Cryogen-free 400-MHz nuclear magnetic resonance spectrometer as a versatile tool for pharmaceutical process analytical technology.

The discovery of new ceramic materials containing Ba-La-Cu oxides in 1986 that exhibited superconducting properties at high temperatures in the range of 35 K or higher, recognized with the Nobel Prize in Physics in 1987, opened a new world of opportunities for nuclear magnetic resonance (NMRs) and magnetic resonance imaging (MRIs) to move away from liquid cryogens. This discovery expands the application of high temperature superconducting (HTS) materials to fields beyond the chemical and medical industries, including electrical power grids, energy, and aerospace. The prototype 400-MHz cryofree HTS NMR spectrometer installed at Amgen's chemistry laboratory has been vital for a variety of applications such as structure analysis, reaction monitoring, and CASE-3D studies with RDCs. The spectrometer has been integrated with Amgen's chemistry and analytical workflows, providing pipeline project support in tandem with other Kinetic Analysis Platform technologies. The 400-MHz cryofree HTS NMR spectrometer, as the name implies, does not require liquid cryogens refills and has smaller footprint that facilitates installation into a chemistry laboratory fume hood, sharing the hood with a process chemistry reactor. Our evaluation of its performance for structural analysis with CASE-3D protocol and for reaction monitoring of Amgen's pipeline chemistry was successful. We envision that the HTS magnets would become part of the standard NMR and MRI spectrometers in the future. We believe that while the technology is being developed, there is room for all magnet options, including HTS, low temperature superconducting (LTS) magnets, and low field benchtop NMRs with permanent magnets, where utilization will be dependent on application type and costs.

Anisotropic NMR data acquisition with a prototype 400 MHz cryogen-free NMR spectrometer.

High-temperature superconducting (HTS) materials have recently been incorporated into the construction of HTS cryogen-free magnets for nuclear magnetic resonance (NMR) spectroscopy. These HTS NMR spectrometers do not require liquid cryogens, thereby providing significant cost savings and facilitating easy integration into chemistry laboratories. However, the optimal performance of these HTS magnets against standard cryogen NMR magnets must be evaluated, especially with demanding modern NMR applications such as NMR in anisotropic media. The stability of the HTS magnets over time and their performance with complex pulse sequence experiments are the main unknown factors of this new technology. In this study, we evaluate the utility of our prototype 400 MHz cryogen-free power-driven HTS NMR spectrometer, installed in the fumehood of a chemistry laboratory, for stereochemical analysis of three commercial natural products (artemisinin, artemether, and dihydroartemisinin) via measurement of anisotropic NMR data, in particular, residual dipolar couplings. The accuracy of measurement of the anisotropic NMR data with the HTS magnet spectrometer is evaluated through the CASE-3D fitting protocol, as implemented in the Mestrenova-StereoFitter software program.

Atroposelective Brønsted Acid-Catalyzed Photocyclization to Access Chiral N-Aryl Quinolones with Low Rotational Barriers.

Herein, a novel route to atropisomeric N-aryl quinolones with low rotational barriers is demonstrated, leveraging a dual photochemical/organocatalytic approach to the required ring closure in up to 94% yield and up to >99% ee. The use of a continuous flow system allows for impurity suppression and enables rapid scale-up to a decagram scale.

Ipso Nitration of Aryl Boronic Acids Using Fuming Nitric Acid.

The ipso nitration of aryl boronic acid derivatives has been developed using fuming nitric acid as the nitrating agent. This facile procedure provides efficient and chemoselective access to a variety of aromatic nitro compounds. While several activating agents and nitro sources have been reported in the literature for this synthetically useful transformation, this report demonstrates that these processes likely generate a common active reagent, anhydrous HNO3. Kinetic and mechanistic studies have revealed that the reaction order in HNO3 is >2 and indicate that the •NO2 radical is the active species.

An algorithm for discovery and determination of exponentially decaying components in nuclear magnetic resonance relaxometry data.

In many branches of physics, the time evolution of various quantities measured in systems passing from excited to equilibrium states, while theoretically very complex, can be in practice well approximated by a sum of exponential decays. Multiexponential relaxometry data analysis is about determining the number of exponential components and their corresponding amplitudes and decay rates, starting from noisy recorded time series, under the assumption of the discreteness of the number of components present. A technique for decomposing a signal modelled as a sum of exponential decays into its components is introduced, consisting of a modified version of the algorithm minimum description length (MDL) + matrix pencil, originally proposed by Lin et al. for the analysis of nuclear magnetic resonance spectroscopy data. The procedure starts by denoising the discrete time-domain signal, and then a number of different decimations are applied, each being followed by an MDL + matrix pencil detection-estimation step, and finally, a postprocessing of the intermediate outcomes is done. The comprised model order estimator eliminates the need of providing prior estimates of the number of components present.

Development of a Commercial Process To Prepare AMG 232 Using a Green Ozonolysis-Pinnick Tandem Transformation.

A robust process to manufacture AMG 232 was developed to deliver drug substance of high purity. Highlights of the commercial process development efforts include the following: (i) use of a novel bench-stable Vilsmeier reagent, methoxymethylene- N, N-dimethyliminium methyl sulfate, for selective in situ activation of a primary alcohol intermediate; (ii) use of a new crystalline and stable isopropyl calcium sulfinate reagent ensuring robust preparation of a sulfone intermediate; (iii) development of a safe ozonolysis process conducted in an aqueous solvent mixture in either batch or continuous manufacturing mode; and (iv) control of the drug substance purity by crystallization of a salt rejecting impurities effectively. The new process was demonstrated to afford the drug substance (99.9 LC area %) in 49.8% overall yield from starting material DLAC (1).

Performance of new 400-MHz HTS power-driven magnet NMR technology on typical pharmaceutical API, cinacalcet HCl.

After years towards higher field strength magnets, nuclear magnetic resonance (NMR) technology in commercial instruments in the past decade has expanded at low and high magnetic fields to take advantage of new opportunities. At lower field strengths, permanent magnets are well established, whereas for midrange and high field, developments utilize superconducting magnets cooled with cryogenic liquids. Recently, the desire to locate NMR spectrometers in nontypical NMR laboratories has created interest in the development of cryogen-free magnets. These magnets require no cryogenic maintenance, eliminating routine filling and large cryogen dewars in the facility. Risks of spontaneous quenches and safety concerns when working with cryogenic liquids are eliminated. The highest field commercially available cryogen-free NMR magnet previously reported was at 4.7 T in 2013. Here we tested a prototype cryogen-free 9.4-T power-driven high-temperature-superconducting (HTS) magnet mated to commercial NMR spectrometer electronics. We chose cinacalcet HCl, a typical active pharmaceutical ingredient, to evaluate its performance towards structure elucidation. Satisfactory standard 1D and 2D homonuclear and heteronuclear NMR results were obtained and compared with those from a standard 9.4-T cryogenically cooled superconducting NMR instrument. The results were similar between both systems with minor differences. Further comparison with different shims and probes in the HTS magnet system confirmed that the magnet homogeneity profile could be matched with commercially available NMR equipment for optimal results. We conclude that HTS magnet technology works well providing results comparable with those of standard instruments, leading us to investigate additional applications for this magnet technology outside a traditional NMR facility.

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products.

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the average content of fluorinated drugs in their formulated drug product forms: tablets or capsules. This method is specific to fluorinated drugs because it detects only the content of fluorine, avoiding interference from the excipients that lack fluorine. The advantages of measuring the active content of fluorinated drugs using low-field 19F TD-NMR versus high-field 19F solid-state (SS) NMR are the simplicity of the method; the low cost; and the non-destructive nature of the technique, with all samples recoverable in intact forms (e.g., powders, tablets, and capsules), making this technique affordable for any laboratory. We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content. Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

Isolation and structure elucidation of unexpected in-process impurities during tetrazole ring formation of an investigational drug substance.

During the formation of a tetrazole ring on an investigational drug, two in-process impurities were detected and analyzed by LC-MS, which suggested that both impurities were drug-related with the same mass-to-charge ratio. To understand and control their formation, both impurities were isolated from the mother liquor of the reaction using a multi-step isolation procedure to obtain a sufficient amount for high-resolution mass spectrometry (HRMS) and NMR structural analysis. HRMS suggested a protonated mass of 577.32 Da for both impurities; however, MS fragmentation patterns provided limited information on their structures. NMR analysis indicated the presence on an additional NH functional group in both isolates with similar spatial and bond correlations to one of the dimethylcarbamoyl moieties and the corresponding aromatic ring. A phenyldimethylcarbamoylamino moiety was supported by the NMR and HRMS data and could be explained based on the 'Schmidt-like' reaction mechanism, which was an unexpected reaction pathway. Because the reaction conditions were fixed because of safety concerns, the crystallization protocol was redesigned to reduce the levels of these impurities significantly. Copyright © 2016 John Wiley & Sons, Ltd.

Application of (19) F time-domain NMR to measure content in fluorine-containing drug products.

It is necessary to show that the active content in the dosage form of drugs is within a certain narrow range of the label claim. In case of fluorinated drugs, the active content can be measured by high field solid state NMR because the excipients lack fluorine. To make NMR reachable to any laboratory, simple to use, and at a low cost, measurement of (19) F nucleus using a 23 MHz (for (1) H) low field benchtop time-domain (TD) NMR was investigated. Three fluorinated drug products, cinacalcet, lansoprazole, and ciprofloxacin, were chosen for this study. The doses for these drug products range from 15 to 500 mg. The average drug content measured using (19) F TD-NMR compares well with the reported label claims for the three drugs tested. (19) F TD-NMR is a simple and non-destructive technique to measure drug content in tablets. In addition, the accessibility and simplicity of the technique makes it an excellent process analytical technology tool for development and manufacturing in the pharmaceutical industry. Copyright © 2015 John Wiley & Sons, Ltd.

Monitoring chemical reactions by low-field benchtop NMR at 45 MHz: pros and cons.

Monitoring chemical reactions is the key to controlling chemical processes where NMR can provide support. High-field NMR gives detailed structural information on chemical compounds and reactions; however, it is expensive and complex to operate. Conversely, low-field NMR instruments are simple and relatively inexpensive alternatives. While low-field NMR does not provide the detailed information as the high-field instruments as a result of their smaller chemical shift dispersion and the complex secondary coupling, it remains of practical value as a process analytical technology (PAT) tool and is complimentary to other established methods, such as ReactIR and Raman spectroscopy. We have tested a picoSpin-45 (currently under ThermoFisher Scientific) benchtop NMR instrument to monitor three types of reactions by 1D (1) H NMR: a Fischer esterification, a Suzuki cross-coupling, and the formation of an oxime. The Fischer esterification is a relatively simple reaction run at high concentration and served as proof of concept. The Suzuki coupling is an example of a more complex, commonly used reaction involving overlapping signals. Finally, the oxime formation involved a reaction in two phases that cannot be monitored by other PAT tools. Here, we discuss the pros and cons of monitoring these reactions at a low-field of 45 MHz by 1D (1) H NMR. Copyright © 2015 John Wiley & Sons, Ltd.

An efficient, regioselective amination of 3,5-disubstituted pyridine N-oxides using saccharin as an ammonium surrogate.

A process for the regioselective amination of unsymmetrical 3,5-substituted pyridine N-oxides has been developed utilizing cheap, readily available saccharin as an ammonium surrogate. High conversions of the corresponding saccharin adducts have been achieved under mild reaction conditions. In situ deprotection under acidic conditions allows for a one-pot process to substituted aminopyridines. High regioselectivities were obtained from a variety of 3,5-disubstituted pyridine N-oxides.

A Multidisciplinary Investigation to Determine the Structure and Source of Dimeric Impurities in AMG 517 Drug Substance.

In the initial scale-up batches of the experimental drug substance AMG 517, a pair of unexpected impurities was observed by HPLC. Analysis of data from initial LC-MS experiments indicated the presence of two dimer-like molecules. One impurity had an additional sulfur atom incorporated into its structure relative to the other impurity. Isolation of the impurities was performed, and further structural elucidation experiments were conducted with high-resolution LC-MS and 2D NMR. The dimeric structures were confirmed, with one of the impurities having an unexpected C-S-C linkage. Based on the synthetic route of AMG 517, it was unlikely that these impurities were generated during the last two steps of the process. Stress studies on the enriched impurities were carried out to further confirm the existence of the C-S-C linkage in the benzothiazole portion of AMG 517. Further investigation revealed that these two dimeric impurities originated from existing impurities in the AMG 517 starting material, N-acetyl benzothiazole. The characterization of these two dimeric impurities allowed for better quality control of new batches of the N-acetyl benzothiazole starting material. As a result, subsequent batches of AMG 517 contained no reportable levels of these two impurities.

Metabolism of MK-0524, a prostaglandin D2 receptor 1 antagonist, in microsomes and hepatocytes from preclinical species and humans.

(3R)-4-(4-Chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl acetic acid (MK-0524) is a potent orally active human prostaglandin D(2) receptor 1 antagonist that is currently under development for the prevention of niacin-induced flushing. The major in vitro and in vivo metabolite of MK-0524 is the acyl glucuronic acid conjugate of the parent compound, M2. To compare metabolism of MK-0524 across preclinical species and humans, studies were undertaken to determine the in vitro kinetic parameters (K(m) and V(max)) for the glucuronidation of MK-0524 in Sprague-Dawley rat, beagle dog, cynomolgus monkey, and human liver microsomes, human intestinal microsomes, and in recombinant human UDP glucuronosyltransferases (UGT). A comparison of K(m) values indicated that UGT1A9 has the potential to catalyze the glucuronidation of MK-0524 in the liver, whereas UGT1A3 and UGT2B7 have the potential to catalyze the glucuronidation in the intestine. MK-0524 also was subject to phase I oxidative metabolism; however, the rate was significantly lower than that of glucuronidation. The rate of phase I metabolism was ranked as follows: rat approximately monkey > human intestine > dog > human liver with qualitatively similar metabolite profiles across species. In all the cases, the major metabolites were the monohydroxylated epimers (M1 and M4) and the keto-metabolite, M3. Use of inhibitory monoclonal antibodies and recombinant human cytochromes P450 suggested that CYP3A4 was the major isozyme involved in the oxidative metabolism of MK-0524, with a minor contribution from CYP2C9. The major metabolite in hepatocyte preparations was the acyl glucuronide, M2, with minor amounts of M1, M3, M4, and their corresponding glucuronides. Overall, the in vivo metabolism of MK-0524 is expected to proceed via glucuronidation, with minor contributions from oxidative pathways.

In vitro bioactivation of dihydrobenzoxathiin selective estrogen receptor modulators by cytochrome P450 3A4 in human liver microsomes: formation of reactive iminium and quinone type metabolites.

Estrogens and selective estrogen receptor modulators (SERMs) are prescribed widely in the clinic to alleviate symptoms in postmenopausal women, and they are metabolized to reactive intermediates, which may elicit adverse effects. As part of our efforts to develop safer SERMs, in vitro covalent protein binding of (2S,3R)-(+)-3-(4-hydroxyphenyl)-2-[4-(2-piperidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (I) was evaluated. Radioactivity from [3H]I became covalently bound to proteins in a fashion that was both time- and NADPH-dependent in human liver microsomes and reached a value of 1106 pmol equiv/mg protein following a 45 min incubation. At least three pathways are involved in the bioactivation of I, namely, oxidative cleavage of the dihydrobenzoxathiin moiety to give a hydroquinone/para-benzoquinone redox couple, hydroxylation at position 5 or 7 of the benzoxathiin moiety leading to an o-quinone intermediate, and metabolism of the piperidine ring to give an iminium ion. The latter reactive intermediate was identified as its bis-cyano adduct when human liver microsomal incubations were performed in the presence of sodium cyanide. Structural modification of I, including a replacement of the piperidine with a pyrrolidine group, led to (2S,3R)-(+)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol (II), which did not form a reactive iminium ion. Following the incubation of II with human liver microsomes, covalent binding to proteins was reduced (461 pmol equiv/mg protein), the residual level of binding apparently due to the formation of a rearranged biphenyl quinone type metabolite. Studies with inhibitory antibodies and chemical inhibitors showed that P450 3A4 was the primary enzyme responsible for oxidative bioactivation of I and II in human liver microsomes. These studies thus demonstrated that gaining an understanding of bioactivation mechanisms may be exploited in terms of guiding structural modifications of drug candidates to minimize covalent protein binding and, hopefully, to lower the potential for drug-mediated adverse effects.

Dioxododecenoic acid: a lipid hydroperoxide-derived bifunctional electrophile responsible for etheno DNA adduct formation.

It has been proposed that 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid [13(S)-HPODE]-mediated formation of 4-oxo-2(E)-nonenal and 4-hydroxy-2(E)-nonenal arises from a Hock rearrangement. This suggested that a 4-oxo-2(E)-nonenal-related molecule, 9,12-dioxo-10(E)-dodecenoic acid (DODE), could also result from the intermediate formation of 9-hydroperoxy-12-oxo-10(E)-dodecenoic acid. A recent report has described the formation of DODE-derived etheno adducts when 13(S)-HPODE was allowed to decompose in the presence of 2'-deoxynucleosides or DNA. However, the regioselectivity of lipid hydroperoxide-derived DODE addition to 2'-deoxyguanosine (dGuo) or other 2'-deoxynucleosides was not determined. The structure of carboxynonanone-etheno-dGuo formed from vitamin C-mediated 13(S)-HPODE decomposition has now been established by a combination of 1H and 13C NMR spectroscopy studies of its bis-methylated derivative. The site of dGuo methylation was first established as being at N-5 rather than at O-9 from NMR analysis of a methyl derivative of the model compound, heptanone-etheno-dGuo. (1)H,(13)C 2D heteronuclear multiple bond correlations were then used to establish unequivocally that the bis-methyl derivative of carboxynonanone-etheno-dGuo was 3-(2'-deoxy-beta-d-erythropentafuranosyl)imidazo-7-(9' '-carboxymethylnona-2' '-one)-9-oxo-5-N-methyl[1,2-a]purine rather than its 6-(9' '-carboxymethylnona-2"-one)-9-oxo-5-N-methyl[1,2-a]purine regioisomer. Therefore, etheno adduct formation occurred by initial nucleophilic attack of the exocyclic N(2) amino group of dGuo at the C-12 aldehyde of DODE to form an unstable carbinolamine intermediate. This was followed by intramolecular Michael addition of the pyrimidine N1 of dGuo to C-11 of the resulting alpha,beta-unsaturated ketone. Subsequent dehydration gave 3-(2'-deoxy-beta-d-erythropentafuranosyl)imidazo-7-(9' '-carboxynona-2' '-one)-9-oxo-[1,2-a]purine (carboxynonanone-etheno-dGuo). An efficient synthesis of DODE was developed starting from readily available 1,8-octanediol using a furan homologation procedure. This synthetic method allowed multigram quantities of DODE to be readily prepared. Synthetic DODE when reacted with dGuo gave carboxynonanone-etheno-dGuo that was identical with that derived from vitamin C-mediated 13(S)-HPODE decomposition in the presence of dGuo.

Unexpected formation of etheno-2'-deoxyguanosine adducts from 5(S)-hydroperoxyeicosatetraenoic acid: evidence for a bis-hydroperoxide intermediate.

Analysis of products from the reaction between 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid and 2'-deoxyguanosine in the presence of FeII, FeIII, or vitamin C by liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry revealed the presence of four DNA adducts. Surprisingly, adducts I and II had mass spectral characteristics identical to those for 1,N2-etheno-2'-deoxyguanosine and heptanone-1,N2-etheno-2'-deoxyguanosine. These adducts have previously been shown to arise from the homolytic decomposition of 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid. It appears that under the reaction conditions, 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid was subjected to a previously unknown peroxidation reaction to give a bis-hydroperoxide intermediate that underwent a Hock rearrangement to produce 3(Z)-nonenal from the omega-terminus. The 3(Z)-nonenal was then converted to 4-hydroperoxy-2-nonenal, a precursor to the formation of 4-oxo-2-nonenal. 4-Oxo-2-nonenal forms heptanone-1,N2-etheno-adducts with 2'-deoxyguanosine, whereas 4-hydroperoxy-2-nonenal forms 1,N2-etheno-2'-deoxyguanosine. Two novel carboxylate adducts were also identified. The structure of the more abundant adduct (III) was characterized as its methyl ester derivative by NMR spectroscopy as 3-(2'-deoxy-beta-D-erythropentafuranosyl)imidazo-7-(5' '-carboxypenta-2' '-one)-9-oxo[1,2-alpha]purine (5-carboxy-2-pentanone-1,N2-etheno-2'-deoxyguanosine). This etheno adduct was formed by the reaction of 2'-deoxyguanosine with 5,8-dioxo-6(E)-octenoic acid. The bifunctional electrophile is proposed to arise from the alpha-terminus during the Hock rearrangement of bis-hydroperoxide derived from 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid. 5-Carboxy-2-pentanone-1,N2-etheno-2'-deoxyguanosine may serve as a biomarker of 5-lipoxygenase-mediated oxidative stress. The less abundant carboxylate adduct IV arose from a quite different pathway and was tentatively characterized as 6-carboxy-3-hydroxy-1-hexene-1,N2-etheno-2'-deoxyguanosine.

In vitro metabolism of a new oxazolidinedione hypoglycemic agent utilizing liver microsomes and recombinant human cytochrome P450 enzymes.

The compound, 5-{4-[3-(4-cyclohexyl-2-propylphenoxy)propoxy]phenyl}-1,3-oxazolidine-2,4-dione (compound A) is a peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist. PPARgamma agonists have proven useful in the treatment of type 2 diabetes, which is characterized by hyperglycemia, insulin resistance and/or abnormal insulin secretion. The metabolism of this oxazolidinedione (OZD) was investigated in male rat, dog, monkey and human liver microsomes, and recombinant human cytochrome P450 enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in the presence of NADPH. Routes of metabolism included monohydroxylation of the cyclohexane ring at multiple positions, monohydroxylation of the n-propyl side chain or the tether linkage, and OZD ring opening, giving rise to the keto amide and alcohol amide entities. Liver microsomes showed subtle qualitative and quantitative metabolic differences among rat, dog, monkey and human preparations. Further, CYP2C8 and CYP2C19 did not display different regioselectivity for hydroxylation on the cyclohexane ring with both of them giving rise to C-3 and C-4 hydroxy metabolites, but they did display different stereoselectivity with CYP2C8 preferring cyclohexane hydroxylation in equatorial positions and CYP2C19 in axial positions.