WO2008114009A1

WO2008114009A1 - 10h-benzo(g)pterdine-2,4-dione compounds for the treatment of proliferative disorders

Info

Publication number: WO2008114009A1
Application number: PCT/GB2008/000962
Authority: WO
Inventors: Shankar Balasubramanian; Mallesham Bejugam
Original assignee: Cambridge Enterprise Ltd
Current assignee: Cambridge Enterprise Ltd
Priority date: 2007-03-22
Filing date: 2008-03-18
Publication date: 2008-09-25
Anticipated expiration: 2009-09-22
Also published as: GB0705517D0

Abstract

This invention pertains generally to the field of G-quadruplex ligands, and more particularly, to certain 10H-benzo[g]pteridine-2,4-dione compounds ('BPD compounds'), as described herein, which, inter alia, (selectively) bind (and stabilize) G-quadruplexes. The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, both in vitro and in vivo, to (selectively) bind (and stabilize) G-quadruplexes, to inhibit telomerase, to regulate cell proliferation, and in the treatment of proliferative disorders, such as cancer. Formula (I):

Description

10H-BENZO[G]PTERIDINE-2,4-DIONE COMPOUNDS AND THEIR USE IN THERAPY

RELATED APPLICATION

This application is related to United Kingdom patent application number 0705517.1 filed 22 March 2007, the contents of each of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention pertains generally to the field of G-quadruplex ligands, and more particularly, to certain 10H-benzo[g]pteridine-2,4-dione compounds ("BPD compounds") which, inter alia, (selectively.) bind (and stabilize) G-quadruplexes. The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, both in vitro and in vivo, to (selectively) bind (and stabilize) G-quadruplexes, to inhibit telomerase, to alter expression of certain genes, to regulate cell proliferation, and in the treatment of proliferative disorders, such as cancer.

BACKGROUND

A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

Ranges are often expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent "about," it will be understood that the particular value forms another embodiment.

This disclosure includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

G-Quadruplexes

Nucleic acid sequences rich in guanine are capable of forming four-stranded structures called G-quadruplexes, stabilized by Hoogsteen hydrogen bonding between a tetrad of guanine bases (see, e.g., Gellert et al., 1962; Guschlbauer et al., 1990). Telomeric repeats in a variety of organisms (see, e.g., Blackburn, 1990; Blackburn, 1991 ) have been shown to form these structures in vitro and high-resolution structures of the human telomeric sequence d(T2AG3)n have been solved by NMR spectroscopy (see, e.g., Wang et al., 1993) and X-ray crystallography (see, e.g., Parkinson et al., 2002). Quadruplexes have also been shown to exist in vivo in Stylonychia lemnae macronuclei (see, e.g., Schaffitzel et al., 2001 ). The formation of these telomeric quadruplexes has been shown to decrease the activity of the enzyme telomerase (see, e.g., Fletcher et al., 1998), which is responsible for elongating telomeres. Since elevated telomerase activity has been implicated in -85% of cancers (see, e.g., Mergny et al., 2002), this has become a significant strategy for drug development (see, e.g., Neidle et al., 2002) and molecules that bind to and stabilize G-quadruplexes have been identified (see, e.g., Mergny et al., 2002).

Recently, there has been growing interest in quadruplex forming sequences elsewhere in the genome. There has been particular focus on the quadruplex formed by the nuclease hypersensitive element of the c-myc promoter (see, e.g., Grand et al., 2002; Seenisamy et al., 2004; Simonsson et al., 1998; Siddiqui-Jain et al., 2002) and structural studies have been performed on this sequence (see, e.g., Phan et al., 2004; Ambrus et al., 2005). Other potential quadruplex-forming sequences include the fragile X syndrome repeat d(CGG)n (see, e.g., Fry et al., 1999; Fry et al., 1994; Fojtik et al., 2004), and the Cystatin B promoter (see, e.g., Saha et al., 2001), which has a region with sequence (CGCG₄CG₄)₄ and is involved in epilepsy. G-rich strands of the human insulin gene can form quadruplexes (see, e.g., Castati et al., 1996), as can the mouse Ms6-hm hypervariable satellite repeat (see, e.g., Weitzmann et al., 2002), with sequence (CAGGG)_n. It has recently been proposed that the promoter regions of the RET protooncogene (see, e.g., Sun et al., 2003), c-kit (Rankin et al., 2005; Fernando et al., 2006), and Ki-ras (see, e.g., Cogoi et al., 2004) can each form a quadruplex. G-rich RNA can also fold into quadruplex structures, e.g. the insulin-like growth factor Il mRNA (see, e.g., Christansen et al., 1994). Efforts have been made to gauge the prevalence of G- quadruplexes in the human genome (see, e.g., Huppert et al., 2005).

The existence of quadruplex nucleic acids in nature is also supported by the observation of a number of naturally occurring quadruplex binding proteins, such as the helicases implicated in Bloom's (see, e.g., Sun et al., 1998) and Werner's (see, e.g., Fry et al., 1999) syndromes, the yeast telomere binding protein Rap1 (see, e.g., Giraldo et al., 1994) and the β subunit of the Oxytrchia nova telomere binding protein (see, e.g., Laporte et al., 1998). The fragile X syndrome mental retardation protein has been shown to target G-quadruplex mRNA with a physiological consequence (see, e.g., Darnell et al., 2001).

The use of a quadruplex-specific small molecule probe (see, e.g., Chang et al., 2004) and also an antibody probe (see, e.g., Schaffitzel et al., 2001) has shown that quadruplex structures can exist at the ends of telomeres.

The most widely studied DNA quadruplexes are those derived from telomeric repeat sequences. Zahler et al. demonstrated that telomeric DNA folded into a quadruplex was not a competent substrate for the enzyme telomerase (see, e.g., Zahler et al., 1991). Hurley and Neidle subsequently demonstrated that a quadruplex stabilising ligand could inhibit the extension of DNA substrate by human telomerase (see, e.g., Sun et al., 1997). Telomerase is critical for immortality in most human cancers (see, e.g., Morin et al.,

1995), as demonstrated in key experiments by the Weinberg lab (see, e.g., Hahn et al., 1999), therefore ligand induced quadruplex stabilisation in telomeric DNA has potential as an anti-cancer therapeutic strategy (see, e.g., Neidle et al., 2002). Biophysical studies on the human telomeric quadruplex have provided valuable insights into its structural (see, e.g., Wang et al., 1993; Parkinson et al., 2002) and dynamic properties (see, e.g., Phan et al., 2003; Ying et al., 2003).

In recent years, a number of first generation of ligands that selectively bind quadruplexes and inhibit the action of human telomerase have been found. It has been shown that quadruplex inhibitors can induce relatively rapid telomere attrition, and cell growth inhibition, usually accompanied by apoptosis (see, e.g., Gowan et al., 2002; Sumi et al., 2004; Shammas et al., 2004(a); Riou et al., 2002; Shammas et al., 2003), although there is one reported case of cell growth effects without changes in telomere length (see, e.g., Leonetti et al., 2004). Additional studies, using synthetic PNA molecules that target the telomere, have shown rather rapid cellular effects (see, e.g., Shammas et al., 2004(b)) and support that targeting the telomeric DNA component of the telomerase complex may be a fruitful strategy. One quadruplex agent has exhibited biological activity in in-vivo models, without cytotoxicity (see, e.g., Gowan et al., 2002).

During the past decade, there has been growing interest in the structure, recognition and function of four stranded DNA G-quadruplexes. The best-studied example is the human telomeric quadruplex, which is associated with the inhibition of telomere elongation by telomerase, an enzyme up-regulated in cancer cells (see, e.g., Zahler et al., 1991). Furthermore, a large number of other putative quadruplex forming sequences have been identified in the human genome (see, e.g., Huppert et al., 2005; see, e.g., Todd et al., 2005). Recent studies have suggested that the formation of quadruplexes in the promoter region of certain genes may regulate the expression of these genes (see, e.g., Simonsson, 2001 ; Siddiqui-Jain, et al., 2002; Rankin et al., 2005; Sun, et al., 2005). Thus there is considerable interest in the design and synthesis of quadruplex stabilizing ligands which have potential to intervene with biological function. A major associated challenge for chemists is to identify small molecule scaffolds that recognize quadruplex DNA with specificity over double-stranded DNA. Many of the quadruplex ligands reported to date are flat structures containing extended aromatic ring systems which are believed to stack on top of the G-quadruplex via π-π stacking interactions (see, e.g., Moore et al., 2006; Kaiser et al., 2006; Dixon et al., 2005; Cookson et al., 2005; Seenisamy et al., 2005).

There is a great need for antiproliferative agents which offer one or more of the following advantages:

(a) improved activity.

(b) improved selectivity (e.g., against tumour cells versus normal cells). (c) complement the activity of other treatments (e.g., chemotherapeutic agents);

(d) reduced intensity of undesired side-effects;

(e) fewer undesired side-effects;

(f) simpler methods of administration;

(g) reduction in required dosage amounts; (h) reduction in required frequency of administration;

(i) increased ease of synthesis, purification, handling, storage, etc.; (j) reduced cost of synthesis, purification, handling, storage, etc.

Thus, one aim of the present invention is the provision of compounds which (selectively) bind G-quadruplexes, which are (selective) G-quadruplex ligands, telomerase inhibitors, antiproliferative agents, anti-cancer agents, etc., and which offer one or more of the above properties and advantages.

The present invention pertains to certain 10H-benzo[g]pteridine-2,4-dione compounds ("BPD compounds"), and the discovery of their surprising and unexpected activity as (selective) G-quadruplex ligands, telomerase inhibitors, antiproliferative agents, anti-cancer agents, etc. SUMMARY OF THE INVENTION

One aspect of the invention pertains to certain compounds, specifically, certain 10H-benzo[g]pteridine-2,4-dione compounds ("BPD compounds"), and their surprising and unexpected activity as (selective) (stabilizing) ligands of G-quadruplxes.

Another aspect of the invention pertains to a pharmaceutical composition comprising a BPD compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

Another aspect of the present invention pertains to a method of stabilizing a G-quadruplex, comprising contacting said G-quadruplex (e.g., within a living cell) with an effective amount of a BPD compound, as described herein. Such a method may be performed in vitro or in vivo.

Another aspect of the present invention pertains to a method of inhibiting telomerase (for example, inhibiting telomerase activity, inhibiting formation of telomerase complexes, inhibiting activity of telomerase complexes, etc.), comprising contacting said telomerase (e.g., within a living cell) with an effective amount of a BPD compound, as described herein. Such a method may be performed in vitro or in vivo.

Another aspect of the present invention pertains to a method of regulating cell proliferation comprising contacting a living cell with an effective amount of a BPD compound, as described herein, preferably in the form of a pharmaceutically acceptable composition.

Another aspect of the present invention pertains to a method of treating a proliferative disorder in a subject comprising administering to said subject a therapeutically-effective amount of a BPD compound, as described herein. In one preferred embodiment, the proliferative disorder is cancer.

Another aspect of the present invention pertains to a BPD compound as described herein for use in a method of treatment of the human or animal body by therapy, for example, in a method of treatment of a proliferative disorder, for example, cancer.

Another aspect of the present invention pertains to use of a BPD compound, as described herein, in the manufacture of a medicament for use in the treatment of a proliferative disorder. In one preferred embodiment, the proliferative disorder is cancer.

Another aspect of the present invention pertains to a method of treatment comprising administering to a patient in need of treatment a therapeutically effective amount of a BPD compound as described herein, preferably in the form of a pharmaceutical composition.

In one embodiment, the treatment is treatment of a proliferative disorder, for example, cancer.

Another aspect of the present invention pertains to a kit comprising (a) a BPD compound as described herein, preferably provided as a pharmaceutical composition and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, written instructions on how to administer the compound.

As will be appreciated by one of skill in the art, features and preferred embodiments of one aspect of the invention will also pertain to other aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the circular dichroism (CD) spectral data (ellipticity (mdeg) versus wavelength (nm)) obtained in the study of the interaction of BPD5 (40 μM) with G-quadruplex DNA of htelo (4 μM), in K⁺ and Na⁺ containing buffer (50 mM Tris.HCI, pH 7.4, 10O mM KCI, and 10O mM NaCI):

(a) without ligand (O, open pentagon) ("Htelo-tris");

(b) in the presence of BPD5, without salt (■, filled square) ("Htelo-tris-BPD5");

(c) folded in the presence of 100 mM potassium without ligand (^*, star) ("Htelo-trisK100"); (d) folded in the presence of 100 mM potassium in the presence of BDP5

(A, filled triangle) "Htelo-trisK100-BPD5");

(e) folded in the presence of 100 mM sodium without ligand (+, plus) ("Htelo-trisNa100");

(f) folded in the presence of 100 mM sodium with BPD5 (•.filled circle) ("Htelo-trisNa100-BPD5").

Figure 2 illustrates the circular dichroism (CD) spectral data (ellipticity (mdeg) versus wavelength (nm)) obtained in the study of the interaction of BPD5 with G-quadruplex DNA of htelo in K⁺ containing buffer (50 mM Tris.HCI, pH 7.4, 100 mM KCI): (a) folded in the presence of 100 mM potassium without ligand (O₁ open hexagon)

("HteloKIOO");

(b) folded in the presence 100 mM of potassium and 0.5 equivalents of BPD5 (♦, filled diamond) ("0.5-BPD5");

(c) same as (b) but with 1.0 equivalents BPD5 (x, cross) ("1.0-BPD5"); (d) same as (b) but with 2.0 equivalents BPD5 (•, filled circle) ("2.0-BPD5");

(e) same as (b) but with 3.0 equivalents BPD5 (■, filled square) ("3.0-BPD5"); (f) same as (b) but with 4.0 equivalents BPD5 (O₁ open pentagon) ("4.0-BPD5");

(g) same as (b) but with 8.0 equivalents BPD5 (+, plus) ("8.0-BPD5"); (h) same as (b) but with 16.0 equivalents BPD5 (^*, star) ("16.0-BPD5").

Figure 3 illustrates the circular dichroism (CD) spectral data (ellipticity (mdeg) versus wavelength (nm)) obtained in the study of the interaction of BPD5 (40 μM) with G- quadruplex DNA of c-kit (4 μM) in K⁺ containing buffer (50 mM Tris.HCI, pH 7.4, 100 mM KCI):

(a) without salt and ligand (o, open circle) ("Ckit"); (b) in the presence of BDP 5 (A, filled triangle) ("Ckit-BPD5");

(c) folded in the presence of 100 mM potassium and BDP 5 (■, filled square) ("Ckit-K100-BPD5");

(d) folded in the presence of 100 mM potassium without ligand (^*, star) ("Ckit-K100"); (e) folded in the presence of 100 mM sodium without ligand (x, cross)

("Ckit-Na100");

(f) folded in the presence of 100 mM sodium and ligand (+, plus) ("Ckit-Na100-BPD5").

Figure 4 is bar graph showing the percentage gene expression of c-kit in MCF-7 cells treated with BPD5 or BPD8. The figure shows the levels of expression for control cells treated with 10% DMSO in water (100% expression, blank bar), and for cells treated with the isoalloxazines BPD5 or BPD8 at 6 hours at 5 μM concentration (shaded bars).

Figure 5 is a bar graph showing the percentage gene expression of c-kit in HGC-27 cells treated with BPD8. The figure shows the levels of expression for control cells treated with 10% DMSO in water (100% expression, blank bar), and for cells treated with BPD8 at 2, 4 and 8 hours at 5 μM concentration (shaded bars).

DETAILED DESCRIPTION OF THE INVENTION

Isoalloxazine, also known as 10H-benzo[g]pteridine-2,4-dione, has the structure shown below. Perhaps the best known example of an isoalloxazine derivative is riboflavin (i.e., Vitamin B2), also shown below. 10H-b

It has been reported that oxidised riboflavin binds to G-quartet with a sub-mircomolar binding affinity (see, e.g., Lauhon et al., 1995).

The present invention pertains generally to compounds which may be described as "10H-benzo[g]pteridine-2,4-dione compounds" ("BPD compounds"), and their surprising and unexpected ability to (selectively) bind (and stabilize) G-quadruplexes and to inhibit telomerase, and their use to inhibit telomerase, to regulate cell proliferation, and in the treatment of proliferative disorders, such as cancer.

Compounds

One aspect of the present invention pertains to compounds of the following formula, and pharmaceutically acceptable salts, solvates, and hydrates thereof (collectively referred to herein as "10H-benzo[g]pteridine-2,4-dione" or "BPD" compounds):

wherein:

R^C6 is independently -H, -G¹, or -G²; R^C7 is independently -H, -G¹, or -G²; R^C9 is independently -H, -G¹, or -G²;

wherein: each -G¹, if present, is independently -Ph, -NHPh, -NR^A1Ph, or -OPh;

wherein:

-R -,A1 is independently saturated aliphatic C^alkyl; and each Ph is independently unsubstituted phenyl or phenyl substituted with one or more groups G²;

and wherein: each -G², if present, is independently:

-F, -Cl, -Br, -I₁ -R*², -CF₃, -OH, -OR^₁ -OCF₃, -SR^, -NH₂, -NHR^, -NR^A2 ₂, -NR^A3R^A4, -L^A1-OH, -L^A1-OR^A2,

-L^A1-NH₂, -L^A1-NHR^A2, -L^-NR^, or -L^A1-NR^A3R^A4, -X¹-L^A1-OH, -X¹-L^A1-OR^A2,

-X¹-L^A1-NH₂, -X'-L^-NHR^, -X^L^-NR^, or -X¹-L^A1-NR^A3R^M;

wherein: each -X¹- is independently -O-, -NH-, or -NR^-; each -R*² is independently saturated aliphatic C_1-4alkyl; each -NR^A3R^M is independently -Q¹; each -L^A1- is independently saturated aliphatic C^alkylene;

and wherein: R^N8B is independently -H or -G³;

wherein: each G³ is independently saturated aliphatic C^alkyl;

and wherein:

R^N1° is independently saturated aliphatic C^alkyl, -G⁴, -|_^10G-G⁵, -Q², -L¹⁰-Q³, or -L^10P-Q⁴;

wherein:

G⁴ is phenyl or Cs-βheteroaryl, and is independently unsubstituted or substituted with one or more substituents -G⁶;

G⁵ is phenyl or C_5-6heteroaryl, and is independently unsubstituted or substituted with one or more substituents -G⁶;

-L^10G- is independently saturated aliphatic C^alkylene; and

-L¹⁰- is independently saturated aliphatic C^alkylene; and -|_^10P- is independently phenylene or Cs-βheteroarylene, and is independently unsubstituted or substituted with one or more substituents -G⁶;

wherein: each -G⁶, if present, is independently: -F, -Cl, -Br, -I, -R^A5, -CF₃, -OH, -OR^A5, -OCF₃, -SR^A5,

-NH₂, -NHR^A5, -NR^A5 ₂, -NR^A6R^A7, -L^-OH, -L^-OR^,

-L^-NH₂, -L^A2-NHR^A5, -L^A2-NR^A5 _2l or -L^-NR^R^, -X²-L^A2-OH, -X'-L^-OR^,

-X²^-NH₂, -X'-L^-NHR*⁵, -X'-L^-NR^, or -X'-L^-NR^⁷;

wherein: each -X²- is independently -O-, -NH-, or -NR^A5-; each -R^A5 is independently saturated aliphatic C_1-4alkyl; each -NR^A6R^A7 is independently -Q⁵; each -I_^A2- is independently saturated aliphatic C_2-8alkylene;

and wherein: each of -L³- and -L⁸- is independently saturated aliphatic C₂-ealkylene;

and wherein:

-NR^3PAR^3PB is independently -Q⁶; -NR^8PAR^8PB is independently -Q⁷;

and wherein: each of -Q¹, -Q², -Q³, -Q⁴, -Q⁵, -Q⁶, and -Q⁷ is independently: -NH₂, -NHR^B1, -NR^B1 ₂, or -NR⁸²R⁸³;

wherein: each R^B1 is independently saturated aliphatic C^alkyl; and in each group -NR⁸²R⁸³, R⁸² and R⁸³, taken together with the nitrogen atom to which they are attached, form a 5-, 6-, or 7-membered non-aromatic ring having exactly 1 ring heteroatom or exactly 2 ring heteroatoms, wherein one of said exactly 2 ring heteroatoms is N, and the other of said exactly 2 ring heteratoms is independently selected from N and O.

In one embodiment, R^C6 is independently -H or -G².

In one embodiment, R^C6 is independently -G¹ or -G².

In one embodiment, R^C6 is independently -H.

In one embodiment, R^C6 is independently -G¹. In one embodiment, R^C6 is independently -G².

In one embodiment, R^C7 is independently -H or -G². In one embodiment, R^C7 is independently -G¹ or -G². In one embodiment, R^C7 is independently -H. In one embodiment, R^C7 is independently -G¹. In one embodiment, R^C7 is independently -G². In one embodiment, R^C9 is independently -H or -G². In one embodiment, R^C9 is independently -G¹ or -G². In one embodiment, R^C9 is independently -H. 5 In one embodiment, R^C9 is independently -G¹. In one embodiment, R^C9 is independently -G².

In one embodiment, R^A1, if present, is independently -Me, -Et, -nPr, or -iPr. In one embodiment, R^A1, if present, is independently -Me. 10

In one embodiment, each -G², if present, is independently -F, -Cl, -Br, -I₁ -R*², -CF₃, -OR^A2, or -OCF₃.

In one embodiment, each R*², if present, is independently -Me, -Et, -nPr, or -iPr. 15 In one embodiment, each R^A2, if present, is independently -Me.

In one embodiment, each -L^A1-, if present, is independently -(CH₂)_ni-, wherein n1 is independently 2, 3, 4, 5, 6, 7, or 8. In one embodiment, n1 is independently 2, 3, 4, 5, or 6. In one embodiment, n1 is independently 2, 3, or 4. 20

In one embodiment, R^N8B is independently -H. In one embodiment, R^N8B is independently -G³.

In one embodiment, G³, if present, is independently -Me, -Et, -nPr, or -iPr. 25 In one embodiment, G³, if present, is independently -Me.

In one embodiment, R^N1° is independently saturated aliphatic C_1-6alkyl, -G⁴, or -|_^10G-G⁵.

In one embodiment, R^N1° is independently -L¹⁰-Q³ or -L^10P-Q⁴.

In one embodiment, R^N1° is independently -G⁴ or -L^10G-G⁵. 30

In one embodiment, R^N1° is independently saturated aliphatic C_halky!.

In one embodiment, R^N1° is independently -G⁴.

In one embodiment, R^N1° is independently -L^10G-G⁵.

In one embodiment, R^N1° is independently -Q². 35 In one embodiment, R^N1° is independently -L¹⁰-Q³.

In one embodiment, R^N1° is independently -L^10P-Q⁴.

In one embodiment, R^N1° is independently -Me, -Et, -nPr, or -iPr. In one embodiment, R^N1° is independently -Me. W In one embodiment, -L¹⁰-, if present, is independently -(CH₂)_n3-, wherein n3 is independently 2, 3, 4, 5, 6, 7, or 8. In one embodiment, n3 is independently 2, 3, 4, 5, or 6. In one embodiment, n3 is independently 2, 3, or 4.

In one embodiment, -L^10G-, if present, is independently -(CH₂)_n2-, wherein n2 is independently 1 , 2, 3, or 4. In one embodiment, n2 is independently 1 or 2. In one embodiment, n2 is independently 1.

In one embodiment, -L^10P- is independently phenylene, oxazol-di-yl, thiazol-di-yl, isoxazol-di-yl, isothiazol-di-yl, pyrazol-di-yl, pyridin-di-yl, pyrimidin-di-yl, or pyrazin-di-yl; and is independently unsubstituted or substituted with one or more substituents -G⁶.

In one embodiment, -L^10P-, if present, is independently phenylene (e.g., 1 ,4-phenylene, 1 ,3-phenylene, 1 ,2-phenylene); and is independently unsubstituted or substituted with one or more substituents -G⁶.

In one embodiment, -G⁴, if present, is independently phenyl or C_5-6heteroaryl, and is independently unsubstituted or substituted with one or more substituents -G⁶.

In one embodiment, -G⁴, if present, is independently phenyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridinyl, pyrimidinyl, or pyrazinyl; and is independently unsubstituted or substituted with one or more substituents -G⁶.

In one embodiment, -G⁴, if present, is independently phenyl; and is independently unsubstituted or substituted with one or more substituents -G⁶.

In one embodiment, -G⁵, if present, is independently phenyl or Cs-eheteroaryl, and is independently unsubstituted or substituted with one or more substituents -G⁶.

In one embodiment, -G⁵, if present, is independently phenyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridinyl, pyrimidinyl, or pyrazinyl; and is independently unsubstituted or substituted with one or more substituents -G⁶.

In one embodiment, -G⁵, if present, is independently phenyl; and is independently unsubstituted or substituted with one or more substituents -G⁶.

In one embodiment, each -L*²-, if present, is independently -(CH₂),*-, wherein n4 is independently 2, 3, 4, 5, 6, 7, or 8. In one embodiment, n4 is independently 2, 3, 4, 5, or 6. In one embodiment, n4 is independently 2, 3, or 4. In one embodiment, each -G⁶, if present, is independently -F₁ -Cl, -Br₁ -I, -R^A5, -CF₃, -OR^A5, or -OCF₃.

In one embodiment, each R^A5, if present, is independently -Me, -Et, -nPr, or -iPr. In one embodiment, each R^A5, if present, is independently -Me.

In one embodiment, -L³- is independently -(CH₂)_n5-, wherein n5 is independently 2, 3, 4, 5, 6, 7, or 8. In one embodiment, n5 is independently 2, 3, 4, 5, or 6. In one embodiment, n5 is independently 2, 3, or 4.

In one embodiment, -L⁸- is independently -(CH₂W. wherein n6 is independently 2, 3, 4, 5, 6, 7, or 8. In one embodiment, n6 is independently 2, 3, 4, 5, or 6. In one embodiment, n6 is independently 2, 3, or 4.

In one embodiment, -L³- and -L⁸- are the same.

In one embodiment, each of -Q¹, -Q², -Q³, -Q⁴, -Q⁵, -Q⁶, and -Q⁷ is independently: -NH₂, -NHR^B1, or -NR⁶²R⁶³.

In one embodiment, each -Q¹, if present, is independently -NH₂, -NHR⁶¹, -NR^B1 _2l or -NR⁶²R⁶³. In one embodiment, each -Q¹, if present, is independently -NH₂, -NHR⁶¹, or -NR⁶²R⁶³.

In one embodiment, -Q², if present, is independently -NH₂, -NHR⁶¹, -NR^B1 ₂, or -NR⁶²R⁶³. In one embodiment, -Q², if present, is independently -NH₂, -NHR⁶¹, or -NR⁶²R⁶³.

In one embodiment, -Q³, if present, is independently -NH₂, -NHR⁶¹, -NR^B1 ₂, or -NR⁶²R⁶³. In one embodiment, -Q³, if present, is independently -NH₂, -NHR⁶¹, or -NR⁶²R⁶³.

In one embodiment, -Q⁴, if present, is independently -NH₂, -NHR⁶¹, -NR^B1 ₂, or -NR⁶²R⁶³. In one embodiment, -Q⁴, if present, is independently -NH₂, -NHR⁶¹, or -NR⁶²R⁶³.

In one embodiment, each -Q⁵, if present, is independently -NH₂, -NHR⁶¹, -NR⁶¹ ₂, or -NR⁸²R⁶³. In one embodiment, each -Q⁵, if present, is independently -NH₂, -NHR⁶¹, or -NR⁶²R⁸³.

In one embodiment, -Q⁶ is independently -NH₂, -NHR⁶¹, -NR⁶¹ ₂, or -NR⁶²R⁶³. In one embodiment, -Q⁶ is independently -NH₂, -NHR⁶¹, or -NR⁸²R⁶³.

In one embodiment, -Q⁷ is independently -NH₂, -NHR⁶¹, -NR^B1 ₂, or -NR⁶²R⁶³. In one embodiment, -Q⁷ is independently -NH₂, -NHR⁶¹, or -NR⁸²R⁶³. In one embodiment, each -Q¹, if present, is independently -NH₂, -NR^B1 _2l or -NHR^B1. In one embodiment, each -Q¹, if present, is independently -NH₂ or -NHR^B1.

In one embodiment, each -Q⁵, if present, is independently -NH₂, -NR^B1 ₂, or -NHR^B1. In one embodiment, each -Q⁵, if present, is independently -NH₂ or -NHR^B1.

In one embodiment, -Q², if present, is independently -NR^B2R^B3.

In one embodiment, -Q³, if present, is independently -NR^B2R^B3. In one embodiment, -Q⁴, if present, is independently -NR^B2R^B3.

In one embodiment, -Q⁶ is independently -NR⁸²R⁶³. In one embodiment, -Q⁷ is independently -NR⁸²R⁶³.

In one embodiment -Q⁶ and -Q⁷ are the same.

In one embodiment, each R^B1, if present, is independently -Me, -Et, -nPr, or -iPr. In one embodiment, each R⁸¹, if present, is independently -Me.

In one embodiment, each -NR⁸²R⁸³, if present, is independently pyrrolidino, imidazolidino, N-CCvsalkyO-imidazolidino, pyrazolidino, N-(C_1-3alkyl)-pyrazolidino, piperidino, N-Cd^alkyl^piperidino, piperizino, morpholino, azepino, diazepino, or N^CvaalkyO-diazepino.

In one embodiment, -Q¹, if present, is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂, -N(nPr)₂, -N(JPr)₂, pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

In one embodiment, -Q¹, if present, is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂, -N(nPr)_2l or -N(JPr)₂.

In one embodiment, -Q⁵, if present, is independently -NH₂, -NHMe₁ -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂, -N(nPr)₂, -N(JPr)₂, pyrrolidino, imidazolidino,

N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

In one embodiment, -Q⁵, if present, is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂, -N(nPr)₂, or -N(JPr)₂. In one embodiment, -Q², if present, is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂, -N(nPr)₂, -N(JPr)₂, pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

In one embodiment, -Q², if present, is independently pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

In one embodiment, -Q³, if present, is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂, -N(nPr)₂, -N(JPr)₂, pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

In one embodiment, -Q³, if present, is independently pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

In one embodiment, -Q⁴, if present, is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂, -N(nPr)₂, -N(JPr)₂, pyrrolidino, imidazolidino,

In one embodiment, -Q⁴, if present, is independently pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

In one embodiment, -Q⁶ is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr),

-N(Me)₂, -N(Et)₂, -N(nPr)₂, -N(JPr)₂, pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

In one embodiment, -Q⁶ is independently pyrrolidino, imidazolidino,

In one embodiment, -Q⁷ is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂, -N(nPr)₂, -N(iPr)₂, pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

In one embodiment, -Q⁷ is independently pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

In one embodiment, the group -NR^N8B-L⁸-NR^8PAR^8PB is independently selected from:

In one embodiment, the group -L -NR R is independently selected from:

In one embodiment, the group -L -G is independently selected from:

In one embodiment, the group -G⁴ is independently selected from:

In one embodiment, the group -L -Q is independently selected from:

In one embodiment, the group -L -Q is independently selected from

Molecular Weight

In one embodiment, the compound has a molecular weight of from 329 to 1200. In one embodiment, the bottom of range is 330; 350; 375; 400; 425; 450. In one embodiment, the top of range is 1100; 1000, 900, 800, 700. In one embodiment, the range is 329 to 700.

Combinations

Each and every compatible combination of the embodiments described above is explicitly disclosed herein, as if each and every combination was individually and explicitly recited.

Some Preferred Embodiments

Examples of some preferred compounds include the following compounds, and pharmaceutically acceptable salts, solvates, and hydrates thereof:

Substantially Purified Forms

One aspect of the present invention pertains to BPD compounds, as described herein, in substantially purified form and/or in a form substantially free from contaminants.

In one embodiment, the substantially purified form is at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight.

Unless specified, the substantially purified form refers to the compound in any stereoisomeric or enantiomeric form. For example, in one embodiment, the substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds. In one embodiment, the substantially purified form refers to one stereoisomer, e.g., optically pure stereoisomer. In one embodiment, the substantially purified form refers to a mixture of enantiomers. In one embodiment, the substantially purified form refers to a equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate). In one embodiment, the substantially purified form refers to one enantiomer, e.g., optically pure enantiomer.

In one embodiment, the contaminants represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1% by weight.

Unless specified, the contaminants refer to other compounds, that is, other than stereoisomers or enantiomers. In one embodiment, the contaminants refer to other compounds and other stereoisomers. In one embodiment, the contaminants refer to other compounds and the other enantiomer.

In one embodiment, the substantially purified form is at least 60% optically pure (i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer or enantiomer), e.g., at least 70% optically pure, e.g., at least 80% optically pure, e.g., at least 90% optically pure, e.g., at least 95% optically pure, e.g., at least 97% optically pure, e.g., at least 98% optically pure, e.g., at least 99% optically pure.

Isomers

Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").

Note that, except as discussed below for tautomeric forms, specifically excluded from the term "isomers," as used herein, are structural (or constitutional) isomers (i.e., isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, -OCH₃, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH₂OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., C_1-7alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hydroxyazo, and nitro/aci-nitro.

keto enol enolate

Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including ¹H, ²H (D), and ³H (T); C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopic form, including ¹⁶O and ¹⁸O; and the like.

Many of the chemical structures shown herein indicate one or more specific stereoisomeric configurations. Similarly, many of the chemical structures shown herein are silent in this respect, and do not indicate any stereoisomeric configuration. Where a chemical structure herein is silent with respect to the stereoisomeric configuration at a position, that structure is intended to depict all possible stereoisomeric configuration at that position, both individually, as if each possible stereoisomeric configuration was individually recited, and also as a mixture (e.g., a racemic mixture) of stereoisomers. Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including mixtures (e.g., racemic mixtures) thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

Salts

It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharrn. ScL. Vol. 66, pp. 1-19.

For example, if the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO^"), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may be cationic (e.g., -NH₂ may be -NH₃ ⁺), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose. Unless otherwise specified, a reference to a particular compound also includes salt forms thereof.

Solvates and Hydrates

It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the compound. The term "solvate" is used herein in the conventional sense to refer to a complex of solute (e.g., compound, salt of compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

Unless otherwise specified, a reference to a particular compound also includes solvate and hydrate forms thereof.

Chemical Synthesis

Methods for the chemical synthesis of BPD compounds of the present invention are described herein. These and/or other well-known methods may be modified and/or adapted in known ways in order to facilitate the synthesis of additional BPD compounds of the present invention.

The 10H-benzo[g]pteridine-2,4-dione ("BPD") compounds described herein may be prepared, for example, by methods in which, first, 2,4-dichloro-i-nitro-benzene (1) is reacted with a suitable amine to form the corresponding (5-chloro-2-nitro-phenyl) amine (2). This amine (2) is then reduced, for example, with zinc dust, and then reacted with alloxan and boric acid to give the corresponding 8-chloro-10-substituted- 10H-benzo[g]pteridine-2,4-dione (3). This compound is then reacted with a suitable chloride to form the corresponding 3,10-disubstituted-8-chloro-10H-benzo[g]pteridine-2,4- dione (4). This compound is then reacted with suitable amine to give the corresponding 3,8,10-trisubstituted-8-chloro-10H-benzo[g]pteridine-2,4-dione (5).

An example of such a method is illustrated in the following scheme: Scheme 1

Rⁱ-NH₂, anhydrous K₂CO₃, DMF, 9O⁰C, 24-36 hours or: R¹-NH₂, neat, 100⁰C, 2-3 hours

i) Zinc dust, AcOH, room temperature, 20-30 minutes ii) Alloxan. H₂O, boric acid, AcOH, room temperature, 0.5 to 2 hours or 100°C, 0.5 to 5 hours

R²CI, anhydrous K₂CO₃, DMF, 80-90⁰C, 2-5 hours

R³NH₂, neat, 100⁰C, 20-30 minutes

Pharmaceutical Compositions

Another aspect of the invention pertains to a pharmaceutical composition comprising a BPD compound as described herein.

Another aspect of the invention pertains to a pharmaceutical composition comprising a BPD compound as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

Another aspect of the invention pertains to a method of preparing a pharmaceutical composition comprising admixing a BPD compound as described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

Examples of suitable pharmaceutically acceptable carriers, diluents, and excipients are described below. Uses

Methods of Selectively Binding a G-Quadruplex

Another aspect of the present invention pertains to a method of selectively binding a G-quadruplex, comprising contacting said G-quadruplex with an effective amount of a BPD compound, as described herein.

Such a method may be performed in vitro or in vivo. In one embodiment, said G-quadruplex is within a living cell. In one embodiment, said G-quadruplex is within a living cell that is not an integral part of a living human or multicellular animal body. In one embodiment, said G-quadruplex is within a living cell that is an integral part of a living human or multicellular animal body.

In one embodiment, the method is a method of selectively binding a G-quadruplex within a living cell, comprising contacting said cell with an effective amount of a BPD compound, as described herein, preferably in the form of a pharmaceutically acceptable composition. Such a method may be performed in vitro or in vivo.

One of ordinary skill in the art is readily able to determine whether or not a candidate compound selectively binding a G-quadruplex.

Methods of Stabilizing a G-Quadruplex

Another aspect of the present invention pertains to a method of stabilizing a

G-quadruplex, comprising contacting said G-quadruplex with an effective amount of a BPD compound, as described herein.

In one embodiment, the method is a method of stabilizing a G-quadruplex within a living cell, comprising contacting said cell with an effective amount of a BPD compound, as described herein, preferably in the form of a pharmaceutically acceptable composition. Such a method may be performed in vitro or in vivo.

One of ordinary skill in the art is readily able to determine whether or not a candidate compound stabilizes a G-quadruplex. Methods of Inhibiting Telomerase

Another aspect of the present invention pertains to a method of inhibiting telomerase (for example, inhibiting telomerase activity, inhibiting formation of telomerase complexes, inhibiting activity of telomerase complexes, etc.), comprising contacting said telomerase with an effective amount of a BPD compound, as described herein.

Such a method may be performed in vitro or in vivo. In one embodiment, said telomerase is within a living cell. In one embodiment, said telomerase is within a living cell that is not an integral part of a living human or multicellular animal body. In one embodiment, said telomerase js within a living cell that is an integral part of a living human or multicellular animal body.

In one emdobiment, the method is a method of inhibiting telomerase within a living cell, comprising contacting said cell with an effective amount of a BPD compound, as described herein, preferably in the form of a pharmaceutically acceptable composition. Such a method may be performed in vitro or in vivo.

One of ordinary skill in the art is readily able to determine whether or not a candidate compound inhibits telomerase activity.

Methods of Inhibiting Gene Expression

Another aspect of the present invention pertains to a method reducing, repressing, or inhibiting gene expression in a cell, comprising contacting a living cell with an effective amount of a BPD compound, as described herein, preferably in the form of a pharmaceutically acceptable composition. Such a method may be performed in vitro or in vivo.

In one embodiment, the gene expression is c-kit gene expression (e.g., expression of the gene that encodes the c-kit receptor).

In one embodiment, the gene expression is vascular endothelial growth factor (VEGF) gene expression (e.g., expression of the gene that encodes the VEGF receptor).

In one embodiment, the gene expression is c-myc gene expression (e.g., expression of the gene that encodes the c-myc receptor).

In one embodiment, the gene expression is BCL-2 gene expression (e.g., expression of the gene that encodes the BCL-2 receptor). In one embodiment, the gene expression is B-raf gene expression (e.g., expression of the gene that encodes the B-raf receptor).

In one embodiment, the gene expression is k-ras gene expression (e.g., expression of the gene that encodes the f k-ras receptor).

In one embodiment, said cell is a living cell that is not an integral part of a living human or multicellular animal body. In one embodiment, said cell is a living cell that is an integral part of a living human or multicellular animal body.

One of ordinary skill in the art is readily able to determine whether or not a candidate compound regulates cell proliferation for any particular cell line.

Methods of Regulating Cell Proliferation

Another aspect of the present invention pertains to a method of regulating (e.g., inhibiting) cell proliferation comprising contacting a living cell with an effective amount of a BPD compound, as described herein, preferably in the form of a pharmaceutically acceptable composition. Such a method may be performed in vitro or in vivo.

In one embodiment, said cell is a living cell that is not an integral part of a living human or multicellular animal body. In one embodiment, said cell is a living cell that \s an integral part of a living human or multicellular animal body.

For example, a sample of cells (e.g., from a tumour) may be grown in vitro and a candidate compound brought into contact with the cells, and the effect of the compound on those cells observed. As examples of "effect," the morphological status of the cells may be determined (e.g., alive or dead), or the expression levels of genes associated with cell cycle regulation determined. Where the candidate compound is found to exert an influence on the cells, this may be used as a prognostic or diagnostic marker of the efficacy of the compound in methods of treating a patient with cells of the same type (e.g., the tumour or a tumour of the same cellular type). Methods of Treatment

Another aspect of the present invention pertains to a method of treatment comprising administering to a patient in need of treatment a therapeutically-effective amount of a BPD compound, as described herein, preferably in the form of a pharmaceutical composition.

Use in Methods of Therapy

Another aspect of the present invention pertains to a BPD compound, as described herein, for use in a method of treatment, for example, a method of treatment of the human or animal body by therapy.

Use in the Manufacture of Medicaments

Another aspect of the present invention pertains to use of a BPD compound, as described herein, in the manufacture of a medicament for use in treatment.

In one embodiment, the medicament comprises the BPD compound.

Disorders Treated - Disorders Mediated by a G-Quadruplex

The BPD compounds described herein are useful in the treatment of disorders that are mediated by a G-quadruplex.

Thus, in one embodiment, the treatment is treatment of a disorder that is mediated by a G-quadruplex.

A disorder that is mediated by a G-quadruplex is a disorder that, for initiation or maintenance or progress of the disorder, requires the formation or destruction of a

G-quadruplex. For such disorders, treatment with a G-quadruplex ligand, e.g., a selective G-quadruplex ligand, e.g., a selective stabilising G-quadruplex ligand, is beneficial.

c-kit Promoter Quadruplex:

C-kit is a tyrosine kinase receptor for the growth-promoting cytokine SCF (stem cell factor) which plays an important biological role in the control of differentiation. Two quadruplex forming sequence stretches have been identified within the c-kit promoter. Expression of mutant c-kit/SCF has been implicated in the genesis of several solid tumour types (see, e.g., Rankin et al., 2005). About 40% of small cell lung cancers express c-kit (see, e.g., Micke et al., 2003), as do essentially all Ewing's sarcoma (see, e.g., Merchant et al., 2002), and up to 85% of gastrointestinal stromal tumours (see, e.g., DeMatteo, 2002). Activating mutations have also been found in a small fraction of acute myeloid leukaemias (see, e.g., Kelly et al., 2002). Interestingly, c-kit activity can be inhibited with the new therapeutic agent Gleevec®, and in Ewing's sarcoma and gastrointestinal stromal tumours, there is at least in vitro evidence that c-kit inhibition can retard tumor growth (see, e.g., DeMatteo, 2002; Druker, 2002). Other targets on the basis of c-kit expression include: prostate cancer (see, e.g., Simak et al., 2000), and adenocarcinoma lung cancers (see, e.g., Micke et al., 2004).

As mentioned above, c-kit is a receptor tyrosine kinase expressed in a number of different normal cell types, whose dysregulated expression or activity can drive the proliferation of cancer cells. In particular, c-kit dysregulation is a common feature of gastro-intestinal stromal tumours (GIST), a leiomyosarcomatous neoplasm with an estimated incidence of 1200 cases per year in the UK and 5000 cases per year in the USA. There is a strong base of evidence that GIST tumour cells almost invariably contain mutations in c-kit.

These mutations cluster around the juxta-transmembrane region of the receptor, or within the activation loop of the kinase domain, dysregulating enzymatic activity. Patient survival with standard cytotoxic treatment regimes is poor. However, clinical trials demonstrate that c-kit inhibition by the small molecule Gleevec® (ST1571 , Novartis) provides marked clinical improvement, and this agent has become the standard treatment for patients with metastatic GIST. Although a fraction of GIST tumours with activation loop mutations respond poorly to Gleevec® from the outset, generally speaking, Gleevec® treatment leads to rapid tumour regression and clinical improvement. However, the tumour mass does not usually regress completely, and the usual clinical course involves a period of quiescence followed by the outgrowth of cells from the persisting tumour mass. Clinical recrudescence of this kind is often accompanied by refractoriness to c-kit inhibition by Gleevec®, through acquired mutations in the kinase activation loop. These clinical observations highlight the need for alternative strategies for the therapeutic inhibition of c-kit activity, both in GIST patients who fail to respond to Gleevec®, or in those who develop recrudescent tumours that are Gleevec®-refractory. Similarly, activation loop mutations in c-kit found in certain cases of acute myeloid leukaemia (incidence -2000 cases/year in the UK) or testicular seminomas (incidence -2000 cases/year in the UK) are also typically resistant to Gleevec® therapy.

Thus, in one embodiment, the treatment is treatment of a disorder (e.g., cancer) that is characterised by expression (or overexpression) of c-kit.

Thus, in one embodiment, the treatment is treatment of a solid tumour, gastro-intestinal stromal tumour (GIST), small cell lung cancer, adenocarcinoma lung cancer, Ewing's sarcoma, acute myeloid leukaemia, prostate cancer, or testicular seminoma; for example, that is characterised by expression (or overexpression) of c-kit. Human Vascular Endothelial Growth Factor (VEGF) Promoter Quadruplex:

A quadruplex sequence has been identified and characterised in the VEGF promoter. The angiogenic switch in cancer cells is often initiated by the expression of VEGF. Vascular endothelial growth factor receptor (VEGF) is a clinically validated target implicated in the induction of new blood vessel formation, which is essential for tumours to grow to more than a limited, small size, before they cause symptoms. Thus, the VEGF promoter quadruplex is a molecular target for the treatment of solid tumors. (See, e.g., Sun et al., 2005; Folkman, 1971.)

Thus, in one embodiment, the treatment is treatment of a disorder (e.g., cancer) that is characterised by expression (or overexpression) of VEGF.

Thus, in one embodiment, the treatment is treatment of a solid tumour; for example, that is characterised by expression (or overexpression) of VEGF.

c-myc Promoter Quadruplex:

A quadruplex forming sequence has been identified within the c-myc promoter.

Overexpression of the c-MYC oncogene is linked with a wide range of cancers that include colon cancer, breast cancer, small-cell lung cancer, osteosarcomas, glioblastomas, and myeloid leukaemia. (See, e.g., Siddiqui-Jain et al., 2002; Pelengaris et al., 2000; Spencer et al., 1991; Marcu et al., 1992; Facchini et al., 1998).

Thus, in one embodiment, the treatment is treatment of a disorder (e.g., cancer) that is characterised by expression (or overexpression) of c-myc.

Thus, in one embodiment, the treatment is treatment of colon cancer, breast cancer, small-cell lung cancer, osteosarcoma, glioblastoma, or myeloid leukaemia; for example, that is characterised by expression (or overexpression) of c-myc.

BCL-2 Promoter Quadruplex:

Quadruplex forming sequences have been identified within the BCL-2 promoter. BCL-2 promoter quadruplexes are important for B-cell and T-cell lymphomas, breast, prostate, cervical, colorectal and non-small cell lung carcinomas. (See, e.g., Dai et al., 2006; Akagi et al., 1994; Joensuu et al., 1994; Tjalma et al., 1998; Pezzella et al., 1993; McDonnell et al., 1992; Baretton et al., 1996; Reed et al., 1994.) Thus, in one embodiment, the treatment is treatment of a disorder (e.g., cancer) that is characterised by expression (or overexpression) of BCL-2.

Thus, in one embodiment, the treatment is treatment of B-cell lymphoma, T-cell lymphoma, breast cancer, prostate cancer, cervical cancer, colorectal cancer, and non-small cell lung cancer; for example, that is characterised by expression (or overexpression) of BCL-2.

B-raf Promoter Quadruplex:

The inventors have identified a quadruplex forming sequence within the B-raf promoter. B-raf encodes an intracellular serine-threonine kinase that is critical for growth signalling through the ras pathway. B-raf activity is dysregulated by mutations in up to 70% of malignant melanoma, an aggressive and hard-to-treat neoplasm of the skin, whose annual incidence is estimated to be about 7500 cases/year in the UK or 30000 cases/year in the USA.

Thus, in one embodiment, the treatment is treatment of a disorder (e.g., cancer) that is characterised by expression (or overexpression) of B-raf.

Thus, in one embodiment, the treatment is treatment of skin cancer or melanoma; for example, that is characterised by expression (or overexpression) of B-raf.

k-ras Promoter Quadruplex:

Thus, in one embodiment, the treatment is treatment of a disorder (e.g., cancer) that is characterised by expression (or overexpression) of k-ras.

Disorders Treated - Proliferative Disorders and Cancer

In one embodiment, the treatment is treatment of a proliferative disorder.

The terms "proliferative condition," "proliferative disorder," and "proliferative disease," are used interchangeably herein and pertain to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells that is undesired, such as, neoplastic or hyperplastic growth.

Thus, in one embodiment, the compounds are anti-proliferative agents. The term "antiproliferative agent," as used herein, pertains to a compound that treats a proliferative disorder (i.e., a compound which is useful in the treatment of a proliferative disorder). In one embodiment, the treatment is treatment of a proliferative disorder characterised by benign, pre-malignant, or malignant cellular proliferation, including but not limited to, neoplasms, hyperplasias, and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (see below), psoriasis, bone diseases, fibroproliferative disorders (e.g., of connective tissues), pulmonary fibrosis, atherosclerosis, smooth muscle cell proliferation in the blood vessels, such as stenosis or restenosis following angioplasty.

In one embodiment, the treatment is treatment of cancer.

Thus, in one embodiment, the compounds are anti-cancer agents. The term "anti-cancer agent" as used herein, pertains to a compound that treats a cancer (i.e., a compound which is useful in the treatment of a cancer). The anti-cancer effect may arise through one or more mechanisms, including but not limited to, the regulation of cell proliferation, the inhibition of cell cycle progression, the inhibition of angiogenesis (the formation of new blood vessels), the inhibition of metastasis (the spread of a tumour from its origin), the inhibition of invasion (the spread of tumour cells into neighbouring normal structures), or the promotion of apoptosis (programmed cell death).

One of ordinary skill in the art is readily able to determine whether or not a candidate compound treats a proliferative disorder, or treats cancer, for any particular cell type.

In one embodiment, the treatment is treatment of: solid tumour cancer, gastrointestinal stromal cancer (GIST), stomach cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma lung cancer, gastrointestinal cancer, thyroid cancer, breast cancer, ovarian cancer, epithelial ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, testicular cancer, testicular seminoma, liver cancer, kidney cancer, renal cell carcinoma, bladder cancer, pancreatic cancer, oesophageal cancer, brain cancer, glioblastoma, glioma, sarcoma, osteosarcoma, Ewing's sarcoma, bone cancer, skin cancer (e.g., head and neck cancer), squamous cancer, Kaposi's sarcoma, melanoma, malignant melanoma, lymphoma, B-cell lymphoma, T-cell lymphoma, leukaemia, myeloid leukaemia, acute myeloid leukaemia, or a tumour of unknown origin.

All histological subtypes of the cancers above are included. For example, a pathologist may determine the histological subtype of a cancer based upon the cell morphology, for example, mucinous, adenocarcinoma, serous, papillary, etc.

The BPD compounds described herein may be used in the treatment of the cancers described herein, independent of the mechanisms discussed herein. Screeninq

Prior to treatment, a patient may be screened in order to determine whether a disease or disorder from which the patient is or may be suffering is one which would be susceptible to treatment with a BPD compound as described herein.

Treatment

The term "treatment," as used herein in the context of treating a disorder, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the disorder, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviation of symptoms of the disorder, amelioration of the disorder, and cure of the disorder. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, use with patients who have not yet developed the disorder, but who are at risk of developing the disorder, is encompassed by the term "treatment." For example, treatment includes the prophylaxis of cancer, reducing the risk of cancer, alleviating the symptoms of cancer, etc.

The term "therapeutically-effective amount," as used herein, pertains to that amount of a compound, or a material, composition or dosage form comprising a compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.

Combination Therapies

The term "treatment" includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. For example, the BPD compounds described herein may also be used in combination therapies, e.g., in conjunction with other agents, for example, cytotoxic agents, anticancer agents, etc. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g., drugs, antibodies (e.g., as in immunotherapy), prodrugs (e.g., as in photodynamic therapy, GDEPT, ADEPT, etc.); surgery; radiation therapy; photodynamic therapy; gene therapy; and controlled diets.

For example, it may be beneficial to combine treatment with a BPD compound as described herein with one or more other (e.g., 1, 2, 3, 4) agents or therapies that regulates cell growth or survival or differentiation via a different mechanism, thus treating several characteristic features of cancer development. In one embodiment, a BPD compound as described herein is combined with one or more (e.g., 1 , 2, 3, 4) additional therapeutic agents, as described below.

One aspect of the present invention pertains to a BPD compound as described herein, in combination with one or more (e.g., 1, 2, 3, 4) additional therapeutic agents, as described below.

The particular combination would be at the discretion of the physician who would select dosages using his/her common general knowledge and dosing regimens known to a skilled practitioner.

The agents (i.e., a BPD compound as described here, plus one or more other agents) may be administered simultaneously or sequentially, and may be administered in individually varying dose schedules and via different routes. For example, when administered sequentially, the agents can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1 , 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s).

The agents (i.e., a BPD compound as described here, plus one or more other agents) may be formulated together in a single dosage form, or alternatively, the individual agents may be formulated separately and presented together in the form of a kit, optionally with instructions for their use, as described below.

Other Uses

The BPD compounds described herein may also be used as cell culture additives to inhibit telomerase, inhibit cell proliferation, etc.

The BPD compounds described herein may also be used as part of an in vitro assay, for example, in order to determine whether a candidate host is likely to benefit from treatment with the compound in question.

The BPD compounds described herein may also be used as a standard, for example, in an assay, in order to identify other active compounds, other anti-proliferative agents, other anti-cancer agents, etc.

Kits

One aspect of the invention pertains to a kit comprising (a) a BPD compound as described herein, or a composition comprising a compound as described herein, e.g., preferably provided in a suitable container and/or with suitable packaging; and

(b) instructions for use, e.g., written instructions on how to administer the compound or composition.

The written instructions may also include a list of indications for which the active ingredient is a suitable treatment.

Routes of Administration

The BPD compound or pharmaceutical composition comprising the BPD compound may be administered to a subject by any convenient route of administration, whether systemically/peripherally or topically (i.e., at the site of desired action).

Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., using an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly.

The Subject/Patient

The subject/patient may be an animal, a mammal, a placental mammal, a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang, gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development, for example, a foetus.

In one preferred embodiment, the subject/patient is a human. Formulations

While it is possible for the BPD compound to be administered alone, it is preferable to present it as a pharmaceutical formulation (e.g., composition, preparation, medicament) comprising at least one BPD compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents. The formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents.

Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound.

The term "pharmaceutically acceptable" as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients. 2nd edition, 1994.

The formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.

The formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof. Formulations may suitably be in the form of liquids, solutions (e.g., aqueous, nonaqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated tablets), granules, powders, lozenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols.

Formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers. Formulations may also suitably be provided in the form of a depot or reservoir.

The compound may be dissolved in, suspended in, or admixed with one or more other pharmaceutically acceptable ingredients. The compound may be presented in a liposome or other microparticulate which is designed to target the compound, for example, to blood components or one or more organs.

Formulations suitable for oral administration (e.g., by ingestion) include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders, capsules, cachets, pills, ampoules, boluses.

Formulations suitable for buccal administration include mouthwashes, lozenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs. Lozenges typically comprise the compound in a flavored basis, usually sucrose and acacia or tragacanth. Pastilles typically comprise the compound in an inert matrix, such as gelatin and glycerin, or sucrose and acacia. Mouthwashes typically comprise the compound in a suitable liquid carrier.

Formulations suitable for sublingual administration include tablets, lozenges, pastilles, capsules, and pills.

Formulations suitable for oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil- in-water, water-in-oil), mouthwashes, lozenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.

Formulations suitable for non-oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), suppositories, pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.

Formulations suitable for transdermal administration include gels, pastes, ointments, 5 creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs.

Tablets may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by

10 compressing in a suitable machine the compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone,

15 cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid); flavours, flavour enhancing agents, and sweeteners. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be

20 coated or scored and may be formulated so as to provide slow or controlled release of the compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with a coating, for example, to affect release, for example an enteric coating, to provide release in parts of the gut other than the stomach.

?5

Ointments are typically prepared from the compound and a paraffinic or a water-miscible ointment base.

Creams are typically prepared from the compound and an oil-in-water cream base. If 30 desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1 ,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the compound through the skin or other affected 55 areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.

Emulsions are typically prepared from the compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may \0 comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabiliser(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. 5

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the compound in most oils likely to be used in

10 pharmaceutical emulsion formulations may be very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate,

15 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

UO Formulations suitable for intranasal administration, where the carrier is a liquid, include, for example, nasal spray, nasal drops, and (for aerosol administration by nebuliser) aqueous or oily solutions of the compound.

Formulations suitable for intranasal administration, where the carrier is a solid, include, .5 for example, those presented as a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.

i0 Formulations suitable for pulmonary administration (e.g., by inhalation or insufflation therapy) include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.

>5 Formulations suitable for ocular administration include eye drops wherein the compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the compound.

Formulations suitable for rectal administration may be presented as a suppository with a 0 suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the compound, such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration (e.g., by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the compound in the liquid is from about 1 ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1 μg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) disorder requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

Dosage

It will be appreciated by one of skill in the art that appropriate dosages of the BPD compounds, and compositions comprising the BPD compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the disorder, and the species, sex, age, weight, disorder, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action that achieve the desired effect without causing substantial harmful or deleterious side-effects. Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.

In general, a suitable dose of the compound is in the range of about 10 μg/m² to 1 g/m² per treatment (e.g., per day), more typically 1 mg/m² to 500 mg/m² per treatment (e.g., per day). Where the compound is a salt, an ester, an amide, a prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

EXAMPLES

The following examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, as described herein.

Chemical Synthesis

All chemicals and anhydrous solvents were purchased from Aldrich, Alfa Aesar, or Fisher Scientific, unless otherwise indicated. Anhydrous reactions were carried out under nitrogen atmosphere. Thin layer chromatography was performed using silica gel 60F_2S4 coated on glass plates which were purchased from Merck. The synthesised compounds were purified by flash column chromatography using silica-gel 60 (0.04-0.063 mm). Developed TLC plates were visualised under UV lamp and stained with iodine. ¹H NMR and ¹³C NMR spectra were recorded on Brucker drx 500 and drx 400 instruments. Chemical shifts were relative to the deuterated solvent peak and are reported in parts per million (ppm). The high resolution mass spectra were recorded on Micromass Q-Tof spectrometer using electrospray ionisation technique. Melting points were determined on Griffin melting point apparatus.

Synthesis 1 N-(5-Chloro-2-nitro-phenyl)-N',N'-dimethyl-benzene-1 ,4-diamine (1a) e

A 50 mL round bottom flask was charged with 2,4-dichloronitrobenzene (2.0 g, 10.41 mmol) and 4-Λ/,Λ/-dimethylamino aniline (1.56 g, 11.45 mmol). The reaction mixture was stirred at 90°C under neat conditions and nitrogen atmosphere for 15 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (20 mL). The crude product was purified by flash chromatography using 5% ethyl acetate in petroleum ether to afford the title compound as a brown solid (0.9 g, 30%). Melting point 228- 230⁰C; ¹H NMR (CDCI₃, 400 MHz) δ_H 9.42 (s, NH), 8.12 (d, ArH, J = 9.13 Hz), 7.12 (dt, 2 X ArH, J = 3.41 Hz, J'= 2.06 Hz), 6.94 (d, ArH, J = 2.17 Hz), 6.76 (dt, 2 X ArH, J = 3.38 Hz, J = 2.2 Hz), 6.61 (dd, ArH, J = 2.17 Hz, J = 11.3 Hz), 3.00 (s, 6H); ¹³C NMR (CHCI₃, 100 MHz) δ_c 145.47 (qC), 145.72 (qC), 142.39 (qC), 131.12 (qC), 127.98 (qC), 127.19 (ArC), 126.19 (ArC), 116.80 (ArC)₁ 115.1 (ArC)₁ 113.22 (ArC), 40.58 (CH₃); HRMS (ESI): Calculated mass for C₁₄H₁₃CIN₃O₂ [M⁺] is 290.0693 [M-H]⁺ and found at 290.0701 [M-H]⁺.

Synthesis 2

(5-Chloro-2-nitro-phenyl)-(4-fluoro-phenyl)-amine (1 b)

A 50 mL round bottom flask was charged with 2,4-dichloronitrobenzene (3.0 g, 15.62 mmol) and 4-fluoroaniline (4 mL). The reaction mixture was stirred at 100⁰C under neat conditions and nitrogen atmosphere for 48 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (15 mL). The crude product was and purified by flash chromatography using 1% ethyl acetate in petroleum ether to afford the title compound as a yellow solid (1.1 g, 27%). NMR (CDCI₃, 400 MHz) δ_H 9.51 (s, NH), 8.11 (d, ArH₁ J = 9.07 Hz), 7.15 (dd, ArH, J = 6.25 Hz, J'= 10.39 Hz), 7.07 (m, 4H), 6.91 (d, ArH₁ J = 2.19 Hz); ¹³C NMR (CHCI₃, 100 MHz) δ_c 159.82 (qC), 144.47 (qC), 142.22 (qC), 132.3 (qC), 131.12 (qC), 129.1 (ArC), 127.19 (ArC), 116.80 (ArC)₁ 115.1 (ArC), 113.22 (ArC); HRMS (ESI): Calculated mass for C₁₂H₈CIFN₂O₂ [M]⁺ is 266.0712 [M]⁺ and found at 267.0791 [M+H]⁺.

Synthesis 3 (5-Chloro-2-nitro-phenyl)-(4-piperidin-1 -yl-phenyl)-amine (1 c)

A 50 ml. round bottom flask was charged with 2,4-dichloronitrobenzene (2.0 g, 10.41 mmol) and 4-Λ/,Λ/-dimethylamino aniline (2.01 g, 11.45 mmol). The reaction mixture was stirred at 100⁰C under neat conditions and nitrogen atmosphere for 2 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (20 ml_). The crude product was purified by flash chromatography using 5% ethyl acetate in petroleum ether to afford the title compound as a brown solid (0.9 g, 27%). Melting point 138- 140⁰C; ¹H NMR (CDCI₃, 400 MHz) δ_H 9.42 (s, NH), 8.10 (d, ArH, J = 9.15 Hz), 7.10 (dt, 2 X ArH₁ J = 3.41 Hz, J' = 2.06 Hz)₁ 6.95 (m, 3 X ArH)₁ 6.62 (dd, ArH, J = 2.18 Hz, J = 11.33 Hz), 3.20 (t, 4H, J = 5.37 Hz), 1.73 (quintet, 4H), 1.59 (m, 2H); ¹³C NMR (CHCI₃, 100 MHz) δ_c 150.89 (qC), 145.36 (qC), 142.38 (qC), 130.75 (qC), 128. 37 (qC), 127.92 (ArC), 126.76 (ArC), 117.09 (ArC), 116.99 (ArC), 115.60 (ArC), 50.31 (CH₂), 25.73 (CH₂), 24.20 (CH₂); HRMS (ESI): Calculated mass for Ci₇H₁₉CIN₃O₂ [M+H]⁺ 332.1157 [M+H]⁺ and found at 332.1160 [M+H]⁺.

Synthesis 4

(5-Chloro-2-nitro-phenyl)-(4-methoxy-phenyl)-amine (1d)

A 50 ml_ round bottom flask was charged with 2,4-dichloronitrobenzene (2.0 g, 10.41 mmol) and p-anisidine (1.41 g, 11.45 mmol). The reaction mixture was stirred at 100⁰C under neat conditions and nitrogen atmosphere for 8 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (20 mL). The crude product was purified by flash chromatography using 2% ethyl acetate in petroleum ether to afford the title compound as an orange solid (0.62 g, 22%). Melting point 228-230⁰C ; ¹H NMR (CDCI₃, 400 MHz) δ_H 9.41 (s, NH), 8.12 (d, ArH, J = 9.12 Hz), 7.18 (dt, 2 X ArH, J = 3.36 Hz= 2.12 Hz), 6.97 (dt, 2 x ArH₁ J = 3.36 Hz, J' = 2.24 Hz), 6.92 (d, ArH, J = 2.22 Hz), 6.64 (dd, ArH, J = 2.16 Hz, J'= 11.33 Hz), 3.84 (s, 3H); ¹³C NMR (CHCI₃, 100 MHz) δ_c 158.32 (qC), 145.16 (qC), 142.48 (qC), 130.92 (qC), 130.31 (qC ), 128.02 (ArC), 127.34 (ArC)₁ 117.25 (ArC), 115.19 (ArC), 114.95 (ArC), 55.53 (CH₃); HRMS (ESI): Calculated mass for C₁₃H₁₁CIN₂O₃ [M]⁺ 279.0539 [M+H]⁺ and found at 279.0531 [M+H]⁺.

Synthesis 5

N'-(5-Chloro-2-nitro-phenyl)-N,N-dimethyl-propane-1 ,3-diamine (1e)

A 50 ml_ round bottom flask was charged with 2,4-dichloronitrobenzene (2.0 g, 10.4 mmol) and Λ/,Λ/-dimethylaminopropyl amine (2 mL, excess). The reaction mixture was stirred at 100⁰C under neat conditions and nitrogen atmosphere for 3 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (15 mL). The crude product was purified by flash chromatography using 10% methanol in chloroform to afford the title compound as a yellow viscous liquid (2.6 g, 97%). ¹H NMR (CDCI₃, 500 MHz) δ_H 8.08 (d, ArH, J = 9.12 Hz), 6.82 (d, ArH, J = 2.09 Hz), 6.60 (dd, ArH, J = 2.11 Hz, J' = 11.23 Hz), 6.35 (bs, NH), 3.37 (m, 2H), 2.82 (t, 2H, J = 7.47 Hz), 2.52 (s, 6H), 1.90

(quintet, 2H); ¹³C NMR (CHCI₃, 125 MHz) δ_c 176.36 (qC), 145.54 (qC), 142.93 (qC), 128.3 (ArC), 116.11 (ArC), 113.14 (ArC)₁ 58.49 (CH₂), 43.33 (CH₃), 40.74 (CH₂), 24.8 (CH₂); HRMS (ESI): Calculated mass for C₁₁H₁₆CIN₃O₂ [M]⁺ 257.1034 [M]⁺ and found at 258.1047 [M+H]⁺.

Synthesis 6 (5-Chloro-2-nitro-phenyl)-(3-pyrrolidin-1-yl-propyl)-amine (1f)

A 50 ml. round bottom flask was charged with 2,4-dichloronitrobenzene (2.0 g, 10.4 mmol) and 3-aminopropyl pyrrolidine (2 mL, excess). The reaction mixture was stirred at 100⁰C under neat conditions and nitrogen atmosphere for 1.5 hours. The reaction mixture was cooled to room temperature and diluted with ethyl acetate (20 mL). The crude product was purified by flash chromatography using 10% methanol in chloroform to afford the title compound as a yellow solid (2.2 g, 76%). Melting point 50-52⁰C; ¹H NMR (CDCI₃, 400 MHz) δ_H 8.48 (s, NH), 8.07 (d, ArH, J = 7.43 Hz), 6.89 (d, ArH, J = 2.14), 6.55 (dd, ArH, J = 2.34 Hz, J¹ = 9.17 Hz), 3.35 (m, 2H) 2.59 (t, 2H, J = 6.66 Hz); 2.50 (m, 4H), 1.89 (quintet, 2H), 1.80 (m, 4H) ¹³C NMR (CHCI₃, 100 MHz) δ_c 145.96 (qC), 142.59 (qC), 130.44 (qC), 128.17 (ArC), 115.37 (ArC), 113.3 (ArC), 54.26 (CH₂), 42.25 (CH₂),

27.63 (CH₂), 23.47 (CH₂); HRMS (ESI): Calculated mass for C₁₃H₁₈CIN₃O₂ [M]⁺ 284.1164 [M+H]⁺ and found at 284.1160 [M+H]⁺.

Synthesis 7 (5-Chloro-2-nitro-phenyl)-[4-(4-methyl-piperazin-1-yl)-phenyl]-amine (1g)

To a stirred suspension of 4-Λ/-methylpiperazinoaniline (1.0 g, 5.22 mmol) and dry potassium carbonate (1.44 g, 10.44 mmol) in anhydrous DMF (5 mL), was added 2,4- dichloronitrobenzene (0.912 g, 4.75 mmol) in DMF (5 mL). The reaction mixture was stirred at 90⁰C under nitrogen atmosphere for 24 hours. The reaction mixture was filtered and washed with DMF (5 mL). The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 5% methanol in chloroform to give the title compound as a brown solid (0.75 g, 46%). Melting point 174-176°C; ¹H NMR (CDCI₃, 400 MHz) δ_H 9.41 (s, NH), 8.10 (d, ArH, J = 9.14 Hz), 7.25 (d, 2 X ArH, J = 10.03 Hz), 6.95 (m, 3 X ArH), 6.62 (dd, ArH, J = 2.34 Hz₁ J' = 13.66 Hz ), 3.24 (t, 4H, J = 5.1 Hz), 2.58 (t, 4H, J = 5.14 Hz), 2.33 (s, 3H); ¹³C NMR (CHCI₃, 100 MHz) δ_c 149.98 (qC), 145.20 (qC), 142.4 (qC), 135.79 (qC), 130.81 (qC ), 129.0 (ArC), 127.99 (ArC), 117.41 (ArC), 116.78 (ArC), 115.05 (ArC), 55.0 (CH₂), 48.83 (CH₂), 46.12 (CH₃); HRMS (ESI): Calculated mass for C₁₇H₁₉CIN₄O₂ [M]⁺ 346.1197 [M]⁺ and found at 347.1248 [M+H]⁺. Svnthesis 8 8-Chloro-10-(4-dimethylamino-phenyl)-10H-benzo[g]pteridine-2,4-dione (2a)

To a stirred solution of compound (1a) (0.5 g, 1.71 mmol) in glacial acetic acid (15 mL) was added Zn dust (1.11 g, 17.1 mmol) in portions, whilst maintaining the temperature below 40⁰C (using a cold water bath). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 20 minutes. The reaction mixture was quickly filtered through a pad of celite and washed with acetic acid (5 mL). To the same acetic acid solution of diamine was added alloxane.H₂O (0.32 g, 2.05 mmol) and boric acid (0.16

10 g, 2.56 mmol). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 30 minutes. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 5% MeOH in CHCI₃ to afford a green solid (0.23 g, 37%). Melting point >270°C; ¹H NMR (CDCI₃ + CD₃OD-d⁴, 400 MHz) δ_H 8.17 (d, ArH, J = 8.7 Hz), 7.52 (dd, ArH, J = 6.61 Hz, J'= 10.93 Hz), 7.04 (dt, 2 X ArH,

15 J = 3.37 Hz, J = 2.2 Hz), 7.01 (d, ArH, J = 2.06 Hz), 6.84 (dt, 2 X ArH, J = 3.28 Hz, J' = 2.15 Hz), 3.03 (s, 6H); HRMS (ESI): Calculated mass for C₁₈H₁₅CIN₅O₂ [M+H]⁺ is 368.0923 [M+H]⁺ and found at 368.0919 [M+H]⁺.

Synthesis 9

20 8-Chloro-10-(4-fluoro-phenyl)-10H-benzo[g]pteridine-2,4-dione (2b)

To a stirred solution of compound (1b) (0.7 g, 2.62 mmol) in glacial acetic acid (15 mL) was added Zn dust (1.71 g, 26.2 mmol) in portions, whilst maintaining the temperature below 40⁰C (using a cold water bath). The reaction mixture was stirred at room

>5 temperature under nitrogen atmosphere for 30 minutes. The reaction mixture was quickly filtered through a pad of celite and washed with acetic acid (5 mL). To the same acetic acid solution of diamine was added alloxane.H₂O (0.504 g, 3.14 mmol) and boric acid (0.25 g, 3.93 mmol). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 5% MeOH in CHCI₃ to afford the title compound as a yellow solid (0.35 g, 39%). Melting point >270°C; ¹H NMR (DMSOd⁶, 400 MHz) δ_H 8.19 (d, ArH₁ J = 4.87 Hz), 7.65 (dd, ArH, J = 6.61 Hz₁ J'= 10.89

Hz), 7.52 (m, 4 X ArH), 6.71 (d, ArH, J = 2.11 Hz); ¹³C NMR (DMSO-d⁶, 100 MHz) δ_c 164.8 (qC), 162.32 (qC), 159.91 (qC), 156.33 (qC), 150.95 (qC), 141.08 (qC), 138.28 (qC), 135.21 (qC), 134.18 (qC), 132.95 (ArC), 129.93 (ArC), 129.84 (ArC), 126.97 (ArC), 117.46 (ArC), 117.22 (ArC), 116.32 (ArC); HRMS (ESI): Calculated mass for C₁₆H₉CIFN₄O₂ [M+H]⁺ is 343.0384 [M+H]⁺ and found at 343.0392 [M+H]^*.

Synthesis 10 8-Chloro-10-(4-piperidin-1 -yl-phenyl)-10H-benzo[g]pteridine-2,4-dione (2c)

To a stirred solution of compound (1c) (0.5 g, 1.5 mmol) in glacial acetic acid (15 mL) was added Zn dust (0.98 g, 15.1 mmol) in portions, whilst maintaining the temperature below 40⁰C (using a cold water bath). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 30 minutes. The reaction mixture was filtered through a pad of celite and washed with acetic acid (5 mL). To the same acetic acid solution of diamine was added alloxane.H₂O (0.29 g, 1.8 mmol) and boric acid (0.14 g, 2.25 mmol). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 3% MeOH in CHCI₃ to afford the title compound as a brown solid (0.51 g, 83%). Melting point >270°C; ¹H NMR (CD₃OD-d⁴, 400 MHz) δ_H 8.18 (d, ArH, J = 8.76 Hz), 7.62 (dd, ArH, J = 2.16 Hz, J' = 10.92 Hz), 7.22 (m, 4 X ArH), 6.79

(d, ArH, J = 2.19 Hz), 3.42 (quintet, 4H), 1.75 (m, 4H)₁ 1.66 (m, 2H); ¹³C NMR (DMSO-d⁶, 125 MHz) δ_c 172.1 (qC), 159.55 (qC), 155.66 (qC), 152.35 (qC), 139.83 (qC), 138.91 (qC), 135.8 (qC), 133.58 (qC), 133.21 (ArC), 128.13 (ArC)₁ 126.19 (ArC), 125.19 (qC), 116.19 (qC), 116.06 (qC), 48.71 (CH₂), 25.23 (CH₂), 23.94 (CH₂); HRMS (ESI): Calculated mass for C₂₁H₁₉CIN₅O₂ [M+H]⁺ is 408.1219 [M+H]⁺ and found at 408.1221 [M+H]⁺. Svnthesis 11 8-Chloro-10-(4-methoxy-phenyl)-10H-benzo[g]pteridine-2,4-dione (2d)

To a stirred solution of compound (1d) (0.2 g, 0.71 mmol) in glacial acetic acid (10 mL) was added Zn dust (0.47 g, 7.1 mmol) in portions, whilst maintaining the temperature below 40⁰C (using a cold water bath). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 20 minutes. The reaction mixture was quickly filtered through a pad of celite and washed with acetic acid (5 mL). To the same acetic acid solution of diamine was added alloxane.H₂O (0.14 g, 0.85 mmol) and boric acid (0.066 g, 1.07 mmol). The reaction mixture was stirred at 100⁰C under nitrogen atmosphere for 0.5 hours. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 4% MeOH in CHCI₃ to afford the title compound as a greenish solid (0.16 g, 64%). Melting point > 270°C; ¹H NMR (DMSO-d⁶, 500 MHz) δ_H 11.49 (s, NH), 8.22 (d, ArH, J = 7.7 Hz), 7.68 (dd, ArH, J = 2.08 Hz, J = 10.79 Hz), 7.36 (d, 2 X ArH, J = 8.86 Hz), 7.26 (d, 2 X ArH, J = 8.88 Hz), 6.74 (d, ArH, J = 2.04 Hz), 3.90 (s, OCH₃) ¹³C NMR (DMSO-d⁶, 125 MHz) δ_c 160.12 (qC), 159.47 (qC), 155.57 (qC), 152.25 (qC), 139.83 (qC), 139.01 (qC), 135.54 (qC), 133.54 (qC), 133.23 (ArC), 128.98 (ArC), 128.04 (qC), 126.27 (ArC), 116.07 (ArC)₁ 115.69 (ArC), 55.64 (CH₃); HRMS (ESI): Calculated mass for C_2IH₁₉CIN₅O₂ [M+H]⁺ is 408.1219 [M+H]⁺ and found at 408.1221 [M+H]\

Synthesis 12 8-Chloro-10-methyl-10H-benzo[g]pteridine-2,4-dione (2e)

To a stirred solution of 4-chloro-2-methylamino nitrobenzene (2.0 g, 10.7 mmol) in glacial acetic acid (30 mL) was added Zn dust (7.0 g, 107.2 mmol) in portions, whilst maintaining the temperature below 40⁰C (using a cold water bath). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 1 hour. The reaction mixture was quickly filtered through a pad of celite and washed with acetic acid (10 mL). To the same acetic acid solution of diamine was added alloxane.H₂O (2.06 g, 12.8 mmol) and boric acid (1.0 g, 16.1 mmol). The reaction mixture was stirred at 100⁰C under nitrogen atmosphere for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 3% MeOH in CHCI₃ to afford the title compound as a yellow solid (0.87 g, 31 %). Melting point >270°C; ¹H NMR (DMSO-d⁶, 5 500 MHz) δ_H 11.43 (s, NH), 8.12 (d, ArH, J = 8.72 Hz), 8.06 (d, ArH, J = 2.06 Hz), 7.65

(dd, ArH, J = 2.08 Hz, J = 10.78 Hz), 3.92 (s, 3H); ¹³C NMR (DMSO-d⁶, 125 MHz) δ_c 159.62 (qC), 155.56 (qC), 151.07 (qC), 139.56 (qC), 138.91 , (qC), 133.37, (qC), 133.19 (ArC)₁ 126.41 (ArC), 116.48 (ArC), 32.12 (CH₃); HRMS (ESI): Calculated mass for C₁₁H₇CIN₄O₂ [M]⁺ is 263.1012 [M+H]⁺ and found at 263.1021 [M+H]\ 10

Synthesis 13 8-Chloro-10-(3-dimethylamino-propyl)-10H-benzo[g]pteridine-2,4-dione (2f)

To a stirred solution of compound (1e) (0.5 g, 1.94 mmol) in glacial acetic acid (8 ml_) was

15 added Zn dust (1.34 g, 19.4 mmol) in portions, whilst maintaining the temperature below 4O⁰C (using a cold water bath). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 30 minutes. The reaction mixture was filtered through a pad of celite and washed with acetic acid (5 mL). To the same acetic acid solution of diamine was added alloxane.H₂O (0.37 g, 2.32 mmol) and boric acid (0.18 g, 2.91 mmol).

10 The reaction mixture was stirred at 100⁰C under nitrogen atmosphere for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 18% MeOH in CHCI₃ to afford the title compound as yellow solid (0.195 g, 30%). Melting point 260⁰C, decomposed; ¹H NMR (DMSO-d⁶, 500 MHz) δ_H 11.5 (S, NH), 8.19 (d, ArH, J = 1.88 Hz), 8.12 (d, ArH₁ J = 8.7 Hz), 7.65 (dd, ArH, J = 1.94

.5 Hz, J = 10.66 Hz₁), 4.55 (t, 2H, J = 6.78 Hz), 3.12 (bs, 2H), 3.10 (s, 6H), 1.9 (t, 2H, J =

6.26 Hz); ¹³C NMR (DMSO-d⁶, 125 MHz) δ_c 160.03 (qC), 159.94 (qC), 156.01 (qC), 140.04 (qC), 139.36 (qC), 134.02 (qC), 135.54 (qC), 133.77 (ArC), 126.78 (ArC), 116.52 (ArC), 55.43 (CH₂), 45.92 (CH₃), 42.94 (CH₂), 23.95 (CH₂); HRMS (ESI): Calculated mass for C₁₅H₁₆CIN₅O₂ [M]⁺ is 334.1076 [M+H]⁺ and found at 334.1065 [M+H]⁺.

!0 Synthesis 14 8-Chloro-10-(3-pyrrolidin-1 -yl-propyl)-10H-benzo[g]pteridine-2,4-dione (2g)

To a stirred solution of compound (1f) (2.1 g, 7.4 mmol) in glacial acetic acid (30 mL) was added Zn dust (4.83 g, 74.0 mmol) in portions, whilst maintaining the temperature below 40⁰C (using a cold water bath). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 30 minutes. The reaction mixture was filtered through a pad of celite and washed with acetic acid (10 ml). To the same acetic acid solution of diamine was added alloxane.H₂O (1.42 g, 8.8 mmol) and boric acid (0.69 g, 11.1 mmol). The reaction mixture was stirred at 100⁰C under nitrogen atmosphere for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 20% MeOH in CHCI₃ to afford the title compound as yellow solid (0.83 g, 33%). Melting point >270°C; ¹H NMR (DMSO-d⁶, 400 MHz) δ_H 8.1 (bs, NH), 7.62 (d, ArH, J = 7.64 Hz), 6.95 (dd, ArH, J = 2.15 Hz, J = 10.58 Hz₁), 6.78 (d, ArH, J = 8.43 Hz), 4.65 (t, 2H, J = 6.82 Hz), 3.98 (t, 2H₁ J = 6.92 Hz), 2.55 (t, 2H, J = 6.42 Hz), 2.46 (m,

2H), 1.98 (quintet, 2H), 1.72 (m, 4H); ¹³C NMR (DMSO-d⁶, 125 MHz) δ_c 160.09 (qC), 159.54 (qC), 155.51 (qC), 141.04 (qC), 139.86 (qC), 134.52 (qC), 135.94 (qC), 133.9 (ArC), 127.7 (ArC), 117.52 (ArC), 54.43 (CH₂), 48.92 (CH₂), 40.94 (CH₂), 23.95 (CH₂), 20.76 (CH₂); HRMS (ESI): Calculated mass for C₁₇H₁₈CIN₅O₂ [M]⁺ is 360.1235 [M+H]⁺ and found at 360.1221 [M+H]⁺.

Synthesis 15 8-Chloro-10-[4-(4-methyl-piperazin-1 -yl)-phenyl]-10H-benzo[g]pteridine-2,4-dione (2h)

To a stirred solution of compound (1g) (0.5 g, 1.44 mmol) in glacial acetic acid (15 mL) was added Zn dust (1.41 g, 21.6 mmol) in portions, whilst maintaining the temperature below 40⁰C (using a cold water bath). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 30 minutes. The reaction mixture was filtered through a pad of celite and washed with acetic acid (5 mL). To the same acetic acid solution of diamine was added alloxane.H₂O (0.28 g, 1.73 mmol) and boric acid (0.132 g, 2.16 mmol). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 15% MeOH in CHCI₃ to afford the title compound as a brown solid (0.61g, 100%). Melting point >270°C; ¹H NMR (DMSO- d⁶, 500 MHz) δ_H 8.18 (s, NH), 8.13 (d, ArH, J = 8.71 Hz)₁ 7.65 (dd, ArH, J = 2.19 Hz, J' = 10.9 Hz), 7.20 (d, 4 X ArH, J = 4.72 Hz)₁ 6.76 (d, ArH, J = 2.19 Hz), 3.27 (t, 4H, J = 4.81 Hz)₁ 2.51 (m, 4H) 2.25 (s, 3H); ¹³C NMR (DMSO-d⁶, 125 MHz) δ_c 159.54 (qC), 155.63 (qC), 152.33 (qC), 151.63 (qC), 139.82 (qC), 138.93 (qC), 135.74 (ArC), 128.15 (ArC), 126.2 (ArC), 125.75 (qC), 116.17 (ArC), 115.9 (ArC), 54.62 (CH₂), 48.63 (CH₃), 40.1 (CH₂); HRMS (ESI): Calculated mass for C₂₁H₁₉CIN₆O₂ [M]⁺ is 422.1258 [M]⁺ and found at 423.1336 [M+H]⁺.

Synthesis 16

8-Chloro-10-(4-dimethylamino-phenyl)-3-(3-dimethylamino-propyl)- 10H-benzo[g]pteridine-2,4-dione (3a)

To a stirred suspension of compound (2a) (0.1 g, 0.27 mmol) and anhydrous potassium carbonate (0.11 g, 0.81 mmol) in anhydrous DMF (5 mL) was added N₁N- dimethylaminopropyl chloride.HCI (0.13 g, 0.81 mmol). The reaction mixture was stirred at 80⁰C under nitrogen atmosphere for 2 hours. The reaction mixture was filtered and washed with DMF (5 mL). The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 15% MeOH in CHCI₃ to afford the title compound as a brown solid (55 mg, 45%). Melting point 250⁰C₁ decomposed; ¹H NMR (CD₃OD-d⁴, 500 MHz) δ_H 8.19 (d, ArH, J = 8.77 Hz), 7.63 (dd, ArH, J = 2.15 Hz, J' = 10.9 Hz), 7.18 (d, 2 X ArH, J = 9.03 Hz), 6.99 (d, ArH, J = 2.11 Hz)₁ 6.82 (d, 2 X ArH₁ J = 9.03 Hz)₁ 4.06 (t, 2H, J = 7.27 Hz), 3.13 (s, 6H), 2.49 (t, 2H, J = 7.4 Hz), 2.30 (s, 6H)₁ 1.91 (quintet, 2H); ¹³C NMR (CD₃OD-d⁴, 125 MHz) δ_c 161.61 (qC), 158.13 (qC), 153.13 (qC), 152.74 (qC), 142.29 (qC), 139.65 (qC), 137.6 (qC), 135.7 (qC), 134.17 (qC), 129.12 (ArC), 128.2 (ArC), 124.52 (ArC)₁ 118.33 (ArC), 114.16 (ArC), 57.88 (CH₂), 45.71 (CH₃), 41.71 (CH₂), 41.11 (CH₃), 26.4 (CH₂); HRMS (ESI): Calculated mass for C₂₃H₂₅CIN₆O₂ [M]⁺ is 453.1823 [M+H]⁺ and found at 453.1811 [M+H]\

Synthesis 17

8-Chloro-10-(4-dimethylamino-phenyl)-3-(3-pyrrolidin-1-yl-propyl)- 10H-benzo[g]pteridine-2,4-dione (3b)

To a stirred suspension of compound (2a) (0.4 g, 1.08 mmol) and anhydrous potassium carbonate (0.45 g, 3.26 mmol) in anhydrous DMF (5 mL) was added

3-chloropropylpyrrolidine (0.48 g, 3.26 mmol) in anhydrous DMF (2 mL). The reaction mixture was stirred at 90°C under nitrogen atmosphere for 2 hours. The reaction mixture was filtered and washed with DMF (10 mL). The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 20% MeOH in CHCI₃ to afford the title compound as a brown solid (0.25 mg, 48%). Melting point 260⁰C, decomposed; ¹H NMR (DMSO-d⁶, 500 MHz) δ_H 8.21 (d, ArH, J = 8.72 Hz), 7.67 (dd, ArH, J = 2.2 Hz, J' = 10.92 Hz)₁ 7.16 (dt, 2 X ArH, J = 2.02 Hz, J¹ = 12.23 Hz)₁ 6.94 (dt, 2 X ArH, J = 2.02 Hz, J' = 12.26 Hz), 6.82 (d, ArH, J = 2.19 Hz), 3.90 (t, 2H₁ J = 7.11 Hz)₁ 3.02 (S₁ 6H), 2.62 (m, 6H), 1.80 (quintet, 2H), 1.70 (m, 4H); ¹³C NMR (DMSO-d⁶, 125 MHz) δ_c 159.19 (qC), 154.88 (qC), 151.06 (qC), 139.08 (qC), 135.82 (qC), 133.8 (qC),

133.25 (ArC), 128.03 (ArC)₁ 126.32 (ArC), 123.15 (qC), 116.29 (ArC), 112.86 (ArC), 53.53 (CH₂), 53.37 (CH₂), 40.01 (CH₃), 23.09 (CH₂); HRMS (ESI): Calculated mass for C₂₅H₂₇CIN₆O₂ [M]⁺ is 479.1859 [M+H]⁺ and found at 489.1876 [M+H]⁺.

Svnthesis 18

8-Chloro-10-(4-dimethylamino-phenyl)-3-(3-morpholin-4-yl-propyl)- 10H-benzo[g]pteridine-2,4-dione (3c)

To a stirred suspension of compound (2a) (0.25 g, 0.68 mmol) and anhydrous potassium carbonate (0.28 g, 2.03 mmol) in anhydrous DMF (6 ml.) was added Λ/-(3-chloropropyl)- morpholine (0.33 g, 2.03 mmol). The reaction mixture was stirred at 80⁰C under nitrogen atmosphere for 2 hours. The reaction mixture was filtered and washed with DMF (5 ml_). The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 8% MeOH in CHCI₃ to afford the title compound as a brown solid (100 mg, 30%). Melting point 240⁰C, decomposed; ¹H NMR (CDCI₃, 400 MHz) δ_H 8.22 (d, ArH, J = 4.96 Hz), 7.47 (dd, ArH, J = 4.84 Hz, J' = 13.58 Hz), 7.08 (dt, 2 X ArH, J = 3.35 Hz, J'= 2.2 Hz), 7.04 (d, ArH, J = 2.12 Hz), 6.87 (dt, 2 X ArH₁ J = 3.31 Hz, J'= 2.2 Hz), 4.15 (t, 4H, J = 7.17 Hz), 3.62 (t, 2H, J = 4.62 Hz), 3.07 (s, 6H), 2.46 (m, 4H), 2.41 (m, 2H), 1.87 (quintet, 2H); ¹³C NMR (CDCI₃, 100 MHz) δ_c 159.45 (qC), 155.21 (qC),

151.33 (qC), 150.65 (qC), 141.74 (qC), 137.65 (qC), 135.86 (qC), 134.1 (qC), 133.46 (qC), 127.7 (ArC), 127.1 (ArC), 122.3 (ArC), 117.17 (ArC), 113.16 (ArC), 67.05 (CH₂), 56..30 (CH₂), 53.49 (CH₂), 40.52 (CH₂), 40.31 (CH₃), 24.18 (CH₂); HRMS (ESI): Calculated mass for C₂₅H₂₇CIN₆O₃ [M]⁺ is 495.1911 [M+H]⁺ and found at 495.1901 [MH-H]⁺.

Synthesis 19

8-Chloro-3-(3-dimethylamino-propyl)-10-(4-fluoro-phenyl)- 10H-benzo[g]pteridine-2,4-dione (3d)

To a stirred suspension of compound (2b) (0.15 g, 0.48 mmol) and anhydrous potassium carbonate (0.18 g, 1.31 mmol) in anhydrous DMF (6 mL) was added N₁N- dimethylaminopropyl chloride. HCI (0.21 g, 1.31 mmol). The reaction mixture was stirred at 90⁰C under nitrogen atmosphere for 4 hours. The reaction mixture was filtered and washed with DMF (5 mL). The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 17% MeOH in CHCI₃ to afford the 5 title compound as a yellow solid (50 mg, 27%). ¹H NMR (CD₃OD-d⁴, 400 MHz) δ_H 8.20 (d, ArH₁ J = 8.76 Hz), 7.67 (dd, ArH, J = 2.14 Hz, J' = 10.89 Hz), 7.47 (m, 4H), 6.95 (d, ArH, J = 2.07 Hz), 4.04 (t, 2H, J = 7.27 Hz)₁ 2.53 (t, 2H₁ J = 7.34 Hz), 2.32 (s, 6H)₁ 1.91 (quintet, 2H); ¹³C NMR (CD₃OD-d⁴, 100 MHz) δ_c 164.8 (qC), 162.33 (qC), 159.91 (qC), 156.34 (qC), 150.95 (qC), 141.09 (qC), 138.28 (qC), 135.21 (qC), 134.18 (qC), 132.92 10 (ArC), 131.12 (qC), 129.93 (ArC), 129.84 (ArC), 126.96 (ArC), 117.46 (ArC), 117.25

(ArC), 116.34 (ArC), 56.39 (CH₂), 43.71 (CH₃). 39.65 (CH₂), 24.64 (CH₂); HRMS (ESI): Calculated mass for C₂₁H₁₉CIFN₅O₂ [M]⁺ is 428.1290 [M+H]⁺ and found at 428.1302 [M+H]⁺.

15 Synthesis 20

8-Chloro-10-(4-piperidin-1-yl-phenyl)-3-(3-pyrrolidin-1-yl-propyl)- 10H-benzo[g]pteridine-2,4-dione (3e)

To a stirred suspension of compound (2c) (0.15 g, 0.37 mmol) and anhydrous potassium

20 carbonate (0.15 g, 1.1 mmol) in anhydrous DMF (6 ml) was added Λ/-(3-chloropropyl) pyrrolidine (0.16 g, 1.1 mmol). The reaction mixture was stirred at 90⁰C under nitrogen atmosphere for 1 hour. The reaction mixture was filtered and washed with DMF (5 mL). The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 15% MeOH in CHCI₃ to afford the title compound as a brown

>5 solid (165 mg, 87%). Melting point 256°C, decomposed; ¹H NMR (CD₃OD-d⁴, 500 MHz) δ_H 8.18 (d, ArH, J = 8.76 Hz), 7.64 (dd, ArH₁ J = 2.16 Hz, J' = 10.92 Hz), 7.19 (s, 4 X ArH), 6.98 (d, ArH, J = 2.12 Hz), 4.09 (dt. 2H, J = 6.38 Hz, J = 2.15 Hz)₁ 3.35 (t, 4H₁ J = 5.23 Hz)₁ 2.83 (m, 4H)₁ 2.71 (m, 2H), 2.0 (m, 6H), 1.84 (m, 6H); ¹³C NMR (CD₃OD-d⁴, 125 MHz) δ_c 172.81 (qC) 161.61 (qC), 158.19 (qC), 154.59 (qC), 152.61 (qC), 142.42 (qC),

IO 139.61 (qC), 137.33 (qC), 135.74 (qC), 134.23 (ArC), 129.25 (ArC)₁ 128.34 (ArC), 126.43

(ArC), 118.25 (ArC)₁ 117.93 (ArC), 63.59 (CH₂), 55.18 (CH₂), 54.6 (CH₂), 53.93 (CH₂). 50.9 (CH₂). 40.85 (CH₂), 28.42 (CH₂), 27.2 (CH₂), 26.7 (CH₂). 25.41 (CH₂), 24.12 (CH₂). 20.73 (CH₂); HRMS (ESI): Calculated mass for C₂₈H₃₂CIN₆O₂ [M+H]⁺ is 519.2252 [M+H]⁺ and found at 519.2269 [M+H]⁺.

Synthesis 21

8-Chloro-10-(4-methoxy-phenyl)-3-(3-pyrrolidin-1 -yl-propyl)- 10H-benzo[g]pteridine-2,4-dione (3f)

To a stirred suspension of compound (2d) (0.12 g, 0.34 mmol) and anhydrous potassium carbonate (0.14 g, 1.01 mmol) in anhydrous DMF (6 mL) was added /V-(3-chloropropyl) pyrrolidine (0.15 g, 1.01 mmol). The reaction mixture was stirred at 90⁰C under nitrogen atmosphere for 1 hour. The reaction mixture was filtered and washed with DMF (5 mL). The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 16% MeOH in CHCI₃ to afford the title compound as a yellow solid (60 mg, 39%). Melting point >270°C; ¹H NMR (CD₃OD-d⁴, 500 MHz) δ_H 8.19 (d, ArH, J = 8.78 Hz)₁ 7.64 (dd, ArH, J = 2.13 Hz, J" = 10.9 Hz), 7.32 (d, 2 X ArH, J = 6.75 Hz),

7.23 (d, 2 XArH, J = 6.84 Hz), 6.92 (d, ArH, J = 2.1 Hz), 4.08 (t, 2H, J = 7.01 Hz), 3.11 (s, 3H), 2.87 (m, 6H), 2.0 (quintet, 2H), 1.89 (quintet, 4H); ¹³C NMR (CD₃OD-d⁴, 125 MHz) δ: 162.63 (qC) 161.56 (qC), 158.03 (qC), 152.52 (qC), 142.49 (qC), 139.65 (qC), 137.65 (qC), 135.68 (qC), 134.26 (ArC), 130.01 (qC), 128.85 (ArC), 128.4 (ArC), 118.06 (ArC), 116.87 (ArC), 56.26 (CH₂), 55.11 (CH₃), 54.48 (CH₂), 40.62 (CH₂), 26.91 (CH₂), 24.07

(CH₂); HRMS (ESI): Calculated mass for C₂₄H₂₅CIN₅O₃ [M+H]⁺ is 466.1655 [M+H]⁺ and found at 466.1640 [M+H]⁺.

Synthesis 22 8-Chloro-3-(3-dimethylamino-propyl)-10-methyl-10H-benzo[g]pteridine-2,4-dione (3g)

To a stirred suspension of compound (2e) (0.1 g, 0.38 mmol) and anhydrous potassium carbonate (0.157 g, 1.14 mmol) in anhydrous DMF (5 mL) was added N₁N- dimethylaminopropyl chloride. HCI (0.18 g, 1.14 mmol). The reaction mixture was stirred at 80⁰C under nitrogen atmosphere for 1 hour. The reaction mixture was filtered and washed with DMF (5 mL). The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 20% MeOH in CHCI₃ to afford the title compound as a yellow solid (0.075 g, 58%). Melting point >270°C; ¹H NMR (CD₃OD- d⁴, 400 MHz) δ_H 8.12 (d, ArH, J = 8.77 Hz), 8.03 (d, ArH, J = 2.0 Hz), 7.67 (dd, ArH, J = 2.06 Hz, J = 10.77 Hz), 4.10 (t, 2H, J = 7.02 Hz), 4.06 (s, 3H), 2.76 (t, 2H, J = 7.5 Hz),

2.67 (s, 6H), 2.03 (quintet, 2H); ¹³C NMR (CD₃OD-d⁴, 100 MHz) δ_H 161.56 (qC) 157.88 (qC), 151.17 (qC), 143.13 (qC), 138.56 (qC), 135.81 (qC), 135.52 (qC), 134.46 (ArC), 128.52 (ArC), 117.57 (ArC), 57.41 (CH₂), 44.6(CH₃), 40.59 (CH₂), 32.89 (CH₃), 25.66 (CH₂); HRMS (ESI): Calculated mass for C₁₆H₁₈CIN₅O₂ [M]⁺ is 348.1233 [M+H]⁺ and found at 348.1221 [M+H]⁺.

Synthesis 23 8-Chloro-10-methyl-3-(3-pyrrolidin-1 -yl-propyl)-10H-benzo[g]pteridine-2,4-dione (3h)

To a stirred suspension of compound (2e) (0.2 g, 0.76 mmol) and anhydrous potassium carbonate (0.21 g, 1.52 mmol) in anhydrous DMF (4 ml) was added 3-chloropropylpyrrolidine (0.34 g, 2.28 mmol) in anhydrous DMF (2 mL). The reaction mixture was stirred at 80⁰C under nitrogen atmosphere for 1 hour. The reaction mixture was filtered and washed with DMF (5 mL). The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 20% MeOH in CHCI₃ to afford the title compound as yellow solid (0.1 g, 36%). Melting point >270°C; ¹H NMR (CD₃OD-d⁴, 400 MHz) δ_H 8.13 (d, ArH₁ J - 8.76 Hz), 8.01 (d, ArH, J = 2.02 Hz), 7.67 (dd, ArH, J = 2.07 Hz, J' = 10.8 Hz), 4.09 (t, 2H, J = 6.58 Hz), 4.01 (s, 3H), 2.83 (m, 6H), 2.01 (m, 2H), 1.85 (m, 4H); ¹³C NMR (CD₃OD-d⁴, 100 MHz) δ_c 161.61 (qC) 157.94 (qC), 151.2 (qC), 143.07 (qC), 138.62 (qC), 135.87 (qC), 135.56 (qC), 134.0 (ArC), 128.48

(ArC), 117.55 (ArC), 54.95 (CH₂), 54.52 (CH₂), 43.6 (CH₂), 40.89 (CH₂), 32.84 (CH₃), 27.23 (CH₂), 24.13 (CH₂); HRMS (ESI): Calculated mass for C₁₈H₂₀CIN₅O₂ [M]⁺ is 373.1245 [M]⁺ and found at 374.1256 [M+H]⁺.

Synthesis 24

8-Chloro-3,10-bis-(3-dimethylamino-propyl)-10H-benzo[g]pteridine-2,4-dione (3i)

To a stirred suspension of compound (2f) (0.335 g, 1.0 mmol) and anhydrous potassium carbonate (0.414 g, 3.0 mmol) in anhydrous DMF (10 mL) was added N₁N- dimethylaminopropyl chloride. HCI (0.476 g, 3.0 mmol). The reaction mixture was stirred at 90⁰C under nitrogen atmosphere for 5 hours. The reaction mixture was filtered and washed with DMF (10 mL). The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using MeOH : CHCI₃ : Et₃N (1: 4: 0.1 ) to afford the title compound as a yellow solid (0.21 g, 50%). Melting point 210-

212°C; ¹H NMR (CD₃OD-d⁴, 400 MHz) δ_H 8.18 (d, ArH, J = 2.1 Hz), 8.15 (s, ArH), 7:65 (dd, ArH, J = 2.02 Hz, J' = 10.78 Hz), 4.75 (t, 2H, J = 7.92 Hz), 4.15 (t, 2H, J = 7.27 Hz), 2.55 (m, 4H), 2.37 (s, 6H), 2.34 (s, 6H), 2.05 (quintet, 2H), 1.95 (quintet, 2H); ¹³C NMR (CD₃OD-d⁴, 125 MHz) δ_c 160.99 (qC) 156.52 (qC), 149.51 (qC), 141.62 (qC), 137.36 (qC), 134.43 (qC), 133.74 (qC), 133.26 (ArC), 127.88 (ArC), 115.94 (ArC), 56.32 (CH₂),

55.48 (CH₂), 43.9 (CH₃), 43.6 (CH₃), 42.98 (CH₂), 39.55 (CH₂), 24.78 (CH₂), 24.16 (CH₂); HRMS (ESI): Calculated mass for C₂₀H₂₇CIN₆O₂ [M]⁺ is 419.1959 [M+H]⁺ and found at 419.1956 [M+H]⁺.

Synthesis 25

8-Chloro-3,10-bis-(3-pyrrolidin-1-yl-propyl)-10H-benzo[g]pteridine-2,4-dione (3j)

To a stirred suspension of compound (2g) (0.4 g, 1.15 mmol) and anhydrous potassium carbonate (0.32 g, 2.3 mmol) in anhydrous DMF (6 ml) was added 3-chloropropylpyrrolidine (0.51 g, 3.45 mmol) in anhydrous DMF (2 mL). The reaction mixture was stirred at 80⁰C under nitrogen atmosphere for 2 hours. The reaction mixture was filtered and washed with DMF (10 ml_). The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using MeOH : CHCI₃ : Et₃N (1 : 4: 0.1 ) to afford the title compound as a yellow solid (0.28 g, 50%). Melting point 248-250⁰C; ¹H NMR (CD₃OD-d⁴, 500 MHz) δ_H 8.20 (d, ArH, J = 8.78 Hz), 8.16 (d, ArH, J = 1.92 Hz), 7.72 (dd, ArH, J = 1.96 Hz, J' = 10.73 Hz), 4.81 (t, 2H, J = 6.79 Hz), 4.16 (t,

2H, J = 6.58 Hz), 3.35 (m, 12H), 2.35 (quintet, 2H), 2.18 (quintet, 2H), 2.12 (quintet, 4H), 2.08 (quintet, 4H); ¹³C NMR (CD₃OD-d⁴, 125 MHz) δ_c 160.29 (qC) 156.29 (qC), 149.98 (qC), 142.05 (qC), 137.39 (qC), 134.66 (ArC), 127.47 (ArC), 115.0 (ArC), 53.78 (CH₂), 53.75 (CH₂), 48.05 (CH₂), 42.05 (CH₂), 38.46 (CH₂), 24.5 (CH₂), 23.89 (CH₂), 22.63 (CH₂); HRMS (ESI): Calculated mass for C₂₄H₃₁CIN₆O₂ [M]⁺ is 471.2281 [M+H]⁺ and found at 471.2269 [M+H]⁺.

Synthesis 26

8-Chloro-10-[4-(4-methyl-piperazin-1 -yl)-phenyl]-3-(3-pyrrolidin-1 -yl-propyl)- 10H-benzo[g]pteridine-2,4-dione (3k)

To a stirred suspension of compound (2h) (0.2 g, 0.47 mmol) and anhydrous potassium carbonate (0.195 g, 1.41 mmol) in anhydrous DMF (8 ml_) was added Λ/-(3-chloropropyl) pyrrolidine (0.21 g, 1.41 mmol) in anhydrous DMF (2 mL). The reaction mixture was stirred at 90⁰C under nitrogen atmosphere for 1.5 hours. The reaction mixture was filtered and washed with DMF (5 mL). The solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 15% MeOH in CHCI₃ and 0.5% Et₃N to afford the title compound as a brown solid (90 mg, 36%). Melting point 264-266°C; ¹H NMR (CD₃OD-d⁴, 500 MHz) δ_H 8.19 (d, ArH, J = 8.77 Hz), 7.65 (dd, ArH, J = 2.15 Hz, J' = 10.91 Hz), 7.25 (m, 4 X ArH), 6.95 (d, ArH, J = 2.12 Hz), 4.12 (t, 2H, J = 6.66 Hz), 3.38 (t, 4H, J = 4.76 Hz), 3.28 (quintet, 6H), 2.65 (t, 4H₁ J = 5.08 Hz), 2.39 (s, 3H), 2.08 (quintet, 2H), 1.98 (m, 4H); ¹³C NMR (CD₃OD-d⁴, 125 MHz) δ_c 161.68 (qC), 158.26 (qC), 153.81 (qC), 152.6 (qC), 142.56 (qC), 139.57 (qC), 137.16 (qC), 135.75 (qC), 134.26 (ArC), 129.93 (ArC), 129.41 (ArC), 129.22 (qC), 127.25 (ArC), 126.31 (qC), 117.81 (ArC), 55.79 (CH₂), 55.09 (CH₂), 54.14 (CH₂), 46.04 (CH₃), 40.1 (CH₂), 26.18 (CH₂), 24.06 (CH₂); HRMS (ESI): Calculated mass for C₂₈H₃₂CIN₇O₂ [M]⁺ is 533.2306 [M]⁺ and found at 534.2384 [M+H]\

Synthesis 27

10-(4-Dimethylamino-phenyl)-3-(3-dimethylamino-propyl)-8-(3-dimethylamino- propylamino)-10H-benzo[g]pteridine-2,4-dione (4a)

A 10 ml. round bottom flask was charged with compound (3a) (60 mg, 0.13 mmol) and Λ/,Λ/-dimethylaminopropyl amine (2 ml_). The reaction mixture was stirred at 100⁰C, under nitrogen atmosphere for 30 minutes. The reaction mixture was cooled to room temperature and cold ether (5 mL) was added to afford an orange solid. The solid was separated by centrifugation and was washed with diethyl ether (3 X 10 mL) to afford the title compound as an orange solid (40 mg, 59%). Melting point 248-250⁰C; ¹H NMR (CD₃OD-d⁴, 400 MHz) δ_H 7.79 (d, ArH, J = 9.11 Hz), 7.16 (d, 2 X ArH, J = 8.95 Hz), 7.01 (d, ArH, J = 8.79 Hz), 6.93 (d, 2 X ArH, J = 8.95 Hz), 5.85 (s, ArH), 3.98 (t, 2H₁ J = 7.82

Hz), 3.07 (m, 2H), 3.03 (s, 6H), 2.41 (t, 2H, J = 7.54 Hz), 2.29 (t, 2H, J = 7.33 Hz), 2.23 (s, 6H), 2.14 (S₁ 6H), 1.85 (quintet, 2H), 1.69 (quintet, 2H); ¹³C NMR (CD₃OD-d⁴, 100 MHz) δ_c 163.09 (qC), 158.63 (qC), 157.22 (qC), 152.75 (qC), 152.6 (qC), 133.73 (qC), 129.0 (ArC), (qC), 127.98 (qC), 125.68 (qC), 114.25 (ArC), 58.06 (CH₂), 57.92 (CH₂), 45.44 (CH₃), 45.32 (CH₃), 42.22 (CH₂), 40.82 (CH₂), 40.62 (CH₃), 26.72 (CH₂); HRMS

(ESI): Calculated mass for C₂₈H₃₈N₈O₂ [M]⁺ is 519.3196 [M+H]⁺ and found at 519.3206 [M+H]⁺.

Svnthesis 28

10-(4-Dimethylamino-phenyl)-3-(3-pyrrolidin-1-yl-propyl)-8-(3-pyrrolidin-1-yl-propylamino)-

10H-benzo[g]pteridine-2,4-dione (4b)

A 10 mL round bottom flask was charged with compound (3b) (90 mg, 0.19 mmol) and 3- aminopropyl pyrrolidine (2 mL). The reaction mixture was stirred at 100⁰C, under nitrogen atmosphere for 20 minutes. The reaction mixture was cooled to room temperature and cold ether (5 mL) was added to afford a red solid. The solid was separated by centrifugation and was washed with diethyl ether (3 X 10 mL) to afford the

10 title compound as a red solid (95 mg, 88%). Melting point 244-246°C, decomposed; ¹H NMR (CD₃OD-d⁴, 500 MHz) δ_H 7.70 (d, ArH, J = 8.82 Hz), 7.19 (d, 2 X ArH, J = 8.77 Hz), 7.01 (d, ArH, J = 8.79 Hz), 6.96 (d, 2 X ArH, J = 8.96 Hz), 5.77 (s, ArH), 4.02 (t, 2H, J = 6.78 Hz), 3.05 (s, 6H), 2.86 (t, 2H, J = 7.06 Hz), 2.70 (m, 6H), 2.61 (t, 4H₁ J = 5.49 Hz), 2.50 (m, 4H), 1.95 (quintet, 2H), 1.82 (m, 10H); ¹³C NMR (CD₃OD-d⁴, 125 MHz) δ_c

15 163.06 (qC), 158.95 (qC), 157.79 (qC), 152.79 (qC), 152.51 (qC), 133.93 (qC), 129.27 (ArC), (qC), 127.40 (qC), 125.59 (qC), 114.28 (ArC), 55.03 (CH₂), 54.99 (CH₂), 54.96 (CH₂), 54.89 (CH₂), 54.67 (CH₂), 54.48 (CH₂), 42.46 (CH₂), 40.64 (CH₃), 40.49 (CH₂), 40.45 (CH₂), 29.29 (CH₂), 27.59 (CH₂), 24.20 (CH₂), 24.03 (CH₂); HRMS (ESI): Calculated mass for C₃₂H₄₂N₈O₂ [M]⁺ is 571.3509 [M+H]⁺ and found at 571.3535 [M+H]⁺.

20

Synthesis 29

10-(4-Dimethylamino-phenyl)-3-(3-morpholin-4-yl-propyl)-8-(3-morpholin-4-yl- propylamino)-10H-benzo[g]pteridine-2,4-dione (4c)

>5 A 10 mL round bottom flask was charged with compound (3c) (60 mg, 0.12 mmol) and 3- aminopropyl morpholine (2 mL). The reaction mixture was stirred at 100⁰C, under nitrogen atmosphere for 90 minutes. The reaction mixture was cooled to room temperature and cold ether (5 ml.) was added to afford a red solid. The solid was separated by centrifugation and was washed with diethyl ether (3 X 10 ml_). The crude product was purified by flash chromatography using MeOH: CHCI₃: Et₃N (1 :4:0.1 ) to afford the title compound as a red solid (25 mg, 35%). Melting point 284-286°C; ¹H NMR (CDCI₃, 500 MHz) δ_H 7.94 (d, ArH₁ J = 9.07 Hz), 7.53 (s, NH), 7.05 (d, 2 X ArH₁ J = 8.97 Hz)₁ 6.83 (d, 2 X ArH, J = 8.99 Hz), 6.75 (dd, ArH, J = 2.21 Hz, J¹ = 11.31 Hz), 5.72 (d, ArH, J = 1.93 Hz), 4.13 (t, 2H, J = 7.27 Hz), 3.64 (t, 8H, J = 4.39 Hz), 3.03 (s, 6H), 2.51 (t, 2H, J = 5.69 Hz), 2.45 (m, 12H), 1.88 (quintet, 2H), 1.73 (quintet, 2H); ¹³C NMR (CD₃OD- d⁴, 125 MHz) δ_c 161.16 (qC), 156.22 (qC), 154.44 (qC), 151.18 (qC), 150.98 (qC),

134.38 (qC), 131.31 (ArC), (qC), 128.93 (qC), 127.81 (ArC), 123.84 (qC), 113.53 (ArC), 67.06 (CH₂), 58.62 (CH₂), 56.36 (CH₂), 53.54 (CH₂), 43.77 (CH₂), 40.41 (CH₃), 39.94 (CH₂), 24.45 (CH₂), 23.15; HRMS (ESI): Calculated mass for C₃₂H₄₂N₈O₄ [M⁺] is 603.3407 [M+H]⁺ and found at 603.3412 [M+H]⁺.

Synthesis 30

3-(3-Dimethylamino-propyl)-8-(3-dimethylamino-propylamino)-10-(4-fluoro-phenyl)-

10H-benzo[g]pteridine-2,4-dione (4d)

A 10 ml. round bottom flask was charged with compound (3d) (25 mg, 0.058 mmol) and Λ/,Λ/-dimethylaminopropyl amine (1 mL). The reaction mixture was stirred at 100⁰C, under nitrogen atmosphere for 30 minutes. The reaction mixture was cooled to room temperature and cold ether (5 mL) was added to afford an orange solid. The solid was separated by centrifugation and was washed with diethyl ether (3 X 10 mL). The crude product was purified by flash chromatography using MeOH: CHCI₃: Et₃N (1 :4:0.1) to afford the title compound as an orange solid (20 mg, 70%). ¹H NMR (CD₃OD-d⁴, 500 MHz) δ_H 7.84 (d, ArH₁ J = 8.67 Hz), 7.42 (m, 4H)₁ 7.05 (d, ArH, J = 7.67 Hz), 5.70 (s, ArH), 4.03 (t, 2H, J = 7.25 Hz)₁ 2.46 (t, 2H, J = 7.5 Hz), 2.31 (m, 2H)₁ 2.27 (s, 6H), 2.23 (s, 6H), 2.19 (m, 2H)₁ 1.83 (quintet, 2H), 1.69 (quintet, 2H); ¹³C NMR (CD₃OD-d⁴, 100 MHz) δ_c 163.01 (qC), 158.48 (qC), 152.32 (qC), 133.65 (qC), 133.56 (qC), 131.41 (ArC)₁

131.33 (ArC), 128.01 (qC), 118.7 (ArC)₁ 118.52 (ArC), 58.06 (CH₂), 57.92 (CH₂), 45.44 (CH₃), 42.22 (CH₂), 40.82 (CH₂), 40.62 (CH₃), 26.72 (CH₂); HRMS (ESI): Calculated mass for C₂₆H₃₂FN₇O₂ [M]⁺ is 494.2680 [M+H]⁺ and found at 494.2696 [M+H]⁺. Svnthesis 31 lO-C^Piperidin-i-yl-phenyO-S-CS-pyrrolidin-i-yl-propyO-δ-CS-pyrrolidin-i-yl-propylamino)-

10H-benzo[g]pteridine-2,4-dione (4e)

5 A 10 ml round bottom flask was charged with compound (3e) (75 mg, 0.144 mmol) and 3- aminopropyl pyrrolidine (2 mL). The reaction mixture was stirred at 100⁰C, under nitrogen atmosphere for 20 minutes. The reaction mixture was cooled to room temperature and cold ether (5 mL) was added to afford a red solid. The solid was separated by centrifugation and was washed with diethyl ether (3 X 10 mL) to afford the

10 title compound as a red solid (80 mg, 91 %). Melting point >270°C; ¹H NMR (CD₃OD-d⁴, 500 MHz) δ_H 7.72 (d, ArH, J = 6.25 Hz), 7.2 (q, 4 X ArH, J = 16.14 Hz, J = 25.46 Hz), 6.97 (d, ArH, J = 6.95 Hz), 5.82 (s, ArH)₁ 4.03 (t, 2H, J = 6.82 Hz), 3.21 (m, 2H), 2.75(m, 8H), 2.49 (m, 8H), 1.95 (quintet, 2H), 1.72 (m, 16H); ¹³C NMR (CD₃OD-d⁴, 125 MHz) δ_c 161.64 (qC), 157.49 (qC), 152.8 (qC), 150.99 (qC), 132.45 (qC), 127.92 (ArC), 126.16

15 (qC), 126.09 (qC), 1 16.61 (ArC), 53.6 (CH₂), 53.38 (CH₂), 53.3 (CH₂), 53.08 (CH₂), 49.57

(CH₂), 47.04 (CH₂), 40.98 (CH₂), 26.12 (CH₂), 25.32 (CH₂), 23.96 (CH₂), 22.77 (CH₂), 22.61 (CH₂); HRMS (ESI): Calculated mass for C₃₅H₄₆N₈O₂ [M]⁺ is 61 1.3841 [M+H]⁺ and found at 61 1.3816 [M+H]⁺.

20 Synthesis 32

10-(4-Methoxy-phenyl)-3-(3-pyrrolidin-1-yl-propyl)-8-(3-pyrrolidin-1-yl-propylamino)-

10H-benzo[g]pteridine-2,4-dione (4f)

A 10 mL round bottom flask was charged with compound (3f) (45 mg, 0.96 mmol) and 3- !5 aminopropyl pyrrolidine (2 mL). The reaction mixture was stirred at 100⁰C, under nitrogen atmosphere for 20 minutes. The reaction mixture was cooled to room temperature and cold ether (5 ml.) was added to afford a red solid. The solid was separated by centrifugation and was washed with diethyl ether (3 X 10 mL) to afford the title compound as a red solid (32 mg, 84%). Melting point >270°C; ¹H NMR (CD₃OD-d\ 500 MHz) δ_H 7.63 (d, ArH, J = 6.73 Hz)₁ 7.37 (d, 2 X ArH, J = 8.6 Hz), 7.22 (d, 2 X ArH, J = 8.8 Hz), 6.98 (d, 2H₁ J = 7.4 Hz)₁ 5.57 (d, ArH, J = 7.4 Hz)₁ 3.99 (t, 2H₁ J = 6.37 Hz)₁ 3.9 (s, 3H), 3.73 (t, 2H, J = 7.82 Hz), 2.72 (m, 6H)₁ 2.61 (m, 6H)₁ 1.98 (quintet, 4H)₁ 1.81 (m, 8H); ¹³C NMR (CD₃OD-d⁴, 125 MHz) δ_c 162.93 (qC), 162.29 (qC), 158.93 (qC), 152.23 (qC), 131.95 (qC), 130.25 (ArC), 129.9 (qC), 127.07 (qC), 116.93 (ArC)₁ 56.3 (CH₃), 54.73 (CH₂), 54.52 (CH₂), 54.52 (CH₂), 54.16 (CH₂), 53.51 (CH₂), 42.39 (CH₂),

40.18 (CH₂), 24.19 (CH₂), 23.96 (CH₂), 22.95 (CH₂); HRMS (ESI): Calculated mass for C₃₁H₃₉N₇O₃ [M]⁺ is 558.3190 [M+H]⁺ and found at 558.3187 [M+H]⁺.

Synthesis 33 3-(3-Dimethylamino-propyl)-8-(3-dimethylamino-propylamino)-10-methyl-

10H-benzo[g]pteridine-2,4-dione (4g)

A 10 mL round bottom flask was charged with compound (3g) (65 mg, 0.186 mmol) and Λ/,Λ/-dimethylaminopropyl amine (2 mL, excess). The reaction mixture was stirred at 100⁰C, under nitrogen atmosphere for 30 minutes. The reaction mixture was cooled to room temperature and cold ether (5 mL) was added to afford an orange solid. The solid was separated by centrifugation and was washed with diethyl ether (3 X 10 mL) to afford the title compound as an orange solid (40 mg, 52%). Melting point 170-172⁰C; ¹H NMR (CD₃OD-d⁴, 400 MHz) δ_H 7.75 (d, ArH₁ J = 9.24 Hz), 7.06 (d, ArH, J = 8.13 Hz)₁ 6.32 (s, ArH), 4.04 (t, 2H, J = 7.33 Hz), 3.97 (s, 3H)₁ 3.29 (t, 2H, J = 7.89 Hz), 2.48 (quintet, 4H), 2.28 (s, 6H), 2.25 (s, 6H), 1.89 (m, 4H); ¹³C NMR (CD₃OD-d\ 100 MHz) δ_c 163.06 (qC), 158.55 (qC), 157.6 (qC), 151.02 (qC), 133.28 (ArC)₁ 126.97 (ArC), 120.0 (ArC), 58.01 (CH₂), 45.48 (CH₃), 45.27 (CH₃), 42.52 (CH₂), 40.78 (CH₂), 32.19 (CH₃), 27.23 (CH₂), 18.29 (CH₂); HRMS (ESI): Calculated mass for C₂₁H₃₁N₇O₂ [M]⁺ is 413.2539 [M]⁺ and found at 414.2617 [M+H]⁺. Svnthesis 34

10-Methyl-3-(3-pyrrolidin-1 -yl-propyl)-8-(3-pyrrolidin-1 -yl-propylamino)- 10H-benzo[g]pteridine-2,4-dione (4h)

5 A 10 ml. round bottom flask was charged with compound (3h) (17 mg, 0.045 mmol) and 3-aminopropylpyrrolidine (1.5 ml_, excess). The reaction mixture was stirred at 100⁰C, under nitrogen atmosphere for 30 minutes. The reaction mixture was cooled to room temperature and cold ether (5 mL) was added to afford an orange solid. The solid was separated by centrifugation and was washed with diethyl ether (3 X 10 mL) to afford the

10 title compound as an orange solid (10 mg, 47%). Melting point 154-156°C; ¹H NMR

(CD₃OD-d⁴, 500 MHz) δ_H 7.75 (d, ArH, J = 9.06 Hz), 7.05 (d, ArH₁ J = 8.86 Hz), 6.50 (s, ArH), 4.06 (t, 2H, J = 7.22 Hz), 3.96 (s, 3H), 3.43 (t, 2H, J = 7.03 Hz), 2.65 (m, 12H), 1.95 (m, 4H), 1.80 (m, 8H); ¹³C NMR (CD₃OD-d⁴, 125 MHz) δ_c 163.16 (qC), 158.62 (qC), 157.66 (qC), 151.08 (qC), 133.34 (ArC), 127.0 (ArC), 55.05 (CH₂), 54.95 (CH₂), 54.90

15 (CH₂), 42.65 (CH₂), 40.84 (CH₂), 30.2 (CH3), 27.91 (CH₂), 24.21 (CH₂), 24.12 (CH₂);

HRMS (ESI): Calculated mass for C₂₅H₃₅N₇O₂ [M]⁺ is 466.2934 [M+H]⁺ and found at 466.2924 [M+H]⁺.

Synthesis 35

20 3,10-Bis-(3-dimethylamino-propyl)-8-(3-dimethylamino-propylamino)-

10H-benzo[g]pteridine-2,4-dione (4i)

A 10 mL round bottom flask was charged with compound (3i) (7 mg, 0.017 mmol) and Λ/,Λ/-dimethylaminopropyl amine (1.5 mL, excess). The reaction mixture was stirred at

>5 100⁰C, under nitrogen atmosphere for 30 minutes. The reaction mixture was cooled to room temperature and cold ether (4 mL) was added to afford an orange solid. The solid was separated by centrifugation and was washed with diethyl ether (3 X 10 mL) to afford the title compound as an orange solid (7 mg, 87%). Melting point 258-260⁰C; ¹H NMR (CD₃OD-d⁴, 500 MHz) δ_H 7.78 (d, ArH, J = 9.14 Hz), 7.08 (d, ArH, J = 8.72 Hz), 6.60 (s, iO ArH), 4.65 (s, 2H), 4.05 (t, 2H, J = 7.38 Hz), 3.45 (t, 2H, J = 3.07 Hz), 2.55 (t, 2H, J = 7.2 Hz), 2.47 (t, 2H₁ J = 7.3 Hz), 2.42 (t, 2H, J = 7.8 Hz), 2.28 (s, 12H), 2.25 (s, 6H), 2.05 (quintet, 2H), 1.9 (m, 4H); ¹³C NMR (CD₃OD-(J⁴, 125 MHz) δ_c 161.72 (qC), 157.2 (qC), 156.24 (qC), 149.38 (qC), 137.23 (qC), 133. 77 (qC), 132.1 (ArC), 125.92 (ArC), 65.47 (CH₂), 56.64 (CH₂), 56.1 (CH₂), 44.87 (CH₃), 44.18 (CH₃), 44.08 (CH₃), 42.43 (CH₃), 40.96 (CH₂), 39.41 (CH₂), 25.96 (CH₂), 23.74 (CH₂); HRMS (ESI): Calculated mass for C₂₅H₄₀N₈O₂ [M]⁺ is 485.3345 [M+H]⁺ and found at 485.3346 [M+H]⁺.

Synthesis 36

3,10-Bis-(3-pyrrolidin-1 -yl-propyl)-8-(3-pyrrolidin-1 -yl-propylamino)- 10H-benzo[g]pteridine-2,4-dione (4j)

A 10 mL round bottom flask was charged with compound (3j) (75 mg, 1.07 mmol) and 3- aminopropylpyrrolidine (2 mL, excess). The reaction mixture was stirred at 100⁰C, under nitrogen atmosphere for 30 minutes. The reaction mixture was cooled to room temperature and cold ether (10 mL) was added to afford an orange solid. The solid was separated by centrifugation and was washed with diethyl ether (3 X 10 mL) to afford the title compound as an orange solid (50 mg, 56%). Melting point > 270⁰C; ¹H NMR (CD₃OD-d⁴, 500 MHz) δ_H 7.76 (d, ArH, J = 9.13 Hz), 7.06 (d, ArH, J = 8.84 Hz), 6.57 (s, ArH), 4.65 (s, 2H)₁ 4.05 (t, 2H, J = 7.26 Hz), 3.44 (t, 2H, J = 6.94 Hz), 2.68 (t, 2H, J = 7.27 Hz), 2.62 (t, 2H, J = 7.51Hz), 2.55 (m, 14H), 2.06 (quintet, 2H), 1.92 (m, 4H), 1.81

(quintet, 4H), 1.76 (quintet, 8H); ¹³C NMR (CD₃OD-d⁴, 125 MHz) δ_c 163.19 (qC), 158.59 (qC), 157.66 (qC), 133.54 (ArC), 127.25 (ArC), 117.26 (ArC), 55.07 (CH₂), 54.97 (CH₂), 54.95 (CH₂), 43.89 (CH₂), 42.63 (CH₂), 40.94 (CH₂), 28.03 (CH₂), 26.37(CH₂), 24.26 (CH₂), 24.23 (CH₂); HRMS (ESI): Calculated mass for C₃₁H₄₆N₈O₂ [M]⁺ is 563.3825 [M+H]⁺ and found at 563.3816 [M+H]⁺. Synthesis 37

10-[4-(4-Methyl-piperazin-1 -yl)-phenyl]-3-(3-pyrrolidin-1 -yl-propyl)-8-(3-pyrrolidin-1 -yl- propylamino)-10H-benzo[g]pteridine-2,4-dione (4k)

A 10 mL round bottom flask was charged with compound (3k) (50 mg, 0.093 mmol) and 3-aminopropylpyrrolidine (2 mL). The reaction mixture was stirred at 100⁰C, under nitrogen atmosphere for 30 minutes. The reaction mixture was cooled to room temperature and cold ether (5 mL) was added to afford a red solid. The crude compound was purified by preparative TLC to afford the title compound as a red coloured semi-solid (20 mg, 35%). ¹H NMR (CD₃OD-d\ 500 MHz) δ_H 7.57 (d, ArH, J = 4.46 Hz), 7.36 (d, 2 X ArH, J = 8.33 Hz), 7.27 (d, 2 X ArH, J = 8.95 Hz)₁ 7.21 (s, ArH), 7.05 (d, ArH, J = 8.44 Hz), 4.07 (t, 2H, J = 5.39 Hz), 3.39 (t, 4H, J = 4.65 Hz), 3.12 (t, 4H, J = 8.23 Hz), 3.06 (m, 8H), 2.85 (t, 4H, J = 6.99 Hz), 2.71 (t, 4H, J = 4.71 Hz), 2.44 (s, 3H), 1.95 (m, 12H); HRMS (ESI): Calculated mass for C₃₅H₄₇N₉O₂ [M]⁺ is 625.3853 [M]⁺ and found at 626.3931 [M+H]⁺.

Study 1

The BIAcore SPR (Surface Plasmon Resonance) method has been used to evaluate the binding affinity of the BPD compounds with G-quadruplex DNA. See, e.g.,

Teulade-Fichou, M.-P., et al., 2003; Ladame et al., 2004; and Moore et al., 2006.

A native quadruplex forming sequence of htelo (d(biotin-[GTTA(GGGTTA)₄GG]) and conserved c-kit (d(biotin-[C₃G₃CG₃CGCGAG₃AG₄AG₂]) as well as a hairpin duplex DNA (d(biotin-[G₂CATAGTGCGTG₃CGT₂AGC]) was used in this study.

The quantitative SPR experimental results revealed that the compounds BPD1 , BPD2, BPD3 and BPD4 moderately bind to G-quadruplex DNA of htelo and c-kit, but they also bind to duplex DNA with a weaker affinity. However, BPD compounds BPD5, BPD6, BPD7 and BPD8 bind tightly in sub-micromolar binding affinity with G-quadruplex DNA of htelo and conserved c-kit, but they don't bind to duplex DNA, up to 200 μM. However, some of the BPD compounds tested were found to be very selective to G-quadruplex DNA of conserved c-kit.

The data are summarised in the following table.

NB = No binding observed at concentrations up to 200 μM of compound.

The data show that the compounds BPD5, BPD6, BPD10, and BPD11 are strong quadruplex binding agents.

BPD9 was subsequently tested against G-quadruplex DNA of htelo and c-kit and the duplex DNA. The calculated dissociation constants (K_d) are 63 ± 2 μM, 7.7 ± 0.2 μM and NB respectively.

The phrases "dissocaiation constant (K_d)" and "binding affinty" are used interchangably herein.

Study 2

The ability of the BPD compounds to stabilize G-quadruplex DNA was assessed by a fluorescence resonance energy transfer (FRET) melting assay, in order to measure the melting transitions.

The sequences shown in the following table were used.

However, due to fluorescence quenching problems with BPD1 , BPD2, BPD3, BPD4, and BPD8, FRET-melting results were not obtained. The FRET-melting results for BPD5, BPD6, BPD7, and BPD10 indicated that both BPD5 and BPD6 showed a high degree of stabilization of G-quadruplex DNA of both htelo and conserved c-kit. However, there was no considerable discrimination in quadruplex stabilization between htelo and conserved c- kit. BPD7 appears to moderately stabilize the G-quadruplex DNA of both htelo and c-kit. This result may be due to the lower PKa of the morpholine nitrogen and cause the ligand to be less soluble in an aqueous environment.

The data are summarised in the following table.

ND = Not done.

These data show that compound BPD5, BPD6, and BPD10 possess excellent G-quadruplex stabilization properties. Studv 3

In order to gain further insight into the molecular interaction of BPD5 with G-quadruplex DNA, detailed CD (Circular Dichroism) spectroscopic studies were performed with the DNA shown in the following table in the presence of BPD5.

Significant CD spectral changes were observed when the BPD5 was incubated with htelo in the presence of 100 mM potassium ions. The anti-parallel form of the quadruplex was suppressed and parallel form was appeared. However, there was not much change when incubated in the presence of 100 mM sodium ions.

See Figure 1. Figure 1 illustrates the circular dichroism (CD) spectral data (ellipticity (mdeg) versus wavelength (nm)) obtained in the study of the interaction of BPD5 (40 μM) with G-quadruplex DNA of htelo (4 μM), in K⁺ and Na⁺ containing buffer (50 mM Tris.HCI, pH 7.4, 100 mM KCI, and 100 mM NaCI):

(a) without ligand (O, open pentagon) ("Htelo-tris");

(c) folded in the presence of 100 mM potassium without ligand (*, star) ("Htelo-trisK100");

(d) folded in the presence of 100 mM potassium in the presence of BDP5 (A, filled triangle) "Htelo-trisK100-BPD5");

(e) folded in the presence of 100 mM sodium without ligand (+, plus) ("Htelo-trisNa100"); (f) folded in the presence of 100 mM sodium with BPD5 (•.filled circle)

("Htelo-trisNa100-BPD5").

Since significant CD spectral changes were observed with 100 mM potassium ions, the dependence upon concentration was studied. These studies revealed that for increasing concentrations of BPD5, the anti-parallel form of the G-quadruplex was slowly suppressed and parallel form appeared.

See Figure 2. Figure 2 illustrates the the circular dichroism (CD) spectral data (ellipticity (mdeg) versus wavelength (nm)) obtained in the study of the interaction of BPD5 with G-quadruplex DNA of htelo in K⁺ containing buffer (50 mM Tris.HCI, pH 7.4, 100 mM KCI): (a) folded in the presence of 100 mM potassium without ligand (O₁ open hexagon) ("HteloKIOO");

5 (c) same as (b) but with 1.0 equivalents BPD5 (x, cross) ("1.0-BPD5");

(d) same as (b) but with 2.0 equivalents BPD5 (•, filled circle) ("2.0-BPD5");

(e) same as (b) but with 3.0 equivalents BPD5 (■, filled square) ("3.0-BPD5");

(f) same as (b) but with 4.0 equivalents BPD5 (O₁ open pentagon) ("4.0-BPD5");

(g) same as (b) but with 8.0 equivalents BPD5 (+, plus) ("8.0-BPD5"); 10 (h) same as (b) but with 16.0 equivalents BPD5 (^*, star) ("16.0-BPD5").

Studies were then performed to determine if any changes could be observed with the G-quadruplex of conserved c-kit. The quadruplex forming sequence of conserved c-kit was incubated with 40 μM BPD5 in the presence of 100 mM Na⁺ or 100 mM or K⁺ in two 15 separate CD experiments. The results indicated that, as the parallel form of the

G-quadruplex of conserved c-kit is already stabilized in the presence of K⁺, little change was observed in both CD spectroscopic experiments.

See Figure 3. Figure 3 illustrates the the circular dichroism (CD) spectral data (ellipticity 20 (mdeg) versus wavelength (nm)) obtained in the study of the interaction of BPD5 (40 μM) with G-quadruplex DNA of c-kit (4 μM) in K⁺ containing buffer (50 mM Tris.HCI, pH 7.4, 10O mM KCI):

(a) without salt and ligand (o, open circle) ("Ckit");

(b) in the presence of BDP 5 (A, filled triangle) ("Ckit-BPD5");

25 (c) folded in the presence of 100 mM potassium and BDP 5 (■, filled square)

("Ckit-K100-BPD5");

(d) folded in the presence of 100 mM potassium without ligand (*, star) ("Ckit-K100");

(e) folded in the presence of 100 mM sodium without ligand (x, cross) 30 ("Ckit-Na100");

These data suggest that compound BPD5 plays an important role in changing the $5 structural properties of the G-quadruplex DNA of htelo.

Study 4

Cell based experiments were performed using MCF-7 (a breast cancer cell line), in the \0 presence of BPD5 and BPD8, in order to determine if these compounds could influence c-kit gene expression. Quantification of c-kit gene expression by real-time PCR. Human MCF-7 cells were grown in DMEM + 10% Fetal Calf Serum, seeded at a cell density of 3.4x10⁵ cells per 6- well plate (9.5 cm² growth area). At time point 0, the medium was supplemented with test compound. Cells were harvested after several hours incubation and total RNA was extracted (Rneasy Mini Kit, Qiagen). RNA (200 ng) was reverse transcribed with SuperScriptlll (Invitrogen) according to the manufacturer's instructions. The cDNA was quantified using primers specific to human c-kit and beta-actin genes in a Roche LC480 LightCylcler, using the SYBR Green Master Mix. Crossing point values (Cp) were

10 calculated by the LightCycler software (v. 1.0). Cp values were used to calculate the fold changes in gene expression according to Pfaffl et al, 2002.

BPD5 and BPD8 (0.5 μM and 5 μM concentration) were incubated with MCF-7 cells for 3 hours and 6 hours. The results showed significant c-kit gene expression changes with

15 BPD5 and BPD8.

The data are summarised in Figure 4 and the following table.

Figure 4 is bar graph showing the percentage gene expression of c-kit in MCF-7 cells

20 treated with BPD5 or BPD8. The figure shows the levels of expression for control cells treated with 10% DMSO in water (100% expression, blank bar), and for cells treated with the isoalloxazines BPD5 or BPD8 at 6 hours at 5 μM concentration (shaded bars).

(^*) Control gene: beta Actin.

!5

These data demonstrate that compounds BPD5 and BPD8 showed a significant repression of c-kit gene expression in MCF-7 cells, as judged by quantitaion of c-kit mRNA levels relative to that of the control gene β-actin.

Study 5

The BIAcore SPR (Surface Plasmon Resonance) method has also been used to evaluate the differences in dissociation constant of the BPD compounds binding to c-kit and c-kit1, as well as htelo. The sequences and methods described in Study 1 were used in this study along with the c-kit1 promoter G-quadruplex sequence ^(DiOtJn-[AG₃AG₃CGCTG₃AG₂AG₃])). The c-kit sequence used in this study (and Study 1 ) is also known in the art as the c-kit2 promoter G-quadruplex sequence.

The quantitative SPR experimental results revealed that the BPD compounds tested showed a preference for binding to the c-kit2 quadruplex, with 3-fold selectivity for c-kit2 over c-kit1 for ligand BPD8 and 14-fold selectivity for c-kit2 over htelo for ligand BPD5 (see Table 6).

The K_d results given below for c-kit and htelo differ slightly from those values calculated in Study 1. The values given in Table 6 were obtained from a greater number of repeat experiments.

nd = not determined

Study 6

The ability of the BPD compounds to stabilize G-quadruplex DNA of c-kit1 was assessed by a fluorescence resonance energy transfer (FRET) melting assay, as described in Study 2, in order to measure the melting transitions.

The c-kit1 sequence shown in the following table was used.

Table 7

DNA Sequence conserved c-kit1 5'-FAM-d(GGG AGG GCG CTG GGA GGA GGG)-TAMRA-3" The data are summarised in the following table.

nd = not determined

Study 7

Further cell based experiments were performed using HGC-27, in the presence of BPD8, in order to determine if this compound could influence c-kit gene expression. HGC-27 is a gastrointestinal stromal tumour cell line which shows much higher basal levels of c-kit oncogene expression compared with MCF-7 (-16-fold higher expression).

Human HGC-27 cells were quantified in a manner identical to the quantification of MCF-7 cells, as described above in Study 4.

BPD8 (5 μM concentration) was incubated with HGC-27 cells for 2, 6 and 24 hours. The results showed significant c-kit gene expression changes with BPD8.

The data are summarised in Figure 5 and the following table.

(^*) Control gene: beta Actin.

These data demonstrate that compound BPD8 shows repression of c-kit gene expression in HGC-27 cells, as judged by quantitaion of c-kit mRNA levels relative to that of the control gene β-actin.

The foregoing has described the principles, preferred embodiments, and modes of operation of the present invention. However, the invention should not be construed as limited to the particular embodiments discussed. Instead, the above-described embodiments should be regarded as illustrative rather than restrictive, and it should be appreciated that variations may be made in those embodiments by workers skilled in the art without departing from the scope of the present invention.

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Claims

1. A compound selected from compounds of the following formula, and pharmaceutically acceptable salts, solvates, and hydrates thereof:

wherein:

R^C6 is independently -H, -G¹, or -G²; R^C7 is independently -H, -G¹, or -G²; R^C9 is independently -H, -G¹, or -G²; 10 wherein: each -G¹, if present, is independently -Ph, -NHPh, -NR^A1Ph, or -OPh;

wherein: 15 -R^A1 is independently saturated aliphatic C^alkyl; and each Ph is independently unsubstituted phenyl or phenyl substituted with one or more groups G²;

and wherein: 20 each -G², if present, is independently:

-F, -Cl, -Br, -I, -R^ω, -CF₃, -OH, -OR^, -OCF₃, -SR^A2,

-NH₂, -NHR^, -NR^₂, -NR^A3R^A4,

-L^A1-OH, -L^-OR^,

-L^A1-NH₂, -L^-NHR^, -L^-NR^, or -L^A1-NR^A3R^A4, .5 -X¹-L^A1-OH, -X'-L^-OR^,

-X¹-L^A1-NH₂, -X¹-L^A1-NHR^A2, -X'-L^-NR^, or -X¹-L^A1-NR^A3R^A4;

wherein: each -X¹- is independently -O-, -NH-, or -NR^-; 50 each -R^ is independently saturated aliphatic C_1-4alkyl; each -NR^A3R^A4 is independently -Q¹; each -L^A1- is independently saturated aliphatic C_2-8alkylene;

and wherein: !5 R^N8B is independently -H or -G³; wherein: each G³ is independently saturated aliphatic C^alkyl;

5 and wherein:

R^N1° is independently saturated aliphatic C^alkyl, -G⁴, -L^10G-G⁵, -Q², -L¹⁰-Q³, or -L^10P-Q⁴;

wherein: 10 G⁴ is phenyl or C_5-6heteroaryl, and is independently unsubstituted or substituted with one or more substituents -G⁶;

G⁵ is phenyl or C₅.₆heteroaryl, and is independently unsubstituted or substituted with one or more substituents -G⁶;

-L^10G- is independently saturated aliphatic C_1-4alkylene; and 15 -L¹⁰- is independently saturated aliphatic C₂-₈alkylene; and

-L^10P- is independently phenylene or Cs-βheteroarylene, and is independently unsubstituted or substituted with one or more substituents -G⁶;

wherein: 20 each -G⁶, if present, is independently:

-F, -Cl, -Br, -I, -R^A5, -CF₃, -OH, -OR^A5, -OCF₃, -SR^A5,

-NH₂, -NHR*⁵, -NR^A5 ₂, -NR^A6R^A7,

-L^-OH, -L^A2-OR^A5,

-L^A2-NH₂, -L^A2-NHR^A5, -L^-NR^, or -L^-NR^R^, ?5 -X'-L^-OH, -X'-L^-OR^,

-X²^-NH₂, -X'-L^-NHR^, -X^L^-NR^, or -X'-L^-NR^⁷;

wherein: each -X²- is independently -O-, -NH-, or -NR^A5-; JO each -R^A5 is independently saturated aliphatic C^alkyl; each -NR^A6R^A7 is independently -Q⁵; each -L*²- is independently saturated aliphatic C_2-8alkylene;

and wherein: !5 each of -L³- and -L⁸- is independently saturated aliphatic C_2-8alkylene;

and wherein:

-NR^3PAR^3PB is independently -Q⁶; -NR^8PAR^8PB is independently -Q⁷; ■0 and wherein: each of -Q¹, -Q², -Q³, -Q⁴, -Q⁵, -Q⁶, and -Q⁷ is independently:

-NH₂, -NHR^B1, -NR^B1 ₂, or -NR^B2R^B3;

5 wherein: each R^B1 is independently saturated aliphatic C_1-4alkyl; and in each group -NR^B2R^B3, R^B2 and R^B3, taken together with the nitrogen atom to which they are attached, form a 5-, 6-, or 7-membered non-aromatic ring having exactly 1 ring heteroatom or exactly 2 ring heteroatoms, wherein one of said 10 exactly 2 ring heteroatoms is N, and the other of said exactly 2 ring heteratoms is independently selected from N and O.

2. A compound according to claim 1 , wherein R^C6 is independently -H or -G².

15 3. A compound according to claim 1 , wherein R^C6 is independently -H.

4. A compound according to any one of claims 1 to 3, wherein R⁰⁷ is independently -H or -G².

-0 5. A compound according to any one of claims 1 to 3, wherein R^C7 is independently -H.

6. A compound according to any one of claims 1 to 5, wherein R^C9 is independently -H or -G².

>5

7. A compound according to any one of claims 1 to 5, wherein R^C9 is independently -H.

8. A compound according to any one of claims 1 to 7, wherein R^A1, if present, is i0 independently -Me, -Et, -nPr, or -iPr.

9. A compound according to any one of claims 1 to 7, wherein R^A1, if present, is independently -Me.

I5 10. A compound according to any one of claims 1 to 9, wherein each -G², if present, is independently -F, -Cl, -Br, -I, -R*², -CF₃, -OR*², or -OCF₃.

11. A compound according to any one of claims 1 to 10, wherein each R*², if present, is independently -Me, -Et, -nPr, or -iPr.

12. A compound according to any one of claims 1 to 10, wherein each R^A2, if present, is independently -Me.

13. A compound according to any one of claims 1 to 12, wherein each -L^A1-, if present, is independently -(CH₂)_ni-, wherein n1 is independently 2, 3, 4, 5, 6, 7, or 8.

14. A compound according to any one of claims 1 to 12, wherein each -L^A1-, if present, is independently -(CH₂)_ni-, wherein n1 is independently 2, 3, or 4.

15. A compound according to any one of claims 1 to 14, wherein R^N8B is independently -H.

16. A compound according to any one of claims 1 to 14, wherein R^N8B is independently -G³.

17. A compound according to any one of claims 1 to 16, wherein G³, if present, is independently -Me, -Et, -nPr, or -iPr.

18. A compound according to any one of claims 1 to 16, wherein G³, if present, is independently -Me.

19. A compound according to any one of claims 1 to 18, wherein R^N1° is independently saturated aliphatic C_1-6alkyl, -G⁴, or -L^10G-G⁵.

20. A compound according to any one of claims 1 to 18, wherein R^N1° is independently -L¹⁰-Q³ or -L^10P-Q⁴.

21. A compound according to any one of claims 1 to 18, wherein R^N1° is independently -G⁴ or -|_^10G-G⁵.

22. A compound according to any one of claims 1 to 18, wherein R^N1° is independently saturated aliphatic C_1-6alkyl.

23. A compound according to any one of claims 1 to 18, wherein R^N1° is independently -G⁴.

24. A compound according to any one of claims 1 to 18, wherein R^N1° is independently -|_^10G-G⁵.

25. A compound according to any one of claims 1 to 18, wherein R^N1° is independently -Q².

26. A compound according to any one of claims 1 to 18, wherein R^N1° is 5 independently -L¹⁰-Q³. '

27. A compound according to any one of claims 1 to 18, wherein R^N1° is independently -L^10P-Q⁴.

10 28. A compound according to any one of claims 1 to 18, wherein R^N1° is independently -Me, -Et, -nPr, or -iPr.

29. A compound according to any one of claims 1 to 18, wherein R^N1° is independently -Me.

15

30. A compound according to any one of claims 1 to 27, wherein -L¹⁰-, if present, is independently -(CH₂)_n3-, wherein n3 is independently 2, 3, 4, 5, 6, 7, or 8.

31. A compound according to any one of claims 1 to 27, wherein -L¹⁰-, if present, is 20 independently -(CH₂)_n3-, wherein n3 is independently 2, 3, or 4.

32. A compound according to any one of claims 1 to 27, wherein -L^10G-, if present, is independently -(CH₂)_n2-, wherein n2 is independently 1 , 2, 3, or 4.

15 33. A compound according to any one of claims 1 to 27, wherein -L^10G-, if present, is independently -(CH₂)_n2-, wherein n2 is independently 1.

34. A compound according to any one of claims 1 to 27, wherein -L^10P-, if present, is independently phenylene, oxazol-di-yl, thiazol-di-yl, isoxazol-di-yl, isothiazol-di-yl,

IO pyrazol-di-yl, pyridin-di-yl, pyrimidin-di-yl, or pyrazin-di-yl; and is independently unsubstituted or substituted with one or more substituents -G⁶.

35. A compound according to any one of claims 1 to 27, wherein -L^10P-, if present, is independently phenylene; and is independently unsubstituted or substituted with

>5 one or more substituents -G⁶.

36. A compound according to any one of claims 1 to 35, wherein -G⁴, if present, is independently phenyl or C_5-6heteroaryl, and is independently unsubstituted or substituted with one or more substituents -G⁶.

0

37. A compound according to any one of claims 1 to 35, wherein -G⁴, if present, is independently phenyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridinyl, pyrimidinyl, or pyrazinyl; and is independently unsubstituted or substituted with one or more substituents -G⁶.

5

38. A compound according to any one of claims 1 to 35, wherein -G⁴, if present, is independently phenyl; and is independently unsubstituted or substituted with one or more substituents -G⁶.

10 39. A compound according to any one of claims 1 to 35, wherein -G⁵, if present, is independently phenyl or C₅.₆heteroaryl, and is independently unsubstituted or substituted with one or more substituents -G⁶.

40. A compound according to any one of claims 1 to 35, wherein -G⁵, if present, is 15 independently phenyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridinyl, pyrimidinyl, or pyrazinyl; and is independently unsubstituted or substituted with one or more substituents -G⁶.

41. A compound according to any one of claims 1 to 35, wherein -G⁵, if present, is .0 independently phenyl; and is independently unsubstituted or substituted with one or more substituents -G⁶.

42. A compound according to any one of claims 1 to 41 , wherein -L^A2-, if present, is independently -(CH₂),*-, wherein n4 is independently 2, 3, 4, 5, 6, 7, or 8.

!5

43. A compound according to any one of claims 1 to 41 , wherein -L^A2-, if present, is independently -(CH₂)_n4-, wherein n4 is independently 2, 3, or 4.

44. A compound according to any one of claims 1 to 41 , wherein each -G⁵, if present, .0 is independently -F, -Cl, -Br, -I₁ -R^A5 _t -CF₃, -OR^A5, or -OCF₃.

45. A compound according to any one of claims 1 to 44, wherein each R^A5, if present, is independently -Me, -Et, -nPr, or -iPr.

5 46. A compound according to any one of claims 1 to 44, wherein each R^A5, if present, is independently -Me.

47. A compound according to any one of claims 1 to 46, wherein -L³- is independently -(CH₂)_n5-, wherein n5 is independently 2, 3, 4, 5, 6, 7, or 8.

48. A compound according to any one of claims 1 to 46, wherein -L³- is independently -(CH₂)_n5-, wherein n5 is independently 2, 3, or 4.

49. A compound according to any one of claims 1 to 48, wherein -L⁸- is independently 5 -(CH₂)_n6-, wherein n6 is independently 2, 3, 4, 5, 6, 7, or 8.

50. A compound according to any one of claims 1 to 48, wherein -L⁸- is independently -(CH₂)_n6-, wherein n6 is independently 2, 3, or 4.

10 51. A compound according to any one of claims 1 to 48, wherein -L³- and -L⁸- are the same.

52. A compound according to any one of claims 1 to 51 , wherein -Q¹, if present, is independently -NH₂, -NHR^B1, or -NR^B1 ₂.

15

53. A compound according to any one of claims 1 to 52, wherein -Q⁵, if present, is independently -NH₂, -NHR^B1, or -NR^B1 ₂.

54. A compound according to any one of claims 1 to 53, wherein -Q², if present, 20 is independently -NR^B2R^B3.

55. A compound according to any one of claims 1 to 54, wherein -Q³, if present, is independently -NR⁸²R⁸³.

25 56. A compound according to any one of claims 1 to 55, wherein -Q⁴, if present, is independently -NR⁶²R⁸³.

57. A compound according to any one of claims 1 to 56, wherein -Q⁶ is independently -NR⁸²R⁸³.

JO

58. A compound according to any one of claims 1 to 57, wherein -Q⁷ is independently -NR⁸²R⁸³.

59. A compound according to any one of claims 1 to 58, wherein -Q⁶ and -Q⁷ are the 55 same.

60. A compound according to any one of claims 1 to 60, wherein each R^B1, if present, is independently -Me, -Et, -nPr, or -iPr.

^[0 61. A compound according to any one of claims 1 to 60, wherein each R^B1, if present, is independently -Me.

62. A compound according to any one of claims 1 to 61 , wherein each -NR^B2R^B3, if present, is independently pyrrolidino, imidazolidino, N-(Ci_-3alkyl)-imidazolidino, pyrazolidino, N-(C_1-3alkyl)-pyrazolidino, piperidino, N-(C_1-3alkyl)-piperidino, 5 piperizino, morpholino, azepino, diazepino, or N^CvsalkyO-diazepino.

63. A compound according to any one of claims 1 to 51 , wherein each -Q¹, if present, is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂, -N(nPr)₂, -N(JPr)₂, pyrrolidino, imidazolidino, N-(methyl)-imidazolidino,

10 pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

64. A compound according to any one of claims 1 to 51 , wherein each -Q¹, if present, is is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂,

15 -N(nPr)₂, or -N(JPr)₂.

65. A compound according to any one of claims 1 to 51 and 63 to 64, wherein each -Q⁵, if present, is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂, -N(nPr)₂, -N(JPr)₂, pyrrolidino, imidazolidino, N-(methyl)-imidazolidino,

20 pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

66. A compound according to any one of claims 1 to 51 and 63 to 64, wherein each -Q⁵, if present, is is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr),

>5 -N(Me)₂, -N(Et)₂, -N(nPr)₂, or -N(JPr)₂.

67. A compound according to any one of claims 1 to 51 and 63 to 66, wherein -Q², if present, is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂, -N(nPr)₂, -N(JPr)₂, pyrrolidino, imidazolidino, N-(methyl)-imidazolidino,

50 pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

68. A compound according to any one of claims 1 to 51 and 63 to 66, wherein -Q², if present, is independently pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, i5 pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

69. A compound according to any one of claims 1 to 51 and 63 to 68, wherein -Q³, if present, is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂, -N(nPr)₂, -N(JPr)₂, pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino,

5 morpholino, azepino, diazepino, or N-(methyl)-diazepino.

70. A compound according to any one of claims 1 to 51 and 63 to 68, wherein -Q³, if present, is independently pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino,

10 morpholino, azepino, diazepino, or N-(methyl)-diazepino.

71. A compound according to any one of claims 1 to 51 and 63 to 70, wherein -Q⁴, if present, is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂, -N(nPr)₂, -N(JPr)₂, pyrrolidino, imidazolidino, N-(methyl)-imidazolidino,

15 pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

72. A compound according to any one of claims 1 to 51 and 63 to 70, wherein -Q⁴, if present, is independently pyrrolidino, imidazolidino, N-(methyl)-imidazolidino,

73. A compound according to any one of claims 1 to 51 and 63 to 72, wherein -Q⁶ is independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂,

25 -N(nPr)₂, -N(JPr)₂, pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

74. A compound according to any one of claims 1 to 51 and 63 to 72, wherein -Q⁶ is 30 independently pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino,

N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

75. A compound according to any one of claims 1 to 51 and 63 to 74, wherein -Q⁷ is J5 independently -NH₂, -NHMe, -NHEt, -NH(nPr), -NH(iPr), -N(Me)₂, -N(Et)₂,

-N(nPr)₂, -N(JPr)₂, pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

76. A compound according to any one of claims 1 to 51 and 63 to 74, wherein -Q⁷ is independently pyrrolidino, imidazolidino, N-(methyl)-imidazolidino, pyrazolidino, N-(methyl)-pyrazolidino, piperidino, N-(methyl)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(methyl)-diazepino.

77. A compound according to any one of claims 1 to 15, wherein the group __{N R} ^N8B__L ⁸__NR ^8PA _R ^8PB J₅ j_ndependently selected from:

78. A compound according to any one of claims 1 to 15 and 77, wherein the group -L³-NR^3PAR^3PB is independently selected from:

^i

79. A compound according to claim 1 , selected from the following compounds and pharmaceutically acceptable salts, solvates, and hydrates thereof:

BPD1

80. A pharmaceutical composition comprising a compound according to any one of claims 1 to 79, and a pharmaceutically acceptable arrier, diluent, or excipient.

5 81. A method of preparing a pharmaceutical composition comprising admixing a compound according to any one of claims 1 to 79 and a pharmaceutically acceptable arrier, diluent, or excipient.

82. A method of selectively binding a G-quadruplex, comprising contacting said

10 G-quadruplex with an effective amount of a compound according to any one of claims 1 to 79.

83. A method of stabilizing a G-quadruplex, comprising contacting said G-quadruplex with an effective amount of a compound according to any one of claims 1 to 79.

I5

84. A method of inhibiting telomerase comprising contacting said telomerase with an effective amount of a compound according to any one of claims 1 to 79.

85. A method of inhibiting cell proliferation comprising contacting a living cell with an 10 effective amount of a compound according to any one of claims 1 to 79.

86. A compound according to any one of claims 1 to 79 for use in a method of treatment of the human or animal body by therapy.

!5 87. A compound according to any one of claims 1 to 79 for use in a method of treatment of a disorder that is mediated by a G-quadruplex.

88. A compound according to any one of claims 1 to 79 for use in a method of treatment of a proliferative disorder. 0

89. A compound according to any one of claims 1 to 79 for use in a method of treatment of cancer.

90. A compound according to any one of claims 1 to 79 for use in a method of

5 treatment of: solid tumour cancer, gastrointestinal stromal cancer (GIST), stomach cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma lung cancer, gastrointestinal cancer, thyroid cancer, breast cancer, ovarian cancer, epithelial ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, testicular

10 cancer, testicular seminoma, liver cancer, kidney cancer, renal cell carcinoma, bladder cancer, pancreatic cancer, oesophageal cancer, brain cancer, glioblastoma, glioma, sarcoma, osteosarcoma, Ewing's sarcoma, bone cancer, skin cancer (e.g., head and neck cancer), squamous cancer, Kaposi's sarcoma, melanoma, malignant melanoma, lymphoma, B-cell lymphoma, T-cell lymphoma,

15 leukaemia, myeloid leukaemia, acute myeloid leukaemia, or a tumour of unknown origin.

91. A compound according to any one of claims 1 to 79 for use in a method of treatment of a disorder that is characterised by expression or overexpression of

20 c-kit.

92. A compound according to any one of claims 1 to 79 for use in a method of treatment of a disorder that is characterised by expression (or overexpression) of VEGF.

15

93. A compound according to any one of claims 1 to 79 for use in a method of treatment of a disorder that is characterised by expression (or overexpression) of c-MYC.

JO 94. A compound according to any one of claims 1 to 79 for use in a method of treatment of a disorder that is characterised by expression (or overexpression) of BCL-2.

95. A compound according to any one of claims 1 to 79 for use in a method of

S5 treatment of a disorder that is characterised by expression (or overexpression) of

B-raf.

96. Use of a compound according to any one of claims 1 to 79 in the manufacture of a medicament for use in treatment, wherein the treatment is a treatment as defined

0 in any one of claims 87 to 95. A method of treatment comprising administering to a patient in need of treatment a therapeutically-effective amount of a compound according to any one of claims 1 to 79, wherein the treatment is a treatment as defined in any one of claims 87 to 95.