Syntheses of perdeuterated indoles and derivatives as probes for the biosyntheses of crucifer phytoalexins

A simple two‐step preparation of [²H₄]indole, a starting material necessary for the synthesis of various crucifer metabolites, starting with readily available ¹H NMR solvent [²H₅]nitrobenzene (99% deuterated) was developed. [4,5,6,7‐²H₄]Indole 99% deuterated at the specified positions was then used to synthesize [4′,5′,6′,7′‐²H₄]indolyl‐3‐acetaldoxime, [4′,5′,6′,7′‐²H₄]1‐methoxyindolyl‐3‐acetaldoxime, [1″,1″,1″,4′,5′,6′,7′‐²H₇]1‐methoxyindolyl‐3‐acetaldoxime, [4′,5′,6′,7′‐²H₄]1‐methoxybrassinin, and [3,3,3,4′,5′,6′,7′‐²H₇]1‐methoxybrassinin. Copyright © 2005 John Wiley & Sons, Ltd.     

Synthesis of tritium‐labeled (R,R)‐4‐methoxyfenoterol

The preparation of 2′,4′,6′‐[³H₃]‐(R,R)‐4‐methoxyfenoterol, a tritium‐labeled derivative of (R,R)‐4‐methoxyfenoterol was demonstrated on a 15 mCi scale providing material with a specific activity of 57 Ci/mmol. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of isotopomers of dopamine labeled with deuterium or tritium

The synthesis of four isotopomers of dopamine labeled with deuterium or tritium is reported. The ring labeled [2′,5′,6′‐²H₃]‐, and [2′,5′,6′‐³H₃]‐dopamine were obtained using acid catalyzed isotopic exchange between dopamine and heavy or tritiated water respectively. Two selectively labeled isotopomers, i.e. [1R‐²H]‐, and [1R‐³H]‐dopamine were synthesized by enzymatic decarboxylation of L ‐DOPA using the enzyme tyrosine decarboxylase (EC 4.1.1.25) from Streptococcus faecalis. Copyright © 2006 John Wiley & Sons, Ltd.     

Enzymatic synthesis of isotopomers of tyramine labeled with deuterium and tritium

The combined chemical and enzymatic methods of synthesis of five isotopomers of L ‐tyrosine, L ‐Tyr, and their derivatives, i.e. corresponding isotopomers of tyramine (TA), labeled with deuterium and tritium have been reported. Two‐step synthesis consists with introduction of deuterium or tritium label into intermediate L ‐Tyr using isotope exchange followed by enzymatic decarboxylation using enzyme tyrosine decarboxylase (EC 4.1.1.25). This way five isotopomers of L ‐tyrosine, i.e. [2‐²H]‐L‐, [2‐³H]‐L‐,[2‐²H/³H]‐L‐, [3′,5′‐²H₂]‐L‐,[3′,5′‐³H₂]‐L ‐Tyr, and six isotopomers of tyramine i.e. [1S‐²H]‐, [1S‐³H]‐, [1S‐²H/³H]‐, [3′,5′‐²H₂]‐, [3′,5′‐²H₂]‐, [2′,6′‐²H₂]‐TA were obtained. Copyright © 2007 John Wiley & Sons, Ltd.     

Labeled oxazaphosphorines for applications in MS studies. Synthesis of deuterium labeled cyclophosphamides and ifosfamides

A variety of deuterated 3‐amino‐1‐propanols were made by the LiAlD₄ or AlD₃ reduction of nitrile or ester precursors. The labeled aminopropanols and/or deuterated bis(2‐chloroethyl)amines were used to synthesize [4,4‐²H₂]‐, [6,6‐²H₂]‐, [α,α.α′,α′‐²H₄]‐, and [4,4,β,β,β′,β′‐²H₆]‐cyclophosphamide. The labeled aminopropanols were also used to synthesize deuterated 3‐(2′‐benzyloxyethylamino)‐1‐propanols [BnOCH₂CH₂NH(CH₂)₃OH] which, along with labeled and unlabeled 2‐chloroethylamines, led to [4,4‐²H₂]‐, [6,6‐²H₂]‐, [α,α‐²H₂]‐, [α′,α′‐²H₂]‐, [α,α.α′,α′‐²H₄]‐, and [6,6,α,α,α′,α′‐²H₆]‐ifosfamide. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis of [2H8]‐enterolactone and [2H10]‐enterodiol

Enterolactone and enterodiol are the main mammalian metabolites of dietary butyrolactone type lignans. The study of biological properties and potential health effects of these compounds requires isotopically labelled compounds as standards for quantitative measurements. An expedient deutero‐labelling method for enterolactone is to use the D₃PO₄·BF₃/D₂O complex at room temperature which will exchange all eight aromatic hydrogens, even from inactivated meta positions, to form [2,4,5,6,2′,4′,5′,6′‐²H₈]‐enterolactone in 74% yield and 99% isotopic purity. [2,4,5,6,9,9,2′,4′,5′,6′‐²H₁₀]‐Enterodiol was prepared from [2,4,5,6,2′,4′,5′,6′‐²H₈]‐enterolactone by reduction with LiAlD₄ which introduces two more deuterium atoms into the molecule. Copyright © 2003 John Wiley & Sons, Ltd.     

Synthesis of tritium labelled mecillinam

The potent β‐lactam antibiotic mecillinam has successfully been labelled with tritium. 2,3,4,7‐Tetrahydro‐1H‐azepin‐1‐carbaldehyde was formed in a ring‐closing metathesis reaction and then condensed with trimethylsilyl protected penicillanic acid to give dehydro‐mecillinam. Pd/C catalysed tritiation then gave [³H₂]‐mecillinam (6‐β (([3,4‐³H₂]hexahydro‐1H‐azepin‐1‐yl)methyleneamino)penicillanic acid) with a radiochemical purity of 96% and a specific activity of 24 Ci/mmol. Copyright © 2003 John Wiley & Sons, Ltd.     

Preparation of deuterium‐labeled biotransformation products of 2,4,6‐trinitrotoluene

Methods for the preparation of deuterium‐labeled analogs to six prominent biotransformation products of the explosive 2,4,6‐trinitrotoluene were developed. These are useful as reference standards for stable isotope dilution techniques and for solid state ²H NMR spectroscopic studies. Although syntheses for most of the target compounds in protiated form had been reported in the past, most of those were found to be poorly suited for the preparation of the deuterated materials. Selective reduction of [²H₅]trinitrotoluene furnished [²H₅]‐4,6‐dinitro‐2‐hydroxylaminotoluene, [²H₅]‐2,6‐dinitro‐4‐hydroxylaminotoluene, [²H₅]‐2‐amino‐4,6‐dinitrotoluene, and [²H₅]‐4‐amino‐2,6‐dinitrotoluene. The syntheses of [²H₁₀]‐2,2′‐azo‐4,4′,6,6′‐tetranitrotoluene and [²H₁₀]‐4,4′‐azo‐2,2′,6,6′‐tetranitrotoluene were accomplished by selective oxidation of [²H₅]‐2‐amino‐4,6‐dinitrotoluene and [²H₅]‐4‐amino‐2,6‐dinitrotoluene, respectively.     

Synthesis of tritium labeled isotopomers of L‐tyrosine

The synthesis of four selectively labeled isotopomers of L ‐tyrosine, (L ‐Tyr), using chemical and enzymatic methods is reported. Four tritium labeled isotopomers of L ‐phenylalanine (L ‐Phe) – [2‐³H]‐, [2′,6′‐³H₂]‐, [3R‐³H]‐ and [3S‐³H]‐ have been synthesized using a combination of chemical and enzymatic methods. The labeled isotopomers of L ‐Phe have been converted into [2 ‐³H]‐, [2′,6′‐³H₂]‐, [3R‐³H]‐, and [3S‐³H]‐L ‐Tyr by using the enzyme L ‐phenyl‐alanine 4′‐monooxygenase. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of 13C‐labeled 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐[13C5] ribofuranosyl 5′‐monophosphate

5‐Aminoimidazole‐4‐carboxamide‐1‐β‐D‐[¹³C₅] ribofuranosyl 5′‐monophosphate ([¹³C₅ ribose] AICAR‐PO₃H₂) ( 6 ) has been synthesized from [¹³C₅]adenosine. Incorporation of the mass‐label into [¹³C₅ ribose] AICAR‐PO₃H₂ provides a useful standard to aid in metabolite identification and quantification in monitoring metabolic pathways. A synthetic route to the ¹³C‐labeled compound has not been previously reported. Our method employs a hybrid enzymatic, and chemical synthesis approach that applies an enzymatic conversion from adenosine to inosine followed by a ring‐cleavage of the protected inosine. A direct phosphorylation of the resulting 2′,3′‐isopropylidine acadesine ( 5 ) was developed to yield the title compound in 99% purity following ion exchange chromatography.     

Type and location of fluorescent probes incorporated into the potent μ‐opioid peptide [Dmt1]DALDA affect potency,receptor selectivity and intrinsic efficacy*

Abstract: The dermorphin‐derived tetrapeptide H‐Dmt‐d ‐Arg‐Phe‐Lys‐NH₂ (Dmt = 2′,6′‐dimethyltyrosine) ([Dmt¹]DALDA) is a highly potent and selective μ‐opioid agonist capable of crossing the blood–brain barrier and producing a potent, centrally mediated analgesic effect when given systemically. For the purpose of biodistribution studies by fluorescence techniques, [Dmt¹]DALDA analogues containing various fluorescent labels [dansyl, anthraniloyl (atn), fluorescein, or 6‐dimethylamino‐2′‐naphthoyl] in several different locations of the peptide were synthesized and characterized in vitro in the guinea‐pig ileum and mouse vas deferens assays, and in μ‐, δ‐ and κ‐opioid receptor‐binding assays. The analogues showed various degrees of μ receptor‐binding selectivity, but all of them were less μ‐selective than the [Dmt¹]DALDA parent peptide. Most analogues retained potent, full μ‐agonist activity, except for one with fluorescein attached at the C‐terminus ( 3a ) (partial μ‐agonist) and one containing β‐(6′‐dimethylamino‐2′‐naphthoyl)alanine (aladan) in place of Phe³ ( 4 ) (μ‐ and κ‐antagonist). The obtained data indicate that the receptor‐binding affinity, receptor selectivity and intrinsic efficacy of the prepared analogues vary very significantly, depending on the type of fluorescent label used and on its location in the peptide. The results suggest that the biological activity profile of fluorescence‐labeled peptide analogues should always be carefully determined prior to their use in biodistribution studies or other studies. One of the analogues containing the atn group ( 2a ) proved highly useful in a study of cellular uptake and intracellular distribution by confocal laser scanning microscopy.     

Multiple labeling of a potent CX3CR1 antagonist for the treatment of multiple sclerosis

Several methods for the preparation of five isotopologues of the CX₃CR1 antagonist 1 were developed. Volatile and radioactive 1‐chloro‐ and 1‐bromo‐ethyl‐benzene was handled in [2′‐¹⁴C] and [3′, 5′‐³H] labeling of 1. d ‐Leucinol ((R)‐2‐amino‐4‐methylpentan‐1‐ol) was labeled as [1‐¹⁴C] and [4‐¹⁴C] via a Wittig reaction using Garner's aldehyde and a Strecker amino acid synthesis with d ‐acylase resolvation, respectively. A [²H₁₀]d ‐leucinol was used for the stable labeled [M + 10] isotopologue. The products were isolated with 97.6–100% stereo chemical and radiochemical purity as for specific activity 768 GBq/mmol and 1.6–2.0 GBq/mmol, respectively.     

Synthesis of [14C]‐ and [13C6]‐labeled potent HIV non‐nucleoside reverse transcriptase inhibitor

5,11‐Dihydro‐11‐ethyl‐5‐methyl‐8‐{2‐{(1‐oxido‐4‐quinolinyl)oxy}ethyl}‐6H‐dipyrido[3,2‐b:2′,3′‐e][1,4]diazepin‐6‐one, (1), labeled with carbon‐14 in the quinoline–benzene ring, in one of the pyridine rings of the dipyridodiazepinone tricyclic moiety, and in the side chain, was prepared in three different syntheses with specific activities ranging from 44 to 47 mCi/mmol (1.63–1.75 GBq/mmol). In the first synthesis, 5,11‐dihydro‐11‐ethyl‐8‐(2‐hydroxyethyl)‐5‐methyl‐6H‐dipyrido[3,2‐b:2′,3′‐e][1,4]diazepin‐6‐one (2) was coupled to 4‐hydroxyquinoline, [benzene‐¹⁴C(U)]‐, using Mitsunobu's reaction conditions, followed by the oxidation of the quinoline nitrogen with 3‐chloroperoxybenzoic acid to give ([¹⁴C]‐(1a)) in 43% radiochemical yield. Second, 3‐amino‐2‐chloropyridine, [2,6‐¹⁴C]‐, was used to prepare 8‐bromo‐5,11‐dihydro‐11‐ethyl‐5‐methyl‐6H‐dipyrido[3,2‐b:2′,3′‐e][1,4]diazepin‐6‐one (8), and then Stille coupled to allyl(tributyl)tin followed by ozonolysis of the terminal double bond and in situ reduction of the resulting aldehyde to alcohol (10). Mitsunobu etherification and oxidation as seen before gave ([¹⁴C]‐(1b)) in eight steps and in 11% radiochemical yield. Finally, carbon‐14 potassium cyanide was used to prepare isopropyl cyanoacetate (12), which was used to transform bromide (8) to labeled aryl acetic acid (13) under palladium catalysis. Trihydroborane reduction of the acid gave alcohol (14) labeled in the side chain, which was used as described above to prepare ([¹⁴C]‐(1c)) in 4.3% radiochemical yield. The radiochemical purities of these compounds were determined by radio‐HPLC and radio‐TLC to be more than 98%. To prepare [¹³C₆]‐(1), [¹³C₆]‐4‐hydroxyquinoline was prepared from [¹³C₆]‐aniline and then coupled to (2) and oxidized as seen before. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis and anticonvulsant activity of 1-(2-(8-(benzyloxy)quinolin-2-yl)-1-butyrylcyclopropyl)-3-substituted urea derivatives

H‐Dmt‐D‐Arg‐Phe‐Lys‐NH₂ ([Dmt¹]DALDA) is a synthetic tetrapeptide with extraordinary selectivity for the mu‐opioid receptor and is an extremely potent analgesic. [Dmt¹]DALDA is unusual in the way that the greater part of its analgesic potency appears to be produced by its actions in the spinal cord. Furthermore, [Dmt¹]DALDA inhibits norepinephrine re‐uptake and is a mitochondria‐targeted antioxidant. Such characteristics may make [Dmt¹]DALDA particularly effective against neuropathic pain. The present study was designed to compare the effects of [Dmt¹]DALDA and morphine on thermal hyperalgesia in an experimental neuropathic pain model. Neuropathic pain was induced in rats by surgical ligation of the L5 spinal nerve, and thermal pain thresholds were assessed by latencies of paw withdrawal to radiant heat. The increase in paw withdrawal latency was greater after the administration of [Dmt¹]DALDA than that of morphine in neuropathic rats at doses that were equianalgesic in naïve animals. We conclude that [Dmt¹]DALDA is more effective than morphine against thermal hyperalgesia in this experimental model of neuropathic pain.     

Isotopically labelled tropane alkaloids. The synthesis of (RS)‐[3′, 3′‐2H2]‐ and (RS)‐[1′‐13C, 3′, 3′‐2H2]‐ hyoscyamines for metabolism studies in plants

A synthetic route to isotopically labelled forms of the tropane alkaloid hyoscyamine, including (RS)‐[3′, 3′,‐²H₂]‐ ( 2a ) and (RS)‐[1′‐¹³C, 3′, 3′,‐²H₂]‐ ( 2b ) hyoscyamines, involving the reaction between phenylacetyl tropine and formaldehyde is described. The isotopically labelled products enable the metabolism of hyoscyamine to be studied in plants such as Datura stramonium. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of 3H and 14C labeled (S)‐3‐(5‐chloro‐2‐methoxyphenyl)‐1,3‐dihydro‐3‐fluoro‐6‐(trifluoromethyl)‐2H‐indol‐2‐one,maxipost™. An agent for post‐stroke neuroprotection

The syntheses of tritium labeled (S)‐3‐(5‐chloro‐2‐[OC³H₃]methoxyphenyl‐1,3‐dihydro‐3‐fluoro‐6‐(trifluoromethyl)‐1H‐indol‐2‐one, and carbon‐14 (S)‐3‐(5‐chloro‐2‐methoxyphenyl)‐1,3‐dihydro‐3‐fluoro‐6‐(trifluoromethyl)‐2H‐[2,3‐¹⁴C₂] indol‐2‐one are reported. The ³H‐labeled compound was prepared in a two‐step synthesis from C³H₃I. The final product was purified via chiral HPLC to yield the desired enantiomer in a 4% radiochemical yield and a specific activity of 60 Ci/mmol. The ¹⁴C‐labeled compound was prepared in a four‐step synthesis from diethyl [carboxylate‐¹⁴C₁,₂] oxalate. The final product was purified via chiral HPLC to yield the desired enantiomer in a 20% radiochemical yield and a specific activity of 28.4 μCi/mg. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of [2H3]‐labelled sulfamethoxazole and its main urinary metabolites

Sulfamethoxazole was labelled at positions 3, 5, and 4′ by H/D exchange in a mixture of 5% ²H₂SO₄ and 95% ²H₂O (v/v) under reflux for 72h with good isotope incorporation and acceptable yield. Subsequently, [²H₃]‐sulfamethoxazole‐N₁‐glucuronide and [²H₃]‐N₄‐acetyl‐sulfamethoxazole were synthesized. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis of 14C‐labelled EM‐800 (SCH 57050) and EM‐652·HCl (SCH 57068·HCl,acolbifene), pure selective estrogen receptor modulators

EM‐800 (SCH 57050) and EM‐652·HCl (SCH 57068·HCl, acolbifene) are orally active pure selective estrogen receptor modulators. The corresponding ¹⁴C₂‐radiolabelled compounds 1 and 2 were synthesized for metabolic studies with uniform labelling of two carbons within the benzene ring of the 2H‐1‐benzopyran moiety by optical resolution of racemic (±)‐[¹⁴C₂]EM‐343 4 . This pivotal intermediate amine was prepared in 6 steps with 38% yield from commercially available [U‐¹⁴C₂]resorcinol ( 3 ). Resolution by selective crystallization of the diastereomeric mixture of (S)‐(+)‐camphorsulfonates salts gave the desired (+)‐[¹⁴C₂]EM‐652·(+)‐CSA 13 . Moreover, the racemic amine 4 was recovered from mother liquors by basic treatment, and resolved again. We obtained salt 13 , at a 52% yield with 97% diastereomeric excess by repeating the resolution–racemization process. Finally, the corresponding dipivaloate (+)‐[¹⁴C₂]EM‐800 1 and hydrochloride salt (+)‐[¹⁴C₂]EM‐652·HCl 2 were prepared at respective specific activities of 19.7 and 24.5 µCi/mg with 96.3% radiochemical purity. Copyright © 2004 John Wiley & Sons, Ltd.     

A chimeric opioid peptide with mixed μ agonist/δ antagonist properties

Abstract: There is evidence to indicate that opioid compounds with mixed μ agonist/δ antagonist properties are analgesics with low propensity to produce tolerance and physical dependence. A chimeric peptide containing the potent and selective μ agonist H‐Dmt‐D‐Arg‐Phe‐Lys‐NH₂ ([Dmt¹]DALDA) (Dmt = 2′,6′‐dimethyltyrosine) and the potent and selective δ antagonist H‐Tyr‐TicΨ[CH₂‐NH]Cha‐Phe‐OH (TICP[Ψ]) (Cha = cyclohexylalanine), connected ‘tail‐to‐tail’ via a short linker, was synthesized using a combination of solid‐phase and solution techniques. The resulting peptide, H‐Dmt→D‐Arg→Phe→Lys‐NH‐CH₂‐CH₂‐NH‐Phe←Cha[NH‐CH₂]ΨTic←Tyr‐H, showed the expected μ agonist/δ antagonist profile in the guinea‐pig ileum and mouse vas deferens assays. Its μ and δ receptor binding affinities were in the low nanomolar range, as determined in rat brain membrane binding assays.     

Synthesis of tertiary 14C‐labelled nonylphenol isomers

The ring‐¹⁴C‐labelled p‐nonylphenol (NP) isomers 4(3′,5′‐dimethyl‐3′‐heptyl)‐phenol (p353NP), 4(3′,6′‐dimethyl‐3′‐heptyl)‐phenol (p363NP) and 4(2′,6′‐dimethyl‐2′‐heptyl)‐phenol (p262NP) were synthesized for application in metabolism and sorption studies. Friedel–Crafts alkylation of ¹⁴C‐labelled phenol and the corresponding tertiary nonylalcohol with BF₃ as catalyst was used. After clean‐up of p262NP and p363NP by preparative thin‐layer chromatography radiochemical yields amounted to 62.8 and 64.6%, specific radioactivities were 332 and 88.2 MBq/mmol, and radiochemical purities 97.6 and 99.0%. For both isomers, a large‐scale synthesis with non‐labelled phenol was additionally developed, which led to pure products (96 and 99%, respectively) without further purification steps. In the case of p353NP, which was formed as a diastereomeric mixture, the crude synthetic product had a radiochemical purity of 96.9% (radiochemical yield: 76.0%; specific activity: 298 MBq/mmol); thus, purification was not necessary. All products were characterized by means of gas chromatography‐mass spectroscopy, ¹H‐ and ¹³C‐NMR, as well as IR. Copyright © 2002 John Wiley & Sons, Ltd.