Patent Application
20230278955
The present invention is directed to novel vitamin D3 derivatives and pharmaceutical or medical use thereof for treating a disease selected from metabolic disease, liver disease, diabetes, cancer, obesity or cardiovascular disease in a subject in need thereof.
Sterol regulatory element-binding proteins (SREBPs) are one of families of transcription factors involved in lipid homeostasis. SREBPs control lipid metabolism in all tissues, by regulating expression of the genes related to biosynthesis and uptake of fatty acids, triglycerides, cholesterol, and phospholipids. Because of their central roles in lipid metabolism, SREBPs are strongly linked to metabolic syndromes. For example, high insulin levels, induced by high calorie diets or obesity, hyper-activate SREBPs, causing triglyceride accumulation and inducing fatty liver diseases. Hyperactivation of SREBPs also increases cholesterol levels and suppresses insulin receptor substrate-2, leading to hyperlipidemia, arteriosclerosis, and insulin resistance. Furthermore, activation of SREBPs is often correlated with the growth of cancers and the ability of hepatitis virus to cause fatty liver diseases (Non Patent Literature 1). The involvement of SREBP activation in multiple diseases has made these transcription factors attractive pharmaceutical targets. To date, the only known “endogenous” molecules that directly inhibit the SREBP activation pathway are sterols.
It is an object of the present invention to provide compounds useful as a SREBP inhibitor and useful for treating a disease such as metabolic disease including non-alcoholic steatohepatitis (NASH); liver disease including fatty liver; diabetes; cancer; obesity; cardiovascular disease; and the like.
The inventors have found novel vitamin D3 derivatives as a SREBP inhibitor. The vitamin D3 derivatives in the present invention are useful for treating a disease such as metabolic disease including non-alcoholic steatohepatitis (NASH); liver disease including fatty liver; diabetes; cancer; obesity; cardiovascular disease; and the like.
In one aspect, the present invention is directed to a compound of the following general formula (I):
wherein RA and RB are each independently selected from hydroxyl, NR1R2 or halogen;
R1 and R2 are each independently selected from hydrogen, C1-4 alkyl, optionally substituted C1-4 alkylcarbonyl, C1-4 alkylsulfonyl, C1-4 alkoxycarbonyl, benzyloxycarbonyl, 3 to 6-membered cycloalkyl-C1-4 alkyl, optionally substituted C6-10 arylcarbonyl, optionally substituted C6-10 arylsulfonyl, optionally substituted 5 to 6-membered saturated heterocyclyl-C1-4 alkyl, 5 to 6-membered heteroaryl or a group of the following formula:
or
R1 and R2 may optionally combine together with the nitrogen atom to which they attach to form a nitrogen-containing oxo-substituted saturated 5- to 6-membered heterocyclic ring which may be optionally fused with a C6-10 aryl ring; and
R3 is hydrogen or ═CH2; or a pharmaceutically acceptable salt thereof.
In another aspect, the present invention is also directed to a method for treating a disease selected from metabolic disease, liver disease, obesity, diabetes, cardiovascular disease, or cancer in a subject, comprising the step of administering to the subject in need thereof a therapeutically effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof or administrating vitamin D, vitamin D3 and known derivatives of vitamin D3 such as 25-OHVitD3, 1,25diOHVitD3, and 24,25-diOHVitD3.
In another aspect, the present invention is also directed to a method for inhibiting SREBPs in a subject, comprising the step of administering to the subject in need thereof a therapeutically effective amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof or administrating vitamin D, vitamin D3 and known derivatives of vitamin D3 such as 25-OHVitD3, 1,25diOHVitD3, and 24,25-diOHVitD3.
In still another aspect, the present invention is also directed to a pharmaceutical composition, comprising as the active ingredient the compound of Formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
In still another aspect, the present invention is also directed to use of the compound of Formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of metabolic disease including non-alcoholic steatohepatitis; liver disease including fatty liver; diabetes; cancer; obesity; or cardiovascular disease.
In still another aspect, the present invention is also directed to the compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of metabolic disease including non-alcoholic steatohepatitis; liver disease including fatty liver; diabetes; cancer; obesity; or cardiovascular disease.
The compound of Formula (I) or a pharmaceutically acceptable salt thereof has SREBP inhibitory activity and may be useful for treating a disease such as metabolic disease including non-alcoholic steatohepatitis (NASH), liver disease including fatty liver, diabetes, cancer, obesity, cardiovascular disease or the like.
The term “alkyl” used herein refers to a straight- or branched-chain hydrocarbon group preferably having 1 to 4 carbon atoms, and includes, for example, methyl, ethyl, normal-propyl, isopropyl, nornal-butyl, isobutyl, tert-butyl, etc.
The term “alkoxy” used herein refers to a monovalent group wherein the above mentioned alkyl group attaches to oxygen atom, and may be a straight- or branched-chain group preferably having 1 to 4 carbon atoms. The alkoxy group includes, for example, methoxy, ethoxy, normal-propoxy, isopropoxy, normal-butoxy, isobutoxy, tert-butoxy, etc.
The term “alkylcarbonyl” used herein refers to a group wherein the above mentioned alkyl group attaches to carbonyl group, and is preferably C1-4 alkylcarbonyl. The alkylcarbonyl group includes, for example, acetyl, ethylcarbonyl, normal-propylcarbonyl, isopropylcarbonyl, normal-butylcarbonyl, isobutylcarbonyl, tert-butylcarbonyl, etc.
The term “alkylsulfonyl” used herein refers to a group wherein the above mentioned alkyl group attaches to sulfonyl group, and is preferably C1-4 alkylsulfonyl. The alkylsulfonyl group includes, for example, methylsulfonyl, ethylsulfonyl, normal-propylsulfonyl, isopropylsulfonyl, normal-butylsulfonyl, isobutylsulfonyl, tert-butylsulfonyl, etc.
The term “alkoxycarbonyl” used herein refers to a group wherein the above mentioned alkoxy group attaches to carbonyl group, and is preferably C1-4 alkoxycarbonyl. The alkoxycarbonyl group includes, for example, methoxycarbonyl, ethoxycarbonyl, normal-propoxycarbonyl, isopropoxycarbonyl, normal-butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, etc.
The term “cycloalkyl” used herein refers to a saturated aliphatic monocyclic hydrocarbon ring preferably having 3 to 6 carbon atoms. The cycloalkyl group includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. A preferable cycloalkyl group is 3 to 6-membered cycloalkyl, and more preferable one is cyclopropyl.
The term “aryl” used herein refers to a monovalent group of monocyclic aromatic hydrocarbon ring or polycyclic aromatic hydrocarbon ring preferably having 6 to 10 carbon atoms. The aryl group includes, for example, phenyl, naphthyl, etc. A preferable aryl is C6-10 aryl, and more preferable one is phenyl or naphthyl.
The term “arylcarbonyl” used herein refers to a group wherein the above mentioned aryl group attaches to carbonyl group, and is preferably C6-10 arylcarbonyl. The arylcarbonyl group includes, for example, benzoyl, naphthylcarbonyl, etc. A preferable arylcarbonyl includes benzoyl, etc.
The term “arylsulfonyl” used herein refers to a group wherein the above mentioned aryl group attaches to sulfonyl group, and is preferably C6-10 arylsulfonyl. The arylsulfonyl group includes, for example, phenylsulfonyl, naphthylsulfonyl, etc. A preferable arylsulfonyl includes phenylsulfonyl, etc.
The term “heterocyclyl” or “heterocyclic” used herein refers to a monovalent group of saturated or partially unsaturated 5 to 6-membered monocyclic group having at least one heteroatom, preferably one or two heteroatom(s), independently selected from nitrogen, oxygen or sulfur and carbon atoms. The heterocyclyl group includes, for example, pyrrolidinyl, oxazolinyl, pyrazolidinyl, piperidyl, piperazinyl, morpholinyl, etc. A preferable heterocyclyl group includes pyrrolidinyl, piperidyl, morpholinyl, etc.
The term “heteroaryl” used herein refers to a monovalent group of aromatic cyclic group having at least one heteroatom independently selected from nitrogen, oxygen or sulfur and carbon atoms, and is preferably 5 to 6-membered heteroaryl group. The heteroaryl group includes, for example, pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, oxazolyl, thiazolyl, imidazolyl, pyridyl, pyrimidinyl, etc. A preferable heteroaryl group includes thiazolyl, pyridyl, etc.
The term “nitrogen-containing oxo-substituted saturated 5 to 6-membered heterocyclic ring which may be optionally fused with a C6-10 aryl ring” used herein refers to the above mentioned heterocyclyl ring containing at least one nitrogen atom in the ring which is substituted with at least one oxo group, and includes, for example, γ-lactam, δ-lactam, phthalimidyl, etc.
The term “halogen” or “halo” used herein refers to fluorine atom, chlorine atom, bromine atom, iodine atom, etc.
The optionally substituted alkylcarbonyl refers to the above mentioned alkylcarbonyl which may be optionally substituted with the same or different at least one halogen. The substituent in the optionally substituted alkylcarbonyl includes the same or different 1 to 9, preferably 1 to 3, halogen atom(s) and in particular three fluorine atoms.
The optionally substituted arylcarbonyl refers to the above mentioned arylcarbonyl which may be optionally substituted with the same or different at least one group selected from the group consisting of halogen, halo-C1-4 alkyl, —S-halo-C1-4 alkyl, C1-4 alkoxy, halo-C1-4 alkoxy, nitro, cyano, C1-4 alkoxycarbonyl, and C6-10 aryl. Preferable substituents in the optionally substituted arylcarbonyl include the same or different 1 to 4 group(s) selected from the group consisting of chloro, fluoro, bromo, methyl, methoxy, trifluoromethyl, methoxycarbonyl, trifluoromethoxy, nitro, cyano, —S—CF1, phenyl, etc.
The optionally substituted arylsulfonyl refers to the above mentioned arylsulfonyl which may be optionally substituted with the same or different at least one group selected from the group consisting of C1-4 alkyl and nitro. A preferable substituent in the optionally substituted arylsulfonyl includes methyl, nitro, etc.
The optionally substituted 5 to 6-membered saturated heterocyclyl-alkyl refers to the above mentioned alkyl substituted with the above mentioned heterocyclyl which may be optionally substituted with the same or different at least one group selected from the group consisting of halogen and hydroxyl. A preferable substituent in the optionally substituted 5 to 6-membered saturated heterocyclyl-alkyl includes fluoro, hydroxy, etc.
In one aspect, the present invention is directed to the following Items or embodiments.
Item 1: A compound of the following general formula (I):
wherein RA and RB are each independently selected from hydroxyl, NR1R2 or halogen;
R1 and R2 are each independently selected from hydrogen, C1-4 alkyl, optionally substituted C1-4 alkylcarbonyl, C1-4 alkylsulfonyl, C1-4 alkoxycarbonyl, benzyloxycarbonyl, 3 to 6-membered cycloalkyl-C1-4 alkyl, optionally substituted C6-10 arylcarbonyl, optionally substituted C6-10 arylsulfonyl, optionally substituted 5 to 6-membered saturated heterocyclyl-C1-4 allyl, 5 to 6-membered heteroaryl or a group of the following formula:
or
R1 and R2 may optionally combine together with the nitrogen atom to which they attach to form a nitrogen-containing oxo-substituted saturated 5 to 6-membered heterocyclic ring which may be optionally fused with a C6-10 aryl ring; and
R3 is hydrogen or ═CH2; or a pharmaceutically acceptable salt thereof.
Item 2: The compound of Item 1, wherein R1 and R2 are each independently selected from hydrogen, C1-4 alkyl, C1-4 alkylcarbonyl which may be optionally substituted with the same or different at least one halogen, C1-4 alkylsulfonyl, C1-4 alkoxycarbonyl, benzyloxycarbonyl, 3 to 6-membered cycloalkyl-C1-4 alkyl, C6-10 arylcarbonyl which may be optionally substituted with the same or different at least one group selected from the group consisting of halogen, halo-C1-4 alkyl, —S-halo-C1-4 alkyl, C1-4 alkoxy, halo-C1_4 alkoxy, nitro, cyano, C1-4 alkoxycarbonyl and C6-10 aryl, C6-10 arylsulfonyl which may be optionally substituted with the same or different at least one group selected from the group consisting of C1-4 alkyl, nitro and di-(C1-4 alkyl)amino, 5 to 6-membered saturated heterocyclyl-C1-4 alkyl which may be optionally substituted with the same or different at least one group selected from the group consisting of halogen and hydroxyl, 5 to 6-membered heteroaryl, or a group of the following formula:
or
R1 and R2 may optionally combine together with the nitrogen atom to which they attach to form a nitrogen-containing oxo-substituted saturated 5 to 6-membered heterocyclic ring which may be optionally fused with a C6-10 aryl ring;
provided that R1 and R2 are not concurrently hydrogen; or a pharmaceutically acceptable salt thereof.
Item 3: The compound of either Item 1 or 2, wherein R3 is ═CH2, or a pharmaceutically acceptable salt thereof.
Item 4: The compound of either Item 1 or 2, wherein R3 is hydrogen, or a pharmaceutically acceptable salt thereof.
Item 5: The compound of any one of Items 1 to 4, having any one of the following formulae:
wherein X is halogen and the other symbols have the same meanings as defined in Item 1.
Item 6: The compound of any one of Items 1 to 5, wherein R2 is hydrogen, or a pharmaceutically acceptable salt thereof.
Item 7: The compound of any one of Items 1 to 5, wherein X is fluoro;
R1 is selected from tert-butoxycarbonyl, benzyloxycarbonyl, acetyl, p-methylphenylsulfonyl, o-nitrophenylsulfonyl, p-trifluoromethylbenzoyl, p-bromobenzoyl, ethylcarbonyl, propylcarbonyl, p-methoxybenzoyl, p-fluorobenzoyl, p-[(trifluoromethyl)thio]benzoyl, 2,3,4,5-tetrafluorobenzoyl, 2,4,5-trifluorobenzoyl, 3,4-dimethoxybenzoyl, 2,3,4-trifluorobenzoyl, 3,4-difluorobenzoyl, 2,4-difluorobenzoyl, 3-chloro-4-fluorobenzoyl, 2-chloro-4-fluorobenzoyl, p-nitrobenzoyl, 2-trifluoromethyl-4-fluorobenzoyl, 3-trifluoromethyl-4-fluorobenzoyl, p-trifluoromethoxybenzoyl, p-cyanobenzoyl, p-methoxycarbonylbenzoyl, p-phenylbenzoyl, 2-morpholinylethyl, 2-(4-fluoropiperidinyl)ethyl, 2-(4-hydroxypiperidinyl)ethyl, 2-pyridyl, 2-thiazolyl, cyclopropylmethyl, ethyl, butyl, methylsulfonyl, trifluoromethylcarbonyl, 5-dimethylamino-1-naphthylsulfonyl, or a group of the following structure:
and
R2 is hydrogen; or
R1 and R2 may combine together with the nitrogen atom to which they attach to form γ-lactam, δ-lactam or phthalimidyl.
Item 8: The compound of Item 5, wherein X is fluoro;
R1 is selected from acetyl, trifluoromethylcarbonyl, tert-butoxycarbonyl, benzyloxy-carbonyl, methylsulfonyl, 5-dimethylamino-1-naphthylsulfonyl, p-methylphenylsulfonyl, o-nitrophenylsulfonyl, p-trifluoromethylbenzoyl, p-bromobenzoyl, p-[(trifluoromethyl)thio]benzoyl, or a group of the following structure:
and
R2 is hydrogen; or
R1 and R2 may combine together with the nitrogen atom to which they attach to form phthalimidyl.
Item 9: The compound of Item 1, having a structure:
Item 10: A method for treating a disease selected from metabolic disease, liver disease, obesity, diabetes, cardiovascular disease, or cancer in a subject, comprising the step of administering to the subject in need thereof a therapeutically effective amount of the compound of any one of Items 1 to 9 or a pharmaceutically acceptable salt thereof or administrating vitamin D, vitamin D3 and known derivatives of vitamin D3, such as 25-OHVitD3, 1,25diOHVitD3, and 24,25-diOHVitD3.
Item 11: The method of Item 10, wherein the disease is obesity through the induction of weight loss, non-alcoholic steatohepatitis (NASH), fatty liver, or cancer.
Item 12: A method for inhibiting SREBPs in a subject, comprising the step of administering to the subject in need thereof a therapeutically effective amount of the compound of any one of Items 1 to 9 or a pharmaceutically acceptable salt thereof or administrating vitamin D, vitamin D3 and known derivatives of vitamin D3, such as 25-OHVitD3, 1,25diOHVitD3, and 24,25-diOHVitD3.
Item 13: A pharmaceutical composition, comprising as the active ingredient the compound of any one of Items 1 to 9 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
Item 14: Use of the compound of any one of Items 1 to 9 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of metabolic disease including non-alcoholic steatohepatitis, liver disease including fatty liver, diabetes, cancer, obesity or cardiovascular disease.
Item 15: Compound of any one of Items 1 to 9 or a pharmaceutically acceptable salt thereof for use in the treatment of metabolic disease including non-alcoholic steatohepatitis, liver disease including fatty liver, diabetes, cancer, obesity or cardiovascular disease.
In one embodiment, one of RA and RB is hydroxyl and the other is NR1R2 or halogen.
In one embodiment, R1 and R2 each independently include hydrogen, tert-butoxycarbonyl, benzyloxycarbonyl, acetyl, p-methylphenylsulfonyl, o-nitrophenylsulfonyl, p-trifluoromethylbenzoyl, p-bromobenzoyl, ethylcarbonyl, propylcarbonyl, p-methoxybenzoyl, p-fluorobenzoyl, p-[(trifluoromethyl)thio]benzoyl, 2,3,4,5-tetrafluorobenzoyl, 2,4,5-trifluorobenzoyl, 3,4-dimethoxybenzoyl, 2,3,4-trifluorobenzoyl, 3,4-difluorobenzoyl, 2,4-difluorobenzoyl, 3-chloro-4-fluorobenzoyl, 2-chloro-4-fluorobenzoyl, p-nitrobenzoyl, 2-trifluoromethyl-4-fluorobenzoyl, 3-trifluoromethyl-4-fluorobenzoyl, p-trifluoromethoxybenzoyl, p-cyanobenzoyl, p-methoxycarbonylbenzoyl, p-phenylbenzoyl, 2-morpholinylethyl, 2-(4-fluoropiperidinyl)ethyl, 2-(4-hydroxypiperidinyl)ethyl, 2-pyridyl, 2-thiazolyl, cyclopropylmethyl, ethyl, butyl, methylsulfonyl, trifluoromethylcarbonyl, 5-dimethylamino-1-naphthylsulfonyl, and a group of the following probe structure:
In another embodiment, R1 and R2 may combine together with the nitrogen atom to which they attach to form for example γ-lactam, δ-lactam or phthalimidyl.
In still another embodiment, R3 is ═CH2.
In still another embodiment, R3 is hydrogen.
The pharmaceutically acceptable salt used herein refers to any salts which are known in the art and do not have excess toxicity. In particular, the pharmaceutically acceptable salt may include a salt with an inorganic acid, an organic acid, an inorganic base, or an organic base. Such an inorganic acid includes hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, and phosphoric acid. Such an organic acid includes acetic acid, trifluoroacetic acid, benzoic acid, p-toluenesulfonic acid, citric acid, oxalic acid, maleic acid, fumaric acid, lactic acid, malic acid, succinic acid, and tartaric acid. Such an inorganic base includes lithium, sodium potassium, magnesium, calcium, aluminum, and zinc. Such an organic base includes arginine and lysine. A preferable pharmaceutically acceptable salt is a salt with an inorganic acid, and in particular hydrochloride.
The pharmaceutically acceptable carrier used herein includes various conventional organic or inorganic carrier substances, for example, substances in solid preparations such as excipients, disintegrators, binders, glidants and lubricants, commonly used in the art, and substances in liquid preparations such as solvents, solubilizing agents, suspending agents, isotonizing agents, buffers and soothing agents, commonly used in the art. Additives commonly used in the art such as preservatives, antioxidants, colorants, and edulcorants may be added to a pharmaceutical composition in the present invention, if needed.
The compound of Formula (I) may be orally or parenterally administered in a therapeutically effective amount to mammals such as mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, cattle, horses, sheep, monkeys, and human. While the therapeutically effective amount of the compound of Formula (I) may vary depending on subjects, diseases, dosage forms, routes of administration, and the like, the therapeutically effective amount of the compound of Formula (I) generally ranges for example from about 0.01 mg through about 0.1 mg to about 1 g through about 10 g per day, which may be administered once or several times in a divided amount.
For the avoidance of doubt, it is confirmed that in the general description above, the proposal of general preferences and options in respect of different features of the compounds, methods, use, and compositions constitutes in the usual way the proposal of general combinations of those general preferences and options for the different features, insofar as they are combinable and compatible and are put forward in the same context.
A method for preparing the compound of Formula (I) or a pharmaceutically acceptable salt thereof is illustrated as below, but is not limited thereto. For example, the schemes as below show illustrative preparation methods for exemplary compounds in the present invention. Compounds obtained in each step may be isolated or purified by known methods including distillation, recrystallization, column chromatography, etc., if needed, and may be also used in the next step without isolation or purification.
The following abbreviations may be used herein for example:
Ac: acetyl
Bz: benzoyl
Ts: toluenesulfonyl
TMS: trimethylsilyl
TES: triethylsilyl
Tf: trifluoromethylsulfonyl
Boc: tert-butoxycarbonyl
Ns: o-nitrobenzenesulfonyl
Cbz: benzyloxycarbonyl
TBS: tert-butyldimethylsilyl
HMDS: bis(trimethylsilyl)amide
PPTS: pyridinium p-toluenesulfonate
NPhTh: phthalimide
TBAF: tetrabutylammonium fluoride
McCl: chloromethylsulfonyl chloride
TPAP: tetrapropylammonium perruthenate
DIBAL: diisobutyl aluminum hydride
TFAA: trifluoroacetic anhydride
DMAP: dimethylaminopyridine
HOBt: 1-hydroxybenzotriazole
TFA: trifluoroacetic acid
NHS: N-hydroxysuccinimide
DCC: N,N′-dicyclohexylcarbodiimide
EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
DMT-MM: 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
DIPEA: N,N-diisopropylethylamine
TEA: triethylamine
THF: tetrahydrofuran
DMSO: dimethylsulfoxide
DMF: dimethylformamide
NMM: N-methylmorpholine
NMO: N-methylmorpholine N-oxide
DIAD: diisopropyl azodicarboxylate
DAST: N,N-diethylaminosulfur trifluoride
Preparation Method Via Coupling Reaction
The compound of Formula (I) wherein R3 is ═CH2 may be prepared according to the following procedure:
In the scheme, X″ is halogen, and the other symbols have the same meanings as defined in Item 1.
Step 1a
A compound of Formula [a1] may be coupled with a compound of Formula [a2] in the presence of a palladium catalyst such as tetrakis(triphenylphosphine)palladium (0) (i.e., Pd(PPh3)4) and a base such as triethylamine in a solvent such as toluene to give a compound of Formula [a3]. The reaction temperature may range from room temperature to about 100° C., preferably about 90° C.
Compound [a1] and Compound [a2] may be prepared according to any one of the methods of preparing intermediate compounds below.
Step 2a
A protecting group such as tert-butyldimethylsilyl and triethylsilyl groups in a compound of Formula [a3] may be deprotected by treatment with hydrogen fluoride with a base such as 3HF.Et3N and HF.pyridine in a solvent such as tetrahydrofuran to give a compound of Formula [a4]. The reaction temperature may be any temperature that the reaction can proceed, preferably room temperature.
Step 3a
A compound of Formula [a3] wherein R1 and R2 are not concurrently hydrogen (e.g. R1 is arylsulfonyl and R2 is hydrogen) may be optionally subjected to Mitsunobu reaction using an organic phosphine compound such as triphenylphosphine and an azocarboxylic acid ester such as diisopropyl azodicarboxylate in a solvent such as tetrahydrofuran. The resulting compound may be sequentially treated or a compound of Formula [a3] may be treated with a thiol such as 1-dodecanethiol in the presence of a base such as sodium hydride in a solvent such as ether including diethylether to give a compound of Formula [a5]. The reaction temperature may range from 0° C. to room temperature, preferably room temperature or a gradually changed temperature starting from 0° C. and raising to room temperature.
Step 4a
A compound of Formula [a5] may be treated with R1X′ wherein X′ is halogen or hydroxyl in the presence of a base such as triethylamine in a solvent such as dichloromethane to give a compound of Formula [a3]. The reaction temperature may be any temperature that the reaction can proceed, preferably 0° C. The resulting Compound [a3] may be then subjected to the deprotection according to Step 2a to give a compound of Formula [a4].
Preparation Method Via Julia Coupling Reaction
The compound of Formula (I) wherein R3 is hydrogen may be prepared according to the following procedure:
In the scheme, RB1 is phthalimidyl or benzyloxy, RB2 is amino or hydroxyl, and the other symbols have the same meanings as defined in Item 1.
Step 1b
A compound of Formula [b1] may be treated with a compound of Formula [b2] in the presence of a base such as lithium bis(trimethylsilyl)amide (i.e., LiHMDS) in a solvent such as tetrahydrofuran to give a compound of Formula [b3]. The reaction temperature may range from −78° C. to room temperature, preferably a gradually changed temperature starting from −78° C. and warming to room temperature.
Compound [b1] and Compound [b2] may be prepared according to any one of the methods of preparing intermediate compounds below.
Step 2b
A compound of Formula [b3] may be treated with a base such as hydrazine hydrate and potassium carbonate in a solvent such as methanol and ethanol to give a compound of Formula [b4]. The reaction temperature may range from room temperature to about 60° C.
Step 3b
A compound of Formula [b4] may be treated with R1X′ wherein X′ is halogen or hydroxyl in the presence of a base such as triethylamine in a solvent such as dichloromethane, followed by the deprotection according to Step 2a to give a compound of Formula [b5]. The temperature of the reaction with R1X′ may be any temperature that the reaction can proceed, preferably 0° C.
Step 4b
A compound of Formula [b4] may be alternatively treated with a fluorination agent such as N,N-diethylaminosulfur trifluoride (i.e., DAST) in a solvent such as dichloromethane, followed by the deprotection according to Step 2a to give a compound of Formula [b6]. The temperature of the fluorination reaction may be any temperature that the reaction can proceed, preferably −78° C.
Preparation Methods for Intermediate Compounds 14a and 14b
In the scheme, R1 has the same meaning as defined in Item 1 and X′ is halogen or hydroxyl.
Preparation Methods for Intermediate Compound 16
In the scheme, Compound 57 may be prepared from Compound 56 according to a common procedure in the art such as the method described in Antonio Mourino et al. Chem. Eur. J. 2010, 16, 1432-1435.
In particular, methods of preparing the compounds of Formula (I) wherein R3 is ═CH2 in the present invention are illustrated in the following schemes using the above prepared intermediate compounds or derivatives thereof which may be prepared in a similar way to the above schemes.
Preparation Methods for Compounds 18a, 18b, 19a and 19b
Derivatives of Compounds 18a and 18b may be also prepared in a similar procedure to the above.
In the scheme, R1 has the same meaning as defined in Item 1.
Preparation methods for Compounds 28, 32, 36, and 39
In the scheme, R1 has the same meaning as defined in Item 1 and X′ is halogen or hydroxyl.
Alternatively, the compound of Formula (I) wherein R1 is a group of the following formula:
in the present invention may be prepared according to the following scheme.
Preparation Methods for Compound 106
In addition, methods of preparing the compounds of Formula (I) wherein R3 is hydrogen in the present invention are illustrated in the following schemes.
Preparation Methods for Intermediate Compound 66
In the scheme, Compound 61 may be prepared from Compound 60 according to a common procedure in the art such as the method described in John H. White et al. Proc. Natl. Acad. Soc. 2008, 105, 8250-8255.
Preparation Methods for Intermediate Compound 67
Preparation Methods for Intermediate Compounds 81 and 93
Preparation Methods for Compounds 72 to 77 and Derivatives Thereof
In the scheme, R1 has the same meaning as defined in Item 1 and X′ is halogen or hydroxyl.
Preparation Methods for Compounds 82 to 84 and Derivatives Thereof
In the scheme, R1 has the same meaning as defined in Item 1 and X′ is halogen or hydroxyl.
Preparation Methods for Compound 98
Unless otherwise stated, preparations were performed under an argon atmosphere using freshly dried solvents. All preparations were monitored by thin-layer chromatography using Merck silica gel 60 F254 pre-coated plates (0.25 mm) and were visualized by UV and p-anisaldehyde staining. Flash column chromatography was performed under pressurization using silica gel (particle size 40-100 μm) purchased from Cica or NH silica gel (NH-DM1020) purchased from FUJI SILYSIA CHEMICAL LTD. 1H NMR spectra were recorded on JNM-ECX 400 or JNM-AL 300. The spectra are referenced internally according to residual solvent signals of CDCl3 (1H NMR; δ=7.26 ppm) or CD3OD (1H NMR; δ=3.34 ppm). Data for 1H NMR spectra are reported as follows: chemical shift (δ ppm) (multiplicity, coupling constant (Hz), integration). Multiplicity and qualifier abbreviations are as follows: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad. Mass spectra were recorded on JEOL JMS-T100X spectrometer with ESI-MS mode using methanol as solvent or JEOL JMS-MS700V spectrometer with FAB-MS mode using DMSO as solvent.
To a solution of BH3—SMe2 (100 mL, 1.05 mol) in THF (200 mL) was added a solution of L-malic acid (50 g, 0.37 mol; purchased from Tokyo Chemical Industry Co., Ltd.) in THF (400 mL) dropwise at 0° C., then the reaction mixture was warmed to room temperature. After stirring for 16 h, the reaction mixture was cooled to 0° C. before adding MeOH (400 mL). After additive 30 min at room temperature, the mixture was evaporated. Moreover, the residue was evaporated with MeOH (200 mL) six times and acetone (200 mL) two times.
To a crude triol 1 in acetone (600 mL) was added p-TsOH.H2O (4.0 g). After stirring for 21 h, Et3N (2 mL) was added. After additive 30 min, the mixture was evaporated. The residue was chromatographed on silica gel (hexane/ethyl acetate; 3:1 to 1:1) to give acetal 2 (31.0 g, 57%, 2 steps) as a colorless oil.
A solution of oxalyl chloride (2.1 mL, 24.1 mmol) in CH2Cl2 (110 mL) was cooled to −78° C. and added dry DMSO (4.3 mL, 60.24 mmol) dropwise. After stirring for 30 min at same temperature, a solution of acetal 2 (1.8 g, 12.1 mmol) in CH2Cl2 (10 mL) was added dropwise, and the reaction mixture was stirred for 1 h. To a suspension was added Et3N (16.8 mL, 120 mmol) dropwise, then the reaction mixture was warmed to room temperature. After additive 1 h, ethyl(triphenylphosphoranylidene)acetate (8.4 g, 24.1 mmol) was added and stirred for 1 day. The reaction was quenched by H2O, and the aqueous layer was extracted with CH2Cl2 three times. The combined mixture was dried over MgSO4 and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 15:1 to 5:1) to give ethyl ester 3 (2.38 g, 91%) as yellow oil.
A solution of ethyl ester 3 (2.55 g, 11.9 mmol) in CH2Cl2 (120 mL) was added diisobutyl aluminum hydride in hexane (27.4 mL, 27.4 mmol, 1.0 M). After stirring for 2 h, the reaction was quenched by careful addition of MeOH (19 mL). The mixture was added sat. Rochelle salt aq. (33 mL) and stirred for 30 min. The organic layer was washed with sat. Rochelle salt aq. three times, and the aqueous layer was extracted with CHCl3 three times. The combined mixture was dried over MgSO4 and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 6:1 to 2:1) to give alcohol 4 (1.80 g, 88%) as a colorless oil.
To a solution of allyl alcohol 4 (636 mg, 3.69 mmol) in pyridine (3.7 mL) was added benzoyl chloride (0.64 mL, 5.54 mmol) dropwise at 0° C., then the reaction mixture was warmed to room temperature. After stirring for 2 h, the reaction mixture was added H2O. The aqueous layer was extracted with ethyl acetate three times and organic layer was washed with brine. The combined mixture was dried over MgSO4 and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 30:1) to give benzoate ester (991 mg, 97%) as colorless oil. 1H NMR (300 MHz, CDCl3) δ 8.05-7.40 (m, 5H), 5.90-5.74 (m, 2H), 4.78 (d, J=4.80 Hz, 1H), 4.21-4.12 (m, 1H), 4.03 (dd, J=7.89, 6.18 Hz, 1H), 1.98 (dd, J=7.89, 7.20 Hz, 1H), 2.48-2.28 (m, 2H), 1.41 (s, 3H), 1.35 (s, 3H). 13C NMR (300 MHz, CDCl3) δ 166.26, 132.89,z 130.64, 129.54, 128.28, 126.98, 109.00, 74.95, 68.75, 65.18, 36.45, 26.81, 25.56.
HR-MS ESI Calcd for C16H20Na1O4[M+Na]+: 299.12593, Found: 299.12835.
[α]25D=+5.40 (c=2.1 in CHCl3)
To a benzoate ester 5 (991 mg, 3.59 mmol) was added acetic acid (15.4 mL) and H2O (2.6 mL). After stirring at room temperature for 12 h, the reaction mixture was evaporated. The residue was chromatographed on silica gel (hexane/ethyl acetate; 1:1) to give diol 6 (825 mg, 97%) as a colorless oil.
1H NMR (300 MHz, CDCl3) δ 8.04-7.39 (m, 5H), 5.91-5.72 (m, 2H), 4.77 (d, J=5.49 Hz, 1H), 4.17 (dddd, J=6.53, 6.53, 6.53, 3.09 Hz, 1H), 3.64 (dd, J=11.31, 3.09 Hz, 1H), 3.46 (dd, J=11.31, 7.23 Hz, 1H), 2.25 (dd, J=6.54 Hz, 2H).
13C NMR (300 MHz, CDCl3) (166.49, 133.00, 131.11, 130.03, 129.55, 128.32, 127.18, 71.28, 66.13, 65.27, 36.24.
HR-MS ESI Calcd for C13H16Na1O4[M+Na]+: 259.09463, Found: 259.09503.
[α]25D=−6.18 (c=2.0 in CHCl3)
A solution of diol 6 (3.81 g, 16.1 mmol) in pyridine (32 mL) was added p-toluenesulfonyl chloride (3.69 g, 19.3 mmol) at 0° C., then the reaction mixture was slowly warmed to room temperature. After stirring for 4 h, the reaction was quenched by H2O. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 5:1 to 1:1) to give tosylate 7 (4.93 g, 78%) as a colorless oil.
1H NMR (300 MHz, CDCl3) δ 8.04-7.31 (m, 9H), 5.85-5.69 (m, 2H), 4.74 (d, J=4.80 Hz, 2H), 4.04 (dd, J=12.69, 6.51 Hz, 1H), 3.96-3.91 (m, 2H), 2.42 (s, 3H), 2.42-2.26 (m, 2H).
13C NMR (300 MHz, CDCl3) δ 166.30, 145.09, 132.98, 132.43, 130.01, 129.91, 129.70, 129.55, 128.32, 128.02, 127.90, 72.92, 68.60, 64.97, 35.82, 21.60.
HR-MS ESI Calcd for C20H22Na1O6S1[M+Na]+: 413.10348, Found: 413.10279.
[α]25D=+5.36 (c=2.0 in CHCl3)
A solution of tosylate 7 (4.93 g, 12.6 mmol) in THF (120 mL) was added NaH (1.01 g, 25.2 mmol, 60%) at 0° C., then the reaction mixture was warmed to room temperature. After stirring for 8 h, the reaction was quenched by sat. aq. NH4Cl. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 1:0 to 6:1) to give epoxide 8 (2.55 g, 93%) as colorless oil.
1H NMR (300 MHz, CDCl3) δ 8.06-7.40 (m, 5H), 5.92-5.77 (m, 2H), 4.79 (d, J=4.80 Hz, 2H), 3.03-2.98 (m, 1H), 2.76 (dd, J=4.83 Hz, 1H), 2.51 (dd, J=4.83, 2.76 Hz, 1H), 2.36-2.33 (m, 2H).
13C NMR (300 MHz, CDCl3) δ 166.24, 132.89, 130.10, 129.82, 129.54, 128.27, 126.97, 65.09, 50.99, 46.51, 35.00.
HR-MS ESI Calcd for C13H14Na1O3 [M+Na]+: 241.08406, Found: 241.08341.
[α]25D=−2.69 (c=2.1 in CHCl3)
A solution of trimethylsilylacetylene (3.60 mL, 25.7 mmol) in THF (18 mL) was added n-BuLi in hexane (8.1 mL, 21.05 mmol, 2.6 M) dropwise at −78° C. and stirred for 30 min. A solution of epoxide 8 (2.55 g, 11.7 mmol) in THF (5 mL) and BF3—OEt2 (1.8 mL, 14 mmol) was added dropwise at the same temperature, then the reaction mixture was warmed to room temperature over 2 h. The reaction was quenched with sat. NH4Cl aq., the aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 30:1 to 10:1) to give alcohol 9 (3.61 g, 98%) as a colorless oil.
1H NMR (300 MHz, CDCl3) δ 8.05-7.39 (m, 5H), 5.93-5.74 (m, 2H), 4.78 (d, J=5.85 Hz, 2H), 3.82-3.78 (m, 1H), 2.51-2.26 (m, 4H), 0.14 (s, 9H).
13C NMR (300 MHz, CDCl3) δ 166.28, 132.87, 131.08, 130.06, 129.52, 128.25, 127.37, 102.74, 87.67, 69.04, 65.19, 38.93, 28.12, −0.03.
HR-MS ESI Calcd for C18H24Na1O3Si1 [M+Na]+: 339.13924, Found: 339.14268.
[α]25D=−2.89 (c=3.5 in CHCl3)
Alcohol 9 (3.61 g) was added imidazole (4.67 g, 68.48 mmol) and tert-butyldimethylsilyl chloride (6.88 g, 45.65 mmol) at room temperature, then DMF was added until the reagents were dissolved. After stirring for 4 h, the reaction mixture was diluted with diethyl ether and quenched with sat. NaHCO3 aq. The aqueous layer was extracted with diethyl ether three times. The combined organic extracts were washed with H2O and brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 100:1 to 20:1) to give silyl ether 10 (4.52 g, 92%) as a colorless oil.
1H NMR (300 MHz, CDCl3) δ 8.07-7.41 (m, 5H), 5.92-5.70 (m, 2H), 4.78 (d, J=5.85 Hz, 2H), 3.91-3.83 (m, 1H), 2.49-2.24 (m, 4H), 0.88 (s, 9H), 0.15 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H).
13C NMR (300 MHz, CDCl3) δ 166.46, 132.98, 131.88, 130.38, 129.71, 128.41, 126.90, 104.36, 86.51, 70.78, 65.53, 39.91, 28.60, 25.90, 18.16, 0.17, −4.36, −4.51.
HR-MS ESI Calcd for C18H24Na1O3Si1 [M+Na]+: 453.22572, Found: 453.22357.
[α]25D=−5.34 (c=2.4 in CHCl3)
A solution of allyl alcohol 10 (8.28 g, 19.22 mmol) in MeOH (64 mL) was added potassium carbonate (7.97 g, 57.67 mmol) at 0° C., then the reaction mixture was cooled to room temperature. After stirring for 18 h, the reaction was quenched by H2O. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 10:1 to 5:1) to give allyl alcohol 11 (4.74 g, 97%) as colorless oil.
1H NMR (300 MHz, CDCl3) δ 5.72-5.69 (m, 2H), 4.1 (d, J=4.1 Hz, 2H), 3.89-3.81 (m, 1H), 2.43-2.24 (m, 2H), 2.31 (dd, J=6.18, 2.73 Hz, 2H), 1.98 (t, J=2.76 Hz, 1H), 0.88 (s, 9H), 0.07 (s, 3H), 0.05 (s, 3H).
A solution of allyl alcohol 11 (1.46 g, 5.73 mmol) in CH2Cl2 (57 mL) was added trichloroacetyl isocyanate (1.4 mL, 11.46 mmol) at 0° C. The reaction was monitored by thin layer chromatography on silica gel plates (eluent: 4:1 ethyl acetate to hexane). After 15 min, the starting material spot completely converted to a new spot, then the reaction mixture was concentrated in vacuo. A solution of the residue in MeOH (36 mL) was added H2O (8.2 mL) and potassium carbonate (3.2 g, 22.93 mmol) at 0° C., then the reaction mixture was warmed to room temperature. After stirring for 2 h, the reaction mixture was added H2O, the aqueous layer was extracted with CH2Cl2 three times, dried over MgSO4, and concentrated in vacuo. The residue was filtered through a pad of silica gel to give carbamate 12.
To a solution of carbamate 12 (5.73 mmol) and Et3N (3.2 mL, 22.93 mmol) in CH2Cl2 (230 mL) was added trifluoroacetic anhydride (1.2 mL, 8.60 mmol) dropwise at 0° C. After stirring for 1 h, the organic layer was washed with H2O and brine. The combined organic extracts were dried over Na2SO4, and concentrated in vacuo to crude isocyanate 13.
A suspension of t-BuOK (0.64 g, 5.73 mmol) and t-BuOH (1.1 mL, 11.46 mmol) in THF (30 mL) was cooled to 0° C. A crude isocyanate 13 (5.73 mmol) in THF (8 mL) was slowly added dropwise. The reaction was monitored by thin layer chromatography on silica gel plates (eluent: 4:1 ethyl acetate to hexane). After stirring for 25 min, the starting material spot completely converted to new spots. The reaction mixture was added acetic acid (0.33 mL, 5.73 mmol) and H2O (19 mL), then Boc2O was added until disappeared amine. After stirring for 13 h, the amine completely converted to product, then the reaction mixture was diluted with ethyl acetate, the aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 100:1 to 20:1) to give Boc amine 14 as a diastereomeric mixture wherein the ratio of 14a:14b was 2:1 determined by 1H NMR.
To a solution of diastereomeric mixture 14 (5.73 mmol) in THF (11 mL) was added tetrabutylammonium fluoride in THF (5.7 mL, 1 M). After stirring for 2.5 h, the reaction mixture was evaporated. The residue was chromatographed on silica gel (hexane/ethyl acetate; 50:1 to 1:1) to give alcohol 15a (0.72 g, 52%) and alcohol 15b (0.39 g, 28%) as a single diastereomer.
15a: 1H NMR (300 MHz, CDCl3) δ 5.90-5.78 (m, 1H), 5.24-5.12 (m, 2H), 4.76 (d, J=8.9 Hz, 1H), 4.42 (br, 1H), 3.82 (br, 1H), 2.51-2.33 (m, 2H), 2.04 (t, J=2.4 Hz, 1H), 1.79-1.60 (m, 2H), 1.45 (s, 9H).
15b: 1H NMR (300 MHz, CDCl3) δ 5.84-5.72 (m, 1H), 5.24-5.11 (m, 2H), 4.72 (br, 1H), 4.25 (br, 1H), 3.93-3.85 (m, 1H), 2.51-2.35 (m, 2H), 2.06 (t, J=2.4 Hz, 1H), 1.78-1.65 (m, 2H), 1.44 (s, 9H).
To the alcohol 15a (35 mg, 0.146 mmol) was added imidazole (60 mg, 0.875 mmol) and tert-butyldimethylsilyl chloride (88 mg, 0.583 mmol) at room temperature, then DMF was added until the reagents were dissolved. After stirring for 2 h, the reaction mixture was diluted with diethyl ether and quenched with sat. NaHCO3 aq. The aqueous layer was extracted with diethyl ether three times. The combined organic extracts were washed with H2O and brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 30:1) to give 14a (52 mg, q.y.) as colorless oil.
1H NMR (300 MHz, CDCl3) δ 5.83-5.72 (m, 1H), 5.18-5.07 (m, 2H), 4.26 (br, 1H), 4.01-3.93 (m, 1H), 2.43-2.27 (m, 2H), 2.01 (t, J=2.4 Hz, 1H), 1.79-1.72 (br, 2H), 1.42 (s, 9H), 0.90 (s, 9H), 0.10 (s, 3H), 0.09 (s, 3H).
By following the same procedure described for 14a, 14b (35 mg, q.y.) was obtained from 15b (23 mg, 0.094 mmol) as colorless oil.
1H NMR (300 MHz, CDCl3) δ 5.83-5.72 (m, 1H), 5.21-5.08 (m, 2H), 4.75 (br, 1H), 4.22-4.13 (m, 1H), 3.94-3.86 (m, 1H), 2.40-2.37 (m, 2H), 2.00 (t, J=2.8 Hz, 1H), 1.86-1.59 (m, 2H), 1.43 (s, 9H), 0.91 (d, J=3.5 Hz, 9H), 0.09 (s, 3H), 0.08 (d, J=1.7 Hz, 3H).
Acetyl chloride (1.1 mL, 15.09 mmol) was slowly added to EtOH (5.0 mL) dropwise at 0° C. After stirring for 30 min, this solution was added to alcohol 15 (0.12 g, 0.50 mmol). After additional stirring for 2 h, the reaction mixture was evaporated. The residue was washed with Et2O, affording amine as a yellow solid. To a solution of above amine (0.50 mmol) in H2O (5 mL) was added acetic acid (0.086 mL, 1.5 mmol), DMT-MM (0.60 g, 2.01 mmol), N-methylmorpholine (0.33 mL, 3.01 mmol) and stirred for 2 h. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 1:2 to 0:1) to give acetamide a and 13 as a single diastereomer. To α-acetamide (about 0.33 mmol) was added imidazole (92 mg, 1.34 mmol) and tert-butyldimethylsilyl chloride (102 mg, 0.67 mmol) at room temperature, and then DMF was added until the reagents were dissolved. After stirring for 1 h, the reaction mixture was diluted with diethyl ether and quenched with sat. aq. NaHCO3. The aqueous layer was extracted with diethyl ether three times. The combined organic extracts were washed with H2O and brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 4:1 to 2:1) to give 20a (82.0 mg, 55%, 3 steps) as a colorless oil.
By following the same procedure described for 20a, 20b (37.2 mg, 25%, 3 steps) was obtained from β-acetamide (about 0.17 mmol) as a colorless oil.
A solution of alcohol 15a (0.62 g, 2.61 mmol) in pyridine (2.5 mL) was added acetic anhydride (2.5 mL) at room temperature. After stirring for 5.5 h, the reaction mixture was evaporated. The residue was chromatographed on silica gel (hexane/ethyl acetate; 10:1 to 5:1) to give acetate ester 23 (0.72 g, 99%).
Acetyl chloride (2.8 mL, 38.58 mmol) was slowly added to EtOH (13.0 mL) dropwise at 0° C. After stirring for 30 min, this solution was added to acetate ester 23 (0.72 g, 2.57 mmol). After additional stirring for 3 h, the reaction mixture was evaporated. The residue was washed with Et2O, affording amine 24 as a yellow solid.
To a solution of crude amine 24 (2.57 mmol) in CH2Cl2 (13 mL) was added Et3N (0.9 mL, 6.17 mmol), o-nitrobenzenesulfonyl chloride (0.68 g, 3.09 mmol) at 0° C., then the reaction mixture was warmed to room temperature. After stirring for 6.5 h, the reaction was quenched by H2O. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was filtered through a pad of silica gel to give acetate ester as colorless oil. To a solution of acetate ester (2.57 mmol) in MeOH (9 mL) was potassium carbonate (0.53 g, 3.86 mmol) at 0° C., then the reaction mixture was warmed to room temperature. After stirring for 15 h, H2O was added, and the aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 4:1 to 1:1) to give nosylate 25 (0.82 g, 99%) as a colorless oil.
To the nosylate 25 (0.82 g, 2.53 mmol) was added imidazole (0.69 g, 10.14 mmol) and tert-butyldimethylsilyl chloride (0.77 g, 5.07 mmol) at room temperature, then DMF was added until the reagents were dissolved. After stirring for 3 h, the reaction mixture was diluted with diethyl ether and quenched with sat. NaHCO3 aq. The aqueous layer was extracted with diethyl ether three times. The combined organic extracts were washed with H2O and brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 15:1 to 5:1) to give 26 (1.03 g, 93%) as a colorless oil.
1H NMR (300 MHz, CDCl3) δ 8.08-7.68 (m, 4H), 5.73 (d, J=6.18 Hz, 1H), 5.55 (ddd, J=13.08, 7.56, 5.52 Hz, 1H), 5.05 (dd, J=12.72, 0.69 Hz, 1H), 4.90 (dd, J=7.56, 0.69 Hz, 1H), 4.24-4.15 (m, 1H), 4.09-4.03 (m, 1H), 2.40 (ddd, J=12.36, 3.09, 2.40 Hz, 1H), 2.32 (ddd, J=12.36, 5.85, 2.07 Hz, 1H), 2.00 (t, J=2.07 Hz, 1H), 1.89 (ddd, J=10.65, 6.87, 2.07 Hz, 1H), 1.76 (ddd, J=10.65, 5.85, 2.73 Hz, 1H), 0.92 (s, 9H), 0.17 (s, 3H), 0.13 (s, 3H).
To a solution of crude amine 24 (21.9 mg, 0.105 mmol) in CH2Cl2 (0.8 mL) was added Et3N (0.035 mL, 0.25 mmol), p-toluenesulfonyl chloride (24.2 mg, 0.126 mmol) at 0° C., then the reaction mixture was warmed to room temperature. After stirring for 14 h, the reaction was quenched by H2O. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was filtered through a pad of silica gel to give acetate ester (23.9 mg, not pure) as colorless oil. To a solution of acetate ester (0.071 mmol) in MeOH (0.7 mL) was potassium carbonate (15 mg, 0.11 mmol) at 0° C., then the reaction mixture was warmed to room temperature. After stirring for 3 h, H2O was added, and the aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 4:1 to 1:1) to give tosylate 29 (19.9 mg, 95%) as a colorless oil.
To the tosylate 29 (19.9 mg, 0.068 mmol) was added imidazole (19 mg, 0.271 mmol) and tert-butyldimethylsilyl chloride (21 mg, 0.135 mmol) at room temperature, then DMF was added until the reagents were dissolved. After stirring for 3 h, the reaction mixture was diluted with diethyl ether and quenched with sat. NaHCO3 aq. The aqueous layer was extracted with diethyl ether three times. The combined organic extracts were washed with H2O and brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 6:1 to 3:1) to give 30 (26.8 mg, 97%) as a colorless oil.
1H NMR (300 MHz, CDCl3) δ 7.73-7.27 (m, 4H), 5.70 (d, J=5.16 Hz, 1H), 5.63 (ddd, J=17.19, 10.29, 6.54 Hz, 1H), 5.12 (dd, J=17.19, 1.05 Hz, 1H), 5.01 (dd, J=10.29, 1.05 Hz, 1H), 4.04-3.90 (m, 2H), 2.42 (s, 3H), 2.25 (ddd, J=16.83, 4.80, 2.73 Hz, 1H), 2.10 (ddd, J=16.83, 8.22, 2.73 Hz, 1H), 1.95 (t, J=2.73 Hz, 1H), 1.85-1.67 (m, 2H), 0.92 (s, 9H), 0.12 (s, 3H), 0.10 (s, 3H).
To a solution of crude amine 24 (26.7 mg, 0.122 mmol) in CH2Cl2 (1.0 mL) was added Et3N (0.041 mL, 0.293 mmol), benzyl chloroformate (0.021 mL, 0.147 mmol) at 0° C., then the reaction mixture was warmed to room temperature. After stirring for 14 h, the reaction was quenched by H2O. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 4:1 to 1:1) to give NHCbz 33 (18.6 mg, 56%) as a colorless oil.
To a solution of NHCbz 33 (16.0 mg, 0.059 mmol) in CH2Cl2 (0.6 mL) was added 2,6-lutidine (0.041 mL, 0.351 mmol) and tert-butyldimethylsilyl trifluoromethanesulfonate (0.046 mL, 0.176 mmol) at room temperature. After stirring for 2 h, the reaction was quenched by sat. NaHCO3 aq. The aqueous layer was extracted with CH2Cl2 three times. The combined organic extracts were dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 10:1 to 3:1) to give 34 (21.4 mg, 94%) as a colorless oil.
Imidazole (12.0 mg, 0.0461 mmol), TBSCl (4 μL, 0.0690 mmol) were successively added to a solution of 4-hydroxy piperidine (306.7 mg, 3.032 mmol) in dichloromethane (10.0 mL) at room temperature and stirred for 1 d. The reaction mixture was washed with H2O, sat. NaHCO3 aq. and brine, dried over MgSO4, and concentrated in vacuo. The residue was evapolated with toluene for removing residual reagents to give crude product (692.3 mg, not pure). To a solution of above crude product (692.3 mg) in acetonitrile (1.5 mL) was added 2-bromoethanol (0.45 mL, 6.367 mmol) and potassium carbonate (0.67 g, 4.851 mmol) at room temperature and then the mixture was heated under reflux for 4 h. The reaction mixture was filtered through a pad of celite and the resulting filtrate was concentrated. Purification by flash chromatography on silica gel (NH silica gel, hexane/ethyl acetate; 10:0 to 1:1) to give 51 (500.9 mg, 64%).
To a solution of vitamin D2 (8.0 g, 20.2 mmol; purchased from Tokyo Chemical Industry Co., Ltd.) in dichloromethane (270 mL) was added methanol (112 mL) and the solution was cooled to −78° C. Ozone was passed through the solution at −78° C. for 3 h. After flushing nitrogen to the solution to remove the residual ozone, the resulting mixture was treated with NaBH4 (4.56 g, 120 mmol) and stirred for 30 min at −78° C., and then allowed to warm to room temperature. The reaction was quenched by the addition of 0.5 M aq. HCl, and extracted with dichloromethane. The organic phase was washed with water and dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 6:1 to 2:1) to give diol 55 (4.46 g, >99%).
1H NMR (CDCl3) δ 4.09 (d, J=2.6 Hz, 1H), 3.64 (dd, J=3.3, 10.6 Hz, 1H), 3.39 (dd, J=6.6, 10.6 Hz, 1H), 1.99 (dd, J=2.6, 13.2 Hz, 1H), 1.88-1.76 (m, 3H), 1.63-1.29 (m. 9H), 1.20-1.12 (m. 2H), 1.03 (d, J=6.6 Hz, 3H), 0.95 (s, 3H)
To a solution of diol 55 (1.00 g, 4.71 mmol) in THF (20 mL) was successively added imidazole (960 mg, 14.14 mmol), triphenylphosphine (1.20 g, 5.65 mmol), iodide (1.32 g, 5.18 mmol) at −20° C. and stirring for 15 min. The reaction mixture was warmed to room temperature. After additional stirring for 2 h, the mixture was cooled to 0° C. before adding sat. NaHCO3 aq. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with sat. Na2S2O3 aq. and H2O, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 10:1) to give iodide 56 (1.41 g, 93%).
This step was carried out according to the method described in Antonio Mourino et al. Chem. Eur. J. 2010, 16, 1432-1435. A suspension of activated zinc (8.32 g, 127.26 mmol) in pyridine (90 mL) were successively added methyl acrylate (13.3 mL, 148 mmol) and NiCl2.6H2O (3.78 g, 15.91 mmol) at room temperature. The mixture was stirred at 60° C. for 1 h and then cooled to 0° C. A solution of iodide 56 (3.42 g, 10.61 mmol) in pyridine (10 mL) was added dropwise. The reaction mixture was stirred for 2 h, and then diluted with ethyl acetate. The mixture was filtered through a pad of celite. The filtrate was washed with 1 M HCl aq. two times, and the aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with H2O and brine, dried over MgSO4, and concentrated in vacuo. The pyridine hydrochloride was removed by filtering through a plug of cotton, and the filtrate was evaporated. The residue was chromatographed on silica gel (hexane/ethyl acetate; 8:1 to 4:1) to give methyl ester 57 (2.77 g, 93%).
Activated magnesium (2.91 g, 119.6 mmol) in dry diethyl ether (46 mL) was added methyl iodide (5.7 mL, 92 mmol) dropwise at 0° C. To a solution of methyl ester 57 (5.17 g, 18.30 mmol) in dry diethyl ether (90 mL) was added above Grignard reagent (46 mL, 2 m) dropwise at −78° C. The mixture was stirred for 15 min at same temperature and then warmed to room temperature. After stirring for 2.5 h, the reaction was quenched by careful addition of sat. NH4Cl aq. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 4:1 to 2:1) to give diol 58 (4.59 g, 89%).
A solution of diol 58 (300 mg, 1.06 mmol) in dry acetonitrile (10 mL) was added molecular sieves 4 angstroms (530 mg) and N-methyl morpholine N-oxide (250 mg, 2.12 mmol) and stirred for 30 min. Tetraisopropylammonium perruthenate (15 mg, 0.043 mmol) was added, then the reaction mixture was warmed to room temperature. After stirring for 2.5 h, the mixture was filtered through a pad of silica gel, and the filtrate was evaporated to give crude ketone. A suspension of (bromomethyl)triphenylphosphonium bromide (2.32 g, 5.31 mmol) in toluene (50 mL) was sonicated for 30 min at room temperature and evaporated with toluene (50 mL) three times at 50° C. To the suspension was added dry THF (7 mL) and cooled to 0° C. Sodium bis(trimethylsilyl)amide in THF (2.8 mL, 5.20 mmol, 1.9 M) was added dropwise. After stirring for 30 min, a solution of above crude ketone (1.06 mmol) in THF (1.2 mL) was added dropwise. After additive 3 h, the reaction was quenched by the addition of a few drops of sat. NH4Cl aq. The mixture was filtered, and the filtrate was evaporated. The residue was chromatographed on silica gel (hexane/ethyl acetate; 20:1 to 10:1) to give bromo olefin 59 (239 mg, 63%, 2 steps).
A solution of bromo olefin 59 (0.51 g, 1.45 mmol) in CH2Cl2 (7.2 mL) was added 2,6-lutidine (0.34 mL, 2.89 mmol) and dropwised triethylsilyl trifluoromethanesulfonate (0.42 mL, 1.88 mmol). After stirring for 2.5 h, the reaction was quenched by sat. aq. NaHCO3. The aqueous layer was extracted with CH2Cl2 three times. The combined organic extracts were dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/dichloromethane; 10:1) to give diol 16 (0.62 g, 91%).
1H NMR (300 MHz, CDCl3) δ 5.64 (s, 1H), 2.89-2.84 (m, 1H), 2.04-1.15 (m, 18H), 1.18 (s, 6H), 0.97-0.62 (m, 12H), 0.56 (s, 3H), 0.56 (q, J=7.89 Hz, 2H).
To a solution of 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acid (0.500 g, 2.17 mmol; purchased from Tokyo Chemical Industry Co., Ltd.) and N-hydroxysuccinimide (0.250 g, 2.17 mmol) in dichloromethane (15 mL) was added N,N′-dicyclohexylcarbodiimide (0.450 g, 2.17 mmol) at 0° C. and the mixture was stirred at room temperature overnight. The precipitation was filtered off and the filtrate was concentrated under reduced pressure to give a crude product. To a solution of 5-methyl L-glutamate (0.380 g, 2.36 mmol) in acetonitrile/H2O (10:3, 13 mL) was added the crude product and triethylamine (0.660 g, 6.52 mmol) and the mixture was stirred at room temperature overnight. The mixture was evaporated and the residue was dissolved in ethyl acetate, washed with aq. HCl (1 M), water, brine, and dried (MgSO4). The solvent was evaporated to afford crude product 102 (0.890 g), which was used in next step without further purification.
To a mixture of crude 102 (0.890 g) and N-Boc-4,7,10-trioxa-1,13-tridecanediamine (0.690 g, 2.15 mmol) in dichloromethane (10 mL) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.50 g, 2.6 mmol), 4-dimethylaminopyridine (0.32 g, 2.85 mmol) and 1-hydroxybenzotriazole monohydrate (0.35 g, 2.59 mmol) at 0° C., and the mixture was stirred at room temperature overnight. The mixture was washed with sat. aq. NaHCO3, water, and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography (ethyl acetate:hexane; 2:1) to yield 103 (0.760 g, 1.13 mmol, 52% for 3 steps).
To a solution of 103 (0.760 g, 1.13 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (2 mL) at 0° C. and the mixture was stirred at room temperature for 2 h. The solvent was evaporated and the residue was dissolved in chloroform, washed with sat. aq. Na2CO3, water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo to afford a crude product (0.60 g). To a solution of the crude product (0.60 g) in N,N′-dimethylformamide (5 mL) was added NHS-Biotin (0.36 g, 1.04 mmol) and triethylamine (0.32 g, 3.12 mmol), and the mixture was stirred at room temperature overnight. The solvent was evaporated and the residue was purified by column chromatography (methanol:chloroform; 1:10) to yield 104 (0.70 g, 0.87 mmol, 77% for steps).
To a mixture of 104 (0.56 g, 0.70 mmol) in tetrahydrofuran (3 mL) and water (1 mL) was added lithium hydroxide monohydrate (59 mg, 1.4 mmol) at 0° C. and the mixture was stirred at room temperature for 2 h. The mixture was acidified with aq. HCl (1 M) and extracted with chloroform three times. The combined extracts were dried over anhydrous magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography (methanol:chloroform; 1:3) to yield 105 (0.46 g, 0.59 mmol, 84%).
This step was carried out according to the method described in John H. White et al. Proc. Natl. Acad. Soc. 2008, 105, 8250-8255. 1,2-Diol 61 (3.69 g, 9.442 mmol) was obtained in 39% yield from D-(−)-Quinic acid 60 (4.72 g, 24.562 mmol).
A solution of diol 61 (7.69 g, 19.680 mmol) in CH2Cl2 (190 mL) was added benzaldehyde dimethyl acetal and pyridinium p-toluenesulfonate at room temperature. After stirring for 7 h, the reaction was quenched by sat. NaHCO3 aq. The aqueous layer was extracted with CH2Cl2 three times. The combined organic extracts were dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/dichloromethane; 50:1) to give benzylidene acetal (12.76 g) including residual reagent (benzaldehyde dimethyl acetal). To a solution of above benzylidene acetal (12.76 g) in THF (480 mL) was added a solution of TBAF3.H2O (65 mL, 1.0 M in THF) at 0° C. After stirring for 5 h, the reaction mixture was quenched with sat. NH4Cl aq. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 50:1 to 2:1) to give alcohol 62 (5.38 g, 75%).
A solution of alcohol 62 (0.57 g, 1.556 mmol) in CH2Cl2 (5.0 mL) was added pyridine (0.50 mL, 6.222 mmol) and chloromethylsulfonyl chloride (0.28 mL, 3.111 mmol) at 0° C. After stirring for 2 h, the reaction was quenched with sat. NH4Cl aq. The aqueous layer was extracted with CH2Cl2 three times. The combined organic extracts were dried over MgSO4, and concentrated in vacuo to give crude monochlate. To a solution of above crude monochlate in DMF (7.8 mL) was added sodium azide (0.51 mg, 7.778 mmol) at room temperature, then the mixture was warmed to 60° C. and stirred for 4.5 h. The reaction mixture was quenched with H2O. The aqueous layer was extracted with ethyl ether three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 50:1) to give azide 63 (0.35 g, 58% from 62).
A solution of trimethylphosphine (1.40 mL, 1.355 mmol, 1.0 m in toluene) and distilled water (0.23 mL) was added to a solution of azide 63 (0.35 g, 0.903 mmol) in THF (9.0 mL) at room temperature. After stirring for 21 h, the mixture was concentrated at 50° C. to give crude amine. To a solution of above crude amine in THF (9.0 mL) was added N-carbethoxy phthalimide (0.50 mg, 2.258 mmol) at room temperature. After stirring for 22 h, to the mixture was added sat. Na2CO3 aq. (1 mL) and stirred vigorously. After additive 10 min, the aqueous layer was extracted with ethyl ether three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 10:1 to 6:1) to give azide 64 (0.31 g, 69% from 63).
A solution of phthalimide 64 (334.0 mg, 1.355 mmol) in ethanol (6.8 mL) was added palladium hydroxide (34 mg, 10 wt %, 20% on Carbon wetted with ca. 50% Water from TCI Co., Ltd.) at room temperature. The reaction vessel was purged and placed under hydrogen and stirred vigorously for 15 h. The reaction mixture was filterd through a pad of celite and filtrate was concentrated. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 10:1 to 1:1) to give diol 65 (177.8 mg, 53%) and starting material (92.6 mg, 34% recovery).
To a solution of 65 (155.2 mg, 0.383 mmol) in methanol (16.7 mL) was added dropwise a solution of sodium periodate (368 mg, 1.722 mmol) in distilled water (2.4 mL) at 0° C. After stirring for 17 h, the reaction mixture was diluted with water and the aqueous layer was extracted with dichloromethane three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 15:1 to 1:1) to give ketone 66 (114.7 mg, 80%) and starting material 65 (18.2 mg, 12% recovery).
1H NMR (300 MHz, CDCl3) δ 7.88-7.82 (m, 2H), 7.77-7.71 (m, 2H), 4.43 (dddd, J=13.07, 12.73, 4.47, 4.13 Hz, 1H), 3.94 (dddd, J=11.01, 10.66, 6.19, 4.47 Hz, 1H), 3.35 (dd, J=14.10, 13.07 Hz, 1H), 2.75-2.63 (m, 2H), 2.58-2.43 (m, 2H), 2.19-2.10 (m, 1H), 0.87 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H).
A solution of diol 58 (0.91 g, 3.211 mmol) in dry dichloromethane (11 mL) was added molecular sieves 4A (1.61 g) and N-methyl morpholine N-oxide (0.75 g, 6.422 mmol) and stirred for 30 min. Tetraisopropylammonium perruthenate (45 mg, 0.128 mmol) was added, then the reaction mixture was warmed to room temperature. After stirring for 2 h, the mixture was filtered through a pad of silica gel, and the filtrate was evaporated to give crude ketone. A suspension of sodium hydride (0.77 g, 19.266 mmol, 60% stabilized by oil) in THF (20 mL) was added triethyl phosphonoacetate (5.8 mL, 28.900 mmol) at 0° C. and warmed to room temperature. To the solution was added a solution of above ketone (3.211 mmol) in THF (12 mL). After stirring for 3 days, the reaction was quenched with H2O and aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 15:1 to 10:1) to give α,β-unsaturated ester 99 (1.09 g, 97%) as a colorless oil.
1H NMR (300 MHz, CDCl3) δ 5.45 (s, 1H), 4.14 (q, J=7.22 Hz, 2H), 3.87-3.82 (m, 1H), 2.13-1.20 (m, 18H), 1.28 (t, J=7.22 Hz, 3H), 1.21 (s, 6H), 0.94 (d, J=6.19 Hz, 3H), 0.57 (s, 3H).
A solution of α,β-unsaturated ester 99 (0.80 g, 2.278 mmol) in CH2Cl2 (23 mL) was added diisobutyl aluminum hydride in hexane (6.8 mL, 6.835 mmol, 1.0 M). After stirring for 2 h, the reaction was quenched by careful addition of MeOH (5 mL). The mixture was added sat. Rochelle salt aq. (8 mL) and stirred for 30 min. The organic layer was washed with sat. Rochelle salt aq. three times, and the aqueous layer was extracted with CHCl3 three times. The combined mixture was dried over MgSO4 and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 5:1 to 1:1) to give allyl alcohol 100 (0.77 g, q.y.) as a colorless oil.
1H NMR (300 MHz, CDCl3) δ 5.21 (dd, J=7.22, 6.88 Hz, 1H), 4.20 (dd, J=6.19, 5.85 Hz, 2H), 2.64-2.59 (m, 1H), 2.01-1.20 (m, 18H), 1.21 (s, 6H), 0.93 (d, J=6.19 Hz, 3H), 0.54 (s, 3H).
Triphenylphosphine (104.0 mg, 0.395 mmol), 2-mercapt benzothiazole (66 mg, 0.395 mmol) were added to a solution of allyl alcohol 100 (75.5 mg, 0.247 mmol) in dichloromethane (0.80 mL) at 0° C., then added dropwise diisopropyl azodicarboxylate (77 μL, 0.395 mmol). After stirring for 1 h, the reaction mixture was quenched with H2O. The aqueous layer was extracted with dichloromethane three times. The combined organic extracts were dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 10:1 to 5:1) to thioether (0.247 mmol) including residual reagent. To a solution of thioether (0.247 mmol) in ethanol (3.3 mL) was added hydrogen peroxide (1.3 mL, 30% solution in water) and hexaammonium heptamolybdate tetrahydrate (97 mg, 0.079 mmol) at room temperature. After stirring for 3 h, the reaction was quenched with 10% Na2S2O3 aq. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 5:1 to 3:1) to give alcohol 101 (0.18 g, 94%).
To a solution of alcohol 101 (1.04 g, 2.127 mmol) in N,N-dimethylformamide (7 mL) was added imidazole (0.43 g, 6.382 mmol) and N,N-dimethylamino-4-pyridine (52 mg, 0.425 mmol) at room temperature, then added dropwise chloro triethylsilane (0.89 mL, 5.318 mmol). After stirring for 6 h, the reaction mixture was quenched with H2O. The aqueous layer was extracted with ethyl ether three times. The combined organic extracts were washed with H2O and brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 40:1 to 20:1) to give benzothiazolyl sulfone 67 (1.28 g, 100%).
1H NMR (300 MHz, CDCl3) δ 8.22-7.56 (m, 4H), 5.01 (dd, J=7.79, 7.79 Hz, 1H), 4.43 (dd, J=14.20, 8.70 Hz, 1H), 4.20 (dd, J=14.20, 6.87 Hz, 1H), 2.56-2.53 (m, 1H), 1.90-1.81 (m, 3H), 1.62-1.15 (m, 15H), 1.17 (s, 6H), 0.93 (t, J=7.79 Hz, 9H), 0.84 (d, J=6.41 Hz, 3H), 0.55 (q, J=7.79 Hz, 6H), 0.25 (s, 3H).
A solution of alcohol 62 (1.22 g, 3.353 mmol) in CH2Cl2 (190 mL) was added molecular sieves 4A and N-methylmorpholine N-oxide at room temperature. After stirring for 15 min, tetrapropylammonium perruthenate was added at 0° C. After additive 1 h, the reaction mixture was filtered through a pad of silica gel and filtrate was concentrated. The residue was chromatographed on silica gel (Hexane/dichloromethane; 15:1 to 5:1) to give ketone 78a (0.48 g, 39%) and 78b (0.63 g, 52%).
A solution of ketone 78a (130.5 mg, 0.360 mmol) in ethanol (3.6 mL) was added sodium borohydride (27.2 mg, 0.720 mmol). After stirring for 3.5 h, the reaction mixture was quenched with H2O and brine, then aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 15:1 to 5:1) to give cis-alcohol 79a (16.5 mg, 21%) and trans-alcohol 62a (41.8 mg, 52%) as a colorless oil.
By following the same procedure described for 79a, cis-alcohol 79b (47.8 mg, 36%) and trans-alcohol 62b (61.5 mg, 47%) was obtained from 78b (79.5 mg, 0.219 mmol) as a colorless oil.
By following the same procedure described for 64, phthalimide 80 (0.70 g, 62%) was obtained from 79a and 79b (0.84 g, 2.291 mmol) as an amorphous solid like foam.
A solution of phthalimide 80 (46.4 mg, 0.094 mmol) in ethanol (1.0 mL) was added palladium hydroxide (5 mg, 10 wt %, 20% on Carbon wetted with ca. 50% Water from TCI Co., Ltd.) at room temperature. The reaction vessel was purged and placed under hydrogen and stirred vigorously for 5.5 h. The reaction mixture was filterd through a pad of celite and filtrate was concentrated. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 10:1 to 1:1) to give diol (19.2 mg, 50%) and starting material (12.7 mg, 27% recovery). To a solution of above diol (19.2 mg, 0.047 mmol) in methanol (2.1 mL) was added dropwise a solution of sodium periodate (46 mg, 0.213 mmol) in distilled water (0.3 mL) at 0° C. After stirring for 7 h, the reaction mixture was diluted with water and the aqueous layer was extracted with dichloromethane three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 15:1 to 2:1) to give ketone 81 (13.9 mg, 79%) as a colorless solid.
1H NMR (300 MHz, CDCl3) δ 7.87-7.70 (m, 4H), 5.05 (dddd, J=12.38, 12.04, 4.47, 4.47 Hz, 1H), 4.52-4.46 (m, 1H), 3.31 (dd, J=13.76, 12.73 Hz, 1H), 2.75-2.47 (m, 3H), 2.02-1.94 (m, 1H), 0.90 (s, 9H), 0.08 (s, 6H).
A solution of alcohol 79a and 79b (0.82 g, 2.2436 mmol) in CH2Cl2 (22.0 mL) was added triethylamine (0.94 mL, 6.735 mmol), N,N-dimethyl-4-aminopyridine (60.5 mg, 0.495 mmol) and benzoyl chloride (0.52 mL, 4.513 mmol) at 0° C., then the mixture was warmed to room temperature and stirred for 2 h. The reaction was quenched with sat. NaHCO3 aq. The aqueous layer was extracted with CH2Cl2 three times. The combined organic extracts were dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 20:1) to give 92 (0.96 mg, 91%) as a colorless solid.
A solution of benzoate ester 92 (0.96 g, 2.047 mmol) in ethyl acetate (41 mL) was added palladium hydroxide (97.8 mg, 10 wt %, 20% on Carbon wetted with ca. 50% Water from TCI Co., Ltd.) at room temperature. The reaction vessel was purged and placed under hydrogen and stirred vigorously for 2 h. The reaction mixture was filtered through a pad of celite and filtrate was concentrated. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 5:1 to 2:1) to give diol (0.77 g, 99%) as a colorless solid. To a solution of above diol (0.77 g, 2.017 mmol) in methanol (84 mL) was added dropwise a solution of sodium periodate (2.00 g, 9.335 mmol) in distilled water (12 mL) at 0° C. After stirring for 1.5 h, the reaction mixture was diluted with water and the aqueous layer was extracted with dichloromethane three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 10:1 to 5:1) to give ketone 93 (0.68 g, 97%) as a colorless solid.
NHBoc VD3 18a and 18b
To a solution of 16 (109 mg, 0.231 mmol) and 14a (36 mg, 0.101 mmol) in toluene (1.0 mL) and Et3N (1.0 mL) was added Pd(PPh3)4 (10-20 mol %) at room temperature, then the reaction mixture was heated at 90° C. After stirring for 3.5 h, the reaction mixture was filtered through a pad of silica gel column and the filtrates were evaporated. The residue was chromatographed on silica gel (hexane/ethyl acetate; 100:1) to give 17a (19 mg, 0.025 mmol, not pure). To a solution of 17a (19 mg, 0.025 mmol) in THF (1 mL) was added 3HF-Et3N (0.050 mL, 0.299 mmol) at room temperature. The reaction was monitored by thin layer chromatography on silica gel plates (eluent: 1:1 ethyl acetate to hexane). The reaction was quenched by sat. NaHCO3, the aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 5:2 to 2:1) to give 1α-NHBoc VD3 18a (7.2 mg, 2 steps 14%).
1H NMR (300 MHz, CDCl3) δ 6.35 (d, J=11.3 Hz, 1H), 5.99 (d, J=11.3 Hz, 1H), 5.22 (s, 1H), 4.95 (s, 1H), 4.51-4.08 (m, 3H), 2.81 (d, J=12.5 Hz, 1H), 2.56 (d, J=12.5 Hz, 1H), 2.35-0.85 (m, 39H), 1.45 (s, 9H), 1.21 (s, 6H), 0.93 (d, J=6.2 Hz, 3H), 0.53 (s, 3H);
HRMS (ESI) Calcd for C32HS3N1Na1O4[M+Na]+: 538.3872, Found: 538.3882.
By following the same procedure described for 18a, 1β-NHBoc VD3 18b (3.5 mg, 13%) was obtained from 16 (59 mg, 0.124 mmol) and 14b (19 mg, 0.054 mmol) as a colorless oil.
1-amino-25(OH)D 19a and 19b
Acetyl chloride (0.080 mL, 1.13 mmol) was slowly added to EtOH (1.1 mL) dropwise at 0° C. After stirring for 30 min, this solution was added to diol 18a (15.9 mg, 0.0308 mmol). After additional stirring for 6 h, the reaction mixture was evaporated. The residue was washed with Et2O, affording 19a (13.01 mg, 93%) as a yellow solid.
1H NMR (300 MHz, MeOH-d4) δ 6.45 (d, J=11.0 Hz, 1H), 6.04 (d, J=11.0 Hz, 1H), 5.28 (s, 1H), 5.17 (s, 1H), 4.19-4.11 (m, 1H), 4.07-4.02 (m, 1H), 2.89 (d, J=10.5 Hz, 1H), 2.57 (d, J=10.5 Hz, 1H), 2.41-1.13 (m, 27H), 0.97 (d, J=6.2 Hz, 3H), 0.58 (s, 3H);
HRMS (ESI) Calcd for C27H46N1O2 [M+H]+: 416.3529, Found: 416.3532. By following the same procedure described for 19a, 19b (2.82 mg, 92%) was obtained from 18b (3.5 mg, 0.00678 mmol) as a yellow solid.
1H NMR (300 MHz, MeOH-d4) δ 6.50 (d, J=11.0 Hz, 1H), 6.01 (d, J=11.0 Hz, 1H), 5.38 (s, 1H), 5.19 (s, 1H), 4.11-4.01 (m, 1H), 3.92-3.86 (m, 1H), 2.89 (d, J=10.5 Hz, 1H), 2.57 (d, J=10.5 Hz, 1H), 2.42-1.14 (m, 27H), 0.97 (d, J=6.2 Hz, 3H), 0.57 (s, 3H).
NHAc VD3 22a and 22b
To a solution of 16 (106 mg, 0.225 mmol) and 20a (40.6 mg, 0.137 mmol) in toluene (1.4 mL) and Et3N (1.4 mL) was added Pd(PPh3)4 (10-20 mol %) at room temperature, then the reaction mixture was heated at 90° C. After stirring for 5 h, the reaction mixture was filtered through a pad of silica gel column and the filtrates were evaporated. The residue was chromatographed on silica gel (hexane/ethyl acetate; 6:1 to 3:1) to give 21a (9.7 mg, 10%). To a solution of 21a (9.7 mg, 0.014 mmol) in THF (0.3 mL) was added 3HF-Et3N (0.030 mL, 0.18 mmol) at room temperature. The reaction was monitored by thin layer chromatography on silica gel plates (eluent: 3:2:1 chloroform to ethyl acetate to methanol). The reaction mixture was quenched with sat. NaHCO3 aq. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 1:10 to 0:1) to give 1α-NHAc VD3 22a (5.87 mg, 90%).
1H NMR (400 MHz, CDCl3) δ 6.38 (d, J=11.0 Hz, 1H), 5.97 (d, J=11.0 Hz, 1H), 5.37 (d, J=8.7 Hz, 1H), 5.20 (s, 1H), 4.96 (s, 1H), 4.77 (dd, J=13.7, 6.0 Hz, 1H), 4.10-4.05 (m, 1H), 2.81 (d, J=12.4 Hz, 1H), 2.57 (d, J=12.4 Hz, 1H), 2.35-0.83 (m, 32H), 1.99 (s, 3H), 1.21 (s, 6H), 0.93 (d, J=6.0 Hz, 3H), 0.52 (s, 3H); HRMS (ESI) Calcd for C29H47N1Na1O3[M+Na]+: 480.3454, Found: 480.3458. By following the same procedure described for 22a, 1-NHAc VD3 22b (10.6 mg, 79%) was obtained from 16 (104 mg, 0.221 mmol) and 20b (37.2 mg, 0.126 mmol) as colorless oil.
1H NMR (400 MHz, CDCl3) δ 6.46 (d, J=9.2 Hz, 1H), 6.36 (d, J=11.0 Hz, 1H), 5.95 (d, J=11.0 Hz, 1H), 5.27 (s, 1H), 4.95 (s, 1H), 4.70-4.65 (m, 1H), 4.18-4.13 (m, 1H), 2.81 (d, J=12.0 Hz, 1H), 2.59 (d, J=12.0 Hz, 1H), 2.36-0.81 (m, 32H), 1.94 (s, 3H), 1.21 (s, 6H), 0.92 (d, J=6.4 Hz, 3H), 0.52 (s, 3H).
1α-NHNs VD3 28
To a solution of 16 (64.0 mg, 0.136 mmol) and 26 (39.3 mg, 0.090 mmol) in toluene (0.9 mL) and Et3N (0.9 mL) was added Pd(PPh3)4 (10-20 mol %) at room temperature, then the reaction mixture was heated at 90° C. After stirring for 5 h, the reaction mixture was filtered through a pad of silica gel column and the filtrates were evaporated. The residue was chromatographed on silica gel (hexane/ethyl acetate; 10:1) to give 27 (12.4 mg, 47%). To a solution of 27 (12.4 mg, 0.015 mmol) in THF (0.3 mL) was added 3HF-Et3N (0.31 mL, 1.92 mmol) at room temperature. The reaction was monitored by thin layer chromatography on silica gel plates (eluent: 1:1 ethyl acetate to hexane). The reaction was quenched by sat. aq. NaHCO3. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 3:1 to 1:2) to give 1α-NHNs VD3 28 (6.51 mg, 90%).
1H NMR (300 MHz, CDCl3) δ 8.20-7.24 (m, 4H), 6.36 (d, J=11.3 Hz, 1H), 5.74 (d, J=11.3 Hz, 1H), 5.41 (d, J=8.2 Hz, 1H), 5.04 (s, 1H), 4.82 (s, 1H), 4.37-4.09 (m, 2H), 2.78 (d, J=11.9 Hz, 1H), 2.55 (d, J=11.9 Hz, 1H), 2.31-0.80 (m, 30H), 1.23 (s, 6H), 0.93 (d, J=6.5 Hz, 3H), 0.51 (s, 3H); HRMS (ESI) Calcd for C33H48N2Na1O6S1 [M+Na]+: 623.3131, Found: 623.3161.
1α-NHTs VD3 32
To a solution of 16 (47.0 mg, 0.099 mmol) and 30 (26.8 mg, 0.066 mmol) in toluene (0.65 mL) and Et3N (0.65 mL) was added Pd(PPh3)4 (10-20 mol %) at room temperature, then the reaction mixture was heated at 90° C. After stirring for 6 h, the reaction mixture was filtered through a pad of silica gel column and the filtrate was evaporated. The residue was chromatographed on silica gel (hexane/ethyl acetate; 10:1) to give 31 (5.1 mg, 10%). To a solution of 31 (5.1 mg, 0.00639 mmol) in THF (0.13 mL) was added 3HF-Et3N (0.091 mL, 0.556 mmol) at room temperature. The reaction was monitored by thin layer chromatography on silica gel plates (eluent: 1:1 ethyl acetate to hexane). The reaction mixture was quenched with sat. NaHCO3 aq. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 2:1 to 1:1) to give 1α-NHTs VD3 32 (2.20 mg, 60%).
1H NMR (300 MHz, CDCl3) δ 7.73 (d, J=8.2 Hz, 2H), 7.29 (d, J=8.2 Hz, 2H), 6.33 (d, J=11.0 Hz, 1H), 5.80 (d, J=11.0 Hz, 1H), 4.93 (s, 1H), 4.64 (s, 1H), 4.37 (d, J=7.2 Hz, 1H), 4.16-4.01 (m, 2H), 2.79-2.73 (m, 2H), 2.43 (s, 3H), 2.24-0.80 (m, 30H), 1.22 (s, 6H), 0.94 (d, J=6.2 Hz, 3H), 0.54 (s, 3H); HRMS (FAB) Calcd for C34H51NNaO4S [M+Na]+: 592.3437, Found: 592.3433.
1α-NHCbz VD3 36
To a solution of 16 (45.7 mg, 0.097 mmol) and 34 (21.4 mg, 0.055 mmol) in toluene (0.55 mL) and Et3N (0.55 mL) was added Pd(PPh3)4 (10-20 mol %) at room temperature, then the reaction mixture was heated at 90° C. After stirring for 6 h, the reaction mixture was filtered through a pad of silica gel column and the filtrates were evaporated. The residue was chromatographed on silica gel (hexane/ethyl acetate; 75:1 to 40:1) to give 35 (5.6 mg, 13%). To a solution of 35 (5.6 mg, 0.0072 mmol) in THF (0.145 mL) was added 3HF-Et3N (0.13 mL, 0.818 mmol) at room temperature. The reaction was monitored by thin layer chromatography on silica gel plates (eluent: 1:1 ethyl acetate to hexane). The reaction mixture was quenched with sat. NaHCO3 aq. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 2:1 to 1:1) to give 1α-NHCbz VD3 36 (1.16 mg, 29%).
1H NMR (300 MHz, CDCl3) δ 7.36 (s, 5H), 6.36 (d, J=11.0 Hz, 1H), 5.97 (d, J=11.0 Hz, 1H), 5.23 (s, 1H), 5.12-5.09 (m, 2H), 4.97 (s, 1H), 4.57-4.46 (m, 1H), 4.12-4.06 (m, 1H), 2.84-0.81 (m, 32H), 1.22 (s, 6H), 0.93 (d, J=6.2 Hz, 3H), 0.51 (s, 3H).
HRMS (FAB) Calcd for C35H51NNaO4 [M+Na]+: 572.3716, Found: 572.3717.
Amine 37
A solution of 1-dodecanethiol (0.0040 mL, 0.01411 mmol) in diethyl ether (0.10 mL) was added NaH (1.00 mg, 0.01881 mmol, 60% stabilized in mineral oil) at 0° C. and stirred for 30 min. To the suspension was added nosylate 27 (7.8 mg, 0.00941 mmol) in diethyl ether (0.10 mL), and then the reaction mixture was warmed to room temperature. After additive 2 h, the reaction was quenched by H2O. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 4:1 to 1:1) to give amine 37 (4.6 mg, 76%).
1H NMR (300 MHz, CDCl3) δ 6.27 (d, J=11.34 Hz, 1H), 5.98 (d, J=11.34 Hz, 1H), 5.17 (s, 1H), 4.91 (s, 1H), 4.16-4.08 (m, 1H), 3.76-3.69 (m, 1H), 2.85-2.77 (m, 1H), 2.49-0.80 (m, 25H), 1.18 (s, 6H), 0.94 (t, J=7.89 Hz, 9H), 0.86 (s, 9H), 0.56 (q, J=7.89 Hz, 6H), 0.54 (s, 3H), 0.59 (s, 6H).
1α-NH(p-CF3Bz) VD3 38
A solution of amine 37 (5.2 mg, 0.00807 mmol) in CH2Cl2 (0.20 mL) was added Et3N (0.0028 mL, 0.02018 mmol), and carefully dropwised p-(trifluoromethyl)benzoyl chloride (0.0014 mL, 0.00969 mmol) at −20° C. After stirring for 1.5 h, the reaction was quenched by sat. NaHCO3 aq. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 30:1 to 15:1) to give benzamide (6.0 mg, 91%, not pure). To a solution of the benzamide (6.0 mg, 7.35 μmol) in THF (0.20 mL) was added 3HF-Et3N (0.14 mL, 0.878 mmol) at room temperature. The reaction was monitored by thin layer chromatography on silica gel plates (eluent: 2:1 ethyl acetate to hexane). The reaction mixture was quenched with sat. aq. NaHCO3. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 2:1 to 1:a1) to give 1α-NH(p-CF3Bz) VD3 38 (2.0 mg, 46%).
1H NMR (400 MHz, CDCl3) δ 7.83 (d, J=8.2 Hz, 2H), 7.67 (d, J=8.2 Hz, 2H), 6.45 (d, J=11.4 Hz, 1H), 6.03 (d, J=8.2 Hz, 1H), 5.99 (d, J=11.4 Hz, 1H), 5.31 (s, 1H), 5.03 (s, 1H), 5.00-4.95 (m, 1H), 4.12-4.06 (m, 1H), 2.84 (d, J=12.8 Hz, 1H), 2.65 (d, J=12.8 Hz, 1H), 2.39-0.81 (m, 30H), 1.21 (s, 6H), 0.93 (d, J=6.4 Hz, 3H), 0.46 (s, 3H); HRMS (ESI) Calcd for C35H4RF3N1Na1O3 [M+Na]+: 610.3484, Found: 610.3458.
1α-NH(p-BrBz) VD3 39
By following the similar procedure described for 38, 1α-NH(p-CF3Bz) VD3 39 (1.77 mg, 19%) purified by flash chromatography (Hexane/EtOAc; 1:3 to 1:10) was obtained from 37 (7.3 mg, 11.33 μmol) as a solid.
1H NMR (400 MHz, CDCl3) δ 7.59-7.52 (m, 4H), 6.45 (d, J=11.0 Hz, 1H), 5.99 (d, J=15.6 Hz, 1H), 5.96 (d, J=11.0 Hz, 1H), 5.29 (s, 1H), 5.01 (s, 1H), 4.98-4.92 (m, 1H), 4.10-4.02 (m, 1H), 2.83 (d, J=12.4 Hz, 1H), 2.64 (d, J=12.4 Hz, 1H), 2.38-0.83 (m, 30H), 1.21 (s, 6H), 0.94 (d, J=5.9 Hz, 3H), 0.49 (s, 3H);
HRMS (ESI) Calcd for C34H48Br1N1Na1O3 [M+Na]+: 622.2695, Found: 622.2683.
1α-NH(p-OMeBz) VD3 40
By following the similar procedure described for 38, 1α-NH(p-OMeBz) VD3 40 (2.46 mg, 52%) purified by flash chromatography (hexane/ethyl acetate; 1:3 to 1:10) was obtained from 37 (5.6 mg, 8.69 μmol) as a solid.
1H NMR (400 MHz, CDCl3) δ 7.68 (d, J=8.72 Hz, 2H), 6.89 (d, J=8.72 Hz, 2H), 6.44 (d, J=11.44 Hz, 1H), 6.01 (d, J=11.44 Hz, 1H), 5.93 (d, J=8.28 Hz, 1H), 5.30 (s, 1H), 5.00 (s, 1H), 4.97-4.92 (m, 1H), 4.13-4.04 (m, 1H), 3.85 (s, 3H), 2.86-0.83 (m, 22H), 2.35 (dd, J=12.80, 7.80 Hz, 1H) 1.20 (s, 6H), 0.93 (d, J=6.4 Hz, 3H), 0.51 (s, 3H);
HR-MS ESI Calcd for C35H51N1Na1O4 [M+Na]+: 572.37158, Found: 572.36923.
1α-NH(p-SCF3Bz) VD3 41
By following the similar procedure described for 38, 1α-NH(p-SCF3Bz) VD3 41 (118.88 mg, 74%) purified by flash chromatography (hexane/ethyl acetate; 3:1 to 1:1) was obtained from 37 (166.2 mg, 0.258 mmol) as a solid.
1H NMR (300 MHz, CDCl3) δ 7.75 (d, J=8.25 Hz, 2H), 7.68 (d, J=8.25 Hz, 2H), 6.45 (d, J=10.98 Hz, 1H), 6.07 (d, J=7.92 Hz, 1H), 5.99 (d, J=10.98 Hz, 1H), 5.30 (s, 1H), 5.01 (s, 1H), 4.95 (dd, J=11.67, 6.87 Hz, 1H), 4.12-4.04 (m, 1H), 2.86-1.15 (m, 23H), 1.21 (s, 6H), 0.93 (d, J=5.82 Hz, 3H), 0.48 (s, 3H); HR-MS ESI Calcd for C35H48F3N1Na1O3S1 [M+Na]+: 642.32047, Found: 642.31973.
1α-NH(2,3,4,5-tetrafluoroBz) VD3 42
By following the similar procedure described for 38, 1α-NH(2,3,4,5-tetrafluoroBz) VD3 42 (2.21 mg, 61%) purified by flash chromatography (NH silica gel, hexane/ethyl acetate; 1:1) was obtained from 37 (3.8 mg, 5.899 μmol) as a solid.
1H NMR (400 MHz, CDCl3) δ 7.80-7.72 (m, 1H), 6.52 (dd, J=11.92, 7.32 Hz, 1H), 6.46 (d, J=11.44 Hz, 1H), 5.97 (d, J=11.44 Hz, 1H), 5.30 (s, 1H), 5.03 (s, 1H), 4.98-4.92 (m, 1H), 4.11-4.04 (m, 1H), 2.86-1.15 (m, 22H), 2.35 (dd, J=13.28, 7.80 Hz, 1H), 1.21 (s, 6H), 0.93 (d, J=5.52 Hz, 3H), 0.48 (s, 3H);
HR-MS ESI Calcd for C34H45F4N1Na1O3[M+Na]+: 614.32333, Found: 614.32730.
1α-NH(2,4,5-trifluoroBz) VD3 43
By following the similar procedure described for 38, 1α-NH(2,4,5-trifluoroBz) VD3 43 (19.10 mg, 89%) purified by flash chromatography (NH silica gel, hexane/ethyl acetate; 1:1) was obtained from 37 (23.5 mg, 0.037 mmol) as a solid.
1H NMR (400 MHz, CDCl3) δ 8.00-7.93 (m, 1H), 6.98-6.92 (m, 1H), 6.62 (dd, J=13.28, 8.24 Hz, 1H), 6.44 (d, J=11.00 Hz, 1H), 5.97 (d, J=11.00 Hz, 1H), 5.29 (s, 1H), 5.01 (s, 1H), 4.99-4.92 (m, 1H), 4.12-4.04 (m, 1H), 2.85-1.15 (m, 22H), 2.35 (dd, J=12.84, 7.80 Hz, 1H), 1.21 (s, 6H), 0.93 (d, J=6.44 Hz, 3H), 0.47 (s, 3H);
HR-MS ESI Calcd for C34H46F3N1Na1O3[M+Na]+: 596.33275, Found: 596.33404.
1α-NH(3,4-dimethoxyBz) VD3 44
By following the similar procedure described for 38, 1α-NH(3,4-dimethoxyBz) VD3 44 (1.76 mg, 78%) purified by flash chromatography (hexane/ethyl acetate; 1:3) was obtained from 37 (2.5 mg, 3.881 μmol) as a solid.
1H NMR (300 MHz, CDCl3) δ 7.46-7.44 (m, 1H), 7.17-7.13 (m, 1H), 6.80 (d, J=8.58 Hz, 1H), 6.44 (d, J=10.65 Hz, 1H), 6.01 (d, J=10.65 Hz, 1H), 5.96 (d, J=8.25 Hz, 1H), 5.30 (s, 1H), 5.01 (s, 1H), 5.00-4.92 (m, 1H), 4.16-4.05 (m, 1H), 3.94 (s, 3H), 3.91 (s, 3H), 2.86-1.15 (m, 23H), 1.21 (s, 6H), 0.93 (d, J=6.18 Hz, 3H), 0.50 (s, 3H);
HR-MS ESI Calcd for C36H53N1Na1O5[M+Na]+: 602.38214, Found: 602.38563.
1α-NH(propionyl) VD3 45
By following the similar procedure described for 38, 1α-NH(propionyl) VD3 45 (1.31 mg, 72%) purified by flash chromatography (chloroform/ethyl acetate/methanol; 18:1:1) was obtained from 37 (2.5 mg, 3.881 μmol) as a solid.
1H NMR (300 MHz, CDCl3) δ 6.39 (d, J=11.34 Hz, 1H), 5.96 (d, J=11.34 Hz, 1H), 5.33 (d, J=8.58 Hz, 1H), 5.20 (s, 1H), 4.96 (s, 1H), 4.85-4.74 (m, 1H), 4.10-4.01 (m, 1H), 2.85-1.15 (m, 23H), 2.21 (q, J=7.56 Hz, 2H), 1.21 (s, 6H), 1.15 (t, J=7.56 Hz, 3H), 0.93 (d, J=6.18 Hz, 3H), 0.51 (s, 3H);
HR-MS ESI Calcd for C30H49N1Na1O3[M+Na]+: 494.36101, Found: 494.36202.
1α-NH(butyryl) VD3 46
By following the similar procedure described for 38, 1α-NH(butyryl) VD3 46 (1.84 mg, 98%) purified by flash chromatography (chloroform/methanol; 20:1) was obtained from 37 (2.5 mg, 3.881 μmol) as a solid.
1H NMR (300 MHz, CDCl3) δ 6.39 (d, J=11.34 Hz, 1H), 5.96 (d, J=11.34 Hz, 1H), 5.33 (d, J=8.25 Hz, 1H), 5.21 (s, 1H), 4.95 (s, 1H), 4.84-4.75 (m, 1H), 4.10-4.00 (m, 1H), 2.84-1.15 (m, 23H), 2.16 (t, J=7.56 Hz, 2H), 1.66 (q, J=7.56 Hz, 2H), 1.21 (s, 6H), 0.95 (t, J=7.56 Hz, 3H), 0.93 (d, J=6.18 Hz, 3H), 0.52 (s, 3H);
HR-MS ESI Calcd for C31H51N1Na1O3[M+Na]+: 508.37666, Found: 508.37541.
1α-NHEt VD3 47
Triphenylphosphine (12.0 mg, 0.0461 mmol), ethanol (4 μL, 0.0690 mmol) and diisopropyl azodicarboxylate (9.0 μL, 0.0461 mmol) were added to a solution of nosylamide 28 (19.1 mg, 0.0230 mmol) in THF (0.43 mL) at room temperature and stirred for 12 h. The reaction mixture was quenched with H2O. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 20:1) to give ethylated nosylamide (13.0 mg, 66%, not pure). To a solution of 1-dodecanethiol (7.2 μL, 0.0303 mmol) in diethyl ether (100 μL) was added sodium hydride (1.2 mg, 0.0288 mmol, 60% stabilized by oil) at 0° C. and stirred for 30 min. The suspension was added to a solution of above ethylated nosylamide (13.0 mg, 0.0152 mmol) in diethyl ether (200 μL). After additive 8 h, the reaction was quenched with H2O. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 10:1 to 1:1) to give ethyl amine (5.8 mg, 57%). HF-Py (83.5 mg, 0.842 mmol) was added to a solution of ethyl amine (5.8 mg, 8.628 μmol) in THF (0.50 mL) at room temperature. The reaction was monitored by thin layer chromatography on silica gel plates (eluent: 3:2:1 chloroform to ethyl acetate to methanol). The reaction mixture was quenched with sat. NaHCO3 aq. The aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (NH silica gel, chloroform/methanol; 100:1) to give 1α-NHEt VD3 47 (3.93 mg, 86%).
1H NMR (300 MHz, CDCl3) δ 6.38 (d, J=11.31 Hz, 1H), 5.99 (d, J=11.31 Hz, 1H), 5.12 (d, J=2.04 Hz, 1H), 4.97 (d, J=2.04 Hz, 1H), 4.13-4.04 (m, 1H), 3.33 (t, J=4.47 Hz, 1H), 2.81 (dd, J=11.67, 3.78 Hz, 1H), 2.68 (dd, J=11.34, 7.23 Hz, 1H), 2.65-1.15 (m, 22H), 2.52 (dd, J=11.34, 7.23 Hz, 1H), 1.21 (s, 6H), 1.08 (t, J=7.20 Hz, 3H), 0.93 (d, J=6.18 Hz, 3H), 0.51 (s, 3H);
HR-MS ESI Calcd for C29H50N1O2[M+H]+: 444.38415, Found: 444.38318.
1α-NHBu VD3 48
By following the similar procedure described for 47, 1α-NHBu VD3 48 (4.70 mg, 36%) purified by flash chromatography (chloroform/methanol; 200:1) was obtained from 28 (20.4 mg, 0.0246 mmol) as a solid.
1H NMR (300 MHz, CDCl3) δ 6.38 (d, J=11.31 Hz, 1H), 5.98 (d, J=11.31 Hz, 1H), 5.13 (d, J=2.40 Hz, 1H), 4.98 (d, J=2.40 Hz, 1H), 4.13-4.02 (t, J=4.11 Hz. 1H), 2.84-1.15 (m, 29H), 1.21 (s, 6H), 0.93 (d, J=6.51 Hz, 3H), 0.89 (t, J=7.20 Hz, 3H), 0.51 (s, 3H);
HR-MS ESI Calcd for C31H54N1O2 [M+H]+: 472.41545, Found: 472.41701.
1α-NH(cyclopropylmethyl) VD3 49
By following the similar procedure described for 47, 1α-NH(cyclopropylmethyl) VD3 49 (8.95 mg, 38%) purified by flash chromatography (chloroform/ethyl acetate/methanol; 6:4:1 to 3:2:2) was obtained from 28 (41.2 mg, 0.0497 mmol) as a solid.
1H NMR (300 MHz, CDCl3) δ 6.41 (d, J=11.34 Hz, 1H), 5.92 (d, J=11.34 Hz, 1H), 5.13 (d, J=2.07 Hz, 1H), 5.01 (d, J=2.07 Hz, 1H), 4.20-4.09 (m, 1H), 3.39 (t, J=4.11 Hz, 1H), 2.85-1.15 (m, 30H), 1.21 (s, 6H), 0.93 (d, J=6.51 Hz, 3H), 0.52 (s, 3H);
HR-MS ESI Calcd for C31H52N1O2 [M+H]+: 470.39980, Found: 470.39797.
1α-NH(2-morpholinoethyl) VD3 50
By following the similar procedure described for 47, 1α-NH(2-morpholinoethyl) VD3 50 (2.56 mg, 15%) purified by flash chromatography (chloroform/ethyl acetate/methanol; 6:4:1 to chloroform/methanol; 1:1) was obtained from 28 (38.9 mg, 0.0469 mmol) as a solid.
1H NMR (300 MHz, CDCl3) δ 6.38 (d, J=11.34 Hz, 1H), 5.98 (d, J=11.34 Hz, 1H), 5.21 (s, 1H), 5.04 (s, 1H), 4.18-4.08 (m, 1H), 3.68 (t, J=4.47 Hz, 4H), 3.48-3.42 (m, 1H), 2.83-1.15 (m, 31H), 1.21 (s, 6H), 0.93 (d, J=6.18 Hz, 3H), 0.50 (s, 3H);
HR-MS ESI Calcd for C33H57N2O3 [M+H]+: 529.43692, Found: 529.43542.
1α-NH(2-(4-hydroxy piperidyl)ethyl) VD3 52
By following the similar procedure described for 47, 1α-NH(2-(4-hydroxy piperidyl)ethyl) VD3 52 (4.22 mg, 20%) purified by flash chromatography (NH silica gel, chloroform/ethyl acetate/methanol; 18:1:1) was obtained from 28 (43.5 mg, 0.0525 mmol) as a solid.
1H NMR (300 MHz, CDCl3) δ 6.36 (d, J=11.34 Hz, 1H), 6.01 (d, J=11.34 Hz, 1H), 5.16 (d, J=2.07 Hz, 1H), 4.98 (d, J=2.07 Hz, 1H), 4.16-4.05 (m, 1H), 3.72-3.62 (m, 1H), 3.34 (t, J=4.47 Hz, 1H), 2.83-1.15 (m, 34H), 1.21 (s, 6H), 0.93 (d, J=5.82 Hz, 3H), 0.51 (s, 3H);
HR-MS ESI Calcd for C34H59N2O3 [M+H]+: 543.45257, Found: 543.44912.
1α-NHMs VD3 53
By following the similar procedure described for 38, 1α-NHMs VD3 53 (1.49 mg, 70%) purified by flash chromatography (chloroform/methanol; 15:1) was obtained from 37 (2.8 mg, 4.347 μmol) as a solid.
1H NMR (300 MHz, CDCl3) δ 6.41 (d, J=11.34 Hz, 1H), 5.95 (d, J=11.34 Hz, 1H), 5.36 (s, 1H), 5.06 (s, 1H), 4.34-4.25 (m, 1H), 4.14-4.04 (m, 1H), 2.83 (d, J=12.4 Hz, 1H), 2.98 (s, 3H), 2.83-1.15 (m, 23H), 1.21 (s, 6H), 0.93 (d, J=6.18 Hz, 3H), 0.52 (s, 3H);
HR-MS ESI Calcd for C28H47N1Na1O4S1 [M+Na]+: 516.31235, Found: 516.31391.
1α-NH(p-fluoroBz) VD3 54
By following the similar procedure described for 38, 1α-NH(p-fluoroBz) VD3 54 (1.89 mg, 48%) purified by flash chromatography (NH silica, hexane/ethyl acetate; 20:1) was obtained from 37 (4.7 mg, 7.296 μmol) as a solid.
1H NMR (400 MHz, CDCl3) δ 7.72 (dd, J=8.72, 5.04 Hz, 2H), 7.08 (t, J=8.72 Hz, 2H), 6.45 (d, J=11.00 Hz, 1H), 6.00 (d, J=11.00 Hz, 1H), 5.94 (d, J=7.80 Hz, 1H), 5.30 (s, 1H), 5.01 (s, 1H), 4.98-4.91 (m, 1H), 4.12-4.04 (m, 1H), 2.84 (dd, J=12.84, 3.68 Hz, 1H), 2.64 (dd, J=12.84, 3.68 Hz, 1H), 2.35 (dd, J=12.84, 7.80 Hz, 1H), 2.21-1.15 (m, 20H), 1.21 (s, 6H), 0.94 (d, J=5.96 Hz, 3H), 0.50 (s, 3H);
HR-MS ESI Calcd for C34H48F1N1Na1O3[M+Na]+: 560.35159, Found: 560.35351.
To a stirred solution of 105 (4.9 mg, 0.0062 mmol) and 19a (1.4 mg, 0.0031 mmol) in methanol (1.0 mL) was added 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.7 mg, 0.0062 mmol) and N,N-diisopropylethylamine (0.54 μL, 0.0031 mmol) at 0° C. and the mixture was stirred at room temperature overnight. The solvent was evaporated and the residue was purified by HPLC to yield 106 (1.9 mg, 0.0016 mmol, 51%).
1H NMR (CD3OD) δ 0.51 (s, 3H), 0.94-3.36 (m, 47H), 3.48-3.64 (m, 20H), 4.03-4.81 (m, 4H), 4.88 (s, 1H), 5.15 (s, 1H), 5.96 (d, 1H), 6.32 (d, 1H), 7.35 (d, 2H), 8.01 (d, 2H);
HRMS (FAB) Calcd for C61H90F3N8NaO10S [M+Na]+: 1207.6429, Found: 1207.6427.
19-nor 1β-NPhth-3β-OTBS VD3 68 and 1α-OTBS-3α-NPhth VD3 69
To a solution of benzothiazolyl sulfone 67 (61.9 mg, 0.102 mmol) in THF (1.0 mL) was added dropwise lithium bis(trimethylsilyl)amide (90 μL, 0.116 mmol, 1.3 M in THF) at −78° C. After stirring for 1 h, a solution of ketone 66 (29.0 mg, 0.078 mmol) in THF (1.0 mL) was added. The mixture was stirred for 2 h at same temperature, then warmed up to room temperature and stirred for 3 h. The reaction mixture was quenched with water and the aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 15:1) to give 68 (12.2 mg, 21%), 69 (24.0 mg, 40%) and starting material 67 (26.9 mg, 44% recovery).
68; 1H NMR (300 MHz, CDCl3) δ 7.85-7.81 (m, 2H), 7.73-7.69 (m, 2H), 6.23 (d, J=11.45 Hz, 1H), 5.73 (d, J=11.45 Hz, 1H), 4.20-4.10 (m, 1H), 3.67 (dddd, J=10.99, 10.76, 5.95, 4.58 Hz, 1H), 2.82-1.15 (m, 25H), 1.18 (s, 6H), 0.95-0.91 (m, 12H), 0.88 (s, 9H), 0.57 (s, 3H), 0.55 (q, J=7.79 Hz, 6H), 0.08 (s, 3H), 0.05 (s, 3H).
69; 1H NMR (300 MHz, CDCl3) δ 7.84-7.80 (m, 2H), 7.72-7.68 (m, 2H), 6.23 (d, J=11.45 Hz, 1H), 5.85 (d, J=11.45 Hz, 1H), 4.14 (dddd, J=12.82, 12.36, 4.12, 4.12 Hz, 1H), 3.58 (dddd, J=10.99, 10.53, 6.42, 4.12 Hz, 1H), 3.03-2.97 (m, 2H), 2.79-2.74 (m, 1H), 2.49 (ddd, J=12.36, 12.36, 10.99 Hz, 1H), 2.22 (dd, J=12.36, 3.66 Hz, 1H), 2.02-1.15 (m, 20H), 1.19 (s, 6H), 0.95 (t, J=7.79 Hz, 9H), 0.93 (d, J=6.41 Hz, 3H), 0.89 (s, 9H), 0.56 (q, J=7.79 Hz, 6H), 0.56 (s, 3H), 0.09 (s, 3H), 0.06 (s, 3H).
19-nor 1β-amino-3β-OTBS VD3 70 and 1α-OTBS-3α-amino VD3 71
To a solution of 68 (16.7 mg, 0.0219 mmol) in ethanol (0.55 mL) was added hydrazine hydrate (5.3 μL, 0.110 mmol) at room temperature. The mixture was stirred for 1.5 h at 60° C. The reaction mixture was filtered through a plug of cotton and filtrate was concentrated to give crude 70.
By following the same procedure described for 70, crude 71 was obtained from 37 (19.5 mg, 0.0256 mmol).
19-nor 1β-NHTs-3β-OH VD3 72 and 1α-OH-3α-NHTs VD3 73
To a solution of crude 70 (ca. 0.011 mmol) in CH2Cl2 (0.60 mL) was added trimethylamine (4.0 μL, 0.028 mmol) and p-toluenesulfonyl chloride (2.5 mg, 0.013 mmol) at 0° C. After stirring for 2.5 h, the reaction mixture was quenched with water and the aqueous layer was extracted with dichloromethane three times, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (NH silica gel, hexane/ethyl acetate; 30:1 to 4:1) to give 1-NHTs-3p-OTBS VD3 (6.4 mg, 73%) as a colorless oil. HF-Py (121.0 mg, 1.221 mmol) was added to a solution of above 1β-NHTs-3β-OTBS VD3 (6.4 mg, 8.139 μmol) in THF (0.60 mL) and the mixture was stirred for 20 h. The reaction mixture was quenched with sat. NaHCO3 aq. and aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 3:1 to 1:2) to give 1β-NHTs-3β-OH VD3 72 (4.29 mg, 94%) as a white solid.
1H NMR (300 MHz, CDCl3) δ 7.76 (d, J=8.26 Hz, 2H), 7.29 (d, J=8.26 Hz, 2H), 6.26 (d, J=11.35 Hz, 1H), 5.69 (d, J=11.35 Hz, 1H), 5.20 (d, J=8.94 Hz, 1H), 3.95-3.87 (m, 1H), 3.54-3.44 (m, 1H), 2.76 (dd, J=11.70, 3.78 Hz, 1H), 2.45-2.36 (m, 1H), 2.43 (s, 3H), 2.28 (dd, J=13.42, 6.88 Hz, 1H), 2.15 (dd, J=11.42, 6.54 Hz, 1H), 2.05-1.20 (m, 21H), 1.23 (s, 6H), 0.94 (d, J=6.19 Hz, 3H), 0.54 (s, 3H).
HR-MS ESI Calcd for C33H51N1Na1O4S1 [M+Na]+: 580.34365, Found: 580.34011. By following the same procedure described for 72, 1α-OTBS-3α-NHTs VD3 73 (131.4 mg, 83%) was obtained from 71 (ca. 0.283 mmol) as a white solid.
1H NMR (300 MHz, CDCl3) δ 7.76 (d, J=8.26 Hz, 2H), 7.30 (d, J=8.26 Hz, 2H), 6.10 (d, J=11.01 Hz, 1H), 5.76 (d, J=11.01 Hz, 1H), 5.20 (d, J=8.60 Hz, 1H), 3.88-3.81 (m, 1H), 3.48-3.40 (m, 1H), 2.74-2.70 (m, 1H), 2.53 (dd, J=13.07, 3.44 Hz, 1H), 2.43 (s, 3H), 2.34-1.20 (m, 23H), 1.22 (s, 6H), 0.94 (d, J=6.19 Hz, 3H), 0.56 (s, 3H).
HR-MS ESI Calcd for C33H51N1Na1O4S1 [M+Na]+: 580.34365, Found: 580.34781.
19-nor 1β-NH(p-SCF3Bz)-3β-OH VD3 74 and 1α-OH-3α-NH(p-SCF3Bz) VD3 75
To a solution of crude 70 (ca. 0.011 mmol) in CH2Cl2 (0.60 mL) was added trimethylamine (4.0 μL, 0.028 mmol) and (p-trifluoromethylthio)benzoyl chloride (2.2 μL, 0.013 mmol) at 0° C. After stirring for 1 h, the reaction mixture was quenched with water and the aqueous layer was extracted with dichloromethane three times, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (hexane/ethyl acetate; 10:1 to 6:1) to give 1β-NH(p-SCF3Bz)-3β-OTBS VD3 (7.6 mg, 82%) as a colorless oil. HFPy (129.3 mg, 1.304 mmol) was added to a solution of above 1β-NH(p-SCF3Bz)-3β-OTBS VD3 (7.6 mg, 9.087 μmol) in THF (0.60 mL) and the mixture was stirred for 20 h. The reaction mixture was quenched with sat. NaHCO3 aq. and aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 3:1 to 1:2) to give 1-NH(p-SCF3Bz)-3β-OH VD3 74 (3.24 mg, 59%) as a white solid.
1H NMR (300 MHz, CDCl3) δ 7.73-7.64 (m, 4H), 7.50 (d, J=8.26 Hz, 1H), 6.34 (d, J=11.35 Hz, 1H), 5.72 (d, J=11.35 Hz, 1H), 4.51-4.46 (m, 1H), 3.31-3.27 (m, 1H), 2.94 (dd, J=13.42, 3.78 Hz, 1H), 2.79-2.74 (m, 1H), 2.60-2.56 (m, 1H), 2.35-1.20 (m, 22H), 1.21 (s, 6H), 0.90 (d, J=6.54 Hz, 3H), 0.50 (s, 3H).
HR-MS ESI Calcd for C34H48F3N1Na1O3S1 [M+Na]+: 630.32047, Found: 630.31864. By following the same procedure described for 74, 1α-OTBS-3α-NHTs VD3 75 (5.06 mg, 65%) was obtained from 71 (ca. 0.013 mmol) as a white solid.
1H NMR (300 MHz, CDCl3) δ 7.80-7.67 (m, 4H), 7.33 (d, J=8.26 Hz, 1H), 6.29 (d, J=11.01 Hz, 1H), 5.84 (d, J=11.01 Hz, 1H), 4.40-4.32 (m, 1H), 4.23-4.18 (m, 1H), 2.76-2.68 (m, 1H), 2.60 (dd, J=14.10, 5.85 Hz, 1H), 2.54-1.21 (m, 22H), 2.37 (dd, J=14.10, 5.85 Hz, 1H), 1.22 (s, 6H), 0.94 (d, J=6.19 Hz, 3H), 0.55 (s, 3H).
HR-MS ESI Calcd for C34H48F3N1Na1O3S1 [M+Na]+: 630.32047, Found: 630.32327.
19-nor 1β-NPhth-3β-OH VD3 76 and 1α-OH-3α-NHPhth VD3 77
HF.Py (150.0 mg, 1.513 mmol) was added to a solution of above 1β-NPhth-3β-OTBS VD3 68 (12.2 mg, 16.005 μmol) in THF (0.60 mL) and the mixture was stirred for 20 h. The reaction mixture was quenched with sat. NaHCO3 aq. and aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 3:1 to 1:1) to give 1β-NPhth-3β-OH VD3 76 (8.54 mg, 100%) as a white solid.
1H NMR (300 MHz, CDCl3) δ 7.86-7.70 (m, 4H), 6.27 (d, J=11.35 Hz, 1H), 5.72 (d, J=11.35 Hz, 1H), 4.24-4.13 (m, 1H), 3.72 (dddd, J=11.01, 10.66, 6.88, 4.47 Hz, 1H), 2.81-2.74 (m, 2H), 2.61 (dd, J=12.07, 4.13 Hz, 1H), 2.39 (ddd, J=12.04, 11.70, 11.70 Hz, 1H), 2.25-1.19 (m, 21H), 1.20 (s, 6H), 0.93 (d, J=6.54 Hz, 3H), 0.56 (s, 3H).
HR-MS ESI Calcd for C34H47N1Na1O4[M+Na]+: 556.34028, Found: 556.34468. By following the same procedure described for 76, 1α-OH-3α-NHPhth VD3 77 (8.76 mg, 94%) was obtained from 69 (13.3 mg, 17.448 μmol) as a white solid.
1H NMR (300 MHz, CDCl3) δ 7.85-7.69 (m, 4H), 6.25 (d, J=11.35 Hz, 1H), 5.86 (d, J=11.35 Hz, 1H), 4.17 (dddd, J=12.38, 12.38, 4.13, 3.78 Hz, 1H), 3.72 (dddd, J=11.01, 11.01, 6.54, 4.13 Hz, 1H), 3.16 (dd, J=12.73, 4.13 Hz, 2H), 3.00 (dd, J=12.38, 12.38 Hz, 1H), 2.77 (dd, J=12.04, 3.78 Hz, 1H), 2.46 (ddd, J=12.04, 11.70, 11.70 Hz, 1H), 2.77 (dd, J=12.38, 3.78 Hz, 1H), 2.17-1.21 (m, 19H), 1.22 (s, 6H), 0.94 (d, J=6.19 Hz, 3H), 0.55 (s, 3H).
HR-MS ESI Calcd for C34H47N1Na1O4[M+Na]+: 556.34028, Found: 556.34111.
19-nor 1α-NPhth-3β-OTBS VD3 82 and 1α-OTBS-3-NPhth VD3 83
To a solution of benzothiazolyl sulfone 67 (202.0 mg, 0.334 mmol) in THF (2.0 mL) was added dropwise lithium bis(trimethylsilyl)amide (0.28 mL, 0.336 mmol, 1.3 m in THF) at −78° C. After stirring for 1 h, a solution of ketone 81 (72.4 mg, 0.194 mmol) in THF (1.0 mL) was added. The mixture was stirred for 2 h at same temperature, then warmed up to room temperature and stirred for 0.5 h. The reaction mixture was quenched with water and the aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 30:1 to 15:1) to give 82 (21.5 mg, 15%), 83 (33.8 mg, 22%) and starting material 67 (98.7 mg, 49% recovery).
82; 1H NMR (400 MHz, CDCl3) δ 7.84-7.68 (m, 4H), 6.14 (d, J=10.99 Hz, 1H), 5.77 (d, J=10.99 Hz, 1H), 4.64 (dddd, J=11.91, 11.91, 4.58, 44.12 Hz, 1H), 4.26-4.22 (m, 1H), 2.89-1.14 (m, 25H), 1.17 (s, 6H), 0.93 (t, J=10.99 Hz, 9H), 0.91 (d, J=6.41 Hz, 3H), 0.89 (s, 9H), 0.54 (q, J=8.24 Hz, 6H), 0.51 (s, 3H), 0.06 (s, 3H), 0.05 (s, 3H).
19-nor 1α-amino-3β-OTBS VD3 84
By following the same procedure described for 70, crude 84 was obtained from 82 (3.8 mg, 4.985 μmol).
19-nor 1α-NHTs-3β-OH VD3 85
By following the same procedure described for 72, 1α-NHTs-3β-OH VD3 85 (1.58 mg, 52%) was obtained from crude 84 (ca. 5.422 μmol) as a white solid.
1H NMR (300 MHz, CDCl3) δ 7.77-7.29 (m, 4H), 6.27 (d, J=11.35 Hz, 1H), 5.59 (d, J=11.35 Hz, 1H), 5.20 (d, J=8.94 Hz, 1H), 4.00-3.93 (m, 1H), 3.69-3.60 (m, 1H), 2.44 (s, 3H), 2.28-1.20 (m, 24H), 1.23 (s, 6H), 0.95 (d, J=6.19 Hz, 3H), 0.56 (s, 3H).
HR-MS FAB Calcd for C33H51N1Na1O4S1 [M+Na]+: 580.3437, Found: 580.3435.
19-nor 1α-NH(p-SCF3Bz)-3β-OH VD3 86
By following the same procedure described for 74, 1α-NH(p-SCF3Bz)-3β-OH VD3 86 (1.70 mg, 56%) was obtained from crude 84 (ca. 4.985 μmol) as a white solid.
1H NMR (300 MHz, CDCl3) δ 7.73-7.65 (m, 4H), 6.42 (d, J=11.35 Hz, 1H), 6.03 (d, J=8.26 Hz, 1H), 5.83 (d, J=11.35 Hz, 1H), 4.55-4.48 (m, 1H), 3.97-3.88 (m, 1H), 2.85-1.20 (m, 25H), 1.21 (s, 6H), 0.93 (d, J=5.85 Hz, 3H), 0.43 (s, 3H).
HR-MS FAB Calcd for C34H48F3N1Na1O3S1 [M+Na]+: 630.3205, Found: 630.3207.
19-nor 1α-NPhth-3β-OH VD3 87
By following the same procedure described for 76, 1α-NPhth-3β-OH VD3 87 (1.46 mg, 52%) was obtained from crude 84 (ca. 5.248 μmol) as a white solid.
1H NMR (300 MHz, CDCl3) δ 7.85-7.69 (m, 4H), 6.29 (d, J=11.01 Hz, 1H), 5.80 (d, J=11.01 Hz, 1H), 4.60-4.49 (m, 1H), 4.34-4.25 (m, 1H), 2.94-1.20 (m, 25H), 1.20 (s, 6H), 0.92 (d, J=6.19 Hz, 3H), 0.51 (s, 3H).
HR-MS FAB Calcd for C34H48N1O4 [M+H]+: 534.3583, Found: 534.3567.
19-nor 1α-NHAc-3β-OH VD3 88
By following the similar procedure described for 76, 1α-NHAc-3β-OH VD3 88 (2.15 mg, 76%) was obtained from crude 84 (ca. 5.422 tmol) as a white solid.
1H NMR (300 MHz, CD3OD) δ 6.25 (d, J=10.32 Hz, 1H), 5.82 (d, J=10.32 Hz, 1H), 4.70-4.55 (m, 1H), 4.06-3.96 (m, 1H), 2.87-1.15 (m, 28H), 1.16 (s, 6H), 0.97 (d, J=5.50 Hz, 3H), 0.56 (s, 3H).
HR-MS FAB Calcd for C28H47N1Na1O3[M+Na]+: 468.3454, Found: 468.3454.
19-nor 1α-NHMs-3β-OH VD3 89
By following the similar procedure described for 76, 1α-NHMs-3β-OH VD3 89 (1.64 mg, 55%) was obtained from crude 84 (ca. 5.422 μmol) as a white solid.
1H NMR (300 MHz, CDCl3) δ 6.32 (d, J=11.01 Hz, 1H), 5.81 (d, J=11.01 Hz, 1H), 4.08-4.00 (m, 1H), 3.84-3.76 (m, 1H), 2.98 (s, 3H), 2.78 (dd, J=13.42, 4.13 Hz, 1H), 2.69 (dd, J=13.42, 4.13 Hz, 1H), 2.49 (dd, J=13.42, 4.13 Hz, 1H), 2.29 (dd, J=13.07, 7.22 Hz, 1H), 2.19 (dd, J=13.07, 7.22 Hz, 1H), 2.05-1.20 (m, 20H), 1.22 (s, 6H), 0.94 (d, J=6.19 Hz, 3H), 0.54 (s, 3H).
HR-MS FAB Calcd for C27H47N1Na1O4S1 [M+Na]+: 504.3124, Found: 504.3121.
19-nor 1α-NH(trifluoroacetyl)-3β-OH VD3 90
By following the similar procedure described for 76, 1α-NH(trifluoroacetyl)-3β-OH VD3 90 (2.04 mg, 75%) was obtained from crude 84 (ca. 5.422 μmol) as a white solid.
1H NMR (300 MHz, CDCl3) δ 6.38 (d, J=11.35 Hz, 1H), 6.13 (d, J=7.91 Hz, 1H), 5.77 (d, J=11.35 Hz, 1H), 4.37-4.25 (m, 1H), 3.95 (dddd, J=7.91, 7.91, 4.13, 4.13 Hz, 1H), 2.80 (dd, J=11.70, 3.78 Hz, 1H), 2.57-1.20 (m, 24H), 1.22 (s, 6H), 0.94 (d, J=6.19 Hz, 3H), 0.51 (s, 3H).
HR-MS FAB Calcd for C28H45F3N1O3[M+H]+: 500.3351, Found: 500.3340.
19-nor 1α-NHDns-3β-OH VD3 91
By following the similar procedure described for 76, 1α-NHDns-3β-OH VD3 91 (1.72 mg, 51%) was obtained from crude 84 (ca. 5.422 μmol) as a white solid.
1H NMR (300 MHz, CDCl3) δ 8.56-7.17 (m, 6H), 6.23 (d, J=11.70 Hz, 1H), 5.60 (d, J=11.70 Hz, 1H), 4.54 (d, J=8.26 Hz, 1H), 3.92-3.85 (m, 1H), 3.65-3.54 (m, 1H), 2.90 (s, 6H), 2.76-1.23 (m, 25H), 1.24 (s, 6H), 0.95 (d, J=6.19 Hz, 3H), 0.53 (s, 3H).
HR-MS FAB Calcd for C38H56N2Na1O4S1[M+Na]+: 659.3859, Found: 659.3860.
19-nor 1-OBz-3-OTBS VD3 94
To a solution of benzothiazolyl sulfone 67 (558.1 mg, 0.924 mmol) and ketone 93 (214.7 mg, 0.616 mmol) in THF (12.3 mL) was added dropwise lithium bis(trimethylsilyl)amide (0.94 mL, 1.232 mmol, 1.3 m in THF) at −78° C. The mixture was stirred for 1 h at same temperature, then warmed up to room temperature and stirred for 0.5 h. The reaction mixture was quenched with water and the aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 75:1 to 10:1) to give 94 (379.1 mg, 83%) as isomer mixture and starting material 67 (112.9 mg, 20% recovery).
19-nor 1β-OBz-3β-OTBS VD3 95 and 1α-OTBS-3α-OBz VD3 96
94 (496.7 mg, 0.674 mmol) was dissolved in methanol (13.5 mL), then the solution was added potassium carbonate (372.5 mg, 2.695 mmol) at room temperature and stirred for 13 h. The reaction mixture was quenched with sat. NH4Cl aq. and the aqueous layer was extracted with dichloromethane three times. The combined organic extracts were dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 15:1 to 5:1) to give 95 (118.0 mg, 44%) and isomer 96 (192.6 mg, 45%).
19-nor 1α-F-3p-OTBS VD3 97
To a solution of 95 (87.2 mg, 0.138 mmol) in dichloromethane (2.8 mL) was added N,N-diethylaminosulfur trifluoride (27.3 μL, 0.207 mmol) at −78° C. After stirring for 1 h, the reaction mixture was quenched with sat. NaHCO3 aq. and the aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 75:1 to 50:1) to give 97 (54.5 mg, 62%).
19-nor 1α-F-3β-OH VD398
HF.Py (120.3 mg, 1.213 mmol) was added to a solution of 97 (54.5 mg, 0.085 mmol) in THF (1.7 mL) and the mixture was stirred for 16 h. The reaction mixture was quenched with sat. NaHCO3 aq. and aqueous layer was extracted with ethyl acetate three times. The combined organic extracts were washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was chromatographed on silica gel (Hexane/ethyl acetate; 10:1 to 1:1) to give 1α-F-3β-OH VD3 98 (12.9 mg, 37%) as a white solid.
1H NMR (300 MHz, CDCl3) δ 6.31 (d, J=11.35 Hz, 1H), 5.80 (q, J=11.35 Hz, 1H), 4.66 (ddddd, J=48.16, 7.91, 7.57, 3.78, 3.78 Hz, 1H), 3.77 (dddd, J=7.91, 7.57, 3.78, 3.78 Hz, 1H) 2.79-1.18 (m, 25H), 1.19 (s, 6H), 0.92 (d, J=6.54 Hz, 3H), 0.53 (s, 3H). HR-MS ESI Calcd for C26H43F1Na1O2[M+Na]+: 429.31448, Found: 429.31441.
Experiment 1: PLAP-BP Assay
The molecules in a chemical library which consists of 280 endogenous lipid related molecules were screened for their ability to inhibit the expression of a luciferase reporter gene. In this gene, three repeats of sterol regulatory elements (SREs) control expression of luciferase. Lipid depletion activates SREBPs, which bind to the SRE domain and work as transcription factors to express luciferase. The SREBP-responsive reporter construct was co-transfected into Chinese hamster ovary K1 (CHO-K1) cells, along with a control β-galactosidase (β-gal) reporter gene. A constitutively active actin promoter in the reporter gene drives the expression of 3-gal. The levels of luciferase expression from the SREBP-responsive reporter gene were normalized to the levels of β-gal expression.
Eleven molecules that decreased the expression levels of the reporter gene by more than 40% were selected as hit molecules from the first screening. A reporter assay developed by Sakai et al. (Mol. Cell 1998, 2, 505-514) was used to determine whether the selected hit molecules affected the ER-Golgi translocation and proteolytic processing of SREBPs, and to eliminate molecules that gave false positive results. In this assay, a secreted alkaline phosphatase, fused with an SREBP-2 fragment lacking the NH2-terminal DNA-binding domain (PLAP-BP2513-1141), was used to monitor translocation and processing, through changes in the fluorescence of a fluorogenic phosphatase substrate. When cells were co-transfected with plasmids encoding PLAP-BP2513-141 and SREBP cleavage-activating protein (SCAP), PLAP phosphatase was secreted, and fluorescence signals were generated. Of the eleven hit molecules, vitamin D3 (VD) derivatives caused a significant decrease in secretion, compared to the DMSO control (
Experiment 1-A: Procedures for Cell Culture
CHO-K1 cells were maintained in a medium A (1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle medium, supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin sulfate, and 5% (v/v) fetal bovine serum) at 37° C. under humidified 5% CO2.
Experiment 1-B: Procedures for PLAP-BP Assay (
On day 0, CHO-K1 cells were added to 96-well plates at 2.0×104 cells per well in medium A. On day 1, the cells were co-transfected with pCMV-PLAP-BP2513-1141 (0.1 μg/well), pCMV-SCAP (0-2.0 μg/well), and β-gal reporter, in which the expression of β-galactosidase was controlled by an actin promoter (pAc-β-gal, 0.1 μg/well) using FuGENE(Registered trademark) HD transfection reagent (Promega), according to the manufacturer's protocol. After 5 h of incubation, the cells were washed with PBS and then incubated in medium B (1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle medium, supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin sulfate, 5% (v/v) lipid-depleted serum, 50 μM compactin, and 50 μM lithium mevalonate), in the absence or presence of VD derivatives (10 μM) or sterols (10 μg/mL of cholesterol and 1.0 μg/mL of 25-hydroxycholesterol). After 20 h of incubation, an aliquot of the medium was assayed for secreted alkaline phosphatase activity. The cells in each well were lysed and used for measurement of β-galactosidase activities. The alkaline phosphatase activity was normalized by the activity of β-galactosidase.
Experiment 2: Luciferase Reporter Assay
25(OH)D inhibited the activation of SREBPs in a dose-dependent manner in the reporter assay, and had an IC50 value of 1.0 μM (
Experiment 2-A: Procedures for Luciferase reporter assay (
On day 0, CHO-K1 cells were added to 96-well plates at 1.0×104 cells per well in medium A. On day 1, the cells were co-transfected with an SRE-1-driven luciferase reporter construct (pSRE-Luc) and pAc-β-gal at a 20/1 ratio, using FuGENE(Registered trademark) HD transfection reagent (Promega), according to the manufacturer's protocol. After 8-12 h of incubation, the cells were washed with PBS buffer, then incubated in medium B containing the specific test compounds. Stock solutions of each compound in DMSO were prepared and added to medium B to give a 200-fold (v/v) dilution (0.5% DMSO). After 20-24 h of incubation, the cells in each well were lysed by freeze-thaw with Reporter Lysis Buffer (Promega), and aliquots were used to measure luciferase and β-galactosidase activities. Luciferase activity was measured using the Steady-Glo(Registered trademark) Luciferase Assay System (Promega), and β-galactosidase activity was measured using the β-galactosidase Enzyme Assay System (Promega). Luciferase activity was normalized to (galactosidase activity.
Experiment 2-B: Procedures for Western-Blot Analysis (
On day 0, CHO-K1 cells were added to 6-well plates at 3.0×105 cells per well in medium A. On day 1, the cells were washed with PBS, and then incubated in medium B in the absence or presence of specific test compounds (5 μM). After 16 h of incubation, the cells were washed three times with cold PBS, and lysed with a buffer A (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% (v/v) Nonidet P40, 0.5% (w/v) sodium deoxycholate, 8 M urea, and protease inhibitor cocktail (Nacalai)). The cell lysate were passed 16 times through a 25G needle and centrifuged at 7,000 g at 4° C. for 10 min. The supernatants were transferred to new tubes, and the pellets were extracted with a buffer A. The resulting buffer was centrifuged at 7,000 g at 4° C. for 10 min, and the supernatants were combined to the previous ones. The resulting lysate was mixed with 0.20 volume of 6×SDS sample buffer (Nacalai) and incubated at room temperature for 30 min. The samples were separated on a 10% SDS-PAGE gel and blotted, using mouse monoclonal antibodies against SREBP-2 (IgG-7D4), SCAP (IgG-9D5), c-Myc (IgG1-MC045), and actin (AC-40). The specific bands were visualized using enhanced chemiluminescence (ECL Prime Western Blotting Detection Reagent, GE Healthcare) on an ImageQuant LAS 500 (GE Healthcare).
SREBP Inhibitory Mechanism
The effect of 25(OH)D on the levels of SCAP was also confirmed by Western blot analysis (
Despite the discovery of the SREBP inhibitory mechanism of hydroxylated VD derivatives, these endogenous molecules per se cannot be used for specific pharmacological intervention of SREBP due to their well-established roles in calcium homeostasis. For example, overdose administration 25(OH)D, which is converted to an active VDR agonist, 1,25(OH)2D, increases serum calcium concentrations, often resulting in formation of kidney stones. One of possible ways to eliminate its VDR activity is to prevent metabolic hydroxylation of the C1 position. Example compounds 18a, 22a, 22b, 28, 32, 36, 38, 39 and 106 are unlikely to be VDR agonists or converted to VDR agonists.
Reporter assays showed that these Example compounds inhibited activation of SREBPs, with IC50 values ranging from 0.5 to 16 μM (Table 1). The inhibition of SREBP activation mediated by Example compounds 22a, 22b, 28, 32, 36, 38, and 39 (
Table 1. Inhibitory effects of 1-N-VD derivatives on the activation of SREBP.
aAll values are the averages of three independent experiments.
The compound of Formula (I) or a pharmaceutically acceptable salt thereof may be useful for treating a disease such as metabolic disease including non-alcoholic steatohepatitis (NASH), liver disease including fatty liver, diabetes, cancer, obesity, cardiovascular disease, etc. Claims 1-13. (canceled)
| Number | Date | Country | |
|---|---|---|---|
| 62096575 | Dec 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15539260 | Jun 2017 | US |
| Child | 18117358 | US |
