KR101935429B1 - Organic nano-system for detecting antiviral agent-resistant virus - Google Patents

Abstract

The present invention relates to an organic nanosystem for antiviral resistance or susceptible virus detection. The 10,12-pentacosidinoinic acid nanoparticles to which the compound specifically binding to the antiviral resistance virus or antiviral susceptible virus of the present invention are bound are red when bound to the target virus and blue when not bound . This can be quickly and conveniently visualized by the naked eye and can be usefully used to quickly plan the treatment of patients infected with influenza virus.

Description

[0002] Organic nano-systems for detecting antiviral agent-resistant viruses [

The present invention relates to an organic nanosystem for antiviral-resistance or susceptible virus detection.

Influenza (influenza) is a respiratory disease that spreads through the respiratory system of people and animals (birds, pigs, dogs, horses, etc.) by the influenza virus. Human influenza occurs every year in the 10-20% of the world, and is highly contagious and tends to spread worldwide on an annual basis. Symptoms of influenza include high fever, headache, muscle aches, inflammation of the throat, pain, cough and other respiratory diseases. Severe cases can lead to deaths of elderly people and chronic disease holders.

If you suspect an influenza infection, you should treat it promptly to prevent dangerous situations and prevent further spread to others. Currently, oseltamivir phosphate (Tamiflu) is mainly used for the treatment of influenza infection. Recently, the occurrence of virus mutant which shows resistance to oseltamivir is increasing. Methods for distinguishing viruses have been reported in a number of publications, for example Marin MJ et al. Discloses a method for distinguishing human influenza virus from avian influenza virus. However, no effective method has been developed to confirm whether a patient suspected of influenza infection is infected with oseltamivir-resistant virus.

Marin MJ et al. (Glyconanoparticles for the plasmonic detection and discrimination between human and avian influenza, Org Biomol Chem. 2013 Nov 7; 11 (41): 7101-7)

The present invention provides 10,12-pentacosadiynoic acid nanoparticles to which a compound specifically binding to an antiviral resistance virus or an antiviral susceptible virus is bound.

The present invention provides a kit for detecting an antiviral resistant virus comprising the nanoparticles and a kit for detecting an antiviral susceptible virus.

The present invention provides a method for providing information on whether the infected virus of the subject is an antiviral resistant virus or an antiviral susceptible virus, comprising contacting the sample isolated from the virus-infected subject with the nanoparticle .

The present invention provides 10,12-pentacosadiynoic acid nanoparticles to which a compound specifically binding to an antiviral resistance virus or an antiviral susceptible virus is bound.

The present invention provides a composition for detecting an antiviral resistant virus comprising 10,12-pentacosinicinoic acid nanoparticles to which a compound specifically binding to an antiviral resistance virus is bound.

The present invention provides an anti-viral agent resistant virus detection kit comprising 10,12-pentacosinicinoic acid nanoparticles to which a compound specifically binding to an antiviral resistance virus is bound.

The present invention relates to a method for screening for a virus, comprising contacting a sample isolated from a virus-infected subject with 10,12-pentacosinicinoic acid nanoparticles bound with a compound that specifically binds to an antiviral resistance virus, And provides a method for providing information on whether the virus is an antiviral resistant virus. In one embodiment of the present invention, the subject may be infected with a virus by a conventional diagnostic method known in the art, and it can be estimated which virus is the virus. Then, 10,12-pentacosidinoinic acid nanoparticles to which a compound that specifically binds to a virus that is resistant to an antiviral agent used in the virus are added, among the viruses of the same species as the presumed virus, are prepared, Is contacted with the nanoparticles, it can be determined whether or not the infected virus of the subject is an antiviral resistant virus.

The present invention also provides a composition for detecting an antiviral susceptible virus comprising 10,12-pentacosinicinoic acid nanoparticles to which a compound specifically binding to an antiviral susceptible virus is bound.

The present invention provides a kit for detecting an antiviral agent susceptible virus comprising 10,12-pentacosidinoic acid nanoparticles to which a compound specifically binding to an antiviral susceptible virus is bound.

The present invention relates to a method for screening for a virus, comprising contacting a sample isolated from a virus-infected subject with a 10,12-pentacosadienoic acid nanoparticle bound with a compound that specifically binds to an antiviral susceptible virus, Provides a method of providing information about whether a virus is an antiviral susceptible virus. In one embodiment of the present invention, the subject may be infected with a virus by a conventional diagnostic method known in the art, and it can be estimated which virus is the virus. Then, 10,12-pentacosadienoic acid nanoparticles to which a compound that specifically binds to a virus showing susceptibility to an antiviral agent used for the virus among the viruses of the same species as the presumed virus are bound, Is contacted with the nanoparticles, it can be determined whether or not the infected virus of the subject is an antiviral susceptible virus.

In the present invention, " susceptibility " means a virus showing a therapeutic effect of an antiviral agent, and " resistance " means a virus showing no therapeutic effect of an antiviral agent. For example, the antiviral resistance virus is an influenza virus that is resistant to oseltamivir (tamiflu (TM)) due to mutation of I223R and / or H275Y, and the antiviral susceptible virus does not mutate I223R and / or H275Y, It may be an influenza virus that shows sensitivity to tamivir. However, the kind of the antiviral agent is not limited thereto, and it may be any antiviral agent. The type of the virus is also not limited to influenza virus and can be any virus.

In one embodiment of the present invention, the virus may be a virus belonging to Flaviviridae . Flaviviridae are divided into three genus.

(1) Flavivirus. This genus includes the Dengue virus group (Dengue virus, Dengue virus type 1, Dengue virus type 2, Dengue virus type 3, Dengue virus type 4), Japanese encephalitis virus group (Alfuy virus, Japanese encephalitis virus, Kookaburra virus, Koutango virus, Kunjin virus , Murray Valley encephalitis virus, St. Louis encephalitis virus, Stratford virus, Usutu virus, West Nile virus), Modoc virus group, Rio Bravo virus group (Apoi virus, Rio Brovo virus, Saboya virus), Ntaya virus group, Group (tick-borne encephalitis virus), Tyuleniy virus group, Uganda S virus group and yellow fever virus group. In addition to this main group, there are some additional flaviviruses that are not classified.

(2) Hepacivirus. This genus contains only one type of hepatitis C virus (HCV). There are various subtypes of hepatitis C virus.

(3) Pestivirus. This genus includes bovine viral diarrhea virus-2 (BVDV-2), pestivirus type 1 (including BVDV), pestivirus type 2 (including porcine cholera virus) and Border Disease Virus.

Examples of antiviral agents identified as being active against hepatitis C flavivirus include:

(1) interferon and ribavirin (Battaglia, A. M. el al., Ann Pharmacother., 2000, 34, 487, Berenguer, M. et al., Antivir. Ther., 1998, 3 (Suppl.

(2) Substrate-based NS3 protease inhibitors including alpha keto amide and hydrazinourea (Attwood et al., PCT WO 98/22496, 1998; Attwood et al., Antivirus Chemistry and Chemotherapy 1999,10,259; Attwood et al. German Patent Publication DE 19914474; Tung et al., PCT WO 98/17679) and inhibitors (Llinas-Brunet et al, Hepat III C inhibitor peptide analogues, PCT WO 99/07734) ending with an electrophile such as boronic acid or phosphate;

(3) 2,4,6-trihydroxy-3-nitro-benzamide derivatives (including Sudo K), including RD3-4082 substituted with 14 carbon chains on the amide and RD3-4078 with para- a non-substrate-based inhibitor such as Sudo K. et al. antivirus Chemistry and Chemotherapy, 1998, 9, 186);

(4) a thiazolidine derivative (Sudo K. et al., Antiviral Research, 1996, 32, 9-18) exhibiting associated inhibition in reverse phase HPLC analysis using NS3 / 4A fusion protein and NS5A / In particular, compounds RD-1-6250, RD4 6205 and RD4 6193 with fused cinnamoyl moieties substituted with long alkyl chains;

(5) Kakiuchi N. et al. J. EBS Letters 421, 217-220; Takeshita N. et al. Thiazolidines and benzanilides identified in Analytical Biochemistry, 1997, 247, 242-246;

(6) Protease (Chu M. et al., Tetrahedron Letters, 1996, 37, 7229-7232) isolated from the fermentation broth of Streptomyces sp., Sch 68631 in SDS PAGE and autoradiography, And phenanthrenequinone (Penicillium grenadii), which is active against the fungi Penicillium griscofuluum, protease isolated from Sch 351633 (Chu M. et al., Bioorganic and Medicinal Chemistry Letters 9, 1949) ;

(7) selective NS3 inhibitors based on glycine (eglin c) in macro molecules separated from leech (Qasim M. A. et al., Biochemistry, 1997, 36, 1598);

(8) HCV helicase inhibitors (Diana G. D. et al., U.S. Patent No. 5,633,358 and Diana G. D. et al., PCT WO 97/36554);

(9) a polymer such as a nucleotide analogue, glyotoxin (Ferrari R. et al. Journal of Virology, 1999, 73, 1649), and natural serrerin (Lohmann V. et al., Virology, 1998, 249, 108) Lase inhibitors;

(Alt M. et al., Hepatology, 1995, 22, 707), which is complementary to sequence stretch in the 5 'non-coding region (NCR) Nucleotides 326-348 containing the ' end ' or nucleotides 371-388 located in the core coding region of HCV RNA (Alt M. et al., Archives of Virology, 1997, 142, 589; Galderisi U. et al., Journal of cellular Physiology, 1999, 181, 2151);

(11) IRES-dependent detoxifying inhibitors (Ikeda N et al., Agent for the prevention and treatment of hepatis C, JP-08268890; Kai Y. et al. Prevention and treatment of virus diseases, JP- 10101591);

(12) Nuclease-resistant ribozyme (Maccjak D. J. et al., Hepatology, 1999, 30, abstract 995);

(13) amantadine, such as rimantadine (Smith, Annual Meeting of the American Gastroenterological Association and AASLD, 1996);

(14) Quinolones such as oproxacin, ciprofloxacin, and berofloxacin (AASLD Abstratcts, Hepatology, Oct. 1994, Program Issue, 20 (4), pt. 2, abstract no.

(15) 2'-deoxy-L-nucleoside (Watanabe et al., WO 01/34618), and 1 -? - 1-ribofuranosyl) -1,2,4-triazole- amide (Lebo fishy ^TM) (Tam WO 01/46212) nucleoside analogs, including (Ismaili et al WO 01/60315;. Storer WO 01/32153); And

(US Pat. No. 5,922,757, Chojkier et al.), Vitamin E and other antioxidants (U.S. Patent No. 5,922,757, (Cho et al.), Chojkier et al.), Squalene, amantadine, bile acid (U.S. Patent No. 5,846,964, Ozeki et al.), N- (phosphonoacetyl) -1-aspartic acid (US Patent No. 5,830,905, Diana et al. (US Pat. No. 5,633,388, Diana et al.), Polyadenylic acid derivatives (US Pat. No. 5,496,546, Wang et al.), 2 ', 3'-dideoxyinosine (US Pat. No. 5,026,687, (US Pat. No. 5,891,874, Colacino et al.), Glucamine (Mueller et al., WO 01/08672), substituted -1,5-imino-D-gluc Toll compounds (Mueller et al., WO 00/47198).

In another embodiment of the present invention, the virus may be a virus belonging to Orthomyxoviridae . The orthomic spirals are divided into the following three genera.

(1) Influenza viruses A and B. This genus includes influenza A and B viruses, each containing eight distinct RNA segments. The major forms of infection in humans are influenza viruses H1N1 and H3N2.

(2) Influenza virus C. This genus includes only one species, influenza C, which contains only seven distinct RNA segments.

(3) Influenza virus D. This genus is Influenza D, the only mite-mediated virus that is structurally and genetically similar to influenza A, B and C.

Examples of antiviral agents identified as being active against influenza A virus include,

(1) Actomycin D (Barry, R. D. et al., "Participation of deoxyribonucleic acid in the multiplication of influenza virus" Nature, 1962, 194, 1139-1140);

(2) Amantadine (Van Voris, L. P. et al., &Quot; antiviruses for the chemoprophylaxis and treatment of influenza " Semin Respir Infect, 1992, 7, 61-70);

(3) 4-amino- or 4-guanidino-2-deoxy-2,3-didehydro-DN-acetylneuroaminic acid-4-amino- or 4-guanidino- , M. et al., "Rational design of potent sialidase-based inhibitors of influenza virus replication" Nature, 1993, 363, 418-423);

(4) ribavirin (Van Voris, L. P. et al., &Quot; antiviruses for the chemoprophylaxis and treatment of influenza ", Semin Respir Infect, 1992, 7, 61-70);

(5) Interferon (Came, PE et al. &Quot; Antibiral activity of an interferon-inducing synthetic polymer " Proc Soc Exp Biol Med, 1969, 131, 443-446; Gerone, PJ et al. &Quot; Inhibition of respiratory virus infections of mice with aerosols "Infect Immun, 1971, 3, 323-327; Takano, K. et al.," Passive interferon protection in mouse influenza "J Infect Dis, 1991, 164, 969-972);

(6) Inactivated influenza A and B virus vaccines ("Clinical studies on influenza vaccine-1978" Rev Infect Dis, 1983, 5, 721-764; Galasso, GT et al. Inactivated influenza vaccine " In: " Infectious influenza vaccine " J Hyg, 1981, Mortality SA, Mortimer EA, eds. Vaccines Philadelphia: Saunders, 1988, 420-434; Meyer, HM, Jr. et al., &Quot; Review of presence vaccines for influenza " Am J Clin Pathol, 1978, 70, 146-152; (ACIP), 1993, 42 (RR-6), 1-14, Palache, AM et al., "Antibodies to the Antibody response after influenza immunization with various vaccine doses: A double-blind, placebo-controlled, multi-center, dose-response study in elderly nursing-home residents and young volunteers " Vaccine, 1993,11,3-9; Potter, C. W. " Inactivated influenza virus vaccine " In: Beare AS, ed. Basic and applied influeza research, Boca Raton, FL: CRC Press, 1982, 119-158)

(7) oseltamivir, zanamivir, and the like.

In another embodiment of the present invention, the virus may be a virus belonging to Paramyxoviridae . The paramagnetic consuming reed consists of two subassemblies, each subassembly contains three and one genus.

(1) Paramyxovinae . This subfamily includes three genera:

a) Paramyxoviruses are represented by Sendai virus and include human parainfluenza viruses 1 and 3;

b) Rubulavirus is represented by Mumps virus, Monkey virus 5, Newcastle disease virus and human parainfluenza virus 2 and 4;

c) measles virus. This genus is represented by the measles virus.

(2) Pneumovirinae. This subspecies contains one genus.

a) Pneumovirus. This genus includes respiratory syncytial virus (RSV) (respiratory syncytial virus (RSV)) and also includes bovine (BRSV), positive RSV (ORSC), goat RSV (CRSV), mouse pneumovirus and turkey non-tracheal virus .

Examples of antiviral agents identified as being active against RSV include,

(1) ribavirin (Hruska, J. F. et al., "In vivo Inhibition of Respiratory Syncytial Virus by Ribavirin" antimicrob Agents Chemother, 1982, 21, 125-130); And

(2) Purified human intravenous IgG-IVIG (Prince, GA et al. "Effectiveness of topically administered antibodies in experimental immunotherapy of respiratory syncytial virus infection in cotton rats" J Virol, 1987, 61, 1851 1954; al. " Immunoprophylaxis and immunotherapy of respiratory syncytial virus infection in cotton rats ", Infect Immun, 1982, 42, 81 87).

In one embodiment of the present invention, the compound may be a compound that binds specifically to the antiviral resistance virus more strongly than the antiviral susceptible virus. For example, the compound may be oseltamivir hexylthiol represented by the following formula (1) or oseltamivir hexylamine represented by the following formula (2).

[Chemical Formula 1] < EMI ID =

However, the types of compounds that can bind to the PCNA nanoparticles of the present invention and bind specifically to the antiviral resistance viruses more than the antiviral susceptible viruses are not limited thereto, and are more specific than the antiviral susceptible viruses Or < RTI ID = 0.0 > a < / RTI >

In another embodiment of the present invention, the compound may be a compound that binds specifically to an antiviral susceptible virus more strongly than an antiviral resistant virus. For example, the compound may be an Oseltamivir Ethyleneglycol EGamine-acid or an Oseltamivir EGamide-ester as described in Formula 4, .

[Chemical Formula 3]

The compound represented by Formula 3 or Formula 4 may be prepared according to the method described in Korean Patent No. 10-1575747.

The types of compounds that can bind to the PCNA nanoparticles of the present invention and bind specifically to the antiviral susceptible viruses more than the antiviral resistance viruses are not limited thereto and may be more specific than antiviral susceptible viruses It may be any compound having strong binding properties.

Whether or not the PCNA nanoparticles of the present invention specifically bind to an antiviral resistant virus or an antiviral susceptible virus can be confirmed by visual observation of color. Although PCDA nanoparticles can be observed somewhat differently depending on the type and concentration of the compound bound to the PCDA nanoparticles, the material used in the kit, the type of sample, other substances contained in the sample, and the amount of virus contained in the sample, If the virus is not bound to the virus or antiviral susceptible virus, it will be blue, but if it is bound, it will be redder. For example, in the upper right diagram of FIG. 4, the PCNA nanoparticles are clearly blue when bound to an antiviral resistance virus and red when they are not bound. In the lower right diagram of FIG. 4, The difference between blue and red is not apparent in the sample as in PBS. However, in this case, it can be seen that when bound to an antiviral resistant virus, it is clearly redder than that of a non-virus-resistant virus. As such, color comparison (colorimetry) allows an ordinary technician to easily distinguish whether the PCDA nanoparticles of the present invention specifically bind to an antiviral resistant virus or an antiviral susceptible virus.

The PCNA nanoparticles of the present invention can be suitably modified to better bind to an antiviral resistance virus or a compound that specifically binds to an antiviral susceptible virus. There is no limitation on the type of modification, and various modification methods known in the art can be applied according to the structure of the compound. For example, the modification can be to bind the cross-linker to the PCDA nanoparticles. For example, when the compound contains an amine group, NHS (N-hydroxysuccinimide) can be bound to PCDA nanoparticles.

The subject to which the invention may be applied may be a human or other animal, such as a bird or a mammal.

Examples of samples to which the present invention can be applied include whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsy, lymphatic fluid, multiple fluid, interstitial fluid, bone marrow, CSF, semen, saliva, mucus, sputum, .

The kit of the present invention may further include items necessary for other detection, and may further include instructions for use.

The kit of the present invention can be implemented in various forms. For example, a detection solution kit, a rapid kit (a kit in which a sample is dropped on a kit by using a lab-on-a-chip so that the sample moves on the kit to quickly check the result), or a plastic stick, And may be implemented in the form of various other types of strips. In one embodiment of the present invention, the kit of the present invention may be a paper stick with one or more pads attached thereto. Here, the pad may be made of any material. The PCDA nanoparticles of the present invention bind to the pad, and color changes may occur upon contact with the sample. To facilitate color comparison, two pads can be attached to one paper stick and only one of the pads can be bound to the PCDA nanoparticles of the present invention.

The existing kits are limited in terms of distribution and use because they are weak against heat and pH using antibodies. In the case of gene analysis, a detection device is required. In the case of the present invention, since the compound is used, It can be stored for a long time and it can be commercialized in various forms.

The 10,12-pentacosidinoinic acid nanoparticles to which the compound specifically binding to the antiviral resistance virus or antiviral susceptible virus of the present invention are bound are red when bound to the target virus and blue when not bound . This can be quickly and conveniently visualized by the naked eye and can be usefully used to quickly plan the treatment of patients infected with influenza virus.

1 is a schematic diagram of a process for preparing PCDA nanoparticles.
2 is a photograph of the produced PCDA nanoparticles taken at UV of 254 nm. The central view was observed with an electron microscope without UV irradiation, and the right side was observed with an electron microscope at 254 nm UV.
FIG. 3 is a schematic diagram showing that the PCDA nanoparticles to which oseltamivir analogue is bound are red or blue, respectively, when bound to oseltamivir resistance virus or not.
The upper right-hand side of FIG. 4 shows the color of PCAM nanoparticles bound with oseltamivir analogue in the presence or absence of oseltamivir resistance virus in PBS, and the right lower side shows oseltamivir resistance This figure shows the color represented by the PCDA nanoparticles bound with the oseltamivir analogue in the presence and absence of the virus.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  1. Preparation of nanoparticles

1-1. Oseltamivir Hexylthiol ( Oseltamivir Hexylthiol ) synthesis

Oseltamivirhexylthiol was synthesized by the scheme below. In the following, the numbers in the parentheses after the compound names refer to the numbers shown at the bottom of the compounds in the following scheme.

S-6- Hydroxyhexyl ethanethioate  (2) Synthesis

Potassium ethanethioate (6.31 g, 55.2 mmol) was slowly added dropwise while stirring a solution of 1-bromohexanol (1) (5.00 g, 27.6 mmol) in DMF (50 mL) at room temperature. The reaction mixture was stirred for 12 hours and then diluted with distilled water (30 mL). The mixture was extracted with Et ₂ O (3 x 30 mL) and the combined organic layers were dried over anhydrous MgSO ₄ , filtered and concentrated. The concentrate was separated using column chromatography (Hexanes: EtOAc = 2: 1 - 1: 1) to give pale yellow liquid Compound 2 (4.20 g, 86%).

( 3R, 5S ) -Ethyl 4- acetamido -5- ( tert - butoxycarbonylamino ) -3- ( pentane -3-yloxy) cyclohex -One- enecarboxylate  (4) Synthesis

Di-tert-butyl dicarbonate (7.84 mL, 34.1 mmol) and triethylamine (6.80 mL, 48.8 mmol) were added dropwise to a solution of Oseltamivir phosphate salt (3) (10.0 g, 24.4 mmol) in MeOH . The reaction mixture was stirred at room temperature for 12 hours. Distilled water (100 mL) was added to the mixture and stirred for 1 hour, and the resulting white solid was filtered and washed with distilled water. The filtrate was dried in a vacuum oven to give compound 4 (5.71 g, 56%) as a white solid.

( 3R, 5S )-4- acetamido -5- ( tert - butoxycarbonylamino ) -3- ( pentane -3-yloxy) cyclohex-1-enecarboxylic acid (5) Synthesis

Compound at room temperature for 4 THF / H ₂ O of (5.70 g, 13.8 mmol): While stirring (10 1, v / v, 30mL) was added a solution of NaOH (663 mg, 16.6 mmol) . The reaction mixture was stirred for 24 hours and then concentrated to remove the reaction solvent. The concentrate was diluted with distilled water (20 mL) and the reaction vessel was cooled to 0 < 0 > C. The mixture was acidified to pH 5 with 1 M HCl aqueous solution, and stirred for 1 hour. The resulting white solid was filtered and washed with distilled water. The filtrate was dried in a vacuum oven to give compound 5 (4.0 g, 75%) as a white solid.

Synthesis of (3R, 5S) -6- (Acetylthio) hexyl-4-acetamido-5- (tert-butoxycarbonylamino) -3- (pentan- 3- yloxy) cyclohex- 1-enecarboxylate

(2.20 g, 12.5 mmol) and 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride (2.79 g, 10.4 mmol) were added to a solution of compound 5 (4.00 g, 10.4 mmol) in CH ₂ Cl ₂ g, 14.6 mmol), 4- (dimethylamino) pyridine (1.52 g, 12.5 mmol) and triethylamine (2.90 mL, 20.8 mmol) were added sequentially. After stirring at room temperature for 24 hours, distilled water was added to the reaction mixture to terminate the reaction. The mixture was extracted with CH ₂ Cl ₂ (3 x 30 mL) and the combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The concentrate was separated using column chromatography (Hexanes: EtOAc = 2: 1 - 1: 1) to obtain a colorless liquid Compound 6 (3.51 g, 62%).

Synthesis of Oseltamivir Hexylthiol (7)

At room temperature, a solution of compound 6 (3.50 g, 6.45 mmol) in MeOH (30 mL) was slowly added with concentrated hydrochloric acid (2.15 mL, 25.8 mmol) while stirring. The reaction vessel was heated at 50 < 0 > C for 72 hours. The reaction mixture was cooled to room temperature and then concentrated to remove the reaction solvent. The concentrate was diluted with MeOH (5 mL) and Et ₂ O slowly added with stirring. Filtered and the resulting white solid was washed with Et ₂ O. The filtrate was dried in a vacuum oven to obtain Compound 7 (oseltamivirhexylthiol) (560 mg, 20%) as a white solid.

The oseltamivirhexylthiol is represented by the following general formula (1).

[Chemical Formula 1]

1-2. Oseltamivir Hexylamine  ( Oseltamivir Hexylamine ) synthesis

Oseltamivirhexylamine was synthesized by the following scheme. In the following, the numbers in the parentheses after the compound names refer to the numbers shown at the bottom of the compounds in the following scheme.

tert -Butyl 6- hydroxyhexylcarbamate  (9) Synthesis

Di-tert-butyl dicarbonate (6.47 mL, 28.2 mmol) was added dropwise with stirring at room temperature to a solution of compound 8 (3.00 g, 25.6 mmol) in MeOH (30 mL). The reaction mixture was stirred at room temperature for 6 hours and then concentrated. The concentrate was separated by column chromatography (Hexanes: EtOAc = 2: 1 - 1: 1) to give pale yellow liquid 9 (4.10 g, 74%).

( 3R, 5S ) -6- ( tert - Butoxycarbonylamino ) hexyl -4- acetamido -5- ( tert -butoxycarbonylamino) -3- (pentan-3-yloxy) cyclohex-1-enecarboxylate (10)

(2.18 g, 10.0 mmol) and 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride (2.44 g, 10.0 mmol) were added to a solution of compound 5 (3.50 g, 9.10 mmol) in DMF 12.7 mmol), 4- (dimethylamino) pyridine (1.33 g, 10.9 mmol) and triethylamine (2.54 mL, 18.2 mmol) were added sequentially. After stirring at room temperature for 24 hours, distilled water was added to the reaction mixture to terminate the reaction. The mixture was extracted with EtOAc (3 x 20 mL), and concentrated after drying combined, filtered over anhydrous Na ₂ SO ₄ organic layers. The concentrate was separated using column chromatography (Hexanes: EtOAc = 2: 1 - 1: 1) to give pale yellow liquid compound 10 (2.41 g, 45%).

Oseltamivir Hexylamine (11) Synthesis

At room temperature, concentrated hydrochloric acid (1.75 mL, 21.0 mmol) was slowly added dropwise while stirring a solution of compound 10 (2.45 g, 4.20 mmol) in MeOH (20 mL). After stirring at room temperature for 24 hours, the reaction mixture was concentrated to remove the reaction solvent. The concentrate was diluted with MeOH (5 mL) and Et ₂ O slowly added with stirring. Filtered and the resulting white solid was washed with Et ₂ O. The filtrate was dried in a vacuum oven to obtain a white solid compound 11 (oseltamivirhexylamine) (1.15 g, 65%).

The oseltamivirhexylamine is represented by the following general formula (2).

(2)

1-3. PCDA  Nanoparticle manufacturing

N-hydroxysuccinimide (NHS) (0.35 g, 3.0 mmol) was added to anhydrous dichloromethane (DCM, 27 mL, 0.1 M) solution of 10,12-pentacosadiynoic acid (hereinafter "PCDA" EDC-HCl (0.57 g, 3.1 mmol) was added and stirred at room temperature for 2 hours. The reaction mixture was extracted with dichloromethane and water for 3 hours and washed with brine solution. The organic layer (organic layer) was evaporated over MgSO _4, filtered, and evaporation of the organic solvent by rotary evaporation to give the PCDA-NHS.

A solution containing 1 mM PCDA in a total of 4 mL of acetone containing PCDA and PCDA-NHS in a ratio of 9: 1 (v / v) was prepared, rapidly injected into 20 mL of deionized water, Lt; / RTI > Then, the mixture was stirred overnight to evaporate the acetone, and the mixture was cooled overnight at 4 ° C. to prepare a nanoparticle mixture in which PCDA nanoparticles and PCDA-NHS nanoparticles were mixed at a ratio of 9: 1 (schematic diagram of FIG. 1). As described above, the binding with the compound can be increased by allowing the PCDA nanoparticles having the crosslinking agent to be mixed.

The left side of FIG. 2 shows that the produced PCDA nanoparticles were photographed at 254 nm UV and appeared blue. FIG. 2 shows the PCDA nanoparticles produced by the electron microscope without UV irradiation, and the right side of FIG. 2 shows the PCDA nanoparticles irradiated with UV at 254 nm.

1-4. PCDA  The nanoparticle- Oseltamivir  Analog bond

1 mL of the PCNA nanoparticle solution synthesized in 1-3 was irradiated with UV at 254 nm for 30 minutes. 1 ml of oseltamivirhexylamine (10 mg / ml) was added to the nanoparticle solution, and the mixture was allowed to bind to PCNA nanoparticles by voltexing for 12 hours or longer.

Example  2. Color comparison according to antiviral susceptibility

As shown in the above 1-3, it was predicted that PCDA nanoparticles exhibiting red color when the PCDA nanoparticles bound to oseltamivir analogue appearing in blue bind to the target (FIG. 3).

To confirm this, influenza virus (antiviral resistant virus) or H275Y mutant (influenza virus-susceptible virus) with H275Y mutation in each well of a 96-well plate was added at 10 ⁵ PFU per 1 mL of PBS. Then, the PCDA nanoparticles bound with the oseltamivir analogue prepared in 1-4 above, the antiviral resistant virus as the binding target, were added to each well containing the antiviral resistance virus or the antiviral susceptible virus, respectively. As a result, when observed with the naked eye, PCDA nanoparticles bound with oseltamivir analogue appeared blue when contacted with antiviral susceptible virus, but turned red when contacted with antiviral resistant virus. The PCDA nanoparticles bound with oseltamivir analogue were also blue when they were contacted with PBS without virus (FIG. 4, top right view).

In addition, the mucus from the nose of a human is collected with a cotton swab and dispersed in saline to prepare a co-mucus sample, and a solution containing an antiviral resistance virus or an antiviral susceptible virus at 10 ⁵ PFU / mL per 50 microliter of the coexisting mucus sample To the wells were added PCDA nanoparticles conjugated with oseltamivir analogue, the binding target of the antiviral resistance virus. As a result, when observed with the naked eye, the PCDA nanoparticles bound with the oseltamivir analogue appeared blue when in contact with the antiviral susceptible virus and red or purple when contacted with the antiviral resistance virus 4 bottom right figure).

Claims (6)

10,12-pentacosadiynoic acid nanoparticles to which a compound of formula (1) or (2) is bound.
[Chemical Formula 1] < EMI ID =

A kit for detecting an antiviral resistant virus comprising a H275 mutant, comprising the nanoparticle of claim 1. The method according to claim 1, wherein the 10,12-pentacosadienoic acid nanoparticles are modified with NHS (N-hydroxysuccinimide), 10,12-pentacosadiynoic acid ) Nanoparticles. delete delete A method of providing information about whether the infected virus is an antiviral resistant virus comprising an H275 mutation, comprising contacting the sample isolated from the virus infected subject with the nanoparticles of claim 1.

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