New morpholine schif base and the use thereof in diseases caused by viral pathogens

Abstract

This invention discloses the novel Formula (I) [4,4'-disulfanediylbis(N-(4-morpholinobenzylidene)aniline)] compound for use in the treatment of diseases caused by viruses, particularly Herpes simplex virus-1, human Poliovirus, Adenovirus and Bovine coronavirus, the form of this compound in a structure conjugated with G- GQDs (glutathione-functionalised graphene quantum dots) and the synthesis method thereof.

Description

[0001] NEW MORPHOLINE SCHIF BASE AND THE USE THEREOF IN DISEASES CAUSED BY VIRAL PATHOGENS

[0002] Technical Field

[0003] This invention relates to a novel synthetic Schiff base and the uses thereof for the treatment of diseases caused by viral pathogens. More specifically, it discloses a composition and the production method thereof using a novel morpholine Schiff base and pharmaceutically acceptable glutathione-functionalised graphene quantum dots as carriers for use in the treatment of diseases caused by viruses, particularly Herpes simplex virus-1 , human poliovirus, Adenovirus and Bovine coronavirus.

[0004] State of the Art

[0005] Viruses are submicroscopic infectious agents that multiply inside living cells and can infect all types of organisms, including animals, plants, bacteria and archaea. They can be defined as DNA and RNA viruses according to their genomic structure and can cause very serious diseases in humans, from the common cold to Ebola and AIDS. Viruses that cause infection invade living cells to replicate themselves and cannot multiply outside the host. The infection process is a cycle that includes the attachment of the virus to the host cell membrane, penetration, opening of the viral structure, gene expression and replication, packaging of all the components formed, and then the release of viruses from the host cell by lysis or budding

[0001] ,

[0006] Most pandemics and epidemics throughout history have been caused by infectious diseases caused by existing or newly emerging pathogenic viruses. In the last 100 years, it has been observed that many large-scale epidemics (pandemics such as Spanish flu, Yellow Fever, AIDS, COVID-19) are caused by viral pathogens [2-5], Antiviral drugs are used to control or treat viral infections. Since viruses are obligate pathogens, the antivirals used must prevent viruses from infecting the host cell and / or stop their replication within the cell without damaging the cells of the host organism. In viral infection treatments, it is important to use broad-spectrum pharmacological agents that can affect both DNA and RNA viruses and subtypes of these genotypes, as in bacteria and fungi. Because due to cross-reactivity of drugs with each other and different half-lives and absorption times, combined treatments applied during infection lead to immune deficiency in patients on the one hand, and cause the emergence of resistant virus strains on the other. Currently, most FDA-approved and commercialised antiviral drugs target the cellular mechanism of the virus using nucleoside analogues, while very few target host cells / cellular mechanisms using non-nucleoside antiviral drugs and are effective only against certain viral strains. Therefore, there is a need to develop target-specific antiviral bioactive molecules and formulations obtained from these molecules that are effective against both DNA and RNA viruses without damaging the host cell, and can be eliminated from the body in a short time [6,7],

[0007] These agents can be small or large molecules or a combination of different strategies and can reduce or eliminate infectious diseases caused by viruses [8],

[0008] In addition, since most antiviral drugs are effective only against certain viral strains, efforts to develop broad-spectrum antiviral agents for various genotypes or subtypes should be considered [7],

[0009] In this context, structures called Schiff bases containing imine or azomethine groups, which are widely used in the field of pharmaceutical sciences due to their biological effects, have attracted attention [9,10,11 ], Researchers have synthesised and studied various Schiff base derivatives for their antiviral potential

[0012] , In literature studies, N- methyl-isatin [3-thiosemicarbazone has been reported as an imine containing a thiosemicarbazone core and has been approved by the FDA (formula a)

[0013] ,

[0010] Formula a. N-methyl-isatin [3-thiosemicarbazone compound

[0013]

[0011] In another study, the cytotoxicity and antiviral activity of Schiff base derivative containing 2-phenylquinazoline and its derivatives were evaluated against Herpes simplex virus-1 , Herpes simplex virus-2, vaccina virus, and vesicular stomatitis virus. Among the studied compounds, the one with -OH substitution was shown to be the most promising in terms of antiviral activity (formula b)

[0014] ,

[0012] Formula b. 3-(benzylideneamino)2-phenylquinazolin-4(3H)-one compound

[0013] Isatin functionalised bis-Schiff bases were tested for antiviral activity against a panel of DNA and RNA viruses. Synthetic bis-Schiff bases were found to be more potent against the tested viruses than commercially available antiviral drugs such as Brivudine, Ribavirin (formula c)

[0015] ,

[0014] Formula c. B is-Sch iff base containing isatin group

[0015] In order to increase these known effects of Schiff bases, when they are derivatised by substituting the morpholine group, moderate anticancer and antimicrobial effects have been reported in the literature [16,17], Morpholine is a six-member heterocyclic structure containing one nitrogen and one oxygen atom. It is characterised by the presence of both amine and ether functional groups.

[0016] Graphene quantum dots (GQDs) are a type of carbon-based planar material with functional groups on their edges for functionalisation. GQDs have attracted great interest in biomedical research due to their good dispersibility in aqueous and organic solvents, biocompatibility and low toxicity and have been used in some applications such as drug delivery [16,17], In summary, there are no effective, protective or therapeutic drugs against pandemic viruses that have not been previously identified in the prior art. Despite some recent successes in the development of antiviral chemical therapeutic agents, there is a need for drugs with broad-spectrum, effective antiviral activity that can be used in the treatment of viral diseases in clinical medicine.

[0017] Description of the Invention

[0018] The aim of the invention is to realise a new antiviral morpholine Schiff base compound and its synthesis in a single step for use in the treatment of diseases caused by viruses.

[0019] Another aim of the invention is to synthesise a new compound that provides strong antiviral activity against Herpes simplex virus-1 , human poliovirus, Adenovirus and Bovine coronavirus and its conjugate with G-GQDs.

[0020] The aim of the invention is to synthesise a new type of bis-sch iff base as a result of the reaction of 4-(4-morpholinyl) benzaldehyde and 4-aminophenyl disulphide and to overcome the problems and inadequacies given above by developing a new production method in conjugating with graphene quantum dots.

[0021] Another aim of the invention is to increase the antiviral effect of the compound even when applied at lower doses with this synthesised conjugated structure.

[0022] The use of morpholine compound for antiviral purposes reveals the originality of our invention. Another advantage of the molecule obtained in the study carried out within the scope of this invention is that it is a tetradentate Schiff base ligand, unlike those previously reported, and provides steric and electronic coordination environments.

[0023] Description of Drawings

[0024] Figure 1: Mass spectrum representation of formula I [ 4,4’-disulfanediylbis(N-(4- morpholinobenzylidenejaniline) ].

[0025] Detailed Description of the Invention

[0026] This invention discloses the novel Formula I [ 4,4’-disulfanediylbis(N-(4- morpholinobenzylidenejaniline) ] compound for use in the treatment of diseases caused by viruses, particularly Herpes simplex virus-1 , human Poliovirus, Adenovirus and Bovine coronavirus, the form of this compound in a structure conjugated with G- GQDs (glutathione-functionalised graphene quantum dots) and the synthesis method thereof.

[0027] This invention relates to the morpholine Schiff base shown by formula I below for use in the treatment of diseases caused by viruses, more specifically; Herpes simplex virus-1 , human Poliovirus, Adenovirus and Bovine coronavirus:

[0028] Formula I

[0029] The new morpholine Schiff base of the invention is shown with formula I and is called [ 4,4’-disulfanediylbis(N-(4-morpholinobenzylidene)aniline) ]. In pharmaceutical compositions containing the formula I compound of the invention, the effective dose for each virus is 10 pg / ml.

[0030] The invention differs from the state of the art with realising a new morpholine Schiff base compound with antiviral effect and its synthesis in a single step.

[0031] The diseases caused by Herpes simplex virus-1 , human Poliovirus, Adenovirus and Bovine coronavirus mentioned herein can be listed as the following disease types.

[0032] Herpes simplex virus-1 (HSV-1 ): HSV-1 usually causes cold-like lesions (cold sores) in the mouth and lips, but can also cause infection in the genital areas (genital herpes). In rare cases, HSV-1 can cause brain inflammation (encephalitis) or eye infections.

[0033] Poliovirus: Poliovirus is a virus that causes poliomyelitis. This disease is an infectious disease that affects the spinal cord and brain membranes and can cause symptoms ranging from muscle weakness to paralysis. In severe cases, it can also affect the respiratory muscles and can be fatal.

[0034] Adenovirus can cause respiratory tract infections, eye infections, intestinal infections, and urinary tract infections. In addition, in some cases, adenovirus can cause serious respiratory problems and pneumonia. Bovine coronavirus is a virus found mainly in the cattle population. It usually causes diarrhoea and respiratory tract infections in cattle. Since this virus is from the same family as SARS-CoV-2, it can be studied as a model virus instead of SARS-CoV-2 in laboratories with a safety level of 2 for safety purposes. For this reason, BcoV was used in our study to determine the effect of our molecule on COVID-19 caused by SARS-CoV-2.

[0035] In a preferred embodiment of the invention, in order to increase the antiviral effect of Formula I, a composition using Graphene quantum dots (GQDs) that exhibits high biocompatibility and low toxicity has been described. In the invention, glutathione- functionalised graphene quantum dots (G-GQDs) (formula II) are preferably used as carriers in the composition.

[0036] Formula II

[0037] An important feature of the invention relates to a conjugate containing a newly developed Schiff base and glutathione-functionalised graphene quantum dots.

[0038] The conjugate (Formula III) of the formula 1 [4,4’-disulfanedylbis(N-(4- morpholinobenzylidene)aniline] compound conjugated with the glutathione- functionalised graphene quantum dots (G-GQDs), which is the subject of the invention, is shown below:

[0039] Formula III

[0040] The antiviral activity of the 4,4’-disulfanediylbis(N-(4-morpholinobenzylidene)aniline) compound has been determined and can be synthesised in a single step, compared to other synthetic molecules studied in the state of the art. However, the hydrophobic structure of the obtained compound, which limits its solubility and reduces its activity in biological environments in biological studies, is a disadvantage. In order to overcome this problem, conjugation was carried out with non-toxic carbon-based quantum dots used as drug carriers. Quantum dots, which prevent accumulation in biological environments and also reduce the dose required for treatment, were used. Therefore, the morpholine Schiff base conjugate shown with formula III, which is the subject of the invention, can be used as an antiviral drug.

[0041] Production method of the conjugate (Formula III) formed by the formula I [4,4’- disulfanedylbis(N-(4-morpholinobenzylidene)aniline] compound conjugated with glutathione functionalised graphene quantum dots, which is the subject of the invention, comprises the process steps of:

[0042] - Synthesising the molecule of Formula I [ 4,4’-disulfanediylbis(N-(4-morpholino benzylidene)aniline) ], o Dissolving 4-morpholinebenzaldehyde (5.23 mmol) with ethanol in a beaker, o Adding 4-aminophenyl disulphide dissolved in ethanol (2.61 mmol) into the beaker, o Setting the reaction temperature to reflux temperature and stirring at this temperature (80-90°C) for 24 hours, o Forming Schiff base,

[0043] - Preparing glutathione-functionalised graphene quantum dots (G-GQDs) (formula II), o Dissolving citric acid (5.20 mmol) and glutathione (0.98 mmol) in deionised water, o Adjusting the reaction temperature to 180-200°C and stirring at this temperature for 6 hours, o Cooling the mixture to room temperature, o Dialysing citric acid, o Applying freeze drying method,

[0044] - Conjugating the formula I compound with G-GQDs (Formula II), o Adding the Formula I compound dissolved in dimethylformamide to the G-GQDs solution dissolved in deionised water, o Stirring the mixture at room temperature for 4 days, o Precipitating the resulting mixture (50 ml) with ethanol, and o Obtaining the conjugate (Formula III) collected by centrifugation.

[0045] In a preferred embodiment, this conjugate is washed with organic solvents and dried in vacuum. In the invention, ethanol and / or acetone are preferably used as organic solvents.

[0046] The chemical reactions and chemical formulas of the Schiff base production method conjugated with glutathione-functionalised graphene quantum dots are shown below:

[0047] Formula I In the first reaction above, the synthesis of the molecule Formula I [4,4’- disulfanediylbis(N-(4-morpholinobenzylidene)aniline) ] is shown. Reaction parameters: ethanol, boiling temperature, 24 hours.

[0048] In the second reaction above, the synthesis of glutathione functionalised graphene quantum dots is given. Reaction parameters: 180-220°C, distilled water, 6 hours.

[0049] Formula III In the reaction above, the conjugation of formula I with glutathione-functionalised graphene quantum dots (formula II) and the formation of conjugate (formula III) are demonstrated.

[0050] Example 1 :

[0051] The preparation of graphene quantum dot (G-GQDs) functionalised with Glutathione and Formula I [4,4’-disulfanediylbis(N-(4-morpholinobenzylidene)aniline)], which is the subject of the invention, and the exemplary synthesis steps of the conjugation of Formula I compound with G-GQDs are given below:

[0052] - Synthesising the molecule of Formula I [ 4,4’-disulfanediylbis(N-(4-morpholino benzylidene)aniline) ]

[0053] In a 100 ml flask, 4-morpholinebenzaldehyde (1 gr, 5.23 mmol) was dissolved in 20 ml of ethanol. Disulphide (0.65 gr, 2.61 mmol) dissolved in 20 ml of ethanol was added and the reaction temperature was adjusted to 80-90°C and stirred for 24 hours at 80- 90°C. The termination of the reaction towards the formation of Schiff base was determined by TLC (CHCh / Hexane: 5 / 1 ). The solution was evaporated to 1 / 3 volume, the precipitated product was filtered, and dried. (Yield 85%, the 1stReaction given above shows this synthesis)

[0054] UV- Vis (CHCh- 1x10-5M, max (nm) (log 8) : 261 (4.61 ), 294 (4.23), 331 (4.30).

[0055] MS (ESI-qTOF): m / z 595.22 [M+1 ]+

[0056] - Preparing glutathione-functionalised graphene quantum dots (G-GQDs) (formula II),

[0057] 1 g citric acid and 0.3 g glutathione were dissolved in 20 ml deionised water. The reaction temperature was set at 200°C and stirred at this temperature for 6 hours. The mixture cooled to room temperature was dialysed to remove unreacted citric acid, glutathione or any by-products that may form and finally dried by freeze drying (2ndReaction given above shows this stage).

[0058] - Conjugation of the formula I compound with G-GQDs (Formula II)

[0059] 100 mg of formula I compound dissolved in 1 ml of dimethylformamide was added to the solution of G-GQDs (50 mg) dissolved in 2 ml of deionised water and the mixture was stirred at room temperature for 4 days. The resulting mixture was precipitated with ethanol, and the conjugate collected by centrifugation was washed with organic solvents and dried in vacuum (formula III).

[0060] Example 2:

[0061] The antiviral analyses of the conjugation of the Formula I compound with G-GQDs, which is the subject of the invention, are detailed below:

[0062] Cytotoxicity test

[0063] The effect of the prepared Formula I and Formula II compounds on cell viability was measured with a substance called MTS depending on the mitochondrial dehydrogenase enzyme activity. In this method, HaCaT (immortalised human keratinocyte cell) was prepared and planted in 96-well culture plates in a medium with 5000 cells in each well. After the incubation period (24 hours), the medium on the cells was removed and compounds at certain concentrations were diluted with the medium and applied to the cells. The response of the cells to the toxicity of the molecules was determined by measuring cell viability after 72 hours. After the incubation period is completed, the MTS substance added to the cells together with the medium causes the formation of coloured formazan crystals as an indicator of cell viability. This colour change was evaluated based on absorbance measurement using an ELISA plate reader. The obtained values were analysed.

[0064] Calculation of infectivity titre with TCID 50

[0065] The cells were removed from the flask and placed in a 15 ml falcon tube and centrifuged at 500xg for 5 minutes. The medium on the cells that settled at the bottom of the falcon tube was discarded and 1 ml of new medium was added to the cells and dissolved with a pipette. Then, the cells were seeded in a 96-well plate as a monolayer within 24±2 hours and incubated at 37°C in a 5% CO2 incubator. After the cells were observed to be monolayers under the inverted microscope, the cells were processed. The Vero cell line was used for Adenovirus and Poliovirus, and the Raw cell line was used for Murine norovirus.

[0066] The substances with a determined non-toxic dose were prepared with virus medium according to the volume to be used. Serial virus dilutions were made in a new 96-well plate with virus medium on a logarithmic scale (Log 10). The medium of the prepared monolayer cells is discarded. It was washed twice with virus medium. Serial dilutions prepared on the cells were transferred to a new 96-well plate and the medium volume was completed to 200 pl with the prepared non-toxic dose of the substance. The plates were incubated for 72 hours in a 37°C 5% CO2 incubator. At the end of the incubation period, the cytopathic effect (CPE) due to the virus suspension was evaluated under an inverted microscope. The obtained results were evaluated by calculating TCID50 according to the formulas below using the Spearman-Karber method.

[0067] M= X k + d [0.5 - (1 / n) (r) ]

[0068] X k : is the highest dilution dose. d: is the difference between dilutions. n: is the number of wells per dilution. r: is the total of the (-) responses.

[0069] Mv =lg (Va / Vc) = Ig(Va)- Ig(Vc)

[0070] Mv : is the antiviral activity value.

[0071] Ig (Va) : is the logarithmic mean of two biological replicates for control tests.

[0072] Ig (Vc): is the logarithmic mean of two biological replicates for the experimental groups.

[0073] The results of the analysis are given in Table 1 below.

[0074] Table 1 : Antiviral activity of the compound of formula 1 [4,4’-disulfanedylbis(N-(4- morpholinobenzylidene)aniline] conjugated with formula II (G-GQDs) REFERENCES

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Claims

CLAIMS1. Morpholine Schiff base, indicated by formula I, for use in the treatment of diseases caused by Herpes simplex virus-1 , human Poliovirus, Adenovirus and Bovine coronavirus.Formula I2. A composition comprising the Formula I compound according to Claim 1 .

3. A composition according to Claim 2, comprising the Formula I compound at a dose of 10 pg / ml.

4. A composition according to Claim 2, comprising pharmaceutically acceptable glutathione-functionalised graphene quantum dots (G-GQDs) (represented byFormula II) as carriers.Formula II5. The conjugate (Formula III) of the formula 1 [4,4’-disulfanedylbis(N-(4- morpholinobenzylidene)aniline] compound conjugated with the glutathione- functionalised graphene quantum dots (G-GQDs).Formula III6. Use of a conjugate (Formula III) according to Claim 5 as an antiviral drug.

7. Production method of the conjugate (Formula III) formed by the formula I [4,4’- disulfanedylbis(N-(4-morpholinobenzylidene)aniline] compound conjugated with glutathione functionalised graphene quantum dots according to Claim 5, comprising the process steps of:- Synthesising the molecule of Formula I [ 4,4’-disulfanediylbis(N-(4-morpholino benzylidene)aniline) ],- Preparing glutathione-functionalised graphene quantum dots (G-GQDs) (formula II), and- Conjugating the formula I compound with G-GQDs (Formula II).

8. Production method according to Claim 7, comprising, in the process step of synthesising the molecule of Formula I [ 4,4’-disulfanediylbis(N-(4-morpholino benzylidene)aniline) ], the process steps of: o Dissolving 4-morpholinebenzaldehyde with ethanol in a beaker, o Adding 4-aminophenyl disulphide dissolved in ethanol into the container,o Setting the reaction temperature to reflux temperature and stirring at reflux temperature for 24 hours, and o Forming Schiff base.

9. Production method according to Claim 8, comprising, in the process step of synthesising the molecule of Formula I [ 4,4’-disulfanediylbis(N-(4-morpholino benzylidene)aniline) ], the process steps of: o Dissolving 5.23 mmol 4-morpholinebenzaldehyde in a beaker, o Adding 2.61 mmol of 4-aminophenyl disulphide dissolved in ethanol into the beaker, o Setting the reaction temperature to reflux temperature and stirring at this temperature (80-90°C) for 24 hours, and o Forming Schiff base.

10. Production method according to Claim 7, comprising, in the process step of preparing glutathione-functionalised graphene quantum dot (G-GQDs) (formula II), the process steps of: o Dissolving citric acid and glutathione in deionised water, o Adjusting the reaction temperature to 180-200°C and stirring at this temperature for 6 hours, o Cooling the mixture to room temperature, o Dialysing citric acid, and o applying the drying process.

11. Production method according to Claim 10, comprising, in the process step of preparing glutathione-functionalised graphene quantum dot (G-GQDs) (formula II), the process steps of: o Dissolving 5.20 mmol citric acid and 0.98 mmol glutathione in deionised water, o Adjusting the reaction temperature to 180-200°C and stirring at this temperature for 6 hours, o Cooling the mixture to room temperature, o Dialysing citric acid, and o applying the drying process.

12. Production method according to Claim 7, comprising, in the process step of conjugating the formula I compound with G-GQDs (Formula II), the process steps of: o Adding the Formula I compound dissolved in dimethylformamide to the G-GQDs solution dissolved in deionised water, o Stirring the mixture at room temperature for 4 days, o Precipitating the resulting mixture with ethanol, and o Obtaining the conjugate (Formula III) collected by centrifugation.

13. Production method according to Claim 12, comprising, in the process step of conjugating the formula I compound with G-GQDs (Formula II), the process steps of precipitating with 50 ml of ethanol.

14. Production method according to Claim 12, comprising washing the resulting conjugate (Formula III) with organic solvents and drying it in vacuum.

15. Production method according to Claim 14, wherein the organic solvent is ethanol and / or acetone.

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