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HK1181047B - Alkaloid aminoester derivatives and medicinal composition thereof - Google Patents

Alkaloid aminoester derivatives and medicinal composition thereof Download PDF

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Publication number
HK1181047B
HK1181047B HK13108338.9A HK13108338A HK1181047B HK 1181047 B HK1181047 B HK 1181047B HK 13108338 A HK13108338 A HK 13108338A HK 1181047 B HK1181047 B HK 1181047B
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HK
Hong Kong
Prior art keywords
group
formula
thiophen
benzo
oxo
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HK13108338.9A
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Chinese (zh)
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HK1181047A1 (en
Inventor
G．阿马里
M．里卡博尼
A．卡利朱里
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奇斯药制品公司
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Priority claimed from PCT/EP2011/058801 external-priority patent/WO2011160918A1/en
Publication of HK1181047A1 publication Critical patent/HK1181047A1/en
Publication of HK1181047B publication Critical patent/HK1181047B/en

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Description

Alkaloid aminoester derivatives and pharmaceutical compositions thereof
Technical Field
The present invention relates to alkaloid aminoester derivatives acting as muscarinic receptor antagonists, processes for their preparation, compositions containing them and therapeutic uses thereof.
Background
Quaternary ammonium salts, which act as muscarinic (M) receptor antagonist drugs, are currently used in bronchodilator-inducing therapies for the treatment of respiratory diseases. Examples of well-known M receptor antagonists are represented, for example, by ipratropium bromide and tiotropium bromide.
Several chemical groups acting as selective M3 receptor antagonist drugs have been developed to treat inflammatory or obstructive airway diseases, such as asthma and Chronic Obstructive Pulmonary Disease (COPD).
Quinuclidine carbamate derivatives and their use as M3 antagonists are disclosed, for example, in WO02/051841, WO03/053966 and WO 2008/012290.
The M and M3 receptor antagonists are currently administered by the inhalation route in order to deliver the drug directly at the site of action, thereby limiting systemic exposure and any undesirable side effects due to systemic absorption.
Therefore, it would be highly desirable to provide M3 receptor antagonists that can act locally, while having high potency and long duration of action. Once absorbed, the drug degrades into inactive compounds that are devoid of any systemic side effects typical of muscarinic antagonists.
Co-pending application WO2010/072338 discloses azoniabicyclo [2.2.2] octane compounds acting as muscarinic receptor antagonists, which compounds further possess the properties required for the above-mentioned treatments.
Surprisingly, it has now been found that the presence of heteroaryl groups in the above-mentioned azoniabicyclo [2.2.2] octane derivatives further improves the duration of action of these compounds according to the details below.
The compounds of the invention therefore behave like soft-drugs (soft-drug) in that they produce a more sustained bronchodilator efficacy in the lungs, but are more consistently and rapidly converted to inactive metabolites after entry into the human plasma.
This behavior yields a great advantage in terms of safety.
Summary of The Invention
The present invention therefore relates to alkaloid aminoester derivatives of general formula (I) acting as muscarinic receptor antagonists, processes for their preparation, compositions comprising them, therapeutic uses and combinations with other pharmaceutically active ingredients, especially for example those currently used in the treatment of respiratory disorders, such as β 2-agonists, corticosteroids, P38MAP kinase inhibitors, IKK2, HNE inhibitors, PDE4 inhibitors, leukotriene modulators, NSAIDs and mucus regulators.
Detailed Description
In particular, the present invention relates to alkaloid aminoester derivatives of general formula (I):
wherein:
R1selected from aryl, heteroaryl, aryl (C)1-C6) Alkyl and heteroaryl (C)1-C6) Alkyl, said group being optionally substituted with one or more substituents selected from: halogen atoms, OH, oxygen (oxo), SH, NH2，NO2，CN，CON(R5)2，NHCOR5，COR5，CO2R5，(C1-C6) Alkylsulfanyl (C)1-C6) Alkylsulfinyl (C)1-C6) Alkylsulfonyl group (C)1-C6) Alkyl radical (C)1-C6) Haloalkyl (C)1-C6) Alkoxy and (C)1-C6) A haloalkoxy group;
R2is H or is selected from (C)1-C6) Alkyl radical (C)3-C8) Cycloalkyl and aryl (C)1-C6) Alkyl, said group being optionally substituted with one or more substituents selected from: halogen atom, OH, oxygen, SH, NH2，NO2，CN，CON(R5)2，NHCOR5，COR5，CO2R5，(C1-C6) Alkylsulfanyl (C)1-C6) Alkylsulfinyl (C)1-C6) Alkylsulfonyl group (C)1-C6) Alkyl radical (C)1-C6) Haloalkyl (C)1-C6) Alkoxy and (C)1-C6) A haloalkoxy group;
R3selected from aryl, heteroaryl, aryl (C)1-C6) Alkyl and heteroaryl (C)1-C6) Alkyl, said group being optionally substituted with one or more substituents selected from: halogen atom, OH, oxygen, SH, NH2，NO2，CN，CON(R5)2，COR5，CO2R5，(C1-C6) Alkylsulfanyl (C)1-C6) Alkylsulfinyl (C)1-C6) Alkylsulfonyl group (C)1-C6) Alkyl radical (C)1-C6) Haloalkyl (C)1-C6) Alkoxy and (C)1-C6) Haloalkoxy and aryl (C)1-C6) An alkoxy group;
R5in each case independently selected from H, (C)1-C6) Alkyl radical (C)2-C6) Alkenyl (C)3-C8) Cycloalkyl, heteroaryl and aryl, said groups being optionally substituted with one or more substituents selected from: halogen atom, OH, oxygen, SH, NH2，NO2，CN，CONH2，COOH；(C1-C6) Alkoxycarbonyl group, (C)1-C6) Alkylsulfanyl (C)1-C6) Alkylsulfinyl (C)1-C6) Alkylsulfonyl group (C)1-C6) Alkyl radical (C)1-C6) Haloalkyl (C)1-C6) Alkoxy and (C)1-C6) A haloalkoxy group;
q represents a group of formula (i) or (ii):
wherein R is4Is a group of formula (Y):
---(CH2)p---P---(CH2)q---W
(Y)
wherein
p is 0 or an integer from 1 to 4;
q is 0 or an integer from 1 to 4;
p is absent or selected from O, S, SO2，CO，NR5CH＝CH，N(R5)SO2，N(R5)COO，N(R5)C(O)，SO2N(R5)，CO(O)N(R5) And C (O) N (R)5)；
W is selected from H, (C)1-C6) Alkyl radical (C)2-C6) Alkenyl (C)3-C8) Cycloalkyl, aryl and heteroaryl, said groups being optionally substituted with one or more substituents selected from: halogen atom, OH, oxygen, SH, NH2，NO2，CN，CON(R5)2，NHCOR5，COR5，CO2R5；(C1-C6) Alkyl radical (C)1-C6) Alkylsulfanyl (C)1-C6) Alkylsulfinyl (C)1-C6) Alkylsulfonyl group (C)1-C6) Alkyl radical (C)1-C6) Haloalkyl (C)1-C6) Alkoxy and (C)1-C6) A haloalkoxy group;
A-is a physiologically acceptable anion;
wherein R is1And R3At least one of which is heteroaryl.
In this specification, unless otherwise specified, the term "halogen" includes fluorine, chlorine, bromine and iodine atoms.
Expression "(C)1-C6) Alkyl "refers to straight or branched chain alkyl groups in which the number of carbon atoms is from 1 to 6. Examples of such groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.
Derived expression "(C)1-C6) Alkoxy "should be interpreted in a similar manner to the alkyl-oxy (e.g., alkoxy) groups mentioned above. Examples of such groups may thus include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.
Also, the expression "(C)1-C6) Alkoxycarbonyl "should be taken in conjunction with (C) mentioned above further bearing a carbonyl group1-C6) Alkoxy, in particular, is to be construed as for example acetoxy (e.g. acetoxycarbonyl), tert-butoxycarbonyl and the like.
Derived expression "(C)1-C6) Haloalkyl "and" (C)1-C6) Haloalkoxy "thus refers to" (C) above1-C6) Alkyl "and" (C)1-C6) Alkoxy ", wherein one or more hydrogen atoms are substituted by one or more halogen atoms, which may be the same or different.
Said (C)1-C6) Haloalkyl and (C)1-C6) Examples of haloalkoxy groups may thus include halogenated, polyhalogenated, and perhalogenated alkyl and alkoxy groups in which all hydrogen atoms are substituted with halogen atoms. As an example, these are especially trifluoromethyl, trifluoromethoxy.
Also, the derived expression "(C)1-C6) Alkylsulfanyl "," (C)1-C6) Alkylsulfinyl "or" (C)1-C6) Alkylsulfonyl "means alkyl-S-, alkyl-SO-or alkyl-SO, respectively2-。
Expression "(C)3-C8) Cycloalkyl "refers to cyclic non-aromatic hydrocarbon groups having 3 to 8 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.
The expression "aryl" refers to a mono-, bi-or tricyclic ring system having 6 to 20 ring atoms, preferably 6 to 15 ring atoms, and wherein at least one ring is aromatic.
The expression "heteroaryl" refers to mono-, bi-or tricyclic ring systems having 5 to 20 ring atoms, preferably 5 to 15 ring atoms, and wherein at least one ring is aromatic and wherein at least one ring atom is a heteroatom or heteroaromatic group (e.g. N, NH, S or O).
Examples of suitable aryl or heteroaryl monocyclic systems include, for example, thiophene, benzene, pyrrole, pyrazole, imidazole, isoxazole, oxazole, isothiazole, thiazole, pyridine, imidazolidine, furan groups and the like.
Examples of suitable aryl or heteroaryl bicyclic systems include naphthalene, biphenylene, purine, pteridine, benzotriazole, quinoline, isoquinoline, indole, isoindole, benzothiophene, dihydrobenzodioxin, dihydrobenzodiazepine, benzoxazinyl, and the like.
Examples of suitable aryl or heteroaryl tricyclic systems include fluorenyl as well as benzo-condensed derivatives of the aforementioned heteroaryl bicyclic systems.
The expression "aryl (C)1-C6) Alkyl "," heteroaryl (C)1-C6) Alkyl means further substituted by aryl or heteroaryl ring respectively (C)1-C6) An alkyl group.
The expression "aryl (C)1-C6) Alkoxy "means (C) further substituted by aryl1-C6) An alkoxy group.
Expression "(C)2-C6) Alkenyl "refers to a straight or branched carbon chain having one or more double bonds. Examples of such groups may thus include ethenyl, propenyl, butenyl, pentenyl, hexenyl and the like.
With respect to R5It is clear to the person skilled in the art that, wherever possible, it may represent H or, in particular, the groups reported above.
Thus, as an example, at R1Is a quilt CON (R)5)2In the case of aryl groups further substituted with a substituent, the substituent also includes CONH2，CONHR5And CON (R)5)(R5) Wherein R is5As listed above.
Advantageously, the physiologically acceptable anion A-Including those selected from: chloride, bromide, iodide, trifluoroacetate, formate, sulfate, phosphate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate, oxalate, succinate, benzoate, and p-toluenesulfonate, preferably chloride, bromide and trifluoroacetate.
Except for the presence of A-In addition to the anion, any time a further basic amino group is present in the compound of formula (I), additional physiologically acceptable anions may be present, in particular those described above. Likewise, in the presence of acidic groups, such as COOH groups, corresponding physiologically acceptable cationic salts may also be present, including, for example, alkali metal or alkaline earth metal ions.
A first group of compounds of the general formula (I) is that in which R1Selected from aryl, heteroaryl and aryl (C)1-C6) Those of alkyl, said groups being optionally substituted with one or more substituents selected from: halogen atom, COR5，CO2R5，CON(R5)2，(C1-C6) Alkyl and (C)1-C6) An alkoxy group; r2Is H or (C)1-C6) An alkyl group; q is a group of formula (i); and R3And R5Having the meanings reported above.
In this case, compounds of the general formula (I) in which R is1Selected from phenyl, benzyl, and thienyl, said groups optionally substituted with one or more substituents selected from the group consisting of: halogen atom, COR5，CO2R5，CON(R5)2，(C1-C6) Alkyl and (C)1-C6) Alkoxy radical, wherein R5Is H or (C)1-C6) An alkyl group; q is a group of formula (i); r2Is H or methyl; and R3Having the meanings reported above.
A second group of compounds of the general formula (I) is that in which R1Selected from aryl, heteroaryl and aryl (C)1-C6) Alkyl, said group being optionally substituted with one or more substituents selected from: halogen atom, COR5，CO2R5，CON(R5)2，(C1-C6) Alkyl and (C)1-C6) An alkoxy group; r2Is H or (C)1-C6) An alkyl group; q is a group of formula (ii); and R3，R4，R5And A-Having the meanings reported above.
In this case, compounds of the general formula (I) in which R is1Selected from phenyl, benzyl and thienyl, said groups being optionally substituted with one or more substituents selected from: halogen atom, COR5，CO2R5，CON(R5)2，(C1-C6) Alkyl and (C)1-C6) Alkoxy radical, wherein R5Is H or (C)1-C6) An alkyl group; q is a group of formula (ii); r2Is H or methyl; and R3，R4And A-Having the meanings reported above.
Another preferred group of compounds of the formula (I) is that in which R3Selected from aryl and heteroaryl, said groups being optionally substituted with one or more substituents selected from: halogen atom, (C)1-C6) Alkoxy, OH and (C)1-C6) An arylalkoxy group; q is a group of formula (i); r2Is H or (C)1-C6) An alkyl group; and R1And R5Having the meanings reported above.
Within this group, an even more preferred group of compounds of formula (I) is that wherein R is3Selected from phenyl, pyridyl, thienyl and benzothienyl, optionally substituted with the substituents described above; q is a group of formula (i); and R2Is H or methyl; and R5Having the meanings reported above.
Another preferred group of compounds of the formula (I) is that in which R3Selected from aryl and heteroaryl, said groups optionally being substitutedSubstituted with one or more substituents selected from: halogen atom, (C)1-C6) Alkoxy, OH and (C)1-C6) An arylalkoxy group; q is a group of formula (ii); r2Is H or (C)1-C6) An alkyl group; and R1，R4，R5And A-Having the meanings reported above.
Within this group, an even more preferred group of compounds of formula (I) is that wherein R is3Selected from phenyl, pyridyl, thienyl and benzothienyl, optionally substituted with the substituents described above; q is a group of formula (ii); r2Is H or methyl and R1，R4And A-Having the meanings reported above.
Another preferred group of compounds of formula (I) are those wherein Q is a group of formula (ii); r4Is a group of formula (Y) wherein P is 0,1,2 or 3, q is 0, P is absent or selected from O, CO and C (O) N (R)5) And W is selected from aryl, (C)2-C6) Alkenyl and heteroaryl, optionally substituted with one or more substituents as described above; and R1，R2，R3，R5And A-Having the meanings reported above.
Within this group, still more preferred are compounds of formula (I) wherein Q is a group of formula (ii); p is 1, q is 0, P is CO and W is selected from phenyl, pyridyl, thienyl, isoxazolyl and thiazolyl, optionally substituted with substituents described above; and R1，R2，R3，R5And A-Having the meanings reported above.
Within this group, even still more preferred are compounds of formula (I) wherein Q is a group of formula (ii); p is 3, q is 0, P is O and W is phenyl optionally substituted by the substituents described above; and R1，R2，R3，R5And A-Having the meanings reported above.
Within this group, even still more preferredIs a compound of formula (I) wherein Q is a group of formula (ii); p is 2, q is 0, P is absent, and W is a phenyl group optionally substituted by the substituents described above; and R1，R2，R3，R5And A-Having the meanings reported above.
Within this group, even still more preferred are compounds of formula (I) wherein Q is a group of formula (ii); p is 1, q is 0, P and-CON (H) -and W are pyridyl optionally substituted by the substituents described above; and R1，R2，R3，R5And A-Having the meanings reported above.
Within this group, still more preferred are compounds of formula (I) wherein Q is a group of formula (ii); p and q are 0, P is absent and W is methyl; and R1，R2，R3，R5And A-Having the meanings reported above.
According to specific embodiments, the present invention provides the following reported compounds as examples:
the compounds of formula (I) show at least two chiral centers marked with asterisks, one in formula I
And another in the Q group of the formula
In addition, it depends inter alia on the R reported before1，R2，R3And R4The meaning indicated, it is clear to the person skilled in the art that additional asymmetric centers may be present in the compounds of the general formula (I). Thus, the present invention also includes any optical stereoisomers, diastereomers, and mixtures thereof, in any proportion.
In a preferred embodiment, the chiral center on the ring of the quinuclidine exhibits the R structure.
In the present invention, since the absolute structures of diastereomers are not defined, they are represented as diastereomers 1,2 or mixtures thereof in the examples.
The invention also provides pharmaceutical compositions of the compounds of formula (I) alone or in combination or admixture with one or more pharmaceutically acceptable carriers and/or excipients.
The invention also provides pharmaceutical compositions suitable for administration by inhalation, such as inhalable powders, propellant-containing metered dose aerosols or propellant-free inhalable formulations.
The invention also provides a compound of formula (I) for use as a medicament.
The invention also provides a compound of formula (I) for use in the treatment of a broncho-obstructive or inflammatory disease, preferably asthma or chronic bronchitis or Chronic Obstructive Pulmonary Disease (COPD).
In a further aspect, the present invention provides the use of a compound of formula (I) for the manufacture of a medicament for the prevention and/or treatment of a broncho-obstructive or inflammatory disease, preferably asthma or chronic bronchitis or Chronic Obstructive Pulmonary Disease (COPD).
The present invention also provides a method of preventing and/or treating a tracheo-obstructive or inflammatory disease, preferably asthma or chronic bronchitis or Chronic Obstructive Pulmonary Disease (COPD), the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I).
The invention also relates to a device which may be a single or multi dose dry powder inhaler, a metered dose aerosol under pressure and a soft mist nebulizer (softmistnebulizer) containing a compound of formula (I).
The invention also relates to kits comprising the above pharmaceutical compositions and devices in suitable vials or containers, which devices may be single-or multi-dose dry powder inhalers, metered dose aerosols and soft mist nebulizers suitable for housing the above vials or containers.
The compounds of general formula (I) can be prepared according to methods known and obvious to those skilled in the art in view of their reaction and operating conditions.
The invention also relates to a process for the preparation of a compound of general formula (I), which process comprises:
(a) alcohol of coupling formula (IX)
With a compound of formula (VIII) to give a compound of general formula (I):
wherein Q has formula (i);
(b) optionally alkylating a compound of formula (I) by an alkylating agent of formula (X):
A-R4
(X)
wherein A is a leaving group selected from the group consisting of halides and sulfonates, and R4As described above, a compound of general formula (I) is obtained, wherein Q has formula (ii), and optionally
(c) Converting a compound of formula (I) into another compound of formula (I) and/or into a pharmaceutically acceptable salt thereof.
The invention also relates to a process suitable for preparing intermediate compounds of the general formula (VIII),
these methods are reported below:
route A-the process comprises the use of a compound of formula (III) wherein LG is a leaving group and K can be either a carboxyl group, either unprotected or in optionally protected form
Alkylating an amine compound of the formula (II) wherein R1And R2Having the meanings reported above.
Route B-the process comprises dissolving and stirring an equimolar mixture of an amine of formula (II) with glyoxylic acid (IV) and boronic acid (V) in a solvent;
route C-the process comprises reacting a compound of formula (VI)
Reaction with (VII)
The operating conditions that can be used in the process of the present invention are described in more detail below and are further reported in scheme 1 below.
The starting materials for the preparation of the compounds of formula (I), that is to say, the compounds of formulae (II) and (III), as well as any reactants in the process, are known and can be readily prepared according to known procedures.
Process for preparing a Compound of formula (I)
According to a particular embodiment of the present invention, the compounds of general formula (I) can be prepared, for example, according to the following synthetic route described in scheme 1.
It may for example be according to three different paths: a, B or C, preparing the compound of the general formula (VIII).
According to scheme a, compounds of formula (VIII) can be prepared by alkylating an amine of formula (II) with a compound of formula (III), wherein LG is a suitable leaving group (e.g., halide, e.g., bromide), and K is a carboxyl group in optionally protected form.
Typically, LG is a halide atom, and more preferablyIt is a bromine atom. With respect to K, it may be either unprotected or the carboxy group in optionally protected form, typically including a carboxyalkyl ester group (e.g., K ═ COO (C)1-C6) Alkyl), preferably carboxymethyl (e.g., COOMe).
The alkylation reaction may be facilitated by the presence of a base, such as an amine selected from triethylamine, pyridine and 4-dimethylaminopyridine, either neat or in a suitable solvent (e.g., acetonitrile). This reaction is typically carried out at a temperature in the range of from about 0 ℃ to about 130 ℃ over a period of from about 1 hour to about 74 hours. The reaction can be carried out under conventional heating (using an oil bath) or under microwave heating. The reaction can be carried out in an open vessel or in a sealed tube.
According to scheme B, the compounds of formula (V) can be prepared by a Petasis-Mannich reaction, e.g. by reacting an equimolar mixture of amine (II), glyoxylic acid (IV) and boronic acid (V) in a suitable solvent (e.g. dichloromethane, acetonitrile) with stirring VIII, following one of the different procedures reported in the literature (e.g. Petasis N.A., Akritopoulou I., tetrahedron Lett.1993,34,583; Follmann, M., Synlett,2005,6, 1009; Kausikk. N., tetrahedron Letterers, 2005,46, 2025). This reaction is typically carried out at a temperature in the range of from about 0 ℃ to about 110 ℃ over a period of from about 1 hour to about 74 hours. The reaction can be carried out under conventional heating (using an oil bath) or under microwave heating. The reaction can be carried out in an open vessel or in a sealed tube.
According to scheme C, compounds of general formulae (VI) and (VII) can be reacted under conditions typical for aromatic nucleophilic substitution to give compound (VIII).
The compound of formula (I) is then prepared by coupling the alcohol (IX) with a compound of formula (VIII), wherein Q is
The operating conditions are chosen on the basis of the reactivity of the compound (VIII) with respect to the alcohol (IX), and of the compatibility of the other groups present in the two reactants (for general reference to the above-mentioned reactions and their operating conditions, see for example Carey, F.A. -Sundeberg, R.J. advanced organic chemistry, 3 rd edition (1990), plenum Press, New York-London, p.145).
In particular, in the case where K is a protected carboxyl group, the protecting group must first be removed before the coupling reaction can take place.
Thus, where K is a carboxylate moiety (e.g., K ═ COOMe), removal of the protecting group is carried out under hydrolysis conditions, typically in the presence of any suitable aqueous base selected from the group consisting of sodium, lithium, and potassium hydroxides.
The reaction is carried out in any suitable solvent, for example in the presence of tetrahydrofuran or dioxane, at Room Temperature (RT), and over a period of time from about 1 hour to about 36 hours.
Alternatively, when starting from a compound of formula (VIII) wherein K is carboxy, standard amidation and peptide coupling conditions may be employed to obtain a compound of formula (I) wherein Q is as defined above. Such conditions include, for example, activation of intermediate (VIII) in the presence of N-Hydroxybenzotriazole (HOBT), for example by one or more equivalent commercially available condensing agents such as carbodiimides (e.g., Dicyclohexylcarbodiimide (DCC) and the like), followed by reaction of the activated intermediate with alcohol (IX) resulting in formation of compound (I), wherein Q is as defined above. An organic base, such as triethylamine, may also be present in the reaction mixture. The activated intermediate may be either isolated, or preformed, or generated in situ, and then suitably reacted with an alcohol of formula (IX). Suitable solvents for the coupling reaction include, but are not limited to, halocarbon solvents (e.g., dichloromethane), tetrahydrofuran, dioxane, and acetonitrile. The reaction is conducted at a temperature ranging from about 0 ℃ up to about 170 ℃ for a period ranging from about 1 hour up to about 72 hours. The reaction can be carried out under conventional heating conditions (using an oil bath) or under microwave irradiation. The reaction can be carried out either in an open vessel or in a sealed tube.
Once obtained, the compounds of general formula (I) can be obtained either as single diastereoisomers or as mixtures of diastereoisomers, in which Q is as defined above. For example, in the case where the alcohol (IX) is characterized by the structure R, the corresponding compound (I) can be obtained in both the structures S-R or R-R, as well as mixtures of diastereomers (R-R and S-R structures).
The mixture of diastereomers may be converted to a compound of formula (I) wherein Q is a group of formula (ii), or may be most conveniently resolved to give two single diastereomers, which may themselves be converted to a compound of formula (I) wherein Q is as defined above. This separation can be accomplished using procedures well known to those skilled in the art. These procedures include, but are not limited to, chromatographic purification, preparative HPLC purification, and crystallization. For example, the two diastereomers can be separated by flash chromatography on silica gel eluted with a suitable solvent or solvent mixture, such as DCM, methanol and the like. In another process of the invention, separation of diastereomers can be carried out by using a column packed with a chiral stationary phase, such as ChiralpackAY or chiralgel od or chiralgel oz, and eluting with acetonitrile and/or with a mixture of acetonitrile and an alcohol, for example. Alternatively, separation of the diastereomers may be most conveniently achieved by crystallization from a suitable solvent (e.g., diethyl ether), either as the free base or after formation of a suitable salt (e.g., (+) -tartaric acid).
The compound of formula (I) wherein Q is a group of formula (I) is then alkylated with a reagent of formula (I) to give a compound of formula (I) wherein Q is a group of formula (ii).
This reaction is described in several different conditions, mainly in the literature. For example, the reaction may be carried out neat or in a suitable solvent selected from acetonitrile, DMF, DMSO and tetrahydrofuran. The reaction is typically carried out at a temperature ranging from about 0 ℃ up to about 170 ℃ for a time ranging from minutes up to about 72 hours. The reaction can be carried out under conventional heating (using an oil bath) or under microwave irradiation. The reaction can be carried out either in an open vessel or in a sealed tube.
Compounds of general formula (I) wherein Q is a group of formula (ii) may be considered end products or may be further reacted to prepare further compounds of general formula (I). Thus, in the formula (I), R1，R2，R3，R4And R5Any suitable part of the group may undergo various reactions to give other final compounds of general formula (I).
Likewise, any free acid group (e.g. carboxyl) or free amino group may suitably be converted into the corresponding pharmaceutically acceptable salt, optionally salifying of a compound of formula (I) wherein Q is a group of formula (ii) may be carried out.
In this case, the operating conditions used for the optional salification of the compounds of the invention are also within the ordinary knowledge of the person skilled in the art.
As reported previously, the compounds of formula (III) are known and, if not commercially available, can be readily prepared according to known methods widely reported in the literature.
For example, compounds of formula (III) wherein LG is a halogen, e.g. bromine, can be prepared by halogenation of a suitably substituted phenyl acetate (e.g. following the procedure reported in Epstein, j.w. in j.med.chem.,1981,24/5,481). Alternatively, the compounds of formula (III) can be prepared starting from appropriately substituted mandelic acid derivatives using conventional procedures (Larock, L.C., comprehensive organic transformation, Secondedification (1999), A review of suitable reactions is given on page 689-700 of John Wiley & Son Inc.).
From all the above, it will be clear to the skilled person that the above-described process for preparing suitable compounds of formula (I) according to the invention, in its full scope of any variants, can be modified conveniently, for example as the case may be, by selecting suitable condensing agents, solvents and protecting groups, in order to adapt to the reaction conditions of particular needs.
More particularly, functional groups present in any compound of formula (III), (IV), (VI) or (VIII) and which may generate unwanted side reactions and by-products need to be suitably protected before condensation reactions occur.
Likewise, once the above reaction is complete, subsequent deprotection of those same protected groups may follow.
In the present invention, unless otherwise specified, the term "protecting group" means a protecting group suitable for protecting the functional group to which it is bonded.
Specifically, protecting groups are used to protect amino, hydroxyl or carboxyl functional groups.
Suitable protecting groups may thus include, for example, benzyl, benzyloxycarbonyl, alkyl or benzyl esters, or other substituents commonly used to protect such functional groups, all of which are well known [ see, for general reference, t.w.green; protective group in organic Synthesis (Wiley, N.Y.1981) ].
The invention also provides pharmaceutical compositions of the compounds of formula (I) in admixture with one or more pharmaceutically acceptable carriers, such as those described in Remington's pharmaceutical sciences handbook, xviied., mackpub., n.y., u.s.a.
In the present invention, the terms active ingredient or active or compound are considered as synonyms for interchangeable use.
Administration of the compounds of the present invention can be accomplished according to patient requirements, for example, oral, nasal, parenteral (subcutaneous, intravenous, intramuscular, intrasternal, and by infusion), by inhalation, rectal, vaginal, topical (topically), topical, transdermal, and ocular administration.
Various solid oral dosage forms may be used to administer the compounds of the present invention, including solid forms such as tablets, gelatin capsules, caplets, granules, cough syrups and bulk powders. The compounds of the present invention may be administered alone or in combination with various pharmaceutically acceptable carriers, diluents (e.g., sucrose, mannose, lactose, starch) and excipients known in the art, including, but not limited to, suspending agents, solubilizers, buffers, binders, disintegrants, preservatives, colorants, flavorants, lubricants and the like. Sustained release capsules, tablets and gels are also advantageous in administering the compounds of the invention.
Various liquid oral dosage forms may also be used to administer the compounds of the present invention, including aqueous and non-aqueous solutions, emulsions, suspensions, syrups and elixirs. These dosage forms may also contain suitable inert diluents known in the art such as water and suitable excipients known in the art such as preservatives, wetting agents, sweeteners, flavoring agents, and agents for emulsifying and/or suspending the compounds of the present invention. The compounds of the invention may be injected, for example, intravenously in the form of an isotonic solution. Other compositions are also possible.
Suppositories for rectal administration of the compounds of the invention can be prepared by mixing the compounds with suitable excipients, for example cocoa butter, salicylates and polyethylene glycols.
Formulations for vaginal administration may be in the form of cream, gel, paste, foam, or spray formulations containing, in addition to the active ingredient, such suitable carriers as are known in the art.
For topical application, the pharmaceutical composition may be in the form of a cream, ointment, liniment, lotion, emulsion, suspension, gel, solution, paste, powder, spray, and drops suitable for application to the skin, eyes, ears, or nose. Topical administration may also involve transdermal administration by means such as transdermal patches.
For the treatment of respiratory diseases, the compounds of the invention are preferably administered by inhalation.
Inhalable compositions include inhalable powders, propellant-containing metered dose aerosols or propellant-free inhalable formulations.
For administration in dry powder form, single-or multi-dose inhalers known from the prior art can be used. In this case, the powder may be filled in gelatin, plastic or other capsules, cartridges or blister packs or containers.
Diluents or carriers which are generally non-toxic and chemically inert to the compounds of the invention, such as lactose or any other additive suitable for modifying the respirable fraction, may be added to the powdered compounds of the invention.
Inhalation aerosols containing propellant gases, for example hydrofluoroalkanes, may contain the compounds of the invention either in solution or dispersion. The propellant-driven formulation may also contain other ingredients, such as co-solvents, stabilizers and optionally other excipients.
Propellant-free inhalable formulations containing the compounds of the invention may be in the form of solutions or suspensions in aqueous, alcoholic or hydroalcoholic media, and they may be delivered by jet or ultrasonic nebulizers or by soft mist nebulizers.
The compounds of the invention may be administered as the sole active ingredient or in combination with other pharmaceutically active ingredients, including those currently commonly used in the treatment of respiratory disorders, such as β 2-agonists, corticosteroids, P38MAP kinase inhibitors, IKK2, HNE inhibitors, PDE4 inhibitors, leukotriene modulators, NSAIDs and mucus regulators.
The invention also provides a combination of a compound of formula (I) with a beta 2-agonist selected from GSK-642444, indacaterol (indacaterol), miwiterodol (milveterol), amoeborol (arformoterol), salbutamol, levalbuterol (levalbuterol), terbutaline, AZD-3199, BI-1744-CL, LAS-100977, mexican, isoproterenol, procaterol, clenbuterol, reproterol, fenoterol and ASF-1020.
The present invention also provides a combination of a compound of formula (I) and a corticosteroid selected from the group consisting of propionate, ciclesonide, mometasone furoate and budesonide.
The present invention also provides a combination of a compound of general formula (I) and a P38 inhibitor selected from the group consisting of samimod (semapimod), talmopimod (talmapimod), pirfenidone, PH-797804, GSK-725, a minute and lobimod (loshapimod).
The invention also provides a combination of a compound of formula (I) and an IKK2 inhibitor.
The present invention also provides a combination of a compound of formula (I) with an HNE inhibitor selected from the group consisting of AAT, ADC-7828, Aeriva, TAPI, AE-3763, KRP-109, AX-9657, POL-6014, AER-002, AGTC-0106, respriva, AZD-9668, zemaira, AATIV, PGX-100, elastinase protein (elafin), SPHD-400, alpha 1-protease inhibitor C and alpha 1-protease inhibitor.
The present invention also provides a combination of a compound of formula (I) with a PDE4 inhibitor selected from the group consisting of AN-2728, AN-2898, CBS-3595, apremilast, ELB-353, KF-66490, K-34, LAS-37779, IBFB-211913, AWD-12-281, simperidone, cilomilast, roflumilast, BAY19-8004 and SCH-351591, AN-6415, indus-82010, TPI-PD3, ELB-353, CC-11050, GSK-256066, oglemilast, OX-914, tetomilast, MEM-1414 and RPL-554.
The invention also provides a combination of a compound of formula (I) and a leukotriene modulator selected from montelukast, zafirlukast and pranlukast.
The present invention also provides a combination of a compound of formula (I) and an NSAID selected from ibuprofen and ketoprofen.
The invention also provides a combination of a compound of formula (I) and a mucus regulator selected from INS-37217, diquafosol, sibenadet, CS-003, talnetant, DNK-333, MSI-1956 and gefitinib.
The dosage of the compounds of the invention depends on a variety of factors including the particular disease to be treated, the severity of the symptoms, the route of administration, the frequency of the dosage intervals, the particular compound used, the efficacy, toxicity profile, and the pharmacokinetic profile of the compound.
Advantageously, the compounds of formula (I) can be administered, for example, in a dose comprising 0.001-1000mg/d, preferably 0.1-500 mg/d.
When the compounds of formula (I) are administered by the inhalation route, they are preferably administered in a dose comprising 0.001-500mg/d, preferably 0.1-200 mg/d.
The compounds of formula (I) may be administered for the prevention and/or treatment of any disease in which an M3 antagonist is active. The diseases include: diseases involving inflammation, such as asthma and COPD, acute rhinitis; diseases involving the gastrointestinal tract, such as gastric ulcers; diseases involving the cardiovascular system, such as acute myocardial infarction; diseases involving the urogenital tract, such as renal colic; against cholinesterase and mushroom poisoning; use in anesthesia; use in ophthalmology.
They also include neurological and psychiatric diseases, such as parkinson's disease and motion sickness.
Preferably, the compounds of formula (I) are administered for the prevention and/or treatment of respiratory diseases, such as asthma and moderate to severe conditions of COPD.
Other respiratory diseases include bronchitis, bronchiolitis, bronchiectasis, acute nasopharyngitis, acute and chronic sinusitis, maxillary sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, epiglottitis, epitopic laryngitis, chronic diseases of tonsils and adenoids, hypertrophy of tonsils and adenoids, peritonsillar abscess, rhinitis, nasal abscess or ulcer, pneumonia, viral and bacterial pneumonia, bronchopneumonia, influenza, extrinsic allergic alveolitis, pneumoconiosis of coal miners, asbestosis, pneumoconiosis, pulmonary disease, respiratory diseases due to chemical fumes, steam and other external agents, emphysema, pleurisy, pneumothorax, lung and mediastinal abscess, pulmonary congestion and blood stasis (pneumostasis), post-inflammatory pulmonary fibrosis, other alveolar and alveolar wall (lung alveolar alveoli) lung diseases, idiopathic fibrositis, Hardy-Reid syndrome, atelectasis, ARDS, acute respiratory disorder, mediastinitis.
The invention will now be further illustrated by the following examples.
I ═ intermediates
C ═ compound
Example 1
Preparation of (3R) -3- (2- (6-methoxypyridin-3-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane trifluoroacetate (C3)
Preparation of 2- (6-methoxypyridin-3-yl) -2- (phenylamino) acetic acid (I1):
a mixture of aniline (0.30ml, 3.27mmol), 6-methoxypyridin-3-ylboronic acid (500mg, 3.27mmol) and 2-oxyacetic acid hydrate (301mg, 3.27mmol) in acetonitrile (20ml) was heated at 100 ℃ for 1 hour under microwave irradiation. The reaction was evaporated to dryness and the crude product was triturated with acetonitrile. The solid was collected by suction filtration to give 2- (6-methoxypyridin-3-yl) -2- (phenylamino) acetic acid (202mg, 23% yield).
Preparation of (R) -quinuclidin-3-yl 2- (6-methoxypyridin-3-yl) -2- (phenylamino) acetate (C2):
a mixture of 2- (6-methoxypyridin-3-yl) -2- (phenylamino) acetic acid (I1) (100mg, 0.39mmol), (R) -quinuclidin-3-hydroxy (59.1mg, 0.46mmol), HOBT (71.2mg, 0.46mmol) and DCC (96mg, 0.46mmol) was dissolved in dry THF (10 ml). The resulting reaction was stirred at room temperature overnight. The solvent was then removed under reduced pressure and the residue was taken up in EtOAc and 2MK2CO3Washing is carried out for 2 times. In Na2SO4The organic phase was dried, filtered and evaporated to dryness.The crude product was purified by flash chromatography (DCM/MeOH ═ 98/2) to afford (R) -quinuclidin-3-yl 2- (6-methoxypyridin-3-yl) -2- (phenylamino) acetate (33mg, 23% yield).
Preparation of (3R) -3- (2- (6-methoxypyridin-3-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane trifluoroacetate (C3):
2-chloro-1-phenylethanone (13.9mg, 0.09mmol) was added to a solution of (R) - ((R) -quinuclidin-3-yl) 2-phenyl-2- (phenylamino) acetate (C2) (60mg, 0.18mmol) in EtOAc (3 mL). The reaction was stirred at room temperature overnight. Evaporation of EtOAc and use of Et2And O, crushing the residue. The solid was collected by filtration and then purified by preparative HPLC to give (3R) -3- (2- (6-methoxypyridin-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azocationic bicyclo [2.2.2]Octane trifluoroacetate (26.4mg, 49% yield).
1HNMR(300MHz,DMSO-d6)ppm8.33-8.39(m,1H),7.92-8.04(m,2H),7.88(dd,1H),7.70-7.82(m,1H),7.54-7.68(m,2H),7.05-7.20(m,2H),6.87-6.88(d,1H),6.69-6.78(m,2H),6.55-6.69(m,1H),5.39-5.42(s,1H),5.20-5.28(m,1H),5.10-5.17(s,2H),3.85-3.86(s,3H),3.73-3.80(m,1H),3.36-3.61(m,5H),2.12-22-2.33-2.44(m,1H),1.84-2.12(m,4H)；
LC-MS(ESIPOS)：486.19(M+)；
Example 2
Preparation of (3R) -3- (2- (2-acetylthiophen-3-ylamino) -2-phenylacetyloxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane trifluoroacetate (C6)
Preparation of 2- (2-acetylthiophen-3-ylamino) -2-phenylacetic acid (I4):
a solution of ethyl 2-bromo-2-phenylacetate (349. mu.l, 2.00mmol) and 1- (3-aminothiophen-2-yl) ethanone (282mg, 2.00mmol) in acetonitrile (2ml) was heated at 100 ℃ for 1h under microwave irradiation. Acetonitrile was evaporated and the residue was dissolved in EtOH (2ml) and sodium hydroxide (80.0mg, 2.00mmol) was added. The reaction was stirred at room temperature for 24 h, then ethanol was evaporated in vacuo and the residue was taken up in water and washed with EtOAc. The pH of the aqueous phase was adjusted to 3 with HCl and the aqueous phase was back-extracted with EtOAc. In Na2SO4The organic phase was dried, filtered and evaporated to dryness to give 2- (2-acetylthiophen-3-ylamino) -2-phenylacetic acid (188mg, 34% yield) which was used in the next step without any further purification.
Preparation of (2-acetyl-thiophen-3-ylamino) -phenyl-acetic acid (R) - (1-aza-bicyclo [2.2.2] oct-3-yl) ester (C5):
a mixture of 2- (2-acetylthiophen-3-ylamino) -2-phenylacetic acid (I4) (188mg, 0.68mmol), (R) -quinuclidin-3-hydroxy (104mg, 0.82mmol), HOBT (125mg, 0.82mmol) and DCC (169mg, 0.82mmol) in dry THF (8ml) was stirred at room temperature overnight. Evaporation of THF and absorption of the crude product with EtOAc and 2MK2CO3Washing is carried out for 2 times. The organic phase was dried (Na)2SO4) Filtered and evaporated to dryness. The crude product was purified by preparative HPLC. The fractions containing the product were combined and evaporated in vacuo. Dissolving the residue in 2MK2CO3And extracted 2 times with EtOAc to give (2-acetyl-thiophen-3-ylamino) -phenyl-acetic acid (R) - (1-aza-bicyclo [2.2.2]Oct-3-yl) ester (48mg, 18% yield).
Preparation of (3R) -3- (2- (2-acetylthiophen-3-ylamino) -2-phenylacetyloxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane trifluoroacetate (C6):
2-chloro-1-phenylethanone (19.3mg, 0.12mmol) was added to a solution of (2-acetyl-thiophen-3-ylamino) -phenyl-acetic acid (R) - (1-aza-bicyclo [2.2.2] oct-3-yl) methyl ester (C5) (35.7mg, 0.09mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight. The solvent was removed under reduced pressure. The crude product was purified by preparative HPLC to give (3R) -3- (2- (2-acetylthiophen-3-ylamino) -2-phenylacetyloxy) -1- (2-oxo-2-phenylethyl) -1-azocationic bicyclo [2.2.2] octane trifluoroacetate (42.1mg, 55% yield).
1HNMR(300MHz,DMSO-d6)ppm9.01-9.04(d,1H),7.91-8.07(m,2H),7.72-7.81(m,1H),7.70-7.71(d,1H),7.56-7.67(m,2H),7.28-7.54(m,5H),6.74-6.75(d,1H),5.68-5.74(d,1H),5.21-5.35(m,1H),5.12-5.17(s,2H),3.94-4.25(m,1H),3.29-3.88(m,5H),2.34(s,3H),2.15-2.25-2.38-2.46(m,1H),1.43-2.15(m,4H)；
LC-MS(ESIPOS)：503.14(M+).
Example 3
Preparation of (3R) -3- (2- (2-carbamoylthiophen-3-ylamino) -2-phenylacetyloxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (C9)
Preparation of 2- (2-carbamoylthiophen-3-ylamino) -2-phenylacetic acid (I7):
a solution of ethyl 2-bromo-2-phenylacetate (349. mu.l, 2.00mmol) and 3-aminothiophene-2-carboxamide (284mg, 2.00mmol) in acetonitrile (2ml) was heated at 100 ℃ for 1h under microwave irradiation. Acetonitrile was evaporated and the residue was dissolved in EtOH (2 ml). Sodium hydroxide (80mg, 2.00mmol) was added and the reaction stirred at room temperature for 24 hours. The ethanol was then evaporated and the residue was taken up in water and washed with EtOAc. The pH of the aqueous phase was adjusted to 3 with HCl, and the product was extracted with EtOAc. In Na2SO4The organic phase was dried, filtered and evaporated to dryness to give 2- (2-carbamoylthiophen-3-ylamino) -2-phenylacetic acid (389mg, 70% yield), which was used in the next step without further purificationThe method is used under the condition of.
Preparation of (R) -quinuclidin-3-yl 2- (2-carbamoylthiophen-3-ylamino) -2-phenyl acetate (C8):
a mixture of 2- (2-carbamoylthiophen-3-ylamino) -2-phenylacetic acid (I7) (389mg, 1.41mmol), (R) -quinuclidine-3-hydroxy (215mg, 1.69mmol), DCC (349mg, 1.69mmol) and HOBT (259mg, 1.69mmol) in dry THF (5ml) was stirred at room temperature overnight. THF was evaporated and the crude product was taken up in EtOAc and 2MK was used2CO3Washing is carried out for 2 times. In Na2SO4The organic phase was dried, filtered and evaporated to dryness. The crude product was purified by preparative HPLC. The fractions were combined with the product and evaporated to dryness. Dissolving the residue in 2MK2CO3And extracted 2 times with EtOAc to give (R) -quinuclidin-3-yl 2- (2-carbamoylthiophen-3-ylamino) -2-phenyl acetate (32mg, 6% yield).
Preparation of (3R) -3- (2- (2-carbamoylthiophen-3-ylamino) -2-phenylacetyloxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (C9):
2-chloro-1-phenylethanone (12.8mg, 0.08mmol) was added to a solution of (R) -quinuclidin-3-yl 2- (2-carbamoylthiophen-3-ylamino) -2-phenylacetate (C8) (32mg, 0.08mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight, then the solvent was removed under reduced pressure and the crude product was purified by preparative HPLC to give (3R) -3- (2- (2-carbamoylthiophen-3-ylamino) -2-phenylacetyloxy) -1- (2-oxo-2-phenylethyl) -1-azocationic bicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (24.3mg, 47% yield).
1HNMR(300MHz,DMSO-d6)ppm8.44-8.62(m,1H)7.89-8.03(m,2H)7.69-7.82(m,1H)7.54-7.67(m,2H)7.29-7.54(m,5H)6.96-7.11(m,1H)6.65-6.79(m,1H)5.49-5.70(m,1H)5.03-5.37(m,3H)4.00-4.23(m,1H)3.66-3.83(m,5H)2.39(m,1H)1.50-2.16(m,4H)；
LC-MS(ESIPOS)：504.13(M+).
Example 4
Preparation of (3R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetyloxy) -1- (2-oxo-2- (thiazol-2-yl) ethyl) -1-azoniabicyclo [2.2.2] -octane trifluoroacetate (diastereomer 1 of C12)
Preparation of 2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetic acid (I10):
a solution of ethyl 2-bromo-2-phenylacetate (600mg, 2.79mmol) and methyl 3-aminothiophene-2-carboxylate (438mg, 2.79mmol) in acetonitrile (20ml) was heated at 100 ℃ for 1h under microwave irradiation. Acetonitrile was evaporated to dryness and in the next step the resulting residue was used without any further purification.
Preparation of methyl 3- (2-oxo-1-phenyl-2- ((R) -quinuclidin-3-yloxy) ethylamino) thiophene-2-carboxylate (diastereoisomers 1 and 2 of C11):
a mixture of 2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetic acid (I10) (404mg, 1.39mmol), (R) -quinuclidine-3-hydroxy (529mg, 4.17mmol), DCC (859mg, 4.17mmol) and HOBT (567mg, 4.17mmol) in dry THF (25ml) was stirred at room temperature overnight. THF was evaporated and the crude product was taken up in EtOAc and 2MK was used2CO3Washing is carried out for 2 times. In Na2SO4The organic phase was dried, filtered and evaporated to dryness. The crude product was purified by flash chromatography (DCM/MeOH ═ 98/2-95/5) to recover C11 diastereomer 1(161 mg; 28% yield), and then C11 diastereomer 2(127g, 35% yield).
Diastereomer 1 of C11:
1HNMR(300MHz,DMSO-d6)ppm：7.76(d,1H),7.64(d,1H),7.22-7.54(m,5H),6.71(d,1H),5.60(d,1H),4.58-4.89(m,1H),3.78(s,3H),2.98(ddd,1H),2.54-2.69(m,3H),2.13-2.27(m,1H),1.96-2.09(m,1H),1.85-1.96(m,1H),1.51-1.73(m,2H),1.21-1.51(m,2H)
LC-MS(ESIPOS)：401.1(M+)
diastereomer 2 of C11:
1HNMR(300MHz,DMSO-d6)ppm：7.78(d,1H)7.66(d,1H)7.17-7.53(m,5H)6.68(d,1H)5.63(d,1H)4.72-5.09(m,1H)3.78(s,3H)2.71-3.04(m,5H)1.10-2.07(m,6H)
LC-MS(ESIPOS)：401.1(M+).
preparation of (3R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetyloxy) -1- (2-oxo-2- (thiazol-2-yl) ethyl) -1-azoniabicyclo [2.2.2] -octane trifluoroacetate (diastereomer 1 of C12):
2-bromo-1- (thiazol-2-yl) ethanone (18.4mg, 0.09mmol) was added to a solution of methyl 3- (2-oxo-1-phenyl-2- ((R) -quinuclidin-3-yloxy) -ethylamino) thiophene-2-carboxylate (diastereomer 1 of C11) (35.7mg, 0.09mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight, and then the solvent was removed under reduced pressure. The crude product was purified by preparative HPLC to give (3R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetyloxy) -1- (2-oxo-2- (thiazol-2-yl) ethyl) -1-azocationic bicyclo [2.2.2] octane trifluoroacetate (16mg, 28% yield).
1HNMR(300MHz,DMSO-d6)ppm8.38(d,1H)8.23(d,1H)7.76(d,1H)7.66(d,1H)7.28-7.58(m,5H)6.73(d,1H)5.66-5.75(m,1H)5.25(s,1H)5.14(s,2H)4.00-4.19(m,1H)3.78(s,3H)3.63-3.75(m,3H)3.41(m,2H)2.33-2.46(m,1H)1.85-2.13(m,4H)；
LC-MS(ESIPOS)：526.13(M+).
Example 5
Preparation of (3R) -1- (2- (4-hydroxyphenyl) -2-oxyethyl) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetyloxy) -1-azoniabicyclo [2.2.2] octane bromide (C13)
Alternative preparation of 2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetic acid (I10):
a mixture of phenylboronic acid (286mg, 2.35mmol), methyl 3-aminothiophene-2-carboxylate (369mg, 2.35mmol) and 2-oxoacetic acid hydrate (216mg, 2.35mmol) in acetonitrile (20ml) was stirred at room temperature for 2 hours. The reaction was filtered and the precipitate was washed with little acetonitrile. The solution was concentrated under reduced pressure and the precipitate was collected again by filtration and washed with a little acetonitrile. The combined precipitate was dried under vacuum overnight to give 2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetic acid (558mg, 82% yield).
Alternative preparation of methyl 3- (2-oxo-1-phenyl-2- ((R) -quinuclidin-3-yloxy) ethylamino) thiophene-2-carboxylate (C11):
2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetic acid (I10) (700mg, 2.40mmol), 1H-benzo [ d ] in dry THF (20ml) was stirred at room temperature][1,2,3]A mixture of triazol-1-ol (487mg, 3.60mmol), (R) -quinuclidin-3-hydroxy (458mg, 3.60mmol) and DCC (744mg, 3.60mmol) was left overnight. Evaporation of THF in DCM and 2MK2CO3The crude product was partitioned between. The organic phase was collected over Na2SO4Dried, filtered and evaporated to dryness. The crude product was purified by flash chromatography (DCM/MeOH ═ 95/5) to afford methyl 3- (2-oxo-1-phenyl-2- ((R) -quinuclidin-3-yloxy) ethylamino) thiophene-2-carboxylate (302mg, 31.4% yield).
Preparation of (3R) -1- (2- (4-hydroxyphenyl) -2-oxyethyl) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetyloxy) -1-azocationic bicyclo [2.2.2] octane bromide (C13):
2-bromo-1- (4-hydroxyphenyl) ethanone (19.9mg,0.09mmol) was added to a solution of methyl 3- (2-oxo-1-phenyl-2- ((R) -quinuclidin-3-yloxy) ethylamino) thiophene-2-carboxylate (C11) (37mg, 0.09mmol) in acetonitrile (2 ml). The reaction was stirred at room temperature overnight and Et was added2O (1ml), and the product was collected by suction filtration to give (3R) -1- (2- (4-hydroxyphenyl) -2-oxyethyl) -3- (2- (2- (methoxycarbonyl) -thiophen-3-ylamino) -2-phenylacetyloxy) -1-azoniabicyclo- [2.2.2]Octane bromide (36.7mg, 64.5% yield).
1HNMR(300MHz,DMSO-d6)ppm7.80-7.92(m,2H),7.72-7.80(m,1H),7.62-7.69(m,1H),7.29-7.57(m,5H),6.83-6.99(m,2H),6.72-6.74(d,1H),5.66-5.72(d,1H),5.17-5.31(m,1H),4.99-5.05(s,2H),3.97-4.21(m,1H),3.78(s,3H),3.36-3.74(m,5H),2.15-2.25-2.34-2.46(m,1H),1.38-2.12(m,4H)；
LC-MS(ESIPOS)：535.39(M+).
Example 6
Preparation of (3R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetyloxy) -1- (2-oxo-2- (thiophen-3-yl) ethyl) -1-azoniabicyclo [2.2.2] -octane bromide (C14)
2-bromo-1- (thiophen-3-yl) ethanone (25.6mg, 0.12mmol) was added to a solution of methyl 3- (2-oxo-1-phenyl-2- ((R) -quinuclidin-3-yloxy) -ethylamino) thiophene-2-carboxylate (C11) (50mg, 0.12mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight and then the solvent was evaporated in vacuo. With Et2O the residue was pulverized and then subjected to preparative HPLC (eluent: CH)3CN/H2O) purification to obtain (3R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetyloxy) -1- (2-oxo-2- (thiophen-3-yl) ethyl) -1-azocationic bicyclo [2.2.2]Octane bromide (44.3mg, 58.6% yield).
1HNMR(300MHz,DMSO-d6)ppm8.42-8.70(m,1H),7.27-7.87(m,9H),6.71-6.73(d,1H),5.66-5.72(d,1H),5.25(br.s.,1H),4.98-5.03(s,2H),4.00-4.21(m,1H),3.78(s,3H),3.46-3.73(m,5H),2.14-2.24-2.34-2.45(m,1H),1.67-2.14(m,4H)；
LC-MS(ESIPOS)：525.33(M+).
Example 7
Preparation of (3R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetyloxy) -1- (3-phenoxypropyl) -1-azoniabicyclo [2.2.2] octane bromide (C15)
(3-Bromopropoxy) benzene (19.7. mu.l, 0.12mmol) was added to a solution of methyl 3- (2-oxo-1-phenyl-2- ((R) -quinuclidin-3-yloxy) -ethylamino) thiophene-2-carboxylate (C11) (50mg, 0.12mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight. Evaporation of the solvent, and application of Et2O the residue was pulverized and then subjected to preparative HPLC (eluent: CH)3CN/H2O) purification to obtain (3R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetyloxy) -1- (3-phenoxypropyl) -1-azoniabicyclo [2.2.2]Octane bromide (25.3mg, 32.9% yield).
1HNMR(300MHz,DMSO-d6)ppm7.78-7.83(d,1H),7.61-7.71(m,1H),7.21-7.57(m,6H),6.83-7.03(m,4H),6.65-6.73(d,1H),5.59-5.68(d,1H),5.02-5.26(m,1H),3.82-4.19(m,4H),3.78(s,3H),3.34-3.73(m,5H),2.81-3.14(m,1H),1.43-2.43(m,7H)；
LC-MS(ESIPOS)：535.43(M+).
Example 8
Preparation of (3R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetyloxy) -1- (2-oxo-2- (pyridin-2-yl) ethyl) -1-azoniabicyclo [2.2.2] -octane 2,2, 2-trifluoroacetate anion (C16)
2-bromo-1- (pyridin-2-yl) ethanone hydrobromic acid (35.1mg, 0.12mmol) was added to a solution of methyl 3- (2-oxo-1-phenyl-2- ((R) -quinuclidin-3-yloxy) ethylamino) thiophene-2-carboxylate (C11) (50mg, 0.12mmol) and TEA (17.4. mu.l, 0.12mmol) in EtOAc (2 ml). The reaction was stirred at room temperature for 2 days, then the solvent was removed in vacuo. With Et2The crude product was crushed and filtered. By preparative HPLC (eluent: CH)3CN/H2O), then purified by preparative HPLC (eluent: CH (CH)3CN/H2O/TFA) was further purified to obtain the title compound (15.3mg, 16.4% yield).
1HNMR(300MHz,DMSO-d6)ppm8.66-8.83(m,1H),7.94-8.25(m,2H),7.70-7.87(m,2H),7.61-7.70(m,1H),7.28-7.57(m,5H),6.72-6.74(d,1H),5.67-5.72(d,1H),5.24-5.30(m,1H),5.22-5.29(s,2H),4.01-4.25(m,1H),3.78(s,3H),3.51-3.92(m,5H),2.16-2.24-2.33-2.46(m,1H),1.40-2.13(m,4H)；
LC-MS(ESIPOS)：520.39(M+).
Example 9
Preparation of (3R) -1- (4-fluorophenethyl) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetyloxy) -1-azoniabicyclo- [2.2.2] octane 2,2, 2-trifluoroacetate (C17)
Reacting 1- (2-bromoethyl) -4-fluorobenzene (25)4 μ l, 0.12mmol) was added to a solution of methyl 3- (2-oxo-1-phenyl-2- ((R) -quinuclidin-3-yloxy) -ethylamino) thiophene-2-carboxylate (C11) (50mg, 0.12mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight. Evaporation of the solvent, and application of Et2And O, crushing the residue. First by preparative HPLC (eluent: CH)3CN/H2O), then purified by preparative HPLC (eluent: CH (CH)3CN/H2O/TFA) to obtain (3R) -1- (4-fluorophenethyl) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2-phenylacetyloxy) -1-azoniabicycloo- [2.2.2]Octane 2,2, 2-trifluoroacetate salt (15mg, 18.9% yield).
1HNMR(300MHz,DMSO-d6)ppm7.78-7.84(d,1H),7.62-7.72(m,1H),7.03-7.57(m,9H),6.66-6.73(d,1H),5.60-5.68(d,1H),5.00-5.35(m,1H),3.82-3.98(m,1H),3.78-3.79(s,3H),3.32-3.62(m,6H),2.76-3.18(m,3H),2.10-2.20-2.32-2.42(m,1H),1.36-2.09(m,4H)；
LC-MS(ESIPOS)：523.39(M+).
Example 10
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2- (thiophen-2-yl) ethyl) -1-azoniabicyclo [2.2.2] octane bromide (C20)
Preparation of 2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetic acid (I18):
a benzene ring [ b ]]Thien-3-ylboronic acid (387mg, 2.17mmol), aniline (202mg, 2.17mmol) and 2-oxyacetic acid hydrate (200mg, 2.17mmol) were dissolved in acetonitrile (12ml), followed by stirring at 100 ℃ under microwave irradiation for 1 hour. The solvent was evaporated and the residue was dissolved in EtOAc and NaHCO3And (5) saturated washing. Adding 2N HCl to the aqueous phase until the pH is about 7, and using EtOAc extraction of the product. With Na2SO4The organic phase was dried, filtered and evaporated to give 2- (benzo [ b ]]Thiophen-3-yl) -2- (phenylamino) acetic acid (295mg, 48% yield).
Preparation of (R) -quinuclidin-3-yl 2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetate (C19):
2- (benzo [ b ]]Thiophen-3-yl) -2- (phenylamino) acetic acid (I18) (295mg, 1.04mmol), (R) -quinuclidin-3-hydroxy (159mg, 1.25mmol), 1H-benzo [ d][1,2,3]Triazol-1-ol hydrate (191mg, 1.25mmol) and DCC (258mg, 1.25mmol) were dissolved in dry THF and stirred at room temperature for 15 hours. The solvent was evaporated and the crude product was dissolved in EtOAc and washed with NaHCO3Water and brine. With Na2SO4The organic phase was dried, filtered and evaporated in vacuo. The crude product was purified by flash chromatography (DCM/MeOH ═ 9/1) to give (R) -quinuclidin-3-yl 2- (benzo [ b)]Thiophen-3-yl) -2- (phenylamino) acetate (180mg, 44% yield).
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2- (thiophen-2-yl) ethyl) -1-azocationic bicyclo [2.2.2] octane bromide (C20):
(R) -Quinuclidin-3-yl 2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetate (C19) (90mg, 0.23mmol) was dissolved in EtOAc (3ml) and 2-bromo-1- (thiophen-2-yl) ethanone (51.7mg, 0.25mmol) was added. The mixture was stirred at room temperature overnight. The crude product was purified by flash chromatography (DCM/MeOH ═ 9/1) to give (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2- (thiophen-2-yl) ethyl) -1-azocationic bicyclo [2.2.2] octane bromide (50mg, 36.5% yield).
1HNMR(300MHz,DMSO-d6)ppm8.21(td,1H)7.99-8.18(m,3H)7.89(d,1H)7.30-7.54(m,3H)7.12(t,2H)6.71-6.91(m,2H)6.63(t,1H)6.35-6.52(m,1H)5.69-5.92(m,1H)5.14-5.35(m,1H)4.81-5.09(m,2H)3.98-4.24(m,1H)3.79(d,1H)3.54-3.67(m,3H)3.12-3.42(m,1H)2.30-2.42(m,1H)1.52-2.12(m,4H)；
LC-MS(ESIPOS)：401.1(M+).
Example 11
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane trifluoroacetate (C21)
2-chloro-1-phenylethanone (39.0mg, 0.25mmol) was added to a solution of (R) -quinuclidin-3-yl 2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) -acetate (C19) (90mg, 0.23mmol) in EtOAc (3 ml). The reaction was stirred at room temperature overnight. The solvent was evaporated and the crude product was purified by preparative HPLC to give (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azocationic bicyclo [2.2.2] octane trifluoroacetate (37.2mg, 26% yield).
1HNMR(300MHz,DMSO-d6)ppm8.08-8.24(m,1H)7.85-8.08(m,4H)7.69-7.84(m,1H)7.56-7.69(m,2H)7.34-7.54(m,2H)7.12(t,2H)6.81(dd,2H)6.63(t,1H)6.30-6.55(m,1H)5.74-5.91(m,1H)5.18-5.34(m,1H)5.15(s,1H)5.06(s,1H)4.00-4.17(m,1H)3.80(d,1H)3.43-3.70(m,3H)3.23-3.43(m,1H)2.37(t,1H)1.50-2.17(m,4H)；
LC-MS(ESIPOS)：401.1(M+).
Example 12
Preparation of (R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) -acetoxy) -1- (2- (4-hydroxyphenyl) -2-oxyethyl) -1-azoniabicyclo [2.2.2] octane bromide (C22)
2-bromo-1- (4-hydroxyphenyl) ethanone (54.8mg, 0.25mmol) was added portionwise to (R) -quinuclidin-3-yl 2- (benzo [ b ] b]Thien-3-yl) -2- (phenylamino) acetate (C19) (100mg, 0.25mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight. The precipitate was filtered and washed with ethyl acetate, then with CH3CN pulverizing it to obtain (R) -3- (2- (benzo [ b ]]Thien-3-yl) -2- (phenylamino) acetoxy) -1- (2- (4-hydroxyphenyl) -2-oxyethyl) -1-azoniabicyclo [2.2.2]Octane bromide (101mg, 65.3% yield).
1HNMR(300MHz,DMSO-d6)ppm9.84-11.39(m,1H),8.07-8.21(m,1H),7.97-8.07(m,1H),7.76-7.95(m,3H),7.34-7.54(m,2H),7.02-7.20(m,2H),6.85-6.96(m,2H),6.72-6.85(m,2H),6.53-6.68(m,1H),6.46(d,1H),5.65-6.02(m,1H),5.15-5.37(m,1H),4.82-5.09(m,1H),3.98-4.20(m,1H),3.43-3.88(m,5H),2.01-2.13-2.33-2.42(m,1H),1.32-2.03(m,4H)；
LC-MS(ESIPOS)：527.24(M+).
Example 13
Preparation of (R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2- (pyridin-2-ylamino) ethyl) -1-azoniabicyclo [2.2.2] -octane chloride (C23)
2-chloro-N- (pyridin-2-yl) acetamide (43.5mg, 0.25mmol) was added portionwise to (R) -quinuclidin-3-yl 2- (benzo [ b)]Thien-3-yl) -2- (phenylamino) acetate (C19) (100mg, 0.25mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight. The solvent is evaporated and the residue is purified by preparative HPLC (eluent: CH)3CN，H2O), purifying the residue. By i-Pr2O pulverizing the product, filtering and vacuum drying at room temperature to obtain (R) -3- (2- (benzene)And [ b ]]Thien-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2- (pyridin-2-ylamino) ethyl) -1-azoniabicyclo [2.2.2]Octane chloride (31mg, 21.6% yield).
1HNMR(300MHz,DMSO-d6)ppm11.04-11.10(s,1H),8.32-8.45(m,1H),8.07-8.18(m,1H),7.96-8.07(m,2H),7.82-7.95(m,1H),7.90(s,1H),7.34-7.50(m,2H),7.22(ddd,1H),7.03-7.17(m,2H),6.71-6.87(m,2H),6.61(t,1H),6.47(d,1H),5.76-5.86(m,1H),5.10-5.32(m,1H),4.20-4.30(s,2H),3.97-4.12(m,1H),3.43-3.90(m,4H),3.09-3.25(m,1H),2.03-2.12-2.30-2.40(m,1H),1.32-2.02(m,4H)；
LC-MS(ESIPOS)：527.24(M+).
Example 14
Preparation of (R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2- (thiophen-3-yl) ethyl) -1-azoniabicyclo [2.2.2] octane bromide (C24)
2-bromo-1- (thiophen-3-yl) ethanone (52.2mg, 0.25mmol) was added portionwise to (R) -quinuclidin-3-yl 2- (benzo [ b ] b]Thien-3-yl) -2- (phenylamino) acetate (C19) (100mg, 0.25mmol) in EtOAc (2 ml). The reaction was stirred at room temperature for 3 hours. The precipitate was filtered, washed with EtOAc, and purified by preparative HPLC (eluent: CH)3CN，H2O) purification to obtain (R) -3- (2- (benzo [ b ]]Thien-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2- (thien-3-yl) ethyl) -1-azacationic bicyclo [2.2.2]Octane bromide (32mg, 21.0% yield).
1HNMR(300MHz,DMSO-d6)ppm8.59-8.64(dd,1H),8.11-8.15(d,1H),8.03(d,1H),7.89-7.91(s,1H),7.74(dd,1H),7.54-7.58(dd,1H),7.34-7.52(m,2H),7.04-7.20(m,2H),6.75-6.91(m,2H),6.56-6.70(m,1H),6.46(d,1H),5.74-5.89(m,1H),5.16-5.32(m,1H),4.77-5.06(m,1H),3.95-4.21(m,1H),3.32-3.86(m,5H),2.05-2.13-2.31-2.42(m,1H),1.31-2.02(m,4H)；
LC-MS(ESIPOS)：516.98(M+).
Example 15
Preparation of (R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2- (thiazol-2-yl) ethyl) -1-azoniabicyclo [2.2.2] octane bromide (C25)
2-bromo-1- (thiazol-2-yl) ethanone (52.5mg, 0.25mmol) was added portionwise to (R) -quinuclidin-3-yl 2- (benzo [ b)]Thien-3-yl) -2- (phenylamino) acetate (C19) (100mg, 0.25mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight. The precipitate was filtered, washed with EtOAc and purified by preparative HPLC (eluent: CH)3CN，H2O) purification to obtain (R) -3- (2- (benzo [ b ]]Thien-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2- (thiazol-2-yl) ethyl) -1-azoniabicyclo [2.2.2]Octane bromide (42mg, 27.5% yield).
1HNMR(300MHz,DMSO-d6)ppm8.35-8.43(m,1H),8.23-8.25(d,1H),8.07-8.17(m,1H),7.98-8.07(m,1H),7.90-7.91(s,1H),7.35-7.54(m,2H),7.02-7.20(m,2H),6.73-6.88(m,2H),6.56-6.70(m,1H),6.46(d,1H),5.77-5.87(m,1H),5.20-5.29(m,1H),4.92-5.20(m,1H),3.97-4.23(m,1H),3.32-3.90(m,5H),2.05-2.16-2.30-2.42(m,1H),1.31-2.04(m,4H)；
LC-MS(ESIPOS)：518.21(M+).
Example 16
Preparation of (R) - ((R) -quinuclidin-3-yl) 2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetate (diastereomer 1 of C19) and (S) - ((R) -quinuclidin-3-yl) 2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetate (diastereomer 2 of C19)
Stirring 2- (benzo [ b ] at room temperature]Thien-3-yl) -2- (phenylamino) acetic acid (I18) (2.75g, 9.71mmol), 1H-benzo [ d][1,2,3]A solution of triazol-1-ol hydrate (1.78g, 11.6mmol) and N, N' -methanadiylidene dicyclohexylamine (2.40g, 11.6mmol) in dry THF (80ml) for 1 hour. (R) -quinuclidin-3-hydroxy (1.48g, 11.6mmol) was added and stirring was maintained overnight. The solvent was evaporated, and the reaction mixture was washed with EtOAc and saturated NaHCO3The residue was partitioned between, the organic layer was washed with brine, over Na2SO4Dried, filtered and evaporated. By flash Chromatography (CH)3CN/MeOH＝75/25+0.2％NH4OH), purifying the residue by first collecting (R) - ((R) -quinuclidin-3-yl) 2- (benzo [ b)]Thien-3-yl) -2- (phenylamino) acetate (1.1g, 28.9% yield, diastereomer 1 of C19), then (R) -quinuclidin-3-yl 2- (benzo [ b ] was collected]Thien-3-yl) -2- (phenylamino) acetate (1.1g, 28.9% yield, mixture of diastereomers) and finally (S) - ((R) -quinuclidin-3-yl) 2- (benzo [ b ]]Thiophen-3-yl) -2- (phenylamino) acetate (0.4g, 10.5% yield, diastereomer 2 of C19).
Diastereomer 1 of C19:1HNMR(300MHz,DMSO-d6)ppm8.06-8.20(m,1H),7.95-8.06(m,1H),7.86(s,1H),7.33-7.50(m,2H),7.01-7.16(m,2H),6.73-6.85(m,2H),6.55-6.67(m,1H),6.38(d,1H),5.72(d,1H),4.61-4.76(m,1H),2.92(ddd,1H),2.55-2.70(m,3H),1.94-2.18(m,2H),1.80-1.94(m,1H),1.31-1.62(m,3H),1.03-1.31(m,1H)；
LC-MS(ESIPOS)：393.00(M+).
diastereomer 2 of C19:1HNMR(300MHz,DMSO-d6)ppm8.13(dd,1H),7.92-8.06(m,1H),7.83(s,1H),7.43(m,2H),6.95-7.18(m,2H),6.67-6.85(m,2H),6.49-6.66(m,1H),6.37(d,1H),5.71(d,1H),4.55-4.93(m,1H),2.98-3.16(m,1H),2.53-2.61(m,4H),2.32-2.48(m,1H),1.59-1.71(m,1H),1.28-1.58(m,3H),0.89-1.15(m,1H)；
LC-MS(ESIPOS)：393.22(M+).
example 17
Preparation of (R) -3- ((R) -2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2- (thiophen-2-yl) ethyl) -1-azoniabicyclo [2.2.2] octane chloride (diastereomer 1 of C20)
2-chloro-1- (thiophen-2-yl) ethanone (32.7mg, 0.20mmol) was added to a solution of (R) - ((R) -quinuclidin-3-yl) 2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetate (diastereomer 1 of C19) (80mg, 0.20mmol) in EtOAc (3 ml). The reaction was stirred at room temperature overnight. Diethyl ether (10ml) was added and the precipitate was collected and dried in vacuo to give (R) -3- ((R) -2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2- (thiophen-2-yl) ethyl) -1-azocationic bicyclo [2.2.2] octane chloride (101mg, 90% yield).
1HNMR(300MHz,DMSO-d6)ppm8.21(dd,1H),7.98-8.15(m,3H),7.91(s,1H),7.37-7.56(m,2H),7.35(dd,1H),7.01-7.19(m,2H),6.82(m,2H),6.57-6.68(m,1H),6.49(d,1H),5.83(d,1H),5.17-5.32(m,1H),5.00(s,2H),3.94-4.21(m,1H),3.46-3.78(m,4H),3.33-3.44(m,1H),2.31-2.39(m,1H),1.65-2.14(m,4H)；
LC-MS(ESIPOS)：517.19(M+).
Example 18
Preparation of (R) -3- ((R) -2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo- [2.2.2] octane chloride (diastereomer 1 of C21)
2-chloro-1-phenylethanone (98mg, 0.64mmol) was added to (R) - ((R) -quinuclidin-3-yl) 2- (benzo [ b [ -b ])]Thien-3-yl) -2- (phenylamino) acetate (diastereomer 1 of C19) (250mg, 0.64mmol) in CH3CN (3 ml). The reaction was stirred at room temperature overnight, then the solvent was evaporated, and Et was used2And O, crushing the residue. By preparative HPLC (eluent: CH)3CN/H2O), purifying the crude product to obtain (R) -3- ((R) -2- (benzo [ b ]]Thien-3-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2]Octane chloride (210mg, 60.3% yield).
1HNMR(300MHz,DMSO-d6)ppm8.12(d,1H),8.04(d,1H),7.92-8.00(m,2H),7.90(s,1H),7.71-7.81(m,1H),7.55-7.67(m,2H),7.46-7.53(m,1H),7.37-7.46(m,1H),7.06-7.19(m,2H),6.75-6.88(m,2H),6.58-6.69(m,1H),6.48(d,1H),5.84(d,1H),5.17-5.36(m,1H),4.96-5.16(m,2H),3.98-4.20(m,1H),3.45-3.74(m,4H),3.33-3.45(m,1H),2.32-2.42(m,1H),1.69-2.22(m,4H)；
LC-MS(ESIPOS)：511.11(M+).
Example 19
Preparation of (R) -3- ((S) -2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2- (thiophen-2-yl) ethyl) -1-azoniabicyclo- [2.2.2] octane chloride (diastereomer 2 of C20)
To a solution of (S) - ((R) -quinuclidin-3-yl) 2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetate (diastereomer 2 of C19) (62mg, 0.16mmol) in ethyl acetate (3ml) was added 2-chloro-1- (thiophen-2-yl) ethanone (25.4mg, 0.16mmol) and the reaction was stirred at room temperature overnight. Diethyl ether (10ml) was added, and the precipitate was filtered and dried in vacuo to give (R) -3- ((S) -2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2- (thiophen-2-yl) ethyl) -1-azocationic bicyclo [2.2.2] octane chloride (75mg, 86% yield).
1HNMR(300MHz,DMSO-d6)ppm8.22(dd,1H),8.15(dd,1H),8.10(dd,1H),8.03(dd,1H),7.92(s,1H),7.38-7.63(m,2H),7.36(dd,1H),7.00-7.20(m,2H),6.72-6.90(m,2H),6.63(t,1H),6.49(d,1H),5.75-5.84(m,1H),5.15-5.40(m,1H),4.89-5.15(m,2H),4.12(dd,1H),3.82(d,1H),3.45-3.72(m,4H),2.03-2.14(m,1H),1.78-2.03(m,2H),1.56-1.77(m,1H),1.29-1.56(m,1H)；
LC-MS(ESIPOS)：517.21(M+).
Example 20
Preparation of (R) -3- ((S) -2- (benzo [ b ] thiophen-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo- [2.2.2] octane chloride (diastereomer 2 of C21)
(S) - ((R) -quinuclidin-3-yl) 2- (benzo [ b) was stirred at room temperature]Thien-3-yl) -2- (phenylamino) acetate (diastereomer 2 of C19) (230mg, 0.586mmol) and 2-chloro-1-phenylethanone (91mg, 0.586mmol) in dry CH3CN (2ml) for 24 hours. The solvent is then evaporated and the residue is purified by preparative HPLC (eluent: CH)3CN/H2O), purifying the residue to obtain (R) -3- ((S) -2- (benzo [ b ]]Thien-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2]OctaneChloride (102mg, 31.8% yield).
1HNMR(300MHz,DMSO-d6)ppm8.16(d,1H),7.96-8.09(m,3H),7.93(s,1H),7.68-7.84(m,1H),7.54-7.68(m,2H),7.25-7.54(m,2H),6.96-7.24(m,2H),6.71-6.92(m,2H),6.56-6.71(m,1H),6.49(d,1H),5.68-5.94(m,1H),5.22-5.38(m,1H),5.01-5.21(m,2H),4.12(dd,1H),3.83(d,1H),3.47-3.74(m,4H),2.03-2.17(m,1H),1.79-2.03(m,2H),1.56-1.79(m,1H),1.31-1.55(m,1H)；
LC-MS(ESIPOS)：511.26(M+).
Example 21
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-7-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] -octane chloride (C28)
Preparation of 2- (benzo [ b ] thiophen-7-yl) -2- (phenylamino) acetic acid (I26):
stirring at room temperature in CH3Benzo [ b ] in CN (20ml)]A mixture of thiophen-7-ylboronic acid (300mg, 1.68mmol), aniline (157mg, 1.68mmol) and 2-oxoacetic acid hydrate (155mg, 1.68mmol) was stirred for 2 hours. The solvent was evaporated and the crude product was purified by flash chromatography (DCM/MeOH ═ 9/1) to afford 2- (benzo [ b)]Thiophen-7-yl) -2- (phenylamino) acetic acid (303mg, 63.5% yield).
Preparation of (R) -quinuclidin-3-yl 2- (benzo [ b ] thiophen-7-yl) -2- (phenylamino) acetate (C27):
stirring 2- (benzo [ b ] at room temperature]Thien-7-yl) -2- (phenylamino) acetic acid (I26) (303mg, 1.07mmol), (R) -quinuclidin-3-hydroxy (136mg, 1.07mmol), DCC (221mg, 1.07mmol) and HOBT (164mg, 1.07mmol) in THF (20ml) for 3 days. THF was evaporated, the crude product was taken up in EtOAc and taken up with 2MK2CO3And then washed 2 times with brine. In Na2SO4The organic phase was dried, filtered and evaporated to dryness. Flash chromatography (EtOAc/MeOH 9/1 to EtOAc/MeOH 9/1+ 0.5% NH4OH), purifying the crude product to obtain (R) -quinuclidin-3-yl 2- (benzo [ b)]Thiophen-7-yl) -2- (phenylamino) acetate (180mg, 42.9% yield).
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-7-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane chloride (C28):
2-chloro-1-phenylethanone (35.4mg, 0.23mmol) was added to (R) -quinuclidin-3-yl 2- (benzo [ b)]Thien-7-yl) -2- (phenylamino) acetate (C27) (90mg, 0.23mmol) in EtOAc (3 ml). The reaction was stirred at room temperature overnight. Remove the solvent in vacuo and use Et2And O, crushing the crude product. By preparative HPLC (eluent: CH)3CN/H2O), purifying the compound to obtain (3R) -3- (2- (benzo [ b ])]Thien-7-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2]Octane chloride (26.6mg, 21.2% yield).
1HNMR(300MHz,DMSO-d6)ppm7.86-8.08(m,4H),7.69-7.84(m,2H),7.54-7.69(m,3H),7.39-7.54(m,2H),6.98-7.18(m,2H),6.68-6.79(m,2H),6.56-6.66(m,1H),6.53(d,1H),5.49-5.66(m,1H),5.22-5.34(m,1H),4.85-5.22(m,1H),3.97-4.28(m,1H),3.32-3.91(m,5H),2.04-2.13-2.34-2.45(m,1H),1.23-2.01(m,4H)；
LC-MS(ESIPOS)：511.26(M+).
Example 22
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-7-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2- (thiophen-2-yl) ethyl) -1-azoniabicyclo [2.2.2] octane chloride (C29)
2-chloro-1- (thiophen-2-yl) ethanone (36.8mg, 0.23mmol) was added to (R) -quinuclidin-3-yl 2- (benzo [ b ] b]Thien-7-yl) -2- (phenylamino) acetate (C27) (90mg, 0.23mmol) in acetonitrile (3 ml). The reaction was stirred at room temperature overnight. Remove the solvent in vacuo and use Et2And O, crushing the crude product. First by preparative HPLC (eluent: CH)3CN/H2O), then the compound was purified by flash chromatography (DCM/MeOH ═ 95/5) to obtain (3R) -3- (2- (benzo [ b ] b)]Thien-7-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2- (thiophen-2-yl) ethyl) -1-azoniabicyclo [2.2.2]Octane chloride (20mg, 16% yield).
1HNMR(300MHz,DMSO-d6)ppm8.15-8.30(m,1H),8.05(d,1H),7.84-7.93(m,1H),7.78(d,1H),7.51(d,1H),7.39-7.63(m,2H),7.34(dd,1H),6.98-7.13(m,2H),6.66-6.77(m,2H),6.56-6.64(m,1H),6.53(d,1H),5.42-5.68(m,1H),5.17-5.38(m,1H),4.74-5.14(m,1H),3.96-4.26(m,1H),3.29-3.92(m,5H),2.33-2.44(m,1H),1.32-2.16(m,4H)；
LC-MS(ESIPOS)：517.19(M+).
Example 23
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-2-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (C32)
Preparation of 2- (benzo [ b ] thiophen-2-yl) -2- (phenylamino) acetic acid (I30):
a mixture of benzo [ b ] thiophen-2-ylboronic acid (500mg, 2.81mmol), aniline (240. mu.l, 2.81mmol) and 2-oxoacetic acid hydrate (259mg,2.81mmol) in acetonitrile (20ml) was stirred at room temperature for 3 days. The solvent was evaporated to dryness and the crude product was used as such in the next step.
Preparation of (R) -quinuclidin-3-yl 2- (benzo [ b ] thiophen-2-yl) -2- (phenylamino) acetate (C31):
2- (benzo [ b ] in THF (30ml) was stirred at room temperature]Thien-2-yl) -2- (phenylamino) acetic acid (I30) (796mg, 2.81mmol), (R) -quinuclidin-3-hydroxy (429mg, 3.37mmol), HOBT (516mg, 3.37mmol) and DCC (696mg, 3.37mmol) were mixed overnight. THF was evaporated in EtOAc and 1MK2CO3The crude product was partitioned between. In Na2SO4The organic phase was dried, filtered and evaporated to dryness. The crude product was purified by flash chromatography (DCM/MeOH ═ 98/2) to give (R) -quinuclidin-3-yl 2- (benzo [ b)]Thiophen-2-yl) -2- (phenylamino) acetate (361mg, 32.7% yield).
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-2-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (C32):
2-chloro-1-phenylethanone (59.1mg, 0.38mmol) was added to (R) -quinuclidin-3-yl 2- (benzo [ b)]Thien-2-yl) -2- (phenylamino) acetate (C31) (150mg, 0.38mmol) in EtOAc (5ml) and stirring the reaction at room temperature for 20 h. EtOAc is removed in vacuo and Et2O (2ml) pulverized the solid. The compound was purified by preparative HPLC to give (3R) -3- (2- (benzo [ b ]]Thien-2-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2]Octane 2,2, 2-trifluoroacetate salt (42.8mg, 17.9% yield).
1HNMR(300MHz,DMSO-d6)ppm7.90-8.06(m,3H),7.81-7.89(m,1H),7.69-7.81(m,1H),7.53-7.67(m,3H),7.29-7.45(m,2H),7.05-7.19(m,2H),6.77-6.89(m,2H),6.61-6.72(m,1H),6.55(br.s.,1H),5.79-5.82(s,1H),5.26-5.43(m,1H),5.13-5.17(s,2H),4.01-4.27(m,1H),3.53-3.87(m,5H),2.23-2.33-2.35-2.45(m,1H),1.72-2.21(m,4H)；
LC-MS(ESIPOS)：511.21(M+).
Example 24
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-2-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2- (thiazol-2-yl) ethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (C33)
2-bromo-1- (thiazol-2-yl) ethanone (42.0mg, 0.20mmol) was added to (R) -quinuclidin-3-yl 2- (benzo [ b)]Thien-2-yl) -2- (phenylamino) acetate (C31) (80mg, 0.20mmol) in acetonitrile (2 ml). The reaction was stirred at room temperature overnight, then a second portion of 2-bromo-1- (thiazol-2-yl) ethanone (12.6mg, 0.06mmol) was added and stirring was maintained for an additional 4 hours. Evaporation of the solvent, and application of Et2O the crude product was pulverized and then purified by preparative HPLC to obtain (3R) -3- (2- (benzo [ b)]Thien-2-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2- (thiazol-2-yl) ethyl) -1-azoniabicyclo [2.2.2]Octane 2,2, 2-trifluoroacetate salt (24mg, 18.6% yield).
1HNMR(300MHz,DMSO-d6)ppm8.38-8.39(d,1H),8.23-8.24(d,1H),7.89-8.00(m,1H),7.77-7.89(m,1H),7.52-7.66(m,1H),7.29-7.46(m,2H),7.00-7.20(m,2H),6.80-6.91(m,2H),6.60-6.71(m,1H),6.55(br.s.,1H),5.78-5.81(s,1H),5.24-5.35(m,1H),5.15-5.19(s,2H),4.10-4.27(m,1H),3.41-3.88(m,5H),2.23-2.31-2.36-2.45(m,1H),1.73-2.17(m,4H)；
LC-MS(ESIPOS)：518.18(M+).
Example 25
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (methyl- (phenyl) amino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] -octane chloride (C36)
Preparation of 2- (benzo [ b ] thiophen-3-yl) -2- (methyl (phenyl) amino) acetic acid (I34):
a mixture of benzo [ b ] thiophen-3-ylboronic acid (300mg, 1.68mmol), N-methylaniline (184. mu.l, 1.68mmol) and 2-oxyacetic acid hydrate (155mg, 1.65mmol) in acetonitrile (20ml) was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography (DCM/MeOH ═ 95/5) to afford 2- (benzo [ b ] thiophen-3-yl) -2- (methyl (phenyl) amino) acetic acid (351mg, 70% yield).
Preparation of (R) -quinuclidin-3-yl 2- (benzo [ b ] thiophen-3-yl) -2- (methyl (phenyl) amino) acetate (C35):
2- (benzo [ b ] in dry THF (20ml) was stirred at room temperature]Thien-3-yl) -2- (methyl (phenyl) amino) acetic acid (I34) (351mg, 1.18mmol), (R) -quinuclidin-3-hydroxy (180mg, 1.42mmol), a mixture of DCC (292mg, 1.42mmol) and HOBT (217mg, 1.42mmol) was left overnight. Evaporation of the solvent, and reaction between EtOAc and 2MK2CO3The crude product was partitioned between. With 2MK2CO3And the organic phase was washed with brine over Na2SO4Dried, filtered and evaporated to dryness. By flash chromatography (MeCN/MeOH 8/2+ 0.2% NH4OH), purifying the crude product to obtain (R) -quinuclidin-3-yl 2- (benzo [ b)]Thiophen-3-yl) -2- (methyl (phenyl) amino) acetate (224mg, 46.7% yield).
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (methyl- (phenyl) amino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] -octane chloride (C36):
2-chloro-1-phenylethanone (42.6mg, 0.27mmol) was added to (R) -quinuclidin-3-yl 2- (benzo [ b)]Thien-3-yl) -2- (methyl (phenyl) amino) acetate (C35) (112mg, 0.27mmol) in acetonitrile (3 ml). The reaction was stirred at room temperature for 3 hours,the solvent was then evaporated in vacuo. With Et2O pulverizing the residue, filtering and drying to obtain (3R) -3- (2- (benzo [ b ]]Thien-3-yl) -2- (methyl (phenyl) amino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2]Octane chloride (112.2mg, 72.6% yield).
1HNMR(300MHz,DMSO-d6)ppm7.94-8.17(m,3H),7.82-7.94(m,1H),7.70-7.82(m,1H),7.55-7.67(m,3H),7.21-7.48(m,4H),7.06(dd,2H),6.80(td,1H),6.28-6.30(s,1H),5.30(s,2H),5.21-5.61(m,1H),4.05-4.39(m,1H),3.49-3.89(m,5H),2.72-2.74(s,3H),2.22-2.43(m,1H),1.94-2.16(m,2H),1.70-1.94(m,1H),1.37-1.70(m,1H)；
LC-MS(ESIPOS)：525.15(M+).
Example 26
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (benzylamino) -acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate anion (C39)
Preparation of 2- (benzo [ b ] thiophen-3-yl) -2- (benzylamino) acetic acid (I37):
benzo [ b ] in acetonitrile (20ml) was stirred at room temperature]Thien-3-ylboronic acid (300mg, 1.68mmol), phenylmethylamine (184. mu.l, 1.68mmol) and 2-oxoacetic acid hydrate (155mg, 1.68mmol) for a mixture of 2 hours. Remove the solvent and use Et2O/CH3CN (9/1) pulverizing the crude product to obtain 2- (benzo [ b ]]Thiophen-3-yl) -2- (benzylamino) acetic acid (303mg, 60.5% yield).
Preparation of (R) -quinuclidin-3-yl 2- (benzo [ b ] thiophen-3-yl) -2- (benzylamino) acetate (C38):
2-Aconitine in dry THF (20ml) was stirred at room temperature(benzo [ b ]]Thien-3-yl) -2- (benzylamino) acetic acid (I37) (303mg, 1.02mmol), (R) -quinuclidin-3-hydroxy (156mg, 1.22mmol), DCC (252mg, 1.22mmol) and HOBT (187mg, 1.22mmol) were mixed overnight. Evaporate the solvent and add in EtOAc and 2MK2CO3The crude product is partitioned with 2MK2CO3And the organic phase was washed with brine over Na2SO4Dried, filtered and evaporated to dryness. By flash Chromatography (CH)3CN/MeOH＝8/2+0.2％NH4OH), purifying the crude product to obtain (R) -quinuclidin-3-yl 2- (benzo [ b)]Thiophen-3-yl) -2- (benzylamino) acetate (53mg, 12.8% yield).
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (benzylamino) -acetoxy) -1- (2-oxo-2-phenylethyl) -1-azocationic bicyclo [2.2.2] octane 2,2, 2-trifluoroacetate anion (C39):
2-chloro-1-phenylethanone (20.1mg, 0.13mmol) was added to (R) -quinuclidin-3-yl 2- (benzo [ b)]Thien-3-yl) -2- (benzylamino) acetate (C38) (53mg, 0.13mmol) in CH3CN (3 ml). The reaction was stirred at room temperature for 5 hours, then a second portion of 2-chloro-1-phenylethanone (6.05mg, 0.04mmol) was added and stirring was maintained overnight. The reaction was heated at 80 ℃ for 30 minutes under microwave irradiation. The solvent was evaporated, and the crude product was purified by preparative HPLC to give (3R) -3- (2- (benzo [ b ]]Thien-3-yl) -2- (benzylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2]Octane 2,2, 2-trifluoroacetate anion (34.1mg, 34.7% yield).
1HNMR(300MHz,DMSO-d6)ppm8.01-8.16(m,3H),7.87-8.01(m,2H),7.69-7.82(m,1H),7.30-7.68(m,9H),5.52-5.96(m,1H),5.24-5.45(m,1H),5.09-5.14(s,2H),3.95-4.39(m,3H),3.74-3.85(m,1H),3.15-3.52(m,4H),2.11-2.23-2.33-2.43(m,1H),1.09-2.10(m,4H)；
LC-MS(ESIPOS)：525.25(M+).
Example 27
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (3-fluorophenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo- [2.2.2] octane 2,2, 2-trifluoroacetate (C42)
Preparation of 2- (benzo [ b ] thiophen-3-yl) -2- (3-fluorophenylamino) acetic acid (I40):
a mixture of benzo [ b ] thiophen-3-ylboronic acid (400mg, 2.25mmol), 3-fluoroaniline (217. mu.l, 2.25mmol), 2-oxoacetic acid hydrate (207mg, 2.25mmol) in acetonitrile (20ml) was stirred at room temperature for 4 hours. The solvent was removed in vacuo and the crude product was used as such in the next step.
Preparation of (R) -quinuclidin-3-yl 2- (benzo [ b ] thiophen-3-yl) -2- (3-fluorophenylamino) acetate (C41):
2- (benzo [ b ] in THF (20ml) was stirred at room temperature]Thien-3-yl) -2- (3-fluorophenylamino) acetic acid (I40) (677mg, 2.25mmol), (R) -quinuclidin-3-hydroxy (343mg, 2.70mmol), HOBT (413mg, 2.70mmol) and DCC (556mg, 2.70mmol) were mixed overnight. THF was evaporated in EtOAc and 1MK2CO3The crude product was partitioned between. In Na2SO4The organic phase was dried, filtered and evaporated to dryness. The crude product was purified by flash chromatography (DCM/MeOH ═ 98/2-95/5) to give (R) -quinuclidin-3-yl 2- (benzo [ b)]Thien-3-yl) -2- (3-fluorophenylamino) acetate (421mg, 45.6% yield in two steps).
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (3-fluorophenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo- [2.2.2] octane 2,2, 2-trifluoroacetate (C42):
2-chloro-1-phenylethanone (39.5mg, 0.26mmol) was added to (R) -quinuclidin-3-yl 2- (benzo [ b)]Thiophen-3-yl) -2- (3-fluorophenylamino) acetate (C41) (105mg, 0.26mmol) in EtOAc (3 ml). The reaction was stirred at room temperature overnight, then a second portion of 2-chloro-1-phenylethanone (11.9mg, 0.07mmol) was added and the reaction stirred at room temperature for an additional 4 hours. Evaporation of EtOAc and use of Et2The crude product was pulverized O (2ml) and the compound was purified by preparative HPLC first and then by flash chromatography (DCM/MeOH ═ 97/3-94/6) to afford (3R) -3- (2- (benzo [ b ] b)]Thien-3-yl) -2- (3-fluorophenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2]-octane 2,2, 2-trifluoroacetate salt (45 mg; 27.4% yield).
1HNMR(300MHz,DMSO-d6)ppm8.09-8.20(m,1H),7.86-8.08(m,4H),7.36-7.82(m,5H),7.02-7.19(m,1H),6.71-6.90(m,1H),6.55-6.69(m,2H),6.32-6.47(m,1H),5.86(m,1H),5.23-5.34(m,1H),5.08-5.15(s,2H),3.98-4.24(m,1H),3.78-3.88(m,1H),3.28-3.65(m,4H),2.05-2.15-2.31-2.44(m,1H),1.31-2.04(m,4H)；
LC-MS(ESIPOS)：529.21(M+).
Example 28
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (2-ethylphenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane bromide (C45)
Preparation of 2- (benzo [ b ] thiophen-3-yl) -2- (2-ethylphenylamino) -acetic acid (I43):
benzo [ b ] thiophen-3-ylboronic acid (500mg, 2.81mmol), 2-oxoacetic acid hydrate (259mg,2.81mmol) were dissolved in acetonitrile (20ml), followed by addition of 2-ethylaniline (346. mu.l, 2.81 mmol). The mixture was stirred at room temperature for 72 hours. The solvent was evaporated and in the next step the crude product was used without any further purification.
Preparation of (R) -quinuclidin-3-yl 2- (benzo [ b ] thiophen-3-yl) -2- (2-ethylphenylamino) acetate (C44):
2- (benzo [ b ] in THF (28ml) was stirred at room temperature]Thien-3-yl) -2- (2-ethylphenylamino) acetic acid (I43) (875mg, 2.81mmol), a mixture of HOBT (430mg, 2.81mmol) and DCC (1.16g, 5.62mmol) for 30 min. Then (R) -quinuclidin-3-hydroxy (715mg, 5.62mmol) was added and the reaction stirred at room temperature overnight, the solvent evaporated and the reaction stirred in EtOAc and saturated Na2CO3The crude product was partitioned between. The organic phase was dried over sodium sulfate, filtered and evaporated to dryness. The crude product was purified by flash chromatography (EtOAc/MeOH ═ 3/1) to collect (R) -quinuclidin-3-yl 2- (benzo [ b)]Thien-3-yl) -2- (2-ethylphenylamino) acetate (86mg, 7.3% yield in two steps).
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (2-ethylphenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azacationic bicyclo- [2.2.2] octane bromide (C45):
2-bromo-1-phenylethanone (44.8mg, 0.22mmol) and (R) -quinuclidin-3-yl 2- (benzo [ b ] in EtOAc (3ml) were heated at 100 ℃ under microwave irradiation]Thien-3-yl) -2- (2-ethylphenylamino) acetate (C44) (86mg, 0.20mmol) for 1 hour. 2-bromo-1-phenylethanone (44.8mg, 0.22mmol) was added and the mixture was heated under microwave irradiation at 100 ℃ for an additional 1 hour. Evaporation of the solvent, and application of Et2O pulverizing the crude product, and collecting (3R) -3- (2- (benzo [ b ]]Thien-3-yl) -2- (2-ethylphenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azacationic bicyclo [2.2.2]Octane bromide (44.6mg, 35.2% yield).
1HNMR(300MHz,DMSO-d6)ppm8.11-8.19(m,1H),8.00-8.08(m,1H),7.91-7.97(m,2H),7.90(s,1H),7.70-7.81(m,1H),7.56-7.65(m,2H),7.49(td,1H),7.38-7.46(m,1H),7.06(dd,1H),6.99(td,1H),6.65(td,1H),6.59(dd,1H),5.90(d,1H),5.35(d,1H),5.21-5.32(m,1H),4.92-5.12(m,2H),3.92-4.15(m,1H),3.44-3.74(m,4H),3.31-3.38(m,1H),2.62(q,2H),2.33-2.43(m,1H),1.73-2.17(m,4H),1.20(t,3H)；
LC-MS(ESIPOS)：539.20(M+).
Example 29
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (3-methoxyphenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo- [2.2.2] octane 2,2, 2-trifluoroacetate (C48)
Preparation of 2- (benzo [ b ] thiophen-3-yl) -2- (3-methoxyphenylamino) acetic acid (I46):
a mixture of 3-methoxyaniline (400mg, 3.25mmol), benzo [ b ] thiophen-3-ylboronic acid (578mg, 3.25mmol) and 2-oxyacetic acid hydrate (299mg, 3.25mmol) in acetonitrile (30ml) was stirred at room temperature overnight. The solvent was removed in vacuo and in the next step the crude product was used as such.
Preparation of (R) -quinuclidin-3-yl 2- (benzo [ b ] thiophen-3-yl) -2- (3-methoxyphenylamino) acetate (C47):
2- (benzo [ b ] in dry THF (30ml) was stirred at room temperature]Thien-3-yl) -2- (3-methoxyphenylamino) acetic acid (I46) (1.02g, 3.25mmol), (R) -quinuclidin-3-hydroxy (496mg, 3.90mmol), DCC (805mg, 3.90mmol) and HOBT (597mg, 3.90mmol) were mixed overnight. THF was evaporated and the crude product was taken up in EtOAc and taken up with 1MK2CO3And then washed 2 times with brine. In Na2SO4The organic phase was dried, filtered and evaporated to dryness. The crude product was purified by flash chromatography (DCM/MeOH ═ 98/2-95/5) to give (R) -quinuclidin-3-yl 2- (benzo [ b)]Thien-3-yl) -2- (3-methoxyphenylamino) acetate (213mg, 15.5% yield in two steps).
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (3-methoxyphenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo- [2.2.2] octane 2,2, 2-trifluoroacetate (C48):
reacting (R) -quinuclidin-3-yl 2- (benzo [ b ]]Thiophen-3-yl) -2- (3-methoxyphenylamino) -acetate (C47) (110mg, 0.26mmol) was dissolved in a mixture of ethyl acetate (2ml) and acetonitrile (1 ml). 2-bromo-1-phenylethanone (57.0mg, 0.29mmol) was added portionwise and the mixture was stirred at room temperature for 3 days. Remove the solvent in vacuo and use Et2And O, crushing the residue. The product was purified by preparative HPLC to give (3R) -3- (2- (benzo [ b ]]Thien-3-yl) -2- (3-methoxyphenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azacationic bicyclo [2.2.2]-octane 2,2, 2-trifluoroacetate salt (50.28mg, 29.5% yield).
1HNMR(300MHz,DMSO-d6)ppm8.07-8.21(m,1H),7.84-8.07(m,4H),7.69-7.81(m,1H),7.56-7.69(m,2H),7.46-7.52(m,1H),7.36-7.46(m,1H),7.02(t,1H),6.49(br.s.,1H),6.33-6.45(m,2H),6.15-6.27(m,1H),5.80-5.83(s,1H),5.20-5.37(m,1H),5.06-5.14(s,2H),3.72-4.21(m,2H),3.65-3.66(s,3H),3.27-3.63(m,4H),2.06-2.15-2.32-.42(m,1H),1.32-2.06(m,4H)；
LC-MS(ESIPOS)：541.13(M+).
Example 30
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (3- (ethoxycarbonyl) phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (C51)
Preparation of 2- (benzo [ b ] thiophen-3-yl) -2- (3- (ethoxycarbonyl) -phenylamino) acetic acid (I49):
to a solution of benzo [ b ] thiophen-3-ylboronic acid (500mg, 2.81mmol), 2-oxoacetic acid hydrate (259mg,2.81mmol) in acetonitrile (20ml) was added ethyl 3-aminobenzoate (419. mu.l, 2.81 mmol). The reaction was stirred at room temperature for 72 hours. The solvent was then evaporated in vacuo. In the next step, the residue was used without any further purification.
Preparation of ethyl 3- (1- (benzo [ b ] thiophen-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy) ethylamino) benzoate (C50):
2- (benzo [ b ]]Thien-3-yl) -2- (3- (ethoxycarbonyl) phenylamino) acetic acid (I49) (998mg, 2.81mmol), HOBT (430mg, 2.81mmol), DCC (1159mg, 5.62mmol) was dissolved in THF (28 ml). The mixture was stirred at room temperature for 30min, then (R) -quinuclidin-3-hydroxy (714mg, 5.62mmol) was added. The resulting reaction was stirred at room temperature overnight. The solvent was evaporated and washed with EtOAc and saturated Na2CO3The crude product was partitioned between. The organic phase was dried over sodium sulfate, filtered and evaporated to dryness. The crude product was purified by flash chromatography (DCM/MeOH ═ 95/5) and 3- (1- (benzo [ b ] was collected]Thien-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy) ethylamino) benzoic acid ethyl ester (222mg, 17.0% yield over two steps).
Preparation of (3R) -3- (2- (benzo [ b ] thiophen-3-yl) -2- (3- (ethoxycarbonyl) phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (C51):
to 3- (1- (benzo [ b ])]To a solution of thiophen-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy) ethylamino) ethyl benzoate (C50) (110mg, 0.24mmol) in ethyl acetate (2ml) was added 2-bromo-1-phenylethanone (51.8mg, 0.26mmol), and the reaction was stirred at room temperature for 3 days. Remove the solvent in vacuo and use Et2O the crude product was pulverized and then purified by preparative HPLC to obtain (3R) -3- (2- (benzo [ b)]Thien-3-yl) -2- (3- (ethoxycarbonyl) phenylamino) -acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2]Octane 2,2, 2-trifluoroacetate salt (28.9mg, 17.5% yield).
1HNMR(300MHz,DMSO-d6)ppm8.10-8.22(m,1H),7.87-8.09(m,4H),7.67-7.83(m,1H),7.55-7.67(m,2H),7.37-7.54(m,3H),7.18-7.31(m,2H),7.01-7.14(m,1H),6.88(br.s.,1H),5.89-5.91(s,1H),5.20-5.34(m,1H),5.08-5.15(s,2H),4.22-4.26(q,2H),3.30-4.17(m,6H),2.08-2.17-2.32-2.43(m,1H),1.42-2.07(m,4H),1.25-1.28(t,3H)；
LC-MS(ESIPOS)：583.21(M+).
Example 31
Preparation of (R) -3- (2- (6- (benzyloxy) pyridin-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo- [2.2.2] octane 2,2, 2-trifluoroacetate (diastereomer 1 of C54)
Preparation of 2- (6- (benzyloxy) pyridin-3-yl) -2- (phenylamino) acetic acid (I52):
to a suspension of 2-oxoacetic acid hydrate (0.40g, 4.37mmol) and aniline (0.40ml, 4.37mmol) in acetonitrile (25ml) was added 6- (benzyloxy) pyridin-3-ylboronic acid (1.00g, 4.37mmol) and the reaction was refluxed for 2 hours. The solvent was evaporated and the resulting dark brown oil was purified by flash chromatography (DCM/MeOH ═ 97/3) to give 2- (6- (benzyloxy) pyridin-3-yl) -2- (phenylamino) acetic acid (460mg, 31.5% yield).
Preparation of (R) -quinuclidin-3-yl 2- (6- (benzyloxy) pyridin-3-yl) -2- (phenylamino) acetate (diastereoisomers 1 and 2 of C53):
to a solution of 2- (6- (benzyloxy) pyridin-3-yl) -2- (phenylamino) acetic acid (I52) (460mg, 1.38mmol) in THF (20ml) was added DCC (341mg, 1.65mmol), 1H-benzo [ d][1,2,3]Triazol-1-ol (223mg, 1.65mmol) and (R) -quinuclidin-3-hydroxy (210mg, 1.65 mmol). The resulting reaction was stirred at room temperature for 15 hours, and then the solvent was evaporated. DCM was added and insoluble material was filtered off. And with Na2CO3The organic phase was washed 2 times with brine over Na2SO4Dried and evaporated. The crude product was purified by flash chromatography (DCM/MeOH-95/5-97/3) first recovering C53 diastereomer 1(71mg, 12% yield), then a mixture of C53 diastereoisomers (84mg, 14% yield) and finally C53 diastereomer 2(21mg, 4% yield).
Preparation of (R) -3- (2- (6- (benzyloxy) pyridin-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (diastereomer 1 of C54):
2-chloro-1-phenylethanone (24.7mg, 0.16mmol) was added to a solution of (R) -quinuclidin-3-yl 2- (6- (benzyloxy) pyridin-3-yl) -2- (phenylamino) -acetate (diastereomer 1 of C53) (71.0mg, 0.16mmol) in EtOAc (4 ml). The reaction was stirred at room temperature overnight. The solvent was evaporated and the crude product was purified by preparative HPLC to give (R) -3- (2- (6- (benzyloxy) pyridin-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (43.1mg, 40% yield).
1HNMR(300MHz,DMSO-d6)ppm8.38(d,1H)7.85-8.06(m,3H)7.75(t,1H)7.59(t,2H)7.29-7.49(m,5H)7.11(dd,2H)6.95(d,1H)6.75(d,2H)6.62(t,1H)5.44(s,1H)5.36(s,2H)5.19-5.29(m,1H)5.11(s,2H)4.06(m,1H)3.30-3.78(m,5H)2.33-2.44(m,1H)1.79-2.18(m,4H)；
LC-MS(ESIPOS)：562.26(M+).
Example 32
Preparation of (3R) -3- (2- (6-hydroxypyridin-3-yl) -2- (phenylamino) -acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (C55)
2-chloro-1-phenylethanone (29.3mg, 0.12mmol) was added to a solution of (R) -quinuclidin-3-yl 2- (6- (benzyloxy) pyridin-3-yl) -2- (phenylamino) acetate (C53) (84mg, 0.19mmol, mixture of diastereomers) in EtOAc (4 ml). The reaction was stirred at room temperature overnight, then a catalytic amount of 10% Pd/C (about 10mg) and 37% HCl (0.50ml) were added and the reaction was hydrogenated at 20psi for 2 hours, the Pd/C was filtered off and the colorless solution was evaporated to dryness. The crude product was purified by preparative HPLC to give (3R) -3- (2- (6-hydroxypyridin-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azocationic bicyclo [2.2.2] octane chloride (20.3mg, 18% yield).
1HNMR(300MHz,DMSO-d6)ppm11.25-11.95(m,1H)7.91-8.08(m,2H)7.71-7.85(m,1H)7.52-7.71(m,5H)7.12(dd,2H)6.72(d,2H)6.63(t,1H)6.38(d,1H)5.20-5.28(m,1H)5.18(d,3H)4.06-4.18(m,1H)3.48-3.82(m,5H)2.18-2.26(m,1H)1.75-2.10(m,4H)；
LC-MS(ESIPOS)：472.16(M+).
Example 33
Preparation of (R) -3- (2- (6- (benzyloxy) pyridin-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] -octane 2,2, 2-trifluoroacetate (C54)
2-chloro-1-phenylethanone (8mg, 0.05mmol) was added to a solution of (R) -quinuclidin-3-yl 2- (6- (benzyloxy) pyridin-3-yl) -2- (phenylamino) acetate (C53) (22mg, 0.05mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight. The solvent was evaporated and the crude product was purified by preparative HPLC to give (R) -3- (2- (6- (benzyloxy) pyridin-3-yl) -2- (phenylamino) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azocationic bicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (14.5mg, 43% yield).
1HNMR(300MHz,DMSO-d6)ppm8.38(d,1H)7.85-8.11(m,3H)7.77(t,1H)7.62(t,2H)7.21-7.51(m,5H)7.11(dd,2H)6.93(d,1H)6.74(d,2H)6.62(t,1H)6.40(s,1H)5.40(s,1H)5.36(s,2H)5.24(s,1H)5.17(s,2H)4.12(dd,1H)3.76-3.88(m,5H)2.09-2.22(m,1H)1.91-2.08(m,2H)1.73-1.89(m,1H)1.53-1.73(m,1H)；
LC-MS(ESIPOS)：562.21(M+).
Example 34
Preparation of (3R) -3- (2- (4-methoxyphenylamino) -2- (thien-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (C58)
Preparation of 2- (4-methoxyphenylamino) -2- (thiophen-3-yl) acetic acid (I56):
a mixture of thiophen-3-ylboronic acid (339mg, 2.65mmol), 2-oxoacetic acid hydrate (244mg, 2.65mmol) and 4-methoxyaniline (326mg, 2.65mmol) was heated at 100 ℃ for 1 hour under microwave irradiation. The precipitate was recovered and washed sequentially with acetonitrile, DCM and Et2And O washing. The compound was dried under vacuum at 40 ℃ to give 2- (4-methoxyphenylamino) -2- (thiophen-3-yl) acetic acid (540mg, 77% yield).
Preparation of (R) -quinuclidin-3-yl 2- (4-methoxyphenylamino) -2- (thiophen-3-yl) acetate (C57):
a mixture of 2- (4-methoxyphenylamino) -2- (thiophen-3-yl) acetic acid (I56) (544mg, 2.01mmol), (R) -quinuclidine-3-hydroxy (614mg, 4.83mmol), DCC (511mg, 2.47mmol) and HOBT (380mg, 2.48mmol) in dry THF (160ml) was stirred at room temperature overnight. The solvent was evaporated and washed with EtOAc and 2M NaHCO3The residue is distributed among the two. With 2MNaHCO3The organic phase was further washed with brine over Na2SO4Dried and evaporated. The residue was purified by flash chromatography (DCM/MeOH ═ 95/5-7/3) to give (R) -quinuclidin-3-yl 2- (4-methoxyphenylamino) -2- (thiophen-3-yl) acetate (40mg, 5% yield).
Preparation of (3R) -3- (2- (4-methoxyphenylamino) -2- (thiophen-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (C58):
a mixture of (R) -quinuclidin-3-yl 2- (4-methoxyphenylamino) -2- (thiophen-3-yl) acetate (C57) (40mg, 0.11mmol) and 2-chloro-1-phenylethanone (16.6mg, 0.11mmol) in EtOAc (5ml) was stirred at room temperature overnight. The solvent was evaporated and the residue was purified by preparative HPLC to give (3R) -3- (2- (4-methoxyphenylamino) -2- (thiophen-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (33mg, 51% yield).
1HNMR(300MHz,DMSO-d6)ppm7.90-8.06(m,2H)7.71-7.84(m,1H)7.52-7.70(m,4H)7.28(dd,1H)6.65-6.81(m,4H)5.40(d,1H)5.05-5.30(m,3H)4.02-4.18(m,1H)3.62(s,3H)3.39-3.70(m,5H)2.35(d,1H)1.50-2.14(m,4H)；
LC-MS(ESIPOS)：562.21(M+).
Example 35
Preparation of (R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (diastereomer 1 of C61)
Preparation of 2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2- (6-methoxypyridin-3-yl) acetic acid (I59):
to a suspension of 2-oxoacetic acid hydrate (156mg, 1.70mmol) and methyl 3-aminothiophene-2-carboxylate (267mg, 1.70mmol) in acetonitrile (15ml) was added 6-methoxypyridin-3-ylboronic acid (260mg, 1.70 mmol). The reaction was refluxed for 2 hours and the solvent was evaporated to give 2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2- (6-methoxypyridin-3-yl) acetic acid (548mg, 100% yield the product was used without any further purification in the next step.
Preparation of (R) -methyl 3- (1- (6-methoxypyridin-3-yl) -2-oxo-2- (quinuclidin-3-yloxy) ethylamino) thiophene-2-carboxylate (diastereomer 1-2 of C60):
to a solution of 2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2- (6-methoxypyridin-3-yl) acetic acid (I59) (548mg, 1.70mmol) in THF (20ml) was added N, N' -methanediylidine dicyclohexylamine (421mg, 2.04mmol), 1H-benzo [ d ] b][1,2,3]Triazol-1-ol (276mg, 2.04mmol) and (R) -quinuclidin-3-hydroxy (259mg, 2.04 mmol). The reaction was stirred at room temperature for 15 hours, and then the solvent was evaporated. The residue was taken up with DCM and the insoluble material was filtered off. With Na2CO3Then the organic phase was washed 2 times with brine over Na2SO4Dried, filtered and evaporated. The crude product was purified by flash chromatography (DCM/MeOH ═ 95/5) first collecting C60 diastereomer 1(140mg, 19% yield) and then a mixture of C60 diastereoisomers 1 and 2 (100mg, 14% yield).
Preparation of (R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azocationic bicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (diastereomer 1 of C61):
to a solution of (R) -methyl 3- (1- (6-methoxypyridin-3-yl) -2-oxo-2- (quinuclidin-3-yloxy) ethylamino) thiophene-2-carboxylate (diastereomer 1 of C60) (60mg, 0.14mmol) in EtOAc (3ml) and acetonitrile (3ml) was added 2-chloro-1-phenylethanone (23.6mg, 0.15mmol) and the reaction was stirred at room temperature for 15 h. The solvent was then evaporated and the crude product was purified by preparative HPLC to give (R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azocationic bicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (48mg, 52% yield).
1HNMR(300MHz,DMSO-d6)ppm8.33(d,1H)7.89-8.05(m,2H)7.52-7.83(m,6H)6.88(d,1H)6.77(d,1H)5.72-5.81(m,1H)5.22-5.36(m,1H)5.13(s,2H)3.97-4.17(m,1H)3.85(s,3H)3.78(s,3H)3.36-3.75(m,5H)2.40-2.47(m,1H)1.83-2.21(m,4H)；
LC-MS(ESIPOS)：550.18(M+).
Example 36
Preparation of (R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2- (thiazol-2-yl) ethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (diastereomer 1 of C62)
To a solution of (R) -methyl 3- (1- (6-methoxypyridin-3-yl) -2-oxo-2- (quinuclidin-3-yloxy) ethylamino) thiophene-2-carboxylate (diastereomer 1 of C60) (60mg, 0.14mmol) in EtOAc (3ml) and acetonitrile (3ml) was added 2-bromo-1- (thiazol-2-yl) ethanone (31.5mg, 0.15mmol) and the reaction was stirred at room temperature for 15 h. The solvent was evaporated and the crude product was purified by preparative HPLC to give (R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2- (thiazol-2-yl) ethyl) -1-azocationic bicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (47mg, 50% yield).
1HNMR(300MHz,DMSO-d6)ppm8.38(d,1H)8.32(d,1H)8.23(d,1H)7.63-7.82(m,3H)6.87(d,1H)6.76(d,1H)5.65-5.79(m,1H)5.23-5.33(m,1H)5.16(s,2H)4.04-4.17(m,1H)3.86(s,3H)3.78(s,3H)3.37-3.75(m,5H)2.39-2.47(m,1H)1.85-2.17(m,4H)；
LC-MS(ESIPOS)：557.09(M+).
Example 37
Preparation of (3R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2- (thiophen-2-yl) ethyl) -1-azoniabicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (C63)
To a solution of methyl 3- (1- (6-methoxypyridin-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy) ethylamino) thiophene-2-carboxylate (C60) (60mg, 0.14mmol) in EtOAc (3ml) and acetonitrile (3ml) was added 2-bromo-1- (thiophen-2-yl) ethanone (31.4mg, 0.15mmol) and the reaction was stirred at room temperature for 15 h. The solvent was evaporated and the crude product was purified by preparative HPLC to give (3R) -3- (2- (2- (methoxycarbonyl) thiophen-3-ylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2- (thiophen-2-yl) ethyl) -1-azacationic bicyclo [2.2.2] octane 2,2, 2-trifluoroacetate (57mg, 61% yield).
1HNMR(300MHz,DMSO-d6)ppm8.27-8.38(m,1H)8.15-8.27(m,1H)7.96-8.12(m,1H)7.61-7.86(m,3H)7.24-7.41(m,1H)6.86(d,1H)6.76(dd,1H)5.70(d,1H)5.14-5.36(m,1H)4.87-5.12(m,2H)4.00(br.s.,1H)3.85(s,3H)3.78(s,3H)3.37-3.75(m,5H)2.37-2.46(m,1H)1.57-2.14(m,4H)
LC-MS(ESIPOS)：556.12(M+).
Example 38
Preparation of (3R) -3- (2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo- [2.2.2] octane chloride (C66)
Preparation of 2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetic acid (I64):
a mixture of 6-methoxypyridin-3-ylboronic acid (1.2g, 7.84mmol), ethyl 3-aminobenzoate (1.29g, 7.84mmol) and 2-oxyacetic acid hydrate (723mg, 7.84mmol) in acetonitrile (70ml) was stirred at room temperature overnight. The solvent was evaporated in vacuo and the crude product was purified by flash chromatography (DCM/MeOH ═ 9/1) to afford 2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetic acid (0.95g, 38% yield).
Preparation of ethyl 3- (1- (6-methoxypyridin-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy) ethylamino) benzoate (C65):
a mixture of 2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetic acid (I64) (951mg, 2.51mmol), (R) -quinuclidin-3-hydroxy (694mg, 5.47mmol), HOBT (837mg, 5.47mmol) and DCC (1.13g, 5.47mmol) in dry THF (35ml) was stirred at room temperature overnight. The solvent was evaporated and washed with DCM and 2MK2CO3The crude product was partitioned between. In Na2SO4The organic phase was dried, filtered and evaporated to dryness. The crude product was purified by flash chromatography (DMC/MeOH ═ 97/3-95/5) to afford ethyl 3- (1- (6-methoxypyridin-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy) ethylamino) benzoate (348mg, 27.5% yield).
Preparation of (3R) -3- (2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo- [2.2.2] octane chloride (C66):
2-chloro-1-phenylethanone (17.6mg, 0.11mmol) was added to 3- (1- (6-methoxypyridin-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy)Yl) ethylamino) ethyl benzoate (C65) (50mg, 0.11mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight and then the solvent was evaporated to dryness. With Et2The residue was pulverized and then subjected to preparative HPLC (eluent: CH)3CN/H2O purification to give (3R) -3- (2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2]Octane chloride (26.3mg, 38.9% yield).
1HNMR(300MHz,DMSO-d6)ppm8.38(t,1H),7.85-8.04(m,3H),7.71-7.82(m,1H),7.55-7.69(m,2H),7.37(br.s.,1H),7.17-7.28(m,2H),7.01(dt,1H),6.88(dd,1H),6.80-6.85(d,1H),5.48-5.51(d,1H),5.21-5.30(m,1H),5.17-5.22(br.s.,2H),4.20-4.36(m,2H),3.97-4.24(m,1H),3.86(s,3H),3.44-3.80(m,5H),2.16-2.24-2.34-2.41(m,1H),1.56-2.15(m,4H),1.27-1.30(t,3H)；
LC-MS(ESIPOS)：558.46(M+).
Example 39
Preparation of (3R) -3- (2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2- (thiophen-2-yl) ethyl) -1-azoniabicyclo [2.2.2] octane chloride (C67)
2-chloro-1- (thiophen-2-yl) ethanone (18.3mg, 0.11mmol) was added to a solution of 3- (1- (6-methoxypyridin-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy) -ethylamino) benzoic acid (C65) (50mg, 0.11mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight. Evaporate the solvent and use Et2O pulverize the residue, filter and dry, purify the product by flash chromatography (DCM/MeOH ═ 97/3-9/1) to obtain (3R) -3- (2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2- (thiophen-2-yl)Ethyl) -1-azacationic bicyclo [2.2.2]Octane chloride (41.6mg, 60.9% yield).
1HNMR(300MHz,DMSO-d6)ppm8.37(t,1H),8.19-8.25(m,1H),8.04-8.12(m,1H),7.86-7.93(m,1H),7.30-7.41(m,2H),7.17-7.29(m,2H),6.96-7.05(m,1H),6.87(d,1H),6.79-6.84(d,1H),5.46-5.49(d,1H),5.16-5.28(m,1H),5.04-5.08(s,2H),4.21-4.37(m,2H),3.97-4.19(m,1H),3.85-3.86(s,3H),3.46-3.83(m,5H),2.13-2.24-2.31-2.41(m,1H),1.58-2.14(m,4H),1.29-1.30(t,3H)；
LC-MS(ESIPOS)：564.39(M+).
Example 40
Preparation of (3R) -3- (2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2- (4-hydroxyphenyl) -2-oxyethyl) -1-azoniabicyclo [2.2.2] octane bromide (C68)
2-bromo-1- (4-hydroxyphenyl) ethanone (24.5mg, 0.11mmol) was added to a solution of ethyl 3- (1- (6-methoxypyridin-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy) ethylamino) benzoate (C65) (50mg, 0.11mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight. Evaporation of the solvent, and application of Et2The residue was pulverized, filtered and dried. The product was purified by flash chromatography (DCM/MeOH ═ 97/3-9/1) to give (3R) -3- (2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2- (4-hydroxyphenyl) -2-oxyethyl) -1-azocationbicyclo [2.2.2]Octane bromide (37.3mg, 50.1% yield).
1HNMR(300MHz,DMSO-d6)ppm10.74(br.s.,1H),8.38(t,1H),7.78-7.95(m,3H),7.36(s,1H),7.18-7.30(m,2H),6.96-7.06(m,1H),6.84-6.96(m,3H),6.77-6.80(d,1H),5.46-5.49(d,1H),5.15-5.30(m,1H),5.01-5.06(s,2H),4.24-4.27(q,2H),4.00-4.18(m,1H),3.85-3.86(s,3H),3.44-3.81(m,5H),2.13-2.23-2.32-2.41(m,1H),1.60-2.13(m,4H),1.28-1.30(t,3H)；
LC-MS(ESIPOS)：574.43(M+).
EXAMPLE 41
Preparation of (3R) -3- (2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2- (thiazol-2-yl) ethyl) -1-azoniabicyclo [2.2.2] octane bromide (C69)
2-bromo-1- (thiazol-2-yl) ethanone (23.4mg, 0.11mmol) was added to a solution of ethyl 3- (1- (6-methoxypyridin-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy) ethylamino) benzoate (C65) (50mg, 0.11mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight, then the solvent was evaporated and Et2O pulverizing the residue, filtering and drying to obtain (3R) -3- (2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2- (thiazol-2-yl) ethyl) -1-azoniabicyclo [2.2.2]Octane bromide (46.5mg, 63.3% yield).
1HNMR(300MHz,DMSO-d6)ppm8.36-8.41(m,2H),8.24(d,1H),7.89(dd,1H),7.36(s,1H),7.18-7.29(m,2H),6.98-7.05(m,1H),6.87(d,1H),6.77-6.79(d,1H),5.46-5.49(d,1H),5.21-5.28(m,1H),5.16-5.20(s,2H),4.26-4.27(q,2H),4.02-4.21(m,1H),3.86(s,3H),3.41-3.82(m,5H),2.15-2.23-2.32-2.42(m,1H),1.64-2.15(m,4H),1.29-1.30(t,3H)；
LC-MS(ESIPOS)：565.38(M+).
Example 42
Preparation of (3R) -3- (2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2- (isoxazol-3-ylamino) -2-oxyethyl) -1-azoniabicyclo [2.2.2] octane chloride (C70)
2-chloro-N- (isoxazol-3-yl) acetamide (18.3mg, 0.11mmol) was added to a solution of ethyl 3- (1- (6-methoxypyridin-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy) ethylamino) benzoate (C65) (50mg, 0.11mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight. Evaporation of the solvent, and application of Et2The residue was pulverized, filtered and dried. By preparative HPLC (eluent: CH)3CN/H2O), purifying the product to obtain (3R) -3- (2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2- (isoxazol-3-ylamino) -2-oxyethyl) -1-azocationic bicyclo [2.2.2]Octane chloride (26.2mg, 38.4% yield).
1HNMR(300MHz,DMSO-d6)ppm11.77(br.s.,1H),8.85-8.96(m,1H),8.32-8.41(m,1H),7.85-7.93(m,1H),7.31-7.39(m,1H),7.15-7.28(m,2H),6.95-7.05(m,1H),6.73-6.95(m,3H),5.45-5.48(d,1H),5.08-5.27(m,1H),3.97-4.38(m,5H),3.85(s,3H),3.39-3.84(m,5H),2.12-2.23-2.31-2.40(m,1H),1.58-2.13(m,4H),1.29-1.30(t,3H)；
LC-MS(ESIPOS)：564.42(M+).
Example 43
Preparation of (3R) -3- (2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1-methyl-1-azoniabicyclo [2.2.2] octane iodide (C71)
Methyl iodide (7.1 μ l, 0.11mmol) was added to a solution of ethyl 3- (1- (6-methoxypyridin-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy) ethylamino) benzoate (C65) (50mg, 0.11mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight. Evaporate the solvent and use Et2The residue was pulverized, filtered and dried. By preparative HPLC (eluent: CH)3CN/H2O), purifying the product to obtain (3R) -3- (2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1-methyl-1-azocationic bicyclo [2.2.2]Octane iodide (24.3mg, 36.7% yield).
1HNMR(300MHz,DMSO-d6)ppm8.35(d,1H),7.74-7.93(m,1H),7.33(t,1H),7.18-7.29(m,2H),6.92-7.05(m,1H),6.86-6.88(d,1H),6.75-6.78(d,1H),5.41-5.44(d,1H),4.96-5.24(m,1H),4.28(q,2H),3.85-3.86(s,3H),3.69-3.94(m,1H),3.40(m,4H),3.06-3.25(m,1H),2.91-2.96(s,3H),2.09-2.19(m,1H),1.77-2.04(m,3H),1.59-1.77-2.25-2.35(m,1H),1.30(t,3H)；
LC-MS(ESIPOS)：454.36(M+).
Example 44
Preparation of (3R) -3- (2- (3- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (3-methylbut-2-enyl) -1-azoniabicyclo [2.2.2] octane bromide (C72)
1-bromo-3-methylbut-2-ene (16.9mg, 0.11mmol) was added to a solution of ethyl 3- (1- (6-methoxypyridin-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy) -ethylamino) benzoate (C65) (50mg, 0.11mmol) in EtOAc (2 ml). The reaction was stirred at room temperature overnight. Evaporate the solvent and use Et2The residue was pulverized, filtered and dried. The product was purified by flash chromatography (DCM/MeOH ═ 97/3-9/1) to yield (3R) -3- (2- (3) c- (ethoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (3-methylbut-2-enyl) -1-azoniabicyclo [2.2.2]Octane bromide (56mg, 84% yield).
1HNMR(300MHz,DMSO-d6)ppm8.35(d,1H),7.78-7.92(m,1H),7.31-7.39(m,1H),7.18-7.30(m,2H),6.94-7.08(m,1H),6.87-6.88(d,1H),6.76-6.78(d,1H),5.42-5.45(d,1H),5.23(t,1H),5.07-5.18(m,1H),4.28(q,2H),3.85-3.86(s,3H),3.70-3.84(m,3H),3.33-3.45(m,2H),3.12-3.25(m,2H),2.81-3.03(m,1H),1.85-2.37(m,3H),1.81-1.82(br.s.,3H),1.70-1.74(s,3H),1.52-1.77(m,2H),1.30(t,3H)；
LC-MS(ESIPOS)：508.44(M+).
Example 45
Preparation of (3R) -3- (2- (3-ethylphenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo- [2.2.2] octane chloride (C75)
Preparation of 2- (3-ethylphenylamino) -2- (6-methoxypyridin-3-yl) acetic acid (I73):
a mixture of 6-methoxypyridin-3-ylboronic acid (200mg, 1.31mmol), 3-ethylaniline (163. mu.l, 1.31mmol) and 2-oxoacetic acid hydrate (120mg, 1.31mmol) in acetonitrile (20ml) was stirred at room temperature overnight. The solvent was evaporated and the crude product was purified by flash chromatography (DCM/MeOH ═ 9/1) to afford 2- (3-ethylphenylamino) -2- (6-methoxypyridin-3-yl) acetic acid (265mg, 70.8% yield).
Preparation of (R) -quinuclidin-3-yl 2- (3-ethylphenylamino) -2- (6-methoxypyridin-3-yl) acetate (C74):
2- (3-ethylphenylamino) -2- (6-methoxypyridin-3-yl) acetic acid (I73) (265mg, 0)93mmol), (R) -quinuclidine-3-hydroxy (129mg, 1.02mmol), 1H-benzo [ d][1,2,3]A mixture of triazol-1-ol hydrate (156mg, 1.02mmol), N, N' -methanediylidine dicyclohexylamine (210mg, 1.02mmol) was dissolved in THF (10ml) and stirred at room temperature overnight. Evaporate the solvent and absorb the crude product with EtOAc and use 2MK2CO3Washing is carried out for 2 times. In Na2SO4The organic phase was dried, filtered and evaporated to dryness to obtain the title compound, which was used without further purification in the next step.
Preparation of (3R) -3- (2- (3-ethylphenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo- [2.2.2] octane chloride (C75):
2-chloro-1-phenylethanone (143mg, 0.93mmol) was added to a solution of (R) -quinuclidin-3-yl 2- (3-ethylphenylamino) -2- (6-methoxypyridin-3-yl) acetate (C74) (366mg, 0.93mmol) in EtOAc (3ml) and the resulting reaction was stirred at room temperature overnight. The solvent is evaporated, and by preparative HPLC (eluent: CH)3CN/H2O), purifying the crude product to obtain (3R) -3- (2- (3-ethylphenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azocationic bicyclo [2.2.2]Octane chloride (69.3mg, 13.6% yield in two steps).
1HNMR(300MHz,DMSO-d6)ppm8.37(t,1H),7.93-8.04(m,2H),7.84-7.93(m,1H),7.76(d,1H),7.53-7.70(m,2H),6.96-7.07(m,1H),6.86-6.88(d,1H),6.58-6.65(m,1H),6.54(d,1H),6.48(d,1H),5.38-5.41(s,1H),5.19-5.30(m,1H),5.10-5.16(s,2H),3.98-4.20(m,1H),3.85-3.86(s,3H),3.62-3.82(m,5H),2.42-2.48(m,2H),2.14-2.22-2.33-2.41(m,1H),1.55-2.13(m,4H),1.13(t,3H)；
LC-MS(ESIPOS)：514.3(M+).
Example 46
Preparation of (3R) -3- (2- (3-fluorophenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo- [2.2.2] octane chloride (C78)
Preparation of 2- (3-fluorophenylamino) -2- (6-methoxypyridin-3-yl) acetic acid (I76):
a mixture of 6-methoxypyridin-3-ylboronic acid (200mg, 1.31mmol), 3-fluoroaniline (126. mu.l, 1.31mmol) and 2-oxoacetic acid hydrate (120mg, 1.31mmol) in acetonitrile (20ml) was stirred at room temperature overnight. The solvent was evaporated and the crude product was purified by flash chromatography (DCM/MeOH ═ 9/1) to afford 2- (3-fluorophenylamino) -2- (6-methoxypyridin-3-yl) acetic acid (48mg, 13.3% yield).
Preparation of (R) -quinuclidin-3-yl 2- (3-fluorophenylamino) -2- (6-methoxypyridin-3-yl) acetate (C77):
a mixture of 2- (3-fluorophenylamino) -2- (6-methoxypyridin-3-yl) acetic acid (I76) (48mg, 0.17mmol), (R) -quinuclidin-3-hydroxy (24.31mg, 0.19mmol), HOBT (29.3mg, 0.19mmol) and DCC (39.4mg, 0.19mmol) was dissolved in THF (10ml) and stirred at room temperature overnight. Evaporation of the solvent and absorption of the crude product with EtOAc and with 2MK2CO3Washing is carried out for 2 times. In Na2SO4The organic phase was dried, filtered and evaporated to dryness to obtain the title compound, which was used without further purification in the next step.
Preparation of (3R) -3- (2- (3-fluorophenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo- [2.2.2] octane chloride (C78):
2-chloro-1-phenylethanone (26.9mg, 0.17mmol) was added to a solution of (R) -quinuclidin-3-yl 2- (3-fluorophenylamino) -2- (6-methoxypyridin-3-yl) acetate (C77) (67.1mg, 0.17mmol) in EtOAc (3mL) and the resulting reaction was stirred at room temperature overnight. The solvent was evaporated and chromatographed by flash chromatography (DCM/MeOH ═ 97-3-9/1) and then purified by preparative HPLC (eluent: CH (CH)3CN/H2O), purifying the crude product to obtain (3R) -3- (2- (3-fluorophenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azocationic bicyclo [2.2.2]Octane chloride (19.5mg, 20.7% yield).
1HNMR(300MHz,DMSO-d6)ppm8.33-8.43(m,1H),7.91-8.03(m,2H),7.86-7.88(dd,1H),7.70-7.81(m,1H),7.54-7.68(m,2H),7.01-7.21(m,1H),6.88(d,1H),6.76(br.s.,1H),6.51-6.61(m,2H),6.32-6.45(m,1H),5.39-5.60(m,1H),5.21-5.34(m,1H),5.12-5.17(s,2H),4.06-4.19(m,1H),3.85-3.86(s,3H),3.58-3.81(m,5H),2.14-2.24-2.32-2.42(m,1H),1.48-2.14(m,4H)；
LC-MS(ESIPOS)：504.28(M+).
Example 47
Preparation of (3R) -3- (2- (2- (methoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo- [2.2.2] octane chloride (C81)
Preparation of 2- (2- (methoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetic acid (I79):
a mixture of 6-methoxypyridin-3-ylboronic acid (200mg, 1.31mmol), methyl 2-aminobenzoate (170. mu.l, 1.31mmol) and 2-oxyacetic acid hydrate (120mg, 1.31mmol) in acetonitrile (20ml) was stirred at room temperature overnight. The solvent was evaporated and the crude product was purified by flash chromatography (DCM/MeOH ═ 9/1) to afford 2- (2- (methoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetic acid (118mg, 28.5% yield).
Preparation of methyl 2- (1- (6-methoxypyridin-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy) ethylamino) benzoate (C80):
2- (2- (methoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetic acid (I79) (118mg, 0.37mmol), (R) -quinuclidin-3-hydroxy (52.2mg, 0.41mmol), 1H-benzo [ d][1,2,3]A mixture of triazol-1-ol hydrate (62.8mg, 0.41mmol) and N, N' -methanadiylidene dicyclohexylamine (85mg, 0.41mmol) was dissolved in dry THF (10ml) and stirred at room temperature overnight. Evaporate the solvent under reduced pressure and absorb the crude product with EtOAc and use 2MK2CO3Washing is carried out for 2 times. In Na2SO4The organic phase was dried, filtered and evaporated to dryness to obtain the title compound, which was used without further purification in the next step.
Preparation of (3R) -3- (2- (2- (methoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azoniabicyclo [2.2.2] octane chloride (C81):
2-chloro-1-phenylethanone (57.7mg, 0.37mmol) was added to a solution of methyl 2- (1- (6-methoxypyridin-3-yl) -2-oxo-2- ((R) -quinuclidin-3-yloxy) ethylamino) benzoate (C80) (159mg, 0.37mmol) in EtOAc (3ml) and the resulting reaction was stirred at room temperature overnight. The solvent was evaporated and purified by flash chromatography first (DCM/MeOH ═ 97/3-9/1) and then by preparative HPLC (eluent: CH)3CN/H2O), and finally purifying the crude product by flash chromatography (DCM/MeOH ═ 95/5) to obtain (3R) -3- (2- (2- (methoxycarbonyl) phenylamino) -2- (6-methoxypyridin-3-yl) acetoxy) -1- (2-oxo-2-phenylethyl) -1-azocationbicyclo [2.2.2]Octane chloride (21.2mg, 9.8% yield).
1HNMR(300MHz,DMSO-d6)ppm8.76(d,1H),8.37(t,1H),7.90-8.08(m,2H),7.83-7.91(m,1H),7.72-7.83(m,2H),7.50-7.66(m,2H),7.30-7.44(m,1H),6.89(d,1H),6.62-6.72(m,2H),5.65-5.70(d,1H),5.22-5.35(m,1H),5.13-5.17(s,2H),3.98-4.26(m,1H),3.86(s,3H),3.85(s,3H),3.52-3.76(m,5H),2.20-2.46(m,1H),1.62-2.17(m,4H)；
LC-MS(ESIPOS)：544.31(M+).
Biological characteristics
Example 48
Radioligand binding assay examples of cloned human muscarinic receptors
In the absence of Ca++/Mg++The CHO-K1 clone expressing the human M1-, M2-, M3-receptor (Euroscreen, SwissprotP11229, P08172, P20309, Genbank: J02960, respectively) was harvested in phosphate-buffered saline and harvested by centrifugation at 1500rpm for 10 minutes at 4 ℃. The pellet was resuspended in ice-cold buffer A (15mM Tris-HClpH7.4, 2mM MgCl)20.3mM EDTA, 1mM AGTA). Cloned cells expressing the M1-, M2-, M3-receptor were homogenized by PBIproltron (set 5, total 15 s). The crude membrane fraction was collected by two successive centrifugation steps at 40000g for 20min at 4 ℃ and separated by a washing step in buffer A.
The pellets obtained from these three cell lines were finally resuspended in buffer C (75mM Tris HClpH7.4, 12.5mM MgCl)20.3mM EDTA, 1mM EGTA, 250mM sucrose) and storing an aliquot at-80 ℃.
On the day of the experiment, frozen membranes of M1-, M2-, and M3-receptors were resuspended in buffer D (50mM Tris-HClpH7.4, 2.5mM MgCl)21mM EDTA). Use of non-selective muscarinic radioligands [3H]N-methylscopolamine (mol. Pharmacol.45: 899-907), labeling the binding sites for M1, M2, and M3. Binding experiments were performed in 96-well plates in duplicate at radioligand concentrations of 0.1-0.3nM (10-point concentration curve). Nonspecific binding was determined in the presence of cold 10. mu.M N-methylscopolamine. Samples (final volume 0.75ml) were incubated for 120min for the binding assay for M1, 60min for the binding assay for M2, and 90min for the binding assay for M3 at room temperature.
The reaction was stopped by rapid filtration through GF/BUnifilter plates and washed twice (0.75ml) with cold buffer using a packard Filtermate harvester. The radioactivity on the filters was measured by a microplate scintillation counter topcount nxt (canberra packard).
In the assays of the invention, Ki values for the tested compounds are determined from the observed IC50 values according to known methods. Lower Ki values indicate that the compounds tested have higher binding affinity for the receptor.
Ki values for the compounds tested in the present invention include 0.1nM to 1 μ M.
The interaction with the M3 muscarinic receptor can be estimated by the results of in vitro studies evaluating the potency of the tested compounds and the resulting shift in inhibitor activity (offset) following intratracheal washing of the antagonist in isolated guinea pigs.
Example 49
In vitro interaction with guinea pig M3 receptor
The potential for antagonist activity in isolated guinea pigs was studied with minor changes following the procedure previously described by Haddadeb et al in BrJPharmacol127,413-420,1999.
Cumulative concentration-response curves of the test antagonists were plotted on the formulation pre-ordered (precontracted) by carbachol until complete inhibition of smooth muscle tone (tone) was achieved. Antagonist concentrations (IC) that produce 50% reversal of carbachol-induced tone contraction50) As a measure of its potential in this biological assay.
In experiments aimed at evaluating the shift in inhibitory effect produced by test compounds, the minimum concentration of test compound known to produce the greatest inhibitory effect was given to carbachol-ordered formulations. Once the tonic contraction is completely reversed, the organ bath solution is refreshed and the formulation is thoroughly washed with fresh Krebs solution. Carbachol (0.3 μ M) was administered again during the next 4 hours (at the 30min interval between washing and the next administration).
The inhibitory effect of the compounds of the invention administered at a concentration of 10nM after carbachol administration was expressed as% recovery of the contractile response to carbachol. The recovery rate 4 hours after washing was less than 50%.
IC of test Compounds50Values include 0.1nM to 300 nM.
Example 50
Plasma stability
To demonstrate compound degradation, the compounds of the present invention were tested for stability in human plasma at 1 and 5 hours. Briefly, 250. mu.M of a 10. mu.l stock solution of the compound in acetonitrile was added to 1ml of human plasma and the samples were incubated at 37 ℃. Plasma (50 μ L) was taken after 0,1 and 5 hours of incubation and added to 140 μ L acetonitrile and verapamil was added as an internal standard (250 ng/ml). The samples were analyzed by HPLC-MS/MS analysis.
Plasma stability was calculated as the percentage remaining after 1 and 5 hours by dividing the peak area at 1 or 5 hours by the peak area at time 0.
After 1 and 5 hours of incubation, some representative compounds of the invention were tested for plasma stability, and the values obtained included 0-25%, indicating that the compounds of the invention are very unstable in human plasma.
Icon symbol
*NMR
s ═ singlet
d ═ double peak
t is triplet
q is quartet
double peak (doublets) double peak (doublettofdublets)
m is multiplet
br is wide

Claims (9)

1. Compounds of general formula (I) and pharmaceutically acceptable salts thereof,
wherein
R1Selected from phenyl, benzyl, and thienyl, said groups optionally substituted with one or more substituents selected from the group consisting of: halogen atom, CON (R)5)2，COR5，CO2R5，(C1-C6) Alkyl radical (C)1-C6) An alkoxy group;
R2is H or (C)1-C6) An alkyl group;
R3selected from phenyl, pyridyl and benzothienyl, said groups being optionally substituted with one or more substituents selected from: OH, (C)1-C6) Alkoxy and phenyl (C)1-C6) An alkoxy group;
R5is H or (C)1-C6) An alkyl group, a carboxyl group,
q represents a group of formula (i):
the group of (i) or (ii):
wherein R is4Is a group of formula (Y):
---(CH2)p---P---(CH2)q---W
(Y)
and wherein
p is 1;
q is 0;
p is CO;
w is selected from phenyl, pyridyl, thienyl and thiazolyl, said groups being optionally substituted with-OH,
or P is 3, q is 0, P is O, W is phenyl;
or P is 2, q is 0, P is absent, W is phenyl; optionally substituted by halogen atoms;
or P is 1, q is 0, P is-con (h) -, W is pyridinyl or isoxazolyl;
or P and q are 0, P is absent, W is methyl;
or P is 1, q is 0, P is absent, W is (C)2-C6) An alkenyl group;
A-is a physiologically acceptable anion;
wherein R is1And R3Is selected from thienyl, pyridyl and benzothienyl, and when Q is a group of formula (i), and R is3When it is phenyl, R1Is not 2- (methoxycarbonyl) thiophen-3-yl.
2. The compound of claim 1, wherein Q is a group of formula (i).
3. A compound according to claim 1 or 2, wherein Q is a group of formula (i); r2Is H or methyl.
4. The compound of claim 1, wherein Q is a group of formula (ii).
5. A pharmaceutical composition comprising a compound of formula (I) as defined in any one of claims 1 to 4 and one or more pharmaceutically acceptable carriers and/or excipients.
6. A pharmaceutical composition comprising a compound of formula (I) according to any one of claims 1 to 4, suitable for administration by inhalation selected from inhalable powders, propellant-containing metered dose aerosols or propellant-free inhalable formulations.
7. Use of a compound of formula (I) according to any one of claims 1 to 4 for the manufacture of a medicament for the prevention and/or treatment of broncho-obstructive or inflammatory diseases selected from asthma or chronic bronchitis or Chronic Obstructive Pulmonary Disease (COPD).
8. A compound of formula (I) as defined in any one of claims 1 to 4 in combination with one or more active ingredients selected from β 2-agonists, corticosteroids, P38MAP kinase inhibitors, IKK2 inhibitors, HNE inhibitors, PDE4 inhibitors, leukotriene modulators, NSAIDs and mucus regulators.
9. A device comprising a pharmaceutical composition according to claim 8 selected from the group consisting of single or multi-dose dry powder inhalers, metered dose aerosols and soft mist nebulizers.
HK13108338.9A 2010-06-22 2011-05-30 Alkaloid aminoester derivatives and medicinal composition thereof HK1181047B (en)

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EP10166898 2010-06-22
EP10166898.6 2010-06-22
PCT/EP2011/058801 WO2011160918A1 (en) 2010-06-22 2011-05-30 Alkaloid aminoester derivatives and medicinal composition thereof

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