WO2022124944A1 - Di(diazoniadispiro[5.2.5.2]hexadecan)-5-nitropyrimidines for the treatment of coronaviral infections - Google Patents
Di(diazoniadispiro[5.2.5.2]hexadecan)-5-nitropyrimidines for the treatment of coronaviral infections Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/499—Spiro-condensed pyrazines or piperazines
Definitions
- This invention is related to the use of compounds of of di(diazoniadispiro [5.2.5.2]hexadecan)-5-nitropyrimidine which are active against coronaviruses, to prevent and treat coronaviral infections in humans and animals, including the SARS-Cov-2 infection.
- coronavirus A human coronavirus was first identified in 1965. Later, coronaviruses became a center of attention due to a breakout of atypical pneumonia, or SARS, in 2002 to 2003 in China. The disease was caused by the SARS-CoV virus. The breakout spread to other countries; in total, 8273 people became infected, and 775 died (9.6% lethality).
- MERS-CoV virus caused MERS, first cases of which were documented in 2012. In 2015, there was a breakout of MERS in South Korea, when 183 people became infected, and 33 people died.
- chloroquine has high antiviral activity not only against type A influenza viruses (which internalize in endosomes), but also against coronaviruses (De Wilde A.H., Jochmans D., Posthuma C.C. etal. Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture, Antimicrob. Agents Chemother., 2014, 58: 4875-4884;Kearney J.
- Chloroquine as a potential treatment and prevention measure for the 2019 novel coronavirus a review, Preprints. 2020. Art. 2020030275), the internalization of which is almost exclusively carried out by means of membrane fusion, i.e., without formation of endosomes (Matsuyama S., Ujike M., Morikawa S. etal. Protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection. PNAS USA, 2005, 102: 12543-12547).
- the carbohydrate aperiodic polymers due to their structural properties have the highest informational capacity. This ensures high specificity of ligand-receptor interactions of oligosaccharide conjugates. It is suggested that glycan structure is coded in eukaryotic genome indirectly. Oligosaccharides synthesis takes place in Golgi apparatus cisterns with the participation of secondary protein matrices which form functional heterogenic glycosyltransferase associations.
- herpes viruses types 1 and 2 herpes viruses (Schmidtke M., Riabova O., Dahse H.-M., Stelzner A., Makarov V. Synthesis, Cytotoxicity and Antiviral Activity of N,N'-bis-5-nitropyrimidyl Derivatives of Di spirotripiperazine. Anti viral Res., 2002, 55: 117-127), human papilloma virus (Selinka H., Florin L., Patel H.D., Freitag K., Schmidtke M., Makarov V.A., Sapp M.
- etal.Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells. J. Virol., 2014, 88, 13221-1323; Milewska A., Nowak P., Owczarek K. et al. Entry of Human Coronavirus NL63 into the Cell. J. Virol., 2018, 92: e01933-17; Szczepanski A., Owczarek K., Bzowska M. et al.
- the purpose of this invention is to rationalize a possibility of use of Formula 1 di (dispirotripiparazine)-pyrimidines to treat human and animal infections caused by coronaviruses, including SARS-Cov-2.
- R is hydrogen or methyl.
- Formula 1 compounds may be represented by bases as well as pharmaceutically acceptable salts, in particular hydrochlorides. These salts may be obtained in situ in the process of synthesis, excretion, or purification of Formula 1 compounds, or they may be prepared intentionally. The compounds may be obtained and used in crystal form. Formula 1 compounds in hard state may be represented by crystalline hydrates as well as by substances without crystal water content.
- a unique mechanism of affecting the viral invasion into a host cell is based on the blocking of viral adsorption to a target cell due to specific inhibition of heparan sulfate proteoglycans.
- This process may be described as blocking of viral adhesion to a host cell; a drug based on the di(dispirotripiperazine)-pyrimidine formula 1 interacts with proteoglycans of host cells, which ensures wide spectrum and generalizability of its antiviral action (Cagno V., Tseligka E.D., Jones S.T., Tapparel C., Heparan Sulfate Proteoglycans and Viral Attachment: True Receptors or Adaptation Bias? Viruses, 2019, 77(7), 596).
- di(dispirotripiperazine)-pyrimidine compounds The mechanism of action of di(dispirotripiperazine)-pyrimidine compounds is probably (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) related to their ability to specifically bind heparan sulfate proteoglycans which leads to dramatic reduction in viral replication. Is has been demonstrated that attachment of investigational substances is being antagonized by heparin.
- the target of diazoniadispiro[5.2.5.2.]hexadecanes is represented by 2 sulfate groups located in adjacent saccharide residues; for example, for GlcA2S-GlcNS6S, GlcA2S-GlcNS3S, IdoA2S-GlcNAc6S, IdoA2S- GlcNH23SS6S, IdoA2S-GlcNS6S, and IdoA2SGlcNS3S, good electrostatic interaction was demonstrated between the negatively charged sulfate group and positively charged atoms of nitrogen in di(dispirotripiperazine)-pyrimidines.
- di(dispirotripiperazine)-pyrimidine formula 1 compounds block key functional groups of heparan sulfate proteoglycans, preventing viral replication and ensuring high antiviral activity.
- drugs based on this mechanism of action anywhere in the world.
- di(dispirotripiperazine)-pyrimidine formula 1 compounds are easily soluble in water and thus might be used in diverse pharmaceutical forms for local (aerosols) or systemic use.
- the objective of this invention was to discover a new medication for the treatment of coronaviral infections, including SARS-Cov-2.
- the work has resulted in a discovery of high antiviral activity of di(dispirotripiperazine) -pyrimidine formula 1 compounds.
- the di(dispirotripiperazine)-pyrimidine formula 1 compounds might be obtained according to the synthesis scheme which follows.
- Hydrochloride benzoylpiperazine 18 g (0.0795 M) is added to a solution of 5.34 g (0.0954 M) KOH in 55 ml of ethanol and mixed for 0.5 hours at 20-22°C. After that, 13.3 ml (0.199 M) of ethilenchlorhydrine are added, and a solution of 11.6 g (0.207 M) KOH in 98 ml of ethanol (rectificated) is being dripped for 1 hour; the temperature of the mass cannot exceed 20 °C. Twenty hours later, the resulting KC1 is being filtered and washed by 25 ml absolute ethanol.
- the filtrate is cooled down to 10°C, after which 58 ml of 12% HCl/EtOH (control pH 2-3) are slowly added, mixed for 1 hour at 5°C, and left for a night in the fridge.
- the precipitate is being filtered, washed with 2x10 ml of absolute ethanol, and dried on air (48 hours) or in a drying oven (4-5 hours at 50-55°C). This process yields 15.6 g (72%) of hydrochloride l-benzoyl-4-( ⁇ -oxyethyl)piperazine with melting point 215°C.
- Hoechst solution 100 ⁇ l was used.
- the plates were read by means of an automatic confocal microscope (Opera Phoenix) which measures the number of infected cells (GFP signal) and their viability (Hoechst signal).
- vervet monkey renal Vero cells A passaged culture of vervet monkey renal Vero cells (American collection of cell cultures and viruses) was used for the experiment.
- the cells were cultivated in a growing medium consisting of DMEM (minimum Eagle medium manufactured by PanEco, Russia) with the addition of 10% embryonic bovine serum (EBS, PanEco, Russia) inactivated by heat, 2pM glutamine (Sigma, USA), and antibiotics (100 U/ml penicillin and 100 pg/ml streptomycin).
- the supporting medium contained all listed ingredients as well as 2% EBS.
- the cells were incubated for 20 hours in a thermostat, at +37°C and with 5 % CO 2 in the atmosphere.
- a transport medium which contained SARS-CoV- 2 extracted from a patient.
- the presence of viral RNA was confirmed by SARS- CoV-2 RNA screening test (Generium, Vladimir region, Russia).
- SARS- CoV-2 RNA screening test Generium, Vladimir region, Russia.
- TCD50 tissue cytopathic dose
- the viral TCD50 was defined as a minimal dilution of virus-containing clinical isolate which led to 50% damage of cellular monolayer, without any degeneration of control cellular monolayer where there was no infection.
- the efficacy of PDSTP 0.1 mg/ml solution was visually assessed on the base of maintenance of a formed cellular monolayer using inverted LOMO microscope.
- Formula 1 compounds (1) and (4) protect epithelial Vero cells from cytopathic action of SARS-CoV-2.
- Example 7 Prevention of SARS-CoV-2 adhesion to pulmonary cells by means of Formula 1 compounds
- lungs were isolated from 2 male guinea pigs. After opening the thorax, granulated ice was poured into the thoracic cavity, and the lungs were removed and placed onto a cooling table at +4°C. Tissue disintegration was conducted according to a standard method. The following media were prepared for gradient centrifuging and recovery of subcellular structures, taking into account adapted guidelines (Gureev A.P., Kokina A.V., Siromiatnikov M.Yu., Popov V.O. Optimization of methods for isolating mitochondria from different tissues of the mouse. News from Voronezh State University, Ser.
- Recovery medium containing 220 mM mannitol, 100 mM saccharose, 1 mM EDTA, 20 mM HEPES, BSA (fatty acid-free) 2 mg/ml. All ingredients were dissolved in deionized water with final pH 7.4.
- washing medium containing 220 mM mannitol, 100 mM saccharose, 1 mM EDTA, 20 mM HEPES. All ingredients were dissolved in deionized water with final pH 7.4.
- Lung tissue was washed in the recovery medium and homogenized in a glass homogenizer at +4 °C.
- the homogenate was transferred into centrifuging tubes, and medium was added up to needed volume.
- the homogenate was centrifuged for 5 minutes at 600g.
- the resulting supernatant was transferred to clean tubes, and washing medium was added up to needed volume.
- the precipitation of free mitochondrial and liposomal fractions was performed for 10 minutes at 14000g. Resulting supernatant was removed, and the precipitate was resuspended in the washing medium. After that, the precipitate from several tubes was transferred to one tube and carefully placed on 23% Percoll solution. Centrifuging in Percoll gradient was performed for 15 minutes at 23000g.
- membrane vesicles were obtained, 6-15 ⁇ m in diameter (12 ⁇ m for up to 72% vesicles), containing receptor structures for binding the virus. Addition of the latter led to agglomeration of structures and change in the solution opacity measured by nephelometer. At the same time, addition of water-soluble PDSTP compound into the medium prevented an increase in the solution opacity. The number of binding sites in testing system was much higher than the number of introduced viral particles.
- Virus-containing transport media were obtained during the examination of patients with SARS-CoV-2 infection; the presence of viral RNA was confirmed by approved RT-PCR tests. Each transport medium was diluted 1 to 1000 times to evaluate its interaction with biologic material; 100 ⁇ l samples were introduced in testing system using a dosing device. In each biologic material, dilutions of transport media from 5 patients were tested. Due to impossibility of preparation of standard solutions and viral cultures, for each sample, relative (percentage) index was calculated; after that, an average was calculated from these indices. The results are presented in the Table 3.
- the mixture was centrifuged for 5 minutes at 14000g which permitted to precipitate vesicles together with adhered viruses.
- the presence of free (for example, due to the drug action) specific RNA was assessed by RT- PCR.
- Example 8 Formula 1 compounds efficacy in vivo
- a SARS-CoV-2 infection model in golden hamsters was used.
- the virus was titrated in a Vero-B cell culture by the number of plaque-forming units and was introduced intranasally in the dose of 4 ⁇ 10 4 TCD50, 26 ⁇ l per animal.
- group I intact animals (negative control)
- group II SARS-CoV-2-infected animals (positive control).
- Group III included animals who received 15 mg/kg PDSTP once daily intraperitoneally for 5 days before the infection (prevention group).
- Group IV included animals who received 15 mg/kg PDSTP once daily intraperitoneally for 5 days after the virus introduction, in days 3 to 7 of the infection, on the background of its manifestations (treatment group).
- the monitoring of infected animals included the assessment of symptoms (sneezing, rhinorrhea), the frequency of which was not statistically different between infected animals. Body weight loss was seen in infected animals. The animals were withdrawn from the experiment on the day 7. On autopsy, lungs and spleen were removed, weighed, and relative weight (percentage of body weight) of these organs was calculated. The right lung was placed into a Petri dish with saline and subjected to diaphanoscopy to count the number of tissue indurations and erythema areas with hemorrhages in the lung parenchyma.
- Table 5 presents biometric characteristics (Me [Q25 ⁇ Q75]) of female golden hamsters on Day 7 of SARS-CoV-2 infection (intranasal infection with SARS-CoV- 2 (26 ⁇ l per animal) 4 ⁇ 10 4 TCD50).
- SARS-CoV-2 infection intranasal infection with SARS-CoV- 2 (26 ⁇ l per animal) 4 ⁇ 10 4 TCD50.
- Table 6 presents quantitative characteristics of diaphanoscopy. It was observed that PDSTP as prevention or treatment decreased the amount of indured, erythematous, and bleeding areas in the lung tissue. In the group of animals treated with the drug, this effect was evident on visual examination and confirmed by counting. In this group, pulmonary tissue was represented by isolated areas of induration, with substantially defined borders, of different sizes but mostly not very large and without tendency to coalesce.
- the areas of changed color were granular on dissection and were not elevated above the adjacent tissue. Most surface area looked like normal tissue, light rose and light red in color. The lungs were slightly indurated, elastic, insignificantly hydropic. The dissected surface was of homogenous color and blood content. On dissection, there was almost no fluid when pressure was applied.
- Figure 1 shows typical observations in lungs at animals' autopsy; quantitative results of diaphanoscopy are reflected in the Table 6. It was observed that PDSTP as prevention or treatment decreased the amount of indured, erythematous, and bleeding areas in the lung tissue. In the group of animals treated with the drug, this effect was evident on visual examination and confirmed by counting.
- pulmonary tissue was represented by isolated areas of induration, with substantially defined borders, of different sizes but mostly not very large and without tendency to coalesce. The areas of changed color were granular on dissection and were not elevated above the adjacent tissue. Most surface area looked like normal tissue, light rose and light red in color.
- the lungs were slightly indurated, elastic, insignificantly hydropic. The dissected surface was of homogenous color and blood content. On dissection, there was almost no fluid when pressure was applied.
- the absorbed dose was calculated as: where D is absorbed dose (mg/kg); RMV is the rat respiratory minute volume MO ⁇ , (L/kg*min), C is mean concentration of the compound in the camera during the inhalation period (mg/L), AT is inhalation time (min).
- D absorbed dose
- RMV the rat respiratory minute volume MO ⁇ , (L/kg*min)
- C mean concentration of the compound in the camera during the inhalation period (mg/L)
- AT inhalation time (min).
- the absorbed dose of the drug was 1.15 mg/kg.
- the LD 50 of the Formula 1 compound (1) after a single intravenous administration in rats was 139.5 (116.7 - 166.6) mg/kg for males and 155.8 (132.8 -182.7) mg/kg for females. Calculated therapeutic dose cannot exceed 1/20 LD 50 .
- the resulting absorbed dose is about 1/100 LD 50 , which ensures its safety; thus, this dose may be multiplied and used for
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Abstract
This invention is related to the usage of di(diazoniadispiro [5.2.5.2]hexadecan)-5-nitropyrimidine derivatives which are active against coronaviruses, for prevention and treatment of coronaviral infections in humans and animals, including the SARS-Cov-2 infection. Usage of the compounds from the general Formula 1: where R is CH3, or its hydrochloric salt, dihydrochloric salt, or dihydrochloride hexahydrate, for the treatment or prevention of coronaviral infection caused by SARS-CoV-2, in humans or animals.
Description
DI(DIAZONIADISPIRO[5.2.5.2]HEXADECAN)-5-NITROPYRIMIDINES FOR THE TREATMENT OF CORONAVIRAL INFECTIONS
This invention is related to the use of compounds of of di(diazoniadispiro [5.2.5.2]hexadecan)-5-nitropyrimidine which are active against coronaviruses, to prevent and treat coronaviral infections in humans and animals, including the SARS-Cov-2 infection.
Description
Although the area of virology is constantly advancing, the issues of viral respiratory infections, mainly associated with diverse coronaviruses, have recently become especially important due to the severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) epidemics and, by and large, SARS- CoV-2 pandemic. Among all viral infective agents in human, coronaviruses hold the third place, after influenza viruses and rhino viruses, and cause in average 12% cases. Coronaviral infections are especially dangerous due to the fact that they often lead to severe viral pneumonias, which are associated with significant pulmonary damage and in many cases fatal.
A human coronavirus was first identified in 1965. Later, coronaviruses became a center of attention due to a breakout of atypical pneumonia, or SARS, in 2002 to 2003 in China. The disease was caused by the SARS-CoV virus. The breakout spread to other countries; in total, 8273 people became infected, and 775 died (9.6% lethality).
The MERS-CoV virus caused MERS, first cases of which were documented in 2012. In 2015, there was a breakout of MERS in South Korea, when 183 people became infected, and 33 people died.
In December 2019, a breakout of pneumonia caused by the SARS-CoV-2 virus started in China; in 2020, it grew into a pandemic affecting all countries of the world. It is obvious that pandemics of respiratory viral infections associated with
coronaviruses went after humans in the past, and we have no reasons to expect this not happening in the future.
Biology of coronaviruses unavoidably results in appearance of new pandemic strains, and it is impossible to predict moments of their appearance, genomic variability and antigenic properties. Thus, epidemics and pandemics of new respiratory coronaviral infections will always start in the absence of specific immune prophylactics and treatment. This defines the importance of preliminary discovery and development of pathogenetic medications and methods of respiratory viral infections treatment and prevention, with biological properties of coronaviruses taken into account. It is also a known fact that the immunity after a coronaviral infection does not usually last long and often do not protect from reinfection; this underlines the need in development of drugs with wide antiviral spectrum which would protect the host from viral infection.
Currently, the basis of pathogenetic therapy for coronaviral infections is chloroquine and some of its derivatives. The original chloroquine is known since 1947 and is indicated for malaria treatment and prevention (Mengesha T., Makonnen E. Comparative efficacy and safety of chloroquine and alternative antimalarial drugs: a meta-analysis from six African countries, East Afr. Med. J. 1999, 76: 314-319; Bello S.O., Chika A., Bello A.Y. Is chloroquine better than artemisin in combination therapy as first line treatment in adult Nigerians with un complicated malaria? A cost effectiveness analysis, Afr. J. Infect. Dis. 2010: 29-42; Waqar T., Khushdil A., Haque K. Efficacy of chloroquine as a first line agent in the treatment of uncomplicated malaria due to Plasmodium Vivax in children and treatment practices in Pakistan: a pilot study. J. Pak. Med. Assoc., 2016, 66: 30-33), lepra treatment (Bezerra E.L., Vilar M.J., da Trindado Neto P.B., Sato E.I. Double- blind, randomized, controlled clinical trial of clofazimine compared with chloroquine in patients with systemic lupus erythematosus. Arthritis Rheum., 2005, 52(1): 3073-3078; Gordon C., Amissah-Arthur M.B., Gayed M. et al. The British
Society for Rheumatology guideline for the management of systemic lupus erythematosus in adults. Rheumatology, 2018,57(1): el-e45), as an anti- inflammatory in patients with rheumatoid arthritis (Schrezenmeier E., Dorner T. Mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology. Nat. Rev. Rheumatol., 2020. 16(3): 155-166), for the treatment of antiphospholipid syndrome (Tektonidou M.G., Andreoli L., Limper M. etal. EULAR recommendations for the management of antiphospholipid syndrome in adults. Ann. Rheun. Dis., 2019, 78: 1296-1304), as well as in patients with Sjogren syndrome (Lee H.J., Shin S., Yoon S.G. et al. The effect of chloroquine on the development of dry eye in Sjogren syndrome animal model. Invest. Ophthalmol. Vis. Sci., 2019, 60: 3708-3716), and some other diseases.
The ability of chloroquine to inhibit acidification of endosomes containing respiratory viruses and thus to block their RNA release and following replication, can hardly be a satisfactory explanation of its antiviral activity. Is has been shown that chloroquine has high antiviral activity not only against type A influenza viruses (which internalize in endosomes), but also against coronaviruses (De Wilde A.H., Jochmans D., Posthuma C.C. etal. Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture, Antimicrob. Agents Chemother., 2014, 58: 4875-4884;Kearney J. Chloroquine as a potential treatment and prevention measure for the 2019 novel coronavirus: a review, Preprints. 2020. Art. 2020030275), the internalization of which is almost exclusively carried out by means of membrane fusion, i.e., without formation of endosomes (Matsuyama S., Ujike M., Morikawa S. etal. Protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection. PNAS USA, 2005, 102: 12543-12547).
Among the 3 biological aperiodic polymers (nucleic acids, polypeptides, carbohydrates), the carbohydrate aperiodic polymers (glycans, oligosaccharides) due to their structural properties have the highest informational capacity. This ensures
high specificity of ligand-receptor interactions of oligosaccharide conjugates. It is suggested that glycan structure is coded in eukaryotic genome indirectly. Oligosaccharides synthesis takes place in Golgi apparatus cisterns with the participation of secondary protein matrices which form functional heterogenic glycosyltransferase associations. Naturally, special structure of such matrix protein molecules and thus their affinity to glycan synthesis enzymes, may quickly and significantly change under the influence of pH dynamics and redox potential in the Golgi apparatus environment. It should be taken into account that all participants of the human cells' interaction with coronaviruses (glycoproteins, glycolipids) are copiously decorated by glycans with terminal sialic acids which are recognized by viral particles as specific receptors.
The issue of glycans' participation in viral adhesion and replication is critical, as this data may be applied in design and development of a wide spectrum drug against viruses, known as well as currently unknown. Such drugs may become a reserve for pathogenetic treatment of new coronaviral infections appearing in the future. It should be noted that several viruses causing pathology in humans (Schmidtke M., Karger A., Meerbach A., Egerer R., Stelzner A., Makarov V. Binding of a N,N'-bisheteryl derivative of dispirotripiperazine to heparan sulfate residues on the cell surface specifically prevents infection of viruses from different families. Virology, 2003, 311: 134-143), including types 1 and 2 herpes viruses (Schmidtke M., Riabova O., Dahse H.-M., Stelzner A., Makarov V. Synthesis, Cytotoxicity and Antiviral Activity of N,N'-bis-5-nitropyrimidyl Derivatives of Di spirotripiperazine. Anti viral Res., 2002, 55: 117-127), human papilloma virus (Selinka H., Florin L., Patel H.D., Freitag K., Schmidtke M., Makarov V.A., Sapp M. Inhibition of transfer to secondary receptors by heparan sulfate -binding drug or antibody induces noninfectious uptake of human papillomavirus. J. of Virology, 2007, 81: 10970-10980), cytomegalovirus (Paeschke R., Woskobojnik I., Makarov V., Schmidtke M., Bogner E. DSTP-27 prevents entry of human cytomegalovirus.
Antimicrob Agents Chemother.2014, 58(4): 1963-1971), some HIV subtypes, respiratory syncytial virus, as well as coronaviruses (MilewskaA., ZarebskiM., NowakP. etal.Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells. J. Virol., 2014, 88, 13221-1323; Milewska A., Nowak P., Owczarek K. et al. Entry of Human Coronavirus NL63 into the Cell. J. Virol., 2018, 92: e01933-17; Szczepanski A., Owczarek K., Bzowska M. et al. Canine Respiratory Coronavirus, Bovine Coronavirus, and Human Coronavirus OC43: Receptors and Attachment Factors, Viruses, 2019, 11: 328) use a common heparan sulfate- dependent mechanism of adhesion to a wall of a host cell. For this reason, blockers of viral/host cell interaction may play an important role in the treatment of infections caused by these viruses.
The purpose of this invention is to rationalize a possibility of use of Formula 1 di (dispirotripiparazine)-pyrimidines to treat human and animal infections caused by coronaviruses, including SARS-Cov-2.
where R is hydrogen or methyl.
Formula 1 compounds may be represented by bases as well as pharmaceutically acceptable salts, in particular hydrochlorides. These salts may be obtained in situ in the process of synthesis, excretion, or purification of Formula 1 compounds, or they may be prepared intentionally.
The compounds may be obtained and used in crystal form. Formula 1 compounds in hard state may be represented by crystalline hydrates as well as by substances without crystal water content.
Activity of general Formula 1 compounds and their pharmaceutically acceptable acidic or basic salts against coronaviruses is studied; it permits using them for the manufacturing of medications based on these compounds, for treatment or prevention of diseases caused by the mentioned viruses.
One of the promising directions in the search for new antiviral compounds is the narrow specificity of action against some components of the viral lifecycle. A unique mechanism of affecting the viral invasion into a host cell is based on the blocking of viral adsorption to a target cell due to specific inhibition of heparan sulfate proteoglycans.
Compounds of di(dispirotripiperazine)-pyrimidine formula 1 discovered during target-driven search of new drugs specifically block heparan sulfate proteoglycans on the walls of host cells and, thus, prevent specific adsorption of viruses, including coronaviruses, to host cells. This process may be described as blocking of viral adhesion to a host cell; a drug based on the di(dispirotripiperazine)-pyrimidine formula 1 interacts with proteoglycans of host cells, which ensures wide spectrum and generalizability of its antiviral action (Cagno V., Tseligka E.D., Jones S.T., Tapparel C., Heparan Sulfate Proteoglycans and Viral Attachment: True Receptors or Adaptation Bias? Viruses, 2019, 77(7), 596).
The mechanism of action of di(dispirotripiperazine)-pyrimidine compounds is probably (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) related to their ability to specifically bind heparan sulfate proteoglycans which leads to dramatic reduction in viral replication. Is has been demonstrated that attachment of investigational substances is being antagonized by heparin. The target of diazoniadispiro[5.2.5.2.]hexadecanes is
represented by 2 sulfate groups located in adjacent saccharide residues; for example, for GlcA2S-GlcNS6S, GlcA2S-GlcNS3S, IdoA2S-GlcNAc6S, IdoA2S- GlcNH23SS6S, IdoA2S-GlcNS6S, and IdoA2SGlcNS3S, good electrostatic interaction was demonstrated between the negatively charged sulfate group and positively charged atoms of nitrogen in di(dispirotripiperazine)-pyrimidines. It has been also demonstrated that similar interaction is possible with a carbonyl group of AUA-GlcNSIdoUA2S-GlcNAc-UA2S-GlcNS-IdoUA2S-GlcNH23S octasaccharide, which represents an area of heparan sulfate essential for coronaviruses to penetrate a host cell. Thus, di(dispirotripiperazine)-pyrimidine formula 1 compounds block key functional groups of heparan sulfate proteoglycans, preventing viral replication and ensuring high antiviral activity. Currently, there are no drugs based on this mechanism of action anywhere in the world.
There is also data that dispirotripiperazine compounds have moderate immunosuppressive effect which can be useful for blocking cytokine storm caused by coronaviral infection (Markland, W. / McQuaid, T. / Kwong, A. D. Antiviral Efficacy of VX-497, a Novel IMPDH Inhibitor, or Ribavirin in Combination with Interferon in Encephalomyocarditis Virus (EMCV) Infected L929 Cells, Antiviral research, 1999, 41(2), 65; Benenson, E. V.; Timina, O. B. Prospidine versus methotrexate pulse in highly active rheumatoid arthritis: A controlled 6-month clinical trial, Clinical Rheumatology. 1994, 13(1), 54-59).
Derivatives of di(dispirotripiperazine)-pyrimidine formula 1 compounds are easily soluble in water and thus might be used in diverse pharmaceutical forms for local (aerosols) or systemic use.
The objective of this invention was to discover a new medication for the treatment of coronaviral infections, including SARS-Cov-2. The work has resulted in a discovery of high antiviral activity of di(dispirotripiperazine) -pyrimidine formula 1 compounds.
Detailed description of the embodiments
The di(dispirotripiperazine)-pyrimidine formula 1 compounds might be obtained according to the synthesis scheme which follows.
Example 1. 4,6-di(3,12-diaza-6,9- diazoniadispiro[5.2.5.2]hexadecan-l-yl)-2- methyl-5- nitropyrimidine tetrahydrochloride dihydrochloride (1)
To a solution of 15.2 g (0.133 M) 1 -formylpiperazine 3 in 267 ml of chloroform, 12.26 g (0.146 M) of sodium bicarbonate are added. The mixture is cooled down to 10°C with water, after which a solution of 17 ml (0.146 M) of benzoylchloride in 27 ml of chloroform is being dripped to the mixture for 0.5 hours. The mixture is then being mixed for 16 hours (nighttime included) at room temperature. The resulting mass is being washed twice, using 150 ml water each time; the organic layer is being dried above sodium sulphate for 0.5 hours, mixing continuously. After evaporating chloroform, 200 ml of hexane are added, the solution is mixed at 4°C, the hexane is decanted. After that, this operation is repeated. After the addition of the 3rd hexane portion, the mixture is being mixed for 0.5 hours. Resulting precipitation is filtrated, washed with 20 ml of hexane and 20 ml of ether, and left to airdry. The process results in obtaining 22.4 g (77%) of 4- benzoylpiperazine-l-carbaldehyde with melting point 87 °C.
10.9 g (0.05 M) of 4-benzoylpiperazine-l-carbaldehyde are being dissolved in 60 ml of mixture of MeOH and concentrated HC1 (HCl:MeOH ration 1:11) and mixed for 24 hours at room temperature. The resulting precipitant is being filtered and washed by 2x5 ml of methanol and 2x10 ml of acetone, then dried in a drying oven for 4 hours at 90°C. This yields 7.55 g (63 %) of hydrochloride 1- benzoylpiperazine with melting point 315 °C.
Hydrochloride benzoylpiperazine 18 g (0.0795 M) is added to a solution of 5.34 g (0.0954 M) KOH in 55 ml of ethanol and mixed for 0.5 hours at 20-22°C. After that, 13.3 ml (0.199 M) of ethilenchlorhydrine are added, and a solution of 11.6 g (0.207 M) KOH in 98 ml of ethanol (rectificated) is being dripped for 1 hour; the temperature of the mass cannot exceed 20 °C. Twenty hours later, the resulting KC1 is being filtered and washed by 25 ml absolute ethanol. The filtrate is cooled down to 10°C, after which 58 ml of 12% HCl/EtOH (control pH 2-3) are slowly added, mixed for 1 hour at 5°C, and left for a night in the fridge. The precipitate is being filtered, washed with 2x10 ml of absolute ethanol, and dried on air (48 hours) or in a drying oven (4-5 hours at 50-55°C). This process yields 15.6 g (72%) of hydrochloride l-benzoyl-4-(β-oxyethyl)piperazine with melting point 215°C.
To a suspension of 13.5 g (0.05 M) hydrochloride 1 -benzoyl-4-(β- oxyethyl)piperazine in 96 ml of chloroform, 8 ml of SOCI2 is being dripped for 0.5 hours, with continuous mixing; at the same time, the temperature of reacting mass is being increased up to 45°C by means of an oil bath. The mixture stays at this temperature for 0.5 hours, then heated to 55°C and left for 0.5 hours, then heated to 70°C and mixed for 3 hours. After that the reacting mass is being cooled down to 20°C and left in a fridge for 16 hours. The precipitate is filtered, washed with 2x30 ml of chloroform, then dried for 3 hours at 40-45°C in a drying oven. This process yields 11.7 g (81%) of hydrochloride l-benzoyl-4-(β-chloroethyl)piperazine with melting point 230°C.
To a suspension of 7.35 g (0.0254 M) hydrochloride 1 -benzoyl-4-(0- chloroethyl)piperazine in 15 ml ethanol (rectificate), a solution of 1.12 g (0.028 M) NaOH in 19 ml of 96% ethanol is added and mixed for 1.5 hours at 20-25°C. After that, NaCl is filtered and washed by 2x5 ml of absolute ethanol. The resulting filtrate is boiled with continuous mixing for 1 our, and then evaporated on a rotary evaporator at 80°C, until dry. The rests are heated to 120°C for 16 hours. After cooling down, 15 ml of distilled water are added and mixed while boiling up to complete dissolution. To this solution, 0.7 g of activated charcoal are added and boiled for 10 minutes. The charcoal is filtered and washed by 2x5 ml of hot water. The mother solution is cooled down and left in the fridge. The precipitate is filtered and washed with water (2x5 ml) and alcohol (2x5 ml), then dried for 2 hours at 100°C. This process yields 3.1 g (45%) of dichloride N,N"-dibenzoyl-N',N"- dispirotripiperazinium in dihydrate form, with melting point >360°C (with decomposition).
A mixture of 3.1 g (0.0057 M) dichloride N,N"-dibenzoyl-N',N"- di spiro tripiperazinium in dihydrate form and 20 ml 10% hydrochloric acid obtained by means of mixing 7 ml of concentrated hydrochloric acid and 13 ml distilled water; the mixture is boiled with mixing for 4 hours. The reacting mixture is cooled using ice bath down to 10-15°C; the precipitating benzoic acid is filtered and washed with water. The filtrate is evaporated on a rotary evaporator until dry. Solid residue is mixed with 10 ml of methanol, the precipitate is filtered and washed with 5 ml of methanol, then dried for 2 hours at 100°C. This yields 1.9 g (82%) of di chloride N',N"-dispirotripiperazinium dihydrochloride dihydrate with melting point >330°C (with decomposition).
To a solution of 1.9 g (0.0047 M) dichloride N' ,N" -dispirotripiperazinium dihydrochloride dihydrate in 3.2 ml water, 0.26 g (0.0108 M) of LiOH is being added in small portions, with continuous mixing at 20°C (pH 9). Then 0.17 g of activated charcoal are added, mixed for 0.5 hours, after which the charcoal is
filtered and washed with 2x1 ml water. The mother solution is diluted with 30 ml of methanol and left for 16 hours at 5°C in the fridge. The resulting precipitate is filtered and washed by 5 ml methanol on the filter, then dried for 2 hours at 100°C. This yields 1.1 g (79%) 3,12-diaza-6,9-diazonidiaspiro[5.2.5.2]hexadecane dichloride with melting point 350 °C (with decomposition).
2. Obtaining 4,6-di(3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecan-l-yl)-2- methyl- 5 -nitropyrimidine tetrachloride dihydrochloridehexahydrate according to Formula 1.
4,6-di(3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecan-l-yl)-2-methyl-5- nitropyrimidine tetrachloride dihydrochloridehexahydrate
To a solution of 4,6-dichloro-2-methyl-5-nitropyrimidine (0.12 M) in 940 ml of ethanol, a solution of 3,12-diaza-6,9-diazonidiaspiro[5.2.5.2]hexadecane dichloride (0.24 M) in 220 ml of water is added with intensive mixing. The suspension is heated for 4 hours at 70°C. After that, the mixture is cooled down to room temperature, the precipitate is filtered and washed with ethanol, then dried for 18 hours at 110°C and left on air for 4x24 hours. The yield of 4,6-di(3,12-diaza-6,9-
diazoniadispiro[5.2.5.2]hexadecan-l-yl)-2-methyl-5-nitropyrimidine tetrachloride dihydrochloridehexahydrate is 87%.
Melting point 216-220°C, with decomposition.
S (m/z): 694.1622 (M+-Cf) C29H53Cl3N11O2
Analysis of microelements (%). Estimated: C29H67Cl6N11O8: C, 38.25; H, 7.42; Cl 23.36; N, 16.92. Measured: C, 38.44; H, 7.38; Cl 22.98; N, 16.78.
Karl Fischer titration. Measured: 13.6 %.
Example 2. 4,6-di(3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecan-l-yl)-2- methyl- 5 -nitropyrimidine tetrachloride dihydrochloride (2)
Crystal 4,6-di(3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecan-l-yl)-2- methyl- 5 -nitropyrimidine tetrachloride dihydrochloridehexahydrate (4.0 g) was dried in the drying oven at 120°C for 16 hours, until the substance weight stopped changing. This yielded 3.58 g (99 %) of dry 4,6-di(3,12-diaza-6,9- diazoniadispiro[5.2.5.2]hexadecan-l-yl)-2-methyl-5-nitropyrimi dine tetrachloride dihydrochloride.
Melting point 216-220°C, with decomposition.
ES (m/z): 694.1622 (M+-Cf) C29H53Cl3N11O2
Analysis of microelements (%). Estimated: C29H55Cl6N11O2: C, 43.40; H, 6.91; C126.51; N, 19.02. Measured: C, 43.42; H, 7.03; Cl 26.48; N, 19.09.
Karl Fischer titration. Measured: 0.4 %.
Example 3. 4,6-di(3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecan-l-yl)-2- methyl- 5 -nitropyrimidine tetrachloride (3)
To a solution of 4,6-dichloro-2-methyl-5-nitropyrimidine (0.12 M) in 940 ml of ethanol, a solution of 3,12-diaza-6,9-diazonidiaspiro[5.2.5.2]hexadecane dichloride (0.24 M) in 220 ml of water is added with intensive mixing. The suspension is heated for 4 hours at 70°C, then 0.24 M of triethylamine are added. The reacting
mass is maintained at 50°C for 1 hour, cooled down to room temperature; the precipitate is filtered and washed with ethanol. The resulting solid matter is cleaned by means of redeposition from aquatic solution using 1:1 acetone/methanol mixture. The product is dried at 110°C for 5 hours. The yield of 4,6-di(3,12-diaza-6,9- diazoniadispiro[5.2.5.2]hexadecan-l-yl)-2-methyl-5-nitropyrimi dine tetrachloride is 83%.
Melting point 196-201 °C, with decomposition.
ES (m/z): 694.1622 (M+-Cf) C29H53Cl3N11O2
Analysis of microelements (%). Estimated: C29H53Cl4N11O2: C, 47.74; H, 7.32; C119.44; N, 21.12. Measured: C, 47.67; H, 7.26; Cl 19.56; N, 21.23.
Example 4. 4,6-di(3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecan-l-yl)-5- nitropyrimidine tetrachloride (4)
To a solution of 4,6-dichloro-5-nitropyrimidine (0.12 M) in 940 ml of ethanol, a solution of 3,12-diaza-6,9-diazonidiaspiro[5.2.5.2]hexadecane dichloride (0.24 M) in 220 ml of water is added with intensive mixing. The suspension is heated for 4 hours at 70°C, then 0.24 M of triethylamine are added. The reacting mass is maintained at 50°C for 1 hour, cooled down to room temperature; the precipitate is filtered and washed with ethanol. The resulting solid matter is cleaned by means of redeposition from aquatic solution using 1 : 1 acetone/methanol mixture. The product is dried at 110°C for 5 hours. The yield of 4,6-di(3,12-diaza-6,9- diazoniadispiro[5.2.5.2]hexadecan-l-yl)-5-nitropyrimidine tetrachloride is 81%.
Melting point 226-230°C, with decomposition.
ES (m/z): 680.1356 (M+-Cf) C28H51Cl3N11O2
Analysis of microelements (%). Estimated: C28H51C14N11O2: C, 47.00; H, 7.18; C119.82; N, 21.53. Measured: C, 47.07; H, 7.08; Cl 19.92; N, 21.37.
Example 5. Antiviral activity of Formula 1 compounds against SARS-Cov- 2 in U2-OS Ace2 cells in vitro
This experiment was performed according to the description in EMBO J. 2020 Oct 13:el06267. doi: 10.15252/embj.2020106267. Investigational substances were beforehand dissolved in water, in concentration of 25 pM. Experimental solution (100 μl) was added to U2-OS Ace2 cells (8xl03 cells per well) in a growing medium (DMEM 10% FCS 1% PS) and incubated at 37 °C for 45-60 minutes. Virus was added in the amount of 10 μl per well (final MOI 0.1). The cells were incubated at 37 °C for 20 hours, fixated with 8% PFA during 30 minutes at room temperature, and then washed with PBS. To dye the cellular nuclei and measure their viability, Hoechst solution (100 μl) was used. The plates were read by means of an automatic confocal microscope (Opera Phoenix) which measures the number of infected cells (GFP signal) and their viability (Hoechst signal).
The results are presented in Table 1.
Table 1. Antiviral activity of Formula 1 compounds against SARS-Cov-2 in U2-OS Ace2 cells in vitro
Example 6. Antiviral activity of Formula 1 compounds against SARS-Cov- 2 in vitro
A passaged culture of vervet monkey renal Vero cells (American collection of cell cultures and viruses) was used for the experiment. The cells were cultivated in a growing medium consisting of DMEM (minimum Eagle medium manufactured by PanEco, Russia) with the addition of 10% embryonic bovine serum (EBS, PanEco, Russia) inactivated by heat, 2pM glutamine (Sigma, USA), and antibiotics (100 U/ml penicillin and 100 pg/ml streptomycin). The supporting medium contained all listed ingredients as well as 2% EBS. The cells were incubated for 20 hours in a thermostat, at +37°C and with 5 % CO2 in the atmosphere.
In the experiments, a transport medium was used which contained SARS-CoV- 2 extracted from a patient. The presence of viral RNA was confirmed by SARS- CoV-2 RNA screening test (Generium, Vladimir region, Russia). To perform the experiment, dilutions were prepared from the transport medium with confirmed activity. The 50% tissue cytopathic dose (TCD50) was assessed according to a standard method, using cell culture in 96-well culture plates with volume of inoculation 2><104 cells per well. Cell cultures were incubated with viral load for 24 hours.
The viral TCD50 was defined as a minimal dilution of virus-containing clinical isolate which led to 50% damage of cellular monolayer, without any degeneration of control cellular monolayer where there was no infection. The efficacy of PDSTP 0.1 mg/ml solution was visually assessed on the base of maintenance of a formed cellular monolayer using inverted LOMO microscope.
In parallel, the viral TCD50 was assessed with Formula 1 compounds added into the medium in different concentrations.
The results of assessment of Formula 1 compounds antiviral activity are presented in Table 2.
Table 2. Antiviral activity of Formula 1 compounds in vitro
Thus, Formula 1 compounds (1) and (4) protect epithelial Vero cells from cytopathic action of SARS-CoV-2.
Example 7. Prevention of SARS-CoV-2 adhesion to pulmonary cells by means of Formula 1 compounds
For this experiment, lungs were isolated from 2 male guinea pigs. After opening the thorax, granulated ice was poured into the thoracic cavity, and the lungs were removed and placed onto a cooling table at +4°C. Tissue disintegration was
conducted according to a standard method. The following media were prepared for gradient centrifuging and recovery of subcellular structures, taking into account adapted guidelines (Gureev A.P., Kokina A.V., Siromiatnikov M.Yu., Popov V.O. Optimization of methods for isolating mitochondria from different tissues of the mouse. News from Voronezh State University, Ser. Chem., Biol., Pharm., 2015, 4, 61-65; Egorova M.V., Afanasiev S.A., Esopoea M.B., Aфаmасьев C.A. Isolation of mitochondria from cells and tissues of animals and humans: modern methodological techniques. Siberian Med. J., 2011, 26(1), 22-28):
1. Recovery medium containing 220 mM mannitol, 100 mM saccharose, 1 mM EDTA, 20 mM HEPES, BSA (fatty acid-free) 2 mg/ml. All ingredients were dissolved in deionized water with final pH 7.4.
2. Washing medium containing 220 mM mannitol, 100 mM saccharose, 1 mM EDTA, 20 mM HEPES. All ingredients were dissolved in deionized water with final pH 7.4.
3. 220 mM mannitol, 100 mM saccharose, 1 mM EDTA, 20 mM HEPES were added to 100% Percoll with final pH 7.4. Percoll solution 23% was prepared from 100% Percoll by its dilution with washing medium.
Lung tissue was washed in the recovery medium and homogenized in a glass homogenizer at +4 °C. The homogenate was transferred into centrifuging tubes, and medium was added up to needed volume. The homogenate was centrifuged for 5 minutes at 600g. The resulting supernatant was transferred to clean tubes, and washing medium was added up to needed volume. During repeated centrifuging, the precipitation of free mitochondrial and liposomal fractions was performed for 10 minutes at 14000g. Resulting supernatant was removed, and the precipitate was resuspended in the washing medium. After that, the precipitate from several tubes was transferred to one tube and carefully placed on 23% Percoll solution. Centrifuging in Percoll gradient was performed for 15 minutes at 23000g. After the centrifuging, 3 phases were separated. Superior layer and firm middle layer were
carefully removed. The inferior layer was resuspended, and the washing medium was added. Next washing was performed by centrifuging for 10 minutes at 18000g. The supernatant was removed, the precipitate was resuspended, transferred to one tube, centrifuged for 5 minutes at 14000g. The supernatant was removed, and the precipitate was resuspended in the washing medium.
As a result, membrane vesicles were obtained, 6-15 μm in diameter (12 μm for up to 72% vesicles), containing receptor structures for binding the virus. Addition of the latter led to agglomeration of structures and change in the solution opacity measured by nephelometer. At the same time, addition of water-soluble PDSTP compound into the medium prevented an increase in the solution opacity. The number of binding sites in testing system was much higher than the number of introduced viral particles.
Virus-containing transport media were obtained during the examination of patients with SARS-CoV-2 infection; the presence of viral RNA was confirmed by approved RT-PCR tests. Each transport medium was diluted 1 to 1000 times to evaluate its interaction with biologic material; 100 μl samples were introduced in testing system using a dosing device. In each biologic material, dilutions of transport media from 5 patients were tested. Due to impossibility of preparation of standard solutions and viral cultures, for each sample, relative (percentage) index was calculated; after that, an average was calculated from these indices. The results are presented in the Table 3.
Table 3. Changes in opacity (units) of biologic testing system for the evaluation of SARS-CoV-2 binding to pulmonary cell receptors
At the second stage, the mixture was centrifuged for 5 minutes at 14000g which permitted to precipitate vesicles together with adhered viruses. The presence of free (for example, due to the drug action) specific RNA was assessed by RT- PCR.
Table 4. Qualitative assessment of SARS-CoV-2 RNA unbound to pulmonary cell receptors in the supernatant after centrifuging, by RT-PCR
Thus, Formula 1 compounds (1) and (4) compounds in a dose-related manner bind to affine structures of biological testing system (lung) and prevent SARS-CoV- 2 particles adhesion.
Example 8. Formula 1 compounds efficacy in vivo To evaluate the efficacy of PDSTP, a SARS-CoV-2 infection model in golden hamsters was used. The virus was titrated in a Vero-B cell culture by the number of plaque-forming units and was introduced intranasally in the dose of 4× 104 TCD50, 26 μl per animal. Several groups of animals from the same litter were formed: group I — intact animals (negative control), group II — SARS-CoV-2-infected animals (positive control). Group III included animals who received 15 mg/kg PDSTP once
daily intraperitoneally for 5 days before the infection (prevention group). Group IV included animals who received 15 mg/kg PDSTP once daily intraperitoneally for 5 days after the virus introduction, in days 3 to 7 of the infection, on the background of its manifestations (treatment group).
The monitoring of infected animals included the assessment of symptoms (sneezing, rhinorrhea), the frequency of which was not statistically different between infected animals. Body weight loss was seen in infected animals. The animals were withdrawn from the experiment on the day 7. On autopsy, lungs and spleen were removed, weighed, and relative weight (percentage of body weight) of these organs was calculated. The right lung was placed into a Petri dish with saline and subjected to diaphanoscopy to count the number of tissue indurations and erythema areas with hemorrhages in the lung parenchyma.
Is has been demonstrated that PDSTP use as a part of prevention or treatment scheme prevents weight loss in animals; these measurements significantly differed from positive control. In the lungs of infected animals, viral pneumonia developed, with multiple areas of heterogenous induration, with indistinct borders and of diverse sizes, but mostly not very large, with a tendency to coalesce, especially in lower areas of the lungs. On the surface of the lungs, there were spots of different colors, from light gray to grayish-rose to light red and dark reddish. These colored areas had granular surface on dissection and were slightly elevated above the adjacent tissue. In the group of positive control, there were almost no normal light rose colored tissue. The lungs seemed thickened, firmly elastic, hydropic. The surface of a lung section looked versicolored, of heterogenous blood content. Under pressure on an indurated area, there was almost no fluid coming. According to the organ gravimetry results, PDSTP almost did not affect the development of viral pneumonia, and relative weights of lungs of infected animals in different groups were not significantly different; at the same time, this tissue had substantial differences from that in intact animals.
It was noted that relative weight of spleen significantly decreased in infected animals which probably reflected developing immunodeficiency. Preventive use of PDSTP intensified this process, while therapeutic use of the drug permitted to maintain spleen parameters at the levels of intact animals.
Table 5 presents biometric characteristics (Me [Q25÷ Q75]) of female golden hamsters on Day 7 of SARS-CoV-2 infection (intranasal infection with SARS-CoV- 2 (26 μl per animal) 4× 104 TCD50). At autopsy, lungs were inspected. Table 6 presents quantitative characteristics of diaphanoscopy. It was observed that PDSTP as prevention or treatment decreased the amount of indured, erythematous, and bleeding areas in the lung tissue. In the group of animals treated with the drug, this effect was evident on visual examination and confirmed by counting. In this group, pulmonary tissue was represented by isolated areas of induration, with substantially defined borders, of different sizes but mostly not very large and without tendency to coalesce. The areas of changed color were granular on dissection and were not elevated above the adjacent tissue. Most surface area looked like normal tissue, light rose and light red in color. The lungs were slightly indurated, elastic, insignificantly hydropic. The dissected surface was of homogenous color and blood content. On dissection, there was almost no fluid when pressure was applied.
Figure 1 shows typical observations in lungs at animals' autopsy; quantitative results of diaphanoscopy are reflected in the Table 6. It was observed that PDSTP as prevention or treatment decreased the amount of indured, erythematous, and bleeding areas in the lung tissue. In the group of animals treated with the drug, this effect was evident on visual examination and confirmed by counting. In this group, pulmonary tissue was represented by isolated areas of induration, with substantially defined borders, of different sizes but mostly not very large and without tendency to coalesce. The areas of changed color were granular on dissection and were not elevated above the adjacent tissue. Most surface area looked like normal tissue, light rose and light red in color. The lungs were slightly indurated, elastic, insignificantly
hydropic. The dissected surface was of homogenous color and blood content. On dissection, there was almost no fluid when pressure was applied.
Thus, these experiments have shown that in case of SARS-CoV-2 infection the therapeutic use of PDSTP is preferable, as it ensures a decrease in congestive/hemorrhagic pulmonary damage which can potentially lead to fibrosis, and also prevents the deficiency of splenic lymphocytes provoked by the infection.
Table 5. Biometric characteristics (Me [Q25-Q75]) of female golden hamsters on Day 7 of SARS-CoV-2 infection. Intranasal infection with SARS-CoV-2 (26 μl per animal) 4× 104 TCD50
Table 6. Morphological characteristics (Me [Q25-Q75]) on diaphanascopy of right lungs of female golden hamsters on Day 7 of SARS-CoV-2 infection. Intranasal infection with SARS-CoV-2 (26 μl per animal) 4× 104 TCD50
Example 9. Formula 1 compound use for inhalation use
To evaluate toxicity on inhalating, 3 L inhalation camera was used; the Compound (1) produced via Formula 1 was introduced into the camera by means of a dry-powder inhaler and a compressor, with volumetric flow rate 80 L/min. Mass- median aerodynamic diameter of aerosol particles was 1.3±0.24 μm, with polydispersion degree 1.4±0.15.
For the study, a period between 5 and 10 minutes from the beginning of inhalation was chosen; at this time, mean concentration of the Formula 1 compound in the camera was homogeneous. The absorbed dose was calculated as:
where D is absorbed dose (mg/kg); RMV is the rat respiratory minute volume MOД , (L/kg*min), C is mean concentration of the compound in the camera during the inhalation period (mg/L), AT is inhalation time (min). For the inhalation of a prototype or a claimed composition during 5 minutes in these conditions, the absorbed dose of the drug was 1.15 mg/kg. According to earlier studies, the LD50 of the Formula 1 compound (1) after a single intravenous administration in rats was 139.5 (116.7 - 166.6) mg/kg for males and 155.8 (132.8 -182.7) mg/kg for females. Calculated therapeutic dose cannot exceed 1/20 LD50. The resulting absorbed dose is about 1/100 LD50, which ensures its safety; thus, this dose may be multiplied and used for protective effect.
Claims
2. Usage according the Claim 1 with a Formula 1 compound as dihydrochloride.
3. Usage according the Claim 1 with a Formula 1 compound as dihydrochloride hexahydrate.
4. Usage according the Claims 1-3, using systemic administration, injections, or inhalation of the compound for the treatment or prevention of coronaviral infection.
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ARTEMENKO A. G. ET AL.: "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", JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY, vol. 60, no. 1, 2007, pages 68 - 77, XP008141706, DOI: 10.1093/jac/dkml72 * |
SERRANO G. ET AL.: "Liposomal Lactoferrin as Potential Preventative and Cure for COVID-19", RMJ, vol. 9, 26 June 2020 (2020-06-26), pages 39 - 44 * |
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