Synthesis and inhibitory evaluation of 3-linked imipramines for the exploration of the S2 site of the human serotonin transporter.

The human serotonin transporter is the primary target of several antidepressant drugs, and the importance of a primary, high affinity binding site (S1) for antidepressant binding is well documented. The existence of a lower affinity, secondary binding site (S2) has, however, been debated. Herein we report the synthesis of 3-position coupled imipramine ligands from clomipramine using a copper free Sonogashira reaction. Ligand design was inspired by results from docking and steered molecular dynamics simulations, and the ligands were utilized in a structure-activity relationship study of the positional relationship between the S1 and S2 sites. The computer simulations suggested that the S2 site does indeed exist although with lower affinity for imipramine than observed within the S1 site. Additionally, it was possible to dock the 3-linked imipramine analogs into positions which occupy the S1 and the S2 site simultaneously. The structure activity relationship study showed that the shortest ligands were the most potent, and mutations enlarging the proposed S2 site were found to affect the larger ligands positively, while the smaller ligands were mostly unaffected.

Basic N-interlinked imipramines show apoptotic activity against malignant cells including Burkitt's lymphoma.

We here report the synthesis of ethylene glycol N-interlinked imipramine dimers of various lengths from the tricyclic antidepressant desipramine via an amide coupling reaction followed by reduction with lithium aluminium hydride. The target molecules were found to be potent inhibitors of cellular viability while inducing cell type specific death mechanisms in three cancer cell lines including a highly chemoresistant Burkitt's lymphoma cell line. Basic amine analogues were found to be important for increased potency. Imipramine and desipramine were also tested for apoptotic activity and were found to be much less active than the novel dimeric compounds. Imipramine dimers were only found to be moderate inhibitors of the human serotonin transporter (hSERT) having IC(50) values in the micromolar region whilst the induction of cell death occurred independently of hSERT expression. These results demonstrate the potential of newly designed and synthesised imipramines derivatives for use against malignant cells, including those resistant to standard chemotherapy.

Anti-invasive adjuvant therapy with imipramine blue enhances chemotherapeutic efficacy against glioma.

The invasive nature of glioblastoma (GBM) represents a major clinical challenge contributing to poor outcomes. Invasion of GBM into healthy tissue restricts chemotherapeutic access and complicates surgical resection. Here, we test the hypothesis that an effective anti-invasive agent can "contain" GBM and increase the efficacy of chemotherapy. We report a new anti-invasive small molecule, Imipramine Blue (IB), which inhibits invasion of glioma in vitro when tested against several models. IB inhibits NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase-mediated reactive oxygen species generation and alters expression of actin regulatory elements. In vivo, liposomal IB (nano-IB) halts invasion of glioma, leading to a more compact tumor in an aggressively invasive RT2 syngeneic astrocytoma rodent model. When nano-IB therapy was followed by liposomal doxorubicin (nano-DXR) chemotherapy, the combination therapy prolonged survival compared to nano-IB or nano-DXR alone. Our data demonstrate that nano-IB-mediated containment of diffuse glioma enhanced the efficacy of nano-DXR chemotherapy, demonstrating the promise of an anti-invasive compound as an adjuvant treatment for glioma.

Synthesis and local anesthetic activity of fluoro-substituted imipramine and its analogues.

A series of fluoro-substituted imipramines and its analogues, 6a-6e, were synthesized and evaluated for their in vitro local anesthetic activity. Compound 6b was found to have potency, onset, and duration of action comparable to those of lidocaine (lidocaine hydrochloride, CAS:6108-05-0). Dissociation constants (pK(a)) of these compounds have been determined to be 7.6-7.9.

Vasoactive properties of antidepressant N-alkyl derivatives.

Hypotension is a principal side effect of antidepressant therapy. In addition to serotonin and noradrenalin reuptake inhibition, some antidepressants have shown ion channel interactions which are thought to be related to the vascular effects of these agents. Methylation of the pharmacophore has shown to change the pharmacological properties of a variety of compounds. The purpose of this work was to evaluate whether methylation of the amino group of imipramine (TCA's) and fluoxetine (SSRI) could change their vasodilator properties. N-methyl imipramine (NMI), N-methyl fluoxetine and (NMF) N-N dimethyl fluoxetine (NNDF) were synthesized and compared with desipramine (DES), imipramine (IMI) and fluoxetine (F) in their ability to relax rat aortic rings pre-contracted with 80mM KCl using an isolated bath preparation. Drugs were evaluated in thoracic aorta rings with and without endothelium. All compounds displayed a vasorelaxant effect. Endothelium-denuded aortic rings showed an increased relaxant response for IMI and derivatives compared with endothelium-intact vessels, while no endothelium-dependent effect was observed with F and its methyl derivatives. Maximal relaxant potency was displayed by dimethylated derivatives (IMI and NMF), while NMI in the TCA series and NNDF in the SSRI series (both with 3 methyl groups), had the least potency to relax either preparation. Endothelium plays an important role inhibiting the vasodilatation induced by IMI and its derivatives. Vascular relaxation is increased in the compounds tested with 2 methyl groups in their structure, while the presence of 3 methyl groups (positive charge) importantly reduced the relaxant potency.

A chimeric ligand approach leading to potent antiprion active acridine derivatives: design, synthesis, and biological investigations.

Human transmissible neurodegenerations including Creutzfeldt-Jakob disease are unique, since they are caused by prions, an infectious agent that replicates without nucleic acids but instead by inducing conversion of a host-resident normal prion protein to a misfolded conformational isoform. For pharmacotherapy of these unusual diseases, tricyclic heterocyclic compounds such as quinacrine have been considered, but with ambiguous success in vivo, so far. On the basis of the synergistic antiprion effects of quinacrine and iminodibenzyl derivatives, we introduce a novel class of potential pharmaceuticals representing structural chimeras of quinacrine and imipramine analogues. We describe the chemical synthesis and bioassays of a focused library of these compounds. The most potent target compound 2a revealed an EC(50) value of 20 nM determined with a cell model of prion disease, thus substantially improving the antiprion efficacy of quinacrine.

Tritiation of imipramine and pseudo irreversible analogues 3-cyanoimipramine and 2-nitroimipramine at high specific activity.

Methods are presented to synthesize and characterize [N-methyl-3H] imipramine 3, [N-methyl-3H] 3-cyanoimipramine 6 and [N-methyl-3H] 2-nitroimipramine 9.

Synthetic strategies to lower affinity for CYP2D6.

There are several models for the CYP2D6 active site with the characteristics of their substrates and inhibitors well defined. Imipramine possesses such characteristics and is both a substrate and an inhibitor of the CYP2D6 enzyme. Possible synthetic strategies to avoid interaction with the enzyme have been investigated, including: attenuation of basicity; and alteration of rigidity and length of the alkyl chain. Imipramine inhibited the 1'-hydroxylation of bufuralol (10 microM), an in vitro marker of CYP2D6 activity, in a CYP2D6 cell line (IC50 = 2.4 microM). Inhibitory potency was attenuated by the removal of the basic centre; imipramine N-oxide had no inhibitory effect on bufuralol 1'-hydroxylation. However, removal of this basic centre, as a strategy to decrease CYP2D6 interaction, may well have a detrimental effect on pharmacological efficacy. Both an increase and decrease in the N-N carbon chain length [2C,4C] caused a reduction in inhibitory potency. In addition, introduction of a carbonyl adjacent to the amino dibenzyl moiety into 2C, 3C and 4C compounds brought about a further reduction in inhibitory potency. These data demonstrate that changes to the molecule, distal to the basic centre, can attenuate the affinity of the molecule for CYP2D6 and are in keeping with the known characteristics of the enzyme.

Ion-pair chromatography of inositol polyphosphates with N-methylimipramine.

A novel counter-ion, N-methylimipramine, was synthesized and utilized in the separation of inositol phosphates by ion-pair chromatography. The structural identity of the counter-ion was documented by nuclear magnetic resonance spectroscopy. This counter-ion was capable of resolving inositol phosphates isocratically by reversed-phase high-performance liquid chromatography. Solvent polarity and ionic strength markedly affected the retention of the polyphosphorylated inositides. pH, however, was less significant in its effects. Injection of inositol trisphosphate paired to N-methylimipramine into a mobile phase containing tetrabutylammonium ions demonstrated free exchange of the inositide between the counter-ions. This counter-ion may therefore prove useful in defining empirically the mechanisms of ion-pair chromatography.

2-Iodoimipramine, a novel ligand for the serotonin transporter.

Iodoimipramine was synthesized by iodinating imipramine with ICI. Iodoimipramine competitively inhibits [3H]imipramine binding with a KI of 0.52 nM and also inhibits [3H]serotonin transport competitively, suggesting that serotonin, imipramine, and iodoimipramine all bind to the same site on the serotonin transporter. Association of [125I]iodoimipramine to platelet membranes in Na+ requires 20 min to reach equilibrium at 25 degrees and 1.5 hr at 0 degrees. [125I] Iodoimipramine binding at equilibrium is saturable and Na+ dependent, with a KD of 0.58 nM and a Bmax of 1.3 pmol/mg at 25 degrees. Serotonin competitively inhibits [125I]iodoimipramine binding, with a KI of 1.3 microM. [125I]Iodoimipramine bound at 0 degrees in the presence of Na+ does not dissociate unless the temperature is raised or Na+ is removed from the medium. At 25 degrees, dissociation of [125I] iodoimipramine from platelet membranes in the presence of Na+ is only partial, with 40% of the ligand remaining persistently bound over 5 hr after a 50-fold dilution.

Synthesis and evaluation of [11C]cyanoimipramine.

[11C]Cyanoimipramine has been prepared by methylation of the desmethyl cyanoimipramine with [11C]methyl iodide. The chemically and radiochemically pure labelled product was obtained with a high specific activity (greater than 300 mCi/mumol). When 11C (or 3H)-cyanoimipramine was intravenously administered in mice, high accumulations were shown in brain and lung. Thirty minutes after injection of the tracer, differences were found in the radioactivity between the cerebral cortex and the cerebellum. The regional distribution of radioactivity in the rat brain 30 min after i.v. injection of [11C]cyanoimipramine was also examined, and the radioactivity was high in receptor rich areas (striatum, cerebral cortex etc.) but low in receptor poor area (cerebellum). The in vivo stability of [3H]cyanoimipramine was quite stable in the mouse brain for at least 30 min. Thirty minutes after injection, the radioactivity in the cerebral cortex of the carrier-added state was reduced as compared with the carrier-free state. Taken together, the in vivo specific binding of [3H]cyanoimipramine in the cerebral cortex was estimated at about 40-50% of the total radioactivity. Furthermore, the distribution of [3H]cyanoimipramine in the mice forced to swim was examined. Significant changes in the distribution of [3H]cyanoimipramine were observed in the cerebral cortex.

Rapid reductive-carboxylation of secondary amines, one pot synthesis of N'-(4-11C-methyl)imipramine.

A new rapid high yield synthesis of radiolabeled N'-(4-11C-methyl)imipramine has been developed using a reductive-carboxylation approach, in which 11CO2 is reacted with either N'-trimethylsilyldesimipramine or N'-lithium derivative of desimipramine, followed by lithium aluminum hydride reduction, to give no carrier added or carrier added 11C-labeled imipramine respectively. The final product is characterized by chromatographic and spectroscopic methods.

Synthesis and some pharmacological properties of a conformationally restricted imipramine analogue.

The synthesis of the semi-rigid imipramine analogue 2-(N,N-dimethylaminomethylene)-2,3,7,8-tetrahydro-1H-quino[1,8-ab] [I]-benzazepine is described. The compound was pharmacologically evaluated in a number of general in vivo screening tests for antidepressive activity. From these preliminary tests the compound appeared to show an imipramine-like activity. However, it did not have an effect on the noradrenaline depletion by 4, alpha-dimethyl-m-tyramine. These results are discussed on the basis of the conformational requirements determining the pharmacological profile of antidepressants.

The production in high yield of N'-(4-[11C]methyl)-imipramine.

A method for the routine production in high yield of N'-(4-[11C]methyl)-imipramine is presented. The label is incorporated by reaction of C-11 methyl iodide (11CH3I) upon desipramine in dimethylsulfoxide. Quaternization of the tertiary amine by 11CH3I is minimized by using an excess of desipramine. The reaction proceeds at room temperature for 10 min and the product is isolated by means of high-performance liquid chromatography (HPLC). The entire production takes only 40 min and results in a radiochemical yield of 60%. About 60 mCi of labeled product are available for medical application; the specific activity, at the time of use, is 50 mCi/mumole. The product was characterized by chromatographic and spectrometric methods.

Nitroimipramines - synthesis and pharmacological effects of potent long-acting inhibitors of [3H] serotonin uptake and [3H] imipramine binding.

A series of nitro derivatives of imipramine have been prepared by nitration of imipramine. Several of the products, especially 2-nitroimipramine (2) and 2,8-dinitro-imipramine (4) were found to be very potent inhibitors of [3H] serotonin uptake and high affinity [3H]imipramine binding in human platelets. In contrast to the parent antidepressant, imipramine, the inhibition of platelet [3H] serotonin uptake and [3H] imipramine binding by the nitro derivatives of imipramine was long-acting and essentially irreversible at low temperatures. These compounds should prove to be valuable tools for subsequent studies on the purification and characterization of the transport protein(s) involved in serotonin uptake and may have novel behavioral and clinical effects.