WO2017069661A1

WO2017069661A1 - Pyrimidyl-di(diazaspiro-alkanes) with antiviral activity

Info

Publication number: WO2017069661A1
Application number: PCT/RU2016/050040
Authority: WO
Inventors: Vadim Albertovich MAKAROV; Vladimir Georgievich Nesterenko; Roman Nikolaevich BOLGARIN; Elena Alexandrovna NOVOSELOVA
Original assignee: Limited Liability Company “Nearmedic Plus”
Priority date: 2015-10-20
Filing date: 2016-09-21
Publication date: 2017-04-27
Also published as: RU2015144872A; RU2633699C2

Abstract

The invention relates to novel pyrimidyl-di(diazaspiro-alkane) derivatives of formula (I) or a pharmaceutically acceptable acid additive salt thereof. The compounds exhibit a wide spectrum of antiviral activity against herpes virus, human immunodeficiency virus, papillomavirus, and hepatitis virus. The invention also relates to use of the compounds of formula (I), a method for preparing thereof, and a method for treating by using the claimed compounds. The general formula is: (I) A represents pyrimidine-4,6-diyl or pyrimidine-2,4-15 diyl that can comprise H, NO₂, CHO, OH, OAlk, halogen, NH(Alk), or N(Alk)₂ as a substituent; Hal represents chlorine or bromine; Alk is a linear or branched substituent having 1 to 4 carbon atoms.

Description

PY RIMIDY L -DI(DIAZASPIRO-A L KA NE S) WIT H A NT IVIRA L ACTIVITY

DE SC RIPTION TE CH NICA L FIE L D

The invention relates to novel biologically active py ri mi dyl -di ( di azaspi ro- alkane) derivatives of general formula (I), a pharmaceutically acceptable salt thereof, which exhibit activity against various viruses, a method for preparing and use thereof as antiviral agents.

BACKGROU ND A RT

The problem of searching for novel antiviral agents is due to a significant spread of viral infections in humans and animals. This is associated with a decrease in the immune protection in the human population, and with broad-scale development of viral resistance to commercially available drugs. The problem of rapid development of resistance is determined by the fact that in general, many used, known antiviral drugs are derivatives of one class of compounds, and by insufficient efficacy and toxicity of drugs.

A promising direction in searching for novel effective antiviral compounds is considered a narrow specificity of action on components of the viral life cycle. One of unique mechanisms of action on the process of viral invasion into a host cell is based on the adsorption of virus to a target cell by means of specific blockage of heparan sulfate (HS) receptors. After the role of heparan sulfate receptors in cellular interactions was discovered, more scientific papers appeared which dealt with the search for different methods for blocking this mechanism of virus adsorption based on competitive binding to the receptors of a host cell. The possibility of using this mechanism of action was studied for a variety of viruses: H SV-1, 2 (Shukla D., Spear P.G. Herpesviruses and heparan sulfate: an intimate relationship in aid of viral entry. J Clin Invest. 2001, 108(4), 503-10) H PV (Selinka HC, Giroglou T, Sapp M. A nalysis of the infectious entry pathway of human papillomavirus type 33 pseudovirions, Virology, 2002, 1, 279- 287; Selinka HC, Giroglou T, Nowak T, Christensen ND, Sapp M. Further evidence that papillomavirus capsids exist in two distinct conformations, J Virol., 2003, 77, 12961-7), H CMV (Paeschke R., Woskobojnik I., Makarov V ., Schmidtke M., Bogner E . DST P-27 prevents entry of human cytomegalovirus. Antimicrob Agents Chemother. 2014, 58(4): 1963-1971), some H IV strains (Patel V, Ferguson M, Minor PD. Antigenic sites on type 2 poliovirus, V irology, 1993, 192, 361-4), RSV (Hallak L K, Spillmann D, Collins PL, Peeples M E . Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection, J Virol, 2000, 74, 10508-13; Donalisio M, Rusnati M, Cagno V, Civra A, Bugatti A, Giuliani A, Pirri G, V olante M, Papotti M, Landolfo S, Lembo D., Inhibition of human respiratory syncytial virus infectivity by a dendrimeric heparan sulfate- binding peptide, Antimicrob Agents Chemother., 2012, 56, 5278-88), H BV, H CV (J iang Y F, He B, Ma J , L i NP, Gong GZ, Cheng D. Comparison of the antiviral effects of entecavir and adefovir dipivoxil in chronic H BV infection: a randomized control trial, Acta Gastroenterol Belg., 2012, 75, 316-21), some E nteroviruses (Tan CW, Chan Y F. E nterovirus 71 receptors: promising drug targets?, Expert Rev Anti Infect Ther. 2013, 11, 547-9), etc. The mechanism of action of diazoniadispiro-alkanes has been studied by the example of a 3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecane derivative. It has been proven that it is associated with a specific property of the compound to bind to heparan sulfate glycoconjugates, which does not allow virus to bind to a host cell and to continue its life cycle, and finally results in a reduction in the viral titer. It has been shown that the binding of diazoniadispiro-alkane is antagonized by heparin. The target for diazoniadispiro-alkanes is two sulfate groups located on adjacent sugar residues, thus, for example, for GlcA2S-GlcNS6S, GlcA2S-GlcNS3S, IdoA2S-GlcNAc6S, IdoA2S-GlcNH23SS6S, IdoA2S-GlcNS6S, and IdoA2SGIcNS3S, and a good electrostatic interaction is observed between negative charges on the sulfate groups and positively charged nitrogen atoms of diazoniadispiro-alkanes. Also, it has been shown that the same kind of interactions can occur with a carboxyl group of octasaccharide a UA-GlcNSIdoUA2S-GlcNAc-UA2S-GlcNS-IdoUA2S-GlcNH23S, which is one of the key regions of heparan sulfate glycoconjugates for viral penetration into a host cell (Schmidtke M., Riabova @, Dahse <§}-©., Stelzner A., Makarov V ., Synthesis, Cytotoxicity and Antiviral Activity of N,N'-bis-5-nitropyrimidyl Derivatives of Dispirotripiperazine, Antiviral Research, 2002, 55, 117-127; Schmidtke, M., Wutzler, P., Makarov, V . Novel opportunities to study and block interactions between viruses and cell surface heparan sulfates, Lett. Drug Design Discov., 2004, 1, 35-44; Artemenko, A.G., Muratov, E .N., K uz'min, V .E ., Kovdienko, N.A., Hromov, A.I., Makarov, V .A., Riabova, O.B., Wutzler, P., Schmidtke, M.,. Identification of individual structural fragments of N,N'-(bis-5-nitropyrimidyl)dispirotripiperazine derivatives for cytotoxicity and anti herpetic activity allows the prediction of new highly active compounds. J our na I of Antimicrobial Chemotherapy, 2007, 60, 68-77; Paeschke R., Woskobojnik L, Makarov V ., Schmidtke M., Bogner E . DSTP-27 prevents entry of human cytomegalovirus. Anti mi crob Agents Chemother. 2014, 58, 1963-1971). Today, in the world there are no drugs with such mechanism of action.

SU M MA RY OF INV E NTION

The object of the present invention is to provide novel pharmacologically active compounds against viral infections, including strains resistant to currently available drugs, which would have a low toxicity, would not cause adverse effects in warm- blooded organisms, and would have mechanism of action that affect the process of viral invasion into a host cell by means of blocking heparan sulfate receptors of the target cell.

This object has been solved by the synthesis of pyri mi dyl-di-(diazadispiro- alkane) derivatives comprising a central pyri mi dine moiety linked with two di azadi spi ro-al kane resi dues, of general f ormul a I

Hal^~ ω

wherein

X and Y are ©£¾, or X is ©S¾ and Y is orX is ©¾©S¾ and Y is ©£¾;

A represents pyri mi di ne-4,6-diyl or pyrimidine-2,4-diyl that can comprise H, N0₂, CHO, OH, OAlk, halogen, NHAlk, or NA Ik₂ as a substituent; wherein Alk is a linear or branched substituent having 1 to 4 carbon atoms; and

Hal is a halogen atom.

Preferred compounds are compounds of formula (I), wherein

X and Y are ©£¾, or X is ©S¾ and Y is orX is ©¾©S¾ and Y is CH₂; A represents pyri mi di ne-4,6-diyl or pyri mi di ne-2,4-diyl that comprises CI, Br,

C H3 or OH as a substituent;

Hal is a halogen atom, chlorine or bromine. Samples of such compounds are compounds of formula (I), wherein X is Y is ©¾; A is py ri mi di ne-4, 6- di y I that can comprise H, N0₂, or Alk as a substituenf Hal is a chlorine or bromine atom, and Alk is methyl,

or compounds of formula (I), wherein X is ©¾, Y is ^is pyrimidine- 4,6-diyl that can comprise (¾ N0₂, or A lk as a substituent; Hal is a chlorine or bromine atom, and Alk is methyl.

Earlier, we have prepared the following compounds: 3,3'-(5-nitropyrimidine-4,6- diyl)bis-3,12-diaza-6,9-dazoniadispiro[5.2.5.2]hexadecane tetrachloride and 3,3'-(2- methyl-5-nitropyrimidine-4,6-diyl)bis-3,12-diaza-6,9- dazoniadispiro[5.2.5.2]hexadecane tetrachloride, whose insignificant antiherpetic activity is disclosed (Schmidtke M., Riabova @, Dahse H.-M., Stelzner A., Makarov V ., Synthesis, Cytotoxicity and Antiviral Activity of N,N'-bis-5-nitropyrimidyl Derivatives of Dispirotripiperazine, Antiviral Research, 2002, 55, 117-127; Artemenko A.G., Muratov E .N., K uz'min V .E ., Kovdienko N.A., Hromov A. I., Makarov V .A., Riabova O.B., Wutzler P., Schmidtke M.,. Identification of individual structural fragments of N,N'-(bis-5-nitropyrimidyl)dispirotripiperazine derivatives for cytotoxicity and antiherpetic activity allows the prediction of new highly active compounds. J ournal of Antimicrobial Chemotherapy, 2007, 60(1), pp. 68-77).

The invention also relates to a pharmaceutically acceptable acid addition salt of said compounds.

The compounds can be prepared and used in a crystalline form.

Synthesized compounds of general formula I and their pharmaceutically acceptable acid or base addition salts have been studied against pathogenic viruses and can be used in the manufacture of medicaments for the treatment or prevention of viral diseases, for example, caused by human immunodeficiency virus, cytomegalovirus, hepatitis C or B virus, herpes virus type 1 and 2, and papillomatosis virus.

BRIE F DESCRIPTION OF DRAWINGS

Fig.1 Compounds 3 and 11 were studied in PsV experiment with HeLaT cells. Two concentrations of each compound were added simultaneously with H PV 16-PsVs virus and were compared with untreated cells. The luciferase activity was measured at 48 hpi.

D ESCRIPTION OF E M BODIME NTS The term "pharmaceutically acceptable salt of compounds of formula (I)" means any salts of an inorganic or organic acid or base, which have a required pharmacological activity of the initial compound. These salts can be prepared in situ during the synthesis, isolation or purification of a compound of formula (I) or can be specially prepared.

Pharmaceutically addition salts with acids are characterized by that they comprise therapeutically active non-toxic addition salt forms with acids which are able to form compounds of formula I. Said addition salts with acids can be prepared by the treatment of compounds, in base form, represented by general formula I by suitable acids, for example, inorganic acids: a hidrohalic acid, in particular, hydrochloric acid, hydrobromic acid; sulfuric acid, nitric acid, or phosphoric acid; and organic acids, for example: acetic acid, hydroxyacetic acid, propionic acid, lactic acid, piruvic acid, oxalic acid, malonic acid, maleic acid, fumaric acid, malic acid, tartaric acid, or citric acid.

The invention also relates to use of a compound of formula (I) or a pharmaceutically acceptable acid addition salt thereof, optionally, in a crystalline form, or a pharmaceutical composition based thereon in the manufactory of a medicament for the preventi on or treatment of vi ral i nf ecti ons.

Another subject matter of the invention is a method for preventing or treating a viral infection, comprising administering or applying to a subject a compound of formula (I), a pharmaceutically acceptable acid addition salt or a pharmaceutical composition based thereon, in an effective amount. A method for treating with the claimed compounds, a pharmaceutical composition or a medicament based thereon also is effective against strains resistant to currently available medicaments.

Experiments in vivo have shown a high activity of py ri mi dy I - di ( di azadi spi ro- alkane) representatives against strains of herpes viruses type 1 and 2, papillomatosis, hepatiti s C , and cytomegal ovi rus.

Examples of preparing compounds according to the invention are disclosed below.

A. 3,12-Diaza-6,9-diazoniadispiro[5.2.5.2]hexadecane dichloride derivatives The initial compound, 3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecane dichloride, was synthesized according to the scheme below.

Sodium bicarbonate in an amount of 12.26 g (0.146 mol) was added to a solution of 15.2 g (0.133 mol) of 1-formylpiperazine 3 in 267 mL of chloroform. The mixture was cooled with water to 10eC, and a solution of 17 mL (0.146 mol) of benzoyl chloride i n 27 mL of chl orof orm was added by drops thereto for 0.5 h. T he mixture was sti rred at room temperature for 16 hours (and was allowed to stand over night). Then, the reaction mixture was washed with 150 mL of water two times, the organic layer was dried over sodium sulfate under stirring for 0.5 h. Chloroform was evaporated, and 200 mL of hexane was added to the residue and stirred, while cooling to 4e<® and then hexane was decanted. The option was repeated once more. After addition of the third portion of hexane, the mixture was stirred for 0.5 h. The formed residue was filtered off, washed with 20 mL of hexane and 20 mL of ether, and dried in air. The yield of 4-benzoyl- piperazine-1-carbaldehyde was 22.4 g (77%), m.p. 87e©

10.9 g (0.05 mol) of 4-benzoyl-piperazine-1-carbaldehyde was dissolved in 60 mL of a mixture of MeOH and concentrated HCI (a ratio of HCI/MeOH was 1 :11) and stirred at room temperature for 24 h. The precipitated residue was filtered off, washed with methanol (2x5 mL) and acetone (1x10 mL), and dried in a dryer at 90e©for 4 h. The yield of 1-benzoyl-piperazine hydrochloride was 7.55 g (63%), m.p. 315e©

Benzoyl-piperazine hydrochloride in an amount of 18 g (0.0795 mol) was added to a solution of 5.34 g (0.0954 mol) of KOH in 55 mL of ethanol and stirred at 20-22eC for 0.5 h. Then, 13.3 mL (0.199 mol) of ethylene chlorohydrin was added thereto, and a solution of 11.6 g (0.207 mol) of K OH in 98 mL of ethanol (rectificate) was added by drops for one hour, avoiding an increase in temperature higher than 20eC. After 20 hours, the formed KCI was filtered off, washed with 25 mL of absolute ethanol. The filtrate was cooled to 10eC, and 58 mL of 12% HCI/EtOH (control of 7® 2-3) was slowly added thereto and stirred for 1 h at 5eC, and then was allowed to stand over night in a refrigerator. The residue was filtered, washed with absolute ethanol (2x10 mL), dried either in air for 2 days or in a dryer at 50-55eC for 4-5 h. The yield of 1 -benzoyl - 4-(b-oxyethyl)piperazine hydrochloride was 15.6 g (73%), m.p. 215eC.

8 mL of SOC was added by drops to a suspension of 13.5 g (0.05 mol) of 1- benzoyl-4-(b-oxyethyl)piperazine hydrochloride in 96 mL of chloroform, while increasing the temperature of the reaction mass to 45eC by heating in oil bath. The reaction mass was hold at this temperature for 0.5 h, then was heated to 55eC and aged for 0.5 h, and then was heated to 70eC, and stirred for 3 h. After that the reaction mass was cooled to 20eC and was allowed to stand in a refrigerator for 16 h. The residue was filtered off, washed with chloroform (2x30 mL) and dried for 3 h at 40-45eC in a dryer. The yield of 1-benzoyl-4-(b-chloroethyl)piperazine hydrochloride was 11.7 g (81 %), m.p. 230eC.

A solution of 1.12 g (0.028 mol) of NaOH in 19 mL of 96% ethanol was added to a suspension of 7.35 g (0.0254 mol) of 1-benzoyl-4-(b-chloroethyl)piperazine hydrochloride in 15 mL of ethanol (rectificate) and stirred at 20-25eC for 1.5 h. Then, NaCI was filtered and washed with absolute ethanol (2x5 mL). The filtrate was boiled under stirring for 1 h and then evaporated in a rotary evaporator at 80eC in a bath to dryness. The residue was heated at 120eC for 16 h and cooled. Distilled water in an amount of 15 mL was added thereto and stirred under boiling to complete dissolution of the residue. Activated carbon in an amount of 0.7 g was added to the mixture, and the mixture was boi led for 10 mi n. T he carbon was fi Itered and washed with hot water (2x5 mL). The stock solution was cooled and allowed to stand in a refrigerator for 16 h. The precipitated residue was filtered off, washed with water (2x5 mL) and alcohol (2x5 mL), and dried at 100e© for 2 h. The yield of N,N"-dibenzoyl-N',N"- dispirotripiperazinia dichloride in dehydrate form was 3.1 g, m.p. > 360e© (with decomposition).

A mixture of 3.1 g (0.0057 mol) of N,N"-dibenzoyl-N',N"-dispirotripiperazine dichloride in dehydrate form and 20 mL of 10% of hydrochloric acid prepared by mixing of 7 mL of concentrated hydrochloric acid with 13 mL of distilled water was boiled under stirring for 4 h. The reaction mixture was cooled in ice bath to 10-15e®, and precipitated benzoic acid was filtered and washed with water. The filtrate was evaporated to dryness in a rotary evaporator. The solid residue was stirred with 10 mL of methanol, the residue was filtered, washed with 5 mL of methanol, and dried at 100e©for 2 h. The yield of N ', N "-di spi rotri pi peraz i ni a di hydrochloride dehydrate was 1.9 g (82%), m.p. > 330e©(with decomposition).

L iOH in an amount of 0.26 g (0.0108 mol) was added to a mixture of 1.9 g (0.0047 mol) of N ', N "-di spi rotri pi perazi ni a di hydrochloride dihydrate in 3.2 mL of water by little portions under stirring at 20e©(pH 9). Then, 0.17 g of activated carbon was added and stirred for 0.5 h, the carbon was filtered off and washed with water (2x1 mL). The stock solution was diluted with 30 mL of methanol and allowed to stand in a refrigerator at 5e©for 16 h. The precipitated residue was filtered off, washed on a filter with 5 mL of methanol, and dried at 100eC for 2 h. The yield of 3,12-diaza-6,9- diazoniadispiro[5.2.5.2]hexadecane dichloride was 1.1 g (79%), m.p. 350e© (with decomposition).

Example 1

3, 3'-( 5- F ormy I py ri mi di ne-4, 6- di y I ) bi s-3, 12- di aza-6, 9- azoniadis iro[5.2.5.2]hexadecane tetrachloride (Compound 1)

A solution of 4,6-dichloro-5-formyl pyrimidine (0.0005 mol) in 4 mL of ethyl alcohol was added to a solution of 3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecane dichloride (0.001 mol) in 3 mL of water. The solution was boiled under stirring for 2 h. Tri ethyl amine (0.002 mol) was added thereto. The heating was stopped after 20 min. The solution was cooled to room temperature and evaporated to dryness, and 35 mL of methanol were added thereto. The precipitated residue was filtered off, washed with methanol and acetone. T he product was obtai ned i n an amount of 17 g.

T he product was dried at 100e©for 20 h.

T he yi el d of a yel I ow crystal I i ne compound was 46% .

E SI-MS/MS - 663

E lemental analysis: C29H52C I4N10O:

Calc: ©, 49.86; <§) 7.50; N, 20.05

Found: C, 49.36; <§) 7.84; N, 20.33 Example 2

3 '-(2-Phenyl-5-formylpyrimidine-4,6-diyl)bis-3,12-diaza-6,9- di

A solution of 4,6-dichloro-2-phenyl-5-formyl pyri mi dine (0.0005 mol) in 6 mL of dioxane was added to a solution of 3,12-diaza-6,9- diazoniadispiro[5.2.5.2]hexadecane dichloride (0.001) in 2 mL of water. The reaction mass was boiled under stirring for 3.5 h. A new residue was slowly precipitated from the solution. Tri ethyl amine (0.002 mol) was added thereto. The heating was stopped after 20 min. The reaction mass was cooled to room temperature. Methanol in an amount of 20 mL was added, and the residue was filtered off and washed with ethanol and acetone. The product was obtained in an amount of 0.29 g.

T he product was dried at 100e©for 20 h.

The yield of a white crystalline compound was 37%.

E SI-MS/MS - 737.

E lemental analysis: C35H56C I4N10O:

Calc: <® 54.26; <§) 7.29; N, 18.08

Found: <® 53.57; <§) 7.26; N, 18.07

Example 3

3,3'-(5-Formyl-6-hydroxypyrimidine-2,4-diyl)bis-3,12-diaza-6,9- diazoniadispiro[5.2.5.2]hexadecane tetrachloride (Compound 3)

A solution of 2-methylsulfanyl-5-formyl-4,6-dichloropyrimidine (0.0005 mol) in 7 mL of dioxane was added to 3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecane dichloride (0.001) in 2 mL of water. The solution was boiled under stirring for 4 h. Oil was precipitated from the reaction mass. Triethylamine (0.002 mol) was added thereto. The heating was stopped after 20 min. The reaction mass was cooled to room temperature. The solution was decanted, and 20 mL of methanol and 5 mL of acetone were added to the remaining oil. The precipitated residue was filtered off and washed with methanol, tetrahydrofuran, and acetone. The product was obtained in an amount of 0.12 g. T he product was dri ed at 100e©f or 20 h.

T he yield of a white crystal I i ne compound was 17%.

E SI-MS/MS - 677.

E lemental analysis: C29H52C I4N10O2:

Calc: <® 48.74; <§) 7.33; N, 19.60

Found: <® 49.07; <§) 7.37; N, 19.62

B. 3,12-Diaza-6,9-diazoniadispiro[5.2.5.3]heptadecane di bromide derivatives The initial 3,12-diaza-6,9-diazoniadispiro[5.2.5.2]heptadecane dibromide was synthesized according to the scheme below, similarly to the synthesis of 3,12-diaza-6,9- diazoniadispiro[5.2.5.2]hexadecane dichloride, as described above.

1-Formylpiperazine 1 (0.1 mol) was dissolved in chloroform (200 mL), and sodium bicarbonate (0.1 1 mol) was slowly added and a solution of benzoyl chloride (0.11 mol) in 20 mL of chloroform was dropped to the resulting solution. The mixture was stirred at room temperature for 16 hours, then was extracted with 100 mL of water two times, and dried over sodium sulfate. Chloroform was evaporated, hexane was added, and the mixture was cooled at 41C. The precipitated residue was filtered off and washed with ether. The yield of 4- benzoyl pi perazine-1- carbaldehyde was 71 %.

4-Benzoylpiperazine-1-carbaldehyde 2 (0.005 mol) was dissolved in 10 mL of a solution prepared from 5.5 mL of concentrated hydrochloric acid and 60 mL of methanol and stirred at room temperature for 24 h. The precipitated residue was filtered off and washed with methanol three times. The yield of 1 -benzoyl pi perazine hydrochloride was 60%.

1-Benzoylpiperazine hydrochloride 3 (0.014 mol) was suspended in 5 mL of ethanol, and a solution of NaOH (0.017 mol) in 15 mL of ethanol was added thereto. The mixture was stirred for 1 h. NaCI was filtered off, the stock solution was evaporated, chloroform was added, NaCI was filtered off once more, and chloroform was evaporated. 1-Benzoylpiperazine was obtained in the form of oil with a yield of 95%. A mixture of 1 -benzoyl pi perazine 4 (0.035 mol), 1,3-dibromopropane (0.0175 mol), and sodium bicarbonate (0.056 mol) was boiled in 60 mL of ethanol for 16 h. The hot reaction mass was filtered from NaBr, evaporated to dryness, chloroform was added, filtered off from NaBr, and evaporated to dryness once more. 1,1 '-Propane- 1,3- diylbis( 4- benzoyl pi perazine) was prepared in the form of oil with a yield of 60%.

A mixture of 1,1 '-propane-1,3-diylbis(4-benzoylpiperazine) (0.005 mol) and 1,2- dibromoethane (0.05 mol) was heated at 1101C for 6 h, and then cooled. The precipitated residue was filtered off and washed with ether. The yield of 3,12- dibenzoyl-3,12-diaza-6,9-diazoniadispiro[5.2.5.3]heptadecane di bromide was 69%.

3,12-Dibenzoyl-3,12-diaza-6,9-diazoniadispiro[5.2.5.3]heptadecane dibromide

(0.005 mol) was boiled with 18 mL of 10% hydrobromic acid for 4 h, then was cooled. Benzoic acid was filtered off, and the stock solution was evaporated to a viscous suspension. 40 mL of methanol and 10 mL of acetone were added, and the precipitated residue was filtered off and was washed with acetone. The yield of 3,12-diaza-6,9- diazoniadispiro[5.2.5.3]heptadecane dibromide di bromohydrate was 70%.

3, 12-D iaza-6,9-diazoniadispi ro[5.2.5.3] heptadecane dibromide di bromohydrate 7 (0.003 mol) was treated with a solution of LiOH (0.006 mol) in 3 mL of water. Then activated carbon was added and stirred for 20 min, the carbon was filtered off, and 10 mL of acetone and 20 mL of methanol were added to the stock solution. The precipitated residue was filtered off and washed with acetone. The yield of 3,12-diaza- 6,9-diazoniadispiro[5.2.5.3] heptadecane dibromide was 88%, m.p. 258-262e©

Example 4

3,3'-(5-Nitropyrimidine-4,6-diyl)bis-3,12-diaza-6,9- diazoniadispiro[5.2.5.3] heptadecane tetrabromide (Compound 4)

A solution of 4,6-dichloro-5-nitropyrimidine (0.000375 mol) in 4 mL of ethyl alcohol was added to a solution of 3, 12-diaza-6,9-diazoniadispiro[5.2.5.3] heptadecane dibromide (0.00075 mol) in 2 mL of water. The reaction mass was boiled under stirring for 3 h. Triethylamine (0.0015 mol) was added. The heating was stopped after 20 min. The reaction mass was cooled to room temperature. Methanol in an amount of 25 mL was added and triturated. The precipitated residue was filtered off and washed with methanol, tetrahydrofuran, and acetone. The product was obtained in an amount of 0.13 g. T he product was dried at 100e©for 20 h.

T he yi el d of a yel I ow crystal I i ne compound was 37% .

E SI-MS/MS - 838.

E lemental analysis: C3oHs5Br4Nii02:

Calc: ® 39.10; <§) 6.02; N, 18.91

Found: ® 39.08; <§) 6.40; N, 18.91

Example 5

3,3'-(2-Methyl-5-nitropyrimidine-4,6-diyl)bis-3,12-diaza-6,9- diazoniadispiro[5.2.5.3]heptadecane tetrabromide (Compound 5)

A solution of 2-methyl-4,6-dichloro-5-nitropyrimidine (0.0005 mol) in 4 mL of ethyl alcohol was added to a solution of 3,12-diaza-6,9- diazoniadispiro[5.2.5.3]heptadecane dibromide (0.001 mol) in 3 mL of water. The reaction mass was boiled under stirring for 3 h. Oil was slowly precipitated from the reaction mass. Tri ethyl amine (0.002 mol) was added. The heating was stopped after 20 min. The reaction mass was cooled to room temperature. Methanol in an amount of 30 mL was added and triturated. The solution was decanted, and 15 mL of methanol and 5 mL were added to the remaining oil. The precipitated residue was filtered off and washed with methanol, tetrahydrofuran, and acetone. The product was obtained in an amount of 0.36 g. T he product was dri ed at 100e©f or 20 h.

T he yi el d of a yel I ow crystal I i ne compound was 77% .

E SI-MS/MS - 856.

E lemental analysis: C3iHs7Br4Nn02:

: ® 39.10; ©, 6.14; N, 16.50 Found: <® 39.23; @ 6.19; N, 16.35

Example 6

3, 3'-( 5- F ormy I py ri mi di ne-4, 6- di y I ) bi s-3, 12- di aza-6, 9- diazoniadispiro[5.2.5.3]heptadecane tetrabromide (Compound 6)

A solution of 5-formyl-4,6-dichloropyrimidine (0.0005 mol) in 4 mL of ethyl alcohol was added to a solution of 3,12-diaza-6,9-diazoniadispiro[5.2.5.3]heptadecane dibromide (0.001 mol) in 3 mL of water. The solution was boiled under stirring for 2 h. Tri ethyl amine (0.002 mol) was added. The heating was stopped after 20 min. The reaction mass was cooled to room temperature, the solution was evaporated to dryness, and 30 mL of methanol were added. The precipitated residue was filtered off and washed with methanol and acetone. The product was obtained in an amount of 0.23 g. The product was dried at 100e©for 20 h.

T he yi el d of a yel I ow crystal I i ne compound was 51 % .

E SI-MS/MS - 821

E lemental analysis: C3iHs6Br4NioO:

Calc: <® 41.17; <§) 6.24; N, 15.49

Found: <® 41.69; <§) 6.26; N, 15.37

Example 7

3,3'-(6-Hydroxy-5-formylpyrimidine-2,4-diyl)bis-3,12-diaza-6,9- diazoniadispiro[5.2.5.3]heptadecane tetrabromide (Compound 7)

A solution of 2-methylsulfanyl-5-formyl-4,6-dichloropyrimidine (0.000375 mol) in 6 mL of dioxane was added to a solution of 3,12-diaza-6,9- diazoniadispiro[5.2.5.3]heptadecane dibromide (0.00075) in 2 mL of water. The solution was boiled under stirring for 4.5 h. Oil was precipitated from the reaction mass. Tri ethyl amine (0.0015 mol) was added. The heating was stopped after 20 min. The reaction mass was cooled to room temperature. The solution was decanted, and 15 mL of methanol and 5 mL of acetone were added to the remaining oil. The precipitated residue was filtered and washed with methanol, tetrahydrofuran, and acetone. The product was obtai ned i n an amount of 0.16 g. T he product was dri ed at 100e©f or 20 h.

The yield of a white crystalline compound was 23%.

E SI-MS/MS - 835

E lemental analysis: C3iHs6Br4Nio02:

Calc: <® 41.69; <® 6.29; N, 15.17

Found: <® 40.95; @ 6.13; N, 15.22

Example 8

3,3'-(2-Phenyl-5-formylpyrimidine-2,4-diyl)bis-3,12-diaza-6,9- diazoniadispiro[5.2.5.3]heptadecane tetrabromide (Compound 8) f

A solution of 2-phenyl-5-formyl-4,6-dichloropyrimidine (0.000375 mol) in 6 mL of dioxane was added to a solution of 3,12-diaza-6,9- diazoniadispiro[5.2.5.3]heptadecane dibromide (0.00075) in 2 mL of water. The solution was boiled under stirring for 3 h. Oil was precipitated from the reaction mass. Tri ethyl amine (0.0015 mol) was added. The heating was stopped after 20 min. The reaction mass was cooled to room temperature. The solution was decanted, 15 mL of methanol and 5 mL of acetone were added to the remaining oil, and the precipitated residue was filtered off and washed with methanol, tetrahydrofuran, and acetone. The product was obtai ned i n an amount of 0.15 g. T he product was dri ed at 100e©f or 20 h.

T he yi el d of a yel I ow crystal I i ne compound was 40% .

E SI-MS/MS - 897.

E lemental analysis: C37H6oBr4NioO:

Calc: <® 45.32; <§) 6.17; N, 14.28

Found: <® 45.29; <§) 6.67; N, 15.27

Example 9

3,3'-(2-Chloropyrimidine-4,6-diyl)bis-3,12-diaza-6,9- diazoniadispiro[5.2.5.3]heptadecane tetrabromide (Compound 9)

A solution of 5-nitro-2,4,6-trichloropyrimidine (0.00025 mol) in 4 mL of methyl alcohol was added to a solution of 3,12-diaza-6,9-diazoniadispiro[5.2.5.3]heptadecane di bromi de (0.0005 mol) i n 2 mL of water. T he sol uti on was boi I ed under sti rri ng for 3 h. Triethylamine (0.001 mol) was added. The heating was stopped after 20 min. The reaction mass was cooled to room temperature. Methanol in an amount of 25 mL was added and triturated. The precipitated residue was filtered off and washed with methanol, tetrahydrofuran, and acetone. The product was obtained in an amount of 0.07 g. T he product was dried at 100e©for 20 h.

T he y i el d a I i ght yel I ow crystal I i ne compound was 30% .

E SI-MS/MS - 875. E lemental analysis: C3oH54Br4CI Nn02

Calc: <© 37.69; <§) 5.69; N, 16.12

Found: <® 37.58; <® 5.62; N, 16.17

B. 3,13-Diaza-7,10-diazoniadispiro[6.2.6.2]octadecane di bromide derivatives The initial compound, 3,13-diaza-7,10-diazoniadispiro[6.2.6.2]octadecane di bromide, was synthesized according to the scheme below.

HBr UGH

Glacial acetic acid in an amount of 25 mL was added to 5.37 g (0.0537 mol) of homopiperazine under cooling with iced water, while the temperature increased up to 40-50eC. When the temperature decreased to 20e(¾ the cooling was stopped. The suspension was stirred to complete dissolution of homopiperazine (about 2 h). Then the solution was cooled to 51C (ice bath), and 6.27 mL (0.0537) of benzoyl chloride were slowly dropped (45 min) so that the temperature of the mass was not higher than 10tC. Then, the solution was stirred at room temperature for 3 h. Acetic acid was evaporated in a rotary evaporator at 80tC. The volume of distillation was 15-16 mL, which corresponded to the 2/3 volume of acetic acid used in the reaction. The residue, which was viscous oil, was diluted with 50 mL of water, cooled in ice bath to 151C under stirring, and alkalized with a 40% solution of NaOH (20 mL) to pH 8-8.5. The precipitated by-product of 1,4-dibenzoyldiazepine was filtered off, and the stock solution was extracted with chloroform (3x40 mL). The pH value of the aqueous layer was checked. If the pH value was lower than 7, it was alkalized once more to pH of 8- 8.5 and extracted with chloroform (2x40 mL). Combined chloroform extracts were dried over Na₂S04. Chloroform was evaporated to obtain 7.13 g (65%) of 1 -benzoyl - 1,4-diazepine in the form of oil.

A mixture of 7.13 g (0.035 mol) of 1-benzoyl-1,4-diazepine, 60 mL of ethanol, 1.51 mL (0.0175 mol) of 1,2-dibromoethane, and 4.7 g (0.056 mol) of sodium bicarbonate was stirred under boiling for 16 h. The reaction mass was cooled, and the inorganic residue was filtered off and washed with 10 mL of alcohol. The stock solution was evaporated to dryness, and 100 mL of water was added and extracted with chloroform (3x70 mL). The reaction mass was dried over Na₂S0₄ under stirring for 0.5 h. Chloroform was evaporated to dryness. 1,1 '-Ethane-1,2-diylbis(4-benzoyl-1,4- di azepi ne) i n the form of oi I was obtai ned i n an amount of 8 g ( 100%) .

A mixture of 8 g (0.0183 mol) of 1,1 '-ethane-1,2-diylbis(4-benzoyl-1,4- diazepine) and 16 mL of 1,2-dibromoethane was stirred and heated at 120tC for 4 h. The viscous paste was cooled to 20tC, 50 mL of ether was added and stirred, and the residue was filtered off and washed with ether (2x10 mL). Then, the paste was washed on a filter under stirring with chloroform (3x50 mL) and ether (2x10 mL). The residue in the form of paste was dried in air for 16 h. The yield of 3,13-dibenzoyl-3,13 diaza- 7,10-diazoniadispiro[6.2.6.2]octadecane dibromide was 7.2 g, m.p. about 3201C.

A mixture of 15.5 g (0.025 mol) of 3,13-dibenzoyl-3,13 diaza-7,10- diazoniadispiro[6.2.6.2]octadecane dibromide and 100 mL of 10% hydrobromic acid was stirred under boiling for 4 h. The mixture was cooled, the precipitated benzoic acid was filtered off and washed with water. Activated carbon in an amount of 1.5 g was added to the stock solution, stirred for 20 min, and filtered. The clarified solution was evaporated in a rotary evaporator to dryness. Methanol (210 mL) and acetone (60 mL) were added to the residue and stirred for 0.5 h. The residue was filtered off and washed with acetone (2x50 mL), and dried in a dryer at 100tC. The yield of 3,13-diaza-7,10- diazoniadispiro[6.2.6.2]octadecane dibromide was 12.66 g (88%), m.p. 310-315e©

L iOH in an amount of 1.06 g (0.44 mol) was added to a solution of 12.66 g

(0.022 mol) of 3,13-diaza-7,10-diazoniadispiro[6.2.6.2]octadecane dibromide in 22 mL of water under stirring at 20tC (pH 10). Then, 1.3 g of activated carbon was added and stirred for 20 h, the carbon was filtered and washed with 3 mL of water. The stock solution was diluted with 145 mL of acetone and 75 mL of methanol. The precipitated curdy residue was filtered under slight vacuum and washed on a filter under stirring with acetone (2x30 mL), washed once more without stirring, and then dried in air. The yield of 3,13-diaza-7,10-diazoniadispiro[6.2.6.2]octadecane dibromide was 6.4 g (70%), m.p. 280e©

Example 10

3,3'-(5-Nitropyrimidine-4,6-diyl)bis-3,13-diaza-7,10- di azoni adi s i ro[6.2.6.2] octadecane tetrabromi de ( C ompound 10)

A solution of 5-nitro-4,6-dichloro-nitropyrimidine (0.00036 mol) in 5 mL of ethyl alcohol was added to a solution of 3,13-diaza-7,10- diazoniadispiro[6.2.6.2]octadecane tetrabromide (0.00072 mol) in 2 mL of water. The reaction mass was boiled under stirring for 4 h. Triethylamine (0.0014 mol) was added. The heating was stopped after 20 min. The reaction mass was cooled to room temperature. Methanol in an amount of 25 mL was added and triturated. The precipitated residue was filtered off and washed with methanol, tetrahydrofuran, and acetone. The product was obtained in an amount of 0.1 g. The product was dried at 100e©for 20 h.

T he yi el d of a yel I ow crystal I i ne compound was 69% .

E SI-MS/MS - 870.

E lemental analysis: C32H59Br4Nn02;

Calc: <® 40.48; <§) 6.26; N, 16.23

Found: <® 41.19; <§) 6.28; N, 16.84

Example 11

3,3'-(2-Methyl-5-nitropyrimidine-4,6-diyl)bis-3,13-diaza-7,10- di azoni adi spi ro[6.2.6.2] octadecane tetrabromi de ( C ompound 11 )

A solution of 2-methyl-5-nitro-4,6-dichloro-nitropyrimidine (0.00036 mol) in 4 mL of ethyl alcohol was added to a solution of 3,13-diaza-7,10- diazoniadispiro[6.2.6.2]octadecane dibromide (0.00072 mol) in 3 mL of water. The reaction mass was boiled under stirring for 3.5 h. Oil was slowly precipitated from the reaction mass. Triethylamine (0.0014 mol) was added. The heating was stopped after 20 min. The reaction mass was cooled to room temperature. Methanol in an amount of 30 mL was added and triturated, the precipitated residue was filtered off and washed with methanol and acetone. The product was obtained in an amount of 0.13 g. The product was dri ed at 100e©f or 20 h.

T he yi el d of a yel I ow crystal I i ne compound was 37% .

E SI-MS/MS - 880.

E lemental analysis: C33H6iBr4Nn02:

Calc: <® 41.44; <§) 6.58; N, 15.99

Found: <® 40.52; <§) 6.52; N, 16.08

Example 12

3, 3'-( 5- F ormy I py ri mi di ne-4, 6- di y I ) bi s-3, 13- di aza-7, 10- di azoni adi spi ro[6.2.6.2] octadecane tetrabromi de ( C ompound 12)

A solution of 2-formyl-4,6-dichloropyrimidine (0.00036 mol) in 4 mL of ethyl alcohol was added to a solution of 3, 13-diaza-7,10-diazoniadispiro[6.2.6.2] octadecane dibromide (0.00072 mol) in 2 mL of water. The solution was boiled under stirring for 2 h. Triethylamine (0.0014 mol) was added. The heating was stopped after 20 min. The reaction mass was cooled to room temperature. The solution was evaporated to dryness, and 20 mL of methanol were added. The precipitated residue was filtered and washed with methanol and acetone. The product was obtained in an amount of 0.27 g. The product was dried at 100e©for 20 h.

The yield a fawn-colored crystalline compound was 40%.

E SI-MS/MS - 853.

E lemental analysis: C33H6oBr4NioO:

Calc: <® 42.50; <§) 6.46; N, 15.02

Found: <® 42.67; <§) 6.43; N, 15.04

Example 13

3,3'-(2-Phenyl-5-formylpyrimidine-4,6-diyl)bis-3,13-diaza-7,10- di azoni adi spi ro[6.2.6.2] octadecane tetrabromi de ( C ompound 13) .

A solution of 2-phenyl-5-formyl-4,6-dichloropyrimidine (0.00024 mol) in 5 mL of dioxane was added to 3,13-diaza-7,10-diazoniadispiro[6.2.6.2] octadecane di bromide (0.00048) in 1 mL of water. The reaction mass was boiled under stirring for 4 h. Oil was slowly precipitated from the solution. Triethylamine (0.00096 mol) was added. The heating was stopped after 20 min. The reaction mass was cooled to room temperature and evaporated to dryness. Methanol in an amount of 20 mL was added, the residue was filtered off and washed with ethanol and acetone. The product was obtained in an amount of 0.03 g. The product was dried at 100e©for 20 h.

The yield a fawn-colored crystalline compound was 6%.

E SI-MS/MS - 929.

E lemental analysis: C39H64Br4NnO:

Calc: <® 46.44; ©, 6.40; N, 13.89

Found: <® 46.48; ©, 6.24; N, 13.32

Description of cytotoxicity and antiviral activity of the compounds according to the invention Evaluation of the cytotoxicity of the synthesized dispirotripiperazine derivatives to GM K cells

2x1ο⁴ GMK cells were cultured in a 96- well flat- bottom microtiter plate (Falcon 3075). The cytotoxicity of the studied compositions was determined by confluent monol ayers of G M K eel I s, formed i n 60 wel I s ( 5% C 0₂, 37eC ) . W hen monol ayers were removed from the nutrient medium, 100 mL of the nutrient medium were added to nine twice-diluted compositions, and the latter were incubated for 72 h at 371C in 5% C0₂. The cells from six wells were used as control without treatment. A Dynex immunoassay system (DIAS, Guernsey, Great Britain), which had been developed for an automated E L ISA method, was used for careful purification, staining, measurement, and evaluation of the viability of the cell monolayers in a cytotoxic preparation, and in an antiviral assay. The staining to evaluate T NF cytotoxical activity was carried out by using crystal violet as disclosed in Nain et al. (1990). At first, the supernatant was aspirated, and the cell monolayers were washed three times with 300 mL of a physiological sodium chloride solution to remove dead cells. Then, the cells were fixed and washed one time with 50 mL of a 0.003% solution of crystal violet (w/v) in 20% methanol for 10 min. After six subsequent washings with 100 mL of water, the monolayers were treated with a lysis buffer (a solution of 0.8979 of sodium citrate and 1.25 mL of 1 N HCI in 98.05 mL of 47.5% ethanol) for 20 min to eluate crystal violet. T hen, opti cal densi ty of separate wel I s was eval uated spectrophotometri cal I y at 540/630 nm and analyzed by using the DIAS system. The viability of the cells was measured as a percentage ratio to the mean optical density based on the results of measuring six wells, which was taken as 100%. The cytotoxic concentration decreasing the cell viability by 50% (CC50) was calculated from a dose-effect curve of the mean values of two paral I el probes i n three studi es.

Evaluation of antiviral activity of the synthesized py ri mi dy I - di ( di azadi spi ro- alkane) derivatives of formula (I)

The antiviral activity was studied according to the method disclosed earlier (©. Schmidtke, U. Schnittler, CD J ahn, H.-M. Dahse, A. Stelzner, A rapid assay for evaluation of antiviral activity against coxsackie virus B3, influenza virus A, and herpes simplex virus type 1, J ournal of V irological Methods 95 (2001) 133-143). When the cells were removed from the growth solution, the confluent monolayers of cells formed for two days in 6-well plates (FA LCON 3046) were inoculated with 1 mL of a viral suspension of a corresponding virus in a nutrient medium on GM K cells, which comprises about 100 mL of plaque-forming units (PFU) in the absence and presence of the studied compositions twice diluted. After adsorption for 1 h at 371C, the seed material was aspirated and mixed with 2 mL of a corresponding growth solution comprising 0.4% agar and a drug at a corresponding concentration. Three untreated control probes of viruses and one non-infected untreated control cell were also studied. The concentrations of the components were determined by duplicating measurements. The probes were incubated at 371C for 72 h until platelets were detected and then fixed and washed with a 0.4% solution of crystal violet in a mixture of formalin (3% of v/v) and ethanol (1.67% v/v) in water. The platelets were counted by using a negatoscope after their removal from agar application. A dependence curve of the mean platelet count in two comparative treated wells at each concentration versus the platelet counts in three untreated virus-infected wells was plotted. The concentration at which the platelet count reduces by 50% (IC50) was calculated from a dose-effect curve for the latest values in three tests of platelet reduction.

The results of the activity of the compounds against herpes virus are given in Table 1. The study of anti-herpetic activity (HSV-1) showed that the most of studied compounds of formula (I) had a high antiviral activity.

Table 1. Cytotoxicity and activity against herpes simplex virus (HSV-1) of the studied diazadispiro compounds.

C C50 C C 10 ICso

I M I M I M I g/mL

Compound 1 >100 >100 4.44 4.61

Compound 2 >100 >100 6.17 7.96

Compound 3 >100 >100 5.19 7.26

Compound 4 >100 >100 2.86 3.11

Compound 5 >100 91.04 1.97 1.98

Compound 6 >100 >100 71.80 79.43

Compound 7 >100 >100 5.33 5.61

Compound 8 >100 100.00 7.83 7.98

Compound 9 >100 >100 7.07 7.75 Compound 10 >100 >100 4.54 4.78

Compound 11 >100 87.89 1.12 1.08

Compound 12 >100 >100 68.63 73.64

Compound 13 100.00 34.91 6.93 6.86

It was shown that pyrimidyl-di(diazadispiro-alkane) derivatives of formula (I) has a high activity against human immunodeficiency virus, and, as was expected, all compounds are equally active against both sensitive and resistant strains. The activity of the compounds against respiratory syncytial virus was also shown. The results are given in Table 2, wherein

E C 50 is a concentration of the compounds, providing 50% protection of cells against death;

CC50 is a concentration resulting in 50% death of cells;

Concentration of a compound (i M) required to reduce the growth of false- i nf ected M T -4 eel I s by 50%, as defi ned by the M T T assay.

Concentration of a compound (i M) required to achieve 50% protection of MT- 4 cells against death caused by HIV - 1, as defined by the MTT assay.

Concentration of a compound (i M) required to reduce the viability of false- infected MD BK cells by 50%, as defined by the MTT assay.

Concentration of a compound (i M) required to achieve 50% protection of

MDBK cells against death caused by BV DV virus, as defined by the MTT assay.

Concentration of a compound (i M) required to reduce the viability of false- infected V ero-76 cells by 50%, as defined by the MTT assay;

^Concentration of a compound (i M) required to prevent by 50% the formation of gaps in V ero-76 monocell layers infected with CV B-5(k), Sb.

Table 2. Cytotoxicity and antiviral activity of the compounds of formula (I) against immunodeficiency virus (including resistant strains) and respiratory syncytial virus.

Compound 4 >100 27 >100 5.0 6.0 4.0 4.0 6.6

Compound 7 >100 41 2.3 6.0 7.0 3.0 6.0 8.0

Compound 3 >100 44 4.0 6.0 6.0 2.0 4.0 6.8

Compound 5 >100 74 >100 7.0 15.0 4.0 7.0 6.8

Compound 11 >100 28 >52 5.6 6.8 2.0 5.0 7.0

Compound 10 >100 80 >100 >100

Compound 8 80 43 >73 >28

94-13 55 33 >100 6.0 6.0 1.2 3.0 4.6

Efavirenz 10 36 >20 0.002 0.03 0.08 12.0 0.01

Azidothymidi ne - 0.02 0.02 0.01 0.02 0.08

Nevi rapine - 0.08 6.3 80 100 5.0

2 ^"-C- methyl -

>100 >100 >100 >100

guanosine

2 ^"-C- methyl -

>100 >100 >100 >47

cytidine

6-Aza-uridine >100 >100 1.4 >0.3

Mycophenolic

>100 2.8 >13 ND

acid

Acycloguanosine >100 >100 >100 >100

The antiviral activity of pyrimidyl-di(diazadispiro-alkanes) of formula (I) against papillomavirus infection was studied by the method based on Pseudovirus-based neutralization Assay (PsV Assay) with H PV 16-PsVs virus that delivers a plasmid encoding Gaussia L uciferase in HeLaT cells in DME M. This method is described in (Selinka et al., J V irol, 2007, 81(20): 10970-80, Inhibition of transfer to secondary receptors by heparan sulfate- binding drug or antibody induces noninfectious uptake of human papillomavirus). The results were read in a Luciferase 48 hpi device adjusted for the determination of Relative L ight Units (RL U). Based on the method disclosed in the above-cited publication, HeLaT cells were treated with H PV 16-PsVs virus and, at the same time, they were treated with two concentrations of py ri mi dyl -di ( di azadi spi ro- alkanes) - 0.5 ι g/mL and 5 ι g/ml . The test was carried by using 96- well plates which were not treated with an experimental compound (n=4) and cells (n=3) treated with the claimed compounds of formula (I). Statistic analysis was carried out by using Two-Way A NOVA Test. The results are shown in Fig.1.

Both studied compounds significantly reduced luciferase activity that characterizes PsV infection. They inhibit completely the viral penetration at a concentration of 5 ι g/mL when compounds were added together with the virus (p<0.001). When compound 3 was used at a concentration of 0.5 ι g/mL, the inhibition of papillomavirus penetration into a cell was more significant than for compound 11, but, in any case, at a concentration of 0.5 ι g/mL, both compounds significantly reduced the infection of a cell with the virus as compared with the cells untreated with a preparation (p<0.0001).

Claims

C L AIM S

1. A compound of formula I:

A represents pyri mi di ne-4,6-diyl or pyrimidine-2,4-diyl that can comprise H,

NO2, CHO, OH, OA Ik, halogen, NH(Alk), or N( A I k)₂ as a substituent, wherein Alk is a linear or branched substituent having 1 to 4 carbon atoms;

Hal is a halogen atom,

or a pharmaceutically acceptable salt thereof.

A represents pyri mi di ne-4, 6-diyl or pyrimidine-2,4-diyl that comprises 0, Br, CH 3, or OH as a substituent;

Hal is a halogen atom, chlorine or bromine;

provided that the compound is not 3,3'-(5-nitropyrimidine-4,6-diyl)bis-3,12- diaza-6,9-diazoniadispiro[5.2.5.2]hexadecane tetrachloride or 3,3'-(2-methyl-5- nitropyrimidine-4,6-diyl)bis-3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecane tetrachloride.

3. A compound of formula (I):

wherein X is Y is A is pyri mi di ne-4, 6-diyl that can comprise H, N0₂, or

Alk as a substituent; Hal is chlorine or bromine, and Alk is methyl.

4. A compound of formula (I):

A Ik as a substituent; Hal is chlorine or bromine, and A Ik is methyl.

5. Use of a compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof

4Ha) wherein

A represents pyri mi di ne-4,6-diyl or pyrimidine-2,4-diyl that can comprise H, N0₂, CHO, OH, OAlk, CI, NHAlk, or NA I k₂ as a substituent;

Hal represents chlorine or bromine;

A Ik is a linear or branched substituent having 1 to 4 carbon atoms, the compounds having antiviral activity,

for the preventi on or treatment of vi ral di seases;

provided that the compound is not 3,3'-(5-nitropyrimidine-4,6-diyl)bis-3,12- diaza-6,9-diazoniadispiro[5.2.5.2]hexadecane tetrachloride or 3,3'-(2- methyl -5- nitropyrimidine-4,6-diyl)bis-3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecane tetrachloride.

6. U se of compounds accordi ng to clai ms 1 -4 for the preventi on and/or treatment of papillomavirus infection in a human or animal.

7. U se of compounds accordi ng to clai ms 1 -4 for the preventi on and/or treatment of immunodeficiency virus infection in a human or animal.

8. U se of compounds accordi ng to clai ms 1 -4 for the preventi on and/or treatment of cytomegalovirus infection in a human or animal.

9. U se of compounds accordi ng to clai ms 1 -4 for the preventi on and/or treatment of hepatitis C or B virus infection in a human or animal.

10. Use of compounds according to claim 2 for the prevention and/or treatment of a vi ral i nf ecti on i n a human or ani mal .

11. Use of compounds according to claim 5 for the prevention and/or treatment of papillomavirus infection in a human or animal.