CA2989161A1

CA2989161A1 - Ethynylxanthines, preparation and use for cancer treatment

Info

Publication number: CA2989161A1
Application number: CA2989161A
Authority: CA
Inventors: Pavels Arsenjans; Jelena VASILJEVA; Ilona Domracheva; Irina Shestakova; Anita GULBE; Iveta KANEPE-LAPSA; Valerjans Kauss; Ivars Kalvins
Original assignee: Latvian Institute of Organic Synthesis
Current assignee: Latvian Institute of Organic Synthesis
Priority date: 2015-03-27
Filing date: 2015-03-27
Publication date: 2016-10-06
Anticipated expiration: 2035-03-27
Also published as: GB2553684A; WO2016159745A1; GB2553684B; GB2553684A8; CA2989161C

Abstract

The present invention relates to novel ethynylxanthine derivatives of formula (I) which exhibit high antiproliferative activity against various tumor cell lines, methods for their synthesis and use for the treatment and/or prevention of cancer.

Description

ETHYNYLXANTHINES, PREPARATION AND USE FOR CANCER
TREATMENT
FIELD OF THE INVENTION
The present invention relates to novel ethynylxanthine derivatives, methods for their synthesis and use for the treatment and/or prevention of cancer.
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 drugs 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

2 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. 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 neuro-oncology (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 (LD50=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.

3 \
õ

N N
Proxyfeine PCT Patent application No. W02008077557 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, !:11 N
I = __ R3 ONN

8-Ethynyl xanthines wherein R1 and R2 represent, e.g., hydrogen, Ci_6alkyl, cycloalkyl, heterocycloalkyl, aryl (wherein these groups may be further substituted), etc.; R3 represents e.g., aryl, hetaryl group.
PCT Patent application No. W02014/143799 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.

.41¨(Ra)n R3, N
,>¨R2 N-Benzy1-8-ethynyl xanthines THE PRESENT INVENTION
We have discovered that certain ethynylxanthine derivatives exhibit high antiproliferative activity against various tumor cell lines. Therefore, these substances

4 may be therapeutically beneficial in the treatment of tumors. These substances may be administered in the form of a pharmaceutical composition, wherein they are present together with one or more phaimaceutically acceptable diluents, carriers, or excipients.
OBJECTS OF THE INVENTION
It is an object of the present invention to provide novel antiproliferative compounds, methods for their synthesis and use for treatment and/or prevention cancer.
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

R. Ny, R3 ONN
I ____________________________________________ R4 1, wherein RI represents hydrogen, Ci_4alkyl, hydroxy-C2_4a1ky1, C1_3alkoxy-C2_4a1ky1, C1-3alkylcarbonyl-Ci_4alkyl or Ci_3alkyl(Ci_3alkyl)amino-C2_4alkyl;
R2 represents C1_4a1ky1, hydroxy-C2_4a1ky1, Ci_4alkylcarbonyl-Ci_4alkyl, C1_3alkoxy-C2-Alkyl, C1_3a1ky1(Ci_3allcyl)amino-C2_4a1ky1 or halo-C2_4a1ky1;
R3 represents Ci_4alkyl, allyl or Ci_3alkoxy-C2_4a1ky1;
with the proviso that it 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;

5 R4 represents Ci_4alkyl, hydroxy-C1_4alkyl, Ci_3alkoxy-Ci_4alkyl, amino-C1_4alkyl, 1-hydroxy- di-(Ci_3alkyl)methyl , 1-amino-di-(Ci_3alkyl)methyl, 1-hydroxy- cyclo-C3_ 6alkyl, 1-amino-cyclo-C3_6alkyl, 1 -(hydroxy-Ci _3alkyl)- cycloC3_6alkyl , C1_ 5 3alkylamino-Ci_3alkyl, Ci_3alkyl(Ci_3alkyl)amino-Ci_3alkyl, di-(Ci_3alkoxy-C2_4alkyl)-amino-Ci_3alkyl, heterocyclyl-Ci _3alkyl, aryl or heteroaryl;
wherein the term "heterocycly1" 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-C ..
4alkylaminocarbonyl, N,N-di-(Ci_3alkyl)aminocarbonyl, CH2OH, trifluoromethyl, CI_ 4alkyl, allyl, C2_4alkynyl, Ch4alkoxy, difluoromethoxy, trifluoromethoxy, cyclo-C3_6alkoxy, hydroxy-Ci_olkyl, C1_3alkoxy-Ci_4alkyl, Ci_3alkoxy-C24a1koxy, di-(Ci_ 3alkyl)amino, di-(Ci_3alkyl)amino-Ci_3alkyl, di-(C1.3alkyl)amino-C2_4alkoxy, Ci_4alkylsulfonylamino and C1_4alkyl-aminosulfonyl;
the telin "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_4a1kyl, Ci_4alkoxy, difluoromethoxy, trifluoromethoxy, cyclo-C3_6alkoxy, Ci_3alkoxy-C1_4alkyl, cyclo-C3.
6a1kylamino and di-(Ci_3a1kyl)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:

6 8-(3-Hydroxy-3-methylbut-1 -yn-1- y1)-1,3 ,7 -trimethy1-1H-purine-2,6(3H,7H)-dione, 8-((1-Hydroxycyclohexyl)ethyny1)-1,3,7-trimethy1-1H-purine-2,6(3H,7H)-dione, 8-((1-Aminocyclohexypethyny1)-3,7-dimethyl-1H-purine-2,6(3H,7H)-dione, 8-(3-(Dimethylamino)prop-1-yn-l-y1)-1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione, 8-(3-(bis(2-methoxyethyDamino)prop-1-yn-l-y1)-1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione, 1,3,7-Trimethy1-8-(3-(pyrrolidin-1-yl)prop-1-yn-1-y1)-1H-purine-2,6(3H,7H)-dione, 1,3,7-Trimethy1-8-(3-(piperidin-1-y1)prop-1-yn-1-y1)-1H-purine-2,6(3H,7H)-dione, 8-(3-(Azepan-1-yl)prop-1-yn-l-y1)-1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione, 1,3,7-Trimethy1-8-(3-morpholinoprop-1-yn-1-y1)-1H-purine-2,6(311,7H)-dione, 8-(3-Hydroxy-3-methylbut-1-yn-1-y1)-3,7-dimethyl-1-(5-oxohexyl)-1H-purine-2,6(3H,7H)-dione, 8-((1-Hydroxycyclohexypethyny1)-3,7-dimethyl-1-(5-oxohexyl)-1H-purine-2,6(3H,7H)-dione, 8-((1-Aminocyclohexypethyny1)-3,7-dimethy1-1-(5-oxohexyl)-1H-purine-2,6(3H,7H)-dione, 8-(3-(Bis(2-methoxyethyl)amino)prop-1-yn-1-y1)-3,7-dimethy1-1-(5-oxohexyl)-1H-purine-2,6(3H,7H)-dione, 3,7-Dimethy1-1-(5-oxohexyl)-8-(3-(pyrrolidin-1-y1)prop-1-yn-1-y1)-1H-purine-2,6(3H,7H)-dione, 3,7-Dimethy1-1-(5-oxohexyl)-8-(3-(piperidin-1-ypprop-1-yn-1-y1)-1H-purine-2,6(3H,7H)-dione, 1,3,7-Trimethy1-8-(phenylethyny1)-1H-purine-2,6(3H,7H)-dione, 1,3,9-Trimethy1-8-(3-(pyrrolidin-1-y1)prop-1-yn-1-y1)-1H-purine-2,6(3H,9H)-dione, 1,3 ,9-Trimethy1-8-(phenylethyny1)-1H-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 manufacturing process of a compound selected from those of Formula I as defined above, comprising reaction of a compound of Formula H:

7 "))'¨Br F'R2 wherein R1, R2 and R3 are as defined for Formula I above, with a compound of Formula 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 Et0Ac), in the presence of CuI and palladium catalyst generated in situ (e.g., from PdC12 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.
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_6a1ky1" represents monocyclic alkyl groups having 3, 4, 5 or 6 carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
The term "heterocycly1" 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, thiomorpholinyl and piperazinyl.
The term "halo" or "halogen" represents fluorine, chlorine, bromine and iodine.

8 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 invention, 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 forms, 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-methylbenzenesulfonic, 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

9 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-metlnylpyrrolidine and toluene (1:1) (Method C) gave the desired products in high yields. Alternative method was elaborated (Method D). Perfoiming the reaction in ethyl acetate and using 2 mol-% of Pd(PPh3)4 and 2 mol-% of PdC12 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-1-yn-1-y1)-1,3,7-trimethy1-1H-purine-2,6(3H,7H)-dione (I-1) in 55% yield and by-product 81(E)-5-hydroxy-2-(1-hydroxy-1-methylethyl)-5-methylhex-1-en-3- ynyl] 1,3,7-trimethyl- 1H-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.
o 0 R1,N/R3R1 ,R3 j I 1-Br + _________________ R4 = _____________________________________________________________ R4 01\1 N

Reaction conditions:
Method A. (Ph3P)2PdC12, CuI, N-methylpyrrolidine or DMF, DIEA, 50 C;
Method B. Pd(OAc)2, Ph3P, CuI, N-methylpyrrolidine or DMAC, DIEA, 55 C;
Method C. Pd(OAc)2, Ph3P, CuI, N-methylpyrrolidine/toluene (1:1), DIEA, 50 C;

Method D. Pd(PPh3)4, PdC12, Ph3P, CuI, 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 1H-purine-2,6(3H,711)-diones. Representative method for the synthesis of compound II wherein RI and are methyl groups shown in Scheme 2. Comrnercially 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 1H-purine-2,6(3H,7H)-diones II, wherein R3 is at purine N(7) (3, 4).

'N
I I Br N N ONN
I (R1 = Me) 3 (R1 = Me) 2 (R1 = MeC(0)(CH2)4) 4 (R1 = MeC(0)(CH2)4) Compounds H, wherein R3 is at purine N(9), can be prepared by bromination of position 8 of respective 8-unsubstituted 1,3,9-substituted 1H-purine-2,6(3H,9H)-diones. Representative method for the synthesis of compound II wherein RI, R2 and R3 are methyl groups (compound 11) is shown in Scheme 3. Thus, 1,3-dimethy1-6-

10 chlorouracil (6) was prepared by the treatment of 1,3-dimethylbarbituric acid (5) in phosphorous oxychloride (POC13) 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-dimethy1-5-nitropyrimidine-2,4(1H,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-1,3-dimethy1-6-methylamino-pyrimidine-2,4(1H,3H)-dione (9) with formic acid gave 1,3,9-trimethy1-3,9-dihydro-purin-2,6(3H,9H)-dione (10). Necessary 8-bromo- 1,3 ,9-trimethy1-3 ,9-dihydropurin-2,6(314,9H)-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 1H-purine-2,6(3 H,9H)-diones.
Scheme 3. Preparation of 8-bromo 1H-purine-2,6(3H,9H)-dione 11 (II, wherein R3 is at purine N(9)).

11 POCI,HNO, N NN O2 MeNH2 N'N NO2 I I \

H, NNH2HCO2H NBS
I I I .=-=Br \ \ \

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 5 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 N,N-dimethylformamide, "DMAC" is defined as N,N-dimethylacetamide, "NMP" is defined as N-methylpyrrolidone, "DMSO" as dimethyl sulfoxide, "HCl" as hydrochloric acid, õaq. NH3" as aqueous ammonia solution, õ"MeCN" as acetonitrile, "DIEA" as diisopropylethylamine, "Et0Ac" as ethyl acetate, "rt" as room temperature.
Intermediate 3.
8-Bromo-1,3,7-trimethy1-1H-purine-2,6(3H,711)-dione (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),

12 dried over sodium sulfate, filtered and evaporated to give the title compound (13.5 g, 99%). 1H NMR (CDC13/TMS, 400 MHz) g (ppm): 3.41 (s, 311, CH3), 3.57 (s, 3H, CH3), 3.97 (s, 3H, CH3).
Intermediate 4 8-Bromo-3,7-dimethy1-1-(5-oxohexyl)-1H-purine-2,6(3H,7/1)-done (4) N

To a round-botton flask containing freshly distilled C112C12 (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%). 1H
NMR
(CDC13/TMS, 400 MHz) (5. (ppm): 1.61-1.67 (m, 4H), 2.14 (s, 3H), 2.49 (t, 2H), 3.54 (s, 3H), 3.95 (s, 311), 3.99 (t, 211).
Intermediate 11.
8-Bromo-1,3,9-trimethy1-1H-purine-2,6(3H,911)-dione (11).

I \
a) 6-Chloro-1,3-dimethylpyrimidine-2,4(1H,3H)-dione (6).
To a suspension of 1,3-dimethylbarbituric acid (7.8 g, 50 mmol) in P0C13 (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 POC13 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 Na2SO4, evaporated to dryness, and the residue crystallized with ether to give the title

13 compound (8.0 g) as a yellow crystals. 1H NMR (400 MHz, DMSO-d6) 8 (ppm): 3.10 and 3.15 (both s, both 311), 6.07 (s, 1H).
b) 6-Ch1oro-1,3-dimethy1-5-nitropyrimidine-2,4(1H,3H)-dione (7).
Compound 6 (15 g, 74.5 mmol) was added portionwise to a cooled solution of conc.
H2SO4 (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 Na2SO4 and concentrated in vacuo to give the title compound (12.0 g) as a yellow solid.

(400 MHz, DMSO-d6) 8 (ppm): 3.07 and 3.26 (both s, both 3T1).
1,3-Dimethy1-6-methylamino-5-nitropyrimidine-2,4(1H,3H)-dione (8).
To a stirred solution of 6-chloro-1,3-dimethy1-5-nitropyrimidine-2,4(1H,3H)-dione (7) (11.0 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 the title compound (12.5 g) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) 8 (ppm): 2.49, 2.75 and 3.35 (all s, all 3H), 7.74 (br s, 1H).
d) 5-Amino-1,3-dimethy1-6-methylamino-pyrimidine-2,4(1H,3H)-dione (9).
To a stirred solution of 1,3-dimethy1-6-methylamino-5-nitropyrimidine-2,4(1H,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) 8 (ppm): 2.89 (d, 3H), 3.31 (s, 3H), 3.40 (s, 311), 4.75 (br s, 1H). MS (EI) m/z: 185 [M].
e) 1,3,9-Trimethy1-1H-purine-2,6(3H,9H)-dione (10).
A mixture of 5-amino-1,3-dimethy1-6-methylamino-pyrimidine-2,4(1H,31])-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 CH2C12, washed with aq. Na2CO3, dried over Na2SO4 and evaporated to dryness. The residue was purified by column chromatography (eluent CH2C12 Me0H, 10:1) to give the title compound (0.5 g) as a white solid. 1H NMR (400 MHz,

14 DMSO-d6) 6 (ppin): 3.22, 3.68 and 3.93 (all s, all 3H), 7.66 (s, 1H). MS (EI) natz: 195 [M+2]+.
1) 8-Bromo-1,3,9-trimethy1-3H-purine-2,6(3H,9H)-dione (H).
A mixture of 1,3,9-trimethy1-1H-purine-2,6(3H,91-1)-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 Na2SO4 and evaporated to dryness. The residue was purified by column chromatography (CH2C12 : Me0H, 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) 8 (ppm): 3.22, 3.69 and 3.88 (all s, all 3H). MS (EI) m/z: 375 [M+21.
Example 1 8-(3-Hydroxy-3-methylbut-1-yn-1-y1)-1,3,7-trimethy1-111-purine-2,6(3H,7H)-dione (I-1) `-N-JI\xN OH
I ____________________________________ LT=

Method A. To a mixture of PdC12 (113 mg, 0.637 mmol), CuI (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-1,3,7-trimethy1-1H-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 Et0Ac (300 mL), washed with brine (80 mL) containing aq. NH3 (0.5 mL) and stirred at rt for the additional min. The aqueous phase was separated and the organic phase was washed with 25 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. 111 NMR (CDC13/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).
30 13C NMR (CDC13/TMS, 100.6 MHz) g(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) ink: 277.2 [M+11+.

Method B. To a mixture of Pd(OAc)2 (100 mg, 0.446 mmol), CuI (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 5 simultaneous barbotation with argon. Then a solution of 8-bromo-1,3,7-trimethy1-1H-purine-2,6(3H,7Th-dione (1.21 g, 4.46 mmol) and 2-methylbut-3-yri-2-ol (0.65 mL, 6.69 mmol) and dry DIM. (4.0 mL) was added and stirring was continued at 55 C
for 24 h. Compound I-1 (31% yield) was isolated as in Method A.
10 Method C. To a mixture of Pd(OAc)2 (100 mg, 0.446 mmol), CuI (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-1,3,7-trimethy1-1H-purine-2,6(3H,711)-dione (1.21 g, 4.46 mmol) and 2-methylbut-3-yn-2-ol (0.65 mL, 6.69

15 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 I-1 (72% yield) was isolated as in Method A.
Method D. A vial charged with Pd(PPh3)4 (346 mg, 0.3 mmol), PdC12 (51 mg, 0.3 mmol), Ph3P (157 mg, 0.6 mmol), CuI (58 mg, 0.3 mmol), 8-bromo-1,3,7-trimethy1-1H-purine-2,6(3H,7H)-dione (4.08 g, 15.0 mmol) and 2-methylbut-3-yn-ol (2.05 mL, 21.0 mmol), DfEA (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 Et0Ac (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 I-1 in 55% yield and 8-[(E)-5-hydroxy-2-(1 -hydroxy-1-methylethyl)-5-methylhex-1- en-3-ynyl] -1,3 ,7-trimethy1-1H-purine-2,6(3H,7H)-dione as a by-product in 26% yield. By-product: 1H NMR
(CDC13/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)

16 (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 2 841-Hydroxycyclohexypethyny1)-1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione (I-2) /
'---N---4,-,-N HO

ON---r\I
I
Yield: 49% (Method C), 54% (Method D), 11% (Method B); mp = 194-196 C. 1H
NMR (CDC13/TMS, 400 MHz) g (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, 311), 3.89 (s, 311). 13C NMR (CDC13/TMS, 100.6 MHz) g (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 (El) nik: 317.5 [M+11+.
Example 3 8-((1-Aminocyclohexyl)ethyny1)-3,7-dimethy1-1H-purine-2,6(3H,7H)-dione (1-3) H2N, / ______________________________________ ) HNA----"Nr I
Yield: 48% (Method D), 43% (Method C), 16% (Method B), 7 % (Method A); mp =
187-189 C. 1H NMR (CDC13/TMS, 400 MHz) g(ppm): 1.20-1.29 (m, 1H), 1.48-1.67 (m, 511), 1.72-1.77 (m, 2H), 1.81 (br s, 211), 1.93-1.97 (m, 2H), 3.39 (s, 3H), 3.55 (s, 3H), 3.98 (s, 3H). 13C NMR (CDC13/TMS, 100.6 MHz) 8 (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) ink:
302.3 [M+11+.
Example 4 8-(3-(Dimethylamino)prop-1-yn-1-y1)-1,3,7-trimethy1-1H-purine-2,6(3H,7H)-dione (1-4)

17 I =
0 N N N¨
I
Isolated as hydrochloride. Yield: 58% (Method D), 47% (Method C), 4% (Method B);
mp = 197-199 C (dec.) 1H NMR (DMSO-d6, 400 MHz) g(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) 5 (ppm): 27.6, 29.3, 33.0, 41.6, 45.7, 76.4, 87.1, 107.6, 132.9, 146.8, 150.7, 154Ø MS
(EI) m/z: 277.5 [M+1].
Example 5 8-(3-(bis(2-methoxyethyl)amino)prop-1-yn-1-y1)-1,3,7-trimethy1-1H-purine-2,6(3H,7H)-dione (1-5) NN
=
ONN __ \N
1 \-0\

Yield: 33% (Method D), 30% (Method C), 10% (Method B); foam. 111 NMR
(CDC13/TMS, 400 MHz) 5(ppm): 2.79 (t, 4H), 3.31 (s, 611), 3.34 (s, 3H), 3.48 (t, 4H), 3.51 (s, 3H), 3.82 (s, 211), 3.96 (s, 3H). 13C NMR (CDC13/TMS, 100.6 MHz) 6.(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) m/z: 364.5 [M+1]+.
Example 6 1,3,7-Trimethy1-8-(3-(pyrrolidin-1-yl)prop-1-yn-1-y1)-1H-purine-2,6(3H,7H)-dione (1-6) NN
I _____ \N
ON
Yield: 57% (Method D), 52% (Method C), 8% (Method A); mp = 149-150 C. 1H
NMR (CDC13/TMS, 400 MHz) c5*(ppm): 1.83-1.86 (m, 4H), 2.68-2.72 (m, 4H), 3.39

18 (s, 3H), 3.56 (s, 3H), 3.75 (s, 2H), 4.00 (s, 3H). 13C NMR (CDC13/TMS, 100.6 MHz) (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)mtz: 302.3 [M4-11+.
Example 7 1,3,7-Trimethy1-8-(3-(piperidin-l-y1)prop-1-yn-1-y1)-1H-purine-2,6(347,7H)-dione (I-7) O
N)CN __ I\
N N N

Yield: 35% (Method C), 30% (Method D); mp = 137-138 C. 1H NMR (CDC13/TMS, 400 MHz) 8(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 (CDC13/TMS, 100.6 MHz) (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) mk: 316.3 [M+1]+.
Example 8 8-(3-(Azepan-1-yl)prop-1-yn-1-y1)-1,3,7-trimethyl-11/-purine-2,6(3H,7H)-dione (1-8) N)N __ I
ON N
Yield: 34% (Method C), foam. 111 NMR (CDC13/TMS, 400 MHz) 8(ppm): 1.61-1.64 (m, 4H), 1.70-1.76 (m, 4H), 2.81 (t, 4H), 3.39 (s, 3H), 3.55 (s, 311), 3.74 (s, 2H), 4.01 (s, 3H). 13C NMR (CDC13/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) mk: 330.3 [M+1]+.
Example 9

19 1,3,7-Trimethy1-8-(3-morpholinoprop-1-yn-1.-y1)-1H-purine-2,6(all,7H)-dione (I-9) 1(111 =
N
\--0 Yield: 38% (Method C), mp =188-190 C. 1H NMR (CDC13/TMS, 400 MHz) (ppm): 2.64 (t, 4H), 3.40 (s, 311), 3.56 (s, 3H), 3.64 (s, 2H), 3.76 (t, 4H), 4.01 (s, 3H).
13C NMR (CDC13/TMS, 100.6 MHz) g (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+11+.
Example 10 8-(3-Hydroxy-3-methylbut-1-yn-1-y1)-3,7-dimethy1-1-(5-oxohexyl)-1H-purine-2,6(3H,7H)-dione (1-10) OH
=
N N

Yield: 47% (Method D), 44% (Method C); foam. 1H NMR (CDC13/TMS, 400 MHz) g (ppm): 1.62-1.66 (m, 4H), 1.65 (s, 6H), 2.12 (s, 3H), 2.48 (t, 2H), 2.72 (br s, 1H), 3.53 (s, 3H), 3.96 (s, 3H), 3.97 (t, 2H). 13C NMR (CDC13/TMS, 100.6 MHz) g(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-1-1]+.
Example 11 84(1-Hydroxycyclohexypethyny1)-3,7-dimethyl-1-(5-oxohexyl)-1H-purine-2,6(3H,7H)-dione (1-11) HO
ONN
I /

Yield: 42% (Method C), mp = 126-128 C. 1H NMR (CDC13/TMS, 400 MHz) g (ppm): 1.27-1.37 (m, 1H), 1.54-1.79 (m, 71I), 2.02-2.08 (m, 2H), 2.13 (s, 3H), 2.48 (t, 2H), 3.54 (s, 3H), 3.97 (s, 311), 3.97-4.01 (2H, m). 13C NMR (CDC13/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 (EI) m/z: 401.6 [M+11+.
Example 12 5 8-((1-Aminoeyclohexyl)ethyny1)-3,7-dimethyl-1-(5-oxohexyl)-1H-purine-2,6(3H,71/)-dione (1-12) -Yield: 45% (Method C), mp > 200 C. 1H NMR (CDC13/TMS, 400 MHz) g (ppm):
1.24-1.34 (m, 1H), 1.61-1.84 (m, 7H), 1.95-2.01 (m, 2H), 2.13 (s, 3H), 2.34-2.37 (m, 10 2H), 2.48 (t, 2H), 3.39 (s, 311), 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 (El) m/z: 400.5 [M+1]+.
15 Example 13 8-(3-(Bis(2-methoxyethyDamino)prop-1-yn-1-y1)-3,7-dimethyl-1-(5-oxohexyl)-1H-purine-2,6(3H,7H)-dione (1-13) S.I

Yield: 35% (Method C), foam. 1H NMR (CDC13/TMS, 400 MHz) g(ppm): 1.60-1.65 20 (m, 4H), 2.10 (s, 3H), 2.47 (t, 2H), 2.34-2.37 (m, 2H), 2.83 (t, 4H), 3.30 (s, 6H), 3.45 (t, 4H), 3.54-3.55 (m, 2H), 3.56 (s, 311), 3.92 (s, 3H), 3.97 (t, 2H). 13C NMR

(CDC13/TMS, 100.6 MHz) g(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) m/z:
448.6 [1\44-1]+.
Example 14

21 3,7-Dimethy1-1-(5-oxohexyl)-8-(3-(pyrrolidin-1-y1)prop-1-yn-l-y1)-1H-purine-2,6(3H,7H)-dione (.14) N NI ___ Isolated as hydrochloride. Yield: 41%, foam. 1H NMR (CDC13/TMS, 400 MHz) (ppm): 1.61-1.70 (m, 4H), 1.82-1.86 (m, 4H), 2.13 (s, 3H), 2.49 (t, 211), 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).

(CDC13/TMS, 100.6 MHz) 8(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) nilz:
386.3 [M+11+.
Example 15 3,7-Dimethy1-1-(5-oxohexyl)-8-(3-(piperidin-1-ypprop-1-yn-1-y1)-1H-purine-2,6(3H,7H)-dione (1-15) )1WµWIL--"N
ONN \N
Yield: 39% (Method C), mp = 182-184 C. 1H NMR (CDC13/TMS, 400 MHz) 8 (ppm): 1.34-1.48 (m, 1H), 1.58-1.66 (m, 411), 1.90-1.93 (m, 3H), 2.11 (s, 3H), 2.21-2.29 (m, 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 (CDC13/TMS, 100.6 MHz) (ppm): 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 (El) mk: 400.2 [M+1]+.
Example 16 1,3,7-Trimethy1-8-(phenylethyny1)-1H-purine-2,6(3H,7H)-dione (I-16) I =
N N

22 1H NMR (CDC13/TMS, 400 MHz) (5 (ppm): Yield: 36% (Method C). 1H NMR
(CDC13/TMS, 400 MHz) 6 (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 [Mr.
Example 17 1,3,9-Trimethy1-8-(3-(pyrrolidin-1-yl)prop-1-yn-1-y1)-1H-purine-2,6(3H,9H)-dione (I-17) -.--.-NN N
_________________________________________ /

I \
Isolated as hydrochloride. 1H NMR (DMSO-d6, 400 MHz) S (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 1,3,9-Trimethy1-8-(phenylethyny1)-1H-purine-2,6(3H,9H)-dione (I-18) = ilk I \
Yield: 75% (Method C), mp > 200 C. 1H NMR (CDC13/TMS, 400 MHz) g (ppm):
3.42 (s, 3H), 3.80 (s, 3H), 4.06 (s, 311), 7.35-7.42 (m, 3H), 7.53-7.55 (m, 211).
13C NMR (DMSO-d6, 100.6 MHz) (5(ppm): 28.1, 31.0, 33.8, 78.1, 93.6, 116.6, 120.1, 128.9, 129.9, 131.2, 131.6, 140.0, 150.8, 156.1. MS (EI) m/z: 295.3 [Mr.
ANTIPROLIFERATIVE ACTIVITY
Anticancer activity of 8-ethynylxanthines was tested on monolayer tumor cell lines:
MDA-MB-435s (human melanoma), H9C2 (rat embrio cardiomyoblast), MCF-7 (human breast adenocarcinoma, estrogen-positive), HepG2 (human hepatocellular carcinoma), SH-SY5Y (human neuroblastoma), C6 (rat glioma), U937 (human histiocytic leukemia), and A549 (human lung carcinoma). The borderline concentration, relevant to the highest tolerated dose, was deteimined for each

23 compound using the NIH 3T3 (Mouse Swiss Albino embryo fibroblasts) cell line.
The basal cytotoxicity was used to predict starting doses for in vivo acute oral LD50 values in rodent. The results of these experiments are summarized in Table 1.
Caffeine, Proxyfeine, Pentoxifylline and Temodar were used as references. Caffeine and Pentoxifylline exhibit no cytotoxic effect on all studied cell lines. Despite the fact that Temodar is widely used drug for brain cancer prevention, in vitro results showed more than a modest activity on studied tumor cell lines (IC50=298 i.tM on SH-neuroblastoma and IC50=31 [tM on U937 lymphoma cell lines). Cancer cells are more sensitive to Proxyfeine (IC50=96-283 [tM). Surprisingly, 8-ethynylcaffeines have an extended ability to suppress cancer cell growth. Thus, caffeine derivatives 1-showed high in vitro antiproliferative effect against majority of cancer cells, moreover, these compounds are active against brain tumors (IC50=8.2+11.5 HM on SH-SY5Y
and C6 cell lines). It should be noted that caffeine 1-3 selectively inhibits lymphoma U937 cell growth (IC50=5.1 1.1M), and derivative 1-6 ¨ estrogen-positive human breast adenocarcinoma MCF-7 (IC50=5.7 [tM). In a series of pentoxifylline derivatives 15 piperidylmethyl derivative 1-15 showed high cytotoxicity against studied cancer cells, especially, on lymphoma U937 (IC50=4.7 [tM) and glioma C6 (IC50=7.1 p,M).
In vitro cytotoxicity assay Monolayer tumor cell line: MDA-MB-435s (human melanoma), H9C2 (rat embrio cardiomyoblast), MCF-7 (human breast adenocarcinoma, estrogen-positive), HepG2 (human hepatocellular carcinoma), SH-SY5Y (human neuroblastoma), C6 (rat glioma), U937 (human histiocytic leukemia), and A549 (human lung carcinoma), and normal cell line NH-1 3T3 (mouse fibroblasts) were cultured in standard medium DMEM (Dulbecco's modified Eagle's medium) containing 1% non-essential amino-acids, 2 mM glutamine and supplemented with 10% fetal bovine serum (FBS, Sigma) ("Sigma"). All cells obtained from the American Type Culture Collection.
About 2-10104 cells/mL (depending on line nature) were placed in 96-well plates immediately after compounds were added to the wells. The control cells without test compounds were cultured on separate plate. The plates were incubated for 72 h, C, 5% CO2. The number of surviving cells was determined using 344,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolinium bromide (MTT). MTT-test: after incubating with preparations culture medium was removed and 200 1.11, of fresh

24 medium with 10 mM HEPES was added in each well of the plate, then 20 p L of MIT (2 mg/mL in HBSS) was added. After incubation (3 h., 37 C, 5%CO2) the medium with myr was removed and 200 jaL of DMSO and 25 pL of glycine buffer (pH 10.5) were added at once to each sample. The samples were tested at 540 nm on Anthos HT 11 photometer.
Basal toxicity test Compounds were tested on NIH 3T3 (normal mouse fibroblasts, "ATCC") cell line according the basal toxicity test (INVITOX Protocol No 64, 1992) and non-toxic compounds were selected. 9,000 NIH 3T3 cells/well were placed into 96-well plates for 24 h and then exposed to the test compound over a range of eight concentration (1-1000 ttg/mL) for 24 h. Upon that, the cells were incubated with the neutral red dye for 4 h and then OD was determined at 540 nm. Alternative LD50 values (LD50 value is the amount of the drug that is taken to kill 50% of the test animals) was calculated according to the formula: log (LD50 [mmol/kg] = 0.435 x log (IC50 [mmo1/1]) =
0.625.
The IC50 values were calculated using the program Graph Pad 5 Prism 3Ø

25 Table 1. In vitro cytotoxicity caused by 8-ethynylxanthines was tested on monolayer tumor cell lines: MDA-MB-435s (human melanoma), t..) o ,-, H9C2 (rat embrio cardiomyoblast), MCF-7 (human breast adenocarcinoma, estrogen-positive), HepG2 (human hepatocellular carcinoma), SH- o, ,-, u, SY5Y (human neuroblastoma), C6 (rat glioma), U937 (human histiocytic leukemia), and A549 (human lung carcinoma). ,o .6.
u, MDA-MB-435s H9C2 MCF-7 HepG2 SH-SY5Y C6 Compound 1050 ICso LDso, mg/kg Caffeine * * * * * * *
* 1670 Proxyfeine 283 * 135 * 111 96 97 * 1891 Pentoxifylline * * * * * * *
* >2000 P

Temodar 515 * * * 298 * 31 * >2000 1-1 119 141 * * 83 261 79 239 1116 , rõ

, 1-2 * * * * * 187 95 * 1329 , , 5.1 52 347 , , , 9.2 42 873 1-5 * * * * * * *
* 1924 1-6 8.6 75 5.7 17 8.6 11.5 26 8.6 262 1-7 14 68 14 60 14 8.5 5.7 17 383 1-8 8.2 8.2 8.2 13.7 8.2 8.2 5.5 8.2 287 1-d n 1-9 * * * * * *
124 * 1676 r 1-10 * 22 169 86 105 277 83 188 2235 .t.) 1-11 * * 259 * 116 183 85 259 1766 u, C:=--, o o o o ,-,

26 t..) o 1-14 7.3 9.8 4.9 12 4.9 17 12 7.3 253 ,-, 1-15 19 52 11.8 26 9.5 7.1 4.7 11.8 698 u, yD

.6.

13.6 85 648 u, 1-17 * * * * 193 * 151 * 1733 aIC50 - Concentration (pM) providing 50% cell killing effect (KIT).
P

.2 . 3 .
, , , n 1-i r .t.) ,-, u, -,i-::--, o o o o ,-,

Claims

32

1. A compound selected from those of Formula I:
wherein R1 represents hydrogen, C1-4alkyl, hydroxy-C2-4alkyl, C1-3alkoxy-C2-4alkyl, C1-3alkylcarbonyl-C1-4alkyl or C1-3alkyl(C1-3alkyl)amino-C2-4alkyl;
R2 represents C1-4alkyl, hydroxy-C2-4alkyl, C1-4alkylcarbonyl-C1-4alkyl, C1-3alkoxy-C2-4alkyl, C1-3alkyl(C1-3alkyl)amino-C2-4alkyl or halo-C2-4alkyl;
R3 represents C1-4alkyl, allyl or C1-3alkoxy-C2-4alkyl;
with the proviso that if R2 is ethyl, R3 is methyl and R4 is 1-hydroxy-ethyl, then R1 is not a hydrogen;
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) atorn 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 C1-4alkyl, hydroxy-C1-4alkyl, C1-3alkoxy-C1-4alkyl, amino-C1-4alkyl, 1-hydroxy-di-(C1-3alkyl)methyl, 1-amino-di-(C1-3alkyl)methyl, 1-hydroxy-cyclo-C3-6alkyl, 1-amino-cyclo-C3-6alkyl, 1-(hydroxy-C1-3alkyl)-cycloC3-6alkyI, C1-3alkylamino-C1-3alkyl, C1-3alkyl(C1-3alkyl)amino-C1-3alkyl, di-(C1-3alkoxy-C2-4alkyl)-amino-C1-3alkyl, heterocyclyl-C1-3alkyl, 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, C1-4alkoxycarbonyl, N-C1-4alkylaminocarbonyl, N,N-di-(C1-3alkyl)aminocarbonyl, CH2OH, trifluoromethyl, 4alkyl, allyl, C2-4alkynyl, C1-4alkoxy, difluoromethoxy, trifluoromethoxy, cyclo-C3-6alkoxy, hydroxy-C1-4alkyl, C1-3alkoxy-C1-4alkyl, C1-3alkoxy-C2-4alkoxy, di-(C1-3alkyl)amino, di-(C1-3alkyl)amino-C1-3alkyl, di-(C1-3alkyl)amino-C2-4alkoxy, C1-4alkylsulfonylamino and C1-4alkyl-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, C1-4alkyl, C1-4alkoxy, difluoromethoxy, trifluoromethoxy, cyclo-C3-6alkoxy, C1-3alkoxy-C1-4alkyl, cyclo-C3-6alkylamino and di-(C1-3alkyl)amino;
its optical isomers, polymorphs and pharmaceutically acceptable acid addition salts and hydrates and solvates thereof with the proviso that 8-(3-hydroxyprop-1-yn-1-yl)-1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione is excluded.

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-C1-4alkyl, amino-C1-4alkyl, 1-hydroxy-di-(C1-3alkyl)methyl, 1-amino-di-(C1-3alkyl)methyl, 1-hydroxy-cyclo-C3-6alkyl, 1-amino-cyclo-C3-6alkyl, 1-(hydroxy-C1-3alkyl)-cycloC3-6alkyl, C1-3alkyl(C1-3alkyl)amino-C1-3alkyl, di-(C1-3alkoxy-C2-4alkyl)-amino-C1-3alkyl, heterocyclyl-C1-3alkyl or aryl.

5. The compound as claimed in Claim 4, wherein R2 and R3 each independently represent C1-4alkyl or C1-3alkoxy-C2-4alkyl with the proviso that if R2 is ethyl, R3 is methyl and R4 is 1-hydroxy-ethyl, then R1 is not a hydrogen.

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-1-yn-1-yl)-1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione, 8-((1-Hydroxycyclohexyl)ethynyl)-1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione, 8-((1-Aminocyclohexyl)ethynyl)-3,7-dimethyl-1H-purine-2,6(3H,7H)-dione, 8-(3-(Dimethylamino)prop-1-yn-1-yI)-1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione, 8-(3-(bis(2-methoxyethyl)amino)prop-1-yn-1-yl)-1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione, 1,3,7-Trimethyl-8-(3-(pyrrolidin-1-yl)prop-1-yn-1-yl)-1H-purine-2,6(3H,7H)-dione, 1,3,7-Trimethyl-8-(3-(piperidin-1-yl)prop-1-yn-1-yl)-1H-purine-2,6(3H,7H)-dione, 8-(3-(Azepan-1-yl)prop-1-yn-1-yl)-1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione, 1,3,7-Trimethyl-8-(3-morpholinoprop-1-yn-1-yl)-1H-purine-2,6(3H,7H)-dione, 8-(3-Hydroxy-3-methylbut-1-yn-1-yl)-3,7-dimethyl-1-(5-oxohexyl)-1H-purine-2,6(3H,7H)-dione, 8-((1-Hydroxycyclohexyl)ethynyl)-3,7-dimethyl-1-(5-oxohexyl)-1H-purine-2,6(3H,7H)-dione, 8-((1-Aminocyclohexyl)ethynyl)-3,7-dimethyl-1-(5-oxohexyl)-1H-purine-2,6(3H,7H)-dione, 8-(3-(Bis(2-methoxyethyl)amino)prop-1-yn-1-yl)-3,7-dimethyl-1-(5-oxohexyl)-1H-purine-2,6(3H,7H)-dione, 3, 7-Dimethyl-1-(5-oxohexyl)-8-(3-(pyrrolidin-1-yl)prop-1-yn-1-yl)-1H-purine-2,6(3H, 7H)-dione, 3,7-Dimethyl-1-(5-oxohexyl)-8-(3-(piperidin-1-yl)prop-1-yn-1-yl)-1H-purine-2,6(3H, 7H)-dione, 1,3,7-Trimethyl-8-(phenylethynyl)-1H-purine-2, 6(3H, 7H)-dione, 1,3,9-Trimethyl-8-(3-(pyrrolidin-1-yl)prop-1-yn-1-yl)-1H-purine-2,6(3H,9H)-dione, 1,3,9-Trimethyl-8-(phenylethynyl)-1H-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 in the treatment or prevention of melanoma, breast adenocarcinoma, hepatocellular carcinoma, neuroblastoma, glioma, lymphoma, and lung cancer.

10. A process for the synthesis of a compound selected from those of Formula I:
wherein R1 represents hydrogen, C1-4alkyl, hydroxy-C2-4alkyl, C1-3alkoxy-C2-4alkyl, C1-3alkylcarbonyl-C1-4alkyl or C1-3alkyl(C1-3alkyl)amino-C2-4alkyl;
R2 represents C1-4alkyl, hydroxy-C2-4alkyl, C1-4alkylcarbonyl-C1-4alkyl, C1-3alkoxy-C2-4alkyl, C1-3alkyl(C1-3alkyl)amino-C2-4alkyl or halo-C2-4alkyl;
R3 represents C1-4alkyl, allyl or C1-3alkoxy-C2-4alkyl;
with the proviso that if R2 is ethyl, R3 is methyl and R4 is 1-hydroxy-ethyl, then R1 is not a hydrogen;

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 C1-4alkyl, hydroxy-C1-4alkyl, C1-3alkoxy-C1-4alkyl, amino-C1-4alkyl, 1-hydroxy-di-(C1-3alkyl)methyl, 1-amino-di-(C1-3alkyl)methyl, 1-hydroxy-cyclo-C3-6alkyl, 1-amino-cyclo-C3-6alkyl, 1-(hydroxy-C1-3alkyl)-cycloC3-6alkyl, C1-3alkylamino-C1-3alkyl, C1-3alkyl(C1-3alkyl)amino-C1-3alkyl, di-(C1-3alkoxy-C2-4alkyl)-amino-C1-3alkyl, heterocyclyl-C1-3alkyl, aryl or heteroaryl;
and its optical isomers, polymorphs and pharmaceutically acceptable acid and base addition salts and hydrates and solvates thereof with the proviso that 8-(3-hydroxyprop-1-yn-1-yl)-1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione is excluded;
comprising reaction of a compound of Formula II:
with a compound of Formula 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-acceptable salt, hydrate or solvate.