in the formula, R₁Represents benzo [ d ]][1,3]Dioxolan-5-yl, substituted phenyl, unsubstituted phenyl, substituted aromatic heterocyclic group or unsubstituted aromatic heterocyclic group;

R₂represents hydrogen, substituted C1-C6 alkyl or unsubstituted C1-C6 alkyl;

R₃represents hydrogen, substituted C1-C6 alkyl or unsubstituted C1-C6 alkyl;

R₂and R₃The same or different;

wherein when said R is₂When represents substituted C1-C6 alkyl, the substituent is hydroxyl, methyl, methoxy, dimethylamino or ethyl;

when said R is₃When represents substituted C1-C6 alkyl, the substituent is hydroxyl, methyl, methoxy, dimethylamino or ethyl;

when said R is₁When represents substituted phenyl, the substituent is fluorine, chlorine, bromine, methoxy, methyl or ethyl;

when said R is₁When representing a substituted aromatic heterocyclic group, the substituent is fluorine, chlorine, bromine, methoxy, methyl or ethyl.

It should be noted that: (1) "phenyl" refers to a group having a benzene ring as a functional group. (2) "aryl" refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. When the ring-constituting atoms contain at least one hetero atom in addition to carbon atoms, an aromatic heterocyclic group is used. (3) "pharmaceutically acceptable salts" refers to those salts that retain the biological effectiveness and properties of the parent compound. The salt comprises: acid addition salts obtained by reaction of the free base of the parent compound with an inorganic acid or with an organic acid; such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid, sulfuric acid, perchloric acid, and the like; such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, fumaric acid, gluconic acid, glutamic acid, isethionic acid, lactic acid, maleic acid, mandelic acid, mucic acid, pamoic acid, pantothenic acid, succinic acid, tartaric acid, malonic acid, or the like; preferably hydrochloric acid or (L) -malic acid; or when the acid proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion, or coordinated with an organic base, a salt is formed; such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like.

Preferably, the active ingredient is at least one of the following compounds:

in any of the above schemes, preferably, the pharmaceutical composition comprises the following components by mass percent, based on 100% of the total mass of the pharmaceutical composition:

the pharmaceutical composition provided by the invention has high specificity and low side effect, improves the capability of an immune system by activating the activity of TLR8, and can effectively treat, relieve and/or prevent various related diseases caused by an immune mechanism, such as cancer, viral infection and the like.

In a second aspect, the present invention provides a method for preparing a pharmaceutical composition according to the first aspect as an agonist of TLR8, comprising the steps of:

sieving the active ingredient and pharmaceutically acceptable adjuvants respectively;

weighing the active ingredients and pharmaceutically acceptable auxiliary materials according to the formula ratio, and uniformly mixing to obtain a first mixture;

compressing the first mixture into a tablet or granulate to obtain a pharmaceutical composition as an agonist of TLR 8.

In a third aspect, the invention provides a use of the pharmaceutical composition as the TLR8 agonist according to the first aspect in the preparation of a medicament for preventing and/or treating tumors and/or viral diseases.

It is noted that an "agonist," also known as an agonist, refers to a class of molecules that enhance the activity of another molecule and promote a response.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the raw materials, instruments, equipment and the like used in the following examples are either commercially available or available by existing methods; the dosage of the experimental reagent is the dosage of the reagent in the conventional experimental operation if no special description exists; the experimental methods are conventional methods unless otherwise specified.

In a first aspect, the embodiment of the present invention provides a pharmaceutical composition as a TLR8 agonist, where the pharmaceutical composition includes the following components in percentage by mass, based on 100% of the total mass of the pharmaceutical composition:

1 to 10 percent of active component;

90-99% of pharmaceutically acceptable auxiliary materials;

the active ingredient is a compound having the structure of formula I:

R₂represents hydrogen, substituted C1-C6 alkyl or unsubstituted C1-C6 alkyl;

R₃represents hydrogen, substituted C1-C6 alkyl or unsubstituted C1-C6 alkyl;

R₂and R₃The same or different;

Further, the active ingredient is at least one of the following compounds:

further, the pharmaceutical composition comprises the following components in percentage by mass based on the total mass of the pharmaceutical composition as 100%:

further, the filler includes at least one of pregelatinized starch, microcrystalline cellulose, starch, lactose, and mannitol.

Further, the disintegrant comprises at least one of sodium carboxymethyl starch, croscarmellose sodium, crospovidone, and low-substituted hydroxypropyl cellulose.

Further, the adhesive comprises at least one of hydroxypropyl cellulose, hypromellose, povidone, and gelatin.

Further, the lubricant comprises at least one of sodium stearyl fumarate, magnesium stearate, aerosil and talcum powder.

Further, the pharmaceutical composition is a common tablet, a dispersible tablet, a chewable tablet, a dry suspension, a granule or a capsule. The pharmaceutical composition provided by the embodiment of the present invention can be prepared into different dosage forms according to clinical needs, such as common tablets, dispersible tablets, chewable tablets, dry suspensions, granules or capsules, and the active ingredient represented by the formula (1) can be contained in a proper amount according to the purpose, object and property of the pharmaceutical composition of the present invention, but usually contained in an amount of 1mg to 5000mg, preferably 10mg to 3,000 mg.

Typical routes of administration of the pharmaceutical compositions of the embodiments of the present invention include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous.

The pharmaceutically effective amount of the pharmaceutical composition of the present embodiment and the administration method or means thereof may be appropriately selected by those skilled in the art depending on the severity of symptoms, treatment regimen, age, body weight, sex, general health status of the patient, and (genetic) race background of the patient. In general, the dosage of the pharmaceutical composition of the present invention (dosage refers to the amount of the active substance) is 0.001 to 10 mg/day/kg body weight, preferably 0.01 to 5 mg/day/kg body weight.

The pharmaceutical compositions may be prepared by methods conventional in the art. For example, when preparing a common tablet, a direct tabletting method can be adopted, namely, all the components are mixed and then tabletted; optionally granulating all the components and tabletting; optionally, a part of the ingredients can be granulated and mixed with the rest ingredients for tabletting. In addition, tablets may be coated by methods commonly used in the art.

Further, the preparation method of the active ingredient comprises the following steps:

(1) synthesis of intermediate III:

reacting the compound II with urea in a first reaction solvent in the presence of alkali to obtain an intermediate III;

(2) synthesis of intermediate IV:

reacting the intermediate III with hydrogen in a second reaction solvent under the action of a catalyst to obtain an intermediate IV;

(3) synthesis of intermediate VI:

heating the intermediate IV and the compound V in a third reaction solvent in the presence of alkali under the action of a catalyst to react to obtain an intermediate VI;

(4) synthesis of intermediate VII:

reacting the intermediate VI with an oxidant in a fourth reaction solvent under the action of a catalyst to obtain an intermediate VII;

(5) synthesis of intermediate VIII:

reacting the intermediate VII with a chlorinated reagent in a fifth reaction solvent in the presence of alkali to obtain an intermediate VIII;

(6) synthesis of Compound I:

and reacting the intermediate VIII with a compound IX in a sixth reaction solvent in the presence of a base to obtain the compound I.

The preparation method of the active ingredient provided by the embodiment of the invention has the advantages of simple and convenient operation, mild conditions, low requirements on equipment conditions, easy realization, simple post-treatment and high yield, and is suitable for industrial large-scale production.

Further, in the step (1), the base includes at least one of sodium methoxide, sodium ethoxide, sodium hydride, sodium tert-butoxide, and potassium tert-butoxide.

Further, in the step (1), the first reaction solvent includes at least one of methanol, ethanol, t-butanol, and N, N-Dimethylformamide (DMF).

Further, in the step (1), the reaction temperature is 0 to 80 ℃ and, for example, the reaction temperature may be 0 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ or 80 ℃.

Further, in the step (2), the catalyst comprises at least one of palladium carbon and raney nickel.

Further, in the step (2), the second reaction solvent includes at least one of methanol, ethanol, and isopropanol.

Further, in the step (2), the reaction temperature is 0 to 80 ℃ and, for example, the reaction temperature may be 0 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ or 80 ℃.

Further, in the step (3), the catalyst comprises at least one of bis (triphenylphosphine) palladium dichloride (II), tetrakis (triphenylphosphine) palladium, bis (dibenzylideneacetone) palladium, palladium acetate, and [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride, and under the action of the catalyst, the intermediate VI can be synthesized quickly.

Further, in the step (3), the base includes at least one of potassium carbonate, cesium carbonate, sodium tert-butoxide, potassium phosphate, and sodium acetate.

Further, in the step (3), the third reaction solvent includes at least one of toluene, dimethyl sulfoxide, N-dimethylformamide, and N, N-dimethylacetamide.

Further, in the step (3), the reaction temperature is 30 to 120 ℃ and, for example, the reaction temperature may be 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃, 110 ℃ or 120 ℃.

Further, in the step (4), the oxidizing agent is sodium periodate.

Further, in the step (4), the catalyst is ruthenium trichloride hydrate.

Further, in the step (4), the fourth reaction solvent includes at least one of ethyl acetate and isopropyl acetate.

Further, in the step (4), the reaction temperature is 20 to 80 ℃ and, for example, the reaction temperature may be 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ or 80 ℃.

Further, in the step (5), the chlorinating reagent comprises at least one of phosphorus oxychloride, phosphorus trichloride and phosphorus pentachloride.

Further, in the step (5), the base includes at least one of triethylamine, N-diisopropylethylamine, and morpholine.

Further, in the step (5), the fifth reaction solvent includes at least one of toluene and acetonitrile.

Further, in the step (5), the reaction temperature is 20 to 120 ℃ and, for example, the reaction temperature may be 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 100 ℃, 110 ℃ or 120 ℃.

Further, in the step (6), the base includes at least one of triethylamine, N-diisopropylethylamine, morpholine, pyridine, potassium carbonate, and cesium carbonate.

Further, in the step (6), the sixth reaction solvent includes at least one of dichloromethane, pyridine, toluene, and N, N-dimethylformamide.

Further, in the step (6), the reaction temperature is 20 to 100 ℃, for example, the reaction temperature may be 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ or 100 ℃.

(1) sieving the active ingredient and pharmaceutically acceptable adjuvants respectively;

(2) weighing the active ingredients and pharmaceutically acceptable auxiliary materials according to the formula ratio, and uniformly mixing to obtain a first mixture;

(3) compressing the first mixture into a tablet or granulate to obtain a pharmaceutical composition as an agonist of TLR 8.

Further, in the step (3), the granulation is dry granulation or wet granulation, the first mixture is granulated to obtain broken dry granules, and the dry granules are granulated by a screen to obtain the first granules.

Further, mixing the first granules with a lubricant to obtain second granules, and subpackaging or encapsulating the second granules to obtain a medicinal composition granular preparation or a capsule preparation.

In a third aspect, the embodiments of the present invention provide a use of the pharmaceutical composition according to the first aspect as a TLR8 agonist in the preparation of a medicament for preventing and/or treating tumors and/or viral diseases.

Further, the tumor is selected from skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, rectal cancer, esophageal cancer, tongue cancer, kidney cancer, cervical cancer, uterine corpus cancer, endometrial cancer, testicular cancer, urinary cancer, melanoma, astrocytic cancer, meningioma, hodgkin's lymphoma, non-hodgkin's lymphoma, acute lymphatic leukemia, chronic lymphatic leukemia, acute myeloid leukemia, chronic myeloid leukemia, adult T-cell leukemia lymphoma, hepatocellular carcinoma, bronchial cancer, small cell lung cancer, non-small cell lung cancer, multiple myeloma, basal cell tumor, seminoma, chondrosarcoma, myosarcoma, fibrosarcoma.

Further, the viral disease is selected from hepatitis A, hepatitis B, and hepatitis C.

The invention is described in further detail with reference to a part of the test results, which are described in detail below with reference to specific examples.

Examples 1-3 provide pharmaceutical compositions as TLR8 agonists in tablet form, including the ingredients in amounts shown in table 1, examples 4-6 provide pharmaceutical compositions as TLR8 agonists in granules, and examples 7-9 provide pharmaceutical compositions as TLR8 agonists in capsule form, including the ingredients in amounts shown in table 3.

TABLE 1 examples 1-3 pharmaceutical composition component content

TABLE 2 examples 4-6 pharmaceutical composition component content

TABLE 3 contents of ingredients of pharmaceutical compositions of examples 7 to 9

Example 1

The content of the ingredients of the pharmaceutical composition as the TLR8 agonist provided in this example is shown in table 1, and the active ingredient of the pharmaceutical composition is (R) -2-amino-7- (3-fluorophenyl) -4- ((1-hydroxy-2-methylhexan-2-yl) amino) -6, 7-dihydropyrido [3,4-d ] pyrimidin-8 (5H) -one.

The preparation method of the pharmaceutical composition provided in this embodiment is as follows:

step (1) preparation of active ingredients:

synthesis of intermediate 1 c:

dissolving compound 1a (297.1g, 1000.0mmol) and compound 1b (60.0g, 1000.0mmol) sodium methoxide (MeONa, 216.0g, 4000.0mmol) in methanol (MeOH, 3000ml), stirring at 25 deg.C for 10 hours, monitoring the reaction by TLC, after the reaction is completed, concentrating under reduced pressure to remove methanol, adding water (500ml) to quench the reaction, extracting the reaction solution after the completion of the reaction twice with ethyl acetate, the volume of ethyl acetate used for each extraction being 1000ml, combining the organic layers, drying the organic layers, concentrating to obtain a pale yellow solid, and recrystallizing with ethyl acetate and petroleum ether to obtain 208.3g of intermediate 1c with a yield of 81.3%, wherein the intermediate 1c is a white solid.

Synthesis of intermediate 1 d:

the intermediate 1c (208g, 812.2mmol) obtained above and 10% palladium on carbon (water content 50% -60%) (20.0g) were dissolved in methanol (MeOH, 1000ml), and were subjected to hydrogenation reaction at 25 ℃ for 8 hours after 2 times of replacement with hydrogen, followed by TLC monitoring reaction, filtration after completion of the reaction, concentration of the filtrate, and column chromatography separation to obtain 113.2g of intermediate 1d, the yield of which was 83.9%, and the intermediate 1d was an off-white solid.

Synthesis of intermediate 1 f:

intermediate 1d (16.6g, 100.0mmol), Compound 1e (17.4g, 100.0mmol), Pd (dppf)₂Cl₂([1,1' -bis (diphenylphosphino) ferrocene)]Palladium dichloride, 3.7g, 5.0mmol), K₂CO₃(27.6g, 200.0mmol) is dissolved in N, N-dimethylformamide (DMF, 200ml), the temperature is raised to 80 ℃, stirring is carried out for 6 hours, TLC is used for monitoring the reaction, the pH is adjusted to 5-6 by dilute hydrochloric acid after the reaction is finished, then ethyl acetate is used for extracting the reaction liquid after the reaction is finished twice, the volume of the ethyl acetate used for each extraction is 200ml, organic layers are combined, the organic layers are dried, filtered, the filtrate is concentrated, and column chromatography separation is carried out to obtain 17.6g of intermediate 1f, the yield is 67.7%, and the intermediate 1f is an off-white solid.

Synthesis of intermediate 1 g:

intermediate 1f (17.0g, 65.4mmol), ruthenium trichloride hydrate (RuCl)₃1.5g, 6.5mmol), sodium periodate (NaIO)₄28.0g, 130.8mmol) in ethyl acetate (EtOAc, 2)00ml) and purified water (200ml), stirring and reacting for 12 hours at 25 ℃, monitoring the reaction by TLC, standing and layering after the reaction is finished to obtain an organic layer 1 and a water layer, extracting the water layer by using ethyl acetate (200ml) to obtain an organic layer 2, combining the organic layer 1 and the organic layer 2, concentrating the organic layer, and carrying out column chromatography separation to obtain 14.4g of an intermediate 1g, wherein the yield is 80.4%, and the intermediate 1g is an off-white solid.

Synthesis of intermediate 1 h:

intermediate 1g (14.0g, 51.1mmol), N-diisopropylethylamine (DIEA, 13.2g, 102.2mmol) were dissolved in Toluene (Toluene, 100ml) and phosphorus oxychloride (POCl) was added at 25 deg.C₃31.1g, 204.4mmol), heating to 80 ℃, stirring for reaction for 4 hours, monitoring the reaction by TLC, adding water (100ml) to quench the reaction after the reaction is finished, drying and concentrating an organic layer, and carrying out column chromatography separation to obtain 11.3g of an intermediate for 1 hour, wherein the yield is 75.7%, and the intermediate for 1 hour is a light yellow oily liquid.

Synthesis of Compound 1:

intermediate 1h (292mg, 1.0mmol), Compound 1i (131mg, 1.0mmol), Triethylamine (Et)₃N, 202mg, 2.0mmol) was dissolved in N, N-dimethylformamide (DMF, 20ml), the reaction was stirred at 60 ℃ for 4 hours, the reaction was monitored by TLC, water (20ml) was added after the reaction was completed to quench the reaction, then the reacted reaction solution after the reaction was completed was extracted twice with ethyl acetate, the volume of ethyl acetate used for each extraction was 30ml, the organic layers were combined, dried, concentrated, and separated by column chromatography to give 167mg of compound 1, the yield was 43.0%, ESI (+) m/z ═ 388.2, and compound 1 was an off-white solid.

Step (2): sieving the active ingredients, microcrystalline cellulose PH101, sodium carboxymethyl starch, and polyvidone K30 with 60 mesh sieve to obtain active ingredients, microcrystalline cellulose PH101, sodium carboxymethyl starch, and polyvidone K30 with particle size meeting the requirement.

And (3): weighing the active ingredients, microcrystalline cellulose PH101, sodium carboxymethyl starch and povidone K30 which meet the particle size requirement and are obtained in the step (2) according to the formula shown in the table 1.

And (4): mixing the active ingredient, the microcrystalline cellulose PH101, the povidone K30 and the sodium carboxymethyl starch in a three-dimensional mixer at 10 revolutions per minute for 30 minutes to obtain a first mixture.

And (5): stearic acid was weighed according to the formula shown in table 1, the magnesium stearate was added to the first mixture, and the mixture was mixed in a three-dimensional mixer at 10 rpm for 5min to obtain a second mixture.

And (6): and (3) pressing the second mixture into tablets by using a rotary tablet press, controlling the tablet hardness to be 5-8 kg, obtaining the pharmaceutical composition tablets serving as the TLR8 agonist, coating the pressed tablets by using a high-efficiency coating machine, wherein the coating material is Opadry series film coating powder of Carlekang company, preparing the coating powder into a coating solution with the mass fraction of 10%, and then coating by using the coating solution, wherein the weight of a coating layer is controlled to be increased by 2%.

And (7): and (5) packaging, installing a mould, a PVC hard sheet and an aluminum foil, and packaging according to 10 sheets/plate.

Example 2

The content of the ingredients of the pharmaceutical composition as the TLR8 agonist provided in this example is shown in table 1, and the active ingredient of the pharmaceutical composition is 2-amino-7- (3-fluorophenyl) -4- ((1, 3-dimethoxypropan-2-yl) amino) -6, 7-dihydropyrido [3,4-d ] pyrimidin-8 (5H) -one.

step (1) preparation of active ingredients:

the synthesis of intermediate 1c, intermediate 1d, intermediate 1f, intermediate 1g and intermediate 1h was carried out according to the synthesis method shown in example 1.

Synthesis of Compound 2:

intermediate 1h (292mg, 1.0mmol), Compound 2a (119mg, 1.0mmol), Triethylamine (Et)₃N, 202mg, 2.0mmol) in N, N-dimethylformamide (DMF, 20ml), heating to 60 deg.C, stirring for 4 hr, monitoring by TLC, adding water (20ml) after the reaction is completed, and quenchingThe reaction mixture was extracted twice with ethyl acetate (30 ml volume) each time, the organic layers were combined, dried, concentrated, and separated by column chromatography to give 195mg of compound 2, 52.0% yield (ESI (+) m/z ═ 376.2), compound 2 was an off-white solid.

The steps (2) to (7) are the same as in example 1.

The dissolution test (the test conditions are the same as the effect example 2) is carried out on the pharmaceutical composition preparation which is obtained in the example 2 and is used as the TLR8 agonist, and the dissolution of the pharmaceutical composition preparation at 30min is 96.3%.

Example 3

The content of the ingredients of the pharmaceutical composition as the TLR8 agonist provided in this example is shown in table 1, and the active ingredient of the pharmaceutical composition is 2-amino-7- (3-fluorophenyl) -4- ((1- (dimethylamino) -3-methoxypropan-2-yl) amino) -6, 7-dihydropyrido [3,4-d ] pyrimidin-8 (5H) -one.

step (1) preparation of active ingredients:

Synthesis of Compound 3:

intermediate 1h (292mg, 1.0mmol), Compound 3a (132mg, 1.0mmol), Triethylamine (Et)₃N, 202mg, 2.0mmol) was dissolved in N, N-dimethylformamide (DMF, 20ml), the reaction was stirred at 60 ℃ for 4 hours, the reaction was monitored by TLC, water (20ml) was added after the reaction was completed to quench the reaction, then the reacted reaction solution after the reaction was completed was extracted twice with ethyl acetate, the volume of ethyl acetate used for each extraction was 30ml, the organic layers were combined, dried, concentrated, and separated by column chromatography to give 208mg of compound 3, yield was 53.6%, ESI (+) m/z ═ 389.2, and compound 3 was an off-white solid.

The steps (2) to (7) are the same as in example 1.

The dissolution test (the test conditions are the same as the effect example 2) is carried out on the pharmaceutical composition preparation which is used as the TLR8 agonist and is obtained in example 3, and the dissolution of the pharmaceutical composition preparation at 30min is 95.8%.

Example 4

The content of the ingredients of the pharmaceutical composition as the TLR8 agonist provided in this example is shown in table 2, and the active ingredient of the pharmaceutical composition is 2-amino-7- (3-fluorophenyl) -4- ((1- (dimethylamino) -5-methoxypentane-3-yl) amino) -6, 7-dihydropyrido [3,4-d ] pyrimidin-8 (5H) -one.

step (1) preparation of active ingredients:

Synthesis of Compound 4:

intermediate 1h (292mg, 1.0mmol), Compound 4a (160mg, 1.0mmol), Triethylamine (Et)₃N, 202mg, 2.0mmol) was dissolved in N, N-dimethylformamide (DMF, 20ml), the reaction was stirred at 60 ℃ for 4 hours, the TLC monitored, after the reaction was completed, water (20ml) was added to quench the reaction, then the reacted reaction solution was extracted twice with ethyl acetate, the volume of ethyl acetate used for each extraction was 30ml, the organic layers were combined, dried, concentrated, and separated by column chromatography to give 188mg of compound 4, the yield was 45.2%, ESI (+) m/z ═ 417.2, and compound 4 was an off-white solid.

Step (2): respectively sieving the active ingredient, the microcrystalline cellulose PH101, the sodium carboxymethyl starch and the hydroxypropyl cellulose through a 60-mesh sieve to obtain the active ingredient, the microcrystalline cellulose PH101, the sodium carboxymethyl starch and the hydroxypropyl cellulose which meet the particle size requirement, and weighing the active ingredient, the microcrystalline cellulose PH101, the sodium carboxymethyl starch and the hydroxypropyl cellulose according to the formula shown in the table 2 for later use.

And (3): dissolving hydroxypropyl cellulose in purified water to obtain a hydroxypropyl cellulose solution with the mass fraction of 5%, adding the active ingredient, microcrystalline cellulose PH101 and sodium carboxymethyl starch into a wet granulator, mixing for 5min, adding the hydroxypropyl cellulose solution with the mass fraction of 5% into the wet granulator, and preparing granules to obtain first granules.

And (4): and (4) putting the first particles obtained in the step (3) into a fluidized bed for drying, wherein the air inlet temperature of the fluidized bed is 50-70 ℃, the material temperature is 30-40 ℃, the drying is carried out until the moisture of the particles is 2% -4%, so as to obtain dry particles, and the obtained dry particles are sieved by a 20-mesh sieve for finishing, so as to obtain second particles.

And (5): weighing magnesium stearate according to the formula shown in table 2, mixing the second granules obtained in step (4) and magnesium stearate in a three-dimensional mixer at a speed of 10 rpm for 15min, and preparing third granules.

And (6): and (4) subpackaging the third granules obtained in the step (5) to obtain the medicinal composition granule preparation serving as the TLR8 agonist.

Example 5

The content of the ingredients of the pharmaceutical composition as the TLR8 agonist provided in this example is shown in table 2, and the active ingredient of the pharmaceutical composition is 2-amino-7- (3-fluorophenyl) -4- ((1-hydroxy-5-methoxypentan-3-yl) amino) -6, 7-dihydropyrido [3,4-d ] pyrimidin-8 (5H) -one.

step (1) preparation of active ingredients:

the synthesis of intermediate 1c, intermediate 1d, intermediate 1f, intermediate 1gg and intermediate 1h was carried out according to the synthesis method shown in example 1.

Synthesis of Compound 5:

intermediate 1h (292mg, 1.0mmol), Compound 5a (133mg, 1.0mmol), Triethylamine (Et)₃N, 202mg, 2.0mmol) was dissolved in N, N-dimethylformamide (DMF, 20ml), the reaction was stirred at 60 ℃ for 4 hours, the reaction was monitored by TLC, water (20ml) was added after the reaction was completed to quench the reaction, then the reacted reaction solution was extracted twice with ethyl acetate, the volume of ethyl acetate used for each extraction was 30ml, the organic layers were combined, dried, concentrated, and separated by column chromatography to give 212mg of compound 5, the yield was 54.5%, ESI (+) m/z was 390.2, and compound 5 was an off-white solid.

The steps (2) to (6) are the same as in example 4.

The dissolution test (the test conditions are the same as the effect example 2) is carried out on the pharmaceutical composition preparation which is obtained in the example 5 and is used as the TLR8 agonist, and the dissolution of the pharmaceutical composition preparation at 30min is measured to be 94%.

Example 6

The content of the ingredients of the pharmaceutical composition as the TLR8 agonist provided in this example is shown in table 2, and the active ingredient of the pharmaceutical composition is 2-amino-7- (3-fluoro-4-methoxyphenyl) -4- ((1-hydroxy-2-methylhexan-2-yl) amino) -6, 7-dihydropyrido [3,4-d ] pyrimidin-8 (5H) -one.

step (1) preparation of active ingredients:

the synthesis of intermediate 1c and intermediate 1d was performed according to the synthesis method shown in example 1.

Synthesis of intermediate 6 b:

intermediate 1d (16.6g, 100.0mmol), Compound 6a (20.5g, 100.0mmol), Pd (dppf)₂Cl₂([1,1' -bis (diphenylphosphino) ferrocene)]Palladium dichloride, 3.7g, 5.0mmol), K₂CO₃(27.6g, 200.0mmol) was dissolved in N, N-dimethylformamide (DMF, 200ml), the reaction was stirred for 8 hours while warming to 80 ℃ and monitored by TLCAfter the reaction is finished, the pH value is adjusted to 5-6 by using diluted hydrochloric acid, then the reaction liquid after the reaction is finished is extracted twice by using ethyl acetate, the volume of the ethyl acetate used in each extraction is 200ml, organic layers are combined, dried, filtered, concentrated, and subjected to column chromatography separation to obtain 19.0g of an intermediate 6b, the yield is 65.5%, and the intermediate 6b is a white-like solid.

Synthesis of intermediate 6 c:

intermediate 6b (19.0g, 65.5mmol), ruthenium trichloride hydrate (RuCl)₃1.5g, 6.5mmol), sodium periodate (NaIO)₄28.0g, 130.8mmol) was dissolved in ethyl acetate (EtOAc, 200ml) and purified water (200ml), the reaction was stirred at 25 ℃ for 12 hours, TLC monitored, after the reaction was complete, the reaction was allowed to stand and separate into layers to give organic layer 1 and an aqueous layer, the aqueous layer was extracted with ethyl acetate (200ml) to give organic layer 2, organic layer 1 and organic layer 2 were combined, the organic layer was concentrated and separated by column chromatography to give 15.3g of intermediate 6c, yield 76.8%, intermediate 6c was a white-like solid.

Synthesis of intermediate 6 d:

intermediate 6c (15.0g, 49.3mmol), N-diisopropylethylamine (DIEA, 12.7g, 98.7mmol) were dissolved in Toluene (Toluene, 100ml) and phosphorus oxychloride (POCl) was added at 25 deg.C₃30.0g of 197.4mmol), heating to 80 ℃, stirring for reaction for 4 hours, monitoring the reaction by TLC, adding water (100ml) to quench the reaction after the reaction is finished, drying an organic layer, concentrating, and carrying out column chromatography separation to obtain 9.8g of intermediate 6d, wherein the yield is 61.8 percent, and the intermediate 6d is light yellow oily liquid.

Synthesis of Compound 6:

intermediate 6d (322mg, 1.0mmol), Compound 1i (131mg, 1.0mmol), Triethylamine (Et)₃N, 202mg, 2.0mmol) was dissolved in N, N-dimethylformamide (DMF, 20ml), the reaction was stirred at 60 ℃ for 4 hours, the reaction was monitored by TLC, water (20ml) was added after the reaction was completed to quench the reaction, then the reacted reaction solution was extracted twice with ethyl acetate, the volume of ethyl acetate used for each extraction was 30ml, the organic layers were combined, dried, concentrated, and separated by column chromatography to give 230mg of compound 6, the yield was 55.2%, ESI (+) m/z was 418.2, and compound 6 was an off-white solid.

The steps (2) to (6) are the same as in example 4.

The dissolution test (the test conditions are the same as the effect example 2) is carried out on the pharmaceutical composition preparation which is obtained in the example 6 and is used as the TLR8 agonist, and the dissolution of the pharmaceutical composition preparation at 30min is 93.8%.

Example 7

The content of the ingredients of the pharmaceutical composition as the TLR8 agonist provided in this example is shown in table 3, and the active ingredient of the pharmaceutical composition is 2-amino-7- (3-fluoro-4-methoxyphenyl) -4- ((1, 3-dimethoxypropan-2-yl) amino) -6, 7-dihydropyrido [3,4-d ] pyrimidin-8 (5H) -one.

step (1) preparation of active ingredients:

the synthesis of intermediate 1c and intermediate 1d was performed according to the synthesis method shown in example 1, and the synthesis of intermediate 6b, intermediate 6c, and intermediate 6d was performed according to the synthesis method shown in example 6.

Synthesis of compound 7:

intermediate 6d (322mg, 1.0mmol), Compound 2a (119mg, 1.0mmol), Triethylamine (Et)₃N, 202mg, 2.0mmol) was dissolved in N, N-dimethylformamide (DMF, 20ml), the reaction was stirred at 60 ℃ for 4 hours, the reaction was monitored by TLC, water (20ml) was added after the reaction was completed to quench the reaction, then the reacted reaction solution was extracted twice with ethyl acetate, the volume of ethyl acetate used for each extraction was 30ml, the organic layers were combined, dried, concentrated, and separated by column chromatography to give 255mg of compound 7, the yield was 63.0%, ESI (+) m/z was 406.2, and compound 7 was an off-white solid.

Step (2): the active ingredients, the pregelatinized starch, the sodium carboxymethyl starch and the povidone K30 are respectively screened by a 60-mesh screen to obtain the active ingredients, the pregelatinized starch, the sodium carboxymethyl starch and the povidone K30 which meet the particle size requirement, and the active ingredients, the pregelatinized starch, the sodium carboxymethyl starch and the povidone K30 are weighed according to the formula shown in Table 3 for later use.

And (3): dissolving povidone K30 in purified water to obtain povidone K30 solution with the mass fraction of 5%, adding the active ingredients, pregelatinized starch and sodium carboxymethyl starch into a wet granulator, mixing, adding the povidone K30 solution with the mass fraction of 5% into the wet granulator, mixing, and preparing granules to obtain the first granules.

And (4): the procedure was exactly the same as in step (4) in example 4.

And (5): weighing magnesium stearate according to the formula shown in table 3, mixing the second granules obtained in step (4) and magnesium stearate in a three-dimensional mixer at a speed of 10 rpm for 20min, and preparing third granules.

And (6): and (4) putting the third granules obtained in the step (5) into a capsule filling machine for filling, and carrying out aluminum-plastic packaging on the obtained capsule to obtain the pharmaceutical composition capsule preparation serving as the TLR8 agonist.

Example 8

The content of the ingredients of the pharmaceutical composition as the TLR8 agonist provided in this example is shown in table 3, and the active ingredient of the pharmaceutical composition is 2-amino-7- (3-fluoro-4-methoxyphenyl) -4- ((1- (dimethylamino) -3-methoxypropan-2-yl) amino) -6, 7-dihydropyrido [3,4-d ] pyrimidin-8 (5H) -one.

step (1) preparation of active ingredients:

Synthesis of compound 8:

intermediate 6d (322mg, 1.0mmol) was combinedSubstance 3a (132mg, 1.0mmol), triethylamine (Et)₃N, 202mg, 2.0mmol) was dissolved in N, N-dimethylformamide (DMF, 20ml), the reaction was stirred at 60 ℃ for 4 hours, the reaction was monitored by TLC, water (20ml) was added after the reaction was completed to quench the reaction, then the reacted reaction solution after the reaction was completed was extracted twice with ethyl acetate, the volume of ethyl acetate used for each extraction was 30ml, the organic layers were combined, dried, concentrated, and separated by column chromatography to obtain 262mg of compound 8, the yield was 62.7%, ESI (+) m/z ═ 419.2, and compound 8 was an off-white solid.

The steps (2) to (6) are the same as in example 7.

The dissolution test (the test conditions are the same as the effect example 2) is carried out on the pharmaceutical composition preparation which is obtained in the example 8 and is used as the TLR8 agonist, and the dissolution of the pharmaceutical composition preparation at 30min is measured to be 92.5%.

Example 9

The content of the ingredients of the pharmaceutical composition as the TLR8 agonist provided in this example is shown in table 3, and the active ingredient of the pharmaceutical composition is 2-amino-7- (3-fluoro-4-methoxyphenyl) -4- ((1- (dimethylamino) -5-methoxypentan-3-yl) amino) -6, 7-dihydropyrido [3,4-d ] pyrimidin-8 (5H) -one.

step (1) preparation of active ingredients:

Synthesis of compound 9:

intermediate 6d (322mg, 1.0mmol), Compound 4a (160mg, 1.0mmol), Triethylamine (Et)₃N, 202mg, 2.0mmol) was dissolved in N, N-dimethylformamide (DMF, 20ml), the temperature was raised to 60 ℃ and the reaction was stirred for 4 hours, the reaction was monitored by TLC, after completion of the reaction, water (20ml) was added to quench the reaction, after which ethyl acetate was usedThe reaction mixture after the completion of the reaction was extracted twice with a volume of ethyl acetate of 30ml for each extraction, and the organic layers were combined, dried, concentrated, and separated by column chromatography to give 225mg of compound 9 in 50.4% yield (ESI (+) m/z) ═ 447.2, and compound 9 was an off-white solid.

The steps (2) to (6) are the same as in example 7.

The dissolution test (the test conditions are the same as the effect example 2) is carried out on the pharmaceutical composition preparation which is used as the TLR8 agonist and is obtained in example 9, and the dissolution of the pharmaceutical composition preparation at 30min is 91.3%.

Effect example 1 in vitro Activity test of TLR8 with active ingredients of examples 1 to 9

The method is carried out in human embryonic kidney cells (HEK293) expressing TLR8 family members by adopting a cell test method, after an active ingredient activates TLR, downstream NF-kB is activated, and then a secretory embryonic alkaline phosphatase reporter gene is activated, and the activity of an embryonic alkaline phosphatase is detected by a Quanti-blue (InvivoGen) reagent so as to reflect the activity of a TLR8 agonist.

The detailed experimental method is as follows:

HEK-BLUE-hTLR8 cell line was purchased from Invivogen, and cultured in DMEM medium containing 4.5g/L glucose (Sigma-Aldrich) and 10% fetal bovine serum at 37 deg.C, 95% humidity and 5% CO₂。

The active ingredient was tested in 10 concentration gradients from 0.5nM to 15. mu.M. TLR8 agonist of known activity was added as a positive control, and 1 μ L dimethyl sulfoxide (DMSO) as a negative control.

The cells were treated as follows: the cells were removed from the culture dish and centrifuged to remove the medium, resuspended in a T-150 flask with 10mL of pre-warmed phosphate buffered saline (PBS, pH7.4), 12mL of pre-warmed medium was added, gently pipetted up and down, and counted under the microscope. 200,000 single cell suspensions per mL were immediately prepared in medium and 200. mu.L/well (40,000/well) were added to 96-well plates. The final concentration of DMSO was 0.5%.

Adding active ingredients, and adding 5% CO at 37 deg.C₂The culture was carried out in an incubator for 24 hours.

Pipette 20. mu.L/well of supernatant into 180. mu.L of 37 ℃ preheated Quanti-Blue, incubate at 37 ℃ for 1.5 hours, and detect absorbance (OD value) with a spectrophotometer at 650 nm. The calculation formula of the activation effect is as follows:

effect% (OD average of dosing group-DMSO group OD average)/(OD average of positive drug group-DMSO group OD average) × 100.

The concentration-effect curves were fitted with Graphpad software and EC calculated₅₀。

Note: the positive control is VTX-2337(motolimod), chemical name: 2-amino-N, N-dipropyl-8- (4- (pyrrolidine-1-carbonyl) phenyl) -3H-benzo [ b ] azepine-4-carboxamide.

EC₅₀Specific results for the values are given in the following table:

+ 1-10 μ M, + 0.1-1 μ M, and ++ < 0.1 μ M

Examples	EC₅₀(TLR8)	Examples	EC₅₀(TLR8)
				Compound 1	++	Compound 6	++
Compound 2	+	Compound 7	+
				Compound 3	+++	Compound 8	+
Compound 4	+	Compound 9	+++
				Compound 5	+++	Positive control	++

As can be seen from the above table, compound 3, compound 5 and compound 9 were the most potent for activating TLR 8.

Effect example 2 determination of dissolution rates of pharmaceutical compositions provided in example 1, example 4 and example 7

Dissolution tests were performed on the formulations of the pharmaceutical compositions provided in example 1, example 4 and example 7, respectively, as TLR8 agonists.

The test method comprises the following steps: reference is made to the second method of determination of dissolution and release (2015 edition "chinese pharmacopoeia", the fourth general rule 0931;

the instrument comprises the following steps: an ultraviolet spectrophotometer and a dissolution tester;

dissolution medium: pH4.5 acetate buffer solution; volume of dissolution medium: 1000 ml; rotating speed: 50 revolutions per minute;

sampling time: 5min, 10min, 15min, 20min, 30 min.

The following test results were obtained:

as can be seen from the above table, the pharmaceutical composition preparations prepared according to examples 1, 4 and 7 have a dissolution rate of more than 90% at 30min, and have a better dissolution rate and a faster dissolution rate.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. The pharmaceutical composition as the TLR8 agonist is characterized by comprising the following components in percentage by mass based on 100% of the total mass of the pharmaceutical composition:

1 to 10 percent of active component;

90-99% of pharmaceutically acceptable auxiliary materials;

the active ingredient is at least one of the following compounds or pharmaceutically acceptable salts thereof:

2. the pharmaceutical composition as the TLR8 agonist according to claim 1, wherein the pharmaceutical composition comprises the following components in percentage by mass based on 100% of the total mass of the pharmaceutical composition:

3. the pharmaceutical composition for use as a TLR8 agonist according to claim 2, wherein the filler comprises at least one of pregelatinized starch, microcrystalline cellulose, starch, lactose, mannitol.

4. The pharmaceutical composition for use as a TLR8 agonist according to claim 2, wherein the disintegrant comprises at least one of sodium carboxymethyl starch, croscarmellose sodium, crospovidone, low substituted hydroxypropyl cellulose.

5. The pharmaceutical composition for use as a TLR8 agonist according to claim 2, wherein the binding agent comprises at least one of hydroxypropyl cellulose, hypromellose, povidone, gelatin.

6. The pharmaceutical composition for use as a TLR8 agonist according to claim 2, wherein the lubricant comprises at least one of sodium stearyl fumarate, magnesium stearate, aerosil, talc.

7. The pharmaceutical composition for use as the TLR8 agonist according to claim 1 or 2, wherein the pharmaceutical composition is in the form of a plain tablet, a dispersible tablet, a chewable tablet, a dry suspension, a granule or a capsule.

8. A method of preparing a pharmaceutical composition as a TLR8 agonist according to claim 1 or 2, comprising the steps of:

9. Use of the pharmaceutical composition as TLR8 agonist according to claim 1 or 2 in the manufacture of a medicament for the prevention and/or treatment of a tumor and/or a viral disease.