ACETYLENE COMPOUNDS
The present invention is concerned with certain adenosine derivatives having an acetylene group attached to the 5' position by a linker group, which are adenosine A1 agonists, and to their use in therapy. In particular, it is concerned with the use of these adenosine derivatives in treating conditions where there is an advantage in decreasing plasma free fatty acid concentration, or reducing heart rate or which subject is suffering from or susceptible to ischaemic heart disease, peripheral vascular disease or stroke or which subject is suffering pain, a CNS disorder, sleep apnoea or emesis.
A variety of compounds which are agonists at the adenosine A1 receptor have been described in the art. These include compounds described in published patent applications W099/24449, WO99/24450, W099/24451 , WO97/43300, W098/16539, WO98/04126, WO98/01459, EP0322242, GB2226027, EP222330, WO98/08855, WO94/17090, W099/67262 and WO00/23447.
The present inventors have surprisingly found that adenosine derivatives with certain acetylene groups attached to the 5' position by a linker group exhibit adenosine A1 agonist activity.
The present invention provides compounds of formula (I):
R1
(I) wherein X represents O or CH2;
Z represents (CH2)P or CH2OCH2 wherein p is 1 , 2 or 3;
R1 represents:
(i) -alkn-C3-9cycloalkyl or -alknC3.9cycloalkenyl, said cycloalkyl or cycloalkenyl group being optionally substituted by one or more substituents selected from OH, halogen, d. 6alkyl, C^alkoxy, C2-6alkenyloxy, C2-6aIkynyloxy and phenyl, wherein "alk" represents C^
3alkylene and n represents 0 or 1 , and said "alk" group may be optionally substituted by a
C3-6cycloalkyl group;
(ii) a phenyl group optionally substituted by one or more substituents selected from halogen, CF3, cyano, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C1-6alkoxy, C1-6alkylOH, CO2H and C02C1-6 alkyl;
(iii) a C .yheterocyclic group containing at least one heteroatom selected from O, N or S, and optionally substituted by one or more substituents selected from: OH, C^alkyl, Cι_ 6alkoxy, CO2C1-4alkyl, COC1-4alkyl, CO2aryl or CO2alknC3-6cycloalkyl, wherein "alk" represents C1-3alkylene and n represents 0 or 1 ;
(iv) a straight or branched Chalky! group optionally substituted by one or more groups selected from phenyl, halogen, hydroxy, C1-6alkoxy and C3-7 cycloalkyl, wherein one or more carbon atoms of the C1-12alkyl group may be optionally replaced by a group independently selected from S(=0)n (where n is 0, 1 or 2) and N; or (v) a fused bicyclic ring
wherein A represents C
4-ecycloalkyl or phenyl and B represents phenyl optionally substituted by C
1-3alkyl, and the bicyclic ring is attached to the purine-6-amino moiety via a ring atom of ring A;
R2 represents C1-3alkyl, halogen, hydrogen or C1-3alkoxy group;
R3 and R4 independently represent hydrogen or a C1-6alkyl group; and pharmaceutically acceptable derivatives thereof.
Further aspects of the invention include:
A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable derivative thereof together with a pharmaceutical carrier and/or excipient.
Use of a compound of formula (I) or a pharmaceutically acceptable derivative thereof for the manufacture of a medicament for the treatment of a patient suffering from a condition where there is an advantage in decreasing plasma free fatty acid concentration, or reducing heart rate.
Use of a compound of formula (I) or a pharmaceutically acceptable derivative thereof for the manufacture of a medicament for the treatment of a patient suffering from or susceptible to ischaemic heart disease, peripheral vascular disease or stroke or which subject is suffering pain, a CNS disorder, sleep apnoea or emesis.
A method of treating a patient suffering from a condition where there is an advantage in decreasing plasma free fatty acid concentration, or reducing heart rate comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof.
A method of treating a patient suffering or susceptible to ischaemic heart disease, peripheral vascular disease or stroke or which subject is suffering pain, a CNS disorder, sleep apnoea, or emesis comprising administering a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof.
Conveniently, R1 may represent alknC3-9cycloalkyl wherein n is 0 or 1 and the said cycloalkyl is either unsubstituted or substituted by one to four substituents selected from OH, halogen, C1-6alkyl, C1-6alkoxy, C -3alkenyloxy, C3-6cycloalkyl and phenyl. Preferably the alknC3-9cycloalkyl group is unsubstituted or monosubstituted on the cycloalkyl moiety by OH, C1-3alkyl, C1-3alkoxy or phenyl, or is mono- or disubstituted by halogen, for example fluorine. Preferably the alknC3-9cycloalkyl group is unsubstituted or monosubstituted on the cycloalkyl moiety by OH or C -3alkyl or C1-3alkoxy, more preferably OH or C1-3alkoxy. Preferably n is zero. Preferably the cycloalkyl ring has 3 to 8 carbon atoms, more preferably 5 or 6 carbon atoms. More preferably the alknC3-9cycloalkyl group is hydroxycyclopentyl or C1-3alkoxycyclohexyl, most preferably C1-3alkoxycyclohexyl.
Conveniently, R1 may represent alknC3-9cycloalkenyl wherein n is 0 or 1 and the said cycloalkenyl is either substituted by at least one substituent selected from OH, halogen, C-i. 6alkyl, C1-6alkoxy, and phenyl, or is unsubstituted. Preferably n is zero. More preferably, the cycloalkenyl group is unsubstituted. Preferably the cycloalkenyl ring has 5 or 6 carbon atoms, more preferably the ring is cyclohexenyl.
Conveniently, R1 may represent phenyl optionally substituted by one, two or three substituents, alternatively one or two substitutents, selected from halogen, for example chlorine and fluorine, and C1-6alkyl. More preferably R1 may represent phenyl optionally substituted by one or two substitutents selected from chlorine, fluorine and methyl. Preferably the phenyl is disubstituted. Preferably the phenyl is disubstituted in the 2,3 or 2,4 or 2,5 positions. In a yet further aspect R1 represents phenyl optionally substituted by one or more substituents selected from halogen, e.g. chlorine and fluorine, and C1-6alkyl;
RR2 rreepprreessents hydrogen or halogen; and R3 and R4 independently represent hydrogen or C1-6alkyl.
Conveniently, R
1 may represent a substituted or unsubstituted
group, the substitutent being selected from C
1-6alkyl, C0
2C
1-4alkyl and C0
2alk
nC
3^cycloalkyl. Preferably the heterocyclic ring is 6 membered and more preferably contains only one O or N heteroatom. Conveniently, the heterocyclic group is unsubstituted or, when substituted, the substituent is CO
2C
1-4alkyl or CO
2alk
nC
3-6cycloalkyl, the heteroatom is N and the substituent is directly attached to said ring nitrogen atom. Preferably when the heterocyclic group is substituted the substituent is CO
2C
1- alkyl, the heteroatom is N and the substituent is directly attached to said ring nitrogen atom. Most preferably when the heterocyclic group is unsubstituted the heteroatom is O. Most preferably when the heterocyclic ring is substituted the heteroatom is N.
Conveniently R
1 may represent a straight or branched C
1-6alkyl wherein one or two of the carbon atoms may be optionally replaced by S(=O)
n groups (where n is 0, 1 or 2). Where a carbon is replaced by a said S(=O)
n group, a carbon atom adjacent to that S(=O)
n group may be optionally replaced by N. Preferably n is 1 or 2, more preferably n is 2.
In a preferred aspect of the invention, R1 represents:
(i) -alknC3-9cycloalkyl, said cycloalkyl group being optionally substituted by one or more substituents selected from OH, halogen, C^alkyl, C1-6alkoxy, C2-6alkenyloxy and phenyl, wherein "alk" represents C1-3alkylene and n represents 0 or 1 ;
(ii) phenyl optionally substituted by one or more substituents selected from halogen and C1-6alkyl; or
(iii) a C4-7heterocyclic group containing at least one heteroatom selected from O or N, optionally substituted by C1-6alkyl, CO2Cι_ alkyl, or CO2alknC3-6cycloalkyl.
In a more preferred aspect of the invention, R1 represents:
(i) -alknC3-7cycloalkyl, said cycloalkyl group being optionally substituted by one or two substituents selected from OH, C1-3alkyl and C1-3alkoxy, wherein "alk" represents Ci.
3alkylene and n represents 0; (ii) phenyl optionally substituted by one to three, more preferably one or two, substituents selected from halogen and C1-6alkyl; or
(iii) a C^heterocyclic group containing at least one heteroatom selected from O or N, optionally substituted by C1-6alkyl, COaC^alkyl, or C02alknC3-6cycloalkyl.
R2 preferably represents hydrogen or halogen. More preferably, R2 represents hydrogen or chlorine. Most preferably, R2 represents hydrogen.
R3 and R4 preferably both represent hydrogen.
X preferably represents O.
Z preferably represents (CH2)P wherein p is 1 , or CH2OCH2.
It is to be understood that the present invention covers all combinations of particular and preferred groups mentioned above.
Examples of compounds of the invention include:
(2R,3R,4S,5R)-2-[6-(2,2-dimethyl-cyclopropylamino)-purin-9-yl]-5-prop-2-ynyloxymethyl- tetrahydro-furan-3,4-diol; (2R,3R,4S,5R)-2-[6-(fA'aA?s-4-methoxy-cyclohexylamino)-purin-9-yl]-5-prop-2- ynyloxymethyl-tetrahydro-furan-3,4-diol;
(2R,3R,4S,5R)-2-[6-(fra/?s-4-ethoxy-cyclohexylamino)-purin-9-yl]-5-prop-2-ynyloxymethyl- tetrahydro-furan-3,4-diol;
(2R,3R,4S,5R)-2-[6-((1S*,5S*,6R*)-bicyclo[3.2.0]heptylamino)-purin-9-yl]-5-prop-2- ynyloxymethyl-tetrahydro-furan-3,4-diol; (2R,3R,4S,5R)-2-[6-(cyclopropylamino)-purin-9-yl]-5-prop-2-ynyloxymethyl-tetrahydro- furan-3,4-diol;
4-[9((2R,3R,4S,5R)-3,4-dihydroxy-5-prop-2-ynyloxymethyl-tetrahydro-furan-2-yl)-9H-purin-
6-yl-amino]-piperidine-1-carboxylic acid ethyl ester;
(2R,3R,4S,5R)-2-[6-(tetrahydro-pyran-4-ylamino)-purin-9-yl]-5-prop-2-ynyloxymethyl- tetrahydro-furan-3,4-diol;
(2R,3R,4S,5R)-2-[6-((1R,2R)-2-hydroxy-cyclopentylamino)-purin-9-yl]-5-prop-2- ynyloxymethyl-tetrahydro-furan-3,4-diol;
(2R,3R,4S,5R)-2-[6-(5-chloro-2-methyl-phenylamino)-purin-9-yl]-5-prop-2-ynyloxymethyl- tetrahydro-furan-3,4-diol; (2R,3R,4S,5R)-2-[6-(3-chloro-2-methyl-phenylamino)-purin-9-yl]-5-prop-2-ynyloxymethyl- tetrahydro-furan-3,4-diol;
(2R,3R,4S,5R)-2-[6-(5-fluoro-2-methyl-phenylamino)-purin-9-yl]-5-prop-2-ynyloxymethyl- tetrahydro-furan-3,4-diol;
(2R,3R,4S,5R)-2-[6-(3-fluoro-2-methyl-phenylamino)-purin-9-yl]-5-prop-2-ynyloxymethyl- tetrahydro-furan-3,4-diol;
(2R,3R,4S,5R)-2-[6-(4-chloro-2-fluoro-phenylamino)-purin-9-yl]-5-prop-2-ynyloxymethyl- tetrahydro-furan-3,4-dioI;
(2R,3R,4S,5R)-2-[6-(5-chloro-2-fluoro-phenylamino)-purin-9-yl]-5-prop-2-ynyloxymethyl- tetrahydro-furan-3,4-diol; (2R,3R,4S,5R)-2-[6-(2,3,4-trifluoro-phenylamino)-purin-9-yl]-5-prop-2-ynyloxymethyl- tetrahydro-furan-3,4-diol;
(2R,3R,4S,5R)-2-[6-(3-chloro-2-methylphenyl)amino)-purin-9-yl]-5-prop-2-ynyltetrahydrofuran-
3,4-diol;
(2R,3R,4S,5R)-2-[6-(3-fluoro-2-methylphenyl)amino)-purin-9-yl]-5-prop-2-ynyltetrahydrofuran- 3,4-diol;
(2R,3R,4S,5R)-2-[6-(tetrahydro-2H-pyran-4-ylamino)-purin-9-yl]-5-prop-2-ynyltetrahydrofuran-
3,4-diol; and ethyl 4-({9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-prop-2-ynyltetrahydrofuran-2-yl]-purin-6- yl}amino)piperidine-1 -carboxylate and pharmaceutically acceptable derivatives thereof.
Further examples of the invention include the compounds of examples 1-13, and 16-19 and pharmaceutically acceptable derivatives thereof.
The compounds of formula (I) contain chiral (asymmetric) centres. The individual stereoisomers (enantiomers and diastereoisomers) and mixtures of these are within the scope of the present invention.
As used herein, the terms "alkyl" and "alkoxy" mean both straight and branched chain saturated hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, propyl and butyl groups. Examples of alkoxy groups include methoxy and ethoxy groups. Other examples include propoxy and butoxy. Alkyl groups may be unsubstituted, or substituted with one to four substituents, preferably one to three substituents selected from phenyl, halogen, hydroxy, C1-6alkoxy and C3-7cycloalkyl.
One to three, preferably one or two, carbon atoms of an alkyl chain may be replaced by a group independently selected from S(=0)n (where n is 0, 1 or 2) and N. When the heteroatom N replaces a carbon atom in a Cι.12alkyl group the N atom will, where appropriate be substituted by one or two substituents selected from hydrogen and d. 6alkyl.
As used herein, the terms "alkenyl", "alkenyloxy", "alkynyl" and "alkynyloxy" mean both straight and branched chain unsaturated hydrocarbon groups. Examples of alkenyl groups include ethylene and propylene. Examples of alkynyl groups include ethynyl and propynyl. Examples of alkenyloxy groups include propenyloxy and ethenyloxy. Examples of alkynyloxy groups include propynyloxy and ethynyloxy.
As used herein, the term "halogen" means fluorine, chlorine, bromine or iodine.
As used herein, the term "aryl" means monocyclic or bicyclic aromatic carbocyclic groups such as phenyl and naphthyl, especially phenyl.
As used herein, the term "cycloalkyl" means an aliphatic group having 3 to 9 carbon atoms, more preferably 3 to 7 carbon atoms, in the ring system unless otherwise defined. The cycloalkyl group can be monocyclic or bicyclic. A bicyclic group may be fused or bridged. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl and cyclopentyl. Other examples of monocyclic cycloalkyl groups are cyclohexyl, cycloheptyl and cyclooctyl. Examples of bicyclic cycloalkyl groups include bicyclo[2.2.1]hept-2-yl.
Preferably, the cycloalkyl group is monocyclic. Cycloalkyl groups may be unsubstituted, or substituted with one to four substituents, preferably one or two substituents as defined hereinabove.
As used herein, the term "cycloalkenyl" means a partially unsaturated aliphatic group having 3 to 9 carbon atoms in the ring system. The cycloalkenyl group can be monocyclic or bicyclic. Preferably, the cycloalkenyl group is monocyclic. Examples of monocyclic cycloalkenyl groups include cyclopentenyl and cyclohexenyl. Cycloalkenyl groups may be unsubstituted, or substituted with one to four substituents, preferably one or two substituents.
As used herein, the term "heterocyclic" means a cyclic group of 4 to 7 carbon atoms wherein one or more of the carbon atoms is/are replaced by heteroatoms independently selected from nitrogen, oxygen or sulfur. The heterocycle may be aromatic or non- aromatic, i.e., may be saturated (i.e. aliphatic), partially or fully unsaturated. This group may optionally be substituted as defined hereinabove. The heteroatom is preferably O or N. The heterocycle is preferably non-aromatic. Examples of heterocyclyl groups include piperidinyl, tetrahydrofuranyl and tetrahydropyranyl. Heterocyclyl groups may be unsubstituted, or substituted with one to four substituents, preferably one or two substituents.
As used herein, the term "pharmaceutically acceptable derivative", means any pharmaceutically acceptable salt, solvate, ester or amide, or salt or solvate of such ester or amide, of a compound of formula (I), or any other compound which upon administration to the recipient is capable of providing (directly or indirectly) a compound of formula (I) or an active metabolite or residue thereof, e.g. a prodrug. Preferred pharmaceutically acceptable derivatives according to the invention are any pharmaceutically acceptable salts, solvates and prodrugs, more preferably pharmaceutically acceptable salts and solvates.
As used herein, the term "pharmaceutically acceptable" means a compound which is suitable for pharmaceutical use.
Pharmaceutically acceptable salts of the compounds of formula (I) include those derived from pharmaceutically acceptable inorganic and organic acids. Examples of suitable acids include hydrochloric, hydrobromic, sulphuric, nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic, salicylic, succinic, toluene-p-sulphonic, tartaric, acetic, citric, methanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acids. Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.
Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". Solvates of the compound of formula (I) are within the scope of the invention.
As used herein, the term "prodrug" means a compound which is converted within the body, e.g. by hydrolysis in the blood, into its active form that has medical effects. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series; and in Edward B.
Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.
The compounds useful in the invention are agonists at the adenosine A1 receptor. Preferably they are selective agonists at the adenosine A1 receptor. By selective it is meant that the affinity for the A1 receptor is at least 2 times, preferably 5 times and more preferably 10 times greater than the other adenosine receptor subtypes. Agonist selectivity of compounds against other human adenosine receptors can be determined using Chinese hamster ovary (CHO) cells transfected with the gene for the relevant human adenosine receptor following a method based on that of Castanon, KV. and
Spevak, W. (1994) Biochem. Biophys. Res. Commun. 198, 626-631. The CHO cells are also transfected with cyclic AMP response elements promoting the gene for secreted placental alkaline phosphatase (SPAP) (Wood, KV. (1995J Curr. Opinion. Biotechnology, 6, 50-58). The effect of test compounds is determined by their effects on basal levels of cAMP (A2a) or on forskolin-enhanced cAMP (A1 and A3) as reflected by changes in levels of SPAP. EC50 values for compounds are determined as a ratio to that of the non- selective agonist N-ethyl carboxamidoadenosine (NECA). "Adenosine receptors: New opportunities for future drugs" (S.A. Poulsen etal, Bioorg. Med. Chem,. 1998, 6, 619-641) summarises disease conditions that may be treated with adenosine A1 agonists.
Compounds according to the invention have applicability as inhibitors of lipolysis i.e. they decrease plasma free fatty acid concentrations. The compounds may thus be used in the treatment of hyperlipidaemias. Furthermore, as a consequence of their anti-lipolytic activity, the compounds have the ability to lower elevated blood glucose, insulin and ketone body levels and therefore may be of value in the therapy of diabetes. Since anti- lipolytic agents have hypolipidaemic and hypofibrinogenaemic activity, the compounds may also show anti-atherosclerotic activity. The assay described by P. Strong et al. in Clinical Science (1993), 84, 663-669 may be used to determine the anti-lipolytic activity of compounds of the invention by their ability to lower the concentration of non-esterified fatty acids (NEFA) in starved rats.
In addition to their anti-lipolytic effect, the compounds of the invention may independently affect cardiac function by reducing heart rate and conduction. The compounds may thus be used in the therapy of a number of cardiovascular disorders, for example cardiac arrythmias, particularly following myocardial infarction, and angina. The compounds may also inhibit renin release and thus be of use in the therapy of hypertension and heart failure.
Furthermore, the compounds of the invention are useful as cardioprotective agents, having applicability in the treatment of ischaemic heart disease. As used herein the term
"ischaemic heart disease" includes damage associated with both myocardial ischaemia and reperfusion, for example, associated with coronary artery bypass grafting (CABG),
percutaneous translumenal coronary angioplasty (PTCA), cardioplegia, acute myocardial infarction, thrombolysis, stable and unstable angina and cardiac surgery including in particular cardiac transplantation. The compounds of the invention additionally are useful for treating ischaemic damage to other organs. The compounds of the invention may also be valuable in the treatment of other disorders arising as a result of widespread atheromatous disease, for example, peripheral vascular disease (PVD) and stroke.
The compounds of the invention may also be useful as CNS agents (e.g. as hypnotics, sedatives, analgesics and/or anti-convulsants particularly finding use in the treatment of epilepsy). They are therefore useful in treating or preventing pain. They may be used to improve the condition of a host, typically of a human being, suffering from pain. They may be employed to alleviate pain in a host. Thus, a compound of formula (I) and its pharmaceutically acceptable acid addition salts may be used as a preemptive analgesic to treat acute pain such as musculoskeletal pain, post operative pain and surgical pain, chronic pain such as chronic inflammatory pain (e.g. rheumatoid arthritis and osteoarthritis), neuropathic pain (e.g. post herpetic neuralgia, diabetic neuropathies associated with diabetes, trigeminal neuralgia, pain associated with functional bowel disorders, e.g. irritable bowel syndrome, non cardiac chest pain and sympathetically maintained pain) and pain associated with cancer and fibromyalgia. The compound of formula (I) may also be used in the treatment or prevention of migraine or of pain associated with migraine, tension headache and cluster headaches, pain associated with functional bowel disorders (e.g. IBS), non cardiac chest pain and non ulcer dyspepsia. The compound of formula (I) may also be used in the treatment of nociceptive pain (e.g. headaches, labour pain, menstrual pain and early post-operative pain).
In addition, the compounds of the invention may find use in the treatment of sleep apnoea.
Furthermore, the compounds of the invention may find use in the treatment of emesis. Treatment of emesis includes treatment of nausea, retching and vomiting. Emesis includes acute emesis, delayed emesis and anticipatory emesis.
Accordingly, the invention provides a compound of formula (I) or a pharmaceutically acceptable derivative thereof for use in therapy, and in particular in the treatment of human or animal subjects suffering from a condition in which there is an advantage in decreasing plasma free fatty acid concentration, or reducing heart rate and conduction, or whereby the therapy involves the treatment of ischaemic heart disease, peripheral vascular disease or stroke or which subject is suffering from pain, a CNS disorder, sleep apnoea or emesis.
In another aspect, the invention provides a method of treatment of a human or animal subject suffering from a condition in which there is an advantage in decreasing plasma free fatty acid concentration, or reducing heart rate and conduction, or which subject is suffering from or susceptible to ischaemic heart disease, peripheral vascular disease or
stroke, or which subject is suffering from pain, a CNS disorder, sleep apnoea or emesis, which method comprises administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof.
In another aspect the invention also provides for the use of a compound of formula (I) or a pharmaceutically acceptable derivative thereof for the manufacture of a medicament for the treatment of human or animal subjects suffering from a condition in which there is an advantage in decreasing plasma free fatty acid concentration, or reducing heart rate and conduction, or which subject is suffering from or susceptible to ischaemic heart disease, peripheral vascular disease (PVD) or stroke, or which patient is suffering from pain, a CNS disorder, sleep apnoea or emesis.
In respect of the above mentioned ischaemic treatment, the methods of the present invention are applicable not only where ischaemia is planned or expected, for example in cardiac surgery, but also in cases of sudden or unexpected ischaemia, for example in heart attack and unstable angina.
In a preferred aspect, the invention provides a compound of formula (I) or a pharmaceutically acceptable derivative thereof for use in therapy, and in particular in the treatment of human or animal subjects of conditions associated with pain including acute pain, chronic pain, inflammatory pain, neuropathic pain, nociceptive pain and pain associated with migraine, tension headaches, cluster headaches and functional bowel disorder. In an alternative embodiment, the invention provides a compound of formula (I) or a pharmaceutically acceptable derivative thereof for use in therapy, and in particular in the treatment of human or animal subjects of conditions associated with pain including acute pain, chronic pain, inflammatory pain, neuropathic pain and pain associated with migraine, tension headaches, cluster headaches and functional bowel disorder.
In another aspect, the invention provides a method of treatment of a human or animal subject suffering from a condition associated with pain including acute pain, chronic pain, inflammatory pain, neuropathic pain, nociceptive pain and pain associated with migraine, tension headaches, cluster headaches and functional bowel disorder; alternatively acute pain, chronic pain, inflammatory pain, neuropathic pain and pain associated with migraine, tension headaches, cluster headaches and functional bowel disorder; which method comprises administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof.
In another aspect the invention also provides for the use of a compound of formula (I) or a pharmaceutically acceptable derivative thereof for the manufacture of a medicament for the treatment of human or animal subjects suffering from a condition associated with pain including acute pain, chronic pain, inflammatory pain, neuropathic pain, nociceptive pain and pain associated with migraine, tension headaches, cluster headaches and functional
bowel disorder. In an alternative embodiment the invention provides for the use of a compound of formula (I) or a pharmaceutically acceptable derivative thereof for the manufacture of a medicament for the treatment of human or animal subjects suffering from a condition associated with pain including acute pain, chronic pain, inflammatory pain, neuropathic pain and pain associated with migraine, tension headaches, cluster headaches and functional bowel disorder.
It will be appreciated that reference to treatment includes acute treatment or prophylaxis as well as the alleviation of established symptoms.
While it is possible that compounds of the invention may be administered as the raw material, it is preferable to present the active ingredient as a pharmaceutical formulation.
In a further aspect, the invention provides a pharmaceutical composition comprising at least one compound of formula (I) or a pharmaceutically acceptable derivative thereof in association with a pharmaceutically acceptable carrier and/or excipient. The carrier and/or excipient must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the receipient thereof.
In another aspect, the invention provides a pharmaceutical composition comprising, as active ingredient, at least one compound of formula (I) or a pharmaceutically acceptable derivative thereof in association with a pharmaceutically acceptable carrier and/or excipient for use in therapy, and in particular in the treatment of human or animal subjects suffering from a condition in which there is an advantage in decreasing plasma free fatty acid concentration, or reducing heart rate and conduction, or which subject is suffering from or susceptible to ischaemic heart disease, peripheral vascular disease or stroke, or which subject is suffering from a CNS disorder, sleep apnoea, pain or emesis.
There is further provided by the present invention a process of preparing a pharmaceutical composition, which process comprises mixing at least one compound of formula (I) or a pharmaceutically acceptable derivative thereof, together with a pharmaceutically acceptable carrier and/or excipient.
Compositions according to the invention may be formulated for topical, oral, buccal, parenteral or rectal administration or in a form suitable for administration by inhalation or insufflation. Oral administration is preferred. The compositions may be adapted for sustained release.
For topical administration, the pharmaceutical composition may be given in the form of a transdermal patch.
Tablets and capsules for oral administration may contain conventional excipients such as binding agents, for example mucilage of starch or polyvinylpyrrolidone; fillers, for example, lactose, microcrystalline cellulose or maize-starch; lubricants, for example, magnesium stearate or stearic acid; disintegrants, for example, potato starch, croscarmellose sodium or sodium starch glycollate; or wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in the art. 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 constitution 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, or carboxymethyl cellulose; emulsifying agents, for example, sorbitan mono-oleate; non-aqueous vehicles (which may include edible oils), for example, propylene glycol or ethyl alcohol; and preservatives, for example, methyl or propyl p-hydroxybenzoates or sorbic acid. The preparations may also contain buffer salts, flavouring, colouring and sweetening agents (e.g. mannitol) as appropriate.
For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.
The compounds of formula (I) or pharmaceutically acceptable derivatives thereof may be formulated for parenteral administration by bolus injection or continuous infusion and may be presented in unit dose form in ampoules, or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.
The compounds of formula (I) or pharmaceutically acceptable derivatives thereof may also be formulated as suppositories, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.
A proposed dose of the compounds of the invention for administration to man (of approximately 70kg body weight) is 0.1 mg to 2g, preferably 1mg to 2g, more preferably 1 mg to 10Omg, of the active ingredient per unit dose which could be administered, for example, 1 to 4 times per day. It will be appreciated that it may be necessary to make routine variations to the dosage, depending on the age and condition of the patient. The dosage will also depend on the route of administration.
The compounds of formula (I) may also be used in combination with other therapeutic agents. The invention thus provides, in a further aspect, a combination comprising a
compound of formula (I) or a pharmaceutically acceptable derivative thereof together with a further therapeutic agent.
When a compound of formula (I) or a pharmaceutically acceptable derivative thereof is used in combination with a second therapeutic agent active against the same disease state the dose of each compound may differ from that when the compound is used alone. Suitable second therapeutic agents for the treatment of pain include, for example, COX-2 inhibitor e.g. celecoxib, rofecoxib, valdecoxib, parecoxib, 4-(4-cycIohexyl-2-methyl-5- oxazolyl)-2-fluorobenzenesulfonamide (JTE-522), MK663, 2-(4-ethoxy-phenyl)-3-(4- methanesulfonyl-phenyl)-pyrazolo[1,5-b]pyridazine; 5HT1 agonists e.g. sumatriptan, naratriptan, zolmitriptan, eletriptan, rizatriptan, frovatriptan, almotriptan, alniditan, dihydroergotamine, donitriptan, PNU-142633, ALX-0646, LY334370, U1092291 , IS159, PNY142633; sodium channel blockers e.g. lamotrigine, R(-)-2,4-diamino-5-(2,3- dichlorophenyl)-6-fluoromethyl pyrimidine, 2,6-diamino-3-(2,3,5-trichlorophenyl)pyrazine, 5- amino-6-[2,3,5-trichlorophenyl]-1 ,2,4-triazine; 5HT3 antagonists e.g. alosetron; gabapentin; pregabalin; EP1 antagonists e.g. ZD6416, ZD6804; and opioids e.g. alfentanil, buprenorphine, codeine, dextropropoxyphene, diamorphine, dihydrocodeine, fentanyl, methadone, morphine, oxycodone, levorphanol, pentazocine, pethidine.
The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the invention. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations by any convenient route.
When administration is sequential, either the adenosine A1 agonist or the second therapeutic agent may be administered first. When administration is simultaneous, the combination may be administered either in the same or different pharmaceutical composition.
When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation. When formulated separately they may be provided in any convenient formulation, conveniently in such manner as are known for such compounds in the art.
When a compound of formula (I) or a pharmaceutically acceptable derivative thereof is used in combination with a second therapeutic agent active against the same disease state the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art. It will be appreciated that the amount of a compound of the invention required for use in treatment
will vary with the nature of the condition being treated and the age and the condition of the patient and will be ultimately at the discretion of the attendant physician or veterinarian.
The compounds of formula (I) and pharmaceutically acceptable derivatives thereof may be prepared by the processes described hereinafter, said processes constituting a further aspect of the invention. In the following description, the groups X, Z, R1, R2and R3 are as defined for compounds of formula (I) unless otherwise stated.
According to a first general process (A), a compound of formula (I) may be prepared by reacting a compound of formula (II):
wherein L represents a leaving group such as a halogen atom (e.g. chlorine), benzotriazolyloxy or a linker group capable of binding to a solid phase polymeric support (e.g. a polystyrene resin) and for example may be -SO2C1-4alkylene and P1 and P2 represent hydrogen, C1-6 straight chain or branched alkyl or a suitable protecting group (e.g. acetyl or a protecting group wherein P1 and P2 together form an alkylidene group); with a compound of formula R1NH2 or a salt thereof under basic or buffered conditions, where R1, R2 , Z and X are as defined for compounds of formula (I).
Accordingly the present invention also provides a process for the preparation of a compound of formula (I) or a pharmaceutically acceptable derivative thereof:
R'
(I)
wherein X represents O or CH2;
Z represents (CH2)P or CH2OCH2 wherein p is 1 , 2 or 3;
R1 represents:
(i) -alkn-C3-9cycloalkyl or -alknC3-9cycloalkenyl, said cycloalkyl or cycloalkenyl group being optionally substituted by one or more substituents selected from OH, halogen, d-
6alkyl, C1-6alkoxy, C2-6alkenyloxy, C2-6alkynyloxy and phenyl, wherein "alk" represents d-
3alkylene and n represents 0 or 1 , and said "alk" group may be optionally substituted by a
C3-6cycloalkyl group;
(ii) a phenyl group optionally substituted by one or more substituents selected from halogen, CF3, cyano, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C1-6alkoxy, d-6alkylOH, C02H and
CO2C1-6 alkyl;
(iii) a C4-7heterocyclic group containing at least one heteroatom selected from O, N or
S, and optionally substituted by one or more substituents selected from: OH, d-ealkyl, d.
6alkoxy, C02d-4alkyl, COC^alkyl, C02aryl or CO2alknC3^cycloalkyl, wherein "alk" represents d-salkylene and n represents 0 or 1 ;
(iv) a straight or branched C1-12alkyl group optionally substituted by one or more groups selected from phenyl, halogen, hydroxy, C1-6alkoxy and C3-7 cycloalkyl, wherein one or more carbon atoms of the C1-12alkyl group may be optionally replaced by a group independently selected from S(=O)n (where n is 0, 1 or 2) and N; or (v) a fused bicyclic ring
wherein A represents C
4-6cycloalkyl or phenyl and B represents phenyl optionally substituted by C
1-3alkyl, and the bicyclic ring is attached to the purine-6-amino moiety via a ring atom of ring A;
R2 represents C1-3alkyl, halogen, hydrogen or C1-3alkoxy group;
R3 and R4 independently represent hydrogen or a C1-6alkyl group; comprising: reacting a compound of formula (II):
wherein R
2, Z and X are as defined for compounds of formula (I), L represents a leaving group and P
1 and P
2 represent hydrogen, C
1-6 straight chain or branched alkyl or a protecting group; with a compound of formula R
1NH
2 or a salt thereof wherein R
1 is as defined for compounds of formula (I) and where required, and in any order: interconverting one substituent to another substituent; and/or removing the protecting groups; and/or forming a pharmaceutically acceptable derivative.
Suitable reaction conditions for the preparation of compounds of formula (I) include the use of a solvent such as an alcohol (e.g. a lower alkanol such as ethanol, isopropanol, t- butanol or 3-pentanol), an ether (e.g. tetrahydrofuran or dioxan), a substituted amide (e.g. dimethylformamide), a halogenated hydrocarbon (e.g. chloroform), an aromatic hydrocarbon (e.g. toluene), dimethyl sulfoxide (DMSO) or acetonitrile. Preferably the reaction is carried out at an elevated temperature (e.g. up to the reflux temperature of the solvent). When a salt of the amine is used (e.g. hydrochloride) a suitable acid scavanger is used. Suitable acid scavengers include, for example, inorganic bases such as sodium, cesium or potassium carbonate, or organic bases such as triethylamine, diisopropylethylamine or pyridine.
Alternatively, compounds of formula (II) may be used to prepare compounds of formula (I) by reaction with the group R1NH2, where R1 is as defined for compounds of formula (I), in the presence of CaCO3 and an appropriate solvent, e.g. ethanol or acetic acid. Preferably when the solvent is ethanol the reaction is heated, for example at reflux. In an alternative aspect the reaction may be carried out in acetic acid in the absence of CaC03, preferably the reaction is heated.
The above reactions may be preceded or followed where appropriate by in situ removal of the P1 and P2 protecting groups. For example when P1 and P2 represent acetyl, this may be effected with an amine such as ammonia or tert-butylamine or an alkoxide such as sodium methoxide in a solvent such as methanol or when P1 and P2 represent an alkylidene by acid hydrolysis, e.g. with trifluoroacetic acid (TFA). Interconversion of P1 and P2 protecting groups may occur at any stage in the preparation of the compounds of formula (II), for example when P1 and P2 represent acetyl, compounds of formula (II) may be prepared from compounds wherein P1 and P2 together represent an alkylidene protecting group by acid catalysed removal of the alkylidene protecting group, e.g. with hydrogen chloride in methanol followed by in situ acylation, for example with acetic anhydride in the presence of a base such as pyridine, in a solvent such as dichloromethane.
Otherwise, interconversion of P1 and P2 protecting groups may occur at any stage during the preparation of compounds of formula (II).
Compounds of formula (II) may be prepared by methods described in WO99/67262. For example, compounds of formula (II) where X = O may be prepared by reacting compounds of formula (III):
wherein P3 represents a suitable protecting group, for example acetyl, or a substituent such as C1-3 alkyl, and P1 and P2 are as defined above, with compounds of formula (IV):
wherein L and R2 are as defined above.
The reaction is conveniently carried out in a suitable solvent, such as acetonitrile in the presence of a silylating agent such as trimethylsilyl trifluoromethane sulfonate and a base such as diazabicyclo[5.4.0]undec-7-ene (DBU). Alternatively the compound of formula (IV) may first be silylated with a suitable silylating agent e.g. hexamethyldisilazane followed by reaction of the silylated intermediate with a compound of formula (III) and a suitable Lewis acid, e.g. trimethylsilyl trifluoromethanesulfonate in a suitable solvent such as acetonitrile.
Compounds of formula (IV) are either known in the art or may be prepared from known compounds using methods analogous to those used to prepare the known compounds of formula (IV).
As described above, the compounds of formula (III) may be prepared from alternative protected compounds by replacement of the alternate P1 and P2 protecting groups with other P1 and P2 groups. These represent an exchanging of one protecting group for another and will be apparent to those skilled in the art.
Compounds of formula (III) may be prepared by methods known in the art, for example as described in J. Chem. Soc, Perkin Trans. 1, 1999. 3597 and WO 97/43300.
Specific optical isomers of a compound of formula (I) may be obtained by conventional methods for example, by synthesis from an appropriate asymmetric starting material using any of the processes described herein, or where appropriate by separation of a mixture of isomers of a compound of formula (I) by conventional means e.g. by fractional distillation, fractional crystallisation or chromatography.
In the general processes described above, the compound of formula (I) obtained may be in the form of a salt, conveniently in the form of a pharmaceutically acceptable salt. Where desired, such salts may be converted into the corresponding free bases using conventional methods.
Pharmaceutically acceptable acid addition salts of the compounds of formula (I) may be prepared by reacting a compound of formula (I) with an appropriate acid in the presence of a suitable solvent such as acetonitrile, acetone, chloroform, ethyl acetate or an alcohol (e.g. methanol, ethanol or isopropanol). Pharmaceutically acceptable base addition salts may be obtained in an analogous manner by treating a solution of a compound of formula (I) with a suitable base. Pharmaceutically acceptable salts may also be prepared from other salts, including other pharmaceutically acceptable salts of the compounds of formula (I), using conventional methods.
The present invention will now be further illustrated by the accompanying examples which should not be construed as limiting the scope of the invention in any way.
Examples
Definitions of abbreviations used herein: N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1 ,3-dimethyl-3,4,5,6-tetrahydro-2(1 H)pyrimidinone (DMPU) dichloromethane (DCM), isopropyl alcohol (IPA), methanol (MeOH); ethyl acetate (EtOAc); tetrahydrofuran (THF), trifluoroacetic acid (TFA), trimethylsilyl (TMS), tetrabutylammonium fluoride (TBAF), diisopropylethylamine (DIPEA), dimethylaminopyridine (DMAP), hydroxybenzotriazole (HOBt) and diazabicyclo[5.4.0]undec-7-ene (DBU); high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), thin layer chromatography (tic).
General Scheme for preparation of compounds of formula (I) when Z is CHpOCH?
Intermediate I
Preparation of 1 -(6-chloro-9H-purin-9-vD-1 -deoxy-2,3-0-isopropylidene-5-O-(2-propynal)- β-D-ribofuranose (Intermediate I)
a) Preparation of 2',3'-0-isopropylidene-5'-0-(2-propynvhinosine
A 60% dispersion of sodium hydride in mineral oil (1.40g, 35.0mmol) was added to an ice cold solution of the inosine (5.00g, 16.2mmol) in DMF (50 mL), under a nitrogen atmosphere, over a period of 30 minutes. DMF (10mL) was added to the resultant thick suspension and stirring was continued for 2 hours. An 80% solution of propargyl bromide in toluene (2.2mL, 20.0mmol) was added at 0°C. Upon addition the ice bath was removed and stirring continued for 4 hours. Glacial acetic acid (2mL) was added and the mixture was left to stir for 18 hours. The resultant suspension was evaporated to dryness and partitioned between ethyl acetate (200mL) and water (100mL). The layers were separated and the aqueous phase was extracted further with ethyl acetate (100mL). The combined organic extracts were washed with water and brine, dried (MgSO4), filtered and evaporated to give an oil which was purified by chromatography on silica using Biotage®, with DCM- MeOH-0.880 ammonia (95:5:1) as eluant, to give the title compound (2.80g, 50%). LC/MS, Rt 2.36 min mass spectrum m/z 347 [MH+]
Rf 0.37 DCM-MeOH-0.880 ammonia (18:2:0.2).
b) Preparation of 1-(6-chloro-9H-purin-9-v0-1-deoxy-2,3-O-isopropylidene-5-O-(2- propynaP-β-D-ribofuranose (Intermediate I)
Phosphorous oxychloride (1.9mL, 21.5mmol) and DMAP (0.953g, 7.8mmol) were added to the inosine derivative (2.700g, 7.8mmol) in acetonitrile (30mL). The mixture was heated at reflux, under a nitrogen atmosphere, for 2 hours then allowed to stand overnight at room temperature. Ethyl acetate (250mL) and water (50mL) were added and the layers separated. The organic layer was washed sequentially with saturated sodium bicarbonate (50mL), water (50mL) and brine (50mL) then dried (MgSO4), filtered and evaporated. The
residue was purified by chromatography on silica using Biotage®, with cyclohexane containing ethyl acetate (20-30%) as eluant, to give the title compound (Intermediate
2.070g, 73%).
LC/MS, Rt 2.82min. mass spectrum m/z 365 [MH+]
Rf 0.25 (cyclohexane-ethyl acetate, 1:1).
General procedure A: Reaction of Intermediate I with aliphatic amines
Approximately 33mg (O.lmmol) of Intermediate I was dissolved in IPA (0.5mL) and heated with the amine, or amine hydrochloride, (2eq) and DIPEA (O.OδmL, O.δmmol) at 90°C for 2.5-6 hours in a reacti-vial. The solvent was removed and the crude product was processed to the next step.
The following intermediates were prepared using General Procedure A
General procedure for acetonide deprotection
The acetonide derivative was dissolved in ice cold TFA-water (9:1) (2mL) and left to stand at 0°C for 4-6 hours. The mixture was then added dropwise to a saturated solution of sodium bicarbonate. Extraction with ethyl acetate, drying (Na2SO ) filtration and evaporation delivered the crude compound which was then purified by reverse phase automated preparative HPLC.
The following Examples were prepared by deprotection of the corresponding acetonide derivative according to the general deprotection procedure.
Example 7: Preparation of (2R, 3R. 4S. 5R)-2-r6-(tetrahvdro-pyran-4-ylamino)-purin-9-yll- 5-prop-2-vnyloxymethyl-tetrahydro-furan-3.4-diol
Intermediate I (459mg, 1.3mmol), DIPEA (1.1 mL) and 4-aminotetrahydropyran hydrochloride (219mg, 1.6mmol) were heated in 2-propanal in a reacti-vial at 90°C for 6 hours. The mixture was cooled to room temperature and evaporated. The residue was partitioned between ethyl acetate and saturated ammonium chloride. The layers were separated and the aqueous phase was extracted with ethyl acetate. The combined organics were washed with saturated sodium bicarbonate, dried (Na2SO4), filtered and evaporated. The residue was purified by chromatography on silica using Biotage®, with cyclohexane containing ethyl acetate (60-80%) as eluant, to give the desired intermediate (427mg, 79%) as a white solid. LC/MS Rt 2.70 min, mass spectrum m/z 430 [MH+].
An ice cold mixture of TFA-water (9:1) (5mL) was added to the acetonide derivative
(427mg, I.Ommol). The mixture was left to stand at 0°C overnight (17 hours) then added dropwise to a saturated solution of sodium bicarbonate, and extracted with ethyl acetate, dried (Na2SO4), filtered and evaporated. The residue was purified by chromatography on silica using Biotage®, with ethyl acetate containing ethanol (0-10%) as eluant, to give the title compound(104mg, 27%).
1H NMR δ (DMSO)1.56-1.90 (4H, m), 3.36-3.46 (2H, m), 3.49 (1 H, t), 3.63 (1 H, dd), 3.73 (1H, dd), 3.85-3.94 (2H, m), 4.01-4.07 (1H, m), 4.12-4.17 (1 H, m), 4.20 (2H, s), 4.26-4.41
(1 H, m), 4.54-4.62 (1H, m), 5.31 (1 H, d, OH), 5.52 (1H, d, OH), 5.91 (1H, d), 7.79 (1H, br.s, NH), 8.23(1 H, s), 8.33 (1 H, s).
Example 8: Preparation of (2R. 3R. 4S. 5R)-2-r6-((1 R. 2R)-2-Hvdroxy-cvclopentylaminoV purin-9-vn-5-prop-2-vnyloxymethyl-tetrahydro-furan-3,4-diol
a) Preparation of (2R, 3R, 4S. 5R)-2-(6-chloro-purin-9-yl)-5-prop-2-vnyloxymethyl- tetrahvdro-furan-3.4-diol
An ice cold mixture of TFA-water (10:1) (187mL) was added to the acetonide derivative (1.700g, 4.7mmol). The mixture was stirred at 0°C for 1.5 hours then evaporated, and azeotroped with toluene. The residue was purified by chromatography on silica with DCM- methanol-ammonia (94:6:1) to give the title compound (1.100g, 73%). Rf 0.13 (DCM-methanol-ammonia, 94:6:1).
b) Preparation of (2R. 3R. 4S. 5R)-2-r6-((1 R. 2RV2-hvdroxy-cvclopentylamino)-purin-9-yll- 5-prop-2-vnyloxymethyl-tetrahydro-furan-3.4-diol
The 6-chloropurine derivative (650mg, 2.0mmol), DIPEA (10mL, 57.6mmol) and (1R, 2R)- 2-amino-cyclopentanol hydrochloride (305mg, 2.2mmoI) were heated in 2-propanol (20mL) at reflux overnight. The mixture was cooled to room temperature and further 2- propanol (25mL) and stirred at room temperature for 3 hours. A precipitate formed which was collected by filtration, washed with 2-propanol and diethyl ether then dried to give the title compound (680mg, 87%) as a white solid, mp 152-158°C.
1H NMR δ (DMSO) 1.38-2.15 (6H, m), 3.46 (1H, t), 3.55-3.82 (2H, m), 3.95-4.05 (2H, m), 4.14(1H, t), 4.18(1H, d), 4.28-4.38 (1H, m), 4.55(1 H, t), 4.83-4.92(1 H, m), 5.89(1 H, d), 7.72(1 H, br.s, NH), 8.26(1 H, s), 8.37(1 H, s).
Elemental analysis (Found: C, 56.0; H, 6.7; N, 15.7. C18H23N5O5 requires C, 54.9; H, 7.0; N, 15.6%).
General procedure for reaction of Intermediate I with aryl amines
Approximately 30mg (O.Oδmmol) of Intermediate I was dissolved in ethanol (0.5mL) and heated at 90°C in a reacti-vial with calcium carbonate (16mg) and the corresponding aniline (3 equivalents) for 14.5 hours. LC/MS analysis indicated that the majority of the material had been converted through to the diol final product under these reaction conditions, and thus a deprotection step was not required. The mixture was diluted with ethyl acetate, filtered and evaporated. The residue was purified by reverse phase automated preparative HPLC to give the diol final product.
The following compounds were made using this general procedure.
Alternative procedure for reaction of Intermediate I with aryl amines
125mg (0.34mmol) of Intermediate I was dissolved in ethanol (3mL) and heated at 90°C in a reacti-vial with calcium carbonate (103mg, 1.03mmol) and the corresponding aniline (3 equivalents) for 16-34 hours. LCMS analysis indicated that the majority of the material had been converted through to the product still containing the acetonide group under these reaction conditions, and thus a deprotection step was required. The mixture was diluted with ethyl acetate, filtered and evaporated. The residue was partially purified by flash chromatography on silica with cyclohexane containing ethyl acetate (3-70%) as eluant.
The partially purified material was dissolved in ice cold TFA-water (9:1) (2mL) and stirred at 0°C for 2-6 hours. The mixture was then added dropwise to a saturated aqueous solution of sodium bicarbonate. Extraction with ethyl acetate, drying (Na2S04) filtration and evaporation delivered the crude compound, which was then purified by reverse phase automated preparative HPLC.
The following examples were prepared using this alternative procedure.
General Scheme for the preparation of compounds of formula (I) when Z is CH?
TBAF, THF
Preparation of (2R.3R.4R.5R)-4-(acetoxy)-2-(6-chloro-9H-purin-9-vh-5-(prop-2- ynyl)tetrahydrofuran-3-yl acetate (Intermediate II)
a) Preparation of (2R.3R.4R,5R)-3,4-0-isopropylidene-2-methoxy-5-(3-trimethylsilylprop- 2-vnyl)tetrahydrofuran
(Tri ethylsilyl)acetylene (5mL, 35.4mmol) was dissolved in THF (20mL) and cooled to - 78°C under a nitrogen atmosphere. A7-BuLi (1.6M in hexanes, 23mL, 36.8mmol) was added dropwise. On completion of the addition, the reaction flask was transferred to an ice-water bath and stirred at 0°C for 2hours, to allow formation of lithium (trimethylsilyl)acetylide.
Meanwhile, 2,6-lutidine (1.5mL, 12.98mmol) was dissolved in DCM (20mL) and cooled to - 78°C under a nitrogen atmosphere. Trifluoromethanesulphonic anhydride (2.1 mL, 12.48mmol) was added dropwise. The resulting yellow-green mixture was stirred for 5mins, and then treated with a solution of methyl 2,3-O-isopropylidene-D-ribofuranoside (2.41g, 11.8mmol) in dichloromethane (5mL). This mixture was stirred at -78°C for ca. 1 hour, and then allowed to warm to room temperature. The solution was concentrated in vacuo to give the triflate derivative of the protected ribofuranoside as a dark red oil.
The triflate was redissolved in a mixture of THF (1 OmL) and 1 ,3-dimethyl-3,4,5,6- tetrahydro-2(1 H)pyrimidinone (DMPU) (5mL), and this solution was cooled to -78°C under a nitrogen atmosphere. The solution of lithium (trimethylsilyl)acetylide in THF/hexanes prepared earlier was cooled and added dropwise. The resulting mixture was stirred at - 78°C for 1hour and then -20°C for 1hour. Saturated aqueous ammonium chloride solution (30mL) was added, and the mixture was allowed to warm to room temperature.
The layers were separated and the aqueous layer was extracted with ethyl acetate (x3). The combined organic extracts were dried (MgS0 ), and concentrated to give the crude material (ca.9g). This was assumed to contain residual DMPU and 2,6-lutidine, so was partitioned between ethyl acetate and water. The layers were separated and the aqueous layer was extracted with further ethyl acetate (x1). The combined organic extracts were dried (MgS04) and concentrated to give the crude material (ca.5.5g). Purification by chromatography on silica (gradient elution, EtOAc/ cyclohexane, 2-3%) gave the product as a yellow oil (1.58g, 5.54mmol, 47%). m/z 302 (M ++H20).
Further elution gave a second product as a colourless oil, determined (by NMR) to be the product resulting from loss of the trimethylsilyl group, identical to that prepared in the next step (0.64g, 3.00mol, 25%).
b) Preparation of (2R,3R.4R.5R)-3.4-Q-lsopropylidene-2-methoxy-5-(prop-2- ynvDtetrahvd rof uran
The TMS-acetylide (1.46g, 5.0mmol) was dissolved in THF (10mL) and cooled to 0°C under a nitrogen atmosphere. A solution of tetrabutylammonium fluoride in THF (1 M, 15mL, 15mmol) was added dropwise. The solution was stirred at 0°C for 45mins, and room temperature for 30mins, at which time tic indicted that the reaction was complete. The mixture was partitioned between diethyl ether and water. The layers were separated and the aqueous layer was extracted with further ether (x2). The combined organic
extracts were dried (MgSO4) and concentrated to give the crude material as a brown oil. Purification by chromatography on silica (gradient elution, EtOAc/cyclohexane, 2-4%) gave the product as a colourless oil (0.975g, 4.59mmol, 92%). m/z 230 (M ++H20).
c) Preparation of (2R.3R.4R,5R)-4-(acetoxy)-2-methoxy-5-(prop-2-vnyl)tetrahydrofuran-3- yl acetate and (2S.3R.4R.5R)-4-(acetoxy)-2-methoxy-5-(prop-2-vnyl)tetrahvdrofuran-3-yl acetate
The isopropylidene-protected ribofuranoside (964mg, 4.54mmol) was dissolved in methanol (15mL), and treated with concentrated HCI (0.3mL). The mixture was heated to reflux for 8 hours, by which time tic analysis showed no remaining starting material.
After standing at room temperature overnight, the mixture was treated with pyridine (2mL) and concentrated in vacuo to give a yellow oil. This was redissolved in DCM (20mL) and treated with 4-(dimethylamino)pyridine (55mg, 0.45mmol), triethylamine (6.4mL, 45.9mmol) and acetic anhydride (1.72mL, 18.2mmoI). The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 2.5hours, by which time tic analysis showed complete reaction. The reaction mixture was partitioned between DCM and water. The layers were separated and the aqueous layer was extracted with further DCM (x2). The combined organic extracts were washed with brine, dried (MgS0 ) and concentrated to give a yellow gum. Purification by chromatography on silica (gradient elution EtOAc/cyclohexane, 10-25%) gave the two anomeric products.
The β-anomer (2R) eluted first and was concentrated to give a colourless oil (δOOmg, 3.12mmol, 68%). m/z 274 (M ++H20).
The α-anomer (2S) then eluted and was concentrated to give a colourless oil (228mg, 0.89mmol, 20%). m/z 274 (M ++H20).
d) Preparation of (2R,3R.4R.5R)-4-(acetoxy)-2-(6-chloro-9H-purin-9-vn-5-(prop-2- ynyl)tetrahvdrofuran-3-yl acetate (Intermediate II)
![Figure imgf000030_0002](https://patentimages.storage.googleapis.com/2c/1c/77/e9161c86d35a0d/imgf000030_0002.png)
A mixture of 6-chloropurine (235mg, 1.52mmol) and hexamethyldisilazane (15mL) was heated under a nitrogen atmosphere for 18hours. After cooling, the mixture was concentrated in vacuo. It was redissolved in toluene and reconcentrated twice, and then
dried in vacuo to give the 6-chloro-9-trimethylsilylpurine as a yellow solid. This was suspended in acetonitrile (10mL), and cooled in an ice-bath. The diactetate-protected ribofuranoside (256mg, 1.Ommol) (β-anomer used, but opposite anomer or a mixture would react similarly) was added as a solution in acetonitile (1 OmL) under a nitrogen atmosphere. Trimethylsilyltriflate (0.9mL, 4.97mmol) was added dropwise with stirring and all reaction materials gradually dissolved. After stirring for 20min at 0°C, the reaction was allowed to warm to room temperature, and stirred for 30min. Finally it was heated to 90°C for 5hrs. After standing at room temperature overnight, the mixture was partitioned between ethyl acetate and saturated aqueous sodium bicarbonate. The layers were separated and the aqueous layer was extracted with further ethyl acetate (x2). The combined organic extracts were washed with saturated brine, dried (MgS0 ) and concentrated to give the crude material as a red-brown gum. Purification by chromatography on silica (gradient elution EtOAc/cyclohexane, 40-50%) gave the product as a pale yellow gum (190mg, 0.502mmol, 50%). m/z 379 (MH
+). However, NMR and LCMS showed that this to be a mixture of two isomers, N-9-linked:N-7-linked = 3:2.
The mixture could not be separated on a preparative scale by standard chromatographic methods and was therefore used in subsequent reactions without further purification.
Reaction of Intermediate II with amines and anilines
General Procedure B - reaction of Intermediate II with an arylamine
Intermediate II (1eq.), the required arylamine (preferably at least 2eq.) and calcium carbonate (equal equivalents to the arylamine, or correspondingly more if the amine is a salt) were weighed into a reactivial and suspended in ethanol. The sealed reactivial was heated at 90°C overnight, or longer if deemed necessary by LC/MS analysis. The reaction mixture was filtered and the filtrate concentrated. If the reaction was performed on a small scale, the residue was purified by preparative reverse phase HPLC. On a larger scale, the crude material was partitioned between a suitable organic solvent (e.g.chloroform, ethyl acetate) and water. The layers were separated and the aqueous extracted further. The combined organic extracts were dried (MgS04), concentrated and purified by chromatography on silica.
General Procedure C - reaction of Intermediate II with an alkylamine
Intermediate II (1eq.) and the required amine (1eq. or more) were suspended in 2- propanol, and diisopropylethylamine (2eq.) was added. If the amine was added as a salt
(e.g.the hydrochloride salt), the quantity of DIPEA was increased accordingly. The resulting mixture was heated to reflux until tic or LC/MS indicated that reaction was complete. If the reaction was performed on a small scale, the reaction mixture was concentrated and then purified by preparative reverse phase HPLC. On a larger scale, the reaction was partitioned between dilute HCI and a suitable organic solvent. The layers were separated and the aqueous extracted further. The combined organic extracts were dried (MgS04), concentrated and purified by chromatography.
(2R.3R.4R.5R)-4-(Acetoxy)-2-f6-r3-chloro-2-methylphenvnamino1-9H-purin-9-ylV5-(prop-2- vnyl)tetrahvdrofuran-3-yl acetate (Intermediate III)
Intermediate II (43mg, 0.114mmol) was reacted with 3-chloro-2-methylaniline (0.041 mL, 0.34mmol) according to general procedure B (0.34mmol CaC03 in ethanol, heated for 13hours). Purification by preparative HPLC gave the desired product (purine N-9 linked to the 1-position of ribose) as a colourless gum, (17mg, 0.036mmol, 32%). m/z 484/486 (MH +), LCMS retention time 3.33min.
The isomeric product (purine N-7-linked) was also obtained as a colourless gum, (8mg, 0.017mmol, 15%). m/z 484/486 (MH +), LCMS retention time 3.22min.
(2R,3R.4R.5RV4-(Acetoxy)-2-{6-r3-fluoro-2-methylphenyl)amino1-9H-purin-9-yl)-5-(prop-2- vnyl)tetrahvdrofuran-3-yl acetate (Intermediate IV)
Intermediate II (43mg, 0.114mmol) was reacted with 3-fluoro-2-methylaniline (0.039mL, 0.34mmol) according to general procedure B (0.34mmol CaCO3 in ethanol, heated for
13hours). Purification by preparative HPLC gave the desired product (purine N-9 linked to the 1-position of ribose) as a colourless gum, (17mg, 0.037mmol, 32%). m/z 468 (MH +), LCMS retention time 3.19min.
The isomeric product (purine N-7-linked) was also obtained as a colourless gum, (11 mg, 0.023mmol, 20%).m/z468 (MH +), LCMS retention time 3.09min.
(2R.3R.4R.5R)-4-(Acetoxy)-2-(6-(tetrahvdro-2H-pyran-4-ylamino)-9H-purin-9-yll-5-(prop-2- vnyl)tetrahvdrofuran-3-yl acetate (Intermediate V)
Intermediate II (43mg, 0.114mmol) was reacted with 4-amino-tetrahydropyran hydrochloride salt (31 mg, 0.23mmol) according to general procedure C (0.45mmol DIPEA in isopropanol, heated for 2.5hours). Purification by preparative HPLC gave the desired product (purine N-9 linked to the 1-position of ribose) as a yellow gum, (18mg, 0.041 mmol, 36%). m/z 444 (MH +), LCMS retention time 2.64min.
The isomeric product (purine N-7-linked) was also obtained as a gum, (6mg, 0.014mmol, 12%).m/z 444 (MH +), LCMS retention time 2.52min.
Ethyl 4-(f9-r(2R.3R.4R,5R)-3.4-bis(acetoxy)-5-(prop-2-vnyl)tetrahvdrofuran-2-vn-9H-purin- 6-yl}amino)piperidine-1-carboxylate (Intermediate VI)
Intermediate II (43mg, 0.114mmol) was reacted with ethyl 4-amino-1-piperidinecarboxylate (39mg, 0.23mmol) according to general procedure B (0.45mmol DIPEA in isopropanol, heated for 2.5hours). Purification by preparative HPLC gave the desired product (purine N-9 linked to the 1-position of ribose) as a yellow gum, (22mg, 0.042mmol, 37%). m/z 4515 (MH +), LCMS retention time 2.91 min.
The isomeric product (purine N-7-linked) was also obtained as a gum, (10mg, 0.020mmol, 14%).m/z 444 (MH +), LCMS retention time 2.81 min.
General Procedure D - Deprotection of diacetate protected material to give diol final products
The diacetate-protected products were treated with a 10% solution of t-butylamine or n- butylamine in methanol. When the reaction was judged complete (by LC/MS analysis), the mixture was concentrated, and all traces of methanol and amine removed in vacuo. If necessary, the resulting diol could be purified by preparative HPLC.
Each of the N-9-linked isomers was deprotected using t-butylamine according to general procedure D, to give the diol products which did not require further purification and were characterised as follows:
Example 16 (2R.3R.4S.5RV2-r6-|-(3-chloro-2-methylphenyl)aminol-purin-9-yll-5-prop-2- vnyltetrahvdrofuran-3.4-diol
Deprotection of the diacetate Intermediate III (17mg, 0.036mmol) gave the diol as a colourless gum (14mg, 0.053mmol). m/z 400 (MH +), LC/MS retention time 2.84min, 1H NMR (MeOD) δ 8.30(1 H.s.CH), 8.15(1H,s,CH), 7.36(1 H.d.CH), 7.25(1 H,d,CH),
7.14(1 H,t,CH), 5.97(1 H,d,CH), 4.72(1 H,t,CH), 4.25(1 H,t,CH), 4.08(1 H,quart,CH), 2.71 and 2.56 (2x1H,2xdd,CH2), 2.23(4H,s,CH and CH3).
Example 17 : (2R.3R.4S.5R)-2-r6-r(3-fluoro-2-methylphenvnaminol-purin-9-vn-5-prop-2- vnyltetrahydrofuran-3,4-diol
Deprotection of the diacetate Intermediate IV (17mg, 0.037mmol) gave the diol as a colourless gum (15mg, 0.038mmol). m/z 384 (MH +), LC/MS retention time 2.69min, 1H NMR (MeOD) δ 8.31(1H,s,CH), 8.16(1 H,s,CH), 7.30(1 H,d,CH), 7.15(1 H,quart,CH), 6.90(1 H,t,CH), 5.97(1 H,d,CH), 4.72(1 H,t,CH), 4.25(1 H,t,CH), 4.08(1 H,quart,CH), 2.71 and 2.56 (2x1H,2xdd,CH2), 2.34 (1H,t,CH) 2.10(3H,s,CH3).
Example 18 : (2R.3R.4S.5R)-2-r6-(tetrahvdro-2H-pyran-4-ylaminoVpurin-9-yll-5-prop-2- vnyltetrahvdrofuran-3,4-diol
Deprotection of the diacetate Intermediate V (8mg, 0.017mmol) gave the diol as a colourless gum (7mg, 0.018mmol). m/z 360 (MH +), LCMS retention time 2.15min, 1H NMR (MeOD) δ 8.20(1 H,s,CH), 8.16(1H,s,CH), 5.91(1H,d,CH), 4.66(1 H,t,CH), 4.22- 4.28(1 H,br,CH), 4.21(1H,t,CH), 4.06(1 H,quart,CH), 3.90(2H,td) & 3.49(2H,dt) (2xCH2), 2.69 and 2.53 (2x1H,2xdd,CH2), 2.33 (1 H,t,CH) 1.93(2H,br dd) & 1.58(2H,ddd) (2xCH2).
Example 19: Ethyl 4-({9-r(2R.3R.4S.5R)-3.4-dihvdroxy-5-prop-2-vnyltetrahvdrofuran-2-yll- purin-6-yl)amino)piperidine-1-carboxylate
Deprotection of the diacetate Intermediate VI (11.5mg, 0.022mmol) gave the diol as a colourless gum (10mg, 0.023mmol). m/z 431 (MH +), LCMS retention time 2.46min, 1H NMR (MeOD) δ 8.20(1H,s,CH), 8.16(1 H,s,CH), 5.91(1 H,d,CH), 4.66(1 H,t,CH), 4.18- 4.28(1 H,br,CH), 4.21(1 H,t,CH), 3.98-4.09(5H,m,CH,CH2,1 each of 2xCH2), 3.98(2H,br,1 each of 2xCH2), 2.69 and 2.54 (2x1H,2xdd,CH2), 2.32 (1H,t,CH) 1.92-2.01 and 1.39- 1.51(2x2H,2xm,2xCH2), 1.17(3H,t,CH3).
LC/MS System
• Column: 3.3cm x 4.6mm ID, 3μm ABZ+PLUS • Flow Rate: 3mL/min
• Injection Volume: 5μl
• Temp: RT
Solvents: A: 0.1 % Formic Acid + 10mMolar Ammonium Acetate.
B: 95% Acetonitrile + 0.05% Formic Acid
Gradient: Time A% B%
0.00 100 0
0.70 100 0
4.20 0 100
5.30 0 100
5.50 100 0
Automated preparative HPLC.
• Column: 10cm x 21.2mm ID, 5μm ABZ+PLUS
• Flow Rate: 4mLΛnin
• Temp: RT
Solvents: A: HPLC water + 0.1% Formic Acid
B: Acetonitrile + 0.05% Formic Acid
Gradient: Time A% B%
0.00 95 5
1.45 95 5
20 10 90
30 10 90
32 95 5
Reporter Gene Experiments in yeast
Agonist activity at human adenosine A1 receptors was measured in yeast cell lines expressing the adenosine A1 receptor together with a chimeric G-protein which linked receptor stimulation to the expression of the enzyme β-galactosidase. Cells were plated out in 96-well plates in culture medium to which was added 2mM 3-AT (3-aminotriazoIe) to limit basal growth in the absence of agonist stimulation, and FDG (fluorescein di-β-D- galactopyranoside) as substrate for β-galactosidase, which converts FDG to fluorescein. For measurement of potency, agonists were added to the appropriate wells at a concentration range of approximately 10"10 - lO^M and incubated at 30°C for 18 hours. At this point the amount of fluorescein generated was measured in a spectrophotometer. From these readings, the concentration-dependence of the stimulation by the agonist can be calculated. One of the agonists tested on each 96-well plate was the standard non- selective agonist, N-ethylcarboxamidoadenosine (NECA), and the potency of all test agonists is expressed relative to that of the NECA standard.
(ECR = equipotent concentration ratio relative to NECA = 1)
Reporter Gene Experiments in CHO cells
Agonist activity at human adenosine A3 receptors was measured in Chinese hamster ovary (CHO) cells containing the CRE/SPAP/HYG (CRE = cyclic AMP response element; HYG = hygromycin resistance; SPAP = secreted placental alkaline phosphatase) reporter gene elements, which upon stimulation of cAMP levels produced SPAP. A cell line was used which was stably transfected with the human adenosine A3 receptor in addition to the above elements. Cells were plated out in 96-well plates in culture medium and incubated at 37°C for 1 hour. For measurement of potency, agonists were added to the appropriate wells at a concentration range of approximately 10"10 - lO^M. 15Min later, cAMP levels
were stimulated by addition of a maximal concentration of forskolin. All cells were then incubated for a further 5 hours at 37°C, and cooled to room temperature, after which a substrate for the phosphatase (para-nitrophenol phosphate, pNPP), which is converted by SPAP to a coloured reagent) was then added and the 96-well plates were read in a plate reader. From these readings, the concentration-dependence of the inhibition by the agonist for forskolin-stimulated SPAP production can be calculated. As with the yeast assays, one of the agonists tested on each 96-well plate was the standard non-selective agonist, N-ethylcarboxamidoadenosine (NECA), and the potency of all test agonists is expressed relative to that of the NECA standard. Table 2: Potencies in the reporter gene assay
*ECR = equipotent concentration ratio relative to NECA = 1 (see description in Reporter Gene Assay).
No toxicological effects are indicated/expected when a compound (of the invention) is administered in the above mentioned dosage range.
Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer or step or group of integers but not to the exclusion of any other integer or step or group of integers or steps.