Active oxygen-responsive thioesters for oral administration, methods of synthesis and uses
By designing acid-stable and alkaline-hydrolyzed ROS-responsive thioester compounds, the problems of instability of existing radiation protection drugs in gastric juice and difficulty in releasing active molecules in the intestine were solved, and the stability of oral administration and the reactive oxygen species scavenging effect were achieved.
Patent Information
- Application Number
- CN202211410375.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Priority Date
- 2022-10-28
- Filing Date
- 2022-11-07
- Publication Date
- 2025-10-17
- Estimated Expiration
- 2042-11-07
AI Technical Summary
Existing radiation protection drugs such as amifostine require intravenous injection, have a short distribution half-life and obvious side effects. Oral drugs are unstable in the strong acid environment of gastric juice and have difficulty in effectively releasing active molecules in the intestine to eliminate reactive oxygen species.
A new class of ROS-responsive thioester compounds was designed, which are stable in acidic gastric juice, hydrolyzed in the alkaline environment of the intestine to release active thiols, and utilize the thioester bond to break under alkaline conditions to form reactive oxygen species scavengers.
It achieves a balance between stability in gastric juice and clearance of reactive oxygen species in the intestine, effectively reducing radiation enteritis and providing a new way of oral radiation protection.
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Figure CN115745857B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of thiol radioprotective drugs, and specifically designs a class of active oxygen responsive thioester compounds, and a synthesis method and application thereof. BACKGROUND
[0002] The widespread use of nuclear energy in aerospace, military, medical and other fields has led to an increasing exposure of humans to ionizing radiation (IR). At the same time, as the main way of cancer treatment, radiotherapy can directly produce a large amount of active oxygen, leading to DNA double-strand breaks, intracellular oxidative stress and apoptosis. However, radiotherapy has significant side effects, especially inhibition of hematopoietic system and causing gastrointestinal dysfunction, which can be fatal to patients. Therefore, the development of radioprotective drugs has become a crucial medical goal.
[0003] In recent years, great progress has been made in the synthesis and screening of radioprotective compounds, such as aminothiols, polyphenols and growth factors, which have been reported to be studied. As a free radical scavenger, aminothiols provide a H atom to scavenge reactive oxygen species (ROS). Amifostine is a radioprotective agent approved by the US Food and Drug Administration for use as a radioprotective agent. As an organic thio-phosphoric acid prodrug, it is dephosphorylated into a free thiol metabolite (WR-1065) by the alkaline phosphatase of the plasma membrane. The main administration method of amifostine is intravenous injection, which not only can be quickly removed from the body, but also has a very short distribution half-life, and has obvious side effects such as vomiting, nausea and hypotension. Therefore, it is of great significance to study orally administrable radioprotective drugs.
[0004] The oral administration method requires that the drug have certain stability, including being able to exist stably in a strong acid environment of gastric juice. SUMMARY
[0005] In order to overcome the foregoing problems, the present application provides a synthesis method and application of a new type of orally administrable ROS-responsive thioester compound. This kind of compound is stable in an acidic environment and can be hydrolyzed to release active molecules rich in free thiols in an alkaline environment of the intestinal tract. In the case of a post-radiation active oxygen burst, the TK group is broken and forms an active molecule capable of scavenging active oxygen, effectively improving radiation enteritis. The orally administrable ROS-responsive thioester molecule is expected to become a new type of radioprotective agent.
[0006] The technical solutions adopted by the present application are as follows:
[0007] A new type of ROS-responsive thioester compound, the structural formula of which is as follows:
[0008]
[0009] wherein R = (CH2) nn = 1-6.
[0010] The ROS-responsive thioester compound has the structural characteristics of moderate molecular weight, containing a thioester bond structural unit (-COS-) and a thio ketone bond.
[0011] The application provides a preparation method of compound I, characterized by comprising the following steps:
[0012]
[0013] The compound IV is obtained by protecting a mercaptoamine compound in an organic solvent by Boe; the organic solvent is tetrahydrofuran, methanol, ethanol, acetone, toluene, pyridine, DMF, acetonitrile, chloroform or the like;
[0014]
[0015] The compound IV is subjected to mercapto acetylation to obtain the compound III;
[0016]
[0017] The compound III is subjected to a Boc removal reaction to obtain the compound II;
[0018]
[0019] The compound II is subjected to a condensation reaction with a thio ketone-containing compound TK to obtain the compound I.
[0020] In the preparation of the compound III, the condensation reagent is DIPEA, the molar ratio of the compound IV to DIPEA is 1:1.0-2.0 mol, and the molar ratio of the compound IV to acetyl chloride is 1:1.0-2.0 mol; the reaction temperature is 10-40 DEG C, and the reaction time is 10-24 h.
[0021] In the preparation of the compound II, the molar ratio of the compound III to trifluoroacetic acid (TFA) as a protonic acid is 1:2.0-20.0 mol; the reaction temperature is 10-40 DEG C, and the reaction time is 1-10 h.
[0022] In the preparation of the compound I, the molar ratio of the compound II to the compound TK is 1:2.0-6.0 mol; the reaction temperature is 10-40 DEG C, the reaction time is 6-24 h; the molar ratio of the compound V to potassium thioacetate is 1:1.0-2.0 mol; the reaction temperature is 0-30 DEG C, and the reaction time is 10-24 h.
[0023] The application further provides another preparation method of the compound I, characterized by comprising the following steps:
[0024] The application further provides another preparation method of the compound I, characterized by comprising the following steps:
[0025] The raw material bromamine hydrochloride is subjected to condensation reaction with a thietone-containing compound TK to obtain compound V;
[0026]
[0027] Compound V is reacted with potassium thioacetate to finally obtain compound I.
[0028] The preparation method of the ROS-responsive thioester compound provided by the application is as follows: in the preparation of compound V, the molar ratio of bromamine hydrochloride to TK is 2.0-4.0:1, the reaction temperature is 10-40 DEG C, and the reaction time is 10-24 h.
[0029] The pH-sensitive drug molecule designed in the application can exist stably in a gastric juice environment and release active groups in an intestinal alkaline environment. Through the hydrolysis reaction of the thioester bond in an alkaline environment, oral administration of the radiation protection compound can be realized. The hydrolysis reaction of the compound in the intestinal tract releases active molecules, and the free thiols formed can scavenge active oxygen.
[0030] The novel thioester compound provided by the application can be used as an effective component of a radiation protection drug for intestinal inflammation. Compared with the prior art, the molecular structure of the application has the following effects: (1) the existence of the thioester bond makes the molecule have pH-sensitive performance, can maintain stability in an acidic environment of gastric juice, and can be hydrolyzed to form thiol active molecules in an intestinal alkaline environment, meeting the conditions of oral radiation protection; (2) the introduction of the thietone group can make the molecule have ROS corresponding performance, and when ROS bursts, the thietone bond is broken to form a mercapto active molecule to scavenge ROS. A representative compound is S, S'-(8, 8-dimethyl-4, 12-dioxo-7, 9-dithia-3, 13-azapentadecane-1, 15) diacetyl thioester, which is used for pH-sensitive analysis and ROS scavenging test.
[0031] Drawings accompanying the specification
[0032] Figure 1 A standard curve of glutathione concentration and absorbance at 412 nm;
[0033] Figure 2 A nuclear magnetic hydrogen spectrum of the compound in the presence of HCl;
[0034] Figure 3 Hydrolysis kinetics of compound I-2 in simulated gastric juice and simulated intestinal juice;
[0035] Figure 4 ROS reactivity rupture of compound I-2 in vitro in PBS. DETAILED DESCRIPTION
[0036] In order to more fully explain the practice of the application, the following S, S'-(8,8-dimethyl-4,12-dioxo-7,9-dithia-3,13-azapentadecane-l,15)dithioacetate preparation examples are provided. While the preparation of S, S'-(8,8-dimethyl-4,12-dioxo-7,9-dithia-3,13-azapentadecane-l,15)dithioacetate is typical, these examples are merely illustrative, and are not intended to limit the scope of the application.
[0037] The following is a synthetic route for S, S'-(8,8-dimethyl-4,12-dioxo-7,9-dithia-3,13-azapentadecane-l,15)dithioacetate:
[0038]
[0039] Reagents and apparatus: Unless otherwise stated, all reagents were purchased from a reagent company and used directly without any purification. Column chromatography was performed using silica gel (300-400 mesh) purchased from Tianjin Kermel Chemical Reagent Co., Ltd. 1 HNMR and 13 CNMR were measured using a Vian Inova 500 MHz nuclear magnetic resonance spectrometer with tetramethylsilane (TMS) as an internal standard and dimethyl sulfoxide (DMSO) as a solvent. The multiplicity of the proton signals is abbreviated as s, d, t, q, p and m, which represent singlet, doublet, triplet, quartet, quintet and multiplet, respectively. UV / Vis absorption spectra were recorded on an Agilent Technologies Cary 500 UV / Vis spectrometer.
[0040] Example 1 Preparation of compound IV (2-tert-butoxycarbonylaminoethyl mercaptan)
[0041] Cysteamine hydrochloride (5.00 g, 44.01 mmol) and sodium bicarbonate (7.39 g, 88.03 mmol) were mixed with Et3N (11.53 g, 52.82 mmol) in a mixed solvent of tetrahydrofuran and water (V / V = 1 / 1, 100 mL). The mixture was stirred at room temperature for 12 hours. The mixture was washed by adding pure water, and the organic phase was dried with anhydrous sodium sulfate. The product compound IV was a white solid.
[0042] Example 2 Preparation of compound III (S-(2-((tert-butoxycarbonyl)amino)ethyl)acetylthio)
[0043] S-(2-((tert-butoxycarbonyl)amino)ethyl)acetylthio (2.19 g, 10 mmol) was dissolved in 50 mL of DCM and an equal volume of TFA was added. The mixture was stirred at room temperature for 2 hours and the product was concentrated under reduced pressure to obtain a brown oil, which gave compound II.
[0044] Preparation of compound II (S-(2-aminoethyl)acetylthio) of example 3
[0045] S-(2-((tert-butoxycarbonyl)amino)ethyl)acetylthio (2.19 g, 10 mmol) was dissolved in 50 mL of DCM and an equal volume of TFA was added. The mixture was stirred at room temperature for 2 hours and the product was concentrated under reduced pressure to obtain a brown oil, which gave compound II.
[0046] Preparation of compound I-1 (S,S'-(7,7-dimethyl-3,11-dioxo-6,8-dithia-2,12- azapentadecane-1,13)diacetylthioester) of example 4
[0047] Propane-2,2-diylbis(thio)diacetate (500 mg, 1.98 mmol) was dissolved in DCM at 0°C, and EDCl (835.66 mg, 4.36 mmol), HOBt (589.03 mg, 4.36 mmol) and DIPEA (563.41 mg, 4.36 mmol) were added successively and stirred for 30 min. S-(aminomethyl)acetylthio (1.93 g, 18.43 mmol), DIPEA (2.38 g) were added successively to the above reaction solution, and stirred at room temperature for 12 h. After stirring at room temperature under nitrogen for 24 h, the solvent was removed by vacuum rotary evaporation. Purification by column chromatography using methanol / chloromethane (1:20) as an eluent gave compound I-1. 1 H NMR (300 MHz, DMSO): δ 8.18 (t, 2H), 4.78 (d, 4H), 2.72 (t, 4H), 2.55 (t, 4H), 2.30 (s, 6H), 1.59 (s, 6H).
[0048] Preparation of compound I-2 (S,S'-(8,8-dimethyl-4,12-dioxo-7,9-dithia-3,13- azapentadecane-1,15)diacetylthioester) of example 5
[0049] Propane-2,2-diylbis(sulfanyl)diacetic acid (500 mg, 1.98 mmol) was dissolved in DCM at 0 °C, and then EDCl (835.66 mg, 4.36 mmol), HOBt (589.03 mg, 4.36 mmol) and DIPEA (563.41 mg, 4.36 mmol) were added successively and stirred for 30 min. S-(2- aminoethyl)acetylmercapto (2.19 g, 18.43 mmol), DIPEA (2.38 g) were added successively to the above reaction solution, and stirred at room temperature for 12 h. After stirring at room temperature for 24 h under nitrogen, the solvent was removed by vacuum rotary evaporation. Purification by column chromatography using methanol / chloromethane (1:20) as an eluent gave compound I-2. 1 H NMR (300 MHz, DMSO): δ 8.13 (t, 2H), 3.18 (d, 4H), 2.90 (t, 4H), 2.73 (t, 4H), 2.37 2.27 (m, 10H), 1.52 (s, 6H).
[0050] Example 6 Preparation of compound I-3 (S,S'-(9,9-dimethyl-5,13-dioxo-8,10-dithia- 4,14-diazapentadecane-l,17)diacetothioester)
[0051] Propane-2,2-diylbis(sulfanyl)diacetic acid (500 mg, 1.98 mmol) was dissolved in DCM at 0 °C, and then EDCl (835.66 mg, 4.36 mmol), HOBt (589.03 mg, 4.36 mmol) and DIPEA (563.41 mg, 4.36 mmol) were added successively and stirred for 30 min. S-(3- aminoethyl)acetylmercapto (2.45 g, 18.43 mmol), DIPEA (2.38 g) were added successively to the above reaction solution, and stirred at room temperature for 12 h. After stirring at room temperature for 24 h under nitrogen, the solvent was removed by vacuum rotary evaporation. Purification by column chromatography using methanol / chloromethane (1:20) as an eluent gave compound I-3. 1 H NMR (300 MHz, DMSO): δ 7.70 (t, 2H), 3.42 (d, 4H), 3.24 (t, 4H), 2.72 (t, 4H), 2.55 2.30 (m, 10H), 2.22 (s, 4H), 1.59 (s, 6H).
[0052] Example 7 Preparation of compound I-4 (S,S'-(10,10-dimethyl-6,14-dioxo-9,11- dithia-5,15-diazapentadecane-l,19)diacetothioester)
[0053] Propane-2,2-diylbis(sulfanyl)diacetic acid (500 mg, 1.98 mmol) was dissolved in DCM at 0 °C, and then EDCl (835.66 mg, 4.36 mmol), HOBt (589.03 mg, 4.36 mmol) and DIPEA (563.41 mg, 4.36 mmol) were added successively with stirring for 30 min. S-(4- aminoethyl)acetylmercapto (2.71 g, 18.43 mmol) and DIPEA (2.38 g) were added successively to the above reaction solution, and stirred at room temperature for 12 h. After stirring at room temperature for 24 h under nitrogen, the solvent was removed by vacuum rotary evaporation. Purification by column chromatography using methanol / chloromethane (1:15) as an eluent gave Compound I-4. 1 H NMR (300 MHz, DMSO): δ 7.70 (t, 2H), 3.24 (d, 4H), 3.02 (t, 4H), 2.72 (t, 4H), 2.55 (s, 4H), 2.30 (s, 6H), 1.87-1.52 (m, 14H).
[0054] Example 8 Preparation of Compound I-5 (S,S'-(11,11-dimethyl-7,15-dioxa-10,12- dithia-6,16-azapentadecane-1,21)diacetothioester)
[0055] Propane-2,2-diylbis(sulfanyl)diacetic acid (500 mg, 1.98 mmol) was dissolved in DCM at 0 °C, and then EDCl (835.66 mg, 4.36 mmol), HOBt (589.03 mg, 4.36 mmol) and DIPEA (563.41 mg, 4.36 mmol) were added successively with stirring for 30 min. S-(3- aminoethyl)acetylmercapto (2.97 g, 18.43 mmol) and DIPEA (2.38 g) were added successively to the above reaction solution, and stirred at room temperature for 12 h. After stirring at room temperature for 24 h under nitrogen, the solvent was removed by vacuum rotary evaporation. Purification by column chromatography using methanol / chloromethane (1:15) as an eluent gave Compound I-5. 1 H NMR (300 MHz, DMSO): δ 7.70 (t, 2H), 3.24 (d, 4H), 3.00 (t, 4H), 2.73 (t, 4H), 2.54 (t, 4H), 2.30 (s, 6H), 1.87 (t, 4H), 1.59-1.50 (m, 10H), 1.29 (m, 4H).
[0056] Example 9 Preparation of Compound I-6 (S,S'-(12,12-dimethyl-8,16-dioxa-11,13- dithia-7,17-azapentadecane-1,23)diacetothioester)
[0057] At 0°C, propane-2,2-diylbis(thio)diacetic acid (500 mg, 1.98 mmol) was dissolved in DCM, and EDCl (835.66 mg, 4.36 mmol), HOBt (589.03 mg, 4.36 mmol), and DIPEA (563.41 mg, 4.36 mmol) were added sequentially, and stirred for 30 minutes. S-(3-aminoethyl)acetylmercaptoethanol (3.23 g, 18.43 mmol) and DIPEA (2.38 g) were added sequentially to the reaction solution, and stirred at room temperature for 12 hours. The mixture was stirred at room temperature under nitrogen for 24 hours, and then the solvent was removed by vacuum rotary evaporation. The product was purified by column chromatography using methanol / chloromethane (1:20) as the eluent to obtain compound I-6. 1 H NMR (300MHz, DMSO): δ7.70(t, 2H), 3.24(d, 4H), 3.04(t, 4H), 2.76(t, 4H), 2.56 (t, 4H), 2.31 (s, 6H), 1.87 (t, 4H), 1.58 (s, 6H), 1.42 (m, 4H), 1.32-1.29 (m, 8H).
[0058] Example 10 pH Sensitive Assay of Compound I-2
[0059] The present invention studies the pH sensitivity of the compound in solution. In order to verify the release process of thiol in the synthetic compound, glutathione was selected as the reference molecule and DTNB was selected as the detection reagent to generate a standard curve ( Figure 1 ). The thiol group can react with DTNB to form yellow 2-nitro-5-mercaptobenzoic acid, whose characteristic absorption peak is at 412nm. The concentration of total thiol groups can be quantitatively analyzed by the change of absorbance value. Next, the stability of the compound under simulated gastric conditions (pH=1.2) was studied. After adding hydrochloric acid, the 1 No differences were observed in the HNMR spectra, which confirmed the stability of the compounds under simulated gastric fluid conditions ( Figure 2 ). In addition, the hydrolysis kinetics of the compound at different pH values were evaluated. Within 100 minutes, almost no hydrolysis of the compound (2.0 mM) was observed in simulated gastric fluid. However, in simulated intestinal fluid (pH = 8.4), the absorbance at 412 nm gradually increased in a time-dependent manner and reached a plateau after 30 minutes ( Figure 3 ). This indicates that the compound of the present invention is stable in simulated gastric fluid, but unstable in simulated intestinal fluid.
[0060] Example 11 ROS response determination of compound I-2
[0061] A solution of H202 at a concentration of 5.0 mM was added to the PBS solution of the compound to trigger the release of free thiols and further quantify the free thiols with DTNB. The absorbance of the compound at 412 nm increased significantly compared to the control group after the addition of H202 Figure 4 ). In addition, higher concentrations of H202 (50 mM) induced the release of free thiols from the compound and the absorbance of the reaction product at 412 nm was positively correlated with time and H202 concentration.
[0062] Example 12 In vitro ROS scavenging assay of Compound I
[0063] HIEC-6 cells were seeded in 6-well plates at a density of 2.0 x 105 5 cells / well and after 24 hours of attachment, the cells were incubated with 0.1 mM of the compound in PBS for 24 hours prior to irradiation (6 and 8 Gy). The cells were then washed three times with PBS and 1 mL of 5 mM DCFH-DA was added for 20 minutes at 37°C. After incubation, the cells were washed three times with PBS and the fluorescence intensity was measured at 490 nm using an Infinite F200 multimode reader. The ROS levels were significantly reduced in the dosed groups compared to the IR group (Table 1).
[0064] Table 1. Ability of the compound to scavenge ROS upon co-incubation with HIEC-6 cells and irradiation at 6 and 8 Gy.
[0065]
[0066] Example 13 Protective effect of Compound I on the intestine
[0067] Male C57BL / 6 mice (6-8 weeks) were orally gavaged with 0.2 mL of 125 mg / kg of the compound solution 30 minutes prior to irradiation. In the control group, the blank solvent was a mixture of 10% dimethyl sulfoxide, 40% polyethylene glycol 300, 5% Tween-80 and 45% saline. The mice were irradiated abdominally with a single dose of 13 Gy gamma rays at a rate of 0.99 Gy / min using a Gammacell 40 Exactor (Atomic Energy of Canada Limited, Chalk River, Ontario, Canada). The colon length of C57BL / 6 mice was measured 3 days after 13 Gy irradiation. The colon length of all groups is shown in Table 2.
[0068] Table 2. Colon length of mice
[0069]
[0070]
[0071] Compared with the control group, the colon length of IR group was significantly shortened. The addition of compound can significantly reverse the radiation-induced colon shortening.
Claims
1. Compound of formula I: in, R=(CH2) n ,n=1~6。 2. A method for preparing the compound according to claim 1, characterized in that: The thiolamine compound is subjected to Boc protection in an organic solvent to obtain compound IV; the compound is subjected to thiol acetylation to obtain compound III; compound III is subjected to a Boc removal reaction to obtain compound II; compound II is reacted with a thioketal-containing compound TK to obtain compound I:
3. The method for preparing the compound according to claim 2, wherein in the preparation of compound IV, the molar ratio of the raw material reactants mercapto hydrochloride and (Boc)2O is 1:(1-2); the reaction temperature is 10-40°C, and the reaction time is 10-24h.
4. The method for preparing the compound according to claim 2, wherein in the preparation of compound III, the molar ratio of compound IV to DIPEA is 1:(1-2), and the molar ratio of compound IV to acetyl chloride is 1:(1-2); the reaction temperature is 10-40°C, and the reaction time is 10-24h.
5. The method for preparing the compound according to claim 2, wherein in the preparation of compound II, the molar ratio of compound III to trifluoroacetic acid is 1:(2-20); the reaction temperature is 10-40°C, and the reaction time is 1-10 h.
6. The method for preparing the compound according to claim 2, wherein in the preparation of compound I, the molar ratio of compound II to compound TK is 1:(2-6); the reaction temperature is 10-40°C, and the reaction time is 6-24 hours.
7. A method for preparing the compound according to claim 1, characterized in that: The bromoamine hydrobromide is reacted with the thioketal-containing compound TK to obtain compound V; the compound V is reacted with potassium thioacetate to obtain compound I:
8. The method for preparing the compound according to claim 7, wherein in the preparation of compound V, the molar ratio of the reaction raw materials to TK is (2-4):1, the reaction temperature is 10-40°C, and the reaction time is 10-24h.
9. Use of the compound according to claim 1 in the preparation of radiation protection and free radical scavenging drugs.