CN111747942A - Spiro-containing derivative with beta 2 receptor agonistic activity and M receptor antagonistic activity and application thereof in medicine - Google Patents

Abstract

A compound shown in a general formula (I) or a stereoisomer, a hydrate, a metabolite, a solvate, a pharmaceutically acceptable salt, a eutectic crystal or a prodrug thereof, a preparation method thereof and application thereof in preparing a medicament for treating airway obstruction diseases, wherein the compound shown in the general formula (I) is

Description

Spiro-containing derivative with beta 2 receptor agonistic activity and M receptor antagonistic activity and application thereof in medicine

Technical Field

The invention relates to a nitrogenous heterocyclic spiro derivative, a preparation method thereof and application thereof in medicine, in particular to a derivative with muscarinic receptor antagonism and β₂-dual active azaspiro derivatives of adrenergic receptor agonism or stereoisomers, hydrates, solvates, metabolites, pharmaceutically acceptable salts, co-crystals or prodrugs thereof, pharmaceutical compositions thereof and their use in medicine.

Background

Bronchodilators, which are widely used in clinics, include muscarinic receptor antagonists and β₂Currently used inhaled muscarinic receptor antagonists include ipratropium bromide, oxitropium bromide, glycopyrrolate, tiotropium bromide, aclidinium bromide and umeclidinium bromide β₂Adrenergic receptor agonists reverse bronchoconstrictor responses to various mediators, such as acetylcholine, by stimulating adrenergic receptors of airway smooth muscle β is currently used₂Adrenergic receptor agonists include salbutamol, salmeterol, arformoterol, formoterol, vilanterol and indacaterol. These drugs, in addition to improving lung function, may also improve the quality of life and reduce the exacerbation of the disease.

As more clinical findings have been made, the combination of muscarinic receptor antagonist and β was demonstrated₂Adrenergic receptor agonists are more effective than either therapeutic agent alone, and muscarinic receptor antagonists are now clinically used with β₂-adrenoceptor agonists to prepare compound preparations for treatment of asthmaFor treating asthma and severe COPD, the compound preparation mainly comprises Anoro Ellipta (umeclidinium bromide/vilanterol), Ultbro Breezhaler (glycopyrronium bromide/indacaterol), ipratropium bromide/salbutamol, etc. Although the compound preparation has better treatment effect than the single preparation, the preparation of the preparation has higher requirements.

Therefore, it is desired to develop a compound having both muscarinic receptor antagonism and β₂These compounds are administered as a single therapeutic agent, providing bronchodilatory action from two distinct and possibly synergistic modes of action₂Adrenergic agonist dual action (MABA) compounds may also be combined with corticosteroid (ICS) anti-inflammatory agents drugs to form two therapeutic agents (MABA/ICS) to provide triple action therapeutic effects (Expert opin investig. drugs (2014)23(4): 453-.

Therefore, there is a need to develop novel compounds with both muscarinic receptor antagonism and β₂Dual active agents of adrenergic stimulation to provide more effective single therapeutic doses or compound formulations, providing more clinical medication options for the patient.

Disclosure of Invention

The invention provides a compound shown in a general formula (I) or a stereoisomer, a hydrate, a metabolite, a solvate, a pharmaceutically acceptable salt, a eutectic crystal or a prodrug thereof,

wherein:

Q₁selected from a bond or-N (R)^q)-；

R^qIs selected from H or C_1-6An alkyl group;

cy is selected from unsubstituted or substituted

When substituted, is optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl or C_1-4Substituted by alkoxy radicals, Q₁And Q₂Direct linking, provided that Cy is selected from the group consisting of

Q₂Is selected from-CH-or-N-, with the proviso that Q₁And Q₂Does not contain N at the same time;

n1, n2, n3 and n4 are each independently selected from 0, 1,2,3 or 4, provided that n1 and n2 are not simultaneously 0 and n3 and n4 are not simultaneously 0;

x, Y, Z are each independently selected from nitrogen, -NR^x1-、-CR^x1R^x2-、-CR^x1R^x2CR^x3R^x4-、-CR^x1＝CR^x2-, -S-, -O-and-C (O) -;

R^x1、R^x2、R^x3or R^x4Each independently selected from H or C_1-4An alkyl group;

in the general formula (I), "- - - - - - - -" is selected from a single bond or a double bond;

R¹is selected from

R^1a、R^1bEach independently selected from H, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-10Carbocyclyl, 3-to 8-membered heterocyclyl, C_3-10carbocyclyl-C_1-4Alkylene or 3-to 8-membered heterocyclyl-C_1-4Alkylene, said alkenyl, alkynyl, alkylene, carbocyclyl or heterocyclyl being optionally further substituted by 0 to 4 substituents selected from F, Cl, Br, I, CH₂F、CHF₂、CF₃、OH、OCH₂F、OCHF₂、OCF₃、NH₂Carboxy, cyano, nitro, C_1-4Alkyl radical, C_1-4Alkoxy, -O (═ O) C_1-4Alkyl, - (═ O) C_1-4Alkyl, -OC_3-6Cycloalkyl or C_1-4Alkyl sulfideSubstituted with a substituent selected from the group consisting of N, O or S, provided that R^1a、R^1bCannot be simultaneously H;

R^1cselected from H, hydroxy, cyano, NH₂、C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylthio or-C (═ O) NH₂NH of said₂、-C(＝O)NH₂Or alkyl is optionally further substituted by 0 to 2 substituents selected from F, Cl, Br, I, hydroxy, cyano or C_1-4Alkyl is substituted by a substituent;

R^1eand R^1fEach independently selected from F, Cl, Br, I, CF₃、NH₂OH, carboxyl, cyano, C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio, -NHC_1-4Alkyl, -N (C)_1-4Alkyl radical)₂、-S(＝O)-C_1-4Alkyl, -S (═ O)₂-C_1-4Alkyl or-C (═ O) O-C_1-4An alkyl group;

R²or R⁴Is independently selected from C_1-6Alkylene radical, C_2-6Alkenylene or C_2-6Alkynylene, said alkylene, alkenylene or alkynylene being optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl radical, C_1-4Alkoxy, phenyl or phenyl-C_1-4Substituted by a substituent of alkylene;

R³each independently selected from F, Cl, Br, I, OH, NH₂Carboxy, cyano, nitro, C_1-4Alkyl radical, C_2-4Alkenyl radical, C_2-4Alkynyl, C_1-4Alkoxy, -OC_3-6Cycloalkyl radical, C_1-4Alkylthio, -S (═ O) -C_1-4Alkyl, -S (═ O)₂-C_1-4Alkyl, -C (═ O) -C_1-4Alkyl, -C (═ O) O-C_1-4Alkyl, -OC (═ O) -C_1-4Alkyl or-C (═ O) NH₂The alkyl, alkoxy, cycloalkyl, alkenyl, alkynyl, NH₂or-C (═ O) NH₂Optionally further substituted by 0 to 4 substituents selected from F, Cl, Br, I, CF₃、C_1-4Alkyl radical, C_1-4Alkoxy or-C (═ O) -C_1-4Alkyl radicalSubstituted with the substituent(s);

R⁵、R⁶each independently selected from H or C_1-4An alkyl group;

represents β -adrenoceptor binding group;

a is 0, 1,2,3,4 or 5;

b is 0, 1,2,3 or 4;

c is selected from 0, 1,2,3 or 4.

In a specific embodiment of the present invention, in the compound represented by the above general formula (I) or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof,

represents β -adrenoceptor binding group;

b is preferably selected from

R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷Or R¹⁸Each independently selected from H, F, Cl, Br, I, CF₃、OH、-CH₂OH, cyano, carboxyl, C_1-4Alkyl radical, C_1-4Alkoxy, -C (═ O) C_1-4Alkyl, -C (═ O) OC_1-4Alkyl, -NHC (═ O) H, -NHS (═ O)₂-C_1-4Alkyl, -NHS (═ O)₂-NH₂or-NHS (═ O)₂-NHC_1-4Alkyl, Q is selected from-CR^q1＝CR^q2-、-CR^q1R^q2CR^q3R^q4-、-O-、-S-、-OCR^q1R^q2-、-CR^q1R^q2O-、-SCR^q1R^q2-、-CR^q1R^q2S-, said R-^q1、R^q2、R^q3Or R^q4Each independently selected from H, F, Cl, Br,I or C_1-4An alkyl group;

b is more preferably selected from

Wherein Q is selected from-CH ═ CH-, -CH₂CH₂-、-O-、-S-、-CH₂O-、-OCH₂-、-C(CH₃)₂O-or-OC (CH)₃)₂-；

B is further preferably selected from

In a preferred embodiment of the present invention, in the compound represented by the above general formula (I) or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof,

Q₁selected from a bond or-N (R)^q)-；

R^qIs selected from H or C_1-6An alkyl group;

cy is selected from unsubstituted or substituted

When substituted, is optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl or C_1-4Substituted by a substituent of alkoxy;

when Cy is a heterocyclic ring containing N, the N atom is connected with Q1;

x, Y, Z are each independently selected from nitrogen, -NR^x1-、-CR^x1R^x2-、-CR^x1R^x2CR^x3R^x4-、-CR^x1＝CR^x2-, -S-, -O-and-C (O) -;

R^x1、R^x2、R^x3or R^x4Each independently selected from H or C_1-4An alkyl group;

in the general formula (I), "- - - - - - - -" is selected from a single bond or a double bond;

R¹is selected from

R^1a、R^1bEach independently selected from H, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-10Carbocyclyl, 3-to 8-membered heterocyclyl, C_3-10carbocyclyl-C_1-4Alkylene or 3-to 8-membered heterocyclyl-C_1-4Alkylene, said alkenyl, alkynyl, alkylene, carbocyclyl or heterocyclyl being optionally further substituted by 0 to 4 substituents selected from F, Cl, Br, I, CH₂F、CHF₂、CF₃、OH、OCH₂F、OCHF₂、OCF₃、NH₂Carboxy, cyano, nitro, C_1-4Alkyl radical, C_1-4Alkoxy, -O (═ O) C_1-4Alkyl, - (═ O) C_1-4Alkyl, -OC_3-6Cycloalkyl or C_1-4Alkylthio containing 1 to 3 heteroatoms selected from N, O or S, with the proviso that R is^1a、R^1bCannot be simultaneously H;

R^1cselected from H, hydroxy, cyano, NH₂、C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylthio or-C (═ O) NH₂NH of said₂、-C(＝O)NH₂Or alkyl is optionally further substituted by 0 to 2 substituents selected from F, Cl, Br, I, hydroxy, cyano or C_1-4Alkyl is substituted by a substituent;

R^1eand R^1fEach independently selected from F, Cl, Br, I, CF₃、NH₂OH, carboxyl, cyano, C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio, -NHC_1-4Alkyl radical、-N(C_1-4Alkyl radical)₂、-S(＝O)-C_1-4Alkyl, -S (═ O)₂-C_1-4Alkyl or-C (═ O) O-C_1-4An alkyl group;

R²or R⁴Is independently selected from C_1-6Alkylene radical, C_2-6Alkenylene or C_2-6Alkynylene, said alkylene, alkenylene or alkynylene being optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl radical, C_1-4Alkoxy, phenyl or phenyl-C_1-4Substituted by a substituent of alkylene;

R³each independently selected from F, Cl, Br, I, OH, NH₂Carboxy, cyano, nitro, C_1-4Alkyl radical, C_2-4Alkenyl radical, C_2-4Alkynyl, C_1-4Alkoxy, -OC_3-6Cycloalkyl radical, C_1-4Alkylthio, -S (═ O) -C_1-4Alkyl, -S (═ O)₂-C_1-4Alkyl, -C (═ O) -C_1-4Alkyl, -C (═ O) O-C_1-4Alkyl, -OC (═ O) -C_1-4Alkyl or-C (═ O) NH₂The alkyl, alkoxy, cycloalkyl, alkenyl, alkynyl, NH₂or-C (═ O) NH₂Optionally further substituted by 0 to 4 substituents selected from F, Cl, Br, I, CF₃、C_1-4Alkyl radical, C_1-4Alkoxy or-C (═ O) -C_1-4Alkyl is substituted by a substituent;

R⁵、R⁶each independently selected from H or C_1-4An alkyl group;

b is selected from

Wherein Q is selected from-CH ═ CH-, -CH₂CH₂-、-O-、-S-、-CH₂O-、-OCH₂-、-C(CH₃)₂O-or-OC (CH)₃)₂-；

B is preferably selected from

a is 0, 1,2,3,4 or 5;

b is 0, 1,2,3 or 4;

c is selected from 0, 1,2,3 or 4.

In a preferred embodiment of the present invention, in the compound represented by the above general formula (I) or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof,

Q₁selected from a bond or-N (R)^q)-；

R^qIs selected from H or C_1-6An alkyl group;

cy is selected from unsubstituted or substituted

When substituted, is optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl or C_1-4Substituted by a substituent of alkoxy;

when Cy is a heterocyclic ring containing N, the N atom is connected with Q1;

x, Y, Z are each independently selected from nitrogen, -NR^x1-、-CR^x1R^x2-、-CR^x1R^x2CR^x3R^x4-、-CR^x1＝CR^x2-, -S-, -O-and-C (O) -;

R^x1、R^x2、R^x3or R^x4Each independently selected from H or C_1-4An alkyl group;

in the general formula (I), "- - - - - - - -" is selected from a single bond or a double bond;

R¹is selected from

R^1a、R^1bEach independently selected from H, C_2-4Alkenyl radical, C_2-4Alkynyl, C_3-7Carbocyclyl, 3-to 6-membered heterocyclyl, C_3-7carbocyclyl-C_1-4Alkylene or 3-to 6-membered heterocyclyl-C_1-4Alkylene, said alkenyl, alkynyl, alkylene, carbocyclyl or heterocyclyl being optionally further substituted by 0 to 4 substituents selected from F, Cl, Br, I, CF₃、OH、NH₂Carboxy, cyano, nitro, C_1-4Alkyl radical, C_1-4Alkoxy, -O (═ O) C_1-4Alkyl, - (═ O) C_1-4Alkyl, -OC_3-6Cycloalkyl or C_1-4Alkylthio containing 1 to 3 heteroatoms selected from N, O or S, with the proviso that R is^1a、R^1bNot H at the same time;

R^1cselected from H, hydroxy, cyano, NH₂、C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio or-C (═ O) NH₂NH of said₂、-C(＝O)NH₂Or alkyl is optionally further substituted by 0 to 2 substituents selected from F, Cl, Br, I, hydroxy, cyano or C_1-4Alkyl is substituted by a substituent;

R^1eand R^1fEach independently selected from F, Cl, Br, I, CF₃、NH₂OH, cyano, C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio, -NHC_1-4Alkyl or-N (C)_1-4Alkyl radical)₂；

R³Each independently selected from F, Cl, Br, I, OH, NH₂Carboxy, cyano, nitro, C_1-4Alkyl radical, C_2-4Alkynyl, C_1-4Alkoxy, -OC_3-6Cycloalkyl radical, C_1-4Alkylthio, -S (═ O) -C_1-4Alkyl, -S (═ O)₂-C_1-4Alkyl, -C (═ O) -C_1-4Alkyl or-C (═ O) O-C_1-4Alkyl, alkynyl, alkoxy, cycloalkyl and NH₂Optionally further substituted with 0 to 4 substituents selected from F, Cl, Br,I、CF₃、C_1-4Alkyl radical, C_1-4Alkoxy or-C (═ O) -C_1-4Alkyl is substituted by a substituent;

R²or R⁴Is independently selected from C_1-6Alkylene radical, C_2-6Alkenylene or C_2-6Alkynylene, said alkylene, alkenylene or alkynylene being optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl radical, C_1-4Alkoxy, phenyl or phenyl-C_1-4Substituted by a substituent of alkylene;

R⁵、R⁶each independently selected from H or C_1-4An alkyl group;

b is selected from

Wherein Q is selected from-CH ═ CH-, -CH₂CH₂-、-O-、-S-、-CH₂O-、-OCH₂-、-C(CH₃)₂O-or-OC (CH)₃)₂-；

B is preferably selected from

a is 0, 1,2,3,4 or 5;

b is 0, 1,2,3 or 4;

c is selected from 0, 1,2,3 or 4.

In a preferred embodiment of the present invention, in the compound represented by the above general formula (I) or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof,

Q₁selected from a bond or-N (R)^q)-；

R^qSelected from H, methyl, ethyl, propyl or isopropylA group;

cy is selected from unsubstituted or substituted

When substituted, is optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl or C_1-4Substituted by a substituent of alkoxy;

when Cy is a heterocyclic ring containing N, the N atom is connected with Q1;

x, Y, Z are each independently selected from nitrogen, -NH-, -CH₂-、-CH₂CH₂-, -CH-, -CH ═ CH-, -S-, -O-, and-C (O) -;

in the general formula (I), "- - - - - - - -" is selected from a single bond or a double bond;

R¹is selected from

R²、R⁴Each independently selected from methylene, ethylene, propylene, butylene or pentylene, said methylene, ethylene, propylene, butylene or pentylene being optionally further substituted with 0 to 5 substituents selected from F, Cl, Br, I, cyano, OH, methyl, ethyl, methoxy or ethoxy;

R³each independently selected from F, Cl, Br, cyano, methyl, ethyl, propyl, isopropyl, ethynyl, CHF₂、CF₃Methoxy, ethoxy, -OCHF₂or-OCF₃；

R⁵、R⁶Each independently selected from H, methyl or ethyl;

b is selected from

a is 0 or 1;

b is 0, 1 or 2;

c is 0, 1 or 2.

In a preferred embodiment of the present invention, in the compound represented by the above general formula (I) or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof, the compound is selected from compounds represented by the general formula (II):

Q₁selected from a bond or-N (R)^q)-；

R^qIs selected from H or C_1-6An alkyl group;

cy is selected from unsubstituted or substituted

When substituted, is optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl or C_1-4Substituted by alkoxy radicals, Q₁And Q₂Direct linking, provided that Cy is selected from the group consisting of

Q₂Is selected from-CH-or-N-, with the proviso that Q₁And Q₂Does not contain N at the same time;

n1, n2, n3 and n4 are each independently selected from 0, 1,2,3 or 4, provided that n1 and n2 are not simultaneously 0 and n3 and n4 are not simultaneously 0;

R²is selected from C_1-6Alkylene radical, C_2-6Alkenylene or C_2-6Alkynylene, said alkylene, alkenylene or alkynylene being optionally further substituted with 0 to 5 substituents selected from F, Cl, Br, I,OH, cyano, C_1-4Alkyl radical, C_1-4Alkoxy, phenyl or phenyl-C_1-4Substituted by a substituent of alkylene;

R³each independently selected from F, Cl, Br, I, OH, NH₂Carboxy, cyano, nitro, C_1-4Alkyl radical, C_2-4Alkenyl radical, C_2-4Alkynyl, C_1-4Alkoxy, -OC_3-6Cycloalkyl radical, C_1-4Alkylthio, -S (═ O) -C_1-4Alkyl, -S (═ O)₂-C_1-4Alkyl, -C (═ O) -C_1-4Alkyl, -C (═ O) O-C_1-4Alkyl, -OC (═ O) -C_1-4Alkyl or-C (═ O) NH₂The alkyl, alkoxy, cycloalkyl, alkenyl, alkynyl, NH₂or-C (═ O) NH₂Optionally further substituted by 0 to 4 substituents selected from F, Cl, Br, I, CF₃、C_1-4Alkyl radical, C_1-4Alkoxy or-C (═ O) -C_1-4Alkyl is substituted by a substituent;

c is selected from 0, 1,2,3 or 4.

In a preferred embodiment of the present invention, in the compound represented by the above general formula (II) or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof,

cy is selected from unsubstituted or substituted

When substituted, is optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl or C_1-4Substituted by a substituent of alkoxy;

when Cy is a heterocyclic ring containing N, the N atom is linked to Q1;

Q₁selected from a bond or-N (R)^q)-；

R^qIs selected from H or C_1-6An alkyl group;

R²is selected from C_1-6Alkylene radical, C_2-6Alkenylene or C_2-6Alkynylene, said alkylene, alkenylene or alkynylene being optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl radical, C_1-4Alkoxy, phenyl or phenyl-C_1-4Substituted by a substituent of alkylene;

R³each independently selected from F, Cl, Br, I, OH, NH₂Carboxy, cyano, nitro, C_1-4Alkyl radical, C_2-4Alkenyl radical, C_2-4Alkynyl, C_1-4Alkoxy, -OC_3-6Cycloalkyl radical, C_1-4Alkylthio, -S (═ O) -C_1-4Alkyl, -S (═ O)₂-C_1-4Alkyl, -C (═ O) -C_1-4Alkyl, -C (═ O) O-C_1-4Alkyl, -OC (═ O) -C_1-4Alkyl or-C (═ O) NH₂The alkyl, alkoxy, cycloalkyl, alkenyl, alkynyl, NH₂or-C (═ O) NH₂Optionally further substituted by 0 to 4 substituents selected from F, Cl, Br, I, CF₃、C_1-4Alkyl radical, C_1-4Alkoxy or-C (═ O) -C_1-4Alkyl is substituted by a substituent;

c is selected from 0, 1,2,3 or 4.

In a preferred embodiment of the present invention, in the compound represented by the above general formula (II) or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof,

cy is selected from unsubstituted or substituted

When substituted, is optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl or C_1-4Substituted by a substituent of alkoxy;

when Cy is a heterocyclic ring containing N, the N atom is linked to Q1;

Q₁selected from a bond or-N (R)^q)-；

R^qSelected from H, methyl, ethyl, propyl or isopropyl;

R²selected from methylene, ethylene, propylene, -CH₂CH(CH₃)-、-CH₂C(CH₃)₂-、-C(CH₃)₂CH₂-、-CH(CH₃)CH₂-, butylene, -CH (CH)₃)CH₂CH₂-、-CH₂CH(CH₃)CH₂-、-CH₂CH(CH₃)CH₂-or pentylene;

R³each independently selected from F, Cl, Br, cyano, methyl, ethyl, propyl, isopropyl, ethynyl, CHF₂、CF₃Methoxy, ethoxy, -OCHF₂or-OCF₃；

c is selected from 0, 1 or 2.

In a preferred embodiment of the invention, the compound is selected from the following structures:

the invention also provides a pharmaceutical composition, which contains a therapeutically effective dose of the compound of the general formula (I) or (II) or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, eutectic crystal or prodrug thereof, and a pharmaceutically acceptable carrier, diluent, adjuvant, vehicle or excipient; the composition may further comprise one or more other therapeutic agents; preferably wherein the other therapeutic agent is selected from one or more of a PDE4 inhibitor, a muscarinic receptor antagonist, a corticosteroid and a β -adrenergic receptor agonist.

The invention also provides the compound of the general formula (I) or (II) or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, eutectic crystal or prodrug thereof, and application of the compound in preparing a medicament for treating airway obstructive diseases, preferably application in preparing a medicament for treating asthma, chronic obstructive pulmonary disease or bronchitis.

The present invention also provides a method for treating an obstructive airways disease, which comprises administering a compound represented by the general formula (I) or (II) above or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof, or a pharmaceutical composition as described above.

The present invention also provides a method of treating asthma, chronic obstructive pulmonary disease or bronchitis, which comprises administering a compound of the general formula (I) or (II) above, or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof, or a pharmaceutical composition as described above.

The ditrifluoroacetate salt in the compound of the embodiment of the present invention can be dissolved in a polar organic solvent (e.g., a mixed solvent of methanol and dichloromethane (v/v. 1/90)), the pH is adjusted to be alkaline by adding an alkaline reagent (e.g., a saturated sodium bicarbonate solution or a saturated sodium carbonate solution), the mixture is stirred and extracted with an organic solvent (e.g., dichloromethane, ethyl acetate, etc.), and the free base form of the corresponding compound can be obtained by concentrating the organic phase under reduced pressure.

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

Carbon, hydrogen, oxygen, sulfur, nitrogen or halogen referred to in the groups and compounds of the invention all include isotopes thereof, and carbon, hydrogen, oxygen, sulfur, nitrogen or halogen referred to in the groups and compounds of the invention are optionally further replaced by one or more of their corresponding isotopes, wherein isotopes of carbon include isotopes of carbon¹²C、¹³C and¹⁴c, hydrogen isotopes including protium (H), deuterium (D, also known as deuterium), tritium (T, also known as deuterium), oxygen isotopes packageComprises¹⁶O、¹⁷O and¹⁸isotopes of O, sulfur including³²S、³³S、³⁴S and³⁶isotopes of S, nitrogen include¹⁴N and¹⁵isotopes of N, F¹⁹Isotopes of F, chlorine including³⁵Cl and³⁷cl, isotopes of bromine including⁷⁹Br and⁸¹Br。

"alkyl" means a straight and branched chain monovalent saturated hydrocarbon group, the backbone comprising 1 to 10 carbon atoms, preferably 1 to 8 carbon atoms, further preferably 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, straight and branched chain groups, most preferably 1 to 2 carbon atoms, examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like; said alkyl group may optionally be further substituted by 0, 1,2,3,4 or 5 groups selected from F, Cl, Br, I, ═ O, -CH₂F、-CHF₂、-CF₃、-OCH₂F、-OCHF₂、-OCF₃Hydroxy, -SR¹⁹Nitro, cyano, isocyano, alkyl, hydroxyalkyl, alkoxy, carbocyclyl, heterocyclyl, C_2-8Alkenyl radical, C_2-8Alkynyl, - (CH)₂)_a-C(＝O)-R¹⁹、-(CH₂)_k-C(＝O)-O-R¹⁹、-(CH₂)_k-C(＝O)-NR¹⁹R^19a、-(CH₂)_k-S(＝O)_j-R¹⁹、-O-C(＝O)-O-R¹⁹or-NR¹⁹R^19aWherein R is substituted by a substituent of (1)¹⁹And R^19aEach independently selected from H, hydroxy, amino, carboxyl and C_1-8Alkyl radical, C_1-8Alkoxy radical, C_2-8Alkenyl radical, C_2-8Alkynyl, 3-to 10-membered carbocyclyl, 4-to 10-membered heterocyclyl, 3-to 10-membered carbocyclyloxy or 4-to 10-membered heterocyclyloxy, k is selected from 0, 1,2,3,4 or 5, and j is selected from 0, 1 or 2. Alkyl, k, j, R as appearing herein¹⁹And R^19aAs defined above.

"alkylene" refers to a straight and branched chain divalent saturated hydrocarbon radical, including- (CH)₂)_v- (v is an integer of 1 to 10), examples of alkylene include, but are not limited to, methylene, ethylene, propylene, butylene, and the like; said alkylene may optionally be further substituted by 0, 1,2,3,4 or 5 substituents selected from F, Cl, Br, I, ═ O, -CH₂F、-CHF₂、-CF₃、-OCH₂F、-OCHF₂、-OCF₃Hydroxy, -SR¹⁹Nitro, cyano, isocyano, alkyl, hydroxyalkyl, alkoxy, carbocyclyl, heterocyclyl, C_2-8Alkenyl radical, C_2-8Alkynyl, - (CH)₂)_a-C(＝O)-R¹⁹、-(CH₂)_k-C(＝O)-O-R¹⁹、-(CH₂)_k-C(＝O)-NR¹⁹R^19a、-(CH₂)_k-S(＝O)_j-R¹⁹、-O-C(＝O)-O-R¹⁹or-NR¹⁹R^19aWhen the number of the substituents in the alkylene group is 2 or more, the substituents may be fused together to form a cyclic structure. Alkylene, as used herein, is defined as above.

"alkoxy" refers to a monovalent radical of an O-alkyl group, where alkyl is as defined herein, and examples of alkoxy include, but are not limited to, methoxy, ethoxy, 1-propoxy, 2-propoxy, 1-butoxy, 2-methyl-1-propoxy, 2-butoxy, 2-methyl-2-propoxy, 1-pentyloxy, 2-pentyloxy, 3-pentyloxy, 2-methyl-2-butoxy, 3-methyl-1-butoxy, 2-methyl-1-butoxy, and the like.

"alkenyl" means a straight or branched chain monovalent unsaturated hydrocarbon group having at least 1, and usually 1,2 or 3 carbon-carbon double bonds, and the main chain includes 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and still more preferably 2 to 4 carbon atoms in the main chain, and examples of alkenyl include, but are not limited to, vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-3-butenyl, 1-hexenyl, 2-hexenyl, and mixtures thereof,3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 1-octenyl, 3-octenyl, 1-nonenyl, 3-nonenyl, 1-decenyl, 4-decenyl, 1, 3-butadiene, 1, 3-pentadiene, 1, 4-hexadiene, and the like; said alkenyl may optionally be further substituted by 0, 1,2,3,4 or 5 substituents selected from F, Cl, Br, I, ═ O, -CH₂F、-CHF₂、-CF₃、-OCH₂F、-OCHF₂、-OCF₃Hydroxy, -SR¹⁹Nitro, cyano, isocyano, alkyl, hydroxyalkyl, alkoxy, carbocyclyl, heterocyclyl, C_2-8Alkenyl radical, C_2-8Alkynyl, - (CH)₂)_a-C(＝O)-R¹⁹、-(CH₂)_k-C(＝O)-O-R¹⁹、-(CH₂)_k-C(＝O)-NR¹⁹R^19a、-(CH₂)_k-S(＝O)_j-R¹⁹、-O-C(＝O)-O-R¹⁹or-NR¹⁹R^19aSubstituted with the substituent(s). Alkenyl as used herein, is defined as above.

"alkenylene" refers to a divalent alkenyl group in which the alkenyl group is as defined above.

"alkynyl" refers to straight and branched chain monovalent unsaturated hydrocarbon radicals having at least 1, and typically 1,2 or 3 carbon-carbon triple bonds, and the backbone includes 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and even more preferably 2 to 4 carbon atoms in the backbone, with examples of alkynyl including, but not limited to, ethynyl, 1-propynyl, 2-propynyl, butynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 4-pentynyl, 3-pentynyl, 1-methyl-2-butynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 3-octynyl, 3-nonynyl, and 4-decynyl, and the like; said alkynyl may optionally be further substituted by 0, 1,2,3,4 or 5 substituents selected from F, Cl, Br, I, ═ O, -CH₂F、-CHF₂、-CF₃、-OCH₂F、-OCHF₂、-OCF₃Hydroxy, -SR¹⁹Nitro, cyano, isocyano, alkyl, hydroxyalkyl, alkoxy, carbocyclylHeterocyclic group, C_2-8Alkenyl radical, C_2-8Alkynyl, - (CH)₂)_a-C(＝O)-R¹⁹、-(CH₂)_k-C(＝O)-O-R¹⁹、-(CH₂)_k-C(＝O)-NR¹⁹R^19a、-(CH₂)_k-S(＝O)_j-R¹⁹、-O-C(＝O)-O-R¹⁹or-NR¹⁹R^19aSubstituted with the substituent(s). Alkynyl, as found herein, is defined as above.

"alkynylene" refers to a divalent alkynyl group in which the alkynyl group is as defined above.

"cycloalkyl" refers to a monovalent saturated carbocyclic hydrocarbon group, typically of 3 to 10 carbon atoms, non-limiting examples including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl, and the like. Said cycloalkyl may optionally be further substituted by 0, 1,2,3,4 or 5 substituents selected from F, Cl, Br, I, ═ O, -CH₂F、-CHF₂、-CF₃、-OCH₂F、-OCHF₂、-OCF₃Hydroxy, -SR¹⁹Nitro, cyano, isocyano, alkyl, hydroxyalkyl, alkoxy, carbocyclyl, heterocyclyl, C_2-8Alkenyl radical, C_2-8Alkynyl, - (CH)₂)_a-C(＝O)-R¹⁹、-(CH₂)_k-C(＝O)-O-R¹⁹、-(CH₂)_k-C(＝O)-NR¹⁹R^19a、-(CH₂)_k-S(＝O)_j-R¹⁹、-O-C(＝O)-O-R¹⁹or-NR¹⁹R^19aSubstituted with the substituent(s). Cycloalkyl as found herein, is as defined above.

"cycloalkylene" refers to a divalent cycloalkyl group, wherein cycloalkyl is as defined above.

"aryl" means a monovalent aromatic hydrocarbon group having a single ring or fused rings, typically 6 to 10 carbon atoms, non-limiting examples of which include phenyl, naphthalen-1-yl or naphthalen-2-yl. Said aryl group may optionally be further substituted by 0, 1,2,3,4 or 5 groups selected from F, Cl, Br, I, ═ O, -CH₂F、-CHF₂、-CF₃、-OCH₂F、-OCHF₂、-OCF₃Hydroxy, -SR¹⁹Nitro, cyano, isocyano, alkyl, hydroxyalkyl, alkoxy, carbocyclyl, heterocyclyl, C_2-8Alkenyl radical, C_2-8Alkynyl, - (CH)₂)_a-C(＝O)-R¹⁹、-(CH₂)_k-C(＝O)-O-R¹⁹、-(CH₂)_k-C(＝O)-NR¹⁹R^19a、-(CH₂)_k-S(＝O)_j-R¹⁹、-O-C(＝O)-O-R¹⁹or-NR¹⁹R^19aSubstituted with the substituent(s). Aryl, as found herein, is defined as above.

"arylene" refers to a divalent aromatic radical, wherein aryl is as defined above.

"heteroaryl" refers to a monovalent aryl group having a single ring or two fused rings and containing at least 1 heteroatom selected from N, O or S in the ring, typically consisting of 5 to 8 atoms, non-limiting examples of which include pyrrolyl, imidazolyl, thiazolyl, thienyl, furyl, pyrazolyl, isoxazolyl, oxazolyl, pyridyl, or pyrazinyl. Said heteroaryl may optionally be further substituted by 0, 1,2,3,4 or 5 substituents selected from F, Cl, Br, I, ═ O, -CH₂F、-CHF₂、-CF₃、-OCH₂F、-OCHF₂、-OCF₃Hydroxy, -SR¹⁹Nitro, cyano, isocyano, alkyl, hydroxyalkyl, alkoxy, carbocyclyl, heterocyclyl, C_2-8Alkenyl radical, C_2-8Alkynyl, - (CH)₂)_a-C(＝O)-R¹⁹、-(CH₂)_k-C(＝O)-O-R¹⁹、-(CH₂)_k-C(＝O)-NR¹⁹R^19a、-(CH₂)_k-S(＝O)_j-R¹⁹、-O-C(＝O)-O-R¹⁹or-NR¹⁹R^19aSubstituted with the substituent(s). Heteroaryl, as found herein, is defined as above.

"heteroarylene" refers to a divalent heteroaryl group, wherein heteroaryl is as defined above.

"carbocyclic group" means a saturated or unsaturated aromatic or non-aromatic ring which may be3-to 10-membered monocyclic, 4-to 12-membered bicyclic or 10-to 15-membered tricyclic ring systems to which the carbocyclic group may be attached an endocyclic or spiro ring, non-limiting examples of which include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopentyl-1-alkenyl, 1-cyclopentyl-2-alkenyl, 1-cyclopentyl-3-alkenyl, cyclohexyl, 1-cyclohexyl-2-alkenyl, 1-cyclohexyl-3-alkenyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, phenyl, naphthyl, cyclohexyl-2-alkenyl, 1-cyclohexyl-3-alkenyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclooctyl,

Said carbocyclyl may optionally be further substituted by 0, 1,2,3,4 or 5 substituents selected from F, Cl, Br, I, ═ O, -CH₂F、-CHF₂、-CF₃、-OCH₂F、-OCHF₂、-OCF₃Hydroxy, -SR¹⁹Nitro, cyano, isocyano, alkyl, hydroxyalkyl, alkoxy, carbocyclyl, heterocyclyl, C_2-8Alkenyl radical, C_2-8Alkynyl, - (CH)₂)_a-C(＝O)-R¹⁹、-(CH₂)_k-C(＝O)-O-R¹⁹、-(CH₂)_k-C(＝O)-NR¹⁹R^19a、-(CH₂)_k-S(＝O)_j-R¹⁹、-O-C(＝O)-O-R¹⁹or-NR¹⁹R^19aSubstituted with the substituent(s). Carbocyclyl as appearing herein, is defined as above.

"heterocyclyl" means a saturated or unsaturated aromatic or non-aromatic ring which may be a 3 to 10 membered monocyclic, 4 to 12 membered bicyclic or 10 to 15 membered tricyclic ring system and which contains 1 to 4 heteroatoms selected from N, O or S, preferably a 3 to 8 membered heterocyclyl group, the optionally substituted N, S ring of which may be oxidized to various oxidation states. The heterocyclic group may be attached at a heteroatom or carbon atom, the heterocyclic group may be attached to a bridged or spiro ring, non-limiting examples of which include epoxyethyl, epoxypropyl, aziridinyl, oxetanyl, azetidinyl, thietanyl, 1, 3-dioxolanyl, 1, 4-dioxolanyl, 1, 3-dioxanyl, azepinyl, oxepinyl, thiepinyl, oxazepinylA group, diazepinyl, thiazepinyl, pyridyl, piperidyl, homopiperidinyl, furyl, thienyl, pyranyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, piperazinyl, homopiperazinyl, imidazolyl, piperidyl, perinyl, morpholinyl, thiomorpholinyl, thiaoxazolidyl, 1, 3-dithianyl, dihydrofuranyl, dihydropyranyl, dithiafuranyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydropyrrolyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydropyranyl, benzimidazolyl, benzopyridyl, pyrrolopyridyl, chromanyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxacyclohexyl, 1, 3-dioxolyl, pyridinyl, N-alkylpyrrolinyl, pyrimidyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyr, Pyrazolinyl, dithianyl, dithienoalkyl, dihydrothienyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 1,2,3, 4-tetrahydroisoquinolinyl, 3-azabicyclo [3.1.0 ]]Hexyl, 3-azabicyclo [4.1.0]Heptyl, azabicyclo [2.2.2]Hexyl, 3H-indolylquinozinyl, N-pyridylurea, 1-dioxothiomorpholinyl, azabicyclo [3.2.1]Octyl, azabicyclo [5.2.0 ] groups]Nonoalkyl oxatricyclo [5.3.1.1 ]]Dodecyl, azaadamantyl and oxaspiro [3.3]]A heptalkyl group. Said heterocyclyl may optionally be further substituted by 0, 1,2,3,4 or 5 substituents selected from F, Cl, Br, I, ═ O, -CH₂F、-CHF₂、-CF₃、-OCH₂F、-OCHF₂、-OCF₃Hydroxy, -SR¹⁹Nitro, cyano, isocyano, alkyl, hydroxyalkyl, alkoxy, carbocyclyl, heterocyclyl, C_2-8Alkenyl radical, C_2-8Alkynyl, - (CH)₂)_a-C(＝O)-R¹⁹、-(CH₂)_k-C(＝O)-O-R¹⁹、-(CH₂)_k-C(＝O)-NR¹⁹R^19a、-(CH₂)_k-S(＝O)_j-R¹⁹、-O-C(＝O)-O-R¹⁹or-NR¹⁹R^19aSubstituted with the substituent(s). Heterocyclyl, as used herein, is defined as above.

"β -adrenoceptor binding group" refers to a group capable of binding to β -adrenoceptor, such asSee for review articles "β -acquired receivers in Comprehensive medical Chemistry, 1990, B.E.Main, p187(Pergamon Press)". the above groups are also described, e.g., in WO/2005092841, US/20050215542, WO/2005070872, WO/2006023460, WO/2006051373, WO/2006087315 and WO/2006032627 non-limiting examples include

R⁴、R⁵Each independently selected from H or C_1-4Alkyl, B is selected from

Wherein Q is selected from-CH ═ CH-, -CH₂CH₂-、-O-、-S-、-CH₂O-、-OCH₂-、-C(CH₃)₂O-or-OC (CH)₃)₂-。

"amino protecting group" refers to a group used for amino protection which is suitable for protecting an amino group from chemical reaction, but which is easily removed after the desired chemical reaction is completed in the rest of the molecule. The protection of amino Groups In the protection group In Organic Synthesis (the university of eastern science and technology, the university of Organic chemistry, book of honor, school of eastern science and technology, published by the university of eastern science and technology, 2004; original book Protective Groups In Organic Synthesis (third edition), Theodora W.Green and Peter G.M.Wuts) is described In detail In the chapter on amino protection. Page 494-653 of protective groups in organic synthesis is incorporated herein by reference as part of the present application.

"amino protecting group" includes, but is not limited to, the following groups: benzyl, p-methoxybenzyl, trityl, tert-butoxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, 2,2, 2-trichloroethoxycarbonyl, ethoxycarbonyl, benzyloxycarbonyl, trifluoroacetyl, acetyl or benzoyl.

"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. Such as: "alkyl optionally substituted with F" means that the alkyl group may, but need not, be substituted with F, and the description includes the case where the alkyl group is substituted with F and the case where the alkyl group is not substituted with F.

"pharmaceutical composition" means a mixture of one or several of the compounds described herein or a physiologically/pharmaceutically acceptable salt thereof with other constituents, wherein the other constituents comprise physiologically/pharmaceutically acceptable carriers and excipients.

"carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

"excipient" refers to an inert substance added to a pharmaceutical composition to further depend on the administration of the compound. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and different types of starch, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oils, polyethylene glycols, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like.

"prodrug" refers to a compound that can be converted under physiological conditions or by solvolysis to a compound of the invention that is biologically active. Prodrugs of the invention are prepared by modifying functional groups in compounds of the invention, which modifications may be removed by routine manipulation or in vivo, to yield the parent compound.

"stereoisomers" refers to isomers resulting from the different arrangement of atoms in a molecule, including cis, trans isomers, enantiomers and conformational isomers.

An "effective dose" refers to an amount of a compound that causes physiological or medical translation in a tissue, system, or subject that is sought, including an amount of the compound that is sufficient to prevent, or alleviate to some extent, one or more symptoms of the condition or disorder being treated when administered to a subject.

"solvates" refers to compounds of the invention or salts thereof, which also include stoichiometric or non-stoichiometric amounts of solvents bound by intermolecular non-covalent forces. When the solvent is water, it is a hydrate.

“IC₅₀"half inhibitory concentration" means the concentration at which half of the maximum inhibitory effect is achieved.

Detailed Description

The following detailed description is provided for the purpose of illustrating the embodiments and the advantageous effects thereof, and is not intended to limit the scope of the present disclosure.

The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or (and) Mass Spectrometry (MS). NMR shift () at 10^-6The units in (ppm) are given. NMR was measured using (Bruker Avance III 400 and Bruker Avance 300) nuclear magnetic spectrometers in deuterated dimethyl sulfoxide (DMSO-d)₆) Deuterated chloroform (CDCl)₃) Deuterated methanol (CD)₃OD), internal standard Tetramethylsilane (TMS).

MS was measured by Agilent 6120B (ESI) and Agilent 6120B (APCI).

HPLC was carried out using an Agilent 1260DAD high pressure liquid chromatograph (Zorbax SB-C18100X 4.6 mm).

The thin layer chromatography silica gel plate adopts HSGF254 of tobacco yellow sea or GF254 of Qingdao, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15 mm-0.20 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.

The column chromatography generally uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.

Known starting materials of the present invention can be synthesized by or according to methods known in the art, or can be purchased from companies such as Tatan technology, Annaiji chemistry, Shanghai Demer, Chengdong chemical, Shaoshan far chemical technology, and Bailingwei technology.

The nitrogen atmosphere means that the reaction flask is connected with a nitrogen balloon with a volume of about 1L.

The hydrogen atmosphere refers to a reaction flask connected with a hydrogen balloon with a volume of about 1L.

The hydrogenation reaction was usually evacuated and charged with hydrogen and repeated 3 times.

In the examples, the reaction was carried out under a nitrogen atmosphere without specific mention.

In the examples, the solution means an aqueous solution unless otherwise specified.

In the examples, the reaction temperature is room temperature, unless otherwise specified.

The room temperature is the most suitable reaction temperature and is 20-30 ℃.

TBS is tert-butyl dimethyl silicon base.

Boc is tert-butyloxycarbonyl.

TFA is trifluoroacetic acid.

HATU is 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (CAS: 148893-10-1).

Intermediate 1: 4- (5-Formylbenzotriazol-1-yl) butyl methanesulfonate (intermediate 1)

4-(5-formylbenzotriazol-1-yl)butyl methanesulfonate

The first step is as follows: 4- (4-Hydroxybutylamino) -3-nitro-benzonitrile (1B)

4-(4-hydroxybutylamino)-3-nitro-benzonitrile

4-fluoro-3-nitrobenzonitrile (10g,60.2mmol) was dissolved in tetrahydrofuran (200mL), diisopropylethylamine (11.7g,90.3mmol) and 4-hydroxybutylamine (8.05g,90.3mmol) were added, and the mixture was stirred at room temperature overnight. The reaction solution was concentrated, the residue was dissolved in water, extracted with ethyl acetate (150 mL. times.3), the organic phases were combined, washed with water (100 mL. times.1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude 4- (4-hydroxybutylamino) -3-nitro-benzonitrile (1B) (14.2g) which was used directly in the next step.

LCMS m/z＝236.0[M+1]⁺.

The second step is that: 3-amino-4- (4-hydroxybutylamino) benzonitrile (1C)

3-amino-4-(4-hydroxybutylamino)benzonitrile

4- (4-hydroxybutylamino) -3-nitro-benzonitrile (1B) (12.0g, 51mmol) was added to ethanol (200mL), 10% palladium on carbon (1.5g) was added, replaced with hydrogen three times, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give 3-amino-4- (4-hydroxybutylamino) benzonitrile (1C) (10.2g, yield: 97.6%).

LCMS m/z＝206.0[M+1]⁺

The third step: 1- (4-hydroxybutyl) -benzotriazole-5-carbonitrile (1D)

1-(4-hydroxybutyl)-benzotriazole-5-carbonitrile

3-amino-4- (4-hydroxybutylamino) benzonitrile (1C) (10.0g, 42.6mmol) was dissolved in 200mL of 6M aqueous hydrochloric acid, and 40mL of aqueous sodium nitrite (4.04g,58.5mmol) was slowly added dropwise in an ice-water bath. After the addition was complete, the mixture was stirred at room temperature for 2 hours. Extraction was performed with dichloromethane (100 mL. times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 1- (4-hydroxybutyl) -benzotriazole-5-carbonitrile (1D) (9.2g, yield: 87.6%).

LCMS m/z＝217.0[M+1]⁺

The fourth step: 1- (4-hydroxybutyl) -benzotriazole-5-formaldehyde (1E)

1-(4-hydroxybutyl)-benzotriazole-5-carbaldehyde

1- (4-hydroxybutyl) -benzotriazole-5-carbonitrile (1D) (9.0g, 41.7mmol) was dissolved in 75mL of formic acid and 25mL of water. 10g of aluminum-nickel alloy was added, and the mixture was heated to 90 ℃ and stirred for 5 hours. The reaction system was concentrated under reduced pressure, and the residue was adjusted to pH 9-10 with 2M aqueous sodium hydroxide solution and stirred at room temperature for 1 hour. Adjusting pH to 6-7 with 2M hydrochloric acid water solution, extracting with ethyl acetate (150mL × 3), combining organic phases, washing with water (100mL × 1), drying over anhydrous sodium sulfate, filtering, and concentrating the filtrate under reduced pressure to obtain 1- (4-hydroxybutyl) -benzotriazole-5-carbaldehyde (1E) (6.0g, 66.7%).

LCMS m/z＝220.0[M+1]⁺

The fifth step: 4- (5-Formylbenzotriazol-1-yl) butyl methanesulfonate (intermediate 1)

4-(5-formylbenzotriazol-1-yl)butylmethanesulfonate

1- (4-hydroxybutyl) -benzotriazole-5-carbaldehyde (1E) (6.0g, 27.4mmol) was dissolved in 150mL of dichloromethane, diisopropylethylamine (6.2g, 48.1mmol) was added, MsCl (4.4g, 38.4mmol) was slowly added dropwise over an ice-water bath, and after the addition, stirring was carried out at 0 ℃ for 2 hours. 200mL of methylene chloride was added, and the mixture was washed with 100mL of water, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 4:1-2:1) to give 4- (5-formylbenzotriazol-1-yl) butyl methanesulfonate (intermediate 1) (6.0g, yield: 74.1%).

LCMS m/z＝298.0[M+1]⁺

Intermediate 2: 4- (5-Formylbenzotriazol-1-yl) ethyl methanesulfonate (intermediate 2)

4-(5-formylbenzotriazol-1-yl)ethylmethanesulfonate

The first step is as follows: 4- (4-Hydroxyethylamino) -3-nitrobenzonitrile (2B)

4-(4-Hydroxyethylamino)-3-nitro-benzonitrile

The starting 4-fluoro-3-nitrobenzonitrile (2A) (10g,60.2mmol) was dissolved in 200mL tetrahydrofuran and DIPEA (11.7g,90.5mmol) and ethanolamine (5.52g,90.4mmol) were added sequentially. After the addition was complete, the mixture was stirred at room temperature overnight. The reaction was concentrated, and the residue was dissolved in water, extracted with ethyl acetate (150 mL. times.3), washed with water, dried over anhydrous sodium sulfate, and spin-dried to give crude 4- (4-hydroxyethylamino) -3-nitrobenzonitrile (2B) (12g), which was used directly in the next step.

LC-MS m/z＝208.0[M+1]⁺

The second step is that: 3-amino-4- (4-hydroxyethylamino) benzonitrile (2C)

3-Amino-4-(4-hydroxyethylamino)benzonitrile

4- (4-hydroxyethylamino) -3-nitrobenzonitrile (2B) (5.0g, 24.15mmol) was added to 100ml of ethanol, 0.75 g of 10% palladium on carbon was added, the mixture was replaced with hydrogen three times, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give 3-amino-4- (4-hydroxyethylamino) benzonitrile (2C) (5g, yield: > 99%).

LC-MS m/z＝178.0[M+1]⁺

The third step: 1- (4-hydroxyethyl) -benzotriazole-5-carbonitrile (2D)

1-(4-hydroxyethyl)-benzotriazole-5-carbonitrile

3-amino-4- (4-hydroxyethylamino) benzonitrile (2C) (10.0g, 56.5mmol) was dissolved in 120mL of 6N hydrochloric acid, and 40mL of an aqueous solution of sodium nitrite (5.85g,84.7mmol) was slowly added dropwise in an ice-water bath. After the addition was complete, the mixture was stirred at room temperature for 4 hours. Extraction was performed with dichloromethane (100 mL. times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 1- (4-hydroxyethyl) -benzotriazole-5-carbonitrile (2D) (6.1g, yield: 57.5%).

LC-MS m/z＝189.0[M+1]⁺

The fourth step: 1- (4-hydroxyethyl) -benzotriazole-5-formaldehyde (2E)

1-(4-hydroxyethyl)-benzotriazole-5-carbaldehyde

1- (4-hydroxyethyl) -benzotriazole-5-carbonitrile (2D) (6.1g, 32.4mmol) was dissolved in 75mL of formic acid and 25mL of water. 8g of aluminum-nickel alloy was added, and the mixture was heated to 90 ℃ and stirred for 5 hours. The reaction system was concentrated under reduced pressure, and the residue was adjusted to pH 9-10 with 2M aqueous sodium hydroxide solution and stirred at room temperature for 1 hour. Adjusting pH to 6-7 with 2M hydrochloric acid water solution, extracting with ethyl acetate (150mL × 3), combining organic phases, washing with water (100mL × 1), drying over anhydrous sodium sulfate, filtering, and concentrating the filtrate under reduced pressure to obtain 1- (4-hydroxyethyl) -benzotriazole-5-carbaldehyde (2E) (4.0g, 64.5%).

LCMS m/z＝192.0[M+1]⁺

The fifth step: 4- (5-Formylbenzotriazol-1-yl) ethyl methanesulfonate (intermediate 2)

4-(5-formylbenzotriazol-1-yl)ethylmethanesulfonate

1- (4-hydroxyethyl) -benzotriazole-5-carbaldehyde (2E) (4.1g, 21.5mmol) was dissolved in 150mL of dichloromethane, diisopropylethylamine (5.4g, 41.8mmol) was added, MsCl (3.6g, 31.4mmol) was slowly added dropwise over an ice-water bath, and after the addition, stirring was performed at 0 ℃ for 2 hours. 200mL of methylene chloride was added, and the mixture was washed with 100mL of water, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 4:1-2:1) to give 4- (5-formylbenzotriazol-1-yl) ethyl methanesulfonate (intermediate 2) (5.5g, yield: 95.3%).

LCMS m/z＝298.0[M+1]⁺

¹H NMR(400MHz,DMSO-d₆)10.16(s,1H),8.74(s,1H),8.07(d,2H),5.18(t,2H),4.74(t,2H),3.09(s,3H).

Example 1:

[6- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propylamino ] spiro [3.3] heptan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 1)

[6-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propylamino]spiro[3.3]heptan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: [6- [ tert-Butoxycarbonyl (methyl) amino ] spiro [3.3] heptan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (1b)

[6-[tert-butoxycarbonyl(methyl)amino]spiro[3.3]heptan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

Tert-butyl N- (2-hydroxyspiro [3.3] heptan-6-yl) -N-methylcarbamate (1a) (0.5g, 2.07mmol) was dissolved in toluene (20mL), sodium hydride (0.311g, 7.77mmol) was added, stirring was carried out for 30min, methyl 2-hydroxy-2, 2-bis (2-thienyl) acetate (0.66g, 2.59mmol) was added, and the mixture was heated to 115 ℃ for reaction for 10 hours. The reaction was cooled to room temperature, quenched with 10mL of water, extracted with ethyl acetate (50mL × 2), the organic phases were combined, washed with a saturated aqueous solution of sodium chloride (20mL × 1), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v) ═ 1:100-1:20) to give [6- [ tert-butoxycarbonyl (methyl) amino ] spiro [3.3] hept-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (1b) (0.30g, yield: 20%).

LCMS m/z＝364.1[M-100+1]⁺

The second step is that: [6- (methylamino) spiro [3.3] heptan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (1c)

[6-(Methylamino)spiro[3.3]heptan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

[6- [ tert-Butoxycarbonyl (methyl) amino ] spiro [3.3] heptan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (1b) (0.30g, 0.60mmol) was dissolved in 1, 4-dioxane (10mL), and reacted at room temperature for 2 hours with an excess of hydrogen chloride gas. Water (20mL) was added, the pH was adjusted to 8-9 with solid potassium carbonate at 0 ℃, extracted with ethyl acetate (20 mL. times.2), the organic phases were combined, washed with a saturated aqueous sodium chloride solution (20 mL. times.1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give [6- (methylamino) spiro [3.3] heptan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (1c) (0.16g, yield: 70%).

LCMS m/z＝364.1[M+1]⁺

The third step: [6- [3- (5-formylbenzotriazol-1-yl) propylmethylamino ] spiro [3.3] heptan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (1d)

[6-[3-(5-Formylbenzotriazol-1-yl)propyl-methyl-amino]spiro[3.3]heptan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

[6- (methylamino) spiro [3.3] heptan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (1c) (0.16g, 0.44mmol) was dissolved in 10mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) propyl methanesulfonate (synthetic method see WO2017114377) (0.19g, 0.66mmol) and diisopropylethylamine (0.219g, 1.7mmol) were added, and heated to 90 ℃ for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [6- [3- (5-formylbenzotriazol-1-yl) propylmethylamino ] spiro [3.3] hept-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (1d) (0.15g, yield: 62%).

LCMS m/z＝551.1[M+1]⁺

The fourth step: [6- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propylamino ] spiro [3.3] heptan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 1)

[6-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propylamino]spiro[3.3]heptan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

Reacting [6- [3- (5-formylbenzotriazol-1-yl) propyl ] methylamino]Spiro [3.3]Heptane-2-yl radical]-2-hydroxy-2, 2-bis (2-thienyl) acetate (1d) (0.15g,0.27mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropyl alcohol, (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride salt (0.12g,0.54mmol) was added dropwise, acetic acid (0.03g,0.53mmol) was added dropwise, and 5g of dry

Molecular sieve, stirring for 1 hour at room temperature, adding sodium triacetoxyborohydride (0.23g,1.10mmol), reacting at room temperature overnight, pouring the reaction solution into 50mL saturated aqueous sodium bicarbonate solution, extracting the aqueous phase with dichloromethane/methanol (v/v ═ 10:1) (50mL × 3), combining the organic phases, washing the organic phase with 50mL water, drying over anhydrous sodium sulfate, concentrating, purifying the residue by preparative chromatography (instrument and preparative column: using Glison GX-281 to prepare liquid phase, model of preparative column is Sunfire C18, 5 μm, inner diameter ═ 30mm ═ length ═ 150 mm). preparation method, crude product is dissolved with methanol and dimethyl sulfoxide, and filtered with 0.45 μm filter membrane, mobile phase system acetonitrile/water (containing 0.1% TFA). gradient elution method, acetonitrile is eluted with 5% gradient elution 60% (elution time 15min), freeze-drying to obtain [6- [3- [5- [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxoethyl-2-quinoline-5-oxo-5H-based) and freeze-5-ethyl quinoline]Amino group]Methyl radical]Benzotriazol-1-yl]Propylamine radical]Spiro [3.3]Heptane-2-yl radical]-2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 1) (0.01g, yield: 5%).

¹H NMR(400MHz,CD₃OD)8.21(t,2H),7.89(d,1H),7.72(d,1H),7.36(d,2H),7.26(d,1H),7.12–7.11(m,2H),7.01(d,1H),6.97–6.95(m,2H),6.56(d,1H),5.42–5.41(m,1H),5.04–5.00(m,1H),4.86–4.83(m,2H),4.50(s,2H),3.67–3.61(m,1H),3.28–3.25(m,2H),2.94(s,3H),2.59–2.55(m,2H),2.49–2.43(m,4H),2.17–2.16(m,6H).

LCMS m/z＝378.2[M/2+1]⁺

Example 2:

[2- [2- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] ethyl ] -2-azaspiro [3.5] non-7-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 2)

[2-[2-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]ethyl]-2-azaspiro[3.5]nonan-7-yl]2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: 7- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-2-azaspiro [3,5] nonane-2-carboxylic acid tert-butyl ester (2b)

Tert-butyl 7-[2-hydroxy-2,2-bis(2-thienyl)acetyl]oxy-2-azaspiro[3.5]nonane-2-carboxylate

7-hydroxy-2-azaspiro [3.5] nonane-2-carboxylic acid tert-butyl ester (1.5g, 6.26mmol) was dissolved in toluene (60mL), sodium hydride (0.180g, 7.52mmol) was added, stirring was carried out for 30 minutes, methyl 2-hydroxy-2, 2-bis (2-thienyl) acetate (1.75g, 6.89mmol) was added, and the mixture was heated to 115 ℃ to react for 10 hours. The reaction was cooled to room temperature, quenched with 10mL of water, extracted with ethyl acetate (50mL × 2), washed with a saturated aqueous sodium chloride solution (20mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v) ═ 1:100-1:20) to give 7- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-2-azaspiro [3,5] nonane-2-carboxylic acid tert-butyl ester (2b) (1.4g, yield: 50%).

The second step is that: 2-Azaspiro [3.5] nonan-7-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (2c)

2-Azaspiro[3.5]nonan-7-yl-2-hydroxy-2,2-bis(2-thienyl)acetate

Tert-butyl 7- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-2-azaspiro [3,5] nonane-2-carboxylate (2b) (0.8g, 1.7mmol) was dissolved in 1, 4-dioxane (10mL), and hydrogen chloride gas was introduced and reacted at room temperature for 2 hours. Water (20mL) was added, the pH was adjusted to 8-9 with solid potassium carbonate at 0 ℃, extracted with ethyl acetate (20 mL. times.2), washed with a saturated aqueous sodium chloride solution (20 mL. times.1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 2-azaspiro [3.5] nonan-7-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (2c) (0.50g, yield: 80%).

LCMS m/z＝364.1[M+1]⁺

The third step: [2- [2- (5-Formylbenzotriazol-1-yl) ethyl ] -2-azaspiro [3.5] non-7-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (2d)

[2-[2-(5-formylbenzotriazol-1-yl)ethyl]-2-azaspiro[3.5]nonan-7-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

2-Azaspiro [3.5] nonan-7-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (2c) (0.1g, 0.275mmol) was dissolved in 10mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) ethyl methanesulfonate (intermediate 2) (0.074g, 0.275mmol) and diisopropylethylamine (0.071g, 0.55mmol) were added and the mixture was heated to 90 ℃ for reaction for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [2- [2- (5-formylbenzotriazol-1-yl) ethyl ] -2-azaspiro [3.5] non-7-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (2d) (0.1g, yield: 70%).

LCMS m/z＝537.1[M+1]⁺

The second step is that: [2- [2- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] ethyl ] -2-azaspiro [3.5] non-7-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 2)

[2-[2-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]ethyl]-2-azaspiro[3.5]nonan-7-yl]2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

Reacting [2- [2- (5-formylbenzotriazol-1-yl) ethyl group]-2-azaspiro [3.5]Non-7-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate (2d) (0.1g,0.186mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropyl alcohol, and (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride salt (0.089g,0.373mmol) was added dropwise, acetic acid was added dropwise thereto 1 drop, and 5g of dry ethanol was added

Molecular sieves, after stirring for 1 hour at room temperature, sodium triacetoxyborohydride (0.197g,0.932mmol) was added and the reaction was allowed to proceed overnight at room temperature, the reaction was poured into 50mL of a saturated aqueous solution of sodium bicarbonate, the aqueous phase was extracted with dichloromethane/methanol (v/v 10:1) (50mL × 3), the organic phases were combined, washed with 50mL of water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was subjected to Pre-HPLC (apparatus and preparative column: A liquid phase was prepared using Glison GX-281, model: Sunfire C18, 5 μm, inner diameter ═ 30mm length ═ 150 mm.) the crude product was dissolved in methanol and dimethyl sulfoxide and filtered through a 0.45 μm filter to prepare a sample solutionoxo-1H-quinolin-5-yl) ethyl]Amino group]Methyl radical]Benzotriazol-1-yl]Ethyl radical]-2-azaspiro [3.5]Non-7-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate ditrifluoroacetate (Compound 2) (0.036g, yield: 20%).

¹H NMR(400MHz,CD₃OD)8.30–8.18(m,2H),7.90(d,1H),7.73(d,1H),7.36(dd,2H),7.27(d,1H),7.13(dd,2H),7.06–6.92(m,3H),6.58(d,1H),5.51–5.39(m,1H),5.05(t,2H),4.92(s,1H),4.50(s,2H),3.99–3.94(m,6H),3.31–3.26(m,2H),1.86–1.55(m,8H).

¹⁹F NMR(376MHz,CD₃OD)-74.84(s,1H).

LCMS m/z＝741.2[M+1]⁺

Example 3:

[2- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -2-azaspiro [3.5] non-7-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 3)

[2-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-2-azaspiro[3.5]nonan-7-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: [2- [3- (5-Formylbenzotriazol-1-yl) propyl ] -2-azaspiro [3.5] non-7-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (3a)

[2-[3-(5-formylbenzotriazol-1-yl)propyl]-2-azaspiro[3.5]nonan-7-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

2-Azaspiro [3.5] nonan-7-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (2c) (0.1g, 0.275mmol) was dissolved in 10ml acetonitrile, 4- (5-formylbenzotriazol-1-yl) propyl methanesulfonate (synthetic method see WO2017114377) (0.077g, 0.275mmol) and diisopropylethylamine (0.071g, 0.55mmol) were added and heated to 90 ℃ for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [2- [3- (5-formylbenzotriazol-1-yl) propyl ] -2-azaspiro [3.5] non-7-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (3a) (0.106g, yield: 70%).

LCMS m/z＝551.2[M+1]⁺

The second step is that: [2- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -2-azaspiro [3.5] non-7-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 3)

[2-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-2-azaspiro[3.5]nonan-7-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

By reacting [2- [3- (5-formylbenzotriazol-1-yl) propyl ] group]-2-azaspiro [3.5]Non-7-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate (3a) (0.102g,0.186mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropyl alcohol, and (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride salt (0.089g,0.373mmol) was added dropwise, acetic acid was added dropwise thereto 1 drop, and 5g of dry ethanol was added

Molecular sieves, stirring for 1 hour at room temperature, adding sodium triacetoxyborohydride (0.197g,0.932mmol) and reacting overnight at room temperature, pouring the reaction mixture into 50mL of saturated aqueous sodium bicarbonate, extracting the aqueous phase with dichloromethane/methanol (v/v ═ 10:1) (50mL × 3), combining the organic phases, washing the organic phase with 50mL of water, drying over anhydrous sodium sulfate, concentrating under reduced pressure, passing the residue through Pre-HPLC (Instrument and preparative column: preparation of the liquid phase using Glison GX-281, preparative column model Sunfire C18, 5 μm, internal diameter ═ 30mm length ═ 30mm 150 mm.) preparation method using methanol and dimethyl sulfoxideThe sulfone was dissolved and filtered through a 0.45 μm filter to prepare a sample solution. Mobile phase system: acetonitrile/water (with 0.1% TFA). Gradient elution method: eluting acetonitrile with 5% gradient 60% (elution time 15min), and lyophilizing to obtain [2- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] 2- [3- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl)]Amino group]Methyl radical]Benzotriazol-1-yl]Propyl radical]-2-azaspiro [3.5]Non-7-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate ditrifluoroacetate (Compound 3) (0.036g, yield: 20%).

¹H NMR(400MHz,CD₃OD)8.30–8.12(m,2H),7.88(d,1H),7.70(d,1H),7.36(d,2H),7.27(d,1H),7.13(dd,2H),7.02(d,1H),6.97(dd,2H),6.55(d,1H),5.44(dd,1H),4.93(s,1H),4.85(s,2H),4.50(t,2H),4.15–3.95(m,2H),3.95–3.75(m,2H),3.39–3.32(m,2H),3.29–3.20(m,2H),2.37–2.25(m,2H),1.85–1.57(m,8H).

LCMS m/z＝755.2[M+1]⁺

Example 4:

[7- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl-methyl-amino ] spiro [3.5] non-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 4)

[7-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl-methyl-amino]spiro[3.5]nonan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: [7- [ tert-Butoxycarbonyl (methyl) amino ] spiro [3.5] non-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (4b)

[7-[tert-butoxycarbonyl(methyl)amino]spiro[3.5]nonan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

N- (2-Hydroxyspiro [3.5] non-7-yl) -N-methyl-carbamic acid tert-butyl ester (4a) (0.5g, 1.9mmol) was dissolved in toluene (60mL), sodium hydride (0.075g, 1.9mmol) was added, stirring was carried out for 30 minutes, methyl 2-hydroxy-2, 2-bis (2-thienyl) acetate (0.94g, 3.8mmol) was added, and the mixture was heated to 115 ℃ for reaction for 10 hours. The reaction was cooled to room temperature, quenched with 10mL of water, extracted with ethyl acetate (50mL × 2), washed with a saturated aqueous solution of sodium chloride (20mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v) ═ 1:100-1:20) to give [7- [ tert-butoxycarbonyl (methyl) amino ] spiro [3.5] non-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (4b) (0.40g, yield: 44%)

The second step is that: [7- (methylamino) spiro [3.5] non-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (4c)

[7-(Methylamino)spiro[3.5]nonan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

[7- [ tert-Butoxycarbonyl (methyl) amino ] spiro [3.5] non-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (4b) (0.40g, 0.81mmol) was dissolved in 1, 4-dioxane (10mL), and reacted at room temperature for 2 hours with hydrogen chloride gas. Water (20mL) was added, the pH was adjusted to 8-9 with solid potassium carbonate at 0 ℃, extracted with dichloromethane (30 mL. times.2), washed with a saturated aqueous sodium chloride solution (20 mL. times.1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give [7- (methylamino) spiro [3.5] non-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (4c) (0.30g, yield: 90%).

LCMS m/z＝392.3[M+1]⁺

The third step: [7- [3- (5-formylbenzotriazol-1-yl) propyl-methyl-amino ] spiro [3.5] non-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (4d)

[7-[3-(5-Formylbenzotriazol-1-yl)propyl-methyl-amino]spiro[3.5]nonan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

[7- (methylamino) spiro [3.5] non-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (4c) (0.30g, 0.77mmol) was dissolved in 10mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) propyl methanesulfonate (synthetic method see WO2017114377) (0.24g, 0.84mmol) and diisopropylethylamine (0.20g, 1.5mmol) were added, and after the addition was completed, the mixture was heated to 90 ℃ for reaction for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [7- [3- (5-formylbenzotriazol-1-yl) propyl-methyl-amino ] spiro [3.5] non-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (4d) (0.20g, yield: 45%).

LCMS m/z＝579.3[M+1]⁺

The fourth step: [7- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl-methyl-amino ] spiro [3.5] non-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 4)

[7-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl-methyl-amino]spiro[3.5]nonan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

Reacting [7- [3- (5-formylbenzotriazol-1-yl) propyl-methyl-amino]Spiro [3.5]Non-2-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate (4d) (0.20g,0.346mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropyl alcohol, and (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride salt (0.135g,0.518mmol) was added thereto, acetic acid (0.031g,0.518mmol) was added thereto dropwise, and 5g of dry acetic acid was added thereto

After stirring for 6 hours at room temperature, sodium triacetoxyborohydride (0.220g,1.04mmol) was added and reacted at room temperature overnight. The reaction solution was poured into 50mL of saturated bicarbonateAqueous sodium solution, aqueous phase was extracted with dichloromethane/methanol (v/v 10:1) (50mL × 3), organic phases were combined, organic phase was washed with 50mL water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was subjected to Pre-HPLC (instrument and preparative column: liquid phase was prepared using glison gx-281, model No. Sunfire C18, 5 μm, inner diameter 30mm length 150 mm.) preparation method in which crude product was dissolved with methanol and dimethyl sulfoxide and filtered through 0.45 μm filter to prepare a sample solution.mobile phase system acetonitrile/water (containing 0.1% TFA.) gradient elution method in which acetonitrile was eluted with 5% gradient for 60% (elution time 15min), lyophilized to give [7- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] to [7- [3- [5- [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl)]Amino group]Methyl radical]Benzotriazol-1-yl]Propyl-methyl-amino]Spiro [3.5]Non-2-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 4) (0.050g, yield: 14%).

¹H NMR(400MHz,CD₃OD)8.22(d,2H),7.91(d,1H),7.75–7.67(m,1H),7.36(dt,2H),7.27(d,1H),7.13(d,2H),7.01(d,1H),6.99-6.95(m,2H),6.56(d,1H),5.43(dd,1H),4.90(s,1H),4.85(t,2H),4.55–4.47(m,2H),3.75–3.64(m,1H),3.26(dd,3H),3.10(d,1H),2.82–2.69(m,3H),2.48(d,2H),2.23(d,2H),1.90(dd,2H),1.71(d,2H),1.67–1.52(m,4H),1.52–1.39(m,2H).

¹⁹F NMR(376MHz,CD₃OD)-74.84.

LCMS m/z＝783.3[M+1]⁺.

Example 5:

[3- [2- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] ethyl ] -3-azaspiro [5.5] undecan-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 5)

[3-[2-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]ethyl]-3-azaspiro[5.5]undecan-9-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: 9- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-3-azaspiro [5.5] undecane-3-carboxylic acid tert-butyl ester (5b)

Tert-butyl 9-[2-hydroxy-2,2-bis(2-thienyl)acetyl]oxy-3-azaspiro[5.5]undecane-3-carboxylate

Tert-butyl 9-hydroxy-3-aza-spiro [5.5] undecane-3-carboxylate (1.00g, 3.71mmol) was dissolved in toluene (60mL), sodium hydride (0.178g, 7.42mmol) was added, stirring was carried out for 30 minutes, methyl 2-hydroxy-2, 2-bis (2-thienyl) acetate (1.42g, 5.57mmol) was added, and the temperature was raised to 115 ℃ for reaction for 10 hours. The reaction was cooled to room temperature, quenched with 10mL of water, extracted with ethyl acetate (50mL × 2), washed with a saturated aqueous solution of sodium chloride (20mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v) ═ 1:100-1:20) to give 9- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-3-azaspiro [5.5] undecane-3-carboxylic acid tert-butyl ester (5b) (1.2g, yield: 66%).

¹H NMR(400MHz,CDCl₃)7.27(dt,2H),7.18(dd,2H),6.97(dd,2H),4.98(dt,1H),4.78(s,1H),3.41–3.29(m,4H),1.74(ddd,2H),1.70–1.60(m,2H),1.51–1.41(m,11H),1.36(dd,4H),1.33–1.23(m,2H).

LCMS m/z＝514.2[M+23]⁺

The second step is that: 3-azaspiro [5.5] undecan-9-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (5c)

3-Azaspiro[5.5]undecan-9-yl-2-hydroxy-2,2-bis(2-thienyl)acetate

Tert-butyl 9- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-3-azaspiro [5.5] undecane-3-carboxylate (5b) (0.60g, 1.22mmol) was dissolved in 1, 4-dioxane (10mL), and reacted at room temperature for 2 hours with excess hydrogen chloride gas. Water (20mL) was added, the pH was adjusted to 8-9 with solid potassium carbonate at 0 ℃, extracted with ethyl acetate (20 mL. times.2), washed with a saturated aqueous sodium chloride solution (20 mL. times.1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 3-azaspiro [5.5] undecan-9-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (5c) (0.22g, yield: 50%).

LCMS m/z＝392.1[M+1]⁺

The third step: [3- [2- (5-Formylbenzotriazol-1-yl) ethyl ] -3-azaspiro [5.5] undecan-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (5d)

[3-[2-(5-formylbenzotriazol-1-yl)ethyl]-3-azaspiro[5.5]undecan-9-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

3-Azaspiro [5.5] undecan-9-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (5c) (0.22g, 0.562mmol) was dissolved in 10mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) ethyl methanesulfonate (intermediate 2) (0.182g, 0.674mmol) and diisopropylethylamine (0.145g, 1.12mmol) were added, and after the addition, the reaction was heated to 90 ℃ for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [3- [2- (5-formylbenzotriazol-1-yl) ethyl ] -3-azaspiro [5.5] undecan-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (5d) (0.14g, yield: 44%).

LCMS m/z＝565.2[M+1]⁺

The fourth step: [3- [2- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] ethyl ] -3-azaspiro [5.5] undecan-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 5)

[3-[2-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]ethyl]-3-azaspiro[5.5]undecan-9-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

Reacting [3- [2- (5-formylbenzotriazol-1-yl) ethyl]-3-azaspiro [5.5]Undec-9-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate (5d) (0.14g,0.25mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropyl alcohol, (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride (0.17g,0.37mmol) was added dropwise, acetic acid (0.022g,0.37mmol) was added dropwise, and 5g of dry

Molecular sieves, stirring for 1 hour at room temperature, adding sodium triacetoxyborohydride (0.16g,0.74mmol), reacting overnight at room temperature, pouring the reaction mixture into 50mL of saturated aqueous sodium bicarbonate solution, extracting the aqueous phase with dichloromethane/methanol (v/v ═ 10:1) (50mL × 3), combining the organic phases, washing the organic phase with 50mL of water, drying over anhydrous sodium sulfate, concentrating under reduced pressure, passing the residue through a Pre-HPLC (Instrument and preparative column: A liquid phase is prepared using Glison GX-281, model is Sunfire C18, 5 μm, internal diameter ═ 30mm length ═ 150 mm.) preparation method, crude product is dissolved with methanol and dimethyl sulfoxide, and filtered through a 0.45 μm filter membrane to prepare a sample solution]Amino group]Methyl radical]Benzotriazol-1-yl]Ethyl radical]-3-azaspiro [5.5]Undec-9-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate ditrifluoroacetate salt (compound 5) (0.11g, 45% yield).

¹H NMR(400MHz,CD₃OD)8.27–8.19(m,2H),7.93(d,1H),7.73(dd,1H),7.37(dd,2H),7.27(d,1H),7.15(dd,2H),7.04–6.95(m,3H),6.57(d,1H),5.44(dd,1H),5.20(t,2H),4.97(dd,1H),4.50(s,2H),3.86(t,2H),3.68–3.43(m,2H),3.31(s,2H),3.29–3.25(m,2H),1.72(ddd,8H),1.49(s,4H).

LCMS m/z＝385.2[M/2+1]⁺

Example 6:

[3- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -3-azaspiro [5.5] undecan-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 6)

[3-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-3-azaspiro[5.5]undecan-9-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: [3- [3- (5-Formylbenzotriazol-1-yl) propyl ] -3-azaspiro [5.5] undecan-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (6a)

[3-[3-(5-formylbenzotriazol-1-yl)propyl]-3-azaspiro[5.5]undecan-9-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

3-Azaspiro [5.5] undecane-9-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (5c) (0.080g, 0.20mmol) was dissolved in 10mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) propyl methanesulfonate (synthetic method see WO2017114377) (0.069g, 0.25mmol) and diisopropylethylamine (0.053g, 0.41mmol) were added, heated to 90 ℃ and reacted for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [3- [3- (5-formylbenzotriazol-1-yl) propyl ] -3-azaspiro [5.5] undecan-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (6a) (0.10g, yield: 85%).

LCMS m/z＝579.2[M+1]⁺

The second step is that: [3- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -3-azaspiro [5.5] undecan-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 6)

[3-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-3-azaspiro[5.5]undecan-9-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

Reacting [3- [3- (5-formylbenzotriazol-1-yl) propyl ] group]-3-azaspiro [5.5]Undec-9-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate (6a) (0.10g,0.17mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropyl alcohol, (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride salt (0.12g,0.26mmol) was added dropwise, acetic acid (0.016g,0.26mmol) was added dropwise, and 5g of dry

Molecular sieves, stirring for 1 hour at room temperature, adding sodium triacetoxyborohydride (0.11g,0.52mmol), reacting overnight at room temperature, pouring the reaction mixture into 50mL of saturated aqueous sodium bicarbonate solution, extracting the aqueous phase with dichloromethane/methanol (v/v ═ 10:1) (50mL × 3), combining the organic phases, washing the organic phase with 50mL of water, drying over anhydrous sodium sulfate, concentrating under reduced pressure, passing the residue through a Pre-HPLC (Instrument and preparative column: A liquid phase is prepared using Glison GX-281, model is Sunfire C18, 5 μm, internal diameter ═ 30mm length ═ 150 mm.) preparation method, crude product is dissolved with methanol and dimethyl sulfoxide, and filtered through a 0.45 μm filter membrane to prepare a sample solution]Amino group]Methyl radical]Benzotriazol-1-yl]Propyl radical]-3-azaspiro [5.5]Undec-9-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate ditrifluoroacetate (Compound 6) (0.030g, yield: 17%).

¹H NMR(400MHz,CD₃OD)8.22(d,2H),7.89(d,1H),7.70(d,1H),7.40(ddd,2H),7.27(d,1H),7.19–7.12(m,2H),7.01(d,1H),6.97(dd,2H),6.55(d,1H),5.43(dd,1H),4.96(dd,1H),4.85(t,2H),4.55–4.46(m,2H),3.48–3.37(m,2H),3.30–3.20(m,4H),3.05(t,2H),2.56–2.45(m,2H),1.92(d,1H),1.71(dd,5H),1.55(d,4H),1.46–1.33(m,1H),1.25(d,1H).

LCMS m/z＝783.3[M+1]⁺

Example 7:

[2- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -2-azaspiro [4.4] non-8-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 7)

[2-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-2-azaspiro[4.4]nonan-8-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: 8- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-2-azaspiro [4.4] nonane-2-carboxylic acid tert-butyl ester (7b)

Tert-butyl 8-[2-hydroxy-2,2-bis(2-thienyl)acetyl]oxy-2-azaspiro[4.4]nonane-2-carboxylate

7-hydroxy-2-azaspiro [4.4] nonane-2-carboxylic acid tert-butyl ester (7a) (0.5g, 2.1mmol) was dissolved in toluene (60mL), sodium hydride (0.099g, 4.1mmol) was added, stirring was carried out for 30 minutes, methyl 2-hydroxy-2, 2-bis (2-thienyl) acetate (0.79g, 3.1mmol) was added, and the mixture was heated to 115 ℃ for reaction for 10 hours. The reaction was cooled to room temperature, quenched with 10mL of water, extracted with ethyl acetate (50mL × 2), washed with a saturated aqueous solution of sodium chloride (20mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v) ═ 1:100-1:20) to give 8- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-2-azaspiro [4.4] nonane-2-carboxylic acid tert-butyl ester (7b) (0.90g, yield: 94%).

LCMS m/z＝486.1[M+23]⁺

The second step is that: 2-Azaspiro [4.4] nonan-8-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (7c)

2-Azaspiro[4.4]nonan-8-yl-2-hydroxy-2,2-bis(2-thienyl)acetate

Tert-butyl 8- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-2-azaspiro [4.4] nonane-2-carboxylate (7b) (0.90g, 1.90mmol) was dissolved in 1, 4-dioxane (10mL), and reacted at room temperature for 2 hours with excess hydrogen chloride gas. Water (20mL) was added, the pH was adjusted to 8-9 with solid potassium carbonate at 0 ℃, extracted with ethyl acetate (20 mL. times.2), washed with a saturated aqueous sodium chloride solution (20 mL. times.1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 2-azaspiro [4.4] nonan-8-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (7c) (0.40g, yield: 57%).

LCMS m/z＝364.1[M+1]⁺

The third step: [2- [3- (5-Formylbenzotriazol-1-yl) propyl ] -2-azaspiro [4.4] non-8-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (7d)

[2-[3-(5-formylbenzotriazol-1-yl)propyl]-2-azaspiro[4.4]nonan-8-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

2-Azaspiro [4.4] nonan-8-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (7c) (0.18g, 0.495mmol) was dissolved in 10mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) propyl methanesulfonate (synthetic method see WO2017114377) (0.168g, 0.594mmol) and diisopropylethylamine (0.128g, 0.99mmol) were added, and after the addition was completed, the mixture was heated to 90 ℃ for reaction for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [2- [3- (5-formylbenzotriazol-1-yl) propyl ] -2-azaspiro [4.4] non-8-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (7d) (0.20g, yield: 73%).

LCMS m/z＝551.1[M+1]⁺

The fourth step: [2- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -2-azaspiro [4.4] non-8-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 7)

[2-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-2-azaspiro[4.4]nonan-8-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

By reacting [2- [3- (5-formylbenzotriazol-1-yl) propyl ] group]-2-azaspiro [4.4]Non-8-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate (7d) (0.14g,0.25mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropyl alcohol, (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride (0.17g,0.38mmol) was added dropwise, acetic acid (0.022g,0.38mmol) was added dropwise, and 5g of dry

Molecular sieves, stirring for 1 hour at room temperature, adding sodium triacetoxyborohydride (0.16g,0.76mmol), reacting overnight at room temperature, pouring the reaction mixture into 50mL of saturated aqueous sodium bicarbonate solution, extracting the aqueous phase with dichloromethane/methanol (v/v ═ 10:1) (50mL × 3), combining the organic phases, washing the organic phase with 50mL of water, drying over anhydrous sodium sulfate, concentrating under reduced pressure, passing the residue through a Pre-HPLC (Instrument and preparative column: A liquid phase is prepared using Glison GX-281, model is Sunfire C18, 5 μm, internal diameter ═ 30mm length ═ 150 mm.) preparation method, crude product is dissolved with methanol and dimethyl sulfoxide, and filtered through a 0.45 μm filter membrane to prepare a sample solution]Amino group]Methyl radical]Benzotriazol-1-yl]Propyl radical]-2-azaspiro [4.4]Non-8-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 7) (0.035g, yield: 14％)。

¹H NMR(400MHz,CD₃OD)8.22(d,2H),7.90(dd,1H),7.71(dt,1H),7.42–7.21(m,3H),7.15–7.06(m,2H),7.05–6.84(m,3H),6.55(dd,1H),5.43(dd,1H),5.32(s,1H),4.85(d,2H),4.58–4.45(m,2H),3.57(s,2H),3.33(d,2H),3.29–3.23(m,2H),3.22–3.11(m,1H),3.04(d,1H),2.46(dt,2H),2.11(dd,2H),1.82(dd,6H).

LCMS m/z＝755.2[M+1]⁺

Example 8:

[2- [4- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] butyl ] -2-azaspiro [4.4] non-8-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 8a)

[2-[4-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]butyl]-2-azaspiro[4.4]nonan-8-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate(isomer 1)

[2- [4- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] butyl ] -2-azaspiro [4.4] non-8-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 8b)

[2-[4-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]butyl]-2-azaspiro[4.4]nonan-8-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate(isomer 2)

The first step is as follows: [2- [4- (5-Formylbenzotriazol-1-yl) butyl ] -2-azaspiro [4.4] non-8-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (8b)

[2-[4-(5-Formylbenzotriazol-1-yl)butyl]-2-azaspiro[4.4]nonan-8-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

2-Azaspiro [4.4] nonan-8-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (7c) (0.15g, 0.413mmol) was dissolved in 10mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) butyl methanesulfonate (intermediate 1) (0.147g, 0.495mmol) and diisopropylethylamine (0.107g, 0.825mmol) were added, and after the addition, the reaction was heated to 90 ℃ for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [2- [4- (5-formylbenzotriazol-1-yl) butyl ] -2-azaspiro [4.4] non-8-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (8b) (0.12g, yield: 51%).

LCMS m/z＝565.2[M+1]⁺

The second step is that: [2- [4- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] butyl ] -2-azaspiro [4.4] non-8-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 8a)

[2-[4-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]butyl]-2-azaspiro[4.4]nonan-8-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate(isomer 1)

[2- [4- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] butyl ] -2-azaspiro [4.4] non-8-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 8b)

[2-[4-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]butyl]-2-azaspiro[4.4]nonan-8-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate(isomer 2)

Reacting [2- [4- (5-formylbenzotriazol-1-yl) butyl ] with]-2-azaspiro [4.4]Non-8-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate (8b) (0.12g,0.21mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropanol, and (R) -5- (2-amino acid was addedYl-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride (0.12g,0.32mmol), acetic acid (0.019g,0.32mmol) was added dropwise, and 5g of dry substance was added

Molecular sieves, stirring for 1 hour at room temperature, adding sodium triacetoxyborohydride (0.14g,0.64mmol), reacting overnight at room temperature, pouring the reaction mixture into 50mL of saturated aqueous sodium bicarbonate solution, extracting the aqueous phase with dichloromethane/methanol (v/v ═ 10:1) (50mL × 3), combining the organic phases, washing the organic phase with 50mL of water, drying over anhydrous sodium sulfate, concentrating under reduced pressure, passing the residue through a Pre-HPLC (Instrument and preparative column: A liquid phase is prepared using Glison GX-281, model is Sunfire C18, 5 μm, internal diameter ═ 30mm length ═ 150 mm.) preparation method, crude product is dissolved with methanol and dimethyl sulfoxide, and filtered through a 0.45 μm filter membrane to prepare a sample solution]Amino group]Methyl radical]Benzotriazol-1-yl]Butyl radical]-2-azaspiro [4.4]Non-8-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 8a) and [2- [4- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] ethyl]Amino group]Methyl radical]Benzotriazol-1-yl]Butyl radical]-2-azaspiro [4.4]Non-8-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 8b) (0.045g, yield: 21%).

(Compound 8a peaked earlier than Compound 8b)

Compound 8a:

¹H NMR(400MHz,CD₃OD)8.19(d,2H),7.92(d,1H),7.70(d,1H),7.37(s,2H),7.26(d,1H),7.10(s,2H),7.01(d,1H),6.96(s,2H),6.53(d,1H),5.47–5.38(m,1H),5.32(d,1H),4.84(d,2H),4.50(s,2H),3.63(s,1H),3.25(d,2H),3.15(dd,4H),2.74(dd,1H),2.21–2.08(m,4H),1.99(dd,2H),1.87(dd,3H),1.78(d,1H),1.70(s,2H).

LCMS m/z＝385.2[M/2+1]⁺

compound 8b:

¹H NMR(400MHz,CD₃OD)8.21(d,2H),7.90(d,1H),7.70(dd,1H),7.37(td,2H),7.27(d,1H),7.15–7.09(m,2H),7.01(d,1H),6.98(dt,2H),6.54(d,1H),5.43(dd,1H),5.32(s,1H),4.79(s,2H),4.51(s,2H),3.53(dd,2H),3.29–3.18(m,4H),3.12(dd,1H),3.08–2.96(m,1H),2.17–1.99(m,4H),1.90–1.69(m,8H).

LCMS m/z＝385.2[M/2+1]⁺

example 9:

[8- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -8-azaspiro [3.6] decan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 9)

[8-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-8-azaspiro[3.6]decan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: 2- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-8-azaspiro [3.6] decane-8-carboxylic acid tert-butyl ester (9b)

Tert-butyl 2-[2-hydroxy-2,2-bis(2-thienyl)acetyl]oxy-8-azaspiro[3.6]decane-8-carboxylate

Tert-butyl 2-hydroxy-8-azaspiro [3.6] decane-8-carboxylate (1.0g, 3.92mmol) was dissolved in toluene (60mL), sodium hydride (0.188g, 7.83mmol) was added thereto, the mixture was stirred for 30 minutes, methyl 2-hydroxy-2, 2-bis (2-thienyl) acetate (1.5g, 5.87mmol) was added thereto, and the mixture was heated to 115 ℃ to react for 10 hours. The reaction was cooled to room temperature, quenched with 10mL of water, extracted with ethyl acetate (50mL × 2), washed with a saturated aqueous solution of sodium chloride (20mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (v: v) ═ 1:100-1:20) to give tert-butyl 2- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-8-azaspiro [3.6] decane-8-carboxylate (9b) (1.7g, yield: 90.9%).

LCMS m/z＝500.1[M+23]⁺

The second step is that: 8-Azaspiro [3.6] decan-2-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (9c)

8-azaspiro[3.6]decan-2-yl-2-hydroxy-2,2-bis(2-thienyl)acetate

Tert-butyl 2- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-8-azaspiro [3.6] decane-8-carboxylate (9b) (0.90g, 1.90mmol) was dissolved in 1, 4-dioxane (10mL), and reacted at room temperature for 2 hours with excess hydrogen chloride gas being introduced. Water (20mL) was added, the pH was adjusted to 8-9 with solid potassium carbonate at 0 ℃, extracted with ethyl acetate (20 mL. times.2), washed with a saturated aqueous sodium chloride solution (20 mL. times.1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 8-azaspiro [3.6] decan-2-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (9c) (0.40g, yield: 57%).

LCMS m/z＝378.1[M+1]⁺

The third step: [8- [3- (5-Formylbenzotriazol-1-yl) propyl ] -8-azaspiro [3.6] decan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (9d)

[8-[3-(5-Formylbenzotriazol-1-yl)propyl]-8-azaspiro[3.6]decan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

8-azaspiro [3.6] decan-2-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (9c) (0.150g, 0.397mmol) was dissolved in 10mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) propyl methanesulfonate (synthetic method see WO2017114377) (0.135g, 0.477mmol) and diisopropylethylamine (0.103g, 0.795mmol) were added, and after the addition was completed, the mixture was heated to 90 ℃ for reaction for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [8- [3- (5-formylbenzotriazol-1-yl) propyl ] -8-azaspiro [3.6] decan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (9d) (0.10g, yield: 45%).

LCMS m/z＝565.2[M+1]⁺

The fourth step: [8- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -8-azaspiro [3.6] decan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 9)

[8-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-8-azaspiro[3.6]decan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

Reacting [8- [3- (5-formylbenzotriazol-1-yl) propyl ] group]-8-azaspiro [3.6]Decan-2-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate (9d) (0.10g,0.18mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropyl alcohol, (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride salt (0.069g,0.27mmol) was added dropwise, acetic acid (0.016g,0.27mmol) was added dropwise, and 5g of dry ethanol was added

Molecular sieves, stirring for 1 hour at room temperature, adding sodium triacetoxyborohydride (0.11g,0.53mmol), reacting overnight at room temperature, pouring the reaction mixture into 50mL of saturated aqueous sodium bicarbonate solution, extracting the aqueous phase with dichloromethane/methanol (v/v ═ 10:1) (50mL × 3), combining the organic phases, washing the organic phase with 50mL of water, drying over anhydrous sodium sulfate, concentrating under reduced pressure, passing the residue through a Pre-HPLC (Instrument and preparative column: A liquid phase was prepared using Glison GX-281, model number Sunfire C18, 5 μm, internal diameter ═ 30mm length ═ 150 mm.) preparation method: crude product was dissolved with methanol and dimethyl sulfoxide, and filtered through a 0.45 μm filter to prepare a sample solution-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl]Amino group]Methyl radical]Benzotriazol-1-yl]Propyl radical]-8-azaspiro [3.6]Decan-2-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate ditrifluoroacetate salt (Compound 9) (0.015g, yield: 8.5%).

¹H NMR(400MHz,CD₃OD)8.21(d,2H),7.89(d,1H),7.70(d,1H),7.37(d,2H),7.27(d,1H),7.16–7.09(m,2H),7.01(d,1H),6.97(ddd,2H),6.55(d,1H),5.43(dd,1H),5.11(dd,1H),4.84(d,2H),4.50(s,2H),3.48(s,2H),3.26(dd,4H),3.13(s,2H),2.49(s,2H),2.37(s,2H),2.02–1.66(m,8H).

LCMS m/z＝769.2[M+1]⁺

Example 10:

[8- [4- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] butyl ] -8-azaspiro [3.6] decan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 10)

[8-[4-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]butyl]-8-azaspiro[3.6]decan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: [8- [4- (5-Formylbenzotriazol-1-yl) butyl ] -8-azaspiro [3.6] decan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (10a)

[8-[4-(5-formylbenzotriazol-1-yl)butyl]-8-azaspiro[3.6]decan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

8-Azaspiro [3.6] decan-2-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (9c) (0.20g, 0.53mmol) was dissolved in 10mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) butyl methanesulfonate (intermediate 1) (0.19g, 0.64mmol) and diisopropylethylamine (0.14g, 1.1mmol) were added, and after the addition, the reaction was heated to 90 ℃ for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [8- [4- (5-formylbenzotriazol-1-yl) butyl ] -8-azaspiro [3.6] decan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (10a) (0.12g, yield: 39%).

LCMS m/z＝579.2[M+1]⁺

The second step is that: [8- [4- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] butyl ] -8-azaspiro [3.6] decan-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 10)

[8-[4-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]butyl]-8-azaspiro[3.6]decan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

Reacting [8- [4- (5-formylbenzotriazol-1-yl) butyl ] with]-8-azaspiro [3.6]Decan-2-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate (10a) (0.12g,0.21mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropanol, (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride (0.081g,0.31mmol) was added dropwise, acetic acid (0.018g,0.31mmol) was added dropwise, and 5g of dry ethanol was added

Molecular sieves, stirred at room temperature for 1 hour, added sodium triacetoxyborohydride (0.13g,0.62mmol) and reacted at room temperature overnight, the reaction was poured into 50mL of saturated aqueous sodium bicarbonate, the aqueous phase was extracted with dichloromethane/methanol (v/v ═ 10:1) (50mL × 3), the organic phases were combined, the organic phase was washed with 50mL of water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, the residue was subjected to Pre-HPLC (Instrument and preparative column: preparation of liquid phase using Glison GX-281, preparative column model Sunfire C18, 5 μm, internal diameter ═ 30mm length ═ 30mm x 150mm)Phase system: acetonitrile/water (with 0.1% TFA). Gradient elution method: eluting acetonitrile with 5% gradient 60% (elution time 15min), and lyophilizing to obtain [8- [4- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] ester]Amino group]Methyl radical]Benzotriazol-1-yl]Butyl radical]-8-azaspiro [3.6]Decan-2-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate ditrifluoroacetate (Compound 10) (0.10g, yield: 48%).

¹H NMR(400MHz,CD₃OD)8.27–8.18(m,2H),7.90(d,1H),7.71(d,1H),7.37(ddd,2H),7.27(d,1H),7.15–7.09(m,2H),7.01(d,1H),6.97(ddd,2H),6.55(d,1H),5.44(dd,1H),5.17–5.05(m,1H),4.81–4.75(m,2H),4.51(s,2H),3.50–3.32(m,2H),3.26(dd,2H),3.22–3.00(m,4H),2.44–2.29(m,2H),2.13–2.03(m,2H),2.04–1.84(m,4H),1.78(dt,6H).

LCMS m/z＝783.3[M+1]⁺

Example 11:

[6- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -6-azaspiro [3.4] oct-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 11)

[6-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-6-azaspiro[3.4]octan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: 2- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-6-azaspiro [3.4] octane-6-carboxylic acid tert-butyl ester (11b)

Tert-butyl 2-[2-hydroxy-2,2-bis(2-thienyl)acetyl]oxy-6-azaspiro[3.4]octane-6-carboxylate

Tert-butyl 2-hydroxy-6-azaspiro [3.4] octane-6-carboxylate (1.0g, 4.40mmol) was dissolved in toluene (60mL), sodium hydride (0.11g, 4.40mmol) was added thereto, the mixture was stirred for 30 minutes, methyl 2-hydroxy-2, 2-bis (2-thienyl) acetate (1.75g, 6.89mmol) was added thereto, and the mixture was heated to 115 ℃ to react for 10 hours. The reaction was cooled to room temperature, quenched with 10mL of water, extracted with ethyl acetate (50mL × 2), washed with a saturated aqueous sodium chloride solution (20mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v) ═ 1:100-1:20) to give 2- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-6-azaspiro [3.4] octane-6-carboxylic acid tert-butyl ester (11b) (0.65g, yield: 33%).

¹H NMR(400MHz,CDCl₃)7.29(dd,2H),7.17(dd,2H),6.98(dd,2H),5.21–5.10(m,1H),4.64(s,1H),3.41–3.21(m,4H),2.52–2.38(m,2H),2.20–2.05(m,2H),1.83(dt,2H),1.45(s,9H).

The second step is that: 6-Azaspiro [3.4] oct-2-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (11c)

6-Azaspiro[3.4]octan-2-yl-2-hydroxy-2,2-bis(2-thienyl)acetate

Tert-butyl 2- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-6-azaspiro [3.4] octane-6-carboxylate (11b) (0.65g, 1.4mmol) was dissolved in 1, 4-dioxane (10mL), and hydrogen chloride gas was introduced and reacted at room temperature for 2 hours. Water (20mL) was added, the pH was adjusted to 8-9 with solid potassium carbonate at 0 ℃, extracted with dichloromethane (30 mL. times.2), washed with a saturated aqueous sodium chloride solution (20 mL. times.1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 6-azaspiro [3.4] oct-2-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (11c) (0.45g, yield: 90%).

LCMS m/z＝350.3[M+1]⁺

The third step: [6- [3- (5-Formylbenzotriazol-1-yl) propyl ] -6-azaspiro [3.4] oct-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (11d)

[6-[3-(5-Formylbenzotriazol-1-yl)propyl]-6-azaspiro[3.4]octan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

6-Azaspiro [3.4] oct-2-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (11c) (0.22g, 0.63mmol) was dissolved in 10mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) propyl methanesulfonate (synthetic method see WO2017114377) (0.20g, 0.69mmol) and diisopropylethylamine (0.15g, 1.3mmol) were added, and after the addition was completed, the mixture was heated to 90 ℃ for reaction for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [6- [3- (5-formylbenzotriazol-1-yl) propyl ] -6-azaspiro [3.4] oct-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (11d) (0.11g, yield: 33%).

LCMS m/z＝537.3[M+1]⁺

The fourth step: [6- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -6-azaspiro [3.4] oct-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 11)

[6-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-6-azaspiro[3.4]octan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

Reacting [6- [3- (5-formylbenzotriazol-1-yl) propyl ] group]-6-azaspiro [3.4]Oct-2-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate (11d) (0.11g,0.205mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropyl alcohol, and (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride salt (0.080g,0.307mmol) was added thereto dropwise, 1 drop of acetic acid was added thereto dropwise, and 5g of dry ethanol was added thereto

Molecular sieves, stirred at room temperature for 6 hours, then sodium triacetoxyborohydride (0.130g,0.615 m) was addedmol), reaction at room temperature overnight, pouring the reaction solution into 50mL saturated aqueous sodium bicarbonate solution, extracting the aqueous phase with dichloromethane/methanol (v/v 10:1) (50mL × 3), combining the organic phases, washing the organic phase with 50mL water, drying over anhydrous sodium sulfate, concentrating under reduced pressure, passing the residue through Pre-HPLC (instrument and preparative column: liquid phase prepared using Glison GX-281, model number Sunfire C18, 5 μm, internal diameter 30mm x 150mm in length.) preparation method, dissolving the crude product with methanol and dimethyl sulfoxide, and filtering with 0.45 μm filter to prepare sample solution, mobile phase system acetonitrile/water (containing 0.1% TFA.) gradient elution method, acetonitrile is eluted with 5% gradient for 60% (elution time 15min), and freeze-drying to obtain [6- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] ethyl]Amino group]Methyl radical]Benzotriazol-1-yl]Propyl radical]-6-azaspiro [3.4]Oct-2-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate ditrifluoroacetate (Compound 11) (0.055g, yield: 28%).

¹H NMR(400MHz,CD₃OD)8.31–8.16(m,2H),7.89(dd,1H),7.71(d,1H),7.36(dd,2H),7.27(d,1H),7.13(dd,2H),7.06–6.91(m,3H),6.55(d,1H),5.45(d,1H),5.20–5.04(m,1H),4.85(d,2H),4.52(d,2H),3.66(s,2H),3.35(s,2H),3.27(d,2H),3.15(s,2H),2.69–2.37(m,4H),2.19(dt,4H).

¹⁹F NMR(376MHz,CD₃OD)-74.77.

LCMS m/z＝741.2[M+1]⁺

Example 12:

[6- [2- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] ethyl ] -6-azaspiro [3.4] oct-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 12)

[6-[2-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]ethyl]-6-azaspiro[3.4]octan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: [6- [3- (5-Formylbenzotriazol-1-yl) ethyl ] -6-azaspiro [3.4] oct-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (12a)

[6-[3-(5-formylbenzotriazol-1-yl)ethyl]-6-azaspiro[3.4]octan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

6-Azaspiro [3.4] oct-2-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (11c) (0.40g, 1.10mmol) was dissolved in 10mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) ethyl methanesulfonate (intermediate 2) (0.36g, 1.3mmol) and diisopropylethylamine (0.30g, 2.3mmol) were added, and after the addition, the mixture was heated to 90 ℃ for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [6- [3- (5-formylbenzotriazol-1-yl) ethyl ] -6-azaspiro [3.4] oct-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (12a) (0.20g, yield: 33%).

LCMS m/z＝523.3[M+1]⁺

The second step is that: [6- [2- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] ethyl ] -6-azaspiro [3.4] oct-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 12)

[6-[2-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]ethyl]-6-azaspiro[3.4]octan-2-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

Reacting [6- [3- (5-formylbenzotriazol-1-yl) ethyl group]-6-azaspiro [3.4]Oct-2-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate (12a) (0.20g,0.383mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropyl alcohol, (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride (0.149g,0.574mmol) was added thereto, and 1 drop of acetic acid was added theretoAdding 5g of dry

Molecular sieves, stirring for 6 hours at room temperature, adding sodium triacetoxyborohydride (0.243g,1.15mmol), reacting overnight at room temperature, pouring the reaction mixture into 50mL of saturated aqueous sodium bicarbonate solution, extracting the aqueous phase with dichloromethane/methanol (v/v ═ 10:1) (50mL × 3), combining the organic phases, washing the organic phase with 50mL of water, drying over anhydrous sodium sulfate, concentrating under reduced pressure, passing the residue through Pre-HPLC (instrument and preparative column: A liquid phase is prepared using Glison GX-281, model of preparative column is Sunfire C18, 5 μm, internal diameter ═ 30mm ═ 150 mm.) the crude product is dissolved in methanol and dimethyl sulfoxide and filtered through 0.45 μm filter membrane to prepare a sample solution]Amino group]Methyl radical]Benzotriazol-1-yl]Ethyl radical]-6-azaspiro [3.4]Oct-2-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 12) (0.050g, yield: 14%).

¹H NMR(400MHz,CD₃OD)8.29–8.20(m,2H),7.92(d,1H),7.77–7.71(m,1H),7.37(dd,2H),7.27(d,1H),7.13(dd,2H),7.04–6.95(m,3H),6.57(d,1H),5.45(dd,1H),5.19–5.07(m,3H),4.55–4.46(m,2H),3.92(dd,2H),3.75–3.36(m,4H),3.27(dd,2H),2.57(s,2H),2.34–2.16(m,4H).

¹⁹F NMR(376MHz,CD₃OD)-74.84(s,1H).

LCMS m/z＝364.2[M/2+1]⁺

Example 13:

[6- [4- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] butyl ] -6-azaspiro [3.4] oct-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 13)

[6-[4-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]butyl]-6-azaspiro[3.4]octan-2-yl]2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: [6- [4- (5-Formylbenzotriazol-1-yl) butyl ] -6-azaspiro [3.4] oct-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (13a)

[6-[4-(5-formylbenzotriazol-1-yl)butyl]-6-azaspiro[3.4]octan-2-yl]2-hydroxy-2,2-bis(2-thienyl)acetate

6-Azaspiro [3.4] oct-2-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (11c) (0.40g, 1.10mmol) was dissolved in 10mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) butyl methanesulfonate (intermediate 1) (0.37g, 1.30mmol) and diisopropylethylamine (0.30g, 1.3mmol) were added, and after the addition, the mixture was heated to 90 ℃ for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [6- [4- (5-formylbenzotriazol-1-yl) butyl ] -6-azaspiro [3.4] oct-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (13a) (0.36g, yield: 57%).

LCMS m/z＝551.3[M+1]⁺

The second step is that: [6- [4- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] butyl ] -6-azaspiro [3.4] oct-2-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 13)

[6-[4-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]butyl]-6-azaspiro[3.4]octan-2-yl]2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

Reacting [6- [4- (5-formylbenzotriazol-1-yl) butyl ] with]-6-azaspiro [3.4]Xin-2-radical]-2-hydroxy-2, 2-bis (2-thienyl) acetate (13a) (0.36g,0.654mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropyl alcohol, (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride (0.254g,0.981mmol) was added dropwise, acetic acid (0.058g,0.981mmol) was added dropwise, and 5g of dried

Molecular sieves, stirred at room temperature for 6 hours, added sodium triacetoxyborohydride (0.42g,1.96mmol) and reacted at room temperature overnight, the reaction mixture was poured into 50mL of saturated aqueous sodium bicarbonate solution, the aqueous phase was extracted with dichloromethane/methanol (v/v ═ 10:1) (50mL × 3), the organic phases were combined, the organic phase was washed with 50mL of water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, the residue was subjected to Pre-HPLC (apparatus and preparative column: Glison GX-281 was used to prepare a liquid phase, model Sunfire C18, 5 μm, internal diameter ═ 30mm ═ 150 mm.) the crude product was dissolved in methanol and dimethyl sulfoxide and filtered through a 0.45 μm filter to prepare a sample solution.A mobile phase system, acetonitrile/water (containing 0.1% TFA.) a gradient elution method, acetonitrile was eluted with a gradient of 60% (elution time 15min) to give [6- [4- [5- [ [ (R) -2-hydroxy-2- (8-hydroxy-2-oxoethyl-2-quinolines-5-oxoethyl-5-based on 5H-5-ethyl) and lyophilized]Amino group]Methyl radical]Benzotriazol-1-yl]Butyl radical]-6-azaspiro [3.4]Oct-2-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate ditrifluoroacetate salt (Compound 13) (0.060g, yield: 9.3%).

¹H NMR(400MHz,CD₃OD)8.27–8.15(m,2H),7.90(d,1H),7.70(d,1H),7.41–7.34(m,2H),7.27(d,1H),7.13(td,2H),7.03-6.95(m,3H),6.54(d,1H),5.43(dd,1H),5.16–5.05(m,1H),4.83(s,2H),4.51(s,2H),3.76–3.50(m,2H),3.25(dd,4H),3.16–3.02(m,2H),2.65–2.47(m,2H),2.24(dd,2H),2.19–2.00(m,4H),1.84–1.68(m,2H).

¹⁹F NMR(376MHz,CD₃OD)-74.92.

LCMS m/z＝755.3[M+1]⁺

Example 14:

[2- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -2-azaspiro [3.3] hept-6-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetic acid (compound 14)

[2-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-2-azaspiro[3.3]heptan-6-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: 6- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-2-azaspiro [3.3] heptane-2-carboxylic acid tert-butyl ester (14b)

Tert-butyl 6-[2-hydroxy-2,2-bis(2-thienyl)acetyl]oxy-2-azaspiro[3.3]heptane-2-carboxylate

Tert-butyl 6-hydroxy-2-azaspiro [3.3] heptane-2-carboxylate (14a) (1.5g, 7.03mmol) was dissolved in toluene (100mL), sodium hydride (0.28g, 7.03mmol) was added, stirring was carried out for 30 minutes, methyl 2-hydroxy-2, 2-bis (2-thienyl) acetate (2.68g, 10.5mmol) was added, and the temperature was raised to 115 ℃ for reaction for 10 hours. The reaction was cooled to room temperature, quenched with 10mL of water, extracted with ethyl acetate (50mL × 2), washed with a saturated aqueous sodium chloride solution (20mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v) ═ 1:100-1:20) to give 6- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-2-azaspiro [3.3] heptane-2-carboxylic acid tert-butyl ester (14b) (1.6g, yield: 52%).

The second step is that: 2-azaspiro [3.3] hept-6-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (14c)

2-Azaspiro[3.3]heptan-6-yl-2-hydroxy-2,2-bis(2-thienyl)acetate

Tert-butyl 6- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-2-azaspiro [3.3] heptane-2-carboxylate (14b) (1.4g, 3.2mmol) was dissolved in 1, 4-dioxane (15mL), and hydrogen chloride gas was introduced and reacted at room temperature for 2 hours. Water (20mL) was added, the pH was adjusted to 8-9 with solid potassium carbonate at 0 ℃, extracted with dichloromethane (30 mL. times.2), washed with a saturated aqueous sodium chloride solution (20 mL. times.1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 2-azaspiro [3.3] hept-6-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (14c) (0.80g, yield: 74%).

LCMS m/z＝336.3[M+1]⁺

The third step: [2- [3- (5-Formylbenzotriazol-1-yl) propyl ] -2-azaspiro [3.3] hept-6-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (14d)

[2-[3-(5-formylbenzotriazol-1-yl)propyl]-2-azaspiro[3.3]heptan-6-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

2-Azaspiro [3.3] hept-6-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (14c) (0.40g, 1.20mmol) was dissolved in 10mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) propyl methanesulfonate (synthetic method see WO2017114377) (0.44g, 1.6mmol) and diisopropylethylamine (0.31g, 2.4mmol) were added, and after the addition was completed, the mixture was heated to 90 ℃ for reaction for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [2- [3- (5-formylbenzotriazol-1-yl) propyl ] -2-azaspiro [3.3] hept-6-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (14d) (0.15g, yield: 24%).

LCMS m/z＝523.3[M+1]⁺

The fourth step: [2- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -2-azaspiro [3.3] hept-6-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetic acid (compound 14)

[2-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-2-azaspiro[3.3]heptan-6-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

By reacting [2- [3- (5-formylbenzotriazol-1-yl) propyl ] group]-2-azaspiro [3.3]Hept-6-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate (14d) (0.15g,0.287mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropyl alcohol, (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride (0.112g,0.431mmol) was added dropwise, acetic acid (0.026g,0.431mmol) was added dropwise, 5g of dried

Molecular sieves, stirring for 6 hours at room temperature, adding sodium triacetoxyborohydride (0.182g,0.861mmol), reacting overnight at room temperature, pouring the reaction mixture into 50mL of saturated aqueous sodium bicarbonate solution, extracting the aqueous phase with dichloromethane/methanol (v/v ═ 10:1) (50mL × 3), combining the organic phases, washing the organic phase with 50mL of water, drying over anhydrous sodium sulfate, concentrating under reduced pressure, passing the residue through a Pre-HPLC (apparatus and preparative column: A liquid phase is prepared using Glison GX-281, model of Sunfire C18, 5 μm, internal diameter ═ 30mm length ═ 150 mm.) preparation method, the crude product is dissolved with methanol and dimethyl sulfoxide, and filtered through a 0.45 μm filter membrane to prepare a sample solution]Amino group]Methyl radical]Benzotriazol-1-yl]Propyl radical]-2-azaspiro [3.3]Hept-6-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetic acid (compound 14) (0.020g, yield: 7.3%).

¹H NMR(400MHz,CD₃OD)8.20(t,2H),7.88(d,1H),7.69(d,1H),7.37(dd,2H),7.27(d,1H),7.12(dd,2H),7.04–6.94(m,3H),6.54(d,1H),5.41(d,1H),5.06–4.99(m,1H),4.83(s,2H),4.50(s,2H),4.35–4.21(m,1H),4.12(s,3H),3.26(d,4H),2.77(s,2H),2.32(d,4H).

¹⁹F NMR(376MHz,CD₃OD)-74.82.

LCMS m/z＝727.2[M+1]⁺

Example 15:

[2- [4- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] butyl ] -2-azaspiro [3.3] hept-6-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 15)

[2-[4-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]butyl]-2-azaspiro[3.3]heptan-6-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: [2- [4- (5-Formylbenzotriazol-1-yl) butyl ] -2-azaspiro [3.3] hept-6-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (15a)

[2-[4-(5-Formylbenzotriazol-1-yl)butyl]-2-azaspiro[3.3]heptan-6-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

2-Azaspiro [3.3] hept-6-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (14c) (0.40g, 1.20mmol) was dissolved in 20mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) butyl methanesulfonate (intermediate 1) (0.46g, 1.6mmol) and diisopropylethylamine (0.31g, 2.4mmol) were added, and after the addition was complete, the reaction was heated to 90 ℃ for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [2- [4- (5-formylbenzotriazol-1-yl) butyl ] -2-azaspiro [3.3] hept-6-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (15a) (0.12g, yield: 19%).

LCMS m/z＝537.3[M+1]⁺

The second step is that: [2- [4- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] butyl ] -2-azaspiro [3.3] hept-6-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 15)

[2-[4-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]butyl]-2-azaspiro[3.3]heptan-6-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

Reacting [2- [4- (5-formylbenzotriazol-1-yl) butyl ] with]-2-azaspiro [3.3]Hept-6-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate (15a) (0.12g,0.224mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropyl alcohol, (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride salt (0.087g,0.335mmol) was added dropwise, acetic acid (0.020g,0.335mmol) was added dropwise, and 5g of dry

Molecular sieves, stirred at room temperature for 6 hours, added sodium triacetoxyborohydride (0.142g,0.671mmol) and allowed to react at room temperature overnight, the reaction mixture was poured into 50mL of saturated aqueous sodium bicarbonate solution, the aqueous phase was extracted with dichloromethane/methanol (v/v ═ 10:1) (50mL × 3), the organic phases were combined, the organic phase was washed with 50mL of water, dried over anhydrous sodium sulfate and concentrated under reduced pressure, the residue was subjected to Pre-HPLC (apparatus and preparative column: Glison GX-281 was used to prepare a liquid phase, model Sunfire C18, 5 μm, internal diameter ═ 30mm length ═ 150 mm.) preparation method: the crude product was dissolved in methanol and dimethyl sulfoxide and filtered through a 0.45 μm filter to prepare a sample solution.mobile phase system: acetonitrile/water (containing 0.1% TFA.) gradient elution method: acetonitrile elution 60% (elution time 15min) from 5% gradient elution) and lyophilized to give [2- [4- [ [5- [2- [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-2-ethyl-5-quinolines-5-oxo-ethyl-5-based H-5]Amino group]Methyl radical]Benzotriazol-1-yl]Butyl radical]-2-azaspiro [3.3]Hept-6-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate ditrifluoroacetate salt (Compound 15) (0.010g, yield: 4.6%).

¹H NMR(400MHz,CD₃OD)8.18(d,2H),7.89(d,1H),7.69(dd,1H),7.37(dd,2H),7.27(d,1H),7.12(dd,2H),7.01(d,1H),6.97(dd,2H),6.53(d,1H),5.41(dd,1H),5.02(dt,1H),4.78(s,2H),4.50(s,2H),4.24(s,1H),4.06(s,2H),3.98(s,1H),3.28–3.23(m,2H),3.23–3.17(m,2H),2.75(d,2H),2.35(s,2H),2.12–2.04(m,2H),1.58(dt,2H).

¹⁹F NMR(376MHz,CD₃OD)-74.82.

LCMS m/z＝741.2[M+1]⁺

Example 16:

[7- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -3-oxa-7-azabicyclo [3.3.1] non-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 16)

[7-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-3-oxa-7-azabicyclo[3.3.1]nonan-9-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: 9- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylic acid tert-butyl ester (16b-a)

Tert-butyl 9-[2-hydroxy-2,2-bis(2-thienyl)acetyl]oxy-3-oxa-7-azabicyclo[3.3.1]nonane-7-carboxylate(isomer 1)

9- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylic acid tert-butyl ester (16b-b)

Tert-butyl 9-[2-hydroxy-2,2-bis(2-thienyl)acetyl]oxy-3-oxa-7-azabicyclo[3.3.1]nonane-7-carboxylate(isomer 2)

Tert-butyl 9-hydroxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylate (16a) (1.43g, 10.0mmol) was dissolved in toluene (120mL), sodium hydride (4.0g, 10.0mmol) was added, stirring was carried out for 30 minutes, methyl 2-hydroxy-2, 2-bis (2-thienyl) acetate (2.80g, 11.0mmol) was added, and the mixture was heated to 115 ℃ for reaction for 10 hours. The reaction was cooled to room temperature, quenched with 10mL of water, extracted with ethyl acetate (80mL × 2), washed with a saturated aqueous sodium chloride solution (20mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether (v/v) ═ 1:100-1:20) to give 9- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylic acid tert-butyl ester (16b-a) (1.10g, yield: 23%) and 9- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylic acid tert-butyl ester (16b-b) (0.8g, yield: 17%).

16 b-a: (developing solvent: petroleum ether/ethyl acetate 5/1, Rf value: 0.3)

¹H NMR(400MHz,CDCl₃)7.30(d,2H),7.21(s,2H),7.03–6.95(m,2H),5.11(t,1H),4.69(s,1H),4.55(d,1H),4.35(d,1H),3.81(d,1H),3.77–3.64(m,3H),3.19(d,1H),3.07(d,1H),1.77(d,2H),1.45(s,9H).

16 b-b: (developing solvent: petroleum ether/ethyl acetate 5/1, Rf value: 0.2)

¹H NMR(400MHz,CDCl₃)7.30(dd,2H),7.20(d,2H),6.99(dd,2H),5.18(t,1H),4.69(s,1H),4.19(d,1H),4.12(d,1H),4.03(t,2H),3.73(t,2H),3.05(d,1H),2.97(d,1H),1.85(d,2H),1.45(s,9H).

The second step is that: 3-oxa-7-azabicyclo [3.3.1] non-9-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (16c)

3-Oxa-7-azabicyclo[3.3.1]nonan-9-yl-2-hydroxy-2,2-bis(2-thienyl)acetate

Tert-butyl 9- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylate (16b-a) (1.10g, 2.4mmol) was dissolved in 1, 4-dioxane (10mL), and reacted at room temperature for 2 hours with hydrogen chloride gas. Water (20mL) was added, the pH was adjusted to 8-9 with solid potassium carbonate at 0 ℃, extracted with dichloromethane (30 mL. times.2), washed with a saturated aqueous sodium chloride solution (20 mL. times.1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 3-oxa-7-azabicyclo [3.3.1] non-9-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (16c) (0.35g, yield: 41%).

LCMS m/z＝366.3[M+1]⁺

The third step: [7- [3- (5-Formylbenzotriazol-1-yl) propyl ] -3-oxa-7-azabicyclo [3.3.1] non-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (16d)

[7-[3-(5-formylbenzotriazol-1-yl)propyl]-3-oxa-7-azabicyclo[3.3.1]nonan-9-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

3-oxa-7-azabicyclo [3.3.1] non-9-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (16c) (0.30g, 0.82mmol) was dissolved in 10mL acetonitrile, 4- (5-formylbenzotriazol-1-yl) propyl methanesulfonate (synthetic method see WO2017114377) (0.26g, 0.90mmol) and diisopropylethylamine (0.32g, 2.5mmol) were added, and after the addition, the mixture was heated to 90 ℃ for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [7- [3- (5-formylbenzotriazol-1-yl) propyl ] -3-oxa-7-azabicyclo [3.3.1] non-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (16d) (0.20g, yield: 43%).

LCMS m/z＝553.3[M+1]⁺

The fourth step: [7- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -3-oxa-7-azabicyclo [3.3.1] non-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 16)

[7-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-3-oxa-7-azabicyclo[3.3.1]nonan-9-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

Reacting [7- [3- (5-formylbenzotriazol-1-yl) propyl ] group]-3-oxa-7-azabicyclo [3.3.1]Non-9-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate (16d) (0.20g,0.362mmol) was dissolved in a mixed solvent of 8mL of dimethyl sulfoxide and 2mL of isopropanol, (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride (0.141g,0.543mmol) was added dropwise, acetic acid (0.032g,0.543mmol) was added dropwise, and 5g of dry ethanol was added

Molecular sieves, stirred at room temperature for 6 hours, added sodium triacetoxyborohydride (0.230g,1.09mmol) and allowed to react at room temperature overnight, the reaction mixture was poured into 50mL of saturated aqueous sodium bicarbonate solution, the aqueous phase was extracted with dichloromethane/methanol (v/v ═ 10:1) (50mL × 3), the organic phases were combined, the organic phase was washed with 50mL of water, dried over anhydrous sodium sulfate and concentrated under reduced pressure, the residue was subjected to Pre-HPLC (apparatus and preparative column: A liquid phase was prepared using Glison GX-281, model number Sunfire C18, 5 μm, internal diameter ═ 30mm length ═ 150 mm.) the crude product was dissolved in methanol and dimethyl sulfoxide and filtered through a 0.45 μm filter to prepare a sample solution]Amino group]Methyl radical]Benzotriazol-1-yl]Propyl radical]-3-oxa-7-azabicyclo [3.3.1]Non-9-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate ditrifluoroacetate (Compound 16) (0.10g, yield: 28%).

¹H NMR(400MHz,CD₃OD)8.25–8.11(m,2H),7.88(d,1H),7.70(d,1H),7.41(d,2H),7.27(d,1H),7.19(d,2H),7.01(dd,3H),6.53(d,1H),5.44(dd,1H),5.27(s,1H),4.85(d,2H),4.55–4.45(m,2H),3.89(t,4H),3.71(d,2H),3.47(d,2H),3.25(dd,4H),2.56(dd,2H),2.18(s,2H).

¹⁹F NMR(376MHz,CD₃OD)-74.73.

LCMS m/z＝757.3[M+1]⁺

Example 17:

[7- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -3-oxa-7-azabicyclo [3.3.1] non-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 17)

[7-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-3-oxa-7-azabicyclo[3.3.1]nonan-9-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

The first step is as follows: 3-oxa-7-azabicyclo [3.3.1] non-9-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (17a)

3-oxa-7-azabicyclo[3.3.1]nonan-9-yl-2-hydroxy-2,2-bis(2-thienyl)acetate

Tert-butyl 9- [ 2-hydroxy-2, 2-bis (2-thienyl) acetyl ] oxy-3-oxa-7-azabicyclo [3.3.1] nonane-7-carboxylate (16b-b) (0.90g, 1.9mmol) was dissolved in 1, 4-dioxane (10mL), and reacted at room temperature for 2 hours with hydrogen chloride gas. Water (20mL) was added, the pH was adjusted to 8-9 with solid potassium carbonate at 0 ℃, extracted with dichloromethane (30 mL. times.2), washed with a saturated aqueous sodium chloride solution (20 mL. times.1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 3-oxa-7-azabicyclo [3.3.1] non-9-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (17a) (0.71g, yield: > 99%).

LCMS m/z＝366.3[M+1]⁺

The third step: [7- [3- (5-Formylbenzotriazol-1-yl) propyl ] -3-oxa-7-azabicyclo [3.3.1] non-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (17b)

[7-[3-(5-formylbenzotriazol-1-yl)propyl]-3-oxa-7-azabicyclo[3.3.1]nonan-9-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate

3-oxa-7-azabicyclo [3.3.1] non-9-yl-2-hydroxy-2, 2-bis (2-thienyl) acetate (17a) (0.40g, 1.0mmol) was dissolved in 10ml of acetonitrile, 4- (5-formylbenzotriazol-1-yl) propyl methanesulfonate (synthetic method see WO2017114377) (0.31g, 1.10mmol) and diisopropylethylamine (0.26g, 2.0mmol) were added, and after the addition, the mixture was heated to 90 ℃ for 7 hours. After completion of the reaction, the reaction mixture was directly concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 1:0 to 20:1) to give [7- [3- (5-formylbenzotriazol-1-yl) propyl ] -3-oxa-7-azabicyclo [3.3.1] non-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate (17b) (0.40g, yield: 73%).

LCMS m/z＝553.3[M+1]⁺

The fourth step: [7- [3- [5- [ [ [ (2R) -2-hydroxy-2- (8-hydroxy-2-oxo-1H-quinolin-5-yl) ethyl ] amino ] methyl ] benzotriazol-1-yl ] propyl ] -3-oxa-7-azabicyclo [3.3.1] non-9-yl ] -2-hydroxy-2, 2-bis (2-thienyl) acetate bistrifluoroacetate salt (Compound 17)

[7-[3-[5-[[[(2R)-2-hydroxy-2-(8-hydroxy-2-oxo-1H-quinolin-5-yl)ethyl]amino]methyl]benzotriazol-1-yl]propyl]-3-oxa-7-azabicyclo[3.3.1]nonan-9-yl]-2-hydroxy-2,2-bis(2-thienyl)acetate ditrifluoroacetate

Reacting [7- [3- (5-formylbenzotriazol-1-yl) propyl ] group]-3-oxa-7-azabicyclo [3.3.1]Non-9-yl](17b) (0.20g,0.362mmol) of (2-hydroxy-2, 2-bis (2-thienyl) acetate was dissolved in a mixed solvent of 8ml of dimethyl sulfoxide and 2ml of isopropanol, (R) -5- (2-amino-1-hydroxyethyl) -8-hydroxyquinolin-2- (1H) -one hydrofluoride (0.141g,0.543mmol) was added dropwise, acetic acid (0.032g,0.543mmol) was added dropwise, and 5g of dry ethanol was added

Molecular sieve, stirring at room temperature for 6 hr, adding IIISodium acetoxyborohydride (0.230g,1.09mmol), reaction overnight at room temperature, pouring the reaction solution into 50mL of saturated aqueous sodium bicarbonate solution, extracting the aqueous phase with dichloromethane/methanol (v/v ═ 10:1) (50mL × 3), combining the organic phases, washing the organic phases with 50mL of water, drying over anhydrous sodium sulfate, concentrating, passing the crude product through Pre-HPLC (instrument and preparative column: using Glison GX-281 to prepare a liquid phase, model Sunfire C18, 5 μm, internal diameter ═ 30mm long ═ 150 mm.) the crude product was dissolved with methanol and dimethylsulfoxide and filtered through a 0.45 μm filter to prepare a sample solution]Amino group]Methyl radical]Benzotriazol-1-yl]Propyl radical]-3-oxa-7-azabicyclo [3.3.1]Non-9-yl]-2-hydroxy-2, 2-bis (2-thienyl) acetate ditrifluoroacetate (Compound 17) (0.030g, yield: 8.4%).

¹H NMR(400MHz,CD₃OD)8.23(s,1H),8.18(d,1H),7.91(d,1H),7.73(d,1H),7.33–7.28(m,2H),7.25(d,1H),7.15(dd,2H),7.01(d,1H),6.93–6.87(m,2H),6.54(d,1H),5.41(d,1H),5.27(d,1H),4.85(t,2H),4.52(d,2H),4.19(d,2H),3.85(d,2H),3.60(d,2H),3.29–3.20(m,2H),3.04–2.92(m,2H),2.79(d,2H),2.43(s,2H),2.30(s,2H).

¹⁹F NMR(376MHz,CD₃OD)-74.32.

LCMS m/z＝757.3[M+1]⁺

Biological test example

Test example 1: inhibitory Activity on human muscarinic M3 receptor

CHO cells (PerkinElmer, ES-212-AF) stably expressing human muscarinic receptor 3(hM3) and apo-Aequorin were cultured in Ham' S F12 medium (Invitrogen 12500-062) containing 10% Fetal Bovine Serum (FBS) (Gibico 10099-141), 400. mu.g/mL G418(sigma G5013) and 250. mu.g/mL Zeocin (Invitrogen ant-zn-5p) at 37 ℃ with 5% CO₂Culturing under the condition to achieve 90-100% fusion. The cells were separated by washing with PBS/5mM EDTA, harvested by centrifugation, and resuspended in phenol-free Ham's F12 medium (Invitrogen 11039-Cells were counted and cell concentration adjusted to 1 × 10⁶cells/mL, 15mL of cell suspension was added to a 50mL centrifuge tube, Coelenterazine-h (promega S2011) was added to a final concentration of 5 μ M, wrapped in tinfoil and protected from light, incubated for 4 hours at 20 ℃ on a rotary shaker, and the cells were diluted with 0.1% BSA/phenol-free Red Ham' S F12 medium to a final concentration of 5.0 × 10⁵cells/mL, cells were placed on a rotary shaker and spun at low speed and incubated at room temperature for at least 1 hour. The compounds of the examples were formulated in DMSO as 10mM stock solution, 0.1% BSA/phenol red free Ham's F12 medium diluted in gradient (log (M): 7, -8, -9, -10, -11), and added to 96 well plates at 50. mu.L per well. A further 50. mu.L of cell suspension (25000 cells/well) was added to each well and incubated for 15 minutes at room temperature. The 96 well plates were placed in a microplate reader (Perkin Elmer, Envision), 50. mu.L of acetylcholine chloride (Sigma A6625) solution at 112.92nM (hM3) was added per well with a microplate reader applicator, luminescence was recorded for 20 seconds, and IC was calculated and analyzed using origin7.5₅₀. Inhibitory Activity of the Compounds of the invention at human muscarinic receptors the IC determined by the above assay₅₀The values are given in table 1 below.

Table 1 results of the inhibitory activity of the test compounds on human muscarinic M3 receptor

And (4) conclusion: the compound has obvious inhibitory activity on human muscarinic M3 receptor.

Test example 2: agonistic activity at the human adrenergic beta 2 receptor

Agonist activity of the compounds of the examples on human adrenergic receptors was determined by LANCE Ultra cAMP Assay.

CHO cells (PerkinElmer, ES-034-CF) stably expressing human adrenergic receptor (h β 2) were cultured in MEM-alpha medium (Invitrogen 12561-056) containing 10% Fetal Bovine Serum (FBS) (Gibico 10099-141) and 250. mu.g/mL Zeocin (Invivogen ant-zn-5p) at 37 ℃ with 5% CO₂Culturing under conditions to achieve 90-100% fusion and detecting the agonism of cAMP in the examples using the LANCE Ultra cAMP Assay kit (PerkinElmer TRF0263) cells were separated with PBS/5mM EDTA, harvested by centrifugation, resuspended in Stimulation Buffer (1 XHBSS, 5mM HEPES,0.5mM IBMX, 0.1% BSA, pH7.4), and adjusted to 6 × 10⁵cells/ml. The compounds of the examples were made up in DMSO as 10mM stock, diluted in a Ststimulation Buffer gradient and added to 384 well plates at 5. mu.l per well. mu.L of cell suspension (3000 cells/well) was added to each well, and after incubation at room temperature for 30 minutes, 5. mu.l of 4 × Eu-cAMP concentrator working solution was added to each well, followed by 5. mu.l of 4 × Ulight-anti-cAMP working solution to each well, and incubation at room temperature for 1 hour. The TR-FRET was detected in 384 well plates using a microplate reader (Perkin Elmer, Envision) and EC was calculated and analyzed using origin7.5₅₀. The agonistic activity of the compound of the present invention on human adrenergic receptors was determined by the above experiment, and the EC was measured₅₀The values are shown in Table 2:

TABLE 2 results of agonist activity of test compounds at the human adrenergic beta 2 receptor

And (4) conclusion: the compounds of the present invention have significant agonist activity at the β 2 adrenergic receptor.

Claims (13)

1. A compound shown in a general formula (I) or a stereoisomer, a hydrate, a metabolite, a solvate, a pharmaceutically acceptable salt, a eutectic crystal or a prodrug thereof,

wherein:

Q₁selected from a bond or-N (R)^q)-；

R^qIs selected from H or C_1-6An alkyl group;

cy is selected from unsubstituted or substituted

When substituted, is optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl or C_1-4Substituted by alkoxy radicals, Q₁And Q₂Direct linking, provided that Cy is selected from the group consisting of

Q₂Is selected from-CH-or-N-, with the proviso that Q₁And Q₂Does not contain N at the same time;

n1, n2, n3 and n4 are each independently selected from 0, 1,2,3 or 4, provided that n1 and n2 are not simultaneously 0 and n3 and n4 are not simultaneously 0;

x, Y, Z are each independently selected from nitrogen, -NR^x1-、-CR^x1R^x2-、-CR^x1R^x2CR^x3R^x4-、-CR^x1＝CR^x2-, -S-, -O-and-C (O) -;

R^x1、R^x2、R^x3or R^x4Each independently selected from H or C_1-4An alkyl group;

in the general formula (I)

Selected from single bonds or double bonds;

R¹is selected from

R^1a、R^1bEach independently selected from H, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-10Carbocyclyl, 3-to 8-membered heterocyclyl, C_3-10carbocyclyl-C_1-4Alkylene or 3-to 8-membered heterocyclyl-C_1-4Alkylene, said alkenyl, alkynyl, alkylene, carbocyclyl or heterocyclyl optionally further substituted with 0 to 4 substituents selected from F, Cl、Br、I、CH₂F、CHF₂、CF₃、OH、OCH₂F、OCHF₂、OCF₃、NH₂Carboxy, cyano, nitro, C_1-4Alkyl radical, C_1-4Alkoxy, -O (═ O) C_1-4Alkyl, - (═ O) C_1-4Alkyl, -OC_3-6Cycloalkyl or C_1-4Alkylthio containing 1 to 3 heteroatoms selected from N, O or S, with the proviso that R is^1a、R^1bCannot be simultaneously H;

R^1cselected from H, hydroxy, cyano, NH₂、C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylthio or-C (═ O) NH₂NH of said₂、-C(＝O)NH₂Or alkyl is optionally further substituted by 0 to 2 substituents selected from F, Cl, Br, I, hydroxy, cyano or C_1-4Alkyl is substituted by a substituent;

R^1eand R^1fEach independently selected from F, Cl, Br, I, CF₃、NH₂OH, carboxyl, cyano, C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio, -NHC_1-4Alkyl, -N (C)_1-4Alkyl radical)₂、-S(＝O)-C_1-4Alkyl, -S (═ O)₂-C_1-4Alkyl or-C (═ O) O-C_1-4An alkyl group;

R²or R⁴Is independently selected from C_1-6Alkylene radical, C_2-6Alkenylene or C_2-6Alkynylene, said alkylene, alkenylene or alkynylene being optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl radical, C_1-4Alkoxy, phenyl or phenyl-C_1-4Substituted by a substituent of alkylene;

R³each independently selected from F, Cl, Br, I, OH, NH₂Carboxy, cyano, nitro, C_1-4Alkyl radical, C_2-4Alkenyl radical, C_2-4Alkynyl, C_1-4Alkoxy, -OC_3-6Cycloalkyl radical, C_1-4Alkylthio, -S (═ O) -C_1-4Alkyl, -S (═ O)₂-C_1-4Alkyl, -C (═ O) -C_1-4Alkyl, -C (═ O) O-C_1-4Alkyl, -OC (═ O) -C_1-4Alkyl or-C (═ O) NH₂The alkyl, alkoxy, cycloalkyl, alkenyl, alkynyl, NH₂or-C (═ O) NH₂Optionally further substituted by 0 to 4 substituents selected from F, Cl, Br, I, CF₃、C_1-4Alkyl radical, C_1-4Alkoxy or-C (═ O) -C_1-4Alkyl is substituted by a substituent;

R⁵、R⁶each independently selected from H or C_1-4An alkyl group;

represents β -adrenoceptor binding group;

a is 0, 1,2,3,4 or 5;

b is 0, 1,2,3 or 4;

c is selected from 0, 1,2,3 or 4.

2. The compound according to claim 1, or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof,

b is selected from

Wherein Q is selected from-CH ═ CH-, -CH₂CH₂-、-O-、-S-、-CH₂O-、-OCH₂-、-C(CH₃)₂O-or-OC (CH)₃)₂-。

3. The compound according to claim 2, or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof,

cy is selected from unsubstituted or substituted

When substituted, is optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl or C_1-4Substituted by a substituent of alkoxy;

when Cy is an N-containing heterocycle, the N atom is attached to Q1.

4. The compound of claim 3, or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal, or prodrug thereof,

R^1a、R^1beach independently selected from H, C_2-4Alkenyl radical, C_2-4Alkynyl, C_3-7Carbocyclyl, 3-to 6-membered heterocyclyl, C_3-7carbocyclyl-C_1-4Alkylene or 3-to 6-membered heterocyclyl-C_1-4Alkylene, said alkenyl, alkynyl, alkylene, carbocyclyl or heterocyclyl being optionally further substituted by 0 to 4 substituents selected from F, Cl, Br, I, CF₃、OH、NH₂Carboxy, cyano, nitro, C_1-4Alkyl radical, C_1-4Alkoxy, -O (═ O) C_1-4Alkyl, - (═ O) C_1-4Alkyl, -OC_3-6Cycloalkyl or C_1-4Alkylthio containing 1 to 3 heteroatoms selected from N, O or S, with the proviso that R is^1a、R^1bNot H at the same time;

R^1cselected from H, hydroxy, cyano, NH₂、C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio or-C (═ O) NH₂NH of said₂、-C(＝O)NH₂Or alkyl is optionally further substituted by 0 to 2 substituents selected from F, Cl, Br, I, hydroxy, cyano or C_1-4Alkyl is substituted by a substituent;

R^1eand R^1fEach independently selected from F, Cl,Br、I、CF₃、NH₂OH, cyano, C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkylthio, -NHC_1-4Alkyl or-N (C)_1-4Alkyl radical)₂；

R³Each independently selected from F, Cl, Br, I, OH, NH₂Carboxy, cyano, nitro, C_1-4Alkyl radical, C_2-4Alkynyl, C_1-4Alkoxy, -OC_3-6Cycloalkyl radical, C_1-4Alkylthio, -S (═ O) -C_1-4Alkyl, -S (═ O)₂-C_1-4Alkyl, -C (═ O) -C_1-4Alkyl or-C (═ O) O-C_1-4Alkyl, alkynyl, alkoxy, cycloalkyl and NH₂Optionally further substituted by 0 to 4 substituents selected from F, Cl, Br, I, CF₃、C_1-4Alkyl radical, C_1-4Alkoxy or-C (═ O) -C_1-4Alkyl substituents.

5. The compound of claim 4, or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal, or prodrug thereof,

Q₁selected from a bond or-N (R)^q)-；

R^qSelected from H, methyl, ethyl, propyl or isopropyl;

R¹is selected from

X, Y, Z are each independently selected from nitrogen, -NH-, -CH₂-、-CH₂CH₂-, -CH-, -CH ═ CH-, -S-, -O-, and-C (O) -;

R²、R⁴each independently selected from methylene, ethylene, propylene, butylene or pentylene, said methylene, ethylene, propylene, butylene or pentylene being optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, cyano, OHMethyl, ethyl, methoxy or ethoxy;

R³each independently selected from F, Cl, Br, cyano, methyl, ethyl, propyl, isopropyl, ethynyl, CHF₂、CF₃Methoxy, ethoxy, -OCHF₂or-OCF₃；

R⁵、R⁶Each independently selected from H, methyl or ethyl;

b is selected from

a is 0 or 1;

b is 0, 1 or 2;

c is 0, 1 or 2.

6. The compound according to claim 1, wherein the compound is selected from the group consisting of compounds of formula (II), and stereoisomers, hydrates, metabolites, solvates, pharmaceutically acceptable salts, co-crystals, and prodrugs thereof,

7. the compound of claim 6, or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal, or prodrug thereof,

cy is selected from unsubstituted or substituted

When substituted, is optionally further substituted by 0 to 5 substituents selected from F, Cl, Br, I, OH, cyano, C_1-4Alkyl or C_1-4Substituted by a substituent of alkoxy;

when Cy is a heterocyclic ring containing N, the N atom is connected with Q1;

Q₁selected from a bond or-N (R)^q)-；

R^qIs selected from H or C_1-6An alkyl group.

8. The compound of claim 7, or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal, or prodrug thereof,

R²selected from methylene, ethylene, propylene, -CH₂CH(CH₃)-、-CH₂C(CH₃)₂-、-C(CH₃)₂CH₂-、-CH(CH₃)CH₂-, butylene, -CH (CH)₃)CH₂CH₂-、-CH₂CH(CH₃)CH₂-、-CH₂CH(CH₃)CH₂-or pentylene;

R³each independently selected from F, Cl, Br, cyano, methyl, ethyl, propyl, isopropyl, ethynyl, CHF₂、CF₃Methoxy, ethoxy, -OCHF₂or-OCF₃；

R^qSelected from H, methyl, ethyl, propyl or isopropyl;

c is 0, 1 or 2.

9. The compound of claim 1, or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof, wherein the compound comprises:

10. a pharmaceutical composition comprising a therapeutically effective dose of a compound of any one of claims 1-9, or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal, or prodrug thereof, and a pharmaceutically acceptable carrier, diluent, adjuvant, vehicle, or excipient; the composition optionally further comprises one or more other therapeutic agents.

11. The pharmaceutical composition of claim 10, wherein the additional therapeutic agent is selected from one or more of a PDE4 inhibitor, an M receptor antagonist, a corticosteroid, and a β -adrenoceptor agonist.

12. Use of a compound of any one of claims 1-9, or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal, or prodrug thereof, in the manufacture of a medicament for the treatment of an obstructive airways disease.

13. Use of a compound of any one of claims 1-9, or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt, co-crystal or prodrug thereof, in the manufacture of a medicament for the treatment of asthma, chronic obstructive pulmonary disease or bronchitis.