Sulfonated calix [4] arene derivative and application thereof in resisting I-type herpes simplex virus

Abstract

The invention discloses a sulfonated calix [4] arene derivative and application thereof in resisting I-type herpes simplex virus (HSV-1), and belongs to the field of biological medicine. The invention relates to a sulfonated calix [4] arene derivative, which has a structural general formula as follows: R is equal to The preparation method comprises the following steps: adding sodium hydride into a raw material C4A in a polar aprotic solvent to react with alkyl halide to obtain a C4AH or C4AD intermediate; and reacting the intermediate with chlorosulfonic acid in a chlorine-containing solvent, and adding an alkaline alcohol solvent for treatment to obtain the sulfonated calix [4] arene derivative. The calix [4] arene derivative has a relatively good inhibition rate on expression of HSV-1gB and gD genes, and does not have obvious cytotoxicity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for preparing sulfonated calix[4]arene and its application in resisting herpes simplex virus type 1 (HSV-1). Background Art

[0002] Herpes simplex virus type 1 (HSV) is a group of enveloped DNA viruses that primarily infect the skin, mucous membranes, and nervous tissue. The existing antiviral ointment, acyclovir, has a short half-life, which can cause some patients to experience localized side effects such as itching and burning pain. Long-term use can also cause the virus to mutate into drug-resistant strains. To address this issue, the design and synthesis of novel anti-herpes virus compounds and their development for the prevention and treatment of herpes viruses are of great significance.

[0003] The glycoproteins that constitute the viral attachment ligand (VAL) can interact with heparan sulfate proteoglycans (HSPGs) expressed on the surface of almost all eukaryotic cells, thereby initiating the first step of viral invasion of cells. For example, glycoprotein B (gB) and glycoprotein D (gD) on the HSV-1 envelope capsid are responsible for viral attachment and fusion with the cytoplasmic membrane. Therefore, simulating the sulfated groups of heparan sulfate (HS) on the cell surface can block the attachment of gB protein to cells and reduce the infectivity of HSV-1. Currently, a variety of HSPG mimetics with anti-HSV-1 properties have been developed. For example, after modifying the sulfonate groups on the surface of gold nanoparticles through Au-S bonds, the activity of HSV-1 can be significantly inhibited in vitro (half inhibitory concentration IC 50 =36.3nM) and showed a broad-spectrum inhibitory effect on multiple viruses. However, due to the unclear clearance mechanism of gold nanoparticles in the human body and possible long-term toxicity, their use as drugs is still limited. In order to avoid potential toxicity risks and reduce the IC 50 A feasible approach is to introduce multiple sulfate groups into the biocompatible backbone to improve the affinity of the compound for HSV-1 while reducing drug toxicity. For example, cyclodextrin modified with mercapto undecyl sulfonate showed excellent biocompatibility and micromolar concentration (half inhibitory concentration IC 50 =15.3μM) of anti-HSV-1 activity. However, the high half-inhibitory concentration hinders their potential clinical translation. Therefore, it is urgent to develop drugs with low IC 50 and drugs with low toxicity risks to cope with the ever-changing drug-resistant strains and enhance the possibility of potential clinical application.

[0004] Calixarenes represent the third generation of supramolecular hosts and are macrocycles composed of phenolic units linked by methylene groups at the 2- and 6-positions. Calixarenes have been extensively studied for biomedical applications in in vitro diagnostics (biosensing), in vivo diagnostics (bioimaging), and therapeutics (drug delivery, gene transfection, therapeutic agents). Most calixarene derivatives are nontoxic, and sulfonated calixarenes, in particular, have been shown to be non-hemolytic at concentrations up to 200 mM, paving the way for their in vivo biological applications.

[0005] In summary, using calixarene as a macrocyclic molecular scaffold and modifying its upper rim with polyvalent sulfonic acid groups can closely mimic HSPGs on the cell surface, thereby blocking HSV-1 invasion. The alkyl chains on the lower rim enhance the amphiphilicity of the calixarene and stabilize its conformation, further strengthening its affinity for VAL. Furthermore, the excellent biocompatibility of calixarene renders it non-toxic, making it a potential drug for the treatment and prevention of HSV-1. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a 50 The invention discloses a method for preparing sulfonated calixarene derivatives with low toxicity risk and anti-HSV-1 properties. Such compounds have a good inhibition rate on the expression of HSV-1gB and gD genes and do not have obvious cytotoxicity.

[0007] The sulfonated calix[4]arene derivative of the present invention has the general structural formula:

[0008]

[0009] SC4AH: R=CH2CH2CH2CH2CH2CH2;

[0010] SC4AD: R=CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2.

[0011] The preparation method of the aforementioned sulfonated calix[4]arene derivative of the present invention is represented by the following reaction equation:

[0012]

[0013] R=CH2CH2CH2CH2CH2CH2 or CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2.

[0014] The method comprises the following steps: adding sodium hydride and alkyl halide to compound C4A in a polar aprotic solvent to react to obtain a C4AH or C4AD intermediate; then reacting the intermediate with sulfuric acid or chlorosulfonic acid in a chlorine-containing solvent, and then adding an alkaline alcohol solvent for treatment to obtain a sulfonated calix[4]arene derivative.

[0015] Furthermore, in the above technical solution, the polar aprotic solvent is selected from DMSO or DMF.

[0016] Furthermore, in the above technical solution, the halogenated hydrocarbon is selected from 1-bromohexane or 1-bromododecane.

[0017] Furthermore, in the above technical solution, the chlorine-containing solvent is selected from dichloromethane, chloroform or 1,2-dichloroethane.

[0018] Furthermore, in the above technical solution, the base is selected from sodium hydroxide or sodium carbonate.

[0019] The invention provides an application of the sulfonated calix[4]arene derivative in the preparation of drugs for treating or preventing antiviral infections.

[0020] Furthermore, in the above technical solution, the virus is HSV-1.

[0021] The present invention also provides a pharmaceutical composition for treating or preventing viral infection, wherein the active ingredient comprises the sulfonated calix[4]arene derivative. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the results of the inhibition test of compound SC4A on HSV-1 gB and gD protein gene expression;

[0023] Figure 2 The figure shows the results of the inhibition test of compound SC4AH on the expression of HSV-1 gB and gD protein genes;

[0024] Figure 3 This is a graph showing the results of the inhibition test of compound SC4AD on the expression of HSV-1 gB and gD protein genes.

[0025] Figure 4 The IC values ​​of compound SC4AD for inhibiting HSV-1 gB and gD protein gene expression are shown in Table 1. 50 picture. DETAILED DESCRIPTION

[0026] The above contents of the present invention are further described in detail below through examples, but this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0027] Example 1

[0028] Preparation of Sulfonated Calix[4]arene (SC4A)

[0029]

[0030] 0.97g of C4A was added to 30mL of concentrated sulfuric acid at 80°C for 4h. A small amount of the reaction mixture was tested for water solubility. If no insoluble matter was found, the reaction was stopped and cooled to room temperature. The cooled reaction mixture was added to 400mL of rapidly stirring anhydrous ether, stirred overnight, and filtered. The resulting solid was prepared into a hydrothermal saturated solution. A sodium hydroxide / ethanol solution was added to precipitate the solid. After centrifugation, the supernatant was discarded and the solid was dried. The process was repeated to obtain 1.76g of the pure product with a yield of 92.5%. 1 H NMR (400MHz, D2O) δ7.46 (s, 8H), 3.91 (s, 8H).

[0031] Example 2

[0032] (1) Preparation of calix[4]arene (C4AH)

[0033]

[0034] To a round-bottom flask, add 25 mL of anhydrous DMF and 0.42 g of sodium hydride. Once bubbles cease in the reaction flask, add 480 mg of C4A and react at room temperature for 2 hours. Slowly add 2 mL of 1-bromohexane and continue the reaction at room temperature for 2 hours. Warm to 90°C, then control the reaction temperature for 8 hours. Cool to room temperature to obtain a yellow suspension. Filter, and the filtrate is spin-dried. Column chromatography (dichloromethane / petroleum ether) yields 661 mg of the product, C4AH, in a 76.8% yield. 1 HNMR (400MHz, CDCl3) δ6.63-6.52(m,12H),4.44(d,J=13.3Hz,4H),3.92-3.83(m,8H),3.1 4(d,J=13.4Hz,4H),1.95-1.85(m,8H),1.45-1.31(m,24H),0.92(dd,J=9.2,4.8Hz,12H).

[0035] (2) Preparation of sulfonated calix[4]arene (SC4AH)

[0036]

[0037] 50mL anhydrous trichloromethane and 2.0g C4AH are added into round-bottomed flask.Reaction flask is slowly added to the trichloromethane solution of chlorosulfonic acid (0.5mL chlorosulfonic acid is dissolved in 60mL anhydrous trichloromethane) in constant pressure dropping funnel in ice bath.After being added dropwise to complete, remove ice bath, and room temperature reaction 3h.Spin-dried for solvent, add and contain sodium hydroxide 360mg absolute ethanol solution 100mL, stir.Treat that solid is separated out completely after centrifugation, solid is mixed with water-heat saturated solution and adds ethanol to separate out solid, abandon supernatant oven dry solid after centrifugation, repeat operation and obtain product 265mg, and yield is 8%. 1 H NMR (400MHz, DMSO-d6) δ7.07(s,8H),4.31(s,4H),3.87(s,8H),3.22(s,4H),1.95–1.89(m,8H),1.36(s,24H),0.90(s,12H).

[0038] Example 3

[0039] (1) Preparation of calix[4]arene (C4AD)

[0040]

[0041] To a round-bottom flask, add 25 mL of anhydrous DMF and 0.42 g of sodium hydride. Once bubbles cease in the reaction flask, add 500 mg of C4A and react at room temperature for 1 hour. Slowly add 2 mL of 1-bromododecane and continue the reaction at room temperature for 2 hours. Heat to 90°C, then control the reaction temperature for 8 hours. Cool to room temperature to obtain a yellow suspension. Continue the reaction at room temperature for 2 hours. Heat to 90°C, then control the reaction temperature for 8 hours. Cool to room temperature to obtain a yellow suspension. Filter, and the filtrate is spin-dried. Column chromatography (dichloromethane / petroleum ether) yields 1.040 g of the product in an 80.2% yield. 1 HNMR(400MHz, CDCl3) δ6.57(dt,J=8.7,6.0Hz,12H),4.44(d,J=13.3Hz,4H),3.87(t,J=7.4Hz,8 H), 3.14 (d, J = 13.4Hz, 4H), 1.97-1.85 (m, 8H), 1.32 (d, J = 38.8Hz, 72H), 0.88 (t, J = 6.8Hz, 12H).

[0042] (2) Preparation of sulfonated calix[4]arene (SC4AD)

[0043] 50mL anhydrous chloroform and 2.0g C4AD are added into a round-bottomed flask. Reaction flask is slowly added to the chloroform solution of chlorosulfonic acid (0.5mL chlorosulfonic acid is dissolved in 60mL anhydrous chloroform) by constant pressure dropping funnel in an ice bath. After being added dropwise to complete, ice bath is removed and room temperature reaction is carried out for 3h. The solvent is spin-dried and added to an absolute ethanol solution 100mL containing sodium hydroxide 360mg / , stirring. After solid is separated out completely, centrifugation is carried out, solid is mixed with water-heat saturated solution and ethanol is added to separate out solid. After centrifugation, supernatant is abandoned and dried solid is obtained. Repeating operation obtains product 1.577g, yield 58.1%.

[0044]

[0045] 1 H NMR (400MHz, DMSO-d6) δ7.07 (s, 8H), 4.32 (d, J = 12.7Hz, 4H), 3.86 (t, J = 7.3Hz, 8H), 3.21 (d, J = 12.3Hz, 4H), 1.91 (s, 8H), 1.24 (s, 72H), 0.84 (t, J = 6.7Hz, 12H).

[0046] Example 4

[0047] In vitro anti-HSV-1 activity test

[0048] (1) Amplification of HSV-1

[0049] Vero cells, freshly revived and passaged 2-3 times, were transferred to 150 mm culture dishes. After cells had uniformly grown into a monolayer, the culture medium was aspirated and washed twice with PBS. 500 μL of HSV-1 viral stock solution was diluted to 7.5 mL with DMEM without FBS and added to the culture dish. Infection was allowed to proceed for 2 hours in a cell culture incubator. After infection, the old culture medium was discarded and 12 mL of DMEM with 2% FBS was added to continue incubation for 48-72 hours. After the cells developed extensive lesions, the culture dish was frozen and thawed once between -80°C and 37°C. Centrifuge in a pre-cooled centrifuge at 4°C, 4000 rpm, for 6 minutes. The supernatant was collected and aliquoted. The newly amplified viral fluid was stored at -80°C until further use.

[0050] (2) TCID of HSV-1 50 Determination

[0051] Vero cells were seeded on 96-well plates. After the cells grew into a monolayer, the culture medium was removed and the cells were washed twice with PBS. The virus stock solution was diluted to 10 -3 to 10 -11100 μL was added to each well. After inoculation of virus at 37°C and 5% CO2 for 2 h, the virus solution was discarded, the cells were washed twice with PBS, and DMEM medium containing 2% FBS was added to continue the culture. The lesion holes were observed and recorded every day. When no lesion holes appeared, the TCID was calculated according to the Reed-Muench method. 50 .

[0052] (3) Compound administration method

[0053] Vero cells were cultured at 1.5 × 10 5 100 cells / well were inoculated in a 24-well plate. After the cells grew into a monolayer, the old culture medium was discarded and washed once with PBS. A blank group, a virus group, and a drug group were set up, with 3 replicates in each group. The compound was diluted to the corresponding concentration with DMEM containing 2% FBS, added to the cells and placed in an incubator. After the compound was fully in contact with the cells for 4 hours, additional compounds of the corresponding concentration were prepared and diluted with 10 4 HSV-1 was cultured in a cell culture incubator for 2 h. After the previous step, the old medium containing the compound was discarded, and the cells were washed twice with PBS. The compound and virus incubation medium were added to the cells for full contact for 4 h. The old medium was then discarded, the cells were washed twice with PBS, and the compound was diluted to the corresponding concentration with DMEM containing 2% FBS and added to the cells to continue culturing for 24 h.

[0054] (4) qPCR detection of the inhibition of HSV-1 gB and gD gene expression by compounds

[0055] RNA extraction: Follow the steps in the RNA extraction kit. Aspirate the old medium from the 24-well plate cultured for 24 hours and wash twice with PBS. Add 300 μL of Buffer CRL to each well and lyse the cells. Transfer all lysates to RNA Columns I and centrifuge at 12,000 rpm for 30 seconds. Discard the waste liquid. Add 500 μL of Buffer RWA to RNA Columns I and centrifuge at 12,000 rpm for 30 seconds. Discard the waste liquid. Add 500 μL of Buffer RW to RNA Columns I and centrifuge at 12,000 rpm for 1 minute. Discard the waste liquid. Return RNA Columns I to the collection tube and centrifuge at 12,000 rpm for 1 minute to prevent alcohol contamination. Carefully transfer RNA Columns I to a new 1.5 ml RNase-free centrifuge tube (self-provided). Add 30 μL of RNase-free ddH2O to the center of the adsorption column membrane. Incubate at room temperature for 1 minute. Centrifuge at 12,000 rpm for 1 minute to elute the RNA. RNA concentration was determined using an ultra-micro-volume UV spectrophotometer.

[0056] Reverse transcription: The reverse transcription kit from Nanjing Novozymes was used and the reaction was performed on ice.

[0057] Table 1 Reverse transcription system

[0058]

[0059] Reverse transcription was performed using a PCR instrument at 50°C for 5 min and 85°C for 5 s.

[0060] qPCR process: Use the Nanjing Novozymes SYBR qPCR Master Mix kit and prepare the qPCR reaction system on an ice box according to Table 2.

[0061] Table 2 qRT-PCR system

[0062]

[0063] After the system is fully mixed, add it to a 384-well PCR plate, and perform three replicates for each sample. Perform qPCR reaction at 95°C, 30s, 1 cycle; 95°C, 10s; 60°C, 30s, 40 cycles. -ΔΔCt The relative expression level of the target gene was calculated by this method.

[0064] Table 3 Primer sequences used

[0065]

[0066] Table 4 Number, chemical structure and yield of typical compounds

[0067]

[0068] Table 5 Inhibition of HSV-1 gB and gD protein gene expression by compounds

[0069]

[0070] Table 5 shows the results of the typical compound inhibition test on HSV-1 gB and gD protein gene expression. It can be seen from the table that SC4AD has a significant inhibitory effect on the expression of HSV-1 gB and gD protein genes.

[0071] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A sulfonated calix[4]arene derivative, characterized in that Its general structural formula is: SC4AH: R=CH2CH2CH2CH2CH2CH2; SC4AD: R=CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2.

2. Use of the sulfonated calix[4]arene derivative according to claim 1 in the preparation of drugs for treating or preventing antiviral infections.

3. Use of the sulfonated calix[4]arene derivative according to claim 2 in the preparation of a drug for treating or preventing antiviral infection, wherein the virus is HSV-1.

4. A pharmaceutical composition for treating or preventing viral infection, wherein the active ingredient comprises the sulfonated calix[4]arene derivative according to claim 1.

5. The pharmaceutical composition for treating or preventing antiviral infection according to claim 4, wherein the virus is HSV-1.

6. The method for preparing a sulfonated calix[4]arene derivative according to claim 1, wherein: The steps include: Compound C4A is treated in a polar aprotic solvent with sodium hydride to remove protons, and then an alkyl halide is added to react to obtain a C4AH or C4AD intermediate; the intermediate is then reacted with sulfuric acid or chlorosulfonic acid in a chlorine-containing solvent, and then treated with an alkaline alcohol solvent to obtain a sulfonated calix[4]arene derivative; Among them: R=CH2CH2CH2CH2CH2CH2 or CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2.

7. The method for preparing a sulfonated calix[4]arene derivative according to claim 6, characterized in that: The polar aprotic solvent is selected from DMSO or DMF.

8. The method for preparing a sulfonated calix[4]arene derivative according to claim 6, characterized in that: The halogenated hydrocarbon is selected from 1-bromohexane or 1-bromododecane.

9. The method for preparing a sulfonated calix[4]arene derivative according to claim 6, characterized in that: The chlorinated solvent is selected from dichloromethane, chloroform or 1,2-dichloroethane.

10. The method for preparing a sulfonated calix[4]arene derivative according to claim 6, characterized in that: The base is selected from sodium hydroxide or sodium carbonate.

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