WO2009037468A1

WO2009037468A1 - Thienopyrimidine compounds and compositions

Info

Publication number: WO2009037468A1
Application number: PCT/GB2008/003180
Authority: WO
Inventors: Allan Jordan; Simon Bedford; Klenke Burkhard; Ian Yule; Karinne Poullennec
Original assignee: Vernalis R&D Ltd
Current assignee: Vernalis R&D Ltd
Priority date: 2007-09-21
Filing date: 2008-09-19
Publication date: 2009-03-26
Anticipated expiration: 2010-03-21
Also published as: JP2010540424A; EP2207780A1; EP2207780B1; GB0718432D0; US8354415B2; US20100298349A1

Abstract

Compounds of formula (I) are A2B wherein R1 and R2 are independently selected from hydrogen, or optionally substituted CrC6 alkyl, C1-C6 alkoxy-( C1-C6 )-alkyl, C3-C8 cycloalkyl, aryl, heteroaryl, aryl-( C1-C6 )-alkyl, or heteroaryl-( C1-C6 )-alkyl; or R-1 and R2 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- or 6-membered ring; R3 and R4 are independently selected from hydrogen, or optionally substituted C1-C6 alkyl, C1-C6 alkoxy-( C1-C6 )-alkyl, C3- C8 cycloalkyl, aryl, heteroaryl, aryl-( C1-C6 )-alkyl, or heteroaryl- C1-C6 )-alkyl; or R3 and R4 taken together with the nitrogen atom to which they are attached form an optionally substituted 5- or 6-membered ring; R5 and R6 are independently selected from hydrogen, C1-C6 alkyl, aryl, aryl-( C1-C6 )-alkyl, - NHR7 -N(-R8)-R9, -NH-(C=O)-R10, -(C=O)-NH-R11, -(C=O)-O-R12, or halo; and R7, R8, R9, R10, R-11, and R12 are independently selected from C1-C6 alkyl, aryl, aryl-( C1-C6 )-alkyl and heteroaryl.

Description

THIENOPYRIMIDINE COMPOUNDS AND COMPOSITIONS

This invention relates to novel thienopyrimidine derivatives having A_2B receptor antagonistic activity, to the use of such compounds in medicine, in relation to the treatment of disorders which are responsive to antagonism of the A2B receptor such as nociception, asthma, COPD, inflammatory disorders, diabetes, diabetic retinopathy and cancer, and to pharmaceutical compositions containing such compounds.

Background to the invention

Adenosine is a naturally occurring purine nucleoside, the effects of which include stimulation of nociception afferents, bronchconstriction, immunosupression, vasodilation, inhibition of platelet aggregation, cardiac depression and inhibition of neurotransmitter release.

Adenosine produces a wide range of pharmacological effects mediated by activation of specific cell surface receptors, which are members of the G- protein coupled receptor family. Four subtypes of adenosine receptors have been identified, designated A-i, A_2A, A_2B and A₃.

The A_2B adenosine receptor subtype is coupled to the G₃ G-protein and stimulates adenylyl cyclase activity. Although significant advancement has been made in the understanding of the molecular pharmacology and physiology of A_2B adenosine receptors, due to the lack of highly potent and selective ligands for this receptor subtype, many questions about the pathophysiological role of A_2B receptors are yet to be resolved (Feoktistov and Biaggioni, Pharmacological Reviews (1997), 49(4), 381-402).

A_2B receptors have been implicated in:

. (i) the regulation of mast cell secretion (Feoktistov and Biaggioni., Journal of Clinical Investigation (1995), 96(4), 1979-86). (ii) pain (Abo-Salem et al., Journal of Pharmacology and Experimental

Therapeutics (2004), 308(1 ), 358-366.). (iii) inflammation (Yang et al., Journal of Clinical Investigation (2006),

116(7), 1913-1923). (iv) cancer (Zeng et al., Drug Development Research (2003), 58(4),

405-411 ). (v) diabetes (Harada et al., Journal of Medicinal Chemistry (2001),

44(2), 170-179).

(vi) gene expression (Boyle et al., Arthritis & Rheumatism (1996), 39(6), 923-930).

(vii) cell growth (Dubey et al., Hypertension (1996), 27(3 Pt 2), 786-93

Hypertension (1996), 27(3 Pt 2), 786-93, Dubey et al.,

Hypertension (1998), 31 (1 Pt 2), 516-21 ).

(viii) intestinal functions (Murthy et al., Journal of Neurochemistry (1995), 64(1), 77-84).

(ix) neurosecretion (Mateo et al., 1995).

(x) vascular tone (Haynes et al., American Journal of Physiology

(1995), 268(5, Pt. 2), H1862-H1868).

(xi) asthma (Feoktistov et al., Trends in pharmacological sciences (1998), 19(4), 148-153; Holgate, British Journal of Pharmacology

(2005), 145(8), 1009-1015). (xii) COPD (Van den Berge et al., Drugs in R&D (2007), 8(1), 13-23).

Thus, there remains a medical need for low molecular weight A_2B antagonists with pharmacokinetic and pharmacodynamic properties making them suitable for use as pharmaceutical agents. There also remains a medical need for new treatments of disorders mediated by the A_2B receptor, particularly nociception, asthma, COPD, inflammatory disorders, diabetes, diabetic retinopathy and cancer. The object of the present invention is to provide such pharmaceutical agents and treatments.

It has now been found that certain thienopyrimidine derivatives show efficacy as A₂B antagonists. Brief description of the invention

The present invention relates to a class of substituted thienopyrimidine compounds useful as A2B antagonists, for example, for the treatment of nociception, asthma, COPD, inflammatory disorders, diabetes, diabetic retinopathy and cancer. A core thieno-pyrimidine bicyclic ring, with substitution on the pyrimidine portion by an amido group in addition to an amino group are principle characterising features of the compounds with which the invention is concerned.

Detailed description of the invention

According to the present invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof:

(I)

wherein

Ri and R₂ are independently selected from hydrogen, or optionally substituted Ci-C₆ alkyl, Ci-C₆ alkoxy-(CrC₆)-alkyl, C₃-C₈ cycloalkyl, aryl, heteroaryl, aryl- (Ci-C₆)-alkyl, or heteroaryl-(C₁-C₆)-alkyl;

or Ri and R₂ taken together with the nitrogen atom to which they are attached form an optionally substituted 5- or 6-membered ring; R₃ and R₄ are independently selected from hydrogen, or optionally substituted Ci-C₆ alkyl, CrC₆ alkoxy-(CrC₆)-alkyl, C3-C₈ cycloalkyl, aryl, heteroaryl, aryl- (CrCβJ-alkyl, or heteroaryl-(C_rC₆)-alkyl;

or R₃ and R₄ taken together with the nitrogen atom to which they are attached form an optionally substituted 5- or 6-membered ring;

R5 and R₆ are independently selected from hydrogen, C₁-C₆ alkyl, aryl, aryl- (Ci-C₆)-alkyl, -NHR₇ -N (-Rg)-R₉, -NH-(C=O)-R₁₀, -(C=O)-NH-R₁₁, -(C=O)-O- R₁₂, or halo; and

R₇, R₈, Rg, R10, R11, and R₁₂ are independently selected from CrC₆ alkyl, aryl, aryl-(CrC₆)-alkyl and heteroaryl.

The active compounds of formula (I) are antagonists of the A_2B receptor and are useful for the treatment, prevention and suppression of disorders mediated by the A₂B receptor. Such disorders include nociception; asthma; chronic obstructive pulmonary disease (COPD); inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, lupus, psoriasis and inflammatory bowel disease; diabetes mellitus or diabetes insipidus; diabetic retinopathy and cancer.

According to a further embodiment of the present invention there is provided the use of a compound of formula (I), or a pharmaceutically acceptable "salt, hydrate, solvate, or prodrug thereof, in the manufacture of a medicament for the treatment of disorders mediated by the adenosine A_2B receptor.

According to a further embodiment of the present invention there is provided a method of treatment of a disorder mediated by the A_2B receptor comprising administration to a subject in need of such treatment an effective dose of the compound of formula (I)₁ or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof. According to a further embodiment of the present invention there is provided a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, and a pharmaceutically acceptable carrier.

As used herein, the term "(C_a-C_b)alkyl" wherein a and b are integers refers to a straight or branched chain alkyl radical having from a to b carbon atoms. Thus when a is 1 and b is 6, for example, the term includes methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl and n-hexyl.

As used herein the term "divalent (C_a-C_b)alkylene radical" wherein a and b are integers refers to a saturated hydrocarbon chain having from a to b carbon atoms and two unsatisfied valences.

As used herein the term "(C_a-C_b)alkenyl" wherein a and b are integers refers to a straight or branched chain alkenyl moiety having from a to b carbon atoms having at least one double bond of either E or Z stereochemistry where applicable. The term includes, for example, vinyl, allyl, 1- and 2-butenyl and 2-methyl-2-propenyl.

As used herein the term "divalent (C_a-C_b)alkenylene radical" refers to a hydrocarbon chain having from a to b carbon atoms, at least one double bond, and two unsatisfied valences.

As used herein the term "cycloalkyl" refers to a saturated carbocyclic radical having from 3-8 carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

As used herein the term "cycloalkenyl" refers to a carbocyclic radical having from 3-8 carbon atoms containing at least one double bond, and includes, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl.

As used herein the term "carbocyclic" refers to a mono- or bi-cyclic radical whose ring atoms are all carbon, and includes monocyclic aryl, cycloalkyl, and cycloalkenyl radicals, provided that no single ring present has more than 8 ring members. A "carbocyclic" group includes a mono-bridged or multiply- bridged cyclic alkyl group.

As used herein the term "aryl" refers to a mono-, bi- or tri-cyclic carbocyclic aromatic radical, illustrative of such radicals are phenyl, biphenyl and napthyl.

As used herein the term "heteroaryl" refers to a mono-, bi- or tri-cyclic aromatic radical containing one or more heteroatoms selected from S, N and O. Illustrative of such radicals are thienyl, benzthienyl, fury], benzfuryl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, benzthiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl, benztriazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl and indazolyl.

As used herein the unqualified term "heterocyclyl" or "heterocyclic" includes "heteroaryl" as defined above, and in particular refers to a mono-, bi- or tricyclic non-aromatic radical containing one or more heteroatoms selected from S, N and O, to groups consisting of a monocyclic non-aromatic radical containing one or more such heteroatoms which is covalently linked to another such radical or to a monocyclic carbocyclic radical, and to a mono-, bi- or tri-cyclic non-aromatic radical containing one or more heteroatoms selected from S, N and O which is mono-bridged or multiply-bridged. Illustrative of such radicals are pyrrolyl, furanyl, thienyl, piperidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl, benzfuranyl, pyranyl, isoxazolyl, benzimidazolyl, methylenedioxyphenyl, ethylenedioxyphenyl, maleimido and succinimido groups.

Unless otherwise specified in the context in which it occurs, the term "substituted" as applied to any moiety herein means substituted with at least one substituent, for example selected from (C₁-C₆)alkyl, (Ci-C₆)alkoxy, hydroxy, hydroxy(Ci-C₆)alkyl, mercapto, mercapto(Ci-C₆)alkyl, (Cr C-6)alkylthio, halo (including fluoro and chloro), trifluoromethyl, trifluoromethoxy, nitro, nitrile (-CN), oxo, phenyl, -COOH, -COOR^A, -COR^A, -SO₂R^A, -CONH₂, -SO₂NH₂, -CONHR^A, -SO₂NHR^A, -CONR^AR^B, -SO₂NR^AR^B, -NH₂, -NHR^A, -NR^AR^B, -OCONH₂, -OCONHR^A , -OCONR^AR^B, -NHCOR^A, -NHCOOR^A, -NR^BCOOR^A, -NHSO₂OR^A, -NR^BSO₂OR^A, -NHCONH₂,

-NR^ACONH₂, -NHCONHR⁶ -NR^ACONHR^B, -NHCONR^AR^B or -NR^ACONR^AR^B wherein R^A and R^B are independently a (CrCe)alkyl group, or R^A and R^B when attached to the same nitrogen may form a cyclic amino ring such as a morpholinyl, piperidinyl or piperazinyl ring. An "optional substituent" or "susbtituent" may be one of the foregoing substituent groups.

As used herein the term "salt" includes base addition, acid addition and quaternary salts. Compounds of the invention which are acidic can form salts, including pharmaceutically or veterinarily acceptable salts, with bases such as alkali metal hydroxides, e.g. sodium and potassium hydroxides; alkaline earth metal hydroxides e.g. calcium, barium and magnesium hydroxides; with organic bases e.g. N-ethyl piperidine, dibenzylamine and the like. Those compounds (I) which are basic can form salts, including pharmaceutically or veterinarily acceptable salts with inorganic acids, e.g. with hydrohalic acids such as hydrochloric or hydrobromic acids, sulphuric acid, nitric acid or phosphoric acid and the like, and with organic acids e.g. with acetic, tartaric, succinic, fumaric, maleic, malic, salicylic, citric, methanesulphonic and p- toluene sulphonic acids and the like.

For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

The term 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term 'hydrate' is employed when said solvent is water. Compounds with which the invention is concerned which may exist in one or more stereoisomeric form, because of the presence of asymmetric atoms or rotational restrictions, can exist as a number of stereoisomers with R or S stereochemistry at each chiral centre or as atropisomeres with R or S stereochemistry at each chiral axis. The invention includes all such enantiomers and diastereoisomers and mixtures thereof.

So-called 'pro-drugs' of the compounds of formula (I) are also within the scope of the invention. Thus certain derivatives of compounds of formula (I) which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of formula (I) having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as 'prodrugs'. Further information on the use of prodrugs may be found in Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and Bioreversible Carriers in Drug Design, Pergamon Press, 1987 (ed. E. B. Roche, American Pharmaceutical Association).

Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of formula (I) with certain moieties known to those skilled in the art as 'pro-moieties' as described, for example, in Design of Prodrugs by H. Bundgaard (Elsevier, 1985).

Also included within the scope of the invention are metabolites of compounds of formula (I), that is, compounds formed in vivo upon administration of the drug. Some examples of metabolites include

(i) where the compound of formula (I) contains a methyl group, an hydroxymethyl derivative thereof (-CH₃ -> -CH₂OH):

(ii) where the compound of formula (I) contains an alkoxy group, an hydroxy derivative thereof (-OR -> -OH); (iii) where the compound of formula (I) contains a tertiary amino group, a secondary amino derivative thereof (-NR¹R² -> -NHR¹ or -NHR²);

(iv) where the compound of formula (I) contains a secondary amino group, a primary derivative thereof (-NHR¹ -> -NH₂);

(v) where the compound of formula (I) contains a phenyl moiety, a phenol derivative thereof (-Ph -> -PhOH); and

(vi) where the compound of formula (I) contains an amide group, a carboxylic acid derivative thereof (-CONH₂ -> COOH).

The group -N(R₁J-R₂

In the compounds in accordance with the invention, R₁ and R₂ are independently selected from hydrogen, or optionally substituted CrC₆ alkyl, CrC₆ alkoxy-(CrC6)-alkyl, C₃-C₈ cycloalkyl, aryl, heteroaryl, aryl-(C-ι-C₆)-alkyl, or heteroaryl-(CrC₆)-alkyl.

In a subclass of compounds with which the invention is concerned, R₁ and R₂ are independently selected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkoxy-(C-i-C₆)- alkyl, C₃-C₈ cycloalkyl, aryl-(Ci-C₆)-alkyl, or heteroaryl-(Ci-C₆)-alkyl.

In a further subclass of compounds with which the invention is concerned, Ri is hydrogen and R₂ is selected from hydrogen, C₁-C₆ alkyl, C₁-C₆ alkoxy-(Cr

C₆)-alkyl, C₃-C₈ cycloalkyl, aryl-(C₁-C₆)-alkyl, or heteroaryl-(C₁-C₆)-alkyl. In such cases, R₂ may be hydrogen, methyl, ethyl, isopropyl, 2-nnethoxy-ethyl, cyclopropyl, cyclopentyl, cyclohexyl, benzyl, 2-phenyl-ethyl, or pyrid-3-yl- methyl.

It is presently preferred that R₁ is hydrogen and R₂ is selected from C₁-C₆ alkyl, or C₃-C₈ cycloalkyl. Particularly preferred are those compounds wherein Ri is hydrogen and R₂ is methyl, ethyl, isopropyl, or cyclopropyl.

In another subclass of compounds with which the invention is concerned, Ri and R₂ taken together with the nitrogen atom to which they are attached form an optionally substituted 5- or 6-membered ring.

Preferred compounds are those wherein Ri and R₂ taken together with the nitrogen atom to which they are attached form an optionally substituted pyrrolidine or piperidine ring.

Particularly preferred are those compounds wherein Ri and R₂ taken together with the nitrogen atom to which they are attached form pyrrolidin-1-yl or piperidin-1-yl.

The group -N(R₃)-R₄

In the compounds in accordance with the invention, R₃ and R₄ are independently selected from hydrogen, or optionally substituted Ci-C₆ alkyl, C₁-C₆ alkoxy-(Ci-C₆)-alkyl, C₃-C₈ cycloalkyl, aryl, heteroaryl, aryl-(Ci-C₆)-alkyl, or heteroaryl-(Ci-C₆)-alkyl.

In a subclass of compounds with which the invention is concerned, R₃ and R₄ are independently selected from hydrogen or heteroaryl-(CrC₆)-alkyl.

In a further subclass of compounds with which the invention is concerned, R₃ is hydrogen and R₄ is heteroaryl-(C-,-C₆)-alkyl. in such cases, R₄ includes methyl- or ethyl- substituted by an optionally substituted 5- or 6-membered heteroaryl ring.

Preferred compounds include those wherein R₃ is hydrogen and R₄ is CrC₆ alkyl substituted by pyridyl. Particularly preferred at present are those compounds wherein R₃ is hydrogen and R₄ is pyrid-3-yl methyl or 1-(pyrid-3-yl)ethyl.

In another subclass of compounds with which the invention is concerned, R₃ and R₄ taken together with the nitrogen atom to which they are attached form an optionally substituted 5- or 6-membered ring.

The groups R₅ and R₆

In the compounds in accordance with the invention, R₅ and R₆ are independently selected from hydrogen, C₁-C₆ alkyl, aryl, aryl-(C₁-C₆)-alkyl, - NHR₇ -NC-Re)-R₉, -NH-(C=O)-R₁₀, -(C=O)-NH-R₁₁, -(C=O)-O-R₁₂, or halo; wherein R₇, Rs, Rg, Rio, Rn, and R₁₂ are independently selected from C₁-C₆ alkyl, aryl, aryl-(C-ι-C₆)-alkyl and heteroaryl.

It is presently preferred that both R₅ and R₆ are hydrogen.

Specific compounds with which the invention is concerned include those of the Examples.

The present invention may be employed in respect of a human or animal subject, more preferably a mammal, more preferably a human subject.

As used herein, the term "treatment" as used herein includes prophylactic treatment.

The compound of formula (I) may be used in combination with one or more additional drugs useful in the treatment of the disorders mentioned above, the components being in the same formulation or in separate formulations for administration simultaneously or sequentially.

It will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the causative mechanism and severity of the particular disease undergoing therapy. In general, a suitable dose for orally administrable formulations will usually be in the range of 0.1 to 3000 mg, once, twice or three times per day, or the equivalent daily amount administered by infusion or other routes. However, optimum dose levels and frequency of dosing will be determined by clinical trials as is conventional in the art.

The compounds with which the invention is concerned may be prepared for administration by any route consistent with their pharmacokinetic properties. The orally administrable compositions may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions. Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants for example potato starch, or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.

For topical application to the skin, the drug may be made up into a cream, lotion or ointment. Cream or ointment formulations which may be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.

The active ingredient may also be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the drug can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.

There are multiple synthetic strategies for the synthesis of the compounds (I) with which the present invention is concerned, but all rely on known chemistry, known to the synthetic organic chemist. Thus, compounds according to formula (I), can be synthesised according to procedures described in the standard literature and are well-known to the one skilled in the art. Typical literature sources are "Advanced organic chemistry", 4^th Edition (Wiley), J March, "Comprehensive Organic Transformation", 2^nd Edition (Wiley), R. C. Larock , "Handbook of Heterocyclic Chemistry", 2^nd Edition (Pergamon), A.R. Katritzky), review articles such as found in "Synthesis", "Ace. Chem. Res." , "Chem. Rev", or primary literature sources identified by standard literature searches online or from secondary sources such as "Chemical Abstracts" or "Beilstein". Such literature methods include those of the preparative Examples herein, and methods analogous thereto.

Scheme 1 represents a method known in the art of organic chemistry in general, by which the compounds of the present invention may be prepared:

Scheme 1 OC °C

pure

1eq R-SnBu₃, dioxane 2eq K₂CO₃, water 2eq NaIO₄

2 x 3eq RI-NH₂ 0.05eq PdCI₂(PPh₃)., 2 x 0.1 eq KMnO₄ t 130 °C 0 min at 100 °C, FCC dioxane / water NIVIP, 2 x 3h a

2 x 2 h at r.t., FCC catch & release on MP-TsOH 5% yield, >95% pure 54% yield, >95% pure

Cl no further purification, used crude °C

pure

EXAMPLES

The following examples illustrate the preparation of specific compounds of the invention and are not intended to be limiting of the full scope of the invention.

Examples 1 to 6 relate to the method indicated in Scheme 1.

Preparative Example 1 Urea Cyclisation Thieno[3,2-d]pyrimidine-2,4-diol

A solid mixture of methyl 3-aminothiophene-2-carboxylate (40.0 g, 254 mmol) and urea (99.4 g, 163 mmol) was stirred and heated to 180 ⁰C for 5 h. The now semi-fluid mixture was cooled to 75-80 °C before water (800 ml) was added. After stirring at room temperature for 2 h the formed precipitate was filtered off, washed with further water and dried at 40 ⁰C /. vac. Thieno[3,2- d]pyrimidine-2,4-diol was obtained as cream-coloured powder (37.15 g, 87% yield) of >95% purity. LC-MS: m/z = 169 [M+H⁺]; RT = 1.29 (LC-MS method 2)

¹H-NMR: δ_H (400 MHz, d₆-DMSO) 6.91 (1 H, d, J 5.02 Hz), 8.04 (1 H, d, J 5.02 Hz), 11.30 (2H, br s) Preparative Example 2 Hydroxy-Chloro exchange 2,4-Dichloro-thieno[3,2-d]pyrimidine

A mixture of Example 1 (20 g, 119 mmol) in phenylphosphonic dichloride (120 ml, 850 mmol) was stirred and heated to 180 ⁰C for 4 h. The resulting dark solution was cooled to 80 ⁰C and transferred slowly by pipette onto stirred ice / water (800ml). After an hour of vigorous stirring, the yellow-orange precipitate was filtered off, washed and dried at 40 ⁰C /. vac. The solid was dissolved in DCM (ca. 20 volumes). The solution was passed through a pad of silica and washed through with ethyl acetate : iso-hexane (1 :1 ). The filtrate was reduced /. vac. to yield 2,4-dichloro-thieno[3,2-d]pyrimidine (14.56 g, 60% yield) as a yellow crystalline product. LC-MS: m/z = 205 [M+H⁺]; RT = 4.36 (LC-MS method 2) 1H-NMR: δ_H (400 MHz, d₆-DMSO) 7.74 (1 H, d, J 5.52 Hz), 8.70 (1 H, d, J 5.52 Hz)

Preparative Example 3

Stille Coupling

2-Chloro-4-(1'-ethoxy-vinyl)-thieno[3,2-d]pyrimidine To a solution of Example 2 (3.0 g, 14.6 mmol) in 1 ,4-dioxane (200 mL) was added a solution of K₂CO₃ (4.0 g, 29.3 mmol) in water (40 mL). The almost clear solution was placed under nitrogen atmosphere and (1-Ethoxyvinyl)- tributylstannane (5.0 mL, 14.6 mmol) and PdCI₂(PPh₃)₂ (500 mg, 0.731 mmol). The mixture was heated to 100 ⁰C and stirred at 100 ⁰C for 30 min under nitrogen atmosphere. After cooling to room temp, the 1 ,4-dioxane was removed /. vac. The residue was re-dissolved in DCM (100 mL) and water (50 mL) and transferred to a separating funnel. The aqueous layer was extracted with DCM (30 mL), the combined organic layers were washed with water (50 mL) and sat. brine (30 mL), dried over Na₂SO₄ and evaporated to dryness /. vac. The residue was purified by flash column chromatography (50 g SiO₂ Isolute^® pre-wetted with 1 CV DCM) eluting with neat DCM collecting 12 mL fractions. Desired product eluted in fractions 10 to 20 with R_f = 0.31 (DCM). By-products eluted in fractions 6 to 9 (R_f = 0.59 in DCM) and fractions 21-31 ((Rf = 0.14 in DCM). Fractions 10 to 20 were combined and evaporated to dryness /. vac. providing 2.28 g (65% yield) 2-chloro-4-(1'-ethoxy-vinyl)- thieno[3,2-d]pyrimidine as yellow powder of 95% purity. LC-MS: m/z = 241 [IVHH⁺]; RT = 3.38 (LC-MS method 1) 1H-NMR: δ_H (400 MHz, d₆-DMSO) 1.49 (3H, t, J 7.03 Hz), 4.11 (2H, q, J 7.03 Hz), 4.88 (1 H, d, J 2.51 Hz), 5.76 (1 H, d, J 2.51 Hz), 7.59 (1 H, d, J 5.52 Hz), 8.62 (1 H, d, J 6.02 Hz)

Preparative Example 4

Ethyl 2-chloro-thieno[3,2-d]pyrimidine-4-carboxylate NaIO₄ (1.07 g, 4.99 mmol) was suspended in water (13 mL) and sonicated until a clear solution (pH ~4) was obtained. This solution was added to a solution of Example 3 (600 mg, 2.49, mmol) in 1 ,4-dioxane (40 mL). KMnO₄ (40 mg, 0.249 mmol) was added and the reaction mixture was stirred at room temp for 2 h. Progress of the reaction was checked by TLC (DCM). If remaining starting material was detected, further KMnO₄ (40 mg, 0.249 mmol) was added and the reaction mixture was stirred at room temp for further 2 h. The mixture was adjusted to pH 7-8 with sat. aqueous K₂CO₃ solution (1-2 mL). The precipitate was filtered off and the residue was rinsed thoroughly with DCM (4 x 20 mL). The combined filtrates were washed with water, dried over Na₂SO₄ and evaporated to dryness /. vac. The residue was purified by flash column chromatography (10 g SiO₂ Isolute^®, pre-wetted with 1CV DCM) eluting with neat DCM collecting 10 mL fractions. Ethyl 2-chloro-thieno[3,2- d]pyrimidine-4-carboxylate (325 mg, 54% yield) was isolated as colourless powder of 95% purity. LC-MS: m/z = 243 [M+H⁺]; RT = 2.92 (LC-MS method 1 )

¹H-NMR: δ_H (400 MHz₁ d₆-DMSO) 1.41 (3H, t, J 7.03), 4.50 (2H, q, J 7.03 Hz), 7.72 (1 H, d, J 5.52 Hz), 8.76 (1 H, d, J 6.02 Hz)

General Procedure 1

To a solution of Example 4 (200 mg, 0.824 mmol) in NMP (4 mL) was added R1 amine (2.47 mmol). The reaction mixture was shaken at 130 ⁰C for 3 h. Further R1 amine (2.47 mmol) was added and shaking at 130 ⁰C was continued for further 3 h. After cooling to room temperature the mixture was transferred to a separating funnel with DCM (20 mL) and washed with water (3 ^χ 10 mL). The organic layer was loaded onto MP-TsOH (1g, Argonaut, pre- wetted with neat DCM). The resin was rinsed with DCM (30 mL) and 2-amino substituted thieno[3,2-d]pyrimidine-4-carboxamide eluted with 0.2 N NH₃ in DCM / MeOH (prepared from 2 mL 7N NH₃ in MeOH + 60 mL DCM + 8 mL MeOH) as intensively yellow fractions. Fractions were combined and evaporated to dryness /. vac.

The crude intermediate was dissolved in 12Λ/ aq HCI (10 mL) and evaporated to dryness /^". vac. at a bath temperature of 80 ⁰C. The residue was re- dissolved in 12Λ/ aq HCI (12 mL) and stirred at 100 ⁰C for 14 h. The reaction mixture was evaporated to dryness /. vac. at a bath temperature of 80 ⁰C, re- dissolved in MeOH and evaporated to dryness again to obtain 2-amino substituted thieno[3,2-d]pyrimidine-4-carboxylic acid as a yellow-brownish solid.

The crude acid was dissolved in MeOH (20 mL) and thionylchloride (360 μL, 4.94 mmol) was added dropwise at room temp. The mixture was stirred at 60 ⁰C for 2.5 h and then evaporated to dryness /. vac. The residue was dissolved in DCM / MeOH (10:1 , 100 mL), washed with sat aqueous NaHCO₃ (50 mL) and sat. brine (50 mL), dried over Na₂SO₄ and evaporated to dryness /^'. vac. The residue was purified by column chromatography (1O g SiO₂ Isolute^® pre- wetted with 1 CV DCM / MeOH 50:1 ) eluting with DCM / MeOH 50:1 collecting 5 mL fractions. Fractions were combined and evaporated to dryness /. vac. providing >50% yield of 2-amino substituted thieno[3,2-d]pyrimidine-4-methyl ester as a yellow solid of >95% purity.

The following analogues were prepared using general procedure 1.

Preparative Example 5

2-[(Pyndin-3-ylmethyl)-amino]-thieno[3,2-d]pyrimidine-4-carboxyl[c acid methyl ester

LC-MS: m/z = 301 [M+H⁺]; RT = 2.44 (LC-MS method 1 ) ¹H-NMR: δ_H (400 MHz, d₆-DMSO) 3.97 (3H, s), 4.60 (2H, d, J 6.02 Hz), 7.28 (1 H, d, J 5.52 Hz), 7.31 (1 H, m), 7.77 (1 H, m), 8.14 (1 H, br s), 8.34 (1 H, d, J 5.52 Hz), 8.42 (1 H, m), 8.60 (1 H, br s)

Preparative Example 6

2-(1-Pyridin-3-yl-ethylamino)-thieno[3,2-d]pyrimidine-4-carboxyIic acid methyl ester

LC-MS: m/z = 315 [M+H⁺]; RT = 2.47 (LC-MS method 1 ) 1H-NMR: δ_H (400 MHz, d₆-DMSO) 1.51 (3H, d, J 7.03 Hz), 3.96 (3H, s), 5.21 (1 H, m), 7.24 (1 H, d, J 5.52 Hz), 7.31 (1 H, m), 7.83 (1 H, dt, J 7.53, 2.01 Hz), 8.19 (1 H, br s), 8.31 (1 H, d, J 5.52 Hz), 8.39 (1 H, dd, J 5.02, 1.51 Hz), 8.66 (1 H, d, J 2.01 Hz)

General Procedure 2

To a solution of the corresponding example 5 or 6 (0.50 mmol) in 1 ,4-dioxane (4 imL) was added the required amine (>10 eq). The mixture was stirred under reflux for 1 h. After cooling to room temp the mixture was evaporated to dryness /. vac. and the residue was purified by flash column chromatography (5 g SiO₂ Isolute^® pre-wetted with EtOAc). Crude material was loaded as a solution in a minimum neat DCM and product was eluted with neat EtOAc collecting 6 mL fractions. Fractions were combined and evaporated to dryness /. vac. providing >80% yield 2-amino substituted thieno[3,2-d]pyrimidin-4- carboxamide as yellow powder of >95% purity.

The following analogues were prepared using general procedure 2.

Example 7

[2-(1-Pyridin-3-yI-ethylamino)-thieno[3,2-d]pyrimidin-4-yl]-pyrrolidin-1-yl- methanone

LC-MS: m/z = 345 [M+H⁺]; RT = 2.52 (LC-MS method 1 ) 1H-NMR: δ_H (400 MHz, d₆-DMSO) 1.52 (3H, d, J 7.03 Hz), 1.81 (4H, m), 3.52 (2H, m), 3.82 (2H, m), 5.17 (1 H, m), 7.19 (1 H, d, J 5.52 Hz), 7.31 (1 H, m), 7.80 (1 H₁ dt, J 8.03, 2.01 Hz), 7.84 (1 H, d, J 7.53 Hz), 8.24 (1 H, d, J 5.52 Hz), 8.40 (1 H, dd, J 5.02, 1.51 Hz), 8.63 (1 H, d, J 2.51 Hz)

Example 8 2-[(Pyridin-3-yImethyl)-amino]-thieno[3,2-d]pyrimidine-4-carboxylic acid ethylamide

LC-MS: m/z = 314 [M+H⁺]; RT = 1.59 (LC-MS method 1 )

¹H-NMR: δ_H (400 MHz, d₆-DMSO) 1.15 (3H, t, J 7.07 Hz), 3.38 (2H, q, J 7.07 Hz), 4.70 (2H, br s), 7.22 (1 H, d, J 5.56 Hz), 7.34 (1 H, dd, J 7.83, 4.80 Hz),

7.80 (2H, m), 8.32 (1 H, d, J 5.56 Hz), 8.43 (1 H, d, J 4.55 Hz), 8.64 (1 H, s),

8.81 (1H, br s)

Example 9

{2-[(Pyridin-3-ylmethyl)-amino]-thieno[3,2-d]pyrimidfn-4-yl}-pyrrolidin-1- yl-methanone

LC-MS: m/z = 340 [M+H⁺]; RT = 2.42 (LC-MS method 1) 1H-NMR: δ_H (400 MHz, d₆-DMSO) 1.81 (4H, m), 3.54 (2H, t, J 6.53 Hz), 3.78 (2H, br s), 4.60 (2H, d, J 6.02 Hz), 7.23 (1 H, d, J 5.52 Hz), 7.32 (1 H, m), 7.74 (1 H, d, J 7.53 Hz), 7.88 (1 H, t, J 6.02 Hz), 8.27 (1 H, d, J 6.02 Hz), 8.43 (1 H, dd, J 4.52, 1.51 Hz), 8.57 (1 H, s)

Example 10

2-[(Pyridin-3-ylmethyl)-amino]-thieno[3,2-d]pyrimidine-4-carboxylic acid cyclopropylamide

LC-MS: m/z = 326 [M+H⁴]; RT = 2.39 (LC-MS method 1 ) 1H-NMR: δ_H (400 MHz, d₆-DMSO) 0.68 (2H, m), 0.77 (2H, m), 2.90 (1 H, m), 4.67 (2H, d, J 6.02 Hz), 7.22 (1 H, d, J 5.52 Hz), 7.31 (1 H, m), 7.76 (2H, m), 8.32 (1 H, d, J 5.52 Hz), 8.42 (1 H, br d, J 4.52 Hz), 8.61 (2H, m)

Example 11 2-[(Pyπdin-3-ylmethyl)-amino]-thieno[3,2-d]pyrimidine-4-carboxylic acid isopropylamide

LC-MS: m/z = 328 [M+H*]; RT = 1.74 (LC-MS method 1 )

¹H-NMR: δ_H (400 MHz, d₆-DMSO) 1.22 (6H, d, J 6.57 Hz), 4.10 (1 H, m), 4.65 (2H, br d, J 5.56 Hz), 7.23 (1 H, d J 5.31 Hz), 7.34 (1 H, m), 7.80 (1 H, dm, J 7.83 Hz), 7.89 (1 H, m), 8.32 (1H, d, J 8.34 Hz), 8.33 (1 H, d, J 5.56 Hz), 8.43 (1 H, dd, J 4.55, 1.26 Hz), 8.64 (1 H, s)

Example 12 2-[(Pyridin-3-yImethyl)-amino]-thieno[3,2-d]pyrimidine-4-carboxylic acid cyclopentylamide

LC-MS: m/z = 354 [M+H⁺]; RT = 1.95 (LC-MS method 1)

¹H-NMR: δ_H (400 MHz, d₆-DMSO) 1.58 (4H, m), 1.71 (2H, m), 1.93 (2H, m), 4.23 (1 H, m), 4.64 (2H, d, J 5.56 Hz), 7.23 (1 H, d, J 5.56 Hz), 7.32 (1 H, dd, J 7.83, 4.80 Hz), 7.77 (1 H, dt, J 7.83, 1.90 Hz), 7.89 (1 H, br s), 8.33 (1 H, d, J 5.56 Hz), 8.35 (1 H, m), 8.42 (1 H, dd, J 4.55, 1.52 Hz), 8.62 (1 H, s)

Example 13

2-[(Pyridin-3-ylmethyl)-amino]-thieno[3,2-d]pyrimidine-4-carboxylic acid methylamide

LC-MS: m/z = 300 [M+H⁺]; RT = 1.46 (LC-MS method 1 ) 1H-NMR: δ_H (400 MHz, d₆-DMSO) 2.88 (3H, d, J 5.05 Hz), 4.72 (2H, m), 7.21 (1 H, d, J 5.56 Hz), 7.34 (1 H₁ dd, J 7.83, 4.80 Hz), 7.79 (2H, dm, J 7.83 Hz), 832 (1 H, d, J 5.56 Hz), 8.43 (1 H, dd, J 4.55, 1.01 Hz), 8.63 (1 H, s), 8.84 (1 H, br s)

Example 14 2-[(Pyridin-3-ylmethyl)-amino]-thieno[3,2-d]pyrimidine-4-carboxylic acid phenethyl -amide

LC-MS: m/z = 390 [M+H⁺]; RT = 2.07 (LC-MS method 1)

¹H-NMR: δ_H (400 MHz, d₆-DMSO) 2.88 (2H, t, J 7.33), 3.58 (2H, m), 4.69 (2H, m), 7.17-7.34 (7H, m), 7.75 (1H, dm, J 7.83 Hz), 7.82 (1H, br s), 8.33 (1H, d, J

5.56 Hz), 8.43 (1 H, dd, J 4.80, 1.26 Hz), 8.61 (1 H, m), 8.85 (1 H, br s)

Example 15

2-[(Pyridin-3-yImethyI)-amino]-thieno[3,2-d]pyrimidine-4-carboxyiic acid cyclohexylamide

LC-MS: m/z = 368 [M+H⁺]; RT = 2.10 (LC-MS method 1) 1H-NMR: δ_H (400 MHz, d₆-DMSO) 1.13-1.45 (5H, m), 1.60 (1 H, m), 1.72 (2H, m), 1.82 (2H₁ m), 3.78 (1 H, m), 4.64 (2H, m), 7.23 (1 H, d, J 5.05 Hz), 7.32 (1 H, m), 7.78 (1 H, d, J 7.33 Hz), 7.90 (1 H, br s), 8.32 (2H, m), 8.42 (1 H, br d, J 4.04 Hz), 8.62 (1 H, s)

Example 16

2-[(Pyridin-3-ylmethyl)-amino]-thieno[3,2-d]pyrimidine-4-carboxylic acid benzylamide

LC-MS: m/z = 376 [M+H⁺]; RT = 1.99 (LC-MS method 1 ) 1H-NMR: δ_H (400 MHz, d₆-DMSO) 4.56 (2H, d, J 6.32 Hz), 4.71 (2H, br s), 7.21-7.31 (3H, m), 7.32-7.35 (4H, m), 7.78 (1 H, dm, J 7.83 Hz), 7.84 (1 H, br s), 8.33 (1 H, d, J 5.56 Hz), 8.41 (1 H, m), 8.63 (1 H, s), 9.37 (1 H, br s)

Example 17

2-[(Pyridin-3-ylmethyl)-amino]-thieno[3,2-d]pyrimidine-4-carboxylic acid (2-methoxy-ethyI)-amide

LC-MS: m/z = 344 [M+H⁺]; RT = 2.35 (LC-MS method 1 ) 1H-NMR: δ_H (400 MHz, d₆-DMSO) 3.30 (2H, m, under DMSO-H₂O), 3.51 (5H, m), 4.67 (2H, br d, J 6.02 Hz), 7.23 (1 H, d, J 5.52 Hz), 7.32 (1 H, m), 7.78 (1 H, dt, J 7.53, 2.01 Hz), 7.85 (1 H, t, J 6.53 Hz), 8.33 (1 H, d, J 5.52 Hz), 8.42 (1 H, dd, J 5.02, 1.51 Hz), 8.63 (2H, br m)

Example 18

2-[(Pyridin-3-ylmethyl)-amino]-thieno[3,2-d]pyrimidine-4-carboxylic acid (pyridin-3-ylmethyl)-amide

LC-MS: m/z = 377 [M+H⁺]; RT = 2.46 (LC-MS method 1 ) 1H-NMR: δ_H (400 MHz, d₆-DMSO) 4.58 (2H, d, J 6.02 Hz), 4.71 (2H, d, J 5.52 Hz), 7.22 (1 H, d J 5.52 Hz), 7.29 (1 H, m), 7.37 (1 H, m), 7.76 (3H, m), 8.33 (1 H, d, J 5.52 Hz), 8.42 (1 H, dd, J 5.02, 1.51 Hz), 8.48 (1H, dd, J 5.02, 1.51 Hz), 8.58 (1 H, m), 8.63 (1 H, m), 9.41 (1 H, br s)

Example 19

Piperidin-1-yl-{2-[(pyridin-3-ylmethyl)-amino]-thieno[3,2-d]pyrimidin-4- yl}-methanone

LC-MS: m/z = 354 [M+H⁺]; RT = 1.66 (LC-MS method 1 ) 1H-NMR: δ_H (400 MHz, d₆-DMSO) 1.31-1.48 (2H, m), 1.58 (4H, m), 3.41 (2H, m), 3.61 (2H, m), 4.57 (2H, d, J 6.06 Hz), 7.25 (1 H, d, J 5.56 Hz), 7.32 (1 H, m), 7.73 (1 H, br d, J 7.33 Hz), 7.96 (1 H, m), 8.28 (1 H, d, J 5.56 Hz), 8.42 (1 H, m), 8.56 (1 H, s)

Crude 2-amino substituted thieno[3,2-d]pyrimidine-4-methyl ester, before purification by column chromatography, could also be used as starting material for the amide formation, however, this left a more difficult final purification. Using this procedure the following product was isolated together with the 2-NH₂ analogue as by-product.

Example 20

2-(1-Pyridin-3-yl-ethylamino)-thieno[3,2-d]pyrimidine-4-carboxylic acid cyclopropylamide

LC-MS: m/z = 340 [M+H⁺]; RT = 2.46 (LC-MS method 1) 1H-NMR: δ_H (400 MHz, CJ₆-DMSO) 0.67 (2H, m), 0.78 (2H, m), 1.51 (3H, d, J 7.03 Hz), 2.90 (1 H, m), 5.33 (1 H, br m), 7.19 (1 H, d, J 5.52 Hz), 7.31 (1 H, m), 7.78 (11-1, br m), 7.84 (1 H, dt, J 8.03, 2.01 Hz), 8.30 (1 H, d, J 5.52 Hz), 8.39 (1 H, dd, J 4.52, 1.51 Hz), 8.49 (1 H, br d, J 4.52 Hz), 8.69 (1 H, br d, J 2.01 Hz)

Example 21 2-Amino-thieno[3,2-d]pyrimidine-4-carboxylic acid cyclopropylamide

LC-MS: m/z = 235 [M+H⁺]; RT = 2.34 (LC-MS method 1 ) 1H-NMR: δ_H (400 MHz, d₆-DMSO) 0.69 (2H, m), 0.76 (2H, m), 2.91 (1 H, m), 6.61 (2H₁ s), 7.19 (1 H, d, J 5.52 Hz), 8.31 (1H, d, J 5.52 Hz), 8.59 (1H, d, J 4.52 Hz)

Example 22. (R)-[2-(1 -Pyridin-3-yl-ethyIamino)-thieno[3,2-d]pyrimidin-4- yl]-pyrrolidin-1-yl-methanone

Prepared as per example 7, using (R)-1-Pyridin-3-yl-ethylamine, which was itself obtained via the resolution protocol described in Smith et al., Journal of the American Chemical Society, 1973, 95, pp 811-818. Example 22 had spectroscopic properties identical to the racemic example 7. Chiral LC RT = 45.40 min

Example 23. (S)-[2-(1 -Pyridin-3-yl-ethylamino)-thieno[3,2-d]pyrimidin-4- yl]-pyrrolidin-1 -yl-methanone

Prepared as per example 7, using (S)-1-Pyridin-3-yl-ethylamine, which was itself obtained via the resolution protocol described in Smith et. al., Journal of the American Chemical Society, 1973, 95, pp 811-818. Example 23 had spectroscopic properties identical to the racemic example 7. Chiral LC RT = 30.40 min

General Procedures

All reagents obtained from commercial sources were used without further purification. Anhydrous solvents were obtained from commercial sources and used without further drying. Flash chromatography was performed with prepacked silica-gel cartridges (Strata Si-1 ; 61 A, Phenomenex, Cheshire, UK or IST Flash II, 54 A, Argonaut, Hengoed, UK). Thin layer chromatography was conducted with 5 x 10 cm plates coated with Merck Type 60 F_2S4 silica-gel. Microwave heating was performed with a Biotage Initiator™ 2.0 instrument.

The compounds of the present invention were characterized by liquid chromatography-mass spectroscopy (LC-MS) using the following methods.

LC-MS Method 1

Instrument: Waters 2695 pump and 2700 sample manager Waters ZQ2000, M/z range 100 to 900 amu

Column: Gemini 5μm, C18 110A, 30 mm x 2mm from Phenomenex. Pt no 00A-4435-B0

Temperature: Ambient Mobile Phase: A - Water + 10 mMol / ammonium formate + 0.04% (v/v) formic acid at pH ca 3.5 B - 100% Acetonitrile + 0.04% (v/v) formic acid

Injection Volume 1OuL Gradient:

Detection: UV detection from 220 to 400nm (1 :3 split MS to UV)

LC-MS Method 2

Column: Gemini 5μm, C18 110A, 30 mm x 2mm from

Phenomenex. Pt no 00A-4435-B0

Temperature: Ambient Mobile Phase: A - Water + 10 mMol / ammonium formate + 0.04% (v/v) formic acid at pH ca 3.5

B - 100% Acetonitrile + 0.04% (v/v) formic acid Injection Volume 5 uL Gradient:

Detection: UV detection from 220 to 400nm

Nuclear magnetic resonance (NMR) analysis was performed with a Bruker DPX400 spectrometer and proton NMR spectra were measured at 400 MHz. The spectral reference was the known chemical shift of the solvent. Proton NMR data is reported as follows: chemical shift (δ) in ppm, followed by the integration, the multiplicity (where s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, dd = doublet of doublets and br = broad), and the coupling constant rounded to the nearest 0.1 Hz.

Some compounds of the invention were purified by preparative HPLC. These were performed on a Waters FractionLynx MS autopurification system, with a Gemini^® 5 μm C18(2), 100 mm * 20 mm i.d. column from Phenomenex, running at a flow rate of 20 cm³min^"1 with UV diode array detection (210-400 nm) and mass-directed collection. Gradients used for each compound are shown in Table 1.

At pH 4: solvent A = 10 mM ammonium acetate in HPLC grade water + 0.08% v/v formic acid. Solvent B = 95% v/v HPLC grade acetonitrile + 5% v/v solvent A + 0.08% v/v formic acid. At pH 9: solvent A = 10 mM ammonium acetate in HPLC grade water + 0.08% v/v ammonia solution. Solvent B = 95% v/v HPLC grade acetonitrile + 5% v/v solvent A + 0.08% v/v ammonia solution.

The mass spectrometer was a Waters Micromass ZQ2000 spectrometer, operating in positive or negative ion electrospray ionisation modes, with a molecular weight scan range of 150 to 1000.

Table 1. Preparative HPLC gradients

% Solvent B for Example No.

0.0 5 5 5 5 5 5

0.5 6 15 15 30 10 25

7.0 25 30 40 40 20 50

7.5 95 95 95 95 95 95

9.5 95 95 95 95 95 95

10 5 5 5 5 5 5

Those compounds of the invention were analyded by chiral HPLC using the method detailed below.

Instrument: Perkin Elmer Series 250 HPLC, equipped with a Perkin Elmer 785A UVΛ/isible detector

Column: ChiralPak AD-H column, 250 x 4.6 mm

Temperature: 30 ⁰C

Mobile Phase: 80% isohexane, 20% iso-propyl alcohol, 0.1 %

Diethylamine, 1 mL/min, 60 min run time Detection: UV detection at 265 nM

Injection Volume 10μL

IUPAC chemical names were generated using AutoNom Standard. Assay Description

The use of a Fluorometric Imaging Plate Reader (FLIPR) to measure calcium flux in Adenosine-receptor expressing cells is a well-established technique. In this assay calcium flux is triggered by receptor activation and measured through the fluorescence of an incorporated calcium-sensitive dye. The potencies shown were determined using expressed human adenosine A_2B receptors in mammalian cell lines. Selectivity values were obtained by using mammalian cell lines expressing the human adenosine A-i, A_2A and A₃ receptors. Compound potency was determined from dose response curves and are reported as IC₅₀ values.

The compounds tested in the above assay were assigned to one of two activity ranges, namely A = IC₅₀ <500 nM, or B = IC₅₀ >500 nM, as indicated in Table 2 below.

Table 2

Example Activity

7 B

8 B

9 A

10 B

11 A

12 B

13 B

14 A

15 A

16 A

17 B

18 B

19 B

20 A

21 B

22 B

23 A

Claims

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

(i)

wherein

Ri and R₂ are independently selected from hydrogen, or optionally substituted CrC₆ alkyl, Ci-C₆ alkoxy-(Ci-C₆)-alkyl, C₃-C₈ cycloalkyl, aryl, heteroaryl, aryl- (Ci-C₆)-alkyl, or heteroaryl-(C₁-C₆)-alkyl;

or Ri and R₂ taken together with the nitrogen atom to which they are attached form an optionally substituted 5- or 6-membered ring;

R₃ and R₄ are independently selected from hydrogen, or optionally substituted Ci-C₆ alkyl, Ci-C₆ alkoxy-(Ci-C₆)-alkyl, C₃-C₈ cycloalkyl, aryl, heteroaryl, aryl- (d-C₆)-alkyl, or heteroaryl-(C_rC₆)-alkyl;

R₅ and R₆ are independently selected from hydrogen, C_rC₆ alkyl, aryl, aryl- (C_rC₆)-alkyl, -NHR₇ -N(-R₈)-R₉, -NH-(C=O)-R₁₀, -(C=O)-NH-Rn , -(C=O)-O- R-_I2, or halo; and Rz, Rβ_> R9, R10, Rii_> and R12 are independently selected from Ci-C₆ alkyl, aryl, aryl-(Ci-C₆)-alkyl and heteroaryl.

2. A compound as claimed in claim 1 wherein Ri is hydrogen, C₁-C₆ alkyl, Ci-C₆ alkoxy-(Ci-C₆)-alkyl, C₃-C₈ cycloalkyl, aryl-(Ci-C₆)-alkyl, or heteroaryl-

(Ci-C₆)-alkyl.

3. A compound as claimed in claim 1 or claim 2 wherein Ri is hydrogen, methyl, ethyl, isopropyl, 2-methoxy-ethyl, cyclopropyl, cyclopentyl, cyclohexyl, benzyl, 2-phenyl-ethyl, or pyrid-3-yl-methyl.

4. A compound as claimed in any of the preceding claims wherein Ri is methyl or ethyl.

5. A compound as claimed in any of the preceding claims wherein R₂ is hydrogen, Ci-C₆ alkyl, Ci-C₆ alkoxy-(Ci-C₆)-alkyl, C₃-C₈ cycloalkyl, aryl-(Cr C₆)-alkyl, or heteroaryl-(Ci-C₆)-alkyl.

6. A compound as claimed in claim 5 wherein R₂ is hydrogen, methyl, ethyl, isopropyl, 2-methoxy-ethyl, cyclopropyl, cyclopentyl, cyclohexyl, benzyl,

2-phenyl-ethyl, or pyrid-3-yl-methyl.

7. A compound as claimed in claim 5 or claim 6 wherein R₂ is methyl or ethyl.

8. A compound as claimed in claim 1 wherein Ri and R₂ taken together with the nitrogen atom to which they are attached form an optionally substituted 5- or 6-membered ring.

9. A compound as claimed in claim 8 wherein Ri and R₂ taken together with the nitrogen atom to which they are attached form a pyrrolidine or piperidine ring optionally substituted by fluoro, chloro, bromo, cyano, methyl, trifluoromethyl, ethyl, hydroxyl, hydroxymethyl, or hydroxyethyl.

10. A compound as claimed in claim 8 or claim 9 wherein Ri and R₂ taken together with the nitrogen atom to which they are attached form pyrrolidin-1-yl or piperidin-1-yl.

11. A compound as claimed in any of the preceding claims wherein R₃ is hydrogen or heteroaryl-(C₁-C₆)-alkyl.

12. A compound as claimed in any of the preceding claims wherein R₄ is hydrogen or heteroaryl-(CrC₆)-alkyl.

13. A compound as claimed in any of claims 1 to 10 wherein R₃ is hydrogen and R₄ is heteroaryl-(Ci-C₆)-alkyl.

14. A compound as claimed in any of the preceding claims wherein R₅ is hydrogen.

15. A compound as claimed in any of the preceding claims wherein R@ is hydrogen.

16. A compound of formula (II) or a pharmaceutically acceptable salt, hydrate or solvate thereof:

(H)

wherein

R-i is -NH-R₃ or an optionally substituted monocyclic 5- or 6-membered nitrogen-containing ring coupled via a nitrogen atom; R₂ is CrC₆ alkyl substituted by an optionally substituted 5- or 6-membered heteroaryl ring; and

R₃ is hydrogen, Ci-C₆ alkyl, Ci-C₆ alkoxy-(Ci-C₆)-alkyl, C₃-C₈ cycloalkyl, aryl- (Ci-C₆)-alkyl, or heteroaryl-(C_rC₆)-alkyl.

17. A compound as claimed in claim 16 wherein Ri is an optionally substituted monocyclic 5- or 6-membered nitrogen-containing ring coupled via a nitrogen atom.

18. A compound as claimed in claim 16 or claim 17 wherein Ri is a pyrrolidine or piperidine ring optionally substituted by fluoro, chloro, bromo, cyano, methyl, trifluoromethyl, ethyl, hydroxy!, hydroxymethyl, or hydroxyethyl.

19. A compound as claimed in any of claims 16 to 18 wherein Ri is pyrrolidin-1-yl or piperidin-1-yl.

20. A compound as claimed in claim 16 wherein Ri is CrC₆ alkylamino or C₃-C₈ cycloalkylamino.

21. A compound as claimed in claim 20 wherein Ri is methylamino, ethylamino, isopropylamino, or cyclopropylamino.

22. A compound as claimed in any of claims 16 to 21 wherein R₂ is methyl- or ethyl- substituted by an optionally substituted 5- or 6-membered heteroaryl ring.

23. A compound as claimed in any of claims 16 to 21 wherein R₂ is methyl- or ethyl- substituted by a 5- or 6-membered heteroaryl ring optionally substituted by fluoro, chloro, bromo, cyano, methyl, trifluoromethyl, ethyl, hydroxyl, hydroxymethyl, or hydroxyethyl.

24. A compound as claimed in any of claims 16 to 21 wherein R₂ is C₁-C₆ alkyl substituted by pyridyl.

25. A compound as claimed in any of claims 16 to 21 wherein R₂ is pyrid-3- ylmethyl or 1-(pyrid-3-yl)ethyl.

26. A pharmaceutical composition comprising a compound as claimed in any of the preceding claims and a pharmaceutically acceptable carrier.

27. The use of a compound as claimed in any of claims 1 to 25 in the manufacture of a medicament for the treatment of disorders mediated by the adenosine A_2B receptor.

28. A method of treating a disorder mediated by the adenosine A_2B receptor comprising the administration to a subject suffering such a disorder an effective amount of a compound as claimed in any of claims 1 to 25.

29. The use as claimed in claim 27, or a method as claimed in claim 28 wherein the disorder mediated by the adenosine A₂₆ receptor is nociception, asthma, COPD, inflammatory disorders, diabetes, diabetic retinopathy, or cancer.