, and respiratory syncytial virus. The present invention also provides a method for treating HIV infection by administering a compound having formula I to a subject in need thereof, thereby reducing the HIV infection.
Identifying Compounds that can Bind to Quadruplex Forming Regions of DNA
Compounds described herein are identified as compounds that can bind to quadruplex forming regions of DNA where a biological activity of this region, often expressed as a “signal,” produced in a system containing the compound is different than the signal produced in a system not containing the compound. While background signals may be assessed each time a new molecule is probed by the assay, detecting the background signal is not required each time a new molecule is assayed.
Examples of quadruplex forming nucleic acid sequences are set forth in the following Table A:










TABLE A






SEQ





ID



SEQUENCE
NO
ORIGIN


















TG4AG3TG4AG3TG4AAGG
1
CMYC



 


GGGGGGGGGGGGGCGGGGGCGGGGGCGGGGGAGGGGC
2
PDGFA


 


G8ACGCG3AGCTG5AG3CTTG4CCAG3CG4CGCTTAG5
3
PDGFB/




c-sis


 


AGGAAGGGGAGGGCCGGGGGGAGGTGGC
4
CABL


 


AGGGGCGGGGCGGGGCGGGGGC
5
RET


 


GGGAGGAAGGGGGCGGGAGCGGGGC
6
BCL-2


 


GGGGGGCGGGGGCGGGCGCAGGGGGAGGGGGC
7
Cyclin




D1/BCL-1


 


CGGGGCGGGGCGGGGGCGGGGGC
8
H-RAS


 


AGAGGAGGAGGAGGTCACGGAGGAGGAGGAGAAGGAGGA
9
CMYB


GGAGGAA


 


(GGA)4
10
VAV


 


AGAGAAGAGGGGAGGAGGAGGAGGAGAGGAGGAGGCGC
11
HMGA2


 


GGAGGGGGAGGGG
12
CPIM


 


AGGAGAAGGAGGAGGTGGAGGAGGAGG
13
HER2/neu


 


AGGAGGAGGAGAATGCGAGGAGGAGGGAGGAGA
14
EGFR


 


GGGGCGGGCCGGGGGCGGGGTCCCGGCGGGGCGGAG
15
VEGF


 


CGGGAGGAGGAGGAAGGAGGAAGCGCG
16
CSRC









In addition to determining whether a test molecule or test nucleic acid gives rise to a different signal, the affinity of the interaction between the nucleic acid and the compound may be quantified. IC50, Kd, or Ki threshold values may be compared to the measured IC50 or Kd values for each interaction, and thereby identify a test molecule as a quadruplex interacting molecule or a test nucleic acid as a quadruplex forming nucleic acid. For example, IC50 or Kd threshold values of 10 μM or less, 1 μM or less, and 100 nM or less are often utilized. In another example, threshold values of 10 nM or less, 1 nM or less, 100 pM or less, and 10 pM or less may be utilized to identify quadruplex interacting molecules and quadruplex forming nucleic acids.
Many assays are available for identifying compounds that have affinity for quadruplex forming regions of DNA. In some of these assays, the biological activity is the quadruplex nucleic acid binding to a compound and binding is measured as a signal. In other assays, the biological activity is a polymerase arresting function of a quadruplex and the degree of arrest is measured as a decrease in a signal. In certain assays, the biological activity is transcription and transcription levels can be quantified as a signal. In another assay, the biological activity is cell death and the number of cells undergoing cell death is quantified. Another assay monitors proliferation rates of cancer cells. Examples of assays are fluorescence binding assays, gel mobility shift assays (see, e.g., Jin & Pike, Mol. Endocrinol. (1996) 10:196-205), polymerase arrest assays, transcription reporter assays, cancer cell proliferation assays, and apoptosis assays (see, e.g., Amersham Biosciences (Piscataway, N.J.)), and embodiments of such assays are described hereafter. Also, topoisomerase assays can be utilized to determine whether the quadruplex interacting molecules have a topoisomerase pathway activity (see, e.g., TopoGEN, Inc. (Columbus, Ohio)).
Gel Electrophoretic Mobility Shift Assay (EMSA)
An EMSA is useful for determining whether a nucleic acid forms a quadruplex and whether a nucleotide sequence is quadruplex-destabilizing. EMSA is conducted as described previously (Jin & Pike, Mol. Endocrinol. 10: 196-205 (1996)) with minor modifications. Generally, synthetic single-stranded oligonucleotides are labeled in the 5′-terminus with T4-kinase in the presence of [γ-32P] ATP (1,000 mCi/mmol, Amersham Life Science) and purified through a sephadex column. 32P-labeled oligonucleotides ( ̃30,000 cpm) are then incubated with or without various concentrations of a testing compound in 20 μl of a buffer containing 10 mM Tris pH 7.5, 100 mM KCl, 5 mM dithiothreitol, 0.1 mM EDTA, 5 mM MgCl2, 10% glycerol, 0.05% Nonedit P-40, and 0.1 mg/ml of poly(dI-dC) (Pharmacia). After incubation for 20 minutes at room temperature, binding reactions are loaded on a 5% polyacrylamide gel in 0.25× Tris borate-EDTA buffer (0.25×TBE, 1×TBE is 89 mM Tris-borate, pH 8.0, 1 mM EDTA). The gel is dried and each band is quantified using a phosphoimager.
DMS Methylation Protection Assay
Chemical footprinting assays are useful for assessing quadruplex structure. Quadruplex structure is assessed by determining which nucleotides in a nucleic acid are protected or unprotected from chemical modification as a result of being inaccessible or accessible, respectively, to the modifying reagent. A DMS methylation assay is an example of a chemical footprinting assay. In such an assay, bands from EMSA are isolated and subjected to DMS-induced strand cleavage. Each band of interest is excised from an electrophoretic mobility shift gel and soaked in 100 mM KCl solution (300 μl) for 6 hours at 4° C. The solutions are filtered (microcentrifuge) and 30,000 cpm (per reaction) of DNA solution is diluted further with 100 mM KCl in 0.1×TE to a total volume of 70 μl (per reaction). Following the addition of 1 μl salmon sperm DNA (0.1 μg/μl), the reaction mixture is incubated with 1 μl DMS solution (DMS:ethanol; 4:1; v:v) for a period of time. Each reaction is quenched with 18 μl of stop buffer (b-mercaptoathanol:water:NaOAc (3 M); 1:6:7; v:v:v). Following ethanol precipitation (twice) and piperidine cleavage, the reactions are separated on a preparative gel (16%) and visualized on a phosphoimager.
Polymerase Arrest Assay
An arrest assay includes a template nucleic acid, which may comprise a quadruplex forming sequence, and a primer nucleic acid which hybridizes to the template nucleic acid 5′ of the quadruplex-forming sequence. The primer is extended by a polymerase (e.g., Taq polymerase), which advances from the primer along the template nucleic acid. In this assay, a quadruplex structure can block or arrest the advance of the enzyme, leading to shorter transcription fragments. Also, the arrest assay may be conducted at a variety of temperatures, including 45° C. and 60° C., and at a variety of ion concentrations.
An example of the Taq polymerase stop assay is described in Han, et at., Nuci. Acids Res. (1999) 27:537-542, which is a modification of that used by Weitzmann, et at., J. Bid. Chem. (1996) 271:20958-20964. Briefly, a reaction mixture of template DNA (50 nM), Tris.HCl (50 mM), MgCl2 (10 mM), DTT (0.5 mM), EDTA (0.1 mM), BSA (60 ng), and 5′-end-labeled quadruplex nucleic acid ( ̃18 nM) is heated to 90° C. for 5 minutes and allowed to cool to ambient temperature over 30 minutes. Taq Polymerase (1 μl) is added to the reaction mixture, and the reaction is maintained at a constant temperature for 30 minutes. Following the addition of 10 μl stop buffer (formamide (20 ml), 1 M NaOH (200 μl), 0.5 M EDTA (400 μl), and 10 mg bromophenol blue), the reactions are separated on a preparative gel (12%) and visualized on a phosphoimager. Adenine sequencing (indicated by “A” at the top of the gel) is performed using double-stranded DNA Cycle Sequencing System from Life Technologies. The general sequence for the template strands is TCCAACTATGTATAC (SEQ ID NO:17)-INSERT-TTAGCGACACGCAATTGCTATAGTGAGTCGTATTA (SEQ ID NO:18), where “INSERT” refers to a nucleic acid sequence comprising a quadruplex forming sequence (See e.g., Table A). Bands on the gel that exhibit slower mobility are indicative of quadruplex formation.
High Throughput Polymerase Arrest Assay
A high throughput polymerase arrest assay has been developed. The assay comprises contacting a template nucleic acid, often DNA, with a primer, which also is often DNA; contacting the primer/template complex with a compound described herein (also referred to as a “test compound”); contacting the primer/template complex with a polymerase; and separating reaction products. The assay often includes the step of denaturing the primer/template complex mixture and then renaturing the complex, which often is carried out before a test molecule is added to the system. Multiple assays often are carried out using varying concentrations of a test compound, such that an IC50 value can be obtained, for example. The reaction products often include extended primers of different lengths. Where a test compound does not significantly interact with a quadruplex structure in the template, the primer often is extended to the end of the template.
Where a test compound significantly interacts with a quadruplex structure in the template, the primer often is extended only to the quadruplex structure in the template and no further. Thus, the reaction mixture often includes at least two reaction products when a test compound interacts with a quadruplex structure in the template, one having a completely extended primer and one having an incompletely extended primer, and these two reaction products are separated. The products may be separated using any convenient separation method, such as mass spectrometry and in one embodiment, capillary electrophoresis.
The reaction products often are identified by detecting a detectable label linked to the primer. The detectable label may be non-covalently linked to the 5′ end of the primer (e.g., a biotin molecule covalently linked to the 5′ end of the primer which is non-covalently linked to an avidin molecule joined to a detectable label). The detectable label may be joined to the primer at any stage of the assay, sometimes before the primer is added to the system, after the primer is extended, or after the products are separated. The detectable label often is covalently linked to the primer using a procedure selected based upon the nature of the chemical groups in the detectable label.
Many methods for covalently linking detectable labels to nucleic acids are available, such as chemically coupling an allylamine-derivatized nucleotide to a succinimidyl-ester derivative of a detectable label, and then generating a primer using the labeled nucleotide. (See, e.g., Nature Biotech (2000) 18:345-348 and http address info.med.yale.edu/genetics/ward/tavi/n_coupling.html). A spacer (often between 5-16 carbon atoms long) sometimes is incorporated between the detectable label and the nucleotide. Any convenient detectable label may be utilized, including but not limited to a radioactive isotope (e.g., 125I, 131I, 35S, 32P, 14C or 3H); a light scattering label (e.g., a spherical gold or silver label; Genicon Sciences Corporation, San Diego, Calif. and U.S. Pat. No. 6,214,560); an enzymic or protein label (e.g., GFP or peroxidase); or another chromogenic label or dye sometimes is utilized. Often, a fluorescent label is utilized (e.g., amino-methyl coumarin (AMCA); diethyl aminomethyl coumarin (DEAC); cascade blue (CB); fluorescein isothiocyanate (FITC); Oregon green (OG); Alexa 488 (A488); rhodamine green (RGr); lanthanide chelate (e.g., europium), carboxy-rhodamine 6G (R6G); tetramethyl rhodamine (TAMRA); Texas Red (TxR); Cy3; Cy3.5; Cy5, Cy5.5 and carboxynaphtofluorescein (CNF), digoxigenin (DIG); and 2,4-dinitrophenyl (DNP)). Other fluorophores and attendant excitation and emission wavelengths are described in Anantha, et al., Biochemistry (1998) 37:2709-2714 and Qu & Chaires, Methods Enzymol (2000) 321:353-369).
In an embodiment, a primer oligonucleotide covalently linked to a fluorescent label is contacted with template DNA. The resulting complex is contacted with a test molecule and then contacted with a polymerase capable of extending the primer. The reaction products then are separated and detected by capillary electrophoresis. A longer primer sequence was used for practicing this embodiment as compared to embodiments where the primer includes no covalently-linked fluorophore or where capillary electrophoresis is not utilized for separation. Deoxynucleotides are added at any stage of the assay before the separation, often when the primer is contacted with the template DNA. The template DNA/primer complex often is denatured (e.g., by increasing the temperature of the system) and then renatured (e.g., by cooling the system) before a test compound is added).
The following is a specific example of the assay embodiment. A 5′-fluorescent-labeled (FAM) primer (P45, 15 nM) was mixed with template DNA (15 nM) in a Tris-HCL buffer (15 mM Tris, pH 7.5) containing 10 mM MgCl2, 0.1 mM EDTA and 0.1 mM mixed deoxynucleotide triphosphates (dNTP's). The FAM-P45 primer (5′-6FAM-AGTCTGAC TGACTGTACGTAGCTAATACGACTCACTATAGCAATT-3′) (SEQ ID NO:19) and the template DNA (5′-TCCAACTATGTATACTGGGGAGGGTGGGGAGGGTGGGGAAGGTT AGCGACACGCAATTGCTATAGTGAGTCGTATTAGCTACGTACAGTCAGTCAGACT-3′) (SEQ ID NO:20) were synthesized and HPLC purified by Applied Biosystems. The mixture was denatured at 95° C. for 5 minutes and, after cooling down to room temperature, was incubated at 37° C. for 15 minutes.
After cooling down to room temperature, 1 mM KCl2 and the test compound (various concentrations) were added and the mixture incubated for 15 minutes at room temperature. The primer extension was preformed by adding 10 mM KCl and Taq DNA Polymerase (2.5 U/reaction, Promega) and incubating at 70° C. for 30 minutes. The reaction was stopped by adding 1 μl of the reaction mixture to 10 μl Hi-Di Formamide mixed and 0.25 μl LIZ120 size standard. Hi-Di Formamide and LIZ120 size standard were purchased from Applied Biosystems. The partially extended quadruplex arrest product was between 61 or 62 bases long and the full-length extended product was 99 bases long. The products were separated and analyzed using capillary electrophoresis. Capillary electrophoresis was performed using an ABI PRISM 3100-Avant Genetic Analyzer. The assay was performed using compounds described above and results are shown in Table 1. μM concentrations reported in Table 1 are concentrations at which 50% of the DNA was arrested in the assay (i.e., the ratio of shorter partially extended DNA (arrested DNA) to full-length extended DNA is 1:1).
Transcription Reporter Assay
In a transcription reporter assay, test quadruplex DNA is coupled to a reporter system, such that a formation or stabilization of a quadruplex structure can modulate a reporter signal. An example of such a system is a reporter expression system in which a polypeptide, such as luciferase or green fluorescent protein (GFP), is expressed by a gene operably linked to the potential quadruplex forming nucleic acid and expression of the polypeptide can be detected. As used herein, the term “operably linked” refers to a nucleotide sequence which is regulated by a sequence comprising the potential quadruplex forming nucleic acid. A sequence may be operably linked when it is on the same nucleic acid as the quadruplex DNA, or on a different nucleic acid. An exemplary luciferase reporter system is described herein.
A luciferase promoter assay described in He, et al., Science (1998) 281:1509-1512 often is utilized for the study of quadruplex formation. Specifically, a vector utilized for the assay is set forth in reference 11 of the He, et al., document. In this assay, HeLa cells are transfected using the lipofectamin 2000-based system (Invitrogen) according to the manufacturer's protocol, using 0.1 μg of pRL-TK (Renilla luciferase reporter plasmid) and 0.9 μg of the quadruplex-forming plasmid. Firefly and Renilla luciferase activities are assayed using the Dual Luciferase Reporter Assay System (Promega) in a 96-well plate format according to the manufacturer's protocol.
Circular Dichroism Assay
Circular dichroism (CD) is utilized to determine whether another molecule interacts with a quadruplex nucleic acid. CD is particularly useful for determining whether a PNA or PNA-peptide conjugate hybridizes with a quadruplex nucleic acid in vitro. PNA probes are added to quadruplex DNA (5 μM each) in a buffer containing 10 mM potassium phosphate (pH 7.2) and 10 or 250 mM KCl at 37° C. and then allowed to stand for 5 minutes at the same temperature before recording spectra. CD spectra are recorded on a Jasco J-715 spectropolarimeter equipped with a thermoelectrically controlled single cell holder. CD intensity normally is detected between 220 nm and 320 nm and comparative spectra for quadruplex DNA alone, PNA alone, and quadruplex DNA with PNA are generated to determine the presence or absence of an interaction (see, e.g., Datta, et al., JACS (2001) 123:9612-9619). Spectra are arranged to represent the average of eight scans recorded at 100 nm/min.
Fluorescence Binding Assay
An example of a fluorescence binding assay is a system that includes a quadruplex nucleic acid, a signal molecule, and a test molecule. The signal molecule generates a fluorescent signal when bound to the quadruplex nucleic acid (e.g., N-methylmesoporphyrin IX (NMM)), and the signal is altered when a test compound competes with the signal molecule for binding to the quadruplex nucleic acid. An alteration in the signal when test molecule is present as compared to when test compound is not present identifies the test compound as a quadruplex interacting compound.
50 μl of quadruplex nucleic acid or a nucleic acid not capable of forming a quadruplex is added in 96-well plate. A test compound also is added in varying concentrations. A typical assay is carried out in 100 μl of 20 mM HEPES buffer, pH 7.0, 140 mM NaCl, and 100 mM KCl. 50 μl of the signal molecule NMM then is added for a final concentration of 3 μM. NMM is obtained from Frontier Scientific Inc, Logan, Utah. Fluorescence is measured at an excitation wavelength of 420 nm and an emission wavelength of 660 nm using a FluroStar 2000 fluorometer (BMG Labtechnologies, Durham, N.C.). Fluorescence often is plotted as a function of concentration of the test compound or quadruplex-targeted nucleic acid and maximum fluorescent signals for NMM are assessed in the absence of these molecules.
Cell Proliferation Assay
In a cancer cell proliferation assay, cell proliferation rates are assessed as a function of different concentrations of test compounds added to the cell culture medium. Any cancer cell type can be utilized in the assay. In one embodiment, colon cancer cells are cultured in vitro and test compounds are added to the culture medium at varying concentrations. A useful colon cancer cell line is colo320, which is a colon adenocarcinoma cell line deposited with the National Institutes of Health as accession number JCRB0225. Parameters for using such cells are available at the http address cellbank.nihs.go.jp/cell/data/jcrb0225. htm.
Formulation of Compounds
As used herein, the term “pharmaceutically acceptable salts, esters and amides” includes but are not limited to carboxylate salts, amino acid addition salts, esters and amides of the compounds, as well as the zwitterionic forms thereof, which are known to those skilled in the art as suitable for use with humans and animals. (See, e.g., Gerge, S. M., et al., “Pharmaceutical Salts,” J. Pharm. Sci. (1977) 66:1-19, which is incorporated herein by reference.)
Any suitable formulation of the compounds described herein can be prepared. In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts. Pharmaceutically acceptable salts are obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids also are made.
A compound may be formulated as a pharmaceutical composition and administered to a mammalian host in need of such treatment. In one embodiment, the mammalian host is human. Any suitable route of administration may be used, including but not limited to oral, parenteral, intravenous, intramuscular, topical and subcutaneous routes.
In one embodiment, a compound is administered systemically (e.g., orally) in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
Tablets, troches, pills, capsules, and the like also may contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Any material used in preparing any unit dosage form is pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.
The active compound also may be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts may be prepared in a buffered solution, often phosphate buffered saline, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The compound is sometimes prepared as a polymatrix-containing formulation for such administration (e.g., a liposome or microsome). Liposomes are described for example in U.S. Pat. No. 5,703,055 (Felgner, et al.) and Gregoriadis, Liposome Technology vols. I to III (2nd ed. 1993).
The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are peprared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
For topical administration, the present compounds may be applied in liquid form. Compounds often are administered as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid. Examples of useful dermatological compositions used to deliver compounds to the skin are known (see, e.g., Jacquet, et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith, et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
Compounds may be formulated with a solid carrier, which include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
Generally, the concentration of the compound in a liquid composition often is from about 0.1 wt % to about 25 wt %, sometimes from about 0.5 wt % to about 10 wt %. The concentration in a semi-solid or solid composition such as a gel or a powder often is about 0.1 wt % to about 5 wt %, sometimes about 0.5 wt % to about 2.5 wt %. A compound composition may be prepared as a unit dosage form, which is prepared according to conventional techniques known in the pharmaceutical industry. In general terms, such techniques include bringing a compound into association with pharmaceutical carrier(s) and/or excipient(s) in liquid form or finely divided solid form, or both, and then shaping the product if required. The compound composition may be formulated into any dosage form, such as tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions also may be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances which increase viscosity, including for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. The suspension may also contain one or more stabilizers.
The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
A useful compound dosage often is determined by assessing its in vitro activity in a cell or tissue system and/or in vivo activity in an animal system. For example, methods for extrapolating an effective dosage in mice and other animals to humans are known to the art (see, e.g., U.S. Pat. No. 4,938,949). Such systems can be used for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) of a compound. The dose ratio between a toxic and therapeutic effect is the therapeutic index and it can be expressed as the ratio ED50/LD50. The compound dosage often lies within a range of circulating concentrations for which the ED50 is associated with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compounds used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose sometimes is formulated to achieve a circulating plasma concentration range covering the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in in vitro assays, as such information often is used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
Another example of effective dose determination for a subject is the ability to directly assay levels of “free” and “bound” compound in the serum of the test subject. Such assays may utilize antibody mimics and/or “biosensors” generated by molecular imprinting techniques. The compound is used as a template, or “imprinting molecule”, to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. Subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated “negative image” of the compound and is able to selectively rebind the molecule under biological assay conditions (see, e.g., Ansell, et al., Current Opinion in Biotechnology (1996) 7:89-94 and in Shea, Trends in Polymer Science (1994) 2:166-173). Such “imprinted” affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix (see, e.g., Vlatakis, et al., Nature (1993) 361:645-647). Through the use of isotope-labeling, “free” concentration of compound can be readily monitored and used in calculations of IC50. Such “imprinted” affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC50. An example of such a “biosensor” is discussed in Kriz, et al., Analytical Chemistry (1995) 67:2142-2144.
Exemplary doses include milligram or microgram amounts of the compound per kilogram of subject or sample weight, for example, about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid described herein, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
The following examples are offered to illustrate but not to limit the invention.
EXAMPLES
The following are exemplary procedures for synthesizing substituted quinobenzoxazines analogs.
Example 1
Preparation of Substituted Quinobenzoxazine Analogs
The general synthetic scheme for the preparation of substituted quinobenzoxazines analogs is shown in Scheme 1.




Ethyl-(2′,3′,4′,5′-tetrafluorobenzoyl)-ethanoate




Potassium ethyl malonate (3.66 g, 21.5 mmol), MgCl2 (2.44 g, 25.7 mmol) and TEA (2.05 g, 20.3 mmol) were mixed in acetonitrile (70 ml) at 10-15° C. for 2.5 hr. 2,3,4,5-tetrafluorobenzoyl chloride (2.00 g, 10.3 mmol) in acetonitrile (10 ml) was added at 0° C. over 15 min followed by a second addition of TEA (0.23 g, 2.3 mmol). After allowing to warm to RT, the mixture was stirred for 16 hr. After removal of volatiles in vacculo Toluene (30 ml) was added and removed in vacuo. Following the addition of toluene (60 ml), HCl 1.5 M (40 ml) was added cautiously, ensuring the temperature did not exceed 25° C. The organic fraction was washed with HCl 1.5 M (2×25 ml) and water (2×25 ml), dried over MgSO4 and reduced to a light orange oil in vacuo ([M+1]+265, 98%).
Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate




Dimethyl acetal dimethyl formamide (0.61 g, 5.1 mmol) was added dropwise to ethyl-(2′,3′,4′,5′-tetrafluorobenzoyl)-ethanoate (0.9 g, 3.41 mmol) dissolved in acetic anhydride (2 ml), under argon. After 30 min solvent was removed in vacuo to leave the product as an orange oil in a quantitative yield ([M+1+] 320).
Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazine-5-carboxylate




Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate (3.4 g, 8.0 mmol) and 2-amino-4-phenyl-phenol (1.5 g, 8.0 mmol) in 20 DMSO (20 ml) was stirred under vacuum at 60° C. for 30 min. K2CO3 (5 g) and MeCN (20 ml) was added and the suspension was heated at 80° C. for 1 hr. After cooling to RT, the mixture was poured into a slight excess of dilute sulfuric acid and filtered. The product was recovered as a yellow-brown solid ([M+1]+420, 65%).
Example 2
Preparation of 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]l-8-phenyl-phenoxazine-5-carboxylic acid




Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazine-5-carboxylate (2.2 g, 5.3 mmol) was refluxed in a mixture of conc. HCl and acetic acid (20 ml each) for 2 hr. After cooling to room temperature cold water (40 ml) was added to the reaction mixture and the resulting precipitate filtered and washed with ether to afford the product as a yellow-brown solid 90% ([M+1]+392).
Example 3
Preparation of Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(napthyl-2′′,3′′-diamino)-prop-2-enoate




Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate (10.53 g, 33 mmol) in acetonitrile (50 ml) was added to a solution of 2,3-diaminonaphthalene (5.22 g, 33 mmol) in acetonitrile (150 ml), maintained at 50° C. under argon. After 3 hours, volatiles were removed in vacuo and the residue was subjected to chromatography over silica (15% EtOAc/Hexane) to yield the product as a yellow solid ([M+1]+433) (55%).
Example 4
Preparation of Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phendiazine-5-carboxylate




Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(napthyl-2′′, 3′′diamino)-prop-2-enoate (600 mg 1.4 mmol) was dissolved in a slurry of K2CO3 in DMF (500 ml), The mixture was stirred vigorously at 100° C. for 1 hour, then allowed to cool to RT. The K2CO3 was removed by filtration and the DMF removed in vacuo to leave a yellow-brown solid in quantitative yield. ([M+1]+393).
Example 5
Preparation of 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phendiazine-5-carboxylic acid




KOH solution (1N, 2.54 ml, 2.56 mmol) was added to a solution of ethyl 1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phendiazine-5-carboxylate (500 mg, 1.28 mmol) in ethanol (400 ml), heated under reflux. After 2 hours the reaction mixture was allowed to cool to RT, then neutralized with HCl solution (1N). The product was collected by filtration as a yellow solid, 89%. ([M+1]+365).
Example 6
Preparation of Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-phenthiazine-5-carboxylate
Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(N-aminobenzyldisulfide)-prop-2-enoate




Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate (17.7 g, 55.3 mmol) in acetonitrile (10 ml) was added to a solution of 1,2-aminothiophenol dimer (5.22 g, 33 mmol) in acetonitrile (100 ml). After 3 hours, volatiles were removed in vacuo and the residue was subjected to chromatography over silica (1% MeOH/DCM) to yield the product as a yellow solid ([M+1]+523) (50%).
Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-phenthiazine-5-carboxylate




Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(N-aminobenzyldisulfide)-prop-2-enoate (2.5 g 3.2 mmol) was dissolved in DMF (120 ml) and heated under reflux for six hours. Removal of DMF in vacuo gave the product as a yellow solid 90% ([M+1]+360).
Example 7
Preparation of 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-phenthiazine-5-carboxylic acid




KOH solution (1N, 3.0, 3.0 mmol) was added to a solution of ethyl 1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-phenthiazine-5-carboxylate (1000 mg, 2.5 mmol) in ethanol (400 ml), heated under reflux. After 2 hours the reaction mixture was allowed to cool to RT, then neutralized with HCl solution (1N). The product was collected by filtration as a yellow solid, ([M+1]+332, 95%)
Example 8
Preparation of 5,6-Difluoro-9-hydroxy-3-oxo-3H-pyrido[3,2,1-kl]pyrimido[g]phenoxazine-2-carboxylic acid 7-nitroquinazoline-4,6-diol




To a solution containing 20 ml of 48% aqueous HBr and 20 ml of AcOH was added 6-methoxy-7-nitro-3,4-dihydroquinazolin-4-one (1.4 g, 6.3 mmol) and the mixture was refluxed overnight. The resulting solution was evaporated to afford the crude phenol as a residue and was used without further purification (1.2 g, 5.8 mmol) (M+1, 208).
7-aminoguinazoline-4,6-diol




The crude product from above (1.0 g, 5.8 mmol) was diluted with 40 ml water and 3 g of Tin II chloride dihydrate was added and the reaction was stirred at room temperature. After 1 h the reaction was neutralized with K2CO3, and extracted with EtOAc (3×50 mL). The combined organic extracts were dried over sodium sulfate and the solvent was removed in vacuo to afford the crude amino alcohol (1.0 g, 5.6 mmol) (M+1, 178).
Ethyl; 5,6-difluoro-9-hydroxy-3-oxo-3H-pyrido[3,2,1-kl]pyrimido[g]phenoxazine-2-carboxylate




To a solution of the tetrafluoroenamine (2.2 g, 6.9 mmol) in DMSO (3 mL) was added the aminophenol (1.0 g, 5.6 mmol) and the reaction mixture was stirred under vacuum (rotary evaporator) at 60° C. for 20 minutes. The reaction mixture was allowed to cool to room temperature and was diluted with acetonitrile (200 mL) and potassium carbonate was added. The mixture was heated to reflux for 5 hours and poured into dilute HOAc/water. The solid product was collected by vacuum filtration and dried to afford the difluoroester as a tan solid (1.3 g, 3.2 mmol) (M+1, 412).
5,6-Difluoro-9-hydroxy-3-oxo-3H-pyrido[3,2,1-kl]pyrimido[g]phenoxazine-2-carboxylic acid




The difluoroester (1.3 g, 3.2 mmol) was dissolved in a 1:1 mixture of glacial acetic acid and 12 M HCl (20 mL) and refluxed for 30 min. The mixture was then cooled to room temperature and poured into water. The solid product was then collected by vacuum filtration and dried to afford the difluoroacid as a tan solid (0.98 g, 2.5 mmol) ([M+1]+392).
Example 9
Preparation of 2-(2-(Ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazin-9-yloxy)acetic acid
Ethyl 5,6-difluoro-9-hydroxy-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate




To a solution of the tetrafluoroenamine (5.8 g, 18.2 mmol), dissolved in DMSO (12 mL), was added 2,4-dihydroxyaniline hydrochloride (2.5 g, 15.5 mmol) and the mixture was heated to 60° C. under vacuum (rotary evaporator) for 20 minutes. The reaction mixture was then diluted with acetonitrile (100 mL) and potassium carbonate (3 g) was added and the mixture was refluxed overnight. The mixture was allowed to cool to room temperature and the solvent was removed in vacuo. A slight excess of 2 M HCl was added to rapidly dissolve the carbonate, and the solid precipitate was filtered and dried to afford the difluoroester as a tan solid (5.0 g, 13.9 mmol) (M+1, 360).
2-(2-(Ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazin-9-yloxy)acetic acid




To a solution of the difluoroester (2.1 g, 5.8 mmol) and tert-butylbromoacetate (2.0 g, 10.3 mmol) in DMF (30 mL) was added potassium carbonate (2.0 g) and the mixture was heated to 60° C. for 1 hour. The reaction was allowed to cool and poured into water (500 mL) and extracted with ethyl acetate (3×100 mL),washed with brine, dried over magnesium sulfate and filtered over a pad of silica gel (30×50 mm), eluting with ethyl acetate. The solvent was removed in vacuo and the resulting material was triturated with hexanes and dried to afford the tert-butyl ester as a tan solid (2.8 g, 5.8 mmol). This material was dissolved in trifluoroacetic acid (40 mL) and stirred at room temperature for 30 minutes. The solvent was removed in vacuo to afford the acid as a tan solid (2.4 g, 5.7 mmol) (M+1, 418).
Example 10
Preparation of 9-(Carboxymethoxy)-5,6-difluoro-3-oxo-3H-Pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid




The difluoroester (2.4 g, 5.7 mmol) was dissolved in a 1:1 mixture of glacial acetic acid and 12 M HCl (40 mL) and refluxed for 1 hour. The mixture was then cooled to room temperature and poured into water. The solid product was then collected by vacuum filtration and dried to afford the difluoroacid as a tan solid (2.0 g, 5.1 mmol) (M+1, 390).
Example 11
Preparation of Ethyl 1,2,3-trifluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phenoxazine-5-carboxylate




To a solution of pentafluoroenamine (8 g, 23.7 mmol), prepared by a similar procedure as for the tetrafluoroenamine dissolved in DMSO (12 mL) was added 3-amino-2-naphthol (3.5 g, 21.9 mmol) and the mixture was heated to 60° C. under vacuum (rotary evaporator) for 2 hours. The reaction mixture was then diluted with acetonitrile (200 mL) and potassium carbonate (8.0 g) was added and the mixture was refluxed overnight. The mixture was allowed to cool to room temperature and the solvent was removed in vacuo. A slight excess of 2 M HCl was added to rapidly dissolve the carbonate, and the solid precipitate was filtered and dried to afford the difluoroester as a tan solid (1.3 g, 3.2 mmol) (M+1, 412).
Example 12
Preparation of 1,2,3-Trifluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phenoxazine-5-carboxylic acid




The trifluoroester (1.3 g, 3.2 mmol) was dissolved in acetic acid (5 mL) and 12 M HCl was added (5 mL) and the reaction mixture was heated to reflux for 2 hours. The mixture was then cooled to room temperature, poured into water and the solid product was collected by vacuum filtration and dried to afford the trifluoroacid as a pale solid (1.0 g, 2.6 mmol) (M+1, 384).
Example 13
Preparation of Ethyl 1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phenoxazine-5-carboxylate




To a solution of the enamine (30 g, 94 mmol) in ethyl acetate (100 mL) was added 3-amino-2-naphthol (10 g, 63 mmol) at room temperature and the mixture was immediately placed on a rotary evaporator and the solvent was removed over 2 hours at a temperature below 0° C. (ice formed on the flask) to produce a yellow solid. To this solid was added ether (200 mL) and the slurry was filtered to afford a yellow solid. This solid was then dissolved in DMF (200 mL) and potassium carbonate was added (16.5 g, 120 mmol) and the mixture was heated to 90° C. for 1 hour. The mixture was allowed to cool to room temperature and water was added (500 mL) and the resulting solid was filtered, washed with water and dried to afford the difluoroester as a tan solid (12.2 g, 30.8 mmol) (M+1, 394).
Example 14
Preparation of 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phenoxazine-5-carboxylic acid




The difluoroester (5 g, 12.7 mmol) was dissolved in methanol (50 mL) and conc HCl was added (20 mL) and the mixture was refluxed for 12 hours. The mixture was allowed to cool to room temperature and the solid was collected by vacuum filtration, washing with methanol to afford the difluoroacid as a light tan powder (3.6 g, 9.9 mmol) (M+1, 366).
Example 15
Preparation of Ethyl 1-fluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phenoxazine-5-carboxylate




To a solution of the enamine, similarly prepared as the tetrafluoroenamine (14 g, 46.3 mmol) in ethyl acetate (100 mL) was added 3-Amino-2-naphthol (5.0 g, 31.2 mmol) at room temperature and the mixture was immediately placed on a rotary evaporator and the solvent was removed over 2 hours at a temperature below 0° C. (ice formed on the flask) to produce a yellow solid. To this solid was added methanol (200 mL) and the slurry was filtered to afford a yellow solid. This solid was then dissolved in acetonitrile (200 mL) and potassium carbonate was added (10.0 g, 72.5 mmol) and the mixture was heated to 80° C. for 1 hour. The mixture was allowed to cool to room temperature and water was added (500 mL) and the resulting solid was filtered, washed with water and dried to afford the fluoroester as a tan solid (6.0 g, 16.0 mmol) (M+1, 376).
Example 16
Preparation of 1-Fluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phenoxazine-5-carboxylic acid




The fluoroester (6.0 g, 16.0 mmol) was dissolved in acetic acid (10 mL) and 12 M HCl was added (10 mL) and the reaction mixture was heated to reflux for 2 hours. The mixture was then cooled to room temperature, poured into water and the solid product was collected by vacuum filtration and dried to afford the fluoroacid as a pale solid (4.8 g, 13.8 mmol) (M+1, 348).
Example 17
Preparation of Ethyl 9-chloro-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate




To a solution of the enamine (14.4 g, 45.3 mmol) in ethyl acetate (200 mL) was added 5-chloro-2-aminophenol (5.0 g, 34.8 mmol) and the solvent was removed in vacuo with a rotary evaporator over 2 hours without heating. Methanol was added and the resulting phenolic enamine was isolated by vacuum filtration. The resulting solid (7.0 g) was dissolved in acetonitrile and potassium carbonate was added and the resulting mixture was heated to reflux for 2 hours. The mixture was then allowed to cool to room temperature and poured into Dilute HCl. The resulting solid was collected by vacuum filtration and dried to afford the difluoroester as a pale yellow solid (5.0 g, 13.3 mmol) (M+1, 378).
Example 18
Preparation of 9-Chloro-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid




The difluoroester (5.0 g, 13.3 mmol) was dissolved in acetic acid (45 mL) and 12 M HCl was added (30 mL) and the reaction mixture was heated to reflux for 2 hours. The mixture was then cooled to room temperature, poured into water and the solid product was collected by vacuum filtration and dried to afford the difluoroacid as a pale solid (4.0 g, 10.6 mmol) (M+1, 350).
Example 19
Preparation of Ethyl 10-chloro-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate




To a solution of the enamine (14.4 g, 45.3 mmol) in ethyl acetate (200 mL) was added 4-chloro-2-aminophenol (5.0 g, 34.8 mmol) and the solvent was removed in vacuo with a rotary evaporator over 2 hours without heating. Methanol was added and the resulting phenolic enamine was isolated by vacuum filtration. The resulting solid (7.5 g) was dissolved in acetonitrile and potassium carbonate was added and the resulting mixture was heated to reflux for 2 hours. The mixture was then allowed to cool to room temperature and poured into Dilute HCl. The resulting solid was collected by vacuum filtration and dried to afford the difluoroester as a pale yellow solid (5.0 g, 13.3 mmol) (M+1, 378).
Example 20
Preparation of 10-Chloro-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid




The difluoroester (2.5 g) was dissolved in acetic acid (25 mL) and 12 M HCl was added (20 mL) and the reaction mixture was heated to reflux for 2 hours. The mixture was then cooled to room temperature, poured into water and the solid product was collected by vacuum filtration and dried to afford the difluoroacid as a pale solid (2.0 g, 5.3 mmol) (M+1, 350).
Example 21
Preparation of Ethyl 5,6-Difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate




To a solution of the enamine (5.7 g, 17.9 mmol) in ethyl acetate (50 mL) was added 2-aminophenol (1.9 g, 17.43 mmol) at room temperature and the mixture was immediately placed on a rotary evaporator and the solvent was removed over 2 hours at a temperature below 0° C. (ice formed on the flask) to produce a yellow solid. To this solid was added ether (25 mL) and the slurry was filtered to afford a yellow solid. This solid was then dissolved in DMF (20 mL) and potassium carbonate was added (2.9 g, 21 mmol) and the mixture was heated to 90° C. for 1 hour. The mixture was allowed to cool to room temperature and water was added (200 mL) and the resulting solid was filtered, washed with water and dried to afford the phenoxazine as a tan solid (2.9 g, 8.45 mmol) (M+1, 344).
Example 22
Preparation of 5,6-Difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid




The difluoroester (5.0 g, 14 mmol) was dissolved in methanol (50 mL) and conc HCl was added (20 mL) and the mixture was refluxed for 2 hours. The mixture was allowed to cool to room temperature and the solid was collected by vacuum filtration, washing with methanol to afford the difluoroacid as a light tan powder (4.2 g, 13.3 mmol, 91%) (M+1, 316).
Example 23
Preparation of Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[h]-phenoxazine-5-carboxylate




To a solution of the enamine (14.0 g, 45.3 mmol) in ethyl acetate (200 mL) was added 1-amino-2-naphthol (5.0 g, 31.3 mmol) and the solvent was removed in vacuo with a rotary evaporator over 2 hours without heating. Methanol was added and the resulting phenolic enamine was isolated by vacuum filtration. The solid was dissolved in acetonitrile and potassium carbonate (10 g) was added and the mixture was heated to reflux for 2 hours. The mixture was then allowed to cool to room temperature and poured into Dilute HCl. The resulting solid was collected by vacuum filtration and dried to afford the difluoroester as a pale yellow solid (5.0 g, 13.3 mmol) (M+1, 376).
Example 24
Preparation of 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[h]-phenoxazine-5-carboxylic acid




The difluoroester (5.5 g) was dissolved in acetic acid (25 mL) and 12 M HCl was added (20 mL) and the reaction mixture was heated to reflux for 2 hours. The mixture was then cooled to room temperature, poured into water and the solid product was collected by vacuum filtration and dried to afford the difluoroacid as a pale solid (5.0 g, 14.4 mmol) (M+1, 348).
Example 25
Preparation of Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[f]-phenoxazine-5-carboxylate




To a solution of the enamine (45 g, 141 mmol) in ethyl acetate (500 mL) was added 2-amino-1-naphthol (15.0 g, 93.8 mmol) and the solvent was removed in vacuo with a rotary evaporator over 2 hours without heating. Methanol was added and the phenolic enamine was isolated by vacuum filtration. The resulting solid was dissolved in acetonitrile (400 mL) and potassium carbonate (25 g) was added and the mixture was heated to reflux for 2.5 hours. The mixture was then allowed to cool to room temperature and poured into Dilute HCl. The resulting solid was collected by vacuum filtration and dried to afford the difluoroester as a pale yellow solid (19.69 g, 50.1 mmol) (M+1, 394).
Example 26
Preparation of 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[f]-phenoxazine-5-carboxylic acid




The difluoroester (15.0 g,38.1 mmol) was dissolved in acetic acid (60 mL) and 12M HCl was added (60 mL) and the reaction mixture was heated to reflux for 2 hours. The mixture was then cooled to room temperature, poured into water and the solid product was collected by vacuum filtration and dried to afford the difluoroacid as a pale solid (11.7 g, 32 mmol) (M+1, 366).
Example 27
Preparation of Ethyl 1,2-Difluoro4-oxo-4H-pyrido[3,2,1-kl]benzo[b]furan[2,3-g]-phenoxazine-5-carboxylate 3-Aminodibenzofuran-2-ol




To a solution of the dibenzofuran (15 g, 70.4 mmol) dissolved in methylene chloride (500 mL) at 0° C. was added BBr3 (200 mL, 1 M in CH2Cl2) via addition funnel. After the addition was complete, the mixture was allowed to come to room temperature over 1 hour and then quenched with water followed by potassium carbonate (40 g). The resulting solid was recovered by vacuum filtration and dried to afford the hydroxyl dibenzofuran as a white solid (13.2 g, 199 mmol) (M+1, 200).
Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[b]furan[2,3-g]-phenoxazine-5-carboxylate




To a solution of the tetrafluoroenamine (15.0 g, 47 mmol) dissolved in DMSO (30 mL) was added the hydroxyl dibenzofuran (12.0 g, 60 mmol) and the mixture was heated to 60° C. under vacuum (rotary evaporator) for 20 minutes. The reaction mixture was then diluted with acetonitrile (200 mL) and potassium carbonate (17 g) was added and the mixture was refluxed for 2.5 hours. The mixture was allowed to cool to room temperature and the solvent was removed in vacuo. A slight excess of 2 M HCl was added to rapidly dissolve the carbonate, and the solid precipitate was filtered and dried to afford the difluoroester as a tan solid (15.0 g, 34.6 mmol) (M+1, 434).
Example 28
Preparation of 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[b]furan[2,3-g]-phenoxazine-5-carboxylic acid




The difluoroester (15.0 g,34.6 mmol) was dissolved in acetic acid (60 mL) and 12 M HCl was added (60 mL) and the reaction mixture was heated to reflux for 2 hours. The mixture was then cooled to room temperature, poured into water and the solid product was collected by vacuum filtration and dried to afford the difluoroacid as a pale solid (13.7 g, 34 mmol) (M+1, 406).
Example 29
Preparation of Ethyl 2-(ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-10-carboxylate




To a solution of the tetrafluoroenamine (7.0 g, 21.9 mmol) dissolved in DMSO (25 mL) was added 4-hydroxy-3-amino benzoic acid (3.0 g, 19.6 mmol) and the mixture was heated to 60° C. under vacuum (rotary evaporator) for 2 hours. The reaction mixture was then diluted with acetonitrile (200 mL) and potassium carbonate (8.0 g) was added and the mixture was refluxed overnight. The mixture was allowed to cool to room temperature and the solvent was removed in vacuo. A slight excess of 2 M HCl was added to rapidly dissolve the carbonate, and the solid precipitate was filtered and dried to afford the difluoroester as a tan solid (6.2 g, 16.0 mmol) (M+1, 388).
Example 30
Preparation of 2-(Ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-10-carboxylic acid




The difluoroester (6.2, 16.0 mmol g) was dissolved in acetic acid (25 mL) and 12 M HCl was added (20 mL) and the reaction mixture was heated to reflux for 2 hours. The mixture was then cooled to room temperature, poured into water and the solid product was collected by vacuum filtration and dried to afford the difluorodi-acid as a pale solid (5.3 g, 14.8 mmol) (M+1, 360).
Example 31
Preparation of Ethyl 5,6-difluoro-10-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate




A solution of the enamine (6.0 g, 18.7 mmol) and 2-amino-4-nitrophenol (3.5 g, 23.3 mmol) in acetonitrile was heated to 80° C. for 15 minutes. Potassium carbonate was then added (8.0 g) and the mixture was heated to reflux overnight. The reaction mixture was then filtered hot and the solvent was removed in vacuo to afford the crude nitroester (5.0 g, 12.8 mmol) (M+1, 389).
Example 32
Preparation of 5,6-Difluoro-10-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid




The crude difluoroester (5.0 g, 12.8 mmol) was dissolved in acetic acid (25 mL) and 12 M HCl was added (20 mL) and the reaction mixture was heated to reflux for 2 hours. The mixture was then cooled to room temperature, poured into water and the solid product was collected by vacuum filtration and dried to afford the difluoroacid as a pale solid (2.0 g, 5.5 mmol) (M+1, 361).
Example 33
Preparation of Ethyl 5,6-difluoro-3-oxo-10-phenyl-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate




A solution of the enamine (5,4 g, 16,9 mmol) and 3-amino-4-hydroxybiphenyl (3.5 g, 18.9 mmol) in acetonitrile was heated to 80° C. for 90 minutes. Potassium carbonate was then added (8.0 g) and the mixture was heated to reflux overnight. The reaction mixture was then filtered hot and the solvent was removed in vacuo to afford the crude difluoroester (3.9 g, 9.3 mmol) (M+1, 420).
Example 34
Preparation of 5,6-Difluoro-3-oxo-10-phenyl-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid




The crude difluoroester (3.6 g, 8.6 mmol) was dissolved in acetic acid (10 mL) and 12 M HCl was added (10 mL) and the reaction mixture was heated to reflux for 2 hours. The mixture was then cooled to room temperature, poured into water and the solid product was collected by vacuum filtration and dried to afford the difluoroacid as a pale solid (2.6 g, 6.6 mmol) (M+1,392).
Example 35
Preparation of Ethyl 1,2-Difluoro-4-oxo-4H-11-sulfonic-acid-pyrido[3,2,1-kl]benzo[h]-phenoxazine-5-carboxylate




To a solution of the tetrafluoroenamine (5.4 g, 16.9 mmol) dissolved in DMSO (30 mL) was added 1-amino-2-hydroxy-4-naphthalenesulfonic acid (4.8 g, 20 mmol) and the mixture was heated to 60° C. under vacuum (rotary evaporator) for 2 hours. To the reaction mixture was added potassium carbonate (10.0 g) and the mixture was heated to 60° C. for 1 hour. The mixture was allowed to cool to room temperature and a slight excess of 2 M HCl was added to rapidly dissolve the carbonate. The aqueous layer was decanted and the remaining organic residue was dissolved in methanol (100 mL) and precipitated with ethyl acetate (200 mL) and the solid precipitate was filtered and dried to afford the sulfonic acid as a brown solid (3.1 g, 6.5 mmol) (M+1, 474).
Example 36
Preparation of 1,2-Difluoro-4-oxo-4H-11-sulfonic-pyrido[3,2,1-kl]benzo[h]-phenoxazine-5-carboxylic acids




The crude difluoroester (1.5 g, 3.2 mmol) was dissolved in acetic acid (10 mL) and 12 M HCl was added (10 mL) and the reaction mixture was heated to reflux for 30 minutes. The solvent was removed in vacuo to afford the sulfonic acid as a brown solid (1.1 g, 2.5 mmol) (M+1, 446)
Example 37
Preparation of 2-(Ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-9-carboxylic acid




A solution of the difluoroenamine (5.2 g, 16.3 mmol) and 4-amino-3-hydroxybenzoic acid (4.0 g, 26.1 mmol) in DMSO was stirred at room temperature for 1.5 hours. Potassium carbonate (8 g) was then added and the reaction mixture was stirred under vacuum (rotary evaporator) for 1 hour. The mixture was then heated to 100° C. for 1 hour and then allowed to cool to room temperature. The reaction mixture was then poured into 1 M H2SO4 (500 mL) and the solids were recovered by vacuum filtration. The resulting solid was dried to afford the crude difluoroacid as a tan solid (5.0 g, 12.9 mmol) (M+1, 388).
Example 38
Preparation of 5,6-Difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2,9-dicarboxylic acid




The crude difluoroester (5.0 g, 12.9 mmol) was dissolved in acetic acid (20 mL) and 12 M HCl was added (20 mL) and the reaction mixture was heated to reflux for 1 hour. The reaction was allowed to cool to room temperature and water was added. The resulting solid was collected by vacuum filtration and dried overnight to afford the di-acid as a tan solid (1.9 g, 5.3 mmol) (M+1, 360).
Example 39
Preparation of Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-fluorenone-5-carboxylate 3-Nitro-2-hydroxyfluorenone




A solution of NO2BF4 (3.52 g, 25.5 mmol) in acetonitrile (100 ml) was added dropwise to a solution of 2-hydroxyfluorenone (5 g, 25.5 mmol) in acetonitrile (400 ml) at ambient temperature. The reaction mixture was then cooled to 0° C. and water (100 ml) was added to precipitate impurities. After filtration, water (200 ml) was added and the precipitate filtered off as a red solid (68%) (M+1, 242).
3-amino-2-hydroxyfluorenone




A mixture of 3-nitro-2-hydroxyfluorenone (1.6 g, 6.6 mmol) and SnCl2 (3 g, 6.6 mmol) was refluxed in 100 ml acetic acid:conc. HCl (1:1) for 1 hour. The mixture was allowed to cool to room temperature and neutralized with ammonium hydroxide. After extracting with EtOAc (3×100 ml), combined organic fractions were dried over magnesium sulfate and evaporated to leave the product as a brown solid (65%) (M+1, 212).
Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-fluorenone-5-carboxylate




A mixture of 3-amino-2-hydroxyfluorenone (0.9 g, 4.26 mmol) and ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate (1.36 g, 4.26 mmol) were heated in DMSO (50 ml) under vacuum for 18 hr. The product was extracted using EtOAc/Brine and the organic layers combined and dried to give the product as a red solid. The solid was dissolved in DMSO (40 ml) containing a large excess of K2CO3 and heated at 100° C. for 30 min. After cooling to room temperature, brine (30 ml) was added and the precipitated product collected as a yellow solid (60% over two steps) (M+1, 446).
Example 40
Preparation of 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-fluorenone-5-carboxylic acid




A mixture of ethyl 1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-fluorenone-5-carboxylate in acetic acid:conc. HCl (1:1) (50 ml each) was heated at reflux for 2 hr. After cooling to room temperature, water (50 ml) was added and the product collected as yellow solid (94%) (M+1, 418).
Example 41
Preparation of Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-anthraquinone-5-carboxylate




A mixture of 3-amino-2-hydroxyanthraquinone (5.54 g, 23.2 mmol) and ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate (8.7 g, 34.8 mmol) were heated in a minimum of DMSO ( ̃10 ml) under vacuum for 24 hr. The product was precipitated by the addition of water (50 ml). The solid was dried overnight in a vacuum oven and dissolved in DMSO (40 ml) containing a large excess of K2CO3 and heated at 100° C. for 30 min. After cooling to room temperature, brine (30 ml) was added and the precipitated product collected as a yellow solid (60% over two steps) (M+1, 474).
Example 42
Preparation of 1,2-Difluoro-4-oxo-4H-Pyrido[3,2,1-kl]-8-anthraquinone-5-carboxylic acid




A mixture of ethyl 1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-anthraquinonenone-5-carboxylate (3.5 g, 6.8 mmol) in acetic acid:conc. HCl (1:1) (50 ml each) was heated at reflux for 2 hr. After cooling to room temperature, water (50 ml) was added and the product collected as yellow solid (94%) (M+1, 446).
Example 43
Preparation of Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazole-5-carboxylate 2-amino(t-butoxy carbonyl)-5-amino hydroquinone




A solution of Boc anhydride (7.17 g,33 mmol) and DIEA (17 ml, 99 mmol) in DMSO (20 ml) was added dropwise at room temperature to stirred solution of 1,4-dihydroxy-2,5-diaminobenzene (7 g, 33 mmol). After stirring for 18 hr, the product was separated between EtOAc and brine and the organic layers combined and dried over MgSO4. After removal of solvent the residue was subjected to column chromatography on silica eluting with 25-50% EtOAc in hexane to give pure product (45%) (M+1, 239).
4-hydroxy-3-amino(t-butoxy carbonyl)-phenoxazole




To solution of 2-amino(t-butoxy carbonyl)-5-amino hydroquinone (4.69 g, 23.3 mmol) dissolved in acetonitrile/water (1:1; 20 ml) was added Na hydrosulfite (large excess) and the mixture stirred at room temp. for 15 min. The acetonitrile was removed in vacuo and the aqueous mixture extracted with EtOAc (3×20 ml). Combined organic layers were dried over MgSO4 and solvent removed in vacuo. The residue was taken up in neat triethyl orthoformate (100 ml), left to stir for 16 hr then heated to reflux for 10 min. The product was precipitated following cooling to room temp. by the addition of water (83%)(M+1, 251).
4-hydroxy-2-amino phenoxazole




4-Hydroxy-3-amino(t-butoxy carbonyl)-phenoxazole (3 g, 12 mmol) was dissolved in neat TFA (100 ml) and allowed to stir at room temperature for 1 hour. TFA was removed in vacuo to leave the final product as a TFA salt (quant.) (M−1, 149)
Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(N-(4-hydroxy-2-amino phenoxazole))-prop)-2-enoate




A solution of ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate (7.34 g, 23 mmol) and 2-amino-4-phenyl-phenol (3.45 g, 23 mmol) in EtOAc (20 ml) containing triethylamine (10 ml) was stirred under vacuum on the rotary evaporator for 3 hours. The EtOAc was removed in vacuo and the residue subjected to column chromatography on silica eluting with 50% EtOAc in hexane to give pure product (72%) (M+1, 425).
Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazole-5-carboxylate




A solution of ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(N-(4-hydroxy-2-amino phenoxazole))-prop-2-enoate (3.5 g, 8.25 mmol) in DMSO (50 m) containing K2CO3 (large excess) was heated at 80° C. for 10 min. After cooling to room temperature, water was added to precipitate the product as a yellow sold (82%) (M+1, 385).
Example 44
Preparation of 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(3′-hydroxy-4′-amino phenyl)-5-carboxylic acid




A mixture of ethyl 1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazole-5-carboxylate (2.3 g, 6 mmol) in acetic acid:conc. HCl (1:1; 100 ml) was heated to reflux for 30 min. After cooling to room temp., volatiles were removed in vacuo to leave the product as a brown solid (82%) (M+1, 347).
Example 45
Preparation of 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(nitro-phenoxazine)-5-carboxylic acid 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(3′-hydroxy-4′-amino-(N-2′′-fluoro-4′′-nitro phenyl)-phenyl))-5-carboxylic acid




A solution of 1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(3′-hydroxy-4′-amino phenyl)-5-carboxylic acid (0.5 g, 1.44 mmol), 3,4-difluoro-nitro benzene (0.5 ml, 4.3 mmol) and DIEA (1 ml) was heated to 90° C. in NMP (50 ml) for 30 min. After cooling to room temp. the product was precipitated by the addition of water and filtered (63%) (M+1, 486).
1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(nitro-phenoxazine)-5-carboxylic acid




A solution of 2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(3′-hydroxy-4′-amino phenyl)-5-carboxylic acid (0.3 g, 0.6 mmol) in DMSO (50 ml) containing an excess of K2CO3 was stirred and heated to 110° C. for 1 hr. After cooling to room temp. the product was precipitated by the addition of 3M HCl and filtered (71%) (M+1, 465).
Example 46
Preparation of 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(amino-phenoxazine)-5-carboxylic acid




A mixture of 1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(nitro-phenoxazine)-5-carboxylic acid (0.1 g, 0.2 mmol) and Tin (II) chloride (0.15 g, 0.6 mmol) in acetic acid:conc. HCl (1:1; 50 ml) was heated to reflux for four hr. After cooling to room temp. the product was precipitated by the addition of water and filtered (72%) (M+1,435).
Example 47
Preparation of Preparation of Amide Derivatives of Substituted Quinobenzoxazine Analogs




To a series of solutions of the fluoroacid (0.5 mmol) in NMP (3.6 mL) was added the amines NHR1R2 (0.5-2.0 mmol) at room temperature. The vessels were sealed and heated on a 90° C. hotplate with constant stirring for 1-2 hours until the reactions were determined to be complete by HPLC/MS analysis. The reaction mixtures were allowed to cool to room temperature and water was added (20 mL). The resulting precipitates were collected by vacuum filtration and dried under vacuum. In cases where 1.0 equivalent of amine was used, the resulting reaction mixtures were used in the next step “as is.” The resulting solids or solutions were treated with HBTU (2.5 eq.) and DIEA in 3.6 mL NMP and allowed to stir for 30 minutes at room temperature under an inert atmosphere. These solutions were added to a series of amines NHR3R4 (2.5 equivalents) in a 96 well format (Whatman Uniplate, 2 mL) and allowed to react for 2 hours. Methanol was then added (50-100 μL) and the plate was filtered (Whatman Unifilter Polypropylene). The resulting liquids were directly chromatographed on reverse HPLC (Waters Xterra 19×50 mm) with mass directed collection (Micromass ZQ, Waters FCII). The fractions were analyzed for purity (MS TIC, UV) and dried by vacuum evaporation (Savant) with an average yield of 5-10 mg). Tables 1 and 2 list exemplary ester-substituted and amide-substituted quinobenzoxazines analogs, respectively.
Example 48
Antitumor Data
Two xenograft models for inoculation were harvested and diluted to a concentration of 50×106 cells/ml or 100×106 cells/ml. Four to six week old nude mice were injected with 0.1 ml of the cell suspension which contains between 5×106 and 10×106 cells. When tumors are of a suitable size compound dosing is commenced. Tumor sizes are measured throughout the treatment period with calipers and body weights also measured.
Antitumor activities for compounds CX-3092 (1204) and CX-1535 (148) are shown in FIGS. 1 and 2, indicating efficacy (slow tumor weight gain) and a lack of toxicity (little body weight change). FIG. 1 shows the antitumor activity of compound 494 in Ramos, a model for fatal childhood leukemia. FIG. 2 shows the antitumor activity of compound 516 in HCT-116, a model of colorectal cancer.
Example 49
Cell Proliferation and/or Cytotoxicity Assay
Cell Culture
Human cervical epithelial cells (HeLa cells) were obtained from American Type Culture Collection (Manassas, Va.). Cells were grown in Eagle's minimum essential medium (MEM, Hyclone, Utah) supplemented with 2 mM Glutamine, 0.1 mM nonessential amino acid, 1 mM Na Pyruvate, 1.5 g/L NaHCO3, 50 mg/L gentamicin, and 10% fetal bovine serum (Hyclone, USA) in a humidified atmosphere of 5% CO2 at 37° C.
MTS Assay
Antiproliferative effects of anticancer drugs were tested by the CellTiter 96 AQueous assay (Promega, Wis.), which is a calorimetric assay for determining the number of viable cells. (See, e.g., Wang, L., et al., Methods Cell Sci (1996) 18:249-255).
Cells (4,500 cells/well) were seeded on 96 well flat bottom plates (Corning, N.Y.) in 100 μl of culture medium without any anticancer drug on day 0, and the culture medium was exchanged for that contained anticancer drugs at various concentrations on day 1. After incubation for 3 days under normal growth conditions (on day 4), the monolayers were washed once in PBS, and the medium was switched to 100 μl of PBS in each of the 96 well plate. After mixing MTS and PMS at the ratio of 20:1,20 μl of MTS/PMS solution was added to each of the 96 well plate and incubated for 4 hours in a humidified atmosphere of 5% CO2 at 37° C. The absorbance was read at 490 mn using FLUOstar Galaxy 96 well plate reader (BMG Labtechnologies, Germany). μM concentrations (MTS data) reported in Tables 1-2 are concentrations at which 50% of antiproliferative cell response is seen. Compounds whose IC50 values were greater than 5 μM were not reported.
Example 50
Measurement of mRNA Values in Cell Assays
Real-time quantitative PCR (QPCR) method was used to detect accurately the changes of the target c-myc and the endogenous reference GAPDH gene copies in the same tube. Cells (15,000 cells/well) were seed on 96 well flat bottom plates (Corning, N.Y.) and incubated under normal growth conditions for overnight. The next day, the culture medium was exchanged for that contained anticancer drugs at various concentrations and incubate for 4 hrs in a humidified atmosphere of 5% CO2 at 37° C. Total RNA (tRNA) was extracted using the RNeasy 96 Kit (QIAGEN, CA). The concentration of the tRNA was determined by the RiboGreen RNA Quantitation Reagent (Molecular Probes, OR).
Reverse-transcription (RT) reaction was occurred using 50 ng of tRNA from each well in a 25 μl reaction containing 1× TaqMan RT buffer, 2.5 uM random hexamers, 5.5 mM MgCl2, 0.5 mM each deoxynucleoside triphosphate (dNTP), 30 U MultiScribe Reverse Transcriptase, and 10 U RNase inhibitor. RT reactions were incubated for 10 min at 25° C., reverse-transcribed for 30 min at 48° C., inactivated for 5 min at 95° C., and placed at 4° C. All RT reagents were purchased from Applied Biosystems, CA.
Real-Time QPCR reaction was performed in a 50 μl reaction containing the 5 μl of cDNA, 1× Universal PCR Master Mix, 1× c-myc Pre-Developed Primers and Probe set, and 0.8× GAPDH Pre-Developed Primers and Probe set. Because of the relative abundance of GAPDH gene in Hela, GAPDH primers and probe concentration were adjusted to get accurate threshold cycles (CT) for both genes in the same tube. The threshold cycle (CT) indicates the fractional cycle number at which the amount of amplified target reaches a fixed threshold. By doing so, the GAPDH amplification was stopped before it can limit the common reactants available for amplification of the c-myc, resulted in a reduction in ΔRn value of GAPDH, but no effect on its CT value, and equal amplification efficiency for both genes. The ΔRn value represents the normalized reporter signal minus the baseline signal. ΔRn increases during PCR as amplicon copy number increases until the reaction approaches a plateau.
The c-myc probe was labeled with 6FAMTM dye-MGB and the GAPDH probe was labeled with VICTM dye-MGB. Preincubation was performed for 2 min at 50° C. to activate AmpErase UNG enzyme and then for 10 min at 95° C. to activate AmpliTaq DNA Polymerase. DNA was amplified for 40 cycles of 15 sec at 95° C. and 1 min at 60° C. Human c-myc and GAPDH cDNA were amplified, detected, and quantitated in real time using the ABI Prism 7000 Sequence Detection system (Applied Biosystems, CA), which was set to detect both 6-FAM and VIC reporter dyes simultaneously.
The data was analyzed by using the ABI PRISM Sequence Detection System and Microsoft Excel. Relative quantitation was done using the standard curve and comparative CT method at the same time, and both methods gave equivalent results. The cycle at which the amplification plot crosses the CT is known to accurately reflect relative mRNA values. (See, Heid, et al., Genome Res. (1996) 6:986-994; Gibson, et al., Genome Res. (1996) 6:995-1001). QPCR reactions were set up in triplicate at each cDNA sample and the triplicate CT values were averaged. All reagents including Pre-Developed Primers and probe set were purchased from Applied Biosystems, CA. μM concentrations (STOP data) reported in Tables 1-2 are concentrations at which 50% inhibition of c-myc mRNA levels are seen. Compounds whose IC50 values were greater than 5 μM were not reported.
It is understood that the foregoing detailed description and accompanying examples are merely illustrative, and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, formulations and/or methods of use of the invention, may be made without departing from the spirit and scope thereof. U.S. patents and publications referenced herein are incorporated by reference.











TABLE 1








Stop Data
MTS Data



Structure
c-Myc μM
Hella μM



















1




4


 


2




2.5


 


3




2.5


 


4




1.76


 


5




1.75
7.20


 


6




1.75


 


7




1.75


 


8




1.75


 


9




1.75


 


10




1.75


 


11




1.75


 


12




1.75


 


13




1.75


 


14




1.75


 


15




1.75


 


16




0.9


 


17




0.75


 


18




0.75


 


19




0.75


 


20




0.75


 


21




0.75


 


22




0.75


 


23




0.75


 


24




0.5
7.00


 


25




0.25
0.20




















TABLE 2








Stop Data
MTS Data



Structure
c-Myc μM
Hella μM



















26




4
0.73


 


27




3
3.80


 


28




3
2.50


 


29




3
2.00


 


30




3
1.80


 


31




3
1.40


 


32




3
0.60


 


33




3
0.29


 


34




3
0.28


 


35




3
0.21


 


36




3
0.16


 


37




2.5
2.80


 


38




2.5
0.26


 


39




2.5


 


40




2.5


 


41




2.5


 


42




2.5


 


43




2.5


 


44




2.5


 


45




2.5


 


46




2.5


 


47




2.5


 


48




2.5


 


49




2.5


 


50




2.5


 


51




2.5


 


52




2.5


 


53




2.5


 


54




2.5


 


55




2.5


 


56




2.5


 


57




2.5


 


58




2.5


 


59




2.5


 


60




2.5


 


61




2.5


 


62




2.5


 


63




2.5


 


64




2.5


 


65




2.5


 


66




2.5


 


67




2.5


 


68




2.5


 


69




2.5


 


70




2.5


 


71




2.5


 


72




2.5


 


73




2.5


 


74




2.5


 


75




2.5


 


76




2.5


 


77




2.5


 


78




2.5


 


79




2.5


 


80




2.5


 


81




2.5


 


82




2.5


 


83




2.5


 


84




2.5


 


85




2.5


 


86




2.5


 


87




2.5


 


88




2.5


 


89




2.5


 


90




2.5


 


91




2.5


 


92




2.5


 


93




2.5


 


94




2.5


 


95




2.5


 


96




2.5


 


97




2.5


 


98




2.5


 


99




2.5


 


100




2.5


 


101




2.25


 


102




1.8
2.20


 


103




1.8


 


104




1.75
2.80


 


105




1.75
2.80


 


106




1.75
2.50


 


107




1.75
1.80


 


108




1.75
0.46


 


109




1.75
0.31


 


110




1.75
0.25


 


111




1.75
0.22


 


112




1.75
0.22


 


113




1.75


 


114




1.75


 


115




1.75


 


116




1.75


 


117




1.75


 


118




1.75


 


119




1.75


 


120




1.75


 


121




1.75


 


122




1.75


 


123




1.75


 


124




1.75


 


125




1.75


 


126




1.75


 


127




1.75


 


128




1.75


 


129




1.75


 


130




1.75


 


131




1.75


 


132




1.75


 


133




1.75


 


134




1.75


 


135




1.75


 


136




1.75


 


137




1.75


 


138




1.75


 


139




1.75


 


140




1.75


 


141




1.75


 


142




1.75


 


143




1.75


 


144




1.75


 


145




1.75


 


146




1.75


 


147




1.75


 


148




1.75


 


149




1.75


 


150




1.75


 


151




1.75


 


152




1.75


 


153




1.75


 


154




1.75


 


155




1.75


 


156




1.75


 


157




1.75


 


158




1.75


 


159




1.75


 


160




1.75


 


161




1.75


 


162




1.75


 


163




1.75


 


164




1.75


 


165




1.75


 


166




1.75


 


167




1.75


 


168




1.75


 


169




1.75


 


170




1.75


 


171




1.75


 


172




1.75


 


173




1.75


 


174




1.75


 


175




1.75


 


176




1.75


 


177




1.75


 


178




1.75


 


179




1.75


 


180




1.75


 


181




1.75


 


182




1.75


 


183




1.75


 


184




1.75


 


185




1.75


 


186




1.75


 


187




1.75


 


188




1.75


 


189




1.75


 


190




1.75


 


191




1.75


 


192




1.75


 


193




1.75


 


194




1.75


 


195




1.75


 


196




1.75


 


197




1.75


 


198




1.75


 


199




1.75


 


200




1.75


 


201




1.75


 


202




1.75


 


203




1.75


 


204




1.75


 


205




1.75


 


206




1.75


 


207




1.75


 


208




1.75


 


209




1.75


 


210




1.75


 


211




1.75


 


212




1.75


 


213




1.75


 


214




1.75


 


215




1.75


 


216




1.75


 


217




1.75


 


218




1.75


 


219




1.75


 


220




1.75


 


221




1.75


 


222




1.75


 


223




1.75


 


224




1.75


 


225




1.75


 


226




1.75


 


227




1.75


 


228




1.75


 


229




1.75


 


230




1.75


 


231




1.75


 


232




1.75


 


233




1.75


 


234




1.75


 


235




1.75


 


236




1.75


 


237




1.75


 


238




1.75


 


239




1.75


 


240




1.75


 


241




1.75


 


242




1.75


 


243




1.75


 


244




1.75


 


245




1.75


 


246




1.75


 


247




1.75


 


248




1.75


 


249




1.75


 


250




1.75


 


251




1.75


 


252




1.75


 


253




1.75


 


254




1.75


 


255




1.75


 


256




1.75


 


257




1.75


 


258




1.75


 


259




1.75


 


260




1.75


 


261




1.75


 


262




1.75


 


263




1.75


 


264




1.75


 


265




1.75


 


266




1.75


 


267




1.75


 


268




1.75


 


269




1.75


 


270




1.75


 


271




1.75


 


272




1.75


 


273




1.75


 


274




1.75


 


275




1.75


 


276




1.75


 


277




1.75


 


278




1.75


 


279




1.75


 


280




1.75


 


281




1.75


 


282




1.75


 


283




1.75


 


284




1.75


 


285




1.75


 


286




1.75


 


287




1.75


 


288




1.75


 


289




1.75


 


290




1.75


 


291




1.75


 


292




1.75


 


293




1.75


 


294




1.75


 


295




1.75


 


296




1.75


 


297




1.75


 


298




1.75


 


299




1.75


 


300




1.75


 


301




1.75


 


302




1.75


 


303




1.5
2.10


 


304




1.13


 


305




1.05


 


306




1
3.20


 


307




1
3.10


 


308




1
3.10


 


309




1
3.00


 


310




1
2.30


 


311




1
2.10


 


312




1
1.90


 


313




1


 


314




1


 


315




1


 


316




1


 


317




1


 


318




1


 


319




1


 


320




1


 


321




1


 


322




1


 


323




1


 


324




1


 


325




1


 


326




1


 


327




1


 


328




1


 


329




1


 


330




1


 


331




1


 


332




1


 


333




1


 


334




1


 


335




1


 


336




1


 


337




1


 


338




1


 


339




0.94


 


340




0.9
8.50


 


341




0.9
0.28


 


342




0.9


 


343




0.9


 


344




0.9


 


345




0.9


 


346




0.9


 


347




0.9


 


348




0.9


 


349




0.9


 


350




0.9


 


351




0.9


 


352




0.9


 


353




0.9


 


354




0.9


 


355




0.89


 


356




0.85


 


357




0.75
8.60


 


358




0.75
5.70


 


359




0.75
4.80


 


360




0.75
4.50


 


361




0.75
4.20


 


362




0.75
4.00


 


363




0.75
3.80


 


364




0.75
3.80


 


365




0.75
3.80


 


366




0.75
3.70


 


367




0.75
3.70


 


368




0.75
3.60


 


369




0.75
3.50


 


370




0.75
3.50


 


371




0.75
3.50


 


372




0.75
3.40


 


373




0.75
3.30


 


374




0.75
3.30


 


375




0.75
2.70


 


376




0.75
2.40


 


377




0.75
2.20


 


378




0.75
2.10


 


379




0.75
1.90


 


380




0.75
1.80


 


381




0.75
1.80


 


382




0.75
1.80


 


383




0.75
0.37


 


384




0.75
0.37


 


385




0.75
0.36


 


386




0.75
0.34


 


387




0.75
0.33


 


388




0.75
0.31


 


389




0.75
0.29


 


390




0.75
0.24


 


391




0.75
0.24


 


392




0.75
0.19


 


393




0.75


 


394




0.75


 


395




0.75


 


396




0.75


 


397




0.75


 


398




0.75


 


399




0.75


 


400




0.75


 


401




0.75


 


402




0.75


 


403




0.75


 


404




0.75


 


405




0.75


 


406




0.75


 


407




0.75


 


408




0.75


 


409




0.75


 


410




0.75


 


411




0.75


 


412




0.75


 


413




0.75


 


414




0.75


 


415




0.75


 


416




0.75


 


417




0.75


 


418




0.75


 


419




0.75


 


420




0.75


 


421




0.75


 


422




0.75


 


423




0.75


 


424




0.75


 


425




0.75


 


426




0.75


 


427




0.75


 


428




0.75


 


429




0.75


 


430




0.75


 


431




0.75


 


432




0.75


 


433




0.75


 


434




0.75


 


435




0.75


 


436




0.75


 


437




0.75


 


438




0.75


 


439




0.75


 


440




0.75


 


441




0.75


 


442




0.75


 


443




0.75


 


444




0.75


 


445




0.75


 


446




0.75


 


447




0.75


 


448




0.75


 


449




0.75


 


450




0.75


 


451




0.75


 


452




0.75


 


453




0.75


 


454




0.75


 


455




0.75


 


456




0.75


 


457




0.75


 


458




0.75


 


459




0.75


 


460




0.75


 


461




0.75


 


462




0.75


 


463




0.75


 


464




0.75


 


465




0.75


 


466




0.75


 


467




0.75


 


468




0.74


 


469




0.73


 


470




0.64


 


471




0.64


 


472




0.62
3.30


 


473




0.62


 


474




0.58
0.37


 


475




0.58
0.24


 


476




0.58


 


477




0.55
2.10


 


478




0.53


 


479




0.5
7.40


 


480




0.5
3.70


 


481




0.5
3.60


 


482




0.5
3.40


 


483




0.5
3.20


 


484




0.5
3.10


 


485




0.5
0.50


 


486




0.5
0.39


 


487




0.5
0.18


 


488




0.5


 


489




0.5


 


490




0.5


 


491




0.45


 


492




0.45


 


493




0.44
0.40


 


494




0.44
0.19


 


495




0.42


 


496




0.41
4.00


 


497




0.41
2.10


 


498




0.41


 


499




0.4


 


500




0.375
5.60


 


501




0.375
4.20


 


502




0.375
4.00


 


503




0.375
4.00


 


504




0.375
3.40


 


505




0.375
3.40


 


506




0.375
3.40


 


507




0.375
3.30


 


508




0.375
3.20


 


509




0.375
3.10


 


510




0.375
3.10


 


511




0.375
3.10


 


512




0.375
3.10


 


513




0.375
3.10


 


514




0.375
2.90


 


515




0.375
2.50


 


516




0.375
2.30


 


517




0.375
2.20


 


518




0.375
2.20


 


519




0.375
2.10


 


520




0.375
1.90


 


521




0.375
1.70


 


522




0.375
1.70


 


523




0.375
1.60


 


524




0.375
1.50


 


525




0.375
1.,2


 


526




0.375
0.90


 


527




0.375
0.79


 


528




0.375
0.75


 


529




0.375
0.75


 


530




0.375
0.72


 


531




0.375
0.48


 


532




0.375
0.44


 


533




0.375
0.40


 


534




0.375
0.40


 


535




0.375
0.31


 


536




0.375
0.31


 


537




0.375
0.29


 


538




0.375
0.28


 


539




0.375
0.28


 


540




0.375
0.27


 


541




0.375
0.27


 


542




0.375
0.23


 


543




0.375
0.20


 


544




0.375
0.20


 


545




0.375
0.15


 


546




0.375
0.10


 


547




0.375
0.10


 


548




0.375
0.10


 


549




0.375


 


550




0.375


 


551




0.375


 


552




0.375


 


553




0.375


 


554




0.375


 


555




0.375


 


556




0.375


 


557




0.375


 


558




0.375


 


559




0.375


 


560




0.37


 


561




0.34


 


562




0.32
0.85


 


563




0.25
0.31


 


564




0.25
0.29


 


565




0.25
0.20


 


566




0.25
0.03


 


567




0.25


 


568




0.22
4.10


 


569




0.18
7.80


 


570




0.18
6.80


 


571




0.18
4.80


 


572




0.18
4.80


 


573




0.18
4.50


 


574




0.18
4.00


 


575




0.18
2.10


 


576




0.18
2.10


 


577




0.18
1.10


 


578




0.18
0.58


 


579




0.18
0.49


 


580




0.18
0.30


 


581




0.18
0.30


 


582




0.18
0.28


 


583




0.18
0.25


 


584




0.18
0.19


 


585




0.18


 


586




0.18


 


587




0.18


 


588




0.18


 


589




0.18


 


590




0.18


 


591




0.13
3.30


 


592




0.1
3.80


 


1467




0.25
0.35


 


1468




0.375
0.35


 


1469




1.75


 


1470




1.75


 


1471




5

















TABLE 3







593






 


594






 


595






 


596






 


597






 


598






 


599






 


600






 


601






 


602






 


603






 


604






 


605






 


606






 


607






 


608






 


609






 


610






 


611






 


612






 


613






 


614






 


615






 


616






 


617






 


618






 


619






 


620






 


621






 


622






 


623






 


624






 


625






 


626






 


627






 


628






 


629






 


630






 


631






 


632






 


633






 


634






 


635






 


636






 


637






 


638






 


639






 


640






 


641






 


642






 


643






 


644






 


645






 


646






 


647






 


648






 


649






 


650






 


651






 


652






 


653






 


654






 


655






 


656






 


657






 


658






 


659






 


660






 


661






 


662






 


663






 


664






 


665






 


666






 


667






 


668






 


669






 


670






 


671






 


672






 


673






 


674






 


675






 


676






 


677






 


678






 


679






 


680






 


681






 


682






 


683






 


684






 


685






 


686






 


687






 


688






 


689






 


690






 


691






 


692






 


693






 


694






 


695






 


696






 


697






 


698






 


699






 


700






 


701






 


702






 


703






 


704






 


705






 


706






 


707






 


708






 


709






 


710






 


711






 


712






 


713






 


714






 


715






 


716






 


717






 


718






 


719






 


720






 


721






 


722






 


723






 


724






 


725






 


726






 


727






 


728






 


729






 


730






 


731






 


732






 


733






 


734






 


735






 


736






 


737






 


738






 


739






 


740






 


741






 


742






 


743






 


744






 


745






 


746






 


747






 


748






 


749






 


750






 


751






 


752






 


753






 


754






 


755






 


756






 


757






 


758






 


759






 


760






 


761






 


762






 


763






 


764






 


765






 


766






 


767






 


768






 


769






 


770






 


771






 


772






 


773






 


774






 


775






 


776






 


777






 


778






 


779






 


780






 


781






 


782






 


783






 


784






 


785






 


786






 


787






 


788






 


789






 


790






 


791






 


792






 


793






 


794






 


795






 


796






 


797






 


798






 


799






 


800






 


801






 


802






 


803






 


804






 


805






 


806






 


807






 


808






 


809






 


810






 


811






 


812






 


813






 


814






 


815






 


816






 


817






 


818






 


819






 


820






 


821






 


822






 


823






 


824






 


825






 


826






 


827






 


828






 


829






 


830






 


831






 


832






 


833






 


834






 


835






 


836






 


837






 


838






 


839






 


840






 


841






 


842






 


843






 


844






 


845






 


846






 


847






 


848






 


849






 


850






 


851






 


852






 


853






 


854






 


855






 


856






 


857






 


858






 


859






 


860






 


861






 


862






 


863






 


864






 


865






 


866






 


867






 


868






 


869






 


870






 


871






 


872






 


873






 


874






 


875






 


876






 


877






 


878






 


879






 


880






 


881






 


882






 


883






 


884






 


885






 


886






 


887






 


888






 


889






 


890






 


891






 


892






 


893






 


894






 


895






 


896






 


897






 


898






 


899






 


900






 


901






 


902






 


903






 


904






 


905






 


906






 


907






 


908






 


909






 


910






 


911






 


912






 


913






 


914






 


915






 


916






 


917






 


918






 


919






 


920






 


921






 


922






 


923






 


924






 


925






 


926






 


927






 


928






 


929






 


930






 


931






 


932






 


933






 


934






 


935






 


936






 


937






 


938






 


939






 


940






 


941






 


942






 


943






 


944






 


945






 


946






 


947






 


948






 


949






 


950






 


951






 


952






 


953






 


954






 


955






 


956






 


957






 


958






 


959






 


960






 


961






 


962






 


963






 


964






 


965






 


966






 


967






 


968






 


969






 


970






 


971






 


972






 


973






 


974






 


975






 


976






 


977






 


978






 


979






 


980






 


981






 


982






 


983






 


984






 


985






 


986






 


987






 


988






 


989






 


990






 


991






 


992






 


993






 


994






 


995






 


996






 


997






 


998






 


999






 


1000






 


1001






 


1002






 


1003






 


1004






 


1005






 


1006






 


1007






 


1008






 


1009






 


1010






 


1011






 


1012






 


1013






 


1014






 


1015






 


1016






 


1017






 


1018






 


1019






 


1020






 


1021






 


1022






 


1023






 


1024






 


1025






 


1026






 


1027






 


1028






 


1029






 


1030






 


1031






 


1032






 


1033






 


1034






 


1035






 


1036






 


1037






 


1038






 


1039






 


1040






 


1041






 


1042






 


1043






 


1044






 


1045






 


1046






 


1047






 


1048






 


1049






 


1050






 


1051






 


1052






 


1053






 


1054






 


1055






 


1056






 


1057






 


1058






 


1059






 


1060






 


1061






 


1062






 


1063






 


1064






 


1065






 


1066






 


1067






 


1068






 


1069






 


1070






 


1071






 


1072






 


1073






 


1074






 


1075






 


1076






 


1077






 


1078






 


1079






 


1080






 


1081






 


1082






 


1083






 


1084






 


1085






 


1086






 


1087






 


1088






 


1089






 


1090






 


1091






 


1092






 


1093






 


1094






 


1095






 


1096






 


1097






 


1098






 


1099






 


1100






 


1101






 


1102






 


1103






 


1104






 


1105






 


1106






 


1107






 


1108






 


1109






 


1110






 


1111






 


1112






 


1113






 


1114






 


1115






 


1116






 


1117






 


1118






 


1119






 


1120






 


1121






 


1122






 


1123






 


1124






 


1125






 


1126






 


1127






 


1128






 


1129






 


1130






 


1131






 


1132






 


1133






 


1134






 


1135






 


1136






 


1137






 


1138






 


1139






 


1140






 


1141






 


1142






 


1143






 


1144






 


1145






 


1146






 


1147






 


1148






 


1149






 


1150






 


1151






 


1152






 


1153






 


1154






 


1155






 


1156






 


1157






 


1158






 


1159






 


1160






 


1161






 


1162






 


1163






 


1164






 


1165






 


1166






 


1167






 


1168






 


1169






 


1170






 


1171






 


1172






 


1173






 


1174






 


1175






 


1176






 


1177






 


1178






 


1179






 


1180






 


1181






 


1182






 


1183






 


1184






 


1185






 


1186






 


1187






 


1188






 


1189






 


1190






 


1191






 


1192






 


1193






 


1194






 


1195






 


1196






 


1197






 


1198






 


1199






 


1200






 


1201






 


1202






 


1203






 


1204






 


1205






 


1206






 


1207






 


1208






 


1209






 


1210






 


1211






 


1212






 


1213






 


1214






 


1215






 


1216






 


1217






 


1218






 


1219






 


1220






 


1221






 


1222






 


1223






 


1224






 


1225






 


1226






 


1227






 


1228






 


1229






 


1230






 


1231






 


1232






 


1233






 


1234






 


1235






 


1236






 


1237






 


1238






 


1239






 


1240






 


1241






 


1242






 


1243






 


1244






 


1245






 


1246






 


1247






 


1248






 


1249






 


1250






 


1251






 


1252






 


1253






 


1254






 


1255






 


1256






 


1257






 


1258






 


1259






 


1260






 


1261






 


1262






 


1263






 


1264






 


1265






 


1266






 


1267






 


1268






 


1269






 


1270






 


1271






 


1272






 


1273






 


1274






 


1275






 


1276






 


1277






 


1278






 


1279






 


1280






 


1281






 


1282






 


1283






 


1284






 


1285






 


1286






 


1287






 


1288






 


1289






 


1290






 


1291






 


1292






 


1293






 


1294






 


1295






 


1296






 


1297






 


1298






 


1299






 


1300






 


1301






 


1302






 


1303






 


1304






 


1305






 


1306






 


1307






 


1308






 


1309






 


1310






 


1311






 


1312






 


1313






 


1314






 


1315






 


1316






 


1317






 


1318






 


1319






 


1320






 


1321






 


1322






 


1323






 


1324






 


1325






 


1326






 


1327






 


1328






 


1329






 


1330






 


1331






 


1332






 


1333






 


1334






 


1335






 


1336






 


1337






 


1338






 


1339






 


1340






 


1341






 


1342






 


1343






 


1344






 


1345






 


1346






 


1347






 


1348






 


1349






 


1350






 


1351






 


1352






 


1353






 


1354






 


1355






 


1356






 


1357






 


1358






 


1359






 


1360






 


1361






 


1362






 


1363






 


1364






 


1365






 


1366






 


1367






 


1368






 


1369






 


1370






 


1371






 


1372






 


1373






 


1374






 


1375






 


1376






 


1377






 


1378






 


1379






 


1380






 


1381






 


1382






 


1383






 


1384






 


1385






 


1386






 


1387






 


1388






 


1389






 


1390






 


1391






 


1392






 


1393






 


1394






 


1395






 


1396






 


1397






 


1398






 


1399






 


1400






 


1401






 


1402






 


1403






 


1404






 


1405






 


1406






 


1407






 


1408






 


1409






 


1410






 


1411






 


1412






 


1413






 


1414






 


1415






 


1416






 


1417






 


1418






 


1419






 


1420






 


1421






 


1422






 


1423






 


1424






 


1425






 


1426






 


1427






 


1428






 


1429






 


1430






 


1431






 


1432






 


1433






 


1434






 


1435






 


1436






 


1437






 


1438






 


1439






 


1440






 


1441






 


1442






 


1443






 


1444






 


1445






 


1446






 


1447






 


1448






 


1449






 


1450






 


1451






 


1452






 


1453






 


1454






 


1455






 


1456






 


1457






 


1458






 


1459






 


1460






 


1461






 


1462






 


1463






 


1464






 


1465






 


1466

## Claims

1. A method for reducing cell proliferation or inducing cell death, comprising contacting a cell with an effective amount of the compound having formula 1,



or pharmaceutically acceptable salts, and esters thereof;
wherein V is H, halo, NR1R2 or NR1—(CR12)n—NR3R4;
A is H, fluoro, or NR12;
Z is O, S, NR1 or CH2;
U is selected from the group consisting of NR1—(CR12)n—NR3R4 and NR1R;
X is OR2, NR1R2, halo, azido, or SR2;
n is 1-6;
wherein R1 and R2 together with N in NR1R2 and R3 and R4 together with N in NR3R4 may independently form an optionally substituted 5-6 membered ring containing N, and optionally O or S;
R is an optionally substituted 5-14 membered heterocyclic ring containing one or more N, O or S; or a C1-10 alkyl or C2-10 alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O, and S, and optionally substituted with an optionally substituted carbocyclic or heterocyclic ring;
R1 and R3 are independently H or a C1-6 alkyl;
R2 and R4 are independently H or a C1-10 alkyl or C2-10 alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O, and S, and optionally substituted with a carbocyclic or heterocyclic ring; or
R2 is an optionally substituted heterocyclic ring, aryl or heteroaryl;
W is selected from the group consisting of










wherein Q, Q1, Q2, and Q3 are independently CH or N;
Y is independently O, CH, C═O or NR1;
and R5 is a substituent at any position on the fused ring; and is H, OR2, C1-6 alkyl, C2-6 alkenyl, each optionally substituted by halo, or ═O; or two adjacent R5 are linked to obtain a 5-6 membered substituted or unsubstituted carbocyclic or heterocyclic ring, optionally fused to an additional substituted or unsubstituted carbocyclic or heterocyclic ring;
wherein each optionally substituted moiety is substituted with one or more halo, OR2, NR1R2, carbamate, C1-10 alkyl, C2-10 alkenyl, each optionally substituted by halo, ═O, aryl or one or more heteroatoms selected from N, O and S; or is substituted with an aryl, a carbocyclic or a heterocyclic ring;
thereby reducing cell proliferation or inducing cell death;
wherein when said contacting is in vivo, said cell is a prostate cancer cell, a colorectal cancer cell, a cervical cancer cell, a leukemia cell, or a lymphoma cell.

2. The method of claim 1, wherein A and X are independently halo.

3. The method of claim 2, wherein said halo is fluoro.

4. The method of claim 1, where V is H.

5. The method of claim 1, wherein X is NR1R2.

6. The method of claim 5, wherein R1 is H and R2 is a C1-10 alkyl optionally containing N, O or S, and optionally substituted with a C3-6 cycloalkyl, aryl or a 5-14 membered heterocyclic ring containing one or more N, O or S.

7. The method of claim 6, wherein said 5-14 membered heterocyclic ring is selected from the group consisting of tetrahydrofuran, 1,3-dioxolane, 2,3-dihydrofuran, tetrahydropyran, benzofuran, isobenzofuran, 1,3-dihydro-isobenzofuran, isoxazole, 4,5-dihydroisoxazole, piperidine, pyrrolidine, pyrrolidin-2-one, pyrrole, pyridine, pyrimidine, octahydro-pyrrolo[3,4-b]pyridine, piperazine, pyrazine, morpholine, thiomorpholine, imidazole, imidazolidine-2,4-dione, benzimidazole, 1,3-dihydrobenzimidazol-2-one, indole, thiazole, benzothiazole, thiadiazole, thiophene, tetrahydro-thiophene 1,1-dioxide, diazepine, triazole, guanidine, diazabicyclo[2.2.1]heptane, 2,5-diazabicyclo[2.2.1]heptane, and 2,3,4,4a,9,9a-hexahydro-1H-β-carboline.

8. The method of claim 5, wherein R1 is H and R2 is an aryl or a 5-14 membered heterocyclic ring containing one or more N, O or S, each optionally substituted with an amino or another heterocyclic ring.

9. The method of claim 8, wherein said 5-14 membered heterocyclic ring is selected from the group consisting of tetrahydrofuran, 1,3-dioxolane, 2,3-dihydrofuran, tetrahydropyran, benzofuran, isobenzofuran, 1,3-dihydro-isobenzofuran, isoxazole, 4,5-dihydroisoxazole, piperidine, pyrrolidine, pyrrolidin-2-one, pyrrole, pyridine, pyrimidine, octahydro-pyrrolo[3,4-b]pyridine, piperazine, pyrazine, morpholine, thiomorpholine, imidazole, imidazolidine-2,4-dione, benzimidazole, 1,3-dihydrobenzimidazol-2-one, indole, thiazole, benzothiazole, thiadiazole, thiophene, tetrahydro-thiophene 1,1-dioxide, diazepine, triazole, guanidine, diazabicyclo[2.2.1]heptane, 2,5-diazabicyclo[2.2.1]heptane, and 2,3,4,4a,9,9a-hexahydro-1H-β-carboline.

10. The method of claim 5, wherein R1 and R2 together with N in NR1R2 form an optionally substituted 5-6 membered ring containing one or more N, O or S.

11. The method of claim 10, where NR1R2 is pyrrolidine, imidazole, pyridine, morpholine, thiomorpholine, piperazine, piperidine or diazepine.

12. The method of claim 1, wherein n is 2-3.

13. The method of claim 1, wherein NR3R4 is an acyclic amine, or guanidinyl or a tautomer thereof.

14. The method of claim 1, wherein NR3R4 is morpholine, thiomorpholine, imidazole, pyrrolidine, piperazine, pyridine or piperidine.

15. The method of claim 1, wherein U is NR1—(CR12)n—NR3R4.

16. The method of claim 15, wherein X is NR1R2, R1 and R2 together with N in NR1R2, and R3 and R4 together with N in NR3R4 each independently form a substituted 5-6 membered ring containing one or more N, O or S.

17. The method of claim 16, wherein X is optionally substituted with amino, carbamate, a C1-10 alkyl containing one or more non-adjacent N, O or S, and optionally substituted with a heterocyclic ring; aryl or a saturated or unsaturated heterocyclic ring, each of which is optionally substituted.

18. The method of claim 16, wherein X is substituted with a heterocyclic ring selected from the group consisting of tetrahydrofuran, 1,3-dioxolane, 2,3-dihydrofuran, tetrahydropyran, benzofuran, isobenzofuran, 1,3-dihydro-isobenzofuran, isoxazole, 4,5-dihydroisoxazole, piperidine, pyrrolidine, pyrrolidin-2-one, pyrrole, pyridine, pyrimidine, octahydro-pyrrolo[3,4-b]pyridine, piperazine, pyrazine, morpholine, thiomorpholine, imidazole, imidazolidine-2,4-dione, benzimidazole, 1,3-dihydrobenzimidazol-2-one, indole, thiazole, benzothiazole, thiadiazole, thiophene, tetrahydro-thiophene 1,1-dioxide, diazepine, triazole, guanidine, diazabicyclo[2.2.1]heptane, 2,5-diazabicyclo[2.2.1]heptane, and 2,3,4,4a,9,9a-hexahydro-1H-β-carboline.

19. The method of claim 16, wherein X and NR3R4 are independently morpholine, thiomorpholine, imidazole, pyrrolidine, piperazine, pyridine or piperidine.

20. The method of claim 19, wherein X and NR3R4 are independently pyrrolidin.

21. The method of claim 20, wherein X is substituted with pyrazine.

22. The method of claim 21, wherein W is naphthalenyl.

23. The method of claim 1, wherein W is benzene, pyridine, biphenyl, naphthalene, phenanthrene, quinoline, isoquinoline, quinazoline, cinnoline, phthalazine, quinoxaline, indole, benzimidazole, benzoxazole, benzthiazole, benzofuran, anthrone, xanthone, acridone, fluorenone, carbazolyl, pyrimido[4,3-b]furan, pyrido[4,3-b]indole, pyrido[2,3-b]indole, dibenzofuran, acridine or acridizine.

24. The method of claim 1, wherein U is NR1R.

25. The method of claim 24, wherein R is a C1-10 alkyl or C2-10 alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O, and S, and is substituted with an optionally substituted carbocyclic or heterocyclic ring.

26. The method of claim 25, wherein U is NR1R, wherein R1 is H, and R is a C1-10 alkyl or C2-10 alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O, and S, and R is substituted with an optionally substituted pyrrolidine or piperazine ring.

27. A method for reducing cell proliferation or inducing cell death, comprising contacting a cell with an effective amount of a compound selected from the group consisting of









































































































































































































































































































































































































and the pharmaceutically acceptable salts thereof;
thereby reducing cell proliferation or inducing cell death;
wherein when said contacting is in vivo, said cell is a prostate cancer cell, a colorectal cancer cell, a cervical cancer cell, a leukemia cell, or a lymphoma cell.

28. The method of claim 1, wherein said compound is



or a pharmaceutically acceptable salt thereof.

29. The method of claim 1, wherein said compound is selected from:



and the pharmaceutically acceptable salts thereof.

30. The method of claim 1, wherein said cell is a prostate cancer cell.

31. The method of claim 1, wherein said cell is a colorectal cancer cell.

32. The method of claim 1, wherein said cell is a cervical cancer cell.

33. The method of claim 1, wherein said cell is a leukemia cell.

34. The method of claim 1, wherein said cell is a lymphoma cell.