CN112028892B - Application of 4-amino-pyrrolotriazine derivative in preparation of anti-pulmonary fibrosis preparation - Google Patents

Abstract

The invention relates to the technical field of anti-pulmonary fibrosis preparations, in particular to application of a 4-amino-pyrrolotriazine derivative in preparation of an anti-pulmonary fibrosis preparation, wherein the 4-amino-pyrrolotriazine derivative has a structural formula shown in the specification, can prevent and/or treat pulmonary fibrosis diseases induced by common chronic inflammation, infection, environmental agents, radiation, chronic symptoms, drugs, chemical poisons and the like, effectively inhibits neuraminidase activity in lung tissues, reduces the content of collagen fibers in the lung tissues, slows down the development degree of pulmonary fibrosis lesions, and can be used as or for preparing drugs capable of preventing and/or treating the pulmonary fibrosis diseases.

Description

Application of 4-amino-pyrrolotriazine derivative in preparation of anti-pulmonary fibrosis preparation

Technical Field

The invention relates to the technical field of anti-pulmonary fibrosis preparations, in particular to application of a 4-amino-pyrrolotriazine derivative in preparation of an anti-pulmonary fibrosis preparation.

Background

Pulmonary Fibrosis (PF) is a diffuse interstitial pulmonary disease characterized by inflammation and extracellular matrix deposition, which is progressive and fatal. The etiology of most patients with pulmonary fibrosis is unknown (idiopathic), the group of diseases is called Idiopathic Interstitial Pneumonia (IIP), and the most common disease type with pulmonary fibrosis as a main manifestation form in the Idiopathic Interstitial Pneumonia (IIP) is Idiopathic Pulmonary Fibrosis (IPF), which is a serious interstitial lung disease that can cause progressive loss of lung function, and is one of the interstitial lung diseases, the incidence of which is the highest and the prognosis of which is very poor. Idiopathic Interstitial Pneumonia (IIP) is classified into the following categories: (1) common interstitial pneumonia: idiopathic pulmonary fibrosis, idiopathic nonspecific interstitial pneumonia, respiratory bronchial interstitial pneumonia, desquamation interstitial pneumonia, cryptogenic interstitial pneumonia, acute interstitial pneumonia; (2) rare idiopathic interstitial pneumonia: idiopathic lymphatic interstitial pneumonia, idiopathic pleuropneumoniae parenchymal elasticity fiber hyperplasia; (3) non-categorical interstitial pneumonia.

Clinically, pulmonary fibrosis is manifested by progressive dyspnea and pulmonary dysfunction, which seriously affect the respiratory function of human body, manifested by dry cough and progressive dyspnea (insufficient conscious qi), and the respiratory function of patients is continuously worsened with the aggravation of illness and pulmonary injury. The morbidity and mortality of the idiopathic pulmonary fibrosis are increased year by year, the 5-year mortality of the idiopathic pulmonary fibrosis can reach 50-70 percent after diagnosis, the mortality is higher than that of most tumors, and the idiopathic pulmonary fibrosis is called a tumor-like disease. Pulmonary fibrosis usually occurs in 40-50 years old, and men are more likely to develop in women. Dyspnea is the most common symptom of pulmonary fibrosis. In mild pulmonary fibrosis, dyspnea often occurs during vigorous activity, and is therefore often overlooked or misdiagnosed as another disease. When pulmonary fibrosis progresses, dyspnea also occurs at rest, and progressive dyspnea may occur in patients with severe pulmonary fibrosis. Other symptoms include dry cough and hypodynamia. Some patients had clubbing and cyanosis. Severe consequences of lung tissue fibrosis lead to structural changes in normal lung tissue and loss of function. When a large amount of fibrous tissue without gas exchange function replaces alveoli, oxygen cannot enter the blood. Patients with breathing disorder, anoxia, acidosis, loss of labor force, and death in the end.

Glucocorticoid is a first-choice treatment method for idiopathic pulmonary fibrosis for a long time, is particularly suitable for treating acute-stage pulmonary fibrosis, but has a weak curative effect on chronic-stage pulmonary fibrosis. In addition, the anti-inflammatory action of the glucocorticoid has short maintenance time and poor long-term curative effect, and a new way for treating idiopathic pulmonary fibrosis is always searched clinically.

The above background disclosure is only for the purpose of assisting understanding of the inventive concept and technical solutions of the present invention, and does not necessarily belong to the prior art of the present patent application, and should not be used for evaluating the novelty and inventive step of the present application in the case that there is no clear evidence that the above content is disclosed at the filing date of the present patent application.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of a 4-amino-pyrrolotriazine derivative in the preparation of an anti-pulmonary fibrosis preparation, wherein the derivative can effectively inhibit neuraminidase activity in lung tissue, reduce collagen fiber content in lung tissue, prevent and/or treat pulmonary fibrosis diseases, and slow down the development degree of pulmonary fibrosis diseases.

In order to achieve the above object, the present invention provides the following aspects [1] to [5 ].

[1] A 4-amino-pyrrolotriazine derivative having the formula (1):

in the formula (1), R is a C7-C8 alkyl group or a C7-C8 aryl group, preferably a benzyl group or a phenethyl group.

In some preferred embodiments, the 4-amino-pyrrolotriazine derivatives include, but are not limited to:

5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid heptyl ester;

5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid octyl ester;

5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid benzyl ester;

5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid phenethyl ester.

In the process of preparing and researching the activity of the 4-amino-pyrrolotriazine derivative, it is unexpectedly found that the 4-amino-pyrrolotriazine derivative can prevent and/or treat pulmonary fibrosis diseases induced by common chronic inflammation, infection, environmental agents, radiation, chronic symptoms, drugs, chemical poisons and the like, effectively inhibit neuraminidase activity in lung tissues, reduce collagen fiber content in lung tissues and slow the development degree of pulmonary fibrosis lesions, and therefore, the 4-amino-pyrrolotriazine derivative can be used as or for preparing drugs capable of preventing and/or treating pulmonary fibrosis diseases.

[2] The 4-amino-pyrrolotriazine derivative described in the item [1] is produced by a method comprising the steps of:

1) 1 weight part of 3-methyl-2-cyanopyrrole-4-formic ether is completely dissolved in 5-6 volume parts of DMF, cooled to minus 25 to minus 22, added with 0.6-0.65 weight part of NaH under stirring and continuously stirred for at least 45min; slowly dripping 150 parts by volume of solution containing 0.15-0.18 mol/LNH into the reaction solution ₂ Stirring the Cl solution in ether at constant temperature for at least 2h, heating to room temperature and stirringReacting for at least 1h; saturated Na ₂ S ₂ O ₃ Quenching the aqueous solution to obtain an organic phase, and separating by silica gel column chromatography to obtain an intermediate product 1;

2) Dissolving the intermediate product 1 in formamide of 5-10 weight times, heating to 165-170 ℃, refluxing for at least 5h, tracking by TLC until the reaction is complete, adding purified water of 2-3 weight times of formamide into the reaction system, and performing suction filtration to obtain an intermediate product 2;

3) Sequentially adding 3.8-4.0 parts by weight of 1-bromo-2-chloroethane and 1 part by weight of potassium carbonate into 14-20 parts by volume of DMF (dimethyl formamide), heating to 60-65 ℃, adding 1 part by weight of intermediate product 2 under stirring, tracking by TLC (thin layer chromatography) until the reaction is complete, filtering off the potassium carbonate while hot, concentrating the filtrate, adding ice water, rapidly stirring for at least 30min, carrying out vacuum drying on the filter cake after suction filtration, and carrying out silica gel column chromatography separation to obtain the 4-amino-pyrrolotriazine derivative.

In some preferred embodiments, in step 1) of preparing the 4-amino-pyrrolotriazine derivative, the 3-methyl-2-cyanopyrrole-4-carboxylic acid ester is an ester of 3-methyl-2-cyanopyrrole-4-carboxylic acid with a C7-C8 alkyl alcohol or a C7-C8 aromatic alcohol, preferably benzyl alcohol or phenethyl alcohol.

In some preferred embodiments, in the step 1) of preparing the 4-amino-pyrrolotriazine derivative, the rotation speed of stirring is not lower than 300r/min.

In some preferred embodiments, in the step 1) of preparing the 4-amino-pyrrolotriazine derivative, the dropping rate of the slow dropping is 5 to 20mL/min.

In some preferred embodiments, in step 1) of preparing the 4-amino-pyrrolotriazine derivative, the temperature increase rate is not higher than 5 ℃/min.

In some preferred embodiments, in step 1) of preparing the 4-amino-pyrrolotriazine derivative, the organic phase includes an organic phase obtained by preliminary separation and an organic phase obtained after the aqueous phase is extracted with diethyl ether at least 3 times; and the organic phase is washed by saturated saline solution, dried by anhydrous sodium sulfate and concentrated before being separated by silica gel column chromatography.

In some preferred embodiments, in step 1) of preparing the 4-amino-pyrrolotriazine derivative, the mobile phase in the silica gel column chromatography separation is ethyl acetate: petroleum ether =1:4 (V: V).

In some preferred embodiments, in the step 2) of preparing the 4-amino-pyrrolotriazine derivative, the temperature increase rate is controlled to be 5 to 10 ℃/min.

In some preferred embodiments, in the step 3) of preparing the 4-amino-pyrrolotriazine derivative, the temperature rising rate is controlled to be 3 to 5 ℃/min.

In some preferred embodiments, in the step 3) of preparing the 4-amino-pyrrolotriazine derivative, the stirring rate is not less than 150r/min.

In some preferred embodiments, in the step 3) of preparing the 4-amino-pyrrolotriazine derivative, the rapid stirring rate is not less than 900r/min.

In some preferred embodiments, in the step 3) of preparing the 4-amino-pyrrolotriazine derivative, vacuum drying means vacuum drying at a temperature of 50 to 60 ℃ for at least 5 hours.

In some preferred embodiments, in step 3) of preparing the 4-amino-pyrrolotriazine derivative, the mobile phase in the silica gel column chromatography separation is ethyl acetate: petroleum ether =1:1 (V: V).

The method adopts a reasonable and feasible synthetic route to prepare and obtain a plurality of 4-amino-pyrrolotriazine derivatives, the raw materials required by the synthetic route are cheap and easy to obtain, the reaction time is short, the reaction conditions are easy to control, the post-treatment is simple, the target product can be obtained with the total yield of more than 50% through three steps of reactions, a new path is opened up for enriching the types of the 4-amino-pyrrolotriazine derivatives, and the obtained derivatives have excellent biological activity.

[3] A composition comprising at least one 4-amino-pyrrolotriazine derivative as described in item [1] or [2] and a pharmaceutically acceptable carrier.

In some preferred embodiments, the composition comprises at least one compound of formula (1) according to the present invention as an active ingredient and at least one inorganic or organic, solid or liquid pharmaceutically acceptable carrier or excipient.

In other preferred embodiments, the pharmaceutically acceptable carrier is a variety of pharmaceutically commonly used adjuvants and/or excipients, including, but not limited to, sugars (such as lactose, glucose, or sucrose), starches (such as corn starch or potato starch), cellulose and its derivatives (such as sodium carboxymethylcellulose, ethyl cellulose, or methyl cellulose), malt, gelatin, talc, solid lubricants (such as stearic acid or magnesium stearate), calcium sulfate, vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, or cocoa butter), polyols (such as propylene glycol, glycerol, sorbitol, mannitol, or polyethylene glycol), alginic acid, emulsifiers, wetting agents (such as sodium lauryl sulfate), colorants, flavors, tableting agents, stabilizers, antioxidants, preservatives, pyrogen-free water, isotonic saline solutions, phosphate buffers, and the like; the carrier can improve the stability, activity, bioavailability and the like of the formula according to needs, or generate acceptable taste or smell in the case of oral administration.

In other preferred embodiments, the composition can be prepared into any conventional form including oral preparations and injection preparations according to a general method in pharmacy. The oral preparation is preferably tablet, granule, pill, powder, syrup, decoction and capsule; more preferably tablets, granules, pills and capsules.

[4] Use of the 4-amino-pyrrolotriazine derivative described in the item [1] or [2] in the preparation of an agent for inhibiting the activity of neuraminidase in lung tissue.

[5] Use of the 4-amino-pyrrolotriazine derivative described in the item [1] or [2] for the preparation of a preparation for pulmonary fibrosis disease resistance.

In some preferred embodiments, the pulmonary fibrotic disease comprises a pulmonary fibrotic disease induced by a common chronic inflammation, infection, environmental agent, radiation, chronic condition, drug, or chemical poison.

The above-described preferred conditions may be combined with each other to obtain a specific embodiment, in accordance with common knowledge in the art.

The raw materials or reagents involved in the invention are all common commercial products, and the operations involved are all routine operations in the field unless otherwise specified.

The invention has the beneficial effects that:

1) The 4-amino-pyrrolotriazine derivative can effectively inhibit the activity of neuraminidase in lung tissues, reduce the content of collagen fibers in the lung tissues, prevent and/or treat pulmonary fibrosis diseases induced by common chronic inflammation, infection, environmental agents, radiation, chronic disease states, drugs, chemical poisons and the like, and slow down the development degree of pulmonary fibrosis lesions;

2) The method adopts a reasonable and feasible synthetic route to prepare and obtain a plurality of 4-amino-pyrrolotriazine derivatives, the raw materials required by the synthetic route are cheap and easy to obtain, the reaction time is short, the reaction conditions are easy to control, the post-treatment is simple, the target product can be obtained with the total yield of more than 50 percent through three steps of reactions, a new path is opened up for enriching the types of the 4-amino-pyrrolotriazine derivatives, and the obtained derivatives have excellent biological activity.

The invention adopts the technical scheme for achieving the purpose, makes up the defects of the prior art, and has reasonable design and convenient operation.

Drawings

These and/or other objects, features, advantages and embodiments of the present invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings in which:

FIG. 1 is a structural formula of the 4-amino-pyrrolotriazine derivative of the present invention;

FIG. 2 is a scheme of the synthesis of 4-amino-pyrrolotriazine derivatives according to the invention;

FIG. 3 is a hydrogen spectrum of heptyl 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate in example 1 of the present invention;

FIG. 4 is a hydrogen spectrum of octyl 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate in example 2 of this invention:

FIG. 5 is a hydrogen spectrum of benzyl 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate in example 3 of this invention:

FIG. 6 is a hydrogen spectrum of 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid phenethyl ester in example 4 of the present invention:

FIG. 7 is a schematic diagram showing the effect of the 4-amino-pyrrolotriazine derivative of the present invention on HYP content in lung tissue of bleomycin-induced pulmonary fibrosis mice.

Detailed Description

Those skilled in the art can appropriately substitute and/or modify the process parameters to implement the present disclosure, but it is specifically noted that all similar substitutes and/or modifications will be apparent to those skilled in the art and are deemed to be included in the present invention. While the products and methods of making described herein have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the products and methods of making described herein may be made and utilized without departing from the spirit and scope of the invention.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The present invention uses the methods and materials described herein; other suitable methods and materials known in the art may be used. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein, and the like, are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

All percentages, parts, ratios, etc., are by weight unless otherwise indicated; additional instructions include, but are not limited to, "wt%" means weight percent, "mol%" means mole percent, "vol%" means volume percent.

Unless otherwise indicated, parts by weight and parts by volume are present in the same reaction step or in the same formulation, i.e., it is understood that "1 part by weight of A … … 2 parts by volume of B" is intended to include the understanding of "1g of A … … mL of B", "5g of A … … 10mL of B", "10mg of A … … 20 μ L of B", or "1kg of A … … L of B", etc., i.e., the terms "parts by weight" and "parts by volume" are understood to refer to the volumes corresponding to equal weights of water at ambient temperature and pressure.

When an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5 (1 to 5)" is described, the described range is understood to include ranges of "1 to 4 (1 to 4)", "1 to 3 (1 to 3)", "1 to 2 (1 to 2) and 4 to 5 (4 to 5)", "1 to 3 (1 to 3) and 5", and the like. Where numerical ranges are described herein, unless otherwise stated, the ranges are intended to include the endpoints of the ranges, and all integers and fractions within the ranges.

When the term "about" is used to describe a numerical value or an end point value of a range, the disclosure should be understood to include the specific value or end point referred to.

Furthermore, "or" means "or" unless expressly indicated to the contrary, rather than "or" exclusively. For example, condition a "or" B "applies to any of the following conditions: a is true (or present) and B is false (or not present), a is false (or not present) and B is true (or present), and both a and B are true (or present).

In addition, the indefinite articles "a" and "an" preceding an element or component of the invention are intended to mean no limitation on the number of occurrences (i.e., occurrences) of the element or component. Thus, "a" or "an" should be understood to include one or at least one and the singular forms of an element or component also include the plural unless the singular is explicitly stated.

It should be noted that, in this document, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation. The use of the phrase "comprising one of the elements does not exclude the presence of other like elements in the process, method, article, or apparatus that comprises the element.

The materials, methods, and examples described herein are illustrative only and not intended to be limiting unless otherwise specified. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

The synthetic route of the 4-amino-pyrrolotriazine derivative is shown as the following formula.

The present invention is described in detail below.

Example 1: 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid heptyl ester:

this example provides a 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid heptyl ester, which is synthesized by the following steps:

1) Completely dissolving 1g of 3-methyl-2-cyanopyrrole-4-heptylic acid ester in 6mL of DMF, cooling to-25 ℃, adding 0.6g of NaH while stirring, and continuously stirring at 300r/min for 45min; 150mL of a solution containing 0.15mol/LNH was slowly added dropwise to the reaction solution at a rate of 10mL/min ₂ Stirring the ether solution of Cl for 2h at constant temperature, heating to room temperature at the temperature of 5 ℃/min, and stirring for reaction for 1h; saturated Na ₂ S ₂ O ₃ Quenching the aqueous solution to obtain an organic phase, extracting the aqueous phase with diethyl ether for 3 times, combining all the organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, concentrating, and separating with silica gel column chromatography (mobile phase is ethyl acetate: petroleum ether =1:4 (V: V)) to obtain 0.992g of intermediate product 1, with a yield of 93.8%;

2) Dissolving 2g of intermediate product 1 in 16g of formamide, heating to 170 ℃ at the speed of 10 ℃/min, refluxing for 5h, tracking by TLC until the reaction is complete, adding 40g of purified water into the reaction system, and performing suction filtration to obtain 1.943g of intermediate product 2, wherein the yield is 88.5%;

3) Sequentially adding 4.0g of 1-bromo-2-chloroethane and 1g of potassium carbonate into 15ml of mixed solution of sodium hydroxide, heating to 64 ℃ at a temperature of 4 ℃/min, adding 1g of intermediate product 2 while stirring at 180r/min, tracking by TLC until the reaction is completed, filtering the potassium carbonate while the potassium carbonate is hot, concentrating the filtrate, adding ice water, quickly stirring for 30min at a speed of 900r/min, performing suction filtration, performing vacuum drying on the filter cake for 5h at a temperature of 60 ℃, and performing silica gel column chromatography separation (the mobile phase is ethyl acetate: petroleum ether =1:1 (V: V)) to obtain 0.664g of 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-heptyl formate with the yield of 61.2%; the total yield is 50.8%.

The hydrogen spectrum of the heptylpyrrolo 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate of this example is shown in FIG. 3.

Example 2: 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid octyl ester:

this example provides octyl 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate, the synthetic procedure is the same as example 1, and the specific steps are as follows:

1) Completely dissolving 1.5g of 3-methyl-2-cyanopyrrole-4-octyl formate in 9mL of DMF, cooling to-22 ℃, adding 0.9g of NaH under stirring, and continuously stirring for 50min at 450r/min; 150mL of a solution containing 0.16mol/LNH was slowly added dropwise to the reaction solution at a rate of 12mL/min ₂ Continuously stirring the Cl ether solution for 2h at constant temperature, heating to room temperature at 4 ℃/min, and stirring for reacting for 1h; saturated Na ₂ S ₂ O ₃ Quenching the aqueous solution to obtain an organic phase, extracting the aqueous phase with diethyl ether for 3 times, combining all the organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, concentrating, and separating with silica gel column chromatography (mobile phase is ethyl acetate: petroleum ether =1:4 (V: V)) to obtain 1.469g of intermediate product 1, with a yield of 92.9%;

2) Dissolving 1g of intermediate product 1 in 10g of formamide, heating to 170 ℃ at the speed of 8 ℃/min, refluxing for 5h, tracking by TLC until the reaction is complete, adding 25g of purified water into the reaction system, and performing suction filtration to obtain 0.972g of intermediate product 2 with the yield of 89.0%;

3) Sequentially adding 3.9g1-bromo-2-chloroethane and 1g of potassium carbonate into 15mLDMF, heating to 64 ℃ at 5 ℃/min, adding 1g of intermediate product 2 while stirring at 300r/min, tracking by TLC until the reaction is completed, filtering off the potassium carbonate while the reaction is hot, concentrating the filtrate, adding ice water, rapidly stirring at 1200r/min for 30min, vacuum-drying the filter cake at 55 ℃ for 5h after suction filtration, and separating by silica gel column chromatography (the mobile phase is ethyl acetate: petroleum ether =1:1 (V: V)) to obtain 0.671g5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-octyl formate with the yield of 62.1%; the total yield is 51.3%.

The hydrogen spectrum of octyl 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate of this example is shown in FIG. 4.

Example 3: 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid benzyl ester:

this example provides 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid benzyl ester, the synthetic procedure is the same as example 1, and the specific steps are as follows:

1) 2g of 3-methyl-2-cyanopyrrole-4-carboxylic acid benzyl ester is completely dissolved in 10mL of DMF, the mixture is cooled to-24 ℃, 1.2gNaH and 360r/min are added under stirring, and the mixture is continuously stirred for 60min; 150mL of a solution containing 0.18mol/LNH was slowly added dropwise to the reaction solution at a rate of 12mL/min ₂ Stirring the ether solution of Cl for 2h at constant temperature, heating to room temperature at the temperature of 3 ℃/min, and stirring for reaction for 1h; saturated Na ₂ S ₂ O ₃ Quenching the aqueous solution to obtain an organic phase, extracting the aqueous phase with diethyl ether for 3 times, combining all the organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, concentrating, and separating with silica gel column chromatography (mobile phase is ethyl acetate: petroleum ether =1:4 (V: V)) to obtain 1.987g of intermediate product 1, with a yield of 93.0%;

2) Dissolving 2g of intermediate product 1 in 20g of formamide, heating to 165 ℃ at the speed of 5 ℃/min, refluxing for 5h, tracking by TLC until the reaction is complete, adding 40g of purified water into the reaction system, and performing suction filtration to obtain 1.950g of intermediate product 2 with the yield of 88.6%;

3) Sequentially adding 8g of 1-bromo-2-chloroethane and 2g of potassium carbonate into 15ml DMF, heating to 64 ℃ at 4 ℃/min, adding 2g of intermediate product 2 while stirring at 300r/min, tracking by TLC (thin layer chromatography) until the reaction is complete, filtering off the potassium carbonate while the potassium carbonate is hot, concentrating the filtrate, adding ice water, rapidly stirring at 1500r/min for 30min, vacuum-drying the filter cake at 55 ℃ for 8h after suction filtration, and separating by silica gel column chromatography (the mobile phase is ethyl acetate: petroleum ether =1:1 (V: V)) to obtain 1.33g5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-benzoic acid benzyl ester, wherein the yield is 61.3%; the total yield is 50.5%.

The hydrogen spectrum of benzyl 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate of this example is shown in FIG. 5.

Example 4: 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid phenethyl ester:

this example provides 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid phenethyl ester, which has the same synthetic steps as in example 1, specifically including the following steps:

1) 2g of 3-methyl-2-cyanopyrrole-4-carboxylic acid phenethyl ester is completely dissolved in 12mL of DMF, cooled to-22 ℃, added with 1.3g of NaH under stirring, and continuously stirred for 45min at 600r/min; 150mL of a solution containing 0.15mol/LNH was slowly added dropwise to the reaction solution at a rate of 20mL/min ₂ Stirring the ether solution of Cl for 2h at constant temperature, heating to room temperature at the speed of 2 ℃/min, and stirring for reaction for 1h; saturated Na ₂ S ₂ O ₃ Quenching the aqueous solution to obtain an organic phase, extracting the water phase with diethyl ether for 3 times, combining all the organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, concentrating, and separating with silica gel column chromatography (the mobile phase is ethyl acetate: petroleum ether =1:4 (V: V)) to obtain 1.989g of intermediate product 1, wherein the yield is 94.2%;

2) Dissolving 2g of intermediate product 1 in 20g of formamide, heating to 168 ℃ at the speed of 8 ℃/min, refluxing for 5h, tracking by TLC until the reaction is complete, adding 50g of purified water into the reaction system, and performing suction filtration to obtain 1.892g of intermediate product 2 with the yield of 86.4%;

3) Sequentially adding 10g of 1-bromo-2-chloroethane and 2.5g of potassium carbonate into 15ml of mixed solution of sodium chloride and potassium carbonate, heating to 63 ℃ at the speed of 3 ℃/min, adding 2.5g of intermediate product 2 while stirring at the speed of 450r/min, tracking by TLC until the reaction is completed, filtering off the potassium carbonate while the potassium carbonate is hot, concentrating the filtrate, adding ice water, rapidly stirring for 30min at the speed of 1000r/min, performing suction filtration, performing vacuum drying on the filter cake for 12h at the temperature of 50 ℃, and performing silica gel column chromatography separation (the mobile phase is ethyl acetate: petroleum ether =1:1 (V: V)) to obtain 1.701g5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-benzoic acid phenethyl ester, wherein the yield is 62.8%; the total yield is 51.1%.

The hydrogen spectrum of 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid phenethyl ester of this example is shown in FIG. 6.

Experimental example 1: in vivo experiments to inhibit neuraminidase activity in lung tissue:

male SPF mice (20-25 g) which are healthy, physiological and uniform in weight are taken to be bred adaptively for 7 days, and then are divided into 11 groups, each group is 10, and the groups are divided into 1 control group, 1 model group and 9 administration groups, the control group is injected with sterilized normal saline with the same amount as that of other groups through a trachea, and the model group and the administration groups are injected with bleomycin with 5mg/kg per kilogram for molding. After 2h, 5mg/kg/d oseltamivir phosphate and high and low doses of the derivatives (5 mg/kg/d and 25 mg/kg/d) of examples 1 to 4 were respectively administered to the mice in the administration group by tracheal injection, and the mice in the control group and the model group were administered with an equal volume of a vehicle, 0.5% sodium carboxymethylcellulose by intragastric administration for 3 weeks. The abdominal aorta was sacrificed by exsanguination 2h after the last dose and lung tissue was taken.

The lung tissue is placed on ice, washed clean with ice-cold physiological saline, the surface water is sucked off by filter paper, weighed, homogenate containing 0.25mmol/L sucrose and 1mmol/L LEDTA is added according to the proportion of 500g/L, supernatant is taken after centrifugation at 2000g multiplied by 10min, supernatant is taken after centrifugation at 80000g multiplied by 100min, and the neuraminidase level in the lung tissue is detected by ELISA method. The test results are shown in table 1.

TABLE 1 neuraminidase Activity in Lung tissue

Experiments prove that the 4-amino-pyrrolotriazine derivatives in examples 1 to 4 of the present application can effectively inhibit the neuraminidase activity in lung tissues, particularly, the benzyl 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate in example 3 and the phenethyl 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate in example 4 have better inhibition effects on the neuraminidase activity in lung tissues and both show dose dependence.

Experimental example 2: pulmonary fibrosis pharmacodynamic experiment:

male ICR mice (25-30 g) which are healthy, physiological and uniform in weight are taken to be bred adaptively for 7 days, and then divided into 11 groups, 20 mice in each group are divided into 1 control group, 1 model group, 1 positive drug group and 8 experimental compound administration groups, the control group is injected with sterilized normal saline with the same amount as that of other groups through a trachea, and the model group and the administration groups are injected with bleomycin with 5mg/kg per kilogram for molding through the trachea. Beginning at 7d after molding, performing intragastric perfusion with equivalent sterilized normal saline every day for the control group and the model group, performing intragastric perfusion with 10mg/kg/d prednisone acetate for the positive drug group, performing intragastric perfusion with 10mg/kg/d derivatives of examples 1-4 for the experimental compound administration group every day, and continuously performing intragastric perfusion for 4 weeks. Mice were sacrificed by cervical dislocation after anesthesia at 14d and 28d, respectively, weight records were weighed, lung tissues were taken and placed on ice, rinsed with ice-cold physiological saline, filter paper was blotted dry to remove surface moisture and weighed, and the lung coefficient was calculated, which = lung weight (mg)/weight (g). The statistical results are shown in tables 2 and 3.

TABLE 2 Effect on mouse body weight

Group of	0d body weight (g)	7d body weight (g)	14d body weight (g)	28d body weight (g)
					Control group	27.8±1.2	31.5±1.6	35.2±1.4	42.1±2.0
Model set	27.6±1.6	26.1±1.5	23.5±1.9	16.0±1.5
					Positive drug group	27.4±1.8	25.0±1.9	27.9±1.8	34.5±1.6
EXAMPLE 1 group	26.5±1.6	24.6±1.5	26.4±1.4	31.9±1.8
					EXAMPLE 2 group	27.2±1.9	23.8±1.7	26.0±1.8	30.4±1.4
EXAMPLE 3 group	27.9±1.2	25.4±1.3	27.6±1.5	33.3±1.3
					Examples4 groups of	26.7±1.6	24.3±1.8	28.2±1.3	35.8±1.8

As can be seen from table 2, compared with the control group, the weight of the mice in the model group is significantly reduced, which indicates that the bleomycin-induced pulmonary fibrosis of the mice seriously affects the growth of the mice, and compared with the mice in the model group, the weight gains of the mice in the positive drug group and the mice in the groups of examples 1 to 4 of the present application are significant, which indicates that the 4-amino-pyrrolotriazine derivatives in the examples 1 to 4 of the present application can effectively improve the body constitution of the mice with bleomycin-induced pulmonary fibrosis, and reduce the reduction degree of the weight of the mice caused by the pulmonary fibrosis, even improve the weight of the mice in the later period, and slowly increase the weight of the mice.

TABLE 3 Effect on pulmonary coefficients in mice

As can be seen from table 3 above, compared with the normal control group, the pulmonary factor of the model group modeled by bleomycin is obviously improved, which indicates that the lung weight/body weight ratio of the model group is improved, and the pulmonary fibrosis is severe, while the pulmonary factors of the mice in the positive drug group and the groups of examples 1 to 4 of the present application are reduced compared with the model group, which indicates that the homotrophic drugs of the 4-amino-pyrrolotriazine derivatives in examples 1 to 4 of the present application are similar to each other, so that the pulmonary fibrosis of the mice induced by the drugs can be improved, and the disease development process of the mice can be slowed down.

Hydroxyproline (HYP) is an amino acid obtained by hydrolysis of connective tissue protein, has a high content (about 13%) in collagen, plays a key role in the stability of collagen, and since collagen contains much HYP, the HYP content can be measured to indirectly reflect the change of the total amount of tissue collagen, and the catabolic condition of collagen can be known, so that the HYP content in lung tissue is detected by a digestion method at 14d and 28d after molding, and the statistical result is shown in fig. 7.

As can be seen from fig. 7, the amount of HYP in the lung tissue of the mice in the model group is significantly increased, and compared with the model group, the amount of HYP in the lung tissue of the mice in the positive drug group and the groups of mice in examples 1 to 4 of the present application shows a significantly decreased trend, indicating that the 4-amino-pyrrolotriazine derivative disclosed in the present application intervenes in the lung tissue of the mice with pulmonary fibrosis caused by bleomycin, and can further improve pulmonary fibrosis, so that the 4-amino-pyrrolotriazine derivative disclosed in the present application can be used as a drug for preventing and/or treating pulmonary fibrosis, or can be used for preparing an anti-pulmonary fibrosis preparation.

Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.

In view of the numerous embodiments of the present invention, the experimental data of each embodiment is huge and is not suitable for being listed and explained herein one by one, but the contents to be verified and the final conclusions obtained by each embodiment are close. Therefore, the contents of the verification of the respective examples are not described one by one, and the excellent points of the present invention will be described only by representative examples 1 to 4 and experimental examples 1 to 2.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

While the above detailed description has shown, described, and pointed out novel features as applied to various embodiments, it will be understood that various omissions, substitutions, and changes in the form and details of the device or method illustrated may be made without departing from the spirit of the disclosure. In addition, the various features and methods described above may be used independently of one another, or may be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of the present disclosure. Many of the embodiments described above include similar components, and thus, these similar components are interchangeable in different embodiments. While the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and obvious modifications and equivalents thereof. Accordingly, the invention is not intended to be limited by the specific disclosure of preferred embodiments herein.

Claims (9)

1. A 4-amino-pyrrolotriazine derivative having the formula (1):

   

   in the formula (1), R is C7-C8 alkyl or C7-C8 aryl.
2. 2. The derivative according to claim 1, characterized in that: the 4-amino-pyrrolotriazine derivatives include, but are not limited to:

   heptylp 5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate;

   5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid octyl ester;

   5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid benzyl ester;

   5-methyl-4- (1-aziridinyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid phenethyl ester.
3. 3. A process for preparing a 4-amino-pyrrolotriazine derivative as claimed in claim 1 or 2, characterized by comprising the steps of:

   1) 1 weight part of 3-methyl-2-cyanopyrrole-4-formic ether is completely dissolved in 5-6 volume parts of DMF, cooled to minus 25 to minus 22, added with 0.6-0.65 weight part of NaH under stirring and continuously stirred for at least 45min; slowly dripping 150 parts by volume of solution containing 0.15-0.18 mol/LNH into the reaction solution ₂ Keeping stirring the Cl ether solution for at least 2h at constant temperature, heating to room temperature, and stirring for reacting for at least 1h; saturated Na ₂ S ₂ O ₃ Quenching the aqueous solution to obtain an organic phase, and separating by silica gel column chromatography to obtain an intermediate product 1;

   2) Dissolving the intermediate product 1 in formamide of 5-10 weight times, heating to 165-170 ℃, refluxing for at least 5h, tracking by TLC until the reaction is complete, adding purified water of 2-3 weight times of formamide into the reaction system, and performing suction filtration to obtain an intermediate product 2;

   3) Sequentially adding 3.8-4.0 parts by weight of 1-bromo-2-chloroethane and 1 part by weight of potassium carbonate into 14-20 parts by volume of DMF (dimethyl formamide), heating to 60-65 ℃, adding 1 part by weight of intermediate product 2 under stirring, tracking by TLC (thin layer chromatography) until the reaction is complete, filtering off the potassium carbonate while hot, concentrating the filtrate, adding ice water, rapidly stirring for at least 30min, carrying out vacuum drying on the filter cake after suction filtration, and carrying out silica gel column chromatography separation to obtain the 4-amino-pyrrolotriazine derivative;

   the route of the process for preparing 4-amino-pyrrolotriazine derivatives of claim 1 or 2 is:

   
4. 4. the method of claim 3, wherein: in the step 1), the 3-methyl-2-cyanopyrrole-4-formic acid ester is an ester of 3-methyl-2-cyanopyrrole-4-formic acid and C7-C8 alkyl alcohol or C7-C8 aromatic alcohol.
5. 5. The method according to claim 3 or 4, characterized in that: in the step 1), the organic phase comprises an organic phase obtained by preliminary separation and an organic phase obtained by extracting the aqueous phase for at least 3 times by using diethyl ether; and the organic phase is washed by saturated saline solution, dried by anhydrous sodium sulfate and concentrated before being separated by silica gel column chromatography.
6. 6. A composition comprising at least one 4-amino-pyrrolotriazine derivative of any of claims 1 to 5 and a pharmaceutically acceptable carrier.
7. 7. The composition of claim 6, wherein: the composition can be prepared into any conventional form according to a general method in pharmacy, and comprises an oral preparation and an injection preparation.
8. 8. Use of a 4-amino-pyrrolotriazine derivative as claimed in any of claims 1 to 5 for the preparation of an agent for inhibiting the activity of neuraminidase in lung tissue.
9. 9. Use of a 4-amino-pyrrolotriazine derivative as claimed in any of claims 1 to 5 for the preparation of a preparation against pulmonary fibrotic diseases.

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