Cd4 mimetic compounds with anti-hiv activity

Abstract

Provided are CD4 mimetic compounds having improved effectiveness in anti-HIV therapy and further improved in vivo kinetics. Provided are compounds represented by the following general formula (I): [in the formula, R 1 represents -C2H4(OC2H4) n -OCH3, or C m H 2m+1 , R 2 represents O or NH, n is 3 to 25, and m is 4 to 22], or salts thereof, and HIV infection inhibitors containing the compounds or salts thereof as active ingredients.

Description

TECHNICAL FIELD

[0001] The present application relates to a novel CD4 mimetic compound having anti-HIV activity. More specifically, the present application relates to a CD4 mimetic compound having improved in vivo kinetics. BACKGROUND

[0002] Human immunodeficiency virus (HIV) is known as a virus that causes acquired immunodeficiency syndrome (AIDS). The infection route of HIV is mainly sexual contact, infection by blood transfusion or blood products, and mother-to-child infection, and there is no risk of infection through the air. As of now, about 749 million people worldwide are infected with HIV, of which 320 million people have died from AIDS-related diseases. At present, AIDS is successfully suppressed by chemotherapy using a pharmaceutical agent, but the eradication of AIDS has not yet been achieved.

[0003] Among HIVs, there are HIV-1 and HIV-2, and further, HIV-1 is classified into subtypes A to K. HIV-1 is more common in the western hemisphere, Europe, Asia, central, southern, and eastern Africa, and HIV-2 is more common in western Africa. HIV-2 has weaker infectivity than HIV-1, which is more common, and is limited in the area of prevalence, and thus the development of anti-HIV drugs and AIDS vaccines is mainly targeted at HIV-1.

[0004] HIV-1 is a retrovirus. The mature virus is a spherical shape with a diameter of 100 to 110 nm, has a core structure of a cannonball type, contains two copies of a single-stranded RNA genome or reverse transcriptase, integrase, etc., and is wrapped in an envelope structure composed of a lipid bilayer membrane and a coat protein. On the surface of the virion, gp120 and gp41, which are coat proteins, exist as a trimer, and they specifically bind to CD4, CXCR4, and CCR5 present on helper T cells or macrophages that are human CD4-positive T cells, and play an important role in the invasion of HIV into host cells.

[0005] Currently, anti-HIV drugs used in the clinic are mainly enzyme inhibitors that inhibit the action of enzymes specific to HIV, such as "reverse transcriptase inhibitors", "protease inhibitors", "integrase inhibitors", and the like. A multi-drug combination therapy (anti-retroviral therapy: ART) in which a plurality of agents are administered has been established, but there are few invasion inhibitors that inhibit the invasion of HIV into cells, and only enfuvirtide, which is a membrane fusion inhibitor, and maraviroc, which is a CCR5 inhibitor, are used clinically. However, these agents need to be administered regularly for life because they can inhibit the growth of viruses but cannot kill viruses. Therefore, the risk of side effects accumulating due to long-term administration, and the high cost of treatment, are problems. Furthermore, because HIV easily causes mutations, the emergence of drug-resistant viruses is a major problem, and vaccine development is also difficult.

[0006] The first stage of HIV-1 invasion into a host cell is the interaction of the coat protein gp120 of HIV-1 with the host cell surface protein CD4 (the first receptor). With this interaction, the structure of gp120 changes greatly, and a region called the V3 loop is exposed. Next, the V3 loop interacts with the second receptor (the co-receptor, CCR5 or CXCR4), and as a result, gp41 is exposed to the surface, penetrates the membrane of the host cell, and invades the host cell through membrane fusion.

[0007] In 2005, a low-molecular compound, NBD-556, which has an effect of inhibiting the above invasion, was reported through a syncytia formation inhibition screening of HIV-1 (Non-Patent Literature 1). NBD-556 is also able to bind to the Phe43-cavity of gp120, which is the interaction site of CD4, and induce a structural change in gp120, and thus is attracting attention as a low-molecular CD4 mimetic (mimic) compound (Non-Patent Literatures 2 to 4). However, NBD-556 has problems such as low anti-HIV activity, high cytotoxicity, and low water solubility, and research on the structure-activity relationship of NBD-556 as a lead compound is being actively conducted (Non-Patent Literatures 5 to 8).

[0008] The inventors and others have so far found that by changing the piperidine ring portion of NBD-556, a compound HAR-171 having two cyclohexyl groups was synthesized, which has lower cytotoxicity than NBD-556 and shows stronger anti-HIV activity (Non-Patent Literature 6). In addition, various novel derivatives of a mono-cyclohexyl type compound designed by introducing one cyclohexyl group in the piperidine ring of NBD-556 to improve hydrophilicity were synthesized, and their anti-HIV activity, cytotoxicity, gp120 structure change induction ability were evaluated, and the prediction of the interaction mode by using the docking simulation of the comprehensive computational chemistry system Molecular Operating Environment (MOE, Chemical Computing Group Inc.) was performed, as a result, a substance having excellent anti-HIV activity and low cytotoxicity could be obtained (Patent Literature 1). It was also found that when these compounds were used with neutralizing antibodies that specifically recognize the V3 loop exposed in the middle of the HIV entry mechanism, a synergistic anti-HIV effect was brought about.

[0009] Among the compounds reported in the above Patent Literature 1, as one of the CD4 mimetic compounds having significantly higher anti-HIV activity and substantially reduced cytotoxicity than NBD-556, N 1 -(4- chlorophenyl)-N 2 -(1-(2-(5-guanidinopentanoylamino)ethyl)-2-cyclohexylpiperidin-4-yl)oxalylamide (hereinafter referred to as YIR-821).

[0010] [Chemical Formula 1]

[0011]

[0012] Patent Literature 1: International Publication No. 2016 / 190331

[0013] Non-Patent Literature 1: Qian, Z. et al., Virology 339, 213-225 (2005)

[0014] Non-Patent Literature 2: Schon, A. et al., Biochemistry 45, 10973-10980 (2006)

[0015] Non-Patent Literature 3: Madani, N. et al., Structure 16, 1689-1701 (2008)

[0016] Non-Patent Literature 4: Hillel, H. et al., Plos Pathogens 5, e1000360 (2009)

[0017] Non-patent literature 5: Yamada, Y. et al., Bioorg. Med. Chem. Lett. 20, 354-358 (2010)

[0018] Non-patent literature 6: Narumi, T. et al., Bioorg. Med. Chem. 19, 6735-6742 (2011)

[0019] Non-patent literature 7: Nguyen, W. et al., Bioorg. Med. Chem. Lett. 22, 7106-7109 (2012)

[0020] Non-patent literature 8: Narumi, T. et al., Bioorg. Med. Chem. 21, 2518-2526 (2013) SUMMARY

[0021] As described above, the present inventors et al. reported a CD4 mimetic compound having high anti-HIV activity, and low cytotoxicity, and capable of bringing about synergistic anti-HIV activity when used in combination with neutralizing antibodies, but various studies were conducted as a subject to obtain a compound further improved in in vivo kinetics by further advancing structure-activity correlation studies.

[0022] In view of the above-described subject, as a result of repeatedly studying various derivatives synthesized from the above-described YIR-821 as a parent compound, the present inventors et al. succeeded in obtaining a novel CD4 mimetic compound having a significantly longer half-life than YIR-821 in vitro and in vivo, and thus a long-term effect can be sustained, by adding a PEG chain or an alkyl chain as a side chain to YIR-821, thereby completing the present application.

[0023] That is, the present application provides the following.

[0024] 1. A compound represented by the following general formula (I) or a salt thereof,

[0025] [Chemical Formula 2]

[0026]

[0027] [In the formula, R 1 represents C2H4(OC2H4) n -OCH3, or C m H 2m+1 , R 2 represents O or NH, n is 3 to 25, and m is 4 to 22].

[0028] 2. The compound according to the above 1 or a salt thereof, represented by the following formula,

[0029] [Chemical Formula 3]

[0030]

[0031] [In the formula, n is 3 to 25].

[0032] 3. The compound or salt thereof according to the above 2, wherein n is 4 to 23.

[0033] 4. An HIV infection inhibitor comprising the compound or salt thereof according to any one of the above 1 to 3 as an effective ingredient.

[0034] 5. A pharmaceutical composition for treating or preventing HIV infection, comprising the HIV infection inhibitor according to the above 4.

[0035] 6. The HIV infection inhibitor according to the above 4, or the pharmaceutical composition according to the above 5, which is administered in combination with an anti-HIV antibody.

[0036] 7. The HIV infection inhibitor or pharmaceutical composition according to the above 6, wherein the anti-HIV antibody is a V3 loop-specific neutralizing antibody on the surface of HIV-1.

[0037] This specification includes the disclosure of Japanese Patent Application No. 2019-195967 on which this application claims priority.

[0038] According to the present application, it is possible to provide a CD4 mimetic compound having high anti-HIV activity, and low cytotoxicity, and a significantly prolonged half-life; and a novel HIV infection inhibitor comprising the same as an effective ingredient. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The blood concentration change of YIR-821 after administration by intravenous injection is shown.

[0040] Figure 2 The blood concentration change of TKB-002·2TFA after administration by intravenous injection is shown.

[0041] Figure 3 The degradation of YIR-821 derivatives releasing alkyl chains over time in vitro is shown.

[0042] Figure 4 The degradation of YIR-821 derivatives releasing PEG chains over time in vitro is shown. DETAILED DESCRIPTION

[0043] Hereinafter, the present application will be described in detail.

[0044] As described above, the present application provides a compound represented by General Formula (I) or a salt thereof,

[0045] [Chemical Formula 4]

[0046]

[0047] [In the formula, n is 3 to 25.] 1 represents C2H4(OC2H4) n -OCH3, or C m H 2m+1 , R 2 represents O or NH, n is 3 to 25, and m is 4 to 22.

[0048] The above compound is a YIR-821 derivative having a PEG chain or an alkyl chain in the side chain of YIR-821. In order to have an activity suitable as a CD4 mimetic compound, low toxicity, and suitable solubility, the PEG chain length or the alkyl chain length must be within a specific range, and therefore, in the above general formula, n is within a range of 3 to 25 in the derivative having a PEG chain, for example, n can be within a range of 4 to 23, 3 to 7, 3 to 5, 9 to 13, 10 to 12, 21 to 25, or 22 to 24. In the derivative having an alkyl chain, m is preferably within a range of 4 to 22, for example, m can be within a range of 5 to 21, 6 to 20, 7 to 19, or 8 to 18.

[0049] In one embodiment, the compound of the present application is a compound having a PEG chain, R 1 is C2H4(OC2H4) n -OCH3, R 2 is NH. The compound of this embodiment is represented by the following formula, and a degradation reaction releasing the PEG chain is difficult to occur, which can result in a longer half-life.

[0050] [Chemical Formula 5]

[0051]

[0052] [In the formula, n is 3 to 25.]

[0053] In another embodiment, the compound of the present application is a compound having a PEG chain, R 1 is C2H4(OC2H4) n -OCH3, R 2 is O. The compound of this embodiment is represented by the following formula, and a reaction releasing the PEG chain through hydrolysis of the -C(=O)O- group can occur at a relatively early stage.

[0054] [Chemical Formula 6]

[0055]

[0056] [In the formula, n is 3 to 25.]

[0057] In one embodiment, the compound of the present application is a compound having an alkyl chain, R 1 is C m H 2m+1 , and R 2 is NH. The compound of this embodiment is represented by the following formula, and the degradation reaction releasing the alkyl chain is difficult to occur, which can lead to a longer half-life.

[0058] [Chemical Formula 7]

[0059]

[0060] [In the formula, m is 4 to 22.]

[0061] In another embodiment, the compound of the present application is a compound having an alkyl chain, R 1 is C m H 2m+1 , and R 2 is O. The compound of this embodiment is represented by the following formula, and the reaction releasing the alkyl chain by hydrolysis of the -C(=O)O- group can occur at a relatively early stage.

[0062] [Chemical Formula 8]

[0063]

[0064] [In the formula, m is 4 to 22.]

[0065] In a preferred aspect of the present application, the compound of the present application is a compound having the following structure, wherein R 1 is C2H4(OC2H4) n -OCH3, R 2 is NH, and n is 4. In this specification, the compound represented by the following formula is referred to as TKB-001.

[0066] [Chemical Formula 9]

[0067]

[0068] In another preferred aspect of the present application, the compound of the present application is a compound having the following structure, wherein R 1 is C2H4(OC2H4) n -OCH3, R 2NH, and n is 11. In the present specification, the compound described below is referred to as TKB-002. As shown in the examples, it was found that the blood half-life of TKB-002 is 3.48 times longer than that of YIR-821, and is stable even in the presence of human liver microsomes.

[0069] [Chemical Formula 10]

[0070]

[0071] In still another preferred embodiment of the present application, the compound of the present application is a compound having the structure described below, wherein R 1 C2H4(OC2H4) n -OCH3, R 2 NH, and n is 23. In the present specification, the compound described below is referred to as TKB-003.

[0072] [Chemical Formula 11]

[0073]

[0074] The compound of the present application can be synthesized using the description in the present specification, and techniques generally used in the art, for example, by a synthetic route starting from YIR-821 as a parent compound, but the synthetic method is not particularly limited. The synthesis of YIR-821 is not limited, but can be performed based on the description in International Publication No. 2016 / 190331, for example.

[0075] The compound of the present application can form a salt with an inorganic or organic base at either or both of the piperidine ring nitrogen atom and the nitrogen atom of the guanidine group. As the salt, there is no particular limitation, but a pharmaceutically acceptable salt is preferred, and for example, a hydrochloride, a sulfate, a phosphate, a succinate, a fumarate, a methanesulfonate, a p-toluenesulfonate, a hydrobromide, an acetate, a trifluoroacetate, or the like can be preferably used.

[0076] The compound of the present application or a salt thereof functions as a CD4 mimetic compound, and can competitively inhibit the binding of Phe43-cavity of gp120 of HIV to CD4. In addition, not being bound by any theory, but the structural change of gp120 caused by the compound of the present application or a salt thereof is different from that produced by the interaction with CD4, and exposes the V3 loop, but does not produce the subsequent binding to the co-receptor, and thus can inhibit the invasion of HIV into the host cell. Therefore, the present application provides an HIV infection inhibitor comprising the above-described compound of the present application or a salt thereof as an effective ingredient.

[0077] The compound of the present application or a salt thereof can also be administered directly as an HIV infection inhibitor, but can be administered as a pharmaceutical composition by appropriately adding, in addition to the active ingredient, carriers, excipients, preservatives, oxidation stabilizers, and the like, which are generally used in pharmaceutical compositions. Therefore, the present application also provides a pharmaceutical composition for treating or preventing HIV infection, which comprises the above-mentioned HIV infection inhibitor.

[0078] The HIV infection inhibitor and the pharmaceutical composition of the present application can be administered orally and non-orally, for example, orally, by injection or infusion into intravenous, intramuscular, transdermal, subcutaneous, intradermal, intraperitoneal, and the like, but are not particularly limited. The administration route can preferably be intravenous, intramuscular, or subcutaneous injection. The preferred administration route for administering the HIV infection inhibitor and the pharmaceutical composition of the present application can be appropriately determined by those skilled in the art.

[0079] The amount of the HIV infection inhibitor of the present application to be administered to a human is not particularly limited depending on the age, body weight, symptoms, and the like of the patient to be administered, and can be, for example, in the range of 100 μg / kg body weight to 100 mg / kg body weight, preferably 500 μg / kg body weight to 50 mg / kg body weight, and more preferably 1 mg / kg to 30 mg / kg body weight per day.

[0080] Further, the HIV infection inhibitor of the present application can be used alone, but is also intended to be used in combination with other anti-HIV agents having inhibitory effects according to different mechanisms. As the other anti-HIV agents, there are no particular limitations, and examples include reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, and the like. As the reverse transcriptase inhibitors, for example, zidovudine, lamivudine, abacavir, tenofovir, emtricitabine, efavirenz, and the like can be mentioned, as the protease inhibitors, for example, atazanavir, dananavir, ritonavir, and the like can be mentioned, and as the integrase inhibitors, for example, raltegravir and the like can be mentioned, but are not intended to be limited thereto. The anti-HIV infection inhibitor and the other anti-HIV agent can be contained in the same or different pharmaceutical compositions. The administration of the HIV infection inhibitor of the present application and the other anti-HIV agent can be simultaneous, consecutive, or completely different. In addition, the administration routes of the HIV infection inhibitor of the present application and the other anti-HIV agent can be the same or different.

[0081] In addition, the HIV infection inhibitor of the present application is also intended to be used in combination with HIV-specific antibodies. As the antibodies, there are no particular limitations, but since the HIV infection inhibitor of the present application causes a structural change in gpl20 by binding to HIV, which can cause the V3 loop to be exposed, it is preferable to use neutralizing antibodies, particularly V3 loop-specific neutralizing monoclonal antibodies and functional fragments thereof for HIV.

[0082] As for anti-HIV monoclonal antibodies, various research and development have been conducted in the field, and as a representative of monoclonal antibodies against the V3 loop of HIV, KD-247 (generic name: suvizumab) is in clinical trials. Details of anti-HIV monoclonal antibodies are described, for example, in Journal of Virology, June 2006, p. 5552-5562; Journal of Virology, June 2006, p. 5563-5570; Chemical and Pharmaceutical Bulletin, 23: 42-54 (2014); Patent No. 2989862; and Patent No. 5526386, and the amino acid sequence of the VH and VL regions of KD-247 is also disclosed in Journal of Virology, June 2006, p. 5552-5562. Those skilled in the art can obtain the KD-247 antibody and other anti-HIV antibodies based on this information.

[0083] Accordingly, the present application provides the above-mentioned HIV infection inhibitor, or the above-mentioned pharmaceutical composition, characterized by being administered in combination with an anti-HIV antibody. Without being limited, in a preferred embodiment, the anti-HIV antibody is a V3 loop-specific neutralizing antibody on the surface of HIV-1.

[0084] The administration of the HIV infection inhibitor of the present application and the anti-HIV antibody can be simultaneous, consecutive, or completely different. In addition, the administration route of the HIV infection inhibitor of the present application and the anti-HIV antibody can be the same or different.

[0085] Examples

[0086] The present application is specifically described by the following examples, but the present application is not limited by these examples.

[0087] [Reference Example 1 Measurement of Blood Half-Life of YIR-821]

[0088] A rhesus monkey (MM616, body weight 8.00 kg) was intravenously injected with 192 mg (10.1 mg / mL) of YIR-821, and blood was collected 21 minutes, 39 minutes, 63 minutes, 91 minutes, 98 minutes, 113 minutes, 143 minutes, 201 minutes, 328 minutes, and 453 minutes after the injection, and the concentration in the blood was measured by HPLC.

[0089] The HPLC conditions are described below.

[0090] Measurement device: JASCO PU-2089plus (JASCO Corporation, Ltd.)

[0091] Chromatography column: Cosmosil 5C18 - ARII column (4.6 x 250 mm, Nacalai Tesque, Inc.)

[0092] Solvent A: H2O containing 0.1% (v / v) TFA

[0093] Solvent B: CH3CN containing 0.1% (v / v) TFA

[0094] Gradient: 20-50% solvent B in solvent A, 30 min

[0095] Flow rate: 1 cm 3 / minute

[0096] Detection: detection by UV at 260 nm

[0097] As a result, the blood concentration of YIR-821 was shifted as shown in Figure 1 Table 1, and the blood half-life was calculated to be 31.0 minutes.

[0098] [Example 1 Synthesis of the compound of the present application 1]

[0099] The compound of the present application having a PEG chain was synthesized by the following synthesis scheme of the steps.

[0100] [Chemical Formula 12]

[0101]

[0102] To a solution of compound 1 (13.0 g, 20.0 mmol) in MeOH (67 mL) was added SOCl2(1.6 mL, 22 mmol) at 0°C. The reaction mixture was stirred at room temperature for 17 hours, and then the mixture was concentrated under reduced pressure to obtain a crude mixture as a white powder. To the crude mixture in CH2Cl2(219 mL) was added piperidine (9.32 g, 109.5 mmol). The reaction mixture was stirred at room temperature for 2 hours, and then purified by silica gel column chromatography (CHCl3 / MeOH = 10 / 1) to obtain compound 3 (N ω -((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-L-arginine methyl ester) as a white powder (7.61 g, yield 87%).

[0103] 1H NMR (500 MHz, CDC13) δ 1.45 (s, 6H), 1.59-1.66 (m, 2H), 1.79-1.82 (m, 1H), 1.87 (s, 3H), 2.09 (s, 3H), 2.51 (s, 3H), 2.57 (s, 3H), 2.95 (s, 2H), 3.16-3.21 (m, 2H), 3.48-3.51 (m, 1H), 3.69 (s, 3H), 6.22 (br, 2H), 6.31 (br, 1H); 13 C NMR (500 MHz, CDC13) δ 12.6, 18.1, 19.4, 25.9, 28.8, 31.5, 41.1, 43.5, 51.0, 52.4, 54.1, 86.6, 117.7, 124.8, 132.5, 133.3, 138.6, 156.4, 159.0, 176.1; [a] D = +4.90 (c 1.02, CHCl3); HRMS (ESI), C 20 H 33 N4O5S + [M+H] + m / z: calcd 441.2166, found 441.2165.

[0104] [Chemical Formula 13]

[0105]

[0106] To a solution of compound 3 (6.81 g, 15.5 mmol) in CH2Cl2(155 mL) was added succinic anhydride (1.64 g, 16.3 mmol) and Et3N (2.38 mL, 17.1 mol) at room temperature. The reaction mixture was stirred at room temperature for 17 hours, then the mixture was concentrated under reduced pressure, purified by column chromatography on silica gel (CHCl3 / MeOH = 5 / 1) to obtain compound 4 ((S)-4-((1-methoxy-1-oxo-5-(3-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidino)pentan-2-yl)amino)-4-oxobutanoic acid) as a white powder (5.04 g, 9.32 mmol, yield 60%).

[0107] 1H NMR (400 MHz, CDC13) δ 1.46 (s, 7H), 1.58 (b, 2H), 1.68-1.75 (m, 1H), 1.89-1.91 (m, 1H), 2.09 (s, 3H), 2.44-2.48 (m, 4H), 2.55-2.66 (m, 6H) 2.78-2.85 (m, 1H), 2.95 (s, 2H), 3.18 (b, 2H), 3.73 (s, 3H), 4.60 (b, 1H), 6.36 (b, 2H), 6.91 (b, 1H); 13 C NMR (500 MHz, CDC13) δ 12.8, 18.2, 19.6, 25.1, 28.9, 29.8, 31.0, 41.0 (2C), 43.5, 52.0, 52.1, 52.2, 53.0, 86.9, 118.0, 125.1, 132.9, 138.9, 156.6, 159.4, 172.8, 173.1, 176.5; [a] D = +22.7 (c 1.02, CHCl3); HRMS (ESI), C 24 H 35 N4O8S - [M-H] - m / z: calcd 539.2181, found 539.2179.

[0108] [Formula 14]

[0109]

[0110] To a solution of compound 4 (540 mg, 1 mmol) in CHCl3(10 mL) was added HOBt-H2O (203 mg, 1.5 mmol), EDCI-H2O (288 mg, 1.5 mmol), m-PEG4-amine-HCl (288 mg, 1 mmol), and DIPEA (697 μL, 4 mmol) at 0 °C. The reaction mixture was stirred at room temperature overnight, then quenched with saturated aqueous NH4Cl, extracted with CH2Cl2, and the organic phase was concentrated to dryness over MgSO4. To the crude mixture in THF (10 mL) was added 1 M aqueous LiOH (2 mL) at room temperature. The reaction mixture was stirred at room temperature for 30 min, then the mixture was filtered, quenched with 1 N aqueous HC1, and extracted with CHCl3. The organic phase was dried over MgSO4and concentrated under reduced pressure to give compound 6 (N 2 -(18-oxo-2,5,8,11,14-pentaoxa-17-azahenicosan-21-oyl)-N ω-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-L-arginine).

[0111] [Chemical Formula 15]

[0112]

[0113] To a solution of compound 7 (49.7 mg, 127.8 μmol) in THF (1.3 mL) was added LiAlH4(16.0 mg, 421.7 μmol) at 0°C. The mixture was stirred at room temperature for 1.5 hours, then a saturated aqueous solution of potassium sodium tartrate was added at 0°C, and the mixture was alkalized with a 1 M aqueous NaOH solution (pH = 13-14). Extraction was performed with CHCl3, and the organic phase was dried over MgSO4and concentrated under reduced pressure. The crude mixture was dissolved in CH2Cl2(1 mL), and HOBt-H2O (10.4 mg, 71.1 μmol), EDCI-H2O (13.6 mg, 71.1 μmol), the above-mentioned compound 6 (49.1 mg), and DIPEA (22 μL, 129.2 μmol) were added at 0°C. The reaction mixture was stirred at room temperature for 16 hours, then the reaction was stopped by adding a saturated aqueous NaHCO3solution, extraction was performed with CHCl3, and the organic phase was dried over MgSO4. The crude product was dissolved in TFA (540 μL) / H2O (30 μL) at 0°C. The mixture was stirred at room temperature for 1 hour, then quenched with a saturated aqueous NaHCO3solution and extracted with CHCl3. The organic phase was dried over MgSO4. Concentration under reduced pressure and purification by HPLC gave the trifluoroacetate salt of compound 10 (N 1 -((2S)-1-((2-(4-(2-((4-chlorophenyl)amino)-2-oxoacetamido)-1- azaspiro[5.5]undec-1-yl)ethyl)amino)-5-guanidino-1-oxopentan-2-yl)-N 4 -(2,5,8,11,14-pentaoxa hexadecan-16-yl)succinamide 2TFA (TKB-001 trifluoroacetate salt)) as a colorless oil (34.3 mg, 30.9 μmol, 24% yield (3 steps)); t R = 11.7 min (linear gradient of B in A, 15 min 30-50%); HRMS (ESI), C 54 H 96 FN 10 O 17 + [M+H] + m / z calcd for C

[0114] [Example 2 Synthesis of a compound of the invention 2]

[0115] In the same manner as in Example 1, compounds having different PEG chain lengths were synthesized.

[0116] [Chemical Formula 16]

[0117]

[0118] To a solution of compound 4 (1.06 g, 1.97 mmol) in CHCl3(17.9 mL) was added HOBt-H2O (301.7 mg, 1.97 mmol), EDCI-H2O (0.378 g, 1.97 mmol), m-PEG11-amine (1.00 g, 1.79 mmol), and DIPEA (0.61 mL, 3.58 mmol) at 0°C. The reaction mixture was stirred at room temperature overnight, then quenched with saturated aqueous NH4Cl, extracted with CH2Cl2, and the organic phase was concentrated to dryness over MgSO4. To the crude mixture in THF (19.7 mL) was added 1 N aqueous LiOH (0.39 mL) at room temperature. The reaction mixture was stirred at room temperature for 30 minutes, then the mixture was filtered, quenched with 1 N aqueous HCl, and extracted with CHCl3. The organic phase was dried over MgSO4and concentrated under reduced pressure to give compound 12 (N 2 -(39-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-38-azatetratriacontane-42-oyl)-N ω -((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-L-arginine).

[0119] [Chemical Formula 17]

[0120]

[0121] In the same manner as in Example 1, the trifluoroacetate salt of the above compound 14 (N 1 -((2S)-1-((2-(4-(2-((4-chlorophenyl)amino)-2-oxoacetamido)-1-azaspiro[5.5]undec-1-yl)ethyl)amino)-5-guanidino-1-oxopentan-2-yl)-N 4 -(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)succinamide 2TFA (TKB-002 trifluoroacetate salt)) as a colorless oil.

[0122] t R= 15.3 min (linear gradient of B in A, 15 min 30-50%); HRMS (ESI), C 55 H 97 ClN9O 17 + [M+H] + m / z calcd 1190.6685, found 1190.6688.

[0123] [Example 3 Synthesis of compounds of the invention 3]

[0124] In the same manner as Example 1, compounds with different PEG chain length were synthesized.

[0125] [Formula 18]

[0126]

[0127] To a solution of compound 4 (49 mg, 91.9 μmol) in CH2Cl2(91 μL) was added m-PEG23-amine (100 mg, 91.9 μmol), EDCI-H2O (19.4 mg, 101 μmol), HOBt-H2O (13.7 mg, 101 μmmol), and DIPEA (32 μL, 184 μmol) at 0 °C. The reaction mixture was stirred at room temperature overnight, then quenched with saturated aqueous NH4Cl, extracted with CH2Cl2, and the organic phase was concentrated to dryness over MgSO4. To the crude mixture in THF (426 μL) was added IN LiOH aqueous solution (92.2 μL) at room temperature. The reaction mixture was stirred at room temperature for 30 min, then the mixture was filtered, quenched with aqueous NH4Cl, and extracted with CHCl3. The organic phase was concentrated to dryness over MgSO4to give compound 16 (N 2 -((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-L-arginine). ω -((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-L-arginine).

[0128] [Formula 19]

[0129]

[0130] In the same manner as Example 1, the trifluoroacetate salt of the above compound 18 was obtained (N 1- ((2S)-1-((2-(4-(2-((4-chlorophenyl)amino)-2-oxoacetamido)-1- azaspiro[5.5]undecan-1-yl)ethyl)amino)-5-guanidino-1-oxopentan-2-yl)-N 4 - (2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71- tetraoxaheptacontan-73-yl)succinamide 2TFA (TKB-003 trifluoroacetate salt) was a colorless oil.

[0131] t R = 16.8 min (linear gradient of B in A, 15 min 30-50%); HRMS (ESI), C 79 H 145 ClN9O 29 + [M + H] + m / z calcd 1718.9831, found 1718.9830.

[0132] [Example 4 Measurement of blood half-life of the compound of the present application]

[0133] In the same manner as in Reference Example 1, 23.4 μmol (33.2 mg / 20 mL) of the trifluoroacetate salt of TKB-002 obtained in Example 2 was intravenously injected into a cynomolgus monkey (MM616, body weight 8.78 kg), and blood was collected before injection, during administration, 10 minutes, 37 minutes, 1 hour, 2.5 hours, 4.5 hours, 6.5 hours, and 24 hours after administration, and the concentration in blood was measured by HPLC using the same conditions as in Reference Example 1.

[0134] As a result, the concentration in blood of the trifluoroacetate salt of TKB-002 was shifted as shown in Figure 2 and the blood half-life was calculated to be 107.9 minutes, which was confirmed to be 3.48 times as long as that of YIR-821.

[0135] [Example 5 Synthesis 4 of the compound of the present application]

[0136] The compound of the present application having an alkyl chain was synthesized by the following synthesis scheme.

[0137] [Chemical Formula 20]

[0138]

[0139] To a solution of compound 19 (2.4 g, 10 mmol) in DCM (50 mL) was added succinic anhydride (1.1 g, 11 mmol) and Et3N (2.77 mL, 20 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours, then concentrated under reduced pressure. The residue was crystallized from acetone to give compound 20 (4-(hexadecylamino)-4-oxobutanoic acid, 3.34 g, yield 98%) as a solid.

[0140] 1 H-NMR (500 MHz, MeOH) δ 0.80-0.83 (t, 3H), 1.20-1.22 (m, 26H), 1.39-1.42 (m, 2H), 2.35-2.37 (t, 2H), 2.48-2.50 (t, 2H), 3.05-3.08 (t, 2H); 13 C NMR (125 MHz, MeOH) δ 14.4, 23.7, 28.0, 30.4 (12C), 31.6, 33.1, 40.5, 174.4, 176.4; HRMS (ESI), C 20 H 38 NO3[M-H] - m / z calcd for C20H39N2O4 340.2857, found 340.2853.

[0141] [Chemical Formula 21]

[0142]

[0143] To a stirred solution of compound 20 (329 mg, 0.963 mmol) and N-hydroxysuccinimide (222 mg, 1.92 mmol) in DME (6.4 mL) and CH2Cl2(6.4 mL) was added EDCI-HCl (308 mg, 1.61 mmol) at room temperature. After 2 h, the reaction mixture was diluted with EtOAc and washed sequentially with water, 1% HC1, 5% aqueous NaHC03, H20, and brine. The organic phase was then dried (Na2S04), concentrated to give the activated ester. The intermediate was dissolved in MeCN (6.42 mL) and DMAP (588 mg, 4.81 mmol) and compound 22 (460 mg, 1.44 mmol) were added at room temperature. The reaction mixture was stirred for 15 h and then acidified to pH 2 with HC1 (0.1 M) at 0 °C. The solution was extracted with EtOAc and the extracts were combined and washed with H20 and brine. The mixture was dried (Na2S04), concentrated. The residue was purified by column chromatography (1% AcOH in CH3Cl / MeOH = 30 / 1) to give compound 23 (2-((4-(hexadecylamino)-4-oxobutanoyl)oxy)-5-((4-nitrophenyl)sulfonamido)pentanoic acid) (355 mg, 57% yield) as a solid.

[0144] 1 H-NMR (500 MHz, MeOH) δ 0.84-0.87 (t, 3H), 1.22 (m, 26H), 1.44-1.46 (m, 2H), 1.64-1.70 (m, 2H), 1.87-1.93 (m, 2H), 2.48-2.52 (m, 2H), 2.68-2.70 (m, 2H), 3.09-3.13 (m, 2H), 3.16-3.20 (m, 2H), 5.00-5.03 (m, 1H), 5.78 (br, 1H), 6.13 (br, 1H), 7.72-7.74 (m, 2H), 7.82-7.84 (m, 1H), 8.09-8.10 (m, 1H); 13 C NMR (125 MHz, CDC13) δ 14.0, 22.6, 26.8, 27.8, 29.2, 29.5 (12C), 30.9, 31.8, 39.8, 43.1, 71.6, 125.2, 128.2, 129.0, 130.9, 132.8, 133.6, 148.0, 171.8, 172.4; HRMS (ESI), C 31 H 52 N3O9S [M+H] + m / z calcd for 640.3273, found 640.3275.

[0145] [Chemical Formula 22]

[0146]

[0147] To a solution of compound 23 (543 mg, 0.846 mmol) in DMF (2.82 mL) was added HOBt-H20 (216 mg, 1.41 mmol), EDCI-HCl (270 mg, 1.41 mmol), compound 8 (111 mg, 0.282 mmol) and NEt3(0.273 mL, 1.97 mmol) at 0°C. The reaction mixture was stirred at room temperature for 12 hours, then quenched by adding saturated aqueous NaHC03solution at 0°C, extracted with CHC13. The organic phase was dried over Na2S04, then concentrated under reduced pressure, purified by column chromatography (CHC13 / MeOH = 10 / 1 ~ 6 / 1) to give compound 24 (1-((2-(4-(2-((4-chlorophenyl)amino)-2-oxoacetamido)-1- azaspiro[5.5]undecan-1-yl)ethyl)amino)-5-((4-nitrophenyl)sulfonamido)-1- oxopentan-2-yl 4-(hexadecylamino)-4-oxobutanoate) (116.5 mg, yield 41%) as a solid.

[0148] 1 H-NMR (500 MHz, MeOH) δ 0.84-0.87 (t, 3H), 1.10-1.14 (m, 3H), 1.23 (m, 28H), 1.34-1.65 (m, 12H), 1.88-1.93 (m, 4H), 2.13-2.34 (m, 3H), 2.53-2.80 (m, 6H), 2.94-3.00 (m, 2H), 3.09 (m, 2H), 3.14-3.37 (m, 4H), 3.93-3.98 (m, 1H), 5.20-5.22 (m, 1H), 5.70-5.84 (m, 2H), 7.29-7.31 (m, 2H), 7.57-7.60 (m, 2H), 7.71-7.73 (m, 2H), 7.82-7.84 (m, 1H), 8.08-8.11 (m, 1H), 9.36 (br, 1H); 13C NMR (125 MHz, CDC13) δ 14.1, 22.2, 22.6, 24.9, 25.1, 26.0, 26.9, 28.4, 28.6, 29.6 (12C), 30.9, 31.4, 31.8, 37.4, 37.7, 38.0, 39.8, 43.1, 44.5, 45.2, 46.1, 46.3, 56.5, 72.9, 120.9 (2C), 125.2 (2C), 128.1, 129.0, 131.0, 132.7, 133.5, 135.0, 148.0, 157.5, 158.9, 169.1, 171.0, 171.7; HRMS (ESI), C 51 H 79 ClN7O 10 S[M+H] + m / z calcd 1016.5292, found 1016.5292.

[0149] [Chemical Formula 23]

[0150]

[0151] To a solution of compound 24 (65.0 mg, 0.064 mmol) in MeCN (0.64 mL) was added PhSH (0.014 mL, 0.14 mmol) and i-Pr2NEt (0.023 mL, 0.14 mmol) at room temperature. The reaction mixture was stirred at room temperature for 43 h, then concentrated under reduced pressure, and purified by column chromatography (CHCl3 / MeOH = 10 / 1 ~ 6 / 1) to give compound 25 (5-amino-1-((2-(4-(2-((4-chlorophenyl)amino)-2-oxoacetamido)-1- azaspiro[5.5]undecan-1-yl)ethyl)amino)-1-oxopentan-2-yl 4-(hexadecylamino)-4- oxobutanoate) (38.5 mg, yield 72%) as a white powder.

[0152] 1 H-NMR (500 MHz, MeOH) δ 0.84-0.87 (t, 3H), 1.10-1.13 (m, 4H), 1.23 (m, 28H), 1.45-1.65 (m, 10H), 1.86-1.99 (m, 5H), 2.14-2.31 (m, 2H), 2.44-2.86 (m, 7H), 2.98-3.01 (m, 1H), 3.14-3.36 (m, 4H), 3.97 (m, 1H), 5.22 (m, 1H), 6.05 (br, 2H), 7.29-7.31 (m, 2H), 7.43 (m, 1H), 7.57-7.59 (m, 2H), 9.39 (br, 1H);13 C NMR (125 MHz, CDC13) δ 14.1, 22.2, 22.6, 24.0, 25.2, 26.0, 27.0, 28.7, 29.3, 29.7 (12C), 31.0, 31.3, 33.9, 37.2, 37.8, 39.8, 44.4, 45.3, 46.2, 46.5, 56.8, 70.5, 72.9, 120.9 (2C), 129.1 (2C), 130.2, 135.0, 157.6, 159.0, 169.5, 171.3, 171.7; HRMS (ESI), C 45 H 76 Cl N6O6 [M+H] + m / z calcd 831.5509, found 831.5505.

[0153] [Chemical Formula 24]

[0154]

[0155] To a solution of compound 25 (39.9 mg, 0.048 mmol) in DMF (0.96 mL) was added i-Pr2NEt (0.01 mL, 0.059 mmol) and 1H-pyrazole-1-carboxamidine hydrochloride (7.7 mg, 0.053 mmol) at room temperature. The reaction mixture was stirred at room temperature for 45 h, then concentrated under reduced pressure and purified by HPLC to give compound 26, 1-((2-(4-(2-((4-chlorophenyl)amino)-2-oxoacetamido)-1- azaspiro[5.5]undecan-1-yl)ethyl)amino)-5-guanidino-1-oxopentan-2-yl 4-(hexadecylamino)- 4-oxobutanoate, trifluoroacetate salt, as a white powder (36.7 mg, 88% yield). Compound 26 was named as trifluoroacetate salt of YIS-527. Compound YIS-527 is a compound with an alkyl chain, wherein R 1 is C 16 H 33 (R 1 represents C m H 2m+1 , m is 16), R 2 is O.

[0156] 1H-NMR (500 MHz, MeOH) δ 0.87-0.90 (t, 3H), 1.26 (m, 26H), 1.48 (m, 3H), 1.54-1.99 (m, 15H), 2.17-2.23 (m, 2H), 2.56-2.78 (m, 5H), 2.91-2.93 (m, 1H), 3.13-3.23 (m, 4H), 3.38 (m, 2H), 3.58-3.78 (m, 4H), 4.16 (m, 1H), 5.07 (m, 1H), 7.33-7.35 (m, 2H), 7.72-7.74 (m, 2H); 13 C NMR (125 MHz, CDC13) δ 14.4, 18.4, 22.4, 22.7, 23.7, 25.0, 25.7, 28.1, 29.7, 30.3 (12C), 31.0, 33.1, 34.9, 36.8, 37.8, 40.6, 41.8, 42.8, 49.8, 51.4, 58.3, 74.6, 79.5, 122.9 (2C), 129.9 (2C), 131.1, 137.4, 158.7, 159.4, 161.4, 162.3, 174.1, 175.1; HRMS (ESI), C 46 H 78 ClN8O6[M+H] + m / z calcd 873.5727, found 873.5724.

[0157] [Example 6 Synthesis of the compound of the present application 5]

[0158] In the same manner as Example 5, the compound of the present application having a PEG chain was synthesized.

[0159] [Formula 25]

[0160]

[0161] Compound 28 (2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate) was synthesized from compound 27 in the manner previously reported (Sheik, D.A. et al. J. Am. Chem. Soc. 9, 1829-1836 (2015)).

[0162] [Formula 26]

[0163]

[0164] Compound 29 (2,5,8,11,14-pentaoxahexadecan-16-ol) was synthesized from compound 28 following the previously reported procedure (Zhao, B. et al. Macromolecules. 38, 9509-9517 (2005)).

[0165] [Formula 27]

[0166]

[0167] Compound 30 (2,5,8,11,14-pentaoxahexadecan-16-yl 4-methylbenzenesulfonate) was synthesized from compound 29 following the previously reported procedure (Wolfe, AL. et al. J. Med. Chem. 56, 6845-6857 (2013)).

[0168] [Formula 28]

[0169]

[0170] To a stirred solution of compound 30 (813 mg, 2 mmol) in DMF (2 mL) was added NaN3(308 mg, 1.61 mmol) at room temperature. The reaction mixture was stirred at 80 °C for 11.5 h, then concentrated under reduced pressure. The precipitate was filtered to give compound 31 (16-azido-2,5,8,11,14-pentaoxahexadecane) (355 mg, yield 57%) as a solid.

[0171] 1 H-NMR (400 MHz, CDC13) δ 3.37-3.40 (m, 5H), 3.53-3.56 (m, 2H), 3.63-3.68 (m, 16H; 13 C NMR (125 MHz, CDC13) δ 50.7, 59.0, 70.0, 70.6 (7C), 71.9; HRMS (ESI), C 11 H 23 N3NaO5 [M + H] + m / z calcd 300.1530, found 300.1526.

[0172] [Formula 29]

[0173]

[0174] Compound 32 (2,5,8,11,14-pentaoxahexadecan-16-amine) was synthesized from compound 31 following the previously reported procedure (Wolfe, AL. et al. J. Med. Chem. 56, 6845-6857 (2013)).

[0175] [Chemical Formula 30]

[0176]

[0177] To a solution of compound 32 (2.4 g, 10 mmol) in DCM (50 mL) was added succinic anhydride (1.1 g, 11 mmol) and Et3N (2.77 mL, 20 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 hours, then concentrated under reduced pressure. The residue was crystallized from acetone to give compound 33 (18-oxo-2,5,8,11,14-pentaoxa-17-azaheneicosan-21-carboxylic acid) (3.34 g, yield 98%) as a yellow oil.

[0178] 1 H-NMR (400 MHz, CDC13) δ 2.53-2.56 (t, 2H), 2.65-2.68 (t, 2H), 3.37 (s, 3H), 3.43-3.46 (m, 2H), 3.53-3.70 (m, 18H); 13 C NMR (125 MHz, CDC13) δ 28.7, 30.9, 39.5, 58.9, 70.2, 70.4 (7C), 71.8, 172.6, 172.7; HRMS (ESI), C 15 H 30 N7O8[M+H] + m / z calcd for C26H45N7O8 552.3296, found 552.3297.

[0179] [Chemical Formula 31]

[0180]

[0181] To a stirred solution of compound 33 (1.05 g, 2.5 mmol) and N-hydroxysuccinimide (575 mg, 5 mmol) in DME (16.7 mL) and CH2Cl2(16.7 mL) was added EDCI-HCl (800 mg, 4.175 mmol) at room temperature. After 2 h, the reaction mixture was diluted with EtOAc and washed with water, 1% HC1, 5% aqueous NaHC03, H20, and brine. The organic phase was then dried (Na2S04), concentrated to give the activated ester. The intermediate was dissolved in MeCN (20 mL), and DMAP (1.83 g, 15 mmol) and compound 22 (1.43 g, 4.5 mmol) were added at room temperature. The reaction mixture was stirred for 12 h, then acidified to pH 2 with HC1 (0.1 M) at 0 °C. The solution was extracted with EtOAc, and the extracts were combined and washed with H20 and brine. The mixture was dried (Na2S04), concentrated. The residue was purified by column chromatography (1% AcOH in CH3Cl / MeOH = 30 / 1) to give compound 35 (2-((4-(hexadecylamino)-4-oxobutanoyl)oxy)-5-((4-nitrophenyl)sulfonamido)pentanoic acid) (36.3 mg, 2% yield) as a yellow oil.

[0182] 1 H-NMR (500 MHz, MeOH) δ 1.65-1.72 (m, 2H), 1.71-1.98 (m, 2H), 2.61-2.64 (m, 2H), 2.68-2.78 (m, 2H), 3.11-3.15 (m, 2H), 3.34 (s, 3H), 3.45-3.68 (m, 20H), 5.01-5.03 (m, 1H), 5.58 (br, 1H), 7.37 (br, 1H), 7.74-7.76 (m, 2H), 7.84-7.86 (m, 1H), 8.10-8.11 (m, 1H); 13 CNMR (125 MHz, CDC13) δ 25.3, 27.7, 29.8, 30.9, 39.8, 43.0, 58.7, 69.2, 69.7 (7C), 71.5, 71.9, 125.4, 131.0, 132.9, 133.4, 133.7, 148.0, 172.2, 172.4, 174.1; HRMS (ESI), C 26 H 42 N3O 14 S[M-H] - m / z calcd 650.2236, found 650.2233.

[0183] [Chemical Formula 32]

[0184]

[0185] To a solution of compound 35 (99.0 mg, 0.152 mmol) in DMF (0.51 mL) was added HOBt-H20 (38.8 mg, 0.25 mmol), EDCI-HCl (48.5 mg, 0.25 mmol), compound 8 (20 mg, 0.05 mmol) and NEt3(0.026 mL, 0.15 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 12 h, then quenched with 0 °C saturated aqueous NaHC03solution, extracted with CHC13. The organic phase was dried over Na2S04, then concentrated under reduced pressure, purified by column chromatography (CHC13 / MeOH = 10 / 1 ~ 6 / 1) to give compound 36 (1-((2-(4-(2-((4-chlorophenyl)amino)-2-oxoacetamido)-1- azaspiro[5.5]undecan-1-yl)ethyl)amino)-5-((4-nitrophenyl)sulfonamido)-1- oxopentan-2-yl 18-oxo-2,5,8,11,14-pentaoxa-17-azahenicosan-21-carboxylate) (21.3 mg, yield 41%) as yellow oil.

[0186] 1 H-NMR (500 MHz, MeOH) δ 1.12-1.43 (m, 9H), 1.60-1.73 (m, 7H), 1.86-1.92 (m, 4H), 2.22-2.36 (m, 2H), 2.58-2.80 (m, 7H), 3.05-3.10 (m, 4H), 3.36 (s, 3H), 3.46-3.72 (m, 20H), 3.95-3.99 (m, 1H), 5.21-5.22 (m, 1H), 7.31-7.32 (m, 2H), 7.57-7.59 (m, 2H), 7.71-7.73 (m, 2H), 7.81-7.84 (m, 1H), 8.10-8.11 (m, 1H), 9.32 (br, 1H); 13 C NMR (125 MHz, CDC13) δ 18.4, 22.1, 22.2, 25.0, 25.1, 25.9, 28.5, 29.6, 30.4, 30.6, 37.4, 39.4, 43.1, 44.3, 50.7, 58.4, 59.0, 69.7 (2C), 70.1 (7C), 70.4, 71.8, 72.8, 120.9 (2C), 125.2 (2C), 129.2, 130.3, 131.0, 132.6, 133.4, 133.6, 135.0, 148.0, 157.4, 159.0, 169.4, 171.4, 171.7; HRMS (ESI), C46 H 69 ClN7O 15 S[M+H] + m / z calculated 1026.4255, found 1026.4251.

[0187] [Chemical Formula 33]

[0188]

[0189] To a solution of compound 36 (21.3 mg, 0.021 mmol) in MeCN (0.21 mL) was added PhSH (0.005 mL, 0.05 mmol) and i-Pr2NEt (0.007 mL, 0.04 mmol) at room temperature. The reaction mixture was stirred at room temperature for 37 h, then concentrated under reduced pressure, purified by column chromatography (CHCl3 / MeOH = 10 / 1 ~ 6 / 1) to give compound 37 (5-amino-1-((2-(4-(2-((4-chlorophenyl)amino)-2-oxoacetamido)-1- azaspiro[5.5]undecan-1-yl)ethyl)amino)-1-oxopentan-2-yl 18-oxo-2,5,8,11,14- pentaaoxahexadocosan-21-carboxylate) (24.3 mg, yield 99%) as a yellow oil.

[0190] 1 H-NMR (500 MHz, MeOH) δ 1.11-1.33 (m, 11H), 1.53-1.72 (m, 7H), 1.83-2.01 (m, 4H), 2.18-2.31 (m, 2H), 2.56-3.02 (m, 9H), 3.26 (s, 3H), 3.53-3.65 (m, 20H), 3.95-3.97 (m, 1H), 4.43 (br, 2H), 5.30-5.32 (m, 1H), 7.31-7.33 (m, 2H), 7.57-7.59 (m, 2H), 9.30 (br, 1H); 13 CNMR (125 MHz, CDCl3) δ 18.4, 22.2, 22.3, 25.0, 25.2, 26.1, 28.6, 29.6, 30.2, 31.5, 37.3, 39.6, 44.6, 45.5, 46.6, 56.5, 58.9, 69.9 (2C), 70.2 (7C), 70.4, 71.6, 72.9, 120.9 (2C), 129.2 (2C), 130.3, 135.0, 157.5, 159.0, 169.5, 170.8, 172.6; HRMS (ESI), C 40 H 66 ClN6O 11[M+H] + m / z calcd 841.4473, found 841.4476.

[0191] [Chemical Formula 34]

[0192]

[0193] To a solution of compound 37 (24.3 mg, 0.029 mmol) in DMF (0.58 mL) was added i-Pr2NEt (0.06 mL, 0.035 mmol) and 1H-pyrazole-1-carboxamidine hydrochloride (4.6 mg, 0.032 mmol) at room temperature. The reaction mixture was stirred at room temperature for 18 hours, then concentrated under reduced pressure to give compound 38 (1-((2-(4-(2-((4-chlorophenyl)amino)-2-oxoacetamido)-1- azaspiro[5.5]undecan-1-yl)ethyl)amino)-5-guanidino-1-oxopentan-2-yl 18-oxo-2,5,8,11,14- penta-oxa-17-azahenicosan-21-carboxylate) trifluoroacetate as a yellow oil (24.1 mg, yield 45%) after purification by HPLC. Compound 38 was named as trifluoroacetate salt of YIS-540. Compound YIS-540 is a compound with PEG chain, wherein in General Formula (I) in the present specification, R 1 is C2H4(OC2H4) n -OCH3(R 1 represents -C2H4(OC2H4) n -OCH3, n is 4), R 2 is O.

[0194] 1 H-NMR (500 MHz, MeOH) δ 1.12-1.26 (m, 3H), 1.42-1.48 (m, 3H), 1.68-2.18 (m, 17H), 2.18-2.31 (m, 2H), 2.68-2.82 (m, 5H), 3.20 (br, 2H), 3.36-3.77 (m, 23H), 4.10 (m, 1H), 5.10 (m, 1H), 7.32-7.34 (m, 2H), 7.57-7.59 (m, 2H); 13C NMR (125 MHz, CDC13) δ 18.1, 21.2, 21.4, 24.5, 28.1, 28.5, 28.7, 29.3, 29.7, 30.1, 33.4, 35.1, 35.3, 39.3, 39.4, 40.5, 41.7, 50.7, 58.6, 69.7, 69.9, 70.0 (7C), 71.4, 72.9, 121.1 (2C), 129.3 (2C), 130.6, 134.8, 157.0, 159.6, 160.4, 172.2, 173.6; HRMS (ESI), C 41 H 68 ClN8O 11 [M+H] + m / z calcd 883.4691, found 883.4694.

[0195] [Example 7 Stability 1 of the compound of the present application]

[0196] Since the alkyl chain is bound to the side chain of YIR-821 via a -C(=O)O- group, it was assumed that the compound (YIS-527) obtained in Example 5 is hydrolyzed in vitro and in vivo. Therefore, in the present example, in vitro release of the alkyl chain was measured.

[0197] As a result, the following degradation reaction was observed in PBS at 37°C, pH 7.4. The results are shown in Table 1. Figure 3 .

[0198] [Formula 35]

[0199]

[0200] [Example 8 Stability 2 of the compound of the present application]

[0201] In the same manner as in Example 7, in vitro release of the PEG chain was measured for the compound (YIS-540) obtained in Example 6 in which the PEG chain is bound to the side chain of YIR-821 via a -C(=O)O- group.

[0202] As a result, the following degradation reaction was observed in PBS at 37°C, pH 7.4. The results are shown in Table 2. Figure 4 .

[0203] [Formula 36]

[0204]

[0205] [Example 9 Activity evaluation 1]

[0206] TKB-001, TKB-002, TKB-003 synthesized in Examples 1 to 3 were evaluated for cytotoxicity and anti-HIV activity together with efavirenz (EFV) which is one of antiviral drugs, NBD-556, and YIR-821. In the present example, TZM-bl cells (purchased from NIH AIDS Reagent Program (https: / / www.aidsreagent.org / Index.cfm)) were used for any evaluation. TZM-bl cells are indicator cells having CD4 / CCR5 / CXCR4 on the surface and luciferase expression by HIV infection. In the cells, β-galactosidase gene linked to HIV-1 LTR sequence is integrated, and when HIV-1 is infected, transcriptional activator Tat is expressed from the tat gene of the infected HIV-1, and by the action of the Tat on the promoter region of the LTR, β-galactosidase is configured to be expressed. By adding a substrate thereto, enzyme cleavage occurs, galactose and luciferin are generated, and thus, by detecting the amount of chemiluminescence emitted by oxidation of luciferin by a luminometer, the number of HIV-1 infected cells can be measured.

[0207] 1) CC 50 (Calculation of cytotoxicity)

[0208] Each compound was stepwise diluted, and then 1 x 10 4 TZM-bl cells were added, and after incubation for 48 hours, Luc reporter reagent was added, and the amount of luminescence was measured (reagent name CTG), and CC 50 value was calculated.

[0209] 2) IC 50 (Calculation of anti-HIV activity)

[0210] Each compound was stepwise diluted, and then 1 x 10 4 TZM-bl cells were added, and 100 TCID 50 of HIV isolate (KP5mvcR) was added, and after incubation for 48 hours, the cells were recovered, Luc reporter reagent was added, and the amount of luminescence was measured, and the infection inhibition rate (IC 50 ) was calculated. HIV isolate (KP5mvcR) is an MVC-resistant HIV clone, which was made by placing MVC-resistant KP-5 Env in the vector of NL43, which was obtained by 48 passages of clinical isolate KP-5 (YTA) in PM1 CCR5 cells in the presence of MVC (Yoshimura K, et al., J. Gen. Virol., 95, 1816-1826, 2014).

[0211] The results (CC50, IC50, and SI (Selectivity index)) of each compound are shown in Table 1 below.

[0212] [Table 1]

[0213]

[0214] As can be seen from the results of Table 1, the compounds TKB-001, TKB-002, and TKB-003 of the present application were all confirmed to have low cytotoxicity and high anti-HIV activity, and thus have the same effectiveness as existing antiviral drugs. In addition, the results of SI indicate that TKB-002 has particularly high safety and good effects.

[0215] [Example 10 Activity Evaluation 2]

[0216] For YIS-527 and YIS-540 synthesized in Examples 5 and 6, cytotoxicity and anti-HIV activity were evaluated in the same manner as in Example 9.

[0217] The results of the activity evaluation of YIS-527 and YIS-540 are shown in Table 2 below together with the results of YIR-821 as a control compound.

[0218] [Table 2]

[0219]

[0220] As can be seen from the results of Table 2, the compounds YIS-527 and YIS-540 of the present application were all confirmed to have low cytotoxicity to the same extent as YIR-821, and high anti-HIV activity.

[0221] [Example 11 Stability Evaluation]

[0222] For TKB-002 synthesized in Example 2, a biological stability evaluation was performed.

[0223] Specifically, the trifluoroacetate salt of TKB-002 was dissolved in 100 mM sodium phosphate buffer (NaPB, pH 7.4) to make a 0.3 mM solution. 183 μL of this solution was mixed with 2 μL of 20 mM NADPH and 5 μL of 20 mg / mL human microsomes (50 Donors (Thermo Fisher Scientific, cat#: HMMCPL, Lot#: PL050E-A)) in 100 mM NaPB (pH 7.4). The mixture was pre-incubated at 37°C for 5 minutes, and then 10 μL of a 20 mM NADPH solution was added thereto, and incubated at 37°C for 60 minutes with gentle stirring.

[0224] Then, the reaction was stopped by adding 200 μL of ethyl acetate, and stirred with a vortex mixer, and centrifuged at 3,000 rpm for 5 minutes. The supernatant was filtered, and the ethyl acetate was removed with nitrogen.

[0225] To this mixture, 50 μL of MeCN containing 0.1% TFA was added, and analyzed by HPLC using the same conditions as in Reference Example 1, Example 4.

[0226] As a result, TKB-002 was detected as a single peak at a position that remained for about 20 minutes under the HPLC conditions used, but showed the same peak intensity as before incubation after incubation with human liver microsomes for 60 minutes, indicating that it is very stable in the presence of human liver microsomes.

[0227] Industrial applicability

[0228] The CD4 mimetic compound of the present application can be provided as a compound having an anti-HIV activity equal to or more than the anti-viral drugs reported previously and the CD4 mimetic compounds developed previously by the present inventors, and having low cytotoxicity and a long half-life in vitro and in vivo, and thus can be expected to have a longer action duration, and thus can reduce the cost of treatment.

[0229] All publications, patents and patent applications cited in this specification are directly incorporated by reference into this specification.

Claims

1. A compound represented by the following general formula (I) or a salt thereof, wherein R 1 represents C2H4(OC2H4) n -OCH3, or C m H 2m+1 , R 2 represents O or NH, n is 3 to 25, and m is 4 to 22.

2. The compound or a salt thereof according to claim 1, represented by the following formula, in the formula, n is 3 to 25.

3. The compound or a salt thereof according to claim 2, n is 4 to 23.

4. An HIV infection inhibitor, containing the compound or a salt thereof according to any one of claims 1 to 3 as an effective ingredient.

5. A pharmaceutical composition for treating or preventing HIV infection, containing the HIV infection inhibitor according to claim 4.

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