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WO2009022104A1 - Dérivés de fascaplysine et leur utilisation dans le traitement d'un cancer - Google Patents

Dérivés de fascaplysine et leur utilisation dans le traitement d'un cancer Download PDF

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WO2009022104A1
WO2009022104A1 PCT/GB2008/002678 GB2008002678W WO2009022104A1 WO 2009022104 A1 WO2009022104 A1 WO 2009022104A1 GB 2008002678 W GB2008002678 W GB 2008002678W WO 2009022104 A1 WO2009022104 A1 WO 2009022104A1
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alkyl
halo
groups
substituents selected
cycloalkyl
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Bhabatosh Chaudhuri
Sachin Govindrao Mahale
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De Montfort University
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De Montfort University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/14—Radicals substituted by nitrogen atoms, not forming part of a nitro radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D209/20—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals substituted additionally by nitrogen atoms, e.g. tryptophane
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention relates to chemical compounds, and to their use in the treatment of cancer.
  • chemotherapeutic anti-cancer agents are one of the standard current treatment protocols for the majority of cancers. However, there is still a need in the art for more effective and less toxic anti-cancer agents.
  • the phenomenon of progressive and uncontrolled cell growth that eventually leads to tumour development and other proliferative disorders underlines the fundamental role of the cell division cycle in cancer.
  • the normal mitotic cell division cycle consists of four distinct phases: the gap phases G 1 and G 2 , the DNA synthesis phase (S) and mitotic (M) phase.
  • S DNA synthesis phase
  • M mitotic phase.
  • a dormant, quiescent cell at G 0 must be re-awakened with the help of growth-promoting factors and ushered into the G 1 phase of the cell cycle.
  • the transition of the cell during the early G 1 to the 171Jd-G 1 phase is driven by growth promoting extra-cellular signals.
  • the cell Towards the end of mid-Gi, the cell crosses the restriction point and enters the late G 1 phase.
  • Cdk1-Cdk12 Cdk catalytic subunits
  • cyclin A-cyclin L1 cyclins
  • the cyclins show a characteristic discontinuous expression over the different phases of the cell cycle.
  • the D-type cyclins (cyclin D1 , D2 and D3) are expressed during early G 1 phase of the cell cycle. Their expression is triggered by the extra-cellular mitogenic signals received from various growth factors.
  • the induction and nuclear localization of D-type cyclins is a consequence of the activation of mitogen-activated protein kinases (MAPK-s) by mitogenic signals.
  • cyclin D1 has been reported to be induced by the oestrogen receptor pathway where the small molecule oestrogen provides the extra-cellular mitogenic signal (Sabbah ef al, 1999; Hong ef al, 1998).
  • Cdk4 and Cdk6 D-type cyclins specifically activate cyclin dependent kinases 4 and 6 (Cdk4 and Cdk6).
  • Cdk4/Cdk6-cyclin D complexes Kato 1997.
  • Cdk6 is thought to be a functional homologue of Cdk4, although recent reports indicate that Cdk6 may also actively participate in differentiation (Grossel and Hinds 2006).
  • Cyclin dependent kinase 4 (Cdk4) is crucially important because a cell's initial commitment to complete a cell division cycle depends on Cdk4 activity. Activation of Cdk4 at Go/d is also essential for the later G ⁇ /S transition in the cell cycle, at least in cells that contain a functional copy of pRb. Moreover, many human cancers are characterized by either over-expression of its activating partner cyclin D1 or loss of p16 INK4a which is a natural Cdk4 inhibitory protein (Bartek ef a/, 1996; Weinberg 1995).
  • Cdk4 protein is either overexpressed or amplified in a wide range of human tumours (Matsumoto ef al, 1999; Kim ef al, 1998). Even the target of Cdk4, the retinoblastoma protein (pRb), is inactivated in many tumours resulting in excessive cell proliferation.
  • Cdkl-s The natural inhibitors of the cyclin dependent kinases (Cdkl-s) negatively control cell cycle progression.
  • Cdkls Two distinct families of Cdkls are known. The first family is INK4 (inhibitor of CdM), which includes the proteins, p16 INK4a , p15 INK4b , p18 INK4c and p19 INK4d . They specifically inhibit Cdk4 and Cdk6 but were initially discovered as inhibitors of Cdk4 only.
  • the second family of proteins which are known to be global inhibitors of Cdk activities, is the CIP/KIP (CDK inhibitory proteins/kinase inhibitory proteins) family and consists of the proteins p21 c ⁇ p1 , p27 KIP1 and p57 KIP2 .
  • the p16 INK4 protein commonly known as p16, is a naturally occurring inhibitor of the cyclin dependent kinases 4 and 6 (Cdk4 and Cdk6), which normally bind to the regulatory D-type cyclins so that they can perform their functional role in the cell cycle. It is estimated that at least 60% of all cancers lack p16 functional activity (Shapiro ef al, 1995; Sakaguchi ef al, 1996).
  • pRb function A correlation between pRb function and p16 expression inside the cells is well known and, in most cases, it is inversely proportional to the functional activity of the pRb protein (Tarn et al, 1994; Li et al, 1994). Generally speaking, cells which lack functional p16 contain active pRb. Conversely, when pRb is inactivated by viral oncogenes, p16 is usually over-expressed. In many cancers, either cyclin D1 is overproduced or p16 is inactive (Kataoka et al, 2000; Campbell et al, 2000; Seike et al, 2000).
  • Cdk4/Cdk6 The most extensively studied substrate for Cdk4/Cdk6 is the product of the tumour suppressor retinoblastoma gene (RB), which acts as a repressor for a number of genes required for GJS transition, and also initiation and completion of DNA synthesis.
  • RB tumour suppressor retinoblastoma gene
  • the phosphorylation of the retinoblastoma protein (pRb) by cyciin D bound Cdk4 (i.e. the Cdk4-cyclin D complex) leads to its functional inactivation and frees the E2F family members to perform their role in transcription.
  • Members of the E2F family initiate a cascade of events that include further phosphorylation of pRb by Cdk2-cyclin E and Cdk2-cyclin A complexes.
  • Cdk4-mediated phosphorylation of pRb compels the cell to commit itself towards completing one full cell division cycle (Bartek ef al
  • retinoblastoma protein pRb/p105 is considered to be one of the key regulators of the cell division cycle.
  • pRb When pRb is unphosphorylated, it is active as a growth suppressor protein, whereas one reason for loss of the norma] pRb function in cells is hyper-phosphorylation of the pRb protein by mutated or over-expressed kinases such as cyclin D-Cdk4/6 complexes (Simin ef al, 2004).
  • Selective Cdk inhibitors are considered to be particularly useful as cancer therapeutics since they may minimise undesirable toxicity or side effects (Garrett and Fattaey 1999; Sausville 2003; Senderowicz 2003; Dai and Grant 2004; Eastman 2004; Liu et al, 2004; Swanton 2004; Hirai et al, 2005; Benson et al, 2005; Senderowicz 2005; Schwartz 2005; Welburn and Endicott 2005).
  • Cdk4-cyclin D1 is a crucially important target for cancer therapy (Yu et al, 2006; Landis et al, 2006; Malumbres and Barbacid 2006).
  • the malignant proliferation could be arrested through enzymatic inhibition of Cdk4. This would indicate that a small molecule, which specifically inhibits the Cdk4 enzyme activity in vitro, prevents cell growth and inhibits tumour volume in vivo could be of immense therapeutic value for the treatment of cancer.
  • Fascaplysin isolated from a marine sponge (Roll et al, 1988; Soni et al, 2000), is a pentacyclic quaternary salt that inhibits specifically Cdk4 causing G 0 ZG 1 arrest of cancer cells.
  • fascaplysin inhibits the phosphorylation of the cellular pRb protein at Cdk4-specific serine residues confirming specific activity against the Cdk4 enzyme.
  • fascaplysin is highly toxic, and the potential for its planar structure to intercalate double-stranded DNA has been suggested as a possible explanation of its toxicity (Hormann et al, 2001 ).
  • Fascaplysin is a selective Cdk4 inhibitor that arrests cycling cells specifically at the G 0 ZG 1 boundary which correlates with the accumulation of hypo-phosphorylated pRb. Fascaplysin prevents the initiation of pRb hyper-phosphorylation which is specifically triggered by Cdk4 activity (Paull et al, 1989; Meijer et al, 1997; Alessi et al, 1998; Carlson et al, 1996; Soni et al, 2000; Soni et al, 2001 ).
  • Fascaplysin was identified in a large screening program as a small molecule that specifically targets enzymatic activity of Cdk4 and inhibits the in vitro phosphorylation of the retinoblastoma protein pRb, the most prominent Cdk4 substrate (Soni et al, 2000). Fascaplysin demonstrates pRb-dependent arrest of U2-OS (osteosarcoma) and HCT-116 (colon carcinoma) cancer cells at the G 0 ZGi phase of the cell cycle. It has also shown similar activity against normal lung fibroblast-derived MRC-5 cells in vitro.
  • fascaplysin will be therapeutically useful as an anticancer agent because it is a highly toxic molecule.
  • the potential for its planar structure to intercalate with double-stranded DNA has been suggested as a possible explanation for its unusual biological activity and toxicity.
  • the DNA binding property of fascaplysin is similar to the structurally related DNA intercalating agents, cryptolepine and ellipticine (Hormann er a/, 2001).
  • Aubry et al (2004, 2005) describe the synthesis of non-planar analogues of fascaplysin which were shown to be selective inhibitors of CDK4 over CDK2.
  • the most active compound 8f from Aubry et al (2005) was reported to have an IC 50 for the inhibition of CDK4 of 50 ⁇ M.
  • Aubry et al (2006) describe the synthesis of non-planar analogues of fascaplysin which were shown to be selective inhibitors of CDK4 over CDK2.
  • the most active compound 9q (referred to herein as CA224) was reported to have an IC 50 for the inhibition of CDK4 of 6 ⁇ M.
  • Mahale et al (2006a) also describe non-planar analogues of fascaplysin which were predicted to bind to the ATP-binding site of CDK4, and which were shown to be selective inhibitors of CDK4 over CDK2.
  • the most active compound CA224 was shown not to intercalate with double-stranded DNA, and to arrest growth of Calu-1 cancer cells at Go/G1 by inhibiting pRb phosphorylation. Both Aubry et al (2006) and Mahale et al (2006a) concluded that CA224 could be the basis for the development of more potent CDK4 specific inhibitors.
  • Mahale et al (2006b) describe a number of non-planar analogues of fascaplysin which were shown to be selective inhibitors of CDK4 and not to interact or intercalate with double-stranded DNA.
  • the most active compound CA199 was reported to have an IC 50 for the inhibition of CDK4-cyclin D1 of 20 ⁇ M, and to inhibit growth of cancer cell lines in a pRb dependent manner at concentrations ranging from 10-40 ⁇ M.
  • Mahale et al concluded that CA199 could be the basis for the development of more potent CDK4 specific inhibitors.
  • Mahale & Chaudhuri (2006) reviewed the development of CDK inhibitors as anticancer agents and emphasized the importance of inhibition of CDK4-cyclin D1 as a target for cancer therapy. Mahale & Chaudhuri discussed the rational design of CDK-4 specific inhibitors derived from the structure of fascaplysin. The best compounds in three classes of fascaplysin analogues were 7a, 9q (referred to herein as CA224) and 12m (referred to herein as CA199). These compounds were reported to have an IC 50 for CDK4 of 50 ⁇ M, 6 ⁇ M and 20 ⁇ M, and a mean IC 50 for growth inhibition in a panel of cancer lines of 50 ⁇ M, 3.5 ⁇ M and 7 ⁇ M, respectively.
  • Example 1 The biological activity of the DE002 series of compounds is presented in Example 1.
  • DE002 inhibits Cdk4 specifically, which is highly significant due to the crucial role of Cdk4 in the development of human cancers, and we have shown that DE002 does not intercalate with double-stranded DNA thus minimising toxicity.
  • DE002 blocks at the G 0 ZG 1 phase of the cell division cycle.
  • DE002 is a powerful antioxidant, as shown by its ability to prevent H 2 O 2 -induced cellular reactive oxygen species (ROS) formation in vitro.
  • Antioxidants such as the flavonoid class of phytochemicals, can be used for cancer therapy, by quenching the oxidant H 2 O 2 and preventing ROS formation.
  • Formation of ROS inside the living cells could have detrimental effects on cells, since ROS can induce DNA strand breaks and also can mediate conformational changes in protein molecules. These changes at molecular level could result in many degenerative diseases that include cancer (Izzotti et al, 2006; Butterfield ef al, 1998).
  • the antioxidant property of DE002 is additionally valuable not only for the treatment of cancer but also for its prevention.
  • DE002 itself a potentially valuable anticancer agent
  • analogues based on the DE002 scaffold may be used to improve the therapeutic index of DE002 for the treatment of cancer.
  • DE002 is a fascaplysin analogue that has a tryptoline (2,3,4,9-tetrahydro-1 H-beta- carboline) structure at the left hand side of the molecule and an unsubstituted phenyl group in the orffto-position at the right hand side of the molecule.
  • tryptoline 2,3,4,9-tetrahydro-1 H-beta- carboline
  • phenyl group in the orffto-position at the right hand side of the molecule.
  • R Sa to R 8h , R 9a to R 9h , R 1Oa to R 1Oh and R 11a to R 11h independently represent, at each occurrence,
  • each aryl independently represents a C 6-10 carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from
  • R 12a to R 12h and R 13a to R 13h independently represent, at each occurrence, (a) H,
  • Het 1 to Het 13 independently represent 4- to 14-membered heterocyclic or 5- to 10-membered heteroaromatic groups containing one or more heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may comprise one, two or three rings, and may be substituted by one or more substituents selected from
  • phenyl which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C 1-4 alkyl and C 1-4 alkoxy) or (k) Het b ;
  • R 14a to R 14h and R 14a to R 14h independently represent, at each occurrence
  • n, p, q, r, s, t, u and v independently represent O, 1 or 2;
  • alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups, as well as the alkyl part of alkoxy groups, may be substituted by one or more halo atoms, and (ii) cycloalkyl and cycloalkenyl groups may comprise one or two rings and may additionally be ring-fused to one or two benzene rings;
  • R represents H or C 1-3 alkyl
  • R is not present; R 8a and R 9a independently represent, at each occurrence,
  • each aryl independently represents a C 6-10 carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from
  • R 12a and R 13a independently represent, at each occurrence
  • each of R 1 to R 4 may independently represent H, halo, or C 1-6 alkyl; and
  • R 5 is either not present, or represents one substituent on the fused benzene ring selected from selected from halo or C 1-6 alkyl.
  • each of R 1 to R 4 may independently represent H, halo, or C 1-3 alkyl.
  • each of R 1 to R 4 may independently represent H or halo.
  • R 5 is not present; and R 6 represents H.
  • R 1 to R 4 represent H.
  • R 1 to R 4 represent H; R 5 is not present; R 6 represents H; and R 7 is not present.
  • the compound of Formula I is, or comprises, biphenyl-2-yl-(4,4a,9,9a-tetrahydro-1 H-beta-carbolin-2-yl)-methanone (DE002).
  • the compound of Formula I is not (3-Methoxy-phenyl)-(1 , 3,4,9- tetrahydro-beta-carbolin-2-yl)-methanone (CA199).
  • AJW089 is a fascaplysin analogue that is similar to CA224 but has an unsubstituted (unmethylated) central amido group and an unsubstituted phenyl group in the ortho- position.
  • a fascaplysin analogue that is similar to CA224 but has an unsubstituted (unmethylated) central amido group and an unsubstituted phenyl group in the ortho- position.
  • R 7a to R 7h , R 8a to R 8h and R 9a to R 9h independently represent, at each occurrence, (a) H, (b) C 1-10 alkyl, C 2-10 alkenyl, C 2- i 0 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH 1 C ⁇ alkoxy, aryl and Het 4 ),
  • each aryl independently represents a C 6-I0 carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from (a) halo,
  • phenyl which latter group is optionally susbtituted by one or more substituents selected from OH, halo, Ci -4 alkyl and Ci -4 alkoxy) or G) Het 8 ;
  • R 1Oa to R 1Oh and R 11a to R 11h independently represent, at each occurrence
  • Het 1 to Het 10 independently represent 4- to 14-membered heterocyclic or 5- to 10-membered heteroaromatic groups containing one or more heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may comprise one, two or three rings, and may be substituted by one or more substituents selected from
  • phenyl which latter group is optionally substituted by one or more substituents selected from OH, halo, C 1-4 alkyl and C 1-4 alkoxy) or (e) Het d ; provided that R 12b or R 13b does not represent H when v or w, respectively is 1 or 2;
  • n, p, q, r, s, t, u, v and w independently represent O, 1 or 2;
  • alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups may be substituted by one or more halo atoms, and
  • cycloalkyl and cycloalkenyl groups may comprise one or two rings and may additionally be ring-fused to one or two benzene rings;
  • each of R 1 to R 4 may independently represent H, halo, CN, NO 2 , Ci -12 alkyl, C 1-
  • R 5 is either not present, or represents one to two substituents on the fused benzene ring selected from halo, CN, NO 2 , C 1 - 12 alkyl, C 1-I2 alkenyl, C 1-12 alkynyl, C 3 .-
  • 2 cycloalkyl or C 4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, C 3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, 0, halo, C 1-4 alkyl and Ci -4 alkoxy) or 0R 8a ;
  • R 6 represents H
  • R 7a and R 8a independently represent, at each occurrence
  • each aryl independently represents a C 6-10 carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from (a) halo,
  • R 1 to R 4 represent H, halo, or C 1-6 alkyl
  • R 5 is either not present, or represents one substituent on the fused benzene ring selected from halo or Ci -6 alkyl.
  • each of R 1 to R 4 may independently represent H, halo, or Ci -3 alkyl.
  • each of R 1 to R 4 may independently represent H or halo.
  • R 5 is not present; and R 6 represents H.
  • R 1 to R 4 represent H.
  • R 5 is not present; R 6 represents H;
  • R 7 is not present.
  • the compound of Formula Il is, or comprises, biphenyl-2-carboxylic acid [2-(1 H-indol-3-yl)-ethyl]-amide (AJW089).
  • the compound of Formula Il is not biphenyI-4-carboxylic acid [2-(1 H- indol-3-yl)-ethyl]-amide.
  • Compounds of Formula I and Formula Il (and Formula III as described below) may contain double bonds and may thus exist as E (entalle) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.
  • Compounds of Formula I and Formula Il may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism.
  • Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques.
  • the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e.
  • a 'chiral pool' method by reaction of the appropriate starting material with a 'chiral auxiliary' which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.
  • Ci -q alkyl (where q is the upper limit of the range) as defined herein may be straight-chain or, when there is a sufficient number (i.e. a minimum of three) of carbon atoms, be branched-chain, and/or cyclic (so forming, in the case of alkyl, a C 3-q cycloalkyl group). Further, when there is a sufficient number (i.e. a minimum of four) of carbon atoms, such groups may also be part cyclic. Further, unless otherwise specified, such alkyl groups may also be saturated or, when there is a sufficient number (i.e. a minimum of two) of carbon atoms and unless otherwise specified, be unsaturated (forming, for example, a C 2 . q alkenyl or a C 2 -q alkynyl group).
  • halo when used herein, includes fluoro, chloro, bromo and iodo.
  • aryl groups that may be mentioned include C 6- io aryl groups. Such groups may be monocyclic, bicyclic or tricyclic and have between 6 and 10 ring carbon atoms, in which at least one ring is aromatic. C 6-10 aryl groups include phenyl, naphthyl and the like, such as 1 ,2,3,4-tetrahydronaphthyl, indanyl and indenyl. The point of attachment of aryl groups may be via any atom of the ring system. However, when aryl groups are bicyclic or tricyclic, they are linked to the rest of the molecule via an atom of the aromatic ring.
  • heteroaryl groups that may be mentioned include those which have between 6 and 10 members. Such groups may be monocyclic, bicyclic or tricyclic, provided that at least one of the rings is aromatic and wherein at least one (e.g. one to four) of the atoms in the ring system is other than carbon (i.e. a heteroatom).
  • Heteroaryl groups that may be mentioned include benzimidazolyl, benzodioxanyl, benzodioxepinyl, benzodioxolyl (including 1 ,3-benzodioxolyl), benzofuranyl, benzofurazanyl, benzothiazolyl, benzothiadiazolyl (including 2,1 ,3-benzothiadiazolyl), benzoxadiazolyl (including 2,1 ,3-benzoxadiazolyl), benzoxazinyl (including 3,4-dihydro- 2H-1,4-benzoxazinyl), benzoxazolyl, benzimidazolyl, benzomorpholinyl, benzoselena- diazolyl (including 2,1 ,3-benzoselenadiazolyl), benzothienyl, chromanyl, cinnolinyl, furanyl, imidazolyl, imidazo[1,2-a]pyr
  • heteroaryl groups may, where appropriate, be located on any atom in the ring system including a heteroatom.
  • the point of attachment of heteroaryl groups may be via any atom in the ring system including (where appropriate) a heteroatom (such as a nitrogen atom), or an atom on any fused carbocyclic ring that may be present as part of the ring system.
  • heteroaryl groups when bicyclic or tricyclic, they are linked to the rest of the molecule via an atom of the aromatic ring.
  • Heteroaryl groups may also be in the N- or S- oxidised form. Heteroatoms that may be mentioned include include phosphorus, silicon, boron, tellurium, selenium and, preferably, oxygen, nitrogen and sulfur.
  • Salts which may be conveniently used in therapy include physiologically acceptable base salts, for example, derived from an appropriate base, such as an alkali metal (eg sodium), alkaline earth metal (eg magnesium) salts, ammonium and NX 4 + (wherein X is C 1-4 alky! salts.
  • physiologically acceptable acid salts include hydrochloride, sulphate, mesylate, besylate, phosphate and glutamate.
  • Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a parent compound, e.g. of Formula I or Il (or Formula III as described below) with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of the invention in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
  • prodrugs of compounds of Formula I or Il (or Formula III as described below).
  • prodrug we include compounds that form a compound of Formula I or Il (or Formula III as described below) in an experimentally-detectable amount, within a predetermined time (e.g. about 1 hour), following oral or parenteral administration. All prodrugs of the compounds of Formula I and Formula Il (and Formula III as described below) are included within the scope of the invention.
  • certain compounds of Formula I or Il may possess no or minimal pharmacological activity as such, but may be administered parenterally or orally, and thereafter be metabolised in the body to form compounds of formula I that possess pharmacological activity as such.
  • Such compounds (which also includes compounds that may possess some pharmacological activity, but that activity is appreciably lower than that of the "active" compounds of Formula I or Il (or Formula III as described below) to which they are metabolised), may also be described as "prodrugs".
  • the compounds of Formula I and Formula Il (and Formula III as described below) and salts thereof may be useful because they possess pharmacological activity, and/or are metabolised in the body following oral or parenteral administration to form compounds which possess pharmacological activity.
  • the compounds of Formula I and Formula Il are therapeutic agents which are typically formulated for administration to an individual as a pharmaceutical composition, i.e. together with a pharmaceutically acceptable carrier, diluent or excipient.
  • a third aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula U as defined above in the second aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • pharmaceutically acceptable is included that the formulation is sterile and pyrogen free. Suitable pharmaceutical carriers are well known in the art of pharmacy.
  • the carriers must be "acceptable” in the sense of being compatible with the therapeutic agent and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free; however, other acceptable carriers may be used, including carboxymethyl cellulose, Tween and polyethyleneglycol (PEG).
  • Alternative carriers include nanoparticles and/or lipids/biodegradable polymers (see, Sinha et al (2006) MoI Cancer Ther. 5(8): 1909-17; Yih & Al-fandi (2006) J Cell Biochem. 97(6): 1184-90; Duncan (2006) Nat Rev Cancer. 6(9): 688-701) which may have advantages in overcoming problems of low solubility in the more traditional carriers.
  • the therapeutic agent will generally be formulated for administration in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions or formulations of the invention are for parenteral administration, more particularly for intravenous administration, for example by injection.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the pharmaceutical composition is suitable for topical administration to a patient, i.e. for dermal administration of the therapeutic agent.
  • the therapeutic agent may be formulated for administration orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form.
  • the therapeutic agent can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications.
  • the compound of invention may also be administered via intracavern ⁇ sal injection.
  • Tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
  • Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
  • Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
  • the therapeutic agents may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • the therapeutic agent can also be administered parenterally, for example, intravenously, intra-artehally, intraperitoneally, intrathecally, intraventricular ⁇ , intrastemally, intracranially, intra-musculariy or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques weli-known to those skilled in the art.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition 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 of the kind previously described.
  • the therapeutic agents may be formulated for administration subcutaneously, rectally, nasally, tracheally, bronchially, by any other parenteral route or via inhalation, in a pharmaceutically acceptable dosage form.
  • compositions may be administered at varying doses.
  • the formulation is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient.
  • the daily dosage level of the therapeutic agent will usually be from 0.25 to 25 g per adult (i.e. from about 5 to 250 mg/kg), administered in single or divided doses.
  • CA224 are efficacious at 100 mg/kg in SCID mice, and we expect that DE002 will show efficacy at still lower concentrations. It is interesting to note that since the fascaplysin analogues of the invention, such as DE002 and CA224, are relatively non-toxic, they can be used at higher concentrations than are possible for most other anti-cancer compounds.
  • the tablets or capsules of the therapeutic agent may contain from 0.1, 0.5, 1, 5 or 10 g of therapeutic agent for administration singly or two or more at a time, as appropriate.
  • the physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.
  • the amount of a compound in a dose is typically an "effective amount", by which we mean an amount of the compound which confers a therapeutic effect on the treated patient.
  • the effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).
  • Preferred pharmaceutical formulations include those in which the active ingredient is present in at least 1% (such as at least 10%, preferably in at least 30% and most preferably in at least 50%) by weight. That is, the ratio of active ingredient to the other components (i.e. the addition of adjuvant, diluent and carrier) of the pharmaceutical composition is at least 1:99 (e.g. at least 10:90, preferably at least 30:70 and most preferably at least 50:50) by weight.
  • the physician, veterinarian or skilled person will be able to determine the actual dosage which will be most suitable for an individual patient, which is likely to vary with the route of administration, the type and severity of the condition that is to be treated, as well as the species, age, weight, sex, renal function, hepatic function and response of the particular patient to be treated.
  • the above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • the therapeutic agent may be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetrafluoroethane (HFA 134A3 or 1 ,1 ,1 ,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas.
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetra
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
  • a lubricant e.g. sorbitan trioleate.
  • Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a therapeutic agent and a suitable powder base such as lactose or starch.
  • Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff' contains a clinically-useful dose of the therapeutic agent for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.
  • the therapeutic agent can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder.
  • the therapeutic agents may also be transdermal ⁇ administered, for example, by the use of a skin patch. They may also be administered by the ocular route, particularly for treating cancers of the eye such as retinoblastoma.
  • the therapeutic agent can be formulated as mi ' cronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.
  • a preservative such as a benzylalkonium chloride.
  • they may be formulated in an ointment such as petrolatum.
  • the therapeutic agent can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • the therapeutic agent may be delivered using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections.
  • An example of such a system is the Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.
  • the therapeutic agent may be administered by a surgically implanted device that releases the drug directly to the required site.
  • a surgically implanted device that releases the drug directly to the required site.
  • Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis.
  • the direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.
  • a therapeutic agent is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.
  • a fourth aspect of the invention provides a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, for use in medicine.
  • a fifth aspect of the invention provides a method of treating cancer in a patient, the method comprising administering to the patient a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention.
  • This aspect of the invention also provides the use of a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, in the preparation of a medicament for treating cancer in a patient.
  • This aspect of the invention also provides a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, for use in treating cancer in a patient.
  • CA224 surprisingly beneficial properties of CA224 that would lead the skilled person to consider CA224 and analogues thereof to be suitable for use in treating cancer.
  • IC 50 for Cdk4-cyclin D1 5.5 ⁇ M.
  • CA224 inhibits these Cdk-s with an IC 50 greater than 500 ⁇ M, i.e. CA224 is highly selective for Cdk4-cyclin D1.
  • CA224 The dual mechanism of action of CA224 proved crucial for the block of growth at the G 0 ZG 1 phase of the cell cycle of cancer cells (Calu-1) with impaired mitotic spindle checkpoint.
  • the G 0 /Gi block seen in Calu-1 cells is imposed as a consequence of Cdk4-cyclin D1 inhibition. It is very likely that the G 0 /Gi arrest is a result of the prevention of pRb phosphorylation at Cdk4-specific serine residues, Ser780, Ser795 and Ser807/811.
  • CA224 induces massive apoptosis in cancer cells which are known to be resistant to chemotherapy and significantly reduces the colony formation efficiency (in a dose-dependent manner) of the lung cancer cells, A549 and Calu-1.
  • CA224 may be a useful compound for the treatment of cancer, or a lead compound for the development of an improved anticancer agent.
  • CA224 is a fascaplysin analogue that has an unsubstituted phenyl group in the ortho- position. Without wishing to be bound by theory, we propose that this feature in a fascaplysin analogue is required for the activity of CA224 and, together with a central amido group having a substituent other than H (methyl group preferred), defines the CA224 series of compounds (Formula I, below).
  • a sixth aspect of the invention provides a method of treating cancer in a patient, the method comprising administering to the patient a compound of Formula III
  • R 7 represents C 1-6 alkyl
  • R 8a to R 8h , R 9a to R 9h and R 1Oa to R 10h independently represent, at each occurrence, (a) H, (b) C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C 1-6 alkoxy, aryl and Het 5 ),
  • each aryl independently represents a C 6-10 carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from (a) halo, (b) CN,
  • phenyl which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C 1-4 alkyl and C 1-4 alkoxy) or G) Het 9 ;
  • R 11a to R 11h and R 12a to R 12h independently represent, at each occurrence
  • phenyl which latter group is optionally substituted by one or more substituents selected from OH, CN, halo, C 1 ⁇ 3 alkyl and C 1-6 alkoxy) or
  • Het 1 to Het 11 independently represent 4- to 14-membered heterocyclic or 5- to 10-membered heteroaromatic groups containing one or more heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may comprise one, two or three rings, and may be substituted by one or more substituents selected from
  • N(R 13e )S(O) 2 R 13f , N(R 139 )(R 13h ), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, C 1-4 alkyl and C 1-4 alkoxy) and Hef, and which C 3-12 cycloalkyl or C 4-I2 cycloalkenyl groups may additionally be substituted by 0, (d) 0R 14a ,
  • phenyl which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C 1-4 alkyl and C 1-4 alkoxy) or (k) Het b ;
  • R 13a to R 13h and R 14a to R 14h independently represent, at each occurrence
  • n, p, q, r, s, t, u, v and w independently represent O, 1 or 2;
  • alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups, as well as the alkyl part of alkoxy groups, may be substituted by one or more halo atoms, and (ii) cycloalkyl and cycloalkenyl groups may comprise one or two rings and may additionally be ring-fused to one or two benzene rings;
  • each of R 1 to R 4 may independently represent H, halo, CN, NO 2 , C 1-12 alkyl, C 1- 12 alkenyl, C 1-12 alkynyl, C 3-I2 cycloalkyl or C 4- - I2 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C 1 .
  • R 6 represents H
  • R 7 represents methyl, ethyl, propyl or isopropyl
  • R 8a and R 9a independently represent, at each occurrence, (a) H, (b) C- I-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, Ci -6 alkoxy and aryi), or
  • each aryl independently represents a C 6-I0 carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from
  • R 11a and R 12a independently represent, at each occurrence, (a) H,
  • each of R 1 to R 4 may independently represent H, halo, or C 1-6 alkyl; and R 5 is either not present, or represents one substituent on the fused benzene ring selected from selected from halo or Ci -6 alkyl.
  • each of R 1 to R 4 may independently represent H, halo, or Ci- 3 alkyl.
  • each of R 1 to R 4 may independently represent H or halo.
  • R 5 is not present; R 6 represents H; and R 7 represents methyl or ethyl.
  • R 1 to R 4 represent H.
  • R 1 to R 4 represent H; R 5 is not present; R 6 represents H; and R 7 is methyl.
  • the compound of Formula III is, or comprises, biphenyl-4-carboxylic acid [2-(1 H-indol-3-yl)-ethyl]-methyl-amide (CA224).
  • This aspect of the invention includes the use of a compound of Formula III as described above in the preparation of a medicament for use in treating cancer in a patient.
  • the invention also includes a Formula III as described above for use in treating cancer in a patient.
  • the patient to be treated may be any individual who would benefit from such treatment.
  • the patient to be treated is a human.
  • the methods and compositions of the invention may be used to treat cancer in mammals including agriculturally important animals such as cows, horses, pigs and sheep as well as domestic pets such as cats and dogs.
  • the methods have uses in both human and veterinary medicine. Due to the multiple sites of action within the cell cycle, a large range of cancers are suitable for treatment by the compounds of Formula I, Il and III, including breast cancer, colorectal cancer, pancreatic cancer, lung cancer, myeloma, glioblastoma or retinoblastoma.
  • Cancer is caused by multi-genetic events (mis-regulation of a single gene rarely, if ever, causes cancer). Therefore, in order for a cancer to be curable, multiple genes/proteins should be targeted. Unlike Cdk4-specific inhibitors or other Cdk-specific inhibitors, the compounds of Formula I 1 Il and III have multiple modes of anticancer activity.
  • Cdk4 inhibitor refractory tumours are well known in the art and include drug-resistant tumours.
  • flavopiridol a pan-Cdk inhibitor which potently inhbits Cdk4
  • flavopiridol itself causes resistance in cancer cells - possibly through upregulation of the telomerase catalytic subunit (Incles et al (2003) MoI Pharmacol. 64(5): 1101-8).
  • Cdk4 inhibitors exert their effect via regulation of pRb, and are not suitable for the treatment of pRb-negative (pRb-mutant or pRb-null) tumours.
  • compounds of Formula I 1 Il and III are effective against Rb-negative cancer cells in vitro. Accordingly, we now consider that compounds of Formula I, Il and III are therapeutically effective in treating pRb-negative cancers. Rb loss is known to occur in many tumour types.
  • a cancer that is pRb-negative we include a cancer that has a high proportion of cells that are pRb-mutant or pRb-null.
  • a cancer that is pRb-negative has at least 50%, or at least 60%, or at least 70%, preferably at least 80%, more preferably at least 85%, yet more preferably at least 90%, and still more preferably at least 95% of cancer cells that are pRb-mutant or pRb-null.
  • a cancer that is pRb-negative has at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.9%, or more of cancer cells that are pRb-mutant or pRb-null. Most preferably, all of the cancer cells in a cancer that is pRb-negative are pRb-mutant or pRb-null.
  • pRb-mutant we mean that the cancer cell does not produce any functional Rb protein or does so at undetectable levels.
  • the function of pRb is the ability to act as a repressor of genes required for G-
  • cancer that is pRb-null we mean that the cancer cell does not produce any Rb protein because it lacks a functional RB gene.
  • a cancer cell may be Rb-mutant or Rb-null
  • Rb-mutant or Rb-null may be due to either insertional, deletional or point mutational events in the Rb gene.
  • the cell is Rb-mutant or Rb-null.
  • Point mutational events may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the Rb gene product.
  • the method comprises the prior step of determining the Rb status of the cancer before administering the compound of Formula I, II, or III to the patient.
  • this embodiment of the invention provides a method of treating Rb- negative cancer in a patient, the method comprising: determining that the cancer is Rb-negative; and administering the compound of Formula I, II, or III to the patient.
  • the invention includes the use of a compound of Formula I, II, or III in the preparation of a medicament for treating cancer in a patient who has previously been determined to have Rb-negative cancer.
  • the cancer to be treated may be a p53+ cancer.
  • All types of cancers may contain both p53-positive (p53+) and p53-negative cells.
  • the p53-negative cells are malignant and are untreatable.
  • all cancers have the potential of becoming p53-negative (p53-null or p53-mutant). Even if a single cell in a cancer was to be p53-negative, and if it was not treatable, then the cancer will not be cured.
  • the cancer to be treated may be a p53-negative cancer, whether p53-null tumours or containing point mutations in the p53 gene (p53- mutant).
  • point mutations in p53 have been found in the majority of cancers, albeit at varying frequencies. Mutations are found in only 10-20% of leukaemias, prostate cancers or hepatocarcinomas, but in as many as 60-70% of ovarian, bladder, head and neck, colon and lung carcinomas. Overall it is estimated that as many as 40-50% of all cancers harbour p53 mutations.
  • p53 inactivation occurs via epigenetic mechanisms including p53 protein degradation in cervical carcinoma mediated by human papilloma virus (HPV) and degradation/inactivation of p53 by mdm-2 in soft tissue sarcomas (Fojo (2002) "p53 as a therapeutic target: unresolved issues on the road to cancer therapy targeting mutant p53" Drug Resistance Updates 5: 209-216).
  • a cancer that is p53-negative we include a cancer that has a high proportion of cells that are p53-mutant or p53-null.
  • a cancer that is p53-negative has at least 50%, or at least 60%, or at least 70%, preferably at least 80%, more preferably at least 85%, yet more preferably at least 90%, and still more preferably at least 95% of cancer cells that are p53-mutant or p53-null.
  • a cancer that is p53-negative has at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.9%, or more of cancer cells that are p53-mutant or p53-null.
  • all of the cancer cells in a cancer that is p53-negative are p53-mutant or p53-null.
  • a cancer cell that is p53-mutant we mean that the cancer cell does not produce any functional p53 protein or does so at undetectable levels.
  • the function of p53 is the ability to act as a repressor of p21.
  • p21 function can be measured using the p21 promoter linked to a reporter gene.
  • cancer that is p53-null we mean that the cancer cell does not produce any p53 protein because of the lack of a functional p53 gene.
  • the method comprises the prior step of determining the p53 status of the cancer before administering the compound of Formula I 1 II, or III to the patient.
  • this embodiment of the invention provides a method of treating p53- negative cancer in a patient, the method comprising: determining that the cancer is p53-negative; and administering a compound of Formula I, II, or III to the patient.
  • the invention thus includes the use of a compound of Formula I 1 II, or III in the preparation of a medicament for treating cancer in a patient who has previously been determined to have p53-negative cancer.
  • a cancer cell may be p53-mutant or p53-null may be due to either insertional, deletional or point mutational events in the p53 gene.
  • the cell is p53-mutant or p53-null.
  • Point mutational events may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the p53 gene product.
  • determining whether a cancer cell is p53-negative or positive or Rb-negative or positive is carried out by RT-PCR on a sample of the cancer tissue or cancer cells using methods that are now well known in the art.
  • Messenger RNA mRNA
  • RT-PCR using primers specific for the 5' and the 3'-ends of RB or p53 genes is used to isolated cDNA that encodes RB and p53.
  • Tissues which harbour null mutations i.e. cells which contain homozygous deletions of the genes
  • will yield no PCR product whereas a product would be obtained from a control tissue that harbours either the wild-type RB or the p53 gene.
  • a tissue harbouring point mutations in the gene will yield a gene product.
  • DNA sequencing confirms inactivating mutations in the genes. Typically, this is followed by sequencing the p53 or Rb transcript to look for inactivating mutations in the genes.
  • inactivating mutations in the p53 or Rb transcript can be detected using any of the other ways well known in the art. By this route, the person of skill in the art can determine whether a cancer expresses any p53 or Rb, and whether any p53 or Rb thus expressed is functional (i.e. whether the cancer is p53- and/or pRb- negative or positive, as defined).
  • Cdk inhibitors when used in combination with conventional cytotoxic drugs, potentiate cell death in a number of tumour models (Tenzer and
  • Cdk inhibitors have been used in combination with several other DNA damaging agents and chemotherapeutic agents like cisplatin, 5- flurouracil, paclitaxel, mitomycin C, doxorubicin and gemcitabine. Highly encouraging outcomes from these different drug combination experiments have been observed in clinical studies. Synergistic effects against tumour growth, sensitization of tumour cells, potentiation of apoptosis and increased cell death are the main effects found after
  • Cdk inhibitors are used in combination with other anticancer molecules (Lara et al,
  • a seventh aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (i) a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, or a compound of Formula III as defined above in the sixth aspect of the invention, and (ii) a further anticancer agent, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the further anticancer agent may be selected from alkylating agents including nitrogen mustards such as mechlorethamine (HN 2 ), cyclophosphamide, ifosfamide, melphalan (L- sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonates such as busulphan; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin
  • nitrogen mustards such as mechlorethamine (HN 2 ), cyclophosphamide, ifosfamide, melphalan (L- sarcolysin) and chlorambucil
  • ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa
  • alkyl sulphonates such as
  • streptozotocin and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazole- carboxamide); antimetabolites including folic acid analogues such as methotrexate
  • pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); and purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2'-deoxycoformycin); natural products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymes such as L-asparaginase; and
  • the clinically used anticancer agents are typically grouped by mechanism of action: Alkylaying agents, Topoisomerase I inhibitors, Topoisomerase Il inhibitors, RNA/DNA antimetabolites, DNA antimetabolites and Antimitotic agents.
  • Alkylaying agents Topoisomerase I inhibitors
  • Topoisomerase Il inhibitors Topoisomerase Il inhibitors
  • RNA/DNA antimetabolites DNA antimetabolites
  • Antimitotic agents The US NIH/National Cancer Institute website lists 122 compounds
  • Alkylating agents including Asaley, AZQ, BCNU, Busulfan, carboxyphthalatoplatinum, CBDCA, CCNU 1 CHIP, chlorambucil, chlorozotocin, c/s-platinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, teroxirone, tetraplatin, thio-tepa, triethylenemel
  • RNA/DNA antimetabolites including L-alanosine, 5-azacytidine, 5-fluorouracil, acivicin, 3 aminopterin derivatives, an antifol, Baker's soluble antifol, dichlorallyl lawsone, brequinar, ftorafur (pro-drug), 5,6-dihydro-5-azacytidine, methotrexate, methotrexate derivative, N-(phosphonoacetyl)-L-aspartate (PALA), pyrazofurin, trimetrexate;
  • DNA antimetabolites including, 3-HP, 2'-deoxy-5-fluorouridine, 5-HP, alpha-TGDR, aphidicolin glycinate, ara-C, 5-aza-2'-deoxycytidine, beta-TGDR, cyclocytidine, guanazole, hydroxyurea, inosine glycodialdehyde, macbecin II, pyrazoloimidazo
  • the further anticancer agent is selected from cisplatin, carboplatin, 5- flurouracil, paclitaxel, mitomycin C, doxorubicin, gemcitabine, Velcade ® , Glivec ® , COX- 2 inhibitors and mitoxantrone. Indeed, Velcade ® , Glivec ® and COX-2 inhibitors are currently being used as combinations in clinical trials in conjunction with Cdk inhibitors.
  • An eighth aspect of the invention provides (i) a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, or a compound of Formula III as defined above in the sixth aspect of the invention, and (ii) a further anticancer agent, for use in medicine.
  • a ninth aspect of the invention provides a method of treating cancer in a patient, the method comprising administering to the patient (i) a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, or a compound of Formula III as defined above in the sixth aspect of the invention, and (ii) a further anticancer agent as defined above in the seventh aspect of the invention.
  • the method comprises administering to the patient a pharmaceutical composition as defined above in the seventh aspect of the invention.
  • a pharmaceutical composition as defined above in the seventh aspect of the invention.
  • the compound of Formula I, Il or III and the further anticancer agent may be administered separately.
  • the compound of Formula I, Il or III and the further anticancer agent can be administered sequentially or (substantially) simultaneously. The may be administered within the same pharmaceutical formulation or medicament or they may be formulated and administered separately.
  • This aspect of the invention includes the use of (i) a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, or a compound of Formula III as defined above in the sixth aspect of the invention, and (ii) a further anticancer agent as defined above in the seventh aspect of the invention, in the preparation of a medicament for treating cancer in a patient.
  • the invention also includes the use of a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, or a compound of Formula III as defined above in the sixth aspect of the invention, in the preparation of a medicament for treating cancer in a patient who is administered a further anticancer agent as defined above in the seventh aspect of the invention.
  • a further anticancer agent as defined above in the seventh aspect of the invention.
  • the patient is administered the further anticancer agent at the same time as the medicament, although the patient may have been (or will be) administered the further anticancer agent before (or after) receiving the medicament containing the compound of Formula I 1 Il or III.
  • invention further includes the use of a further anticancer agent as defined above in the seventh aspect of the invention in the preparation of a medicament for treating cancer in a patient who is administered a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, or a compound of Formula Ui as defined above in the sixth aspect of the invention.
  • the patient is administered the compound of Formula I, Il or III at the same time as the medicament, although the patient may have been (or will be) administered the compound of Formula I 1 Il or III before (or after) receiving the medicament containing the further anticancer agent.
  • the invention also includes (i) a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, or a compound of Formula III as defined above in the sixth aspect of the invention, and (ii) a further anticancer agent as defined above in the seventh aspect of the invention, for use in treating cancer in a patient.
  • a tenth aspect of the invention provides a method of identifying an anticancer agent, or a lead compound for the identification of an anticancer agent, the method comprising: providing a candidate compound which is a compound of Formula IV;
  • R 9a to R 9h , R 1Oa to R 1Oh , R 11a to R 11h and R 12a to R 12h independently represent, at each occurrence,
  • each aryl independently represents a C 6-10 carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from
  • phenyl which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C 1-4 alkyl and C 1-4 alkoxy) or G) Het 11 ;
  • R 13a to R 13h and R 14a to R 14h independently represent, at each occurrence
  • Het 1 to Het 13 independently represent 4- to 14-membered heterocyclic or 5- to
  • phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C 1-4 alkyl and C 1-4 alkoxy) or
  • n, p, q, r, s, t, u and v independently represent O, 1 or 2;
  • alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups may be substituted by one or more halo atoms, and
  • cycloalkyl and cycloalkenyl groups may comprise one or two rings and may additionally be ring-fused to one or two benzene rings;
  • Candidate compounds of Formula IV that could be mentioned include compounds wherein R 1 represents phenyl.
  • the candidate compound of Formula IV is a compound of Formula I, as defined in the first aspect of the invention.
  • the candidate compound of Formula IV is not (3-Methoxy-phenyl)- (1 ,3,4,9-tetrahydro-D-carbolin-2-yl)-methanone (CA199).
  • An eleventh aspect of the invention provides a method of identifying an anticancer agent, or a lead compound for the identification of an anticancer agent, the method comprising: providing a candidate compound which is a compound of Formula V
  • R 9a to R 9h , R 1Oa to R 10h , R 11a to R 11h , R 12a to R 12h and R 13a to R 13h independently represent, at each occurrence,
  • each aryl independently represents a C 6-10 carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from
  • phenyl which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C 1-4 alkyl and C 1-4 alkoxy) or
  • R 14a to R 14h and R 15a to R 15h independently represent, at each occurrence
  • Het 1 to Het 8 independently represent 4- to 14-membered heterocyclic or 5- to 10-membered heteroaromatic groups containing one or more heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may comprise one, two or three rings, and may be substituted by one or more substituents selected from (a) halo,
  • n, p, q, r, s, t, u, v and w independently represent O, 1 or 2;
  • alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups, as well as the alkyl part of alkoxy groups, may be substituted by one or more halo atoms, and (ii) cycloalkyl and cycloalkenyl groups may comprise one or two rings and may additionally be ring-fused to one or two benzene rings; and determining whether the candidate compound exhibits at least one anticancer activity which is not dependent upon Cdk4 inhibition.
  • Candidate compounds of Formula V that can be mentioned include those compounds wherein R 1 represents phenyl.
  • the candidate compound of Formula V is a compound of Formula Il as defined in the second aspect of the invention.
  • the compound of Formula V is not biphenyl-4-carboxylic acid [2- ( 1 H-indol-3-yl)-ethyl]-amide.
  • the candidate compound of Formula V is a compound of Formula III as defined in the sixth aspect of the invention.
  • the compound of Formula V is not biphenyl-4-carboxylic acid [2- (1 H-indol-3-yl)-ethyl]-methyl-amide (CA224).
  • screening assays which are capable of high throughput operation are particularly preferred.
  • the candidate compound may be a drug-like compound or lead compound for the development of a drug-like compound.
  • drug-like compound is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament.
  • a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, which may be of less than 5000 Daltons, and which may be water-soluble.
  • a drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes or the blood.brain barrier, but it will be appreciated that these features are not essential.
  • lead compound is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
  • the identified compound may be modified, and the modified compound tested for at least one anticancer activity which is not dependent upon Cdk4 inhibition.
  • the at least one anticancer activity is selected from:
  • Blocking cells at the G 2 M phase of the cell division cycle Preferably greater than 40% of cells should maintain a block at G 2 /M (Stark & Taylor (2006) MoI
  • the cells are synchronised either post G 0 /G 1 at d/S (using hydroxyurea, mimosine or thymidine as blocking agents at G 1 ZS) or G 2 /M (using nocodazole or paclitaxel as blocking agents at G 2 /M), and, after release of cells from synchrony, the ability of the candidate compound to effect cell block at G 2 /M is assessed.
  • G 1 ZS in the presence of a candidate compound having the desired ability
  • cells proceed forward in the cell cycle and block at G 2 ZM.
  • the cells remain at G 2 ZM and do not proceed further through the cell cycle.
  • H 2 DCF diacetate H 2 DCFDA
  • Cancer cells i.e. colon cancer LS174T, non-small cell cancer A549 cells
  • a density of 20,000 cellsZ well in a 96-well tissue culture grade black-well plate are seeded at a density of 20,000 cellsZ well in a 96-well tissue culture grade black-well plate.
  • Cells are allowed to recover for 24h at 37 0 C in a humidified CO 2 incubator. Cells are incubated with different concentrations of the candidate compound (e.g., 2.5- 80 ⁇ M) for 60 min. The compound is removed, cells are briefly washed with PBS, and 10 ⁇ M H 2 DCFDA in PBS is added. The plate is incubated at 37°C in the CO 2 incubator and after 30 min H 2 O 2 is added at a final concentration of 500 ⁇ M. The oxidation of intracellular non-fluorescent H 2 DCF to highly fluorescent DCF is measured using a fluorimeter (Bio-Tek) using an excitation of 485 nM and emission of 528 nM.
  • the candidate compound e.g., 2.5- 80 ⁇ M
  • Blank values indicating the fluorescence of the dye in PBS are subtracted from all samples.
  • the % of control is calculated in comparison with fluorescence exhibited by cells in presence of H 2 O 2 alone.
  • 50,000 cells/well of H4IIE rat hepatoma cells may be seeded in a 96-well microtitre plate. The cells are allowed to attach and the medium changed. Cells are first incubated with different concentrations of the test compound for 60 min, then the medium containing the compound is removed and the cells washed with PBS twice and fresh medium added. H 2 DCFDA is added at a final concentration of 5 ⁇ M and the cells are incubated further for 30 min.
  • the oxidation of intracellular non-fluorescent H 2 DCF to highly fluorescent DCF can be measured after addition of H 2 O 2 (500 ⁇ M) at an excitation of 485 nm and an emission of 525 nm to measure the inhibition by the test compound of H 2 O 2 mediated ROS formation.
  • cancer cells may be seeded at a density of 100,000-150,000 cells/well in a 6-well tissue culture grade plate. Cells may be treated in the same way as mentioned above and a qualitative reduction in fluorescence in compound-treated cells may be assessed using fluorescence microscopy (Olympus BH Series).
  • the screening methods may also comprise the additional step of determining the solubility of the identified compound or modified compound in one or more clinically acceptable solvents. Identification of the optimal solvent would be especially useful.
  • Suitable test solvents include phospholipids and nanoparticle technology as discussed above. Many other suitable test solvents are known in the art (see, for example, Remington: The Science and Practice of Pharmacy, 19 th Edition (1995), ISBN: 0-912734-04-3).
  • the identified compound or the modified compound is further tested for the ability to inhibit the growth of cancer cells in vitro.
  • Various approaches are known in the art for conducting a cell proliferation assay. For example, trypan blue is a simple way to evaluate cell membrane integrity and thus isolate viable cells which may be counted directly using light microscopy.
  • the uptake and incorporation of radioactive substances usually tritium-labelled thymidine, or bromodeoxyuridine, could be used as an indicator of the number of viable cells.
  • cell proliferation may be assayed using the colorimetric MTT assay which relies on the reduction of a tetrazolium component MTT by the mitochondria of viable cells.
  • paclitaxel paclitaxel, doxorubicin, cisplatin and multi-drug resistant lines
  • the identified compound or the modified compound is further tested for the ability to reduce the efficiency of colony formation of cancer cells in vitro. This may be used to assess the effect of the candidate compounds on the long term survival of cancer cells.
  • a colony formation assay may be invaluable to understand the anticancer potential of a candidate compound (Wu Wei et al, 1983) since the loss of a cancer cell's ability to form a colony could possibly indicate its permanent exit from the cell cycle.
  • Any colony formation protocol known in the art may be used in the context of the present invention (see, for example, Gohji et al (1987) J Urol. 137(3): 539-43). For example, a typical method of performing a colony formation assay is follows.
  • Cells are plated at a concentration of 500 cells per well in 2 ml of complete medium in 35 mm or 6-well plates. The plates are incubated for a 24 h stabilization period and further incubated with different concentrations of candidate compound for 24 h. Plates are then gently washed with PBS, replaced with fresh complete medium and incubated at 37 0 C. After 10-12 days of incubation (when the colonies are visible), the plates are gently washed with PBS and the cell colonies fixed in methanolracetic acid (2:1) fixative for 20 mi ⁇ at room temperature. The plates are washed 2X with 2 ml of distilled water per well and then air dried for 15 min.
  • Suitable cancer cell lines include non-small cell lung cancer, pancreatic cancer, colon cancer, prostate cancer, breast cancer and myeloma-derived cell lines.
  • the screening methods preferably also comprise the further step of assessing the identified compound or modified compound for efficacy in an assay of angiogenesis, tumour invasion and/or cell migration. Additionally, the screening methods may also comprise the further step of assessing the identified compound or modified compound in pharmacokinetic, pharmacodynamic and toxicological studies in animal models.
  • a number of assays for determining the ability of a candidate compound to inhibit angiogenesis are commercially available. Both in vitro and in vivo assays are well known in the art and are described, for example, by Auerbach et a/ (2003).
  • Examples include the in vivo Matrigel plug and corneal neovascularisation assays, in vivo or in vitro chick chorioallantoic membrane (CAM) assays, in vitro cellular (proliferation, migration, tube formation) and organotypic (aortic ring) assays, and the chick aortic arch and Matrigel sponge assays.
  • CAM chick chorioallantoic membrane
  • cellular proliferation, migration, tube formation
  • organotypic (aortic ring) assays and the chick aortic arch and Matrigel sponge assays.
  • the screening methods preferably also comprise the further step of assessing the identified compound or modified compound for efficacy in an animal model of cancer.
  • the model may be a model for any of the cancers mentioned above.
  • the animal model of cancer is a mouse model.
  • the animal model of cancer may be a model of an Rb-negative cancer.
  • the model may be a xenograft or allograft model (Voskoglou-Nomikos et a ⁇ (2003) Clinical Cancer Research 9: 4227-4239).
  • the screening methods of the invention may further comprise the step of synthesising, purifying and/or formulating the identified compound.
  • the invention also includes a method for preparing an anticancer compound of Formula IV or Formula V, the method comprising identifying a compound using the screening methods described above and synthesising, purifying and/or formulating the identified compound.
  • the invention includes a method of making a pharmaceutical composition comprising the step of mixing the compound identified using the screening methods described above with a pharmaceutically acceptable carrier.
  • FIG. 1 Systematic representation of the cell division cycle. This figure shows various cyclin dependent kinases and their respective cyclin partners, their positions at different phases of the cell division cycle, where they function and where their activities are required for the controlled progression of the cell cycle.
  • Fascaplysin analogues were made by releasing bonds a, b and changing double bond c into a single bond leading to derivatives of the four series of compounds (see, Aubry et al, 2004; Aubry et al, 2005; Aubry et al, 2006).
  • Figure 3 Representative histogram of a flow cytometric analysis.
  • the histogram obtained after the FACS analysis of NCI-H 1299 asynchronous cells shows the percentage of cells at the different phases of a normal cell cycle.
  • Figure 4 Representative standard curve of Bradford protein assay showing linear increase in absorbance with increase in BSA concentration.
  • FIG. 5 Coomassie blue staining of Cdk enzymes purified on glutathione- agarose beads.
  • the gel was loaded with lysates from cells co-infected with Cdk4/GST-cyclin D1 (lane 1 ), purified Cdk4/GST-cyclin D1 (lane 2; a distinct band of size 52 kD indicates GST-cycli ⁇ D1), lysates from cells co-infected with Cdk2/GST- cyclin A (lane 3), purified Cdk2/GST-cyclin A (lane 4; a distinct band of size 73 kD indicates GST-cyclin A), lysates from cells co-infected with Cdk2/GST-cyclin E (lane 5), purified Cdk2/GST-cyclin E (lane 6; a distinct band of size 68 kD indicates GST-cyclin E), lysates from cells co-infected with His-Cdk4/GST-cyclin D1 (lane 7),
  • FIG. 6 Western blot analyses of purified Cdk enzymes.
  • Cdk4/GST-cyclin D1, Cdk2/GST-cyclin A, His-Cdk2/GST-cyclin E and His-Cdk4/GST-cyclin D1 were purified on glutathione agarose beads. 30 ⁇ g of each sample was loaded on 10% agarose gel in the lanes indicated in the figure above. The resolved proteins were transferred on Immobilon-P membrane and probed with specific antibodies for Cdk4, Cdk2, cyclin D1 and cyclin A.
  • Western analyses confirmed the presence of Cdks and cyclins in the holoenzyme complexes of the active enzymes
  • Figure 7. The effect of Cdk enzyme concentrations on rates of reaction by monitoring ATP depletion. Above graphs show that ATP depletion increases with increasing concentrations of Cdk enzymes, thus increasing the fold difference in relative light units (RLU) between the control and blank reactions.
  • RLU relative light units
  • Figure 8 IC 50 determination of flavopiridol, fascapfysin, CINK4 and indirubin 5 suiphonic acid in the in vitro Cdk4-cyclin D1 enzyme assay.
  • the percent enzyme inhibition values represent the mean and standard deviation (from the mean) from three independent experiments.
  • Figure 9 IC 50 determination of flavopiridol, fascaplysin, roscovitine and indirubin 5 suiphonic acid in the in vitro Cdk2-cyclin A enzyme assay.
  • the percent enzyme inhibition values represent the mean and standard deviation (from the mean) from three independent experiments.
  • Figure 10 IC 50 determination of flavopiridol, fascaplysin, roscovitine and indirubin 5 suiphonic acid in the in vitro Cdk2-cyclin E enzyme assay.
  • the percent enzyme inhibition values represent the mean and standard deviation (from the mean) from three independent experiments.
  • Figure 13 FACS analysis of serum-starved Calu-1 cells released in the presence of CA199 and Western blot analysis of proteins from asynchronous Calu-1 cells treated with DE002. The results support the inhibition of the Cdk4-cyclin D1 enzyme at the cellular level in the mitotic spindle checkpoint-deficient Calu-1 cells after treatment with DE002.
  • Figure 13A shows FACS analyses of untreated or control cells (A), serum-starved cells for 24 h (B), serum-starved ceils released in the presence of DE002 at the IC 50 concentration for 24 h (C), and serum-starved cells released in the presence of DE002 at the IC 7O concentration for 24 h (D).
  • FIG. 13B shows on Western blots the status of pRb phosphorylation in Cdk4-specific serine residues after a 24 h treatment of asynchronously growing Calu-1 cell with DE002.
  • the phospho-specific polyclonal antibodies Ser780, Ser795 and Ser807/811 detect pRb phosphorylated at Serine residues 780, 795 and 807/811 respectively.
  • the monoclonal antibody against pRb (4H) detects the total phosphorylated and unphosphorylated pRb protein.
  • Figure 13B shows proteins from untreated cells (C), from cells treated with IC 50 concentration of fascaplysin, 24 h (T1 ), from cells treated with IC 50 concentration of DE002, 24 h (T2), and from cells treated with IC 70 concentration of DE002, 24 h (T3).
  • Figure 14 FACS analysis showing that DE002 does not prevent late G1- b
  • Figure 15 Flow cytometric analysis of asynchronous A549 and NCI-H1299 (mitotic spindle checkpoint-proficient, human non-small cell lung carcinoma (NSCLC) cell lines.
  • DE002 treatment resulted in the majority of cells undergoing cell cycle blockage in the G 2 ZM phase of the cell cycle (4n DNA content) in both cell lines.
  • the Figure shows untreated A549 cells as control (A); A549 cells after treatment with IC 50 concentration of DE002 for 24 h (B); A549 cells after treatment with IC 70 concentration of DE002 for 24 h (C); untreated NCI-H 1299 cells as control (D); NCI- HI 299 cells after treatment with IC 50 concentration of DE002 for 24 h (E).
  • FIG. 16 FACS analysis of NCI-H358 NSCLC cells, synchronised at G 2 /M and G 1 ZS and released in the presence of DE002.
  • Nocodazole and hydroxyurea were used to synchronize NCI-H358 cells at the G 2 ZM and G 1 ZS phases of the cell division cycle.
  • Synchronised cells were released either in fresh medium or in fresh medium containing IC 50 concentration of DE002. After release from block in the presence of DE002, cells either maintain the block at G 2 /M (after release from the nocodazole block) or move forward in the cell cycle and then get blocked later at the G2ZM phase (after release from the hydroxyurea block).
  • the figure shows untreated cells as control (A); cells treated with 1 ⁇ M the nocodazole for 18 h (B); cells treated with 1 ⁇ M nocodazole for 18 h and released in fresh medium (C); cells treated with 1 ⁇ M nocodazole for 18 h and released in the presence of [IC 50 ] DE002, 12 h (D).
  • the figure shows untreated cells as control (E); cells treated with 250 ⁇ M hydroxyurea for 18 h (F); cells treated with 250 ⁇ M hydroxyurea for 18 h and released in fresh medium (G); cells treated with 250 ⁇ M hydroxyurea for 18 h and released in the presence of [IC 50 ] DE002, 18 h (H).
  • DE002 selectively kills SV40-transformed cells as revealed by FACS analysis.
  • DE002 treatment of BNL CL2 (mouse embryonic normal hepatic) cells for 48 h at IC 50 and IC 70 concentrations resulted in prominent G2/M arrest with less than 8% cells showing apoptosis. This can be seen through comparison of histograms (A, B and C) in the upper panel of the figure.
  • BNL SV A.8 mouse embryonic SV40 transformed hepatic cells; upper panel of the figure underwent apoptotic cell death upon incubation with DE002.
  • the levels of apoptosis are depicted as the percentage of cells appearing in the SUb-G 1 peak. 43% and 51 % of cells were found in the sub-G1 peak after 48 h exposure to [IC 50 ] DE002 (E) and [IC 70 ] DE002 (F).
  • the untreated control cells (D) do not show any cells in the SUb-G 1 peak.
  • FIG. 18 Selective killing of SV40-transformed mouse embryonic hepatic cells.
  • BNL CL2 (normal) and BNL SV A.8 (SV40-transformed) cells were incubated with different concentrations of DE002 for 48 h. Apoptosis was selectively seen only in SV40-transformed cells but not in the parent line which is the normal counterpart.
  • A The left hand graph shows percentage cell death measured by the trypan blue dye exclusion method in the two cell lines (BNL CL2 and BNL SV A.8) after treatment with increasing concentrations of DE002 for 48 h.
  • the right hand graph shows cell growth inhibition of BNL CL2 and BNL SV A.8 cells after treatment with DE002 for 48 h, as measured by the MTT assay.
  • B Fluorescence microscopic pictures of DAPI-stained cells captured at 40X magnification. Untreated BNL SV A.8 cells (a); BNL SV A.8 cells treated with [IC 50 ] of DE002 for 48 h (b); Untreated BNL CL2 cells (c); BNL CL2 cells treated with [IC 50 ] of DE002 for 48 h (d). A minimum of 500 nuclei were counted for each sample. The fragmented nuclei and apoptotic cells are indicated with arrows Figure 19. DE002 does not intercalate with pBlueScript plasmid DNA.
  • topoisomerase I catalysed DNA unwinding assay and compared with the results obtained using fascaplysin and the DNA-intercalating agent, camptothecin.
  • the unwinding/relaxation assays were carried out as described.
  • the final products of topoisomerase I relaxation assay were resolved on a 0.8% agarose gel and were stained with 0.5 mg/ml ethidium bromide in order to visualise with UV illumination.
  • Lane 1 contained the control pBlueScript DNA showing the supercoiled form.
  • Lane 2 contained the control relaxation reaction with topoisomerase I enzyme without any compound.
  • Lanes 3, 4, 5, 6 and 7 contained the topoisomerase I relaxation reaction carried in the presence of camptothecin, fascaplysin and DE002 at the concentrations indicated in the figure.
  • FIG 20 Topoisomerase I treated plasmid DNA was further subjected to DE002 treatment. The reaction products were resolved on a 0.8% agarose gel and stained with 0.5 mg/ml ethidium bromide in order to visualise DNA with UV illumination.
  • the figure shows relaxed pBlueScript plasmid DNA (lane 1 ), relaxed plasmid DNA treated with 1 ⁇ M, 10 ⁇ M and 100 ⁇ M of DE002 (lanes 2, 3 and 4 respectively) and relaxed plasmid DNA treated with fascaplysin 1 ⁇ M (lane 5).
  • DE002 does not displace ethidium bromide from the minor groove of double-stranded DNA molecules.
  • the ability of fascaplysin, DE002 (a non-planar analogue of fascaplysin) and actinomycin D (a known DNA intercalator) to interact with the minor groove of DNA was determined by the fluorescence-based ethidium bromide displacement assay. The final concentration of ethidium bromide in the assay was 1.3 ⁇ M.
  • the Figure shows representative curves with increasing concentrations of fascaplysin (filled squares) actinomycin D (unfilled squares) and DE002 (filled triangles). The results represent means and standard deviations from three independent experiments.
  • FIG. 22 A549 and LS174T cells analysed by Western blotting.
  • Equal amounts of total proteins (40 ⁇ g as estimated by Bradford protein estimation assays) from cell lysates were resolved on a 10% SDS- polyacrylamide gel and then transferred on to an Immobilon-P Transfer Membrane.
  • the membranes were probed with the following antibodies: mouse monoclonal antibody cyclin B1 (CR-UK, Cat.
  • FIG. 23 Cell-free tubulin polymerisation assay in vitro. Purified tubulin was used to test the ability of DE002 to inhibit tubulin polymerisation in vitro. The assay measures the increase in optical density as a result of tubulin assembly or polymerisation. Nocodazoie and paclitaxel were used in the assay, as controls, as a known inhibitor and enhancer of tubulin polymerisation, respectively. DE002 was tested at four different concentrations which had already show inhibition of in vitro cell growth. The change in Vmax value was used as an indicator of tubulin — ligand interactions. The polymerisation curves indicate that 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M and 25 ⁇ M concentrations of DE002 reduced the Vmax value from 19 mOD/min (control) to 12.5,
  • FIG. 24 Inhibition of tubulin polymerisation and enhancement of tubulin de- polymerisation in vivo.
  • Tubulin polymerisation assay was performed in A549 (whole cells) after treatment with DE002 for 30 min at the concentrations indicated in the figure.
  • Supernatant and pellet represent unassembled and assembled tubulin, respectively.
  • Tubulin polymerisation is detectable by the increase of tubulin amounts in the pellet and its disappearance from the supernatant.
  • the Western blots show dose- dependent inhibition of tubulin polymerisation after the simultaneous treatment of cells with paclitaxel and DE002. This results in the accumulation of unassembled tubulin in the supernatant.
  • DE002 also acts as an enhancer for tubulin de-polymerisation in a dose-dependent manner when paclitaxel-stabilized tubulin was subjected to DE002 treatment for 30 min.
  • the proteins were resolved on a 10% SDS-polyacrylamide gel and then transferred on to an Immobilon-P Transfer Membrane.
  • the membranes were probed with a specific mouse monoclonal ⁇ -tubuiin antibody B-7 (Santa Cruz Biotechnology, Cat. No. sc-5286) at a 1:1000 dilution.
  • HRP conjugated mouse secondary antibody (Santa Cruz Biotechnology, Cat. No. sc-2302) was used at a dilution of 1:2500 to illuminate the protein bands.
  • Figure 25 Long-term survival of cancer cells after treatment with DE002. A549 and Calu-1 cells were investigated for their long-term survival efficiency after treatment with different concentrations of DE002. The colony formation efficiency is expressed as the percentage of colonies formed in the treated cultures compared with untreated cultures.
  • A549 cells The representative plates show untreated A549 cells (a); A549 cells treated with 0.25 ⁇ M DE002 (b); A549 cells treated with 0.5 ⁇ M DE002 (c); A549 cells treated with 1 ⁇ M DE002, (d); A549 cells treated with 0.8 ⁇ M fascaplysin (IC 50 ) (e); untreated Calu-1 cells (f); Calu-1 cells treated with 1.25 ⁇ M DE002 (g); Calu-1 cells treated with 2.5 ⁇ M DE002 (h); Calu-1 cells treated with 5 ⁇ M DE002 (i); Calu-1 cells treated with 1 ⁇ M fascaplysin (IC 50 ) G)-
  • Figure 26 Long term survival of SV40 transformed mouse embryonic hepatic cells after the treatment with DE002.
  • the cells were treated with different concentrations of DE002 for 24 h and then incubated in drug free medium for 12 days. The colonies were fixed in methanol : acetic acid (2:1 ) and stained with 1% crystal violet. Representative plates were scanned using the gel documentation system.
  • the figure shows BNL CL2 cells (a) untreated cells; (b) treated with DE002, IC 20 (0.25 ⁇ M); (c) treated with DE002 IC 30 (0.4 ⁇ M); (d) treated with DE002 IC 50 (0.6 ⁇ M) and (e) treated with DE002 IC 70 (0.9 ⁇ M).
  • BNL SV A.8 cells (f) untreated cells; (g) treated with DE002, IC 20 (0.25 ⁇ M); (h) treated with DE002 IC 30 (0.4 ⁇ M); (i) treated with DE002 IC 50 (0.6 ⁇ M) and 0) treated with DE002 IC 70 (0.9 ⁇ M).
  • DE002 inhibits H 2 O 2 -mediated ROS formation in rat hepatoma cells, H4IIE.
  • H2DCF rat hepatoma cells
  • the oxidation of intracellular non-fluorescent H2DCF to highly fluorescent DCF was measured after addition of H 2 O 2 (500 ⁇ M) to H4IIE cells.
  • the cells were pre- incubated with different concentrations of DE002 for 60 min in order to assess the antioxidant potential of DE002.
  • IC 50 represents the concentration at which 50% of ROS formation was inhibited by DE002 treatment.
  • the results represent means and standard deviations (from the mean value) from three independent experiments.
  • FIG. 28 CA224 does not intercalate DNA.
  • the ability of CA224 to intercalate DNA was investigated using a topoisomerase I catalysed DNA unwinding/relaxation assay and compared with the effects of fascaplysin.
  • DNA relaxation assays were carried out as described. Lane 1 contains the control pBlueScript DNA and shows the supercoiled form as seen as a single band in the figure. Lane 2 contains the control relaxation reaction with topoisomerase I enzyme in the absence of any compound. Lanes 3, 4, 5, 6 and 7 contain the topoisomerase I relaxation reaction carried out in the presence of CA224, camptothecin and fascaplysin at the concentrations indicated in the figure.
  • CA224 does not displace ethidium bromide from the minor groove of double-stranded DNA.
  • the ability of CA224 to interact with the minor groove of double-stranded DNA was determined by a fluorescence based ethidium bromide displacement assay. The assay was performed as described with increasing concentrations of fascaplysin (filled squares), actinomycin D (unfilled squares) and CA224 (filled triangles). The results represent means and standard deviations from three independent experiments.
  • the IC 50 is the concentration of compound at which 50% displacement of bound ethidium bromide is observed.
  • Figure 3OA and 3OB FACS analysis and Western blotting in Calu-1 cells (mitotic spindle checkpoint-deficient cells) after treatment with CA224 indicates the inhibition of enzyme Cdk4-cycli ⁇ Df at the cellular level.
  • Figure 3OA shows untreated cells as control (A); cells starved of serum for 24 h (B); serum-starved cells released in the presence of IC 50 concentration of CA224 at for 24 h (C); serum-starved cells released in the presence of IC 70 concentration of CA224 for 24 h (D).
  • Figure 3OB shows the status of pRb phosphorylation at Cdk4-specific serine residues after cells were treated with CA224 for 24 h.
  • the rabbit polyclonal antibodies Ser780, Ser795 and Ser807/811 detect the phosphorylated pRb at Serine 780, Serine 795 and Serine 807/811 residues while the mouse monoclonal pRb (4H) detects the total phosphorylated and unphosphorylated pRb protein.
  • untreated cells as control (lane C); cells treated with IC 50 concentration of fascaplysin, 24 h (lane T1 ); cells treated with IC 50 concentration of CA224, 24 h (lane T2) and cells treated with IC 70 concentration of CA224, 24 h (lane T3).
  • FIG. 31 Response of the mitotic spindle checkpoint-proficient human lung cancer cell lines A549 and NCI-M 299 to CA224 treatment.
  • Flow cytometric analysis of asynchronous cells show that the majority of cells are arrested in the G 2 /M phase of the cell cycle (4n DNA content) in both cell lines.
  • FIG 32 The pro-metaphase block induced in NCI-H358 cells with nocodazole and paclitaxel treatment, was maintained by CA224. Cells synchronized by nocodazole and paclitaxel were released either in fresh medium or in fresh medium containing IC 50 concentration of CA224 compound exhibit greater tendency of CA224 to block the cell growth at G 2 /M.
  • control cells (B) treated with 1 ⁇ M nocodazole for 18 h; (C) treated with 1 ⁇ M nocodazole for 18 h and released in fresh medium; (D) treated with 1 ⁇ M nocodazole for 18 h and released in the presence of CA224, IC 50 ; (E) treated with 50 nM paclitaxel for 18 h; (C) treated with 50 nM paclitaxel for 18 h and released in fresh medium; (D) treated with 50 nM paclitaxel for 18 h and released in the presence of CA224, IC 50 .
  • FIG 33 Analysis of NCI-H358 cells using the flow cytometer.
  • Cells synchronized at Gi/S by treatment with hydroxyurea were released either in fresh medium or in fresh medium containing IC 50 concentration of CA224.
  • Treated cells exhibit greater tendency towards cell growth block at G 2 /M.
  • untreated cells act as control (A); cells treated with 250 ⁇ M hydroxyurea for 18 h (B); cells treated with 250 ⁇ M hydroxyurea for 18 h and released in fresh medium (C); cells treated with 250 ⁇ M hydroxyurea for 18 h and released in the presence of IC 50 concentration of CA224 (D).
  • FIG 34 Selective apoptosis in SV-40 transformed cells by CA224, analysed by FACS.
  • BNL CL2 mouse embryonic normal hepatic cells
  • BNL SV A.8 mouse embryonic SV-40 transformed hepatic cells
  • the apoptosis was quantified by measuring the percentage of cells that appeared under the sub-G-i peak, 31% and 44% cells were found in the SUb-G 1 peak after 48 h exposure with CA224, at the IC 50 (histogram E) and IC 70 (histogram F) concentrations, respectively.
  • the untreated cells (D) do not show any apoptosis.
  • FIG. 35 Western blot analysis of p53+ cells, A549 and LS174T.
  • the treatment with CA224 at the IC 50 concentration for 24 h resulted in the induction of the tumour suppressor protein, p53.
  • the Cdk inhibitor, P 21 CIP1/WAF1 (p21 ) was also induced.
  • the levels of p27 KIP1 (p27) were elevated while cyclin B1 and Cdk1 levels were down-regulated.
  • FIG. 36 Western blot analysis of lysates from MiaPaCa-2 cells.
  • CA224 treatment (IC 50 ) of MiaPaCa-2 cells for 24 h did not alter p53, p21 and p27 expression levels.
  • Cdk1 protein levels did not change while cyclin B1 was up-regulated.
  • the phosphorylation status of Cdk1 at Tyr15 remained unaffected after treatment.
  • FIG 37 In vitro polymerisation assay using purified tubulin.
  • the ability of CA224 to inhibit tubulin polymerisation in vitro was investigated as described. Paclitaxel and nocodazole were used in the assay as a known enhancer and inhibitor of tubulin polymerisation, respectively.
  • CA224 was tested for a range of concentrations at which it shows inhibition of in vitro cell growth. The change in Vmax value was used as an indicator of tubulin — ligand interactions.
  • the polymerisation curves indicate that 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M and 25 ⁇ M concentrations of CA224 reduced the Vmax value from 19 mOD/min (control) to 6.2, 2.1 , 1.1 and 0.4 mOD/min, respectively.
  • the curves shown are average of three independent experiments.
  • FIG 38 In vivo tubulin polymerisation assay in A549 cells. Western blots show the response of CA224 to tubulin polymerisation in the presence of paclitaxel and the effect of CA224 on paclitaxel-stabilized tubulin. The assay is performed in whole cells (A549) after 30 min compound treatment at the concentrations indicated in Figure. Supernatant and pellet represent unassembled and assembled tubulin respectively. Tubulin polymerisation is detectable by the increase of tubulin in the pellet and its disappearance from the supernatant. Simultaneous treatment of paclitaxel and CA224 show inhibition of tubulin polymerisation by CA224 in a dose-dependent manner and results in accumulation of unassembled tubulin in the supernatants. We observed that CA224 also acts as an enhancer for tubulin de-polymerisation in a dose-dependent manner when paclitaxel-stabilized tubulin was subjected to CA224 treatment.
  • FIG 39 CA224 activity in colony formation assays. A549 and Calu-1 cells were investigated for their long-term survival efficiency after treatment with different concentrations of CA224. The colony formation efficiency is expressed as the percentage of colonies formed in the treated cultures compared with untreated cultures.
  • A The representative plates show untreated A549 cells (a); cells treated with CA224, 3.12 ⁇ M (b); cells treated with CA224 6.25 ⁇ M (c); cells treated with CA224, 12.5 ⁇ M (d); cells treated with fascaplysin 0.8 ⁇ M (e); untreated Calu-1 cells (T); cells treated with CA224, 3.12 ⁇ M (g); cells treated with CA224 6.25 ⁇ M (h); cells treated with CA224, 12.5 ⁇ M (i); cells treated with fascaplysin 1 ⁇ M G).
  • B The curves represent colony formation efficiencies of A549 and Calu-1 cells with increasing concentrations of CA224. All results represent the means and standard deviations of three independent experiments.
  • Figure 40 Induction of apoptotic cell death analysed by DAPI staining and FACS.
  • A549 cells with increasing concentrations of CA224 for 24 h shows the dose-dependent induction of fragmented nuclei, disrupted cell membrane and apoptotic cell death.
  • A The fluorescence microscopic images captured at 40X magnification after staining with DAPI. Untreated A549 cells (a); cells treated with CA224, 1 ⁇ M (b); cells treated with CA224, 2.5 ⁇ M (c); cells treated with CA224, 5 ⁇ M (d); cells treated with CA224, 10 ⁇ M (e). The pre-apoptotic and apoptotic cells are indicated with arrows.
  • B The percentage apoptosis, induced in three cancer cell lines by treatment with CA224, determined by FACS analysis. The percentage of cells undergoing apoptosis is calculated from the percentage of cells that appear in the SUb-G 1 peak during cell cycle analysis. The number of apoptotic cells increase with the concentration of CA224 used and also the time of incubation.
  • Figure 41 Tumour growth inhibition curves for CA224, AJW089, DE002 and flavopiridol (used as a positive control) in the in vivo HCT116 xenograft model.
  • Figure 42 Animal weight profile in the HCT116 SCID mice xenograft model.
  • the body weights of animals in different treatment and control groups were monitored by taking measurements daily during the treatment schedule. By considering the body weight at the start of treatment as 100%, the percentage weight loss was calculated on subsequent days of treatment.
  • FIG. 44 Tumour growth inhibition curves for CA224, AJW089, DE002 and flavopiridol (used as a positive control) in the in vivo NCI-H460 tumour model.
  • Figure 45 Animal weight profile in the NCI-H460 xenograft model.
  • the body weights of animals in different treatment and control groups were monitored by taking the measurements daily during the treatment schedule. By considering the body weight at the start of the treatment as 100%, the percentage weight loss was calculated on subsequent days of treatments.
  • Figure 46 Average growth inhibition percentage of CA224, AJW089, DE002 and flavopiridol in NCI-H460 xenograft model. Stars indicate significant tumour growth inhibition. The tumour growth inhibition above 50% was considered as significant activity.
  • FIG. 47 Pictures of NCI-H460 tumour tissues exhibiting the growth inhibition of compounds CA224, DE002, AKW089 and flavopiridol in SCID mice. These pictures of the tumours were obtained after treatment of tumour-bearing animals with
  • H460 cells were injected subcutaneously into the dorsal side of SCID mice at a tune of
  • FIG. 48 Control and treatment groups of SCID mice showing NCI-H460 tumour growth inhibition following treatment with CA224, DE002, AKW089 and flavopiridol. These pictures were obtained after treatment with CA224, DE002 and flavopiridol. The treatments were continued for 9 consecutive days intraperitoneal ⁇ when tumour growth had reached about 4-6 mm in diameter after about 6 days.
  • Reagents used for the assays Purified Cdk enzyme complexes and their appropriate substrates, kinase buffer, DMSO, 5X Pl solution, ATP, Kinase-GloTM reagent, Cdk inhibitory compounds and distilled water.
  • Fascaplysin and its structural analogues, flavopiridol, CINK4, indirubin-5-sulphonic acid-sodium salt and Purvalanol A were dissolved in 100% DMSO as 10 mM stock solutions, aliquoted in micro-centrifuge tubes and stored at -80 0 C. Multiple " freeze thawing of the 10 mM stocks was avoided for all compounds. Compounds were further diluted in kinase buffer in order to obtain the desired serial dilutions (usually 10-fold) for determination of IC 50 -S (i.e. the concentrations of compounds at which a kinase activity was inhibited by 50%).
  • the kinase assays were carried out in a 96-well format in solid white polystyrene plates (Fisher Scientific, Cat. No. DPS-134-050A).
  • the purified enzyme complexes and substrates were diluted in kinase buffer in order to obtain the desired enzyme and substrate concentrations.
  • the kinase assays were performed in a total volume of 50 ⁇ l kinase buffer (Table 1 ). Initially 10 ⁇ l of kinase buffer containing kinase substrate, i.e.
  • ATP solution (10 mM stock solution in sterile distilled water) was diluted to 30 ⁇ M in 5X Pl solution
  • the assays were initiated by adding 200 ng (except 100 ng in case of Cdk1-cyclin B1 and 75 ng in case of Cdk9-cyclin T1 ) of active enzyme complexes per reaction and the plates were incubated for 30 min at 30 0 C in a humidified incubator.
  • ICgn concentrations The measured Relative Luminescence Units (RLU) from the control, blank and test samples were used to determine the ATP depletion in kinase reactions.
  • We also observed that the ATP depletion in all substrate blanks reactions containing only GST-pRb152 or Histone H1 or GST-CTD or MBP but no enzyme was negligible, i.e. less than 1-3%.
  • the potency to inhibit enzyme activity was calculated at different concentrations and the IC 50 (the concentration at which 50% enzyme activity is inhibited) was calculated by extrapolation.
  • Cdk inhibitory compounds reported in the literature, i.e. fascaplysin, flavopiridol, roscovitine CINK4 and indirubin-5- sulphonic acid-sodium salt (Knockaert et al, 2002; Soni et al, 2000; Soni ef a/, 2001; Meijer ef a/, 1997) were used to validate the chemiluminescent assays.
  • the IC 50 concentrations of these known compounds determined using this assay compared favourably with the IC 50 values published in the literature.
  • the cancer cell lines were used for screening the fascapiysin analogues in cell proliferation assays, FACS analyses, Western blotting, apoptosis assays, colony formation assays and anti-oxidation assays.
  • Cells with an early passage number were used for all these assays (cells were grown in the culture for a maximum of 10 passages and then fresh vials were revived from liquid nitrogen).
  • cells were washed and re-suspended in ice-cold cryo- protectant solution (95% fetal bovine serum (FBS) and 5% DMSO).
  • the tubes were transferred to an iso-propanol bath and then placed at -80 0 C in order to achieve controlled freezing of the cells (1°C/min). On the next day the cells were transferred to a liquid nitrogen cryo-can and stored under the liquid phase.
  • NSCLC non-small cell lung carcinoma
  • the colon carcinoma line LS174T (pRb + , p53 + ), the prostate carcinoma line PC3 (pRb + , p53-null) and the pancreatic cancer line MiaPaca-2 (pRb + , p53-mutant) were cultured in RPMI-1640 medium, supplemented with 10% FBS, 2 mM L Glutamine solution and 100 ⁇ g/ml NormocinTM.
  • the BNL CL.2 and BNL SV A.8 (normal mouse embryonic liver cells and its SV40 transformed subline) were grown in RPMI-1640 medium supplemented with 15% FBS, 1 mM sodium pyruvate solution and 1% PSN solution (penicillin, streptomycin and neomycin mixture).
  • the large T antigen of SV40 virus is responsible for functional inactivation of the tumour suppressor proteins pRb and p53.
  • H4IIE rat hepatoma cells
  • DMEM Dulbecco's modified Eagles's medium
  • Cell proliferation assay Reagents used for cell proliferation assays 2 mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) stock solution (Sigma-Aldrich, Cat. No. M2003), DMSO, trypan blue, trypsin-EDTA solution, sterile PBS, PRMI-1640 or DMEM, FBS, NormocinTM or PSN solution, 100X non essential amino acids solution and 100 mM sodium pyruvate solution.
  • MTT 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide
  • DMSO trypan blue
  • trypsin-EDTA solution sterile PBS
  • PRMI-1640 or DMEM sterile PBS
  • FBS NormocinTM or PSN solution
  • 100X non essential amino acids solution 100 mM sodium pyruvate solution.
  • Cell number/ml average from four large squares * dilution factor* 10 4 .
  • the cells were seeded in 96-well plates (Sarstedt, Cat. No. 83.1835), at densities between 5,000-10,000 cells per well, depending on the doubling time of the individual cell line, in 180 ⁇ l of complete growth medium. The wells at the extreme four corners were omitted in order to avoid the edge effect and variations in the assay.
  • the plated cells were allowed to stabilize at 37°C for 24 h in a CO 2 incubator. All the compounds were dissolved in 100% DMSO and 10 rtiM stock solutions were prepared. After 24 h of stabilization, the stock solutions of compounds were diluted in medium without any serum and antibiotics.
  • the absorbance was measured at a wavelength of 540 nm using a 96-well plate- reader (Biotek Synergy HT). Each concentration was tested in triplicate and the average of the three readings was calculated. The IC 50 concentration of compound was considered as the concentration at which 50% of cell growth was inhibited as compared to the control wells which did not contain any drug. The wells containing medium but no cells were treated as reagent blanks and both the mean control and mean test readings were corrected for the reagent blank before using them to determine the % growth inhibition. The % cell growth inhibition for each concentration of a compound was calculated using the following formula:
  • % cell growth inhibition (corrected mean control - corrected mean test) x 100 corrected mean control
  • Ethidium bromide is a commonly used nucleic acid stain that intercalates with the double-stranded DNA molecules and is also a known chemical mutagen. If excited at 260 nm, ethidium bromide can emit fluorescence at 600 nm. The fluorescence of ethidium bromide increases 25-fold upon binding with double stranded DNA molecules and when the DNA bound ethidium bromide is displaced, quenching of the fluorescence can be observed.
  • the pBlueScript DNA was used for binding studies in this modified assay based on the method previously described by Geall et al, 1999 and Brotz-Oesterhelt et al, 2003.
  • Ethidium bromide was dissolved in sterile distilled water as a 10 mg/ml stock solution and stored at 4°C.
  • the DH5 ⁇ E. coli cells, transformed with pBlueScript plasmid DNA, were grown in LB medium broth containing 25 ⁇ g/ml ampicillin.
  • the plasmid DNA was isolated using the Maxi (QIAGEN-tip 500) preparation protocol following the manufacturer's instructions.
  • Reagents used for the EtBr displacement assay 10 mg/ml EtBr solution, purified pBlueScript plasmid DNA, DMSO, EtBr displacement assay buffer, test compounds.
  • % fluorescence corrected mean RFU in control - corrected mean RFU in test * 100 corrected mean RFU in control
  • the graphs of % fluorescence against the concentration were plotted in Excel and the concentration that showed 50% reduction in % fluroscence was considered as the IC 50 in the EtBr displacement assay.
  • Topoisomerase I catalysed DNA relaxation or unwinding assay in vitro
  • topological isomers of DNA molecules are essential for many vital cellular processes like DNA replication, transcription, chromosomal segregation and recombination. This can be achived by breaking and re-uniting (re-ligating) the DNA strands sequentially. This process is catalysed by a key class of enzymes known as topoisomerases.
  • topoisomerases There are two types of topoisomerases, DNA topoisomerase I (topo I) which acts on only one strand and induces single-strand nicks in the DNA followed by their religation while DNA topoisomerase Il (topo II) acts on both the strands resulting in DNA breaks and sequential reunions (Giles and Sharma 2005; Leppard and Champoux 2005).
  • Reagents used for DNA unwinding assays Topoisomerase I enzyme, 10 mg/ml EtBr solution, purified pBlueScript plasmid DNA, agarose, DNA unwinding assay buffer, phenol, chloroform, EDTA, Tris-EDTA buffer, SDS, absolute ethanol, 10X DNA loading buffer and test compounds.
  • a double-stranded plasmid DNA mainly exists in a super- coiled form and a small fraction of it exists in a nicked circular or linear form.
  • the super-coiled DNA when treated with topo I or topo Il enzyme in vitro can become relaxed and be converted into sequential topological isomers which differ in their mobility when separated on an agarose gel.
  • Compounds having affinity for DNA molecules and that can intercalate into DNA can hamper the topoisomerase I catalysed DNA relaxation process, thus allowing the assessment of the ability of compounds to intercalate DNA in an in vitro assay (Antony et al, 2005; Das et al, 2006; Fortune et al, 1998).
  • fascaplysin and its structural analogues were intercalate into plasmid DNA.
  • the pBlueScript plasmid DNA was used for the assay. Each reaction contained 5 nM super-coiled pBlueScript plasmid DNA and 10 units of topoisomerase I active enzyme.
  • relaxed plasmid DNA was first prepared by treating with topo I enzyme for 30 min and then used as an initial substrate for the assay. DNA relaxation assays were performed in the presence or absence of compounds in 40 ⁇ l of DNA unwinding assay buffer.
  • reaction mixtures were treated with 3 ⁇ l of 250 mM EDTA and extracted with phenol/chloroform.
  • the DNA was dissolved in Tris-EDTA buffer, pH 8.
  • the samples (20 ⁇ l) were treated with 2 ⁇ l of 2.5% SDS, mixed with 2.5 ⁇ l agarose gel- loading buffer (10X) and subjected to electrophoresis on a 0.8% agarose gel without ethidium bromide (separating the DNA in the presence of ethidium bromide would convert the relaxed DNA into the supercoiled form).
  • DNA bands were stained with 1 ⁇ g/ml ethidium bromide and visualised using a UV illuminator.
  • the compounds were compared with fascaplysin which is a known DNA intercalating molecule. Camptothecin which is a known topoisomerase I inhibitor was used to test the activity and inhibition of the enzyme.
  • Flow cytometric analyses Information about the normal cell cycle and the effects on the cell cycle due to drug treatment can be assessed by studying the DNA content in drug-treated mammalian cells. Since DNA content of cells at different phases of the cell cycle differ substantially, cells can be differentiated on the basis of the amount of DNA present and hence percentage of cells in different phases of cell cycle can be determined. For example, if cells are stained with nucleic acid specific stains, like propidium iodide, all cells in the d phase should take up the same amount of stain and should fluoresce in a single channel (or single peak). Measurements of DNA content can be performed using a Fluorescence Activated Cell Sorter (FACS).
  • FACS Fluorescence Activated Cell Sorter
  • a FACS machine measures the fluorescence from the DNA-binding fluorochrome which infers the total DNA content.
  • the instrument used for these studies was the Beckman-Coulter EPICS® ALTRATM and the instrument was calibrated before every use with the help of 'fluorescent calibration beads' supplied by Beckman-Coulter.
  • the single peaks for forward scatter, side scatter and the rest of the filters including PMT 4 (excitation 495, emission 637) were calibrated in such a way that the coefficient of variation (CV) falls below 2%.
  • a typical cell cycle picture obtained by FACS analysis is shown in Figure 3.
  • Reagents used for FACS analyses Propidium iodide (Sigma-Aldrich, Cat. No. P4170), RNase A (Sigma-Aldrich, Cat. No. R6513), PBS, absolute ethanol, different cell cycle and anticancer compounds and cancer or normal cells cultured in vitro.
  • Experimental procedures Single cell suspensions for FACS analyses were made after harvesting the cells. Cells were seeded in such a way that no cultures became confluent or super-confluent before cell harvesting.
  • mammalian cells are starved of serum growth factors, their growth tends to be arrested in early G 1 phase of the cell cycle (Soni et al, 2001).
  • cells were serum starved by incubating in medium containing 0.1 % FBS for 24 h. They were then released in complete growth medium supplemented with 10% FBS in the presence or absence of test compound for a further 24 to 48 h. The cells released in the fresh medium were used to check any abnormal effect on cells due to serum starvation.
  • Mimosine block experiments Mimosine, a non-essential amino acid, inhibits DNA polymerase ⁇ in eukyaryotic cells and thereby blocks them at the d/S boundary of the cell cycle (Ji et al, 1997).
  • To block cells at the G 1 ZS boundary cells were seeded in 25 cm 2 tissue culture flasks. When the cultures became 40-50% confluent, they were treated with mimosine as follows.
  • Mimosine was prepared as a 10 mM stock solution in 100% DMSO, diluted in sterile growth medium without serum and added to cultures at 200 ⁇ M as a final concentration for 32 h. Cells were washed twice with fresh medium and then incubated in fresh medium in the absence or presence of test compounds for a further 18 h or 36 h.
  • Nocodazole was dissolved in 100% DMSO as a 10 mM stock solution and then stored at -80 0 C until required.
  • the NCI-H358 (p53-null) cells were seeded in 25 cm 2 tissue culture flasks. Cells were allowed to grow until 40-50 % confluency had been reached; then nocodazole (diluted in pre-warmed growth medium without FBS) was added to culture flasks at a final concentration 1 ⁇ M (a sublethal concentration at which cells can re-enter cell cycle without any damage or apoptosis). The flasks were returned to the incubator and incubated for 18 h. After this, the cells were harvested, fixed and stained with propidium iodide for FACS analysis. The rest of the cells were released in fresh medium in the absence or presence of test compound and incubated for a further 12 h. All samples were harvested, fixed, stained and subjected to FACs analysis.
  • Paclitaxel block experiments Paclitaxel was dissolved in 100% DMSO as a 10 mM stock solution and further diluted in pre-warmed growth medium without FBS. The paclitaxel block experiments were performed as described above.
  • Floating cells were collected by centrifugation of the growth medium at 3,000 rpm for 5 min. Attached cells from the flasks were harvested by trypsinization. All the collected cells (floating and attached) were washed once with PBS and counted using a haemocytometer. 1 * 10 6 cells were fixed in 1 ml of 70% chilled (-20 0 C) ethanol for at least 1 h. Cells were stored at -20 0 C until the staining.
  • cells were centrifuged for 5 min at 3,000 rpm at room temperature and the pellet re- suspended in 1 ml of PBS containing 50 ⁇ g/ml propidium iodide and 0.5 mg/ml DNase free Ribonuclease (RNase A Sigma Cat. No. R6513). The cells were stained for 1 h in the dark at 4°C and then analysed by FACS.
  • Cell lysis buffer Sigma-Aldrich, Cat. No. C-2978
  • protease inhibitor cocktail Sigma-Aldrich, Cat. No. P8340
  • Bradford reagent BIO-RAD protein assay
  • the cells were incubated at room temperature and allowed to lyse for a period of 2 h.
  • the lysates were centrifuged at 14,000 rpm for 10 min at 4°C and the clear supernatants were assayed for protein content using the Bradford method.
  • appropriate dilutions of sample and standard (BSA 1 mg/ml solution) were made in sterile distilled water. 10 ⁇ l of diluted sample or standard was added to 790 ⁇ l of sterile distilled water, then 200 ⁇ l of Bradford reagent was added to each tube and mixed well. The tubes were incubated at room temperature for 10 min.
  • Reagents used 6X SDS sample buffer, 30% acrylamide/Bis solution (BIO-RAD, Cat. No. 161-0156), Tris-HCI pH 8 & pH 6.8, SDS, ammonium persulphate (APS), TEMED, pre-stained protein marker, distilled water and butanol.
  • TEMED 20 ⁇ l For each experiment APS solution was made fresh. The components were added in the sequence indicated above and the solution poured between the two glass plates immediately after the addition of APS and TEMED. Dual vertical gel electrophoresis apparatus (BIO-RAD, MINI PROTEIN IITM) was used to perform the gel electrophoresis. 1 ml of water saturated butanol was poured on top of the running gel. The gel was allowed to polymerise at room temperature for 30 min. The water- saturated butanol was removed and the upper part of the polymerised gel was washed with distilled water. The 4% stacking gel was prepared using the following composition of chemicals. 4% Stacking gel: Distilled water 2.5 ml
  • Reagents used for transfer and membrane blocking Cathode buffer, Anode I buffer, Anode Il buffer, blocking grade non-fat dry milk powder (BIO-RAD, Cat. No. 170 6404), appropriate primary and secondary antibodies, developer solution (Kodak, Cat. No. 190 0943), fixer solution (Kodak, Cat. No. 190 1875) and ECL detection reagent kit (Santa Cruz Biotechnology, Cat. No. sc-2048)
  • the Immobilon-P membrane was labelled to mark the side on which proteins will be transferred to and then slowly placed on the gel, air bubbles were removed.
  • 3X Whatman ® chromatography papers (the same size as the gel) were soaked in Anode Il buffer and placed on top of the gel.
  • Another a set of 3X Whatman ® chromatography papers were soaked in Anode I buffer and placed on top of the previous Whatman ® papers and air bubbles were removed.
  • the top part of the blotter was carefully placed and the transfer was carried out at a constant current of 0.8 mA/cm 2 of gel surface (6 mA per gel).
  • BIO-RAD power pack 200 was used to maintain the low voltage required to avoid heating of the gels.
  • the transfer was carried out for 1 h and 30 min at room temperature. After transfer, the membrane was removed and washed in PBS containing 0.1% Tween 20 for 10 min with gentle shaking. The PBS/0.1% Tween 20 was poured off and 10 ml of blocking solution, 5% milk powder in PBS was added to the membrane, and incubated with gentle shaking for 1 h. The membrane was rinsed in PBS/0.2 % Tween 20 solution and washed 1 x 15 min followed by 3 * 5 min in PBS/0.2 % Tween 20 with gentle shaking. The membrane was incubated with primary antibody diluted in 1% milk powder in PBS/0.2% Tween 20 solution.
  • the membranes were probed with different primary antibodies (at 4°C overnight) as described: C-22 (Santa Cruz Biotechnology, Cat. No. sc-260) at 1 :1000 dilution to detect Cdk4; M-20 (Santa Cruz Biotechnology, Cat. No. sc-718) at 1:1000 dilution to detect cyciin D1 ; Cdk2 human (CR-UK, Cat. No. AN21.2) at 1 :4000 dilution to detect Cdk2; cyciin A (CR-UK, Cat. No.E23.1 ) at 1 :5000 dilution to detect cyciin A; cdc2 (New England Biolabs, Cat. No.
  • sc-2301 is used at a 1 :2500 dilution in the case of a mouse monoclonal primary antibody or an anti-rabbit antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Cat. No. sc-2301) is used at a 1:2000 dilution in the case of a rabbit polyclonal primary antibody.
  • the secondary antibody was diluted in PBS/0.2% Tween 20 containing 1% milk powder and incubated with the membrane for 1 h 30 min at room temperature with gentle shaking. After probing with secondary antibody, the membrane was washed 2 * 10 min in PBS/0.2% Tween 20 followed by 2 * 10 min in PBS with gentle shaking. Finally the membrane was rinsed with sterile distilled water.
  • the protein bands can be visualised by chemiluminescence using the ECL detection kit (Santa Cruz Biotechnology, Cat. No. sc-2048).
  • the membrane was placed on cling film and 2 ml of ECL detection reagent was added drop-wise onto the membrane. The membrane was incubated with the reagent for 1 min and the excess fluid was removed. The chemiluminescence was detected using either Gel documentation system (BIO- RAD, UNIVERSAL HOOD Il S. N. 76S) or by exposing to HyperfilmTM ECLTM (Amersham Pharmacia Biotech). Immediately after the treatment with ECL detection reagent, the membrane was exposed to HyperfilmTM ECLTM for various times (e.g. 30 s to 5 min) and developed.
  • tubulin polymerisation and depolymerisation assays in vivo were performed with some modifications to the basic procedures described previously (Jordan et al, 2002; Giannakakou et. al., 1997; Hua et al, 2001 ).
  • A549 cells were plated at a concentration of 10,000 cells per well in 1 ml complete growth medium in 24-well (15 mm) plates. The plates were incubated for 24 h to allow cell attachment and stabilization.
  • the cells were treated simultaneously with 10 nM paclitaxel and different concentrations of test compound for 30 min.
  • the cells were treated with 10 nM paclitaxel for 30 min.
  • the cell monolayer was washed twice with sterile PBS and fresh growth medium containing different concentrations of compound was added. The plates were further incubated for 30 min, the cell monolayer were washed twice with sterile PBS at room temperature and then 100 ⁇ l tubulin extraction buffer supplemented with 2 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Sigma-Aidrich Cat. No. P8340) was added per well. The plates were incubated for 10 min at room temperature and then the cell suspensions were transferred to microcentrifuge tubes.
  • the cell lysates were incubated at room temperature for 5 min and then centrifuged at 16,000 rpm for 10 min in order to separate the soluble and polymerised tubulin fractions.
  • Each supernatant and pellet fraction was mixed with 6X SDS sample buffer, heated for 7 min at 95°C and resolved on a 10% SDS-polyacrylamide gel.
  • the resolved proteins were Western blotted and tubulin-specific proteins were illuminated with a mouse monoclonal ⁇ -tubulin antibody B-7 (Santa Cruz Biotechnology, Cat. No. sc-5286) used at a 1:1000 dilution and HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Cat. No. sc-2302) used at a 1:2500 dilution.
  • Tubulin polymerisation assay in vitro
  • the purified bovine brain tubulin and the cytoDYNAMIXTM Screen was obtained commercially (Cytoskeleton Inc. Denver USA) and the polymerisation assays were carried out according to the method described previously (Ferlini et al, 2005; Jordan et al, 2002).
  • the tubulin polymerisation assay is based on an adaptation of the original methods of Lee et al, (1997) which demonstrated that light is scattered by microtubules to an extent that is proportional to the concentration of the microtubule polymer in the solution.
  • Reagents used for tubulin polymerisation General tubulin buffer, tubulin glycerol buffer, tubulin polymerisation buffer, purified bovine brain tubulin (Cytoskeleton Inc. Denver USA, Cat. No. BK006 / CDS03), 200 mM GTP solution, DMSO and test compounds.
  • test compounds to inhibit tubulin polymerisation in vitro was determined according to the manufacturer's instructions.
  • the tubulin polymerisation assays were performed in 96-well plates. Prior to starting the assay, the plate was pre-warmed at 37°C for 30 min (warming the plate is essential for higher polymerisation activity as well as reproducible results).
  • Tubulin polymerisation buffer (TP buffer) was prepared by the addition of the following components and then incubation on ice for 10 min: 1.5 ml of general tubulin buffer (80 mM PIPES pH 6.9, 2 mM MgCI 2 , 0.5 mM EGTA), 500 ⁇ l of tubulin glycerol buffer (15% glycerol in general tubulin buffer) and 20 ⁇ l of GTP stock solution (200 mM).
  • the TP buffer was made fresh each time and used within 4 h of preparation due to unstable nature of GTP.
  • 1 ml of general tubulin buffer was made warm by incubating at 37°C for 10 min and then used for the different dilutions of paclitaxel, nocodazole and test compounds.
  • 10 ⁇ l of general tubulin buffer was pipetted into two wells.
  • 10 ⁇ l of 10 ⁇ M paclitaxel 10 ⁇ M paclitaxel
  • 10 ⁇ M nocodazole or 10X concentrations of the test compounds were added to the respective wells.
  • the purified bovine brain tubulin (10 mg/ml) was defrosted by placing it on a room temperature water bath for exactly 1 min or until thawed and then placed on ice (the fast thawing is important because concentrated tubulin may start polymerising).
  • the concentrated tubulin protein was diluted 1 :3.3 in ice cold TP buffer to obtain 3 mg/ml solution. 100 ⁇ l of diluted tubulin was added in each well and the plate immediately placed in a temperature regulated Biotech spectrophotometer previously adjusted to 37°C. The absorbance was recorded using the kinetic set-up described below.
  • DAPO staining The post apoptotic characterstic of cells like degeneration of nuclear membrane and nuclear fragmentation were detected using 4'-6-Diamidino-2-phenylindole (DAPI) staining followed by observations under a fluorescence microscope (Sandra et a/, 2006).
  • DAPI is known to form complexes with natural double-stranded DNA.
  • DAPI binds to DNA, its fluorescence is strongly enhanced making it possible to observe the nuclear morphology of cells under a fluorescence microscope.
  • DAPI Sigma-Aldrich, Cat. No. D8417
  • glycerol glycerol
  • acetic acid glycerol
  • ethanol glycerol
  • ethanol glycerol
  • the mitotic index (percentage of cells in mitosis) of cultures followed by the treatment with test compound was determined by fluoroscence microscopic observations after staining the cells with DAPI (Augustin, et al, 1997).
  • A549 cells were seeded at a density of 50,000 cells per well in 6 well tissue culture plates. After 24 h, the medium was replaced with fresh medium containing the desired concentration of test compound. 20 h later, attached and floating cells were collected and combined (by trypsinization and centrifugatron) The cells were fixed in chilled (-20 0 C) acetic acid:ethanol (1:3) fixative for 10 min. One drop of cell suspension was dropped on a glass slide in order to disrupt the cell membrane and was allowed to air dry for 10 min.
  • the smear was mounted in a mounting medium containing 1 ⁇ g/ml DAPI in 50% glycerol and covered with a coverslip.
  • the slides were observed under a fluorescence microscope and at least 1000 nuclei per slide were observed.
  • the relative percentages of cells in M (mitosis) phase as compared with control cultures were calculated and the mitotic indices were determined.
  • Reagents used for colony formation assays PBS, complete growth medium, trypsin- EDTA solution, 1% crystal violet solution (Sigma-Aldrich, Cat. No. C3886), methanol, acetic acid, DMSO and distilled water.
  • the plates were washed 2X with 2 ml of distilled water per well and then air dried for 15 min. 1 ml of 1 % crystal violet solution was added to each well and the colonies were allowed to stain for 15 min. The staining solution was removed, the plates were washed 3X with distilled water and then air dried for 30 min. The colonies were evaluated by visual counts and the number of colonies in treated cultures were expressed as a percentage of the control cultures. Representative plates were scanned using the gel documentation system.
  • H 2 DCF diacetate (Molecular probes Cat. No. D399)
  • H 2 O 2 DMEM H 2 O 2 DMEM
  • FBS FBS
  • L-glutamine L-glutamine
  • non essential amino acid solution H 2 O 2 DMEM
  • H4IIE rat hepatoma cells
  • 50,000 cells/well were seeded in a 96-well microtitre plate. The cells were allowed to attach and then the medium was changed. Cells were first incubated with different concentrations of test compounds for 60 min, then the medium containing compounds was removed and the cells were washed with PBS twice and fresh medium was then added. H 2 DCF diacetate was added at a final concentration of 5 ⁇ M and the cells were further incubated for 30 min.
  • H 2 O 2 mediated ROS formation The oxidation of intracellular non-fluorescent H 2 DCF to highly fluorescent DCF was measured after addition *of H 2 O 2 (500 ⁇ M) at an excitation of 485 nm and an emission of 525 nm to measure the inhibition by the test compound of H 2 O 2 mediated ROS formation (Srinivas et al, 2004; Steffan et al, 2005).
  • Senescence is the condition where cells can no longer replicate themselves through processes like entering the normal cell cycle and cellular mitosis. Cells lose the ability to divide during senescence, their physical appearance or morphology also changes during this process and can be observed microscopically. Senescence is also associated with changes in cellular gene expression patterns which results in the detection of ⁇ -galactosidase activity by using cellular staining techniques. In these studies ⁇ -galactosidase staining of cells was used to detect the senescent cells (Serrano et al, 1997). Reagents used: complete growth medium, trypsin EDTA solution, sterile PBS, DMSO, senescent cell staining kit (Sigma-Aldrich Cat. No. CS0030), glycerol.
  • MiaPaCa-2 (40,000 cells /well) or LS174T (30,000/well) or A549 (25,000/well) cells were seeded in 6-well plates in 2 ml complete growth medium. Cells were incubated for 24 h (to allow for stabilization) followed by 96 h of treatment with test compound (at IC 3O and IC 50 concentrations). After compound treatment, the growth medium was removed by aspiration and the cells were washed twice with 1 ml PBS. 1.5 ml of fixation buffer was added to each well and the plates were incubated at room temperature for 10 min. While cells were being fixed, the staining mixture was prepared as per the manufacturer's instructions.
  • the cells were washed 3 times with 1 ml PBS and 1 ml of staining mixture was added to each well. The plates were incubated at 37°C without CO 2 for 48 h. The cells were observed under the microscope and the percentage of cells expressing ⁇ -galactosidase was determined. The staining mixture was removed and replaced with 70% glycerol solution and stored at 4°C for long term storage.
  • mice Swiss albino mice were used to determine the maximum tolerated dose for all three compounds. In this study 6 animals per group were administered with compounds at different doses for five days (Q1 D x 5) via intraperitoneal route. Animals were monitored for weight loss, morbidity symptoms and mortality up to two weeks by the end of treatment. Significant weight loss was considered when mean animal weight dropped by >10% and was considered highly significant when the drop was >20%.
  • the compound AJW089 was partially soluble in 10% polyethyleneglycol 400 (PEG 400). An appropriate quantity of this compound was mixed by gradual addition of 10% PEG solution and was triturated until a homogenous suspension was achieved.
  • PEG 400 polyethyleneglycol 400
  • HCT-116 experiments A group of 60 SCID (strain-CBySmn.CB17-Pr/ccfc sc/c y j , The Jackson Laboratory, Stock # 001803) male mice weighing 18-25 g and 6-8 weeks old were used for the studies.
  • Human colon carcinoma, HCT-116 (ATCC, Cat. No. CCL- 247) cells were grown in McCoy's 5A medium supplemented with 10% FBS (Sigma- Aldrich). 6.6 * 10 6 of cultured cells in 0.2 ml of suspension were injected subcutaneously into dorsal side of SCID mice. When the tumour growth reached to about 4-6 mm in diameter (about 5 days), the animals were randomly divided into eight groups, each containing 7 mice. The treatments were continued for 9 consecutive days intraperitoneal ⁇ .
  • NCI-H460 experiments A group of 65 Severely Combined Immune-Deficient (SClD strain-CBySmn.CBI 7-PrZCdC 5 ⁇ AJ, The Jackson Laboratory, Stock # 001803) female mice weighing 15-24 g and 6-8 weeks old were used.
  • Human non-small-cell lung carcinoma NCI-H460 (ATCC, Cat. No. HTB-177) cells grown in RPMI-1640 medium supplemented with 10% FBS (Sigma-Aldrich). The cultured cells were injected subcutaneously into the dorsal side of SCID mice at a tune of 5.3 * 10 6 cells in 0.2 ml of suspension. When the tumour growth reached about 4-6 mm in diameter (about 6 days), the animals were randomly divided into eight groups, each containing 6 or 7 mice. The treatments were continued for 9 consecutive days intraperitoneally.
  • Tumour weight measurements Tumour size was recorded at 2-5 day intervals. Tumour weight (mg) was estimated according to the formula for a prolate ellipsoid: ⁇ Length (mm) x [width (mm) 2 ] x 0.5 ⁇ assuming specific gravity to be one and ⁇ to be 3. Tumour growth in compound treated animals is calculated as T/C (Treated/Control) x 100% and Growth inhibition Percent (% Gl) was [100-% T/C] (Tashiro et al 1989; Mohammad et al, 1998; Mohammad et al, 1996).
  • Body weight measurements The body weights of animals in different treatment and control groups were monitored by taking the measurements daily during the treatment schedule. By considering the body weight at the start of the treatment as 100%, the percent weight loss was calculated on subsequent days of treatments.
  • Chemiluminescent-based Cdk assays Sf9 cells, co-infected with baculoviruses carrying the gene of interest, were lysed. Cdk complexes, present in the whole cell lysates, were purified by incubation with glutathione-agarose beads. 10 ⁇ l from each sample was resolved on SDS-PAGE and stained with Coomassie blue. Distinct bands representing GST-tagged cyclins and associated Cdks can be seen in Figure 5. The cyclins are tagged with GST (molecular weight 27 kD).
  • the ATP concentrations for the standardisation of all kinase assays were kept constant, i.e. the final ATP concentration in the kinase reaction was always 6 ⁇ M.
  • the kinase substrates GST-pRb152, Histone H1 , GST-CTD and MBP were used at the concentrations described above. The substrate concentrations were chosen so that less than 5% ATP depletion occurred at a given substrate concentration.
  • the optimum enzyme concentration was determined by performing titration experiments using a varied set of enzyme concentrations. The optimum concentrations of different enzymes were also determined by testing them for their inhibition with known Cdk inhibitory molecules. In the case of Cdk1-cyclin B1 , the active enzyme complexes were used at 100 ng per reaction according to the manufacturer's instructions. Determination of optimum enzyme concentration
  • cyclin dependent kinases Cdk-s
  • Cdk cyclin dependent kinases
  • Known cyclin dependent kinase (Cdk) inhibitors were used as standard controls. Inhibitors were tested at a minimum of 5 different concentrations. The concentration at which 50% enzyme activity was inhibited (IC 50 ) was calculated and compared with the IC 50 values published in the literature (Table 3 and Figures 8 to 12). The IC 50 concentrations of the known compounds calculated using this assay compare favourably with the IC 50 values published in the literature confirming that the assays are accurate.
  • Example 1 Biological activity of the DE002 series of fascaplysin analogues
  • the desired product was extracted into CHCI 3 , washed with saturated brine solution (2 x 25 mL) and H 2 O (2 x 25 mL), aqueous washings being re-extracted with CH 2 CI 2 (3 x 50 mL), the combined organic phases were then dried over anhydrous sodium or magnesium sulphate, filtered and isolated under reduced pressure.
  • the analogues listed in Table 4 were tested in a number of different bio-chemical assays. Their potency to inhibit different Cdks in vitro was evaluated first. This was followed by further analysis on their ability to inhibit cancer cell growth in vitro. The DNA-binding properties of these compounds were also explored.
  • CA210 CA211 CA212
  • IC 50 is the concentration of a compound at which 50% cell growth was inhibited.
  • CA199 and DE002 inhibited the growth of cancer cells at low micro-molar concentrations (Table 6) and they were found to be the most active molecules in the series with an average IC 50 of 7 ⁇ M and 0.75 ⁇ M, respectively.
  • the compounds CA198 and CA211 also inhibit the in vitro cell growth at low micro molar concentrations.
  • the inhibition of cell growth was independent of the presence or absence of the tumour suppressor proteins p53 and pRb (see Table 7 for indication of the p53 and pRb status of the cancer cell lines).
  • High potency of DE002 in a cell line which lacks pRB activity i.e.
  • NCI-H358 which is pRb-null
  • Cdk4 inhibition may not be the only cellular target for the mechanism of action of these molecules.
  • the presence of ceUular pRb is essential for mediation of the action of Cdk4 inhibitors.
  • IC 50 values are in ⁇ M. The IC 50 values represent means and standard deviations from three independent experiments.
  • Table 7 The four best compounds in the DE002 series were evaluated in 9 different cancer cell lines and one normal line. IC 50 concentrations, expressed in ⁇ M, for in vitro cell growth inhibition induced after exposure to CA199, CA211 , CA198 and DE002 for 48 h. All results represent means and standard deviation from three independent experiments. The tissue origin and the p53 or pRb status are indicated in brackets.
  • Calu-1 cells were treated with IC 50 and IC 70 concentrations of DE002 after release from cell synchronization.
  • Cells were starved of serum for 24 h using 0.1% FBS. This resulted either in the partial (at IC 50 ) or full maintenance (at IC 70 ) of the G 0 ZG 1 block. Since the maintenance of the G 0 ZG 1 block after serum starvation requires Cdk4 enzyme to be inactive, these results may indicate that DE002 is likely to inhibit cellular Cdk4 at these concentrations ( Figure 13A) thereby maintaining the G 0 ZG 1 block.
  • the Calu-1 cell line was chosen to check Cdk4-specific pRb phosphorylation.
  • the pRb phosphorylation status was tested in DE002-treated Calu-1 cells to seek affirmation of DE002's ability to inhibit cellular Cdk4-cyclin D1 enzyme.
  • the Western blot results obtained after DE002 (IC 50 ) treatment of Calu-1 cells for 24 h show that pRb remains unphosphorylated at serine residues Ser780, Ser795 and Ser807/811 which are specifically phosphorylated by Cdk4 enzyme while pRb levels in DE002 treated cells remain unchanged ( Figure 13B).
  • Mimosine a non-protein amino acid inhibits DNA polymerase ⁇ in eukaryotic cells and thereby blocks them at the d/S boundary of the ceil cycle (Ji C et a/, 1997). Since the function of the Cdk4 enzyme is crucial while cells progress through the early Gi phase of the cell cycle, we hypothesised that if DE002 selectively inhibits Cdk4, it would not affect the progression of cells which have already passed early G 1 and are blocked at the G 1 ZS boundary. We therefore studied the effect of DE002 on mimosine-treated cells which are blocked at G 1 ZS.
  • Calu-1 cells were blocked with mimosine for 32 h and were then released in the presence of (a) fresh medium, (b) DE002, (c) roscovitine (a Cdk2-specific inhibitor) and fascaplysin (a Cdk4-specific inhibitor which also intercalates DNA).
  • roscovitine a Cdk2-specific inhibitor
  • fascaplysin a Cdk4-specific inhibitor which also intercalates DNA
  • ICgn concentration of DE002 induced profound G?ZM block in two asvnchronouslv growing cancer cells, A549 (p53+) and NCI-H 1299 (p53-null)
  • NSCLC non-small cell lung carcinoma
  • DE002 blocks NCI-H358 cells at G 2 ZM after release from hvdroxyurea-mediated G 1 ZS cell synchronization
  • NCI-H358 cells were treated with 250 ⁇ M hydroxyurea for18 h to block cells at the G 1 ZS boundary (77% cells observed at G 1 ZS; Figure 16, histogram F), at a stage of the cell cycle where Cdk2-specific inhibitors normally act.
  • G 1 ZS boundary 77% cells observed at G 1 ZS; Figure 16, histogram F
  • cells proceed from G 1 ZS, confirming that DE002 does not inhibit cellular Cdk2 as indicated by the results from the in vitro Cdk2 enzyme assay. Released cells ultimately accumulate at G 2 ZM (74%; Figure 16, histogram H).
  • DE002 selectively induces apoptotic cell death in SV40 large T antigen transformed normal mouse embryonic liver cells
  • SV40 large T antigen inactivates the two tumour suppressor proteins, p53 and pRb, and thereby through inactivation of two major tumour suppressors transforms normal cells into tumorigenic ones (Herzig et al, 1999; Pipas and Levine 2001 ).
  • IC 50 of DE002 in BNL CL2 is -7.0 ⁇ M
  • IC 50 of DE002 in BNL SV A.8 is -8.2 ⁇ M
  • IC 70 of DE002 in BNL CL2 is 9.8 ⁇ M
  • IC 70 of DE002 in BNL SV A.8 is 11 ⁇ M (see Table 5 and Figure 18, Panel A, right- hand graph).
  • DE002 acts as a strong antiproliferative agent in both normal and SV40-transformed cells.
  • DE002 selectively kills SV40- transformed cells at concentrations where normal cells are totally unaffected. This would suggest that cancer cells, which are analogous to SV40-transformed cells, will be much more sensitive to apoptosis than untransformed normal cells when treated with DE002.
  • Results obtained from the ethidium bromide displacement assay indicate that none of the DE002 series of fascaplysin analogues interact with the minor groove of double- stranded DNA because they do not displace the DNA-bound ethidium bromide (Table 5).
  • Representative curves of fascaplysin, DE002 and actinomyci ⁇ D are shown in Figure 21 which exhibits the interactions of these compounds with pBlueScript plasmid DNA. As seen in Figure 21 , 100 ⁇ M of DE002 was incapable of displacing 1.3 ⁇ M ethidium bromide from pBlueScript DNA. Less than 5% displacement of bound ethidium bromide was observed at 100 ⁇ M.
  • A549 a NSCLC cell line
  • LS174T a colon carcinoma line
  • Both cells lines bear functional copies of the tumour suppressor gene p53 and are therefore referred to as p53+ cells.
  • A549 and LS174T contain functional copies of the retinoblastoma protein.
  • Treatment with DE002 results in a 10-fold induction of p53.
  • the non-specific Cdk inhibitory protein P 21 CIP1/WAF1 (p21 ) is induced since p53 is a transcriptional activator that can activate p21 gene transcription by binding to the p53 protein-specific enhancer sequences in the p21 gene promoter.
  • the levels of another pan-Cdk inhibitor, p27 KIP1 (p27) are also elevated after DE002 treatment ( Figure 22).
  • the proteins Cdk1 and cyclin B1 are down-regulated in treated cells when compared with proteins from untreated control cells. Repression of the expression of the cyclin B1 and Cdk1 proteins (Figure 22) is a possible explanation of the profound G 2 /M block observed in A549 cells (portrayed in Figure 15) and LS174T cells (results not shown). Elevated levels of p21 and p27 not only can reinforce the G 2 /M block but may also play a major role in the G 0 ZG 1 block which is observed in cells treated with higher concentrations (i.e. IC 70 ) of DE002.
  • DE002 was evaluated for its effect on the cell cycle in MiaPaCa-2 cells. Similar cell cycle results to those obtained from other cancer cell lines (A549, NCI-H1299, NCI- H358, and BNL SV A.8) were obtained.
  • MiaPaCa-2 cells are also arrested at G 2 /M phase of the cell cycle followed by the treatment with IC 50 and IC 70 concentration of DE002 for 24 h. Approximately 52 and 55% cells were found to be blocked at the G 2 /M phase using IC 50 and IC 70 concentrations of DE002, whereas in control cultures only 18% cells appeared in the G 2 /M phase (data not shown).
  • A549 lung cancer, NSCLC
  • the mitotic spindle checkpoint in A549 cells is normal and hence these cells should be sensitive to anti- microtubule agents.
  • the assembled (cytoskeletal) and unassembled (cytosolic) forms of tubulin were determined from their accumulation and disappearance from pellet and supernatant fractions of the cell lysates treated with DE002 and further evaluated with Western blotting.
  • A549 cells have the normal mitotic spindle checkpoint and bear functional copies of the pRb and p53 proteins.
  • Calu-1 cells have an impaired mitotic spindle checkpoint, contain a functional pRb protein but are p53-null.
  • DE002 selectively reduces long-term survival in SV40 large T-antigen transformed normal mouse embryonic liver cells BNL-CL2.
  • DE002 The ability of DE002 to reduce colony formation efficiency of mouse embryonic hepatic cells (BNL CL2) and SV40 transformed mouse embryonic hepatic cells (BNL SV A.8) was evaluated in a colony formation assay.
  • DE002 was tested at IC 2 O, IC 30 , IC 50 and IC 70 concentrations in both the cell lines. These concentrations were determined using MTT cell proliferation assay. The representative plates are shown in Figure 26. The results indicate that even at the low concentrations of DE002 (IC 2 o) the efficiency of colony formation is selectively reduced in SV40 transformed cells. Less than 50% of colonies were observed at IC 2O concentrations DE002. At IC 50 and IC 70 concentrations, more than 90% cells lost their ability to form colonies.
  • DE002 was tested for its ability to induce senescence in A549 cells.
  • Non-toxic concentrations of DE002 IC 50 and IC 30 ) were used to find out the effect of this compound on senescence.
  • DE002 inhibits H 2 O 2 -induced cellular ROS formation in H4IIE cells
  • the structure of DE002 resembles the structure of an indole derivative. Indoles constitute some of the most powerful antioxidants known in nature (Andreadou et al, 2002; Mor et al, 2003; Olgen et al, 2002)
  • H4IIE rat hepatoma cells were used to test the possible antioxidant potential of DE002. It was found that DE002 inhibits 50% of H 2 O 2 -mediated ROS formation at -20 ⁇ M concentration. The results are shown in Figure 27. It was found that the pre- incubation of H4IIE cells with DE002 protects cells from H 2 O 2 -mediated ROS induction. Formation of reactive oxygen species (ROS) inside the living cells could have detrimental effects on cells, since ROS can induce DNA strand breaks and also can mediate conformational changes in protein molecules. These changes at molecular level could result in many degenerative diseases that include cancer (Izzotti et al, 2006; Butterfield et al, 1998). Hence the antioxidant property of DE002 can have vital importance not only for the treatment of cancer but also for prevention of the disease.
  • ROS reactive oxygen species
  • Silibinin a naturally occurring flavonoid undergoing clinical trials at the National Cancer Institute also acts as an anti-oxidant.
  • DE002 inhibits H 2 O 2 -mediated ROS formation (IC 50 ⁇ 20 ⁇ M) whereas silibinin does not (IC 50 >200 ⁇ M) (Dehmlow et al, 1996), H 2 O 2 being one of the most powerful pro-oxidants known.
  • the well known anti-oxidant, / ⁇ -acetylcysteine has an IC 50 >100 ⁇ M for inhibition of H 2 O 2 - mediated ROS formation
  • DE002 a non-planar analogue of fascaplysin, is a compound that shows novel effects on cancer cells.
  • DE002 was initially identified on the basis of an in vitro screen for the Cdk4 enzyme, and was found to inhibit Cdk4 specifically (it does not inhibit Cdk2, Cdk1 and Cdk9). Besides, Cdk4-specific inhibition, DE002 shows some unique properties.
  • DE002 has a profile which is uniquely distinct from any anticancer compound reported in the literature.
  • DE002 and other compounds within the DE002 series having a similar structural scaffold, are clinically useful compounds for the treatment of cancer.
  • Example 2 Biological activity of the CA224 and AJW089 series of fascaplysin analogues
  • CA224 specifically inhibits Cdk4-cyclin D1 enzyme in vitro
  • CA224 inhibits Cdk4-cyclin D1 at an IC 50 of approximately 6 ⁇ M, while compounds AJW089, MS014, AJW099, AJW090 and AJW102 also inhibited Cdk4-cyclin D1 in vitro with relatively high potencies (IC 50 of 7 ⁇ M, 15 ⁇ M, 11 ⁇ M, 15 ⁇ M and 9 ⁇ M, respectively).
  • IC 50 7 ⁇ M, 15 ⁇ M, 11 ⁇ M, 15 ⁇ M and 9 ⁇ M, respectively.
  • IC 50 values are presented in ⁇ M concentrations. All the fascaplysin analogues were dissolved in 100% DMSO solution and were further diluted in the kinase assay buffer or the ethidium bromide displacement assay buffer. The IC 50 values represent means and standard deviations from three independent experiments.
  • CA224 and AJW089 inhibited the growth of cancer cells at low micro-molar concentrations (Table 10 and Table 1 1 ) and were found to be the most active molecules with an average IC 50 of 4 ⁇ M and 2 ⁇ M respectively.
  • IC 50 values are denoted in ⁇ M.
  • the in vitro cell growth inhibition was determined by the MTT assay. All compounds including fascaplysin were dissolved in 100% DMSO and the resultant solutions were then further diluted in fresh growth medium just before addition to the cultured cells.
  • the IC 50 values represent means and standard deviations from three independent experiments. IC 50 is the concentration at which 50% of cell growth was inhibited after treatment with compound.
  • the cell growth inhibition was determined by the MTT assay.
  • the IC 50 values represent means and standard deviations from three independent experiments. IC 50 is the concentration at which 50% of cell growth was inhibited after treatment with compound.
  • PC-3 (prostate; p53-null, pRb+) 47 + 3 6.2 + 1.1 15 + 1.5 4.5 + 0.7
  • MiaPaCa-2 pancreatic; p53His273mut, pRb+) 31 + 2.2 4 + 0.3 10.2 + 0.9 3.7 + 1.1
  • NCI-H 1299 (NSCLC; p53-null, pRb+) 21 + 0.9 2.5 + 0.3 11.5 + 1.6 1.78 H H 0.4
  • NCI-H358 (NSCLC; p53-null, pRb null) 26 + 2 2.2 + 0.6 14 + 1.4 1 .45 H H 0.2
  • CA224 was tested for its ability to intercalate DNA molecules using topoisomerase I catalysed unwinding/relaxation assay (Figure 28).
  • Relaxation of supercoiled pBlueScript plasmid DNA was carried out in the presence of fascaplysin, CA224 and camptothecin. Fascaplysin shows inhibition of DNA relaxation catalysed by the enzyme topoisomerase I indicating the intercalating nature of a planar molecule.
  • the non-planar compound, CA224 does not show any inhibition of DNA relaxation even at high concentrations up to 100 ⁇ M (Figure 28).
  • CA224 selectively inhibits Cdk4-cyclinD1 (Table 9). Thus it should also block the growth of asynchronously growing cells at the G 1 phase of the cell cycle and should maintain the G 0 IGi block induced by serum starvation.
  • Cell lines with varied mitotic spindle checkpoint and p53 status were used to investigate the role of CA224 in blocking specific phases of the cell division cycle.
  • CA224 retains the GnZG 1 block in serum-starved p53-null Calu-1 cells
  • Calu-1 cells In Calu-1 cells, the mitotic spindle checkpoint is impaired and since CA224 also inhibits tubulin polymerisation in vitro with higher potency than it inhibits the enzyme Cdk4- cyciin D1 (see below), Calu-1 cells were used to study the Cdk4 inhibitory property of CA224.
  • CA224 at the ICgn concentration induces profound G 2 ZM block in two asynchronous cancer cell lines (p53+ A549 and p53-null NCI-H1299 cells) Incubation of A549 cells with CA224 at the IC 50 concentration for 24 h induces a profound block at G 2 /M as indicated by the percentage of cells at G 2 ZM. As seen in Figure 31, at the IC 50 concentration of CA224, 89% cells appeared to be arrested in the G 2 ZM phase (B) and at the IC 70 concentration 91% cells blocked at G 2 ZM (C).
  • NCI-H358 (p53-null) cells were treated with nocodazole (1 ⁇ M: a sub-optimal concentration) for only 18 h in order to induce a partial block at G 2 /M so that treated cells are minimally stressed.
  • the blocked cells were released either in fresh medium for 12 h (they readily re-enter the cell cycle without any apoptosis) or in the presence of CA224 for 12 h (cells not only maintain the G 2 /M block but also >50% of G 0 ZG 1 and S phase cells enter G 2 /M ( Figure 32, histograms A,B, C and D).
  • CA224 blocks NCI-H358 cells in G?/M after release from hydroxyurea-mediated G1/S cell synchronization
  • SV40 large T antigen inactivates both the tumour suppressor proteins, p53 and pRb, and thereby transforms normal cells into tumorigenic cells.
  • the primary characteristic of an ideal anti-cancer compound is to selectively kill rapidly proliferating cancerous cells while leaving the normal cells unaffected.
  • the selective cell death induction in cancer cells by CA224 treatment is very significant.
  • tumour suppressor protein plays a crucial role in controlling cell proliferation.
  • p53 can induce p21 which is a cell cycle inhibitory protein, and thereby blocking cell growth.
  • Intracellular accumulation of p53 can also result in cyclin B1 depletion which could result in the G 2 /M arrest of cells.
  • CA224 was analysed for its effects on the cell cycle of MiaPaCa-2 cells which are p53 mutated. FACS analysis shows that CA224 also blocks MiaPaCa-2 cells at the G 2 /M phase of the cell cycle. At the IC 50 concentration of CA224, approximately 48% cells were found in G 2 /M while in control cultures (i.e. cells that were untreated) only 16% cells appeared in G 2 /M (data not shown).
  • Cell free tubulin polymerisation assays in vitro indicate inhibition of tubulin polymerisation by novel fascaplysin analogues including CA224 CA224 inhibits growth of cancer cells in vitro at concentrations that are lower than the concentration for inhibition of the enzyme Cdk4-cyclin D1.
  • FACS analyses shown above
  • mitotic index experiments have already confirmed that CA224 blocks cell growth at the pro-metaphase of the cell cycle.
  • the resistance of cells containing an impaired mitotic spindle checkpoint to CA224- mediated growth inhibition suggests that CA224 has another cellular target other than Cdk4. It is possible that it has a role as an anti-microtubule agent.
  • CA224 decreased the Vmax from 17 mOD/min to 6.2, 2.1 , 1.1 and 0.4 mOD/min at 2.5, 5, 10 and 25 ⁇ M, respectively. As a consequence of decreased Vmax, up to 80% reduction in final polymer mass is observed.
  • CA224 inhibits paclitaxel-mediated tubulin polymerisation and enhances tubulin de- polymerisation in vivo
  • CA224 also enhances the depolymerisation of the stabilized tubulin protein.
  • the polymerised and depolymerised (soluble) forms of tubulin can be perceived from the accumulation or disappearance (observed by Western blotting) of tubulin protein from the pellets and vice versa from the supernatant fractions of lysed cells treated with CA224 (at IC 50 concentrations).
  • CA224 shows prevention of tubulin polymerisation (mediated by paclitaxel) in a dose dependent manner ( Figure 38).
  • results show enhancement of tubulin de-polymerisation with increasing concentrations of CA224 ( Figure 38).
  • the concentration at which 50% colony formation efficiency is observed is comparatively lower than the IC 50 concentration for cell growth inhibition in the MTT assay, indicating that large number of cells lose the ability to form colonies or do not survive for a long time after CA224 treatment.
  • the percentage of apoptotic cells is determined by the number of ce/is appearing in the sub-Gi peak.
  • the SUb-G 1 peak in these cells was compared to that in control cultures.
  • the percentage apoptosis in three different cell lines is presented in Figure 4OB. The results indicate that long-term exposure (72 h) of cells to CA224 can induce massive cell death both in p53+ and p53-null cancer cells.
  • A549 cells treated with different concentrations of CA224 for 24 h were analysed by nuclear staining (i.e. DAPI staining).
  • DAPI staining i.e. DAPI staining
  • Example 3 Anti-tumour activity of CA224, AJW089 and DE002 against xenografts human cancer cells in SCID mice
  • CA224, AJW089 and DE002 are the three most potent molecules which inhibit cancer cell growth (Table 12). These three compounds were therefore chosen to test their activity against models of human cancers in vivo. Table 12. IC 50 -S of CA224, AJW089 and DE002 in nine different cancer cell lines and a normal cell line.
  • SCID mice lacking both T and B immune cells are an established modef system to study the in vivo efficacy of molecules against human cancers (Bankert et at, 2002; Kelland 2004).
  • the human tumour cells can grow in SCID mice without any graft rejection because the mice are immuno-deficient.
  • Human tumour cells grown in cell culture or a small piece of tumour of human origin can be transplanted subcutaneously and grown for a few days to form a small tumour. The small tumours can then be treated with potential anti-cancer compounds for evaluation of their in vivo efficacy.
  • MTD is the highest daily dose of a compound that does not cause over- toxicity (which sometimes can translate to death) in laboratory mice or rats.
  • the studies which allowed determination of MTD were performed in Swiss albino mice for two weeks. The concentration at which the three compounds would be tested in vivo was thus ascertained. Loss in animal body weight was considered as a measure of over-toxicity for the test compound. The concentration of the compound at which >10% weight loss was observed was determined and designated as MTD. However, since a weight loss of up to 20% of the initial weight is usually harmless and animals usually recover once the treatment is stopped, the MTD figures used were an underestimation of the true value.
  • the MTD was found to be -500mg/kg.
  • CA224 and DE002 were tested at two different concentrations, VA of MTD and Vi of MTD.
  • AJW089 was tested at lower than VA of MTD concentrations due to its poor solubility.
  • mice Toxicity and MTD findings study in Swiss albino mice. Each group contained 6 mice and were injected with one dose intra-peritoneally (i.p.) on 5 consecutive days, using different concentrations of CA224, AJW089 and DE002. The body weight of the animals was then monitored for two weeks and the drop in relative mean animal weight was calculated.
  • CA224 and DE002 significantly inhibit the growth ofHCT116 xenografts
  • Flavopiridol is a known Cdk inhibitor and its ability to inhibit tumour growth in vivo has been widely reported (Zhai et a/, 2002; Drees et al, 1997; Senderowicz and Sausville 2000). Hence flavopiridol was used as a standard compound in the in vivo experiments.
  • the compounds CA224 and DE002 showed statistically significant (p ⁇ 0.05) tumour growth inhibition (Table 14; Figure 41 and Figure 43) at Vz MTD as well as V* MTD concentrations.
  • CA224 and DE002 exhibited approximately 80% tumour growth inhibition as compared to the untreated group. These results indicate their strong anti-tumour properties in vivo.
  • the weight loss observed in treated animals was found to be ⁇ 10% of the starting weights of the animals ( Figure 42). This loss of weight can be considered to be statistically insignificant indicating that compound treatment cause no major toxicity or harm to the animals.
  • the body weight of the animals at the beginning of treatment was measured and this was considered to be 100%.
  • the percentage weight loss or gain was calculated using the initial weight as reference.
  • NCI-H460 is a drug resistant lung cancer cell line derived from NSCLC, a form of cancer which is mostly untreatable using the drugs currently available in the clinic.
  • NCI-H460 cells demonstrate aggressive growth and spread of cancer cells in vivo in SCID mice. Most anti-cancer compounds which inhibit cancer cell growth in vitro fail to inhibit NCI-H460 tumours in vivo due to the aggressive nature of these tumours.
  • CA224, DE002 and AJW089 were tested for their in vivo efficacy in the SCID mice-NCI-H460 model.
  • Flavopiridol the pan-Cdk inhibitor which is effective in a variety of mouse tumour models, was used as the standard compound in these in vivo experiments.
  • CA224 and DE002 both proved to be highly efficacious against NCI-H460 at Vz MTD and ⁇ A MTD concentrations.
  • AJW089 failed to exhibit significant activity in this particular in vivo model (Table 16; Figure 44) due to its low solubility in the solvent and hence low bioavailability.
  • AD Animal died Effect on animal body weight
  • tumour growth inhibition after compound treatment was calculated.
  • the tumour growth inhibition values were derived from the data presented above. The results indicate more that 75% tumour growth inhibition following CA224 and DE002 treatment (Figure 46). After the experimental schedule, the animals were sacrificed and photographs were taken to demonstrate in situ tumour growth inhibition (Figure 48). Representative animals from each group were dissected and tumour tissues (Figure 47) were photographed and preserved for further experiments.
  • CA224 and DE002 are highly efficacious against human tumours derived from HCT116 and NCI-H460 cell in in vivo.
  • the two compounds show minimal toxicity in animal models and can be tolerated up to 1000 mg/kg concentrations.
  • CA224 and DE002 have proven their efficacy as a new class of anticancer compounds via these studies.
  • the use of more soluble salts of AJW089, or the use of different solvents, is expected to show that this compound is also a highly effective anticancer agent in vivo.
  • Flavopiridol induces G1 arrest with inhibition of cyclin dependent kinase (CDK) 2 and Cdk4 in human breast carcinoma cells. Cancer Res, 56: 2973-2978.
  • CDK cyclin dependent kinase
  • Flavopiridol (L86-8275): selective antitumor activity in vitro and activity in vivo for prostate carcinoma cells. 1997. Clin Cancer Res. 3(2):273-9.
  • Paclitaxel-resistant human ovarian cancer cells have mutant beta-tubulins that exhibit impaired paclitaxel-driven polymerization. J Biol Chem,. 272(27): 17118-17125.
  • Fascaplysin a selective CDK4 inhibitor, exhibit anti-angiogenic activity in vitro and in vivo. Cancer Chemother Pharmacol, [Epub ahead of print].

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Abstract

L'invention concerne un procédé destiné à traiter un cancer, consistant à administrer un composé représenté par la formule (I), (II) ou (III) à un patient.
PCT/GB2008/002678 2007-08-13 2008-08-07 Dérivés de fascaplysine et leur utilisation dans le traitement d'un cancer Ceased WO2009022104A1 (fr)

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CN113332282A (zh) * 2021-06-16 2021-09-03 南方海洋科学与工程广东省实验室(湛江) 一种Fascaplysin类化合物的应用
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US8716308B2 (en) 2008-01-11 2014-05-06 Albany Molecular Research, Inc. (1-azinone)-substituted pyridoindoles
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US9073925B2 (en) 2009-07-01 2015-07-07 Albany Molecular Research, Inc. Azinone-substituted azabicycloalkane-indole and azabicycloalkane-pyrrolo-pyridine MCH-1 antagonists, methods of making, and use thereof
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WO2014041125A1 (fr) * 2012-09-13 2014-03-20 Baden-Württemberg Stiftung Gmbh Inhibiteurs spécifiques de la protéine p21 comme agents thérapeutiques
US10568889B2 (en) 2014-04-29 2020-02-25 Emory University Prostaglandin receptor EP2 antagonists, derivatives, compositions, and uses related thereto
EP3137074A4 (fr) * 2014-04-29 2017-11-15 Emory University Antagonistes du récepteur ep2 des prostaglandines, dérivés, compositions et utilisations associés
US11077120B2 (en) 2014-04-29 2021-08-03 Emory University Prostaglandin receptor EP2 antagonists, derivatives, compositions, and uses related thereto
JP2019515643A (ja) * 2015-09-23 2019-06-13 ミネルバ バイオテクノロジーズ コーポレーション 幹細胞分化剤のスクリーニング方法
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CN105524061A (zh) * 2015-12-15 2016-04-27 中国人民解放军第二军医大学 多靶点抗肿瘤活性的吴茱萸碱衍生物及其制备与应用
CN105669666A (zh) * 2016-01-08 2016-06-15 华东师范大学 小分子化合物yf-452及其在制备抗血管新生药物中的应用
US12240829B2 (en) 2019-03-20 2025-03-04 Emory University Prostaglandin receptor EP2 antagonists, derivatives, and uses related thereto
CN113332282A (zh) * 2021-06-16 2021-09-03 南方海洋科学与工程广东省实验室(湛江) 一种Fascaplysin类化合物的应用

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