CN111233929B - Deuterated nucleoside analogue and preparation method and application thereof - Google Patents

Abstract

The invention provides a compound shown as a formula I, or a pharmaceutically acceptable salt or prodrug thereof, wherein R ₁ ～R ₃₅ Each independently selected from hydrogen or deuterium, and R ₁ ～R ₃₅ At least one of which is selected from deuterium. The invention further provides a preparation method and application of the compound. Experiments prove that the compound has obvious inhibition effect on SARS-CoV-2 and longer half-life than Reidesvir, and the invention has very good application prospect in preparing medicaments for treating and/or preventing the diseases caused by SARS-CoV-2.

Description

Deuterated nucleoside analogue and preparation method and application thereof

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to a deuterated nucleoside analogue, and a preparation method and application thereof.

Background

Coronaviruses (CoV) are a group of pathogenic microorganisms that pose serious hazards to humans. To date, a total of 7 human-infecting coronaviruses, SARS-CoV, MERS-CoV-2 (a novel coronavirus, also 2019-nCoV), HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1, have been discovered in humans. The latter four coronaviruses cause only mild cold or diarrhea symptoms.

Whether SARS in 2003, MERS in 2012, or covi-19 (a disease caused by a novel coronavirus) outbreak lasting up to 2020, poses serious threats and impacts on people's life health worldwide. Severe Acute Respiratory Syndrome (SARS) is an acute respiratory infectious disease caused by SARS coronavirus (SARS-CoV), and the main clinical manifestations are as follows: the incubation period is 1 to 16 days, and is usually 3 to 5 days. The fever is the first symptom with acute onset and strong infectivity, the fever can be aversion to cold, the body temperature is usually over 38 ℃, the fever is irregular or atonic fever, heat is remained, and the like, the fever is Cheng Duowei for 1 to 2 weeks; with headache, muscular soreness, asthenia, and diarrhea. Dry cough and little sputum appear after 3 to 7 days of onset, blood streak sputum occasionally appears, and the sign of the lung is not obvious. The disease reaches the peak in 10-14 days, the symptoms of infection and poisoning such as fever, hypodynamia and the like are aggravated, frequent cough, shortness of breath, dyspnea and dyspnea appear, and asthma and palpitation are forced to rest in bed when the patient moves slightly. This stage is prone to secondary infections of the respiratory tract. After the course of disease is 2-3 weeks, fever gradually recedes, and other symptoms and physical signs are relieved or even disappear. The absorption and recovery of the inflammatory changes of the lung are slow, and the inflammatory changes of the lung can be completely absorbed and recovered after the normal body temperature is about 2 weeks. Mild patients have mild clinical symptoms. Severe patients are in serious condition and are prone to respiratory distress syndrome. The disease condition of children patients is lighter than that of adults. There are a few patients who do not have fever as the first symptom, especially patients with recent surgical history or underlying disease. In 2003, data published by the world health organization showed that 919 people died due to SARS worldwide, the fatality rate was approximately 11%. Middle East Respiratory Syndrome (MERS) is a serious respiratory disease caused by MERS-CoV, and the main symptoms include fever, cough, dyspnea, and the like, and digestive system symptoms such as diarrhea, nausea, vomiting, and the like may also occur, which may cause serious complications such as pneumonia and renal failure. MERS-CoV has led to infections in more than 2468 worldwide, deaths in more than 851, with MERS patient mortality around 38% according to the World Health Organization (WHO) statistics since 2012 (431/1139). According to the statistics of the World Health Organization (WHO), the mortality rate of MERS patients is about 38% (431/1139).

The disease caused by the novel coronavirus (COVID-19) is caused by infection with the novel coronavirus (SARS-CoV-2).

The general symptoms of SARS-CoV-2 infection are: fever, weakness, dry cough, and gradually dyspnea; some patients have slight onset symptoms and even no obvious fever. The severe symptoms are: acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis, and hemorrhagic blood coagulation dysfunction. SARS-CoV-2 virus positive can be detected in nasopharyngeal swab, sputum, respiratory tract secretion, blood and excrement of SARS-CoV-2 infected patient, and the chest imaging of patient shows that it has multiple small spot images and change of interstitium in early stage, and has obvious extrapulmonary zone. Further, the lung develops into a double-lung and multiple-wear glass shadow and an infiltrative shadow, and the severe cases may have lung consolidation and pleural effusion rarely.

According to the diagnosis and treatment scheme (trial fifth edition) of pneumonia infected by novel coronavirus, no specific antiviral drug exists for treating the novel coronavirus infected patients clinically at present, isolation and support treatment is generally adopted for infected patients, but the atomization inhalation of alpha-interferon and lopinavir/ritonavir can be tried. The ongoing clinical trial drugs for 2019-nCoV include Chinese patent drugs (such as Shuanghuanglian) or anti-other virus drugs (such as Reddisivir (Remdisivir), abidol, darunavir and the like), which have the inhibiting effect on 2019-nCoV in vitro cell tests. Among them, the structure of Remdisivir (Remdisivir) is shown below, and has shown a better therapeutic effect in 1 U.S. patient infected with a novel Coronavirus (First Case of 2019Novel Coronavir in the United states, michelle, holshue, chase et al., new England,2020January 31DOI. Formally introduced into china in month 2 of 2020, a phase III clinical trial of novel coronavirus infection is currently underway.

Remdisivir (Remdisivir) is a novel adenosine analog monophosphate amide prodrug that is converted within the cell to the pharmacologically active form of Nucleoside Triphosphates (NTPs), which inhibit viral RNA polymerase and inhibit viral nucleic acid synthesis. Phase III clinical studies are currently underway for the treatment of ebola virus (Ebolavirus) infections. It has also been found to have broad-spectrum antiviral activity against various RNA viruses such as Respiratory Syncytial Virus (RSV), vernoninfrus, lassa fever virus, MERS-CoV, SARS, nipah virus (Nipahvirus) and the like in vitro.

The Rudexilvir compound is disclosed in Chinese patent CN103052631A (2016.2.24), and subsequently disclosed are application patents CN105899216A (2016.08.24), CN 107073005A (2017.08.18), CN107074902A (2017.08.18), CN108348526A (2018.07.31) and crystal form patent CN110636884A (2019.12.31).

The hydrogen in the active molecular group of the medicine is replaced by isotope deuterium, which is nontoxic and nonradioactive and is stabilized about 6-9 times than a carbon-hydrogen bond, so that the metabolic site can be sealed to prolong the half-life period of the medicine, the treatment dosage is reduced, and the pharmacological activity of the medicine is not influenced, thereby being considered as an excellent modification method.

At present, no research report on the deuterated products of the ridciclovir compounds exists, and in view of the important role of the ridciclovir compounds in controlling the epidemic situation of the novel coronavirus, the research on the deuterated products of the ridciclovir compounds further improves the in vivo metabolism of the ridciclovir compounds and expands the therapeutic window range has important clinical significance.

Disclosure of Invention

The invention aims to provide a deuterated nucleoside analogue, a preparation method and application thereof, in particular to a deuterated Redexilvir, a preparation method and application thereof.

The invention provides a compound shown in a formula I, or pharmaceutically acceptable salt thereof, or prodrug thereof,

R ₁ ～R ₃₅ each independently selected from hydrogen or deuterium, and R ₁ ～R ₃₅ At least one of which is selected from deuterium.

Further, the compound is shown as a formula II,

wherein R is ₁ ～R ₂₂ Each independently selected from hydrogen or deuterium, and R ₁ ～R ₂₂ At least one of which is selected from deuterium.

Further, the compound is shown as a formula III,

wherein R is ₁₃ ～R ₂₁ Each independently selected from hydrogen or deuterium, and R ₁₃ ～R ₂₁ At least one of which is selected from deuterium.

Further, in the formula III,

R ₁₃ ～R ₁₇ all being deuterium, R ₁₈ ～R ₂₁ Each independently selected from hydrogen or deuterium;

or R ₁₉ ～R ₂₁ All being deuterium, R ₁₃ ～R ₁₈ Each independently selected from hydrogen or deuterium;

or R ₁₈ Selected from deuterium, R ₁₃ ～R ₁₇ And R ₁₉ ～R ₂₁ Each independently selected from hydrogen or deuterium.

Further, the compound is selected from the following structures:

further, the compound synthesis route is as follows:

wherein, the synthetic route comprises the following steps:

(1) Carrying out esterification reaction on the compound A and the compound B to obtain a compound C;

(2) Carrying out N-phosphorylation reaction on the compound C and the compound D; then adding a compound E to carry out nucleophilic substitution reaction to obtain a compound F;

(3) Reacting the compound F with the compound G under the action of a catalyst and an acid-binding agent to generate a compound H;

(4) Deprotecting the compound H to obtain a target product, a compound shown in formula III;

wherein R is ₁₃ ～R ₂₁ As defined in claim 3, R ₃₆ Selected from hydrogen or deuterium.

Further, the air conditioner is characterized in that,

in the step (1), the catalyst used in the esterification reaction is thionyl chloride, the molar ratio of the thionyl chloride to the compound A is 1.0-2.0, the reaction temperature is 20-80 ℃, and the reaction time is 4-8 h;

in the step (2), the organic solvent is dichloromethane; dripping the compound D and triethylamine at-78 ℃, reacting at 0-25 ℃ for 3-6 h, and monitoring the completion of the conversion of the compound C by HPLC, wherein the HPLC conditions are as follows: mobile phase 10% acetonitrile (containing 0.05% triethylamine): 90% of water (containing 0.05% of triethylamine), the time is 10min, the flow rate is 1.0mL/min, the column temperature is 30 ℃, the column is Welch Ultimate AQ-C18,5um,4.6x200mm, then 4-nitrophenol and triethylamine are added at 0-10 ℃, the temperature is kept and stirred for 2-4 h at 10-20 ℃, the reaction solution is decompressed and concentrated at 20-25 ℃ to remove the organic solvent, the concentrate is separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 8:1) to obtain a crude racemate product of the compound F, the crude product is pulped and filtered by diisopropyl ether, wherein the using volume of the diisopropyl ether is 2-6 times of the weight of the crude product, preferably 4 times of the volume, and the filter cake is dried for 4h under vacuum at 40-50 ℃ to obtain the optically pure compound F;

in the step (3), the used organic solvent can be acetonitrile, tetrahydrofuran and toluene, preferably acetonitrile, the used catalyst can be magnesium chloride, zinc chloride, stannic chloride and stannous chloride, preferably magnesium chloride, the acid-binding agent can be triethylamine, pyridine and N, N-diisopropylethylamine, preferably N, N-diisopropylethylamine, the reaction temperature is 50-80 ℃, the reaction time is 0.5-2H, the reaction liquid is decompressed and concentrated to remove the organic solvent, ethyl acetate is added into the concentrate, the obtained solution is washed by 5% sodium citrate, saturated ammonium chloride solution, 5% sodium bicarbonate solution and saturated salt solution in sequence, and the compound H is obtained by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) after the organic phase is concentrated;

in the step (4), the organic solvent can be selected from acetonitrile, tetrahydrofuran and acetone, the reagent is hydrochloric acid, the molar ratio of the hydrochloric acid to the compound H is 1.0-1.5, the reaction temperature is 0-15 ℃, and the reaction time is 1-3H. After the reaction is finished, dropwise adding saturated sodium bicarbonate aqueous solution at 0-5 ℃ to adjust the pH value to 7-8, extracting the reaction solution by ethyl acetate, washing an organic phase by saturated saline solution, decompressing and concentrating to obtain a crude product, and separating and purifying by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain the target compound, namely the compound shown in the formula III.

The present invention provides the use of a compound, or a pharmaceutically acceptable salt or prodrug thereof, in the manufacture of a medicament against a coronavirus selected from MERS-CoV, SARS-CoV-2, more preferably the virus is SARS-CoV-2.

The invention provides application of a compound or a pharmaceutically acceptable salt thereof or a prodrug thereof in preparing a medicament for preventing and/or treating a coronavirus infection disease, wherein the coronavirus infection disease is selected from MERS, SARS and COVID-19, and the COVID-19 is preferred.

The invention provides a pharmaceutical composition, which is characterized in that: the pharmaceutical composition is a preparation prepared from the compound, or pharmaceutically acceptable salt or prodrug thereof and pharmaceutically acceptable auxiliary materials.

On the basis of maintaining the effectiveness of the compound on SARS-CoV-2, the compound obviously prolongs the half-life period, reduces the requirement on dosage, reduces side effects and enlarges the treatment window range, so the compound has very good prospect for preparing the medicine for treating the diseases related to coronavirus infection

Deuterium in the context of the present invention is an isotope of hydrogen (H), also known as deuterium, and the symbol of the element is generally D or ² H, the deuterium nucleus has one proton and one neutron with a relative atomic weight of 2.

The term "deuteron" as used herein refers to a product wherein at least one hydrogen in the molecule is replaced by deuterium.

The pharmaceutically acceptable salt is a product obtained by directly salifying the free base of the compound and an inorganic or organic acid. Wherein the inorganic or organic acid is selected from hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, picric acid, citric acid, maleic acid, methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, etc.

It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.

Detailed Description

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products. EXAMPLE 1 preparation of Compound 1 of the present invention

The synthetic route is as follows:

the preparation method comprises the following steps:

adding L-alanine-3,3,3-d 3 (A-1, 1.00g, 0.011mol) and 2-ethylbutanol (B, 10 mL) into a reaction bottle, stirring the reaction system at 0 ℃ for 5min, slowly adding thionyl chloride (1.44g, 0.012mol), heating to 60 ℃, stirring and keeping the temperature and stirring for 6h, reducing concentration to remove most of solvent, cooling the concentrate to room temperature, adding 30mL of ethyl acetate and stirring for 60min, and performing suction filtration to obtain a white-like solid, namely the compound C-1 (2.11 g, the yield is 90.2%, and the mass spectrometric detection abundance is 99.3atom D).

The mixture of Compound C-1 (1.50g, 0.007mol) and dichloromethane (30 mL) was cooled to-78 deg.C with stirring, and benzyloxydichlorophosphate (D, 1.48g, 0.007mol) was added, followed by slow dropwise addition of triethylamine (0.71g, 0.007mol). The system was warmed to 20 ℃ and stirred at the same temperature for 5h, and HPLC monitored for completion (10% acetonitrile (containing 0.05% TFA): 90% water (containing 0.05% TFA), 10min,1.0mL/min,30 ℃, welch Ultimate AQ-C18,5um, 4.6x200mm). The system was cooled to 0 ℃ and 4-nitrophenol (E, 0.88g, 0.006 mol) was added, then triethylamine (0.71g, 0.007mol) was slowly added dropwise, the reaction warmed to 20 ℃ and stirred for 3h. After the reaction, the solvent was removed by concentration under reduced pressure at 20 to 25 ℃, and the concentrate was separated by silica gel column chromatography (petroleum ether-ethyl acetate = 8:1) to obtain colorless semisolid (2.27g, 71.5%). The obtained semi-solid is stirred with 10mL of diisopropyl ether for 24h and then is filtered, the filter cake is a compound F-1, and the compound F-1 is dried in vacuum for 4h at 40 ℃ to obtain 0.86g, and the yield is 38.0%. ¹ H-NMR(400MHz,DMSO-d6): δ8.37-8.29(m,2H),7.55-7.49(m,2H),7.48-7.40(m,2H),7.29-7.21 (m,3H),6.71(m,1H),4.12-4.07(m,1H),4.01(d,J＝5.7Hz,2H),1.43(m, 1H),1.31-1.21(m,4H),0.80(t,J＝7.4Hz,6H)；LCMS m/z 454.2[M+H].

Taking a compound F-1 (0.65g, 0.001mol), a compound G (0.33g, 0.001mol), magnesium chloride (0.10g, 0.001mol) and 10mL of acetonitrile, stirring at 50 ℃ for 10min, then adding N, N-diisopropylethylamine (0.39g, 0.003mol), stirring for 20min, cooling to 20 ℃, concentrating at 30-40 ℃ under reduced pressure to remove the acetonitrile, then adding 100mL of ethyl acetate, washing the reaction solution with a 5% citric acid aqueous solution (20 mL), a saturated ammonium chloride aqueous solution (20 mL), a 5% sodium bicarbonate aqueous solution (20 mL) and a saturated saline solution (20 mL), concentrating the organic phase under reduced pressure, and performing column chromatographyThe product was purified by chromatography (petroleum ether-ethyl acetate = 4:1) to give compound H-1 (0.47g, 73.0%) as an off-white solid. ¹ H-NMR(400MHz,methanol-d4):δ7.89 (s,1H),7.28–7.23(m,2H),7.09–7.05(m,3H),6.83(m,2H),5.30(d,J＝ 6.6Hz,1H),4.91(m,1H),4.57-4.51(m,1H),4.35-4.22(m,2H),4.00(m, 1H),3.89(m,1H),3.80(m,1H),1.60(s,3H),1.49-1.43(m,1H),1.40(s, 3H),1.36-1.23(m,4H),0.86(t,J＝7.4Hz,6H)；LCMS m/z 646.1[M+H].

Taking a compound H-1 (0.40g, 0.0006 mol) and 10mL THF, stirring the system, cooling to 5 ℃, then dropwise adding concentrated hydrochloric acid (0.07g, 0.0007 mol), keeping the temperature for 5 ℃, stirring for 2H, adding 5mL water, adjusting the pH to 7-8 by using a saturated sodium bicarbonate solution, extracting the system by using ethyl acetate (10mL x 2), washing by using saturated saline water (5 mL), decompressing and concentrating to obtain a white-like solid, namely a compound 1 crude product, and purifying by using column chromatography (petroleum ether-ethyl acetate = 2:1) to obtain 0.27g, wherein the yield is 75.0%. ¹ H NMR(400MHz,CD3OD):7.81(s，1H)，7.31-7.25 (m，2H)，7.20-7.07(m，3H)，6.91-6.80(m,2H)，1.49-1.38(m，1H)，1.32-1.21(m,4H),0.84(m,6H).LCMS m/z 606.1[M+H].

EXAMPLES 2-7 preparation of Compounds 2-7

The preparation of the compounds 2 to 7 selects different substrates A series and D series, and adopts the synthetic route and the operation steps of the compound 1 to prepare the compound, which is shown in the table 1.

TABLE 1 preparation of Compounds 2 to 7

The beneficial effects of the compounds of the present invention are demonstrated by the following experimental examples.

Experimental example 1 Effect of the Compound of the present invention on SARS-CoV-2 in HAE cell System

1. Experimental methods

HAE cells were plated in 384-well plates and cultured for 24 hours.

SARS-CoV-2 was inoculated onto the culture plate, and the cells were cultured at 37 ℃ for 72 hours. Compounds 1-7 of the present invention and Remdisivir were administered separately. And 24 hours later, detecting the virus factors. And calculate the median effective concentration EC50.

2. The results are shown in Table 2

TABLE 2 effectiveness of the compounds of the invention against SARS-CoV-2

Compound (I)	EC50(um)
		Remdisivir	0.050
Compound 1	0.061
		Compound 2	0.051
Compound 3	0.073
		Compound 4	0.066
Compound 5	0.053
		Compound 6	0.064
Compound 7	0.070

From the above results, it can be seen that the in vitro efficiency of compounds 1 to 7 of the present invention, i.e., deuterium substitution of specific structure of Remdisivir, against SARS-CoV-2 is comparable to Remdisivir, wherein the in vitro efficiency of compounds 2 and 5 against SARS-CoV-2 is almost unchanged, and the effectiveness of Remdisivir is retained.

EXAMPLE 2 study of the half-Life of the Compound of the present invention in animals

Taking 10 SD rats, adopting tail vein injection to carry out administration of the compounds 1-7 and Remdisivir, collecting blood at 8 time points for each animal, drawing a blood concentration curve, and calculating the half-life period of the drug according to the result.

The test results are shown in Table 3.

TABLE 3 in vivo half-life results for the compounds of the invention in animals

Through half-life test results, it can be seen that the compound 1 and the compound 2 of the present invention realize the effect of prolonging the half-life of Remdisivir by deuterium substitution at a specific position, and the prolonging effects are that the compound 1 is prolonged by 13.2% and the compound 2 is prolonged by 26.7%, respectively. The half-life of the compounds 3-5 is basically equivalent to that of Remdisivir, the compounds 3 and 5 are slightly longer, and the half-life of the compounds 6-7 is slightly shorter than that of Remdisivir.

In conclusion, on the basis of keeping the effectiveness on SARS-CoV-2, the compounds 1 and 2 of the invention remarkably prolong the half-life period, reduce the requirement on dosage, reduce side effects and enlarge the treatment window range, therefore, the invention has very good prospect for preparing the medicine for treating the diseases related to coronavirus infection.

Claims (6)

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following structures:

2. use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for combating a coronavirus selected from MERS-CoV, SARS-CoV-2.

3. Use according to claim 2, wherein the coronavirus is SARS-CoV-2.

4. Use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention and/or treatment of a disease caused by a coronavirus infection selected from MERS, SARS, COVID-19.

5. The use according to claim 4, wherein the coronavirus infectious disease is COVID-19.

6. A pharmaceutical composition characterized by: the pharmaceutical composition is a preparation prepared from the compound or the pharmaceutically acceptable salt of the compound of claim 1 and pharmaceutically acceptable auxiliary materials.