ZA200405886B - Pyranones useful as ATM inhibitors. - Google Patents

Description

PYRANONES USEFUL AS ATM INHIBITORS

The present invention relates to compounds which act as ATM inhibitors, their use and synthesis.

Human DNA is constantly under attack from reactive oxygen intermediates principally from by-products of oxidative metabolism. Reactive oxygen species are capable of producing

DNA single-strand breaks and, where two of these are generated in close proximity, DNA double strand breaks (DSBs). In addition, single- and double-strand breaks can be induced when a DNA replication fork encounters a damaged template, and are generated by exogenous agents such as 1onising radiation (IR) and certain anti-cancer drugs (e.g. bleomycin, etoposide, camptothecin). DSBs also occur as intermediates in site- specific V(D)J recombination, a process that is critical for the generation of a functional vertebrate immune system. If

DNA DSBs are left unrepaired or are repaired inaccurately, mutations and/or chromosomal aberrations are induced, which in turn may lead to cell death. To combat the serious threats posed by DNA DSBs, eukaryotic cells have evolved several mechanisms to mediate their repair. Critical to the process of DNA repair is the slowing down of cellular proliferation to allow time for the cell to repair the damage. A key protein in the detection of DNA DSBs and in the signalling of this information to the cell cycle machinery is the kinase ATM (ataxia telangiectasia mutated) (Durocher and Jackson (2001)

DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme? Curr Opin Cell Biol., 13:225-31, Abraham (2001) Cell cycle checkpoint signaling through the ATM and ATR kinases.

Genes Dev., 15; 2177-96).

The ATM protein is an ~350 kDa polypeptide that is a member of the phosphatidylinositol (PI) 3-kinase family of proteins by virtue of a putative kinase domain in its carboxyl-terminal region (Savitsky et al (1995) A single ataxia telangiectasia . gene with a product similar to PI-3 kinase. Science, 268:1749- 53). Classical PI 3-kinases, such as PI 3-kinase itself, are involved in signal transduction and phosphorylate inositol lipids that act as intracellular second messengers (reviewed in Toker and Cantley (1997) Signalling through the lipid products of phosphoinositide-3-0OH kinase, Nature, 387: 673-6).

However, ATM bears most sequence similarity with a subset of the PI 3-kinase family that comprises proteins which, like

ATM, are involved in cell cycle control and/or in the detection and signalling of DNA damage (Keith and Schreiber (1995) PIK-related kinases: DNA repair, recombination, and cell cycle checkpoints, Science, 270; 50-1, Zakian (1995)

ATM-related genes: what do they tell us about functions of the human gene? Cell, 82; 685-7). Notably there is no evidence to date that any members of this subset of the PI 3-kinase family are able to phosphorylate lipids. However, all members of this family have been shown to possess serine/threonine kinase activity. ATM phosphorylates key proteins involved in a variety of cell-cycle checkpoint signalling pathways that are initiated in response to DNA DSBs production (see below).

These downstream effector proteins include p53, Chk2,

NBS1/nibrin, BRCAl and Rad 17 (Abraham, 2001)

ATM is the product of the gene mutated in ataxia- , telangiectasia (A-T) (Savitsky et al (1995)). A-T is a human autosomal recessive disorder present at an incidence of around . 1 in 100,000 in the population. A-T is characterised by a number of debilitating symptoms, including progressive cerebellar degeneration, occulocutaneous telangiectasia,

growth retardation, immune deficiencies, cancer predisposition and certain characteristics of premature ageing (Lavin and

Shiloh (1997), The genetic defect in ataxia-telangiectasia.

Annu. Rev. Immunol., 15:177-202; Shiloh (2001), ATM and ATR: networking cellular responses to DNA damage, Curr. Opin.

Genet. Dev., 11:71-7). At the cellular level, A-T is characterised by a high degree of chromosomal instability, radio-resistant DNA synthesis, and hypersensitivity to ionizing radiation (IR) and radiomimetic drugs. In addition, .

A-T cells are defective in the radiation induced Gi1-S, S, and

G2-M cell cycle checkpoints that are thought to arrest the cell cycle in response to DNA damage in order to allow repair of the genome prior to DNA replication or mitosis (Lavin and

Shiloh, 1997). This may in part reflect the fact that A-T cells exhibit deficient or severely delayed induction of p53 in response to IR. Indeed, pS53-mediated downstream events are also defective in A-T cells following IR exposure. ATM therefore acts upstream of p53 in an IR-induced DNA damage signalling pathway. A-T cells have also been shown to accumulate DNA double-strand breaks (dsbs) after ionizing radiation, suggesting a defect in dsb repair.

It is clear that ATM is a key regulator of the cellular response to DNA DSBs. Therefore the inhibition of this kinase through small molecules will sensitise cells to both ionising radiation and to chemotherapeutics that induce DNA DSBs either directly or indirectly. ATM inhibitors may thus be used as adjuncts in cancer radiotherapy and chemotherapy. To date the only reported inhibitors of ATM (caffeine and wortmannin;

Sarkaria, et al., (1999) Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer

Res., 59:4375-82; Banin, et al., (1998) Enhanced phosphorylation of p53 by ATM in response to DNA damage.

Science, 281:1674-1677) do cause radiosensitisation but it is unclear whether this mechanism of action is mediated throu gh

ATM inhibition as these small molecules are very non-speci fic in action as kinase inhibitors. .

ATM function in response to ionising radiation induced DNA damage has been shown to be tissue specific. For example, while fibroblasts derived from Atm null mice are radiosensitive Atm null neurons are radioresistant through a lack of IR induced apoptosis (Herzog et al., (1998)

Requirement for Atm in ionizing radiation-induced cell dea th in the developing central nervous system. Science, 280: 1 089- 91). Therefore, inhibitors of ATM have the potential to b-e radio-protective in specific cellular contexts.

ATM inhibitors may also prove useful in the treatment of retroviral mediated diseases. It has been demonstrated theat

ATM function 1s reguired to allow stable retroviral DNA transduction under certain conditions (Daniel et al. (2001)

Wortmannin potentiates integrase-mediated killing of lymphocytes and reduces the efficiency of stable transduct_ion by retroviruses. Mol. Cell Biol., 21: 1164-72). Therefore ATM inhibitors have the potential to block retroviral DNA integration.

ATM is known to play a crucial role in controlling the leragth of telomeric chromosomal ends (Metcalfe et al. (1996)

Accelerated telomere shortening in ataxia telangiectasia. Nat ,

Genet., 13 :350-3). Telomeric ends in most normal cell tyspes shorten at each cell division. Cells with excessively . shortened telomeres are unable to divide. Inhibitors of &ATM may therefore, have utility in preventing cancer progression by limiting the growth potential of cancerous or pre-cancezrous cells. Furthermore, ATM does not appear to be part of the telomerase enzyme itself (Metcalfe et al. (1996)) Therefore it 1s likely that ATM inhibitors will work synergistically with anti-telomerase drugs. 5

Cells derived from A-T patients or from mice null for ATM grow slower in culture than genetically matched ATM positive cells.

Therefore an ATM inhibitor may have growth inhibitory/anti- proliferative properties in its own right. Therefore an ATM inhibitor may be used as a cytostatic agent in the treatment of cancer.

A-T patients display immuno-deficiencies, demonstrating that

ATM 1s required for generation of a fully functional immune system. Inhibitors of ATM may, therefore, be used in modulating the immune system.

In summary ATM inhibitors have the potential to sensitise tumour cells to ionising radiation or DNA DSB inducing chemotherapeutics, to modulate telomere length control mechanisms, to block retroviral integration, modulate the immune system and to protect certain cell types from DNA damage induced apoptosis.

The present inventors have now discovered compounds which exhibit inhibition of ATM.

Accordingly, the first aspect of the invention provides a compound of formula I: ,

R'

RA. _P_ _N :

NL? or Rm

Q

Y and isomers, salts, solvates, chemically protected forms, and prodrugs thereof, wherein: one of P and Q is O, and the other of P and Q is CH, where there is a double bond between whichever of Q and P is CH and the carbon atom bearing the R3 group;

Y is either O or S:

R! and R? are independently hydrogen, an opt_ionally substituted

Ci-7 alkyl group, C3. heterocyclyl group, or Cs ag aryl group, or may together form, along with the nitrogeen atom to which they are attached, an optionally substituted heterocyclic ring having from 4 to 8 ring atoms;

R® is a phenyl or pyridyl group, attached by= a first bridge group selected from -S-, -S(=0)-, -S(=0),-, -0-, -NR"- and

CR“'R*~ to an optionally substituted Cs.,o ca rboaryl group, in which one aromatic ring atom may be replaced by a nitrogen ring atom; the phenyl or pyridyl group and optionally substituted Cs» carboaryl group being optionally further lirmked by a second bridge group, which is bound adjacent the fDirst bridge group on both groups so as to form an optionally substituted Cs_- ring fused to both the phenyl or pyridyl group arnd the Cs_z ‘ carboaryl group, the phenyl or pyridyl group being further optionally substituted; wherein RY is selected from hydrogen, an est-er group, an optionally substituted C;.; alkyl group, an optionally substituted Csi, heterocyclyl group and an optionally substituted Cs_y aryl group: and R°! and R® are independently selected from hydrogen, an optionally substituted C;.; alkyl group, an optionally substituted Ci3.po heterocyclyl group and an optionally substituted Cs.;o aryl group.

Therefore, when P is O and Q is CH, the compound is of formula (la):

R' 3

R Oo N

Nz or ’ "

Y and when P is CH and Q is O, the compound is of formula (Ib): . R' 3

R

= AN (Ib)

O

Y

A second aspect of the invention provides a composition comprising a compound of the first aspect and a pharmaceutically acceptable carrier or diluent.

A third aspect of the invention provides the use of a compound of the first aspect in a method of therapy.

A fourth aspect of the invention provides the use of a compound of the first aspect in the preparation of a medicament for inhibiting the activity of ATM.

o

A fifth aspect of the invention pro vides for the use of a compound as defined in the first as pect of the invention in the preparation of a medicament for use as an adjunct in cancer therapy or for potentiating tumour cells for treatment with lonising radiation or chemothe rapeutic agents.

A sixth aspect of the invention pro-vides for the use of a compound as defined in the first as-pect of the invention in the preparation of a medicament for the treatment of retroviral mediated diseases or disease ameliorated by the inhibition of ATM, which include acquired immunodeficiency syndrome.

A further aspect of the invention provides an active compound as described herein for use in a method of treatment of the human or animal body, preferably im the form of a pharmaceutical composition.

Another aspect of the invention provides a method of inhibiting ATM in vitro or in vivo, comprising contacting a cell with an effective amount of arm active compound as described herein.

Definitions

Cy-7 alkyl: The term “C;_5 alkyl”, a s used herein, pertains to a monovalent moiety obtained by remo=ing a hydrogen atom from a

Cy-7 hydrocarbon compound having from 1 to 7 carbon atoms, which may be aliphatic or alicyclic, or & combination thereof, and : which may be saturated, partially wunsaturated, or fully unsaturated. '

Examples of saturated linear C,., alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, and n-pentyl (amyl) .

Examples of saturated branched C;.5 alkyl groups include, but are not limited to, iso-propyl, iso-butyl, sec-butyl, tert-butyl, and neo-pentyl.

Examples of saturated alicyclic C;-7 alkyl groups (also referred to as "Cs.5 cycloalkyl” groups) include, but are not limited to, groups such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, as well as substituted groups (e.g., groups which comprise such groups), such as methylcyclopropyl, dimethylcyclopropyl, methylcyclobutyl, dimethylcyclobutyl, methylcyclopentyl, dimethylcyclopentyl, methylcyclohexyl, dimethylcyclohexyl, cyclopropylmethyl and cyclohexylmethyl.

Examples of unsaturated C,.; alkyl groups which have one or more carbon-carbon double bonds (also referred to as “Cy.7alkenyl” groups) include, but are not limited to, ethenyl (vinyl, -

CH=CH), 2-propenyl (allyl, -CH-CH=CH,;), isopropenyl (-C(CH3)=CHz), butenyl, pentenyl, and hexenyl.

Examples of unsaturated C;_; alkyl groups which have one or more carbon-carbon triple bonds (also referred to as “Cj.4 alkynyl” groups) include, but are not limited to, ethynyl (ethinyl) and 2-propynyl (propargyl).

Examples of unsaturated alicyclic (carbocyclic) C;.; alkyl groups which have one or more carbon-carbon double bonds (also referred to as “C3.; cycloalkenyl” groups) include, but are not limited to, unsubstituted groups such as cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl, as well as substituted groups (e.g., groups which comprise such groups) such as cyclopropesnylmethyl and cyclohexenylmethyl.

C3-20 heterocyclyl: The term “Ci. heterocyclyl”, as used herein, } pertains to a monovalent moiety obtained by removing a hydrogen atom frorm a ring atom of a Cio heterocyclic compound, said compound having one ring, or two or more rings (e.g., spiro, fused, bricidged), and having from 3 to 20 ring atoms, atoms, of which from 1 to 10 are ring heteroatoms, and wherein at least one of said ring(s) is a heterocyclic ring.

Preferably, each xing has from 3 to 7 ring atoms, of which from 1 to 4 are r.ing heteroatoms. “Cj.” denotes ring atoms, whether carbon ateoms or heteroatoms.

Examples of Cj3.20 meterocyclyl groups having one nitrogen ring atom include, but are not limited to, those derived from aziridine, azetid ine, pyrrolidines (tetrahydropyrrole), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole), 2H-pyrrole or 3H-pyrrole (is opyrrole, 1scazole), piperidine, dihydropyridine, tetrahydropyridine, and azepine.

Examples of Cj_z0 heterocyclyl groups having one oxygen ring atom include, but are not limited to, those derived from oxirane, oxetane, oxolane (tetrahydrofuran), oxole (dihydrofuran), oxane (tetrahydropyran), dihydropyran, pyran (Ce), and oxepin. Examples of substituted Cj.;9 heterocyclyl groups include swmgars, in cyclic form, for example, furanoses and pyranoses, imcluding, for example, ribose, lyxose, xylose, ] galactose, sucrosse, fructose, and arabinose.

Examples of Cj.20 heterocyclyl groups having one sulphur ring atom include, but are not limited to, those derived from thiirane, thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran), and thiepane.

Examples of Cj.;0 heterocyclyl groups having two oxygen ring atoms include, but are not limited to, those derived from dioxolane, dioxane, and dioxepane.

Examples of Cj. heterocyclyl groups having two nitrogen ring atoms include, but are not limited to, those derived from 1lmidazolidine, pyrazolidine (diazolidine), imidazoline, pyrazoline (dihydropyrazole), and piperazine.

Examples of Cj3.0 heterocyclyl groups having one nitrogen ring atom and one oxygen ring atom include, but are not limited to, those derived from tetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole, dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, and oxazine.

Examples of C3.;0 heterocyclyl groups having one oxygen ring atom and one sulphur ring atom include, but are not limited to, those derived from oxathiolane and oxathiane {thioxane).

Examples of Cj3.0 heterocyclyl groups having one nitrogen ring atom and one sulphur ring atom include, but are not limited to, those derived from thiazoline, thiazolidine, and thiomorpholine.

Other examples of Cs;.psheterocyclyl groups include, but are not limited to, oxadiazine and oxathiazine.

Examples of heterocyclyl groups which additionally bear one or more oxo (=0) groups, include, but are not limited to, those derived from:

Cs heterocyclics, such as furanone, pyrone, pyrrolidone (pyr rolidinone), pyrazolone (pyrazolinone), imidazolidone, thia zolone, and isothiazolone;

Ce heterocyclics, such as piperidinone (piperidone), ] pipe:ridinedione, piperazinone, piperazinedione, pyridazinone, and pyrimidinone (e.g., cytosine, thymine, uracil), and barbeituric acid; fused heterocyclics, such as oxindole, purinone (e.g., guamaine), benzoxazolinone, benzopyrone (e.g., coumarin); cyclic anhydrides (-C(=0)-0-C(=0)- in a ring), including but not limited to maleic anhydride, succinic anhydride, and glutaric anhydride; cyclic carbonates (-0-C(=0)-0- in a ring), such as ethylene carbonate and 1,2-propylene carbonate; imides (-C(=0)-NR-C(=0)- in a ring), including but not limi. ted to, succinimide, maleimide, phthalimide, and glut arimide; lactones (cyclic esters, -0-C(=0)- in a ring), including, but not limited to, fB-propiolactone, y-butyrolactone, d-valerolactone (2-piperidone), and €-caprolactone; lactams (cyclic amides, -NR-C(=0)-~ in a ring), including, but not limited to, PB-propiolactam, y-butyrolactam (2~-pyrrolidone), o-valerolactam, and e-caprolactam; cyclic carbamates (-O-C(=0)-NR- in a ring), such as 2-oxazolidone; cyclic ureas (-NR-C{=0)-NR- in a ring), such as 2-irnidazolidone and pyrimidine-2,4-dione (e.g., thymine, uraccil).

Cs-2 aryl: The term “Cs.z9 aryl”, as used herein, pertains to a . monoovalent moiety obtained by removing a hydrogen atom from an aromnatic ring atom of a Cs.yp aromatic compound, said compound hav.ing one ring, or two or more rings (e.g. fused), and having from 5 to 20 ring atoms, and wherein at least one of said ring(s) is an aromatic ring. Preferably, each ring has from 5 to 7 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”, in which case the group may conveniently be referred to as a “Cs-39 carboaryl” group.

Examples of Cs.50 aryl groups which do not have ring hetercatoms (i.e. Cs.p0 carboaryl groups) include, but are not limited to, those derived from benzene (i.e. phenyl) (Cg), naphthalene (Cio), anthracene (C4), phenanthrene (C4), naphthacene (Cis), and pyrene (Cis) -

Examples of aryl groups which comprise fused rings, one of which is not an aromatic ring, include, but are not limited to, groups derived from indene and fluorene.

Alternatively, the ring atoms may include one or more heteroatoms, including but not limited to oxygen, nitrogen, and sulphur, as in “heteroaryl groups”. In this case, the group may conveniently be referred to as a “Cs.;0 heteroaryl” group, wherein "Cs_0” denotes ring atoms, whether carbon atoms or heteroatoms. Preferably, each ring has from 5 to 7 ring atoms, of which from 0 to 4 are ring heteroatoms.

Examples of Cs.;0 heteroaryl groups include, but are not limited to, Cs heteroaryl groups derived from furan (oxole), thiophene (thiole), pyrrole (azole), imidazole (1,3-diazole), pyrazole (1,2-diazole}, triazole, oxazole, isoxazole, thiazole, isothiazole, oxadiazole, and oxatriazole; and Cg heteroaryl groups derived from isoxazine, pyridine (azine), pyridazine (1,2-diazine), pyrimidine (1,3-diazine; e.g., cytosine,

thymine, uracil), pyrazine (1,4-diazine), triazine, tetrazole, and oxadiazole (furazan).

Examples of Cs.;0 heteroaryl groups which comprise fused rings, include, but are not limited to, Co heterocyclic groups derived from benzofuran, isobenzofuran, indole, 1isoindole, purine (e.g., adenine, guanine), benzothiophene, benzimidazole; Cig heterocyclic groups derived from quinoline, isoquinoline, benzodiazine, pyridopyridine, quinoxaline; C3 heterocyclic groups derived from carbazole, dibenzothiophene, dibenzofuran;

Cia heterocyclic groups derived from acridine, xanthene, phenoxathiin, phenazine, phenoxazine, phenothiazine.

The above C,-7 alkyl, Cs; heterocyclyl, and Cs. aryl groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below.

Halo: -¥, -Cl, -Br, and -1I.

Hydroxy: -OH.

Ether: -OR, wherein R is an ether substituent, for example, a

Ci1-7 alkyl group (also referred to as a C;-7 alkoxy group, discussed below), a Cj.; heterocyclyl group (also referred to as a Ci3.20 heterocyclyloxy group), or a Cs-pp aryl group (also referred to as a Cs-z9 aryloxy group), preferably a C;-7 alkyl group. :

Ci-7 alkoxy: -OR, wherein R is a Cj; alkyl group. Examples of

C,-7 alkoxy groups include, but are not limited to, -OCH; (methoxy), -OCH;CH; (ethoxy) and -OC(CH3); (tert-butoxy).

Ci-2 alkdioxylene: The term "C,., alkdioxylene," as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms from each of two different alcohol groups of a

Ci.2 hydrocarbon diol compound having from 1 or 2 carbon atoms, i.e. CH, (OH), and HO-CH,-CH,-OH, to form -0O-CH,-O- and -0O-CH;-

CHz-O-. This bidentate moiety may be the substituent group of a single atom or of two adjacent atoms.

Oxo (keto, -one): =0. Examples of cyclic compounds and/or groups having, as a substituent, an oxo group (=0) include, but are not limited to, carbocyclics such as cyclopentanone and cyclohexanone; heterocyclics, such as pyrone, pyrrolidone, pyrazolone, pyrazolinone, piperidone, piperidinedione, piperazinedione, and imidazolidone; cyclic anhydrides, including but not limited to maleic anhydride and succinic anhydride; cyclic carbonates, such as propylene carbonate; imides, including but not limited to, succinimide and maleimide; lactones (cyclic esters, -0-C(=0)- in a ring), including, but not limited to, B-propiolactone, y-butyrolactone, d-valerolactone, and e-caprolactone; and lactams (cyclic amides, -NH-C(=0)- in a ring), including, but not limited to, PB-propiolactam, y-butyrolactam (2- pyrrolidone), &-valerolactam, and e-caprolactam.

Imino (imine): =NR, wherein R is an imino substituent, for example, hydrogen, C;.; alkyl group, a Ci.zoheterocyclyl group, or a Cs.po aryl group, preferably hydrogen or a C;.; alkyl group.

Examples of imino groups include, but are not limited to, =NH, =NMe, =NEt, and =NPh.

Formyl (carbaldehyde, carboxaldehyde): -C(=0)H.

AMENDED SHEET

Acyl (keto): -C(=0)R, wherein R is an acyl substituent, for example, a Cy.salkyl group (also referred to as Ci-7 alkylacyl or

Ci-7 alkanoyl), a Cj.p0 heterocyclyl group (also referred to as

C3-20 heterocyclylacyl), or a Cs.y90 aryl group (also referred to as Cs-20 arylacyl), preferably a C;., alkyl group. Examples of acyl groups include, but are not limited to, -C{(=0)CH; (acetyl), -C(=0)CH,CH3 (propionyl), -C(=0)C(CHs); (butyryl), and -C(=0)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): -COOH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): -C(=0)OR, wherein R is an ester substituent, for example, a

Ci-7 alkyl group, a Ci. heterocyclyl group, or a Cs.y aryl group, preferably a Cy.;alkyl group. Examples of ester groups include, but are not limited to, -C(=0)0OCH3, -C(=0)0CH,CH;, -C(=0)OC(CH3)3, and -C(=0)OPh.

Acyloxy (reverse ester): -OC(=0)R, wherein R is an acyloxy substituent, for example, a C;.; alkyl group, a Cj. heterocyclyl group, or a Cs.;p aryl group, preferably a C;_;alkyl group. Examples of acyloxy groups include, but are not limited to, -0C(=0)CHj3 (acetoxy), -OC(=0)CH,;CH;, -~OC(=0)C (CHj3) 3, -0C (=0) Ph, and -0C (=0) CH,Ph.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): -C(=0)NR'R?, wherein R' and R? are independently amino substituents, as defined for amino groups. Examples of amido ) groups include, but are not limited to, -C(=0)NH,, -C(=0)NHCHs;, -C(=0)N(CH3)2, —-C(=0O)NHCH3CH;, and -C(=0)N(CH,CH3),, as well as : amido groups in which R' and R?, together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.

Acylamido (acylamino): -NR'C(=0)R?, wherein R! is an amide substituent, for example, hydrogen, a C; - alkyl group, a Cs 20 heterocyclyl group, or a Cs. aryl group, preferably hydrogen or a C;.; alkyl group, and R? is an acyl substituent, for example, a C;.; alkyl group, a Ci.;o heterocyclyl group, or a Cs. aryl group, preferably hydrogen or a C; ; alkyl group. Examples of acylamide groups include, but are not limited to, -NHC (=O) CH; , -NHC(=0)CH,CHs;, and -NHC(=0)Ph. R! and R? may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl and phthalimidyl: oO oo hey succinimidyl maleimidyl phthalimidyl

Thioamido (thiocarbamyl): -C(=S)NR'R?, wherein R! and R? are independently amino substituents, as defined for amino groups.

Examples of thiocamido groups include, but are not limited to, -C(=8)NH;, -C(=S)NHCH;, -C(=S)N(CHs3),, and -C(=S)NHCH,CH,.

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms and one carbon atom, hy

N\

NN

Amino: -NR'R?, wherein R! and R? are independently amino substituents, for example, hydrogen, a C;.; alkyl group (also

AMENDED SHEET referred to as C,.5 alkylamino or di-Cj.7 alkylamino), a Cj. heterocyclyl group, or a Cs-20 aryl group, preferably H or a

Ci-7alkyl group, or, in the case of a "cyclic" amino group, R! and R?, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Examples of amino groups include, but are not limited to, -NH,, -NHCH3;, -NHC (CH3) 5, -N (CH3) 5, -N (CH,CH3) 5, and -NHPh.

Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Imino: =NR, wherein R is an imino substituent, for example, hydrogen, a C;.; alkyl group, a C3.20 heterocyclyl group, or a

Cs-z0 aryl group, preferably H or a C;; alkyl group.

Amidine: -C(=NR)NR;, wherein each R is an amidine substituent, for example, hydrogen, a C,.; alkyl group, a C3.p9 heterocyclyl group, or a Cs.yo aryl group, preferably H or a C4 alkyl group.

An example of an amidine group is -C(=NH)NH,.

Nitro: -NO,.

Nitroso: -NO.

Azido: -Nj.

Cyano (nitrile, carbonitrile): -CN.

Isocyano: -NC.

Cyanato: -OCN.

Isocyanato: -NCO.

Thiocyano (thiocyanato): -SCN.

Isothiocyano (isothiocyanato): -NCS.

Sulfhydryl (thiol, mercapto): -SH.

Thioether (sulfide): -SR, wherein R is a thioether substituent, for example, a C;.; alkyl group (also referred to as a Cy; alkylthio group), a Cs;.»0 heterocyclyl group, or a Cs. 2 aryl group, preferably a C;.; alkyl group. Examples of Ci., alkylthio groups include, but are not limited to, -SCH; and -SCH,CH;.

Disulfide: -SS-R, wherein R is a disulfide substituent, for example, a C;.; alkyl group, a Ci;.yo heterocyclyl group, or a C=.z0 aryl group, preferably a C,.; alkyl group (also referred to herein as C;.; alkyl disulfide). Examples of C;.; alkyl disulfide groups include, but are not limited to, -SSCH; and -SSCH,CHs.

Sulfone (sulfonyl): -S(=0)3;R, wherein R is a sulfone substituent, for example, a C;.; alkyl group, a Ci.a0 heterocyclyl group, or a Cs.po aryl group, preferably a Ci, alkyl group. Examples of sulfone groups include, but are noft limited to, -S(=0),CH; (methanesulfonyl, mesyl), -S(=0).CF, (triflyl) ’ -S (=0) 2CH,CH3;, -S (=0) 2C4 Fy (nonaflyl) , -S (=0) 2CH,CF3 (tresyl), -S(=0);Ph (phenylsulfonyl), 4-methylphenylsulfonyl (tosyl), 4-bromophenylsulfonyl (brosyl), and 4 -nitrophenylsulfonyl (nosyl).

Sulfine (sulfinyl, sulfoxide): -S(=0)R, wherein R is a sulfime substituent, for example, a C;.; alkyl group, a Ci_z0

AMENDED SHEET heterocyclyl group, or a Cs. aryl group, preferably a C;., alkyl group. Examples of sulfine groups include, but are not limited to, -S(=0)CH; and -S(=0)CH,CH;.

Sulfonyloxy: -0S(=0).R, wherein R is a sulfonyloxy substituent, for example, a C;.; alkyl group, a Cs.,0 heterocyclyl group, or a

Cs-20 aryl group, preferably a C;.; alkyl group. Examples of sulfonyloxy groups include, but are not limited to, -0S(=0),CH, and -0S(=0),CH,CH;.

Sulfinyloxy: -0S(=0)R, wherein R is a sulfinyloxy substituent, for example, a C;.; alkyl group, a Cis.zo heterocyclyl group, or a

Cs.20 aryl group, preferably a C;.; alkyl group. Examples of sulfinyloxy groups include, but are not limited to, -0S(=0)CH; and -0S (=0) CH,CH,;.

Sulfamino: -NR'S(=0),0H, wherein R' is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, -NHS(=0),0H and -N (CH3) S(=0) ,0H.

Sulfinamino: -NR'S(=0O)R, wherein R! is an amino substituent, as defined for amino groups, and R is a sulfinamino substituent, for example, a C;.; alkyl group, a Ci.a0 heterocyclyl group, or a Cs.o aryl group, preferably a C;.5 alkyl group. Examples of sulfinamino groups include, but are not limited to, -NHS(=0)CH; and -N(CH;)S (=0)CgHs.

Sulfamyl: -S(=0)NR'R?, wherein R' and R® are independently amino substituents, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, -S(=0)NH,, -S(=0)NH (CH3) , -S(=O)N(CHs)2, -S(=0)NH(CH,CHs), -S(=0)N(CH,CH;),, and -S(=0)NHPh.

AMENDED SHEET sulfinamino groups include, but are not limited to, -NHS(=0)CH; and -N(CHj3) S{=0) C¢Hs.

Sulfamyl: -S(=0)NR'R?, wherein R! and R? are indepenciently amino substituents, as defined for amino groups. Example s of sulfamyl groups include, but are not limited to, -S (=0)NH,, ~S(=0)NH(CH3), -S(=0)N(CH3),, -S(=0)NH(CH;CH3), =-S(=C)N(CH,CH;)2, and -S(=0)NHPh.

Sulfonamino: -NR!'S(=0),R, wherein R! is an amino sub stituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a Cj;-7 alkyl group, a Cs.pp heterocyclyl group, or a Cs. aryl group, preferably a Cis alkyl group. Examples of sulfonamino groups includ e, but are not limited to, -NHS(=0),CH; and -N(CH3)S(=0),CeHs. A special class of sulfonamino groups are those derived from sultams - in these groups one of R!' and R is a Cs.5 aryl group, preferably phenyl, whilst the other of R! and R is ca bidentate group which links to the Cs-;0 aryl group, such as a bidentate group derived from a C;.; alkyl group. Examples of such groups include, but are not limited to: 0..,0° 0 —N 2, 3-dihydro-tenzo([d]isothiazole-1, 1-dioxide-2-yl 4) 0“ \ 0 1, 3-dihydro-benzo(clisothiazole-2, 2-dioxide-1-yl

Phosphoramidite: -OP(OR!)-NR?,, where R! and R? are phosphoramidite substituents, for example, -H, a (optionally substituted) C;.; alkyl group, a Cs.»p heterocyclyl group, or a

Cs-20 aryl group, preferably -H, a C;.; alkyl group, or a Cs.ap aryl group. Examples of phosphoramidite groups include, but are not limited to, -OP(OCH;CH;)-N(CH;),, -OP(OCH,CH;)-N(i-Pr),, and -OP (OCH,CH,CN)-N(i-Pr),.

Phosphoramidate: -OP(=0) (OR!) -NR?,, where R' and R? are phosphoramidate substituents, for example, -H, a (optionally substituted) C;.; alkyl group, a Ci. heterocyclyl group, or a

Cs.20 aryl group, preferably -H, a Ci.; alkyl group, or a Cs. aryl group. Examples of phosphoramidate groups include, but are not limited to, -OP(=0) (OCH;CH3)~-N(CH3),, -OP(=0) (OCH,CH,) -

N(i-Pr),, and -OP(=0) (OCH,CH,CN) -N(i-Pr),.

In many cases, substituents may themselves be substituted.

For example, a C;.; alkoxy group may be substituted with, for example, a C;.; alkyl (also referred to as a C;.; alkyl-C;.,alkoxy group), for example, cyclohexylmethoxy, a Ci.;p heterocyclyl group (also referred to as a Cj3.;¢ heterocyclyl-C; , alkoxy group), for example phthalimidoethoxy, or a Cs.zo aryl group (also referred to as a Cs.zoaryl-Ci.salkoxy group), for example, benzyloxy.

AMENDED SHEET

Cs-2_ring

The Cs.; ring in R® has at least two carbon-carbon double bond, by virtue of its fusion to a benzene or pyr idine ring and a

Cs-20 carboaryl group. The nitrogen ring atom of the pyridyl group, if present, does not form part of the Cs.; ring.

Thus, the Cs.; ring may be a Cs.; sulphur con taining heterocycle, a Cs.; oxygen heterocycle, a Cs_, nitrogen containing heterocycle or a Cs.; cyclic group containing at least 5 carbon ring atoms.

The second bridge group may typically be a single bond (resulting in a Cg ring), or have 1 or 2 atoms in a chain (resulting in C¢ and C; rings respectively), which atoms are usually selected from C, S, © and N, with substitution as appropriate.

Cs-7 sulphur containing heterocycle

The Cs.; sulphur containing heterocycle in R™ will have at least two carbon-carbon double bonds, by virtue o= its fusion to a benzene or pyridine ring and a Cs.zg carboarysl group. Examples of relevant Cg.; sulphur containing heterocy«les include, but are not limited to:

S

Cs: 1) thiophene

S S S S S

«0 0 0 OO

Oo S N

H 0 thiaine 4-thiaoxaine 1,4-dithiaine 4H-4-azathmaine 4-oxothiaine

AMENDED SHEET

S S S S S

0) S N

H

. . 0 thiaine 4-thiaoxaine 1,4-dithiaine 4H-4-azathiaine 4-oxothiaine

S S S S

— - N 4,5-dihydrothiaepine thiaepine 4-oxa-5-hydrothiaepine 4H-4-aza-5-hydrothiaepine a Ss a

O

4-azathiaepine 4-oxo-5-hydrothiaepine 5-hydro-1,4-dithiaepine

The Cs.; sulphur containing heterocycle may be substituted (when possible) by the substituent group listed above.

The groups shown above may in particular be substituted on the sulphur atom in the first bridge group by one or two oxo (=0) groups.

Cs-1_oxygen containing heterocycle

The Cs-; oxygen containing heterocycle in R?® will have at least two carbon-carbon double bonds, by virtue of its fusion to a benzene or pyridine ring and a Cs-zp carboaryl group. Examples of relevant Cs. oxygen containing heterocycles include, but are not limited to:

PAS

Cs: \ J furan 0 0 O Oo 0 «0 O00 0 © S N ] 0) 4H-pyran 1.4-dioxin 4-thiaoxaine 4H-1,4-Oxazine 4-pyrone (p-Isoxazine)

Oo 0 O Oo

O00 OO — O N

H

4,5-dihydrooxaepine oxaepine 5-hydro-1,4-dioxaespine 4H-4-aza-5-hydro-oxaepine 0 0] 0)

I 3) AR 1 Bb

Oo 4-azaoxaepine 4-oxo-4,5-hydrooxaepine 4-thia-5-hydro-oxaepine

The Cs.7 oxygen containing heterocycle may be substituted (when possible) by the substituent groups Misted above.

Cs. nitrogen containing heterocycle

The Cs.; nitrogen containing heterocycle in R® will have at least two carbon-carbon double bonds, by virtue of its fusion to a benzene or pyridine ring and a Cs. carboaryl group.

Examples of relevant Cs.» nitrogen cortaining heterocycles include, but are not limited to (illuastrated with RY = H):

N

1H-Pyrrole }

H H H H H

N N N N N

«0 0000 ° ° y i 1,4-Dihydro-pyridine 4H-[1,4]0xazine 4H-[1,4]Thiazine 1.4-Dihydro-pyrazine 0 4H-[1,4]Thiazine 1-oxide 0 OOO — oO N 4,5-Dihydro-1H-azepine 1H-Azepine 4,7-Dihydro-[1,4]oxazepine 4,5-Dihydro-1H-[1 4]diazepine

N N N

3 OO

BERGEN

Oo . 1H-[1,4|Diazepine 1-5-Dihydro-azepin-4-one 4,7-Dihydro-[1,4]thiazepine

The Cs.; nitrogen containing heterocycle may be substituted (when possible) by the substituent group listed above. In particular, the nitrogen atom in the first bridge group may be substituted by RN.

Cs-7_cyclic group containing at least 5 carbon ring atoms

The Cs.7 cyclic group containing at least 5 carbon ring atoms in R® will have at least two carbon-carbon double bonds, by virtue of being fused to a benzene or pyridine ring and a Cs_y carboaryl group. Examples of relevant Cs. cyclic group containing at least 5 carbon ring atoms include, but are not limited to: :

Cs: \ J]

Cyclopersta-1,3-diene 0 0 0 0 i

Oo S N 'e} Cyclohexa-1.4-diene 4H-Pyran 4H-Thiopyran 1.4-Dihydro-pyridine 4H-Thiopyran 1-oxide «OO 0 ®) N

H

Cyclohepta-1, 4-diene Cyclohepta-1,3,5-triene 2,5-Dihydro-oxepine 2,5-Dihydro-1H-azepine 0 4H-Mzepine Cyclohepta-2,5-dienone ~~ 2,5-Dihydro-thiepine

The Cs. cyclic group containing at least 5 carbon ring atoms may be substituted (when possible) by the substituent group listed abowe.

Possible R® Structures

Accordingly, when the phenyl or pyridyl group is linked to a :

Cs-290 carboarryl group, R’ can be of the following structure, wherein the phenyl or pyridyl group and the Cs.;o carboaryl group are Zillustrated as benzene rings, without being limited thereto, amd where X may be 0, S, S(=0), S{=0),, NR" and

CREIR®?:

SW! 3 :

S

QL CQ QUO

EN NN

X NT Ny

H O

OU

X X (3 0) oO

S S 0) 0 sScXsSecRsPcHNgle!

H with substitution as appropriate on the above core structures

When the phenyl or pyridyl group is linked to a Cs. carboaryl group in which one aromatic carbon ring atom has been replaced by an aromatic nitrogen ring atom, then R’ can be any of the structures shown above, where the benzene ring represents a

Cs-20 carboaryl group containing a nitrogen ring atom, for example: gi

NS

X

~~ = x Na

X

N~ | N S = ©

XX NN ~

X X N X

H 0

N

9

Nao I J ~

X NT TX where X is as defined above.

If the first group in R® is a pyridyl group, rather than the phenyl group as illustrated above, the nitrogen ring atom may be at any available position of the ring.

The first bridge may be situated at any possible position of the phenyl group in R’ and the optional second bridge group may be situated on either adjacent atom of the phenyl group (if possible). Therefore, the resulting R’ group (as a whole) may be a radical at a number of possible positions on the benzene ring bound to the central moiety, for example the following possible R’ group (unsubstituted xanthenyl): o 4 7 2 may be a radical at the 1, 2, 3 or 4 positions.

Includes Other Forms

Included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (-COOH) also includes the anionic (carboxylate) form (-COO"), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N*HR!R?), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group.

Similarly, a reference to a hydroxyl group also includes the anionic form (-O07), a salt or solvate thereof, as well as conventional protected forms of a hydroxyl group.

Isomers, Salts, Solvates, Protected Forms, and Prodrugs

Certain compounds may exist 1n one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z- forms; c¢c-, t-, and r- forms; endo- and exo-forms; R-, S$S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and B-forms:; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).

Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers”, as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, -OCH;, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, =-CH,0H.

Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta- chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., Cy; alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

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

H 0 _ NN ,OH HW \ pe

TN pe PAIN pe PRN keto enol enolate

Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including *H, 2H (D), and ’H (T); C may be in any isotopic form, including °c, 3c, and ''C; 0 may be in any isotopic form, including '®0 and %0; and the like.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation

(e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, and protected forms of thereof, for example, as discussed below.

It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, “Pharmaceutically Acceptable Salts”, J. Pharm.

Sci., Vol. 66, pp. 1-19.

For example, if the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO0"), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na' and K', alkaline earth cations such as Ca’" and Mg‘, and other cations such as Al**. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NHs') and substituted ammonium ions {(e.g.,

NH3;R*, NH,R;', NHR3*, NR;'). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is

N{(CH3)q'.

formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulphuric, sulphurous, nitric, nitrous, phosphoric, and phosphorous. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: acetic, propionic, succinic, glycolic, stearic, palmitic, lactic, malic, pamoic, tartaric, citric, ascorbic, maleic, hydroxymaleic, phenylacetic, glutamic, aspartic, benzoic, cinnamic, pyruvic, salicyclic, sulfanilic, 2-acetyoxybenzoic, fumaric, phenylsulfonic, toluenesulfonic, methanesulfonic, ethanesulfonic, ethane disulfonic, oxalic, pantothenic, isethionic, wvaleric, lactobionic, and gluconic. Examples of suitable polymeric anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

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

It may be convenient or desirable to prepare, purify, and/or handle the active compound in a chemically protected form.

The term “chemically protected form”, as used herein, pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions, that is, are in the form of a protected or protecting group (also known as a masked or masking group or a blocked or blocking group).

AMENDED SHEET are in the form of a protected or protecting group (also known as a masked or masking group or a blocked or blocking group) -

By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting group may be removed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example, Protective Groups in Organic Synthesis (T. Green and P. Wuts, Wiley, 1999).

For example, a hydroxy group may be protected as an ether (-

OR) or an ester (-0OC(=0)R), for example, as: a t-butyl ether; a benzyl, benzhydryl (diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester (-0OC(=0)CHsj, -0OAC) .

For example, an aldehyde or ketone group may be protected as an acetal or ketal, respectively, in which the carbonyl group (>C=0) 1s converted to a diether (>C(OR);,), by reaction with, for example, a primary alcohol. The aldehyde or ketone group 1s readily regenerated by hydrolysis using a large excess of water in the presence of acid.

For example, an amine group may be protected, for example, as an amide or a urethane, for example, as: a methyl amide (-NHCO-CH3): a benzyloxy amide (-NHCO-OCH,C¢Hs, -NH-Cbz); as a t-butoxy amide (-NHCO-OC(CHj3)3, -NH-Boc); a 2-biphenyl-2- propoxy amide (-NHCO-OC(CHj;),CgH4CeHs, -NH-Bpoc), as a 9- fluorenylmethoxy amide (-NH-Fmoc), as a 6-nitroveratryloxy amide (-NH-Nvoc), as a 2-trimethylsilylethyloxy amide (-NH-

Teoc), as a 2,2,2-trichloroethyloxy amide (-NH-Troc), as an allyloxy amide (-NH-Alloc), as a 2 (-phenylsulphonyl)ethyloxy amide (-NH-Psec); or, in suitable cases, as an N-oxide (>NO-°).

For example, a carboxylic acid group may be protected as an ester for example, as: an Cj; alkyl ester (e.g. a methyl ester; a t-butyl ester); a C,.; haloalkyl ester (e.g., a Ci-7 trihaloalkyl ester); a triCi- alkylsilyl-C;.7 alkyl ester; or a

Cs-20 aryl-C;.; alkyl ester (e.g. a benzyl ester; a nitrobenzyl ester); or as an amide, for example, as a methyl amide.

For example, a thiol group may be protected as a thioether (-SR), for example, as: a benzyl thioether; an acetamidomethyl ether (-S-CH,;NHC(=0)CHj;).

It may be convenient or desirable to prepare, purify, and/or handle the active compound in the form of a prodrug. The term “prodrug”, as used herein, pertains to a compound which, when metabolised (e.g. in vivo), yields the desired active compound. Typically, the prodrug is inactive, or less active than the active compound, but may provide advantageous handling, administration, or metabolic properties.

For example, some prodrugs are esters of the active compound (e.g. a physiologically acceptable metabolically labile ester). During metabolism, the ester group (-C(=0)OR) is cleaved to yield the active drug. Such esters may be formed by esterification, for example, of any of the carboxylic acid groups (-C(=0)OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required. Examples of such metabolically labile esters “include those wherein R is C,.; alkyl (e.g. -Me, -Et); C4 aminoalkyl (e.g. aminoethyl; 2-(N,N-diethylamino)ethyl;

2-(4-morpholino)ethyl); and acyloxy-C,., alkyl (e.g. acyloxymethyl; acyloxyethyl; e.g. pivaloyloxymethyl; acetoxymethyl; l-acetoxyethyl; 1-(l-methoxy-l-methyl)ethyl- carbonxyloxyethyl; 1-(benzoyloxy)ethyl; isopropoxy- carbonyloxymethyl; l-isopropoxy-carbonyloxyethyl; cyclohexyl- carbonyloxymethyl; l-cyclohexyl-carbonyloxyethyl; cyclohexyloxy-carbonyloxymethyl; l-cyclohexyloxy- carbonyloxyethyl; (4-tetrahydropyranyloxy) carbonyloxymethyl; 1-(4-tetrahydropyranyloxy)carbonyloxyethyl; (4-tetrahydropyranyl)carbonyloxymethyl; and l1-(4-tetrahydropyranyl)carbonyloxyethyl).

Also, some prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound. For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.

Further Preferences

The following preferences may be different for different aspects of the present invention, and may be combined together.

In compounds of formula I, it is preferred that P is O and Q is CH, i.e. that the compound is of formula Ia.

Y 1s preferably O.

In formula I, when R! and R? form, along with the nitrogen atom to which they are attached, a heterocyclic ring having from 4 to 8 atoms, this may form part of a Cy.20 heterocyclyl group defined above (except with a minimum of 4 ring atoms), which must contain at least one nitrogen ring atom. It is preferred

Single rings having one nitrogen atom include azetidine, pyrrolidine (tetrahydropyrrole), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole), piperidine, dihydropyridine, tetrahydropyridine, and azepine; two nitrogen atoms include imidazolidine, pyrazolidine (diazolidine), imidazoline, pyrazoline (dihydropyrazole), and piperazine; one nitrogen and one oxygen include tetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole, dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, and oxazine; one nitrogen and one sulphur include thiazoline, thiazolidine, and thiomorpholine.

Preferred rings are those containing one heteroatom in addition to the nitrogen, and in particular, the preferred heteroatoms are oxygen and sulphur. Thus preferred groups include morpholino, thiomorpholino, thiazolinyl. Preferred groups without a further heteroatom include pyrrolidino.

The most preferred groups are morpholino and thiomorpholino.

As mentioned above, these heterocyclic groups may themselves be substituted; a preferred class of substituent is a Cj. alkyl group. When the heterocyclic group is morpholino, the substituent group or groups are preferably methyl or ethyl, and more preferably methyl. A sole methyl substituent is most preferably in the 2 position.

As well as the single ring groups listed above, rings with bridges or cross-links are also envisaged. Examples of these types of ring where the group contains a nitrogen and an oxygen atom are:

AMENDED SHEET

As well as the single ring groups listed above, rings with bridges or cross-links are also envisaged. Examples of these types of ring where the group contains a nitrogen and an oxygen atom are: 0 Oo © oc (7) —N ~ Way g

These are named 8-oxa-3-aza-bicyclo[3.2.1]Joct-3-yl, 6-oxa-3- aza-bicyclo(3.1.0]hex-3-yl, 2-oxa-5-aza-bicyclo(2.2.1]hept-5- yl, and 7-oxa-3-aza-bicyclo[4.1.0]hept-3-yl, respectively.

In R?, the phenyl or pyridyl group is preferably a phenyl group.

Rand R®? are preferably H.

RY is preferably H, or an ester.

Preferred substituents of the phenyl or pyridyl ring in R3 include, but are not limited to, halo, hydroxy, C;-7 alkyl, Ci_—, alkoxy, acyl, acyloxy, amino, nitro, cyano, thiol and C,-7 alkylthio, with halo and hydroxy being most preferred.

Preferred substituents of the phenyl or pyridyl ring or the C s. »¢ carboaryl group in R® also include, but are not limited to, acylamido, sulfonamino, ether, ester, amido, amino and acyl.

In the acylamido group, the amide substituent is preferably hydrogen, and the acyl substituent is preferably selected from ester (where the ester substituent is alkyl or aryl), C,.4

A particularly preferred acyl susbtituent on the acylamido group is of formula III:

R’ * N 1] ~ ~R* ( ) wherein n is 1 to 4, preferably 1 or 2, and R?® and R* are independently hydrogen, an optionally substituted C;.; alkyl group, Ci3.pp heterocyclyl group, or Cs.;p aryl group, or may together form, along with the nitrogen atom to which they are attached, an optionally substituted heterocyclic ring having from 4 to 8 ring atoms.

In the sulfonamino group, the amino substituent is preferably hydrogen and the sulfonamino substituent selected from C;_- alkyl and Cs.z¢ aryl.

In the ether group, the ether substituent is preferably C,.; alkyl (optionally substituted by amino, Ci.;, heterocyclyl, thioether and Cs.,0 aryl). A particularly preferred ether substituent is of formula III (defined above).

In the amido group, the amido substituents are preferably independently selected from hydrogen and C;.; alkyl (optionally substituted by C;.;0 heterocyclyl, Cs.a0 aryl and amino). A particularly preferred amido substituent is of formula III (defined above).

In the acyl group, the acyl substituent is preferably Ci. heterocyclyl.

These substituents are preferably either para to the radical position in the phenyl or pyridyl group, and when the first

AMENDED SHEET

In the acyl group, the acyl substituent is preferably Cj-z0 heterocyclyl.

These substituents are preferably either para to the radical

BS) position in the phenyl or pyridyl group, and when the first bridge group is ortho the radical position in the phernyl or pyridyl group, para to the first bridge group in the Cs-20 aryl group (especially when that group is phenyl).

Preferred structures for R’ include, but are not limit ed to the following ‘core’ groups, which may bear substitution at appropriate positions, where * indicates the preferred radical position (which is typically adjacent the first bridge group on the phenyl group):

See J ®. )

X * X * xX . with the most preferred core structures being:

Cr CL

Be x >

H

94 9g g 7 \

JZ s as

Particularly preferred R® groups include: 0 R, ,O

J 2S.

R NH O NH OR

B® $ ( _ ~

S S

O O

Jy Us

R N R

® @ S

S wherein R stands for the appropriate substituent group, as defined above.

Acronyms

For convenience, many chemical moieties are represented using well known abbreviations, including but not limited to, methyl (Me), ethyl (Et), n-propyl (nPr), iso-propyl (iPr), n-butyl (nBu), tert-butyl (tBu), n-hexyl (nHex), cyclohexyl (cHex), phenyl (Ph), biphenyl (biPh), benzyl (Bn), naphthyl (naph),

methoxy (MeO), ethoxy (EtO), benzoyl (Bz), acety=l (Ac), 1,3- bis (diphenylphosphino) propane (dppf).

For convenience, many chemical compounds are reporesented using well known abbreviations, including but not limi ted to, methanol (MeOH), ethanol (EtOH), iso-propanol (i -PrOH), methyl ethyl ketone (MEK), ether or diethyl ether (Et,0 ), acetic acid (AcOH), dichloromethane (methylene chloride, DCMI), trifluoroacetic acid (TFA), dimethylformamide ([DMF), tetrahydrofuran (THF), and dimethylsulfoxide (DMISO).

Synthesis Routes

Compounds according to the first aspect of the invention, of formula Ia, where Y=0, may be synthesised by thes coupling of a

Z-chloro-6-amino-pyran-4-one to an appropriate aarylboronic acid or arylboronate ester using a palladium cat-alysed coupling reaction, e.g. Suzuki coupling. Compouands where Y=S can be derived from the corresponding compound wshere Y=0.

Synthesis of 2-chloro-6-amino-pyran-4-ones

These may be synthesised by the following route:

Ca o 0 0 puNEE Vw =A oe 0 (a) (b) NT To ) 0 cl l, (©) _R 0 R Cl Oo N o) R? (1)

In step (a) CCls is added across the carbon-carb on double bond of diketene by free-radical addition to yield 4—chloro- 4(2,2,2,~trichloro-ethyl)-oxetan-2-one (1). Sui_table initiators include peroxide, such as BCHPO ((biss-4-t- butylcyclohexyl)peroxydicarbonate).

In step (b), the amine R'R?NH opens the lactone ring by nucleophilic attack at the carbonyl centre. The oxy anion generated then displaces the chlorine atom on the o-carbon to give rise to a PB-keto-amide intermediate. Further elimination of HCl finally give the 5,5-dichloro-1-amino-pent-4-ene-1, 3- dione. Suitable conditions for this step include inorganic base such as sodium hydrogen carbonate and solvent such as dry dichloromethane.

In step (c), ring closure takes place by displacement of one of the 5-chloro groups by the oxygen of the amide moiety to form the pyran-4-one ring, which reaction is catalysed by a

Lewis acid, such as perchloric acid.

Arylboronic Acids and Arylboronate esters

Some appropriate arylboronic acids and arylboronate esters are commercially available. Other appropriate arylboronic acids and arylboronate esters may be synthesised by using one of the following routes, in which the starting materials are commercially available or readily synthesised. For example, a synthesis route to thioxanthenone is described in Archer, S., et al., J. Med. Chem., 25, 220-227, 1982, and the conversion of thioxanthenone to thiothanxene is described in Mlotkowska,

B.L., et al., J. Heterocyclic Chem., 28, 731-736, 1991. Other routes are shown in the examples, and include routes where the central Cs.; ring is synthesised by ring closure from an appropriate carboxylic acid, optionally followed by reduction of the remaining keto group.

Synthesis of aryl boronate esters

O_o xX B (a) _ —_— X= TfO, Br,

R R

(a): PdCi2dppf, dppf, Pinacol diborane, KOAc where R is the remainder of the R® group

Aryl boronate esters may be formed by Pd(0)-catalysed cross coupling reaction of the appropriate aryl triflate or aryl halide with tetra{alkoxy)diboron, e.g. pinacol diboron.

Suitable conditions include the use of a catalyst, such as

PdCl,dppf, extra ligands, such as dppf, potassium acetate as a base, in a solvent such as dioxane, DMF or DMSO.

Examples of this method are to be found in T Ishiyama, et al.,

Tet. Lett., vol. 38, no. 19, 3447-3450, 1997 and A Giroux, et . al., Tet. Lett., vol. 38, no. 22, 3841-3844, 1997.

Synthesis of aryl boronic acids

HO_ _OH

B

(J Cl

R R

(a):t-Buli, (E1O),B where R is the remainder of the R® group

Boronic acids may be generated via lithiation of the aromatic ring by tert-butyl lithium followed by the reaction of the anion formed with alkyl borate such as triethyl borate to give the desired aryl boronic acid.

Palladium Catalysed Coupling

The coupling of the arylboronic acid or arylboronate ester to the 2-chloro-6-amino-pyran-4-one can be carried out using the normal conditions, e.g. a palladium catalyst (Pd (PPhy)g,,

Pd (dppf) Cl,) and base (Na,CO;, NaOCH,CH;, T1OH, N(CH,CH3)s,

K3POy4) .

Compounds according to the first aspect of the invention, of formula Ib, where Y=0 may be synthesised according to the following method, wherein R represents the rest of R’:

Oo OH S

ANS a SH

R ———— R

OH S OH S

N R' " SEY c NSN —_— R —— R | 2

R

O SEt . 1 R pr _R R [ d N e No 2 0

In step (a), CS, is added to the acetophenone derivative, in the presence of a base, such as potassium tert-butoxide, to yield a 3-aryl-3-hydroxy-dithiocacrylic acid.

In step (b), iodoethane undergoes nucleophliic attack by the activated thioacid, to yield the ethyl ester. Activation of the thioacid can be achieved by the use of base, for example, a mixture of tetrabutylammonium hydrogen sulphate and sodium hydroxide.

In step (c), an amine displaces the ethyl group, which is followed in step (d) by reaction of the remaining thio group with iodoethane (via the tautomeric compound).

The final step (e) is a condensation with ethyl bromoacetate to yield the ring-closed 4-amino-6-aryl-pyran-2-one.

Conversion of Y from O to S

This conversion may be achieved using Lawesson’s reagent in an organic solvent, such as toluene, followed by the appropriate purification steps. Protection of groups sensitive to

Lawesson’s reagent can be carried out before it is used, followed by deprotection once the pyranthione has been synthesised.

Use of Compounds of the Invention

The present invention provides active compounds, specifically, active 2Z2-aryl-6-amino-pyran-4-ones, 2-aryl-6-amino-pyran-4- thiones, 4-amino-6-aryl-pyran-2-ones and 4-amino-6-aryl-pyran- 2-thiones.

The term “active”, as used herein, pertains to compounds which are capable of inhibiting ATM activity, and specifically includes both compounds with intrinsic activity (drugs) as well as prodrugs of such compounds, which prodrugs may themselves exhibit little or no intrinsic activity.

One assay which may be used in order to assess the ATM inhibition offered by a particular compound is described in the examples below.

The present invention further provides a method of inhibiting

ATM in a cell, comprising contacting said cell with an effective amount of an active compound, preferably in the form of a pharmaceutically acceptable composition. Such a method may be practised in vitro or in vivo.

For example, a sample of cells (e.g. from a tumour) may be grown in vitro and an active compound brought into contact with said cells in conjunction with agents that have a known curative effect, and the enhancement of the curative effect of the compound on those cells observed.

The present invention further provides active compounds which inhibit ATM activity as well as methods of inhibiting ATM activity comprising contacting a cell with an effective amount of an active compound, whether in vitro or in vivo.

The invention further provides active compounds for use in a method of treatment of the human or animal body. Such a method may comprise administering to such a subject a therapeutically-effective amount of an active compound, preferably in the form of a pharmaceutical composition.

The term “treatment” as used herein in the context of treating a condition, pertains generally to treatment and therapy,

whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a

BS) halt in the rate of progress, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e. prophylaxis) is also included.

The term “therapecutically-effective amount” as used herein, pertains to that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio.

The present inventors have. found that compounds of the present invention can efficiently repress retroviral vector transduction in one-step, cell based integration assays (termed LUCIA) and inhibit HIV-1 infection in 4-day replication assays at sub-micromolar concentrations. Further, in contrast to the observations of Daniel et al., where it was concluded that the effect of ATM on retroviral integration would only be seen in a DNA-PK-deficient background, this effect works in the presence of functional DNA-PK activity.

Initial linkage of linear retroviral DNA with host cell chromosomal DNA is catalysed by viral integrase (IN) and results in short staggered DNA strand breaks in the host cell

DNA at the site of attachment (Brown, P.O. (1990) Integration of retroviral DNA. Curr Top Microbiol Immunol, 157, 19-48).

These gapped DNA intermediates are shown to be sensed as sites . of DNA damage by the host cell and repaired by the ATM pathway to complete the process of integration and allow productive infection to occur. Compounds of the invention prevent the repair of gapped DNA intermediates by the ATM pathway and thus brevent complete integration of retroviral DNA into th.e host genome.

As described above, the invention provides a compound as defined in the first aspect of the invention for use i n the treatment of retroviral infection and the use of such a compound in the manufacture of a medicament for use in the treatment of retroviral infection.

Also provided by the invention is a method of treatmen t of a retroviral infection comprising administering a compou nd as defined in the first aspect of the invention to an individual in need thereof.

An exemplary compound of the invention which is shown to be useful in the treatment of retroviral infection is 2-

Thianthren-1-yl~6-morpholin-4-yl-pyran-4-one (4).

Retroviral mediated diseases which may be treated as d._escribed above include HIV infection and acquired immunodeficie ncy syndrome (AIDS) and Human T-cell Leukaemia virus (HTLV) infection and its associated diseases adult T-cell leukaemia/lymphoma (ATLL) and tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM) .

Compounds of the invention may be used in combination with other retroviral therapies to suppress virus replicati on, for example in a ‘highly active anti-retroviral therapy’ o-r HAART treatment.

The invention provides a pharmaceutical composition comprising a compound as described herein and one or more other anti- retroviral agents.

The invention also provides a composition comprising a compound as defined in the first aspect of the invention and one or more other anti-retroviral agents for treatment of a retroviral infection and the use of such a composition in the manufacture of a medicament for use in the treatment of a retroviral infection.

Suitable anti-retroviral agents which inhibit retroviral replication, for example retroviral protease inhibitors (PI) such as Sequinavir, Indinavir, Ritonavir and Nelfinavir, nucleoside retroviral reverse transcriptase inhibitors such as 3'-azido-3'deoxythymidine (AZT; Zidovudine), 2', 3'-

Dideoxycytosine (ddC; Zalcitabine), 2', 3'-Dideoxyinosine (ddI; Didanosine)and 3TC; (Lamivudine), and non-nucleoside retroviral reverse transcriptase inhibitors such as

Nevirapine, Delavirdine and Efavirenz.

Administration

The active compound or pharmaceutical composition comprising the active compound may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose): rectal; vaginal; : parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular,

subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneowmusly or intramuscularly.

The subject may be a eukaryote, an animal, a vesrtebrate animal, a mammal, a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a Frrimate, simian (e.g. a monkey or ape), a monkey (e.g. marmosett, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, gibloon), or a human.

Formulations

While it is possible for the active compound to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g. formulation) comprising at least one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, &=djuvants, excipients, diluents, fillers, buffers, stabiliisers, preservatives, lubricants, or other materials wvell known to those skilled in the art and optionally other t-herapeutic or prophylactic agents.

Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one active compound, as defined above, together wit h one or more pharmaceutically acceptable carriers, excipilent.s, buffers, adjuvants, stabilisers, or other materials, as described herein.

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

Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical

Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into assoclation the active compound with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, losenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.

Formulations suitable for oral administration (e.g. by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each comtaining a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous OT non-aqueous liquid; or as an oil-in-water liguid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary:- or as a paste.

A tablet may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may oe prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g. povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g. lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc, silica); disintegrants (e.g. sodium starch glycolate, cross-linked povidone, cros:s-linked sodium carboxymethyl cellulose); surface-acti ve or dispersing or wetting agents (e.g. sodium lauryl sul fate); and preservatives (e.g. methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid). Moulded tablets may be made by moulding in a suitable machine a mixture of the powdiered compound moistened with an inert liquid diluent. The tabelets may optionally be coated or scored and may be formulated so as to provide slow : or controlled release of the active co mpound therein using, for example, hydroxypropylmethyl cellu lose in varying proportions to provide the desired rel ease profile. Tablets may optionally be provided with an ent eric coating, to provide release in parts of the gut other than the stomach.

Formulations suitable for topical administration (e.g. transdermal, intranasal, ocular, buccal, and sublingual) may be formulated as an ointment, cream, suspension, lotion, powder, solution, past, gel, spray, aerosol, or oil.

Alternatively, a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active compounds and optionally one or more excipients or diluents.

Formulations suitable for topical administration in the mouth include losenges comprising the active compound in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active compound in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active compound in a suitable liquid carrier.

Formulations suitable for topical administration to the eye also include eye drops wherein the active compound 1s dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active compound.

Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or : oily solutions of the active compound.

Formulations suitable for adm inistration by inhalation include those presented as an aerosol spray from a pressurised pack, with the use of a suitable pr opellant, such as dichlorodifluoromethane, tric hlorofluoromethane, dichoro- tetrafluoroethane, carbon dio xide, or other suitable gases.

Formulations suitable for top-ical administration via the skin include ointments, creams, an d emulsions. When formulated in an ointment, the active compo-und may optionally be employed with either a paraffinic or a water-miscible ointment base.

Alternatively, the active compounds may be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream ba se may include, for example, at least about 30% w/w of a poly-hydric alcohol, i.e., an alcohol having two or more hydroxyl asroups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.

When formulated as a topical emulsion, the oily phase may optionally comprise merely am emulsifier (otherwise known as an emulgent), or it may complises a mixture of at least one emulsifier with a fat or an ©il or with both a fat and an oil.

Preferably, a hydrophilic emualsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include botth an oil and a fat. Together, the emulsifier (s) with or witthout stabiliser (s) make up the so-called emulsifying wax, ard the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.

Suitable emulgents and emulsion stabilisers include Tween 60,

Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low.

Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required.

Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

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

Formulations suitable for parenteral administration (e.g. by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, 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, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.

Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's

Solution, or Lactated Ringer’s Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 pg/ml, for example from about 10 ng/ml to about 1 ug/mml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condit®on requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may lobe prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs.

Dosage

It will be appreciated that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

Administration in vivo can be effected in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) throughout the course of treatment.

Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject ) being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

In general, a suitable dose of the active compound is in the range of about 100 pg to about 250 mg per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, prodr-ug, or the like, the amount administered is calculated on tthe basis of the parent compound and so the actual weight tmo be used is increased proportionately.

EXAMPLES

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

In these examplles, reference is made to the following figures.

Figure 1 shows that compound 4 can sensitise cells to ionising radiation. The % survival of HeLa cells was measured with increasing ioniising radiation, in the absence of compound 4 (®m), and at two different concentrations of compound 4, 0.5 uM (A) and 2 uM (es).

Figure 2 shows that compound 4 can sensitise cells to etopside. The % survival of LoVo cells was measured with increasing concentrations of etopside, in the absence of compound 4 (WM). and in the presence of 10uM of compound 4 (e).

Figure 3 shows that compound 4 can sensitise cells to camptothecin. The % survival of LoVo cells was measured with increasing concentrations of camptothecin, in the absence of compound 4 (MW), and in the presence of 10pM of compound 4 (ee).

Figure 4 shows that compound 4 can sensitise cells to doxorubicin. "The % survival of LoVo cells was measured with increasing concentrations of doxorubicin, in the absence of compound 4 (MB), and in the presence of 10uM of compound 4 (e).

Figure 5 shows that compound 4 can inhibit recombinant retroviral vector infections. The inhibition of retroviral transduction by the ATM inhibitor Compound 4 was assessed by performing HIV-1 based LUCIA on Jurkat T-cells in the presence of increasing concentrations of compound 4 (¢). Data are presented as transduction efficiency (luciferase signal) relative to untreated control cells. The ICsy concentration for HIV-1 infections by compound 4 is around 1 uM. Drug cytotoxicity (0) was determined by MTS formazan dye reduction . "assays and data are presented as the percentage of viable cells remaining after drug treatment. No significant cytotoxicity was observed over the concentration range of

Compound 4 tested.

Figure 6 shows that Compound 4 does not inhibit HIV-1 RT. The inhibition of HIV-1 RT was assessed by performing chemilluminescent HIV-1 reverse transcriptase assays in the presence of increasing amounts of compound 4 (4). No significant anti-RT activity for compound 4 is observed over the concentration range used. Control RT inhibition using nevirapine (0) is also shown.

Figure 7 shows that Compound 4 acts synergistically with AZT to inhibit HIV-1 infections. HIV-1 based LUCIA was performed on Hela cells with increasing concentrations of Compound 4 in the absence (A) or presence of 0.1 pM (OJ), 0.4 pM (*) or 1.2 uM (0) AZT. Data are presented as transduction efficiency (as determined by luciferase activity) relative to untreated control cells. The combined presence of both Compound 4 and

AZT shows enhanced anti-HIV activity when compared to each drug alone.

Figure 8 shows that Compound 4 inhibits HIV-1 replication. 4- day HIV-1 replication assays were performed on C1866 cells in the presence of increasing concentrations of Compound 4 (4) or

AZT (0). HIV-1 titres were quantified by p24 antigen ELISA and data are shown as the percentage of HIV-1 p24 in cell-free supernatants relative to untreated control cells. (A)

Replication assays performed using wild type HIV-1 strain (HIV-1yxsz wt). {B) Replication assays performed using an AZT resistant HIV-1 strain (HIV-1luxp, AZTres). Compound 4 inhibits

HIV-1 replication equally well in both wild-type and AZT resistant HIV-1 strains. (C) Control drug cytotoxicity (A) was determined by XTT dye reduction assays. Data are presented as the percentage of viable cells remaining after drug treatment. No significant cytotoxicity was observed over the effective Compound 4 concentration range shown to inhibit

HIV-1 replication.

A) Chemical Examples

General Experimental Methods

Thin layer chromatography was carried out using Merck

Kieselgel 60 F;s54 glass backed plates. The plates were visualized by the use of a UV lamp (254 nm). Silica gel 60 (particle sizes 40-63 pu) supplied by E.M.Merck was employed for flash chromatography. 'H NMR spectra were recorded at 300

MHz on a Bruker DPX-300 instrument. Chemical shifts were referenced to tetramethylsilane.

Purification and identification of libraries samples

The samples were purified on Gilson LC units.

Mobile phase A - 0.1% aqueous TFA, Mobile phase B -

Acetonitrile, Flow rate 6 ml/min., Gradient - typically starting at 90% A/10% B for one minute, rising to 97% B after 15 minutes, holding there for 2 minutes, then back to the starting conditions. Column: Jones Chromatography Genesis 4p

C18 column, 10 mm x 250 mm. Peak acquisition based on UV detection at 254 nm.

Mass Specs were recorded on a Finnegan LCQ instrument in positive ion mode.

Mobile phase A - 0.1% aqueous formic acid, Mobile phase B -

Acetonitrile, Flow rate 2 ml/min., Gradient - starting at 95%

A/5% B for one minute, rising to 98% B after 5 minutes, : holding there for 3 minutes, then back to the starting conditions. Column - Phenomenex 5p Luna C18 column, 4.6 mm x 50 mm

UV detection at 254 nm, PDA detection scanning from 210 to 600 nm.

Mass spectra of Other Compounds

Mass spectra of non-library compounds and building blocks were recorded on a Micromass 2ZQ instrument (single quadrupole, operating in electrospray ionisation mode), using a Waters 600

HPLC Pump and 2700 Autosampler.

Mobile Phase A: 0.1% Formic acid in water, Mobile phase B: 0.1% Formic acid in acetonitrile, Flow rate: 2.0 ml/min.,

Gradient: 538 to 95%B over 3mins, hold 3mins. Column: Varies, but always C18 50 mm x 4.6 mm (Currently Genesis C18 4 p.

Jones Chromatography). PDA detection: Waters 996, scan range 210-400 nm.

Synthesis of 2-Chloro-6-morpholin-4-yl-pyran-4-one (3)

Ch Cl 0

Cl Oo 0)

Lo ag MOK a. Ia! hs Av] Cl NT a” Yo” ON 0O 0) (_o 0 Ls 1 2 3 4-Chloro-4-(2,2,2-trichloro-ethyl) ~oxetan-2-one (1)

A solution of BCHPO (bis-4-t-butylcyclohexyl)peroxydicarbonate (11.8 g) and diketene (83.5 ml) in CCl, (300 ml) was added dropwise over 120 minutes to a refluxing solution of CCl,, and was stirred for a further 1 hour. The resulting pale yellow solution was cooled and azeotroped with DCM. The resulting residue was stirred with hexane (3 x 150 ml) for 10 minutes and the liquor was decanted off through a celite pad. The filtered liquors were combined and concentrated in vacuo to give 1 as a pale yellow oil (125.0 g, 52.9%). 5,5-Dichloro-l1-morpholin-4-yl-pent-4-ene-~1,3-dicne (2)

Two separate solutions of 1 (62.5 g, 0.26 mmol) and morpholine (24.0 g, 0.28 mol) in DCM (120 ml) were added simultaneously to a mixture of NaHCO; (44.0 g, 0.52 mol) in dry DCM (300 ml).

The reaction was maintained at 15°C over 140 minutes with stirring. The reaction was filtered, washed with DCM (3 x 100 ml) and the combined organic layers were concentrated in vacuo to a slurry which was then passed through a short silica pad, and further washed with DCM (4 x 100 ml). The combined organic layers were concentrated in vacuo, suspended in hexane (400 ml) and stirred for 1 hour, filtered and dried to give a cream solid. The solid was suspended in TBME (100 ml), stirred for 15 minutes, filtered, washed with TBME and dried to give 2 as a white powder (47.8 g, 72%). m/z (LC-MS, ESP): 252 (M'

2-Chloro-6-morpholin-4-yl-pyran-4-one (3)

To a suspension of 2 (11.3 g, 44.9 mmol) in dioxane was added perchloric acid (11.4 ml, 0.14 mol) and the reaction was } heated at 90°C under N;, for 1 hour. The reaction was cooled, neutralised with 2M NaOH (75 ml) and filtered. The aqueous layer was extracted with DCM (4 x 30 ml) and the organic layers were combined and dried over MgSO,. The organic layer was further treated with charcoal and filtered through celite.

The dark yellow filtrate was evaporated in vacuo, and the resulting solid was triturated with hexane (50 ml) and dried to give 3 (7.3 g, 75%) as a light yellow powder. m/z (LC-MS,

ESP): 216 (M' +1). H-NMR (300MHz, DMSO-d¢): 3.3 (t, 4H), 3.65 (t, 4H), 5.4 (d, 1H), 6.25 (d, 1H).

Example 1: Synthesis of 2-Thianthren-1-yl-6-morpholin-4-yl- pyran—-4-one (4)

To

Sher

S oh 4 2~-Chloro-6-morpholin-4-yl-pyran-4-one (3) (863 mg, 4 mmol), thianthrene-1l-boronic acid (1.145 g, 4.4 mmol), and ground potassium carbonate (1.105 g, 8 mmol) were suspended in dioxane (10 ml) and degassed (sonication for 5 minutes then saturated with Nz). Pd(PPh3)s (231 mg, 0.2 mmol) was then added and the reaction mixture was then heated at 90°C for 24 hours under a vigorous stirring and a N; atmosphere. The solvent was removed in vacuo and the residue was then suspended in water 50 ml) and extracted with ethyl acetate (3 x 100 ml). The organics were combined, washed with saturated brine and dried over sodium sulphate. The solvent was removed in vacuo and the residue was purified by column chromatography (silica; ethyl acetate:ethanocl; 9:1) to give the title compound as a white solid (70 mg, 4%). 'H-NMR (300MHz,DMSO-de¢) : Oy = 3.44 (4H, t,

J 5Hz); 3.76 (4H, t, J 5Hz); 5.57 (1H, d, J 2Hz):; 6.30 (1H, d,

J 2Hz); 7.43 (2H, m); 7.53 (lH, t, 8Hz); 7.66 (3H, m); 8.49 (1H, dd, J land 8 Hz). m/z (LC-MS, ESP) : 396 (M' +1).

Example 2: Synthesis of 2-Phenoxathiin-4-yl-6-morpholin-4-yl- pyran-4-one (5)

To her 6] sal 5 2-Chloro-6-morpholin-4-yl-pyran-4-one (3) (863 mg, 4 mmol), phenoxathiin-4-boronic acid (1.07 g, 4.4 mmol), and ground potassium carbonate (1.1 g, 8 mmol) were suspended in dioxane (10 ml) and degassed (sonication for 5 minutes then saturated with N;). Pd(PPhs3)s (231 mg, 0.2 mmol) was then added and the reaction mixture was then heated at 90°C for 24 hours under a vigorous stirring and a N; atmosphere. The solvent was removed in vacuo and the residue was then suspended in water (50 ml) and extracted with ethyl acetate (3 x 50 ml). The organics were combined, washed with saturated brine and dried over sodium sulphate. The solvent was removed in vacuo and the residue was purified by column chromatography (silica; ethyl acetate:ethanol; 9:1) to give the title compound as a white solid (620 mg, 41%). 'H-NMR (300MHz, DMSO-dg¢) : & = 3.38 (4H, t, J SHz); 3.71 (4H, t, J SHz); 5.49 (1H, d, J 2Hz); 6.49 (1H, d, J 2Hz); 7.06 (1H, dd, J 1 and 8Hz); 7.26 (4H, m); 7.46 (1H, dd, J 1.5 and 8Hz); 7.55 (1H, dd, J 1.5 and 8 Hz). m/z (LC-

MS, ESP) : 380 (M' +1).

Example 3: Synthesis of 2-Dibenzofuran-l1-yl-6-morpholin-4-yl- pyran-4-one (6)

To

SYS

Oo 6 2-Chloro-6-morpholin-4-yl-pyran-4-one (3) (22 mg, 0.1 mmol), 4- dibenzofuran-l-boronic acid (28 mg, 0.13 mmol), and caesium carbonate (65 mg, 0.2 mmol) were suspended in dioxane (0.5 ml) and degassed (sonication for 5 minutes then saturated with Nj).

Pd (PPh3)s (5 mg, 0.005 mmol) was then added and the reaction mixture was then heated at 90°C for 24 hours under a vigorous stirring and a N; atmosphere. The reaction mixture was purified by preparative HPLC to give the title compound (2.1 mg; 6%). m/z (LC-MS, ESP): 348 (M' +1).

Example 4: Synthesis of 2-Dibenzothiophen-1-yl-6-morpholin-4- yl-pyran—-4-one (7) (oo

Svs 0] 7 2-Chloro-6-morpholin-4-yl-pyran-4-one (3) (740 mg, 3.43 mmol), dibenzothiophene-1l-boronic acid (860 mg, 3.77 mmol), and ground potassium carbonate (964 mg, 6.86 mmol) were suspended in dioxane (10 ml) and degassed (sonication for 5S minutes then . saturated with Np). Pd(PPhj3), (200 mg, 0.17 mmol) was then added and the reaction mixture was then heated at 90°C for 24 ’ hours under a vigorous stirring and a N; atmosphere. The solvent were removed in vacuo and the residue was then suspended in water (50 ml) and extracted with ethyl acetate (3

Xx 50 ml). The organics were combined, washed with saturated brine and dried over sodium sulphate. The solvent was removed in vacuo and the residue was purified by column chromatography (silica; ethyl acetate:ethanol; 9:1) to give the title compound as a white solid (80 mg, 6%). "H-NMR (300MHz, DMSO- de): Oy = 3.49 (4H, t, J SHz); 3.76 (4H, t, J 5SHz); 5.53 (1H, d, J 2Hz); 6.63 (1H, d, J 2Hz); 7.59 (2H, m); 7.69 (1H, t, J 8BHz); 7.96 (1H, dd, J 1 and 7.5Hz); 8.11 (1H, m); 8.47 (1H, m); 8.57 (1H, dd, J 1 and 8 Hz). m/z (LC-MS, ESP): 364 (M' +1).

Example 5: Synthesis of 2-(2-Phenylsulfanyl-phenyl)-6- morpholin-4-yl-pyran-4-one (9) (a) 2-phenylsulfido-benzene boronic acid (8)

HO_ _OH

B

IC

8

To a cooled (-78°C), stirred solution of diphenyl sulphide (1.66 ml, 10 mmol) in 30ml anhydrous THF, was added dropwise under a nitrogen atmosphere 7 ml t-BuLi. Upon addition of t-

BuLi the solution turned orange then brown. The mixture was allowed to warm to room temperature and then left stirring for 3 hours. The mixture was cooled (-78°C). Triethyl borate (2.03 ml, 12 mmol) was added dropwise to the cooled yellow solution turning the solution lime coloured. During this addition, the temperature was monitored and not allowed to rise above -75°C. The solution was then left to warm to room temperature and left stirring for 2 hours. Water was added to the reaction mixture and the aqueous were extracted with diethyl ether. The agueous layer (pH 14) was acidified to pH

1 with (1 M HCl) and the product was extracted into diethyl ether. The organics were dried over magnesium sulphate and the organics were evaporated off in vacuo, yielding an oily residue (690 mg, 30%), which was used without further 5 purification. (b) 2-(2’-Phenylsulfido-phenyl) -6-morpholin-4-yl-pyran-4-one (9)

To

O No a

DA

9 2-Chloro-6-morpholin-4-yl-pyran-4-one (3) (582 mg, 2.7 mmol), 2-phenylsulphido-benzene boronic acid (8) (690 g, 3 mmol), and ground potassium carbonate (819 mg, 5.94 mmol) were suspended in dioxane (10ml) and degassed (sonication for 5 minutes then saturated with N;). Pd(PPhs)s (156 mg, 0.13 mmol) was then added and the reaction mixture was then heated at 90°C for 24 hours under a vigorous stirring and a N; atmosphere. The solvent was removed in vacuo and the residue was purified by preparative

HPLC to give the title compound (27mg, 3%). 'H-NMR (300MHz,

DMSO-dg): Oy = 3.37 (4H, t); 3.76 (4H, t) 5.45 (1H, d); 6.31 (1H, d); 7.32-7.55 (9H, m). m/z (LC-MS, ESP): 366 (M' +1).

Example 6: Synthesis of 2-(1-Fluoro-9-oxo-9H-thioxanthen-4- yl) -6-morpholin-4-yl-pyran-4-one (13) 0. 0

OH SF

OH 0" Ck

S S F

== C1) = CT ] © F O F 10 11 o_O F ] 4 0

S Oo Oo NS c d_,

S

O F P O

12 13 a: H,50,, Thiosalicylic acid; b: Tf,O, pyridine; c¢: bis(pinacolato)diboron, PdCLdppf, dppf, dioxane, 100°C; d: chloropyranone, Pd(PPh,),, dioxane, 90°C (a) 1-Fluoro~4-hydroxy-thioxanthen-9-one (10)

Thiosalicylic acid (46.26 g, 0.3 mol) and 4-fluorophenol (56.05 g, 0.5 mol) were dissolved in conc. HS804 (750 ml) and the mixture was stirred under nitrogen for 24 hours. The reaction mixture was then poured onte ice (1.5 L) and the yellow precipitate was filtered and washed with water (300 ml). The precipitate was dried at 50°C for 24 hours and was used without further purification (31.4 g, 42.5%). m/z (LC-MS,

ESP): 247 (M" +1).

(b) 1-fluoro-9-oxo-thioxanthen-4-yl trifluoromethane sulfo nate 1-Fluoro-4-hydroxy-thioxanthen-9-one (4.92 g, 20 mmol) was dissolved in dry pyridine (100 ml) and cooled to 0°C under a . nitrogen atmosphere. Triflic anhydride (3.66 ml, 22.3 mmol ) was added drop wise to the stirred solution over 5 minutes-.

The reaction was left overnight and was then poured onto weater (300 ml) and the precipitate formed was filtered. The soli.d was purified through a plug of silica (ethyl acetate:hexarme; 1:9) to give the title compound as a white fluffy solid (L .72 g, 22.4%). m/z (LC-MS, ESP): 379 (MY +1). (c) 1-Fluoro-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2—yl) - thioxanthen-9-one (12) 1-fluoro-9-oxo-thioxanthen-4-yl trifluoromethane sulfonate (11) (378 mg, 1 mmol), bis (pinacolato)diboron (305 mg, 1.2 mmol), and ground potassium acetate (294mg, 3mmol) were suspended in dioxane (5 ml) and degassed (sonication for minutes then saturated with N;). PdCl,dppf (40 mg, 0.050 mmol) and dppf (27.7 mg, 0.05 mmol) was then added and the reaction mixture was then heated at 100°C for 24hrs under a vigororus stirring and a N; atmosphere. The solvent was removed in wacuo and the residue was purified by column chromatography (si lica:; ethyl acetate:ethanol; 9:1) to give an oil which was used without further purification (116 mg, 32%). m/z (LC-MS, E SP): 357 (M' +1).

(d) 2-(1-Fluoro-9-oxo-thioxanthen-4-yl)-6-morpholin-4-yl- pyran-4-one (13) 2-Chloro-6-morpholin-4-yl-pyran-4-one (3) (100 mg, 0.46 mmol), 1-Fluoro-4-(4,4,5, 5~tetramethyl- (1, 3,2]dioxaborolan-2-yl)-

thioxanthen-9-one (12) (110 mg, 0.31 mmol), and ground potassium carbonate (63mg, 0.62 mmol) were suspended in dioxane (5 ml) and degassed (sonication for 5 minutes then saturated with Nj). Pd(PPh3), (18 mg, 0.016 mmol) was then added and the reaction mixture was then heated at 90°C for 24 hours under a vigorous stirring and a N; atmosphere.

The solvent were removed in vacuo and the residue was then suspended in water (50 ml) and extracted with ethyl acetate (3 x 50 ml). The organics were combined, washed with saturated brine and dried over sodium sulphate.

The solvent was removed in vacuo and the residue was purified by column chromatography (silica: ethyl acetate:ethanol; 9:1) to give the title compound as a white solid (5 mg, 4%). m/z (LC-MS, ESP): 410 (M* +1).

Example 7: Synthesis of 2-(1-Fluoro-9H-thioxanthe=n-4-yl)-6- morpholin-4-yl-pyran-4-one (17) 0, _o0

OH OH 0x -

Oo F F F 14 15.

PLO ou A - = —_— S

DA

16 F 17 a: BH;-THF; b: Tf,0, pyridine; c: bis(pinacolato)diboron, PdCl,dppf, dp pf, dioxane, 100°C; d: chloropyranone, Pd(PPh,),, dioxane, 90°C (a) 1-Fluoro-9H-thioxanthen-4-o0l (14) 1-Fluoro-4-hydroxy-thioxanthen-9-one (4.93 g, 20 mmol) was dissolved in THF (50 ml) and cooled down to 0°C umder a N, atmosphere. Borane-tetrahydrofuran complex (1M, 6:0 ml, 60 mmol) was added drop wise to the stirred solutiom over 10 minutes. The reaction was left to react overnight. and was then quenched with acetone (100 ml). The mixture was e=vaporated to dryness and the residue was taken into water (200 ml). The product was extracted in ethyl acetate (3 x 100 mil) and the organics were combined, dried over sodium sulphat.e and evaporated in vacuo. The residue was purified by column chromatography (silica, hexane:ethyl acetate, 9:1.) to give a white solid which is readily oxidised by air (2.1.9 g, 47%). H-

AMENDED SHEET

NMR (300MHz, DMSO-dg¢): &y = 3.86 (2H, s); 6.73 (1H, m); 6.95 (1H, m); 7.24 (2H, m) 7.47 (2H, m); 10.07 (1H, s). (b) 1-fluoro-9H-thioxanthen-4-yl trifluoromethane sulfonate (15) 1-Fluoro-9H-thioxanthen-4-0l (1.66 g, 7.15 mmol) was dissolved in dry pyridine (35 ml) and cooled to 0°C under a nitrogen atmosphere. Triflic anhydride (2.22 g, 7.87 mmol) was added dropwise to the stirred solution over 5 minutes. The reaction was left to react for 4 hours at room temperature and was then pour onto water (350 ml). The milky solution was extracted with DCM (3 x 200 ml), the organics were combined and dried over magnesium sulphate. The solvent was removed in vacuo and the solid obtained was purified through a plug of silica (ethyl acetate:hexane; 3:97) to give the title compound as a white fluffy solid (2.55 g, 98%). H-NMR (300MHz, DMSO-dg): Oy = 3.86 (2H, s); 7.3-7.6 (6H, m) (c) 1-Fluoro-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)- 9H-thioxanthe (16) l1-fluoro-9H-thioxanthen-4-yl trifluoromethane sulfonate (1 g, 2.75 mmol), bis(pinacolato)diboron (840 mg, 3.30 mmol), and ground potassium acetate (809 mg, 8.25 mmol) were suspended in dioxane (7 ml) and degassed (sonication for 5 minutes then saturated with Nj). PdCl.dppf (0.112 mg, 0.138 mmol) and dppf (77 mg, 0.138 mmol) was then added and the reaction mixture was then heated at 100°C for 24 hours under a vigorous stirring and a N, atmosphere. The solvent were removed in vaccuo and the residue was purified by column chromatography (silica; ethyl acetate:ethanol; 9:1) to give an oil which was used without further purification (460 mg, 49%).

AMENDED SHEET

(d) 2-(1-Fluoro-9H-thioxanthen-4-yl)-6-morph-olin-4-yl-pyran-4- one (17) 2-Chloro-6-morpholin-4-yl-pyran-4-one (3) (252 mg, 1.17 mmol), ' 1-Fluoro-4-(4,4,5,5-tetramethyl-(1,3,2]dioxaaborolan-2-yl)-~9H- thioxanthe (400 mg, 1.17 mmol), and ground pootassium carbonate (239 mg, 2.34 mmol) were suspended in dioxarae (7 ml) and degassed (sonication for 5 minutes then satuarated with Nj).

Pd (PPh3), (67 mg, 0.059 mmol) was then added and the reaction mixture was then heated at 90°C for 24hrs urader a vigorous stirring and a N; atmosphere. The solvent were removed in vaccuo and the residue was purified by colurmn chromatography (silica; ethyl acetate:ethanol; 9:1) to give an off white solid which was triturated in ether and gave the title compound as a white solid (72.3 mg, 16%). 'F3-NMR (300MHz, DMSO- de): 64 = 3.41 (4H, t); 3.71 (4H, t) 5.50 (1H, d):; 6.21 (1H, dy; 7.25-7.35 (3H, m); 7.52-7.62 (3H, m). m_/z (LC-MS, ESP): 396 (M* +1).

Example 8: Synthesis of 2-Thianthren-1l-yl-6 -morpholin-4-yl- pyran-4-thione (18) o Fo oN_J o_N_

S Toluene, reflux S

SH Cro (18) 2-Thianthren-1-yl-6-morpholin-4-yl-pyran-4—one (4) (140 mg, 0.354 mmol) was dissolved in toluene (5 ml) . Lawesson's reagent (215 mg, 0.53 mmol) was added to the solution and the ’ mixture was refluxed overnight under nitrocggen with stirring. ‘

The toluene was evaporated off in vacuo ancd the residue was purified via column chromatography (silica, dichloromethane) to give the desired compound (18) as dark orange solid (27 mg,

18%) .'H-NMR (300MHz, DMSO-d¢): &; = 3.56 (4H, t, J SHz); 3.73 (4H, t, J 5Hz); 7.83 (1H, d, J 2Hz): 7.76 (1H, d, J 2Hz):; 7.30-7.80 (7H, m). m/z (LC-MS, ESP):412 (M' +1).

Example 9: 2~(7-Amino~9H-thioxanthen-4-yl)-6-morpholin~4-yl- pyran-4-one N-amide derivatives )

ON

Be hd 0 OMe

S Ss

OH , — nee ow — 00 0 O,N O,N © o} re ~o0 0 OH joes

O,N HN H,N o. 0

OH OT¢ B joe® (J) — IX pe I 3S

BocNH BocNH BocNH g o_N_ C o_N_J 0 Uo i

SER. DEE:

BocNH H,N (19) (20) 2-(2-Methoxy-phenylsulfanyl)-5-nitro-benzoic acid 2-Methoxythiophenol (9.9ml, 81.29mmol) was added to a solution of KOH (18.24g, 325.18mmol) in water (80ml) degassed for 15 minutes. 2-Bromo-5-nitrobenzoic acid (20.0g, 81.29mmol) and copper bronze (258mg, 4.06mmol) were added to the reaction mixture, which was refluxed overnight. The reaction was stopped and the mixture was filtered t hrough a celite pad and washed with 2M NaOH then water (50ml). The filtrate was acidified (pH 1) with concentrated HCl . The precipitate formed ’ was filtered and dried overnight in a vacuum oven (50°C) to give the crude title compound (26.0 g) as a pale yellow solid.

The product was used without further pourification. 5-Methoxy-2-nitro-thioxanthene-9-one 2-(2-Methoxy-phenylsulfanyl)-5-nitro-toenzoic acid (13.00g, 42 .58mmol) was suspended in methanesul_phonic acid (100ml) and heated at 100°C. The crude mixture was slowly poured onto ice with vigorous stirring then neutralized with conc. ammonia solution. The precipitate was filtered and washed with water.

The yellow/ lime colored solid was dried under vacuum at 50°C to give the crude title compound which was used without any further purification (12g, 98%). m/z (LC-MS, ESP), RT=4.89 min, (M'+1)= 288. 5-Methoxy-2-nitro 9H-thioxanthene

To a cooled (0°C) suspension of 5-methoxy-2-nitro- thioxanthene-9-one (24.46g, 85.13mmol ) in anhydrous tetrahydrofuran (40ml) under nitrogen atmosphere, was added drop wise borane-THF complex (170ml, '1.0M in THF). The mixture was allowed to warm to room temperatu.re with stirring overnight. The reaction mixture was c ooled (0°C) and the excess borane was guenched with aceto ne. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (1:1, dichloromethane/ hexane) to give the title k compound (11.59g, 50%) as a bright ye llow amorphous solid.

HNMR (300MHz, DMSO-d¢): dw= 3.86 (3H, s), 4.03 (2H, s), 7.00 : (2H, dd), 7.28 (1H, t), 7.73 (1H, d), 8.05 (1H, dd), 8.28 (1H, d).

5-Methoxy-9H-thioxanthen-2-ylamine 5-Methoxy-2-nitro 9H-thioxanthene (11.59g, 42.40mmol) was suspended in ethyl acetate (250ml). SnCl;.2H,0 (47.84qg, 212mmol) was added and the clear yellow solution was stirred at 50°C overnight. The reaction was the quenched with NaOH (2M) and then extracted with ethyl acetate (3x300ml). The organics were washed with saturated brine (100ml), dried over magnesium sulphate and the solvents were removed in vacuo to give the title compound (10.32g, 100%) as viscous yellow oil.

The oil was used without further purification. . 'HNMR (300MHz,

DMSO-dg) : Oy= 3.83 (3H, s), 3.67 (2H, s), 5.14 (2H, bs), 6.43 (1H, dd), 6.61 (1H, d), 6.89 (1H, d), 6.89 (1H, d), 7.06 (1H, d), 7.18 (1H, t). m/z (LC-MS, ESP), RT=3.88 min, (M' +1)= 244. 7-Amino-9H-thixanthen-4-ol 5-Methoxy-9H-thioxanthen-2-ylamine (10.32g, 41.09mmol) and pyridine hydrochloride (49.0g, 424mmol) were heated at 200°C under nitrogen atmosphere for 5 hours. The black reaction mixture was allowed to cool down to room temperature and water (50ml) was then added. The mixture was neutralized with NaOH (2M) to pH 7 then extracted with dichloromethane (4x100ml).

The organics were washed with saturated brine, dried over

MgS0O,4) and concentrated in vacuo to give a black oil. This oil was purified by flash chromatography (dichloromethane) to give the title compound (7.78g, 80%) as dark brown oil which was used without further purification. HNMR (300MHz, DMSO-dg): &y= 3.61 (2H, s), 5.08 (2H, bs), 6.42 (1H, dd), 6.58 (1H, d), 6.69 (1H, d), 6.81 (1H, d) 6.95-7.06 (2H, m), 9.88 (1H, bs); m/z (LC-MS, ESP), RT= 3.23 min, (M' +1)= 230. (5~-Hydroxy-9H-thioxanthen-2-yl)-carbamic acid tert-butyl ester

To a solution of 7-amino-9H-thixanthen-4-ol (7.77 g, 81.32 mmol) in THE (14 ml) was added dropwise di-tert-butyl dicarbonate (17.74 mg, O.49 mmol) in THF (4 ml). The reaction was stirred at room temperature under nitrogen atmosphere.

Upon completion of the rmmeaction the solvent was evaporated.

The residue was taken up in methanol (50ml), and sodium } hydroxide (4.06g, 10l1.1&mmol) was added. The dark brown mixture was refluxed fow 20 minutes. The solvent was evaporated in vacuo and the oil was taken up in water, extracted with ethyl acestate, dried over MgSO, and evaporated in vacuo to give the crude product. The dark brown oil was purified by flash chromatography (dichloromethane) to give the title compound (4.2g, 38%), as a cream coloured amorphous solid. 'HNMR (300MHz, DMSO-d¢): &y= 3.74 (2H, s), 6.74 (1H, d), 6.87 (1H, 4d), 7.04 (1H, tt), 7.23-7.33 (2H, m), 7.57 (1H, bs}, 10.03 (1H, bs). (5-Trifluoromethanesul f onyl-9H-thioxanthen-2-yl)-carbamic acid tert-butyl ester

To a cooled (0°C) golden colored solution of (5-hydroxy-9H- thioxanthen-2-yl)-carba mic acid tert-butyl ester (4.0gqg, 12.14dmmol) in anhydrous pyridine (8ml) under nitrogen atmosphere was added tr-ifluoromethanesulphonic anhydride (2.36ml, 13.35mmol) drosp wise. The solution turned deep orange upon addition of trifluworomethanesulphonic anhydride. The reaction was allowed to warm to room temperature. After 10 minutes of stirring at this temperature the solution was poured into water (20mlL). The product was extracted with ethyl acetate. The organics were washed with saturated brine, dried over MgSO, and co ncentrated in vacuo to give the title , compound (5.6g, 100%) @=s a dark orange solid.

{5-(4,4,5,5-Tetramethyl-[1,3,2)dioxaborolan-2-yl) -9H- thioxanthen~-2-yl]-carbamic acid tert-butyl ester (5-Trifluoromethanesulfonyl-9H-thioxanthen-2-yl)-carbamic acid tert-butyl ester (3.31g, 7.17mmol), bis(pinacolato)diboron (2.18g, 8.e6mmol) and potassium acetate (2.11g, 21.5mmol) in 1,4-dioxane (20ml) was degassed for 15 minutes. To the yellow suspension was then added PdCl1, (dppf) (293mg, 0.36mmol) and dppf (199mg, O.36mmol). The dark red mixture was heated to 90°C under a N; atmosphere for 48 hours. The crude mixture was purified by flash chromatography (dichloromethane) to give viscous brown oil (3.15qg), which was used without any further purification. [5-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-9H-thioxanthen-2- yl] -carbamic acid tert-butyl ester (19) {5-(4,4,5,5-Tetramethyl-[1,3,2}dioxaborolan-2-yl)}-9H- thioxanthen-2-yl]-carbamic acid tert-butyl ester (1.02g, 2.32mmol), 2-chloro-6-morpholin-4-yl-pyran-4-one (3) {0.60qg, 2.78mmol) and K;CO; (0.64g, 4.64mmol) were dissolved in dry 1,4-dioxane (ml). The mixture was degassed for 15 minutes and

Pd (PPhs}s (0.13g, 0.12mol) was then added The dark brown mixture was heated to 90°C under an atmosphere of N, for 24 hour. The reaction mixture was concentrated in vacuo and water (50ml) was added. The brown solid was filtered and washed with water (1.21g, 88%). m/z (LC-MS, ESP), RT = 4.6 minutes, (M'+1l)= 493. 2-(7-Amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one (20)

To a solution of [5-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-9H- thioxanthen-2-yl]-carbamic acid tert-butyl ester (19) (1.08 gq, 2.19 mmol) in dichloromethane (10 ml) was added trifluoroacetic acid (2 ml) and left under stirring at room temperature overnight. The solvent was dried in vacuo reveal. ing a viscous dark brown liquid. Saturated sodium bicarbonate solution (20 ml) was added to the residue, which was left to stir for 20 mins. The brown precipitate was . filtemred, washing with water and left to dry in the vacuum oven overnight (0.77g, 90%). HNMR (300MHz, DMSO-dg): &y= 3.40 (4H, uw), 3.70 (4H, t), 3.77 (2H, s), 5.23 (2H, bs), 5.50 (1H, d), 6 .17 (1H, d), 6.44 (1H, dd), 6.65 (1H, d), 7.09 (1H, d), 7.3% (1H, tt), 7.47-7.59 (2H, m); m/z (LC-MS, ESP), RT= 3.51 minuteses, (M'+1)= 392. 2-(7-_Amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one

N-ami.de derivatives (a) 'To a small test tube was added 2-(7-amino-9H-thioxanthen- 4-yl) -6-morpholin-4-yl-pyran-4-one (20) (20mg, 0.05mmol), dry dimet hylacetamide (0.5ml), triethylamine (0.01ml, 0.08mmol) and t he desired acid chloride (0.08mmol) with stirring overn.ight. The reaction was purified by preparative HPLC to give the desired products, which are shown below:

To ot

JT

H

Il I Ba el 5 te ne a

BREAN EN

IE Fc a EO 23 i 3.58 493

TT

Aa 3 _0 } 95 3.66 521 cr 26 _N 90 3.53 498 gy 27 I3 90 4.16 487

PZ

28 ON 90 3.43 498 lJ } 29 95 4.44 | 527 h Ch Bb - } he } )

NS i} 33 o 90 4.13 516

Ng 34 0 90 3.64 479 or Halll

HO 4.12 517 ak

N,

N . / 36 JA 3.43 546

TL

37 ~ 3.91 555

CI

38 ! 0” 4.16 587 pe ~o .

39 I 90 3.59 507 ~o . 40 Q 3.5 493

Mg. 41 > 90 4.1 569 i

JG . 42 O— 85 4.31 515 43 CTL 4.16 539 : 95 4.46 573 44 N oO

F - 45 O~n 95 4.02 — 46 S 95 4.72 586

N \

Ban 47 7 95 3.67 488

NO oA ”

Q 95 3.42 493

HO . :

AN 95 3.38 505

HO

D . lo} 50 Q FF 3.48 565

FF

(b) To a small test tube was added 2-(7-amino-9H-thioxanthen- 4-yl)-6-morpholin~-4-yl-pyran-4-one (20) (20mg, 0.05mmol), dry dimethylacetamide (0.5ml), triethylamine (8pl, 0.06mmol) and chloroacetyl chloride (4pl, 0.06mmol) with stirring overnight. ’

The appropriate amine or thiol (20mg or 20pl) was then added and left to stir at room temperature overnight. The reaction was purified by preparative HPLC to give the desired products, which are shown below: o oN.

Dolo

Oo

OI

R N

H

Compound TR Purity | Retention Time | M'+1

Mins) 52 (Te 95 2.98 519 wh) 53 (NT 95 2.98 533

NL

54 HO 95 2.98 538

J

C

OH

55 AOS. 95 3.23 566

Je

I I I

H

To

H

BES

HEE vr /

HEE v

H

BEEN

) _ - B

H oJ 63 ISR 95 3.18 570 ~o ) iS _ B

N 37 >.

H

65 ~ 95 ] 3.1 506

SoM

H

66 HA 95 2.96 67 0 95 2.97 563

NPN

H

OC “a

H

{c) To a small test tube was added 2-(7-amino-9H-thioxanthen- 4-yl)-6-morpholin-4-yl-pyran-4-one (20) (20mg, 0.05mmol), dry dimethylacetamide (0.5ml), triethylamine (8upl, 0.06émmol) and 3-bromopropionyl chloride (5pl, 0.05mmol) with stirring overnight. The appropriate amine or thiol (20mg or 20pl, hydrochloride salts were freed by addition of triethylamine) was then added and left to stir at room temperature overnight.

The reaction was purified by preparative HPLC to give the desired products, which are shown below: 0 on)

S | . 0

JT

R N

H

Compound R Purity | Retention Time | M® +1 (Mins) 3.17 5 32

N

70 HN 2.88 5 33

Love ~. 7 SN 95 2.98 5 47

LN. 72 TU 95 2.95 1 5 52

Ho Nao, 73 ~o 95 3.19 580

Sg oNe, oA ”

H

76 95 3.08 518

Ch 77 2.81 =61 (A 78 H 95 2.83 47 79 3.2 S46

Ne. 9 .84 .591

CC [Cea TT

No~N, 81 i! 95 3.3 584

BEE i} tt \Y

N- NH

83 ™ 3.06 520

SN, 84 HN A. 90 2.98 464 85 : oY y 95 2.89 577

LN, 86 0, 95 3.14 | 548

Neo.

Example 10: 2-(4-Hydroxy-9H-thioxanthen-1yl)-6-morpholin-4- yl-pyran-4-one ether derivatives

OH OH

SH S S

Cl — COD — C0 —

O 0 Br Br oBOC OBOC HO oo

S S o NJ

CL) — (J — 3

Br BL $®

Oo 0 0

VI 87 (87) 1-Bromo-4-hydroxy-thioxanthen-9-one

Thiosalicylic acid (20.0g, 129.71mmol) and 4-bromophenol (35.99, 207.53mmol) were suspended in conc. H;SO, (200ml) and stirred for 48 hours. The red solution was slowly poured onto ice (500ml) with vigorous stirring. The resulting yellow precipitate was filtered, and dried in a vacuum oven (50°C) to give the title compound (24.23g, 61%) as a yellow amorphous i solid. m/z (LC-MS, ESP), RT=4.39 min, (M-1)= 305-307. 1-Bromo-9H-thioxanthen-4-ol

To a cooled (0°C) suspension of 1l-bromo-4-hydroxy-thioxanthen- 9-one (24.23g, 78.88mmol) in anhydrous tetrahydrofuran (400ml)

under nitrogen atmosphere, was added dropwise borane-THF complex (237ml, 1M in THF). The cloudy mixture was allowed to warm to room temperature and was left stirring overnight. The suspension dissolved gradually as the reaction p rogressed giving a yellow solution. The reaction mixture w as cooled (0°C) and the excess borane was quenched with acetone. The yellow solution was evaporated In vacuo. The resulting oil was purified by flash chromatography (4:1, hexane / ethyl acetate) to give the title compound (11.50g, 50%). m/z (L.C-MS, ESP},

RT=4.84 min, (M -1)= 291-293.

Carbonic acid tert-butyl ester 1-(4,4,5,5-tetra=methyl- [1,3,2]dioxaborolan-2-yl)-9H- thioxanthen-4-yl ester

To a stirred sclution of 1-bromo-9H-thioxanthen—4-o0l (11.50qg, 39.22mmol) in pyridine (7ml) was added triethylaamine (8.15ml, 58.83mmol). To the solution was added dropwise di-tert-butyl dicarbonate (9.41g, 3.14mmol) in pyridine (4ml). After 1 hour of stirring the crude reaction mixture was poured into water (100ml) and extracted with dichloromethane (3x10O0ml). The organics were washed with sat. brine (50ml), dra ed over MgSO4 and the solvent was evaporated in vacuo to give the title compound (10.40g, 67%) as a clear viscous oil. - HNMR (300MHz,

DMSO-d¢) : 6y= 1.53 (9H, s), 4.09 (2H, s), 7.15-7 .65 (6H, m).

Carbonic acid tert-butyl ester 1-(4,4,5,5-tetrarnethyl- [1,3,2]dioxaborolan-2~yl)-9H-thioxanthen-4-yl ester :

Carbonic acid tert-butyl ester 1-(4,4,5,5-tetrarmethyl- [1,3,2)dioxaborolan-2-yl)-9H-thioxanthen-4-yl ester (5.00g, 12.71mmol), anhydrous potassium acetate (3.74g, 38.13mmol), 1,1'-bis (diphenylphosphino) ferrocene (352mg, 0. 64mmol) and bis(pinacolato)diboron (3.87g, 15.25mmol) were suspended in anhydrous dioxane (8ml) under nitrogen atmosphe re. The mixture was degassed for 10 minutes and dichloro(l,1'-

bis (diphenylphosphino) ferrocenelpalladium(II) dichloromethane adduct (514mg, 0.64mmol) was added to the mixture. The reaction was heated at 90°C under nitrogen atmosphere for 24 hours. The crude reaction mixture was purified by flash chromatography (dichloromethane), to give the title compound (3.029) as a crude brown oil which was used without further purification. 2-(4-Hydroxy-9H-thioxanthen-1yl)-6-morpholin-4-yl-pyran-4-one (87)

Carbonic acid tert-butyl ester 1-(4,4,5,5~tetramethyl- [1,3,2]dioxaborolan-2-yl)-9H-thioxanthen-4-yl ester (3.00g, 6.81mmol), 2-chloro-6-morpholin-4-yl-pyran-4-one (1.22g, 5.67mmol) and potassium carbonate (2.07g, 14.98mmol) were suspended in anhydrous dioxane (6ml) under nitrogen atmosphere. The solution was degassed for 15 minutes. To the solution tetrakis(triphenylphosphino) palladium (291mg, 5% eq.) was added. The mixture was degassed for a further 5 minutes. The reaction was heated at 90°C under nitrogen atmosphere for 24 hours. The solvent was evaporated in vacuo and the crude mixture was purified by column chromatography (9:1, ethyl acetate/ethanol), to yield the title compound (421mg, 16%) as a light yellow amorphous solid. 'H NMR (300MHz,

DMSO-d6): &;= 3.33 (4H, t), 3.67 (4H, t), 3.88 (2H, s), 5.45 (1H, d), 6.05 (1H, d), 6.87 (1H, d), 7.24-7.65 (5H, m), 10.62 (1H, bs); m/z (LC-MS, ESP), RT=3.96 min, (M'+1)= 394. 2- (4-Hydroxy-9H~thioxanthen-1yl) -6-morpholin~4-yl-pyran-4-one . ether derivatives (a) To a mixture of 2-(4-hydroxy-9H-thioxanthen-1yl)-6- morpholin-4-yl-pyran-4-one (87) (20mg, 0.05mmol) and potassium carbonate (16mg, 0.1lmmol) in N,N-dimethylformamide (0.5ml)

was added dibromoethane (22nl, 0.25mmol). After 4 hours the appropriate amine or thiol (0.254mmol, 5ecy) was added to the solution, and the compounds isolated are shown below:

R To g 6) N

S

DE

Compound R ~ [purity | Retermtion Time M* +1 (Mins) 88 0) 95 3.26 505 tC

OL. 89 y 95 3.00 506

Ne

Oo. 90 [ 95 3.13 520

N

Y

Oo. 91 al rr 95 3.03 525 o., 92 al 7 95 3.3 553

C o.,

93 7 95 3.02 481 iS

Ny 94 jt 95 2.76 480 iS

OL. 95 oT OH 85 3.03 511 y iS

CN

96 3.15 491

Y o.. 97 J 2.88 534 . o., : 2.83 520 )

N

OL, ) 95 3.28 519 q

O.. 100 Sn 95 3.08 465 \

101 0” 95 3.38 557

B

Ng 102 CN 90 3.7 | 521

NO at oN (b) 2-(4-hydroxy-9H-thioxanthen-1 yl)-6-morpholin-4-yl-pyran-4- one (87) (20 mg, 0.0508 mmol), pot assium carbonate (44mg, 0.315 mmol) and N,N-dimethylformamide ( 0.5ml) was added to 2, 3 or 4-picolyl chloride hydrochloride (0.25mmol), respectively.

The reactions were stirred at rocem temperature for 2 hours.

The crude reaction mixtures were submitted for purification by preparative HPLC without any further workup, and the compounds produced are shown below: h 0 ro 4 oO N

S J

J

J

Compound Purity Retention M'+1

Time (Mins) 103 ZN 95 4.07 485

Ny 104 3.52 485

JL

Oo.

105 NZ 90 3.37 485 gy o. :

Example 11: N-Acyl 2-(l1-Amino-9H-thioxanthen-4-yl) -6- morpholin-4-yl-pyran-4-one derivatives

OH OBn

SH S

Lr —CCC

OH = = 0] le) F oO F

OBn OBn 0) [sos

O NPMB NPMB N

OH OTf B”

NBOC NBOC NBOC

OCHN

BOC C3 To H,N No © NA 0 nN

I J [TU ® 0 ® o) " (106) 1-Fluoro-4-hydroxy-thioxanthen-9-one

To a solution of 2-thiosalicylic acid (39.32g, 255 mmol) in concentrated sulfuric acid (700 ml) was added 4-fluorophenol (32.0 gq, 280 mmol). The red solution was then stirred at room temperature for 18 hours. Upon completion, the mixture was poured directly onto 4 litres of crushed ice and the resulting red solid was filtered off, and then suspended in water (1

L)and treated with ammonia solution until pH 6 attained whereupon the precipitate was re-filtered to give the title compound as an orange solid (44.48 g, 70.8%) m/z (LC-MS, ESP): 247 [M+H]*, R/T = 3.99 mins: 4-Benzyloxy-1-fluoro-thioxanthen-9-one

K,CO; (21.0 g, 150 mmol) wa s added to a stirred suspension of 1-Fluoro-4-hydroxy-thioxanthen-9-one (18.47 g, 75.0 mmol) in methanol (100 mL) followed by benzylbromide (16 mL, 75.0 mmol) which was added in slow stream via syringe. The resulting mixture was then heated to reflux for 90 minutes and then cooled to room temperature before it was poured onto cruched ice (0.5 L). The resultinsg precipitate was filtered off and dried (P,0s) to give the ti tle compound as a yellow solid (16.7 g, 66.1%) m/z (LC-MS, ESP) : 337 [M+H]", R/T = 5.22 mins 4-Benzyloxy-1-(4-methoxy-b-enzylamino)-thioxanthen-9-one

To a solution of 4-methoxy benzyl amine (1.63 g, 11.89 mmol) in dry pyridine (10 ml) was a dded 4-Benzyloxy-1-fluoro- thioxanthen-9-one (1 g, 2. 97 mmol) in a single portion. The nixture was then heated tos reflux (140 °C) for 18 hrs. The resulting hot orange suspe=nsion was allowed to cool to room temperature before being pooured onto 100 ml of crushed ice.

The precipitate was filterred off and washed with copious amounts of water to give tthe title compound as a red/orange solid (1.35 g, 89.6%). m/= (LC-MS, ESP): 454 [M +H]' R/T = 6.09 mins.

(4-Benzyloxy-9H-thioxanthen~-1-yl) - (4-methoxy-benzyl)-amine

To a cooled (0°C) suspension of 4-benzyloxy-1-(4-methoxy- benzylamino)-thioxanthen-9-one (8.16 g, 18.00 mmol) in dry THF (150 ml) was added Borane-THF complex (90 mmol, 90 ml 1M in

THF) in a dropwise fashion. The reaction was allowed to slowly warm to room temperature.and stirred for a further 16 hours to give a homogeneous yellow solution. To mixture was then cooled (0°C) and diluted slowly with acetone (150 ml) and then stirred for 60 minutes at room temperature. The solvent was removed 1n vacuo to give a crude residue that was diluted in CH;Cl, (100 ml) and the washed with a saturated solution of

NaHCO; (100 ml), dried using MgS0O4, filtered and concentrated in vacuo to give the title compound as a mild amber oil (7.90 g, 99.8%) m/z (LC-MS, ESP): 438 [M+H]', R/T = 5.01 mins. 1-Amino-9H-thioxanthen-4-ol (4-Benzyloxy-9H-thioxanthen-1-yl)-(4-methoxy-benzyl)-amine (14.51 g, 33.00 mmol) was mixed thoroughly with solid pyridine hydrochloride (190 g, 165.00 mmol) before being heated to 150°C and stirred at this temperature for a further 12 hours.

Upon completion the reaction was cooled slightly before being poured into an beaker of ice/water. The brown precipitate was removed by filtration and the filtrate adjusted to pH 11 with

NH3;0OH solution before being extracted with CHCl, (3x100 ml).

The combined organic phases were then washed with water (1x100 ml) and brine (1x100 ml) then dried using MgS04, filtered and concentrated in vacuo to give the title compound as a thick brown oil (7.50 g, 99.1%) m/z (LC-MS, ESP): 229 [M+H]*, R/T = 4.15 mins. (4-Hydroxy-9H-thioxanthen-1-yl)-carbamic acid tert-butyl ester

To a solution of l-amino-9H-thioxanthen-4-o0l (7.57 g, 33.00 mmol) in dry THE (50 ml) was added di-tertiary butyl dicarbonate (20 g, 91.64 mmol) in a single portion. The reaction was st irred at room temperature for 4 hours before the addition of methanol (50 mL) and solid NaOH (10 g, 250 mmol). The res-ulting slurry was stirred at room temperature for 1 hr before= the addition of H,0 (250 ml) and EtOAc (250 ml). The orgammic extract was removed and the remaining aqueous extracted further with EtOAc (2x50 ml). The combined organics were t—hen dried using MgS0O4, filtered and concentrated iri vacuo to give the title compound a dark amber oil (10.87 g, 92 %) m/z (LC-MS, ESP): 328 [M-H]", R/T = 4.73 mins

Trifluoro-methanesulfonic acid 1-tert-butoxycarbonylamino-9H- thioxanthen-4-s1 ester

To a cooled (0TC) solution of (4-Hydroxy-9H-thioxanthen-1-yl)- carbamic acid tert-butyl ester (10.05 g, 30.50 mmol) in dry pyridine (70 mIl) was added trifluoromethanesulphonic anhydride (8 ml, 48.77 mrnol) in a slow stream via syringe over 10 mins.

The brown mixtwire was stirred at 0°C for a further 30 mins before the add_ition of water in a dropwise fashion. The mixture was extracted with EtOAc (3x100 mL), the organic extracts combined, dried using MgSO4, filtered and concentrated i n vacuo to give a pale brown oil. Purification of the crude r esidue was accomplished by flash chromatography (Si0,) using Hesxanes:EtOAc (4:1) to give a mild amber oil that was purified by flash chromatography (SiOz) (Hexanes then 3:1 -

Hexanes:EtOAc) to give a mild amber oil (9.42 g, 67.0%) m/z (LC-MS, ESP): 460 [M-H]", R/T = 5.52 mins. [4-(4,4,5,5-Testramethyl~(1,3,2]dioxaborolan-2-yl) -9H~ thioxanthen-1—yl]-carbamic acid tert-butyl ester "To a solution of Trifluoro-methanesulfonic acid l-tert- butoxycarbonyl amino-9H-thioxanthen-4-yl ester (3.05 g, 6.60 mmol) in dry dioxane (10 ml) was added bis{pinacolato)diboron (2.0 g, 7.92 mmol) and anhydrous potassium acetate (1.9 g, 19.80 mmol). The reaction was then degassed (sonication for 20 min then saturated with N,) before the addition of dichloro{l, 1’ -bis(diphenylphosphino) ferrocene] palladium(II) dichloromethane adduct (0.26 g). The reaction mixture was degassed for a further 20 minutes before a reflux condenser was attached to the reaction vessel which was then heated to 100°C and stirred vigorously for 24 hours. The brown reaction mixture was then poured onto a silica pad prepared in hexanes and eluted with CH,Cl,:Hexanes (1:1). The collected eluent was concentrated in vacuo to give crude title compound as a dark brown oil that was used without further purification (2.90 qg,). {4- (6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-9H-thioxanthen-1- yl]-carbamic acid tert-butyl ester [4-(4,4,5,5-Tetramethyl-{1, 3, 2]dioxaborolan-2-yl)-9H- thioxanthen-1-yl]l-carbamic acid tert-butyl ester (2.90 g, 6.50 mmol) was introduced to a solution of 2-Chloro-6-morpholin-4- yl-pyran-4-one (3) (1.4 g, 6.50 mmol) in anhydrous dioxane (6 mL). Powdered K,CO; (2.01 g, 14.50 mmol) was added and the mixture degassed (sonication for 20 mins then saturated with

N,). To the degassed solution was added Tetrakis (triphenylphosphine) palladium (0.39 g) before it was degassed for a further 20 minutes. A reflux condenser was attached to the reaction vessel which was submerged into an oil bath maintained at 100°C for 14 hours whereupon the golden mixture was cooled and diluted with EtOAc (50 ml) and then washed with water (20 ml) and saturated brine (20 ml). Organic extract was dried using MgSO4, filtered and concentrated in vacuo to give the title compound as a light brown oil that was used without further purification. m/z (LC-MS, ESP): 493 ([M+H]', R/T = 4.41 mins. 2-(1-Amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran-4-one (106)

To a solution of [4-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-9H- thioxanthen~-1-yl]-carbamic acid tert-butyl ester (3.25 g) in

CHCl, (25 ml) was added trifluoroacetic acid (5 ml). The mixture was stirred at room temperature for 18 hrs wereupon 1it was cooled (0°C) and quenched by dropwise addition of saturated NaHCO; until the pH 9 was attained. The mixture was then extracted using CH;Cl; (3x20 mL), the combined organic extracts were then dried (MgSOQO,), filtered and concentrated in vacuo to give a semi-crystalline solid that was applied onto a thin silica pad and eluted with EtOAc (100%) going to

EtOAc:MeOH (9:1). The eluent was concentrated in vacuo to give the title comp.und as a mild amber oil (1.46 g, 56.4% over three steps) m/z (LC-MS, ESP): 393 [M+H]', R/T = 3.79 mins

N-Acyl 2-(1-Amino-9H-thioxanthen-4-yl)-6-morpholin-4-yl-pyran- 4-one derivatives (a) To a stirred solution of 2-(l1-Amino-9H-thioxanthen-4-yl)- 6-morpholin-4-yl-pyran-4-one (106) (39 mg, 0.1 mmol) in anhydrous N,N-dimethylformamide (1 ml), N- ethyldiisopropylamine (0.4 ml, 2.31 mmol) and O-(7- azabenzotriazol-1l-yl1l)-N,N,N',N'- tetramethyluroniumhexafluorophosphate (50 mg, 1.3 mmol) were added. The appropriate carboxylic acid (0.1 mmol) was then added and the mixture stirred at room temperature overnight.

The compound was then purified by preparative HPLC to give the desired compounds, which are shown below:

©

HN To on] :

DER

107 /=0 95 | 487 a 108 9 "95 | 546

CL

109 Ox 95 555 110 = 95 580

NN

7 S 112 “TL 95 555

S

NF ; i 114 NX 95 498 bo 116 = 95 479

BEE

BEEN

118 0 3 85 493

Ct Tr BB 119 Na 95 498

Y

} }

Cr 121 : 0 95 557 0 ~o FF -. 122 } ! 95 544 _L = . 123 95 569

Nan 124 DS 95 507 [lo] 125 95 569 [e] 4 a 3 " - — - ) B

(b) To a solution of Z-(l~-amino-8H-thioxanthen-4-yl)-6- morpholin-4-yl-pyran-&-one (106) (25 mg, 0.06 mmol) and pyridine (0.5 mmol) im CHCl; (1 mL) was added the appropriate sulfonyl chloride (0.2 mmol) in a single portion. The } reaction was stirred at room temperature overnight. The resulting reaction mixture was then purified by preparative

HPLC to give the desiwed compounds, which are shown below: y 0=$=0

HN To on.

DoT

DE

T

131 Q 97 57

Example 12: 2-Morpholin-4-yl-6-(11-oxo0-10,11-dihydro- dibenzo[b,f]thiepin-4-yl) ~-pyran-4-one ~ ~ 0 = — UR — 0} © 0

Hy 0 otf _ Og — CCH — CH 0 Oo 0

To © on_J

N oar \_/ © (132) [2-(2-Methoxy-phenylsulfanyl) -phenyl]-acetic acid 2-Methoxythiophenol (2.8g, 20mmol) was added to a solution of potassium hydroxide (4.6g, 80mmol) in water (50ml) and the mixture was degassed for 15 minutes. 2-Iodophenylacetic acid (5.24g, 20mmol) and copper bronze (64mg, 1lmmol) were then added to the reaction mixture, which was refluxed overnight.

The solution was cooled down, filtered and the precipitate washed with water (50ml). The filtrate was acidified with conc

HCl (pH 1), extracted with dichloromethane (3x100ml). The organics were combined, extracted with saturated brine, dried over sodium sulphate and evaporated in vacuo to give the title compound as a pale brown oil which solidified overnight. The compound was used without any further purification (5.10qg,

6-Methoxy-1. 1H-dibenzo(b,f]thiepin-10-one [2- (2-Methoxy-phenylsulfanyl)-phenyl]}-acetic acid (5.40g, 20mmol) wass dissolved in methanesulfonic acid (50ml) and the mixture wass heated for 2 hours at 90°C under stirring and a nitrogen atmosphere. The reaction mixture was cooled to room temperature and poured onto ice with stirring. The black precipitate was filtered and dried over night in a vacuum oven (50°C). The compound was used without any further purification (4.60g, 91%). 6-Hydroxy—-_11H-dibenzo([b,f]thiepin-10-one 6-Methoxy—-11H-dibenzo[b,f]jthiepin-10-one (1.54g, 6mmol) and pyridine h-ydrochloride (10g) were heated for 2 hours at 200°C with stirr.ing and N; atmosphere. The reaction was cooled down to room temmperature and then triturated in water (200ml) The pale green precipitate was filtered and dried overnight in a vacuum ove n (50°C) (1.40g, 96%). 'HNMR (300MHz, DMSO-d¢): y= 4.20 (2H, s), 7.05-7.69 (7H, m), 10.56 (1H, s).

Trifluoro--methanesulfonic acid 11-oxo-10,11-dihydro- dibenzofb, f]thiepin-4-yl ester 6-Hydroxy=--11H-dibenzo[b, f]thiepin-10-one (242mg, lmmol) was dissolved in dry pyridine (5ml) and trifluoromethanesuphonic anhydride (0.17ml, lmmol) was added drop wise to the stirred solution aat 0°C under N, atmosphere. The reaction mixture was left to resact for 4 hrs and was then poured into water. (50ml) :

The organic were extracted with dichloromethane (3x50ml), washed with 0.2N HCl, dried over magnesium sulphate and : evaporatecd in vaccuo to give a dark brown solid. This solid was purifiied by column chromatography (dichloromethane/hexane, 3:7, Rf=0_15) to give the title compound as a pale brown solid (0.37g, 100%). 'HNMR (300MHz, DMSO-de): dy= 3.70 (2H, s), 7.31- 7.8 (7H, rn).

6-(4,4,5,5-Tetramethyl-[1,3,2)dioxaborolan-2-yl)-11H- dibenzo[b,f]jthiepin-10-one

Trifluoro-methanesulfonic acid 11-oxo0-10,11-dihydro- dibenzolb, f)thiepin-4-yl ester (0.374 g, lmmol), bis (pinacolato)diboron (305mg, 1.2mmol) and potassium acetate (294mg, 3mmol) were dissolved in 1,4-dioxane (5mL) and the mixture was degassed for 5 min. Pd(dppf)Cl, (40mg, 0.05mmol) and dppf (28mg, 0.05mmol) were added to the vessel, and the reagents heated to 100°C under nitrogen with stirring for 12 hours. The reaction mixture was purified by flash chromatography (dichloromethane/hexane, 1:4) and the black residue was used without further purification (0.359). 2-Morpholin-4-yl-6-(11-ox0-10,11-dihydro-dibenzo(b,f]thiepin- 4-yl) -pyran-4-one (132) 2-Chloro-6-morpholin-4-yl-pyran-4-one (3) (215mg, lmmol), 6- (4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-11H- dibenzo[b, f]thiepin-10-one (352mg, lmmol), and ground potassium carbonate (276mg, 2mmol) were suspended in 1,4- dioxane (10ml) and degassed for 5 minutes. Pd(PPhj3)s (57mg, 0.05mmol) was then added and the reaction mixture was then heated at 90°C for 4hours under a vigorous stirring and a N; atmosphere. The solvent was removed in vaccuo and the residue was then suspended in water (100ml). The organics were extracted with dichloromethane (3x100ml), combined, washed with saturated brine and dried over sodium sulphate. The solvent was removed in vaccuo and the residue was purified by column chromatography (silica; ethyl acetate:ethanol; 9:1) to give the title compound as a pale brown solid (0.12g, 29%). 14NMR (300MHz, DMSO-dg): &= 3.40 (4H, t), 3.70 (4H, t), 4.43 (2H, s), 5.55 (1H, d), 6.29 (1H, d), 7.25-7.55 (5H, m), 7.78-

7.81 (1H, m), 8.20-8.22 (1H, m); m/z (LC-MS, ESP), RT=4.12 min, (M'+1)= 406.

Example 13: 2-(10,11-Dihydro-dibenzo[b,f)thiepin-4-yl)-6- morp holin-4-yl-pyran-4-one ~ 7 s./ "0 We OH _

Oo 4 oT f gC __ 0 3 \_/ © (133) 4-Mesthoxy-10,11-dihydro-dibenzo(b,f]thiepine

Hydr-razine hydrate (4ml) and potassium hydroxide (2.72g, 48mmol) was added to 6-methoxy-11H-dibenzo[b, f]thiepin~-10-~one (4.1L.0g, 16mmol) in ethylene glycol (20ml) and the reaction mixture was heated at 175°C for 3 hours. The reaction mixture was cooled down to room temperature and water was added (100ml). The white solution was extracted with ether (3x=200ml), the organics were combined, washed with water (100ml), brine (100ml) and dried over magnesium sulphate. The solwent was removed in vacuo to give an oil which solidify uporna standing to give a brown solid which was used without any further purification (2.45g, 63%). : 10, _11-Dihydro-dibenzo(b,f]thiepin-4-o0l 4-Me2thoxy-10,11-dihydro-dibenzo(b, flthiepine (2.42g, 10mmol) and pyridine hydrochloride (15g) were heated with stirring at 180°°C for one hour. Water (100ml) was added to the reaction mixture and the organics were extracted with ethyl acetate (3x100 ml). The organics were combined and washed with 2N HCl (50ml), brine (50 ml), and dried over magnesium sulphate. The solvent were removed in vacuo and the residue was purified by column chromatography (1:9; dichloromethane:hexane) to give the desired compound as a white solid (1.55g, 68%). 'HNMR (300MHz, DMSO-dg¢): &y= 3.13-3.23 (4H, m), 6.70 (2H, t), 6.97- 7.15 (4H, m), 7.38 (1H,s) 9.78 (1H, s); m/z (LC-MS, ESP),

RT=4.59 min, (M'+1l)= 229.

Trifluoro-methanesulfonic acid 10,11-dihydro- dibenzo[b,f]thiepin-4-yl ester 10,11-Dihydro~-dibenzo(b, f]thiepin-4-0l (1.26g, 5.5mmol) was dissolved in dry pyridine (5ml) and trifluoromethanesuphonic anhydride (1.12ml, 6.6mmol) was added drop wise to the stirred © solution at 0°C under N, atmosphere. The reaction mixture was left to react for 4 hrs and was then poured into water. {100ml) The organic were extracted with dichloromethane {3x50ml), washed with 0.2N HCl, dried over magnesium sulphate and evaporated in vaccuo to give a dark brown solid. This solid was purified by flash chromatography (dichloromethane) to give an oil (1.1g, 56%). HNMR (300MHz, DMSO-dg¢): 8y= 3.25- 3.29 (2H, m), 3.37-3.41 (2H, m), 7.12-7.17 (1H, m), 7.21-7.31 (3H, m), 7.38-7.41 (3H,s). 2-(10,11-Dihydro-dibenzo(b,f]thiepin-4-yl1)-4,4,5,5- tetramethyl-[1,3,2]dioxaborolane

Trifluoro-methanesulfonic acid 10,11-dihydro- dibenzo[b, f]lthiepin-4-yl ester (1.08g, 3mmol), bis(pinacolato)diboron (914mg, 3.6mmol) and potassium acetate (883mg, 9mmol) were dissolved in 1,4-dioxane (10mL) and the mixture was degassed for 5 minutes. Pd{(dppf)Cl, (121mg, 0.15mmol) and dppf (83mg, 0.15mmol) were added to the vessel,

and the reagents heated to 100°C under nitrogen with stirring for 12 hours.

The reaction mixture was purified by flash chromatography (dichloromethane) and the black residue was used without further purification (0.879). ns) 2-(10,11-Dihydro-dibenzo([b,f]thiepin-4-yl)-6-morpholin-4-yl- pyran-4-one 2-Chloro-6-morpholin-4-yl-pyran-4-one (3) (1.12g, 5.2mmol), 2- (10,11-Dihydro-dibenzo|b, flthiepin-4-yl)-4,4,5,5-tetramethyl- (1,3,2)dioxaborolane (880mg, 2.6mmol), and ground potassium carbonate (720mg, 5.2mmol) were suspended in 1,4-dioxane (10ml) and degassed for 5 minutes. bis(tri-t- butylphosphine)palladium (66mg, 0.13 mmol) was then added and the reaction mixture was then heated at 90°C for 4 hours under 1.5 a vigorous stirring and a N; atmosphere.

The solvent was removed in vaccuo and the residue was then suspended in water (100ml). The organics were extracted with dichloromethane (3x100ml), combined, washed with saturated brine and dried over sodium sulphate.

The solvent was removed in vaccuo and the residue was purified by column chromatography (silica; ethyl acetate:ethanol; 9:1) to give a pale brown solid (50mg, 5%). HNMR (300MHz, DMSO-d¢): y= 3.24-3.32 (6H, m), 3.44 (2H, t), 3.66 (4H, t), 5.50 (1H, d), 6.10 (1H, d), 7.08-7.51 (7H, m); m/z (LC-MS, ESP), RT= 4.48min, (M'+1)= 392.

WO003/070726 PCT/GB03/00770

Example 14: 2-Morpholin-4-yl-6-(10H-phenothiazin-4-~yl)-pyran- 4-one

Foe 0 CL) — CL

S S

OH OH OH

0.0 Jog? NL

N o) To " A o_N_J _ O07 °N s s J o..° .B. sl UF Q © 0 0’ TF ——

F (134) o

Oo N

S

©) (135) 10H-Phenothiazin-4-ol

To a solution of 3-phenylamino-phenol (5 g, 26.9%mmol) in 1,2- dichlorobenzene (50 ml) was added Sg sulfur(1.82 g, 56.76 mmol) in a single portion and iodine (0.1 g, 0.39 mmol) which was added in three portions over 10 minutes. A reflux condenser was attached to the reaction vessel which was heated to 185°C under a nitrogen atmosphere. The mixture was stirred at this temperature for 4 hours and then allowed to cool to room temperature. The reaction mixture was filtered to remove a black precipitate and the filtrate diluted with Et;0 (100 ml) and washed with water (2x100 ml). The organic layer was separated and the volatile solvents removed to give a deep green oil that was purified by flash column chromatography (S10,) (Hexanes then 8:1-Hexanes:EtOAc) to give a pale yellow solid (2.38 g, 40.96%) m/z (LC-MS, ESP) 216 [M+H}', R/T = 4.12 mins. : : 4-Hydroxy-phenothiazine-10-carboxylic acid tert-butyl ester

To a solution of 10H-Phenothiazin-4-o0l (0.77 g, 3.58 mmol) in anhydrous pyridine (10 ml) was added di-tertiary butyl dicarbonate (3.12 g, 14.31 mmol) in a single portion. The solution was heated to 80°C and stirred under a nitrogen atmosphere for 60 minutes before being allowed to cool to room temperature and treated with water (20 ml) and extracted with

EtOAc (2x30 ml). The organic layers were then washed with water (20 ml), dried using MgSO4, filrtered and concentrated in vacuo to give an amber oil. The crude residue was treatyed with MeOH (15 ml) and solid NaOH (0.65 g, 16.25 mmol). The mixture was heated to 80°C for 60 minutes then cooled to room temperature and neutralised to pH7 with 1M HCl solution. The resulting suspension was then filtered and dried to give the title compound as a beige solid (1.13 g, 100%) that was used without further purification. m/z (LC-MS, ESP): 315 [M-H]", R/T = 4.72 mins. 4-Trifluoromethanesulfonyloxy-phenothiazine-10-carboxylic acid tert-butyl ester

Trifluoromethanesulfonic anhydride (2.95 ml, 17.09 mmol) was added in a dropwise fashion over 10 minutes to a cooled (0°C) stirred solution of 4-Hydroxy-phenothiazine-10-carboxylic acid tert-butyl ester (3.60 g, 11.41 mmol) in pyridine (40 ml).

The reaction mixture was stirred at 0°C for 1 hour before the addition of water (80 ml). The mixture was extracted using

EtOAc (2x60 ml). The organic extracts were then dried using

MgSO, filtered and concentrated in vacuo to give a dark brown

0il. The crude residue was then purified by flash chromatography (SiO;) (4:1-Hexanes:EtOAc) to yield a yellow oil (5.02 g, 98.24%) m/z (LC-MS, ESP): 348 [M+H-BOC]", R/T = 5.61 mins 4-(4,4,5,5-Tetramethyl-[1,3,2]dioxabocrolan-2-yl)- phenothiazine-10-carboxylic acid tert-butyl ester

To a stirred solution of 4-trifluoromethanesulfonyloxy-~ phenothiazine-10-carboxylic acid tert-butyl ester (3.0 g, 6.7 mmol) in anhydrous dioxane (10 ml) was added bis (pinacolato)diboron (2.05 g, 8.06 mmol) and potassium acetate (1.96 g, 20.01 mmol). The reaction was then degassed (sonication for 20 minutes then saturated with N;)} before the addition of dichloro(l,1’ -bis(diphenylphosphino) ferrocene] palladium (II) dichloromethane adduct (0.27 g, 0.33 mmol). The reaction mixture was degassed for a further 20 minutess before a reflux condenser was attached to the reaction vessel which was then heated to 90°C and stirred vigorously for 72 hours.

The dark brown reaction mixture was then allowed to cool to room temperature before it was applied to a thick silica pad prepared in hexanes and eluted with hexanes:CH,Cl,-(2:1). The eluent was concentrated in vacuo to give a dark brown oil (2.85 g, 100%) that was used for the next transformation with no further purification. m/z (LC-MS, ESP): 326 [M+H-BOC]*, R/T = 5.86 mins 4-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-phenothiazine-10- carboxylic acid tert-butyl ester (134)

Powdered potassium carbonate (2.03 g, 14.68 mmol) and 2-

Chloro-6-morpholin-4-yl-pyran-4-one (1.44 g, 6.70 mmol) were added to a stirred solution of 4-(4,4,5,5-tetramethyl- (1, 3,2]dioxaborolan-2-yl)-phenothiazine-10~-carboxylic acid tert-butyl ester (2.85 g, 6.70 mmol) in anhydrous dioxane (20 ml) and the mixture degassed (sonication for 20 minutes then saturated with N;) thoroughly. Tetrakis (triphenylphosphine) palladium was then added in a single portion and the mixture degassed (sonication for 20 minutes then saturated with Nj) once again before a reflux condenser was attached and the mixture heated to 100°C under a nitrogen atmosphere for 20 hours. Water (30 ml) was added and the mixture extracted with

EtOAc (3x30 ml). The organic extracts were then dried using

MgS0O,, filtered and concentrated in vacuo to yleld a dark brown, crystalline solid (3.21 g, 100%) that was taken forward with no further purification. m/z (LC-MS, ESP): 479 [M+H]", R/T = 4.55 mins 2-Morpholin-4-yl-6-(10H-phenothiazin-4-yl)-pyran-4-one (135)

To a stirred solution of 4-(6-Morpholin-4-yl-4-oxo-4H-pyran-2- yl) -phenothiazine-10-carboxylic acid tert-butyl ester (3.65 g, 7.63 mmol), in CH,Cl, (30 ml) was added trifluoroacetic acid in a single portion. The mixture was stirred at room temperature for 20 hours whereupon the reaction was concentrated in vacuo to give a thick syrup that was basified in a dropwise fashion with saturated NaHCO; (40 ml). The dark green mixture was then stirred at room temperature for 18 hours. The mixture was filtered and the filtrant retained, washed with water and dried to give the title compound as a dark green solid (2.89qg, 83.74% over 3 steps) m/z (LC-MS, ESP): 479 [M+H]*, R/T = 4.05 mins

Example 15: 4-Morpholin-4-yl-6-thianthren-l-yl-pyran-2-one oO Oo O

S x oh oe

CL) — CL 1) — CL 0) — (oo © S © S 0 NS

Coch ond! = C0 —x

To

Ox NJ To ak Rens

OY RE [0]

Thianthrene-]l-carboxylic acid t-Butyl lithium (1.7M in hexane, 55.1ml, 93.6mmol) was ad ded drop wise to a stirred solution of thianthrene (16.9g, 78 mmol) in dry THF (250ml) at -78°C over 30 minutes under an iner t atmosphere (N;). The reaction mixture was allowed to warm to room temperature and the resulting reddish solution was 1 eft stirring for 24 hours. The mixture was then cooled down t o - 78°C and carbon dioxide (from dry ice pellet and dried by- passing over some activated A4 sieves) was bubbled into t_he solution for 1 hour. The reaction was warmed up back to room temperature with CO; still bubbling through it for anothe r hour. Water (10ml) was then added carefully to the soluti_on and the pH was adjusted to 1 (pH paper) with 2N HCl. The solvent was removed in vacuo and the yellow solid formed was filtered and dried overnight in a vacuum desiccators. The solid was then recrystalised from methanol to give the dessired product as a pale yellow crystalline solid (11.9g, 59%). ‘HNMR

(300MHz, CDCls3): 64 = 7.25 (3H, m); 7.50 (4H, m). m/z (LC-MS,

ESP): RT= 4.53min, (M™ - 1)= 259 l1-Thianthren-1-yl-ethanone

Methyl lithium (1.6M in ether, 57ml, 90mmol) was added drop wise to a stirred solution of thianthrene-l-carboxylic acid (11.71g, 45mmol) in dry tetrahydrofuran (200ml) at -78°C over 30 minutes under an inert atmosphere (N;). The reaction mixture was allowed to warm to room temperature and (very thick white suspension present) was left stirring for 4 hours. Water (10ml) was then added carefully to the solution and the pH was adjusted to 1 (pH paper) with 2N- HCl. The solvent was removed in vacuo and the yellow solid formed was filtered and dried overnight in a vacuum desiccators. The solid was then purified by column chromatography (ethyl acetate/hexane; 1:9) and was recrystalised from ethanol to give the desired product (6.58q, 57%). 'HNMR (300MHz, CDCl3): 8 = 2.65 (3H, s); 7.26 (3H, m); 7.47 (2H, m); 7.62 (2H, d). m/z (LC-MS, ESP) RT= 4.95 min; (M' + 1)= 259 3-Oxo-3-thianthren-1l-yl-dithiopropionic acid

A solution of CS; (1.55ml, 25.5mmol) and 1l-thianthren-1-yl- ethanone (6.59g, 25.5mmol) in dry tetrahydrofuran (20ml) was added drop wise to a solution of potassium t-butoxide (5.73qg, 51lmmol) in dry tetrahydrofuran (50ml) under N; at 0°C. A red coloration and the formation of a precipitate were observed.

The mixture was left under vigorous stirring over the weekend and was then poured onto water (200ml) and extracted with ether (3x100ml). The aqueous was acidified with 2N H,S504 to pH 1 (Whatmann pH paper) and the extracted with ether (3x100ml).

The organic were dried over magnesium sulphate and the solvent was evaporated in vacuo to give the desired product as a dark orange resin (5.00g, 59%). m/z (LC-MS, ESP), RT= 5 .11; (M -1)= 3-Oxo-3-thianthren-1-yl-dithiopropionic acid ethyl ester

Tetrabutylammonium hydrogen sulphate (5.1g, 15mmol ) and sodium hydroxide (1.2g, 30mmol) were dissolved in water ( 50ml) A solution of 3-oxo-3-thianthren-1l-yl-dithiopropioni c¢ acid (5.02g, 15mmol) in dichloromethane (50ml) was adde d to the solution in one portion and was stirred vigorously for 30 minutes. The aqueous layer was removed and iodoetheane (4ml) was added to the dichloromethane solution that wass then stirred for lhr. The solvent was removed in vacuo and the residue was taken into water (200ml) The organics were extracted with ether (3x100ml), dried over magnesi um sulphate and evaporated in vacuo. The residue was then puri fied by column chromatography (ethyl acetate:hexane; 1:4) to give the desired compound as a bright yellow solid (4.00 g, 73%). THNMR (300MHz, CDCl3): Oy = 1.43 (3H, t), 3.33 (3H, qq), &.57 (1H, s), 7.26 (3H, m), 7.51 (3H, m), 7.60 (1H, m), 15.09 (1.H, s); m/z (LC-MS, ESP), RT=6.50 min, (M'-1)= 361. 3-Morpholin-4-yl-l-thianthren-1-yl-3-thioxo-propari-1-one

Morpholine (0.96ml, 1llmmol) was added to a solution of 3-oxo- 3-thianthren-1l-yl-dithiopropionic acid ethyl estem (3.99qg, 1lmmol) in ethanol (20ml). The reaction was refluxed for 8 hours and was then cooled to room temperature. The precipitate : formed was filtered and dried to give the desired product as a bright orange solid (3.50g, 82%). m/z (LC-MS, ESPD), RT= 4.8land 5.33min same (M+1l)= 388 3-Ethylsulfanyl-3-morpholin-4-yl-1-thianthren-1-y_l-propenone 3-Morpholin-4-yl-l-thianthren-1-yl-3-thioxo~propam-1l-one (3.49g, 9mmol), iodoethane (0.8ml, 10mmol), and g:xrinded potassium carbonate (1.38g, 10Ommol) were suspended in acetone (20ml) and the mixture was refluxed for 24 hours. The solvent was removed in vacuo and the residue was taken into water (50ml) . The organics were extracted into dichloromethane (3x100ml), dried over magnesium sulphate and evaporated in vacuo. The crude product was purified by column chromatography (ethyl acetate/hexane) to give the desired product as a yellow solid (2.26g, 60%). 'HNMR (300MHz, CDCl3): &y= 1.34 (3H, t), 2.96 (2H, 4), 3.73 (4H, m), 3.84 (4H, m), 7.21 (3H, m), 7.47 (4H, m); m/z (LC-MS, ESP), RT= 5.0lmin, (M'+1)= 416. 4-Morpholin~4-yl-6-thianthren-1-yl-pyran-2-one (136)

A suspension of activated zinc dust (0.65g, 10mmol), ethyl bromoacetate (0.56ml, 5mmol) and a few crystals of iodine in dry tetrahydrofuran (20ml) were heated at 50°C for one hour with stirring under a N; atmosphere. A solution of 3- ethylsulfanyl-3-morpholin-4-yl-l-thianthren-1-yl-propenone (1.04g, 2.5mmol) in dry tetrahydrofuran (20ml) was added drop wise with stirring and the mixture was refluxed for 12 hours under a N; atmosphere. The mixture was then poured over ice cold dilute 3% H;SO, (50ml), the aqueous layer was extracted - with ethyl acetate (3x50ml), the combined extracts were dried over magnesium sulphate and the solvent was evaporated in vacuo. The residue was purified by column chromatography (ethyl acetate/hexane) to give the desired product (0.35g, 35%). 'HNMR (300MHz, CDClj): &y= 3.45 (4H,t), 3.85 (4H, t), 5.35 (1H, 4d), 6.29 (1H, d), 7.26 (3H, m), 7.50 (3H, m) 7.61 (1H, m); m/z (LC-MS, ESP), RT= 4.50min, (M'+1l)= 396.

Example 16: 6-(6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)- thianthrene-2-carboxylic acid amide Derivatives

COOH COOH COOH

(A

O-- Cl, Clo oT

F f F HOOC SOH x

Br Br 04-0 — pe ;e o LL } 3 JCI = CL oo 0 0 lo) 3 gt phe , 0 — ro LT lo] o 0 5 (137) (138) 3-Chlorosulfonyl-4-fluoro-benzoic acid

Chlorosulphonic acid (100 ml, 1.5 mol) was gradually added to 4-fluorobenzoic acid (43g, 0.307mol) with stirring. The clear dark yellow mixture was heated to 150°C for 24 hours. The yellow solution was cooled back to room temperature and poured onto ice with vigorous stirring. The white precipitate was filtered and pressed dry. The solid was dried overnight in a desiccator under vacuum and over activated silica (54.65qg, 75%). Mp: 116-117°C; m/z (LC-MS, ESP), RT= 4.03min, (M-1)= 237-239 (ratio 1:3). 4-Fluoro-3-sulfino-benzoic acid

Sodium sulphite (130g, 1.034mol) was added slowly to a solution of 3-chlorosulfonyl-4-fluoro-benzoic acid (49.39g, 0.207mol) in water (150ml) at 0°C with a vigorous stirring.

After the addition was completed the reaction was »warmed back to room temperature for 1 hour and the pH of the s-olution was kept around pH 6-7 with 2N sodium hydroxide soluti on. The white milky suspension was filtered and the solid washed with 2N sodium hydroxide solution (150ml) and then wate r (100ml).

The filtrate was then cooled in an ice bath and co ncentrated

HCl was added until no more precipitate was formed (pH<1l). The white precipitate was then filtered, pressed dry a nd left in a dessicator overnight under vacuum and over activat ed silica (27.92g, 66%). m/z (LC-MS, ESP), RT= 0.98min, (M -1)= 203 §-(2-Bromo-phenylsulfanyl)-3-sulfino-benzoic acid 2-Bromobenzenethiol (25g, 132 mmol) was added to a solution of 4-fluoro-3-sulfino-benzoic acid (13.5g, 66mmol) amd NaOH pellets (llg, 264mmol) in water (30ml). The yellowr mixture was then degassed for 10 minutes and then heated to 14 0°C for 48 hours. The reaction was then cooled to 0°C and acidified to pH 4-5 (pH paper) with concentrated HCl. The precipit-ate formed was filtered, washed with hexane and was dried in a vacuum dessicator over activated silica overnight (20.69cy, 84%). m/z (LC-MS, ESP), RT= 3.67min, (M-1)= 373. 6-Bromo-thianthrene-2-carboxylic acid 4-(2-bromo-phenylsulfanyl)-3-sulfino-benzoic acid (14g, 38mmol) was added slowly to a stirred solution of methanesulphonic acid (160ml). The purple solutiora was heated to 60°C for 3 hours. The reaction was cooled down to room temperature and was poured into ice (300ml) where an off-white precipitate appeared. The solid was filtered and washed with water (100ml) and then dried in a vacuum dessicatcr over activated silica (9.48g, 73%). 'HNMR (300MHz, CDCl_3): &y= 7.29 (1H, t), 7.59 (1H, dd), 7.70 (1H,dd) 7.74 (lH, d) . 7.87 (1H, dd), 8.03 (1H, d).m/z (LC-MS, ESP), RT= 4.99min, (M-1)= 339

6-Bromo-thianthrene-2-carboxylic acid methyl ester

To 6-bromo-thianthrene-2-carboxylic acid (9g, 28mmol) in methanol (180ml) was slowly added conc. HSO4 (5 ml). The milky white suspension was heated to 80°C until all the solid had gone into solution (2hrs). The suspension was concentrated in vacuo. Water (100ml) was added and the organics were then extracted with dichloromethane (3 x 70 ml), dried over MgSO4 and evaporated in vacuo, yielding to a yellow solid. (4.48q, 45%). 'HNMR (300MHz, CDCl3): &y= 3.94 (3H, s); 7.13 (1H, t), 7.44 (1H, dd), 7.54 (1lH,dd) 7.61 (1H, d), 7.93 (1H, dd), 8.13 (1H, dy). 6-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-thianthrene- 2-carboxylic acid methyl ester 6-Bromo-thianthrene-2-carboxylic acid methyl ester (1g, 2.8mmol), bis(pilnacolato)diboron (0.86g, 3.4mmol) and potassium acetate (0.12g, 0.14mmol) in 1,4-dioxane (15ml) was degassed for 15 minutes. To the yellow suspension was then added PdCl; (dppf) (78mg, 0.14mmol) and dppf (0.83g, 8.5mmol).

The dark red mixture was heated to 90°C under a N, atmosphere ) for 48 hours. The crude mixture was purified by flash chromatography (dichloromethane) to give viscous brown oil (1.13g), which was used without any further purification. 6- (6-Morpholin-4~yl-4-oxo-4H-pyran-2-yl)-thianthrene-2- carboxylic acid methyl ester (137) 6-(4,4,5,5~-Tetramethyl~[1, 3, 2]dioxaborolan-2-yl)-thianthrene- 2-carboxylic acid methyl ester (1.1g, 2.83mmol), 2-chloro-6- morpholin-4-yl-pyran-4-one (0.73g, 3.4mmol) and K,CO; (0.8g, 5.66mmol) were dissolved in dry 1,4-dioxane (7ml). The mixture was degassed for 15 mins and Pd(PPhi3)s (0.16 g, 5 mol 3%) was then added The dark brown mixture was heated to 90°C under an atmosphere of N; for 24 hour. The reaction: mixture was concentrated in vacuo and water (100ml) w&Es added. The brown solid was filtered and washed with water «(1.23g, 96%). m/z (LC-MS, ESP), RT= 4.49 min, (M'+1l)= 454. 6- (6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl) -#hianthrene-2- carboxylate sodium salt (138) 6- (6-Morpholin-4-yl-4-oxo-4H-pyran-2-yl)-®thianthrene-2- carboxylic acid methyl ester (1.1g, 2.43mrnol) and NaOH Pellets (97mg, 2.43mmol) were dissolved in methanol (40ml). The brown suspension was heated to 80°C under N; for- 24 hours. The solvent was removed in vacuo and the resicldue was triturated with diethyl ether. The product was collexcted by filtration as a fine dark brown powder (1.11g, 99%). m/ z (LC-MS, ESP),

RT=3.90 min, (M -1)= 438 6-(6-Morpholin-4~yl-4-oxo~-4H-pyran-2-yl)- thianthrene-2- carboxylic acid amide Derivatives

Z20 6- (6-morpholin-4~yl-4-oxo-4H-pyran-2-yl)- thianthrene-2- carboxylate sodium salt (138) (20mg, 0.04mmol), HBTU (18mg, 0.05mmol), di-isopropylethylamine (9pl, 0 .05Smmol), the appropriate amine (0.04mmol) and dry dime thylacetamide (0.5ml). The dark brown mixture was stirr ed at room temp for 2 hours and then purified by preparative HP LC to give the desired products, which are shown below:

=

So A] : S [1 (0) 0]

R

Compound Purity | Retention Time | M'+1 (Mins) 139 x 35 3.65 453 140 oo 95 3.7 467

LN

141 95 4.11 495 hh ” 0 Ns,

A

0 Ns.

154 ) 95 4.77 535

N_ 155 N. 95 4.3 507 ] = i} ] or

NL

157 95 4.14 507 158 95 537 oA

PN

159 95 4.02 537

[0]

AN

160 0 95 3.69 509 161 ® 95 4.31 521

Ns } )

H

163 ort 95 3.72 552

N - y

O

N

164 ~ Ne 95 3.12 537

N .

NS

165 ~ Ne 95 3.49 524

N . oJ 166 nN. 95 3.29 550 ~

167 H 95 3.23 536 or 168 Nn. 95 3.22 550 o

AN

169 Nn ) 95 3.62 572

AN

170 kA 95 3.43 530 “ °N

J

171 n 95 3.27 530 z= > _N 172 | Ho 95 3.21 530 a

A

N

173 N He 95 3.26 544

Oh . 174 ) | } 95 3.3 558

SA

175 LN 95 3.05 579

NS

176 N 95 3.17 533

RS ~. 177 PPA 95 3.19 552 oJ 178 0™ 95 3.18 566

NN

179 NN 95 3.44

SUS

180 Ee. 95 3.49 651 on x © 181 PN 3.29 538

J

B) Biological Examples

Materials and Methods

In vitro ATM inhibition Assays

In order to assess the inhibitory action of the compounds against ATM in vitro, the following assay was used to determine IC.; values.

ATM protein was immunoprecipitated from Hela cell nuclear extract using rabbit polyclonal anti-sera raised to the C- terminal ~500 amino-acid residues of the human ATM protein.

The immunoprecipitation was performed according to the methodology described by Banin, S. et al. (1998). 10 ul of immunoprecipitated ATM in Buffer C (50 mM Hepes, pH 7.4, 6 mM

MgCl, 150 mM NaCl, 0.1 mM sodium orthovanadate, 4 mM MnCl2, 0.1 mM dithiothreitol, 10% glycerol) was added to 32.5 pl of buffer C containing 1 ug of the ATM substrate GSTpS53N66 in a

V-bottomed 96 well polypropylene plate. The GSTpS53N66 substrate is the amino terminal 66 amino acid residues of human wild type p53 fused to glutathione S-transferase. ATM : phosphorylates p53 on the residue serine 15 (Banin, S. et al. .(1998)). Varying concentrations of inhibitor were then added.

All compounds were diluted in DMSO to give a final assay concentration of between 100pM and 1 nM, with DMSO being at a final concentration of 1%. After 10 minutes of incubation at 37°C, the reactions were initiated by the addition of 5 pl of 500 uM Na-ATP. After 1 hour with shaking at 37°C, 150 pl of phosphate buffered saline (PBS) was added to the reaction and the plate centrifuged at 1500 rpm for 10 minutes. 5 pl of the reaction was then transferred to a 96 well opaque white plate containing 45 pl of PBS to allow the GSTp53N66 substrate to bind to the plate wells. The plate was covered and incubated at room temperature for 1 hour with shaking before discarding the contents. The plate wells were washed twice by the addition of PBS prior to the addition of 3% (w/v) bovine serum albumin (BSA) in PBS. The plate was incubated at room temperature for 1 hour with shaking before discarding the contents and washing twice with PBS. To the wells, 50 pl of a 1:10,000 dilution of primary phosphoserine-15 antibody (Cell

Signaling Technology, #9284L) in 3% BSA/PBS was added to detect the phosphorylation event on the serine 15 residue of p53 elicited by the ATM kinase. After 1 hour of incubation at room temperature with shaking, the wells were washed four times with PBS prior to the addition of an anti-rabbit HRP conjugated secondary antibody (Pierce, 31462) with shaking for 1 hour at room temperature. The wells were then washed four times with PBS before the addition of chemiluminescence reagent (NEN Renaissance, NEL105). The plate was then shaken briefly, covered with a transparent plate seal and transferred to a TopCount NXT for chemiluminescent counting. Counts per second, following a one second counting time, were recorded for each reaction.

The enzyme activity for each compound is then calculated using the following equation:

$ Inhibition = 100- (LB of unknown - mean negative com x100) (mean positive cpm - mean negative cpm)

Sensitisation of Cells to ionising radiation or DNA double } strand break chemotherapies

To test the efficacy of the ATM inhibitor compound 4 on its ability to sensitise cells to ionising radiation or to DNA double strand break inducing chemotherapeutics, clonogenic survival assays were performed using the Hela or ILoVo human tumour derived cell lines. The HeLa line was used for ionising radiation studies while LoVo was used for studies with chemotherapeutic agents. Enough cells to give ~100 colonies per treatment were seeded into 6 well dishes 4-6 hours prior to the addition of compound 4 at the concentrations shown on the graphs. After 1 hour, a concentration range of either etoposide (figure 2), camptothecin (figure 3) or doxorubicin (figure 4) was added.

For ionising radiation treatment (figure 1), after 1 hour of incubation with compound 4, cells were irradiated at 1Gy/min using a Faxitron 43855D X-ray cabinet. For all treatments, after a further 16 hours incubation, drug containing media was removed and fresh media added prior to a further incubation of 10 days before the staining of colonies with Giemsa. All compounds were solubilised in DMSO, with a final concentration on cells of no more than 0.1%. Resulting colonies containing >50 cells were counted as positives.

Recombinant retroviral vectors and virus preparation.

The WW /LTR /Vpr replication deficient HIV-1 gag/pol expressing packaging constructs were designed based on the vector LAP2GPH - (Haselhorst et al., 1998 Development of cell lines stably expressing human immunodeficiency virus type 1 proteins for studies in encapsidation and gene transfer. J Gen Virol, 79,

231-7.). A HIV-1 integrase mutant packaging construct, which codes for a D64V amino acid change in the integrase gene, was made by site directed mutagenesis (Quikchange mutagenesis

System, Stratagene). The HIV-1 luciferase transfer vector,

HIV~Luc, was constructed by inserting the firefly luciferase gene in-between two HIV-1 LTR sequences and a ¥ HIV-1 RNA packaging signal sequence. The VSV G envelope expression plasmid has been described previously (Naldini et al., (1996)

In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science, 272, 263-7). HIV-1 recombinant retroviral stocks were produced using a modification of the three-plasmid expression system described by Naldini et al., 1996. 6 x10°® human kidney 293T cells were co-transfected with 10 pg packaging construct WT or integrase

D64V mutant, 8 pg HIV-Luc transfer vector and 5 pug VSV G envelope protein expression plasmids using Lipofectamine-2000 reagent (Gibco-BRL). 48 hours post transfection retrovirus- containing cell culture supernatants were harvested, filtered through 0.45 pM cellulose acetate membranes and stored at - 80°C. Recombinant HIV-1 viral titres were estimated using the

HIV-1 p24 gag antigen ELISA kit from Beckman-Coulter, according to the manufacturers’ instructions.

Retroviral transductions (LUCIA).

For the HIV-1 based luciferase assays (LUCIA), Jurkat T-cells (suspension cultures) were transduced with HIV-Luc recombinant virus containing supernants at an MOI of 0.5 in the presence of 8 ng/ml polybrene at 37°C for 1 hour. Cells were washed and then plated in multiple wells (3 x10? cells/well) of a 96-well opaque-white tissue culture plate (Corning) containing different concentrations of inhibitors. For Hela cells {adherent cells) were plated and allowed to attach for 24 hours before exposure to virus containing supernatants. Cells were incubated at 37°C for 48 hours post virus addition and

Luciferase activity was quantified on a Packard TopCount-NXT microplate scintillation counter using Bright-Glo luciferase assay reagent (Promega Corp.). The standard error (S.E.) given for all guantified transduction experiments is calculated from at least three independent experiments. Cytotoxicity was evaluated in parallel with LUCIA (but without virus) using the commercially available CellTiter-96 AQueous One solution cell proliferation assay (Promega Corp.), according to the manufacturers’ instructions.

HIV-1 4-day replication assays

C8166 T-cells were washed and infected with HIV-1 (strains

HXB24: and HXB2pzrres, RT amino acid changes 67N, 70R, 215F, 219Q) at low multiplicity of infection for one hour at room temperature. The cells were then washed and distributed (5 x 10° cells/well) in triplicate into the wells of 96-well cell culture plates containing different concentrations of inhibitors. The plates were then incubated at 37°C for 4 days.

The cell-free culture fluid was then harvested and assayed for levels of p24 viral antigen using a commercially available

ELISA kit (Murex), according to the manufacturers’ instructions. Cytotoxicity was evaluated by distributing (5 x 10? cells/well) uninfected C8166 T-cells into triplicate wells of 96-well cell culture plates containing different concentrations of inhibitor and incubating the plates at 37°C.

After 4 days, 25ul of XTT, which is metabolised by viable but not dead cells was added and the plates incubated for a . further 3 hours at 37°C. Finally, the absorbance was read at a wavelength of 450nm.

Results

In vitro ATM assays

Compounds were assayed for ATM inhibition activity using the method described above. Some results are detailed below in

Table 1 as IC¢y values (the concentration at which 50% of the enzyme activity is inhibited). These are determined over a range of different concentrations, normally from 100 pM down to 1 nM. Such ICsy values are used as comparative values to identify increased compound potencies.

TABLE 1 [ow

EL

The following compounds had ICyg values of less than 200nM: 19- 43, 44-87, 93, 102, 106-107, 109-113, 115-117, 119, 122-124, 126-131, 133-135, 137-140, 142-182.

The following compounds had ICsgo values of less than 2uM, in addition to those listed above: 88-92, 94-101, 103-105, 108, 114, 118, 120-121, 125, 132, 136 and 141.

Sensitisation of Cells to ionising radiation or DNA double strand break chemotherapies

The data shown in figures 1-4 clearly show that inhibiting ATM with compound 4 has a significant effect on sensitising tumour derived cell lines to DNA double strand break inducing agents.

Retroviral transductions (LUCIA)

Compound 4 (known as Ku0064) was tested for its ability to repress retroviral infections using HIV-1 based LUCIA (Fig.5).

It was found to efficiently inhibit HIV-1 LUCIA at low micromolar concentrations in Jurkat T-cells (Fig. 5) as well as all other ATM proficient cell lines tested. The 50% inhibitory concentration (ICsp) for Compound 4 in LUCIA was around 1-2 pM in Jurkat cells (Fig. 5) and in the range of 1 to 10 uM for all other cell lines tested.

Compound 4 was also tested for cytotoxic and growth inhibition effects in parallel to LUCIA to ensure that this was not the reason for the observed reduction in transduction efficiency.

At concentrations up to 10 uM, compound 4 exposure showed no significant cytotoxic effects on Jurkat cells during the course of the assay (Fig. 5).

HIV-1 based LUCIA was performed on Hela cells in the presence of increasing concentrations of both compound 4 and the nucleoside analog reverse transcriptase inhibitor, 3'-azido- 3’ -deoxythymidine (AZT). :

Fig. 7 shows that the combination of compound 4 and AZT was found to act more effectively in inhibiting HIV-1 infection than either drug alone. Fig. 7 provides an example in which increasing concentrations of AZT was found to enhance the effectiveness of a compound of the present invention in inhibiting HIV-1 infections.

HIV Replication Inhibition

A replication competent HIV-1 strain (HIVuyxs,) was used to infect C8166 T-cells in the absence or presence of increasi_ng concentrations of compound 4 (Figure 8), in order to demonstrate the effectiveness of compounds of the present invention in a system where HIV replication occurs. After 4- days of virus replication, the amount of HIV-1 in cell cult-ure supernatants was quantified by p24 antigen ELISA. As a control, the RT inhibitor AZT was used in parallel experiments. Fig. 8A shows the inhibition of HIV-1 replication of a wild type HIV-1 strain (HIV; wt) by comp-ound 4 and AZT. The ICso concentration of Compound 4 for HIV-1 replication inhibition was 0.1 pM. AZT showed an ICsp of O .002 pM. 4-day replication assays were performed using an AZT drug resistant strain of HIV-1 (HIVys, AZTres) in the absence or presence of increasing concentrations of compound 4 (Fig. 8B,

Table 2). The 1C¢y concentration of Compound 4 for HIV-1 replication inhibition on the AZT resistant strain was 0.06 nM. AZT showed an ICso of 0.05 pM (Table 2), thereby demonstrating a 25-fold resistance to AZT when compared to the wild type strain.

Compound 4 inhibited HIV-1 replication equally well on wilcdl- type HIV-1 (ICs = 0.1 uM; Fig. 8A, Table 2) as on the AZT resistant HIV-1 strain (ICsy = 0.06 uM; Fig. 8B, Table 2).

These data provide evidence that compounds of the present invention may be effective in both the treatment of wild-type and acquired AZT resistant HIV-1 infections and, by implication, HIV-1 strains resistant to other drugs that target viral proteins.

Compound 4 was also tested for cytotoxic and growth inhibition effects on CB166 cells in parallel to the HIV-1 replication assays to ensure that this was not the reason for the observed effects of viral titre (Fig. 8C). Exposure of C8166 cells to

Compound 4 showed no significant cytotoxic effects during the course of the assay or over the effective concentration range (less than 1 pM) shown to inhibit HIV-1 replication. The 50% cytotoxic concentration (CCsg) of compound 4 on C8166 cells was estimated to be greater than 20 pM. Table 2 summarises the experiments showing the anti-HIV-1 activity of Compound 4 in 4-day replication assays. Interestingly, the ICsg in LUCIA (1 pM; Fig 5) was observed to be 10-fold higher than in the replication assays (0.1 pM; Fig 8A). This difference can be explained by the fact that in replication assays multiple rounds of infection occur and each round provides the potential for HIV-1 inhibition. Therefore, the inhibitory effect of Compound 4 becomes compounded in HIV-1 replication assays. The estimated ICsp concentrations in replication assays may therefore represent a more accurate reflection of the extent of inhibition that may be seen in HIV-1 infected patients. ae

Wild type AZT resistant

Compound 4 | oa | 6.06 (25 fold resistant)

Table 2

Claims (15)

Claims

1. A compound of formula I: R' RA _P_ _N ~N 2 Or R (1) Q Y and isomers, salts, solvates, chemically protected forms, and prodrugs thereof, wherein: one of P and Q is O, and the other of P and Q is CH, where there is a double bond between whichever of Q and P is CH and the carbon atom bearing the R?® group; Y is either O or S; R! and R? are independently hydrogen, an optionally substituted Ci-7 alkyl group, Cs.z0 heterocyclyl group, or Cs.3¢ aryl group, or may together form, along with the nitrogen atom to which they are attached, an optionally substituted heterocyclic ring having from 4 to 8 ring atoms; R?® is a phenyl or pyridyl group, attached by a first bridge group selected from -S-, -5(=0)-, -5§(=0),-, -O0-, -NR- and CR'R“*~ to an optionally substituted Cs-9 carboaryl group, in which one aromatic ring atom may be replaced by a nitrogen ring atom; the phenyl or pyridyl group and optionally substituted Cs.yg carboaryl group being optionally further linked by a second bridge group, which is bound adjacent the first bridge group on both groups so as to form an optionally substituted Cs; ring fused to both the phenyl or pyridyl group and the Cs-3g carboaryl group, the phenyl or pyridyl group being further optionally substituted; wherein RY is selected from hydrogen, an ester group, an optionally substituted Cj.» alkyl group, an optionally substituted C;.;o heterocyclyl group and an optionally substituted Cs. aryl group; and R®* and R®® are independently selected from hydrogen, an optionally substituted C;.; alkyl group, an optionally substituted C;.;o heterocyclyl group and an optionally substituted Cs. aryl group.

2. A compound according to claim 1, of formula Ia: R' RA__O_ _N Np? or R (1a) Y

3. A compound according to either claim 1 or claim 2, wherein Y is O.

4. A compound according to any one of claims 1 to 3, wherein R!' and R? form, along with the nitrogen atom to which they are attached, a heterocyclic ring having 6 ring atoms.

5. A compound according to claim 4, wherein R!' and R?® form, along with the nitrogen atom to which they are attached, a group selected from morpholino and thiomorpholino

6. A compound according to any one of claims 1 to 5, wherein the phenyl or pyridyl group is a phenyl group.

7. A compound according to any one of claims 1 to 6, wherein the phenyl or pyridyl ring or the Cs.;, carboaryl group in R? bear a substituent selected from the group consisting of acylamido, sulfonamino, ether, ester, amido and acyl. AMENDED SHEET

8. A co mpound according to any one of claims 1 to 7, wherein R> is selescted from the following optionally substituted groups CO CLIC (LI Ss o H Cr

9. A composition comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

10. A compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof for use in a method of therapy.

11. The use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the activity of

ATM.

12. The use of a compound according to any one of claims 1 to 8 or a phkrarmaceutically acceptable salt thereof in the preparation of a medicament for use as an adjunct in cancer therapy or for potentiating tumour cells for treatment with ionising radiation or chemotherapeutic agents.

13. The use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of retroviral mediated diseases or disease ameliorated by the inhibition of ATM, which include acquired immunodeficiency syndrome.

14. A method of inhibiting ATM in vitro, comprising contacting a cell with an effective amount of a compound according to any one of claims 1 to 8.

15. A compound of formula I specifically described as any one of compounds 4 to 7, 9, 13, 17, 18 to 182. AMENDED SHEET