CN116003258A - 12-O-octanoyl-phorbol ester derivative and preparation method and application thereof - Google Patents

Abstract

The embodiment of the invention discloses a 12-O-octanoyl-phorbol ester derivative, a preparation method and application thereof. The 12-O-octanoyl-phorbol ester derivative comprises a compound shown in a general formula (I) and pharmaceutically acceptable salts. The compound shown in the general formula (I) provided by the invention has obvious anti-HIV-1 activity, and the Therapeutic Index (TI) is in the range of 2125.86-73549.55. Can be used as medicine for resisting HIV-1.

Description

12-O-octanoyl-phorbol ester derivative and preparation method and application thereof

Technical Field

The embodiment of the invention relates to the technical field of medicines, in particular to a 12-O-octanoyl-phorbol ester derivative, a preparation method and application thereof.

Background

Human immunodeficiency virus (Human immunodeficiency virus, HIV) is the causative agent of acquired immunodeficiency syndrome (Acquired immunodeficiency syndrome, AIDS). HIV was first discovered from 1981 to date, and aids has become one of the most serious diseases experienced by humans. HIV belongs to the group of human lentiviruses in the genus lentivirus of the family retrovirus, and is classified as type 1 and type 2. HIV-1 is currently predominantly prevalent worldwide. HIV-1 infected patients are clinically primarily dependent on antiretroviral therapy (antiretroviral therapy, ART). Since the first anti-HIV drug zidovudine (AZT) was introduced in 1987, humans have been continually explored in the field of anti-HIV-1 research, leading to the development of a life-long controllable chronic disease from the absolute disease of AIDS.

The current antiretroviral therapy can effectively inhibit viral replication, greatly reduce the mortality of aids patients, prolong the life span of patients and improve the quality of life thereof. However, upon interruption of ART, viral replication bounces. Latent viral reservoirs become a major obstacle to clearance of HIV-1. "activation and killing" is a strategy for curing HIV-1 by activating the HIV-1 virus in a latent state with an activator followed by death of cells activated and expressing viral proteins by virus-mediated cytopathic or immune-mediated clearance in combination with ART [ Mbonye U and Karn J.the Molecular Basis for Human Immunodeficiency Virus Latency. Annu Rev Virol,2017,4 (1): 261-285.Kim Y,Anderson J L and Lewin S R.Getting the"Kill"into"Shock and Kill": strategies to Eliminate Latent HIV. Cell Host Microbe,2018,23 (1): 14-26 ].

The existing anti-HIV-1 medicines can not thoroughly clear the HIV-1in a patient, and side effects and drug resistance problems caused by long-term use are increasingly prominent. Therefore, the development of new anti-HIV-1 drugs with new targets is urgent. Natural products are one of the important sources of new drugs and drug lead compounds. The natural products with anti-HIV activity are obtained from plants, microorganisms and marine organisms, which has positive significance in searching safe and effective anti-AIDS drugs and exploring and developing new therapeutic targets. Studies show that phorbol ester compounds have dual functions. During the acute infection phase, HIV-1 replication can be inhibited. On the other hand, the compounds can be used as latent activators to activate the expression of HIV-1in latent cells [ Kulkosky J, culnan D M, roman J, et al Prostratin: activation of latent HIV-1expression suggests a potential inductive adjuvant therapy for HAART.Blood,2001,98 (10): 3006-3015. Dela Torre-Tarazona H E, jimenez R, bueno P, et al.4-Deoxyphorbol inhibits HIV-1infection in synergism with antiretroviral drugs and reactivates viral reservoirs through PKC/MEK activation synergizing with vorinostat.Biochemical Pharmacology,2020,177:113937 ]. To date, no report exists in the prior art on the preparation method and application of 12-O-octanoyl-phorbol ester derivatives.

Disclosure of Invention

Therefore, the embodiment of the invention provides a 12-O-octanoyl-phorbol ester derivative, a preparation method and application.

In order to achieve the above object, the embodiment of the present invention provides the following technical solutions:

according to a first aspect of the embodiment of the invention, the invention provides a 12-O-octanoyl-phorbol ester derivative, which comprises a compound shown in a general formula (I) and pharmaceutically acceptable salts;

according to a second aspect of embodiments of the present invention, there is provided a process for the preparation of 12-O-octanoyl-phorbol ester derivatives as described above,

(1) The preparation routes of the compounds HPB-5074 and HPB-507A are as follows:

the preparation method of the compounds HPB-5075 and HPB-5076 comprises the following preparation routes:

further, the preparation method of the 12-O-octanoyl-phorbol ester derivatives comprises the following steps:

preparation of Compound 1 by chemoselective triphenylmethyl protection of 20-OH starting from Phorbol and isolation of the product;

condensing compound 1 with n-octanoic acid to form an ester to produce compound 2 and isolating the product;

compound 2 was prepared by selective removal of 13-OH ester groups at weak basicity (ph=8.5) to prepare compound 3 and isolation of the product;

removing Tr protecting group from the compound 3 under the action of perchloric acid to prepare a compound 4 and separating a product;

compound 4 Compound HPB-507A is prepared by chemoselective acetylation of 20-OH and isolation of the product;

compound 3 compound 5 was prepared by 13-OH acetylation and the product isolated;

removing Tr protecting group from the compound 5 under the action of perchloric acid to prepare a compound HPB-5074 and separating a product;

removing Tr protecting group from the compound 2 under the action of perchloric acid to prepare a compound HPB-5075 and separating a product;

compound HPB-5075 Compound HPB-5076 was prepared by chemoselective acetylation of 20-OH and isolation of the product.

According to a third aspect of embodiments of the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of a 12-O-octanoyl-phorbol ester derivative as described above and a pharmaceutically acceptable carrier and/or excipient.

Further, the weight percentage of the 12-O-octanoyl-phorbol ester derivatives is 0.1-99.5%, preferably 0.5-90%.

The carrier and/or excipient is pharmaceutically acceptable, non-toxic and inert to humans and animals. It will be appreciated that the pharmaceutically acceptable carriers and/or excipients are one or more solid, semi-solid and liquid diluents, fillers and pharmaceutical formulation adjuvants. The pharmaceutical composition of the invention is prepared into various dosage forms, such as liquid preparations (injection) and solid preparations (tablets and capsules) by adopting a method accepted in the pharmaceutical and food fields. The medicine of the present invention may be used in treating AIDS via intravenous injection, intravenous drip, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. or through oral administration.

According to a fourth aspect of the embodiment of the invention, the invention provides an application of the 12-O-octanoyl-phorbol ester derivatives in preparing anti-AIDS drugs.

According to a fifth aspect of embodiments of the present invention, the present invention provides the use of a pharmaceutical composition as described above for the preparation of an anti-aids drug.

The embodiment of the invention has the following advantages:

in the process of researching natural product anti-HIV-1, the invention discovers that the compound of the formula (I) inhibits the replication of HIV-1 virus in nanomolar concentration and has obvious anti-HIV-1 virus activity; can activate the expression of latent HIV-1 virus in cell line, and the activation effect is stronger than that of control compound Prostratin or equal to PMA. Can be used as medicine for resisting HIV-1.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.

FIG. 1 shows the evaluation of the cytotoxic activity of compound HPB-507A and compound HPB-5074 against C8166 cell line.

FIG. 2 shows HIV-1 virus inhibition rates of HPB-5074, HPB-507A, HPB-5075, HPB-5076 at different concentration gradients.

FIG. 3 shows the reactivation effect of HPB-5074 on latent HIV-1; FIG. 3A and FIG. 3B are the percent changes in the flow test for GFP positive cells at different concentrations of HPB-5074 (1 nM, 10nM, 100 nM) and PMA (10 nM) for J-Lat C1124 hours, 48 hours, respectively, and FIG. 3C is the percent changes in the flow test for GFP positive cells at different concentrations of HPB-5074 (1 nM, 10nM, 100 nM), PMA (10 nM) and Prostratin (1. Mu.M) for J-Lat A2 hours.

FIG. 4 is a graph showing the effect of HPB-5074 on T cell activation; 1 μM HPB-5074, prostartin treatment of PBMC for 48 hours, FIGS. 4A and 4B are flow assays for CD, respectively ⁴⁺ T cells and CD8 ⁺ Expression changes of CD25, CD69, HLA-DR in T cells.

FIG. 5 is the effect of combination on HPB-5074 latent activation; FIG. 5A shows the percentage of GFP-positive cells in each group as measured by flow cytometry using HPB-5074 (100 nM) alone or in combination with 1. Mu.M Prostratin, SAHA, JQ1 for 24 hours and FIG. 5B shows the percentage of GFP-positive cells in each group as measured by flow cytometry using HPB-5074 (100 nM) alone or in combination with 1. Mu.M DRV, ETR, RAL, T-20 for 48 hours.

Detailed Description

Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

Preparation of the compounds of the invention:

the preparation routes of the compounds HPB-507A and HPB-5074 are as follows:

the preparation method comprises the following steps:

compound 1: the compound phorbol (3.64 g,10.0 mmol) was dissolved in pyridine (30 mL) and catalytic amounts of DMAP (122 mg,1.0 mmol), triphenylchloromethane (11.4 g,40 mmol) were added sequentially at room temperature and reaction 18h, TLC showed the end of the reaction. EtOAc (300 mL), 2N H was added ₂ SO ₄ Washing with/brine (10:1, 150 mL), na ₂ SO ₄ After drying and concentration, silica gel column chromatography gave compound 1 (4.85 g) as a white solid in 80% yield.

Compound 2: compound 1 (220 mg,0.36 mmol) was dissolved in anhydrous DCM (20 mL), DMAP (878.0 mg,7.2 mmol), n-octanoic acid (520.0 mg,3.6 mmol), EDCI (690.0 mg,3.6 mmol) was added sequentially at room temperature, stirring was performed for 6h, TLC showed that the reaction was complete, concentrated under reduced pressure, silica gel column chromatography gave compound 2 in 90% yield.

Compound 3: compound 2 (430.0 mg,0.5 mmol) was dissolved in MeOH (20 mL) and anhydrous K was added ₂ CO ₃ (30 mg) pH of the reaction system was 8 to 9, stirred at room temperature for 6 hours, TLC showed complete reaction of starting material, at 0.1. 0.1M H ₂ SO ₄ Regulating pH to 7, concentrating under reduced pressure to dry, and performing silica gel column chromatography to obtain compound 3 with a yield of 70%.

Compound 4: compound 3 was dissolved in MeOH (20 mL) and 1 drop of HClO was added ₄ (70%, w/w), ph=5, stirring at room temperature for 0.1h, tlc showed complete reaction, naOAc was used to adjust ph=7, concentrated to dryness under reduced pressure, and silica gel column chromatography gave compound 4 in 80% yield.

Compound HPB-507A: compound 4 (250.0 mg,0.5 mmol) was dissolved in anhydrous DCM/THF (20 mL,1:1, V/V), cooled at 0deg.C for 15min, and DIPEA (2.0 eq), DMAP (0.1 eq), ac were added sequentially ₂ O (2.1 eq) was stirred at 0deg.C for 6h, TLC showed complete reaction, concentrated to dryness under reduced pressure, and chromatographed on silica gel to give compound HPB-507A in 75% yield.

Compound 5: compound 3 (366.0 mg,0.5 mmol) was dissolved in anhydrous DCM/THF (20 mL,1:1, V/V), cooled at 0deg.C for 15min, and DIPEA (2.0 eq), DMAP (0.1 eq), ac were added sequentially ₂ O (2.1 eq) was stirred at 0deg.C for 6h, TLC showed complete reaction, concentrated to dryness under reduced pressure and chromatographed on silica gel to give compound 5 in 75% yield.

Compound HPB-5074: compound 5 was dissolved in MeOH (20 mL) and 1 drop of HClO was added ₄ (70%, w/w) pH=5, stirring at room temperature for 0.1h, TLC showed complete reaction, naOAc was used to adjust pH=7, vacuum concentrating to dryness, silica gel column chromatography to give compound HPB-5074, 80% yield。

NMR, MS of HPB-507A:

¹ H NMR(600MHz,CDCl ₃ )δ7.61(s,1H),5.71(t,J＝8.0Hz,1H),5.55(s,1H),5.41(d,J＝10.3Hz,1H),4.46(q,J＝12.4Hz,1H),3.40–3.17(m,2H),2.65–2.37(m,2H),2.32(dd,J＝14.8,7.4Hz,2H),2.19–1.96(m,8H),1.79(d,J＝1.6Hz,3H),1.70–1.61(m,2H),1.56(s,9H),1.44–1.14(m,20H),0.93–0.70(m,8H).； ¹³ C NMR(125MHz,CDCl ₃ )δ208.70,173.74,173.69,170.74,160.81,160.75,136.69,135.49,132.87,132.74,131.86,127.75,88.55,78.08,76.49,76.42,75.51,73.60,73.54,69.40,65.55,56.06,42.89,39.32,39.25,38.99,38.91,36.11,36.06,34.57,31.68,29.69,29.32,29.23,29.11,28.95,28.92,25.61,25.17,23.80,22.57,21.08,20.97,16.74,14.39,14.12,14.06,10.11,1.01.EIMS m/z:575

[M+H] ⁺ .

NMR, MS of HPB-5074:

¹ H NMR(600MHz,CDCl ₃ )δ7.59(s,1H),5.67(d,J＝3.6Hz,1H),4.82(d,J＝9.8Hz,1H),4.72(s,1H),4.46(s,2H),3.13(d,J＝37.4Hz,2H),2.54(d,J＝19.1Hz,1H),2.47–2.28(m,4H),2.17(s,1H),2.13–2.07(m,1H),2.05(s,3H),1.80(s,3H),1.62(dd,J＝15.3,7.6Hz,2H),1.36–1.21(m,10H),1.20(s,3H),1.05(s,3H),1.02(d,J＝6.4Hz,2H),0.88(dd,J＝14.3,7.2Hz,5H). ¹³ C NMR(125MHz,CDCl ₃ )δ208.57,176.74,170.75,160.24,135.85,133.49,132.60,87.49,79.02,73.27,69.45,60.75,56.66,43.39,39.24,39.00,35.05,34.32,31.64,29.09,28.88,27.76,24.97,22.56,22.22,20.94,17.13,15.95,14.03,10.14.EIMS m/z:533[M+H] ⁺ .

the preparation routes of the compounds HPB-5075 and HPB-5076 are as follows:

the preparation method comprises the following steps:

compound HPB-5075: compound 2 was dissolved in MeOH (20 mL) and 1 drop of HClO was added ₄ (70%, w/w), pH=5, stirring at room temperature for 0.1h, TLC indicationThe reaction was complete, ph=7 was adjusted with NaOAc, concentrated to dryness under reduced pressure, and chromatographed on silica gel to give compound HPB-5075 in 80% yield.

Compound HPB-5076: compound HPB-5075 (310.0 mg,0.5 mmol) was dissolved in anhydrous DCM/THF (20 mL,1:1, V/V), cooled at 0deg.C for 15min, and DIPEA (2.0 eq), DMAP (0.1 eq), ac were added sequentially ₂ O (2.1 eq) was stirred at 0deg.C for 6h, TLC showed complete reaction, concentrated to dryness under reduced pressure, and chromatographed on silica gel to give compound HPB-507A in 75% yield.

NMR, MS of HPB-5075:

¹ H NMR(600MHz,CDCl ₃ )δ7.57(s,1H),5.72–5.54(m,2H),5.39(d,J＝10.3Hz,1H),4.00(dd,J＝37.3,12.6Hz,2H),3.24(d,J＝8.8Hz,2H),2.90(d,J＝12.8Hz,1H),2.53(dd,J＝58.0,19.1Hz,2H),2.40–2.21(m,5H),2.14(dq,J＝12.8,6.4Hz,1H),1.90(s,1H),1.74(s,3H),1.61(dt,J＝15.0,7.3Hz,4H),1.37–1.13(m,23H),1.05(d,J＝5.1Hz,1H),0.95–0.67(m,9H). ¹³ C NMR(125MHz,CDCl ₃ )δ209.20,176.42,173.61,160.87,140.51,132.80,129.21,76.50,73.61,68.00,65.33,56.00,42.84,38.93,38.45,36.26,34.54,34.34,31.67,31.59,29.00,28.92,28.91,28.87,25.69,25.18,24.48,23.80,22.58,22.56,16.80,14.39,14.05,10.08.EIMS m/z:617[M+H] ⁺ .

NMR, MS of HPB-5076:

¹ H NMR(600MHz,CDCl ₃ )δ7.60(s,1H),5.84–5.62(m,2H),5.40(d,J＝10.3Hz,1H),4.45(q,J＝12.4Hz,2H),3.24(d,J＝5.6Hz,2H),2.54(d,J＝19.1Hz,1H),2.41(d,J＝21.6Hz,2H),2.36–2.25(m,4H),2.12(dd,J＝10.3,6.4Hz,1H),2.04(s,3H),1.77(s,3H),1.61(dd,J＝15.0,7.5Hz,4H),1.38–1.17(m,25H),1.03(d,J＝5.1Hz,1H),0.87(t,J＝7.3Hz,10H). ¹³ C NMR(151MHz,CDCl ₃ )δ208.84,176.37,173.56,170.77,160.95,135.43,132.82,78.06,76.41,73.48,69.42,65.24,56.04,42.91,39.28,38.88,36.13,34.53,34.30,31.67,31.59,29.00,28.91,28.90,28.87,25.68,25.17,24.45,23.80,22.58,22.55,20.96,16.76,14.36,14.04,10.10.EIMS m/z:533[M+H] ⁺ .

example 2

Tablet: 10mg of any one of the compounds obtained in example 1, 180mg of lactose, 55mg of starch and 5mg of magnesium stearate.

The preparation method comprises the following steps: the compound, lactose and starch were mixed, uniformly wetted with water, the wetted mixture was sieved and dried, sieved again, magnesium stearate was added, and the mixture was tabletted, each tablet weighing 250mg, with a compound content of 10mg.

Example 3

The injection comprises the following components: 2mg of any one of the compounds obtained in example 1, 10mg of sodium chloride and sterilized water for injection.

The preparation method comprises the following steps: dissolving the compound and sodium chloride in appropriate amount of injectable water, filtering to obtain filtrate, and packaging into ampoule under aseptic condition to obtain injection.

Example 4

The capsule comprises the following components: 10mg of any one of the compounds obtained in example 1, 187mg of lactose and 3mg of magnesium stearate.

The preparation method comprises the following steps: the compound was mixed with adjuvants, sieved, mixed homogeneously, and the resulting mixture was filled into hard gelatin capsules each weighing 200mg and the active ingredient 10mg.

Pharmacological action of the compounds of the invention against HIV:

the compounds HPB-507A, HPB-5074, HPB-5075 and HPB-5076 can inhibit the replication of HIV-1 experimental strains and drug-resistant strains with different targets in nanomolar concentrations, and the antiviral activity is superior to that of a control compound Prostratin. Meanwhile, the compounds can obviously reactivate HIV-1expression in an HIV-1 latent cell line, and the activating effect is stronger than that of a control compound Prostratin or equivalent to that of PMA. The HPB-5074 has more remarkable latent activating effect when combined with other types of latent activators, and the anti-HIV-1 medicine does not influence the latent activating effect.

The pharmacological test results of the compounds of the present invention are described below by using the test examples of the present invention, which are used to demonstrate the application of the compounds of the present invention in anti-AIDS drugs.

Experimental example 1

Compounds HPB-507A, HPB-5074, HPB-5075, HPB-5076 were tested for in vitro cytotoxicity and anti-HIV-1 activity.

Determination of C8166 Fine Compounds by MTT colorimetryCell toxicity, calculated survival and concentration at 50% of cells toxic (50%cytotoxic concentration,CC ₅₀ ). Measuring the protective effect of the compound on HIV-1 induced cytopathy, calculating the inhibition rate and the concentration at which syncytia formation is inhibited by 50% (50%effective concentration,EC) ₅₀ ). Then, the therapeutic index TI value (CC ₅₀ /EC ₅₀ )。

Cytotoxicity experiments. In 96-well plates, the test compounds were 5-fold diluted for a total of 6 gradients. Mu.l of compound was added to each well and 3 multiplex wells were set. Then 4X 10 is added to each well ⁴ 100 μl of each C8166 cell suspension. Prostratin is a positive control drug. A negative control containing only cells and a blank control containing only medium were set at the same time. 37 ℃,5% CO ₂ The cells were incubated in an incubator for 3 days with 20. Mu.l MTT solution (5. Mu.g/ml) per well for 4 hours, 100. Mu.l supernatant per well was discarded, and a 12% SDS-50% DMF solution was added thereto for overnight incubation. The OD value is measured by a ELx800 enzyme label instrument, and the measurement wavelength is 570nm/630nm. Drawing a dose response curve according to experimental results, and calculating CC ₅₀ Values.

Syncytia formation inhibition experiments. After 5-fold gradient dilution of the test compound, 100 μl per well is added to the 96-well plate. Then 100. Mu.l of a solution containing 4X 10 were added to each well ⁴ Individual C8166 cells and HIV-1 _ⅢB Is a suspension of (2)

(moi=0.03), 3 compound wells were set. Control wells containing only the cytoviral suspension were also set up, and Prostratin was used as positive drug control. 37 ℃,5% CO ₂ After 3 days of incubation, under an inverted microscope (100×), 5 non-overlapping fields were selected and the number of syncytia counted. Drawing a dose response curve according to experimental results

(FIG. 2), per Reed&Muench method to calculate EC of compound ₅₀ Values.

TABLE 1 Compound pair HIV-1 _ⅢB Is an antiviral activity of (a)

From the experimental results in table 1 above, it can be seen that: the tested compounds can inhibit the replication of HIV-1 virus in a lower nanomolar range, and have remarkable anti-HIV-1 virus activity.

Experimental example 2

Inhibition of replication of HIV-1 test and drug resistant strains in cells by the compounds HPB-507A, HPB-5074.

100 μl of compound diluted at a doubling ratio was added to each well, and 3 multiplex wells were set. Then 100. Mu.l of a solution containing 4X 10 were added to each well ⁴ Individual C8166 cells and HIV-1 _ⅢB Or HIV-1 _74V (NRTIs)、HIV-1 _RF/V82F/184V (PIs)、pNL4-3 _{GP41(36G)V38A,N42T} Suspension of (FIs) (moi=0.03). Control wells containing only the cytovirus suspension and blank control wells containing only the medium were simultaneously set, and Prostratin was the positive control drug. 37 ℃,5% CO ₂ Culturing for 4 days. Culture supernatants were collected and virus was lysed with Triton X-100 at a final concentration of 0.5%. Enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) for detecting expression of p24 antigen in supernatant, and calculating inhibition rate and EC ₅₀ Values.

TABLE 2 inhibitory Activity of Compounds against replication of test strains and different drug resistant strains

From the experimental results in table 2 above, it can be seen that: the compounds HPB-507A and HPB-5074 can inhibit the replication of HIV-1 experimental strain and drug-resistant strain viruses at nanomolar level, and the anti-HIV-1 virus activity is superior to that of a control compound Prostratin.

Experimental example 3

Compound HPB-5074 activates expression of latent HIV-1.

HPB-5074 was tested for HIV-1 reactivation effect in two latent cell lines. J-Lat is a human T lymphocyte cell line. The J-Lat cell is an HIV-1 latent cell model obtained by infecting human Jurkat T cells with HIV-1 pseudoviruses carrying the expression enhanced green fluorescent protein and then sorting and stimulating. Expression of HIV-1 is at low level replication without stimulation by an activator. After stimulation of J-Lat cells with a latent activator, the effect of latent activation can be assessed by detecting the expression of green fluorescent protein.

In 24 hole plate, seed is spread 2.5×10 ⁵ J-Lat C11 cells per well. The concentrations of compound HPB-5074 were 1nM, 10nM and 100nM, respectively. The concentration of control drug PMA was 10nM.37 ℃ and 5% CO ₂ The cells were collected from each well after culturing for 24 hours and 48 hours, respectively. Cells were resuspended in 200ul of PBS and washed 2 times. The percentage of GFP positive cells in the cells was detected by a flow-on-machine and the data was analyzed using software FlowJo V10.

Flow results showed that HPB-5074 at concentrations of 10nM and 100nM had significant latent reactivation effect when J-Lat cells were treated for 24 hours (FIG. 3A) and 48 hours (FIG. 3B). Furthermore, GFP expression increased with increasing concentration and with increasing time.

In 24 hole plate, seed is spread 2.5×10 ⁵ J-Lat A2 cells per well. The concentrations of compound HPB-5074 were 1nM, 10nM and 100nM, respectively. The concentrations of control drugs PMA and Prostratin were 10nM and 1. Mu.M, respectively. 37 ℃ and 5% CO ₂ Culturing for 48h, and collecting cells of each well. The cells were resuspended by 2 washes with PBS, 200. Mu.l. The percentage of GFP positive cells in the cells was detected by a flow-on-machine and the data was analyzed using software FlowJo V10.

Flow results showed that HPB-5074 at concentrations of 1nM, 10nM and 100nM had significant latent reactivation effect when J-Lat A2 cells were treated for 48 hours (FIG. 3C). Furthermore, the latent reactivation effect of 10nM HPB-5074 is better than that of 1. Mu.M protriatin.

Experimental example 4

Toxicity of Compound HPB-5074 to human PBMC and effect on T cell activation.

The white blood cell concentrate of healthy people is separated by adopting a density gradient centrifugation method to obtain peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC) [ the ethical committee of scientific research of people involved in the Kunming animal research of China academy of sciences, batch number: KIZRKX-2021-013]. Compound HPB-5074 and positive control drug Prostratin were 5-fold diluted in 96-well plates for a total of 6 gradients of 100 μl per well, with 3 replicate wells set. Then, 100. Mu.l of 5X 10 was added to each well ⁶ PBMC suspension per ml. Setting upNegative control wells containing only cell suspension and blank control wells containing only medium, 37 ℃,5% CO ₂ Culturing. On day 4, each well was supplemented with 100 μl of the corresponding concentration of drug-containing medium. On day 7, 30 μl MTT solution was added per well. Incubation was carried out at 37℃for 4 hours, 150. Mu.l of supernatant was discarded per well, 150. Mu.l of 12% SDS-50% DMF solution was added, and incubation was carried out at 37℃overnight. OD was measured by Biotek ELx800 microplate reader at 570nm/630nm. Calculation of cell viability and CC ₅₀ The value, i.e. the drug concentration at which 50% of the C8166 cells are toxic.

TABLE 2 toxicity of Compounds to PBMC

Cytotoxicity test results show that the compound HPB-5074 has smaller toxicity influence on PBMC.

To exclude the latent activator from extensive and sustained immune activation of T cells, especially with CD8 ⁺ T cell depletion is associated. The effect of HPB-5074 on immune cells was further assessed. Flow cytometry detected HPB-5074 versus CD4 ⁺ T cells and CD8 ⁺ The effect of T cell activation markers (cell surface receptors CD25, CD69 and HLA-DR) expression. 1 μM HPB-5074 and Prostratin treated PBMC,37℃C, 5% CO respectively ₂ Culturing for 48 hours. Cells were collected, washed 2 times with PBS, added with a premix containing antibodies specific for three receptors, CD25, CD69 and HLA-DR, and incubated at 4℃for 30 minutes in the absence of light. The cells were washed 1 time with PBS and resuspended in 200 ul. Expression of each set of three cell surface receptors was detected by a flow-on-machine and the data was analyzed using the software FlowJo V10.

Flow results showed that after 48 hours of treatment of cells with HPB-5074 and prostatin, the expression of the early activation marker CD69 molecule of T cells was slightly increased, but the expression of the late activation marker CD25 molecule was not significantly increased (fig. 4). Indicating that HPB-5074 does not cause extensive cell activation.

Experimental example 5

It is difficult to effectively activate latent HIV-1expression in vivo using a single activator, and thus HPB-5074 is used in combination with other latent activators to test the efficacy of the combination.

In 24 hole plate, seed is spread 2.5×10 ⁵ J-Lat C11 cells per well. 100nM HPB-5074 was co-processed with 1. Mu.M Prostratin, SAHA, JQ 1J-Lat C11 cells for 24 hours, respectively, and the percentage of GFP positive cells in each group was detected using flow cytometry.

As shown in FIG. 5A, the percentage of 100nM HPB-5074 that activates GFP expression when combined with 1. Mu.M SAHA or JQ1 was significantly increased over the percentage of GFP expression when each individual drug was used. And when the compound is combined with the latent activator Prostratin of the same type, the latent activating effect is not increased. Thus, the compounds of the present invention may be used in combination with other types of latent activators to exert better latent activation.

Experimental example 6

Effect of anti-HIV-1 virus drugs on latent reactivation of HPB-5074.

Following activation of HIV-1expression using a latent activator, antiretroviral drug therapy is also required to block new rounds of infection by activated HIV-1. To confirm whether antiviral drugs would affect the ability of HPB-5074 to be latent reactivated, the latent activation effect of HPB-5074 was tested for the presence of antiviral drugs at different targets.

In 24 hole plate, seed is spread 2.5×10 ⁵ J-Lat C11 cells per well. 100nM HPB-5074 was treated with 1. Mu.M DRV (Darunavir), ETR (Etravirine), RAL (raltegravir), T-20 (enfuvirtide) for 48 hours, respectively. The percentage of GFP positive cells in each group was separately detected using flow cytometry.

As a result, as shown in FIG. 5B, the percentage of activation of GFP expression by 100nM HPB-5074 in combination with 1. Mu.M antiretroviral drug was not significantly reduced from that of each individual drug. Therefore, when the compound of the present invention is used together with an antiviral drug, the latent activating effect is not weakened.

While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (6)

1. The 12-O-octanoyl-phorbol ester derivatives are characterized by comprising compounds shown in a general formula (I) and pharmaceutically acceptable salts;

   
2. 2. the process for producing 12-O-octanoyl-phorbol ester derivatives according to claim 1, wherein,

   (1) The preparation routes of the compounds HPB-5074 and HPB-507A are as follows:

   

   the preparation method of the compounds HPB-5075 and HPB-5076 comprises the following preparation routes:

   
3. 3. the method for preparing 12-O-octanoyl-phorbol ester derivatives according to claim 2, comprising the steps of:

   preparation of Compound 1 by chemoselective triphenylmethyl protection of 20-OH starting from Phorbol and isolation of the product;

   condensing compound 1 with n-octanoic acid to form an ester to produce compound 2 and isolating the product;

   compound 2 was prepared by selective removal of 13-OH ester groups at weak basicity (ph=8.5) to prepare compound 3 and isolation of the product;

   removing Tr protecting group from the compound 3 under the action of perchloric acid to prepare a compound 4 and separating a product;

   compound 4 Compound HPB-507A is prepared by chemoselective acetylation of 20-OH and isolation of the product;

   compound 3 compound 5 was prepared by 13-OH acetylation and the product isolated;

   removing Tr protecting group from the compound 5 under the action of perchloric acid to prepare a compound HPB-5074 and separating a product;

   removing Tr protecting group from the compound 2 under the action of perchloric acid to prepare a compound HPB-5075 and separating a product;

   compound HPB-5075 Compound HPB-5076 was prepared by chemoselective acetylation of 20-OH and isolation of the product.
4. 4. A pharmaceutical composition comprising a therapeutically effective amount of a 12-O-octanoyl-phorbol ester derivative of claim 1 and a pharmaceutically acceptable carrier and/or excipient.
5. 5. The use of 12-O-octanoyl-phorbol ester derivatives according to claim 1in the preparation of anti-aids drugs.
6. 6. The use of the pharmaceutical composition of claim 4 for the preparation of anti-aids drugs.

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