Synthesis of carbon‐14 and stable isotope labeled Avagacestat: a novel gamma secretase inhibitor for the treatment of Alzheimer's disease

Bristol‐Myers Squibb and others are developing drugs that target novel mechanisms to combat Alzheimer's disease. γ‐Secretase inhibitors are one class of potential therapies that have received considerable attention. (R)‐2‐(4‐Chloro‐N‐(2‐fluoro‐4‐(1,2,4‐oxadiazol‐3‐yl)benzyl)phenylsulfonamido)‐5,5,5‐trifluoropentanamide (Avagacestat) is a γ‐secretase‐inhibiting drug that has been investigated by Bristol‐Myers Squibb in preclinical and clinical studies. An important step in the development process was the synthesis of a carbon‐14‐labeled analog for use in a human absorption, distribution, metabolism, and excretion study and a stable isotope labeled analog for use as a standard in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. Carbon‐14 labeled Avagacestat was synthesized in seven steps in a 33% overall yield from carbon‐14 labeled potassium cyanide. A total of 5.95 mCi was prepared with a specific activity of 0.81 μCi/mg and a radiochemical purity of 99.9%. ¹³C₆‐Labeled Avagacestat was synthesized in three steps in a 15% overall yield from 4‐chloro[¹³C₆]aniline. A total of 585 mg was prepared with a ultraviolet purity of 99.9%.     

Efficient syntheses of isotopically labeled PD0198961, a novel synthetic coagulation factor Xa inhibitor

PD0198961 was investigated as a potent and selective inhibitor of coagulation factor Xa for the treatment of thrombotic disorders. Radioactive and stable isotope‐labeled PD0198961 were synthesized for absorption, distribution, metabolism and elimination studies of the compound in animals and for use as mass spectral internal standards in support of bioanalytical assays, respectively. [¹⁴C]PD0198961 was prepared from [¹⁴C]CuCN in four radiosynthetic steps in an overall yield of 48% with a radiochemical purity of >99%. The cyanation reaction of an aromatic bromide with inorganic [¹⁴C]cyanide as a key radiolabeling step was investigated. The deuterium‐labeling was accomplished in a different reaction sequence from the ¹⁴C labeling. This convergent process introduced stable isotope labeling via cis‐2,6‐dimethyl[²H₅]piperidine, which was synthesized by catalytic hydrogenation of 2,6‐dimethylpyridine with deuterium gas. Copyright © 2009 John Wiley & Sons, Ltd.     

The syntheses of [13C6] and [phenyl‐14C(U)]BMS‐816336, an inhibitor of 11β‐hydroxysteroid dehydrogenase type 1, for type 2 diabetes

Type 2 diabetes is a significant worldwide health problem. To support the development of BMS‐816336 as an inhibitor of 11β‐hydroxysteroid dehydrogenase type 1 for type 2 diabetes, the synthesis of carbon‐14 labeled material was required for use in metabolic profiling. [Phenyl‐¹⁴C(U)]BMS‐816336 was synthesized in 8 steps and 22% radiochemical yield from commercially available [¹⁴C(U)]bromobenzene. The radiochemical purity of [phenyl‐¹⁴C(U)]BMS‐816336 was 100% having a specific activity of 84.4 μCi/mg or 28.8 mCi/mmol for a total of 8.9 mCi. It was also necessary to synthesize [¹³C₆]BMS‐816336 for use as a liquid chromatography/mass spectrometry standard. [¹³C₆]BMS‐816336 was also prepared in 8 labeled steps in 26% yield from [¹³C₆]bromobenzene.     

The synthesis of 14C‐labeled, 13CD2‐labeled saxagliptin,and its 13CD2‐labeled 5‐hydroxy metabolite

¹⁴C‐labeled saxagliptin, ¹³CD₂‐labeled saxagliptin, and its ¹³CD₂‐labeled 5‐hydroxy metabolite were synthesized to further support development of the compound for biological studies. This paper describes new syntheses leading to the desired compounds. A total of 3.0 mCi of ¹⁴C‐labeled saxagliptin was obtained with a specific activity of 53.98 μCi/mg (17.13 mCi/mmol). The radiochemical purity determined by HPLC was 99.29%, and the overall radiochemical yield was 3.0% based upon 100 mCi of [¹⁴C]CH₂I₂ starting material. By following similar synthetic routes, 580.0 mg of ¹³CD₂‐labeled saxagliptin and 153.1 mg of ¹³CD₂‐labeled 5‐hydroxysaxagliptin metabolite were prepared. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis and stability of a carbon‐14‐labeled 3‐hydroxy‐3‐methylglutaryl coenzyme‐A reductase inhibitor

Inhibition of 3‐hydroxy‐3‐methylglutaryl coenzyme‐A reductase (HMGR) is an effective method of lowering plasma low‐density lipoprotein cholesterol levels. Hemi‐calcium (3R,5S,E)‐7‐(4‐(4‐fluorophenyl)‐6‐isopropyl‐2‐(methyl(1‐methyl‐1H‐1,2,4‐triazol‐5‐yl)amino)pyrimidin‐5‐yl)‐3,5‐dihydroxyhept‐6‐enoate (1) is a cholesterol‐lowering statin drug that effectively inhibits HMGR. An important step in the development of this compound was the synthesis of a carbon‐14‐labeled analog for use in preclinical absorption, distribution, metabolism and excretion studies. The synthesis of a carbon‐14‐labeled analog of the cholesterol‐lowering statin drug 1 is described. The carbon‐14‐labeled compound [¹⁴C]‐1 was prepared in 11 steps from [¹⁴C]‐labeled urea. The overall radiochemical yield for the synthesis was 22% and the radiochemical purity of [¹⁴C]‐1 was 99.9% immediately after synthesis. It was found that [¹⁴C]‐1 with a specific activity of 43.2 µCi/mg decomposed at a rate of about 1.9%/month when stored at ?78°C under argon. Three samples of [¹⁴C]‐1 were prepared to study the chemical stability of the molecule. One sample had a specific activity of 3.8 µCi/mg and the other two contained radical inhibitors, L ‐ascorbic acid (1% by weight, specific activity of 10.5 µCi/mg) or BHT (1% by weight, specific activity of 9.8 µCi/mg). For these samples the decomposition rates were decreased to 0.5%/month, 0.2%/month and 0.1%/month, respectively. Copyright © 2010 John Wiley & Sons, Ltd.     

Syntheses of AZ12320927 labeled with H‐3, C‐14, and H‐2

In support of a program to develop a treatment for depression, three isotopically labeled forms of the 5‐HT_1B antagonist AZ12320927 were synthesized. A tritium labeled version was synthesized for autoradiography using Ir‐catalyzed hydrogen–tritium exchange. A C‐14 labeled version was prepared for use in metabolism studies in four‐steps from [u‐¹⁴C]p‐nitrophenol. A stable isotope labeled version was synthesized for use as an internal standard for LC/MS/MS quantitation in two steps from chromenone 1. Copyright © 2008 John Wiley & Sons, Ltd.     

Carbon‐14‐and carbon‐13‐labeled phosphoric acid [2‐4‐(4‐cyanophenyl)‐thiazol‐2‐yl]‐(2,4‐difluorophenyl)‐1‐[1,2,4]triazol‐4‐yl‐methylpropoxymethyl] monoester dilysine salt,a prodrug of ravuconazole

4‐Bromobenzoic acid [carboxyl‐¹⁴C] and 4‐(2‐bromoacetyl) [Ar‐¹³C₆]benzonitrile were transformed into the title compounds containing [ring¹⁴C‐thiazol‐4‐yl] and [Ar‐¹³C₆‐benzonitrile]. ¹⁴C‐Ravuconazole was prepared in 37% yield and Purity >99%. ¹³C₆‐Ravuconazole was made in 56% overall yield and Purity of >98%. Each labeled compound was converted by additional reaction steps to the corresponding labeled prodrug. Copyright © 2011 John Wiley & Sons, Ltd     

Synthesis of isotopically labeled daclatasvir for use in human clinical studies

Daclatasvir is a novel hepatitis C virus NS5A inhibitor developed by Bristol‐Myers Squibb and marketed as Daklinza®. The need to support the development of daclatasvir required the synthesis of carbon‐14 labeled material for use in human absorption, distribution, metabolism, and excretion studies. A total of 7.53 mCi of [¹⁴C]‐daclatasvir was synthesized in eight steps from commercially available [¹⁴C]‐copper cyanide. The radiochemical purity was 99.6%, and specific activity was 3.86 μCi/mg. To support a human absolute bioavailability study, 5.56 g of [¹³C₂, ¹⁵N₄]‐daclatasvir was synthesized in four steps.     

Synthesis of a stable‐isotope‐labeled biotinylated pentasaccharide conjugate (EP217609), a dual‐effect anticoagulant drug

EP217609 is a neutralizable dual‐effect anticoagulant under clinical investigation in cardiopulmonary bypass during cardiac surgery. Stable‐isotope‐labeled EP217609 was synthesized as an internal standard for mass spectrometry in support of bioassays. EP217609 was obtained in six steps starting from three building blocks in an overall yield of 42%, with a chemical purity of >99%. Thus, coupling between the N‐protected labeled biotin‐lysine 4 , prepared in three steps from [¹³C,¹⁵N]‐l ‐lysine 2 , and the pentasaccharide‐spacer‐amine 6 was first performed. Removal of the Cbz protective group to 8 followed by coupling of the activated peptidomimetic building‐block 10 gave the immediate precursor of EP217609, which could be obtained after catalytic hydrogenolysis of 1,2,4‐oxadiazol‐5(2H)‐one into amidine.     

An alternative and robust synthesis of [13C4]Baraclude® (entecavir)

Stable isotope‐labeled [¹³C₄]entecavir (1) was prepared in 11 steps. Commercially available [¹³C]guanidine hydrochloride and diethyl[1,2,3‐¹³C₃]malonate were condensed to yield 2‐amino[2,4,5,6‐¹³C₄]pyrimidine‐4,6‐diol (8). This was converted to the desired purine (7) in five steps. Introduction of the chiral epoxide was followed by subsequent deprotection to give [¹³C₄]entecavir (1), in an overall yield of 5.7% from labeled precursors. The chemical purity of the title compound was determined to be >99% by HPLC. The isotopic distribution was determined by mass spectrometry to be 282[M + 4], 98.4%; 281[M + 3], 1.6%; and 278[M + 0], <0.1%. Copyright © 2013 John Wiley & Sons, Ltd.     

The 14C, 13C and 15N syntheses of MON 37500, a sulfonylurea wheat herbicide

[¹⁴C] and [¹³C]MON 37500 labeled in the imidazopyridine (Im) ring system were synthesized in seven steps in an overall yield of 39 and 28%, respectively, from [2‐¹⁴C] and [2‐¹³C]chloroacetic acid. [¹⁴C]MON 37500 labeled in the pyrimidine (Pd) ring system was synthesized from [2‐¹⁴C]diethyl malonate in four steps in 48% yield. Lastly, [¹⁵N]MON 37500 was prepared in three steps in an overall yield of 28% from [¹⁵N]ammonium chloride. Copyright © 2005 John Wiley & Sons, Ltd.     

Synthesis of isotope‐labeled HSP90 inhibitor: [13CD3] and [14C]‐TAK‐459

[¹³CD₃]‐TAK‐459 (1A), an HSP90 inhibitor, was synthesized from [¹³CD₃]‐sodium methoxide in three steps in an overall yield of 29%. The key intermediate [¹³CD₃]‐2‐methoxy‐6‐(4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolan‐2‐yl)pyridine was synthesized in two steps from 2,6‐dibromopyridine and stable isotope‐labeled sodium methoxide. [¹⁴C]‐TAK‐459 (1B) was synthesized from [¹⁴C(U)]‐guanidine hydrochloride in five steps in an overall radiochemical yield of 5.4%. The key intermediate, [¹⁴C]‐(R)‐2‐amino‐7‐(2‐bromo‐4‐fluorophenyl)‐4‐methyl‐7,8‐dihydropyrido[4,3‐d]pyrimidin‐5(6H)‐one, was prepared by microwave‐assisted condensation.     

Synthesis of empagliflozin,a novel and selective sodium‐glucose co‐transporter‐2 inhibitor,labeled with carbon‐14 and carbon‐13

Empagliflozin, (2S,3R,4R,5S,6R)‐2‐[4‐chloro‐3‐[[4‐[(3S)‐oxolan‐3‐yl]oxyphenyl]methyl]phenyl]‐6‐(hydroxymethyl)oxane‐3,4,5‐triol was recently approved by the FDA for the treatment of chronic type 2 diabetes mellitus. Herein, we report the synthesis of carbon‐13 and carbon‐14 labeled empagliflozin. Carbon‐13 labeled empagliflozin was prepared in five steps and in 34% overall chemical yield starting from the commercially available α‐D‐glucose‐[¹³C₆]. For the radiosynthesis, the carbon‐14 atom was introduced in three different positions of the molecule. In the first synthesis, Carbon‐14 D‐(+)‐gluconic acid δ‐lactone was used to prepare specifically labeled empagliflozin in carbon‐1 of the sugar moiety in four steps and in 19% overall radiochemical yield. Carbon‐14 labeled empagliflozin with the radioactive atom in the benzylic position was obtained in eight steps and in 7% overall radiochemical yield. In the last synthesis carbon‐14 uniformly labeled phenol was used to give [¹⁴C]empagliflozin in eight steps and in 18% overall radiochemical yield. In all these radiosyntheses, the specific activities of the final compounds were higher than 53 mCi/mmol, and the radiochemical purities were above 98.5%.     

Synthesis of stable isotope‐labeled epothilone D using a degradation–reconstruction approach

The stabilization of microtubules using epothilones represents a novel mechanism of action to treat Alzheimer's disease. Epothilone D is one such microtubule‐stabilizing drug that has been investigated by Bristol‐Myers Squibb. An important step in the development process was the synthesis of a stable isotope‐labeled analog for use in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. A novel synthetic route to stable isotope‐labeled epothilone D is described. The synthetic route was based on a strategy to degrade epothilone B and then use that key intermediate to reconstruct stable isotope‐labeled epothilone D. Epothilone B was treated with potassium osmate and sodium periodate. The thiazole moiety in epothilone B was efficiently cleaved to give (1S,3S,7S,10R,11S,12S,16R)‐3‐acetyl‐7,11‐dihydroxy‐8,8,10,12,16‐pentamethyl‐4,17‐dioxabicyclo[14.1.0]heptadecane‐5,9‐dione. The epoxide in the macrocyclic ring of that intermediate was cleanly removed by treatment with tungsten hexachloride and n‐butyllithium to give the corresponding olefin (4S,7R,8S,9S,16S,Z)‐16‐acetyl‐4,8‐dihydroxy‐5,5,7,9,13‐pentamethyloxacyclohexadec‐13‐ene‐2,6‐dione. Bis(triethylsilyl) protection produced (4S,7R,8S,9S,16S,Z)‐16‐acetyl‐5,5,7,9,13‐pentamethyl‐4,8‐bis(triethylsilyloxy)‐oxacyclohexadec‐13‐ene‐2,6‐dione. This intermediate was coupled to a stable isotope‐labeled thiazole using a Wittig reaction as the key step to provide ¹³C₅, ¹⁵N‐labeled epothilone D. In summary, the synthesis was completed in nine total steps, only six of which involved isotopically labeled reagents. A total of 168 mg of ¹³C₅, ¹⁵N‐labeled epothilone D was prepared in an 8% overall yield from ¹³C₂, ¹⁵N‐labeled thioacetamide and ¹³C₃‐labeled ethyl bromopyruvate.     

Synthesis of two potent glucocorticoid receptor agonists labeled with carbon‐14 and stable isotopes

Two potent glucocorticoid receptor agonists were prepared labeled with carbon‐14 and with stable isotopes to perform drug metabolism, pharmacokinetics, and bioanalytical studies. Carbon‐14 labeled (1) was obtained from an enantiopure alkyne (5) via a Sonogashira coupling to a previously reported 5‐amino‐4‐iodo‐[2‐¹⁴C]pyrimidine [¹⁴C]‐(6), followed by a base‐mediated cyclization (1) in 72% overall radiochemical yield. Carbon‐14 labeled (2) was prepared in five steps employing a key benzoic acid intermediate [¹⁴C]‐(13), which was synthesized in one pot from enolization of trifluoromethylketone (12), followed by bromine–magnesium exchange and then electrophile trapping reaction with [¹⁴C]‐carbon dioxide. A chiral auxiliary (S)‐1‐(4‐methoxyphenyl)ethylamine was then coupled to this acid to give [¹⁴C]‐(15). Propargylation and separation of diastereoisomers by crystallizations gave the desired diastereomer [¹⁴C]‐(17) in 34% yield. Sonogashira coupling to iodopyridine (10) followed by cyclization to the azaindole [¹⁴C]‐(18) and finally removal of the chiral auxiliary gave [¹⁴C]‐(2) in 7% overall yield. For stable isotope syntheses, [¹³C₆]‐(1) was obtained in three steps using [¹³C₄]‐(6) and trimethylsilylacetylene‐[¹³C₂] in 26% yield, while [²H₅]‐(2) was obtained by first preparing the iodopyridine [²H₅]‐(10) in five steps. Then, Sonogashira coupling to chiral alkyne (24) and cyclization gave [²H₅]‐(2) in 42% overall yield.     

Syntheses of [2H3, 15N], [14C]Nexavar™ and its labeled metabolites

Nexavar?, Sorafenib tosylate (BAY 43‐9006 tosylate) is a potent small molecule Raf kinase inhibitor for the treatment of hyperproliferative disorders such as cancer. Both radiolabeled and stable isotope labeled compounds were required for drug absorption, distribution, metabolism and excretion (ADME) and quantitative mass spectrometry bio‐analytical studies. Nexavar? labeled with carbon‐14 in the carboxamide group was prepared in two steps in an overall radiochemical yield of 42% starting from 4‐chloro‐N‐methyl‐2‐pyridine‐[¹⁴C]carboxamide. The [²H₃,¹⁵N] version of Nexavar? was prepared in 75% yield based on 4‐chloro‐N‐[²H₃]methyl‐2‐pyridine‐[¹⁵N]carboxamide. The pyridine N‐oxide metabolite labeled with carbon‐14 as well as with deuterium and nitrogen‐15 and was synthesized by oxidation in yields of 59% and 87%, respectively. Starting from [²H₂, ¹³C]formaldehyde the N‐hydroxymethyl metabolite was labeled with carbon‐13 and deuterium in one step in a 45% overall yield. Copyright © 2006 John Wiley & Sons, Ltd.     

Synthesis of [2H, 13C] and [14C] labeled vasopressin V1a/V2 receptor antagonist RWJ‐676070 and its stable labeled N‐des‐benzoyl metabolite

Stable isotope‐labeled ([¹³C₂, D₃]) and carbon 14‐labeled vasopressin receptor antagonist RWJ‐676070, a spirobenzazepine amide, were prepared in separate syntheses for use in drug metabolism studies. The stable isotopically labeled sample was prepared starting from [¹³C₂, D₆]dimethyl sulfate and [¹³C]copper (I) cyanide in nine steps with a 14% overall isotopic yield. The ¹⁴C label was introduced in five steps starting from [¹⁴C]potassium cyanide to provide material having a specific activity of 58 mCi/mmol (2.15 GBq/mmol). Selective hydrolysis of the metabolically labile amide bond in [¹³C₂, D₃]RWJ‐676070 gave the corresponding labeled metabolite in one step. Copyright © 2008 John Wiley & Sons, Ltd.     

Carbon-13 labeling of ibrutinib for human microdosing

Ibrutinib is an oral medication for the treatment of B cell malignancies. During its clinical development, a stable isotopologue of ibrutinib was required for the assessment of the drug's absolute oral bioavailability via intravenous microdosing. The following work describes a 10-step, gram-scale production of carbon-13 labeled ibrutinib from [¹³C₆]phenol (¹³C₆, 99%) in 31% overall yield with >99% chemical purity and >99% enantiomeric excess (ee), suitable for intravenous microdosing in humans.     

Synthesis and preliminary biological evaluation of a 99mTc‐labeled hypericin derivative as a necrosis avid imaging agent

Mono‐[¹²³I]iodohypericin and mono‐[¹²³I]iodohypericin monocarboxylic acid are iodine‐123‐labeled hypericin derivatives which have shown great promise in preclinical studies as necrosis avid imaging agents in animal models of infarction. In view of the more attractive properties of a ^99mTc‐labeled hypericin derivative, we have synthesized a conjugate of protohypericin monocarboxylic acid with S‐benzoylmercaptoacetyldiglycyl‐diaminopentane in an overall yield of 15%. The conjugate was labeled with technetium‐99m by exchange labeling at pH 10 in a labeling yield of 95% followed by photocyclization to yield ^99mTc‐mercaptoacetyldiglycyl‐1,5‐diaminopentylene hypericincarboxamide (^99mTc‐13). The negatively charged ^99mTc‐13 complex was purified by reversed phase high‐pressure liquid chromatography and the log P_7.4 was determined to be 2.36. In normal NMRI mice, the complex showed slow hepatobiliary clearance while plasma clearance was rapid. The tracer was evaluated in rats with reperfused hepatic infarction by ex vivo autoradiography, gamma counting and histochemical techniques. Unlike the radioiodinated hypericin derivatives, the new tracer agent did not show preferential uptake in necrotic tissue on autoradiography and gamma counting techniques. Conjugation of hypericin with a ^99mTc‐chelate, resulting in a change in size, charge and lipophilicity, had a profound effect on the necrosis avidity of the tracer agent. The results show that ^99mTc‐13 is not suitable for imaging necrosis. Copyright © 2008 John Wiley & Sons, Ltd.