WO2020240199A1 - Therapeutic compounds - Google Patents

Abstract

The invention provides a product which is a compound of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof: (I) wherein: X is selected from: hydrogen; a methyl group which is optionally substituted, wherein any substituent groups present are independently selected from hydroxyl, halo and NR'₂, where each R' is independently selected from hydrogen and methyl; and an ethyl group which is either unsubstituted or is substituted with from one to five F substituent groups; Y is selected from: hydrogen; a methyl group which is optionally substituted, wherein any substituent groups present are independently selected from hydroxyl, halo and NR'₂, where each R' is independently selected from hydrogen and methyl; and an ethyl group which is either unsubstituted or is substituted with from one to five F substituent groups; R_A is hydrogen, or is a C1-2 alkyl group which is optionally substituted, wherein any substituent groups present are independently selected from hydroxyl, halo and NR'₂, where each R' is independently selected from hydrogen and methyl.

Description

THERAPEUTIC COMPOUNDS

TECHNICAU FIEUD

The present invention relates to therapeutic compounds, pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, both in vitro and in vivo, to inhibit cell proliferation, and in particular the use of such compounds and compositions in the treatment of proliferative disorders such as cancer.

BACKGROUND

Temozolomide (TMZ) is an oral chemotherapy drug, also known as 3,4-dihydro-3- methyl-4-oxoimidazo[5, l-d]-l,2,3,5-tetrazine-8-carboxamide; M & B 39831 ; 8- carbamoyl-3-methylimidazo[5, l-d]-l,2,3,5-tetrazin-4(3H)-one; methazolastone; CCRG- 81045; NSC-362856; Temodal; or Temodar.

TMZ acts as a methylating agent and is primarily used as a treatment of brain cancers (e.g. glioma). It is used as a second-line treatment for astrocytoma and a first-line treatment for glioblastoma multiforme (GBM).

TMZ is a prodrug, being cleaved in a multi-step pathway firstly to liberate an unstable monomethyltriazene (MTIC), which then suffers proteolytic fragmentation to generate a highly-reactive methylating agent (methanediazonium ion) and 5-aminoimidazole-4- carboxamide (see, e.g., Arrowsmith et al., 2002, J. Med. Chem.. Vol. 45, pp. 5458-5470). Support for this process comes from the isolation of MTIC from the degradation of temozolomide in aqueous sodium carbonate solution (see, e.g., Stevens et al., 1984, J. Med. Chem., Vol. 27, pp. 196-201). There is only a small pH window, close to physiological pH, where ring-opening of temozolomide is accompanied by fragmentation of MTIC in a methylating mode. The methanediazonium active species derived from MTIC (or temozolomide) is believed to covalently methylate guanine residues of DNA in tracts of three or more guanines (see, e.g., Hartley et al., 1988, Carcinogenesis. Vol. 9, pp. 669-674; Clark et al., 1995, J. Med. Chem.. Vol. 38, pp. 1493-1504). The significant site of DNA methylation is the O- 6 position of guanine residues. Tumours which express high levels of the DNA repair protein 0(6)-methylguanine methyltransferase (MGMT; also known as ATase) are inherently resistant to the drug (see, e.g., Wedge et al., 1996, Br. J. Cancer. Vol. 74, pp. 1030-1036; Lee et al., 1994, Br. J. Cancer. Vol. 69, pp. 452-456.) 0-6 guanine methylation is a cytotoxic (antitumour) lesion since it provokes base mis-pairing with thymine during DNA replication. Unless repaired by MGMT, mis-pairing on the daughter strand is recognised by mismatch repair proteins which trigger futile cycles of thymine excision and re-insertion leading to persistent DNA strand breaks.

The promoter methylation status (at cytosine C-5 in CpG sequences of the MGMT gene) is a powerful predictor of clinical outcome in glioblastoma patients (see, e.g., Hegi et al., 2004, Clin. Cancer Res. Vol. 10, pp. 1871-1874; Hegi et al., 2005, New England J. Med., Vol. 352, pp. 997-1003).

Temozolomide is the subject of granted claim 13 of US Patent No 5,260,291 to Uunt et al. granted 09 November 1993.

As set out in Svec et al, ACS Chem Biol, 2018, Vol. 13, pp. 3206-3216, even in the era of personalized medicine and immunotherapy, TMZ remains the standard-of-care for glioblastoma (GBM). This paper describes the construction of novel C8-substituted imidazotetrazines and concludes from assays of these compounds that the C8 amide is not required for anticancer activity, and indeed compounds lacking an H-bond donor or acceptor (or both) at C8 can still retain activity comparable to that of TMZ against cancer cells in culture.

WO 2009/077741 describes certain 3-substituted-4-oxo-3,4-dihydro-imidazo[5, 1- d] [l,2,3,5]tetrazine-8-carboxylic acid amide (3TM) compounds of general formula: In WO 2010/149968, it is explained in that tumours with the MGMT gene switched off, as in some brain tumours, are unable to repair the 0-6 guanine lesions and are particularly sensitive to temozolomide . Conversely, most common tumours with the MGMT repair gene switched on, leading to high cellular levels of MGMT, can repair the 0-6 guanine lesions and are resistant to the drug. This epigenetic feature considerably restricts the spectrum of action of temozolomide and its penetration of the cancer market.

WO 2010/149968 generically describes 3 -substituted-8-substituted-3H-imidazo[5, 1 - d] [ l ,2,3,5]tetrazin-4-one compounds (38TM compounds) within a broad general formula:

Specific compounds disclosed in WO 2010/149968 for which data are provided include:

MM-004 WW-027

WW-028

WO 2010/149968 therefore concludes that, unlike TMZ, these compounds have good activity against tumour cell lines regardless of the MGMT and MMR (Mis-Match Repair) status of the cell line.

However, there remains a need for further candidates for successful treatment of proliferative disorders such as cancer. SUMMARY OF THE INVENTION

The present invention provides new therapeutic compounds which have, surprisingly, been found to be more effective than TMZ against a range of human cancer cell lines_. The compounds of the invention have a broad anti-tumour spectrum of activity, including against colorectal, breast, melanoma, nasopharyngeal and GBM cell lines.

Therefore compounds of the invention have useful therapeutic activity against major killer tumour types which are currently inherently resistant to temozolomide. The invention also provides compounds with a good selectivity index, i.e. showing significantly greater activity against cancer cell lines compared to non-transformed fibroblasts.

The invention also provides compounds with properties that make them suitable for use as a therapeutic agent, including stability at low pH, excellent blood brain barrier (BBB) penetration, and excellent oral bioavailability.

Furthermore, these compounds of the invention are also, surprisingly, more effective than specific compounds described in WO 2010/149968. In particular, the compounds of the invention have been shown to be more effective against colorectal cancer cell lines (e.g. HCT1 16).

In a first aspect, the invention provides a product which is a compound of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof:

wherein

X is selected from: hydrogen; a methyl group which is optionally substituted, wherein any substituent groups present are independently selected from hydroxyl, halo and NR'₂, where each R' is independently selected from hydrogen and methyl; and an ethyl group which is either unsubstituted or is substituted with from one to five F substituent groups;

Y is selected from: hydrogen; a methyl group which is optionally substituted, wherein any substituent groups present are independently selected from hydroxyl, halo and NR'₂, where each R' is independently selected from hydrogen and methyl; and an ethyl group which is either unsubstituted or is substituted with from one to five F substituent groups;

R_A is hydrogen, or is a C 1-2 alkyl group which is optionally substituted, wherein any substituent groups present are independently selected from hydroxyl, halo and NR'₂, where each R' is independently selected from hydrogen and methyl.

These compounds of formula (I) differ from the specific compounds described in WO 2010/149968 and furthermore, as set out in the examples, surprisingly have improved properties as compared to WO 2010/149968 compounds. For example, significantly greater inhibitory effectiveness was shown against the human GBM U373V line and against the isogenic line transfected with MGMT, U373M.

In a second aspect, the invention provides a pharmaceutical formulation comprising a product according to the first aspect in combination with a pharmaceutically acceptable carrier, excipient or diluent.

The products and pharmaceutical formulations of the invention can be used, both in vitro and in vivo, to inhibit cell proliferation. In particular, the products and pharmaceutical formulations of the invention can be used in the treatment of proliferative disorders such as cancer.

The compounds of the invention show in vitro anti-tumour activity and selectivity in relation to: MGMT+/- GBM, MMR- CRC, breast, nasopharyngeal, lung, and melanoma human-derived carcinoma cell lines. The compounds of the invention also show activity and selectivity in relation to MRC-5 fibroblasts.

In a third aspect, therefore, the invention provides the product according to the first aspect or the pharmaceutical formulation according to the second aspect for use in a method of treating a proliferative condition. In the method an effective amount of the product according to the first aspect or the pharmaceutical formulation according to the second aspect may be provided to a patient having said proliferative condition via any suitable mode of administration, e.g. oral or parenteral.

In one embodiment, the proliferative condition is cancer. In one embodiment, the proliferative condition is colorectal, brain, breast, lung, melanoma, or nasopharygeal cancer. In one such embodiment, the proliferative condition may be colorectal cancer or brain cancer.

The following beneficial therapeutic properties have been identified for the products of the invention:

• Enhanced potency compared to TMZ

o Activity in TMZ-sensitive U373V cells has been achieved at a level 14.5- fold greater than TMZ

• Overcomes major mechanisms of inherent and acquired TMZ-resistance:

o Activity in U373M MGMT+ cell line has been achieved at a level 73.8- fold greater than TMZ

o Activity in MMR- HCT 1 16 cell line has been achieved at a level 54.6- fold greater than TMZ

• Broad anti-tumour spectrum of activity, including colorectal, breast, melanoma, nasopharyngeal and GBM cell lines

• Excellent selectivity index (activity against all cancer cell lines tested compared to non-transformed fibroblasts)

o A selectivity index of greater than 5-fold has been achieved.

Furthermore, the products of the invention have been found to have favourable properties in terms of being suitable for use as a therapeutic agent:

• Good acid stability

o Products are stable at pH 3, which allows parenteral administration

• Plasma-protein-binding of < 1%

• Good microsomal stability

o Products have no P450-liability

• Products are not P-glycoprotein substrates

• Excellent blood brain barrier (BBB) penetration

• Excellent oral bioavailability

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides therapeutic products which are compounds of formula (I), or pharmaceutically acceptable salts, hydrates or solvates thereof. These are 3-substituted-8-substituted-3H-imidazo[5, l-d] [ l,2,3,5]tetrazin-4-one compounds, where the substituent at the 3 -position is a propargyl group and where the substituent at the 8-position is a thiazole group.

The thiazole ring can optionally be substituted at its 4- and/or 5- positions. Specifically, the thiazole ring can optionally be mono-substituted at its 4- and/or 5- positions. In one embodiment, the thiazole ring is optionally mono-substituted at either its 4-position or at its 5- position, but not both. However, significantly, according to the present invention these 4- and 5- positions on the thiazole ring are not sterically bulky. Small groups have been found to give the best results.

The combination of a non-sterically bulky thiazole at the 8 -position and a propargyl group at the 3 -position gives rise to products that have a good selectivity index and have a broad antitumour spectrum of activity. Significantly, these compounds have properties that make them suitable for use as a therapeutic agent, including stability at low pH, excellent blood brain barrier (BBB) penetration, and excellent oral bioavailability.

These compounds are, in particular, surprisingly effective against colorectal cancer cell lines.

In one embodiment, the compound of formula (I) has a molecular weight, MW, of 400Da or less, or 380Da or less, such as 350Da or less, or 325Da or less; preferably 300Da or less, e.g. 290Da or less. It may be that the compound of formula (I) has a molecular weight, MW, of from 250 to 380Da or from 250 to 325Da, e.g. from 250 to 300Da or from 250 to 290Da.

In formula (I), X is selected from: hydrogen; a methyl group which is optionally substituted, wherein any substituent groups present are independently selected from hydroxyl, halo and NR'₂, where each R' is independently selected from hydrogen and methyl; and an ethyl group which is either unsubstituted or is substituted with from one to five F substituent groups. When X is a methyl group, preferred substituent groups may be hydroxyl and NR'_2, e.g. hydroxyl and NH₂, and in particular mono-substitution with such groups may be contemplated. Mono-substitution with Cl or Br may also usefully be contemplated. In another useful embodiment, the methyl group has mono-, di- or tri- substitution with F; in one embodiment X is trifluoromethyl. When X is an ethyl group, in one embodiment X is pentafluoroethyl.

In one embodiment, X is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl, Cl, Br, and NR'₂, where each R' is independently selected from hydrogen and methyl; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

In one embodiment, X is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl and NH₂; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

In one embodiment, X is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl and NH₂; and a methyl group which is substituted with one, two or three F substituent groups.

In one embodiment, X is selected from: hydrogen; unsubstituted methyl; hydroxymethyl, aminomethyl, trifluoromethyl, unsubstituted ethyl; and pentafluoroethyl.

In formula (I), Y is selected from: hydrogen; a methyl group which is optionally substituted, wherein any substituent groups present are independently selected from hydroxyl, halo and NR'₂, where each R' is independently selected from hydrogen and methyl; and an ethyl group which is either unsubstituted or is substituted with from one to five F substituent groups. When Y is a methyl group, preferred substituent groups may be hydroxyl and NR'₂ e.g. hydroxyl and NH₂, and in particular mono-substitution with such groups may be contemplated. Mono-substitution with Cl or Br may also usefully be contemplated. In another useful embodiment, the methyl group has mono-, di- or tri substitution with F; in one embodiment Y is trifluoromethyl. When Y is an ethyl group, in one embodiment Y is pentafluoroethyl. In one embodiment, Y is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl, Cl, Br, and NR'₂, where each R' is independently selected from hydrogen and methyl; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

In one embodiment, Y is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl and NH₂; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

In one embodiment, Y is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl and NH₂; and a methyl group which is substituted with one, two or three F substituent groups.

In one embodiment, Y is selected from: hydrogen; unsubstituted methyl; hydroxymethyl, aminomethyl, trifluoromethyl, unsubstituted ethyl; and pentafluoroethyl.

In one embodiment, X and Y in combination have no more than three carbon atoms, such as no more than two carbon atoms. It may be that X and Y in combination have no more than three carbon atoms and no more than three substituent groups. In one embodiment X and Y in combination have no more than three carbon atoms and no more than two substituent groups. It may be that X and Y in combination have no more than two carbon atoms and no more than two substituent groups.

In one embodiment, one or both of X and Y is hydrogen. In one embodiment, X and Y in combination have no more than one carbon atom.

In formula (I), R_A is hydrogen, or is a C 1-2 alkyl group which is optionally substituted, wherein any substituent groups present are independently selected from hydroxyl, halo and NR'₂, where each R' is independently selected from hydrogen and methyl. When R_A is a methyl group, preferred substituent groups may be hydroxyl and NR'₂ e.g. hydroxyl and NH₂, and in particular mono-substitution with such groups may be contemplated. Mono-substitution with Cl or Br may also usefully be contemplated. In another useful embodiment, the methyl group has mono-, di- or tri-substitution with F; in one embodiment R_A is trifluoromethyl. When R_A is an ethyl group, in one embodiment R_A is pentafluoroethyl .

In one embodiment, R_A is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl, Cl, Br, and NR'₂, where each R' is independently selected from hydrogen and methyl; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; an ethyl group which is substituted with one substituent group selected from hydroxyl, Cl, Br, and NR'₂, where each R' is independently selected from hydrogen and methyl; and an ethyl group which is substituted with from one to five F substituent groups.

In one embodiment, R_A is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl, Cl, Br, and NR'₂, where each R' is independently selected from hydrogen and methyl; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

In one embodiment, R_A is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl and NH₂; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

In one embodiment, R_A is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl and NH₂; and a methyl group which is substituted with one, two or three F substituent groups.

In one embodiment, R_A is selected from: hydrogen; unsubstituted methyl; hydroxymethyl, aminomethyl, trifluoromethyl, unsubstituted ethyl; and pentafluoroethyl. In one embodiment, each of X, Y, and R_A are independently selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl, Cl, Br, and NR'₂, where each R' is independently selected from hydrogen and methyl; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

In one embodiment, each of X, Y, and R_A are independently selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl and NH₂; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

In one embodiment, R_A is hydrogen and each of X and Y are independently selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl and NH₂; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

In one embodiment, R_A is hydrogen and each of X and Y are independently selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl and NH₂; and a methyl group which is substituted with one, two or three F substituent groups.

Specific examples of compounds of formula (I), which may optionally be provided in the form of pharmaceutically acceptable salts, hydrates or solvates thereof, are :

(I-A) (I-B) (I-C) (I-D)

Further specific examples of compounds of formula (I), which may optionally be provided in the form of pharmaceutically acceptable salts, hydrates or solvates thereof, are compounds (I-E), (I-F) and (I-G), as shown in the examples. Salts

It may be convenient or desirable to prepare, purify, and/or handle a corresponding pharmaceutically acceptable salt of the compound. Examples of pharmaceutically acceptable salts are discussed in Berge et al, 1977, "Pharmaceutically Acceptable Salts," J. Pharm. ScL Vol. 66, pp. 1 - 19. In one embodiment, the compound of formula (I) is provided in the form of a salt of an organic or mineral acid.

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

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

Alternatively, if the compound is anionic, or has a functional group which may be anionic, then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R²⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

Unless otherwise specified, a reference to a particular compound also includes salt forms thereof.

Hydrates and Solvates

It may be convenient or desirable to prepare, purify, and/or handle a corresponding pharmaceutically acceptable hydrate or solvate of the compound.

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

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

Chemically Protected Forms

It may be convenient or desirable to prepare, purify, and/or handle the compound in a chemically protected form. The term "chemically protected form" is used herein in the conventional chemical sense and pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions under specified conditions (e.g., pH, temperature, radiation, solvent, and the like). In practice, well known chemical methods are employed to reversibly render unreactive a functional group, which otherwise would be reactive, under specified conditions. In a chemically protected form, one or more reactive functional groups 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; 4th Edition; John Wiley and Sons, 2006).

A wide variety of such "protecting," "blocking," or "masking" methods are widely used and well known in organic synthesis. For example, a compound which has two non equivalent reactive functional groups, both of which would be reactive under specified conditions, may be derivatized to render one of the functional groups "protected," and therefore unreactive, under the specified conditions; so protected, the compound may be used as a reactant which has effectively only one reactive functional group. After the desired reaction (involving the other functional group) is complete, the protected group may be "deprotected" to return it to its original functionality.

A hydroxy group may be protected as an ether (-OR) or an ester (-OC(=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 (-OC(=0)CH₃, -OAC) .

An amine group may be protected, for example, as an amide (-NRCO-R) or a urethane (- NRCO-OR), for example, as a methyl amide (-NHCO-CH₃); a benzyloxy amide (-NHCO- OCH₂C₆H₅, -NH-Cbz); as a t-butoxy amide (-NHCO-OC(CH₃)₃, -NH-Boc); a 2-biphenyl- 2-propoxy amide (-NHCO-OC(CH₃)₂C₆H₄C₆H₅, -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), or as a 2(-phenylsulfonyl)ethyloxy amide (-NH- Psec).

Deuterated Forms The compound may be provided in a deuterated form, in which one or more hydrogen atoms are replaced by deuterium.

In one embodiment, the compound is one where R_A is hydrogen and the deuterated form replaces this hydrogen with deuterium. Thus there may be a deuterated propargyl group at the 3-position.

Unless otherwise specified, a reference to a particular compound also includes deuterated forms thereof.

Synthesis

Med. Chem. Commun., 2016, 7, 2332 describes synthetic routes to 3-substituted imidazo tetrazines structurally related to temozolomide.

ACS Chem. Biol. 2018, 13, 1 1, 3206-3216 describes synthetic routes to 8-substituted imidazo tetrazines structurally related to temozolomide.

The skilled person can synthesise the products of the invention based on these and other known synthetic routes.

Detailed syntheses for a number of 3-substituted-8-substituted-3H-imidazo[5, l- d] [ l,2,3,5]tetrazin-4-one compounds are provided in the Examples below. These can be readily modified to manufacture other 3-substituted-8-substituted-3H-imidazo[5, l- d] [ l,2,3,5]tetrazin-4-one compounds within the scope of the claims.

Formulations

The invention provides a pharmaceutical formulation comprising a product according to the first aspect in combination with a pharmaceutically acceptable carrier, excipient or diluent.

One or more other pharmaceutically acceptable ingredients may optionally be included. These include, but are not limited to, pharmaceutically acceptable adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents. The formulation may optionally further comprise other active agents, for example, other therapeutic or prophylactic agents.

If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound.

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

Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients. 5th edition, 2005.

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

Formulations may suitably be in the form of liquids, solutions (e.g., aqueous, non- aqueous), suspensions (e.g. , aqueous, non-aqueous), emulsions (e.g., oil-in-water, water- in-oil), elixirs, syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated tablets), granules, powders, lozenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols.

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

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

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

Use in Methods of Inhibiting Cell Proliferation

The products according to the first aspect are beneficial in that they: (a) regulate (e.g., inhibit) cell proliferation; (b) inhibit cell cycle progression; (c) promote cell death; or (d) a combination of one or more of these.

Therefore the products of the invention may be used in a method of regulating (e.g., inhibiting) cell proliferation (e.g., proliferation of a cell), inhibiting cell cycle progression, promoting cell death, or a combination of one or more these, in vitro or in vivo, by contacting a cell with an effective amount of a product according to the first aspect.

In one embodiment, the method is a method of regulating (e.g., inhibiting) cell proliferation (e.g., proliferation of a cell), in vitro or in vivo, comprising contacting a cell with an effective amount of a product according to the first aspect.

In one embodiment, the method is performed in vitro. In one embodiment, the method is performed in vivo.

In one embodiment, the product is provided in the form of a pharmaceutical formulation according to the second aspect.

Any type of cell may be treated, including but not limited to, cancer cells derived from lung, gastrointestinal (including, e.g., bowel, colon), breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, or skin tumours, or nasopharyngeal tumours.

The skilled reader will be readily able to determine whether or not a candidate compound regulates (e.g., inhibits) cell proliferation, etc. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described herein. For example, a sample of cells (e.g., from a tumour) may be grown in vitro and a compound brought into contact with said cells, and the effect of the compound on those cells observed. As an example of an "effect," the morphological status of the cells (e.g., alive or dead, etc.) may be determined. Where the compound is found to exert an influence on the cells, this may be used as a prognostic or diagnostic marker of the efficacy of the compound in methods of treating a patient carrying cells of the same cellular type.

Methods of Treatment

The invention provides the product according to the first aspect, or the pharmaceutical formulation according to the second aspect, for use in a method of treating a proliferative condition. In the method an effective amount of the product according to the first aspect or the pharmaceutical formulation according to the second aspect may be provided to a patient having said proliferative condition via any suitable mode of administration, e.g. oral or parenteral.

The term "proliferative condition," as used herein, pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth.

In one embodiment, the treatment is treatment of: a proliferative condition characterised by benign, pre-malignant, or malignant cellular proliferation, including but not limited to, neoplasms, hyperplasias, and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (see below), psoriasis, bone diseases, fibroproliferative disorders (e.g., of connective tissues), pulmonary fibrosis, atherosclerosis, smooth muscle cell proliferation in the blood vessels, such as stenosis or restenosis following angioplasty.

In one embodiment, the treatment is treatment of cancer.

In one embodiment, the treatment is treatment of lung cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, stomach cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, thyroid cancer, breast cancer, ovarian cancer, endometrial cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, renal cell carcinoma, bladder cancer, pancreatic cancer, brain cancer, glioma, sarcoma, osteosarcoma, bone cancer, nasopharyngeal cancer, squamous carcinoma of the head or neck, skin cancer, squamous cancer, Kaposi's sarcoma, melanoma, malignant melanoma, lymphoma, or leukaemia.

In one embodiment, the treatment is treatment of a carcinoma, for example a carcinoma of the bladder, breast, colon (e.g., colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermal, liver, lung (e.g., adenocarcinoma, small cell lung cancer and non-small cell lung carcinomas), oesophagus, gall bladder, ovary, pancreas (e.g., exocrine pancreatic carcinoma), stomach, cervix, thyroid, prostate, skin (e.g., squamous cell carcinoma); a hematopoietic tumour of lymphoid lineage, for example leukaemia, acute lymphocytic leukaemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non- Hodgkin's lymphoma, hairy cell lymphoma, or Burkett's lymphoma; a hematopoietic tumour of myeloid lineage, for example acute and chronic myelogenous leukaemias, myelodysplastic syndrome, or promyelocytic leukaemia; a tumour of mesenchymal origin, for example fibrosarcoma or habdomyosarcoma; a tumour of the central or peripheral nervous system, for example astrocytoma, neuroblastoma, glioma or schwannoma; melanoma; seminoma; teratocarcinoma; osteosarcoma; xenoderoma pigmentoum; keratoctanthoma; thyroid follicular cancer; or Kaposi's sarcoma.

In one embodiment, the treatment is treatment of solid tumour cancer. In one embodiment, the treatment is treatment of haematological cancer.

In one embodiment, the treatment is treatment of lung cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, renal cancer, prostate cancer, oesophageal cancer, squamous carcinoma of the head or neck, or glioma.

In one embodiment, the treatment is treatment of colorectal cancer or glioma.

In one embodiment, the cancer is characterised by cancer stem cells.

In one embodiment, the cancer is MGMT- cancer. In one embodiment, the cancer is MGMT+ cancer. In one embodiment, the cancer is MMR proficient cancer. In one embodiment, the cancer is MMR deficient cancer. In one embodiment, the cancer is temozolomide resistant or temozolomide refractory.

In one embodiment, the cancer is inherently temozolomide resistant or inherently temozolomide refractory.

In one embodiment, the cancer is temozolomide resistant or temozolomide refractory following exposure to (e.g., treatment with) temozolomide. The anti-cancer effect may arise through one or more mechanisms, including but not limited to, the regulation of cell proliferation, the inhibition of cell cycle progression, the inhibition of angiogenesis (the formation of new blood vessels), the inhibition of metastasis (the spread of a tumour from its origin), the inhibition of invasion (the spread of tumour cells into neighbouring normal structures), or the promotion of cell death (e.g. programmed cell death). The compounds of the present invention may be used in the treatment of the cancers described herein, independent of the mechanisms discussed herein.

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 halt in the rate of progress, alleviation of symptoms of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, use with patients who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term "treatment."

For example, treatment includes the prophylaxis of cancer, reducing the incidence of cancer, alleviating the symptoms of cancer, etc.

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

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

Other Uses

The products according to the first aspect may also be used as cell culture additives to inhibit cell proliferation.

The products according to the first aspect may also be used as part of an in vitro assay, for example, in order to determine whether a candidate host is likely to benefit from treatment with the product in question.

Modes of Administration

The products according to the first aspect and the pharmaceutical formulations according to the second aspect may be administered to a patient by any convenient route of administration, whether systemically/peripherally or topically (i.e ., at the site of desired action).

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

The Patient

The subject/patient may be a mammal. The subject/patient may, for example, be canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), simian (e.g., a monkey or an ape), or may be a human. In one preferred embodiment, the subject/patient is a human. In one embodiment, the human is one year old or more, or five years old or more, such as ten years old or more, or twelve years old or more, or sixteen years old or more, or eighteen years old or more. Thus in one embodiment, the subject/patient is an adult human.

Dosage

It will be appreciated that appropriate dosages of products according to the first aspect and the pharmaceutical formulations according to the second aspect can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, 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, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.

In general, a suitable dose of the compound is in the range of about 10pg to about 250 mg (more typically about lOOpg to about 25 mg) per kilogram of body weight of the subject per day. Where the compound is a salt, an ester, an amide, a prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

Examples

General methodology for assays:

A. MTT assays

Cells were seeded into 96-well plates at a density of 6.5 x 10² /well and allowed to attach overnight. Following this overnight incubation, the test compounds in nutrient medium (from lOOmM stock solutions) were added to the wells (n = 4-8; final well concentrations 0.5-1,000mM).

MTT assays were performed at the time of agent addition and then following 6 days of incubation (37°C, 5% C0₂). Thus, cell growth following exposure to the test compounds could be determined.

Sterile filtered MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to the wells (50 mΐ; 0.4 mg/ml in phosphate buffered saline). The plates were re incubated for 4 hours allowing metabolism of MTT by viable cells to insoluble formazan crystals. Medium and unconverted MTT were aspirated and DMSO ( 150 mΐ) was added to each well. Ensuring formazan solubilization, absorbance was read on an Anthos Labtec Systems plate reader (550 nm) and data transferred to a computer using Deltasoft 3™ software.

Agent concentrations causing 50% growth inhibition (GI₅₀) were calculated by interpolation.

B. Clonogenic assays

Clonogenic assays, measuring tumour cell survival and subsequent proliferative ability following agent exposure, were performed. Exponentially growing cells were seeded in triplicate at a density of 200 cells/well in 6- well plates and allowed to attach overnight.

Following this overnight incubation, the test compounds were added to the wells (2, 10, 20, and 50mM), while control wells received vehicle alone. After 24 hours of exposure, well contents were aspirated, cells washed and agent-free media introduced. Plates were incubated for 14 days at 37 °C in an atmosphere of 5% C0₂. Cells were washed (3x in phosphate buffered saline), fixed with pre-chilled methanol (20 min), stained with 0.5% methylene blue in 1 : 1 methanol/H ₂ O (v/v) for 10 min, washed thoroughly in dH₂0 and air-dried. Cell colonies containing more than 50 cells were counted as survival colonies. Toxicity was estimated by dividing the mean number of colonies following treatment with agent by the mean number of colonies in the absence of agent (survival fraction).

C. pH stability assays

The pH stability assay involves the incubation of test compounds at 1 mM in phosphate buffer solution (PBS) at 37°C at different pH values for 120 minutes. At 5 set time points (0, 10, 20, 30, 60 and 120 minutes) small aliquots are withdrawn and crashed in acetonitrile containing the internal standard (Leucine Enkephalin). The test compound is quantified in samples using LC-MS/MS and its half-life (ti_/2) in acidic, neutral and basic conditions is derived from the percentage of parent compound remaining over time.

1. Each incubation reaction is composed as follows:

• 495 pL PBS at the pH value to be assessed: (a) pH 3, (b) pH 5, (c) pH 7.4

• 5 pL 100 mM test compound

2. 495 pL PBS pipetted into a deep well incubation plate in duplicate

3. Pre-incubate the plate for 10 minutes at 37°C.

4. Add 5 pL 100 mM test compound to each well and mix 5x.

5. Incubate for 0, 10, 20, 30, 60 and 120 minutes at 37°C.

6. At each time-point, mix 5x and then remove 65 pL of incubate into a fresh plate (Sample Plate V-bottom) containing 65pL of 0.5 mM Leucine Enkephalin internal standard in acetonitrile.

7. Seal the plate.

8. Heat seal the analysis plate.

9. Store in fridge at 4°C until ready for analysis by uHPLC-MS/MS. D. Parallel artificial membrane permeability assay (PAMPA)

A parallel artificial membrane permeability assay (PAMPA) measures passive diffusion. The PAMPA assay measures the levels of compound in the donor and acceptor compartments. The drug is placed in the donor compartment and the concentrations are measured in both the acceptor and donor compartments after the incubation. If the drug has diffused across the hydrophobic membrane, it will be detected in the acceptor compartment.

A pre-made PAMPA pre-coated plate system from Corning is utilised (the Corning Gentest Pre-coated PAMPA plate system). The membrane present is constructed of structured layers of phospholipids to mimic the hydrophobic membrane. This assay has been fully automated and the end point consists of LC-MS/MS readouts. A calibration curve is generated for each compound to which the donor and acceptor concentrations are measured. Caffeine is used as a positive control and Famotidine as a negative control.

Compound requirements: lOmM DMSO, 20pL

Internal Standard: 1 mM Leucine Enkephalin in acetonitrile

Buffer: 10% Methanol in PBS

Final DMSO %: 2 %

Replicates: 3

Incubation Time: 500 rpm for 300 min at 25°C

Analysis Method: UPLC-MS/MS

The Biomek NX^P liquid handler workstation, is used, with the ‘PAMPA final 28 compounds’ method from the Physical Sciences Project. Permeability (Pe), in units of cm/sec, is determined according to the equation:

Pe = dM/dt* l/A*Cd(0)

where dM/dt represents the steady-state appearance rate of the test drug in the acceptor compartment (in units of mol/s), A is the exposed membrane area (in units of cm²) and CD(0) is the initial drug concentration in the donor compartment (in units of mol/cm³).

Recovery is calculated as follows: % Recovery = (Total compound mass in donor and receiver compartments at the end of the incubation / Initial compound mass in the donor compartment) x 100

E. MDCK-WT/MDR1 Permeability Assay

Wild type MDCK (Madin-Darby canine kidney) cells when transfected with either the Multi Drug Resistance gene-1 (MDR1 ; P-gp) or Breast Cancer Resistance gene (BCRP) are used as a model of brain penetration and to understand the impact of drug transporters on drug uptake into the brain. When cultured as a monolayer, MDCKII cells differentiate to form tight junctions between cells that mimic epithelial barriers (e.g. BBB).

The PrediPort™-WT/MDRl/BCRP Kit from ReadyCell S.L. (Barcelona, Spain) is utilised; this is a ready-to-use cell-based assay for rapid in vitro assessment of drug’s permeability and MDR1 substrate assessment. Differentiated and polarized MDCK cells, wild type and MDR1 or BCRP transfected, are plated on a 96-transwell permeable system as a single monolayer to allow for automated high throughput screening of compounds. Drug transport is assessed in both directions (apical to basolateral (A-B) and basolateral to apical (B-A)) across the cell monolayer. The buffer used for the assay does not include HEPES, so as to minimise the inhibitory effect on uptake transporters.

Test compound concentrations are quantified using a calibration curve following analysis by LC-MS/MS, and the apparent permeability coefficient (Papp) and efflux ratio of the compound across the monolayer are calculated in addition to the net efflux ratio (MDCKII_MDRI/ BCRP / MDCKII_WT) . The efflux ratio is used as an indicator of active efflux.

Compound requirements: lOmM DMSO, 100pL

Test Article Concentrations: 10 mM

Buffer: HBSS buffer at pH7.4 or 6.5-7.4

Replicates: Triplicate

Incubation Time: 2 hours at 37°C in a C0₂ incubator

Analysis Method: LC-MS/MS

Controls: Quinidine; propranolol (high permeability

marker) Cell monolayer integrity marker: Lucifer yellow

The permeability coefficient (Papp) is calculated from the following equation:

where dQ/dt is the amount of compound in basal (A-B) or apical (B-A) compartment as a function of time (nmol/s) . CO is the initial concentration in the donor (apical or basal) compartment (Mean of T=0) (nmol/mL) and A is the area of the transwell (cm²).

The efflux ratio is calculated as:

The net efflux ratio is calculated as:

Efflux ratio_MD

Efflux ratio _WT

Example 1 : Synthesis 1.1 : Reference Example - Formation of “Comparative 1”

(3-propargyl variant of TMZ)

Synthesis of 3-(Propar₂yl)imidazof5,l-dJ-l,2,3,5-tetrazin-4-oxo-8-carboxamide

Comparative 1

Triphosgene (8.89 g, 30 mmol) was added to a biphasic mixture of propargylamine (5.8 mL, 91 mmol) in dichloromethane ( 150 mL) and saturated aqueous sodium hydrogen carbonate ( 150 mL) in an ice bath. The reaction mixture was stirred vigorously for 25 min and extracted with dichloromethane (2 x 150 mL). The organic layer was dried over MgS0₄ and concentrated at atmospheric pressure at 40 °C to approximately 10 mL to give a solution that is ca. 12.6% w/w of the isocyanate; d_H (400MHz; CDC1₃) 4.00 (2 H, d, J 2.5, CH₂), 2.42 ( 1 H, t, J 2.5, CH); 5_C ( 100MHz; CDC1₃) 125.8 (C), 78.2 (C), 72.9 (CH), 32.5 (CH₂). The dichloromethane solution was used in the next step without further purification.

Propargyl isocyanate in dichloromethane ( 10.4 g; 12.6% w/w) was added dropwise to a suspension of 5-diazoimidazole-4-carboxamide ( 1.44 g, 8 mmol) in DMSO ( 14 mL) at room temperature in the dark under argon. The reaction mixture was left to stir for 22 h, poured onto ice and allowed to warm to room temperature . The resulting precipitate was collected by vacuum filtration and dried under vacuum over P₂0₅ resulting in title compound ( 1 .53, 67%) as a pale pink solid. Further purification could be achieved by flash column chromatography (0-2% MeOH in dichloromethane) to give the title compound as a cream coloured solid; mp 163 °C; (Found: [M+Na]⁺, 241.0446. C₈H₆N₆0₂ ⁺+Na⁺ requires 241.0444); _max (EtOH)/nm 324 (log e 2.90); v_max (ATR^cm^-1 3296, 3264, 3 124, 2989, 2793, 2129, 1736, 1690, 1634, 1580, 1449, 1412, 1362, 1341 , 1303, 1282, 1256, 1210, 1 177, 1 1 18, 1071 , 1037; d_H (400 MHz; DMSO-r/₆) 8.86 ( 1 H, s, CH, imidazole H-6), 7.82 ( 1 H, br s, NH), 7.70 ( 1 H, br s, NH), 5.13 (2 H, d, J 2.5, CH₂), 3.52 ( 1 H, t, J 2.5, CH); d_€ ( 100 MHz; DMSO-r/₆) 161.4 (C), 138.5 (C), 134.2 (C), 13 1.3 (C), 129.2 (CH), 77.3 (C), 76.5 (CH), 38.5 (CH₂); m/z (El) 241 [M+Na]⁺, 100%).

1.2: Reference Example - Formation of an Intermediate

Synthesis of 3-(Propar₂yl)imidazof5,l-dJ-l,2,3,5-tetrazin-4-oxo-8-thiocarboxamide

Phosphorus pentasulfide ( 1.27 g, 2.86 mmol), 3-(propargyl)imidazo[5,

2,3,5- tetrazin-4-oxo-8-carboxamide (780 mg, 3.58 mmol) and hexamethyldisiloxane ( 1 .7 mL, 7.87 mmol) were stirred in dichloromethane (20 mL) at reflux for 16 h. The reaction mixture was poured onto a flash column and purified ( 10% acetonitrile in dichloromethane) to give the title compound as a bright orange solid (896 mg, 84%); mp 133 °C; (Found: [M+Na]⁺, 257.0223. C₈H₆N₆OS⁺Na⁺ requires 257.0216); _max (EtOH)/nm 208 (log e 4.05), 265 (log e 3.92), 330 (log e 3.89); v_max (ATR)/cm^_13370, 3252, 3172, 3114, 2993, 2925, 2794, 2361, 2342, 2130, 1744, 1605, 1543, 1479, 1453, 1416, 1389, 1351, 1301, 1271, 1247, 1204, 1160, 1075, 1038, 1001; d_H (400 MHz; DMSO- ₆) 9.98 (1 H, br s, NH), 9.48 (1 H, br s, NH), 8.85 (1 H, s, CH, imidazole H-6), 5.13 (2 H, d, J2.5, CH₂), 3.53 (1 H, t, 2.5, CH); 5_C (100 MHz; DMSO- ₆) 187.8 (C), 138.5 (C), 134.7 (C), 134.1 (C), 128.6 (CH), 77.3 (C), 76.5 (CH), 38.5 (CH₂); m/z (ESI+) 257 [M+Na]⁺, 100%), 491 (30), 766 (11).

1.3: Example of the Invention

(I-E)

To a solution of 3-(propargyl)imidazo[5,l-£/]-l,2,3,5-tetrazin-4-oxo-8-thiocarboxamide (397 mg, 1.70 mmol) in dry acetonitrile (18 mL) was added l-bromobutan-2-one (256 mg, 1.70 mmol) and the reaction mixture was stirred for 64 h at room temperature under argon. The reaction mixture was concentrated under vacuum and purified by flash column chromatography (0-10% acetonitrile in dichloromethane) to give the title compound as a green-yellow solid (74 mg, 15%); mp 74-76 ^°C; (Found: M+Na⁺, 309.0529. Ci₂H₁₀N₆OS+Na⁺ requires 309.0529); _max (EtOH)/nm 270 (log e 2.84), 366 (log e 2.74); v_max (ATR)/cm^_13285, 3124, 2970, 2934, 2128, 1740, 1508, 1448, 1372, 1348, 1307, 1266, 1230, 1076, 1020, 1003; d_H (400 MHz; CDC1₃) 8.48 (1 H, s, CH, H-6), 7.11 (1H, t, J 1.0, CH, H-14), 5.14 (2 H, d, J2.5, CH₂), 2.98 (2 H, dq, J7.5, 1.0, CH₂), 2.44 (1 H, t, J2.5, CH), 1.37 (3 H, t, J7.5, CH₃); d_€ (100 MHz; CDC1₃) 161.8 (C), 157.3 (C), 138.5 (C), 134.8 (C), 130.7 (C), 129.7 (CH), 115.1 (CH), 76.0 (CH), 74.5 (C), 39.0 (CH₂), 25.1 (CH₂), 13.5 (CH₃); m/z (ESI) 309 (M+Na⁺, 100%), 287 (M+H⁺, 43%).

(ESI+) 257 [M+Na]⁺, 100%), 491 (30), 766 (11). 1.4: Example of the Invention

Synthesis of 8-(4-Methylthiazol-2-yl)-3-(yroyarsyl)imidazot5,l-dl-l,2,3,5-tetrazin-4- one

To a solution of 3-(propargyl)imidazo[5, l -£/]- l ,2,3,5-tetrazin-4-oxo-8-thiocarboxamide (65 mg, 0.28 mmol) in dry acetonitrile (2.8 mL) was added 1 -chloroacetone (0.22 mL, 1.39 mmol) and the reaction mixture was stirred for 48 h at room temperature under argon. The reaction mixture was evaporated to dryness and purified by flash column chromatography (0- 1 % propan-2-ol in dichloromethane) to give the title compound as a green solid (28 mg, 37%); mp 168 ^°C; (Found: M+Na⁺, 295.0374. CnH₈N₆OS+Na⁺ requires 295.0373); _max (EtOH)/nm 269 (log e 4.70), 366 (log e 4.90); v_max (ATR)/cm^_1 3281 , 3 104, 2970, 2929, 2361 , 2342, 2138, 1980, 1736, 1655, 1560, 15 1 1 , 1446, 1372, 1350, 1305, 1263, 1233 , 1 173, 1076, 1019; d_H (400 MHz; CDC1₃) 8.47 ( 1 H, s, CH, H-6), 7.08 ( 1 H, s, CH, H- 14), 5.14 (2 H, d, J 2.5, CH₂), 2.59 (3 H, s, CH₃), 2.43 ( 1 H, t, J 2.5, CH); 5c ( 100 MHz; CDC1₃) 157.5 (C), 155.6 (C), 138.5 (C), 134.7 (C), 130.6 (C), 129.7 (CH), 1 16.4 (CH), 76.0 (C), 74.5 (CH), 39.0 (CH₂), 17.5 (CH₃); m/z (ESI) 273 (M+H⁺, 100%), 295 (M⁺+Na⁺, 14%).

(ESI+) 257 [M+Na]⁺, 100%), 491 (30), 766 ( 1 1) .

1.5: Example of the Invention

Synthesis of 8-(Thiazol-2-yl)-3-(propargyl)imidaz.o[5,l-dI-l,2,3,5-tetrazin-4-one

(I-A) To a solution of 3-(propargyl)imidazo[5,l-£/]-l,2,3,5-tetrazin-4-oxo-8-thiocarboxamide (300 mg, 1.28 mmol) in dry acetone (2.9 mL) was added 2-bromo-l, 1-dimethoxyethane (0.3 mL, 2.56 mmol) and HC1 in 1,4-dioxane (4 M, 1.6 mL) and the reaction mixture was stirred for 5 h at 50 ^°C under argon. The reaction mixture was evaporated to dryness and purified by flash column chromatography (0-0.5% methanol in dichloromethane) to give the title compound as an orange solid (114 mg, 32%); mp 153 °C; (Found: M+H⁺, 259.0397. C_IOH₆N₆OS+H⁺ requires 259.0397); _max (EtOH)/nm 268 (log e 2.58), 360 (log e 2.80); v_max (ATR)/cm^_13225, 3134, 3123, 3092, 2972, 2956, 2922, 2852, 2115, 1727, 1503, 1452, 1427, 1396, 1373, 1344, 1302, 1274, 1233, 1143, 1118, 1069, 1056, 1014; d_H (400 MHz; CD₃CN) 8.49 (1 H, s, CH, H-6), 8.00 (1 H, d, J3.2, CH, H-13), 7.66 (1 H, d, J 3.2, CH, H-14), 5.09 (2 H, d, J 2.5, CH₂), 2.73 (1 H, t, J 2.5, CH); 5_C (100 MHz; CD₃CN) 160.0 (C), 145.7 (CH), 139.8 (C), 134.1 (C), 132.2 (C), 130.8 (CH), 122.4 (CH), 77.5 (C), 75.1 (CH), 39.4 (CH₂); m/z (ESI) 539 (2M+Na⁺, 100%), 281 (M+H⁺, 65%), 259 (M +H⁺, 51%).

1.6: Reference Example - Formation of an Intermediate

Synthesis _ o _ 8-(4-Hvdroxy-4-(trifluoromethyl)-5,5-dihydrothiazol-2-yl)-3-

(proparsyl)imidazof5,l-dJ-l,2,3,5-tetrazin-4-one

To a solution of 3-(propargyl)imidazo[5,l-r/]-l,2,3,5-tetrazin-4-oxo-8-thiocarboxamide (450 mg, 1.92 mmol) in dry acetonitrile (12 mL) was added 3-bromo-l, 1, 1- trifluoroacetone (404 mg, 2.11 mmol) and the reaction mixture was stirred for 4 h at 50 ^°C under argon. The reaction mixture was evaporated to dryness and purified by flash column chromatography (0-4% methanol in dichloromethane) to give the title compound as a beige solid (328 mg, 50%); mp 162-163 °C; (Found: M+H⁺, 345.0381. C_IIH₇F₃N₆0₂S+H⁺ requires 345.0376); v_max (ATR)/cm^_13264, 3133, 2969, 2926, 2133, 1723, 1578, 1524, 1495, 1459, 1440, 1369, 1350, 1296, 1279, 1228, 1162, 1122, 1074, 1018; d_H (400 MHz; CD₃CN) 8.54 (1 H, s, CH, H-6), 5.23 (1 H, s, OH), 5.14 (2 H, d, J 2.5, CH₂), 3.80 ( 1 H, d, J 13.0, CHH), 3.50 ( 1 H, d, J 13.0, CHH), 2.77 ( 1 H, t, J 2.5, CH); 5c ( 100 MHz; CD₃CN) 168.3 (C), 139.4 (C), 134.9 (C), 13 1.5 (C), 130.7 (CH), 129. 1 - 120.6 (CF₃, q, J 277.5), 107.5- 106.6 (C, q, J 30. 1 ), 77. 1 (C), 75.4(CH), 39.9 (CH₂), 38.4 (CH₂); 5_F (376 MHz; CD₃CN) -82.71 (s, CF₃); m/z (ESI) 345 (M+H⁺, 100%).

1.7: Example of the Invention

To a solution of 8-(4-hydroxy-4-(trifluoromethyl)-5,5-dihydrothiazol-2-yl)-3 - (propargyl)imidazo[5, l -<2] - l ,2,3,5-tetrazinone ( 108 mg, 0.3 1 mmol) and dry triethylamine (0.065 mL, 0.47 mmol) in dry acetonitrile ( 1.5 mL) in an ice bath was added trifluoroacetic anhydride (0.065 mL, 0.47 mmol) under argon. The reaction mixture was allowed to warm to room temperature and left to stir for 2 h. The reaction mixture was evaporated to dryness and purified by column chromatography (0-3 % acetonitrile in dichloromethane) to give the title compound as a beige crystalline solid (89 mg, 88%); mp 133 ^°C; (Found: M+Na⁺, 349.0088. CnH₅F₃N₆OS+Na⁺ requires 349.0090); _max (EtOH)/nm 264 (log e 3.32), 352 (log e 3.77); v_max (ATR)/cm^-1 3264, 3 133, 1723, 1578, 1524, 1495, 1459, 1440, 1369, 1350, 1296, 1279, 1228, 1 162, 1 122, 1074, 1018; d_H (400 MHz; CDC1₃) 8.53 ( 1 H, s, CH, H-6), 7.94 ( 1 H, s, CH, H- 14), 5.17 (2 H, d, J 2.5, CH₂), 2.46 ( 1 H, t, J 2.5, CH); 5_C ( 100 MHz; CDC1₃) 160.4 (C), 147.4- 146.3 (C, q, J 37.0), 138.2 (CH), 133.0 (C), 13 1.4 (C), 129.9 (CH), 124.4- 1 16.3 (CF₃, q, J 269.0), 122.8 (CH), 75.8 (C), 74.7 (CH), 39.2 (CH₂); d_R (376 MHz; CD₃CN) -64.44 (s, CF₃); m/z (ESI) 349 (M+Na⁺, 100%).

1.8: Reference Example - Formation of an Intermediate

Synthesis of 8-(4-Hydroxy-4-(4, 4,5,5, 5-pentafluoroethyl)-5,5-dihydrothiazol-2-yl)-3-

To a solution of 3-(propargyl)imidazo[5,l-<7]-l,2,3,5-tetrazin-4-oxo-8-thiocarboxamide (115 mg, 0.49 mmol) in dry acetonitrile (5 mL) was added l-bromo-4,4,5,5,5- pentafluorobutan-2-one (0.08 mL, 0.54 mmol) and the mixture was stirred for 18 h at room temperature under argon. The reaction mixture was evaporated to dryness and purified by flash column chromatography (0-4% methanol in dichloromethane) to give the title compound as a red solid (84 mg, 44%); mp 108-110 °C; (Found: M+H⁺, 395.0350. C_I2H₇F₅N₆0₂S+H⁺ requires 395.0344); _max (EtOH)/nm 328 (log e 3.73); v_max (ATR)/cm^_13302, 3132, 2359, 2130, 1748, 1606, 1556, 1455, 1422, 1378, 1343, 1310, 1273, 1216, 1188, 1157, 1074; d_H (400 MHz; CDC1₃) 8.59 (1 H, s, CH, H-6), 5.15 (2 H, d, J 2.5, CH₂), 5.07 (1 H, br s, OH), 3.84 (1 H, d, J 12.8, CHH), 3.55 (1 H, d, J 12.8, CHH), 2.46 (1 H, t, J 2.5, CH); 5_C (100 MHz; CD₃CN) 167.3 (C), 137.9 (C), 133.4 (C), 131.5 (C), 130.1 (CH), 123.7-114.4 (CF₃, qt, J35.0, 273.0), 115.7-109.5 (CF₂, tq, J35.0, 273.0), 107.2-106.7 (C, t, .724.5), 75.5 (C), 75.0 (CH), 39.4 (CH₂), 38.3 (CH₂); d_R (376 MHz; CDC1₃) -78.87 (3 F, s, CF₃), -123.82 (2 F, s, CF₂); m/z (ESI) 811 (2M+Na⁺, 100%).

1.9: Example of the Invention

Synthesis of 8-(4-(4, 4,5,5, 5-Pentafluoroethyl)-thiazol-2-yl)-3-(propargyl)imidazof 5,1- d]-l,2,3,5-tetrazin-4-one

To a solution of 8-(4-hydroxy-4-(4,4,5,5,5-pentafluoroethyl)-5,5-dihydrothiazol-2-yl)-3- (propargyl)imidazo[5,l-r/]-l,2,3,5-tetrazin-4-one (98 mg, 0.25 mmol) and dry triethylamine (0.05 mL, 0.37 mmol) in dry acetonitrile (2.5 mL) in an ice bath was added trifluoroacetic anhydride (0.05 mL, 0.37 mmol) under argon. The reaction mixture was warmed to room temperature and left to stir for 2 h. The reaction mixture was evaporated to dryness and purified by flash column chromatography (dichloromethane) to give the title compound as a colourless crystalline solid (74 mg, 79%); mp 106 ^°C; (Found: M+Na⁺, 399.0061. Ci₂H₅F₅N₆OS+Na⁺ requires 399.0058); _max (EtOH)/nm 264 (log e 3.78), 351 (log e 4.05); v_max (ATR)/cm^_1 3295, 3122, 2938, 2360, 2342, 2245, 2132, 1743, 1581, 1581, 1560, 1496, 1452, 1420, 1371, 1348, 1304, 1267, 1192, 1143, 1095, 1075, 1021; d_H (400 MHz; CDC1₃) 8.51 (1 H, s, CH, H-6), 7.98 (1 H, s, CH, H-14), 5.17 (2 H, d, J 2.5, CH₂), 2.46 (1 H, t, J 2.5, CH); 5_C (100 MHz; CDC1₃) 160.4 (C), 146.2- 145.6 (C, t, J 28.5), 138.2 (C), 133.1 (C), 131.5 (C), 129.9 (CH), 124.2 (CH, t, J 4.5), 120.6-112.1 (CF₃, qt, J 38.5, 270), 113.5-107.3 (CF₂, tq, J 38.5, 270), 75.8 (C), 74.7 (CH), 39.2 (CH₂); 5_F (376 MHz; CDC1₃) -83.57 (3 F, t, J2.3, CF₃), -112.79 (2 F, q, J2.3, CF₂); m/z (ESI) 775 (2M+Na⁺, 100%).

1.10: Example of the Invention

Synthesis of 8-(4-Bromomethyl-thiazol-2-yl)-3-(nronarsyl)imidazof5,l-dl-l,2,3,5- tetrazin-4-one

To a solution of 3-(propargyl)imidazo[5,l-r/]-l,2,3,5-tetrazin-4-oxo-8-thiocarboxamide (990 mg, 4.23 mmol) in dry acetonitrile (42 mL) was added 1,3-dibromo propan-2-one (9.13 mg, 4.23 mmol) and the reaction mixture was stirred for 17 h at room temperature under argon. The reaction mixture was evaporated to dryness and purified by flash column chromatography (0-1% methanol in dichloromethane) to give the title compound as a yellow solid (593 mg, 40%); mp 157-160 °C; (Found: M+H⁺, 350.9658. CnH₈BrN₆OS+H⁺ requires 350.9658); _max (EtOH)/nm 263 (log e 4.74), 362 (log e 4.95); v_max (ATR)/cm^_1 3245, 3118, 3100, 2924, 2127, 1723, 1579, 1551, 1507, 1458, 1439, 1426, 1372, 1354, 1286, 1252, 1230, 1213, 1156, 1120, 1085, 1033, 1020; d_H (400 MHz; CDC1₃) 8.50 (1 H, s, CH, H-6), 7.53 (1 H, s, CH, H-14), 5.16 (2 H, d, J2.5, CH₂), 4.73 (2 H, s, CH₂), 2.45 (1 H, t, J 2.5, CH); 5_C (100 MHz; CDC1₃) 158.7 (C), 154.8 (C), 138.4 (C), 134.0 (C), 131.0 (C), 129.8 (CH), 120.4 (CH), 75.9 (C), 74.6 (CH), 39.1 (CH₂), 26.9 (CH₂); m/z (ESI) 352/350 (M⁺+H⁺, 100/98%), 374/372 (M⁺+Na⁺, 23/23%), 727/725 (2M⁺+Na⁺, 14/24%).

1.11: Example of the Invention

Synthesis of 8-(4-Hydroxy methyl-thiazol-2-yl)-3-(proparsy l)imidaz.o[5,l -dl-1, 2,3,5- tetraz.in-4-one

To a solution of 8-(4-bromomethyl-thiazol-2-yl)-3-(propargyl)imidazo[5,l-£]-l, 2,3,5- tetrazinone (144 mg, 0.41 mmol) in /V,/V-dimethylformamide (1.0 mL) under argon was added silver trifluoroacetate (136 mg, 0.62 mmol) in one portion and the reaction mixture was stirred for 7 h at room temperature under argon. The reaction mixture was diluted with ether (5 mL), washed with hydrochloric acid (1 M, 3 x 5 mL) and the aqueous layer was then extracted with dichloromethane (3 x 5 mL), washed with brine (20 mL), dried over MgS0₄ and concentrated under vacuum and purified by flash column chromatography (0-3% methanol in dichloromethane) to give the title compound as a green solid (70 mg, 60%); mp 135-137 °C; (Pound: M+Na⁺, 311.0321. CnH₈N₆0₂S+Na⁺ requires 311.0322); _max (EtOH)/nm 273 (log e 3.91), 365 (log e 4.02); v_max (ATR)/cm^_1 3413, 3279, 3262, 3125, 3093, 2977, 2947, 1736, 1519, 1508, 1450, 1434, 1370, 1353, 1301, 1251, 1229, 1170, 1151, 1076, 1048; d_H (400 MHz; CDC1₃) 8.47 (1 H, s, CH, H-6), 7.40 (1 H, s, CH, H-14), 5.12 (2 H, d, J2.5, CH₂), 4.90 (2 H, d, J0.9, CH₂OH), 3.65 (1

H, br s, OH), 2.45 (1 H, t, 72.5, CH); 5_C (100 MHz; CDC1₃) 158.9 (C), 158.6 (C), 138.4 (C), 134.0 (C), 130.8 (C), 129.8 (CH), 117.4 (CH), 75.9 (C), 74.6 (CH), 61.0 (CH₂), 39.1 (CH₂); m/z (ESI) 311 (M⁺+Na⁺, 100%), 289 (M⁺+H⁺, 63%), 599 (2M⁺+H⁺, 35%).

I.12: Example of the Invention

Synthesis of 8-(5-Methylthiazol-2-yl)-3-(proparsyl)imidazof5,l-dl-l,2,3,5-tetrazin-4- one To a solution of 3-(propargyl)imidazo[5, l -£/]- l ,2,3,5-tetrazin-4-oxo-8-thiocarboxamide ( 135 mg, 0.62 mmol) in dry acetone (6 mL) was added 2-bromopropionaldehyde ( 150 mg, 1.23 mmol) and the reaction mixture was stirred for 16 hours at reflux under argon. The reaction mixture was evaporated to dryness and purified by flash column chromatography (0-2% methanol in dichloromethane) to give the title compound as a bright yellow solid (42 mg, 25%); mp 167 °C; (Found: M+Na⁺, 295.0377.

CnH₈N₃OS+Na⁺ requires 295.0373); _max (EtOH)/nm 239 (log e 1.83), 366 (log e 2.01); v_max (ATR)/cm^_1 3240, 3 127, 3071 , 2126, 1736, 1521 , 1485, 1436, 1413, 1378, 1345, 1307, 1266, 1237, 1 170, 1 138, 1 1 15 , 1072, 1034, 1017; d_H (400 MHz; CDC1₃) 8.46 ( 1 H, s, CH, H-6), 7.74 ( 1 H, q, J 1. 1 , CH, H- 13), 5.14 (2 H, d, J 2.5, CH₂), 2.58 (3 H, d, J 1. 1 , CH₃), 2.44 ( 1 H, t, J 2.5, CH); 5_C ( 100 MHz; CDC1₃) 157.0 (C), 143.4 (CH), 138.6 (C), 136.8 (C), 134.8 (C), 130.4 (C), 129.6 (CH), 76.0 (C), 74.5 (CH), 39. 1 (CH₂), 12.2 (CH₃); m/z (ESI) 567 (2M+Na⁺, 100%), 273 (M⁺+H⁺, 63%), 295 (M⁺+Na⁺, 3 1 %).

Example 2: MTT Assay - comparison with TMZ

Three compounds according to the invention - Compounds (I-A), (I-B) and (I-C) -were tested in vitro on the cell lines MCF-7, MDA-MB-468, A549, MDA-MB-435, HT29 and HK- 1. TMZ was also tested on these cell lines.

GI₅₀ values (mM) were established. The results are shown in Figure 1.

Comments

It can be seen that Compounds (I-A), (I-B) and (I-C) according to the invention are each significantly more effective against all tested cell lines than TMZ.

The results also show that the thiazole group at the 8-position can be unsubstituted or can be substituted with a sterically small group, and the compound remains effective . Example 3A: MTT Assay - comparison with TMZ and TMZ variant

Three compounds according to the invention - Compounds (I-A), (I-B) and (I-C) - were tested in vitro on the cell lines U373V, U373M, HCT-116 and MRC-5.

TMZ was also tested on these cell lines, as well as Comparative 1 which had the same alkyne group (propargyl) at the 3 -position as Compounds (I-A), (I-B) and (I-C) but which retained the amide at the 8-position as in TMZ.

GI_5O values (mM) were established. The results are as follows:

Comments

It can be seen that Compounds (I-A), (I-B) and (I-C) according to the invention are each significantly more effective against all tested cell lines than TMZ. In addition, Compounds (I-A), (I-B) and (I-C) according to the invention are each significantly more effective against all tested cell lines than the variant of TMZ which modifies the 3-position but not the 8-position substituent group, i.e. that lacks the thiazole group of the invention.

The results show that the thiazole group at the 8-position can be unsubstituted or can be substituted with a sterically small group, and the compound remains effective.

Example 3B: MTT Assay - comparison with TMZ and TMZ variant

Two further compounds according to the invention - Compounds (I-D) and (I-E) - were tested in vitro on the human derived glioblastoma cell lines U373V and U373M, and the human colorectal cell line HCT-116. TMZ was also tested on these cell lines, as well as Comparative 1 which had the same alkyne group (propargyl) at the 3 -position as Compounds (I-D) and (I-E) but which retained the amide at the 8-position as in TMZ.

GI_5O values (mM) were established. The results are as follows:

Comments

It can be seen that Compounds (I-D) and (I-E) according to the invention are both significantly more effective against all tested cell lines than TMZ.

In addition, Compounds (I-D) and (I-E) according to the invention are both significantly more effective against all tested cell lines than the variant of TMZ which modifies the 3- position but not the 8-position substituent group, i.e. that lacks the thiazole group of the invention.

The results show that the thiazole group at the 8-position can be substituted with a sterically small group, and the compound remains effective.

Example 3C: MTT Assay - comparison of fluorinated variants with TMZ

Two fluorinated compounds according to the invention - Compounds (I-F) and (I-G) - were tested in vitro on the human derived glioblastoma cell lines U373V and U373M, and the human colorectal cell line HCT-116.. TMZ was also tested on these cell lines. GI_5O values (mM) were established. The results are as follows:

Comments

It can be seen that Compounds (I-F) and (I-G) according to the invention are both significantly more effective against all tested cell lines than TMZ.

The results show that the thiazole group at the 8-position can be substituted with a sterically small fluorinated group, and the compound remains effective.

Example 3D: Comparison with WO 2010/149968 data

WO 2010/149968 describes a number of specific compounds as having a U373V GI₅₀ of less than 75mM, namely compounds: WW-001, WW-002, WW-003, WW-004, WW-005, WW-006, WW-007, WW-008, WW-009, WW-01 1, WW-012, WW-013, WW-014, WW- 015, WW-016, WW-021, WW-023, WW-024, WW-025, WW-026, WW-027, WW-028, WW-029, WW-030, WW-031, WW-032, WW-033, WW-034, WW-035, WW-036, WW-

037, WW-038, WW-039, WW-040, WW-041, WW-042, WW-044, WW-045, WW-046, WW-047, WW-048, WW-049, WW-050, WW-05 1, WW-052, WW-054, WW-055, WW- 058, WW-062, WW-063. In WO 2010/149968 specific data is provided for WW-028 :

which has thiazole group at the 8-position plus an alkyne group at the 3 -position, but the thiazole group has a bulky substituent, namely a further thiazole group.

WW-028 is described as having a U373V GI₅₀ of 50.0mM.

It is noteworthy from Example 3A that the Compounds (I-A), (I-B) and (I-C) according to the invention have a U373V GI₅₀ of very much less: ranging from 3.59 to 24.4 mM.

It is likewise noteworthy from Example 3B that the Compounds (I-D) and (I-E) according to the invention have a U373V GI₅₀ of very much less: ranging from 6.84 to 23.3 mM.

It is also noteworthy from Example 3C that fluorinated variants, Compounds (I-F) and (I- G), according to the invention have a U373V GI₅₀ of very much less: ranging from 27.1 to 28.2 mM.

Similarly, in WO 2010/149968 each of the following compounds was described as having a U373M GI₅₀ of less than 60mM: WW-001, WW-002, WW-003, WW-004, WW- 005, WW-006, WW-007, WW-008, WW-009, WW-012, WW-013, WW-014, WW-015, WW-016, WW-021, WW-023, WW-024, WW-025, WW-026, WW-027, WW-028, WW- 029, WW-030, WW-031, WW-032, WW-033, WW-034, WW-035, WW-036, WW-037, WW-038, WW-039, WW-040, WW-042, WW-046, WW-047, WW-048, WW-049, WW- 050, WW-051, WW-052, WW-055, WW-058, WW-062, WW-063.

In WO 2010/149968 specific data is provided for WW-028 :

which has thiazole group at the 8-position plus an alkyne group at the 3 -position, but the thiazole group has a bulky substituent, namely a further thiazole group. WW-028 is described as having a U373M GI₅₀ of 35. I mM.

It is noteworthy from Example 3A that the Compounds (I-A), (I-B) and (I-C) according to the invention have a U373M GI₅₀ of very much less: ranging from 4.09 to 14.8 mM.

It is likewise noteworthy from Example 3B that the Compounds (I-D) and (I-E) according to the invention have a U373M GI₅₀ of very much less: ranging from 3.99 to 9.67pM.

Therefore the compounds according to the present invention, with their specific combination of the non-bulky thiazole group at the 8 -position plus the non-bulky alkyne group at the 3 -position are surprisingly significantly more effective than the preferred compounds of WO 2010/149968. This was not foreseeable from WO 2010/149968.

Example 4: MTT Assay - effect of substituent location on thiazole group

Two compounds according to the invention - Compounds (I-B) and (I-D) - were tested in vitro on the cell lines U373V, U373M and HCT-1 16.

GI₅₀ values (mM) were established. The results are shown in Figure 2.

Comments

It can be seen that Compounds (I-B) and (I-D) according to the invention are both effective against all tested cell lines.

This shows that the thiazole group at the 8-position can be substituted on the 4-position or on the 5 -position on the thiazole ring, and the compound remains effective.

Example 5: MTT Assay - effect of thiazole group plus alkyne group

Four compounds were tested on the cell lines U373V, U373M and HCT-1 16. GI₅₀ values (pM) were established.

The tested compounds were:

• TMZ

• Compound (I-B) • Comparative 1 which had the same alkyne group (propargyl) at the 3 -position as Compound (I-B) but retained the amide at the 8-position as in TMZ

• Comparative 2 which retained the methyl group substituent at the 3-position as in TMZ but had the same thiazole substituent at the 8-position as Compound (I-B).

The results are as follows:

Comments

It can be seen that Compound (I-B) according to the invention is significantly more effective against all tested cell lines than TMZ. In addition, Compound (I-B) according to the invention is significantly more effective against all tested cell lines than the two variants of TMZ, which each modified one but not both of the 3 -position and the 8 -position substituent groups.

Therefore it is the combination of the thiazole substituent at the 8-position and the alkyne group substituent at the 3 -position that gives rise to the beneficial properties of the present invention.

Example 6: MTT Assay - effect of substituent size on thiazole

Five compounds were tested on the MMR-deficient human colon cancer cell line HCT- 1 16. GI_5O values (mM) were established.

The tested compounds were:

• TMZ

• Compound (I-A)

• Compound (I-B)

• Compound (I-C)

• Comparative 3 which had a propargyl at the 3 -position and thiazole substituent at the 8-position, but had a bulky iso-butyl group at the 4-position on the thiazole ring

Comparative 3 is equivalent to WW-020 as disclosed in WO 2010/149968.

The results are as follows:

Comments

It can be seen that Compounds (I-A), (I-B) and (I-C) according to the invention are each significantly more effective than TMZ.

In addition, Compounds (I-A), (I-B) and (I-C) according to the invention are each, surprisingly, more effective than the variant which has a sterically bulky substituent on the thiazole ring.

The results show that the thiazole group at the 8-position can be unsubstituted or can be substituted with a sterically small group, and the compound remains effective, but that a sterically bulky group decreases the efficacy.

Example 7: Clonogenic Assay - clonogenic survival following brief exposure of cells to imidazotetrazine analogues

Clonogenic assays were carried out to determine drug activity.

Two compounds according to the invention - Compounds (I-A) and (I-B) - were tested in vitro on the cell lines U373V, U373M and HCT-1 16.

In this regard, each compound was tested in cells at a number of different concentrations. Cells were exposed to the drug for 24 hours then left to proliferate until colonies formed. The colonies were fixed and stained with methylene blue, and colonies counted.

TMZ was also tested, as well as Comparative 1, which had the same alkyne group (propargyl) at the 3 -position as Compound (I-B) but retained the amide at the 8 -position as in TMZ.

The results are shown in Figure 3. Comments

It can be seen that Compounds (I-A) and (I-B) according to the invention are each significantly more effective at reducing colonies than TMZ, for all tested cell lines.

In addition, Compounds (I-A) and (I-B) according to the invention are both more effective at reducing colonies at a 20 mM concentration than the Comparative 1 compound.

Compound (I-B) is surprisingly effective at significantly reducing the number of colonies even at a 10 pM concentration.

Example 8: pH stability test

The stability of TMZ and the compounds of formula (I-A), (I-B) and (I-C) was tested at different pH values: (a) 3, (b), 5.5, (c) 7.4.

The results are as follows:

Comments

The stability at low pH for all compounds is beneficial, in that it permits for parenteral administration.

Stability at acidic pH values also suggests that the compounds could be suitable for oral delivery. Example 9: Permeability test:

The compound of formula (I-A) was tested using a number of assays:

(I-A)

The results are as follows:

1) Parallel Artificial Membrane Permeability Assay (PAMPA)

NB. P_a < 1 indicates poor permeability

2) MDCK-WT/MDR1 Permeability Assay

PSA: 72.99

cLogP: 0.529

Comments

In permeability studies, (utilising PAMPA artificial membranes and MDCKII W/T MDR- 1 isogenic kidney cells), it was revealed that, in contrast to TMZ, compound (I-A) according to the invention is not a P-glycoprotein substrate.

Experimental evidence for compound (I-A) according to the invention also inferred excellent brain penetrance (> 20) and oral bioavailability (> 10). Example 10- Growth inhibition of carcinoma cell lines as determined by 7-dav MTT assay - comparison with TMZ

Three compounds according to the invention - Compounds (I-A), (I-B) and (I-C) - were tested in vitro on an extended panel of human tumour cell lines. TMZ was also tested on these cell lines.

The cell lines tested were: (a) HK-1, nasopharangeal carcinoma; (b) MDA-MB-435, malignant melanoma cell line; (c) MCF-7, ER+ breast carcinoma; (d) MDA-MB-468, triple negative breast carcinoma;€ A549, lung carcinoma; (f) HT-29, CRC, MGMT+ and MMR+.

GI_5O values (mM) were established. The results are as follows:

Comments

It can be seen that Compounds (I-A), (I-B) and (I-C) according to the invention are each significantly more effective against all tested cell lines than TMZ.

This included significantly more effective results against the two breast cancer cell lines (oestrogen receptor (ER) positive) MCF-7 and triple negative (ER, progesterone, HER2) MDA-MB-468.

Example 11 - Physical properties of selected imidazotetrazines

a Determined by ^:H NMR spectroscopy in phosphate buffers;

b Calculated using ChemDraw vl6.0.1.4. It can be appreciated that the compounds of the invention are small structures, and with cLogP values indicating amphiphilic character.

The polar surface area (PSA) is a measure of the molecular surface arising from polar atoms (nitrogen and oxygen and any attached hydrogens); lower values are seen to predict more efficient blood brain barrier passage. The values for the compounds of the invention are below that for TMZ, which is known by PET studies to access tumors in the brains of humans in clinical studies.

The half-life (t ½) values show stability, with compound (I-A) having the highest stability at pH 7.4.

Conclusion

Replacing the substituents at the 3-(methyl) and 8-(carboxamide) positions of TMZ with a propargyl group and a non-sterically bulky thiazole residue, respectively, has been shown to lead to compounds having enhanced growth-inhibitory activity against a surprisingly extensive panel of human tumor cell lines, including against human derived glioblastoma and colorectal cell lines.

Notably, this enhanced activity is observed against MGMT-expressing and MMR- deficient cell lines, which are molecular features that confer resistance to TMZ. Therefore the compounds of the invention unexpectedly overcome the problems normally associated with MGMT repair and MMR deficiency.

The compounds of the invention therefore overcome the known issue of GBM tumours having resistance towards TMZ.

The compounds of the invention are not disclosed in WO 2010/149968, and are surprisingly more effective than the specific compounds of WO 2010/149968, including having a noticeably greater inhibitory activity against GBM tumours. The compounds of the invention also beneficially show a broad spectrum of activity.

Claims

1. A product which is a compound of formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof:

wherein

X is selected from: hydrogen; a methyl group which is optionally substituted, wherein any substituent groups present are independently selected from hydroxyl, halo and NR'₂, where each R' is independently selected from hydrogen and methyl; and an ethyl group which is either unsubstituted or is substituted with from one to five F substituent groups;

Y is selected from: hydrogen; a methyl group which is optionally substituted, wherein any substituent groups present are independently selected from hydroxyl, halo and NR'₂, where each R' is independently selected from hydrogen and methyl; and an ethyl group which is either unsubstituted or is substituted with from one to five F substituent groups;

R_A is hydrogen, or is a C l -2 alkyl group which is optionally substituted, wherein any substituent groups present are independently selected from hydroxyl, halo and NR'₂, where each R' is independently selected from hydrogen and methyl.

2. A pharmaceutical formulation comprising a product as defined in claim 1 in combination with a pharmaceutically acceptable carrier, excipient or diluent.

3. The product of claim 1 or pharmaceutical formulation of claim 2 for use in a method of treating a proliferative condition.

4. The product or pharmaceutical formulation for use in treatment according to claim 3, wherein the proliferative condition is cancer.

5. The product or pharmaceutical formulation for use in treatment according to claim 4, wherein the cancer is selected from the group consisting of: glioma, colorectal cancer, lung cancer, breast cancer, ovarian cancer, melanoma, renal cancer, prostate cancer, oesophageal cancer, and squamous carcinoma of the head or neck.

6. The product, the pharmaceutical formulation, or the product or pharmaceutical formulation for use in treatment according to any one of the preceding claims, wherein X is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl, Cl, Br, and NR'₂, where each R' is independently selected from hydrogen and methyl; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

7. The product, the pharmaceutical formulation, or the product or pharmaceutical formulation for use in treatment according to claim 6, wherein X is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl and NH₂; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

8. The product, the pharmaceutical formulation, or the product or pharmaceutical formulation for use in treatment according to any one of the preceding claims, wherein Y is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl, Cl, Br, and NR'₂, where each R' is independently selected from hydrogen and methyl; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

9. The product, the pharmaceutical formulation, or the product or pharmaceutical formulation for use in treatment according to claim 8, wherein Y is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl and NH₂; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

10. The product, the pharmaceutical formulation, or the product or pharmaceutical formulation for use in treatment according to any one of the preceding claims, wherein X and Y in combination have no more than three carbon atoms.

11. The product, the pharmaceutical formulation, or the product or pharmaceutical formulation for use in treatment according to claim 10, wherein X and Y in combination have no more than three carbon atoms and no more than three substituent groups.

12. The product, the pharmaceutical formulation, or the product or pharmaceutical formulation for use in treatment according to claim 11, wherein X and Y in combination have no more than two carbon atoms and no more than two substituent groups.

13. The product, the pharmaceutical formulation, or the product or pharmaceutical formulation for use in treatment according to any one of the preceding claims, wherein one or both of X and Y are hydrogen.

14. The product, the pharmaceutical formulation, or the product or pharmaceutical formulation for use in treatment according to any one of the preceding claims, wherein R_{A IS} selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl, Cl, Br, and NR'₂, where each R' is independently selected from hydrogen and methyl; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; an ethyl group which is substituted with one substituent group selected from hydroxyl, Cl, Br, and NR'₂, where each R' is independently selected from hydrogen and methyl; and an ethyl group which is substituted with from one to five F substituent groups.

15. The product, the pharmaceutical formulation, or the product or pharmaceutical formulation for use in treatment according to claim 14, wherein R_A is selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl and NH₂; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

16. The product, the pharmaceutical formulation, or the product or pharmaceutical formulation for use in treatment according to any one of the preceding claims, wherein

R_A is hydrogen and each of X and Y are independently selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl and NH₂; a methyl group which is substituted with one, two or three F substituent groups; an unsubstituted ethyl group; and an ethyl group which is substituted with from one to five F substituent groups.

17. The product, the pharmaceutical formulation, or the product or pharmaceutical formulation for use in treatment according to any one of the preceding claims, wherein R_A is hydrogen and each of X and Y are independently selected from: hydrogen; an unsubstituted methyl group; a methyl group which is substituted with one substituent group selected from hydroxyl and NH₂; and an unsubstituted ethyl group.

18. The product, the pharmaceutical formulation, or the product or pharmaceutical formulation for use in treatment according to any one of the preceding claims, wherein the product is a compound of any one of the formulae set out below, or a pharmaceutically acceptable salt, hydrate or solvate thereof:

(I-A) (I-B) (I-G)

19. The product, the pharmaceutical formulation, or the product or pharmaceutical formulation for use in treatment according to any one of the preceding claims, wherein the product is a compound of any one of the formulae set out below, or a pharmaceutically acceptable salt, hydrate or solvate thereof:

(I-C) (I D)