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1. WO2020222666 - USE OF A CHIRAL 2-AZABICYCLOALKANE DERIVATIVE WITH DANSYL SUBSTITUENT AS VP24 PROTEASE INHIBITOR TO INHIBIT THE REPLICATION OF HSV-1 VIRUS

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Use of a chiral 2-azabicycloalkane derivative with dansyl substituent as VP24 protease inhibitor to inhibit the replication of HSV-1 virus

The subject of the invention is the use of a chiral 2-azabicycloalkane derivative with dansyl substituent for the treatment and prevention of herpes type 1 by inhibiting the replication of HSV 1 virus.

The herpes simplex virus type 1 (HSV-1) is an enveloped, neurotropic virus with genetic material in the form of double stranded DNA. It is one of the most widespread human pathogens and it is estimated that up to 90% of the population may carry it (Fatahzadeh M., Schwartz R.A., J Am Acad Dermatol. 2007, 57, 737-763). Typically, HSV-1 causes oral mucosal infections and is less often associated with genital infections. The infection is easily transmitted through direct contact such as kissing or sexual intercourse. The symptoms of infection are usually mild and are limited to fluid-filled blisters and ulcers (“cold sore”). However, HSV-1 can also cause much more serious diseases - viral keratitis can lead to blindness, while viral encephalitis can even lead to death (Farooq A.V., Shukla D., Surv Ophthalmol. 2012, 57, 448-462; Tyler K.L., Herpes 2004, 11, Suppl. 2:57A-64A; Whitley R.J., Antiviral Res. 2006, 71, 141-148).

The infection starts in epithelial cells, from where the virus travels to the nucleus of the spinal neuron using retrograde transport. Inside the neuron, the virus becomes dormant (latent); such infection is asymptomatic but persists for the rest of the patient’s life. The virus may reactivate, which may be spontaneous or occur in response to stress factors such as fever, menstruation or sunlight; the state of the patient’s immune system determines the frequency of relapse (Arvin A. M. Human herpesviruses biology, therapy, and immunoprophylaxis , New York: Cambridge University Press 2007). The ability to become latent is the most characteristic feature of the Herpesviridae family (Wilson A. C., Mohr T, Trends Microbiol. 2012, 20, 604-611).

Current therapies against herpes viral infections are based on the use of nucleoside analogues: acyclovir and its derivatives. The action of these compounds is based on interfering with the process of viral DNA synthesis, as a result of which no new infectious virions are formed, and the spread of infection is stopped. The main disadvantage of this type of drugs is their low bioavailability, although more modem derivatives of acyclovir, such as ganciclovir or valacyclovir partially solve this problem (Mubareka S., Leung V., Aoki F.Y., Vinh D.C., Expert Opin Drug Safety 2010, 9, 643-658). For the treatment of strains resistant to nucleoside analogues, foscarnet is used, which uses a different mechanism to inhibit the viral activity of DNA polymerase, however, strains of herpes vims that are also resistant to this inhibitor have already been observed Blot N., Schneider P., Young P., Janvresse C., Dehesdin D., Tron P., Vannier J.P., Bone Marrow Transplant. 2000, 26, 903-905).

Over the last 20 years viral proteases have more and more often become the target for new antiviral compounds. They have already found clinical application primarily as one of the main components of combined Highly Active Antiretroviral Therapy (HAART) (Hughes P.J., Cretton-Scott E., Teague A., Wensel T.M., P&T 2011, 36, 332-345). Another important example, even though lesser known, of the use of viral protease inhibitors in the clinic are hepatitis C NS3/NS4a protease inhibitors (McCauley J.A., Rudd M.T., Curr Opin Pharmacol. 2016, 30, 84-92). Compounds with similar mechanism of action have also been developed for human rhinovirus and coronavirus SARS (Patick A.K., Brothers M.A., Maldonado F., Binford S., Maldonado O., Fuhrman S., Petersen A., Smith G.J., Zalman L.S., Bums-Naas L.A., Tran J.Q., Antimicrob Agents Chemother. 2005, 49, 2267-2275; Yang S., Chen S. L, Hsu M.F., Wu J.D., Tseng C.T., Liu Y.F., Chen H.C., Kuo C.W., Wu C.S., Chang L.W., JMed Chem. 2006, 49, 4971-4980). Attempts have also been made to develop such inhibitors for viruses of the Herpesviridae family, but no clinical trials have ever been conducted for them (Shimba N., Nomura A.M., Marnett A.B., Craik C.S., J Virol. 2004, 78- 6657-6665; Tong L., Qian C., Massariol M.J., Deziel R., Yoakim C., Lagace L., Nat Struct Biol. 1998, 5, 819-826).

HSV-1 Protease VP24 is an ORF UL26 product. This gene is a rare case of overlapping transcription units among viruses - a smaller UL26.5 unit is entirely contained within a larger UL26 unit (Liu F. Y., Roizman B., J Virol. 1991, 65, 5149-5156; Liu F.Y., Roizman B., J Virol. 1991, 65, 206-212). The resulting polyprotein is then cut at the R site (between 247 and 248 amino acid), and at the M site (between 610 and 611 amino acid) (Dilanni C.L., Drier D.A., Deckman I.C., McCann P.J., Liu F., Roizman B., Colonno R.J., Cordingley M.G., J Biol Chem. 1993, 268, 2048- 2051). The first cut results in the serine protease VP24, while the second one generates the main capsid scaffold protein VP21.

This scaffolding is essential at the initial stage of forming the viral capsid. At further stages the scaffolding is removed for the genetic material of the virus to be packed inside (Knipe D.M., Howley P.M., Fields Virology , 6 wyd. Philadelphia, PA: Wolters Kluwer/Lippincott Williams & Wilkins Health; 2013). If the scaffolding is not removed, capsids do not develop, and new infectious virions are not produced. It has been proven that a virus devoid of the VP24 protease gene is not capable of replication (Gao M., Matusick-Kumar L., Hurlburt W., DiTusa S.F., Newcomb W.W., Brown J.C., McCann P.J., Deckman T, Colonno R.J., J Virol. 1994, 68, 3702-3712).

There are currently no reports of effective protease VP24 inhibitors for the herpes virus. Such inhibitors were developed for the protease of Kaposi's sarcoma-associated herpesvirus, but their efficacy was only found in biochemical tests, and their effect on virus replication was not confirmed (Shimba N., Nomura A.M., Marnett A.B., Craik C.S ., J Virol. 2004, 78, 6657-6665).

The invention concerns chiral 2-azabicycloalkane derivative with dansyl substituent for use as a drug especially for the treatment and prevention of herpes type 1 by inhibiting the replication of HSV-1 virus.

The object of the invention is presented in more detail in performance examples and in the following figures:

Fig. 1 presents the scheme of obtaining chiral 2-azabicycloalkane derivative with dansyl substituent, formula 1,

Fig. 2 presents the X-ray structure of chiral 2-azabicycloalkane derivative with dansyl substituent,

Fig. 3 presents the cytotoxicity of chiral 2-azabicycloalkane derivative with dansyl substituent. Cell viability was presented as % of the value of the control sample, not treated with an inhibitor,

Fig. 4 presents the antiviral activity of the chiral 2-azabicycloalkane derivative with dansyl substituent. The amount of viral DNA in the medium was determined by real time PCR,

Fig. 5 presents the antiviral activity of chiral 2-azabicycloalkane derivative with dansyl substituent. The amount of viral DNA in the medium was determined by real time PCR (A), while the number of functional virus particles was determined by plaque forming test (B),

Fig. 6 presents the mechanism of antiviral activity of chiral 2-azabicycloalkane derivative with dansyl substituent. A series of functional tests involving the addition of an inhibitor at various stages of viral infection allows to determine at what stage the inhibition occurs. The antiviral activity is presented as the number of copies of viral DNA in one millilitre of medium (A) and the number of units forming plaque in one millilitre of medium (B),

Fig. 7 presents the effect of chiral 2-azabicycloalkane derivative with dansyl substituent on the spread of HSV-1 infection. The cells were labelled for the presence of virus (green), F-actin (red) and DNA (blue).

The activity of chiral 2-azabicycloalkane derivative with dansyl substituent, formula 1, has been tested for its ability to inhibit VP24 protease expressed by HSV-1 in vitro and to inhibit HSV-1 replication in cellular tests.

Example 1 : Obtaining the compound 5-(dimethylamino)-N-((lS,4S,5R)-2-((S)-l-phenyl ethyl)-2-azabicyclo[3.2. l]octan-4-yl)naphthalene-l -sulfonamide

(EWDE39/55BF)

Axo-2-azabicycloalkane amine (( 1 L',4L', 5/^)-2-[(L')- 1 -phenyl ethyl ]-4-amine-2-azabicyclo[3.2. ljoctane, (0.54 mmol), is dissolved in dry dichloromethane (20 mL). Dansyl chloride (0.54 mmol) and powdered potassium hydroxide (0.97 mmol, 80% molar exccess) are then added. The reaction mixture is stirred at room temperature for 24 h and then extracted 3 times using water-CH?Cb system of solvents. The combined organic phases are dried over anhydrous NaiSCb. After removal of drying agent, the solvent is evaporated under reduced pressure. The resulting crude product is purified by column chromatography on silica using chloroform/methanol (95/5 v/v ) as an eluent. The product with formula 1 is obtained in the form of a yellow solid with the yield of 49%, and melting point at 61-63 °C. /a] D20 = +30.2 ( c 0,6; CH2Ch). Ή NMR (400 MHz, CDCh): S 1.10 (m, 4H); 1.22-1.27 (m, 4H); 1.41 (s, 1H); 2.09 (m, 2H); 2.23 (m, 1H); 2.86 (s, 10H); 3.64 (q, 1H, J= 3.0 Hz); 7.07-7.17 (m, 8H); 7.39-7.50 (m; 3H); 8.14-8.16 (m, 1H); 8.22-8.24 (m, 1H); 8.45-8.47 (m,

1H). 13C NMR (100 MHz, CDCh): d 20.2, 27.1, 27.9, 39.8, 41.6, 45.5, 48.8, 53.0, 62.3, 63.2, 115.2, 118.8, 127.3-130.2, 134.9, 135.9, 144.2, 145.0, 186.0. FT-IR: 635, 789, 1144, 1162, 1316, 1453, 1575, 1613, 2787, 2934, 3309, 3393 cm 1; HRMS (ESI-TOF) m/z: [M+H]+ 464.2375; calculated for [CiTHasNsOiSf: 464.2366. X-ray analysis of the product is presented in fig. 2.

Example 2: Determination of the activity of chiral 2-azabicycloalkane derivative with dansyl substituent for VP24 protease of HSV-1.

In order to determine the activity of chiral 2-azabicycloalkane derivative with dansyl substituent, the enzymatic activity of VP24 protease of HSV-1 virus was measured using HPLC method. For this purpose 50 pg/ml recombinant protease VP24 of HSV-1 virus in Tris buffer (500 mM NaCl, 50 mM Tris, 2 mM imidazole, 5% glycerol, pH 7.5) was prepared and incubated for 1 hour at room temperature in the presence of chiral 2-azabicycloalkane derivative with dansyl substituent at 100 mM in DMSO. The control sample was incubated only with DMSO. After that time HSV-1 protease VP24 substrate (with HTYLQASEKFKMW-ME sequence) at 100 pM in DMSO was added to the samples and HPLC analysis of the mixture was performed. Final DMSO concentration in all samples did not exceed 2%. Measurements were repeated at equal intervals. The percentage of inhibition was determined on the basis of signal surface area from the resulting product for the sample analysed in the presence of chiral 2-azabicycloalkane derivative with dansyl substituent relative to the signal from the resulting product in the control sample. The values have been standardised by subtracting the base line values (Table 1).

Table 1: Summary of results of protease VP 24 inhibition of HSV-1 virus by chiral 2-azabicycloalkane derivatives with dansyl substituent.

Example 3 : Impact of chiral 2-azabicycloalkane derivative with dansyl substituent on cell viability.

The cytotoxicity of the compound was tested using the XTT test. The analysis was performed on Vero E6 cell line ( Cercopithecus aethiops kidney epithelial cells, ATCC CRL-1586), which is permissive to HSV-1. This test is based on a study of the activity of mitochondrial enzymes by determining their ability to reduce the substrate to a coloured product. Cell viability was determined by measuring the absorbance at 450 nm against the control sample, that is cells grown in a medium with no additives. The cells were grown for 2 days in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 3% inactivated Fetal Bovine Serum (FBS) and penicillin and streptomycin, with the addition of the tested compound.

Within the tested concentration range, the compound under analysis shows no considerable toxicity to cells.

Example 4: Effect of chiral 2-azabicycloalkane derivative with dansyl substituent on the replication of herpes type 1 (HSV-1).

After determining the concentration of the compound, which had a toxic effect on cells, its impact on HSV-1 replication was studied. Cells were preincubated with 10 pg/ml of inhibitor for 30 min and then, still in the presence of the inhibitor, infected with HSV-1, strain 17 with TCID50 (tissue culture infective dose of 50%) of 400 per millilitre. After 2 hours unbound virus particles were removed by means of washing 3 times with PBS solution and incubated, also in the presence of the inhibitor, for another 2 days. After this time, viral DNA was isolated from the cellular medium and used as a matrix in a quantitative real-time PCR reaction (primer sequences: forward 5 '-CAT CAC CGA CCC GGA GAG GGA C-3', reverse 5'-GGG CCA GGC GCT TGT TGG TGT A-3', sonda 5'-FAM CCG CCG AAC TGA GCA GAC ACC CGC GC BHQ1-3'). The ability of the compound to inhibit HSV-1 replication was determined as a logarithmic decrease in the number of viral DNA copies per millilitre of medium.

The tested compound of chiral 2-azabicycloalkane derivative with dansyl substituent as protease VP24 inhibitor shows antiviral activity and that it effectively inhibits HSV-1 virus multiplication about 10,000 times (4 log).

Example 5: Determination of the effective concentration of chiral 2-azabicycloalkane derivative with dansyl substituent.

To determine the effective dose of the chiral 2-azabicycloalkane derivative with dansyl substituent, the test as above was performed using different inhibitor concentrations in the range 0.1 - 100 mM. The results were analysed using real-time PCR and the plaque formation test.

The IC50 value determined by real-time PCR is 3.947 ± 1.443 pg/ml, while by the plaque formation test it equals 4.346 ± 0.5612 pg/ml. The value of selectivity index determined by real-time PCR is >25.33, whereas that determined by the plaque formation test is >23.

Example 6: Mechanism of antiviral activity of chiral 2-azabicycloalkane derivative with dansyl substituent.

The mechanism of antiviral activity of the inhibitor has been tested by means of a series of functional tests based on the use of the inhibitor at different stages of viral infection:

• Test I (inactivation test) - does the inhibitor EWDE39/ 55BF interact directly with the virus, making it incapable of cellular infection?

• Test II (interaction test) - does the inhibitor EWDE39/ 55BF interact directly with the cell, making it resistant to virus infection?

• Test III (adsorption test) - does the inhibitor EWDE39/55BF interfere with the interaction of the virus with its adhesion factor on the cell surface, preventing the virus from attaching to the cell?

• Test IV (folding, packaging and release test) - does the inhibitor EWDE39/55BF interfere with late stages of viral infection (e.g. folding, packaging, release) preventing the formation of new infectious virions.

It was established that inhibition of virus replication occurs in the late stages of infection. A second inhibition mechanism is also possible at the stage of virus adsorption.

Example 7: Impact of chiral 2-azabicycloalkane derivative with dansyl substituent on the spread of HSV-1 infection.

Cells were incubated for one hour in the presence of 10,000 times diluted viral stock or cellular control lysate (mock). Unbound virus particles were then removed by washing with PBS solution and the medium with 10 mM inhibitor or pure medium as a control was applied to the cells and incubated for another 16 hours. After that time the cells were fixed and subjected to immunohistochemical staining for the presence of virus (anti-VP5 HSV antibodies), F-actin (phalloidin) and DNA (DAPI). The preparations were imaged using confocal microscope ZEISS LSM 710. Fig.7 shows single cross sections. A significant reduction in the spread of HSV-1 infection by the inhibitor in the form of chiral 2-azabicycloalkane derivative with dansyl substituent is visible in relation to the control, not treated with the inhibitor.

Example 8: Impact of chiral 2-azabicycloalkane derivative with dansyl substituent on the retention of HSV-1 capsids inside cell nuclei.

Cells were infected with HSV-1 at TCID5o= 10000 for 16 h in the presence of the inhibitor. Then, slides were fixed and stained for major capsid protein VP5, f-actin, and nuclear DNA. The preparations were imaged using EVOS cell imaging system. Subsequently, quantification of the fluorescence accumulated in the nucleus compared to the total signal recorded for a particular cell was performed. As the inhibitor is expected to hamper the maturation of viral capsids, and only mature capsids are able to exit the cell nucleus, a retention of viral capsids inside the nuclei should be observed in the presence of the inhibitor. As indicated in Figure 8, the signal in the nucleus was much higher in the cells treated with protease inhibitors, compared to the control cells.

Example 9: In vivo antiviral activity of chiral 2-azabicycloalkane derivative with dansyl substituent.

Chiral 2-azabicycloalkane derivative with dansyl substituent was applied onto the scarified skin at 4 mM concentration. For toxicity studies, the inhibitor was administered to the scarified skin twice daily for 4 days. None of the tested inhibitors exhibited any systemic or local toxicity during the observation time. For antiviral activity analysis, the inhibitor was administered in two settings: before and after HSV-1 infection, or only after the infection, in which case a placebo formulation was applied prior to the infection. The treatment was repeated twice daily for 4 following days, and the animals were observed for 14 days. A commercially available ointment containing 5% (w/v) ACV was used as a positive control. On day 9, all infected animals receiving no treatment died. On the last day of observation,

46,7% of mice treated with EWDI/39/55BF before and after the infection were still alive and did not develop any disease symptoms, compared to 66.7% survival achieved in animals treated with ACV. The results are demonstrated in Fig. 9.

Example 10: Determination of the activity of chiral 2-azabicycloalkane derivative with dansyl substituent towards VP24 protease of HSV-1 - kinetic studies.

In order to determine the activity of chiral 2-azabicycloalkane derivative with dansyl substituent, kinetic studies of VP24 protease of HSV-1 were performed. The assay was carried out in the 0.1 M HEPES, 0.5 M NaCl, 0.03% Triton X-100 buffer (pH 7.5) supplemented with 5% DMSO at 37°C. The enzyme activity was measured using internally quenched fluorescent substrate (ABZ-Ile-Arg-Leu-Ala-Tyr(3-N02)-NH2) in the range of concentrations from 41.8 mM to 5.22 pM. The VP24 protease of HSV-1 at the concentration of 10 pg/ml was preincubated at RT for 15 min with the chiral 2-azabicycloalkane derivative with dansyl substituent used at concentrations ranging from 25 pM to 5 pM or without inhibitor. Subsequently the substrate was added and the fluorescence intensity (kex 310 nm/kem 410 nm) was measured for 2h. Each point was analysed in triplicates. Based on obtained data the noncompetitive enzyme inhibition was assumed with the inhibition constant value ofKi = 293.9 ± 138.4 pM.

Halina J. Winohradnik European Patent Attorney