WO2009127815A1

WO2009127815A1 - Aminoalyl-imidazotetrazines for treatment of cancer

Info

Publication number: WO2009127815A1
Application number: PCT/GB2009/000957
Authority: WO
Inventors: Richard Thomas Wheelhouse; Dimitrios Pletsas
Original assignee: The University Of Bradford
Priority date: 2008-04-18
Filing date: 2009-04-14
Publication date: 2009-10-22
Also published as: GB0807103D0

Abstract

The present invention relates to imidazotetrazines of Formula (I) and their use in the treatment of cancer.

Description

AMINOALYL-IMIDAZOTETRAZINES FOR TREATMENT OF CANCER

Field of the Invention

The present invention relates to compounds, in particular imidazotetrazines, useful in the treatment of cancer.

Background to the Invention

Mitzolomide, a member of the N(3) substituted family of 8-carbamoylimidazo[5,l-d]- [1,2,3,5] tetrazin-4(3H)-ones, had curative activity against murine tumour models but it proved unpredictably myelosuppressive in humans so was replaced by the less toxic analogue Temozolomide. The range of tumours that can be treated with Temozolomide is strictly limited, some tumours are tolerant of the drug and others are actively resistant. Temozolomide (Temodal®, Temodar®) is currently licensed for the treatment of malignant melanoma and glioma.

Temozolomide is a DNA methylating agent and clinical response depends on two major DNA repair systems. Susceptibility to the drug depends on the presence of active mismatch repair (MMR). Without this a cell line is not susceptible to the drug so the tumour cannot respond to the drug. A low level of MMR is notably characteristic of certain tumours including colon, ovarian and bladder tumours. There is a need for new DNA modifying agents whose therapeutic effect is independent of active MMR in the tumour cells.

The principal route of active resistance to temozolomide is O6-alkylguanine-DNA alkyltransferase (MGMT) which repairs the drug-damaged sites on DNA so reverses the effect of the drug. There is a need for new DNA alkylating compounds with activity independent of MGMT within the tumour cells.

The present inventors have prepared DNA alkylating agents whose activity is independent of the MMR and MGMT status of the cell lines without the use of an adjuvant. Such agents can be used to treat those tumours that were previously either not susceptible to DNA methylating agents or which were actively resistant to them.

Statements of Invention

According to a first aspect of the invention there is provided a compound of formula I

wherein R and R¹ are each independently selected from hydrogen or R¹²; or R and R¹ taken together with the atoms to which they are attached form a carbocycle or a heterocycle, optionally substituted with one or more R¹²;

A is selected from N, O or S;

X is selected from the group consisting of hydrogen, halogen, halide, and hydrocarbyl optionally substituted with R¹²;

Y, which may be absent, is selected from the group consisting of hydrogen, halogen, halide and hydrocarbyl optionally substituted with R¹²,

wherein each R¹² is independently selected from halogen, trifiuoromethyl, cyano, nitro, oxo, =NR¹³, -OR¹³, -C(O)R¹³, -C(O)OR¹³, -OC(O)R¹³, -S(O)₁R¹³, - N(R¹³)R¹⁴, -C(O)N(R¹³)R¹⁴, -SO₂N(R¹³)R¹⁴ and R¹⁵;

wherein R¹³ and R¹⁴ are each independently selected from hydrogen or R¹⁵; wherein R¹⁵ is selected from hydrocarbyl and -(CH₂)_q-heterocyclyl, and each R¹⁵ is optionally and independently substituted with one or more of halogen, cyano, amino, hydroxy, C_1-6 alkyl and C_1-6 alkoxy;

n is 0, 1, 2, 3, 4 or 5; q is O, 1, 2, 3, 4, 5 or 6;

or a pharmaceutically acceptable salt or prodrug thereof.

The compounds of the invention can exist in different forms, such as free acids, free bases, esters and other prodrugs, salts and tautomers, for example, and the invention includes all variant forms of the compounds.

Hydrocarbyl The term "hydrocarbyl" as used herein includes reference to moieties consisting exclusively of hydrogen and carbon atoms; such a moiety may comprise an aliphatic and/or an aromatic moiety. The moiety may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Examples of hydrocarbyl groups include Ci_-6 alkyl (e.g. Ci, C₂, C₃ or C₄ alkyl, for example methyl, ethyl, propyl, isopropyl, n- butyl, sec-butyl or tert-butyl); Ci_-6 alkyl substituted by aryl (e.g. benzyl) or by cycloalkyl (e.g. cyclopropylmethyl); cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl); alkenyl (e.g. 2-butenyl); alkynyl (e.g. 2-butynyl); aryl (e.g. phenyl, naphthyl or fluorenyl) and the like.

Alkyl

The terms "alkyl" and "C_1-6 alkyl" as used herein include reference to a straight or branched chain alkyl moiety having 1, 2, 3, 4, 5 or 6 carbon atoms. This term includes reference to groups such as methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyl or tert-butyl), pentyl, hexyl and the like. In particular, alkyl may have 1, 2, 3 or 4 carbon atoms. Alkenyl

The terms "alkenyl" and "C₂-6 alkenyl" as used herein include reference to a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6 carbon atoms and having, in addition, at least one double bond, of either E or Z stereochemistry where applicable. This term includes reference to groups such as ethenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-ρentenyl, 1-hexenyl, 2-hexenyl and 3-hexenyl and the like.

Alkynyl

The terms "alkynyl" and "C_2-6 alkynyl" as used herein include reference to a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6 carbon atoms and having, in addition, at least one triple bond. This term includes reference to groups such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1- hexynyl, 2-hexynyl and 3-hexynyl and the like.

Alkoxy

The terms "alkoxy" and "C_1-6 alkoxy" as used herein include reference to -O-alkyl, wherein alkyl is straight or branched chain and comprises 1, 2, 3, 4, 5 or 6 carbon atoms. In one class of embodiments, alkoxy has 1 , 2, 3 or 4 carbon atoms. This term includes reference to groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentoxy, hexoxy and the like.

Cycloalkyl

The term "cycloalkyl" as used herein includes reference to an alicyclic moiety having 3, 4,

5, 6, 7 or 8 carbon atoms. The group may be a bridged or polycyclic ring system. More often cycloalkyl groups are monocyclic. This term includes reference to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo[2.2.2]octyl and the like.

Aryl The term "aryl" as used herein includes reference to an aromatic ring system comprising

6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring carbon atoms. Aryl is often phenyl but may be a polycyclic ring system, having two or more rings, at least one of which is aromatic. This term includes reference to groups such as phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl and the like. The term aryl includes heteroaryl.

Carbocyclyl

The term "carbocyclyl" as used herein includes reference to a saturated (e.g. cycloalkyl) or unsaturated (e.g. aryl) ring moiety having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon ring atoms. In particular, carbocyclyl includes a 3- to 10-membered ring or ring system and, in particular, a 5- or 6-membered ring, which may be saturated or unsaturated.

A carbocyclic moiety is, for example, selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo[2.2.2]octyl, phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl and the like.

Heterocyclyl

The term "heterocyclyl" as used herein includes reference to a saturated (e.g. heterocycloalkyl) or unsaturated (e.g. heteroaryl) heterocyclic ring moiety having from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen, oxygen, phosphorus, silicon and sulphur. In particular, heterocyclyl includes a 3- to 10-membered ring or ring system and more particularly a 5- or 6-or 7- membered ring, which may be saturated or unsaturated.

A heterocyclic moiety is, for example, selected from oxiranyl, azirinyl, 1 ,2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thiopyranyl, thianthrenyl, isoben- zofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidyl, piperazinyl, pyridazinyl, moφholinyl, thiomorpholinyl, especially thiomorpholino, indolizinyl, isoindolyl, 3H-indolyl, indolyl, benzimidazolyl, cumaryl, indazolyl, triazolyl, tetrazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl, benzothiophenyl, dibenzothiophenyl, phthalazinyl, naphthyridinyl, quinoxalyl, quinazolinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, furazanyl, phenazinyl, phenothiazinyl, phenoxazinyl, chromenyl, isochromanyl, chromanyl and the like. Heterocycloalkyl

The term "heterocycloalkyl" as used herein includes reference to a saturated heterocyclic moiety having 3, 4, 5, 6 or 7 ring carbon atoms and 1, 2, 3, 4 or 5 ring heteroatoms selected from nitrogen, oxygen, phosphorus and sulphur. The group may be a polycyclic ring system but more often is monocyclic. This term includes reference to groups such as azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, oxiranyl, pyrazolidinyl, imidazolyl, indolizidinyl, piperazinyl, thiazolidinyl, morpholinyl, thiomorpholinyl, quinolinidinyl and the like.

Heteroaryl

The term "heteroaryl" as used herein includes reference to an aromatic heterocyclic ring system having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen, oxygen and sulphur. The group may be a polycyclic ring system, having two or more rings, at least one of which is aromatic, but is more often monocyclic. This term includes reference to groups such as pyrimidinyl, furanyl, benzo[b]thiophenyl, thiophenyl, pyrrolyl, imidazolyl, pyrrolidinyl, pyridinyl, benzo[b]furanyl, pyrazinyl, purinyl, indolyl, benzimidazolyl, quinolinyl, phenothiazinyl, triazinyl, phthalazinyl, 2H- chromenyl, oxazolyl, isoxazolyl, thiazolyl, isoindolyl, indazolyl, purinyl, isoquinolinyl, quinazolinyl, pteridinyl and the like.

Halogen

The term "halogen" as used herein includes reference to F, Cl, Br or I. In particular, halogen may be F or Cl.

Halide

The term "halide" as sued herein refers to a moiety containing a halogen as the anion. Examples include a hydrogen halide such as HBr and HI.

Substituted The term "substituted" as used herein in reference to a moiety means that one or more, especially up to 5, more especially 1, 2 or 3, of the hydrogen atoms in said moiety are replaced independently of each other by the corresponding number of the described substituents. The term "optionally substituted" as used herein means substituted or unsubstituted.

It will, of course, be understood that substituents are only at positions where they are che- mically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without inappropriate effort whether a particular substitution is possible.

For example, amino or hydroxy groups with free hydrogen may be unstable if bound to carbon atoms with unsaturated (e.g. olefinic) bonds. Additionally, it will of course be understood that the substituents described herein may themselves be substituted by any substituent, subject to the aforementioned restriction to appropriate substitutions as recognised by the skilled man.

Independently

Where two or more moieties are described as being "each independently" selected from a list of atoms or groups, this means that the moieties may be the same or different. The identity of each moiety is therefore independent of the identities of the one or more other moieties.

X may be cyclohydrocarbyl, for example aryl or heteroaryl, optionally substituted with R¹². Preferably X is aryl, for example phenyl, optionally substituted with R¹².

X is preferably a group

wherein D, E, J, K and L are independently selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, oxo, =NR¹⁶, -OR¹⁶, -C(O)R¹⁶, -C(O)OR¹⁶, -OC(O)R¹⁶, - S(O)₁R¹⁶, -N(R¹⁶)R¹⁷, -C(O)N(R¹⁶)R¹⁷, -SO₂N(R¹⁶)R¹⁷ and R¹⁸ wherein R¹⁶ and R¹⁷ are each independently selected from hydrogen or R¹⁸;

wherein R¹⁸ is selected from hydrocarbyl and -(CH₂)_r-heterocyclyl, and each R¹⁸ is optionally and independently substituted with one or more of halogen, cyano, amino, hydroxy, C_1-6 alkyl and Cj_-6 alkoxy;

r is 0, 1, 2, 3, 4, 5 or 6.

In an embodiment of the invention D and/or E and/or J and/or K and/or L are hydrogen. In a preferred embodiment of the invention D, E, J, K and L are all hydrogen.

In an embodiment of the invention D or E is nitro.

In an embodiment of the invention J or K is an amine or an amide. Examples include amide, thioamide and sulphonamide.

In one embodiment L is an electron withdrawing group. For example L may be selected from groups comprising fluorine, cyano and nitro. L may be selected from nitro, fluorine atoms, fluoroalkyl, fluoroaryl and fluoroheteroaryl. In an embodiment of the invention L is nitro.

Thus in one embodiment the invention provides a compound of formula I wherein X is the group

wherein L is as hereinbefore defined. Compounds according to the invention wherein L is an electron rich group have been shown to be superior in terms of activity and effect. Thus in a preferred aspect of the invention X is the group

wherein D or E and/or L are independently selected from electron rich groups including amino, sulfide, hydroxyl, alkoxy or hydrocarbyl for example alkyl (e.g. C 1-6 alkyl).

D or E and/or L may be selected -OR¹⁶, -C(O)R¹⁶, -C(O)OR¹⁶, -OC(O)R¹⁶, -S(O)₁R¹⁶, - N(R¹⁶)R¹⁷, -C(O)N(R¹⁶)R¹⁷, -SO₂N(R¹⁶)R¹⁷ and R¹⁸.

hi an embodiment of the invention L is an electron rich group for example amino, sulfide, hydroxyl, alkoxy or hydrocarbyl for example alkyl (e.g. C 1-6 alkyl such as methyl).

J and K may be hydrogen.

In a preferred compound of the invention R and R¹ taken together with the atoms to which they are attached form a heterocycle, optionally substituted with one or more R¹². The heterocycle may be a five membered heterocycle containing from 1 to 5 heteroatoms independently selected from N, S or O wherein each substitutable carbon or heteroatom in the heterocycle is optionally and independently substituted by one or more R¹². Preferably the heterocycle is a five membered heterocycle containing from 1 to 2 N atoms wherein each substitutable carbon or N in the heterocycle is optionally and independently substituted by one or more R¹². Thus in a preferred aspect of the invention the compound is of formula (H)

(H)

wherein A, X, Y and n are as defined herein;

R⁴ is selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, oxo, =NR¹⁹, -OR¹⁹, - C(O)R¹⁹, -C(O)OR¹⁹, -OC(O)R¹⁹, -S(O)₁R¹⁹, -N(R¹⁹)R²⁰, -C(O)N(R¹⁹)R²⁰, -SO₂N(R¹⁹)R²⁰ and R²¹

wherein R¹⁹ and R²⁰ are each independently selected from hydrogen or R²¹;

wherein R²¹ is selected from hydrocarbyl and -(CH₂)_t-heterocyclyl, and each R²¹ is optionally and independently substituted with one or more of halogen, cyano, amino, hydroxy, C_1-6 alkyl and Ci_-6 alkoxy;

t is O, 1, 2, 3, 4, 5 or 6;

In a further preferred aspect of the invention the compound is of formula (lib)

(lib) wherein A, X, Y and n are as defined herein;

wherein R²¹ is selected from hydrocarbyl and -(CthVheterocyclyl, and each R²¹ is optionally and independently substituted with one or more of halogen, cyano, amino, hydroxy, C_1-6 alkyl and Ci_-6 alkoxy;

t is 0, 1, 2, 3, 4, 5 or 6;

In a preferred compound of the invention the compound is of formula II or Hb and R⁴ is amide for example CONH₂.

In a preferred compound of the invention A is selected from O or S. Preferably A is O.

In a preferred compound of the invention n is 1 , 2 or 3 for example 1.

In a preferred aspect of the invention Y is hydrogen, halogen, halide or an aliphatic hydrocarbyl group for example alkyl (e.g. C 1-6 alklyl).

In a further preferred aspect of the invention Y is cylohydrocarbyl for example aryl such as heteroaryl. Thus the compound of the invention may be of formula III

(III) wherein R, R¹, A, X and n are as defined herein;

B is selected from N, O or S;

R² and R³ are each independently selected from hydrogen or R²²; or R² and R³ taken together with the atoms to which they are attached form a carbocycle or a heterocycle, optionally substituted with one or more R²²;

wherein each R²² is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, =NR²³, -OR²³, -C(O)R²³, -C(O)OR²³, -OC(O)R²³, -S(O)₁R²³, - N(R²³)R²⁴, -C(O)N(R²³)R²⁴, -SO₂N(R²³)R²⁴ and R²⁵;

wherein R²³ and R²⁴ are each independently selected from hydrogen or R²⁵;

wherein R²⁵ is selected from hydrocarbyl and -(CH₂)_u-heterocyclyl, and each R²⁵ is optionally and independently substituted with one or more of halogen, cyano, amino, hydroxy, Ci_-6 alkyl and C_1-6 alkoxy;

m is O, 1, 2, 3, 4 or 5

or a salt thereof.

In a preferred compound of the invention A and B are independently selected from O or S. Preferably A and B are O.

In a preferred compound of the invention m is 1, 2 or 3 for example 1. Preferably n is 1 and m is 1.

In a preferred compound of the invention R and R and/or R and R taken together with the atoms to which they are attached form a heterocycle, optionally substituted with one or more R¹². The heterocycle may be a five membered heterocycle containing from 1 to 5 heteroatoms independently selected from N, S or O wherein each substitutable carbon or heteroatom in the heterocycle is optionally and independently substituted by one or more R¹². Thus in a preferred aspect the invention provides a compound of formula (IV)

wherein R⁴, A, B, X, n and m are as defined herein;

R⁵ is selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, oxo, =NR²⁶, -OR²⁶, - C(O)R²⁶, -C(O)OR²⁶, -OC(O)R²⁶, -S(O))R²⁶, -N(R²⁶)R²⁷, -C(O)N(R²⁶)R²⁷, -SO₂N(R²⁶)R²⁷ and R²⁸

wherein R²⁶ and R²⁷ are each independently selected from hydrogen or R²⁸;

wherein R is selected from hydrocarbyl and -(CH₂)_u-heterocyclyl, and each R is optionally and independently substituted with one or more of halogen, cyano, amino, hydroxy, C_1-6 alkyl and C₁^ alkoxy;

u is O, 1, 2, 3, 4, 5 or 6;

In a preferred compound of the invention the compound is of formula IV and R is amide for example CONH₂.

In an embodiment of the invention the compound is an anilinoethyl imidazotetrazine.

In a further embodiment of the invention the compound is an anilino (e.g. diethylanilino) linked bis imidazotetrazine. Compounds of the invention may exist in different forms, such as free acids, free bases, esters and other prodrugs, salts and tautomers, for example, and the disclosure includes all variant forms of the compounds.

The extent of protection includes counterfeit or fraudulent products which contain or purport to contain a compound of the invention irrespective of whether they do in fact contain such a compound and irrespective of whether any such compound is contained in a therapeutically effective amount.

Included in the scope of protection are packages which include a description or instructions which indicate that the package contains a species or pharmaceutical formulation of the invention and a product which is or comprises, or purports to be or comprise, such a formulation or species. Such packages may be, but are not necessarily, counterfeit or fraudulent.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

It will of course be appreciated that, where appropriate, each compound may be in the form of the free compound, an acid or base addition salt, or a prodrug.

Where appropriate, compounds of the invention may be in the form of pharmaceutically acceptable salts. The term "pharmaceutically acceptable" as used herein includes reference to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. This term includes acceptability for both human and veterinary purposes. Pharmaceutically acceptable salts can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., US, 1985, p. 1418, the disclosure of which is hereby incorporated by reference; see also Stahl et al, Eds, "Handbook of Pharmaceutical Salts Properties Selection and Use", Verlag Helvetica Chimica Acta and Wiley-VCH, 2002.

The disclosure thus includes pharmaceutically-acceptable salts of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof, for example the conventional non-toxic salts or the quaternary ammonium salts which are formed, e.g. from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3- phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen- containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others. The invention includes prodrugs for the active pharmaceutical species of the invention, for example in which one or more functional groups are protected or derivatised but can be converted in vivo to the functional group, as in the case of esters of carboxylic acids convertible in vivo to the free acid, or in the case of protected amines, to the free amino group. The term "prodrug," as used herein, represents in particular compounds which are rapidly transformed in vivo to the parent compound, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987; H Bundgaard, ed, Design of Prodrugs, Elsevier, 1985; and Judkins, et al. Synthetic Communications, 26(23), 4351-4367 (1996), each of which is incorporated herein by reference.

Prodrugs therefore include drugs having a functional group which has been transformed into a reversible derivative thereof. Typically, such prodrugs are transformed to the active drug by hydrolysis. As examples may be mentioned the following:

Prodrugs also include compounds convertible to the active drug by an oxidative or reductive reaction. As examples may be mentioned oxidative activation (e.g. N- and O- dealkylation, oxidative deamination, N-oxidation or epoxidation) and reductive activation (e.g. azo reduction, sulfoxide reduction, disulfide reduction, bioreductive alkylation or nitro reduction).

Also to be mentioned as metabolic activations of prodrugs are nucleotide activation, phosphorylation activation and decarboxylation activation. For additional information, see "The Organic Chemistry of Drug Design and Drug Action", R B Silverman (particularly Chapter 8, pages 497 to 546), incorporated herein by reference.

The use of protecting groups is fully described in 'Protective Groups in Organic Chemistry^', edited by J W F McOmie, Plenum Press (1973), and 'Protective Groups in Organic Synthesis^', 2nd edition, T W Greene & P G M Wutz, Wiley-Interscience (1991).

Thus, it will be appreciated by those skilled in the art that, although protected derivatives of compounds of the disclosure may not possess pharmacological activity as such, they may be administered, for example parenterally or orally, and thereafter metabolised in the body to form compounds of the invention which are pharmacologically active. Such derivatives are therefore examples of "prodrugs". All prodrugs of the described compounds are included within the scope of the disclosure.

It will be appreciated that all isomeric and tautomeric forms of the compounds are encompassed by the present invention.

The compounds of the disclosure may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. All diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means (e.g. HPLC, chromatography over silica). All stereoisomers are included within the scope of the disclosure. Where a single enantiomer or diasteromer is disclosed, the disclosure also covers the other enantiomers or diastereomers, and also racemates; in this regard, particular reference is made to the specific compounds listed herein.

Geometric isomers may also exist in the compounds of the present disclosure. The present disclosure contemplates the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond and designates such isomers as of the Z or E configuration, wherein the term "Z" represents substituents on the same side of the carbon-carbon double bond and the term "E" represents substituents on opposite sides of the carbon-carbon double bond.

The disclosure therefore includes all variant forms of the defined compounds, for example any tautomer or any pharmaceutically acceptable salt, ester, acid or other variant of the defined compounds and their tautomers as well as substances which, upon administration, are capable of providing directly or indirectly a compound as defined above or providing a species which is capable of existing in equilibrium with such a compound.

Synthesis

By way of illustration, a compound of the invention may be prepared according to the following general Schemes:

Analogue W X^" Y^" a H H H b H CH₃ H

C H CH₃ NO₂ d H F H e H Cl H f H OMe H g H OMe NO₂ Reagents and conditions: (i) CuCI, AcOH, Δ; (ii) NH₂NH₂ XH₂O / IPA, Δ; (iii) NaNO₂ AcOH, H₂O/CH₂CI₂, 0 ⁰C; (iv) PhMe, Δ, [>95%]; (v) DMSO, RT, days.

Scheme 1: Route to Diethylanilino-linked Bis-imidazotetrazines

In addition to the bi-functional derivatives 6, the analogous mono-functional agents 5 were also prepared; an example with an aliphatic amine 8 is also shown.

5 5

Reagents and conditions: (i) CuCI, AcOH, Δ; (ii) NH₂NH₂IxH₂O / IPA, Δ; (iii) NaNO₂, AcOH, H₂O/CH₂CI₂, O DC; (iv) PhMe, Δ, [>95%]; (v) DMSO, RT, days; (vi) TFA, CH₃CN, O DC, 2 h.

Scheme 2: Route to Anilinoethyl Imidazotetrazines. Z^" =

BOCNH ' Z^" = NCO

Reagents and conditions: (i) NH₂NH₂ xH2O / IPA, Δ; (ii) NaNO₂, AcOH, H₂O/CH₂CI₂, O ⁰C; (iii) PhMe, Δ, [>95%]; (iv) DMSO, 30 ⁰C, 36 h; (vi) HBr, CH₃CN, RT, 12 h.

Scheme 3. Preparation of Compounds with Aliphatic Sidechains

Reagents and conditions: (i) HNO₃ / H₂SO₄, 0-10 ⁰C; (ii) NH₂(CH₂J₂CO₂Me / Et₃N / CH₃CN, Δ; (iii) NH₂NH₂ XH₂O / IPA, Δ; (iv) NaNO₂, AcOH, H₂O/CH₂CI₂, 0 ⁰C; (v) PhMe, Δ; (vi) 2 / DMSO, RT, 24 h; (vii) HNO₃ / H₂SO₄, 0-70 ⁰C 12 h; (viii) SOCI₂ / DME / DMF, 50 ⁰C, 18 h; (ix) 27 / Et₃N / THF / 100 ⁰C, 12 h; (x) -20 ⁰C, 12 h.

Scheme 4: Route to Precursors of CB 1954 analogues

It will be understood that the processes detailed above are solely for the purpose of illustrating the invention and should not be construed as limiting. A process utilising similar or analogous reagents and/or conditions known to one skilled in the art may also be used to obtain a compound of the invention.

Any mixtures of final products or intermediates obtained can be separated on the basis of the physico-chemical differences of the constituents, in a known manner, into the pure final products or intermediates, for example by chromatography, distillation, fractional crystallisation, or by the formation of a salt if appropriate or possible under the circumstances.

The present inventors have found that yields of the compounds of the invention can be improved by purifying the crude isocyanate prior to reacting it with 5-diazoimidazole-4- carboxamide. Thus, in a further aspect the invention provides a process for the preparation of a compound according to the invention, the process comprising the steps of

(i) contacting a compound of formula V with an ether

^X' (V)

wherein R' is hydrogen, C 1-6 alkyl (for example methyl) optionally substituted with NCO; and wherein X' has the same meaning as X as defined herein; (ii) contacting the product of (i) with a diazoimidazole.

The diazoimidazole may be S-diazoimidazole^-carboxamide.

In a preferred process of the invention R' is hydrogen or methyl.

In a further preferred process of the invention the compound of formula V is

OCN /\ /\ NCO

N'

X'

wherein X' is as defined herein.

Preferably (i) is carried out with heating. The ether extract is separated from an oily residue and then evaporated to give the product (i).

Preferably step (ii) is performed in the presence of a solvent such as DMSO. The DMSO is generally dry DMSO at room temperature. Step (ii) may be carried out under nitrogen and may be carried out in the absence of light.

Administration & Pharmaceutical Formulations

In other aspects the invention provides a compound, or pharmaceutically acceptable salt or prodrug thereof, as hereinbefore described for use as a medicament. The use of compounds of the invention as medicaments may be desirable over known DNA alkylating agents due, inter alia, to the former being more effective in the treatment of resistant cancers and cancers which were previously shown not to be susceptible to DNA alkylating agents.

In further aspects, there is provided a pharmaceutical formulation comprising a compound, or pharmaceutically acceptable salt or prodrug, as hereinbefore described.

The formulation may contain at least one additional pharmaceutically acceptable component, e.g. an excipient, diluent or carrier. Preferably, the formulation is intended for parenteral administration.

According to a further aspect of the invention there is provided a pharmaceutical composition comprising a compound, or pharmaceutically acceptable salt or prodrug thereof, according to the invention. A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.

The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or

Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration. The selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required for to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

In an aspect of the present invention, the compound, or pharmaceutical composition, of the invention may be used to treat cancer. According to a further aspect of the invention there is provided a compound according to the invention, or a pharmaceutically acceptable salt or prodrug thereof, for use in the treatment of cancer in a patient.

Another aspect of the invention concerns the use of a compound according to the invention, or a pharmaceutically acceptable salt or prodrug thereof, for the manufacture of a medicament for the treatment of cancer.

According to a further aspect of the invention there is provided a method to treat cancer in a subject comprising administering an effective amount of a compound according to the invention. In a preferred method of the invention said subject is human.

As used herein, the term "cancer" refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed . cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term "cancer" includes malignancies of the various organ systems, such as those affecting, for example, lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumours, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, ovarian, bladder, colon, skin, prostate, breast and central nervous system for example the brain.

In a preferred aspect a compound or composition according to the invention may be useful in the treatment of colon, prostate, ovary and skin.

The term "carcinoma" also includes carcinosarcomas, e.g., which include malignant tumours composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation. Further types of cancer include leukaemia, skin, intracranial and brain cancer.

A pharmaceutical formulation comprising a compound of the present invention may be administered in combination, either sequentially or at a substantially similar time as a chemotherapeutic agent.

As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and may be performed either for prophylaxis or during the course of clinical pathology. Desirable effects include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, lowering the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.The term, "treatment" as used herein is intended to include the treatment and prevention of the indicated conditions/disorders

There is further provided a package or kit of parts comprising:

(1) a compound as described herein; together with

(2) instructions to use the agent in a method described herein. The package defined herein may comprise more than one dosage unit, in order to provide for repeat dosing. If more than one dosage unit is present, such units may be the same, or may be different in terms of the dose of active agent composition and/or physical form.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

An embodiment of the invention will now be described by way of example only and with reference to the following Figures and Materials and Methods;

Figure 1. IC₅₀ values (μM) of selected compounds against A2780 (MMR+) and A2780- cp70 (MMR-) cells in the absence (MGMT+) and presence (MGMT-) of the MGMT inactivator PaTrin2 (10 mM). All data are the means of at least three determinations. TMZ, temozolomide Ia; MTZ, mitozolomide Ib. * IC₅₀ >250 μM.

Figure 2. Hammett Plot for the Bi-functional Imidazotetrazines 6a, b, d, e, f.

EXAMPLE

Synthesis of compounds

Twenty novel imidazotetrazines have been achieved, Table 1. These were prepared by variants on the standard imidazotetrazine synthesis (Scheme) where a diazoimidazole 2 reacts with an isocyanate 3 to yield the imidazotetrazine. The target compounds all included an aminoethyl (CH₂CH₂NR₂) group in the 3-substituent. The requisite β- aminoisocyanate precursors 4 are inherently unstable compounds as they bear conventionally incompatible functional groups. Preparation, isolation and purification of these required development work to devise special production and handling procedures. The methods developed are broadly applicable and may be applied to extend further the range of accessible structures in the imidazotetrazine class.

The novel compounds may be considered under three categories: Group 1, mono- functional agents 5; Group 2, bi-functional agents 6; Group 3, CB 1954 analogues 7.

Table 1. Summary of Compounds Prepared CONH₂

Cpd R^A W^A X^A Cpd W^B X^B _γb Cpd R^C x^c

5a CH₃ H H H 6a H H H 7a CH₃ H

5b CH₃ H CH₃ H 6b H CH₃ H 7b H H

5c CH₃ NO₂ CH₃ H 6c NO₂ CH₃ NO₂ 7c H NO;

5d CH₃ H Cl H 6d H F H

5e H H Cl H 6c H Cl H Others

5h CH₃ H OMe NO₂ 9 V I

5i BOC H Cl H

5k CH₃ H NO₂ H Experimental Synthesis

Dimethyl 3,3'-(4-chlorophenylazanediyl)dipropanoate 1Oe General Method A: Double Michael addition to anilines. 4-Chloroaniline (10 g, 78.4 mmol) was mixed with methyl acrylate (67.5 g, 784 mmol, 10 eq), cuprous chloride (1.24 g, 12.5 mmol, 0.16 eq) and AcOH (100 mL) and heated under reflux at 140 °C for 48 h. The reaction mixture was allowed to reach room temperature and water (300 mL) was added with strong agitation. The batch was allowed to stand in the fridge overnight and the water layer was decanted leaving the oil behind. The oil was washed with more water (2 x 30OmL), diluted with diethylether (200 mL), washed with water (300 mL), dried over MgSO₄ and evaporated to give diester 1Oe as a light brown oil (19.O g, 88%).

Dimethyl 3,3'-(/Molylazanediyl)dipropanoate 10b Prepared according to General method A. Diester 10b was obtained as an orange oil (25.5 g, 98%).

Dimethyl 3,3'-(4-fluorophenylazanediyl)dipropanoate 1Od

Prepared according to General Method A. Diester 1Od was obtained as a yellow oil (0.99 g, 75%).

3,3'-(4-Chlorophenylazanediyl)dipropanehydrazide lie General Method B: Preparation of Bis-hydrazides

Diester 1Oe (19 g, 63.4 mmol) was mixed with hydrazine hydrate (12.6 g, 0.25 mol, 4 eq) in propan-2-ol (60 mL) for 48 h at room temperature. The resulting solid was collected by filtration, washed with propan-2-ol and dried in vacuo to give hydrazide lie as white solid (18.8 g, 99%).

3,3'-(Phenylazanediyl)dipropanehydrazide lla Prepared according to General Method B. Hydrazide 11a was obtained as a white solid (6.3 g, 72%).

3,3'-(p-Tolylazanediyl)dipropanehydrazide llb Prepared According to General Method B. Hydrazide lib was obtained as a white solid (24.1 g, 94%).

3,3'-(4-FluorophenylazanediyI)dipropanehydrazide 1 Id Prepared according to General Method B. Hydrazide Hd was obtained as a white solid (0.63 g, 64%): m.p. 131.4 ⁰C.

3,3'-(4-Methoxyphenylazanediyl)dipropanehydrazide 1 If

Prepared according to General Method B. Hydrazide Hf was obtained as a white solid (20.5 g, 95%).

3,3'-(2,2'-(4-ChlorophenyIazanediyl)bis(ethane-2,l-diyl))bis-(4-oxo-3,4- dihydroimidazo[5,l-d] [l,2,3_»5]tetrazine-8-carboxamide) 6e General Method C: Preparation of Bis-imidazotetrazines Hydrazide lie (1.0 g, 3.18 mmol, 1 eq) was dissolved in dichloromethane (10 mL). Water (10 mL) was added followed by HCl (2.5 mL, 37 %). The mixture was cooled in an ice/CaCl₂ bath and a solution OfNaNO₂ (0.57 g, 8.26 mmol, 2.6 eq) in water (10 mL) was added with strong agitation below 5 °C. After the addition, the reaction was allowed to reach room temperature and stirred overnight. The organic layer was separated, dried over MgSO₄ and evaporated to give the azide 12e as a crude oil, identified by IR. The oil was diluted with toluene (100 mL) and heated under reflux for 2 h under N₂. The volatile components were removed to give the crude isocyanate 13e as an oil. Diethylether (150 mL) was added and the mixture heated with strong agitation. The hot solution was decanted leaving a residue of oily imputies behind. The ether was evaporated to leave pure isocyanate as pale yellow oil (v_max 2260s). The isocyanate (0.29g, 1.03 mmol) was mixed with diazo-IC 2 (0.3 g, 2.17 mmol, 2.1 eq) in dry DMSO (0.1 mL) under N₂ at room temperature in the absence of light for 24 h. The bis-imidazotetrazine was purified by flash column chromatography eluting with CHCl₃: AcOH (1 :1) to give imidazotetrazine 6e as a light brown solid (0.1 g, 6%): m.p. 143-144 °C.

3,3'-(2,2'-(Phenylazanediyl)bis(ethane-2,l-diyl))bis-(4-oxo-3,4-dihydroimidazo[5,l- d] [l,2,3,5]tetrazine-8-carboxamide) 6a Prepared according to General Method C. The product was purified by flash column chromatography (5% AcOHZCH₃CN) and imidazotetrazine 6a was obtained as a yellow solid (0.38 g, 60%): m.p. 194-195 °C.

3,3'-(2,2'-(p-TolylazanediyI)bis(ethane-2,l-diyI))bis-(4-oxo-3,4-dihydroimidazo[5,l- d] [l,2,3,5]tetrazine-8-carboxamide) 6b

Prepared by General Method C. The product was purified by flash column chromatography (20-60% AcOH/CHCl₃) and imidazotetrazine 6b was obtained as yellow solid (0.25 g, 13%): m.p. 195-196 °C.

3,3'-(2,2'-(4-Fuorophenylazanediyl)bis(ethane-2,l-diyl))bis-(4-oxo-3,4- dihydroimidazo[5,l-d] [l,2,3,5]tetrazine-8-carboxamide) 6d

Prepared according to General Method C. Imidazotetrazine 6d was obtained as dark yellow solid (0.04 g, 42%): m.p. 290-291 ⁰C. Found: C, 45.40; H, 3.63; N, 33.44. C₂₀H₁₈FN₁₃O₄-0.35 H₂O 0.3 AcOH requires: C, 45.89; H, 3.47; N, 34.79 %.

3,3'-(2,2'-(4-MethoxyphenylazanediyI)bis(ethane-2,l-diyl))bis-(4-oxo-3,4- dihydroimidazo[5,l-d] [l,2,3,5]tetrazine-8-carboxamide) 6f

Prepared according to General Method C. The product was purified by flash column chromatography (10% AcOH/CHCl₃) and 6f was obtained as red solid (0.2 g, 11%): m.p. 178-179 °C. Found: C, 45.66; H, 3.94; N, 33.54. C₂₁H₂₁Ni₃O₅ ^«0.75 H₂O requires: C, 47.10; H, 3.95; N, 34.00 %.

3,3'-(2,2'-(4-Methoxy-2-nitrophenylazanediyl)bis(ethane-2,l-diyI))bis-(4-oxo-3,4- dihydroimidazo[5,l-d][l,2,3_»5]tetrazine-8-carboxamide) 6g

General Method D: Preparation of Nitroanilino Bis-imidazotetrazines Hydrazide Hf (1.0 g, 4.82 mmol) was dissolved in dichloromethane (10 mL). Water (4 rnL) was added followed by HCl (4 mL, 37%). A solution OfNaNO₂ (1.33 g, 19.3 mmol, 4 eq) in water (4 mL) was added with strong agitation at 0 ⁰C, allowing the batch to exotherm to 15 ⁰C. The reaction mixture was stirred for 30 min at room temperature and the organic layer was separated, dried over MgSO₄ and evaporated to give the crude azide 12g as an oil, identified by IR. The oil was diluted with toluene (100 mL) and heated under reflux for 1 h under N₂. The volatile components were removed in vacuo to give the crude isocyanate 13g as an oil. Diethylether (150 mL) was added and the mixture was heated with strong agitation. The hot ether solution was decanted leaving an oily residue of impurities behind. The ether layer was evaporated to leave pure isocyanate as pale yellow oil v_max 2260s). The isocyanate (0.21 g, 0.70 mmol) was mixed with diazo-IC 2 (0.19 g, 1.39 mmol, 2 eq) in dry DMSO (0.3 mL) under N₂. The reaction mixture was stirred at room temperature in the absence of light for 24 h. The product was purified by flash column chromatography (5% AcOHZCH₃CN) and imidazotetrazine 6g was obtained as an orange/yellow solid (0.21 g, 26%): m.p. 139-140 °C. Found: C, 41.60; H, 3.97; N, 31.20. C₂₁H₂₀N₁₄O₇-I^ H₂O 0.4 AcOH requires: C, 43.45; H, 3.47; N, 33.78 %.

3,3'-(2,2'-(4-Methyl-2-nitrophenylazanediyl)bis(ethane-2,l-diyl))bis-(4-oxo-3,4- dihydroimidazo[5,l-d] [l,2,3,5]tetrazine-8-carboxamide) 6c

Prepared according to General method D. The product was purified by flash column chromatography (5-10% ACOH/CH₃CN) and imidazotetrazine 6c was obtained as an orange solid (0.42 g, 41%): m.p. 147-148 °C.

Methyl 3-(4-chlorophenylamino)propanoate 14e

General Method E: Single Michael Addition to Anilines.

4-Chloroaniline (20.0 g, 157 mmol) was mixed with methyl acrylate (54.0 g, 627 mmol, 4 eq), cuprous chloride (1.56 g, 15.68 mmol, 0.1 eq) and AcOH (30 mL) and heated under reflux at 160 °C for 2 h. The solvents were removed by evaporation and the residue partitioned between chloroform (500 mL) and water (500 mL). The organic layer was dried (MgSO₄) in the presence of charcoal and filtered through celite. The solvents were removed leaving an oily residue. Petroleum ether (200 mL) was added and heated to reflux. The solvent was decanted and the product precipitated upon standing. The solid was collected by filtration, washed with petroleum ether and dried to give ester 14e (23.7 g, 71 %).

Methyl 3-((4-chlorophenyl)(methyl)amino)propanoate 14d Prepared according to General Method E. Ester 27 was obtained as a yellow oil (9.1 g,

%).

Methyl 3-((4-fluorophenyl)(methyl)amino)propanoate 14f Prepared according to General Method E. Ester 14f was obtained as an orange oil (2.22 g,

Methyl 3-(fer/-butoxycarbonyl-(4-chlorophenyl)amino)propanoate 14i Ester 14e (2.27 g, 10.6 mmol) was mixed with di-tert-butyl carbonate (9.3 g, 42.6 mmol, 4 eq) in the absence of solvent. The mixture was heated at 100 ⁰C for 18 h. The mixture was partitioned between H₂O and petroleum ether. The organic phase was washed with H₂O (5 x 50 mL), dried and evaporated to give ester 14i as an oil (3.2 g, 97%).

3-(4-Chlorophenylamino)propanehydrazide 15e

General Method F: Preparation of Monohydrazides.

Ester 14e (3.7g, ll.δmmol, 1 eq) was mixed with hydrazine hydrate (5.9g, l lδmmol, 10 eq) in propan-2-ol (1OmL) for 48h at room temperature. The volatile components were removed by evaporation to leave hydrazide 15e as colourless oil (3.5 g, 95%).

3-(Methyl(phenyl)amino)propanehydrazide 15a

Prepared according to General Method F. Hydrazide 15a was obtained as a colourless oil

(12.7 g, 98%).

3-(Methyl(p-tolyl)amino)propanehydrazide 15b

Prepared by General Method F. Hydrazide 15b was obtained as a colourless oil (6.4 g, 93%).

3-((4-Chlorophenyl)(methyl)amino)propanehydrazide 15d Prepared according to General method F. Hydrazide 15d was obtained as white solid (4.0 g, 44%):

3-((4-Fluorophenyl)(methyl)amino)propanehydrazide 15f

Prepared according to General method F. Hydrazide 15f was obtained as a yellow oil (2.16 g, 97%).

3-((4-Methoxyphenyl)(methyI)amino)propanehydrazide l5j Prepared according to General Method F. Hydrazide 15j was obtained as an oil which crystallised upon standing (4.5 g, 95%).

tert-Butyl 4-chlorophenyI(3-hydrazinyl-3-oxopropyl)carbamate 15i Prepared according to General Method F. Hydrazide 15i was obtained as a colourless oil (3.5 g, 95%).

3-(2-(4-Chlorophenylamino)ethyl)-4-oxo-3,4-dihydroiinidazo[5,l-d][l,2,3,5]tetrazine- 8-carboxamide 5e General Method G: Preparation of Mono-imidazotetrazines

Hydrazide 15e (1.0 g, 4.68 mmol) was dissolved in dichloromethane (20 mL). Water (20 mL) was added followed by HCl (2.5 mL, 37%). The mixture was cooled in an ice/CaCl₂ bath and a solution Of NaNO₂ (0.39 g, 56.2 mmol, 1.2 eq) in water (10 mL) was added with strong agitation at below -5 °C. The ice bath was removed and dichloromethane (20 mL) was added. The reaction mixture was stirred for 40 min and the organic layer was separated, dried over MgSO₄ and evaporated to give the crude azide 16e as an oil, identified by IR. The oil was diluted with toluene (100 mL) and heated under reflux for 1 h under N₂. The volatile components were removed to give the crude isocyanate 17e as a oil. Diethylether (15OmL) was added and the mixture heated with strong agitation. The hot ether solution was decanted leaving an oily residue of impurites behind. The ether layer was evaporated to leave pure isocyanate as pale yellow oil (v_max 2260s). The isocyanate (0.1 g, 0.48 mmol) was mixed with diazo-IC 2 (0.07 g, 0.48 mmol, 1 eq) in dry DMSO (0.3 mL) under N₂ at room temperature in the absence of light for 24 h. The reaction was quenched with water (10 mL) and the resultant solid collected by fitrlation and washed with copious amounts of water to leave imidazotetrazine 5e as a brown solid (0.16 g, 29%): m.p.160-161 ⁰C.

3-(2-(Methyl(phenyl)amino)ethyl)-4-oxo-3,4-dihydroimidazo[5,l-d][l,2,3,5]tetrazine- 8-carboxamide 5a Prepared according to Genral Method G. The product was purified by flash column chromatography (2% MeOH/CHCl₃) and imidazotetrazine 5a was obtained as a white solid (0.34 g, 21%): m.p. 186-187 ⁰C. Found: C, 53.08; H, 4.70; N, 30.07. C₁₄H]₅N₇O₂O.! CHCl₃ requires: C, 53.67; H, 4.83; N, 31.29 %. 3-(2-(MethyI-(p-toIyl)amino)ethyl)-4-oxo-3,4-dihydroimidazo[5,l- d][l,2,3,5]tetrazine-8-carboxamide 5b

Prepared according to General Method G. The product was purified by flash column chromatography (ethylacetate) and imidazotetrazine 5b was obtained as a white solid (0.3 g, 20%): m.p. 172-173 °C. Found: C, 55.53; H, 5.37; N, 29.77. Ci₅Hi₇N₇O₂O.05 hexane requires: C, 55.04; H, 5.23; N, 29.95 %.

3-(2-((4-Chlorophenyl)(methyl)amino)ethyl)-4-oxo-3,4-dihydroimidazo[5,l- d][l,2,3,5]tetrazine-8-carboxamide 5d

Prepared by General Method G. The product was precipitated with water, collected by filtration and the crude solid dried and suspended in chloroform (20 mL). The impurities were removed by filtration and the filtrate evaporated to yield imidazotetrazine 5d as a yellow solid (0.084 g, 25%): m.p.154-155 °C. Found: C, 44.65; H, 3.89; N, 24.98. C₁₄H₁₄ClN₇O₂-OJ CHCl₃ -0.15 AcOH requires: C, 48.35; H, 4.06; N, 28.19 %.

/er/-Butyl 2-(8-carbamoyl-4-oxoimidazo[5,l-d][l,2,3,5]tetrazin-3(4H)-yl)ethyl(4- chlorophenyl)carbamate 5i

Prepared according to General Method G. The product was purified with flash column chromatography (10% ACOH/CHCI₃) and imidazotetrazine 5i was obtained as a white solid (0.15 g, 51%): m.p. 177-178 °C. Found: C, 49.48; H, 4.70; N, 22.05. C₁₈H₂₀ClN₇O₄-0.05 CHC1₃ O.O5 Et₂O requires: C, 49.83; H, 4.65; N, 22.60 %.

3-(2-(MethyI(4-methyl-2-nitrophenyI)amino)ethyl)-4-oxo-3,4-dihydroimidazo[5,l- d][l,2,3j5]tetrazine-8-carboxamide 5c

Prepared according to General Method D. On completion of the reaction, the mixture was quenched with propan-2-ol (10 mL) and the solid collected by filtration and washed with propan-2-ol then methanol to leave inidazotetrazine 5c as an orange solid (0.43 g, 24%): m.p.133-134 °C. Found: C, 47.95; H, 4.20; N, 30.14. C₁₅H₁₆N₈O₄-0.15 H₂O requires: C, 48.39; H, 4.33; N, 30.09 %.

3-(2-((4-Fluoro-2-nitrophenyl)(methyl)amino)ethyI)-4-oxo-3,4-dihydroimidazo[5,l- d] [l,2,3_»5]tetrazine-8-carboxamide 5g Prepared according to General Method D. The product was purified with flash column chromatography (ethyl acetate) and imidazotetrazine 5g was obtained as a red solid (0.343 g, 17%): m.p. 156-157 °C.

3-(2-((4-Methoxy-2-nitrophenyl)(methyI)amino)ethyl)-4-oxo-3,4-dihydroimidazo[5,l- d] [l,2,3,5]tetrazine-8-carboxamide 5h

Prepared according to General Method D. The product was purified by flash column chromatography (10% AcOH/CHCb). The solid was washed with 50% MeOH/H₂O and imidazotetrazine 5h was obtained as a purplish red solid (0.21 g, 26%): m.p. 166-168 °C. Found: C, 46.48; H, 4.19; N, 28.47. C₁₅H₁₆N₈O₅-O^ MeOH requires: C, 46.39; H, 4.15; N, 28.85 %.

8-CarbamoyI-3-(2' (p-fluoro(methylanilino))ethylimidazo[5,l-d] [1,2,3,51 tetrazin- 4(3H)-one 5f Prepared according to General Method D. The reaction mixture was suspended in water (30 ml) and filtered. The solid on the filter was washed with copious amounts of water until the washings came through colourless, then with ether. Column chromatography (10% acetic acid in chloroform on silica) gave an orange solid. The imidazotetrazine was then dissolved in chloroform, filtered, the chloroform evaporated and the residue collected as a slurry in ether and filtered.

8-Carbamoyl-3-(2-(4-nitromethylanilino))ethylimidazo[5,l-d][l,2,3,5]tetrazin-4(3H)- one 5k

Hydrazide 15a (0.3g, 1.6 mmol) was dissolved in a mixture of DCM (10 ml) and HCl (10 ml, 14.8%), the solution was stirred in a CaCl₂-ice bath at 0 ⁰C, a solution of NaNO₂ (0.66 g, 9.6 mmol) in H₂O (10 ml) was added gradually and the exthothermic reaction was kept at 0-5 ⁰C. A further amount of DCM (15ml) was added, the DCM layer separated, washed with two portions of H₂O (20ml) dried with on MgSO₄, then filtered. Formation of the azide 16k was confirmed by IR and ¹H NMR. The DCM solution of the crude azide 16k was stirred under nitrogen at rt overnight. The isocyanate formation was confirmed by IR. The DCM was evaporated under reduced pressure at low temperature (an ice bath was used to lower the temperature) and the crude isocyanate 17k collected as a yellow oil (0.27 g, 90 %). The crude mixture of isocyanate 17k (0.27 g, 1.22 mmol) was diluted with DMSO (1.5 ml), under nitrogen then added to a suspension of diazo-IC (0.17 g, 1.22 mmol) in DMSO (1.5 ml), the mixture was stirred at r.t protected from light for 48 h. The reaction mixture was then suspended in water (30 ml) and filtered. The solid on the filter was washed with copious amounts of water until the washings came through colourless, then with ether. Column chromatography on silica with gradient elution with 5-20 % acetic acid in chloroform was used for the purification. The imidazotetrazine 5k was then dissolved in DMF and the solution filtered, water was added the imidazotetrazine precipitated as a yellow solid which was collected by filtration, washed with copious amount of water and dried, m.p. 179-180 ⁰C.

JV-Nitroso-iV-(3-azido-3-oxopropyl)carbainic acid-/-butyl ester 30 The hydrazide 29 (1.8 g, 8.86 mmol) was suspended in a mixture of water (10 ml) and DCM (10 ml) and left to stir in an ice bath at 0 °C, HCl (5 ml, 10 M) was added gradually followed by NaNO₂ (3.67 g, 53.13 mmol) solution in water (10 ml) added dropwise, keeping the exothermic reaction at 0-5 °C. Excess DCM (10 ml) was added and then the organic layer was separated, washed with water, dried over anhydrous MgSO₄, filtered and evaporated to give a yellow oil (1.22 g, 68%). Presence of the azide 30 was confirmed by IR band 2150s cm^"1.

7V-Nitroso-./V-(2-isocyanatoethyl)carbamic acid /-butyl ester 31 The azide 30 (1.22g, 5.7 mmol) was dissolved in anhydrous DCM (15 ml) and stirred under nitrogen at 30 ⁰C for 48 h. Isocyanate formation was confirmed by IR, which showed 2275 s cm^"1. The DCM was evaporated under reduced pressure at low temperature and the isocyanate 31 collected as a yellow oil (0.8 g, 60 %).

8-CarbamoyI-3-((iV-r-butoxycarbonyl-N-nitroso)-2-aminoethyl) imidazo [5,1- d] [1,2,3,5] tetrazin-4(3H)-one 32

The isocyanate 31 (0.80 g, 4.37 mmol) was added to a suspension of diazo-IC 2 (0.6 g, 4.37 mmol) in DMSO (1.8 ml), the mixture was stirred at 30 ⁰C protected from light. The reaction was complete after 36 h as monitored by ¹H NMR. Water (10 ml) was added and the mixture filtered. The residue was washed with copious amounts of water until the washings came through colourless. The solid was washed with /-propanol and left to dry on the filter. The solid was suspended in chloroform (20 ml), filtered, and the chloroform filtrate evaporated to dryness to give the imidazotetrazine 32 as a pale yellow solid (0.11 g, 14 % yield), mp 154-157 °C. Found: C, 36.54; H, 3.78; N, 26.96%. Ci₂H₁₆N₈O₅-0.5 CHCl₃, requires C, 36.44; H, 4.04, N, 27.20%.

3-(2-aminoethyl) imidazo [5,1-d] [1,2,3,5] tetrazin-4(3H)-one Hydrobromide salt 8 HBr (48%, 0.65 g, 8.03mmol) was added over 30 min to a stirred solution of BOC- protected imidazotetrazine 32 (0.1 g, 0.31 mmol) in acetonitrile (6 ml). The mixture was stirred at r.t. overnight and a precipitate formed. The solid was collected by filtration and dried under vacuum. The solid was re-suspended in chloroform, filtered and then evaporated to give imidazotetrazine hydrobromide salt 8 (0.088 g, 88 %) m.p. 145-148 °C. Found: C, 23.24; H, 2.94; N, 25.95%. C₇H₉N₇O₂-V₃HBr. 0.5H₂O-V₃CH₃CN, requires C, 23.22; H, 3.10; N, 25.86%.

3-Isopropyl-4-oxo-3,4-dihydroimidazo[5,l-d][l,2,3,5]tetrazine-8-carboxamide 9

Isopropyl isocyanate (0.3 g, 3.45 mmol) (Aldrich) was reacted with diazo-IC 2 (0.43 g, 3.14 mmol) in dry DMSO (1 mL) for 24 h at room temperature. The reaction mixture was quenched with water and the product purified by flash column chromatography (3% methanol/ether). The imidazotetrazine 9 was obtained as a white crystalline solid (0.35g, 55%): m.p. 129-130 °C.

3-Fluorobenzamide 18 3-Fluorobenzoic acid (54.4 g, 0.39 mol) was suspended in DME (150 mL), thionyl chloride (98.4g, 0.83 mol, 2.1 eq) and DMF (1 mL). The reaction mixture was stirred at 50 °C for 18 h. The reaction was quenched with ammonia solution (35% aq., 500 g) and the volatile components were removed by evaporation. The crude solid was suspended in ethylacetate and heated under reflux for 2 h with vigorous agitation. The mixture was filtered hot to remove the inorganics and the volume was reduced to approximately 150 mL. Hexane (150 mL) was added and the resulting precipitate was collected by filtration and washed with ethylacetate :hexane (1:4) (46.4 g, 86%): m.p. 133-134 °C.

3-Fluoro-4-nitrobenzamide 19 Amide 18 (20 g, 144 mmol) was dissolved in cone, nitric acid (40 mL) and added dropwise to a mixture of cone, sulphuric acid (70 mL) and cone, nitric acid (10 mL) while keeping the temperature <10 ⁰C. The mixture was partitioned between water (1 L) and ethylacetate (IL) and the organic phase separated, dried (MgSO₄) and evaporated to an oil. This was suspended in ethylacetate (100 mL) and heated. Hexane (100 mL) was added and the mixture was cooled to -20°C overnight. The resulting crystalline solid was collected by filtration and washed with hexane to give nitroamide 19 as white crystals (11.5 g, 44%): m.p. 129-130 °C

5-Chloro-2,4-dinitrobenzoic acid 25

3-Chlorobenzoic acid (50 g, 0.32 mol) was suspended in a pre-cooled mixture of fuming nitric acid (150 mL) and cone, sulphuric acid (190 mL). The mixture was heated at 70 °C overnight. The reaction mixture was diluted with water (600 mL) and the precipitated solid was collected by filtration and dried. The crude solid was suspended in dichloromethane heated to reflux and filtered hot to give benzoic acid 25 as a white solid (26.7 g, 34%): m.p.176-177 °C

5-Chloro-2,4-dinitrobenzamide 26

Acid 25 (26.7 g, 0.11 mol) was mixed with thionylchloride (140 g, 1.1 mol, 10 eq) and heated at 100 °C for 3 h. Volatile components were removed by evaporation and the crude residue was dissolved in tetrahydrofuran and cooled to -20 ⁰C. Ammonia solution (35% aq, 7Og) in tetrahydrofuran (300 mL) was added slowly keeping the temperature <0 °C. The reaction mixture was partitioned between brine and ethylacetate. The organic layer was washed with brine, dried (MgSO₄) and evaporated to dryness. The resulting solid was triturated with chloroform to give amide 26 as a solid (19.1 g, 72%).

Methyl 3-((5-carbamoyl-2-nitrophenyI)(methyl)amino)propanoate 20a

3-(Methylamino)propanoate 27 was prepared by slow addition of methylamine (3.8 g, 122 mmol, 1 eq) to methylacrylate (11.6 g, 135 mmol, 1.1 eq) while keeping the temperature <-20 ⁰C. The mixture was allowed to stand in the freezer overnight. The mixture was evaporated to leave an oil containing methyl 3-(methylamino)propanoate in a 1 :1 mixture with dimethyl-3,3'-(methylazanediyl)dipropanoate that was used directly. Fluorobenzamide 19 (0.25g, 1.36mmol, 1 eq) was mixed with 3- (methylamino)propanoate (50%, 1.2 g, 5.4 mmol, 4 eq) and triethylamine (1.1 g, 10.8 mmol, 8 eq) in CH₃CN (25 mL) and heated at 100 °C overnight. The mixture was evaporated to dryness and the residue recrystallised from methanol :diethylether:petroleum ether (1 :2:3) to obtain aniline 20a as a yellow solid (0.32 g, 84%): m.p. 125-126 ⁰C.

Methyl 3-(5-carbamoyl-2-nkrophenylamino)propanoate 20b β- Alanine methyl ester (0.2 g, 1.09 mmol) was mixed with triethylamine (0.17 g, 1.62 mmol, 1.5 eq) in CH₃CN (5 mL) and added to a solution of fluoroamide 19 in CH₃CN (10 mL). The mixture was heated under reflux for 2 h. The volatile components were removed and the crude residue re-suspended in ethyl acetate (20 mL), filtered and evaporated to dryness. The crude product was recrystallised from CHaCN/ether to give ester 20b as orange crystals: m.p. 154-155.

Methyl 3-(5-carbamoyl-2,4-dinitrophenylamino)propanoate 20c

A mixture of amide 26 (0.2 g, 0.81 mmol), β-alaninemethylester hydrochloride (0.15 g, 1.06 mmol, 1.3 eq) and triethylamine (0.15 g, 1.22 mmol, 1.5 eq) in CH₃CN was heated under reflux overnight. The volatile components were removed by evaporation and the residue suspended in ethylacetate. The mixture was filtered and the filtrate reduced to dryness. The crude solid was recrystallised from CH₃CN/diethyl ether. The solid was collected by filtration and washed with water to give ester 20c as a yellow solid (0.23 g, 90%). m.p.194-195 °C.

3-((3-Hy draziny 1-3-oxopropy l)(methy l)amino)-4-nitrobenzamide 21a Ester 20a (2.0 g, 7.11 mmol) was mixed with hydrazine hydrate (1.2 g, 24.0 mmol, 3.4 eq) in CH₃CN (30 mL) was heated at 60 °C overnight. The solids were collected by filtration and washed with CH₃CN to give hydrazide 21a as a white solid (1.1 g, 55%).

3-(3-Hydrazinyl-3-oxopropylamino)-4-nitrobenzaiiiide 21b

Ester 20b (2.7 g, 10.1 mmol) was mixed with hydrazine hydrate (1.52 g, 30.3 mmol, 3 eq) in propan-2-ol (20 mL) and stirred at room temperature overnight. The resultant solid was collected by filtration and washed with propan-2-ol to give hydrazide 21b as a yellow solid (2.1 Ig, 78%). 5-(3-Hydrazinyl-3-oxopropylamino)-2,4-dinitrobenzamide 21c

To a solution of ester 20c (2.0 g, 6.41 mmol) in tetrahydrofuran (100 mL) was added hydrazine hydrate (10.0 g, 200 mmol, 20 eq) in CH₃CN (100 mL). The mixture was heated at 115 ⁰C overnight. The mixture was evaporated to an oily residue which was suspended in methanol, a solid formed that was collected by filtration and washed with methanol to give hydrazide 21c as a yellow/green solid (0.67 g, 34%): m.p. 279-280⁰C.

3-(2-((5-CarbamoyI-2-nitrophenyl)(methyl)amino)ethyl)-4-oxo-3,4- dihydroimidazo[5,l-d][l,2,3»5]tetrazine-8-carboxamide 7a Prepared by General Method G. The product was purified by flash column chromatography (CH₃CN) and imidazotetrazine 7a was obtained as a yellow solid (0.12 g, 9%): m.p. 189-190 ⁰C; HRMS: (M+NH₄)⁺ requires 419.1534, found 419.1540.

3-(2-(5-Carbamoyl-2-nitrophenylamino)ethyl)-4-oxo-3,4-dihydroimidazo[5,l- d] [l,2,3,5]tetrazine-8-carboxamide 7b

Prepared by General Method G. The product was purified by flash column chromatography (CH₃CN) and imidazotetrazine 7b was obtained as a light yellow solid (0.22 g, 41%): m.p.187-188 ⁰C.

3-(2-(5-Carbamoyl-2,4-dinitrophenylamino)ethyl)-4-oxo-3,4-dihydroimidazo[5,l- d][l,2,3,5]tetrazine-8-carboxamide 7c

Prepared by General Method G. The product was purified with flash column chromatography (CH₃CN) and imidazotetrazine 7c was obtained as a yellow solid (15 mg, 4%): m.p. 220-221 ⁰C. HRMS: (M+NH₄)⁺ requires 450.1229, found 450.1226.

Experimental - Chemosensitivity assays

The cell lines employed included A2780 (human ovarian carcinoma), the MMR deficient derivative Cp70 and RTl 12 (a NQO2-rich human bladder carcinoma cell line). Cells were plated into 96 well culture plates at 1 x 10³ cells per well and incubated over night at 37⁰C in a CO₂ enriched (5%) atmosphere to enable cells to adhere to the plate. Culture media was removed and replaced with fresh media containing test compound concentrations ranging from 0 (controls) to a 250 μM. Following 5 days incubation at 37⁰C, cell survival was determined using the MTT assay. All compounds were dissolved in DMSO (with the exception of salt 8 which is water soluble) and the final concentration of DMSO in the culture plates was <0.1% (v/v). Patrin-2 was used as an inhibitor of MGMT and cells were incubated with test compounds in the presence or absence of 10 μM Patrin-2 as described above. For all NQO2 studies, test compounds were incubated in the presence or absence of an NRH analogue (reduced l-(carbamoylmethyl)pyridinium-3- carboxamide,100 μM) and cell survival following continuous 5 day exposure was determined as described above.

Evaluation the Chemosensitising Effectiveness New compounds were screened for chemosensitivity in a test system based on that reported by Margisson et al. (MoI. Can. Ther. 2004, 3, 123-127). The A2780 ovarian carcinoma cell line and its MMR-deficient variant A2780-cp70 were used. MGMT status of the cells was modified chemically using the MGMT inactivator PaTrin2, at a dose previously-shown to elicit complete inactivation of that repair protein. IC₅₀ data for compounds from Class 1 and 2 are presented in Figure 1, Tables 2 and 3.

All new agents were more active than temozolomide and mitozolomide. Dependence on MMR can be examined by comparing the red bars with the pale blue bars (i.e. MMR+/MMR- with MGMT inactivated in both cases). For temozolomide the IC₅₀ was >27-fold lower in the MMR-proficient cell line. For the new bi-functional agents 6, this ratio was reduced to 5-5.8-fold and for the mono-functional agents 5, 2.8-10-fold. The extent of MGMT-mediated resistance can be assessed by comparing the lilac and the red bars (i.e. MGMT-/MGMT+ with MMR competent in both cases). Here, temozolomide was >30-fold more potent when MGMT was inactivated whilst for the new agents, this ratio was 0.5-5-fold for the bi-functional and 1.1-2.9-fold for the mono-functional agents. Importantly, in the absence of MMR, all compounds showed activity greater than temozolomide irrespective of the MGMT status of the cells (yellow bars and pale blue bars) showing that MMR-dependent toxicity and MGMT-mediated resistance are now only a minor determinants of the anti-tumour effect.

The putative CB 1954 precursors were evaluated in a test system using RTl 12 cells in the presence and absence of a synthetic analogue of the non-biogenic cofactor NRH which activates latent NQO2 in tumour cells (Knox et al, Cancer Res., 2000, 60, 4179—4186). In this system, CB 1954 showed a 290-fold potentiation whilst the tetrazinone analogue 7c was 22-fold more potent against the cells with activated NQO2.

SAR The data in Figure 1 show that all compounds prepared are more active than temozolomide and the bi-functional agents 6 are generally more potent than the mono- functional agents 5. Within the mono-functional series, there is a general insensitivity to the effects of MMR and MGMT such that the activity of compounds 5a and 5b is approaching independence of both repair systems. Presence of a methyl group on the aniline nitrogen has little effect (compare 5d and 5e). There are sufficient examples for the bi-functional agents to allow exploration of the relationship between anti-tumour activity and the electronic effect of the ring para substituent "X" using the Hammett sigma-pαrα constant, see Figure 2. Here a strong connection is seen between sigma and IC₅₀ - with an IC₅₀ optimum for the compound 6b (X=CH₃), irrespective of the MMR and MGMT status of the model. Furthermore, the effects of MMR and MGMT are also at their minima for this compound.

Table 2 In vitro Chemosensitivity Data for Bifunctional Imidazotetrazines

Table 3 In vitro Chemosensitivity Data for Monofunctional Imidazotetrazines

7b

82 16 167 16

7c

126 25 6.85 0.35

Table 4 In vitro Chemosensitivity and NQO2 activation of Imidazotetrazines that are potential prodrugs of CB 1954

Compound IC₅₀ (μM)

HCTlIo⁺ HCT116-'

5a 200 200

5b 40.2 29.2

5d 55 35.8

5e 13.2 9.5

5k 161 120

6a 5.5 4.8

6b 6.2 4.4

6d 28 17

6f 7.6 9.9

Cisplatin 11.8 1.9

Table 5 In vitro Chemosensitivity if compounds against P53 wild type (+/+) and P53 mutant (-/-) HCTl 16 colon carcinoma cell lines.

Claims

1. A compound of formula I

A is selected from N, O or S;

wherein each R¹² is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, =NR¹³, -OR¹³, -C(O)R¹³, -C(O)OR¹³, -OC(O)R¹³, -S(O),R¹³, - N(R¹³)R¹⁴, -C(O)N(R¹³)R¹⁴, -SO₂N(R¹³)R¹⁴ and R¹⁵;

wherein R¹³ and R¹⁴ are each independently selected from hydrogen or R¹⁵;

wherein R¹⁵ is selected from hydrocarbyl and -(CH₂)_q-heterocyclyl, and each R¹⁵ is optionally and independently substituted with one or more of halogen, cyano, amino, hydroxy, C_1-6 alkyl and Ci_-6 alkoxy; n is 0, 1, 2, 3, 4 or 5; q is O, 1, 2, 3, 4, 5 or 6;

or a pharmaceutically acceptable salt or prodrug thereof.

2. A compound as claimed in claim 1 wherein X is cyclohydrocarbyl optionally substituted with R¹².

3. A compound as claimed in claim 2 wherein X is aryl or heteroaryl optionally substituted with R¹².

4. A compound as claimed in claim 3 wherein X is aryl optionally substituted with R¹².

5. A compound as claimed in claim 1 wherein X is the group

wherein D, E, J, K and L are independently selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, oxo, =NR¹⁶, -OR¹⁶, -C(O)R¹⁶, -C(O)OR¹⁶, -OC(O)R¹⁶, - S(O)iR¹⁶, -N(R¹⁶)R¹⁷, -C(O)N(R¹⁶)R¹⁷, -SO₂N(R¹⁶)R¹⁷ and R¹⁸

wherein R¹⁶ and R¹⁷ are each independently selected from hydrogen or R¹⁸;

wherein R¹⁸ is selected from hydrocarbyl and -(CH₂)_r-heterocyclyl, and each R¹⁸ is optionally and independently substituted with one or more of halogen, cyano, amino, hydroxy, Ci_-6 alkyl and Ci_-6 alkoxy;

r is O, 1, 2, 3, 4, 5 or 6.

6. A compound as claimed in claim 5 wherein D or E is nitro.

7. A compound as claimed in claim 5 wherein J or K is an amine or an amide.

8. A compound as claimed in claim 5 wherein L is an electron withdrawing group.

9. A compound as claimed in claim 8 wherein L is selected from groups comprising fluorine, cyano and nitro.

10. A compound as claimed in claim 9 wherein L is nitro.

11. A compound as claimed in claim 5 wherein X is the group

12. A compound as claimed in claim 1 wherein X is the group

wherein D or E and/or L are independently selected from electron rich groups including amino, sulfide, hydroxyl, alkoxy or hydrocarbyl; D or E and/or L may be selected -OR¹⁶, -C(O)R¹⁶, -C(O)OR¹⁶, -OC(O)R¹⁶, -S(O))R¹⁶, - N(R¹⁶)R¹⁷, -C(O)N(R¹⁶)R¹⁷, -SO₂N(R¹⁶)R¹⁷ and R¹⁸.

13. A compound as claimed in claim 12 wherein L is an electron rich group.

14. A compound as claimed in claim 13 wherein L is selected from amino, sulfide, hydroxyl, alkoxy or hydrocarbyl.

15. A compound as claimed in any preceding claim wherein R and R¹ taken together with the atoms to which they are attached form a heterocycle, optionally substituted with one or more R¹².

16. A compound as claimed in claim 15 the heterocycle is a five membered heterocycle containing from 1 to 2 N atoms wherein each substitutable carbon or N in the heterocycle is optionally and independently substituted by one or more R¹².

17. A compound as claimed in claim 16 wherein the compound is of formula (II)

(II)

wherein R⁴ is selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, oxo, =NR¹⁹, -OR¹⁹, -C(O)R¹⁹, -C(O)OR¹⁹, -OC(O)R¹⁹, -S(O)₁R¹⁹, -N(R¹⁹)R²⁰, -C(O)N(R¹⁹)R²⁰, - SO₂N(R¹⁹)R²⁰ and R²¹

wherein R¹⁹ and R²⁰ are each independently selected from hydrogen or R²¹; wherein R²¹ is selected from hydrocarbyl and -(CFkVheterocyclyl, and each R²¹ is optionally and independently substituted with one or more of halogen, cyano, amino, hydroxy, Ci_-6 alkyl and C_1-6 alkoxy;

t is O, 1, 2, 3, 4, 5 or 6.

18. A compound as claimed in claim 17 wherein R⁴ is amide.

19. A compound as claimed in any preceding claim wherein A is selected from O or S.

20. A compound as claimed in claim 19 wherein A is O.

21. A compound as claimed in any preceding claim wherein n is 1 , 2 or 3.

22. A compound as claimed in any preceding claim wherein Y is hydrogen, halogen, halide or an aliphatic hydrocarbyl group.

23. A compound as claimed in claim 22 wherein Y is cylohydrocarbyl.

24. A compound as claimed in claim 23 wherein Y is heteroaryl.

25. A compound as claimed in claim 24 wherein the compound is of formula III

wherein B is selected from N, O or S; R² and R³ are each independently selected from hydrogen or R²²; or R² and R³ taken together with the atoms to which they are attached form a carbocycle or a heterocycle, optionally substituted with one or more R²²;

wherein each R²² is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, =NR²³, -OR²³, -C(O)R²³, -C(O)OR²³, -OC(O)R²³, -S(O),R²³, - N(R²³)R²⁴, -C(O)N(R²³)R²⁴, -SO₂N(R²³)R²⁴ and R²⁵;

wherein R²⁵ is selected from hydrocarbyl and -(CH₂)_u-heterocyclyl, and each R²⁵ is optionally and independently substituted with one or more of halogen, cyano, amino, hydroxy, Cj_-6 alkyl and Ci_-6 alkoxy;

m is O, 1, 2, 3, 4 or 5

or a salt thereof.

26. A compound as claimed in claim 25 wherein A and B are independently selected from O or S.

27. A compound as claimed in claim 26 wherein A and B are O.

28. A compound as claimed in claim 25 wherein R² and R³ taken together with the atoms to which they are attached form a heterocycle, optionally substituted with one or more R¹².

29. A compound as claimed in claim 28 wherein the heterocycle is a five membered heterocycle containing from 1 to 5 heteroatoms independently selected from N, S or O wherein each substitutable carbon or heteroatom in the heterocycle is optionally and independently substituted by one or more R¹².

30. A compound as claimed in claim 29 wherein the compound is of formula (FV)

(IV)

wherein R⁵ is selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, oxo, =NR²⁶, -OR²⁶, -C(O)R²⁶, -C(O)OR²⁶, -OC(O)R²⁶, -S(O)iR²⁶, -N(R²⁶)R²⁷, -C(O)N(R²⁶)R²⁷, - SO₂N(R²⁶)R²⁷ and R²⁸

wherein R²⁸ is selected from hydrocarbyl and -(CH₂)_u-heterocyclyl, and each R²⁸ is optionally and independently substituted with one or more of halogen, cyano, amino, hydroxy, C_1-6 alkyl and Ci_-6 alkoxy;

u is O, 1, 2, 3, 4, 5 or 6;

31. A compound as claimed in claim 30 wherein R⁵ is amide.

32. A compound as claimed in any preceding claim wherein the compound is an anilinoethyl imidazotetrazine.

33. A compound as claimed in any one of claims 1 to 31 wherein the compound is an anilino linked bis imidazotetrazine.

34. A compound as claimed in claim 33 wherein the compound is a diethylanilino linked bis imidazotetrazine.

35. A process for preparing a compound as claiming in any preceding claim the process comprising the steps of

(i) contacting a compound of formula V with an ether

X' (V)

wherein R' is hydrogen or C 1-6 alkyl optionally substituted with NCO; and wherein X' has the same meaning as X as defined in any one of the preceding claims;

(ii) contacting the product of (i) with a diazoimidazole.

36. A process as claimed in claim 35 wherein R' is hydrogen or methyl.

37. A process as claimed in claim 35 wherein the compound of formula V is

OCN. NCO

N'

X¹

38. A compound, or pharmaceutically acceptable salt or prodrug thereof, as claimed in any one of claims 1 to 33 for use as a medicament.

39. A pharmaceutical formulation comprising a compound, or pharmaceutically acceptable salt or prodrug, as claimed in any one of claims 1 to 33 and at least one an excipient, diluent or carrier.

40. A compound, or pharmaceutically acceptable salt or prodrug, as claimed in any one of claims 1 to 33, or a pharmaceutical formulation as claimed in claim 39, for use in the treatment of cancer.

41. Use of a compound, or a pharmaceutically acceptable salt or prodrug thereof, as claimed in any one of claims 1 to 33 in the manufacture of a medicament for the treatment of cancer.

42. A method to treat cancer in a subject comprising administering an effective amount of compound, or a pharmaceutically acceptable salt or prodrug thereof, as claimed in any one of claims 1 to 33.