Synthetic aldimines, pharmaceutical compositions against htlv-1 infection and uses

Abstract

The present technology relates to synthetic aldimines and pharmaceutical compositions containing synthetic aldimines, isolated or in combination, with immunomodulatory activity of the antiviral response for the treatment of viral infections caused by the HTLV-1 virus (human T-lymphotropic virus 1).

Description

1 / 23 “SYNTHETIC ALDIMINES, PHARMACEUTICAL COMPOSITIONS AGAINST HTLV-1 INFECTION AND USES”

[001] The present technology relates to synthetic aldimines and pharmaceutical compositions containing synthetic aldimines, isolated or in combination, with immunomodulatory activity of the antiviral response for the treatment of viral infections caused by the HTLV-1 virus (Human T Lymphotropic virus 1).

[002] HTLV-1 belongs to the Retrovindae family, subfamily Orthoretrovirinae and Deltaretrovirus genus. It is an oncogenic virus that preferentially infects CD4+ T lymphocytes and was the first retrovirus associated with human diseases to be discovered.

[003] It is estimated that around 5 to 10 million people are infected with HTLV-1 worldwide. This data originates from mathematical models based on 1.5 billion individuals living in areas endemic for HTLV-1 with solid epidemiological data. However, this number may be even higher, since epidemiology in certain countries, such as India and China, for example, is not very precise. In Brazil, data from blood donation candidates estimate that there are 2.5 million infected people, making it the country with the highest number of infections in the world.

[004] HTLV transmission occurs from mother to child through breastfeeding, the birth canal, unprotected sexual intercourse, and contact with contaminated blood through the sharing of needles and syringes, as well as through intravenous drug use and blood transfusions. Approximately 5% of individuals infected with HTLV-1 will develop some associated pathology, with adult T-cell leukemia / lymphoma (ATL) and HTLV-associated myelopathy / tropical spastic paraparesis (HAM / TSP) being the most common. Petition 870180167425, dated 12 / 26 / 2018, page 12 / 40 2 / 23

[005] ATL was first described in the late 1970s as a T-cell lymphoproliferative neoplasm affecting monoclonal populations of T cells that express CD3+, CD4+, CD8-, and CD25+ markers on the cell surface. ATL can occur in four different forms: latent and chronic, with slower progression; lymphomatous and acute, which present more aggressive clinical states. There is no standard treatment for ATL, which is based on disease classification and prognostic factors. Treatment options include interferon alpha (IFN-α) and zidovudine (AZT) cocktail therapy, multi-agent chemotherapy, and hematopoietic stem cell transplantation.

[006] HAM / TSP is a neuroinflammatory disease of the central nervous system associated with perivascular and parenchymal infiltration of HTLV-1-infected T cells and activation of cytotoxic T cells. HAM / TSP is characterized by chronic meningomyelitis of the white and gray matter of the spinal cord, followed by axonal degeneration, affecting the lumbar and thoracic spinal cord. Urinary tract symptoms, such as increased urinary frequency, nocturia, dysuria, burning sensation on urination, hesitancy, sensation of incomplete bladder emptying, and incontinence, are the first signs of myelopathy. Existing treatments for HAM / TSP are based on the symptomatology of infected patients; there is still no effective drug or cocktail for systemic use, and it is not yet possible to prevent the development of the demyelinating condition induced by HTLV-1.The use of antispastic drugs, analgesics, laxatives, and bladder control medications helps in the rehabilitation of patients with HAM / TSP. Glucocorticoids reduce motor disability due to their anti-inflammatory properties. Furthermore, the use of corticosteroids has reduced spasticity in these patients. Petition 870180167425, dated 12 / 26 / 2018, page 13 / 40 3 / 23

[007] Other inflammatory infections caused by HTLV-1, such as uveitis, infectious dermatitis, polymyositis, chronic respiratory diseases, HTLV-1-associated arthropathies, Sjögren's syndrome, and certain acute myeloid leukemias, are also reported, causing a major impact on both the lives of infected individuals and health systems.

[008] Although HTLV was discovered more than 30 years ago and causes serious pathologies that greatly impact the quality of life of infected individuals, little progress has been made regarding anti-HTLV therapies. In contrast, in infections with the human immunodeficiency virus, HIV, antiretroviral therapies have evolved not only to prolong the life of the infected individual, but also to drastically reduce the transmission of the virus, transforming AIDS (acquired immunodeficiency syndrome) from a death sentence into a controllable chronic disease. Although HIV-1 and HTLV-1 are retroviruses with tropism for CD4+ T lymphocytes, their biology and pathogenesis are quite different, which makes the use of anti-HIV cocktails ineffective in treatments against HTLV.

[009] Drugs, in general, can have two fields of action: one based on a target, such as some viral protein; the other based on assisting the host cell in fighting infection, acting on blocking some stage of viral multiplication or even regulating components of the antiviral cellular immune response, aiming to block infection.

[0010] The immune system defends the host from pathogens through innate immunity (detection of pathogens through the recognition of pathogen-associated molecular patterns - PAMPs - by pattern recognition receptors - PRRs), which triggers innate antiviral mechanisms. A large part of these mechanisms are mediated by Petition 870180167425, dated 12 / 26 / 2018, page 14 / 40 4 / 23 interferons, leading to the activation of the adaptive immune response, which provides a more targeted and antigen-specific action, as well as a longer-lasting one.

[0011] Among the studies carried out in an attempt to obtain possible drugs to be used in the treatment against HTLV, the substances found in the literature target the inhibition of some viral protein, whether structural or enzymatic, mostly plant extracts, or immunomodulation based on cytokines and corticosteroids already in current use for other pathologies.

[0012] The crude extract of Citrus bergamia (BSext) and two components of this extract, nomilin and limonin, have inhibitory activity against the gene expression of tax / rex (BALESTRIERI, E. et al.; Antiviral activity of seed extract from Citrus bergamia towards human retroviruses. Bioorganic & Medicinal Chemistry, v. 19, p. 2084-2089. 2011).

[0013] Miradenolide, isolated from Alomia myriadenia, was able to inhibit gag-pol gene expression at a concentration of 1μM in MT2 cells permanently infected with HTLV-1 and reduce the expression of the structural proteins gp46 and p19, as well as the number of viral particles on the surface of these cells (MARTINS, C. et al. A reduction of viral mRNA, proteins and induction of altered morphogenesis reveals the anti-HTLV-1 activity of myriadenolide labdane-diterpene in vitro.

[0014] Azidothymidine (AZT), a thymidine analogue that inhibits HIV reverse transcriptase activity, prevented HTLV-1 infection (MACCHI, B. et al. Susceptibility of primary HTLV-1 isolates from patients with HTLV-1-associated myelopathy to reverse transcriptase inhibitors. Viruses, v. 3, p. 469-483.

[0015] Tenofovir disoproxil fumarate (TDF), an adenine analogue, and PCOANs (nucleoside 2'-oxa-3'aza carbohydrate phosphonates) prevented cell-to-cell transmission of the virus and also inhibited the activity of Petition 870180167425, dated 12 / 26 / 2018, p. 15 / 40 5 / 23 reverse transcriptase (MACCHI, B. et al. Susceptibility of primary HTLV-1 isolates from patients with HTLV-1-associated myelopathy to reverse transcriptase inhibitors. Viruses, v. 3, p. 469-483. 2011);

[0016] Treatment with lamivudine and zidovudine showed improvement in HAM / TSP symptoms, contributing to the patient's quality of life (HASSAN, S.; AMER, S.; ZERVOS, M. Tropical spastic paraparesis treated with combivir (lamivudine-zidovudine). Journal of Clinical Neuroscience, v. 20, p. 759-760. 2013);

[0017] The use of the anti-CCR4 monoclonal antibody mogamulizumab in patients with chronic ATL achieved clinical remission, with rapid normalization of lymphocyte count and reduction of HTLV proviral load (COOK, LB et al. HTLV-1 Proviral Load after Two Months' Treatment with Anti-CCR4 Monoclonal Antibody Mogamulizumab Predicts a Molecular Response to Disease and Durable Clinical Remission in Leukaemic Subtypes of Adult T-Cell Leukaemia / Lymphoma. Blood, v. 128, n. 22, p. 5356. 2016).

[0018] The use of corticosteroids increases the production of interferons (gamma, alpha and βία), which have cytostatic and antiviral activity, and are used as immunomodulatory therapy aimed at altering the progression of HAM / TSP, but have shown limited action (GONÇALVES, DU et al. Epidemiology, treatment, and prevention of Human T-cell Leukemia Virus Type 1-associated diseases. Clinical Microbiology Reviews, v. 23, n. 3, p. 577-589. 2011; MOENS, B. et al. Ascorbic acid has superior ex vivo antiproliferative, cell death-inducing and immunomodulatory effects over IFN-α in HTLV-1-associated myelopathy. Neglected Tropical Diseases, v. 6, n. 7, p. 1-15. 2012; HASSAN, S.; AMER, S.; ZERVOS, M. Petition 870180167425, dated 12 / 26 / 2018, p. 16 / 40 6 / 23 Tropical spastic paraparesis treated with combivir (lamivudine-zidovudine). Journal of Clinical Neuroscience, vol. 20, p. 759-760. 2013).

[0019] Amantadines are derived from adamantanes, which are widely used in the pharmaceutical industry due to their pharmacophoric properties (ability to interact with a cellular receptor). Substances that have an incorporated adamantane core are more lipophilic, possessing greater permeability through the blood-brain barrier, improving the delivery of the substance to the cellular target.

[0020] Among the uses of amantadine as an antiviral, described in the state of the art, the following stand out: (i) against the Influenza virus, amantadine prevents the acidification of the endosome, blocking the release of viral RNA into the cell cytoplasm, thus interrupting its multiplication cycle (HOFFMANN, CE; NEUMAYER, EM; HAFF, RF; GOLSBY, RA Mode of action of the antiviral activity of amantadine in tissue culture. Journal of Bacteriology, v. 90, n. 3, p. 623-628. 1965; KAGAN, BL Lysosomotropic agents in AIDS treatment. West Journal of Medicine, v. 146, n. 2, p. 234. 1987; WANKA, L.; IQBAL, K.; SCHREINER, PR The lipophilic bullet hits the targets: medicinal chemistry of adamantane derivatives. Chemical Reviews, v. 113, n. 5, p. 3516-3604. 2013); (ii) against the influenza virus, Kontarov and colleagues (2013) demonstrated that the 1-boroadamantane derivative BG12 exhibits antiviral inhibitory activity through direct action on viral polymerase.In this work, the authors sought to study the mechanism involved in the interaction of the compound with the viral membrane using a phospholipid monolayer of phosphatidylcholine. It was verified that at a concentration of 10-6M there is a potential difference in the monolayer, a phenomenon that can lead to a decrease in the degree of interaction of the viral membrane with the cell membrane, favoring its use as an antiviral for enveloped viruses (KONTAROV, NA; et al. Investigation of the inhibition action of antiviral preparation 1Petition 870180167425, of 12 / 26 / 2018, p. 17 / 40). 7 / 23 boraadamantane regarding the flu virus. International Journal of Biomedicine, v. 3, n. 1, p / 44-46. 2013); (iii) Against the Hepatitis C virus, amantadine inhibits the action of the viral p7 protein, responsible for the flow of calcium ions from the endoplasmic reticulum to the cytoplasm, essential for the formation of infectious particles (GRIFFIN, SD et al. The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, amantadine. FEBS Letters, v. 535, p. 34-38). (iv) Against HIV, the use of amantadine was initially suggested in the early stages of infection (KAGAN, BL. Lysosomotropic agents in AIDS treatment. West Journal of Medicine, v. 146, n. 2, p. 234. 1987). Currently, a derivative of amantadine, memantine, is used in the treatment of HIV-associated dementia (ZHAO, Y. et al.; ACTG 301 TEAM. Memantine for AIDS dementia complex). open-label report f ACTG 301. HIV Clinical Trials, v.11, n.1, p. 59-67. 2010; WANKA, L.; IQBAL, K.; SCHREINER, PR The lipophilic bullet hits the targets: medicinal chemistry of adamantane derivatives. Chemical Reviews, vol. 113, no. 5, p. 3516-3604. 2013).

[0021] Several researchers are searching for new therapies using molecules extracted from plants, chemical substances used in anti-HIV therapies, and immunomodulatory molecules. However, to date, there is no effective treatment for the infections. The severe chronicity caused by HTLV-1 makes the search for possible antiviral agents necessary.

[0022] The present technology describes the use of amantadine derivatives, which have the amantadine chemical nucleus conjugated to (hetero)aromatic nuclei from condensation reactions with different aldehydes. In the prior art, no similar technology has been found that describes the amantadine derivatives and pharmaceutical compositions as described in the present technology and that have activity against HTLV-1 infection. Petition 870180167425, dated 12 / 26 / 2018, page 18 / 40 8 / 23

[0023] The present technology relates to pharmaceutical compositions comprising synthetic aldimines derived from amantadine, isolated or in combination, with immunomodulatory activity of the antiviral response for the treatment of viral infections caused by the HTLV-1 virus (Human T Lymphotropic virus 1). As highlighted above, immunomodulation has been used as palliative treatment for patients infected with HTLV-1, using cytokines and corticosteroids already in current use for other pathologies. These approaches differ from the present technology, since the chemical substances described herein modulate the antiviral immune response in cells infected with HTLV-1 in a directive manner. It is also noteworthy that the synthetic aldimines, the subject of the present technology, are obtained in only one reaction step, with yields in the range of 70-90% after 2 minutes of microwave irradiation (MII). BRIEF DESCRIPTION OF THE FIGURES

[0024] Figure 1 represents the analysis of cell viability in the face of the use of the compounds of the present technology, by determining the subdiploid DNA content, by flow cytometry, using hypotonic fluorochrome solution (HFS), which lyses the cell membrane allowing the DNA to be intercalated by Propidium Iodide (PI) and measuring the pro-apoptotic potential of the aldimines tested in MT2 cells (permanently infected by HTLV-1). 13A: 3F2; 13B: 3F3; and 13C: 3E10. The diluent control (ethanol) was considered to correspond to 100% cell viability. The dotted black line indicates 100% viability and the red line 80%. CCelula: Cell Control; CDiluente: Diluent Control. The 1000μM concentration showed slight cell mortality in 3F2 and 3F3, while in 3E10 this effect was observed at the 100μM concentration.However, in statistical analysis with OneWay ANOVA test and Tukey's post-hoc test (p<0.05), there was no statistical difference between the three derivatives tested and the viability control. Petition 870180167425, dated 12 / 26 / 2018, page 19 / 40 9 / 23

[0025] Figure 2 represents the analysis of cell viability by determining the subdiploid DNA content after the use of aldimines in Jukart cells. 14A: 3F2; 14B: 3F3; and 14C: 3E10. The diluent control was considered to correspond to 100% cell viability. The dotted black line indicates 100% viability and the red line 80%. CCelula: Cell Control; CDiluente: Diluent Control. The letters indicate statistical difference, with identical letters meaning no significant difference and different letters indicating a significant difference. For 3F2, statistical analysis with OneWay ANOVA and Tukey's post-hoc test showed a statistical difference (p<0.05) between the diluent control and the other samples, except at the 1000μM concentration. This was also statistically significant (p<0.05) for all samples, except for the 0.0001μM concentration, which in turn was statistically equal to the 100μM concentration.For 3F3, the same test showed a statistically significant difference (p<0.05) between the groups: diluent control and 1000μM concentration versus the other groups (cell control and treatment at concentrations from 100μM to 0.000μM). Finally, for 3E10, the test showed a statistically significant difference (p<0.05) between the groups: diluent control and 100μM concentration versus the other groups (cell control and treatment at concentrations from 10μM to 0.000μM).

[0026] Figure 3 represents the analysis of cellular mitochondrial activity by MTT, measuring cell viability after the use of aldimines in MT2 cells. 15A: 3F2; 15B: 3F3; and 15C: 3E10. The diluent control was considered to correspond to 100% cell viability. The dotted black line indicates 100% viability and the red line 80%. CCelula: Cell Control; CDiluente: Diluent Control; CMorte: Death Control. The letters indicate statistical difference, with identical letters meaning no significant difference and different letters indicating a significant difference. For 3F2, statistical analysis with a OneWay test was performed. Petition 870180167425, dated 12 / 26 / 2018, page 20 / 40 10 / 23 ANOVA and Tukey's post-hoc test showed a significant difference (p<0.05) between the cell death control versus the other treatments, and between the cell control and the treatments at concentrations of 10μM and 0.1μM versus the concentration of 0.0001μM. For 3F3, statistical analysis showed a statistically significant difference (p<0.05) between the cell death control versus the other treatments and the treatment at a concentration of 1000μM versus the other concentrations. And for 3E10, the same test showed a statistically significant difference (p<0.05) between the cell death control and the treatment at a concentration of 1000μM versus the other treatments.

[0027] Figure 4 represents the analysis of cellular mitochondrial activity by MTT measuring cell viability after the use of aldimines in Jurkat cells. 16A: 3F2; 16B: 3F3; and 16C: 3E10. The diluent control was considered to correspond to 100% cell viability. The dotted black line indicates 100% viability and the red line 80%. CCelula: Cell Control; CDiluente: Diluent Control; CMorte: Death Control. The letters indicate statistical difference, with identical letters meaning no significant difference and different letters indicating a significant difference.Statistical analysis using OneWay ANOVA and Tukey's post-hoc test showed a statistically significant difference (p<0.05) between the groups for 3F2: diluent control versus cell control, death control, and concentrations of 1000μM, 100μM, 10μM, and 0.1μM; concentration of 1000μM versus the others; concentration of 100μM versus diluent control, 1000μM, 0.00μM, 0.000μM, and death control; and death control versus the others. For 3F3, there was a statistically significant difference (p<0.05) between the death control and the treatment at the 1000μM concentration compared to the other treatments. For 3E10, the test showed a statistically significant difference (p<0.05) between the control for mortality and the treatments at concentrations of 1000μM and 100μM compared to the other treatments. Petition 870180167425, dated 12 / 26 / 2018, page 21 / 40 11 / 23

[0028] Figure 5 represents the analysis of the modulation of innate immunity genes to the antiviral response in MT2 cells treated with the three aldimines and using a commercial microarray system for analyzing the antiviral immune response. 17A: 3F2; 17B: 3F3; and 17C: 3E10. 1: treatment at a concentration of 1μM; and 2: treatment at a concentration of 0.1μM. Genes marked in red were upregulated, those in blue were downregulated, and those in black were not regulated, compared to the diluent control at a concentration of 1μM and a gene regulation cutoff point of 2 times.

[0029] Figure 6 represents the analysis of the modulation of innate immunity genes to the antiviral response in Jurkat cells treated with the three aldimines. 18A: 3F2; 18B: 3F3; and 18C: 3E10. Treatment with the three derivatives was performed at a concentration of ^M. Genes marked in red were positively regulated, those in blue negatively regulated, and those in black were not regulated, compared to the diluent control at a concentration of ^M and a gene regulation cutoff point of 2 times. DETAILED DESCRIPTION OF THE TECHNOLOGY

[0030] The present technology relates to synthetic aldimines and pharmaceutical compositions containing synthetic aldimines, isolated or in combination, with immunomodulatory activity of the antiviral response for the treatment of viral infections caused by the HTLV-1 virus (Human T Lymphotropic virus 1).

[0031] More specifically, synthetic aldimines are characterized by having the following chemical structure, where the R subunit is an aromatic, heteroaromatic, or aliphatic aldehyde: Petition 870180167425, dated 12 / 26 / 2018, p. 22 / 40 12 / 23 R = alkyl, aryl, or heteroaryl

[0032] Preferably, synthetic aldimines have the following chemical structures:

[0033] Pharmaceutical compositions against HTLV-1 infection comprise the synthetic aldimines defined above, alone or in combination, and exhibit immunomodulatory activity of the antiviral response.

[0034] The concentration of synthetic aldimines in pharmaceutical compositions varies from 0.0001 μM to 1000 μM, with the preferred concentrations for immunomodulation being between 0.1 μM and 1 μM.

[0035] The synthetic aldimines and pharmaceutical compositions of the present technology can be used to prepare a medicament for the treatment of infections caused by the HTLV-1 virus.

[0036] The present technology can be better understood by the following examples, which are not limiting to the technology. EXAMPLE 1. OBTAINING ALDIMINES

[0037] Aldimines were obtained by condensation between different aromatic aldehydes and amantadine, using ethanol as solvent. Ethanolic solutions containing equimolar amounts of the respective aldehydes and amantadine were irradiated in a DISCOVER CEM® microwave reactor under the following conditions: temperature of Petition 870180167425, dated 12 / 26 / 2018, page 23 / 40 13 / 23 The reaction was carried out at 80°C; maximum power of 200 watts; ramp time of 2 minutes; reaction time of 2 minutes; maximum stirring; and under cooling. Subsequently, the reaction products were purified by recrystallization using specific solvents for each product obtained. Once purified, the aldimines were duly characterized by infrared (IR) and hydrogen and carbon nuclear magnetic resonance (1H and 13C NMR, respectively) spectroscopies. IR spectra were obtained on KBr pellets using a Spectrum RX I spectrophotometer. 1H (200 MHz) and 13C (50 MHz) NMR spectra were obtained using a Bruker DPX 200 AVANCE spectrometer, using dimethyl sulfoxide [(CD3)2SO] or chloroform (CDCl3) as deuterated solvents. Chemical shifts (3) were expressed in parts per million (ppm) and referenced by the signs of the respective solvents or the internal standard tetramethylsilane (TMS).

[0038] The aromatic aldimine 3E10 was obtained by condensation between the heteroaromatic aldehyde and amantadine. The reaction product was purified by recrystallization using ethanol as solvent.

[0039] The infrared spectrum obtained for aldimine 3E10 shows characteristic Csp2-H bond stretching bands at 3146 and 3096 cm-1, and Csp3-H bands at 2906 and 2850 cm-1. The presence of the -NO2 group is evidenced by the strong absorption bands at 1522 and 1362 cm-1. Bands resulting from C=N and C=C bond stretching are also observed at 1570 and 1488 cm-1.

[0040] In the 1H NMR spectrum of the aforementioned compound, two multiplets are observed in the regions between 31.56-1.79 and 32.05-2.16, with integration corresponding to a total of 15 hydrogens, attributed to the aliphatic hydrogens of the molecule. A pair of doublets centered at 37.20 and 37.74, with scalar coupling constants equal to 3.7 Hz, integrated for one hydrogen each, are attributed to the hydrogens of the furan ring. Petition 870180167425, dated 12 / 26 / 2018, page 24 / 40 14 / 23 The symbol for the hydrogen in the -CH=N- group is observed as a syllable in $8,22.

[0041] The 13C NMR spectrum obtained for aldimine 3E10 shows a total of 9 signals, as expected. In a more shielded region of the spectrum, 4 signals ($28.8, 35.8, 42.4 and 58.7) are observed, referring to the aliphatic carbons of the adamantane ring. The signals of the hydrogenated carbons of the furanic ring are observed at $114.0 and $115.8. Three other signals appear in a more unshielded region of the spectrum, at $144.6, $151.9 and $153.5, which correspond to the carbons of the -CH=N- group, together with the two non-hydrogenated carbons of the furanic ring.

[0042] Aromatic aldimine 3F2 was obtained by condensation between the aromatic aldehyde and amantadine. The reaction product was purified by recrystallization using ethanol as solvent.

[0043] The infrared spectrum obtained for aldimine 3F2 shows characteristic Csp2-H bond stretching bands at 3058 and 3020 cm-1, and Csp3-H bands at 2914, 2882 and 2848 cm-1. The absorption band from the C=N bond stretching is observed at 1638 cm-1, along with C=C bond stretching bands, which appear at 1578, 1490 and 1450 cm-1.

[0044] In the 1H NMR spectrum obtained for compound 3F2, two multiplets are observed in a more shielded region, between 1.57-1.89 and 2.20-2.28, with integration corresponding to the 15 hydrogens of the aliphatic portion of the molecule. Two other multiplets are observed in a more unshielded region, between 7.27-7.47 and 7.65-7.81, integrated for three and two hydrogens, respectively, attributed to the 5 hydrogens of the aromatic ring of the compound. The signal referring to the hydrogen of the CH=N- group is observed as a singlet integrated for one hydrogen, at 8.27. Petition 870180167425, dated 12 / 26 / 2018, page 25 / 40 15 / 23

[0045] The 13C NMR spectrum obtained for aldimine 3F2 shows a total of 9 signals. The signals corresponding to the carbons of the aliphatic portion of the molecule are observed at δ29.7, 36.7, 43.3 and 57.6. Four other signals appear in a less shielded region of the spectrum, at δ 128.0, 128.6, 130.2 and 137.4, corresponding to the carbons of the aromatic ring. Finally, the signal corresponding to the carbon of the -CH=N- group is observed at δ 155.0.

[0046] The aromatic aldimine 3F3 was obtained by condensation between the aromatic aldehyde and amantadine. The reaction product was purified by recrystallization, using ethanol as solvent.

[0047] The infrared spectrum obtained for aldimine 3F3 shows an absorption band at 3418 cm-1, characteristic of OH bond stretching. Bands resulting from C sp2-H and C sp3-H bond stretching are observed at 3048 and 3004, 2908 and 2850 cm-1. The absorption band resulting from C=N bond stretching is observed at 1630 cm-1.

[0048] In the 1H NMR spectrum of the compound in question, two multiplets are observed in the regions between δ 1.61-1.90 and δ 2.10-2.25, with integration corresponding to a total of 15 hydrogens, attributed to the hydrogens of the adamantane ring. In a region characteristic of aromatic compounds, a triplet centered at δ6.82, a doublet centered at δ 6.92, and a multiplet between δ 7.18 and 7.35 are observed, corresponding to the 4 hydrogens of the aromatic portion of the compound. The signal referring to the hydrogen of the -CH=N- group is observed as a singlet at δ 8.31. Finally, at δ 14.52, a broadened signal is observed corresponding to the hydrogen of the OH group.

[0049] The 13C NMR spectrum obtained for compound 3F3 shows 4 signals at δ 29.5, 36.4, 43.0 and 57.2, referring to the carbons of the aliphatic portion of the molecule. Another five signals are observed at δ 117.5, 118.0, 119.0, 131.4 and 132.1, a region characteristic of aromatic compounds. Petition 870180167425, dated 12 / 26 / 2018, page 26 / 40 16 / 23 The signal for the carbon in the -CH=N- group is verified at δ 159.3, along with the signal for the carbon directly bonded to oxygen, which appears at δ 162.5. EXAMPLE 2. TOXICITY OF ALDIMINES 3F2, 3F3 AND 3E10 IN HTLV-1, MT2, and JURKAT cells permanently infected

[0050] Initially, it was necessary to verify whether the substances synthesized in example 1 exhibit toxicity to cells in an in vitro system. MT2 cells (a permanently transformed T cell line infected with the HTLV-1 virus) and Jurkat cells (a T cell line also transformed but not infected with HTLV-1) were used as an uninfected control.

[0051] Two cell mortality parameters were analyzed: the first allows measuring the percentage of apoptotic cells by flow cytometry (determination of subdiploid DNA content using a hypotonic fluorochrome solution - HFS, which lyses the cell membrane allowing the DNA to be intercalated by propidium iodide). This analysis (HFS) evaluates the subdiploid DNA content present in the cell, since apoptotic cells have a DNA content lower than 2n and normal cells have 2n or 4n. The second method allows measuring the activity of the mitochondrial enzyme succinate dehydrogenase in viable cells that reduce tetrazolium (yellow coloration) to its insoluble form (purple), forming formazan crystals (MTT). Both analyses were conducted using the mean and standard deviation of three independent experiments.For both HFS and MTT, MT2 or Jukart cells were incubated for 24h with aldimines (3F2, 3F3 and 3E10) at concentrations of 1000μM, 100μM, 10μM, 1μM, 0.1μM, 0.01μM, 0.001μM and 0.0001M. In addition, a cell control (without substances that could interfere with cell viability) and a control of the diluent used were performed. Petition 870180167425, dated 12 / 26 / 2018, page 27 / 40 17 / 23 in the evaluated compounds (ethanol) at the highest concentration of the assays (1000 μM) and a cell death control (in order to ensure that the cells would completely lose viability under a given condition).

[0052] For the HFS assay, 1x106 MT2 cells or Jukart cells were used, with cell pellets resuspended in 300 μl of HFS (propidium iodide 50 μg / ml in 0.1% sodium citrate and Triton X-100 0.1%) and incubated for 4 h at 4°C for flow cytometry analysis. After delineating the population in point plots of size versus granularity, the nuclei were analyzed in histograms and the subdiploid DNA content was determined using the FlowJo program, and statistical analyses were performed using GraphPad Prism 6.0, as shown in Figures 1 and 2.

[0053] The results presented in Figure 1, for MT2 cells, and Figure 2, for Jurkat cells, demonstrate that cells treated with aldimines did not show viability lower than 80% after 24 hours of treatment, at any of the concentrations tested (1000μM to 0.000μM), when compared to the diluent control (absolute ethanol) at a concentration of 1000μM. Since ethyl alcohol can alter the cell profile and the aldimines were diluted in it, it was chosen as a parameter in the toxicity assessment. For the treatment of MT2 cells, no statistically significant difference was obtained between the treatments (p<0.05). However, for the treatment with Jurkat cells, the graphs show that the diluent control and the substances at a concentration of 1000μM had a significant reduction in subdiploid DNA content (p<0.05) when compared to the other groups in the treatment with 3F2 and 3F3.For 3E10, this difference was between the diluent control and the 100μM concentration (p<0.05). It is also possible to observe that the cell control exceeded the 100% viable cell level established by the diluent control. Although statistically significant, the highlighted numbers... Petition 870180167425, dated 12 / 26 / 2018, page 28 / 40 18 / 23 Figure 2 shows that this difference was small, and that, in relation to the untreated control, the drugs did not present cytotoxicity using this early mortality analysis model.

[0054] For the MTT assay, 5.0 x 10⁴ MT2 or Jukart cells were used, which were incubated with 20 μL of MTT solution (5 mg / ml) for 4 hours in the dark. Then, 120 μL of the supernatant was removed from each well and the same amount of acidified isopropanol (1% 4M hydrochloric acid in isopropanol) was added. The cells were thoroughly homogenized to solubilize the formazan crystals and subsequently subjected to spectrophotometric analysis at a wavelength of 595 nm. The results were analyzed using the GraphPad Prism 6.0 statistical software and are shown in Figures 3 and 4.

[0055] The assessment of cell viability by colorimetric method performed through the MTT assay analyzes cellular mitochondrial activity, that is, it is possible to infer about the late death of the cell. According to the graphs represented in Figure 3, aldimine 3F2 caused less damage to MT2 cells when compared to the other amantadine derivatives after 24 hours of treatment. None of the concentrations showed viability less than 80%, although in relation to the cell control, the 10μM and 0.000M concentrations showed a statistically significant difference at the 0.000M concentration. Both 3F3 and 3E10 at the highest concentration (1000μM) showed toxicity in MT2, when compared to the diluent control, with a statistically significant difference (p<0.05).Figure 3 shows that mitochondrial activity in the cells was altered in the presence of ethanol (concentration 1000μM) compared to the control cell; however, this difference was not statistically significant. For Jurkat cells, according to Figure 4, compounds 3F2 and 3F3 showed viability lower than 80% only at the 1000μM concentration when compared to the control. Petition 870180167425, dated 12 / 26 / 2018, pages 29 / 40 19 / 23 diluent after 24 hours of treatment (p<0.05). However, the diluent control alone showed a statistically significant reduction (p<0.05) in mitochondrial activity when compared to the cell control in the experiment performed with 3F2. For 3F3, the statistical difference between the 100μM concentration and the death control was not significant between themselves, but was significant between the other groups compared (p<0.05). For 3E10, both the 100μM concentration and the 100μM concentration showed no statistical difference from the death control, and both were statistically different (p<0.05) from the others. EXAMPLE 3. REGULATION OF THE ANTIVIRAL ANTI-HTLV-1 INNATE IMMUNE RESPONSE USING ALDIMINES 3F2, 3F3 AND 3E10

[0056] Having verified that the substances are not toxic to the target cells, the influence of aldimines on the messenger RNA expression of 84 genes constituting the antiviral immune response cascade was investigated. To this end, 1x106 MT2 cells or Jukart cells were incubated with the 3F2 (A), 3F3 (B) and 3E10 (C) derivatives at concentrations 0.0M (1) and 0.0M (2) and subsequently subjected to total RNA extraction using the RNeasy mini kit system (Qiagen catalog number 74104). The total RNAs were then subjected to gene expression analysis of cellular antiviral activity using the RT2 Profiler PCR Array Antiviral Response system (Qiagen catalog number PAHS-122Z) according to the manufacturer's protocol. After the reaction, the baseline and threshold were defined and the data plotted on a spreadsheet provided by the manufacturer for data analysis using commercial software, as shown in Figure 5 (MT2 cells; HTLV-1 infected) and Figure 6 (Jurkat cells; uninfected).

[0057] The evaluation of antiviral activity against HTLV-1 in MT2 cells was performed using the real-time PCR system (RT2 Profiler PCR Array Antiviral Response - Qiagen catalog number PAHS-122Z) in Petition 870180167425, dated 12 / 26 / 2018, pages 30 / 40 20 / 23 an array of 84 genes involved in the innate antiviral response. After sample validation, they were subjected to PCR array analysis and analyzed using the website http: / / pcrdataanalysis.sabiosciences.com / pcr / arrayanalysis.php. The geometric mean of the samples was calculated and normalized using the normalizing genes ACTB (Actin, beta), B2M (Beta-2-microglobulin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), HPRT1 (Hypoxanthine phosphoribosyltransferase 1), and RPLPO (Ribosomal protein, large, PO). The cutoff point used for the positive or negative regulation of gene expression modulation was 2.0 times the control. After analyzing the results, of the 84 genes evaluated in the antiviral response array, 32 (38%) were regulated in the six samples tested (three derivatives analyzed at two different concentrations).Table 1 shows the results of the analyzed genes and their regulation in relation to diluent control for MT2 and Jurkat cells. Jurkat cells were used in this experiment as a comparison parameter between a cell model permanently infected with the HTLV-1 virus (MT2) and an uninfected one (Jurkat), since both are transformed T lymphocytes (lymphoblasts). Scatter plotter graphs were made for each sample with the 32 regulated genes (Figure 5 [MT2 cells; HTLV-1 infected] and Figure 6 [Jurkat cells; uninfected]).

[0058] Table 1. Messenger RNA regulation of genes involved in the innate antiviral immune response in MT2 and Jurkat cells. Substance and concentration Jurka Jurka Jurka MT2 MT2 MT2 ttt Transcript Description 3F2 3F3 3E10 1μ Ο,ΐμ 1μ Ο,ΐμ 1μ Ο,ΐμ ΙμΜ Μ Μ ΙμΜ Μ Μ ΙμΜ Μ M Petition 870180167425, dated 12 / 26 / 2018, pages 31 / 40 21 / 23 Fold Regulation Toll-like receptors CD80 2.7 NR NR 3.7 NR NR NR NR -2.0 CD80 molecule CXCL10 -3.3 NR NR -2.8 NR NR NR NR 2.4 Chemokine (CXC motif) ligand 10 CXCL11 NR NR -3.3 NR NR -3.3 NR NR 4.7 Chemokine (CXC motif ligand 11 CXCL9 NR NR NR NR NR -2.2 8.3 NR NR Chemokine (CXC motif ligand 9 IL15 NR -2.5 NR NR NR NR 2.0 NR 4.9 Interleukin 15 IL18 -3.2 NR -7.0 NR NR NR NR NR 2.2 Interleukin 18 (interferon-gammainducing factor) IL1B -4.8 -6.7 -3.4 -2.5 -6.5 -6.1 NR 2.6 Interleukin 1, beta IL6 NR NR NR NR NR NR NR NR 2.1 Interleukin 6 (interferon, beta 2) IL8 NR -4.4 -3.4 -2.2 -5.8 NR -5.4 -3.7 3.7 Interleukin 8 IRAKI NR NR NR NR NR NR -22 -2.1 NR Interleukin-1 receptor-associated kinase 1 IRI3 NR NR NR NR NR NR NR NR 2.6 Interferon regulatory factor 3 IRI7 NR NR NR NR NR NR NR NR -2.4 Interferon regulatory factor 7 MAP2K1 NR NR NR NR NR NR NR NR NR -3.6 Mitogen-activated protein kinase kinase 1 MAP2K3 NR NR NR NR NR NR NR NR NR 3,3 Mitogen-activated protein kinase kinase 3 MAPK8 NR NR NR NR NR NR 2.5 NR -2.1 Mitogen-activated protein kinase 8 MYD88 NR NR NR NR NR NR NR NR 2.6 Myeloid differentiation primary response gene (88) Nuclear factor of kappa light NR NR NR NR NR NR NR NR -2.6 polypeptide gene enhancer in B- NFKB1 cells 1 RELA NR NR NR NR NR NR NR NR -2.6 V-rel reticuloendotheliosis viral oncogene homolog A (avian) SPP1 -2.4 -6.9 NR -3.5 NR NR NR NR 2.5 Secreted phosphoprotein 1 TBK1 NR NR NR NR NR 3.7 NR 3.0 TANK-binding kinase 1 TLR3 NR NR 2.2 NR NR NR -7.7 NR 25 Toll-like receptor 3 TLR9 NR -2.6 -4.6 NR -4.1 NR -34.5 -2.3 NR Toll-like receptor 9 Receptores NOD ATG5 NR NR NR NR NR NR NR NR -2,4 ATG5 autophagy related 5 homolog (S. cerevisiae) OAS2 NR 2,6 2,4 NR 2,7 NR NR NR 3,1 2'-5'-oligoadenylate synthetase 2, Petition 870180167425, de 26 / 12 / 2018, pág. 32 / 40 22 / 23 Substância e concentracão Descrição do transcrito Jurka Jurka Jurka MT2 MT2 ttt MT2 3F2 3F3 3E10 ΙμΜ 1μ M 0.1μ M ΙμΜ 1μ M 0.1μ M ΙμΜ 1μ M 0.1μ M Fold Regulation 69 / 71kDa PYCARD NR -7.5 -3.5 NR NR -2.4 -2.4 -2.3 NR PYD and CARD domain containing SUGT1 NR NR NR NR NR NR NR 3.0 SGT1, suppressor of G2 allele of SKP1 (S. cerevisiae) Receptores RIG-I CASP10 NR NR NR NR -3.2 NR NR NR NR Caspase 10, apoptosis-related cysteine ​​peptidase CCL5 NR NR NR 3.5 NR NR NR NR 2.5 Chemokine (CC motif ligand 5 DDX3X NR NR NR NR NR NR NR -2.9 DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked TRADD NR NR 2.2 NR 2.2 2.1 NR NR NR TNFRSFlA-associated via death domain TRIM25 NR NR NR NR NR NR -3.0 NR -4.1 Tripartite motif containing 25 Type I Interferon signaling pathway IFNAR1 NR NR NR NR NR NR NR NR NR -2.1 Interferon (alpha, beta and omega) receptor 1

[0059] Figures 5 (MT2) and 6 (Jurkat) show that aldimines modulated the analyzed genes differently, acting in a dose-dependent manner. Table 1 shows that, of the 32 genes modulated in the studied antiviral pathways, twenty-two genes (22 - 69%) belong to Toll-like receptor pathways, four genes (4 - 12.5%) belong to NOD-like receptor pathways, five genes (5 - 15.6%) to RIG-I-like receptor pathways, and one gene (1 - 3%) to type I Interferon signaling pathways. Considering that Jurkat cells represent the uninfected control and MT2 cells the HTLV-1 infected cells, the Petition 870180167425, dated 12 / 26 / 2018, pp. 33 / 40 Compounds 23 / 23 (at concentrations of 1μM and / or 0.1μM) 3F2 and 3F3 induced modulation in Jurkat in 6 genes versus 11 in MT2, and compound 3E10 induced modulation in Jurkat in 5 genes versus 29 in MT2.

[0060] This dataset proves that immunomodulation and interaction with the triggering of antiviral pathways occurred in greater numbers in cells infected by HTLV-1, confirming its specificity. Considering that HTLV-1 infection causes dysregulation of the immune system leading to clinical outcomes with very poor prognosis, inducing neoplastic and / or demyelinating disease as the infection progresses and viral chronicity worsens, these compounds studied here have the potential to be used in the treatment of infected individuals. Petition 870180167425, dated 12 / 26 / 2018, pages 34 / 40

Claims

1 / 1 CLAIMS 1. The use of aldimine 3E10, defined by the structure characterized by being in the preparation of a medicine for the treatment of infections caused by the HTLV-1 virus. Petition 870260085474, dated 08 / 21 / 2026, page 6 / 6

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