WO2016159746A1

WO2016159746A1 - Novel ethynylxanthines, their preparation and use as inhibitors of matrix metalloproteinases and angiogenesis

Info

Publication number: WO2016159746A1
Application number: PCT/LV2015/000002
Authority: WO
Inventors: Pavels Arsenjans; Jelena VASILJEVA; llona DOMRACHEVA; Irina Shestakova; Anita GULBE; Iveta KANEPE-LAPSA; Valerjans Kauss; Ivars Kalvins
Original assignee: Latvian Institute Of Organic Synthesis
Priority date: 2015-03-27
Filing date: 2015-03-27
Publication date: 2016-10-06
Also published as: GB2553685B; GB2553685A8; GB2553685A

Abstract

The present invention relates to a novel ethynylxanthine derivatives of formula (I) which exhibit ability selectively suppress matrix metalloproteinases and therefore may act as inhibitors of angiogenesis, methods for their synthesis and use for the treatment and/or prevention of various diseases and disorders caused by overexpression of matrix metalloproteinases by administration of such substances.

Description

NOVEL ETHYNYLXANTHINES, THEIR PREPARATION AND USE AS INHIBITORS OF MATRIX METALLOPROTEINASES AND ANGIOGENESIS FIELD OF THE INVENTION

The present invention relates to a novel ethynylxanthine derivatives acting as matrix metalloproteinase inhibitors, methods for their synthesis and use for the treatment and/or prevention of various diseases and disorders caused overexpressed matrix metalloproteinases especially as angiogenesis inhibitors to prevent and/or to treat cancer methastasis and other diseases in which inhibition of angiogenesis is desired and/or required.

BACKGROUND OF THE INVENTION

Cancer is one of the main causes of death among economically developed countries (Jermal, CA Cancer J. Clin., 2011, 61 69-90). According to the International Health Organization, more than 7 million people diagnosed with various forms of cancer, die each year (Jermal, CA Cancer J. Clin., 2010, 60, 277-300; Siegel, CA Cancer J. Clin. 2012, 62, 10-29). Unfortunately, the number of cancer diagnosis in past few years has increased almost twice. In clinical practice, the treatment of cancer involves a wide range of chemotherapy drugs. Besides, most of them exhibit various side effects, high toxicity and moderate selectivity. Therefore, a new generation of selective, low toxic anticancer agent development is one of the main tasks in medicinal chemistry and pharmaceutical industries. Despite the advanced studies in the elaboration of anticancer drugs, the treatment outcome for brain malignant tumors still remains a challenge. The majority of promising antitumor drugs used against brain tumors in clinical trials, unfortunately, had limited impact on human survival, due to inadequacy of efficient drug delivery to the target in the central nervous system (CNS) at a sufficient concentration (Siegal, Neuro Oncol, 2013 10.1093/neuonc/not016; Lampson, Drug Discov. Today, 2009, 14, 185-191; Huse, Nat. Rev., 2010, 10, 319- 331 ).

Increased interest in xanthines stems from the fact that this heterocyclic system occurs in a number of natural substances; xanthine derivatives are able to cross through the blood brain barrier (BBB). It is an important class of compounds with a wide range of pharmacological effects, including anticancer, anti-HIV, anticoagulant, antispasmodic and antibacterial activity. Nowadays, in clinical practice, a number of anticancer drags based on the structure of the natural purine analogues were developed (cladribine, fludarabine, mercaptopurine, thioguanine, clofarabine, nelarabine, etc.). These are the first line therapy agents to cure hematologic malignant diseases. Agents in the therapeutic effect of complete remission constitute 80% after monochemotherapy course. Such compounds act as antimetabolites by replacing the natural nucleoside in DNA and RNA synthesis such as multi-cellular enzyme inhibitors. Unfortunately, these drugs showed a wide range of side effects, and high treatment costs limit the possibilities in clinical practice.

To minimize side effects currently being developed antitumor drugs based on caffeine core. Caffeine derivatives possess CNS expression as calcium agonist or antagonist effect. Recent studies show that caffeine-containing coffee daily use is able to lower mouth and brain cancer formation up to 39% (Michaud, Am. J. Clin. Nutr. 2010, 92, 1145-50; Kang, Cancer. Res. 2010, 70, 1173-83). Also, it reduces the risk of women cervical cancer development. Caffeine reduces a chance of prostate cancer formation in men by 60%. The same effect was observed in ability to prevent breast, colon and hepatic cancer (Hepatology, 2007, 46, 430-435). Caffeine enhances doxorubicin, cisplatin activity in metastatic carcinomas, lymphomas, bone and soft tissue sarcomas (Hayashi, Anticancer Res., 2005, 25, 2399-2406). Also, caffeine effectively inhibits breast cancer resistance protein (BCRP) multidrug resistance (MDR) on MCF-7 and MCF-7 MX100 (mitoxantrone-resistant) cell lines. One of the advantages of caffeine derivatives action is the ability to cross through the BBB, allowing substances to come into the CNS. It opens the possibility to cure malignant diseases, such as neuroblastoma and glioblastoma multiforme, in the brain (Vartanyan, Psychopharm. Biol. Narc, 2005, 5, 1093-1095). Drugs used in neurooncology (temodar, carmustine (BCNU), lomusthine (CCNU), etc.) have a limited ability to cross through the BBB and are highly toxic. In a series of caffeine analogues, such as 8-(3-(dimethylamino)propoxy)caffeine (proxyfeine), antitumor agents were developed, however introduction in the clinic of the EU and the US interferes, due to high levels of toxicity (LDso=355 mg/kg), as well as a large number of serious side effects. Proxyfeine (RU 2166948, 20.05.2001) is used in chemotherapy for cancer patients at high risk of brain metastases and the rehabilitation of the metastatic lesions to the brain, as well as the early stages of cancer metastasis prevention in Russia and other countries.

Proxyfeine

PCT Patent application No. WO2008077557 discloses preparation of 8-ethynyl xanthine derivatives as selective A2A receptor antagonists and their use as medicines, for example, in the treatment of dopamine-related movement disorders,

wherein R] and R₂ represent, e.g., hydrogen, C_1-6alkyl, cycloalkyl, heterocycloalkyl, aryl (wherein these groups may be further substituted), etc.; R₃ represents e.g., aryl, hetaryl group.

PCT Patent application No. WO2014/143799 A2, 2014 discloses preparation of N7- benzyl 8-ethynyl xanthine derivatives as agents for treatment of short transient receptor potential channel 5 (TrpC5) disorders A2A receptor antagonists and their use as medicines, for example, in the treatment of dopamine-related movement disorders,

N-Benzyl-8-ethynyl xanthines

Compounds such as caffeine (Liu, J. Cell Physiol. 2010, 224, 775-85; Camouse, Expert Rev. Anticancer Ther. 2005, 5, 1061-8; US8435541 B2; Yang, Clin. 0002

4

Experiment. Metastasis, 2005, 21, 719-735), proxyfeine, pentoxifylline (Kara, Curr. Ther. Res. Clin. Exp., 2008, 69, 488-502), and Temodar showed no ability to selectively inhibit human matrix metalloproteinases at low concentrations. According to our data novel ethynylxanthines are potent and selective matrix metalloproteinases inhibitors.

THE PRESENT INVENTION

We have discovered that certain ethynylxanthine derivatives exhibit ability selectively suppress matrix metalloproteinases and threfore may act as inhibitors of angiogenesis. Therefore, these substances may be therapeutically beneficial in the treatment of diseases dependent on extended angiogenesis expeccially formation of methastasis by the progerssion of different types of cancer . These substances may be administered in the form of a pharmaceutical composition, wherein they are present together with one or more pharmaceutically acceptable diluents, carriers, or excipients.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide novel pharmaceutical compounds, which are inhibitors of matrix metalloproteses and therefore can be used as angiogenesis inhibitors, methods for their synthesis and the treatment and/or prevention of various diseases and disorders caused by overexpression of matrix metallopreoteases by administration of such substances.

Yet additional objects will become apparent hereinafter, and still further objects will be apparent to one skilled in the art.

SUMMARY OF THE INVENTION

What we therefore believe to be comprised by our invention may be summarized inter alia in the following words:

A compound selected from those of Formula I

0002

5 wherein

R¹ represents hydrogen, Q^alkyl, hydroxy-C₂_₄alkyl, Ci_3alkoxy-C₂_₄alkyl, C|. 3alkylcarbonyl-Ci-4alkyl or C₁.₃alkyl(Ci.₃alkyl)amino-C₂_₄alkyl;

R² represents

4alkyl, Ci-₃alkyl(Ci-₃alkyl)amino-C2-₄alkyl or halo-C₂-₄alkyl;

R³ represents Q^alkyl, allyl or Q-aalko y^^alkyl; with the proviso that if substituent R³ is at purine N(7) atom the dotted line between N(7) and C(8) represents no bond, and the dotted line between C(8) and N(9) represents chemical bond;

and

with the proviso that if substituent R³ is at purine N(9) atom the dotted line between N(7) and C(8) represents chemical bond, and the dotted line between C(8) and N(9) represents no bond;

R⁴ represents Q^alkyl, hydroxy-Q^alkyl, Ci_₃alkoxy-C₁. alkyl, amino-C^alkyl, 1- hydroxy-di-(C_1-3alkyl)methyl, l-amino-di-(Ci_-3alkyl)methyl, l-hydroxy-cyclo-C_3- ₆alkyl, l-amino-cyclo-C_3-6alkyl, l-(hydroxy-C_1-3alkyl)-cycloC_3-6alkyl, Q. ₃alkylamino-C_1-3alkyl, C^alkyliC^alky^amino-Ci-salkyl, di-(C]_-3alkoxy-C_2-4alkyl)- amino-Ci_-3alkyl, heterocyclyl-Ci-₃alkyl, aryl or heteroaryl; wherein the term "heterocyclyl" represents saturated 4-7 membered heterocycle containing one or two heteroatoms selected from oxygen, sulfur and nitrogen, wherein the

heterocyclyl may be azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, tetrahydrofuryl, morpholinyl, thiomorpholinyl and piperazinyl; the term "aryl" represents phenyl or phenyl substituted by one or more substituents selected independently from halogen, cyano, Q^alkoxycarbonyl, N-Q. ₄alkylaminocarbonyl, N,N-di-(Ci_-3alkyl)aminocarbonyl, CH₂OH, trifluoromethyl, Ci_ ₄alkyl, allyl, C₂-₄alkynyl, C_1- alkoxy, difluoromethoxy, trifluoromcthoxy, cyclo- C-₃.₆alkoxy, hydroxy-C i ^alkyl, C i .₃alkox y-C i .₄alkyl , C]_-3alkoxy-C2-₄alkoxy, di-(C|. ₃alkyl)amino, di-(C i _₃alkyl)amino-C] _₃alkyl, di-fC i _₃alkyl)amino-C₂-₄alkoxy, C i .₄alkylsulfonylamino and C^alkyl-aminosulfonyl; the term "heteroaryl" represents an aromatic 5 or 6 membered ring comprising one to three heteroatoms selected from oxygen, sulfur and nitrogen, wherein the heteroaryl may be unsubstituted or optionally substituted by one or more substituents selected independently from halogen, cyano, trifluoromethyl, C_1-4alkyl, Ci_-4alkoxy, difluoromethoxy, trifluoromefhoxy, cyclo-C₃_₆alkoxy, Ci_₃alkoxy-C_1-4alkyl, cyclo-C₃_ ₆alkylamino and di-(Ci_-3alkyl)amino; its optical isomers, polymorphs and pharmaceutically acceptable acid addition salts and hydrates and solvates thereof.

Specific compounds of Formula I within the present invention include but are not limited to:

8-(3-Hydroxy-3-memylbut-l-yn-l-yl)-13,7- methyl-lH-purine-2,6(3H_>7H)-dione, 8-(( 1 -Hydroxycyclohexyl)ethynyl)- 1 ,3 ,7-trimethyl- lH-purine-2,6(3H,7H)-dione, 8 (l-Aminocyclohexyl)ethynyl)-3,7-dimethyl-lH-purine-2,6(3H,7H)-dione,

8-(3-(Dimethylamino)prop- 1 -yn- 1 -yl)- 1 ,3 ,7-trimethyl- 1 H-purine-2,6(3H,7H)-dione, 8-(3-(bis(2-methoxyethyl)amino)prop- 1 -yn- 1-yl)- 1 ,3,7-trimethyl- lH-purine- 2,6(3H,7H)-dione,

1 ,3 ,7-Trimethyl-8-(3-(pyrrolidin- 1 -yl)prop- 1 -yn- 1 -yl)- lH-purine-2,6(3H,7H)-dione, l,3,7-Trime l-8-(3-(piperidin-l-yl)prop-l-yn-l-yl)-lH-purine-2,6(3H,7H)-dione, 8-(3-(Azepan-l-yl)prop- 1-yn- 1-yl)- 1 ,3 ,7-trimethyl- lH-purine-2,6(3H,7H)-dione, l,3,7-Trimethyl-8-(3-morpholinoprop-l-yn-l-yl)-lH-purine-2,6(3H,7H)-dione, 8-(3-Hydroxy-3-methylbut-l-yn-l-yl)-3,7-dimethyl-l-(5-oxohexyl)-lH-purine- 2,6(3H,7H)-dione,

8-(( 1 -Hydroxycyclohexyl)ethynyl)-3 ,7-dimethyl- 1 -(5-oxohexyl)- 1 H-purine

2,6(3H,7H)-dione,

8-((l-Aminocyclohexyl)ethynyl)-3,7-dimethyl-l-(5-oxohexyl)-lH-purine- 2,6(3H,7H)-dione, 8-(3· (Bis(2-methoxyethyl)amino)prop- 1-yn- l-yl)-3,7-dimethyl- 1 -(5-oxohexyl)- I II- purine-2,6(3H,7II)-dione,

3,7-Dimethyl- 1 -(5-oxohexyI)-8-(3-(pyrrolidin- 1 -yl)prop- 1-yn- 1-yl)- 1 H-purine 2,6(3H,7H)-dione,

3,7-Dimethyl- 1 -(5-oxohexyl)-8-(3-(pipcridin- 1 - yl)prop- 1-yn-l -yl)- 1 H-purine- 2,6(3H,7H)-dione,

l,3,7-Trimethyl-8-(phenylethynyl)-lH-purine-2,6(3H,7H)-dione,

1 ,3,9-Trimethyl-8-(3-(pyrrolidin- 1 -yl)prop- 1 -yn- 1 -yl)- 1 H-purine-2,6(3H,9H)-dione, 1 ,3 ,9-Trimethyl-8-(phenylethynyl)- 1 H-purine-2,6(3H,9H)-dione

and optical isomers, polymorphs, and pharmaceutically-acceptable acid addition salts, hydrates, and solvates thereof.

The invention also relates to a process for the synthesis or preparation of a compound selected from those of Formula I as defined above, comprising reaction of a compound of Formula II :

wherein R¹, R² and R³ are as defined for Formula I above, with a compound of Formula III:

≡÷-R⁴

III wherein R⁴ is as defined for Formula I above, optionally in the presence of base in an appropriate solvent (e.g., DIEA in DMF, NMP, DMAC or EtOAc), in the presence of Cul and palladium catalyst generated in situ (e.g., from PdCl₂ or Pd(OAc)₂ and PPh₃) or commercially available (Ph₃P)₄Pd to yield a compound of Formula I, which may be converted, if desired, into an optical isomer, polymorph, pharmaceutically-acceptable salt, hydrate or solvate. 2

8

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "Ci_₄alkyl" represents straight or branched chain alkyl groups having 1, 2, 3 or 4 carbon atoms, examples of such alkyl groups include methyl, ethyl, n-propyl, 2-propyl, n-butyl, 2-butyl, iso-butyl and tert-butyl.

The term "cyclo-C3-₆alkyl" represents monocyclic alkyl groups having 3, 4, 5 or 6 carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The term "heterocyclyl" represents a saturated 4-7 membered heterocycle containing one or two heteroatoms selected from oxygen, sulfur and nitrogen, examples of such heterocyclyl groups include azetidinyl, pyrrolidinyl, piperidinyl, azepanyl,

tetrahydrofuryl, morpholinyl, miomorpholinyl and piperazinyl.

The term "halo" or "halogen" represents fluorine, chlorine, bromine and iodine.

In addition, using methods known to those skilled in the art, analogs and derivatives of the compounds of the invention can be created which have improved therapeutic efficacy, i.e., higher potency and/or selectivity at a specific targeted receptor type, greater ability to penetrate mammalian blood-brain barriers, fewer side effects, etc.

It will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. It is to be understood that the present invention encompasses any racemic, optically-active, tautomeric, or stereoisomeric form of a compound of the mvention, which possesses the useful properties described herein.

For therapeutic use, the compounds of Formula I can be in the form of a pharmaceutically acceptable salt or a solvate. The term "pharmaceutically acceptable" refers here to the therapeutically active non-toxic salt forais, which the compounds of Formula I are able to form. The latter can conveniently be obtained by treating the base form with such appropriate acids as inorganic acids such as hydrochloric acid, hydrobromic acid; sulfuric acid; nitric acid; phosphoric acid and the like; or organic acids such as acetic, propanoic, hydroxyacetic, 2-hydroxypropanoic, oxopropanoic, oxalic, malonic, succinic, maleic, fumaric, malic, tartaric, methanesulfonic, benzenesulfonic, 4-methylbenzencsulfonic, 2-hydroxybenzoic, and like acids. Conversely, the salt may be converted to the free base by treatment with alkali. Scheme 1 describes the preparation of compounds of Formula I of the present invention. All of the starting materials II are prepared by representative procedures described in Schemes 2 and 3, by procedures well known to one of ordinary skill in organic chemistry or can be obtained commercially. All of the final compounds of the present invention are prepared by procedures described in these charts or by procedures analogous thereto, which procedures would be well known to one of ordinary skill in organic chemistry. All of the variables used in the schemes are as defined below or as in the claims.

We have found that product yields in palladium catalyzed cross-coupling of terminal acetylenes and 8-bromoxanthines strongly depends on the nature of catalyst and solvent. In accordance with experimental data, reaction of terminal acetylenes III with 8-bromoxanthines under routine experimental conditions (Methods A and B) in general led to the formation of the corresponding 8-ethynylxanthines I in very low yields. Suprisingly, we have found that use of the mixture of N-methylpyrrolidine and toluene (1:1) (Method C) gave the desired products in high yields. Alternative method was elaborated (Method D). Performing the reaction in ethyl acetate and using 2 mol- % of Pd(PPh₃)4 and 2 mol-% of PdCl₂ decreases the cost of reaction. It should be noted that in the representative example the treatment of 8-bromocaffeine with two equivalents of 2-methylbut-3-yn-2-ol under conditions of Method D led to the formation of desired 8-(3-hydroxy-3-methylbut-l-yn-l-yl)-l,3,7-trimethyl-lH-purine- 2,6(3H,7H)-dione (1-1) in 55% yield and by-product 8-[(E)-5-hydroxy-2-(l-hydroxy- l-memylemyl)-5-memylhex-l-en-3-ynyl]-l,3,7-trimethyl-lH-purine-2,6(3H,7H)- dione in 26% yield similar to the method elaborated previously (Arsenyan, Tetrahedron Lett., 2013, 54, 6524-6528).

Scheme 1. General procedure toward compounds of Formula I .

Reaction conditions:

Method A. (Ph₃P)₂PdCl₂, Cul, N-methylpyrrolidine or DMF, DEEA, 50 °C;

Method B. Pd(OAc)₂, Ph₃P, Cul, N-methylpyrrolidine or DMAC, DIEA, 55 °C;

Method C. Pd(OAc)₂, Ph₃P, Cul, N-methylpyrrolidine/toluene (1:1), DIEA, 50 °C; Method D. Pd(PPh₃)₄, PdCl₂, Ph₃P, Cul, ethyl acetate, DIEA, 40 °C.

Compounds II, wherein R³ is at purine N(7), can be prepared by bromination of position 8 of corresponding 8-unsubstituted 1,3,7-substituted lH-purine-2,6(3H,7H)- diones. Representative method for the synthesis of compound II wherein R 1 and R 3 are methyl groups shown in Scheme 2. Commercially available caffeine (1) and pentoxifylline (2) are brominated in position 8 by N-bromosuccinimide (NBS) in dichloromethane in analogy to published procedure [Synlett, 2012, 23, 1191-1198]. Both products 3 and 4 were isolated in almost quantitative yields.

Scheme 2. General procedure for the preparation of 8-bromo lH-purine-2,6(3H,7H)- diones II, wherein R³ is at purine N(7) (3, 4).

1 (R¹ = Me) 3 (R¹ = Me)

2 (R¹ = MeC(0)(CH₂)₄) 4 (R = MeC(0)(CH₂)₄)

Compounds II, wherein R³ is at purine N(9), can be prepared by bromination of position 8 of respective 8-unsubstituted 1 ,3,9-substituted lH-purine-2,6(3H,9H)- diones. Representative method for the synthesis of compound II wherein R¹, R² and R³ are methyl groups (compound 11) is shown in Scheme 3. Thus, l,3-dimethyl-6- chlorouracil (6) was prepared by the treatment of 1 ,3-dimethylbarbituric acid (5) in phosphorous oxychloride (POCl₃) with water followed by heating under reflux. Then treatment of compound 6 with a mixture of fuming nitric acid and sulfuric acid resulted in formation of 6-chloro- 1 ,3-dimethyl-5-nitropyrimidine-2,4( 1 H,3H)-dione (7). Next, choro substituent was substituted by methylamino moiety to give intermediate 8 and nitro group was reducted by hydrogen using palladium on charcoal as a catalyst. Finally, condensation of 5-amino-l,3-dimethyl-6-methylamino- pyrimidine-2,4(lH,3H)-dione (9) with formic acid gave l,3,9-trimethyl-3,9-dihydro- purin-2,6(3H,9H)-dione (10). Necessary 8-bromo-l,3,9-trimethyl-3,9-dihydropurin- 2,6(3H,9H)-dione (11) was obtained in the reaction of compound 10 with N- bromosuccinimide (NBS) in acetonitrile. The procedures shown in the Scheme 3 are general and can be used to prepare analogous 8-bromo 1,3,9-substituted lH-purine- 2,6(3H,9H)-diones. Scheme 3. Preparation of 8-bromo lH-purine-2,6(3H,9H)-dione 11 (II, wherein R³ is at purine N(9)).

6

9 10 11 It will be appreciated that in the above transformations it may be necessary or desirable to protect any sensitive groups in the molecule of the compound in question in order to avoid undesirable side reactions.

EXPERIMENTAL PART

The compounds and their preparation of the present invention will be better understood in connection with the following examples, which are intended as an illustration of and not a limitation upon the scope of the invention. Hereinafter, "DMF" is defined as Ν,Ν-dimethylformamide, "DMAC" is defined as N,N-dimethylacetamide, "NMP" is defined as N-methylpyrrolidone, "DMSO" as dimethyl sulfoxide, "HQ" as hydrochloric acid, „aq. NH ' as aqueous ammonia solution, „"MeCN" as acetonitrile, "DIEA" as diisopropylethylamine, "EtOAc" as ethyl acetate, "it" as room temperature.

Intermediate 3.

8-Bromo-l,3,7-trimethyl"lH-pu ione (3)

To a round-botton flask containing freshly distilled CH₂C1₂ (150 mL) was added caffeine (10.0 g, 0.05 mol) and NBS (17.6 g, 0.10 mol). When the solids had dissolved in solvent, water (50 mL) was added and the reaction mixture was stirred for 5 days. Then cold 2 M aq.NaOH (30 mL) was added and the mixture was shaken till decolorization. The organic layer was separated, washed with water (2x200 mL), dried over sodium sulfate, filtered and evaporated to give the title compound (13.5 g, 99%). 1H NMR (CDCI₃ TMS, 400 MHz) (ppm): 3.41 (s, 3H, CH₃), 3.57 (s, 3H, CH₃), 3.97 (s, 3H, CH₃).

Intermediate 4

8-Bromo-3,7-dimethyl-l-(5 -dione (4)

To a round-botton flask containing freshly distilled CH₂C1₂ (150 mL) was added pentoxifylline (13.4 g, 0.05 mol) and NBS (17.6 g, 0.10 mol). When the solids had dissolved, water (50 mL) was added and the reaction mixture stirred for 5 days. Then cold 2 M aq. NaOH (30 mL) was added and the mixture was shaken till decolorization. The organic layer was separated, washed with water (2x200 mL), dried over sodium sulfate, filtered and evaporated to give the title compound (16.95 g, 95%). ^lH NMR 00002

13

(CDCI₃ TMS, 400 MHz) f)^' (ppm): 1.61-1.67 (m, 4H), 2.14 (s, 3H), 2.49 (t, 2H), 3.54 (s, 3H), 3.95 (s, 3H), 3.99 (t, 2H).

Intermediate 11.

8-Bromo-l,3,9-trimethyI-l//-purine-2,6i3H,9//)-dione (11).

a) 6-ChIoro-l,3-dimethyIpyrimidine-2,4(lH,3H)-dione (6).

To a suspension of 1,3-dimethylbarbituric acid (7.8 g, 50 mmol) in POCl₃ (60 mL) was slowly added water (2.5 mL, 0.14 mol) and the reaction mixture was refluxed for 1 h under nitrogen atmosphere. An excess of POCl₃ was distilled off under reduced presssure and the residue was quenched with 40 mL of ice water. The mixture was extracted with chloroform (2x100 mL). The organic phase was dried over anhydrous Na₂S0₄, evaporated to dryness, and the residue crystallized with ether to give the title compound (8.0 g) as a yellow crystals. ^]H NMR (400 MHz, DMSO- d₆) δ (ppm): 3.10 and 3.15 (both s, both 3H), 6.07 (s, 1H).

b) 6-Chloro-l,3-dimethyl-5-nitropyrimidine-2,4(lH,3H)-dione (7).

Compound 6 (15 g, 74.5 mmol) was added portionwise to a cooled solution of cone. H₂S0₄ (40 mL) . The reaction temperature was maintained below 10 °C. Fuming nitric acid (15 mL) was added dropwise to the above reaction mixture and it was stirred for 2 h at the same temperature. The reaction nixture was poured onto the ice cold water (500 mL) and extracted with chloroform (2x260 mL). The combined organic extracts were washed with water (260 mL), dried over anhydrous Na₂S0₄ and concentrated in vacuo to give the title compound (12.0 g) as a yellow solid. 1H NMR (400 MHz, DMSO-d₆) δ (ppm): 3.07 and 3.26 (both s, both 3H).

c) l,3-Dimethyl-6-methylamino-5-nitropyrimidine-2,4(lH,3H)-dione (8).

To a stirred solution of 6-chloro-l,3-dimethyl-5-nitropyrimidine-2,4(lH,3H)-dione (7) (1 1.0 g, 50.1 mmol) in chloroform (90 mL) was added dropwise a solution of 40% aq. methylamine (7.76 mL, 100.2 mmol) in dichloromethane (20 mL) under nitrogen atmosphere. After stirring at room temperature for 1 h the reaction mixture was concentrated under reduced pressure. The residue was crystallized from ether to give 15 000002

14 the title compound (12.5 g) as a yellow solid. Ή NMR (400 MHz, DMSO- d_fi) δ (ppm): 2.49, 2.75 and 3.35 (all s, all 3H), 7.74 (br s, I H).

d) 5-Amino-l^-dimethyl-6-methylaminc»-pyriinidiiie-2,4(l//,3//)-dione (9).

To a stirred solution of l,3~dimethyl-6-methylamino-5-nitropyrimidine~2,4(lH,3H)- dione (8) (2 g, 10 mmol) in wet methanol (140 mL) was added 10% Pd-C ( 1 g) under hydrogen balloon atmosphere at room temperature. After overnight stirring, the reaction mixture was filtered and the filtrate was concentrated to give the title compound (1.5 g). 1H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.89 (d, 3H), 3.31 (s, 3H), 3.40 (s, 3H), 4.75 (br s, 1H). MS (EI) m/z: 185 [M]⁺.

e) l,3,9-Trimethyl-LH-purine-2,6(3H,9H)-dione (10).

A mixture of 5-amino-l,3-dimethyl-6-methylamino-pyrimidine-2,4(lH,3H)-dione (9) (1.5 g) and formic acid (10 mL) was refluxed for 3 h under nitrogen atmosphere. An excess of formic acid was evaporated under reduce pressure. The residue was extracted with CH₂CI₂, washed with aq. Na₂C0₃, dried over Na₂S0₄ and evaporated to dryness. The residue was purified by column chromatography (eluent CH₂C1₂ : MeOH, 10:1) to give the title compound (0.5 g) as a white solid. ^!H NMR (400 MHz, DMSO-d₆) δ (ppm): 3.22, 3.68 and 3.93 (all s, all 3H), 7.66 (s, 1H). MS (EI) m/z: 195 [M+2]⁺.

f) 8-Bromo-l,3,9-trimethyl-lH-purine-2,6(3H,9H)-dione (11).

A mixture of l,3,9-trimethyl-lH-purine-2,6(3H,9H)-dione (10) (1.0 g, 5.15 mmol) and NBS (1.2 g, 6.7 mmol) in dry MeCN (40 mL) was stirred for 3 h at room temperature. Water (30 mL) and CH₂C1₂ (150 mL) was added, the organic phase was separated, dried over Na₂S0₄ and evaporated to dryness. The residue was purified by column chromatography (CH₂C1₂ : MeOH, 9:1) to give the title compound (0.57 g) as a white solid with m.p. >200 °C. 1H NMR (400 MHz, DMSO-d₆) δ (ppm): 3.22, 3.69 and 3.88 (all s, all 3H). MS (EI) m/z: 375 [M+2]⁺.

Example 1

8 3-Hydroxy-3-methyIbut-l-yn-l-yl)-l,3,7-trimethyI-lH-piirine-2,6(3H,7H)- dione (1-1)

0002

Method A. To a mixture of PdCl₂ (113 mg, 0.637 mmol), Cul (242 mg, 1.27 mmol), and triphenylphosphine (333 mg, 1.27 mmol) was added dry NMP or DMF (40 mL). The reaction mixture was allowed to stir at 40 °C for 15 min with simultaneous barbotation with argon. Then a solution of 8~bromo-l ,3,7-trimethyl TH-purine- 2,6(3H,7H)-dione (1.73 g, 6.37 mmol) and 2~methylbut-3-yn-2-ol (0.93 mL, 9.56 mmol) and dry DIEA (3.6 mL) was added and stirring was continued at 50 °C for 24 h. After cooling to rt, the reaction mixture was poured to EtOAc (300 mL), washed with brine (80 mL) containing aq. N¾ (0.5 mL) and stirred at rt for the additional 30 min. The aqueous phase was separated and the organic phase was washed with brine (3 x 80 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel using a mixture of water (containing 0.1% HC1) - MeCN (5% - 70%) as eluent to give the title compound in 42% yield, mp = 180-182 °C. Ή NMR (CDC1 TMS, 400 MHz) (ppm): 1.63 (s, 6H), 3.36 (s, 3H), 3.51 (s, 3H), 3.52 (br s, 1H), 3.90 (s, 3H). ¹³C NMR (CDCI₃/TMS, 100.6 MHz) (ppm): 28.0, 29.8, 30.8, 32.9, 65.1, 69.6, 102.5, 107.5, 135.0, 147.4, 151.5, 154.6. MS (EI) mlz: 277.2 [M+l]⁺.

Method B. To a mixture of Pd(OAc)₂ (100 mg, 0.446 mmol), Cul (169 mg, 0.868 mmol), and triphenylphosphine (233 mg, 0.890 mmol) dry NMP or DMAC (40 mL) was added. Reaction mixture was allowed to stir for 15 min at 40 °C with simultaneous barbotation with argon. Then a solution of 8-bromo-l,3,7-trimethyl-lH- purine-2,6(3H,7H)-dione (1.21 g, 4.46 mmol) and 2-methylbut-3-yn-2-ol (0.65 mL, 6.69 mmol) and dry DIEA (4.0 mL) was added and stirring was continued at 55 °C for 24 h. Compound 1-1 (31% yield) was isolated as in Method A.

Method C. To a mixture of Pd(OAc)₂ (100 mg, 0.446 mmol), Cul (169 mg, 0.868 mmol), and triphenylphosphine (233 mg, 0.890 mmol) dry NMP (10 mL) was added. Reaction mixture was allowed to stir for 15 min at 40 °C with simultaneous barbotation with argon. Then solution of 8-bromo-l,3,7-trimethyl-lH-purine- 2,6(3H,7H)-dione (1.21 g, 4.46 mmol) and 2-methylbut-3-yn-2-ol (0.65 mL, 6.69 mmol) and dry DIEA (4.0 mL) in NMP (10 mL) and toluene (20 mL) was added and stirring was continued at 50 °C for 24 h. Compound 1-1 (72% yield) was isolated as in Method A. Method D. A vial charged with Pd(PPh₃)₄ (346 mg, 0.3 mmol), PdCl₂ (51 mg. 0.3 mmol), Ph₃P (157 mg, 0.6 mmol), Cul (58 mg. 0.3 mmol), 8-bromo- 1,3,7- trimethyl-lH- purine-'2,6(3H,7H)-dione (4.08 g, 15.0 mmol) and 2-methylbut-3~yn-2- ol (2.05 mL, 21.0 mmol), DIE A (5.0 mL) and ethyl acetate (70 mL) was stirred for 15 min at 40 °C with simultaneous barbotation with argon. Then reaction mixture was stirred for additional 2-4 h. After cooling to rt, the reaction mixture was filtered through a silica gel pad and washed with EtOAc (200 mL). Then solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel using mixture of water (containing 0.1% aq. HC1) - MeCN (5% - 70%) as eluent to give compound 1-1 in 55% yield and 8-[(£)-5- hydroxy-2-(l -hydroxy- l-methylethyl)-5-methylhex- l-en-3-ynyl]- 1 ,3,7-trimethyl- 1H- purine-2,6(3H,7H)-dione as a by-product in 26% yield. By-product: 1H NMR (CDC1 TMS, 400 MHz) <?(ppm): 1.32 (s, 6H), 1.52 (s, 6H), 3.35 (s, 3H), 3.49 (s, 3H), 4.03 (s, 3H). 5.13 (br s, 2H), 6.65 (s, 1H). ¹³C NMR (CDC1₃/TMS, 100.6 MHz) δ (ppm): 28.1, 29.7, 29.9, 31.0, 33.7, 65.3, 70.7, 78.6, 95.9, 107.9, 109.6, 146.5, 147.3, 151.2, 155.0, 156.1.

Example 2

8-((l-Hydroxycyclohexyl)ethynyl)-l,3,7-trimethyl-lH-purine-2,6(3H,7H)-dione (1-2)

Yield: 49% (Method C), 54% (Method D), 11% (Method B); mp = 194-196 °C. *H NMR (CDC1 TMS, 400 MHz) δ (ppm): 1.24-1.33 (m, 1H), 1.48-1.57 (m, 3H), 1.66- 1.75 (m, 4H), 1.94-2.02 (m, 2H), 3.30 (br s, 1H), 3.34 (s, 3H), 3.49 (s, 3H), 3.89 (s, 3H). ¹³C NMR (CDCI3/TMS, 100.6 MHz) (ppm): 22.9, 24.8, 27.9, 29.7, 32.9, 39.2, 68.6, 71.5, 102.0, 107.4, 135.1, 147.3, 151.4, 154.5. MS (EI) mlz: 317.5 [M+lf.

Example 3

8-((l-AminocycIohexyI)ethynyI)-3,7-<Iimethyl-lH-purine-2,6(3//,7H)-dione (1-3)

Yield: 48% (Method D), 43% (Method C), 16% (Method B), 7 % (Method A); mp = 187- 189 °C. Ή NMR (CDCI₃ TMS, 400 MHz) <5(ppm): 1.20-1.29 (m, 1H), 1.48-1.67 (m, 5H), 1.72-1.77 (m, 2H), 1.81 (br s, 2H), 1.93-1.97 (m, 2H), 3.39 (s, 3H), 3.55 (s, 3H), 3.98 (s, 3H). ¹³C NMR (CDCI3/TMS, 100.6 MHz) <S(ppm): 23.2, 25.1, 27.9, 29.7, 33.0, 39.6, 50.4, 70.9, 104.5, 107.6, 135.8, 147.6, 151.5, 154.8. MS (EI) ml_Z 302.3 [M+l]⁺.

Example 4

8 3 Dimethylamino)prop-l-yn-l-yl)-l,3,7-trimethyl-lH-purine-2,6(3H,7H)- dione (1-4)

Isolated as hydrochloride. Yield: 58% (Method D), 47% (Method C), 4% (Method B); mp = 197-199 °C (dec.) 1H NMR (DMSO-d₆, 400 MHz) (ppm): 2.88 (s, 6H), 3.21 (s, 3H), 3.38 (s, 3H), 3.96 (s, 3H), 4.50 (s, 2H). ¹³C NMR (DMSO-d₆, 100.6 MHz) δ (ppm): 27.6, 29.3, 33.0, 41.6, 45.7, 76.4, 87.1, 107.6, 132.9, 146.8, 150.7, 154.0. MS (EI) mlz: 277.5 [M+l].

Example 5

8-(3-(bis(2-methoxyethyl)amino)prop-l-yn-l-yl)-l,3_>7-trimethyl-lH-purine- 2,6(3H,7H)-dione (1-5)

Yield: 33% (Method D), 30% (Method C), 10% (Method B); foam. Ή NMR (CDCI3 TMS, 400 MHz) £(ppm): 2.79 (t, 4H), 3.31 (s. 6H), 3.34 (s, 3H), 3.48 (t, 4H), 3.51 (s, 311), 3.82 (s, 2H), 3.96 (s, 3H). ^UC NM (CDC1₃/TMS, 100.6 MHz) <5> (ppm): 27.8, 29.6, 33.0, 44.1. 53.5, 58.7, 70.9, 73.2, 94.1, 107.5, 135.4, 147.5, 151.4, 154.7. MS (EI) mlz: 364.5 [M+l]⁺. Example 6

1 _»7-Trimethyl-8-(3-(pyrroUdin-l-yl)prop-l-yn-l-yl)-lH-piiriiie-2,6(3//,7H)- dione (1-6)

Yield: 57% (Method D), 52% (Method C), 8% (Method A); mp = 149-150 °C. ^lH NMR (CDC1₃ATMS, 400 MHz) S(ppm): 1.83-1.86 (m, 4H), 2.68-2.72 (m, 4H), 3.39 (s, 3H), 3.56 (s, 3H), 3.75 (s, 2H), 4.00 (s, 3H). ¹³C NMR (CDC1₃/TMS, 100.6 MHz) δ (ppm): 23.8, 27.9, 29.7, 33.1, 43.6, 52.7, 72.6, 94.5, 107.6, 135.4, 147.6, 151.5, 154.8. MS (EI) mlz: 302.3 [M+l]⁺. Example 7

1 ,7-Trimethyl-8-(3-(piperidin-l-yl)prop-l-yn-l-yl)-lH-purine.2,6(3H,7H)-dione (1-7)

Yield: 35% (Method C), 30% (Method D); mp = 137-138 °C. 1H NMR (CDC1₃/TMS, 400 MHz) (ppm): 1.40-1.48 (m, 2H), 1.60-1.67 (m, 4H), 2.54-2.60 (m, 4H), 3.39 (s, 3H), 3.55 (s, 3H), 3.60 (s, 2H), 4.00 (s, 3H). ¹³C NMR (CDC1 TMS, 100.6 MHz) δ (ppm): 23.6, 25.8, 27.9, 28.0, 29.8, 48.3, 53.4, 73.2, 94.2, 107.6, 135.5, 147.6, 151.5, 154.8. MS (EI) mlz: 316.3 [M+l]⁺. Example 8

8-(3-(Azepan-l-yl)prop-l-yn-l-yl)-l,3,7-trimethyI-lH-purine-2,6(3H,7i/)-dione

Yield: 34% (Method C), foam. Ή NMR (CDCl TMS, 400 MHz) (ppm): 1.61-1.64 (m, 4H), 1.70- 1.76 (m, 4H), 2.81 (t, 4H), 3.39 (s, 3H), 3.55 (s, 3H), 3.74 is, 211), 4.01 (s, 3H). ¹³C NMR (CDCl TMS, 100.6 MHz) <5 (ppm): 26.6, 27.9, 28.0, 29.7, 33.1, 48.7, 55.3, 72.9, 93.2, 107.6, 135.4, 147.6, 151.5, 154.8. MS (EI) m/z: 330.3 [M+l]⁺.

Example 9

l,3,7-Trimethyl-8-(3-morpholinoprop-l-yn-l-yl)-lH-purine-2,6(3H,7H)-dione (I-

9)

Yield: 38% (Method C), mp =188-190 °C. 1H NMR (CDC1₃ TMS, 400 MHz) δ (ppm): 2.64 (t, 4H), 3.40 (s, 3H), 3.56 (s, 3H), 3.64 (s, 2H), 3.76 (t, 4H), 4.01 (s, 3H). ¹³C NMR (CDCl TMS, 100.6 MHz) (ppm): 28.0, 29.7, 33.2, 47.8, 52.3, 66.7, 73.7, 93.1, 107.7, 135.2, 147.6, 151.5, 154.8. MS (EI) m/z: 318.3 [M+l]⁺.

Example 10

8-(3-Hydroxy-3-methylbut-l-yn-l-yl)-3,7-dimethyl-l-(5-oxohexyl)-lH-purine- 2,6(3H,7H)-dione (1-10

Yield: 47% (Method D), 44% (Method C); foam. Ή NMR (CDC1₃ TMS, 400 MHz) δ (ppm): 1.62- 1.66 (m, 4H), 1.65 (s, 6H), 2.12 (s, 311), 2.48 (t, 2H), 2.72 (br s, 1H), 3.53 (s, 3H), 3.96 (s, 3H), 3.97 (t, 2H). ¹³C NMR (CDCl TMS, 100.6 MHz) (ppm): 20.9, 27.3, 29.7, 29.9, 30.9, 33.0, 40.9, 43.1, 65.4, 70.0, 102.2, 107.7, 135.1, 147.6, 151.2, 154.6, 208.7. MS (EI) m/z: 361.1 [M+l]⁺. 00002

20

Example 11

8 il-Hydroxycyclohexyl)et nyl)-3,7-diinethyl-l-(5-oxoJiexyl)-1H-purine- 2,6(3H,7//)-clione (1-1

Yield: 42% (Method C), mp = 126-128 °C. 1H NMR (CDCI₃/TMS, 400 MHz) δ (ppm): 1.27-1.37 (m, IH), 1.54-1.79 (m, 7H), 2.02-2.08 (m, 2H), 2.13 (s, 3H), 2.48 (t, 2H), 3.54 (s, 3H), 3.97 (s, 3H), 3.97-4.01 (2H, m). ¹³C NMR (CDCI3 TMS, 100.6 MHz) (ppm): 20.9, 23.0, 24.9, 27.4, 29.7, 29.9, 33.1, 39.3, 40.9, 43.1, 69.0, 72.0, 101.5, 107.8, 135.3, 147.6, 151.2, 154.6, 208.6. MS (El) /z: 401.6 [M+l]⁺.

Example 12

8-((l-Aminocyclohexyl)ethynyl)-3,7-dimethyl-l-(5-oxohexyl)-lH-purine- 2,6(3H,7H)-dione (1-1

Yield: 45% (Method C), mp > 200° C. 1H NMR (CDCb TMS, 400 MHz) δ (ppm): 1.24-1.34 (m, IH), 1.61-1.84 (m, 7H), 1.95-2.01 (m, 2H), 2.13 (s, 3H), 2.34-2.37 (m, 2H), 2.48 (t, 2H), 3.39 (s, 3H), 3.49 (s, 3H, 3.93-3.97 (m, 2H), 4.01 (s, 3H), 9.28 (br s, 2H). ¹³C NMR (CDC1₃/TMS, 100.6 MHz) £(ppm): 20.9, 22.5, 24.1, 27.3, 25.1, 29.7, 29.9, 33.5, 35.9, 41.0, 43.1, 53.6, 75.7, 93.9, 108.0, 133.8, 147.4, 150.9, 154.4, 208.6. MS (EI) mlz: 400.5 [M+l]⁺.

Example 13

8-(3-(Bis(2-methoxyethyl)amino)prop-l-yn-l-yl)-3,7-dimethyl-l-(5-oxohexyl)-lH- purine-2,6(3H,7H)-dione (1-13)

Yield: 35% (Method C), foam. Ή NMR (CDCl TMS, 400 MHz) <J(ppm): 1.60-1.65 (m, 4H), 2.10 (s, 3H), 2.47 (t, 2Η), 2.34-2.37 (m, 2Η), 2.83 (t, 4H), 3.30 (s, 6Η), 3.45 (t, 4H), 3.54-3.55 (m, 2H), 3.56 (s, 3H), 3.92 (s, 3H), 3.97 (t, 2H). ¹³C NMR (CDCI₃/TMS, 100.6 MHz) (ppm): 20.9, 27.4, 29.8, 31.9, 40.7, 43.1, 54.2, 58.8, 63.0, 66.5, 71.0, 76.9, 107.2, 110.3, 143.1, 147.3, 148.1, 151.3, 155.1, 208.6. MS (EI) mlz: 448.6 [M+l]⁺.

Example 14

3,7-Dimethyl-l-(5-oxohexyl)-8-(3-(pyrrolidin-l-yl)prop-l-yn-l-yI)-lH-purine- 2,6(3H,7H)-dione (1-14

Isolated as hydrochloride. Yield: 41%, foam. 1H NMR (CDCI3AMS, 400 MHz) δ (ppm): 1.61-1.70 (m, 4H), 1.82-1.86 (m, 4H), 2.13 (s, 3H), 2.49 (t, 2H), 2.64-2.68 (m, 4H), 3.48 (d, 2H), 3.58 (s, 3H), 3.93 (s, 3H), 4.01 (t, 2H), 6.78 (br s, 1H). ¹³C NMR (CDCI₃AMS, 100.6 MHz) £(ppm): 20.8, 23.9, 27.3, 29.6, 29.9, 33.6, 40.9, 43.0, 43.3, 52.7, 77.6, 85.1, 108.4, 133.0, 147.4, 151.0, 154.5, 208.5. MS (EI) mlz: 386.3 [M+l]⁺.

Example 15

3,7-Dimethyl-l-(5-oxohexyl)-8-(3-(piperidin-l-yl)prop-l-yn-l-yl)-lH-purine- 2,6(3H,7H)-dione (1-1

Yield: 39% (Method C), mp = 182-184 °C. Ή NMR (CDCI3/TMS, 400 MHz) δ (ppm): 1.34-1.48 (m, 1H), 1.58-1.66 (m, 4H), 1.90-1.93 (m, 3H), 2.1 1 (s, 3H), 2.21 - 2.29 (ra, 2H), 2.47 (t, 2H), 2.94-2.97 (m, 2H), 3.51 (s, 3H), 3.60-3.63 (m, 2H), 3.97 (t, 2H), 4.07 (s, 3H), 4.22 (s, 2H). ¹³C NMR (CDCI₃ TMS, 100.6 MHz) S (pp ): 20.8, 21.5, 22.7, 27.3, 29.6, 29.9, 33.7, 40.9, 43.0, 46.7, 52.6, 78.7, 84.6, 108.5, 133.1, 147.5, 151.0, 154.5, 208.5. MS (EI) m/z: 400.2 [M+l]⁺.

Example 16

l,3,7-Trimethyl-8-(phen l -dione (1-16)

*H NMR (CDC1₃ TMS, 400 MHz) δ (ppm): Yield: 36% (Method C). 1H NMR (CDC1₃ TMS, 400 MHz) £ (ppm): 3.43 (s, 3H), 3.61 (s, 3H), 4.10 (s, 3H), 7.39-7.46 (m, 3H), 7.61-7.63 (m, 2H). MS (EI) m/z: 295.5 [M]⁺.

Example 17

l,3,9-Trimethyl-8-(3-(pyrrolidin-l-yI)prop-l-yn-l-yI)-lH-purine-2,6(3H,9H)- dione (1-17)

Isolated as hydrochloride. 1H NMR (DMSO-d₆, 400 MHz) δ (ppm): 1.86-1.99 (m, 4H), 3.07-3.20 (m, 4H), 3.25 (s, 3H), 3.72 (s, 3H), 3.93 (s, 3H), 4.35 (s, 2H). MS (EI) m/z: 302.1 [M]⁺.

Example 18

l,3,9-Trimethyl-8-(phenylethyiiyl)-lH-purine-2,6(3H,9H)-dione (1-18)

Yield: 75% (Method C), mp > 200 °C. Ή NMR (CDCI3 TMS, 400 MHz) (ppm): 3.42 (s, 3H), 3.80 (s, 3H), 4.06 (s, 311), 7.35-7.42 (m, 3H), 7.53-7.55 (m, 2H). "C NMR (DMSO-d₆, 100.6 MHz) <5> (ppm): 28.1, 31.0, 33.8, 78.1, 93.6, 1 16.6, 120.1, 128.9, 129.9, 131.2, 131.6, 140.0, 150.8, 156.1. MS (EI) m/z: 295.3 [M]⁺.

ANGIOGENESIS INHIBITION

Matrix metalloproteinases MMPs, also designated matrixins, hydrolyze components of the extracellular matrix. These proteinases play a central role in many biological processes, such as embryogenesis, normal tissue remodeling, wound healing, and angiogenesis, and in diseases such as atheroma, arthritis, cancer, and tissue ulceration (Nguyen, Int. J. Biochem. Cell Biol. 2001, 33, 960-70). Matrix metalloproteinase MMP-2 (gelatinase A) is a very active at degrading extracellular matrix. Inhibition of matrix metalloproteinase enzymes is one of the well established target to prevent formation of cancer metastasis (Forsyth, J. Neurooncol., 1998, 36, 21-29; Itoh, Cancer Res., 1998, 58, 1048-1051; Di Carlo, Prevent. Res, line 18. 2012, P&R Public. 34). According to our data, reference compounds such as caffeine, proxyfeine, pentoxifylline, and Temodar showed no ability to inhibit any of studied human matrix metalloproteinases at low concentrations, if detected with the use of MMP Inhibitor Fluorimetric Profiling kit (Biomol, USA). In opposite to that novel caffeine derivatives 1-1, 1-9, and 1-12 exhibit ability to selectively inhibit MMP 2 (Gelatinase A) up to 52%. Besides, (aminocyclohexyl)ethynyl derivative 1-3 inhibits MMP-10 activity up to 53%.

These findings support the potential of therapeutic use of invented novel xanthine derivatives to treat diseases, progression of which is dependent of increased angiogenesis controlled by matrix metalloproteases - such as metastatic cancers, arthritis and others.

MMP assay

Inhibitors of matrix metalloproteinase enzymes were detected with the use of MMP Inhibitor Fluorimetric Profiling kit (Biomol, USA) accordingly to manufacturer's instructions. MMP activity assays were performed in 96-well plates using the

TM

recombinant human MMP-1-10, 12, 13 and 14 catalytic domains and OmniMMP fluorogenic substrate Mca-Pro- Leu Gly Leu-Dpa-Ala-Arg-NH₂. The test compounds

(20 μΜ) were dissolved in DMSO. The compound NNGH (N-isobutyl-N-(4- methoxyphenylsulfonyl)-glycylhydroxamic acid) was used as a prototypic control inhibitor. The rate of substrate hydrolysis was determined by fluorescence intensity measuring for 10 minutes at 37 °C temperature using a fluorescence plate reader (Tecan infinite M1000, Austria) with excitation at 328 nm and emission at 420 nm. Data analysis was performed using program Graph Pad Prism® 3.0.

Table 1. Matrix metalloproteinases inhibition caused by ethynyl xanthines I-l - 1-18

Compound MMP 1 MMP 2 MMP 3 MMP 7 MMP 8 MMP 9 MMP 10 MMP 12 MMP 13 MMP 14

NNGH* (1.3 μΜ) 100 100 100 7.2 100 100 100 100 100 100

Caffeine 8.0 0 7.2 2.3 3.2 0 0 3.2 0 9.3

Proxyfeine 1.3 0 2.0 2.5 0 0.5 0 4.6 0 7.3

Pentoxifylline 0 0 0 1.1 0 0 0 3.5 0.6 8.4

Temodar 6.5 0 3.8 8.5 6.5 23.1 3.1 0 5.8 13.4

I-l 0 47.4 1.1 0 9.8 3.8 6.6 2.4 0 20.9

1-2 3.9 0 7.5 1.2 10.6 0 6.5 0 11.2 0

1-3 0 23.7 0 0 31.0 12.0 53.3 0 0 48.1

1-4 0 4.5 0 0 0 0 0 0 0 10.6

1-5 0.1 0 0 8.4 8.9 0 2.6 2.0 11.8 13.7

1-6 0 0 4.8 0 4.4 0 0 6.3 12.0 9.4

1-7 1.7 0 2.8 0 1.6 3.3 6.2 8.4 5.4 9.2

1-8 0.6 0 0 1.8 10.2 0 0 9.8 13.9 7.0

1-9 5.9 51.8 1.1 5.6 10.4 18.9 12.7 24.0 0 14.6

1-10 1.44 0 6.5 2.1 1.4 0.2 5.4 5.72 1.9 7.1

1-11 0 0 0 2.6 0.5 0 2.8 6.69 0.2 9.4

1-12 0 39.8 0 4.2 3.9 8.3 0 0 2.0 31.5

1-14 0 19.4 0 0 9.1 7.3 0 0 1.6 19.7

1-15 0 0 0 0 0 0 0 0 0 0

1-16 0 0 20.2 1.7 4.7 2.3 1.8 3.5 0 0

1-17 0 18.4 0 4.1 9.9 10.1 0 0 1.6 9.7

1-18 0 23.4 0 9.3 19.7 14.1 0 0 16.0 24.9

* N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid

Claims

A compound selected from those of Formula I:

wherein

R¹ represents hydrogen, C^alkyl, hydroxy-C₂-₄alkyl, C₁.₃alkoxy-C2_-4alkyl, Q,

or C₁.₃alkyl(Ci_3alkyl)amino-C₂-₄alkyl;

R² represents Q^alkyl, hydroxy-C₂_₄alkyl, C₁.₄alkylcarbonyl-C₁.₄alkyl, Ci.₃alkoxy-C_2- ₄alkyl, Ci-3alkyl(C₁.3alkyl)amino-C₂_₄alkyl or halo-C_2-4alkyl;

R³ represents C_1-4alkyl, allyl or Ci_-3alkoxy-C₂.₄alkyl; with the proviso that if substituent R³ is at purine N(7) atom the dotted line between N(7) and C(8) represents no bond, and the dotted line between C(8) and N(9) represents chemical bond;

and

R⁴ represents C₁_₄alkyl, hydroxy-C₁_₄alkyl,

1- hydroxy-di-CCi-salky methyl, l-amino-di-(Ci.₃alkyl)methyl, l-hydroxy-cyclo-C₃_ ₆alkyl, 1 -amino-cyclo-Cs-ealkyl, l-(hydroxy-C_1-3alkyl)-cycloC₃-₆alkyl, C_\ . ₃alkylamino-Ci_-3alkyl, Ci_₃alkyl(Ci-3alkyl)aniino-Ci-₃alkyl, di-(Ci-₃alkoxy-C₂.₄alkyl)- amino-C₁.3a.kyI, heterocyclyl-Q^alkyl, aryl or heteroaryl; wherein the term "heterocyclyl" represents saturated 4-7 membered heterocycle containing one or two heteroatoms selected from oxygen, sulfur and nitrogen, wherein the heterocyclyl may be azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, tetrahydrofuryl, morpholinyl, thiomorpholinyl and piperazinyl; the term "aryl" represents phenyl or phenyl substituted by one or more substituents selected independently from halogen, cyano, Ci.₄alkoxycarbonyl, N-Q- ₄alkylaminocarbonyl, N^-dHQ-salky^aminocarbonyl, CH₂OH, trifluoromethyl, Ci. ₄alkyl, allyl, C₂.₄alkynyl, Ci_₄alkoxy, difluoromethoxy, trifluoromethoxy, cyclo- C₃-₆alkoxy, hydroxy-Ci^alkyl, Q.salkoxy-C alkyl, C₁_₃alkoxy-C_2-4alkoxy, di-(C₁. ₃alkyl)amino,

di-(C₁_3alkyl)amino-C₂.₄alkoxy, Ci-₄alkylsulfonylamino and Ci-₄alkyl-aminosulfonyl; the term "heteroaryl" represents an aromatic 5 or 6 membered ring comprising one to three heteroatoms selected from oxygen, sulfur and nitrogen, wherein the heteroaryl may be unsubstituted or optionally substituted by one or more substituents selected independently from halogen, cyano, trifluoromethyl, Ci_₄alkyl, Ci_₄alkoxy, difluoromethoxy, trifluoromethoxy, cyclo-C3-6alkoxy,

cyclo-C3_ ₆alkylamino and di-(C_1-3alkyl)amino; its optical isomers, polymorphs and pharmaceutically acceptable acid addition salts and hydrates and solvates thereof.

2. The compound as claimed in Claim 1, wherein R³ is at purine N(7) atom.

3. The compound as claimed in Claim 1, wherein R³ is at purine N(9) atom.

4. The compound as claimed in Claims 1, 2 and 3, wherein R⁴ represents hydroxy- C_1-4alkyl, ammo-C_halky!, l-hydroxy-di-(Ci.₃alkyl)methyl, ] -amino-di-(Ci- 3alkyl)methyl, 1 -hydroxy-cyclo-C₃.(,alkyl, l-amino-cyclo-C₃.₆alkyl, l-(hydroxy- Ci-₃alkyl)-cycloC₃-₆alkyl, C_1-3alkyl(Ci_3alkyl)amino-Ci_3alkyl, di-(Ci.₃alkoxy- C_2-4alkyl)-amino-Ci_₃alkyl, heterocyclyl-C i .₃alkyl or aryl.

5. The compound as claimed in Claim 4. wherein R 2 and R 3 each independently represent

or C i .lalkoxy-C -.talkyl .

6. The compound as claimed in Claim 5, wherein R² and R³ each represent methyl.

7. The compound as claimed in any of Claims 1 to 6, wherein R¹ represents methyl or 5-oxohexyl.

8. The compound as claimed in Claim 1, which is selected from:

8-(3-Hydroxy-3-methylbut-l-yn-l-yl)-l,3,7-trimethyl-lH-purine-2,6(3H,7H)- dione,

8-((l -Hydroxycyclohexyl)ethynyl)- 1 ,3 ,7-trimethyl- 1 H-purine-2,6(3H,7H)-dione, 8-((l-Aminocyclohexyl)ethynyl)-3,7-dimethyl-lH-purine-2,6(3H,7H)-dione, 8-(3-(Dimethylamino)prop- 1 -yn- 1 -yl)- 1 ,3 ,7-trimethyl- 1 H-purine-2,6(3H,7H)- dione,

8-(3-(bis(2-methoxyethyl)amino)prop- 1 -yn- 1-yl)- 1 ,3 ,7-trimethyl- 1 H-purine- 2,6(3H,7H)-dione,

1 ,3 ,7-Trimethyl-8-(3-(pyrrolidin- 1 -yl)prop- 1 -yn- 1 -yl)- lH-purine-2,6(3H,7H)- dione,

1 ,3,7-Trimethyl-8-(3-(piperidin- l-yl)prop- 1 -yn- 1-yl)- lH-purine-2,6(3H,7H)- dione,

8-(3-(Azepan-l-yl)prop-l-yn-l-yl)-l,3,7-trimemyl-lH-purine-2,6(3H,7H)-dione, 1 ,3 ,7-Trimethyl-8-(3-morpholinoprop- 1 -yn- 1 -yl)- 1 H-purine-2,6(3H,7H)-dione, 8-(3-Hydroxy-3-methylbut-l-yn-l-yl)-3,7-dimethyl-l-(5-oxohexyl)-lH-purine- 2,6(3H,7H)-dione,

8-(( 1 -Hydroxycyclohexyl)ethynyl)-3 ,7-dimethyl- 1 -(5-oxohexyl)- 1 H-purine- 2,6(3H,7H)-dione,

8-(( 1 - Aminocyclohexyl)ethynyl)-3 ,7-dimethyl- 1 -(5-oxohexyl)- 1 H-purine- 2,6(3H,7H)-dione,

8-(3-(Bis(2-methoxyethyl)amino)prop- 1 -yn- 1 -yl)-3 ,7-dimethyl- 1 (5-oxohexyl)- lH-purine-2,6(3H,7H)-dione,

3,7-Dimethyl-l-(5-oxohexyl)-8-(3-(pyrrolidin-l-yl)prop-l -yn-l-yl)-lH-purine- 2,6(3H,7H)-dione. 3 ,7-Dimethyl- 1 -(5-oxohexyl)-8-(3-(piperidin- l-yl)prop- 1 -yn- 1 -yl)- lH-purine- 2,6(311.711)-dione.

1,3 J-Trimethyl-8-(phenylethynyl)-lH-purine-2,6(3H,7H)-dione,

1 ,3 ,9-Trimethyl-8-(3 -(pyrrol idin- 1 -yl)prop- 1 -yn- 1 -yl)-1 H-purinc-2,6(3H,9H)- dione,

1 ,3 ,9-Trirnethyl-8-(phcnylethynyl )- 1 H-purine-2,6(3H,9H)-dione and

optical isomers, polymorphs, and pharmaceutically-acceptable acid addition salts, hydrates, and solvates thereof.

9. The compound as claimed in any of Claims 1 to 8 for use as matrix metalloproteinase inhibitors for the treatment of disease in which inhibition of matrix metalloproteasis is desired and/or required.

10. The compound as claimed in any of Claims 1 to 8 for use in the manufacture of a medicament for the treatment of disease in which inhibition of angiogenesis is desired and/or required.

11. A process for the synthesis of a compound selected from those of Formula I:

wherein

R¹ represents hydrogen, Q^alkyl, hydroxy-C₂_₄alkyl, C^alkoxy-C^alkyl, C_\. salkylcarbonyl-Ci^alkyl or C_1-3alkyl(C_1-3alkyl)amino-C₂_₄alkyl;

R² represents Ci_₄alkyl, hydroxy-C2-₄alkyl,

4alkyl, Ci_-3alkyl(C₁_₃alkyl)ammo-C2_-4alkyl or halo-C₂„₄alkyl;

R³ represents

ally! or Ci_₃alkoxy-C2-₄alkyl; with the proviso that if substituent R³ is at purine N(7) atom the dotted line between N(7) and C(8) represents no bond, and the dotted line between C(8) and N(9) represents chemical bond;

and

with the proviso that if substituent R is at purine N(9) atom the dotted line between N(7) and C(8) represents chemical bond, and the dotted line between C(8) and N(9) represents no bond;

R⁴ represents Ci_₄alkyl, hydroxy-Ci- alkyl,

amino-C].₄alkyl, 1- hydroxy-di-(Ci_₃alkyl)methyl,

l-hydroxy-cyclo-C₃- ealkyl, l-amino-cyclo-C₃.₆alkyl, l-(hydroxy-C]_-3alkyl)-cycloC₃-₆alkyl, Q. ₃alkylamino-C₁.₃alkyl, Ci.₃alkyl(Ci_₃alkyl)amino-C^alkyl, di-(C₁.₃alkoxy-C₂-₄alkyl)- amino-Ci-salkyl, heterocyclyl-Ci_₃alkyl, aryl or heteroaryl; and its optical isomers, polymorphs and pharmaceutically acceptable acid and base addition salts and hydrates and solvates thereof; comprising reaction of a compound of Formula II:

with a compound of Formula III:

≡^R⁴

III

optionally in the presence of base in an appropriate solvent (e.g., DIEA in DMF, NMP, DMAC or EtOAc), in the presence of Cul and palladium catalyst (e.g. Pd(PPh₃)₄) or palladium catalyst generated in situ (e.g., from PdCl₂ or Pd(OAc)₂ and PPh₃) to yield a compound of Formula I, which may be converted, if desired, into an optical isomer, polymorph, pharmaceutically-aeceptable salt, hydrate or solvate.