Patent Application
20240174675
Pharmacological stimulation of lower airway repair has significant potential for treating a variety of conditions in which alveolar destruction and maladaptive remodeling are causative of disease. The alveolus, the primary unit of mammalian gas exchange, is composed of two epithelial cell types: large squamous alveolar type 1 cells (AEC1s), which provide surface area for gas exchange, and cuboidal alveolar type 2 cells (AEC2s), which secrete surfactant.1 In addition, AEC2s have been identified as the primary progenitor cell type responsible for repopulating the alveolar epithelium.2 AEC2s clonally proliferate over adulthood, asymmetrically dividing to give rise to AEC1s and AEC2s.2 It has been additionally demonstrated that idiopathic pulmonary fibrosis (IPF) is caused by exhaustion of the stem cell capacity of AEC2s.3 Diminished AEC2 proliferation results in denuded alveolar basement membranes, which ultimately promotes colonization of the lower airway by hyperplastic upper airway-derived epithelial cells and extracellular matrix-secreting myofibroblasts.3 Additionally, it has been demonstrated that restoring AEC2 proliferation through treatment with exogenous factors (IL-6 or hyaluronic acid) inhibits disease severity in mouse models of IPF.4 In addition to IPF, acute respiratory distress syndrome (ARDS)—the acute loss of alveolar epithelial barrier function—is caused by damage to and insufficient reparative growth by AEC2 cells.5
The present disclosure provides, in various embodiments, a compound that is useful, for example, in promoting specific proliferation of AEC2s relative to other cell types in the lung. The compound in some embodiments is of formula (I) or pharmaceutically acceptable salt thereof.
In formula (I), each — represents a single bond that, when optionally present, form a fused cyclopropyl ring.
Subscript m1 is an integer that is 0 when Z1 is H, and is 1 when Z1 is other than H. Subscript n1 is an integer selected from 0, 1, 2, and 3.
R1 is selected from H, C1-C10-alkyl, and —C1-C10-alkyl-(C6-C10-aryl), and is optionally substituted with one to six —OH. R2 is C1-C10-alkyl substituted with one to six —OH.
Alternatively, per additional embodiments, the compound is formula (II) or a pharmaceutically acceptable salt thereof:
In formula (II), W is CH or N.
Subscript o is an integer selected from 1, 2, and 3.
R3 is selected from C1-C6-alkyl, C1-C6-hydroxyalkyl, and —(CH2CH2O)xH (wherein x is an integer selected from 1, 2, 3, 4, and 5). R4 is C2-C8-alkynyl.
R5a, R5b, R5c, and R5d are independently selected from H, C1-C6-alkyl, halo, —NRARB (wherein RA and RB are independently selected from H and C1-C10-alkyl), —C(O)OH, —B(OH)2, —C(O)NRARB, —C(O)ORA, and —C(O)-L2-Z2[(CH2)n2—NR6R7]m2. L2 is a C2-C12-alkyl wherein one or more —CH2— are optionally and independently replaced by a moiety selected from —O—, —C(O)—, and —NH—. Z2 is selected from H, C6-C10-aryl, and 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S). At least one of R5a, R5b, R5c, and R5d is other than H.
R6 is selected from H, C1-C10-alkyl, and —C1-C10-alkyl-(C6-C10-aryl), and is optionally substituted with one to six —OH. R7 is C1-C10-alkyl substituted with one to six —OH.
Subscripts m2 is an integer that is 0 when Z1 is H, and is 1 when Z1 is other than H. Subscript n2 is an integer selected from 0, 1, 2, and 3.
Further, when W is CH, then W5a and R5d are not selected from —C(O)OH, —C(O)OMe, and —C(O)OEt.
Alternatively, per additional embodiments, the compound is of formula (III) or a pharmaceutically acceptable salt thereof:
In formula (III), X3 is —O— or —NH—. L3 is a bond or C2-C12-alkyl wherein one or more —CH2— are optionally and independently replaced by a moiety selected from —O—, —C(O)—, and —NH—. Z3 is selected from H, —N3, C6-C10-aryl, 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), and (3- to 14-membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S). Heteroaryl and heterocycloalkyl are optionally substituted with 1 to 6 substituents selected from the group consisting of halo, NO2, OH, CN, and C1-C6-haloalkyl.
Subscript m3 is an integer that is 0 when Z3 is H or —N3, and is 1 when Z is other than H or —N3. Subscript n3 is an integer selected from 0, 1, 2, and 3.
R8 is selected from H, C1-C10-alkyl or —C1-C6-alkyl-(C6-C10-aryl), and is optionally substituted with one to six —OH. R9 is C1-C10-alkyl substituted with one to six —OH. R10 is C1-C6-haloalkyl. Each R9 is independently selected from H, C1-C6-alkyl, and halo.
Subscript o3 is an integer selected from 0, 1, 2, and 3. Subscript p3 is an integer selected from 0, 1, 2, and 3. Subscript q3 is an integer selected from 0, 1, 2, and 3.
The compound of the present disclosure does not include any of the following compounds:
In another embodiment, the present disclosure provides a pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof as described herein.
The present disclosure also provides, in an embodiment, a method for selectively increasing the proliferation of cuboidal alveolar type 2 (AEC2) cells in a subject in need thereof, or for restoring diminished proliferation of AEC2 cells in a subject in need thereof. The method comprises administering to the subject a compound or pharmaceutically acceptable salt thereof as described herein.
In an embodiment, the present disclosure provides a method for inhibiting dipeptidyl peptidase IV (DPP4) in a subject in need thereof. The method comprises administering to the subject a compound or pharmaceutically acceptable salt thereof as described herein.
Another embodiment of the present disclosure is a method for treating a pulmonary disease in a subject suffering therefrom. The method comprises administering to the subject a compound or pharmaceutically acceptable salt thereof as described herein.
Also provided in various embodiments is a compound or pharmaceutically acceptable salt thereof as described herein for use in selectively increasing the proliferation of cuboidal alveolar type 2 (AEC2) cells in a subject in need thereof, or for restoring diminished proliferation of AEC2 cells in a subject in need thereof.
In additional embodiments, the present disclosure provides a compound or pharmaceutically acceptable salt thereof as described herein for use in inhibiting dipeptidyl peptidase IV (DPP4) in a subject in need thereof.
In still additional embodiments, the present disclosure provides a compound or pharmaceutically acceptable salt thereof as described herein for use in treating a pulmonary disease in a subject suffering therefrom.
The present disclosure satisfies a long-felt need for drug-like compounds that stimulate reparative proliferation of pulmonary stem- and progenitor-cell populations. Compounds of the present disclosure promote specific proliferation of AEC2s relative to other cell types in the lung (e.g., pulmonary fibroblasts) and thereby exhibit disease-modifying efficacy in a number of lower airway diseases. Furthermore, the compounds are useful as inhibitors of dipeptidyl peptidase IV (DPP4).
“Alkyl” refers to straight or branched chain hydrocarbyl including from 1 to about 20 carbon atoms. For instance, an alkyl can have from 1 to 10 carbon atoms or 1 to 6 carbon atoms. Exemplary alkyl includes straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, and the like, and also includes branched chain isomers of straight chain alkyl groups, for example without limitation, —CH(CH3)2, —CH(CH3)(CH2CH3), —CH(CH2CH3)2, —C(CH3)3, —C(CH2CH3)3, —CH2CH(CH)2, —CH2CH(CH3)(CH2CH3), —CH2CH(CH2CH3)2, —CH2C(CH3)3, —CH2C(CH2CH3)3, —CH(CH 3)CH(CH3)(CH2CH3), —CH2CH2CH(CH3)2, —CH2CH2CH(CH3)(CH2CH3), —CH2CH2CH(CH2CH3) z, —CH2CH2C(CH3)3, —CH2CH2C(CH2CH3)3, —CH(CH3)CH2CH(CH3)2, —CH(CH3)CH(CH3)CH(CH3)2, and the like. Thus, alkyl groups include primary alkyl groups, secondary alkyl groups, and tertiary alkyl groups. An alkyl group can be unsubstituted or optionally substituted with one or more substituents as described herein.
The term “haloalkyl” is an alkyl as defined herein that is substituted with 1, 2, 3, 4, 5, or 6 halo. An illustrative haloalkyl is —CF3.
Each of the terms “halogen,” “halide,” and “halo” refers to —F or fluoro, —Cl or chloro, —Br or bromo, or —I or iodo.
“Alkyne or “alkynyl” refers to a straight or branched chain unsaturated hydrocarbon having the indicated number of carbon atoms and at least one triple bond. Examples of a (C2-C8)alkynyl group include, but are not limited to, acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne, 2-heptyne, 3-heptyne, 1-octyne, 2-octyne, 3-octyne and 4-octyne. An alkynyl group can be unsubstituted or optionally substituted with one or more substituents as described herein.
“Aryl” when used alone or as part of another term means an optionally fused carbocyclic aromatic group having the number of carbon atoms designated or if no number is designated, up to 14 carbon atoms, such as a C6-C14-aryl or C6-C10-aryl. Illustrative aryl groups are phenyl, naphthyl, biphenyl, phenanthrenyl, naphthacenyl, and the like (see e.g. Lang's Handbook of Chemistry (Dean, J. A., ed) 13th ed. Table 7-2 [1985]). An exemplary aryl is phenyl. “Aryl” can be optionally fused with a cycloalkyl ring, as herein defined. An aryl group can be unsubstituted or optionally substituted with one or more substituents as described herein.
The term “heteroatom” refers to N, O, and S. Compounds of the present disclosure that contain N or S atoms can be optionally oxidized to the corresponding N-oxide, sulfoxide, or sulfone compounds.
“Heteroaryl,” alone or in combination with any other moiety described herein, refers to a monocyclic aromatic ring structure containing 5 to 10, such as 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10 atoms, containing one or more, such as 1-4, 1-3, or 1-2, heteroatoms independently selected from the group consisting of O, S, and N. Heteroaryl is also intended to include oxidized S or N, such as sulfinyl, sulfonyl and N-oxide of a tertiary ring nitrogen. A carbon or heteroatom is the point of attachment of the heteroaryl ring structure such that a stable compound is produced. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrazinyl, quinoxalyl, indolizinyl, benzo[b]thienyl, quinazolinyl, purinyl, indolyl, quinolinyl, pyrimidinyl, pyrrolyl, pyrazolyl, oxazolyl, thiazolyl, thienyl, isoxazolyl, oxathiadiazolyl, isothiazolyl, tetrazolyl, imidazolyl, triazolyl, furanyl, benzofuryl, and indolyl. A heteroaryl group can be unsubstituted or optionally substituted with one or more substituents as described herein.
“Heterocycloalkyl” is a saturated or partially unsaturated non-aromatic monocyclic, bicyclic, tricyclic or polycyclic ring system, optionally spiro-fused, that has from 3 to 14, such as 3 to 6, atoms in which 1 to 3 carbon atoms in the ring are replaced by heteroatoms of O, S or N. A heterocycloalkyl is optionally fused with aryl or heteroaryl of 5-6 ring members, and includes oxidized S or N, such as sulfinyl, sulfonyl and N-oxide of a tertiary ring nitrogen. The point of attachment of the heterocycloalkyl ring is at a carbon or heteroatom such that a stable ring is retained. Examples of heterocycloalkyl groups include without limitation morpholino, tetrahydrofuranyl, dihydropyridinyl, piperidinyl, pyrrolidinyl, piperazinyl, dihydrobenzofuryl, and dihydroindolyl. A heterocycloalkyl group can be unsubstituted or optionally substituted with one or more substituents as described herein.
The term “nitrile” or “cyano” can be used interchangeably and refer to a —CN group which is bound to a carbon atom of a heteroaryl ring, aryl ring and a heterocycloalkyl ring.
The term “oxo” refers to a ═O atom bound to an atom that is part of a saturated or unsaturated moiety. Thus, the ═O atom can be bound to a carbon, sulfur, or nitrogen atom that is part of a cyclic or acyclic moiety.
A “hydroxyl” or “hydroxy” refers to an —OH group.
The substituent —CO2H may be replaced with bioisosteric replacements such as:
and the like, wherein R has the same definition as RA as defined herein. See, e.g., T
Compounds described herein can exist in various isomeric forms, including configurational, geometric, and conformational isomers, including, for example, cis- or trans-conformations. The compounds may also exist in one or more tautomeric forms, including both single tautomers and mixtures of tautomers. The term “isomer” is intended to encompass all isomeric forms of a compound of this disclosure, including tautomeric forms of the compound. The compounds of the present disclosure may also exist in open-chain or cyclized forms. In some cases, one or more of the cyclized forms may result from the loss of water. The specific composition of the open-chain and cyclized forms may be dependent on how the compound is isolated, stored or administered. For example, the compound may exist primarily in an open-chained form under acidic conditions but cyclize under neutral conditions. All forms are included in the disclosure.
Some compounds described herein can have asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms. A compound as described herein can be in the form of an optical isomer or a diastereomer. Accordingly, the disclosure encompasses compounds and their uses as described herein in the form of their optical isomers, diastereoisomers and mixtures thereof, including a racemic mixture. Optical isomers of the compounds of the disclosure can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, simulated moving bed technology or via chemical separation of stereoisomers through the employment of optically active resolving agents.
Unless otherwise indicated, the term “stereoisomer” means one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. Thus, a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound, or greater than about 99% by weight of one stereoisomer of the compound and less than about 1% by weight of the other stereoisomers of the compound. The stereoisomer as described above can be viewed as composition comprising two stereoisomers that are present in their respective weight percentages described herein.
If there is a discrepancy between a depicted structure and a name given to that structure, then the depicted structure controls. Additionally, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it. In some cases, however, where more than one chiral center exists, the structures and names may be represented as single enantiomers to help describe the relative stereochemistry. Those skilled in the art of organic synthesis will know if the compounds are prepared as single enantiomers from the methods used to prepare them.
As used herein, and unless otherwise specified to the contrary, the term “compound” is inclusive in that it encompasses a compound or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof. Thus, for instance, a compound of the present disclosure includes a pharmaceutically acceptable salt of a tautomer of the compound.
In this description, a “pharmaceutically acceptable salt” is a pharmaceutically acceptable, organic or inorganic acid or base salt of a compound described herein. Representative pharmaceutically acceptable salts include, e.g., alkali metal salts, alkali earth salts, ammonium salts, water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1-methene-bis-2-hydroxy-3-naphthoate, einbonate), pantothenate, phosphate/diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts. A pharmaceutically acceptable salt can have more than one charged atom in its structure. In this example, the pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterions.
The terms “treat”, “treating” and “treatment” refer to the amelioration or eradication of a disease or symptoms associated with a disease. In certain embodiments, such terms refer to minimizing the spread or worsening of the disease resulting from the administration of one or more prophylactic or therapeutic agents to a patient with such a disease.
The terms “prevent,” “preventing,” and “prevention” refer to the prevention of the onset, recurrence, or spread of the disease in a patient resulting from the administration of a prophylactic or therapeutic agent.
The term “effective amount” refers to an amount of a compound as described herein or other active ingredient sufficient to provide a therapeutic or prophylactic benefit in the treatment or prevention of a disease or to delay or minimize symptoms associated with a disease. Further, a therapeutically effective amount with respect to a compound as described herein means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or prevention of a disease. Used in connection with a compound as described herein, the term can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease, or enhances the therapeutic efficacy of or is synergistic with another therapeutic agent.
A “patient” or subject” includes an animal, such as a human, cow, horse, sheep, lamb, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig. In accordance with some embodiments, the animal is a mammal such as a non-primate and a primate (e.g., monkey and human). In one embodiment, a patient is a human, such as a human infant, child, adolescent or adult. In the present disclosure, the terms “patient” and “subject” are used interchangeably.
“Inhibitor” means a compound which prevents or reduces the expression, catalytic activity, and/or localization (i.e., local concentration) of DPP4.
As described generally above, the present disclosure provides a compound of formula (I) or pharmaceutically acceptable salt thereof:
In formula (I), each — represents a single bond that, when optionally present, form a fused cyclopropyl ring.
L1A is —NHCH2— or —CH(NH2)—.
X1 is selected from —O—, —S—, —S(O)—, S(O)2—, and —NH—. L1B is a C2-C12-alkyl wherein one or more —CH2— are optionally and independently replaced by a moiety selected from —O—, —C(O)—, and —NH—. Z1 is selected from H, C6-C10-aryl, and 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S).
Subscript m1 is an integer that is 0 when Z1 is H, and is 1 when Z1 is other than H. Subscript n1 is an integer selected from 0, 1, 2, and 3.
R1 is selected from H, C1-C10-alkyl, and —C1-C10-alkyl-(C6-C10-aryl), and is optionally substituted with one to six —OH.
R2 is C1-C10-alkyl substituted with one to six —OH. In various embodiments, R2 is substituted with 1, 2, 3, 4, 5, or 6—OH. An illustrative R2 is shown below with one example of several of its diastereomers:
respectively.
In various embodiments, L1A is —NHCH2. In other embodiments, L1A is —CH(NH2)—.
In some embodiments, X1 is O.
In additional embodiments, Z1 is H. In other embodiments, Z1 is C6-C10-aryl or 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S). An example of C6-C10-aryl is phenyl, per one embodiment. An example of heteroaryl is triazolyl, per another embodiment.
In various embodiments, optionally in combination with any other embodiment herein described, n1 is 1 or 2.
In additional embodiments, optionally in combination with any other embodiment herein described, R1 is C1-C10-alkyl optionally substituted with one to six —OH. For example, in an illustrative embodiment, R1 is C1-C6-alkyl.
In still additional embodiments, optionally in combination with any other embodiment herein described, R2 is C2-C6-alkyl substituted with one to five —OH, or R2 is C2-C6-alkyl substituted with three to five —OH. In an exemplary embodiment, R2 is:
Further embodiments provide for a compound of formula (I) wherein:
In some embodiments, the optional single bonds represented by “—” are absent. In other embodiments, they are present so as to form a fused cyclopropyl ring. These embodiments are illustrated by the following structures:
respectively.
As described herein, the moiety L1B is a C2-C12-alkyl in which one or more —CH2— groups are optionally and independently replaced by —O—, C(O)—, and —NH—. In some embodiments, 1, 2, 3, 4, or 5—CH2— groups are replaced. It should be understood, in accordance with chemical principles, that replacement forms only stable compounds, such as when replacement occurs at adjacent —CH2— groups. Examples of L1B include the following:
In other embodiments, the compound is of formula (II) or a pharmaceutically acceptable salt thereof:
In formula (II), W is CH or N.
Subscript o is an integer selected from 1, 2, and 3.
R3 is selected from C1-C6-alkyl, C1-C6-hydroxyalkyl, and —(CH2CH2O)xH (wherein x is an integer selected from 1, 2, 3, 4, and 5). R4 is C2-C8-alkynyl.
R5a, R5b, R5c, and R5d are independently selected from H, C1-C6-alkyl, halo, —NRARB (wherein RA and RB are independently selected from H and C6-C10-alkyl), —C(O)OH, —B(OH)2, —C(O)NRARB, —C(O)ORA, and —C(O)-L2-Z2—[(CH2)n2—NR6R7]m2. L2 is a C2-C12-alkyl wherein one or more —CH2— are optionally and independently replaced by a moiety selected from —O—, —C(O)—, and —NH—. Z2 is selected from H, C9-C10-aryl, and 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S). At least one of R5a, R5b, R5c, and R5d is other than H.
In addition, when W is CH, then R5a and R5d are not selected from —C(O)OH, —C(O)OMe, and —C(O)OEt.
R6 is selected from H, C1-C10-alkyl, and —C1-C10-alkyl-(C6-C10-aryl), and is optionally substituted with one to six —OH. R7 is C1-C10-alkyl substituted with one to six —OH. In various embodiments, R7 is substituted with 1, 2, 3, 4, 5, or 6—OH. An illustrative R7 is shown below with one example of several of its diastereomers:
respectively.
Subscripts m2 is an integer that is 0 when Z1 is H, and is 1 when Z1 is other than H. Subscript n2 is an integer selected from 0, 1, 2, and 3.
In embodiments, W is CH. In other embodiments, W is N.
In various embodiments, one, two, or three of R5a, R5b, R5c, and R5d is H. In some embodiments, each of R5b and R5d is H. In other embodiments, R5c is halo, such as chloro. In still other embodiments, R5a is —C(O)OH or —C(O)ORA. In further embodiments, R5a is —C(O)-L2-Z2—[(CH2)n2—NR6R7]m2. All these embodiments and their combinations are contemplated.
In connection with embodiments wherein one of R5a, R5b, R5c, and R5d, such as R5a, is —C(O)-L2-Z2—[(CH2)n2—NR6R7]m2, an additional embodiment provides for Z2 as phenyl or triazolyl. In exemplary embodiments, Z2 is triazolyl.
In additional embodiments, optionally in combination with any other embodiment herein described, n2 is 1 or 2.
In still further embodiments, optionally in combination with any other embodiment herein described, R6 is C1-C10-alkyl optionally substituted with one to six —OH. In an embodiment, R6 is C1-C6-alkyl.
In additional embodiments, optionally in combination with any other embodiment herein described, R7 is C2-C6-alkyl substituted with one to five —OH or three to five —OH. For example, in illustrative embodiments R7 is:
In still further embodiments, the present disclosure provides a compound of formula (II) wherein:
As described herein, in various embodiments, the moiety L2 is a C2-C12-alkyl in which one or more —CH2— groups are optionally and independently replaced by —O—, C(O)—, and —NH—. In some embodiments, 1, 2, 3, 4, or 5—CH2— groups are replaced. It should be understood, in accordance with chemical principles, that replacement forms only stable compounds, such as when replacement occurs at adjacent —CH2— groups. Examples of L2 include the following:
In various embodiments, o is 1 or 2. In a specific embodiment, o is 1.
Optionally in combination with any other embodiment are various embodiments in which Z2 is C6-C10-aryl or 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S). In an embodiment, Z2 is C6-C10-aryl, such as phenyl.
In additional embodiments, the compound is of formula (III) or a pharmaceutically acceptable salt thereof:
In formula (III), X3 is —O— or —NH—. L3 is a bond or C2-C12-alkyl wherein one or more —CH2— are optionally and independently replaced by a moiety selected from —O—, —C(O)—, and —NH—. Z3 is selected from H, —N3, C6-C10-aryl, 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), and (3- to 14-membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S). Heteroaryl and heterocycloalkyl are optionally substituted with 1 to 6 substituents selected from the group consisting of halo, NO2, OH, CN, and C1-C6-haloalkyl.
Subscript m3 is an integer that is 0 when Z3 is H or —N3, and is 1 when Z3 is other than H or —N3. Subscript n3 is an integer selected from 0, 1, 2, and 3.
R8 is selected from H, C1-C10-alkyl or —C1-C10-alkyl-(C6-C10-aryl), and is optionally substituted with one to six —OH. R9 is C1-C10-alkyl substituted with one to six —OH. R10 is C1-C6-haloalkyl. Each R11 is independently selected from H, C1-C6-alkyl, and halo.
Subscript o3 is an integer selected from 0, 1, 2, and 3. Subscript p3 is an integer selected from 0, 1, 2, and 3. Subscript q3 is an integer selected from 0, 1, 2, and 3.
In some embodiments, X3 is 0. In other embodiments, X3 is —NH—.
As described herein, the moiety L3 is a C2-C12-alkyl in which one or more —CH2-groups are optionally and independently replaced by —O—, C(O)—, and —NH—. In some embodiments, moiety L3 is a C5-C12-alkyl, C5-C10-alkyl, C7-C12-alkyl, C8-C12-alkyl, or C9-C12-alkyl. In some embodiments, optionally in combination with any embodiment described herein, 1, 2, 3, 4, or 5—CH2— groups are replaced. It should be understood, in accordance with chemical principles, that replacement forms only stable compounds, such as when replacement occurs at adjacent —CH2— groups. Examples of L3 include the following:
In various embodiments, Z3 is —N3. Accordingly, in these embodiments, m3 is 0.
In other embodiments, Z3 is C6-C10-aryl or 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S). An illustrative Z3 is phenyl or triazolyl.
In some embodiments, optionally in combination with any other embodiment herein described, n3 is 1 or 2.
In further embodiments, optionally in combination with any other embodiment herein described, R8 is C1-C10-alkyl optionally substituted with one to six —OH. In an illustrative embodiment, R8 is C1-C6-alkyl.
In additional embodiments, optionally in combination with any other embodiment herein described, R9 is C2-C6-alkyl substituted with one to five —OH, such as three to five —OH. In an exemplary embodiment, R9 is:
The present disclosure provides in embodiments a compound of formula (III) wherein:
It should be understood that, notwithstanding the general definitions described herein, the compound does not include any of the following compounds:
The present disclosure provides, in still additional embodiments, specific compounds or their pharmaceutically acceptable salts as set forth in Tables 1-4 below.
The disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds as described herein, or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof in admixture with a pharmaceutically acceptable carrier. In some embodiments, the composition further contains, in accordance with accepted practices of pharmaceutical compounding, one or more additional therapeutic agents, pharmaceutically acceptable excipients, diluents, adjuvants, stabilizers, emulsifiers, preservatives, colorants, buffers, flavor imparting agents.
In one embodiment, the pharmaceutical composition comprises a compound selected from those illustrated in any table disclosed herein or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof, and a pharmaceutically acceptable carrier.
The pharmaceutical composition of the present disclosure is formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular subject being treated, the clinical condition of the subject, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
The “therapeutically effective amount” of a compound or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof that is administered is governed by such considerations, and is the minimum amount necessary to regenerate AEC2 cell proliferation, or to inhibit DPP4, or both. Such amount may be below the amount that is toxic to normal cells, or the subject as a whole. Generally, the initial therapeutically effective amount of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure that is administered is in the range of about 0.01 to about 200 mg/kg or about 0.1 to about 20 mg/kg of patient body weight per day, with the typical initial range being about 0.3 to about 15 mg/kg/day. Oral unit dosage forms, such as tablets and capsules, may contain from about 0.1 mg to about 1000 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In another embodiment, such dosage forms contain from about 50 mg to about 500 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In yet another embodiment, such dosage forms contain from about 25 mg to about 200 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In still another embodiment, such dosage forms contain from about 10 mg to about 100 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In a further embodiment, such dosage forms contain from about 5 mg to about 50 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In any of the foregoing embodiments the dosage form can be administered once a day or twice per day.
The compositions of the present disclosure can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
Suitable oral compositions as described herein include without limitation tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, syrups or elixirs.
In another aspect, also encompassed are pharmaceutical compositions suitable for single unit dosages that comprise a compound of the disclosure or its pharmaceutically acceptable stereoisomer, salt, or tautomer and a pharmaceutically acceptable carrier.
The compositions of the present disclosure that are suitable for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions. For instance, liquid formulations of the compounds of the present disclosure contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically palatable preparations of the compound.
For tablet compositions, a compound of the present disclosure in admixture with non-toxic pharmaceutically acceptable excipients is used for the manufacture of tablets. Examples of such excipients include without limitation inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known coating techniques to delay disintegration and absorption in the gastrointestinal tract and thereby to provide a sustained therapeutic action over a desired time period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
For aqueous suspensions, a compound of the present disclosure is admixed with excipients suitable for maintaining a stable suspension. Examples of such excipients include without limitation are sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia.
Oral suspensions can also contain dispersing or wetting agents, such as naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
Oily suspensions may be formulated by suspending a compound of the present disclosure in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide a compound of the present disclosure in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
Pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monoleate, and condensation reaction products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monoleate. The emulsions may also contain sweetening and flavoring agents.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable, an aqueous suspension or an oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The compounds of the present disclosure may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the compounds with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the compound. Such materials are cocoa butter and polyethylene glycols.
Compositions for parenteral administrations are administered in a sterile medium. Depending on the vehicle used and concentration the concentration of the compounds in the formulation, the parenteral formulation can either be a suspension or a solution containing dissolved compound. Adjuvants such as local anesthetics, preservatives and buffering agents can also be added to parenteral compositions.
Compounds of the present disclosure are useful as inhibitors of DPP4. They are further useful in selectively promoting the proliferation AEC2 cells, without affecting myofibroblast activation or proliferation, which is undesirable in most disease contexts. The compounds, in various embodiments, promote alveolar repair as therapy for diseases whose etiology derives from epithelial degeneration and maladaptive remodeling. Exemplary indications include but are not limited to idiopathic pulmonary fibrosis (IPF), acute respiratory distress syndromes (ARDS), and infant respiratory distress syndromes (IRDS).
An advantage of the compounds of the present disclosure is their ability to modulate the regenerative capacity of AEC2 cells. Against this property are drugs approved for IPF, for example, that inhibit the activation and proliferation of pulmonary fibroblasts and myofibroblasts, which are the source of scar tissue production in the diseased lung. In contrast, the compounds of the present disclosure promote repair of the alveolus by directly targeting the source of disease in IPF: this is the ineffective self-renewal of damaged AEC2 cells. Therefore, AEC2-targeting compounds of the presenting disclosure offer additional disease modifying efficacy as a single agent or as combination therapy with an approved IPF drug (e.g., Pirfenidone).
The present disclosure, in various embodiments, thus provides a method for selectively increasing the proliferation of cuboidal alveolar type 2 (AEC2) cells in a subject in need thereof, or for restoring diminished proliferation of AEC2 cells in a subject in need thereof. The method comprises administering to the subject a compound or pharmaceutically acceptable salt as disclosed herein.
Also provided in another embodiment is a method for inhibiting dipeptidyl peptidase IV (DPP4) in a subject in need thereof. The method comprises administering to the subject a compound or pharmaceutically acceptable salt as disclosed herein.
The present disclosure provides, in various embodiments, a method for treating a subject suffering from a disease or condition whose etiology derives from epithelial degeneration, maladaptive remodeling, and/or ineffective self-renewal of damaged AEC2 cells. In some embodiments, the disease is a pulmonary disease or lung condition. In additional embodiments, the disease or condition includes those selected from Idiopathic pulmonary fibrosis (IPF), Acute respiratory distress syndrome (ARDS), Chronic Obstructive Pulmonary Disease (COPD), Emphysema, Silicosis, Asbestosis, Pneumoconiosis, Aluminosis, Bauxite fibrosis, Berylliosis, Siderosis, Stannosis, Pulmonary Talcosis, Labrador lung (mixed dust Pneumoconiosis), Sarcoidosis, Hypersensitivity pneumonitis (HP)/extrinsic allergic alveolitis (EAA), Chronic Bronchitis, Desquamative interstitial pneumonia (DIP), Respiratory bronchiolitis interstitial lung disease (RBILD), Acute interstitial pneumonia (AIP), Nonspecific interstitial pneumonia (NSIP), Cryptogenic organizing pneumonia (COP=idiopathic BOOP), Secondary organizing pneumonia (BOOP), Lymphoid interstitial pneumonia (LIP), Idiopathic interstitial pneumonia: unspecified, Hypereosinophilic lung diseases, Tuberculosis (TB), Pulmonary Edema, Interstitial Lung Disease, Bronchopulmonary Dysplasia (BPD), Coronavirus, COVID-19, Cryptogenic Organizing Pneumonia (COP), Cystic Fibrosis (CF), E-cigarette or Vaping Use-Associated Lung Injury (EVALI), Hantavirus Pulmonary Syndrome (HPS), Histoplasmosis, Influenza, Legionnaires' Disease, MAC Lung Disease, Alpha-1 Antitrypsin Deficiency, Aspergillosis, Lymphangioleiomyomatosis (LAM), Middle Eastern Respiratory Syndrome (MERS), Nontuberculous Mycobacterial Lung Disease (N™), Lung cancer, Pulmonary Embolism, Goodpasture syndrome, idiopathic pulmonary hemosiderosis, alveolar hemorrhage syndrome of undetermined origin, alveolar hemorrhage syndrome of determined origin, Sporadic pulmonary lymphangioleiomyomatosis (S-LAM), Pulmonary lymphangioleiomyomatosis in tuberous sclerosis (TSC-LAM), Alveolar proteinosis, Pulmonary amyloidosis, Primary pulmonary lymphoma, Primary ciliary dyskinesia (without or with situs inversus), Rare cause of hypersensitivity pneumonitis (all causes other than farmer's lung disease and pigeon breeder's lung disease), Pulmonary arteriovenous malformations in hereditary hemorrhagic telangiectasia (HHT), interstitial lung disease in systemic sclerosis, interstitial lung disease in rheumatoid arthritis, interstitial lung disease in idiopathic inflammatory myopathies (polymyositis, dermatomyositis, anti-synthetase syndrome), interstitial lung disease in Sjögren syndrome, interstitial lung disease in mixed connective tissue disease (MCTD), interstitial lung disease in overlap syndromes, interstitial lung disease in undifferentiated connective tissue disease, and Bronchiolitis obliterans (in non-transplanted patients).
In other embodiments, the disease is chosen from inflammatory diseases, and other diseases and disorders. The disease or disorder includes those selected from Infectious colitis, Ulcerative colitis, Crohn's disease, Ischemic colitis, Radiation colitis, Peptic ulcer, Intestinal cancer, Intestinal obstruction, Rheumatoid arthritis, Psoriatic arthritis, Hashimoto thyroiditis, Systemic lupus erythematosus, Multiple Sclerosis, Graves' Disease, Type 1 Diabetes Mellitus, Psoriasis, Ankylosing spondylitis, Scleroderma, Myositis, Gout, Antiphospholipid Antibody Syndrome (APS), Vasculitis, Dilated cardiomyopathy, Hypertrophic cardiomyopathy, Restrictive cardiomyopathy, Left-sided heart failure, Right-sided heart failure, Systolic heart failure, Diastolic heart failure (heart failure with preserved ejection fraction), Atrial Septal Defect, Atrioventricular Septal Defect, Coarctation of the Aorta, Double-outlet Right Ventricle, d-Transposition of the Great Arteries, Ebstein Anomaly, Hypoplastic Left Heart Syndrome, Interrupted Aortic Arch, Pulmonary Atresia, Single Ventricle, Tetralogy of Fallot, Total Anomalous Pulmonary Venous Return, Tricuspid Atresia, Truncus Arteriosus, Ventricular Septal Defect, Polycystic kidney disease, Diabetes Insipidus, Goodpasture's Disease, IgA Vasculitis, IgA Nephropathy, Lupus Nephritis, Adult Nephrotic Syndrome, Childhood Nephrotic Syndrome, Hemolytic Uremic Syndrome, Medullary Sponge Kidney, Kidney dysplasia, Renal artery stenosis, Renovascular hypertension, Renal tubular acidosis, Alport syndrome, Wenger's granulomatosis, Alagille syndrome, Cystinosis, Fabry disease, Focal segmental glomerulosclerosis (FSGS), Glomerulonephritis, aHUS (atypical hemolytic uremic syndrome), Hemolytic uremic syndrome (HUS), Henoch-Schönlein purpura, IgA nephropathy (Berger's disease), Interstitial nephritis, Minimal change disease, Nephrotic syndrome, Thrombotic thrombocytopenic purpura (TTP), Granulomatosis with polyangiitis (GPA), Eczema, Psoriasis, Cellulitis, Impetigo, Atopic dermatitis, Epidermolysis Bullosa, Lichen Sclerosis, Ichthyosis, Vitiligo, Acral peeling skin syndrome, Blau syndrome, Primary cutaneous amyloidosis, Cutaneous abscess, Pressure Ulcers, Blepharitis, Furunculosis, Full or partial thickness burns, Capillaritis, Cellulitis, Corneal Abrasion, Corneal Erosion, Xerosis, Lichen Planus, Lichen Simplex Chronicus, Venous Ulcer (Stasis Ulcer), Adult Still's disease, Agammaglobulinemia, Alopecia areata, Autoimmune angioedema, Autoimmune dysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis, Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune urticaria, Axonal & neuronal neuropathy (AMAN), Baló disease, Bullous pemphigoid, Celiac disease, Chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or Eosinophilic Granulomatosis (EGPA), Cicatricial pemphigoid, Cogan's syndrome, Cold agglutinin disease, Coxsackie myocarditis, CREST syndrome, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus, Eosinophilic esophagitis (EoE), Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Granulomatosis with Polyangiitis, Guillain-Barre syndrome, Hashimoto's thyroiditis, Henoch-Schonlein purpura (HSP), Herpes gestationis or pemphigoid gestationis (PG), Hypogammaglobulinemia, IgG4-related sclerosing disease, Immune thrombocytopenic purpura (ITP), Inclusion body myositis (IBM), Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Linear IgA disease (LAD), Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, Multifocal Motor Neuropathy (MMN) or MMNCB, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neonatal Lupus, Neuromyelitis optica, Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism (PR), PANDAS, Paraneoplastic cerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Pars planitis (peripheral uveitis), Parsonage-Turner syndrome, Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia (PA), POEMS syndrome, Polyarteritis nodosa, Polyglandular syndromes type I, II, III, Polymyalgia rheumatica, Polymyositis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progesterone dermatitis, Pure red cell aplasia (PRCA), Pyoderma gangrenosum, Raynaud's phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Relapsing polychondritis, Restless legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjögren's syndrome, Sperm & testicular autoimmunity, Stiff person syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia (SO), Takayasu's arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Thyroid eye disease (TED), Alagille Syndrome, Alcohol-Related Liver Disease, Autoimmune Hepatitis, Biliary Atresia, Cirrhosis, Lysosomal Acid Lipase Deficiency (LAL-D), Liver Cysts, Liver Cancer, Newborn Jaundice, Non-Alcoholic Fatty Liver Disease, Non-Alcoholic Steatohepatitis, Primary Biliary Cholangitis (PBC), Progressive Familial Intrahepatic Cholestasis (PFIC), Osteoporosis, Paget's Disease, Osteonecrosis, Osteoarthritis, Low Bone Density, Gout, Fibrous Dysplasia, Marfan Syndrome, and Osteogenesis Imperfecta.
Numbered references in the preceding sections are as follows:
Additional embodiments of the present disclosure are set forth in the following non-limiting examples. All compounds of the present disclosure are made by procedures analogous to those illustrated below.
Step 1. (2R,3R,4R,5S)-6-(hexylamino)hexane-1,2,3,4,5-pentaol
To a stirred solution of hexan-1-amine (2.0 g, 19.76 mmol) and D-glucose (3.56 g, 19.76 mmol) in methanol (20 mL) was added Raney-Ni (1.2 g) at room temperature. The reaction mixture was heated at 65° C. under H2 gas pressure (160 psi) for 16 h. After consumption of the starting materials, the reaction mixture was filtered through celite bed. The filtrate was concentrated under reduced pressure to afford 4.5 g of (2R,3R,4R,5S)-6-(hexylamino)hexane-1,2,3,4,5-pentaol as an off-white solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.2].
Step 2. (2R,3R,4R,5S)-6-(hexyl(prop-2-yn-1-yl)amino)hexane-1,2,3,4,5-pentaol (Int-1):
To a stirred solution of (2R,3R,4R,5S)-6-(hexylamino)hexane-1,2,3,4,5-pentaol (1.0 g, 3.77 mmol) in THF (15 mL) was added 3-bromoprop-1-yne (80% solution in toluene, 0.84 mL, 5.65 mmol) and the reaction mixture was stirred at 80° C. for 16 h. After consumption of the starting materials, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to afford 0.5 g of (2R,3R,4R,5S)-6-(hexyl(prop-2-yn-1-yl)amino)hexane-1,2,3,4,5-pentaol as yellow colour gum. [TLC system: MeOH:DCM (1:9); Rf value: 0.3].
Step 1. Tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-hydroxyadamantan-1-yl)carbamate:
A mixture of (2S)-1-(((1S,3R,5S)-3-hydroxyadamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (200 g, 659 mmol, 1.00 eq) and (Boc)2O (165 g, 758 mmol, 1.15 eq) in THF (400 mL) and t-BuOH (400 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 70° C. for 12 hrs under N2 atmosphere. The solution was concentrated under reduced pressure, and MTBE (200 mL) was added. The mixture was filtered to give tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1 S,3R,5S)-3-hydroxyadamantan-1-yl)carbamate (239 g, 592 mmol, 89.8% yield, 99.7% purity) as a white solid. TLC system: PE:EA (0:1); Rf: 0.40.
Step 2. (1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl methanesulfonate (Int-2):
To a solution of compound tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-hydroxyadamantan-1-yl)carbamate (230 g, 570 mmol, 1.00 eq) and N,N,N′,N′-tetramethyl-1,6-hexanediamine (216 g, 1.25 mol, 2.20 eq) in DCM (2.30 L) and cooled to 0-10° C., methanesulfonyl chloride (97.9 g, 855 mmol, 1.50 eq) were added at 0-10° C. The reaction was stirred for 2 hrs at 15-20° C. The reaction mixture was quenched by adding ice water (1.00 L) at 0° C., and extracted with EA (2.00 L). The combined organic layers were washed with brine 1.00 L, dried over Na2SO4, filtered and concentrated under reduced pressure to give (1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl methanesulfonate (440 g, crude) as a light yellow oil. TLC system: PE:EA (10:1); Rf: 0.60).
Step 1. Tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-hydroxyethoxy)ethoxy)adamantan-1-yl)carbamate:
To a stirred solution of (1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl methanesulfonate (3.58 g, 7.43 mmol) and 2,2′-oxybis(ethan-1-ol) (35.2 mL, 371.67 mmol) in acetonitrile (10 mL) was added molecular sieves 4 Å (4 g) and the mixture was stirred at 70° C. for 16 h. After completion, the reaction mixture was diluted with water (500 mL) and resulting mixture was extracted with ethyl acetate (3×500 mL). The combined organic layer was dried over anhydrous sodium sulfate and evaporated to give crude product. The crude was then purified by silica gel column chromatography (230-400 mesh) using 3% methanol in dichloromethane to afford tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1 S,3R,5S)-3-(2-(2-hydroxyethoxy)ethoxy)adamantan-1-yl)carbamate (2.7 g) as a brown gum. TLC system: MeOH:DCM (1:9); Rf. 0.4.
Step 2. 2-(2-(((1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethoxy)ethyl methanesulfonate:
To a stirred solution of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-hydroxyethoxy)ethoxy)adamantan-1-yl)carbamate (2.7 g, 5.49 mmol) in dichloromethane (25 mL) was added triethylamine (1.67 g, 16.47 mmol) followed by methanesulfonyl chloride (1.26 g, 10.98 mmol) in dichloromethane (5 mL) at −10° C. The resultant mixture was stirred at −10° C. for 1 h. After completion, the reaction mixture was diluted with water (100 mL) and resulting mixture was extracted with dichloromethane (2×300 mL). The combined organic layer was dried over anhydrous sodium sulfate and evaporated to give 2-(2-(((1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethoxy)ethyl methanesulfonate (3.1 g) as a pale yellow foamy solid. TLC system: MeOH:DCM (0.5:9.5); Rf. 0.4 (TLC was eluted 2 times).
Step 3. Tert-butyl ((1S,3R,5S)-3-(2-(2-azidoethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (Int-3):
To a stirred solution of 2-(2-(((1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethoxy)ethyl methanesulfonate (3.1 g, 5.44 mmol) in N,N-dimethylformamide (10 mL), sodium azide (0.71 g, 10.88 mmol) was added and the reaction mixture was stirred at 70° C. for 16 h. After completion, the reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (3×300 mL). The combined organic layer was dried over anhydrous sodium sulfate and evaporated to give tert-butyl ((1S,3R,5S)-3-(2-(2-azidoethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (2.5 g) as a yellow gum. TLC system: MeOH:DCM (0.5:9.5); Rf. 0.3 (TLC was eluted 2 times).
To a mixture of (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (2.0 g, 11.08 mmol), prop-2-yn-1-amine (0.3 g, 5.54 mmol) and acetic acid (2.0 mL) in methanol (16 mL) was added sodium cyanoborohydride (0.7 g, 11.08 mmol). The reaction mixture was stirred at 60° C. for 16 h. After completion (reaction monitored by LCMS), the reaction mixture was evaporated and the residue was washed with methanol (2×20 mL), diethyl ether (2×20 mL) and dried to afford (2R,2′R,3R,3′R,4R,4′R,5S,5'S)-6,6′-(prop-2-yn-1-ylazanediyl)bis(hexane-1,2,3,4,5-pentaol) (1.3 g) as an off-white solid.
To the stirred solution of tert-butyl ((1S,3R,5S)-3-(2-(2-azidoethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (1.0 g, 1.94 mmol) in methanol (10 mL), triphenylphosphine (0.76 g, 2.9 mmol) was added and the reaction mixture was stirred at 70° C. for 16 h. After completion of reaction, the reaction mixture was evaporated, residue was purified by silica gel column chromatography (basified by triethylamine) using 6% methanol in dichloromethane to afford tert-butyl ((1S,3R,5S)-3-(2-(2-aminoethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (0.28 g) as pale yellow solid. TLC system: MeOH:DCM (1:9); Rf. 0.2.
Step 1. Tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((S,3R,5S)-3-hydroxyadamantan-1-yl)carbamate:
To a stirred suspension of (2S)-1-(((1S,3R,5S)-3-hydroxyadamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (15 g, 49.44 mmol) in toluene (150 mL) were added triethylamine (13.78 mL, 98.88 mmol) and di-tert-butyl dicarbonate (17.0 mL, 74.16 mmol) and the reaction mixture was stirred at 100° C. for 16 h. After completion, the reaction mixture was evaporated to give crude residue, which was then purified by silica gel (230-400 mesh) column chromatography using 3% methanol in dichloromethane to afford tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-hydroxyadamantan-1-yl)carbamate (17.0 g) as an off-white solid. TLC system: MeOH:DCM (0.5:9.5); Rr 0.2 (TLC was eluted 2 times).
Step 2. (1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl methanesulfonate:
To a stirred solution of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-hydroxyadamantan-1-yl)carbamate (0.5 g, 1.24 mmol) in dichloromethane (10 mL) was added triethylamine (0.38 g, 3.72 mmol) followed by methanesulfonyl chloride (0.21 g, 1.86 mmol) in dichloromethane (2.5 mL) at −10° C. The reaction mixture was stirred at −10° C. for 30 min. After completion, the reaction mixture was diluted with water (10 mL) and resulting mixture was extracted with dichloromethane (2×30 mL). The combined organic layer was dried over anhydrous sodium sulfate and evaporated to give (1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl methanesulfonate (0.59 g) as a pale yellow foamy solid. TLC system: MeOH:DCM (0.5:9.5); Rf. 0.3 (TLC was eluted 2 times).
Step 3. Tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-hydroxyethoxy)ethoxy)adamantan-1-yl)carbamate (Int-6):
To a stirred solution of (1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl methanesulfonate (3.58 g, 7.43 mmol) and 2,2′-oxybis(ethan-1-ol) (35.2 mL, 371.67 mmol) in acetonitrile (10 mL) was added molecular sieves 4 Å (4 g) and the mixture was stirred at 70° C. for 16 h. After completion, the reaction mixture was diluted with water (500 mL) and resulting mixture was extracted with ethyl acetate (3×500 mL). The combined organic layer was dried over anhydrous sodium sulphate and evaporated to give crude product. The crude was then purified by silica gel column chromatography (230400 mesh) using 3% methanol in dichloromethane to afford tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-hydroxyethoxy)ethoxy)adamantan-1-yl)carbamate (2.7 g) as a brown gum. TLC system: MeOH:DCM (1:9); Rf. 0.4.
Step 1. 4-(2-(hexylamino)ethyl)phenol:
To a solution of 4-(2-aminoethyl)phenol (15 g, 109 mmol) and hexanal (10 g, 100 mmol) in EtOH (100 mL) was added AcOH (1 drop). The reaction mixture was stirred at 25° C. for 12 hrs. Then NaBH4 (2 g, 54 mmol) was added, and the reaction mixture was stirred at 25° C. for 1 h. The reaction mixture was concentrated under pressure at 40° C. The residue was diluted with water and extracted with EtOAc (200 mL×2). The organic layers were washed with brine (50 mL), then dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under vacuum to give crude product. The crude product was purified via Flash column chromatography, eluting with (CH2Cl2/MeOH, from 0% to 10%) to give 4-(2-(hexylamino)ethyl)phenol (6 g, 24% yield) as a yellow oil. ES MS M/Z=222 (M+1).
Step 2. Benzyl hexyl(4-hydroxyphenethyl)carbamate:
To a solution of 4-[2-(hexylamino)ethyl]phenol (6.2 g, 28 mmol) and DIEA (5.41 g, 42 mmol) in DCM (100 mL) was added a solution of CbzCl (4.29 g, 25 mmol) in DCM (10 mL) at 0° C. The reaction mixture was stirred at 0° C. for 1 h. Then the mixture was diluted with H2O (100 mL) and extracted with DCM (100 mL×2). The DCM phase was dried over Na2SO4 and concentrated. The residue was purified via Flash column chromatography, eluting with (PE/EtOAc, from 5% to 20%) to give benzyl hexyl(4-hydroxyphenethyl)carbamate (7.63 g, 76% yield) as a yellow oil. ES MS M/Z=356 (M+1).
Step 3. Benzyl (4-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)phenethyl)(hexyl)carbamate:
To a mixture of benzyl hexyl(4-hydroxyphenethyl)carbamate (8.63 g, 24.3 mmol), 2-(2-hydroxyethyl)isoindole-1,3-dione (4.64 g, 24.3 mmol), PPh3 (12.73 g, 48.6 mol) in THF (50 mL) was added a solution of ADDP (12.24 g, 48.6 mmol) in THF (30 mL) at 25° C. under N2 atmosphere. The reaction mixture was stirred at 70° C. under N2 atmosphere for 12 hrs. The reaction mixture was concentrated under pressure at 40° C. The residue was purified via Flash column chromatography, eluting with (PE/EtOAc, from 10% to 50%) to give benzyl (4-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)phenethyl)hexyl)carbamate (10.4 g, 81% yield) as a yellow oil. ES MS M/Z=529 (M+1).
Step 4. 2-(2-(4-(2-(hexylamino)ethyl)phenoxy)ethyl)isoindoline-1,3-dione:
To a solution of benzyl (4-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)phenethyl)hexyl)carbamate (4.86 g, 9.20 mmol) in MeCN (10 mL) was added TMSI (5 g, 35.7 mmol). The reaction mixture was stirred at 25° C. for 10 min. The reaction mixture was concentrated under pressure at 40° C. The residue was purified via Flash column chromatography and eluting with (CH2Cl2/MeOH, from 5% to 10%) to give 2-(2-(4-(2-(hexylamino)ethyl)phenoxy)ethyl)isoindoline-1,3-dione (2.56 g, 70% yield) as a yellow solid. ES MS M/Z=395 (M+1).
Step 5. 2-(2-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)phenoxy)ethyl)isoindoline-1,3-dione:
To a solution of 2-(2-(4-(2-(hexylamino)ethyl)phenoxy)ethyl)isoindoline-1,3-dione (2.92 g, 7.41 mmol) and (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (4.00 g, 22.23 mmol) in MeOH (20 mL) was added AcOH (10 drops). The reaction mixture was stirred at 25° C. for 1 h. Then NaBH3CN (1.86 g, 29.64 mmol) was added, and the reaction mixture was stirred at 50° C. for 12 hrs. To the reaction mixture was added (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (1.33 g, 14.82 mmol) every 12 hrs until 2-(2-(4-(2-(hexylamino)ethyl)phenoxy)ethyl)isoindoline-1,3-dione was disappeared (total about 4 equivalent of (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal was added into the reaction mixture, total reaction time was about 3 days). The reaction mixture was concentrated under pressure at 40° C. The residue was purified via Flash column chromatography and eluted with (DCM/MeOH, from 5% to 10%) to give impure 2-(2-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)phenoxy)ethyl)isoindoline-1,3-dione (4 g, 87% yield) as a colorless oil. ES MS M/Z=559 (M+1).
Step 6. (2R,3R,4R,5S)-6-((4-(2-aminoethoxy)phenethyl)(hexyl)amino)hexane-1,2,3,4,5-pentaol (Int-7):
To a solution of 2-(2-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)phenoxy)ethyl) isoindoline-1,3-dione (3.5 g, 6.27 mmol) in EtOH (20 mL) was added Hydrazine hydrate (10 mL). The reaction mixture was stirred at 80° C. for 4 hrs. The reaction mixture was concentrated under pressure at 40° C. The residue was purified via Flash column chromatography and eluted with (MeOH/DCM, from 20% to 50%) and give (2R,3R,4R,5S)-6-((4-(2-aminoethoxy)phenethyl)(hexyl)amino)hexane-1,2,3,4,5-pentaol (2.5 g, 93% yield) as a colorless oil. ES MS M/Z=429 (M+1).
To a mixture of (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (1.0 g, 5.54 mmol), prop-2-yn-1-amine (0.61 g, 11.08 mmol) and acetic acid (1.0 mL) in methanol (20 mL) was added sodium cyanoborohydride (0.35 g, 5.54 mmol) at 0° C. The reaction mixture was stirred at room temperature for 16 h. After completion (reaction monitored by LCMS), the reaction mixture was evaporated and residue was washed with diethyl ether (2×20 mL) and dried to afford (2R,3R,4R,5S)-6-(prop-2-yn-1-ylamino)hexane-1,2,3,4,5-pentaol (1.0 g, crude) as an off-white solid.
Step 1. Tert-butyl ((1S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-hydroxyadamantan-1-yl)-2-oxoethyl)carbamate:
To a stirred suspension of (1S,3S,5S)-2-((2S)-2-amino-2-((1S,3R,5S)-3-hydroxyadamantan-1-yl)acetyl)-2-azabicyclo[3.1.0]hexane-3-carbonitrile (2 g, 6.34 mmol) in dichloromethane (40 mL) was added triethylamine (1.8 mL, 12.68 mmol) followed by di-tert-butyl dicarbonate (2.2 mL, 9.51 mmol) at 0° C. and the reaction mixture was stirred at room temperature for 3 h. After completion, the reaction mixture was evaporated to give crude residue, which was then purified by silica gel (230-400 mesh) column chromatography using 3% methanol in dichloromethane to afford tert-butyl ((1S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-hydroxyadamantan-1-yl)-2-oxoethyl)carbamate (2.5 g) as an off-white solid. TLC system: MeOH:DCM (0.5:9.5); Rr 0.4.
Step 2. (1R,3S,5S)-3-((S)-1-((tert-butoxycarbonyl)amino)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)adamantan-1-yl methanesulfonate:
To a stirred solution of tert-butyl ((1S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-hydroxyadamantan-1-yl)-2-oxoethyl)carbamate (2.5 g, 6.02 mmol) in dichloromethane (50 mL) was added triethylamine (2.5 mL, 18.06 mmol) followed by methanesulfonyl chloride (1.03 g, 9.03 mmol) in dichloromethane (2.5 mL) at −10° C. and the mixture was stirred for 30 min. After completion, water (50 mL) was added to the reaction mixture and extracted with dichloromethane (2×100 mL). The combined organic layer was dried over anhydrous sodium sulfate and evaporated to give (1R,3S,5S)-3-((S)-1-((tert-butoxycarbonyl)amino)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)adamantan-1-yl methanesulfonate (3.2 g) crude as a white foamy solid. TLC system: 100% EtOAc; Rf. 0.4.
Step 3. Tert-butyl ((1S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)-2-oxoethyl)carbamate (Int-9):
To a stirred solution of (1R,3S,5S)-3-((S)-1-((tert-butoxycarbonyl)amino)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)adamantan-1-yl methanesulfonate (3.2 g, 6.48 mmol) and ethane-1,2-diol (18.2 mL, 324.13 mmol) in acetonitrile (30 mL) was added molecular sieves 4 Å (3.5 g), the resultant mixture was stirred at 70° C. for 16 h. After completion, the reaction mixture was concentrated and the crude was filtered through Buchner funnel, diluted with water (100 mL) and extracted with ethyl acetate (3×100 mL). The combined organic layer was dried over anhydrous sodium sulfate and evaporated to afford tert-butyl ((1 S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)-2-oxoethyl)carbamate (2.0 g) crude as an off-white foamy solid. TLC system: 100% EtOAc, Rf. 0.3.
tert-butyl ((1S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-(2-(2-hydroxyethoxy)ethoxy)adamantan-1-yl)-2-oxoethyl)carbamate (Int-9B) was prepared according to the same route.
To a suspension of N-hexyl-D-glucamine (840 mg, 1.05 eq) and potassium carbonate (594 mg, 1.5 eq) in methanol (8 mL), tert-butyl 4-(bromomethyl)benzylcarbamate (820 mg, 1.0 eq) was added portion wise at 0° C. After 4 hours of stirring at room temperature, the reaction mixture was filtered via a pad of Celite with dichloromethane. The filtrate was then washed by water can concentrated in vacuo to provide the crude product as a yellow oil which was used for the next step without further purification. The above crude (700 mg) was dissolved with 4 M HCl in dioxane (4.5 mL) and methanol (0.5 mL) at 0° C. After stirring at room temperature for 3 hours, the volatiles were removed under reduced pressure to afford a white powder of (2R,3R,4R,5S)-6-((4-(aminomethyl)benzyl)(hexyl)amino)hexane-1,2,3,4,5-pentaol hydrochloride which was used for next step without further purification.
Step 1. 2-(2-(2-hydroxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate:
To a stirred solution of compound 2,2′-(ethane-1,2-diylbis(oxy))bis(ethan-1-ol) (15 g, 99.88 mmol) in DCM (150 mL) was added silver oxide (27.7 g, 119.85 mmol) followed by potassium iodide (1.6 g, 9.98 mmol) under nitrogen atm at rt. The resultant reaction mixture was stirred at rt for 10 min. p-TsCl (19 g, 99.88 mmol) was added and the resultant reaction mixture was stirred at rt for 16 h. The progress of the reaction was monitored by TLC. After completion of starting material, reaction mixture was concentrated under reduced pressure to get crude compound. The crude compound was purified by column chromatography using silica gel (230-400 mesh) eluting with 10% EtOAC-petether to get 11 g of compound 5 as a colorless liquid. [TLC system: EtOAC:petether (2:8); Rf value: 0.5].
Step 2. 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol:
To a stirred solution of compound 2-(2-(2-hydroxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (11 g, 36.14 mmol) in DMF (200 mL) was added sodium azide (4.7 g, 72.28 mmol) at rt. The resultant reaction mixture was heated at 120° C. for 6 h. The progress of the reaction was monitored by TLC. After completion of starting material, reaction mixture was concentrated under reduced pressure to get crude compound. The crude compound was dissolved in diethyl ether (150 mL) and the solid precipitate was removed by filtration through celite pad. The filtrate was collected and concentrated under reduced pressure to get crude compound. The crude compound was purified by column chromatography using silica gel (230-400 mesh) eluting with 10% EtOAC-petether to get 5 g of compound 6 as an off-white solid. [TLC system: EtOAC:petether (3:7); Rf value: 0.3].
Step 3. 1-azido-2-(2-(2-bromoethoxy)ethoxy)ethane (Int-11):
To a stirred solution of CBr4 (4.1 g, 12.56 mmol) in DCM (40 mL) was added Ph3P (3.3 g, 12.56 mmol). Reaction mixture was cooled to 0° C., compound 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol (2 g, 11.42 mmol) was diluted with DCM (15 mL) and added to reaction mixture slowly drop wise. The reaction mixture was stirred at rt for 8 h. After completion of starting material, the reaction mixture was quenched with water (20 mL) and extracted with DCM (2×50 mL). The combined organic layer was concentrated under reduced pressure to get crude compound. The crude compound was purified by column chromatography using silica gel (230-400 mesh) eluting with 15% EtOAC-petether to get 1.2 g of Int-11 as a colour less liquid. [TLC system: EtOAC:petether (1:1); Rf value: 0.7].
Step 1. 8-bromo-7-(but-2-yn-1-yl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione:
To a stirred solution of compound 8-bromo-3-methyl-3,7-dihydro-1H-purine-2,6-dione (10.0 g, 40.81 mmol) in DMF (100 mL) was added DIPEA (7.13 mL, 40.81 mmol) and 1-bromobut-2-yne (5.43 g, 40.81 mmol) portion wise at room temperature. The reaction mixture was stirred at room temperature for 16 h. After completion of starting material, the reaction mixture was quenched with cold water and the precipitated solid was filtered off, dried under vacuum to get 9.3 g of compound 8-bromo-7-(but-2-yn-1-yl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione as an off-white solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.7].
Step 2. Tert-butyl (R)-(1-(7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)piperidin-3-yl)carbamate (Int-13):
To a stirred solution of 8-bromo-7-(but-2-yn-1-yl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione (4.5 g, 15.15 mmol) in DMF (45 mL) were added tert-butyl (R)-piperidin-3-ylcarbamate (3.33 g, 16.66 mmol) and K2CO3 (6.28 g, 45.45 mmol) and the reaction mixture was stirred at 90° C. for 16 h. After completion of starting material, the reaction mixture was quenched with cold water and the precipitated solid was filtered off, dried under vacuum to get 5.0 g of tert-butyl (R)-(1-(7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)piperidin-3-yl)carbamate as an off-white solid. [TLC system: EtOAc:pet-ether (1:1); Rf value: 0.3].
Step 1. Methyl 6-fluoro-2-methylnicotinate:
To a stirred solution of 6-fluoro-2-methylnicotinic acid (3.0 g, 19.34 mmol) in THF (36 mL) and methanol (9 mL) at 0° C. was added TMS diazomethane solution 2 M in toluene (12.57 mL, 25.14 mmol) and the reaction mixture was allowed to warm to room temperature over 2 h. After completion of the reaction, volatiles were removed under reduced pressure to get the crude compound. The crude compound was purified by column chromatography (using silica gel 100-200 mesh, 0-10% EtOAc in pet-ether as an eluent) to afford 1.6 g of compound methyl 6-fluoro-2-methylnicotinate as colorless liquid product. [TLC system: EtOAc:pet-ether (3:7); Rf value: 0.8].
Step 2. Methyl 2-(bromomethyl)-6-fluoronicotinate:
To a stirred solution of methyl 6-fluoro-2-methylnicotinate methyl 6-fluoro-2-methylnicotinate (1.6 g, 9.46 mmol) in 1,2-dichloroethane (16 mL), were added N-Bromosuccinimide (1.68 g, 9.46 mmol) followed by catalytic benzoyl peroxide (0.11 g, 0.47 mmol) portion wise. The reaction mixture was stirred at 70° C. for 16 h. After completion of the starting material, solvent was evaporated under reduced pressure, quenched with water (100 mL) and extracted the compound with dichloromethane (100 mL*2). The combined organic layer were washed with brine solution (50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure to get the crude compound. The crude compound was purified by column chromatography (using silica gel 230-400 mesh, 0-4% EtOAc in pet-ether as an eluent) to afford 1.2 g of compound methyl 2-(bromomethyl)-6-fluoronicotinate as colorless liquid product. [TLC system: EtOAc:pet-ether (1:9); Rf value: 0.7].
Step 3. Methyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-fluoronicotinate (Int-14):
To a stirred solution of methyl 2-(bromomethyl)-6-fluoronicotinate methyl 2-(bromomethyl)-6-fluoronicotinate (0.6 g, 2.42 mmol) in DMF (6 mL), were added compound tert-butyl (R)-(1-(7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)piperidin-3-yl)carbamate (1.01 g, 2.42 mmol) and K2CO3 (0.33 g, 2.42 mmol) and the reaction mixture was stirred at 70° C. for 16 h. After completion of starting material, the reaction mixture was quenched with cold water and the precipitated solid was filtered off, dried under vacuum to get 1 g of compound methyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-fluoronicotinate as an off-white solid. [TLC system: EtOAc:pet-ether (1:1); Rf value: 0.4].
Sep 1. Methyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-(methylamino)nicotinate:
To a stirred solution of compound methyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-fluoronicotinate (1.1 g, 1.88 mmol) in DMF (11 mL) was added K2CO3 (0.39 g, 2.82 mmol) followed by compound methanamine (1.1 mL, 2.26 mmol) and the reaction mixture was stirred at rt for 4 h. After completion of starting material (TLC monitoring), the reaction mixture was quenched with cold water and the precipitated solid was filtered off, dried under vacuum to get 0.82 g of compound methyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-(methylamino)nicotinate as an off-white solid. [TLC system: EtOAc:Pet ether (1:9); Rf value: 0.3].
Step 2 (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-(methylamino)nicotinic acid (Int-15):
To a stirred solution of compound methyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-(methylamino)nicotinate (0.6 g, 1.01 mmol) in MeOH (3 mL), THF (3 mL) and water (1 mL) was added NaOH (0.081 g, 2.02 mmol) at 0° C. The reaction mixture was stirred at 50° C. for 3 h. After completion of starting material (TLC monitoring), the reaction mixture was concentrated under reduced pressure to get crude compound. The crude compound was diluted with water, acidified with 1N HCl. The precipitated solid was filtered off and dried under vacuum to afford 0. g of compound as an off-white solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.1].
Step 1. 1.7-diazidoheptane:
To the solution of compound 1,7-dibromoheptane (10 g, 38.76 mmol) in DMF (100 mL) was added NaN3 (5.04 g, 77.52 mmol) portion wise at room temperature. The reaction mixture was stirred at 60° C. for 16 h. After completion of the reaction (TLC monitoring). The reaction mixture was diluted with water (300 mL) and extracted with EtOAc (2×100 mL). The combined organic layer was dried over anhydrous sodium sulfate and evaporated to get crude. The obtained crude was purified by silica gel (100-200 mesh) column chromatography using 3% EtOAc in pet-ether to afford 1,7-diazidoheptane (6.5 g) as colorless oil compound. [TLC system: EtOAc:pet-ether (1:9); R value: 0.8].
Step 2. 7-azidoheptan-1-amine (Int-16):
To the solution of compound 1,7-diazidoheptane (6.5 g, 35.67 mmol) in 1N HCl (100 mL) was added EtOAc (32.5 mL) and Et2O (32.5 mL) followed by TPP (4.68 g, 17.84 mmol) and the reaction mixture was stirred at rt for 16 h. After completion of the reaction (TLC monitoring), the reaction mixture was diluted with water (200 mL) and washed with Et2O (200 mL). The aqueous layer was basified with saturated NaHCO3 and extracted with EtOAc (2×300 mL). The combined organic layer was dried over anhydrous sodium sulfate and evaporated to afford 7-azidoheptan-1-amine (2.5 g) as colorless oil compound. [TLC system: MeOH:DCM (1:9); Rf value: 0.1].
To the solution of compound prop-2-yn-1-amine (0.2 g, 1.81 mmol) and (2R,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propanal (0.97 g 3.62 mmol) in MeOH (20 mL) and was added AcOH (0.1 mL) and the reaction mixture was stirred at 60° C. for 1 h. The reaction mixture was allowed to come to rt and then added NaCNBH3 (0.68 g 10.86 mmol) at portion wise at 0° C. The reaction mixture was stirred at 60° C. for 16 h. After completion of the reaction (TLC monitoring), the reaction mixture was concentrated under reduced pressure to get a crude mass. The crude compound washed with Et2O (50 mL) and dried under vacuo to afford compound (1R,1′R,2S,2'S)-3,3′-(prop-2-yn-1-ylazanediyl)bis(1-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propane-1,2-diol) (0.9 g) as off-white solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.1].
Step 1. Methyl (R)-6-((7-azidoheptyl)amino)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)nicotinate:
To the of solution of compound methyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-fluoronicotinate (3.5 g, 6.00 mmol) in DMF (30 mL) was added compound 3 (1.41 g, 9.00 mmol) and K2CO3 (1.24 g, 9.00 mmol) and the reaction mixture was stirred at 80° C. for 16 h. After completion of the reaction (TLC monitoring), the reaction mixture was diluted with water (100 mL) and extracted with EtOAc (2×100 mL). The combined organic layer was dried over anhydrous sodium sulfate and evaporated to get crude. The obtained crude was purified by silica gel (100-200 mesh) column chromatography using 30% EtOAc in pet-ether to afford methyl (R)-6-((7-azidoheptyl)amino)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)nicotinate (2.8 g) as off-white solid. [TLC system: EtOAc:pet-ether (1:1); Rf value: 0.5].
Step 2. Methyl 6-((7-(4-((bis((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)amino)methyl)-1H-1,2,3-triazol-1-yl)heptyl)amino)-2-((7-(but-2-yn-1-yl)-8-((R)-3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)nicotinate (Int-18):
To the solution of compound methyl (R)-6-((7-azidoheptyl)amino)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)nicotinate (0.5 g, 0.69 mmol) and (1R,1′R,2S,2'S)-3,3′-(prop-2-yn-1-ylazanediyl)bis(1-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propane-1,2-diol) (0.97 g 3.62 mmol) in t-BuOH (10 mL) and H2O (1 mL) and was added CuSO4.5H2O (0.207 g, 0.83 mmol) and Na-ascorbate (0.204 g 1.03 mmol) and the reaction mixture was stirred at rt for 16 h. After completion of the reaction (TLC monitoring). The reaction mixture was concentrated under reduced pressure to get a crude mass. The crude compound was washed with Et2O (50 mL) and dried under vacuo to afford compound methyl 6-((7-(4-((bis((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)amino)methyl)-1H-1,2,3-triazol-1-yl)heptyl)amino)-2-((7-(but-2-yn-1-yl)-8-((R)-3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)nicotinate (0.9 g) as off-white solid. The crude compound was taken to the next step without further purification. [TLC system: MeOH:DCM (1:9); Rf value: 0.1].
To a stirred solution of methyl 5-chloro-2-methylbenzoate methyl 5-chloro-2-methylbenzoate (10.0 g, 54.16 mmol) in 1,2-dichloroethane (100 mL) were added N-bromosuccinimide (9.64 g, 54.16 mmol) and catalytic benzoyl peroxide (0.66 g, 2.71 mmol) portion wise. The reaction mixture was stirred at 80° C. for 5 h. After completion of the starting material the reaction mixture was evaporated under reduced pressure, quenched with water and extracted with ethyl acetate (3×100 mL). The organic layer was washed with brine solution (150 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure to get the crude compound. The crude compound was purified by column chromatography (using silica gel 230-400 mesh, 100% pet-ether as an eluent) to afford 8 g of compound methyl 2-(bromomethyl)-5-chlorobenzoate as colorless liquid. [TLC system: pet-ether; Rf value: 0.7].
Step 1. 8-bromo-7-(but-2-yn-1-yl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione:
To a stirred solution of compound 8-bromo-3-methyl-3,7-dihydro-1H-purine-2,6-dione (10.0 g, 40.81 mmol) in DMF (100 mL) was added DIPEA (7.13 mL, 40.81 mmol) and 1-bromobut-2-yne (5.43 g, 40.81 mmol) portion wise at room temperature. The reaction mixture was stirred at room temperature for 16 h. After completion of starting material, the reaction mixture was quenched with cold water and the precipitated solid was filtered off, dried under vacuum to get 9.3 g of compound 8-bromo-7-(but-2-yn-1-yl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione as an off-white solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.7].
Step 2. Methyl 2-((8-bromo-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate:
To a stirred solution of compound 8-bromo-7-(but-2-yn-1-yl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione (2.0 g, 6.73 mmol) in DMF (20 mL) were added compound methyl 2-(bromomethyl)-5-chlorobenzoate (1.77 g, 6.73 mmol) and K2CO3 (2.79 g, 20.19 mmol) and the reaction mixture was stirred at 50° C. for 16 h. After completion of starting material, the reaction mixture was quenched with cold water and the precipitated solid was filtered off, dried under vacuum to get 2.5 g of compound methyl 2-((8-bromo-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate as an off-white solid. [TLC system: EtOAc:pet-ether (1:1); Rf value: 0.7].
Step 3. Ethyl methyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate:
To a stirred solution of compound methyl 2-((8-bromo-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate (2.5 g, 5.21 mmol) in DMF (25 mL) were added compound tert-butyl (R)-piperidin-3-ylcarbamate (1.25 g, 6.25 mmol) and K2CO3 (2.16 g, 15.63 mmol) and the reaction mixture was stirred at 65° C. for 8 h. After completion of starting material, the reaction mixture was quenched with cold water and the precipitated solid was filtered off, dried under vacuum to get 2.0 g of compound ethyl methyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate as an off-white solid. [TLC system: EtOAc:pet-ether (1:1); Rf value: 0.3].
Step 4. (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoic Acid (Int-20):
To a stirred solution of compound ethyl methyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate (0.5 g, 0.83 mmol) in MeOH (5 mL) and water (1 mL) was added LiOH·H2O (0.11 g, 2.5 mmol) at 0° C. The reaction mixture was stirred at room temperature for 16 h. After completion of starting material, the reaction mixture was concentrated under reduced pressure to get crude compound. The crude compound was diluted with water, acidified with 1N HCL. The precipitated solid was filtered off and dried under vacuum to afford 0.4 g of compound (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoic acid as an off-white solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.3].
Step 1. Methyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate
To the stirred suspension of compound (R)-3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoic acid (1.35 g, 4.05 mmol) in DCM (20 mL), was added Et3N (1.69 mL, 12.15 mmol) and BOP—Cl (1.54 g, 6.07 mmol) followed by compound methyl 3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (1.0 g, 4.05 mmol). The reaction was stirred at RT for 16 h and the progress of reaction was monitored by TLC. After completion of reaction, reaction mixture was concentrated under reduced pressure to get crude compound. The crude compound was purified by column chromatography over silica gel (Davisil) (using 0-60% EtOAc in Pet Ether as an eluent) to afford 1.6 g of compound methyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate as white solid. [TLC system: EtOAc: Pet Ether (6:4); Rf value: 0.5].
Step 2. (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylic acid (Int-21):
To a solution of compound methyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.5 g, 0.88 mmol) in MeOH (10 mL) and THF (10 mL) was added 4 M NaOH solution (2.7 mL, 5.5V) at 0° C. Then reaction was stirred at RT for 2 h. After completion of reaction, reaction mixture was concentrated and residue was acidified with 10% HCl up to pH−4 then reaction mixture was concentrated to get residue which was dried by co-evaporation with ACN and toluene to afford 0.450 g of compound (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylic acid as off white solid.
To the stirred suspension of compound (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylic acid (0.45 g, 0.81 mmol) in DCM (10 mL), was added Et3N (0.341 mL, 2.45 mmol) and BOP-Cl (0.312 g, 1.22 mmol) followed by compound 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol (0.214 g, 1.22 mmol). The reaction mixture was stirred at RT for 16 h. The progress of reaction was monitored by TLC. After completion of reaction, reaction mixture was concentrated to get crude compound which was purified by column chromatography over silica gel (Davisil) (using 0-60% EtOAc in Pet Ether as an eluent) to afford 0.4 g of compound 2-(2-(2-azidoethoxy)ethoxy)ethyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate as colorless gum. [TLC system: EtOAc: Pet Ether (7:3); Rf value: 0.7].
To a stirred solution of 2-(2-(2-azidoethoxy)ethoxy)ethyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.45 g, 0.64 mmol) in 1,4-dioxane (4.5 mL) was added 4 M HCl in 1,4-dioxane (1 mL) dropwise at 0° C. and the reaction mixture was stirred at room temperature for 2 h. After completion of starting material, the reaction mixture was concentrated under reduced pressure to get crude compound. The crude compound was purified by reverse phase preparative-HPLC to afford 0.1 g of compound Int-23 as an off-white solid[TLC system: EtOAc: Pet Ether (7:3); Rf value: 0.2].
Step 1. Heptyl (3-aminoadamantan-1-yl)carbamate:
Adamantane-1,3-diamine dihydrochloride [500 mg, 2.09 mmol], heptyl chloroformate [336 mg, 1.88 mmol] and TEA [316 mg, 3.14 mmol] were added to DCM [15 mL]. The reaction mixture was stirred for 2 h at rt under N2. The mixture was filtered and washed three times with DCM (5 mL). The filtrate was concentrated to afford methyl 6-chloro-4-methoxypyridazine-3-carboxylate (270 mg, 14% yield) as a white solid. ES MS M/Z=309 (M+1).
Step 2. Heptyl (3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)carbamate (1):
Heptyl N-(3-aminoadamantan-1-yl)carbamate [300 mg, 0.97 mmol], (2S)-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile [150 mg, 0.87 mmol], K2CO3 [402 mg, 2.91 mmol] and potassium iodide [161 mg, 0.97 mmol] were added to MeCN [10 mL]. The reaction mixture was stirred for 2 h at rt. The mixture was concentrated and the residue was purified by column chromatography on silica gel, eluting with (DCM:MeOH=20:1) to give heptyl (3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)carbamate (1, 160 mg, 90% yield) as a yellow oil. ES MS M/Z=445 (M+1). 1H NMR (400 MHz, DMSO-d6) 6.79 (s, 1H), 4.73 (dd, J=7.3, 3.8 Hz, 1H), 3.85 (t, J=6.4 Hz, 2H), 3.65-3.55 (m, 1H), 3.51-3.38 (m, 2H), 3.36 (d, J=7.3 Hz, 1H), 3.29 (s, 1H), 2.17-2.06 (m, 4H), 2.05-1.95 (m, 2H), 1.72 (d, J=17.5 Hz, 6H), 1.49 (d, J=12.8 Hz, 8H), 1.26 (s, 8H), 0.86 (t, J=6.9 Hz, 3H).
Step 1. Benzyl (3-hydroxyadamantan-1-yl)carbamate:
To a solution of 3-aminoadamantan-1-ol (5 g, 29.9 mmol) and DIEA (5.79 g, 44.9 mmol) in DCM (20 mL) was added a solution of CbzCl (5.08 g, 29.9 mmol) in DCM (20 mL) at 0° C. The reaction mixture was stirred at 25° C. for 1 h. The mixture was diluted with water and extracted with DCM (100 mL). The organic layers were dried over Na2SO4 and concentrated under pressure at 40° C. The residue was purified via Flash column chromatography, eluted with (MeOH/DCM, from 5% to 10%) to give benzyl (3-hydroxyadamantan-1-yl)carbamate (8.5 g, 94% yield) as a white solid. ES MS M/Z=302 (M+1).
Step 2. 3-(((benzyloxy)carbonyl)amino)adamantan-1-yl methanesulfonate:
To a solution of benzyl (3-hydroxyadamantan-1-yl)carbamate (3.01 g, 10 mmol) and triethylamine (3 g, 30 mmol) in DCM (20 mL) was added a solution of methanesulfonyl chloride (1.71 g, 15 mmol) in DCM (2 mL) at 0° C. The reaction mixture was stirred at 0° C. for 1 h. The mixture was diluted with water and extracted with DCM (30 mL). The organic layers were dried over Na2SO4 and concentrated under pressure at 30° C. to give 3-(((benzyloxy)carbonyl)amino)adamantan-1-yl methanesulfonate (3.3 g, 87% yield) as a white solid, which was used to the next step without further purification. ES MS M/Z=402 (M+23).
Step 3. S-(3-aminoadamantan-1-yl) ethanethioate:
A solution of 3-(((benzyloxy)carbonyl)amino)adamantan-1-yl methanesulfonate (2.5 g, 6.59 mmol) in thiolacetic acid (15 mL) was stirred at 100° C. for 12 h. The reaction mixture was concentrated under pressure at 45° C. The residue was purified via Flash column chromatography, eluted with MeOH/DCM (from 5% to 10%) to give S-(3-aminoadamantan-1-yl) ethanethioate (1 g, 67% yield) as a yellow solid. ES MS M/Z=226 (M+1).
Step 4. S-(3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl) ethanethioate:
To a mixture of S-(3-aminoadamantan-1-yl) ethanethioate (113 mg, 0.5 mmol), K2CO3 (207 mg, 1.5 mmol) and KI (8 mg, 0.05 mmol) in MeCN (5 mL) was added 2-chloro-1-(pyrrolidin-1-yl)ethan-1-one (58 mg, 0.4 mmol). The reaction mixture was stirred at 75° C. for 6 h. The reaction mixture was concentrated under pressure at 40° C. The residue was purified via Flash column chromatography, eluted with MeOH/DCM (from 5% to 10%) to give S-(3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl) ethanethioate (128 mg, 71% yield) as a yellow oil. ES MS M/Z=362 (M+1).
Step 5. (2S)-1-((3-mercaptoadamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile:
To a solution of S-(3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl) ethanethioate (1 g, 2.77 mmol) in MeOH/H2O (V/V=20:1, 10 mL) was added K2CO3 (1.14 g, 8.31 mmol). The reaction mixture was stirred at 25° C. for 30 min. The reaction mixture was dried over Na2SO4 and concentrated under pressure at 35° C. The residue was purified via Flash column chromatography, eluted with MeOH/DCM (from 10% to 20%) to give (2S)-1-((3-mercaptoadamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (300 mg, 33% yield) as a yellow oil. ES MS M/Z=320 (M+1)
Step 6. Tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)(3-mercaptoadamantan-1-yl)carbamate:
A mixture of (2S)-1-((3-mercaptoadamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (400 mg, 1.25 mmol), Boc2O (545 mg, 2.5 mmol) and TEA (631 mg, 6.25 mmol) in toluene (10 mL) was stirred at reflux for 12 h. The mixture was concentrated. The residue was purified via Flash column chromatography, eluted with MeOH/DCM (from 5% to 10%) to give tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)(3-mercaptoadamantan-1-yl)carbamate (400 mg, 76% yield) as a colorless oil. ES MS M/Z=442 (M+23)
Step 7. Tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)(3-(heptylthio)adamantan-1-yl)carbamate:
A mixture of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)(3-mercaptoadamantan-1-yl)carbamate (170 mg, 0.40 mmol), 1-iodoheptane (138 mg, 0.61 mmol) and potassium carbonate (224 mg, 1.62 mmol) in MeCN (3 mL) was stirred at 50° C. for 15 hrs. The mixture was concentrated and the residue was purified by column chromatography on silica gel, eluting with (PE:EtOAc=2:1) to give tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)(3-(heptylthio)adamantan-1-yl)carbamate (50 mg, 27% yield) as a yellow solid. ES MS M/Z=540 (M+23).
Step 8. (2S)-1-((3-(heptylthio)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (2):
A solution of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)(3-(heptylthio)adamantan-1-yl)carbamate (50 mg, 0.10 mmol) in TFA/DCM (V/V=1:3, 4 mL) was stirred at room temperature for 30 min. The mixture was concentrated and the residue was purified by Prep-HPLC (Gemini-C18 150×21.2 mm, 5 um, mobile phase: ACN—H2O, gradient: 30˜70%) to give (2S)-1-((3-(heptylthio)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (32 mg, 80% yield) as a white solid. ES MS M/Z=418 (M+1). 1H NMR (400 MHz, CD3OD) 8.40 (s, 1H), 4.81 (t, J=5.2 Hz, 1H), 3.99-3.87 (m, 2H), 3.74-3.68 (m, 1H), 3.56-3.49 (m, 1H), 2.56 (t, J=7.2 Hz, 2H), 2.35-2.24 (m, 4H), 2.23-2.14 (m, 2H), 2.01-1.94 (m, 2H), 1.92-1.78 (m, 8H), 1.73-1.65 (m, 2H), 1.57-1.50 (m, 2H), 1.43-1.36 (m, 2H), 1.35-1.24 (m, 6H), 0.90 (t, J=6.8 Hz, 3H).
To a solution of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(heptylthio)adamantan-1-yl)carbamate (20 mg, 0.03 mmol) in DCM (3 mL) under nitrogen atmosphere, was added dropwise 3-Chloroperbenzoic acid (10 mg, 0.05 mmol) at −40° C. The reaction mixture was stirred at −40° C. for 30 min. TFA (1 mL) was added slowly. The reaction mixture was stirred at room temperature for addition 30 min. The reaction mixture was concentrated under pressure. Then the crude product was purified via Genal-Prep-HPLC (Gemini-C18 150×21.2 mm, 5 um, mobile phase: ACN—H2O, 0.1% FA, gradient: 5˜50%) to give the product (2S)-1-((3-(heptylsulfinyl)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (6.5 mg, 38.90%) as a white solid. MS (ESI): mass calcd. for C24H39N3O2S 433.66, m/z found 433.8 [M+H]+. 1H NMR (400 MHz, CD3OD) 8.38 (s, 1H), 4.82-4.79 (m, 1H), 3.95-3.84 (m, 2H), 3.75-3.70 (m, 1H), 3.56-3.50 (m, 1H), 2.72-2.66 (m, 2H), 2.43 (s, 2H), 2.31-2.25 (m, 2H), 2.22-2.14 (m, 2H), 2.01-1.72 (m, 14H), 1.57-1.45 (m, 2H), 1.42-1.35 (m, 2H), 1.33-1.31 (m, 4H), 0.92-0.89 (m, 3H).
Step 1. tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)(3-((2-hydroxyethyl)thio)adamantan-1-yl)carbamate
To a mixture of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)(3-mercaptoadamantan-1-yl)carbamate (350 mg, 0.83 mmol) and K2CO3 (572 mg, 4.15 mmol) in MeCN (15 mL) was added 2-iodoethanol (1.42 g, 8.3 mmol). The reaction mixture was stirred at 25° C. for 12 h. The reaction mixture was concentrated under pressure. The residue was purified via Flash Chromatography and was eluted with CH2Cl2/MeOH (10:1) to give the product tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)(3-((2-hydroxyethyl)thio)adamantan-1-yl)carbamate (340 mg, 88%) as a yellow oil. MS (ESI): mass calcd. for C24H37N3O4S 463.25, m/z found 486.2 [M+Na]+.
Step 2. tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)(3-((2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)thio) adamantan-1-yl)carbamate
To a mixture of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)(3-((2-hydroxyethyl)thio)adamantan-1-yl)carbamate (340 mg, 0.73 mmol) and Et3N (221 mg, 2.19 mmol) in DCM (20 mL) was added 4-nitrophenyl chloroformate (220 mg, 1.09 mmol). The reaction mixture was stirred at 25° C. for 3 hrs. H2O (20 mL) was added. The residue was extracted with DCM (30 mL). The DCM phase was concentrated to give tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)(3-((2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)thio)adamantan-1-yl)carbamate (600 mg, crude) as a yellow oil, which was used to the next step without further purification. MS (ESI): mass calcd. for C31H40N4O8S 628.26, m/z found 629.7 [M+H]+.
Step 3. tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-((2-(((2-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)phenoxy)ethyl)carbamoyl)oxy)ethyl)thio)adamantan-1-yl)carbamate
To a solution of (2R,3R,4R,5S)-6-((4-(2-aminoethoxy)phenethyl)(hexyl)amino)hexane-1,2,3,4,5-pentaol (410 mg, 0.95 mmol) in THF (15 mL) was added TEA (303 mg, 3 mmol), followed by a solution of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)(3-((2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)thio)adamantan-1-yl)carbamate (903 mg, 1.43 mmol) in THF (5 mL). The reaction mixture was stirred at 25° C. for 4 hrs. The reaction mixture was concentrated under pressure. The residue was purified via Flash Chromatography and was eluted with CH2Cl2/MeOH (5:1) to give the product tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-((2-(((2-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)phenoxy)ethyl)carbamoyl)oxy)ethyl)thio) adamantan-1-yl)carbamate (220 mg, 25%) as a yellow oil. MS (ESI): mass calcd. for C47H75N5O11S 917.52, m/z found 918.4 [M+H]+.
Step 4. 2-(((1R,3S,5S)-3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)thio)ethyl (2-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)phenoxy)ethyl)carbamate
To a solution of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-((2-(((2-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)phenoxy)ethyl)carbamoyl)oxy)ethyl)thio) adamantan-1-yl)carbamate (400 mg, 0.43 mmol) in DCM (15 mL) was added ZnBr2 (1086 mg, 4.8 mmol). The reaction mixture was stirred at 25° C. for 8 hrs. The solution was filtered, the filtrate was concentrated and purified via Prep-TLC (CH2Cl2/MeOH)=(5:1) to give impure product, then the impure product was purified via Genal-Prep-HPLC (Gemini-C18 150×21.2 mm, 5 um, mobile phase: ACN—H2O, 0.1% FA, gradient: 5-50%) to give the product 2-(((1R,3S,5S)-3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)thio)ethyl (2-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)phenoxy)ethyl)carbamate (V2209613) (20 mg, 5%) as a white solid. MS (ESI): mass calcd. for C42H67N5O9S 817.47, m/z found 818.4 [M+H]+. 1H NMR (400 MHz, MeOD) δ 8.53 (s, 2H), 7.24 (d, J=8.1 Hz, 2H), 6.94 (d, J=8.2 Hz, 2H), 4.77-4.86 (m, 1H), 4.24-3.97 (m, 5H), 3.94-3.78 (m, 4H), 3.77-3.63 (m, 4H), 3.61-3.45 (m, 3H), 3.44-3.33 (m, 5H), 3.22-3.15 (m, 2H), 3.10-2.95 (m, 2H), 2.88-2.73 (m, 2H), 2.40-2.10 (m, 6H), 2.02-1.92 (m, 2H), 1.88-1.66 (m, 11H), 1.45-1.30 (m, 6H), 1.00-0.90 (m, 3H).
Step 1. Tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)carbamate:
To a stirred solution of (1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl methanesulfonate (Int-2, 0.59 g, 1.22 mmol) and ethane-1,2-diol (3.8 g, 61.25 mmol) in acetonitrile (3.0 mL) was added molecular sieves 4 Å (2.0 g) and the resultant mixture was stirred at 70° C. for 16 h. After completion, the reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (3×50 mL). The combined organic layer was dried over anhydrous sodium sulfate and evaporated to give crude product. The crude was then purified by silica gel column chromatography using 3% methanol in dichloromethane and further purified by RP-HPLC using following conditions: column/dimensions: X-BRIDGE-C18 (19*250, 5 um), mobile phase A:10 mM ammonium bicarbonate in water, Mobile phase B: ACN Gradient (Time/% B): 00/10, 3/10, 7/35, 20/75, 20.1/100, 22/100, 22.1/10, 24/10, flow rate: 17 ml/min. The desired fraction was evaporated to afford tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)carbamate (0.1 g) as an off-white solid. TLC system: MeOH: DCM (1:9); Rf: 0.4.
Step 2. (2S)-1-(((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (5):
To a stirred suspension of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)carbamate (0.09 g, 0.2 mmol) in acetonitrile (5 mL), 1 M tin(IV) chloride solution in heptane (0.8 mL, 0.8 mmol) was added and the mixture was stirred at room temperature for 3 h. After completion, the reaction mixture was evaporated, the residue was co-evaporated with methanol (10 mL) and washed with diethyl ether (2×5 mL) to give crude product. The crude product was then purified by C-18 column chromatography using 30% methanol in 10 mM ammonium bicarbonate in water. The desired fraction was lyophilized to afford (2S)-1-(((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (0.055 g) as an off-white solid. TLC system: MeOH:DCM (0.5:9.5); Rf. 0.1. LCMS M/Z 348.37 (M+1)1H NMR (400 MHz, DMSO-d6) δ 4.74-4.72 (m, 1H), 4.46 (bs, 1H), 3.63-3.32 (m, 8H), 2.17-2.11 (m, 4H), 2.03-1.99 (m, 2H), 1.68 (t, J=6.0 Hz, 1H), 1.60-1.40 (m, 12H).
Step 1. Benzyl (3-((methylthio)methoxy)adamantan-1-yl)carbamate:
A mixture of benzyl (3-hydroxyadamantan-1-yl)carbamate (3 g, 9.97 mmol) and Ac2O (35.1 g, 29.9 mmol) in DMSO (30 mL) was stirred at room temperature overnight. The reaction mixture was diluted with EtOAc (100 mL) and washed with water (30 mL×3). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel, eluting with (PE/EtOAc=8/1) to give benzyl (3-((methylthio)methoxy) adamantan-1-yl)carbamate (2.6 g, 72.2% yield) as a pale yellow oil. ES MS M/Z=384 (M+23).
Step 2. ((3-(((benzyloxy)carbonyl)amino)adamantan-1-yl)oxy)methyl octanoate:
To a solution of benzyl (3-((methylthio)methoxy) adamantan-1-yl)carbamate (2.5 g, 6.93 mmol) in CDCl3 (20 mL) was added SOCl2 (1.99 g, 13.86 mmol) at 0° C. slowly. The mixture was stirred at 0° C. for 30 min. The mixture was concentrated and dissolved in THF (20 mL). To the solution was added TEA (1.40 g, 13.86 mmol) and octanoic acid (1.99 g, 13.86 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (50 mL) and washed with water (20 mL×2). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel, eluting with (PE/EtOAc=8/1) to give ((3-(((benzyloxy)carbonyl)amino)adamantan-1-yl)oxy)methyl octanoate (1.9 g, 60% yield) as a pale yellow oil. ES MS M/Z=480 (M+23).
Step 3. ((3-aminoadamantan-1-yl)oxy)methyl octanoate:
To a solution of -(((benzyloxy)carbonyl)amino)adamantan-1-yl)oxy)methyl octanoate (1.7 g, 3.72 mmol) in MeOH (10 mL) was added Pd/C (500 mg). The reaction mixture was stirred at room temperature under H2 atmosphere overnight. The mixture was filtered and the filtrate was evaporated under reduced pressure to give ((3-aminoadamantan-1-yl)oxy)methyl octanoate (1.1 g, 91.4% yield) as a colorless oil. ES MS M/Z=324 (M+1).
Step 4. ((3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)methyl octanoate (7):
A mixture of ((3-aminoadamantan-1-yl)oxy)methyl octanoate (1 g, 3.1 mmol), (S)-1-(2-chloroacetyl) pyrrolidine-2-carbonitrile (533 mg, 3.1 mmol), potassium carbonate (513 mg, 3.72 mmol) and Potassium iodide (257 mg, 1.55 mmol) in Acetonitrile (15 mL) was stirred at 50° C. under N2 atmosphere for 5 hrs. The mixture was diluted with EtOAc (30 mL) and washed with water (10 mL×2). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel, eluting with (PE/EtOAc=6/1) to give ((3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)methyl octanoate (mCMP694) (400 mg, 28% yield) as a pale yellow oil. ES MS M/Z=460 (M+1). 1H NMR (400 MHz, CDCl3) 5.39 (s, 2H), 4.87-4.73 (m, 1H), 3.66-3.39 (m, 4H), 2.36-2.25 (m, 6H), 2.24-2.13 (m, 2H), 1.83 (s, 3H), 1.80-1.68 (m, 6H), 1.66-1.56 (m, 6H), 1.33-1.24 (m, 8H), 0.88 (t, J=7.2 Hz, 3H).
Step 1. Tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)adamantan-1-yl)carbamate:
To a solution of compound (1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl methanesulfonate (137 g, 285 mmol, 1.00 eq) in THF (1.40 L), TEA (66.1 g, 653 mmol, 2.29 eq) and 2,2′-(ethane-1,2-diylbis(oxy))bis(ethan-1-ol) (722 g, 4.81 mol, 16.8 eq) were added. The reaction was heated to 70° C. for 48 hrs. The reaction mixture was diluted with H2O (2.00 L) and extracted with EA (2.00 L×2). The combined organic layers were washed with 5% aqueous solution of citric acid (2.00 L), then the combined organic layers were washed with saturated aqueous solution of NaHCO3 (2.00 L), dried over Na2SO4, filtered and concentrated under reduced pressure <45° C. The residue was purified by column chromatography (SiO2, PE/EA=1/1 to 0/1) to give compound tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)adamantan-1-yl)carbamate (175 g, 327 mmol, 57.4% yield, N/A purity) as a light yellow oil. TLC System: EA only; Rf. 0.15.
Step 2. 2-(2-(2-(((1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethoxy)ethoxy)ethyl methanesulfonate:
To a solution of compound tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)adamantan-1-yl)carbamate (162 g, 302 mmol, 1.00 eq) and N,N,N′,N′-tetramethyl-1,6-hexanediamine (115 g, 667 mmol, 2.21 eq) in DCM (1600 mL), MsCl (54.9 g, 479 mmol, 37.1 mL, 1.59 eq) were added at 0-10° C. The reaction was stirred for 2 hrs at 0-10° C. The reaction mixture was quenched by addition of ice water (1.00 L) at 0° C., and then titrated with 5% aqueous solution of citric acid (2.00 L) until the pH=5˜6, and extracted with EA (3.00 L). The combined organic layers were washed with NaHCO3 (1.00 L), dried over Na2SO4, filtered and concentrated under reduced pressure to give compound 2-(2-(2-(((1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethoxy)ethoxy)ethyl methanesulfonate (180 g, crude) as a light yellow oil. TLC system: EA; Rf: 0.30.
Step 3. tert-butyl ((1S,3R,5S)-3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate:
To a solution of compound 2-(2-(2-(((1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethoxy)ethoxy)ethyl methanesulfonate (90.0 g, 146 mmol, 1.00 eq) in DMF (1.00 L) was added NaN3 (22.3 g, 343 mmol, 2.34 eq) at 20˜30° C. under N2, then the mixture was stirred at 70° C. for 12 hrs. The reaction was added to saturated Na2CO3 aqueous solution (2.00 L), then extracted with EA 2.00 L. The combined organic layers were washed with brine 2.00 L, dried over Na2SO4, filtered and concentrated under reduced pressure to give compound tert-butyl ((1S,3R,5S)-3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (153 g, crude) as a light yellow oil. TLC system: EA; Rf: 0.30.
Step 4. Tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-(2-(4-((hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)adamantan-1-yl)carbamate:
A mixture of compound tert-butyl ((1S,3R,5S)-3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (152 g, 271 mmol, 1.00 eq), (2R,3R,4R,5S)-6-(hexyl(prop-2-yn-1-yl)amino)hexane-1,2,3,4,5-pentaol (98.8 g, 325 mmol, 1.20 eq) and CuI (6.84 g, 35.9 mmol, 0.13 eq) in THF (1.52 L) was stirred 12 hrs at 25° C. The reaction mixture was diluted with H2O (2.00 L) and extracted with EA (2.00 L). The combined organic layers were washed with brine (1.00 L), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by reversed-phase HPLC (0.1% NH3·H2O) to give compound tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-(2-(4-((hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)adamantan-1-yl)carbamate (100 g, 115 mmol, 42.7% yield, 41.3% purity) as a yellow oil. TLC system: DCM:MeOH=5:1; Rf: 0.20.
Step 5. (2S)-1-(((1S,3R,5S)-3-(2-(2-(2-(4-((hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (8):
To a solution of compound tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-(2-(4-((hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)adamantan-1-yl)carbamate (40.0 g, 46.3 mmol, 1.00 eq) in ACN (320 mL) was added HCl (1 M, 560 mL, 12.1 eq) at 0° C. The reaction mixture was stirred at 25° C. for 18 hrs. The reaction mixture was extracted with DCM (1.00 L×2). The aqueous phase was adjusted to pH 9˜10 by Sat. NaHCO3 and K2CO3 and extracted with DCM (1.00 L×2). The combined organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure to give 13 (24.5 g, 31.0 mmol, 66.9% yield, 96.7% purity) as a light yellow oil. LCMS M/Z M+1=764.7. 1H-NMR (400 MHz, CDCl3) δ 7.73 (s, 1H), 4.92-4.70 (m, 1H), 4.52 (br t, J=4.5 Hz, 2H), 3.94-3.82 (m, 5H), 3.81-3.71 (m, 4H), 3.71-3.63 (m, 3H), 3.56 (br d, J=19.3 Hz, 9H), 3.47-3.33 (m, 3H), 2.77-2.58 (m, 2H), 2.53 (td, J=7.7, 12.7 Hz, 1H), 2.47-2.37 (m, 1H), 2.36-2.01 (m, 6H), 1.99 (s, 1H), 1.75-1.40 (m, 14H), 1.23 (br s, 6H), 0.84 (br t, J=6.7 Hz, 3H).
Similarly, Compound 15 was prepared from Int-1 and Int-5. Compounds 20 and 21 were prepared from saxagliptin according to the route for compound 8.
Step 1. Tert-butyl ((1S,3R,5S)-3-(2-(2-(4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate:
Mixture of tert-butyl ((1 S,3R,5S)-3-(2-(2-azidoethoxy)ethoxy) adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (0.6 g, 1.16 mmol), (2R,2′R,3R,3′R,4R,4′R,5S,5'S)-6,6′-(prop-2-yn-1-ylazanediyl)bis(hexane-1,2,3,4,5-pentaol) (0.66 g, 1.74 mmol), copper sulfate pentahydrate (0.35 g, 1.39 mmol) and sodium ascorbate (0.34 g, 1.74 mmol) in tert-Butyl alcohol (10 mL) and water (2 mL) mixture was stirred at room temperature for 16 h. After completion (monitored by LCMS), the reaction mixture was filtered through celite and washed with methanol and water (1:1) (100 mL) and filtrate was evaporated to give crude product. The crude was then purified by reverse phase C18 column chromatography using 50% methanol in 0.1% formic acid in water. The desired product containing fractions were collected and evaporated to give tert-butyl ((1S,3R,5S)-3-(2-(2-(4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (0.46 g, 44%) as an off-white solid.
Step 2. (2S)-1-(((1S,3R,5S)-3-(2-(2-(4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (9):
To a stirred suspension of tert-butyl ((1S,3R,5S)-3-(2-(2-(4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (0.35 g, 0.39 mmol) in acetonitrile (15 mL) was added 1 M tin(IV) chloride solution in heptane (1.55 mL, 1.55 mmol) and the reaction mixture was stirred at room temperature for 3 h. After completion (reaction monitored by LCMS), the reaction mixture was evaporated and the residue was co-evaporated with methanol (10 mL), washed with diethyl ether (2×5 mL) to give crude product. The crude was then purified by C-18 column chromatography using 30% methanol in 10 mM ammonium bicarbonate in water. The desired fraction was lyophilized to afford (2S)-1-(((1S,3R,5S)-3-(2-(2-(4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (0.076 g) as a pale yellow solid. LCMS M/Z 800.59 (M+1) 1H NMR (400 MHz, DMSO-d6) δ 7.95 (s, 1H), 4.75-4.72 (m, 1H), 4.70-4.10 (m, 12H), 3.81-3.72 (m, 6H), 3.63-3.39 (m, 22H), 2.17-1.91 (m, 6H), 1.56-1.44 (m, 12H).
Step 1. Tert-butyl ((1S,3R,5S)-3-(2-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate:
To a mixture of tert-butyl ((1S,3R,5S)-3-(2-(2-aminoethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (0.21 g, 0.43 mmol), (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (0.19 g, 1.07 mmol) and acetic acid (1.0 mL) in methanol (10 mL) was added sodium cyanoborohydride (0.034 g, 0.52 mmol). The reaction mixture was stirred at 70° C. for 24 h. After completion of reaction (reaction monitored by LCMS), the reaction mixture was evaporated to get crude. The obtained crude was purified by reverse phase C18 column chromatography using 50% methanol in 0.1% formic acid in water. The desired product containing fractions were collected and evaporated to give tert-butyl ((1S,3R,5S)-3-(2-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (0.28 g) as colorless gum.
Step 2. (2S)-1-(((1S,3R,5S)-3-(2-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)ethoxy)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (10):
To the stirred solution of tert-butyl ((1S,3R,5S)-3-(2-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (0.22 g, 0.27 mmol) in water (5 mL), 2N aqueous hydrochloric acid was added and the reaction mixture was stirred at room temperature for 24 h. After completion of reaction (monitored by LCMS), the reaction mixture was purified by RP prep HPLC using following conditions: Column/dimensions: X-BRIDGE(19*150), 5 um, mobile phase A: 10 mM ammonium bicarbonate in water (aq), mobile phase B: ACN, Gradient (Time % B): 0/2, 3/2, 16/36, 16.1/100, 18/100, 18.1/2, 20/2, flow rate: 17 ml/min. The desired fraction was lyophilized to afford (2S)-1-(((1S,3R,5S)-3-(2-(2-(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)ethoxy)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (0.14 g, 72.5%) as off-white solid. LCMS: M/Z 717.52 [M−1] 1H NMR (400 MHz, DMSO-d6): δ 4.74-4.70 (m, 1H), 4.51-4.46 (m, 4H), 4.35-4.29 (m, 4H), 4.19 (d, J=6.4 Hz, 2H), 3.65-3.55 (m, 7H), 3.49-3.33 (m, 16H), 2.75-2.50 (m, 5H), 2.17-1.95 (m, 6H), 1.70 (bs, 1H), 1.59-1.45 (m, 12H).
Step 1. Tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)ethoxy)adamantan-1-yl)carbamate:
To a mixture of tert-butyl ((1S,3R,5S)-3-(2-(2-aminoethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (0.22 g, 0.45 mmol), (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (0.081 g, 0.45 mmol) and acetic acid (1.0 mL) in methanol (50 mL) was added sodium cyanoborohydride (0.03 g, 0.45 mmol). The reaction mixture was stirred at 60° C. for 24 h. After completion (reaction monitored by LCMS), the mixture was evaporated under reduced pressure to give crude product. The crude was then purified by reverse phase preparative HPLC using following conditions. Column/dimensions: X-BRIDGE(19*150), 5 um, mobile phase A: 10 mM ammonium bicarbonate in water (aq), mobile phase B: ACN, gradient (Time/% B): 0/10, 1/10, 20/50, 20.10/95, 22.10/95, 22.20/10, 24/10 flow rate: 17 ml/min. The desired fractions were collected and lyophilized to afford tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)ethoxy)adamantan-1-yl)carbamate (0.035 g) as an off-white solid.
Step 2. (2S)-1-(((1S,3R,5S)-3-(2-(2-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)ethoxy)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (11):
To the stirred suspension of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)ethoxy)adamantan-1-yl)carbamate (0.03 g, 0.046 mmol) in dichloromethane (1 mL), trifluoroacetic acid was added at 0° C. and the reaction mixture was stirred at room temperature for 1 h. After completion (monitored by LCMS), the reaction mixture was evaporated under reduced pressure to give crude product. The crude was then purified by reverse phase preparative HPLC using following conditions: Column/dimensions: X-BRIDGE(19*150), 5 um, mobile phase A: 10 mM ammonium bicarbonate in water (aq), mobile phase B: ACN, gradient (Time/% B): 0/10, 1/10, 11/45.20, 11.10/95, 13.10/95, 13.20/10, 15/10, flow rate: 17 ml/min. The desired fraction was lyophilized to afford (2S)-1-(((1S,3R,5S)-3-(2-(2-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)ethoxy)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (0.012 g, 47.2%) as an off-white solid. LCMS: M/Z 555.29 (M+1) 1H NMR (400 MHz, DMSO-d6) δ 4.73-4.29 (m, 6H), 3.63-3.13 (m, 17H), 2.67-2.60 (m, 4H), 2.16-1.99 (m, 6H), 1.59-1.45 (m, 12H).
Step 1. (2R,3R,4R,5S)-6-(ethylamino)hexane-1,2,3,4,5-pentaol:
To the mixture of (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (5 g, 2.77 mmol) and 2 M ethylamine in THF (20 mL, 41.6 mmol) in methanol (150 mL) was added raney nickel (5 g) and the reaction mixture was stirred at 70° C. for 16 h under 150 psi hydrogen gas. After completion (monitored by LCMS), the reaction mixture was filtered through a pad of celite and washed with methanol (50 mL). The filtrate was evaporated to give solid, which was triturated with diethyl ether (50 mL) and dried to afford (2R,3R 4R,5S)-6-(ethylamino)hexane-1,2,3,4,5-pentaol (5 g, 86%) as an off-white solid.
Step 2. Tert-butyl (4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate:
To a solution of (2R,3R,4R,5S)-6-(ethylamino)hexane-1,2,3,4,5-pentaol (1.0 g, 4.78 mmol) in methanol (10 mL) was added potassium carbonate (0.99 g, 7.17 mmol) followed by tert-butyl (4-(bromomethyl)benzyl)carbamate (1.44 g, 4.78 mmol) portion wise at 0° C. and the mixture was stirred at room temperature for 16 h. After completion (monitored by LCMS), the reaction mixture was evaporated to give crude. The crude product was then purified by reverse phase C18 flash column chromatography using 18% of 0.1% formic acid in water and methanol as a mobile phase. The desired product containing fractions were collected and evaporated to give tert-butyl (4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate formate (1.3 g) as a colorless foamy solid.
Step 3. (2R,3R,4R,5S)-6-((4-(aminomethyl)benzyl)(ethyl)amino)hexane-1,2,3,4,5-pentaol hydrochloride:
To a stirred solution of tert-butyl (4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate formate (0.25 g, 0.527 mmol) in methanol (2.5 mL) was added 4 M hydrochloric acid in 1.4 dioxane slowly at 0° C. and the resultant mixture was stirred at room temperature for 2 h. After completion (monitored by LCMS), the reaction mixture was evaporated to give (2R,3R,4R,5S)-6-((4-(aminomethyl)benzyl)(ethyl)amino)hexane-1,2,3,4,5-pentaol hydrochloride (0.19 g, crude) as a green semi solid.
Step 4. 2-(2-(((1R,3S,5S)-3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethoxy)ethyl (4-nitrophenyl) carbonate:
To a stirred solution of (2S)-1-(((1S,3R,5S)-3-(2-(2-hydroxyethoxy)ethoxy)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (0.2 g, 0.407 mmol) in dichloromethane (4 mL) was added triethylamine (0.164 g, 1.628 mmol) subsequently 4-nitrophenyl carbonochloridate (0.09 g, 0.447 mmol) in dichloromethane (1 mL) at −10° C. and the mixture was stirred at room temperature for 2 h. After completion, the reaction mixture was taken as such to next step.
Step 5. 2-(2-(((1R,3S,5S)-3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethoxy)ethyl (4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate:
To a stirred suspension of (2R,3R,4R,5S)-6-((4-(aminomethyl)benzyl)(ethyl)amino)hexane-1,2,3,4,5-pentaol hydrochloride (0.17 g, 0.47 mmol) in acetonitrile (5 mL) was added triethylamine (0.24 g, 2.34 mmol) followed by 2-(2-(((1R 3S,5S)-3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethoxy)ethyl (4-nitrophenyl) carbonate (0.26 g, 0.39 mmol) in dichloromethane (5 mL) at 0° C. and the mixture was stirred at room temperature for 16 h. After completion (monitored by LCMS), the reaction mixture was evaporated to give crude, which was then purified by reverse phase C18 column chromatography using 40% of 0.1% formic acid in water and acetonitrile as a mobile phase. The desired product containing fractions were collected and evaporated to give 2-(2-(((1R,3S,5S)-3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethoxy)ethyl (4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate (0.3 g, crude) as an yellow semi solid.
Step 6. 2-(2-(((1R,3S,5S)-3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethoxy)ethyl (4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate (12-formate salt):
To a stirred suspension of 2-(2-(((1R,3S,5S)-3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethoxy)ethyl (4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate (0.29 g, 0.34 mmol) in dichloromethane (4 mL), trifluroacetic acid was added at 0° C. and the reaction mixture was stirred at room temperature for 3 h. After completion (monitored by LCMS), the reaction mixture was concentrated and the residue was washed with diethyl ether (3×10 mL) and dried to give crude product. The crude product was then purified by reverse phase preparative HPLC using following conditions: Column/dimensions: SUNFIREC18 (19*150*5μ), mobile phase A: 0.1% FA in water (aq), mobile phase B: acetonitrile, gradient (time/% B): 0/5, 2/5, 10/20, 10.1/100, 14/100, 14.1/5, 16/5, flow rate: 17 ml/min. The desired fraction was lyophilized to afford 2-(2-(((1R,3S,5S)-3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethoxy)ethyl (4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate formate (0.038 g) as an off-white solid. LCMS M/Z 746.72 (M+1) 1H NMR (400 MHz, DMSO-d6): δ 8.17 (s, 2H), 7.73 (s, 1H), 7.27 (d, J=7.6 Hz, 2H), 7.17 (d, J=8.0 Hz, 2H), 4.75-4.74 (m, 3H), 4.16-4.06 (m, 4H), 3.75-3.35 (m, 21H), 2.61-2.50 (m, 1H), 2.46-2.41 (m, 3H), 2.19-1.99 (m, 6H), 1.60-1.52 (m, 12H), 0.94 (t, J=7.2 Hz, 3H).
Step 1. Tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)carbamate:
To a solution of (1R,3S,5S)-3-((tert-butoxycarbonyl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl methanesulfonate (1.25 g, 2.59 mmol) in MeCN (5 mL) was added ethane-1,2-diol (8.04 g, 129.6 mmol). The reaction mixture was stirred at 70° C. for 12 hrs. The reaction mixture was concentrated under pressure at 40° C. The residue was purified via Flash column chromatography and eluted with (MeOH/DCM, from 1% to 10%) to give tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)carbamate (860 mg, 74% yield) as a yellow oil. ES MS M/Z=470 (M+23).
Step 2. Tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(((4-nitrophenoxy)carbonyl)oxy)ethoxy)adamantan-1-yl)carbamate:
To a solution of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)carbamate (860 mg, 1.92 mmol) and TEA (1000 mg, 10 mmol) in DCM (20 mL) was added 4-nitrophenyl chloroformate (800 mg, 4 mmol). The reaction mixture was stirred at 25° C. for 3 hrs. The mixture was quenched with H2O (20 mL) and extracted with DCM (30 mL). The organic layer was dried over Na2SO4 and concentrated under pressure at 30° C. to give tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl((1S,3R,5S)-3-(2-(((4-nitrophenoxy)carbonyl)oxy)ethoxy) adamantan-1-yl)carbamate (1.2 g, crude) as a yellow oil, which was used to the next step without further purification. ES MS M/Z=635 (M+23).
Step 2. Tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(((2-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)phenoxy)ethyl)carbamoyl)oxy)ethoxy)adamantan-1-yl)carbamate:
To a solution of (2R,3R,4R,5S)-6-((4-(2-aminoethoxy)phenethyl)(hexyl)amino)hexane-1,2,3,4,5-pentaol in THF (10 mL) was added TEA (210 mg, 2.1 mmol), followed by a solution of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(((4-nitrophenoxy)carbonyl)oxy)ethoxy)adamantan-1-yl)carbamate (428 mg, 0.7 mmol) in THF (1 mL). The reaction mixture was stirred at 25° C. for 4 hrs. The reaction mixture was concentrated under pressure at 40° C. The residue was purified via Flash column chromatography and eluted with (MeOH/DCM, from 10% to 50%) to give tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1 S,3R,5S)-3-(2-(((2-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)phenoxy)ethyl)carbamoyl)oxy)ethoxy)adamantan-1-yl)carbamate (436 mg, 69% yield) as a yellow oil. ES MS M/Z=902 (M+1).
Step 3. 2-(((1R,3S,5S)-3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethyl (2-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)phenoxy)ethyl)carbamate (13):
To a solution of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(((2-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)phenoxy)ethyl)carbamoyl)oxy)ethoxy)adamantan-1-yl)carbamate (436 mg, 0.48 mmol) in DCM (15 mL) was added ZnBr2 (1086 mg, 4.8 mmol). The reaction mixture was stirred at 25° C. for 12 hrs. The mixture was filtered and the filtrate was concentrated under pressure at 35° C. The residue was purified via Prep-TLC (CH2Cl2/MeOH=5:1) to give impure product, which was purified via Genal-Prep-HPLC (Gemini-C18 150×21.2 mm, 5 um, mobile phase: ACN—H2O, 0.1% FA, gradient: 5˜50%) 2-(((1R,3S,5S)-3-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)amino)adamantan-1-yl)oxy)ethyl (2-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethyl)phenoxy)ethyl)carbamate (13) (50 mg, 12% yield) as a white solid. ES MS M/Z=802 (M+1). 1H NMR (400 MHz, CD3OD) 8.48 (s, 2H), 7.22 (d, J=8.5 Hz, 2H), 6.92 (d, J=8.6 Hz, 2H), 4.83-4.78 (m, 1H), 4.17-4.07 (m, 3H), 4.05-3.95 (m, 2H), 3.91-3.74 (m, 4H), 3.74-3.60 (m, 6H), 3.55-3.50 (m, 1H), 3.50-3.43 (m, 2H), 3.41-3.32 (m, 4H), 3.26-3.16 (m, 2H), 3.05-2.94 (m, 2H), 2.40-2.09 (m, 6H), 1.85-1.65 (m, 12H), 1.58 (s, 2H), 1.42-1.30 (m, 6H), 0.97-0.89 (m, 3H).
Step 1. Mixture of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)(1S,3R,5S)-3-(2-(2-(4-((((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)carbamate and tert-butyl ((1S,3R,5S)-3-(2-(2-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate:
Mixture of tert-butyl ((1S,3R,5S)-3-(2-(2-azidoethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (1.0 g, 1.94 mmol), (2R,3R,4R,5S)-6-(prop-2-yn-1-ylamino)hexane-1,2,3,4,5-pentaol (0.64 g, 2.90 mmol), copper (11) sulfate pentahydrate (0.58 g, 2.33 mmol) and sodium ascorbate (0.58 g, 2.91 mmol) in tert-butyl alcohol (10 mL) and water (2 mL) mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was filtered through celite and washed with methanol and water (1:1) (100 mL) and the filtrate was evaporated to give crude. The crude was then purified by reverse phase C18 column chromatography using 50% methanol in 0.1% formic acid in water. The desired product containing fractions were collected and evaporated to give mixture of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-(4-((((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)carbamate and tert-butyl ((1S,3R,5S)-3-(2-(2-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (0.59 g, crude) as a brown solid.
Step 2. Tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-(4-((((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)carbamate:
Mixture of tert-butyl ((1S,3R,5S)-3-(2-(2-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)(2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate (0.44 g, 0.77 mmol), (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (0.11 g, 0.61 mmol) and acetic acid (0.5 mL) in methanol (25 mL) was added sodium cyanoborohydride (0.048 g, 0.77 mmol) and the mixture was stirred at 60° C. for 6 h. After completion, the reaction mixture was evaporated to give crude product which was then purified by RP preparative HPLC using following conditions. Column/dimensions: X-BRIDGE C18 (19*250) Sum, mobile phase A: 10 mM ABC in water (aq), mobile phase B: 100% ACN, gradient (time/% B):0/20, 3/20, 18/40, 18.1/100, 20/100, 20.1/20, 24/20, flow rate: 17 ml/min. The desired product containing fraction was evaporated to afford tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-(4-((((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)carbamate (0.1 g) as an off-white solid.
Step 3. (2S)-1-(((1S,3R,5S)-3-(2-(2-(4-((((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (14):
To the stirred solution of tert-butyl (2-((S)-2-cyanopyrrolidin-1-yl)-2-oxoethyl)((1S,3R,5S)-3-(2-(2-(4-((((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)carbamate (0.1 g, 0.122 mmol) in acetonitrile and water (1:1) mixture (4 mL), 2N aqueous hydrochloric acid (1.22 mL, 2.44 mmol) was added and the reaction mixture was stirred at room temperature for 24 h. After completion of reaction (monitored by LCMS). The reaction mixture was evaporated to get crude. Obtained crude was purified by RP preparative HPLC using following conditions. Column/dimensions: X BRIDGE C18 (19×250 mm), 5μ, mobile phase A: 10 mM ABC in water, mobile phase B: acetonitrile, gradient (time/% B): 0/5, 4/5, 12/75, 12.1/5, 15/5. flow rate: 16 ml/min. The desired peak fraction was lyophilized to afford (2S)-1-(((1S,3R,5S)-3-(2-(2-(4-((((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)adamantan-1-yl)glycyl)pyrrolidine-2-carbonitrile (0.063 g, 73.3%) as off-white solid. LCMS M/Z 636.49 (M+1) 1H NMR (400 MHz, DMSO-d6) δ 7.92 (s, 1H), 5.10 (bs, 1H), 4.73-4.65 (m, 2H), 4.49-4.43 (m, 3H), 4.3 (bs, 2H), 3.80-3.36 (m, 19H), 2.64-2.60 (m, 2H), 2.16-2.01 (m, 6H), 1.56-1.46 (m, 12H).
To the stirred solution of tert-butyl ((1S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)-2-oxoethyl) carbamate (0.30 g, 0.65 mmol), DCM (3 mL), TFA (3 mL) was added drop wise at 0° C. and the reaction mixture was stirred at room temperature for 3 h. After completion of reaction (monitored by LCMS), the reaction mixture was concentrated and wash with diethyl ether (2×50 mL) and evaporated to get crude. The obtained crude was basified with NaHCO3 (20 mL) and extracted with DCM (3-50 mL) dried over anhydrous Na2SO4 and concentrated. The crude was purified by reverse phase C-18 column chromatography using 27% ACN and 10 mM ABC in water. The desired product containing fractions were collected and lyophilized to afford (1S,3S,5S)-2-((2S)-2-amino-2-((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)acetyl)-2-azabicyclo[3.1.0]hexane-3-carbonitrile (0.053 g) as an off-white solid. LCMS M/Z 360.46 (M+1) 1H NMR (400 MHz, DMSO-d6) δ 5.10-5.07 (m, 1H), 4.46-4.43 (m, 1H), 3.89-3.87 (m, 1H), 3.45-3.31 (m, 5H), 2.21-2.14 (m, 3H), 1.77-1.32 (m, 14H), 0.97 (m, 1H), 0.72-0.70 (s, 1H).
To a solution of tert-butyl ((1S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)-2-oxoethyl)carbamate (36 mg, 1.0 eq) in dichloromethane (0.21 mL), triethylamine (0.04 mL, 4.0 eq) and 4-nitrophenyl chloroformate (17.1 mg in 0.13 mL dichloromethane) were added at 0° C. After stirring for 4 hours at room temperature, the mixture was added to a solution of (2R,3R,4R,5S)-6-((4-(aminomethyl)benzyl)(hexyl)amino)hexane-1,2,3,4,5-pentaol (46 mg, 1.2 eq) and triethylamine (0.08 mL, 6.0 eq) in acetonitrile (0.2 mL+0.05 mL*2 wash) at 0° C. The reaction was stirred for 12 hours before the addition of saturated aqueous NaHCO3. The mixture was then extracted with dichloromethane for three times. The combined organic layer was washed with brine and concentrated in vacuo to provide the crude which was used for the next step without further purification. The above crude was treated with trifluoracetic acid (0.1 mL) and dichloromethane (0.2 mL). After stirring for 4 hours, the volatiles were removed under reduced pressure. The crude was purified by C18 reverse phase prep HPLC to afford the formic salt of 2-(((1R,3S,5S)-3-((S)-1-amino-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)adamantan-1-yl)oxy)ethyl (4-((hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)phenyl)glycinate (17.4 mg, 25%) as white powder. ES MS M/Z=501.56 (M+1—adamantane) 1H NMR (500 MHz, MeOD) δ 7.51 (d, J=7.8 Hz, 2H), 7.42 (d, J=7.9 Hz, 2H), 5.21 (dd, J=10.7, 2.3 Hz, 1H), 4.49-4.30 (m, 4H), 4.23-4.12 (m, 4H), 3.94 (td, J=6.2, 2.7 Hz, 1H), 3.83-3.75 (m, 2H), 3.73-3.60 (m, 5H), 3.32-3.27 (m, 2H), 3.11 (qdd, J=13.0, 10.1, 6.1 Hz, 2H), 2.65 (ddd, J=13.7, 10.6, 5.7 Hz, 1H), 2.39-2.26 (m, 3H), 2.03 (dq, J=9.0, 5.7 Hz, 1H), 1.90-1.56 (m, 16H), 1.41-1.23 (m, 11H), 1.14 (dt, J=9.0, 6.4 Hz, 1H), 1.00-0.87 (m, 4H).
Similarly, compounds 17 and 18 were synthesized from Int-9.
Step 1. Tert-butyl (4-(azidomethyl)benzyl)carbamate:
To a stirred solution of tert-butyl (4-(bromomethyl)benzyl)carbamate (2.0 g, 6.66 mmol) in DMF (20 mL), sodium azide (0.86 g, 13.33 mmol) was added at rt and the reaction mixture was stirred at 80° C. for 2 h. After completion, the reaction mixture was quenched with ice cold water solid was filtered through Buchner funnel and dried to afford tert-butyl (4-(azidomethyl)benzyl)carbamate (1.5 g) as an off-white solid. TLC system: EtOAc: pet ether (0.3:0.7); Rf. 0.4.
Step 2. Tert-butyl (4-(aminomethyl)benzyl)carbamate:
To a stirred solution of tert-butyl (4-(azidomethyl)benzyl)carbamate (1.3 g, 4.95 mmol) in methanol (15 mL), triphenylphosphine (1.9 g, 7.43 mmol) was added and the reaction mixture was stirred at 80° C. for 2 h. After completion, the reaction mixture was evaporated and purified by grace column chromatography using 8-10% MeOH in DCM to afford tert-butyl (4-(aminomethyl)benzyl)carbamate (0.9 g) as a gummy. TLC system: MeOH:DCM (0.1:0.9) Rf. 0.2.
Step 3. Tert-butyl (4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate:
To a stirred solution of tert-butyl (4-(aminomethyl)benzyl)carbamate (0.88 g, 3.72 mmol) and (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal (1.6 g, 9.30 mmol) in methanol (26 mL) was acetic acid (0.8 mL) was added at 0° C. and the resultant mixture was stirred at rt for 20 min. Then sodium cyanoborohydride (0.49 g, 7.81 mmol) was added at 0° C. and the reaction mixture was heated at 80° C. for 16 h. After completion, the reaction mixture was concentrated and purified by RP column chromatography using 25% MeOH and 0.1% FA in water. The pure fraction was evaporated to afford tert-butyl (4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl) carbamate (1.0 g) as a white solid.
Step 4. (2R,2′R,3R,3′R,4R,4′R,5S,5'S)-6,6′-((4-(aminomethyl)benzyl) azanediyl)bis(hexane-1,2,3,4,5-pentaol) hydrochloride:
To the stirred solution of tert-butyl (4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate formate (0.8 g, 1.41 mmol) in methanol (4.0 mL), 4 M hydrochloric acid in 1.4 dioxane (8.0 mL) was added at 0° C. and the reaction mixture was stirred at room temperature for 2 h. After completion of reaction (monitored by LCMS), the reaction mixture was evaporated to afford (2R,2′R,3R,3′R,4R,4′R,5S,5'S)-6,6′-((4-(aminomethyl)benzyl)azanediyl)bis(hexane-1,2,3,4,5-pentaol) hydrochloride (0.95 g, crude) as a white gummy solid.
Step 5. Tert-butyl ((1S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-(2-(((4-nitrophenoxy)carbonyl)oxy)ethoxy)adamantan-1-yl)-2-oxoethyl)carbamate:
To the stirred solution of tert-butyl ((1S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)-2-oxoethyl)carbamate (0.8 g, 1.74 mmol) in dichloromethane (10 mL), triethylamine (0.9 mL, 1.91 mmol) was added and then 4-nitrophenyl carbonochloridate (0.38 g, 6.96 mmol) in in dichloromethane (2 mL) was added at −10° C. and the reaction mixture was stirred at room temperature for 2 h. After completion of the reaction, reaction mixture as such taken to next step.
Step 6. 2-(((1R,3S,5S)-3-((S)-1-((tert-butoxycarbonyl)amino)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)adamantan-1-yl)oxy)ethyl (4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl) carbamate:
To the stirred solution of (2R,2′R,3R,3′R,4R,4′R,5S,5'S)-6,6′-((4-(aminomethyl)benzyl)azanediyl)bis(hexane-1,2,3,4,5-pentaol) hydrochloride (0.96 g, 1.92 mmol), in ACN (10 mL) TEA (1.3 mL, 9.60 mmol), was added at 0° C. Then tert-butyl ((1S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-(2-(((4-nitrophenoxy)carbonyl)oxy)ethoxy)adamantan-1-yl)-2-oxoethyl)carbamate (1.17 g, 1.60 mmol), was added drop wise to the reaction mixture at 0° C. and stirred 80° C. for 16 h. After completion of the reaction the reaction mixture was concentrated and purified by RP C18 column chromatography using 25% MeOH and 0.1 M FA in water. The desired product containing fractions were collected and evaporated to afford 2-(((1R,3S,5S)-3-((S)-1-((tert-butoxycarbonyl)amino)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)adamantan-1-yl)oxy)ethyl (4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate (0.8 g) as yellow gum.
Step 7. 2-(((1R,3S,5S)-3-((S)-1-amino-2-((1S,3S,5S)-3-cyano-2-azabicyclo [3.1.0]hexan-2-yl)-2-oxoethyl)adamantan-1-yl)oxy)ethyl (4-((bis((2S,3R,4R,5R)-2,3,4,5,6 pentahydroxyhexyl)amino)methyl)benzyl) carbamate trifluoroacetate salt (22):
To the stirred solution of 2-(((1R,3S,5S)-3-((S)-1-((tert-butoxycarbonyl)amino)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)adamantan-1-yl)oxy)ethyl (4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate (0.80 g, 0.84 mmol), DCM (8 mL), TFA (4 mL) was added drop wise at 0° C. and the reaction mixture was stirred at room temperature for 1 h. After completion of reaction (monitored by LCMS), the reaction mixture was concentrated and wash with diethyl ether (2×100 mL) and evaporated to get crude. The obtained crude was purified by RP SUNFIRE-C18 (150*19*5μ) using 20% ACN and 0.1% TFA in water. The desired product containing fractions were collected and lyophilized to afford of 2-(((1R,3S,5S)-3-((S)-1-amino-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)adamantan-1-yl)oxy)ethyl (4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate trifluoracetate salt (0.184 g) as an off-white solid. LCMS M/Z 850.68 (M+1) 1H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H), 8.17 (s, 2H), 7.79 (t, J=6.0 Hz, 1H), 7.48 (d, J 8.0 Hz, 2H), 7.32 (d, J=8.0 Hz, 2H), 5.60 (bs, 1H), 4.95 (bs, 1H), 5.27-5.24 (m, 1H), 4.95 (m, 2H), 4.37-4.02 (m, 16H), 3.72-3.52 (m, 12H), 3.16 (m, 4H), 2.32-2.23 (m, 3H), 2.07 (m, 1H), 1.73-1.46 (m, 12H), 1.04 (m, 1H), 0.75 (s, 1H).
Step 1. Tert-butyl ((1S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-(2-(((4-nitrophenoxy)carbonyl)oxy)ethoxy)adamantan-1-yl)-2-oxoethyl)carbamate:
To the stirred solution of tert-butyl ((1S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-(2-hydroxyethoxy)adamantan-1-yl)-2-oxoethyl)carbamate (0.8 g, 1.74 mmol) in DCM (10 mL) was added TEA (0.9 mL, 6.96 mmol) followed by 4-nitrophenyl carbonochloridate (0.38 g, 1.91 mmol) in DCM (0.5 mL) dropwise at −10° C. stirred at room temperature for 2 h. After completion, the reaction mixture was taken to next step to give tert-butyl ((1S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-(2-(((4-nitrophenoxy)carbonyl)oxy)ethoxy)adamantan-1-yl)-2-oxoethyl)carbamate (1.17 g) as yellow solution. TLC system: 100% EtOAc, Rf: 0.4.
Step 2. 2-(((1R,3S,5S)-3-((S)-1-((tert-butoxycarbonyl)amino)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)adamantan-1-yl)oxy)ethyl (4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate:
To the stirred solution of tert-butyl (2R,3R,4R,5S)-6-((4-(aminomethyl)benzyl)(ethyl)amino)hexane-1,2,3,4,5-pentaol hydrochloride (0.81 g, 2.24 mmol), in ACN (10 mL) TEA (1.5 mL, 11.22), was added at 0° C. Then tert-butyl ((1S)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-1-((1S,3R,5S)-3-(2-(((4-nitrophenoxy)carbonyl)oxy)ethoxy)adamantan-1-yl)-2-oxoethyl)carbamate (1.17 g, 1.87 mmol), was added drop wise to the reaction mixture at 0° C. and stirred 80° C. for 16 h. After completion of the reaction the reaction mixture was concentrated and purified by RP C18 column chromatography using 27% ACN and 0.1 M FA in water. The desired product containing fractions were collected and evaporated to afford 2-(((1R,3S,5S)-3-((S)-1-((tert-butoxycarbonyl)amino)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)adamantan-1-yl)oxy)ethyl (4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate (1.0 g) as a yellow gum.
Step 3. 2-(((1R,3S,5S)-3-((S)-1-amino-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)adamantan-1-yl)oxy)ethyl (4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate formate (23):
To the stirred solution of 2-(((1R,3S,5S)-3-((S)-1-((tert-butoxycarbonyl)amino)-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)adamantan-1-yl)oxy)ethyl(4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate (1 g, 1.22 mmol) in DCM (10 mL) was added TFA (10 mL) dropwise at 0° C. and the resultant mixture was stirred at room temperature for 1 h. After completion (monitored by LCMS), the reaction mixture was concentrated and washed with diethyl ether (2×100 mL) to give crude product. The obtained crude was then purified by RP chromatography using C-18 column and 20% CAN: 0.1 M FA in water as an eluent. The desired product containing fractions were collected and lyophilized to afford 2-(((1R,3S,5S)-3-((S)-1-amino-2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-oxoethyl)adamantan-1-yl)oxy)ethyl(4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)benzyl)carbamate formate (0.59 g) as an off-white solid. LCMS M/Z 712.50 (M−1) 1H NMR (400 MHz, DMSO-d6) δ 8.13 (s, 1H), 7.74 (t, J=6.0 Hz, 1H), 7.38 (d, J=7.6 Hz, 2H), 7.24 (d, J=7.6 Hz, 2H), 5.22 (m, 1H), 4.18-4.02 (m, 10H), 3.89 (s, 1H), 2.17-3.66-3.52 (m, 4H), 3.49-3.47 (m, 4H), 2.92-2.71 (m, 4H), 2.50 (s, 3H), 2.27-2.21 (m, 3H), 1.96 (m, 1H), 1.71-1.43 (m, 12H), 1.09-1.02 (m, 4H), 0.74 (s, 1H).
Step 1. (R)-(2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)phenyl)boronic acid:
To a stirred solution of tert-butyl (R)-(1-(7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)piperidin-3-yl)carbamate (0.1 g, 0.24 mmol) and (2-(bromomethyl)phenyl)boronic acid (0.08 g, 0.36 mmol) in DMF (1 mL) was added K2CO3 (0.03 g, 0.24 mmol) and the reaction mixture was stirred at room temperature for 16 h. After completion of starting material, the reaction mixture was diluted with EtOAc (20 mL), washed with water (20 mL) and brine solution (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to get a crude mass. The crude residue was purified by reverse phase preparative-HPLC to afford 0.06 g of (R)-(2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)phenyl)boronic acid as an off-white solid. [TLC system: EtOAc:pet-ether (8:2); Rf value: 0.5].
Step 2. (R)-(2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)phenyl)boronic acid (27):
To a stirred solution of (R)-(2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)phenyl)boronic acid (0.06 g, 0.11 mmol) in 1,4-dioxane (0.6 mL) was added 4 M HCl in 1,4-dioxane (0.12 mL) dropwise at 0° C. and the reaction mixture was stirred at room temperature for 3 h. After completion of starting material, the reaction mixture was concentrated under reduced pressure and triturated with diethyl ether to afford 0.025 g of compound 27 (HCl salt) as an off-white solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.2]. LC MS M/Z=451.46 (M+1). 1H NMR (400 MHz, DMSO-d6) δ 8.22 (s, 2H), 8.12 (s, 2H), 7.55 (d, J=6.8 Hz, 1H), 7.27-7.14 (m, 2H), 6.91 (d, J=7.6 Hz, 1H), 5.25 (s, 2H), 5.00-4.88 (m, 2H), 3.69 (d, J=2.4 Hz, 1H), 3.49-3.40 (m, 5H), 3.22-3.14 (m, 2H), 2.00-1.90 (m, 2H), 1.79 (s, 3H), 1.71-1.61 (m, 2H).
Step 1. (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-fluoronicotinic acid
To a stirred solution of methyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-fluoronicotinate (0.9 g, 1.56 mmol) in THF (9 mL) and water (2 mL) was added LiOH·H2O (0.129 g, 3.08 mmol) at 0° C. The reaction mixture was stirred at rt for 4 h. After completion of starting material, the reaction mixture was concentrated under reduced pressure to get crude compound. The crude compound was diluted with water, acidified with 1N HCL. The precipitated solid was filtered off and dried under vacuum to afford 0.72 g of compound (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-fluoronicotinic acid as an off-white solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.3].
Step 2. Hexyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-fluoronicotinate:
To a stirred solution of compound (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-fluoronicotinic acid (0.72 g, 1.26 mmol) in DMF (15 mL) were added C6H13Br (0.25 g, 1.51 mmol) and K2CO3 (0.52 g, 3.78 mmol) and the reaction mixture was stirred at rt for 16 h. After completion of starting material, the reaction mixture was quenched with cold water and the precipitated solid was filtered off, dried under vacuum to get 0.3 g of compound hexyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-fluoronicotinate as an off-white solid. [TLC system: EtOAc:Pet ether (1:1); Rf value: 0.8].
Step 3. Hexyl (R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-fluoronicotinate (30):
To a stirred solution of compound hexyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-fluoronicotinate (0.30 g, 0.46 mmol) in 1,4-dioxane (3 mL) was added 4 M HCl in 1,4-dioxane (2 mL) at 0° C. and the reaction mixture was stirred at room temperature for 2 h. After completion of starting material, the reaction mixture was concentrated under reduced pressure to get a crude compound. The crude compound was purified by reverse phase preparative-HPLC to afford 0.181 g of compound hexyl (R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-fluoronicotinate as an off white solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.3]. LC MS M/Z=554.87 (M+1) 1H NMR (400 MHz, DMSO-d6) δ 8.48 (t, J=8.4 Hz, 1H), 8.12 (d, J=3.6 Hz, 3H), 7.22-7.19 (dd, J=8.4 Hz, 2.4 Hz, 1H), 5.49 (s, 2H), 4.94 (m, 2H), 4.34 (t, J=6.4 Hz, 2H), 3.71 (m, 1H), 3.50-3.40 (m, 5H), 3.21-3.16 (m, 2H), 2.02-1.89 (m, 1H), 1.89-1.77 (m, 1H), 1.76-1.65 (m, 7H), 1.44-1.41 (m, 2H), 1.34-1.31 (m, 4H), 0.89-0.86 (m, 3H).
To a stirred solution of compound (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-(methylamino)nicotinic acid (0.5 g, 0.86 mmol) in 1,4-dioxane (3 mL) was added 4 M HCl in 1,4-dioxane (2 mL) at 0° C. and the reaction mixture was stirred at room temperature for 2 h. After completion of starting material, the reaction mixture was concentrated under reduced pressure to get a crude compound. The crude compound was purified by reverse phase preparative-HPLC to afford 0.157 g of compound hexyl (R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-(methylamino)nicotinic acid as an off white solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.3]. LC MS M/Z=481.28 (M+1) 1H NMR (400 MHz, DMSO-d6) δ 8.24 (m, 3H), 7.87 (d, J=7.6 Hz, 1H), 6.33 (d, J=8.4 Hz, 1H), 5.37 (s, 2H), 5.00-4.88 (m, 2H), 3.68-3.38 (m, 6H), 3.18-3.12 (m, 2H), 2.45 (m, 3H), 2.02-1.90 (m, 2H), 1.77 (s, 3H), 1.70-1.62 (m, 2H).
Step 1. (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-(methylamino)nicotinic acid:
To a stirred solution of compound (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-(methylamino)nicotinic acid (1.0 g, 1.72 mmol, sharing same intermediate with mCMT478) in DMF (10 mL) were added C6H13Br (0.34 g, 2.06 mmol) and K2CO3 (0.71 g, 5.16 mmol) and the reaction mixture was stirred at rt for 16 h. After completion of starting material, the reaction mixture was quenched with cold water and the precipitated solid was filtered off, dried under vacuum to get 0.7 g of compound (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-(methylamino)nicotinic acid as an off-white solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.8].
Step 2. Hexyl (R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-(methylamino)nicotinate (32):
To a stirred solution of compound hexyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-(methylamino)nicotinate (0.41 g, 0.62 mmol) in 1,4-dioxane (4 mL) was added 4 M HCl in 1,4-dioxane (3 mL) at 0° C. and the reaction mixture was stirred at room temperature for 2 h. After completion of starting material, the reaction mixture was concentrated under reduced pressure to get a crude compound. The crude compound was purified by reverse phase preparative-HPLC to afford 0.161 g of compound hexyl (R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-(methylamino)nicotinate as an off white solid. [TLC system: MeOH:DCM (1:9); Rr value: 0.3]. LC MS M/Z=565.79 (M+1) 1H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 3H), 7.87 (d, J=8.4 Hz, 1H), 7.21 (br s, 1H), 6.34 (d, J=8.8, 1H), 5.36 (s, 2H), 4.99-4.88 (m, 2H), 4.22 (t, J=6.4 Hz, 2H), 3.70-3.59 (m, 1H), 3.49-3.39 (m, 5H), 3.18-3.11 (m, 2H), 2.44 (s, 3H), 2.02-1.99 (m, 2H), 1.90-1.61 (m, 7H), 1.42-1.31 (m, 6H), 0.89 (s, 3H).
To the solution of compound methyl 6-((7-(4-((bis((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)amino)methyl)-1H-1,2,3-triazol-1-yl)heptyl)amino)-2-((7-(but-2-yn-1-yl)-8-((R)-3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)nicotinate (0.9 g, 0.70 mmol) in H2O (10 mL) and then was added conc. HCl (2 mL) at 0° C. and the reaction mixture was stirred at room temperature for 2 h. After completion of the reaction (LCMS monitoring), the reaction mixture was concentrated under reduced pressure to get a crude mass. The crude purified by RP-HPLC to afford compound methyl 2-((8-((R)-3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-((7-(4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)heptyl)amino)nicotinate (0.041 g) as off white solid. LC MS M/Z=1003.62 (M+1) 1H NMR (400 MHz, DMSO d6) δ 8.95 (s, 1H), 8.34 (s, 1H), 8.21 (m, 3H), 7.84 (d, J=8.7 Hz, 1H), 7.39 (brs, 1H), 6.31 (d, J=8.8 Hz, 1H), 5.34 (br m, 4H), 4.93 (m, 2H), 4.56 (m, 2H), 4.39 (t, J=6.9 Hz, 2H), 4.13 (m, 2H), 3.79 (s, 3H), 3.59 (m, 6H), 3.43 (m, 11H), 3.15 (m, 6H), 2.86 (s, 2H), 2.04 (m, 1H), 1.91 (m, 1H), 1.78 (m, 7H), 1.16 (m, 8H), 0.89 (m, 2H).
Step 1. 6-((7-(4-((bis((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)amino)methyl)-1H-1,2,3-triazol-1-yl)heptyl)amino)-2-((7-(but-2-yn-1-yl)-8-((R)-3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)nicotinic acid:
To the solution of compound methyl 6-((7-(4-((bis((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)amino)methyl)-1H-1,2,3-triazol-1-yl)heptyl)amino)-2-((7-(but-2-yn-1-yl)-8-((R)-3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)nicotinate (0.350 g, 0.27 mmol) in MeOH (5 mL) and H2O (1 mL) and was added NaOH (0.054 g, 1.35 mmol) and the reaction mixture was stirred at rt for 16 h. After completion of the reaction (LCMS monitoring). The reaction mixture was concentrated under reduced pressure to get a crude mass. The crude compound was washed with Et2O (50 mL) and dried in vacuo to afford compound 6-((7-(4-((bis((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)amino)methyl)-1H-1,2,3-triazol-1-yl)heptyl)amino)-2-((7-(but-2-yn-1-yl)-8-((R)-3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)nicotinic acid (0.3 g) as off-white solid. The curded compound take for next step.
Step 2. 2-((8-((R)-3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-((7-(4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)heptyl)amino)nicotinic acid (35):
To the solution of compound 6-((7-(4-((bis((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)amino)methyl)-1H-1,2,3-triazol-1-yl)heptyl)amino)-2-((7-(but-2-yn-1-yl)-8-((R)-3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)nicotinic acid (0.3 g, 0.24 mmol) in H2O (10 mL) was added con·HCl (0.6 mL) at 0° C. and the reaction mixture was stirred at room temperature for 2 h. After completion of the reaction (LCMS monitoring), the reaction mixture was concentrated under reduced pressure to get a crude mass. The crude purified by RP prep HPLC to afford compound 2-((8-((R)-3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-6-((7-(4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)heptyl)amino)nicotinic acid (0.014 g) as off white solid. LC MS M/Z=987.62 (M+1) 1H NMR (400 MHz, DMSO) δ 7.93 (s, 1H), 7.79 (d, J=8.6 Hz, 1H), 7.01 (br s, 1H), 6.22 (d, J=8.6 Hz, 1H), 5.31 (s, 2H), 4.59-4.26 (m, 14H), 3.77 (m, 4H), 3.55 (m, 9H), 3.38 (m, 7H), 2.90 (m, 5H), 2.50 (m, 2H), 1.74 (m, 9H), 1.27 (m, 1H), 1.12 (m, 6H), 0.91 (m, 2H).
Step 1. Isopropyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate:
To a stirred solution of compound (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoic acid (0.5 g, 0.85 mmol) in DMF (5 mL) were added 2-bromopropane (0.15 g, 1.28 mmol) and K2CO3 (0.35 g, 2.56 mmol) and the reaction mixture was stirred at room temperature for 16 h. After completion of starting material, the reaction mixture was quenched with cold water. The precipitated solid was filtered off, dried under vacuum and washed with diethyl ether to afford 0.4 g of compound isopropyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate as an off-white solid. [TLC system: EtOAc:pet-ether (3:7); Rf value: 0.7].
Step 2. Isopropyl (R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate (37):
To a stirred solution of compound isopropyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate (0.4 g, 0.64 mmol) in 1,4-dioxane (4 mL) was added 4 M HCl in 1,4-dioxane (4 mL) at 0° C. and the reaction mixture was stirred at room temperature for 3 h. After completion of starting material, the reaction mixture was concentrated under reduced pressure to get a crude mass. The crude compound was purified by reverse phase preparative-HPLC to afford 0.19 g of compound isopropyl (R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate as off-white solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.2]. LC MS M/Z=527.49 (M+1) 1H NMR (400 MHz, DMSO-d6) δ 7.83 (d, J=2.0 Hz, 1H), 7.54 (dd, J=8.4, 2.0 Hz, 1H), 7.08 (d, J=8.4 Hz, 1H), 5.32 (s, 2H), 5.22-5.16 (m, 1H), 4.88 (s, 2H), 3.66-3.58 (m, 2H), 3.38 (s, 3H), 2.99 (t, J=10.4 Hz, 1H), 2.84-2.72 (m, 2H), 1.87-1.57 (m, 8H), 1.37 (d, J=6.0 Hz, 6H), 1.27-1.26 (m, 1H).
Similarly, hexyl (R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate (38) was synthesized.
Step 1. Nonyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate:
To a stirred solution of (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoic acid (0.35 g, 0.6 mmol) in DMF (3.5 mL) were added 1-bromononane (0.15 g, 0.72 mmol) and K2CO3 (0.25 g, 1.8 mmol) and the reaction mixture was stirred at room temperature for 16 h. After completion of starting material, the reaction mixture was quenched with cold water. The precipitated solid was filtered off, dried under vacuum and washed with diethyl ether to afford 0.25 g of nonyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate as an off-white solid. [TLC system: EtOAc:pet-ether (1:1); Rf value: 0.7].
Step 2. Nonyl (R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate (39):
To a stirred solution of nonyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate (0.35 g, 0.49 mmol) in 1,4-dioxane (2 mL) was added 4 M HCl in 1,4-dioxane (2 mL) at 0° C. and the reaction mixture was stirred at room temperature for 3 h. After completion of starting material, the reaction mixture was concentrated under reduced pressure to get a crude mass. The crude compound was purified by reverse phase preparative-HPLC to afford 0.13 g of nonyl (R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate (39) as a brown solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.3]. LC MS M/Z=611.64 (M+1). 1H NMR (400 MHz, DMSO-d6) δ 7.85 (d, J=2.4 Hz, 1H), 7.55 (dd, J=8.4, 2.0 Hz, 1H), 7.08 (d, J=8.4 Hz, 1H), 5.32 (s, 2H), 4.88 (s, 2H), 4.32 (t, J=6.4 Hz, 2H), 3.66-3.58 (m, 2H), 3.37 (s, 3H), 3.01-2.96 (m, 1H), 2.82-2.72 (m, 2H), 1.87-1.60 (m, 10H), 1.41-1.24 (m, 13H), 0.84 (t, J=6.4 Hz, 3H).
Similarly, (R)-2-((8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chloro-N-pentylbenzamide (40) was synthesized.
Step 1. 2-(2-bromoethoxy)ethyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate:
To a stirred solution of (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoic acid (1.0 g, 1.71 mmol) and 1-bromo-2-(2-bromoethoxy)ethane (1.98 g, 8.55 mmol) in DMF (10 mL) was added K2CO3 (0.71 g, 5.13 mmol) and the reaction mixture was stirred at room temperature for 16 h. After completion of starting material, the reaction mixture was quenched with cold water and the precipitated solid was filtered off, dried under vacuum to get 1.0 g of 2-(2-bromoethoxy)ethyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate as an off-white solid. [TLC system: EtOAc:pet ether (3:7); Rf value: 0.6].
Step 2. 2-(2-azidoethoxy)ethyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate:
To a stirred solution of 2-(2-bromoethoxy)ethyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate (3) (1.0 g, 1.36 mmol) in DMF (10 mL) was added NaN3 (0.44 g, 6.79 mmol) and the reaction mixture was stirred at room temperature for 16 h. After completion of starting material, the reaction mixture was quenched with water and extracted with ethyl acetate (3×25 mL). The combined organic layer was washed with brine solution (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to get crude compound. The crude compound was purified by column chromatography (using silica gel 100-200 mesh, 30% ethyl acetate in pet-ether as an eluent) to afford 0.7 g of 2-(2-azidoethoxy)ethyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate as an off-white solid. [TLC system: EtOAc:pet ether (1:1); Rf value: 0.4].
Step 3. 2-(2-(4-((hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl 2-((7-(but-2-yn-1-yl)-8-((R)-3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate:
To a stirred solution of 2-(2-azidoethoxy)ethyl (R)-2-((7-(but-2-yn-1-yl)-8-(3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate (0.3 g, 0.43 mmol) and (2R,3R,4R,5S)-6-(hexyl(prop-2-yn-1-yl)amino)hexane-1,2,3,4,5-pentaol 5 (0.26 g, 0.86 mmol) in a mixture of DCM and water (6 mL, 1:1) were added CuSO4.5H2O (0.13 g, 0.52 mmol) and Sodium ascorbate (0.13 g, 0.65 mmol). The reaction mixture was stirred at room temperature for 8 h. After completion of starting material, the reaction mixture was quenched with water and extracted with DCM (3×20 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to furnish 0.45 g of crude 2-(2-(4-((hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl 2-((7-(but-2-yn-1-yl)-8-((R)-3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate as yellow color solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.5]. This crude compound was used in the next step without any further purification.
Step 4. 2-(2-(4-((hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl 2-((8-((R)-3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate (41):
To a stirred solution of 2-(2-(4-((hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl 2-((7-(but-2-yn-1-yl)-8-((R)-3-((tert-butoxycarbonyl)amino)piperidin-1-yl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)methyl)-5-chlorobenzoate (0.45 g, 0.45 mmol) in 1,4-dioxane (4.5 mL) was added 4 M HCl in 1,4-dioxane (1 mL) dropwise at 0° C. and the reaction mixture was stirred at room temperature for 2 h. After completion of starting material, the reaction mixture was concentrated under reduced pressure to get crude compound. The crude compound was purified by reverse phase preparative-HPLC to afford 0.1 g of compound 41 (HCl salt) as an off-white solid. [TLC system: MeOH:DCM (1:9); Rf value: 0.2]. LC MS M/Z=901.25 (M+H). 1H NMR (400 MHz, DMSO-d6) δ 9.5 (s, 1H), 8.31 (d, J=7.6 Hz, 1H), 8.23 (s, 3H), 7.84 (s, 1H), 7.57 (dd, J=8.4, 2.0 Hz, 1H), 7.08 (d, J=8.4 Hz, 1H), 5.33 (s, 3H), 5.01-4.87 (m, 2H), 4.63 (s, 2H), 4.41 (s, 4H), 4.12-4.03 (m, 1H), 3.93-3.91 (m, 2H), 3.79 (m, 2H), 3.74-3.60 (m, 3H), 3.39 (m, 8H), 3.20-3.17 (m, 4H), 2.99-2.98 (m, 2H), 2.02-1.91 (m, 2H), 1.78-1.63 (m, 7H), 1.25 (s, 6H), 0.86 (s, 3H).
To a solution of compound 2-(2-(2-azidoethoxy)ethoxy)ethyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.4 g, 0.565 mmol) in DCM (10 mL) was added 4 M HCl in 1,4-dioxane at 0° C. Then reaction was stirred at RT for 3 h. After completion of reaction, reaction mixture was concentrated and residue was basified with saturated NaHCO3 solution and extracted with EtOAc (20 mL×2) and dried over anhydrous sodium sulfate and evaporated to get crude which was purified by column chromatography over silica gel (Davisil) (using 0-8% MeOH in DCM as an eluent) to afford 30 mg of 2-(2-(2-azidoethoxy)ethoxy)ethyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate as pale brown gum. [TLC system: MeOH:DCM (1:9); Rf value: 0.5]. LC MS M/Z=608.64 (M+1). 1H NMR (400 MHz, DMSO-d6) δ 7.45-7.43 (m, 2H), 4.96-4.93 (m, 2H), 4.37-4.36 (m, 2H), 4.25-4.11 (m, 2H), 3.94-3.93 (m, 2H), 3.75-3.71 (m, 2H), 3.60-3.58 (m, 6H), 3.37-3.34 (m, 2H), 3.31 (m, 1H), 2.73-2.58 (m, 2H), 2.51-2.49 (m, 2H), 1.80 (bs, 2H).
Step 1. 2-(2-(2-(4-((((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate:
To a stirred solution of compound 2-(2-(2-azidoethoxy)ethoxy)ethyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.6 g, 0.847 mmol) and compound 6-(prop-2-yn-1-ylamino)hexane-1,2,3,4,5-pentaol (0.371 g, 1.695 mmol) in t-BuOH (5 mL) and water (1 mL) was added CuSO4, 5·H2O (0.254 g, 1.017 mmol) and Na-ascorbate (0.251 g, 1.271 mmol). The resulting mixture was stirred at RT for 16 h. After completion of reaction, reaction mixture was filtered. The filtrate was concentrated under reduced pressure to get crude compound. The crude compound was purified by RP-Prep-HPLC to afford 60 mg of compound 2-(2-(2-(4-((((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate as brown sticky solid.
Step 2. 2-(2-(2-(4-((((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (44):
To a solution of compound 2-(2-(2-(4-((((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.060 g, 0.064 mmol) in DCM (1 mL) was added 4 M HCl in 1,4-dioxane (0.6 mL). The reaction mixture was stirred at RT for 3 h. After completion of reaction, reaction mixture was filtered and concentrated to get crude compound. The crude compound was purified by RP-Prep-HPLC to afford 0.0157 g of 2-(2-(2-(4-((((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate as an off white solid. LC MS M/Z=827.41 (M+1) 1H NMR (400 MHz, DMSO-d6) δ 7.89 (s, 1H), 7.45-7.41 (m, 2H), 5.10-4.85 (m, 3H), 4.60-4.10 (m, 10H), 4.00-3.30 (m, 20H), 3.10-3.05 (m, 1H), 2.71-2.50 (m, 5H), 2.10-1.40 (m, 2H).
Step 1. 6-(ethylamino)hexane-1,2,3,4,5-pentaol:
To a solution of compound 2,3,4,5,6-pentahydroxyhexanal (5.0 g, 27.75 mmol) and compound ethanamine (20.8 mL, 41.62 mmol) in MeOH (30 mL) was added Raney-Ni (5.0 g). The reaction mixture was stirred at 60° C. under H2 atmosphere (150 psi) for 16 h. After completion of reaction, reaction mixture was filtered through celite-bed and filtrate was concentrated to get 5.0 g compound 6-(ethylamino)hexane-1,2,3,4,5-pentaol (crude) as off white solid which was used for next step without purification.
Step 2. 6-(ethyl(prop-2-yn-1-yl)amino)hexane-1,2,3,4,5-pentaol:
To a solution of compound 6-(ethylamino)hexane-1,2,3,4,5-pentaol (2.0 g, 9.563 mmol), compound 3-bromoprop-1-yne (0.85 mL, 11.47 mmol) in THF (30 mL) was added and the reaction mixture was heated at 60° C. for 16 h. After completion of reaction, reaction mixture was filtered and filtrate was concentrated to get 0.450 g compound 6-(ethyl(prop-2-yn-1-yl)amino)hexane-1,2,3,4,5-pentaol as brown sticky compound which was used for next step without purification.
Step 3. 2-(2-(2-(4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate:
To a solution of compound 2-(2-(2-azidoethoxy)ethoxy)ethyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.6 g, 0.847 mmol) and compound 6-(ethyl(prop-2-yn-1-yl)amino)hexane-1,2,3,4,5-pentaol (0.420 g, 169 mmol) in t-BuOH (10 mL) and water (2 mL) was added CuSO4, 5·H2O (0.254 g, 1.017 mmol) and Na-ascorbate (0.251 g, 1.271 mmol). The resultant reaction mixture was stirred at RT for 16 h. After completion of reaction, reaction mixture was filtered and concentrated to get crude compound. The crude compound was purified by Prep-HPLC to afford 0.240 g of compound 2-(2-(2-(4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate as brown sticky compound.
Step 4. 2-(2-(2-(4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (45):
To a solution of compound 2-(2-(2-(4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.240 g, 0.280 mmol) in DCM (3 mL) was added 4 M HCl in 1,4-dioxane (2.4 mL). The reaction mixture was stirred at RT for 2 h. After completion of reaction, reaction mixture was filtered and concentrated under reduced pressure to afford crude compound. The crude compound was purified by trituration using diethyl ether to afford 175 mg of 2-(2-(2-(4-((ethyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate as off white solid. LC MS M/Z=855.65 (M+1) 1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.29 (d, J=5.8 Hz, 1H), 8.10-8.00 (m, 3H), 7.58 (q, J=9.0 Hz, 2H), 5.49 (s, 1H), 4.95-4.93 (m, 3H), 4.57-3.38 (m, 28H), 3.18-2.66 (m, 8H), 1.31-1.26 (m, 3H).
Step 1. 2-(2-(2-(4-((hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate:
To a solution of 2-(2-(2-azidoethoxy)ethoxy)ethyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (460 mg, 0.65 mmol) in THF (3 mL) was added 2R,3R,4R,5S)-6-(hexyl(prop-2-yn-1-yl)amino)hexane-1,2,3,4,5-pentaol (prepared in Example 9 step 4) (295 mg, 0.97 mmol), Na ascorbate (193 mg, 0.97 mmol), CuSO4.5H2O (242.5 mg, 0.97 mmol) and H2O (2 mL). The mixture was stirred at room temperature for 19 h. The reaction mixture was filtered and the filtrate was purified by Prep-HPLC (Gemini-C18 150×21.2 mm, 5 um, mobile phase: ACN(0.1% FA)-H2O (0.1% FA), gradient: 20˜70%) to give 2-(2-(2-(4-((hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (140 mg, 21.2%) as a yellow solid. MS (ESI): mass calcd. for C44H64F6N8O12 1010.45, m/z found 1011 [M+1].
Step 2. 2-(2-(2-(4-((hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (46):
To a 2-(2-(2-(4-((hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate in DCM (5 mL) was added TFA (0.5 mL). The mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under pressure at 40° C. and the residue was purified by Prep-HPLC (Gemini-C18 150×21.2 mm, 5 um, mobile phase: ACN(0.1% TFA)-H2O(0.1% TFA), gradient: 20-70%) to give 2-(2-(2-(4-((hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (65.4 mg, 51.8%) as a yellow solid. MS (ESI): mass calcd. for Chemical Formula: C39H56F6N8O10 910.40, m/z found 911.4 [M+1]. 1H NMR (400 MHz, MeOD) δ 8.28 (s, 1H), 7.36 (m, 1H), 7.27-7.17 (m, 1H), 5.07 (m, 1H), 4.99 (m, 1H), 4.58 (m, 4H), 4.45 (m, 2H), 4.29 (m, 4H), 4.11-3.94 (m, 2H), 3.92-3.87 (m, 3H), 3.77 (m, 4H), 3.64 (m, 7H), 3.27-3.03 (m, 5H), 3.00-2.92 (m, 1H), 2.84 (m, 1H), 1.83 (s, 2H), 1.36 (s, 6H), 0.91 (t, J=6.5 Hz, 3H).
An alternative synthesis of compound (46) is described below.
Step 1. 2-(2-(2-azidoethoxy)ethoxy)ethyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate
To a solution of 2-(2-(2-azidoethoxy)ethoxy)ethyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (Int-22, 300 g, 424 mmol) in ethyl acetate (1 L) was added HCl in ethyl acetate (4 M, 1 L) at room temperature. The mixture was stirred for 2 hours and concentrated to give a crude reside. It was redissolved in EA (1.00 L), quenched with aq. NaHCO3 to adjust pH=8, the organic layer was separated, dried over with Na2SO4, filtered and the filtrate was concentrated to give compound 2-(2-(2-azidoethoxy)ethoxy)ethyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (720 g, 1.12 mol, 88.1% yield, 94.5% purity) as yellow oil. 1H NMR: DMSO-d6 7.92 (s, 1H), 7.52-7.33 (m, 2H), 5.00-4.95 (m, 2H), 4.48-4.25 (m, 10H), 3.94-3.81 (m, 2H), 3.81-3.78 (m, 2H), 3.71-3.54 (m, 14H), 3.39-3.35 (m, 2H), 2.57-2.56 (m, 3H), 2.38-2.33 (m, 4H), 1.40-1.39 (m, 4H), 1.24-1.17 (m, 6H), 0.83 (t, J=6.8 Hz, 3H).
Step 2. 2-(2-(2-(4-((hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (46)
A solution of Compound 2-(2-(2-azidoethoxy)ethoxy)ethyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (240 g, 395 mmol, 1.00 eq) and (2R,3R,4R,5S)-6-(hexyl(prop-2-yn-1-yl)amino)hexane-1,2,3,4,5-pentaol (Int-1, 144 g, 474.63 mmol, 1.20 eq) in THF (2.00 L) and H2O (1.00 L) was prepared. CuSO4.5H2O (99.0 g, 396 mmol, 1.00 eq) and Na-ascorbate (78.9 g, 398 mmol, 1.01 eq) was added to the mixture at 10-25° C. and the mixture was stirred at 25° C. for 10 hrs. The mixture of three batches was diluted with NH3·H2O/aq·NaCl=1V/2V (1.50 L), extracted with EA (1.50 L×2), washed with NH3·H2O/aq·NaCl=1V/2V (1.50 L×3), until the aqueous phase color turned colorless form blue. The organic layer was dried over with Na2SO4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (0.1% FA condition), the mixture was concentrated to remove MeCN, and then quenched with NaHCO3 to adjust pH=9, extracted with EA (10.0 L×2), dried over with Na2SO4, filtered. The filtrate was concentrated under reduced pressure to give 2-(2-(2-(4-((hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (46) (290 g, 306 mmol, 25.9% yield, 96.3% purity) as yellow solid. MS (ESI): mass calcd. for Chemical Formula: C39H56F6N8O10 910.40, m/z found 911.5 [M+1]. 1H NMR (DMSO-d6) 7.92 (s, 1H), 7.52-7.33 (m, 2H), 5.00-4.95 (m, 2H), 4.48-4.25 (m, 10H), 3.94-3.93 (m, 2H), 3.81-3.79 (m, 2H), 3.71-3.39 (m, 14H), 3.35-3.27 (m, 2H), 2.72-2.54 (m, 3H), 2.38-2.33 (m, 4H), 1.55-1.33 (m, 4H), 1.24-1.15 (m, 6H), 0.83 (t, J=8.0 Hz, 3H).
Step 3. 2-(2-(2-(4-((hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate tartrate salt
To a solution of 2-(2-(2-(4-((hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (260 g, 285 mmol) in ethanol (1 L) was added tartaric acid (43.7 g, 291 mmol, 1.02 eq.) in ethanol (1 L). The mixture was stirred at 60° C. for 5 hrs. The mixture was concentrated under reduced pressure to give 2-(2-(2-(4-((hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate tartrate salt as yellow solid (293.3 g, 93.7% yield, 96.8% pure). 1H NMR DMSO-d6 8.01-7.95 (m, 1H), 7.55-7.47 (m, 2H), 5.02-4.90 (m, 2H), 4.48 (t, J=8.0 Hz, 2H), 4.34-3.7 (m, 9H), 3.81-3.48 (m, 15H), 3.40-3.35 (m, 2H), 2.97-2.58 (m, 5H), 2.46-2.33 (m, 4H), 1.43-1.42 (m, 2H), 1.27-1.20 (m, 6H), 0.83 (t, J=6.0 Hz, 3H).
Step 1. Synthesis of (ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl)bis(4-methyl benzene sulfonate)
To the stirred solution of 2,2′-(ethane-1,2-diylbis(oxy))bis(ethan-1-ol) (15.0 g, 99.88 mmol) in DCM (300 mL), Et3N (55.58 mL, 399.54 mmol) was added at 0° C. followed by tosyl Chloride (76.16 g, 399.54 mmol). The reaction mixture was stirred at room temperature for 16 h. After completion of reaction, reaction mixture was quenched with cold water (500 mL) and extracted with CH2Cl2 (2×500 mL). Combined organic layer was washed with cold water (500 mL), brine (500 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure to get crude compound. The crude compound was purified by column chromatography over silica gel (100-200 mesh) (using 0-50% EtOAc in Pet Ether as an eluent) to afford 37.0 g of (ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) as off white solid. [TLC system: EtOAc: Pet Ether (4:6); Rf value: 0.6].
Step 2. Synthesis of 1,2-bis(2-azidoethoxy)ethane
To a stirred solution of (ethane-1,2-diylbis(oxy))bis(ethane-1,2-diylbis(4-methylbenzenesulfonate) (5.0 g, 10.90 mmol) in DMF (50 mL), sodium azide (2.83 g, 43.61 mmol) was added. Then reaction mixture was heated at 70° C. for 16 h. After completion of reaction, reaction mixture was concentrated, and the residue was stirred in diethyl ether and filtered. Filtrate was concentrated to get 2.0 g of 1,2-bis(2-azidoethoxy)ethane as colorless oil. [TLC system: EtOAc: Pet Ether (2:8); Rf value: 0.6].
Step 3. 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine hydrochloride
To a vigorously stirred solution of 1,2-bis(2-azidoethoxy)ethane (3.0 g, 14.98 mmol) in Et2O (22 mL) and THF (4.4 mL) was added 1N HCl (17.6 mL) solution followed by PPh3 (3.9 g, 14.98 mmol) in Et2O (22 mL). The reaction mixture was stirred at room temperature for 12 h. After completion of reaction, aqueous layer was separated and washed with diethyl ether (2×100 mL). The aqueous layer was concentrated and residue was co-evaporated with acetonitrile (2-3 times) to get 2.0 g of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine hydrochloride as colorless oil. [TLC system: MeOH:DCM (0.5:9.5); Rf value: 0.4].
Step 4. tert-butyl (R)-(4-(1-((2-(2-(2-azidoethoxy)ethoxy)ethyl)carbamoyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-yl)carbamate
To a stirred solution of (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylic acid (2.9 g, 5.27 mmol) and 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine hydrochloride (1.66 g, 7.90 mmol) in DMF (30 mL) was added HATU (3.0 g, 7.90 mmol) followed by DIPEA (3.39 mL, 26.35 mmol) at 0° C. The reaction mixture was stirred at room temperature for 45 min. The progress of the reaction was monitored by TLC. After completion of reaction, reaction mixture was quenched with cold water (2×50 mL) and extracted with EtOAc (2×100 mL). The combined organic layer was washed with cold water (100 mL), brine (100 mL), dried over anhydrous Na2SO4 and concentrated to get crude compound. The crude compound was purified by column chromatography over silica gel (Davisil) (using 0-70% EtOAc in Pet Ether as an eluent) to afford 3.0 g of. tert-butyl (R)-(4-(1-((2-(2-(2-azidoethoxy)ethoxy)ethyl)carbamoyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-yl)carbamate as an off white solid. [TLC system: EtOAc: Pet Ether (7:3); Rf value: 0.5].
Step 5. (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-N-(2-(2-(2-azidoethoxy) ethoxy)ethyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxamide
To a solution of tert-butyl (R)-(4-(1-((2-(2-(2-azidoethoxy)ethoxy)ethyl)carbamoyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-yl)carbamate (2.4 g, 3.339 mmol) in CH2Cl2 (24 mL) was added 4 M HCl in 1,4-dioxane (24.0 mL) at 0° C. The reaction mixture was stirred at room temperature for 6 h. After completion of reaction, reaction mixture was concentrated and residue was basified with saturated NaHCO3 solution and extracted with 10% MeOH in DCM (2×200 mL). The combined organic layer was dried over anhydrous Na2SO4 and concentrated to get 2.2 g (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-N-(2-(2-(2-azidoethoxy) ethoxy)ethyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxamide as an oil. [TLC system: MeOH:DCM (0.5:9.5); Rf value: 0.4].
Step 6. 7-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-N-(2-(2-(2-(4-((hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxamide
To a solution of (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-N-(2-(2-(2-azidoethoxy) ethoxy)ethyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxamide (2.2 g, 3.627 mmol) and compound 8 (1.65 g, 5.440 mmol) in t-BuOH (66 mL) and water (22 mL) was added CuSO4, 5.1120 (1.08 g, 4.352 mmol) and Na-ascorbate (1.07 g, 5.440 mmol). The reaction mixture was stirred at room temperature for 2 h. After completion of reaction, reaction mixture was filtered and concentrated to get crude compound. The crude compound was purified by RP-Prep-HPLC afford 0.573 g of 7-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-N-(2-(2-(2-(4-((hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxamide as an off white solid.
LC MS M/Z=910.51 (M+1) 1H NMR (400 MHz, DMSO-d6) δ 8.02-7.98 (m, 1H), 7.91 (s, 1H), 7.45-7.41 (m, 2H), 4.98-4.87 (m, 2H), 4.47-4.46 (m, 4H), 4.42-4.13 (m, 4H), 3.92 (s, 2H), 3.80 (s, 2H), 3.72 (s, 3H), 3.62-3.56 (m, 2H), 3.59-3.32 (m, 12H), 2.67-2.56 (m, 4H), 2.40-2.33 (m, 4H), 1.40 (bs, 2H), 1.23-1.15 (m, 6H), 0.83 (t, J=7.2 Hz, 3H).
To a solution of compound 2-(2-(2-azidoethoxy)ethoxy)ethyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.1 g, 0.164 mmol) and compound (2R,3R,4R,5S)-6-(((2R,3S,4S,5S)-2,3,4,5,6-pentahydroxyhexyl)(prop-2-yn-1-yl)amino)hexane-1,2,3,4,5-pentaol (0.126 g, 0.329 mmol) in t-BuOH (3.0 mL) and water (0.6 mL) was added CuSO4,5·H2O (0.05 g, 0.197 mmol) and Na-ascorbate (0.048 g, 0.246 mmol). Then reaction was stirred at RT for 16 h. After completion of reaction, reaction mixture was filtered and concentrated to get crude compound. The crude compound was purified by RP-Prep-H-PLC to afford 20 mg of 2-(2-(2-(4-((bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl 7-((R)-3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate as off white solid compound. LC MS M/Z=991.57 (M+1) 1H NMR (400 MHz, DMSO-d6) δ 7.94 (s, 1H), 7.45-7.43 (m, 2H), 5.00-4.95 (m, 2H), 4.87-4.14 (m, 18H), 3.93-3.47 (m, 23H), 3.39-3.16 (m, 8H), 2.61-2.58 (m, 2H).
Step 1. 2-morpholinoethyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate:
To a stirred solution of (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylic acid (0.5 g, 0.91 mmol) in 1.2 dichloroethane (10 mL) and triethylamine (0.37 g, 3.64 mmol) was added bis(2-oxo-3-oxazolidinyl)phosphinic chloride (0.46 g, 1.82 mmol) at 0° C. and the resulting mixture was stirred at 0° C. for 1 h. Then 2-morpholinoethan-1-ol (0.24 g, 1.82 mmol) was added at 0° C. and the reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (2×50 mL). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to give crude product. The obtained crude residue was then purified by column chromatography over silica gel (230-400 mesh) using 4% methanol in dichloromethane as a gradient to afford 2-morpholinoethyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.51 g, 84.7%) as an off-white solid. TLC system. MeOH:DCM (1:9); Rf: 0.3.
Step 2. 2-morpholinoethyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate:
To a stirred solution of 2-morpholinoethyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.51 g, 0.77 mmol) in 1.4 dioxane (5.1 mL), hydrogen chloride solution 4.0 M in dioxane (5.1 mL) was added at 0° C. and the reaction mixture was stirred at room temperature for 4 h. After completion, the reaction mixture was concentrated under reduced pressure to give crude product. The obtained crude product was then purified by RP preparative HPLC using following conditions. Column/dimensions: X-BRIDGE-C18 (19*250) 5 um, mobile phase A; 10 mM ammonium bicarbonate in water, mobile phase B: acetonitrile, gradient (time/% B): 0/5, 1/5, 8/40, 13.5/40, 13.51/100, 18/100, 18.1/5, 20.5/5, flow rate: 18 ml/min. The desired fraction was lyophilized to afford 2-morpholinoethyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.25 g, 57.7%) as an off-white semi solid. TLC system. MeOH:DCM (1:9); Rf: 0.05.
Step 3. (R)-4-(1-((2-morpholinoethoxy)carbonyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-aminium (2R,3S)-3-carboxy-2,3-dihydroxypropanoate-Tartrate salt (49):
Mixture of 2-morpholinoethyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.25 g, 0.44 mmol) and L(+)-tartaric acid (0.73 g, 0.49 mmol) in demineralized water (2.5 mL) was sonicated to get clear solution. The resulting solution was lyophilized to afford (R)-4-(1-((2-morpholinoethoxy)carbonyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-aminium (2R,3S)-3-carboxy-2,3-dihydroxypropanoate (0.32 g) as an off-white solid. LCMS M/Z 564.46. 1H NMR (400 MHz, DMSO) δ 7.52 (dd, J=9.5, 4.0 Hz, 2H), 5.03-4.87 (m, 2H), 4.35-4.08 (m, 5H), 3.95-3.88 (m, 4H), 3.67 (s, 2H), 3.56 (t, J=4.6 Hz, 4H), 2.89 (s, 3H), 2.82-2.58 (m, 4H), 2.44 (t, J=4.7 Hz, 4H).
Step 1. tert-butyl (R)-(4-(1-((2-(2-morpholinoethoxy)ethyl)carbamoyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-yl)carbamate:
To a stirred solution of (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylic acid (0.4 g, 0.73 mmol), 2-(2-morpholinoethoxy)ethan-1-amine (0.127 g, (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (0.334 g, 0.88 mmol) in N,N-dimethylformamide (4 mL) was slowly added N,N-diisopropylethylamine (0.47 g, 3.65 mmol) at 0° C. and the reaction mixture was stirred at at room temperature for 4 h. After completion, the reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (3×100 mL). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to give crude product. The obtained crude residue was then purified by column chromatography over silica gel (230-400 mesh) using 4% methanol in dichloromethane as a gradient to afford tert-butyl (R)-(4-(1-((2-(2-morpholinoethoxy)ethyl)carbamoyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-yl)carbamate (0.4 g, 78%) as a brown solid. TLC system. MeOH:DCM (1:9); Rf: 0.3.
Step 2. (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-N-(2-(2-morpholinoethoxy)ethyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxamide:
To a stirred solution of tert-butyl (R)-(4-(1-((2-(2-morpholinoethoxy)ethyl)carbamoyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-yl)carbamate (0.4 g, 0.566 mmol) in 1.4 dioxane (4 mL), 4.0 M HCl in dioxane (4 mL) was added at 0° C. and the mixture was stirred at room temperature for 4 h. After completion, the reaction mixture was concentrated under reduced pressure to give crude product. Obtained crude was then purified by RP preparative HPLC using following conditions. Column/dimensions: Column/dimensions: X-select C18 (19*250 mm), 5 um, mobile phase A: 10 MM ammonium bicarbonate in water, mobile phase B: acetonitrile, gradient (time?% B): 0/10, 1/10, 10/40, 15.51/61.5, 15.6/100, 18/100, 18.1/10, 20/10, flow rate: 17 ml/min. The desired fraction was lyophilized to afford (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-N-(2-(2-morpholinoethoxy)ethyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxamide (0.16 g, 46.6%) as a colorless gum. TLC system. MeOH:DCM (1:9); Rf: 0.05.
Step 3. (R)-4-(1-((2-(2-morpholinoethoxy)ethyl)carbamoyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-aminium (2R,3R)-3-carboxy-2,3-dihydroxypropanoate (tartrate salt) (50):
To a stirred solution of (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-N-(2-(2-morpholinoethoxy)ethyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxamide (0.16 g, 0.26 mmol) in dichloromethane (4 mL) and methanol (1 mL) mixture was added L(+) tartaric acid (0.039 g, 0.26 mmol), the mixture was stirred at room temperature for 6 h. The solvent was evaporated under reduced pressure, the residue was then washed with n-pentane (2×5 mL) and re-dissolved demineralized water (1.4 mL). The resulting solution was lyophilized to afford (R)-4-(1-((2-(2-morpholinoethoxy)ethyl)carbamoyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-aminium (2R,3R)-3-carboxy-2,3-dihydroxypropanoate (0.175 g) as an off-white solid. LCMS M/Z 607.51. 1H NMR (400 MHz, DMSO) δ 8.08-7.95 (m, 1H), 7.56-7.44 (m, 2H), 5.03-4.87 (m, 2H), 4.26-4.07 (m, 3H), 3.88 (s, 4H), 3.37 (t, J=6.1 Hz, 13H), 2.87 (d, J=6.7 Hz, 2H), 2.72-2.65 (m, 2H), 2.46-2.36 (m, 7H).
Step 1. 3-morpholinopropyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate
To a stirred solution of (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylic acid (0.3 g, 0.545 mmol) in 1.2 dichloroethane (6 mL) and triethylamine (0.165 g, 1.63 mmol), was added bis(2-oxo-3-oxazolidinyl)phosphinic chloride (0.207 g, 0.817 mmol) at 0° C. and the resulting mixture was stirred at 0° C. for 1 h. Then 3-morpholinopropan-1-ol (0.119 g, 0.817 mmol) was added at 0° C. and the reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (2×30 mL). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to give crude product. The obtained crude residue was then purified by column chromatography over silica gel (230-400 mesh) using 4% methanol in dichloromethane as a gradient to afford 3-morpholinopropyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.21 g, 57.2%) as an off-white solid. TLC system. MeOH:DCM (1:9); Rf: 0.3.
Step 2. 3-morpholinopropyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate:
To a stirred solution of 3-morpholinopropyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.21 g, 0.31 mmol) in 1.4 dioxane (2 mL) was added 4.0 M in dioxane (2 mL) at 0° C. and the reaction mixture was stirred at room temperature for 4 h. After completion, the reaction mixture was concentrated under reduced pressure to give crude product. Obtained crude was purified by RP preparative HPLC using following conditions. Column/dimensions: X select C18 (19*250 mm) 5μ, mobile phase A: 10 mM ammonium bicarbonate in water, mobile phase B: acetonitrile, gradient (time/% B): 0.01/25, 1/25, 9/40, 13/40, 13.1/100, 18/100, 18.1/25, 20/25 flow rate: 18 ml/min. The desired fraction was lyophilized to afford 3-morpholinopropyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.125 g, 69.8%) as a colorless gum. TLC system. MeOH:DCM (1:9); Rf: 0.05.
Step 3. (R)-4-(1-((3-morpholinopropoxy)carbonyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-aminium (2R,3R)-3-carboxy-2,3-dihydroxypropanoate (51):
To a stirred solution of 3-morpholinopropyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.125 g, 0.216 mmol) in mixture of dichloromethane (4 mL) and methanol (1.0 mL) was added L(+)-tartaric acid (0.032 g, 0.216 mmol) and the mixture was stirred at room temperature for 6 h. The reaction mixture was concentrated, washed with n-pentane (2×5 mL). The resulting residue was dissolved in water and lyophilized to afford (R)-4-(1-((3-morpholinopropoxy)carbonyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-aminium (2R,3R)-3-carboxy-2,3-dihydroxypropanoate (0.077 g) as off-white solid. LCMS M/Z 578.22. 1H NMR (400 MHz, DMSO) δ 7.56-7.46 (m, 2H), 4.94 (d, J=11.9 Hz, 2H), 4.28 (t, J=6.6 Hz, 5H), 3.90 (s, 6H), 3.56 (s, 4H), 2.86 (s, 3H), 2.72-2.66 (m, 2H), 2.38 (dd, J=13.7, 6.5 Hz, 6H), 1.89-1.80 (m, 2H).
Step 1. 2-(2-morpholinoethoxy)ethyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate:
To a stirred solution of (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine—To a mixture of 1-carboxylic acid (0.3 g, 0.54 mmol) in 1.2 dichloroethane (6 mL) and triethylamine (0.27 g, 2.7 mmol) was added bis(2-oxo-3-oxazolidinyl)phosphinic chloride (0.28 g, 1.09 mmol) at 0° C. and the resulting mixture was stirred at 0° C. for 1 h. Then 2-(2-morpholinoethoxy)ethan-1-ol (0.191 g, 1.09 mmol) was added at 0° C. and the reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (2×30 mL). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to give crude product. The obtained crude residue was then purified by column chromatography over silica gel (230-400 mesh) using 4% methanol in dichloromethane as a gradient to afford 22-(2-morpholinoethoxy)ethyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.32 g, 83.1%) as an off-white solid. TLC system. MeOH:DCM (1:9); Rf: 0.3.
Step 2. 2-(2-morpholinoethoxy)ethyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate:
To a stirred solution of 2-(2-morpholinoethoxy)ethyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.32 g, 0.45 mmol) in 1.4 dioxane (3.2 mL) was added 4.0 M HCl in dioxane (3.2 mL) at 0° C. and the mixture was stirred at room temperature for 4 h. After completion, the reaction mixture was concentrated under reduced pressure to give crude product. The crude product was then purified by reverse phase preparative HPLC using following conditions. Column/dimensions: X-bridge-C18 (19*250 mm) 5 um, mobile phase A: 10 mM ammonium bicarbonate in water, mobile phase B: acetonitrile, gradient (time/% B): 0/5, 1/5, 12/75, 14/75, 14.05/98, 16.50/98, 16.51/5, 19/5, flow rate: 18 ml/min. The desired fraction was lyophilized to afford 2-(2-morpholinoethoxy)ethyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.14 g, 51.1%) as a colorless gum. TLC system. MeOH:DCM (1:9); Rf: 0.05.
Step 3. (R)-4-(1-((2-(2-morpholinoethoxy)ethoxy)carbonyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-aminium (2R,3R)-3-carboxy-2,3-dihydroxypropanoate (Tartrate salt) (52):
Mixture of 2-(2-morpholinoethoxy)ethyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.14 g, 0.23 mmol) and L(+)-tartaric acid (0.038 g, 0.25 mmol) in demineralized water (1.4 mL) was sonicated to get clear solution. The resulting solution was lyophilized to afford (R)-4-(1-((2-(2-morpholinoethoxy)ethoxy)carbonyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-aminium (2R,3R)-3-carboxy-2,3-dihydroxypropanoate (0.172 g) as an off-white solid. LCMS M/Z 608.46. 1H NMR (400 MHz, DMSO) δ 7.53 (q, J=9.6 Hz, 2H), 4.95 (q, J=18.0 Hz, 2H), 4.40-4.05 (m, 6H), 3.92 (d, J=12.6 Hz, 5H), 3.68 (t, J=4.8 Hz, 5H), 3.50 (t, J=4.6 Hz, 4H), 2.73 (s, 2H), 2.46 (d, J=5.7 Hz, 3H), 2.38 (s, 4H).
Step 1. 5-morpholinopentyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate:
To a stirred solution of (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylic acid (1.0 g, 1.82 mmol) in 1.2 dichloroethane (20 mL) and triethylamine (1.27 g, 9.1 mmol), was added bis(2-oxo-3-oxazolidinyl)phosphinic chloride (0.93 g, 3.64 mmol) at 0° C. and the resulting mixture was stirred at 0° C. for 1 h. Then 5-morpholinopentan-1-ol (0.94 g, 5.45 mmol) was added at 0° C. and the mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (2×30 mL). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to give crude product. The obtained crude residue was then purified by column chromatography over silica gel (230-400 mesh) using 4% methanol in dichloromethane as a gradient to afford 5-morpholinopentyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.6 g, 46.9%) as a colorless gum. TLC system. MeOH:DCM (1:9); Rf: 0.3.
Sep 2. 5-morpholinopentyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate:
To a stirred solution of 5-morpholinopentyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.6 g, 0.85 mmol) in 1.4 dioxane (6 mL) was added 4.0 M HCl in dioxane (6 mL) at 0° C. and the mixture was stirred at room temperature for 4 h. After completion, the reaction mixture was concentrated under reduced pressure to give crude product. The crude product was then purified by RP preparative HPLC using following conditions. Column/dimensions: X select C18 (19*250 mm) 5 um, mobile phase A: 10 mM ammonium bicarbonate in water, mobile phase B: acetonitrile, gradient (time/% B): 0/20, 1/20, 12/50, 16.5/50, 16.6/100, 19/100, 19.1/20, 21/20, flow rate: 18 ml/min. The desired fraction was lyophilized to afford 5-morpholinopentyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.29 g, 56.3%) as a colorless gum. TLC system. MeOH:DCM (1:9); Rf: 0.05.
Step 3. (R)-4-(1-(((5-morpholinopentyl)oxy)carbonyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-aminium (2R,3R)-3-carboxy-2,3-dihydroxypropanoate (Tartrate salt) (53):
Mixture of 5-morpholinopentyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.19 g, 0.31 mmol) and L(+)-tartaric acid (0.052 g, 0.34 mmol) in demineralized water (1.9 mL) was sonicated for 2 min to get clear solution. The resulting solution was lyophilized to afford (R)-4-(1-(((5-morpholinopentyl)oxy)carbonyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-aminium (2R,3R)-3-carboxy-2,3-dihydroxypropanoate (0.23 g) as an off-white solid. LCMS M/Z 606.48. 1H NMR (400 MHz, DMSO) δ 7.51 (td, J=10.2, 6.9 Hz, 2H), 5.00-4.88 (m, 2H), 4.24 (td, J=7.2, 3.4 Hz, 5H), 3.99-3.87 (m, 5H), 3.68 (s, 2H), 3.54 (t, J=4.5 Hz, 5H), 2.90 (s, 2H), 2.80-2.64 (m, 2H), 2.38-2.26 (m, 6H), 1.68 (p, J=7.3 Hz, 2H), 1.55-1.34 (m, 4H).
Step 1. 4-morpholinobutyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate:
To a stirred solution of (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylic acid (0.5 g, 0.91 mmol) in 1.2 dichloroethane (10 mL) and triethylamine (0.37 g, 3.64 mmol) was added bis(2-oxo-3-oxazolidinyl)phosphinic chloride (0.35 g, 1.36 mmol) at 0° C. and the mixture was stirred at 0° C. for 1 h. Then 4-morpholinobutan-1-ol (0.22 g, 1.36 mmol) was added at 0° C. and the reaction mixture was stirred at room temperature for 16 h. After completion, the reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (2×30 mL). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to give crude product. The crude residue was then purified by column chromatography over silica gel (230-400 mesh) using 4% methanol in dichloromethane as an eluent to afford 4-morpholinobutyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.45 g, 71.6%) as an off-white solid. TLC system. MeOH:DCM (1:9); Rf: 0.3.
Step 2. 4-morpholinobutyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate:
To a stirred solution of 4-morpholinobutyl (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.45 g, 0.65 mmol) in 1.4 dioxane (4.5 mL) was added 4.0 M HCl in dioxane (4.5 mL) at 0° C. and the mixture was stirred at room temperature for 4 h. After completion, the reaction mixture was concentrated under reduced pressure to give crude product. The crude product was then purified by RP preparative HPLC using following conditions. Column/dimensions: X-bridge C 18 (19*250 mm) 5μ, mobile phase A: 10 MM ammonium bicarbonate in water, mobile phase B: acetonitrile (org), gradient (time/% B): 0/20, 2/20, 10/45, 14/45, 14.10/100, 16/100, 16.10/20, 18/20. flow rate: 17 ml/min. The desired fraction was lyophilized to afford 4-morpholinobutyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.16 g, 41.7%) as colorless gum. TLC system. MeOH:DCM (1:9); Rf: 0.05.
Step 3. (R)-4-(1-((4-morpholinobutoxy)carbonyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-aminium (2R,3R)-3-carboxy-2,3-dihydroxypropanoate (Tartrate salt) (54):
Mixture of 5-morpholinopentyl 4-morpholinobutyl (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylate (0.09 g, 0.15 mmol) and L(+)-tartaric acid (0.025 g, 0.167 mmol) in demineralized water (1 mL) was sonicated to get clear solution. The resulting solution was lyophilized to afford (R)-4-(1-((4-morpholinobutoxy)carbonyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-aminium (2R,3R)-3-carboxy-2,3-dihydroxypropanoate (0.115 g) as an off-white solid. LCMS M/Z 592.49. 1H NMR (400 MHz, DMSO) δ 7.53 (q, J=8.2 Hz, 2H), 4.94 (d, J=11.9 Hz, 2H), 4.25 (dd, J=7.7, 5.3 Hz, 5H), 3.93 (s, 5H), 3.64 (d, J=6.5 Hz, 5H), 2.87 (d, J=7.4 Hz, 2H), 2.78-2.64 (m, 2H), 2.45-2.13 (m, 6H), 1.68 (d, J=8.2 Hz, 2H), 1.54 (q, J=7.4 Hz, 2H).
Step 1. Tert-butyl (R)-(4-(1-((2-methyl-2-azaspiro[3.3]heptan-6-yl)carbamoyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-yl)carbamate:
To a stirred solution of (R)-7-(3-((tert-butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoyl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxylic acid (0.5 g, 0.91 mmol), 2-methyl-2-azaspiro[3.3]heptan-6-amine hydrochloride (0.18 g, 0.91), (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (0.415 g, 0.1.09 mmol) in N,N-dimethylformamide (5 mL) was slowly added N,N-diisopropylethylamine (0.59 g, 4.55 mmol) at 0° C. and the reaction mixture was stirred at at room temperature for 4 h. After completion, the reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (3-100 mL). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to give crude product. The obtained crude residue was then purified by column chromatography over silica gel (230-400 mesh) using 4% methanol in dichloromethane as an eluent to afford tert-butyl (R)-(4-(1-((2-methyl-2-azaspiro[3.3]heptan-6-yl)carbamoyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-yl)carbamate (0.3 g, 50.2%) as an off-white solid. TLC system. MeOH:DCM (1:9); Rf. 0.3.
Step 2. (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-N-(2-methyl-2-azaspiro[3.3]heptan-6-yl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxamide:
To a stirred solution of tert-butyl (R)-(4-(1-((2-methyl-2-azaspiro[3.3]heptan-6-yl)carbamoyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-yl)carbamate (0.3 g, 0.45 mmol) in 1.4 dioxane (3 mL), was added 4.0 M HCl in dioxane (3 mL) at 0° C. and the mixture was stirred at room temperature for 4 h. After completion, the reaction mixture was concentrated under reduced pressure to give crude product. The obtained crude was then purified by RP preparative HPLC using following conditions. Column/dimensions: X-select C 18 (19*250 mm) 5μ, mobile phase A: 10 MM ammonium bicarbonate in water, mobile phase B: acetonitrile (org), gradient (time/% B): 0/10, 1/10, 10/40, 16/63.2, 16.1/100, 19/100, 19.1/10, 22/10, flow rate: 17 ml/min. The desired fraction was lyophilized to afford (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-N-(2-methyl-2-azaspiro[3.3]heptan-6-yl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxamide (0.09 g, 35.4%) as an off-white solid. TLC system. MeOH:DCM (1:9); Rf: 0.05.
Step 3. (R)-4-(1-((2-methyl-2-azaspiro[3.3]heptan-6-yl)carbamoyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-aminium (2R,3R)-3-carboxy-2,3-dihydroxypropanoate (Tartrate salt) (55):
Mixture of (R)-7-(3-amino-4-(2,4,5-trifluorophenyl)butanoyl)-N-(2-methyl-2-azaspiro[3.3]heptan-6-yl)-3-(trifluoromethyl)-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine-1-carboxamide (0.14 g, 0.25 mmol), in dichloromethane (4 mL) and methanol (I mL) mixture L(+) tartaric acid (0.038 g, 0.25 mmol) was added and the reaction mixture was stirred at room temperature for 6 h. The reaction mixture was concentrated, residue was washed with n-pentane (2×5 mL) and residue was dissolved demineralized water (3 mL). The resulting solution was lyophilized to afford (R)-4-(1-((2-methyl-2-azaspiro[3.3]heptan-6-yl)carbamoyl)-3-(trifluoromethyl)-5,6-dihydroimidazo[1,5-a]pyrazin-7(8H)-yl)-4-oxo-1-(2,4,5-trifluorophenyl)butan-2-aminium (2R,3R)-3-carboxy-2,3-dihydroxypropanoate (0.155 g) as an off-white solid. LCMS M/Z 559.46. 1H NMR (400 MHz, DMSO) δ 8.40-8.22 (m, 1H), 7.48 (q, J=9.2 Hz, 2H), 4.91 (d, J=14.9 Hz, 2H), 4.26 (dd, J=16.4, 8.0 Hz, 3H), 4.11 (s, 1H), 3.89 (s, 3H), 3.83 (s, 3H), 2.78 (d, J=7.1 Hz, 2H), 2.70-2.56 (m, 3H), 2.45-2.28 (m, 7H).
DPP4 activity assay. Human DPP4 activity assay data were obtained using a DPP4 Activity Assay Kit (Sigma-Aldrich, MAK088) according to the manufacturer's instructions. Briefly, 10 μL of DPP4 Assay Buffer were transferred per well in low volume 384-well plates before transferring 10 μL of test compound dissolved in DPP4 assay buffer. To each well was added 5 μL of Master Reaction Mix containing a fluorescent substrate that becomes fluorescent upon cleavage by the enzyme. Fluorescence intensity measurements were recorded at 1-minute time intervals over the course of 20 minutes using an Envision Multimode Plate Reader (PerkinElmer). Results are presented in Table 4 below.
AEC2 proliferation assay. Primary human AEC2s were plated at a density of 1,500 cells per well in black 384-well plates (Greiner) coated with 10 ug/mL Laminin (Life Technologies) in 50 μL of Small Airway Epithelial Cell Growth Medium (Lonza) without EGF, retinoic acid and with 5% BPE. 100 nL of test compound dissolved in DMSO was then delivered using a Biomek FX instrument (Beckman Coulter) fitted with a pintool head (V & P Scientific). After 96 hours of growth at 37° C., cells were fixed with 4% paraformaldehyde, washed three times with PBS, and then immunostained for KI-67 positivity (1:1000, Abcam, ab15580) overnight at 4° C. After three additional washes, the cells were incubated with secondary AlexaFluor conjugated secondary antibody for 1 hour at room temperature and then exposed to 10 μg/mL Hoechst 33342 (Life Technologies). Plates were sealed, and then quantitative high content imaging carried out on CellInsight CX5 HCS instrument (ThermoFisher). The ACE2 proliferation concentration curve for compound 46 is depicted in
1H NMR
1H NMR (400 MHz, DMSO-d6) 6.79 (s, 1H), 4.73 (dd, J = 7.3, 3.8 Hz, 1H), 3.85 (t, J = 6.4 Hz, 2H), 3.65-3.55 (m, 1H), 3.51- 3.38 (m, 2H), 3.36 (d, J = 7.3 Hz, 1H), 3.29 (s, 1H), 2.17- 2.06 (m, 4H), 2.05-1.95 (m, 2H), 1.72 (d, J = 17.5 Hz, 6H), 1.49 (d, J = 12.8 Hz, 8H), 1.26 (s, 8H), 0.86 (t, J = 6.9 Hz, 3H).
1H NMR (400 MHz, CD3OD) 8.40 (s, 1H), 4.81 (t, J = 5.2 Hz, 1H), 3.99-3.87 (m, 2H), 3.74- 3.68 (m, 1H), 3.56-3.49 (m, 1H), 2.56 (t, J = 7.2 Hz, 2H), 2.35-2.24 (m, 4H), 2.23- 2.14 (m, 2H), 2.01-1.94 (m, 2H), 1.92-1.78 (m, 8H), 1.73- 1.65 (m, 2H), 1.57-1.50 (m, 2H), 1.43-1.36 (m, 2H), 1.35- 1.24 (m, 6H), 0.90 (t, J = 6.8 Hz, 3H).
1H NMR (400 MHz, DMSO- d6) δ 4.74-4.72 (m, 1H), 4.46 (bs, 1H), 3.63-3.32 (m, 8H), 2.17-2.11 (m, 4H), 2.03-1.99 (m, 2H), 1.68 (t, J = 6.0 Hz, 1H), 1.60-1.40 (m, 12H).
1H NMR (400 MHz, CDCl3) 4.73 (d, 1H), 3.91 (d, 1H), 3.71-3.52 (m, 4H), 3.38 (t, 2H), 2.38 (s, 2H), 2.27-2.10 (m, 4H), 1.83-1.73 (m, 10H), 1.58 (s, 2H), 1.50 (t, 2H), 1.28 (s, 8H), 0.88 (t, J = 7.2 Hz, 3H).
1H NMR (400 MHz, CDCl3) 5.39 (s, 2H), 4.87-4.73 (m, 1H), 3.66-3.39 (m, 4H), 2.36- 2.25 (m, 6H), 2.24-2.13 (m, 2H), 1.83 (s, 3H), 1.80-1.68 (m, 6H), 1.66-1.56 (m, 6H), 1.33- 1.24 (m, 8H), 0.88 (t, J = 7.2 Hz, 3H).
1H-NMR (400 MHz, CDCl3) δ 7.73 (s, 1H), 4.92-4.70 (m, 1H), 4.52 (br t, J = 4.5 Hz, 2H), 3.94-3.82 (m, 5H), 3.81-3.71 (m, 4H), 3.71-3.63 (m, 3H), 3.56 (br d, J = 19.3 Hz, 9H), 3.47-3.33 (m, 3H), 2.77-2.58 (m, 2H), 2.53 (td, J = 7.7, 12.7 Hz, 1H), 2.47-2.37 (m, 1H), 2.36-2.01 (m, 6H), 1.99 (s, 1H), 1.75-1.40 (m, 14H), 1.23 (br s, 6H), 0.84 (br t, J = 6.7 Hz, 3H).
1H NMR (400 MHz, DMSO- d6) δ 7.95 (s, 1H), 4.75-4.72 (m, 1H), 4.70-4.10 (m, 12H), 3.81- 3.72 (m, 6H), 3.63-3.39 (m, 22H), 2.17-1.91 (m, 6H), 1.56- 1.44 (m, 12H).
1H NMR (400 MHz, DMSO- d6): δ 4.74-4.70 (m, 1H), 4.51- 4.46 (m, 4H), 4.35-4.29 (m, 4H), 4.19 (d, J = 6.4 Hz, 2H), 3.65-3.55 (m, 7H), 3.49-3.33 (m, 16H), 2.75-2.50 (m, 5H), 2.17-1.95 (m, 6H), 1.70 (bs, 1H), 1.59-1.45 (m, 12H).
1H NMR (400 MHz, DMSO- d6) δ 4.73-4.29 (m, 6H), 3.63- 3.13 (m, 17H), 2.67-2.60 (m, 4H), 2.16-1.99 (m, 6H), 1.59- 1.45 (m, 12H).
1H NMR (400 MHz, DMSO- d6): δ 8.17 (s, 2H), 7.73 (s, 1H), 7.27 (d, J = 7.6 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 4.75- 4.74 (m, 3H), 4.16-4.06 (m, 4H), 3.75-3.35 (m, 21H), 2.61- 2.50 (m, 1H), 2.46-2.41 (m, 3H), 2.19-1.99 (m, 6H), 1.60- 1.52 (m, 12H), 0.94 (t, J = 7.2 Hz, 3H).
1H NMR (400 MHz, CD3OD) 8.48 (s, 2H), 7.22 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 4.83-4.78 (m, 1H), 4.17- 4.07 (m, 3H), 4.05-3.95 (m, 2H), 3.91-3.74 (m, 4H), 3.74- 3.60 (m, 6H), 3.55-3.50 (m, 1H), 3.50-3.43 (m, 2H), 3.41- 3.32 (m, 4H), 3.26-3.16 (m, 2H), 3.05-2.94 (m, 2H), 2.40- 2.09 (m, 6H), 1.85-1.65 (m, 12H), 1.58 (s, 2H), 1.42-1.30 (m, 6H), 0.97-0.89 (m, 3H).
1H NMR (400 MHz, DMSO- d6) δ 7.92 (s, 1H), 5.10 (bs, 1H), 4.73-4.65 (m, 2H), 4.49- 4.43 (m, 3H), 4.3 (bs, 2H), 3.80-3.36 (m, 19H), 2.64-2.60 (m, 2H), 2.16-2.01 (m, 6H), 1.56-1.46 (m, 12H).
1H NMR (400 MHz, MeOD) δ 8.44 (br, 2H), 8.13 (s, 1H), 4.82 (t, 1H), 4.60 (t, 2H), 4.24 (d, 2H), 4.08 (dd, 1H), 3.97 (d, 1H), 3.91-3.87 (m, 2H), 3.74- 3.56 (m, 12H), 3.01 (d, 2H), 2.89-2.82 (m, 2H), 2.37 (br, 2H), 2.27 (t, 2H), 2.20-2.07 (m, 2H), 1.84-1.68 (m, 12H), 1.61 (s, 2H), 1.31 (s, 6H), 0.91 (t, 3H).
1H NMR (400 MHz, DMSO- d6) δ 5.10-5.07 (m, 1H), 4.46- 4.43 (m, 1H), 3.89- 3.87 (m, 1H), 3.45- 3.31 (m, 5H), 2.21- 2.14 (m, 3H), 1.77-1.32 (m, 14H), 0.97 (m, 1H), 0.72- 0.70 (s, 1H).
1H NMR (500 MHz, MeOD) δ 7.55-7.37 (m, 4H), 5.23 (dd, J = 10.7, 2.3 Hz, 1H), 4.39-4.26 (m, 5H), 4.15 (t, J = 5.0 Hz, 2H), 3.94 (td, J = 6.2, 2.7 Hz, 1H), 3.68 (t, J = 5.1 Hz, 2H), 3.28-3.12 (m, 4H), 2.65 (ddd, J = 14.1, 10.8, 5.7 Hz, 1H), 2.43-2.27 (m, 3H), 2.05 (dq, J = 9.0, 5.6 Hz, 1H), 1.91-1.56 (m, 12H), 1.36 (t, J = 7.3 Hz, 7H), 1.15 (q, J = 6.9 Hz, 1H), 1.02-0.94 (m, 1H).
1H NMR (500 MHz, MeOD) δ 7.53 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 7.9 Hz, 2H), 5.23 (dd, J = 10.7, 2.3 Hz, 1H), 4.52 (s, 2H), 4.33 (d, J = 20.1 Hz, 3H), 4.15 (t, J = 5.0 Hz, 2H), 3.98- 3.88 (m, 5H), 3.68 (t, J = 5.1 Hz, 2H), 3.37 (m, 4H), 2.65 (ddd, J = 13.8, 10.8, 5.7 Hz, 1H), 2.42-2.29 (m, 3H), 2.05 (dq, J = 8.9, 5.7 Hz, 1H), 1.90- 1.58 (m, 13H), 1.36-1.29 (m, 1H), 1.15 (q, J = 6.8 Hz, 1H),
1H NMR (500 MHz, MeOD) δ 7.51 (d, J = 7.8 Hz, 2H), 7.42 (d, J = 7.9 Hz, 2H), 5.21 (dd, J = 10.7, 2.3 Hz, 1H), 4.49-4.30 (m, 4H), 4.23-4.12 (m, 4H), 3.94 (td, J = 6.2, 2.7 Hz, 1H), 3.83-3.75 (m, 2H), 3.73- 3.60 (m, 5H), 3.32-3.27 (m, 2H), 3.11 (qdd, J = 13.0, 10.1, 6.1 Hz, 2H), 2.65 (ddd, J = 13.7, 10.6, 5.7 Hz, 1H), 2.39- 2.26 (m, 3H), 2.03 (dq, J = 9.0, 5.7 Hz, 1H), 1.90-1.56 (m, 16H), 1.41-1.23 (m, 11H), 1.14 (dt, J = 9.0, 6.4 Hz, 1H), 1.00-0.87 (m, 4H).
1H NMR (400 MHz, MeOD) δ 8.41 (br, 2H), 8.20 (s, 1H), 5.20 (dd, 1H), 4.61 (t, 2H), 4.36 (d, 2H), 4.13 (t, 2H), 3.97 (t, 3H), 3.71-3.64 (m, 5H), 3.49 (s, 4H), 3.91-3.12 (m, 2H), 3.01-2.91 (m, 2H), 2.63-2.59 (m, 1H), 2.31-2.33 (m, 3H), 2.03-1.98 (m, 1H), 1.80-1.57 (m, 14H), 1.32 (s, 6H), 1.10 (dd, 1H), 0.95- 0.90 (m, 4H).
1H NMR (400 MHz, MeOD) δ 8.38 (br, 2H), 8.21 (s, 1H), 5.19 (dd, 1H), 4.63 (t, 2H), 4.42 (s, 2H), 4.18-4.15 (m, 2H), 3.98 (t, 3H), 3.70-3.56 (m, 13H), 3.18- 3.16 (m, 2H), 3.03-2.93 (m, 2H), 2.63-2.59 (m, 1H), 2.37- 2.31 (m, 3H), 2.01-1.98 (m, 1H), 1.76-1.61 (m, 14H), 1.35 (s, 6H), 1.11 (dd, 1H), 0.94-0.91 (m, 4H).
1H NMR (400 MHz, DMSO- d6) δ 8.54 (s, 1H), 8.17 (s, 2H), 7.79 (t, J = 6.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 5.60 (bs, 1H), 4.95 (bs, 1H), 5.27-5.24 (m, 1H), 4.95 (m, 2H), 4.37- 4.02 (m, 16H), 3.72- 3.52 (m, 12H), 3.16 (m, 4H), 2.32-2.23 (m, 3H), 2.07 (m, 1H), 1.73-1.46 (m, 12H), 1.04 (m, 1H), 0.75 (s, 1H).
1H NMR (400 MHz, DMSO- d6) δ 8.13 (s, 1H), 7.74 (t, J = 6.0 Hz, 1H), 7.38 (d, J = 7.6 Hz, 2H), 7.24 (d, J = 7.6 Hz, 2H), 5.22 (m, 1H), 4.18- 4.02 (m, 10H), 3.89 (s, 1H), 2.17-3.66-3.52 (m, 4H), 3.49- 3.47 (m, 4H), 2.92-2.71 (m, 4H), 2.50 (s, 3H), 2.27-2.21 (m, 3H), 1.96 (m, 1H), 1.71-1.43 (m, 12H), 1.09-1.02 (m, 4H), 0.74 (s, 1H).
1H NMR (400 MHz, CDCl3) 4.73 (d, 1H), 3.97 (d, 1H), 3.71-3.52 (m, 4H), 3.38 (t, 2H), 2.38 (s, 2H), 2.27-2.10 (m, 4H), 1.83-1.70 (m, 10H), 1.58 (s, 2H), 1.53-1.57 (m, 2H), 1.33- 1.22 (m, 8H), 0.90-0.86 (m, 3H).
1H NMR (400 MHz, DMSO- d6) δ 8.22 (s, 2H), 8.12 (s, 2H), 7.55 (d, J = 6.8 Hz, 1H), 7.27- 7.14 (m, 2H), 6.91 (d, J = 7.6 Hz, 1H), 5.25 (s, 2H), 5.00-4.88 (m, 2H), 3.69 (d, J = 2.4 Hz, 1H), 3.49-3.40 (m, 5H), 3.22- 3.14 (m, 2H), 2.00-1.90 (m, 2H), 1.79 (s, 3H), 1.71-1.61 (m, 2H).
1H NMR (400 MHz, DMSO- d6) δ 8.13 (br, 3H), 7.87 (d, 2H), 7.38 (d, 2H), 5.10 (s, 2H), 4.93 (dd, 2H), 3.67 (d, 1H), 3.49-3.33 (m, 5H), 3.21-3.08 (m, 2H), 2.01- 1.90 (m, 2H), 1.79 (s, 3H), 1.70-1.66 (m, 2H).
1H NMR (400 MHz, DMSO- d6) δ 13.7 (br. s, 1H), 8.46 (t, J = 8.4 Hz, 1H), 8.12 (br. s, 3H), 7.16-7.14 (m, 1H), 5.45 (s, 2H), 4.98-4.86 (m, 2H), 3.71-3.69 (m, 1H), 3.51-3.48 (m, 1H), 3.39 (s, 3H), 3.37 (s, 1H), 3.22- 3.16 (m, 2H), 2.00-1.99 (m, 1H), 1.95-1.91 (m, 1H), 1.78 (s, 3H), 1.75-1.62 (m, 2H).
1H NMR (400 MHz, DMSO- d6) δ 8.48 (t, J = 8.4 Hz, 1H), 8.12 (d, J = 3.6 Hz, 3H), 7.22- 7.19 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 5.49 (s, 2H), 4.94 (m, 2H), 4.34 (t, J = 6.4 Hz, 2H), 3.71 (m, 1H), 3.50-3.40 (m, 5H), 3.21- 3.16 (m, 2H), 2.02-1.89 (m, 1H), 1.89-1.77 (m, 1H), 1.76- 1.65 (m, 7H), 1.44- 1.41 (m, 2H), 1.34-1.31 (m, 4H), 0.89-0.86 (m, 3H).
1H NMR (400 MHz, DMSO- d6) δ 8.24 (m, 3H), 7.87 (d, J = 7.6 Hz, 1H), 6.33 (d, J = 8.4 Hz, 1H), 5.37 (s, 2H), 5.00-4.88 (m, 2H), 3.68-3.38 (m, 6H), 3.18-3.12 (m, 2H), 2.45 (m, 3H), 2.02-1.90 (m, 2H), 1.77 (s, 3H), 1.70-1.62 (m, 2H).
1H NMR (400 MHz, DMSO- d6) δ 8.18 (s, 3H), 7.87 (d, J = 8.4 Hz, 1H), 7.21 (br s, 1H), 6.34 (d, J = 8.8, 1H), 5.36 (s, 2H), 4.99- 4.88 (m, 2H), 4.22 (t, J = 6.4 Hz, 2H), 3.70-3.59 (m, 1H), 3.49-3.39 (m, 5H), 3.18- 3.11 (m, 2H), 2.44 (s, 3H), 2.02- 1.99 (m, 2H), 1.90-1.61 (m, 7H), 1.42-1.31 (m, 6H), 0.89 (s, 3H).
1H NMR (400 MHz, DMSO-d6) δ 12.26 (br. s, 1H), 8.19 (s, 3H), 7.82 (d, J = 8.8 Hz, 1H), 7.30 (br. s, 1H), 6.29 (d, J = 8.8 Hz, 1H), 5.35 (s, 2H), 5.00-4.86 (m, 2H), 3.68-3.65 (m, 2H), 3.38 (s, 4H), 3.14-3.09 (m, 2H), 2.87 (br. s, 2H), 2.03-2.01 (m, 1H), 1.90 (m, 1H), 1.76-1.61 (m, 5H), 1.14-1.11 (m, 2H), 0.93- 0.91 (m, 2H), 0.68 (t, J = 7.2 Hz, 3H).
1H NMR (400 MHz, DMSO d6) δ 8.95 (s, 1H), 8.34 (s, 1H), 8.21 (m, 3H), 7.84 (d, J = 8.7 Hz, 1H), 7.39 (brs, 1H), 6.31 (d, J = 8.8 Hz, 1H), 5.34 (br m, 4H), 4.93 (m, 2H), 4.56 (m, 2H), 4.39 (t, J = 6.9 Hz, 2H), 4.13 (m, 2H), 3.79 (s, 3H), 3.59 (m, 6H), 3.43 (m, 11H), 3.15 (m, 6H), 2.86 (s, 2H), 2.04 (m, 1H), 1.91 (m, 1H), 1.78 (m, 7H), 1.16 (m, 8H), 0.89 (m, 2H).
1H NMR (400 MHz, DMSO) δ 7.93 (s, 1H), 7.79 (d, J = 8.6 Hz, 1H), 7.01 (br s, 1H), 6.22 (d, J = 8.6 Hz, 1H), 5.31 (s, 2H), 4.59-4.26 (m, 14H), 3.77 (m, 4H), 3.55 (m, 9H), 3.38 (m, 7H), 2.90 (m, 5H), 2.50 (m, 2H), 1.74 (m, 9H), 1.27 (m, 1H), 1.12 (m, 6H), 0.91 (m, 2H).
1H NMR (400 MHz, DMSO-d6) δ 7.79 (d, J = 2.0 Hz, 1H), 7.35 (dd, J = 8.4, 2.4 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 5.00 (s, 2H), 4.88 (s, 2H), 3.66-3.58 (m, 2H), 3.39 (s, 3H), 3.00 (t, J = 10.0 Hz, 1H), 2.84-2.72 (m, 2H), 1.87-1.61 (m, 8H), 1.24- 1.21 (m, 1H).
1H NMR (400 MHz, DMSO-d6) δ 7.83 (d, J = 2.0 Hz, 1H), 7.54 (dd, J = 8.4, 2.0 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 5.32 (s, 2H), 5.22-5.16 (m, 1H), 4.88 (s, 2H), 3.66-3.58 (m, 2H), 3.38 (s, 3H), 2.99 (t, J = 10.4 Hz, 1H), 2.84-2.72 (m, 2H), 1.87-1.57 (m, 8H), 1.37 (d, J = 6.0 Hz, 6H), 1.27-1.26 (m, 1H).
1H NMR (400 MHz, DMSO- d6) δ 7.84 (d, 1H), 7.54 (dd, 1H), 7.07 (d, 1H), 5.32 (s, 2H), 4.88 (s, 2H), 4.31 (t, 2H), 3.65- 3.57 (m, 2H), 3.37 (s, 3H), 3.01-2.96 (m, 1H), 2.82-2.68 (m, 2H), 1.87-1.59 (m, 9H), 1.42-1.20 (m, 7H), 0.88 (t, 3H).
1H NMR (400 MHz, DMSO- d6) δ 7.85 (d, J = 2.4 Hz, 1H), 7.55 (dd, J = 8.4, 2.0 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 5.32 (s, 2H), 4.88 (s, 2H), 4.32 (t, J = 6.4 Hz, 2H), 3.66-3.58 (m, 2H), 3.37 (s, 3H), 3.01-2.96 (m, 1H), 2.82-2.72 (m, 2H), 1.87-1.60 (m, 10H), 1.41-1.24 (m, 13H), 0.84 (t, J = 6.4 Hz, 3H).
1H NMR (400 MHz, DMSO-d6) δ 8.56 (t, J = 5.2 Hz, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.37 (dd, J = 8.4, 2.4 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 5.14 (s, 2H), 4.88 (s, 2H), 3.65-3.57 (m, 2H), 3.37 (s, 3H), 3.27-3.23 (m, 2H), 2.98 (t, J = 10.4 Hz, 1H), 2.88-2.71 (m, 2H), 1.87-1.52 (m, 9H), 1.39-1.27 (m, 5H), 0.89 (t, J = 6.8 Hz, 3H).
1H NMR (400 MHz, DMSO- d6) δ 9.5 (s, 1H), 8.31 (d, J = 7.6 Hz, 1H), 8.23 (s, 3H), 7.84 (s, 1H), 7.57 (dd, J = 8.4, 2.0 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 5.33 (s, 3H), 5.01-4.87 (m, 2H), 4.63 (s, 2H), 4.41 (s, 4H), 4.12-4.03 (m, 1H), 3.93-3.91 (m, 2H), 3.79 (m, 2H), 3.74- 3.60 (m, 3H), 3.39 (m, 8H), 3.20-3.17 (m, 4H), 2.99-2.98 (m, 2H), 2.02-1.91 (m, 2H), 1.78-1.63 (m, 7H), 1.25 (s, 6H), 0.86 (s, 3H).
1H NMR (400 MHz, DMSO- d6) δ 7.45-7.43 (m, 2H), 4.96- 4.93 (m, 2H), 4.37-4.36 (m, 2H), 4.25-4.11 (m, 2H), 3.94- 3.93 (m, 2H), 3.75-3.71 (m, 2H), 3.60-3.58 (m, 6H), 3.37- 3.34 (m, 2H), 3.31 (m, 1H), 2.73-2.58 (m, 2H), 2.51-2.49 (m, 2H), 1.80 (bs, 2H).
1H NMR (400 MHz, DMSO- d6) δ 7.45-7.43 (m, 2H), 4.96- 4.93 (m, 2H), 4.37-4.36 (m, 2H), 4.25-4.11 (m, 2H), 3.94- 3.93 (m, 2H), 3.75-3.71 (m, 2H), 3.60-3.58 (m, 6H), 3.37- 3.34 (m, 2H), 3.31 (m, 1H), 2.73-2.58 (m, 2H), 2.51-2.49 (m, 2H), 1.80 (bs, 2H).
1H NMR (400 MHz, DMSO-d6) δ 7.89 (s, 1H), 7.45-7.41 (m, 2H), 5.10-4.85 (m, 3H), 4.60- 4.10 (m, 10H), 4.00-3.30 (m, 20H), 3.10-3.05 (m, 1H), 2.71- 2.50 (m, 5H), 2.10-1.40 (m, 2H).
1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.29 (d, J = 5.8 Hz, 1H), 8.10-8.00 (m, 3H), 7.58 (q, J = 9.0 Hz, 2H), 5.49 (s, 1H), 4.95-4.93 (m, 3H), 4.57-3.38 (m, 28H), 3.18-2.66 (m, 8H), 1.31-1.26 (m, 3H).
1H NMR (400 MHz, MeOD) δ 8.28 (s, 1H), 7.36 (m, 1H), 7.27- 7.17 (m, 1H), 5.07 (m, 1H), 4.99 (m, 1H), 4.58 (m, 4H), 4.45 (m, 2H), 4.29 (m, 4H), 4.11-3.94 (m, 2H), 3.92- 3.87 (m, 3H), 3.77 (m, 4H), 3.64 (m, 7H), 3.27-3.03 (m, 5H), 3.00-2.92 (m, 1H), 2.84 (m, 1H), 1.83 (s, 2H), 1.36 (s, 6H), 0.91 (t, J = 6.5 Hz, 3H).
1H NMR (400 MHz, DMSO-d6) δ 8.02-7.98 (m, 1H), 7.91 (s, 1H), 7.45-7.41 (m, 2H), 4.98- 4.87 (m, 2H), 4.47-4.46 (m, 4H), 4.42-4.13 (m, 4H), 3.92 (s, 2H), 3.80 (s, 2H), 3.72 (s, 3H), 3.62-3.56 (m, 2H), 3.59- 3.32 (m, 12H), 2.67-2.56 (m, 4H), 2.40-2.33 (m, 4H), 1.40 (bs, 2H), 1.23- 1.15 (m, 6H), 0.83 (t, J = 7.2 Hz, 3H).
1H NMR (400 MHz, DMSO-d6) δ 7.94 (s, 1H), 7.45-7.43 (m, 2H), 5.00-4.95 (m, 2H), 4.87- 4.14 (m, 18H), 3.93-3.47 (m, 23H), 3.39-3.16 (m, 8H), 2.61- 2.58 (m, 2H).
1H NMR (400 MHz, DMSO) δ 7.52 (dd, J = 9.5, 4.0 Hz, 2H), 5.03-4.87 (m, 2H), 4.35- 4.08 (m, 5H), 3.95-3.88 (m, 4H), 3.67 (s, 2H), 3.56 (t, J = 4.6 Hz, 4H), 2.89 (s, 3H), 2.82- 2.58 (m, 4H), 2.44 (t, J = 4.7 Hz, 4H).
1H NMR (400 MHz, DMSO) δ 8.08-7.95 (m, 1H), 7.56- 7.44 (m, 2H), 5.03-4.87 (m, 2H), 4.26-4.07 (m, 3H), 3.88 (s, 4H), 3.37 (t, J = 6.1 Hz, 13H), 2.87 (d, J = 6.7 Hz, 2H), 2.72-2.65 (m, 2H), 2.46- 2.36 (m, 7H).
1H NMR (400 MHz, DMSO) δ 7.56-7.46 (m, 2H), 4.94 (d, J = 11.9 Hz, 2H), 4.28 (t, J = 6.6 Hz, 5H), 3.90 (s, 6H), 3.56 (s, 4H), 2.86 (s, 3H), 2.72-2.66 (m, 2H), 2.38 (dd, J = 13.7, 6.5 Hz, 6H), 1.89-1.80 (m, 2H).
1H NMR (400 MHz, DMSO) δ 7.53 (q, J = 9.6 Hz, 2H), 4.95 (q, J = 18.0 Hz, 2H), 4.40- 4.05 (m, 6H), 3.92 (d, J = 12.6 Hz, 5H), 3.68 (t, J = 4.8 Hz, 5H), 3.50 (t, J = 4.6 Hz, 4H), 2.73 (s, 2H), 2.46 (d, J = 5.7 Hz, 3H), 2.38 (s, 4H).
1H NMR (400 MHz, DMSO) δ 7.51 (td, J = 10.2, 6.9 Hz, 2H), 5.00-4.88 (m, 2H), 4.24 (td, J = 7.2, 3.4 Hz, 5H), 3.99-3.87 (m, 5H), 3.68 (s, 2H), 3.54 (t, J = 4.5 Hz, 5H), 2.90 (s, 2H), 2.80-2.64 (m, 2H), 2.38- 2.26 (m, 6H), 1.68 (p, J = 7.3 Hz, 2H), 1.55-1.34 (m, 4H).
1H NMR (400 MHz, DMSO) δ 7.53 (q, J = 8.2 Hz, 2H), 4.94 (d, J = 11.9 Hz, 2H), 4.25 (dd, J = 7.7, 5.3 Hz, 5H), 3.93 (s, 5H), 3.64 (d, J = 6.5 Hz, 5H), 2.87 (d, J = 7.4 Hz, 2H), 2.78-2.64 (m, 2H), 2.45-2.13 (m, 6H), 1.68 (d, J = 8.2 Hz, 2H), 1.54 (q, J = 7.4 Hz, 2H).
1H NMR (400 MHz, DMSO) δ 8.40-8.22 (m, 1H), 7.48 (q, J = 9.2 Hz, 2H), 4.91 (d, J = 14.9 Hz, 2H), 4.26 (dd, J = 16.4, 8.0 Hz, 3H), 4.11 (s, 1H), 3.89 (s, 3H), 3.83 (s, 3H), 2.78 (d, J = 7.1 Hz, 2H), 2.70-2.56 (m, 3H), 2.45-2.28 (m, 7H).
To assess the time course of the plasma and lung exposure of compounds disclosed herein, rodents were dosed IT with an exemplary compound of the present disclosure. The plasma and lung samples were taken at different time points. The drug levels were measured by LCMS.
When dosed IT to mouse, compound 46 exhibited significantly higher plasma and lung exposure profiles compared to retagliptin (
Table 5 presents the pharmacokinetic parameters of compound 46 in mouse and rat.
LPS from E. coli I111:B4 (Sigma) was used to induce acute lung injury in mice. Body weight matched (19 gram-22 gram) female C57BL/6J mice at 9-11 weeks of age were selected to use in ALI model.
For oral delivery, DPP4 inhibitors were dissolved in PBS resulting in clear solution. Vehicle control or DPP4 inhibitors were dosed at 10 ml/kg through oral gavage once or twice a day selected based on PK profile. For intratracheally delivered DPP4 inhibitors, compounds were dissolved in PBS resulting in clear solution. Vehicle control or DPP4 inhibitors were dosed at 2 ml/kg through a 22 g flexible catheter every other day.
LPS (1.5 mg/kg for testing orally delivered DPP4 inhibitors and 1.2 mg/kg for testing intratracheally delivered DPP4 inhibitors) or PBS in sham group was intratracheally injection into mice lung at day 0. DPP4 inhibitors or vehicle control were given to mice starting from one day before LPS injection (day −1).
All animals were sacrificed at day 3.5 after LPS injection. Bronchoalveolar lavage fluid (BALF) was retrieved using standard method. 1 ml 4% formalin was used to inflate the lung, which was subsequently fixed in 4% formalin for 24 hours and preserved in 70% EtOH until histological process.
For readouts, total protein content in BALF was quantified using BCA assay; pulmonary inflammation and damage were assessed using H & E staining.
Bleomycin (Hospira) was used to induce lung fibrosis in mice. Body weight matched (24 gram˜28 gram) male C57BL/6J mice at 10-12 weeks of age were selected to use in bleomycin model.
For oral delivery, DPP4 inhibitors were dissolved in PBS resulting in clear solution. Vehicle control or DPP4 inhibitors were dosed at 10 ml/kg through oral gavage once or twice a day selected based on PK profile. For intratracheally delivered DPP4 inhibitors, compounds were dissolved in PBS resulting in clear solution. Vehicle control or DPP4 inhibitors were dosed at 2 ml/kg through a 22 g flexible catheter every four days.
0.5 U/kg Bleomycin or PBS in sham group was intratracheally injected into mice lung at day 0. DPP4 inhibitors or vehicle control were given to mice starting from one day before bleomycin injection (day −1).
All animals were sacrificed at day 20 after bleomycin injection. Bronchoalveolar lavage fluid (BALF) was retrieved using standard method. 1 ml 4% formalin was used to inflate the lung, which was subsequently fixed in 4% formalin for 24 hours and preserved in 709% EtOH until histological process.
For readouts, body weight was measured every day; total protein content in BALF was quantified using BCA assay; pulmonary fibrosis was assessed using Masson's trichrome staining.
In a mouse ALI model, Compound 46 showed a minimal efficacious dose of 0.02 mg/kg every other day via intratracheal administration. Compound 46 was also efficacious in the bleomycin induced pulmonary fibrosis model, displaying rescue of key metrics including body weight, BALF protein content, fibrotic area, and histological scoring (
Additional assessment of the efficacy of compound 46 was determined in combination with the standard of care drug Nintedanib in the rodent bleomycin model (
Single cell RNA-sequencing from the lungs of mice was performed to understand which cell types are proliferative in response to compound 46 treatment. After treatment with compound (0.5 mg/kg IT, animals sacrificed 2 and 4 days after dosing), only cycling and transitional AEC2s were induced to proliferate among other cellular populations, including other epithelial cell types but excluding actively expanding immune cells (
This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/139,956, filed on Jan. 21, 2021, which application is incorporated in its entirety as if fully set forth herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/70259 | 1/20/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63139956 | Jan 2021 | US |
