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We previously identified a spiro [piperidine-4,1-pyrido [3,4-b]indole] class of co-potentiators that function in synergy with existing CFTR potentiators such as VX-770 or GLGP1837 to restore channel activity of a defined subset of minimal function cystic fibrosis transmembrane conductance regulator (CFTR) mutants. Here, structure-activity studies were conducted to improve their potency over the previously identified compound, 20 (originally termed CP-A01). Targeted synthesis of 37 spiro [piperidine-4,1-pyrido [3,4-b]indoles] was generally accomplished using versatile two or three step reaction protocols with each step having high efficiency. Structure-activity relationship studies established that analog 2i, with 6'-methoxyindole and 2,4,5-trifluorobenzyl substituents, had the greatest potency for activation of N1303K-CFTR, with EC50 ∼600 nM representing an ∼17-fold improvement over the original compound identified in a small molecule screen.

Photoswitches capable of accessing two geometric states are highly desirable, especially if their design is modular and incorporates a pharmacophore tethering site. We describe a redox isomerization strategy for synthesizing p-formylazobenzenes from p-nitrobenzyl alcohol. The resulting azo-aldehydes can be readily converted to photoswitchable compounds with excellent photophysical properties using simple hydrazide click chemistry. As a proof of principle, we synthesized a photoswitchable surfactant enabling the photocontrol of an emulsion with exceptionally high spatiotemporal precision.

Pendrin is a transmembrane chloride/anion antiporter that is strongly upregulated in the airways in rhinoviral infection, asthma, cystic fibrosis and chronic rhinosinusitis. Based on its role in the regulation of airway surface liquid depth, pendrin inhibitors have potential indications for treatment of inflammatory airways diseases. Here, a completely regioselective route to tetrahydro-pyrazolopyridine pendrin inhibitors based on 1,3-diketone and substituted hydrazine condensation was been developed. Structure-activity relationships at the tetrahydropyridyl nitrogen were investigated using a focused library, establishing the privileged nature of N-phenyl ureas and improving inhibitor potency by greater than 2-fold.

The Cadogan cyclization is a robust but harsh method for the synthesis of 2 H-indazoles, a valuable class of nitrogen heterocycles. Although nitrene generation by exhaustive deoxygenation is widely accepted as the operating mechanism in the reductive cyclization of nitroaromatics, non-nitrene pathways have only been theorized previously. Here, 2 H-indazole N-oxides were synthesized through an interrupted Cadogan/Davis-Beirut reaction and are presented as direct evidence of competent oxygenated intermediates; mechanistic implications for both reactions are discussed. Isolation and characterization of these N-oxides enabled a formal Cadogan cyclization at room temperature for 2 H-indazole synthesis.

BackgroundCurrent modulator therapies for some cystic fibrosis-causing CFTR mutants, including N1303K, have limited efficacy. We provide evidence here to support combination potentiator (co-potentiator) therapy for mutant CFTRs that are poorly responsive to single potentiators.MethodsFunctional synergy screens done on N1303K and W1282X CFTR, in which small molecules were tested with VX-770, identified arylsulfonamide-pyrrolopyridine, phenoxy-benzimidazole and flavone co-potentiators.ResultsA previously identified arylsulfonamide-pyrrolopyridine co-potentiator (ASP-11) added with VX-770 increased N1303K-CFTR current 7-fold more than VX-770 alone. ASP-11 increased by ~65% of the current of G551D-CFTR compared to VX-770, was additive with VX-770 on F508del-CFTR, and activated wild-type CFTR in the absence of a cAMP agonist. ASP-11 efficacy with VX-770 was demonstrated in primary CF human airway cell cultures having N1303K, W1282X and G551D CFTR mutations. Structure-activity studies on 11 synthesized ASP-11 analogs produced compounds with EC50 down to 0.5 μM.ConclusionsThese studies support combination potentiator therapy for CF caused by some CFTR mutations that are not effectively treated by single potentiators.

A concise, one-step route to indazolones from primary alkyl amines and o-nitrobenzyl alcohols is reported. The key step in this readily scalable indazolone forming process involves base-mediated in situ o-nitrobenzyl alcohol → o-nitrosobenzaldehyde conversion. Although this functional group interconversion is known to be useful for 2 H-indazole synthesis, its reactivity was modulated for indazolone formation.

Reaction options, alkoxide vs hydroxide vs amine addition to the key intermediate (o-nitrosoimine) generated in the Davis-Beirut reaction of an o-nitrobenzylamine substrate, are reported to explain the nucleophilic addition selectivity of this one-pot indazole-forming process. The hydroxide addition/deprotection pathway as well as the fate of the resulting o-nitrosobenzaldehyde were both uncovered with several o-nitrobenzylamine substrates, and design elements required for an efficient double Davis-Beirut reaction, inspired by new mechanistic insights, were defined.

The discovery of a new variation on the Davis-Beirut reaction is described in which an atypical heterocyclic framework (the anthranil or benzo[c]isoxazole framework) is formed as the result of diversion of a key reactive intermediate away from its expected reactivity-a potentially general approach to reaction design and development. Experimental and computational support for the proposed mechanism and origins of altered reactivity are described.

Constipation is a common condition for which current treatments can have limited efficacy. By high-throughput screening, we recently identified a phenylquinoxalinone activator of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that stimulated intestinal fluid secretion and normalized stool output in a mouse model of opioid-induced constipation. Here, we report phenylquinoxalinone structure-activity analysis, mechanism of action, animal efficacy data in acute and chronic models of constipation, and functional data in ex vivo primary cultured human enterocytes. Structure-activity analysis was done on 175 phenylquinoxalinone analogs, including 15 synthesized compounds. The most potent compound, CFTRact-J027, activated CFTR with EC50 ∼ 200 nM, with patch-clamp analysis showing a linear CFTR current-voltage relationship with direct CFTR activation. CFTRact-J027 corrected reduced stool output and hydration in a mouse model of acute constipation produced by scopolamine and in a chronically constipated mouse strain (C3H/HeJ). Direct comparison with the approved prosecretory drugs lubiprostone and linaclotide showed substantially greater intestinal fluid secretion with CFTRact-J027, as well as greater efficacy in a constipation model. As evidence to support efficacy in human constipation, CFTRact-J027 increased transepithelial fluid transport in enteroids generated from normal human small intestine. Also, CFTRact-J027 was rapidly metabolized in vitro in human hepatic microsomes, suggesting minimal systemic exposure upon oral administration. These data establish structure-activity and mechanistic data for phenylquinoxalinone CFTR activators, and support their potential efficacy in human constipation.

We previously identified phenylquinoxalinone CFTRact-J027 (4) as a cystic fibrosis transmembrane conductance regulator (CFTR) activator with an EC50 of ∼200 nM and demonstrated its therapeutic efficacy in mouse models of constipation. Here, structure-activity studies were done on 36 synthesized phenylquinoxalinone analogs to identify compounds with improved potency and altered metabolic stability. Synthesis of the phenylquinoxalinone core was generally accomplished by condensation of 1,2-phenylenediamines with substituted phenyloxoacetates. Structure-activity studies established, among other features, the privileged nature of a properly positioned nitro moiety on the 3-aryl group. Synthesized analogs showed improved CFTR activation potency compared to 4 with EC50 down to 21 nM and with greater metabolic stability. CFTR activators have potential therapeutic indications in constipation, dry eye, cholestatic liver diseases, and inflammatory lung disorders.

Two new substituted polyoxotantalate clusters, [Ti2 Ta8 O28 ](8-) and [Ti12 Ta6 O44 ](10-) , considerably expand the pH range where tantalates persist in aqueous solution. The structures of [Ti2 Ta8 O28 ](8-) and [Ti12 Ta6 O44 ](10-) are reported as tetramethylammonium salts after synthesis at hydrothermal conditions in aqueous solution. These Ti-substituted polyoxotantalate clusters have analogues among recently discovered niobates, but are slightly larger and more persistent in solution. Most importantly, they exhibit a much wider range of pH stability than the familiar hexatantalate cluster, which is the only other tantalate known to be stable at highly basic pH conditions. These molecules are kinetically stable to near-neutral pH, making them excellent synthons for further development into materials and catalysts, and an significant advance in adapting tantalates for use in aqueous solutions.

Two new substituted polyoxotantalate clusters, [Ti2 Ta8 O28 ](8-) and [Ti12 Ta6 O44 ](10-) , considerably expand the pH range where tantalates persist in aqueous solution. The structures of [Ti2 Ta8 O28 ](8-) and [Ti12 Ta6 O44 ](10-) are reported as tetramethylammonium salts after synthesis at hydrothermal conditions in aqueous solution. These Ti-substituted polyoxotantalate clusters have analogues among recently discovered niobates, but are slightly larger and more persistent in solution. Most importantly, they exhibit a much wider range of pH stability than the familiar hexatantalate cluster, which is the only other tantalate known to be stable at highly basic pH conditions. These molecules are kinetically stable to near-neutral pH, making them excellent synthons for further development into materials and catalysts, and an significant advance in adapting tantalates for use in aqueous solutions.

Two new rhodium-substituted polyoxoniobates, [H2RhNb9O28](6-) (RhNb9) and [Rh2(OH)4Nb10O30](8-) (Rh2Nb10) are reported. The two distinct Rh(III)-substituted niobate clusters behave differently when the pH is raised with TMAOH: the Rh2Nb10 is stable until pH ∼ 12.7, but RhNb9 dissociates to form RhNb5 and RhNb10, similar to some of our other metal-substituted niobates, such as the MNb9 ions (M = Cr or Mn), which transform to MNb10 when the solution pH is raised.

Two new rhodium-substituted polyoxoniobates, [H2RhNb9O28](6-) (RhNb9) and [Rh2(OH)4Nb10O30](8-) (Rh2Nb10) are reported. The two distinct Rh(III)-substituted niobate clusters behave differently when the pH is raised with TMAOH: the Rh2Nb10 is stable until pH ∼ 12.7, but RhNb9 dissociates to form RhNb5 and RhNb10, similar to some of our other metal-substituted niobates, such as the MNb9 ions (M = Cr or Mn), which transform to MNb10 when the solution pH is raised.

Polyoxoniobate clusters that are stable in acidic solutions are rare and particularly useful in industrial processes. Here we report a new pentaphosphate niobate polyoxometalate cluster (TMA)9H3Nb9P5O41·28H2O (Nb9P5) that is stable over a wide pH range and that can be converted reversibly into the peroxo form.

Two new peroxoniobophosphate clusters were isolated as tetramethylammonium (TMA) salts having the stoichiometries: TMA5[HNb4P2O14(O2)4]⋅9 H2O and TMA3[H7Nb6P4O24(O2)6]⋅7 H2O. The former is stable over the pH range: 3

Two new peroxoniobophosphate clusters were isolated as tetramethylammonium (TMA) salts having the stoichiometries: TMA5[HNb4P2O14(O2)4]⋅9 H2O and TMA3[H7Nb6P4O24(O2)6]⋅7 H2O. The former is stable over the pH range: 3

Caps in α-Keggin-type polyoxometalates [PM2Nb12O40](9-) (M: Nb[double bond, length as m-dash]O or V[double bond, length as m-dash]O) can be removed in basic condition to produce uncapped [PNb12O40](15-). Transmetalation or capping occurs from the reaction of [PNb14O42](9-) or [PNb12O40](15-) with either Sb2O3 or V2O5 to form [PSb2Nb12O40](9-) or [PV2Nb12O42](9-), respectively.

In prior studies, aqueous Hf sulfate-peroxide solutions were spin-coated, dehydrated, patterned by electron-beam lithography, ion-exchanged (OH(-) for SO4(2-)), and finally converted to HfO2 hard masks via annealing. The atomic-level details of the underlying aqueous chemistries of these processes are complex and yet to be understood. Yet a thorough understanding of this specific chemical system will inspire development of design rules for other aqueous-precursor-to-solid-state metal oxide systems. Often-observed crystallization of the Hf18 polyoxometalate from aqueous Hf sulfate-peroxide precursor solutions has led us to believe that Hf18 may represent an important intermediate step in this process. However, via detailed solution studies described here (small-angle X-ray scattering, electrospray ionization mass spectrometry, and Raman spectroscopy), we ascertained that Hf18 is in fact not a prenucleation cluster of Hf sulfate coatings. Rather, the Hf tetramers, pentamers, and hexamers that are the core building blocks of Hf18 are robustly persistent over variable compositions and aging time of precursor solutions, and therefore they are likely the rudimentary building blocks of the deposited thin-film materials. These Hf clusters are capped and linked by sulfate and peroxide anions in solution, which probably prevents crystallization of Hf18 during the rapid dehydration process of spin-coating. In fact, crystallization of Hf18 from the amorphous gel coating would be detrimental to formation of a high-density conformal coating that we obtain from precursor solutions. Therefore, this study revealed that the well-known Hf18 polyoxometalate is not likely to be an important intermediate in the thin-film process. However, its subunits are, confirming the universal importance of deriving information from the solid state, albeit judiciously and critically, to understand the solution state.

The mammalian sperm acrosome reaction (AR) is essential to fertilization, and the egg zona pellucida (ZP) is generally believed to be an in vivo initiator of the fertilizing sperm AR. Previously a neuronal glycine receptor/[C1.sup.-] channel (GlyR) was detected on the plasma membrane of mammalian sperm and earlier pharmacological studies suggested that this receptor/channel is important to the ZP-initiated AR. Here, sperm from mice with mutations in the neuronal GlyR [Alpha] or [Beta] subunits (spasmodic and spastic) were shown to be deficient in their ability to undergo the AR initiated in vitro by glycine or by solubilized ZP from mouse eggs. However, both spontaneous and calcium ionophore (A23187)-initiated AR were unaffected. The ZP-initiated AR in wild-type sperm was maximal after 2 h of capacitation, but capacitation of sperm from spasmodic mice for up to 3 h did not result in significant ZP-initiated AR. Similar results were observed when sperm from wild-type and spastic mice were compared. Testis from mice with the [Beta] subunit mutation contained truncated [Beta] subunit mRNAs. Moreover, a monoclonal antibody against GlyR completely blocked ZP initiation of AR in normal mouse sperm. Our results are consistent with an essential role for the sperm GlyR in the ZP-initiated AR. [C] 2000 Academic Press Key Words: glycine receptor/[C1.sup.-] channel; acrosome reaction; spasmodic mouse; spastic mouse; zona pellucida; hyperekplexia.