Search

Caution is warranted when in vitro results of biomarkers labeled with tritium were perfunctorily used to criticize in vivo data and conclusions derived with the same tracers labeled with positron emitters and positron emission tomography (PET). This concept is illustrated herein with the PET utilization of [18F]FDDNP, a biomarker used for in vivo visualization of β-amyloid and tau protein neuroaggregates in humans, later contradicted by in vitro data reported with [3H]FDDNP. In this investigation, we analyze the multiple factors involved in the experimental design of the [3H]FDDNP in vitro study that led to the erroneous interpretation of results. The present work describes full details on the synthesis, characterization, purity, and kinetics of radiolytic stability of [3H]FDDNP. The optimal in vitro conditions for detecting tau and β-amyloid protein aggregates using macroscopic and microscopic autoradiography with both [18F]FDDNP and [3H]FDDNP are also presented. Macroscopic autoradiography determinations were performed with [3H]FDDNP of verified purity using established methods described previously in the literature. The autoradiographic results using phosphate buffered saline (PBS) with less than 1 % EtOH and pure, freshly prepared [3H]FDDNP compared with the earlier reported data using [3H]FDDNP of undetermined purity and PBS in 10 % EtOH demonstrate the critical importance of rigorous experimental design for meaningful in vitro determinations. [18F]FDDNP binding to both amyloid plaques and neurofibrillary tangles was confirmed by amyloid and tau immunohistochemical stains of adjacent tissues. This work illustrates the sensitivity of in vitro techniques to various experimental conditions and underscores that conclusions obtained from translational in vitro to in vivo determinations must always be performed with extreme care to avoid wrong interpretations that can be perpetuated and assumed without further analysis.

Caution is warranted when in vitro results of biomarkers labeled with tritium were perfunctorily used to criticize in vivo data and conclusions derived with the same tracers labeled with positron emitters and positron emission tomography (PET). This concept is illustrated herein with the PET utilization of [The present work describes full details on the synthesis, characterization, purity, and kinetics of radiolytic stability of [The autoradiographic results using phosphate buffered saline (PBS) with less than 1 % EtOH and pure, freshly prepared [This work illustrates the sensitivity of in vitro techniques to various experimental conditions and underscores that conclusions obtained from translational in vitro to in vivo determinations must always be performed with extreme care to avoid wrong interpretations that can be perpetuated and assumed without further analysis.

Deposition of aggregates of hyperphosphorylated tau protein is a hallmark of tauopathies like Alzheimer and many other neurodegenerative diseases. A sensitive and selective method of in vivo detection of tau-aggregate presence and distribution could provide the means of an early diagnosis of tau-associated diseases. Furthermore, the use of selective molecular probes that enable histochemical differentiation of protein aggregates post-mortem would be advantageous for the insight into the properties of tau protein aggregates. We chose to design new molecular probes based on the structure of 2-(1-(6-((2-[18F]fluoroethyl)(methyl)amino)-2-naphthyl)ethylidene)malononitrile to investigate their likelihood of fitting into VQIVYK tau protein binding channel model. In a modular approach, using cross-coupling reactions, we synthesized a series of candidates, radiolabeled them with fluorine-18 radioisotope, and determined their physicochemical and in vitro binding properties. Herein we report the synthesis of a serie...

In this work, we report the synthesis, X-ray structures, and optical properties of two DDNP analogs containing an unsaturated spacer inserted between the donor N,N-dimethylamino group and the naphthalene ring. The new compounds have longer distances between the donor and acceptor groups and are intended to serve as molecular probes for the assessment of spatial and polarity requirements for binding to protein aggregates found in the central nervous system (CNS) of patients with neurodegenerative diseases.

The positron-emission tomography (PET) probe 2-(1-[6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene) (FDDNP) is used for the noninvasive brain imaging of amyloid-β (Aβ) and other amyloid aggregates present in Alzheimer’s disease and other neurodegenerative diseases. A series of FDDNP analogs has been synthesized and characterized using spectroscopic and computational methods. The binding affinities of these molecules have been measured experimentally and explained through the use of a computational model. The analogs were created by systematically modifying the donor and the acceptor sides of FDDNP to learn the structural requirements for optimal binding to Aβ aggregates. FDDNP and its analogs are neutral, environmentally sensitive, fluorescent molecules with high dipole moments, as evidenced by their spectroscopic properties and dipole moment calculations. The preferred solution-state conformation of these compounds is directly related to the binding affinities. The extreme cases were a nonplanar analog t -butyl-FDDNP, which shows low binding affinity for Aβ aggregates (520 nM K i ) in vitro and a nearly planar tricyclic analog cDDNP, which displayed the highest binding affinity (10 pM K i ). Using a previously published X-ray crystallographic model of 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP) bound to an amyloidogenic Aβ peptide model, we show that the binding affinity is inversely related to the distortion energy necessary to avoid steric clashes along the internal surface of the binding channel.

Understanding the molecular mechanisms associated with the development of dementia is essential for designing successful interventions. Dementia, like cancer and cardiovascular disease, requires early detection to potentially arrest or prevent further disease progression. By the time a neurologist begins to manage clinical symptoms, the disease has often damaged the brain significantly. Because successful treatment is the logical goal, detecting the disease when brain damage is still limited is of the essence. The role of chemistry in this discovery process is critical. With the advent of molecular imaging, the understanding of molecular mechanisms in human neurodegenerative diseases has exploded. Traditionally, knowledge of enzyme and neurotransmitter function in humans has been extrapolated from animal studies, but now we can acquire data directly from both healthy and diseased human subjects. In this Account, we describe the use of molecular imaging probes to elucidate the biochemical and cellular bases of dementia (e.g., Alzheimer's disease) and the application of these discoveries to the design of successful therapeutic interventions. Molecular imaging permits observation and evaluation of the basic molecular mechanisms of disease progression in the living brains of patients. 2-Deoxy-2-[(18)F]fluoro-d-glucose is used to assess the effect of Alzheimer's disease progression on neuronal circuits projecting from and to the temporal lobe (one of the earliest metabolic signs of the disease). Recently, we have developed imaging probes for detection of amyloid neuropathology (both tau and beta-amyloid peptide deposits) and neuronal losses. These probes allow us to visualize the development of pathology in the living brain of dementia patients and its consequences, such as losses of critical neurons associated with memory deficits and other neuropsychiatric impairments. Because inflammatory processes are tightly connected to the brain degenerative processes, inflammation is now emerging as an important target for new molecular imaging probes. The combination of molecular probes targeting various processes of dementia is a useful tool for detailed monitoring of disease mechanism, progression, and diagnosis, as well as for the development of rational strategies for promising therapeutic interventions.

Naphthalene derivatives bearing electron-accepting and electron-donating groups at the 2,6-positions belong to the family of D-π-A push-pull dyes. It has been found that these compounds, e.g., 2-(1-(6-((2-(fluoro)ethyl)(methyl)amino)naphthalen-2-yl)ethylidene)malononitrile (FDDNP), show not only interesting optical properties, such as solvatochromism, but they have the potential to label protein aggregates of different compositions formed in the brain of patients suffering from neurodegenerative diseases like Alzheimer’s (AD). In continuation of our research we set our goal to find new FDDNP analogs, which would inherit optical and binding properties but hopefully show better specificity for tau protein aggregates, which are characteristic for neurodegeneration caused by repetitive mild trauma. In this work we report on the synthesis of new FDDNP analogs in which the acceptor group has been formally replaced with an aromatic five- or six-membered heterocycle. The heterocyclic moiety was annealed to the central naphthalene ring either by classical ring closure reactions or by modern transition metal-catalyzed coupling reactions. The chemical characterization, NMR spectra, and UV/vis properties of all new compounds are reported.

In the presence of strong acids in aqueous and anhydrous media alkyl aryl ketones bearing a strong electron-releasing group in the aromatic ring undergo protiodeacylation. In a reaction with triflic acid in aromatic solvents, the transfer of the liberated acyl group to aromatic solvent molecules is observed. The effects of the nature and the position of the activating group and the strength of the acid and the solvent, on the course of this ipso-substitution were investigated.

The biomarker 2-(1-{6-[(2-[(18)F]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile ([(18)F]FDDNP) is used as a positron emission tomography (PET) imaging probe for Alzheimer's disease and other neurodegenerative diseases. A high-yield and fully automated synthesis of [(18)F]FDDNP--along with the synthesis and characterization of non-radioactive FDDNP, a fluorescent probe derived from 2-(1,1-dicyanopropenyl-2)-6-dimethylaminonaphthalene (DDNP)--are reported. Radiofluorination of the tosyloxy precursor 2-{[6-(2,2-dicyano-1-methylvinyl)-2-naphthyl](methyl)amino}ethyl-4-methylbenzenesulfonate (DDNPTs) with K(18)F/Kryptofix 2.2.2. yielded chemically (>99%) and radiochemically (>99%) pure [(18)F]FDDNP in high radiochemical yields (40-60%; n> 120), with specific activities ranging from 4 to 8 Ci/mumol at the end of synthesis (90 minutes). Both remote, semiautomated and automated synthesis procedures are described. Either approach provides a reliable method for production of large quantities (110-170 mCi from 500 mCi of [(18)F]fluoride) of [(18)F]FDDNP allowing for multiple PET experiments in the same day or for distribution of the tracer from a single cyclotron facility to PET imaging centers at various geographical distances.

This article presents a comprehensive review of the in vitro and in vivo detection of neurofibrillary tangles (NFTs) and beta-amyloid senile plaques (SPs), neuropathological lesions found in the brains of the Alzheimer's disease (AD) patients, using FDDNP and its analogs. FDDNP and its analogs have excellent ability to bind to NFTs and SPs in vitro as shown by binding assays, confocal fluorescence microscopy with stained AD brain tissue and digital autoradiography with [18F]FDDNP. [18F]FDDNP-PET molecular imaging permits detection of these pathologies in living subjects. The discovery of a new binding site to Abeta(1-40) fibrils as a result of FDDNP binding also opens a therapeutic opportunity for early treatment of Alzheimer's disease. FDDNP shares a previously unrecognized common binding site on Abeta(1-40) fibrils and senile plaques with non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., naproxen and ibuprofen). Naproxen, ibuprofen and even FDDNP significantly inhibit aggregation of the Abeta(1-40) peptide in the micromolar range. This new binding site on Abeta(1-40) fibrils also offers a molecular template for design of anti-aggregation drugs without the secondary effects of NSAIDs. Therefore it is anticipated that a new vision for prevention, early diagnosis and treatment of Alzheimer's disease would be rapidly developing.

The authors used 2-(1-{6-[(2-[ 18 F]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile ([ 18 F]FDDNP), a hydrophobic radiofluorinated derivative of 2-{1-[6-(dimethylamino)-2-naphthyl]ethylidene}malononitrile (DDNP), in conjunction with positron emission tomography to determine the localization and load of neurofibrillary tangles (NFTs) and β-amyloid senile plaques (APs) in the brains of living Alzheimer disease (AD) patients. Previous work illustrated the in vitro binding characteristics of [ 18 F]FDDNP to synthetic β-amyloid(1–40) fibrils and to NFTs and APs in human AD brain specimens. In the present study, greater accumulation and slower clearance was observed in AP- and NFT-dense brain areas and correlated with lower memory performance scores. The relative residence time of the probe in brain regions affected by AD was significantly greater in patients with AD (n=9) than in control subjects (n=7; p=0.0007). This noninvasive technique for monitoring AP and NFT development is expected to facilitate diagnostic assessment of patients with AD and assist in response-monitoring during experimental treatments.

The course of the reaction between [1-(6-acetyl-2-naphthyl)piperidin-4-yl]methyl 4-methylbenzenesulfonate (1) and pyrrolidine, a model nucleophile, in DMF in the presence of anhydrous potassium carbonate at ambient and elevated temperatures is investigated. At elevated temperatures a 1-azabicyclo[2.2.1]heptane derivative is identified as the intermediate in the piperidine-to-pyrrolidine ring contraction process. The formation of formate, carbamate, and hydroxy derivatives, as a consequence of DMF and K2CO3 participating in the reaction, is documented.

Products formed in the reaction of 1-(6-acetyl-2-naphthyl)-4- [(4-methylphenyl)sulfonyloxy] methylpiperidine with spiperone ketal, 8-{3-[2-(4-fluorophenyl)-1,3-dioxolan-2-yl]propyl}-1-phenyl-1,3,8-triazaspiro[4.5] decan-4-one, at room and elevated temperatures were compared and the respective structure were assigned based on 1H and 13C NMR spectra. Piperidine to pyrrolidine ring transformation at elevated temperature is documented. © 1998 John Wiley & Sons, Ltd.

To prepare reactive dyes matching the optical properties of 2-(1,1-dicyano-propenyl-2)-6-dimethylaminonaphthalene (DDNP), 2-ethylaminoethanol or 4-piperidinemethanol were used in a Bucherer reaction with 1-(6-hydroxy-2-naphthyl)-1-ethanone. The two amines were chosen to mimic the dimethylamino group’s steric and electronic (inductive) effects and to provide a reactive site for ligand attachment. The intermediate hydroxy derivatives were transformed into fluorescent reactive p-toluenesulfonic acid esters which in turn were conjugated to the test ligand spiperone. Knoevenagel condensation with malononitrile afforded Vis wavelength fluorescent probes.

Functionalization of a viscosity-sensitive visible wavelength fluorophore 2-(1,1-dicyanopropenyl-2)-6-dimethylaminonaphthalene (DDNP), with the intent to incorporate its favorable optical properties into a probe for structural and functional imaging by fluorescence microscopy, is described. Spiperone, a highly potent ligand for the dopamine D2 receptors, was conjugated via an ethylpiperazine moiety to the fluorophore giving fluorescent probes that can be excited in the UV and Vis range.

The recrystallization of [{Ti(C5Me5)F3}4CaF2] 1 and [{Ti(C5Me4Et)F3}4CaF2] 2 in the presence of an excess of hmpa (hexamethylphosphoramide) resulted in the formation of [{Ti(C5Me5)F3}4(CaF2)(hmpa)] 3 and [{Ti(C5Me4Et)F3}4(CaF2)(hmpa)] 4, respectively. The adducts 3 and 4 were spectroscopically characterized and the molecular structure of 4 was determined, showing a co-ordination of calcium by eight fluorine atoms from two [Ti2(C5Me4Et)2F7]– moieties and by an oxygen atom of an hmpa ligand. Adduct formation and solution behavior of 2 in different solvents was studied by 1H and 19F NMR and variable-temperature NMR. A temperature dependent equilibrium between 2 and a solvated form 2·solv (solv = solvent molecule) is proposed. With increase of the temperature the equilibrium between 2 and 2·solv was observed to be entropy-shifted to the non-solvated form.

6, 7-Dihydrocyclopenta[b]pyran-2, 5-dione 1 was converted with hydrazines to fused pyridine-2(1H)-ones of type a. In contrast, 2H-1-benzopyran-2, 5-diones 2–4 gave under the same reaction conditions 5-hydrazonobenzopyrans of type b. Calculated heats of formation matched experimental findings.

A novel FDDNP analog, namely 1-[6-(3-fluoropyrrolidin-1-yl)-2-naphthyl]ethylidenemalononitrile, was synthesized to probe the influence of the applied structural changes at the donor-side of the molecule on tau protein aggregate binding. Reported is also a synthetic procedure, which can be directly applied to the preparation of the [18F]-radiolabeled compound.

From the corresponding heterocyclic amino acids 2 and 9a the heterocyclic systems imidazo[1,5-a]pyridine (3) and imidazo[1,5-a]quinoline (10) are easily accessible. From compound 7 the tricyclic system 11 was prepared and from compound 17a a pyridyl-1,2,4-triazinone (18) could be obtained.

The title N-oxides were prepared from several carboxamide oximes on treatment with 1,3-dicarbonyl derivatives.

Functionalization of a viscosity-sensitive visible wavelength fluorophore 2-(1,1-dicyanopropenyl-2)-6-dimethylaminonaphthalene (DDNP), with the intent to incorporate its favorable optical properties into a probe for structural and functional imaging by fluorescence microscopy, is described. Spiperone, a highly potent ligand for the dopamine D2 receptors, was conjugated via an ethylpiperazine moiety to the fluorophore giving fluorescent probes that can be excited in the UV and Vis range.

To prepare reactive dyes matching the optical properties of 2-(1,1-dicyano-propenyl-2)-6-dimethylaminonaphthalene (DDNP), 2-ethylaminoethanol or 4-piperidinemethanol were used in a Bucherer reaction with 1-(6-hydroxy-2-naphthyl)-1-ethanone. The two amines were chosen to mimic the dimethylamino group’s steric and electronic (inductive) effects and to provide a reactive site for ligand attachment. The intermediate hydroxy derivatives were transformed into fluorescent reactive p-toluenesulfonic acid esters which in turn were conjugated to the test ligand spiperone. Knoevenagel condensation with malononitrile afforded Vis wavelength fluorescent probes.

The course of the reaction between [1-(6-acetyl-2-naphthyl)piperidin-4-yl]methyl 4-methylbenzenesulfonate (1) and pyrrolidine, a model nucleophile, in DMF in the presence of anhydrous potassium carbonate at ambient and elevated temperatures is investigated. At elevated temperatures a 1-azabicyclo[2.2.1]heptane derivative is identified as the intermediate in the piperidine-to-pyrrolidine ring contraction process. The formation of formate, carbamate, and hydroxy derivatives, as a consequence of DMF and K2CO3 participating in the reaction, is documented.

From the corresponding heterocyclic amino acids 2 and 9a the heterocyclic systems imidazo[1,5-a]pyridine (3) and imidazo[1,5-a]quinoline (10) are easily accessible. From compound 7 the tricyclic system 11 was prepared and from compound 17a a pyridyl-1,2,4-triazinone (18) could be obtained.

Deposition of conformationally altered proteins prominently characterizes pathogenesis and pathomorphology of a number of neurodegenerative disorders. 2-(1-{6-[(2-[F-18]fluoroethyl) (methyl)amino]-2-naphthyl} ethylidene) malononitrile ([F-18]FDDNP), a hydrophobic, viscosity-sensitive, solvent-sensitive, fluorescent imaging probe has been used with positron emission tomography to visualize brain pathology in the living brain of Alzheimer disease (AD) patients. Its non-radiofluorinated analog FDDNP was shown to label senile plaques and neurofibrillary tangles (NFTs) in brain tissue sections. This work aimed at evaluating FDDNP labeling of various protein deposits in fixed, paraffin-embedded brain tissue sections of selected neurodegenerative disorders: AD, cerebral amyloid angiopathy (CAA), transmissible spongiform encephalopathies, progressive supranuclear palsy (PSP), Pick disease (PiD), Parkinson disease, dementia with Lewy bodies, multiple system atrophy (MSA). Cerebral hypertensive vascular hyalinosis (HVH) was used as negative control. Significant agreement between amyloid histochemical properties and FDDNP labeling of the deposits was established. FDDNP labeling showed high positive predictive value for birefringence in senile plaques and NFTs in AD, prion plaques and amyloid deposits in CAA. No FDDNP labeled structures were observed in HVH, PSP, PiD or MSA tissue sections. Our findings may be of significant value for the detection of neuropathological aggregates with [F-18]FDDNP in some of these disorders in the living brain of human subjects.

Dopamine reuptake transporter binding kinetics of 2β-carbomethoxy-3β-(4-[ 18 F]fluoromethylphenyl)tropane (p-FWIN) and 2β-carbomethoxy-3β-(2-[ 18 F]fluoromethylphenyl)tropane (o-FWIN) were determined in vervet monkeys using positron emission tomography (PET). Ligand localization was rapid and specific to the striatum with kinetic estimates comparable with those of 11 C-labeled WIN 35,428 (CWIN). Binding was more specific with p-FWIN than with CWIN or o-FWIN. The relatively longer half-life of the 18 F radiolabel enabled longer acquisition times with p-FWIN, resulting in less variability in the kinetic estimates.

An efficient method is described for the cleavage of enolisable aryl methyl and aryl ethyl ketones using an excess of KOH in DMF at an elevated temperature. It presents a general hydrolytic method yielding aromatic carboxylic acids, and is complementary to the widely used oxidative methods for ketone cleavage.

Transformation of the amino group of amino acids into an amidine and subsequent treatment with hydrazine leads to 1,2,4-triazine-6(1H)-ones. The method described is a new synthetic approach. Unsaturated amino acids gave imidazol-5-one derivatives after the same reaction sequence

Senile plaques (SPs) and neurofibrillary tangles (NFTs) are hallmark pathologies accompanying the neurodegeneration involved in Alzheimer's disease (AD), for which beta-amyloid (Abeta) peptide is a major constituent of SPs. Our laboratories previously developed the hydrophobic, fluorescent molecular-imaging probe 2-(1-(6-[(2-[(18)F]fluoroethyl)(methyl)amino]-2-naphthyl)ethylidene)malononitrile ([(18)F]FDDNP), which crosses the blood-brain barrier and determines the localization and load of SPs and NFTs in vivo in AD patients. In this report, we used fluorimetric and radioactive binding assays to determine the binding affinities of FDDNP and its analog, 1-(6-[(2-[(18)F]fluoroethyl)(methyl)amino]naphthalen-2-yl)ethanone ([(18)F]FENE), to synthetic fibrils of Abeta(1-40). FDDNP and FENE both appeared to bind to two kinetically distinguishable binding sites on Abeta(1-40) fibrils. Fluorescence titrations yielded apparent K(d) values of 0.12 and 0.16 nm for high-affinity binding sites for FDDNP and FENE, respectively, and apparent K(d) values of 1.86 and 71.2 nm for the low-affinity binding sites. The traditional radioactive binding assays also produced apparent K(d) values in the low nanomolar range. The presence of two kinetically distinguishable binding sites for FDDNP and FENE suggests multiple binding sites for SPs and identifies the parameters that allow for the structural optimization of this family of probes for in vivo use. The high-affinity binding of the probes to multiple binding sites on fibrils are consistent with results obtained with digital autoradiography, immunohistochemistry, and confocal fluorescence microscopy using human brain specimens of AD patients.

Treatment of 4-ethoxymethylene-2-phenyl-5(4H)-oxazolone with activated methylene compounds under acidic or basic conditions leads to 2H-pyran-2-ones and fused pyran-2-ones. On the other hand, methyl (3-benzoylamino-5-methoxycarbonyl-2-oxo-2H-pyran-6-yl)-acetate (4) has also been prepared by a one-pot synthesis from dimethyl 1,3-acetonedicarboxylate, diethoxymethyl acetate, hippuric acid and acetic anhydride

Transformation of the amino group of amino acids into an amidine and subsequent treatment with hydrazine leads to 1,2,4-triazine-6(1H)-ones. The method described is a new synthetic approach. Unsaturated amino acids gave imidazol-5-one derivatives after the same reaction sequence

1,3-Dipolar cycloaddition of diazomethane to 6-chloro substituted azolopyridazines 1-4 produces isomeric pairs of 7-methyl-7H- 9-12 and 8-methyl-8H-pyrazolo[4,3-d]azolopyridazines 13-16. The nucleophilic substitution of chlorine at position 6 affords the corresponding 6-amino 17, 20, 23, 26, 29,32,35 and 38, 6-methoxy 18, 21, 24, 27, 30,33, 36, and 39 and 6-hydrazino derivatives 19, 22, 25, 28, 31, 34, 37, and 40 of these tricyclic systems.

Reactions de dimethylaminomethyleneamino-4 pyrido [2,3-d] pyrimidinioolate-3 et de s-triazolo [3,4-f]- et tetrazolo [5,1-f] pyrido [2,3-d] pyrimidines: obtention des composes a methylene actif de depart substitues sur le methylene actif par [azolyl-3 pyridyl-2aminomethylene]

Syntheses of some new pyridotriazolo- and pyridotetrazolopyrimidines are described. These tricyclic systems and tetrazoloquinazoline react with some carbon nucleophiles, generated from compounds with reactive methylene groups, exclusively at position 2 of the fused pyrimidine ring to give the corresponding open-chain enamines.

Derivatives of pyrimido(1,2-b)indazole and s-triazino(1,2-b)indazole were prepared by several different routes and some transformations are described.

Syntheses of isomeric azolopyridopyrimidines are described. As starting material the corresponding pyridopyrimidines were used. It could be established that in many cases a transformation of a functional group with the purpose to form an annelated five-membered ring proceeded with ring opening at the pyrimidine part. Subsequent ring closure with one carbon synthons gave then the desired tricyclic heterocycles.

Syntheses of 1-methylimidazo[1,2-c]quinazolinium iodide (3) and 1-methylimidazo[2,1-f]pyrido[3,2-d]pyrimidinium iodide (4) are described. These tricyclic compounds react with nucleophiles derived from active methylene compounds to give the corresponding open-chain enamines.

2-Azidothiazolo[5,4-b]pyridine and 2-azidothiazolo[4,5-c]pyridine were synthesized. Azido-tetrazolo isomerizations of these compounds were investigated and in both cases also the tetrazolo form could be obtained. In solution, equilibria at different temperatures were determined.

The transformations of 7-methyl-7H- and 8-methyl-8H-pyrazolo[4, 3-d]tetrazolo[1, 5-b]pyridazines 1, 2, 9 and 10 into 8-methyl-8H- and 9-methyl-9H-pyrazolo[3, 4-H]-s-triazolo[4, 3-b]pyridazines 7 and 8, and 1-methyl-1H-and 2-methyl-2H-imidazo[1, 2-b]pyrazolo[3, 4-d]pyridazines 13 and 14 are described.