A [3+3] Aldol-SNAr-Dehydration Approach to 2-Naphthol and 7-Hydroxyquinoline Derivatives.

A one-pot [3+3] aldol-SNAr-dehydration annulation sequence was utilized to fuse hindered phenols onto aromatic substrates. The transformation joins doubly activated 1,3-disubstituted acetone derivatives (dinucleophiles) with C5-activated 2-fluorobenzaldehyde SNAr acceptors (dielectrophiles) in the presence of K2CO3 in DMF at 65-70 °C to form polysubstituted 2-naphthols and 7-hydroxyquinolines. The reaction is regioselective in adding the most stable anionic center to the aldehyde followed by SNAr closure of the less stabilized anion to the electron-deficient aromatic ring. Twenty-seven examples are reported, and a probable mechanism is presented. In two cases where SNAr activation on the acceptor ring was lower (a C5 trifluoromethyl group on the aromatic ring or a 2-fluoropyridine), diethyl 1,3-acetonedicarboxylate initiated an interesting Grob-type fragmentation to give cinnamate esters as the products.

Domino Aldol-SNAr-Dehydration Sequence for [3+3] Annulations to Prepare Quinolin-2(1H)-ones and 1,8-Naphthyridin-2(1H)-ones.

A domino aldol-SNAr-dehydration [3+3] annulation strategy has been utilized to fuse six-membered cyclic amides onto aromatic substrates. 2-Arylacetamides have been reacted with 2-fluorobenzaldehyde derivatives activated toward SNAr reaction by an electron-withdrawing substituent (NO2, CN, CF3, CO2Me) at C5 to prepare 3,6-disubstituted quinolin-2(1H)-ones. Additionally, 3-substituted 1,8-naphthyridin-2(1H)-ones have been similarly derived from 2-fluoronicotinaldehyde. Fifteen examples are reported, and two possible mechanistic scenarios are presented and discussed.

Synthesis and Elimination Pathways of 1-Methanesulfonyl-1,2-dihydroquinoline Sulfonamides.

A series of new Morita-Baylis-Hillman acetates were prepared and reacted with methanesulfonamide (K2CO3, DMF, 23 °C) to produce tertiary dihydroquinoline sulfonamides in high yields. Subsequent efforts to eliminate the methylsulfonyl group from these derivatives (K2CO3, DMF, 90 °C) as a route to quinolines were met with mixed results. Although dihydroquinoline sulfonamides prepared from ethyl acrylate and acrylonitrile generally underwent elimination to give excellent yields of quinolines, those generated from 3-buten-2-one failed to undergo elimination and instead decomposed. The failure of these ketone substrates to aromatize presumably derives from the enolizable methyl ketone at C-3. Finally, the attempted aromatization of the acrylate-derived 6,7-difluoro-1,2-dihydroquinoline sulfonamide demonstrated that other interesting processes could occur in preference to the desired elimination.

Domino Nitro Reduction-Friedländer Heterocyclization for the Preparation of Quinolines.

The Friedländer synthesis offers efficient access to substituted quinolines from 2-aminobenzaldehydes and activated ketones in the presence of a base. The disadvantage of this procedure lies in the fact that relatively few 2-aminobenzaldehyde derivatives are readily available. To overcome this problem, we report a modification of this process involving the in situ reduction of 2-nitrobenzaldehydes with Fe/AcOH in the presence of active methylene compounds (AMCs) to produce substituted quinolines in high yields. The conditions are mild enough to tolerate a wide range of functionality in both reacting partners and promote reactions not only with phenyl and benzyl ketones, but also with ß-keto-esters, ß-keto-nitriles, ß-keto-sulfones and ß-diketones. The reaction of 2-nitroaromatic ketones with unsymmetrical AMCs is less reliable, giving a competitive formation of substituted quinolin-2(1H)-ones from the cyclization of the Z Knoevenagel intermediate which appears to be favored when certain large groups are adjacent to the AMC ketone carbonyl.

Domino Aza-Michael-SNAr-Heteroaromatization Route to C5-Substituted 1-Alkyl-1H-Indole-3-Carboxylic Esters.

A new synthesis of C5-substituted 1-alkyl-1H-indole-3-carboxylic esters is reported. A series of methyl 2-arylacrylate aza-Michael acceptors were prepared with aromatic substitution to activate them towards SNAr reaction. Subsequent reaction with a series of primary amines generated the title compounds. Initially, the sequence was expected to produce indoline products, but oxidative heteroaromatization intervened to generate the indoles. The reaction proceeded under anhydrous conditions in DMF at 23-90 °C using equimolar quantities of the acrylate and the amine with 2 equiv. of K2CO3 to give 61-92% of the indole products. The reaction involves an aza-Michael addition, followed by SNAr ring closure and heteroaromatization. Since the reactions were run under nitrogen, the final oxidation to the indole likely results from reaction with dissolved oxygen in the DMF. Substrates incorporating a 2-arylacrylonitrile proved too reactive to prepare using our protocol. The synthesis of the reaction substrates, their relative reactivities, and mechanistic details of the conversion are discussed.

Synthesis and Characterization of N-Isopropylacrylamide Microspheres as pH Sensors.

Swellable polymer microspheres that respond to pH were prepared by free radical dispersion polymerization using N-isopropylacrylamide (NIPA), N,N'-methylenebisacrylamide (MBA), 2,2-dimethoxy-2-phenylacetylphenone, N-tert-butylacrylamide (NTBA), and a pH-sensitive functional comonomer (acrylic acid, methacrylic acid, ethacrylic acid, or propacrylic acid). The diameter of the microspheres was between 0.5 and 1.0 µm. These microspheres were cast into hydrogel membranes prepared by mixing the pH-sensitive swellable polymer particles with aqueous polyvinyl alcohol (PVA) solutions followed by crosslinking with glutaric dialdehyde for use as pH sensors. Large changes in the turbidity of the PVA membrane were observed as the pH of the buffer solution in contact with the membrane was varied. These changes were monitored by UV-visible absorbance spectroscopy. Polymer swelling of many NIPA copolymers was reversible and independent of the ionic strength of the buffer solution in contact with the membrane. Both the degree of swelling and the apparent pKa of the polymer microspheres increased with temperature. Furthermore, the apparent pKa of the polymer particles could be tuned to respond sharply to pH in a broad range (pH 4.0-7.0) by varying the amount of crosslinker (MBA) and transition temperature modifier (NTBA), and the amount, pKa, and hydrophobicity of the pH-sensitive functional comonomer (alkyl acrylic acid) used in the formulation. Potential applications of these polymer particles include fiber optic pH sensing where the pH-sensitive material can be immobilized on the distol end of an optical fiber.

Dihydroquinolines, Dihydronaphthyridines and Quinolones by Domino Reactions of Morita-Baylis-Hillman Acetates.

An efficient synthetic route to highly substituted dihydroquinolines and dihydronaphthyridines has been developed using a domino reaction of Morita-Baylis-Hillman (MBH) acetates with primary aliphatic and aromatic amines in DMF at 50-90 °C. The MBH substrates incorporate a side chain acetate positioned adjacent to an acrylate or acrylonitrile aza-Michael acceptor as well as an aromatic ring activated toward SNAr ring closure. A control experiment established that the initial reaction was an SN2'-type displacement of the side chain acetate by the amine to generate the alkene product with the added nitrogen nucleophile positioned trans to the SNAr aromatic ring acceptor. Thus, equilibration of the initial alkene geometry is required prior to cyclization. A further double bond migration was observed for several reactions targeting dihydronaphthyridines from substrates with a side chain acrylonitrile moiety. MBH acetates incorporating a 2,5-difluorophenyl moiety were found to have dual reactivity in these annulations. In the absence of O2, the expected dihydroquinolines were formed, while in the presence of O2, quinolones were produced. All of the products were isolated in good to excellent yields (72-93%). Numerous cases (42) are reported, and mechanisms are discussed.

Tetrahydroquinoline units in flexible heteroarotinoids (Flex-Hets) convey anti-cancer properties in A2780 ovarian cancer cells.

SHetA2 (NSC 721689), our lead Flex-Het anti-cancer agent, consists of a thiochroman (Ring A) and a 4-nitrophenyl (Ring B) linked by a thiourea bridge. In this work, several series of new analogs having a tetrahydroquinoline (THQ, Ring A) unit connected by a urea or thiourea linker to a 4-substituted phenyl (Ring B) have been prepared and evaluated relative to SHetA2 in terms of binding affinity with mortalin and inhibition of A2780 ovarian cancer cells. Six of the derivatives equaled or exceeded the efficacy shown by SHetA2. Compounds 1a-d (series 1), lacking a methyl on the Ring A nitrogen and the gem-dimethyls on the adjacent carbon, showed only weak activity. Salt 2, the quaternized N,N-dimethyl iodide salt analog of 1a, also possessed very modest growth inhibition in the cell line studied. Series 3 compounds, which had a C3 ketone and an N-methyl replacing the sulfur in Ring A, were most successful. Compound 3a [Ring A = 1,2,2,4,4-pentamethyl-3-oxo-1,2,3,4-tetrahydroquinolin-6-yl; urea linker; Ring B = 4-nitrophenyl] had slightly lower potency (IC50 3.8 µM), but better efficacy (94.8%) than SHetA2 (IC50 3.17 µM, efficacy 84.3%). In addition, 3c and 3d [urea and thiourea linkers, respectively; Ring B = 4-(trifluoromethyl)phenyl] and 3e and 3f [urea and thiourea linkers, respectively; Ring B = 4-(trifluoromethoxy)phenyl] were also evaluated since these agents possessed electron-withdrawing groups with H-bonding capability. All displayed good activity. Compounds 3c and 3e showed improvement in both potency and efficacy compared to SHetA2. In general, when the linker group between Rings A and B was a urea, efficacy values slightly exceeded those with a thiourea linker in the carbonyl-containing THQ systems 3a-g. In contrast, when Ring A possessed the 1,2,2,4,4-pentamethyl-3-hydroxytetrahydroquinolin-6-yl unit (4a-f, series 4), very modest potency and efficacy was observed. Model compound 5, an exact N-methyl THQ analog of SHetA2, demonstrated less potency (IC50 4.5 µM), but improved efficacy (91.7%). Modeling studies were performed to rationalize the observed results.

Naphthalenes and Quinolines by Domino Reactions of Morita-Baylis-Hillman Acetates.

An efficient synthetic route to highly functionalized naphthalenes and quinolines has been developed using domino reactions between Morita-Baylis-Hillman (MBH) acetates and active methylene compounds (AMCs) promoted by anhydrous K2CO3 in dry N,N-dimethylformamide (DMF) at 23 °C. The substrates incorporate allylic acetates positioned adjacent to a Michael acceptor as well as an aromatic ring activated toward a SNAr ring closure. A control experiment indicated that the initial reaction was an SN2'-type displacement of a side chain acetoxy by the AMC anion to afford the alkene product bearing the added nucleophile trans to the SNAr aromatic ring acceptor. Thus, equilibration of the alkene geometry of the initial product was required prior to cyclization. Products were isolated in good to excellent yields. Numerous cases (24) are reported, and several mechanistic possibilities are discussed.

Swellable Copolymers of N-isopropylacrylamide and Alkyl Acrylic Acids for Optical pH Sensing.

Swellable polymers that respond to pH (including a portion of the physiological pH range) have been prepared from N-isopropylacrylamide (NIPA) copolymerized with acrylic acid, methacrylic acid, ethacrylic acid or propacrylic acid by dispersion polymerization. When the swellable polymer particles are dispersed in a polyvinyl alcohol (PVA) hydrogel membrane, large changes occur in the turbidity of the membrane (which is measured using an absorbance spectrometer) as the pH of the buffer solution in contact with the hydrogel membrane is varied. The swelling of the NIPA copolymer is nonionic, as the ionic strength of the buffer solution in contact with the PVA membrane was increased from 0.1 to 1.0 M without a decrease in the swelling. For many of these NIPA copolymers, swelling was also reversible in both low- and high ionic strength pH-buffered media and at ambient and physiological temperatures. The composition of the formulation used to prepare these copolymers of NIPA can be correlated to the enthalpy and entropy of the pH-induced swelling.

Small Molecule Inhibitors of the BfrB-Bfd Interaction Decrease Pseudomonas aeruginosa Fitness and Potentiate Fluoroquinolone Activity.

The iron storage protein bacterioferritin (BfrB) is central to bacterial iron homeostasis. The mobilization of iron from BfrB, which requires binding by a cognate ferredoxin (Bfd), is essential to the regulation of cytosolic iron levels in P. aeruginosa. This paper describes the structure-guided development of small molecule inhibitors of the BfrB-Bfd protein-protein interaction. The process was initiated by screening a fragment library and followed by obtaining the structure of a fragment hit bound to BfrB. The structural insights were used to develop a series of 4-(benzylamino)- and 4-((3-phenylpropyl)amino)-isoindoline-1,3-dione analogs that selectively bind BfrB at the Bfd binding site. Challenging P. aeruginosa cells with the 4-substituted isoindoline analogs revealed a dose-dependent growth phenotype. Further investigation determined that the analogs elicit a pyoverdin hyperproduction phenotype that is consistent with blockade of the BfrB-Bfd interaction and ensuing irreversible accumulation of iron in BfrB, with concomitant depletion of iron in the cytosol. The irreversible accumulation of iron in BfrB prompted by the 4-substituted isoindoline analogs was confirmed by visualization of BfrB-iron in P. aeruginosa cell lysates separated on native PAGE gels and stained for iron with Ferene S. Challenging P. aeruginosa cultures with a combination of commercial fluoroquinolone and our isoindoline analogs results in significantly lower cell survival relative to treatment with either antibiotic or analog alone. Collectively, these findings furnish proof of concept for the usefulness of small molecule probes designed to dysregulate bacterial iron homeostasis by targeting a protein-protein interaction pivotal for iron storage in the bacterial cell.

4H-Benzo[d][1,3]oxazin-4-ones and Dihydro Analogs from Substituted Anthranilic Acids and Orthoesters.

A one-pot route to 2-alkyl and 2-aryl-4H-benzo[d][1,3]oxazin-4-ones (also known as 4H-3,1-benzoxazin-4-ones) has been developed and studied. The method involves the reaction of aryl-substituted anthranilic acids with orthoesters in ethanol catalyzed by acetic acid. Additionally, we have also investigated the reaction under microwave conditions. Not all of the substrates were successful in yielding the target heterocycles as some of the reactions failed to undergo the final elimination. This process led to the isolation of (±)-2-alkyl/aryl-2-ethoxy-1,2-dihydro-4H-benzo[d][1,3]oxazin-4-ones. The formation of the dihydro analogs correlated with the electron density on the aromatic ring: Electron-donating groups favored the 4H- benzo[d][1,3]oxazin-4-ones, while electron-withdrawing groups tended to favor the dihydro product. Substituting a pyridine ring for the benzene ring in the substrate acid suppressed the reaction.

Quinazolin-4(3H)-ones and 5,6-Dihydropyrimidin-4(3H)-ones from ß-Aminoamides and Orthoesters.

Quinazolin-4(3H)-ones have been prepared in one step from 2-aminobenzamides and orthoesters in the presence of acetic acid. Simple 2-aminobenzamides were easily converted to the heterocycles by refluxing in absolute ethanol with 1.5 equivalents of the orthoester and 2 equivalents of acetic acid for 12⁻24 h. Ring-substituted and hindered 2-aminobenzamides as well as cases incorporating an additional basic nitrogen required pressure tube conditions with 3 equivalents each of the orthoester and acetic acid in ethanol at 110 °C for 12⁻72 h. The reaction was tolerant towards functionality on the benzamide and a range of structures was accessible. Workup involved removal of the solvent under vacuum and either recrystallization from ethanol or trituration with ether-pentane. Several 5,6-dihydropyrimidin-4(3H)-ones were also prepared from 3-amino-2,2-dimethylpropionamide. All products were characterized by melting point, FT-IR, ¹H-NMR, 13C-NMR, and HRMS.

Syntheses of 1-Aryl-5-nitro-1H-indazoles and a General One-Pot Route to 1-Aryl-1H-indazoles.

An efficient route to substituted 1-aryl-1H-indazoles has been developed and optimized. The method involved the preparation of arylhydrazones from acetophenone or benzaldehyde substituted by fluorine at C2 and nitro at C5, followed by deprotonation and nucleophilic aromatic substitution (SNAr) ring closure in 45-90%. Modification of this procedure to a one-pot domino process was successful in the acetophenone series (73-96%), while the benzaldehyde series (63-73%) required a step-wise addition of reagents. A general one-pot protocol for 1-aryl-1H-indazole formation without the limiting substitution patterns required for the SNAr cyclization has also been achieved in 62-78% yields. A selection of 1-aryl-1H-indazoles was prepared in high yield by a procedure that requires only a single laboratory operation.

Direct Synthesis of Thioethers from Carboxylates and Thiols Catalyzed by FeCl3.

A new and efficient method has been developed for the synthesis of thioethers from carboxylates and thiols. The reaction proceeds via a Fe(III)-catalyzed direct displacement of carboxylates from benzylic or allylic esters by heterocyclic thiols. Short reaction times, good to excellent yields of products, and few side reactions are the significant features of the new protocol.

Modified 2,4-diaminopyrimidine-based dihydrofolate reductase inhibitors as potential drug scaffolds against Bacillus anthracis.

The current Letter describes the synthesis and biological evaluation of dihydrophthalazine-appended 2,4-diaminopyrimidine (DAP) inhibitors (1) oxidized at the methylene bridge linking the DAP ring to the central aromatic ring and (2) modified at the central ring ether groups. Structures 4a-b incorporating an oxidized methylene bridge showed a decrease in activity, while slightly larger alkyl groups (CH2CH3 vs CH3) on the central ring oxygen atoms (R(2) and R(3)) had a minimal impact on the inhibition. Comparison of the potency data for previously reported RAB1 and BN-53 with the most potent of the new derivatives (19 b and 20a-b) showed similar values for inhibition of cellular growth and direct enzymatic inhibition (MICs 0.5-2 µg/mL). Compounds 29-34 with larger ester and ether groups containing substituted aromatic rings at R(3) exhibited slightly reduced activity (MICs 2-16 µg/mL). One explanation for this attenuated activity could be encroachment of the extended R(3) into the neighboring NADPH co-factor. These results indicate that modest additions to the central ring oxygen atoms are well tolerated, while larger modifications have the potential to act as dual-site inhibitors of dihydrofolate reductase (DHFR).

OSU-6: A Highly Efficient, Metal-Free, Heterogeneous Catalyst for the Click Synthesis of 5-Benzyl and 5-Aryl-1H-tetrazoles.

OSU-6, an MCM-41 type hexagonal mesoporous silica with mild Brönsted acid properties, has been used as an efficient, metal-free, heterogeneous catalyst for the click synthesis of 5-benzyl and 5-aryl-1H-tetrazoles from nitriles in DMF at 90 °C. This catalyst offers advantages including ease of operation, milder conditions, high yields, and reusability. Studies are presented that demonstrate the robust nature of the catalyst under the optimized reaction conditions. OSU-6 promotes the 1,3-dipolar addition of azides to nitriles without significant degradation or clogging of the nanoporous structure. The catalyst can be reused up to five times without a significant reduction in yield, and it does not require treatment with acid between reactions.

Evaluation of New Dihydrophthalazine-Appended 2,4-Diaminopyrimidines against Bacillus anthracis: Improved Syntheses Using a New Pincer Complex.

The synthesis and evaluation of ten new dihydrophthalazine-appended 2,4-diaminopyrimidines as potential drugs to treat Bacillus anthracis is reported. An improved synthesis utilizing a new pincer catalyst, dichlorobis[1-(dicyclohexylphosphanyl)-piperidine]palladium(II), allows the final Heck coupling to be performed at 90 °C using triethylamine as the base. These milder conditions have been used to achieve improved yields for new and previously reported substrates with functional groups that degrade or react at the normal 140 °C reaction temperature. An analytical protocol for separating the S and R enantiomers of two of the most active compounds is also disclosed. Finally, the X-ray structure for the most active enantiomer of the lead compound, (S)-RAB1, is given.

The structure and competitive substrate inhibition of dihydrofolate reductase from Enterococcus faecalis reveal restrictions to cofactor docking.

We are addressing bacterial resistance to antibiotics by repurposing a well-established classic antimicrobial target, the dihydrofolate reductase (DHFR) enzyme. In this work, we have focused on Enterococcus faecalis, a nosocomial pathogen that frequently harbors antibiotic resistance determinants leading to complicated and difficult-to-treat infections. An inhibitor series with a hydrophobic dihydrophthalazine heterocycle was designed from the anti-folate trimethoprim. We have examined the potency of this inhibitor series based on inhibition of DHFR enzyme activity and bacterial growth, including in the presence of the exogenous product analogue folinic acid. The resulting preferences were rationalized using a cocrystal structure of the DHFR from this organism with a propyl-bearing series member (RAB-propyl). In a companion apo structure, we identify four buried waters that act as placeholders for a conserved hydrogen-bonding network to the substrate and indicate an important role in protein stability during catalytic cycling. In these structures, the nicotinamide of the nicotinamide adenine dinucleotide phosphate cofactor is visualized outside of its binding pocket, which is exacerbated by RAB-propyl binding. Finally, homology models of the TMP(R) sequences dfrK and dfrF were constructed. While the dfrK-encoded protein shows clear sequence changes that would be detrimental to inhibitor binding, the dfrF-encoded protein model suggests the protein would be relatively unstable. These data suggest a utility for anti-DHFR compounds for treating infections arising from E. faecalis. They also highlight a role for water in stabilizing the DHFR substrate pocket and for competitive substrate inhibitors that may gain advantages in potency by the perturbation of cofactor dynamics.

Structure-activity relationship for enantiomers of potent inhibitors of B. anthracis dihydrofolate reductase.

BACKGROUND: Bacterial resistance to antibiotic therapies is increasing and new treatment options are badly needed. There is an overlap between these resistant bacteria and organisms classified as likely bioterror weapons. For example, Bacillus anthracis is innately resistant to the anti-folate trimethoprim due to sequence changes found in the dihydrofolate reductase enzyme. Development of new inhibitors provides an opportunity to enhance the current arsenal of anti-folate antibiotics while also expanding the coverage of the anti-folate class. METHODS: We have characterized inhibitors of B. anthracis dihydrofolate reductase by measuring the K(i) and MIC values and calculating the energetics of binding. This series contains a core diaminopyrimidine ring, a central dimethoxybenzyl ring, and a dihydrophthalazine moiety. We have altered the chemical groups extended from a chiral center on the dihydropyridazine ring of the phthalazine moiety. The interactions for the most potent compounds were visualized by X-ray structure determination. RESULTS: We find that the potency of individual enantiomers is divergent with clear preference for the S-enantiomer, while maintaining a high conservation of contacts within the binding site. The preference for enantiomers seems to be predicated largely by differential interactions with protein residues Leu29, Gln30 and Arg53. CONCLUSIONS: These studies have clarified the activity of modifications and of individual enantiomers, and highlighted the role of the less-active R-enantiomer in effectively diluting the more active S-enantiomer in racemic solutions. This directly contributes to the development of new antimicrobials, combating trimethoprim resistance, and treatment options for potential bioterrorism agents.