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Pharmaceutical Chemistry Journal, Vol. 44, No. 12, March, 2011 (Russian Original Vol. 44, No. 12, December, 2010)

SYNTHESIS AND ANTI-INFLUENZA ACTIVITY OF SYNTHETIC RIBONUCLEASES L. S. Koroleva,1, 2 N. S. Svishcheva,1 E. A. Burakova,1 N. V. Gribkova,3 N. P. Schmeleva,3 L. M. Rustamova,3 V. M. Sabynin,3 and V. N. Sil’nikov1 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 44, No. 12, pp. 31 – 34, December, 2010. Original article submitted December 28, 2009.

The acute toxicity and antiviral activity of synthetic ribonucleases with various structures have been studied. It is established that synthetic ribonucleases exhibit low toxicity with respect to MDCK cell cultures and inhibit to different extents the reproduction of influenza A and B type viruses. Key words: synthetic ribonucleases, antiviral activity.

overuse of these drugs led to the emergence of influenza virus strains that were resistant to them. This was largely due to the fact that the known drugs affect either directly or indirectly the most variable part of the virus, the viral proteins. However, the viral genetic RNA contains significantly more conserved portions. It can be assumed that drugs that selectively interfere with viral RNA can provide a basis for designing highly effective anti-influenza drugs that do not engender resistant strains.

Most antiviral drugs are targeted at different proteins [1]. There are currently two groups of anti-influenza drugs that exhibit proven clinical effectiveness: M2-protein blockers (amantadine, rimantadine) and inhibitors of another viral protein, neuraminidase (zanamivir, oseltamivir) [2]. The 1 2 3

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Medical Sciences, Novosibirsk, Russia. Novosibirsk State University, Novosibirsk, Russia;. Research Institute for Epidemiology and Microbiology, Minsk, Republic of Belarus.

O H2N

N H

R

O

NH2

R

OH

I, II

III, IV

NH2 O

O H N

NH2

R =

R

H N

N H

R

NH2 O

O H2 N

R

H N

O

NH2

O

N+

N+ C6H13

N+

N+ C6H13

N+

N+ C12H25

V

R =

O

OH

VI

N

HN HO

N+ H N

H2N

O N H

O

N

N+ C12H25

O O

VIII

IX

VII

NH

679 0091-150X/11/4412-0679 © 2011 Springer Science+Business Media, Inc.

680

L. S. Koroleva et al.

TABLE 1. Physicochemical Properties of Synthetic Ribonucleases ComYield,* % pound

I

72

II

87

III

29

IV

48

V

51

VI

59

VII

65

PMR spectrum,** d, ppm (J/Hz)

MALDI-MS, m/z

Empirical formula, calculated MW

1.31 (m, 4H, -O-CH2(CH2)2CH2-O-), 1.52 (m, 8H, -NH-CH2CH2CH2-O-, -CHCH2CH2(CH2)2Lys), 1.70 (m, 8H, -CHCH2CH2CH2CH2Lys), 2.75 (m, 4H, -CH(CH2)3CH2Lys), 3.16 (m, 4H, -NH-CH2CH2CH2-O-), 3.36 (m, 8H, -NH-CH2CH2CH2-O-, -O-CH2(CH2)2CH2-O-), 3.71 (m, 2H, -CH(CH2)4Lys) 1.24 (m, 16H, -NH-(CH2)2-(CH2)8-(CH2)2-NH-), 1.42 (m, 8H, -NH-CH2CH2-(CH2)8-, -CHCH2CH2(CH2)2Lys), 1.54 (m, 4H, -CH(CH2)2CH2CH2Lys), 1.69 (m, 4H, -CHCH2(CH2)2CH2Lys), 2.75 (m, 4H, -CH(CH2)3CH2Lys), 3.10 (m, 4H, -NH-CH2CH2-(CH2)8-), 3.70 (m, 2H, -CH(CH2)4)Lys 1.39 (m, 4H, -O-CH2(CH2)2CH2-O-), 1.58 (m, 4H, -NH-CH2CH2CH2-O-), 3.10 (m, 4H, -NH-CH2CH2CH2-O-) 3.31 (m, 8Í, -O-CH2(CH2)2CH2-O-, -NH-CH2CH2CH2-O-), 3.66 – 3.81 (m, 4H, -CH-CH2Ser), 3.87 (m, 2H, -CH-CH2Ser) 1.18 (m, 16H, -NH-(CH2)2-(CH2)8-(CH2)2-NH-), 1.42 (m, 4H, -NH-CH2CH2-), 3.14 (m, 4H, -NH-CH2CH2-), 3.77 – 3.94 (m, 2H, -CH-CH2Ser), 3.97 (m, 4H, -CH-CH2Ser) 1.32 (q, 2H, CH-CH2-CH2Lys, J 6.6, 13.7), 1.58 (t, 2H, NH2e-CH2-CH2Lys, J 7.9), 1.80 (q, 2H, CH-CH2Lys, J 9, 16.2), 2.87 (t, 2H, NH2e-CH2Lys, J 8.3), 3.18 (m, 2H, CH2His), 3.64 (s, 3H, O-CH3His), 3.91 (t, 1H, CHLys, J 6.7), 4.76 (m, 1H, CHHis), 7.20 (s, 1H, H(5)), 8.51 (s, 1H, H(2)) 1.10 (t, 3H, CH3Thr, J 7.3), 2.85 (m, 2H, CH2His), 3.63 (t, 3H, O-CH3His, J 3.8), 3.77 (d, 1H, CH-CH3, J 6.0), 4.05 (t, 1H, CHHis, J 6.3), 4.83 (m, 1H, CHThr), 7.25 (s, 1H, H(5)), 8.52 (s, 1H, H(2)) 0.71 (t, 3H, CH3-C9, J 6.2), 1.10 (m, 14H, CH3-(CH2)7), 1.45 (m, 2H, O-CH2-CH2), 3.02 (m, 4H, CH2His, CH2Tyr), 3.83 (m, 2H, CH2Gly), 3.95 (m, 3H, O-CH2, CHTyr), 4.09 (m, 1H, CHHis), 6.64 (d, 2H, CHaromTyr, J 8.1), 6.93 (d, 2H, CHaromTyr, J 8.2), 7.15 (s, 1H, H(5)), 8.47 (s, 1H, H(2))

461.38 [M+H]+

C22H48N6O4 460.37

457.68 [M+H]+

C24H52N6O2 456.42

400.63 [M+Na]+

C16H34N4O6 378.25

375.72 [M+H]+ 397.67 [M+Na]+ 375.72 [M+H]+ 397.67 [M+Na]+

C18H38N4O4 374.29

375.72 [M+H]+ 397.67 [M+Na]+

C18H38N4O4 374.29

516.70 [M + H]+

C27H41N5O5 515.31

C18H38N4O4 374.29

*

Yields are calculated based on 4,9-dioxa-1,12-diaminododecane (for I and III); on 1,12-diaminododecane (for II and IV); on starting protected amino acid (Lys, Thr) (for V and VI); on GlyOC10H21 trifluoroacetate. ** DMSO-d6, for I – II; D2O, III – VII.

Ribonucleases from various organisms offer an alternative to commonly accepted chemotherapy and are promising drugs for treating several viral and oncological diseases [3, 4]. A series of RNAases, the antiviral or antitumor activity of which was confirmed clinically, are presently known. The most well-known of these are RNAase A and an onconase, RNAase from oocytes of the frog Rana pipiens [5]. However, the drawbacks inherent to natural enzymes, i.e., the high molecular weight, existence of effective inhibitors of natural RNAases, low stability, complexity of preparing homogeneous drugs, and high cost have prompted research aimed at developing low-molecular-weight organic TABLE 2. Toxicity of Synthetic Ribonucleases in MDCK Cell Culture Compound

MTC, g/mL

I II III IV V VI VII VIII IX

25.0 12.5 25.0 25.0 25.0 25.0 12.5 50.0 12.5

catalysts of RNA hydrolysis that mimic the functions of natural RNAases [6]. A broad array of compounds of various structures that can catalyze the hydrolysis of phosphodiester bonds in RNA were synthesized over several years in the Organic Synthesis Laboratory of the Institute of Chemical Biology and Fundamental Medicine (Siberian Branch, Russian Academy of Medical Sciences) [7 – 11]. In particular, several of the synthetic ribonucleases were highly capable of cleaving extensive fragments of influenza virus mRNA under physiological conditions [12]. The goal of the present work was to synthesize and evaluate the activity of novel synthetic ribonucleases with respect to reproduction of influenza viruses in Madin—Darby canine kidney (MDCK) cell culture. We determined the toxicity and antiviral activity of synthetic ribonucleases I – IX in MDCK cell culture with respect to models of epidemic virus strains influenza A/Ivatsvichi/115/07 (H3N2) and B/Minsk/140/07. We studied synthetic ribonucleases of different structures: 1) peptide-like molecules containing various functionally significant amino acids bound by various linkers (I – VI); 2) dipeptides (V – VI); 3) the decyl ester of a tripeptide (VII); 4) two quaternized 1,4-diazabicyclo[2.2.2]octane units bound by a linker to alkyl substituents of different length (VIII – IX).

Synthesis and Anti-Influenza Activity of Synthetic Ribonucleases

EXPERIMENTAL CHEMICAL PART We used dicyclohexylcarbodiimide, N-hydroxysuccinimide, di-tert-butylpyrocarbonate, N,N-diisopropylethylamine (Aldrich, USA), Na-Boc-glycine, L-serine, Na-Boc-OBzl-L-threonine, L-lysine, and N-Boc-L-phenylalanine (FisherBiotech, USA). The other chemical reagents and solvents (chemically pure and high purity) were obtained domestically and purified as necessary by standard methods [13]. The hydrogenation catalyst was Pd (5%) on activated carbon (Aldrich, USA).

TABLE 3. Antiviral Activity of Synthetic Ribonucleases in MDCK Cell Culture with Respect to Influenza A/Ivatsevichi/115/07 (H3N2) Virus Percent inhibition of virus reproduction by drug, % ± m Drug concentration, mg/mL

I

II

III

IV

V

VI

VII

VIII

IX

25 12.5 6.25 3.125 12.5 6.25 3.125 25 12.5 6.25 3.125 25 12.5 6.25 3.125 25 12.5 6.25 3.125 25 12.5 6.25 3.125 12.5 6.25 3.125 50 25 12.5 6.25 6.25 3.125 1.56

with simultaneous placement of virus and drug on a monolayer

with placement of drug 60 min before infection

48 ± 9.9 59 ± 1.4 22 ± 4.2 64 ± 8.4 25 ± 2.8 39 ± 5.6 41 ± 4.2 13 ± 1.4 14 ± 0.7 26 ± 1.4 12 ± 1.4 9 ± 1.4 27 ± 2.8 15 ± 1.4 11 ± 2.8 13 ± 1.4 14 ± 2.8 22 ± 4.2 12.5 ± 0.7 0 13 ± 1.2 23 ± 1.0 37 ± 1.2 0 0 0 0 0 18 ± 1.4 11 ± 1.4 28 ± 2.8 0 0

64 ± 0.7 59 ± 9.1 66 ± 2.8 76 ± 5.6 25 ± 1.4 32 ± 1.4 53 ± 4.2 24 ± 5.6 46 ± 5.6 48 ± 5.6 31 ± 4.2 7 ± 1.4 35 ± 2.8 33 ± 1.4 36 ± 5.6 23 ± 2.8 36 ± 2.8 46 ± 4.2 48 ± 5.6 5 ± 1.2 14 ± 1.0 20 ± 2.0 18 ± 1.6 33 ± 1.4 43 ± 5.6 27 ± 5.6 0 11 ± 1.4 15 ± 4.2 17 ± 4.7 30 ± 4.2 2 ± 0.7 0

681

NMR spectra were recorded in D2O and DMSO-d6 (Aldrich, USA) on Bruker AM-400 and Bruker AV-300 spectrometers (Germany). The internal standards for PMR and 13 C NMR spectra were TMS or resonances of residual solvent protons. Chemical shifts are given on the d scale. High-resolution mass spectra were taken on a Bruker Reflex III MALDI TOF instrument (Bruker Analytical Systems, Inc., Germany). Column chromatography used Kieselgel 60 silica gel (63 – 100 mm, Merck, Germany) (eluted by an EtOH gradient 0 ® 10 vol% in CH2Cl2). The reversed-phase sorbent was Preparative C18 125 Å (55 – 105 mm, Waters, USA) (eluted by an EtOH gradient 0 > 20 vol% in H2O containing 0.05% CF3COOH). Ribonucleases (VIII – IX) were synthesized as before [7]. Protected amino acids and N-hydroxysuccinimide esters of amino acids were synthesized and protecting groups were removed using standard methods [14]. The general method for synthesizing peptides (V – VI) was analogous to that published [11]. The O-decyl ester of Na-Boc-glycine was synthesized as before [9]. General method for preparing synthetic ribonucleases I – IV. A solution of the appropriate diamine (2.3 mmol) and N,N-diisopropylethylamine (5.1 mmol, 0.87 mL) in EtOAc (10 mL) was stirred and treated with a solution of the N-hydroxysuccinimide ester of Boc-protected amino acid (5.1 mmol) in EtOAc (5 mL). The mixture was stirred for 24 h at 22°C; treated with N,N-dimethylethylenediamine (1 mmol, 110 mL); stirred for 30 min; and washed successively with citric acid solution (2%, 20 mL), water (20 mL), saturated NaHCO3 solution (20 mL), and saturated NaCl solution (20 mL). The organic layer was dried over anhydrous Na2SO4. The solvent was removed in vacuo. The solid was dissolved in CF3COOH:CH2Cl2 (1:1, 5 mL) and

TABLE 4. Antiviral Activity of Synthetic Ribonucleases in MDCK Cell Culture with Respect to Influenza B/Minsk/140/07 Virus Percent inhibition of viral reproduction by drug Drug concentration, mg/mL

with placement of drug 60 min before infection

78 ± 1.7 100 100 100

96 ± 2.1 100 100

93 ± 2.8 100

90 ± 1.4

6.25 3.125

100

93 ± 7.0

25

60 ± 2.0

98 ± 2.9

12.5

54 ± 4.0

6.25

82 ± 8.7

96 ± 3.6 100

3.125

82 ± 2.5

98 ± 2.0

II

12.5

III

6.25 3.125 25 12.5

IV

with simultaneous placement of virus and drug on a monolayer

97 ± 4.9 96 ± 8.4

682

L. S. Koroleva et al.

stored for 1 h at room temperature. The solvent and CF3COOH were evaporated in vacuo. The solid was evaporated with EtOH (3 ´ 10 mL). The desired products were purified using reversed-phase column chromatography with elution by a gradient of EtOH in H2O (0 > 20% + 0.05% CF3COOH). Fractions containing the desired product were evaporated and dried in vacuo. Table 1 lists the yields and physicochemical properties of the compounds. EXPERIMENTAL BIOLOGICAL PART The toxicity was studied using a 2-day MDCK cell culture that was rinsed twice with Hanks medium. The studied compounds were added to growth medium without serum at concentrations of 50, 25, 12.5, 6.25, 3.125, and 1.56 mg/mL. The results were calculated after 36 – 72 h. The toxicity of the drugs was evaluated using a four-plus system that analyzed the cell morphology and microscopic integrity of a monolayer under an inverting microscope. The maximum tolerated concentration (MTC) was taken as one half of the drug dose that had no cytotoxic activity after incubation for 72 h [15]. Antiviral activity was studied in MDCK cell culture with respect to models of epidemic influenza A/Ivatsevichi/115/07 (H3N2) viral strains (I – IX) and B/Minsk/140/07 (II – IV) by comparing reproduction of influenza virus reproduction in MDCK cell culture with and without addition of the drug. Three independent experiments were carried out in triplicate for each compound. Antiviral activity of synthetic ribonucleases was determined by placing simultaneously the studied drug and influenza virus and by placing the drug 60 min before infection of the cell monolayer. The multiplicity of infection was 100 – 1000 TCD50/0.1 mL [15]. The influenza virus reproduction was evaluated by determining the influenza virus antigen expression level in a monolayer of MDCK cell culture using IFA 20 h after infection of the monolayer [16]. The percent inhibition of virus reproduction was calculated using the formula: OD 450 exp– OD 450 cell control ×100% OD 450 virus control–OD 450 cell control where OD450 is the optical density measured at 450 nm. It was found that synthetic ribonucleases typically had low toxicities for MDCK cell culture (Table 2) and exhibited antiviral activity with respect to influenza A and B viruses (Tables 3 and 4). The degree of manifestation of the antiviral effect varied depending on the used concentrations and administration regime. Compound I, which suppressed influenza A virus reproduction with both simultaneous virus placement and with addition to cells before infection, exhibited the greatest antiviral activity. Compounds II – V were less active with respect to influenza A/Ivatsevichi/115/07 (H3N2) influenza virus with placement of the drug 60 min be-

fore cell infection. Compounds VI – IX suppressed insignificantly virus reproduction only with placement beforehand. Ribonucleases II – IV inhibited influenza B virus reproduction more strongly up to complete suppression of virus reproduction in MDCK cell culture. Reproduction of influenza B/Minsk/140/07 virus was suppressed with both simultaneous placement of the drug and influenza virus and placement of the drug 60 min before infection of the MDCK cell culture monolayer. Thus, the results confirmed that novel antiviral drugs targeting viral RNA that are based on synthetic ribonucleases can be prepared. ACKNOWLEDGMENTS The work was performed with financial support of the Russian Foundation for Basic Research (Projects 07-04-00990-a, 08-04-90038-Bel a, 09-04-01483-a), the SB RAS Integrated Project No. 88, and the Belarusian Foundation for Basic Research (Project B08R-067). REFERENCES 1. O. I. Kiselev, I. G. Marinich, and A. A. Sominina (eds.), Influenza and Other Respiratory Viral Infections: Epidemiology, Prophylaxis, Diagnosis, and Therapy [in Russian], St. Petersburg (2003), pp. 96 – 108. 2. I. A. Leneva, R. G. Glushkov, and T. A. Gus?kova, Khim.-farm. Zh., 38(11), 8 – 14 (2004). 3. A. Benito, M. Ribo, and M. Vilanova, Mol. BioSystems, 1, 294 – 302 (2005). 4. J. Matougek, Comp. Biochem. Physiol. Part C: Toxicol. Pharmacol., 129, 175 – 191 (2001). 5. A. A. Makarov and O. N. Ilinskaya, FEBS Lett., 540, 15 – 20 (2003). 6. M. A. Zenkova (ed.), Artificial Nucleases, Nucleic Acids and Molecular Biology, Vol. 13, Springer Verlag, Berlin (2004), pp. 111 – 128. 7. E. A. Burakova and V. N. Silnikov, Nucleosides, Nucleotides, Nucleic Acids, 23, No. 6 – 7, 915 – 920 (2004). 8. N. S. Zhdan, I. L. Kuznetsova, A. V. Vlasov, et al., Bioorg. Khim., 25, 723 – 732 (1999). 9. L. S. Koroleva, A. A. Donina, N. V. Tamkovich, et al., Izv. Akad. Nauk, Ser. Khim., 11, 2596 – 2605 (2005). 10. D. A. Konevetz, I. E. Beck, N. G. Beloglazova, et al., Tetrahedron, 55, 503 – 512 (1999). 11. I. L. Kuznetsova, N. S. Zhdan, M. A. Zenkova, et al., Izv. Akad. Nauk, Ser. Khim., 2, 435 – 442 (2004). 12. N. Beloglazova, A. Vlassov, D. Konevetc, et al., Nucleosides Nucleotides, 18, 1463 – 1465 (1999). 13. A. J. Gordon and R. A. Ford, A Chemist’s Companion, Wiley-Interscience, New York (1972). 14. A. A. Gershkovich and V. K. Kibirev, Synthesis of Peptides. Reagents and Methods [in Russian], Naukova Dumka, Kiev (1987). 15. Primary study of antiviral properties of synthetic and natural compounds, Methodical Recommendations, Minsk (1986). 16. I. A. Leneva, N. I. Fadeeva, T. A. Gus’kova, et al., Khim.-farm. Zh., 38(9), 4 – 8 (1994).