Synthesis of a dual carbon-14-labeled calcitonin gene-related peptide receptor antagonist for use in a human absorption-distribution-metabolism-elimination study.

Oral calcitonin gene-related peptide (CGRP) receptor antagonists have been shown to be effective in the acute and preventive treatment of migraine. CGRP receptor antagonists offer safety advantages over triptans because they are not active vasoconstrictors, which reduces cardiovascular risks. Bristol Myers Squibb discovered a high affinity CGRP receptor antagonist BMS-927711 for the treatment of migraine now FDA approved as Nurtec® ODT (rimegepant). Dual-labeled [14 C]-BMS-927711 was prepared and used in a human absorption-distribution-metabolism-elimination (ADME) study. A dual-labeled analog of BMS-927711 was required to fully track the compound's metabolic transformation. The carbon-14-labeled synthesis of both right side and left side portions of [14 C]-BMS-927711 is described.

Synthesis of carbon-14 and stable isotope labeled Censavudine.

Censavudine is a nucleoside reverse transcriptase inhibitor (NRTI) explored clinically by Bristol Myers Squibb for the treatment of human immunodeficiency virus-1 (HIV-1). As part of the development process, a carbon-14 labeled analog was synthesized for use in a human absorption, distribution, metabolism, and excretion (ADME) study. A stable isotope labeled analog was also synthesized for use as a mass spectrum internal standard in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. Carbon-14 labeled Censavudine was synthesized in 10 steps in a 9% overall yield from carbon-14 labeled trimethylsilylacetylene. A total of 4.44 mCi of material was prepared with a specific activity of 0.25 µCi/mg. The radiochemical and UV purities were 99% and it met all of the specifications for use in a human clinical study. Deuterium labeled Censavudine was synthesized in two steps in a 68% overall yield from [D4 ]-thymine. A total of 237 mg were prepared with a UV purity of 99%.

Synthesis of tritium labeled 30-KDa PEG and conjugation to form [3 H]Pegbelfermin.

Non-alcoholic steatohepatitis (NASH) is the most chronic liver condition in the western population and is fueled by the obesity and type 2 diabetes epidemic. Pegbelfermin (1), a PEGylated human fibroblast growth factor 21 (FGF21) analogue, has previously been shown to improve markers of metabolism and liver fibrosis in obese patients with type 2 diabetes. Radiolabeled Pegbelfermin was needed to access the accumulation of intact drug and metabolized PEG. In an effort to accomplish both goals with one labeled synthesis, the isotopic label was positioned in the PEG. A total of 21 mCi of tritium labeled Pegbelfermin was synthesized having a specific activity of 21.6 Ci/mmol for use in animal studies.

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 [(14) C]-daclatasvir was synthesized in eight steps from commercially available [(14) C]-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 [(13) C2 , (15) N4 ]-daclatasvir was synthesized in four steps.

The syntheses of isotopically labelled CB-1 antagonists for the treatment of obesity.

BMS-725519, BMS-811064, and BMS-812204 are potent and selective central cannabinoid receptor antagonists that have been investigated for the treatment of human obesity. To further understand their biotransformation profiles, radiolabelled and stable-labelled products were required. This paper describes the utility of [14 C]1,1-carbonyldiimidazole as a radiolabelling reagent for the syntheses of carbonyl-labelled [14 C]BMS-725519, [14 C]BMS-811064, and [14 C]BMS-812204. The syntheses of stable-labelled [13 C6 ]BMS-725519 and [13 CD313 CD2 ]BMS-812204 synthesized from of [13 C6 ]4-chloroacetophenone and [13 CD313 CD2 ]iodoethane, respectively, are also described.

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 (13)C5, (15)N-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 (13)C5, (15)N-labeled epothilone D was prepared in an 8% overall yield from (13)C2, (15)N-labeled thioacetamide and (13)C3-labeled ethyl bromopyruvate.

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%. (13) C6 -Labeled Avagacestat was synthesized in three steps in a 15% overall yield from 4-chloro[(13) C6 ]aniline. A total of 585 mg was prepared with a ultraviolet purity of 99.9%.

Pd(II)-catalyzed C-H iodination using molecular I2 as the sole oxidant.

Pd-catalyzed ortho-C-H iodination directed by a weakly coordinating amide auxiliary using I2 as the sole oxidant was developed. This reaction is compatible with a wide range of heterocycles including pyridines, imidazoles, oxazoles, thiazoles, isoxazoles, and pyrazoles.

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

Stable isotope-labeled [(13) C4 ]entecavir (1) was prepared in 11 steps. Commercially available [(13) C]guanidine hydrochloride and diethyl[1,2,3-(13) C3 ]malonate were condensed to yield 2-amino[2,4,5,6-(13) C4 ]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 [(13) C4 ]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%.

Practical and efficient strategy for evaluating oral absolute bioavailability with an intravenous microdose of a stable isotopically-labeled drug using a selected reaction monitoring mass spectrometry assay.

A strategy of using selected reaction monitoring (SRM) mass spectrometry for evaluating oral absolute bioavailability with concurrent intravenous (i.v.) microdosing a stable isotopically labeled (SIL) drug was developed and validated. First, the isotopic contribution to SRM (ICSRM) of the proposed SIL drug and SIL internal standard (IS) was theoretically calculated to guide their chemical synthesis. Second, the lack of an isotope effect on drug exposure was evaluated in a monkey study by i.v. dosing a mixture of the SIL and the unlabeled drugs. Third, after the SIL drug (100 µg) was concurrently i.v. dosed to humans, at T(max) of an oral therapeutic dose of the unlabeled drug, both drugs in plasma specimens were simultaneously quantified by a sensitive and accurate SRM assay. This strategy significantly improves bioanalytical data quality and saves time, costs, and resources by avoiding a traditional absolute bioavailability study or the newer approach of microdoses of a radio-microtracer measured by accelerator mass spectrometry.

Calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry assays and its application in supporting microdose absolute bioavailability studies.

A methodology for the accurate calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays and its application in supporting microdose absolute bioavailability studies are reported for the first time. For simplicity, this calculation methodology and the strategy to minimize the isotopic interference are demonstrated using a simple molecule entity, then applied to actual development drugs. The exact isotopic interferences calculated with this methodology were often much less than the traditionally used, overestimated isotopic interferences simply based on the molecular isotope abundance. One application of the methodology is the selection of a stable isotopically labeled internal standard (SIL-IS) for an LC-MS/MS bioanalytical assay. The second application is the selection of an SIL analogue for use in intravenous (i.v.) microdosing for the determination of absolute bioavailability. In the case of microdosing, the traditional approach of calculating isotopic interferences can result in selecting a labeling scheme that overlabels the i.v.-dosed drug or leads to incorrect conclusions on the feasibility of using an SIL drug and analysis by LC-MS/MS. The methodology presented here can guide the synthesis by accurately calculating the isotopic interferences when labeling at different positions, using different selective reaction monitoring (SRM) transitions or adding more labeling positions. This methodology has been successfully applied to the selection of the labeled i.v.-dosed drugs for use in two microdose absolute bioavailability studies, before initiating the chemical synthesis. With this methodology, significant time and cost saving can be achieved in supporting microdose absolute bioavailability studies with stable labeled drugs.

Quantification of 4-beta-hydroxycholesterol in human plasma using automated sample preparation and LC-ESI-MS/MS analysis.

It has recently been proposed that plasma levels of 4ß-hydroxycholesterol (4ßHC) may be indicative of cytochrome P450 3A4 (P450 3A) activity and therefore could be used to probe for P450 3A-mediated drug-drug interactions. With this in mind, we describe a highly sensitive and precise liquid chromatography-electrospray ionization-tandem mass spectrometry method for the measurement of 4ßHC in human plasma with a lower limit of quantification established at 2 ng/mL using 50 µL of plasma. The entire sample preparation scheme including saponification and derivatization of 4ßHC to the corresponding dipicolinyl ester (DPE) was completed in less than 8 h using an automated sample preparation scheme enabling higher-throughput capabilities. Chromatographic resolution of 4ßHC from 4α-hydroxycholesterol and other endogenous isobaric species was achieved in 11-min using an isocratic gradient on a C18 column. Because of endogenous concentrations of 4ßHC in plasma, a stable isotope labeled (SIL) analogue, d7-4ßHC, was used as a surrogate analyte and measured in the standard curve and quality control samples prepared in plasma. A second SIL analogue, d4-4ßHC, was used as the internal standard. The intraday and interday accuracy for the assay was within 6% of nominal concentrations, and the precision for these measurements was less than 5% relative standard deviation. Rigorous stability assessments demonstrated adequate stability of endogenous 4ßHC in plasma and the corresponding DPE derivative for the analysis of clinical study samples. The results from clinical samples following treatment with a potent P450 3A inducer (rifampin) or inhibitor (ketoconazole) are reported and demonstrate the potential future application for this highly precise and robust analytical assay.

Concerted vs stepwise mechanisms in dehydro-Diels-Alder reactions.

The Diels-Alder reaction is not limited to 1,3-dienes. Many cycloadditions of enynes and a smaller number of examples with 1,3-diynes have been reported. These "dehydro"-Diels-Alder cycloadditions are one class of dehydropericyclic reactions which have long been used to generate strained cyclic allenes and other novel structures. CCSD(T)//M05-2X computational results are reported for the cycloadditions of vinylacetylene and butadiyne with ethylene and acetylene. Both concerted and stepwise diradical routes have been explored for each reaction, with location of relevant stationary points. Relative to 1,3-dienes, replacement of one double bond by a triple bond adds 6-6.5 kcal/mol to the activation barrier; a second triple bond adds 4.3-4.5 kcal/mol to the barrier. Product strain decreases the predicted exothermicity. In every case, a concerted reaction is favored energetically. The difference between concerted and stepwise reactions is 5.2-6.6 kcal/mol for enynes but diminishes to 0.5-2 kcal/mol for diynes. Experimental studies on intramolecular diyne + ene cycloadditions show two distinct reaction pathways, providing evidence for competing concerted and stepwise mechanisms. Diyne + yne cycloadditions connect with arynes and ethynyl-1,3-cyclobutadiene. This potential energy surface appears to be flat, with only a minute advantage for a concerted process; many diyne cycloadditions or aryne cycloreversions will proceed by a stepwise mechanism.

LC-MS/MS bioanalysis of plasma 1, 14-tetradecanedioic acid and 1, 16-hexadecanedioic acid as candidate biomarkers for organic anion-transporting polypeptide mediated drug-drug interactions.

AIM: A robust LC-MS/MS assay was developed to quantify endogenous 1, 14-tetradecanedioic acid (TDA) and 1, 16-hexadecanedioic acid (HDA) in human plasma as potential biomarkers for evaluating drug-drug interactions mediated by the hepatic drug transporters, organic anion-transporting polypeptides. RESULTS: This assay was validated using fit-for-purpose approach over standard curve range of 2.5-1000 nM for TDA and HDA using analyte-free charcoal-stripped human plasma as the surrogate matrix. Chromatographic separation condition was successfully optimized to separate TDA from an interference peak while maintaining both analytes in neutral forms to minimize carryover issue. CONCLUSION: The described assay is currently applied to a clinical study for evaluating TDA/HDA as potential substitute biomarkers for drug-drug interaction studies.

Overcoming interference with the detection of a stable isotopically labeled microtracer in the evaluation of beclabuvir absolute bioavailability using a concomitant microtracer approach.

The oral absolute bioavailability of beclabuvir in healthy subjects was determined using a microdose (100µg) of the stable isotopically labeled tracer via intravenous (IV) infusion started after oral dosing of beclabuvir (150mg). To simultaneously analyze the concentrations of the IV microtracer ([13C6]beclabuvir) and beclabuvir in plasma samples, a liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) method was initially developed. Surprisingly beclabuvir significantly interfered with the IV microtracer detection when using the selected reaction monitoring (SRM) in the assay. An interfering component from the drug substance was observed using a high resolution mass spectrometer (HRMS). The mass-to-charge (m/z) of the interfering component was -32ppm different from the nominal value for the IV microtracer and thus could not be differentiated in the SRM assay by the unit mass resolution. To overcome this interference, we evaluated two approaches by either monitoring an alternative product ion using the SRM assay or isolating the interfering component using the parallel reaction monitoring (PRM) assay on the HRMS. This case study has demonstrated two practical approaches for overcoming interferences with the detection of stable isotopically labeled IV microtracers in the evaluation of absolute bioavailability, which provides users the flexibility in using either LC-MS/MS or HRMS to mitigate unpredicted interferences in the assay to support microtracer absolute bioavailability studies.

Bioanalysis of dried saliva spot (DSS) samples using detergent-assisted sample extraction with UHPLC-MS/MS detection.

Dried saliva spot (DSS) sampling is a non-invasive sample collection technique for bioanalysis that can be potentially implemented at the patient's home. A UHPLC-MS/MS assay was developed using detergent-assisted sample extraction to quantify BMS-927711, a drug candidate in development for the treatment of migraines, in human DSS. By implementing DSS sampling at the patients' home, the bioanalytical sample collection for pharmacokinetic evaluation can be done at the time of the acute migraine attack without the need for clinical visits. DSS samples were prepared by spotting 15 µL of liquid saliva onto regular Whatman FTA™ DMPK-C cards and verified with a UV lamp (at λ 254 nm or 365 nm) during DSS punching. The 4-mm DSS punches in a 96-well plate were sonicated with 200 µL of [(13)C2, D4]-BMS-927711 internal standard (IS) solution in 20/80 MeOH/water for 10 min, followed by sonication with 50 µL of 100 mM NH4OAc with 1.0% Triton-X-100 (as detergent) prior to liquid-liquid extraction with 600 µL EtOAc/Hexane (90:10). UHPLC-MS/MS was performed with an Aquity(®) UPLC BEH C18 Column (2.1 × 50 mm, 1.7 µm) on a Triple Quad™ 5500 mass spectrometer. The assay was linear with a concentration range from 2.00 to 1000 ng mL(-1) for BMS-927711 in human saliva. The intra- and inter-assay precision was within 8.8% CV, and the accuracy was within ±6.7% Dev of the nominal concentration values. This UHPLC-MS/MS assay has been successfully applied to determine the drug's pharmacokinetics within a clinical study. For the first time, we observed BMS-927711 exposure in human DSS, confirming the suitability of this sampling technique for migraine patients to use at home. Detergent-assisted extraction with Triton-X-100 could be very useful in DSS or other dried matrix spot (DMS) assays to overcome low or inconsistent analyte recovery issues.

Sensitive and accurate liquid chromatography-tandem mass spectrometry methods for quantitative determination of a novel hepatitis C NS5B inhibitor BMS-791325 and its active metabolite in human plasma and urine.

BMS-791325 is a novel hepatitis C NS5B inhibitor which is currently in clinical development. To support pharmacokinetic (PK) assessments, sensitive, accurate, precise, and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed and validated for the quantitation of BMS-791325 and its active N-demethyl metabolite (BMS-794712) in human plasma and urine. Plasma and urine samples were extracted with methyl-t-butyl ether followed by an LC-MS/MS analysis which was conducted in a multiple reaction monitoring (MRM) mode for the simultaneous detection of the two analytes in human plasma (0.1-50 ng/mL) and in human urine (5-2500 ng/mL). Intra-run precision (3.0% R.S.D.), inter-run precision (5.3% R.S.D.), and accuracy (±4.7% deviation) from plasma and urine quality control samples provide evidence of the methods accuracy and precision. Selectivity, stability in matrices, extraction recovery, matrix effect on LC-MS detection, and interference of coadministered drugs (famotidine and ritonavir) were all acceptable. Reproducibility of the plasma method was demonstrated by reanalysis of a portion of study samples. The results of cross-validations demonstrated the equivalency of two methods validated in two labs. The plasma method was applied to the analysis of several thousand clinical study samples for PK evaluations of the drug in normal healthy subjects and in patients. The urine method was used in the first in human study to evaluate renal clearance and urinary recovery.

Multiplexed LC-MS/MS method for the simultaneous quantitation of three novel hepatitis C antivirals, daclatasvir, asunaprevir, and beclabuvir in human plasma.

Dual or triple combination regimens of novel hepatitis C direct-acting antivirals (DAA, daclatasvir, asunaprevir, or beclabuvir) provide high sustained virological response rates and reduced frequency of resistance compared to clinical monotherapy. To support pharmacokinetic (PK) assessments in clinical studies, a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitation of daclatasvir, asunaprevir, beclabuvir (BMS-791325) and its active metabolite (BMS-794712) in human plasma was developed and validated. Human plasma samples were extracted with methyl-t-butyl ether followed by an LC-MS/MS analysis, which was conducted in a multiple reaction monitoring (MRM) mode. The lower limits of quantitation (LLOQ) were 1 ng/mL for daclatasvir, asunaprevir, and BMS-794712, and 2 ng/mL for beclabuvir. Intra-run precision (≤4.5% CV), inter-run precision (≤2.9% CV), and accuracy (±5.3% deviation) based on different concentration levels (low, geometric mean, mid and high) of the quality control samples (QCs) provided evidence of the methods accuracy and precision. Selectivity and matrix effect on LC-MS/MS detection, stability in plasma, and potential interference of coadministered drugs (ribavirin and interferon) were all evaluated and the results were acceptable. Method reproducibility was demonstrated by the reanalysis of a portion of study samples. The cross-validation results for QCs demonstrated the equivalency between this method and two single-analyte methods which were previously validated for quantitation of daclatasvir in human plasma. This approach of using a multiplexed LC-MS/MS method for the simultaneous quantitation of three DAAs is time- and cost-effective, and can maintain good data quality in sample analysis.

Thermal isomerization of (3-butenyl)cyclopropane to norbornane.

[reaction: see text] (3-Butenyl)cyclopropane isomerizes thermally to norbornane and gives rise to many other products. Deuterium and carbon-13-labeled versions of (3-butenyl)cyclopropane have been prepared and isomerized, establishing that the formation of norbornane involves cleavage of the cyclopropyl C2-C3 bond.

Suprafacial and Antarafacial Paths for the Thermal Vinylcyclopropane-to-Cyclopentene Rearrangement of 1-Ethenylbicyclo[4.1.0]heptane to Bicyclo[4.3.0]non-1(9)-ene.

The gas phase thermal rearrangement of 1-ethenylbicyclo[4.1.0]heptane at 338 degrees C gives the expected vinylcyclopropane-to-cyclopentene product, bicyclo[4.3.0]non-1(9)-ene. The analogous rearrangement of 1-(2'-(E)-d-ethenyl)bicyclo[4.1.0]heptane takes place with the allylic moiety being utilized in both suprafacial and antarafacial stereochemical ways, for both endo and exo isomers of 8-d-bicyclo[4.3.0]non-1(9)-ene are formed. The product ratio, defined by deuterium NMR in the presence of Ag(fod) and Yb(fod)(3) shift reagents, corresponds to (79 +/- 2)% suprafacial (sr + si) and (21 +/- 2)% antarafacial (ar + ai) reaction stereochemistry.