Patent Application
20230101819
The present disclosure provides carboxamides and sulfonamides as SMYD protein inhibitors, such as SMYD3 and SMYD2 inhibitors, and therapeutic methods of treating conditions and diseases wherein inhibition of SMYD proteins such as SMYD3 and SMYD2 provides a benefit.
Epigenetic regulation of gene expression is an important biological determinant of protein production and cellular differentiation and plays a significant pathogenic role in a number of human diseases. Epigenetic regulation involves heritable modification of genetic material without changing its nucleotide sequence. Typically, epigenetic regulation is mediated by selective and reversible modification (e.g., methylation) of DNA and proteins (e.g., histones) that control the conformational transition between transcriptionally active and inactive states of chromatin. These covalent modifications can be controlled by enzymes such as methyltransferases (e.g., SMYD proteins such as SMYD3 and SMYD2), many of which are associated with genetic alterations that can cause human disease, such as proliferative disorders. Thus, there is a need for the development of small molecules that are capable of inhibiting the activity of SMYD proteins such as SMYD3 and SMYD2.
In one aspect, the present disclosure provides carboxamido and sulfonamide compounds represented by Formulae I-XVIII below, and the pharmaceutically acceptable salts and solvates thereof, collectively referred to herein as “Compounds of the Disclosure.”
In another aspect, the present disclosure provides a Compound of the Disclosure and one or more pharmaceutically acceptable carriers.
In another aspect, the present disclosure provides a method of inhibiting SMYD proteins, such as SMYD3 or SMYD2, or both, in a mammal, comprising administering to the mammal an effective amount of at least one Compound of the Disclosure.
In another aspect, the present disclosure provides methods for treating a disease, disorder, or condition, e.g., cancer, responsive to inhibition of SMYD proteins, such as SMYD3 or SMYD2, or both, comprising administering a therapeutically effective amount of a Compound of the Disclosure.
In another aspect, the present disclosure provides the use of Compounds of the Disclosure as inhibitors of SMYD3.
In another aspect, the present disclosure provides the use of Compounds of the Disclosure as inhibitors of SMYD2.
In another aspect, the present disclosure provides the use of Compounds of the Disclosure as inhibitors of SMYD proteins.
In another aspect, the present disclosure provides a pharmaceutical composition for treating a disease, disorder, or condition responsive to inhibition of SMYD proteins, such as SMYD3 or SMYD2, or both, wherein the pharmaceutical composition comprises a therapeutically effective amount of a Compound of the Disclosure in a mixture with one or more pharmaceutically acceptable carriers.
In another aspect, the present disclosure provides Compounds of the Disclosure for use in treating cancer in a mammal, breast, cervical, colon, kidney, liver, head and neck, skin, pancreatic, ovary, esophageal, lung, and prostate cancer.
In another aspect, the present disclosure provides a Compound of the Disclosure for use in the manufacture of a medicament for treating cancer in a mammal.
In another aspect, the present disclosure provides kit comprising a Compound of the Disclosure.
Additional embodiments and advantages of the disclosure will be set forth, in part, in the description that follows, and will flow from the description, or can be learned by practice of the disclosure. The embodiments and advantages of the disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention as claimed.
One aspect of the present disclosure is based on the use of Compounds of the Disclosure as inhibitors of SMYD proteins. In view of this property, the Compounds of the Disclosure are useful for treating diseases, disorders, or conditions, e.g., cancer, responsive to inhibition of SMYD proteins.
One aspect of the present disclosure is based on the use of Compounds of the Disclosure as inhibitors of SMYD3. In view of this property, the Compounds of the Disclosure are useful for treating diseases, disorders, or conditions, e.g., cancer, responsive to inhibition of SMYD3.
One aspect of the present disclosure is based on the use of Compounds of the Disclosure as inhibitors of SMYD2. In view of this property, the Compounds of the Disclosure are useful for treating diseases, disorders, or conditions, e.g., cancer, responsive to inhibition of STAYD2.
In one embodiment, Compounds of the Disclosure are compounds having Formula I:
and the pharmaceutically acceptable salts or solvates, e.g., hydrates, there wherein:
A is selected from the group consisting of 1,2,3-triazolyl, 1-imidazolyl, 1-isoquinolinyl, 1-pyrazolyl, 2-(1,2,3,4-tetrahydroquinolinyl), 2-benzo[d]imidazolyl, 2-benzo[d]thiazolyl, 2-chromenyl-4-one, 2-furanyl, 2-imidazo[1,2-b]pyridazinyl, 2-imidazolyl, 2-indolyl, 2-naphthalenyl, 2-pyrazinyl, 2-pyridyl, 2-pyrimidinyl, 2-pyrrolidinyl, 2-pyrrolyl, 2-quinolinyl, 2-quinoxalinyl, 2-thiazolo[5,4-d]pyridinyl, 2-thiazolyl, 2-thiophenyl, 3-(1,2,3,4-tetrahydroisoquinoline), 3-(1,2,4-oxadiazolyl), 3-imidazo[1,2-a]pyrimidinyl, 3-indazolyl, 3-indolyl, 3-isothiazolyl, 3-pyrazolyl, 3-pyridazinyl, 3-pyridinyl-2-one, 3-pyridyl, 3-pyrrolo[3,2-b]pyridinyl, 3-quinolinyl, 4-(2,2-difluorobenzo[d][1,3]dioxolyl), 4-cyclohexanyl-1-amine, 4-imidazolyl, 4-indolinyl-2-one, 4-indolyl, 4-isothiazolyl, 4-oxazolyl, 4-piperidinyl, 4-pyrazolyl, 4-pyridyl, 5-(1,3-dihydro-2H-benzo[d]imidazolyl-2-one), 5-(1,3-dihydro-2H-pyrrolo[2,3-c]pyridinyl-2-one), 5-(1,3-dihydro-2H-pyrrolo[2,3-c]pyridinyl-2-one), 5-(2,2-difluorobenzo[d][1,3]dioxolyl), 5-(2,4-dihydro-3H-1,2,4-triazolyl-3-one), 5-4H-furo[3,2-b]pyrrolyl, 5-benzo[c][1,2,5]oxadiazolyl, 5-benzo[d][1,3]dioxolyl, 5-benzo[d]oxazolyl-2(3H)-one, 5-bicyclo[2.2.1]heptyl-2-ene, 5-indolinyl-2,3-dione, 5-indolinyl-2-one, 5-indolyl, 5-isoindolinyl-1-one, 5-isoxazolyl, 5-pyrazolo[3,4-c]pyridinyl, 5-pyrazolyl, 5-pyrimidinyl, 5-thiazolyl, 6-(1,2,3,4-tetrahydronaphthalenyl), 6-(3,4-dihydroquinolinyl-2(1H)-one), 6-(3,4-dihydroquinoxalinyl-2(1H)-one), 6-(4,5-dihydropyridazinyl-3 (2H)-one), 6-benzo[b][1,4]oxazinyl-3-one, 6-benzo[d]imidazolyl, 6-benzo[d]oxazolyl-2(3H)-one, 6-benzo[d]thiazolyl, 6-chromenyl-2-one, 6-imidazo[2,1-b]thiazole, 6-indazolyl, 6-indolinyl-2-one, 6-indolyl, 6-isoquinolinyl, 6-quinolinyl, 6-quinoxalinyl, 6-quinoxalinyl-2(1H)-one, 7-(3,4-dihydroquinolinyl-2(1H)-one), 7-(3,4-dihydroquinoxalin-2(1H)-one), 7-benzo[b][1,4]oxazinyl-3-one, 7-indolinyl-2-one, 8-benzo[b][1,4]oxazinyl-3-one, cyclopropanyl, phenyl, 4-(prop-1-en-1-yl)-imidazole, 1-butanyl-imidazole, sec-butylcyclopropane, 2-(ethylsulfonyl)propanyl, 1-isobutylpyrrolidine, 4-pyridyl 1-oxide, and 5-benzo[c][1,2,5]oxadiazolyl 1-oxide,
each of which is optionally substituted with one, two, or three substituents independently selected from the group consisting of halo, hydroxy, alkoxy, amino, alkylamino, dialkylamino, (amino)alkyl, (alkylamino)alkyl, (dialkylamino)alkyd, C1-6 alkyl, haloalkyl, hydroxyalkyl, (carboxamido)alkyl, (cycloalkyl)alkyl, optionally substituted C3-12 cycloalkyl, optionally substituted C6-14 aryl, optionally substituted 5- to 14-membered heteroaryl, optionally substituted 4- to 14-membered heterocyclo, aralkyl, —N(H)C(═O)R6, —C(═O)R7, and —S(═O)2R8;
Y is selected from the group consisting of —C(R5a)(R5b)C(═O)—, —C(═O)—, and —S(═O)2—;
B is selected from the group consisting of C1-10 alkylenyl, optionally substituted C3-12 cycloalkylenyl, optionally substituted C6-14 arylenyl, optionally substituted 4- to 14-membered heterocyclenyl, and —C(H)R1R2,
with the proviso that B is not optionally substituted pyrrolidinenyl;
X is selected from the group consisting of —N(R3)—, —S(═O)2—, —S(═O)2N(R3)—, —N(R3)S(═O)2—, —S(═O)2C(R4)(H)—, —C(═O)—, —C(═O)N(R3)—, —N(R3)C(═O)—, —C(═O)O—, —OC(═O)—, —C(—O)C(R4)(H)N(R3)—, —N(OC(═O)C(R4)(H)—, and —C(═O)C(R4)(H)—; or X is absent, i.e., Z forms a bond with B;
Z is selected from the group consisting of hydrogen, optionally substituted C1-6 alkyl, fluoroalkyl, hydroxyalkyl, (amino)alkyl, (alkylamino)alkyl, (dialkylamino)alkyl, (cycloalkylamino)alkyl, (heterocyclo)alkyl, (cycloalkyl)alkyl, (amino)(hydroxy)alkyl, (amino)(aryl)alkyl, (hydroxy)(aryl)alkyl, (aralkylamino)alkyl, (hydroxyalkylamino)alkyl, alkoxyalkyl, optionally substituted C6-11 aryl, optionally substituted 4- to 14-membered heterocyclo, optionally substituted 5- to 14-membered heteroaryl, optionally substituted C3-12 cycloalkyl, aralkyl, and (heteroaryl)alkyl; or
Z is —CH(R9a)(R9b);
R9 is selected from the group consisting of hydrogen. C1-6 alkyl, fluoroalkyl, hydroxyalkyl, (amino)alkyl, (alkylamino)alkyl, (dialkylamino)alkyl, alkoxyalkyl, optionally substituted C6-14 aryl, optionally substituted 4- to 14-membered heterocyclo, optionally substituted 5- to 14-membered heteroaryl, optionally substituted C3-12 cycloalkyl, aralkyl, and (heteroaryl)alkyl;
R9b is selected from the group consisting of optionally substituted C6-14 aryl, optionally substituted 4- to 14-membered heterocyclo, optionally substituted 5- to 14-membered heteroaryl, optionally substituted C3-12 cycloalkyl, aralkyl, and (heteroaryl alkyl;
R1 is selected from the group consisting of hydrogen, C1-6 alkyl, amino)alkyl, (alkylamino)alkyl, (dialkylamino)alkyl, hydroxyalkyl, alkoxyalkyl, aryloxyalkyl, optionally substituted C3-12 cycloalkyl, optionally substituted 4- to 14-membered heterocyclo, optionally substituted C6-14 aryl, aralkyl, and alkoxycarbonyl;
R2 is selected from the group consisting of C1-6 alkyl, optionally substituted C3-12 cycloalkyl, optionally substituted C6-14 aryl, optionally substituted 5- to 14-membered heteroaryl, optionally substituted 4- to 14-membered heterocyclo, and (heteroaryl alkyl;
R3 is selected from the group consisting of hydrogen and C1-4 alkyl; and
R4 is selected from the group consisting of hydrogen. C1-4alkyl, hydroxy, amino, alkylamino, dialkylamino, cycloalkylamino, (amino)alkyl, (alkylamino)alkyl, (dialkylamino)alkyl, and hydroxyalkyl.
R5a is selected from the group consisting of hydrogen and C1-4 alkyl;
R5b is selected from the group consisting of hydrogen, C1-4 alkyl, and 4- to 14-membered heterocyclo;
R6 is C1-4 alkyl,
R7 is C1-4 alkyl; and
R8 is selected from the group consisting of C1-4 alkyl, amino, alkylamino, and dialkylamino,
wherein —X—Z is attached to any available carbon or nitrogen atom of B, R1, or R2, e.g., when R2 is C1-6 alkyl, e.g., ethyl, a hydrogen atom of that ethyl group is replaced with —X—Z to give —CH2CH2—X—Z or
or
when R2 is optionally substituted C3-12 cycloalkyl, e.g., cyclohexyl, a hydrogen atom of the cyclohexyl group is replaced with —X—Z to give:
or
when R2 is optionally substituted 4- to 14-membered heterocyclo, e.g., piperidinyl, the hydrogen atom attached to the piperidinyl nitrogen atom is replaced with —X—Z to give:
or
when R2 is optionally substituted C6-14 aryl, phenyl, a hydrogen atom on that phenyl group is replaced with —X—Z to give:
In another embodiment, Compounds of the Disclosure are compounds having Formula I, and the pharmaceutically acceptable salts or solvates, hydrates, thereof, wherein Z is selected from the group consisting of hydrogen, optionally substituted C1-6 alkyl, fluoroalkyl, hydroxyalkyl, (amino)alkyl, (alkylamino)alkyl, (dialkylaminoalkyl, (cycloalkylamino)alkyl, (heterocyclo)alkyl, (cycloalkyl)alkyl, (amino)(hydroxy)alkyl, (amino)(aryl)alkyl, (hydroxy)(aryl)alkyl, (aralkylamino)alkyl, (hydroxyalkylamino)alkyl, alkoxyalkyl, optionally substituted C6-14 aryl, optionally substituted 4- to 14-membered heterocyclo, optionally substituted 5- to 14-membered heteroacyl, optionally substituted C3-12 cycloalkyl, aralkyl, and (heteroaryl)alkyl.
In another embodiment, Compounds of the Disclosure are compounds having Formula I, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein X is absent.
In another embodiment, Compounds of the Disclosure are compounds having Formula I, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein X is absent; B is optionally substituted 4- or 6- to 14-membered heterocyclenyl; and Z is selected from the group consisting of hydrogen, optionally substituted C1-6 alkyl, fluoroalkyl, hydroxyalkyl, optionally substituted C6-14 aryl, optionally substituted 4- to 14-membered heterocyclo, optionally substituted 5- to 14-membered heteroaryl, optionally substituted C3-12 cycloalkyl, aralkyl, and (heteroaryl)alkyl.
In another embodiment, Compounds of the Disclosure are compounds having Formula I. and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein B is optionally substituted 4- or 6- to 14-membered heterocyclenyl; X is absent; and Z is —CH(R9a)(R9b).
In another embodiment, Compounds of the Disclosure are compounds having Formula I, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein B is optionally substituted 4- or 6- to 14-membered heterocyclenyl; X is absent; and Z is —CH(R9a)(R9b), wherein:
R9a is selected from the group consisting of hydrogen, C1-6 alkyl, and optionally substituted C3-12 cycloalkyl; and
R9b is selected from the group consisting of optionally substituted C6-14 aryl, optionally substituted 5- to 14-membered heteroaryl, optionally substituted C3-12 cycloalkyl, aralkyl, and (heleroaryl)alkyl.
In another embodiment, Compounds of the Disclosure are compounds having Formula I, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein B is C1-10 alkylenyl. In another embodiment, X is selected from the group consisting of —N(R3)C(═O)C(R4)(H)— and —N(R3)C(═O)—. In another embodiment, Z is selected front the group consisting of C1-6 alkyl and (amino)alkyl.
In another embodiment, Compounds of the Disclosure are compounds having Formula I, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein B is optionally substituted C6-14 arylenyl. In another embodiment, B is divalent form of optionally substituted phenyl.
In another embodiment, Compounds of the Disclosure are compounds having Formula II:
and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein X is absent and Z is (amino)alkyl; and A and Y are as defined above in connection with Formula I.
In another embodiment, Compounds of the Disclosure are compounds having Formula I, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein B is optionally substituted C3-12 cycloalkylenyl.
In another embodiment, Compounds of the Disclosure are compounds having Formula III, Formula IV, or Formula V:
and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein R10a, R10b, R11a, and R11b are each independently selected from the group consisting of hydrogen and C1-4 alkyl; and A, Y, X, and Z are as defined above in connection with Formula I. In another embodiment, X is —N(R)C(═O)— and Z is (amino)alkyl. In another embodiment, X is —N(R3)— and Z is hydrogen.
In another embodiment, Compounds of the Disclosure are compounds having Formula I, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein B is optionally substituted 4- to 14-membered heterocyclenyl.
In another embodiment, Compounds of the Disclosure are compounds having Formula VI, Formula VII, or Formula VIII:
and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein R12a, R12b, R13a, and R13b are each independently selected from the group consisting of hydrogen and C1-4 alkyl; and A, Y, X, and Z are as defined above in connection with Formula I. In another embodiment, X is selected from the group consisting of —C(═O)C(R4)(H)—, —C(═O)—, and —S(═O)2—; and R4 is selected from the group consisting of hydrogen and amino. In another embodiment, Z is selected from the group consisting of (amino)alkyl, (akylamino)alkyl, (dialkylamino)alkyl, (heterocyclo)alkyl, optionally substituted C3-12 cycloalkyl, optionally substituted C6-14 aryl, aralkyl, and (heteroaryl)alkyl.
In another embodiment, Compounds of the Disclosure are compounds having Formula VI, Formula VII, or Formula VIII, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein R12a, R12b, R13a, and R13b are each independently selected from the group consisting of hydrogen and C1-4 alkyl; A is 5-indolinyl-2-one that is optionally substituted with one or two substituents selected from the group consisting of halo, hydroxy, alkoxy, amino, alkylamino, dialkylamino, (amino)alkyl, (alkylamino)alkyl, (dialkylamino)alkyl, C1-6 alkyl, haloalkyl, and hydroxyalkyl; Y is —C(═O)—; X is —S(═O)2—; and Z is as defined above in connection with Formula I. In another embodiment, A is 6-chloro-5-indolinyl-2-one, i.e.,
In another embodiment, Z is selected from the group consisting of (amino)alkyl, (alkylamino)alkyl, (dialkylamino)alkyl, (heterocyclo)alkyl, optionally substituted C3-12 cycloalkyl, optionally substituted C6-14 aryl, aralkyl, and (heteroaryl)alkyl.
In another embodiment, Compounds of the Disclosure are compounds having Formula I, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein B is —C(H)R1R2. In this embodiment, a hydrogen atom of R1 and R2 is replaced with —X—Z.
In another embodiment, Compounds of the Disclosure are compounds having Formula IX, Formula X, or Formula XI:
and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof. In another embodiment, R1 is selected from the group consisting of hydrogen, C1-6 alkyl, alkoxycarbonyl, and optionally substituted C6-14 aryl. In another embodiment, R1 is selected from the group consisting of hydrogen and methyl. In another embodiment, X is —C(═O)C(R4)(H)— and R1 is amino. In another embodiment, X is selected from the group consisting of:
and
In another embodiment, Z is C1-6 alkyl. In another embodiment, Z is methyl.
In another embodiment, Compounds of the Disclosure are compounds having any one of Formula I-XI, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein Y is —C(R5a)(R5b)C(═O)—. In another embodiment, R5a and R5b are each independently selected from the group consisting of hydrogen and methyl.
In another embodiment, Compounds of the Disclosure are compounds having any one of Formula I-XI, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein Y is —S(═O)2—.
In another embodiment, Compounds of the Disclosure are compounds having any one of Formula I-XI, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein Y is —C(═O)—.
In another embodiment, Compounds of the Disclosure are compounds having any one of Formula I-XI, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, hereof, wherein A is selected from the group consisting of 1,2,3-triazolyl, 1,2,4-triazolyl, 2-(1,2,3,4-tetrahydroquinolinyl), 2-indolyl, 2-thiazolyl, 3-(1,2,4-oxadiazolyl), 3-isothiazolyl, 5-(1,3-dihydro-2H-benzo[d]imidazolyl-2-one), 5-(1,3)-dihydro-2H-pyrrolo[2,3-b]pyridinyl-2-one), 5-(1,3-dihydro-2H-pyrrolo[2,3-c]pyridinyl-2-one), 5-(2,2-difluorobenzo[d][1,3]dioxolyl), 5-benzo[d]oxazolyl-2(3H)-one, 5-indolinyl-2-one, 6-benzo[b][1,4]oxazinyl-3-one, and 6-isoquinolinyl.
In another embodiment, Compounds of the Disclosure are compounds having any one of Formula I-XI, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein A is 5-indolnyl-2-one.
In another embodiment, Compounds of the Disclosure are compounds having Formula XII:
and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein:
A1 is selected from the group consisting of 1,2,3-triazolyl, 1-imidazoyl, 1-pyrazolyl, 2-(4,2,3,4-tetrahydroquinolinyl), 2-benzo[d]imidazolyl, 2-benzo[d]thiazolyl, 2-chromenyl-4-one, 2-furanyl, 2-imidazo[1,2-b]pyridazinyl, 2-imidazoiyl, 2-indolyl, 2-naphthalenyl, 2-pyrazinyl, 2-pyridyl, 2-pyrrolidinyl, 2-pyrrolyl, 2-quinoxalinyl, 2-thiophenyl, 3-(1,2,3,4-tetrahydroisoquinoline), 3-(1,2,4-oxadiazolyl), 3-imidazo[1,2-a]pyrimidinyl, 3-indazolyl, 3-indolyl, 3-isothiazolyl, 3-pyrazolyl, 3-pyridazinyl, 3-pyridinyl-2-one, 3-pyridyl, 3-pyrrolo[3,2-b]pyridinyl, 3-quinolinyl, 4-(2,2-difluorobenzo[d][1,3]dioxolyl), 4-cyclohexanyl-1-amine, one, 4-indolyl, 4-isothiazolyl, 4-oxazolyl, 4-piperidinyl, 4-pyrazolyl, 4-pyridyl, 4-quinolinyl, 5-(1,3-dihydro-2H-benzo[d]imidazolyl-2-one), 5-(1,3-dihydro-2H-pyrrolo[2,3-b]pyridinyl-2-one), 1,3-dihydro-2H-pyrrolo[2,3-c]pyridinyl-2-one), 5-(2,2-difluorobenzo[d][1,3]dioxolyl), 5-(2,4-dihydro-3H-1,2,4-triazolyl-3-one), 5-4H-furo[3,2-b]pyrrolyl, 5-benzo[c][1,2,5]oxadiazolyl, 5-benzo[d][1,3]dioxolyl, 5-benzo[d]oxazolyl-2(3H)-one, 5-bicyclo[221]heptyl-2-ene, 5-indolinyl-2-one, 5-indolyl, 5-isoindolinyl-one, 5-isoxazolyl, 5-pyrazolo[3,4-c]pyridinyl, 5-pyrazolyl, 5-pyrimidinyl, 5-thiazolyl, 6-(1,2,3,4-tetrahydronaphthalenyl, 6-(3,4-dihydroquinolinyl-2(1H)-one), 6-(3,4-dihydroquinoxalinyl-2(1H)-one), dihydropyridazinyl-3(2H)-one), 6-benzo[b][1,4]oxazinyl-3-one, 6-benzo[d]6-benzo[d]oxazolyl-2(3H)-one, 6-benzo[d]thiazolyl, 6-chromenyl-2-one, 6-imidazo[2,1-b]thiazole, 6-indazolyl, 6-indolinyl-2-one, 6-indolyl, 6-isoquinolinyl, 6-quinoxalinyl, 6-quinoxalinyl-2(1H)-one, 7-(3,4-dihydroquinolinyl-2(1H)-one), 7-(3,4-dihydroquinoxalin-2(1H)-one), 7-benzo[b][1,4]oxazinyl-3-one, 7-indolinyl-2-one, 7-quinolinyl, 8-benzo[b][1,4]oxazinyl-3-one, cyclopropanyl, phenyl, 4-(prop-1-en-1-yl)-imidazole, 1-butanyl-imidazole, sec-butylcyclopropane, 2-(ethylsulfonyl)propanyl, 1-isobutylpyrrolidine, 4-pyridyl 1-oxide, and 5-benzo[c][1,2,5]oxadiazolyl 1-oxide,
each of which is optionally substituted with one, two, or three substituents independently selected from the group consisting of halo, hydroxy, alkoxy, amino, alkylamino, dialkylamino, (amino)alkyl, (alkylamino)alkyl, (dialkylamino)alkyl, C1-6 alkyl haloalkyl, hydroxyalkyl, (carboxamido)alkyl, (cycloalkyl)alkyl, optionally substituted 03.12 cycloalkyl, optionally substituted C6-14 aryl, optionally substituted 5- to 14-membered heteroaryl, optionally substituted 4- to 14-membered heterocyclo, and aralkyl;
B1 is selected from the group consisting of optionally substituted C3-12 cycloalkylenyl and optionally substituted 4- to 14-membered heterocyclenyl;
X1 is selected from the group consisting of —N(R3a), —S(═O)2—, —S(O)2N(R3a)—, —N(R3a)S(O)2—, —S(═O)2C(R4a)(H)—, —C(═)—, —C(═O)N(R3a)—, —N(R3a)C(═O)—, —C(═O)O—, —OC(═O)—, —C(═O)C(R4a)(H)N(R3a), —N(R3a)C(═O)C(R4a)(H)—, and —C(═O)C(R4a)(H)—; or X1 is absent, i.e., Z1 forms a bond with B1;
Z1 is selected from the group consisting of hydrogen, optionally substituted C1-6 alkyl, fluoroalkyl, hydroxyalkyl, (amino)alkyl, (alkylamino)alkyl, (dialkylamino)alkyl, (cycloalkylamino)alkyl, (heterocyclo)alkyl, (cycloalkyl)alkyl, (amino)(hydroxy)alkyl, (amino)(aryl)alkyl, (hydroxy)(aryl)alkyl, (aralkylamino)alkyl, (hydroxyalkylamino)alkyl, alkoxyalkyl, optionally substituted C6-14 aryl, optionally substituted 4- to 14-membered heterocyclo, optionally substituted 5- to 14-membered heteroaryl, optionally substituted C3-12 cycloalkyl, aralkyl, and (heteroaryl)alkyl;
R3a is selected from the group consisting of hydrogen and C1-4 alkyl; and
R4a is selected from the group consisting of hydrogen, C1-4 alkyl, hydroxy, amino, alkylamino, and dialkylamino.
In another embodiment, Compounds of the Disclosure are compounds having Formula XII, or a pharmaceutically acceptable salt or hydrate thereof, wherein A1 is 5-indolinyl-2-one. In another embodiment, B1 is optionally substituted C3-12 cycloalkylenyl. In another embodiment, X1 is selected from the group consisting of —S(═O)2— and —C(═O)—. In another embodiment, Z1 is selected from the group consisting of (amino)alkyl, (alkylamino)alkyl, and (dialkylamino)alkyl.
In another embodiment, Compounds of the Disclosure are compounds having Formula XIII:
or a pharmaceutically acceptable salt or hydrate thereof, wherein
X2 is selected from the group consisting of —N(R3b)—, —S(═O)2—, —S(═O)2N(R3b)—, —N(R3b)S(═O)—; —S(═O)2C(R4)(H)—, —C(═O)—, —C(═O)N(R3b)—, —N(R3h)C(═O)—, —C(═C)O—, —OC(═CCU)—, —C(═O)C(R4b)(H)N(R3b)—, —N(R3h)C(═O)C(R4b)(H)—, and —C(═O)C(R4b)(H)—; or X is absent, i.e., Z2 forms a bond with the nitrogen atom;
Z2 is selected from the group consisting of hydrogen, optionally substituted C1-6 alkyl, fluoroalkyl, hydroxyalkyl, (amino)alkyl, (alkylamino)alky-1, (dialkylamino)alkyl, (cycloalkylamino)alkyl, (heterocyclo)alkyl, (cycloalkyl)alkyl, (amino)(hydroxy)alkyl, (amino)(aryl)alkyl, (hydroxy)(aryl)alkyl, (aralkylamino)alkyl, (hydroxyalkylamino))alkyl, alkoxyalkyl, optionally substituted C6-14 aryl, optionally substituted 4- to 14-membered heterocycle, optionally substituted 5- to 14-membered heteroaryl optionally substituted C3-12 cycloalkyl, aralkyl, and (heteroaryl)alkyl;
R1a is selected from the group consisting of hydrogen, C1-6 alkyl, and optionally substituted C6-14 aryl;
R3b is selected from the group consisting of hydrogen and C1-4 alkyl; and
R4b is selected from the group consisting of hydrogen, C1-4 alkyl, hydroxy, amino, alkylamino, and dialkylamino.
In another embodiment, Compounds of the Disclosure are compounds having Formula XIII, or a pharmaceutically acceptable salt or hydrate thereof, wherein X2 is selected from the group consisting of —S(═O)2— and —C(═O)—. In another embodiment, Z2 is selected from the group consisting of (amino)alkyl, (alkylamino)alkyl, and (dialkylamino)alkyl. In another embodiment, X is absent; and Z2 is hydrogen. In another embodiment, R1 is selected from the group consisting of hydrogen and methyl.
In another embodiment, Compounds of the Disclosure are compounds having Formula I, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein Y is —C(═O)— and A is selected from the group consisting of 5-indolinyl-2-one and 1,2,3-triazolyl.
In another embodiment, Compounds of the Disclosure are compounds having Formula XIV:
and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein A, X, and Z are as defined above in connection with Formula I. In another embodiment, X is selected from the group consisting of S(═O)2— and —S(═O)2C(R4)(H)—. In another embodiment, X is —S(═O)2—. In another embodiment, X is —S(═O)2CH2—. In another embodiment, Z is selected from the group consisting of optionally substituted C6-14 aryl, optionally substituted 4- to 14-membered heterocyclo, and optionally substituted C3-12 cycloalkyl. In another embodiment, Z is optionally substituted 4- to 14-membered heterocycle. In another embodiment, Z is an optionally substituted piperidinyl, wherein the nitrogen atom is attached to X or the 4-carbon atom is attached to X. In another embodiment, A is 5-indolinyl-2-one that is optionally substituted with one or two substituents selected from the group consisting of halo, hydroxy, alkoxy, amino, alkylamino, dialkylamino, (amino)alkyl, (alkylamino)alkyl, (dialkylamine)alkyl, C1-6 alkyl, haloalkyl, and hydroxyalkyl. In another embodiment, A is 6-chloro-5-indolinyl-2-one, i.e.,
In another embodiment, a Compound of the Disclosure is N-((1R,3r,5S)-8-((4-(benzylamino)piperidin-1-yl)sulfonyl)-8-azabicyclo[3,2,1]octan-3-yl)-6-chloro-2-oxoindoline-5-carboxamide or (i-chloro-2-oxo-N-((1R,3r,5S)-8-(((1-(4,4,4-trifluorobutyl)piperidin-4-yl)methylsulfonyl)-8-azabicyclo[3.2.1]octan-3-yl)indoline-5-carboxamide, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof.
It will be understood by those of ordinary skill in the art that compounds having Formula XIV can be drawn in various ways, e.g.,
In another embodiment, Compounds of the Disclosure are compounds having Formula I, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein A is 1,2,3-triazolyl which may be optionally substituted with one substituent, and Y is —C(═O)—.
In another embodiment, Compounds of the Disclosure are compounds having having Formula XV:
and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein:
R is selected from the group consisting of C1-6 alkyl and C3-12 cycloalkyl;
B is optionally substituted 4- or 6- to 14-membered heterocyclenyl, B is:
(wherein the nitrogen atom is attached to —X—Z); and
X and 7 are as defined above in connection with Formula I. In another embodiment, X is absent. In another embodiment, Z is selected from the group consisting of hydrogen, C1-6 alkyl, C3-12 cycloalkyl, aralkyl, and (heteroaryl)alkyl, or Z is —CH(R9a)(R9b), In another embodiment, Z is selected from the group consisting of aralkyl and (heteroaryl)alkyl. In another embodiment, Z is (heteroaryl)alkyl that is substituted with an aralkyl, e.g.,
or (heteroaryl)alkyl,
In another embodiment, Compounds of the Disclosure are compounds having having Formula XVI:
and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein:
R″ is selected from the group consisting of C1-6 alkyl and C3-12 cycloalkyl;
B is optionally substituted 4- or 6- to 14-membered heterocyclenyl, e.g., B is:
(wherein the nitrogen atom is attached to —X—Z); and
X and Z are as defined above in connection with Formula I. In another embodiment, X is absent. In another embodiment, Z is selected from the group consisting of hydrogen, C1-6 alkyd, C3-12 cycloalkyl, aralkyl, and (heteroaryl)alkyl, or Z is —CH(R9a)(R9b). In another embodiment, Z is selected from the group consisting of aralkyl and (heteroaryl)alkyl. In another embodiment, Z is (heteroaryl)alkyl that is substituted with an aralkyl, e.g.,
or (heteroaryl)alkyl, e.g.,
In another embodiment, Compounds of the Disclosure are compounds having having Formula XVII:
and the pharmaceutically acceptable salts or solvates, e.g., hydrates thereof, wherein A, X, and Z are as defined above in connection with Formula I. In another embodiment, X is absent. In another embodiment, Z is selected from the group consisting of hydrogen, C1-6 alkyl, C3-12 cycloalkyl, aralkyl, and (heteroaryl)alkyl, or Z is —CH(R9a)(R9b). In another embodiment, Z is selected from the group consisting of aralkyl and (heteroaryl)alkyl. In another embodiment, Z is aralkyl. In another embodiment, Z is (heteroaryl)alkyl. In another embodiment, Z is (heteroaryl)alkyl that is substituted with an aralkyl, e.g.,
or (heteroaryl)alkyl, e.g.,
In another embodiment, Compounds of the Disclosure are compounds having having Formula XVIII:
and the pharmaceutical acceptable salts or solvates, e.g., hydrates, thereof, wherein:
R′″ is selected from the group consisting of aralkyl and (heteroaryl)alkyl; and
A is as defined above in connection with Formula I. In another embodiment, A is selected from the group consisting of 1,2,3-triazolyl, 3-pyridazinyl, 2-pyridyl, and 2-imidazolyl, each of which is optionally substituted with one substituent selected from the group consisting of C1-6 alkyl and C3-6 cycloalkyl. In another embodiment, A is selected from the group consisting of:
In another embodiment, R′″ is aralkyl. In another embodiment, R′″ is (heteroaryl)alkyl. In another embodiment, R′″ is benzyl wherein the phenyl group is optionally substituted with one or two substituents, e.g., —CH2(4-Cl-Ph), —CH2(3-Cl-Ph), and —CH2(4-CF3-Ph).
In another embodiment, Compounds of the Disclosure are compounds of Table 1, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, or different pharmaceutically acceptable salt thereof. The chemical names of the compounds of Table 1 are provided in Table 1A.
In another embodiment, Compounds of the Disclosure are compounds of Table 3, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, or different pharmaceutically acceptable salt thereof. The chemical names of the compounds of Table 3 are provided in Table 3A.
In another embodiment, Compounds of the Disclosure are compounds of Table 4, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, or different pharmaceutically acceptable salt thereof. The chemical names of the compounds of Table 4 are provided in Table 4A.
In another embodiment, Compounds of the Disclosure are compounds of Table 5, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, or different pharmaceutically acceptable salt thereof.
In another embodiment, Compounds of the Disclosure are compounds of Table 6, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, or different pharmaceutically acceptable salt thereof. The chemical names of the compounds of Table 6 are provided in Table 6A.
In another embodiment, Compounds of the Disclosure are compounds of Tables 1, 1A, 3, 3A, 4, 4A, 5, 6 and 6A, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, or different pharmaceutically acceptable salt thereof.
In another embodiment, Compounds of the Disclosure are compounds selected from the group consisting of:
It should be appreciated that the Compounds of the Disclosure in certain embodiments are the free base, various salts, and hydrate forms, and are not limited to the particular salt listed in Tables 1 and 3-6.
In another embodiment, a Compound of the Disclosure is a compound having Formulae I-WM, provided that the compound is not N-(14(4-acetamidophenyl)sulfonyl)piperidin-4-yl)benzamide:
In some embodiments, the disclosure relates to pharmaceutical compositions comprising N-(1-((4-acetamidophenyl)sulfonyl)piperidin-4-yl)benzamide and a pharmaceutically acceptable carrier.
In some embodiments, the disclosure relates to a method of inhibiting SMYD proteins, such as SMYD3 or SMYD2, or both, in a subject, comprising administering to a subject in need thereof an effective amount of N-(1-((4-acetamidophenyl) sulfonyl)piperidin-4-yl)benzamide.
For the purpose of the present disclosure, the terms used in connection with A or A1 have the chemical structures set forth in Table 2, each of which may be optionally substituted with one or more substituents, e.g., 1, 2, 3, 4, or 5 substituents, depending on the nature of the group and the number of available positions. For example, when A or A1 is 2-furanyl there are three carbon atoms for available for substitution. When A or A1 is 2-naphthalenyl there are seven carbon atoms available for substitution. Substitution may occur at arty available carbon or nitrogen atom. Optional substituents include, but are not limited to, halo, hydroxy, alkoxy, amino, akylamino, dialkylamino, (amino)alkyl, (alkylamino)alkyl, (dialkylamino)alkyl, C1-6 alkyl, haloalkyl, hydroxyalkyl, (carboxamido)alkyl, (cycloalkyl)alkyl, optionally substituted C3-12 cycloalkyl, optionally substituted C6-14 aryl, optionally substituted 5- to 14-membered heteroaryl, optionally substituted. 4- to 14-membered heterocyclo, or aralkyl.
For the purpose of the present disclosure, the term “alkyl” as used by itself or as part of another group refers to a straight- or branched-chain aliphatic hydrocarbon containing one to twelve carbon atoms (i.e., C1-12 alkyl) or the number of carbon atoms designated (i.e., a C1 alkyl such as methyl, a C2 alkyl such as ethyl, a C3 alkyl such as propyl or isopropyl, etc.). In one embodiment, the alkyl group is chosen from a straight chain C1-10 alkyl group. In another embodiment, the alkyl group is chosen from a branched chain C3-10 alkyl group. In another embodiment, the alkyl group is chosen from a straight chain C1-6 alkyl group. In another embodiment, the alkyl group is chosen front a branched chain C3-6 alkyl group. In another embodiment, the alkyl group is chosen from a straight chain C1-4 alkyl group. In another embodiment, the alkyl group is chosen from a branched chain C3-4 alkyl group. In another embodiment, the alkyl group is chosen from a straight or branched chain C3-4 alkyl group. In another embodiment, the alkyl group is partially or completely deuterated, i.e., one or more hydrogen atoms of the alkyl group are replaced with deuterium atoms. Non-limiting exemplary C1-10 alkyl groups include methyl (including —CD3), ethyl, propyl, isopropyl, butyl, see-butyl, tert-butyl, iso-butyl,-pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Non-limiting exemplary C1-4 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, and iso-butyl.
For the purpose of the present disclosure, the term “optionally substituted alkyl” as used by itself or as part of another group means that the alkyl as defined above is either unsubstituted or substituted with one, two, or three substituents independently chosen from nitro, haloalkoxy, aryloxy, aralkyloxy, alkylthio, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, ureido, guanidino, carboxy, alkoxycarbonyl, and carboxyalkyl. In one embodiment, the alkyl is a C1-6 alkyl. In another embodiment, the alkyl is a C1-4 alkyl. In one embodiment, the optionally substituted alkyl is substituted with two substituents. In another embodiment, the optionally substituted alkyl is substituted with one substituent. Non-limiting exemplary optionally substituted alkyl groups include —CH2CH2NO2, —CH2CH2CO2H, —CH2CH2SO2CH1, —CH2CH2COPh, and —CH2C6H11.
For the purpose of the present disclosure, the term “alkylenyl” as used herein by itself or part of another group refers to a divalent form of an alkyl group as defined above. In one embodiment, the alkylenyl is a divalent form of a C1-6 alkyl. In one embodiment, the alkylenyl is a divalent form of a C1-4 alkyl, Non-limiting exemplary alkylenyl groups include —CH2CH2—, —CH2CH2CH2—, —CH2CH(CH3)CH2—, and —CH2C(CH3)2CH2—.
For the purpose of the present disclosure, the term “cycloalkyl” as used by itself or as part of another group refers to saturated and partially unsaturated (containing one or two double bonds) cyclic aliphatic hydrocarbons containing one to three rings having from three to twelve carbon atoms (i.e., C3-12 cycloalkyl) or the number of carbons designated. In one embodiment, the cycloalkyl group has two rings. In one embodiment, the cycloalkyl group has one ring. In another embodiment, the cycloalkyl group is chosen from a C3-8 cycloalkyl group. In another embodiment, the cycloalkyl group is chosen from a C3-6 cycloalkyl group. Non-limiting exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decant), adamantyl, cyclohexenyl, spiro[3.3]heptane, and bicyclo[3.3.1]nonane.
For the purpose of the present disclosure, the term “optionally substituted cycloalkyl” as used by itself or as part of another group means that the cycloalkyl as defined above is either unsubstituted or substituted with one, two, or three substituents independently chosen from halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, cycloalkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyl, aralkyloxy, alkylthio, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, ureido, guanidino, carboxy, carboxyalkyl, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl optionally substituted heterocyclo, alkoxyalkyl (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialkylamino)alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (heterocyclo)alkyl, or (heteroaryl)alkyl. In one embodiment, the optionally substituted cycloalkyl is substituted with two substituents. In another embodiment, the optionally substituted cycloalkyl is substituted with one substituent. In one embodiment, the optionally substituted cycloalkyl is an (amino)cycloalkyl. For the purpose of the present disclosure, the term “(amino)cycloalkyl” as used by itself or as part of another group means that the optionally substituted cycloalkyl as defined above is substituted with one amino or alkylamino group, and optionally one or two additional substituents. In one embodiment, the optionally substituted cycloalkyl is an (amino)cyclohexyl. For the purpose of the present disclosure, the term “(amino)cyclohexyl” as used by itself or as part of another group means that the optionally substituted cycloalkyl as defined above is a cyclohexyl group substituted with one amino or alkylamino group, and optionally one or two additional substituents. Non-limiting exemplary optionally substituted cycloalkyl groups include:
Non-limiting exemplary (amino)cycloalkyl groups include:
Non-limiting exemplary (amino)cyclohexyl groups include:
For the purpose of the present disclosure, the term “optionally substituted cyclohexyl” as used by itself or as part of another group means that the optionally substituted cycloalkyl as defined above is an optionally substituted cyclohexyl group.
For the purpose of the present disclosure, the term “cycloalkylenyl” as used herein by itself or part of another group refers to a divalent form of an optionally substituted cycloalkyl group as defined above. In one embodiment, the cycloalkylenyl is a “cyclohexylenyl” The term “cyclohexylenyl” as used herein by itself or part of another group refers to a divalent form of an optionally substituted cyclohexyl group, Non-limiting exemplary cycloalkylenyl groups include:
For the purpose of the present disclosure, the term “1,4-cyclohexylenyl” as used herein by itself or part of another group refers to a cyclohexylenyl as defined above wherein the 1- and 4-positions of the cyclohexyl ring are substituted, Non-limiting exemplary 1,4-cyclohexylenyl groups include:
For the purpose of the present disclosure, the term “(cycloalkylenyl)alkyl” as used herein by itself or part of another group refers to an alkyl group substituted with a divalent form of an optionally substituted cycloalkyl Group. In one embodiment, the cycloalkylenyl is a divalent for of optionally substituted cyclohexyl. In one embodiment, the alkyl is C1-4 alkyl. Non-limiting exemplary (cycloalkylenyl)alkyl groups include:
For the purpose of the present disclosure, the term “cycloalkenyl” as used by itself or part of another group refers to a partially unsaturated cycloalkyl group as defined above. In one embodiment, the cycloalkenyl has one carbon-to-carbon double bond. In another embodiment, the cycloalkenyl group is chosen from a C4-8 cycloalkenyl group. Exemplary cycloalkenyl groups include cyclopentenyl and cyclohexenyl.
For the purpose of the present disclosure, the term “optionally substituted cycloalkenyl” as used by itself or as part of another group means that the cycloalkenyl as defined above is either unsubstituted or substituted with one, two, or three substituents independently chosen from halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, monohydroxyalkyl, dihydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, ureido, guanidino, carboxy, carboxyalkyl, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialkylamino)alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (heterocyclo)alkyl, and (heteroaryl)alkyl. In one embodiment, the optionally substituted cycloalkenyl is substituted with two substituents. In another embodiment, the optionally substituted cycloalkenyl is substituted with one substituent. In another embodiment, the cycloalkenyl is unsubstituted.
For the purpose of the present disclosure, the term “alkenyl” as used by itself or as part of another group refers to an alkyl group as defined above containing one, two or three carbon-to-carbon double bonds. In one embodiment, the alkenyl group is chosen from a C296 alkenyl group. In another embodiment, the alkenyl group is chosen from a C2-4 alkenyl group. Non-limiting exemplary alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, sec-butenyl, pentenyl, and hexenyl.
For the purpose of the present disclosure, the term “optionally substituted alkenyl” as used herein by itself or as part of another group means the alkenyl as defined above is either unsubstituted or substituted with one, two or three substituents independently chosen from halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, ureido, guanidino, carboxy, carboxyalkyl, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted heterocycle.
For the purpose of the present disclosure, the term “alkynyl” as used by itself or as part of another group refers to an alkyl group as defined above containing one to three carbon-to-carbon triple bonds. In one embodiment, the alkynyl has one carbon-to-carbon triple bond. In one embodiment, the alkynyl group is chosen from a C2-6 alkynyl group. In another embodiment, the alkynyl group is chosen from a C2-4 alkynyl group. Non-limiting exemplary alkynyl groups include ethynyl, propynyl, butynyl, 2-butynyl, pentynyl, and hexynyl groups.
For the purpose of the present disclosure, the term “optionally substituted alkynyl” as used herein by itself or as part of another group means the alkynyl as defined above is either unsubstituted or substituted with one, two or three substituents independently chosen from halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, ureido, guanidino, ear boxy, carboxyalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclo.
For the purpose of the present disclosure, the term “haloalkyl” as used by itself or as part of another group refers to an alkyl group substituted by one or more fluorine, chlorine, bromine and/or iodine atoms, in one embodiment, the alkyl group is substituted by one, two, or three fluorine and/or chlorine atoms: In another embodiment, the haloalkyl group is chosen from a C1-4 haloalkyl group. Non-limiting exemplary haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3,3,3-trifluoropropyl, 4,4,4-trifluorobutyl, and trichloromethyl groups.
For the purpose of the present disclosure, the term “fluoroalkyl” as used by itself or as part of another group refers to an alkyl group substituted by one or more fluorine atoms. In one embodiment, the alkyl group is substituted by one, two, or three fluorine atoms. In another embodiment, the fluoroalkyl group is chosen from a C1-4 fluoroalkyl group. Non-limiting exemplary fluoroalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1-difluorethyl, 2,2-di fluoroethyl, 2,2,2-trifluoroethyl, 3,3,3-trifluoropropyl, and 4,4,4-trifluorobutyl.
For the purpose of the present disclosure, the term “hydroxyalkyl” as used by itself or as part of another group refers to an alkyl group substituted with one or more, e.g., one, two, or three, hydroxy groups. In one embodiment, the hydroxyalkyl group is a monohydroxyalkyl group, i.e., substituted with one hydroxy group. In another embodiment, the hydroxyalkyl group is a di hydroxyalkyl group, i.e., substituted with two hydroxy groups. In another embodiment, the hydroxyalkyl group is chosen from a C1-4 hydroxyalkyl group. Non-limiting exemplary hydroxyalkyl groups include hydroxymethyl, hydroxyethyl, hydroxypropyl and hydroxybutyl groups, such as 1-hydroxyethyl, 2-hydroxyethyl, 1,2-dihydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 3-hydroxybutyl, 4-hydroxybutyl, 2-hydroxy-1-methylpropyl, and 1,3-dihydroxyprop-2-yl.
For the purpose of the present disclosure, the term “alkoxy” as used by itself or as part of another group refers to an optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl or optionally substituted alkynyl attached to a terminal oxygen atom. In one embodiment, the alkoxy group is chosen from a C1-4 alkoxy group. In another embodiment, the alkoxy group is chosen from a C1-4 alkyl attached to a terminal oxygen atone, e.g., methoxy, ethoxy, tert-butoxy, —OCH2C≡CH, —OCH2C≡CCH3, and —OCH2CH2CH2—C≡CH.
For the purpose of the present disclosure, the term “alkylthio” as used by itself or as part of another group refers to a sulfur atom substituted by an optionally substituted alkyl group. In one embodiment, the alkylthio group is chosen from a C1-4 alkylthio group. Non-limiting exemplary alkylthio groups include —SCH3, and —SCH2CH3.
For the purpose of the present disclosure, the term “alkoxyalkyl” as used by itself or as part of another group refers to an alkyl group substituted with an alkoxy group. Non-limiting exemplary alkoxyalkyl groups include methoxymethyl, methoxyethyl, methoxypropyl, methoxybutyl, ethoxymethyl, ethoxyethy, ethoxypropyl, ethoxybutyl, propoxymethyl, iso-propoxymethyl, propoxyethyl, propoxypropyl, butoxymethyl, tert-butoxymethyl, isobutoxymethyl, sec-butoxymethyl, pentyloxymethyl, —CH2OCH2C≡CH and —CH2OCH2CH2CH2C≡CH.
For the purpose of the present disclosure, the term. “haloalkoxy” as used by itself or as part of another group refers to a haloalkyl attached to a terminal oxygen atom. Non-limiting exemplary haloalkoxy groups include fluoromethoxy, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.
For the purpose of the present disclosure, the term “heteroalkyl” as used by itself or part of another group refers to a stable straight or branched chain hydrocarbon radical containing 1 to 10 carbon atoms and at least two heteroatoms, which can be the same or different, selected from O, N, or S, wherein: 1) the nitrogen atom(s) and sulfur atom(s) can optionally be oxidized; and/or 2) the nitrogen atom(s) can optionally be quaternized. The heteroatoms can be placed at any interior position of the heteroalkyl group or at a position at which the heteroalkyl group is attached to the remainder of the molecule. In one embodiment, the heteroalkyl group contains two oxygen atoms. In one embodiment, the heteroalkyl contains one oxygen and one nitrogen atom, e.g., a (hydroxyalkylamino)alkyl group, e.g., —CH2N(CH3)CH2CH2CH2OH. In one embodiment, the heteroalkyl contains two nitrogen atoms. Non-limiting exemplary heteroalkyl groups include —CH7OCH2CH2OCH3, —OCH2CHOCH2CH2OCH3, —CH2NHCH2CH2OCH2, —OCH2CH2NH2, —NHCH2CH2N(H)CH3, —NHCH2CH2OCH3, —CH2CCH2CH2NH2, —CH2OCH2CH2N(H)CH2CH3, and —OCH2CH2OCH3.
For the purpose of the present disclosure, the term “aryl” as used by itself or as part of another group refers to a monocyclic or bicyclic aromatic ring system having from six to fourteen carbon atoms (i.e., C6-14 aryl). Non-limiting exemplary aryl groups include phenyl (abbreviated as “Ph”), naphthyl, phenanthryl, anthracyl, indenyl, azulenyl, biphenyl, biphenylenyl, and fluorenyl groups. In one embodiment, the aryl group is chosen from phenyl or naphthyl. In one embodiment, the aryl group is phenyl.
For the purpose of the present disclosure, the term “optionally substituted aryl” as used herein by itself or as part of another group means that the aryl as defined above is either unsubstituted or substituted with one to five substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, aralkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyl, aralkyloxy, (aralkyloxy)alkyl, alkylthio, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, ureido, guanidino, carboxy, carboxyalkyl, heteroalkyl, optionally substituted alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, haloalkoxy)alkyl, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialkylamino)alkyl, (cyano)alkyl, (carboxamido)alkyl, (carboxamido)alkyl-O—, mercaptoalkyl, (heterocyclo)alkyl, (heterocyclo)alkyl-O—, (cycloalkylamino)alkyl, (hydroxyalkylamino)alkyl, (amino)(heteroaryl)alkyl, (heterocycloamino)alkyl (amino)(hydroxy)alkyl, (heteroaryl)alkyl, (heteroaryl)alkyl-O—, —N(R43)(R44), —CH2N(R43)(R44), —CH2N(H)C(═O)—R45, and —N(H)C(═O)—R45, wherein R43 is hydrogen, C1-4alkyl, optionally substituted aryl, or optionally substituted heteroaryl; R44 is alkoxyalkyl, (heterocyclo)alkyl, (amino)alkyl, (alkylamino)alkyl, aralkyl, or (dialkylamino)alkyl; and R45 is alkyl, alkoxyalkyl, (heterocyclo)alkyl, (amino)alkyl, (alkylamino)alkyl, (dialkylamino)alkyl, optionally substituted aryl, optionally substituted heteroaryl, aralkyl, or (heteroaryl)alkyl. In another embodiment, the optionally substituted aryl is substituted with one to five substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyl, aralkyloxy, (aralkyloxy)alkyl, alkylthio, carboxamido, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, ureido, guanidino, carboxy, carboxyalkyl, heteroalkyl, optionally substituted alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, (C1-4 haloalkoxy)alkyl, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialkylamino)alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (heterocyclo)alkyl, (cycloalkylamino)alkyl, (hydroxyalkylamino)alkyl, (amino)(heteroaryl)alkyl, (heterocycloamino)alkyl (amino)(hydroxy)alkyl, (heteroaryl)alkyl, —N(R43)(R44), —CH2N(H)C(═O)—R45, and —N(H)C(═O)—R45.
In one embodiment, the optionally substituted aryl is an optionally substituted phenyl. In one embodiment, the optionally substituted phenyl has four substituents. In another embodiment, the optionally substituted phenyl has three substituents. In another embodiment, the optionally substituted phenyl has two substituents. In another embodiment, the optionally substituted phenyl has one substituent. In another embodiment, the optionally substituted phenyl has at least one amino, alkylamino, dialkylamino, (amino)alkyl, (alkylamino)alkyl, (dialkylamino)alkyl, (amino)(heteroaryl)alkyl, or (amino)(hydroxy)alkyl substituent. Non-limiting exemplary substituted aryl groups include 2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 3-fluorophenyl, 3-chlorophenyl, 4-methylphenyl, 4-ethylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 2,6-di-fluoroethenyl, 2,6-di-chlorophenyl, 2-methyl, 3-methoxyphenyl, 2-ethyl, 3-methoxyphenyl, 3,4-di-methoxyphertyl, 3,5-di-fluorophertyl triethylphenyl, 3,5-dimethoxy, 4-methylphenyl, 2-fluoro-3-chlorophenyl, 3-chloro-4-fluorophenyl, and 2-phenylpropan-2-amine. The term optionally substituted aryl is meant to include aryl groups having fused optionally substituted cycloalkyl and fused optionally substituted heterocyclo rings. Examples include:
For the purpose of the present disclosure, the term “arylenyl” as used herein by itself or part of another group refers to a divalent form of an optionally substituted aryl group as defined above. In one embodiment, the arylenyl is a divalent form of an optionally substituted phenyl. In one embodiment, the arylenyl is a divalent form of phenyl. Non-limiting exemplary alkylenyl groups include:
For the purpose of the present disclosure, the term “aryloxy” as used by itself or as part of another group refers to an optionally substituted aryl attached to a terminal oxygen atom. A non-limiting exemplary aryloxy group is PhO—.
For the purpose of the present disclosure, the term “heteroaryloxy” as used by itself or as part of another group refers to an optionally substituted heteroaryl attached to a terminal oxygen atom.
For the purpose of the present disclosure, the term “aralkyloxy” or “arylalkyloxy” as used by itself or as part of another group refers to an aralkyl group attached to a terminal oxygen atom. A iron-limiting exemplary aralkyloxy group is PhCH2O—.
For the purpose of the present disclosure, the term “(aralkyloxy)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with an aralkyloxy group. In one embodiment, the alkyl is a C1-4 alkyl. Non-limiting exemplary “(aralkyloxy)alkyl” groups include —CH2OCH2(3-F-Ph) and —CH2OCH2CH2CH2(2-OMe-Ph).
For the purpose of the present disclosure, the term “heteroaryl” or “heteroaromatic” refers to monocyclic and bicyclic aromatic ring systems having 5 to 14 ling atoms (i.e., a 5- to 14-membered heteroaryl) and 1, 2, 3, or 4 heteroatoms independently chosen from oxygen, nitrogen, or sulfur. In one embodiment, the heteroaryl has three heteroatoms. In another embodiment, the heteroaryl has two heteroatoms. In another embodiment, the heteroaryl has one heteroatom. In one embodiment, the heteroaryl has 5 ring atoms, e.g.; thienyl, a 5-membered heteroaryl having four carbon atoms and one sulfur atom. In another embodiment, the heteroaryl has 6 ring atoms, e.g., pyridyl, a 6-membered heteroaryl having five carbon atoms and one nitrogen atom. Von-limiting exemplary heteroaryl groups include thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, benzofuryl, pyranyl, isobenzofuranyl, benzooxazonyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolyl phthalazinyl, naphthyridinyl, cinnolinyl, quinazolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenoxazinyl, thiazolyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl, and phenoxazinyl. In one embodiment, the heteroaryl is chosen from thienyl (e.g., thien-2-yl and thien-3-yl), furyl (e.g., 2-furyl and 3-furyl), pyrrolyl (e.g., 1H-pyrrol-2-yl and 1H-pyrrol-3-yl), imidazolyl (e.g., 2H-imidazol-2-yl and 2H-imidazol-4-yl), pyrazolyl (e.g., 1H-pyrazol-3-yl, lii-pyrazol-4-yl, and 1H-pyrazol-5-yl), pyridyl (e.g., pyridin-2-yl, pyridin-3-yl, and pyridin-4-yl), pyrimidinyl pyrimidin-2-yl, pyrimidin-4-yl, and pyrimidin-5-yl), thiazolyl (e.g., thiazol-2-yl, thiazol-1-yl, and thiazol-5-yl), isothiazolyl (e.g., isothiazol-3-yl, isothiazol-4-yl, and isothiazol-5-0, oxazolyl (e.g., oxazol-2-yl, oxazol-4-yl, and oxazol-5-yl) and isoxazolyl (e.g., isoxazol-3-yl, isoxazol-4-yl, and isoxazol-5-yl). The term “heteroaryl” is also meant to include possible N-oxides. Exemplary N-oxides include pyridyl N-oxide.
For the purpose of the present disclosure, the term “optionally substituted heteroaryl” as used by itself or as part of another group means that the heteroaryl as defined above is either unsubstituted or substituted with one to four substituents, e.g., one or two substituents, independently chosen from halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aralkyl, aryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, ureido, guanidino, carboxy, carboxyalkyl, alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclo, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialkylamino)alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (heterocyclo)alkyl, (heteroaryl)alkyl, —N(R43)(R44), or —N(H)C(═O)—R45, wherein R43 is hydrogen or C1-4 alkyl, R44 is alkoxyalkyl, (heterocyclo)alkyl, (amino)alkyl, (alkylamino)alkyl, or (dialkylamino)alkyl; and R43 is alkyl, optionally substituted aryl, or optionally substituted heteroaryl. In one embodiment, the optionally substituted heteroaryl has one substituent. In one embodiment, the substituent is amino, alkylamino, dialkylamino, (amino)alkyl, hydroxyalkyl amino, (alkylimino)alkyl, (dialkylamino)alkyl, (heterocyclo)alkyl, —N(R43)(R44), or —N(H)C(═O)—R45. In another embodiment, the substituent is aralkyl or (heteroaryl)alkyl. Examples include:
In one embodiment, the optionally substituted is an optionally substituted pyridyl, i.e., 2-, 3-, or 4-pyridyl. Any available carbon or nitrogen atom can be substituted. The term optionally substituted heteroaryl is meant to include heteroaryl groups having fused optionally substituted cycloalkyl and fused optionally substituted heterocyclo rings. Examples include:
For the purpose of the present disclosure, the term “heteroarylenyl” as used herein by itself or part of another group refers to a divalent form of an optionally substituted heteroaryl group as defined above. In one embodiment, the heteroarylenyl is a divalent form of an optionally substituted pyridyl. Non-limiting exemplary heteroarylenyl groups include:
For the purpose of the present disclosure, the term “heterocycle” or “heterocyclo” as used by itself or as part of another group refers to saturated and partially unsaturated (e.g., containing one or two double bonds) cyclic groups containing one, two, or three rings having front three to fourteen ring members (i.e., a 3- to 14-membered heterocyclo) and at least one heteroatom. Each heteroatom is independently selected from the group consisting of oxygen, sulfur, including sulfoxide and sulfuric, and/or nitrogen atoms, which can be quaternized. The term “heterocyclo” is meant to include cyclic ureido groups such as imidazolidinyl 2 one, cyclic amide groups such as β-lactam, γ-lactam, δ-lactam and ε-lactam, and cyclic carbamate groups such as oxazolidinyl-2-one. The term “heterocyclo” is also meant to include groups having fused optionally substituted aryl groups, e.g., indolinyl, indolinyl-2-one, benzo[d]oxazolyl-2(3H)one. In one embodiment, the heterocyclo group is chosen from a 4-, 5-, 6-, 7- or 8-membered cyclic group containing one ring and one or two oxygen and/or nitrogen atoms. In one embodiment, the heterocyclo group is chosen from a 5- or 6-membered cyclic group containing one ring and one or two nitrogen atoms. In one embodiment, the heterocyclo group is chosen from a 8-, 9-, 10-, 11-, or 12-membered cyclic group containing two rings and one or two nitrogen atoms. The heterocyclo can be optionally linked to the rest of the molecule through a carbon or nitrogen atom. Non-limiting exemplary heterocyclo groups include 2-oxopyrrolidin-3-yl, 2-imidazolidinone, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, 8-azabicyclo[3.2.1]octane (nortropane), 6-azaspiro[2,5]octane, 5-azaspiro[3,4]octane, indolinyl, indolinyl-2-one, 1,3-dihydro-2H-benzo[d]imidazol-2-one.
For the purpose of the present disclosure, the term “optionally substituted heterocyclo” as used herein by itself or part of another group means the heterocyclo as defined above is either unsubstituted or substituted with one to four substituents independently selected from halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, aralkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyl, aralkyloxy, alkylthio, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, ureido, guanidino, carboxy, carboxyalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclo, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialkylamino)alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (heterocyclo)alkyl, and (heteroaryl)alkyl. Substitution may occur on any available carbon or nitrogen atom, and may form a spirocycle. In another embodiment, the optionally substituted heterocyclo is substituted with one to four substituents independently selected from halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyl, aralkyloxy, alkylthio, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, ureido, guanidino, carboxy, carboxyalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclo, alkoxyalkyl, (amino)alkyl, hydroxyalkylamino, (alkylamino)alkyl, (dialkylamino)alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (heterocyclo)alkyl, and (heteroaryl)alkyl. In one embodiment, the optionally substituted heterocyclo is substituted with at least one amino, alkylamino, or dialkylamino group. Non-limiting exemplary optionally substituted heterocyclo groups include:
For the purpose of the present disclosure, the term “heterocyclenyl” as used herein by itself or part of another group refers to a divalent form of an optionally substituted heterocyclo group as defined above. In one embodiment, the heterocyclenyl is a divalent form of an optionally substituted azetidine. In one embodiment, the heterocyclenyl is a divalent form of an optionally substituted piperidinyl. Non-limiting exemplary heterocyclenyl groups include:
For the purpose of the present disclosure, the term “optionally substituted pyrrolidinyl” as used by itself or as part of another group means that the optionally substituted heterocyclo as defined above is an optionally substituted pyrrolidinyl group.
For the purpose of the present disclosure, the term “optionally substituted pyrrolidinenyl” as used herein by itself or part of another group refers to a divalent form of an optionally substituted pyrrolidinyl group as defined above. Non-limiting exemplary optionally substituted pyrrolidinenyl groups include:
For the purpose of the present disclosure, the term “amino” as used by itself or as part of another group refers to —NH2.
For the purpose of the present disclosure, the term “alkylamino” as used by itself or as part of another group refers to —NHR22 wherein R22 is C1-6 alkyl. In one embodiment, R22 is C alkyl. Non-limiting exemplary alkylamino groups include —N(H)CH3 and —N(H)CH2CH3.
For the purpose of the present disclosure, the term “dialkylamino” as used by itself or as part of another group refers to —NR23aR23b, wherein R23a and R23b are each independently C1-6 alkyl. In one embodiment, R23a and R23b are each independently C1-4 alkyl. Non-limiting exemplary dialkylamino groups include —N(CH3)2 and —N(CH3)CH2CH(CH3)2.
For the purpose of the present disclosure, the term “hydroxyalkylamino” as used by itself or as part of another group refers to —NR24aR24b, wherein R24a is hydrogen or C1-4 alkyl, and R24b is hydroxyalkyl. Non-limiting exemplary hydroxyalkylamino groups include —N(H)CH2CH2OH, —N(H)CH2CH2CH2OH, —N(CH)CH2CH2OH, and —N(CH3)CH2CH2CH2OH.
For the purpose of the present disclosure, the term “(hydroxyalkylamino)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with an hydroxyalkylamino group. In one embodiment, the alkyl is a C1-4 alkyl. A non-limiting exemplary (hydroxyalkylamino)alkyl group is —CH2N(CH3)CH2CH2CH2OH.
For the purpose of the present disclosure, the term “cycloalkylamino” as used by itself or as part of another group refers to —NR25aR25b, wherein R25a is optionally substituted cycloalkyl and R25b is hydrogen or C1-4 alkyl.
For the purpose of the present disclosure, the term “heterocycloamino” as used by itself or as part of another group refers to —NR25cR25d, wherein R25c is optionally substituted heterocyclo and R25d is hydrogen or C1-4 alkyl. Non-limiting exemplary heterocycloamino groups include:
For the purpose of the present disclosure, the term “(heterocycloamino)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with an heterocycloamino group. In one embodiment, the alkyl is a C1-4 alkyl.
For the purpose of the present disclosure, the term “aralkylamino” as used by itself or as part of another group refers to —NR26aR26b, wherein R26a is aralkyl and R26b is hydrogen or C1-4 alkyl. Non-limiting exemplary aralkylamino groups include —N(H)CH2Ph and —N(CH3)CH2Ph.
For the purpose of the present disclosure, the term “(amino)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with an amino group. In one embodiment, the alkyl is a C1-4 alkyl, Non-limiting exemplary (amino)alkyl groups include —CH2NH2, —C(NH2)(H)CH3, —CH2CH2NH2, —CH2C(NH2)(H)CH3, —CH2CH2CH2NH2, —CH2CH2CH2CH2NH2, and —CH2C(CH3)2CH2NH2.
For the purpose of the present disclosure, the terra “(alkylamino)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with an alkylamino group. In one embodiment, the alkyl is a C1-4 alkyl. A non-limiting exemplary (alkylamino)alkyl group is —CH2CH2N(H)CH3.
For the purpose of the present: disclosure, the term “(dialkylamino)alkyl” as used by itself or as part of another group refers to an alkyl group substituted by a dialkylamino group. In one embodiment, the alkyl is a C1-4 alkyl. Non-limiting exemplary (dialkylamino)alkyl groups are —CH2CH2N(CH3)2.
For the purpose of the present disclosure, the term “(cycloalkylamino)alkyl” as used by itself or as part of another group refers to an alkyl group substituted by a cycloalkylamino group. In one embodiment, the alkyl is a C1-4 alkyl, Non-limiting exemplary (cycloalkylamino)alkyl groups include —CH2N(H)cyclopropyl, —CH2N(H)cyclobutyl, and —CH2N(H)cyclohexyl.
For the purpose of the present disclosure, the term “(aralkylamino)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with an aralkylamino group. In one embodiment, the alkyl is a C1-4 alkyl. A non-limiting exemplary (aralkylamino)alkyl group is —CH2CH2CH2N(H)CH2Ph.
For the purpose of the present disclosure, the term “(hydroxyalkylamino)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with an hydroxyalkylamino group. A non-limiting exemplary (hydroxyalkylamino)alkyl group is —CH2CH2NHCH2CH2OH.
For the purpose of the present disclosure, the term “(cyano)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with one or more cyano, e.g., —CN, groups. In one embodiment, the alkyl is a C1-4 alkyl. Non-limiting exemplary (cyano)alkyl groups include —CH2CH2CN, —CH2CH2CH2CN, and —CH2CH2CH2CH2CN.
For the purpose of the present disclosure, the term “(amino)(hydroxy)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with one amino, alkylamino, dialkylamino, car heterocyclo group and one hydroxy group. In one embodiment, the alkyl is a C1-6 alkyl. In another embodiment, the alkyl is a C1-4 alkyl. Non-limiting exemplary (amino)(hydroxy)alkyl groups include:
For the purpose of the present disclosure, the term “(amino)(carboxamido)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with one amino, alkylamino, or dialkylamino, and one carboxamido group. In one embodiment, the alkyl is a Cd alkyl. Non-limiting exemplary (amino)(carboxamido)alkyl groups include:
For the purpose of the present disclosure, the term “(amino)(aryl)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with one amino, alkylamino, or dialkylamino group and one optionally substituted aryl group. In one embodiment, the alkyl is a C1-6 alkyl. In one embodiment, the optionally substituted aryl group is an optionally substituted phenyl. Non-limiting exemplary (amino)(aryl)alkyl groups include:
For the purpose of the present disclosure, the term “(amino)(heteroaryl)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with one amino, alkylamino, or dialkylamino group and one optionally substituted heteroaryl group. In one embodiment, the alkyl is a C1-6 alkyl. In one embodiment, the alkyl is a C1-4 alkyl. In one embodiment, the optionally substituted heteroaryl group is an optionally substituted pyridyl. Non-limiting exemplary (amino)(heteroaryl)alkyl groups include:
For the purpose of the present disclosure, the term “(cycloalkyl)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with one optionally substituted cycloalkyl group. In one embodiment, the alkyl is a C1-4 alkyl. In one embodiment, the cycloalkyl is a C3-6 cycloalkyl. In one embodiment, the optionally substituted cycloalkyl group is substituted with an amino or (amino)alkyl group. Non-limiting exemplary (cycloalkyl)alkyl groups include:
For the purpose of the present disclosure, the term “(hydroxy)(aryl)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with one hydroxy group and one optionally substituted aryl group. In one embodiment, the alkyl is a C1-6 alkyl. In one embodiment, the optionally substituted aryl group is an optionally substituted phenyl, Non-limiting exemplary (hydroxy)(aryl)alkyl groups include:
For the purpose of the present disclosure, the term “carboxamido” as used by itself or as part of another group refers to a radical of formula —C(═O)NR26aR26b, wherein R26a and R26b are each independently hydrogen, optionally substituted alkyl, optionally substituted aryl, aralkyl, (heteroaryl)alkyl, or optionally substituted heteroaryl, or R26a and R26b taken together with the nitrogen to which they are attached from a 3- to 8-membered heterocyclo group. In one embodiment, R26a and R26b are each independently hydrogen or optionally substituted alkyl. Non-limiting exemplary carboxamido groups include —CONH2, —CON(H)CH3, −CON(CH3)2, and —CON(H)Ph.
For the purpose of the present disclosure, the term “(carboxamido)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with a carboxamido group, Non-limiting exemplary (carboxamido)alkyl groups include —CH2CONH2, —C(H)CH3—CONH2, and —CH2CON(H)CH3.
For the purpose of the present disclosure, the term “sulfonamido” as used by itself or as part of another group refers to a radical of the formula —SO2NR27aR27b, wherein R27a and R27b are each independently hydrogen, optionally substituted alkyl, or optionally substituted aryl, or R21a and R27b taken together with the nitrogen to which they are attached from a 3- to 8-membered heterocyclo group. Non-limiting exemplary sulfonamido groups include —SO2NH2, —SO2N(H)CH3, and —SO2N(H)Ph.
For the purpose of the present disclosure, the term “alkylcarbonyl” as used by itself or as part of another group refers to a carbonyl group, i.e., —C(═O)—, substituted by an alkyl group. A non-limiting exemplary alkylcarbonyl group is —COCH.
For the purpose of the present disclosure, the term “arylcarbonyl” as used by itself or as part of another group refers to a carbonyl group, i.e., —C(═O)—, substituted by an optionally substituted aryl group, A non-limiting exemplary arylcarbonyl group is —COPh.
For the purpose of the present disclosure, the term “alkylsulfonyl” as used by itself or as part of another group refers to a sulfonyl group, i.e., —SO2—, substituted by any of the above-mentioned optionally substituted alkyl groups. A non-limiting exemplary alkylsulfonyl group is —SO2CH3.
For the purpose of the present disclosure, the term “arylsulfonyl” as used by itself or as part of another group refers to a sulfonyl group, i.e., —SO2—, substituted by any of the above-mentioned optionally substituted aryl groups. A non-limiting exemplary arylsulfonyl group is —SO2Ph.
For the purpose of the present disclosure, the term “mercaptoalkyl” as used by itself or as part of another group refers to any of the above-mentioned alkyl groups substituted by a —SH group.
For the purpose of the present disclosure, the term “carboxy” as used by itself or as part of another group refers to a radical of the formula —COOH.
For the purpose of the present disclosure, the term “carboxyalkyl” as used by itself or as part of another group refers to any of the above-mentioned alkyl groups substituted with a —COOH. Anon-limiting exemplary carboxyalkyl group is —CH2CO2H.
For the purpose of the present disclosure, the term “alkoxycarbonyl” as used by itself or as part of another group refers to a carbonyl group, i.e., —C(═O)—, substituted by an alkoxy group. Non-limiting exemplary alkoxycarbonyl groups are —CO2Me and —CO2Et.
For the purpose of the present disclosure, the term “aralkyl” or “arylalkyl” as used by itself or as part of another group refers to art alkyl group substituted with one, two, or three optionally substituted aryl groups. In one embodiment, the aralkyl group is a C1-4alkyl substituted with ore optionally substituted aryl group. In another embodiment, the aralkyl group is a C1 or C2 alkyl substituted with one optionally substituted phenyl group. In another embodiment, the aralkyl group is a C1 alkyl substituted with one optionally substituted phenyl group, i.e., a benzyl group wherein the phenyl is optionally substituted. Non-limiting exemplary aralkyl groups include benzyl, phenethyl, —CHPh2, —CH2(4-OH-Ph), and —CH(4-F-Ph)2.
For the purpose of the present disclosure, the term “ureido” as used by itself or as part of another group refers to a radical of the formula —NR30a—C(═O)—NR30bR30c, wherein R22a is hydrogen, alkyl, or optionally substituted aryl, and R30b and R30c are each independently hydrogen, alkyl, or optionally substituted aryl, or R30b and R30c taken together with the nitrogen to which they are attached form a 4- to 8-membered heterocyclo group. Non-limiting exemplary ureido groups include —NH—C(C═O)—NH2 and —NH—C(C═O)—NHCH3.
For the purpose of the present disclosure, the term “guanidino” as used by itself or as part of another group refers to a radical of the formula —NR28a—C(═NR29)—NR28bR28c, wherein R28a, R28b, and R28c are each independently hydrogen, alkyl, or optionally substituted aryl, and R29 is hydrogen, alkyl, cyano, alkylsulfonyl, alkylcarbonyl, carboxamido, or sulfonamido. Non-limiting exemplary guanidino groups include —NH—C(C═NH)—NH2, —NH—C(C═NCN)—NH2, and —NH—C(C═NH)—NHCH3.
For the purpose of the present disclosure, the term “(heterocyclo)alkyl” as used by itself or as part of another group refers to an alkyl group substituted with one, two, or three optionally substituted heterocyclo groups. In one embodiment, the (heterocyclo)alkyl is a C1-4 alkyl substituted with one optionally substituted heterocyclo group. The heterocyclo can be linked to the alkyl group through a carbon or nitrogen atom. Non-limiting exemplary (heterocycle)alkyl groups include:
For the purpose of the present disclosure, the term “(heteroaryl)alkyl” or “heteroaralkyl” as used by itself or as part of another group refers to an alkyl group substituted with one, two, or three optionally substituted heteroaryl groups. In one embodiment, the (heteroaryl)alkyl group is a C1-4 alkyl substituted, with one optionally substituted heteroaryl group, Non-limiting exemplary (heteroaryl)alkyl groups include:
For the purpose of the present disclosure, the term “alkylcarbonylamino” as used by itself or as part of another group refers to an alkylcarbonyl group attached to an amino A non-limiting exemplary alkylcarbonylamino group is —NHCOCH3.
The present disclosure encompasses any of the Compounds of the Disclosure being isotopically-labelled (i.e., radiolabeled) by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2H (or deuterium (D)), 3H, 11C, 13C, 14C, 15N, 18O, 17O, 31P, 32P, 35S, 18F, and 36Cl, respectively, e.g., 3H, 11C, and 14C, In one embodiment, provided is a composition wherein substantially all of the atoms at a position within the Compound of the Disclosure are replaced by an atom having a different atomic mass or mass number. In another embodiment, provided is a composition wherein a portion of the atoms at a position within the Compound of the disclosure are replaced, i.e., the Compound of the Disclosure is enriched at a position with an atom having a different atomic mass or mass number.” Isotopically-labelled Compounds of the Disclosure can be prepared by methods known in the art.
Compounds of the Disclosure may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. The present disclosure is meant to encompass the use of all such possible forms, as well as their racemic and resolved forms and mixtures thereof. The individual enantiomers can be separated according to methods known in the an in view of the present disclosure. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that they include both F and Z geometric isomers. All tautomers are intended to be encompassed by the present disclosure as well.
As used herein, the term “stereoisomers” is a general term for all isomers of individual molecules that differ only in the orientation of their atoms in space. It includes enantiomers and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereomers).
The term “chiral center” or “asymmetric carbon atom” refers to a carbon atom to which four different groups are attached.
The terms “enantiomer” and “enantiomeric” refer to a molecule that cannot be superimposed on its mirror image and hence is optically active wherein the enantiomer rotates the plane of polarized light in one direction and its mirror image compound rotates the plane of polarized light, in the opposite direction.
The term “racemic” refers to a mixture of equal parts of enantiomers and which mixture is optically inactive.
The term “absolute configuration” refers to the spatial arrangement of the atoms of a chiral molecular entity (or group) and its stereochemical description, e.g., R or S.
The stereochemical terms and conventions used in the specification are meant to be consistent with those described in Pure & App. Chem 68:2193 (1996), unless otherwise indicated.
The term “enantiomeric excess” or “ee” refers to a measure for how much of one enantiomer is present compared to the other. For a mixture of R and S enantiomers, the percent enantiomeric excess is defined as |R—S|*100, where R and S are the respective mole or weight fractions of enantiomers in a mixture such that R+S=1. With knowledge of the optical rotation of a chiral substance, the percent enantiomeric excess is defined as ([α]obs/[α]max)*100, where [α]obs the optical rotation of the mixture of enantiomers and [α]max is the optical rotation of the pure enantiomer. Determination of enantiomeric excess is possible using a variety of analytical techniques, including NMR spectroscopy, chiral column chromatography or optical polarimetry.
The terms “enantiomerically pure” or “enantiopure” refer to a sample of a chiral substance all of whose molecules (within the hinds of detection) have the same chirality sense.
The terms “enantiomerically enriched” or “enantioenriched” refer to a sample of a chiral substance whose enantiomeric ratio is greater than 50:50. Enantiomerically enriched compounds may be enantiomerically pure.
The terms “a” and “an” refer to one or more.
The term “about,” as used herein, includes the recited number±10%. Thus, “about 10” means 9 to 11.
The present disclosure encompasses the preparation and nee of salts of the Compounds of the Disclosure, including non-toxic pharmaceutically acceptable salts, Examples of pharmaceutically acceptable addition salts include inorganic and organic acid addition salts and basic salts. The pharmaceutically acceptable salts include, but are not limited to, metal salts such as sodium salt, potassium salt, cesium salt and the like; alkaline earth metals such as calcium salt, magnesium salt and the like; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N′-dibenzylethylenediamine salt and the like; inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulphate and the like, organic acid salts such as citrate, lactate, tartrate, maleate, fumarate, mandelate, acetate, dichloroacetate, trifluoroacetate, oxalate, formate and the like; sulfonates such as methanesulfonate, benzenesulfonate, p-toluenesulfonate and the like; and amino acid salts such as arginate, asparaginate, glutamate and the like. The term “pharmaceutically acceptable salt” as used herein, refers to any salt, e.g., obtained by reaction with an acid or a base, of a Compound of the Disclosure that is physiologically tolerated in the target patient (e.g., a mammal, e.g., a human).
Acid addition salts can be formed by mixing a solution of the particular Compound of the Disclosure with a solution of a pharmaceutically acceptable non-toxic acid such as hydrochloric acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalic acid, dichloroacetic acid, or the like. Basic salts can be formed by mixing a solution of the compound of the present disclosure with a solution of a pharmaceutically acceptable non-toxic base such as sodium hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate and the like.
The present disclosure encompasses the preparation and use of solvates of Compounds of the Disclosure. Solvates typically do not significantly alter the physiological activity or toxicity of the compounds, and as such may function as pharmacological equivalents. The term “solvate” as used herein is a combination, physical association and/or salvation of a compound of the present disclosure with a solvent molecule such as, e.g. a disolvate, monosulfate or hemisolvate, where the ratio of solvent molecule to compound of the present disclosure is about 2:1, about 1:1 or about 1:2, respectively. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding, in certain instances, the solvate can be isolated, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. Thus, “solvate” encompasses both solution-phase and isolatable solvates. Compounds of the Disclosure can be present as solvated forms with a pharmaceutically acceptable solvent, such as water, methanol, ethanol, and the like, and it is intended that the disclosure includes both solvated and unsolvated forms of Compounds of the Disclosure. One type of solvate is a hydrate. A “hydrate” relates to a particular subgroup of solvates where the solvent molecule is water. Solvates typically can function as pharmacological equivalents. Preparation of solvates is known in the art. See, for example, M. Cairn et al, J. Pharmaceut. Sci., 93(3):601-611 (2004), which describes the preparation of solvates of fluconazole with ethyl acetate and with water. Similar preparation of solvates, hemisolvates, hydrates, and the like are described by E. C. van Tender et al., AAPS Pharm. Sci. Tech., 5(1):Article 12 (2004), and A. I. Bingham et al., Chem. Commun. 603-604 (2001), A typical, non-limiting, process of preparing a solvate would involve dissolving a Compound of the Disclosure in a desired solvent (organic, water, or a mixture thereof) at temperatures above 20° C. to about 25° C., then cooling the solution at a rate sufficient to form crystals, and isolating the crystals by known methods, e.g., filtration. Analytical techniques such as infrared spectroscopy can be used to confirm the presence of the solvent in a crystal of the solvate.
Since Compounds of the Disclosure are inhibitors of SMYD proteins, such as SMYD3 and SMYD2, a number of diseases, conditions, or disorders mediated by SMYD proteins, such as SMYD3 and SMYD2, can be treated by employing these compounds. The present disclosure is thus directed generally to a method the treating a disease, condition, or disorder responsive to the inhibition of SMYD proteins, such as SMYD3 and SMYD2, in an animal suffering from, or at risk of suffering from, the disorder, the method comprising administering to the animal an effective amount of one or more Compounds of the Disclosure.
The present disclosure is further directed to a method of inhibiting SMYD proteins in an animal in need thereof, the method comprising administering to the animal a therapeutically effective amount of at least one Compound of the Disclosure.
The present disclosure is further directed to a method of inhibiting SMYD3 in an animal in need thereof, the method comprising administering to the animal a therapeutically effective amount of at least one Compound of the Disclosure.
The present disclosure is further directed to a method of inhibiting SMYD2 in an animal in need thereof, the method comprising administering to the animal a therapeutically effective amount of at least one Compound of the Disclosure.
As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to eliminating, reducing, or ameliorating a disease or condition, and/or symptoms associated therewith. Although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated. As used herein, the terms “treat,” “treating,” “treatment,” and the like may include “prophylactic treatment,” which refers to reducing the probability of redeveloping a disease or condition, or of a recurrence of a previously-controlled disease or condition, in a subject who does not have, but is at risk of or is susceptible to, redeveloping a disease or condition or a recurrence of the disease or condition. The term “treat” and synonyms contemplate administering a therapeutically effective amount of a Compound of the Disclosure to an individual in need of such treatment.
Within the meaning of the disclosure, “treatment” also includes relapse prophylaxis or phase prophylaxis, as well as the treatment of acute or chronic signs, symptoms and/or malfunctions. The treatment can be orientated symptomatically, for example, to suppress symptoms. It can be effected over a short period, be oriented over a medium term, or can be a long-term treatment, for example within the context of a maintenance therapy.
The term “therapeutically effective amount” or “effective dose” as used herein refers to an amount of the active ingredient(s) that is(are) sufficient, when administered by a method of the disclosure, to efficaciously deliver the active ingredient(s) for the treatment of condition or disease of interest to an individual in need thereof. In the case of a cancer or other proliferation disorder, the therapeutically effective amount of the agent may reduce (i.e., retard to some extent and preferably stop) unwanted cellular proliferation; reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., retard to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., retard to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; modulate protein methylation in the target cells; and/or relieve, to some extent, one or more of the symptoms associated with the cancer. To the extent the administered compound or composition prevents growth and/or kills existing cancer cells, it may be cytostatic and/or cytotoxic.
The term “container” means any receptacle and closure therefore suitable Air storing, shipping, dispensing, and/or handling a pharmaceutical product.
The term “insert” means information accompanying a pharmaceutical product that provides a description of how to administer the product, along with the safety and efficacy data required to allow the physician, pharmacist, and patient to make an informed decision regarding use of the product. The package insert generally is regarded as the “label” for a pharmaceutical product.
The term “disease” or “condition” or “disorder” denotes disturbances and/or anomalies that as a rule are regarded as being pathological conditions or functions, and that can manifest themselves in the form of particular signs, symptoms, and/or malfunctions. As demonstrated below, Compounds of the Disclosure inhibit SMYD proteins, such as SMYD3 and SMYD2 and can be used in treating diseases and conditions such as proliferative diseases, wherein inhibition of SMYD proteins, such as SMYD3 and SMYD2 provides a benefit.
In some embodiments, the Compounds of the Disclosure can be used to treat a “SMYD protein mediated disorder” (e.g., a SMYD3-mediated disorder or a SMYD2-mediated disorder). A SMYD protein mediated disorder is any pathological condition in which a SMYD protein is know to play a role. In some embodiments, a SMYD-mediated disorder is a proliferative disease.
In some embodiments inhibiting SMYD proteins, such as SMYD3 and SMYD2, is the inhibition of the activity of one or more activities of SMYD proteins such as SMYD3 and SMYD2. In some embodiments, the activity of the SMYD proteins such as SMYD3 and SMYD2 is the ability of the SMYD protein such as SMYD3 or SMYD2 to transfer a methyl group to a target protein (e.g., histone). It should be appreciated that the activity of the one or more SMYD proteins such as SMYD3 and SMYD2 may be inhibited in vitro or in vivo. Exemplary levels of inhibition of the activity one or more SMYD proteins such as SMYD3 and SMYD2 include at least 10% inhibition, at least 20% inhibition, at least 30% inhibition, at least 40% inhibition, at least 50% inhibition, at least 60% inhibition, at least 70% inhibition, at least 10% inhibition, at least 90% inhibition, and up to 100% inhibition.
The SMYD (SET and MYND domain) family of lysine methyltransferases (KMTs) plays pivotal roles in various cellular processes, including gene expression regulation and DNA damage response. The family of human SMYD proteins consists of SMYD1, SMYD2; SMYD3, SMYD4 and SMYD5. SMYD1, SMYD2, and SMYD3 share a high degree of sequence homology and, with the exception of SMYD5, human SMYD proteins harbor at least one C-terminal tetratrico peptide repeat (TPR) domain. (See e.g., Abu-Farha et al. J. Mol Cell Biol. (2011) 3 (5) 301-308). The SMYD proteins have been found to be linked to various cancers (See e.g., Hamamoto et al, Nat Cell. Biol. 2004, 6: 731-740), Hu et al. Cancer Research 2009, 4067-4072, and Komatsu et al. Carcinogenesis 2009, 301139-1146.)
SMYD3 is a protein methyltransferase toured to be expressed at high levels in a number of different cancers (Hamamoto, R., et al., Nat. Cell Biol., 6(8):731-40 (2004)). SMYD3 likely plays a role in the regulation of gene transcription and signal transduction pathways critical for survival of breast, liver, prostate and lung cancer cell lines (Hamamoto, R., et al., Nat. Cell Biol., 6(8):731-40 (2004); Hamamoto, R., et al., Cancer Sci., 97(2):113-8 (2006); Van Alter, G. S., et al., Epigenetics, 7(4):340-3 (2012); Liu, C., et al., J. Natl. Cancer Inst., 105(22):1119-28 (2013); Mazur, P. K., et al., Nature, 510(7504):283 (2014)).
Genetic knockdown of SMYD3 leads to a decrease in proliferation of a variety of cancer cell lines (Hamamoto, R., et al., Nat. Cell Biol., 6(8):731-40 (2004); Hamamoto, R., et al., Cancer Sci., 97(2):113-8 (2006); Van Allen, G. S., et al., Epigenetics, 7(41:340-3 (2012); Liu, C., et al., J. Natl. Cancer Inst., 105(22):1719.28 (2013); Mazur, P. K., et al., Nature, 510(7504):283.7 (2014)). Several studies employing RNAi-based technologies have shown that ablation of SMYD3 in hepatocellular carcinoma cell lines greatly reduces cell viability and that its pro-survival role is dependent on its catalytic activity (Hamamoto, R., et al., Nat. Cell Biol., 6(8):731-40 (2004); Van Aller, G. S., et al., Epigenetics, 7(4):340-3 (2012)). Moreover, SMYD3 has also been shown to be a critical mediator of transformation resulting from gain of function mutations in the oncogene, KRAS for both pancreatic and lung adenocarcinoma in mouse models. The dependence of KRAS on SMYD3 was also shown to be dependent on its catalytic activity (Mazur, P. K., et al., Nature, 510(7504):283-7 (2014)). SMYD3 function has also been implicated in colorectal cancers and RNAi mediated knockdown of SMYD3 has been shown to impair colorectal cell proliferation, (Peserico et al., Cell Physiol. 2015 Feb. 28. doi: 10.1002/jcp.249; 5. [Epub ahead of print]).
Furthermore, SMYD3 function has also been shown to play a role in immunology and development. For instance, de Almeida reported that SMYD3 plays a role in generation of inducible regulatory T cells (iTreg) cells, in a mouse model of respiratory syncytial virus (RSV) infection, a model in which iTreg cells have a critical role in regulating lung pathogenesis, SMYD3−/− mice demonstrated exacerbation of RSV-induced disease related to enhanced proinflammatory responses and worsened pathogenesis within the lung (de Almeida et al. Mucoscal Immunol. 2015 Feb. 11. doi: 10.1038/mi.2015.4. [Epub ahead of print]). In addition, as to development, Proserpio et al. have shown the importance of SMYD3 in the regulation of skeletal muscle atrophy, (Proserpio et al, Genes Dev. 2013 Jun. 1; 27(11):1299-3121, while Fujii et al. have elucidated the role of SMYD3 in cardiac and skeletal muscle development (Fujii et al. PLoS One, 2011; 6(8):02349).
SMYD2 (SET and MYND domain-containing protein 2) was first characterized as protein that is a member of a sub-family of SET domain containing proteins which catalyze the site-specific transfer of methyl groups onto substrate proteins. SMYD2 was initially shown to have methyltransferase activity towards lysine 36 on historic H3 (H3K36) but has subsequently been shown to have both historic and non-histone methyltransferase activity.
SMYD2 has been implicated in the pathogenesis of multiple cancers. It has been shown to be over-expressed, compared to matched normal samples, in tumors of the breast, cervix, colon, kidney, liver, head and neck, skin, pancreas, ovary, esophagus and prostate, as well as hematologic malignancies such as AML, B- and T-ALL, CLL and MCL, suggesting a role for SMYD2 in the biology of these cancers. More specifically, studies using genetic knock-down of SMYD2 have demonstrated anti-proliferative effects in esophageal squamous cell carcinoma (ESCC), bladder carcinoma and cervical carcinoma cell lines. (See e.g., Komatsu et al., Carcinogenesis 2009, 30, 1139, and Cho et al., Neoplasia. 2012 June; 14(6):476-86). Moreover, high expression of SMYD2 has been shown to be a poor prognostic factor in both ESCC and pediatric ALL. (See e.g., Komatsu et al. Br J Cancer. 2015 Jan. 20; 112(2):357-64, and Sakamoto et al., Leuk Res. 2014 April; 38(4):496-502). Recently, Nguyen et al., have shoe that a small molecule inhibitor of SMYD2 (LLY-507) inhibited the proliferation of several esophageal, liver and breast cancer cell lines in a dose-dependent manner. (Nguyen et al. J Biol Chem. 2015 Mar. 30. pii: jbc.M114.626861. [Epub ahead of prat]).
SMYD2 has also been implicated in immunology. For instance, Xu et al. have shown that SMYD2 is a negative regulator of macrophage activation by suppressing Interleukin-6 and TNF-alpha production. (Xu et al., J Biol Chem. 2015 Feb. 27:290(9):5414-23).
In one aspect, the present disclosure provides a method of treating cancer in a patient comprising administering a therapeutically effective amount of a Compound of the Disclosure. While not being limited to a specific mechanism, in some embodiments, Compounds of the Disclosure can treat cancer by inhibiting SMYD proteins, such as SMYD3 and SMYD2. Examples of treatable cancers include, but are not limited to, adrenal cancer, acinic cell carcinoma, acoustic neuroma, acral lentigious melanoma, acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm, adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressive NK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer, bladder cancer, blastoma, hone cancer, Brenner tumor, Brown tumor, Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma in situ, carcinosarcoma, cartilage tumor, comentoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid plexus papilloma, clear-cell sarcoma of the kidney, craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine gland neoplasm, endodermal sinus tumor, enteropathy-associated T-cell lymphoma, esophageal cancer, fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of the bone, glial tumor, glioblastoma multiforme, glioma, gliomatosis cerebri, glucagonoma, gonadoblastoma, granulosa cell tumor, gynandroblastoma, gallbladder cancer, gastric cancer, hairy cell leukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma, hematological malignancy, hepatoblastoma, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna, lethal midline carcinoma, leukemia, leydig cell tumor, liposarcoma, lung cancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma, acute lymphocytic leukemia, acute myelogeous leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non-small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant triton tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, medullary carcinoma of the breast, medullary thyroid cancer, medulloblastoma, melanoma, meningioma, merkel cell cancer, mesothelioma, metastatic urothelial carcinoma, mixed Mullerian tumor, mucinous tumor, multiple myeloma, muscle tissue neoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, ocular cancer, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic nerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma, pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, preimary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma periotonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, rectal cancer, sarcoma, Schwannomatosis, seminoma Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer, small blue round cell tumors, small cell carcinoma, soft tissue sarcoma, somatostatinoma, soot wart, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma. Sezary's disease, small intestine cancer, squamous carcinoma, stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroid cancer, transitional cell carcinoma, throat cancer, urachal cancer, urogenital cancer, urothelial carcinoma, usual melanoma, uterine cancer, verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms' tumor.
In another embodiment, the cancer is breast, cervix, colon, kidney, liver, head and neck, skin, pancreas, ovary, esophagus, or prostate cancer.
In another embodiment, the cancer is a hematologic malignancy such as acute myeloid leukemia (AML), B- and T-acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or mantle cell lymphoma (AML).
In another embodiment, the cancer is esophageal squamous cell carcinoma (ESCC), bladder carcinoma, or cervical carcinoma.
In another embodiment, the cancer is a leukemia, for example a leukemia selected from acute monocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia and mixed lineage leukemia (MU), In another embodiment the cancer is NUT-midline carcinoma. In another embodiment the cancer is multiple myeloma. In another embodiment the cancer is a lung cancer such as small cell lung cancer (SCLC). In another embodiment the cancer is a neuroblastoma. In another embodiment the cancer is Burkitt's lymphoma. In another embodiment the cancer is cervical cancer. In another embodiment the cancer is esophageal cancer. In another embodiment the cancer is ovarian cancer. In another embodiment the cancer is colorectal cancer. In another embodiment, the cancer is prostate cancer. In another embodiment, the cancer is breast cancer.
In another embodiment, the present disclosure provides a therapeutic method of modulating protein methylation, gene expression, cell proliferation, cell differentiation and/or apoptosis in vivo in the cancers mentioned above by administering a therapeutically effective amount of a Compound of the Disclosure to a subject in need of such therapy.
Compounds of the Disclosure can be administered to a mammal in the form of a raw chemical without any other components present. Compounds of the Disclosure can also be administered to a mammal as part of a pharmaceutical composition containing the compound combined with a suitable pharmaceutically acceptable carrier. Such a carrier can be selected from pharmaceutically acceptable excipients and auxiliaries. The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable vehicle” encompasses any of the standard pharmaceutical carriers, solvents, surfactants, or vehicles. Suitable pharmaceutically acceptable vehicles include aqueous vehicles and nonaqueous vehicles. Standard pharmaceutical carriers and their formulations are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 19th ed. 1995.
Pharmaceutical compositions within the scope of the present disclosure include all compositions where a Compound of the Disclosure is combined with one or more pharmaceutically acceptable carriers. In one embodiment, the Compound of the Disclosure is present in the composition in an amount that is effective to achieve its intended therapeutic purpose. While individual needs may vary, a determination of optimal ranges of effective amounts of each compound is within the skill of the art. Typically, a Compound of the Disclosure can be administered to a mammal, e.g., a human, orally at a dose of from about 0.0025 to about 1500 mg per kg body weight of the mammal, or an equivalent amount of a pharmaceutically acceptable salt or solvate thereof, per day to treat the particular disorder. A useful oral dose of a Compound of the Disclosure administered to a mammal is from about 0.0025 to about 50 mg per kg body weight of the mammal, or an equivalent amount of the pharmaceutically acceptable salt or solvate thereof. For intramuscular injection, the dose is typically about one-half of the oral dose.
A unit oral dose may comprise from about 0.01 rug to about 1 g of the Compound of the Disclosure, e.g., about 0.01 mg to about 500 mg, about 0.01 mg to about 250 mg, about 0.01 mg to about 100 mg, 0.01 mg to about 50 mg, e.g., about 0.1 mg to about 10 mg, of the compound. The unit dose can be administered one or more times daily, e.g., as one or more tablets or capsules, each containing from about 0.01 mg to about 1 g of the compound, or an equivalent amount of a pharmaceutically acceptable salt or solvate thereof.
A pharmaceutical composition of the present disclosure can be administered to any patient that may experience the beneficial effects of a Compound of the Disclosure. Foremost among such patients are mammals, e.g., humans and companion animals, although the disclosure is not intended to be so limited. In one embodiment, the patient is a human.
A pharmaceutical composition of the present disclosure can be administered by any means that achieves its intended purpose. For example, administration can be by the oral, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, intranasal, transmucosal, rectal, intravaginal or buccal route, or by inhalation. The dosage administered and route of administration will vary, depending upon the circumstances of the particular subject, and taking into account such factors as age, gender, health, and weight of the recipient, condition or disorder to be treated, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
In one embodiment, a pharmaceutical composition of the present disclosure can be administered orally. In another embodiment, a pharmaceutical composition of the present disclosure cart be administered orally and is formulated into tablets, dragees, capsules, or an oral liquid preparation. In one embodiment the oral formulation comprises extruded multiparticulates comprising the Compound of the Disclosure.
Alternatively, a pharmaceutical composition of the present disclosure can be administered, rectally, and is formulated in suppositories.
Alternatively, a pharmaceutical composition of the present disclosure can be administered by injection.
Alternatively, a pharmaceutical composition of the present disclosure can be administered transdermally.
Alternatively, a pharmaceutical composition of the present disclosure can be administered by inhalation or by intranasal or transmucosal administration.
Alternatively, a pharmaceutical composition of the present disclosure can be administered by the intravaginal route.
A pharmaceutical composition of the present disclosure can contain from about 0.01 to 99 percent by weight, e.g., from about 0.25 to 75 percent by weight, of a Compound of the Disclosure, e.g., about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% by weight of a Compound of the Disclosure.
A pharmaceutical composition of the present disclosure is manufactured in a manner which itself will be known in view of the instant disclosure, for example, by means of conventional mixing, granulating, dragee-making, dissolving, extrusion, or lyophilizing processes. Thus, pharmaceutical compositions for oral use can be obtained by combining the active compound with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
Suitable excipients include fillers such as saccharides (for example, lactose, sucrose, mannitol or sorbitol), cellulose preparations, calcium phosphates (for example, tricalcium phosphate or calcium hydrogen phosphate), as well as binders such as starch paste (using, for example, maize starch, wheat starch, rice starch, or potato starch), gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxy ethylcellulose, and/or polyvinyl pyrrolidone. If desired, one or more disintegrating agents can be added, such as the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
Auxiliaries are typically flow-regulating agents and lubricants such as, for example, silica, talc, stearic acid or salts thereof (e.g., magnesium stearate or calcium stearate), and, polyethylene glycol. Dragee cores are provided with suitable coatings that are resistant to gastric juices. For this purpose, concentrated saccharide solutions can be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate can be used. Dye stuffs or pigments can be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.
Examples of other pharmaceutical preparations that can be used orally include Rush-fit capsules made of gelatin, or soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain a compound in the form of granules, which can be mixed with tillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers, or in the form of extruded multiparticulates, in soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils or liquid paraffin. In addition, stabilizers can be added.
Possible pharmaceutical preparations for rectal administration include, for example, suppositories, which consist of a combination of one or more active compounds with a suppository base. Suitable suppository bases include natural and synthetic triglycerides, and paraffin hydrocarbons, among others. It is also possible to use gelatin rectal capsules consisting of a combination of active compound with a base material such as, for example, a liquid triglyceride, polyethylene glycol, or paraffin hydrocarbon.
Suitable formulations for parenteral administration include aqueous solutions of the active compound in a water-soluble form such as, for example, a water-soluble salt, alkaline solution, or acidic solution. Alternatively, a suspension of the active compound can be prepared as an oily suspension. Suitable lipophilic solvents or vehicles for such as suspension may include fatty oils (for example, sesame oil), synthetic fatty acid esters (for example, ethyl oleate), triglycerides, or a polyethylene glycol such as polyethylene glycol-400 (PEG-400). An aqueous suspension may contain one or more substances to increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. The suspension may optionally contain stabilizers.
In another embodiment, the present disclosure provides kits which comprise a Compound. of the Disclosure (or a composition comprising a Compound of the Disclosure) packaged in a manner that facilitates their use to practice methods of the present disclosure. In one embodiment, the kit includes a Compound of the Disclosure (or a composition comprising a Compound of the Disclosure) packaged in a container, such as a sealed bottle or vessel, with a label affixed to the container or included in the kit that describes use of the compound or composition to practice the method of the disclosure. In one embodiment, the compound or composition is packaged in a unit dosage form. The kit further can include a device suitable for administering the composition according to the intended route of administration.
Compounds of the Disclosure are prepared using methods known to those skilled in the art in view of this disclosure, or by the illustrative methods shown in the General Schemes below. In the Genera Schemes, A, R12a, R12b, R13a, R13b, and L of Formulae A-D are as defined in connection with Formula VI, unless otherwise indicated. In any of the General Schemes, suitable protecting can be employed in the synthesis, for example, when Z is (amino)alkyl or any other group that may group that may require protection. (See, Wuts, P. G. M.; Greene, T. W., “Green's Protective Groups in Organic Synthesis”, 4th Ed., J. Wiley & Sons, N Y, 2007).
Compound A is converted to compound B (i.e., a compound having Formula VI, wherein X is —S(═O)2—) by coupling with a suitable sulfonyl chloride (Z—SO2Cl) in the presence of a suitable base such as TFA or DIPEA in a suitable solvent such as dichloromethane, acetonitrile, or DMF.
Compound A is converted to compound C (i.e, a compound having Formula VI, wherein X is —C(═O)—) by coupling with a suitable acide chloride (Z—COCl) in the presence of a suitable base such as TFA or DIPEA in a suitable solvent such as dichloromethane, acetonitrile, or DMF, or by coupling with a suitable carboxylic acid (Z—CO2H) in the presence of a suitable coupling reagent such as HATU and a suitable base such as TFA or DIPEA in a suitable solvent such as dichloromethane, acetonitrile, or DMF.
Compound A is converted to compound D (i.e., a compound having Formula VI, wherein X is —C(═O)C(R4)(H)—) by coupling with a suitable carboxylic acid (Z—(C(H)R4CO2H) in the presence of a suitable coupling reagent such as HATU and a suitable base such as TFA or DIPEA in a suitable solvent such as dichloromethane, acetonitrile, or DMF.
General methods and experimental procedures for preparing and characterizing Compounds of the Disclosure are set forth in the general schemes above and the examples below. Wherever needed, reactions were heated using conventional hotplate apparatus or heating mantle or microwave irradiation equipment. Reactions were conducted with or without stirring, under atmospheric or elevated pressure in either open or closed vessels. Reaction progress was monitored using conventional techniques such as TLC, HPLC, UPLC, or LCMS using instrumentation and methods described below, Reactions were quenched and crude compounds isolated using conventional methods as described in the specific examples provided. Solvent removal was carried out with or without heating, under atmospheric or reduced pressure, using either a rotary or centrifugal evaporator.
Compound purification was carried out as needed using a variety of traditional methods including, but not limited to, preparative chromatography under acidic, neutral; or basic conditions using either normal phase or reverse phase HPLC or flash columns or Prep-TLC plates. Compound purity and mass confirmations were conducted using standard HPLC and/or UPLC and/or MS spectrometers and or LCMS and/or GC equipment (i.e., including, but not limited to the following instrumentation: Waters Alliance 2695 with 2996 PDA detector connected with ZQ detector and ESI source; Shimadzu LDMS-2020; Waters Acquity H Class with PDA detector connected with SQ detector and ESI source; Agilent 1100 Series with PDA detector; Waters Alliance 2695 with 2998 PDA detector; AB SCIEX API 2000 with ESI source; Agilent 7890 GC). Exemplified compounds were dissolved in either MeOH or McCN to a concentration of approximately 1 mg/mL and analyzed by injection of 0.5-10) μL into an appropriate LCMS system using the methods provided in the following table. In each case the flow rate is 1 ml/min.
Compound structure confirmations were carded out using standard 300 or 400 MHz NMR spectrometers with nOe's conducted whenever necessary.
The following abbreviations are used herein:
To a solution of 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (20.0 g, 87.23 mmol) in DCM (200 mL) was added HATU (36.46 g, 95.95 mmol), TFA (17.62 g, 174.46 mmol) and N,O-dimethylhydroxylamine (8.93 g, 9159 mmol). The mixture was stirred at 25° C. for 16 h. The mixture was washed with H2O and the DCM layer was evaporated and the residue purified by silica column (PE/EA=1:1) to give the test-butyl 4-(methoxy(methyl)carbamoyl)piperidine-1-carboxylate (21.0 g, yield: 88.5%). 1H NMR (400 MHz, CDCl3): δ=4.27-4.01 (m, 2H), 3.71 (s, 3H), 3.19 (s, 3H), 2.85-2.70 (m, 3H), 1.75-1.62 (m, 4H), 1.46 (s, 9H).
To a solution of tert-butyl 4-(methoxy-(methyl)carbamoyl)piperidine-1-carboxylate (10.0 g, 36.76 mmol) in THF (150 mL) was added MeMgBr (24.5 mL, 73.52 mmol) at −78° C. The mixture was stirred at 25° C. for 16 h. TLC showed the reaction worked well. The mixture was quenched with aq. NH4Cl and H2O (100 mL) was added. The mixture was extracted with DCM (100 mL×3). The DCM layer was dried and evaporated to give the tert-butyri 4-acetylpiperidine-1-carboxylate (7.9 g, yield: 94.7%). 1H NMR (400 MHz, CDCl3): δ=4.10 (br. s., 2H), 2.88-2.70 (m, 2H), 2.46 (tt, J=3.6, 11.5Hz, 1H), 2.17 (s, 3H), 1.84 (d, J=11.5Hz, 2H), 1.58-1.48 (m, 2H), 1.48-1.41 (m, 9H).
To a solution of tert-butyl 4-acetylpiperidine-1-carboxylate (7.9 g, 34.8 mmol) in MeOH (100 mL) was added NH4OAc (10.72 g, 139.2 mmol) and AcOH (1 mL). The mixture was stirred al: 25° C. for 2 h, then was added NaBH3CN (2.63 g, 41.76 mmol). The mixture was stirred at 25° C. for 16 h. The mixture was evaporated and 2N NaOH (50 mL) was added to the residue. The solution was extracted with DCM (100 mL×3). The DCM layer was washed with H2O and brine dried and evaporated to give the tert-butyl 4-(1-aminoethyl)piperidine-1-carboxylate (7.93 g, yield: 100%). 1H NMR (400 MHz, CDCl3): δ=4.15 (br. s., 2H), 2.77-2.57 (m, 3H), 1.76-1.59 (m, 2H), 1.46 (s, 9H), 1.33-10 (m, 5H), 1.06 (d, J=6.5Hz, 3H).
To a solution of tert-butyl 4-(1-aminoethyl)piperidine-1-carboxylate (7.93 g, 34.8 mmol) in THF (80 mL) was added 1C2CO3 (9.6 g, 69.6 mmol) and Cbz-Cl (7.1 g, 41.76 mmol). The mixture was stirred at 25° C. for 16 h. The mixture diluted with H2O and extracted with EA (50 mL×3) and the EA layer evaporated to give tert-butyl 4-(1-(((benzyloxy)carbonyl)amino)ethyl)piperidine-1-carboxylate (10.1 g, yield: 80.2%). 1H NMR (400 MHz, DMSO-d6): δ=7.42-7.23 (m, 5H), 7.16 (d, J=8.8Hz, 1H), 5.00 (s, 2H), 3.94 (d, J=11.8Hz, 2H), 3.45-3.34 (m, 1H), 2.71-2.55 (m, 2H), 1.67-1.42 (m, 3H), 1.38 (s, 9H), 1.13-0.89 (m, 5H).
To a solution of tert-butyl 4-(1-(((benzyloxy)carbonyl)amino)ethyl)piperidine-1-carboxylate (10.1 g, 27.87 mmol) in EA (50 mL) was added HCl/EA (50 mL). The mixture was stirred at 25° C. for 16 h. Solvents were then evaporated to give the benzyl (1-(piperdin-4-yl)ethyl)carbamate as a white solid. (8.3 g, yield: 100%). 1H NMR (400 MHz, DMSO-d6): δ=8.88 (br. s., 1H), 8.50 (d, J=9.5Hz, 1H), 7.42-7.30 (m, 5H), 7.26 (d, J=8.8Hz, 1H), 5.04-4.98 (m, 2H), 3.46-3.41 (m, 1H), 3.24 (d, J=11.8Hz, 2H), 2.84-2.71 (m, 2H), 1.74 (d, J=13.6Hz, 2H), 1.53 (d, J=3.8Hz, 1H), 1.40-1.28 (m, 2H), 1.05-0.99 (m, 3H).
To a solution of benzyl (1-(piperidin-4-yl)ethyl)carbamate (7.0 g, 23.43 mmol) in Devi (100 mL) was added HATU (8.9 g, 23.43 mmol), TFA (4.73 g, 46.86 mmol) and (S)-2-((tert-butoxycarbonyl)amino)propanoic acid (4.43 g, 23.43 mmol). The mixture was stirred at 25 for 5 h. The mixture was washed with H2O and the DCM layer evaporated. The residue was purified by silica column (PE/EA-1:1) to give the tert-butyl N-[(1S)-2-[4-[1-(benzyloxy carbonylamino)ethyl]-1-piperidyl]-1-methyl-2-oxo-ethyl]carbamate (8.9 g, yield: 87.7%). 1H NMR (400 MHz, CDCl3): δ=7.37 (s, 5H), 5.58 (br, s., 1H), 5.15-5.03 (m, 2H), 4.60 (dd, J=7.4, 14.7Hz, 3H), 3.96-3.86 (m, 1H), 3.68 (br. s., 1H), 2.99 (br. s., 1H), 2.54 (d, J=11.0Hz, 1H), 1.91-1.63 (m, 3H), 1.44 (br. s., 914), 1.28 (d, 6.8Hz, 414), 1.21-1.07 (m, 4H).
To a solution of tert-butyl N-[(1S)-2-[4-[1-(benzyloxycarbonylamino)ethyl]-1-piperidyl]-1-methyl-2-oxo-ethyl]carbamate (8.9 g, 20.55 mmol) in MeOH (100 mL) was added Pd/C (900 mg). The mixture was stirred at 25° C. for 16 hours under H2 (50 psi). The reaction mixture was filtered and the filtrate was evaporated to give the tert-butyl ((2S)-1-(4-(1-aminoethyl)piperidin-1-yl)-1-oxopropan-2-yl)carbamate. (5.6 g, yield 91.2%). 1H NMR (400 MHz, MeOD-d6): δ 5.61 (t, J=7.2Hz, 1H), 4.71-4.56 (in. 2H), 3.91 (br. s., 1H), 3.06-2.96 (m, 1H), 2.77-2.71 (m, 1H), 2.59-2.50 (m, 1H), 1.85-1.68 (m, 2H), 1.44 (d, J=3.0Hz, 9H), 1.35-1.22 (m, 4H), 1.19-1.04 (m, 4H); LCMS (m/z): 300.2 [M+H]+.
To a solution of 3-((tert-butoxycarbonyl)amino)propanoic acid (8.38 g, 44.30 mmol, 1.10 Eq) and TFA (8.2 g, 80 mmol, 2.0 Eq) in DCM (500 mL) was added HATU (15.31 g, 40.27 mmol, 1.00 Eq) in one portion at 20° C. The mixture was stirred at 20° C. for 30 minutes. Then benzyl N-(4-aminocyclohexyl)carbamate (10.00 g, 40.27 mmol, 1.00 Eq) was added in one portion at 20° C. The mixture was stirred at 20° C. for 12 hr at which point LCMS analysis showed the reaction was complete. The mixture was washed with water (400 mL×3) and extracted with DCM (600 mL×3). The combined organic layer was concentrated in vacuum. The residue was purified by recrystallization (from minimum MeOH) to afford tert-butyl N-[3-[[4-(benzyloxycarbonylamino) cyclohexyl]amino]-3-oxo-propyl]carbamate (14.60 g. 34.80 mmol, 86.42% yield) as white solid, 1H NMR (400 MHz, DMSO-d6) δ 7.74 (d, J=7.78Hz, 1H), 7.31-7.40 (m 5H), 7.20 (d, 7.78Hz. 1H). 6.73 (t, J=5.40Hz, 1H), 5.00 (s, 2 PI), 3.44 (d, J=6.78Hz, 1H), 3.23 (dd, J=7.40, 3.14Hz, 1H), 3.10 (q, J=6.61Hz, 2H), 2.18 (t, J=7.40Hz, 2H), 1.78 (br. s., 4H), 1.37 (s, 9H), 1.16-1.27 (m, 4H); LCMS (m/z): 320.2 [M+H−100]+.
To a solution of tert.-butyl N-[3-[[4-(benzyloxycarbonylamino) cyclohexyl]amino]-3-oxo-propyl]carbamate (14.60 g, 34.80 mmol, 1.00 Eq) in MeOH (500 mL) was added Pd/C (5 g) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (50 psi) at 50° C. for 12 hours. LCMS showed the starting material was consumed completely. The reaction mixture was filtered and concentrated to give tert-butyl N-[3-[(4-aminocyclohexyl) amino]-3-oxo-propyl]carbamate (9.80 g, 34.34 mmol, 98.67% yield) as yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 7.74 (d, J=7.53Hz, 1H), 6.63-6.84 (m, 1H), 3.43 (br. s., 1H), 3.09 (q, =6.78Hz, 2H), 2.60 (br. s., 1H), 2.18 (t, J=3=7.28Hz, 2H), 1.70-1.82 (m, 4H), 1.37 (s, 9H), 1.08-1.20 (m, 4H); LCMS (m/z): 230.2 [M+H−56]+.
To a mixture of 6-chloro-2-oxoindoline-5-carboxylic acid (100.00 mg, 472.59 umol, 1.00 Eq), HATU (179.69 mg, 472.59 umol, 1.00 Eq) and TFA (47.82 rug, 472.59 umol, 1.00 Eq) in DCM (10 mL) was added tert-butyl ((2S)-1-(4-(1-aminoethyl)piperidin-1-yl)-1-oxopropan-2-yl)carbamate (141.50 mg, 472.59 umol, 1.00 Eq). The mixture was stirred at 20° C. for 3 hours. LCMS showed the reaction was complete, Water (5 mL) was added to the reaction and the aqueous phase was extracted with DCM (10 mL×3). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuum to afford tert-butyl ((2S)-1-(4-(1-(6-chloro-2-oxoindoline-5-carboxamido)ethyl)piperidin-1-yl)-1-oxopropan-2-yl)carbamate (100.00 mg, crude). LCMS (m/z):493.2 [M+H]+.
To a solution of tert-butyl ((2S)-1-(4-(1-(6-chloro-2-oxoindoline-5-carboxamido)ethyl)piperidin-1-yl)-1-oxopropan-2-yl)carbamate (100.00 mg, 202.84 umol, 1.00 Eq) in DCM (10 mL) was added dropwise TFA (3 mL) at 0° C. The resulting solution was then stirred for 3 hours at 25° C. TLC showed the reaction was complete. The mixture was evaporated and purified by prep-HPLC to afford N-(1-(1-(S)-2-aminopropanoyl)piperidin-4-yl)ethyl)-6-chloro-2-oxoindoline-5-carboxamide (38.50 mg, yield: 48.31%). 1H NMR (400 MHz, MeOD-d4): =7.29 (s, 1H), 6.95 (s, 1H), 4.56 (d, J=12.5Hz, 1H), 4.45-4.38 (m, 1H), 3.91 (d, J=12.3Hz, 2H), 3.54 (s, 2H), 3.15 (br. s., 2H), 2.72-2.63 (m, 1H), 2.01-1.85 (m, 2H), 1.75 (br. s., 1H), 1.46 (dd, J=6.9, 11.7Hz, 3H), 1.20-1.18 (m, 4H). LCMS (m/z): 393.2 [M+H]+.
To a stirred solution of 2-oxoindoline-5-carboxylic acid (0.177 g, 1.00 mmol) in DMF (2 mL), were added EDCI.HCl (0.24 g, 1.25 mmol), HOBt (0.168 g, 1.25 mmol) and triethylamine 0.35 mL, 2.5 mmol). The solution was stirred for 10 min at 0° C. tert-Butyl ((2S)-1-(4-(1-aminoethyl)piperidin-1-yl)-1-oxopropan-2-yl)carbamate (0.25 g. 0.83 mmol) was added and the reaction stirred at rt for 6 h. The progress of the reaction was monitored by TLC. After complete consumption of starting material, the reaction was quenched with water and extracted with ethyl acetate. The organic layer was separated, washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain a crude residue which was purified by column chromatography to afford tert-butyl ((2S)-1-oxo-1-(4-(1-(2-oxoindoline-5-carboxamido)ethyl)piperidin-1-yl)propan-2-yl)carbamate (0.11 g, 28%). LCMS: 359.25 (M-Boc)+.
To a stirred solution of tert-butyl ((25)-1-oxo-1-(4-(1-(2-oxoindoline-5-carboxamido)ethyl)piperidin-1-yl)propan-2-yl)carbamate (0.1 g, 0.47 mmol) in dioxane (2 mL) was added 4 M dioxane:HCl solution (4 nip at 0° C. and the reaction mixture stirred at rt for 5 h. The progress of the reaction was monitored by TLC. After complete consumption of tert-butyl ((2S)-1-oxo-1-(4-(1-(2-oxoindoline-5-carboxamido)ethyl) piperidin-1-yl)propan-2-yl)carbamate, the solvent was removed under reduced pressure to obtain a crude residue which was purified by repeated washing with ether and pentane obtain N-(1-(1-((S)-2-aminopropanoyl)piperidin-4-yl)ethyl)-2-oxoindoline-5-carboxamide hydrochloride (0.08 g, 93%). 1H NMR (400 MHz, DMSO-d6): δ 10.63 (s, 1H), 8.06 (q, J=11.0, 8.4Hz, 4H), 7.77-7.70 (m, 214), 6.85 (d, J=8.5Hz, 1H), 4.38-4.35 (m, 2H), 3.85 (d, J=13.9Hz, 2H), 3.53 (s. 2H), 3.09-2.90 (m, 1H), 2.57 (dd, J=25.3, 12.8Hz, 1H), 1.75 (dd, J=26.2, 12.6Hz, 3H), 1.3-1.28 (m, 3H), 1.12 (d, J=6.8 Hz, 4H), 1.02 (d, J=12.5Hz, 1H); LCMS: 359.25 (M+1)+.
To a stirred solution of 2-oxo-2,3-dihydrobenzo[d]oxazole-6-carboxylic acid (0.2 g, 0.66 mmol) in DMF (1.5 mL) was added EDCI.HCl (0.191 g, 1.00 mmol), HOBt (0.091 g, 0.66 mmol) and diispropylethylamine (0.34 mL, 2.00 mmol). The solution was stirred for 10 min at 0° C. After that, tert-butyl ((2S)-1-(4-(1-aminoethyl)piperidin-1-yl)-1-oxopropan-2-yl)carbamate (0.142 g, 0.80 mmol) was added and the reaction stirred at rt for 12 h. The progress of the reaction was monitored by TLC. After complete consumption of starting material, the reaction was quenched with water and extracted with ethyl acetate. The organic layer was separated, washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain a crude residue which was purified by column chromatography to afford tert-butyl ((2S)-1-oxo-1-(4-(1-(2-oxo-2,3-dihydrobenzo[d]oxazole-6-carboxamido)ethyl)piperidin-1-yl)propan-2-yl)carbamate (0.104 g, 33%). LCMS: 361.05 (M−Boc)+.
To a stirred solution of ((2S)-1-oxo-1-(4-(1-(2-oxo-2,3-dihydrobenzo[d]oxazole-6-carboxamido)ethyl)piperidin-1-yl)propan-2-34)carbamate (0.03 g, 0.08 mmol) in dioxane (1 ML) at 0° C. was added 4M dioxane:HCl solution (1 mL). The reaction mixture was stirred at rt for 3 h. The progress of the reaction was monitored by TLC. After complete consumption of starting material, the solvent was removed under reduced pressure to obtain a crude residue which was purified by repeated washing with ether and hexane, to obtain N-(1-(1-((S)-2-aminopropanoyl)piperidin-4-yl)ethyl)-2-oxo-2,3-dihydrobenzo[d]oxazole-6-carboxamide hydrochloride (0.008 g, 36%). 1H NMR (400 MHz, DMSO-d6): δ 10.66 (s, 1H), 8.72 (d. J=12.3Hz, 1H). 8.42 (q, J=8.1, 7.1Hz, 2H), 7.74 d, J=8.0Hz, 2H), 6.86 (d, J=8.1Hz, 1H), 3.54 (s, 2H), 3.25 (d, J=12.4Hz, 2H), 3.16 (t, H=6.1Hz, 2H), 2.82 (q, J=11.8Hz, 2H), 2.62 (s, 1H), 1.79 (d, J=13.4 Hz, 2H), 1.35-1.32 (m, 2H); LCMS: 274.15 (M+H)+
To a stirred solution of 2-oxo-2,3-dihydro-1H-benzo[d]imidazole-5-carboxylic acid (0.2 g, 0.66 mmol) in DMF (1.5 mL) was added EDCI.HCl (0,191 g, 1.00 mmol), HOBt (0.091 g, 0.66 mmol) and diispropylethylamine (0.34 mL, 2.00 mmol), The solution was stirred for 10 min at 0° C. After that tert-butyl ((2S)-1-(4-(1-aminoethyl)piperidin-1-yl)-1-oxopropan-2-yl)carbamate (0.142 g, 0.80 mmol) was added and the reaction stirred at rt for 12 h. The progress of the reaction was monitored by TLC. After complete consumption of starting material, the reaction was quenched with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na7SO4 and concentrated under reduced pressure to obtain a crude residue which was purified by column chromatography to afford tert-butyl ((2S)-1-oxo-1-(4-(1-(2-oxo-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamido)ethyl)piperidin-1-yl)propan-2-yl)carbamate (0.140 g, 45%). LCMS: 360.2 (M-Boc)+.
To a stirred solution of tert-butyl ((2S)-1-oxo-1-(4-(1-(2-oxo-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamido)ethyl)piperidin-1-yl)propan-2-yl)carbamate (0.1 g, 0.21 mmol) in dioxane (2 mL) at 0° C. was added 4M dioxane:HCl solution (1 mL). The reaction mixture was stirred at rt for 3 h. The progress of the reaction was monitored by TLC. After complete consumption of the starting material, the solvent was removed under reduced pressure to obtain a crude residue which was purified by repeated washing with ether and hexane to obtain N-(1-(1-((S)-2-aminopropanoyl)piperidin-4-yl)ethyl)-2-oxo-2,3-dihydro-M-benzo[d]imidazole-5-carboxamide hydrochloride (0.060 g, 69%). 1H NMR (400 MHz, DMSO-d6): δ 10.85 (d, J=16.4Hz, 2H), 8.10 (p, J=8.9, 7.6Hz, 4H), 7.55-7.52 (m, 1H), 7.44 (q, 1=2.0Hz, 1H), 6.95 (d, 1=8.1Hz, 1H), 4.44-4.24 (m, 2H), 3.88-3.86 (m, 2H), 3.09-2.91 (m, 1H), 2.6-2.57 (m, 1H), 1.83-1.64 (m, 3H), 1.33-0.99 (m, 814); LCMS: 360.25 (M+1)+.
To a solution of 1H-1,2,4-triazole-5-carboxylic acid (2.00 g, 17.69 mmol, 1.00 Eq) in DMF (100 mL) was added TFA (2.68 g, 26.53 mmol, 1.50 Eq), BOP-C1 (4.95 g, 19.46 mmol, 1.10 Eq), and tert-butyl 4-aminopiperidine-1-carboxylate (3.90 g, 19.46 mmol, 1.10 Eq). The reaction mixture was stirred at 20° C. for 12 hr. The reaction mixture was concentrated and dissolved in MeOH, filtered, the organic layer was concentrated and purified by silica gel column chromatography to give tert-butyl 4-(1H-1,2,4-triazole-5-carbonylamino)piperidine-1-carboxylate (3.13 g, 10.60 mmol, 59.9% yield) as a yellow solid. LCMS (m/z): 240.1 [M+H−56]+.
To a solution of tert-butyl 4-(1H-1,2,4-triazole-5-carbonylamino)piperidine-1-carboxylate (3.13 g, 10.60 mmol, 1.00 Eq) in DCM (50 mL) was added TFA (10 mL), The reaction mixture was stirred at 20 CC for 5 hr. The reaction mixture was concentrated and lyophilized to afford N-(4-piperidyl)-1H-1,2,4-triazole-5-carboxamide (6.00 g, 19.40 mmol, 91.51% yield) as a light yellow solid. LCMS (mix); 196.2 [M+H]+
To a mixture of 4-((tert-butoxycarbonyl)amino)butanoic acid (203.00 mg, 998.87 umol, 1.00 q) and HATU (379.80 mg, 998.87 umol, 1.00 Eq) in DCM (10 mL) was added Et3N (202.15 mg, 2.00 mmol, 2.00 Eq) in one portion at 20° C. The mixture was stirred at 20° C. for 30 min. Then N-(piperidin-4-yl)-1H-1,2,4-triazole-5-carboxamide (195.00 mg, 998.87 umol, 1.00 Eq) was added in one portion at 20° C. The mixture was stirred at 20° C. for 12 h. LCMS showed the reaction was complete. The reaction mixture was washed with water (40 mL×3) and extracted with DCM (50 mL×3). The combined organic layer was concentrated under vacuum. The residue was purified by prep-HPLC to afford tert-butyl (4-(4-(1H-1,2,4-triazole-5-carboxamido)piperidin-1-yl)-4-oxobutyl)carbamate (200.00 mg, 525.71 umol, 52.63% yield) as white solid. 1H NMR (400 MHz, MeOD-d4) δ 8.45 (s, 1H) 4.54 (d, 1=13.30Hz, 1H) 4.13-4.21 (m, 1H) 4.01 (d, J=13.80Hz, 1H) 3.23-3.30 (m, 1H) 3.11 (1, J=6.78Hz, 2H) 2.85 (t, J=11.67Hz, 1H) 2.46 (t, J=7.53Hz, 2H) 1.97-2.07 (m, 2H) 1.78 (quin, J=7.15Hz, 2H) 1.53-1.65 (m, 2H) 1.38-1.53 (m, 9H); LCMS (m/z): 381.2 [M+H]+.
To a mixture of tert-butyl (4-(4-(1H-1,2,4-triazole-5-carboxamido)piperidin-1-yl)-4-oxobutyl)carbamate (200.00 mg, 525.71 umol, 1.00 Eq) in DCM (20 nit) was added TEA (5 mL) dropwise at 0° C. The mixture was stirred at 0° C. for 30 min. The reaction then warmed slowly to 20° C. and stirred at this temperature for another 12 h showed the reaction complete. The mixture was concentrated under reduced pressure at 60° C. The residue was purified by prep-HPLC to afford N (1 (4 aminobutanoyl)piperidin-4-yl)-1H-1,2,4-triazole-5-carboxamide (104.20 mg, 50.26% yield) as colorless oil (TFA salt). 1H NMR (400 MHz, MeOD-d4) δ 8.49 (s, 1H) 4.54 (d, 1=13.55Hz, 1H) 4.12-4.21 (m, 1H) 4.00 (d, J=13.80Hz, 1H) 3.25 (t, J=11.80Hz. 1H) 3.02 (t, J=7.28Hz, 2H) 2.86 (t, J=11.67Hz, 1H) 2.60 (t, J=6.90Hz, 2H) 1.92-2.08 (m, 4H) 1.46-1.73 (m, 2H); LCMS (m/z): 281.1 [M+H]+
To a stirred solution of 1-benzyl-3-methyl-1H-pyrazole-5-carboxylic acid (0.150 g, 0.69 mmol) in DMF (5 mL) was added HATU (0.393 g, 1.0 mmol) and diisopropylethylamine (0.24 mL, 1.4 mmol). The solution was stirred for 10 ruin at 0° C. tert-Butyl ((1r,4r)-4-aminocyclohexyl)carbamate (0.147 g, 0.69 mmol) was added and the reaction stirred at rt for 2 h. The progress of the reaction was monitored by TLC. After complete consumption of starting material, the reaction was quenched with water and extracted with ethyl acetate. The organic layer was separated, washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain a crude residue which was purified by column chromatography to afford tert-butyl ((1r,4r)-4(1-benzyl-3-methyl-1H-pyrazole-5-carboxamido)cyclohexyl)carbamate (0.08 g, 25%). LCMS: 313.1 (M−100)+.
To a stirred solution of tert-butyl ((1r,4r)-4-(1-benzyl-3-methyl-1H-pyrazole-5-carboxamido)cyclohexyl)carbamate (0.05 g, 0.121 mmol) in dioxane (1 mL) at 0° C. was added 4 M dioxane:HCl (1.5 mL), The reaction mixture was stirred at rt for 1 h. The progress of the reaction was monitored by TLC After complete consumption of the starting material, the solvent was removed under reduced pressure to obtain a crude residue. The material was purified by repeated washing with ether and pentane to obtain N-((1r,4r)-4-aminocyclohexyl)-1-benzyl-3-methy-1H-pyrazole-5-carboxamide hydrochloride (0.03 g, 51%). 1H NMR (400 MHz, DMSO-d6): δ 8.26 (d, J=7.8Hz, 1H), 7.94 (d, J=5.3Hz, 3H), 7.33-7.10 (m, 5H), 6.66 (s, 1H), 5.60 (s, 2H), 2.96 (d, J=10.9 Hz, 1H), 2.16 (s, 3H), 1.96 (d, J=10.4Hz, 2H), 1.83 (d, J=11.1Hz, 2H), 1.47-1.26 (m, 4H); LCMS: 313.2 (M+H)+.
To a stirred solution of tert-butyl ((1r,4r)-4-aminocyclohexyl)carbamate (0.090 g, 0.420 mmol) in DMF (2 mL) was added EDCI (0.096 g, 0.504 mmol), HOBT (0.068 g. 0.504 mmol) and DIPEA (0.3 mL) and the solution stirred for 10 min at 0° C. 1-cyclopropyl-1H-1,2,3-triazole-4-carboxylic acid (0.064 g, 0.420 mmol) was then added and the reaction mixture stirred at rt for 2 hr. The progress of the reaction was monitored by TLC. After complete consumption of starting material, the reaction was quenched with water and extracted with ethyl acetate. The organic layer was separated, washed with brine, dried using Na2SO1 and concentrated under reduced pressure to obtain a residue which was purified by column chromatography to afford tert-butyl ((1r,4r)-4-(1-cyclopropyl-1H-1,2,3-triazole-4-carboxamido)cyclohexyl)carbamate (0.035 g, 24%). LCMS: 250.1 (M−100)4 observed.
To a stirred solution of tert-butyl ((1r,4r)-4-(1-cyclopropyl-1H-1,2,3-triazole-d-carboxamido)cyclohexyl)carbamate (0.035 g, 0.10 mmol) in methanol (3 mL) was added 4M methanol:HCl (3 mL) at 0° C. and the reaction stirred at rt for 16 hr. The progress of the reaction was monitored by TLC. After complete consumption of starting material, the solvent was removed under reduced pressure and the residue was purified by washings with diethyl ether and DCM to obtain N-((1r,4r)-4-aminocyclohexyl)-1-cyclopropyl-1H-1,2,3-triazole-4-carboxamide hydrochloride (0.013 g, 49%). 1H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.31 (d, J=8.3Hz, 1H), 7.97 (s, 2H), 4.1-4.05 (m, J=7.5, 3.9Hz, 1H), 3.75-3.66 (m, 1H), 3.01-2.88 (m, 1H), 1.97 (d, J=10.5Hz, 2H), 1.83 (d, J=9.5Hz, 2H), 1.55-4.34 (m, 4H), 1.25-1.07 (m, 4H); LCMS: 250.05 (M+H)+.
To a stirred solution of 2-oxoindoline 5 carboxylic acid (0.7 g, 3.95 mmol) in DMF (5 mL) was added EDCI.HCI (1.13 g, 5.92 mmol), HOBt (0.8 g, 5.92 mmol) and triethylamine (1.7 mL, 11.8 mmol). The solution was stirred for 10 min at 0° C. After that tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (0.93 g, 4.34 mmol) was added and the reaction stirred at rt for 16 h. The progress of the reaction was monitored by TLC. After complete consumption of starting material, the reaction was quenched with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain a crude residue which was purified by preparative HPLC to afford tert-butyl 4-((2-oxoindoline-5-carboxamido)methyl)piperidine-1-carboxylate (0.130 g, 8%) LCMS: 274.1 (M−Boc)+.
To a stirred solution of tert-butyl 4-(2-oxoindoline-5-carboxamido)methyl)piperidine-1-carboxylate (0.03 g, 0.08 mmol) in dioxane (1 mL) at 0° C. was added 4M dioxane:HCl solution (1 nit). The reaction mixture was stirred at rt for 3 h. The progress of the reaction was monitored by TLC. After complete consumption of starting material, the solvent was removed under reduced pressure to obtain a crude residue which was purified by repeated washing with ether and hexane to obtain 2-oxo-N-(piperidin-4-ylmethyl)indoline-5-carboxamide hydrochloride (0.008 g, 36%). 1H NMR (400 MHz, L)MSO-d6): δ 10.66 (s, 1H), 8.72 (d, J==12.3Hz, 1H), 8.42 (q, J=8.1, 7.1 Hz, 2H), 7.74 (d, J=8.0Hz, 2H), 6.86 (d, J=8.1Hz, 1H). 3.54 (s, 2H), 3.25 (d, J=12.4 Hz, 2H), 3.16 (t, J=0.1=6.1Hz, 2H), 2.82 (q, J=11.8Hz, 2H), 2.62 (s, 1H), 1.79 (d, J=13.4Hz, 2H), 1.35-1.32 (m, 2H); LCMS: 274.15 (M+H)+.
Into a 100-mL round-bottom flask was placed 2,2,2-trichlomethyl N-[1R,3S,5S)-8-azabicyclo[3.2.1]octan-3-yl]carbamate (780 mg, 2.59 mmcl, 1.00 equiv), dichloromethane (10 mL), TEA (0.93 g) added dropwise at 0° C. Then benzyl 4-[(chlorosulfonyl)methyl]piperidine-1-carboxylate (1 g. 3.01 mmol, 1.17 equiv) was added in several portions. The resulting solution was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum. The residue was chromatographed on a silica gel column with dichloromethane/methanol. (20:1-10:1). This resulted in 1.3 g (84%) of benzyl 4-[[(1R,3r,5S)-3-[[(2,2,2-trichloroethoxy)carbony]lamino]-8-azabicyclo[3.2.1]octane-8-sulfonyl]methyl]piperidine-1-carboxylate as a white solid. 1H NMR (400 MHz, CDCl3) δ: 7.41-7.32 (m, 5H), 5.32-5.10 (m, 2H), 4.48 (s, 4.27-4.12 (m, 4H). 4.00-3.96 (m, 1H), 2.93-2.80 (m, 4H), 2.30-2.07 (m, 5H), 1.98-1.91 (m, 6H), 1.30-1.26 (m. 3H) ppm, LCMS (method A, ESI): RT=1.32 min, m/z=620.2 [M+Na]+.
Into a 100-mL round-bottom flask was placed benzyl 4-[[(1R,3r,5S)-3-[[(2,2,2-trichloroethoxy)carbonyl]amino]-8-azabicyclo[3.2.1]octane-8-sulfonyl]methyl]piperidine-1-carboxylate (1 g, 1.68 mmol, 1.00 equiv), acetic acid (15 mL), water (1 mL), and zinc (1.63 g, 24.92 mmol, 14.88 equiv). The resulting mixture was stirred for 2 h at room temperature and then diluted with 30 mL of H2O. The solids were filtered out. The pH value of the filtrate was adjusted to 9 with NaOH (40%, aq.), The resulting solution was extracted with 3×30 mL of dichloromethane and the organic layers combined, dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in 0.7 g (99%) of benzyl 4-[[(1R,3r,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-sulfonyl]methyl]piperidine-1-carboxylate as a white solid. LCMS (method D, ESI): RT=0.85 min, m/z=422.3 [m+H]+.
Into a 25-mL round-bottom flask was placed 2-oxo-2,3-dihydro-1,3-benzoxazole-6-carboxylic acid (100 mg, 0.56 mmol. 2.35 equiv), N,N-dimethylformamide (10 mL), HOBT (135 mg, 2.00 equiv) and EDCI (191 mg, 2.00 equiv), Then benzyl 4-[[(1R,3r,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-sulfonyl]methyl]piperidine-1-carboxylate (100 mg, 0.24 mmol, 1.00 equiv) was added in several portions. After complete addition, TFA (250 mg, 5.00 equiv) was added dropwise. The resulting solution was stirred for 1 h at room temperature. The mixture was concentrated under vacuum and the residue diluted with 10 mL of H2O. This mixture was extracted with 3×10 mL of ethyl acetate and the organic layers combined. The combined extracts were washed with 2×30 mL of brine, dried, and concentrated. The residue was chromatographed on a silica gel column with dichloromethane/methanol (1011). This resulted in 100 mg (72%) of benzyl 4-[[(1R,3r,5S)-3-(2-oxo-2,3-dihydro-1,3-benzoxazole-6-amido)-8-azabicyclo[3.2.1]octane-8-sulfonyl]methyl]piperidine-1-carboxylate as yellow oil, LCMS (method D, ESI): RT=1.38 min, m/z=583.0 [M+H]+.
Into a 25-mL round-bottom flask was placed benzyl 4-[[(1R,3r,5S)-3-(2-oxo-2,3-dihydro-1,3-benzoxazole-6-amido)-8-azabicyclo[3.2.1]octane-8-sulfonyl]methyl]piperidine-1-carboxylate (100 mg, 0.17 mmol, 1.00 equiv) and hydrochloric acid (12N, 10 ML). The resulting solution was stirred for 4 It at room temperature and then concentrated under vacuum. The residue was applied onto Pre-HPLC with the following conditions, Column: X Bridge C18, 19*150 mm, 5 um; Mobile Phase A: Water/0.05% TFA, Mobile Phase B: ACN; Flow rate: 20 mL/min; Gradient: 30% B to 70% B in 10 min; 254 nm. The product was dissolved again into hydrochloric acid (5 mL, 12N) and concentrated under vacuum. This resulted in 7.3 mg (9%) of 2-oxo-N-[(1R,3r,5S)-8-[(piperidin-4-ylmethane)sulfonyl]-8-azabicyclo[3.2.1]octan-3-yl]-2,3-dihydro-1,3-benzoxazole-6-carboxamide as a white solid, 1H NMR (300 MHz, D2O) δ: 7.52-7.49 (m, 2H), 7.18-7.16 (m, 1H), 4.21 (s, 2H). 4.08-4.03 (m, 1H), 3.38-3.34 (m, 2H), 3.20-3.18 (m, 2H), 3.02-2.93 (m, 2H), 2.24-1.94 (m, 11H), 1.54-1.51 (m, 2H) ppm. LCMS (method D, ESI): RT=1.65 min, m/z=449.2 [M−HCl+H]+.
Into a 100-mL round-bottom flask was placed ethyl acetate (10 mL), water (10 mL), methyl 3-amino-4-hydroxybenzoate (1 g, 5.98 mmol, 1.00 equiv), and sodium bicarbonate (553 mg, 1.10 equiv). This was followed by the addition 2-bromopropanoyl bromide (1.3 g, 6.02 mmol, 1.00 equiv) which was added dropwise with stirring, at 0° C. The resulting solution was stirred for 30 min at room temperature. The mixture was then washed with 2×30 mL of H2O and 1×30 mL of brine. The mixture was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. This resulted in 1.5 g (83%) of methyl 3-(2-bromopropanamido)-4-hydroxybenzoate as a brown solid. 1H NMR (300 MHz, CDCl3) δ: 8.89 (s, 1H), 8.43 (s, 1H), 7.89-7.82 (m, 2H), 7.04 (d, J=8.41Hz, 1H), 4.65 (q, J=14.1Hz, 1H), 3.07 (s, 3H), 2.01 (d, J=7.2Hz, 3H) ppm. LCMS (method D, ESI): RT=1.30 min, m/z=302.0 [M+H]+.
Into a 100-mL round-bottom flask was placed N,N-dimethylformamide (10 mL), methyl 3-(2-bromopropanamido)-4-hydroxybenzoate (1.5 g, 4.96 mmol, 1.00 equiv), and potassium carbonate (880 mg, 1.30 equiv). The resulting mixture was stirred for 15 h at room temperature. The mixture was then diluted with 30 mL of H2O. The solids were collected by filtration. This resulted in 1 g (91%) of methyl 2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxylate as a brown solid. 1H NMR (400 MHz, CDCl3) δ: 8.29 (s, 1H), 7.72 (q, J=8.4Hz, 1H), 7.56 (d, 2Hz, 1H), 7.03 (d, J=8.4Hz, 1H), 4.77 (q, J=14Hz, 1H), 3.93 (s, 3H), 1.64 (d, J=7.2Hz, 3H)ppm. LCMS (method C, ESI): RT=0.96 min, m/z=222.0 [M+H]+.
into a 100-mL round-bottom flask was placed tetrahydrofuran (15 methanol (15 mL), water (10 mL), and methyl 2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzo[b][1,4]oxazine-6-carboxylate (1 g, 4.52 mmol, 1.00 equiv). This was followed by the addition of a solution of sodium hydroxide (362 mg, 2.00 equiv) in 5 ml H2O which was added dropwise with stirring at 0° C. The resulting solution was stirred for 10 min at 0° C. in an ice/salt bath. The resulting solution was allowed to react, with stirring, for an additional 15 h at room temperature. The reaction mixture was concentrated under vacuum. The residue was diluted with 30 mL of H2O and the pH adjusted to 3-4 with hydrochloric acid (1 N). The solids were collected by filtration. This resulted in 900 mg (96%) of 2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxylic acid as a white solid. 1H NMR (400 MHz, CD3OD) δ: 7.68 (q, J=8.4Hz, 1H), 7.59 (d, J=2Hz, 1H), 4.47 (s, 1H), 7.03 (d, J=8.4Hz, 1H), 4.75 (q, J=13.6Hz, 1H), 1.55 (d, J=6.8Hz, 3H) ppm, LCMS (method A, ESI): RT=1.08 min, m/z=208.0 [M+H]+.
Into a 100-mL round-bottom flask was placed N,N-dimethylformamide (50 mL), 2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxylic acid (500 mg, 2.41 mmol, 1.00 equiv), EDCI (223 trig, 2.00 equiv), HOBT (652 mg, 2.00 equiv), and tert-butyl (1R,3r,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate (710 mg, 3.14 mmol, 1.30 equiv). This was followed by the addition of TFA (1232 mg, 5.0 equiv) which was added dropwise with stirring at 0° C. The resulting solution was stirred for 14 h at room temperature. The reaction mixture was then diluted with 50 mL of EA. The resulting mixture was washed with 3×30 mL of H2O and 1×30 mL of brine. The mixture was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (4:1). This resulted in 800 mg (80%) of tert-butyl (1R,3r,5S)-3-(2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxamido)-8-azabicyclo[3.2.1]octane-8-carboxylate as a white solid. 1H NMR (400 MHz, CDCl3) δ: 8.66 (s, 1H), 7.52 (d, J=1.6Hz, 1H), 7.22 (q, J=8.4Hz, 1H), 7.02 (d, J=8.4Hz, 1H), 6.51 (d, J=6.8Hz, 1H), 1.72 (q, 13.6Hz, 1H), 1.39-4.20 (m, 3H), 2.45-2.25 (m, 2H), 2.20-2.10 (m, 2H), 1.95-1.75 (m, 1H), 1.60 (d, J=7.2Hz, 3H), 1.50 (s, 9H) ppm. LCMS (method C. BSI): RT=1.08 min, m/z=416.0 [M+H]+.
Into a 100-mL round-bottom flask was placed dichloromethane (20 mL) and tert-butyl (1R,3r,5S)-3-(2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,1]oxazine-6-carboxamido)-8-azabicyclo[3.2.1]octane-8-carboxylate (800 mg, 1.93 mmol, 1.00 equiv). To the above, hydrogen chloride (gas) was introduced. The resulting solution was stirred for 4 h at room temperature and then was concentrated under vacuum. The residue was diluted with 40 mL of H2O. The pH was adjusted to 8 with saturated aqueous sodium carbonate and the resulting mixture extracted with 3×40 mL of DCM. The organic layers were combined and washed with 2×10 mL of brine. The extract was concentrated under vacuum. This resulted in 500 ma (82%) of N-[(1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-yl]-2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxamide as a light yellow solid. 1/1 NMR (400 MHz, CDCl3) δ: 7.42 (q, J=8.4Hz, 1H), 7.37 (d, J=2Hz, 1H), 7.04 (d, J=8.4Hz, 1H), 4.72 (q, J=13.6Hz, 1H), 4.12 (t, J=6.4Hz, 1H), 3.69 (s, 2H), 2.22-2.15 (m, 4H), 2.10-1.95 (m, 4H), 1.51 (d, J=6.8Hz, 3H) ppm. LCMS (method A, ESI): RT=0.93 min, m/z=364.0 [M+H]+.
Into a 100-mL 3-necked round-bottom flask was placed N,N-dimethylformamide (10 ml), N-[(1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-yl]-2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxamide (250 mg, 0.79 mmol, 1.00 equiv), and TFA (231 mg, 3.00 equiv), This was followed by the addition of a solution of benzyl 4-[(chlorosulfonyl)methyl]piperidine-1-carboxylate (657 mg, 1.98 mmol, 2.50 equiv) in 2 ml N,N-dimethylformamide which was added dropwise with stirring at −20° C. The resulting solution was stirred for 30 min at −20° C. The mixture was allowed to react, with stirring, for an additional 15 h at room temperature. The mixture was diluted with 50 mL, of EA and washed with 2×20 mL of water and 2×20 mL of brine. The organic phase was dried over anhydrous sodium sulfate and filtered. The residue was chromatographed on a silica gel column with dichloromethane/methanol (20:1). This resulted in 60 mg (12%) of benzyl 4-[[(1R,3r,5S)-3-(2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxamido)-8-azabicyclo[3.2.1]octane-8-sulfonyl]methyl]piperidine-1-carboxylate as a white solid, LCMS (method B, ESI): RT=1.43 min, m/z=611.0 [M+H]+.
Benzyl 4-[[(1R,3 r,5S)-3-(2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxamido)-8-azabicyclo[3.2.1]octane-8-sulfonyl]methyl]piperidine-1-carboxylate (60 mg) was purified by Chiral-Prep-HPLC with the following conditions (Chiral_HPLC): Column, CHIRALPAK IA; mobile phase, MTBE:EtOH=50:50; Detector, 254 nm. This resulted in 28 mg (47%) of benzyl 4-[[(1R,3r,5S)-3-[(2R)-2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxamido]-8-azabicyclo[3.2.1]octane-8-sulfonyl]methyl]piperidine-1-carboxylate as a white solid. ee value: 100%
Into a 50-mL round-bottom flask was placed benzyl 4-[[(1R,3r,5S)-3-[(2R)-2-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxamido]-8-azabicyclo[3.2.1]octane-8-sulfonyl]methyl]piperidine-1-carboxylate (28 mu, 0.05 mmol, 1.00 equiv) and hydrochloric acid (12N, 10 mL). The resulting solution was stirred for 4 h at room temperature. The resulting mixture was washed with 2×10 mL of DCM and the aqueous layer concentrated under vacuum. The crude product was purified by Prep-HPLC with the following conditions (Prep_HPLC_MC5): Column, X Select C18, 19*250 mm, 5 um; mobile phase, Water/0.05% TFA, Mobile Phase B: ACN; Flow rate: 30 mL/min; Gradient: 23% B to 42% B in 11.5 min; Detector, 254 nm. The resulting fractions were concentrated under vacuum. The solids were dissolved in 2 ml hydrochloric acid (12 N) and again concentrated under vacuum. This resulted in 4.9 mg (21%) of (2R)-2-methyl-3-oxo-[(1R,3r,5S)-8-[(piperidin-4-ylmethane)sulfonyl]-8-azabicyclo[3.2.1]octan-3-yl]-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxamide hydrochloride as a white solid. 1H NMR (400 MHz, D2O) δ: 7.32 (d, J=8Hz, 1H), 7.21 (s, 1H), 7.01 (d, J=8.4Hz, 4.79-4.72 (m, 1H), 4.21 (s, 2H), 4.04 (s, 1H), 3.37 (d, 12.8Hz, 2H), 3.20 (d, J=6.4Hz, 2H), 2.95 (t, J=10.4Hz, 2H), 2.25-2.15 (m, 3H), 2.14-2.00 (m, 6H), 1.95 (d, 14.8Hz, 2H), 1.65-1.40 (m, 5H) ppm. LCMS (method A, ESI): RT=1.49 min, m/z=477.3[M−NCl+H]+.
Into a 250-mL 3-necked round-bottom flask was placed left-butyl (1R,3r,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate (5 g, 22.09 mmol, 1.00 equiv), water (100 mL), and NaHCO3 (4.83 g, 149.50 mmol, 2.60 equiv). The solution was cooled to 0° C. and 2,2,2-trichloroethyl chloroformate (5.63 g, 26.57 mmol, 1.20 equiv) added dropwise over 10 mins. The resulting solution was stirred at room temperature overnight. The reaction mixture was extracted with 3×100 mL of dichloromethane and the organic layers combined. The combined extracts were washed with 3×100 mL of brine, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting, residue was washed with 3×100 mL of hexane. This resulted in 8_32 g (94%) of tert-butyl (1R,3r,5S)-3-[[(2,2,2-trichloroethoxy)carbonyl]amino]-8-azabicyclo[3.2.1]octane-8-carboxylate as a white solid. 1H NMR (300 MHz, CDCl3) δ: 5.30 (brs, 1H), 4.73 (s, 2H), 4.23 (brs, 2H), 4.00-3.89 (m, 2H), 2.30-2.17 (m, 2H), 2.12-2.03 (m, 2H). 1.90-1.80 (m, 2H), 1.78-1.69 (m, 2H), 1.46 (s, 9H) ppm. LCMS (method C, ESI): RT=1.27 min, m/z=386.0 [M+H−15]+.
Into a 250-mL round-bottom flask was placed tert-butyl (1R,3r,5S)-3-[[(2,2,2-trichloroethoxy)carbonyl]amino]-8-azabicyclo[3.2.1]octane-8-carboxylate (4 g, 9.96 mmol, 1.00 equiv) and dichloromethane (40 ink). To this hydrogen chloride (gas) was introduced. The resulting solution was stirred for 1 h at room temperature. The mixture was then concentrated under vacuum. This resulted in 3.3 g (98%) of 2,2,2-trichloroethyl N-[(1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-yl]carbamate hydrochloride as a white solid. 1H NMR (300 MHz, D2O) δ: 4.72 (s, 2H), 4.09 (brs, 2H), 3.83-3.75 (m, 1H), 2.28-1.95 (m, 8H) ppm.
Into a 25-mL round-bottom flask was placed 2,2,2-trichloroethyl N-[(1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-yl]carbamate hydrochloride (1.0 g. 2.96 mmol, 1.00 equiv) and dichloromethane (15 mL). This was followed by the addition of TFA (1.5 g, 14.82 mmol, 5.01 equiv) dropwise with stirring at 0° C. To this was then added text-butyl N-[1-(chlorosulfonyl)piperidin-4-yl]carbamate (1.8 g, 6.02 mmol, 2.04 equiv) in several batches at 0° C. The resulting solution was stirred for 14 h at 20° C. The reaction mixture was diluted with 35 ml, of dichloromethane and washed with 3×10 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (1:2). This resulted in 1.5 g (90%) of 2,2,2-trichloroethyl N-[(1R,3r,5S)-8-(4-[[(tert-butoxy)carbonyl]amino]piperidine-1-sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl]carbamate as a white solid. 1H NMR (400 MHz, CDCl3) δ: 5.22 (brs, 1H), 4.75 (s, 2H), 4.46 (brs, 1H), 4.13 (brs, 2H), 4.01-3.95 (m, 1H), 3.70 (d, J=12.0Hz, 2H), 3.58 (bis, 1H), 2.84 (t, J=11.2Hz, 2H), 2.33-2.14 (m, 4H), 2.10-1.98 (m, 2H), 1.96-1.84 (m, 3H), 1.65-1.50 (m, 3H), 1.47 (s, 9H) ppm. LCMS (method D, ESI): RT=1.58 min, m/z=507.0 [M+H−56]+.
Into a 100-mL round-bottom flask was placed 2,2,2-trichloroethyl N-[(1R,3r,5S)-8-(4-[[(tert-butoxy)carbonyl]amino]piperidine-1-sulfonyl)-8-azabicyclo[3,2,1]octan-3-yl]carbamate (1.0 g, 1.77 mmol, 1.00 equiv), AcOH (15 mL), zinc (1.73 g, 26.45 mmol, 14.92 equiv) and water (1 mL). The resulting mixture was stirred for 1 h at 25° C. The mixture was then diluted with 30 mL of H2O and the solids were filtered out. The pH was adjusted to 9 with sodium carbonate (aq. sat.). The resulting solution was extracted with 5×30 ml, of dichloromethane and the organic layers combined. After concentration this resulted in 500 mg (73%) of tert-butyl N-[1-[(1R,3r,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-yl]carbamate as a white solid. LCMS (method A, ESI): RT=1.08 min, m/z=333.0 [M+H−56]+.
Into a 25-mL round-bottom flask was placed tert-butyl N-[1-[(1R,3r,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-yl]carbamate (200 mg, 0.51 mmol, 1.00 equiv), dichloromethane (5 mL), 2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid (109 mg, 0.62 mmol, 1.20 equiv), EDCI (118 mg, 0.62 mmol, 1.20 equiv), and HOBT (104 mg, 0.77 mmol, 1.50 equiv). This was followed by the addition of TFA (260 mg, 2.57 mmol, 4.99 equiv) dropwise with stirring at 0° C. The resulting solution was stirred for 14 h at 20° C. The reaction mixture was then diluted with 10 mL of dichloromethane and was washed with 2×5 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (1:1). This resulted in 243 mg (86%) of tert-butyl N-[1-[(1R,3r,5S)-3-(2-oxo-2,3-dihydro-1H-indole-5-carboxamido)-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-yl]carbamate as a off-white solid. LCMS (method A, ESI): RT=1.27 min, m/z=448.0 [M+H−100]+.
Into a 25-mL round-bottom flask was placed tert-butyl N-[1-[(1R,3r,5S)-3-(2-oxo-2,3-dihydro-1H-indole-5-carboxamido)-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-yl]carbamate (241 mg, 0.44 mmol, 1.00 equiv) and dichloromethane (5 mL). To this hydrogen chloride (gas) was introduced. The resulting solution was stirred for 2 h at 15° C. The mixture was then concentrated under vacuum. This resulted in 200 mg (94%) of N-[(1R,3r,5S)-8-(4-aminopiperidine-1-sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl]-2-oxo-2,3-dihydro-1H-indole-5-carboxamide hydrochloride as a yellow solid. 1H NMR (400 MHz, CD3OD): 7.70 (d, J=8.0Hz, 2H), 6.97 (d, J=8.0Hz, 1H), 4.14 (s, 3H), 3.86 (d, J=13.2Hz, 2H), 3.61 (s, 2H), 3.31-3.25 (m, 1H), 2.91 (t, J=12.4Hz, 2H), 2.37-2.25 (m, 2H), 2.21-1.98 (m, 8H), 1.76-4.63 (m, 2H) ppm. LCMS (method A, ESI): RT=1.07 min, m/z=448.3 [M+H]+.
Into a 25-mL round-bottom flask was placed N-[(1R,3r,5S)-8-(4-aminopiperidine-1-sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl]-2-oxo-2,3-dihydro-1H-indole-5-carboxamide hydrochloride (60 mg, 0.12 mmol, 1.00 equiv), methanol (5 mL), and benzaldehyde (13 mg, 0.12 mmol, 0.99 equiv). The mixture was stirred for 0.5 h at 20° C. To this NaBH3CN (7.8 mg, 0.12 mmol, 1.00 equiv) was added in hatches. The resulting solution was stirred for 2 h at 70° C. The reaction mixture was concentrated under vacuum. The residue was diluted with 5 mL of H2O and extracted with 2×5 mL of dichloromethane. The organic layers combined and concentrated. The crude product was purified by Prep-HPLC with the following conditions: Column, X Bridge C18, 19*150 mm, 5 um; Mobile Phase A:Water/0.05% TFA, Mobile Phase B: ACN; Flow rate: 20 mL/min; Gradient: 30% B to 70% B in 10 min.; Detector, 254 nm. This resulted in 14.8 mg (18%) of N-[(1R,3r,5S)-8-[4-(benzylamino)piperidine-1-sulfonyl]-8-azabicyclo[3.2.1]octan-3-yl]-2-oxo-2,3-dihydro-1H-indole-5-carboxamide trifluoroacetic acid as a white solid. 1H NMR (300 MHz, CD3OD): 7.70 (d, J=7.8Hz, 2H), 7.69-7.48 (m, 5H), 6.97 (d, J=8.4Hz, 1H), 4.28 (s, 2H), 4.14 (s, 3H), 3.92 (d, J=12.6Hz, 2H), 3.60 (s, 3.54-3.35 (m, 1H), 2.90 (t, 2H), 2.35-2.22 (m, 4H), 2.21-2.10 (m, 4H), 2.09-1.98 (m, 1.85-1.68 (m, 2H) ppm. LCMS (method A, ESI): RT=2.26 min, m/z=538.4 [M+H]+.
Into a 25-mL round-bottom flask was placed 6-chloro-2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid (170 mg, 0.80 mmol, 1.00 equiv), dichloromethane (10 mL), HOBT (216 mg, 1.60 mmol, 2.00 equiv). EDCI (306 rug, 1.60 mmol, 2.00 equiv), and tert-butyl N-1-[(1R,3r,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-ylcarbamate (375 mg, 0.97 mmol, 1.20 equiv). This was followed by the addition of TEA (400 mg, 3.95 mmol, 5.00 equiv) dropwise with stirring at 0° C. The resulting solution was stirred for 2 h at room temperature. The reaction mixture was diluted with 10 mL of dichloromethane and washed with 2×5 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (3:1). This resulted in 300 mg (64%) of tert-butyl N-[1-[(1R,3R,5S)-3-(6-chloro-2-oxo-2,3-dihydro-1H-indole-5-carboxamido)-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-yl]carbamate as a red solid. LCMS (method C, ESI): RT=0.88 min, m/z=582.0 [M+H]+.
Into a 25-mL round-bottom flask was placed tert-butyl N-[1-[(1R,3r,5S)-3-(6-chloro-2-oxo-2,3-dihydro-1H-indole-5-carboxamido)-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-yl]carbamate (300 mg, 0.52 mmol, 1.00 equiv) and hydrogen chloride/dioxane (10 mL, saturated, this solution was made by introducing hydrogen chloride gas into 1,4-dioxane under 0° C. for 6 hours). The resulting solution was stirred for 4 h at room temperature. The mixture was then concentrated under vacuum. This resulted in 170 mg (64%) of N-[(1R,3r,5S)-8-(4-aminopiperidine-1-sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl]-6-chloro-2-oxo-2,3-dihydro-1H-indole-5-carboxamide hydrochloride as a red solid. LCMS (method A, ESI). RT=0.96 min, m/z=482.0 [M+H]+.
Into a 25-mL round-bottom flask was placed N-[(1R,3r,5S)-8-(4-aminopiperidine-1-sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl]-6-chloro-2-oxo-2,3-dihydro-1H-indole-5-carboxamide hydrochloride (50 mg, 0.10 mmol, 1.00 equiv), methanol (5 mL), and benzaldehyde (12.3 rag, 0.12 mmol, 1.20 equiv). The mixture was stirred for 0.5 h at 20° C. To the above NaBH3CN (7.3 mg, 0.12 mmol, 1.20 equiv) was added in batches. The resulting solution was stirred for 1 h at 70° C. The reaction mixture was then concentrated under vacuum and the crude product purified by Prep-HPLC with the following conditions: Column: X Select C18, 19*250 mm, 5 um; Mobile Phase A: Water/0.05% TFA, Mobile Phase B: ACN; Flow rate: 30 mL/min; Gradient: 5% B to 36% B in 12.5 min; Detector: 254 nm. This resulted in 28 mg (42%) of N-[(1R,3r,5S)-8-[4-(benzylamino)piperidine-1-sulfonyl]-8-azabicyclo[3.2.1]octan-3-yl]-6-chloro-2-oxo-2,3-dihydro-1H-indole-5-carboxamide trifluoroacetate as a white solid. 1H NMR (400 MHz, CD3OD): 7.53-7.49 (m, 5H), 7.33 (s, 1H), 6.97 (s, 1H), 4.29 (s, 2H), 4.16 (s, 3H), 3.91 (d, J=12.8Hz, 2H), 3.57 (s, 2H), 3.41-3.37 (m, 1H), 2.89 (t, J=10.8Hz, 2H), 2.37-2.21 (m, 4H), 2.20-2.08 (m, 4H), 2.05-1.96 (m, 2H), 1.80-1.70 (m, ppm, LCMS (method A, ESI): RT=1.31 min, m/z=572.2 [M+H]+.
Into a 250-mL round-bottom flask, was placed water (120 mL). This was followed by the addition of tert-butyl (1R,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate (2 g, 8.84 mmol, 1.00 equiv), sodium bicarbonate (1.92 g, 22.85 mmol, 2.59 equiv). To the mixture was added. 2,2,2-trichloroethyl chloroformate (2.28 g, 10.76 mmol, 1.22 equiv) dropwise with stirring at 0° C. The resulting solution was stirred tor 18 h at 20° C. The resulting solution was extracted with 3×150 mL of ethyl acetate and the organic layers combined and dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in 4.16 g (crude) of tert-butyl (1R,5S)-3-[[(2,2,2-trichloroethoxy)carbonyl]amino]-8-azabicyclo[3.2.1]octane-8-carboxylate as a white solid. 1H NMR (300 MHz, CDCl3): δ 4.75 (s, 2H), 4.25 (s, 2H), 4.00-3.90 (m, 1H), 2.29-2.00 (m, 4H), 2.89-2.71 (m, 4H), 1.4.5 (s, 9H) ppm.
Into a 100 mL round-bottom flask, was placed dichloromethane (20 mL), tert-butyl (1R,3S,5S)-3-[[(2,2,2-trichloroethoxy)carbonyl]amino]-8-azabicyclo[3.2.1]octane-8-carboxylate (2 g, 4.98 mmol, 1.00 equiv). Then hydrogen chloride gas was introduced into mixture. The resulting solution was stirred for 12 h at 80° C. The resulting mixture was concentrated under vacuum. This resulted in 1.7 g (crude) of 2,2,2-trichloroethyl N-[(1R,3S,5S)-8-azabicyclo[3.2.1]octan-3-yl]carbamate as a yellow solid. LCMS (method D, ESI): RT=0.86 min, m/z=303.2 [M+H]+.
Into a 100-mL round-bottom flask, was placed dichloromethane (30 mL), 2,2,2-trichloroethyl N-[(1R,3S,5S)-8-azabicyclo[3.2.1]octan-3-yl]carbamate (2.4 g, 7.96 mmol, 1.00 equiv), TFA (3.2 g, 31.62 mmol, 3.97 equiv). Then benzyl 4-[(chlorosulfonyl)methyl]piperidine-1-carboxylate (4 g, 12.05 mmol, 1.51 equiv) was added by dropwise at 0° C. The resulting solution was stirred for 12 h at 10° C. The resulting mixture was washed with 3×30 ml, of water and 1×30 mL of brine. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column with dichloromethane/methanol (20:1). This resulted in 2.8 g (59%) of benzyl 4-[[(1R,3S,5S)-3-[[(2,2,2-trichloroethoxy)carbonyl]amino]-8-azabicyclo[3.2.1]octane-8-sulfonyl]methyl]piperidine-1-carboxylate as a yellow solid. 1H NMR (300 MHz, CDCl2): δ 7.41-7.30 (m, 5H), 5.15 (s, 2H), 4.75 (s, 2H), 4.30-4.15 (m, 4H), 4.05-3.90 (m, 1H), 2.95-2.76 (m, 4H), 2.35-2.10 (m, 4H), 2.10-1.90 (m, 5H), 1.57 (s, 1H), 1.40-1.20 (m, 3H) ppm. LCMS (method D, ESI): RT=1.15 mm, m/z=596.1 [M+H]+.
Into a 250-mL round-bottom flask, was placed benzyl 4-[[(1R,5S)-3-[[(2,2,2-trichloroethoxy)carbonyl]amino]-8-azabicyclo[3.2.1]octane-8-sulfonyl]methyl]piperidine-1-carboxylate (1.5 g, 2.51 mmol, 1.00 equiv). This was followed by the addition of hydrochloric acid (12 N, 140 nit) at 10° C. The resulting solution was stirred for 12 h at 50° C. The resulting mixture was concentrated under vacuum. This resulted in 1.1 g (88%) of 2,2,2-trichloroethyl N-[(1R,5S)-8-[(piperidin-4-ylmethane)sulfonyl]-8-azabicyclo[3.2.1]octan-3-yl]carbamate hydrochloride as a yellow solid. LCMS (method D, ESI): RT=0.67 min, m/z=464.0 [M+H]+.
Into a 100-mL round-bottom flask, was placed dichloromethane (40 mL). This was followed by the addition of methanol (20 mL), 2,2,2-trichloroethyl N-[(1R,5S)-8-[(piperidin-4-ylmethane)sulfonyl]-8-azabicyclo[3.2.1]octan-3-yl]carbamate hydrochloride (300 mg, 0.60 mmol, 1.00 equiv), 4,4,4-trifluorobutanol (227 mg, 1.80 mmol, 3.00 equiv). Then NaBH3CN (303 mg, 4.81 mmol, 8.00 equiv) was added into by batchwise. To the mixture was added acetic acid (1 mL). The resulting solution was stirred for 6 h at 10° C. The resulting mixture was concentrated under vacuum. The resulting solution was extracted with 3×1100 ml, of dichloromethane and the organic layers combined. The resulting mixture was washed with 1×100 mL of brine. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by flash chromatography with eluent (PE/EtOAc=2/1 to 100% EtOAc). This resulted in 295 mg (86%) of 2,2,2-trichloroethylN-[(1R,5S)-8-([[1-(4,4,4-trifluorobutyl)piperidin-4-yl]methane]sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl]carbamate as yellow oil. 1H NMR (300 MHz, CDCl3): δ 4.75 (s, 2H), 4.30 (s, 2H), 4.02-3.05 (m, 1H), 3.10-3.02 (m, 2H), 3.00-2.95 (m, 2H), 2.58-2.50 (m, 2H), 2.32-4.78 (m, 17H), 1.60-1.48 (m, 2H) ppm, LCMS (method D, ESI): RT=0.97 min, m/z==572.0 [M+H]+.
Into a 100-mL round-bottom flask, was placed 2,2,2-trichloroethyl N-[(1R,3S,5S)-8-([[1-(4,4,4-trifluorobutyl)piperidin-4-yl]methane]sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl]carbamate (50 mg, 0.09 mmol, 1.00 equiv). This was followed by the addition of acetic acid (15 mL), water (1 mL) and Zn (90 mg). The resulting solution was stirred for 12 h at 10° C. The solids were filtered out. The pH value of the solution was adjusted to 8 with sodium carbonate (sat. aq.). The resulting solution was extracted with 3×50 mL of ethyl acetate and the organic layers combined and concentrated under vacuum. This resulted in 25 mg (72%) of (1R,3S,5S)-8-([[1-(4,4,4-trifluorobutyl)piperidin-4-yl]methane]sulfonyl)-8-azabicyclo[3.2.1]octan-3-amine as a yellow solid. LCMS (method B, ESI): RT=1.24 min; m/z=398.0 [M+H]+.
Into a 100-mL round-bottom flask, was placed N,N-dimethylformamide (10 mL), (1R,3S,5S)-8-([[1-(4,4,4-trifluorobutyl)piperidin-4-yl]methane]sulfonyl)-8-azabicyclo[3:2.1]octan-3-amine (50 mg, 0.13 mmol, 1.00 equiv), 6-chloro-2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid (46 mg, 0.22 mmol, 1.73 equiv), 1H-1,2,3-benzotriazol-1-ol (35 mg, 0.26 mmol, 2.06 equiv), EDCI (50 mg, 0.26 mmol, 2.07 equiv), TEA (0.3 mL). The resulting solution was stirred for 12 h at 10° C. The solids were filtered out. The resulting mixture was diluted with 10 mL of water. The resulting solution was extracted with of 2×10 mL dichloromethane and the organic layers combined. The organic phase was dried over anhydrous sodium sulfite and concentrated under vacuum. The crude reside was purified by Prep-HPLC with the following conditions (2 #-Waters 2767-2 (HPLC-08)): Column, Xbridge Prep Phenyl, 5 urn, 19×150 mm; mobile phase, Water with 50 mmol ammonium bicarbonate and acetonitrile (10.0% acetonitrile up to 33.0% in 2 min, up to 53.0% in 8 min, up to 100.0% in 1 min, down to 10.0% in 1 min; Detector. UV 254 nm. This resulted in 5.7 mg (8%) of 6-chloro-2-oxo-N-[(1R,3S,5S)-8-([[1-(4,4,4-trifluorobutyl)piperidin-4-yl]methane]sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl]-2,3-dihydro-1H-indole-5-carboxamide as a light pink solid. 1HNMR (300 MHz, CD3OD): δ 7.32 (s, 1H), 695 (s, 1H), 4.30-4.10 (m, 3H), 3.35 (s, 2H), 3.10-2.90 (m, 4H), 2.50-2.40 (m, 2H), 2.40-190 (m, 15H), 1.85-1.70 (m, 2H), 1.51-1.35 (m, 214) ppm. LCMS (method B, ESI): RT=1.67 min, m/z=591.1 [M+H]+.
Into a 100-mL round-bottom flask was placed dichloromethane (50 mL), 2,2-difluoro-2H-1,3-benzo[d][1,3]dioxole-5-carboxylic acid (1.5 g, 7.42 mmol, 1.00 equiv), tert-butyl (1R,5S,7S)-7-amino-3-azabicyclo[3,3,2]decane-3-carboxylate (2.0 g, 7.86 mmol, 1.06 equiv), HATU (5.65 g), and TFA (2.25 g, 22.24 mmol, 3.00 equiv). The resulting solution was stirred for 12 h at 25° C. The resulting mixture was washed with 3×50 mL of H2O. The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was chromatographed on a silica gel column with PE: EA (1:1). This resulted in 3.0 g (92%) of tert-butyl(1R,5r,7S)-7-(2,2-difluoro-2H-1,3-benzo[d][1,3]dioxole-5-carboxamido)-3-azabicyclo[3.3.2]decane-3-carboxylate as a white solid. 1HNMR (300 MHz, DMSO): δ 8.20-8.15 (m, 1H), 7.77 (s, 1H), 7.68 (d, J=8.7Hz, 1H), 7.52 (d, J=8.4Hz, 1H), 4.08-3.90 (m, 1H), 3.50-3.10 (m, 2H), 2.15-1.80 (m, 8H), 1.50-1.30 (m, 9H) ppm. LCMS (method C, ESI): RT-1.25 min, m/z=411.2 [M+H]+.
Into a 100-mL round-bottom flask was placed a solution of tert-butyl (1R,3r,5S)-3-(2,2-difluoro-2H-1,3-benzo[d][1,3]dioxole-5-carboxamido)-8-azabicyclo[3.2.1]octane-8-carboxylate (1.5 g, 3.65 mmol, 1.00 cquiv) in methanol (30 mL), Hydrogen chloride gas was introduced into the solution at 0° C.; for 1 h. The resulting solution was stirred for another 1 h at 20° C. The mixture was then concentrated under vacuum. This resulted in 1.3 g (crude) of N-[(1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-yl]-2,2-difluoro-2H-1,3-benzo[d][1,3]dioxole-5-carboxamide as a white solid. LCMS (method A, ESI): RT=0.80 min, m/z=311.2 [M+H]+.
Into a 250-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed a solution of N-[(1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-yl]-2,2-difluoro-2H-1,3-benzo[d][1,3]dioxole-5-carboxamide (900 mg, 2.90 mmol, 1.00 equiv) in THF (150 mL). This was followed by the addition of LiHMDS (1M in THF, 10 mL) dropwise with stirring at −60° C. To this was added 4-oxocyclohexane-1-sulfonyl chloride (700 mg, 3.56 mmol, 1.23 equiv) in portions at 60° C. The resulting solution was allowed to warm to room temperature and stirred for another 12 hours at 25° C. The resulting mixture was concentrated under vacuum. The residue was extracted with 3×60 mL of dichloromethane and the organic layers combined, dried over anhydrous sodium sulfate and concentrated. The residue was chromatographed on a C18 gel column with H2O/CH3CN=3:5. This resulted in 260 mg (19%) of 2,2-difluoro-N-[(1R,3rS,5S)-8-[(4-oxocyclohexane)sulfonyl]-8-azabicyclo[3.2.1]octan-3-yl]-2H-1,3-benzo[d][1,3]dioxole-5-carboxamide, as a white solid. LCMS (method B. ESI): RT=1.08 min, m/z=471.0 [M+H]+.
Into a 250-mL round-bottom flask was placed methanol (130 mL), 2,2-difluoro-N-[(1R,3r,5S)-8-[(4-oxocyclohexane)sulfonyl]-8-azabicyclo[3.2.1]octan-3-yl]-2H-1,3-benzo[d][1,3]dioxole-5-carboxamide (200 mg, 0.43 mmol, 1.00 equiv), HCOONH4 (1080 mg, 17.13 mmol, 40.29 equiv), and acetic acid (24 mg, 0.40 mmol, 0.94 equiv. Then NaBH3CN (50 mg, 0.79 mmol, 1.87 equiv) was added batchwise. The resulting solution was stirred for 2 h at 20° C. The mixture was then concentrated under vacuum. The residue was slurried with 150 mL of EtOAc and then filtrated. The filtrate was concentrated under vacuum. The crude product was purified by Prep-HPLC with the following conditions: Column, X Bridge C18, 19*150 mm, 5 um; mobile phase, Mobile Phase A: Water/0.05% TFA, Mobile Phase B: ACN: Flow rate: 20 mL/min; Detector, 254 nm. This resulted in 15.2 mg (6%) of N-[(1R,3r,5S)-8-[(4-aminocyclohexane)sulfonyl]-8-azabicyclo[3.2.1]octan-3-yl]-2,2-difluoro-2H-1,3-benzo[d][1,3]dioxole-5-carboxamide trifluoroacetic acid as a white solid. 1H NMR (300 MHz, D2O): δ 7.46-7.44 (m, 2H), 7.19 (d, J=6Hz, 1H). 4.18 (s, 2H), 4.05 (t, J=6.0Hz, 1H), 3.48-3.10 (m, 2H), 2.30-1.80 (m, 13H), 1.65-1.38 (m, 3H) ppm. LCMS (method D, ESI): RT=1.55 min, m/z=472.0 [M+H]+.
Into a 50-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed dichloromethane (10 mL), 5-ethyl-1,2-thiazole-3-carboxylic acid (44 mg, 0.28 mmol, 1.00 equiv), tert-butyl N-[1-[(1R,3r,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-yl]carbamate (100 mg, 0.34 mmol, 1.21 equiv), HOBT (52 mg, 0.38 mmol, 1.36 equiv)), and EDCI (150 mg, 0.79 mmol, 2.80 equiv). This was followed by the addition of a solution of triethylamine (80 mg, 0.79 mmol, 2.80 equiv) in dichloromethane (1 ml) which was added dropwise with stirring at 0° C. The resulting solution was stirred for 15 hours at 20° C. The reaction was quenched by the addition of 50 mL of water and extracted with 2×100 Mt of dichloromethane. The organic layers were combined and washed with 1×50 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (1:2). This resulted in 120 mg (81%) of tert-butyl N-[1-[(1R,3r,5S)-3-(5-ethyl-1,2-thiazole-3-carboxamido)-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-yl]carbamate as a solid. LCMS (method A, ESI): RT=1.61 min, m/z=528.0[M+H]+.
Into a 50-mL round-bottom flask was placed tert-butyl N-[1-[(1R,3r,5S)-3-(5-ethyl-1,2-thiazole-3-carboxamido)-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-yl]carbamate (120 mg, 0.23 mmol, 1.00 equiv) and hydrogen chloride/dioxane (10 mL, saturated, this solution was made by introducing hydrogen chloride gas into 1,4-dioxane under 0° C. for 6 hours). The resulting solution was stirred for 3 hours at 20° C. The mixture was then concentrated under vacuum. This resulted in 57.8 mg (55%) of N-[(1R,3r,5 S)-8-(4-aminopiperidine-1-sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl]-5-ethyl-1,2-thiazole-3-carboxamide hydrochloride as a solid. 1H NMR (300 MHz, D2O) δ: 7.45 (s, 1H), 4.15-4.02 (m, 3H), 3.80-3.78 (m, 2H), 3.38-3.22 (m, 1H), 2.98-2.82 (m, 4H), 2.30-2.18 (m, 2H), 2.11-1.87 (m, 8H), 1.71-1.52 (m, 2H), 1.30-1.20 (m, 3H) ppm. LCMS (method A, ESI): RT=1.81 min, m/z=428.2 [M−HCl+H]+.
Into a 100-mL round-bottom flask was placed methyl 3-oxo-3,4-dihydro-2H-1,4-benzo[b][1,4]oxazine-6-carboxylate (1 g, 4.83 mmol, 1.00 equiv), methanol (15 mL), tetrahydrofuran (15 mL), and water (15 mL). This was followed by the addition of a solution of sodium hydroxide (386 mg, 9.65 mmol, 2.00 equiv) in 5 ml H2O which was added dropwise with stirring at 0° C. The solution was stirred for 20 mm at 0° C. in an ice/salt bath. The resulting solution was allowed to react, with stirring, for an additional 18 h at room temperature. The reaction mixture was then concentrated under vacuum. The residue was diluted with 50 mL of H2O and The pH adjusted to 3-4 with hydrochloric acid (1 N). The resulting mixture was extracted with 3×50 mL of ethyl acetate. The organic layers were combined and washed with 2×30 mL of water and 1×30 mL of brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under vacuum. This resulted in 850 mg (91%) of 3-oxo-3,4-dihydro-2H-1,4-benzo[b][1,4]oxazine-6-carboxylic acid as a brown solid. 1H NMR (300 MHz, CD3OD) δ: 7.68 (q, J=8.4Hz, 1H), 7.61 (d, J=1.8Hz, 1H), 7.02 (d, J=8.4Hz, 1H), 4.68 (s, 2H) ppm. LCMS (method A, ESI): RT=1.01 min, m/z 194.0 [M+H]+.
Into a 100-mL round-bottom flask was placed dichloromethane (30 mL), 2,2,2-trichloroethyl N-[(1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-yl]carbamate hydrochloride (900 mg, 2.66 mmol, 1.00 equiv), and TFA (1.37 g, 13.54 mmol, 5.00 equiv). This was followed by the addition of a solution of tert-butyl N-[1-(chlorosulfonyl)piperidin-4-yl]carbamate (1.6 g, 5.35 mmol, 2.00 equiv) in 2 ml dichloromethane which was added dropwise with stirring at 0° C. The resulting solution was stirred for 14 h at 10° C. The mixture was then washed with 3×30 mL of brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under vacuum. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (1:1). This resulted in 1.1 g (73%) of 2,2,2-trichloroethyl N-[(1R,3r,5S)-8-(4-[[(tert-butoxy)carbonyl]amino]piperidine-1-sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl]carbamate as a white solid. 1H NMR (400 MHz, CDCl3) δ: 5.22 (s, 1H), 4.75 (s, 2H), 4.47 (s, 1H), 4.14 (s, 2H), 3.98 (d, J=6Hz, 1H), 3.70 (d, J=12Hz, 2H), 3.58 (s, 1H), 2.84 (t, J=11.2Hz, 2H), 2.27-2.25 (m, 4H), 2.03 (d, J=10.8Hz, 2H), 1.95-1.87 (m, 4H), 1.58 (s, 2H), 1.55-1.40 (m, 9H) ppm. LCMS (method C, ESI): RT=1.24 min, m/z=563.0 [M+H]+.
Into a 100-mL round-bottom flask was placed 2,2,2-trichloroethyl N-[(1R,3r,5S)-8-(4-[[(tert-butoxy)carbonyl]amino]piperidine-1-sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl]carbamate (1.1 g, 1.95 mmol, 1.00 equiv), Zn (1.9 g, 15.00 equiv), AcOH (15 mL), and water (1 mL). The resulting mixture was stirred for 3 h at 10° C. The solids were filtered out. The pH was adjusted to 8 with sodium carbonate (aq. sat.). The resulting solution was extracted with 4×50 mL of ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. This resulted in 750 mg (crude) of tert-butyl N-[1-[(1R,3r,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-yl]carbamate as a light yellow crude solid. LCMS (method C, ESI): RT=0.61 min, m/z=389.0 [M+H]+.
Into a 100-mL round-bottom flask was placed N,N-dimethylformamide (10 mL), 3-oxo-3,4-dihydro-2H-1,4-benzo[b][1,4]oxazine-6-carboxylic acid (55 mg, 0.28 mmol, 1.10 equiv), EDCI (98 mg, 0.51 mmol, 2.00 equiv), HOBT (70 mg, 0.52 mmol, 2.00 equiv), and tert-butyl N-1-[(1R,3r,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-ylcarbamate (100 mg, 0.26 mmol, 1.00 equiv). This was followed by the addition of TFA (131 mg, 1.29 mmol, 5.00 equiv) which was added dropwise with stirring at 0° C. The resulting solution was stirred for 15 h at 10° C. The reaction mixture was diluted with 10 mL of H2O and extracted with 3×10 rut of ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was chromatographed on a silica gel column with dichloromethane/methanol (10:1). This resulted in 100 mg (69%) of tert-butyl N-[1-[(1R,3r,5S)-3-(3-oxo-3,4-dihydro-2H-1,4-benzo[b][1,4]oxazine-6-carboxamido)-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-yl]carbamate as a white solid. LCMS (method A, ESI): RT=1.32 min, m/z=586.0 [M+Na]+.
Into a 50-mL round-bottom flask was placed dichloromethane (10 mL) and tert-butyl N-[1-[(1R,3r,5S)-3-(3-oxo-3,4-dihydro-2H-1,4-benzo[b][1,4]oxazine-6-carboxamido)-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-yl]carbamate (100 mg, 0.18 mmol, 1.00 equiv). To the above hydrogen chloride (gas) was introduced. The resulting solution was stirred for 3 h at 10° C. The reaction mixture was then concentrated under vacuum. The crude product (80 mg) was purified by Prep-HPLC with the following conditions (Prep_HPLC_MC5): Column, X Select C18, 19*250 mm, 5 um; mobile phase, A: Water/0.05% TFA, Mobile Phase B: ACN; Flow rate: 30 mL/min; Gradient: 5% B to 36% B in 12.5 min; Detector, 254 nm. The isolated purified product was dissolved in 2 ml concentrated hydrochloric acid and this solution concentrated under vacuum. This resulted in 45.7 mg (52%) of N-[(1R,3r,5S)-8-(4-aminopiperidine-1-sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl]-3-oxo-3,4-dihydro-2H-1,4-benzo[b][1,4]oxazine-6-carboxamide hydrochloride as a white solid, 1H NMR (400 MHz, D2O) δ: 7.28 (q, J=8.4Hz), 7.18 (s, 1H), 6.98 (d, J=8.4Hz, 1H), 4.60 (s, 2H), 4.10-3.95 (m, 3H), 3.74 (d, J=13.2Hz, 2H), 3.35-3.25 (m, 2.88 (t, J=12Hz, 2H), 2.25-2.18 (m, 2H), 2.10-1.98 (m, 6H), 1.91 (d, J=14.8Hz, 2H), 1.67-1.52 (m, 2H) ppm. LCMS (method A, ESi): RT=1.40 min, m/z=464.0 [M−HCl+H]+.
Into a 250-mL round-bottom flask was placed ethyl 2,4-dioxohexanoate (10 g, 58.08 mmol, 1.00 equiv), benzene (100 mL), CH3COONH4 (13.4 g. 173.84 mmol, 2.99 equiv), and acetic acid (10 mL). The resulting solution was stirred at 80° C. overnight. The reaction mixture was cooled and concentrated under vacuum. The residue was diluted with 200 mL of ice-water and the pH adjusted to 8 with Na2CO3 (aq. Sat.). The resulting mixture was extracted with 3×100 ink of ethyl acetate and the organic layers combined, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (1:10-1:5). This resulted in 7 g (70%) of ethyl 2-amino-4-oxohex-2-enoate as yellow oil. 1H NMR (400 MHz, CDCl3) δ: 5.92 (s, 1H), 4.36-4.30 (m, 2H), 2.49-2.44 (m, 2H), 1.28-1.24 (m, 3H), 1.14-1.11 (m, 3H) ppm.
Into a 250-mL round-bottom flask was placed ethyl 2-amino-4-oxohex-2-enoate (4 g, 23.37 mmol, 1.00 equiv), tetrahydrofuran (50 mL), and P2S5 (2.6 g, 11.70 mmol, 0.50 equiv). The mixture was stirred overnight at room temperature. Then the mixture was concentrated and the residue was dissolved in ethyl acetate (20 mL). This solution was cooled to 0° C. and H2O2 (30%, 10 mL) was added dropwise. The resulting mixture was stirred for 10 min at room temperature. To the mixture was added activated charcoal. After filtration, the filtrate was diluted with H2O (20 mL). This was extracted with EA (20 mL×3). The organic layers were combined, dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in 2.44 g (56%) of ethyl 5-ethyl-1,2-thiazole-3-carboxylate as brown oil. LCMS (method A, ESI): RT=1.36 min, m/z=186.1 [M+H]+.
Into a 100-mL round-bottom flask was placed ethyl 5-ethyl-1,2-thiazole-3-carboxylate (244 g. 13.17 mmol, 1.00 equiv), methanol (10 mL), water (10 mL), tetrahydrofuran (10 mL) and LiOH.H217 (1.66 g, 39.56 mmol, 3.00 equiv). The resulting solution was stirred for 1 h at room temperature. The reaction mixture was then concentrated under vacuum. The residue was diluted with 30 mL of H2O and extracted with 5×30 mL of dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in 1.44 g (70%) of 5-ethyl-1,2-thiazole-3-carboxylic acid as a brown solid. 1H NMR (400 MHz, CDCl3) δ: 7.63 (s, 1H), 3.04-2.96 (m, 2H), 1.42-1.19 (m, 3H) ppm. LCMS (method D, ESI): RT=1.09 min, m/z=158.2 [M+H]+.
into a 250-mL round-bottom flask was placed 5-ethyl-1,2-thiazole-3-carboxylic acid (1.5 g, 9.54 mmol, 1.00 equiv), EDCI (2.92 g, 15.23 mmol, 1.60 equiv), 1H-1,2,3-benzotriazol-1-ol (2.1 g, 15.54 mmol, 1.63 equiv), dichloromethane (20 mL), and (2S)-tert-butyl 4-amino-2-methylpiperidine-1-carboxylate (2.45 g, 11.43 mmol, 1.20 equiv). Then TEA (2.89 g, 28.56 mmol, 2.99 equiv) was added dropwise. The resulting solution was stirred overnight at room temperature. The reaction mixture was diluted with 30 mL of H2O and extracted with 3×30 mL of dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was chromatographed on a combi-flash with eluent (EA:PE=1/1). This resulted in 1.5 g of (2S)-tert-butyl 4-(5-ethyl-1,2-thiazole-3-carboxamido)-2-methylpiperidine-1-carboxylate as a brown oil. LCMS (method D, ESI): RT=1.60 min, m/z=376.1 [M+Na]+.
Tert-Butyl(2S)-4-(5-ethyl-1,2-thiazole-3-carboxamido)-2-methylpiperidine-1-carboxylate (470 mg, 1.33 mmol, 1.00 equiv was purified by Chiral-Prep-HPLC with the following conditions: Column, CHIRALCEL OJ-3, mobile phase, Hex (0.2% IPA): EtOH=70:30; Detector, 254 nm. This resulted in 200 mg (43%) of ten-butyl (2S,4S)-4-(5-ethyl-1,2-thiazole-3-carboxamido)-2-methylpiperidine-1-carboxylate as a yellow solid. ee value: 100%
Into a 25-ml, round-bottom flask was placed tert-butyl (2S,4S)-4-(5-ethyl-1,2-thiazole-3-carboxamido)-2-methylpiperidine-1-carboxylate (200 mg, 0.57 mmol, 1.00 equiv), dichloromethane (10 mL). To the above hydrogen chloride (gas) was introduced. The resulting solution was stirred for 1 h at room temperature. The resulting mixture was then concentrated under vacuum. This resulted in 150 mg (91%) of 5-ethyl-N-[(2S,4S)-2-methylpiperidin-4-yl]-1,2-thiazole-3-carboxamide hydrochloride as a white solid. LCMS (method C, ESI): RT=0.49 min, m/z=254.4 [M−HCl+H]+.
Into a 25-mL round-bottom flask was placed 5-ethyl-N-[(2S,4S)-2-methylpiperidin-4-yl]-1,2-thiazole-3-carboxamide hydrochloride (150 mg, 0.52 mmol, 1.00 equiv) and dichloromethane (10 mL). Then TEA (260 mg, 5.00 equiv) added dropwise followed by tert-butyl N-[1-(chlorosulfonyl)piperidin-4-yl]carbamate (750 mg, 2.51 mmol, 4.85 equiv) which was added in several portions. The resulting mixture was stirred for 2 h at room temperature. The mixture was concentrated under vacuum and the residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (1/1). This resulted in 150 mg (56%) of tert-butyl N-[1-[(2S,4S)-4-(5-ethyl-1,2-thiazole-3-amido)-2-methylpiperidine-1-sulfonyl]piperidin-4-yl]carbamate, as a yellow solid. LCMS (method C, ESI): RT=1.57 min, m/z=516.2 [M+H]+.
Into a 25-nit round-bottom flask was placed tert-butyl N-[1-[(2S,4S)-4-(5-ethyl-1,2-thiazole-3-carboxamido)-2-methylpiperidine-1-sulfonyl]piperidin-4-yl]carbamate (150 mg, 0.29 mmol, 1.00 equiv) and dichloromethane (10 mL). To the above hydrogen chloride (gas) was introduced. The resulting solution was stirred for 1 h at room temperature. The mixture was then concentrated under vacuum. This resulted in 90 mg (68%) of N-[(2S,4S)-1-(4-aminopiperidine-1-sulfonyl)-2-methylpiperidin-4-yl]-5-ethyl-1,2-thiazole-3-carboxamide hydrochloride as a white solid. 1H NMR (300 MHz, D2O) δ:7.46 (s, 1H), 4.06-4.01 (m, 1H), 3.71-3.55 (m, 4H), 3.33-3.20 (m, 2H), 2.95-2.82 (m, 4H), 2.04-1.97 (m, 4H), 1.80-1.57 (m, 4H), 1.30-1.22 (m, 6H) ppm. LCMS (method A, ESI): RT=1.74 min, m/z=416.2 [M−HCl+H]+.
tert-Butyl (2S)-4-(5-ethyl-1,2-thiazole-3-carboxamido)-2-methylpiperidine-1-carboxylate (470 mg, 1.33 mmol, 1.00 equiv) was purified by Chiral-Prep-HPLC with the following conditions: Column: CHIRALCEL OJ-3-; mobile phase, Hex (0.2% IPA): EtOH=70:30 Detector, 254 nm. This resulted in 100 mg (21%) of tert-butyl (2S,4R)-4-(5-ethyl-1,2-thiazole-3-amido)-2-methylpiperidine-1-carboxylate as a yellow solid. ee value: 100%.
Into a 25-mL round-bottom flask was placed tert-butyl (2S,4R)-4-(5-ethyl-1,2-thiazole-3-carboxamido)-2-methylpiperidine-1-carboxylate (100 mg, 0.28 mmol, 1.00 equiv) and dichloromethane (10 mL). To the above hydrogen chloride (gas) was introduced. The resulting solution was stirred for 1 h at room temperature. The mixture was concentrated under vacuum. This resulted in 70 mg (85%) of 5-ethyl-N-[(2S,4R)-2-methylpiperidin-4-yl]-1,2-thiazole-3-carboxamide hydrochloride as a white solid. LCMS (method C, ESI): RT=0.49 min, m/z=254.2 [M−HCl+H]+.
Into a 25-mL round-bottom flask was placed 5-ethyl-N-[(2S,4R)-2-methylpiperidin-4-yl]-1,2-thiazole-3-carboxamide hydrochloride (70 mg, 0.24 mmol. 1.00 equiv) and dichloromethane (10 mL). TFA (120 mg) was added dropwise at 0° C. tert-Butyl N-[1-(chlorosulfonyl)piperidin-4-yl]carbamate (350 mg, 1.17 mmol, 4.85 equiv) was then added in several portions. The resulting solution was stirred for 2 h at room temperature. The mixture was then concentrated under vacuum. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (1/1). This resulted in 70 mg (56%) of tert-butyl N-[1-[(2S,4R)-4-(5-ethyl-1,2-thiazole-3-carboxamido)-2-methylpiperidine-1-sulfonyl]piperidin-4-yl]carbamate as a yellow solid. LCMS (method C, ESI): RT=1.54 min, m/z=538.2 [M+Na]+.
Into a 25-mL round-bottom flask was placed tert-butyl N-[1-[(2,S,4R)-4-(5-ethyl-1,2-thiazole-3-carboxamido)-2-methylpiperidine-1-sulfonyl]piperidin-4-yl]carbamate (70 mg, 0.14 mmol, 1.00 equiv) and dichloromethane (10 mL). To the above hydrogen chloride (gas) was introduced. The resulting solution was stirred for 1 h at room temperature. The mixture was then concentrated under vacuum. The crude product was purified by Prep-HPLC with the following conditions: Column: X Bridge RP, 19*150 mm, 5 um; Mobile Phase A:Water/0.05% NH4HCO3, Mobile Phase B: ACN; Flow rate: 30 mL/min; Gradient: 25% B to 45% B in 8 min; 254 nm. This resulted in 13.8 mg (24%) of N-[(2S,4R)-1-(4-aminopiperidine-1-sulfonyl)-2-methylpiperidin-4-yl]-5-ethyl-1,2-thiazole-3-carboxamide as a white solid, 1H NMR (400 MHz, CD3OD) δ: 7.57 (s, 1H), 4.40-4.25 (m, 1H), 4.25-4.15 (m, 1H), 3.67-3.62 (m, 3H), 3.30-3.15 (m, 1H), 3.06-3.00 (m, 2H), 2.89-2.79 (m, 3H), 1.98-1.85 (m, 5H), 1.80-1.58 (m, 2H), 1.51-1.40 (m, 1H), 1.40-1.36 (m, 6H) ppm. LCMS (method A, ESI): RT=1.68 min, m/z=438.1 [M+Na]+.
Into a 1-L round-bottom flask was placed methanol (600 mL), HCOONH4 (32 g. 507.45 mmol, 36.08 equiv) and tert-butyl (2S)-2-methyl-4-oxopiperidine-1-carboxylate (3 g, 14.07 mmol, 1.00 equiv). NaCNBH3 (1.7 g, 27.05 mmol, 1.92 equiv) was added batchwise slowly at 0-5° C. The resulting solution was stirred for 16 hours at 25° C. The reaction mixture was then diluted with 250 mL of ethyl acetate and washed with 3×250 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in 2.5 g (83% of tert-butyl (2S)-4-amino-2-methylpiperidine-1-carboxylate as colorless oil. 1H-NMR (400 MHz, CDCl3) δ: 4.13-4.11 (m, 1H), 3.98-3.97 (m, 1H), 3.49-3.28 (m, 2H), 2.24-2.10 (m, 2H), 1.76-1.75 (m, 2H), 1.45 (s, 9H), 1.27 (d, J=6.0Hz, 3H) ppm. LCMS (method D, ESI): RT=1.04 min, m/z=215.0 [M+H]+.
Into a 250-mL round-bottom flask was placed water (50 mL), tetrahydrofuran (50 sodium carbonate (3.7 g, 34.91 mmol, 2.99 equiv), and tert-butyl (2S)-4-amino-2-methylpiperidine-1-carboxylate (2.5 g, 11.67 mmol, 1.00 equiv). Then benzyl chloroformate (4 g, 23.45 mmol, 2.01 equiv) was added dropwise at 0-5° C. The resulting solution was stirred for 16 hours at 25° C. The resulting mixture was concentrated under vacuum. The residue was diluted with 100 mL of ethyl acetate and washed with 3×100 mL of brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (1/1). This resulted in 2 g (49% of tert-butyl (2S)-4-[[(benzyloxy)carbonyl]amino]-2-methylpiperidine-1-carboxylate as colorless oil. 1H-NMR (300 MHz, CD3OD) δ: 7.36-7.30 (m, 5H), 5.09 (s, 2H), 4.10-4.08 (m, 1H), 3.76-3.70 (m, 2H), 3.27-3.17 (m, 1H), 1.97-1.78 (m, 3H), 1.62-1.55 (m, 1H), 1.41 (s, 9H), 1.25 (d, 8.0Hz, 314) ppm. LCMS (method D, ESI): RT=1.57 min, m/z 349.3 [M+H]+.
Into a 25-mi, round-bottom flask was placed tert-butyl (2S)-4-[[(benzyloxy)carbonyl]amino]-2-methylpiperidine-1-carboxylate (400 mg, 1.15 mmol, 1.00 equiv) and dichloromethane (6 mL). Trifluoroacetic acid (3 mL) was then added dropwise at 0-5° C. The resulting solution was stirred for 30 min at 25° C. The mixture was concentrated under vacuum which resulted in 300 mg (crude) of benzyl N-[(2S)-2-methylpiperidin-4-yl]carbamate as a yellow liquid. LCMS (method A, ESI): RT=1.07 min, m/z=249.1 [M+H]+.
In to a 50-mL, round-bottom flask was placed benzyl N-[(2S)-2-methylpiperidin-4-yl]carbamate (300 mg, 1.21 mmol, 1.00 equiv) and triethylamine (600 mg, 5.93 mmol, 4.00 equiv in dichloromethane (30 mL). This was followed by the addition of 4-acetamidobenzene-1-sulfonyl chloride (720 mg, 3.08 mmol, 2.00 equiv) dropwise with stirring at 0° C. The resulting solution was stirred for 16 hours at 25° C. The mixture was then concentrated under vacuum. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (2/1). This resulted in 300 mg (56%) of benzyl N-[(2S)-1-[(4-acetamidobenzene)sulfonyl]-2-methylpiperidin-4-yl]carbamate as a yellow solid. LCMS (method D, ESI): RT=1.40 min, m/z=446.2 [M+H]+.
Into a 50-mL round-bottom flask was placed benzyl N-[(2S)-1-[(4-acetamidobenzene)sulfonyl]-2-methylpiperidin-4-yl]carbamate (300 mg, 0.67 mmol, 1.00 equiv) and trifluoroacetic acid (10 mL). The resulting solution was stirred for 1 hour at 60° C. in an oil bath. The mixture was then concentrated under vacuum. This resulted in 280 mg (crude) of N-[4-[(2S)-4-amino-2-methylpiperidine-1-sulfonyl]phenyl]acetamide as yellow oil. LCMS (method D, ESI): RT=0.96 min, m/z=312.2 [M+H]+.
N-[4-[(2S)-4-amino-2-methylpiperidine-1-sulfonyl]phenyl]acetamide (200 mg, 0.64 mmol, 1.00 equiv) was separated by Prep-SEC with the following conditions: Column, Lux 5u Cellulose-44.6*150 mm, 5 um Chiral-A (LUX-4); mobile phase, 25% IPA with MeOH: Detector, UV 254/220 nm. This resulted in 100 mg (100%) of N-[4-[(2S,4S)-4-amino-2-methylpiperidine-1-sulfonyl]phenyl]acetamide as a yellow solid and 40 mg (98%) of N-[4-[(2S,4R)-4-amino-2-methylpiperidine-1-sulfonyl]phenyl]acetamide as a yellow solid. ee value: 100%.
Into a 10-mL round-bottom flask was placed 2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid (48 mg, 0.27 mmol, 2.00 equiv), 1-hydroxybenzotrizole (40 mg, 1.26 mmol, 2.00 equiv), trethylamine (50 mg, 0.49 mmol, 4.00 equiv), N-[4-[(2S,4S)-4-amino-2-methylpiperidine-1-sulfonyl]phenyl]acetamide (40 mg, 0.13 mmol, 1.00 equiv), and dichloromethane (4 mL). N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (56 mg, 0.29 mmol, 2.00 equiv) was added batchwise at 0-5° C. The resulting solution was stirred for 16 hours at 25° C. The mixture was then washed with 3×5 mL of brine and the organic layer concentrated under vacuum. The crude product was purified by Prep-HPLC with the following conditions (2 #-Waters 2767-2 (HPLC-08)): Column, Xbridge Shield RP 18, Sum, 19*150 mm; mobile phase, water with 50 mmol NH4HCO3 and CH3CN (10.0% CH3CN up to 28.0% in 2 min, up to 46.0% in 10 min, up to 100.0% in 1 min, down to 10.0% in 1 min); Detector, UV 254 nm. This resulted in 2.6 mg (4%) of N-[(2S,4S)-1-[(4-acetamidobenzene)sulfonyl]-2-methylpiperidin-4-yl]-2-oxo-2,3-dihydro-1H-indole-5-carboxamide as a white solid. 1H-NMR (400 MHz, CD3OD) δ: 7.84-7.78 (m, 4H), 7.74-7.71 (m, 2H), 6.95 (d, J=8.0Hz, 1H), 3.95-3.90 (m, 2H), 3.62-3.51 (m, 1H), 3.33-3.32 (m, 2H), 3.15-3.04 (m, 1H), 2.19 (s, 3H), 2.06-1.98 (m, 1H), 1.93-1.88 (m, 1H), 1.73-1.68 (m, 2H), 1.40 (d, J=8.0Hz, 3H) ppm. LCMS (method D, ESI): RT=2.62 min, m/z=471.2 [M+H]+.
Into a 50-mL round-bottom flask was placed 5-ethyl-2,3-dihydropyridazin-3-one (100 mg, 0.81 mmol, 1.00 equiv) and POCl3 (5 mL). The resulting solution was stirred for 2 hours at 80° C. in an oil bath. The mixture was then concentrated under vacuum. The residue was extracted with 1×100 mL of dichloromethane and the organic layer washed with 50 mL of sodium bicarbonate (aq. sat.) and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was chromatographed on a silica get column with ethyl acetate/petroleum ether (30:100). This resulted in 80 mg (69%) of 3-chloro-54-ethylpyridazine as yellow oil. TLC, Rf=0.2 (PE:EA 10:1).
Into a 30-mL pressure tank reactor (100 mL) was placed 3-chloro-5-ethylpyridazine (80 mg, 0.55 mmol, 1.00 equiv), methanol (10 mL), triethylamine (112 mg, 1.11 mmol, 2.02 equiv), and Pd(dppf)Cl2 (148 mg). To the above CO (gas) was introduced and maintained at 30 atm. The resulting solution was stirred for 15 h at 80° C. The solids were filtered out. The filtrate was extracted with 2×100 mL of ethyl acetate and the organic layers combined, washed with 50 mL of brine, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (30:100). This resulted in 80 tug (86%) of methyl 5-ethylpyridazine-3-carboxylate as yellow oil. LCMS (method C, ESI): RT=0.78 rain, m/z 167.0 [M+H]+.
Into a 10-mL round-bottom flask was placed methyl 5-ethylpyridazine-3-carboxylate 80 mg, 0.48 mmol, 1.00 equiv) and C2H5OH (5 mL). This was followed by the addition of a solution of LiOH.H2O (100 mg, 2.4 mmol, 5.00 equiv) in water (1 mL) which was added dropwise with stirring at 0° C. The resulting solution was stirred for 3 hour at 20° C. The reaction was then quenched by the addition of 50 mL of water. The pfl was adjusted to 5 with hydrochloric acid (6N). The mixture was extracted with 2×100 nit of dichloromethane and the combined organic layers dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in 60 mg (34.7%) of 5-ethylpyridazine-3-carboxylic acid as black oil. LCMS (method D, ESI): RT 0.90 min, m/z=153.0 [M+H]+.
Into a 50-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed dichloromethane 20 mL) and 5-ethylpyridazine-3-carboxylic acid (60 mg, 0.39 mmol, 1.00 equiv). To the above was added tert-butyl N-[1-[(1R,3r,5S)-3-amino-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-yl]carbamate (150 mg, 0.39 mmol, 0.98 equiv), HOBT (79 mg, 0.58 mmol, 1.49 equiv)), and EDCI (223 mg, 1.17 mmol, 2.99 equiv). This was followed by the addition of a solution of triethylamine (118 mg, 1.17 mmol, 2.99 equiv) in dichloromethane (2 ml) which was added dropwise with stirring at 0° C. over 3 mm. The resulting solution was stirred for 15 hours at 20° C. The mixture was extracted with 2×100 mL of dichloromethane and the organic layers combined, washed with 50 mL of water and 50 mL of brine, and concentrated under vacuum. The residue was chromatographed on a silica gel column with ethyl acetate/petroleum ether (50:100). This resulted in 50 mg (24%) of tert-butyl N-[1-[(1R,3r,5 S)-3-(5-ethylpyridazine-3-carboxamido)-8-azabicyclo[3.2.1]octane-8-sulfonyl]piperidin-4-yl]carbamate as yellow oil. LCMS (method D, ESI): RT=1.46 min, m/z=523.0 [M+H]+.
Into a 25-mL round-bottom flask was placed tert-butyl N-[1-[(1R,3r,5S)-3-(5-ethylpyridazine-3-carboxamido)-8-azabicyclo[3,2,1]octane-8-sulfonyl]piperidin-4-yl]carbamate (50 mg, 0.10 mmol, 1.00 equiv) and hydrogen chloride/dioxane (10 mL, saturated, this solution was made by introducing hydrogen chloride gas into 1,4-dioxane under 0° C. for 6 hours). The resulting solution was stirred for 4 h at 20° C. The mixture was then concentrated under vacuum. The crude product was purified by Prep-HPLC with the following conditions: Column: X Select C18, 19*250 mm, 5 um; Mobile Phase A: Water/0.05% TFA, Mobile Phase B: ACN; Flow rate: 30 mL/min; Gradient: 12% B to 52% B in 11.5 min; 254 nm. This resulted in 17.9 mg (35%) of N-[(1R,3r,5S)-8-(4-aminopiperidine-1-sulfonyl)-8-azabicyclo[3.2.1]octan-3-yl]-5-ethylpyridazine-3-carboxamide trifluoroacetate as a yellow solid, 1H NMR (300 MHz, CD3OD) δ: 9.29 (s, 8.19 (s, 1H), 4.37-4.28 (m, 4.21-4.11 (s, 2H), 3.86 (d, J=15.0Hz, 2H), 3.30-3.20 (m, 1H), 2.99-2.80 (m, 4H), 2.45-2.30 (m, 2H), 2.18-2.00 (m, 8H), 1.79-1.61 (m, 2H), 1.40-1.29 (m, 3H) ppm. LCMS (method D, ESI): RT=1.27 min, m/z=423.2[M+H]+.
Into a 50-mL round-bottom flask was placed methyl 2-oxo-2,3-dihydro-1H-indole 5-carboxylate (800 mg, 4.18 mmol, LOU equiv) and methanol (10 mL), This was followed by the addition of a solution of NAM (670 rag, 16.75 mmol, 4.00 equiv) in water (10 mL) dropwise with stirring at 0° C. The resulting solution was stirred for 14 h at 20° C. The mixture was then concentrated under vacuum and the residue taken up in 20 mL of H2O. This was washed with 2×5 mL of dichloromethane. The pH was adjusted to 4 with hydrochloric acid (1 N) and extracted with 5×50 mL of ethyl acetate and the organic layers combined. Concentration resulted in 592 mg (80%) of 2-oxo-2,3-dihydro-1H indole-5-carboxylic acid as a yellow solid. 1H NMR (400 MHz, DMSO) δ: 12.5 (brs, 1H), 10.7 (s, 1H), 7.82 (d, J=8.4Hz, 1H), 7.76 (s, 1H), 6.88 (d, J=8.0Hz, 1H), 3.54 (s, 2H) ppm. LCMS (method A, ESI): RT=0.97 min, m/z=178.0 [M+H]+.
Into a 25-mL round-bottom flask was placed tert-butyl 4-aminopiperidine-1-carboxylate (300 nag, 1.50 mmol, 1.00 equiv), dichloromethane (10 mL), 2-oxo-2,3-dihydro-1H-indole-5-carboxylic acid (319 mg, 1.80 mmol, 1.20 equiv), EDCI (344 mg, 1.79 mmol, 1.20 equiv), and HOBT (304 mg, 2.25 mmol, 1.50 equiv). This was followed by the addition of TFA (454 mg, 4.49 mmol, 3.00 equiv) dropwise with stirring at 0° C. The resulting solution was stirred for 14 h at 20° C. The solids were collected by filtration. This resulted in 393 mg (73%) of tert-butyl 4-(2-oxa-2,3-dihydro-1H-indole-5-amido)piperidine-1-carboxylate as a yellow solid. LCMS (method C, ESI): RT=0.78 min, m/z=304.0 [M+H−56]+.
Into a 25-mL round-bottom flask was placed tert-butyl 4-(2-oxo-2,3-dihydro-1H-indole-5-carboxamido)piperidine-1-carboxylate (250 mg, 0.70 mmol, 1.00 equiv) and hydrogen chloride/dioxane: (3 mL, saturated, this solution was made by introducing hydrogen chloride gas into 1,4-diaxane under 0° C. for 6 hours). The resulting solution was stirred for 2 h at 20° C. The mixture was then concentrated under vacuum. This resulted in 200 mg (97%) of 2-oxo-N-(piperidin-4-yl)-2,3-dihydro-1H-indole-5-carboxamide hydrochloride as a light yellow solid. 1H NMR (400 MHz, D2O) δ: 7.65 (s, 2H), 6.95 (s, 1H), 4.04 (t, J=10.4Hz, 1H), 3.54 (s, 2H), 3.42 (d, J=13.2Hz, 2H), 3.12-3.01 (m, 2H), 2.13 (d, J=14.0Hz, 2H), 1.81-1.65 (m, 2H) ppm. LCMS (method A, ESI): RT=0.89 min, m/z=260.0 [M+H]+.
Into a 50-mL round-bottom flask was placed 2-oxo-N-(piperidin-4-yl)-2,3-dihydro-1H-indole-5-carboxamide hydrochloride (80 mg, 0.27 mmol, 1.00 equiv) and NMP (16 mL). This was followed by the addition of TFA (82 mg, 0.81 mmol, 3.00 equiv) dropwise with stirring at 0° C. To this was then added benzyl 4-[(chlorosulfonyl)methyl]piperidine-1-carboxylate (135 mg, 0.41 mmol, J=1.50 equiv) in several hatches at 0° C. The resulting solution was stirred for 2 h at 20° C. The mixture was concentrated under vacuum. The residue was chromatographed on a silica gel column with dichloromethane/methanol (50:1-20:1). The collected fractions were combined and concentrated under vacuum. This resulted in 100 mg (67%) of benzyl 4-[[4-(2-oxo-2,3-dihydro-1H-indole-5-carboxamdo)piperidine-1-sulfonyl]methyl]piperidine-1-carboxylate as a yellow solid. LCMS (method C, ESI): RT=1.04 min, m/z=555.0 [M+H]+.
Into a 25-mL round-bottom flask was placed benzyl 4-[[4-(2-oxo-2,3-dihydro-1H-indole-5-carboxamido)piperidine-1-sulfonyl]methyl]piperidine-1-carboxylate (80 mg, 0.14 mmol, 1.00 equiv) and hydrochloric acid (12 N, 5 mL). The resulting solution was stirred for 2 h at 20° C. The mixture was then concentrated under vacuum. The crude product was purified by Prep-HPLC with the following conditions: Column: X Bridge C18, 19*150 mm, 5 um; Mobile Phase A: Water/0.05% TFA, Mobile Phase B: ACN; Flow rate: 30 mL/min; Gradient: 15% B to 43% B in 10 mint Detector: 254 nm. This resulted in 13.2 mg (17%) of 2-oxo-N-[1-[(piperidin-4-ylmethane)sulfonyl]piperidin-4-yl]-2,3-dihydro-1H-indole-5-carboxamide trifluoroacetate as a white solid. 1H NMR (400 MHz, D2O) δ: 7.61 (s, 2H), 7.00 (d, J=4.4Hz, 1H), 4.00-3.90 (m, 1H), 3.75-3.66 (m, 2H), 3.60 (s, 2H), 3.43-3.37 (m, 2H), 3.15 (d, J=6.4Hz, 2H), 3.05-2.92 (m, 4H), 2.31-2.18 (m, 1H), 2.15-1.97 (m, 4H), 1.69-1.50 (m, 4H) ppm. LCMS (method A, ESI): RT=1.03 min, m/z=421.1 [M+H]+.
Oxalyl chloride (87.9 g, 693 mmol) was added to a cold solution of N,N-dimethylformamide (42.3 g. 578 mmol) in CHCl3 (150 mL) and the reaction was stirred, at room temperature for 30 min, followed by heating at 40 for a further 1 h. After chilling the reaction to −10 ethyl 2-diazoacetate (63.0 g, 552 mmol) was added and the mixture was stirred at room temperature for 2 h. The mixture was then concentrated and the residue was diluted with ether (200 mL), the solid was collected by filtration and dissolved in 10% aq. HOAc (200 mL), then stirred for a further 1h. The resulting mixture was extracted with ethyl acetate (300 mL×3) and the organic was washed with saturated Na2CO3 aq. (300 mL) and brine (300 mL), dried over Na2SO4, filtered and concentrated to give crude ethyl 2-diazo-3-oxopropanoate (27 g, 32.8%) as red oil, which was used for next step without further purification. 1H-NMR (400 MHz, CD3OD) δ ppm: 9.67 (s, 1H), 4.33 (q, J=7.2Hz, 2H), 1.32 (t, J=7.2, 3H).
To a solution of ethyl 2-diazo-3-oxopropanoate (27 g, 189 mmol) in EtOH (100 mL) was added acetic acid (28.3 g, 472 mmol). Cyclopropanamine (10.7 g, 189 mmol) was added slowly and, the mixture was stirred at room temperature overnight. The solvent was removed and saturated Na2CO3 aq. was added to the residue to bring the pH to 8. The mixture was extracted with ethyl acetate (200 mL×3), washed with brine (100 mL), dried over Na2SO4, filtered and concentrated. The resulting residue was purified by flash chromatography (PE:EA-2:1) to give crude ethyl 1-cyclopropyl-1H-1,2,3-triazole-4-carboxylate (18.5 g, 54.0%) as yellow oil. ESI-LCMS (m/z): 182.2 [M+H]+.
To a solution of ethyl 1-cyclopropyl-1H-1,2,3-triazole-4-carboxylate (18.5 g, 102 mmol) in THE (80 mL)/H2O (40 mL) was added lithium hydroxide hydrate (4.5 g, 107 mmol), the resulting mixture was stirred at room temperature for 3 hr. The solvent was removed and the residue was dissolved in H2O (50 mL), extracted with EA (100 mL). The organic phase was discarded and the water phase was acidified with 2N HCI until the pH=5, The aqueous solution was extracted with DCM:MeOH=10:1 (1.5 L). The organic layer was dried and concentrated to afford 6.3 g of 1-cyclopropyl-1H-1,2,3-triazole-4-carboxylic acid as white solid. The aqueous layer was concentrated to afford another 11.4 g crude product, which was used for next step without further purification. ESI-LCMS (mix): 154.1 [M+H]+.
A solution of 1-cyclopropyl-1H-1,2,3-triazole-4-carboxylic acid (2 g, 13.0 mmol) thionyl chloride (10 mL) was stirred at 65° C. for 2h. The reaction mixture was concentrated under reduced pressure. Then the reaction residue was diluted with DMF (5 mL) and added dropwise to the solution of tert-butyl 3-aminoazetidine-1-carboxylate (2.23 g, 13.0 mmol) and DIPEA (4.19 g, 32.5 mmol) in DCM (15 mL) under 0° C. The resulting mixture was stirred at room temperature overnight. The solvent was removed and the residue was diluted with ethyl acetate (200 mL), washed with water (10 mL×3) and brine (50 mL), dried over Na2SO4, filtered and concentrated. The resulting residue was purified by Flash chromatography (DCM:NH3 in MeOH(7N)=100:0˜50:1) to give tert-butyl 3-(1-cyclopropyl-1H-1,2,3-triazole-4-carboxamido)azetidine-1-carboxylate (3 g, 75.1%) as a yellow solid. ESI-LCMS (m/z): 252.2 [M−55]−.
A solution of tert-butyl 3-(1-cyclopropyl-1H-1,2,3-triazole-4-carboxamido)azetidine-1-carboxylate (3.0 g, 9.8 mmol) in HCl/MeOH (20 mL) was stirred at 50° C. for 2h. After completion, the solvent was removed in vacuo. The residue was dissolved in NH3/MeOH (7 mon, 20 nit) and stirred for 30 min. The solvent was removed and the residue was purified by Flash chromatography (DCM: NH3 in MeOH(7N)=100:0˜30:1-15:1) to give N-(azetidin-3-yl)-1-cyclopropyl-1H-1,2,3-triazole-4-carboxamide (1.6 g, 80%) as a white solid. ESI-LCMS (m/z): 208.1 [M+H]+. 1H-NMR (400 MHz, CD3OD) δ ppm: 8.38 (s, 1H), 4.92-4.86 (m, 1H), 3.98-3.97 (m, 1H), 3.82-3.72 (m, 4H), 1.26-1.20 (m, 4H).
To a solution of 5-chloropyridazin-3-ol (1.0 g, 7.7 mmol) in toluene/H2O (100: 5, 50 mL) was added sequentially cyclopropylboronic acid (987 mg, 11.5 mmol), K3PO4 (4.51 g, 23.0 mmol), diacetoxypalladium (86.2 mg, 384 umol) and tricyclohexylphosphine (107 mg. 384 μmol). The reaction mixture, was stirred at 100° C. under N2 atmosphere for 20 h. then concentrated in vacuum to remove the solvent Water (20 mL) was added and the solution was acidified with hydrochloric acid (4 M) to pH=3. The solution was extracted with EtOAc (200 mL×3) and the combined organic layer were washed with saturated NaCl solution, dried over Na2SO4, concentrated to give a brown residue then purified by silica-gel chromatography (PE:EA=1:1) to afford the desired product (350 mg, 34% yield) as white solid. ESI-LCMS (m/z): 137.1 [M+1]+.
A solution of 5-cyclopropylpyridazin-3-ol (350 mg, 2.6 mmol) in phosphorous oxychloride (10 mL) was stirred at 80° C. for 2 h. The remaining POO; was removed under vacuum then the residue was cooled and added to 20 g of ice. The reaction mixture was neutralized with saturated NaHCO3 solution and extracted with EtOAc (40 mL×3), the combined organic extract is washed with brine (100 mL×2), dried over Na2SO4 and the solvent is removed under vacuum, the resulting residue was purified by silica-gel chromatography (PE:EA=2:1) to afford the product 3-chloro-5-cyclopropylpyridazine as a colorless oil (200 mg, 50% yield). ESI-LCMS (m/z): 155.2 [M+1]+.
Potassium acetate (284 mg, 2.9 mmol) was added to a solution of 3-chloro-5-cyclopropylpyridazine (150 mg, 1.0 mmol) in ethanol (10 mL). The mixture was degassed, then Pd(dppf)Cl2 (35.4 mg, 0.05 mmol) was added. The resulting mixture was heated under an atmosphere of CO at 70T for 20 hr. The reaction mixture was filtrated and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA=1:1) to obtain the desired product ethyl 5-cyclopropylpyridazine-3-carboxylate (100 mg, 54% yield, colorless oil). ESI-LCMS (m/z): 193.1 [M+H]+; 1HNMR. (400 MHz, CD3OD) δ ppm: 9.06 (d, J=2.4Hz, 1H), 7.81 (d, J=2.4Hz, Hi), 7.39 (q, J=6.8Hz, 2H), 2.11-1.92 (m, 1H), 1.34 (t, J=6.8Hz, 3H), 0.96-0.93 (m, 2H).
To a solution of methyl 5-cyclopropylpyridazine-3-carboxylate (185 mg. 1.03 mmol) in THF/H2O (8 mL/2 mL) was added lithium hydroxide hydrate (64.6 mg, 1.54 mmol). The reaction mixture was stirred at room temperature for 3h. Progress of the reaction was monitored by TLC and LCMS. After completion, the mixture was adjusted to pH=5 with 1N HCl, then concentrated directly to 5-cyclopropylpyridazine-3-carboxylic acid (170 mg, 94.6%) as yellow solid and used in next step directly. ESI-LCMS (m/z): 165 [M+1]+.
A mixture of 2-chloro-3-methoxybenzaldehyde (2.38 g, 14 mmol) in THF (50 mL) was stirred at 0° C., and a solution of methylmagnesium bromide (5.6 mL, 16.7 mmol, 3M) in ether was added. The resulting mixture was stirred overnight. The reaction mixture was diluted with 1 N HO (50 mL) and extracted with ethyl acetate (50 mL×3), the combined organic phase was washed with brine, dried over sodium sulfate and concentrated to afford 1-(2-chloro-3-methoxyphenyl)ethanol (2.60 g, 99.6%) as a yellow oil. 1H NMR (400 MHz. (D3OD) δ ppm: 7.49 (d, J=8.4Hz, 1H), 6.90-6.85 (m, 2H), 5.25 ((4, J=6.4, 12.8Hz, 1H), 3.81 (s, 3H), 1.48 (d, J=6.4Hz, 3H).
A mixture of 1-(2-chloro-3-methoxyphenyl)ethonol (2.0 g, 10.7 mmol) and manganese(IV) oxide (4.65 g, 53.5 mmol) in DCM (50 mL) was heated to 40° C. and stirred overnight. The reaction mixture was filtered through Celite and the filtrate was concentrated to afford 1-(2-chloro-3-methoxyphertyl)ethanone (1.83 g, 89.8%) as a yellow oil. ESI-LCMS (m/z): 185.0 [M+1]+.
A mixture of 1-(2-chloro-3-methoxyphenyl)ethanone (540 mmol, 2.92 mmol) and aluminum trichloride (972 mg, 7.29 mmol) in monochlorobenzene (10 ML) was stirred at 120° C. for 2 hrs. After cooling to rt, the reaction mixture was added dropwise into 1N HCl in a water bath and the mixture was extracted with ethyl acetate (20 mL×3). The combined organic phase was washed with brine, dried over sodium sulfate and concentrated. The residue was purified by chromatography (PE:EA=5:1) to afford 1-(2-chloro-3-hydroxyphenyl)ethanone (400 mg, 80.3%) as a yellow solid ESI-LCMS (m/z): 171.0[M+H]+.
A mixture of 1-(2-chloro-3-hydroxyphenyl)ethanone (330 mg, 1.93 mmol), bromoethanol (482 mg, 3.86 mmol) and K2CO3 (800 mg, 5.79 mmol) in DMF (10 mL) was heated to 80° C. overnight. Water was added and the mixture was extracted with ethyl acetate (30 mL×3), the combined organic phase was washed with brine, dried over sodium sulfate and concentrated. The residue was purified by chromatography (DCM:MeOH=50:1) to afford 1-(2-chloro-3-(2-hydroxyethoxy) phenyl)ethanone (400 mg, 79.2%) as a yellow oil. ESI-LCMS (m/z): 251.1 [M+H]+.
A mixture of 1-(2-chloro-3-(2-hydroxyethoxy)phenyl)ethanone (214 mg, 1 mmol), tert-butyl azetidin-3-ylcarbamate (206 mg, 1.20 mmol), acetic acid (120 mg, 2.00 mmol) and NaBH3CN (125 mg, 2.00 mmol) in MeOH (10 ml) was stirred at 70° C. overnight. The reaction mixture was concentrated and adjusted pH=8-9 with a saturated solution of Na2CO3. The resulting mixture was extracted with DCM (30 mL×3) and the combined organic phase was washed with brine, dried over sodium sulfate and concentrated. The residue was purified by prep-TLC (DCM:MeOH=20:1) to afford tert-butyl (1-(1-(2-chloro-3-(2-hydroxyethoxy)phenyl)ethyl)azetidin-3-yl) carbamate (220 mg, 52.7%) as a yellow oil, ESI-LCMS (m/z): 371.2 [M+1]+.
A mixture of tert-butyl (1-(1-(2-chloro-3-(2-hydroxyethoxy)phenyl)ethyl and azetidin-3-yl)carbamate (200 mg, 539 umol) in a solution of HCl/MeOH (10 mL, 3N) was stirred at rt for 3 hrs. The reaction mixture was concentrated under reduced pressure to afford 2-(3-(1-(3-aminoazetidin-1-yl)ethyl)-2-chlorophenoxy)ethanol (120 mg, 68.9%) as a yellow oil. ESI-LCMS (m/z): 271.2 [M+1]+.
A mixture of 1-cyclopropyl-1H-1,2,3-triazole-4-carboxylic acid (22.9 mg, 150 μmol), 2 (3-(1-(3-aminoazetidin-1-yl)ethyl)-2-chlorophenoxy)ethanol (40.6 mg, 0.15 mmol), HATU (85.4 mg, 225 μmol) and DIPEA (38.6 mg, 300 μmol) in DMF (2 mL) was stirred at rt for 2 h. water was added and the mixture was extracted with ethyl acetate (10 mL×3), the combined organic phase was washed with brine, dried over sodium sulfate and concentrated. The residue was purified by Pre-TLC (DCM:MECH=20:1) to afford N-(1-(1-(2-chloro-3-(2-hydroxyethoxy)phenyl)ethyl)azetidin-3-yl)-4-cyclopropyl-1H-1,2,3-triazole-4-carboxamide (15 mg, 24.6%) as a wtite solid, ESI-LCMS (m/z): 406.1 [M+23]+. 1HNMR (400 MHz, CD3OD) δ ppm: 8.38 (s, 1H), 7.43 (d, J=8.4Hz, 1H), 7.00-6.93 (m, 2H), 4.66-4.59 (m, 1H), 4.06-3.95 (m, 4H), 3.89-3.86 (m, 2H), 3.83-3.79 (m, 1H), 3.53-3.50 (m, 1H), 3.22 (t, J=7.2Hz, 1H), 3.04 (t, J=7.2Hz, 1H), 1.30-1.19 (m, 7H).
N-(1-(1-(2-chloro-3-(2-hydroxyethoxy)phenyl)ethyl)azetidin-3-yl)-1-cyclopropyl-1H-1,2,3-triazole-4-carboxamide (350 mg, 862 μmol) was separated by chiral HPLC to obtain two single enantiomers, (isomer1:160 mg, white solid. Ret time 5.02 min; isomer 2:170 mg, Ret time 6.51 min), absolute stereochemistry is undefined, the isomer (Ret time:5.02 min) was assumed to be S configuration, (S)—N-(1-(1-(2-chloro-3-(2-hydroxyethoxy)phenyl)ethyl)azetidin-3-yl)-1-cyclopropyl-1H-1,2,3-triazole-4-carboxamide (160 mg, 45.8%) as a white solid. ESI-LCMS (m/z): 406.2 [M+1]+. 1HNMR (400 MHz, CD3OD) δ ppm: 8.38 (s, 1H), 7.44 (d, J=8.4Hz, 1H), 6.99-6.94 (m, 2H), 4.63 (1, J=7.2Hz, 1H), 4.06-3.98 (m, 4H), 3.89-3.86 (m, 2H), 3.82 (t, J=7.2 Hz, 1H), 3.52 (t, J=7.2Hz, 1H), 3.22 (t, J=7.2Hz, 1H), 3.05 (t, J=7.2Hz, 1H), 1.27-1.21 (m, 7H). SEC conditions: Instrument: SEC-80 (Thar, Waters) t; Column: REGISCELL 20*250 mm, 5 um (Dacel): Column temperature: 35° C.; Mobile phase: CO2/MEOH(0.2% methanol amina)=60/40: Flow rate: 80 g/min: Back pressure; 100 bar; Detection wavelength: 214 nm; Cycle time: 8.8 min; Sample solution: 350 mg dissolved in 15 ml Methanol; Injection volume: 3 mL.
To a solution of 1H-pyrazole-4-carbaldehyde (70 mg, 728 μmol) in MeCN (5 mL) was added 1-(bromomethyl)-4-chlorobenzene (149 mg, 728 umol) and cesium carbonate (172 mg, 1.15 mmol), The mixture was stirred at r.t. for 2h. The mixture was concentrated, diluted with EA and water. The organic phase was washed with brine (10 ml×2), dried over Na2SO4 and concentrated to give 1-(4-chlorobenzyl)-1H-pyrazole-4-carbaldehyde (162 mg, 101%) as a white solid, which was used in the next step without farther purification. ESI-LCMS (m/z): 221[M+H]+.
A mixture of N-(azetidin-3-yl)-1-cyclopropyl-1H-1,2,3-triazole-4-carboxamide (62 mg, 299 μmol), 1-(4-chlorobenzyl)-1H-pyrazole-4-carbaldehyde (65.9 mg, 299 μmol), and sodium cyanoborohydride (56.3 mg, 897 μmol) in MeOH (10 mL) was stirred at 60° C. for 16h. The mixture was cooled and concentrated. The residue was diluted with EA, washed with water (10 mL and brine (10 mL×2), dried over Na2SO4 and concentrated. The residue was purified by prep-HPLC to give N-(1-((1-(4-chlorobenzyl)-1H-pyrazol-4-yl)methyl)azetidin-3-yl)-1-cyclopropyl-1H-1,2,3-triazole-4-carboxamide (70 mg, 56.9%) as a white solid, ESI-LCMS (m/z): 412[M+H]+; 1HNMR (400 MHz, CD3OD) δ ppm: 838 (s, 1H), 7.67 (s, 1H), 7.50 (s, 1H), 7.35 (d, J=8.0Hz, 2H), 7.21 (d, J=8.0Hz, 2H), 5.31 (s, 2H), 4.65-4.61 (m, 1H), 4.00-3.96 (m, 1H), 3.71-3.67 (m, 2H), 3.61 (s, 2H), 3.22 (t, J=8.0Hz, 2H), 1.30-1.24 (m, 2H), 1.21-1.19 (m, 2H).
A mixture of 1-(4-chlorobenzyl)-1H-pyrazole-4-carbaldehyde (200 mg, 0.9063 mmol), tert-Butyl (azetidin-3-yl)carbamate hydrochloride (377 mg, 1.81 mmol) and acetic acid (54.4 mg, 0.9063 mmol) were dissolved in MeOH (40 ml). After stirred, for 2h at r.t., sodium cyanoborohydride (142 mg, 2.26 mmol) was added to the solution. The mixture was stirring over night at r.t. Then, the solution was concentrated under reduced pressure and the residue purified by flash column chromatography (eluant:DCM:MeOH=10:1 (600 ml)). To furnish tert-butyl (1-((1-(4-chlorobenzyl)-1H-pyrazol-4-yl)methyl)azetidin-3-yl)carbamate (273 mg, 54.2%) as colorless oil. ESI-LCMS (m/z): 377[M+H]+.
tert-Butyl (1-((1-(4-chlorobenzyl)-1H-pyrazol-4-yl)methyl)azetidin-3-yl)carbamate (273 mg, 0.4908 mmol) was dissolved in HCl in MeOH (3M) (20 ml). The solution was stirring over night at 50° C. After cooled to r.t., the solution was concentrated to afford 1-((1-(4-chlorobenzyl)-1H-pyrazol-4-yl)methyl)azetidin-3-amine (200 mg, 71.7%) as yellow solid, which was used for next step without purification. ESI-LCMS (m/z): 277 [M+H]+.
5-cyclopropylpyridazine-3-carboxylic acid (60 mg, 0.3654 mmol) and HATU (250 mg, 0.6577 mmol) were dissolved in DMF (6 ml). After stirred for 1h at r.t., 1-((1-(4-chlorobenzyl)-1H-pyrazol-4-yl)methyl)azetidin-3-amine (219 mg, 0.3836 mmol) and triethylamine (110 mg, 1.09 mmol) were added to the solution. Then, the mixture was stirring for 4h at r.t. Then, ethyl acetate (100 ml) was added and the ethyl acetate layer was washed by brine (50 mL×1), dried by Na2SO4, and concentrated. The residue was purified by Prep-HPLC to afford N-(1-((1-(4-chlorobenzyl)-1H-pyrazol-4-yl)methyl)azetidin-3-yl)-5-cyclopropylpyridazine-3-carboxamide (72 mg, 46.7%) as white solid. ESI-LCMS (m/z): 423[M+H]+; 1HNMR 1400 MHz, CD3OD) δ ppm: 9.15 (s, 1H), 7.86 (s, 1H), 7.69 (s, 1H), 7.51 (s, 1H), 7.36-7.32 (m, 2H), 7.20 (d, J=8.4Hz, 2H), 5.32 (s, 2H), 4.72-4.68 (m, 1H), 3.74-3.70 (m, 2H), 3.63 (s, 2H), 3.30-3.26 (m, 2H), 2.12-2.08 (m, 1H), 1.33-1.28 (m, 2H), 1.04-1.00 (m, 2H).
To a solution of 4-bromopicolinic acid (10.0 mmol, 2.02 g), HATU (15.0 mmol, 5.7 g), HOAT (15.0 mmol, 2.04 g) and DIPEA (20.0 mmol, 2.58 g) in DMF (50 ml) was added tert-butyl 3-aminoazetidine-1-carboxylate (10.0 mmol, 1.72 g) and stirred at r.t. overnight. Added ethyl acetate (300 ml), washed with water (150 ml×6), dried over Na2SO4, concentrated and the residue was crystallized with ethyl acetate:petroleum ether=1:5 to give pale yellow powder (2.63 g, 74%). ESI-LCMS (m/z): 356 [M+H]+.
To a solution of tert-butyl 3-(4-bromopicolinamido)azetidine-1-carboxylate (16.2 mmol, 5.8 g), cyclopropylboronic acid (32.4 mmol, 22.8 g), K3PO4 (48.5 mmol, 10.2 g) in dioxane (50 mL) was added Pd(dppf)Cl2 (1.61 mmol, 1.2 g). The mixture was degassed under reduced pressure while stirring and recharged with argon gas, this procedure was repeated for seven times and then heated to 100° C. overnight. The solvent was removed and the residue was purified by column chromatography (eluant:ethyl acetate:petroleum ether=1:3) to give tert-butyl 3-(4-cyclopropylpicolinamide)azetidine-1-carboxylate as a white solid (2.6 g, 50.5%). EST-LCMS (m/z): 318 [M+H]+.
tert-Butyl 3-(4-cyclopropylpicolinamido)azetidine-1-carboxylate (1.1 g, 3.46 mmol) in 3N HCl in MeOH (50 ml) was stirred at room temperature for 16h. LCMS showed the reaction was completed, the solvent was removed under reduced pressure, then NH in MeOH (7N, 10 ml) was added and concentrated. The crude product was purified by flash column chromatography (40 g silica gel column, eluted with DCM:NH3 in MeOH (7N)=10:1) to give the desired product N-(azetidin-3-yl)-4-cyclopropylpicolinamide (537 mg, 71.5%) as a white solid.
A mixture of N-(azetidin-3-yl)-4-cyclopropylpicolinamide (50 mg, 0.2301 mmol), 1-(4-chlorobenzyl)-1H-pyrazole-4-carbaldehyde (50.7 mg, 0.2301 mmol) and HOAc (34.5 mg. 0.6903 mmol) in MeOH (10 ml) was stirred at r.t. for 16 h. LCMS showed the reaction was completed, the solvent was removed under reduced pressure, NH3 in MeOH (7N, 10 ml) was added and concentrated, the crude product, which was purified by Prep-TLC (eluant:DCM:NH3 in MeOH (7N)=20:1) to give the desired product N-(1-((1-(4-chlorobenzyl)-1H-pyrazol-4-yl)methyl)azetidin-3-yl)-4-cyclopropylpicolinamide (30 mg, 30.9%) as a colorless oil. ESI-LCMS (m/z): 422[M+H]+, 1HNMR (400 MHz, CD3OD) ppm: 8.44 (d, J=5.2Hz, 1H), 7.76 (s, 1H), 7.68 (s, 1H), 7.51 (s, 7.35-7.32 (m, 2H), 7.28-7.26 (m, 1H), 7.20 (d, J=8.4Hz, 2H), 5.32 (s, 2H), 4.67-4.63 (m, 1H), 3.73-3.69 (m, 2H), 3.62 (s, 2H), 3.26-3.22 (m, 2H), 2.05-2.01 (m, 1H), 1.20-1.15 (m, 2H), 0.91-0.87 (m, 2H).
To a solution of 5-cyclopropyl-1H-imidazole (5.16 g, 47.7 mmol) in anhydrous THF (50 mL) was added NaH (2.85 g, 71.5 mmol) portion-wise at 0° C. under nitrogen atmosphere and the mixture was stirred 0° C. for 0.5 h. To the reaction mixture was added. SEM-Cl (11.9 g, 71.5 mmol) dropwise at 0° C. under nitrogen atmosphere and the mixture was stirred at 0° C. for 2 h. The mixture was concentrated in vacuo and the residue was purified with column chromotograph on silica gel (petroleum ether:ethyl acetate=2:1) to afford 4-cyclopropyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole (2.10 g, 18.4%) as a yellow liquid. ESI-LCMS (m/z): 239[M+H]+.
Into the stirred solution of 4-cyclopropyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole (400 mg, 1.7 mmol) in THF (20 mL) was added BuLi (0.7 mL, 2.5 M) at −70° C., the mixture was stirred at −70° C. for 1 h, then solid CO2 was added at −70° C., stirred for the next 1 h, acidified with HCl (1M). Concentrated in vacuum to obtained desired product (350 tug, yellow oil, Y: 74%), ESI-LCMS (m/z). 283[M+H]+.
Into the stirred solution of 4-cyclopropyl-1-((2-((trimethylsilyl)ethoxy)methyl)-1H-imidazole-2-carboxylic add (350 mg, 1.23 mmol), tert-butyl 3-aminoazetidine-1-carboxylate (316 mmol, 1.84 mmol) and HATU (700 mg, 1.84 mmol) in DMF (10 mL) was added DIPEA (478 mg, 17 mmol). The mixture was stirred at r.t. for 2 h. Concentrated in vacuum to remove the solvent, the residue was purified by Pre-TLC (petroleum ether:ethyl acetate 1:1) to afford tert-butyl 3(4-cyclopropyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-2-carboxamido)azetidine-1-carboxylate as a colorless oil (200 mg, 37%). ESI-LCMS (m/z): 437[M+H]+.
Into the stirred solution of tert-butyl 3-(4-cyclopropyl-14(2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-2-carboxamido)azetidine-1-carboxylate (200 mgn 0.5 mmol) in DCM (10 mL) was added TFA (444 mg, 4.6 mmol). The mixture was stirred at r.t. for 2 h. Concentrated in vacuum to remove the solvent to obtained the residue, basified with saturated NaHCO3, extracted with EtOAc (30 mL×3), combined the organic layer, dried over Na2SO4, concentrated in vacuum to obtained the crude N-(azetidin-3-yl)-4-cyclopropyl-1H-imidazole-2-carboxamide, which was used without further purification (80 mg, brown oil, Y: 84%). ESI-LCMS (m/z): 207[M+H]+.
Into the stirred solution of N-(azetidin-3-yl)-4-cyclopropyl-1H-imidazole-2-carboxamide (400 mg, 1.9 mmol) and 1-(4-chlorobenzyl)-1H-pyrazole-4-carbaldehyde (425 mg, 1.9 mmol) in MeOH (20 mL) was added NaBH3CN (600 mg, 9.6 mmol). The mixture was stirred at 60° C. for 20 h. The product was purified by reversed phase prep-HPLC (TFA, CH3CN:H2O=5%-95%) to afford N-(1-((1-(4-chlorobenzyl)-1H-pyrazol-4-yl)methyl)azetidin-3-yl)-4-cyclopropyl-1H-imidazole-2-carboxamide (49 mg, 6%). ESI-LCMS (m/z): 411[M+H]+; 1HNMR (400 MHz, CD3OD) δ ppm: 7.92 (s, 1H), 7.67 (s, 1H), 7.36-7.34 (m, 2H), 7.24 (d, J=8.8Hz, 2H), 6.91 (brs, 1H), 5.36 (s, 2H), 4.77-4.74 (m, 1H), 4.31-4.26 (m, 4H), 4.16-4.11 (m, 2W, 1.91-1.87 (m, 1H), 0.93-0.90 (m, 2H), 0.75-0.72 (m, 2H).
To a mixture of 1H-pyrazole-4-carbaldehyde (100 mg, 1.04 mmol) and 1-(chloromethyl)-4-methoxybenzene (162 mg, 1.04 mmol) in CH3CN (6 mL) was added potassium carbonate (287 mg, 2.08 mmol), the resulting mixture was stirred at reflux for 1 h. The mixture was poured into water (20 rut) and extracted with EtOAc (3×20 The organic layer was dried over Na2SO4 and concentrated to give 1-(4-methoxybenzyl)-1H-pyrazole-4-carbaldehyde (220 mg, 93.3%) as a colorless oil. ESI:LCMS (m/z): 217[M+H]+.
To a mixture of N-(azetidin-3-yl)-1-cyclopropyl-1H-1,2,3-triazole-4-carboxamide (50 mg, 0.2412 mmol) and 1-(4-methoxybenzyl)-1H-pyrazole-4-carbaldehyde (62.5 mg, 0.2894 mmol) in methanol (5 mL) was added acetic acid (724 μg, 0.01206 mmol), the mixture was stirred at r.t. for 15 min, then sodium cyanoborohydride (30.3 mg, 0.4824 mmol) was added, the resulting mixture was stirred at 40° C. overnight. The mixture was concentrated under reduced pressure. The residue was dissolved in aqueous solution of Na2CO and extracted with EtOAc (3×20 mL). The organic layer was dried over Na2SO4 and concentrated. The residue was purified by prep-TLC (CH2Cl2/NH3MeOH=20/1) to give 1-cyclopropyl-N-(1-((1-(4-methoxybenzyl)-1H-pyrazol-4-yl)methyl)azetidin-3-yl)-1H-1,2,3-triazole-4-carboxamide: (53 mg, 53.9%) as a white solid. ESI-LCMS (m/z): 408[M+H]−; 1HNMR (400 MHz, CD3OD) δ ppm: 8.39 (s, 1H), 7.62 (s, 1H), 7.48 (s, 1H), 7.19 (d, J=8.4Hz, 2H), 6.90 d, J=8.4Hz, 2H), 5.24 (s, 2H), 4.64-4.62 (m, 1H), 3.99-3.98 (m, 1H), 3.79 (s, 3H), 3.73-3.69 (m, 2H), 3.63 (s, 2H), 3.27-3.23 (m, 2H), 1.28-1.22 (m, 4H).
S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), Tris, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), and Tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) were purchased from Sigma-Aldrich at the highest level of purity possible. 3H-SAM was purchase from American Radiolabeled Chemicals with a specific activity of 80 Ci/mmol. 384-well opaque white OptiPlates and SPA beads (Perkin Elmer, catalog #RPNQ0013) were purchased from PerkinEimer.
N-terminally GST-tagged MEKK2 (MAP3K2) protein corresponding to reference sequence AAF63496.3 was purchased from Life Technologies (catalog; #PV4010). This protein was expressed in High Five insect cells and purified to >85% purity. Protein identity was confirmed by MS/MS analysis after proteolytic digestion. The protein sequence used was:
Full-length human SMYD3 isoform 1 (BAB86333) was inserted into a modified pET21b plasmid containing a His6 tag and TEV and SUMO cleavage sites. Because two common variants of SMYD3 exist in the population, site directed mutagenesis was subsequently performed to change amino acid 13 from all asparagine to a lysine, resulting in plasmid pEPZ533. A lysine at position 13 conforms to the more commonly occurring sequence (NP_001161212).
E. coli (BL21 codon plus RIL strain, Stratagene) were transformed with plasmid pEPZ553 by mixing competent cells and plasmid DNA and incubating on ice for 30 minutes followed by heat shock at 42° C. for 1 minute and cooling on ice for 2 minutes Transformed cells were grown and selected on LB agar with 100 μg/mL ampicillin and 17 μg/mL chloramphenicol at 37° C. overnight. A single clone was used to inoculate 200 mL of LB medium with 100 μg/mL ampicillin and 17 μg/mL chloramphenicol and incubated at 37° C. on an orbital shaker at 180 rpm. Once in log growth, the culture was diluted 1:100 into 2 L of LB medium and grown until OD600 was about 0.3 after which the culture was incubated at 15° C. and 160 rpm. Once OD600 reached about 0.4, IPTG was added to a final concentration of 0.1 mM and the cells were grown overnight at 15 GC and 160 rpm. Cells were harvested by centrifugation at 8000 rpm, for 4 minutes at 4° C. and stored at −80° C. for purification,
Expressed full-length human His-lagged SMYD3 protein was purified from cell paste by Nickel affinity chromatography after equilibration of the resin with Buffer A (25 mM Tris, 200 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol, pH7.8). The column was washed with Buffer B (Buffer A plus 20 mM imidazole) and His-tagged SMYD3 was eluted with Buffer C (Buffer A plus 300 mM imidazole). The His tag, TEV and SUMO cleavage sites were removed generating native SMYD3 by addition of ULP1 protein at a ratio of 1:200 (ULP1:SMYD3). Imidazole was removed by dialysis overnight in Buffer A. The dialyzed solution was applied to a second Nickel column and the native SMYD3 protein was collected from the column flow-through. The flow-through was dialyzed in Buffer D (25 mM iris, 5% glycerol, 5 mM β-mercaptoethanol, 50 mM NaCl, pH7.8) and ULP1 was removed using a Q sepharose fast flow column. SMYD3 was eluted in Buffer A and further purified using an S200 size-exclusion column equilibrated with Buffer A. SMYD3 was concentrated to 2 mg/mL with a final purity of 89%.
General Procedure for SMYD3 Enzyme Assays on MEKK2 protein Substrate
The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (ltd) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid, handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P far the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume Slue. The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (100) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).
Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.
Where top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
SMYD3 biochemical assay data for representative Compounds of the Disclosure are presented in Tables 1A and 3A in the column titled “SMYD3 Biochem IC50 (μM).”
293T/17 adherent cells were purchased from ATCC (American Type Culture Collection), Manassas, Va., USA. MEM/Glutamax medium, Optimem Reduced Serum medium, penicillin-streptomycin, 0.05% trypsin and ix D-PBS were purchased from Life Technologies, Grand island, NY, USA. PBS-10× was purchased from Ambion, Life Technologies, Grand Island, N.Y., USA. PBS with Tweet 20 (PBST (10×)) was purchased from KPL, Gaithersburg, Md., USA. Tet System FBS-approved FBS US Source was purchased from Clontech, Mountain View, Calif., USA. Odyssey blocking buffer, 800CW goat anti-rabbit IgG (H+L) antibody, 680CW Goat anti-mouse IgG (H+L) and Licor Odyssey infrared scanner were purchased from Licor Biosciences, Lincoln, Nebr., USA. Tri-methyl-Lysine [A260]-MEKK2 antibody, MEKK2 and SMYD3 plasmids were made at Epizyme. Anti-flag monoclonal mouse antibody was purchased from Sigma, St. Louis, Mo., USA. Methanol was purchased from VWR, Franklin, Mass., USA. 10% Tween 20 was purchased from KPL, Inc. Gaithersburg, Md., USA. Fugene was purchased from Promega, Madison, Wis., USA. The Biotek ELx405 was purchased from BioTek, Winooski, Vt., LISA. The multidrop combi was purchased from Thermo Scientific, Waltham, Mass., USA.
293T/17 adherent cells were maintained in growth medium (MEM/Glutamax medium supplemented with 10% v/v Tot System PBS and cultured at 37° C. under 5% CO2.
Cell treatment. In Cell Western (ICW) for detection of trimethyl-lysine-MEKK2 and MEKK2.
293T/17 cells were seeded in assay medium at a concentration of 33,333 cells per cm2 in 30 mL medium per T150 flask and incubated at 37° C. under 5% CO2. Plasmids were prepared for delivery to cells by first mixing 1350 μL Opti-MEM with Eugene (81 μL) in a sterile Eppendorf and incubated for five minutes at room temperature (RT). MEKK2-flag (13.6 ug/T150) MEKK2 p3XFlag-CMV-14 with C-3XFlag and SMYD3 (0.151 ug/T150) SMYD3 p3XFlag-CMV-14 without C-3XFlag plasmids were aliquotted to a 1.7 ml, sterile microfuge tube. The gene ID for MEKK2 and SMYD3 is NM_006609.3 and Q9H7B4, respectively. Entire volume of Opti-MEM/Fugene mixture was then added to a microfuge tube containing DNA plasmid, mixed and then incubated ×15 minutes at RT. The medium on the 2931/17 cells was refreshed, and the DNA/Fugene complex is added aseptically to each flask, rocked gently, and incubated at 37 C for 5 hours. Medium was then removed, and cells were washed once with PBS in the flask. Trypsin 0.05% (3 mL) was added and cells incubated for three minutes. Room temperature MEM+10% Tct system PBS was added and cells were mixed gently, and counted using the Vi-cell. Cells were seeded at 100,000 cells/mL in 50 μL MEM/10% Tet FBS/Pen/Strep to a 384 well black/clear poly-T)-lysine coated plate containing test agent diluted in DMSO. The final top concentration of test compound was 40 μM. The total concentration of DMSO did not exceed 0.2% (v/v). Plates were incubated×30 minutes at RT in low-airflow area, followed by incubation at 37° C. under 5% CO2 for 24 hours. Medium was aspirated from all wells of assay plates prior to fixation and permeabilization with ice cold (−20° C.) methanol (90 ullwell) for ten minutes. Plates were rinsed with PBS three times on BioTek ELx405. PBS was removed with a final aspiration, and Odyssey blocking buffer (50 μL/well) was added to each well and incubated for one hour at RT. Primary antibody solution was prepared (anti-trimethyl-MEKK2 at 1:600 dilution plus mouse anti-flag antibody at 1:10,000 dilution in diluent (Odyssey Blocking buffer+0.1% Tween 20)) and 20 μL per well was dispensed using the Multidrop Combi. Assay plates were then sealed with foil, and incubated overnight at 4° C. Plates were washed five times with PBS-Tween (1×) on Biotek ELx405 and blotted on paper towel to remove excess reagent. Detection antibody solution (IRDye 800 CW goat anti-rabbit IgG diluted 1:400 in diluent (Odyssey Blocking buffer+0.1% Tween 20), plus IRDye 680CW goat anti-mouse IgG at 1:500 in diluent (Odyssey Blocking buffer+0.1% Tween 20) was added (20 μL/well) and incubated in dark for one hour at RT. Plates were then washed four times with PBS-T (1×) on ELx405. A final rinse with water was performed (115 μL/well×three washes on the ELx405), Plates were then centrifuged upside down, on paper towel, at 200×g to remove excess reagent. Plates were left to dry in dark for one hour. The Odyssey imager was used to measure the integrated intensity of 700 and 800 wavelengths at resolution of 84 am, medium quality, focus offset 4.0, 700 channel intensity=3.5 to measure the MEKK2-flag signal, 800 channel intensity=5 to measure the Trimethyl-MERK2 signal of each well.
First, the ratio for each well was determined by:
Each plate included fourteen control wells of DMSO only treatment (Minimum Inhibition) as well as fourteen control wells for maximum inhibition (Background). The average of the ratio values for each control type was calculated and used to determine the percent inhibition for each test well in the plate. Reference compound was serially diluted two-fold in MIND for a total of nine test concentrations, beginning at 40 μM. Percent inhibition was calculated (below).
Non-linear regression curves were generated to calculate the IC50 and dose-response relationship using triplicate wells per concentration of compound.
SMYD3 cell assay data for representative Compounds of the Disclosure are presented in Tables 1A and 3A in the column titled “SMYD3 Cell IC50 (μM).”
S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), and Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich at the highest level of purity possible. 3H-SAM was purchase from American Radiolabeled Chemicals with a specific activity of 80 Ci/mmol, 384-well streptavidin Flashplates were purchased from PerkinElmer.
Peptide was synth sized with a N-terminal linker-affinity tag motet and a C-terminal amide cap by 21st Century Biochemicals. The peptide was high high-performance liquid chromatography (HPLC) purified to greater than 95% purity and confirmed by liquid chromatography mass spectrometry (LC-MS). The sequence was ARTKQTARKSTGGKAPRKQLATKAARKSA (K-Biot)-amide. (SEQ ID NO: 3)
Full length SMYD2 (NP_064582.2) was cloned into a pFastbac-Htb-lic vector with an N-terminal His6 tab and FLAG tab, preceded by a TEV protease cleavage site. The protein was expressed in Sf9 insect cells. Cells were resuspended in lysis buffer (2.5 mM HEPES-NaOH. pH 7.5, 200 mM NaCl, 5% glycerol, and 5 mM β-ME) and lysed by sonication. The protein was purified by Ni-NTA (Qiagen), followed by TEV cleavage to remove the His6 tag, subtractive Ni-NTA (Qiagen), and gel filtration chromatography using an S200 column (GE Healthcare). Purified protein was stored in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1 mM TCEP.
The assays were all performed in a buffer consisting of 20 mM Bicine (pH-7.6), 1 mM TCEP, 0.005% Bovine Skin Gelatin, and 0.002% Tween20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a polypropylene 384-well V-bottom plates (Greiner) using a Platemate Plus outfitted with a 384-channel head (Thermo Scientific). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMTYD2, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD2 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD2 for 30 ruin at room temperature, then a cocktail (10 ul) containing, 3H-SAM and peptide was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD2 was 1.5 nM, 3H-SAM was 10 nM, and peptide was 60 nM, SAH in the minimum signal control wells was 1000 uM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radioactive SAM (10 ul) to a final concentration of 600 uM, which dilutes the 3H-SAM to a level where its incorporation into the peptide substrate is no longer detectable. 50 ul of the reaction in the 384-well polypropylene plate was then transferred to a 384-well Flashplate and the biotinylated peptides were allowed to bind to the streptavidin surface for at least 1 hour before being washed three times with 0.1% Tween20 in a Biotek ELx405 plate washer. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled peptide bound to the Flashplate surface, measured as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).
Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.
Where top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. I is the compound concentration.
SMYD2 biochemical assay data for representative Compounds of the Disclosure are presented in Tables 4A and 6A in the column titled “SMYD2 Biochem IC50 (μM).”
Having now fully described this invention, it will be understood by those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any embodiment thereof.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
All patents and publications cited herein are fully incorporated by reference herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62048773 | Sep 2014 | US | |
| 62146799 | Apr 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15510586 | Mar 2017 | US |
| Child | 16269338 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16864891 | May 2020 | US |
| Child | 17532422 | US | |
| Parent | 16269338 | Feb 2019 | US |
| Child | 16864891 | US |
