CN117881657A - Phenyl-sulfamoyl-benzoic acid derivatives as ERAP1 modulators - Google Patents

Phenyl-sulfamoyl-benzoic acid derivatives as ERAP1 modulators Download PDF

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CN117881657A
CN117881657A CN202280053138.0A CN202280053138A CN117881657A CN 117881657 A CN117881657 A CN 117881657A CN 202280053138 A CN202280053138 A CN 202280053138A CN 117881657 A CN117881657 A CN 117881657A
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compound
alkyl
cancer
halo
mmol
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马丁·奎贝尔
杰森·约翰·希尔斯
约翰·福特里尔
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Greywolf Treatment Ltd
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Greywolf Treatment Ltd
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Priority claimed from PCT/GB2022/052013 external-priority patent/WO2023007188A1/en
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Abstract

The present invention relates to compounds of formula (I) or a pharmaceutically acceptable salt or hydrate thereof,wherein: the radical X-Y being-NHSO 2 -; z is a monocyclic or polycyclic cycloalkyl group, or a monocyclic or polycyclic heterocycloalkyl group, each of which is optionally substituted with one or more groups selected from haloalkyl, alkyl, alkenyl, alkynyl and- (CR) 16 R 17 ) m R 18 Wherein m is 0 to 6; l is a direct bond or a group (CR) 14 R 15 ) n Wherein n is 1 or 2; r is R 1 Is H, CN, cl or F or alkyl; r is R 2 Selected from COOH and tetrazolyl; r is R 3 Selected from H, halo, alkoxy, and alkyl; r is R 4 Selected from H and halo; r is R 5 Selected from H, alkyl, haloalkyl, SO 2 -alkyl, cl, alkoxy, OH, CN, hydroxyalkyl, alkylthio, heteroaryl, cycloalkyl, heterocycloalkyl and haloalkoxy; r is R 6 Is H; r is R 7 Selected from H, CN, haloalkyl, halo, SO 2 -alkyl, SO 2 NR 12 R 13 Heteroaryl, CONR 10 R 11 And alkyl, wherein the heteroaryl is optionally substituted with one or more groups selected from alkyl, halo, alkoxy, CN, haloSubstituted alkyl and OH; r is R 9 Selected from H, alkyl, and halo; r is R 10 、R 11 、R 12 And R is 13 Each independently is H or alkyl; r is R 14 And R is 15 Each independently is H, halo, or alkyl; r is R 16 And R is 17 Each independently is H, halo, haloalkyl or alkyl; and each R is 18 Independently selected from OH, CN, alkoxy and halo. Other aspects of the invention relate to such compounds for use in immunooncology and related fields of application.

Description

Phenyl-sulfamoyl-benzoic acid derivatives as ERAP1 modulators
Technical Field
The present invention relates to compounds capable of modulating ERAP 1. The compounds have potential therapeutic applications in the treatment of a variety of disorders including proliferative disorders, viral disorders, immune disorders, and inflammatory disorders.
Background
ERAP1 (endoplasmic reticulum aminopeptidase 1; also known as APPILS or ARTS 1) is an aminopeptidase which is important in the production of partial antigens and neoantigens as part of the antigen presentation pathway 1 . The antigen presentation pathway begins with the proteolytic cleavage of a protein into peptides by the proteasome. These peptides are transported to the endoplasmic reticulum where a portion of the peptides are processed by ERAP1 and then bound to the major histocompatibility complex class I (MHC class I) 1 . Then, the antigen bound to MHC class I is transported to the cell surface and presented to CD8 + T cells and are recognized as self or non-self substances. Neoantigens are antigens that are specific for cancer and can be recognized by the immune system as foreign, causing destruction of cancer cells. The production of neoantigens is either a direct result of somatic mutations in cancer cell DNA to produce mutant proteins, or an indirect result of somatic mutations in protein processing and expression. Those cancers with higher mutation rates and correspondingly higher levels of neoantigens inhibit checkpoints than cancers with lower numbers of neoantigens The response rate of the agent immunotherapy anti-PD-1 antibody (such as pembrolizumab, nivolumab), anti-PD-L1 antibody (such as atrazumab, avilamab, dewaruzumab) and anti-CTLA 4 antibody (such as ipilimumab, tremelimumab) is much higher 2，3 。
The role of ERAP1 in the antigen presentation pathway is to cleave a portion of the peptide by its aminopeptidase activity to produce antigen of optimal length for binding to MHC class I and neoantigen. ERAP1 also excessively cleaves some neoantigens, thereby preventing them from binding to MHC class I and presenting on the cell surface 4 . Removal of ERAP1 activity has been shown to alter antigen and neoantigen antigen repertoires, resulting in some antigen/neoantigen presentation and novel enhancement of antigen/neoantigen presentation 5 . Furthermore, removal of ERAP1 causes CD8 in mouse cancer model 4 + T cell dependent tumor rejection 4 。
Thus, modulators of ERAP1 activity, whether used alone or in combination with current cancer immunotherapeutic agents (including checkpoint inhibitors), are useful in cancer therapy because modulators of ERAP1 activity alter and make more visible to the immune system the antigens and neoantigens presented on the surface of cancer cells, thereby rendering the tumor challenged and destroyed.
It has also been shown that the knockdown of ERAP1 reduces the level of regulatory-like T cells and enhances natural killer cell killing of cancer cells 6,7 . This suggests that modulators of ERAP1 activity may enable effective cancer treatment by modulating the visibility of cancer cells and generating a stronger anti-tumor immune response. The peptide treatment of ERAP1 in antigen presentation may also be applied to infectious viral diseases.
Maben et al (J.Med. Chem.2020;63, 103-121) disclose compounds that selectively inhibit ERAP1 but not its paralogues ERAP2 and IRAP. WO 2020/104822, WO 2020/225569, WO 2021/094763 and WO2022/064187 (Grey Wolf Therapeutics Limited) disclose a range of arylsulfonamide compounds capable of modulating ERAP 1.
The present invention seeks to provide further compounds capable of modulating ERAP 1. Such compounds have potential therapeutic applications in the treatment of a variety of disorders including proliferative disorders, immune disorders, and inflammatory disorders.
Disclosure of Invention
The first aspect of the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt or hydrate thereof,
wherein:
the radical X-Y being-NHSO 2 -；
Z is a monocyclic or polycyclic cycloalkyl group, or a monocyclic or polycyclic heterocycloalkyl group, each of which is optionally substituted with one or more groups selected from haloalkyl, alkyl, alkenyl, alkynyl and- (CR) 16 R 17 ) m R 18 Wherein m is 0 to 6;
l is a direct bond or a group (CR) 14 R 15 ) n Wherein n is 1 or 2;
R 1 selected from H, CN, cl, F and alkyl;
R 2 selected from COOH and tetrazolyl;
R 3 selected from H, halo, alkoxy, and alkyl;
R 4 selected from H and halo;
R 5 selected from H, alkyl, haloalkyl, SO 2 -alkyl, cl, alkoxy, OH, CN, hydroxyalkyl, alkylthio, heteroaryl, cycloalkyl, heterocycloalkyl and haloalkoxy;
R 6 is H;
R 7 selected from H, CN, haloalkyl, halo, SO 2 -alkyl, SO 2 NR 12 R 13 Heteroaryl, CONR 10 R 11 And alkyl, wherein the heteroaryl is optionally substituted with one or more substituents selected from alkyl, halo, alkoxy, CN, haloalkyl, and OH;
R 8 selected from H, alkyl, haloalkyl and halo;
R 9 selected from H, alkyl, and halo;
R 10 、R 11 、R 12 and R is 13 Each independently selected from H and alkyl;
R 14 and R is 15 Each independently selected from the group consisting of H, halo, and alkyl;
R 16 and R is 17 Each independently selected from the group consisting of H, halo, haloalkyl, and alkyl; and
each R is 18 Independently selected from OH, CN, alkoxy and halo.
The invention also includes enantiomers of the compounds of formula (I), as well as mixtures of enantiomers, including racemic mixtures.
Advantageously, the presently claimed compounds are capable of modulating ERAP1, thereby making the compounds therapeutically interesting in the treatment of various disorders, for example in the fields of oncology and immunooncology. In particular, the compounds according to the invention exhibit excellent efficacy against ERAP 1.
A second aspect of the invention relates to a pharmaceutical composition comprising at least one compound as described above and a pharmaceutically acceptable carrier, diluent or excipient.
A third aspect of the invention relates to the use of a compound as described above in medicine.
A fourth aspect of the invention relates to the use of a compound as described above for the treatment or prophylaxis of a condition selected from the group consisting of a proliferative condition, an immune condition, a viral condition and an inflammatory condition.
A fifth aspect of the invention relates to the use of a compound as described above for the preparation of a medicament for the treatment or prophylaxis of a condition selected from the group consisting of a proliferative condition, an immune condition, a viral condition and an inflammatory condition.
A sixth aspect of the invention relates to the use of a compound as described above for the prevention or treatment of a condition caused by, associated with or associated with any aberrant ERAP1 activity.
A seventh aspect of the invention relates to the use of a compound as described above for the preparation of a medicament for the prevention or treatment of a condition caused by, associated with or associated with aberrant ERAP1 activity.
An eighth aspect of the invention relates to a method of treating a mammal suffering from a condition ameliorated by the modulation of ERAP1, wherein the method comprises administering to the mammal a therapeutically effective amount of a compound as described above.
A ninth aspect of the invention relates to the use of a compound as described above for the treatment or prophylaxis of a condition ameliorated by the modulation of ERAP 1.
A tenth aspect of the invention relates to the use of a compound as described above in the manufacture of a medicament for the treatment or prevention of a condition ameliorated by the modulation of ERAP 1.
An eleventh aspect of the invention relates to a method of treating or preventing a disorder selected from the group consisting of a proliferative disorder, an immune disorder, a viral disorder, and an inflammatory disorder in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a compound as described above.
Detailed Description
The present invention relates to bisaryl sulfonamide compounds that are capable of modulating ERAP 1.
"alkyl" is defined herein as a straight or branched chain alkyl group, preferably C 1-20 Alkyl, more preferably C 1-12 Alkyl, even more preferably C 1-10 Alkyl or C 1-6 Alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl. Preferably, alkyl is C 1-4 An alkyl group.
"cycloalkyl" is defined herein as a cyclic alkyl ring, preferably C 3-7 Cycloalkyl groups, more preferably C 3-6 Cycloalkyl groups. Preferred examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl, or fused bicyclic ring systems such as norbornane.
"halogen" is defined herein as chlorine, fluorine, bromine or iodine.
"haloalkyl" is defined herein as a compound which is substituted with one or more halogen atoms (which mayThe same or different) (e.g., fluorine, chlorine, bromine, and iodine) substituted straight or branched alkyl groups as defined above (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl). Preferably, the haloalkyl is C 1-20 Haloalkyl, more preferably C 1-12 Haloalkyl, even more preferably C 1-10 Haloalkyl or C 1-6 Haloalkyl or C 1-3 A haloalkyl group. Preferred examples are CF 3 And CHF 2 Wherein CF is 3 Particularly preferred.
"alkoxy" is defined herein as an oxygen atom bonded to an alkyl group as defined above, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy and hexoxy. Preferably, the alkoxy group is C 1-20 Alkoxy, more preferably C 1-12 Alkoxy, even more preferably C 1-10 Alkoxy or C 1-6 Alkoxy or C 1-3 An alkoxy group. A particularly preferred example is methoxy (-OCH) 3 )。
As used herein, the term "alkenyl" refers to straight and branched carbon chains having at least one carbon-carbon double bond. In some embodiments, alkenyl groups may include C 2 -C 12 Alkenyl groups. In other embodiments, the alkenyl group comprises C 2 -C 10 、C 2 -C 8 、C 2 -C 6 Or C 2 -C 4 Alkenyl groups. In one embodiment of alkenyl groups, the number of double bonds is 1 to 3; in another embodiment of alkenyl groups, the number of double bonds is 1. Other ranges of carbon-carbon double bonds and carbon numbers are also contemplated, depending on the position of the alkenyl moiety on the molecule. "C 2 -C 10 Alkenyl "groups may include more than one double bond in the chain.
As used herein, the term "alkynyl" refers to straight and branched carbon chains having at least one carbon-carbon triple bond. In some embodiments, alkynyl groups may include C 2 -C 12 Alkynyl groups. In other embodiments, alkynyl groups include C 2 -C 10 、C 2 -C 8 、C 2 -C 6 Or C 2 -C 4 Alkynyl groups. In one embodiment of alkynyl, the number of triple bonds is 1 to 3; in another embodiment of alkenyl groups, the number of triple bonds is 1. Particularly preferred alkynyl is-C.ident.CH.
As used herein, the term "polycyclic group" refers to a group comprising two or more cyclic groups, which may be fused, unfused, bridged or spiro.
As used herein, the term "aryl" refers to C 6-12 Aromatic groups, which may be benzo-fused groups, such as phenyl or naphthyl.
"heteroaryl" is defined herein as a monocyclic or bicyclic C 2-12 An aromatic ring, which includes one or more heteroatoms (which may be the same or different), such as oxygen, nitrogen, or sulfur. Examples of suitable heteroaryl groups include thienyl, furyl, pyrrolyl, pyridyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, and the like, and their benzo derivatives, such as benzofuranyl, benzothienyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, and the like; or pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, tetrazolyl, etc., and their benzo derivatives, such as quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, etc. Particularly preferred heteroaryl groups include 1H-imidazol-5-yl, 1H-imidazol-4-yl, 1H-imidazol-2-yl, 1H-pyrrol-1-yl, 1H-pyrrol-2-yl, 1H-pyrrol-3-yl, 1H-pyrrol-4-yl, 1H-pyrrol-5-yl, 1H-pyrazol-1-yl, 1H-pyrazol-5-yl, 1H-pyrazol-3-yl, 1H-pyrazol-4-yl, oxazol-2-yl, oxazol-4-yl, oxazol-5-yl, 1H-1,2, 4-triazol-3-yl, 1H-1,2, 4-triazol-5-yl 1H-1,2, 4-triazol-1-yl, 1H-1,2, 3-triazol-4-yl, 1H-1,2, 3-triazol-5-yl, 1H-1,2, 3-triazol-1-yl, thiazol-5-yl, thiazol-4-yl, 1H-1,2,3, 4-tetrazol-4-yl, 2H-1,2,3, 4-tetrazol-5-yl oxazol-5-yl, oxazol-4-yl, oxazol-2-yl, isoxazol-3-yl, isoxazol-4-yl, isoxazol-5-yl, isothiazol-3-yl, isothiazol-4-yl, isothiazol-5-yl, pyridazin-3-yl, pyridazin-4-yl, pyrazinyl, 1,3, 4-oxadiazole -2-yl, 1,3, 4-oxadiazol-5-yl, 1,2, 5-oxadiazol-3-yl, 1,2, 5-oxadiazol-4-yl, 1,2, 3-oxadiazol-5-yl, 1,2, 4-oxadiazol-3-yl, 1,2, 4-oxadiazol-5-yl, isoxazol-4-yl and isoxazol-3-yl.
"heterocycloalkyl" means a cyclic aliphatic radical containing one or more heteroatoms selected from nitrogen, oxygen and sulfur, optionally interrupted by one or more- (CO) -groups in the ring and/or optionally including one or more double bonds in the ring. Preferably, the heterocycloalkyl group is monocyclic or bicyclic. Preferably, the heterocycloalkyl group is C 3-7 Heterocycloalkyl, more preferably C 3-6 A heterocycloalkyl group. Alternatively, the heterocycloalkyl is C 4-7 Heterocycloalkyl, more preferably C 4-6 A heterocycloalkyl group. Preferred heterocycloalkyl groups include, but are not limited to, piperazinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, tetrahydrofuranyl, and tetrahydropyranyl.
When the compounds of the present invention contain one or more chiral centers, the present invention includes all enantiomers and diastereomers thereof, as well as mixtures thereof.
For example, the point of attachment to the Z group (shown here as a simple 5-membered monocyclic alkyl group for purposes of illustration, but generally applies equally to the Z group) may exist in one of the following configurations:
The invention includes compounds of any of the above configurations, as well as mixtures thereof, including racemic mixtures. Those skilled in the art will appreciate that the absolute stereochemistry (R-and S-) of chiral centers, expressed in terms of x, will depend on the nature of the Z group and the nature and position of any substituents on the Z group.
In a preferred embodiment, the compound is in the form of a mixture of R-and S-enantiomers. In a preferred embodiment, the mixture is a racemic mixture, i.e. a 50:50 mixture of compounds of the R-and S-enantiomers.
As known to those skilled in the art, the racemic mixtures can be used to prepare enantiomerically pure R-and S-forms by separation of the enantiomers using standard methods, such as chemical resolution using optically active acids, or column chromatography or reverse phase column chromatography using a substantially optically active (or "chiral") stationary phase. Racemic mixtures can also be used to prepare enantiomerically enriched mixtures of the S-and R-forms. Mixtures enriched in the R-or S-enantiomer may also be obtained from suitable enantiomer-enriched precursors.
In a preferred embodiment of the invention, the compound is in the form of a mixture comprising enantiomers, wherein a weight to weight ratio of at least about 2:1 or more, preferably at least about 5:1 or more, most preferably at least about 10:1 or more, is advantageous for showing enantiomers (eutomer) with significant in vitro and/or in vivo activity.
In one embodiment, the compound is in the form of a mixture comprising the S-enantiomer and the R-enantiomer, wherein the weight to weight ratio of R-enantiomer to S-enantiomer is greater than 1.05:1, more preferably greater than 2:1, even more preferably greater than 5:1, even more preferably greater than 10:1.
In one embodiment, the compound is in the form of a mixture comprising the S-enantiomer and the R-enantiomer, which is substantially enriched in the R-enantiomer.
In one embodiment, the compound is in the form of a mixture comprising the S-enantiomer and the R-enantiomer, wherein the weight to weight ratio of S-enantiomer to R-enantiomer is greater than 1.05:1, more preferably greater than 2:1, even more preferably greater than 5:1, even more preferably greater than 10:1.
In one embodiment, the compound is in the form of a mixture comprising the S-enantiomer and the R-enantiomer, which is substantially enriched in the S-enantiomer.
A compound of formula (I)
One aspect of the present invention relates to compounds of formula (I), and pharmaceutically acceptable salts and hydrates thereof,
wherein:
the radical X-Y being-NHSO 2 -or-SO 2 NH-；
Z is a monocyclic or polycyclic cycloalkyl group, or a monocyclic or polycyclic heterocycloalkyl group, each of which is optionally substituted with one or more groups selected from haloalkyl, alkyl, alkenyl, alkynyl and- (CR) 16 R 17 ) m R 18 Wherein m is 0 to 6;
l is a direct bond or a group (CR) 14 R 15 ) n Wherein n is 1 or 2;
R 1 selected from H, CN, cl, F and alkyl;
R 2 selected from COOH and tetrazolyl;
R 3 selected from H, halo, alkoxy, and alkyl;
R 4 selected from H and halo;
R 5 selected from H, alkyl, haloalkyl, SO 2 -alkyl, cl, alkoxy, OH, CN, hydroxyalkyl, alkylthio, heteroaryl, cycloalkyl, heterocycloalkyl and haloalkoxy;
R 6 is H;
R 7 selected from H, CN, haloalkyl, halo, SO 2 -alkyl, SO 2 NR 12 R 13 Heteroaryl, CONR 10 R 11 And alkyl, wherein the heteroaryl is optionally substituted with one or more substituents selected from alkyl, halo, alkoxy, CN, haloalkyl, and OH;
R 8 selected from H, alkyl, haloalkyl and halo;
R 9 selected from H, alkyl, and halo;
R 10 、R 11 、R 12 and R is 13 Each independently selected from H and alkyl;
R 14 and R is 15 Each independently selected from the group consisting of H, halo, and alkyl;
R 16 and R is 17 Each independently selected from the group consisting of H, halo, haloalkyl, and alkyl; and
each R is 18 Independently selected from OH, CN, alkoxy and halo.
In a preferred embodiment, X-Y is-NHSO 2 -i.e. the compound has the formula:
in a preferred embodiment, L is a direct bond.
In another preferred embodiment, L is (CR 14 R 15 ) n And n is 1 or 2. More preferably, n is 1.
In a preferred embodiment, R 14 And R is 15 Each independently selected from H, cl and Me. More preferably, R 14 And R is 15 All are H.
In another preferred embodiment, L is (CH 2 ) n And n is 1 or 2, more preferably n is 1.
In a highly preferred embodiment, L is CH 2 Or CH (Me), even more preferably CH 2 。
In the following embodiments, wherein Z is a heterocycloalkyl group containing one or more heteroatoms selected from O, S and N, more preferably one or more heteroatoms selected from O and N. Still more preferably, the heterocycloalkyl group contains one or more O atoms. Even more preferably, the heterocycloalkyl group contains one O atom.
In a preferred embodiment, Z is a monocyclic cycloalkyl or a monocyclic heterocycloalkyl, each of which is optionally substituted. Preferably, Z is 3, 4, 5, 6 or 7 membered monocyclic cycloalkyl or monocyclic heterocycloalkyl, each of which is optionally substituted. More preferably, Z is 4, 5 or 6 membered monocyclic cycloalkyl or monocyclic heterocycloalkyl, even more preferably 4 or 5 membered monocyclic cycloalkyl or monocyclic heterocycloalkyl, each of which is optionally substituted. More preferably, Z is a monocyclic cycloalkyl or monocyclic heterocycloalkyl selected from cyclobutyl, cyclopentyl, cyclohexyl, oxetanyl, tetrahydro-2H-pyranyl, azetidinyl and tetrahydrofuranyl. More preferably, Z is a monocyclic cycloalkyl or monocyclic heterocycloalkyl selected from cyclobutyl, cyclopentyl, oxetanyl, azetidinyl and tetrahydrofuranyl.
In a preferred embodiment, Z is 4-membered monocyclic cycloalkyl or 4-membered monocyclic heterocycloalkyl, each of which is optionally substituted.
In a preferred embodiment, Z is an optionally substituted 4-membered monocyclic cycloalkyl. In a preferred embodiment, Z is cyclobutyl optionally substituted by one or more substituents selected from CN, halo, alkyl, haloalkyl, OH and alkoxy. More preferably, Z is optionally one or more selected from CN, F, me, OH, OMe and CF 3 A cyclobutyl group substituted by a substituent of (a). In a highly preferred embodiment, Z is unsubstituted cyclobutyl.
In a preferred embodiment, Z is optionally substituted 4-membered monocyclic heterocycloalkyl. In a preferred embodiment, Z is oxetanyl or azetidinyl, each of which is optionally substituted with one or more substituents selected from alkyl and halo. More preferably, Z is oxetanyl or azetidinyl, each optionally substituted with one or more substituents selected from Me and F.
In a preferred embodiment, Z is 5-membered monocyclic cycloalkyl or 5-membered monocyclic heterocycloalkyl, each of which is optionally substituted.
In a preferred embodiment, Z is an optionally substituted 5-membered monocyclic cycloalkyl. In a preferred embodiment, Z is cyclopentyl optionally substituted with one or more substituents selected from CN, alkynyl, halo, alkyl, OH, and alkoxy. More preferably, Z is cyclopentyl optionally substituted by one or more substituents selected from CN, F, -C≡ CH, me, OH, OMe.
In a preferred embodiment, Z is an optionally substituted 5-membered monocyclic heterocycloalkyl.
In a preferred embodiment, Z is an optionally substituted polycyclic cycloalkyl or an optionally substituted polycyclic heterocycloalkyl, wherein said polycyclic group is fused, unfused, bridged or spiro.
In a preferred embodiment, Z is an optionally substituted spiro group. As used herein, a spiro group refers to a polycyclic group in which two rings are connected through a common atom. The spiro group may be a full carbocycle (both carbon) or a heterocycle (having one or more non-carbon atoms). Preferably, each ring is independently a 3, 4, 5, 6 or 7 membered ring, optionally containing one or more heteroatoms selected from O, N and S. More preferably, each ring is independently a 3, 4, 5 or 6 membered ring, optionally containing one or more heteroatoms selected from O and N. Even more preferably, each ring is independently a 3, 4 or 5 membered ring, optionally containing one or more heteroatoms selected from O and N.
In a preferred embodiment, Z is an optionally substituted bicyclic spiro group, for example an optionally substituted carbocyclic bicyclic spiro group or an optionally substituted heterocyclic bicyclic spiro group. More preferably, the bicyclic spiro group is selected from the following: spiro [3,3] heptane, spiro [2,4] heptane, spiro [2,3] hexane, 1-oxaspiro [2,3] hexane, 4-oxaspiro [2,3] hexane, 2-oxaspiro [3,3] heptane, 1-oxaspiro [3,3] heptane, 4-oxaspiro [2,4] heptane, 5-oxaspiro [2,4] heptane and 1-oxaspiro [2,4] heptane. Even more preferably, the bicyclic spiro group is selected from spiro [3,3] heptane, spiro [2,4] heptane, spiro [2,3] hexane and 2-oxaspiro [3,3] heptane.
In another preferred embodiment, Z is a polycyclic group which is a fused cycloalkyl or fused heterocycloalkyl, each of which is optionally substituted. As used herein, fused cycloalkyl or fused heterocycloalkyl refers to a polycyclic group wherein two or more rings are connected by two adjacent atoms. Preferably, each ring is independently a 3, 4, 5, 6 or 7 membered ring optionally containing one or more heteroatoms selected from O, N and S. More preferably, each ring is independently a 3, 4 or 5 membered ring optionally containing one or more heteroatoms selected from O and N.
In another preferred embodiment, Z is a polycyclic group which is an unfused polycyclic cycloalkyl or an unfused polycyclic heterocycloalkyl, each of which is optionally substituted. As used herein, unfused cycloalkyl or unfused heterocycloalkyl refers to a polycyclic group wherein two or more rings are connected by a direct bond. Preferably, each ring is independently a 3, 4, 5, 6 or 7 membered ring optionally containing one or more heteroatoms selected from O, N and S. More preferably, each ring is independently a 3, 4 or 5 membered ring optionally containing one or more heteroatoms selected from O and N. Preferably, for this embodiment, Z is an unfused bicyclic cycloalkyl or unfused bicyclic heterocycloalkyl, or unfused cycloalkyl-heterocycloalkyl, each of which is optionally substituted. In a particularly preferred embodiment, Z is selected from cyclopropyl-cyclobutyl, cyclopentyl-cyclopropyl, cyclobutyl-cyclobutyl, cyclopropyl-cyclopropyl and cyclopentyl-cyclopentyl. More preferably, Z is 2-cyclopropyl-cyclobutyl or 3-cyclopropyl-cyclobutyl, even more preferably 2-cyclopropyl-cyclobutyl.
In a preferred embodiment, Z is a fused cycloalkyl or fused heterocycloalkyl group selected from: bicyclo [3.1.0] hexane, bicyclo [4.2.0] octane, decalin, bicyclo [4.1.0] heptane, bicyclo [3.2.0] heptane, octahydropentalene, octahydro-1H-indene and (1 s,2s,3s,4s,6s,7 s) -cubane, each of which is optionally substituted.
In another preferred embodiment, Z is a polycyclic group which is a bridged cycloalkyl or bridged heterocycloalkyl, each of which is optionally substituted. As used herein, bridged cycloalkyl or bridged heterocycloalkyl refers to a polycyclic group in which two (or more) rings are connected by two non-adjacent atoms. Preferably, each ring is independently a 3, 4, 5, 6 or 7 membered ring optionally containing one or more heteroatoms selected from O, N and S. More preferably, each ring is independently a 4, 5 or 6 membered ring optionally containing one or more heteroatoms selected from O and N.
In a preferred embodiment, Z is a bridged cycloalkyl or bridged heterocycloalkyl group selected from: bicyclo [1.1.1] pentane, bicyclo [2.1.1] hexane, 2-oxabicyclo [2.2.1] hexane, 5-oxabicyclo [2.1.1] hexane, bicyclo [2.2.1] heptane, bicyclo [3.2.1] octane, bicyclo [2.2.2] octane, 2-oxabicyclo [2.2.2] octane, 7-oxabicyclo [2.2.1] heptane and 2-oxabicyclo [2.2.1] heptane, each of which is optionally substituted. More preferably, Z is a bridged cycloalkyl selected from the group consisting of bicyclo [1.1.1] pentane and 2-oxabicyclo [2.1.1] hexane, each of which is optionally substituted.
In a preferred embodiment, Z is a bicyclic cycloalkyl or a bicyclic heterocycloalkyl, each of which is fused, bridged or spiro, and each of which is optionally substituted.
In the present disclosure, the Z group is optionally substituted with one or more groups selected from halogen, haloalkyl, alkyl, alkenyl, alkynyl and- (CR) 16 R 17 ) m R 18 Wherein m is 0 to 6. Preferably m is 0, 1, 2 or 3, more preferably 0 or 1, even more preferably 0.
In a preferred embodiment, R 16 And R is 17 Each independently selected from H, cl, F, C 1 -C 4 Alkyl and C 1 -C 1 A haloalkyl group. More preferably, R 16 And R is 17 Each independently selected from H, cl, F, CF 3 And Me. Even more preferably, R 16 And R is 17 All are H.
In a preferred embodiment, the Z groups are selected from halogen, C 1-4 -haloalkyl, C 1-4 -alkyl, C 1-4 -alkenyl, C 1-4 Alkynyl and- (CR) 16 R 17 ) m R 18 Wherein m is 0, 1, 2 or 3. More preferably, m is 0.
In a preferred embodiment, R 18 Selected from OH, CN, OMe, cl, br and F.
In a preferred embodiment, the Z groups are selected from one or more of Me, CF 3 、OH、CN、F、OMe、-C≡CH、-CH 2 -C.ident.CH and-CH 2 And the group substitution of CN.
In a preferred embodiment, Z is selected from:
wherein each Q is independently selected from alkyl, alkoxy, haloalkyl, alkynyl, halo, OH, and CN. More preferably, each Q is independently selected from Me, OMe, CF 3 F, OH, C.ident.CH and CN.
In a preferred embodiment, Z is selected from:
wherein each Q is independently selected from alkyl, alkoxy, haloalkyl, alkynyl, halo, OH, and CN. More preferably, each Q is independently selected from Me, OMe, CF 3 F, OH, C.ident.CH and CN.
In a preferred embodiment, L-Z is selected from:
in a preferred embodiment, Z is selected from:
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preferably, for the above embodiments, L is a direct bond. In a preferred embodiment, L is a direct bond and Z is a group selected from Z1 to Z-72 described above.
In another preferred embodiment, L is a group (CR 14 R 15 ) n And L-Z is selected from the following:
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in a highly preferred embodiment, Z is selected from:
in a preferred embodiment, R 1 Selected from H, CN, F and alkyl.
In a preferred embodiment, R 1 Selected from H, CN and alkyl.
In a preferred embodiment, R 1 Is H.
In a preferred embodiment, R 2 Is COOH.
In a particularly preferred embodiment, X is NH and Y is SO 2 . In another preferred embodiment, X is SO 2 And Y is NH. Preferably, X is NH and Y is SO 2 。
In one preference In embodiments of (2), R 3 Selected from H, cl, F, OMe and Me. More preferably, R 3 Selected from H and F. Even more preferably, R 3 H.
In a preferred embodiment, R 4 Selected from H, cl and F. More preferably, R 4 Selected from H and F.
In a preferred embodiment, R 5 Selected from alkyl, haloalkyl, SO 2 -alkyl, cl, alkoxy, OH, CN, hydroxyalkyl, alkylthio, heteroaryl, cycloalkyl, heterocycloalkyl and haloalkoxy.
In a preferred embodiment, R 5 Selected from alkyl, alkoxy and cycloalkyl. More preferably, R 5 Is cycloalkyl, more preferably cyclopropyl, cyclobutyl or cyclopentyl.
In a particularly preferred embodiment, R 5 Selected from OMe, OEt, me, et and cyclopropyl.
In another preferred embodiment, R 5 Selected from H, me, CF 3 、CHF 2 、SO 2 -Me、Cl、MeO、OH、CH 2 OH, SMe, cyclopropyl, triazolyl, oxetanyl and CN. More preferably, R 5 Selected from H, CN, me, SO 2 -Me、CF 3 And CHF 2 、CH 2 OH, SMe, cyclopropyl, 3, 4-triazol-1-yl, oxetan-3-yl. More preferably, R 5 Selected from H, CN, me, SO 2 -Me、CF 3 And CHF 2 。
In another preferred embodiment, R 5 Selected from OMe, me, et, pr and Cl, more preferably OMe or Et.
In a particularly preferred embodiment, R 5 Selected from OMe, et and cyclopropyl.
In a particularly preferred embodiment, R 5 Is cyclopropyl.
In another preferred embodiment, R 5 Is OMe.
In another preferred embodiment, R 5 Is Et.
In a preferred embodiment of the present invention,R 7 selected from H, CN, haloalkyl, cl, F, SO 2 -alkyl, CONR 10 R 11 、SO 2 NR 16 R 17 Heteroaryl and alkyl, wherein the heteroaryl is optionally substituted with one or more substituents selected from alkyl, halo, alkoxy, CN, haloalkyl and OH.
In a preferred embodiment, R 7 Selected from H, CN, haloalkyl, cl, F, SO 2 -alkyl, CONR 10 R 11 、SO 2 NR 12 R 13 Heteroaryl and alkyl, wherein the heteroaryl is optionally substituted with one or more substituents selected from alkyl, halo, alkoxy, CN, haloalkyl and OH.
In a preferred embodiment, R 7 Heteroaryl optionally substituted with one or more substituents selected from alkyl, halo, alkoxy, CN, haloalkyl and OH.
In a preferred embodiment, R 7 Is heteroaryl selected from the group consisting of pyridyl, thienyl, imidazolyl, pyrimidinyl, pyrazolyl, pyrazinyl, pyridazinyl, thiazolyl, isothiazolyl, triazinyl, pyrrolyl, furanyl, oxazolyl, isoxazolyl, oxadiazolyl, tetrazolyl and triazolyl, wherein each heteroaryl is optionally substituted with one or more substituents selected from the group consisting of alkyl, halo, alkoxy, CN, haloalkyl and OH.
In a preferred embodiment, R 7 Is heteroaryl selected from imidazolyl, pyrazolyl, pyrazinyl, pyridazinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, tetrazolyl, and triazolyl, wherein each heteroaryl is optionally substituted with one or more substituents selected from alkyl, halo, alkoxy, CN, haloalkyl, and OH.
In a preferred embodiment, R 7 Is selected from 1H-imidazol-5-yl, 1H-imidazol-4-yl, 1H-imidazol-2-yl, 1H-pyrrol-1-yl, 1H-pyrrol-2-yl, 1H-pyrrol-3-yl, 1H-pyrrol-4-yl, 1H-pyrrol-5-yl, 1H-pyrazol-1-yl, 1H-pyrazol-5-yl, 1H-pyrazol-3-yl, 1H-pyrazol-4-yl, oxazol-2-yl-a group, oxazol-4-yl, oxazol-5-yl, 1H-1,2, 4-triazol-3-yl, 1H-1,2, 4-triazol-5-yl, 1H-1,2, 4-triazol-1-yl, 1H-1,2, 3-triazol-4-yl, 1H-1,2, 3-triazol-5-yl, 1H-1,2, 3-triazol-1-yl, thiazol-5-yl, thiazol-4-yl, 1H-1,2,3, 4-tetrazol-4-yl, 2H-1,2,3, 4-tetrazol-5-yl, tetrazol-1-yl, oxazol-5-yl, oxazol-4-yl, oxazol-2-yl, isoxazol-3-yl, isoxazol-4-yl, isoxazol-5-yl, isothiazol-3-yl, isothiazol-4-yl, isothiazol-5-yl, pyridazin-3-yl, pyridazin-4-yl, pyrazin-2, 3-oxazol-4-yl, 2-oxazol-3-yl, 2, 4-oxazol-5-yl, 2-oxazol-3-yl, 2, 3-oxazol-4-yl, 2, 3-oxazol-4-yl, wherein each heteroaryl is optionally substituted with one or more substituents selected from alkyl, halo, CN, alkoxy, haloalkyl, and OH.
In a highly preferred embodiment, R 7 Is a heteroaryl selected from the group consisting of 1H-pyrazol-5-yl, 1H-pyrazol-3-yl, 1H-pyrazol-4-yl, oxazol-2-yl, 1H-1,2, 3-triazol-4-yl, 1H-1,2, 3-triazol-5-yl, thiazol-5-yl, 1H-1,2,3, 4-tetrazol-4-yl, 2H-1,2,3, 4-tetrazol-5-yl, tetrazol-1-yl, isoxazol-4-yl, isoxazol-5-yl, isothiazol-5-yl, pyridazin-3-yl, pyridazin-4-yl, pyrazinyl and 1,3, 4-oxadiazol-2-yl, wherein each heteroaryl is optionally substituted with one or more substituents selected from Me, F, cl, CN and MeO.
In a highly preferred embodiment, R 7 Is a heteroaryl group selected from 1H-1,2,3, 4-tetrazol-4-yl, tetrazol-1-yl and 2H-1,2,3, 4-tetrazol-5-yl, each of which is optionally substituted with one or more substituents selected from Me, F, cl, CN and MeO.
In a preferred embodiment, R 7 Selected from H, CN, haloalkyl, cl, F, SO 2 -alkyl, CONR 10 R 11 Heteroaryl and alkyl, wherein the heteroaryl is selected from the group consisting of pyrazol-3-yl, pyrazol-4-yl, pyrazol-5-yl, isothiazol-3-yl, isothiazol-4-yl, isothiazol-5-yl, 1,2, 4-triazol-5-yl, tetrazol-1-yl, tetrazol-5-yl, isoxazolOxazol-3-yl, isoxazol-4-yl and isoxazol-5-yl, each optionally substituted with one or more substituents selected from alkyl, halo, alkoxy, CN, haloalkyl and OH.
In a preferred embodiment, R 7 Selected from CN, haloalkyl, SO 2 -alkyl, SO 2 NR 16 R 17 、CONR 10 R 11 And tetrazolyl.
In a preferred embodiment, R 7 Selected from CN, haloalkyl, SO 2 -alkyl, SO 2 NR 12 R 13 、CONR 10 R 11 And tetrazolyl.
In a preferred embodiment, R 7 Selected from CF 3 、CN、CONH 2 1H-1,2,3, 4-tetrazol-1-yl, SO 2 NH 2 And SO 2 Me。
In a preferred embodiment, R 7 Selected from H, CN, CF 3 、CHF 2 、Cl、F、SO 2 -Me、CONH 2 、SO 2 NH 2 Heteroaryl and Me. More preferably, R 7 Selected from H, CN, me, SO 2 -Me、CONH 2 、SO 2 NH 2 Tetrazolyl, CF 3 And CHF 2 。
In another preferred embodiment, R 7 Selected from CN, haloalkyl, SO 2 -alkyl, SO 2 NH 2 、CONR 10 R 11 And tetrazolyl. More preferably, for this embodiment, R 7 Selected from CF 3 、CN、SO 2 NH 2 1H-1,2,3, 4-tetrazol-1-yl, CONH 2 And SO 2 Me, more preferably selected from CF 3 CN, 1H-1,2,3, 4-tetrazol-1-yl and SO 2 Me。
In a preferred embodiment, R 7 Is haloalkyl or heteroaryl, more preferably tetrazolyl, isothiazolyl or isoxazolyl, each of which is optionally substituted.
In a preferred embodiment, R 7 Selected from tetrazol-1-yl, isothiazol-5-yl and isoxazol-4-yl, each of which is optionallySubstituted with one or more substituents selected from alkyl, halo, alkoxy, CN, haloalkyl and OH.
In a preferred embodiment, R 7 Selected from CN, CF 3 Tetrazol-1-yl, isothiazol-5-yl and 5-methyl-isoxazol-4-yl.
In another preferred embodiment, R 7 Is haloalkyl, more preferably CF 3 。
In a particularly preferred embodiment, R 7 Is CN.
In another preferred embodiment, R 7 Is SO 2 -alkyl, more preferably SO 2 Me。
In another preferred embodiment, R 7 Is SO 2 NR 16 R 17 More preferably SO 2 NH 2 。
In another preferred embodiment, R 7 Is SO 2 NR 12 R 13 More preferably SO 2 NH 2 。
In a preferred embodiment, R 8 Selected from H, alkyl, haloalkyl and Cl.
In another preferred embodiment, R 8 Selected from H, alkyl and halo.
In a preferred embodiment, R 8 Selected from H, cl, F and Me. More preferably, R 8 Selected from H, cl and F.
In another preferred embodiment, R 8 Selected from alkyl and halo. More preferably, R 8 Selected from Me, cl and F.
In a preferred embodiment, R 8 Is H or haloalkyl, more preferably H or CF 3 Even more preferably H.
In a particularly preferred embodiment, R 8 Is Cl.
In a preferred embodiment, R 9 Selected from H, F, cl and Me, more preferably H and F. Even more preferably R 9 H.
In a preferred embodiment，R 1 、R 3 、R 4 、R 6 、R 8 And R is 9 All are H.
In a preferred embodiment, R 10 And R is 11 Each independently is H or Me. More preferably, R 10 And R is R 11 All are H.
In a preferred embodiment, R 12 And R is 13 Each independently is H or Me. More preferably, R 12 And R is R 13 All are H.
In a particularly preferred embodiment:
X-Y is NH-SO 2 ；
R 1 Is H;
R 2 is COOH;
R 3 is H or F;
R 4 is H or F;
R 5 selected from OMe, OEt, me, et and cyclopropyl, more preferably from OMe, cyclopropyl and Et;
R 6 is H;
R 7 selected from CN, tetrazol-1-yl, isothiazol-5-yl and 5-methyl-isoxazol-4-yl;
R 8 selected from H, cl and F;
R 9 selected from H, me, cl, and F; and
l and Z are as defined above.
In a particularly preferred embodiment:
X-Y is NH-SO 2 ；
L is selected from direct bond, CH 2 And CHMe;
R 1 is H;
R 2 is COOH;
R 3 is H or F;
R 4 is H or F;
R 5 selected from OMe, OEt, me, et and cyclopropyl, more preferably from Ome, cyclopropyl and Et;
R 6 is H;
R 7 selected from CN, tetrazol-1-yl, isothiazol-5-yl and 5-methyl-isoxazol-4-yl;
R 8 selected from H, cl and F;
R 9 selected from H, me, cl, and F; and
z is as defined above, and is more preferably selected from Z-1, Z-2 and Z-3.
In a preferred embodiment, the compounds of the invention have the formula (Ia):
Wherein L and Z are as defined according to any of the above embodiments.
In a preferred embodiment, the compound of formula (I) is selected from the following formulae and pharmaceutically acceptable salts and hydrates thereof. In the structures described herein, where the absolute stereochemistry of the bond is known (e.g., derived from a true chiral starting material), the partitioning is represented by (R) or (R) in a conventional manner. In the case of an unknown absolute configuration, the bond is drawn in the plane (flat), with the addition of known descriptors, such as "trans racemate", "trans relative configuration", "trans diastereoisomer D1", etc.:
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in a preferred embodiment, the compounds of the invention exhibit an IC of 100nM to 500nM, more preferably less than 100nM, for the decapeptide WRVYEKC (Dnp) ALK-acid (where Dnp is dinitrophenyl maleimide) (10 mer) 50 . Further details of the assay are described in the accompanying examples.
In a preferred embodiment, the compounds of the present invention are selected from the group consisting of compounds 1, 4, 6, 8-13,16-37, 39-42, 44-51, 53-65, 68-94, 96-100, 102, 103, 105-111, 113-115, 117-126, 128, 130-134, 135-138 and 140-145.
In a more preferred embodiment, the compounds of the present invention are selected from the following compounds 1, 8-12, 16-18, 20, 22-25, 27, 29, 33-35, 41, 42, 44, 47-48, 50, 51, 56-64, 69-74, 76-90, 92, 94, 96-100, 102, 103, 105-110, 113-115, 117-121, 123-126, 128, 131, 134, 136-138, 140-142 and 144.
Therapeutic application
Another aspect of the invention relates to the use of a compound as described herein in medicine. As described in more detail below, the compounds have particular utility in the fields of oncology and immunooncology.
Another aspect of the invention relates to the use of a compound as described herein in the treatment or prevention of a disorder selected from the group consisting of a proliferative disorder, an immune disorder, an inflammatory disorder, and a viral disorder.
In a preferred embodiment, the compounds of the invention modulate ERAP1.
In one embodiment, the compound inhibits the activity of ERAP1.
In an alternative embodiment, the compound increases the activity of ERAP1.
In one embodiment, the compounds of the invention may alter the repertoire of presented antigens.
One aspect of the invention relates to the use of a compound as described herein in the treatment of a proliferative disorder. Preferably, the proliferative disorder is cancer or leukemia.
The cancer may be selected from: basal cell carcinoma, biliary tract carcinoma; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasms; kidney (renal) cancer; laryngeal carcinoma; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous carcinoma); melanoma; a myeloma; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancer; vulvar cancer; lymphomas, including hodgkin's lymphomas and non-hodgkin's lymphomas, as well as B-cell lymphomas (including low grade/follicular non-hodgkin's lymphomas (NHL)); small Lymphocytes (SL) NHL; intermediate grade/follicular NHL; middle-grade diffuse NHL; advanced immunoblastic NHL; higher lymphoblastic NHL; advanced small non-lytic cell NHL; large mass (NHL); mantle cell lymphoma; AIDS-related lymphomas; macroglobulinemia in fahrenheit; chronic Lymphocytic Leukemia (CLL); acute Lymphoblastic Leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; other carcinomas and sarcomas; and post-transplant lymphoproliferative disorders (PTLD), abnormal vascular proliferation associated with plaque hemorrhoids hamartomas (phakomatos), oedema (e.g., oedema associated with brain tumors), and migraines syndrome.
Without wishing to be bound by theory, it is understood that the ERAP1 modulator is capable of altering the repertoire of antigens and neoantigens of cancer cells by at least 10% as determined using immunopeptidography and mass spectrometry. About 50% of this change is up-regulation in presentation of certain antigens and neoantigens, while the other 50% are completely new antigensAnd presentation of neoantigens. Both of these changes result in increased tumor visibility into the immune system, thereby causing CD8 + T cell repertoire and CD8 + Measurable changes in T cell activation state. CD8 + This change in T cell response leads to immune-mediated tumor clearance and can potentially be enhanced by combination with cancer therapies such as antibody checkpoint inhibitors (e.g., anti-PD-1).
Without wishing to be bound by theory, it is understood that the modulator of ERAP1 causes killing of cancer cells by Natural Killer (NK) cells due to disruption of the interaction between the lectin-like receptor CD94-NKG2A on killer cells Ig-like receptor (KIR) or NK cells and the classical or atypical MHC-I-peptide (pMHC-I) complex on cancer cells.
In a preferred embodiment, the condition is cancer and the compound increases the visibility of cancer cells to the immune system by altering the repertoire of antigens and neoantigens presented to the immune system.
Another aspect of the invention relates to a method of improving the visibility of cancer cells to the immune system of a subject by altering the repertoire of antigens and neoantigens presented to the immune system, the method comprising administering to the subject a compound of formula (I).
In a preferred embodiment, the compound increases CD8 + T cell response to cancer cells.
In a preferred embodiment, the compounds of the invention are used for the treatment of the following diseases: diseases in which uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune responses or inappropriate cellular inflammatory responses are mediated by the ERAP1 pathway, particularly where uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune responses or inappropriate cellular inflammatory responses are mediated by the ERAP1 pathway.
In a preferred embodiment, the disorder of uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune response or inappropriate cellular inflammatory response is selected from hematological tumors, solid tumors and/or metastases thereof.
More preferably, the compounds are used to treat a disorder selected from the group consisting of: leukemia and myelodysplastic syndrome, malignant lymphomas, head and neck tumors including brain tumors and brain metastases, breast tumors including non-small cell lung tumors and small cell lung tumors, gastrointestinal tumors, endocrine gland tumors, breast tumors and other gynaecological tumors, urological tumors including kidney tumors, bladder tumors and prostate tumors, skin tumors and sarcomas and/or metastases thereof.
The compounds may kill cancer cells, reduce the number of proliferating cells in cancer and/or reduce the volume or size of tumors (including cancer cells). The compounds can reduce the number of metastatic cancer cells.
In one embodiment, the compounds are useful for (or are useful for) treating cancer in a subject previously afflicted with cancer. The compounds are useful for reducing the likelihood of cancer recurrence, or the likelihood of regressing. The compounds may induce neoantigens in recurrent cancer or further cancer to which the subject already has an existing immune response. As such, the compounds may enhance or potentiate an immune response against cancer.
In one embodiment, the compounds are used to prevent cancer. The compounds are useful for preventing the development of cancer. That is, the compounds may stimulate an immune response, such as a vaccine response, against future cancers. The compound can stimulate an immune response against the neoantigen in the subject. Once a subject has cancer, the subject can be treated again with the compound (or a different compound) to stimulate the production of the same neoantigen, thereby eliciting a preexisting immune response in the subject to the neoantigen to treat or prevent the cancer.
The same or different compounds may be used before and after the subject has suffered from cancer.
In one embodiment, the compounds are useful for preventing cancer.
In one embodiment, the subject may have previously had cancer, may have a family history of cancer, may have a high risk of having cancer, may have a genetic predisposition to having cancer, or may have been exposed to a carcinogen. In one embodiment, the subject may be in remission of cancer.
One embodiment provides antigen presenting cells, such as Dendritic Cells (DCs), that are produced ex vivo. Antigen presenting cells may be generated ex vivo to present neoantigens, such as neoantigens produced by compounds according to the invention. The compounds are useful in methods of ex vivo production of antigen presenting cells that present neoantigens, and wherein the cells are useful as anti-cancer vaccines.
Antigen presenting cells such as dendritic cells may be pulsed or loaded with neoantigens, or genetically modified (by DNA or RNA transfer) to express one, two or more neoantigens. Methods for preparing dendritic cell vaccines are known in the art. Neoantigens may be produced from normal tissues of a subject, wherein ERAP1 is modulated with a compound according to the invention. The source of normal tissue may be fibroblasts or B cells, for example, cells that can be readily expanded in vitro. Alternatively, RNA from cancer, total RNA enriched in poly A) + RNA, or mRNA may be used. Poly a + RNA can also be amplified to generate enough antigen for DC loading to limit the ex vivo culture step.
In one embodiment, dendritic cells that have been treated with a compound as described above can be used to treat a subject. The dendritic cells can be contacted with the compound ex vivo and then the dendritic cells can be administered to a subject. Thus, the compounds can be used in vitro or in vivo, for example, for in situ treatment, or for ex vivo treatment followed by administration of the treated cells to a subject.
Another aspect of the invention relates to the use of a compound as described above in the treatment of an immune disorder. In a preferred embodiment, the immune disorder is an autoimmune disorder.
Examples of autoimmune disorders include, but are not limited to: rheumatoid Arthritis (RA), myasthenia Gravis (MG), multiple Sclerosis (MS), systemic Lupus Erythematosus (SLE), autoimmune thyroiditis (hashimoto's thyroiditis), graves' disease, inflammatory bowel disease, autoimmune uveitis, polymyositis and certain types of diabetes, systemic vasculitis, polymyositis-dermatomyositis, systemic sclerosis (scleroderma), sjogren's syndrome, ankylosing spondylitis and related spinal arthropathy, rheumatic fever, allergic pneumonia, allergic bronchopulmonary aspergillosis, inorganic pneumoconiosis (inorganic dust rimethylamine), sarcoidosis, autoimmune hemolytic anemia (autoimmune rimethyla anemia), immune platelet disorders, cold diseases such as cold fibrinogen, psoriasis, behcet's disease, avian bullet-like chorioretinopathy and autoimmune polycystic adenomatosis.
Polymorphisms in the ERAP1 gene that affect ERAP1 enzymatic activity are closely related to increased risk of autoimmunity, including ankylosing spondylitis, psoriasis, behcet's disease, and avian bullet-like chorioretinopathy 11 . ERAP1 variants that reduce ERAP1 enzymatic activity may prevent disease, whereas ERAP1 variants that have been reported to have increased activity are associated with increased risk of disease 12 . This suggests that modulation of ERAP1 activity may be an effective treatment for autoimmune diseases.
Thus, in a preferred embodiment, the immune disorder is selected from ankylosing spondylitis, psoriasis, behcet's disease and avian bullet-like chorioretinopathy.
In a preferred embodiment, the immune disorder is ankylosing spondylitis. Ankylosing Spondylitis (AS) is an arthritis in which there is a long-term inflammation of the spinal joint. Typically, the joints where the spine connects to the pelvis are also affected. Sometimes involving other joints such as the shoulders or buttocks. Between 0.1% and 1.8% of people are affected by ankylosing spondylitis and usually have had attacks in young and young adults. Although the cause of ankylosing spondylitis is unknown, it involves a combination of genetic and environmental factors. Over 90% of the affected individuals have a specific human leukocyte antigen known as the HLA-B27 antigen. 13 Furthermore, together with HLA-B27, certain variants of ERAP1 are clearly associated with increased or decreased risk of disease, providing evidence of a clear role for modulated antigen presentation in disease. 18 Ankylosing spondylitis cannot be cured, but is treated at presentThe treatment is only used to improve symptoms and prevent deterioration. Drugs used to date include NSAIDs, steroids, DMARDs (e.g. sulfasalazine) and biological agents (e.g. infliximab).
In a preferred embodiment, the immune disorder is Behcet's Disease (BD). Behcet's Disease (BD) is an inflammatory condition that affects multiple parts of the body. The most common symptoms include painful canker sores, genital ulcers, ocular inflammation and arthritis. The etiology is not well defined, but genetic studies indicate that the risk of disease increases in patients carrying HLA-B51 along with the specific variant of ERAP1, although environmental factors play a role. 19 The disease is mainly characterized by autoinflammation of blood vessels, and is therefore sometimes referred to as autoinflammatory disease. Behcet's disease is currently incurable, but can be controlled with drugs that reduce inflammation at the affected area of the body, such as corticosteroids, immunosuppressants, or biotherapeutic agents targeting biological processes involved in the inflammatory process. In a preferred embodiment, the immune disorder is shotgun elastic-like chorioretinopathy. Avian bullet chorioretinopathy (also known as avian bullet uveitis or HLA-A29 uveitis) is a rare form of bilateral posterior uveitis that infects the eye. Bird gun-like chorioretinopathy causes severe progressive inflammation of the choroid and retina. Symptoms include mosquito, blurred vision, dysphotopsia (sparkling sensation in the line of sight), loss of color vision, and night blindness. Avian bullet-like chorioretinopathy is considered an autoimmune disease. The disease has strong correlation with human leukocyte antigen Haplotype (HLA) -A29. This suggests a role for T lymphocytes in pathogenesis. Avian bullet chorioretinopathy is associated with IL-17, a marker cytokine for TH17 cells, which plays an important role in autoimmunity. 15,16 Whole genome association studies have determined HLA-A29:02 as the primary risk factor and that both ERAP1 and ERAP2 are associated with shotgun elastic-like chorioretinopathy. 17,20 Genetic variants within the ERAP1 and ERAP2 loci regulate enzymatic activity and mRNA and protein expression. ERAP2 is an aminopeptidase which, together with ERAP1 cleaves peptides together in the endoplasmic reticulum and loads these peptides on HLA molecules for presentation to T cells of the immune system.
In a preferred embodiment, the immune disorder is psoriasis. Psoriasis is a chronic skin disease in which skin cells accumulate rapidly on the surface of the skin, forming itching and sometimes painful squamous and red patches. The etiology is not well defined but includes environmental and genetic factors. HLA-C06 is closely related to the risk of disease, and variants in ERAP1 (possibly together with HLA-C06) are also closely related to disease. 21 Psoriasis is not curable, and current treatments are only used to ameliorate symptoms and prevent exacerbations. Drugs used in therapy include steroids, methotrexate, sulfasalazine and biological agents (e.g., etanercept).
Another aspect of the invention relates to the use of a compound as described above for the treatment or prophylaxis of a viral disorder. The ERAP1 modulators, such as the compounds described herein, are capable of altering the antigen repertoire of a variety of viruses, which allows identification and destruction of virus-infected cells. Thus, the ERAP1 modulator has potential therapeutic applications in the treatment of viral infections and diseases. ERAP1 modulates certain viral antigens, including antigens from Human Papilloma Virus (HPV), human Cytomegalovirus (CMV), hepatitis C (HCV), and Human Immunodeficiency Virus (HIV) 8 ,9 ,10 . In addition, knockdown of ERAP1 in HPV infected cells alters the repertoire of presented HPV antigens, thereby causing passage through CD8 + Stronger recognition of T cells 8 。
In a preferred embodiment, the viral disorder is a viral disease or viral infection selected from HIV, HPV, CMV and HCV.
In a preferred embodiment, the viral disorder is HIV.
In a preferred embodiment, the viral disorder is HPV.
In a preferred embodiment, the viral disorder is CMV.
In a preferred embodiment, the viral disorder is HCV.
Another aspect relates to the use of a compound described herein in the prevention or treatment of a disorder caused by aberrant activity to ERAP1, a disorder associated with aberrant activity to ERAP1, or a disorder accompanied by aberrant activity to ERAP 1.
Another aspect relates to the use of a compound described herein in the prevention or treatment of an ERAP 1-related disease or disorder.
Yet another aspect relates to the use of a compound described herein in the manufacture of a medicament for the prevention or treatment of a disorder caused by any aberrant activity to ERAP1, a disorder associated with any aberrant activity to ERAP1, or a disorder accompanied by any aberrant activity to ERAP 1.
As used herein, the phrase "preparation of a drug" includes: in addition to the use of the components of the invention in any stage of the preparation of such a medicament, the use of the components of the invention directly as a medicament is also included.
Another aspect relates to the use of a compound as described above in the manufacture of a medicament for the treatment or prophylaxis of a condition selected from the group consisting of a proliferative condition, an immune condition, a viral condition and an inflammatory condition.
Yet another aspect relates to the use of a compound described herein in the manufacture of a medicament for the prevention or treatment of an ERAP 1-related disease or disorder.
Another aspect of the invention relates to a method of treating an ERAP 1-related disease or disorder in a subject. As described in detail below, the methods according to this aspect of the invention are accomplished by administering to a subject in need of treatment a therapeutically effective amount of a compound of the invention as described above (the compound itself, or more preferably, as part of a pharmaceutical composition, in admixture with, for example, a pharmaceutically acceptable carrier).
Yet another aspect of the invention relates to a method of treating a subject having a condition ameliorated by the modulation of ERAP1, wherein the method comprises administering to the subject a therapeutically effective amount of a compound of the invention.
Another aspect relates to a method of treatment for alleviating a condition by modulating ERAP1, wherein the method comprises administering to a subject a therapeutically effective amount of a compound according to the invention.
Preferably, the subject is a mammal, more preferably a human.
The term "method" refers to means, techniques and procedures for accomplishing a given task including, but not limited to, those known to, or readily developed by those skilled in the chemical, pharmaceutical, biological, biochemical and medical arts.
As used herein, the term "treating" includes eliminating, substantially inhibiting, slowing, or reversing the progression of a disease or disorder, substantially ameliorating a clinical symptom of a disease or disorder, or substantially preventing the appearance of a clinical symptom of a disease or disorder.
As used herein, the term "preventing" refers to a method of preventing a condition or disease from occurring in an original organism.
The term "therapeutically effective amount" means that the amount of the compound administered will alleviate one or more symptoms of the disease or disorder being treated to some extent.
For any compound used in the present invention, a therapeutically effective amount is also referred to herein as an effective therapeutic dose, which can be estimated initially by cell culture assays. For example, a dose may be administered to an animal model to achieve a circulating concentration range that includes IC as determined by cell culture 50 Or IC (integrated circuit) 100 . Such information can be used to more accurately determine useful doses for humans. The initial dose can also be estimated from in vivo data. With these preliminary guidelines, one of ordinary skill in the art will be able to determine an effective dose for use in humans.
In addition, by standard pharmaceutical techniques performed in cell culture or experimental animals (e.g., by determining LD 50 And ED 50 ) Toxicity and efficacy of the compounds described herein can be determined. The dose ratio between toxicity and efficacy is the therapeutic index and can be expressed as LD 50 With ED 50 The ratio between them. Compounds exhibiting high therapeutic indices are preferred. From thisThe data obtained in these cell culture experiments and animal studies can be used to determine the dosage range that does not produce toxicity for human use. The dosage of the compound is preferably at a circulating concentration (including ED 50 ) Is within the scope of (2). The dosage may vary within this range depending upon the dosage form employed and the route of administration employed. The exact dosage form, route of administration and dosage may be selected by each physician according to the patient's condition (see, e.g., fingl et al,1975,The Pharmacological Basis of Therapeutics,chapter1,page1).
The dosages and intervals can be individually adjusted so as to provide a plasma level of the active compound sufficient to maintain therapeutic effect. Typical patient dosages for oral administration range from about 50 mg/kg/day to 2000 mg/kg/day, typically from about 100 mg/kg/day to 1000 mg/kg/day, preferably from about 150 mg/kg/day to 700 mg/kg/day, and most preferably from about 250 mg/kg/day to 500 mg/kg/day. Preferably, a therapeutically effective serum level is achieved by administering multiple doses per day. In the case of topical administration or selective absorption, the effective local concentration of the drug may be independent of plasma concentration. Those of skill in the art are able to optimize therapeutically effective topical dosages without undue experimentation.
As used herein, "ERAP 1-related disease or disorder" refers to a disease or disorder characterized by inappropriate ERAP1 activity. Inappropriate activity refers to an increase or decrease in ERAP1 activity caused by a change in the sequence of the ERAP1 protein relative to wild-type ERAP1 (Uniprot ID Q9NZ 08) as determined by enzymatic or cellular assays. Inappropriate activity may also be due to overexpression of ERAP1 in diseased tissue compared to healthy adjacent tissue.
Preferred diseases or conditions for which the compounds described herein are useful in prophylaxis include proliferative disorders, viral disorders, immune disorders and inflammatory disorders as described above.
Accordingly, the present invention further provides the use of a compound as defined herein in the manufacture of a medicament for the treatment of a disease in which modulation of ERAP1 is desired. These diseases include proliferative disorders, viral disorders, immune disorders, and inflammatory disorders as described above.
In a preferred embodiment, the compound activates the ERAP1 conversion of (L) -leucine-7-amido-4-methylcoumarin (L-AMC) to (L) -leucine and the fluorescent molecule 7-amino-4-methylcoumarin. Although the same assay may also determine inhibitors of ERAP1 cleavage of amide bonds in L-AMC, for the purposes of this application this assay is referred to as the "L-AMC activator assay". The potency of all activators was calculated and expressed as the ratio of the enzymatic activity of ERAP1 to its baseline level (i.e., EC 50 ) The activator concentration required was increased by 50%.
In a preferred embodiment, the compounds exhibit an EC of less than about 25 μm in the L-AMC activator assay 50 Values. More preferably, the compounds exhibit an EC of less than about 10. Mu.M, more preferably less than about 5. Mu.M, even more preferably less than about 1. Mu.M, even more preferably less than about 0.1. Mu.M, even more preferably less than about 0.01. Mu.M in the L-AMC activator assay 50 Values.
In a preferred embodiment, the compound inhibits the ability of ERAP1 to hydrolyze the decapeptide substrate wrvyekcdnp alk. The peptide has minimal fluorescence because the fluorescence of the N-terminal tryptophan residue is quenched by the Dinitrophenol (DNP) residue within the peptide. However, as ERAP1 hydrolyzes the N-terminal amide bond and releases tryptophan, this internal quenching is lost and the reaction is monitored by an increase in tryptophan fluorescence during the assay. For the purposes of this application, this assay is referred to as a "10mer inhibition assay" and, as is well known to those skilled in the art, compound potency is calculated and expressed as IC 50 。
In a preferred embodiment, the compounds exhibit an IC of less than about 25. Mu.M in a 10mer assay 50 Values. More preferably, the compounds exhibit an IC in a 10mer assay of less than about 10 μm, more preferably less than about 5 μm, even more preferably less than about 1 μm, even more preferably less than about 0.1 μm, even more preferably less than about 0.01 μm 50 Values. In a preferred embodiment, the compounds exhibit an IC of about 100nM to about 500nM, more preferably less than 100nM, in a 10mer assay 50 Values.
Pharmaceutical composition
For use in the present invention, a compound described herein, or a physiologically acceptable salt, ester or other physiologically functional derivative thereof, may be prepared as a pharmaceutical formulation comprising the compound or a physiologically acceptable salt, ester or other physiologically functional derivative thereof, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic and/or prophylactic ingredients. The carrier or carriers must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The pharmaceutical compositions may be used for human or animal use in human medicine and veterinary medicine.
Examples of such suitable excipients for use in the various forms of pharmaceutical compositions described herein can be found in "Handbook of Pharmaceutical Excipients (handbook of pharmaceutical excipients), 2 nd edition, (1994)", edited by a Wade and PJ Weller. The carrier, or each carrier if more than one is present, must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.
Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences (rest pharmaceutical science), mark (Mack) publishing company (a.r. gennaro editions, 1985).
Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.
The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include any suitable one or more of a binder, lubricant, suspending agent, coating agent, solubilizing agent, buffering agent, flavoring agent, surfactant, thickener, preservative (including antioxidant), etc., as well as a substance included to make the formulation isotonic with the blood of the intended recipient as a carrier, excipient, or diluent, or any suitable binder, lubricant, suspending agent, coating agent, solubilizing agent, buffering agent, flavoring agent, surfactant, thickener, preservative (including antioxidant), etc., as well as a substance included to make the formulation isotonic with the blood of the intended recipient in addition to the carrier, excipient, or diluent.
Examples of suitable binders include starch, gelatin, natural sugars (e.g., glucose, lactose anhydrous, lactose free flow, beta-lactose, corn flavoring agents), natural and synthetic gums (e.g., acacia, tragacanth or sodium alginate), carboxymethylcellulose, and polyethylene glycol.
Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
Preservatives, stabilizers, dyes, and even flavoring agents may be added to the pharmaceutical compositions. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.
Pharmaceutical formulations include those suitable for oral administration, topical administration (including transdermal, buccal and sublingual administration), rectal or parenteral administration (including subcutaneous, intradermal, intramuscular and intravenous administration), nasal and pulmonary administration (e.g. by inhalation). Where appropriate, the formulations may conveniently be presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active compound with the liquid carrier and/or finely divided solid carrier and then, if necessary, shaping the product into the desired formulation.
Pharmaceutical formulations suitable for oral administration, wherein the carrier is a solid, are most preferably provided in the form of unit dose formulations, such as pills, capsules or tablets each containing a predetermined amount of the active compound. Tablets may be made by compression or molding, optionally with one or more additional ingredients. The compressed tablet may be prepared by: the active compound in free-flowing form (e.g. powder or granules) may optionally be compressed after mixing with binders, lubricants, inert diluents, lubricants, surfactants or dispersants in a suitable machine. Molded tablets may be obtained by molding the active compound with an inert liquid diluent. The tablets may optionally be coated and, if uncoated, optionally scored. The capsules can be prepared by the following method: the active compound alone or in a mixture with one or more additional ingredients is filled into a capsule shell, which is then sealed in the usual manner. Cachets are similar to capsules in which the active compound and any one or more additional ingredients are sealed in a rice paper wrapper. The active compounds can also be formulated as dispersible granules, which can be suspended in water, for example, prior to application, or sprinkled on food. The capsule may be enclosed in a pouch, for example. Formulations suitable for oral administration wherein the carrier is liquid may be provided as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water liquid emulsions.
Formulations for oral administration include controlled release dosage forms (e.g., tablets) in which the active compound is formulated in a suitable controlled release matrix or coated with a suitable controlled release film. Such formulations may be particularly convenient for prophylactic use.
Pharmaceutical formulations suitable for rectal administration, wherein the carrier is a solid, are most preferably provided in the form of unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories may be conveniently formed by mixing the active compound with one or more softened or melted carriers, followed by cooling and shaping in a mold. Pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of the active compounds in an aqueous or oleaginous carrier.
Injectable formulations may be suitable for bolus injection or continuous infusion. Such formulations are conveniently provided in unit-dose or multi-dose containers which are sealed after introduction of the formulation until needed. Alternatively, the active compound may be in powder form, which is reconstituted with a suitable carrier, such as sterile, pyrogen-free water, prior to use.
The active compounds may also be formulated as long-acting depot formulations (long-acting depot preparation) which can be administered by intramuscular injection or by implantation, for example subcutaneously or intramuscularly. The depot formulation may comprise, for example, a suitable polymeric or hydrophobic material, or an ion exchange resin. Such long acting formulations are particularly convenient for prophylactic use.
Formulations suitable for pulmonary administration via the buccal space are provided such that particles containing the active compound and desirably ranging in diameter from 0.5 microns to 7 microns are delivered in the bronchial tree of the recipient.
As a possibility, such formulations are in the form of finely divided powders, which may be conveniently provided in penetrable capsules for inhalation devices (e.g. capsules of a suitable gelatin), or in the form of self-propelled formulations comprising the active compound, a suitable liquid or gaseous propellant and optionally other ingredients, such as surfactants and/or solid diluents. Suitable liquid propellants include propane and chlorofluorocarbons, and suitable gaseous propellants include carbon dioxide. Self-propelled formulations in which the active compound is dispensed in the form of droplets of a solution or suspension may also be used.
Such self-propelled formulations are similar to those known in the art and can be prepared by established procedures. Suitably, the self-propelled formulation is provided in a container provided with a manually operable valve or an automatically operated valve having the desired spray characteristics; advantageously, the valve is metered so that a fixed volume, for example 25 microliters to 100 microliters, is delivered per operation of the valve.
As a further possibility, the active compound may be in the form of a solution or suspension for a nebulizer or atomizer, whereby accelerated air flow or ultrasonic agitation is employed to generate a fine mist of droplets for inhalation.
Formulations suitable for nasal administration include those generally similar to those described above for pulmonary administration. When such a formulation is dispensed, the formulation should desirably have a particle size in the range of 10 microns to 200 microns to be able to stay in the nasal cavity; this can be achieved by suitably employing a powder of suitable particle size or selecting a suitable valve. Other suitable formulations include: coarse powder having a particle size in the range of 20 microns to 500 microns for rapid inhalation administration through the nostrils from a container near the nose; and nasal drops comprising an aqueous or oily solution or suspension of 0.2% w/v to 5% w/v of the active compound.
Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1M, and preferably 0.05M phosphate buffer or 0.8% physiological saline. Furthermore, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcohol/water solutions, emulsions or suspensions, including physiological saline and buffered media. Parenteral carriers include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, sodium lactate ringer's injection or fixed oils. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
Formulations suitable for topical administration may be provided, for example, in the form of a gel, cream or ointment. Such formulations may be applied, for example, to a wound or ulcer, the formulation being applied directly to the surface of the wound or ulcer or carried on a suitable support (e.g., bandage, gauze, mesh, etc.) and then applied over the area to be treated.
Liquid or powder formulations may also be provided which may be sprayed or sprinkled directly onto the site to be treated, such as a wound or ulcer. Alternatively, the formulation may be sprayed or sprinkled onto a carrier such as a bandage, gauze, mesh, etc., and then applied to the site to be treated.
According to a further aspect of the present invention there is provided a method of preparing a pharmaceutical or veterinary composition as described above, the method comprising combining one or more active compounds with a carrier, for example by mixing.
In general, the above formulations are prepared by the following method: the active agent is homogeneously and intimately associated with a liquid carrier and/or a finely divided solid carrier, and the product is then, if necessary, shaped. The invention extends to a method of preparing a pharmaceutical composition comprising combining or combining a compound described herein with a pharmaceutically or veterinarily acceptable carrier or excipient.
Salts/esters
The compounds of the invention may be present in the form of salts or esters, particularly in the form of pharmaceutically and veterinarily acceptable salts or esters.
Pharmaceutically acceptable salts of the compounds of the invention include suitable acid addition or base salts thereof. A review of suitable pharmaceutically acceptable salts can be found in Berge et al, J Pharm Sci,66,1-19 (1977). Salts are formed with, for example, the following acids: strong mineral acids such as mineral acids, for example hydrohalic acids (e.g., hydrochloric, hydrobromic and hydroiodic), sulfuric, phosphoric, sulfuric, bisulfate, hemisulfate, thiocyanate, persulfate and sulfonic acids; strong organic carboxylic acids, such as unsubstituted or substituted (e.g., substituted with halogen) alkane carboxylic acids having from 1 to 4 carbon atoms, such as acetic acid; saturated or unsaturated dicarboxylic acids, such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid or tetraphthalic acid; hydroxycarboxylic acids such as ascorbic acid, glycolic acid, lactic acid, malic acid, tartaric acid, or citric acid; amino acids, such as aspartic acid or glutamic acid; benzoic acid; or organic sulphonic acids, e.g. unsubstituted or substituted (e.g. by halogen) 1 -C 4 ) Alkylsulfonic acid, or arylsulfonic acid, such as methanesulfonic acid or p-toluenesulfonic acid. Pharmaceutically and veterinarily unacceptable salts may still be valuable as intermediates.
Preferred salts include, for example, acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptonate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, pamoate (palmoate), pectate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, lactoaldehyde, pivalate (pivalate), camphorite, undecanoate and succinate; organic sulfonates such as methanesulfonate, ethanesulfonate, 2-isethionate, camphorsulfonate, 2-naphthalenesulfonate, benzenesulfonate, p-chlorobenzenesulfonate and p-toluenesulfonate; and inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, hemisulfate, thiocyanate, persulfate, phosphate, and sulfonate. More preferably, the salt is the hydrochloride salt.
Depending on the functional groups being esterified, esters are formed by using organic acids or alcohols/hydroxides. Organic acids include carboxylic acids, such as unsubstituted or substituted (e.g., halogen substituted) alkane carboxylic acids having from 1 to 12 carbon atoms (e.g., acetic acid); saturated or unsaturated dicarboxylic acids, such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid or tetraphthalic acid; hydroxycarboxylic acids such as ascorbic acid, glycolic acid, lactic acid, malic acid, tartaric acid, or citric acid; amino acids, such as aspartic acid or glutamic acid; benzoic acid; or organic sulfonic acids, e.g. unsubstituted or substituted (e.g. by halogen) (C 1 -C 4 ) Alkyl or aryl sulphonic acids, such as methane sulphonic acid or p-toluene sulphonic acid. Suitable hydroxides include inorganic hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide. Alcohols include unsubstituted or substituted (e.g., substituted with halogen) alkane alcohols having from 1 to 12 carbon atoms.
Enantiomers/tautomers
In all aspects of the invention previously discussed, the invention includes (as appropriate) all enantiomers, diastereomers and tautomers of the compounds of the invention. Those skilled in the art will recognize compounds having optical properties (one or more chiral carbon atoms) or tautomeric properties. The corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.
Enantiomers are characterized by the absolute configuration of their chiral centers and are expressed in terms of the R-and S-ordering rules of Cahn, lngo and Prelog. This convention is well known in the art (see, e.g., 'Advanced Organic Chemistry',3 rd edition,ed.March,J.,John Wiley and Sons,New York,1985)。
The chiral center-containing compounds of the present invention may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques so that the individual enantiomers may be used alone.
Stereoisomers and geometric isomers
Some compounds of the invention may exist in stereoisomeric and/or geometric form, e.g., they may have one or more asymmetric centers and/or geometric centers and thus may exist in two or more stereoisomeric and/or geometric forms. The present invention encompasses the use of all individual stereoisomers and geometric isomers of those compounds, as well as mixtures thereof. The term as used in the claims includes these forms provided that the forms retain the appropriate functional activity (although not necessarily to the same extent).
The invention also includes all suitable isotopic variations of the compounds or pharmaceutically acceptable salts thereof. Isotopic variations of the compounds of the present invention or pharmaceutically acceptable salts thereof are defined as those in which at least one atom is replaced by an atom having the same atomic number but an atomic weight different from the atomic weight usually found in nature. Examples of isotopes that can be incorporated into agents and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, e.g., respectively 2 H、 3 H、 13 C、 14 C、 15 N、 17 O、 18 O、 31 P、 32 P、 35 S、 18 F and F 36 Cl. Certain isotopic variations of the agents and pharmaceutically acceptable salts thereof are useful in drug and/or matrix tissue distribution studies, such as those into which a radioisotope (e.g. 3 H or 14 C) Isotopic variation of (c). Tritium (i.e. 3 H) And carbon-14 (i.e 14 C) Isotopes are particularly preferred. Furthermore, the use of isotopes (e.g. deuterium, i.e 2 H) Performing a substitution may provide a degree of therapeutic advantage due to its greater metabolic stability, e.g., an increase in vivo half-life or a reduction in dosage requirements, which may be preferred in some circumstances. For example, the invention includesA compound of formula (I) wherein any hydrogen atom is replaced by a deuterium atom. In general, isotopic variations of the agents of the present invention and pharmaceutically acceptable salts thereof can be prepared by conventional means using appropriate isotopic variations of the suitable drugs.
Atropisomers
Some of the compounds of the present invention may exist in atropisomer form. Atropisomers are stereoisomers produced by hindered rotation about a single bond, where a sufficiently high rotational barrier is created due to energy differences caused by spatial strain or other contributors to separate individual conformational isomers. The present invention includes all such atropisomers.
Prodrugs
The invention also includes compounds of the invention in the form of prodrugs, i.e., covalently bound compounds of the active parent drug that are released in vivo. Such prodrugs are typically compounds of the invention wherein one or more appropriate groups are modified such that the modification is reversible upon administration to a human or mammalian subject. Reversion is typically performed by enzymes naturally occurring in such subjects, but there is also the possibility that: a second agent is administered with such a prodrug to reverse in vivo. Examples of such modifications include esters (e.g., any of those described above), wherein reversion can be by esterases and the like. Other such systems are known to those skilled in the art.
Solvates of the formula
The invention also includes the compounds of the invention in the form of solvates. The term as used in the claims includes these forms. Preferably, the solvate is a hydrate.
Polymorphs
The invention also relates to various crystalline, polymorphic and (anhydrous) hydrated forms of the compounds of the invention. Such methods are well established in the pharmaceutical arts: compounds in any such form may be isolated by slightly altering the purification method and/or the isolation form of the solvent used to synthetically prepare such compounds.
Mode of administration
The pharmaceutical compositions of the present invention may be suitable for rectal administration, nasal administration, bronchial administration, topical administration (including oral and sublingual administration), vaginal or parenteral administration (including subcutaneous, intramuscular, intravenous, intraarterial and intradermal administration), intraperitoneal or intrathecal administration. Preferred formulations are those for oral administration. The formulations may conveniently be presented in unit dosage form (i.e., in discrete portions comprising a unit dose), or may be presented in unit doses in multiple units or sub-units. As an example, the formulation may be in the form of tablets and sustained release capsules, and may be prepared by any method well known in the pharmaceutical arts.
The orally administered formulation of the present invention may be provided in the following form: discrete units containing a predetermined amount of active agent, such as capsules, pills (gellules), drops, cachets, pills or tablets; powder or granules; solutions, emulsions or suspensions of active agents in aqueous or non-aqueous liquids; or an oil-in-water emulsion or a water-in-oil emulsion; or a bolus, etc. Preferably, these compositions contain from 1mg to 250mg of active ingredient per dose, and more preferably from 10mg to 100mg of active ingredient.
For compositions for oral administration (e.g., tablets and capsules), the term "acceptable carrier" includes excipients such as common excipients, for example binders such as syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metal stearates, glycerol stearate stearic acid, silicone oil, paraffin wax, oil and colloidal silica. Flavoring agents such as peppermint, oil of wintergreen, cherry flavoring and the like may also be used. It may be advantageous to add a colorant to make the dosage form easily identifiable. Tablets may also be coated by methods known in the art.
Tablets may be made by compression or molding, optionally with one or more additional ingredients. The compressed tablet may be prepared by: the active agent in free-flowing form (e.g., powder or granules) is compressed in a suitable machine, optionally after mixing with a binder, lubricant, inert diluent, preservative, surfactant or dispersant. Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.
Other formulations suitable for oral administration include: lozenges comprising the active agent in a flavored base (typically sucrose and acacia or tragacanth); pastilles comprising the active agent in an inert base such as gelatin and glycerol (rimethyl), or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.
Other forms of administration include solutions or emulsions that can be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally, or intramuscularly, prepared from sterile or sterilizable solutions. The injectable form generally contains from 10mg to 1000mg, preferably from 10mg to 250mg, of active ingredient per dose.
The pharmaceutical compositions of the invention may also be in the form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
An alternative to transdermal application is through the use of skin patches. For example, the active ingredient may be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycol or liquid paraffin. The active ingredient may also be incorporated in an ointment consisting of white wax or white soft paraffin base at a concentration of between 1 and 10% by weight, such stabilizers and preservatives being added as required.
Dosage of
One of ordinary skill in the art can readily determine the appropriate dosage to administer one of the compositions of the present invention to a subject without undue experimentation. In general, the physician can determine the actual dosage which will be most suitable for an individual patient, and this will depend on a variety of factors, including the activity of the particular compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are examples of average cases. There may of course be individual examples in which higher or lower dosage ranges should be used, these dosage ranges being within the scope of the invention.
The dosage may be further adjusted according to the mode of administration of the compound. For example, to achieve an "effective dose" for acute treatment, parenteral administration of the compound is generally preferred. Although intramuscular intravenous injection is also useful, 5% dextrose in water or physiological saline solution of the intravenous infusion compound, or similar formulations with suitable excipients, are most effective. Typically, the parenteral dosage is from about 0.01mg/kg to about 100mg/kg; preferably between 0.1mg/kg and 20mg/kg in such a way as to maintain the concentration of the drug in the plasma at a concentration effective to regulate ERAP 1. The compound is administered 1 to 4 times daily at a level to achieve a total daily dose of about 0.4 mg/kg/day to about 400 mg/kg/day. One of ordinary skill in the art can readily determine the therapeutically effective precise dosage of the compounds of the present invention, as well as the optimal route of administration of the compounds, by comparing the blood level of the agent to the concentration required to have a therapeutic effect.
The compounds of the invention may also be administered orally to a patient in a manner that provides a concentration of the drug sufficient to achieve one or more of the therapeutic metrics disclosed herein. Typically, pharmaceutical compositions comprising the compounds are administered at an oral dosage of between about 0.1mg/kg and about 50mg/kg in a manner consistent with the patient's condition. Preferably, the oral dosage may be about 0.5mg/kg to about 20mg/kg.
When the compounds of the invention are administered according to the present invention, no unacceptable toxicological effects are expected. The compounds of the present invention may have good bioavailability and may be detected using one of several biological detection techniques to determine the concentration of the compound required to achieve a given pharmacological effect.
Combination of
Another aspect of the invention relates to combinations comprising a compound described herein and one or more additional active agents. In a particularly preferred embodiment, one or more compounds of the invention are administered in combination with one or more additional active agents (e.g., existing commercially available drugs). In this case, the compounds of the invention may be administered sequentially, simultaneously or sequentially with one or more other active agents.
Drugs are generally more effective when administered in combination. In particular, combination therapy is advantageous in order to avoid overlapping of the major toxicities, mechanisms of action and mechanisms of resistance. Furthermore, it is also desirable to administer the most drug at the maximum tolerated dose of the drug with the shortest time interval between such doses. The main advantages of the combination with chemotherapeutic agents are that by biochemical interactions, additive effects or possibly synergistic effects can be promoted and the occurrence of drug resistance can also be reduced.
By studying the activity of test compounds with agents known or suspected to play an important role in the treatment of a particular disorder, a beneficial combination may be suggested. The method may also be used to determine the order of administration of the drugs, i.e., before, simultaneously or after administration. This scheduling may be characteristic of all of the active agents identified herein.
In a preferred embodiment, the additional active agent is an immunotherapeutic agent, more preferably a cancer immunotherapeutic agent. An "immunotherapeutic agent" refers to a treatment that uses the subject's own immune system to combat a disease such as cancer.
In a preferred embodiment, the compounds of the invention inhibit the activity of ERAP1 and the compounds are administered in combination with immunotherapy.
The compounds may increase the sensitivity of cancer cells to immunotherapy. Immunotherapy may be mediated by T cells. In one embodiment, the compound can increase CD8 in a tumor + Number of T cells.
In one embodiment, the compounds are useful for treating cancers that respond poorly or non-respond to immunotherapy.
In a preferred embodiment, the additional active agent is a molecule, co-stimulatory antibody, chemotherapeutic agent, radiotherapeutic agent, targeted therapeutic agent or antibody, in particular a monoclonal antibody, capable of immune checkpoint intervention.
In a preferred embodiment, the additional active agent is a molecule capable of immune checkpoint intervention.
Immune checkpoint molecules include CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP, CD47, CD48, 2B4, B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, IDO, CD39, CD73, A2aR and milk fat philins.
Immune checkpoint molecules include inhibitory and activating molecules, and interventions can be applied to either type of molecule described above or to both types of molecules.
Immune checkpoint inhibitors include, but are not limited to, for example, PD-1 inhibitors, PD-L1 inhibitors, LAG-3 inhibitors, TIG-3 inhibitors, TIGIT inhibitors, BTLA inhibitors, and CTLA-4 inhibitors. Co-stimulatory antibodies deliver positive signals through immunomodulatory receptors including, but not limited to ICOS, CD137, CD27 OX-40, and GITR.
In a highly preferred embodiment, the additional active agent is an antibody checkpoint inhibitor. Suitable examples of antibody checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA 4 antibodies.
In a preferred embodiment, the antibody checkpoint inhibitor is an anti-PD-1 antibody, more preferably selected from the group consisting of pembrolizumab, cimip Li Shan antibody and nivolumab.
In a preferred embodiment, the antibody checkpoint inhibitor is an anti-PD-L1 antibody, more preferably selected from the group consisting of alemtuzumab, avilamab and Dewaruzumab.
In a preferred embodiment, the antibody checkpoint inhibitor is an anti-CTLA 4 antibody, more preferably selected from ipilimumab and tremelimumab.
In a preferred embodiment, the immunotherapy is an anti-cancer vaccine or virus, such as an oncolytic virus.
In a preferred embodiment, the immunotherapy is a cell-based therapy. In one embodiment, the cell-based therapy can be a T cell therapy, such as an adoptive T cell therapy, or a therapy using CAR-T cells.
Adoptive cell-based immunotherapy may include the following: irradiated autologous or allogeneic tumor cells, tumor lysates or apoptotic tumor cells, antigen presenting cell-based immunotherapy, dendritic cell-based immunotherapy, adoptive T cell metastasis, adoptive CAR T cell therapy, autoimmune enhancement therapy (AIET), cancer vaccines, and/or antigen presenting cells. Such cell-based immunotherapy may be further modified to express one or more gene products, thereby further modulating the immune response, e.g., expressing cytokines such as GM-CSF, and/or expressing Tumor Associated Antigen (TAA) antigens such as MAGE-1, gp-100, patient-specific neoantigen vaccines, and the like.
In other embodiments, the immunotherapy may include non-cell based immunotherapy. In one embodiment, a composition comprising an antigen, with or without a vaccine enhancing adjuvant, may be used. Such compositions exist in many well known forms, such as peptide compositions, oncolytic viruses and recombinant antigens including fusion proteins.
In an alternative embodiment, immunomodulatory interleukins may be used, such as IL-2, IL-6, IL-7, IL-12, IL-17, IL-23, and modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms). Immunomodulatory cytokines such as interferon, G-CSF, imiquimod, T F a, and the like, as well as their modulators (e.g., blocking antibodies or more potent or longer lasting forms) may also be used. In another embodiment, immunomodulatory chemokines, such as CCL3, CCL26, CXCL7, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or more durable forms) may be used. In another embodiment, immune modulatory molecules that target immunosuppression, such as STAT3 signaling modulators, fkappaB signaling modulators (FkappaB rimethyla modulators), and immune checkpoint modulators, may be used.
In another embodiment, immunomodulatory drugs may be used, such as immunosuppressants, glucocorticoids, cytostatic agents, immunophilins and their modulators (e.g., rapamycin, calcineurin inhibitors, tacrolimus, cyclosporine (rimethyllami)), pimecrolimus, abbe limus, guanrolimus, sirolimus, everolimus, temsirolimus, zotarolimus, etc.), hydrocortisone (Coripol), cortisone acetate, prednisone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone (rimethamine), fludrocortisone acetate, deoxycorticosterone acetate (doca), aldosterone, non-glucocorticoid steroids, pyrimidine synthesis inhibitors, flumidex, teriflunomide, folic acid analogues, triadimefon methotrexate, anti-thymocyte globulin, anti-lymphocyte globulin, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin, catechin, opium, EVIPDH inhibitor, mycophenolic acid, myriocin, fingolimod, NF-xB inhibitor, raloxifene, tegaserod Luo, denomab, F-xB signaling cascade inhibitor (an F-xB rimethyla cascade inhibitor), alcohol-stopping sulfur, olmesartan, dithiocarbamate, proteasome inhibitor, bortezomib, MG132, prol, PI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomide, fraapine, non-steroid anti-inflammatory drug (NSAID), arsenic trioxide, dehydroxymethyl oxyquinone mycin (DHMEQ), I3C (indole-3-methanol)/DIM (diindolylmethane) (13C/DIM), bay 1 1-7082, luteolin, cell-penetrating peptide SN-50, IKBa-super repressor overexpression (super repressor overexpression), FKB trap Oligodeoxynucleotides (ODNs) or derivatives or analogues of any of these.
In yet another embodiment, an immunomodulatory antibody or protein may be used. For example, the number of the cells to be processed, antibodies that bind to CD40, toll-like receptor (TLR), OX40, GITR, CD27, or 4-lBB, T cell bispecific antibodies, anti-IL-2 receptor antibodies, anti-CD 3 antibodies, OKT3 (Mo Luoshan antibodies), oxybutyzumab, tiapride, wegener, anti-CD 4 antibodies, celecoxib, keliximab, zanalimab, anti-CDl a antibodies, efalizumab, anti-CD 18 antibodies, erlizumab, luo Weizhu mab, anti-CD 20 antibodies, alfuzumab, oxbevacizumab, ofatuzumab, palcoxib, rituximab, anti-CD 23 antibodies, lu Xishan antibodies, anti-CD 40 antibodies, tenelizumab, tolizumab, anti-CD 40L antibodies, lu Lizhu mab, anti-CD 62L antibodies, alexidizumab, anti-CD 80 antibodies, ganciclibab, anti-CD 147 antibodies, ganciclibizumab, ganaxizumab, anti-CD 23 antibodies, oxuja B lymphocyte stimulator (BLyS) inhibitory antibodies, belimumab, CTLA4-lg fusion proteins, abamectin, beracetipran, anti-CTLA 4 antibodies, ipilimumab, trimesamumab, anti-eosinophil chemokine 1 antibodies, bai Ti mumab, anti-a 4 integrin antibodies, natalizumab, anti-IL-6R antibodies, tozumab, anti-LFA-1 antibodies, ondimomab, anti-CD 25 antibodies, basiliximab, darifenacin, enomumab, anti-CD 5 antibodies, alzomumab, anti-CD 2 antibodies, cerlizumab, nereimomab, famuzumab, attlizumab (atlizumab), altlizumab, cetrorhizumab, rituximab, golimumab, ma Simo, xyloside mab, raloxib, pegzhuzumab, rayleigh bevacizumab, luo Weizhu mab, talbevacizumab, attomoab, valaciumab, vipamomab, albesipran, alfasin, li Naxi pu, IL-2 receptor antagonists, anakinra, anti-IL-5 antibodies, meperimab, igE inhibitors, omalizumab, talbevacizumab, IL12 inhibitors, IL23 inhibitors, and you-tec mab.
In one embodiment, the subject may be undergoing or has previously undergone treatment with a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to: alkylating agents, such as thiotepa and CYTOXAN cyclophosphamide; alkyl sulfonates such as busulfan, imperoshu and piposhu; aziridines such as benzodopa (benzodopa), carboquinone, midopopa (meturedopa) and You Liduo bar (uredopa); ethyleneimines and methyl melamines, including altretamine, triton, triethylenephosphoramide, triethylenethiophosphamide and trimethylol melamine; annonaceous acetogenins (e.g., bullatacin and bullatacin ketone); camptothecins (including the synthetic analog topotecan); bryostatin; olopatadine (caly statin); CC-1065 (including adoxolone, calzelone and bizelone analogues thereof); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatin; the class of duocarmycins (including synthetic analogs KW-2189 and CB 1-TM 1); soft corallool (eleutherobin); a podocarpine (pancratistatin); sarcandyl alcohol (sarcandylin); spongostatin (spongostatin); nitrogen mustards, such as chlorambucil, napthalene mustards, chlorophosphamide (cholosphamide), estramustine, ifosfamide, dichloromethyl diethylamine, chlorambucil hydrochloride (mechlorethamine oxide hydrochloride), melphalan, noverichin, chlorambucil cholesterol (phenesterine), prednisolone, triamcinolone, uracil mustards; nitroureas such as carmustine, chlorourea, fotemustine, lomustine, nimustine and ranimustine; antibiotics, such as enediyne (enedyyne) antibiotics (e.g., calicheamicin, particularly calicheamicin γii and calicheamicin ωii (see, e.g., agnew, chem. Intl. Ed. Engl.,33:183-186 (1994)); anthracyclines (dynastins), including dactinomycin A, bisphosphonates, such as chlorophosphonate, epothilone (esperamicin), and neomycin (neocerzistatin) chromophores and related chromoprotein enediynes, aclacinomycin (aclacinomycin), actinomycin, aflatoxin (authamycin), diazoserine, bleomycin, actinomycin, kararatio of carboxin (carbacin), carminomycin (carminomycin), dactinomycin (carminomycin), carcinomycin (carzinophilin), chromomycin (chromamycin), actinomycin D, daunorubicin, ditropin, 6-diaza-5-O-norleucine, ADRIAMYCIN doxorubicin (including morpholino doxorubicin, cyano morpholino doxorubicin, 2-pyrrolopyrromycin and deoxydoxorubicin), epirubicin, epothilone, doxycycline; mitomycins, such as mitomycin C, mycophenolic acid, norgamycin, olivomycin (olivomycin), pervomycin, prednisomycin (potfiromycin), puromycin, tri-iron doxorubicin, rodobicin, streptozotocin, tubercidin, ubenimex, jingstatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as, for example, dimethyl folic acid, methotrexate, pterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine (thiamiprine), thioguanine; pyrimidine analogs such as, for example, ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, deoxyfluorouridine, enocitabine, fluorouridine; androgens, such as carbo Lu Gaotong (calasterone), drotasone propionate, epithioandrosterol, emandrane, and testosterone; anti-adrenal agents such as aminoglutethimide (amiglutethimide), mitotane, and trilostane; folic acid supplements, such as folinic acid; acetoglucurolactone; aldehyde phosphoramidate glycoside (aldophosphamide glycoside); aminolevulinic acid; enuracil; amsacrine; bei Xibu (bestrebicil); bisantrene (bisantrene); edatraxate (edatraxate); dimecoxin; deaquinone (diaziquone); eformitine (elformithin); ammonium elegance; epothilones; etodolac (etoglucid); gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids (maytansinoids) such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mo Pai darol (mopidanmol); diamine nitroacridine (nitroane); prastatin; egg ammonia nitrogen mustard (phenol); pirarubicin; losoxantrone; podophylloic acid (podophyllinic acid); 2-ethyl hydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, eugene, oreg.); carrying out a process of preparing the raw materials; rhizopus extract; benzofurans (sizofurans); germanium spiroamine; tenuazonic acid (tenuazonic acid); triiminoquinone; 2,2',2 "-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verructuracin a, barytropin a, and snake (anguidine)); urethane (rimethyl); vindesine; dacarbazine; mannitol nitrogen mustard; dibromomannitol; dibromodulcitol; pipobromine; a gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes (rimeth), such as TAXOL paclitaxel (Bristol-Myers Squibb Oncology, prinston, new jersey), ABRAXANE free of Cremophor (Cremophor-free), albumin modified nanoparticle formulations of TAXOL (American Pharmaceutical Partners, schaumberg, 111.), and TAXOTERE docetaxel (Rhone-Poulenc Rorer, france antoni); chlorambucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE vinorelbine; can kill tumors (novantrone); teniposide; eda traxas; daunomycin; aminopterin; capecitabine (xeloda); ibandronate (ibandronate); irinotecan (Camptosar, CPT-11) (a treatment regimen comprising irinotecan with 5-FU and folinic acid); topoisomerase inhibitor RFS2000; difluoromethyl ornithine (DMFO); retinoids, such as retinoic acid; capecitabine (capecitabine); combretastatin; folinic acid (LV); oxaliplatin, including oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-Sub>A, raf, H-Ras, EGFR (e.g., erlotinib (TarcevSub>A)) and VEGF-A that reduce cell proliferation, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. In addition, the method of treatment may further comprise the use of radiation. In addition, the method of treatment may further comprise using photodynamic therapy.
Method
Another aspect of the invention relates to a process for preparing a compound of formula (I) as described herein.
In one aspect, the present invention relates to a process for preparing a compound of formula (I), wherein R 2 Is COOH, X is NH, Y is SO 2 And Z, L, R 1 、R 3 、R 4 -R 9 As defined above, the method comprises the steps of:
(i) By using Cs in a solvent 2 CO 3 And Z-L-OH treating the compound of formula (II) to convert the compound of formula (II) to the compound of formula (III);
(ii) Reducing the compound of formula (III) to form a compound of formula (IV);
(iii) Treating a compound of formula (IV) with a compound of formula (V) to form a compound of formula (VI); and
(iv) Hydrolyzing the compound of formula (VI) to form the compound of formula (I).
Preferably, step (i) is performed in acetonitrile, more preferably at room temperature. Those skilled in the art will appreciate that other organic solvents will also be suitable. Preferably, step (ii) comprises heating the compound of formula (III) and Fe, NH 4 Cl and EtOH/H 2 O (preferably v/v=4/1). Preferably, step (iii) is carried out at room temperature in the presence of pyridine and dichloromethane. Preferably, the hydrolysis step (iv) is carried out at room temperature in the presence of a THF solution of LiOH.
Preferred conditions for each step are set forth in scheme 1 and the accompanying examples below.
In another aspect, the invention relates to a process for preparing a compound of formula (I), wherein R 2 Is COOH, X is NH, Y is SO 2 ，R 7 Tetrazolyl and Z, L, R 1 、R 3 、R 4 -R 6 、R 8 And R is 9 As defined above, the method comprises the steps of:
(i) Protecting NH in a compound of formula (VII) with a suitable protecting group PG 2 A group to give a compound of formula (VIII);
(ii) By using Cs in a solvent 2 CO 3 And Z-L-OH treating the compound of formula (VIII) to convert the compound of formula (VIII) to the compound of formula (IX);
(iii) Removing the protecting group PG from the compound of formula (IX) to give a compound of formula (X);
(iv) By trimethoxy methane, naN 3 And HOAc treating the compound of formula (X) to form a compound of formula (XI);
(v) Reducing the compound of formula (XI) to form a compound of formula (XII);
(vi) Treating a compound of formula (XII) with a compound of formula (V) to form a compound of formula (XIII); and
(vii) Hydrolyzing the compound of formula (XIII) to form the compound of formula (I).
Preferably, the protecting group PG is Boc. Those skilled in the art will appreciate that other amine protecting groups are also suitable (see Green t., "Protective Groups in Organic Synthesis", chapter 1, j. Wiley&Sons, inc.,1991,10-14). More preferably, step (i) comprises using (Boc) in DCM at room temperature 2 O, DMAP and TEA solution treatment of the compound of formula (VII). Preferably, step (ii) comprises heating the compound of formula (VIII) and Fe, NH 4 Cl and EtOH/H 2 O (preferably v/v=4/1). Preferably, step (iii) comprises treating the compound of formula (IX) with an acid, more preferably an EtOAc solution of HCl. Preferably, step (iv) comprises reacting compound (X), trimethoxymethane, naN 3 And HOAc to a temperature of at least 80 ℃. Preferably, step (v) comprises heating the (XI) compound and Fe, NH 4 Cl and EtOH/H 2 O (preferably v/v=4/1). Preferably, step (vi) is carried out at room temperature in the presence of pyridine and dichloromethane. Preferably, the hydrolysis step (vii) is carried out at room temperature in the presence of a solution of LiOH in THF.
Preferred conditions for each step are set forth in scheme 2 below and in the examples appended hereto.
The invention is further illustrated by the following non-limiting examples.
Examples
When the preparation of the starting materials is not described, then these are commercially available, known in the literature or readily available to those skilled in the art by standard methods. When it is pointed out that compounds are prepared using a similar process to the previous examples or intermediates, those skilled in the art will appreciate that the reaction time, the number of equivalents of reagents, solvents, concentrations and temperatures may be adjusted for each particular reaction and that a different operation (work-up) or purification technique may be required or desired.
Abbreviations (abbreviations)
AcOH: acetic acid; chloroform-d (deuterated chloroform); ca.: about; DMSO-d 6 (deuterated dimethyl sulfoxide); methanol-d 4 (deuterated methanol); boc (t-butoxycarbonyl); boC (Boc) 2 O (di-tert-butyl dicarbonate); DMF (N, N-dimethylformamide); DCM (dichloromethane); PE (petroleum ether); ESI (electrospray atmospheric pressure ionization); IPA: isopropyl alcohol; TEA (triethylamine); TFA (trifluoroacetic acid); dioxane (1, 4-dioxane); THF (tetrahydrofuran); etOH (ethanol); h 2 O (water); meCN (acetonitrile); etOAc (ethyl acetate); g (g); h (hours); nm (nanometers); 1 h NMR (proton nuclear magnetic resonance); hz (hertz); LC-MS (liquid chromatography-mass spectrometry); MS (mass spectrometry); mg (milligrams); MHz (megahertz); min (min); mL (milliliters); mmol (millimoles); ppm (parts per million); r is R t (retention time); RT (room temperature); TLC (thin layer chromatography); v/v (volume/volume); m/z (mass to charge ratio); HCl (hydrochloric acid); k (K) 3 PO 4 (tripotassium phosphate); HOAc (acetic acid); HCl (hydrochloric acid); cuCl (copper (I) chloride); SOCl 2 (thionyl chloride); cs (cells) 2 CO 3 (cesium carbonate); NH (NH) 4 Cl (ammonium chloride); fe (iron); DIPEA (N, N-diisopropylethylamine); MW (microwaves); pd (dppf) Cl 2 ([ 1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride); xphos Pd G3: methane sulphonic acid (2-dicyclohexylphosphino-2, 4, 6-triisopropyl-1, 1-biphenyl) [2- (2-amino-1, 1-biphenyl) ]Palladium (II) (CAS: 1445085-55-1); pd-174: allyl (2-di-tert-butylphosphine-2, 4, 6-triisopropyl-1, 1' -biphenyl) palladium (II) triflate (CAS: 1798782-25-8); pdCl 2 (AmPhos) 2 : dichloro (di-tert-butyl- (4-dimethylaminophenyl) palladium (II) (CAS: 887919-35-9); dppf:1, 1-ferrocenediyl-bis (diphenylphosphine); naH (sodium hydride); DMAP (4-dimethylaminopyridine); naN) 3 (sodium azide); liOH (lithium hydroxide); NH (NH) 2 -NH 2 (hydrazine hydroxide solution); DEA (diethylamine); DAST (diethylaminosulfur trifluoride); pd/C (carbon)Palladium supported); NBS (N-bromosuccinimide); PE (petroleum ether). Aq (aqueous solution); LC (liquid crystal): liquid chromatography; HPLC: high performance liquid chromatography; m: molar, molecular ion; UV: ultraviolet rays; UPLC: ultra-high performance liquid chromatography. br: a broad peak; d: bimodal; ESI: electrospray ionization; m: multiple peaks; meOH: methanol; min: minutes; PDA: a photodiode array; q: a quartet; s: unimodal, solid; t: a triplet; TBME: tert-butyl methyl ether. Other abbreviations are intended to express their commonly accepted meanings.
General scheme
Scheme 1
Reagent: (a) Cs (cells) 2 CO 3 、MeCN、RT；(b)Fe、NH 4 Cl、EtOH/H 2 O (v/v=4/1), 80 ℃; (c) pyridine, DCM, RT; (d) LiOH, THF, RT.
Scheme 2
Reagent: (a) (Boc) 2 O、DMAP、TEA、DCM、RT；(b)Cs 2 CO 3 MeCN, RT; (c) HCl 4mol/L in EtOAc, RT; (d) Trimethoxymethane, naN 3 、HOAc、80℃；(e)Fe、NH 4 Cl、EtOH/H 2 O (v/v=4/1), 80 ℃; (f) pyridine, DCM, RT; (g) LiOH (aq), THF, RT.
General experimental conditions
All starting materials and solvents were obtained from commercial sources or prepared according to literature citations. Unless otherwise indicated, the reaction mixture was magnetically stirred at room temperature (about 20 ℃) and the reaction was carried out. Column chromatography was performed on an automated flash chromatography system (e.g., biotage Isolera Rf system) using a pre-packed silica (40 μm) column, unless otherwise indicated. Recording using a Bruker AVANCE 400MHz spectrometer 1 H NMR spectrum. 1 Data for H are reported as chemical shifts (ppm) and multiplets (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet). Chemical shifts are expressed in parts per million using the center peak of the residual protic solvent or the tetramethylsilane internal standard as a reference. The spectra were recorded at 298K unless otherwise noted.
Using Waters ACQUITYAn analysis UPLC-MS experiment was performed by an H-Class system equipped with an ACQUITYDA detector and an ACQUITY QDa mass detector to determine retention times and associated mass ions, running one of the analysis methods described below. Analysis LC-MS experiments were performed using an Agilent 1200 series HPLC system coupled to an Agilent 1956, 6100 or 6120 series single quadrupole mass spectrometer running one of the analysis methods described below to determine retention times and associated mass ions.
General method for preparative HPLC:
HPLC instrument: shimadzu 20AP UV detector: SPD-20A. UV wavelength: 214nm and 254nm.
Condition 1: mobile phase a: water; mobile phase B: acetonitrile.
Condition 2: mobile phase a: water containing 0.1% trifluoroacetic acid; mobile phase B: acetonitrile.
Condition 3: mobile phase a: water containing 0.1% formic acid; mobile phase B: acetonitrile.
Condition 4: mobile phase a: water containing 0.1% ammonium hydroxide; mobile phase B: acetonitrile.
Chromatographic column: agilent 10Prep-C18 250x21.2mm. Chromatographic column temperature: ambient temperature
LC gradient: 20% to 85% in 20 minutes; then 85% to 100% in 0.01 min; then hold at 100% for 5 minutes; then 100% to 20% in 0.01 min; hold at 20% for 5 min.
LC flow: 20mL/min binary pump.
Using a signal fromThe "structure to name" conversion of Professional 17 (PerkinElmer) yields the naming of the structure.
The analysis method is as follows:
method 1-acid method (Shimadzu 3 minutes)
Chromatographic column: shimadzu LC-20AD series, binary pump, diode array detector. Agilent Poroshell 120EC-C18,2.7 μm, 4.6X10 mm column
And (3) detection: 2020, quadrupole LC/MS, ion source: API-ESI, TIC: 100-900 m/z, dry gas flow: 15L/min, atomizer pressure: 1.5L/min, dry gas temperature: 250 ℃, vcap:4500V. The sample was dissolved in methanol at a concentration of 1 to 10. Mu.g/mL, and then filtered through a 0.22 μm filter. Sample injection amount: 1-10 mu L. A detector: 214nm and 254nm. Detection wavelength: 214nm and 254nm.
Solvent: a:0.05% v/v formic acid in water, B: meCN solution of 0.05% v/v formic acid
Gradient:
T(min) A(％) B(％) flow (mL/min)
0.00 80 15 1.5
0.28 80 15 1.5
2.38 10 90 1.5
2.39 0 100 1.5
2.69 0 100 1.5
2.70 85 15 1.5
3.00 85 15 1.5
Method 2-acid 5 min method (Shimadzu 5 min)
Chromatographic column: shimadzu LC-20AD series, binary pump, diode array detector. Agilent Poroshell 120EC-C18,2.7 μm, 4.6X50 mm column.
And (3) detection: 2020, quadrupole LC/MS, ion source: API-ESI, TIC: 100-900 m/z, dry gas flow: 15L/min, atomizer pressure: 1.5L/min, dry gas temperature: 250 ℃, vcap:4500V. The sample was dissolved in methanol at a concentration of 1 to 10. Mu.g/mL, and then filtered through a 0.22 μm filter. Sample injection amount: 1-10 mu L. Detection wavelength: 214nm and 254nm.
Solvent: a:0.05% formic acid in water (v/v), B: meCN solution of 0.05% formic acid (v/v).
Gradient:
T(min) A(％) B(％) flow (mL/min)
0.00 80 15 1.0
0.50 80 15 1.0
4.00 15 85 1.0
4.01 0 100 1.0
4.50 0 100 1.0
4.51 85 15 1.0
5.00 85 15 1.0
Method 3 acid method (Waters QDa 3 minutes)
Chromatographic column: waters QDa, binary pump, diode array detector. Waters CORTECS UPLC, C18,1.6 μm, 2.1X10 mm column.
And (3) detection: QDa quadrupole LC/MS, ion source: API-ES, TIC: 70-900 m/z, fragmentation voltage: 70, dry gas flow: 12L/min, atomizer pressure: 36psi, dry gas temperature: 350 ℃, vcap:3000V. The sample was dissolved in methanol at a concentration of 1 to 10. Mu.g/mL, and then filtered through a 0.22 μm filter. Sample injection amount: 1-10 mu L. A detector: 214nm and 254nm.
Solvent: a:0.05% formate in water (v/v), B: meCN solution of 0.05% formate (v/v).
Gradient:
T(min) A(％) B(％) flow (mL/min)
0.00 80 20 0.6
1.80 20 80 0.6
2.65 20 80 0.6
2.80 80 20 0.6
3.00 80 20 0.6
Method 4 acid method for 3 minutes
Chromatographic column: waters ACQUITYCSH C18，1.7μm，2.1×30mm，40℃
And (3) detection: UV PDA 210-400nm, purity at 254nm, ACQUITYESI
Solvent: a:0.1% v/v formic acid in water, B: meCN (MeCN)
Gradient:
Time ％A ％B flow (ml/min)
0.00 98 2 0.77
2.50 0 100 0.77
3.00 0 100 0.77
Method 5 acid method for 4 minutes
Chromatographic column: YMC TRI ART C18,1.6 μm, 2.1X150 mm
And (3) detection: UV PDA 210-400nm, purity at 254nm, ACQUITYESI
Solvent: a:0.1% v/v formic acid in water, B: meCN (MeCN)
Gradient:
Time ％A ％B flow (mL/min)
0.00 97 3 0.8
0.2 97 3 0.8
2.70 2 98 0.8
3.00 0 100 1.0
3.50 0 100 1.0
3.51 97 3 0.80
4.00 97 3 0.80
Compound synthesis: the compounds of the invention may be prepared by methods well known to those skilled in the art, as described in the general synthetic schemes, and using appropriate intermediates.
Intermediate 1. 3- (chlorosulfonyl) -4-methoxybenzoic acid methyl ester
Step 1:3- (chlorosulfonyl) -4-methoxybenzoic acid methyl ester: 4-Methoxybenzoic acid (5.0 g,32.9 mmol) and chlorosulfonic acid (9.5 g,82.3 mmol) were combined in SOCl 2 The mixture in (30 mL) was stirred at room temperature for 12h. The reaction mixture was concentrated to give a crude product. The crude product was purified by silica gel chromatography (eluting with 1/2 EtOAc/PE) to give the title compound as a white solid (5.34 g,21.36mmol, 65%). 1 H NMR(400MHz,DMSO-d 6 )δ11.15(s,1H),8.29(d,J＝2.3Hz,1H),7.91(dd,J＝8.6,2.3Hz,1H),7.08(d,J＝8.7Hz,1H),3.83(s,3H)。
Intermediate 2. 3- (chlorosulfonyl) -4-ethylbenzoic acid
Step 1:3- (chloro)Sulfonyl) -4-ethylbenzoic acid: 4-ethylbenzoic acid (4.5 g,30.0 mmol) and chlorosulfonic acid (8.77 g,75.0 mmol) were combined in SOCl 2 The mixture in (45 mL) was heated at 85℃for 12h. After cooling to room temperature, the reaction mixture was concentrated to give the crude product. The crude product was purified by silica gel chromatography (eluting with 1/3 EtOAc/PE) to give the title compound (2.2 g,8.87mmol, 30%) as a yellow solid. 1 H NMR(400MHz,DMSO-d 6 )δ13.38(s,1H),8.36(d,J＝1.9Hz,1H),7.84(dd,J＝7.9,2.0Hz,1H),7.33(d,J＝7.9Hz,1H),3.09(q,J＝7.5Hz,2H),1.20(t,J＝7.5Hz,3H)。
Intermediate 3. 3- (chlorosulfonyl) -4-cyclopropylbenzoic acid methyl ester
Step 1: methyl 4-cyclopropylbenzoate: cyclopropylboronic acid (120.56 g,1401 mmol), K at room temperature 3 PO 4 (396.2 g,1869 mmol) and tricyclohexylphosphine (26.16 g,93.4 mmol) were added to a solution of methyl 4-bromobenzoate (200 g,934 mmol) in toluene (1800 ml) and water (400 ml). By N 2 The mixture was purged for 45 minutes, then palladium (II) acetate (10.46 g,46.7 mmol) was added. The mixture was heated at 80 ℃ for 6 h. The reaction mixture was cooled to room temperature, filtered through a celite bed, and washed with water (4.0 l) and EtOAc (2×5.0 l). The combined organic layers were washed with water (5L) and dried over Na 2 SO 4 Dried and concentrated under reduced pressure to give 1201g of crude material. The crude material was suspended in n-hexane (8L) and stirred at 0deg.C for 3h. The mixture was filtered through celite and concentrated under reduced pressure to give the title ester as a brown oil (800 g,45 mmol, quantitative, purity 98%) which was used directly in the next step.
* A total of 5 parallel reactions (2X 100g and 3X200g scale) were performed and treated in combination. 1 H NMR(400MHz,DMSO)δ7.83-7.81(d,J＝8.0Hz,2H),7.20-7.18(d,J＝8.4Hz,2H),3.85(s,3H),2.03-1.96(m,1H),1.06–1.01(m,2H),0.77-0.74(m,2H).
Step 2:3-Bromo-4-cyclopropylbenzoic acid methyl ester: to a solution of the ester of step 1 (250 g,1420 mmol) in TFA (3000 ml) was added NBS (252.8 g,1420 mmol) in portions at room temperature. The reaction mixture was heated at 50 ℃ for 16 h. The mixture was cooled at room temperature, diluted with ice water (5.0L) and extracted with n-hexane (2x5.0l). The combined organic layers were treated with saturated Na 2 CO 3 Solution (2L) followed by brine solution (2L) with Na 2 SO 4 Dried and concentrated under reduced pressure. The crude material was purified by normal phase chromatography on silica gel (0-50% DCM in hexanes) to give the title ester as an oil (013 g,1744mmol,39%, 95.7% purity).
* A total of 4 parallel reactions (1 x100g, 1x200g and 2x250g scale) were performed and the combination thereof was treated. 1 H NMR(400MHz,DMSO)δ8.06(s,1H),7.83(d,J＝8.0Hz,1H),7.12(d,J＝8.4Hz,1H),3.87(s,3H),2.22(m,1H),1.12-1.07(m,2H),0.81-0.77(m,2H).
Step 3:3- (benzylthio) -4-cyclopropylbenzoic acid methyl ester: to a solution of the ester of step 2 (100 g,393 mmol) in 1, 4-dioxane (1400 ml) was added Xantphos (11.39 g,19.6 mmol) and Pd at room temperature 2 (dba) 3 (9.01 g,9.8 mmol). By N 2 The reaction mixture was purged for 30 minutes. DIPEA (145.1 ml,787 mmol) and benzyl mercaptan (51.34 g,413 mmol) were added and the reaction mixture was heated to 110℃for 16 h. The reaction mixture was cooled to room temperature, filtered through celite bed, washed with hexane (3.0L) and the combined organic filtrates were washed with distilled water (5.0L). The combined organic layers were taken up with Na 2 SO 4 Dried, filtered and concentrated under reduced pressure. The crude product was purified by normal phase chromatography on silica gel (0-40% dcm in hexanes) to give the title mercaptoether as a yellow solid (425 g,1429mmol,84%, 97% purity).
* A total of 4 parallel reactions (1 x100g and 3x110g scale) were performed and treated in combination. LCMS:3.11min, MS: ES+299.53 (M+1); 1 H NMR(400MHz,DMSO)δ7.86(s,1H),7.68(d,J＝8Hz,1H),7.38(m,5H),7.05(d,J＝8.0Hz,1H),4.26(s,2H),3.82(s,3H),2.18(m,1H),1.03(m,2H),0.72(m,2H)。
step 4:3- (chloro)Sulfonyl) -4-cyclopropylbenzoic acid methyl ester: to a stirred solution of the thiol ether of step 3 (142 g,184 mmol) in a mixture of AcOH (114.3 ml), water (68.8 ml) and MeCN (3124 ml) was added 1, 3-dichloro-5, 5-dimethylhydantoin (187.6 g,952 mmol) in one portion at 0 ℃. The mixture was stirred at the same temperature for 0.5h. The reaction mixture was diluted with distilled water (10L) and extracted with EtOAc (2×5.0L). The combined organic layers were washed with distilled water (2x5.0), with Na 2 SO 4 Dried, filtered and concentrated under reduced pressure. The crude product was purified by silica gel normal phase column chromatography using 6-10% EtOAc in hexanes, followed by trituration with n-hexane to give the title sulfonyl chloride as a white crystalline solid (260 g,970mmol,68%, purity 98%).
* A total of 3 parallel reactions (at a scale of 142 g) were performed and treated in combination. LCMS:2.480min, MS: ES+275.1 (M+1); 1 H NMR(400MHz,DMSO)δ11.81(br,3H),8.35(s,1H),7.77(d,J＝8.0Hz,1H),6.84(d,J＝8.4Hz,1H),3.83(s,3H),3.14(m,1H),1.03(d,J＝6.8Hz,2H),0.75(d,J＝4.4Hz,2H)。
Intermediate 4. 5- (chlorosulfonyl) -4-cyclopropyl-2-fluorobenzoic acid methyl ester
Step 1: 4-chloro-2-fluoro-5-nitrobenzoic acid methyl ester: to a solution of 4-chloro-2-fluoro-5-nitrobenzoic acid (10.0 g,45.6 mmol) in MeOH (200 mL) at 0deg.C was added SOCl 2 (30 mL). The resulting mixture was heated to reflux overnight. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc and taken up in saturated Na 2 CO 3 Wash with water and brine. The organic layer was taken up with Na 2 SO 4 Drying and concentration gave the crude product. The crude product was purified by silica gel chromatography (eluting with 1/10 EtOAc/PE) to give the title compound as an off-white solid (9.82 g,42.1mmol, 92%). 1 H NMR(400MHz,DMSO-d 6 )δ8.59(d,J＝6.9Hz,1H),8.06(d,J＝10.2Hz,1H),3.94(s,3H)。
Step 2:4-Cyclopropyl-2-fluoro-5-nitrobenzoic acid methyl ester: the ester of step 1 (6.0 g,25.8 mmol), cyclopropylboronic acid (6.60 g,77.4 mmol), K are reacted at 90 ℃ 3 PO 4 (10.92 g,51.6 mmol) and Pd (dppf) Cl 2 A mixture of (1.92 g,2.58 mmol) in THF (50 mL) was heated in a sealed tube for 16h. The resulting mixture was filtered through celite, concentrated and purified by silica gel chromatography (eluting with 1/5 EtOAc/PE) to give the title compound as an off-white solid (5.4 g,22.6mmol, 88%). 1 H NMR(400MHz,DMSO-d 6 )δ8.42(d,J＝6.8Hz,1H),7.27(d,J＝12.1Hz,1H),3.93(s,3H),2.46-2.34(m,1H),1.25-1.12(m,2H),1.08-0.94(m,2H)。
Step 3: 5-amino-4-cyclopropyl-2-fluorobenzoic acid methyl ester: the ester of step 2 (1.0 g,4.2 mmol), iron powder (1.17 g,21.0 mmol) and NH 4 A mixture of Cl (0.45 g,8.4 mmol) in a mixture of ethanol and water (5:1) was heated at 85℃for 2h. The resulting mixture was filtered through celite and concentrated to give the title compound as a grey solid (0.85 g,4.1mmol, 98%). UPLC-MS (method 1) M/z 210.10 (M+H) + At 1.32 minutes.
Step 4:5- (chlorosulfonyl) -4-cyclopropyl-2-fluorobenzoic acid methyl ester: to methyl 5-amino-4-cyclopropyl-2-fluorobenzoate (0.85 g,4.1 mmol) in concentrated HCl (3 mL) and H at 0deg.C 2 To a solution in a mixture of O (9.0 mL) NaNO was added in portions 2 (0.56 g,8.2 mmol). The mixture was stirred at 0 ℃ for 30 minutes. To CuCl (40.8 mg,0.41 mmol) at 0deg.C in H 2 SOCl was added dropwise to the solution in O (3 mL) 2 (1.75 mL). The solution was then added dropwise to the reaction described above, and the mixture was stirred at 0 ℃ for 1h. With EtOAc (50 mL) and H 2 The reaction was diluted with O (50 mL) and the aqueous layer was extracted with EtOAc (50 mL. Times.2). The organic layer is treated by Na 2 SO 4 Drying and concentration gave the title compound (0.65 g,2.2mmol, 54%) as a yellow oil. 1 H NMR(400MHz,DMSO-d 6 )8.29(d,J＝8.1Hz,2H),6.64(d,J＝13.0Hz,2H),3.92-3.82(m,1H),3.82(s,3H),1.14-0.98(m,2H),0.90-0.71(m,2H)。
Intermediate 5. 5- (chlorosulfonyl) -4-cyclopropyl-3-fluorobenzoic acid methyl ester
Step 1: 3-bromo-5-fluoro-4-hydroxybenzoic acid methyl ester: to a solution of methyl 3-fluoro-4-hydroxybenzoate (5.0 g,29.4 mmol) in HOAc (50 mL) at 0deg.C was added Br 2 (4.64 g,29.4 mmol). The resulting mixture was stirred at room temperature for 4h. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc and taken up in saturated Na 2 CO 3 Wash with water and brine. The organic layer was taken up with Na 2 SO 4 Drying and concentration gave the crude product. The crude product was purified by silica gel chromatography (eluting with 1/3 EtOAc/PE) to give the title compound (5.8 g,23.4mmol, 80%) as a yellow solid. UPLC-MS (method 1) M/z 248.95 (M-H) - At 1.516 minutes. 1 H NMR(400MHz,DMSO-d 6 )δ11.38(s,1H),7.89(t,J＝1.7Hz,1H),7.69(dd,J＝10.9,2.0Hz,1H),3.85(s,3H)。
Step 2: 3-fluoro-4-hydroxy-5- ((4-methoxybenzyl) thio) benzoic acid methyl ester: methyl 3-bromo-5-fluoro-4-hydroxybenzoate (5.8 g,23.4 mmol), (4-methoxyphenyl) methyl mercaptan (7.2 g,46.8 mmol), DIPEA (6.0 g,46.8 mmol), xantphos (2.7 g,4.68 mmol) and Pd 2 (dba) 3 A solution of (2.14 g,2.34 mmol) in dioxane (50 mL) was heated at 110deg.C for 16 hours. The resulting mixture was filtered through celite, concentrated and purified by silica gel chromatography (eluting with 1/10 EtOAc/PE) to give the title compound (2.85 g,8.85mmol, 38%) as a white solid. UPLC-MS (method 3) M/z 321.0 (M-H) - At 1.654 minutes.
Step 3: methyl 3-fluoro-5- ((4-methoxybenzyl) thio) -4- (((trifluoromethyl) sulfonyl) oxy) benzoate: to a solution of methyl 3-fluoro-4-hydroxy-5- ((-4-methoxybenzyl) thio) benzoate (2.85 g,8.85 mmol) in DCM (50 mL) at 0deg.C was added Tf 2 O (4.99 g,17.7 mmol). Pyridine (1.39 g,17.7 mmol) was added and the resulting mixture was stirred at room temperature for 1h. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc and taken up in saturated Na 2 CO 3 Water and saltWashing with water. The organic layer was taken up with Na 2 SO 4 Drying and concentration gave the crude product. The crude product was purified by silica gel chromatography (eluting with 1/15 EtOAc/PE) to give the title compound as a yellow oil (1.43 g,3.15mmol, 36%). 1 H NMR(400MHz,DMSO-d 6 )δ7.72-7.46(m,3H),7.32-7.24(m,2H),6.94-6.83(m,2H),4.17(s,2H),3.86(s,3H),3.78(s,3H)。
Step 4: methyl 4-cyclopropyl-3-fluoro-5- ((4-methoxybenzyl) thio) benzoate: methyl 3-fluoro-5- ((4-methoxybenzyl) thio) -4- (((trifluoromethyl) sulfonyl) -oxy) benzoate (1.2 g,2.64 mmol), cyclopropylboronic acid (0.45 g,5.28 mmol), K was heated at 100deg.C 2 CO 3 (1.09 g,7.92 mmol) and Pd (PPh) 3 ) 4 (0.3 g,0.26 mmol) in dioxane (30 mL) for 16h. The resulting mixture was filtered through celite, concentrated and purified by silica gel chromatography (eluting with 1/10 EtOAc/PE) to give the title compound (0.52 g,1.5mmol, 57%) as a white solid. 1 H NMR(400MHz,DMSO-d 6 )δ7.73-7.51(m,1H),7.45-7.30(m,1H),7.23(s,2H),6.98-6.86(m,2H),4.29(s,2H),3.87(s,3H),3.76(s,3H),2.12-1.97(m,1H),1.09-0.99(m,2H),0.84-0.75(m,2H)。
Step 5:3- (chlorosulfonyl) -4-cyclopropyl-5-fluorobenzoic acid methyl ester: to methyl 4-cyclopropyl-3-fluoro-5- ((4-methoxybenzyl) thio) benzoate (0.52 g,1.5 mmol), HOAc (0.1 g,1.65 mmol) and H at-5 ℃ 2 O (0.189 g,10.5 mmol) to a mixture of MeCN (10 mL) was added 1, 3-dichloro-5, 5-dimethylimidazoline-2, 4-dione (0.44 g,2.25 mmol) and the solution was stirred at the same temperature for 1h. The mixture was extracted with EtOAc (100 mL) and dried over Na 2 SO 4 Drying and concentration gave the title compound (0.41 g,1.4mmol, 93%) as a yellow oil as a crude product. The crude product was used directly in the next step without further purification.
Intermediate 6 preparation of 2- (cyclopentyloxy) -5- (5-methylisoxazol-4-yl) aniline
Step 1: synthesis of 4-bromo-1- (cyclopentyloxy) -2-nitrobenzene. Cyclopentanol (5.8 g,68.18mmol,1.5 eq.) and Cs 2 CO 3 (22.1 g,68.68mmol,1.5 eq.) was added to acetonitrile (100 mL) and stirred at room temperature. 4-bromo-1-fluoro-2-nitrobenzene (10.0 g,45.45mmol,1.0 eq.) is added in portions and the resulting reaction mixture is stirred at room temperature for 6h. The reaction mixture was diluted with water (200 mL) and extracted with EtOAc (3×150 mL). The combined organic layers were taken up with Na 2 SO 4 Dried, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography using 1% EtOAc in n-hexane to give the title compound (8.5 g, 65%) as a pale yellow liquid. 1 H NMR(400MHz,DMSO-d 6 ):δ1.58-1.62(m,3H),1.63 -1.72(m,3H),1.87-1.98(m,2H),5.02-5.05(m,1H),7.33(d,J＝9.2Hz,1H),7.77-7.79(m,1H),8.07(d,J＝2.4Hz,1H)。
Step 2: synthesis of 2- (4- (cyclopentyloxy) -3-nitrophenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolan. The bromide of step 1 (4.0 g,13.97mmol,1.0 eq.) and KOAc (4.1 g,41.93mmol,3.0 eq.) were stirred in dioxane (40 mL) at room temperature. To this solution, add part by part B 2 Pin 2 (5.3 g,20.95mmol,1.5 eq.) and under N 2 The resulting reaction mixture was purged under gas for 10 minutes. Adding PdCl 2 (dppf) DCM (1.1 g,1.397mmol,0.1 eq.) and the reaction mixture was stirred at 90℃for 16h. The reaction mixture was filtered through a pad of celite and washed with EtOAc (100 mL). The filtrate was concentrated under reduced pressure and the crude material was purified by column chromatography using 5% etoac in n-hexane to give the title dioxolane (3.0 g, 64%) as a pale yellow liquid. 1 H NMR(400MHz,DMSO-d 6 ):δ1.31(s,12H),1.57-1.59(m,2H),1.59 -1.73(m,4H),1.90-1.95(m,2H),5.07-5.09(m,1H),7.36(d,J＝8.4Hz,1H),7.83-7.86(m,1H),7.98(d,J＝1.2Hz,1H)。
Step 3: synthesis of 2- (cyclopentyloxy) -5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) aniline. Dioxyborolane of step 2 (3.0 g,9.0mmol,1.0 eq.) was stirred in MeOH (40 mL) at room temperature. To the direction of thePd/C (2.4 g, w/80%) was added to the solution and the resulting reaction mixture was taken up in H 2 Purged under gas (hydrogen balloon) and stirred at room temperature for 16h. The reaction mixture was filtered through a pad of celite and washed with EtOAc (100 mL). The filtrate was concentrated under reduced pressure to give the title aniline (2.6 g, 84%) as a pale brown viscous liquid. LCMS [ ESI, M+1 ]]:304.7。 1 H NMR(400MHz,DMSO-d 6 ):δ1.29(s,12H),1.56-1.58(m,2H),1.69-1.72(m,4H),1.84-1.88(m,2H),4.57(s,2H),4.77-4.79(t,J＝5.6Hz,1H),6.74(d,J＝8.0Hz,1H),6.85-6.88(m,1H),6.98(d,J＝1.2Hz,1H)。
Step 4: synthesizing 2- (cyclopentyloxy) -5- (5-methyl isoxazol-4-yl) aniline. Dioxahexacyclic ring H in 30mL microwave glass bottle at room temperature 2 Aniline of step 3 (0.2 g,0.66mmol,1.0 eq.) and 4-iodo-5-methylisoxazole (0.17 g,0.79mmol,1.2 eq.) are stirred in O (4:1) (12.5 mL). K is added to the solution 2 CO 3 (0.3 g,2.31mmol,3.5 eq.) and under N 2 The resulting reaction mixture was purged under gas for 10 minutes. PdCl is added to 2 (dppf) DCM (0.05 g,0.06mmol,0.1 eq.) was added to the reaction mixture and sealed with a cap. The resulting reaction mixture was heated under microwaves at 100 ℃ for 1h. An additional 4 batches were prepared on the same scale and the crude reaction mixtures were combined for work-up and purification. The reaction mixture was filtered through a pad of celite and washed with EtOAc (20 mL). The combined filtrates were diluted with water (25 mL) and extracted with EtOAc (3X 25 mL). The combined organic layers were taken up with Na 2 SO 4 Dried, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography using 15% EtOAc in n-hexane as the mobile phase to give the title aniline as a pale brown viscous liquid (0.25 g, 30%). LCMS [ ESI, M+1 ]]:259.7. 1 H NMR(400MHz,DMSO-d 6 ):δ1.57(s,2H),1.73(s,4H),1.86-1.88(d,2H),2.49(s,3H),4.74(s,2H),4.78(s,1H),6.63(d,J＝2.0Hz,1H),6.76(d,J＝1.6Hz,1H),6.81(d,J＝8.0Hz,1H),8.66(s,1H)。
Intermediate 7 preparation of 2- (cyclopentyloxy) -4-fluoro-5- (5-methylisoxazol-4-yl) aniline
Step 1: synthesis of 1-bromo-4- (cyclopentyloxy) -2-fluoro-5-nitrobenzene. Cyclopentanol (4.3 g,50.41mmol,1.0 eq.) and NaH (60%) (4.0 g,100.82mmol,2.0 eq.) were stirred in anhydrous THF (100 mL) at 0 ℃. 4-bromo-1-fluoro-2-nitrobenzene (10.0 g,42.01mmol,1.0 eq.) is added in portions to the solution at 0deg.C, and the resulting reaction mixture is slowly heated and stirred at room temperature for 6h. The reaction mixture was quenched with ice-cold water (150 mL) and extracted with EtOAc (3X 200 mL). The combined organic layers were taken up with Na 2 SO 4 Dried, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography using 1% etoac in n-hexane to give the title bromide as a pale yellow solid (9.0 g, 70%). LCMS [ ESI, M+1 ]]:305.9。 1 H NMR(400MHz,DMSO-d 6 ):δ1.56-1.75(m,6H),1.89-1.93(m,2H),5.06-5.09(t,J＝5.6Hz,1H),7.54(d,J＝10.8Hz,1H),8.31(d,J＝7.6Hz,1H)。
Step 2: synthesis of 2- (4- (cyclopentyloxy) -2-fluoro-5-nitrophenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolan. Step 1 bromide (4.0 g,13.15mmol,1.0 eq.) and KOAc (3.8 g,39.45mmol,3.0 eq.) were stirred in dioxane (80 mL) at room temperature. To this solution was added B 2 Pin 2 (6.0 g,23.67mmol,1.8 eq.) and under N 2 The resulting reaction mixture was purged under gas for 10 minutes. The resulting reaction mixture PdCl 2 (dppf) DCM (1.0 g,1.315mmol,0.1 eq.) was stirred at 100℃for 5h. The reaction mixture was filtered through a pad of celite and washed with EtOAc (100 mL) and the combined filtrates were concentrated under reduced pressure. The crude product was purified by column chromatography using 3% etoac in n-hexane to give the title dioxolane (3.0 g, 65%) as a pale brown viscous liquid. 1 H NMR(400MHz,DMSO-d 6 ):δ1.29(s,12H),1.59-1.61(m,2H),1.64 -1.75(m,4H),1.91-1.96(m,2H),5.10(d,J＝5.2Hz,1H),7.23(d,J＝11.2Hz,1H),8.07(d,J＝6.0Hz,1H)。
Step 3: synthesis of 2- (cyclopentyloxy) -4-fluoro-5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) aniline. Pd was added at room temperatureTo a stirred solution of step 2 of dioxolane (3.0 g,8.54mmol,1.0 eq.) in MeOH (60 mL) was added/C (2.4 g, w/80%). The reaction mixture obtained is reacted in H 2 The gas (balloon) was purged and stirred for 6h. The reaction mixture was filtered through a pad of celite and washed with MeOH (200 mL). The combined filtrates were concentrated under reduced pressure to give the title aniline as a pale brown viscous liquid (2.5 g, 91%). LCMS [ ESI, M+1 ]]：322.7。LCMS[ESI,M+1]:322.7。 1 H NMR(400MHz,DMSO-d 6 ):δ1.25(s,12H),1.56-1.58(m,2H),1.71-1.72(m,4H),1.89(s,2H),4.66(s,2H),4.80-4.82(t,J＝5.2Hz,1H),6.60(d,J＝11.2Hz,1H),6.88(d,J＝6.4Hz,1H)。
Step 4: synthesizing 2- (cyclopentyloxy) -4-fluoro-5- (5-methyl isoxazol-4-yl) aniline. At room temperature in dioxane H 2 Aniline of step 3 (1.0 g,31.13mmol,1.0 eq.) and 4-iodo-5-methylisoxazole (0.97 g,46.69mmol,1.5 eq.) are stirred in O (4:1) (30 mL). K is added to the solution 3 PO 4 (0.86 g,40.46mmol,1.5 eq.) and under N 2 The resulting reaction mixture was purged under gas for 15 minutes. X-phos Pd G2 (0.24G, 0.3.11mmol,0.1 eq.) was added to the reaction mixture and sealed with a cap. The reaction mixture was heated conventionally at 110℃for 1h, then diluted with water (200 mL) and extracted with EtOAc (3X 100 mL). The combined organic layers were taken up with Na 2 SO 4 Dried, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography with 7% etoac in n-hexane to give the title aniline as a pale brown viscous liquid (0.290 g, 34%). LCMS [ ESI, M+1 ]]:277.7。 1 H NMR(400MHz,DMSO-d 6 ):δ1.57-1.61(m,2H),1.73-1.75(m,4H),1.89-1.92(m,2H),2.49(s,3H),4.61(s,2H),4.80-4.83(t,J＝5.2Hz,1H),6.64(d,J＝8.0Hz,1H),6.80(d,J＝8.4Hz,1H),8.58(s,1H)。
Intermediate 8 preparation of 4-chloro-2- (cyclopentyloxy) -5- (5-methylisoxazol-4-yl) aniline
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Step 1: synthesis of 1-bromo-2-chloro-4- (cyclopentyloxy) -5-nitrobenzene. A stirred solution of cyclopentanol (5.0 g,58.95mmol,1.5 eq.) and NaOtBu (5.6 g,58.95mmol,1.5 eq.) in anhydrous DMF (100 mL) was prepared at room temperature. To the solution was added 1-bromo-2-chloro-4-fluoro-5-nitrobenzene (10.0 g,39.30mmol,1.0 eq.) in portions, and the resulting reaction mixture was stirred at room temperature for 2h. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (3×150 mL). The combined organic layers were taken up with Na 2 SO 4 Dried, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography using 1% ethyl acetate in n-hexane to give the title bromide as a pale yellow solid (7.8 g, 62%). 1 H NMR(400MHz,DMSO-d 6 ):δ1.56-1.60(m,2H),1.62-1.74(m,4H),1.87–1.92(m,2H),5.12–5.14(t,J＝5.6Hz,1H),7.70(s,1H),8.31(s,1H)。
Step 2: synthesis of 2- (2-chloro-4- (cyclopentyloxy) -5-nitrophenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolan. The bromide of step 1 (3.0 g,9.35mmol,1.0 eq.) and KOAc (2.7 g,28.07mmol,3.0 eq.) were stirred in dioxane (40 mL) at room temperature. To this solution, add part by part B 2 Pin 2 (3.5 g,14.02mmol,1.5 eq.) and under N 2 The resulting reaction mixture was purged under gas for 10 minutes. PdCl is then added 2 (dppf) DCM (0.76 g,0.93mmol,0.1 eq.) and the resulting reaction mixture was stirred at 90℃for 16h. The reaction mixture was filtered through a pad of celite, the bed was washed with EtOAc (100 mL), and the combined filtrates were concentrated under reduced pressure. The crude product was purified by column chromatography using 5% ethyl acetate in n-hexane to give the title dioxolane as a yellow liquid (1.6 g, 47%). 1 H NMR(400MHz,DMSO-d 6 ):δ1.30(s,12H),1.57–1.61(m,2H),1.64 -1.75(m,4H),1.90–1.96(m,2H),5.15–5.18(t,J＝5.6Hz,1H),7.44(s,1H),8.06(s,1H)。
Step 3: synthesis of 4-chloro-2- (cyclopentyloxy) -5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) aniline. Dioxyborolane of step 2 (1.2 g,3.55mmol,1.0 eq.) was stirred in EtOAc (30 mL) at room temperature. To this solution was added SnCl 2 .2H 2 O(3.6g，17.77mmol,5.0 eq.) the resulting reaction mixture was stirred at room temperature for 16h, then diluted with water (200 mL) and extracted with EtOAc (3×200 mL). The combined organic layers were taken up with Na 2 SO 4 Dried, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography using 10% ethyl acetate in n-hexane to give the title aniline as a pale brown viscous solid (1.0 g, 98%). LCMS [ ESI, M+1 ]]:338.8。 1 H NMR(400MHz,DMSO-d 6 ):δ1.23–1.30(m,12H),1.55(s,2H),1.70-1.72(d,4H),1.87–1.88(d,2H),4.74(s,2H),4.81(s,1H),6.73(s,1H),6.95(s,1H)。
Step 4: synthesizing 4-chloro-2- (cyclopentyloxy) -5- (5-methyl isoxazol-4-yl) aniline. Dioxahexacyclic ring H in 30mL glass bottle at room temperature 2 Aniline of step 3 (1.0 g,2.96mmol,1.0 eq.) and 4-iodo-5-methylisoxazole (0.17 g,2.96mol,1.0 eq.) were stirred in O (4:1) (12.5 mL). K is added to the solution 2 CO 3 (1.4 g,10.36mmol,3.5 eq.) and under N 2 The resulting reaction mixture was purged under gas for 10 minutes, and then PdCl was added 2 (dppf). DCM (0.24 g, 0.298 mmol,0.1 eq.) and sealed with a cap. The resulting reaction mixture was then stirred in a microwave at 100 ℃ for 1h, then filtered through a celite pad and the bed was washed with EtOAc (25 mL). The combined filtrates were diluted with water (25 mL) and extracted with EtOAc (3X 20 mL). The combined organic layers were taken up with Na 2 SO 4 Dried, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography using 15% ethyl acetate in n-hexane to give the title aniline as an off-white color (0.282 g, 33%). LCMS [ ESI, M+1 ]]:293.7。 1 H NMR(400MHz,DMSO-d 6 ):δ1.57(s,2H),1.73(s,4H),1.88(s,2H),2.32(s,3H),4.83(s,2H),4.91(s,1H),6.60(s,1H),6.90(s,1H),8.57(s,1H)。
Intermediate 9 preparation of 2- ((cyclopentyloxy) -5- (isothiazol-5-yl) aniline
Step 1:5- (tributylstannyl) isothiazole. To a solution of 5-bromoisothiazole (4.0 g,24.38 mmol) in THF (200 mL) was added dropwise a solution of n-BuLi (1.6M in hexane (22.86 mL,36.58 mmol) under nitrogen at-78deg.C. The reaction mixture was stirred at-78 ℃ for 30 minutes, then tributyltin chloride (11.90 g,36.58 mmol) was added dropwise at-78 ℃. The reaction mixture was stirred for a further 1h at-78 ℃ and then quenched into saturated ammonium chloride solution (500 mL) and extracted with diethyl ether (2 x250 mL). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by manual column chromatography using neutral alumina eluting with hexane to 3% ethyl acetate in hexane as the mobile phase to give the title isothiazole (10.00 g, 54.8%) as a pale yellow liquid.
* The reaction was carried out in two parallel batches (4.0 g x2=8.0 g) and the workup and purification were combined. LCMS:2.989min, ms: es+374.20,376.10 (M, m+2): 1 H NMR(400MHz,DMSO)δ8.72(s,1H),7.45-7.44(m,1H),1.54-1.48(m,6H),1.31-1.23(m,6H),1.17-1.13(m,6H),0.847(t,J＝7.2Hz,9H)。
Step 2:5- (4- (cyclopentyloxy) -3-nitrophenyl) isothiazole. In a 100ml sealed tube with N 2 A solution of 4-bromo-1- (cyclopentyloxy) -2-nitrobenzene (intermediate 6 step 1) (2.00 g,6.98 mmol) in 1, 4-dioxane (40 mL) was purged with gas for 10 min. (5- (tributylstannyl) isothiazole) of step 1 (3.92 g,10.48 mmol) and Tetrakis (0.806 g,0.698 mmol) were added and taken up with N 2 The gas was purged for an additional 5 minutes. The tube was capped, heated at 110deg.C for 16h, then poured into water (100 mL) and extracted with ethyl acetate (2X 100 mL). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by FCC using neutral alumina (20% ethyl acetate in hexane as the mobile phase) to give the title isothiazole as an off-white solid (1.4 g, 68.99%). LCMS:2.785min, MS: ES+291.2 (M+1); 1 H NMR(400MHz,DMSO)δ8.60(d,J＝1.6Hz,1H),8.26(d,J＝2.0Hz,1H),7.97(dd,J＝8.8,2.4Hz,1H),7.84(d,J＝1.2Hz,1H),7.47(d,J＝8.8Hz,1H),5.13(t,J＝5.6Hz,1H),1.95-1.92(m,2H),1.77-1.60(m,6H)。
step 3:2- (cyclopentyloxy) -5- (isothiazol-5-yl) aniline. At room temperature, go to the stepTo a solution of 2 isothiazole (0.700 g,2.41 mmol) in methanol-water (9:1) (14 mL) was added iron powder (0.675 g,12.05 mmol) and ammonium chloride (0.645 g,12.0 mmol). The reaction mixture was stirred for 3h at 90 ℃ then filtered through celite, the bed was washed with ethyl acetate (2×50 mL), the filtrate was diluted with water (50 mL) and the organic layer was separated. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was triturated in n-pentane: diethyl ether (7:3) (10 mL) to give the title aniline as a pale brown solid (0.299 g, 47.6%). LCMS: 2.7197 min, MS: ES+261.6 (M+1); 1 H NMR(400MHz,DMSO)δ8.49(s,1H),7.51(s,1H),6.95-6.83(m,3H),4.89(s,2H),4.82(s,1H),1.89(s,2H),1.74(s,4H),1.57(s,2H)。
Intermediate 10 preparation of 2- (cyclopentyloxy) -4-fluoro-5- (isothiazol-5-yl) aniline
Step 1:5- (4- (cyclopentyloxy) -2-fluoro-5-nitrophenyl) isothiazole. In a 100mL sealed tube with N 2 A solution of 1-bromo-4- (cyclopentyloxy) -2-fluoro-5-nitrobenzene (intermediate 7 step 1) (2.00 g,6.57 mmol) in 1, 4-dioxane (40 mL) was purged with gas for 10 min. 5- (tributylstannyl) isothiazole (intermediate 9 step 1) (3.69 g,9.86 mmol) and Tetrakis (0.759 g,0.657 mmol) were added and taken up with N 2 The gas was purged for an additional 5 minutes. The reaction mixture was sealed with a cap, heated at 110 ℃ for 16h, then poured into water (100 mL) and extracted with ethyl acetate (2 x75 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by FCC using neutral alumina (12% ethyl acetate in hexane as mobile phase) to give the title isothiazole (0.9 g, 44.4%) as a white solid. LCMS 2.968min, ms: es+309.1 (m+1); 1 H NMR(400MHz,DMSO)δ8.63(t,J＝2.0Hz,1H),8.59(d,J＝8.0Hz,1H),8.01(d,J＝2.0Hz,1H),7.58(d,J＝12.8Hz,1H),5.15(t,J＝5.6Hz,1H),1.98-1.93(m,2H),1.79-1.59(m,6H)。
step 2:2- (cyclopentyloxy) -4-fluoro-5- (isothiazol-5-yl) aniline. At room temperature, step 1 is followed by isothiazideTo a solution of oxazole (0.700 g,2.27 mmol) in methanol in water (9:1) (14 mL) was added iron powder (0.235 g,11.35 mmol) and ammonium chloride (0.603 g,11.3 mmol). The reaction mixture was stirred for 3h at 90 ℃, filtered through celite, the bed was washed with ethyl acetate (2×50 mL), the filtrate was diluted with water (75 mL) and the organic layer was separated. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by FCC (10% ethyl acetate in hexane as mobile phase) using neutral alumina to give a brown solid. The solid was diluted with DCM (2 mL) and precipitated with n-pentane (10 mL), filtered and dried in vacuo to give the title aniline (0.262 g, 41.5%) as a pale brown solid. LCMS: 2.169 min, MS: ES+279.6 (M+1); 1 H NMR(400MHz,DMSO)δ8.54(d,J＝1.6Hz,1H),7.58(d,J＝1.6Hz,1H),7.05(d,J＝7.6Hz,1H),6.90(d,J＝12.8Hz,1H),4.87(t,J＝5.6Hz,1H),4.75(s,2H),1.93-1.89(m,2H),1.77-1.74(m,4H),1.58-1.56(m,2H)。
Intermediate 11 preparation of 4-chloro-2- (cyclopentyloxy) -5- (isothiazol-5-yl) aniline
Step 1:5- (2-chloro-4- (cyclopentyloxy) -5-nitrophenyl) isothiazole. In a 100mL sealed tube with N 2 A solution of 1-bromo-2-chloro-4- (cyclopentyloxy) -5-nitrobenzene (intermediate 8 step 1) (2.00 g,6.23 mmol) in 1, 4-dioxane (40 mL, 20V) was purged with gas for 10 min. 5- (tributylstannyl) isothiazole (intermediate 9 step 1) (3.50 g,9.35 mmol) and Tetrakis (0.321 g,0.623 mmol) were added and taken up with N 2 The gas was purged for an additional 5 minutes. The reaction mixture was sealed with a cap, heated at 110 ℃ for 16h, then poured into water (100 mL) and extracted with ethyl acetate (2 x100 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by FCC (8% ethyl acetate in hexane as mobile phase) using neutral alumina to give the title isothiazole (1.1 g, 54.3%) as a white solid. LCMS:2.936min, MS: ES+325.1 (M+1); 1 H NMR(400MHz,DMSO)δ8.64(d,J＝1.6Hz,1H),8.42(s,1H),7.92(d,J＝2.0Hz,1H),7.71(s,1H),5.21(s,1H),1.97-1.92(m,2H),1.78-1.59(m,6H)。
step 2: 4-chloro-2- (cyclopentyloxy) -5- (isothiazol-5-yl) aniline. To a solution of step 1 isothiazole (0.700 g,2.15 mmol) in methanol-water (9:1) (14 mL) was added iron powder (0.603 g,10.77 mmol) and ammonium chloride (0.576 g,10.77% mmol) at room temperature. The reaction mixture was stirred at 90 ℃ for 3h, then cooled and filtered through celite bed and the bed was washed with ethyl acetate (2 x50 mL). The combined filtrates were washed with water (100 mL), and the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
Trituration of the crude product with n-pentane in diethyl ether (7:3) (10 mL) afforded the title aniline as a pale brown solid (0.313 g, 49.4%). LCMS:2.954min, MS: ES+295.6 (M+1); 1 H NMR(400MHz,DMSO)δ8.56(s,1H),7.59(s,1H),7.04(s,1H),6.96(s,1H),5.03(s,2H),4.88(s,1H),1.92-1.90(m,2H),1.77-1.74(m,4H),1.58(s,2H)。
intermediate 12 preparation of 4-chloro-2-cyclobutoxy-5- (5-methylisoxazol-4-yl) aniline
Step 1: synthesis of 1-bromo-2-chloro-4-cyclobutoxy-5-nitrobenzene. Preparation of cyclobutanol (CAS# 2919-23-5, angene) (1.71 g,0.0237mol,1.0 eq.) and Cs at room temperature 2 CO 3 (15.45 g,0.4745mol,2 eq.) in ACN (60 mL). 1-bromo-2-chloro-4-fluoro-5-nitrobenzene (CAS# 111010-08-3, combi block) (6 g,0.0237mol,1.0 eq) was added to the reaction mixture in portions and stirred at room temperature for 6h. The reaction mixture was poured into ice cold water, and the precipitate was collected and dried to give the title ether (5 g, 69.2%) as a pale yellow solid. 1 H NMR(400MHz,DMSO-d 6 ):δ8.34(s,1H),7.48(s,1H),4.99-4.85(m,1H),2.53-2.43(m,2H),2.08-2.00(m,2H),1.84-1.79(m,1H),1.66-1.59(m,1H)。
Step 2: synthesizing 5-bromo-4-chloro-2-cyclobutoxy aniline. The ether of step 1 (4 g,0.013mol,1 eq.) and iron powder (3.67 g, 0.065) were stirred at room temperature5mol,5 eq) in acetic acid (40 ml) and then heated to 80 ℃ for 2h. The mixture was cooled, filtered through a celite pad, and washed with EtOAc (2×100 mL). The filtrate was concentrated under reduced pressure and the crude residue was purified by column chromatography using 2% etoac in n-hexane as the mobile phase to give the title aniline as a pale pink solid (3.1 g, 85.9%). 1 H NMR(400MHz,DMSO-d 6 ):δ6.90(s,1H),6.76(s,1H),5.13(s,1H),4.71-4.64(m,1H),2.44-2.36(m,2H),2.09-2.00(m,2H),1.81-1.79(m,1H)1.76-1.74(m,1H)。
Step 3: synthesis of 4-chloro-2-cyclobutoxy-5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) aniline. A solution of step 2 aniline (1.2 g,0.0043mol,1.0 eq.) and KOAc (2.14 g,0.0021mol,5.0 eq.) in dioxane (12 ml, 10V) was stirred at room temperature. To this solution, add part by part B 2 Pin 2 (2.21 g,0.0087mol,2.0 eq.) and purging the resulting reaction mixture under argon for 20 minutes. Adding PdCl 2 (dppf) DCM (0.63 g,0.00077mol,0.2 eq.) the resulting reaction mixture was purged under argon for 10 minutes and then stirred at 80℃for 16h. The reaction mixture was cooled, then filtered through a celite pad, and washed with EtOAc (2×100 mL). The filtrate was concentrated under reduced pressure and the crude residue was purified by column chromatography using 4% etoac in n-hexane as the mobile phase to give the title dioxolane as a pale pink solid (0.9 g,2.78mmol, 65%). UPLC-MS (method 5) M/z 324.2 (M+H) + At 2.72 minutes.
Step 4: synthesizing 4-chloro-2-cyclobutoxy-5- (5-methyl isoxazol-4-yl) aniline. Dioxyboropentalene (0.7 g,2.16mol,1.0 eq.) from step 3 and 4-iodo-5-methylisoxazole (CAS# 7064-38-2, enamine) (0.0.45 g,2.16mol,1.0 eq.) were combined in dioxane at room temperature with H 2 The solution in O (8:2) was added to a 30mL microwave glass bottle. Adding K 2 CO 3 (1.04 g,7.58mol,3.5 eq.) and the resulting reaction mixture was purged under argon for 20 minutes. PdCl is added to 2 (dppf) DCM (0.17 g,0.216mol,0.1 eq.) was added to the reaction mixture and heated under microwaves at 110℃for 1h. The resulting reaction mixture was passed through celiteThe pad was filtered and washed with EtOAc (2×100 mL). The filtrate was concentrated under reduced pressure and the crude material was purified by column chromatography using 5% etoac in n-hexane as the mobile phase to give the title aniline as a pale pink solid (0.25 g, 41.5%). UPLC-MS (method 5) M/z 279.1 (M+H) + At 2.46 minutes. 1 H NMR(400MHz,DMSO-d 6 ):δ8.57(s,1H),6.75(s,1H),6.63(s,1H),4.97(s,2H),4.75-4.71(m,1H),2.28(s,1H),2.10-2.05(m,2H),1.81-1.78(m,1H),1.66-1.63(m,1H),1.31-1.18(m,2H)。
Intermediate 13 preparation of 4-chloro-2- (3, 3-difluorocyclobutoxy) -5- (5-methylisoxazol-4-yl) aniline
Step 1: synthesis of 1-bromo-2-chloro-4- (3, 3-difluorocyclobutoxy) -5-nitrobenzene. 3, 3-Difluorocyclobutan-1-ol (CAS# 637031-88-0, angene) (2.56 g,0.0237mol,1.0 eq.) and Cs are stirred at room temperature 2 CO 3 (15.45 g,0.4745mol,2 eq.) in MeCN (60 mL). 1-bromo-2-chloro-4-fluoro-5-nitrobenzene (CAS# 111010-08-3, combi block) (6 g,0.0237mol,1.0 eq) was added in portions and the resulting reaction mixture was stirred at room temperature for 6h. The reaction mixture was poured into ice-cold water, the precipitate was filtered and dried to give the title ether as a pale yellow solid (5 g,14.6mmol, 62%). 1 H NMR(400MHz,DMSO-d 6 ):δ8.39(s,1H),7.60(s,1H),5.05-5.02(m,1H),3.31-3.25(m,2H),2.81-2.74(m,2H)。
Step 2: synthesizing 5-bromo-4-chloro-2- (3, 3-difluoro cyclobutoxy) aniline. A solution of diethyl ether (4 g,0.012mol,1 eq) and iron powder (3.28 g,0.058mol,5 eq) from step 1 in acetic acid (40 ml) was prepared at room temperature and then stirred at 80℃for 2h. The reaction mixture was filtered through a celite pad and washed with EtOAc (2×100 mL). The combined filtrates were concentrated under reduced pressure and the crude residue was purified by column chromatography using 2% etoac in n-hexane as the mobile phase to give the title aniline as a pale pink solid (3.1 g,10mmol, 76%). UPLC-MS (method 5) M/z 311.8 (M+H) + At 2.20 minutes; 1 H NMR(400MHz,DMSO-d 6 ):δ6.93(S,1H),6.88(S,1H),5.28(s,2H),4.80-4.73(m,2H),3.24-3.13(m,2H),2.80-2.67(m,2H)。
step 3: synthesis of 4-chloro-2- (3, 3-difluorocyclobutoxy) -5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) aniline. A solution of step 2 aniline (1.2 g,0.0038mol,1.0 eq.) and KOAc (2.14 g,0.0021mol,5.0 eq.) was stirred in dioxane (12 ml) at room temperature. Add B in portions 2 Pin 2 (2.21 g,0.0087mol,2.3 eq.) the resulting reaction mixture was purged under argon for 20 minutes. Adding PdCl 2 (dppf) DCM (0.63 g,0.00077mol,0.2 eq.) and the reaction mixture was purged under argon for 10 minutes then stirred at 80℃for 16h. The reaction mixture was filtered through a celite pad and washed with EtOAc (2×100 mL). The combined filtrates were concentrated under reduced pressure and the crude residue was purified by column chromatography using 4% etoac in n-hexane as the mobile phase to give the title dioxolane as a pale pink solid (0.7 g, 50.4%). UPLC-MS (method 5) M/z 360.1 (M+H) + At 2.63 minutes.
Step 4: synthesis of 4-chloro-2- (3, 3-difluorocyclobutoxy) -5- (5-methylisoxazol-4-yl) aniline. Dioxa-hexacyclic ring H in a 30mL microwave glass bottle at room temperature 2 A stirred solution of step 3 of dioxolane (0.7 g,1.94mol,1.0 eq.) and 4-iodo-5-methylisoxazole (CAS# 7064-38-2, enamine) (0.400 g,1.94mol,1.0 eq.) was stirred in O (8:2). Adding K 2 CO 3 (0.94 g,6.82mol,3.5 eq.) the resulting reaction mixture was purged under argon for 20 minutes. Adding PdCl 2 (dppf) DCM (0.17 g,0.216mol,0.1 eq.) and sealed with a cap. The resulting reaction mixture was heated under microwaves at 110 ℃ for 1h, cooled, filtered through a pad of celite, and washed with EtOAc (2×100 mL). The combined filtrates were then concentrated under reduced pressure and the crude residue was purified by column chromatography using 5% etoac in n-hexane as the mobile phase to give the title aniline as a pale pink solid (0.26 g,0.72mmol, 37%). UPLC-MS (method 5) M/z 315.1 (M+H) + At 2.35 minutes. 1 H NMR(400MHz,DMSO-d 6 ):δ8.58(S,1H),6.86(s,1H),6.63(s,1H),5.12(m,2H),4.83-4.80(m,1H),3.23-3.18(m,2H),2.79-2.74(m,2H)。2.37(S,3H)。
Intermediate 14 preparation of 4-chloro-2-cyclobutoxy-5- (isothiazol-5-yl) aniline
Step 1: synthesis of 4-chloro-2-cyclobutoxy-5- (isothiazol-5-yl) aniline. In a 10ml sealed tube with N 2 A solution of intermediate 12 step 2 (1.20 g,4.34 mmol) in 1, 4-dioxane (12 mL) was purged with gas for 10 min. 5- (tributylstannyl) isothiazole (intermediate 9 step 1;2.4g,6.52 mmol) and Tetrakis (0.500 g,0.43 mmol) were added using N 2 The gas was purged for an additional 5 minutes and then heated at 110℃for 16 hours. The mixture was cooled, poured into water (100 mL) and extracted with ethyl acetate (2 x100 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by FCC using neutral alumina (5% ethyl acetate in hexane as the mobile phase) to give the title aniline as an off-white solid (0.33 g, 27.5%). LCMS:2.467min, MS: ES+281.1 (M+1); 1 H NMR(400MHz,DMSO)δ8.57(d,J＝1.6Hz,1H),7.60(d,J＝1.6Hz,1H),7.05(s,1H),6.80(s,1H),5.10(s,2H),4.79-4.76(m,1H),2.50-2.41(m,2H),2.14–2.04(m,2H),1.84–1.77(m,1H),1.69–1.62(m,1H)。
intermediate 15 preparation of 4-chloro-2- (3, 3-difluorocyclobutoxy) -5- (isothiazol-5-yl) aniline
Step 1: synthesis of 4-chloro-2- (3, 3-difluorocyclobutoxy) -5- (isothiazol-5-yl) aniline. In a 10ml sealed tube with N 2 A solution of intermediate 13 step 2 (1.50 g,4.84 mmol) in 1, 4-dioxane (12 mL) was purged with gas for 10 min. Intermediate 9, step 1 (2.68 g,7.22 mmol) and Tetrakis (0.555 g,0.48 mmol) were added, further with N 2 The gas was purged for 5 minutes and then heated at 110℃for 16 hours. After cooling, the reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (2 x100 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by FCC using neutral alumina (5% ethyl acetate in hexane as the mobile phase) to give the title aniline as an off-white solid (0.33 g, 22%). UPLC-MS (method 5): M/z 317.1 (M+H) + At 2.37 minutes; 1 H NMR(400MHz,DMSO)δ8.57(d,J＝1.2Hz,1H),7.61(d,J＝1.2Hz,1H),7.06(s,1H),6.92(s,1H),5.25(brs,2H),4.85(brs,1H),3.29-3.18(m,2H),2.84–2.72(m,2H)。
example 1:3- (N- (4-chloro-5-cyano-2- (cyclopentyloxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
Step 1: 2-chloro-4-fluoro-5-nitrobenzonitrile: to H of 2-chloro-4-fluorobenzonitrile (20.0 g,129.0 mmol) at 0deg.C 2 SO 4 Fuming nitric acid (20 mL) was added dropwise to the (98%, 40 mL) solution. The resulting mixture was stirred at 0 ℃ for 30 minutes. The solution was poured into ice water, and the white precipitate formed was collected by filtration and washed with water to give the title compound as a white solid (20.7 g,103.5mmol, yield 80%). 1 H NMR(400MHz,DMSO-d 6 )δ8.95(d,J＝7.7Hz,1H),8.28(d,J＝10.9Hz,1H)。
Step 2: 2-chloro-4- (cyclopentyloxy) -5-nitrobenzonitrile: 2-chloro-4-fluoro-5-nitrobenzamidine (8.6 g,43.0 mmol), cyclopentanol (1.85 g,215.0 mmol) and Cs were reacted at room temperature 2 CO 3 A mixture of (28.0 g,86.0 mmol) in MeCN (100 mL) was stirred overnight. The reaction mixture was filtered through celite. The filtrate was concentrated and passed through Biotage Isolera One (C 18 Column with 10% -90% MeCN/H 2 O elution) to give the title compound as a white solid (5.2 g,19.5mmol, 45% yield). 1 H NMR(400MHz,DMSO-d 6 )δ8.62(s,1H),7.81(s,1H),5.26(td,J＝5.6,2.8Hz,1H),2.04-1.90(m,2H),1.88-1.69(m,2H),1.73-1.56(m,4H)。
Step 3: 5-amino-2-chloro-4- (cyclopentyloxy) benzonitrile: 2-chloro-4- (cyclopentyloxy) -5-nitrobenzonitrile (3.9 g,14.7 mmol), iron powder (4.9 g,88.2 mmol) and NH 4 A mixture of Cl (1.6 g,29.4 mmol) in a mixture of EtOH and water (v/v=5/1, 120 mL) was heated at 85℃for 2h. The resulting mixture was filtered through celite and concentrated to give the title compound as a yellow solid (2.36 g,10.0mmol, yield 68%). UPLC-MS (method 3) M/z 235,237 (M-H) - At 2.03 minutes.
Step 4:3- (N- (4-chloro-5-cyano-2- (cyclopentyloxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester: to a solution of 5-amino-2-chloro-4- (cyclopentyloxy) benzonitrile (2.36 g,10.0 mmol) and pyridine (1.7 g,21.6 mmol) in DCM (100 mL) was added methyl 3- (chlorosulfonyl) -4-cyclopropylbenzoate (intermediate 3;2.96g,10.8 mmol) at room temperature, and the solution was stirred at RT overnight. The solvent was removed in vacuo and the crude product was purified by Biotage Isolera One (C 18 Column with 10% -90% MeCN/H 2 O elution) to give the title compound as a white solid (1.20 g,2.5mmol, yield 25%). UPLC-MS (method 1) M/z 473.05 (M-H) - At 2.383 minutes.
Step 5:3- (N- (4-chloro-5-cyano-2- (cyclopentyloxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid: to 3- (N- (4-chloro-5-cyano-2- (cyclopentyloxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester (1.2 g,2.5 mmol) in THF and H 2 To a solution in a mixture of O (v/v=1:1, 40 mL) LiOH (360 mg,15.0 mmol) was added and the reaction was stirred at RT overnight. The solvent was removed in vacuo and the crude product was purified by Biotage Isolera One (C 18 Column with 10% -90% MeCN/H 2 O eluted, containing 0.1% HCOOH) to give the title compound as a red solid (1.06 g,2.3mmol, 92% yield). UPLC-MS (method 1) M/z 459.05 (M-H) - At 2.150 minutes. 1 H NMR(400MHz,DMSO-d 6 )δ13.22(s,1H),10.06(s,1H),8.32(t,J＝1.4Hz,1H),8.00(dd,J＝8.2,1.9Hz,1H),7.67(s,1H),7.26(s,1H),7.11(d,J＝8.3Hz,1H),4.77-4.80(m,1H),2.68-2.60(td,J＝8.4,4.2Hz,1H),1.76-1.73(m,2H),1.47-1.45(dt,J＝7.9,3.3Hz,1H),1.44-1.38(m,5H),0.99-0.96(m,2H),0.79-0.78(m,2H)。
The following examples were prepared by a method analogous to example 1, substituting, if necessary, appropriate starting materials and intermediates. When intermediates 1 and 2 are used and following general scheme 1, the sulfonamide is formed in the presence of the free acid, thus directly preparing the target acid without the need for penultimate ester hydrolysis.
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Example 20:3- (N- (2- (cyclopentyloxy) -5- (isothiazol-5-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
Step 1:3- (N- (2- (cyclopentyloxy) -5- (isothiazol-5-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester: a mixture of 2- (cyclopentyloxy) -5- (isothiazol-5-yl) aniline (intermediate 9) (100 mg, 380. Mu. Mol, 99% pure), methyl 3- (chlorosulfonyl) -4-cyclopropylbenzoate (157 mg, 570. Mu. Mol) and pyridine (92.0. Mu.L, 1.14 mmol) in DCM (2 mL) was heated to 35℃and stirred for 3 days. The mixture was concentrated onto silica and purified by silica gel chromatography (12 g column, 0-100% EtOAc/isohexane) to give the title compound (171 mg,340 μmol,89%, 99% purity) as a pale yellow solid. UPLC-MS (method 4) M/z 499.4 (M+H) + ,497.2(M-H) - At 2.01 minutes. 1 H NMR(500MHz,DMSO-d 6 )δ9.81(s,1H),8.53(d,J＝1.8Hz,1H),8.41(d,J＝1.9Hz,1H),8.00(dd,J＝8.2,1.9Hz,1H),7.58(d,J＝1.8Hz,1H),7.51–7.45(m,2H),7.15(d,J＝8.3Hz,1H),7.01(d,J＝9.2Hz,1H),4.76–4.70(m,1H),3.84(s,3H),2.80–2.73(m,1H),1.82–1.75(m,2H),1.56–1.40(m,6H),1.06–0.98(m,2H),0.84–0.77(m,2H)。
Step 2:3- (N- (2- (cyclopentyloxy) -5- (isothiazol-5-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid: the product of step 1 above (171 mg, 340. Mu. Mol, 99% purity) and LiOH H were combined 2 O (58.0 mg,1.38 mmol) in THF/MeOH/H 2 The mixture in O (4:1:1, 2.1 mL) was stirred at 40℃overnight. H for mixture 2 O (5 mL) was diluted, acidified to pH 4 with 1M HCl (aq), and extracted with EtOAc (3X 15 mL). The organic extracts were combined, washed with brine (15 mL), dried (MgSO 4 ) And the solvent was removed in vacuo. The residue was loaded onto silica and purified by silica gel chromatography (12 g column, 0-10% MeOH/DCM) followed by trituration with TBME to give the title compound as a pale yellow solid (97.3 mg,199 μmol,59%, 99% purity). UPLC-MS (method 4) M/z 485.3 (M+H) + ,483.2(M-H) - At 1.83 minutes. 1 H NMR(500MHz,DMSO-d 6 )δ13.23(s,1H),9.76(s,1H),8.52(d,J＝1.8Hz,1H),8.41(d,J＝1.9Hz,1H),7.98(dd,J＝8.2,1.9Hz,1H),7.57(d,J＝1.8Hz,1H),7.49–7.45(m,2H),7.12(d,J＝8.2Hz,1H),7.01(d,J＝8.8Hz,1H),4.76–4.70(m,1H),2.79–2.71(m,1H),1.83–1.74(m,2H),1.58–1.41(m,6H),1.05–0.97(m,2H),0.83–0.76(m,2H)。
The following examples are prepared by a method analogous to example 20, substituting, if necessary, appropriate starting materials and intermediates:
example 23:3- (N- (2- (cyclopentyloxy) -5- (isothiazol-5-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
Step 1:3- (N- (2- (cyclopentyloxy) -5- (5-methylisoxazol-4-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester: a mixture of 2- (cyclopentyloxy) -5- (5-methylisoxazol-4-yl) aniline (intermediate 6) (100 mg, 383. Mu. Mol, 99% pure), methyl 3- (chlorosulfonyl) -4-cyclopropylbenzoate (160 mg, 582. Mu. Mol) and pyridine (100. Mu.L, 1.24 mmol) in DCM (2 mL) was heated to 35℃and stirred over the weekend. The mixture was concentrated onto silica and purified by silica gel chromatography (12 g column, 0-100% EtOAc/isohexane) to give the title compound (178 mg,0.35mmol,91% purity 97%) as a pale orange foam. UPLC-MS (method 4): M/z 497.3 (M+H) + ,495.3(M-H) - At 1.96 minutes.
Step 2:3- (N- (2- (cyclopentyloxy) -5- (5-methylisoxazol-4-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid: the product of step 1 above (180 mg, 352. Mu. Mol, 97% purity) and HCl (4M dioxane solution) (500. Mu.L, 2.00 mmol) were combined in dioxane/H 2 The mixture in O (2:1, 1.5 mL) was heated to 50deg.C and stirred overnight. Concentrated HCl (500. Mu.L, 37% w/w) and THF (0.50 mL) were added and the mixture was heated to 70℃and stirred for 24h. Concentrated HCl (500. Mu.L, 37% w/w) was added and the reaction stirred at 70℃for 24h. By H 2 The mixture was diluted with O (10 mL) and extracted with EtOAc (3X 20 mL). The organic extracts were combined, washed with brine (10 mL), dried (MgSO 4 ) And concentrated onto silica. The crude product was partially purified by silica gel chromatography (12 g column, 0-10% MeOH/DCM) followed by preparative HPLC (Waters X-Select CSH C 18 The ODB preparation column was prepared with a column,purification was performed at 5 μm,30mm X100 mm,40-70% (0.1% formic acid (aq))/MeCN to give the title compound as a white fluffy solid (33 mg, 68. Mu. Mol,19%, purity 99%). UPLC-MS (method 4): M/z 483.4 (M+H) + ,481.3(M-H) - At 1.80 minutes. 1 H NMR(500MHz,DMSO-d 6 )δ13.23(br s,1H),9.61(s,1H),8.70(s,1H),8.37(d,J＝1.8Hz,1H),7.96(dd,J＝8.2,1.9Hz,1H),7.30(d,J＝2.2Hz,1H),7.23(dd,J＝8.5,2.3Hz,1H),7.11(d,J＝8.3Hz,1H),6.97(d,J＝8.6Hz,1H),4.78–4.57(m,1H),2.83–2.67(m,1H),2.44(s,3H),1.84–1.71(m,2H),1.59–1.38(m,6H),1.06–0.95(m,2H),0.86–0.76(m,2H)。
The following examples were prepared by a method analogous to example 23, substituting, if necessary, appropriate starting materials and intermediates:
Example 8:3- (N- (2- (cyclopentyloxy) -5- (1H-tetrazol-1-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
Step 1: (4-fluoro-3-nitrophenyl) carbamic acid tert-butyl ester: a mixture of 4-fluoro-3-nitroaniline (1.56 g,10.0 mmol), di-tert-butyl dicarbonate (4.36 g,20.0 mmol), TEA (4.04 g,40.0 mmol) and DMAP (0.622 g,5.0 mmol) in DCM (20 mL) was stirred at room temperature for 12h. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc and taken up in saturated Na 2 CO 3 Wash with water and brine. The organic layer was taken up with Na 2 SO 4 Drying and concentration gave the crude product. The crude product was purified by silica gel chromatography (eluting with 1/10 EtOAc/PE) to give the title compound as a yellow oil (2.0 g,7.81mmol, 39%). 1 H NMR(400MHz,DMSO-d 6 )δ8.13(dd,J＝6.7,2.7Hz,1H),7.76(ddd,J＝8.9,4.0,2.7Hz,1H),7.62(dd,J＝10.9,8.9Hz,1H),1.39(s,9H)。
Step 2: (4- (cyclopentyloxy) -3-nitrophenyl) carbamic acid tert-butyl ester: the carbamate of step 1 (0.76 g,3.0 mmol), cyclopentanol (0.52 g,6.0 mmol) and Cs were reacted with each other 2 CO 3 A mixture of (1.85 g,6.0 mmol) in MeCN (20 mL) was stirred at room temperature for 12h. The reaction mixture was filtered through celite and the filtrate was concentratedAnd pass through Biotage Isolera One (C) 18 Column with 10% -90% MeCN/H 2 O elution) to give the title compound (0.61 g,1.89mmol, 63%).
Step 3: 4-cyclopentyloxy-3-nitroaniline: a solution of the carbamate of step 2 (0.61 g,1.89 mmol) in HCl EtOAc (4M, 3 mL) was stirred at room temperature for 2h. The resulting mixture was concentrated and dried under vacuum to give the title compound (0.22 g,0.97mmol, 51%). UPLC-MS (method 3) M/z 223.0 (M+H) + At 0.94 minutes.
Step 4:1- (4-cyclopentyloxy-3-nitrophenyl) -1H-tetrazole: step 3 aniline (0.22 g,1.0 mmol), trimethyl orthoformate (0.63 g,6.0 mmol) and NaN 3 A mixture of (0.13 g,2.0 mmol) in HOAc (5 mL) was heated at 80deg.C for 2h. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc and taken up in saturated Na 2 CO 3 Wash with water and brine. The organic layer was taken up with Na 2 SO 4 Drying and concentration gave the crude product. The crude product was purified by Biotage Isolera One (C 18 Column with 10% -90% MeCN/H 2 O elution) to give the title compound (0.24 g,0.88mmol, 88%). UPLC-MS (method 3) M/z 276.0 (M+H) + At 1.22 minutes.
Step 5: 2-cyclopentyloxy-5- (1H-tetrazol-1-yl) aniline: tetrazole of step 4 (0.24 g,0.88 mmol), iron powder (0.29 g,5.28 mmol) and NH 4 Cl (0.09 g,1.78 mmol) in EtOH and water (24 mL, etOH/H) 2 The mixture of O (v/v=5/1) was heated at 85 ℃ for 2h. The resulting mixture was filtered through celite and concentrated to give the title compound (0.17 g,0.69mmol, 78%). UPLC-MS (method 3) M/z 246.0 (M+H) + At 1.21 minutes.
Step 6: methyl 4-cyclopropyl-3- (N- (2-isopropoxy-5- (1H-tetrazol-1-yl) phenyl) sulfamoyl) benzoate: to a solution of step 5 aniline (0.17 g,0.69 mmol) and pyridine (0.11 g,1.38 mmol) in dry DCM (5 mL) was added methyl 3- (chlorosulfonyl) -4-cyclopropylbenzoate (intermediate 3) (0.18 g,0.68 mmol) and the solution stirred at RT for 4h. The solvent was removed in vacuo and the crude product was purified by Biotage Isolera One (C 18 The column is provided with a plurality of holes,with 10% -90% MeCN/H 2 O elution) to give the title compound (0.16 g,0.33mmol, 52%). UPLC-MS (method 3) M/z 482.0 (M-H) - At 1.99 minutes.
Step 7:3- (N- (2- (cyclopentyloxy) -5- (1H-tetrazol-1-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid. A mixture of the ester of step 6 (0.16 g,0.33 mmol) and LiOH (0.069 g,1.67 mmol) in THF (5 mL) and water (5 mL) was stirred at room temperature for 2 hours. THF was removed under reduced pressure and the pH of the aqueous solution was adjusted to 3 with 2M HCl. The mixture was extracted with EtOAc (20 mL. Times.3), and dried over Na 2 SO 4 Dried and concentrated in vacuo to give the title compound as a white solid (0.048 g,0.108mmol, 31%). UPLC-MS (method 1) M/z 468.05 (M-H) - At 1.823 minutes. 1 H NMR(400MHz,DMSO-d 6 )δ13.20(s,1H),9.95(s,2H),8.37(d,J＝1.9Hz,1H),7.98-7.96(dd,J＝8.2,1.9Hz,1H),7.78(d,J＝2.7Hz,1H),7.62-7.60(dd,J＝8.9,2.7Hz,1H),7.15-7.12(dd,J＝11.4,8.7Hz,2H),4.73(m,1H),2.73(td,J＝8.5,4.3Hz,1H),1.79-1.78(m,2H),1.50-1.43(m,6H),1.01-0.97(m,2H),0.80-0.77(m,2H)。
The following examples were prepared by a method analogous to example 8, substituting, if necessary, the appropriate starting materials and intermediates:
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Example 52:3- (N- (2- ((2-oxaspiro [3.3] hept-6-yl) oxy) -4-chloro-5-cyanophenyl) sulfamoyl) -4-cyclopropylbenzoic acid
Step 1:4- ((2-oxaspiro [ 3.3)]Hept-6-yl) oxy) -2-chloro-5-nitrobenzonitrile: 2-chloro-4-fluoro-5-nitrobenzonitrile (example 1, procedure1) (0.150 g,0.75 mmol), 2-oxaspiro [3.3]]A mixture of hept-6-ol (0.085 g,0.75 mmol) and NaH (0.022 g,0.90mmol, 60%) in THF (5 mL) was stirred at RT for 12h. The reaction mixture was filtered through celite. The filtrate was concentrated and purified by silica gel chromatography (eluting with 50% etoac/PE) to give the title compound (0.180 g,0.40mmol, 53% yield, 65% purity) as a yellow solid. 1 H NMR(400MHz,DMSO-d 6 )δ8.67(s,1H),7.61(s,1H),4.97(p,J＝6.8Hz,1H),4.64(s,2H),4.55(s,2H),2.87(ddd,J＝10.3,6.8,3.2Hz,2H),2.34-2.23(m,2H)。
Step 2:4- ((2-oxaspiro [ 3.3)]Hept-6-yl) oxy) -5-amino-2-chlorobenzonitrile: 4- ((2-oxaspiro [ 3.3)]Hept-6-yl) oxy) -2-chloro-5-nitrobenzonitrile (0.180 g,0.41mmol, 65%), iron powder (0.114 g,2.04 mmol) and NH 4 A mixture of Cl (0.044 g,0.82 mmol) in a mixture of EtOH and water (3 mL, etOH/H 2 O (v/v=5/1)) was heated at 85 ℃ for 2h. The resulting mixture was filtered through celite and concentrated to provide the crude product. The crude product was purified by Biotage Isolera One (C 18 Column with 10% -90% MeCN/H 2 O elution) to give the title compound as a yellow oil (0.071 g,0.27mmol, 66% yield). UPLC-MS (method 3) M/z 265.0 (M+H) + At 1.089 minutes.
Step 3:3- (N- (2- ((2-oxaspiro [3.3 ])]Hept-6-yl) oxy) -4-chloro-5-cyanophenyl-sulfamoyl) -4-cyclopropylbenzoic acid methyl ester: to 4- ((2-oxaspiro [ 3.3)]To a solution of hept-6-yl) oxy) -5-amino-2-chlorobenzonitrile (0.071 g,0.27 mmol) in pyridine (0.043 g,0.54 mmol) was added methyl 3- (chlorosulfonyl) -4-cyclopropylbenzoate (0.074 g,0.27 mmol) and the solution stirred at RT for 16h. The solvent was removed in vacuo and the crude product was purified by Biotage Isolera One (C 18 Column with 10% -90% MeCN/H 2 O elution) to give the title compound (0.073 g,0.15mmol, yield 55%) as a white solid. UPLC-MS (method 3) M/z 501.00 (M-H) - At 1.570 minutes.
Step 4:3- (N- (2- ((2-oxaspiro [3.3 ])]Hept-6-yl) oxy) -4-chloro-5-cyanophenyl-sulfamoyl) -4-cyclopropylbenzoic acid: 3- (N- (2- ((2-oxaspiro [3.3 ])]Hept-6-yl) oxy) -4-chloro-5-cyanophenyl-sulfamoylA mixture of methyl 4-cyclopropylbenzoate (0.055 g,0.11 mmol) and LiOH (0.013 g,0.55 mmol) in a mixture of THF (1 mL) and water (1 mL) was stirred at RT for 16 hours. THF was removed under reduced pressure and the pH of the aqueous solution was adjusted to 3 with 2M HCl. The mixture was extracted with EtOAc (20 mL. Times.3) and dried over Na 2 SO 4 Dried and concentrated in vacuo to give the crude product. The crude product was purified by Biotage Isolera One (C 18 Column with 10% -90% MeCN/H 2 O elution) to give the title compound (0.026 g,0.053mmol, 48% yield) as a white solid. UPLC-MS (method 1) M/z 487.00 (M-H) - At 1.750 minutes. 1 H NMR(400MHz,DMSO-d 6 )δ13.28(s,1H),10.18(s,1H),8.30(d,J＝1.8Hz,1H),7.99(dd,J＝8.2,1.8Hz,1H),7.75(s,1H),7.18-7.10(m,2H),4.63-4.51(m,1H),4.55(s,2H),4.43(s,2H),2.71(td,J＝8.3,4.2Hz,1H),2.62(ddd,J＝10.1,6.9,3.3Hz,2H),1.73(ddd,J＝10.1,7.0,3.3Hz,2H),1.02(dt,J＝8.2,3.3Hz,2H),0.91-0.78(m,2H)。
The following examples were prepared by methods analogous to examples 1 or 52, substituting, if necessary, the appropriate starting materials and intermediates:
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example 50:3- (N- (4-chloro-2- (3, 3-difluorocyclobutoxy) -5- (isothiazol-5-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
Step 1:3- (N- (4-chloro-2- (3, 3-difluorocyclobutoxy) -5- (isothiazol-5-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester: a solution of intermediate 15 (165 mg, 521. Mu. Mol), methyl 3- (chlorosulfonyl) -4-cyclopropylbenzoate (215 mg, 781. Mu. Mol; intermediate-3) and pyridine (126. Mu.L, 1.56 mmol) in DCM (2 mL) was stirred at RT for 4h. The reaction mixture was diluted with DCM (7 mL) and water (7 mL) and the phases separated. The aqueous phase was extracted with DCM (2×7 mL) and the combined organic phases were concentrated in vacuo. The residue was purified by silica gel chromatography (12 g column, 0-100% etoac/isohexane) to give the title compound (236 mg,0.41mmol, yield 78%, purity 96%) as a white solid. UPLC (method 4) M/z 556.3 (M+H) + ,554.9(M-H) - At 1.94 minutes. 1 H NMR (500 mhz, dmso) δ10.24 (s, 1H), 8.61 (d, j=1.9 hz, 1H), 8.41 (s, 1H), 8.00 (d, j=8.2 hz, 1H), 7.65 (s, 1H) 7.61 (s, 1H), 7.15 (m, 2H), 4.83-4.67 (m, 1H), 3.85 (s, 3H), 3.16-3.02 (m, 2H), 2.88-2.77 (m, 1H), 2.54-2.49 (m, 2H), 1.10-1.01 (m, 2H), 0.88-0.78 (m, 2H). Both protons are masked by the solvent.
Step 2:3- (N- (4-chloro-2- (3, 3-difluorocyclobutoxy) -5- (isothiazol-5-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid: the product from step 1 above (236 mg,0.41mmol, 96% purity) and LiOH H were combined 2 A solution of O (71.4 mg,1.70 mmol) in a mixture of THF (2 mL), water (0.5 mL) and MeOH (0.5 mL) was stirred at RT for 18h. The reaction mixture was diluted with EtOAc (5 mL) and water (5 mL) and then acidified to pH-4. The layers were separated and the aqueous layer was extracted with EtOAc (2×5 mL). The organic phases were combined and concentrated in vacuo. The residue was purified by silica gel chromatography (12 g column, 0-5% (5% acoh in MeOH)/DCM) to give the title compound (169 mg,0.29mmol, 68% yield, 92% purity) as a white solid. UPLC-MS (method 4) M/z 541.7 (M+H) + ,539.3(M-H) - At 1.80 minutes. 1 H NMR(500MHz,DMSO)δ13.27(s,1H),10.20(s,1H),8.58(d,J＝1.8Hz,1H),8.41(d,J＝1.9Hz,1H),7.98(dd,J＝8.1,1.9Hz,1H),7.64(s,1H),7.61(d,J＝1.9Hz,1H),7.17–7.10(m,2H),4.82–4.69(m,1H),3.15–3.04(m,2H),2.85–2.75(m,1H),2.62–2.52(m,2H),1.09–1.01(m,2H),0.86–0.79(m,2H)。
Example 51:3- (N- (4-chloro-2-cyclobutoxy-5- (isothiazol-5-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
The title compound (2.6 mg, 4.0. Mu. Mol, purity 78%) was prepared by a method similar to example 50 using intermediate 14 instead of intermediate 15.UPLC-MS (method 4) M/z 506.4 (M+H) + ,504.2(M-H) - At 1.91 minutes. 1 H NMR(500MHz,DMSO)δ13.26(s,1H),10.05(s,1H),8.58(d,J＝1.8Hz,1H),8.37(d,J＝1.9Hz,1H),8.03–7.97(m,1H),7.65–7.61(m,2H),7.15(d,J＝8.3Hz,1H),6.97(s,1H),4.65(app.p,J＝7.3Hz,1H),2.85–2.76(m,1H),2.29–2.21(m,2H),1.77–1.47(m,4H),1.08–1.01(m,2H),0.85–0.80(m,2H)。
Example 60:3- (N- (4-chloro-2-cyclobutoxy-5- (5-methylisoxazol-4-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
Step 1:3- (N- (4-chloro-2-cyclobutoxy-5- (5-methylisoxazol-4-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester: a solution of intermediate 12 (120 mg, 431. Mu. Mol), methyl 3- (chlorosulfonyl) -4-cyclopropylbenzoate (215 mg, 781. Mu. Mol) and pyridine (126. Mu.L, 1.56 mmol) in DCM (2 mL) was stirred at RT for 4h. The reaction mixture was diluted with DCM (7 mL) and water (7 mL) and the phases were separated. The aqueous phase was extracted with DCM (2×7 mL) and the combined organic phases were concentrated in vacuo. The residue was purified by silica gel chromatography (12 g column, 0-100% etoac/isohexane) to give the title compound (193 mg,0.366mmol, 86% yield, 98% purity) as a pale red solid. UPLC-MS (method 4) M/z 517.4 (M+H) + ,515.2(M-H) - At 1.97 minAnd (3) a clock. 1 H NMR(500MHz,DMSO-d 6 )δ9.97(s,1H),8.60(s,1H),8.32(d,J＝1.9Hz,1H),8.01(d,J＝8.2Hz,1H),7.25(s,1H),7.17(d,J＝8.3Hz,1H),6.91(s,1H),4.59(p,J＝6.9Hz,1H),3.84(s,3H),2.81(s,1H),2.31(s,3H),2.26–2.19(m,2H),1.72–1.65(m,2H),1.65–1.48(m,2H),1.10–1.02(m,2H),0.89–0.82(m,2H)。
Step 2:3- (N- (4-chloro-2-cyclobutoxy-5- (5-methylisoxazol-4-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid: to a solution of the product of step 1 above (193 mg, 366. Mu. Mol, 98% purity) in dioxane (800. Mu.L) and water (400. Mu.L) was added concentrated HCl (aq) (400. Mu.L, 6.05 mmol), and the mixture was heated to 70℃and stirred overnight. Concentrated HCl (aq) (400. Mu.L, 6.05 mmol) was additionally added and stirring was continued for 7h at 70 ℃. After cooling to RT, the mixture was diluted with water (20 mL) and extracted with EtOAc (3×30 mL). The combined organic extracts were washed with brine (20 mL), dried (MgSO 4 ) And the solvent was removed in vacuo. The residue was loaded onto silica and purified by silica gel chromatography (24 g column, 0-10% meoh/DCM) followed by trituration with TBME to give the title compound as a pale brown solid (48.8 mg,92.2 μmol, 25% yield, 95% purity). UPLC-MS (method 4) M/z 503.0 (M+H) + ,501.2(M-H) - At 1.84 minutes. 1 H NMR(500MHz,DMSO-d 6 )δ13.22(s,1H),9.93(s,1H),8.60(s,1H),8.31(d,J＝1.9Hz,1H),7.99(dd,J＝8.2,1.9Hz,1H),7.24(s,1H),7.15(d,J＝8.2Hz,1H),6.91(s,1H),4.64–4.55(m,1H),2.84–2.75(m,1H),2.30(s,3H),2.28–2.19(m,2H),1.75–1.66(m,2H),1.66–1.57(m,1H),1.57–1.48(m,1H),1.09–1.01(m,2H),0.88–0.81(m,2H)。
Example 61:3- (N- (4-chloro-2- (3, 3-difluorocyclobutoxy) -5- (5-methylisoxazol-4-yl) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
The title compound (57.3 mg, 102. Mu. Mol, 96% purity) was prepared by a method similar to example 60 using intermediate 13 instead of intermediate 12.UPLC-MS (square)Method 4) M/z 539.0 (M+H) + ,537.2(M-H) - At 1.74 minutes. 1 H NMR(500MHz,DMSO-d 6 )δ13.23(s,1H),10.08(s,1H),8.59(s,1H),8.33(d,J＝1.9Hz,1H),7.97(dd,J＝8.2,1.9Hz,1H),7.28(s,1H),7.12(d,J＝8.2Hz,1H),7.10(s,1H),4.78–4.67(m,1H),3.14–3.02(m,2H),2.84–2.75(m,1H),2.59–2.44(m,2H),2.28(s,3H),1.08–1.01(m,2H),0.87–0.80(m,2H)。
Example 67:3- (N- (4-chloro-5-cyano-2- (cyclopentyloxy) phenyl) sulfamoyl) -4-cyclopropyl-2-fluorobenzoic acid
Reagent: (a) Pd/C, H 2 、MeOH、RT；(b)NBS、MeCN、RT；(c)Pd 2 (dppf)Cl 2 ，K 3 PO 4 dioxane/H 2 O(v/v＝10:1)、100℃；(d)NaNO 2 、CuCl、SOCl 2 、HCl(cn)、THF/H 2 O (v/v=10/1), RT; (e) pyridine, DCM, RT; (f) LiOH (aq), THF, RT.
Step 1: 3-amino-2-fluorobenzoic acid methyl ester: a mixture of methyl 2-fluoro-3-nitrobenzoate (4.0 g,4.2 mmol) and Pd/C (0.5 g, 10%) in methanol was stirred at 25℃under a hydrogen atmosphere for 2h. The catalyst was removed by filtration through celite and the filtrate was concentrated to give the title compound as a yellow solid (3.38 g,2.0mmol, yield 47%). UPLC-MS (method 1) M/z 170.10 (M+H) + At 1.32 minutes.
Step 2: 3-amino-4-bromo-2-fluorobenzoic acid methyl ester: to a solution of the aniline of step 1 (3.38 g,20.0 mmol) in MeCN (20 mL) was added NBS (3.2 g,18.0 mmol) at 0deg.C. The resulting mixture was stirred at room temperature for 16h. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc and taken up in saturated Na 2 CO 3 Wash with water and brine. The organic layer was taken up with Na 2 SO 4 Drying and concentration gave the crude product. The crude product was purified by silica gel chromatography (eluting with 1/10 EtOAc/PE) to give the title compound as a yellow solid (4.69 g,18.9mmol, 94% yield). UPLC-MS (method)1)m/z 248.0,250.0(M-H) - At 1.516 minutes. 1 H NMR(400MHz,DMSO-d 6 )δ7.17(dd,J＝8.7,1.3Hz,1H),6.81(dd,J＝9.4,8.7Hz,1H),5.62(s,2H)。
Step 3: 3-amino-4-cyclopropyl-2-fluorobenzoic acid methyl ester: bromide in step 2 (4.69 g,18.9 mmol), cyclopropylboronic acid (4.47 g,56.7 mmol), K 3 PO 4 (12.03 g,56.7 mmol) and Pd (dppf) Cl 2 (1.39 g,1.90 mmol) in dioxane and H 2 The solution in the mixture of O (v/v=10:1, 50 mL) was heated in a sealed tube at 110 ℃ for 16h. The resulting mixture was filtered through celite, concentrated and purified by silica gel chromatography (eluting with 1/10 EtOAc/PE) to give the title compound as a yellow solid (2.84 g,13.6mmol, 24% yield). 1 H NMR(400MHz,DMSO-d 6 )δ6.80(dt,J＝22.2,9.0Hz,1H),6.60(d,J＝8.4Hz,1H),5.17(s,2H),2.32-2.21(m,1H),0.87-0.74(m,2H),0.59-0.45(m,2H)。
Step 4:3- (chlorosulfonyl) -4-cyclopropyl-2-fluorobenzoic acid methyl ester: at 0deg.C, to step 3 aniline (0.627 g,3.0 mmol) in concentrated HCl (2 mL) and H 2 NaNO was added in portions to a solution in a mixture of O (4.0 mL) 2 (0.414 g,6.0 mmol). The mixture was stirred at 0 ℃ for 30 minutes. To CuCl (30.0 mg,0.03 mmol) at 0deg.C in H 2 SOCl was added dropwise to the solution in O (1 mL) 2 (2.0 mL). The solution was then added dropwise to the reaction described above, and the mixture was stirred at 0 ℃ for 1h. With EtOAc (50 mL) and H 2 The reaction was diluted with O (50 mL) and the aqueous layer was extracted with EtOAc (50 mL. Times.2). The combined organic layers were taken up with Na 2 SO 4 Drying and concentration gave the title compound as a yellow oil (0.153 g,1.17mmol, 78% yield). The crude product was used directly in the next step without further purification.
Step 5:3- (N- (4-chloro-5-cyano-2- (cyclopentyloxy) phenyl) sulfamoyl) -4-cyclopropyl-2-fluorobenzoic acid methyl ester: to a solution of the sulfonyl chloride of step 4 (0.083 g,0.35 mmol) in pyridine (1 mL) was added 5-amino-2-chloro-4- (cyclopentyloxy) benzonitrile (example 1, step 3;0.354g,1.5 mmol) and the solution was stirred at RT overnight. The solvent was removed in vacuo and the crude product was purified by Biotage Isolera One(C 18 Column with 10% -90% MeCN/H 2 O elution) to give the title compound (0.73 g,0.15mmol, 43% yield) as a white solid. UPLC-MS (method 3) M/z 491.05 (M-H) - At 1.883 minutes.
Step 6:3- (N- (4-chloro-5-cyano-2- (cyclopentyloxy) phenyl) sulfamoyl) -4-cyclopropyl-2-fluorobenzoic acid: to the ester of step 5 (0.073 g,0.15 mmol) in THF and H 2 To a solution in a mixture of O (v/v=1:1, 4 ml) LiOH (35.5 mg,1.5 mmol) was added and the reaction was stirred at RT overnight. The solvent was removed in vacuo and the crude product was purified by Biotage Isolera One (C 18 Column with 10% -90% MeCN/H 2 O elution and purification with 0.1% HCOOH gave the title compound as a red solid (22.6 mg,0.047mmol, 31% yield). UPLC-MS (method 1) M/z 477.00 (M-H) - At 2.083 minutes. 1 H NMR(400MHz,DMSO-d 6 )δ10.13(s,1H),7.74(s,1H),7.60(t,J＝7.9Hz,1H),7.29(s,1H),6.87(d,J＝8.4Hz,1H),4.81(dq,J＝6.5,3.0Hz,1H),2.02(td,J＝8.3,4.2Hz,1H),1.80(dt,J＝11.8,6.7Hz,2H),1.60-1.46(m,2H),1.49–1.40(m,4H),1.13-1.04(m,2H),0.84-0.75(m,2H)。
Example 70: (S) -3- (N- (4-chloro-5-cyano-2- (1-cyclobutylethoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
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Step 1: (S) -2-chloro-4- (1-cyclobutylethoxy) -5-nitrobenzonitrile: to a suspension of NaH (60% mineral oil solution) (257 mg,6.42 mmol) in THF (15 mL) was added dropwise (S) -1-cyclobutylethan-1-ol (400. Mu.L, 3.62 mmol) at 0deg.C. The mixture was warmed to RT and stirred for 30 min. The product of example 1, step 1 (650 mg,3.21mmol, 99% purity) was added to THF (5 mL) and the mixture was heated to 60 ℃ and stirred overnight. The reaction was carefully quenched with water (20 mL) and extracted with EtOAc (3X 50 mL). The combined organic extracts were washed with brine (20 mL), dried (MgSO 4 ) And the solvent was removed in vacuo. The residue was loaded onto silica and passed through silica gelPurification by chromatography (24 g column, 0-50% EtOAc/isohexane) afforded the title compound as a yellow oil (196 mg, 649. Mu. Mol, 20% yield, 93% purity). UPLC (method 4): t R 1.88 minutes. 1 H NMR(500MHz,DMSO-d 6 )δ8.62(s,1H),7.90(s,1H),4.94–4.85(m,1H),2.61–2.51(m,1H),1.98–1.70(m,6H),1.17(d,J＝6.0Hz,3H)。
Step 2: (S) -5-amino-2-chloro-4- (1-cyclobutylethoxy) benzonitrile: a mixture of the product from step 1 above (198 mg, 656. Mu. Mol, 93% purity), ammonium chloride (211 mg,3.94 mmol) and zinc (257 mg,3.94 mmol) in THF (6 mL) and water (2 mL) was stirred at RT overnight. The mixture is passed throughFiltered, washed with EtOAc and the filtrate extracted with EtOAc (3X 15 mL). The combined organic extracts were washed with brine (15 mL), dried (MgSO 4 ) And the solvent was removed in vacuo to give the title compound as a viscous brown gum (160 mg, 625. Mu. Mol, 95% yield, 98% purity). UPLC-MS (method 4) M/z 251.2 (M+H) + At 1.80 minutes. 1 H NMR(500MHz,DMSO-d 6 )δ7.14(s,1H),6.96(s,1H),5.23(s,2H),4.60–4.51(m,1H),2.61–2.51(m,1H),2.02–1.72(m,6H),1.12(d,J＝6.0Hz,3H)。
Step 3: (S) -3- (N- (4-chloro-5-cyano-2- (1-cyclobutylethoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester: a mixture of the product from step 2 above (160 mg, 625. Mu. Mol, 98% pure), methyl 3- (chlorosulfonyl) 4-cyclopropylbenzoate (258 mg, 938. Mu. Mol) and pyridine (160. Mu.L, 1.99 mmol) in DCM (5 mL) was heated to 35℃and stirred for 2 days. The mixture was concentrated onto silica and purified by silica gel chromatography (12 g column, 0-50% etoac/isohexane) to give the title compound (267 mg,535 μmol, 86% yield, 98% purity) as a white solid. UPLC-MS (method 4): M/z 487.2 (M-H) - At 2.04 minutes. 1 H NMR(500MHz,DMSO-d 6 )δ10.09(s,1H),8.31(d,J＝1.9Hz,1H),8.02(dd,J＝8.2,1.9Hz,1H),7.66(s,1H),7.43(s,1H),7.18(d,J＝8.2Hz,1H),4.60–4.51(m,1H),3.85(s,3H),2.75–2.66(m,1H),2.21–2.12(m,1H),1.81–1.74(m,1H),1.72–1.55(m,5H),1.06–0.99(m,2H),0.91–0.82(m,2H),0.78(d,J＝6.0Hz,3H)。
Step 4: (S) -3- (N- (4-chloro-5-cyano-2- (1-cyclobutylethoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid: the product of step 3 above (267 mg, 535. Mu. Mol, 98% purity) was combined with LiOH H 2 A mixture of O (92.0 mg,2.19 mmol) in THF/MeOH/water (4:1:1, 3 mL) was stirred overnight at 40 ℃. The mixture was diluted with water (10 mL), acidified to pH 4 with 1M HCl (aq), and extracted with EtOAc (3X 15 mL). The combined organic extracts were washed with brine (10 mL), dried (MgSO 4 ) And the solvent was removed in vacuo. The residue was loaded onto silica and purified by silica gel chromatography (24 g column, 0-100% etoac/isohexane) followed by trituration with TBME/isohexane to give the title compound (127 mg,262 μmol, yield 49%, purity 98%) as a white solid. UPLC-MS (method 4): M/z 473.3 (M-H) - At 1.88 minutes. 1 H NMR(500MHz,DMSO-d 6 )δ13.23(s,1H),10.05(s,1H),8.31(d,J＝1.9Hz,1H),7.99(dd,J＝8.3,1.9Hz,1H),7.64(s,1H),7.42(s,1H),7.15(d,J＝8.3Hz,1H),4.60–4.51(m,1H),2.76–2.66(m,1H),2.21–2.12(m,1H),1.82–1.74(m,1H),1.73–1.65(m,2H),1.65–1.56(m,3H),1.06–1.00(m,2H),0.87–0.81(m,2H),0.80(d,J＝6.0Hz,3H)。
The following examples were prepared by a method analogous to example 70, substituting, if necessary, appropriate starting materials and intermediates:
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example 73:3- (N- (4-chloro-5-cyano-2- (((1 r,2 s) -2-fluorocyclopentyl) oxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
Reagent: (a) Cs (cells) 2 CO 3 、MeCN、RT；(b)DAST、DCM、-78℃；(c)Fe、NH 4 Cl、EtOH/H 2 O (v/v=4/1), 80 ℃; (d) pyridine, DCM, RT; (e) LiOH (aq), THF, RT.
Step 1: 2-chloro-4- (((1 r,2 r) -2-hydroxycyclopentyl) oxy) -5-nitrobenzonitrile: prepared according to step 2 of example 1 from 2-chloro-4-fluoro-5-nitrobenzonitrile (example 1, step 1).
Step 2: 2-chloro-4- (((1 r,2 s) -2-fluorocyclopentyl) oxy) -5-nitrobenzonitrile: to a solution of the alcohol of step 1 (0.200 g,0.71 mmol) in DCM (5 mL) was added diethylaminosulfur trifluoride (0.686 g,4.26 mmol) at-78deg.C and the solution was stirred at RT for 16h. The solvent was removed in vacuo and the crude product was purified by silica gel chromatography (eluting with 1/5 EtOAc/PE) to give the title compound as a white solid (0.070 g,0.25mmol, 35% yield). 1 H NMR (400 MHz, chloroform-d) δ8.22 (s, 1H), 7.32 (s, 1H), 5.25-5.12 (t, j=5.1 hz, 1H), 4.89 (dt, j=9.1, 4.3hz, 1H), 2.14 (dt, j=13.1, 7.8hz, 6H).
Step 3: 5-amino-2-chloro-4- (((1 r,2 s) -2-fluorocyclopentyl) oxy) benzonitrile: prepared from 2-chloro-4- (((1R, 2S) -2-fluorocyclopentyl) oxy) -5-nitrobenzonitrile according to step 3 of example 1. UPLC-MS (method 1) M/z 255.10 (M+H) + At 1.800 minutes.
Step 4:3- (N- (4-chloro-5-cyano-2- (((1 r,2 s) -2-fluorocyclopentyl) oxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester: prepared according to step 4 of example 1 from step 3 aniline and intermediate 3. UPLC-MS (method 1) M/z 491.00 (M-H) - At 2.183 minutes.
Step 5:3- (N- (4-chloro-5-cyano-2- (((1 r,2 s) -2-fluorocyclopentyl) oxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid: prepared from the ester of step 4 according to step 5 of example 1. UPLC-MS (method 1) M/z 477.00 (M-H) - At 1.950 minutes. 1 H NMR(400MHz,DMSO-d 6 )δ13.19(s,1H),10.22-10.01(m,1H),8.34(s,1H),7.98(d,J＝8.2Hz,1H),7.63(s,1H),7.49(s,1H),7.12(d,J＝8.3Hz,1H),5.03(dd,J＝53.7,4.0Hz,1H),4.82-4.72(m,1H),2.67(s,1H),1.90-1.69(m,4H),1.64-1.42(m,2H),1.05-0.96(m,2H),0.81(d,J＝3.9Hz,2H)。
Example 91:3- (N- (4-chloro-5-cyano-2- (spiro [3.3] hept-1-yloxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid enantiomer E1
Example 77 (85 mg, 173. Mu. Mol, 99% purity) was dissolved in 2.5mL DCM/MeOH mixture by sonication, filtered, and then UV detected by chiral SFC separation (Waters prep 100, with PDA and QDA at all wavelengths, 40 ℃,120 bar, on a ChiralPak IH 250X21mm, 5. Mu.M column, flow 65mL/min, 25% MeOH/CO 2 Elution). The clean fractions were pooled, rinsed with methanol/DCM and concentrated in vacuo to give the title compound as a viscous white solid (55 mg, 90. Mu. Mol, 52% yield, 80% purity) containing 20% w/w DMSO. SFC (Waters UPC) 2 ChiralPak IH 4.6x250 mm,5 μm column, flow 4mL/min, 25% (0.1% ammonia in MeOH)/CO 2 Elution) t R 2.02 minutes. Other analytical data were consistent with example 77.
Example 92:3- (N- (4-chloro-5-cyano-2- (spiro [3.3] hept-1-yloxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid enantiomer E2
The title compound (37 mg, 75. Mu. Mol, yield 43%, purity 99%) was obtained as a white solid from the chiral separation performed in example 91. SFC (Waters UPC) 2 ChiralPak IH 4.6x250mm,5 μm column, flow 4mL/min, 25% (0.1% ammonia in MeOH)/CO 2 ) Elution t R 2.40 minutes. Other analytical data were consistent with example 77.
Example 93:3- (N- (4-chloro-5-cyano-2- (2, 2-dimethylcyclobutoxy) phenyl) -sulfamoyl) -4-cyclopropylbenzoic acid enantiomer E1
Example 80 (108 mg, 225. Mu. Mol, 99% purity) was dissolved in 3.5mL DCM/MeOH mixture by sonication, filtered, and then UV detected by chiral SFC separation (Waters prep 100, with PDA and QDA at all wavelengths, 40 ℃,120 bar, on a ChiralPak IH 250X21mm, 5. Mu.M column, flow 65mL/min, 20% MeOH/CO 2 Elution). The clean fractions were pooled, rinsed with methanol/DCM and concentrated in vacuo to give the title compound as a white solid (47 mg, 99. Mu. Mol, 44% yield, 99% purity). SFC (Waters UPC) 2 ChiralPak IH 4.6X250mm,5 μm column, flow 4mL/min, 20% (0.1% ammonia in MeOH)/CO 2 ) Elution t R 2.42 minutes. Other analytical data are consistent with example 80.
Example 94:3- (N- (4-chloro-5-cyano-2- (2, 2-dimethylcyclobutoxy) phenyl) -sulfamoyl) -4-cyclopropylbenzoic acid enantiomer E2
The title compound (47 mg, 99. Mu. Mol, yield 44%, purity 99%) was obtained as a white solid from the chiral separation performed in example 93. SFC (Waters UPC) 2 ChiralPak IH 4.6X1250 mm,5 μm column, flow 4mL/min, 20% (0.1% ammonia in MeOH)/CO 2 ) Elution t R 2.80 minutes. Other analytical data are consistent with example 80.
Example 99: diastereoisomer D1 of 3- (N- (4-chloro-5-cyano-2- ((trans-2-methylcyclopentyl) oxy) -phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
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Example 90 (477 mg, 934. Mu. Mol, 93% purity) was dissolvedIn dimethyl sulfoxide (2.8 mL), filtered and purified by reverse phase preparative HPLC (Waters 2767Sample Manager, waters 2545Binary Gradient Module, waters Systems Fluidics Organiser, waters 515ACD pump, waters 515Makeup pump, waters 2998Photodiode Array Detector, waters QDa) on a Waters X-Select CSH C18 ODB preparative column,5 μm,30mm x100mm, flow 40 mL/min, UV at all wavelengths using PDA and QDA and ELS detectors, elution with a 0.1% aqueous formic acid-MeCN gradient over 12.5 minutes. In the whole process, 2mL/min MeOH was provided At a column dilution pump (At-column dilution pump), which was included in the following MeCN percentages. Gradient information: 0.0-0.5min,50% mecn;0.5-10.5min from 50% mecn to 80% mecn;10.5-10.6min from 80% mecn to 100% mecn;10.6-12.5min, hold at 100% mecn. The clean fractions were concentrated in vacuo in Genevac. The residue was dissolved in MeOH (30 mg/mL) by sonication, filtered and then separated by chiral SFC (Waters prep 100 with PDA and QDA detector, 40 ℃,120 bar, chiralpak IH 5. Mu.M, 21mm x250mm column; flow 65mL/min,18% MeOH (0.1% TFA), 82% CO 2 ). The clean fractions were pooled, rinsed with MeOH, and concentrated to dryness using a rock evaporator. The residue was redissolved in methanol transferred to the final vial, concentrated in vacuo on Biotage V10, and dried in vacuo to give the title compound as a white solid (117 mg,246 μmol, yield 26%, purity 99%). SFC (Waters UPC) 2 ChiralPak IH 4.6x250 mm,5 μm column, flow 4mL/min, 25% (0.1% ammonia in EtOH)/CO 2 ) Elution t R 2.66 minutes. Other analytical data are consistent with example 90.
Example 100: diastereoisomer D2 of 3- (N- (4-chloro-5-cyano-2- ((trans-2-methylcyclopentyl) oxy) -phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
The title compound (137 mg, 288. Mu. Mol, yield 30%, purity 99%) was obtained as a white solid from the chiral separation performed in example 93. SFC (Waters UPC) 2 ChiralPak IH 4.6x250 mm,5 μm column, flow 4mL/min, 25% (0.1% ammonia in EtOH)/CO 2 ) Elution t R 2.90 minutes. Other analytical data are consistent with example 90.
Example 101:3- (N- (4-chloro-5-cyano-2- (2, 4-tetramethylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
The title compound was prepared by a method analogous to example 70, substituting appropriate starting materials and intermediates if necessary. UPLC (method 4): M/z 501.2 (M-H) - At 2.03 minutes. 1 H NMR (500 mhz, DMSO) delta 13.20 (s, 1H), 9.94 (s, 1H), 8.37 (d, j=1.9 hz, 1H), 7.98 (dd, j=8.2, 1.9hz, 1H), 7.63 (s, 1H), 7.08 (d, j=8.3 hz, 1H), 7.02 (s, 1H), 4.29 (s, 1H), 1.42 (d, j=11.5 hz, 1H), 1.35 (d, j=11.4 hz, 1H), 1.11 (s, 6H), 0.95-0.85 (m, 2H), 0.81-0.75 (m, 8H) (1 protons are blocked by DMSO signals).
Example 103: diastereoisomer D1 of 3- (N- (4-chloro-5-cyano-2- ((trans-2-methoxycyclopentyl) -oxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
Step 1:3- (N- (4-chloro-5-cyano-2- ((trans-2-methoxycyclopentyl) -oxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester diastereomer D1: acCl (100. Mu.L, 1.41 mmol) was added dropwise to anhydrous MeOH (3.0 mL) and the resulting solution was allowed to stand at RT for 10 min to form a dry solution of HCl/MeOH. Example 85 (71.5 mg, 146. Mu. Mol) was suspended in HCl/MeOH solution and the resulting mixture was stirred at RT for 4 days. The mixture was concentrated in vacuo and the residue was dissolved in MeOH (6.7 mg/mL) and sonicatedFiltration followed by chiral SFC (Waters prep 15, 40℃at 210-400nm by DAD, 120bar, C 4 10X250mm,5 μm column, flow 15mL/min,30% MeOH/CO 2 UV detection below) was used for separation. The clean fractions were pooled and concentrated in vacuo to give the title compound as an off-white solid (24.3 mg, 48.1. Mu. Mol, 33% yield). UPLC-MS (method 4): M/z 503.2 (M-H) - At 1.73 minutes. SFC (Waters UPC) 2 C4,4.6X250mm,5 μm column, flow 4mL/min,30% (0.1% ammonia in MeOH)/CO 2 )t R 2.45min。
Step 2:3- (N- (4-chloro-5-cyano-2- ((trans-2-methoxycyclopentyl) oxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid diastereomer D1: the product of step 1 above (24.3 mg, 48.1. Mu. Mol) was mixed with a solution of 1M LiOH (aq) (238. Mu.L, 238. Mu. Mol) in THF (1 mL) and the resulting solution was stirred at RT for 20h. The mixture was diluted with water (1 mL) and concentrated in vacuo to remove THF. The resulting aqueous solution was acidified with 1M HCl (0.3 mL) and the resulting white precipitate was collected by filtration, washing with water. The solid was dissolved in MeCN and concentrated in vacuo to give the title compound as a white powder (22 mg,44 μmol, 91% yield, 98% purity). UPLC-MS (method 4) M/z 490.9 (M+H) + ,489.2(M-H) - At 1.70 minutes. 1 H NMR(500MHz,DMSO)δ13.26(s,1H),10.15(s,1H),8.32(d,J＝1.9Hz,1H),7.99(dd,J＝8.2,1.9Hz,1H),7.69(s,1H),7.35(s,1H),7.13(d,J＝8.3Hz,1H),4.65(dt,J＝6.5,3.3Hz,1H),3.43(dd,J＝6.9,4.4Hz,1H),3.13(s,3H),2.73–2.60(m,1H),1.96–1.86(m,1H),1.76–1.66(m,1H),1.56–1.37(m,3H),1.27–1.16(m,1H),1.03–0.95(m,2H),0.84–0.78(m,2H)。
Example 104: diastereoisomer D2 of 3- (N- (4-chloro-5-cyano-2- ((trans-2-methoxycyclopentyl) -oxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
Step 1:3- (N- (4-chloro-5-cyano-2- ((trans-2-methoxycyclopentyl) -oxy) phenyl) sulfamoyl) -4-cyclopropaneMethyl benzoate diastereomer D2: the title compound (24.6 mg, 48.7. Mu. Mol, yield 34%) was obtained as an off-white solid from the chiral separation performed in example 103. SFC (Waters UPC) 2 ，C 4 4.6X250mm,5 μm column, flow 4mL/min,30% (0.1% ammonia in MeOH)/CO 2 )t R 2.78 minutes. Other analytical data were consistent with example 103, step 1.
Step 2:3- (N- (4-chloro-5-cyano-2- ((trans-2-methoxycyclopentyl) oxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid diastereomer D2: the title compound (22 mg, 43. Mu. Mol, yield 87%) was obtained as a white powder using a method similar to example 103, step 2. Analytical data were consistent with example 103, step 2.
Example 105:3- (N- (4-chloro-5-cyano-2- ((cis-2-methylcyclopentyl) oxy) -phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
The title compound was prepared by a method analogous to example 70, substituting appropriate starting materials and intermediates if necessary. UPLC-MS (method 4): M/z 473.2 (M-H) - At 1.87 minutes. 1 H NMR(500MHz,DMSO-d 6 )δ13.19(br s,1H),9.99(br s,1H),8.35(d,J＝1.8Hz,1H),7.96(d,J＝8.2Hz,1H),7.58(s,1H),7.41–7.25(m,1H),7.09(d,J＝8.3Hz,1H),4.82–4.69(m,1H),2.05–1.91(m,1H),1.91–1.82(m,1H),1.64–1.56(m,1H),1.56–1.47(m,1H),1.46–1.20(m,4H),1.00–0.90(m,2H),0.84–0.75(m,2H),0.69(d,J＝6.8Hz,3H)。
Example 106:3- (N- (4-chloro-5-cyano-2- (spiro [2.3] hex-4-yloxy) phenyl) -sulfamoyl) -4-cyclopropylbenzoic acid. 1.50 diethylamine salt enantiomer E1
Example 56 (22 mg, 46.5. Mu. Mol) was dissolved in 50mg/mL DCM/MeOH, sonicatedThe mixture was filtered and then passed through chiral SFC (Waters prep 100 with PDA and QDA detector, 40 ℃,120 bar, chiralpak IH,21X250mm,5 μm column, flow 65mL/min, (25% (0.1% diethylamine/MeOH)/CO) 2 ) And (5) separating. The clean fractions were pooled, rinsed with MeOH, and concentrated in vacuo to give the title compound as a clear colorless glass (5.3 mg,8.9 μmol, 19% yield, 98% purity). UPLC-MS (method 4) M/z 473.0 (M+H) + ,471.2(M-H) - At 1.82 minutes. 1 H NMR(500MHz,DMSO)δ8.38(d,J＝1.8Hz,1H),7.75(dd,J＝8.1,1.9Hz,1H),7.32(s,1H),6.76(d,J＝8.1Hz,1H),6.63(s,1H),4.82(t,J＝6.5Hz,1H),3.30–3.23(m,1H),2.85(q,J＝7.2Hz,6H),2.47(td,J＝5.7,3.1Hz,1H),2.06–1.91(m,2H),1.91–1.80(m,1H),1.14(t,J＝7.2Hz,9H),1.02–0.81(m,3H),0.72–0.60(m,2H),0.60–0.43(m,2H),0.42–0.30(m,1H)。SFC(Waters UPC 2 ChiralPak IH 4.6X250mm,5 μm column, flow 4mL/min,25% (0.1% ammonia MeOH)/CO 2 )t R 2.12 minutes.
Example 107:3- (N- (4-chloro-5-cyano-2- (spiro [2.3] hex-4-yloxy) phenyl) -sulfamoyl) -4-cyclopropylbenzoic acid, 1.65 diethylamine salt enantiomer E2
The title compound (8.4 mg, 14. Mu. Mol, yield 30%, purity 98%) was obtained as a transparent colorless glass from the chiral separation performed in example 106. SFC (Waters UPC) 2 ChiralPak IH 4.6X250mm,5 μm column, flow 4mL/min,25% (0.1% ammonia MeOH)/CO 2 )t R 2.48 minutes. Other analytical data are consistent with example 106.
Example 108:3- (N- (4-chloro-5-cyano-2- (2-cyclopropylcyclobutoxy) -phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
By a method similar to that of example 70The title compound is prepared, if necessary replaced by appropriate starting materials and intermediates. UPLC-MS (method 4): M/z 485.2 (M-H) - At 1.88 minutes. 1 H NMR(500MHz,DMSO-d 6 ) (10:1 mixture of diastereomers), δ13.23 (br s, 1H), 10.18 (br s, 1H), 8.28 (d, J=1.9 Hz,1H minor), 8.27 (d, J=1.8 Hz,1H major), 7.99 (dd, J= 8.2,1.9Hz,1H major), 7.96 (dd, J= 8.1,1.8Hz,1H minor), 7.72 (s, 1H), 7.22 (s, 1H), 7.15 (d, J=8.4 Hz,1H major), 7.14 (d, J=8.4 Hz,1H minor), 4.77 (q, J=7.0 Hz,1H minor), 4.49 (q, J=7.2 Hz,1H major), 2.84-2.79 (m, 1H minor), 2.79-2.69 (m, 1H major), 2.20-2.12 (m, 1H minor), 2.12-2.03 (m, 1H major), 1.80-1.70 (m, 1H minor), 1.67-1.57 (m, 1H, major), 1.54-1.44 (m, 1H minor), 1.44-1.34 (m, 1H major), 1.30-0.91 (m, 4H), 0.91-0.71 (m, 2H), 0.39-0.30 (m, 1H major), 0.30-0.22 (m, 1H major), 0.15-0.04 (m, 1H minor), 0.00-0.08 (m, 1H major), 0.11-0.21 (m, 1H), 0.31-0.42 (m, 1H minor), 0.50-0.61 (m, 1H minor).
Example 114: diastereoisomer D1 of 3- (N- (4-chloro-5-cyano-2- (3-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
Step 1:3- (N- (4-chloro-5-cyano-2- (3-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester diastereomer D1: methyl 3- (N- (4-chloro-5-cyano-2- (3-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoate (example 88, 219mg, 461. Mu. Mol) was dissolved in 5:1 EtOH: DCM (17.6 mg/mL) and then passed through SFC (Lux iC5, 21.2x250mm,5 μm column, 40 ℃,125 bar, flow 50mL/min,20% EtOH/CO) 2 ) Purification was performed to give the title compound (147 mg, 309. Mu. Mol, 67% yield) as a colourless glass. UPLC-MS (method 4): M/z 475.2 (M+H) + ,473.2(M-H) - At 1.98 minutes. SFC (Lux iC5,4.6x250mm,5 μm column, 40 ℃,125 bar, flow 4mL/min,20% (0.1% ammonia/EtOH)/CO) 2 )t R 10.1 minutes.
Step 2:3- (N- (4-chloro-5-cyano)-2- (3-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid diastereomer D1: the product of step 1 above (147 mg, 309. Mu. Mol) was dissolved in THF (5 mL) and treated with 1M LiOH (aq) (1.55 mL,1.55 mmol). The resulting mixture was stirred at RT for 24h. The mixture was diluted with water (2 mL) and concentrated in vacuo to remove THF. The resulting solution was acidified with 1M HCl (aq), and the white precipitate was collected by filtration, washed with water, and then dried in vacuo to give the title compound as a white solid (130 mg,279 μmol, 90% yield, 99% purity). UPLC-MS (method 4) M/z 461.3 (M+H) + ,459.2(M-H) - At 1.80 minutes. 1 H NMR(500MHz,DMSO)δ13.26(br s,1H),10.16(br s,1H),8.31(d,J＝1.8Hz,1H),7.99(dd,J＝8.2,1.9Hz,1H),7.70(s,1H),7.14(d,J＝8.2Hz,1H),7.08(s,1H),4.47(p,J＝7.3Hz,1H),2.76–2.68(m,1H),2.44–2.36(m,2H),1.88–1.75(m,1H),1.25–1.15(m,2H),1.05–0.99(m,2H),0.97(d,J＝6.6Hz,3H),0.84–0.77(m,2H)。
Example 115: diastereoisomer D2 of 3- (N- (4-chloro-5-cyano-2- (3-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid
Step 1:3- (N- (4-chloro-5-cyano-2- (3-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester diastereomer D2: the title compound (19 mg, 40.4. Mu. Mol, yield 9%) was obtained as a colorless glass from the isolation performed in step 1 of example 114. SFC (Lux iC5,4.6x250m,5 μm column, 40 ℃,125 bar, flow 4mL/min,20% (0.1% ammonia/EtOH)/CO) 2 )t R 9.17 minutes. Other analytical data are consistent with example 114, step 1.
Step 2:3- (N- (4-chloro-5-cyano-2- (3-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid diastereomer D2: the title compound (18.0 mg, 37.1. Mu. Mol, yield 92%, purity 95%) was prepared by a method analogous to example 114, step 2, substituting the appropriate starting materials and intermediates if necessary. UPLC-MS (method 4) M/z 461.6 (M+H) + ,459.2(M-H) - At 1.81 minutes. 1 H NMR(500MHz,DMSO)δ13.22(s,1H),10.18(s,1H),8.30(d,J＝1.9Hz,1H),7.99(dd,J＝8.2,1.9Hz,1H),7.70(s,1H),7.13(d,J＝8.3Hz,1H),7.04(s,1H),4.82(p,J＝6.5Hz,1H),2.80–2.69(m,1H),2.21–2.09(m,1H),1.95–1.87(m,2H),1.84–1.74(m,2H),1.07(d,J＝7.1Hz,3H),1.04–0.98(m,2H),0.84–0.78(m,2H)。
Example 120:3- (N- (4-chloro-5-cyano-2- (2-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid stereoisomer D1
Step 1:3- (N- (4-chloro-5-cyano-2- (2-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester: the title compound (1.2 g,2.53 mmol) was prepared by a method analogous to that of example 70, steps 1-3, substituting appropriate starting materials and intermediates if necessary.
Step 2:3- (N- (4-chloro-5-cyano-2- (2-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester stereoisomer D1: the product from step 1 above (1.2 g,2.53 mmol) was dissolved in a mixture of MeOH (11 mL) and THF (5 mL), then sonicated, filtered and passed through chiral SFC (Waters prep 100 with PDA and QDA detectors, 40 ℃,120 bar, chiralpak IH,21X250mm,5 μm column, flow 65mL/min,25% MeOH/CO 2 ) The separation was performed to obtain three fractions. The first eluted fraction was concentrated in vacuo to give the title compound (379 mg,0.774mmol, 31% yield, 97% purity (3 wt% MeOH)) as a colorless glass. UPLC-MS (method 4) M/z 475.3 (M+H) + ,473.3(M-H) - At 1.99 minutes. 1 H NMR(500MHz,DMSO)δ10.23(s,1H),8.31(d,J＝1.9Hz,1H),8.02(dd,J＝8.3,1.9Hz,1H),7.71(s,1H),7.17(d,J＝8.3Hz,1H),7.11(s,1H),4.31(q,J＝7.1Hz,1H),3.84(s,3H),2.80–2.71(m,1H),2.23–2.05(m,2H),1.80–1.70(m,1H),1.26–1.14(m,1H),1.13–1.00(m,3H),0.96(d,J＝6.7Hz,3H),0.90–0.84(m,1H),0.84–0.76(m,1H)。SFC(Waters UPC 2 ChiralPak IH 4.6X250mm,5 μm column, flow 4mL/min,25% (0).1% ammonia/MeOH)/CO 2 )t R 1.76 minutes.
Step 3:3- (N- (4-chloro-5-cyano-2- (2-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid stereoisomer D1: the product from step 2 above (379 mg, 774. Mu. Mol, 97% purity) and LiOH H were combined 2 A solution of O (134 mg,3.19 mmol) in THF (4 mL), water (1 mL) and MeOH (1 mL) was stirred at RT for 24h. The resulting mixture was concentrated in vacuo and the resulting aqueous solution was diluted with water (10 mL). The solution was acidified with 1M HCl (aq) and the resulting white precipitate collected by filtration and washed with water (5 mL). The solid was dried in vacuo to give the title compound as a white solid (275 mg, 591. Mu. Mol, 76% yield, 99% purity). UPLC-MS (method 4) M/z 461.1 (M+H) + ,459.2(M-H) - At 1.82 minutes. 1 H NMR(500MHz,DMSO)δ13.24(s,1H),10.19(s,1H),8.31(d,J＝1.9Hz,1H),7.99(dd,J＝8.2,1.9Hz,1H),7.69(s,1H),7.14(d,J＝8.3Hz,1H),7.09(s,1H),4.32(q,J＝7.1Hz,1H),2.80–2.72(m,1H),2.24–2.07(m,2H),1.76(q,J＝9.4Hz,1H),1.28–1.19(m,1H),1.14–1.01(m,3H),0.97(d,J＝6.7Hz,3H),0.89–0.82(m,1H),0.81–0.75(m,1H)。
Example 121:3- (N- (4-chloro-5-cyano-2- (2-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid stereoisomer D2
Step 1:3- (N- (4-chloro-5-cyano-2- (2-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester stereoisomer D2: the second eluted fraction of step 2 of example 120 was further purified by chiral SFC (Waters prep 100 with PDA and QDA detector, 40 ℃,120 bar, lux C4, 21X250mm,5 μm column, flow 65mL/min,30% MeOH/CO) to obtain two fractions. The first eluted fraction was passed through chiral SFC (Waters prep 100 with PDA and QDA detector, 40 ℃,120 bar, chiralpak IH,21X250mm,5 μm column, flow 65mL/min,25% MeOH/CO) 2 ) Further purification gave the title compound (275 mg,0.538mmol, yield 21%, purity 93% (7 w)t％MeOH))。SFC(Waters UPC 2 ChiralPak IH 4.6X250mm,5 μm column, flow 4mL/min,25% (0.1% ammonia/MeOH)/CO 2 )t R 1.96 minutes. Other analytical data are consistent with example 120, step 2.
Step 2:3- (N- (4-chloro-5-cyano-2- (2-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid stereoisomer D2: the title compound (173 mg, 375. Mu. Mol, yield 70%) was prepared by a method analogous to example 120, step 3, substituting the appropriate starting materials and intermediates if necessary. Analytical data are consistent with example 120.
Example 122:3- (N- (4-chloro-5-cyano-2- (2-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid stereoisomer D3
Step 1:3- (N- (4-chloro-5-cyano-2- (2-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester stereoisomer D3: the second eluted fraction of example 121 step 1 was further purified by chiral SFC (Waters prep 100 with PDA and QDA detector, 40 ℃,120bar, lux C4, 21X250mm,5 μm column, flow 65mL/min,30% MeOH/CO) to give the title compound as a white solid (51 mg,106mmol, yield 4%, purity 99%). UPLC (method 4) M/z 475.3 (M+H) + ,473.3(M-H) - At 1.97 minutes. 1 H NMR(500MHz,DMSO)δ10.20(s,1H),8.33(d,J＝1.9Hz,1H),8.00(dd,J＝8.2,1.9Hz,1H),7.68(s,1H),7.14(d,J＝8.3Hz,1H),7.07(s,1H),4.65(q,J＝7.3Hz,1H),3.84(s,3H),2.73–2.65(m,2H),2.18–2.09(m,1H),2.01(p,J＝10.0Hz,1H),1.74(p,J＝8.9Hz,1H),1.27–1.23(m,1H),1.08–0.95(m,2H),0.90–0.78(m,2H),0.64(d,J＝7.2Hz,3H)。SFC(Waters UPC 2 ChiralPak IH 4.6X250mm,5 μm column, flow 4mL/min,25% (0.1% ammonia/MeOH)/CO 2 )t R 2.00 minutes.
Step 2:3- (N- (4-chloro-5-cyano-2- (2-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid stereoisomer D3: by analogy with example 120The procedure of step 3 gave the title compound (12 mg, 26. Mu. Mol, yield 24%, purity 97%) if necessary with appropriate starting materials and intermediates. UPLC-MS (method 4) M/z 461.4 (M+H) + ,459.2(M-H) - At 1.81 minutes. 1 H NMR(500MHz,DMSO)δ13.22(s,1H),10.17(s,1H),8.33(d,J＝1.9Hz,1H),7.98(dd,J＝8.2,1.9Hz,1H),7.68(s,1H),7.12(d,J＝8.3Hz,1H),7.08(s,1H),4.66(q,J＝7.2Hz,1H),2.74–2.65(m,2H),2.18–2.10(m,1H),2.06–1.97(m,1H),1.79–1.69(m,1H),1.30–1.22(m,1H),1.09–0.94(m,2H),0.89–0.77(m,2H),0.65(d,J＝7.2Hz,3H)。
Example 123:3- (N- (4-chloro-5-cyano-2- (2-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid stereoisomer D4
Step 1:3- (N- (4-chloro-5-cyano-2- (2-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester stereoisomer D4: through chiral SFC (Waters prep 100 with PDA and QDA detector, 40 ℃,120 bar, chiralpak IH,21X250mm,5 μm column, flow 65mL/min,25% MeOH/CO) 2 ) The third eluted fraction of step 2 of example 120 was further purified to give the title compound (85 mg,177mmol, yield 7%, purity 99%) as a white solid. SFC (Waters UPC) 2 ChiralPak IH 4.6X250mm,5 μm column, flow 4mL/min,25% (0.1% ammonia/MeOH)/CO 2 )t R 2.27 minutes. Other analytical data were consistent with example 122, step 1.
Step 2:3- (N- (4-chloro-5-cyano-2- (2-methylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid stereoisomer D4: the title compound (67 mg, 135. Mu. Mol, yield 76%, purity 93% (6 wt% MeCN)) was prepared by a method analogous to example 120, step 3, if necessary replaced by appropriate starting materials and intermediates. Analytical data were consistent with example 122.
Example 125: (R) -4-cyclopropyl-3- (N- (2- ((2, 2-dimethylcyclopentyl) oxy) -4-fluoro-5- (1H-tetrazol-1-yl) phenyl) sulfamoyl) benzoic acid
Step 1: (tert-butoxycarbonyl) (2, 4-difluoro-5-nitrophenyl) carbamic acid tert-butyl ester: DMAP (0.18 g,1.4 mmol) was added to a stirred solution of 2, 4-difluoro-5-nitroaniline (2.5 g,14 mmol) and di-tert-butyl dicarbonate (7.8 g,36 mmol) in DCM (25 mL) and the resulting mixture stirred at RT for 18h. Concentrating the reaction mixture toOn top of this and purified by silica gel chromatography (80 g column, 0-50% etoac/isohexane) to give the title compound as a clear colorless oil (2.62 g,6.6mmol, 46% yield, 94% purity). 1 H NMR(500MHz,CDCl 3 )δ8.05(t,J＝7.6Hz,1H),7.10(dd,J＝10.2,8.9Hz,1H),1.45(s,18H)。
Step 2: (R) - (tert-butoxycarbonyl) (4- ((2, 2-dimethylcyclopentyl) oxy) -2-fluoro-5-nitrophenyl) carbamic acid tert-butyl ester: with Cs 2 CO 3 (1.71 g,5.25 mmol) A mixture of the product from step 1 above (1.39 g,3.50mmol, 94% purity) and (R) -2, 2-dimethylcyclopent-1-ol (400 mg,3.50 mmol) in DMF (8 mL) was worked up in portions. The resulting mixture was stirred at RT for 4 days. (R) -2, 2-dimethylcyclopent-1-ol (350 mg,3.07 mmol) was additionally added and stirring was continued for an additional 3 days. The mixture was partitioned between TBME (40 mL) and water (40 mL) and the phases separated. Organic dependent water (40 mL), saturated NaHCO 3 (aq) (40 mL) and brine (2X 40 mL) and then washed with MgSO 4 Dried, filtered, and concentrated in vacuo to give a pale yellow oil (1.51 g). The crude product was purified by silica gel chromatography (24 g column, 25-75% DCM/heptane) to give the title compound as a pale yellow oil (1.14 g,1.92mmol, yield 55%, purity 79%). UPLC-MS (method 4) M/z 491.4 (M+Na) + At 2.31 minutes. 1 H NMR(500MHz,CDCl 3 )δ7.85(d,J＝8.0Hz,1H),6.77(d,J＝11.3Hz,1H),4.24–4.19(m,1H),2.26–2.13(m,1H),1.90–1.66(m,3H),1.45(s,18H),1.54–1.41(m,2H),1.16(s,3H),1.03(s,3H)。
Step 3: (R) -1- (4- ((2, 2-dimethylcyclopentyl) oxy) -2-fluoro-5-nitrophenyl) tetrazole: the product from step 2 above (1.14 g,1.92mmol, 79% purity) was dissolved in DCM (12 mL) and treated with TFA (2 mL,26.0 mmol). The resulting mixture was stirred at RT for 18h, then concentrated in vacuo and azeotroped with DCM (30 mL) to give a pale orange solid (891 mg). The solid was dissolved in triethyl orthoformate (22.0 mL,132 mmol) and treated with AcOH (1 mL,17.5 mmol). The resulting mixture was heated at 80℃for 1h, followed by dropwise addition of azido trimethylsilane (600. Mu.L, 4.52 mmol). Heating was continued for 2h. Sodium acetate (300 mg,3.66 mmol) was added and heating continued for 10 min. The mixture was concentrated in vacuo and the residue was taken up in TBME (25 mL) and saturated NaHCO 3 (aq) (15 mL). The phases were separated and the organic phase was successively saturated with NaHCO 3 (aq) (15 mL) and brine (15 mL) and then washed with MgSO 4 Dried, filtered and concentrated in vacuo to give a pale yellow oil (750 mg). The oil was dissolved in AcOH (12 mL,210 mmol) and treated with trimethyl orthoformate (1.53 mL,14.0 mmol). Sodium azide (300 mg,4.61 mmol) was added and the resulting mixture was heated at 80℃for 2h. The mixture was concentrated in vacuo and the residue was taken up in TBME (25 mL) and saturated NaHCO 3 (aq) (15 mL). The phases were separated and the organic phase was successively saturated with NaHCO 3 (aq) (15 mL) and brine (15 mL) and then washed with MgSO 4 Dried, filtered and concentrated in vacuo to give the title compound as a pale yellow oil (662 mg,1.67mmol, 87% yield, 81% purity). UPLC-MS (method 4) M/z 322.0 (M+H) + ,318.6(M+OH-HF) - At 1.77 minutes. 1 H NMR(500MHz,CDCl 3 )δ9.03(d,J＝2.4Hz,1H),8.50(d,J＝7.7Hz,1H),7.03(d,J＝12.3Hz,1H),4.32(dd,J＝6.0,2.8Hz,1H),2.32–2.21(m,1H),1.91–1.73(m,3H),1.57–1.47(m,1H),1.19(s,3H),1.17(s,3H)。
Step 4: (R) -2- ((2, 2-dimethylcyclopentyl) oxy) -4-fluoro-5- (tetrazol-1-yl) aniline: the product of step 3 above (662 mg,1.67mmol, purity 81%) was reacted with NH 4 A solution of Cl (893 mg,16.7 mmol) in THF (6 mL) and water (2 mL) was combined. The obtained rapid stirringThe suspension was cooled in an ice bath and batched with zinc (1.09 g,16.7 mmol). The resulting mixture was warmed to RT and stirred for 4h. The mixture was diluted with EtOAc (20 mL) and the phases separated. The aqueous phase was extracted with EtOAc (2X 20 mL) and the extract was passed through Filtered, combined and concentrated in vacuo to give a dark brown oil (574 mg). The oil was partitioned between DCM (8 mL) and water (4 mL) and the phases separated. The aqueous phase was extracted with DCM (2X 2 mL), the organic phases were combined, concentrated in vacuo and the residue purified by silica gel chromatography (12 g column, 0-10% EtOAc/DCM) to give the title compound as an off-white solid (416 mg,1.36mmol, 81% yield, 95% purity). UPLC-MS (method 4) M/z 292.3 (M+H) + At 1.64 minutes. 1 H NMR(500MHz,CDCl 3 )δ8.98(d,J＝2.3Hz,1H),7.37(d,J＝7.3Hz,1H),6.76(d,J＝12.3Hz,1H),5.66(br s,2H),4.21(dd,J＝6.1,3.4Hz,1H),2.28–2.17(m,1H),1.90–1.68(m,4H),1.55–1.46(m,1H),1.15(s,3H),1.06(s,3H)。
Step 5: (R) -methyl 4-cyclopropyl-3- (N- (2- ((2, 2-dimethylcyclopentyl) oxy) -4-fluoro-5- (tetrazol-1-yl) phenyl) sulfamoyl) benzoate: the product of step 4 above (206 mg, 672. Mu. Mol, 95% purity) was mixed with methyl 3- (chlorosulfonyl) -4-cyclopropylbenzoate (233 mg, 848. Mu. Mol) in DCM (4 mL) and treated with pyridine (172. Mu.L, 2.13 mmol). The resulting solution was left to stand at RT for 18h. The solution was washed sequentially with 1M HCl (aq) (2X 5 mL) and water (5 mL), dried (MgSO) 4 ) And purified directly by silica gel chromatography (12 g column, 0-100% etoac/isohexane) to give the title compound (340 mg,623 μmol, 93% yield, 97% purity) as a pale pink solid. UPLC-MS (method 4) M/z 530.4 (M+H) + ,528.3(M-H) - At 1.90 minutes. 1 H NMR(500MHz,DMSO)δ9.88(s,1H),9.83(d,J＝1.4Hz,1H),8.39(d,J＝1.9Hz,1H),7.99(dd,J＝8.3,1.9Hz,1H),7.58(d,J＝8.0Hz,1H),7.34(d,J＝12.7Hz,1H),7.11(d,J＝8.3Hz,1H),4.31(dd,J＝6.3,4.2Hz,1H),3.84(s,3H),2.67–2.58(m,1H),2.07(dq,J＝13.8,6.6Hz,1H),1.57–1.45(m,3H),1.31–1.14(m,2H),1.01–0.91(m,2H),0.89(s,3H),0.84–0.77(m,2H),0.76(s,3H)。
Step 6: (R) -4-cyclopropyl-3- (N- (2- ((2, 2-dimethylcyclopentyl) oxy) -4-fluoro-5- (tetrazol-1-yl) phenyl) sulfamoyl) benzoic acid: the product of step 5 above (337 mg, 617. Mu. Mol, 97% purity) was dissolved in THF (6 mL) and treated with 1M LiOH (aq) (3.09 mL,3.09 mmol). The resulting mixture was stirred at RT for 3 days. The mixture was diluted with water (2 mL) to give a clear solution which was concentrated in vacuo to remove THF. The resulting solution was acidified using 1M HCl (aq) and the pale pink precipitate collected by filtration, washed with water and dried under vacuum to give the title compound as an off-white powder (306 mg,582 μmol, 94% yield, 98% purity). UPLC-MS (method 4): M/z 516.1 (M+H) + ,514.2(M-H) - At 1.73 minutes. 1 H NMR(500MHz,DMSO)δ13.22(br s,1H),9.90(br s,1H),9.82(d,J＝1.4Hz,1H),8.38(d,J＝1.9Hz,1H),7.96(dd,J＝8.2,1.9Hz,1H),7.58(d,J＝8.1Hz,1H),7.32(d,J＝12.6Hz,1H),7.07(d,J＝8.3Hz,1H),4.31(dd,J＝6.4,4.2Hz,1H),2.64(s,1H),2.12–2.02(m,1H),1.57–1.45(m,3H),1.33–1.15(m,2H),0.99–0.86(m,2H),0.89(s,3H),0.82–0.74(m,2H),0.78(s,3H)。
Example 130:3- (N- (4-chloro-5-cyano-2- (2-cyclopropylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid stereoisomer D1
Step 1:3- (N- (4-chloro-5-cyano-2- (2-cyclopropylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid: the title compound (162 mg, 333. Mu. Mol) was prepared by a method similar to example 70, substituting appropriate starting materials and intermediates as necessary.
Step 2:3- (N- (4-chloro-5-cyano-2- (2-cyclopropylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester stereoisomer D1: Separation 1:the product from step 1 above was dissolved in MeOH (25 mg/mL) by sonication, filtered, then UV detected by chiral SFC (Waters prep 15 by DAD at 210-400nm, 40 ℃,120 bar, chiralpak IH 10X150mm, 5 μm column, flowThe amount was 15mL/min, 20% (0.1% DEA/MeOH)/CO 2 ) And (5) separating. The clean fractions were pooled, rinsed with MeOH and concentrated in vacuo to give three fractions, each contaminated with DEA.Separation 2:the first eluted fraction was dissolved in MeOH (40 mg/mL) by sonication, filtered, and then purified by chiral SFC (Waters prep 100 with PDA and QDA detector, 40 ℃,120 bar, chiralpak IH,21mm x250mm,5 μm column, flow 65mL/min,20% (0.1% TFA/MeOH)/CO 2 ) And (5) separating. The clean fractions were pooled, rinsed with MeOH and concentrated in vacuo to give two fractions, each of which was esterified during solvent evaporation.
Step 3:3- (N- (4-chloro-5-cyano-2- (2-cyclopropylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid stereoisomer D1: the first eluted fraction from isolate 2 in step 2 above was subjected to a procedure similar to that of step 4 of example 70 to give the title compound (47 mg, 96. Mu. Mol, yield 29%, purity 99%). UPLC-MS (method 4) M/z 487.2 (M+H) + ,485.2(M-H) - At 1.89 minutes. 1 H NMR(500MHz,DMSO)δ13.24(br s,1H),10.17(br s,1H),8.26(d,J＝1.9Hz,1H),7.99(dd,J＝8.2,1.9Hz,1H),7.72(s,1H),7.22(s,1H),7.15(d,J＝8.3Hz,1H),4.49(q,J＝7.1Hz,1H),2.80–2.71(m,1H),2.09(q,J＝8.6Hz,1H),1.62(q,J＝9.8Hz,1H),1.44–1.34(m,1H),1.27–1.19(m,1H),1.17–1.09(m,1H),1.09–1.00(m,2H),0.88–0.79(m,2H),0.79–0.71(m,1H),0.39–0.30(m,1H),0.30–0.23(m,1H),-0.00–-0.09(m,1H),-0.13–-0.21(m,1H)。SFC(Waters UPC 2 ChiralPak IH 4.6X250mm,5 μm column, flow 4mL/min,20% (0.1% ammonia/MeOH)/CO 2 )t R 3.17 minutes, 76% enantiomeric excess.
Example 131:3- (N- (4-chloro-5-cyano-2- (2-cyclopropylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid stereoisomer D2
The second eluted fraction from separation 2 in step 2 of example 130 was subjected to a procedure similar to that of example120 procedure of step 3 gave the title compound (61 mg, 124. Mu. Mol, yield 37%, purity 99%). SFC (Waters UPC) 2 ChiralPak IH 4.6X250mm,5 μm column, flow 4mL/min,20% (0.1% ammonia/MeOH)/CO 2 )t R 3.41 minutes, 98% enantiomeric excess. Other analytical data are consistent with example 130.
Example 132:3- (N- (4-chloro-5-cyano-2- (2-cyclopropylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid stereoisomer D3
The second eluted fraction from isolate 1 in step 2 of example 130 was subjected to a procedure similar to step 3 of example 120 to give the title compound (3.8 mg, 7.3. Mu. Mol, yield 2%, purity 94%). UPLC-MS (method 4): M/z 485.2 (M-H) - At 1.88 minutes. 1 H NMR(500MHz,DMSO)δ13.22(br s,1H),10.19(br s,1H),8.28(d,J＝1.9Hz,1H),7.96(dd,J＝8.2,1.9Hz,1H),7.72(s,1H),7.14(d,J＝8.3Hz,1H),7.10(s,1H),4.77(q,J＝7.0Hz,1H),2.86–2.77(m,1H),2.21–2.09(m,2H),1.97–1.88(m,1H),1.80–1.68(m,1H),1.54–1.42(m,1H),1.16–1.09(m,1H),1.09–1.01(m,1H),0.99–0.92(m,1H),0.83–0.75(m,1H),0.37–0.21(m,1H),0.15–0.01(m,1H),-0.13–-0.22(m,1H),-0.33–-0.43(m,1H),-0.51–-0.61(m,1H)。SFC(Waters UPC 2 ChiralPak IH 4.6X250mm,5 μm column, flow 4mL/min,20% (0.1% ammonia/MeOH)/CO 2 )t R 4.31 minutes, 98% enantiomeric excess.
Example 133:3- (N- (4-chloro-5-cyano-2- (2-cyclopropylcyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid stereoisomer D4
The third eluted fraction from separation 1 in example 130 step 2 was subjected to a procedure similar to example 120 step 3 to give the title compound (3.3 mg, 6.6. Mu. Mol)Yield 1%, purity 98%). SFC (Waters UPC) 2 ChiralPak IH 4.6X250mm,5 μm column, flow 4mL/min,20% (0.1% ammonia/MeOH)/CO 2 )t R 5.05 minutes, 90% enantiomeric excess. Other analytical data are consistent with example 132.
The following examples were prepared by methods analogous to example 1 or example 52, substituting, if necessary, appropriate starting materials and intermediates for:
in addition, the following chiral resolution was performed and individual enantiomers or diastereomers were advanced (advanced) to the final example by a method similar to example 1 or example 52, substituting appropriate starting materials and intermediates as necessary:
example 85 intermediate→example 103 and example 104 (alternative route 2)
Intermediate in vitro trans-ester 3- (N- (4-chloro-5-cyano-2- ((2-methoxycyclopentyl) oxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester (105 mg) was purified on a Unichiral CND-5H column (4.6x250 mm), eluting with 90% N-hexane/10% ethanol/0.1% TFA (1 mL/min), 25℃and 254nm UV detection. The appropriate fractions were pooled and reduced in vacuo to give peak 1 (51 mg), RT 13.18min (99.8%) and peak 2 (45 mg), RT 15.48min (99.9%). Advancing the ester of peak 1 gives trans diastereomer D1 (example 103) and advancing the ester of peak 2 gives trans diastereomer D2 (example 104).
7
Example 84 intermediate → example 116 and example 117
Intermediate in vitro racemic trans-aniline 5-amino-2-chloro-4- ((2-ethynylcyclopentyl) oxy) benzonitrile from example 84 (150 mg) was purified on a uniciral CMD-5H column (4.6x250 mm), eluted with 90% n-hexane/10% ethanol (1 mL/min), 25 ℃,254nm uv detection. The appropriate fractions were pooled and reduced in vacuo to give peak 1 (55 mg), RT 13.10min (96.3%) and peak 2 (46 mg), RT 23.69min (98.7%). The aniline of peak 1 was advanced to give the trans diastereomer D1 (example 116), and the aniline of peak 2 was advanced to give the trans diastereomer D2 (example 117).
Example 126 and example 127
The racemic cis acid 3- (N- (4-chloro-5-cyano-2- ((2-ethylcyclopentyl) oxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid (131 mg) was purified on a Unichiral CMD-5H column (4.6X250 mm), eluted with 60% N-hexane/40% isopropanol/0.1% TFA (1 mL/min), 25℃and 254nm UV detection. The appropriate fractions were pooled and reduced in vacuo to give peak 1 (example 126, cis diastereomer D1, 31 mg), RT 6.99min (100%) and peak 2 (example 127, cis diastereomer D2, 22 mg), RT 10.79min (99.3%).
Example 118 results in example 128 and example 129
The racemic trans acid 3- (N- (4-chloro-5-cyano-2- (2-hydroxycyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid (150 mg) was purified on a Unichiral CMD-5H column (4.6X250 mm), eluting with 70% N-hexane/30% ethanol (1 mL/min), at 25℃and 254nm UV detection. The appropriate fractions were pooled and reduced in vacuo to give peak 1 (example 128, trans diastereomer D1, 47 mg), RT 8.48min (97.0%) and peak 2 (example 129, trans diastereomer D2, 49 mg), RT10.51min (99.6%).
Example 113 intermediate → examples 135 and 136
EXAMPLE 113 intermediate racemic cis-aniline 5-amino-2-chloro-4- (2-hydroxycyclobutoxy) benzonitrile (213 mg) was purified on a Unichiral CMD-5H column (4.6X250 mm), eluting with 80% n-hexane/20% ethanol (1 mL/min), 25℃and 254nm UV detection. The appropriate fractions were pooled and reduced in vacuo to give peak 1 (73 mg), RT 12.49min (99.8%) and peak 2 (82 mg), RT 14.58min (99.8%). The aniline of peak 1 was advanced to give cis diastereomer D1 (example 135) and the aniline of peak 2 was advanced to give cis diastereomer D2 (example 136).
Example 119 intermediate → examples 138 and 139
Example 119 intermediate in vitro racemic trans-ester 3- (N- (4-chloro-5-cyano-2- (2-methoxycyclobutoxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester (182 mg) was purified on a Unichiral CNZ-5H column (4.6X250 mm), eluting with 80% N-hexane/20% ethanol/0.1% TFA (1 mL/min), 25℃and 254nm UV detection. The appropriate fractions were pooled and reduced in vacuo to give peak 1 (72 mg), RT 13.76min (100%) and peak 2 (67 mg), RT 16.49min (99.7%). Advancing the ester of peak 1 gives trans diastereomer D1 (example 138) and advancing the ester of peak 2 gives trans diastereomer D2 (example 139).
Example 134 intermediate → examples 140 and 141
Example 134 intermediate in vitro racemic ester 3- (N- (4-chloro-5-cyano-2- ((3, 3-difluorocyclopentyl) oxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid methyl ester (150 mg) was purified on a Unichiral CND-5H column (4.6x250 mm), eluting with 90% N-hexane/10% ethanol/0.1% TFA (1 mL/min), 25 ℃,254nm UV detection. The appropriate fractions were pooled and reduced in vacuo to give peak 1 (32 mg), RT 19.69min (> 95%) and peak 2 (27 mg), RT 21.70min (> 95%). The ester of peak 1 was advanced to give enantiomer E1 (example 140) and the ester of peak 2 was advanced to give enantiomer E2 (example 141).
Example 102 intermediate → examples 142 and 143
Example 102 intermediate racemic cis-aniline 5-amino-2-chloro-4- ((2-methoxycyclopentyl) oxy) benzonitrile (187 mg) was purified on a uniciral CMZ-5H column (4.6x250 mm), eluting with 95% n-hexane/5% ethanol (1 mL/min), 25 ℃,254nm UV detection. The appropriate fractions were pooled and reduced in vacuo to give peak 1 (59 mg), RT 24.14min (100%) and peak 2 (56 mg), RT 25.64min (98.8%). The aniline of peak 1 was advanced to give cis diastereomer D1 (example 142) and the aniline of peak 2 was advanced to give cis diastereomer D2 (example 143).
Example 137 intermediate → example 144 and example 145
Example 137 intermediate racemic cis-aniline 5-amino-2-chloro-4- ((2-methoxycyclobutyl) oxy) benzonitrile (250 mg) was purified on a uniciral CMZ-5H column (4.6x250 mm), eluted with 90% n-hexane/10% ethanol (1 mL/min), 25 ℃,254nm UV detection. The appropriate fractions were pooled and reduced in vacuo to give peak 1 (110 mg), RT 16.56min (98.8%) and peak 2 (86 mg), RT 17.85min (97.8%). The aniline of peak 1 was advanced to give cis diastereomer D1 (example 144), and the aniline of peak 2 was advanced to give cis diastereomer D2 (example 145).
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Example 109 3- (N- (4-chloro-5-cyano-2- (((1R, 2R) -2-methoxycyclopentyl) oxy) phenyl) sulfamoyl) -4-cyclopropylbenzoic acid.
Step 2: 2-chloro-4- (((1 r,2 r) -2-methoxycyclopentyl) oxy) -5-nitrobenzonitrile: 2-chloro-4- (((1R, 2R) -2-hydroxycyclopentyl) oxy) -5-nitrobenzophenoneNitrile (0.141 g,0.5 mmol), meI (0.141 g,1.0 mmol) and Ag 2 A mixture of O (0.23 g,1.0 mmol) in MeCN (5 mL) was heated at 80℃for 12h. The reaction mixture was filtered through celite. The filtrate was concentrated and purified by silica gel chromatography (eluting with 1/10 EtOAc/PE) to give the title compound (0.091 g,0.31mmol, 62% yield) as a yellow oil. 1 H NMR (400 MHz, chloroform-d) δ8.17 (d, j=1.4 hz, 1H), 7.40 (d, j=1.4 hz, 1H), 4.78-4.68 (m, 1H), 3.36 (d, j=1.4 hz, 3H), 2.22-2.13 (m, 1H), 2.11-2.00 (m, 3H), 1.87-1.72 (m, 4H).
The procedure of step 1 was the same as in example 52
The experimental procedure of steps 3, 4 and 5 is the same as in example 1
The following examples were prepared by a method analogous to example 109, substituting, if necessary, appropriate starting materials and intermediates:
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biological investigation
The following tests may be used to illustrate the commercial use of the compounds of the present invention.
Biological assay 1: ERAP 1-mediated amide substrate hydrolysis as measured in biochemical systems
Materials and solutions
1X Assay Buffer (AB): 25mM Bis-Tris propane, 0.05% w/v hydroxypropyl methylcellulose, pH 7.75, with optimal grade water
Decapeptide WRVYEKC (Dnp) ALK-acid (where Dnp is dinitrophenyl maleimide) (10 mer)
L-leucine 7-amino-4-methylcoumarin (L-AMC)
Purified ERAP1 (37-941) -10His (ERAP 1)
Test procedure:
12.5. Mu.L of the 1 XAB solution of ERAP1 enzyme was mixed with 250nL of the DMSO solution of the test compound. 12.5. Mu.L of a 1 XAB solution of 240. Mu. M L-AMC or 100. Mu.M 10-mer of 1 XAB solution was added to the reaction and incubated at 23℃for 1h. For detection, plates were read at either excitation 365nm and emission 442nm (L-AMC), or excitation 279nm and emission 355nm (10 mer). Determination of Compound IC using the 4 parameter equation 50 . The results of the compounds selected according to the invention are shown in table 1.
OVA antigen presentation assay
As previously described [ Reeves et al, (2014) proc.Natl. Acad. Sci. USA 111;17594-17599]The cellular effects of representative compounds according to the invention on antigen presentation were determined by assessing their effect on ovalbumin-specific peptide (SIINFEKL) presentation to T cells. Briefly, siHa cells were transiently transfected with a plasmid encoding mouse H2Kb and an ER-targeted N-terminal extended precursor peptide derived from ovalbumin (MRYMILGLLALAAVCSAAIVMKSIINFEHL) using Lipofectamine 3000. Cells were harvested 6h post-transfection and transfected SiHa cells were seeded on compound response curves spanning 12-point concentrations to quantify ERAP1 inhibitor IC 50 . SiHa cells were cultured for 48h in the presence of the compound. Subsequently, B3Z cells were isolated [ Karttunen et al, (1992) Proc.Natl. Acad. Sci. USA 89;6020-6024]Adding into cell culture for continuous culture for 4 hr; the B3Z T cell hybridoma encodes a TCR that specifically recognizes the SIINFEHL/H2Kb complex on the cell surface, which triggers a signaling cascade upon activation, resulting in transcription of the LacZ gene under the control of the IL-2 promoter. The conversion of chloro- β -D-galactopyranoside (CPRG) to chlorophenol red was quantified by measuring absorbance at 570nm, thereby measuring intracellular β -galactosidase activity as a reading of T cell activation.
Immune peptide histology
Using, for example, purcell and colleagues [ Purcell et al, (2019) Nat Protoc.14;1687-1707] to determine the effect of representative compounds according to the invention on global antigen treatment. Briefly, 500 million SiHa cells were treated with compound for 24h or siRNA for 72h, then harvested, lysed and MHC binding peptides isolated by immunoaffinity capture. Peptides were eluted with 10% (v/v) acetic acid and isolated from MHC-1 and beta 2-microglobulin by HPLC, and then analyzed by LC-MS/MS.
Various modifications and alterations to this aspect of this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.
Table 1: activity of selected Compounds according to the invention
IC 50 vs decapeptide WRVYEKC (Dnp) ALK-acid (wherein Dnp is dinitrophenyl maleimide) (10 mer); high (< 1 nM), medium%>1nM to<5nM, low%>5nM)。
Reference to the literature
1.Serwold et al,(2002),ERAAP customizes peptides for MHC class I molecules in the endoplasmic reticulum；Nature:419,p480.
2.Snyder et al,(2014),Genetic Basis for Clinical Response to CTLA-4Blockade in Melanoma；NEJM:371,p2189.
3.Van Allen et al,(2015),Genomic correlates of response to CTLA-4blockade in metastatic melanoma；Science:348,p124.
4.James et al,(2013),Induction of Protective Antitumor Immunity through Attenuation of ERAAP Function；J Immunol:190,p5839.
5.Niranjana et al,(2016),ERAAP Shapes the Peptidome Associated with Classical and Nonclassical MHC Class I Molecules；J Immunol:197,p1035.
6.Pepelyayeva et al,(2018),ERAP1 deficient mice have reduced Type 1regulatory T cells and develop skeletal and intestinal features of Ankylosing Spondylitis；Sci.Reports:8:p12464.
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11.Reeves et al,(2018),The role of polymorphic ERAP1 in autoinflammatory disease,Biosci.Rep.:29,p38.
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13.Sheehan,NJ(January 2004).“The ramifications of HLA-B 2 7”.Journal of the Royal Society of Medicine.97(1):10–4.
14.Smith,JA(January 2015).“Update on ankylosing spondylitis:current concepts in pathogenesis”.Current allergy and asthma reports.15(1):489.
15.Kuiper JJW,Mutis T,de Jager W,de Groot-Mijnes JD,Rothova A(2011).“Intraocular interleukin-17 and proinflammatory cytokines in HLA-A29-associated birdshot chorioretinopathy”.Am J Ophthalmol.152(2):177–182
16.Kuiper JJW,Emmelot ME,Rothova A,Mutis T(2013).“Interleukin-17 production and T helper 17 cells in peripheral blood mononuclear cells in response to ocular lysate in patients with birdshot chorioretinopathy”.Mol Vis.19:2606–14
17.Kuiper JJW,van Setten J,Ripke S,Van’t Slot R,Mulder F,Missotten T,Baarsma GS,Francioli LC,Pulit SL,de Kovel CG,Ten Dam-van Loon N,den Hollander AI,Huis In Het Veld P,Hoyng CB,Cordero-Coma M,Martín J,V,Arya B,Thomas D,Bakker SC,Ophoff RA,Rothova A,de Bakker PI,Mutis T,Koeleman BP(2014).“A genome-wide association study identifies a functional ERAP2haplotype associated with birdshot chorioretinopathy”.Hum Mol Genet.23(22):6081–6087
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Claims (57)

1. A compound of formula (I) or a pharmaceutically acceptable salt or hydrate thereof,
wherein:
the radical X-Y being-NHSO 2 -；
Z is a monocyclic or polycyclic cycloalkyl group, or a monocyclic or polycyclic heterocycloalkyl group, each of which is optionally substituted with one or more groups selected from haloalkyl, alkyl, alkenyl, alkynyl and- (CR) 16 R 17 ) m R 18 Wherein m is 0 to 6;
l is a direct bond or a group (CR) 14 R 15 ) n Wherein n is 1 or 2;
R 1 Selected from H, cl, F, CN and alkyl;
R 2 selected from COOH and tetrazolyl;
R 3 selected from H, halo, alkoxy, and alkyl;
R 4 selected from H and halo;
R 5 selected from H, alkyl, haloalkyl, SO 2 -alkyl, cl, alkoxy, OH, CN, hydroxyalkyl, alkylthio, heteroaryl, cycloalkyl, heterocycloalkyl and haloalkoxy;
R 6 is H;
R 7 selected from H, CN, haloalkyl, halo, SO 2 -alkyl, SO 2 NR 12 R 13 Heteroaryl, CONR 10 R 11 And alkyl, wherein the heteroaryl is optionally substituted with one or more substituents selected from alkyl, halo, alkoxy, CN, haloalkyl, and OH;
R 8 selected from H, alkyl, haloalkyl and halo;
R 9 selected from H, alkyl, and halo;
R 10 、R 11 、R 12 and R is 13 Each independently selected from H and alkyl;
R 14 and R is 15 Each independently selected from the group consisting of H, halo, and alkyl;
R 16 and R is 17 Each independently selected from the group consisting of H, halo, haloalkyl, and alkyl; and
each R is 18 Independently selected from OH, CN, alkoxy and halo.
2. The compound of claim 1, wherein L is a direct bond.
3. The compound of claim 1, wherein L is (CH 2 ) n And n is 1 or 2.
4. The compound of claim 1, wherein L is CH 2 Or CH (Me), more preferably CH 2 。
5. The compound of any one of the preceding claims, wherein Z is 3-7 membered monocyclic cycloalkyl or 3-7 membered monocyclic heterocycloalkyl, each of which is optionally substituted.
6. A compound according to any one of the preceding claims, wherein Z is 4-membered monocyclic cycloalkyl or 4-membered monocyclic heterocycloalkyl, each of which is optionally substituted.
7. The compound of any one of the preceding claims, wherein Z is optionally substituted 4-membered monocyclic cycloalkyl.
8. The compound of claim 7, wherein Z is selected from:
wherein each Q is independently selected from alkyl, alkoxy, haloalkyl, alkynyl, halo, OH, and CN.
9. The compound according to any one of claims 1 to 6, wherein Z is optionally substituted 4-membered monocyclic heterocycloalkyl.
10. The compound of any one of claims 1 to 5, wherein Z is 5-membered monocyclic cycloalkyl or 5-membered monocyclic heterocycloalkyl, each of which is optionally substituted.
11. The compound of claim 10, wherein Z is an optionally substituted 5-membered monocyclic cycloalkyl.
12. The compound of claim 11, wherein Z is selected from:
wherein each Q is independently selected from alkyl, alkoxy, haloalkyl, alkynyl, halo, OH, and CN.
13. The compound of claim 10, wherein Z is optionally substituted 5-membered monocyclic heterocycloalkyl.
14. The compound of any one of claims 1 to 4, wherein Z is an optionally substituted polycyclocycloalkyl or an optionally substituted polycycloheterocycloalkyl, wherein the polycycle groups are fused, unfused, bridged or spiro.
15. The compound of claim 14, wherein Z is a bicyclic cycloalkyl or a bicyclic heterocycloalkyl, each of which is fused, unfused, bridged or spiro, and each of which is optionally substituted.
16. The compound of claim 14 or claim 15, wherein Z is selected from:
17. the compound of any one of the preceding claims, wherein Z is selected from:
18. the compound of claim 17, wherein L is a direct bond.
19. The compound according to any one of claims 1 to 17, wherein L-Z is selected from:
20. the compound of any one of the preceding claims, wherein R 2 Is COOH.
21. A compound of formula (la) according to any preceding claim wherein R 4 Selected from H and F.
22. The compound of any one of the preceding claims, wherein R 5 Selected from alkyl, haloalkyl, SO 2 -alkyl, cl, alkoxy, OH, CN, hydroxyalkyl, alkylthio, heteroaryl, cycloalkyl, heterocycloalkyl and haloalkoxy, and more preferably selected from alkyl, haloalkoxy and cycloalkyl.
23. The compound of any one of the preceding claims, wherein R 5 Selected from OMe, OEt, me, et and cyclopropyl, and more preferably selected from OMe, et and cyclopropyl, and even more preferably cyclopropyl.
24. The compound of any one of the preceding claims, wherein R 5 Is cyclopropyl.
25. The compound of any one of the preceding claims, wherein R 7 Selected from H, CN, haloalkyl, cl, F, SO 2 -alkyl, CONR 10 R 11 Heteroaryl and alkyl, wherein the heteroaryl is selected from the group consisting of pyrazol-3-yl, pyrazol-4-yl, pyrazol-5-yl, isothiazol-3-yl, isothiazol-4-yl, isothiazol-5-yl, 1,2, 4-triazol-5-yl, tetrazol-1-yl, tetrazol-5-yl, isoxazol-3-yl, and iso-thiazol-4-ylOxazol-4-yl and isoxazol-5-yl, each of which is optionally substituted with one or more substituents selected from alkyl, halo, alkoxy, CN, haloalkyl and OH.
26. The compound according to any one of claims 1 to 25, wherein R 7 Selected from CN, tetrazol-1-yl, isothiazol-5-yl and isoxazol-4-yl, each of which is optionally substituted with one or more substituents selected from alkyl, halo, alkoxy, CN, haloalkyl and OH.
27. The compound of claim 26, wherein R 7 Selected from CN, CF 3 Tetrazol-1-yl, isothiazol-5-yl and 5-methyl-isoxazol-4-yl.
28. The compound of any one of the preceding claims, wherein R 7 Is CN.
29. The compound of any one of the preceding claims, wherein R 8 Selected from H and halo, more preferably H, cl and F.
30. The compound of any one of the preceding claims, wherein R 8 Is Cl.
31. The compound of any one of the preceding claims, wherein R 9 Is H, me, F or Cl, more preferably H.
32. The compound of any one of the preceding claims, wherein R 1 Is H.
33. The compound of any one of the preceding claims, wherein R 1 、R 3 、R 4 And R is 9 All are H.
34. A compound according to claim 1, wherein:
X-Y is NH-SO 2 ；
R 1 Is H;
R 2 is COOH;
R 3 is H or F;
R 4 is H or F;
R 5 selected from OMe, OEt, me, et and cyclopropyl, more preferably from OMe, cyclopropyl and Et;
R 6 Is H;
R 7 selected from CN, tetrazol-1-yl, isothiazol-5-yl and 5-methyl-isoxazol-4-yl;
R 8 selected from H, cl and F;
R 9 selected from H, me, cl, and F; and
l and Z are as defined in any one of claims 1 to 19.
35. The compound of claim 1, having formula (Ia):
wherein L and Z are as defined in any one of claims 1 to 19.
36. A compound according to any one of the preceding claims, selected from the following:
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
and pharmaceutically acceptable salts and hydrates thereof.
37. A pharmaceutical composition comprising a compound of formula (I) as defined in any one of claims 1 to 36 in admixture with a pharmaceutically acceptable excipient, diluent or carrier and optionally one or more additional active agents.
38. Use of a compound of formula (I) as defined in any one of claims 1 to 36 in medicine.
39. Use of a compound of formula (I) as defined in any one of claims 1 to 36 for the treatment or prophylaxis of a condition selected from the group consisting of proliferative disorders, immune disorders, viral disorders and inflammatory disorders.
40. The use of a compound of claim 39, wherein the compound modulates ERAP1.
41. The use of a compound according to claim 39 or claim 40, wherein the disorder is a proliferative disorder, preferably cancer or leukemia.
42. The use of a compound according to any one of claims 39 to 41, wherein the compound kills cancer cells, reduces the number of proliferating cells in cancer, reduces the volume or size of a tumor comprising cancer cells, and/or reduces the number of metastatic cancer cells.
43. The use of a compound according to any one of claims 39 to 42, wherein the compound is for the prevention of cancer, wherein preferably the compound induces neoantigens to which a subject has an existing immune response.
44. The use of a compound according to claim 43, wherein the compound is for use in a subject suffering from or susceptible to cancer, wherein the compound stimulates a neoantigen-directed immune response in the subject, and wherein a second compound (which may be the same or different from the first compound) is subsequently used to stimulate the same neoantigen as the first compound, thereby directing the subject to mount an immune response against the cancer.
45. The use of a compound according to any one of claims 39 to 44, wherein the subject has previously suffered from cancer, has a family history of cancer, has a high risk of developing cancer, has a genetic predisposition to developing cancer, has been exposed to a carcinogen, and/or is in remission.
46. An in vitro or in vivo method for producing antigen presenting cells presenting neoantigens, comprising inducing neoantigens in said antigen presenting cells with a compound of formula (I) as defined in any one of claims 1 to 36, wherein preferably said antigen presenting cells are dendritic cells.
47. An immunogenic composition comprising antigen presenting cells obtained or obtainable by the method of claim 46.
48. Use of an immunogenic composition according to claim 47 in the treatment or prevention of cancer in a subject, wherein preferably the immunogenic composition is a vaccine.
49. The use of a compound according to any one of claims 39 to 45, wherein the compound is used in combination with an immunotherapy, wherein preferably the subject is suffering from cancer, and the compound increases the sensitivity of cancer cells to the immunotherapy.
50. The use of a compound according to claim 49, wherein the immunotherapy is an immune checkpoint intervention, preferably an antibody checkpoint inhibitor.
51. The use of a compound of claim 50, wherein the antibody checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA 4 antibody.
52. The use of a compound according to claim 39 or claim 40, wherein the disorder is an immune disorder, and is preferably selected from ankylosing spondylitis, behcet's disease, psoriasis and shotgun elastic-like chorioretinopathy.
53. The use of a compound according to claim 39 or claim 40, wherein the disorder is an inflammatory disorder, more preferably an autoinflammatory disorder.
54. The use of a compound according to claim 39 or claim 40, wherein the viral disorder is an infectious viral disease selected from HIV, HPV, CMV and HCV.
55. The use of a compound according to any one of claims 39 to 45 or 49 to 51, wherein the disorder is cancer, and wherein the compound increases the visibility of cancer cells to the immune system by altering the repertoire of antigens and neoantigens presented to the immune system.
56. The use of a compound of claim 55, wherein the compound increases the response of cd8+ T cells to the cancer cells.
57. A combination comprising a compound according to any one of claims 1 to 36 and another active agent.
CN202280053138.0A 2021-07-30 2022-07-29 Phenyl-sulfamoyl-benzoic acid derivatives as ERAP1 modulators Pending CN117881657A (en)

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