Inhibition of Bfl-1 with N-aryl maleimides.

High-throughput screening of 66,000 compounds using competitive binding of peptides comprising the BH3 domain to anti-apoptotic Bfl-1 led to the identification of 14 validated 'hits' as inhibitors of Bfl-1. N-Aryl maleimide 1 was among the validated 'hits'. A chemical library encompassing over 280 analogs of 1 was prepared following a two-step synthesis. Structure-activity studies for inhibition of Bfl-1 by analogs of N-aryl maleimide 1 revealed a preference for electron-withdrawing substituents in the N-aryl ring and hydrophilic amines appended to the maleimide core. Inhibitors of Bfl-1 are potential development candidates for anti-cancer therapeutics.

Inhibition of serotonin and norepinephrine reuptake and inhibition of phosphodiesterase by multi-target inhibitors as potential agents for depression.

Compounds possessing more than one functional activity incorporated into the same molecule may have advantages in treating complex disease states. Balanced serotonin/norepinephrine reuptake inhibitors (SNRIs) (i.e., (R)- and (S)-norduloxetine) were chemically linked to a PDE4 inhibitor via a five carbon bridge. The new dual SNRI/PDE4 inhibitors (i.e., (R)-15 and (S)-15) showed moderately potent serotonin reuptake inhibition (IC(50) values of 442 and 404 nM, respectively) but low reuptake inhibition of norepinephrine (IC(50) values of 2097 and 2190 nM, respectively) in vitro. The dual SNRI/PDE4 inhibitors (i.e., (R)-15 and (S)-15) also inhibited PDE4D2 (i.e., K(i) values of 23 and 45 nM, respectively). Due to their synergistic functional activity, SNRI/PDE4 inhibitors may be effective in treating diseases such as depression.

Synthesis and pharmacological evaluation of 6-naltrexamine analogs for alcohol cessation.

A series of substituted aryl amide derivatives of 6-naltrexamine, 3 designed to be metabolically stable were synthesized and used to characterize the structural requirements for their potency to binding and functional activity of human mu (mu), delta (delta) and kappa (kappa) opioid and nociceptin (NOP) receptors. Binding assays showed that 4-10 had subnanomolar K(i) values for mu and kappa opioid receptors. Functional assays for stimulation of [(35)S]GTPgammaS binding showed that several compounds acted as partial or inverse agonists and antagonists of the mu and delta, kappa opioid or NOP receptors. The compounds showed considerable stability in the presence of rat, mouse or human liver preparations and NADPH. The inhibitory activity on the functional activity of human cytochrome P450s was examined to determine any potential inhibition by 4-9. Only modest inhibition of CYP3A4, CYP2C9 and CYP2C19 was observed for a few of the analogs. As a representative example, radiolabeled 6 was examined in vivo and showed reasonable brain penetration. The inhibition of ethanol self-administration in rats trained to self-administer a 10% (w/v) ethanol solution, utilizing operant techniques showed 5-8 to have very potent efficacy (ED(50) values 19-50 microg/kg).

Inhibition of kappa opioid receptors attenuated increased cocaine intake in rats with extended access to cocaine.

OBJECTIVE: Previous studies demonstrated that the dynorphin/kappa opioid system was up-regulated upon repeated cocaine self-administration. In the present study, we tested the hypothesis that increased cocaine self-administration with extended access was associated with increased activity of the kappa opioid system in rats. MATERIALS AND METHODS: Rats self-administered 0.5 mg/kg per injection of cocaine on a fixed-ratio (FR) schedule in either 1-h (short access, ShA) or 6-h (long access, LgA) sessions. After cocaine intake in the LgA rats increased to a maximum, the effects of kappa opioid receptor antagonists and a partial agonist were tested on cocaine intake in ShA and LgA rats. RESULTS: Cocaine self-administration increased under FR and progressive-ratio (PR) schedules in LgA rats. Nor-BNI (15-30 mg/kg), a kappa receptor antagonist, decreased cocaine intake in LgA rats under a PR schedule (ShA, +1.7%; LgA, -27.4% from baseline), whereas naltrexone (0.3-10 mg/kg) and SG-II-49 (0.025-0.1 mg/kg), a nonspecific opioid receptor antagonist and a partial agonist, respectively, decreased cocaine intake in both groups (PR data: SG-II-49, ShA -28.6%, LgA -19.8%; naltrexone, ShA -34.6%, LgA -11.8% compared with vehicle data). CONCLUSIONS: The present study demonstrated that the antagonism of kappa opioid receptors attenuated only the increased cocaine intake in LgA rats under a PR schedule, whereas the antagonism of micro and kappa receptors decreased cocaine intake in both ShA and LgA groups. The data suggest that increased motivation for cocaine in rats with extended access may be related to increased kappa opioid activity and may contribute to compulsive use.

1alpha,25-dihydroxyvitamin D3 interacts with curcuminoids to stimulate amyloid-beta clearance by macrophages of Alzheimer's disease patients.

Patients with Alzheimer's disease (AD) suffer from brain amyloidosis related to defective clearance of amyloid-beta (Abeta) by the innate immune system. To improve the innate immune system of AD patients, we studied immune stimulation of macrophages by 1alpha,25(OH)2-vitamin D3(1,25D3) in combination with curcuminoids. AD patients' macrophages segregate into Type I (positively stimulated by curcuminoids regarding MGAT-III transcription) and Type II (not stimulated). In both Type I and Type II macrophages, 1,25D3 strongly stimulated Abeta phagocytosis and clearance while protecting against apoptosis. Certain synthetic curcuminoids in combination with 1,25D3 had additive effects on phagocytosis in Type I but not Type II macrophages. In addition, we investigated the mechanisms of 1,25D3 and curcuminoids in macrophages. The 1,25D3 genomic antagonist analog MK inhibited 1,25D3 but not curcuminoid effects, suggesting that 1,25D3 acts through the genomic pathway. In silico, 1,25D3 showed preferential binding to the genomic pocket of the vitamin D receptor, whereas bisdemethoxycurcumin showed preference for the non-genomic pocket. 1,25D3 is a promising hormone for AD immunoprophylaxis because in Type I macrophages combined treatment with 1,25D3 and curcuminoids has additive effects, and in Type II macrophages 1,25D3 treatment is effective alone. Human macrophages are a new paradigm for testing immune therapies for AD.

Immune defects in Alzheimer's disease: new medications development.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by the accumulation of intracellular and extracellular aggregates. According to the amyloid beta (Abeta) hypothesis, amyloidosis occurring in the brain is a leading cause of neurodegeneration in AD. Defects in the innate immune system may decrease the clearance of Abeta in the brain. Macrophages of most AD patients do not transport Abeta into endosomes and lysosomes, and monocytes from AD patients do not efficiently clear Abeta from AD brain. After stimulation with Abeta, mononuclear cells of normal subjects display up-regulated transcription of MGAT3, which encodes beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase, and Toll-like receptor (TLR) genes. Monocytes of AD patients generally down-regulate these genes. A commonly used, naturally occurring material from a spice that enhances certain key functions defective in cells of innate immunity of many AD patients has shown epidemiologic rationale for use in AD treatment. Bisdemethoxycurcumin, a natural curcumin, is a minor constituent of turmeric (curry), and it enhances phagocytosis and clearance of Abeta in cells from most AD patients. We confirmed the effectiveness of a synthetic version of the same compound. In mononuclear cells of most AD patients, bisdemethoxycurcumin enhanced defective phagocytosis of Abeta and increased the transcription of MGAT3 and TLR genes. The potency of bisdemethoxycurcumin as a highly purified compound in facilitating the clearance of Abeta in mononuclear cells suggests the promise of enhanced effectiveness compared to curcuminoid mixtures. Bisdemethoxycurcumin appears to enhance immune function in mononuclear cells of AD patients and may provide a novel approach to AD immunotherapy.

Curcuminoids inhibit multiple human cytochromes P450, UDP-glucuronosyltransferase, and sulfotransferase enzymes, whereas piperine is a relatively selective CYP3A4 inhibitor.

Curcuminoid extract and piperine are being evaluated for beneficial effects in Alzheimer's disease, among other intractable disorders. Consequently, we studied the potential for herb-drug interactions involving cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes. The curcuminoid extract inhibited SULT > CYP2C19 > CYP2B6 > UGT > CYP2C9 > CYP3A activities with IC(50) values ranging from 0.99 +/- 0.04 to 25.3 +/- 1.3 microM, whereas CYP2D6, CYP1A2, and CYP2E1 activities were less affected (IC(50) values > 60 microM). Inhibition of CYP3A activity by curcuminoid extract was consistent with competitive inhibition (K(i) = 11.0 +/- 1.3 microM), whereas inhibition of both CYP2C9 and CYP2C19 activities were consistent with mixed competitive-noncompetitive inhibition (10.6 +/- 1.1 and 7.8 +/- 0.9 microM, respectively). Piperine was a relatively selective noncompetitive inhibitor of CYP3A (IC(50) 5.5 +/- 0.7 microM, K(i) = 5.4 +/- 0.3 microM) with less effect on other enzymes evaluated (IC(50) > 29 microM). Curcuminoid extract and piperine inhibited recombinant CYP3A4 much more potently (by >5-fold) than CYP3A5. Pure synthetic curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) were also evaluated for their effects on CYP3A, CYP2C9, UGT, and SULT activities. All three curcuminoids had similar effects on CYP3A, UGT, and SULT activity, but demethoxycurcumin (IC(50) = 8.8 +/- 1.2 microM) was more active against CYP2C9 than either curcumin or bisdemethoxycurcumin (IC(50) > 50 microM). Based on these data and expected tissue concentrations of inhibitors, we predict that a p.o. administered curcuminoid/piperine combination is most likely to inhibit CYP3A, CYP2C9, UGT, and SULT metabolism within the intestinal mucosa.

Synthesis and biological evaluation of alpha- and beta-6-amido derivatives of 17-cyclopropylmethyl-3, 14beta-dihydroxy-4, 5alpha-epoxymorphinan: potential alcohol-cessation agents.

Substituted aryl and aliphatic amide analogues of 6-naltrexamine were synthesized and used to characterize the binding to and functional activity of human mu-, delta-, and kappa-opioid receptors. Competition binding assays showed 11-25 and 27-31 bound to the mu (K(i) = 0.05-1.2 nM) and kappa (K(i) = 0.06-2.4 nM) opioid receptors. Compounds 11-18 possessed significant binding affinity for the delta receptor (K(i) = 0.8-12.4 nM). Functional assays showed several compounds acted as partial or full agonists of delta or kappa receptors while retaining an antagonist profile at the mu receptor. Structure-activity relationship for aryl amides showed that potent compounds possessed lipophilic groups or substituents capable of hydrogen bonding. Metabolic stability studies showed that 11, 12, and 14 possessed considerable stability in the presence of rat, mouse, or human liver preparations. The ED 50 of inhibition of 10% ethanol self-administration in trained rats, using operant techniques for 11, was 0.5 mg/kg.

An aminoacridine derivative for radionuclide therapy: DNA binding properties studied in a novel cell-free in vitro assay.

Radiolabelled DNA-binding compounds can be used to increase the efficiency of radionuclide cancer therapy of disseminated disease. In this work, the aminoacridine compound N-[3-(acridine-9-ylamino)-propyl]-3-iodobenzamide (A3) labelled with the Auger-emitting nuclide 125I using Chloramine-T was studied. Optimal labelling conditions of 125I-A3 were investigated and the interaction with DNA was studied using a novel cell-free in vitro assay with naked human genomic DNA in agarose plugs. This novel assay showed to be simple and reliable. The results verify that 125I-A3 specifically binds DNA with low dissociation and is potent in causing double-strand breaks, yielding 1.0-1.4 breaks per decay. In conclusion, 125I-A3 is a most suitable DNA-binding compound for future therapeutic studies of Auger-electron emitters like 125I.

Synthesis and radioiodination of some daunorubicin and doxorubicin derivatives.

Daunorubicin and doxorubicin are efficient agents for cancer treatment. Their clinical efficacy is, however, hampered by their indiscriminant toxicity. This problem may be circumvented by encapsulating the drugs in liposomes and selectively targeting the tumor cells using tumor targeting agents. Furthermore, the antitumor effect could be enhanced by attaching the Auger electron emitter, (125)I, to daunorubicin and doxorubicin derivatives. In this context a number of ester, amide, and amine derivatives of daunorubicin and doxorubicin were synthesized. Benzoic acid ester derivatives of daunorubicin were synthesized by nucleophilic esterification of the 14-bromodaunorubicin with the potassium salt of the corresponding benzoic acid, resulting in good yields. Nicotinic acids and benzoic acids, activated with a succinimidyl group, were coupled to the amino group of daunorubicin to give the corresponding amide derivatives. Amine derivatives were obtained by the reductive amination of aromatic aldehydes with daunorubicin hydrochloride. The stannylated ester and amide derivatives were used as precursors for radioiodination. Radiolabeling with (125)I was performed using chloramine-T as an oxidant. The optimized labeling resulted in high radiolabeling yields (85-95%) of the radioiodinated daunorubicin and doxorubicin derivatives. Radioiodination of the amines was conducted at the ortho position of the activated phenyl rings providing moderate radiochemical yields (55-75%).