Amyloid disease prevention by transthyretin native state complexation with carborane derivatives lacking cyclooxygenase inhibition

Misfolding and subsequent aggregation of any of a number of proteins leads to the accumulation of amyloid fibrils, which have been associated with a variety of diseases. One such amyloidogenic protein is transthyretin (TTR), a 55-kDa homotetrameric protein found in the blood plasma and cerebrospinal fluid where it binds and transports thyroxine. In humans, the T119M-TTR variant has been shown to be protective against familial amyloid polyneuropathy, a TTR amyloid disease, through kinetic stabilization of the unliganded tetrameric structure. Studies have indicated that a diverse range of small molecules may also bind TTR in the thyroxine-binding pocket and subsequently kinetically stabilize the protein's native conformation in vitro, preventing the misfolding that has been implicated in the progression of several diseases. However, cyclooxygenase inhibition is a common unwanted side effect among such small-molecule kinetic stabilizers. The recent development of transthyretin stabilizers not subject to cyclooxygenase inhibition may prove attractive for the long-term treatment of TTR misfolding diseases in humans. Such compounds are attained by incorporating aromatic carborane icosahedra at strategic points in their structures.     

Biochemical and structural evaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors

To develop potent transthyretin (TTR) amyloidogenesis inhibitors that also display high binding selectivity in blood, it proves useful to systematically optimize each of the three substructural elements that comprise a typical inhibitor: the two aryl rings and the linker joining them. In the first study, described herein, structural modifications to one aryl ring were evaluated by screening a library of 2-arylbenzoxazoles bearing thyroid hormone-like aryl substituents on the 2-aryl ring. Several potent and highly selective amyloidogenesis inhibitors were identified that exhibit minimal thyroid hormone nuclear receptor and COX-1 binding. High resolution crystal structures (1.3-1.5 A) of three inhibitors (2f, 4f, and 4d) in complex with TTR were obtained to characterize their binding orientation. Collectively, the results demonstrate that thyroid hormone-like substitution patterns on one aryl ring lead to potent and highly selective TTR amyloidogenesis inhibitors that lack undesirable thyroid hormone receptor or COX-1 binding.     

N-methylsansalvamide a peptide analogues. Potent new antitumor agents

Sansalvamide A, a cyclic depsipeptide isolated from a marine fungus of the genus Fusarium, is composed of four hydrophobic amino acids (Phe, two Leu, Val) and one hydroxy acid ((S)-2-hydroxy-4-methylpentanoic acid; O-Leu) with five stereogenic centers all having S-stereochemistry. We have recently synthesized the corresponding cyclic peptide (Gu, W.; Liu, S.; Silverman, R. B. Organic Lett. 2002, 4, 4171-4174) and found that it too has antitumor activity. N-Methylation can enhance potency and selectivity for peptides. Consequently, here we synthesize 12 different N-methylated sansalvamide A peptide analogues and show that for several different tumor cell lines three of these analogues are more potent than the natural product; in pancreatic cells, sansalvamide A shows little activity, but the N-methylsansalvamide peptides are potent cytotoxic agents.     

Toward optimization of the linker substructure common to transthyretin amyloidogenesis inhibitors using biochemical and structural studies

To develop potent and highly selective transthyretin (TTR) amyloidogenesis inhibitors, it is useful to systematically optimize the three substructural elements that compose a typical TTR kinetic stabilizer: the two aryl rings and the linker joining them. Herein, we evaluated 40 bisaryl molecules based on 10 unique linker substructures to determine how these linkages influence inhibitor potency and selectivity. These linkers connect one unsubstituted aromatic ring to either a 3,5-X 2 or a 3,5-X 2-4-OH phenyl substructure (X = Br or CH 3). Coconsideration of amyloid inhibition and ex vivo plasma TTR binding selectivity data reveal that direct connection of the two aryls or linkage through nonpolar E-olefin or -CH 2CH 2- substructures generates the most potent and selective TTR amyloidogenesis inhibitors exhibiting minimal undesirable binding to the thyroid hormone nuclear receptor or the COX-1 enzyme. Five high-resolution TTR.inhibitor crystal structures (1.4-1.8 A) provide insight into why such linkers afford inhibitors with greater potency and selectivity.     

Diflunisal in the management of sprains.

A double-blind randomized trial was carried out in 31 patients suffering from acute, minor ligamentous injuries to compare the efficacy of diflunisal in the relief of pain with that of oxyphenbutazone. Patients received either 500 mg diflunisal twice daily or 200 mg oxyphenbutazone 3-times daily for 3 days. The results of subjective assessments showed tha by Day 3 spontaneous pain had either completely resolved or markedly improved in all patients, and that diflunisal was significantly better than oxyphenbutazone on Days 1 and 3 in relieving pain on movement of the joint.     

Potent inhibition of NTPase/helicase of the West Nile Virus by ring-expanded ("fat") nucleoside analogues

A series of ring-expanded ("fat") nucleoside analogues (RENs) containing the 6-aminoimidazo[4,5-e][1,3]diazepine-4,8-dione ring system have been synthesized and screened for inhibition of NTPase/helicase of the West Nile Virus (WNV). To assess the selectivity of RENs against the viral enzymes, a truncated form of human enzyme Suv3((Delta)(1)(-)(159)) was also included in the study. Ring-expanded nucleosides 16, 17, and 19, which possess the long C(12), C(14), and C(18) side-chains, respectively, at position 6, as well as the ring-expanded heterocycle 39, which contains aralkyl substitution at position 1, were all found to have excellent profiles of activity and selectivity toward the viral versus human enzymes against the West Nile Virus (IC(50) ranging 1-10 muM). Compound 30, while being an equally potent inhibitor of WNV, was found to be somewhat less selective, whereas compound 36, which is an alpha-anomeric counterpart of 30, exhibited potent and selective inhibition of WNV (IC(50) 1-3 muM). The same compounds that showed potent inhibition of viral helicase activity completely failed to show any activity against the viral NTPase reaction even up to 500 muM. However, at concentrations >500 muM of RENs and the ATP concentrations >10 times the K(m) value of the enzyme, a significant activation of NTPase activity was observed. This activating effect underwent further dramatic enhancement (>1000%) by further increases in ATP concentration in the reaction mixture, suggesting that the viral helicase and NTPase reactions are not coupled. A tentative mechanistic model has been proposed to explain the observed results.     

Potent antimalarial febrifugine analogues against the plasmodium malaria parasite

Although febrifugine (1) and isofebrifugine (2), alkaloids isolated from roots of the Dichroa febrifuga plant, show powerful antimalarial activity against Plasmodium falciparum, strong side effects such as the emetic effect have precluded their clinical use against malaria. However, their antimalarial potency makes them attractive substances as leads for developing new types of chemotherapeutic antimalarial drugs. Thus, we have evaluated the in vitro antimalarial activity of the analogues of febrifugine (1) and isofebrifugine (2). The activities of the analogues derived from Df-1 (3) and Df-2 (4), condensation products of 1 and 2 with acetone, respectively, were also obtained. The 3' '-keto derivative (7, EC(50) = 2.0 x 10(-8) M) of 1 was found to exhibit potential antimalarial activity with high selectivity against P. falciparum in vitro. The in vitro activities of the reduction product (8, EC(50) = 2.0 x 10(-8) M) of 1 at C-2' and its cyclic derivatives 9 and 10 (EC(50) = 3.7 x 10(-9) and 8.6 x 10(-9) M, respectively) were found to be strongly active and selective. Additionally, the Dess-Martin oxidation product of 3 was found to be strongly active with high selectivity against P. falciparum. A structure-activity relationship study (SAR) demonstrates that the essential role played by the 4-quinazolinone ring in the appearance of activity and the presence of a 1' '-amino group and C-2', C-3' ' O-functionalities are crucial in the activity of 1. For 7, 8, and 9, prepared as racemic forms, an in vivo study has also been conducted.     

Potent inhibition of human liver aldehyde oxidase by raloxifene.

The selective estrogen receptor modulator, raloxifene, has been demonstrated as a potent uncompetitive inhibitor of human liver aldehyde oxidase-catalyzed oxidation of phthalazine, vanillin, and nicotine-Delta1'(5')-iminium ion, with K(i) values of 0.87 to 1.4 nM. Inhibition was not time-dependent. Raloxifene has also been shown to be a noncompetitive inhibitor of an aldehyde oxidase-catalyzed reduction reaction of a hydroxamic acid-containing compound, with a K(i) of 51 nM. However, raloxifene had only small effects on xanthine oxidase, an enzyme related to aldehyde oxidase. In addition, several other compounds of the same therapeutic class as raloxifene were examined for their potential to inhibit aldehyde oxidase. However, none were as potent as raloxifene, since IC(50) values were orders of magnitude higher and ranged from 0.29 to 57 micro M. In an examination of analogs of raloxifene, it was shown that the bisphenol structure with a hydrophobic group on the 3-position of the benzthiophene ring system was the most important element that imparts inhibitory potency. The relevance of these data to the mechanistic understanding of aldehyde oxidase catalysis, as well as to the potential for raloxifene to cause drug interactions with agents for which aldehyde oxidase-mediated metabolism is important, such as zaleplon or famciclovir, is discussed.     

Potent and selective inhibition of varicella-zoster virus (VZV) by nucleoside analogues with an unusual bicyclic base

We herein report the discovery of an entirely new category of potent antiviral agents based on novel deoxynucleoside analogues with unusual bicyclic base moieties. Target structures, previously known as byproducts in Pd-catalyzed coupling of terminal alkynes with 5-iodo-nucleosides, are recognized herein for the first time to be potent and selective inhibitors of varicella-zoster virus (VZV) in vitro. As an unusual structure-activity relationship we noted the absolute requirement of a long alkyl side chain, with an optimum length of C(8)-C(10), for antiviral activity. We thus report the synthesis and characterization of a series of chain-modified analogues and their extensive in vitro evaluation. The lead compounds have a ca. 300-fold enhancement in anti-VZV activity over the reference compound acyclovir, with no detectable in vitro cytotoxicity. The novel structure of these compounds, coupled with their ease of synthesis, excellent antiviral profile, and promising physical properties, makes them of great interest for possible antiviral drug development.     

Potent inhibition of the CFTR chloride channel by suramin

After phosphorylation by protein kinase A and in the presence of ATP, the cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel. In this study we have examined the effects of suramin on the CFTR Cl- current (I(CFTR)) in excised inside-out macropatches from Xenopus oocytes expressing human CFTR; glibenclamide, the standard inhibitor of I(CFTR), and some congeners were tested in comparison. I(CFTR) was activated by addition of the catalytic subunit of protein kinase A and MgATP to the bath. Suramin inhibited I(CFTR) with an IC50 value of 1 microM and a Hill coefficient close to 1; the inhibition showed little voltage dependence and was easily reversed upon washout of the drug. In comparison, glibenclamide inhibited I(CFTR) with an IC50 value of approximately 20 microM. When tested against I(CFTR) in whole oocytes, bath application of suramin was ineffective whereas glibenclamide was about four times weaker than in the inside-out patch configuration. The data show that suramin is the most potent inhibitor of CFTR yet described and suggest that the compound approaches its site of action from the cytosol.     

Synthesis, structure, and activity of diclofenac analogues as transthyretin amyloid fibril formation inhibitors.

Twelve analogues of diclofenac (1), a nonsteroidal antiinflammatory drug and known inhibitor of transthyretin (TTR) amyloid formation, were prepared and evaluated as TTR amyloid formation inhibitors. High activity was exhibited by five of the compounds. Structure-activity relationships reveal that a carboxylic acid is required for activity, but changes in its position as well as the positions of other substituents are tolerated. High-resolution X-ray crystal structures of four of the active compounds bound to TTR were obtained. These demonstrate the significant flexibility with which TTR can accommodate ligands within its two binding sites.     

Potent gastric acid inhibition in the management of Barrett's oesophagus

Barrett's oesophagus is the consequence of excessive and prolonged gastro-oesophageal reflux. The therapeutic objectives in Barrett's oesophagus include the reduction of gastro-oesophageal reflux in order to relieve symptoms and the prevention of the biologic progression to adenocarcinoma. The first objective may be achieved with standard proton pump inhibitor (PPI) therapy, which is the base of the medical therapy in this type of patients, but this therapy has been found not to be associated with normalization of the intraluminal pH of the oesophagus in many patients with Barrett's oesophagus. This condition seems to be necessary in order to reduce mucosal cell proliferation in some studies. Therefore, it has been proposed that patients with Barrett's oesophagus need profound acid inhibition with high-dose PPI. This therapeutic approach provides symptom relief, but there is no direct evidence that it is associated with Barrett's oesophagus regression or progression to adenocarcinoma. Nevertheless, recent and preliminary data suggest that long-term PPI therapy may reduce the risk of developing disease progression. Profound acid inhibition is also combined with endoscopy ablative or resection therapy in patients with Barrett's oesophagus. This therapeutic approach should be still regarded as experimental and more data are needed before its therapeutic role in patients with Barrett's oesophagus is established.     

Diflunisal in the management of sprains

Summary

A double-blind randomized trial was carried out in 31 patients suffering from acute, minor ligamentous injuries to compare the efficacy of diflunisal in the relief of pain with that of oxyphenbutazone. Patients received either 500?mg diflunisal twice daily or 200?mg oxyphenbutazone 3-times daily for 3 days. The results of subjective assessments showed that by Day 3 spontaneous pain had either completely resolved or markedly improved in all patients, and that diflunisal was significantly better than oxyphenbutazone on Days 1 and 3 in relieving pain on movement of the joint.     

Prostaglandin-H synthase inhibition by malonamides. Ring-opened analogues of phenylbutazone

Recent reports of serious concern regarding the safe clinical use of phenylbutazone and its hydroxylated metabolite (oxyphenbutazone) as antiinflammatory agents have prompted the further investigation of ring-opened (malonamide) derivatives as potentially preferable therapeutic derivatives. Earlier reports have claimed reduced toxicity among similar derivatives. These studies reveal the relative degree of prostaglandin-H (PGH) synthase inhibitory activity among a series of malonamide derivatives. Contrary to observations in the pyrazolidinedione series, incorporation of a nonpolar butyl side chain in these malonamides was not beneficial but, rather, detrimental to enzyme-inhibitory activity. Although none of the reported nonbutylated malonamides was as potent an inhibitor of this enzyme as phenylbutazone, they all showed some inhibitory activity. PGH synthase inhibitory activity was especially pronounced in the bis(p-hydroxy anilide) derivatives, even extending to succinamide and adipamide derivatives. Of some interest is the observation that all of these p-hydroxy anilide derivatives were more potent inhibitors of this enzyme than acetaminophen.     

Further SAR studies of piperidine-based analogues of cocaine. 2. Potent dopamine and serotonin reuptake inhibitors

The synthesis and monoamine transporter activity of additional members of a series of 3,4-disubstituted piperidines (truncated analogues of the WIN series) are described. All members of this series were prepared from arecoline hydrobromide in optically pure form and were evaluated for their ability to inhibit high affinity uptake of dopamine (DA), serotonin (5-HT) and norepinephrine (NE) into rat brain nerve endings (synaptosomes). Most of the compounds prepared in this series are reasonably potent DAT inhibitors (K(i) values of 4-400 nM) and have selectivity for the 5-HT transporter relative to both the NE transporter (3-9-fold) and to the DAT ( approximately 25-fold). In the present series, (-)-methyl 1-methyl-4beta-(2-naphthyl)piperidine-3beta-carboxylate (6) was found to be the most potent piperidine-based ligand, exhibiting K(i)'s of 21 nM and 7.6 nM at the DAT and 5-HTT, respectively. While the 5-HTT activity of compound 6 is comparable to that of the antidepressant medication fluoxetine, it is less selective. As is apparent from the data presented, the naphthyl substituted piperidines 6-9, which differ in their stereochemistry, show different degrees of selectivity for the three transporters. Consistent with results reported in the literature for the tropane analogues, removal of the methyl group from the nitrogen atom of 9 leads to a further enhancement in 5-HTT activity. To examine the in vivo effects of these piperidines, preliminary behavioral screening was carried out on piperidine 14. Despite its 2.5-fold greater DAT activity compared to cocaine, piperidine 14 was found to be about 2. 5-fold less potent in increasing distance traveled in mice. However, consistent with its DAT activity, piperidine 14 was found to be about 2.5-fold more potent than cocaine in enhancing stereotypic movements. Further studies of these piperidine-based ligands may provide valuable insights into the pharmacological mechanisms underlying the enhancement in distance traveled versus stereotypic movements. The present results have important implications for better understanding the structural motifs required in the design of agents with specific potency and selectivity at monoamine transporters.     

Nitric oxide synthase inhibition by dimaprit and dimaprit analogues.

1. The similarity in molecular structure between the histamine H2-agonist dimaprit (3-dimethylamino-propyl-isothiourea) and the endogenous nitric oxide synthase (NOS) substrate L-arginine prompted us to study the effect of dimaprit and some dimaprit analogues on NOS activity. Dimaprit and some of its analogues were tested in an in vitro assay which measures the conversion of [3H]-L-arginine to [3H]-L-citrulline. Dimaprit inhibits rat brain NOS (nNOS) concentration dependently with an IC50 of 49+/-14 microM. 2. Removal of one or both of the methyl groups from the non-isothiourea nitrogen of dimaprit improved nNOS inhibitory properties. Aminopropylisothiourea is the most potent compound (IC50 = 4.1+/-0.9 microM) of the series followed by methylaminopropylisothiourea (IC50 = 7.6 +/- microM). 3. The observed effect of aminopropylisothiourea and methylaminopropyl-isothiourea are probably not due to the compounds themselves but to the corresponding mercaptoalkylguanidines, rearrangement products formed in aqueous solutions. This hypothesis is strengthened by the finding that aminobutylisothiourea is not active since a rearrangement to mercaptobutylguanidine does not occur. 4. Remarkably, nitrosylation of the isothiourea group of dimaprit decreases nNOS inhibitory activity, while nitrosylation of the guanidine analogue of dimaprit increases the inhibition of nNOS activity. 5. The pharmacological profile of dimaprit includes inhibition of nNOS. The nNOS inhibitory activity occurs in the same concentration range as the H2-agonist and H3-agonist activity of this compound.     

Batrachotoxin and alpha-scorpion toxin stabilize the open state of single voltage-gated sodium channels

The combined effects of batrachotoxin (BTX) and either scorpion (Leiurus quinquestriatus quinquestriatus) venom (LqqV) or alpha-scorpion toxin (alpha-LqqTX) purified from LqqV on single voltage-gated Na channels were studied in planar lipid bilayers. In the presence of BTX, LqqV caused the channels to remain open at membrane potentials at least 50 mV more hyperpolarized than with BTX alone. alpha-LqqTX mimicked the effect of LqqV, suggesting that this toxin is the active component of the venom. LqqV did not significantly alter single-channel conductance, voltage-dependent block by saxitoxin, or voltage-dependent block by Ca2+, indicating that the venom preferentially affects gating rather than ion permeation. The results indicate that a cooperative interaction between alpha-LqqTX and BTX strongly favors the open state of the Na channel by causing a large hyperpolarizing shift in the voltage dependence of activation. This effect on activation gating is not predicted from the individual effects of the toxins.