CN110724174B - Pyrrolotriazine compound, composition and application thereof - Google Patents

Abstract

The invention relates to a pyrrolotriazine compound, a composition and application thereof, wherein the pyrrolotriazine compound has a structure shown in formula (I)

Wherein R is₁–R₁₁Is as defined in claim 1. The pyrrolotriazine compound has excellent antiviral effect, particularly on feline coronavirus, and can be used for preparing medicines for treating diseases infected by the feline coronavirus, such as feline infectious peritonitis.

Description

Pyrrolotriazine compound, composition and application thereof

Technical Field

The invention relates to the field of virus infection medicines, in particular to a pyrrolotriazine compound, a composition and application thereof.

Background

Feline coronavirus (FCoV) is a positive-stranded RNA virus, which is 80-160 nm in diameter, is enveloped, single-tube RNA, a common infectious virus, and is transmitted through feline feces. FCoV infected cats have a 5-10% chance of becoming infectious peritonitis (FIP), a chronic, progressive, infectious disease of cats characterized primarily by peritonitis and large volumes of pleural effusion. The mortality rate of sick cats is higher, and most medicaments have no treatment effect on the sick cats. Feline coronavirus is an RNA virus which is easy to mutate, has low enzyme activity of a fault repair mechanism in a replication process, so that the feline coronavirus has a very high mutation generation speed, and a vaccine is difficult to develop because the vaccine is developed and researched according to a fixed gene or protein of the virus. At present, the cat infectious peritonitis is mainly treated by conservative treatment and is matched with broad-spectrum antibiotics and antiviral drugs to prevent secondary infection, but the drugs have large side effects and cannot cure the secondary infection. Since there is no effective symptomatic treatment drug for feline infectious peritonitis, there is a need to develop a corresponding therapy.

Disclosure of Invention

Based on the above, there is a need for pyrrolotriazine compounds, compositions and uses thereof, which have antiviral effects, especially against feline coronavirus.

A pyrrolotriazine compound with a structure shown as a formula (I)

Wherein R is₁And R₂Each independently is OR₁₂Or NR₁₃R₁₄(ii) a Or R₁、R₂And R₁And R₂The linked P together form a substituted or unsubstituted 5-8 membered heterocyclic ring;

R₁₂selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or

R₁₃And R₁₄One of them is

The other is H or

R₃-R₁₁、R₁₅、R₁₆、R₁₇And R₁₈Each independently selected from: H. substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aromatic group, substituted or unsubstituted heteroaromatic group, ketone group, carbonyl group, carboxyl group, ester group, alkoxycarbonyl group, aryloxycarbonyl group, amino group, cyano group, carbamoyl group, haloformyl group, isocyano group, isocyanate group, thiocyanate group, isothiocyanate group, hydroxyl group, nitro group or halogen;

R₁₉and R₂₀Each independently selected from: H. a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted heteroaromatic group.

In one embodiment, the compound has a structure shown in formula (II):

in one embodiment, R₁₂Selected from phenyl, naphthyl, anthracenyl, pyridyl or

The phenyl, naphthyl, anthracenyl or pyridinyl group is optionally further substituted by one or more of the following groups: C1-C6 alkyl, C1-C6 alkoxy, 3-8 membered cycloalkyl, hydroxy, cyano or halogen;

R₁₃and R₁₄One of them is

The other is H;

R₁₅、R₁₆、R₁₇and R₁₈Each independently selected from: H. C1-C8 alkyl or 3-8 membered cycloalkyl; the C1-C8 alkyl or 3-8 membered cycloalkyl is optionally further substituted with one or more of the following: C1-C6 alkyl, C1-C6 alkoxy, 3-8 membered cycloalkyl, hydroxy, cyano or halogen;

R₁₉and R₂₀Each independently selected from: H. C1-C8 alkyl or 3-8 membered cycloalkyl.

In one embodiment, R₁₂Selected from phenyl, naphthyl, anthracenyl, pyridyl or

In one embodiment, R₁₃And R₁₄One of them is

The other is H.

In one embodiment, the phenyl, naphthyl, anthracenyl or pyridinyl is optionally further substituted with methoxy or halogen, and the substitution is para.

In one embodiment, R₁₅、R₁₆、R₁₇And R₁₈Each independently selected from: h or C1-C6 alkyl.

In one embodiment, R₁₉And R₂₀Each independently selected from: h or C1-C6 alkyl.

In one embodiment, R₁₉And R₂₀Each independently selected from: isopropyl, tert-butyl, 1-ethylbutyl.

In one embodiment, R is₁And R₂Each taken independently from the structure:

in one embodiment, the pyrrolotriazine compound has a structure shown in formula (III)

R₂₁Selected from: H. substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aromatic group, substituted or unsubstituted heteroaromatic group, ketone group, carbonyl group, carboxyl group, ester group, alkoxycarbonyl group, aryloxycarbonyl group, amino group, cyano group, carbamoyl group, haloformyl group, isocyano group, isocyanate group, thiocyanate group, isothiocyanate group, hydroxyl group, nitro group or halogen.

In one embodiment, R is₂₁Selected from C1-C8 alkyl, aryl or heteroaryl, said C1-C8 alkyl, aryl or heteroaryl being optionally further substituted with one or more of the following: C1-C6 alkyl, C1-C6 alkoxy, 3-8 membered cycloalkyl, hydroxy, cyano or halogen.

In one embodiment, R is₂₁Selected from aryl or heteroaryl, said aryl or heteroaryl being optionally further substituted by halogen.

In one embodiment, R is₂₁Is para-halogen substituted phenyl.

In one embodiment, R is₂₁For para-chlorine extractionA substituted phenyl group.

In one embodiment, R is₅Is H, hydroxy, halogen or C1-C6 alkyl.

In one embodiment, R is₅H, hydroxyl, F or methyl.

In one embodiment, R is₇Selected from: H. C1-C6 alkyl, cyano or halogen.

In one embodiment, R is₇Selected from: H. methyl, ethyl, cyano or F.

In one embodiment, R₉-R₁₁Each independently H, C1-C6 alkyl or halogen.

In one embodiment, R is₉-R₁₁Are all H.

The preparation method of the pyrrolotriazine compound comprises the following steps:

providing a compound represented by the formula (I-1);

reacting a compound shown as a formula (I-1) with a compound shown as a formula (I-2) to obtain a compound shown as a formula (I);

wherein X represents a leaving group.

In one embodiment, X is

Or a halogen.

In one embodiment, the step of providing the compound of formula (I-1) comprises the steps of:

reacting the formula (I-3) with the formula (I-4) to obtain a formula (I-1);

wherein M represents halogen.

A pyrrolotriazine composition comprises a first component and a second component, wherein the first component is the pyrrolotriazine compound, and the second component is an interferon inducer.

In one embodiment, the interferon inducer is one or more of a TLR7 agonist and a STING agonist. These two agonists are associated with innate immunity and are stimulatory genes for interferon IFN, which can upregulate IFN expression. For example, the mechanism of action of STING agonists: generally, the DNA of eukaryotic cells can be immobilized and moved in the nucleus, and only when an unexpected condition occurs, the DNA can flow into the cytoplasm, for example, the cell is damaged, and apoptosis begins, for example, the invasion of external pathogens (viruses, bacteria and the like) and the introduction of exogenous DNA fragments and the like. At this time, the DNA sensor in the cytoplasm is found in time, and corresponding remedial measures are started. The DNA sensors can directly resist the replication of virus or bacteria on one hand, and can rapidly and directly activate the immunity of the body on the other hand. The cell will respond according to these reliable signals through different pathways: such as activation of downstream inflammatory factors by NF κ B, or downstream release of cytokines such as IL-1 β by Caspase 1 activation, or induction of autophagy mediated by p62 and NDP52, such as induction of apoptosis by Caspase 9 (apoptosis) or activation of apoptosis by RIP3 (necrosis). Therefore, the interferon inducer can be used as an adjuvant of the medicament to kill viruses together.

In one embodiment, the TLR7 agonist is PF-4878691, Gardiquimod, or Resiquimod; the STING agonist is a compound with CAS number 2138498-18-5 or a compound with CAS number 2138299-29-1.

In one embodiment, the pyrrolotriazine composition further comprises pharmaceutically acceptable auxiliary materials.

A salt of a pyrrolotriazine compound is prepared from the pyrrolotriazine compound.

The application of the pyrrolotriazine compound or the pyrrolotriazine composition in preparing antiviral drugs.

In one embodiment, the antiviral drug is a drug for treating a disease caused by feline coronavirus infection.

In one embodiment, the disease caused by feline coronavirus infection is feline infectious peritonitis.

A method of treating a disease caused by feline coronavirus infection comprising the steps of:

an effective amount of the above-mentioned pyrrolotriazine compound or the above-mentioned pyrrolotriazine composition is administered.

The pyrrolotriazine compound has excellent antiviral effect, particularly on feline coronavirus, and can be used for preparing medicines for treating diseases infected by the feline coronavirus, such as feline infectious peritonitis.

In the pyrrolotriazine compound, furan is used for replacing pyrrolotriazine as a parent nucleus, and phosphate groups are innovatively introduced into furan substituents, so that compared with the traditional nucleoside compound, the water solubility is improved, and the defects that the traditional medicine is difficult to dissolve, the lipid-water distribution coefficient clogP is less than 0, the performance of penetrating through cell membranes is poor, the in-vivo bioavailability is low and the like are overcome. In addition, the nucleoside medicament shows that the medicinal effect can be embodied only after phosphorylation by phosphokinase in vivo, a phosphorylation step is a speed-determining step, the level of phosphorylation directly influences the exertion of the medicinal effect, the nucleoside medicament has larger defects in the medicament forming property, and the pyrrolotriazine compound well improves the defects, improves the membrane penetration rate and the bioavailability, greatly improves the medicinal effect, reduces the dosage and reduces the possibility of side effects.

Drawings

FIG. 1 shows a graph of the change in body weight before and after treatment in 20 test cats (vertical axis unit: kg);

FIG. 2 shows the change in the number of leukocytes before and after treatment of 20 test cats (vertical axis unit: 1X 10)⁹One/liter);

figure 3 shows the change in white to bulb ratio before and after treatment for 20 test cats.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all 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 terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Definitions and general terms

Unless stated to the contrary, the following terms used in the specification and claims have the following meanings.

The term "optionally substituted with one or more substituents" in the present invention means substituted with one or more substituents, or unsubstituted. In particular, "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "C1-C8 alkyl is optionally substituted with one or more hydroxy groups" means that the hydroxy group may, but need not, be present, and this specification includes the case where C1-C8 alkyl is substituted with hydroxy groups and the case where C1-C8 alkyl is not substituted with hydroxy groups.

"alkyl" refers to a saturated aliphatic hydrocarbon group, including straight and branched chain groups. C₁-C₆Alkyl refers to an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl. C₁-C₄Alkyl refers to an alkyl group containing 1 to 4 carbon atoms. In one embodiment, C₁-C₄The alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl or sec-butyl. Alkyl groups may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment.

"cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbyl substituent. 3-8 membered cycloalkyl is meant to include 3 to 8 carbon atoms. In one embodiment, the 3-8 membered monocyclic cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. Polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups. Cycloalkyl groups may be optionally substituted with one or more substituents.

"Heterocyclyl" means a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent in which one or more ring atoms are selected from nitrogen, oxygen, or S (O)_m(wherein m is an integer of 0 to 2), preferably a nitrogen or oxygen heteroatom; but not the ring moiety of-O-, -O-S-or-S-, the remaining ring atoms being carbon. 4-10 membered heterocyclyl is a ring containing 4 to 10 ring atoms, of which 1-3 are heteroatoms; preferably, the heterocyclyl ring contains 5 to 6 ring atoms of which 1-2 are heteroatoms. In one embodiment, the monocyclic heterocyclyl is dihydrofuranyl, tetrahydrofuranyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, or homopiperazinyl, and the like.

"aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group having a conjugated pi-electron system, preferably 6 to 10 membered, more preferably phenyl and naphthyl, most preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, and the aryl group may be substituted or unsubstituted.

A 5-10 membered "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms, 5 to 10 ring atoms, wherein the heteroatoms include oxygen, sulfur, and nitrogen. Heteroaryl is preferably 5-or 6-membered, for example furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, the ring to which the parent structure is attached being a heteroaryl ring. Heteroaryl groups may be optionally substituted or unsubstituted.

The substituent "amino" in the present invention includes primary, secondary and tertiary amino groups, and specifically, the amino group includes-NR₂₀R₂₁Wherein R is₂₀And R₂₁Is a hydrogen atom or any optional group such as: H. a substituted or unsubstituted straight-chain alkyl group, a substituted or unsubstituted branched-chain alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted heteroaromatic group, and the like.

Alkoxy groups include-O- (alkyl) and-O- (cycloalkyl). Wherein the alkyl and cycloalkyl groups are as defined above. In one embodiment, C₁-C₄Alkoxy is methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy or cyclobutyloxy. Alkoxy groups may be optionally substituted or unsubstituted.

"carbonyl" means "-CO-"; "carboxy" means-COOH; "ester group" means "-COOR₂₂", carbamoyl means" -CONR₂₂R₂₃Wherein R is₂₂And R₂₃Is any optional group, for example: H. a substituted or unsubstituted straight-chain alkyl group, a substituted or unsubstituted branched-chain alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted heteroaromatic group, and the like.

The compounds of the present invention may exist in unsolvated forms as well as solvated forms containing pharmaceutically acceptable solvents such as water, ethanol, and the like, i.e., both solvated and unsolvated forms.

Indicates the attachment site to the parent nucleus.

In the present invention, a certain substitutable site may be substituted with one or more substituents, and when a plurality of substituents are present at the substitutable site, the plurality of substituents may be the same as or different from each other.

"pharmaceutical composition" means a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, in admixture with other chemical components, as well as other components. Such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

Excipients, which may be present in the composition, may be one or more buffers, stabilizers, anti-adherents, surfactants, wetting agents, lubricants, emulsifiers, binders, suspending agents, disintegrants, fillers, adsorbents, coating (enteric or slow release) preservatives, antioxidants, opacifying agents, glidants, processing aids, colorants, sweeteners, fragrances, flavoring agents and other known additives.

"pharmaceutically acceptable salt", i.e., "pharmaceutically acceptable salt", refers to an organic or inorganic salt of a pharmaceutically acceptable compound.

When the compound is acidic or includes sufficiently acidic bioisosteres, the appropriate "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic base including inorganic and organic bases. The salts are derived from inorganic bases containing aluminum, ammonium, calcium, copper, iron, lithium, magnesium, manganese, potassium, sodium, octyl, and the like. Particular embodiments include ammonium, calcium, magnesium, potassium, and sodium salts. Salts are derived from pharmaceutically acceptable organic non-toxic bases including salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as arginine, betaine, caffeine, choline, N, N.sup.1-dibenzylethylenediamine, ethylenediamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, reduced glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, meglumine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

When the compound is basic or includes sufficiently basic bioisosteres, salts can be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, sulfuric, succinic, tartaric, p-toluenesulfonic acid and the like. Particular embodiments include citric acid, hydrobromic acid, hydrochloric acid, phosphoric acid, sulfuric acid, maleic acid, tartaric acid. Other exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, phosphate, acid phosphate, isonicotinic acid, lactic acid, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, fumarate, maleate, gentisate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, methylsulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (e.g., 1, 1' -methylene-bis- (2-hydroxy-3-naphthoate)).

In addition, the pharmaceutical preparation containing the compound may be tablets, capsules, oral liquids, pills, granules, powders, ointments, patches, suppositories, buccal tablets, eye drops, eye ointments, ear drops, sprays, aerosols, inhalants, injections, and the like.

The term "therapeutically effective amount" refers to the amount of an effective compound or pharmaceutical agent that is the minimum amount necessary to ameliorate, cure or treat one or more symptoms of a disease or disorder.

In addition, the compounds and pharmaceutical compositions of the present invention may be administered alone or in combination with other agents. For combination therapy with more than one active agent, when the active agents are in separate dosage formulations, the active agents may be administered separately or in combination. In addition, administration of one agent may be performed before, simultaneously with, or after administration of another agent. When administered in combination with other agents, the "effective amount" of the second agent will depend on the type of drug used.

Route of administration

One or more compounds of the invention are administered by any route suitable for the diseased cat being treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), and parenteral (including subcutaneous, intramuscular), and the like.

The compounds or pharmaceutical compositions of the present invention may also be included in a kit.

It should be noted that the reagent of the present invention is not specified as a specific source, and is a conventional reagent purchased in the market.

The invention will be further illustrated with reference to specific examples. These examples are for illustrative purposes only and do not limit the scope and spirit of the present invention.

Example 1

Preparation of intermediate 1

The preparation of (2R) -isopropyl 2- ((perfluorophenoxy) (phenoxy) phosphoryl) aminopropionate is described below:

to a reaction flask was added 10g (0.6mol) L-alanine isopropyl ester hydrochloride, 100ml Dichloromethane (DCM) and 12g triethylamine (Et)₃N), cooled to-78 ℃, stirred for 10 minutes, and then a dichloromethane solution (13g/50ml) of phenyl dichlorophosphate was slowly added dropwise. After the completion of the dropping, the temperature was then raised to-20 ℃ and the reaction was continued with stirring for 1 hour. 13g of pentafluorophenol are then added dropwise to the reaction solution in 50ml of dichloromethane. After the addition was complete, the reaction was stirred by heating to 25 ℃ and monitored by thin layer chromatography. After the reaction was completed, the precipitate was removed by suction filtration. The organic phase is concentrated under reduced pressure to give 26g of crude product. The crude product was recrystallized from 20ml of ethyl acetate and 100ml of petroleum ether to yield 10.3g of a white product, intermediate 1.¹H NMR(400MHz，DMSO-d₆)δ(ppm):7.43-7.40(m，2H)，7.25-7.23(m，3H)，6.88(d，1H)，4.91-4.85(m，1H)，3.95-3.87(m，1H)，1.28(d，3H)，1.16(d，6H)

Example 2

Preparation of intermediate 2

Preparation of (3R,4R,5R) -2- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -4- (benzyloxy) -5- (benzyloxymethyl) -3-fluorotetrahydrofuran-2-ol as follows:

3.4g of 7-bromopyrrolo [2,1-f ] was added to the reaction flask][1,2,4]Triazine-4-amine, 4.5mL trimethylchlorosilane (TMSCl) and 70mL Tetrahydrofuran (THF), stirred at room temperature for 2 hours. The reaction mixture was then cooled to-78 ℃ and 55mL of a 1.0M n-butyllithium hexane solution (n-BuLi) was added dropwise, and after completion of the addition, the mixture was stirred for 30 minutes, and then a solution of 5.4g of (3R,4R,5R) -4- (benzyloxy) -5- (benzyloxymethyl) -3-fluorodihydrofuran-2 (3H) -one in 70mL of THF was added dropwise. After the addition, the reaction was stirred for 30 minutes, and then quenched by the addition of 42mL of acetic acid. The reaction was concentrated, diluted with 500mL of ethyl acetate and washed with 250mL of saturated NaCl solution. The organic layer was over anhydrous MgSO₄Dried, filtered and concentrated under reduced pressure. The resulting viscous liquid was subjected to silica gel column chromatography to obtain 2.6g of a white solid product.¹H NMR(400MHz,CDCl₃)δ8.06(s,1H),7.87(s,1H),7.82(s,1H),7.65(s,1H),7.26(m,10H),6.94(m,1H),6.72(m,1H),6.09(m,1H),5.33(m,1H),4.66(m,6H),4.72(m,2H).

Preparation of (2R,3R,4R,5R) -2- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -4- (benzyloxy) -5- (benzyloxymethyl) -3-fluorotetrahydrofuran-2-carbonitrile as follows:

2g of (3R,4R,5R) -2- (4-aminopyrrolo [2, 1-f) was added to the reaction flask][1,2,4]Triazin-7-yl) -4- (benzyloxy) -5- (benzyloxymethyl) -3-fluorotetrahydrofuran-2-ol, 100mL dichloromethane3.4mL of Trimethylnitrilosilane (TMSCN), 2.79g of trimethylsilyl trifluoromethanesulfonate (TMSOTf). The solution was stirred at-20 ℃ for 4 hours. After the reaction, 50mL of saturated NaHCO was added to the reaction mixture₃To the solution, 300mL of ethyl acetate and 100mL of water were added. The organic layer was separated and washed with 100mL saturated NaCl solution, MgSO₄Dried, filtered and concentrated under reduced pressure. The resulting viscous liquid was subjected to silica gel column chromatography to give 0.82g of a white solid.¹H NMR(400MHz,CDCl₃)δ8.02(s,1H),7.95(s,1H),7.31(m,10H),7.01(d,1H),6.93(d,1H),6.88(d,1H),6.71(d,1H),5.86(d，1H),5.56(d,1H),4.72(m,7H),3.87(m,2H),3.71(m,2H).MS m/z 474[M+H]。

Preparation of (2R,3R,4R,5R) -2- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -3-fluoro-4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-carbonitrile is as follows:

to a reaction flask was added 1.1g of (2R,3R,4R,5R) -2- (4-aminopyrrolo [2, 1-f)][1,2,4]Triazin-7-yl) -4- (benzyloxy) -5- (benzyloxymethyl) -3-fluorotetrahydrofuran-2-carbonitrile and 120mL dichloromethane were cooled to 0 ℃ with stirring. 77mL of 1.0M BBr₃The dichloromethane solution of (2) was added to the reaction solution, and stirred for 2 hours. The reaction was then cooled to-78 deg.C, 34mL triethylamine was added, followed by 200mL methanol, and the temperature was raised to room temperature. The reaction mixture was concentrated under reduced pressure, and 200mL of a saturated sodium bicarbonate solution and 200mL of dichloromethane were added to the mixture to extract a liquid, and the organic phase was concentrated under reduced pressure. The concentrated solution was subjected to silica gel column chromatography to obtain 0.34g of a white solid, i.e., intermediate 2.¹H NMR(400MHz,CD₃OD)δ8.08(s,1H),7.27(d,1H),7.16(d,1H),5.41(d,1H),4.20(m,2H),3.98(d,1H),3.76(d,1H).MS m/z 294[M+H]。

Example 3

Preparation of intermediate 3

Preparation of 7- ((2S, 3R,4R,5R) -4- (benzyloxy) -5- ((benzyloxy) methyl) -3-fluoro-2-methyltetrahydrofuran-2-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-amine is as follows:

3.6g of the starting nucleoside and 200mL of anhydrous THF were added to the reaction flask, the mixture was cooled to 0 ℃ with stirring, 20mL of a 3N solution of methylmagnesium chloride in THF was added, and the reaction was stirred overnight. After the reaction, 70mmol of acetic acid is added to quench the reaction, and the reaction solution is concentrated under reduced pressure to obtain viscous liquid. The crude material was dissolved in 150mL of anhydrous dichloromethane, 2mL of methanesulfonic acid was added, stirred at room temperature for 12 hours, and then quenched by the addition of 35mmol of triethylamine. The mixture was concentrated under reduced pressure and the residue was subjected to silica gel column chromatography to give 1.53g of methyl-substituted nucleoside.

¹H NMR(400MHz,CD₃Cl)δ7.86(s,1H),7.26-7.39(m,10H),6.76(d,1H),6.71(d,1H),6.22(s,2H),5.52(d,1H),4.41-4.74(m,4H),4.20-4.25(m,1H),3.64-4.01(m,3H),1.75(d,3H).MSm/z 463[M+H].

Preparation of (2R,3R,4R,5S) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -4-fluoro-2- (hydroxymethyl) -5-methyltetrahydrofuran-3-ol is described below

Intermediate 3 was prepared using the same procedure as example 2.

¹H NMR(400MHz,D₂O)δ7.88(s,1H),7.23(d,1H),6.88(d,1H),5.36(d,1H),3.97-4.11(m,2H),3.82(d,1H),3.65(d,1H),1.66(d,3H).MS m/z 283[M+H]

Example 4

Preparation of intermediate 4

Preparation of (2R,3R,4S,5R) -2- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -3, 4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-carbonitrile is as follows:

to the reaction flask was added 5g of 7-bromopyrrolo [2,1-f ] [1,2,4] -triazin-4-amine and 10mL of anhydrous THF. 7mL of TMSCl was added with stirring. The reaction was cooled to-78 ℃ and 10mL of a 1.0M n-butyllithium hexane solution were then added slowly. After the completion of the dropping, the mixture was stirred at-78 ℃ for 10 minutes, and then 10g of lactone was added by syringe. After 1 hour of reaction, the reaction was quenched by the addition of 30mL of acetic acid. The reaction solution was concentrated under reduced pressure, 400mL of methylene chloride and 200mL of water were added, the liquid was extracted, the organic layer was separated and washed with 100mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated under reduced pressure. The concentrated product was subjected to silica gel column chromatography and eluted with 20% ethyl acetate in petroleum ether to give 3.6g of a white solid. MS M/z553[ M + H ].

2g of the above nucleoside product and 80mL of anhydrous methylene chloride were added to a reaction flask, stirred and cooled to 0 ℃ and 2mL of TMSCN was added, and the reaction was stirred for 10 minutes. 8mL of TMSOTf was slowly added to the reaction and the reaction was stirred for 1 hour. The reaction mixture was then added to 200mL of dichloromethane, and 200mL of saturated aqueous sodium bicarbonate solution was added. The extract was separated, and the organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The concentrated solution was eluted with 20% ethyl acetate in petroleum ether and separated by silica gel column chromatography to give 1.75g of a white product.

¹H NMR(400MHz,CD₃Cl)δ7.95-7.89(d,1H),7.29-7.44(m,13H),7.12-7.20(m,1H),6.83-6.88(m,1H),6.71-6.77(m,1H),6.42(bs,2H),5.11(d,0.5H),4.97(d,0.5H),4.32-4.86(m,7H),4.09-4.19(m,2H),3.62-3.91(m,2H).MS m/z 562[M+H].

Intermediate 4 was prepared using the same procedure as example 2.

¹H NMR(400MHz,DMSO)δ7.92(s,1H),7.81-8.00(s,2H),6.86-6.90(m,2H),

6.09(d,1H),5.18(s,1H),4.91(s,1H),4.64(t,1H),4.03-4.07(m,1H),3.95(s,1H),

3.49-3.65(m,2H).MS m/z 292.2[M+H],

Example 5

Preparation of Compound 1

(2S) -isopropyl-2- (((((2R, 3R,4R,5S) -5- (4-aminopyrrole [2,1-f ] - [1,2,4] triazin-7-yl) -4-fluoro-3-hydroxy-5-methyltetrahydrofuran-2-yl) methoxy (phenoxy) phosphate) amino) propionate

To the reaction flask were added 1.0g of the nucleoside compound (intermediate 3) and 50mL of tetrahydrofuran, and cooled in an ice bath. 6.1mL of 1M tert-butylmagnesium chloride was slowly added dropwise. After the addition, the reaction was stirred at room temperature for 60 minutes. Then, 1.8 phosphate ester compound (intermediate 1) was added and the reaction solution was stirred at room temperature for 24 hours. After the reaction was completed, the reaction mixture was added to 100mL of ethyl acetate, washed with 20mL of an aqueous sodium bicarbonate solution, separated by extraction, and the organic phase was dried over sodium sulfate and concentrated in vacuo. Purification by silica gel column chromatography (methanol/dichloromethane ═ 1/10 solution) afforded 1.2g of a white solid, compound 1.¹HNMR(400MHz,CD₃CN)δ7.86(s,1H),7.16-7.44(m,5H),6.70-6.82(m,2H),6.13(,s,2H),5.36(d,1H),4.91-5.00(m,1H),3.85-4.45(m,6H),3.57(m,1H),1.74(m,3H),1.17-1.35(m,9H).MS m/z 552[M+H].

Example 6

Preparation of compound 2:

(2s) -isopropyl-2- (((((((2 r,3r,4r,5r) -5- (4-aminopyrrole [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-4-fluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphate) amino) propanoate

Compound 2 was prepared by the same procedure as in example 5.

¹H NMR(400MHz,CD₃OD)δ7.92(m,1H),7.34-7.16(m,5H),6.99-6.91(m,2H),5.60(m,1H),4.51-4.16(m,4H),4.13-3.91(m,3H),1.34-1.19(m,9H).MS m/z 563.0[M+H].

Example 7

Preparation of compound 3:

isobutyl (S) -2- (((2R,3S,4R,5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) amino) propionate was prepared as described below

Compound 3 was prepared by the same procedure as in example 5.

¹H NMR(400MHz,CD₃OD)δ7.85(s,1H),7.33-7.25(m,2H),7.20-7.11(m,3H),6.90(d,1H),6.86(d,1H),4.91(t,1H),4.80(d,1H),4.43-4.35(m,1H),4.32-4.23(m,1H),4.18(t,1H),3.83(q,2H),1.26(d,3H),1.19(d,6H).MS m/z 561.0[M+H]

Example 8

Preparation of compound 4:

(2R,3R,4R,5S) -2- (4-Aminopyrrolo [2, 1-fluoro ] [1,2,4] triazin-7-yl) -5- (((4- (4-chlorophenyl) -2-oxo-1, 3, 2-dioxophosphazen-2-yl) oxy) methyl) -3-fluoro-4-hydroxytetrahydrofuran-2-methyl

To a reaction flask, 1.40g of the nucleoside compound (intermediate 3), 2mL of triethylamine and 100mL of tetrahydrofuran were added, and the temperature was lowered to-20 ℃. Then 1.40g of 2-chloro-4-phenyl-1, 3, 2-dioxaphospho-2-one is added. The reaction was then stirred at-20 ℃ for 2 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, added to 200mL of ethyl acetate and 100mL of water, concentrated hydrochloric acid was added dropwise to adjust the pH of the aqueous phase to 5, the organic phase was separated by extraction, dried over sodium sulfate and concentrated in vacuo. The concentrate was purified by silica gel column chromatography (ethyl acetate/petroleum ether-2/1 solution)To 0.95g of a white solid, Compound 4.¹H NMR(400MHz,CD₃CN)δ7.88(s,1H)，7.46-7.47(m,4H),6.71-6.83(m,2H),6.14(,s,2H),5.38(d,1H),4.92-5.01(m,1H),4.52-4.55(m,1H),3.86-4.46(m,8H),2.23-2.28(m,1H),2.13-2.17(m,1H),MS m/z 514.2[M+H]

Example 9

Preparation of compound 5:

(2R,3R,4R,5R) -2- (4-Aminopyrrolo [2, 1-fluoro ] [1,2,4] triazin-7-yl) -5- (((4- (4-chlorophenyl) -2-oxo-1, 3, 2-dioxophosphazen-2-yl) oxy) methyl) -3-fluoro-4-hydroxytetrahydrofuran-2-carbonitrile

To a reaction flask, 1.50g of nucleoside compound (intermediate 2), 2mL of triethylamine and 100mL of tetrahydrofuran were added, and the temperature was lowered to-20 ℃. Then 1.45g of 2-chloro-4-phenyl-1, 3, 2-dioxaphospho-2-one is added. The reaction was then stirred at-20 ℃ for 2 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, added to 200mL of ethyl acetate and 100mL of water, concentrated hydrochloric acid was added dropwise to adjust the pH of the aqueous phase to 5, the organic phase was separated by extraction, dried over sodium sulfate and concentrated in vacuo. Preparative HPLC was performed on 100mg of the concentrate to give 16mg of Compound 5. LCMS M/z 525[ M + H ].

Example 10

Preparation of Compound 6

(2R,3R,4S,5R) -2- (4-Aminopyrrolo [2, 1-fluoro ] [1,2,4] triazin-7-yl) -5- (((4- (4-chlorophenyl) -2-oxo-1, 3, 2-dioxophosphazen-2-yl) oxy) 3, 4-dihydroxytetrahydrofuran-2-carbonitrile

To the reaction flask was added 0.80g of nucleoside (intermediate 4), 1mL of triethylamine and 60mL of tetrahydrofuran, and the temperature was reduced to-20 ℃. Then 0.65g of 2-chloro-4-phenyl-1, 3, 2-dioxaphospho-2-one is added. The reaction was then stirred at-20 ℃ for 2 hours. After the reaction, the reaction mixture was concentrated under reduced pressure, added to 100mL of ethyl acetate and 50mL of water, concentrated hydrochloric acid was added dropwise to adjust the pH of the aqueous phase to 5, the organic phase was separated by extraction, dried over sodium sulfate and concentrated in vacuo. Preparative HPLC of 50mg of the concentrate gave 12mg of Compound 6. LCMS M/z 523[ M + H ].

Example 11

To the reaction flask were added 4.40g of p-nitrophenyl phosphate, 3mL of triethylamine, and 100mL of dichloromethane. 4.60g of isopropyl chloromethyl carbonate were then added. The reaction was then stirred at 45 ℃ for 2 hours. And after the reaction is finished, adding the reaction mixture into 200mL of ethyl acetate and 100mL of water, dropwise adding concentrated hydrochloric acid to adjust the pH value of the water phase to 3-4, extracting and separating the liquid, drying the organic phase with sodium sulfate, and concentrating the organic phase in vacuum. The residue was separated by silica gel column chromatography using ethyl acetate/petroleum ether 1/3 as eluent to give 6.57g of intermediate 5 (p-nitrophenyl bis (isopropoxycarbonyloxymethyl phosphate), MS M/z 452[ M + H ]

Example 12

Preparation of compound 7:

to the reaction flask were added 0.85g of the nucleoside compound (intermediate 3) and 40mL of tetrahydrofuran, and cooled in an ice bath. 3.2mL of 1M tert-butylmagnesium chloride were slowly added dropwise. After the addition, the reaction was stirred at room temperature for 80 minutes. Then, 2.3g of the phosphate ester compound (intermediate 5) was added, and the reaction was stirred at room temperature for 24 hours. After the reaction was completed, the reaction mixture was added to 150mL of ethyl acetate, washed with 50mL of an aqueous sodium bicarbonate solution, separated by extraction, and the organic phase was dried over sodium sulfate and concentrated in vacuo. Purification by silica gel column chromatography (ethyl acetate/petroleum ether-1/5 solution) gave 0.35g of a white solid, compound 7.

¹H NMR(400MHz,CD₃CN)δ7.88(s,1H),6.72-6.83(m,2H),6.14(,s,2H),5.75-5.62(m,4H),5.37(d,1H),4.94-5.02(m,1H),4.85-4.89(m,2H),4.12-4.45(m,2H),3.58(m,1H),1.73(m，3H),1.18-1.34(m,12H).MS m/z 595.5[M+H]

Example 13

Preparation of compound 8:

compound 8 was prepared by the same procedure as in example 13.

¹H NMR(400MHz,CD₃OD)δ7.92(m,1H),6.99-6.91(m,2H),5.75-5.62(m,4H),5.60(m,1H),4.85-4.89(m,2H),4.51-4.16(m,2H),4.13-3.91(m,2H),1.18-1.34(m,12H).MS m/z 606.5[M+H]

Example 14

Preparation of compound 9:

compound 9 was prepared by the same procedure as in example 13.

¹H NMR(400MHz,CD₃OD)δ7.85(s,1H),6.90(d,1H),6.86(d,1H),5.75-5.62(m,4H),4.85-4.89(m,2H),4.64(t,1H),4.43-4.35(m,1H),4.32-4.23(m,1H),,4.13-3.91(m,2H),1.18-1.34(m,12H).MS m/z 604.5[M+H]

The method for measuring the antiviral activity comprises the following steps:

protection assay for feline infectious peritonitis virus cells feline kidney (CRFK) cells and FIPV-79-1146 (feline infectious abdominovirus) were used. Colloquially, the virus and cells are mixed and incubated for 7 days in the presence of the test compound. The virus was pre-titrated to allow control wells to undergo 85% to 95% apoptosis due to virus replication. Since the test compound inhibits viral replication, an antiviral effect is observed in the presence of the test compound. Each assay plate contained a cell control well (cell only), a virus control well (cell and virus), a compound toxicity control well (cell only and compound), a compound colorimetric control well (compound only), and a test well (compound, cell)And viruses). Determination of EC of cells by MTS (MTS (3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) cell proliferation kit₅₀(concentration of 50% cell survival) and cytotoxic CC₅₀(concentration that resulted in 50% cell death).

Cell preparation:

cat kidney (CRFK) cells (commercially available from the American Type Culture Collection (ATCC)) were grown in DMEM medium (2.0mM L-glutamine, 100 units/ml penicillin and 100. mu.g/ml streptomycin) supplemented with 10% Fetal Bovine Serum (FBS). Using standard cell culture techniques at 1: the cells were subcultured twice a week at a split ratio of 10. Total cell number and percent viability were determined using a hemocytometer and trypan blue exclusion. For cells to be used in the assay, cell viability must be greater than 95%. 1X10 the day before the measurement⁴Concentration of individual cells/well cells were seeded in 96-well tissue culture plates.

Preparing virus:

the virus used for the assay was feline infectious abdominal virus FIPV-79-1146 (commercially available from the American Type Culture Collection (ATCC)) grown in CRFK cells, producing the virus and serving as a stock virus pool. For each assay, a pre-titrated aliquot of the virus was removed from-70 ℃ conditions and thawed to room temperature in a biosafety cabinet. The virus was then suspended and diluted into tissue culture medium such that the amount of virus added to each well was an amount determined to produce between 85% and 95% cell kill 4-5 days after infection.

Cell staining:

after 5 days of infection, cell viability was determined and compound toxicity quantified using MTS cell proliferation kit staining. Cell viability and cytotoxicity of the compounds were determined by quantifying the soluble formazan product produced by MTS metabolism by the mitochondrial enzyme that metabolises active cells. 20-25. mu.L of MTS reagent was added to each well, followed by 5% CO at 37 ℃₂Microtiter plates were incubated for 4-6 hours under conditions and cell viability was then determined. The plates were read spectrophotometrically at 490/650nm using a SpectraMax Plus plate reader.

And (3) data analysis:

EC for viral inhibition using in silico compounds₅₀And cytotoxic CC₅₀. Table 1 below shows the results of the activity of the test compounds.

TABLE 1

Test article	EC50(μM)	CC50(μM)
			Compound 1	c	d
Compound
	2	1.24	d
Compound
	3	0.031	c
Compound 4				c	d
	Compound
5		0.84	d
	Compound
6		0.046	d
	Compound 7			c	d
Compound 8		c	d
	Compound
9		0.016	c
	Intermediate 4			0.78	d

a is 0.1 to 1 mu M b: 1-3 mu M c, 3 mu M-5 mu M d: greater than 5 μ M

From the measured values in table 1 above, it can be seen that after the intermediate 4 is subjected to phosphorylation, the antiviral activity of three different phosphate compounds (compound 3, compound 6 and compound 9) is improved by tens of times, and such an activity improvement can greatly reduce the dosage of the drug and improve the drug-forming property.

Pharmacodynamic experiment of feline infectious peritonitis

The experimental method comprises the following steps:

compound 9 (20 mg) was dissolved in 5mL of a mixed solution of 30% propylene glycol, 50% PEG400 (polyethylene glycol 400) and 20% ethanol to prepare a solution for administration, and the solution was administered by injection. The preparation was stored in a refrigerator at 4 ℃ and allowed to warm to room temperature before injection, and the corresponding amount was aspirated with a 1ml disposable syringe to remove air bubbles. The injection is performed subcutaneously in a part with soft skin, loose subcutaneous tissue and less blood vessels, such as the neck or the inner side of the thigh. The administration dose is set to 0.2mg/kg, which is 10% and 5% of the conventional dose of the drugs (the conventional dose is 2mg/kg and 4mg/kg), and the injection frequency is once a day. Blood is collected every 1 week, blood routine and corresponding biochemical index measurement are carried out, the end of the test takes the restoration of the biochemical index of the blood of the cat as an end mark, and the core indexes of discussion are body weight, white blood cells and white sphere ratio.

Table 2 below is a numbered, clinical form of Feline Infectious Peritonitis (FIP) for the 20 cats tested.

TABLE 2

FIG. 1 shows a graph of weight change (vertical axis in kg) before and after treatment in 20 cats; FIG. 2 shows the change in the number of leukocytes (vertical axis unit: 1X 10) in test cats before and after treatment⁹One/liter); FIG. 3 shows the change in white to globular ratio before and after treatment.

As can be seen in FIG. 1, the weight of the cats was greater after treatment than before treatment, and as can be seen in FIG. 2, the number of leukocytes was greater after treatment than before treatment. And as can be seen from figure 3, the white to globular ratio levels were lower in 20 test cats at the start of treatment and then by treatment, the white to globular ratio reached a normal level above 0.70 around week 5 of treatment; the number of leucocytes is increased to a normal level; the weight of each test cat rises again, which proves that the medicament has no obvious toxic effect.

In the experiment, all cats are cured without relapse, and the curative effect is obviously better than the 20% relapse rate in the article. According to corresponding test control, urine yellowing occurs 48 hours after the cat injects the intermediate 4, the phenomenon occurs after 10 hours after the cat injects the compound 9, which is a typical symptom that the wet abdominal cats begin to discharge ascites, and shows that the compound 9 has faster effect, and from this treatment test, the pyrrolotriazine compound provided by the invention can achieve better curative effect with a dosage equivalent to one tenth of the reported dosage, has outstanding drug effect improvement, and greatly improves the drug property.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (7)

1. A pyrrolotriazine compound is characterized by having the following structure

2. The process for producing a pyrrolotriazine compound according to claim 1, comprising the steps of:

3. the process according to claim 2, characterized in that the intermediate (5) is prepared by:

4. a pyrrolotriazine composition comprising a first component which is the pyrrolotriazine compound of claim 1 and a second component which is an interferon inducer.

5. The composition of claim 4, wherein the interferon inducer is one or more of a TLR7 agonist and a STING agonist.

6. Use of the pyrrolotriazine compound of claim 1 for the preparation of an antiviral medicament for the treatment of diseases caused by feline coronavirus infection.

7. Use of a pyrrolotriazine composition as claimed in claim 4 or 5 for the preparation of an antiviral medicament for the treatment of diseases caused by feline coronavirus infection.

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