ETHYNYLXANTHINES, PREPARATION AND USE AS CALCIUM
ION CHANNEL MODULATORS
FIELD OF THE INVENTION
The present invention relates to novel ethynylxanthine derivatives as calcium ion channel modulators, methods for their synthesis and use for the treatment and/or prevention of various diseases and disorders.
BACKGROUND OF THE INVENTION
Calcium ion channels are membrane-spanning, multi-subunit proteins that allow calcium entry from the external milieu and concurrent depolarization of the cell's membrane potential. Also, calcium plays a central role in neurotransmitter release. Calcium channels have been implicated in pathologies related to various diseases and disorders, including essential tremor, pain, neuropathic pain, schizophrenia, Parkinson's disease, depression, anxiety, epilepsy, bipolar disorder, sleep disorders, sleep disturbances, psychosis, cardiac arrhythmia, hypertension, certain types of cancer, diabetes, infertility, sexual dysfunction, etc. The known therapeutic regimens for such treating of diseases and disorders suffer from numerous problems and a number of side effects (Proft, Mol. Pharm. Fast Forward, 2014, DOI: 10.1124/mol.114.096008; Bose, Cell Death Disease, 2015, 6, el648; Calcium Channels: Properties, Functions and Regulation, Eds. M. Figgins, Nova Sci. Publ.; Taylor, Cancer Res., 1992, 52, 2413-2418; Monteith, J. Biol. Chem., 2012, 38, 31666- 31673; Hagenacker, Cell Calcium, 2008, 43, 215-227). Accordingly, a more physiological way, to develop additional Ca2+ channel blockers/antagonists, preferably those with higher potency, high selectivity and fewer side effects, searching of remedies against these diseases and disorders is highly desirable (Arranz- Tagarro, Expert Opin. Ther. Pat., 2014, 24, 959-77). 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. Caffeine derivatives possess CNS expression as calcium agonist or antagonist effect.
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 (LD5o=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,
8-ethynyl xanthines wherein R] and R2 represent, e.g., hydrogen, C1-6alkyl, 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.
¾enzyl-8-ethynyl xanthines
THE PRESENT INVENTION
We have determined that certain ethynylxanthine derivatives exhibit ability to act as calcium ion channel modulators. Therefore, these substances may be therapeutically beneficial in the treatment of conditions which involve treatment and/or prevention of various diseases and disorders including dysregulated calcium ion channel activity such as essential tremor, pain, neuropathic pain, schizophrenia, Parkinson's disease, depression, anxiety, epilepsy, bipolar disorder, sleep disorders, sleep disturbances, psychosis, cardiac arrhythmia, hypertension, certain types of cancer, diabetes, infertility, sexual dysfunction 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 calcium ion channel modulators, methods for their synthesis and the treatment and/or prevention of various diseases and disorders include, but not limited to essential tremor, pain, neuropathic pain, schizophrenia, Parkinson's disease, depression, anxiety, epilepsy, bipolar disorder, sleep disorders, sleep disturbances, psychosis, cardiac arrhythmia, hypertension, certain types of cancer, diabetes, infertility, sexual dysfunction in which calcium ion channel activity modulation is desired and/or required 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
I
wherein
R1 represents hydrogen, Ci-4alkyl, hydroxy-C2-4alkyl, Ci-3alkoxy-C2-4alkyl, Cj_ 3alkylcarbonyl-C1-4alkyl or C1-3alkyl(C1-3alkyl)amino-C2-4alkyl;
R2 represents C^alkyl, hydroxy-C2-4alkyl, C1-4alkylcarbonyl-C1-4alkyl, C1_3alkoxy-C2- 4alkyl, C1-3alkyl(C1-3alkyl)amino-C2-4alkyl or halo-C2-4alkyl;
R represents C1-4alkyl, allyl or C1-3alkoxy-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 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;
R
4 represents Ci
-4alkyl, hydroxy-C
1-4alkyl, C
1-3alkoxy-C
1.
4alkyl, amino-C
1_
4alkyl, 1-
l-hydroxy-cyclo-C3.
6alkyl, l-amino-cyclo-C
3.
6alkyl, l-(hydroxy-Ci-
3alkyl)-cycloC
3-
6alkyl, Cj.
3alkylamino-C i -
3alkyl, C j _
3alkyl(C ( _
3alkyl)amino-C i -
3alkyl , di-(C
1-
3alkoxy-C
2.
4alkyl)- amino-Ci-
3alkyl, heterocyclyl-C i - 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.N-di-iCi-salky aminocarbonyl, CH
2OH, trifluoromethyl, Q_
4alkyl, allyl, C
2-4alkynyl, C
1-4alkoxy, difluoromethoxy, trifluoromethoxy, cyclo- C
3- alkoxy,
C
1-3alkoxy-C
2-4alkoxy, di-(C!-
3alkyl)amino, di-(C ^alky amino-C ^alkyl, di-(C ^alky amino-C^alkoxy, Q^alkylsulfonylamino and C
1-4alkyl-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, C
1-4alkyl, C^alkoxy, difluoromethoxy, trifluoromethoxy, cyclo-C3
-6alkoxy, C
1-3alkoxy-C
1.
4alkyl, cyclo-C3. alkylamino and di-CQ-salky 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)-l,3,7-trimethyl-lH-purine-2,6(3H,7H)-dione, 8-((l-Hydroxycyclohexyl)ethynyl) ,3,7-trime l-lH-purine-2,6(3H,7H)-dione, 8-((l-Aminocyclohexyl)ethynyl)-3,7-dimethyl-lH-purine-2,6(3H,7H)-dione,
8-(3-(Dimemylamino)prop-l-yn-l-yl)-l,3,7-trimemyl-lH-purine-2,6(3H,7H)-dione, 8-(3-(bis(2-methoxyethyl)amino)prop- 1-yn- l-yl)-l ,3,7-trimethyl-lH-purine- 2,6(3H,7H)-dione,
1 ,3,7-Trimethyl-8-(3-(pyrrolidin- 1 -yl)prop- 1 -yn- 1 -yl)- 1 H-purine-2,6(3H,7H)-dione,
1 ,3,7-Trimethyl-8-(3-(piperidin- 1 -yl)prop- 1 -yn- 1 -yl)- 1 H-purine-2,6(3H,7H)-dione,
8-(3 -(A/.epan- 1 -yl)prop- 1-yn- 1 -yl)- 1 ,3,7-trimethyl- 1 H-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- 1 -yn- 1 ~yl)-3,7-dimethyl- l-(5~oxohexyl)~ IH-purine- 2,6(3 H,7H)-dione,
8-((l-Hydroxycyclohexyl)ethynyl)-3,7-dimethyl-l-(5-oxohexyl)-lH-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-memoxyethyl)amino)prop-l-yn-l-yl)-3,7-dimethyl-l-(5-oxohexyl)-lH- purine-2,6(3H,7H)-dione,
3 ,7-Dimethyl- 1 -(5-oxohexyl)-8-(3 -(pyrrolidin- 1 -yl)prop- 1-yn- 1 -yl)- lH-purine- 2,6(3H,7H)-dione,
3,7-Dimethyl- 1 -(5-oxohexyl)-8-(3-(piperidin- 1 -yl)prop- 1-yn- 1 -yl)- lH-purine- 2,6(3H,7H)-dione,
1 ,3 ,7-Trimethyl-8-(phenylethynyl)- 1 H-purine-2,6(3H,7H)-dione,
l,3,9-Trimemyl-8-(3-(pyrrolidin-l-yl)prop-l-yn-l-yl)-lH-purine-2,6(3H,9H)-dione, l,3,9-Trimethyl-8-(phenylethynyl)-lH-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. DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term "C1-4alkyl" 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, thiomorpholinyl 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 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-bromoxanfhines 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-mefhylpyrrolidine 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- 1 -yn- 1 -yl)- 1 ,3 ,7-trimethyl- 1 H-purine-
2,6(3H,7H)-dione (1-1) in 55% yield and by-product 8-[ (E)-5-hydroxy-2-( 1 -hydroxy - l-methylethyl)-5-methylhex- 1 -en-3-ynyl]- 1 ,3 ,7-trimethyl- 1 H -purine-2,6(3 H,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
II III I
Reaction conditions:
Method A. (Ph3P)2PdCl2, Cul, N-methylpyrrolidine or DMF, DIEA, 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 R 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 and R 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 R
3 is at purine N(7) (3, 4).
1 (R1 = Me) 3 (R1 = Me)
2 (R1 = MeC(0)(CH2)4) 4 (R1 = MeC(0)(CH2)4)
Compounds II, wherein R 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 R 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-l,3-dimethyl-5-nitropyrimidine-2,4(lH,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- 1, 3 ,9-trimemyl-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)).
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, "HO" as hydrochloric acid,„aq. NH3" as aqueous ammonia solution,„"MeCN" as acetonitrile, "DIEA" as diisopropylethylamine, "EtOAc" as ethyl acetate, "rt" as room temperature.
Intermediate 3.
8-Bromo-l,3,7-trimethyl-m- one (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) δ (ppm): 3.41 (s, 3H, CH3), 3.57 (s, 3H, CH3), 3.97 (s, 3H, CH3). Intermediate 4
8-Bromo-3,7-dimethyI-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%). 1H NMR (CDC13 TMS, 400 MHz) £(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-trimethyl-lH-purine-2,6(3 ,9H)-dione (11).
a) e-Chloro-l^-dimethylpyrimidine^^dH^^-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 (2x 100 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. 1H 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) (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) δ (ppm): 2.49, 2.75 and 3.35 (all s, all 3H), 7.74 (br s, 1H).
d) 5-Amino-l,3-dimethyl-6-methylamino^yrimidine-2,4(l^,3£T)-dione (9).
To a stirred solution of l,3-dimemyl-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) ml v. 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 CH2C12, 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. Ή NMR (400 MHz,
DMSO~d6) δ (ppm): 3.22, 3.68 and 3.93 (all s, all 3H), 7.66 (s, 1 H). MS (EI) mlz: 195 |M+2]+.
f) 8-Bromo-l,3,9-trimethyl-l /-purine-2,6(3H,9 /)-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) mlz: 375 [M+2]+.
Example 1
8-(3-Hydroxy-3-methylbut-l-yn-l-yl)-l,3,7-trimethyl-lH-purine-2,6(3H,7H)- dione (1-1)
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-lH-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. NH3 (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. 1H 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). 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- 1 ,3 ,7-trimethyl- 1 H- 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- 1, 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), DIEA (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-[(E)-5- hydroxy-2-( 1 -hydroxy- 1 -methylethyl)-5-methylhex- 1 -en-3-ynyl]- 1 ,3 ,7-trimethyl- 1 H- purine-2,6(3H,7H)-dione as a by-product in 26% yield. By-product: Ή NMR (CDCI3/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, I H). 13C NMR (CDC1 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, 1 55.0, 156.1.
Example 2
8 (l-Hydroxycyclohexy ethynyI)-l^,7 rimethyl-l /-piirine-2,6 3//,7//)-dione
(1-2)
Yield: 49% (Method C), 54% (Method D), 11% (Method B); mp = 194-196 °C. 1H NMR (CDC1 TMS, 400 MHz) £(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) m/z: 317.5 [M+l]+.
Example 3
8-((l-Aminocyclohexyl)eth e-2,6(3H,7H)-dione (I-3)
Yield: 48% (Method D), 43% (Method C), 16% (Method B), 7 % (Method A); mp = 187-189 °C. 1H NMR (CDC13ATMS, 400 MHz) (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) m/z: 302.3 [M+l]+.
Example 4
8-(3-(Dimethylamino)prop-l-yn-l-yl)-l,3,7-trimethyl-lH
r-purine-2,6(3-^,7//)- dione (1-4)
Isolated as hydrochloride. Yield: 58% (Method D), 47% (Method C), 4% (Method B); mp = 197-199 °C (dec.) Ή 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) m/z 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. 1H NMR (CDC13 TMS, 400 MHz) £(ppm): 2.79 (t, 4H), 3.31 (s, 6H), 3.34 (s, 3H), 3.48 (t, 4H), 3.51 (s, 3H), 3.82 (s, 2H), 3.96 (s, 3H). 13C NMR (CDC13/TMS, 100.6 MHz) £(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+l]+.
Example 6
l^^-Trimethyl-S S-ipyrrolidin-l-y prop-l-yn-l-y -lff-purine- ^iS^TH - dione (1-6)
Yield: 57% (Method D), 52% (Method C), 8% (Method A); mp = 149-150 °C. 1H NMR (CDCI3/TMS, 400 MHz) δ (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). "C NMR (CDCI3 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, 1 1.5, 154.8. MS (EI) mlz: 302.3 [M+l]+. Example 7
l,3,7-Trimethyl-8-(3~(piperidin-l-yl)pro
(1-7)
Yield: 35% (Method C), 30% (Method D); mp = 137-138 °C. 1H NMR (CDCI3 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 (CDCI3/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-trimethyl-lH-purine-2,6(3H,7fir)-dione
(1-8)
Yield: 34% (Method C), foam. 1H NMR (CDCI3/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 (s, 2H), 4.01 (s, 3H). 13C NMR (CDCI3/TMS, 100.6 MHz) < (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) mlz: 330.3 [M+l]+.
Example 9
l,3,7-Trimethyl-8-(3-morpholinoprop-l-yn-l-yl)-lH-puriiie-2,6(3H,7H)-dion
Yield: 38% (Method C), mp =188-190 °C. Ι NMR (CDCI3 TMS, 400 MHz) δ (ppm): 2.64 (t, 4H), 3.40 (s, 3H), 3.56 (s, 3H), 3.64 (s, 2H), 3.76 (t, 411), 4.01 (s, 3H). 13C NMR (CDC1 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) ml v. 318.3 [M+lf.
Example 10
8-(3-Hydroxy-3-methyIbut-l-yn-l-yl)-3,7-dimethyl-l-(5-oxohexyl)-lHr-purine- 2,6(3i/,7H)-dione (1-10
Yield: 47% (Method D), 44% (Method C); foam. 1H NMR (CDCI3 TMS, 400 MHz) δ (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 (CDCI3/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+lf.
Example 11
8-((l-Hydroxycyclohexyl)ethynyl)-3,7-dimethyl-l-(5-oxohexyI)-lH-purine- 2,6(3H,7/7)-dione (1-1
Yield: 42% (Method C), mp = 126-128 °C. 1H NMR (CDCI3 TMS, 400 MHz) δ (ppm): 1.27-1.37 (m, III), 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) δ (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) mlz: 401.6 [M+lf.
Example 12
8-iil-Aminocyclohexyl)ethynyl)-3,7-dimethyl-l-i5-oxohexyl)-lf/-p
2,6(3//,7/i dione (1-1
Yield: 45% (Method C), mp > 200° C. 1H NMR (CDC13 TMS, 400 MHz) £ (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, 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^^H^-dione (1-13)
Yield: 35% (Method C), foam. 1H NMR (CDCI3 TMS, 400 MHz) (ppm): 1.60-1.65 (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, 3H), 3.92 (s, 3H), 3.97 (t, 2H). 13C NMR (CDC1 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-yl)-lH
r-purine- 2,6(3H,7H)-dione (1-14)
Isolated as hydrochloride. Yield: 41 %, foam. Ή NMR (CDCI3/TMS, 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 (CDC13ATMS, 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) ml v. 386.3 [M+l]+.
Example 15
3,7-DimethyI-l-(5-oxohexyl)-8-(3-(piperidin-l-yl)prop-l-yn-l-yl)-li-r-purine- 2,6(3H,7H)-dione (1-1
Yield: 39% (Method C), mp = 182-184 °C. 1H NMR (CDC1 TMS, 400 MHz) δ (ppm): 1.34-1.48 (m, 1H), 1.58-1.66 (m, 4H), 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 (CDCI3 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 (EI) ml v. 400.2 [M+l]+.
Example 16
l,3,7-Trimethyl-8-(phen -dione (I-16)
1H NMR (CDQ TMS, 400 MHz) δ (ppm): Yield: 36% (Method C). 1H NMR (CDCI3 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) mlz: 295.5 [MJ+.
Example 17
lA9-Trimethyl-8-(3-(pyrrolidin-l^
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) mlz: 302.1 [M]+.
Example 18
l,3,9-Trimethyl-8-(phen -dione (I-18)
Yield: 75% (Method C), mp > 200 °C. 1H NMR (CDC13A S, 400 MHz) (ppm): 3.42 (s, 3H), 3.80 (s, 3H), 4.06 (s, 3H), 7.35-7.42 (m, 3H), 7.53-7.55 (m, 2H). 13C NMR (DMSO-d6, 100.6 MHz) (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) mlz: 295.3 [M]+.
CALCIUM CHANNEL AGONIST-ANTAGONIST MODULATION
Calcium channel agonist-antagonist modulation caused by 8-ethynylxanthines was tested on rat cardiomyoblast (H9C2), human neuroblastoma (SH-SY5Y), and smooth muscle (A7R5) cell lines (Table 2). According to our results, reference compounds such as caffeine, pentoxifylline, and Temodar showed moderate or no ability to affect on calcium ion channel activity in these cells, however, proxyfeine exhibits antagonist properties on neuroblastoma and smooth muscle cells. Suprisingly, in a series of studied ethynylxanthines selective calcium ion channel tissue modulators were found (Table 1). Thus, caffeine derivative 1-2 is selective calcium channel antagonist on brain tumor SH-SY5Y cell line (Κ½ο=0.057 mM), however
pentoxifylline derivative 1-15 exhitibs antagonist properties in cardiomyocytes H9C2 cells (IC50=0.075 niM).
These results support terapeutical potential of invented compounds to treat diseases with disregulated calcium chanel activity such as essential tremor, pain, neuropathic pain, schizophrenia, Parkinson's disease, depression, anxiety, epilepsy, bipolar disorder, sleep disorders, sleep disturbances, psychosis, cardiac arrhythmia, hypertension, certain types of cancer, diabetes, infertility, sexual dysfunction, etc.
Table 1. Calcium channel agonist-antagonist modulators on H9C2 (rat cardiomyoblast), SH-SY5Y (human neuroblastoma), A7R5 (smooth muscle) caused by 8-ethynylxanthines.
Compound H9C2, mM SH-SY5Y, mM A7R5, mM
Caffeine AG * *
Proxyfeine >2 0.67 0.52
Pentoxifylline 1.5 >2 >2
Temodar 1.5 * *
1-1 AG 0.35 >2
1-2 0.74 0.057 0.25
1-3 AG AG AG
1-4 0.56 1.0 >2
1-5 AG AG 0.4
1-6 0.43 0.52 0.41
1-7 0.58 0.6 0.52
1-8 0.36 0.18 0.38
1-9 AG AG 1.0
1-10 0.89 1.0 0.6
1-11 AG 0.57 0.52
1-14 0.30 0.87 0.47
1-15 0.075 0.35 0.28
1-16 0.67 1.0 0.29
1-17 0.77 0.58 0.60
1-18 AG 0.59 0.38
- calcium channel agonist; * - no activity.
Intracellular Ca + measurements
Materials: Fluo-4 NW Calcium Assay Kit was purchased from Invitrogen (Sweden). All other reagents were purchased from Aldrich or Sigma.
Cell cultures: H9C2 cells derived from the embryonic rat ventricle, SHSY5Y cells, human neuroblastoma, and A7R5 cells, rat smooth muscle cells. Cell lines were obtained from European Collection of Animal Cell Cultures. Cell lines were grown in Dulbecco's modified Eagle medium (DMEM) containing 1% non-essential amino- acids, 2 mM glutamine and supplemented with 10% fetal bovine serum (FBS, Sigma), at 37°C in a 5% C02. Cells were passaged once a week using 0.25% trypsin, 0.53 mM EDTA solution and grown in 75-mm plastic culture flasks until confluent and seeded in 96-well plates for experiments. Cells were seeded into 96-well plate at a density of 30,000 cells per well and incubated for 72 h.
9+ 9
Intracellular free Ca concentration [Ca ]i was measurement in confluent monolayers of cells with the Ca -sensitive fluorescent indicator Fluo 4 NW.
The model for investigation of Ca channel blocking activity caused by xanthine derivatives was the analysis of the effect of caffeine on intracellular Ca2+ mobilization in the case of H9C2, analysis of the effect of carbachol on intracellular Ca2+ mobilization in SHSY5Y cells and analysis of the effect of KC1 on intracellular Ca mobilization in A7R5 cells. The cells were pre-incubated in the dark for 15 min with tested compounds at concentrations from 0.01 to 2 mM. Application of caffeine 1.2 mM for H9C2, carbachol 30 μΜ for SHSY5Y and KC1 50 mM for A7R5 to Fura- 4NW loaded cells stimulated intracellular Ca2+ mobilization. For investigation of Ca2+ channel agonist activity of compounds Fura-4NW loaded cells were stimulated by the addition of compounds (concentration range from 0.01 to 2 mM). Changes in [Ca2+]i were measured from the fluorescence emitted at 516 nm due to alternate excitation at 494 nm using the fluorescence spectrophotometer (Thermo Ascient, Finland). IC50 values for the tested compounds were calculated using GraphPad Prism® 3.0 software.